ijfs#1336_bozza ok ital. j. food sci., vol. 31, 2019 459 paper factors affecting the spectrophotometric quantification of flavonoids in wine o. corona*1, m. squadrito1 and a. tirelli2 3dipartimento scienze agrarie, alimentari e forestali, università di palermo, viale delle scienze 4, 90128 palermo, italy 3dipartimento di scienze per gli alimenti, la nutrizione e l’ambiente, università di milano, via celoria 2, 20133 milano, italy *corresponding author: tel.: +3909123897058; fax: +39091484035 e-mail address: onofrio.corona@unipa.it abstract the quantification of flavonoids in wine and grape skin extract by spectrophotometric evaluation at 280 nm wavelength provides essential information to oenologist concerning wine composition and evolution, and it is commonly applied in wine labs. the measurement of the absorption peak height at 280 nm reported by di stefano and guidoni (1989) allows to selectively quantify flavonoids with minor interferences. however, it has proved to be susceptible to so2 at low ph or acetone in unpurified grape skin extracts. moreover, the effect of ph on flavonoids quantification in wine, either containing so2 or not, has not been assessed. the effect of so2, purification, ph and dilution solvent on spectrophotometric quantification of flavonoids in red wine samples has been evaluated in this work. so2 can overrate the flavonoids content in red wine when ethanol and clions are contained in acid dilution solvents. a wine sample dilution with a strong acid solvent is mandatory to attain a reliable quantification of flavonoids due to the low anthocyanins absorption at 280 nm in water solution. a minor effect arises from the ethanol content. eventually, flavonoids can be quantified in so2-containing wine diluted with a strong acid solution but a 7% overrating should be expected. keywords: polyphenol index, flavonoids, anthocyanins, so2; hyperchromic effect, bathochromic effect ital. j. food sci., vol. 31, 2019 460 1. introduction the amount of polyphenols, especially flavan-3-ols and anthocyanins, affects astringency, bitter taste and green/woody properties of red wine (gibbins and carpenter, 2013; soares et al., 2015). their fast quantification in wine-making and wine ageing is crucial in winery since it makes it possible to carefully address the oenological choices involving the duration and conditions of maceration and ageing. flavonoids are quantitatively the main phenol fraction in red wine by far; therefore, many analytical methods are based on the quantification of total polyphenols (aleixandre-tudo et al., 2017). however, many of them are poorly selective or accurate (folin and denis, 1912; singleton and rossi, 1965) and require quite complex analytical approaches (garcía-guzmán et al., 2015) or even expensive analytical instrumentation fo r quality control laboratories (kennedy and jones, 2001). the spectrophotometric methods are still among the fastest, easy to apply and cheapest for the oenologist; therefore, they are usually applied in the winery laboratories (aleixandre-tudo et al., 2017). the spectrophotometric analytical approach reported by di stefano and guidoni (1989) is widespread and routinely applied in the wineries to achieve a fast and reliable evaluation of flavonoids in grape extract and wine. it is based on the absorption spectrum obtained in the wavelength range 230–700 nm of a diluted sample. the peak height measured at 280 nm (e280) subtracted from the absorbance measured at its valley-to-valley baseline returns the absorbance value mainly due to the flavonoids (e’280). such an approach allows to avoid the interference due to compounds without an absorption peak at 280 nm like aromatic amino acids, nucleosides and nucleotides (somers and ziemelis, 1985). wine dilution with strong acid solutions allows to quantify the total anthocyanin content based on the height of the absorbance peak at about 520 nm of the spectra. an easier wine dilution with distilled water is commonly applied to assess the total flavonoids based on the e’280. however, there is a lack of information about the analytical factors affecting the accuracy of this approach, in spite of its widespread use at wine control laboratories. corona et al. (2015) pointed out the interference exerted by so2 (as an undissociated molecular form) in quantifying flavonoids extracted from the grape berry and dissolved in a strong acid solvent like ethanol-hydrochloric acid mixture (etoh-hcl). a further interference can arise from residual amounts of acetone used as extraction solvent of phenols. both so2 and acetone can be easily removed by solid phase extraction (spe) packed with a c18 resin (corona at al., 2015). it is well-known that sulfites can negatively affect the spectrophotometric quantification of anthocyanins at wine ph and acetaldehyde is needed to effectively remove the so2 bound to anthocyanins (usseglio-tomasset et al., 1982; mazza et al., 1999). recently, so2 proved capable of forming sulfonated adducts of flavan-3-ols over wine aging (arapitsas et al., 2014). the binding involves the c4 position of the flavan ring and only monomeric flavan-3-ols and the terminal flavanol unit of proanthocyanindins are expected to undergo sulfonation in time. sulfonation of elongation flavanol units has not been reported, possibly owing to steric hindrance issues. the spectrophotometric properties of flavanol-sulfite adducts are unknown, as well as their role in flavonoid quantification. however, their low relative abundance has to be considered, especially in young wine (arapitsas et al., 2018). poor information is available about the role exerted by so2 in wine concerning the quantification of flavonoids based on the e’280 value, especially when strongly acidic solutions (ph < 1) are used as a dilution solvent to attain the quantification of anthocyanins in the meantime. moreover, there is a lack of information about how the composition and acidity of the dilution solvent affect the quantification of total flavonoids assessed using the e’280 value. ital. j. food sci., vol. 31, 2019 461 in this work the effects of so2, ethanol concentration, acid and ph on the absorbance values e280 and e’280 assessed in diluted red wine samples were assessed to monitor their role on the quantification of wine flavonoids. 2. materials and methods 2.1. chemicals methanol, ethanol, sulphuric acid, hydrochloric acid, tartaric acid, ethanol, sodium hydroxide, citric acid monohydrate, potassium phosphate monobasic, sodium phosphate dibasic, hydrogen peroxide solution (30% w/w in water), ethyl acetate, polyvinylpolypyrrolidone (pvpp) and bromocresol green methyl red indicator were purchased from sigma-aldrich (st. louis, mo, usa). sodium metabisulphite and phosphoric acid were purchased from j.t. baker (deventer, holland). seeds and white and red grape tannins were provided by bono and ditta s.p.a. italian grape juice from campobello di mazara (trapani, italy). 2.2. wine samples sixty different commercial red wine samples produced in the years 2013–2015 were collected at the market and submitted for the evaluation of total anthocyanin and total flavonoid contents. moreover, the spectrophotometric response obtained following different dilution conditions of eight samples of red wine obtained from nero d’avola grape (vintages 2013–2015), nerello mascarese grape (vintage 2015), cabernet sauvignon grape (vintage 2014 and 2015) and merlot (vintage 2014) was assessed. all measurements were performed in triplicate. 2.3. purification of flavonoids half millilitre of wine sample was diluted with 5 ml h2so4 5 mm and loaded into a 400 mg c18 spe cartridge (sep-pak, waters, milan, italy) previously conditioned with 2 ml methanol and then 3 ml h2so4 5 mm. the polar compounds were eluted with 3 ml h2so4 5 mm to drying and discarded, then the phenols were collected into a 25 ml volumetric flask by eluting with 3 ml methanol and brought to volume with one of the following solvents: h2o, 0.5 m h2so4, ethanol:h2o:12 m hcl 70:30:1 (v/v/v) (etoh-hcl). the same solutions were also used for diluting 0.5 ml of the wine samples to 25 ml in volumetric flasks. triplicate preparations were carried out. 2.4. determination of total flavonoids flavonoids were purified by treatment with spe procedure. the uv-visible absorption spectra in the range 230–700 nm wavelength of either unpurified or purified flavonoids were recorded, and the absorption values at 280 nm (e280) were measured. triplicate preparations were carried out. the e’280 value was also measured according to di stefano and guidoni (1989) and modified by corona et al. (2015). the total flavonoid content was calculated according to di stefano and guidoni (1989) and corona et al. (2010) as follows: total flavonoids (as mg/l (+)-catechin equivalent): 82.4 × e’280 × 50. ital. j. food sci., vol. 31, 2019 462 2.5. absorbance parameters of white grape skin extract buffered solutions at ph 1.1, 3.0, 5.0 and 7.0 were prepared according to küster et al. (1979) and used for dissolving 30 mg/l grape skin extract. their uv-visible absorption spectra in the range 230-400 nm wavelength were recorded and the values of λmax, e280 and e’280 were measured. triplicate preparations were carried out. 2.6. purification of anthocyanins from red grape skin extract phenols from red grape skin extract were obtained from the skin of 50 berries by using a tartaric buffer (5 g tartaric acid, 22 ml naoh 1 n, 2 g na2s2o5, 125 ml ethanol 95–96%, brought to 1 l with h2o). anthocyanins were obtained from the extract as follows. three millilitres of h2so4 0.5 m and 6 g of pvpp were added to 60 ml of skin extract. the mixture was stirred for 2 min, then centrifuged at 2000 g × 10 min and the pvpp was recovered and then rinsed with 20 ml of h2so4. the mixture was centrifuged as above and the pvpp was recovered. the anthocyanins absorbed on the pvpp were dissolved by dispersing the pvpp into 15 ml etoh-hcl solution and centrifuging at 2000 g × 10 min. the addition of etoh-hcl solution and the centrifugation were carried out four times again, and all the five supernatants were collected and blended in a 100 ml evaporation flask. the ethanol contained in the anthocyanins solution was removed by vacuum-drying and the water solution was transferred in a 100 ml extraction funnel. the residual flavan3-ols were removed by a triplicate extraction with 10 ml ethyl acetate each. the purified anthocyanin extract was transferred in a 100 ml evaporation flask and the residual ethyl acetate was removed by vacuum drying. finally, the dried anthocyanins were dissolved with 50 mm h2so4 10 ml and recovered. 2.7. absorbance parameters of anthocyanins one millilitre of either red skin extract or purified anthocyanins solution was diluted to 25 ml with buffer solutions at ph 1.1, 3.0, 5.0 and 7.0 prepared according to küster et al. (1979) or with 0.1 m hcl solutions containing 10, 20, 40 or 80% ethanol. their uv-visible absorption spectra in the range 230–700 nm wavelength was recorded, and the values of maximum absorption wavelengths in the range 275-282 nm (λmaxuv) and in the range 510-550 nm (λmaxvis) were measured, as well as their absorption values (e280, e’280, e520). 2.8. determination of so2 in wine samples the so2 content in wine was carried out according to the functional eec in 2376 (1990) standard procedures. triplicate determinations were carried out. 2.9 statistical analysis analysis of variance (anova) and tukey’s honestly significant difference (hsd) test to calculate significant differences between treatments were carried out. all tests were performed at a significance level of p < 0.05 using the statistical program spss (ver. 13, ibm, armonk, ny, usa). ital. j. food sci., vol. 31, 2019 463 3. results and discussion a fast quantification of total flavonoids and anthocyanins in wine can be achieved by measuring the spectrophotometric values e’280 and e520 of the sample diluted with an acid solution. however, such an approach can overrate the flavonoid content owing to the presence of gallic acid. so2 has an absorption peak close to 280 nm (276 nm) and diluting wine in strong acid solutions might increase the e’280 value, thus inducing a major overrating of the flavanol concentration (corona et al., 2015). wine dilution with ethanol-hcl can further increase the absorbance of so2 owing to the bathochromic and hyperchromic effects induced by ethanol and cl-, respectively. so2 can be removed from the wine sample by spe packed with a c18 resin (corona et al., 2015). to assess the effect of sample purification on the spectrophotometric quantification of flavonoids, the analytical responses of 61 spe-treated and untreated red wine samples, both of them diluted in an ethanol-hcl solution, were compared (fig. 1). figure 1. comparison of e’280 values obtained for wine samples and their corresponding spe-treated wine (n=3). all the samples were diluted with etoh-hcl solution. a good correlation (r2 = 0.979) was obtained; however, the slope of the regression line shows the e’280 values of wine were 6-7% higher than the corresponding spe-treated wine. such an overrating was expected as spe purification removes the polar phenols unretained on the spe resin, namely gallic acid, tyrosine and tyrosol (di stefano and guidoni, 1989). however, the role of so2 is hard to assess in unknown samples, even though it was proved in the previous work of corona et al. (2015). therefore, a known addition of so2 in real wine samples is expected to increase the e’280 value, but such an interference is hard to quantify owing to the occurrence of different ph values as well as quality and content of so2-binding compounds (ethanal, anthocyanins, pyruvate or other carbonyl compounds). to better focus the interference of so2 on the quantification of flavonoids, the absorption spectra obtained from red wine samples containing different concentrations of so2 either submitted or not to spe purification of flavonoids and diluted with acid solutions with different ph values were compared (table 1). following the purification step, the e280 values of the acid-diluted samples decreased by up to -20% in accordance with the work of somers and zimelis (1985) (table 1). ital. j. food sci., vol. 31, 2019 464 table 1. value of λmax, e280 (as au) e’280 (as au), flavonoids (as mg/l (+)-catechin equivalent) and so2 (mg/l) in wine and spe-treated wine samples diluted with different solutions. wine and vintage year wine spe-treated wine so2 level in wine h2o 5 × 10 -1 m h2so4 ethanol-hcl h2o 5 × 10-1 m h2so4 ethanol-hcl total free nero d'avola 13 λmax 276.0±0.00 a 277.5±0.71b 278.5±0.71b 278.0±0.00a 279.0±0.00a 280.0±0.00a 59.5±1.2 17.3±0.3 e280 0.428±0.01 a 0.466±0.02b 0.484±0.00b 0.378±0.00a 0.400±0.00b 0.390±0.00ab e'280 0.129±0.00 a 0.168±0.00b 0.175±0.00b 0.126±0.00a 0.162±0.00b 0.163±0.00b total flavonoids 1068±11a 1384±87b 1445±9b 1037±12a 1337±22b 1345±11b nero d'avola 13 λmax 277.0±0.00 a 277.0±0.00a 279.0±0.00a 278.5±0.71a 279.0±0.00a 280.0±0.00a 61.1±1.1 17.9±0.1 e280 0.450±0.00 a 0.504±0.01b 0.543±0.00c 0.384±0.07a 0.446±0.01b 0.435±0.00b e'280 0.148±0.00 a 0.186±0.01b 0.201±0.01b 0.125±0.00a 0.184±0.01b 0.187±0.00b total flavonoids 1219±29a 1533±33b 1660±10c 1034±19a 1514±11b 1542±10b nero d'avola 14 λmax 276.0±0.00 a 276.5±0.71b 278.0±0.00b 278.0±0.00a 278.0±0.00a 280.0±0.00a 1.3±0.0 0.6±0.0 e280 0.384±0.01 a 0.403±0.00ab 0.419±0.01b 0.342±0.00a 0.358±0.00b 0.345±0.00a e'280 0.114±0.00 a 0.131±0.00ab 0.140±0.00b 0.106±0.00a 0.136±0.00b 0.130±0.00b total flavonoids 938±22a 1076±43b 1154±43b 873±4a 1122±22c 1071±11b nero d'avola14 λmax 276.0±0.00 a 276.5±0.71ab 278.0±0.00b 278.0±0.00a 278.5±0.71a 280.0±0.00a 1.3±0.0 0.6±0.0 e280 0.415±0.00 a 0.431±0.00ab 0.456±0.02b 0.368±0.00a 0.395±0.01a 0.397±0.00a e'280 0.122±0.00 a 0.139±0.01ab 0.160±0.01b 0.120±0.00a 0.146±0.01b 0.154±0.01b total flavonoids 1007±33a 1145±20ab 1314±120b 992±11a 1203±20b 1273±9c nero d'avola 15 λmax 276.0±0.00 a 277.0±0.00a 279.0±0.00b 278.0±0.00a 279.0±0.00a 280.0±0.00a 24.3±1.2 8.7±0.5 e280 0.415±0.00 a 0.432±0.01a 0.447±0.01a 0.379±0.00a 0.385±0.00b 0.389±0.02b e'280 0.122±0.01 a 0.146±0.01ab 0.157±0.01b 0.124±0.00a 0.151±0.00b 0.155±0.01b total flavonoids 1005±11a 1204±20b 1291±43b 1018±4a 1245±21b 1276±22b nero d'avola 15 λmax 278.0±0.00 a 278.5±0.71a 279.0±0.00a 279.0±0.00a 279.0±0.00a 280.0±0.00a 34.3±4.2 11.3±2.4 e280 0.357±0.00 a 0.379±0.00a 0.381±0.01a 0.323±0.00a 0.319±0.00a 0.319±0.00a e'280 0.110±0.00 a 0.112±0.00a 0.113±0.01a 0.105±0.00a 0.107±0.00a 0.108±0.00a total flavonoids 910±4a 923±15a 930±11a 869±11a 884±11a 892±20a ital. j. food sci., vol. 31, 2019 465 nerello mascalese 15 λmax 276.0±0.00 a 277.0±0.00a 278.0±0.00a 278.0±0.00a 278.0±0.00a 279.5±0.71a 35.7±4.2 13.0±2.9 e280 0.344±0.01 a 0.346±0.00a 0.360±0.01a 0.285±0.01a 0.292±0.00a 0.286±0.00a e'280 0.125±0.00 a 0.130±0.00a 0.144±0.00b 0.119±0.01a 0.118±0.00a 0.126±0.00a total flavonoids 1030±22a 1069±10a 1184±22b 982±20a 970±21a 1038±11b cabernet sauvignon 15 λmax 276.0±0.00 a 277.0±0.00a 279.0±0.00b 278.0±0.00a 277.0±0.00a 279.0±0.00a 86.8±7.2 27.9±4.1 e280 0.565±0.00 a 0.591±0.01b 0.626±0.00c 0.514±0.01a 0.585±0.00c 0.542±0.01b e'280 0.185±0.00 a 0.217±0.01b 0.236±0.01c 0.176±0.01a 0.212±0.00b 0.215±0.01b total flavonoids 1524±28a 1786±30b 1948±15c 1453±15a 1736±48b 1772±6b λmax 276.0±0.00 a 277.5±0.71b 278.5±0.71b n.d. n.d. n.d. 62.1±1.3 22.5±0.75 e280 0.416±0.00 a 0.473±0.01b 0.491±0.01b nero d'avola 14 e'280 0.127±0.00 a 0.171±0.01b 0.174±0.01b total flavonoids 1045±15b 1407±23b 1430±17b λmax 276.0±0.00 a 276.5±0.71ab 278.0±0.00b n.d. n.d. n.d. 27.2±1.5 10.8±0.05 e280 0.398±0.00 a 0.432±0.00b 0.479±0.00b nero d'avola 15 e'280 0.107±0.00 a 0.137±0.00b 0.141±0.01b total flavonoids 884±8a 1130±18b 1161±12b λmax 276.0±0.00 a 277.0±0.00a 278.0±0.00a n.d. n.d. n.d. 33.4±1.7 15.3±0.61 e280 0.369±0.00 a 0.402±0.00b 0.429±0.00b cabernet sauvignon 14 e'280 0.124±0.00 a 0.152±0.00b 0.160±0.00b total flavonoids 1022±10a 1253±12b 1322±31b λmax 276.0±0.00 a 277.0±0.00a 279.0±0.00b n.d. n.d. n.d. 89.7±9.1 30.4±0.60 e280 0.413±0.01 a 0.454±0.01b 0.492±0.01b merlot 14 e'280 0.132±0.01 a 0.166±0.01b 0.172±0.01b total flavonoids 1084±38a 1368±19b 1414±10b n = 3 samples; mean value ± standard deviation. n.d.: not determined. different letters in the same row indicate significant differences between wine samples or spe-treated wine samples (tukey’s hsd test, p < 0.05). ital. j. food sci., vol. 31, 2019 466 it is mainly due to the loss of purines, pyrimidines, nucleosides, amino acids and aromatic alcohols occurred following the purification procedure. therefore, the e280 value confirms to be an unsuitable index of the phenol content in wine. the quantification of flavonoids based on the e’280 value shows the major role of the dilution solvent on the measured values. the e’280 values obtained by water dilution of the wine samples were always significantly lower than the sample diluted with acid solutions. the e’280 value increases as the ph decreases, thus suggesting that the so2 plays a role. moreover, the hyperchromic effect detectable only in the wine samples diluted with ethanol-hcl solution further supports such a conclusion. however, a comparable or even higher increase of the e’280 value can be detected after the removal of so2 by spe, even in the samples containing negligible so2 level (nero d’avola 14). neither the bathochromic nor the hyperchromic effects detected in the wine samples, nero d’avola 14, ought to be observed when ethanolhcl instead of a h2so4 solution is used as dilution solvents. moreover, no increase of the e’280 value should be detected in the spe-treated samples following dilution with h2so4 if so2 had a major role. nonetheless, the calculated flavonoids content strongly increases as the ph of the dilution solvent decreases, even in the spe-treated wine samples (table 1). all these data highlight the minor contribution of so2 to the e’280 value and quantification of flavonoids in wine, while ph and solvent composition strongly affect the analytical response. since the variation of e’280 values also occurs with the spe-treated samples where the hydrophobic compounds eluted with methanol from the c18 resin are contained, phenols are likely involved in this behaviour. however, flavan-3-ols, either monomer or polymer, are not expected to be affected by ph values lower than 7, as their pka exceeds 9. therefore, ph variations in the range 0-7 should attain negligible dissociation effects whatever the alcohol content of the adopted diluting solvent (danilewicz, 2003; friedman and jürgens, 2000). as expected trials carried out at ph values spanning from 1 to 7 with different ethanol content did not show any significative effect on the e’280 values recorded for grape phenols extracted from white skin (table 2) and comparable results were obtained when phenols extracted from grape seeds were evaluated (data not shown). therefore, flavan-3-ols and proanthocyanidins, as well as other colourless skin or seed phenols (phenolic acids, flavonols, hydroxystilbenes), can hardly be responsible for the e’280 variations induced by ph and solvent differences. consequently, the role of anthocyanins was investigated. the absorption spectra of anthocyanins are affected by the ph. moreover, the e280 value of their flavilium ion is higher than its neutral form occurring in wine at ph values lower than 6 (março et al., 2011). the e’280 value obtained assessing anthocyanins from red grape skin extract treated by spe packed with pvpp significantly decreases as the ph increases. the e’280 values increase more than 15% by wine acidification (ph 3–4) down to ph 1 (table 1). such a change is lower than it occurs when the absorption peak at 520–540 nm is considered (see elmaxvis in table 3); nonetheless, it can strongly affect the spectrophotometric evaluation of flavonoid by the e’280, especially when wines containing a high amount of anthocyanins are considered (table 3). as the role of ethanol on the absorbance of anthocyanins in diluted wine is well-known (lee et al., 2005), the increase in e520 values following the increased ethanol concentration was expected. ethanol does not affect the e’280 value and only an ethanol level as high as 80% (v/v) shows a minor role. same results were obtained when hcl was replaced with h2so4 (data not shown). if grape skin extract is concerned, increasing e’280 values are clearly visible when the ethanol content increases due to the hyperchromic effect arising from the presence of so2 in the skin extract (table 3). ital. j. food sci., vol. 31, 2019 467 table 2. effect of ph and dilution solvent on the analytical response parameters of phenols extracted from white grape skin. λmax e280 e'280 ph 1.1 283±0.6a 1.193±0.00a 0.279±0.00a 3.0 284±0.6a 1.197±0.01a 0.280±0.00a 5.0 283±0.6a 1.195±0.00a 0.284±0.00a 7.0 283±0.6a 1.195±0.00a 0.284±0.00a ethanol %, 0.1 m hcl 0% 283±0.6a 1.145±0.00a 0.277±0.00ab 10% 283±0.6a 1.146±0.00a 0.279±0.00b 20% 282±0.0a 1.143±0.01a 0.275±0.00ab 40% 282±0.6a 1.143±0.00a 0.274±0.00ab 80% 282±0.0a 1.149±0.00a 0.270±0.00a n = 3 samples; mean value ± standard deviation. different letters in the same column indicate significant difference between dilution solvents (tukey’s hsd test, p < 0.05). table 3. effect of ph, dilution solvent and purification on some spectrophotometric parameters of grape skin anthocyanins. n = 3 samples; mean value ± standard deviation. different letters in the same column indicate significant difference between treatments (tukey’s hsd test, p < 0.05). 4. conclusions our data highlight that the dilution of wine with water to assess the total flavonoid content by the e’280 value prevents from the spectrophotometric interference of so2 in the analytical response unless it occurs at concentration values exceeding the permitted λmaxuv e280 e'280 λmaxvis eλmaxvis ph purified anthocyanin 1.1 277.0±0.0b 0.466±0.01c 0.221±0.02c 519±0.0a 0.618±0.01d 3.0 277.0±0.0b 0.414±0.00b 0.186±0.00bc 519±0.0a 0.464±0.00c 5.0 276.5±0.7b 0.371±0.00a 0.155±0.00b 519±0.0a 0.192±0.00b 7.0 275.0±0.0a 0.351±0.01a 0.099±0.00a 551±0.0b 0.088±0.00a ethanol %, 0.1 m hcl purified anthocyanin 0% 277.0±0.0a 0.436±0.00a 0.241±0.00b 519±0.0a 0.609±0.00a 20% 278.0±0.0a 0.437±0.00a 0.243±0.00b 528±0.0b 0.650±0.00b 40% 279.0±0.0a 0.435±0.00a 0.246±0.00b 536±0.0c 0.684±0.00c 80% 280.0±0.0a 0.431±0.00a 0.229±0.00a 544±0.0d 0.730±0.00d ethanol %, 0.1 m hcl red grape skin extract 0% 278.0±0.0a 0.854±0.04a 0.315±0.00a 520±0.0a 0.305±0.01a 20% 278.0±0.0a 0.882±0.01a 0.331±0.01a 527±0.0b 0.336±0.00ab 40% 279.0±0.0a 0.892±0.02a 0.334±0.01a 535±0.0c 0.358±0.01bc 80% 280.0±0.0a 0.916±0.03a 0.341±0.01a 542±0.0d 0.381±0.01c ital. j. food sci., vol. 31, 2019 468 amounts in wine. however, under such conditions the interference exerted by the polar non-flavonoid phenols and acid equilibrium of anthocyanins induces a biased quantification. on the other hand, wine dilution with etoh-hcl can induce an overrated quantification due to both the interference of so2 in low ph solutions and the hyperchromic effect exerted by cl-. such interferences can be avoided by carrying out the spe purification of wine and then diluting the sample with an acid solution (ph < 1). acknowledgments the authors thank prof. rocco di stefano for his valuable technical suggestions and dr fabio ditta of the “bono and ditta s.p.a. italian grape juice” campobello di mazara (sicily) for providing grape seed tannins. references aleixandre-tudo j.l., buica a., nieuwoudt h., aleixandre j.l. and du toit w. 2017. spectrophotometric analysis of phenolic compounds in grapes and wines. j. agric. food chem. 65:4009-4026. arapitsas p., speri g., angeli a., perenzoni d. and mattivi f. 2014. the influence of storage on the “chemical age” of red wines. metabolomics 10:816-832. arapitsas p., guella g. and mattivi f. 2018. the impact of so2 on wine flavanols and indoles in relation to wine style and age. sci. rep-uk 8:858. corona o., squadrito m., borsa d. and di stefano r. 2010. behaviour of some compounds with λmax at 280 nm in the determination of total flavonoids of grape skin extracts made from a hydroalcoholic so2-rich solvent. ital. j. food sci. 22:347-351. corona o., squadrito m., vento g., tirelli a. and di stefano r., 2015. over-evaluation of total flavonoids in grape skin extracts containing sulphur dioxide. food chem. 172:537-542. danilewicz j.c. 2003. review of reactions mechanism of oxygen and proposed intermediate reduction products in wine: central role of iron and copper. am. j. enol. vitic. 54:73-85. di stefano, r. and guidoni s. (1989). la determinazione dei polifenoli totali nei mosti e nei vini. vignevini 16:47-52. eec, 1990. european communities. commission regulation no. 2676/90 on “community analysis methods to use in wine sector”. official journal european communities l272/3.10.90. folin, o., and denis, w., 1912. on phosphotungstic-phosphomolybdic compounds as color reagents. j. biol. chem. 12:239-243. friedman m. and jürgens h.s., 2000. effect of ph on the stability of plant phenolic compounds. j. agric. food chem. 48:2101-10. garcía-guzmán j.j., hernández-artiga m.p., palacios-ponce de león l. and bellido-milla d. 2015. selective methods for polyphenols and sulphur dioxide determination in wines. food chem. 182:47-54. gibbins h.l. and carpenter g.h. 2013. alternative mechanisms of astringency – what is the role of saliva?. j. text. stud. 44:364-375. kennedy j.a. and jones g.p. 2001. analysis of proanthocyanidin cleavage products following acid-catalysis in the presence of excess phloroglucinol. j. agric. food chem. 49:1740-1746. küster f.w., thiel a. and fischbeck k. 1979. tabelle logaritmiche per chimici, farmacisti, medici e fisici. 11a ediz., milano, hoepli. lee j., durst r. w. and wrolstad r.e. 2005. determination of total monomeric anthocyanin pigment contentof fruit juices, beverages, natural colorants, and wines by the ph differential method: collaborative study. j. aoac int. 88:12691278. março p.h., poppi r.j., scarminio i.s. and tauler r. 2011. investigation of the ph effect and uv radiation on kinetic degradation of anthocyanin mixtures extracted from hibiscus acetosella. food chem. 125:1020-1027. ital. j. food sci., vol. 31, 2019 469 mazza g., fukumoto l., delaquis p., girard b. and ewert. b. 1999. anthocyanins, phenolics, and color of cabernet franc, merlot, and pinot noir wines from british columbia. j. agric. food chem. 47:4009-4017. usseglio-tomasset l., ciolfi g. and di stefano r. 1982. the influence of the presence of anthocyanins on the aseptic activity of sulfur dioxide towards yeasts. vini d’italia 24:86-94. singleton v.l. and rossi j.a. 1965. colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. am. j. enol. vitic. 16:144-158. soares s., kohl s., thalmann s., mateus n., meyerhof w. and de freitas v. 2015. different phenolic compounds activate distinct human bitter taste receptors. j. agric. food chem. 61:1525-1533. somers t.c. and ziemelis g. 1985. spectral evaluation of total phenolics components in vitis vinifera: grapes and wines. j. sci. food agric. 36:1275-1284. paper received july 21, 2018 accepted december 18, 2018 ijfs#1301_bozza ital. j. food sci., vol. 31, 2019 224 paper comparative study on total polyphenols content of tunisian wild rhus pentaphylla fruit extracts and the evaluation of their biological activities h. fadhil*1, f. mraihi1, j.k. cherif1,2 and m. sökmen3,4 1laboratory of application of chemistry to natural resources and substances and to the environment, lacresne, faculty of sciences bizerte, 7021 bizerte, university of carthage, tunisia 2preparatory institute for the studies of engineers of tunis, 1008 montfleury, 2 rue jawaharlal nehru tunis, tunisia 3king saud university, college of science, riyad 11451, saudi arabia 4konya food and agriculture university, faculty of engineering and architect, department of bioengineering, 42080 konya, turkey *corresponding author: tel.: +21695953982 e-mail address: hajerfdhil@gmail.com abstract this study was focused on determining the total polyphenols content and biological activities from wild rhus pentaphylla fruits at two different maturity stages, (stage 1: greenyellow fruits and stage 2: purple-red fruits highly significant (p≤0.01) increase was observed in total phenolic contents of fruits ranging from 158.65±0.06 to 177.57±0.06 mg eq.ag.100 mg-1dw. in contrast, both total flavonoid (from 157.34±0.07 to 152.69±0.14 mg eq.cat.100 mg-1dw) and condensed tannins contents (from 200.26±0.30 to 131.23±0.24 mg eq.cat.100 mg-1dw) showed dramatic decreases (p≤0.01). evaluation of biological activities has provided good effects. our results show the importance of using this wild fruits in pharmaceutical industries. keywords: rhus pentaphylla, ultrasonic extraction, total polyphenol content, antioxidant activity, antimicrobial potential ital. j. food sci., vol. 31, 2019 225 1. introduction the human body is constantly exposed to a multitude of microbes (bacteria, viruses, parasites, fungi) (tille, 2013). although it has a complex defense system that allows it to meet or host these microbes without allowing them to invade its tissues, it is sometimes necessary to use antimicrobial agents, antivirals etc. (hamblin and hasan, 2004). but in some cases, these agents are unable to treat certain infectious diseases (atanasov et al.,2015). faced with this incapacity, a trend has developed in recent years with the aim of finding new natural sources of plant-based bioactive molecules (carina, 2012). the genus rhus pentaphylla consists flowering species belonging to anacardiaceae family, often grows in non-agricultural areas and widely used in foods (lee et al., 2010). they are recognized in traditional medicine by their therapeutic interests (anticancer, antiinflammatory, antidiarrhea), and it provides bioproducts that have desirable biological activities: antifungal, anti-inflammatory, antimalarial, antimicrobial, antitumorigenic, antioxidant, antiviral and hypoglycemic (abed, 2013). the genus has several varieties in spain, in the region of malaga, as well, it is divided into northern africa, which is common in the west of algeria and morocco (rached, 2009). in tunisia, the genus rhus is represented by two species: rhus tripartita (ucria) and rhus pentaphylla which spreads in north and center of the country (itidel et al., 2013). r. pentaphylla fruits, fresh or dried are slightly acidic (lahsissene et al., 2010), and have a pleasant taste (rached, 2009), also it has been used in the treatment of diarrhea (lahsissene et al., 2010). this genus has also been introduced as a medicinal vascular plant protection and cardiovascular diseases preventive in traditional medicine (sabzghabaee et al., 2014). owing to its richness in tannins, flavonoids and coumarin, these natural molecules have anti-substantial activity of butyrylcholinesterase, and therefore can be used in the treatment of alzheimer's disease (ghouila et al., 2014). this immense family of polyphenols has an important role in food quality through its antioxidant properties; contribute taste, astringency, flavor, color, longterm stability, and their antimicrobial activities (larcher et al., 2013). so in order to search for new drugs to contribute the bacteria and viruses, the aim of this study is to investigate the total polyphenols content of wild r. pentaphylla fruits collected at different maturity stages and to evaluate their biological activities. 2. material and methods 2.1. fruit sampling r. pentaphylla were collected from march to june 2015 in kroumirie mountains (ain draham municipality, governorate of jendouba situated at an altitude of 602 m, at 36°44’57 north latitude and 08°41’12 34’’ east longitude) tunisia (el makni and el aouni, 2011). an experienced plant taxonomist presented during collection. at this point, 3 kg of fruits at two ripening maturities stages, green-yellow color fruits (un-ripe stage; s1) and purple-red color fruits (fully-ripe stage; s2) were collected at the same time to ensure subsequent maturity classes would develop on the clone. the collected fruits were transported in the same cooling and light conditions to chemistry department of faculty of sciences bizerte. the different stage of fruits was immediately washed with distilled water and lyophilized at -80°c for 48 hours. chemical analyses were conducted on the composite lyophilized fruit samples collected over time. ital. j. food sci., vol. 31, 2019 226 2.2. chemicals, standards, and reagents gallic acid, (±)-catechin hydrate (c15h14o6),and 2,2-diphenyl-1-picrylhydrazyl (dpph) were purchased from univar and sigma-aldrich (france).folin-ciocalteu’s reagent was purchased from carlo erba reagents. 2,4,6-tri (2-pyridyl)-s-triazine (c18h12n6) was purchased from acros organics. butylated hydroxytoluene (bht, c15h24o), and iodonitrotetrazolium chloride (c19h13c11n5o2) were purchased from fluka (turkey). aluminium chloride (alcl3), vanillin, all other used chemicals and solvents (methanol, ethanol, acetone, hexane, hcl and dmso) were purchased from merck (darmstadt, germany). 2.3. extract preparation r. pentaphylla fruits at different maturity stages were processed separately. soxhlet, maceration and ultrasonic extraction techniques were recommended in the literature (mollica et al., 2018; günaydın et al., 2017). approximately, 6 g of freeze-dried fruits were stirred with 60 ml methanol: water (80:20,v/v) with vortex (ika ms 3 basic) at 2300 rpm for 1 min. the mixtures were then extracted three times in an ultrasonic bath for 15 minutes (elma transsonic digital, t=25 °c, and f= 150w). the collected extracts were centrifuged at 4000 rpm (eppendorf centrifuge and rotor packages model 5810), for 10 minutes, then filtered through a whatman no. 4 paper and the organic phase (methanol) were evaporated under vacuum evaporator (heidolph). the resulting aqueous phases were washed with water to dissolve the extracted material from the glassware. afterwards the aqueous material was lyophilized and stored until use. the obtained lyophilized materials were dissolved in a known volume of methanol and dmso for the quantification of polyphenols content, and the determination of antioxidant and microbial activities, respectively. 2.4. determination of total phenolics content the total phenolic contents of each extract were determined by the modified folin ciocalteu method described by singleton (singleton et al., 1999; blainski et al., 2013). briefly, 1 ml of methanol extracts was added to 1 ml of folin ciocalteu reagent diluted with distilled water (1:9). the samples were left in room temperature for 5 minutes to develop color; the reaction was then stopped by adding 10 ml of na2co3 (7%). after homogenization and vortex, the mixture was heated for 10 minutes at 40°c, and then incubated in dark for 30 min. measurement of the absorbance was carried out in uv spectrophotometer (thermo scientific evolution 201) at 765 nm. total phenol concentration was calculated from the regression equation of the calibration curve established using different concentrations from stock solution of gallic acid (12-152 μg.ml1). the results were expressed as mg of gallic acid equivalent per gram of dry weight (mg eq. ga. 100 mg-1dw). 2.5. determination of flavonoids content flavonoids content of r. pentaphylla fruit extracts were carried out by following colorimetric assay using aluminum trichloride (alcl3) (zhishen et al., 1999). 0.3 ml of nano2 (5%) was added to 1 ml of methanol extract of each sample. after 6 min, 0.3 ml of 10% methanol solution of alcl3.6h2o was added to the extract samples. to the mixtures, 2 ml of naoh (1m) and 10 ml of h2o were added, followed by keeping them in dark for 2 hours. absorbance was read at 520 nm after incubation in the dark at room temperature ital. j. food sci., vol. 31, 2019 227 for 10 min. the results were expressed in mg catechin equivalent 100 mg-1 dry weight (mg eq.cat.100 mg-1dw). 2.6. determination of condensed tannins content determination of condensed tannins is based on phenolic compounds condensation with vanillin under acidic conditions, which will provide a brown compound (price et al., 1978). briefly, 100 mg of lyophilized plant materials were dissolved in 1ml of methanol and added to 2 ml of 1% vanillin in 70% sulfuric acid h2so4 (1 g vanillin in 77.77 ml of h2so4 completed with distilled water to prepare 100 ml). the entire mixture was placed in a water bath for 15 min at 30°c protected from light. finally the absorbance of the mixture was read at a wavelength λ = 500 nm using a thermo scientific evolution 201 uvvisible spectrophotometer. the results were expressed in mg catechin equivalent 100 mg-1 dry weight (mg eq.cat.100 mg-1dw). 2.7. antioxidant activities 2.7.1 dpph assay among the tests to determine the radical scavenging ability, 2,2-diphenyl-1-picrylhydrazyl (dpph) analysis is one of the most widely used. indeed, the dpph assay is rapid, simple, stable, and economical to measure the antioxidant capacity of foods or plant extracts (jabbari and jabbari, 2016). briefly for each extract, a 50 mg.ml-1 stock solution had been prepared with methanol as solvent, and if necessary sequential dilutions were made (sokmen et al., 2004). then, for all extracts solution, 50 µl was added to an equal volume of freshly dpph radical solution (4.10-3mm) and allowed to stand for 30 min in the dark at room temperature. the experiment was repeated for three times. the entire mixture was placed in the dark at room temperature for 30 min, and the absorbance of the mixture was read at λ = 517 nm using a uv-visible spectrophotometer. bht was used as the standard control. 2.7.2 frap assay ferric reducing antioxidant power (frap) was performed according to the methods of benzie and strain (1999) with slight modifications (pulido et al., 2000) were used as an evaluation of the reducing power of samples. the stock solution (50 mg.ml−1) of each extract was prepared in methanol. a portion of 50μl solution was mixed with 1.5 ml of frap reagent in a test tube and vortexed. blank samples were prepared with methanol and deionized water. both samples and blank were incubated in a water bath for 30 minutes at 37°c and the absorbance of the samples was determined against blank at 593 nm. series of stock solution at 62.5, 125, 250, 500 and 1000 μm were prepared using an aqueous solution of feso4.7h2o as the standard curve. the values obtained were expressed as µm of ferrous equivalent fe (ii) per gram of freeze-dried sample. increases in absorbance due to the formation of a colored tptz-fe2+complex were monitored at 593 nm. a trolox per gram curve (0.10.8 µm) was used as a standard. 2.8. antimicrobial/antifungal tests the same procedure given above was employed for the extraction. the evaluation of antimicrobial activities of r. pentaphylla extracts was carried out using three gram-positive bacteria (staphylococcus aureus atcc 25923, enterococcusfaecalis atcc 29212, listeria ital. j. food sci., vol. 31, 2019 228 monocytogenes atcc 19115) and two gram-negative bacteria (escherichiacoli atcc 25922and pseudomonasaeruginosa atcc 27853) and against one fungal strain (candidaalbicans atcc 10231). broth dilution method micro-dilution technique was one of the most basic methods for antimicrobial susceptibility testing (balouiri el al., 2016). the used pre-culture contain muller-hinton broth medium (mhb), although a pre-culture mcfarland 0.5~108 ufc.ml-1 (colony forming unit) ~ 0.2 od (optical density). the ad of 2 ml of sodium chloride (nacl) or 0.9% phosphate buffered saline (pbs) to vials followed by the addition of bacteria with a cotton rod, and the optical density is measured at λ = 600 nm to obtain an absorbance 0.2 for all used bacterial strains. pbs is used as a blank. each well of a sterile 96-well microtitre plate-shaped v was aliquoted with an equal volume of mueller-hinton broth and fruits extract initially dissolved in dmso. we conducted a series of dilution, for the first eleven well, we added 100 µl of fruit extracts and we add then 100 µl of muller hinton broth solution (ca2+, mg2+). concentrations (5; 2.5; 1.25mg.ml-1; and 4.88μg.ml-1) are taken into a waterfall and the last well was used for the addition of control’s bacteria. after 10 to 15 min, the microplate was incubated in the oven at 37°c for 24 hours. to each well 40µl of colored indicator iodonitrotetrazolium, c19h13c11n5o2 chloride (200µg.ml-1) was added. 3. statistical analysis all results are expressed as (±sd) of total phenolic, flavonoid and tannins contents. statistical analysis was performed using a one-way analysis of variance (anova) with a tukey’s hsd post hoc test. correlation tests were used to determine relationships among the polyphenols content and antioxidant activities during ripening stages of r. pentaphylla fruits. significance overall and within any correlation (confirmed by linear regression test) was accepted at p<0.05. 4. results and discussion 4.1. phenolic contents r. pentaphylla fruits showed a highly significant (p≤0.01) increase in total phenolic content during ripening stage, the order of total phenolic content (table 1), of samples during ripening is [(s1)<(s2)]. the concentration increased from 158.65 for the unripe fruits to 177.57 mg eq.ag.100 mg1dw for the fully-ripe fruits. from the obtained results, it can be seen that the phenolic contents of fruits could also be affected by experimental conditions and various factors such as variety, growing condition, maturity, season, amount of sunlight received. this result was in agreement with previous work (marinova et al., 2005; navarro et al., 2006), where phenolic compounds increase during the last stage especially for red colored varieties, and it’s increasing with maturation. ital. j. food sci., vol. 31, 2019 229 table 1. total polyphenols content and antioxidant activities at different maturity stages of r. pentaphylla fruits extract. 1st stage: un-ripe 2nd stage: fully-ripe total polyphenols content tpc ( mg eq.ag. 100 mg-1dw) 158.65±0.06**a 177.57±0.06**a tfc ( mg eq.cat.100mg-1dw) 157.34±0.07**b 152.69±0.14**b ctc ( mg eq.cat.100mg-1dw) 200.26±0.3**c 131.23±0.24**c antioxidant activities dpph ( ic50 mg.ml -1) 0.42 ˂ 0.5 frap (μmol fe2+.g-1dw) 64.36 81.59 values are expressed as (±sd) (n=2), **highly significant (p≤0.01), a: statistic measurement between un-ripe and fully-ripe fruits for phenolic contents, b: statistic measurement between un-ripe and fully-ripe fruits for flavonoid contents, c: statistic measurement between un-ripe and fully-ripe fruits for tannins contents. 4.2. flavonoid contents fruit ripening are related to important biochemical changes such as color, texture, taste and other quality traits modify. as seen, ripening stage caused a decrease in total flavonoids content; the results of the distribution of tfc in relation to fruit maturity/ripening stages are presented in table 1. the concentration of flavonoid varies in the following order: [(s1)> (s2)], so as the fruit maturity progressed, there is a highly significant (p≤0.01) decrease from 157.34 to 152.69 mg eq.cat.100 mg-1 dw of fc in r. pentaphylla fruits. this decrease in tfc with advanced maturity is in agreement with the study of tlili et al. (2014) who reported that the decrease of flavonoid content during ripening may be due to metabolic production of other phenolic compounds or degradation via enzyme action. 4.3. condensed tannins content as seen in table 1, maturity stages of r. pentaphylla fruits had a marked effect on the amounts of condensed tannins. the variations of flavan-3-ol content during ripening follow this order [(s1) > (s2)] of ripening stages (a highly significant (p≤0.01) decrease). the higher concentrations of condensed tannins are found in s1 un-ripe stage (200.26mg eq.cat.100mg-1dw) approximately two times more than those of fully-ripe (s2), flavan-3ol present a drastic declined during fruit ripening (kennedy et al., 2000). this change in tannins content in the later stages of ripening could be explained by the fact that different phenolic acids might have condensed to form complex phenolic compounds such as tannins and lignin (ben ahmed et al., 2009). a significant difference (p < 0.05) was demonstrated by anova with a tukey’s hsd post hoc test (table 1), which confirm that the variation of this polyphenols content (phenolic, flavonoid, and condensed tannins contents) was influenced by the stage of maturation and/or their interaction according to fruit shape. 4.4. dpph assay methanol extracts of r. pentaphylla fruits crude were investigated through in vitro models radical scavenging activity using dpph method. inhibitory concentration ic50 value is the most critical value that reflects the antioxidant action of the tested species. if this value is low, the tested sample has a high antioxidant activity. the ic50 value of standard synthetic antioxidant bht was ranging 0.17 mg.ml-1. table 1 shows that the 1st stage (un-ripe) of r. pentaphylla exhibited better antioxidant action about 0.42 mg.ml-1, then the 2nd stage (fully ital. j. food sci., vol. 31, 2019 230 ripe). the antioxidant power of r. pentaphylla fruits is due to a wealth of flavonoid and tannins, which have been reported to have multiple biological effects, including antioxidant activity due to the redox properties of their phenolic hydroxy groups and the structural relationships between the different parts of the chemical structures (riceevans and miller, 1996). 4.5. frap assay the ferric reducing antioxidant power (frap) was measured for each extract obtained at stages 1 and 2. results were given in table 1. the obtained values had been expressed as µm of ferrous equivalent fe (ii) per gram dry weight. from the results given in table 1, the 2nd stage of r. pentaphylla exhibited better antioxidant principles then 1st stage (81.59 and 64.36 μmol fe2+.g-1dwrespectively). the order of total activity is the same as total phenol content. a further investigation by palafox-carlos et al. (2012), who suggest that the physiological and ripening process in fruit may affect directly the presence of phenolic compounds content and their antioxidant activity which increased during ripening. 4.6. correlation between total polyphenols content and antioxidant activity as result for this study, there was a positive correlation between the polyphenols content (r = 0.876), the two test (dpph and frap) and the maturity stages, but this correlation is not significant (linear regression test p = 0.0513); the results of the regression is the same in the case of multiple regression (case of this test) and that when we made the regression one by one. 4.7. antimicrobial potentials antimicrobial potential of r. pentaphylla extracts obtained from the two stages was tested separately. the results obtained from the first and the second stages using micro-dilution technique were given in table 2. table 2. antimicrobial activity of different crude extracts of r. pentaphylla. mic(mg.ml-1) methanol/water bacterial strains first stage second stage escherichia coli 1.25 1.25 staphylococcus aureus enterococcus faecalis pseudomonas aeruginosa 1.25 listeria monocytogenes the evaluation of in vitro antibacterial activity of methanol/water extract of r. pentaphylla fruits at different maturity stages against the in-use bacteria, showed that the two stages of fruits extract exhibited antibacterial activity against three positive grams (enterococcusfaecalis, listeria monocytogenes, and staphylococcus aureus) and two negative grams (pseudomonas aeruginosa and escherichia coli) bacteria at different concentrations. mic values of the two stages are ranged 1.25 mg.ml-1 for the tested microorganisms. methanol/water extracts of both stages of r. pentaphylla exhibited important antibacterial effects against escherichia coli and pseudomonasaeruginosa. from the obtained results ital. j. food sci., vol. 31, 2019 231 escherichia coli was found to be the most sensitive organisms for the two stages. the results prove that there might be synergism between polyphenols content and antimicrobial (cushni and lamb, 2005). however, r. pentaphylla showed no antifungal effect against candida albicans at its different maturity stages. 4. conclusions quantitative analysis of widely r. pentaphylla fruit extracts revealed richness in secondary metabolites such as phenolic, flavonoids and condensed tannins. a highly significant difference was demonstrated which confirm that the variation of this polyphenols content was influenced by the stage of maturation and/or their interaction according to fruit shape. dpph test showed that the first stage has the higher antioxidant activity, which is not the case for frap test. a good correlation between the polyphenols content, the two test (dpph and frap) and the maturity stages. as a result of this study, the validation of bioactivity of this fruit as agro alimentary in operating pharmacy was confirmed. in fact, fruit extracts at different maturity stages present a good potential as antibacterial compounds against the tested microorganisms comparing with the commercial antibiotics. so we could suggest that this wild fruits could be used in the treatment of infectious diseases caused by resistant microbes. acknowledgements this study was financially supported by the tunisian ministry of higher education. the authors wish to acknowledge the university of carthage for providing financial support for the research work. references abed d. 2013. etude bibliographique sur la phytochimie de quelques espèces du genre rhus, université kasdimerbah, ouargla. atanasov ag., waltenberger b., pferschy-wenzig em., linder t., wawrosch c., uhrin p. and rollinger jm. 2015. discovery and resupply of pharmacologically active plant-derived natural products: a review. biotechnology advances 33:1582-1614. balouiri m., sadiki m. and ibnsouda sk. 2016. methods for in vitro evaluating antimicrobial activity:a review. j. pharma anal 6:71-79. blainski a., lopes gc. and de mello jcp. 2013. application and analysis of the folinciocalteu method for the determination of the total phenolic content from limoniumbrasiliense l. molecules 18:6852-6865. ben ahmed c., ben rouina b., sensoy s. and boukhriss m. 2009. saline water irrigation effects on fruit development, quality, and phenolic composition of virgin olive oils, cv. chemlali. j. agri. food chem 57:2803-2811. benzie if. and strain jj. 1999. ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. methods in enzymology 299:15-27. carina c.m. 2012. phytochemical and pharmacological investigations of brideliaretusaspreng. cushnie tt. and lamb aj. 2005. antimicrobial activity of flavonoids. inter j. antimicrobial agents 26:343-356. el mokni r. and el aouni mh. 2011. sparaxis tricolore, sparaxis tricolor (curt.) ker-gawl. 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2014. phytochemicals and antioxidant activities of rhustripartitum (ucria) fruits depending on locality and different stages of maturity. food chemistry 160:98-103. zhishen j., mengcheng t. and jianming w. 1999. the determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. food chem. 64:555-559. paper received june 13, 2018 accepted september 21, 2018 ijfs#1557_bozza ital. j. food sci., vol. 32, 2020 91 paper co-extrusion of collagen casings. effects of preparation, brining, and heating on strength, rheology and microstructure s. barbut*, m. ioi and m. marcone department of food science, university of guelph, guelph, on, canada, n1g 2w1 *corresponding author: tel.: 1 5198244120 ext. 53669 email: sbarbut@uoguelph.ca abstract properties of five collagen preparations were investigated to enhance understanding of inline co-extrusion casing formation. the first study revealed that 30% nacl and 5 min brining provided maximum strength. the second study showed 100% difference in tensile strength between preparations; when adjusted to % protein, the difference was smaller but still existed. extrusion force and elastic modulus (g’) also varied; appear to be acid dependent. denaturation temperature of raw dispersions was between 36.7 and 38.9˚c. upon salt brining, it substantially increased to 63.3 65.3˚c. polarized light microscopy revealed numerus intact segments of connective tissue fibers and some cellulose fibers. keywords: casings, collagen, dispersions, films, mechanical properties ital. j. food sci., vol. 32, 2020 92 1. introduction a sausage casing is an essential component in the transformation of comminuted meat into a finished product. in the past 100 years there have been a number of technologies that helped improve processing, handling and functional properties of casings’ uniformity, hygiene, strength, flexibility and stability during storage (osburne, 2000; savic and savic, 2016). before the early twentieth century almost all sausages were produced with natural casings, derived from animal intestines. although natural casings are still considered the ‘gold standard’, advances in casing technology have led to different types of engineered casing. today, casings are produced with various materials, ranging from regenerated biopolymers (e.g., collagen, cellulose) to thermoplastic materials (e.g., polyvinyl alcohol polymers). manipulation of these materials has made it possible to tailor casings with specific functional attributes (wang, 1986; barbut, 2015). until recently, modern casings fabrication required specialized production facilities due to the complexity of the process (savic and savic, 2016; karmas, 1974). the fairly recent development of co-extrusion has eliminated the need to prefabricate, and store casings prior to stuffing. co-extrusion is a continuous operation of sausage production, where a thin layer of casing material is extruded on the comminuted meat” rope” coming out of the stuffer. immediately after formation, the casing material undergoes stabilization, through dehydration and/or cross-linking to enhance its mechanical strength. a brine application (spray, drip, immersion) dehydrates the casing and allows the material to conform to the shape of the meat. further stabilization is commonly performed through cross-linking (e.g., employing the aldehyde in liquid smoke) and air-drying as they both help provide the mechanical strength needed during linking, cooking and packaging (morgan et al., 1988). co-extruded casings can be produced with a collagen gel of fibrous and soluble collagenous material or a dispersion of a hydrocolloid gum such as alginate (morgan et al., 1988; harper et al., 2013). the collagen is typically derived from cattle hides and is mainly composed of collagen (ratanavaraporn et al., 2008). generally speaking, collagen casing production involves corium separation, decalcification and regeneration. during collagen separation, hides are washed and limed (ph 11 to 13) to remove impurities. calcium is later removed to promote uniform swelling of collagen fibrils and then the material is chopped and ground. following decalcification, collagen undergoes regeneration where swelling is promoted via the use of acids (savic and savic, 2016). in commercial products, hydrochloric acid (hcl) is the most commonly used swelling agent, but other acids such as acetic are also used (ratanavaraporn et al., 2008). overall, this is a lengthy (few weeks) and complicated process involving quite a few steps; some are proprietary. prior to extrusion (i.e., at the meat processing plant), the material is mixed one last time to reorient the fibers to improve film strength (savic and savic, 2016; barbut, 2010). although one of the first patents for collagen co-extrusion technology was introduced about 50 years ago, the technology has not been used on a large commercial scale for the following 30-40 years. only recently large dedicated plants, for in-line co-extrusion, have been constructed. today there is still very little published information about the topic (e.g., collagen, composition, viscosity, shear thinning) and even basic data about the effect of raw material source (e.g., animal age, breed), processing parameters during extraction (e.g., type of acid used), and conditions during application (e.g., drying, cross-linking). however, there has been some effort made to understand the properties of certain coextruded collagen films and the effects of different additives (tomihata et al., 1994; ital. j. food sci., vol. 32, 2020 93 olde damink et al., 1995; o’sullivan et al., 2006; wolf et al., 2006; harper et al., 2012; oeschsle et al., 2014; oeschsle et al., 2016; barbut and ioi, 2019). overall, the properties of prepared collagen dispersions and processing conditions of co-extrusion casing production needs more attention. today the co-extrusion process is highly automated and as such also helps to improve food safety and reduce waste (osburn, 2000; barbut, 2015). the main goal of this research was to evaluate different processing conditions that can be used to partially dehydrate collagen films. the study is believed to be the first to look at the differences between commercially prepared collagen dispersions that are used in the production of co-extruded meat products. differences were evaluated by studying the collagen dispersions and films’ mechanical, microstructure and thermomechanical properties. the results should help researchers and manufacturers to better understand co-extrusion through material selection and process controls. 2. materials and methods 2.1. collagen dispersions collagen dispersions were evaluated and identified as collagen 1 through 5 (c1, c2, c3, c4, c5), as they are proprietary blends (see explanation in the introduction). information that was provided to the researchers, by the two major manufacturers in the world, and compositional analysis can be found in table 1 (note: manufacturers did not want to provide any specific information on product number/identity as some of the dispersions are still being modified). protein content was determined in triplicate by the dumas method (fp528, leco, st joseph, mi) using a nitrogen factor 6.25. moisture content is reported as the moisture lost when drying (oven 650g, fisher scientific, toronto, on) from 5 g samples heated at 105˚c for 24 h. table 1. commercial collagen dispersion specifications and the work needed for extrusion (i.e. measurement of resistance to flow) through a 7mm die: c1 (collagen 1), c2 (collagen 2), c3 (collagen 3), c4 (collagen 4) and c5 (collagen 5). collagen ph moisture content protein (%) swelling agenta work of extrusionb (nm) work of extrusionb (nm/%protein) dispersion (%) film (%) c1 2.06 93.6 74.4 5.14 5.16±0.19 1.00±0.04 c2 2.21 95.7 77.8 3.57 3.19±0.21 0.89±0.06 c3 2.01 93.6 74.5 3.68 hcl 4.04±0.05 1.10±0.01 c4 2.67 94.1 74.7 4.37 hcl/acetic acid 3.63±0.01 0.83±0.01 c5 2.04 93.7 76.9 4.82 4.04±0.13 0.84±0.03 aswelling agent as reported by the manufacturer (see text). bn = 9. 2.2. partially dehydrated film formation the method of film formation was adapted from harper et al. (2013) who worked with alginate solutions. briefly, collagen dispersions were first cooled to 4˚c to reduce the ital. j. food sci., vol. 32, 2020 94 adhesiveness during film formation. the collagen dispersions were also degassed using a vacuum packaging machine (multivac canada inc., woodbridge, on) at 7.3 kpa for 25 s, 50 s and 75 s, consecutively (settings 4, 6 and 8, respectively). this was performed to remove gas bubbles that were incorporated during processing, as they can create weak spots in the films. following the degassing stage, dispersions were mixed to assure homogeneity of the samples (dispersions were mixed by rolling the material, placed in plastic bags, 10 times in adjacent directions). later, approximately 3 g portions of the collagen were rolled on a stainless steel board between two layers of plastic sheets with a stainless steel roller. the roller had a recess of 0.50 mm in order to achieve uniform film thickness. the top plastic sheet was removed and the film was then placed in a salt bath on the remaining plastic sheet. the first study was performed with one collagen sample (c2), to evaluate the effects of brine concentration and contact time on the textural properties of the films (note: c2 was selected as it is one of the most popular dispersions used by the industry). brine solutions consisted of 15, 20, 25 and 30% nacl in deionized water. films were immersed in the brine for 1.0, 2.5, 5.0 and 10.0 min intervals. after formation, films were covered again with a plastic sheet to prevent further dehydration before testing. after establishing the salt concentration and time (see data and conclusions in discussion), the second study evaluated all five dispersions and their films, which were dehydrated in 30% nacl brine for 5 min. these conditions ensured that the films were strong enough to be removed from the plastic sheet and tested. 2.3. extrusion force of collagen dispersions the collagen dispersions were evaluated by using a texture analyzer (ta-xt2i, texture technologies corporation, scarsdale, ny) with a forward extrusion fixture (ta 93, texture technologies). approximately 50 g samples were loaded into the cell (100 ml capacity) fitted with a 7 mm opening die, and brought to 4˚c. the plunger compressed the dispersion at a rate of 1 mm/s. from the generated force-distance curve (n = 9 per dispersion) the work of extrusion was calculated once the readings had stabilized (i.e., after pushing down about 10-35 mm). 2.4. mechanical properties and film thickness a standard method for testing film’s tensile properties (astm, 2010) was performed on the partially dehydrated films. films were evaluated by using the texture analyzer (taxt2i, texture technologies) equipped with grippers that were set at a distance of 50 mm, trigger force of 5 g, test speed of 2 mm/s, and an overall test distance of 25 mm. the film’s thickness was determined by measuring each film at the top, center and bottom using a digital micrometer (testing machines inc., islandia, ny). the films were cut into 75 mm × 25 mm strips (jdc precision sample cutter, thwang-albert instrument comp., philadelphia, pa). the average thickness and width of the films were used for the tensile stress calculations. from the generated stress–strain curve, the tensile strength (maximum stress the film endured prior to breaking) and the percent elongation (maximum elongation the film reached prior to breaking) were determined. a total of eighteen films were tested for each of the treatments (six films per each of the three trials). a puncture test was also performed with the texture analyzer. in this test, a 5 mm diameter ball probe was used to puncture films mounted in a film extensibility fixture, which has circular opening of 10 mm diameter (ta-108s5, texture technologies). the test ital. j. food sci., vol. 32, 2020 95 speed was 1 mm/s and the trigger force was set to 5 g. the distance to puncture and work of puncture were determined from the generated force distance curve. a total of eighteen films were tested for each of the treatments (six films per trial). 2.5. light microscopy collagen dispersions were prefixed in 10% neutral buffered formalin for 10 h at room temperature and then dehydrated in 70% isopropanol for 2 h, 95% for 1 h, and 100% for 4 h. the dehydrated samples were dipped in xylene, prior to embedding in paraffin. samples were cut into 4-6 μm cross sections. masson stain was used to differentiate collagen from other meat proteins. in another set of sections, periodic-acid schiff (pas) stain was used to differentiate carbohydrates, specifically cellulose fibers. a light microscope (olympus bx 60, olympus corporation, centre valley, pa) was used to examine the samples. representative images (a total of six images per treatment) were taken using image pro plus (version 6.0, media cybernetics inc., bethesda, md) software. 2.6. rheology of film forming dispersion rheological analysis was performed on the collagen dispersions using a bohlin cs50 (malvern instruments ltd, worcestershire, uk) with a 25 mm din coaxial cylinder bob and cup fixture. the bob was lowered into 13 g of collagen that was preloaded into the bottom of the cup. excess collagen was removed and mineral oil was then applied on the top to keep the exposed surface from drying. the temperature of the collagen was increased from 20° to 55˚c at 1.25˚c/min, held for 2 min and returned to 20˚c, at the same rate. continuous oscillating shear (1 hz and 0.0012 strain) was applied during testing. test parameters were determined in a pre-trial to evaluate the linear range. the elastic modulus (g’) was recorded (bohlin zetasizer series software, version 6.32, malvern instruments ltd.) and used to determine the changes in stiffness of the dispersions (helary et al., 2009). 2.7. differential scanning calorimetry (dsc) the melting profiles of the collagen dispersions and partially dehydrated films were evaluated using a differential scanning calorimeter (dsc q2000, ta instruments, new castle, de). samples (~10 mg) were placed in anodized-aluminum hermetically sealed pans. temperature was ramped from 20° to 80˚c at a rate of 5˚c/min. samples were then held at 80˚c for 2 mins and then cooled back to 20˚ at 5˚c/min. the same thermal profile was used to rescan samples for identifying reversible peaks. the melting behavior was studied between 30° and 80°c by integrating the endothermic peak (ta universal analysis 2000 software, ta instruments) to determine the onset temperature, melting temperature and enthalpy. a total of three dispersions or films were tested for each of the treatments. 2.8. amino acid analysis collagen dispersion’s amino acid profile was analyzed (advanced protein technology center, toronto, on). approximately 0.01 g of each collagen dispersion was weighed. hcl (6n) and norleucine (internal standard) were added to each sample and then ital. j. food sci., vol. 32, 2020 96 hydrolyzed for 24 h at 110˚c. after hydrolysis, an aliquot was transferred to another tube for derivatization. 2.9. experimental design and statistical analysis the experiment used to compare the different dispersions was designed as a complete randomized block with three independent trials. each trial consisted of six measurements, per dispersion, for the mechanical properties of the films (tensile and puncture tests). the statistical analysis was performed using sas version 9.2 (sas inst., cary, nc). a general linear model was used for the analysis of variance (anova). the film type means and interactions were compared by using tukey's multiple comparison analysis (p ≤ 0.05). for setting up dehydration conditions (salt concentration and time) the results from dispersion c2 are reported as averages and standard deviations. 3. results and discussion 3.1. mechanical properties during formation and stabilization of co-extruded casings, the newly produced sausage is exposed to a variety of different stresses (e.g., moving on a conveyer belt, vibration). it is therefore crucial to impart sufficient strength shortly after extrusion so that the sausages can also undergo subsequent treatment, such as smoking, drying and cooking (kobussen et al., 2012). in an industrial setting, the newly formed casings are stabilized by first removing some of the water through brining. dehydration is driven by osmosis, which helps to increase the density of the collagen fibers and thus shortening the distance among collagen molecules to help improve the mechanical stability of the casing (kobussen et al., 2000; visser, 2012). in the first study we established the best conditions for the brining procedure by evaluating the effects of brine concentration (15 to 30% nacl) and brining time (1 to 10 min) using a dispersion that is widely used by the industry (collagen 2). the overall settings used were based on guidelines described in several industry protocols (kobussen et al., 2000). in order to evaluate the mechanical properties of the wet films, (i.e., as applied to the sausage), tensile and puncture tests were performed (fig. 1). ital. j. food sci., vol. 32, 2020 97 figure 1. mechanical properties of collagen films (collagen 2) produced with increasing concentration of nacl and contact time (n=18). mechanical testing demonstrated that there were no significant interactions (p>0.05) between the brine concentration and contact time. overall, there were some significant differences (p<0.05) in the film’s mechanical properties when modifying the salt concentration or contact time (tables 2 and 3). tensile strength, percent elongation and work to break increased with raising salt concentration and contact time. this indicates that there is a need for a certain level of dehydration to stabilize the collagen network in the films. these results also demonstrate that processors would be able to modify the mechanical properties of their casings by altering the concentration or exposure time. table 2. mechanical properties of collagen films (collagen 2) produced with increasing concentration of nacl. means were averaged across contact times; 1.0, 2.5, 5.0, 10.0 min. concentration (%) tensile strength1 (mpa) percent elongation1 (%) distance to break2 (mm) work of break2 (nmm) 15 0.23c 21.0a 4.0a 2.2b 20 0.26bc 21.1a 4.0ab 2.4b 25 0.32b 21.3a 3.8b 2.4b 30 0.40 a 22.7a 3.7b 2.8a 1tensile test. 2puncture test. means, within a column, followed by a similar letter are not significantly different (p>0.05). ital. j. food sci., vol. 32, 2020 98 table 3. mechanical properties of collagen films (collagen 2) produced with increasing contact times to nacl. means were averaged across concentrations; 15, 20, 25, 30% nacl. time (min) tensile strength1 (mpa) percent elongation1 (%) distance to break2 (mm) work of break2 (nmm) 1.0 0.19c 20.0b 3.9a 1.9b 2.5 0.28b 21.5ab 4.0a 2.4a 5.0 0.36a 23.1a 3.9ab 2.8a 10.0 0.39a 21.4ab 3.7b 2.6a 1tensile test. 2puncture test. means, within a column, followed by a similar letter are not significantly different (p>0.05). studying the effects of dehydration conditions helped us to select the best conditions for processing the different commercially prepared dispersions. the higher salt concentration was selected for the evaluations because it provides the most strength and thus helps avoid damaging the casings during further processing. overall, the film produced with 30% salt had the highest tensile strength and work of puncture (p<0.05), across all contact times (table 2). this is also the salt concentration currently used in a number of the industrial settings. although this salt concentration is prepared at the beginning of the day, processors must ensure that it is maintained, because over time it will be diluted (i.e., moisture coming out from the casings; (kobussen et al., 2012)). the dehydration effect can also be seen in the data concerning moisture content in the films after exposure to the brine solution (table 1). table 3 shows that there was no significant difference in mechanical properties between 5 and 10 min. therefore, a 5 min exposure time was selected for use in the follow up evaluation (second study). it should be mentioned that under commercial conditions, exposure times have been reported to be even shorter (bontjer et al., 2011). this is usually due to production pressures. in any case, the products are not rinsed after the industrial brining process so high salt concentration remains on the surface. in the second study, the properties of the actual films produced from the other commercial collagen dispersions were investigated. overall, there were some significant differences in the tensile strength and percent elongation among the five preparations (table 4). films produced with c4 had the lowest tensile strength and percent elongation of the protein films (0.15 mpa and 16.33%, respectively). as will be discussed below, these observations may be correlated to the sample’s protein content, collagen structure, acid used for swelling, and the overall dispersions’ composition. the mechanical properties of the partially dehydrated films were also compared after adjusting for protein concentration (table 5), to examine the effect of protein content (i.e., important for processors dealing with cost per unit). these data reveal differences in the tensile strength, distance at break and work to break; therefore, it would appear that differences among the dispersions are influenced by more than just protein content (assuming protein content affects mechanical properties linearly). it should be noted that meat processors use the dispersions as they arrive to the plant (i.e., no dilution or ingredients added) and therefore cost per unit is critical for them. ital. j. food sci., vol. 32, 2020 99 table 4. mechanical properties of partially dehydrated films: c1 (collagen 1), c2 (collagen 2), c3 (collagen 3), c4 (collagen 4) and c5 (collagen 5). collagen tensile strength1 (mpa) percent elongation1 (%) distance at break2 (mm) work to break2 (nm) thickness (mm) c1 0.33a±0.03 24.38a±3.82 6.12a±0.51 3.72a±0.88 0.43a±0.01 c2 0.29ab±0.05 18.57ab±2.42 5.57a±0.41 2.75ab±0.48 0.37b±0.00 c3 0.19cd±0.02 19.22ab±2.35 5.26a±0.47 1.99b±0.41 0.41a±0.01 c4 0.15d±0.03 16.33b±1.37 5.33a±0.42 1.89b±0.34 0.41a±0.02 c5 0.24bc±0.02 23.42a±1.81 5.91a±0.24 1.81b±0.26 0.42a±0.01 1tensile test. 2puncture test. means, within a column, followed by a similar letter are not significantly different (p>0.05). table 5. comparing mechanical properties of partially dehydrated films: c1 (collagen 1), c2 (collagen 2), c3 (collagen 3), c4 (collagen 4) and c5 (collagen 5) based on adjusting percent protein. collagen tensile strength 1 (mpa/%protein) percent elongation1 (%/%protein) distance at break2 (mm/%protein) work to break2 (nm/%protein) c1 0.046b±0.00 3.30a±0.52 0.83c±0.07 0.50ab±0.12 c2 0.061a±0.01 3.94a±0.51 1.18a±0.09 0.58a±0.10 c3 0.035bc±0.00 3.51a±0.43 0.96bc±0.09 0.36bc±0.07 c4 0.029c±0.01 3.12a±0.26 1.02ab±0.08 0.36bc±0.07 c5 0.037bc±0.00 3.57a±0.28 0.90bc±0.04 0.28c±0.04 1tensile test. 2puncture test. means, within a column, followed by a similar letter are not significantly different (p>0.05). meat processors must also consider the extrusion properties of the dispersion so that they can adjust the co-extrusion equipment. it is interesting to note that today most collagen suppliers do not provide that information. in the current study, the forward extrusion test was performed to provide insight into whether the five dispersions show differences in flow behaviour. overall, the values varied by as much as 60% (table 1). it appeared that samples with lower ph (c1, c3 and c5) required a higher work of extrusion. when the values were adjusted to %protein, c1 and c3 were still the highest. it may suggest that a greater degree of conformational changes increases the stiffness of the dispersion. the conformational changes discussed are a result of lowering the ph from 5 to 2 (away from isoelectric point of 8.26 and 4.56 for collagen and collagen hydrolysate, respectively), which has been reported to increase fiber hydration and swelling (wolf et al., 2006; oeschsle et al., 2014). oeschsle et al. (2014) demonstrated that collagen entanglement depends strongly on the ph as well as acid used, and indicated that entanglement increases with lowering ph values below the isoelectric point. lower tensile strength and elasticity could also be the result of excessive alkaline modification during corium separation. if alkaline modification, or liming, is not controlled then the extracted collagen may be of low molecular weight, which will not ital. j. food sci., vol. 32, 2020 100 contribute as much to the regeneration of collagen structures (savic and savic, 2016). these authors also indicated that intact fibrillar structures produce higher strength and elasticity in collagen casings. commercially prepared collagen dispersions are sometimes modified by the addition of functional ingredients, such as fillers (e.g., cellulose), plasticizers (glycerol), cross-linking agents (smoke condensate), as well as colourants (barbut and ioi, 2019). the combination of collagen (commonly used at 3 to 8%) and other modifiers (ranges from 0 to 10%) typically results in dispersions with 3 to 25% dry matter (kobussen et al., 2000). with such a wide range, one can expect quite a lot of variation among dispersions. in the dispersions evaluated here (representing some of the most commonly used by the industry) dry matter ranged from 4.3 to 6.4% (table 1). 3.2. light microscope imaging light microscopy was used to identify and characterize the homogeneity and condition of the fibers within the collagen dispersion. as previously discussed, collagen dispersions are mixtures of soluble and insoluble collagen. the proportion is affected by the origin of the material, method of extraction (e.g., extent of liming) and processing (e.g., degree of chopping). masson trichrome stain was used to differentiate collagen from other proteins, and also help study the condition of the collagen fibers in relation to their mechanical and thermo-mechanical properties. since cellulose was added to some of the samples, pas stain was also used to identify and characterize carbohydrate fillers (carson, 1997) found in the dispersions. overall, cellulose fibers are one of the most commonly used additives, as the fibers can contribute to the film’s strength and elasticity. mathew et al. (2012) observed that physical entanglements of cellulose nanofibers could increase the tensile strength of dried collagen films. it appears that some of the dispersions studied here contained a certain amount of cellulose (see micrographs below). the micrographs (fig. 2) show some differences in the proportions of long and short fibers, as well as the homogeneity of the collagen network (i.e., shown as areas of varying stain intensity throughout the network) among the dispersions. collagen 3 (c3) appeared to have the greatest homogeneity and as previously mentioned required the highest work of extrusion (1.10 j/% protein) (table 1). therefore, the work of extrusion may be attributed to the homogeneity. helary et al. (2009) also reported that areas of nonhomogeneity affect the mechanical properties of collagen hydrogels. as can be seen, collagens c2 and c5 appeared to have more small circular pockets, which may be small air bubbles that stayed within the dispersions. all of the dispersions show insoluble fibers (some show typical birefringence, as can be seen on the right side of fig. 2), of pretty much similar size range and morphology. the fibers are suspended in the soluble collagen network (i.e., background stained blue by the masson stain). some of the insoluble fibers were identified as cellulose because they had a ribbon-like morphology with twists down their length (reddy and yang, 2004; cranston and gray, 2008). the ribbon-like fibers also picked up the pas carbohydrate stain, providing further evidence that they are cellulose (fig. 3). it is interesting to note that the stained material on the interface between the collagen matrix and cellulose appeared to be darker (fig. 3). this may indicate that collagen might have developed interfacial interactions with some of the cellulose fibers. overall, commercial collagen dispersions often have 0.5% cellulose fiber. since there does not appear to be major differences in the cellulose concentration, the differences in mechanical properties may not be attributed to the addition of cellulose. ital. j. food sci., vol. 32, 2020 101 3.3. thermo-mechanical properties understanding collagen melting temperatures is very important to the meat processors because they often cook the sausages in the co-extruded casings. the rheological tests demonstrated that between 30˚c and 40˚c all of the dispersions start to display a rapid decrease in elasticity (fig. 4). it was observed that the collagen samples with higher ph (table 1; c4 and c2) began to lose their elasticity at a higher temperature. this may be attributed to the fact that collagen undergoes significant conformational change as one lowers the ph from 5 to 2. as previously mentioned, conformational changes result in increased hydration and swelling (wolf et al., 2006), therefore this may have reduced the thermal stability of these dispersions. figure 2. light micrograph images of commercial collagen dispersions: collagen 1 under regular illumination (a); collagen 1 under polarized light (b); collagen 2 under regular illumination (c); collagen 2 under polarization (d); collagen 3 under regular illumination (e); collagen 3 under polarized light (f); collagen 4 under regular illumination (g); collagen 4 under polarized light (h); collagen 5 under regular illumination (i); collagen 5 under polarized light (j). black bar represents 100 μm. ital. j. food sci., vol. 32, 2020 102 figure 3. light microscope image of a commercial collagen dispersion stained with periodic acid schiff. see text for explanation. black bar represents 100 μm. figure 4. rheological thermographs (20 to 55˚c at 1.25˚c/min) of five commercial collagen dispersions: c1 (collagen 1), c2 (collagen 2), c3 (collagen 3), c4 (collagen 4) and c5 (collagen 5). dsc scans were performed on the collagen dispersions and partially dehydrated films. the collagen dispersions exhibited an endothermic peak that started between 33.5 to 35.4˚c, with a maximum at 36.7 to 38.9˚c, and had a denaturation enthalpy of approximately 3.1 to 5.3 j/g (table 6). once the initial dsc run was completed the samples were cooled down to 4˚c, before a secondary run was performed to look for irreversible changes. the second run of all dispersions resulted in no endothermic peaks, indicating that irreversible denaturation occurred. this is similar to friess and lee’s (1996) observation of insoluble collagen fibers. the dsc denaturation temperatures are in the same range as the rheological measurements discussed above. similar to the rheological observations, the collagen with ital. j. food sci., vol. 32, 2020 103 lower ph displayed lower thermal stability in the dsc thermograms. the dispersions with a ph closer to 2 (collagens c1, c3 and c5) appeared to have slightly lower denaturation temperatures. as mentioned before, conformational changes at a lower ph may result in a greater hydration (wolf et al., 2006). gioffre et al. (2011) observed similar thermal denaturation behavior when the ph of wet gelatin films was decreased. the range of onset temperature values corresponds to the rheological transitions observed (fig. 4) and therefore confirms that the collagen in these dispersions has been significantly modified during the preparation procedure (extraction of collagen from hides, the liming process, and the follow up chopping and acidification procedures). it should be mentioned that in native collagen samples denaturation transitions are seen in the 60˚c range (bernal and stanley, 1986). dsc was also performed on partially dehydrated films (brined with 30% salt) to see if there was any effect on the thermal stability of the brined collagen. overall, dehydrating the films greatly increased the stability. the thermograms of the films showed an endothermic peak that started between 58.2 to 60.3˚c, with a maximum at 63.9 to 65.3˚c and a denaturation enthalpy of approximately 1.7 and 4.1 j/g (table 6). these denaturation temperatures are fairly similar to those reported by bernal and stanley (1986) who examined native bovine tendons, and reported denaturation at 61.55˚c. gioffre et al. (2011) also observed an increase in the denaturation temperature when wet gelatin films were dried. thus the difference between the denaturation temperature of the raw collagen dispersions and the partially dehydrated films is the result of some protein denaturation, higher salt concentration and a decrease in moisture content (i.e. 95 to 75%; table 1). fiber assembly may also help explain the increased thermal stability of the partially dehydrated and salted films. mcpherson et al. (1986) suggested that stronger association of collagen fiber structure is correlated with increased denaturation temperature. it has also been demonstrated that high ionic strength conditions result in a greater degree of packed collagen fibers and assembly (williams et al., 1978). overall, since the ionic strength is raised during film dehydration (salt migration into the film), there may be collagen fiber assembly, resulting in a higher thermal stability of the films. table 6. analysis of endothermic peaks from differential scanning calorimetry (dsc) thermograms. five commercial collagen samples were tested as collagen dispersions and partially dehydrated/brined films: c1 (collagen 1), c2 (collagen 2), c3 (collagen 3), c4 (collagen 4) and c5 (collagen 5). collagen treatment onset temperature (˚c) temperature of denaturation (˚c) enthalpy ∆h (j/g) c1 dispersion 33.54±0.21 36.71±0.51 5.33±0.61 c2 dispersion 34.59±0.15 38.44±0.06 3.05±0.31 c3 dispersion 34.26±0.01 38.09±0.08 4.12±0.10 c4 dispersion 35.41±0.11 38.94±0.02 3.93±0.26 c5 dispersion 33.45±0.10 37.30±0.21 4.45±0.03 c1 film 59.90±0.23 64.87±0.12 3.07±0.55 c2 film 58.40±0.21 63.88±0.57 1.76±0.38 c3 film 60.32±1.61 65.00±0.68 3.05±0.21 c4 film 58.22±0.24 63.94±0.61 3.06±0.82 c5 film 58.30±0.40 65.34±0.37 4.19±0.37 ital. j. food sci., vol. 32, 2020 104 3.4. amino acid analysis in addition to the protein content, the protein quality may also influence the mechanical and thermal behaviour of the collagen fibers. analyzing the amino acid profile (table 7) was done to examine potential correlations between the protein quality and performance of the collagen dispersions. collagen fiber’s properties vary as a result of the formation of cross-links between overlapped collagen molecules. cross-links between molecules and fibers can be formed via a number of different mechanisms: schiff base cross-links from enzymatic oxidation (lysyl oxidase), or through non-enzymatic processes like glycation (glucose, lysine and arginine). in general, lysine, glutamic acid and hydroxyl groups have been classified as reactive groups because they project radially from collagen molecules, thus providing sites for intermolecular and interfibrillar cross-links to occur (avery and bailey, 2008). overall, there appeared to be some differences in the amino acid makeup of the dispersions (e.g., lys, glu, oh-pro, arg), but there were no observable trends. table 7. amino acid profile (relative molecular mass) of commercial collagen dispersions. amino acid relative molecular mass (%) collagen 1 collagen 2 collagen 3 collagen 4 collagen 5 asx (asp+asn) 4.6 5.0 5.1 5.3 4.7 glx (glu+gln) 7.5 6.9 7.8 8.2 6.9 oh-pro 14.1 13.1 14.3 14.1 12.8 ser 3.9 3.9 3.9 3.9 3.9 gly 23.1 22.9 22.4 22.2 23.4 his 1.1 0.9 1.1 1.1 0.9 arg 10.6 9.5 10.5 10.6 9.4 thr 1.7 1.8 1.8 1.8 1.8 ala 7.0 8.0 6.9 6.9 7.8 pro 15.8 15.1 15.3 15.1 16.1 tyr 0.6 0.6 0.7 0.7 0.5 val 1.7 2.1 1.7 1.8 2.1 met 2.1 1.7 2.1 1.9 1.8 ile 1.0 1.3 1.1 1.1 1.1 leu 1.5 2.0 1.4 1.4 1.9 phe 1.5 2.2 1.9 1.9 2.1 lys 2.2 2.8 2.0 2.0 2.9 note: values based on a single determination. 4. conclusion manipulating the dehydration conditions (brine concentration; 15 to 30% nacl, and contact time; 1 to 10 min) resulted in differences in mechanical properties when the brine concentrations were averaged across contact times and contact times were averaged across salt concentrations. this indicates that meat processors can adjust the performance of their casings through the modification of brine concentration and contact time. ital. j. food sci., vol. 32, 2020 105 the results also show differences among commercial collagen dispersions and provide actual values (currently not available in the literature), as well as some potential explanations for the differences. the mechanical evaluation of dispersions and films demonstrated that there are significant differences in flow behavior of the raw dispersions as well as tensile strength and percent elongation of the partially dehydrated films (i.e., after the common brining process). it was suggested that a higher degree of intact fibers can result in a collagen film with higher tensile strength and elasticity. collagen dispersions with ph values closer to 2 seem to exhibit lower thermal stability, as conformational changes in the fiber structure occur at lower ph. furthermore, the process of partially dehydrating the collagen fibers greatly increases the temperature of denaturation. overall, this paper provides research and industry personnel with a better understanding of the parameters important for co-extrusion of collagen dispersions into casings, through material selection and manipulation of brining operations. acknowledgements the author would like to thank the ontario ministry of agriculture, food and rural affairs for financial support. references astm. 2010. standard test method for tensile properties of this plastic sheeting. method d882-10, philadelphia, pa. avery n. and bailey a. 2008. restraining cross-links responsible for the mechanical properties of collagen fibers: natural and artificial. in “collagen structure and mechanics”. p. fratzl (ed.), p. 81. springer science, new york, ny. barbut s. 2010. microstructure of natural, extruded and co-extruded collagen casings before and after heating. ital. j. food sci. 22:126. barbut s. 2015. principles of meat processing. in “the science of poultry and meat processing”, isbn-978-088955-625-6. www.poultryandmeatprocessing.com (free download). barbut s. and ioi m. 2019. an investigation of the mechanical, microstructural and thermo-mechanical properties of collagen films cross-linked with smoke condensate and glutaraldehyde. ital. j. food sci. in press. bernal v.m. and stanley d.w. 1986. changes in the melting characteristics of bovine tendon collagen induced by a bacterial collagenase. j. food sci.51(3):834. bontjer m.b.h., kuijpers m.w.j.t. and van den berg k.w. 2011. method for preparing food products by co-extrusion, in particular sausage and food products obtained with this method. european patent wo 2006 135238. carson f.l. 1997. “histotechnology a self-instructional text” 2nd ed. american society of clinical pathology, chicago, il, usa. cranston e.d. and gray d.g. 2008. birefringence in spin-coated films containing cellulose nanocrystals. colloids surf. a physicochem. eng. asp.325:44. friess w. and lee g. 1996. basic thermoanalytical studies of insoluble collagen matrices. biomaterials 17:2289. gioffre m., torricelli p., panzavolta s., rubini k. and bigi a. 2011. role of ph on stability and mechanical properties of gelatin films. j. bioact. compat. polym. 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(eds.), p. 275. american chemical society, washington, usa. visser p.r. 2012. casings for foodstuffs, u.s. patent no. 20120114807a1. wang p.y. 1986. meat processing. in “encyclopedia of food engineering”. c.w. hall, a.w. frall, and a.l. rippen (eds.), pp. 545-550. avi publishing company, inc., westport, ct, usa. williams b.r., gelman r.a., poppke d.c. and piez k.a.. 1978. collagen fibril formation optimal in vitro conditions and preliminary kinetic results. j. biol. chem. 253(18):6578. wolf k.l., sobral p.j.a. and telis v.r.n. 2006. characterization of collagen fibers for biodegradable films production. iufost 13:801. paper received march 19, 2019 accepted july 1, 2019 review ital. j. food sci., vol. 27 2015 129 keywords: omega-3 fatty acids, polyunsaturated fatty acid, biological properties, health important bioactive properties of omega-3 fatty acids rui xu college of food science and technology, hebei normal university of science and technology, qinhuangdao, 066004, china corresponding author: tel. +86-03352039075, fax +86-03352039075, email: robust100@163.com abstract good health has been linked with healthy diet. n-3 fatty acids are required for proper functioning of many physiological systems. there is a large body of evidence documenting the effects of polyunsaturated fatty acids with the first double bond at the third position from methyl-terminal on health benefits. scientific evidence is accumulating to substantiate the role omega-3 fatty acids play in conditions such as cardiovascular disease, certain cancers and other diseases. the availability of n-3 fatty acids to various tissues is of major importance to health and depends on dietary intake for both normal development and in the prevention and management of chronic diseases. in this review we will summarize the biological properties of omega-3 fatty acids. mailto:robust100@163.com 130 ital. j. food sci., vol. 27 2015 introduction long-chain polyunsaturated fatty acid (pufa) with the first double bond at the third position from the methyl-terminal (so called omega-3 fatty acids or n-3 fatty acids) can be found in plants and fish. n-3 fatty acids refer to a group of three fats called alpha-linolenic acid (ala), eicosapentaenoic acid (epa) and docosahexaenoic acid (dha). because these essential fatty acids cannot be synthesized in the human body, they must be derived from dietary sources. they are required for the structure of cell membranes and, because they are unsaturated, they help keep membranes flexible. the peculiar properties of the dha molecule make it a critical component of nerve and retinal cells. flaxseed, hemp, canola and walnuts are generally rich sources of the ala. fish provide varying amounts of n-3 fatty acids in the form of dha and epa. the n-3 pufa are obtained predominately from fish, seafood, meat and eggs (meyer et al., 2003) and in recent years from enriched food products such as bread, milk and margarine. the role played by essential fatty acids in the human body has been the subject of volumes of international research in recent years. the results indicate that n-3 fatty acids may be of value in the treatment of various medical conditions (uauy and valenzuela, 2000). research has been carried out in animal models, tissue cultures and human beings. a present study was to produce pork with enhanced nutritive value for humans, in terms of fatty acid profile. when fish oil was included in the diet, higher levels of epa, dpa and dha were measured in the subcutaneous fat (morel et al., 2013). the positive effects of essential fatty acids are attributed to their ability to reduce inflammation (simopoulos, 2002). many studies show that fish oil with high content of n-3 polyunsaturated fatty acids (pufas) plays an important role in human health and disease. but the effects of fish oil with high content of pufas on gut microbiota, which are also known play a significant role in several human diseases, is not clear. yu et al. (2014) evaluated the effects of fish oil with high content of n-3 pufas on gut microbiota. they found that fish oil treatment resulted in a decrease in helicobacter, uncultured bacterium clone wd2_aaf07d12 (genbank: eu511712.1), clostridiales bacterium, sphingomonadales bacterium and pseudomonas species firmicutes, and several uncultured bacteria. fish oil with a high content of n-3 pufas are capable of producing significant changes in the gut microbiota that may, at least in part, explain the health benefits or injury induced by fish oil use. in recent years, the number of studies describing the health-promoting benefits of n-3 fatty acids has increased. some of the reported activities attributed to the n-3 fatty acids include improving serum cholesterol profiles, stabilizing arrhythmias, reducing inflammation, improving insulin sensitivity in patients with type 2 diabetes, and enhancing the immune response. in this paper, we will discuss diverse effects of an essential component of a healthy diet, long chain n-3 pufa. because modern diets are relatively deficient in this special type of fat, there is a great potential for improving many aspects of health by adding it to the diet. cardiovascular disease cardiovascular disease (cvd) includes all diseases of the blood vessels and circulatory system such as coronary heart disease (chd), ischemic heart disease (ihd), myocardial infarction (mi) and stroke. cvd is the leading cause of death in canada and the united states. since the original epidemiological observations of low cvd mortality in populations with high consumption of fish there has been continuous interest of scientific communities in the possibility of lowering cvd risk by n-3 fatty acids. both epa and dha play a role in modification of lipid and lipoprotein metabolism. n-3 fatty acids have synergistic and additive effects on plasma lipids when co-administered with statins (davidson et al., 2007). increased triglyceride levels are seen among individuals with hiv who receive antiretroviral therapy. a study reported the results of a research that examined the effects of a controlled dietary study supplemented with 4 g of fish oil daily to reduce triglyceride levels in hiv (capili and anastasi, 2013). another practical implication of such studies is the proven safety of n-3 fatty acids add on therapy to statins. both dha and epa have profound effect on platelet function. not only membrane stabilizing effect but also competition of n-3 fatty acids for cyklooxygenase activity with arachidonic acid, which lowers production of platelet activating eicosanoids play an important role (larson et al., 2008). in a study in diabetic patients woodman and co-workers demonstrated administration of highly purified epa/dha was associated with a decrease in platelet aggregability by 30 % (woodman et al., 2003). the platelet effects seem to be mediated mostly by epa (din et al., 2008). oliveira et al.(2014) suggested that fish oil upregulate the expression of the cholesteryl ester transfer protein (cetp) gene. another possibly cardioprotective action of n-3 fatty acids can be modulation of immune response and anti-inflammatory properties. as demonstrated in vitro, dha lowers cytoadhesive molecules expression on endothelial cells and monocytes (mori and beilin, 2004). similarly, levels of interleukin 6, interleukin 1β and tissue necrosis factor α decrease after epa/dha administration (bhatnagar and durrington, 2003). consumption of n-3 fatty acids of 8 g/d ital. j. food sci., vol. 27 2015 131 was associated with a significant reduction of inflammatory marker levels in patients with severe heart failure (mehra et al., 2006). some studies have shown that n-3 fatty acids may increase the susceptibility of ldl to oxidation (sorensen et al., 1998), whereas others have not (higdon et al., 2001). it therefore remains to be established whether ldl oxidative status in vivo is affected by omega-3 fatty acids and, if so, whether this has any adverse clinical implications cancer cancer is another disease that has generated great interest in evaluating the usefulness of n-3 fatty acids. cancer and its treatments are associated with significant long-term side effects such as cachexia, cognitive impairment, distress, pain and fatigue that all warrant supportive care. many experimental studies have shown the role played by n-3 fatty acids in suppressing the development of most cancer processes, including breast, colon, prostate, liver and pancreatic cancers (simopoulos, 2009). in addition, there is evidence that epa and dha exert a potent antiangiogenic effect, inhibiting the production of some of the main angiogenic mediators (spencer et al., 2009). this explains the great interest in establishing fatty acid ingestion in adequate proportions. epa has been suggested to play a protective role in hormone-related cancers, particularly breast and prostate cancers. in animal experiments, epa and dha have consistently inhibited the proliferation of malignant breast and prostate cancers; however, epidemiological studies examining the role of n-3 fatty acids in cancer have not been consistent (terry et al., 2003). fish oils can have a benefit in reversing cancerrelated cachexia by decreasing the protein degradation in cachectic muscle (tisdale, 2003), suggesting that there may be a potential place for n-3 fatty acids in cancer therapy as well as in prevention. xue et al. (2014) suggested that dha exerted its anticancer activity through downregulation of wnt/β-catenin signaling. thus, it should call for further studies to assess the effectiveness of fish oil as a dietary supplement in the prevention and treatment of breast cancer. in the last decade, many clinical trials examining the effects of n-3 fatty acids supplementation on cancer cachexia have been conducted (ries et al., 2012). some randomized controlled trials confirmed these results, especially at dosages of 1.5–2 g epa-enriched enteral liquid formula administrated for at least 8 weeks in gastrointestinal or pancreatic cancers (ryan et al., 2009). clinical studies are emerging to support providing long chain n-3 fatty acids to prevent muscle loss, minimize side effects and improve chemotherapy response in patients with cancer (murphy et al., 2013). a research suggests that marine n-3 fatty acid may increase appetite (signe et al., 2013). this finding would be potentially beneficial for patients with compromised nutritional status. bone the n-3 and n-6 polyunsaturated fatty acids are the immediate precursors to a number of important mediators of immunity, inflammation and bone function, with products of n-6 generally thought to promote inflammation and favour bone resorption. western diets generally provide a 10 to 20-fold deficit in n-3 pufas compared with n-6, and this is thought to have contributed to the marked rise in incidence of disorders of modern human societies, such as heart disease and osteoporosis. osteoporosis is a disease in which bone mass is low and the risk of bone fractures is high. regarding bone diseases, studies in animals have shown that the ingestion of n-3 fatty acids could influence bone formation and resorption (poulsen, 2007). ala may help prevent bone loss and osteoporosis by blocking the production of tumor necrosis factor α, which promotes bone resorption and inhibits bone formation (boyce et al., 2005). when bone metabolism was measured in the volunteers, the highala diet reduced bone resorption without reducing bone formation (zhao et al., 2007). the decrease in bone resorption may have been due to a decrease in the dietary n-6/n-3 ratio as a result of the high-ala diet. tarlton et al. (2013) found that there was a significant 40–60% reduction in keel bone breakage rate, and a corresponding reduction in breakage severity in the n-3 supplemented hens. the biomechanical and biochemical evidence suggests that increased bone turnover has enhanced the bone mechanical properties, and that this may suggest potential benefits for human osteoporosis. however, some studies found no effect of n-3 fatty acids consumption on measures of bone formation and resorption among postmenopausal women (dodin et al., 2005; brooks et al., 2004). these results suggest that the benefit of n-3 fatty acids on bone metabolism is not sufficient to overcome the bone remodeling that occurs during menopause. emotional distress most newly diagnosed and recurrent cancer patients presented with a significant level of mental distress. commonly, distress involves anxiety and depression. a study reported that the antidepressant protective role of n-3 fatty acids might be exerted through production of eicosanoids that are able to reduce the excessive pro-inflammatory cytokine production in depressed patients (kiecolt -glaser et al., 2007). animal studies in which rats were fed an n-3 fatty acids rich diet indicated 132 ital. j. food sci., vol. 27 2015 a better habituation to chronic restraint stress as they showed less stress-induced weight loss, compared to both control and n-3 deficient rats (hennebelle et al., 2012). several clinical trials addressing non-cancer populations have also suggested beneficial effects of n-3 fatty acids on anxiety and depression (lucas et al., 2009; arbabi et al., 2014). however, no trials were found exploring the potential benefits of plant-derived epa. similar positive results were found by tajalizadekhoob et al. (2011) in elderly patients. although the potential benefits of n-3 fatty acids on emotional distress per se have not been examined as a primary endpoint in cancer patients, one trial did study depressive symptoms in lung cancer patients. the trial reported an inverse association between n-3 fatty acids intake, as well as serum n-3 fatty acids and minor depression (kobayakawa et al., 2005). though controversy exists as to whether epa, dha or both are responsible for the efficacy of n-3 fatty acids in depression, results from a randomized controlled trials suggested epa to be a more effective fatty acid component in the treatment of mild to moderate depression (mozaffari-khosravi et al., 2013). there is also evidence that the pathophysiology of major depression is influenced by changes in fatty acid intake. in 2009, dinan et al. evaluated the levels of arachidonic acid, il-6 and tnf α in depressed responders and non-responders to antidepressive treatment. there were significant differences in the epa and arachidonic acid ratio between controls and responders versus non-responders. one research reinforces these observations in major depression and bipolar disorder, and low dha levels may even predict suicidal behaviour (mcnamara et al., 2008). it has been shown that dha content in brain tissue is decreased in patients with neuronal alterations, as in alzheimer’s disease. obesity obesity, a chronic low-grade inflammatory condition, is considered to be a metabolic disorder, whose prevalence is increasing dramatically in most developed countries over the last 20 years. obesity is associated with an increased risk of cvd, type 2 diabetes and a number of cancers. one study suggests that at baseline men with high fish consumption were less likely to be overweight, however no data about association of n-3 fatty acids intake and changes in bmi (body mass index) were provided in this 12-year follow-up cohort (he et al., 2002). the effects of a combination of n-3 fatty acids and a dietary energy restriction in obese volunteers were also examined. parra et al. (2008) showed that satiety was increased after consumption of the n-3 fatty acids-enriched meals. researches have demonstrated that diabetes induces learning and memory deficits (jia et al., 2014). the results suggested that the principle mechanisms involved in the antidiabetic and neuroprotective effect of fish oil were its antioxidant, anti-inflammatory and anti-apoptosis potential, supporting a potential role for fish oil as an adjuvant therapy for the prevention and treatment of diabetic complications. dietary fish oil showed better tissue preservation that was supported by histopathological observations (jangale et al., 2013). thus, the diet proved to be beneficial in preventing tissue injury and alleviating diabetic insults in the livers of diabetic rats. a recent research explored insulin signaling in the newborn rat heart. a diet rich in fish oil improves cardiac akt-related signaling in the offspring of diabetic rats (nasu-kawaharada et al., 2013). hill et al. (2007) found that exercise and n-3 fatty acids supplementation resulted in a significant reduction of body fat. however, munro and garg (2012) described that dietary supplementation with n-3 fatty acids did not promote additional weight loss when combined with a verylow-energy diet for 4 weeks. the most possible cause for the disparities in these results may be related to phenotypical characteristics of the subjects included in the study. indeed, mechanisms underlying this differential response in body weight in obese humans remain a challenging point still to be addressed in future. it should be stated that the effects of n-3 fatty acids on fat mass and weight regulation might be difficult to address due to important differences in how the studies were designed as well as the inclusion criteria and source for n-3 fatty acids supplementation. therefore, effectiveness of the n-3 fatty acids supplementation might be related to dietary and exercise patterns and gender aspects might also be relevant. however, the effects of these fatty acids on insulin sensitivity remain controversial (kabir et al., 2007; navas-carretero et al., 2009; hirabara et aal., 2013), the fact is that insulin resistance is usually linked to other pathological conditions such as hypertrigliceridemia, overweight and cardiovascular diseases and might be difficult to study on itself. thus, further studies are needed to evaluate this aspect of n-3 fatty acids in insulinemia management. nutritional recommendations for the consumption of n-3 fatty acids as we have seen, there are a number of pathologies in which the n-3 fatty acids play an important role, thus reflecting the importance of ensuring their adequate dietary intake. recommendations for dietary intakes of n-3 pufa vary considerably from the consumption of two fish meals a week to epa plus dha intakes of 500 mg/d (kris-etherton et al., 2002) and the japanese recommend consumption of n-3 pufa of 1.6 g/d (sugano, 1996). an approximate estimation of the consumption of n-3 fatty acids ital. j. food sci., vol. 27 2015 133 in europe is 0.1-0.5 g/d. these datas are high in comparison to the estimated intake of dha and epa in the united states (0.1-0.2 g/d), but low in comparison with the data corresponding to japan (up to 2 g/d), where fish is one of the most commonly consumed foods (carrero et al., 2005). in spain, a study carried out by the ministry of agriculture, fisheries and food, showed that the fact that the spanish population consumes levels of n-3 close to the recommended level (1.52 g/d). however, the percentage of energy contributed by epa+dha with respect to total energy in the diet was lower than the recommended value (0.5%). ala, the precursor of n-3 fatty acids, can be converted to longchain n-3 pufa. the minimum intake of n-3 pufa needed for beneficial effects depends on the intake of other fatty acids. dietary amounts of linoleic acid (la) as well as the ratio of la to ala appear to be important for the metabolism of ala to n-3 pufa. indu and ghafoorunissa (1992) showed that a ratio of 4 is appropriate for conversion. this ratio is also consistent with a study by de lorgeril et al. (1994). on comparing the recommendations for n-3 consumption of the different organizations and the existing consumption data, the results show that the consumption of n-3 fatty acids is generally low. the recommendations of the american heart association (aha) are that adults should consume fish at least twice a week. likewise, patients with coronary disease should consume 1 g of epa+dha daily; while patients with hypertriglyceridemia should consume 2-4 g/d of epa+dha (bagga et al., 2002). taking the above into account, the recommended amounts must be adjusted according to the specific needs for the different diseases, and other important dietary factors. in a sense, an increase in n-3 fatty acids in the diet may be regarded as important. from the above we can deduce that a considerable increase in fish consumption is required. maternal levels of n-3 fatty acids during pregnancy determine the levels present in the developing infant. the n-3 fatty acids dha is critical in supporting infant growth and development, and dha levels in newborns are correlated with birth weight and head circumference. it has been suggested that women and their infants may benefit if the mother is supplemented with dha during pregnancy. breast milk contains about 0.5-2.0% ala and about 0.1-0.4% dha (innis, 2000). ala constitutes 75-80% of the total n-3 fatty acids in breast milk (silva et al., 2005), supporting a role for ala in the growth and development of infants. the diets of western countries have contained increasingly larger amounts of la. essential fatty acids also have antibacterial actions and are found in breast milk (das, 2006). indeed, breast milk is rich in la and contains more of ala than of any other omega-3 fatty acid (silva et al., 2005). conclusions our message is that omega-3 fats can contribute to a longer and healthier life. most studies indicate that the consumption of n-3 fatty acids should be more than that presently found in the general population, with a view 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with indigenous korean wine yeasts encapsulated in ca-alginate beads after air-blast drying d.-h. kim1, s.-b. lee2 and h.-d. park*2, 3 1agricultural biotechnology research center, kyungpook national university, 80 daehakro, daegu 702-701, south korea 2school of food science and biotechnology, kyungpook national university, 80 daehakro, daegu 702-701, south korea 3institute of fermentation biotechnology, kyungpook national university, 80 daehakro, daegu 41566, south korea *corresponding author: tel.: +82 539505774; fax: +82 539506772 e-mail address: hpark@knu.ac.kr abstract the aim of this study was to test the possibility of using yeast cells encapsulated in calcium alginate (ca-alginate) beads as a starter for wine fermentation. characteristics of korean campbell early wines fermented by five free yeast cell types and those encapsulated in 2% ca-alginate beads were compared using physicochemical analyses and sensory evaluation tests. the encapsulated yeast cells were shown to ferment korean campbell early grapes with a similar efficiency as that exhibited by the five free yeast celltypes. after fermentation, the characteristics of free cells and encapsulated cells did not show significant differences in terms of content of reducing sugars, soluble solids, total acids, organic acids, and free sugars, as well as in terms of viable cell numbers and other physicochemical properties. the encapsulated cells did, however, produce more alcohol than the free cells. encapsulation in 2% ca-alginate beads was furthermore found to decrease the production of negative volatile compounds. the sensory evaluation of wines fermented by free cells compared with those fermented by ca-alginate bead-encapsulated cells yielded similar scores for the following properties: color, taste, flavor, and overall preference. overall, no significant differences were observed between the two grape wines, and yeast cells encapsulated in 2% ca-alginate beads therefore showed high stability and served as an effective yeast starter for wine fermentation. keywords: campbell early grape, wine, calcium alginate bead, yeast cell immobilization, air-blast drying ital. j. food sci., vol. 30, 2018 536 1. introduction the campbell early grape (vitis labrusca cultivar) is a major grape type used in winemaking and constitutes about 70% of the total grape production in korea (seo et al., 2007; hong and park, 2013). although the demand for domestic wine in korea is increasing, the quality of korean wines remains unreliable owing to high acidity and low sugar content in the wines as well as color weakness of the campbell early grape (kim et al., 2017; lee and kim 2006; park et al., 2004). owing to the short history of wine consumption and the wine industry in korea, studies on these wines are in the preliminary stages (lee et al., 2006; seo and yook, 2007). several researchers have recently attempted to address the problems associated with indigenous yeast and winemaking using campbell early grapes that have adapted to the korean environment (hong and park, 2013; lee et al., 2004; park et al., 2004). the wine fermentation process depends on the ability of yeast to convert grape sugars into alcohol and other compounds (padilla et al., 2016; romano et al., 2003). several studies have reported that indigenous yeasts can improve the sensory properties and the quality of local wines (charoenchai et al., 1997; esteve-zarzoso et al., 1988; hong and park, 2013; lee et al., 2016). however, most korean wines are fermented using an imported yeast starter (choi et al., 2011; kim et al., 2007). the identification of a suitable korean indigenous yeast in winemaking is an important goal for the local wine industry. previously, saccharomyces cerevisiae d8, m12, s13, hanseniaspora uvarum s6 (previously ss6), and issatchenkia orientalis kmbl5774 were isolated from korean grapes to improve the wine quality and were found to enhance the local wine quality (hong and park, 2013; kim, 2006; seo et al., 2007). techniques for drying microorganisms include freeze-drying, spray-drying, and fluidized bed-drying (barbosa et al., 2015; poddar et al., 2014). however, each of these techniques is associated with reduced microorganism viability. recently, alternative drying processes have gained interest owing to their lower costs and faster processing times, as compared to freeze-drying (poddar et al., 2014; santivarangkna et al., 2007). immobilized yeast strains have been optimized based on properties such as survival rate, fermentation ability, viability, and ease of handling without the need for specialized laboratories (caylak and sukan, 1998). a potential application of such immobilized yeasts is the use of korean indigenous yeasts as starter cultures in the local wine industry. different encapsulation techniques have been studied for the preservation and subsequent application of yeast cells (akin, 1987; cassidy et al., 1996; colagrande et al., 1994). over the last three decades, cell immobilization specific to winemaking has been extensively studied owing to the technical and economic advantages such systems may offer over free cell systems (margaritis et al., 1983; stewart and russell, 1986; tsakiris et al., 2004). the use of yeast immobilized in gel-forming materials such as calcium alginate (ca-alginate), agar, carrageenan, cellulosic materials, and pectic acid in winemaking has been well documented (colagrande et al., 1994), and of the various reported methods, immobilization of microbial cells by entrapment in ca-alginate gels is the most widely used approach. this is an attractive technique for various biotechnology, biomedicine, and food technology applications (de vos et al., 2009; kregiel et al., 2013; rokstad et al., 2014). ca-alginate encapsulation offers several advantages over the use of free cells: increased functional efficiency with high cell concentrations in the reactor, easy separation of the immobilized cells in the settling tank, short fermentation lag period, and increased stability of the fermentation system (bardi et al., 1996). several studies in yeast have reported improved survival rates after reduction of moisture content (beker and rapoport, 1987; lievense et al., 1992; lievense et al., 1994), indicating that reduced moisture content enhances the preservation of yeast cells. ital. j. food sci., vol. 30, 2018 537 we previously showed that the immobilization of yeast cells via encapsulation in caalginate beads yielded high survival rates and excellent storability (kim et al., 2017). the aim of this study was thus to investigate the feasibility of using air-blast-dried ca-alginate bead-encapsulated cells compared to the use of free yeast cells in wine fermentation. 2. materials and methods 2.1. strains and medium saccharomyces cerevisiae d8 (kacc 93245p), m12 (kacc 93246p), and s13 (kacc 93247p); hanseniaspora uvarum s6 (previously, ss6 and kacc 93248p); and issatchenkia orientalis kmbl5774 (pichia kudriavzevii kmbl5774 and kacc 93124p) were obtained from the food microbial biotechnology laboratory, department of food science and biotechnology, kyungpook national university (daegu, south korea). the yeasts were cultured aseptically in 100 ml ypd broth [2% yeast extract, 1% peptone, and 2% glucose (w/v); 2% (w/v) agar was added for solid medium] in a rotary shaker (jssi-300c, js research inc., gongju, south korea) at 30°c for 24 h. all yeast strains were stored in 15% glycerol at -80°c. ca-alginate beads were prepared using a previously described method (kim et al., 2017). in this study, 2% ca-alginate bead-encapsulated cells were used and the encapsulated cells showed at least a 51% survival rate when stored at 4°c for 3 months (kim et al., 2017). 2.2. release and viability of encapsulated ca-alginate beads beads containing yeast cells were rehydrated in 10 ml ypd broth and the release of the yeast cells from the 2% ca-alginate beads was monitored for 48 h using an optical microscope (nikon eclipse, te2000-u, melville, ny, usa). after the yeast cells were released from the beads, a 1 ml sample of the resulting yeast culture was serially diluted in a 0.85% (w/v) nacl solution. 2.3. viable cell count samples were serially diluted with 0.85% nacl using the appropriate dilution factors. each sample was spread onto ypd agar medium plates. the plates were cultured at 30°c for 48 h, and the number of white colonies that formed on the ypd agar was counted to provide the number of colony forming units (cfu) (dilutions gave 30 to 300 cfu/ml). 2.4. vinification processing campbell early grapes (vitis labrusca cultivar) were obtained from sangju, kyungpook province, south korea in 2016. the vinification process was assessed using a general winemaking method (hong and park, 2013). the grapes were washed, stemmed, crushed, and treated with potassium metabisulfite (k2s2o5; 200 ppm) to inhibit harmful bacterial and yeast growth. the sugar content off the grape must was raised from 16.5° brix (w/v; ph 3.2) to 24° brix by the addition of sucrose. following inoculation with the two types of yeast cells, namely a 24 h yeast pre-culture or cells immobilized in ca-alginate beads [5% inoculum (w/v)], fermentation was carried out at 18°c for 14 days in glass bottles (5,000 ml volume) equipped with an airlock using 3,000 ml of grape must. post fermentation, ital. j. food sci., vol. 30, 2018 538 free yeast cells, 2% ca-alginate bead-encapsulated cells, and other lees were eliminated by centrifugation at 6,000 × g for 10 min. 2.5. standard chemical analysis the ph of the wine was measured using a ph meter (mettler-toledo gmbh, schwerzenbach, switzerland). the soluble solid content (°brix) was measured using a refractometer (ra250, atago, tokyo, japan), the alcohol content was measured at 15°c using a vinometer, and the total acid content and reducing sugars were quantified using the aoac method (caputi, 1995). total phenolic compounds (tpc) were assessed using the folin-ciocalteau phenol reagent method (singleton and rossi, 1965) and the free sugar content was determined by high-performance liquid chromatography (hplc) using a sugar-pak i column (⌀ 6.5 × 300 mm, waters, milford, ma) and a ca-edta buffer (50 mg/l) at a flow rate of 0.5 ml/min. organic acids were quantified by hplc using a shodex rspak kc-811 column (⌀ 8 × 300 mm, showa denko kk, kawasaki, japan). the column was run with a mobile phase of 0.1% phosphoric acid at a flow rate of 1 ml/min at 40°c. organic acids were detected using a refractive-index detector. acetaldehyde, methanol, and various alcohols (fusel oil) were assessed using gas chromatography (6890n gc; agilent, santa clara, ca, usa) and a flame ionization detector (fid). after distillation, samples were filtered through a membrane filter (millex-hv, 0.45 µm, millipore co., bedford, ma, usa) before injection. separation was performed with an hp-ffap column (⌀ 0.25 mm × 30 m, film thickness = 0.25 µm; agilent technologies, santa clara, ca, usa) using helium as a carrier gas with a constant flow of 1 ml/min. the chromatographic oven temperature was initially kept at 60°c for 4 min, was then increased to 210°c, at 6°c/min, and was then maintained at 210°c for 2 min. the quantitative determination of volatile compounds was performed using the relative area calculated as the ratio. samples were assessed in terms of hunter's color values (l*, lightness; a*, red-green; and b*, yellow-blue) using a vertical-type spectrophotometer (cm3600d, konica minolta, inc., tokyo, japan). all measurements were replicated three times and average values (n = 3) were calculated. 2.6. sensory evaluation sensory evaluation of wines was performed by a panel of twenty trained experts. the color, flavor, taste, and overall preference of the wines were evaluated on a scale of 1 to 5, where 5 was the best score. overall preference according to the taste and flavor was evaluated using the mean value of a hedonic scale from 1 (very poor, dislike extremely) to 5 (excellent, like extremely) (piggott, 1988). 2.7. statistical analysis data were expressed as mean±standard deviation (sd) of triplicate experiments. statistical significance was determined by a student’s t-test for independent means, using microsoft excel (microsoft, redmond, wa, usa). a one-way analysis of variance (anova) and duncan's multiple range tests were used to determine differences between means. the critical level for statistical significance was set at p < 0.05. ital. j. food sci., vol. 30, 2018 539 3. results and discussion 3.1. characteristics of grapes fermented by free cells and ca-alginate bead-encapsulated yeast cells we previously investigated the effects of ca-alginate beads and protective agents on the survival rates of five yeast strains using an air-blast drying method, which demonstrated that 2% ca-alginate beads soaked in protective agents (10% skimmed milk and 10% solutions of various sugars) helped to protect free cells from the environment. specifically, in this study, s. cerevisiae d8 cells with 10% sucrose, s. cerevisiae m12 cells with 10% raffinose, s. cerevisiae s13 cells with 10% trehalose, h. uvarum s6 cells with 10% trehalose, and i. orientalis kmbl5774 cells with 10% glucose, all encapsulated in ca-alginate beads, exhibited the highest survival rates (90.67%, 87.73%, 92.05%, 90.81%, and 87.16% viability, respectively) after air-blast drying at 37°c for 5 h (kim et al., 2017). changes in the ph of wine fermented by encapsulated cells compared to those of wines fermented by free yeast cells were shown in fig. 1. ph has a marked effect on microorganisms: in a study by bae (2002), it was suggested that a ph <3.2 results in sourness during wine fermentation, while a ph <4.0 is recommended for preventing contamination by other harmful bacteria. in this study, the ph of all samples ranged from 3.41 to 3.65 during fermentation. at the beginning of fermentation, the ph values of all samples were 3.59–3.63 and decreased slowly during fermentation to final values of 3.47– 3.52. the changes in total acid content in encapsulated cell-fermented wine compared to free cell-fermented wine were also shown in fig. 1, where it can be seen that the total acid content in all wines increased from 0.41–0.43 to 0.61–0.69 during fermentation. the total acid content of wines fermented using free cells was found to increase slightly after two days, while that of the wines fermented using 2% ca-alginate bead-encapsulated cells was found to increase only slightly after three days. figure 1. changes in ph and total acid content during fermentation. (a) free cells, and (b) cells encapsulated in 2% ca-alginate beads. open circles, s. cerevisiae d8; filled circles, s. cerevisiae m12; open squares, s. cerevisiae s13; filled squares, h. uvarum s6; open triangles, i. orientalis kmbl5774. seo and yook (2007) have reported that the total acid content during fifteen days of campbell early wine fermentation with a commercial wine dry yeast starter was 0.44–0.81. similar to this, our study demonstrated low total acidity levels in wine fermented using ital. j. food sci., vol. 30, 2018 540 encapsulated cells for fourteen days. lee and kim (2006) reported that the korean campbell early grape has a high level of acidity due to the presence of malic acid and tartaric acid, and investigated six different fermentation processes in terms of their capacity to reduce the levels of acidity. based on the findings of the study, both carbonic maceration and precipitation methods were recommended for obtaining high quality wines. the levels of soluble solids in the wines fermented using encapsulated cells were shown to decrease sharply after three days. in contrast, the soluble solid levels in the wines fermented by free cells decreased after two days (fig. 2). we previously showed that the free and immobilized cell populations exhibited similar growth patterns; however, slight differences were observed at the early stage of the fermentation (kim et al., 2017). it should be noted that, as the cell population in the beads increased, the diameter of the beads increased significantly due to the elastic properties of the alginate gel (vives et al., 1993). kim et al., (2017) studied characteristics of red wine fermentation using campbell early and different sugars. the results revealed that wine fermentation with added glucose was faster, and it yielded a higher alcohol content, than that obtained with any other sugars. the wine fermented by the addition of sucrose and high fructose corn syrup showed results similar to that obtained by the addition of glucose. similar results were obtained in our experiments; the soluble solid content of the two wines used in this study decreased upon the addition of sucrose, although the fermentation starting points differed. due to conversion into alcohol, the reducing sugar content of all wine samples decreased sharply (from 25° brix to 0.17° brix) during fermentation. the reducing sugar content and the soluble solid content of samples fermented using 2% ca-alginate bead-encapsulated cells decreased more slowly than that of samples fermented using free cells owing to pore size and different fermentation starting points (figs. 2 and 3) (kim et al., 2017). figure 2. changes in soluble solid contents during fermentation. (a) free cells, and (b) cells encapsulated in 2% ca-alginate beads. open circles, s. cerevisiae d8; filled circles, s. cerevisiae m12; open squares, s. cerevisiae s13; filled squares, h. uvarum s6; open triangles, i. orientalis kmbl5774. most of the reducing sugar content and soluble solid content were consumed during the fermentation. changes in alcohol content in the presence of bead-encapsulated cells compared to that in free cells during fermentation were shown in fig. 3. the alcohol ital. j. food sci., vol. 30, 2018 541 content of wines fermented using free cells ranged from 9.20±0.06% to 12.97±0.09%, while that of wines fermented using encapsulated cells ranged from 9.75±0.04% to 13.43±0.24%. although ca-alginate bead-encapsulated cells fermented at a slightly slower rate than the free cells, their alcohol production was higher than that of free cells. roukas et al., (1991) reported that free and immobilized s. cerevisiae cells produce the same maximum ethanol concentration under similar fermentation conditions. singh et al., (1998) also reported that the concentrations of ethanol produced in free cell and immobilized cell (ca-alginate) batch fermentations were comparable. however, holcberg and maralith (1981) reported that the rate of ethanol production by cells entrapped in agar, alginate, and polyacrylamide gels was higher than that of free cells. norton et al., (1995) reported that the resistance of yeast to ethanol was significantly higher in immobilized cells than in free cells. the findings reported here show that alcohol production using 2% ca-alginate beadencapsulated cells was higher than that using free cells. in both free cell and encapsulated cell systems, h. uvarum s6 and i. orientalis kmbl5774 exhibited slower alcohol production rates and lower maximal alcohol levels compared with s. cerevisiae d8, m12, and s13 (fig. 3). figure 3. changes in alcohol and reducing sugar content during fermentation. (a) free cells, and (b) cells encapsulated in 2% ca-alginate beads. open circles, s. cerevisiae d8; filled circles, s. cerevisiae m12; open squares, s. cerevisiae s13; filled squares, h. uvarum s6; open triangles, i. orientalis kmbl5774. a high cell density of a non-saccharomyces (h. uvarum) yeast has been identified in v. vinifera grape must during the first 4-6 days of fermentation, until the ethanol content reached 4-7% (v/v). accordingly, it has been suggested that the inoculation with pure cultures of h. uvarum cells may enhance ethanol production during fermentation (rojas et al., 2003). in agreement with these reports (moreira et al., 2008; rojas et al., 2003), our findings revealed that h. uvarum s6 produced approximately 9.2±0.06 to 9.75±0.04% (v/v) alcohol. in a previous study, the effects of co-fermentation with various inoculation ratios (s. cerevisiae w-3: non-s. cerevisiae; i. orientalis kmbl5774) were investigated and led to the use of a mixed culture being recommended for better wine quality owing to the low alcohol production capacity of i. orientalis kmbl5774 (kim et al., 2008). in our study, however, a low maximal alcohol content [11.20±0.06 to 11.47±0.29% (v/v)] was achieved by single fermentation and the use of ca-alginate beads encapsulating a single strain (i. orientalis kmbl5774) was therefore studied. ital. j. food sci., vol. 30, 2018 542 the total phenolic compound levels in all the samples ranged from 0.13% to 0.15% (fig. 4), while lee et al., (2006) reported that the total content of phenolic compounds of campbell early wine in korea was approximately 0.12%. the markedly higher total phenolic compound levels observed in the present study may be attributed to different conditions such as vintage, area, and climate (huglin, 1978; huglin and schneider, 1998; seguin, 1975; winkler et al., 1975). no major differences in total phenolic content were observed between free cell and encapsulated cell fermentations. figure 4. changes in total phenolic compound contents during fermentation. (a) free cells, and (b) cells encapsulated in 2% ca-alginate beads. open circles, s. cerevisiae d8; filled circles, s. cerevisiae m12; open squares, s. cerevisiae s13; filled squares, h. uvarum s6; open triangles, i. orientalis kmbl5774. 3.2. yeast cell release from 2% ca-alginate beads and viable cell count during fermentation encapsulated cells (initial cell concentration: 1.0±0.1 × 108 cfu/ml; stored at 4°c for 3 months) and cultured free cells (initial cell concentration: 1.0±0.2 × 108 cfu/ml) were inoculated into 5% (w/v) grape must. at the beginning of the fermentation, the free cells began to grow immediately, while the narrow and complex interior structure of the 2% ca-alginate beads hindered the release of yeast cells from encapsulation following budding from 0 h (figs. 5 and 6) (kim et al., 2017). accordingly, viable cell numbers in the 2% ca-alginate bead samples increased more slowly than those in the free cell samples. as the cell population grows within the beads, the bead diameters increase significantly due to the elastic properties of the alginate gel (babu et al., 1992; vives et al., 1993). this effect results in a delay in the rate at which the maximal cell population (108 cfu/ml) is reached (fig. 6). klinkenberg et al. (2001) reported that the viable cell count (lactobacillus lactis ssp. lactis) inside beads coated with alginate during milk fermentation remained significant at the beginning of fermentation, and that the alginate beads had a significant effect on the rate of cell release after 48 h of fermentation. in our study, however, the encapsulated cells were released slowly after 6 h and the release rate increased sharply after 48 h. in five to six days, the wines fermented by cells encapsulated in 2% ca-alginate beads exhibited maximal populations similar to those exhibited by the free cell-fermented wines (fig. 5). ital. j. food sci., vol. 30, 2018 543 figure 5. optical microphotograph (×100 magnification) of yeast cells released from 2% ca-alginate beads at different time intervals up to 48 h. figure 6. changes in viable cell count during fermentation. (a) free cells, and (b) cells encapsulated in 2% ca-alginate beads. open circles, s. cerevisiae d8; filled circles, s. cerevisiae m12; open squares, s. cerevisiae s13; filled squares, h. uvarum s6; open triangles, i. orientalis kmbl5774. ital. j. food sci., vol. 30, 2018 544 3.3. physicochemical properties of wines fermented by free cells and encapsulated cells after fermentation, sucrose, glucose, galactose, and fructose were identified as the sugar components in the wines and of these sugars, sucrose was found to be present at the highest concentration (1.32±0.01 g/l to 2.05±0.43 g/l) in both wine samples (table 1). the two different fermentation processes were therefore found to yield similar sugar profiles. free sugars are the major components of grape soluble solids and are related to the final alcohol content of wine (conde et al., 2007). using hplc, shiraishi et al., (2010) showed that the major free sugars in grape wine (vitis spp.) were glucose, fructose, and sucrose. kliewer (1966) have also reported the presence of glucose, fructose, galactose, sucrose, maltose, melibiose, raffinose, and stachyose in vitis spp. to determine the organic acid composition of the wines, the levels of malic acid, tartaric acid, citric acid, succinic acid, and acetic acid were assessed in the wines (table 2). malic acid and tartaric acid, which contribute 70–90% of the total acidity of grapes, significantly influence the sensory properties of wine (beelman and gallander, 1979; ruffner, 1982). differences in organic acid content of both wines were negligible, nevertheless wines fermented using encapsulated cells exhibited slightly lower levels than those fermented using free cells. the wine fermented by i. orientalis kmbl5774, which has previously been shown to be able to rapidly degrade malic acid in medium where it represents the sole carbon and energy source (seo et al., 2007), exhibited lower levels of malic acid than the other wines. as expected, the malic acid concentrations were lower in wines fermented with i. orientalis kmbl5774 than in wines fermented with other strains; however, other than malic acid, the levels of other organic acids did not differ markedly between free celland encapsulated cell-fermented wines. lee and kim (2006) studied the de-acidification of wine made from campbell early grapes, and recommended carbonic maceration and cold fermentation to decrease the organic acid content of campbell early wine. several studies have shown that the process of malolactic fermentation results in the degradation of malic acid into lactic acid and carbon dioxide, the consequence of which is a reduction in total acidity (de-acidification) in the wine by strains of lactic acid bacteria (lab) of the genera oenococcus, leuconostoc, lactobacillus, and pediococcus (boulton et al., 2013; volschenk et al., 1997; viljakainen and laaso, 2000). as shown in table 3, the aldehyde content of wines fermented using free and encapsulated cells ranged from 44.88±0.49 to 65.28±4.32 mg/l and from 41.28±2.11 to 65.28±4.32 mg/l, respectively. only small differences in aldehyde levels (lower in those fermented using encapsulated cells), if any, were observed between the free celland encapsulated cell-fermented wines. acetaldehyde is considered to be a leakage product of alcohol fermentation by yeast, and s. cerevisiae and kloeckera apiculata have been shown to produce 0.5–286 mg/l and 9.5–66 mg/l acetaldehyde (geroyiannaki et al., 2007). geroyiannaki et al., (2007) also reported that white and red grape pomace yielded 345 mg/l and 317 mg/l acetaldehyde, respectively. the lowest reported acetaldehyde level in wine was for cagaita wine produced using encapsulated cells (1.031 mg/l; s. cerevisiae ufla ca11), whereas the free cell equivalent yielded 1.378 mg/l acetaldehyde (oliveira et al., 2011). all wines in this study (both free celland encapsulated cellfermented) yielded acetaldehyde levels well below the official limit of 700 mg/l (korea, 2012). during wine production, methanol arises as a result of pectin methyl esterase activity during grape crushing (masino et al., 2008). ital. j. food sci., vol. 30, 2018 545 table 1. free sugar contents (g/l) in wines fermented using 2% ca-alginate bead cells compared to those in wines fermented using free cells after fermentation. type s. cerevisiae d8 s. cerevisiae m12 s. cerevisiae s13 h. uvarum s6 i. orientalis kmbl5774 free cells bead cells p-value free cells bead cells pvalue free cells bead cells p-value free cells bead cells p-value free cells bead cells p-value sucrose 1.87±0.03 1.80±0.02 0.0420 1.71±0.01 1.72±0.05 0.6413 1.32±0.01 1.34±0.01 0.0512 1.45±0.05 1.32±0.06 0.0396* 2.05±0.13 1.96±0.07 0.3489 glucose 1.11±0.08 1.09±0.04 0.7200 1.22±0.12 1.11±0.09 0.2321 1.07±0.08 1.01±0.10 0.4266 1.45±0.11 1.25±0.05 0.0477* 1.17±0.06 1.13±0.04 0.6547 galactose 1.31±0.25* 1.10±0.08* 0.2459 1.22±0.07 1.10±0.08 0.1382 1.25±0.13 1.22±0.07 0.7296 1.29±0.06* 1.03±0.10* 0.0185* 1.32±0.07* 1.10±0.10* 0.0347* fructose 0.11±0.01 0.13±0.01 0.1340 0.13±0.01 0.13±0.03 0.8383 0.15±0.01 0.15±0.03 0.9068 0.11±0.00 0.12±0.00 0.0181* 0.10±0.01 0.10±0.02 0.8731 total free sugars 4.40±0.26 4.12±0.11 0.1662 4.28±0.17 4.06±0.11 0.1382 3.79±0.13 3.72±0.18 0.5917 4.30±0.09 3.71±0.12 0.0026** 4.60±0.05 4.29±0.07 0.0021** all data are expressed as mean±sd (n = 3). *p < 0.05 and **p < 0.01 are considered to be statistically significant by student’s t-test. table 2. organic acid contents (g/l) in wines fermented by 2% ca-alginate bead cells compared to those in wines fermented using free cells after fermentation. type s. cerevisiae d8 s. cerevisiae m12 s. cerevisiae s13 h. uvarum s6 i. orientalis kmbl5774 free cells bead cells p-value free cells bead cells pvalue free cells bead cells p-value free cells bead cells p-value free cells bead cells p-value lactic acid 0.78±0.02 0.77±0.05 0.7145 0.75±0.04 0.79±0.04 0.3038 0.74±0.05 0.85±0.03 0.0342* 0.82± 0.03 0.89±0.04 0.0598 0.82±0.02 0.94±0.05 0.0238* citric acid 0.14±0.06 0.20±0.02 0.2028 0.14±0.03 0.16±0.03 0.6491 0.21±0.04 0.16±0.03 0.1406 0.26±0.03 0.14±0.05* 0.0224* 0.21±0.01 0.16±0.04 0.1133 tartaric acid 0.91±0.05 0.90±0.07 0.8561 0.83±0.02 0.86±0.03 0.1907 0.89±0.02 0.85±0.02 0.0920 0.95±0.07 0.93±0.09 0.8188 0.93±0.07 * 0.92±0.10* 0.9261 malic acid 1.72±0.01 1.70±0.01 0.0296* 1.89±0.04 1.71±0.06 0.0120* 1.63±0.01 1.65±0.03 0.4034 1.70±0.04 1.69±0.03 0.7625 1.33±0.03 1.03±0.06 0.0013** succinic acid 0.13±0.02 0.11±0.07 0.6800 0.12±0.03 0.11±0.01 0.4822 0.13±0.03 0.10±0.04 0.3218 0.09±0.06 0.09±0.04 0.9776 0.09±0.04 0.10±0.03 0.8686 acetic acid 0.16±0.01 0.15±0.03 0.7050 0.11±0.01 0.13±0.04 0.4306 0.10±0.01 0.13±0.09 0.6319 0.16±0.01 0.12±0.03 0.1030 0.15±0.03 0.10±0.02 0.0714 all data are expressed as mean±sd (n = 3). *p < 0.05 and **p < 0.01 are considered to be statistically significant by student’s t-test. ital. j. food sci., vol. 30, 2018 546 table 3. aldehyde, methanol, and fusel oil contents (mg/l) in wines fermented by 2% ca-alginate bead cells compared to those in wines fermented using free cells after fermentation. type s. cerevisiae d8 s. cerevisiae m12 s. cerevisiae s13 h. uvarum s6 i. orientalis kmbl5774 free cells bead cells p-value free cells bead cells p value free cells bead cells p-value free cells bead cells p-value free cells bead cells p-value aldehyde 64.17±2.13 64.27±3.32 0.9670 61.90±3.11 53.07±3.15 0.0259 65.28±4.32 54.66±3.45 0.0292* 44.88±0.49 41.28±2.11 0.0451* 52.01±1.26 47.56±1.65 0.0206* methanol 112.84±2.11 108.69±3.21 0.1346 110.26±3.22 110.28±2.01 0.9932 109.77±4.13 105.17±3.89 0.2329 105.39±1.25 106.30±1.02 0.3839 105.33±3.28 108.53±1.57 0.2021 ethyl acetate 121.79±1.36 119.12±1.27 0.0678 126.2±2.14 115.74±2.35 0.0047** 125.33±1.46 113.35±1.43 0.0006# 158.06±3.52 148.61±3.29 0.0274* 132.19±1.14 130.09±3.21 0.3458 1-propanol 191.67±5.21 155.83±2.74 0.0005# 172.78±3.22 106.20±1.25 0.0000# 155.05±2.14 97.98±1.98 0.0000# 158.25±1.58 121.10±2.04 0.0000# 139.07±2.95 117.35±3.05 0.0003# iso-butanol 361.79±2.56 389.12±1.07 0.0001# 346.22±3.14 345.74±2.65 0.8495 385.33±2.36 373.35±2.47 0.0037** 458.06±4.02 448.61±2.69 0.0277* 432.19±1.02 435.09±1.24 0.0352* iso-amyl alcohol 391.81±3.56 379.17±1.32 0.0045 ** 348.16±2.14 333.12±1.96 0.0009# 387.61±3.02 348.65±2.58 0.0001# 443.71±2.04 396.66±2.65 0.0000# 417.87±3.02 405.28±2.79 0.0061** all the data are expressed as mean±sd (n = 3). *p < 0.05, **p < 0.01 and #p < 0.001are considered to be statistically significant by student’s t-test. ital. j. food sci., vol. 30, 2018 547 the methanol contents in all the wine samples in this study were found to range from 105.17±3.89 mg/l to 112.84±2.11 mg/l (table 3), which are well below the standard maximal value for alcoholic beverages (1,000 mg/l), and were not affected by the caalginate bead system. according to mateos et al., (2006), the major compounds that contribute to the overall volatile effects defining wine aroma are ethyl acetate and higher alcohols such as 1-propanol, isobutyl alcohol, and iso-amyl alcohol. as shown in table 3, the fusel oil analysis conducted in this study revealed that iso-amyl alcohol, which contributes to wine quality (kourkoutas et al., 2001) was present at 333.12±1.96 to 443.71±2.04 mg/l in the wines, and was the most prevalent fusel oil component. the ethyl acetate, 1-propanol, and iso-butanol concentrations ranged from 113.35±1.43 to 158.06±3.52 mg/l, from 97.98±1.98 to 191.67±5.21 mg/l, and from 346.22±1.96 to 443.71±2.04 mg/l, respectively. h. uvarum s6 cells (both free and encapsulated cells) yielded the highest levels of iso-butanol and iso-amyl alcohol during fermentation compared with the other yeast types. oliveira et al., (2011) have reported hanseniaspora and issatchenkia (nonsaccharomyces) as high ester producers. in this study, the wines fermented using encapsulated cells generally contained lower levels of fusel oil components than the wines fermented using the free cells. in terms of hunter's color values, there were no marked differences between the free cell and encapsulated cell fermented wines. the l* values ranged from 38.15±0.01 to 38.97±0.01, the a* values ranged from 7.24±0.04 to 7.66±0.16, and the b* values ranged from 0.00±0.02 to 0.20±0.02 (table 4). there were no marked differences between the free cells and 2% ca-alginate bead cells-fermented wines. 3.4. sensory evaluation the sensory characteristics of wines fermented using free and encapsulated cells were evaluated by a panel of twenty assessors. preferences in terms of color, flavor, taste, and overall preference were determined on a scale of 1 (poorest) to 5 (best) (fig. 7). in terms of flavor and taste, the wines fermented with h. uvarum s6 and i. orientalis kmbl5774 (both free and encapsulated cell fermentations) obtained the highest scores, while the wines fermented with s. cerevisiae d8, m12, and s13 obtained the lowest scores. in agreement with previous reports (mateos et al., 2006; torrens et al., 2008), differences in volatile compound levels between the wines seemed to correlate with the sensory evaluation. among the wines in this study, high levels of acetaldehyde and low levels of ethyl acetate resulted in lower wine quality scores. when comparing wines fermented by free cells with those fermented by encapsulated cells, however, scores for color, taste, flavor, and overall preference did not differ, which indicates that encapsulated yeast cells represent suitable alternative starters for wine fermentation. ital. j. food sci., vol. 30, 2018 548 table 4. hunter's color values for wines fermented by 2% ca-alginate bead cells compared to those for wines fermented using free cells after fermentation. type s. cerevisiae d8 s. cerevisiae m12 s. cerevisiae s13 h. uvarum s6 i. orientalis kmbl5774 free cells bead cells p-value free cells bead cells pvalue free cells bead cells p-value free cells bead cells p-value free cells bead cells p-value l* 38.97±0.01 38.97±0.02 1.0000 38.69±0.14 38.61±0.01 0.4276 38.44±0.03 38.44±0.01 1.0000 38.17±0.04 38.15±0.01 0.4481 38.28±0.02 38.28±0.01 1.0000 a* 7.66±0.16 7.58±0.03 0.4426 7.24±0.04 7.26±0.03 0.5265 7.46±0.13 7.32±0.03 0.1433 7.55±0.01 7.52±0.03 0.1757 7.47±0.04 7.47±0.03 1.0000 b* 0.01±0.02 0.00±0.02 0.5734 0.20±0.02 0.20±0.01 1.0000 0.13±0.02 0.14±0.02 0.5734 0.20±0.02 0.20±0.01 1.0000 0.05±0.21 0.07±0.12 0.8930 δe 38.92±0.01 39.72±0.03 0.0000# 38.64±0.01 39.12±0.13 0.0237* 38.46±0.02 39.15±0.04 0.0000# 38.19±0.02 38.91±0.03 0.0000# 38.30±0.01 39.00±0.02 0.0000# all the data are expressed as mean±sd (n = 3). *p < 0.05 and #p < 0.001 are considered to be statistically significant by student’s t-test. figure 7. radar plot of the sensory evaluation scores for wine fermented by 2% ca-alginate bead cells compared to those for wines fermented by free cells after fermentation. the results reflects the means of scores from 20 semi-trained panelist. ital. j. food sci., vol. 30, 2018 549 4. conclusions in this study, fermentation characteristics of free yeast cell and yeast encapsulated in caalginate beads (immobilized yeast) were compared. there was no significant difference between free yeast cell and immobilized yeast in terms of reducing sugars content, soluble solids, total acids, organic acids, free sugars, viable cell count and other physiochemical properties. however, immobilized yeast produced slightly higher alcohols and a lower total concentration of volatile acids (compound) than free yeast cell. these results suggest that yeast encapsulated in ca-alginate beads have a potential to be used as alternative yeast for wine fermentation as it is cost effective compared to freeze-dried yeast. acknowledgements this study was supported by the rural development administration, republic of korea (research grant pj012425022018). references akin c. 1987. biocatalysis with immobilized cells. biotechnol. genet. eng. 5:319-367. babu g.r.v., wolfram j.h. and chapatwala k.d. 1992. conversion of 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lider. 1974. general viticulture, p, 710. university of california press, berkeley, usa. paper received december 28, 2017 accepted may 5, 2018 ital. j. food sci., vol. 30, 2018 487 paper effect of infrared thermal pre-treatment of sesame seeds (sesamum indicum l.) on oil yield and quality m. krajewskaa, b. ślaska-grzywna*a and d. andrejkoa adepartment of biological bases of food and feed technology, university of life sciences in lublin, faculty of production engineering, lublin, poland *e-mail address: beata.grzywna@up.lublin.pl abstract the aim of the study was to determine the effect of thermal infrared treatment of indian sesame seeds (sesamum indicum l.) on the yield and quality of extracted oil. the infrared radiation treatment of the seeds was applied for 30 s, 60 s, 90 s, 120 s, and 150 s at a temperature of 180ºc. the mean moisture and fat content in the seeds was 7,46 and 45,61%, respectively. the extracted oil was assessed in terms of the hydrolysis degree (av), primary oxidation degree (pv), oxidative stability in the rancimat test, and content of carotenoid and chlorophyll pigments. the study has shown that the infrared thermal treatment of sesame seeds contributed to an increase in the oil extraction efficiency, compared to the control sample. the highest increase in the oil yield was found in the case of seeds heated for 120 s. simultaneously, there was a reduction of the quality of these oils accompanied by increased oxidative stability. the induction time in oils extracted from infrared-treated seeds increased gradually together with the increase in the length of the heating process. the longest induction time (9,53 h) was noted in sesame oil obtained from seeds heated for 150 s. keywords: antioxidant additives, acid value, peroxide value, thermal infrared treatment, oxidative stability. ital. j. food sci., vol. 30, 2018 488 1. introduction freshly pressed and non-refined vegetable oil is characterised by a delicate flavour and aroma typical for the seeds used in the process. it should not have an unpleasant bitter taste, as this may imply that the oil is not fresh. such oils are biologically active, because they are pressed at low temperatures. hence, they are also referred to as ‘virgin’, which means that they have the same chemical composition after extrusion as that in the seeds of plants from which they originate. these oils are not supplemented with pigments, antioxidants, and preservatives and are not devoid of phospholipids or tocopherols (vitamin e) as is the case of the common refined edible oils (pala, 2001). cold-pressed oils contain mainly highly active antioxidants (tocopherols, polyphenols, carotenoids, feldspar), polyunsaturated fatty acids from the n-3 and n-6 groups, and bioactive sterols (roszkowska et al., 2014). the method of oil extraction with the use of a screw press is quite common. the method has been widely popularised as a simple and ecologically friendly technology that does not require excessive energy costs and investment expenditures. unfortunately, there are some limitations to the method, e.g. low yields of the process (considerable oil content left in the pomace) and difficulties in achieving a stable quality of the product. investigations have evidenced that the process of fragmentation or heating the seeds prior to oil extraction increases the efficiency of the pressing process (gupta, 2011). a very important step in the oil extraction process is the hydrothermal pre-treatment of oil-bearing plants. in the oil industry, pre-treatment (t 30 40°c) is applied to achieve a homogenous temperature and humidity of the seed mass and to ensure a proper fragmentation process. in turn, the basic conditioning (roasting) of the seed pulp (t 80 100°c) is applied to facilitate fat extraction and to increase the oil yield (gupta, 2011). currently, investigations are being carried out to assess the potential of other alternative seed pre-treatment methods that are more time and energy efficient. an example of such methods is the infrared thermal treatment technique, which provides a higher temperature (over 160°c) within a shorter time than the conventional convection heating (azadmard-damirchi et al., 2011). infrared radiation penetrates and heats the product inside, thereby leading to partial evaporation of water from the seed interior accompanied by an increase in intracellular pressure. the disintegration of the cellular structure expands the surface area of the oil release thus contributing to enhancement of extraction yields (azadmard-damirchi et al., 2011, yang et al., 2013). the infrared thermal treatment of seeds applied prior to the pressing process not only increases the yield but also shortens the heating time, which is accompanied by reduced energy consumption (azadmard-damirchi et al., 2011). as shown by literature data, the pressing method and seed pre-treatment exert a significant effect on the quality, nutritional value, and oxidative stability of extracted oil (niu et al., 2013, yang et al., 2013). the range of qualitative changes in such oils depends both on the pre-treatment and extraction parameters and on the species of pressed seeds. therefore, the aim of the study was to determine the effect of infrared thermal treatment of indian sesame seeds (sesamum indicum l.) on the yield and quality of extracted oil. ital. j. food sci., vol. 30, 2018 489 2. materials and methods the research was conducted on indian sesame seeds (sesamum indicum l.) harvested in 2016 and oil extracted from the seeds. to balance the humidity of the seeds, the material was stored in a glass jar without a lid at room temperature (20±2˚c) for 14 days. the infrared thermal treatment of the seeds was applied for 30, 60, 90, 120, and 150 s at a temperature of 180ºc. the choice of the temperature range was based on analysis of previous investigations conducted by andrejko et al. (2011). the seeds were heated with the use of an original laboratory device for thermal treatment of bulk plant materials. the device is equipped with two heating heads with four 400 w ecs-1 radiators manufactured by elcer. these temperature radiators supplied by electricity (230 v) have a small fraction of visible radiation (dark radiators) in the spectrum and heat all plane points uniformly (plane radiators). the average temperature of the filament is approx. 500oc and the wavelength is λ = 2,5-3,0 μm. the belt conveyor carrying the research material is powered by a dc motor equipped with a voltage regulator facilitating smooth adjustment of the belt travel speed in the range from 5·10-3m·s-1 to 7·102m·s-1 (the time that the seeds stay in the heating zone ranges from 15 to 200 s, respectively). after cooling, the seeds were pressed in a duo screw press manufactured by farmet (czech republic) with a capacity of 18-25 kg·h-1, motor power 2,2 kw, and a screw speed of 1500 r·min-1. a 0,4 kg seed batch was pressed with the use of a 10-mm diameter nozzle. before the process, the screw press was heated to a temperature of 500c, which was measured with an ama-digit thermometer. after the pressing process, the oil was left for natural sedimentation for 5 days in refrigeration conditions (t 10±1ºc). next, the oil was analysed. the oil extraction efficiency was calculated based on the weight of extracted oil, the weight of the seed sample, and the percentage content of oil determined in the seeds. the extraction efficiency “w” was calculated with formula (1) (rotkiewicz et al., 2002): 𝑊 = 𝑚!" 𝑍!" ∙ 100 [%] where: mol mass of extruded oil, kg, zol mass of oil masa contained in seeds during pressing, kg. the seed moisture was determined with the oven-drying method using a radwag max 50/1/wh moisture analyser at a temperature of 120°c in accordance with standard pnen iso 665:2004. the fat content in the seeds and pomace was determined with the soxtec 8000 device following the soxhlet method based on application asn 310 and in accordance with standard pn-en iso 659:201020. the acid value (av) defines the amount of free fatty acids in cold-pressed oils. the test was carried out with the aocs official method cd 3d-63 (official method, 2000). the peroxide value (pv) defines the amount of primary oxidation products in coldpressed oils (malheiro et al., 2013). the test was carried out with the aocs official method 965.33 (official method, 1999). the oxidative stability was determined with the rancimat accelerated oxidation test, which measures the induction time by detection of volatile acids formed during oil oxidation (mathäus, 1996). the test was carried out with the aocs method (official method, 1989) with the use of the 893 professional biodiesel rancimat device manufactured by metrohm. oil samples (2,50 ± 0,01g) were accurately weighed, placed in a measurement vessel, and subjected to air flow of 20 l/h at a temperature of 120˚c. the results were expressed as the ital. j. food sci., vol. 30, 2018 490 induction time, which was automatically determined from the curve inflection point with the use of stabnet1.0 software provided by the company. the overall colour was determined with a spectrophotometric method in accordance with standard pn-a-86934:1995. measurements of the absorbance of the oil samples were performed after dilution at two wavelengths in the visible range: λ=442nm for the carotenoid pigment group and λ=668nm for the chlorophyll pigment group. the absorbance values were pooled and expressed as an integer the determination was carried out in triplicate. the arithmetic mean of the repetitions was taken as the result. the statistical analysis of the results was carried out in the statistica 10 package. the analysis of variance anova was employed to determine the significance of the differences between the values. inference was carried out at a significance level of 0,05. the mean confidence intervals were analysed in detail with tukey’s test. 3. results and discussion 3.1. seed moisture and fat content the initial mean moisture and fat levels in the sesame seeds were 7,46 and 45,61%, respectively (table 1). the seed moisture content was higher than the value reported by gharby et al. (2017) (6%), whereas the fat content was lower than that determined by nzikou et al. (2009) (54%). 3.2. chemical determinations of sesame oil selected chemical determinations that are indicators of the quality of the extracted sesame oil assessed immediately after pressing are shown in table 1. the acid value (av) was 1,52 mg koh/g and the peroxide value (pv) was 0,58 meq o2/kg. this indicates that the analysed oil fulfilled the quality requirements for cold pressed oils specified in the codex alimentarius in terms of the acid value and peroxide value (av ≤ 4 mg koh/g, pv ≤ 10 meq o2/kg) (2009). the acid value of the analysed oil was lower than that reported by rao et al. (1955), i.e. 2,5 mg koh/g. similarly, the peroxide value determined in this study was significantly different from the results described by yoshida and takagi (1997) (1,42 meq o2/kg), ogbonna and ukaan (2013) (3,95 meq o2/kg), and gharby et al. (2017) (2,7 meq o2/kg) for cold-pressed sesame oil. the differences between the results of this study and those reported by other authors may be associated with the quality of the seeds used. the raw material, e.g. the degree of damage, has a significant effect on the yield and quality of pressed oils (krygier et al. 2000). table 1. mean values of moisture and fat content in sesame seeds as well as the acid value (av), peroxide value (pv), and induction time in the extracted oil. sesame seeds sesame oil moisture (± sd) (%) 7,46±0,05 av (± sd) (mg koh/g) 1,52±0,02 fat content (± sd) (%) 45,61±0,63 pv (± sd) (meq o2/kg) 0,58±0,02 induction time (± sd) (h) 3,91±0,05 oxidation is primarily responsible for deterioration of the quality of fats, disagreeable odour and flavour of fat products, and reduction of their nutritional value. therefore, ital. j. food sci., vol. 30, 2018 491 oxidative stability is one of the most important indicators of the quality of oils, especially those extracted at low temperatures, which contain natural antioxidants (e.g. tocopherols, carotenoids, sterols, phospholipids, and phenolic compounds). concurrently, they contain undesirable compounds with prooxidant activity (e.g. metals, chlorophylls), which are removed in the refining process (koski et al., 2002; zine et al., 2013). the rancimat test revealed (tab. 1) that the induction time of the analysed oil was 3,91 h, and this value differed from results reported by other authors. in their investigations, kamal-eldin and appelqvist (1995) as well as gharby et al. (2017) demonstrated higher induction times of sesame oil, i.e. 6,1 and 28,5h, respectively. these differences may be associated with the different sesame species and rancimat test parameters (t 100°c) used in the investigations. 3.3. effect of seed thermal treatment on oil extraction efficiency and oil content in pomace the thermal treatment contributed to an increase in the oil yield (table 2) and exerted an effect on the traits of the sesame oil (figs. 1-4). the extraction efficiency of the sesame seed oil gradually increased together with the seed heating time until it reached 120 s. the highest extraction yield (56,25%) was achieved after the 120 s thermal treatment. in comparison with the control sample, the increase in the extraction efficiency was 12,68% (p ≤ 0,05). the longer heating time (150 s) also contributed to an increase in the oil yield, in comparison with the control sample; however, the difference over two-fold lower (5,24%) than in the case of seeds treated thermally for 90 and 120 s (table 2). table 2. mean extraction efficiency of the sesame oil relative to the fat content in the pomace. heating time of seeds in t 180°c (s) the fat content in the pomace (± sd) (%) the oil yield (± sd) (%) 0 26,31e ±0,21 43,57a ±0,04 30 25,73e ±0,18 45,12b ±0,04 60 24,65d ±0,23 47,31c ±0,04 90 22,05b ±0,37 55,6e ±0,04 120 21,25a ±0,24 56,25f ±0,03 150 23,77c ±0,23 48,81d ±0,04 mean values in the column denoted by different letters differ significantly statistically at p ≤ 0,05. the highest fat content, i.e. 26,31%, was determined in the pomace from the thermally non-treated seeds (table 2). at the longer 120 s thermal treatment of the seeds, the pomace fat content declined from the value of 25,73% noted in the case of seeds heated for 30 s to 21,25% obtained at the heat treatment lasting 120 s. at the 150 s heating time, the fat content in the pomace increased to 23,77% (table 2). analogous changes in “bakara” cultivar rapeseed were reported in the investigations conducted by rękas et al. (2015). the oil yield from these seeds was in the range from 53,9% in non-heated samples to 65,3% in the case of seeds heated with microwaves for 3 minutes. the oil extraction efficiency in seeds heated for 7 minutes was reduced to 59,8%. the pomace fat content was 25,5% in non-heated seeds, 20,2% in the case of seeds heated for 3 minutes, and 22,2% in seeds heated for 7 minutes. ital. j. food sci., vol. 30, 2018 492 the higher fat content in pomace and the lower differences in the oil pressing yields in the case of the 150 s heating time, compared to the control sample, can be attributed to the insufficient moisture level caused by the excessive time of the thermal treatment. similar conclusions were formulated in the study conducted by kachel-jakubowska (2008). 3.4. effect of seed thermal treatment on the physicochemical traits of the extracted oils to assess the effect of the infrared seed treatment applied prior to the pressing process on the physicochemical traits of the oils, the basic quality parameters were evaluated (figs. 14). the acid value of the analysed oils ranged from 1,52 mg koh/g in the oil extracted from the non-heated seeds to 2,02 mg koh/g in the oil from seeds treated with heat for 150 s (fig. 1). figure 1. acid values of oils pressed from sesame seeds relative to the time of seed thermal pre-treatment “t”; different letters above the bars indicate statistically significant differences. the highest increase in the hydrolysis degree was observed in the oil extracted from seeds treated with 150 s heating. furthermore, the oils were characterised by a low peroxide value (fig. 2), which determines the primary products of oil oxidation. the peroxide value ranged from 0,58 meq o2/kg in the non-heated seeds to 1,75 meq o2/kg in the oil from seeds treated thermally for 150 s. all the analysed oils met the requirements for the acid value and peroxide value in cold-pressed oils specified in the codex alimentarius (2009). a similar effect of seed thermal treatment on changes in av and pv of extracted oils was observed by other authors. in investigations of sesame oil from seeds heated at 160°c for 10, 20, and 25 minutes, yoshida and takagi (1997) demonstrated a pv range from 1,42 (non-heated seeds) to 5,38 meq o2/kg (seeds heated for 25 minutes). kraljić et al. (2013) evidenced that pv of oil from different rapeseed species heated at a temperature of 80°c for 30 minutes ranged from 1,6 (non-heated seeds) to 2,6 mg koh/g (heated seeds -toccata cultivar), from 1,5 (non-heated seeds) to 2,3 mg koh/g (heated seeds oase cultivar), and from 1,3 (non-heated seeds) to 2,3 mg koh/g (heated seeds remy cultivar). in turn, the av of these oils was in the range from 0,2 meq ital. j. food sci., vol. 30, 2018 493 o2/kg (non-heated seeds) to 0,25 meq o2/kg (heated seeds -toccata cultivar), from 0,29 (non-heated seeds) to 0,36 meq o2/kg (heated seeds oase cultivar), and from 0,43 (nonheated seeds) to 0,44 meq o2/kg (heated seeds remy cultivar). in investigations of oil from sesame seeds heated with microwaves at a temperature of 100°c for 15 minutes, abou-gharbia et al. (2000) showed a pv range from 0,46 (non-heated seeds) to 0,98 mg koh/g (heated seeds). the analysis of the oxidative stability of the extracted oils (fig. 3) proved that the parameters of the seed thermal treatment applied before the extraction process extended the oil induction time. figure 2. peroxide values of oils pressed from sesame seeds relative to the time of seed thermal pretreatment “t”; different letters above the bars indicate statistically significant differences. figure 3. induction time of oils pressed from sesame seeds relative to the time of seed thermal pre-treatment “t”; different letters above the bars indicate statistically significant differences. ital. j. food sci., vol. 30, 2018 494 the value of this parameter increased with the longer seed heating time and ranged from 3,91 h for the oil from the non-heated seeds to 9,6 h for the oil extracted from seeds heated for 150 s. the 150 s thermal pre-treatment of the seeds contributed to an almost three-fold increase in the oil induction time, i.e. by 5,69 h. the literature provides reports of the increase in the oxidative stability of oil extracted from thermally treated seeds. in their analyses of oil extracted from sesame seeds heated with microwaves at 100°c for 15 minutes, abou-gharbia et al. (2000) noted an induction time in the range from 9,49 (oil from non-heated seeds) to 18,68 h (oil from heated seeds). although there was a statistically significant increase in the ln value as a result of heating, this value was low (1,8 meqo2/kg) even for oil extracted from seeds heated for 150 s. this did not influence the oxidative stability of the analysed oils. similarly, flaczyk et al. (2004) did not find a direct relationship between the content peroxides in oils and their oxidative stability. the higher content of natural antioxidants in the oils obtained from the heated seeds may have been a determinant of the long induction time in the analysed oil. figure 4. colour of oils extracted from sesame seeds (1000 x (a442nm + a668nm)) relative to the time of thermal pre-treatment of the seeds “t”; different letters above the bars indicate statistically significant differences. the analysis of the colour of the sesame oil (fig. 4) revealed that the oil from the nonheated seeds was the lightest and clearest (overall colour 203). it contained the lowest content of carotenoid and chlorophyll pigments in comparison with the oils extracted from the heated seeds. the thermal treatment of the seeds caused significant darkening of the extracted oil and an increase in absorbance at the wavelengths of 442 and 668 nm. the colour of the oil from the heated seeds was in the range from 211 after the 30 s thermal treatment to 249,67 after heating the seeds for 150 s. therefore, the oil extracted from seeds receiving the longest thermal pre-treatment was the darkest and the least clear. the same effect of thermal seed treatment on changes in the colour of extracted oils was reported by gharbia et al. (2000). in investigations of oil extracted from sesame seeds heated with microwaves at 100°c for 15 minutes, the authors demonstrated a three-fold higher content of total carotenoid and chlorophyll pigments in oil from heated seeds than that in oil ital. j. food sci., vol. 30, 2018 495 extracted from non-heated seeds. the darker colour of the oils extracted from pre-heated seeds can be explained by the presence of products of maillard reaction and phospholipid and chlorophyll degradation products derived from the thermal seed treatment (azadmard-damirchi et al., 2011; lamorska and tys, 2011). 4. conclusions based on the research results, the following conclusions were formulated: infrared thermal treatment of sesame seeds exerts a significant effect on the oil extraction efficiency. the highest increase in the oil yield was noted in the case of seeds heated for 120 s. the longer heating time of 150 s reduced the efficiency of the extraction process. infrared thermal treatment of sesame seeds causes a significant increase in the oxidative stability of oil. the longest induction time in the rancimat test (9,53 h) was recorded for the oil extracted from seeds heated for 150 s. thermal seed pre-treatment contributes to an increase in the acid value and the peroxide value, a higher absorbance value at wavelengths of 442 and 668 nm, and a darker overall colour of oil. all the oils analysed in the study fulfil the quality standards in terms of the acid value (av ≤ 4 mg koh/g) and the peroxide value (pv ≤ 15 meq o2/kg). references abou-gharbia h.a., shehata a.a.y. and shahidi f. 2000. effect of processing on oxidative stability and lipid classes of sesame oil. food research international. 33(5):331-340. andrejko d., grochowicz j., goździewska m. and kobus z. 2011. influence of infrared treatment on mechanical strength and structure of wheat grains. food bioprocess technology 4:1367-1375. aoac official method 965.33. peroxide value of oils and fats. 1999. aocs official and tentative methods of analysis. fat stability. active oxygen method. method 1989. aocs official method cd 3d-63. acid value. 2000. azadmard-damirchi s., alirezalu k. and fathi achachlouei b. 2011. microwave pretreatment of seeds to extract high quality vegetable oil. world academy of science, engineering and technology 57:72-74. codex alimentarius, fao/who. codex standard for named vegetable oils. 2009. flaczyk e., rudzińska m., górecka d., szczepaniak b., klimczak s. and korczak j. 2004. evaluation of selected quality indexes of stored "extra virgin" olives. oilseed crops 25(1):213-224. gharby s., harhar h., bouzouba z., asdadi a., el yadini a. and charrouf z. 2015. chemical characterization and oxidative stability of seeds and oil of sesame grown in morocco. journal of the saudi society of agricultural sciences 16(2):105-111. gupta s.k. 2011. technological innovations in major world oil crops, volume 1. breeding, springer science & business media. kachel-jakubowska m. 2008. the impact of drying on quality of winter rape seeds. agricultural engineering 1 (99):127132. kamal-eldin a. and appelqvist l.a. 1995. the effects of extraction methods on sesame oil stability. journal of the american oil chemists' society 72(8):967-969. koski a., psomiadou e., tsimidou m., hopia a., kefalas p., wähälä k. and heinonen m. 2002. oxidative stability and minor constituents of virgin olive oil and cold-pressed rapeseed oil. eur. food res. technol. 214:294-298. kraljić k., škevin d., pospišil m., obranović m., neđeral s. and bosolt t. 2013. quality of rapeseed oil produced by conditioning seeds at modest temperatures. journal of the american oil chemists' society. 90(4):589-599. ital. j. food sci., vol. 30, 2018 496 krygier k., wroniak m., grześkiewicz s. and obiedziński m. 2000. study on effect of destroyed seeds content on the quality of cold pressed rapeseed oil. oilseed crops. 21:587-596. lamorska l. and tys j. 2011. physicochemical and health properties of fats. acta agrophysica 5:51-55. malheiro r., rodrigues n., manzke g., bento a., pereira j.a. and casal s. 2013. the use of olive leaves and tea extracts as effective antioxidants against the oxidation of soybean oil under microwave heating. ind. crop. prod. 44:37-43. mathäus b. 1996. determination of the oxidative stability of vegetable oils by rancimat and conductivity and chemiluminescence measurements. j. am. oil chem. soc. 73:1039-1043. niu y., jiang m., wan c., yang m. and hu s. 2013. effect of microwave treatment on sinapic acid derivatives in rapeseed and rapeseed meal. j. am. oil chem. soc. 80:307-313. nzikou j.m., matos l., bouanga-kalou g., ndangui c.b., pambou-tobi n.p.g., kimbonguila a. and desobry s. 2009. chemical composition on the seeds and oil of sesame (sesamum indicum l.) grown in congo-brazzaville. advance journal of food science and technology 1(1):6-11. ogbonna p.e. and ukaan s.i. 2013. chemical composition and oil quality of seeds of sesame accessions grown in the nsukka plains of south eastern nigeria. afr. j. agric. res. 8 (9):797-803. pala v., krogh v., muti p., chajès v., riboli e, micheli a. and berrino f. 2001. erythrocyte membrane fatty acids and subsequent breast cancer: a prospective italian study. journal of the national cancer institute. 93(14):1088-1095. pn-a-86934:1995. plant and animal oils and fats. spectrophotometric determination of general color. pn-en iso 659:2010. oil seeds. determination of oil content. pn-en iso 665:2004. oil seeds. determination content of water and volatile substances. rao r.k., krishna m.g., zaheer s.h. and arnold l.k. 1955. alcoholic extraction of vegetable oils. i. solubilities of cottonseed, peanut, sesame, and soybean oils in aqueous ethanol. journal of the american oil chemists society 32(7):420-423. rękas a., wiśniewska k. and wroniak m. 2015. effect of microwave heat treatment of rapeseeds on oil yield and quality of pressed oil. food: science technology quality 22(3):107-122. roszkowska b., dąbrowska a. and batyk i.m. 2014. amaranth its chemical composition and health-promoting value. journal of health sciences 4(10):183-188. rotkiewicz d., tańska m. and konopka i. 2002. seed size of rapeseed as a factor determining their technological value and quality of oil. oilseed crops. 23:103-112. yang m., huang f., liu c., zheng c., zhou q. and wang h. 2013. influence of microwave treatment of rapeseed on minor components content and oxidative stability of oil. food and bioprocess technology 6(11):3206-3216. yoshida h. and takagi s. 1997. effects of seed roasting temperature and time on the quality characteristics of sesame (sesamum indicum l.) oil. journal of the science of food and agriculture 75(1):19-26. zine s., gharby s. and el hadek m. 2013. physicochemical characterization of opuntia ficus-indica seed oil from morocco. biosci. biotechnol. res. asia 10(1):1-7. paper received september 12, 2018 accepted february 20, 2018 ijfs#1179_bozza ital. j. food sci., vol. 31, 2019 67 paper characterization of staphylococcus aureus isolates from traditional dairy products of small-scale alpine farms v. filipello*1, m. tilola1, l. zani2, b. bertasi1, m.v. luini3 and g. finazzi1 1istituto zooprofilattico sperimentale della lombardia e dell'emilia romagna, via a. bianchi 9, 25124 brescia, italy 2department of food and drug, università degli studi di parma, parco area delle scienze 27/a, 47124 parma, italy 3istituto zooprofilattico sperimentale della lombardia e dell'emilia romagna, sezione di lodi, via a. einstein, 26900 lodi, italy *e-mail address: virginia.filipello@izsler.it abstract this study investigated the prevalence of staphylococcus aureus in raw milk dairy products handcrafted in traditional alpine small-scale farms, and characterised the enterotoxigenicity and resistance to methicillin. among the analysed samples, 69% exceeded the international microbiological recommendations. the highest counts were observed for cheese or fatty products (~106 cfu/g). conversely, lower contamination levels concerned raw milk and whey cheese (~102 cfu/g). a total of 163 s. aureus isolates were collected, and the prevalence of mrsa was low (1.7%) but not negligible. the finding of enterotoxins genes in 67% of the isolates is of concern for the public health. keywords: alpine small-scale dairies, dairy products, molecular characterisation, staphylococcal enterotoxins, staphylococcus aureus ital. j. food sci., vol. 31, 2019 68 1. introduction staphylococcal food poisoning (sfp) is one of the most common foodborne diseases worldwide caused by the ingestion of food contaminated with preformed staphylococcal enterotoxins (ses) produced by staphylococcus aureus (hennekinne and dragacci, 2012). sfp is generally characterised by self-limiting gastrointestinal symptoms, but occasionally the disease can be more severe or even fatal (benkerroum, 2017). s. aureus is ubiquitous in the environment and it is also one of the major causes of bovine mastitis (boss et al., 2016). therefore, raw milk and raw milk dairy products may be contaminated with s. aureus, due to the shedding of large segments of the organism into milk (d’amico and donnelly, 2011; rola and osek, 2016). moreover, cheese-makers may carry enterotoxin-producing s. aureus in their noses or on their hands, and the lack of proper hygienic measures during food processing increases the probability of contamination with s. aureus, especially in small-scale artisanal dairies (andré et al., 2008). indeed, dairy products are among the foods most commonly involved in sfp outbreaks (benkerroum, 2017; de buyser and lafarge, 2001). to date, 23 different ses have been described and many s. aureus strains harbour more than one ses gene. ses can be divided into classic types (i.e. a to e) and new variants classified at present as ses or ses-like (sels) based on their ability to cause emesis. ses are synthesised when s. aureus cell density reaches 105-108 cfu g-1. however, all of these toxins are heat-stable and can therefore be still present in the food even when the microorganism is inactivate or the contamination level is reduced by processing (benkerroum, 2017). among s. aureus strains, those that are methicillin-resistant (mrsa) have spread in the last decades as hospital-acquired pathogens (ha-mrsa) throughout the world, causing serious life-threatening infections not responding to a lot of antimicrobial treatments. more recently, community-acquired (ca-mrsa) and livestock-associated (la-mrsa) mrsa have also emerged (bardiau et al., 2013). mrsa have been identified in different foods worldwide, and several food-borne mrsa outbreaks have been reported demonstrating the zoonotic risk of transmission to humans (doulgeraki and nychas, 2017). the screening of s. aureus isolates from food of animal origin is therefore essential to estimate the mrsa emergence and the related zoonotic hazard (bardiau et al., 2013). in alpine regions, in particular in the lombardy region, raw milk dairy products are handcrafted in small-scale artisanal dairies built in pastures. these products and practices are closely linked to environmental, economic and tourist aspects, important for the safeguard and development of alpine culture and society (della torre, 2017). in this context, traditional cheeses represent appealing products to the new trends of searching for natural and authentic foods, and many of them have been awarded with the protected designation of origin (pod) label (lombardia, 2014). however, since the hygienic conditions of traditional plants are very diverse, a specific surveillance plan for the safety of cheese produced in pastures has been developed (italian ministry of health, 2017). data on s. aureus isolates recovered from small-scale alpine dairies are however scarce. the aims of this study were to investigate the prevalence of s. aureus and to characterise isolates from the production chain of artisanal raw milk dairy products. in particular, we tested the isolates for the presence of enterotoxins genes and for resistance to methicillin. ital. j. food sci., vol. 31, 2019 69 2. material and methods 2.1. retrospective database analysis izsler database was asked to obtain data on the prevalence and level of contamination of s. aureus for all milk and dairy products samples referred to our laboratory throughout 2016. samples with ≥ 102 cfu g-1 s. aureus counts were considered as exceeding international microbiological recommendations (reg. ce n. 2073/2005). 2.2. sample selection and s. aureus isolation and identification s. aureus isolates were collected from products tested within the alpine pastures surveillance plan carried out in the lombardy region. the samples were collected in 2016 from a total of 40 small-scale dairies. for s. aureus isolation, serial dilution of each sample homogenate were plated on baird parker agar + rabbit plasma fibrinogen (rpf agar) (biolife italiana, milano, italy) and incubated at 37°c for 48 h. up to 5 characteristic colonies for each sample were planted on blood agar to confirm s. aureus hemolytic property. the species identification was confirmed with pcr of the nuc gene as described by brakstad et al. (brakstad and maeland, 1992). dna was obtained by boiling a suspension of the isolates in 2 ml of demineralised water for 5 min at 99°c. the suspension was then centrifuged at 13 000 g for 5 min and supernatant was used for all the following pcr assays. 2.3. detection of meca and mecc (methicillin resistance) the detection of meca and mecc (meca homologue) was carried out by means of two pcr protocols using specific primers as reported by pichon et al. (pichon et al., 2012). briefly, for both meca and mecc the pcr reaction mix (final volume 20 μl) contained 1x hotstartaq master mix (qiagen inc, hilden, germany), 0.5 μm of each primer, and 1 μl dna. the thermic profile was 95°c for 15 min, followed by 35 cycles of 94°c for 30 s, 58°c for 40 s, and 72°c for 1 min. the final elongation step was performed at 72°c for 10 min. the amplified pcr products were distinguished by electrophoresis in a 2.5% agarose gel (agarose multi purpose, roche -120 v for 40 minutes), stained with eurosafe nucleic acid stain (euroclone, 1x). 100 bp dna ladder (invitrogen, 0.5 µg/µl) was included. 2.4. staphylococcal enterotoxins two multiplex pcr protocols were used as described in bianchi et al. (bianchi et al., 2014) to detect sea, seb, sec, sed, see, seg, seh, sei, selj, selp, and ser ses genes. the electrophoresis conditions were the same for detection of meca and mecc. 3. results and discussion 3.1. retrospective database analysis among the 4177 samples of milk or milk-derived products of different origin analysed by izsler for the presence of coagulase positive staphylococci during 2016, 145 were from small alpine pastures dairies. while for the other dairy products those exceeding the international microbiological recommendations were 22% (867/4032), for the traditional alpine products the proportion increased to 69% (100/145). the level of contamination varied between the different products tested. it is interesting to note that, in general, raw ital. j. food sci., vol. 31, 2019 70 milk has lower contamination values than the final products (fig. 1). this could be due to an exponential growth of s. aureus in the early phases of cheese-making, when the milk is heated to about 40°c, which is the optimum temperature range for s. aureus growth and enterotoxin production (hennekinne et al., 2012). in addition, secondary events of contamination from the cheese-maker’s skin may happen due to inappropriate hygienic procedures. the only product with low level of contamination is whey cheese; as for its production, the whey is heated above 85°c. figure 1. distribution of the s. aureus counts in the different products analysed. 3.2. isolates out of a total of 81 samples (n=23 from raw milk, n=7 from curd, n=39 goat or bovine cheese, n=11 butter, n=1 cream), 172 coagulase positive staphylococci isolates have been collected. a total of 163 (95%) isolates were confirmed as s. aureus by the nuc pcr, and used for further characterisation (s1). 3.3. mrsa isolates among the 163 isolates analysed, 3 (1.7%) were mrsa (meca+; s1). none of the isolates was found to be mecc positive. the isolation frequency of mrsa raw milk and dairy products in the present study is consistent with the low prevalence estimates previously reported. studies from greece and italy have revealed mrsa prevalence estimates of 3% (papadopoulos et al., 2018), 3.8% (cortimiglia et al., 2016) and 0.7% (giacinti et al., 2017). however, given the fact that traditional herding systems on alpine pastures should be extensive and characterized by low rates of antimicrobials administration, the results of this study raise some concern. ital. j. food sci., vol. 31, 2019 71 3.4. ses genes detection at least one ses gene was found in 67% of the isolates (n=110) and 29 different ses genes profiles were distinguished (table 1; s1). table 1. enterotoxins gene profiles. the number at the end of each line represents the number of isolates bearing a specific enterotoxins gene profile. the number at the bottom of each column represents the number of isolates bearing a specific enterotoxin gene. enterotoxins genes sea seb sec sed see ser seg seh sei selj selp no. of isolates ! 21 ! ! ! ! ! 1 ! ! 4 ! ! 4 ! ! ! 2 ! ! ! 5 ! ! ! ! 13 ! ! ! 2 ! ! 1 ! ! ! 4 ! ! ! 1 ! 9 ! 4 ! ! ! 1 ! 9 ! ! ! 1 ! ! 1 ! ! 4 ! ! ! 10 ! ! ! ! 1 ! 1 ! 1 ! ! 2 ! 2 ! ! 1 ! 1 ! 1 ! 2 ! ! ! ! 1 58 10 9 51 1 38 7 8 9 36 1 sea was detected in 53% (n=58) of the isolates, followed by sed (n=51; 46%), ser (n=38; 35%) and selj (n=36; 33%) genes. sea and sed are the ses most frequently associated with sfp, and they have caused outbreaks linked to the consumption of dairy products (hummerjohann and graber, 2014; johler et al., 2015; sabike and edris, 2014). twenty-five isolates (23%) contained the ses gene pattern sed, sej, ser, which are carried on ital. j. food sci., vol. 31, 2019 72 the same plasmid (benkerroum, 2017), with more than half of them (14/25) additionally carrying sea. these patterns have been correlated with genotype b s. aureus as identified by rs-pcr, which has been reported to be a particularly virulent bovineassociated type of s. aureus, and the one most widespread in switzerland and central european countries (hummerjohann et al., 2014). for sed and ser, the exhibition of emetic activity is well established (schubert and bania, 2017), while the situation for selj remains unclear (benkerroum, 2017). nevertheless, all ses and sels belong to the family of superantigens, molecules able to stimulate t-cell proliferation (5000-fold more than in a conventional immune response), driving a massive release of cytokines that cause a life threatening systemic inflammation and toxic shock (tss). however, to date it is not clear whether exposure to ses/sels via food can lead to tss, and it has been suggested that it is the dose that makes the difference between evolution in tss or spf in case of ses/sels ingestion (benkerroum, 2017). seh, which also has been linked to milk-based sfp outbreaks (bianchi et al., 2014), was detected in 8 isolates (7%). improper handling and storage of raw milk and cheese in the early stages of processing contaminated with s. aureus can result in the production of ses, which is also dependent on the initial dose of s. aureus contamination (sabike et al., 2014). based on our data, the contamination of raw milk averaged 102 cfu g-1, while the average contamination of cheese was 106 cfu g-1, indicating that the alpine pasture process of cheese-making allows exponential growth of s. aureus, that reaches a concentration critical for the production of ses (105-108 cfu g-1;(benkerroum, 2017). indeed, one of the samples included in our study was referred to our laboratory for the suspect involvement in a sfp episode. it was an aged cheese (isolate 20.1) which proved positive for sea even if the count of s. aureus was 103 cfu g-1 (data not shown) suggesting that the s. aureus population declined during aging. in the european union, milk-derived products are examined for enterotoxin content only when the number of coagulase-positive staphylococci exceeds 105 cfu g-1 (reg. ce n. 2073/2005). in the light of our findings, this measure may not be appropriate with regard to aged cheese. moreover commercial kits commonly used for ses detection are only available for classical ses (i.e. a to e), leading to an underestimation of the actual incidence of new ses and sels. conversely, the production of ses/sels depends on the expression of the ses/sels genes, which is dependent on a complex regulatory system influenced by specific environmental conditions (i.e. temperature, ph, aw, eh, and salt concentration). it is therefore possible that even when the s. aureus contamination reaches critical levels, the ses/sels are not produced, highlighting again the modest value of s. aureus count as indicator of the presence or absence of ses in food (benkerroum, 2017). indeed, this situation is routinely observed in our laboratory (data not shown). 4. conclusions despite the high overall s. aureus prevalence (69%) in dairy products manufactured in alpine small-scale farms, the estimated mrsa prevalence in our study was low (1.7%) but not negligible. it is therefore necessary to keep monitoring foods and apply control measures against s. aureus in herds to minimise the dissemination of mrsa in animals and subsequently in the community. milk and milk products are considered to be of particular significance as a staphylococcal enterotoxin (se) source. given the high levels of contamination found in many of the products analysed, the presence of enterotoxigenic strains of s. aureus should raise concern. indeed, the technologies used in alpine pasture dairies are not effective in hindering and limiting the proliferation of s. aureus in the early phases of production, and ital. j. food sci., vol. 31, 2019 73 both human and animal sources can be responsible for contamination. within this scope, in traditional dairies major benefits could derive from the application of basic good manufacturing practices, starting from control of the health status of cows and milking hygiene. focused educational interventions and further studies aimed at assessing the routes of transmission of s. aureus in small-scale alpine farms could have a great impact on the quality and safety of these precious and peculiar productions. references andré m.c.d.p.b., campos m.r.h., borges l.j., kipnis a., pimenta f.c. and serafini á.b. 2008. comparison of staphylococcus aureus isolates from food handlers, raw bovine milk and minas frescal cheese by antibiogram and pulsed-field gel electrophoresis following smai digestion. food control 19(2):200-207. doi: doi.org/10.1016/j.foodcont.2007.03.010. bardiau m., yamazaki k., duprez j.-n., taminiau b., mainil j. g. and ote i. 2013. genotypic and phenotypic characterization of methicillin-resistant staphylococcus aureus (mrsa) isolated from milk of bovine mastitis. letters in applied microbiology 57(3):181–186. doi: doi.org/10.1111/lam.12099. benkerroum n. 2017 staphylococcal enterotoxins and enterotoxin-like toxins with special reference to dairy products: an overview. critical reviews in 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doi.org/10.1089/fpd.2016.2210. paper received march 1, 2018 accepted july 27, 2018 ijfs#1233_bozza ital. j. food sci., vol. 31, 2019 332 paper antioxidant properties, proximate analysis, phenolic compounds, anthelmintic and cytotoxic screening of teucrium sandrasicum; an endemic plant for turkey a. kaska*1 and r. mammadov2 1department of science and mathematics, faculty of education, pamukkale university, 20100, denizli, turkey 2department of biology, faculty of arts and science, pamukkale university, 20100, denizli, turkey *corresponding author: tel.: +902582961184; fax: +902582963535 e-mail address: akaska@pau.edu.tr abstract this work was designed to evaluate the phenolic compounds and biological activities (antioxidant, cytotoxic and anthelmintic) of teucrium sandrasicum extracts (ethanol and acetone) as well as to determine proximate parameters (such as proteins, carbohydrates, fat). the phenolic contents were identified using hplc. the ethanol extracts exhibited higher free radical scavenging and antioxidant activities than acetone extracts. the reducing power, metal chelating and radical cation activities were found to be statistically different between the acetone and ethanol extracts. t. sandrasicum exhibited cytotoxic, anthelmintic activities with rich nutrient contents. based on these results, this plant may be considered as a potentially useful source for the food and pharmaceutical industry. keywords: biological activities, medicinal plants, proximate, teucrium sandrasicum ital. j. food sci., vol. 31, 2019 333 1. introduction plants are good sources of natural functional compounds that are medically and biologically important and can be used in various fields, such as food ingredients, medicinal and pharmacological applications. for this reason, there has recently been an increase in the volume of research on the isolation and identification of these compounds. many of these investigations especially relate to the designation of the biological activities of these compounds, such as antioxidant and cytotoxic activities (nickavar and esbati, 2012; al-dabbas, 2017). the beneficial effect of medicinal plants on diseases have been revealed by a considerable number of researchers (cakilcioglu and turkoglu, 2010). medicinal plant species of the genus teucrium have a wealth of phenolic compounds with powerful biological activities and many plants of the teucrium genus have been used in the food industry, as natural preservatives and pharmaceutical applications (canadanovic-brunet et al., 2006; saroglu et al., 2007; bagci et al., 2010). these plants are used to reduce inflammation and relieve indigestion and are also used as herbal medicines for coughs, asthma and stomach pain (amiri, 2010). in addition, teucrium plants are well known for their hypoglycemic, antiseptic, antispasmodic and anthelmintic activities (gharaibeh et al., 1989; saroglu et al., 2007; rehman et al., 2016). the teucrium genus is a member of the lamiaceae family, of which there are more than 340 species widespread throughout the world (mahmoudi and nosratpour, 2013). turkish flora includes 34 teucrium species (dirmenci, 2012), eight of which are endemic (davis, 1982). teucrium sandrasicum is one of the endemic species of the teucrium genus and the aerial parts of this plant are widely used in the daily diet (aksoy-sagirli et al., 2015). in previous limited research, several t. sandrasicum extracts (water, methanol, ethyl acetate, hydro-methanolic) have been evaluated for phenolic compounds, antioxidant activities and antiproliferative effects on various cell lines (aksoy-sagirli et al., 2015; karagoz et al., 2015; tarhan et al., 2016). according to the literature, the biological activities of plant materials are strongly based on the nature of extracting solvents, such as polarities. therefore, the separate examination of plant extracts, obtained from different solvents, will make a significant contribution to medicinal plant studies and their pharmaceutical applications (canadanovic-brunet et al., 2006; stankovic et al., 2011). consequently, more research is required on the biological activities of this aromatic and medicinal plant. within this scope, we therefore consider that t. sandrasicum is a plant worthy of additional investigation. furthermore a thorough investigation of the current literature indicates that no scientific reports have been published to date concerning the antioxidant capacities, and cytotoxic or anthelmintic properties of the ethanol and acetone extracts of t. sandrasicum. with these points in mind, the objectives of the present study are to evaluate the antioxidant capacities, the cytotoxic, anthelmintic activities and the total phenolic and flavonoid contents of the ethanol and acetone extracts of t. sandrasicum, as well as the chemical composition of the ethanol extracts. in addition, the other objective of this study was to determine the proximate content of this medicinal plant. 2. material and methods 2.1. plant materials t. sandrasicum was collected at an altitude of 1600 m from sandras mountain (between denizli-muğla, turkey), in july 2017. the plant material was identified by dr. mehmet çiçek from department of biology, faculty of arts and sciences, pamukkale university, ital. j. food sci., vol. 31, 2019 334 denizli, turkey. a voucher specimen (t. sandrasicum; herbarium no: 2017-145) has been deposited in the private herbarium of dr. m. çiçek (pau) at pamukkale university, denizli, turkey. 2.2. preparation of the plant extracts the air dried aerial parts of t. sandrasicum were ground to a fine powder and extracted with ethanol and acetone. each powdered sample (30 g) were mixed with 300 ml of solvents. extraction was carried out by shaking at 50°c for 6 h in a temperature controlled shaker and the mixture was filtered using filter paper (whatman no.1). this procedure was repeated twice. the solvent was evaporated using a rotary evaporator (ika rv10d, staufen, germany) under vacuum at 40-50°c. samples were lyophilized (labconco freezone, kansas city, mo) and stored at -20°c until tested. all experiments were carried out in triplicates. 2.3. chemicals β-carotene, linoleic acid, 2,2-diphenyl-1-picryl hydrazyl radical (dpph), 2,2'-azino-bis (3ethylbenzothiazoline-6-sulphonic acid) (abts), phosphate buffer, iron (iii) chloride, quercetin, sodium phosphate, sodium carbonate, potassium ferrocyanide, gallic acid, methanol, chloroform, ethanol, and acetone were purchased from sigma-aldrich. butylated hydroxy toluene (bht), folin-ciocalteu reagent, tween 20 was purchased from merck (darmstadt, germany). other chemicals and solvents were of analytical grade. 2.4. determination of phenolic compounds 2.4.1 total phenolic content the total phenolic content of the t. sandrasicum extracts was evaluated using the folinciocalteu method (slinkard and singleton, 1977). in this method, the extract solution (1 mg/ml) was mixed with folin-ciocalteu reagent (1 ml) and distilled water (46 ml). after resting at room temperature for 3 min, 3ml of 2% sodium carbonate solution was added to the mixture and mixed gently. the mixture was incubated at room temperature for 2 h. following this procedure the absorbance was confirmed as 760 nm and the outcomes were shown as mg of the gallic acid equivalents (gae) per gram of extract. 2.4.2 total flavonoid content the total flavonoid content was evaluated using to method of arvouet-grand et al. (1994). one milliliter solution of alcl3 in methanol (2%) was combined with the equivalent quantity of extract solution. after about 10 min the absorbance of the reaction mixtures were determined as 415 nm.. the flavonoid content was calculated from a quercetin standard curve and expressed as milligram of quercetin equivalents (qe) per gram of extract. ital. j. food sci., vol. 31, 2019 335 2.5. determination of antioxidant activity 2.5.1 β-carotene/linoleic acid method in this method, antioxidant capacity was determined using the method of amin and tan (2002). β-carotene stock solution was prepared as follows: 2 mg β-carotene was dissolved in 10 ml chloroform. linoleic acid (20 µl) and 200 µl of 100% tween 20 was added for one milliliter of the solution. a rotary evaporator was used to remove the chloroform. then the remaining residue was added to 100 ml of distilled water and the 1 ml extracts were combined with this emulsion (24 ml). a spectrophotometer was immediately used to measure the initial absorbances at 470 nm. the reaction mixture was incubated for 2 hours at 50o c. following this, the measurement of the absorbance of this mixture was repeated, and a synthetic antioxidant (bht) was applied as the positive control. the total antioxidant activity (aa) was calculated in following way: aa=[1(asamp-aco)/(aosamp-aoco)] x 100 (asamp and aco: absorbance at the initial time of the incubation of samples and control, respectively and aosamp and aoco: absorbance in the samples and control at 120 min) 2.5.2 phosphomolybdenum method the total antioxidant property of t. sandrasicum extracts was determined using the phosphomolybdenum method according to prieto et al. (1999). various concentrations of the extracts (0.1-1.0 mg/ml) were mixed reagent solution (0.6 m sulfuric acid, 28 mm sodium phosphate and 4 mm ammonium molybdate). the reaction mixture (3 ml) and extracts (0.3 ml) were dispersed into test tubes and the tubes were placed at 95o c for 90 min. the absorbances of the mixtures were measured at 695 nm using a spectrophotometer. the antioxidant capacity of the extracts was expressed as µg ascorbic acid equivalents (aa) per milligram of extract. 2.6. evaluation of radical scavenging 2.6.1 free radical scavenging activity (dpph) the radical scavenging activity of the t. sandrasicum extracts was determined using the dpph, as described by meriga et al. (2012) with slight modifications. extracts of different concentrations (1 ml) were combined with 4 ml of methanolic dpph (0.004%) solution. after vortexing the reaction mixture, the decrease in absorbance of each extract and/or control (bht) were measured at 517 nm after 30 minutes. results were expressed as ic50 (the concentration of the sample that is required to scavenge 50% of dpph radicals). 2.6.2 abts radical cation scavenging activity experiments were performed in accordance with the method used by shalaby and shanab (2013) with slight modifications. abts (7mm) and potassium persulphate (2.45 mm) solutions were combined and stored in a dark room for 12-16 h prior to use. before the analysis, the abts solution was diluted with ethanol to an absorbance of 0.700±0.05 at 734 nm. after the addition of 4.5 ml of the abts reaction mixture to the various concentrations (50-400µg/ml) of the extracts (1 mg/ml), the mixture was kept at room temperature for 15 min. there was a reading of 734 nm for the absorbances of the samples. ital. j. food sci., vol. 31, 2019 336 the radical-scavenging activity of the extracts was estimated based on the abts color reduction, by calculating the ic50 (concentration in μg/ml that cause 50% inhibition of abts radicals). 2.7. ferric ion reducing power activities the method described by oyaizu (1986) with slight modifications was used to conduct the reducing power capacity of the extracts. different concentrations of the samples (1 ml) were combined with 0.2m phosphate buffer (1 ml) and 1% potassium ferricyanide (1 ml). the mixture was kept at 50o c for 20 min. trichloroacetic acid (1 ml, 10%) was added to reaction mixture. the aliquot of the upper layer (1.5 ml) was combined with distilled water (1.5 ml ) and ferric chloride (0.1%). after 10 min the absorbance was read, at 700 nm. the activity was expressed as mg of ascorbic acid equivalents (aa) per milliliter of extract. 2.8. metal chelating activity the metal chelating power of the t. sandrasicum extracts was determined using the method of karpagasundari and kulothungan (2014) with slight modifications. one milliliter of the extract and 3.2 ml of deionized water were mixed with 2 mm fecl2 (0.1 ml) solution. after 30 s, 5 mm of ferrozine (0.2 ml) was added. the reaction was activated by adding ferrozine and then the mixture was left to stand for 10 min after which the absorbances of the solutions were measured at 562 nm. the metal chelating activity was calculated in following way: chelating ability (%) = [(acoasamp) / aco] × 100, (aco : absorbance of the control and asamp : absorbance of the extract) 2.9. proximate analysis the t. sandrasicum plant samples were analyzed to determine proteins, fat, carbohydrates, ash and energy, according to the protocols mentioned in aoac (1995). the macrokjeldahl method was applied to evaluate the crude protein content of the samples. the crude fat was evaluated with a soxhlet apparatus for which a known weight of the powdered sample was extracted with petroleum ether. the volume of ash was established by burning at 650±15°c and total carbohydrates were calculated by difference. energy was calculated based on the following equation: energy(kilocalorie)=4×(g protein+ g carbohydrate)+ 9×(g fat). all parameters were made in triplicate. 2.10. quantification of phenolic compounds by hplc reversed-phase high performance liquid chromatography (rp-hplc, shimadzu scientific instruments) was used for the determination of the phenolic compound. detection and quantification was made using a diode array detector (spd-m20a), a lc-20at pump, a cto-10asvp column heater, a sil-10acht auto sampler, a scl-10avp system controller and a dgu-14a degasser. separations were carried out using a c-18 reversed-phase column (agilent zorbax eclipse, 250 x 4.6 mm length, 5µm particle size). the ital. j. food sci., vol. 31, 2019 337 chromatograms were examined at 278 nm. the mobile phases were a: 3.0 % formic acid in dh2o and b: methanol. the samples were dissolved in methanol and this solution (20 µl) was injected into the column. the phenolic composition of the t. sandrasicum ethanolic extract was determined according to the method of caponio et al. (1999) with slight modifications. gallic, 3,4 dihydroxybenzoic, 4-hidroxybenzoic, 2,5 dihydroxybenzoic, chlorogenic , vanillic, caffeic, p-coumaric, ferulic, cinnamic acid and quercetin epicatechin, rutin were used as standards. the amount of individual phenolic compound was determined, based on peaks. the quantity of each phenolic compound was expressed as µg/g of the extract. 2.11. cytotoxic activity the potential cytotoxic capacity of the t. sandrasicum extracts was evaluated using the brine shrimp lethality test. (meyer et al., 1982). artemia salina eggs (10 mg) were incubated in 500 ml artificial seawater and under artificial light for 48 h at 28ºc. after incubation, ten nauplii were collected with a pasteur pipette and placed into test tubes containing brine solution. in the experiments, 0.5ml of plant extract (1000, 500, 100, 50 and 10 ppm) was mixed with 4.5 ml of brine solution. the number of survivors was counted in each concentration of the extracts and the control after about 24 h. the larvae were considered dead if no movement of the appendage was observed within 10 sec. to determine the lc50 values, the data was analyzed using the epa probit analysis program (version 1.5) (finney, 1971). 2.12. anthelmintic activity the anthelmintic activity of the t. sandrasicum extracts was determined using the methods of dash et al. (2002) with slight modifications. tubifex tubifex (annelida) was used in the experiments. the average size of tubifex tubifex was 1-2 cm and 6 worms were placed in a petri dish containing 20 ml test solutions of ethanol and acetone extracts. test samples of the extracts were prepared at different concentrations (2.5, 5, 7.5, 10 mg/ml) in distilled water. albendazole (2.5, 5, 7.5, 10 mg/ml) was used as a reference standard, while distilled water was the negative control. the worms were observed, and the time taken for paralysis and the time taken death was noted in minutes. the mean time for paralysis was logged when movement was lost, or no movement could be perceived apart from when the worm was forcefully shaken. the time of death was recorded of each worm after ascertaining that the worm failed to move when shaken or when given external stimuli. 2.13. statistical analysis all analyses were performed in triplicate and the results presented as mean±se (standard error) and the results analyzed using the minitab statistical package program. to see how the groups differed from each other, the variations between the different extracts were tested with analysis of variance (anova) and tukey (p<0.05), and the different groups were shown with different letters in the same column. if there were only two groups then a t-test was used. 3. results and discussion antioxidant activity determination methods depend upon various parameters such as the concentration and the structure of the compound to be analyzed. for this reason, there is ital. j. food sci., vol. 31, 2019 338 no standard method for determining the antioxidant activity of a compound and one single method cannot fully describe the antioxidant activity (du et al., 2009; jabrikaroui et al., 2012). consequently, we used six complementary antioxidant methods (radical scavenging (dpph and abts), total antioxidant (β-carotene /linoleic acid and phosphomolybdenum), metal chelating and reducing power activities) to evaluate the true antioxidant potential of the extracts. 3.1. total phenolic and flavonoid content the total phenolic and flavonoid contents in the ethanol and acetone extracts from t. sandrasicum have been determined in the present study. our results revealed that in the ethanol extract, the total phenolic content with 107.94±0.59 mggae/g was higher than that of the acetone extracts with 78.2±1.5 mggae/g and this was found to be statistically different (t=18.21, df=10, p<0.001). the phenolic content for the acetone extract determined in the present study was lower than that determined by stankovic et al. (2010) (acetone extract of t. chamaedrys). in addition, the variable amounts of total phenolic content in the different extracts may be due to solvent polarity (marinova and yanishlieva, 1997). the total flavonoid content was found 65.96±0.19 and 51.61±0.56 mgqes/g in acetone and ethanol extracts respectively and these were statistically different (t=24.30, df=9, p<0.001). these results obtained are in line with those of tarhan et al. (2016) who found total flavonoid content varied from 30.23-95.12 mg/g in ethyl acetate, water and hydromethanolic extracts from t. sandrasicum. in addition, similar to our study, bakari et al. (2015) found acetone extract to have a higher total flavonoid content than the ethanol extract in t. polium. 3.2. antioxidant activities 3.2.1 total antioxidant activity (β-carotene-linoleic acid and phosphomolybdenum methods) β-carotene/linoleic acid is used to measure antioxidant activity. antioxidants minimize the oxidation of lipid components in cell membranes, or inhibit the conjugated diene hydroperoxides, known to be carcinogenic, generating from linoleic acid oxidation (tepe et al., 2007). in this study, the antioxidant activity of the ethanol extract from t. sandrasicum (80.18±1.34%) was better than the acetone (73.61±0.95 %) extract (fig. 1). these results are in line with those of bakari et al. (2015) who found ethanol extract exhibited higher antioxidant properties than the acetone extract in t. polium. in addition, total antioxidant activity for the ethanol and acetone extracts determined in this study were higher than those reported by bakari et al. (2015) (t. polium). in this study, there were statistically differences among the antioxidant contents of the different extracts of t. sandrasicum and bht (f2,24= 109.76, p<0.001) (fig. 1). although the synthetic antioxidant (bht) showed the highest antioxidant activity (over 90 %), the ethanol and acetone extracts were as effective as standard and they seemed to reduce the oxidation of linoleic acid, a key concern for the food industry. the antioxidant activity of the samples was also evaluated using the phosphomolybdenum assay, according to the method of prieto et al. (1999). similar to the β-carotene-linoleic acid test system, in this method the ethanol extract from t. sandrasicum showed stronger antioxidant capacities than the acetone extract (table 1). ital. j. food sci., vol. 31, 2019 339 figure 1. antioxidant activity of t. sandrasicum extracts. ethanol extract of t. sandrasicum; acetone extract of t. sandrasicum, bht: standard antioxidant. (different groups were shown with different letters on each boxplot) table 1. antioxidant properties of t sandrasicum extracts. sample dpph (ic50, µg ml -1)* abts (ic50, µg ml -1)* phosphomolybdenum (µg/mg)* power reducing (mg/ml)* ethanol 122.60±1.35 b 174.86±1.52 a 104.03±3.3 a 0.32±0.01 a acetone 184.76±6.07 a 119.11±6.22 b 74.7±6 b 0.24±0.03 b bht 31.64±1.52 c 12.89±1.20 c nt nt bht: standard antioxidant, nt: not tested. *values are mean of three replicate determinations (n=3)±standard error. mean values followed by different letters in a column are significantly different (p<0.05). the antioxidant activities were found to be statistically different between the ethanol and acetone extracts (t=4.31, df=12, p<0.001). as previously reported by nickavar and esbati (2012) and cakir et al. (2003), our results also showed that the high antioxidant capacities of the ethanol extract of t. sandrasicum is due to the presence of high phenolic content. 3.3. radical scavenging activity (dpph and abts) a stable free radical of a deep shade of purple on scavenging dpph becomes yellow. the level of yellowing indicates the scavenging potential of the extracts, in terms of hydrogen donating ability. consequently, dpph is generally used as a substrate to ascertain antioxidant capacity (duh et al., 1999). the results of the dpph in present study are given in table 1. the higher dpph radical scavenging activities were associated with the lower ic50 values. the ethanol extract exhibited higher scavenging activity than the acetone extract and there were statistically differences among the radical scavenging activities of ital. j. food sci., vol. 31, 2019 340 the different extracts of t. sandrasicum and bht (f2,24= 434.32, p<0.001). the present study has demonstrated that the ethanol and acetone extracts from t. sandrasicum have a radical scavenging capacity and the key role of the phenolic content as scavengers of free radicals has been emphasized in several other reports (komali et al., 1999). when the acetone extract of t. sandrasicum was compared with t. montanum (stankovic et al., 2011) and t. polium (bakari et al., 2015) of which the dpph free radical scavenging activities were found to be 108.10 µg/ml and 13 µg/ml respectively, the dpph free radical scavenging activity of the acetone extract of t. sandrasicum was lower than those of these species. the abts scavenging capacity of plant extracts from t. sandrasicum were determined and the results are presented in table 1. the values of ic50 were in the following order: bht ˂ acetone extracts˂ ethanol extracts. aksoy-sagirli et al., (2015) used abts for the determination of radical scavenging activity in methanol extracts from t. sandrasicum. in the present study we also used abts to investigate scavenging activity in ethanol and acetone extracts from t. sandrasicum and found that the ethanol and acetone extracts of t. sandrasicum have radical scavenging activity. free radicals, which are produced in the human body by chemicals or metabolic processes are capable of oxidizing biomolecules (halliwell and gutteridge, 1989) and exposure to free radicals causes cell damage, which may increase the risk of various diseases, such as cancer, heart diseases and diabetes. as with antioxidants, by inhibiting the formation of free radicals, free radical scavengers naturally protect cells from the damage caused by harmful molecules (percival, 1998). the present study reveals that t. sandrasicum extracts could serve as free radical scavengers and due to these properties, they may be used as an ingredient in food. 3.4. ferric ion reducing power activities the reducing ability describes how easily one substance can give electrons to another. a powerful reducing agent is inclined to give electrons. the reducing power method measures the ability of components that act as antioxidants to reduce ferric ion (singh et al., 2012). in the present study, the reducing ability of ethanol and acetone extracts from t. sandrasicum were measured, and the results of this activity demonstrated that the ethanol extract showed a higher reduction ability than those of the acetone extracts (table 1). the reducing power activities were found to be statistically different between the ethanol and acetone extracts (t=2.34, df=9, p<0.05). according to these results, the ethanol and acetone extracts of t. sandrasicum possess antioxidant capacity. this is because the reducing capacity of a compound serves as potential antioxidant activity (singh et al., 2012). in addition, the reducing power of ethanol extract may be due to the high level of phenolic content, which acts as an electron donor. similarly, numerous studies advocate an association between the reducing power and the total phenolic content (goncalves et al., 2013). 3.5. metal chelating properties the metal chelating ability of the studied t. sandrasicum extracts were determined by measuring the iron-ferrozine complex. the metal chelating property of the ethanol and acetone extracts from t. sandrasicum were evaluated and these results showed that the ethanol extract (55.85±4.22 %) exhibited better metal chelating activity when compared with the acetone extract (26.67±1.48 %). although the synthetic metal chelator (edta) exhibited the highest chelating activity (over 80 %), the ethanol and acetone extracts inhibited complex of ferrous, ferrozine and this revealed that they exhibit chelating activity (fig. 2). the metal chelating capacity is important, because this activity reduces the ital. j. food sci., vol. 31, 2019 341 amount of catalyzing transition metal in lipid peroxidation (duh et al., 1999). for this reason, the presence of the chelating properties of the extracts contribute directly to their antioxidant properties. figure 2. metal chelating activity of t. sandrasicum extracts. ethanol extract of t. sandrasicum; acetone extract of t. sandrasicum, edta: standard antioxidant. (different groups were shown with different letters on each bar) 3.6. proximate analysis the evaluation, determining the moisture, crude protein, crude fat, ash, carbohydrate, fibre and energy, as a proximate analysis of the aerial parts of t. sandrasicum, is presented in table 2. when compared with earlier studies, the protein content of t. sandrasicum was found to be lower than those of t. muscatense (rehman et al., 2016) and t. polium (hussain et al., 2013). in contrast with the study of rehman et al. (2016), the carbohydrate content of t. sandrasicum was found to be lower than t. muscatense. the fat content and the energy value of the t. sandrasicum were lower than the fat content and the energy value of the t. polium (hussain et al., 2013). table 2. proximate analysis of t. sandrasicum. constituents aerial parts ash (g/100 g dw) 4.76±0.52 carbohydrate (g/100 g dw) 17.06±1.28 proteins (g/100 g dw) 2.43±0.10 fat (g/100 g dw) 1.10±0.12 moisture (g/100 g fw) 42.17±0.9 fibre (g/100 g dw) 28.48±0.1 energy (kcal/100 g dw) 87.86 ital. j. food sci., vol. 31, 2019 342 the plants species, especially medicinal plants are also used as food or a food supplement and evaluating their nutritional contents can help to understand the significance of these plant species as a dietary supplement and for pharmaceutical applications. proximate analysis of this plant plays a decisive role in assessing its nutritional significance and revealed that this species is good source of nutrients as well as can contribute towards nutritional requirements (pandey et al., 2006; adnan et al., 2010). 3.7. phenolic composition it has been established that the lamiaceae species comprise a range of secondary metabolites, including phenolic acids and flavonoids. in present study, phenolic compositions of ethanol extract of t. sandrasicum were identified using hplc method. phenolic compound that were determined in ethanol extract are listed in table 3 and the main phenolics were identified as caffeic acid and rutin (fig. 3). table 3. phenolic components in the ethanol extract of t. sandrasicum. no phenolic component approximate rt (min) µg/g* 1 gallic acid 6.8 917.35±0.08 2 3,4 dihydroxybenzoic acid 10.7 92.47±0.01 3 4-hydroxybenzoic acid 15.7 1066.40±0.08 4 2,5 dihydroxybenzoic acid 17.2 61.06±0.02 5 chlorogenic acid 18.2 462.02±0.05 6 vanilic acid 19.2 563.29±0.09 7 epicatechin 21.3 1648.12±1.02 8 caffeic acid 22.7 22727.28±5.06 9 p-coumaric acid 26.1 1.47±0.05 10 ferulic acid 30.1 72.06±0.01 11 rutin 45.6 3392.28±1.06 12 cinnamic acid 71.1 315.40±0.03 13 quercetin 70.4 2893.08±0.02 *based on dry weights figure 3. hplc chromatograms of phenolic components in the ethanol extracts of t. sandrasicum. ital. j. food sci., vol. 31, 2019 343 some phenolic compounds determined in present study, such as ferulic, gallic, caffeic, vanillic, chlorogenic acids and rutin, in previous studies were obtained from lamiaceae plants (canadanovich-brunet et al., 2006; roby et al., 2013; kaska et al., 2018). in addition, previously studies have shown that, from these phenolics, caffeic acid exhibits anticarcinogenic properties that act as a carcinogenic inhibitor (magnani et al., 2014). in brief, nowadays the identification and measurement of plants phenolic compounds are considered to be effective mechanisms for ascertaining the importance of plants for human health (amarowicz et al., 2010). this is because phenolic content can directly contribute to the antioxidant capacity of the plants (duh et al., 1999). 3.8. cytotoxic activity the brine shrimp cytotoxicity test is a practical and economic method for the investigation and assessment of toxicity, antifungal and pesticidal effects of plants. lc50 values of less than 1000 µg/ml are regarded as bioactive when using the brine shrimp lethality test to calculate the toxicity of plant extracts (meyer et al., 1982). the lethality of ethanol and acetone extracts were 389.661 and 658.032 µg/ml respectively, and the extracts possessed high cytotoxic activities against brine shrimp. the lethality of these extracts from t. sandrasicum indicates the presence in this species of potent cytotoxic components, which require further investigation. the present study suggests the need for further investigations of this plant, in order to ascertain the potential cytotoxic compound. 3.9. anthelmintic activity in the present study, an investigation was made of the anthelmintic activities of t. sandrasicum extracts (ethanol and acetone). the results presented in table 4 show that the ethanol and acetone extracts obtained from t. sandrasicum is active against tubifex tubifex. table 4. in vitro anthelmintic activity of t. sandrasicum. type of extract concentration used (mg/ml) time (min) taken for paralysis (x±s.e.)* time (min) taken for death (x±s.e.)* control (distilled water) ethanol 2.5 37.33±1.58 a 48.5±1.63 a 5 21±1.93 b 31.67±1.38 b 7.5 15.67±1.20 bc 21.67±0.62 c 10 10.17±0.75 c 14±0.82 d acetone 2.5 22.33±1.52 a 31.5±1.43 a 5 17.83±0.54 b 21±0.48 b 7.5 12±0.45 c 16±0.45 c 10 9±0.37 c 12.65±0.56 d albendazole (standard) 2.5 52.33±2.73 a 61±3.33 a 5 33±1.48 b 48.5±3.48 b 7.5 21.17±2.01 c 35.5±3.21 c 10 13±0.68 d 20.5±1.57 d values are mean±s.e. of six worms. *mean values followed by different letters in a column are significantly different (p<0.05). ital. j. food sci., vol. 31, 2019 344 all doses of the t. sandrasicum extracts (ethanol and acetone) showed better anthelmintic activity, in terms of promoting paralysis and causing death, than the standard. the strong anthelmintic activity of t. sandrasicum extracts may be due to the existence of rich polyphenolic compounds. both humans and animals have benefitted from a broad range of medicinal plants in the treatment of parasitic infections in. anthelmintics, derived from plant sources, present some advantages, such as pharmacological effectiveness and lower toxicity for animals and humans (peixoto et al., 2013). nowadays, there is an increasing interest in studies on the screening of new and effective medicinal plants that have anthelmintic properties. t. sandrasicum extracts possess wormicidal activity and could be effective against the parasitic infection of humans and animals. hence, the lethality of the potent anthelmintic components in this species requires further investigation. 4. conclusions the results revealed in the present study have shown that the acetone and ethanol extracts from this plant have strong antioxidant properties. they have also shown that the plant possesses rich phenolic and flavonoid compounds, making it a good source of nutrients. furthermore, t. sandrasicum extracts have been shown to have cytotoxic and anthelmintic activities. the present study therefore suggests that this plant could be considered as a source of natural agents in the food industry and can be used as a new anthelmintic and cytotoxic agent for pharmacological applications. further investigation is required to isolate and identify the antioxidant, anthelmintic and cytotoxic components found in this plant. this will in turn increase information on the usability of the plant for the food industry and pharmacological applications. acknowledgments we thank all the lab members of the secondary metabolites lab. we also state that there is no conflict of interest among the authors. references adnan m., hussain j., shah m.t., shinwari z.k., ullah f., bahader a., khan n., khan a.l. and watanabe t. 2010. proximate and nutrient composition of 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biscuit; dough; storage; texture impact of buckwheat flour granulation and supplementation level on the quality of composite wheat/buckwheat ginger-nut-type biscuits b. filipčev*, o. šimurina and m. bodroža-solarov institute of food technology, university of novi sad, bul. cara lazara 1, 21000 novi sad, serbia *corresponding author: tel. +381 21 485 3778, email: bojana.filipcev@fins.uns.ac.rs abstract effects of gradual wheat flour substitution with buckwheat flour in ginger-nut biscuit formulation were investigated regarding dough characteristics, physical and textural characteristics of final product assessed after baking and 30 days of storage. buckwheat flour was added at 30, 40, 50% levels and two granulations (fine and coarse). addition of buckwheat flour significantly increased dough hardness and decreased adhesiveness. spread significantly increased in biscuits with 40% and 50% of coarse buckwheat flour. biscuits containing coarse flour were harder and more fracturable than the control, whereas those with fine flour tended to be softer and less fracturable. textural properties were significantly correlated to protein stability to heat and retrogradation tendency of starch in biscuit dough as well as moisture content. mailto:bojana.filipcev@fins.uns.ac.rs 496 ital. j. food sci., vol. 27 2015 introduction ginger-nut biscuit (gnb) is a sweet biscuit containing honey and aromatic spices (cinnamon, ginger, cloves), which is very popular globally. by this term, variety of sweet biscuit types are described. they may range from thin (less than 3 mm thick), crisp varieties with smooth surfaces to thicker (over 3 mm thick), smaller in diameter and softer varieties with prominent cracks on top surface. the large variation in ingredients and their proportions contributed to a wide range of biscuit types. for example, the finest quality nürnberg lebkuchen does not even contain flour but nuts and candid fruits deposited on thin wafer base whereas the polish pierniki toruńskie is made from the highest quality flour. the similar feature in all of them is that they all contain honey and spices. in serbia, ginger-nut biscuits are traditionally produced by artisans, although their production has been industrialized (gavrilović, 2003). in appearance, they mostly resemble the gingersnaps from the united states: usually circularly shaped, over 6 mm thick with cracks on the top surface and soft crumb. formulations of gnb typically include 3550% honey, 28-32% sugar, 0-5% fat on a flour weight basis (gavrilović, 2003), whereas the levels of major ingredients in common sweet biscuits are 30-75% sugar, 15-50% fat and 7-20% water (manley, 2000). according to pyler (1998), basic ratios of flour, fat and sugar in cookies vary from 100:30:30; 100:50:50 to 100:50:variable depending on processing type but may considerably deviate. gnb flour can be specified as a wheat flour with good bread-making performance (quality group b, peak amylogram value 300 b.u.), preferably of higher extractions (ash contents 0.8-1.15% d.b.) due to higher protein, hydrocolloid and enzyme content as opposed to the quality requirements in common biscuit production. the flour in common biscuits is usually soft wheat flour with lower protein content and moisture absorption and ash contents 0.34-0.38% d.b. (pyler, 1998) and weak gluten (pareyt and delcour, 2008). in contrast to common biscuits whose final moisture content is low and ranges between 1-5% (chevallier et al., 2000, 2002), the lowest recommended moisture content in gnb is 7% (gavrilović, 2003). below this value, the quality deteriorates due to increased hardness, fracturability and crumbling. the crumb of gnb is porous with denser or looser pore structure, soft and elastic. its upper surface is cracked which is a common pattern with formulations high in sugar and low in fat and is attributed to sugar recrystallization on the surface area (pareyt et al., 2009). gnb is traditionally perceived as healthier in relation to other types of sweet biscuits probably because they contain honey and low amount of fat. as such, they can be seen as a convenient medium for providing further improvement in nutritional value and functionality by replacing a part of wheat flour with other nutritionally more valuable cereal or noncereal flour. one such ingredient having an excellent reputation for its nutritious quality and abundance in bioactive compounds is common buckwheat. buckwheat is most commonly used for producing flour and groats. while groats are mainly used for porridge and in various ethnic dishes, buckwheat flour is used as an ingredient in a variety of baked, cooked and extruded products: breads made from wheat-buckwheat flour blends at different ingredient proportions, flat bread, pasta (pizzoccheri in italy), noodles (soba in japan, extruded noodles in china and korea), pancakes, breakfast cereals (based on extrudates made from buckwheat, rice, and/or corn blends), and biscuits (buckwheat added at 2030% wheat flour basis). results from previous studies (filipčev et al., 2012) revealed that buckwheat flour has a potential as an ingredient in gnb formulation but a main disadvantage was related to the coarse granulation of commercially available buckwheat flour which reflected gritty texture of the final product. therefore, this study was conducted to investigate the effect of substitution level and granulation of wholegrain buckwheat flour on dough characteristics and gnb physical and textural characteristics. the effects of three substitution levels (30%, 40%, 50%) and two flour granulation sizes (fine (fbf) and coarse (cbf)) were studied. materials and methods materials commercially available wheat flour (wf) type 850 (ash content 0.81% dry basis, moisture content 12.53%), and wholegrain buckwheat flour (ash 2.20% d. b., moisture content 12.31%) were used in the experiment. other ingredients honey, vegetable fat (from sunflower), sugar, nahco 3 , lecithin and spice (cinnamon) were obtained from a local food store (novi sad, serbia). the purchased buckwheat flour was coarsely granulated. to obtain finer granulation, it was remilled on a falling number 3100 mill (perten instruments). the granulation of used flours is given in table 1. water absorption capacity of flours flour sample (5 g) was mixed with an excess of distilled water (25 ml), kept at ambient temperature for 30 min and then centrifuged at ital. j. food sci., vol. 27 2015 497 2000 x g for 15 min. water absorption capacity was expressed as g of water bound by 100 g of dry matter. syneresis degree of wheat and buckwheat flours the method described by singh et al. (2003) was used. flour suspensions (6 % w/v) were heated in a water bath to 90°c and held 20 min at this temperature. cooled flour paste (30 mg) was poured into a centrifuge tube. the tubes were stored for 1 and 10 days at 4°c. syneresis was measured as % of water expelled after centrifugation of sample. biscuit making gnb was prepared by substituting wheat flour with buckwheat (0, 30, 40, 50 %). formulae are found in table 2. firstly, a basic dough was formed by warming a mixture of honey, sugar and water to 80°c and adding part of the flour (75% of total) to a hot mixture. the mixture was mixed to obtain a thick, homogenous mass. after cooling to 40°c, basic dough was sprinkled over with flour, covered with a plastic foil, and left to rest at ambient temperature for two days. other ingredients (remaining flour amount, spices, raising agents dissolved in water, fat and lecithin) were added and mixed using a kitchen mixer with a spiral hook for 10 min. the amount of added water was adjusted table 1 particle size distribution of used flours. particle size wheat flour coarse buckwheat fine buckwheat (weight %) (wf) flour (cbf) flour (fbf) >350 mm 0.00 55.30 5.74 >250 mm 0.02 9.40 11.32 >180 mm 0.40 4.76 11.11 >150 mm 1.32 3.80 8.90 >105 mm 22.54 10.14 18.86 >85 mm 26.85 9.80 30.76 bottom 48.87 6.80 13.31 table 2 ginger nut biscuits formulations. ingredients, g control biscuit supplemented biscuit supplemented with cbfa with fbfa wheat flour 100 70 60 50 70 60 50 buckwheat flour 0 30 40 50 30 40 50 honey 50 50 50 sugar 10 10 10 vegetable fat 5 5 5 nahco 3 2.1 2.1 2.1 spice blend 2 2 2 lecithin 1 1 1 water 20 18 17 16 20 21 22 a cbf-coarse buckwheat flour; fbf-fine buckwheat flour to obtain a maximally soft dough with acceptable handling characteristics. the consistency of dough was evaluated subjectively by an experienced baker. dough moisture content ranged between 17.8-21.7%. after mixing, the dough was sheeted on a pastry break to 10-mm thickness (sfogliatrice mignon, maestrino, pd, italy). the dough was cut to a diameter of 60 mm and baked for 15 min in a deck oven at 170°c. after 1 hour of cooling at room temperature, the biscuits were packed in polyethylene bags and stored at room temperature. texture profile analysis of biscuit dough dough characteristics were evaluated using a texture analyzer (ta.xtplus stable micro systems ltd., surrey, uk) equipped with a 30kg load cell. texture profile analysis (tpa) was employed to measure dough properties as described by (gallagher et al., 2005). doughs were prepared as for the baking test and cut into round pieces (60 mm). a 36-mm cylindrical aluminium probe was used in two compression cycles at test speed 1.0 mm/s. pre and posttest speeds were 2.0 mm/s. the force was measured at 45% compression. the recovery period between the strokes was 5 s. the following 4 parameters were recorded: hardness, adhesiveness, cohesiveness and springiness. seven measurements per each biscuit type were made. measurements were performed on dough after the resting period. 498 ital. j. food sci., vol. 27 2015 analysis of thermo-mechanical properties of biscuit dough by mixolab mixing and pasting properties of the ginger nut biscuit doughs were studied on mixolab (chopin, tripette and renaud, paris, france). this device measures in real time the torque produced by dough during mixing at conditions of controlled temperature regime which include dough heating to 90°c, maintenance at constant temperature and dough cooling to 50°c. in this way, the behaviour of both proteins and starch under dual mechanical shear stress and temperature constraint can be measured (rosell et al., 2006). usually, individual flours or flour blend slurries are analysed in this way. in our study, previously prepared gnb dough was subjected to analysis using a modified mixolab protocol. for the assays, 80 g of dough was inserted to mixolab bowl, followed by the next regime: mixing speed 80 rpm, tank temperature: 30°c, 1st plateau temperature 30°c, duration of first plateau 5 min, heating rate 4.0°c/min, 2nd plateau temperature 90°c, duration of 2nd plateau 7 min, cooling rate 4.0°c/min, 3 rd plateau temperature 50 °c, duration of 3rd plateau 5 min. main derived parameters from the mixolab curves are: development (c1) or maximum torque reached during mixing at 30°c, protein weakening (c2) or the minimum torque produced as a consequence of heating and mechanical stress, maximal torque (c3) produced during the heating stage as a consequence of starch gelatinization, minimal torque at the stage of cooling (c4) and the torque after cooling at 50°c (c5). measurements were replicated twice for each biscuit dough type after the resting period. textural analysis of ginger nut biscuits hardness and fracture of gnb were measured 24 h post-bake by penetration test on ta.xtplus texture analyzer (stable micro systems, england, uk). the test mode was force in compression. a 5 kg load cell was used. the sample was placed on the platform with a holed plate and centrally punctured with a 2 mm cylinder probe through the sample at test-speed 0.5 mm/s. hardness of the sample was calculated from the area under the curve whereas fracturability was calculated from the linear distance. four biscuits from each treatment were punctured five times in an ‘x’ pattern avoiding the outer 1 cm to prevent from edge effects. biscuit dimensions and density diameter (width) and height (thickness) of biscuits were measured using a vernier calliper. diameter was calculated as an average of long and short diameter. spread was calculated from the ratio of width and height. for the measurements, twelve randomly chosen biscuits were taken. density was calculated as a ratio of biscuit mass and volume. since biscuits had a regular shape, their volume was approximated to the volume of cylinder according to formula v=p*(r/2)2*h where h is biscuit height and r is average biscuit diameter. moisture content moisture was calculated according to aoac method 926.5 (2000). two composite samples of each biscuit type were analyzed in duplicates. the composite samples were prepared by homogenization of six individual biscuit samples. statistical analysis an analysis of variance (anova) of data was performed by using a statistica 7.1 statistical software package (statsoft inc., tulsa, oklahoma). tukey’s post-hoc test was used to compare the means at 95% confidence interval. correlation analysis was conducted using spearman’s rank correlation coefficient applied to mean values for each biscuit. results and discussion water absorption capacity (wac) and syneresis degree of used flours buckwheat flour showed higher wac in comparison to wf (table 3). this might be due to various reasons: higher hydration capacity of buckwheat starch in comparison to wheat starch (qian et al., 1998); presence of other hydrophilic constituents in the wholegrain bucktable 3 water absorption capacity (wac) and syneresis (%) of wheat and buckwheat flour. flour wac (g/100 g dry matter) syneresis (%) 1 day 10 days wheat flour 63.96a 9.27a 14.57a buckwheat flour, coarse 101.10b 39.27b 46.86c buckwheat flour, fine 104.14b 40.20b 42.67b a,b,c figures followed by the same letters in a column are not significantly different (p>0.05). wheat flour (fibers). in contrast, baljeet et al. (2010) reported that buckwheat flour had lower wac, higher oil absorption capacity, higher foaming capacity and higher least gelation concentration than wheat. wac of fbf increased but not significantly in comparison to cbf which might be explained by the fact that milling does not tend to increase the amount of damaged starch in buckwheat. it has been shown by qian and co-workers (1998) that ital. j. food sci., vol. 27 2015 499 buckwheat flour has lower amounts of damaged starch than wheat. the syneresis value (%) of cooked pastes from wheat and buckwheat flour significantly differed. wf showed much less syneresis than buckwheat. during storage, syneresis of the pastes increased. the highest syneresis was observed in the paste made from cbf (46.9%), followed by fbf (42.7%). the retrogradation properties of the flours are generally attributed to composition, ratio and interactions of flour constituents: proteins, starch, lipids, fibers. gnb dough characteristics the addition of buckwheat significantly increased dough hardness in comparison to the control (p<0.05) (fig.1). the dough hardness increased remarkably in the case of finely ground buckwheat flour. tpa adhesiveness showed a declining trend which was significant in comparison to the control. dough springiness and cohesiveness also tended to decline with increased proportion of buckwheat, but there were no significant differences between the samples. fbf increased dough cohesiveness in comparison to cbf. fig. 1 dough hardness, adhesiveness, cohesiveness and springiness for different formulations of gnb. a,b,c ,… a, b,… values followed by same letters of the same case are not significantly different (p>0.05). these effects can be related to the particularities of buckwheat flour and flour particle size. buckwheat is characterized by resistant starch, which may contribute to low viscoelastic properties of dough and increased hardening (qian et al., 1998; de francischi et al., 1994; li et al., 1997; qian and kuhn, 1999; yoshimoto et al., 2004; hatcher et al., 2008). the observed variations in dough hardness and adhesiveness were mainly due to the effect of buckwheat flour granularity. even though wac between fbf and cbf did not significantly differ, it seems that finer flour particles were able to absorb more water during mixing, thereby forming more cohesive and harder dough. lowering of dough adhesiveness and cohesiveness as a consequence of increasing amounts of coarse wheat flour was reported earlier (singh gurjal et al. 2003). this coincides with our findings. furthermore, dough made with wholegrain buckwheat flour was reported to have lower adhesiveness in comparison to the majority of other buckwheat flour fractions (ikeda and kishida, 1992). parameters related to thermo-mechanical behaviour of gnb dough made with coarse and fine buckwheat flour is given in table 4. bucktable 4 dough mixolab parameters for different formulations of gnb (cbf-coarse buckwheat flour; fbf-fine buckwheat flour). sample c1 c2 c3 c4 c5 c1-c2 c5-c4 (nm) (nm) (nm) (nm) (nm) (nm) (nm) control 3.45a 2.73e 3.04f 1.65e 1.66b 0.72a 0.005a cbf 30% 3.99b,c 1.20d 1.40c 1.39c 2.71e 2.79c 1.32d 40% 4.31c 0.86c 1.53c 1.54d 2.24d 3.45e 0.70b 50% 3.68a,b 0.68a 1.81d 1.62e 2.69e 3.00d 1.06c fbf 30% 4.22c 1.30d 1.30b 0.97b 1.85c 2.92d 0.88c 40% 4.19c 0.75b 2.00e 0.91b 1.52b 3.44e 0.61b 50% 5.32d 0.85c 1.10a 0.63a 0.95a 4.47e 0.32b a,b,c ,… figures followed by the same letters in a column are not significantly different (p>0.05). 500 ital. j. food sci., vol. 27 2015 wheat supplemented dough was characterized with higher maximal torque during mixing (c1) and lower resistance to thermal and mechanical stresses (low c2 and high c1-c2) than the control dough. these effects seem to be more pronounced in the case of dough supplemented with coarse buckwheat flour. both buckwheat flours affected these parameters in a similar way. this behaviour is probably due to dilution of gluten by addition of non-glutenous buckwheat flour which contributed to the formation of weaker protein network. the control dough showed higher maximal peak during heating (c3) than the buckwheat supplemented doughs. the decrease in peak viscosity of the buckwheat supplemented doughs may be attributed to lower swelling of starch granules and poorer gelatinization which is probably due to native characteristics of buckwheat starch and limited water amount in gnb doughs. similar results were reported by hadnađev and co-workers (2008), even in systems with higher moisture content, resembling bread dough. the buckwheat doughs exhibited lower breakdown torque (c4) which indicates lower stability of warm gel. final torque (c5) and total setback values (c5-c4) are parameters used to characterize starch retrogradation that occurs during cooling to 50°c. final torque (c5) was lower in the control dough and dough made with fine buckwheat flour. the total setback was the lowest in the control dough followed by fbf dough. hence, regarding the ability to resist retrogradation, the tested gnb doughs follow the order: wheat dough > fine buckwheat flour dough > coarse buckwheat flour dough. biscuit dimension, density and spread data related to the biscuit dimension and density is displayed in fig. 2. majority of the variables showed significant differences as compared to the control especially at higher replacement levels (40% and 50%). the height and diameter decreased with increasing levels of both fbf and cbf. despite general shrinkage, the interrelation between both dimensions was such that spread increased significantly at 40% and 50% of cbf addition whereas in other cases, spread did not significantly vary from the control. there was a strong inverse correlation between spread and height (r=-0.84, p<0.01) showing that reduction in height dominantly affected spread whereas spread and diameter were not significantly correlated (r=-0.17, p>0.01). the spread of the biscuit during baking is caused by expansion of dough by leavening and gravitational flow (hoseney and rogers, 1994). it depends on factors that control dough viscosity: the amount of water free to act as solvent and the strength of dough (ram and singh, 2004). it also depends on partitioning of available water between the ingredients. the lesser the amount of water held by ingredients, the more the amount of water is available to dissolve sugar, decrease dough viscosity and increase spread (pareyt and delcour, 2008). coarse flour has been reported to contribute to greater spread (singh gurjal et al., 2003; manley, 1991). substitution of wheat flour with a finely ground light and dark buckwheat flour (granulation size 100-150 mm) in sugarsnap cookie formulations lowered the cookie spread (maeda et al., 2004). it was also found that buckwheat flour of similar granulation dosed at 10-40% level (flour basis) decreased the spread in sugar snap cookies (baljeet et al., 2010). results obtained in this study confirmed the relation between spread and flour granularity: in relation to the control, the addition of cbf increased the spread. higher biscuit spread is considered advantageous in biscuit making (pareyt and delcour, 2008). but, in contrast to majority of biscuits, the main quality requirement for gnb is well developed and soft crumb (gavrilović, 2003). therefore, high spread may not be necessarily considered as preferable in gnb making since the increased thickness (height) is needed to develop a porous and soft crumb. regarding biscuit density, a general trend was fig. 2 effect of gnb formulation on biscuit dimensions (height, diameter, spread) and density. a,b,c ,… a, b,… values followed by same letters of the same case are not significantly different (p>0.05). ital. j. food sci., vol. 27 2015 501 fig. 4 effect of gnb formulation on biscuit hardness and fracturability measured after 24 h and 30 days. a,b,c ,… a, b,… values followed by same letters of the same case are not significantly different (p>0.05). that density increased in the combined formulations. there was a strong inverse correlation between the biscuit dimensions and density r=0.97 (p<0.01) and -0.76 (p>0.05) for height and diameter, respectively, confirming that higher density reduces biscuit development. biscuit moisture content minimal moisture content required for the retention of freshness in gnb is 7%. moisture contents below this value render biscuits unacceptable owing to their increased tendency to dry out during storage. the moisture content of biscuits was significantly affected by the level of flour substitution with buckwheat flour as well as its granulation: increased buckwheat doses and coarse flour tended to decrease it whereas fbf increased the moisture content (fig. 3). as a result, the composite biscuits with 40% and 50% of cbf were significantly lower in moisture (8.00-8.50%, p<0.05, respectively). other formulations had higher moisture contents with the highest registered in gnb made with fbf and the control (≥ 10.0% moisture content). the water content in gnb formula was significantly correlated to biscuit hardness (r=-0.84, p<0.01), dough cohesiveness (r=0.84, p<0.01) and c5 (r=0.92, p<0.01). these results indicate that higher moisture content is advantageous in this category of biscuits as it contributes to softer texture, more cohesive and stable dough. over time, a decrease in moisture contents was observed in all samples. the most marked moisture content decrease (by 8%-10.7%, p<0.05) were in the biscuits substituted with fbf and much less in other samples (by 2.6%-3.8%), in spite of the lower syneresis degree of fbf. although subjected to the high moisture loss, these biscuits remained higher in moisture content than the control due to higher initial moisture. during storage, moisture migration and evaporation occurs because gluten and starch undergoes such transformations which result in water release (willhoft, 1971; senti and dimler, 1960). biscuit texture characteristics granulation of buckwheat flour affected hardness and fracturability of gnb. in general, two trends were observed: as the level of cbf increased, hardness also increased, whereas the addition of fbf decreased hardness (fig. 4). however, the produced results were similar to the control except in the case of gnb made with 50% cbf, which produced significantly higher hardness. fracturability showed a similar trend (fig. 4). baljeet and others (2010) said that the addition of finely ground buckwheat flour decreased the hardness of composite biscuits. increased hardness has been usually related to increased association of wheat proteins as correlated by gaines (1990) in sugar snap cookies and hatcher et al. (2008) in the buckwheat supplemented soba noodles. in contrast to common biscuits, good quality wheat flour is required in the production of ginger nut biscuits. consequently, its texture is related to the quality of flour proteins with the ability to provide unique viscoelastic and network-forming properties. the results of this study showed a significant correlation (r=0.74, p<0.05) between the biscuit hardness and a mixolab parameter fig. 3 effect of gnb formulation and storage time on biscuit moisture content. a,b,c ,… a, b,… values followed by same letters of the same case are not significantly different (p>0.05). 502 ital. j. food sci., vol. 27 2015 related to the quality of proteins, c1-c2. as already noted, c1-c2 indicates protein weakening during heating: higher difference means increased protein weakening i.e. lower stability to heat. gavrilović (2003) suggested that gnb hardness is also affected by starch pasting properties. in this study, significant correlation (r=0.84 and 0.81, p<0.01) was found only between hardness and mixolab parameters which indicates starch gel retrogradation (c5 and c5-c4 (total setback), respectively) i.e. higher biscuit hardness can be related to higher setback and c5 values which are indicators of higher susceptibility to staling. the ability of the buckwheat starch to increase hardness of gnb can be counteracted by using ingredients able to retain more water (such as an observed decreasing trend for hardness in the biscuits made with fbf (fig. 4)). actually, it was found that the moisture content significantly affected biscuit hardness; there was a significant inverse correlation between hardness and formula water content i.e. biscuit moisture content (r=-0.84 (p<0.01) and -0.74 (p<0.05), respectively). according to gavrilović (2003), fracturability of gnb can be related to the presence of partially dehydrated gluten in the dough matrix. charles and co-workers (2004) proposed that the presence of discontinuous phase in gluten network was related to an increase in fracturability in flaky snack. here in the study, significant correlation was found between the biscuit fracturability and two mixolab parameters related to the behaviour of protein during heating (c2 and c1c2): c2 denotes the minimum torque produced during heating as a consequence of the beginning of protein weakening (r=-0.84, p<0.01) and c1-c2 can be associated with protein stability (r=0.95, p<0.01). lower value of c2 and higher value for c1-c2 indicate lower protein stability. in other words, increasing fracturability of gnb can be related to all such factors that may lower the protein stability or discontinue the gluten network such as the addition non-gluten ingredient like buckwheat, especially in the form of coarse flour. there was also a significant correlation between biscuit fracturability and setback value (c5-c4) (r=0.78, p<0.05 ). over the 30-day trial period, a significant increase in hardness and fracturability occurred for gnb made with fbf whereas all others showed insignificant increases as related to the initial values (fig. 4). but, there were no significant differences in hardness and fracturability within all biscuits measured after 30 days of storage. on the basis of the above mentioned high correlation between the texture and mixolab parameters that indicate starch susceptibility toward retrogradation, it could be thought that the lower the susceptibility of starch in biscuit dough to retrograde, the lower the biscuit hardness, fracturability and presumably its tendency towards staling. but, the results obtained after storage showed that gnb made with fbf, which initially gave the softest biscuit and which paste had lower syneresis degree, significantly increased in hardness (in relation to the initial values). this could be due to higher moisture evaporation and more compact structure in comparison to the granular structure of gnb made with cbf. it is also worth noting that gnb dough represents a low-moisture system containing up to 25% moisture and, in addition, interfering ingredients like sugar and fat which might have caused different behaviour and tendencies regarding starch pasting. interestingly, dough hardness was strongly positively correlated with biscuit hardness and fracturability after storage (r=0.84 and r=0.81, p<0.01). in the literature, there is little data on the functional properties of buckwheat flour; more data exist on buckwheat starch. furthermore, data on the ability of buckwheat starch to retrograde are rather contrasting. in the study of qian and colleagues (1998), besides increased hardness and stability, buckwheat starch gels exhibited lowered retrogradation as compared to cereal starches, even after storage. lower susceptibility to retrogradation was also suggested in reports on thermomechanical properties of buckwheat flour slurries or wheat/buckwheat blends slurries (chopin’s mixolab user’s manual 2005, banu et al., 2010). in another study, however, a stronger retrogradation peak was observed in buckwheat thermogram than that in wheat (liu et al., 2006). zheng and associates (1998) reported that buckwheat starch had a higher peak viscosity and setback value than maize and wheat starches which may suggest higher retrogradation tendencies in buckwheat starch since setback viscosity indicates the degree of starch retrogradation, mainly its amylase fraction. starch pasting properties depend on the hydration level (zhou et al., 2009). it was concluded that buckwheat starch gelatinization temperatures and enthalpies increased along with the decrease of water content, which can be associated with an increased retrogradation tendency. moreover, there is little data to relate starch pasting behaviour with the final properties in a biscuit-like products. it was reported that the addition of corn flour produced harder cookies than did the addition of potato flour, although corn flour had been shown to give lower syneresis (retrogradation) than potato flour (singh et al., 2003). some authors did not find any correlation between pasting properties of batters and characteristics of layer cakes (gómez et al., 2010). others suggested that dehulled buckwheat flour, although showing a tendency to retrograde, can be used in buckwheat enriched products (mariotti et al., 2008). inital. j. food sci., vol. 27 2015 503 glett and his team (2009) proposed that only specialty buckwheat flour with low paste viscosity is suitable for mixing with wheat flour to produce bread and cookies. much earlier, lorenz and dilsaver (1982) mentioned that inclusion of native buckwheat starches in cake formulations did not produce cakes of acceptable quality. conclusions the results indicated that the addition of buckwheat significantly increased dough hardness and decreased dough adhesiveness. springiness and cohesiveness of buckwheat doughs were reduced but no significant difference within the samples was observed. the dimensions of gnb decreased, but since the biscuit height was more affected by the rising doses of buckwheat, the spread increased in the composite biscuits (significant difference was noted with cbf at 40% and 50% replacement level). hardness and fracturability of gnb increased with increasing doses of cbf whereas fbf decreased hardness and fracturability. however, a significant change was noted only with the addition of 50% cbf. hardness and fracturability of gnb significantly correlated to the quality of proteins; lower stability of proteins to heat was associated to increased hardness and fracturability in gnb. tendency of starch gel in biscuit dough to retrograde was found to be in significant positive correlation with biscuit hardness and fracturability, however, after 30 days of storage, hardness and fracturability increased most markedly in the biscuits made with fbf which were initially softer than the others. biscuit hardness was in significant inverse relation with the formula water content which might additionally support the importance of protein quality and starch pasting behaviour to the quality of gnb. comparing gnb and common biscuits made from short and semi-sweet dough from literature, it seems that gnb exhibits different behaviour. better quality characteristics are obtained when more water is added to the dough and increased spread cannot be regarded as positive since well-developed crumb is more advantageous for better textural properties. the addition of fbf to gnb formulation is appropriate at the investigated doses. however, since they have been inclined to excessive drying out, the use of humectants or suitable edible coatings would be appropriate. acknowledgements the authors acknowledge the financial support of the ministry of education, science and technological development of the republic of serbia (project iii 46005). references aoac 2000. „ official methods of analysis of aoac international“. aoac, maryland, usa. baljeet, s.y., ritika, b. y. and roshan, l.y. 2010. studies on functional properties and incorporation of buckwheat flour for biscuit making. int. food res. j. 17: 1067-1076. banu, i., vasilean, i. and aprodu, i. 2010. evaluation of rheological behaviour of whole rye and buckwheat blends with whole wheat flour using mixolab. it. j. food sci. 22(1): 83-89. charles, a.l., fu, h-y. and huang, t-c. 2004. role of intact starch granule and gluten on texture of taiwanese flaky snack. j. text. study 35: 311-323. chevallier, s., colonna, p., buléon, a. and della valle, g. 2000. physicochemical behaviours of sugars, lipids and gluten in short dough and biscuit. j. agric. food chem. 48: 1322-1326. chevallier, s., della valle, g., colonna, p., broyart, b. and trystram, g. 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agric. 22: 176. yoshimoto, y., egashira, t., hanashiro, i., ohinata, h., takase, y. and takeda, y. 2004. molecular structure and some phycisochemical properties of buckwheat starches. cereal chem. 81 (4): 515-520. zheng, g.h., sosulski, f.w. and tyler, r.t. 1998. wet milling, composition and functional properties of starch and protein isolated from buckwheat groats. food res. int. 30: 493-502. zhou, y-g., li, d., wang, l-y., li, y., yang, b-n., bhandari, b., chen, x. d. and mao, z-h. 2009. effect of water on thermal behaviors of common buckwheat flour and starch. j. food eng. 93, 242-248. ijfs#147_wahyono_bozza   ital. j. food sci., vol 28, 2016 298 paper improving bread quality using co-cultures of saccharomyces cerevisiae, torulaspora delbrueckii jk08, and pichia anomala jk04 agung wahyono1, sae-byuk lee1, woo-won kang2 and heui-dong park*1 1school of food science and biotechnology, kyungpook national university, republic of korea 2department of food & food-service industry, kyungpook national university, republic of korea *corresponding author: hpark@knu.ac.kr   abstract co-cultured saccharomyces cerevisiae, torulaspora delbrueckii jk08, and pichia anomala jk04 were used as a leavening agent. the leavening ability, crumb structure, texture profile, crumb aroma, and sensory properties of bread were evaluated. the leavening ability of the co-cultures tested was lower than for s. cerevisiae alone. leavening containing a cocultures produced bread crust and crumb that were slightly yellow in colour and bright. generally, co-cultured bread produced a larger diversity and higher abundance of volatile organic compounds. superior colour properties, favorable aroma, and decent textural and structural features resulted in higher sensorial ratings for co-cultured bread. we suggest the use of co-culture as leavening agents for improved bread quality. keywords: bread quality, co-culture, pichia anomala, saccharomyces cerevisiae, torulaspora delbrueckii   ital. j. food sci., vol 28, 2016 299 1. introduction bread is a food that not only has a long history, but also a long future. over 12,000 years ago, the first bread was probably developed by deliberate experimentation using wheat flour and water. product development and process innovation in bread making is still an active field. consumer interest in bringing unique and alternative breads to the table is driving the production of a wide variety of breads (cauvain, 2012; mondal and datta, 2008). a multitude of studies conducted over the 20th century focused on improving bread quality and have explored the immense terrain of recipes (gardner et al., 2002; rosell et al., 2009; movahed et al., 2012; nilufer-erdil et al., 2012), process innovations (karaoğlu et al., 2006; bosmans et al., 2013) and the use of novel microorganisms as leavening agents (caballero et al., 1995; plessas et al., 2005; choi and choi, 2009; mo and sung, 2014). although the experience of bread quality is highly personal, it may be described as the sum of the sensory pleasures associated with flavour, taste, texture, and appearance (cauvain, 2012). the technological role of yeast in bread making has been well established. the main role of yeast is to produce gas by degrading the sugars available in the flour or that are added to the recipe. the gas produces air bubbles that are contained by the stretchy gluten proteins in the flour, which causes the dough to rise and produces an aerated structure in the resulting bakery product. during fermentation, the yeast produces abundant alcohols and other volatile compounds that impart unique tastes and flavours to the bread (stear, 1990; gélinas, 2009; ali et al., 2012; pacheco et al., 2012). over the last two decades, there has been great interest in using new strains of baker’s yeast that produce particular aromas, anti-molding properties, or desirable nutritional characteristics to fulfill the needs of the baking industry (gélinas, 2009). furthermore, the use of co-cultures as leavening agents has been reported to confer favorable effects in baking products. examples include the use of lactobacillus plantarum or pediococcus cerevisiae, combined with saccharomyces cerevisiae, as a co-culture for improving bread quality and delaying staling (elhariry et al., 2011); employing lactic acid bacteria and s. cerevisiae as a starter to enhance the nutritional content and shelf life of cassava-wheat bread (ogunbanwo et al., 2008); the use of lactobacillus delbrueckii ssp. bulgaricus or lactobacillus helveticus mixed with the lactose-fermenting yeast kluyveromyces marxianus to improve bread quality and increase shelf-life (plessas et al., 2008); and the use of starter culture combinations of lactobacillus fermentum, s. cerevisiae, and candida krusei to enhance aroma in ghanaian maize dough fermentation (annan et al., 2003). torulaspora delbrueckii and pichia anomala have unique traits that improve the quality of bakery products. as reported by hernandez-lopez et al. (2003), t. delbrueckii strains igc5321 and igc5323 exhibited higher leavening ability and co2 production than s. cerevisiae after exposure to hyperosmotic and freeze-thaw stress. mo and sung (2014) reported that p. anomala skm-t enhanced flavour properties and extended shelf life. the use of t. delbrueckii, p. anomala, and s. cerevisiae as co-cultured leavening agents has not yet been reported. here, we investigated the effects of co-cultured s. cerevisiae, t. delbrueckii jk08, and p. anomala jk04 as a leavening agent on bread quality. leavening ability, structural crumb features, texture profile, crumb aroma, and sensory properties of bread were evaluated.   ital. j. food sci., vol 28, 2016 300 2. materials and methods 2.1. microorganism strains and bread ingredients t. delbrueckii jk08 (td) and p. anomala jk04 (pa) were isolated from korean traditional starter (nuruk), and then identified and collected at the institute of fermentation biotechnology, kyungpook national university, daegu, south korea. the strains were sub-cultured in yeast extract-peptone-dextrose (ypd, difco, le pont de claix, france) agar (oxoid, hampshire, united kingdom) and stored at 2-4°c. s. cerevisiae (sc) was isolated from compressed instant baker’s yeast (saf-instant s.i., lesaffre, marcq, france). commercial wheat flour (beksul, cheiljedang, seoul, south korea) contained 13.93% protein and 0.47% ash, with a ph of 4.35. sugar and salt were purchased from a supermarket in sangju, south korea. 2.2. preparation of cultures preparation of cultures was described previously (wahyono et al., 2015). the strains were sub-cultured in 5% ypd broth and cultivated in a rotary shaker (jssi-300c, js research, gongju, south korea) at 30°c for 48 h, with shaking at 180 rpm. the yeast cells were collected by centrifugation (hanil supra 22k, hanil, incheon, south korea) at 4000 × g for 10 min at 4°c. the supernatant was discarded, and the pellet was re-suspended in distilled water, vortexed thoroughly, and stored at 4 °c. cell density was measured using a hemocytometer (neubauer chamber, celeromics, cambridge, united kingdom). to measure cell density, 10-1 to 10-4 dilutions of culture were prepared; 10 μl of diluted culture was loaded into the hemocytometer chamber. the chamber was observed at 100× magnification using a binocular microscope (cx31rtsf, olympus, tokyo, japan). cells in four different regions of the chamber were counted. cell density was calculated using the following formula: 𝑐 = ! !·!  ×  10  000 (1) where c is the concentration (expressed as number of cells per millilitre), n is the number of cells, n is number of squares counted, and d is the dilution factor. number 10 000 represents a constant converter to millilitre. 2.3. leavening ability the leavening ability of co-cultures was measured as described previously (wahyono et al., 2015). bread dough containing 20 g flour, 20 ml water, and 4 × 108 yeast cells per ml of water was mixed thoroughly in a 100-ml graduated cylinder and then incubated at 30°c for 210 min. this was done in triplicate and each sample was observed every 30 min. the maximum leavening rate (ml/h) was calculated from the highest volume reached in 210 min divided by the time the highest volume was first recorded. 2.4. bread-making procedure baking was carried out in a bread maker (national sd-bt102, panasonic, osaka, japan) using the 4-h standard bread-making setting. the basic recipe consisted of wheat flour, salt, sugar, and drinkable water. a single culture sc and co-cultures of s. cerevisiae + t.   ital. j. food sci., vol 28, 2016 301 delbrueckii jk08 (sctd), s. cerevisiae + p. anomala jk04 (scpa), and s. cerevisiae + t. delbrueckii jk08 + p. anomala jk04 (sctdpa) were used as leavening agents. the working ratio for co-cultures of s. cerevisiae + t. delbrueckii jk08 (sctd) and s. cerevisiae + p. anomala jk04 (scpa) were 50:50, respectively. the co-culture ratio for s. cerevisiae + t. delbrueckii + p. anomala jk04 (sctdpa) was 30:35:35. in the bread dough, the sc cell density was adjusted to approximately 5 × 108 cells per ml of water added to the dough. for td and pa, the cell densities were approximately 109 cells per ml of water added. dough compositions are given in table 1. the bread maker protocol included a first mixing for 20 min, resting for 25 min, and a second mixing for 10 min, followed by a first fermentation at 35°c for 50 min. a third mixing was performed for 3 min, followed by a second fermentation at 35°c for 40 min, and then a fourth mixing for 2 min. proofing was done at 40°c for 50 min and finally, loaves were baked at 210°c for 40 min. baking was performed in triplicate. after baking, the bread loaves were tempered at ambient temperature (28-30°c) before analysis. 2.5. moisture content, specific volume, and bread yield efficiency the moisture content of the bread crumb was determined by the oven-drying method (czuchajowska et al., 1989). specific volume was determined by the seed displacement method (aacc 10-05, 2000). bread yield efficiency was calculated as described by movahed et al. (2012) using the following formula: 𝑃! = !! !! ×  100 (2) where p2 is bread yield efficiency, w3 is bread weight and w2 is flour weight. 2.6. chromaticity of bread crust and crumb the crust and crumb colour were measured using the cr-400 chroma meter (konicaminolta, tokyo, japan); l (lightness), a (redness), and b (yellowness) values (hunter colour) were measured for six regions of bread crust and crumb. the whiteness index (wi) was calculated according to hsu (2003) and lin et al. (2009). 2.7. texture profile analysis texture profile analysis (tpa) was carried out in triplicate for two slices of bread. tpa was performed using a texture analyzer (ct3 4500, brookfield, middleboro, usa). bread samples were sliced to approximately 25-mm thickness. hardness, cohesiveness, springiness, and chewiness of the center of the bread slices were measured (blandino et al., 2013). the settings and conditions were carried out as described previously by ulziijargal et al. (2013), with some modifications: the acrylic cylindrical probe had a 38.1 mm diameter (ta4/1000), the pretest speed was 2 mm/s, the test speed was 2 mm/s, the post-test speed was 2 mm/s, the distance was 10 mm (40% compression), and the trigger load was 50 g. 2.8. bread crumb image analyses the structural features of the bread crumb were analyzed using imagej software (1.47v, national institutes of health, bethesda, md, usa). structural features included bread cell   ital. j. food sci., vol 28, 2016 302 density, mean cell area, and the fraction of cell area to total area. bread crumb images were captured using a scanner (epson perfection v370 photo, epson, japan) at a resolution of 800 × 800 dpi. images were calibrated to reflect actual size using a known scale, were cropped to 60 × 60 mm, filtered using a bandpass filter, and converted into binary images using the convert to mask feature for differentiating between the cell and non-cell area. before analyzing particles (cells), the particle size was set from 0.01 mm2 to infinity and the circularity was set from 0 to 1. this particle size corresponds to a particle diameter of 0.1 mm, which can be resolved by the human eye (pongjaruvat et al., 2014). 2.9. analysis of volatile compounds in bread crumbs volatile compounds were analyzed as described previously by plessas et al. (2008), using gas chromatography and mass spectrometry (7890a gc-ms; agilent, santa clara, ca, usa) with a flame ionization detector (fid). the separation was performed with a dbwax column (60 m × 250 μm × ɸ 0.25 mm) (waters, milford, ma, us). the detector was an agilent 5975c inert xl msd with triple-axis detector. helium was used as a carrier gas with a constant flow of 1 ml/min. using solid-phase microextraction technique (spme), 1 g of each bread sample was put into a 20-ml vial accessible to the spme needle through the vial septum. then, the vial was submerged in a water bath at 60°c and the spme fiber (50/30 μm dvb/car/pdms, supelco, bellefonte, pa, usa) was exposed to the headspace of the vial for 60 min. when the extraction process was finished, the spme fiber was inserted into the injector port (set at 280°c) of the gas chromatograph (gc) for thermal desorption of volatile compounds for 5 min in splitless mode. the gc temperature program was set as follows: 35°c for 5 min, increased by 5°c/min to 50°c (held for 5 min), increased by 5.5°c/min to 230°c (held for 5 min). volatile compound identification was based on comparison of gc retention times and peak areas with spectral data from the wiley9nist 0.8 library (wiley9nist 0.8 library, mass spectral search program, version 5.0, usa). 2.10. sensory evaluation bread samples were prepared from a freshly baked loaf (6-8 h after baking). the bread samples were cut about 50 × 20 × 25 mm and served on a small paper plate. sensory evaluations were conducted by 15 semi-trained consumers who were students and professors at kyungpook national university, south korea. the sensory attributes tested included appearance, colour, flavour, mouthfeel, and overall acceptability. the sensory attribute scale used for assessing the bread was as follows: 1, extremely dislike; 4, neither like nor dislike; and 7, extremely like (ulziijargal et al., 2013). 2.11. statistical analysis to examine statistical significance, the data were analyzed using one-way analysis of variance (anova) followed by duncan’s multiple range test at the p < 0.05 level of significance. the correlation among structural features and textural profiles of bread crumb was analyzed using a 2-tailed pearson correlation at p < 0.05. the analysis was carried out using spss for windows (ver. 19, ibm, new york, new york, usa). the graphs were constructed using microsoft excel (2007v, microsoft, redmond, washington, usa).   ital. j. food sci., vol 28, 2016 303 3. results and discussion 3.1. leavening ability the leavening ability of co-cultures was compared to that of single cultures (sc) in lean dough containing wheat flour and water (fig. 1). the leavening rates were significantly different among the cultures tested (p < 0.05). for sc, the dough was greatly leavened after only 30 min of incubation, but for co-cultures, the dough was greatly leavened after 60 min of incubation. the leavening rates for sc, sctd, scpa, and sctdpa were 55.4, 41.6, 36.5, and 31.6 ml/h, respectively. in a previous study, we found that the leavening rate for single cultures of t. delbrueckii jk08 and p. anomala jk04 were 8.67 and 2.29 ml/h, respectively. the lower performance of t. delbrueckii jk08 and p. anomala jk04 may be due to slower growth relative to s. cerevisiae (wahyono et al., 2015). bely et al. (2008) also reported lower performance for t. delbrueckii 27828 and t. delbrueckii 31703 in terms of fermentation rate in comparison to s. cerevisiae. on the contrary, hernandez-lopez et al. (2003) reported that t. delbrueckii strains igc5321 and igc5323 exhibited higher leavening ability and co2 production than s. cerevisiae after exposure to hyperosmotic and freeze-thaw stress. we demonstrated that incorporating td and pa with sc produced a co-culture with greatly improved leavening ability for a longer leavening period (> 120 min). elhariry et al. (2011) revealed that incorporating s. cerevisiae and l. plantarum in a sourdough system delivered favorable effects such as improving the leavening ability, as well as the sensory and physical properties of the bread. table 1: list of ingredients used in bread making. ingredients quantity yeast addition (total cells)a s. cerevisiae t. delbrueckiijk08 p. anomalajk04 wheat flour (g) 280 sugar (g) 16.8 salt (g) 5.6 water (ml) 200 leavening agent; sc 1 × 1011 sctd 5 × 1010 1 × 1011 scpa 5 × 1010 1 × 1011 sctdpa 3 × 1010 7 × 1010 7 × 1010 acalculated according to the yeast concentration and ratio determined in the bread-making procedures in the materials and methods.   ital. j. food sci., vol 28, 2016 304 figure 1: leavening rates for co-cultures or a single culture in lean dough. results are means±sd of triplicate. 3.2. physical properties the co-cultures did not affect the moisture content or bread yield, but significantly affected the bread-specific volume (p < 0.05, table 2). the specific volumes of breads produced with the cultures tested were in the range of 4.01-4.37 cm3/g. in comparison to breads leavened with co-cultures, the bread leavened with sc produced the greatest specific volume (4.37 cm3/g), which is consistent with the observation that sc exhibited the highest leavening activity (55.4 ml/h). we previously reported that lower leavening abilities for t. delbrueckii jk08 and p. anomala jk04 produced significantly lower specific volumes in comparison to s. cerevisiae (wahyono et al., 2015). these results strongly suggest a connection between the yeast’s leavening ability and the specific volume of the resulting bread. no significant differences were observed in bread-specific volume for the co-cultures tested. even though there were significant differences in leavening rates for the co-cultures tested, the time period for fermentation in the bread-making process was not long enough for optimum leavening activity for sctd, scpa, sctdpa (120, 150, and 180 min, respectively). however, the specific volume of breads produced by co-cultures is well within the range of standard bread, with specific volumes that ranged from 3.5 to 6 cm3/g (ulziijargal et al., 2013). the co-cultures significantly affected the chromaticity of the bread crust (table 3). sctd leavened bread showed the greatest l, b, and wi values. the l value for sctd was significantly greater than that for sc or scpa. the b value for sctd was only significantly greater than that for sc. the wi value for sctd was significantly higher than those for the other cultures tested. sctdpa produced the highest a value, which was significantly greater than that of sc or sctd. in contrast, sc-leavened bread produced the lowest l, a, b, and wi values. these results were consistent with previous work that demonstrated greater l and b values for bread crust when t. delbrueckii jk08 was used as a leavening agent (wahyono et al., 2015). for crumb colour, the co-cultures significantly affected the l and b values, but not the a and wi values (table 3). scpa produced the greatest l value, which was significantly greater than that of sc. it also produced the greatest b value, which was significantly higher than that of sc or sctd. the greatest l and b values of bread crumb arose when p. anomala jk04 was used as a leavening agent, while lower l and 0,0   10,0   20,0   30,0   40,0   50,0   60,0   70,0   80,0   90,0   100,0   0   30   60   90   120   150   180   210   v ol um e    ( m l)   time  (min)   sc   sctd   scpa   sctdpa     ital. j. food sci., vol 28, 2016 305 b values (darker colour) were produced when s. cerevisiae was used (wahyono et al., 2015). in short, td produced greater lightness and unsaturated yellowish colour in the bread crust, and pa produced similarly a coloured bread crumb. on the other hand, sc produced a darker and saturated yellowish colour in bread crust and crumb. hernandez-lopez et al. (2003) reported that commercial baker’s yeast (s. cerevisiae) exhibited greater maltase and invertase activity than t. delbrueckii igc5321. consequently, sc produced more reactive saccharides, which may contribute to darker colour formation. the saccharides and nitrogen-containing substances involved in the browning reaction create the dark-coloured pigment melanoidin, which confers a darker colour to the bread crust and crumb (stear, 1990). table 2: physical properties of bread leavened with co-cultures or a single culturea. yeast crumb moisture content (%) specific volume (cm3/g) bread yield efficiency (%) sc 46.33±0.50a 4.37±0.15a 147.97±0.75a sctd 46.58±0.59a 4.08±0.05b 148.86±1.00a scpa 46.83±0.35a 4.15±0.01b 147.85±0.21a sctdpa 46.61±0.16a 4.01±0.09b 148.13±0.75a ameans with the same superscript letter in a column are not significantly different at the level p < 0.05. table 3: chromaticity of bread crust and crumb leavened with co-cultures or a single culturea. yeast crust colour crumb colour l a b whiteness index (wi) l a b whiteness index (wi) sc 38.22±1.22c 6.26±0.72c 15.85±0.97b 35.90±0.88b 56.85±2.88b -2.52±0.11a 8.53±0.78c 55.93±2.76a sctd 41.63±0.93a 6.92±0.67bc 17.77±0.24a 38.59±1.00a 59.08±0.11ab -2.56±0.05a 9.25±0.38bc 57.97±0.03a scpa 39.39±0.34bc 7.79±0.26ab 17.00±0.28a 36.57±0.35b 60.22±0.75a -2.42±0.02a 10.34±0.13a 58.82±0.71a sctdpa 40.12±0.83ab 8.23±0.22a 17.66±0.46a 37.02±0.64b 58.37±1.55ab -2.43±0.10a 9.58±0.47ab 57.21±1.40a ameans with the same superscript letter in a column are not significantly different at the level p < 0.05. 3.3. structural features of bread crumb the structural parameters of bread crumb are expected to influence its mechanical behavior. by using image analysis, the structural features of bread crumb can be quantified (zghal et al., 2002). hence, we carried out an image analysis of bread crumb and the results are presented in table 4. the digital binary images of bread crumb from which the structural features could be extracted are shown in fig. 2. the use of co-cultures significantly affected the cell density and the mean cell area of bread crumb, but not the fraction of cell area to total area. the cell density of bread crumb leavened with sctd (58.89 1/cm) was the greatest among cultures tested, and was significantly greater than that leavened with sc (50.04 1/cm). inversely, the mean cell area of bread crumb leavened with sc (0.90 mm2) was the largest, significantly larger than that leavened with sctd (0.73 mm2). these results suggest that as mean cell area increases, cell density decreases. the   ital. j. food sci., vol 28, 2016 306 large mean cell area for sc-leavened bread is consistent with its high specific volume. in other words, bread leavened with sc was more porous than bread produced using cocultures, probably because of superior leavening ability and higher co2 production (wahyono et al., 2015). alternatively, pongjaruvat et al. (2014) reported that high specific volume is tightly correlated with cell density and cell area fraction. we performed correlation analysis for structural features and mechanical parameters (tpa) of bread crumb. we found that the cell density, mean cell area, and fraction of cell area to total area were correlated with cohesiveness, but not hardness, springiness, or chewiness (table 5). structural features were not strongly correlated with overall bread quality, which is more strongly affected by attributes such as odor and appearance (lampignano et al., 2013). figure 2: the digital binary images of breads crumb leavened with co-cultures or a single culture. table 4: structural features of bread crumb leavened with co-cultures or a single culture quantified by image processinga. yeast cell density (1/cm2) mean cell area (mm2) fraction of cell area to total area (%) sc 50.04±5.59b 0.90±0.10a 44.87±0.40a sctd 58.89±2.92a 0.73±0.03b 42.83±2.06a scpa 52.55±3.37ab 0.85±0.09ab 44.34±1.87a sctdpa 53.38±3.59ab 0.84±0.04ab 44.57±1.03a ameans with the same superscript letter in a column are not significantly different at the level p < 0.05.   ital. j. food sci., vol 28, 2016 307 table 5: pearson correlation of structural features and textural profiles of bread crumb. hardness springiness cohesiveness chewiness cell density mean cell area springiness 0.360 cohesiveness -0.485 -0.391 chewiness 0.981** 0.353 -0.319 cell density 0.450 0.365 -0.633* 0.380 mean cell area -0.435 -0.432 0.732** -0.340 -0.950** cell fraction -0.175 -0.326 0.658* -0.043 -0.273 0.541 **correlation is significant at the 0.01 level (2-tailed). *correlation is significant at the 0.05 level (2-tailed). 3.4. texture profile analyses tpa was used to evaluate the textural properties of bread crumb (fig. 3). the co-cultures altered hardness, chewiness and cohesiveness, but these changes were insignificant, except for cohesiveness. sctd-leavened bread was of lower cohesiveness than bread leavened with other cultures (fig. 3), consistent with our previous study, demonstrating that t. delbrueckii jk08 produces bread crumb with lower cohesiveness (wahyono et al., 2015). as stated earlier, the cohesiveness of bread crumb was the only parameter that correlated with its structural features. according to scanlon and zghal (2001), the crumb textural properties were largely determined by the bread crumb structural features. fine and uniformly-sized cells produce a softer texture. here we have demonstrated that a greater mean cell size conferred greater cohesiveness and vice versa. however, this result should be further evaluated in light of previous work that established that crumb cohesiveness is controlled by moisture content and the strength of networks surrounding the cell pore (cauvain, 2004). we have shown that the use of co-cultures produced bread of quality and textural properties comparable to bread leavened using a single culture. figure 3: texture profiles of bread leavened with co-cultures or a single culture. (a) hardness and chewiness of bread crumb. (b) springiness and cohesiveness of bread crumb. results are means ± sd of triplicate. 0   200   400   600   800   1000   1200   sc   sctd   scpa   sctdpa   h ar dn es s/ ch ew in es s   (g )   leavening  agent   hardness   chewiness   a 0,00   0,20   0,40   0,60   0,80   1,00   1,20   sc   sctd   scpa   sctdpa   sp ri n gi n es s/ co he si ve n es s   leavening  agent   springiness   cohesiveness   b *   ital. j. food sci., vol 28, 2016 308 (*) significantly different at the level p < 0.05. 3.5. volatile compounds of bread crumb a total of 54 volatile compounds were identified in the bread crumb leavened with sctdpa, whereas 50, 47, and 49 volatile compounds were identified in the bread crumb leavened with sc, sctd, and scpa, respectively (table 6). sctdpa-leavened bread not only produced more unique volatile compounds, but in greater abundance, as indicated by the greater peak area. in most cases, the bread leavened by co-cultures produced more volatile compounds than that of a single culture. the volatile compounds were predominately alcohols, aldehydes and esters. isobutyl alcohol (i-buoh), isoamyl alcohol (i-amoh), and phenethyl alcohol (pea) were the predominant alcohol groups. i-buoh levels were highest in sctdpa bread (20,169) and the lowest in sc bread (1,986). inversely, i-amoh levels were the highest in scpa bread (11,168) and lowest in sctdpa bread (6,265). as reported by kim et al. (2013), p. anomala y197-13 produced high i-amoh levels and conferred a banana flavour that significantly affected the flavour and taste of turbid rice wine. watanabe et al. (1990) reported that bread containing high levels of i-amoh was less favorable than bread containing high levels of i-buoh. the bread leavened with p. anomala skm-t exhibited a higher pea content and was preferred over s. cerevisiae (mo and sung, 2014) due to the favorable honey and flower odor of pea (jensen et al., 2011). the predominant aldehydes in bread crumb were n-hexanal, furfural, and benzaldehyde. the bread leavened with the co-cultures containing pa (scpa and sctdpa) produced the highest amounts of these compounds. n-hexanal was the most abundant aldehyde and conferred a green flavour. the second most abundant was benzaldehyde which produces an almond odor (birch et al., 2013a). fulfural is characterized by a burnt and sweet, caramel-like, odor (prost et al., 2012). the other volatile compounds that contributed to either favorable (n-octanal, n-decanal) or unfavorable (n-heptenal) odors were comparable among all cultures tested. esters typically have pleasant, fruity, or sweet odors (birch et al., 2013b). in most cases, sctdpa leavened bread contained a greater abundance and higher diversity of ester compounds. compounds including isoamyl acetate (fruity), ethyl caproate (fruity, wine, apple, banana, brandy), ethyl octanoate (fatty, fruity), ethyl decanoate, and ethyldodecanoate were enhanced in the bread leavened with co-cultures containing pa (scpa and sctdpa). sc and td enhanced particular compounds such as ethyl acetate and methyl salicylate, respectively. in alcoholic beverages, ethyl acetate can produce unfavorable sensory qualities. mixed cultures of s. cerevisiae and p. anomala (mutant type) produced higher ethyl acetate-hydrolyzing esterase activities. this enzyme is crucial in the formation of acetate ester, which delivers superior flavour (kurita, 2008). the bread leavened by co-cultures was obviously superior to that leavened by a single culture with regard to the volatile compound content. these compounds were produced mainly from the metabolism of yeasts during dough fermentation and flour lipid oxidation (birch et al., 2013b). these processes are influenced by the availability of free, reactive amino acids, sugars, alcohols, enzyme activity, and the degree of polymerization and hydration of substrates due to mixing to baking (stear, 1990). sadoudi et al. (2012) reported that the use of co-cultures altered the production of volatile compounds in wine, because co-culture interactions influenced the entire metabolic pathway.   ital. j. food sci., vol 28, 2016 309 table 6: volatile compounds contained in the bread crumb leavened with co-cultures or a single culture. no group rt compound flavour description peak area* sc sctd scpa sctdpa 1 acids 30.40 acetic acid acid, pungent (a) 1,973 1,838 2,483 4,043 2 33.28 isobutyric acid 339 275 287 408 3 35.61 2-methylbutanoic acid sweaty (e) 680 603 514 939 4 40.00 2-methylpropanoic acid sweat, butter(a) 81 59 83 176 5 alcohols 18.63 isobutyl alcohol 1,986 7,494 13,946 20,169 6 23.30 isoamyl alcohol banana (f) 8,011 8,174 11,168 6,265 7 26.30 2-ethyl-1-decanol nd nd nd 301 8 27.69 2-methyl-3-pentanol 146 180 67 88 9 27.90 1-hexanol flower (a) 465 610 527 674 10 28.14 2-nonanol 153 159 nd 87 11 28.63 3-ethoxy-1-propanol fruity (a) 87 236 nd 116 12 32.76 1-dodecanol 228 163 280 684 13 35.42 2-furanmethanol 309 552 615 1,857 14 40.86 phenethyl alcohol honey, flower (a) 9,506 12,292 8,391 12,643 15 aldehydes 18.01 n-hexanal green (d) 5,369 19,874 37,144 71,305 16 22.38 n-heptanal fatty, rancid (d) 233 262 528 302 17 25.99 n-octanal citrus (d) 207 263 333 495 18 27.11 2-heptenal 600 1,086 1,594 2,335 19 30.88 furfural 1,646 2,068 2,764 7,529 20 31.79 n-decanal citrus (d) 151 230 237 379 21 32.56 benzaldehyde almond (d) 3,507 5,475 6,389 8,374 22 33.64 5-methyl-2-furfural 132 47 131 529 23 38.80 2,4-decadienal fatty, waxy (b) 38 94 103 224 24 alkenes 8.96 2,4-dimethyl-1-heptene 62 nd nd 396 25 29.86 3-ethyl-2-methyl-1,3-hexadiene 36 58 83 120 26 benzenes 15.92 methyl benzene 269 330 551 413 27 24.95 ethenylbenzene 762 134 8,026 6,265 28 29.97 1,3-bis(1,1-dimethylethyl)benzene 381 563 85 1,503 29 esters 9.10 ethyl acetate pineapple (a) 1,688 120 635 5,590 30 14.66 isobutyl acetate ethereal, fermented nd nd nd 134   ital. j. food sci., vol 28, 2016 310 odor (b) 31 19.87 isoamyl acetate fruity (e) 305 666 5,290 7,923 32 24.16 ethyl caproate fruity, wine, apple, banana, brandy (c) 305 51 4,641 3,903 33 25.43 n-hexyl acetate 37 34 124 256 34 30.10 ethyl octanoate fatty, fruity (a) 625 796 4,646 12,307 35 30.75 amyl caproate 7 nd 84 351 36 34.96 ethyl decanoate 204 171 4,066 9,553 37 36.12 ethyl-9-decenoate nd nd 104 1,658 38 38.27 methyl salicylate 220 1,686 316 1,876 39 38.91 2-phenethyl acetate roasty(e) 235 101 209 868 40 39.22 ethyldodecanoate 336 281 1,069 1,407 41 furans 24.06 2-pentylfuran floral, fruity (d) 309 246 887 566 42 31.99 2-acetylfuran 282 339 450 1,243 43 ketones 12.73 2,3-butanedione buttery, caramel (d) 353 592 460 542 44 22.29 2-heptanone nd nd 58 67 45 25.89 3-hydroxy-2-butanone butterscotch (d) 867 744 922 1,149 46 26.37 1-octen-3-one mushroom (g) 48 263 419 765 47 phenols 40.61 butylatedhydroxytoluene 1,585 2,788 4,507 2,683 48 pyrazines 25.33 methylpyrazine 185 179 228 546 49 27.33 2,6-dimethylpyrazine hazelnut (e) 68 73 219 314 50 27.49 ethylpyrazine 58 95 95 223 51 terpenes 37.04 (-)-.beta.-bisabolene 220 nd 341 222 52 others 28.90 dimethyl trisulfite 51 120 217 263 53 37.64 naphthalene 79 143 446 288 54 41.42 1,4-methanobenzocyclodecene 75 139 265 275 *the values of volatile compounds calculated from the peak area divided by 1000. nd, not detected a. jensen et al. (2011); b. mo and sung (2014); c. daigle et al. (1999); d. birch et al. (2013a); e. prost et al. (2012); f. kim et al. (2013); g. birch et al. (2013b) 3.6. sensory properties the average results of the sensory evaluation of appearance, colour, flavour, mouthfeel, and overall acceptability are shown in fig. 4. in most cases, the co-cultures slightly enhanced all the sensory attributes, except for appearance. all attributes produced satisfactory scores in the range of 4.73–5.57 out of a total 7 points. on average, the co   ital. j. food sci., vol 28, 2016 311 cultures produced marked improvement over the single culture, which scored in the range of 4.07-5.71. sctdpa leavened bread was superior in overall acceptability (5.57), which is attributable to higher ratings in flavour (5.27) and mouthfeel (5.30). high flavour ratings are probably due to the high abundance of favorable volatile compounds (table 6). sctd leavened bread had a superior colour rating (5.53). these results demonstrate that incorporating sc, td, and pa as leavening agents conferred beneficial characteristics to bread. sc contributed to improving bread appearance through greater leavening ability, and td and pa contributed to enhanced flavour, colour, and mouthfeel. figure 4. radar plot of the sensory properties of bread leavened with co-cultures or a single culture. result reflects the means of scores from 15 semi-trained panelists. 4. conclusions we have shown that the use of mixed cultures of s. cerevisiae, t. delbrueckii jk08, and p. anomala jk04 enhanced bread quality. the bread leavened by the co-cultures produced textural and structural properties comparable to single cultures of s. cerevisiae. the cocultured bread had a superior aroma and enhanced sensorial qualities. thus, the use of cocultures as leavening agents has great promise in fulfilling the consumer need for unique and high-quality bread. acknowledgements we thank the directorate general of human resources for science, technology and higher education, the republic of indonesia, and the department of food and food service industry at kyungpook national university, republic of korea, for supporting this study. the authors wish to thank dr. d. silveri, dr. v. galli and mr. l. bartoli for the technical assistance and dr. francesca melini for the technical and linguistic assistance in the revision of this paper. references aacc 10-05. 2000. rapeseed displacement method. in: approved methods of the aacc. 10th ed. st. paul :american association of cereal chemists. 0,00   1,00   2,00   3,00   4,00   5,00   6,00   7,00   appearance   color   flavor  mouthfeel   overal   sc   sctd   scpa   sctdpa     ital. j. food sci., vol 28, 2016 312 ali a., shehzad a., khan m.r., shabbir m.a. and amjid m.r. 2012. yeast, its types and role in fermentation during bread making process-a review. pak. j. food sci. 22: 171-179. <http://psfst.com/__jpd_fstr/63f9e07b9aa310709ce11c70ac9bf266.pdf> annan n.t., poll l., dedeh s.s., plahar w.a. and jakobsen m. 2003. influence of starter culture combinations of lactobacillus fermentum, saccharomyces cerevisiae and candida krusei 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marshall*3 1department of nutrition, university of the incarnate world, san antonio, texas 78209, usa 2department of medical biochemistry, i. horbachevsky ternopil state medical university, ternopil 46001, ukraine 3eurofins microbiology laboratories, inc., 2000 mackenzie court, fort collins, colorado 80528, usa *corresponding author: douglasmarshall@eurofinsus.com abstract this study evaluated commercially available dip solutions of nisin alone, buffered sodium citrate combined with sodium diacetate (bscsd), and combined solution of the three antimicrobials for potential to control growth of surface-inoculated listeria monocytogenes during vacuum packaged refrigerated storage of model beef frankfurters. none of the treatments prevented growth of l. monocytogenes during frankfurters storage. the combined treatments slowed growth of l. monocytogenes better than individual treatments. failure to completely eliminate l. monocytogenes on frankfurters or inhibit outgrowth during storage might be attributed to high initial l. monocytogenes inoculum levels, insufficient quantities of antimicrobials to interact with all the target cells, low nisin activity at high ph, or presence of resistant subpopulations, such as nisin-resistant strains. the model conditions used in the experimental setup, such as elimination of natural microbiota from frankfurters and nutrient-rich diluent used for listeria introduction on the surface, could also contributed to enhanced survival and growth of the pathogen. keywords: ready-to-eat meats, listeria monocytogenes, nisin, sodium citrate, sodium diacetate, refrigerated storage ital. j. food sci., vol. 32, 2020 353 1. introduction recontamination of cooked ready-to-eat (rte) meat products with l. monocytogenes is a major food safety concern. in the u.s.a., l. monocytogenes caused an estimated 2,500 cases of foodborne illnesses with 20% mortality annually (mead et al., 1999). a study performed over a decade later (scallan et al., 2011) also listed l. monocytogenes as the third leading cause of death due to foodborne illnesses at 19% mortality rate, only behind nontyphoidal salmonella spp. and toxoplasma gondii. in addition, a significant annual monetary loss is incurred by the food industry (usda-ers, 2014). u.s.-based food industries were forced to recall food products due to possible l. monocytogenes contamination on seven different occasions in december, 2018 alone (us fda, 2018). the national health plan of reducing listeriosis cases to 0.25 person per 100,000 population has not yet been achieved since 2005 (buckner, 2008). l. monocytogenes can survive under normally limiting and extreme physicochemical conditions (ryser and donnelly, 2001). moreover, l. monocytogenes is ubiquitous in nature. though l. monocytogenes is susceptible to cooking, i.e. temperatures above 70°c (lunden et al., 2003), postprocessing recontamination during cooked frankfurter cooling, case splitting, and packaging is difficult to avoid. at present, rte meat facilities inspected by the u.s. food safety and inspection service (fsis) operate under a ‘zero tolerance’ policy, which means any food contaminated with detectable levels of l. monocytogenes is deemed adulterated (usda-fsis, 2014). therefore, hurdles are required to prevent initial contamination and inhibit growth of this pathogen during storage. one such hurdle is to use post-cooking application of antimicrobials to prevent growth of l. monocytogenes, which is accepted as one of three alternatives to control l. monocytogenes (u.s. code of federal regulations, title 9, part 430, section 430.4) there are many potential methods to prevent spoilage and pathogenic bacteria from growth on rte meats (tokarskyy and marshall, 2010). for example, salts of organic acids (lactates, acetates, diacetates, sorbates, and benzoates) have been studied as antilisterial agents on meat products (barmpalia et al., 2005; samelis et al., 2005; sivarooban et al., 2007; stopforth et al., 2005; zhu et al., 2005). activity of these compounds depends on agent concentration, product composition (ph, water activity, fat, nitrite, and salt content), storage temperature and packaging atmosphere (cleveland et al., 2001; geornaras et al., 2006a; lunden et al., 2003; martinis et al., 1997; nilsson et al., 1997). the contamination level of the food product is another factor that influences the activity of these antimicrobials (bedie et al., 2001; wederquist and sofos, 1994). nisin is a generally recognized as safe (federal register, 1988) bacteriocin that is produced by lactococcus lactis subsp. lactis (naidu, 2000). nisin has greatest antimicrobial activity in the ph range of 3.0-3.5 (fang and lin, 1994) with rapid activity loss at greater ph values (montville and chen, 1998). therefore, it is recommended to use nisin at a ph not greater than 5.5, especially in the presence of sodium chloride and nitrite in meat products (martinis et al., 1997; ukuku and shelef, 1997). studies have shown that nisin is effective in reducing l. monocytogenes counts on rte meat products (delves-broughton, 2005; fang and lin, 1994; tokarskyy and marshall, 2008; ukuku and shelef, 1997). however, the use of nisin has been limited in food products due to the potential for emergence of nisin-resistant strains of l. monocytogenes (crandall and montville, 1998; liu et al., 2002). to overcome this problem, nisin has been combined with other treatments to achieve antilisterial effects (delves ital. j. food sci., vol. 32, 2020 354 broughton, 2005; geornaras et al., 2006a; samelis et al., 2005; tokarskyy and marshall, 2008; zhu et al., 2005). buffered sodium citrate combined with sodium diacetate (bscsd) is a mixture of citric acid, sodium citrate, and sodium diacetate (hull, 2007). buffered sodium citrate (bsc) is approved in the us for cured (9 cfr 318.7 [c] (4)) and uncured (9 cfr 381.7 [f] (4)) processed whole-muscle meat and poultry products (usda-fsis, 1996). bsc is used to enhance flavor in meat and poultry products and the recommended usage level is 1.0 to 1.3% by weight of total formulation (usda-fsis, 1996). in muscle-food products, bsc increases ionic strength, which in turn increases water holding capacity, lowers water activity, and causes less purge loss (ceylan et al., 2003; hull, 2007). sodium diacetate is primarily used as flavor enhancer in meat products at no more than 0.25% of product formulation (usda-fsis, 2000). sodium diacetate has been reported to have antilisterial properties in combination with lactates or nisin in meat and poultry products (samelis et al., 2002; samelis et al., 2005). bscsd has been shown to be effective in controlling germination and outgrowth of clostridium perfringens during cooling of cooked meat and poultry products (thippareddi et al., 2003) and a single study showed possibility for inhibiting l. monocytogenes on beef frankfurters (ceylan et al., 2003). these antimicrobials have been used as individual solutions (patel et al., 2006; samelis et al., 2005) or as separate solutions applied sequentially to food surfaces (geornaras et al., 2006a; geornaras et al., 2006b). most previous studies have used salts of organic acids and nisin as additives in the meat formulation, while relatively fewer studies have evaluated their efficacy as dipping solutions post-processing. little is known about the potential of these agents as processing aids applied as surface treatments rather than as ingredients. therefore, the present study was designed to investigate dip solutions of nisin alone, bscsd alone or the two in various combined treatment sequences on ability to control l. monocytogenes attached on rte beef frankfurters during storage at 4 or 10°c. 2. materials and methods 2.1. preparation of inoculum a cocktail for inoculation was prepared using five strains of l. monocytogenes. strains were acquired from american type culture collection (atcc, manassas, va, usa) and included atcc 15313 (rabbit isolate, england), atcc 51414 (raw milk associated with listeriosis outbreak, massachusetts, usa), atcc 43256 (mexican-style cheese isolate, california, usa) atcc 19115 (human isolate), and atcc 7644 (human isolate). strains were maintained on trypticase soy agar slants with 0.6 % yeast extract (tsaye; bd diagnostic systems, sparks, md) at 4°c with monthly transfers to fresh tsaye slants. to prepare study inocula, each strain was streaked separately onto tsaye plates and incubated 35°c for 24 h. a single colony of each strain from a tsaye plate was transferred into separate 30 ml portions of trypticase soy broth with 0.6% yeast extract (tsbye) and incubated at 35°c for 24 h to obtain stationary phase cultures (approximately 9 log10 cfu/ml). a multistrain cocktail was prepared by combining 2-ml portions of each turbid broth in a sterile test tube and vortexed for 10 sec. a 2-ml portion of this mixed-strain cocktail was then dispersed into 2 l of 0.1% peptone water (bd diagnostic systems) to achieve a final l. monocytogenes inoculum solution containing approximately 6 log10 cfu/ml. ital. j. food sci., vol. 32, 2020 355 2.2. frankfurter inoculation the experimental setup was rather a model than real-life in order to simulate the worst possible conditions for listeria survival and growth, such as destruction of frankfurters natural microbiota to avoid competition, use of stationary culture of pathogen in nutrientrich medium, modifying of frankfurters texture by autoclaving, overnight adaptation and attachment of listeria to the meat surface before application of antimicrobials. briefly, lowfat frankfurters without added antimicrobial agents were purchased from a commercial distributor and transported in a cooler on ice packs directly to the laboratory. the proximate composition of the frankfurters remained unknown. the frankfurters were immediately frozen and stored at -20oc until use but no longer than for a month. for each replicate, 44 frankfurters (11 treatments x 4 sampling days) for 4°c storage and 66 frankfurters (11 treatments x 6 sampling days) for 10°c storage were thawed at 4°c overnight. after thawing, the frankfurters were aseptically cut in half perpendicular to the longitudinal axis (calculated: 2.5 cm diameter; 6 cm length; 57 cm2 surface area; 22 ± 2 g). each replicate portion of halved frankfurters were vacuum packaged (multivac a300/16; kansas city, mo) in vacuum bags (vacuum pouches; prime source, kansas city, mo) at 999 mbar vacuum for 1 sec with a 2.5 sec seal. the vacuum-packaged frankfurters were autoclaved for 15 min at 121°c to destroy indigenous microorganisms (dorsa et al. 1993), and cooled for approximately 2 h at 25 ± 2°c in a laminar flow hood before inoculation with l. monocytogenes. a solution of sterile 0.1 % peptone water was used as a negative control. up to 20 halved frankfurters were placed into 4 l sterile steamer strainers (progressive international, kent, wa). each batch of 20 was dipped in containers with either 2 l of negative control buffer or 2 l of the five-strain inoculum of l. monocytogenes for 5 min with constant agitation. after dipping, the strainers were removed and drained for 10 min into empty sterile containers. the inoculated frankfurters were aseptically transferred to plastic storage bags with a zip closure (ziploc, s. c. johnson and son inc., racine, wi) and stored over night at 4°c to allow for low temperature adaptation and maximum adherence of l. monocytogenes to product surfaces. this practice is similar to commercial frankfurter manufacturing where cooked links can be hydrocooled after cooking or rack cooled overnight in a low temperature cooler. 2.3. antimicrobial treatments commercially-available antimicrobials were secured from the u.s. food ingredients suppliers and prepared according to manufacturer’s instructions but using sterilized distilled water for antimicrobial dissolution “as is”. nisin (1,000 iu/mg, profood international, inc., chicago, il, usa) and buffered sodium citrate combined with sodium diacetate (bscsd, world technology ingredients, inc., jefferson, ga, usa) were used to prepare eleven treatment dipping solutions. the exact composition of bscsd was not declared by the supplier, but according to hull (2007), it may have contained 65 to 95% sodium citrate and 5 to 35% of sodium diacetate. the inoculated frankfurter samples were dipped into 2-l portions of the following individual agent treatment solutions: 1) 0.1% sterile peptone water (no antimicrobials, l. monocytogenes positive control); 2) 2000 iu/ml nisin; 3) 4000 iu/ml nisin; 4) 6000 iu/ml nisin; 5) 2.5% (w/v) bscsd; 6) 3.0% (w/v) bscsd; 7) 3.5% (w/v) bscsd. in addition, two sequential treatment solution treatments were conducted; 8) 6000 iu/ml nisin followed by 3.5% (w/v) bscsd (nisin-bscsd) and 9) 3.5 % (w/v) bscsd followed by 6000 iu/ml nisin (bscsd-nisin). finally, a combined ital. j. food sci., vol. 32, 2020 356 solution treatment was used: 10) 6000 iu/ml nisin and 3.5% (w/v) bscsd; along with a negative control: 11) non-inoculated samples dipped in 0.1% peptone water. dipping was conducted by using sterile steamer strainers as used earlier for frankfurter inoculation. for a single treatment, not more than 20 frankfurter samples were dipped into 2.0 l of treatment solution for 5 min and drained for 10 min before vacuum packaging. for sequential treatments, samples were dipped into the first solution for 5 min, drained for 10 min, and then dipped into another bucket containing the second solution for 5 min followed by draining for 10 min. treated half-frankfurter samples were aseptically individually placed into separate vacuum bags (prime source, 15.24 cm x 21.59 cm) and vacuum packaged (multivac a300 /16). the packages from each treatment were randomly selected into two batches, which were stored at 4 or 10°c for 42 and 20 days, respectively. 2.4. bacterial enumeration frankfurters stored at 4°c were analyzed on days 0, 14, 28 and 42, and those stored at 10°c were analyzed on days 0, 4, 8, 12, 16 and 20. twelve hours post-treatment was designated as day 0. on each sampling day, two packages from each treatment were randomly selected and analyzed for microbial counts. twenty-three milliliters of 0.1% peptone water was added to each frankfurter sample (1:1 ratio) in a stomacher filter bag (thermo-fisher scientific, fairlawn, nj). the samples were homogenized for 2 min using a stomacher (stomacher 400 lab blender, seward medical, london, uk) and the homogenate was serially diluted with 0.1% peptone water. an aliquot of 0.1 ml was taken from each dilution, and spread plated on duplicate modified oxford agar (bd diagnostic systems) plates followed by incubation at 35°c for 48 h. colonies that appeared 2 to 3 mm in diameter and grayish black with a halo were enumerated as l. monocytogenes. 2.5. ph measurements the surface ph of treated frankfurters was determined using corning pinnacle 530 ph meter (corning, ny). half-cut frankfurter samples were dipped into treatment solutions for 5 min and then drained for 10 min before ph measurement. the ph values of the antimicrobial solutions were also measured prior to application. 2.6. statistical analysis three replicate experiments were performed for each storage temperature. at each sampling point, duplicate samples from each treatment were removed from storage. plate counts were converted to log10 cfu/g. least-square means of bacterial counts for each treatment were estimated and significance of differences were determined using analysis of variance using general linear model of statistical analysis system 9.1 (sas 2002). all differences were reported at a significance level of p ≤ 0.05. in addition, the logarithm of the l. monocytogenes counts from each of the two storage temperatures were modeled as a function of time using the baranyi model (baranyi and roberts, 1994). for curve fitting, the program dmfit (provided by dr. j. baranyi, ifr (institute of food research, reading, uk) was used (baranyi, 2005). four baranyi model parameters were measured: 1) lag phase; 2) µmax, which expresses the maximum specific growth rate (per day); 3) y0, which represents the lower asymptote, corresponding to the initial bacterial counts (log10 cfu/g); and 4) yend, represents the upper asymptote, ital. j. food sci., vol. 32, 2020 357 corresponding to the maximum bacterial counts (log10 cfu/g) when the growth curve forms an upper plateau at the stationary phase of growth. the lag phase is formally separated from the exponential and the stationary phase, which can be regarded as part of the potential growth curve defined by the model. the main difference between this model and other sigmoid curves is the mid-phase curve is similar to a linear curve, unlike other classical sigmoid curves that have a pronounced curvature. 3. results and discussion 3.1. ph of frankfurters dipped in antimicrobial solutions table 1 shows treatment solution and frankfurter surface ph values. bscsd solutions had ph values above 5.6, which is not considered inhibitory to growth of l. monocytogenes (usda-ars, 2019, pathogen modeling program online). nisin solutions had growth inhibitory ph values below 4.0 (usda-ars, pathogen modeling program online). despite the low ph of the nisin solutions, no significant (p>0.05) change in the surface ph of frankfurters was observed after dipping in any of the treatment solutions (table 1). table 1. ph values of antimicrobial solutions and treated frankfurters. ph mean ±sd a treatment treatment solution surface ph of treated frankfurters positive control 6.3±0.07 6.0±0.12 ab nisin (2000 iu/ml) 3.9±0.02 5.8±0.03 b nisin (4000 iu/ml) 3.5±0.42 5.8±0.11 b nisin (6000 iu/ml) 3.7±0.07 5.8±0.03 b bscsd (2.5%) 5.8±0.03 5.8±0.05 b bscsd (3.0%) 5.8±0.01 5.8±0.10 b bscsd (3.5%) 5.6±0.19 6.1±0.06 a nisin-bscsd 3.9±0.10 5.6±0.03 5.8±0.02 b bscsd-nisin 5.8± 0.02 3.7±0.11 5.7±0.12 b combined 5.6±0.11 5.8±0.08 b aall means are duplicate measurements from three different experiments. means within a column followed by the same letter(s) are not significantly different (p>0.05). similar ph results were seen by patel et al. (2006) after dipping turkey frankfurters in solutions of nisin, sodium lactate, or sodium diacetate, either alone or in combination. bedie et al. (2001) also did not observe significant changes in the ph of frankfurters when sodium acetate, sodium diacetate, or sodium lactate were added as ingredients. these and other studies (samelis et al., 2005; schlyter et al., 1993; shelef and addala, 1994) reveal that ph reduction is not a major contributor to the antilisterial property of these antimicrobials. instead, activity within the meat system accounts for antilisterial activity (doores et al., 2005; tokarskyy and marshall, 2008). ital. j. food sci., vol. 32, 2020 358 3.2. effects of nisin, bscsd, and combined solutions tables 2 and 3 show effects of 2000, 4000, or 6000 iu nisin/ml, 2.5, 3.0, or 3.5% bscsd, sequential nisin-bscsd treatment, sequential bscsd-nisin treatment, and a combined nisin-bscsd treatment solution on surface inoculated frankfurters stored at 4 and 10°c. table 2. effect of nisin, bscsd, and combined solutions on growth of l. monocytogenes on beef frankfurters stored at 4°c*. treatment l. monocytogenes population (mean log10 cfu/g ± sd) time (day) 0 14 28 42 positive control 4.4±0.4 a 8.0±0.1 a 8.5±0.2 a 9.0±0.2 a nisin (2000 iu) 2.2±1.1 b 7.0±0.1 bc 8.2±0.3 a 9.0±0.1 a nisin (4000 iu) 2.1±0.8 b 7.2±0.3 bc 8.1±0.1ab 9.0±0.2 a nisin (6000 iu) 2.3±0.2 b 7.0±0.1 bc 8.2±0.2 a 9.0±0.2 a bscsd (2.5%) 4.0±0.5 ab 7.3±0.1 bc 8.2±0.2 a 8.9±0.3ab bscsd (3.0%) 3.2±1.1 ab 7.3±0.0 b 8.4±0.3 a 8.8±0.2ab bscsd (3.5%) 3.3±0.8 ab 7.1±0.1 bc 8.3±0.3 a 8.7±0.0 b nisin bscsd 3.1±0.8 ab 6.9±0.3 c 7.5±0.4 bc 8.7±0.1 b bscsd nisin 2.7±1.2 ab 7.3±0.2 bc 7.5±0.4 bc 9.0±0.1 a combined 2.7±1.3 ab 6.3±0.2 d 7.4±0.3 c 8.9±0.3 ab *means within a column followed by the same letter(s) are not significantly different (p>0.05). minimum detection limit was 2.0 log10 cfu/g. table 3. effects of nisin, bscsd, and combined solutions on the growth of l. monocytogenes on beef frankfurters stored at 10°c*. treatment l. monocytogenes population (mean log10 cfu/g ± sd) time (day) 0 4 8 12 16 20 positive control 4.7±0.5 a 7.7±0.5 a 8.6±0.1 abc 8.7±0.1 ab 8.8±0.1 a 9.0±0.1 abc nisin (2000 iu/ml) 2.9±0.3 bc 5.6±0.1 d 7.9±0.5 abcd 8.3±0.2 bc 8.4±0.2 b 8.6±0.1 d nisin (4000 iu/ml) 2.8±0.2 c 6.5±0.2 bcd 7.9±0.4 abcd 8.3±0.3 bc 8.5±0.3 ab 8.7±0.1 cd nisin (6000 iu/ml) 2.2±1.0 c 6.1±0.7 d 8.0±0.1 abcd 8.0±0.1 cd 7.9±0.1 c 8.5±0.3 d bscsd (2.5%) 4.2±0.9 ab 7.5±0.7 ab 8.9±0.2 a 9.0±0.2 a 8.6±0.1 ab 9.1±0.2 ab bscsd (3.0%) 4.5±0.5 a 7.4±0.6 abc 8.5±0.8 abc 9.0±0.2 a 8.6±0.1 ab 9.3±0.2 a bscsd (3.5%) 4.5±0.4 a 7.4±0.5 abc 8.8±0.6 ab 8.8±0.3 a 8.6±0.1 ab 9.0±0.2 abc nisin bscsd 3.0±0.5 bc 6.3±0.4 cd 7.7±0.3 cd 7.9±0.3 cd 8.4±0.1 b 8.8±0.1 bcd bscsd -nisin 2.7±0.5 c 6.3±0.2 cd 7.9±0.3 bcd 8.0±0.1 cd 8.6±0.2 ab 8.9±0.1 bc combined 2.7±0.6 c 6.1±0.3 d 7.5±0.1 d 7.7±0.1 d 8.3±0.2 b 8.5±0.1 d *means within a column followed by the same letter(s) are not significantly different (p>0.05). minimum detection limit was 2.0 log10 cfu/g. ital. j. food sci., vol. 32, 2020 359 treatment and sampling of negative control beef frankfurters followed the same procedures as inoculated samples and showed no l. monocytogenes contamination (results not shown). positive-control untreated samples supported l. monocytogenes growth to populations at or exceeding 8 log10 cfu/g by 14 days of storage at 4°c (table 2) and 8 days at 10°c (table 3). treatment with nisin alone initially reduced (p<0.05) l. monocytogenes counts by approximately 2 logs at both storage temperatures; however, there was no observed (p>0.05) dose-response relationship. although treatment with bscsd alone appeared to reduce counts initially at both temperatures, these reductions were not significant (p>0.05). sequential nisin-bscsd and bscsd-nisin treatments and combined nisin-bscsd treatment initially reduced (p<0.05) counts by approximately 2 logs at 10°c (table 3) but not (p>0.05) at 4°c (table 2). this is suggestive that warmer temperature application may increase antimicrobial activity of the combined treatments. despite some initial count reductions, none of the treatments were able to prevent growth of l. monocytogenes during frankfurter storage at 4 or 10°c under proposed model conditions (tables 2 and 3). by 14 days at 4°c, all treatments were able to keep l. monocytogenes counts significantly lower (p<0.05) than the untreated control, but the count reduction was small (around 1 log) (table 2). the combined nisin-bscsd treatment achieved a larger 1.7 log reduction (p<0.05) at 14 days of storage at 4°c. sequential and combined nisin-bscsd treatments were able to keep counts around 1 log lower (p<0.05) than the untreated control for up to 28 days at 4°c. no meaningful count differences were observed among the treatments on the last day of testing, 42 days at 4°c. similar trends were observed when frankfurters were stored at 10°c, with no meaningful treatment differences observed by 16 days of storage. based on previous reports we expected that sequential or combined nisin-bscsd treatments would be more effective than treatment with either antimicrobial preparation alone (ceylan et al., 2003; cleveland et al., 2001; delves-broughton, 2005; juneja and thippareddi, 2004; samelis et al., 2001; samelis et al., 2005; thippareddi et al., 2003; zhu et al., 2005). the combined application of antimicrobials (either sequential or as a single solution) did not influence l. monocytogenes counts, except day 14 at 4°c, where nisin-bscsd yielded significantly lower counts. perhaps application of low-ph nisin solution before bscsd application influenced activity at this time point. it is noteworthy that surviving l. monocytogenes populations after antimicrobial treatments were able to initiate growth under model conditions. baranyi model growth curves at 4 and 10°c are shown in figures 1 and 2, respectively. growth rate kinetics of the bacterium at the two storage temperatures are shown in tables 4 and 5. there were no lag-phase growth differences observed with any treatment at 4 or 10°c based on the baranyi model (results not shown). in pork bologna formulated with 1.8% sodium lactate and 0.125% sodium diacetate, barmpalia et al. (2005) observed a l. monocytogenes lag phase of 13.78 days and 5.02 days at 4 and 10°c, respectively, using the same baranyi model. geornaras et al. (2006b) found a 10.2 day lag phase at 10°c for commercial cooked sausages treated with 1.5% potassium lactate plus 0.05% sodium diacetate and no lag phase for untreated sausages. result differences in lag phase observations may be due to variations in experimental protocol including number of data points taken during the lag phase, different starting inocula populations, type of product, presence or absence of indigenous bacteria that could be inhibitory to l. monocytogenes, or differences in absorption rates of antimicrobial solutions due to specific surface characteristics (barmpalia et al., 2004; geornaras et al., 2006a; geornaras et al., 2006b; samelis et al., 2001, 2002, 2005). ital. j. food sci., vol. 32, 2020 360 figure 1. growth of l. monocytogenes on vacuum packaged beef frankfurters dipped in solutions of 6000 iu/ml nisin, 3.5% bscsd, and combined solution, stored at 4°c. lm control, l. monocytogenes inoculated on beef frankfurters dipped in 0.1% peptone water with no antimicrobials. data points are not actual experimental values but aids for better visualization of baranyi graphs. table 4. growth kinetics of l. monocytogenes inoculated on the surface of beef frankfurters treated with nisin alone, bscsd alone, nisin-bscsd in sequence, bscsd-nisin in sequence, both combined, then vacuum packaged and stored at 4°c for 42 days. treatment maximum specific growth rate (µmax; per day ±standard error) y0 a (log10 cfu/g) yend b(log10 cfu/g) r 2 lm control 0.26±0.030 4.4 8.7 0.98 nisin (2000 iu/ml) 0.35±0.045 2.2 8.6 0.93 nisin (4000 iu/ml) 0.37±0.046 2.1 8.5 0.93 nisin (6000 iu/ml) 0.34±0.041 2.2 8.6 0.94 scsd (2.5%) 0.23±0.025 4.0 8.5 0.96 scsd (3.0%) 0.30±0.057 3.1 8.6 0.86 scsd (3.5%) 0.27±0.059 3.2 8.6 0.84 nisin + scsd 0.28±0.039 3.1 8.0 0.91 scsd + nisin 0.34±0.046 2.7 8.2 0.90 combined 0.14±0.046 3.3 -c 0.89 alower asymptote estimated by the baranyi model. bupper asymptote estimated by the baranyi model. cno value could be estimated when the upper part of the growth curve ceased without forming an upper asymptote that corresponds to the stationary phase. ital. j. food sci., vol. 32, 2020 361 figure 2. growth of l. monocytogenes on vacuum packaged beef frankfurters dipped in solutions of 6000 iu/ml nisin, 3.5% bscsd, and combined solution, stored at 4°c. lm control, l. monocytogenes inoculated on beef frankfurters dipped in 0.1% peptone water with no antimicrobials. data points are not actual experimental values but aids for better visualization of baranyi graphs. table 5. growth kinetics of l. monocytogenes inoculated on the surface of beef frankfurters treated with nisin, bscsd, alone or in sequence or combination, then vacuum packaged and stored at 10°c for 20 days. treatment maximum specific growth rate (µmax; per day ±standard error) y0 a (log10 cfu/g) yend b (log10 cfu/g) r 2 lm control 0.77±0.020 4.7 8.8 0.95 nisin (2000 iu/ml) 0.66±0.017 2.9 8.4 0.98 nisin (4000 iu/ml) 0.95±0.021 2.8 8.4 0.97 nisin (6000 iu/ml) 1.00±0.04 2.1 8.1 0.92 scsd (2.5%) 0.84±0.037 4.2 8.9 0.89 scsd (3.0%) 0.70±0.037 4.6 8.9 0.87 scsd (3.5%) 0.74±0.027 4.5 8.8 0.92 nisin + scsd 0.79±0.031 3.0 8.2 0.93 scsd + nisin 0.89±0.027 2.7 8.4 0.96 combined 0.83±0.29 2.7 8.1 0.94 alower asymptote estimated by the baranyi model. bupper asymptote estimated by the baranyi model. cno value could be estimated when the upper part of the growth curve ceased without forming an upper asymptote that corresponds to the stationary phase. ° ital. j. food sci., vol. 32, 2020 362 the y0 values of control samples were similar, 4.7 and 4.4 log10 cfu/g at 4 and 10°c, respectively (tables 4 and 5). treatment count reductions were addressed earlier when discussing results shown in tables 2 and 3. maximum specific growth rate (µmax) of nisin-treated frankfurters ranged from 0.339-0.368 day-1 while that of bscsd was 0.230-0.274 day-1 and control was 0.263 day-1 at 4°c. this implies that surviving nisin-treated l. monocytogenes grew faster than non-stressed populations. others (luchansky and call, 2004; rutherford, 2004; samelis et al, 2005) found similar results of greater µmax values with treated samples than with control samples. those researchers postulated that nisin could have slowed the growth of other bacteria thus reducing the bacterial competition against l. monocytogenes, which allowed it to grow at a faster rate than when in the presence of competitor organisms. in contrast, the present study used sterile frankfurters to avoid possible confounding effects of any indigenous microflora, which implies another potential, as yet unproven, reason for nisinresistant growth stimulation. for example, it is possible that a fast-growing, nisinresistant strain was in the inoculum mix. also, perhaps nisin-exposed cells are primed to multiply quickly as a stress-response survival strategy, especially in case of absence of indigenous microbiota. nisin was more effective than bscsd in both initial population size reduction and growth inhibition during storage (tables 1 and 2). combining nisin with bscsd did not improve nisin activity. similar results were reported when nisin was used with sodium diacetate (fang and lin, 1994; samelis et al., 2005; stopforth et al., 2005), sodium acetate (samelis et al., 2002), potassium benzoate (geornaras et al., 2006b; samelis et al., 2005), potassium sorbate (samelis et al., 2005), sodium lactate (fang and lin, 1994), lactic acid (stopforth et al., 2005), acetic acid (stopforth et al., 2005) or grape seed extract (sivarooban et al., 2007). consistent with our findings in most combinations, nisin appeared to be the main contributing antimicrobial factor and lacked significant combined activity with other antimicrobials. the mode of action of nisin involves pore formation in the cytoplasmic membrane, which leads to rapid removal of free amino acids, adenosine triphosphate, and cations from the cell (abee et al., 1994). the antilisterial effect of nisin occurs immediately after cells are exposed to the bacteriocin resulting in cell death (el-khateib et al., 1993). present results are in agreement with previous studies (geornaras et al., 2005; nilsson et al., 1997; samelis et al., 2002; samelis et al., 2005) showing initial reductions of l. monocytogenes populations following treatment with nisin and subsequent cell recovery and growth during storage. the inability of nisin to maintain its antilisterial activity has previously been observed in rte meat products (luchansky and call 2004; samelis et al., 2005). some apparent reasons for loss of nisin activity include insufficient quantities of nisin to interact with all the target cells, nisin activity dependent on low ph, increased resistance of some strains of l. monocytogenes, or uneven distribution of nisin within the food (bouttefroy et al., 2000; henning et al., 1986; muriana, 1996; samelis et al., 2005, martinis et al., 1997). it is known that l. monocytogenes is prone to spontaneous development of resistance to nisin due to mutations (crandall and montville, 1998; martinis et al., 1997; liu et al., 2002); however, the strains used in the present study are not believed to be nisin resistant. addressing these limitations with nisin, tokarskyy and marshall (2008) reported synergistic activity between nisin, lactic acid and monolaurin against l. monocytogenes growth when lactic acid was able to increase membrane fluidity and hence increase nisin activity. additionally, model conditions used in this study were favorable for listeria growth and different from real-life situations. model conditions included destruction of frankfurters natural microbiota to avoid ital. j. food sci., vol. 32, 2020 363 competition, use of stationary culture of pathogen in nutrient-rich medium for inoculation, modifying of frankfurters texture by autoclaving, overnight adaptation and attachment of listeria to the meat surface before application of antimicrobials. dipping in 2.5, 3.0 and 3.5% bscsd had little impact on l. monocytogenes growth at 4 or 10°c (tables 4 and 5). thippareddi et al. (2003) reported that bscsd of ≥ 1.0 % was effective in reducing c. perfringens populations in roast beef or injected pork. the antimicrobial mechanism of bscsd may be similar to organic acid esters, which lower the intracellular ph within the microbial cells, alters cell membrane permeability that affect substrate transport, and inhibits the electron transport system required for energy regeneration (thippareddi et al., 2003). bscsd activity increases as product ph decreases (ceylan et al., 2003; hull, 2007). previous studies with bscsd against l. monocytogenes in meat products are limited. one study observed inhibition of l. monocytogenes by 0.2% sodium diacetate, and stimulated growth with 1% buffered sodium citrate (15 parts sodium citrate, 1 part citric acid w/w) in cooked cured ham products (stekelenburg and kant-muermans, 2001). in contrast to present results, ceylan et al. (2003) found significant inhibition of l. monocytogenes using 1% buffered sodium citrate in combination with 0.1% sodium diacetate in beef frankfurters stored at 3.9°c. the most likely explanation for disparate results is whether agents are added to product formulation (growth inhibition) compared to use as dipping agents (not inhibitory). the present protocol also allowed for l. monocytogenes to attach and adapt during overnight storage at 4°c, a process not unlike commercial practice where frankfurter links potentially exposed to l. monocytogenes environmental contamination are often cooled overnight prior to packaging the next day. 4. conclusion the results of this study demonstrate that dip antimicrobial treatments with nisin and/or bscsd were not effective in preventing low-temperature growth of l. monocytogenes on the surface of frankfurters under proposed model conditions. traditional use of such antimicrobials either as direct product ingredients or as in-package solutions remain as more appropriate application methods. effort to increase activity by combining the different antimicrobial agents did not increase activity. acknowledgments the project was funded, in part, by the beef checkoff through the national cattlemen’s beef association and the mississippi beef council. references abee, t., rombouts, f., hugenholtz, j., guihard, g. and le-tellier, l. 1994. mode of action of nisin z against listeria monocytogenes scott a grown at high and low temperatures. appl. environ. microbiol. 60:1962-1968. baranyi, j. and roberts, t.a. 1994. a dynamic approach to predicting bacterial growth in food. int. j. food microbiol.23:277-294. baranyi, j. 2005. dmfit version 2.0: an excel add-in fitting sigmoid curves. available at: www.combase.cc/index.php/ en/8-category-en-gb/21-tools ital. j. food sci., vol. 32, 2020 364 barmpalia, i. m., geornaras, i., belk, k. e., scanga, j. a., kendall, p. a., smith, g. c. and sofos, j. n. 2004. control of listeria monocytogenes on 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www.fda.gov/safety/recalls/archiverecalls/2018/default.htm?page=1 wederquist, h. and sofos, j.n. 1994. listeria monocytogenes inhibition in refrigerated vacuum packaged turkey bologna by chemical additives. j. food sci. 59:498-500. zhu, m., du, m., cordray, j. and ahn, d.u. 2005. control of listeria monocytogenes contamination in ready-to-eat meat products. compr. rev. food sci. f. 4:34-42. paper received august 21, 2019 accepted january 17, 2020 ijfs#1004_bozza ital. j. food sci., vol. 30, 2018 456 paper effects of temperature on biofilm formation and quorum sensing of aeromonas hydrophila m.f.r. mizana, i.k. jahida, b, s.y. parkc, j.l. silvad, t.j. kime, j. myoung*f and s.-d. ha*a a department of food science and technology, advanced food safety research group, brain korea 21 plus, chung-ang university, 72–1 nae-ri, daedeok-myun, anseong, gyunggi-do 456-756, south korea b department of microbiology, jessore science and technology university, jessore-7408, bangladesh c department of seafood and aquaculture science, institute of marine industry, gyeongsang national university, togyeong 53024, south korea d department of food science, nutrition and health promotion, mississippi state university, mississippi state, mississippi 39762, usa e food and nutrition department, college of education, health and human sciences, wisconsin’s polytechnic university, 712 broadway st s, menomonie, wi 54751, united states f korea zoonosis research institute, chonbuk national university, deokjin-dong 1ga, deokjin-gu, jeonju-si, jeollabuk-do, south korea *e-mail addresses: jinjong.myoung@jbnu.ac.kr; sangdoha@cau.ac.kr abstract aeromonas hydrophila is an emerging foodborne pathogen that causes infections more frequently in summer than in winter. this study evaluated the effects of temperature (437°c) on the biofilm formation and quorum sensing abilities of a. hydrophila on microtiter plates, stainless steel (ss), and crab surfaces. the incubation of the bacterium in luriabertani broth at temperatures of 20-25°c significantly (p < 0.05) enhanced the biofilm formation and intra-species quorum sensing ability (via c4-ahl and c6-ahl). fieldemission electron microscopy revealed that the bacterium colonized the surface of crab and formed biofilms at 25°c. thus, the present study demonstrates that temperature control in food processing environments may reduce a. hydrophila biofilm formation. therefore, the study has significant applications in food processing plants. keywords: aeromonas hydrophila, temperature, biofilm, quorum sensing, crab surface ital. j. food sci., vol. 30, 2018 457 1. introduction aeromonas hydrophila has recently received much attention as an emerging opportunistic and foodborne pathogen and a causative agent of various human infections such as gastrointestinal tract infections, wound and soft tissue infections, and blood-borne dyscrasias (daskalov, 2006). the incidence of these infections is higher during summer, owing to elevated temperatures (janda and abbott, 2010). the importance of a. hydrophila in food safety (daskalov, 2006), fish diseases, and human infections (janda and abbott, 2010) as well as its role in quorum sensing and biofilm formation (chopra et al., 2009) have been studied. the bacterium has been isolated from various fresh and estuarine water samples and animals living in these waters, including fish, crab, shrimp, and other mollusks (ottaviani et al., 2011; deng et al., 2014). microbial biofilms are sessile microbial communities that are attached to either biotic or abiotic surfaces. biofilm formation is a common phenomenon in nature; for instance, biofilms are formed on foods and food-contact surfaces as well as in waste treatment plants. the effect of temperature on the production of biofilms and virulence factors has been studied using various microorganisms, including enterococcus spp., salmonella spp., and listeria monocytogenes (di bonaventura et al., 2008; jahan and holley, 2014). although salmonella is commonly associated with human and animals, it can also be found in the environment (chronicle, 1997; giaouris et al., 2005). as these are environmental microorganisms, such as a. hydrophila, temperature is expected to modulate their survival and ability to form biofilms. quorum sensing is a density-dependent process by which microorganisms coordinate and control intraspecies and interspecies communication (fuqua et al., 1994). several authors have reviewed the importance of quorum sensing in relation to food microbiology (skandamis and nychas, 2012; mizan et al., 2015). a. hydrophila carries quorumsensing genes, including ahyi (encoding n-3-butanoyl-dl-homoserine lactone [c4-ahl]) and ahyr (encoding n-3-hexanoyl homoserine lactone synthase [c6-ahl]) (swift et al., 1997). in addition, the bacterium produces autoinducer 2 (ai-2) for inter-species communication (kozlova et al., 2008). intra-species quorum sensing in a. hydrophila (via c4-ahl and c6-ahl) has been reported to control biofilm formation, motility, and virulence factors (khajanchi et al., 2009). temperature is known to play an important role in governing the rate of microbial activity as well as for the propagation of biofilms and settlement of organisms in aquatic systems (rao, 2010). biofilm formation is influenced by several factors, available nutrients, and organic matter. melo and bott (1997) reported that the development and maturation of biofilm is dependent on temperature, nutrient availability, and water flow rate. as a. hydrophila is common in aquatic environments, it may face adverse environmental conditions such as fluctuations in temperature, humidity, and ph (daskalov, 2006). survey studies have shown that aeromonads are found in high numbers in late summer/early autumn (temperatures are around 20–25°c) but are rarely detected during the cold months (gavriel et al., 1998). the present study focused on the effects of temperatures on biofilm formation and quorum sensing by a. hydrophila on the surface of microtiter plates, stainless steel (ss), and crab shells. in the food industry, biofilm formation is a major concern as an important source of food contamination. therefore, the results of this study will provide potential approaches to avoid contamination, as they reveal the temperatures favorable for a. hydrophila biofilm formation and quorum sensing. ital. j. food sci., vol. 30, 2018 458 2. materials and methods 2.1. bacterial strains, culture media, and growth conditions in the present study, the following strains were used: a. hydrophila kctc 11533 (isolated from surface water) and kccm 32586 (a clinical isolate), vibrio harveyi strain bb120 and bb170, and chromobacterium violaceum cv026. the bioreporter strain cv026 was provided by the animal, plant, and fisheries quarantine and inspection agency, korea. luriabertani (lb) broth (difco laboratories, detroit, mi, usa) was used for the bacterial cultivation and violacein production assay. prior to each experiment, frozen culture aliquots (100 µl) were thawed and inoculated into 5 ml of lb broth. the cultures were incubated at 30°c and 220 rpm for 24 h. these starter cultures were subsequently inoculated into fresh lb broth and cultured to a final optical density of 1.0 at 600 nm (od600), followed by their dilution (1:50) for biofilm formation experiments and quorum sensing assay. 2.2. quantitative biofilm formation assay in microtiter plates the quantitative biofilm assay was performed as previously described (o’toole, 2011; mizan et al., 2016). a. hydrophila was cultured in lb broth for 24 h without shaking, followed by its dilution at 1:50 in lb broth. a total of 100 µl aliquots were placed in the wells of 96-well polystyrene microtiter plates (becton, dickinson and company, franklin lakes, nj, usa) and the microtiter plates were incubated at 4, 10, 15, 20, 25, 30, 35, and 37°c for 72 h without shaking. biofilm formation was normalized to planktonic growth and determined using the following equation (1), according to teh et al. (2010): (1) where bfi is the biofilm formation index, ab is od595 of the crystal violet (cv)-stained attached microorganisms, cw is od595 of the stained blank wells containing sterile (microorganism-free) medium only, gb is od600 of the cells in suspended culture, and gw is od600 of the blank well. biofilm production was classified into three categories according to martinez-medina et al. (2009): weak (0.1 > bfi ≤ 0.5), moderate (0.5 > bfi ≤ 1), and strong (bfi > 1). 2.3. determination of biofilm formation on ss surfaces austenitic ss coupons (type 302, 2 × 2 × 0.1 cm; chung-ang scientific inc., seoul, korea) were processed as described by shen et al. (2012). thereafter, a. hydrophila cell suspensions were diluted at 1:50 and inoculated into 7 ml of fresh lb in 50 ml falcon tubes containing a completely submerged ss coupon. the tubes were incubated without shaking at 4, 10, 15, 20, 25, 30, 35, and 37°c for 24 h to allow biofilm formation on ss coupons. following incubation, each ss coupon was transferred into a small petri dish (55 × 12 mm) containing 1 ml of 0.1% peptone water (pw; oxoid, hampshire, uk) and agitated by rotation in clockwise and anticlockwise direction using sterile forceps. agitation was always performed by the same person; thus, it was assumed that the same amount of pressure was applied to all coupons (jahid and ha, 2014). the cells were separated, vortexed, and diluted in pw for enumeration. cell number was quantified using bacto r2a agar (difco, usa) following incubation for 24 h. ital. j. food sci., vol. 30, 2018 459 2.4. preparation of inocula for crab surfaces cultures grown in lb broth were centrifuged (10,000 ×g for 10 min at 4°c) and the pellets were washed twice with dulbecco’s phosphate-buffered saline (dpbs). the pellets were re-suspended in a suitable amount of dpbs to obtain an absorbance of 1.0 at 600 nm wavelengths. to determine the cell density, serial dilutions were performed and plated on aeromonas selective medium (oxoid, hampshire, uk). these inocula were used to inoculate the crab surfaces. 2.5. biofilm formation on crab surfaces and detachment of cell population crabs (corystes cassivelaunus) used in this study were purchased from a local grocery store in anseong, korea. a delimited area (cm2) of crab shell was dissected and processed immediately, as described by jahid et al. (2015). the shells were incubated at different temperatures (4, 10, 15, 20, 25, 30, 35, and 37°c) without shaking. the detachment of microbial populations from shell surfaces was performed as described by jahid et al. (2014), with minor modifications. briefly, the crab surfaces were washed twice with pbs to free planktonic cells and placed in 10 ml of pw in a sterile stomacher bag (nasco whirlpak, usa). these were processed using a stomacher (bagmixer, interscience, saint nom, france) at maximum speed for 2 min to free the biofilm-forming microbes from the crab shells. a. hydrophila was enumerated after serial dilution and spread plating on aeromonas selective medium containing ampicillin. the plates were incubated at 30°c for 48 h. colonies were counted and the results for biofilm production were expressed as colonyforming unit (cfu)/cm2 for biofilm populations. for each of three independent experiments, two plates per dilution were assessed to obtain the final data. 2.6. quantification of violacein production to quantify violacein production, the procedures described by kim et al. (2013) and jahid et al. (2015) were followed. a. hydrophila was cultivated in lb broth at different temperatures for 24 h and the supernatant was collected by centrifugation at 15,000 ×g for 15 min, followed by its filter sterilization using 0.22 µm filters (millipore corporation, billerica, ma, usa). lb agar was prepared, cooled, and poured using the open side of a 1 ml pipette tip to make a well. a loop full of c. violaceum cv026 overnight culture was spread on the wall of the well and treated with 100 µl of supernatant from each condition at 28°c for 24 h in an upside up of petri dish. next, whole cv026 cells grown on the plate were collected and solubilized with 250 µl of dimethyl sulfoxide (dmso; sigma aldrich). the mixture was vortexed to ensure the release of violacein pigment. after centrifugation at 15,000 ×g for 15 min, the absorbance of 200 µl of colored dmso from cv026 cells was measured at 585 nm wavelength using a microplate reader (spectra max 190; molecular devices). 2.7. autoinducer-2 (ai-2) bioassay the secretion of ai-2 by a. hydrophila during its incubation with crab surfaces at different temperatures (4-37°c) was assessed with minor modifications in previously described procedures (soni et al., 2008). a. hydrophila was inoculated on crab surfaces in cyanobacteria bg-11 freshwater solution (sigma aldrich, inc., st. louis, mo, usa) and incubated at different temperatures without shaking. the cultures were centrifuged at 15,000 ×g for 10 min. the supernatant from the cell-free culture was passed through 0.2 µm tuffryn syringe filters and stored at -20°c. the cell-free supernatants were tested for ital. j. food sci., vol. 30, 2018 460 the presence of ais that induce luminescence of v. harveyi reporter strain bb170. this strain carries sensor 2, but not sensor 1, and is capable of sensing ai-2 but not ai-1. v. harveyi strain bb170 was grown overnight at 30°c with aeration in the autoinducer bioassay (ab) broth and diluted to 1:1,000 in ab medium (bassler et al., 1993). next, 4.5 ml of diluted v. harveyi strain bb170 and 500 µl of the cell-free supernatant from each sample (a. hydrophila supernatant incubated with crab at different temperatures) was added to 50 ml falcon tubes and shaken for 16 h at 220 rpm to allow the reporter strain to produce luminescence. a total of 100 µl samples were transferred to white microtiter plates and the luminescence was measured using a computer-controlled microplate luminometer (glomax® 96 microplate luminometer for luminescence, promega, madison, wi, usa). v. harveyi strain bb120 that produces ai-1 and ai-2 was used as a positive control. control v. harveyi strains were grown overnight at 30°c with shaking at 220 rpm in lb broth and 1 ml of cell-free supernatant from each culture was prepared as described above. 2.8. field-emission scanning electron microscopy a. hydrophila biofilm formation was observed on crab surfaces at 4, 25, and 37°c by fieldemission scanning electron microscopy (fesem). the inoculation and incubation procedures were the same as those described above. fesem samples were processed according to the previously described procedures (mizan et al., 2016). the dehydrated samples were sputter-coated with platinum and visualized with an fesem microscope (sigma, carl zeiss, germany) at an accelerated voltage of 5 kv and a working distance of 5 mm. digitized images of biofilms were collected for further analysis. 2.9. statistical analysis biofilm formation and quorum sensing of a. hydrophila at different temperatures (4–37°c) were analyzed by analysis of variance (anova) using sas software (version 9.2; sas institute inc., cary, nc, usa) for a completely randomized design to determine the significance of differences due to temperature variation. the mean separation was evaluated with duncan’s multiple-range test when the effect was significant (p < 0.05). 3. results and discussion 3.1. impact of temperature on biofilm formation temperature is one of the major factors that affect bacterial growth. most of the clinically important pathogens are mesophiles that grow well at optimum temperatures between 25°c and 40°c (murray et al., 2003). the optimum growth temperature is 20°c for some aeromonas species and 37°c for others (ewing et al., 1961). maalej et al. (2004) reported a decline in a. hydrophila population to a level below the detection level at 23°c and 5°c. the optimum temperature for a. hydrophila infection in goldfish (carassius auratus) is 17– 25°c (rahman et al., 2001). the results from the examination of the biofilm formation on microtiter plates at different temperatures are shown in table 1. a significant increase in the biofilm production was observed at 20–25°c for a. hydrophila strains 11533 and 32586. biofilm formation declined at temperatures over 25°c (i.e., 30-37°c) or below 20°c. rachid et al. (2000) reported the formation of dense biofilms with an increase in temperature. ital. j. food sci., vol. 30, 2018 461 studies have shown that a. hydrophila may attach and produce biofilm on to ss surfaces (lynch et al., 2002), glass (whiteley et al., 1997), and vegetables (jahid et al., 2014) in laboratory settings. lynch et al. (2002) reported that a. hydrophila produces a thin biofilm that may cover 40-50% of ss surface. biofilm formation by a. hydrophila strains 11533 and 32586 on ss coupons is presented in table 1. the trend observed was similar to that reported with microtiter plates. a significant increase (p < 0.05) in biofilm formation was observed at 20-25°c, indicative of the optimum temperature range for biofilm formation. similar trend was observed for both a. hydrophila strains, although they had different origins (i.e., clinical versus environmental). biofilms on fish surfaces and bacteria from marine water source may contaminate seafoodprocessing facilities. vibrio, aeromonas, listeria, and salmonella isolated from seafood are known to cause foodborne illness and form biofilms (takahashi et al., 2009; aberoum and jooyandeh, 2010; norhana et al., 2010; jahid et al., 2015; mizan et al., 2017). the ability of a. hydrophila to produce biofilms on crab surfaces at 4, 10, 15, 20, 25, 30, 35, and 37°c is shown in table 1. biofilm formation was significantly lower (p < 0.05) at 4, 10, and 15°c than at 20-37°c. a significant increase in the biofilm formation was observed for both strains at 20-30°c, while the biofilm growth gradually reduced at 37°c. no significant difference (p > 0.05) was observed in the biofilm formation between the two a. hydrophila strains. table 1. viable counts of biofilm cells of aeromonas hydrophila in luria-bertani (lb) medium with different temperature from 4°c to 37°c. support microtiter plates stainless steel surfaces crab surfaces temperatur e (°c) 11533 (bfi±sem**) 32586 (bfi±sem) 11533 (log cfu/cm2 ±sem) 32586 (log cfu/cm2 ±sem) 11533 (log cfu/cm2 ±sem) 32586 (log cfu/cm2 ±sem) 4 0.29±0.02de 0.22±0.03d 4.56±0.26c 3.62±0.28d 2.05±0.10h 1.66±0.17f 10 0.38±0.03cd 0.47±0.01bc 4.89±0.15bc 5.14±0.22bc 2.76±0.09g 2.81±0.07e 15 0.79±0.02ab 0.63±0.04ab 5.26±0.11b 5.50±0.32b 4.74±0.26f 4.45±0.15d 20 0.91±0.08a 0.72±0.03a 6.23±0.07a 6.22±0.24a 5.98±0.03ab 4.77±0.14d 25 1.06±0.04a 0.70±0.07a 6.35±0.18a 6.72±0.24a 6.08±0.11a 5.55±0.09ab 30 0.34±0.05de 0.45±0.06bc 5.46±0.25b 5.29±0.12b 5.88±0.07abc 5.67±0.17a 35 0.24±0.04e 0.36±0.02cd 5.16±0.08bc 5.34±0.08b 5.43±0.24cd 4.87±0.09c 37 0.16±0.02e 0.38±0.06cd 2.65±0.26e 3.26±0.27de 4.96±0.30de 4.73±0.23d **the values are mean ± sem (standard error mean) of 3 independent experiments. the values with same letters within a column were not significant (p < 0.05) according to duncan’s multiple-range test. 3.2. violacein production the modulation in the quorum sensing ability of a. hydrophila at different temperatures has been previously investigated (medina-martinez et al., 2006). the biosensor c. violaceum strain cv026 (a cvil transposon mutant) with quorum sensing regulation defect fails to produce violet color in the absence of ahl, which controls violacein production. however, cv026 produces color if ahl is externally supplied. the mutant cv026 produces color when grown in the presence of a. hydrophila, as violacein is produced in response to ahl secreted by a. hydrophila. the intensity of the color is proportional to ahl concentration. violacein production was significantly lower (p < 0.05) at 4-15°c than at 20-25°c. ahl produced by strain 11533 was significantly higher (p < 0.05) than that ital. j. food sci., vol. 30, 2018 462 produced by strain 32586 (fig. 1). jahid et al. (2015) stated that the production of ahl by a. hydrophila is strain-dependent. ponce-rossi et al. (2016) reported a. hydrophila atcc7966 to be negative for ahl production, while a. hydrophila embrapa 029 was shown to produce ahl. both a. hydrophila strains produced the highest ahl levels at 25°c (fig. 1). medinamartinez et al. (2006) noted that the optimum temperature for ahl production was 22°c, and no ahl production was observed at 37°c. at 12°c, however, c4-hsl production was detected after 70 h of incubation. figure 1. violacein production in a. hydrophila at different temperatures. values shown are the mean ± sem of three independent experiments. within each treatment, the values marked with the same letter are not significantly different according to duncan’s multiple-range test (p < 0.05). 3.3. ai-2 production although ai-2 production and biofilm formation may be associated with each other (mizan et al., 2016), no strong relationship was observed between ai-2 levels and biofilm formation, as the procedure for ai-2 measurement was too sensitive and variable to compare independent experiments (fig. 2). temperature modulates cell density, and quorum sensing is dependent on cell density. ai-2 production by a. hydrophila was analyzed in crab fresh water samples incubated at different temperatures. as shown in fig. 2, ai-2 production by a. hydrophila strains was origin-dependent; more strains are necessary to justify the results. the environmental strain (kctc 11533) produced lower levels of ai-2 than the clinical strain (kccm 32586). a significant (p < 0.05) increase in ai2 production was observed in both a. hydrophila strains at 20–30°c (fig. 2). further studies on the role of food composition and environment in the production of ai-2 and c4-hsl may increase our understanding of the synthesis and accumulation of these signal molecules in food products. ital. j. food sci., vol. 30, 2018 463 figure 2. ai-2 production in a. hydrophila at different temperatures. values shown are the mean ± sem of three independent experiments. within each treatment, the values marked with the same letter are not significantly different according to duncan’s multiple-range test (p < 0.05). 3.4. analysis of crab samples with fesem aberoum and jooyandeh (2010) reported that processing facilities of seafood products may act as a source of contamination and that a. hydrophila is commonly isolated from unprocessed and processed seafood products. tsai and chen (1996) observed that a. hydrophila contaminated 50% of oysters purchased from native marts of taiwan. biofilm formation may differ, owing to differences in the growth surface and temperature (noori et al., 2016). jahan and holley (2014) observed dense biofilm formation (enterococcus spp.) at temperatures lower than the optimum growth temperature. noori et al. (2016) reported that higher temperatures induced extensive biofilm formation, whereas lower temperatures resulted in the attachment of the bacterial cells (v. parahaemolyticus) as monolayers on crab surface. mizan et al. (2015) observed a. hydrophila cell attachment on crab shell. biofilm formation on crab samples incubated at 4, 25, and 37°c (according to biofilm formation strength) is presented in fig. 3. a. hydrophila failed to form biofilms on crab surfaces at 4°c; the bacterium attached on the crab surface (fig. 3a). in contrast, a. hydrophila formed a strong three-dimensional structure at 25°c, (fig. 3b). at 37°c, the bacterium formed biofilms on the crab surface (fig. 3c). poncerossi et al. (2016) evaluated biofilm formation in two strains of a. hydrophila at the same temperature but different incubation times. these authors found that the biofilm formation was maximum at 48 h and decreased at 72 h. almeida et al. (2017) studied salmonella and observed that maximum biofilm formation may occur after 36 h incubation in contrast to other times evaluated. ital. j. food sci., vol. 30, 2018 464 figure 3. fesem images of a. hydrophila biofilm formation on crab surfaces at different temperatures. the image shown is a representative result for strain kctc 11533. (a) 4°c, (b) 25°c and (c) 37°c. 4. conclusions the two a. hydrophila strains, one originally isolated from surface water (strain kctc 11532) and the other from clinical sample (strain kccm 32586), showed significant variations in the tested phenotypes (i.e., biofilm formation and quorum sensing), indicating strain-specific regulation. strain kctc 11533 was found to produce high concentrations of ahl and lower concentrations of ai-2. strain kccm 32586, on the other hand, showed high ai-2 production and lower ahl activity. therefore, the phenotypic properties differed between the two strains. such studies will elucidate the effect of quorum sensing on the regulation of virulence factors produced by opportunistic pathogens such as a. hydrophila on microtiter plates, ss, and crab surfaces. however, the experimental scope of the study is limited to only one strain of environmental and clinical origin. further studies are warranted to extend the application of this study in food quality and safety regulations. acknowledgements this research was supported by mid-career research program of the national research foundation of korea (nrf) funded by the ministry of education, science, and technology (2016r1a2b4007960). references aberoum a. and jooyandeh h., 2010. a review on occurrence and characterization of the aeromonas species from marine fishes. world j. fish mar. sci. 2:2078e4589. almeida f.a., pimentel‑filho n.j., pinto u.m., mantovani h.c., oliveira l.l. and vanetti m.c. 2017. acyl homoserine lactone‑based quorum sensing stimulates biofilm formation by salmonella enteritidis in anaerobic conditions. arch. microbiol. 199(3):475-486. bassler b.l., wright m., showalter r.e. and silverman m.r. 1993. intercellular signaling in vibrio harveyi: sequence and function of genes regulating expression of luminescence. mol. microbiol. 9(4):773-786. ital. j. food sci., vol. 30, 2018 465 chronicle c. 1997. salmonellosis (public health concerns for the farm family and staff). 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skandamis p.n. and nychas g.j. 2012. quorum sensing in the context of food microbiology. appl. environ. microbiol. 78:5473–5482. soni k.a., jesudhasan p.r., cepeda m., williams b., hume m., russell w.k., jayaraman a. and pillai s.d. 2008. autoinducer ai-2 is involved in regulating a variety of cellular processes in salmonella typhimurium. foodborne pathog. dis. 5:147-153. swift s., karlyshev a.v., fish l., durant e.l., winson m.k., chhabra s.r., williams p., macintyre s. and stewart g.s. 1997. quorum sensing in aeromonas hydrophila and aeromonas salmonicida: identification of the luxri homologs ahyri and asari and their cognate nacylhomoserine lactone signal molecules. j. bacteriol. 179(17):5271-5281. takahashi h., miya s., igarashi k., suda t., kuramoto s. and kimura, b. 2009. biofilm formation ability of listeria monocytogenes isolates from raw ready-to-eat seafood. j. food prot. 72:1476-1480. teh k.h., flint s. and french n. 2010. biofilm formation by campylobacter jejuni in controlled mixed-microbial populations. int. j. food microbiol. 143(3):118-124. paper received october 27, 2017 accepted april 4, 2018 ijfs#1087_bozza ital. j. food sci., vol. 30, 2018 381 paper calcium carbonate effect on alkyl esters and enzymatic activities during olive processing f. caponio*, g. squeo, m. curci, r. silletti, v.m. paradiso, c. summo, c. crecchio and a. pasqualone department of soil, plant and food science (disspa), university of bari aldo moro, via amendola 165/a, 70126 bari, italy *e-mail address: francesco.caponio@uniba.it abstract the effect of coadjuvants during olive oil processing on the oxidative enzymes and the content of fatty acid alkyl esters (faae) has been investigated. two italian olive cultivars, at different ripening degree, were processed immediately after harvesting or after 5 and 12 days of storage. the results highlighted a general decrease of faae and a significant increase in the ppo and pod activities due to the coadjuvant use. the increased oxidases activity could lead to a reduction of oils phenolic compounds. keywords: fatty acid alkyl esters, extra virgin olive oil, oxidases, olive processing, technological coadjuvant ital. j. food sci., vol. 30, 2018 382 1. introduction over the years, the goals of the oil industry have gradually changed. after the introduction of the centrifugal decanters, which made the oil extraction process continuous and led to cost decreases, solving the issues linked to the traditional production, the newest aim was to ensure the highest quality of virgin olive oils (voo) obtained. in fact, suddenly appeared that oils had lower content of phenolic compounds compared to those obtained with the traditional method (pressure), because of the olive paste leaching by the added water, an essential step in order to ensure satisfactory extraction yields by centrifugation (ranalli and angerosa, 1996). it is common knowledge that phenolic compounds, besides affecting voo sensory notes (bitter and pungent notes are directly related to the total phenolic content) are the main responsible of the health benefits associated to the voo consumption (martín-peláez et al., 2013). furthermore, often happened that the olive pomace after the separation step is too much wet, with moisture content even higher than 60%, and thus not appreciated by the pomace oil factories (sánchez moral et al., 2006). usually oil producers overcome this drawback submitting olive pomace to a second centrifugation step by means of threephase decanter (caponio et al., 2015; pasqualone et al., 2016). this allows even to recover an additional amount of olive oil called “ripasso” rising, however, the overall costs of production. moreover, according to the council regulation 1513/2001 (official journal of the european communities, 2001), that oil has to be classified as “crude olive-pomace oil”. nowadays, innovative decanters, such as those equipped with variable dn system, are available on the market. respect to older machines, these decanters allow real-time setting of several working parameters as a function of the raw material characteristics (maturity degree), as well as of the expected virgin olive oil quality, e.g. in terms of phenolic compounds (squeo et al., 2017a). currently, the focus was moved towards the optimisation of the process efficiency (that is maximise the extraction yields) especially during processing of the so-called “difficult pastes” (cert et al., 1996; uceda et al., 2006; caponio et al., 2014), without jeopardising voo quality. different approaches were tested to solve such an issue and, besides working on the malaxation parameters (timetemperature), numerous researches were recently carried out regarding the use of physic processing aids (not forbidden by the european laws). among them, micronized natural talc (mnt) and calcium carbonate have shown a significant positive effect on the extraction process efficiency while the influence on the chemical and organoleptic features of the voo was not univocally pointed out (ben brahim et al., 2015; caponio et al., 2016). besides, calcium carbonate is less expansive and does not involve any health risk for oil-mill operators than the use of mnt (espínola et al., 2009). the employment of technological coadjuvants was even proposed in order to avoiding the need of a second centrifugation step (caponio et al., 2015). the european commission (official journal of the european communities, 2011), aiming at the protection of the highest voo quality and in order to prevent illegal mixtures with low-quality oils, have introduced the determination of fatty acids alkyl esters (faae) as a quality parameter for the extra virgin olive oils (evoo) classification. alkyl esters originate from the esterification of fatty acids and low molecular weight alcohols, methanol and ethanol, arising from the progressive pectin degradation during the olive ripening and from the bad and/or prolonged storage of drupes, respectively (biedermann et al., 2008; jabeur et al., 2015; beltran et al., 2016). that is, faae content in evoo is strongly linked to the quality of the raw material and is considered a clear marker of the sanitary state and/or of the handling procedures of the olive fruits before processing. moreover, due to the high stability, these compounds were even ital. j. food sci., vol. 30, 2018 383 proposed as an efficient tool for discovering mixture with mild deodorised olive oils (pérez-camino et al., 2008). afterwards, the focus was moved towards the content of the fatty acids ethyl esters (faee) only (official journal of the european communities, 2013). in this framework, greater attention would be given to the correlation between the use of coadjuvants in olive oil mill and alkyl esters in the obtained oils. the only study present in literature (squeo et al., 2017b) reports that the use of calcium carbonate on coratina cv. olives processed immediately after harvesting led to a general reduction of faae compared to the untreated samples, evidencing a higher susceptibility of methyl esters (fame) than ethyl esters. furthermore, previous papers have reported as its use led to a decrease in the oil phenolic content (squeo et al., 2016; tamborrino et al., 2017) but, despite the great relevance of phenolics, still today this side effect is not studied and understood. as far as we know, no information are available about the effect of the coadjuvants on the most important olive enzymatic activities and, in particular, polyphenol oxidase (ppo) and peroxidase (pod) which are responsible of the oxidation of phenolic compounds in the first stages of extraction and during the malaxation step (garcía-rodríguez et al., 2011). hence, the aim of this research was to assess the influence of calcium carbonate on the faae content and enzymatic activities involved in the oxidation of the phenolic compounds, in order to reach a deeper knowledge about the possible side effects of such coadjuvant during olive processing. in particular, two italian olive oil cultivars, having different maturation degree, were considered, both processed immediately after harvesting and after 5 and 12 days of storage, with the addition of calcium carbonate at different level and particle size. 2. materials and methods 2.1. materials and reagents calcium carbonate (caco3) was kindly furnished by omya spa (milan, italy). two different mean particle sizes were considered: 2.7 μm (calcipur®2) and 5.7 μm (calcipur®5). all the reagents used for the analytical determination were for analytical purpose or hplc and gc grade. 2.2. sampling olives of coratina and nociara cultivars having the following features respectively were used for the experiment: pigmentation index (defined as in squeo et al., 2016) 2.32 and 4.40, respectively; moisture content 52.92% and 47.15%; total oil content 21.20% and 19.66% (official journal of the european communities, 1991). for each cultivar a homogenous lot of olives were used, which was further divided in 18 batches of about 30 kg each. five trials were carried out on olives processed immediately after harvesting: one, the control (c), without caco3 addition and the others by means of coadjuvant addition (two different particle sizes: 2.7 μm and 5.7 μm) at two different percentages of use (2% and 4% respect to the olives weight), as follows: • without calcium carbonate addition (c, control), • with 2% of calcipur®2 (ca2-2%), • with 4% of calcipur®2 (ca2-4%), • with 2% of calcipur®5 (ca5-2%), ital. j. food sci., vol. 30, 2018 384 • with 4% of calcipur®5 (ca5-4%). the remaining batches were stored in reticular plastic bins for 5 and 12 days (t5 and t12) and processed without any treatment (control, c) or by the use of 2% of caco3 (2.7 μm) as follows: • without calcium carbonate addition (ct5 and ct12, control), • with 2% of calcipur®2 (ca2t5-2% and ca2t12-2%). when expected, calcium carbonate was added at the beginning of the malaxation phase. oil extraction was performed by means of a small industrial plant (oliomio mini 50, moritem s.r.l., tavarnelle val di pesa, florence, italy) of a maximum capacity of 30 kg h-1. after crushing by a fixed hammer crusher, olives paste was malaxed at 20±1°c for 20 min and sent to a 2-phase decanter for the oil separation. two oil samples were collected for each extraction. the samples were then further finished by centrifugation (sl 16r model, thermo scientific, waltham, ma, usa) at 8,867 × g, 5 min, 4°c. finished oils were poured in 100 ml dark glass bottles, leaving an head-space of about 1 cm, hermetically sealed, and stored at room temperature (18-20°c) until were analyzed. all the trials were repeated twice. 2.3. routine analyses the determination of free fatty acids, peroxide value, spectrophotometric extinctions at 232 and 270 nm, and fatty acid composition were carried out as reported by the official journal of the european communities (official journal of the european communities, 1991). 2.4. determination of faae the analyses of the methyl and ethyl esters of fatty acids were carried out according to the official method (official journal of the european communities, 2011). the gas chromatographic system was made up of a 7890b agilent technologies (santa clara, ca, usa) gas-chromatograph equipped with a flame ionization detector (fid). the column used was a capillary fused silica db-5ht (length 15 m, i.d. 0.32 mm, film thickness 0.10 µm). the operating conditions were as follows: oven temperature, 80°c for 1 min and then increased from 20°c min-1 to 140°c, then increased from 5°c min-1 to 335°c and maintained for 20 min. the detector temperature was 350°c. helium was used as the carrier gas, with a flow through the column of 2 ml min-1 in on-column mode. each sample was analysed twice. 2.5. enzymatic activity assessment ten g olive pastes were homogenized with 150 ml cold acetone (-20°c) in a waring blender homogenizer at highest speed for 30 s. powder extract was filtered under vacuum, then further extracted twice with 20 ml of cold acetone, finally washed with diethyl ether, dried at room temperature and finally stored at -20°c (garcía-rodríguez et al., 2011). enzymatic extracts of polyphenoloxidase (ppo, ec 1.14.18.1) and peroxidase (pod, ec 1.11.1.7) were prepared according to (peres et al., 2016): 0.4 g acetone powder were resuspended in 5 ml extraction buffer (0.05 m potassium phosphate, 1 m kcl, ph 6.2) and 2% polyvinilpyrrolidone (pvp) and stirred for 30 min at 400 rpm at 4°c; the suspension was centrifuged at 15,777 × g for 30 min at 4°c and filtered (0.45 µm). for both enzymatic assays the reaction medium consisted of 50 mm sodium phosphate buffer ph 6.2, containing 0.5 ml of filtered crude extracts (above described) in a final volume of 2.5 ml. ppo activity was evaluated using catechol (30 mm) as substrate, ital. j. food sci., vol. 30, 2018 385 following the increase in absorbance at 420 nm, during 1 min. one unit of ppo was defined as the quantity of enzyme that causes the absorbance variation of 0.001 min-1 ml-1, at 25°c; results were expressed as u g-1 fw (fresh weight). pod activity was evaluated using guaiacol (30 mm) and h2o2 (4 mm) as substrates. in particular, guaiacol was incubated for 5 min at 25°c, then h2o2 was added and the absorbance at 470 nm was measured after 2 min. one unit of pod was defined as the consumption of µmol of guaiacol min-1 ml-1, at 25°c. results were expressed as u g-1 fw (fresh weight) (peres et al., 2016). 2.6. total phenolic content for the extraction of phenols, 1 g of oil was dissolved in 1 ml of hexane and 5 ml of methanol/water (70:30, v/v). the mixture was vortexed for 10 min and centrifuged at 6000 rpm for 10 min at 4°c (sl 16r centrifuge, thermo fisher scientific inc., waltham, ma, usa). the methanolic phase was recovered, centrifuged again at 9000 rpm for 5 min at 4°c, and finally filtered through 0.45mm pores filters. the quantification of the phenolic compounds was carried out by means of the folin-ciocalteau method. briefly, 100µl of extract were mixed with 100µl of folin-ciocalteu reagent. after 4 min, 800µl of na2co3 solution 5% (w/v) was added to the mixture and heated in a water bath at 40°c for 20 min. after being cool down for 15 min, the absorbance was measured at 750 nm by an agilent cary 60 spectrophotometer (agilent technologies, santa clara, usa). the total phenolic content was expressed as gallic acid mg equivalents (mg kg-1). 2.7. statistical analysis unbalanced nested anova was used to test the effects of the type of calcium carbonate and the percentages of addition on the fatty acids alkyl esters amount (table 2). two-way anova was used to test the effects of the use of calcium carbonate and the olives days of storage on the fatty acids alkyl esters amount (table 3) while three-way anova was used for testing the influence of the experimental conditions on the enzymatic activities (figure 1). in all cases, tukey post-hoc test for multiple comparisons was carried out on the experimental data by means of minitab 17 software (minitab inc., state college, pa, usa). 3. results and discussion 3.1. influence on the alkyl esters amount table 1 reports the characteristics of the oils extracted from the olives, without carbonate addition (c), immediately after harvesting. the samples fulfilled the european limits for the evoo classification (official journal of the european communities, 2013). the fatty acids profiles matched those typical for olive oils and a variability among the cultivar studied was observed confirming, as it is well known, that fatty acids composition is strongly affected by the genotype (rotondi et al., 2010). nociara cv. oil was richer in palmitic (c16:0), palmitoleic (c16:1), linoleic (c18:2), and linolenic (c18:3) acids than coratina cv. oil. the latter was richer in oleic (c18:1) and eicosenoic (c20:1) acids. overall, nociara cv. oil showed a higher extent of the primary oxidative degradation, likely due to the higher pigmentation index (almost two-fold than coratina) of the drupes and to the higher amount of polyunsaturated fatty acids. indeed, the oxidative susceptibility of the oils rises with the increase of the fatty acids unsaturation degree (choe and min, 2006). ital. j. food sci., vol. 30, 2018 386 the mean values, standard deviations, and statistical analysis of the oils alkyl esters content obtained from non-stored olives are reported in table 2. table 1. basic analytical characteristics of the oils obtained from olives processed immediately after harvesting (n=2). parameter coratina nociara mean value sd mean value sd ffa 0.31 0.02 0.43 0.04 pv 6.5 0.1 9.8 0.1 k232 1.829 0.016 2.215 0.023 k270 0.115 0.005 0.128 0.003 fatty acid composition (%) < c14:0 c16:0 10.09 0.01 14.08 0.12 c16:1 0.68 0.02 1.73 0.04 c17:0 0.06 0.01 0.05 0.00 c17:1 0.08 0.01 0.10 0.01 c18:0 2.12 0.04 1.91 0.08 c18:1 78.97 0.05 67.91 0.02 c18:2 6.77 0.07 12.95 0.04 c18:3 0.55 0.01 0.67 0.03 c20:0 0.34 0.01 0.37 0.03 c20:1 0.33 0.02 0.23 0.02 ffa, free fatty acids (g 100 g-1); pv, peroxide value (meq o2 kg-1); k232, specific absorption at 232 nm; k270, specific absorption at 270 nm; c16:0, palmitic acid; c16:1, palmitoleic acid; c17:0, heptadecanoic acid; c17:1, heptadecenoic acid; c18:0, stearic acid; c18:1, oleic acid; c18:2, linoleic acid; c18:3, linolenic acid; c20:0, arachidic acid; c20:1, eicosenoic acid. table 2. mean values, standard deviations and results of unbalanced nested anova followed by tukey’s hsd test of fatty acids alkyl esters determined in oils obtained from olives processed immediately after harvesting. oils were obtained by adding or not calcium carbonate (n=4). cultivar trial fame (mg kg-1) faee (mg kg-1) faae (mg kg-1) coratina c 4.53±0.22 a 3.75±0.22 a 8.29±0.41 a ca2-2% 3.90±0.16 ab 3.01±0.27 b 6.91±0.34 b ca2-4% 3.94±0.17 ab 3.58±0.16 a 7.52±0.19 ab ca5-2% 3.83±0.30 b 3.95±0.25 a 7.77±0.25 ab ca5-4% 3.66±0.48 b 3.62±0.36 a 7.27±0.83 b nociara c 3.73±0.02 a 6.31±0.31 a 10.04±0.31 a ca2-2% 3.34±0.44 a 5.65±0.25 a 8.99±0.41 a ca2-4% 3.46±0.34 a 6.08±0.32 a 9.53±0.36 a ca5-2% 3.49±0.07 a 6.07±0.59 a 9.56±0.64 a ca5-4% 3.75±0.38 a 6.00±0.48 a 9.75±0.72 a different letters in the same column for the same cultivar indicate significant differences (p ≤ 0.05). c, control without calcium carbonate addition; ca2, calcium carbonate 2.7 µm; ca5, calcium carbonate 5.7 µm; fame, fatty acids methyl esters; faee, fatty acids ethyl esters; faae, fatty acids alkyl esters. ital. j. food sci., vol. 30, 2018 387 the initial levels of faae in the control samples were different for nociara or coratina cvs.: nociara oils were richer particularly in ethyl esters and, as a consequence, in the total amount of alkyl esters. the different starting conditions might be imputable to the different maturity degree of the drupes. by the progressive advancement of the maturity, fruits become softer and more susceptible to alterations, such as yeast growth, leading to a higher availability of ethanol by anaerobic fermentation (conde et al., 2008; di serio et al., 2017). coadjuvant use showed a different effect depending on the processed cultivar. indeed, no statistical influence was found on the faae amount considering nociara cv., while a significant effect was highlighted in the case of coratina cv. in particular, respect to the control, fame amount was significantly reduced by ca5, regardless the percentage used, while faee were significantly lowered by ca2-2%. as a result, the total faae amount was significantly lower in the case of ca2-2% and ca5-4%. in the light of this, it is possible to suppose a cultivar-dependent effect or, more probably, dependent on the different maturation degree of the cultivar studied. indeed, the different amount of water or interfering compounds such as high mass polymeric substances (mafra et al., 2001) might be determinant in modulating the action of calcium carbonate (aguilera et al., 2010). in order to have a deeper understanding about the coadjuvant effect on faae, the same olives have been processed after 5 and 12 days of storage. it is known that faae amount rises during olive storage as a consequence of degradation and fermentative processes (jabeur et al., 2015). table 3 reports the mean values, the standard deviations, and the statistical analysis of the alkyl esters of oils obtained from stored olives added or not with calcium carbonate during malaxation. table 3. mean values, standard deviations and results of two-way anova followed by tukey’s hsd test of the fatty acids alkyl esters of oils obtained from olives stored for 5 (t5) and 12 (t12) days after harvesting. oils were obtained by adding or not calcium carbonate (n=4). cultivar trial fame (mg kg-1) faee (mg kg-1) faae (mg kg-1) coratina c t5 6.93±0.24 c 17.70±1.23 c 24.63±1.45 c ca2t5-2% 6.89±0.18 c 10.99±0.31 d 17.88±0.22 d c t12 17.36±0.79 a 48.74±1.64 a 66.11±1.49 a ca2t12-2% 12.75±0.21 b 27.60±0.25 b 40.35±0.44 b nociara c t5 8.14±0.21 c 38.61±0.51 b 46.75±0.69 b ca2t5-2% 7.73±0.26 c 31.00±0.47 c 38.73±0.36 c c t12 23.55±0.96 a 174.96±2.01 a 198.51±2.82 a ca2t12-2% 21.99±0.34 b 174.42±1.88 a 196.41±1.96 a different letters in the same column for the same cultivar indicate significant differences (p ≤ 0.05). c, control without calcium carbonate addition; ca2, calcium carbonate 2.7 µm; ca5, calcium carbonate 5.7 µm; fame, fatty acids methyl esters; faee, fatty acids ethyl esters; faae, fatty acids alkyl esters. as expected, the variable days of storage had a highly significant effect on the final alkyl esters amount in both the cultivars (in all cases p < 0.001, data not shown). in particular, the faae increment was mainly due to the increase of faee, in accordance with the findings of (jabeur et al., 2015), confirming the role of ethyl esters of fatty acids as a marker of the raw material quality. however, the increment of these compounds was definitely more evident for nociara cv. instead of coratina one, probably due to the higher ital. j. food sci., vol. 30, 2018 388 maturity degree and/or greater substrate availability for the fermentative activities (gómez-coca et al., 2016), showing the higher susceptibility of the nociara cv. olives respect to the coratina cv. fruits. in fact, it is noteworthy to highlight that in the period from 5 to 12 days of storage, the increase of the total alkyl esters and ethyl esters content was extraordinary in the case of nociara cv. rising of about 325% and 353%, respectively. turning on the action of the coadjuvant, a highly significant effect of calcium carbonate addition was observed (in all cases p < 0.01) and, interestingly, the results were different for the cultivars studied. similarly to what previously observed on fresh fruits, the strongest reduction caused by the coadjuvant in terms of faae was observed during coratina olives processing, leading to a decrease of about 26% and 43% for fame and faee, respectively, in the samples obtained after 12 days of storage. that is, the coadjuvant use at 12 days storage, in these experimental conditions, lowered the faee amount under the maximum eu limit for the extra virgin olive oil classification (official journal of the european communities, 2016) moving from about 50 mg kg-1 to less than 30 mg kg-1. after 5 days storage, it was already observed a significant reduction of faee and faae, even if sharper than what happened after 12 days. in the case of nociara cv., a weaker effect of the calcium carbonate was observed, lowering the amount of faee and faae after 5 days of storage and the amount of fame after 12 days. no significant effect was highlighted on the great content of faee and faae after 12 days of storage. based on the behaviour observed in the case of coratina cv. it seemed possible to suppose that the alkyl esters reduction by calcium carbonate was substrate dependent, i.e. the higher the content the higher the reduction. however, such hypothesis was not confirmed by the results obtained from the trials carried out on nociara cv. 3.2. enzymatic activity evaluation figure 1 depicted the enzymatic activities of polyphenol oxidase (ppo, figure 1a) and peroxidase (pod, figure 1b) measured on the coratina and nociara cvs. olive pastes added or not with calcium carbonate during malaxation. ital. j. food sci., vol. 30, 2018 389 figure 1. polyphenol oxidase (ppo, a) and peroxidase (pod, b) activities measured on the olives pastes of nociara and coratina cvs. obtained from fresh olives (t0) and after 12 days of storage (t12) at ambient temperature added (ca) or not (c) with calcium carbonate. different letters indicate significant variations as determined by three-way anova followed by tukey’s hsd test for multiple comparisons. ppo activity (fig. 1a) of fresh fruits was not affected by the coadjuvant use in both the cultivars. at t12, the enzyme activity was significantly higher than t0, in particular in the case of coratina cv. the increased activity might be due to the progressive structure degradation of the fruits, due to the storage, and to the consequently increase in the substrate availability. this hypothesis seems to be confirmed by the higher enzymatic activity observed in the case of coratina cv., which is known to be rich in phenolics (rotondi et al., 2010). concerning the coadjuvant treatment, it is noteworthy that the use of calcium carbonate significantly increases the ppo activity in both the varieties at t12. the most plausible explanation might be the shift of the olives paste ph to optimal values for the enzyme activity due to the basic hydrolysis of the salt. pod catalyses the oxidation of phenolic compounds using hydrogen peroxide or others organic peroxides from the medium as oxidant agents (gajhede, 2001; kader et al., 2002). at t0, the enzyme activity was higher in the case of nociara cv. respect to coratina one, likely due to the higher value of peroxides (table 1) necessary for the pod action, as previously stated. calcium carbonate significantly affected the enzyme activity in the case of nociara cv. while no statistical difference was observed for coratina. at t12, the pod activity was significantly higher in both the cases respect to t0, and a further significant increase, due to the use of the processing aid, was highlighted. overall, our findings might explain the reduction of the phenolic compounds of the investigated samples with the addition of calcium carbonate (table 4), as also observed in previous studies (squeo et al., 2016; tamborrino et al., 2017). moreover, if these behaviours will be confirmed by further studies, even the action of the calcium carbonate, currently assumed as being merely physical, might be rethought. ital. j. food sci., vol. 30, 2018 390 table 4. mean values of phenolic compounds of the samples under investigation of oils obtained from fresh olives (t0) and after storage for 5 (t5) and 12 (t12) days after harvesting. trial coratina cv. nociara cv. ct0 470 188 ca2t0-2% 463 146 ca2t0-4% 406 127 ca5t0-2% 316 164 ca5t0-4% 324 121 c t5 311 133 ca2t5-2% 262 113 c t12 182 127 ca2t12-2% 139 105 c, control without calcium carbonate addition; ca2, calcium carbonate 2.7 µm; ca5, calcium carbonate 5.7 µm. 4. conclusions the results reported highlighted that the use of calcium carbonate modifies the amount of fatty acids alkyl esters as well as the activity of selected enzymes. coadjuvant addition during malaxation led to a general reduction of faae and an increase in the ppo and pod activities, the former useful for the virgin olive oil classification, the latter involved in the phenolic content of voo. the effect of the coadjuvant seems to be cultivar dependent or, more realistically, linked to the raw material ripening degree and oxidative degradation. in the light of this, further studies are needed to confirm such results considering more thoroughly the effect of the olives maturation degree and even concerning 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tsuda*1,2, s. okuda2, t. haraguchi2 and k. kodama2 1faculty of agriculture and life science, hirosaki university, 3 bunkyo-cho, hirosaki city, aomori 036-8561, japan 2faculty of life and environmental sciences, prefectural university of hiroshima, 562, nanatsuka, shobara city, hiroshima 727-0023, japan *corresponding author: tel.: +89172362111; fax: +81172393750 e-mail address: tsudah@hirosaki-u.ac.jp abstract the highest production of exopolysaccharides (epss) by lactobacillus buchneri gm3701 and lb. plantarum rb-3 was 216 and 79.0 mg/l, when incubated in 10% glucose media at 25°c for 6 d and 5% glucose media at 25°c for 4 d, respectively. the epss consisted of mainly glucose. bacterial growth in the media supplemented with the epss was investigated using various bacteria, including lactobacillus, staphylococcus and escherichia strains. the eps enhanced the growth of lb. farciminis hm2001. this result suggests that the growth of some lactic acid bacteria can be enhanced by the supplementation with an eps produced by lactobacillus strains. keywords: exopolysaccharide, growth enhancement, lactobacillus, yeast extract ital. j. food sci., vol. 31, 2019 234 1. introduction exopolysaccharides (epss) are long-chain carbohydrate polymers that are released by a wide range of microorganisms, including fungi and bacteria (donot et al., 2012). epss are present outside of the cell wall, and they exhibit great diversity, not only in their sugar composition but also in their linkage, branching, and substitution (chapot-chartier and kulakauskas, 2014). epss can be bound or unbound to the cell wall, and cellbound epss are distinguished into capsular polysaccharides (cps) (caggianiello et al., 2016). the physiological role of bacterial epss is not yet completely understood. epss may be associated with cell protection against unfavourable environmental conditions, like desiccation, the presence of oxygen or toxic compounds, low temperatures, high osmotic pressures, and bacteriophage attack, and they may contribute to the uptake of metal ions, biofilm formation, and cell adhesion mechanisms (caggianiello et al., 2016; cerning, 1990; sanchez et al., 2006). on the other hand, liu et al. (2017) reported that epss produced by lactobacillus plantarum inhibited the biofilm formation of pseudomonas, escherichia, salmonella, and staphylococcus. generally, it is thought to be very unlikely that bacteria can use epss as an energy source; however, there are some studies that have reported that epss are degraded by lactic acid bacteria (lab). pham et al. (2000) reported that epss produced by lactobacillus rhamnosus were degraded by the enzymes of this strain and that some reducing sugars were liberated. additionally, some bifidobacterium strains can breakdown plant cell wall polysaccharides (van den broek et al., 2008). furthermore, the growth of lab and bifidobacterium strains were enhanced by supplementing the cultures with the eps produced by lactobacilli (hongpattarakere et al., 2012; korakli et al., 2002; ruijssenaars et al., 2000; tsuda and miyamoto, 2010). it is unclear if this enhancement was because of the utilization of monosaccharides degraded from the epss. to begin with, there are only a few reports about the influence of epss on the growth of lab, and more studies are necessary to better understand the influence of epss on the growth of lab. we should point out that crudely purified epss may contain mannan from the yeast extract in media, and the mannan may be used by some bacteria. in this study, epss, produced by a lactobacillus strain, were investigated for their yields and monosaccharide components. furthermore, the influence of epss on growth was evaluated using lactobacillus, streptococcus, staphylococcus, aerococcus, and escherichia strains. 2. material and methods 2.1. bacterial strains in the present study, 22 bacterial strains, including lactobacillus, lactococcus, enterococcus, streptococcus, staphylococcus, aerococcus, paenibacillus, and escherichia coli, were used (table 1). the strains were isolated from wagyu milk, japanese pickles and fermented sushi at our laboratory unless otherwise stated (tsuda et al., 2012; tsuda, 2015). the lactic acid bacteria were incubated in tyg broth (10 g/l tryptone, 5.0 g/l yeast extract, 5.0 g/l glucose, 1.0 g/l tween 80, and 0.1 g/l l-cysteine hcl monohydrate, ph 6.8 ± 0.2). the other strains were also incubated in tyg broth to compare the growth rate. all strains were stored in 10% reconstituted skim milk at -20°c. an inoculum of 1% was used for all tests. ital. j. food sci., vol. 31, 2019 235 table 1. strains used in the present study. species strain no. source lactobacillus reuteri puhm1004 wagyu milk lb. coryniformis sab01 japanese pickles lb. sakei subsp. sakei sab04 japanese pickles lb. delbruckii subsp. bulgaricus nbrc 13953 * lb. alimentarius em2001 fermented sushi lb. casei hm3701 fermented sushi lb. buchneri gm3701 fermented sushi lb. farciminis hm2001 fermented sushi lb. acidipiscis jam3706 fermented sushi lb. plantarum jab2001 fermented sushi lb. plantarum rb3 japanese pickles lb. plantarum puhm1023 wagyu milk enterococcus faecalis puhm1006 wagyu milk lactococcus lactis puhm1014 wagyu milk streptococcus thermophilus nbrc 13957 * str. salivarius ab3002 fermented sushi str. pluranimalium puhm1022 wagyu milk aerococcus viridans puhm5301 wagyu milk staphylococcus auricularis puhm5201 wagyu milk sta. aurigulas puhm5203 wagyu milk paenibacillus turicensis puhm5101 wagyu milk escherichia coli nbrc 102203 * *: nbrc: nite biological resource center. 2.2. effects of the incubation conditions on exopolysaccharide (eps) production lb. buchneri gm3701 and lb. plantarum rb-3 were used as eps-producing lab. the eps productivities were tested using the 22 strains in table 1, and the cultures of the two strains showed ropiness. therefore, strains gm3701 and rb-3 were selected as eps producers. the ropiness was confirmed by inserting a sterile wire loop and pulling ropes from the media. a clear zone area using the indian ink method was used as a simplified indicator of the eps yield. twenty microliter of lab culture was put on a glass slide, and a few drops of indian ink were added and mixed. a cover slip was placed over the mixture, and then, the prepared slide was observed microscopically with immersion oil. a clear zone area for each individual cell was obtained as follows: the cell area was subtracted from the clear zone area, and then, the obtained figures were divided by the cell numbers. this assay was performed with at least 10 clumps. the effect of the incubation temperature on eps production was investigated at 25, 30, and 37°c. glucose, fructose, sucrose, and lactose were supplemented in ty broth (10 g/l tryptone, 5.0 g/l yeast extract, 1.0 g/l tween 80, and 0.1 g/l l-cysteine hcl monohydrate, ph 6.8 ± 0.2) as carbon sources at 25, 50, and 100 g/l. all assays were performed at least three times. ital. j. food sci., vol. 31, 2019 236 2.3. preparation of the epss a modified version of the method from lindsay et al. (2003) was used to prepare epss from bacterial culture. epss in the bacterial culture were precipitated with two volumes of cold ethanol, followed by stirring for 1 h at 4°c. the precipitated epss were collected by suction filtration, and the collected epss were dissolved in deionized water. the epss were again precipitated with two volumes of cold ethanol and subsequently lyophilized. furthermore, epss from the tyg broth were prepared using the same method. the lyophilized epss were analysed for their carbohydrate and protein content. the total amount of carbohydrates in the lyophilized epss was determined with the phenolsulphuric acid method using glucose as the standard (dubois et al., 1956). the protein content was determined using the protein-dye binding method with bovine serum albumin as the standard (bradford, 1976). all assays were performed at least three times. 2.4. monosaccharide analysis of the epss the lyophilized epss were hydrolysed in 2 m trifluoroacetic acid (tfa) at 120°c for 2 h. after hydrolysis, the water and tfa were removed with a centrifugal concentrator (cc181, tomy, tokyo, japan), and the dry sample was dissolved in water. the hydrolysed eps solution was spotted onto silica gel thin layer chromatography plate (merck, tokyo, japan), along with standard solutions for glucose, galactose, mannose, arabinose, xylose, and rhamnose. the plate was developed in 1-butanol:2-propanol:h2o (3:12:4), dried, sprayed with a phenol-sulfuric solution, and then heated at 110°c for 15 min to visualize brown spots (adachi, 1965; huebner et al., 2007). the monosaccharides of the epss were further analysed as follows. the sugar composition was determined by high pressure liquid chromatography with refractive-index detection (column: sugar-d (nacalai tesque, kyoto, japan); mobile phase: 85% acetonitrile; flow rate: 1.0 ml/min; temperature: 30°c). glucose, mannose, n-acetylglucosamine, arabinose, xylose, and rhamnose were used as the standards. 2.5. influence of the epss on bacterial growth growth in media with epss as the sole carbon source was tested with the 22 strains. glucose medium was used as a control. the growth rate was determined using a modified version of the method from tsuda and miyamoto (2010). briefly, the tested strains were inoculated into ty broth (10 g/l tryptone, 5.0 g/l yeast extract, 1.0 g/l tween 80, and 0.1 g/l l-cysteine hcl monohydrate, ph 6.8 ± 0.2) containing 0.2% (w/v) glucose and the lyophilized epss, and the cultures were incubated for 24 h at 37°c. the optical density at 660 nm (od660) of the culture was measured at 0 and 24 h. all assays were performed at least three times. the growth rate against glucose was determined using the following equation: growth rate = (log od660 of tye at 24 h – log od660 of tye at 0 h) / (log od660 of tyg at 24 h – log od660 of tyg at 0 h) tye: ty broth containing the epss produced by strains gm3701 or rb-3 tyg: ty broth containing glucose ital. j. food sci., vol. 31, 2019 237 2.6. statistical analysis to identify differences, a one-way analysis of variance (anova) was applied to the means, and the student-newman-keuls test (p<0.05) was applied using statview 5.0 software (sas institute, cary, nc, usa). 3. results and conclusions 3.1. effects of the incubation conditions on eps production lb. buchneri gm3701 and lb. plantarum rb-3 are eps-producing lab strains. ropiness was confirmed with a loop, and the clear zone surrounding the cell was confirmed by the indian ink method. the effects of the incubation temperature and carbon source, which included glucose, fructose, sucrose, and lactose (100 g/l), on eps production were investigated with lb. buchneri gm3701 and lb. plantarum rb-3 (fig. 1). concerning strain gm3701, the eps yield was higher at 25 and 30°c than at 37°c for all of the sugars (p<0.05). there were no differences among the four tested sugars at 25°c, and the eps yield in the glucose media was higher at 30°c than in the sucrose or lactose media (p<0.05). concerning strain rb-3, the eps yield was higher at 25 and 30°c than at 37°c for all of the sugars (p<0.05). the eps yield in the glucose media was higher than in the sucrose media, and there were no differences among the four tested sugars at 30°c (p<0.05). from these results, it was presumed that a suitable incubation temperature and sugar for eps production by strains gm3701 and rb-3 were 25°c and glucose, respectively. therefore, these incubation conditions were applied in the series of tests. subsequently, the effect of the carbon concentration (25, 50, or 100 g/l) on eps production was investigated with glucose at 25°c (fig. 2). eps production by gm3701 was higher at 100 g/l glucose after 5, 6, and 7 days than at 25 or 50 g/l (p<0.05), and the production by rb-3 was higher at 50 and 100 g/l glucose after 4 days than at 25 g/l (p<0.05). the eps yields are likely to decrease after reaching a maximum, as many studies have reported, and this is caused by enzymes, such as glycohydrolase, that are produced by bacteria (pham et al., 2000). however, it is unclear whether the degraded epss were used for growth in that paper. 3.2. characteristics of the epss the eps-producing strain was incubated at the above condition, and then, the epss were purified by ethanol precipitation and lyophilized. similarly, the epss from the tyg broth were lyophilized. the polysaccharide (ps) yield from the tyg broth was 50.8 mg/l, and the eps yields from strains gm3701 and rb-3 were 340 and 146 mg/l, respectively (table 2). the carbohydrate and protein contents in these lyophilized epss are shown in table 2. all of the epss contained more than 76% carbohydrates and less than 9.6% protein. these results confirmed that the lyophilized epss were not proteinaceous slime. the monosaccharide analysis of the epss was done using seven monosaccharides that are known as constituents of epss (glucose, galactose, mannose, n-acetylglucosamine, arabinose, xylose, and rhamnose) as standards. the pss from the tyg broth consisted mainly of mannose. ital. j. food sci., vol. 31, 2019 238 figure 1. effects of the incubation temperature (1: 25°c, 2: 30°c, 3: 37°c) and carbon source on eps production by lb. buchneri gm3701 (a) and lb. plantarum rb-3 (b). bars represent the standard deviation from the mean (n=3). ♦: glucose, ■: fructose, ▲: sucrose, ×: lactose. ital. j. food sci., vol. 31, 2019 239 figure 2. effect of the glucose concentration on eps production by l. buchneri gm3701 (a) and l. plantarum rb-3 (b). bars represent the standard deviation from the mean (n=3). ◊: 25 g/l, □: 50 g/l, ∆: 100 g/l. table 2. eps yields and the carbohydrate and protein concentrations in the lyophilized epss. strain eps yield carbohydrate protein composition of the eps (%) (mg/l) (%) (%) glucose galactose mannose rhamnose gm3701 340 76.0 9.6 73.2 23.7 trace* rb-3 146 83.2 3.5 27.2 58.6 14.1 tyg broth 50.8 83.0 2.8 12.0 82.8 *: trace means less than 10%. the correct yields and monosaccharide components of the epss produced by the lab strains were estimated by subtracting the ps values, while taking the carbohydrate concentration into consideration (table 3). the calculated eps yields for the strains gm3701 and rb-3 were 216 and 79.0 mg/l, respectively. glucose was found to be a major component of the eps produced by strain gm3701, and glucose, mannose, and rhamnose ital. j. food sci., vol. 31, 2019 240 were found to be the predominant sugar residues in the eps produced by the strain rb-3 (table 3). glucose and rhamnose are typical components of many epss produced by lab. the quantities of the hetero-epss produced by lab vary greatly. the production of eps is 50-350 mg/l for str. thermophilus, 80-600 mg/l for lc. lactis subsp. cremoris, 60-150 mg/l for lb. delbrueckii subsp. bulgaricus, 50-60 mg/l for lb. casei (cerning, 1995), and approximately 140 mg/l for lb. plantarum (staaf et al., 2000; tsuda and miyamoto, 2010). the highest recorded yields for hetero-epss are 2775 mg/l for lb. rhamnosus rw9595m (macedo et al., 2002) and 2500 mg/l for lb. kefiranofaciens wt-2b (maeda et al., 2004). the eps yields from lb. buchneri gm3701 and lb. plantarum rb-3 were 216 and 79.0 mg/l, respectively, and these values were thought to be a normal value for lactobacillus epss. table 3. eps yields and monosaccharide components of lyophilized epss, obtained by subtracting the values for the epss from the tyg broth. strain eps yield composition of the eps (%) (mg/l) glucose mannose rhamnose gm3701 216 70.9 trace* trace rb-3 79.0 41.7 29.0 28.3 *: trace means less than 10%. the ps from the tyg broth was thought to be mannan. it is well known that purified epss are contaminated with the mannan from the yeast cells in yeast extract. glucose and rhamnose are the usual components of many epss produced by lab (caggianiello et al., 2016; donot et al., 2012; sanchez et al., 2006), and the mechanisms of glucose incorporation into the polysaccharide chain are well known (de vuyst et al., 2001). there are some reports about epss composed of glucose and mannose that are produced by lactobacillus (hashiguchi et al., 2011; sanchez et al., 2006). 3.3. growth enhancement by the epss the growth rates of the epss against glucose for the 22 strains are shown in fig. 3. the highest growth rate was observed with lb. farciminis hm2001 (p<0.05). all of the 22 tested strains showed an od660 of more than 0.3 when they were incubated in tyg broth for 24 h. the growth rates of the epss against glucose were calculated, and all of the tested strains showed a growth rate of less than 0.1, except for strain hm2001. the epss produced by strains gm3701 and rb-3 showed growth rates of 0.146 and 0.113 with strain hm2001, respectively. although the monosaccharide composition of the epss was different between the two epss (table 3), the growth of lb. farciminis hm2001 was enhanced by supplementation with either of the eps produced by the lab. the growth enhancement of this strain did not occur following supplementation with the ps from the tyg broth (data not shown). this suggested that the epss produced by lb. buchneri gm3701 and lb. plantarum rb-3 enhanced the growth of strain hm2001. on the other hand, the epss produced by lb. plantarum rb-3 did not enhance the growth of the three tested lb. plantarum strains. ital. j. food sci., vol. 31, 2019 241 figure 3. growth rates of the epss against glucose for the 22 strains. therefore, no species specificity was shown for growth enhancement by epss in this study. we think that strain hm2001 may have the enzymes that degrade the epss produced by lab, and the degraded sugars may be utilized by strain hm2001; the eps or degraded carbohydrate chains may stimulate the growth by working like an extracellular signalling molecule. another factor, such as the charge and linkage types of the eps and the combinations of the enzymes that degrade epss, may be involved in the utilization of epss. russo (2012) reported that β-d-glucan in epss enhanced the growth of lb. plantarum and lb. acidophilus. therefore, a lot of β-d-glucan may exist in the epss used in this study. further work is needed for a better understanding of the physiological importance of epss. references adachi s. 1965. thin-layer chromatography of carbohydrates in the presence of bisulfite. j. chromatogr. 17:295-299. bradford m.m. 1976. a rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72:248-254. van den broek l.a.m., hinz s.w.a., beldman g., vincken j.-p. and voragen a.g.j. 2008. bifidobacterium carbohydrasestheir role in breakdown and synthesis of (potential) prebiotics. mol. nutr. food res. 52:146-163. caggianiello g., kleerebezem m. and spano g. 2016. exopolysaccharides produced by lactic acid bacteria: from healthpromoting benefits to stress tolerance mechanisms. appl. microbiol. biotechnol. 100:3877-3886. cerning j. 1990. exocellular 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microbiol. 40:194-199. russo p., lopez p., capozzi v., de palencia p.f., duenas m.t., spano g. and fiocco d. 2012. beta-glucans improve growth, viability and colonization of probiotic microorganisms. int. j. mol. sci. 13:6026-6039. sanchez j.i., martinez b., guillen r., jimenez-diaz r. and rodriguez a. 2006. culture conditions detemine the balance between two different exopolysaccharides produced by lactobacillus pentosus lps26. appl. environ. microbiol. 72:74957502. staaf m., yang z., huttunen e. and widmalm g. 2000. structural elucidation of the viscous exopolysaccharide produced by lactobacillus helveticus lb161. carbohydr. res. 326:113-119. tsuda h. and miyamoto t. 2010. production of exopolysaccharide by lactobacillus plantarum and the prebiotic activity of the exopolysaccharide. food sci. technol. res. 16:8792. tsuda h., matsumoto t. and ishimi y. 2012. selection of lactic acid bacteria as starter cultures for fermented meat products. food sci. technol. res. 18:713-721. tsuda h. 2015. identification of lactic acid bacteria from raw milk of wagyu cattle and their tolerance to simulated digestive juice. milk sci. 64:207-214. de vuyst l., de vin f., vaningelgem f. and degeest b. 2001. recent developments in the biosynthesis and applications of heteropolysaccharides from lactic acid bacteria. int. dairy j. 11:687-707. paper received july 3, 2018 accepted october 1, 2018 ijfs#1617_bozza ital. j. food sci., vol. 32, 2020 234 paper occurrence of listeria spp. and antibiotic resistance profiles of listeria monocytogenes from raw meat at retail in turkey p. şanlibaba*1, b. uymaz tezel2, g.a. çakmak3, r. keski̇n4 and m. akçeli̇k5 1ankara university, engineering faculty, department of food engineering, 50th year settlement, 06830 gölbaşı, ankara, turkey 2çanakkale onsekiz mart university, bayramiç vocational school, food technology program, 17700 bayramiç, çanakkale, turkey 3akdeniz university, department of food engineering, faculty of engineering, 07058 antalya, turkey 4department of food engineering, engineering faculty, ankara university, 50th year settlement, 06830 gölbaşı, ankara, turkey 5ankara university, faculty of science, department of biology, 06100 ankara, turkey *corresponding author: sanlibab@ankara.edu.tr abstract a total of 190 raw meat samples were collected in ankara to examine the presence of listeria spp. and l. monocytogenes and its antibiotic resistance. of the examined samples, 57 were positive for listeria spp. and among them, 23 were identified as l. monocytogenes. among l. monocytogenes strains, 86.96% of isolates were positive for the presence of the hlya gene. all l. monocytogenes strains were resistant to ampicillin, fosfomycin, nalidixic acid, linezolid, and clindamycin. multi-drug resistance was observed in 73.91% l. monocytogenes strains. in conclusion, the presence of l. monocytogenes in raw meat may be indicative of poor hygiene or cross-contamination. keywords: listeria monocytogenes, prevalence, antibiotic resistance, raw meat ital. j. food sci., vol. 32, 2020 235 1. introduction listeria spp. are gram-positive, facultatively anaerobic, non-spore forming, rod-shaped bacteria with low g + c content and motile at 10–25°c (nayak et al., 2015; coroneo et al., 2016). so far, twenty-one species of the genus have been identified (ncbi, 2019). of them, listeria monocytogenes has been known as the main causative agent of listeriosis in human and other mammals since the 1920s, while l. ivanovii is an animal pathogen and rarely infects humans (doyle et al., 2001; fallah et al., 2012). the overall rate of listeriosis in humans is low but it can be lethal for high-risk groups like pregnant women, unborn or newly delivered infants, elderly people, severe underlying disease conditions like immune-suppression, organ transplants, patients undergoing treatment for cancer, and aids (khen et al., 2015; abay et al., 2017; du et al., 2017). it is difficult to control of contamination by l. monocytogenes as it can grow at refrigeration temperature, low oxygen levels, high salt concentration (20% w/v), wide ph values ranging from 4.3 to 9.6, low water content, and hypoxic conditions (almeida et al., 2013; du et al., 2017; kurpas et al., 2018; noll et al., 2018). this pathogen is also able to survive in vacuum-packed food and modified atmospheres (lambertz et al., 2012). l. monocytogenes is a widely distributed in the environment such as soil, contaminated silage, feces of some animals, and in non-treated water. therefore, it can easily contaminate food products of both animal and plant origin (pesavento et al., 2010; şanlibaba et al., 2018a). l. monocytogenes is also commonly found in food processing environments and various food items including ready-to-eat (rte) foods, milk and dairy products, meat and its products, unwashed raw vegetables, seafood products, and poultry products (fallah et al., 2012). l. monocytogenes is of particular concern in raw, undercooked or rte foodstuffs because processed foods are easily contaminated with raw foods in the foodprocessing environment or at homes (al-nabusi et al., 2015; oyelami et al., 2018). raw meat, such as red meat and chicken, may become contaminated with l. monocytogenes either environmentally or during shipping and prolonged storage, particularly if they are stored at above 4°c. the additional handling of raw meats at the retail also results in the transmission of l. monocytogenes to raw meats mainly via slicing, weighing, and packaging (kovacevic et al., 2012; kurpas et al., 2018). the main concern in raw meat is that it contaminated with l. monocytogenes can infect processed foods (pesavento et al., 2010). antimicrobial resistance (amr) in foodborne pathogens, especially multidrug resistance, is a great concern for human and animal health at both national and international levels (alonso-hernando et al., 2012; du et al., 2017). it has been reported that about 33000 deaths occur each year in the european union due to amr food-borne pathogens (anon., 2019a). l. monocytogenes is naturally susceptible to a wide range of antibiotics that act on gram-positive bacteria (gomez et al., 2014; wang et al., 2015; byrne et al., 2016; mohamed et al., 2016). however, since the first documented report on multi-drug resistant l. monocytogenes strain isolated from a patient with meningoencephalitis in france in 1988 (wang et al., 2013; noll et al., 2018), many strains isolated from food and environmental and clinical samples have shown resistance to one or more antibiotics used for treating listeriosis (şanlibaba et al., 2018a). the use of antimicrobials in veterinary medicine is the main cause of the development of amr foodborne bacterial pathogens including l. monocytogenes, as amr pathogens can easily be transported from animal to human via food consumptions (conter et al., 2009). the genes responsible for antibiotic resistance could be transferred through movable genetic elements such as conjugative transposons, mobilizable plasmids, and self-transferable plasmids to other foodborne bacteria in the gastrointestinal tract. in listeria spp., efflux pumps have also been reported ital. j. food sci., vol. 32, 2020 236 as the resistant mechanism (lungu et al., 2011). enterococcus spp. and staphylococcus spp. serve as a reservoir of resistance genes for l. monocytogenes (natratilova et al., 2004). many studies have been focused on the prevalence of l. monocytogenes from raw meat and their antibiotic resistance from the different parts of the world (dimic et al., 2010; pesavento et al., 2010; indrawattana et al., 2011; osaili et al., 2011; gomez et al., 2014; al-nabusi et al., 2015). however, most of the earlier studies carried out in turkey (akpolat, 2004; yücel et al., 2005; ceylan et al., 2008; erol and ayaz, 2011; dogruer et al., 2015; abay et al., 2017; kocaman and sarimehmetoğlu, 2017; şanlibaba et al., 2018a; şanlibaba et al., 2018b) have been focused on rte foods, dairy products, vegetables, cooked meat, and poultry. the information on the occurrence of listeria spp., particularly l. monocytogenes strains isolated from raw meat and their antimicrobial resistance, is limited. therefore, the primary aim of this study was: i) to determine the incidence of listeria spp., particularly l. monocytogenes, in raw meat samples (chicken meat and red meat) sold in ankara, turkey, ii) to identify the isolated strains by phenotypic and genotypic methods, and iii) to assess resistance in the isolated strains against 34 different antibiotics used for treating listeriosis. 2. materials and methods 2.1. collection of samples a total of 190 raw meat samples were randomly purchased from various supermarkets and butchers in the capital city of turkey over the period of january to april 2018. sampling locations were randomly selected. the samples were collected only once from each place. these samples consisted of: 1) 80 samples of red meat (minced beef, sliced lamb, and meat cubes) and, 2) 110 samples of chicken meat (legs and wings). all of the samples were non-frozen and kept at refrigeration condition in the retail outlets. while chicken samples analyzed were prepackaged including vacuum and normal atmosphere of packaging, red meat samples were non-packaged. all of the samples were checked for expiry dates and transported to the laboratory under aseptic and refrigerated conditions (+4°c) on the sampling day. 2.2. isolation and identification of listeria spp. the two-stage enrichment method, described by the international organization for standardization (en iso, 11290-1), was used for isolation and identification of listeria spp. briefly, 25 g of each sample was aseptically weighed and mixed with 225 ml half strength fraser broth (mercktm, germany) containing selective supplements as the first enrichment culture in a stomacher bag and homogenized in a stomacher (seward 400, usa) for 2 min. the homogenized sample was incubated at 30±1°c for 24±2 h. thereafter, 0.1 ml of preenriched fraser broth was inoculated into 10 ml of full-strength fraser broth containing selective supplements for second enrichment culture and incubated at 37°c for 48±2 h. after the enrichment procedure, a loopful each of the halfand full-strength fraser broths was plated on the chromogenic listeria agar (aloa agar) (mercktm, germany) and polymixin acriflavine lithium chloride ceftazidime aesculin mannitol (palcam) agar (mercktm, germany). the plates were incubated at 37°c for 24–48 h. the light blue colonies surrounded by an opaque halo on aloa agar and gray-green colonies surrounded by ital. j. food sci., vol. 32, 2020 237 diffuse black zone on palcam agar were considered to be of listeria spp. five presumptive colonies were picked up and further purified on tryptic soy agar supplemented with 0.6% of yeast extract (tsa-ye) (sigmatm, germany) as a non-selective medium. subsequently, the pinpoint colonies of tsa-ye were subjected to identification procedures, which included gram’s staining, catalase reactions, oxidase tests, carbohydrate utilization, camp tests with s. aureus and r. equi, and motility at 20-25°c. the isolated listeria species and the reference strains used in this study were inoculated on tryptic soy broth supplemented with 0.6% of yeast extract (tsb-ye) (sigmatm, germany) and brain heart infusion (bhi) broth (mercktm, germany) and incubated at 35°c for 24 h. all of the strains used in this study were stored at –20°c with 30% (v/v) glycerol (mercktm, germany) throughout the study period. the reference strains were obtained from the culture collection of food microbiology laboratory, department of food engineering, ankara university, ankara, turkey. 2.3. molecular identification of listeria spp. and l. monocytogenes the isolates were subjected to polymerase chain reaction (pcr) analysis to confirm their identity as listeria spp. the genomic dna of the strains grown at 35°c overnight in tsbye was extracted using a genomic dna extraction kit (thermo fisher scientifictm), according to the manufacturer’s instructions. the following reference strains were used: l. monocytogenes atcc7644, listeria innocua atcc12612, listeria seeligeri slcc3945, listeria welshimeri atcc35897. the primer pairs designated as u1 (5'– cagcmgccgcggtaatwc–3') and li1 (5'–ctccataaaggtgaccct–3'), amplifying a 938-bp region in the 16s rrna gene sequence of the listeria genus, were used (usman et al., 2016). in order to detect the presence of the hlya gene, an additional pcr was performed, with dg69 (5'–gtgccgccaagaaaaggtta–3') and dg74 (5'cgccacacttgagatat–3') as primers specifically amplifying a 636-bp fragment of the hlya gene (fallah et al., 2013). a standardized pcr protocol was followed for the bacterial lysates, in a final volume of 50 µl reaction mixture containing 5 µl pcr buffer, 1 µl 2 mm dntp mix, 1 µl of each forward and reverse primers, 34.75 µl of sterile distilled water, 0.25 µl of tag dna polymerase, 4 µl of 25 mm mgcl2, and 3 µl of the dna template (blaiotta et al., 2002). the pcr amplification was performed in a programmed thermocycler (techne tc–512, staffordshire, uk). the pcr conditions were as follows: an initial hold of 2 min at 95°c, followed by 35 cycles each of 45 s denaturation at 95°c, 45 s annealing at 55°c and 2 min extension at 72°c, followed by a final extension for 7 min at 72°c and hold at 4°c. the pcr products were electrophoresed on 1% agarose gels and then stained with ethidium bromide solution (0.5 µg/ml). an o’gene rulertm 10000 bp dna molecular weight ladder (fermentastm, finland) was used as a standard. the gels were visualized under uv light using a kodak gel logic 200 imaging system (kodak, usa). amplified pcr fragments were purified by the pcr purification kit (thermo fisher scientifictm) and were sequenced by refgen biotechnology (ankara, turkey). basic local alignment search tool blast) was used to compare the sequences against the nucleotide database in national centre for biotechnology information (ncbi) (www. ncbi.nlm.nih.gov.tr/blast). ital. j. food sci., vol. 32, 2020 238 2.4. antibiotic resistance of l. monocytogenes strains resistance to different antibiotics was determined for all listeria isolates by the disc diffusion method using mueller-hinton agar (mercktm, germany) as the medium, containing 0.5% defibrinated sheep blood, as described by the clinical and laboratory standards institute (clsi) (2011). the selected antibiotics are the ones, commonly used in veterinary and human medicine against listeriosis. the following antibiotic discs were used: penicillin g (10 µg/disc), oxacillin (1 µg/disc), cefotaxime (30 µg/disc), fosfomycin (50 µg/disc), cephalothin (30 µg/disc), furazolidone (50 µg/disc), piperacillin (30 µg/disc), cefuroxime (30 µg/disc), cefoxitin (30 µg/disc), ampicillin (10 µg/disc), amoxicillin/clavulanic acid (20/10 µg/disc), erythromycin (15 µg/disc), clarithromycin (15 µg/disc), tetracycline (30 µg/disc), tigecycline (15 µg/disc), moxifloxacin (5 µg/disc), neomycin (10 µg/disc), ciprofloxacin (5 µg/disc), enrofloxacin (5 µg/disc), levofloxacin (5 µg/disc), nalidixic acid (30 µg/disc), linezolid (30 µg/disc), kanamycin (30 µg/disc), streptomycin (300 µg/disc), gentamicin (120 µg/disc), vancomycin (30 µg/disc), teicoplanin (30 µg/disc), meropenem (10 µg/disc), imipenem (10 µg/disc), clindamycin (2 µg/disc), trimethoprim (5 µg/disc), trimethoprim/sulfamethoxazole (1.25/23.75 µg/disc), chloramphenicol (30 µg/disc), and rifampicin (5 µg/disc). after the incubation at 35°c for 24 h, the diameters of the inhibition zones around each disc were measured. on the basis of the comparative results, the strains were categorized as susceptible, intermediate, or resistant as per the criteria established by clsi (2011). the breakpoints given for staphylococcus species were used to determine the antibiotic resistance profile of l. monocytogenes, as currently there are no resistance criteria given in the clsi guidelines for listeria spp. (wang et al., 2013; khen et al., 2015; du et al., 2017; kuan et al., 2017). e. coli atcc25922, s. aureus atcc6538, and l. monocytogenes atcc7644 were used as quality control strains. 2.5. dendogram construction method the sequences were aligned with the multiple sequence alignment by clustalw and neighbor-joining method was used for phylogenetic tree. 2.6. statistical analysis all statistical analyses were carried out using spss 16 package. the analysis of one-way variance (anova) followed by tukey’s test was applied to determine the differences in the prevalence of listeria spp. between the red meat and chicken samples, and also between the antibiotic resistance of l. monocytogenes strains. the statistical significance was set at p<0.05. 3. results 3.1. occurrence and incidence of listeria spp. and l. monocytogenes a total of 190 samples were examined for the presence of listeria spp. using a two-step selective enrichment method as recommended by en iso 11290-1. the occurrence of listeria spp. and l. monocytogenes in raw meat samples marketed in turkey is presented in table 1. listeria spp. was detected in 57 (30.00%) of the samples. the 16s rrna sequence ital. j. food sci., vol. 32, 2020 239 analysis indicated l. monocytogenes (12.10%; 23/190) to be the most prevalent in the raw meat samples, followed by l. innocua (11.05%; 21/190), l. welshimeri (6.31%; 12/190), and l. seeligeri (0.52%; 1/190). using neighbour joining method, phylogenetic relationships of 57 listeria spp. were allowed to group into two main clusters. cluster 1 was composed of 1 isolate. fifty-six isolates were belonged to cluster 2 (fig. 1). in addition, l. monocytogenes strains were also screened for the virulence-associated hlya gene. among the 23 l. monocytogenes strains, only 20 (86.96%) were positive for the presence of the hlya gene. lp6, lp16, and lp54 strains of l. monocytogenes, isolated from raw chicken samples, were identified as atypical strains since they were devoid of the hlya gene. the prevalence of listeria spp. in the raw chicken meat samples was as follows: 14.54% (16/110) for l. monocytogenes, 11.81% (13/110) for l. innocua, 6.36% (7/110) for l. welshimeri, and 0.90% (1/110) for l. seeligeri. on the other hand, the prevalence in the raw red meat samples was as follows: 10.00% (8/80) for l. innocua, 8.75% (7/80) for l. monocytogenes, and 6.25% (5/80) for l. welshimeri. the samples of raw chicken had the highest occurrence of listeria spp. (33.63%, 37/190), followed by red meat (25.00%, 20/190). in the prevalence of l. monocytogenes was significantly higher (p<0.05) in the raw chicken meat than that in the raw red meat. table 1. prevalence of listeria species in raw meat samples. food samples number of samples (n) number of positive samples n (%) listeria spp. l. monocytogenes l. innocua l. welshimeri l. seeligeri raw red meat 80 20 (25.00) 7 (8.75) 8 (10.00) 5 (6.25) 0 ( – )a raw chicken meat 110 37 (33.63) 16 (14.54) 13 (11.81) 7 (6.36) 1 (0.90) totals 190 57 (30.00) 23 (12.10) 21 (11.05) 12 (6.31) 1 (0.52) a not detected. 3.2. antibiotic resistance of l. monocytogenes strains the antimicrobial resistance of the 23 l. monocytogenes strains against 34 different antibiotics was examined using the disk diffusion method according to clsi (2011) (table 2). of the l. monocytogenes isolates, 23 were resistant to ampicillin, fosfomycin, nalidixic acid, linezolid, and clindamycin. frequent resistance was seen against piperacillin (86.96%, 20/23), oxacillin (82.61%, 19/23), kanamycin (82.61%, 19/23), neomycin (78.26%, 18/23), penicillin g (73.92%, 17/23), amoxicillin/clavulanic acid (73.92%, 17/23), levofloxacin (73.92%, 17/23), teicoplanin (73.92%, 17/23), moxifloxacin (69.57%, 16/23), ciprofloxacin (69.57%, 16/23), and furazolidone (52.17%, 12/23). furthermore, resistance to enrofloxacin (47.83%, 11/23), rifampicin (17.39%, 4/23), streptomycin (17.39%, 4/23), tigecycline (13.04%, 3/23), cefuroxime (13.04%, 3/23), cephalothin (13.04%, 3/23), trimethoprim/sulfamethoxazole (13.04%, 3/23), and cefotaxime (8.69%, 2/23) was also observed. at least one strain was resistant against either of tetracycline, gentamicin, and meropenem (4.35%). in contrast, all strains were susceptible to cefoxitin, erythromycin, clarithromycin, vancomycin, imipenem, trimethoprim, and chloramphenicol. the differences between the antibiotic resistance of l. monocytogenes strains were not found to be statistical significance (p > 0.05). ital. j. food sci., vol. 32, 2020 240 figure 1. dendrogram showing the evolutionary relationships among listeria isolates based on the 16s rrna sequence analysis. ital. j. food sci., vol. 32, 2020 241 table 2. antibiotic susceptibility and resistance (%) of l. monocytogenes strains isolated from raw red meat and chicken meat samples. antimicrobial agenta l. monocytogenes strains totals (n:23) raw red meat (n: 7) raw chicken meat (n:16) sb ib rb sb ib rb sb ib rb n % n % n % n % n % n % n % n % n % penicillins penicillin g -c 2 28.57 5 71.43 2 12.50 2 12.50 12 75.00 2 8.69 4 17.39 17 73.92 oxacillin 1 14.29 6 85.71 3 18.75 13 81.25 4 17.39 19 82.61 ampicillin 7 100.00 16 100.00 23 100.00 amoxicillin/clavulanic acid 1 14.29 2 28.57 4 57.14 2 12.50 1 6.25 13 81.25 3 13.04 3 13.04 17 73.92 piperacillin 2 28.57 5 71.43 1 6.25 15 93.75 3 13.04 20 86.96 cephalosporins cephalothin 4 57.14 1 14.29 2 28.57 15 93.75 1 6.25 19 82.61 1 4.35 3 13.04 cefotaxime 5 71.43 2 28.57 13 81.25 1 6.25 2 12.50 18 78.27 2 13.04 2 8.69 cefuroxime 6 85.71 1 14.29 14 87.50 2 12.50 20 86.96 3 13.04 cefoxitin 7 100.00 16 100.00 23 100.00 macrolides erythromycin 6 85.71 1 14.29 10 62.50 6 37.50 16 69.57 7 30.43 clarithromycin 5 71.43 2 28.57 14 87.50 2 12.50 19 82.61 4 17.39 tetracyclines tetracycline 5 71.43 1 14.29 1 14.29 14 87.50 2 12.50 19 82.61 3 13.04 1 4.35 tigecycline 6 85.71 1 14.29 14 87.50 2 12.50 20 86.96 3 13.04 quinolones ciprofloxacin 2 28.57 5 71.43 2 12.50 3 18.75 11 68.75 2 8.69 5 21.74 16 69.57 levofloxacin 1 14.29 2 28.57 4 57.14 1 6.25 2 12.50 13 81.25 2 8.69 4 17.39 17 73.92 nalidixic acid 7 100.00 16 100.00 23 100.00 moxifloxacin 3 42.86 4 57.14 4 25.00 12 75.00 4 17.39 3 13.04 16 69.57 enrofloxacin 4 57.14 2 28.57 1 14.29 3 18.75 3 18.75 10 62.50 7 30.43 5 21.74 11 47.83 monurol fosfomycin 7 100.00 16 100.00 23 100.00 a the diameters of the zones were compared with the diameters of the clinic laboratory standards institute (clsi 2011). bs : susceptible, i : intermediately resistant, r : resistant. cnot detected. ital. j. food sci., vol. 32, 2020 242 table 2. antibiotic susceptibility and resistance (%) of l. monocytogenes strains isolated from raw red meat and chicken meat samples (continued). antimicrobial agenta l. monocytogenes strains totals (n:23) raw red meat (n: 7) raw chicken meat (n:16) sb ib rb sb ib rb sb ib rb n % n % n % n % n % n % n % n % n % oxazolidinones linezolid 7 100.00 16 100.00 23 100.00 aminoglycosides kanamycin 1 14.29 6 85.71 3 18.75 13 81.25 4 17.39 19 82.61 streptomycin 4 57.14 2 28.57 1 14.29 12 75.00 1 6.25 3 18.75 16 69.57 3 13.04 4 17.39 gentamicin 5 71.43 2 28.57 10 62.50 5 31.25 1 6.25 15 65.22 7 30.43 1 4.35 neomycin 1 14.29 6 85.71 4 25.00 12 75.00 5 21.74 18 78.26 glycopeptides vancomycin 7 100.00 16 100.00 23 100.00 teicoplanin 2 28.57 5 71.43 4 25.00 12 75.00 6 26.08 17 73.92 carbapenems meropenem 5 71.43 1 14.29 1 14.29 11 68.75 5 31.25 16 69.57 6 26.08 1 4.35 imipenem 7 100.00 15 93.75 1 6.25 22 95.65 1 4.35 lincosamides clindamycin 7 100.00 16 100.00 23 100.00 sulfonamides/trimethoprim trimethoprim 4 57.14 3 42.86 14 87.50 2 12.50 18 78.26 5 21.74 trimethoprim/sulfamethoxazole 5 71.43 1 14.29 1 14.29 10 62.50 4 25.00 2 12.50 15 65.22 5 21.74 3 13.04 amphenicol chloramphenicol 7 100.00 16 100.00 23 100.00 rifamycins rifampicin 2 28.57 3 42.86 2 28.57 9 56.25 5 31.25 2 12.50 11 47.83 8 34.78 4 17.39 nitrofurans furazolidone 2 28.57 5 71.43 8 50.00 1 6.25 7 43.75 10 43.48 1 4.35 12 52.17 the diameters of the zones were compared with the diameters of the clinic laboratory standards institute (clsi 2011). bs : susceptible, i : intermediately resistant, r : resistant. cnot detected. ital. j. food sci., vol. 32, 2020 243 multi-drug resistance, i.e., resistance to three or more antimicrobial agents, was observed in 73.91% (17/23) l. monocytogenes strains. while 69.56% (16/23) of the l. monocytogenes strains were resistant to four antibiotics, and 60.86% (14/23) of the isolates were resistant to five antibiotics. on the whole, 13 of 23 (56.52%) l. monocytogenes strains showed resistance to more than six antibiotics. 4. discussion 4.1. prevalence of listeria spp. and l. monocytogenes from raw meat samples as a result of its psychotropic nature, l. monocytogenes is of great concern to the meat industry. l. monocytogenes contamination can occur in raw meat during processing and storage (abay et al., 2017). this initial contamination can spread, propagate, and increase during further processing of meat (yang et al., 2017). normally, only cooked meat is eaten, thus the existence of l. monocytogenes in raw meat could be problematic only if the meat is eaten raw or insufficiently cooked. we should not underestimate the risk of listeriosis in this type of food. there is no criterion for routine microbiological testing of raw meat for the presence of l. monocytogenes in turkey (anon., 2019b). among the 190 samples tested, 57 were positive for listeria spp. and the isolation rate of l. monocytogenes from raw meat samples (12.10%) was higher than our expectations. the presence of l. monocytogenes in raw meat may be attributed to: i) fecal contamination during evisceration, ii) food handlers, and iii) cross-contamination during processing, transportation or marketing (al-nabusi et al., 2015; gomez et al., 2015). this study was also aimed to detect the presence of the hlya gene in l. monocytogenes strains. this gene is one of the most virulent factors associated with l. monocytogenes and essential for intracellular infection (moreno et al., 2014; usman et al., 2016). it was interesting to note that this virulence gene was detected in 86.96% of the l. monocytogenes strains isolated from the raw chicken samples and the rest 13.04% were devoid of it. these strains were named as atypical strains. the presence of this gene in the pathogens thriving on meat suggests a serious risk to human health (zeinali et al., 2015). to the best of our knowledge, this is the first report on the isolation of atypical l. monocytogenes strains, devoid of the hlya gene from foods in turkey. our results are consistent with those reported by kaur et al. (2007), osaili et al. (2011), moreno et al. (2014), usman et al. (2016) and zeinali et al. (2015), who identified some atypical l. monocytogenes strains from food. the hlya gene has an important role in the invasion process of l. monocytogenes (zeinali et al. 2015). the occurrence of some mutations alternating the genes responsible for pathogenesis may be the reason of the absence of hlya. this may explain why some species do not cause infections. further studies are needed to determine the presence of other virulence genes in these strains. the overall incidence of listeria spp. in all raw meat samples was 30%, which is higher than that reported in italy (21.4%) (pesavento et al., 2010), but lower than that documented in japan (58.7%) (indrawattana et al., 2011), iran (34.7%) (fallah et al., 2012), canada (70%) (al-nabusi et al., 2015), and nigeria (80%) (oyelami et al., 2018). our study showed that the overall incidence of l. monocytogenes in raw meat was 12.10%. the prevalence of l. monocytogenes was higher in raw chicken meat (14.54%) than raw red meat (8.75%). the rate of l. monocytogenes contamination in raw meat, from different parts of the world, was found to be 23.3% in morocco (ennaji et al., 2008), 15.4% in japan (indrawattana et al., 2011), 18.2% in jordan (osaili et al., 2011), 14.1% in ital. j. food sci., vol. 32, 2020 244 iran (fallah et al., 2012), 12% in china (wang et al., 2013), 43.8% in canada (alnabusi et al., 2015), and 28% in nigeria (oyelami et al., 2018). these differences suggest that l. monocytogenes contamination rates may be affected by geographical location, weather conditions, environmental conditions, the actions of food handlers, monitoring studies and isolation methods. in previous studies carried out in turkey (akpolat, 2004; yücel et al., 2005; ceylan et al., 2008; erol and ayaz, 2011; dogruer et al., 2015; kocaman and sarimehmetoğlu, 2017), the contamination rate of l. monocytogenes in raw meat samples was found between 4.7% and 32.76%, which is partially consistent with our study. related to these past results, we observed no increase in the incidence of l. monocytogenes in raw meats from turkey. the reason for this consistency in the incidence of l. monocytogenes in raw meats may be the compliance with the good manufacturing practices (gmps), and hazard analysis and critical control point (haccp) systems. 4.2. antibiotic resistance in l. monocytogenes strains earlier, l. monocytogenes was considered to be susceptible to a wide range of antibiotics (lungu et al., 2011; alonso-hernando et al., 2012). however, there has been an increasing number of reports about l. monocytogenes strains resistant to one or more antibiotics from food, clinical, and environmental products since 1988 (gomez et al., 2014; wang et al., 2015). many strains of l. monocytogenes have been reported to be completely or partly resistant to fluoroquinolones, cephalosporins, aztreonam, pipemidic acid, fosfomycin, and macrolides, and other antibiotics, especially those of the third and fourth generations (ruiz-bolivar et al., 2011; wang et al., 2013; gomez et al., 2014; bryne et al., 2016). in the current study, the antimicrobial resistance tests of l. monocytogenes strains revealed that all of the l. monocytogenes strains were resistant to nalidixic acid, fosfomycin, ampicillin, linezolid, and clindamycin. we observed that 69.57%, 73.92%, 69.57% and 47.83 of l. monocytogenes strains were resistant to ciprofloxacin, levofloxacin, moxifloxacin, and enrofloxacin, respectively, which belong to the fluoroquinolones class of antibiotics. these high rates of resistance against fluoroquinolones could be ascribed to the frequent use of these antibiotics in animal feeds to treat infections (fallah et al., 2012; wang et al., 2015). however, unexpectedly our isolates of l. monocytogenes showed low resistance to cephalothin (13.04%), cefuroxime (13.04%), and cefotaxime (8.69%), and all of them were susceptible to cefoxitin (100%). the members of penicillin group antibiotics include ampicillin, oxacillin, penicillin g, amoxicillin, and piperacillin. they are the most active β-lactam compounds that inhibit the synthesis of bacterial cell wall peptidoglycan (etabu and arikekpar, 2016). l. monocytogenes is naturally susceptible to β-lactams (lungu et al., 2011; byrne et al., 2016). clinicians usually treat human listeriosis with the standard antibiotic therapy that includes high doses of penicillin, ampicillin, and amoxicillin alone or combined with gentamicin (korsak et al., 2012; gomez et al., 2014; shi et al., 2015; olaimat et al., 2018). in the present study, l. monocytogenes strains showed high resistance to ampicillin (100%), piperacillin (86.96%), oxacillin (82.61%), penicillin g (73.92%), and amoxicillin/clavulanic acid (73.92%). this finding is highly significant as far as the treatment of human listeriosis is concerned. however, our results were not similar to the previous studies conducted in turkey (terzi et al., 2015; kocaman and sarimehmetoğlu, 2017), which reported low resistance to these antibiotics. thus our observations are of great medical concern. trimethoprim alone or combined with ital. j. food sci., vol. 32, 2020 245 sulfamethoxazole is generally used in the case of allergy to beta-lactams and rifampin, erythromycin, vancomycin, linezolid, and meropenem can also be used as possible alternatives (al-nabusi et al., 2015; şanlibaba et al. 2018a). it is worth noting that our results showed that resistance to trimethoprim/sulfamethoxazole (13.04%) and meropenem (4.35%) was found to be low in this study. in addition, resistance to trimethoprim was not observed in this study, in line with wang et al. (2015), obaidat et al. (2015), and kuan et al. (2017). clindamycin interferes with bacterial protein synthesis in a similar way to erythromycin and chloramphenicol (shi et al., 2015). therefore, owing to the similar mode of action, a cross-resistance among clindamycin, erythromycin, and chloramphenicol can sometimes be detected (ruiz-bolivar et al., 2011; moreno et al., 2014). in this study, no resistance to chloramphenicol and erythromycin was observed; this concords with the findings of yücel et al. (2005), ennaji et al. (2008), and chen et al. (2010). in contrast, resistance to clindamycin was 100%. this, to the best of our knowledge, seems to be the first report from turkey showing the cross-resistance of these antibiotics. these results also concord with the theory of a specific enzyme that inactivates clindamycin, as previously reported for staphylococcus spp. (ruiz-bolivar et al., 2011; moreno et al., 2014). rifampin is the main antibiotic used in the treatment against mycobacterium tuberculosis and gram-positive bacteria (ruiz-bolivar et al., 2011). it has also been recommended to treat listeriosis (olaimat et al., 2018). fortunately, the resistance rate (17.39%), found in our work, does not seem to be alarming and is in agreement with fallah et al. (2013). vancomycin is one of the last therapeutic options for the treatment of human listeriosis especially in case of bacteremia and endocarditis (obaidat et al., 2015). fortunately, all l. monocytogenes strains were found susceptible to the vancomycin in accordance with the results of rahimi et al. (2010), okada et al. (2011), wang et al. (2015), al-nabusi et al. (2015), and bryne et al. (2016). the resistance of the isolates against vancomycin is a contrary finding to that obtained by ieren et al. (2013) and fallah et al. (2013). tetracycline and tigecycline are the members of tetracyclines, whose target of antimicrobial activity in bacteria is the ribosome (etebu and arikekpar, 2016). tetracycline resistance has most frequently been reported in listeria spp. of different origins (pesavento et al., 2010; fallah et al., 2012; korsak et al., 2012). this might have arisen due to the extensive and prolonged use of these antimicrobials in human medicine and as growth promoters in animals (bryne et al., 2016). in contrast, these results could not be confirmed by us, because only one and two of the examined l. monocytogenes strains were resistant to tetracycline and tigecycline, respectively. the rare occurrence of resistance to tetracycline (4.35%) and tigecycline (13.04%) is in accordance with rahimi et al. (2010), al-nabusi et al. (2015), and noll et al. (2018). tetracyclines are not used as the first drug of choice for listeriosis treatment and their use is also not recommended in children and pregnant women (ruiz-bolivar et al., 2011). while l. monocytogenes strains showed resistance to kanamycin (82.61%) and neomycin (78.26%), low resistance to streptomycin (17.39%) and gentamicin (4.35%) was also observed. the high frequency of resistance to kanamycin and neomycin was unexpected and might be in part due to the excessive use of these antibiotics in veterinary medicine in turkey. these results are in agreement with alonso-hernando et al. (2012) and shi et al. (2015). however, some authors have reported high sensitivity of l. monocytogenes to kanamycin (okada et al., 2011; wang et al., 2013; jamali et al., 2015; wu et al., 2015). in the present study, there was no significant association between the different l. monocytogenes strains in terms of antibiotic resistance (p< 0.05). ital. j. food sci., vol. 32, 2020 246 the prevalence of multidrug resistance l. monocytogenes strains isolated from raw meat was 73.91% (data not shown). the multi-resistance patterns reported in other countries are as follows: 16.4% in iran (rahimi et al., 2010), 48% in colombia (ruiz-bolivar et al., 2011), 64.3% in nigeria (ieren et al., 2013), 2.9% in spain (gomez et al., 2014), 21.25% in china (shi et al., 2015), 21% in germany (noll et al., 2018), and 81% in malaysia (kuan et al., 2017). these differences among multidrug resistance in l. monocytogenes strains could result from the differences in the use of antimicrobials at the regional level. the result of our study suggested that the overall incidence of antibiotic resistance in l. monocytogenes is alarming. further, the high resistance rate observed against antibiotics commonly used to treat listeriosis, such as penicillin, oxacillin, ampicillin, piperacillin, and amoxicillin/clavulanic acid, is also a great concern. 5. conclusions our results regarding the occurrence of l. monocytogenes in raw meat and the presence of virulence-associated genes in the strains indicate an alarming situation to the public health. the counts of l. monocytogenes in raw meat at the retail level are crucial for contaminating with cooked foods. retail centers must be controlled legally monitored. this study is the first report of hlya negative l. monocytogenes strains isolated from food in turkey. in this study, we have demonstrated that all l. monocytogenes strains were resistant to ampicillin, fosfomycin, nalidixic acid, linezolid, and clindamycin against gram-positive bacteria. the controlled use of 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effect of antioxidants and packing conditions on storage stability of cereal bar fortified with hydrolyzed collagen from seabass skin s. benjakul*a, s. pisuchpenb, n. o’brienc and s. karnjanapratumd adepartment of food technology, faculty of agro-industry, prince of songkla university, hat yai, songkhla 90112, thailand bdepartment of material product technology, faculty of agro-industry, prince of songkla university, hat yai, songkhla 90112, thailand cschool of food and nutritional sciences, university college cork, cork, ireland dfaculty of agro-industry, king mongkut's institute of technology ladkrabang, ladkrabang, bangkok 10520, thailand *corresponding author: tel.: +6674286334; fax: +6674558866 e-mail address: soottawat.b@psu.ac.th abstract effect of antioxidants (green tea powder (gt) and citric acid (ca)) and packing conditions on storage stability of cereal bar fortified with hydrolyzed collagen (hc) from seabass skin was studied up to 6 months of storage at 25°c in dark. up to 3 months of storage, the addition of antioxidants impeded lipid oxidation, especially those cereal bars packed in polypropylene with normal heat seal (pp). changes in moisture content, water activity, color, texture, pv, tbars and formation of volatiles were effectively retarded when samples were packed under n2 gas in laminated polyethylene/aluminium foil bag (lf) for 6 months of storage. keywords: hydrolyzed collagen, cereal bar, antioxidants, packing condition, physicochemical parameters ital. j. food sci., vol. 31, 2019 348 1. introduction the market for functional foods has been continuously growing (vicentini et al., 2016). fish hydrolyzed collagen (hc), has been demonstrated to contain low-molecular-weight peptides with a wide range of bioactivities (gómez-guillén et al., 2011). bioactivity and health enhancing potentials have led to the use of hc, especially from marine animal, for developing functional foods, cosmetics and pharmaceutical products (rustad et al., 2011; zhuang et al., 2009). with increasing consumer demand for health promoting food, fish hc is of increasing interest as a bioactive ingredient because of its associated nutraceutical aspects (thiansilakul et al., 2007). it can be fortified in drinks to enhance bioactivity such as antioxidant activity (chuaychan et al., 2016). however, fish hc addition can potentially bring about a fishy odor in the finished product, leading to rejection by consumers. recently, bioactive hc powder without fishy odor was developed using enzymatic hydrolysis of seabass skin (benjakul et al., 2017). thus, it could be fortified at higher level imparting increased bioactivities. cereal bars are eaten regularly due to their nutritive values and ease of consumption. several kinds of cereal bars have been produced to serve the growing functional food market (dean et al., 2007; talens et al., 2012). from our preliminary study, the relationship between health concerns of the consumers and purchase intention for cereal bar fortified with hc was investigated. the results indicated that hc powder can be used as supplement for the development of functional cereal bar. focus group results suggested that production of cereal bars consisting of many grains with high levels of hc powder (5%) was feasible. in general, cereal bars have a high oil content, mainly from nuts and some grains, which are susceptible to oxidation during storage. furthermore, hc powder is hygroscopic and absorbs water easily. these changes directly affect physical, chemical and sensory properties of the cereal bars fortified with hc powder during the extended storage. antioxidants have been employed in foods to prevent lipid oxidation, which can result in off-odor and toxicity in foods (rostamzad et al., 2011). due to safety concern, synthetic antioxidants have been commonly replaced by natural antioxidants. antioxidative compounds such as citric acid and green tea powder were reported to retard lipid oxidation in food products (rostamzad et al., 2011; lorenzo and munekata, 2016). moreover, packaging materials and storage conditions are important factors governing the shelf-life of foods via lowering water migration as well as oxygen permeability (nilsuwan et al., 2016; bakkalbaşı et al., 2012). lipid oxidation can be inhibited by using a packaging material with good protection and barrier properties or by storing the products in atmospheres containing a low oxygen content (nilsuwan et al., 2016; bakkalbaşı et al., 2012). the application of antioxidants along with appropriate packaging condition could be an effective means for the shelf-life extension of cereal bars fortified with hc powder. the objective of this study was to investigate the effect of some antioxidants and packaging conditions on quality changes of cereal bars fortified with hc powder during storage at 25°c. 2. materials and methods 2.1. enzymes/materials/chemicals alcalase (ec 3.4.21.62) (food grade) from bacillus licheniformis and papain (e.c. 3.4.22.2) from papaya (carica papaya) latex were gifted from siam victory chemicals co, ltd. (bangkok, thailand). citric acid was procured from chemipan corporation co., ltd. ital. j. food sci., vol. 31, 2019 349 (bangkok, thailand). rolled oat meal (mcgarrett, pk trading (thailand) co., ltd, bangkok, thailand), all dried fruits, nuts and grains (my choice, central food retail company ltd, nonthaburi, thailand), green tea powder (changtong factory, hat yai, songkhla, thailand), strawberry flavour, corn syrup, honey and ingredients for preparing cereal bar were purchased from a local market in hat yai, songkhla, thailand. ammonium thiocyanate, 2-thiobarbituric acid and 1,1,3,3-tetramethoxypropane were obtained from sigma (st. louis, mo, usa). 2.2. production of hydrolyzed collagen (hc) from seabass skin hc from seabass (lates calcarifer) skin was prepared using two-step enzymatic hydrolysis process as described by benjakul et al. (2017). briefly, skins of seabass (lates calcarifer) were washed and cut into small pieces (3.0 × 3.0 cm2). the skins were then pretreated to remove non-collagenous proteins by soaking in 0.10 m naoh with a skin/alkaline solution ratio of 1:10 (w/v) for 3 h. alkali-treated skins were washed until neutral or faintly basic ph of wash water was obtained. the pretreated skins were subsequently immersed in 0.1 m citric acid with a skin/solution ratio of 1:10 (w/v) for 2 h. the swollen skins were washed until wash water became neutral or faintly acidic. the prepared skins were subjected to hydrolysis. to the pretreated skins, deionized (di) water was added at a ratio of 1:5 (w/v). the ph of mixture was adjusted to 7.0 using 1.0 m naoh and 1.0 m hcl and incubated at 40°c for 15 min. papain (3% of solid content) was added and the hydrolysis was conducted at 40°c for 3 h with continuous stirring. the reaction was terminated by heating at 90°c for 15 min. for the second step of hydrolysis, alcalase (2% solid content of fish skin) was added into the mixture, in which ph was readjusted to 8. hydrolysis was performed at 50°c for 2 h. after inactivation at 90°c for 15 min, the resulting hc was filtered through 2 layers of cheesecloth and 2 layers of fiber grass filter to remove the debris. the filtrate was then fed to a filter unit, which consisted of 4 filter cartridges (crc-20-bp-5, c.c.k, taiwan) and 2 carbon capsules (block carbon treatton 20, treatton, taiwan) using a diaphragm pump (r/o-450, treatton, taiwan). feed flow rate was 2 l/min. the resulting filtrate was then concentrated to obtain 10% solid using an alcohol recovery evaporator (euro best technology co., ltd, pathumthani, thailand) at 50°c. hc concentrate was subjected to drying using a spray-dryer (labplant sd-06 basic, north yorkshire, england) equipped with a spry-drying chamber and a two-liquid-nozzle spray nozzle (0.5mm in size). the air inlet temperature was set at 200°c and outlet temperature was controlled at 90°c. the obtained powder was packed in laminated polyethylene/aluminium foil bag and sealed under vacuum. hc powder was kept at 4°c until used. 2.3. preparation of cereal bar fortified with hydrolyzed collagen (hc) the formulation and preparation of cereal bar fortified with hc are shown in fig.1. briefly, all dry ingredients were mixed together in a dough mixer (kitchenaid casserole multifunctional 5k, kitchenaid, benton harbor, mc, usa) at a low speed for 3 min. liquid ingredients were separately mixed and preheated at 75°c for 5 min. liquid mixture was then added to the dry mixture. the mixture was subsequently stirred at a low speed for 3 min. the resulting sticky cereal mixture (35 g) was placed on wax paper, transferred and pressed into aluminium baking tray (2.0×5.0 cm2) with the height of 1.5 cm. the cereal bar in aluminium tray was baked in an electric oven (mamaru mr-1214, mamaru (thailand) co., ltd., bangkok, thailand) at 180°c for 10 min. after baking, the cereal bars were allowed to cool at room temperature for 1 h. the resulting cereal bars were referred ital. j. food sci., vol. 31, 2019 350 to as ‘con’ cereal bar. for another portion of cereal bar mixture, green tea powder (1.0% of total weight) and citric acid (0.01% of total weight) were mixed with cereal bar mixture in the same manner. bars were formed and baked as previously described. the resulting cereal bars were named as ‘gt+ca’ cereal bar. figure 1. diagram of preparation of cereal bar fortified with hydrolyzed collagen (hc). 2.4. packing of cereal bars both ‘con’ and ‘gt+ca’ cereal bars were packed under two different packing conditions. for the first group, the samples were placed in polypropylene (pp) pouch and heat sealed using an impulse sealer with a magnet model me-300him (s.n. mark ltd., park, nonthaburi, thailand). the second group was placed in laminated polyethylene/aluminium foil bag with nitrogen gas flush (lf) (2.8-4.0% o2) before being sealed using a vacuum sealer v-300 (fnb machinery & solutions, bangkok, thailand). pp bag (3.0×7.0 cm2) had 89 µm thickness with a water vapor and oxygen permeabilities of 6.09 g.mm/day.m2.mmhg (25°c) and 2.770 (mlo2/day.pack), respectively. lf bag (3.0×7.0 cm2) had 85 µm thickness with a water vapor and oxygen permeabilities of 5.76 g.mm/day.m2.mmhg (25°c) and 0.002 (ml o2/day.pack), respectively. the thickness of dry ingredients almond slices (25%) rolled oat meal (20%) dried berry mix (15%) pumpkin seed (10%) puffed rice (10%) hc powder (5%) liquid ingredients glucose syrup (8%) honey (4.5%) strawberry flavor (2.5%) mix and preheat at 75°c for 5 min mix in a dough mixer for 3 min mix dry and liquid ingredients in a dough mixer at a low speed for 3 min press the prepared mixture (35 g) into an aluminium baking tray bake at 180°c for 10 min cool at room temperature for 1 h ital. j. food sci., vol. 31, 2019 351 packaging material was measured using a micrometer (mitutoyo, model id-c112pm, serial no. 00320, mitutoyo corp., kawasaki-shi, japan). water vapor (wvp) and oxygen permeabilities were determined using astm method (shiku et al., 2004) and ambient oxygen ingress rate (aoir) method (larsen et al., 2000), respectively. oxygen content in packaging was investigated using map-pak combi gas analyzer (agc instruments, co clare, ireland). the packaged samples were stored in the dark at the controlled temperature (25°c) for 6 months. at 0, 1, 2, 3, 4, 5 and 6 months of storage, the samples were taken for analyses. 2.5. physicochemical analyses 2.5.1 determination of moisture content and water activity the samples were analyzed for moisture content using an oven method (aoac, 2002). water activity (aw) was measured using a water activity meter (4tev, aqualab, pullman, wa, usa). 2.5.2 measurement of color parameters the cereal bar was ground using a blender (model mx-898n, panasonic, panasonic sdn. bhd., kuala lumpur, malaysia) for 3 min. the color parameters of samples were then determined using a colorimeter (colorflex, hunter lab reston, va, usa). the color values were reported in the cie system, including l*, a* and b*, representing lightness, redness/greenness and yellowness/blueness, respectively. total difference of color (∆e*) was calculated as described by takeungwongtrakul et al. (2015). ∆e∗ = ∆l∗! + ∆a∗! + ∆b∗! (1) where ∆l*, ∆a*and ∆b*are the differences between the corresponding color parameter of the sample and that of day 0. 2.5.3 measurement of textural properties hardness and crispiness of cereal bar were determined by a texture analyzer (stable micro systems, godalming, surrey, uk) using a test speed of 1.0 mm/s with a load cell of 50 kg. a special pasta blade and plate (probe ta 47, 60 mm x 20 mm) were used to imitate the biting action of a tooth. the maximum force required to break cereal bar and the distance at break were calculated for each sample. ten measurements were made for each sample. 2.5.4 determination of lipid oxidation 2.5.4.1 peroxide value (pv) pv was determined in oil extracted from the cereal bar using the bligh and dyer method (bligh and dyer 1959). pv was determined using the ferric thiocyanate method (takeungwongtrakul et al., 2015). a standard curve was prepared using cumene hydroperoxide with the concentration range of 0.5–2 ppm. pv was expressed as µg cumene hydroperoxide/g sample. ital. j. food sci., vol. 31, 2019 352 2.5.4.2 thiobarbituric acid reactive substances (tbars) tbars were determined using a distillation tba method as described by karnjanapratum and benjakul (2015b). ten grams of sample, 97.5 ml of deionized water and 2.5 ml of 6 n hcl were transferred to a kjeldahl flask. the mixture was heated and the distillate (200 ml) was collected. to determine tbars, the distillate (0.2 ml) was added to 1 ml of tbar solution (0.375% thiobarbituric acid, 15% tca and 0.25m hcl) and heated in boiling water for 10 min. after cooling with running water and centrifugation at 5000xg for 10 min at room temperature, the absorbance of the pink solution was read at 532 nm. tbars value was calculated from a standard curve of malondialdehyde (mda) (0-10 mg/l) and expressed as µg mda/g sample. 2.5.5 volatile compounds the volatile compounds in the cereal bar samples were determined at 0, 3 and 6 months of storage using solid-phase microextraction gas chromatography mass spectrometry (spme gc–ms) following the method of takeungwongtrakul and benjakul (2017). volatiles were expressed as the abundance (peak area). 2.6. sensory evaluation the packaged samples were taken for sensory evaluation at 0, 3 and 6 months. sensory evaluation was performed by 50 untrained panelists. they were asked to evaluate for appearance, color, odor, flavor, taste, texture and overall likeness using a nine-point hedonic scale, in which a score of 1 = not like very much, 5 = neither like nor dislike and 9 = like extremely. the samples were labelled with random three-digit codes. panelists were instructed to rinse their mouth with drinking water after each sample evaluation and the order of presentation of the samples was randomized (meilgaard et al., 2006). the samples with likeness score less than 7 (moderately like) were not used for further analyses. 2.7. microbiological analysis total viable microbial count was enumerated by pour plating on plate count agar (pca, difco laboratories inc., detroit, mi, usa) at 37°c for 48 h. yeast and mold counts were determined by pour plating using potato dextrose agar (pda, laboratories inc. detroit, mi, usa) at 30°c for 72 h. 2.8. statistical analysis completely randomized design was used. experiments were run in triplicate using three lots of samples. data were subjected to analysis of variance (anova). comparison of means was carried out by the duncan’s multiple range tests (steel and torrie, 1980). statistical analysis was performed using the statistical package for social science (spss 11.0 for windows, spss inc., chicago, il, usa). ital. j. food sci., vol. 31, 2019 353 3. results and discussion 3.1. changes in moisture content and water activity during storage moisture content and water activity of hc fortified cereal bars without (con) and with (gt+ca) addition of antioxidants, packed under different conditions during storage are shown in fig. 1. figure 1. moisture content (a) and water activity (b) of hc fortified cereal bars packed under different conditions during storage. con; cereal bar without antioxidants. gt+ca; cereal bar with added antioxidants (1.0% green tea powder, 0.01% citric acid). pp; polypropylene bag with normal heat seal. lf; laminated polyethylene and aluminium foil bag with modified atmosphere pack (n2 flush) before sealing. bars represent the standard deviation (n=3). moisture contents of all samples during storage up to 6 months were in the range of 3.925.26% (w/w) (fig. 1a). at day 0, moisture contents of con sample (4.16%) was significantly higher than that of gt+ca sample (3.92%) (p<0.05). antioxidants (gt+ca) were added in the powder form, thereby lowering the moisture content of cereal bars. sharp increase in moisture content was found for the samples packed in pp within the first month of storage (p<0.05), regardless of incorporation of antioxidants. thereafter, the con and gt+ca samples packed in pp bag had constant moisture content up to 3 months of storage (p>0.05). for the samples packed in lf bag, the moisture contents increased ital. j. food sci., vol. 31, 2019 354 linearly up to 3 months (p<0.05), irrespective of incorporation of antioxidant. however, no changes were found between 3-6 months of storage (p>0.05). it was noted that the con sample had higher moisture content than gt+ca sample throughout the storage of 6 months. these results suggested that packaging played a profound role in preventing the migration of water into the cereal bars. lf bag showed the higher water vapor barrier property with a water vapor permeability of 5.67 g.mm/day.m2.mmhg (25°c), compared with pp bag (6.09 g.mm/day.m2.mmhg). a similar result was observed for the change in water activity of cereal bars packed in different packing conditions during storage (fig. 1b). at day 0, the con sample had higher water activity than gt+ca sample (p<0.05). water activity of all samples continuously increased, especially those packed in pp bag, which showed the higher increasing rates, compared with those packed in lf bag, regardless of antioxidants used. water activity of all samples during 6 months of storage was in the range of 0.48-0.53. cereal bars with water activity lower than 0.6 are microbiologically safe under storage condition tested. freitas and moretti (2005) studied the stability of cereal bar with high protein using different packaging materials at room temperature. increase in moisture content was observed during storage period tested and the best packaging with respect to water vapor permeability was the one containing aluminium (freitas and moretti, 2005). senhofa et al. (2015) found that the water activity of muesli stored in different packaging varied with packing materials and moisture permeability. in the present study, the packing condition had higher impact than the addition of antioxidants on moisture content and water activity change of cereal bars during storage. 3.2. changes in color during storage color of hc fortified cereal bars without (con) and with addition of antioxidants (gt+ca) packed under different packing conditions during 6 months of storage is presented in table 1. for day 0, con sample showed the lighter color as indicated by higher l* values, compared to that of gt+ca sample (p<0.05). gt+ca sample was more greenish but less yellowish in color as indicated by the lower a* and b* values, respectively, compared to the con sample (p<0.05). this could be related to green color of green tea powder used as antioxidant in the formulation, which contributed to the green color of product. overall, the color of cereal bars was changed during storage, especially those packed in pp bag. the darker color with less yellowness as indicated by the decreased l* and b* values was observed for the con sample packed in pp bag after storage for 3 months (p<0.05), compared to those found at day 0. a similar result was obtained for the gt+ca sample packed in pp bag, in which less yellowness and redness indicated by lower a* and b* values were noted after 3 months of storage (p<0.05). on the other hand, a lower rate of change in color of samples during storage was observed when lf bag was used, particularly in combination of gt+ca addition. these results were in accordance with ∆e* value. increase in ∆e* value at the higher rate was observed from the sample packed in pp bag, especially those of con sample, during the storage. the addition of antioxidants (green tea and citric acid) could retard the discoloration of cereal bar during extended storage as evidenced by the retarded changes in lightness (l* value) and yellowness (b* value), compared with those of the con sample. moreover, lower o2 permeability of lf bag with better water vapor barrier property could reduce chemical reactions, particularly lipid oxidation and maillard reaction. maillard reaction is predominant at room temperature in low-moisture products (aw 0.5-0.7) with high protein content (baptisma and carvalho, 2004). lipid oxidation also resulted in propagation of the maillard reaction (becker et al., 2009). lipid oxidation occurred to a lower extent ital. j. food sci., vol. 31, 2019 355 when oxygen content in packaging was lowered and packaging material with low water vapor permeability was used (bakkalbaşı et al., 2012; nilsuwan et al., 2016). table 1. changes in color (l*, a*, b* and ∆e* values) of hc fortified cereal bars packed under different conditions during the storage. color storage time (month) con gt+ca pp lf pp lf l* 0 67.94±0.96a 62.60±0.86a 1 65.56±0.70a 68.14±1.00a 61.83±1.54ab 61.82±1.43ab 2 64.70±2.86a 66.91±1.03a 61.53±2.13ab 61.80±0.92ab 3 61.23±2.65b 65.46±1.42a 61.33±3.50ab 60.52±2.21ab 4 nd. 65.30±2.00a nd. 60.10±3.72ab 5 nd. 60.63±1.31b nd. 60.10±1.39ab 6 nd. 56.81±2.42c nd. 58.28±0.89b a* 0 5.11±0.28abc 2.99±0.66a 1 5.73±0.68ab 6.28±1.34a 2.18±0.14bc 2.65±0.44ab 2 4.28±1.33bc 5.42±1.14ab 2.16±0.64bc 2.52±0.21ab 3 3.66±0.98c 5.17±0.23abc 1.46±0.55c 1.99±0.32bc 4 nd. 5.13±0.56abc nd. 1.68±0.21c 5 nd. 4.31±0.40bc nd. 1.60±0.33c 6 nd. 4.31±0.39bc nd. 1.55±0.47c b* 0 26.22±0.78a 22.29±0.46a 1 23.34±1.62bcd 24.97±0.37ab 22.57±0.74a 22.28±1.06a 2 23.02±1.02bcd 24.84±0.41ab 20.67±0.29bc 22.14±0.61ab 3 22.19±1.02cd 24.49±0.39ab 20.69±0.57bc 21.89±0.99ab 4 nd. 23.43±1.98bc nd. 21.33±0.64abc 5 nd. 21.57±0.89cd nd. 20.20±1.04cd 6 nd. 21.22±1.54d nd. 19.22±1.01d ∆e* 0 1 3.79±0.97a 1.72±1.14a 1.15±0.90a 0.85±0.86a 2 4.63±2.18b 1.75±0.94a 2.11±1.28b 0.94±0.48b 3 7.96±1.84c 3.02±0.61b 2.55±2.64b 2.34±1.49c 4 nd. 3.84±1.61b nd. 2.98±2.90c 5 nd. 8.70±0.39c nd. 3.54±0.85d 6 nd. 12.23±1.65d nd. 5.49±0.58e values and mean ± sd (n = 3). nd.; not detected. con; cereal bar without antioxidants. gt+ca; cereal bar with added antioxidants (1.0% green tea powder, 0.01% citric acid). pp; polypropylene plastic with normal heat seal. lf; laminated polyethylene and aluminium foil bag with modified atmosphere pack (n2 flush) before sealing. different lowercase letters for the same color attribute within the same column indicate significant difference (p < 0.05). the generated lipid oxidation products, especially aldehydes and ketones, could be a carbonyl source for condensation with amines. as a consequence, browning discoloration via maillard reaction could take place. additionally, with increasing storage time, ital. j. food sci., vol. 31, 2019 356 maillard reaction proceeded to higher extent. thus, browning occurred to a higher degree, particularly for the con sample packed in pp bag. therefore, the addition of antioxidants and packing condition used played an important role in maintaining color of cereal bar during storage. the result indicated that lf bag showed higher preventive effect on color changes of cereal bar fortified with hc than pp bag during the extended storage. 3.3. changes in hardness and crispiness during storage hardness and crispiness of cereal bars without (con) and with addition of antioxidants (gt+ca) stored under different packing conditions during 6 months of storage are shown in fig. 2. figure 2. hardness (a and c) and crispiness (b and d) of hc fortified cereal bars packed under different conditions during storage. con; cereal bar without antioxidants. gt+ca; cereal bar with added antioxidants (1.0% green tea powder, 0.01% citric acid). pp; polypropylene bag with normal heat seal. lf; laminated polyethylene and aluminium foil bag with modified atmosphere pack (n2 flush) before sealing. bars represent the standard deviation (n=3). the highest hardness (195.90-206.39 n) was observed for both con (fig. 2a) and gt+ca samples (fig. 2c) at day 0 of storage (p<0.05). within the first 3 months of storage, hardness decreased for all samples packed in both pp bag and lf bag. there was no difference between cereal bars packed in pp bag and lf bag at the same storage time (p>0.05). after 3 months of storage, hardness of all samples packed in lf bag remained unchanged (p>0.05), excepted that of gt+ca samples after 6 months of storage, which had decreased hardness (p<0.05). this phenomenon was related to the slight increase in moisture content during storage (fig. 1a). increased moisture content more likely negatively affected the textural property of cereal bar. lf bag with better moisture barrier property packaging material prevented and reduced water vapor migration from environment to product, compared with pp bag. this was related with the lower crispiness of cereal bars packed in pp bag, compared to those packed in lf bag, ital. j. food sci., vol. 31, 2019 357 particularly within the first 3 months of storage (fig. 2b and 2d). with extended storage, the samples packed in pp bag had marked decrease in crispiness (p<0.05). there was no change in crispiness for samples packed in lf bag throughout 6 months of storage (p<0.05), except those containing gt+ca, which had a slight decrease after 3 months of storage (p<0.05). similar results were also reported for biscuits fortified with microencapsulated oil, in which high moisture content was related with lowered hardness (takeungwongtrakul and benjakul, 2017). the results indicated that packing condition had higher impact than added antioxidants on textural properties of cereal bars fortified with hc during storage of 6 months. therefore, lf bag could be used for packing the cereal bars to maintain their textural property during storage. 3.4. changes in peroxide value (pv) and thiobarbituric acid reactive substances (tbars) during storage pv and tbars of cereal bars without (con) and with addition of antioxidants (gt+ca) stored under different packing conditions during 6 months of storage are depicted in fig. 3. at day 0 of storage, gt+ca sample had lower pv than that of con (fig. 3a) (p<0.05). this result suggested that lipid oxidation could occur during cereal bar preparation and the addition of antioxidants, green tea powder and citric acid, could prevent lipid oxidation to some degree as indicated by the lower pv of gt+ca sample. pv of all samples notably increased within the first 2 months of storage (p<0.05) and remained unchanged during 2-3 months (p>0.05). after 3 months of storage, the highest pv was found for the con packed in pp bag, compared with others (p<0.05). in general, pv of samples packed in pp bag was higher than that of samples packed in lf bag (p<0.05). furthermore, the addition of antioxidants could suppress the formation of hydroperoxide. nitrogen gas was purged into the sample packed in lf bag. this could also prevent oxidation in the sample in conjunction with antioxidants. in the low oxygen atmosphere, the oxidation of lipid occurs at negligible level. this result was in agreement with bakkalbaşı et al. (2012) who found that higher content of oxygen present in packaging increased lipid oxidation of walnuts during storage, especially at higher storage temperature. after 3 months of storage, sample packed in lf bag was further stored. it was found that the sharp decreases in pv were observed for both samples, con and gt+ca samples. this was more likely caused by the decomposition of hydroperoxides to the secondary products (karnjanapratum and benjakul, 2015a). the lower pv was observed for gt+ca sample, compared with those of con sample (p<0.05). addition of antioxidants in cereal bars had a preventive effect on lipid oxidation by retarding the radical chain reaction. along with the nitrogen atmosphere, the oxidation of lipids in nuts or other ingredients used in formulation could be impeded more effectively. changes in tbars of con and gt+ca samples kept under different packing conditions during storage of 6 months are shown in fig. 3b. the increase in tbars indicated formation of secondary lipid oxidation products. marked increases in tbars were observed for all samples within the first month of storage (p<0.05). tbars of con sample packed in pp bag sharply decreased during 1-3 months, while gt+ca sample packed in pp bag showed gradual decrease after 2 months of storage (p<0.05). the decrease in tbars with extended storage was probably due to the loss in those volatile secondary products (yarnpakdee et al., 2012). green tea and citric acid added in cereal bars could prevent the formation of tbars during storage of sample packed in pp bag, where oxygen was present to some extent. however, lower increase in tbars value was observed for those packed in lf bag within the first month of storage (p<0.05), compared to those packed in pp bag. tbars of samples packed in lf bag were slightly changed during 1-2 months of storage (p<0.05). during 2-3 months of storage, tbars sharply ital. j. food sci., vol. 31, 2019 358 decreased in all samples (p<0.05). after 3 months of storage, drastic decrease in tbars in sample packed in lf bag was observed. however, lower tbars value was noticeable in the gt+ca sample, compared to con sample. during 4-6 months of storage, tbars value remained constant for both samples. thus, the addition of antioxidants could prevent lipid oxidation of cereal bar during storage to some degree. moreover, lf bag was able to effectively retard the early stages as well as the advanced stage of oxidation. as a result, the quality of cereal bar could be maintained during storage. figure 3. pv (a) and tbars (b) values of hc fortified cereal bars packed under different conditions during storage. con; cereal bar without antioxidants. gt+ca; cereal bar with added antioxidants (1.0% green tea powder, 0.01% citric acid). pp; polypropylene bag with normal heat seal. lf; laminated polyethylene and aluminium foil bag with modified atmosphere pack (n2 flush) before sealing. bars represent the standard deviation (n=3). 3.5. volatile compounds volatile compounds in con and gt+ca samples stored under different packing conditions at 0, 3 and 6 months of storage are displayed in table 2. the types and abundance of volatile compounds detected in samples varied with antioxidants and packing conditions used as well as storage times. at day 0 of storage, 21 volatile compounds were identified for both samples, including 7 alcohols, 2 aldehydes, 3 ketones, 5 acids and 4 esters. it was noted that the abundance of total volatile compounds in gt+ca sample (308.83 × 106) was lower than that of con sample (501.83 × 106). some ital. j. food sci., vol. 31, 2019 359 volatiles were not detected in gt+ca sample, while they were found in con sample. 1penten-3-ol was detected in gt+ca sample, indicating the presence of green odor/aroma in green tea (lee et al., 2013). most acids and ester volatile compounds were reported as fruity aroma (mohamed el hadi et al., 2013), which might be related to the mixed dried fruit and strawberry flavor used for preparing cereal bars. several derivatives of volatiles can be formed by the oxidation of lipids. 1-butanol 3-methyl-, 3-hexen-1-ol, 1,2propanediol, 3-furanmethanol and benzyl alcohol were found in the con sample with higher abundance, compared with those of gt+ca sample. aldehydes and ketones are among the main contributors to flavor and their concentration was related to lipid oxidation (sae-leaw et al., 2016; takeungwongtrakul and benjakul, 2017). 2furan-carboxaldehyde, 5-hydroxymethylfurfural, 2-butanone, 3-hydroxy-, 2-propanone, 1hydroxy-, ethanone, 1-(2-furanyl)-, butanoic acid ethyl ester, dipropylene glycol diacetate, 1,2-propanediol 1-acetate, 1,2-propanediol 2-acetate and some acids were detected in the con sample with higher abundance, compared with those of gt+ca sample. this result was in accordance with higher pv and tbars of con sample, compared to those of gt+ca sample at day 0 (fig. 3). the results suggest that green tea and citric acid used in gt+ca sample could prevent lipid oxidation during cereal bar manufacture. after 3 months of storage, marked increase in volatile compounds was observed for all samples packed in both pp and lf bags indicating that lipid oxidation took place in the cereal bars within 3 months of storage. auto-oxidation could occur, especially in cereal, nuts and grains. decomposition of lipid hydroperoxides is a complicated process and produces a multitude of constituents that may have biological effects and cause flavor deterioration in fat-containing foods (el-magoli et al., 1982). this decomposition proceeds by homolytic cleavage of lo-oh to form alkoxy radicals lo·. these radicals undergo carbon-carbon cleavage to form breakdown products including aldehydes, ketones, alcohols, esters and furans (el-magoli et al., 1982). new volatiles including 10 alcohols, 2 aldehydes, 1 ketone, 2 acids and 10 esters were identified for all samples tested. notably, cereal bars packed in pp bag showed higher volatile abundance (706-921×106), compared to those packed in lf bag (419-505× 106). 1,2-propanediol, 3-hexen-1-ol and 1hexanol were found as the major alcohols in all cereal bars. hexanal and 2-furancarboxaldehyde were dominant aldehydes, whereas 2-propanone, 1-hydroxywas the predominant ketone. aldehydes and ketones are known as the major contributors to the development of lipid oxidation off-odor and off-flavor (takeungwongtrakul and benjakul, 2017). aliphatic alcohols such as 1-pentene-3-ol and 1-octen-3-ol contribute to off-flavor and they are produced by oxidative deterioration of food lipid (sae-leaw et al., 2016). acetic acid was the major acid and butanoic acid ethyl ester was found as the high abundant ester in all samples, indicating the fruity flavor of cereal bars (mohamed el hadi et al., 2013). less volatile compounds were found for gt+ca sample, compared with the con sample, regardless of packing condition used. this might be due to the influence of antioxidants used in gt+ca sample, which could prevent lipid oxidation. in addition, some new volatiles were detected only for those packed in pp bag such as 2butanol and 2-penten-1-ol. this correlated well with the higher pv, compared with those packed in lf bag. the lowest abundance together with less types of volatiles was generally found for gt+ca sample packed in lf bag. ethyl maltol, propanoic acid, propanoic acid ethyl ester, 2-hydroxypropyl propionate and 3-furancarboxylic acid methyl ester were found in all samples, excepted for gt+ca sample packed in lf bag. this was more likely owing to the higher barrier properties with lower oxygen content in lf bag as well as antioxidants added, which could prevent lipid oxidation during storage. after 6 months of storage, some compounds were not found and new volatiles were generated. this was possibly due to the volatilization or decomposition of those aforementioned compounds. simultaneously, new compounds were formed. the further ital. j. food sci., vol. 31, 2019 360 oxidation of lipids or some other reactions could change the abundance of volatile compounds during storage (andŕes et al., 2004). among alcohols, 2-butanol, 1propanol,2-methyl-, 2-propanol, 1-methoxy-, 1-pentanol, 1-octanol, 2-propanol, 1,1’oxybis-, benzene ethanol and maltol were not detected, while cyclohexanol was found as new volatile alcohol for both of con and gt+ca samples packed in lf bag. 2-propanone was generated as new ketone with disappearance of 2-butanone, 3-hydroxyand 2(3h)furanone, dihydro-. marked increase in volatile compounds was observed for both con and gt+ca samples with extended storage (6 month), compared with those of samples stored for 3 months. it was noted that gt+ca sample had lower abundance with less types of volatile compounds, compared to those of con sample. the result indicated that lipid oxidation of gt+ca sample packed in lf bag could be reduced to some extent. therefore, cereal bar fortified with hc with and without addition of antioxidants could be packed with lf bag to improve oxidative stability during 6 months of storage. 3.6. sensory properties sensory properties of hc fortified cereal bar in the absence (con) and the presence of antioxidants (gt+ca) stored under different packing conditions are shown in fig. 4. at day 0, the likeness scores of all attributes tested, including appearance, color, odor, flavor, taste, texture and overall likeness for the con and gt+ca samples were in the range of 7.5-8.2. significant decrease in likeness score of both con (fig. 4a) and gt+ca samples (fig. 4c) was observed for those packed in pp bag, especially after 3 months of storage (p<0.05). likeness scores for all attributes of both samples stored in pp bag were lower than 7 (6.0-6.8) after 3 months of storage. there were no differences in likeness score of all attributes tested between both con (fig. 4b) and gt+ca samples (fig. 4d) packed in lf bag during storage of 6 months (p>0.05). this result suggested that the packing condition had more impact than antioxidants on sensory properties during storage. the addition of antioxidants (gt+ca) had no marked influence on sensory qualities during storage. con and gt+ca samples packed in pp were discarded at month 3 since likeness scores for all attributes tested were less than 7. lower water vapor barrier property (6.09 g.mm/day.m2.mmhg) with higher content of o2 (18.0-18.8%) of pp bag could favor physical and chemical changes of cereal bar, whereas lf bag (2.8-4.0% o2 content with water vapor permeability of 5.76 g.mm/day.m2) showed superior property in keeping cereal bars. water vapor from environment could migrate through packaging material and consequently increased the moisture content of product. this affected the appearance, color and texture of cereal bar. in addition, the high content of o2 induced chemical change, especially lipid oxidation, during storage. volatile compounds from lipid oxidation products were related to flavor deterioration known as rancidity (yarnpakdee et al., 2012). moreover, aldehyde and ketone compounds from lipid oxidation were more likely involved in a yellowish discoloration via the maillard reaction (yarnpakdee et al., 2012). those reactions decreased the likeness score for appearance, odor, flavor and taste of cereal bars packed in pp bag. similar results were reported for sensory property of stored muesli in different packaging (senhofa et al., 2015). paper bag with low barrier property caused the greatest decrease of sensory quality during storage, compared with laminated low density polyethylene/aluminium foil container (senhofa et al., 2015). also, lf bag was flushed with n2 to replace air before sealing. this could help in retardation of lipid oxidation. therefore, the packing conditions including packaging material and oxygen content, had direct impact on sensory properties of cereal bar fortified with hc during the storage. lf bag with nitrogen gas could improve the sensory quality of cereal bars during 6-month storage, regardless of antioxidant addition. ital. j. food sci., vol. 31, 2019 361 table 2. volatile compounds of hc fortified cereal bars packed under different conditions during storage. volatile compounds abundance (×106) month 0 month 3 month 6 con gt+ca pp lf lf con gt+ca con gt+ca con gt+ca alcohols 2-butanol nd. nd. 0.86 0.46 nd. nd. nd. nd. 1-propanol, 2-methylnd. nd. 1.99 1.69 1.53 1.31 nd. nd. 2-propanol, 1-methoxynd. nd. 0.42 0.36 0.35 0.28 nd. nd. 1-butanol nd. nd. 1.53 1.4 1.26 1.29 nd. nd. 1-penten-3-ol 1.29 nd. 2.71 2.69 nd. nd. nd. nd. 1-butanol, 3-methyl1.7 nd. 7.01 4.57 6.7 5.32 8.26 7.34 1-pentanol nd. nd. 11.86 10.62 11.85 10.81 nd. nd. 2-penten-1-ol nd. nd. 1.59 1.4 nd. nd. 5.88 6.14 1-hexanol 3.98 3.1 31.97 31.09 29.49 23.48 207.7 117.33 3-hexen-1-ol 90.1 41.21 89.01 65.77 64.47 72.34 303.91 295.12 cyclohexanol nd. nd. nd. nd. nd. nd. 9.17 6.61 2,3-butanediol nd. nd. 4.31 2.97 4.01 3.43 7.47 9.02 1-octanol nd. nd. 0.32 0.69 0.31 nd. nd. nd. 1,2-propanediol 258.83 167.47 342.48 255.17 175.83 111.76 71.61 94.19 3-furanmethanol 2.8 2.28 4.98 4.56 3.23 5.87 46.86 55.63 2-propanol, 1,1'-oxybisnd. nd. 0.3 nd. 0.29 nd. nd. nd. benzyl alcohol 1.71 1.14 1.63 1.55 1.39 0.94 26.18 18.18 benzeneethanol nd. nd. 0.21 nd. 0.2 nd. nd. nd. maltol nd. nd. 0.85 1.07 0.62 0.69 nd. nd. ethyl maltol nd. nd. 1.19 1.16 1.09 nd. 21.73 21.79 aldehydes 2-cyclopentenylethanal nd. nd. nd. nd. nd. nd. 234.1 51.68 hexanal nd. nd. 9.61 9.3 6.66 2.87 23.07 9.74 2-furan-carboxaldehyde 16.17 5.37 6.72 5.82 4.83 4.32 65.28 62.25 benzaldehyde nd. nd. 1.12 0.83 0.82 0.8 9.21 0.94 ital. j. food sci., vol. 31, 2019 362 5-hydroxymethylfurfural 2.52 nd. 0.19 nd. nd. nd. nd. nd. ketones 2-propanone nd. nd. nd. nd. nd. nd. 17.8 10.42 2-butanone, 3-hydroxy3.89 nd. 1.9 1.9 1.88 1.37 nd. nd. 2-propanone, 1-hydroxy7.6 7.53 14.94 12.05 12.75 12.58 57.82 53.37 ethanone, 1-(2-furanyl)0.84 nd. 1.61 1.32 1.25 1.22 17.16 9.45 2(3h)-furanone, dihydrond. nd. 0.76 0.72 0.93 0.76 nd. nd. 2-butanone, 4-phenylnd. nd. nd. nd. nd. nd. 9.49 6.42 4h-pyran-4-one, 3-hydroxy-2-methylnd. nd. nd. nd. nd. nd. 24.24 19.88 4h-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methylnd. nd. nd. nd. nd. nd. 10.43 12.99 acids acetic acid 66.98 48.95 33.92 29.5 26.33 26.14 170.36 143.56 propanoic acid 5.51 5.01 0.99 1.21 1.4 nd. 7.5 5.92 butanoic acid nd. nd. 0.86 0.75 0.71 0.52 nd. nd. pentanoic acid 0.41 nd. 0.56 0.55 0.41 0.36 7.19 5.58 hexanoic acid 14.87 8.86 1.87 1.79 1.75 1.7 29.73 15.26 hexanoic acid, 2-ethylnd. nd. 0.61 0.6 0.53 0.5 nd. nd. butanoic acid, 2-methyl10.71 9.91 3.79 3.79 3.65 3.55 17.36 13.38 esters acetic acid ethyl ester nd. nd. 51.44 35.61 13.23 7.67 76.07 50.32 propanoic acid, ethyl ester nd. nd. 1.48 0.75 0.46 nd. nd. nd. butanoic acid, ethyl ester 3.48 0.95 237.54 176.75 103.38 100.49 495.47 467.95 butanoic acid, 2-methyl-, ethyl ester nd. nd. 12.01 7.81 5.15 4.77 48.12 47.71 butanoic acid, 3-methyl-, ethyl ester nd. nd. 4.04 1.92 1.41 1.35 9.79 5.84 hexanoic acid, ethyl ester nd. nd. 2.55 1.48 0.93 0.91 14.61 13.02 butanoic acid, 2-ethyl-, methyl ester nd. nd. nd. nd. nd. nd. 25.15 22.09 benzyl acetate nd. nd. nd. nd. nd. nd. 4.09 6.17 methyl isobutyrate nd. nd. 2.09 1.72 1.64 1.28 nd. nd. formic acid, octyl ester nd. nd. nd. nd. nd. nd. 7.82 5.43 hexanoic acid, ethyl ester nd. nd. 2.55 1.48 0.93 0.91 13.02 14.61 dipropylene glycol, diacetate 0.32 nd. 9.07 8.75 nd. nd. nd. nd. 1,2-propanediol, 1-acetate 4.57 3.78 5.8 5.55 5.15 5.04 33.97 nd. ital. j. food sci., vol. 31, 2019 363 2-hydroxypropyl propionate nd. nd. 4.14 4.11 3.46 nd. 34.89 32.69 1,2-propanediol, 2-acetate 3.55 3.27 2.79 2.64 2.54 2.27 11.31 11.25 acetic acid, phenylmethyl ester nd. nd. 0.34 0.32 0.29 0.28 nd. nd. ethane-1,1-diol dibutanoate nd. nd. nd. nd. nd. nd. 11.88 9.6 3-furancarboxylic acid, methyl ester nd. nd. 0.51 0.44 0.22 nd. 9.64 7.4 nd.; not detected. con; cereal bar without antioxidants. gt+ca; cereal bar with added antioxidants (1.0% green tea powder, 0.01% citric acid). pp: polypropylene plastic bag with normal heat seal. lf: laminated polyethylene and aluminium foil bag with modified atmosphere pack (n2 flush). figure 4. sensory characteristics of hc fortified cereal bars packed under different conditions during storage. con; cereal bar without antioxidants. gt+ca; cereal bar with added antioxidants (1% green tea powder, 0.01% citric acid). pp; polypropylene plastic with normal heat seal. lf; laminated polyethylene and aluminium foil bag with modified atmosphere pack (n2 flush) before sealing. bars represent the standard deviation (n=3). ital. j. food sci., vol. 31, 2019 364 3.7. microbiological properties total viable count and yeast/mold counts of cereal bars stored under different packaging conditions were determined every month during storage of 6 months. total viable counts for mesophilic aerobic microorganisms were less than 103 cfu/g sample for all samples tested during storage up to 6 months. on the other hand, yeast and mold counts were less than 10 cfu/g sample. the number of microorganisms was in accordance with the expected microbiological quality of processed foods, particularly cereal and cereal products (fda, 2013). the result was in line with low water activity of the cereal bars, which was less than 0.6 during storage (fig. 1b). this low water activity limits the microbial growth. senhofa et al. (2015) studied the storage stability of muesli in different types of packaging materials. mesophilic aerobic bacteria, yeast and mold growth in muesli samples during storage was influenced by the presence of air and its diffusion through packaging material (senhofa et al., 2015). these results indicated that cereal bars with and without addition of antioxidants in all packaging conditions used were handled properly with appropriate storage condition, thus providing the product with microbiological safety and quality for consumers throughout the storage of 6 months. 4. conclusions addition of antioxidants and packing conditions had profound impact on changes in quality and sensory properties of cereal bars fortified with hc during 6 months of storage at 25°c. addition of green tea powder and citric acid as antioxidants could prevent some physicochemical changes and retard lipid oxidation to some degree, especially those cereal bars packed in pp bag. based on sensory acceptance, cereal 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padova, department of agronomy, food, natural resources, animals and environment (dafnae), viale dell’università 16, 35020 legnaro, pd, italy *corresponding author: simone.vincenzi@unipd.it abstract in the present paper we analyzed the stilbene accumulation in grape canes of seven autochthonous grape varieties from veneto region, italy, in comparison to two international cvs. in addition, we investigated the effect of pruning time on the stilbenes accumulation rate during the storage. taking into account the effect of both pruning time (october, november and december) and storage time (from zero to twelve weeks at room temperature), cultivar verdiso and incrocio manzoni 13.0.25 showed the highest accumulation of trans-resveratrol, trans-piceatannol, and trans-ɛ-viniferin, in particular when the canes were harvested in october, highlighting the importance of the cultivar but also the effect of the pruning time on the accumulation of stilbenes in grape canes. keywords: autochthonous varieties, grape canes, piceatannol, resveratrol, viniferin ital. j. food sci., vol. 32, 2020 311 1. introduction the winemaking industry is responsible for large part of grape waste as pomace, grape canes, seeds and stems. some waste as pomace and seeds are valued from food industry being popular as source of antioxidant polyphenols or for grape seed oil production. at the same time, there is still a need for alternative sources of resveratrol, as can be seen by the recent permission of resveratrol as a novel food ingredient in the european union (commission implementing decision (eu) 2016/1190). stilbenoids are a small family of plant secondary metabolites derived from the phenylpropanoid pathway. they act as plant phytoalexins displaying different bioactivities and thus making them compounds of high current interest (fernandez-mar et al., 2012). in the vitaceae, stilbenoids accumulate in response to various biotic and abiotic stresses such as the attack of pathogen erysiphe necator, plasmopara viticola, botrytis cinerea and uv-c irradiation (schnee et al., 2008; schnee et al., 2013; pezet et al., 2004; alonso-villaverde et al., 2011; adrian and jeandet, 2012; gruau et al., 2015; yin et al., 2016). they can also be induced in response to plant hormones, such as ethylene and jasmonate (d’onofrio et al. 2009; jiang et al., 2015). in grapevine, the stilbene trans-resveratrol has attracted particular attention, not only because of its antimicrobial activity, but also due to its health benefits to humans, as antioxidant, anticarcinogenic, anti-inflammatory, cardioprotective and neuroprotective, among others (flamini et al., 2013; bavaresco et al., 2012; shen et al., 2009). stilbenoids accumulate in different parts of grapevine, however, wang et al. (2010) found the highest concentration of trans-resveratrol in grape canes. grape canes waste is generated during winter annual pruning and represents a large source of waste derived from the viticulture industry, with an estimated volume of 1 to 3 t/ha year depending upon plantation density, climate, and vigor of the grape variety (devesa-rey et al., 2011; ewald et al., 2017). currently, emission protection regulations mostly prohibit the burning of grape canes, which was the traditional way of disposal of these woody residues (ewald et al., 2017). grape canes can be considered as an unexploited source of stilbenoids, as proposed by several authors (vergara et al., 2012; lambert et al., 2013; gorena et al., 2014; houillé et al., 2015; guerrero et al., 2016; ewald et al., 2017). different content of stilbenoids have been found in grape canes of vitis vinifera stored at 40 to 45°c or at room temperature (20±3°c). vergara et al. (2012) compared the stilbenoid content in canes of several grape varieties cultivated in different regions and in two different years in chile, finding trans-resveratrol in the range 446 to 6533 mg/kg dw, with the highest content found in gewurztraminer variety. lower values of trans-resveratrol were reported by lambert et al. (2013) comparing grape canes harvested from 16 different varieties in france. these authors found the lowest content of trans-resveratrol in chardonnay (190 mg/kg dw) and the highest content in pinot noir cultivar (1526 mg/kg dw) while the trans-piceatannol and trans-ɛ-viniferin content were significantly higher in all cultivars when compared with vergara et al. (2012). guerrero et al. (2016) described that most abundant stilbenoid was trans-viniferin in all cultivars, which reached the highest concentration in gewürztraminer cultivar. while ewald et al. (2017) found the higher levels of trans-resveratrol and trans-viniferin in pinot blanc and sauvignon blanc harvested in germany (3199-3329 mg/kg dw, respectively). zhang et al. (2011) studied the content of trans-resveratrol in grape canes of many different grape varieties, including local varieties cultivated in the seven major chinese grape producing regions finding high variability. ital. j. food sci., vol. 32, 2020 312 beside the genetic determinants, several other factors could explain these different results, such as the climate, the solvent used for the extraction of stilbenoids (rayne et al., 2008), the temperature and time of grape canes storage (houillé et al., 2015). in addition, other factors, such as the pruning time, could affect the stilbene accumulation rate in canes. this factor has never taken in account before, in fact in many articles this data is not reported at all, and, when present, show to be highly variable, with pruning times varying from 1 to 4 months after the grape harvest. up to date there are no data available concerning stilbenoid content in grape cane waste of italian grape varieties. in the present paper the stilbene accumulation in grape canes of seven autochthonous grape varieties from veneto region, one of the most important wine producing regions in italy, has been studied. in addition, the effect of pruning time on the stilbenes accumulation rate during the storage was taken in account. 2. materials and methods 2.1. plant materials grape canes of vitis vinifera l. from veneto region white varieties, such as bianchetta, glera vcr sel. lungo, incrocio manzoni 6.0.1.3, verdiso; and red varieties incrocio manzoni 13.0.25, marzemino biotipo 13, raboso, were collected randomly from plants from a conventional vineyard at oenological school of conegliano, province of treviso, italy (i.s.i.s.s – istituto statale di istruzione secondaria superiore “g.b. cerletti”) (latitude 45° 87’ 69” n and longitude 12° 28’ 53” e). as reference, international varieties sauvignon blanc inra 316 and pinot noir, grown in the same vineyard, were chosen because according to lambert et al. (2013) these were the cultivars with the highest content of stilbenes. the canes were collected monthly in october (11th), november (15th) and december (13th) (autumn-winter 2016-2017) from 30 selected plants for each variety. about 1.5 kg for each sampling and for each variety were obtained. the canes were cut into 10-20 cm long pieces and stored for three, six, nine and twelve weeks in well-aerated conditions in the dark, at room temperature. for control, a sample was immediately extracted after each pruning sampling point. 2.2. stilbenoid extraction the stilbenoid extraction was performed according to the procedure described by rayne et al. (2008) with some modifications. briefly, the grape canes were ground with a coffee grinder (imetec, azzano san paolo, bg, italy). three-stage extraction (in the dark to avoid stilbene isomerization) was performed by continuous stirring at room temperature using an 8:1 (v/w) 80% ethanol:sample ratio over a 60-min period for each extraction. during the first extraction, 250 µl of t-oh-stilbene 200 µg/ml in ethanol were added as internal standard. the extracts were vacuum filtered at 1.6 μm on glass microfibre filter (gf/a, whatman) and combined and the solvent removed by rotary evaporation (büchi model r-114, flawil, switzerland), then stored at -20°c. all the extractions were performed in triplicate. before quantification, the extracts were defrosted at room temperature and homogenized. an aliquot of the extract (500 µl) was transferred to eppendorf tubes and 500 µl of methanol were added. after centrifugation at 4000 × g for 1 min, part of the supernatant (500 µl) was transferred to hplc vials. ital. j. food sci., vol. 32, 2020 313 2.3. hplc analysis the analysis of stilbenoids was performed according to the procedure described by vincenzi et al. (2013) with some modifications. stilbenes were separated on a c18 lichrospher column (4 mm x 250 mm, 5 μm, agilent technologies, milano, italy) at 40°c, using an hplc system (waters corporation, milford, ma, usa) equipped with a dual band uv detector waters 2487 (waters corporation, milford, ma, usa). the mobile phase gradient was 0.5% v/v formic acid in deionized water (solvent a) and 2% v/v formic acid in methanol (solvent b). the gradient program was 0 to 10% (solvent b) in 3 min, followed by 10 to 30% (solvent b) in 5 min, 30 to 44% (solvent b) in 35 min, 44 to 55% (solvent b) in 2 min, 55 to 75% (solvent b) in 15 min and 75 to 100% (solvent b) in 1 min. after washing for 2 min with solvent b, the column was re-equilibrated with solvent a. the flow rate was 1.0 ml/min and injection volume 20 μl. detection was performed at 306 nm for trans-isomers for transresveratrol, trans-piceatannol and trans-ɛ-viniferin and at 285 nm for the corresponding cisisomers. all the stilbene standards were obtained in trans form from extrasynthese (genay cedex, france). the cis-isomers were obtained by uv-exposition of the corresponding trans-isomers, and were loaded in hplc for the identification of the retention times. the concentration of individual stilbenes (both transand cis-forms) was calculated on the basis of peak areas using calibration curves of commercially available standards of transresveratrol, trans-piceatannol and trans-ɛ-viniferin, and correcting the value for the internal standard recovery. data were analysed by the waters breezetm chromatography software (version 3.30). the limits of detection (lod) and quantification (loq) were performed according to the procedure described by (shrivastava and gupta, 2011). 2.4. statistical analysis within each factor the results were evaluated by one-way analysis of variance (anova), and values were analyzed by tukey’s test using the software statistica 12.0 (statsoft inc, tolson, usa). results were expressed as mean values ± standard deviation (sd) and the value of p < 0.05 was considered statistically significant. for the global analysis of all data in order to take in account the interactions among different variables, a manova test was applied and values were analyzed by wilks’s test using the software xlstat (addinsoft). 3. results and discussion considering that stilbenoid accumulation in cut canes depends on activation of related genes followed by active synthesis of resveratrol and its derivatives, as already reported by houillé et al. (2015) and billet et al. (2018), it is expected that different grape varieties respond in different way after the injury for both total amount of stilbenoid produced and rate of their accumulation. also, the climate and other environmental factors can affect the way the canes respond during the storage period, for this reason the canes of the seven varieties taken in consideration in this study were collected in the same year from plants grown in the same vineyard. the evolutions of stilbenoids during the storage time for samples of different varieties harvested at different times is reported in figs. 1, 2 and 3. ital. j. food sci., vol. 32, 2020 314 figure 1. content of trans-resveratrol (a), trans-piceatannol (b) and trans-ɛ-viniferin (c) on grape canes harvested in october (autumn−winter 2016−2017) from white varieties, bianchetta, glera, incrocio manzoni 6.0.1.3, sauvignon blanc, verdiso, and red varieties incrocio manzoni 13.0.25, marzemino, pinot noir, raboso, and stored at room temperature for three, six, nine, and twelve weeks. results represent the mean ± sd of triplicate assays. ital. j. food sci., vol. 32, 2020 315 figure 2. content of trans-resveratrol (a), trans-piceatannol (b) and trans-ɛ-viniferin (c) on grape canes harvested at pruning time in november (autumn−winter 2016−2017) from white varieties, bianchetta, glera, incrocio manzoni 6.0.1.3, sauvignon blanc, verdiso, and red varieties incrocio manzoni 13.0.25, marzemino, pinot noir, raboso, and stored at room temperature for three, six, nine, and twelve weeks. results represent the mean ± sd of triplicate assays. ital. j. food sci., vol. 32, 2020 316 figure 3. content of trans-resveratrol (a), trans-piceatannol (b) and trans-ɛ-viniferin (c) on grape canes harvested at pruning time in december (autumn−winter 2016−2017) from white varieties, bianchetta, glera, incrocio manzoni 6.0.1.3, sauvignon blanc, verdiso, and red varieties incrocio manzoni 13.0.25, marzemino, pinot noir, raboso, and stored at room temperature for three, six, nine, and twelve weeks. results represent the mean ± sd of triplicate assays. ital. j. food sci., vol. 32, 2020 317 a manova test was also applied to study the effect of different variables on the stilbenoids accumulation (table 1). table 1. results of manova analysis on the total dataset. storage harvest variety storage* harvest storage* variety harvest* variety storage* harvest* variety f-value 49,240 5,227 4,579 6,615 4,387 2,624 2,313 gdl1 12 6 24 21 96 48 150 gdl2 421 318 462 457 477 474 478 p-value < 0,0001 < 0,0001 < 0,0001 < 0,0001 < 0,0001 < 0,0001 < 0,0001 as a first observation, the basal level of trans-piceatannol and trans-resveratrol in fresh canes of pinot noir was quite low (less than 40 mg/kg dw) and below the detection level in the other varieties, whereas the dimer trans-ɛ-viniferin was already present at concentrations 10 to 30 times higher in all the samples (supplementary table 1). these results confirm the literature data (gorena et al., 2014; houillé et al., 2015; billet et al., 2018), only vergara and colleagues (2012) found very high content of transresveratrol (between 2500 and 3500 mg/kg) already at time zero. during the storage a large increase of stilbenoids was observed in all samples, confirming that the main variable driving their accumulation in grape canes is the storage time (highest f value in table 1). however, a significant effect of the harvest time was also found (table 1). collectively, the canes pruned in october showed a gradual increase of stilbenes during all the storage period, whereas the canes collected in november showed a notable peak of accumulation of stilbene content after 6 weeks of storage for almost all the varieties (figs. 1 and 2). houillé et al. (2015) found the same results, i.e. a peak of accumulation of trans-resveratrol and trans-piceatannol after 6 weeks of storage, on eight cultivars (collected in december) and stored for two, four, six, eight and ten weeks. on the other hand, the pruning carried out in december demonstrated a different behavior in the accumulation of stilbenes (fig. 3). for many varieties, in particular for verdiso and incrocio manzoni 13.0.25 (im 13.0.25) the peak of stilbene accumulation was retarded to 9 weeks of storage. this behavior of canes harvested at different times seems to show a slower stilbenoid response in canes harvested in october, probably due to the high quantity of stilbene synthase enzymes still present in the woody tissue, which under regulate the induction of the related genes after the injury. the synthesis of stilbenoids is instead more rapid with the evolution of canes toward winter dormancy until november, then the accumulation rate slowdown in canes harvested in december. this could explain the significant effect of the interaction storage*harvest time (table 1). the different cultivars showed different behaviors, and different responses to harvest time and storage conditions, as confirmed by the significant effect of variety, storage*variety and harvest*variety in the manova test (table 1). among the samples harvested in october, cultivar verdiso, im 13.0.25 and marzemino showed the highest increase of trans-resveratrol, trans-piceatannol, and trans-ɛ-viniferin after twelve weeks at rt (fig. 1). among the white varieties, verdiso is one of the last to be harvested. similarly, im 13.0.25 and marzemino are, among red varieties, those with the later harvest. it is not clear if this common behavior could be related to the similar accumulation rate of stilbenes in pruned canes. regarding resveratrol, even though after 9 ital. j. food sci., vol. 32, 2020 318 weeks pinot noir was the cultivar with the highest content, confirming the high stilbene metabolism of this variety, after 12 weeks the content in im 13.0.25 canes increased again reaching the highest value among all the varieties taken in consideration. even for piceatannol, im 13.0.25 presented concentration usually higher than pinot noir. the canes harvested in november demonstrated an increase for all stilbene compounds when maintained for twelve weeks at rt. again, the cultivar im 13.0.25 presented a constant increase of trans-resveratrol content reaching, after 12 weeks of storage, a value up to 2016±365 mg/kg dw, comparable with those found in october. in this group, a high increase of the content of trans-piceatannol (633±64 mg/kg dw) and trans-ɛ-viniferin (2193±213 mg/kg dw) was detected even on the cultivar bianchetta (fig. 2). compared to pinot noir, which reached after 6 weeks the maximum content of piceatannol and resveratrol among all varieties, bianchetta and im 13.0.25 were able to reach the same or higher quantities for both compounds after a storage of 12 weeks. in grape canes harvested in december pinot noir showed a very small accumulation of resveratrol and piceatannol, while again verdiso and im 13.0.25 were able to reach a high content of both compounds (fig. 3) (supplementary tables 2-5). our findings confirm the results previously found by guerrero et al. (2016), which highlight the importance of the cultivar in the accumulation of stilbenes in grape canes after pruning. in addition, for the first time, we demonstrated how the pruning time affects both the quantity and the accumulation rate of stilbenes in pruned canes. the information on the maximum stilbene content recoverable from canes of different grapevine cultivars could be interesting for grape producers in order to obtain cane extracts with high stilbenes concentration from their own grape canes waste. these extracts can be the base for the purification of stilbenes to be used in the food or cosmetic industries, with a big economic income considering the value of food-grade resveratrol is about 2000-3000 us$/kg (zhang et al., 2011). however, the crude cane extract could be also reused by grape growers in the same vineyard in an idea of circular economy. in fact, stilbenes have shown antifungal activity against different fungi. until now, the antifungal activity in vitro of the crude cane extracts from pinot noir, gamaret and divico cultivars against plasmopara viticola, erysiphe necator and botrytis cinerea was reported by schnee et al. (2013). recently, the direct antifungal activity of crude cane extract from pinot noir against b. cinerea was studied by monitoring the mycelium growth on nutrient agar medium, and also in grapevine plants in vitro and in vivo by de bona et al. (2019). 4. conclusion the information on the maximum stilbene content recoverable from canes of different grapevine cultivars could be interesting for grape producers, but many factors have to be taken in to account to obtain the highest yield of these compounds. first, the storage time is the main factor driving the increase of stilbenoids in grape canes, confirming the literature data, but we demonstrated for the first time that the total amount of stilbenoids and their rate of accumulation depends significantly on the pruning time. in addition the data reported in the present study confirm the importance of the cultivar in the accumulation of stilbenes in grape canes after pruning. finally, this study showed that the cultivars verdiso and incrocio manzoni 13.0.25 possess a high potential of stilbene accumulation, mainly when the canes were harvested in october. ital. j. food sci., vol. 32, 2020 319 acknowledgements financial support was provided by national scientific and 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2011. occurrence and estimation of transresveratrol in one-year-old canes from seven major chinese grape producing regions. molecules 16:2846-2861. paper received july 17, 2019 accepted november 26, 2019 ijfs#1058_bozza ital. j. food sci., vol. 30, 2018 393 paper italian wines in china’s e-commerce market: focus on piedmont region products a. dal vecchio, s. massaglia*, v.m. merlino, d. borra and m. hao department of agricultural, forest and food sciences, university of turin, largo paolo braccini 2, 10095 grugliasco, to, italy *e-mail address: stefano.massaglia@unito.it abstract in this study an analysis was conducted on the market performance of italian wines, in particular from piedmont region, and consumer preference in the chinese e-commerce market. the chinese e-commerce platform company, taobao, and the professional online wine businesses, yesmywine, wine9 and juxian, were investigated. chinese consumers were willing to buy famous wines at a high price (i.e. barolo). furthermore, sparkling wines, from piedmont and emilia romagna, and typical red wines, from veneto and tuscany, were frequently chosen. however, italian wineries did not pay much attention to the chinese e-commerce market, missing potential for increasing business. keywords: chinese consumers, e-commerce, italian wine ital. j. food sci., vol. 30, 2018 394 1. introduction china is currently one of the world’s most dynamic markets for wine imports (masset et al., 2016). the country ranks fourth in world-class destinations for wine, with 2.1 billion euros and 6.3 million hectolitres of imported wine. for many decades, chinese perceived grape wine was as a symbol of western-sophistication and wealth, often consumed only by the elite. this trend has changed significantly only in the past decade (huang, 2000). the demand was mostly from high-level chinese bureaucrats and wealthy businessmen for top ranked bordeaux wines. these wines were purchased as gifts and were rarely consumed for pleasure (yang and paladino, 2015; lockshin et al., 2016; seidemann et al., 2016). in the last ten years, economic factors such as the rural exodus to urban areas with its ensuing access to more expensive goods, together with an increase in wealth, has spurred a wider-range of fine wine consumption (masset et al., 2016). the interest and enjoyment of foreign wines has become especially prominent in the largest cities (shanghai, beijing, shenzhen, guangzhou or hong kong) where a mix of expatriates, western-educated chinese and an open-minded population have an appetite for imported goods (mu et al., 2017; trotta et al., 2015; mitry et al., 2009; lee, 2009). this recent trend has also affected china’s social structure, which has been influenced by younger consumers who tend to purchase wine by using new e-commerce platforms, and it’s continuing to grow. for example, the demand for wine consumption in december 2012 for the younger target group was 31%, and reached all the way to 45% in march 2015 (li et al., 2011). new data from 2015 shows china's per capita consumption of wine increased from 1.9 to 2 liters and the market potential is expanding (bacca, 2015). before the 2012 nation-wide crackdown on corruption in china, a large extent of wine consumption depended on group-purchases by public institutions, and the products tended to be highend. however, after the chinese government ban on lavish banquets with great displays of wealth, this led to an almost immediate reduction in the purchase of very expensive wines from france. italian wine imports also fell due to the chinese anti-corruption laws, by almost 27%, but increased 7% in 2014, followed by an increase of 14% in 2015. the outcome has been a growth of individual interest in wine and increasing efforts from almost every wine producing country to tap into the growing chinese market for western wines (datamonitor international, 2015; lannes et al., 2012; lockshin, 2014; marketline, 2015; lockshin et al., 2016). additionally, the accession of china to the world trade organization in 2005 stimulated imports of wine due to a decrease from 43% to 14% in tariffs (masset et al., 2016; ianchovichina and martin, 2006). wine imports have risen by more than 400% to 289 million litres from 2008 to 2013 with france accounting for a market share of around 53% (geffroy, 2014). this data corresponding to a growing business volume in this period of 60%. from 2014 to 2015, china's imports of international wine had a further increase (from 1.17 to 1.84 billion euros), with france still in the top spot. in 2016, imports from france grew by 11% or 900 million euros (http://www.inumeridelvino.it/). it is 42% of the market, which becomes 45% when considering bottled wine and sparkling wine. starting in 2009, bottled wine began to play an important role in total imports, while imported bulk wine gradually decreased. this reflects the maturity of the consumer`s taste, and, on the other hand, reflects the upgrade of the chinese wine industry chain (lockshin et al., 2016). currently, italy represents 12% of china’s wine import market. italian imports increased by 33%; in terms of value, this translates to a 24% increase or 20.3 billion euros. huge business opportunities for imported wine in the chinese market stem mainly from the reduction in tariffs, an immature chinese wine brand market (only major domestic brands, such as changyu and a few others, cover china's low-end wine market) and an increasing demand by consumers for high-end products. data show that, before 2012, ital. j. food sci., vol. 30, 2018 395 french wine always held the largest share of china's imported wine market, and maintained a sustained growth. but since the second half of 2012, this growth has slowed down and decreased. this shows that chinese consumers are increasing the diversity of their taste preference and willing to try a variety of wines from sources around the world. clearly this trend offers abounding business opportunities for wine suppliers and brand operators. this is especially evident in large and medium-sized markets. imported wine sale volume in retail channels have also increased in the past 30 years. in china there are differences among commercial supermarket retail formats; there are supermarkets targeted to high-class consumer groups, such as the ole store of vanguard group, and city supermarket; there are supermarkets targeted to mass consumption like carrefour, wal-mart, rt-mart, lotus, and membership warehouse storage supermarkets such as sam's club and metro; there are neighborhood community supermarkets such as vingo shop and seven-11. all supermarket levels cooperate with traditional importers and distributors. in order to optimize the costs, supermarkets choose the large importers who have more varieties. the supermarkets take the entrance fee and commissions from each wine. overall the retail price of wine includes the supermarket storage cost and its profit. however in 2013, with the development of an e-commerce platform, the price is more competitive and transparent because there no entrance fees or other costs in these platforms. as a result, massive consumer supermarkets like carrefour choose to compete on price, so now they prefer to display low-grade wine to get a competitive price (qin, 2017). the rapid development of chinese e-commerce in recent years, in particular the development of comprehensive platforms, has led to a rapid development of imported wine e-commerce (zeng and szolnoki, 2016; fang et al., 2017). on the comprehensive platform, importers and distributors set up their own webpage directly for the consumers. the vertical e-business includes business-to-customer professional wine platforms such as yesmywine, wine9 and jiuxian. the vertical wine platforms work just like importers and also cooperating with exclusive importers for some particular brands. due to the different supply channels, wine from e-commerce has a lower cost. n overview of the chinese consumer of italian wine was provided using the search indexes and sale volumes provided by the taobao online platform. market performance and the evolution of italian and piedmont wines in the chinese e-market were also analyzed. 2. methodology two kinds of wine e-commerce platforms in china were used in this study: the chinese website for online shopping, taobao, and professional online wine business websites (yesmywine, wine9 and juxian). in china, one of the mainstream comprehensive websites is taobao and tmall from alibaba group. taobao was founded in 2003 by alibaba’s founder jack ma. taobao supports business-to-customer (b2c) and customer-tocustomer (c2c) retail by providing a platform for business and individual entrepreneurs to open online stores (gong, 2016; ren et al., 2012). every individual or company can open their own online shop in taobao: consumers can buy almost everything they need online, often at much lower prices than the ones of traditional retail channels. sellers on taobao list items for sale with a posted price and without a time limit excluding auctions with a fixed time-frame. this mechanism broadly allowed the use of posted price format both in online and offline retailing (chen et al., 2015). despite the posted price in taobao, compared to e-bay's online auctions, the price formation process includes several factors related to the communicated information by the seller, but above all the reputation ital. j. food sci., vol. 30, 2018 396 deriving from the feedback received: reputable sellers are found to sell more products or services at higher prices (chen et al., 2015; zhang et al., 2016; qiu et al., 2016; fan et al., 2014; melnik and alm, 2002). this aspect influences also the recommender system which represents one important advantage of online shopping, which, contrary to offline market, suggests to the consumer products they might be interested in (mo and chen, 2015). in 2011, taobao split into two websites: taobao and tmall. only authenticated brands can join tmall. this new b2c platform integrates thousands of brands, manufacturers, and provides the quality assurance of goods and a seven-day without reason return service (yu et al., 2013). today it has 500 million registered users and more than 60 million people visit the network daily. over 48,000 items are sold every minute on this platform (gong, 2016). as currently happens for the most used online wine sales platforms (mcgechan, 2013), taobao members can use computers or smart phones to visit its website or application to shop for goods. sellers using taobao can show their goods to customers who can select products and order from this "market". customers can also correspond and bargain with the sellers, similar to traditional markets in china (lu et al., 2009). taobao is an incredibly user-friendly platform, providing a shopping guide for users to learn about selecting products and making online payments (gong, 2016). in contrast, tmall takes advantage of the "shopping mall" model by renting out online booths to brands for a fee (zou et al., 2014). china is new to the concept of c2c. taobao brings the small businesses to the internet by adopting a “small businesses to consumers” model, whereas tmall uses a “brand to consumers” model (hu & checchinato, 2015). in 2014, alibaba launched tmall-international, which directly supplies the imported goods from abroad for domestic consumers. similar to the original tmall, the tmall-international platform only allows authenticated foreign suppliers to join. the suppliers can directly communicate with chinese consumers. tmall lends them warehouses located in bonded areas (the tax free zones), and the foreign suppliers send their goods to the warehouse. due to the cooperation between tmall-international and customs, each package passes the customs separately and quickly. however, due to strict policies and regulations, this system can’t be used with wine and cigarettes for now. payments are done by means of alipay for both platforms, which is not only an intermediary payment tool, but also protects the legitimate rights and interests for both the seller and the buyer. alipay was created in 2003 by the alibaba group team as an offline payment method designed to increase the use of taobao, improving the security of payments. unlike paypal, which takes high proportion commission when cooperates with some e-commerce site, alipay is free for both sellers and buyers (li and liu, 2007). additionally, taobao provides the online chatting platform, aliwangwang, to ensure a successful transaction and increase transparency (gao et al., 2016; holsapple et al., 2014). taobao’s reputation system is the most popular and successful one in china (lin and li, 2005): among the reputation growth strategies, taobao also provides a lot of information about its activities on its website by regularly posting information on the number of buyers and sellers transacting. in addition, taobao provides details on the total transaction volumes, for each product category. taobao also makes publicly available several indices: the taobao index provides information on searches, transactions and characteristics of buyers at product category level. the taobao interest index tracks searches, bookmarks, and transactions by category by day and by week (chu and manchanda, 2015). data from alibaba official software indextao were used for our analysis. indextao software can analyze users data related that had searched a specific keyword in taobao. all the data with the distribution curve have to go through an indexation process, so here the "search index" (daily keywords search data) is an indexation of search volume, ital. j. food sci., vol. 30, 2018 397 reflecting search trends, and it is not equivalent to the times this keyword had been searched on taobao (li et al., 2014). users are considered as potential consumers of the specific keyword. italian wine performance was analyzed from january 2013 to december 2015 using “italian wine” and “italian red wine” keywords. data obtained are reliable, because each alipay is associated with users personal bank account and id card. to protect privacy, alibaba doesn’t disclose any of the searches and the specific number of associated data. chinese wine consumers characteristics based on the platform data were analyzed: consumers registration information were extracted using taobao search index to analyze users characteristics (age, gender, wine consumption levels based on the user’s shopping record within six monthshobbies, and the geographical distribution). for the analysis of age range, indextao introduced the concept of the preference index (pi), the ratio between the proportion of all the potential consumers in a specific age range and the proportion of the same consumer group of taobao users. for example, the preference index of age group 18-24 is equal to the proportion of 18-24 potential consumers in all potential consumers (alibaba) divided to the proportion of 18-24 consumers in the taobao users. age data is divided from 18 to 24 years, 25-29 years, 30-34 years, 35-39 years, 40-59 years, 50-59 years and more than 60 years. the level of consumption is defined by the frequency of choosing a product, either expensive or not, on the website within six months. the range is defined by low, under average, medium, over average and high level of consumption. data from yesmywine were employed to analyze italian wine market performance, available wines on the platform and the sales volume of italian and piedmont wines. the same research was carried out using the wine9 and juxian platforms. many chinese industries began to create their own vertical business-to-customer (b2c) websites. china now has many professional online websites for the imported wines. the selected b2c online wine business websites presented different wine products and established a double service, both online and offline sales platforms. yesmywine, a website founded in 2008, has already become one of the biggest b2c wine specific platforms in china focusing on imported wine. its success can be attributed to an all-round, three-dimensional, interactive customer information management system (gao and li, 2015). it keeps a close relationship with customers through wine tasting events and other programs. according to a recent study, consumers who buy wine on yesmywine were 18-35 year-old, middle class, located in first or second-tier cities, and frequently used the internet (li, 2016). this consumer group has some common characteristics: they are located in large cities and a majority of them are sensitive to the price. they pursue high consumption efficiency and a high-quality lifestyle. in the chinese wine market, the majority of the offline wine shops are supplied by distributors, from importers to secondary and tertiary distributors. the cost increases due to changing hands in the supply chain. some of the wines on yesmywine come from direct purchase abroad since yesmywine works as an importer. the other parts come from primary suppliers, or directly from the importers. currently yesmywine directly purchases from 11 countries, including grands crus classés wine from bordeaux, olivier leflaive from burgundy, spier from south africa, craneford from australia, beronia from spain, henri abele from france, rio bueno from chile, fabiano from italy, erswnwe praelat from germany, and also some unknown brands from various wineries. wine can be searched according to price and production areas. wine9 was founded in 2009 and is under the jurisdiction of three divisions: the wine9 official website, a comprehensive platform (online store on tabobao and tmall) and an offline department (for wholesale). in 2015, wine9 began to import beer, chocolate, food, and health products. today it has 4 million registered users online and 80 offline franchisees. ital. j. food sci., vol. 30, 2018 398 another professional wine business is jiuxian. on the webpage layout of jiuxian, the wine is sorted according to sales volume. jiuxian uses low prices for their products, which determines their selection of wines, mostly the easily accepted cheap sparkling wine, or oem (original equipment manufacturer) wines, such as rosso&rosso series. jiuxian only has a small selection of italian docg (controlled and guaranteed designation of origin) red wines. 3. results from the taobao index data analysis, si of keyword “italian red wine” (fig. 1) reached 688 in 2014 and received the highest index of 1187 in 2015. from 2013 to 2014, the average search index was about 100, while in 2015 after the peak, si of “italian red wine”, reached about 200 on average. fig. 2 reported the search index of the keyword “italian wine”. from 2013 to 2014 the si had been maintained at a low level with an average of about 50. in 2014 the index appeared in a peak reaching 571. from 2014 to 2015, the si had been maintained at a high level. there was three index peaks: 571, 588 and 704. figure 1. the si of keyword “italian red wine”. figure 2. the si of keyword “italian wine”. ital. j. food sci., vol. 30, 2018 399 figs. 3 and 4 show the geographical distribution of consumers who searched the keywords “italian red wine” and “italian wine”, respectively. potential users were concentrated in southeastern coastal regions of china. in both cases, guangdong province had the highest proportion, while the second was zhejiang province. the proportions of the two key words were 18.73% and 15.92%, respectively. figure 3. geographic distribution of potential consumers under the keyword “italian red wine”. figure 4. geographic distribution of potential consumers under the keyword “italian wine”. preference index (pi) was used for consumers’ age description: no users older than 60 years searched for “italian wine” online. results of pi showed that consumers from the ages of 18-24 have no obvious or slightly low preference to italian wines. the age groups 35-39 and 40-59 have a low preference for italian wine, but the age group 50-59 has the highest preference. on the view of distribution, the 25-29 and 30-35 age groups accounted for 47% of the population in the "italian red wine" keyword. regarding the “italian wine” keyword, the latter two age groups accounted for 52.7%. however, a higher percentage ital. j. food sci., vol. 30, 2018 400 does not mean a higher preference. this means the potential customers are concentrated mostly in the 25-34 age range. due to people in this age range accounting for a large percentage of the e-commerce users, they have very large base. we can see the preference index of this age as average or slightly over average, and they have a preference use for the keyword “italian wine” instead “italian red wine”. in terms of gender, males are proportionately higher than that of females, but the difference is not significant. figs. 5 and 6 reveal the level of consumption (the frequency of choosing a product, either expensive or not, on the website within six months) chinese wine consumers reported for both keywords. potential consumers for italian wine were concentrated in the average, over average and high-class levels, which mean they have a good acceptability of high price commodities. figure 5. consumption level of potential consumers under keyword “italian red wine”. figure 6. consumption level of potential consumers under keyword “italian wine”. ital. j. food sci., vol. 30, 2018 401 figs 7. and 8 show the same trend of hobbies of italian wine and italian red wine. the taobao users who search italian wine also have an interest in the outdoors, sports, photography, home (quality life) and collecting. yesmywine presents a total of 6,824 wines from 13 different countries (fig. 9) (http://www.yesmywine.com/). the top three countries were france with 3,111 wines, australia with 684 wines, and italy with 600 wines. figure 7. hobbies of potential consumers of “italian red wine”. figure 8. hobbies of potential consumers of “italian wine”. yesmywine had 600 italian units of wine with a combination of sales (red, sparkling and white wines). this does not mean 600 varieties of wine. from the point view of price rank, yesmywine has 8 price rankings (table 1). in the 1-49¥ (1-7€) range, there were 13 italian wines. out of this total, 6 were sparkling wines, 6 regional red wines, and 1 white wine. in the 50-99¥ (7-14€) range, there were 116 italian wines, including 52 sparkling wines. of the 41 red wines, there were regional wines such as chianti, barbera d’asti, montepulciano d’abruzzo, nero d’avola, and primitivo. 19 white wines, and 4 rose wines. in the 100-199¥ (14-27€) range, there were 130 varieties, including 72 red wines, 44 sparkling wines and 14 white wines. this section had 72 red wines, all which were based on the regional red wines. in this price range appeared dolcetto d’alba, barbera d’asti, barbera d’alba, nero d’avola, primitivo, nebbiolo, chianti, chianti classico, and chianti riserva. in the 200-299¥ (27-42€) range, there were 108 wines, including 70 red wines, and 20 were combination sales. this price section was still strong in regional red wines, showcasing amarone, barbaresco, dolcetto d’alba, ital. j. food sci., vol. 30, 2018 402 barbera d’alba, chianti, chianti classico, langhe doc, corvina di valpolicella, montepulciano doc, and freisa d’asti. the sparkling wines had 32 varieties, but most of them were combination sales. figure 9. origin of wine sales on yesmywine. table 1. italian wine numbers in 8 price ranges. price rank in yuan price rank in euro total number red wine sparkling wine white wine 1-49¥ 0-7€ 13 6 6 1 50-99¥ 7-14€ 116 41 56 19 100-199¥ 14-27€ 130 72 44 14 200-299¥ 27-42€ 108 70 32 6 300-499¥ 42-69€ 80 55 23 2 500-799¥ 69-111€ 61 54 7 7 800-999¥ 111-138€ 22 21 0 1 over 1000¥ 138€ 70 59 4 7 in the 300-499¥ (42-69€) range, there were 80 commodities, including 55 red wines. except for the combination of red wines, regional wines such as brunello di montalcino, dolcetto d’asti, and roero rarely appeared. wgvt (wine grape varietal table) and gi (geographical indication), mainly were doc (controlled designation of origin) and docg (controlled and guaranteed designation of origin). this range has many brands of barolo, amarone, and nebbiolo. there were also 23 kinds of sparkling wines. combination sales accounted for 90% of the entire range. other single commodities were the proseccos. in the 500-799¥ (69-111€) range, there were a total of 61 commodities, including 48 red wines, of which 11 were amarone, 8 barolo, 7 brunello di montalcino, 5 ital. j. food sci., vol. 30, 2018 403 barbareco, 4 barbera, and 3 chianti. the remaining few wines were doc regional red, as well as 7 white wines. sparkling wine in this range only represented the franciacorta by berlicchi. in the 800-999¥ (111-138€) range, italian wine had a total of 22 items in which 21 were red wines, 4 brunello di montalcino, 3 barolo, 3 amarone, 4 kinds of tuscany igt, 1 barbaresco, 1 langhe doc, 1 barbera d`alba, and 4 group combination sales. only 1 white wine combination was sold. in the over 1000¥ (138€) range, there were 70 kinds of wines. in addition to the combined products, 59 were red wine, except for the famous brunello di montalcino, barolo, and amarone. other wines came from well-known wineries. in fig. 10, italian wine has been classified by geographical division. italy’s piedmont region has the largest number 151 kinds of wines, accounting for 25% of the total. tuscany and veneto both rank 2nd. some regions have a few wines on yesmywine, like campagnia, which has only one wine, which is not labeled on the figure. in other northern italian regions, aosta valley, trentino alto adige, and liguria do not have wines on yesmywine. as for southern italian regions, our research did not find wines from molise and basilicata on yesmywine. however, wine from major regions can be found on yesmywine. the most important region, piedmont, presented a total of 151 commodities on yesmywine. there are 109 red wines of which 32 brands are barolo, 22 barbera, 21 barbaresco, 11 dolcetto, 8 lange doc, 8 combination sales, 4 nebbiolo, 1 roero, 1 freisa, and 1 grignolino. sparkling wines include 38 products, mainly the moscato variety. white wine were 16, mainly chardonnay, gavi and combination sales. figure 10. italian wine geographical subdivision. the sale volume data was collected from each sale page. in the italian wine section, wines are ranked by sale volume. the wines sold from each italian region are reported in table 2 comparing the scenarios in the two considered years (2014 and 2016). ital. j. food sci., vol. 30, 2018 404 table 2. wines sold from each italian region in 2014 and 2016. italian wines sold in 2014 wines sold in 2016 region of origin n. of labels region of origin n. of labels veneto 9 emilia romagna 10 emilia romagna 6 marche 5 piedmont 2 veneto 4 marche 1 piedmont 2 in 2014, in the top 25 wines, 9 wines come from veneto, 6 from emilia romagna, 2 from piedmont. the average price was 74.16¥ (10€). the first wine sold for volume was an igt white malvasia sparkling sweet wine from emilia romagna (price at 78 ¥, 21,1582 bottes sold), followed by a veneto igt (price at 52 ¥, 11,2627 bottles sold), and by another veneto igt (price at 49 ¥, 106,920 bottles sold), which was the cheaper product in this classification. this ranking comprised mainly of wines from emilia romagna and veneto. the most expensive wine was an abruzzo doc (price at 135 yuan, 5,092 bottles sold), which ranked 14th. data collected from yesmywine in 2016 showed a different scenario compared to 2014. the best-selling wine in terms of volume remained the same as 2014, with an increase in price (88 ¥) and volume (237.839 bottles sold, +11% with respect to 2014). moreover, new labels originating from the traditional brands were included in the rankings. marche wine held the highest price (159 ¥). of the top 25 wines in 2016 were 4 wines coming from veneto, 10 from emilia romagna, 2 from piedmont, and 5 from marche. number of italian wines sold by yesmywine platform classified in the price ranges is reported in table 2, comparing the two considered years (2014 and 2016). none of the other wines were included in the higher price range in both the years (table 3). the cheap wines still dominated the top sellers of italian wines. in 2014 there were 12 sparkling wines, 12 red vdt or igt wines, 1 white garganega. in 2016, 18 sparkling wines in total, 5 red vdt or igt wine, 1 white garganega, and 1 rose merlot were sold. the cheap sparkling wines from emilia romagna and piedmont, as well as the vdt wine from veneto, were very popular choices. comparing 2016 with 2014, sparkling wine increased in 2016 and, on the contrary, red wines decreased from 12 to 5 products. the sparkling wine was very profitable and the red wine sold as usual. the average price of the top 25 piedmont wines was 302¥ (42€). it was 4 times compared to the italian wines, due to the high price of nebbiolo. table 3. classification of italian wines in function of price range. the comparison between the scenarios in 2014 and 2016 is reported. price rank in yuan price rank in euro total number in 2014 % total number in 2014 % 1-49¥ 0-7€ 4 16% 4 16% 50-99¥ 7-14€ 16 64% 19 76% 100-199¥ 14-27€ 5 20% 2 8% the top 25 piedmont wines in 2014 were divided: 11 sparkling wines and 14 red wines. moscato was the most popular variety (average price was 108¥, 14€). the most popular red variety was nebbiolo with an average price of 728¥ (101€). in 2016, the top 25 piedmont best sellers had changed. this may be due to some wines being sold out. the ital. j. food sci., vol. 30, 2018 405 new data from february 2016 shows the same trend. there were 10 sparkling wines and 15 red wines. in comparison with the top 25 of italian wines, the piedmont ones had a higher price. moscato was still the most popular white variety with an average price equal to 101¥ (14€), while nebbiolo was the most popular red variety with an average price of 811¥ (112.5€). the best seller of piedmont wine from both considered years was a sweet sparkling wine (80% malvasia, 20% moscato) with an increased sales volume, growing from 4,843 to 11,311 bottles (+57%). however, the increase of red wine was very slow, with sales volume only several hundred, even though some new red wines appeared in 2016. overall, yesmywine has sold a very important number of italian wines. most of them are cheap red wines and sparkling wines. some of these wines are only produced for foreign market and can’t be found in the italian market. some of the bulk wines are bottled in china. the real bottled and high quality italian wines don't have a gratifying performance in the chinese e-commerce market. yesmywine does not assume the obligation to promote italian wines. for each product, yesmywine describes the wines in detail, including brand, winery information, origin, and also a brief introduction of the country. however, it is possible to find on the wine page some false information. this is especially true regarding information on the labeling of wine-producing areas, classification, the winery’s name, and the verification method or the introduction of the wine. it is not comprehensive because oftentimes when the information is translated from italian to chinese it can lose the correct meaning. in 2014, the best wine seller on wine9 was moscato from piedmont. the first (price 168 ¥) has sold 13,372 bottles, while the second one (79 ¥) sold 13,456 bottles. sale volumes were much higher than the third. in fact, in third place was a red wine from veneto (389 ¥) with sales volume equal to 5,022. this is not only because of the lower price of moscato, but also due to moscato being an easy to drink wine with low alcohol percentage. in china, moscato is popular for both male and female consumers. moscato in 2014 had a beautiful blue bottle and label, according to the consumer`s comments. they consider moscato to be the “best party wine”. in 2016, the ranking of the top 25 italian wines changed. this time the first place was a moscato spumante from emilia romagna (78 ¥, 7,097 bottles sold). piedmont wines positioned in last place. wine9 had a higher price in top 25 sellers wine compared to that of yesmywine. wine9 chose to cooperate with several famous wine brands like zonin, masi, banfi, and citra and has paid more attention to italian red wines. the regional red wines from veneto and tuscany were also popular. in 2014, 7 piedmont products were sold on the wine9 website (table 4). moscato, gavi and barbera varieties represented this rank with different prices (with a maximum of 1,299 ¥ of 2 barbera, to 79 ¥ of moscato2). however, in 2016, only moscato and one brand of malvasia were the only piedmont wines that could be found on wine9. table 4. piedmont wines on wine9.com. rank classification variety price/yuan sales volumes (bottles) 1 / moscato 1 168 13772 2 / moscato 2 79 13456 3 docg gavi 1 398 4469 4 docg barbera 1 238 3702 5 docg moscato 3 99 3629 6 docg gavi 2 298 3304 7 doc barbera 2 1299 3140 ending in 2014, 7 from piedmonts wines on wine9.com. ital. j. food sci., vol. 30, 2018 406 table 4. continues. rank classification variety price/yuan sales volumes (bottles) 1 / malvasia 1 118 1821 2 / moscato 4 89 1820 3 / moscato 5 78 1742 4 / moscato 6 88 698 ending in 2016, 4 piedmonts wines on wine9.com analyzing the ranking of 25 italian wines sold through jiuxian, in 2014 the top 5 wines sold were piedmont table wines sold at a maximum price of 59 ¥ and a minimum of 29 (piedmont table wine is the lowest in the top 25 of italian wines). in general piedmont wine accounted for 52% of italian wine sold. the most expensive wine sold in 2014, and ranked 17th, was a docg in apulia at 269 ¥. in 2016, piedmont wines were reduced by 36%, which fell to the last place in the ranking. however, regarding table wines, they were replaced in 2016 by important docgs. nebbiolo, with high prices (1099 ¥) was in 22nd place. at the top of this ranking was a lambrusco of emilia romagna (168 ¥), followed by a merlot veneto and, third, a piedmont muscat (79 ¥). in 2016, piedmont wines sold on jiuxian decreased by -50% (from 18 to 9 products) (table 5). table 5. piedmont wine on jiuxian.com. rank classification variety price /yuan 1 wgvt grapes mixture 1 59 2 / grapes mixture 2 69 3 wgvt merlot, sangiovese 1 49 4 wgvt merlot 1 29 5 wgvt moscato 7 39 6 docg moscato 8 128 7 / moscato 9 79 8 / moscato 10 69 9 / moscato 11 59 10 / moscato 12 69 11 wgvt dolcetto, barbera, pinot nero 1 99 12 wgvt chardonary, pinot grigio 1 59 13 wgvt barbera 3 79 14 / malvasia 2 96 15 moscato 13 118 16 doc barbera 4 88 17 doc croatina1 398 18 doc barbera 5 699 piedmont wine present on jiuxian.com in 2014. ital. j. food sci., vol. 30, 2018 407 table 5. continues. rank classification variety price /yuan 1 docg moscato 8 79 2 / mix 1 118 3 / malvasia 1 79 4 / prosecco 1 158 5 / moscato 14 149 6 docg nebbiolo 1 1099 7 / moscato 13 118 8 / moscato 9 89 9 / moscato 4 78 piedmont wine present on jiuxian.com in 2016. this development saw the elimination of table wines and the affirmation of sparkling wines. furthermore, there was an introduction of quality wines, such as the nebbiolo variety selling at the price of 1,099 ¥. in table 6 a comparison between the sales ranks of piedmont wines on the various online sales platforms is reported. ital. j. food sci., vol. 30, 2018 408 table 6. comparison between ranks of piedmonts wines sold by the different considered online platforms in the two years (2014 and 2016). yesmywine jiuxian wine9 2014 2016 2014 2016 2014 2016 rank variety price/yuan variety price/yuan variety price/yuan variety price /yuan variety price/yuan variety price/yuan 1 malvasia 68 sparkling and sweet malvasia 65 grapes mixture 1 59 moscato 8 79 moscato 1 168 malvasia 1 118 2 martini 107 fragolino 98 grapes mixture 2 69 mix 1 118 moscato 2 79 moscato 4 89 3 moscato 89 moscato 2 69 merlot, sangiovese 1 49 malvasia 1 79 gavi 1 398 moscato 5 78 4 moscato 2 79 grapes mixture 1 68 merlot 1 29 prosecco 1 158 barbera 1 238 moscato 6 88 5 barolo 259 sweet white sparkling wine 59 moscato 7 39 moscato 14 149 moscato 3 99 \ \ 6 moscato fruit 105 moscato sparkling 110 moscato 8 128 nebbiolo 1 1099 gavi 2 298 \ \ 7 moscato rose 148 moscato 15 212 moscato 9 79 moscato 13 118 barbera 2 1299 \ \ ital. j. food sci., vol. 30, 2018 409 4. discussion and conclusions our research has confirmed the rapid evolution and development of the wine market, in particular italian and piedmont wines, in china. the use of online sales has made this market even more dynamic in the short-term and becoming increasingly accessible by more target consumers. the more important wine e-commerce platforms are yesmywine, wine9, jiuxian, and the comprehensive platform, taobao. italian wine is ranked fifth in the wine market in china, after france, chile, spain and australia, but italian sparkling wine ranks first. in fact, our research has found that buying sparkling wine is experiencing a period of strong expansion, especially for red sparkling wines. this was evident when comparing 2014 with 2016. consumer profiles from taobao data described young consumers being especially attracted to medium-high level products in terms of price. this result is confirmed also in fountain and zhu (2016). the evolution of red wine typology purchased from 2014 to 2015 also justifies the changes in the italian wine rankings sold through the other online sales platforms considered in this work. the data confirmed that the chinese red wine market has grown by 66% from 2009 to 2014 (euromonitor, 2015), and 92% of wine consumption in 2013 was red (hermoso, 2014). potential consumers are those who are looking up wine on e-commerce platforms, but not necessarily purchasing the products. it's worth noting that consumers looking for "italian wine" on taobao do not mean that they are buying it (mitry et al., 2009; lee, 2009). most of the potential consumers are concentrated in the southeastern coast of china because are concentrated mainly in big cities. however, the society in rural china has changed under the network economy and the e-commerce developments (geng et al., 2016). in recent years, the imported italian bulk wine has had a slight increase in volume; however it still maintains a high average price among the important countries. this phenomenon ensures that the chinese market does not have low-end italian wines and protects the high-end italian wine market. our analysis of the top 25 sellers from professional online platforms shows that chinese consumers prefer sparkling wine from piedmont and emilia romagna. the typical original red wines from veneto and tuscany are more saleable. the data shows piedmont sparkling wines are much more popular in china compared to the piedmont red wines. however, the tendency of chinese consumers to increasingly buy expensive wines, compared to previous years (zeng and szolnoki, 2016), is confirmed by our results that highlight as consumers are willing to pay a high price for piedmont red wines like barolo. as reported in masset et al. (2016), the increased interest in fine wine consumption in china is linked to improved economic opportunities and political transparency has led the population to adopt traits from western lifestyles. at the same time, chinese government policies have altered consumer behavior by favoring healthier red wine consumption. finally, a more educated and wealthy class has emerged a fondness for fine wine (sun, 2009). compared to wine9 and jiuxian, yesmywine has more italian wine and better descriptions of its wine, but problems still exist. italian wineries should enhance cooperation with the chinese importers like yesmywine. this includes supervising the marketing process and assisting the importers with the wine descriptions on their websites. this could be done by wine producers in italy translating its brand and wine in chinese for the convenience of local consumers, and try to associate the wine with its cultural and historical background, an element loved by chinese consumers. additionally, italian wine producers should try to explain how the wine can be combined with various chinese dishes on the description pages. if there is no appropriate chinese dish to pair ital. j. food sci., vol. 30, 2018 410 with the wine, try to introduce some italian typical dishes, and combine with italian food culture to attract consumers (lockshin et al., 2016). traditionally, wine is developed in each locality as an accompaniment to local foods. wine is now traded globally, creating challenges for matching wine with cultural customs and cuisines typical of countries where wine is not a traditional beverage. furthermore, imported wine is also considered a status symbol in some countries (lockshin, et al., 2012; 2016). to buy wine in china is more than just the product, but it’s also buying the foreign wine culture (china market, 2011). the translation of wine brands and labels plays a key role in conveying the wine’s message to potential consumers and these translations must deal effectively with culture-specific items (champney, 2014). parallel, china’s e-business environment is ripe full of opportunities for wine sellers due to the following reasons: first, china does not have a mature wine retail terminal chain compared with italy. second, china has a mature internet platform system and a huge user-base. third, china is mature in thirdparty payment providers and has low logistics costs. a statistically significant difference between online and offline border effects in china has been demonstrate by li et al. (2016). in online trade, unlike offline trade, local governments face difficulties in controlling border effect and to restrict online trade: this effect due to the existence of government protectionism in the offline market should be used by italian producer of wines. data from taobao showed that the 25-35 age group has more preference for italian wines than french ones. social media has significantly influenced this age group, which is a big opportunity for wine producers to advertise their products. this is seen as an effective tool compared to conventional advertising since it provides quick and informative details and gives rise to an increased number of responses (eisend & kuster, 2011; lockshin et al., 2016). in 2015, wine9 gradually ended cooperation with the importers and began to purchase and import wines all on their own for the purpose of increased profits. this reform had a negative impact on the piedmont wine industry. the change also created new problems of developing a professional online wine platform. for example, they didn`t have experience with wine selection or strong financial support that large importers have. when they skipped the importers, wine9 had difficulty with wine selection and the financial support was not enough to support the diversity of wine. these reasons caused the varieties to decrease piedmont wine on wine9. however, this phenomenon just shows how important the process is for the development of professional online wine platforms and the cooperation with importers. acknowledgements we would like to thank all of the producers, operators, colleagues and collaborators who actively participated in this research project. the authors are also grateful to winston gilcrease unesco chair, university of turin for his assistance in editing the final version of this paper. references bacca 2015. i consumi di vino nel mondo 2014 aggiornamento oiv. http://www.inumeridelvino.it/2015/05/iconsumi-di-vino-nel-mondo-2014-aggiornamento-oiv.html#more-16451 champney a.g. 2014. wine translation in the chinese market. communicating in a connected world, 78. chen l., johnson g. a. and luo y. 2015. great and small walls of china: distance & chinese e-commerce. china market 2011. national strategies on the influence of imported wine to the chinese wine industry. volume 45. ital. j. food sci., vol. 30, 2018 411 chu j. and manchanda p. 2015. quantifying cross and direct network effects in online c2c platforms. marketing science, forthcoming; ross school of business paper no. 1248. data from offical jiuxian website. http://www.jiuxian.com/ data from offical wine9 website. http://www.wine9.com/ data from offical yesmywine website. http://www.yesmywine.com/ datamonitor international (2015). wine in china. http://www.datamonitor.com/store/product/china_wine?productid=mlip1635-0005/ eisend m. and kuster f. 2011. the effectiveness of publicity versus advertising: a meta-analytic investigation of its moderators. journal of the academy of marketing science 39(6): 906. euromonitor 2015. passport wine in china report. june 2015. fan, y. ju, j. and xiao m. 2014. reputation premium and reputation management: evidence from the largest e-commerce platform in china. fang y. yang h. and zhang x. 2017. development process, current status and future trends of chinese grape and wine industry. in the wine value chain in china (pp.. 269-281). fountain j. and zhu m. 2016. young chinese consumers’ wine socialization, current wine behavior and perceptions of wine. in: capitello r. 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valorisation (biogest-siteia), university of modena and reggio emilia, via amendola 2, 42122 reggio emilia, italy cdepartment of agricultural and food sciences (distal), university of bologna, via fanin 44, 40127 bologna, italy *corresponding author: tel.: +39 (0)522522085; fax: +39 (0)522522027 email: giovanna.minelli@unimore.it abstract the effects of including extruded linseed in pig diets supplemented with either polyphenol-rich red grape skin extract (3 g kg-1) or synthetic antioxidants (200 mg kg-1 αtocopheryl acetate plus 0.21 mg kg-1 of selenium) on shelf-life of pork stored in modified atmosphere packaging (map) at different oxygen concentrations (0 and 70%) were evaluated. linseed reduced n-6/n-3 fatty acid ratio in lipids of backfat and loin. color parameters, ph, weight losse, oxidative stability (tbars), did not differ between antioxidants neither in raw, nor in cooked, nor in stored muscle. high oxygen concentration in map increased tbars and ∆e, yielding redder meat. keywords: antioxidant, extruded linseed, fatty acid composition, grape skin, modified atmosphere packaging, pork ital. j. food sci., vol. 32, 2020 152 1. introduction in recent years, consumers’ awareness of the cause-effect relationship between dietary fat composition and health has prompted research into modify omega-6/omega-3 polyunsaturated fatty acids (pufa) ratio in meat and meat products (corino et al., 2008; riley et al., 2000). it is known that in western countries omega-6 fatty acids are the majority of pufa in the food supply, whereas the consumption of omega-3 fatty acids is very low (simopoulos, 2002). among the factors that can affect the deposition of lipids and their fatty acid (fa) composition in pig meat, diet composition and slaughter weight play a pivotal role (lo fiego et al., 2010; wood et al., 2008). thus pork can be a source of omega-3 pufa whether pigs are fed with diets containing linseed or its by-products, feeds rich in αlinolenic acid (c18:3n-3, ala) (corino et al., 2014; guillevic et al., 2009b; hoz et al., 2003; musella et al., 2009). on the other hand, the enrichment in pufa can negatively affect the oxidative stability of pork. lipid oxidation impairs the acceptability of meat modifying sensory attributes and nutritional quality (jakobsen and bertelsen, 2000). to prevent the oxidative phenomena, synthetic antioxidants are usually added in pig diets, vitamin e, as αtocopheryl acetate, being among the most common (dunshea et al., 2005). recently, consumers have shown an ever-increasing interest in natural antioxidants obtained from plant sources (haak et al., 2008; lorenzo et al., 2014). winery by-products are rich in phenolic compounds whose antioxidant activity has been widely studied (brenes et al., 2016). further, anti-inflammatory, anti-carcinogenic, cardioprotective and vasodilatory properties have been ascribed to polyphenols (teixeira et al., 2014). in the po valley, where italian pig production is concentrated, the cultivation of the grapevine is also widespread. wine industry, like many other sectors of the italian agri-food industry, produces a lot of waste products with consequent environmental problems. for instance, wastes represent almost one third of the total of the grapes used in wineries, posing serious storage and disposal problems (teixeira et al., 2014). the commitment to sustainable agriculture requires the reduction or elimination of these wastes. in light of all this, stakeholders of the pig industry have shown a strong interest in the possibility of utilize these by-products in the feeding of pigs. nonetheless, the effect of grape skin extract in pig feeding on pork quality still needs to be elucidated (brenes et al., 2016). usually fresh meat is commercialized packed in expanded polystyrene trays wrapped with stretchable cling films, characterized by high oxygen transmission rate, in order to maintain the typical bright color. in these conditions, fresh meat has a very short shelf-life due to microbial proliferation and a rapid brown discoloration (due to metmyoglobin formation) that normally takes place before unacceptable bacterial growth has occurred. in order to further increase the shelf-life of meats modified atmospheres packaging (map), usually characterized by variations in the content of n2, co2 and o2 in the package, is extensively used (spanos et al., 2016). modified atmosphere packaging in an atmosphere high in oxygen (up to 80%) extends display life, prolonging the stability of the oxymyoglobin red pigments, but it promotes lipid oxidation (mcmillin, 2017). the aims of this study were to investigate the effect of inclusion of extruded linseed in pig diets on carcass lipid fatty acid composition and to evaluate the protective effect of either supranutritional doses of vitamin e and selenium (se) or grape skin extracts on the quality and shelf-life of pork packaged in modified atmospheres with or without oxygen in the inner gaseous atmosphere, during refrigerated storage. ital. j. food sci., vol. 32, 2020 153 2. materials and methods 2.1. animals and diets all the experimental procedures were in accordance to the italian legislation (d.lgs 4 marzo 2014 n. 26 art. 2 punto f). twelve castrated male italian large white pigs, evenly divided according to weight into three groups of four subjects each, were housed in 9 m2 concrete floored pens. starting from 79.4±7.4 kg body weight (bw) and till slaughtering, at 135.4±9.7 kg bw, the subjects were fed restricted for 70 days on 8.5% of metabolic weight (bw0.75) either on a barley-soya bean meal feed (c group) or the same feed where 5% barley was substituted with 5% extruded linseed. linseed diets were supplemented either with supranutritional levels of synthetic antioxidants (200 ppm of α-tocopheryl acetate and 0.21 ppm of se (le group) or with 3 g kg-1 feed of red grape skin extract (enocianina fornaciari, reggio emilia, italy) (lgse group), providing 29.8 ppm of polyphenols (expressed as gallic acid equivalent). grape skin extract is a natural product used in the food industry as a supplement, nutraceutical or food coloring, included in finished products at concentrations ranging from 2 to 4g kg-1, according to the manufacturer’s suggestions, that we complied with. the diets were isoenergetic and isoproteic and with the same lysine/digestible energy ratio. water was always available through nipple drinkers and the diet was distributed in liquid form (water:feed ratio 3:1). grape skin extract was diluted in the water of the diet. composition of experimental diets and their fatty acid proportions are shown in table 1. 2.2. slaughtering and sampling the pigs were slaughtered on the same day in a commercial slaughterhouse, where the subjects were electrically stunned, in agreement with the council regulation (ec) no 1099/2009 on the protection of animals at the time of the killing. after slaughtering, each carcass was graded using fat-o-meater, at level of 3/4 last rib, at 8 cm from the splitting line of the carcass (d.m. mipaaf october 24, 2018 gu n. 298, december 24, 2018) and dissected in commercial cuts. at dissection the whole left longissimus thoracis et lumborum (ltl) muscle and a sample of subcutaneous adipose tissue at the last rib level, were removed from each carcass. the samples were stored at +4°c and sent to the laboratory for subsequent analyses. at the lab, 24 h post mortem, individual ltl muscles were sliced and, on a subsample taken at the last rib, some physical parameters and oxidative stability were determined as described below. a second subsample of ltl muscle, destined to chemical analyses, was vacuum packed (elegen, scandiano, reggio emilia, italy) and stored at -20°c. eventually, six slices for each ltl muscle were packaged in modified atmosphere for the shelf-life study. individual backfat samples were vacuum packed and stored at -20°c for the subsequent fatty acid composition determination. 2.3. physical parameters of raw and cooked muscle the values of ph were measured at 24 h post mortem using a portable crison ph-meter equipped with a xerolite electrode (crison instruments, alella, spain). the ph probe was calibrated using two buffers (ph 4.0 and 7.0). ital. j. food sci., vol. 32, 2020 154 table 1. composition of experimental diets (as fed basis). diet (a) c le lgse ingredients extruded linseed % 0.0 5.0 5.0 barley % 89.5 84.4 84.5 solvent extracted soybean meal % 7.0 7.0 7.0 l-lysine % 0.3 0.3 0.3 l-threonine % 0.1 0.1 0.1 calcium carbonate % 1.2 0.8 1.2 dicalcium phosphate % 1.0 1.0 1.0 sodium chloride % 0.4 0.4 0.4 minerals and vitamins premix(b) % 0.5 0.5 0.5 vitamin e + selenium(c) % 0.0 0.5 0.0 red grape skin extract % 0.0 0.0 0.3 calculated nutrient composition(d) digestible energy (de) kcal/kg 3082 3151 3154 calcium % 0.78 0.76 0.80 phosphorus % 0.52 0.53 0.53 digestible phosphorus % 0.24 0.25 0.25 lysine % 0.85 0.87 0.87 digestible lysine % 0.73 0.75 0.75 lysine/de ratio g/mcal 2.75 2.75 2.75 analyzed composition (e) dry matter % 90.5 90.6 90.7 crude protein % as fed 13.2 13.7 13.6 crude fat " 1.6 3.5 3.5 crude fiber " 5.0 5.0 4.9 adf " 7.0 6.8 7.0 ndf " 20.6 19.6 20.8 adl " 1.3 1.2 1.4 crude ash " 5.2 5.1 5.0 fas(f) composition (% of total fas) c 14:0 (myristic) 0.59 0.26 0.32 c 16:0 (palmitic) 27.53 15.94 16.70 c 16:1 (palmitoleic) 0.08 0.02 0.02 c 18:0 (stearic) 1.30 2.86 2.79 c 18:1n-9 (oleic) 12.47 16.10 15.96 c 18:2n-6 (linoleic) 52.16 35.50 35.44 c 18:3n-3 (α-linolenic) 5.81 29.24 28.65 c 20:1 (eicosenoic) 0.06 0.04 0.11 (a)c, control (extruded linseed, 0 g kg-1); le, (extruded linseed, 50 g kg-1, vitamin e (α-tocopheryl acetate), 200 mg and se 0.21 mg kg-1); lgse, (extruded linseed, 50 g kg-1, red grape skin extract 3 g kg-1). (b)the vitamins and minerals of the diet provided by premix (kg-1): vitamin a, 15,000 ui; vitamin d3, 2000 iu; vitamin e (αtocopheryl acetate), 50 mg; vitamin k, 2.5 mg; vitamin b1, 2.0 mg; vitamin b2, 5.0 mg; calcium dpantothenate, 15.0 mg; niacin, 25.0 mg; vitamin b12, 0.036 mg; vitamin b6, 4.0 mg; folic acid, 1.0 mg; biotin, 0.15 mg; choline chloride, 346.0 mg; zn (zno), 100.0 mg; cu (cuso4), 15.0 mg; mn (mno), 25.0 mg; fe (feso4), 150.0 mg; i (ca(io3)2), 1.5 mg; co (coco3), 0.4 mg; se (na2seo3), 0.1 mg. (c)providing vitamin e (αtocopheryl acetate) 200 mg and se 0.21 mg kg-1 as fed, supported on caco3. (d)according to sauvant et al. (2004). (e)according to the association of official analytical chemists (1995). (f)fatty acids ital. j. food sci., vol. 32, 2020 155 the sample surface color was determined using a minolta cm-600d spectrophotometer (konica minolta holdings, inc, osaka, japan) with a window diameter of 8 mm and d65 as illuminant source. before color measuring, the spectrophotometer was calibrated against a white plate supplied by the manufacturer. color measurements complied the cielab color system, where the three fundamental coordinates are l*“lightness”, a* “redness”, b*“yellowness” values. further, chroma (c), referred to as saturation index and color intensity, was calculated as: (a*2+b*2)0.5, and hue angle (h), spectral color, was calculated as follows: tan-1 (b*/a*). the average values were the mean of three measurements in different areas of the surface of the sample, avoiding the zones of visible fat. drip loss was determined according to honikel (1998), slightly modified. a slice of fresh muscle (about 100 g) was hooked and then suspended in an inflated bag, ensuring that the sample did not make contact with the bag. the weight loss percentage after 48 h of storage at 4°c was calculated. cooking loss was calculated as weight difference between raw and cooked samples. in brief, slices of about 2.5 cm thickness, weighing approximately 100 g, placed in vacuum plastic bags, were put in water-bath till the core temperature reached 70°c. the internal temperature of samples was controlled during cooking with a temperature probe. the samples were weighed after cooling. cooking losses were expressed as a percentage of the initial sample weight. the warner–bratzler shear force (wbsf) was determined on the cooked samples. the samples were cut, parallel to the longitudinal orientation of the muscle fibres according to honikel (1998) method, into six cylindrical cores (ø 1.50 cm). each core was sheared with a wbsf device attached to a zwick z50 kn testing machine (model bt1-fb050tn, zwick roell, kennesaw, ga usa) with a 1kn load cell equipped with the v-shaped blade with a triangular hole of 60° at a speed of 250 mm/min. the peak force (average value of 6 measurements for each sample) was expressed as kg. 2.4. chemical composition of muscle after thawing, samples of ltl muscle were analyzed in duplicate to determine the moisture, ether extract with previous acid hydrolysis, and crude protein, according to the methods of the association of analytical chemists (aoac, 1995). results were expressed as percentage of wet matter. 2.5. fatty acid profile of backfat and muscle fatty acid (fa) profile of lipids in thawed samples of subcutaneous adipose tissue and ltl muscle was determined using a trace™gc ultra (thermo electron corporation, rodano, milano, italy) equipped with a flame ionization detector, a ptv injector, and a tr-fame column (thermo scientific, rodano, milano), 30 m long, 0.25 mm i.d., 0.2 μm film thickness. total lipids were extracted from the samples of subcutaneous adipose tissue (iupac, 1979) and from ltl muscle (folch et al., 1957). then, an aliquot of 25 mg was subjected to methylation by means of a methanolic solution of potassium hydroxide (koh 2n) according to ficarra et al. (2010), using tridecanoic acid (c13:0) (larodan fine chemicals ab, malmö, sweden) as internal standard. the injection of the fatty acid methyl ester sample (1 μl) was performed in split mode with a split flow of 10 ml/min, operating at a constant flow of 1 ml/min of helium as carrier gas. the temperature of injector and detector was kept at 240°c. the temperature program used for the analysis started from 140°c, was maintained for 2 min, then increased to 250°c, at a rate of 4°c min−1, and kept at this temperature for 5 min. the peaks of the fatty acid methyl esters ital. j. food sci., vol. 32, 2020 156 (fames) were recorded and integrated using chrom-card software (vers. 2.3.3, thermo electron corporation, rodano, milano, italy) and identified by comparison with the retention times of standard solutions with known quantities of various methyl esters (supelco® 37 component fame mix, pufa standard n.2, animal source, supelco, bellafonte, pa, usa and single fames standard, larodan fine chemicals ab, malmö, sweden). for quantification purposes, the response factor was calculated, and the method of the internal standard was used. the amount of each fame in the sample was expressed as fame relative percentage with respect to the total amount of fames. iodine value (iv) of backfat was calculated adopting the equation proposed by lo fiego et al. (2016): iv = 85.703 + [c14:0] x 2.740 [c16:0] x 1.085 [c18:0] x 0.710 + [c18:2n-6] x 0.986. 2.6. oxidative stability of raw and cooked muscle oxidative stability was evaluated by the 2-thiobarbituric acid reactive substances (tbars) measurements according to siu and draper (1978). in detail, each sample of muscle was minced and an aliquot of 2.5 g was homogenized in 12.5 ml of distilled water at 9500 rpm for 2 min, using an ultra-turrax tissue homogenizer (ika, germany), and then vortexed for 1 min at high speed. samples were centrifuged for 20 min at 2000 rpm at 4°c with 12.5 ml of 10% trichloroacetic acid (tca) (sigma-aldrich, milan, italy) and the supernatant decanted through a paper filter (whatman 541). four ml of clear filtrate were transferred into 15 ml pyrex screw cap test tubes and added of 1 ml of 0.06m 2-thiobarbituric acid (tba). a distilled water-tca-tba reagent blank was prepared and treated like the samples. the samples were heated in a water bath at 80°c for 90 min and then cooled. absorbance at 532 nm was measured against a blank sample on two replicates of each sample on a jasco spectrophotometer (model v550, uv/vis, tokyo, japan) immediately after cooling. tbars were expressed as mg of malondialdehyde (mda) per kg of muscle using tetraethoxypropane (tep) (sigma-aldrich, milan, italy) as a standard. 2.7. packaging and shelf-life study from each loin, 6 slices (approximately 2-2.5 cm thick), designated for the evaluation of the shelf-life in two different modified atmospheres, were individually weighed and packed in a total of 72 high barrier trays lidded with a pet/evoh/pe film (aerpack system, kindly supplied by coopbox group, italy). the whole package oxygen transmission rate (otr) was < 0.1 cm3 day-1 (air, 25°c). map was performed using a semiautomatic vacuum compensation thermosealing machine (ca.ve.co, italy). two different gaseous mixtures were used: 70%n2/30%co2 (n2) and 70%o2/30%co2 (o2). gas composition of the headspace was analysed before opening packages using a hwd-gc equipped with a concentric crt i column (6ft x 1/4”; outer column: activated molecular sieve; inner column: porous polymer mixture) (alltech italia, s.r.l, italy). gascromatographic conditions: gas carrier helium (65 ml min-1); temperature 55°c; analysis time 5 min. a septum was glued onto the surface of the lid and a 50 ml gas aliquot was withdrawn with a gastight syringe and injected in the gascromatograph. the calibration was performed by injecting separately pure gases as external standard (supplied by sapio; pureness 99.9) and calculating the response factor for each one. all samples packaged in modified atmosphere were stored in the dark at 3±1°c for a maximum of 8 days. twenty-four trays (12 subjects x 2 different modified atmospheres) were removed from the refrigerators at day 4, 6 and 8 of storage, weighed to calculate weight losses and then submitted to color, ph and tbars determination, as described ital. j. food sci., vol. 32, 2020 157 above (sections 2.3 and 2.6). color measurements were carried out after allowing a 30 min blooming, following pack opening. moreover, overall color variation (∆e) was calculated as (∆l*2 + ∆a*2 + ∆b*2)0.5 where ∆l*, ∆a*and ∆b* are the difference between time 0 (at 24 h post mortem) and the values l*, a* and b* respectively at 4 (∆e0_4), 6 (∆e0_6) and 8 (∆e0_8) days of refrigerated storage. 2.8. statistical analysis statistical analysis was performed by means of analysis of variance using the glm procedure of sas (sas institute inc., cary, nc, usa). the statistical model for performance, carcass characteristics, qualitative characteristics and chemical composition of fresh ltl muscle, fatty acid composition of subcutaneous adipose tissue and ltl muscle, used dietary treatments (dt) (c, le and lgse) as independent variables. the data from samples stored in map were statistically analyzed within storage day including in the model also the packaging atmosphere (p) (o2 or n2) and the interaction dtxp. the interaction was never statistically significant (p>0.05) and was thus eliminated from the model. the two degrees of freedom of dietary treatments were a priori splitted into two orthogonal contrasts comparing, respectively, control group vs average of extruded linseed groups (c vs le+lgse/2) and antioxidant supplemented groups between them (le vs lgse). 3. results 3.1. performance and carcass characteristics no effect of dietary treatments (dt) was found on farm performances (average daily gain 0.8±0.1 kg, slaughter weight 135.4±9.7 kg) and carcass characteristics (carcass weight 113.7±8.3 kg, dressing percentage 83.9±1.2, backfat thickness 24.3±3.3 mm and lean meat content 54.3±2.1%) (data not reported in tables). guillevic et al. (2009a) and haak et al. (2008), feeding linseed to finishing light weight pigs, could not detect either any difference in on farm and abattoir performances. 3.2. fresh meat quality the effect of dt on qualitative characteristics of meat is reported in table 2. according to corino et al. (2014), muscle ph is not affected by linseed feeding. our results confirm the inferences of their meta-analysis. the control group showed a slightly higher b* and chroma values (p <0.05), denoting a greater yellowness and color intensity than linseed groups. corino et al. (2008) did not find significant differences related to the values of b* in pig fed with linseed. while our chroma values agree with data reported by juárez et al. (2001), but there was no obvious explanation for this trend, therefore it requires further investigation. no difference was found between le and lgse groups. further, drip and cooking losses, oxidative stability in raw and cooked muscle, shear force and chemical composition (moisture, crude protein and lipid contents of ltl muscle) were affected neither by linseed nor by the antioxidants. corino et al. (2008) and haak et al. (2008) found that linseed inclusion did not influence these qualitative traits in meat of light pigs either. moreover, boler et al. (2009) reported that vitamin e reduces lipid ital. j. food sci., vol. 32, 2020 158 oxidation but has no effect on any carcass characteristics and loin quality. eventually, the lack of effects of linseed feeding on chemical parameters of the muscle (moisture, crude protein and lipid contents) confirms the findings of hoz et al. (2003) on tenderloin. table 2. qualitative characteristics and chemical composition of longissimus thoracis et lumborum muscle from pigs fed with the experimental diets. items dietary treatment(a) p-value r-mse(b) c (n=4) le (n=4) lgse (n=4) cvs (le+lgse)/2 le vs lgse ph post mortem (24h) 5.58 5.53 5.59 0.679 0.236 0.067 l* 55.83 56.26 55.70 0.878 0.616 1.526 a* 1.97 0.74 0.56 0.127 0.847 1.284 b* 12.88 12.08 11.18 0.044 0.177 0.870 chroma 13.13 12.15 11.20 0.049 0.232 1.049 hue angle 81.98 86.75 87.17 0.142 0.908 5.047 drip loss (%) 3.46 3.13 3.39 0.763 0.731 1.032 cooking loss (%) 17.05 19.78 16.98 0.358 0.110 2.236 tbars(c) (raw muscle) 0.045 0.069 0.091 0.134 0.396 0.034 tbars(c) (cooked muscle) 0.386 0.405 0.279 0.698 0.341 0.178 shear force (kg) 4.99 4.87 4.85 0.669 0.957 0.471 chemical composition (%) moisture 72.30 71.96 72.04 0.544 0.896 0.785 crude protein 23.39 23.16 23.44 0.845 0.575 0.688 lipids 1.36 1.74 1.27 0.571 0.145 0.418 (a)c, control (extruded linseed, 0 g kg-1); le, (extruded linseed, 50 g kg-1; vitamin e (α-tocopheryl acetate), 200 mg and se 0.21 mg kg-1); lgse, (extruded linseed, 50 g kg-1; red grape skin extract 3 g kg-1). (b)root mean square error. (c)tbars (thiobarbituric acid reactive substances) expressed in mg of malondialdehyde (mda) per kilogram of muscle. 3.3. adipose tissue and intramuscular fatty acid composition table 3 shows lipid content and fatty acid composition of subcutaneous adipose tissue. no differences attributable to extruded linseed dietary inclusion or type of antioxidant were found for lipid content of backfat. the proportion of total saturated (sfa), monounsaturated (mufa) and polyunsaturated (pufa) fatty acids was not affected by the inclusion of linseed in the diet, in agreement with musella et al. (2009) who detected no difference in the percentage of main fa classes in ham covering trimmed fat between control and linseed-fed pigs. the total content of n-6 pufa, although tendentially lower in linseed fed subjects, was not significantly influenced by dietary treatments (p>0.05) either. this is likely due to the limited number of experimental units. overall, among n-6 pufa and mufa, only γlinolenic (c18:3n-6) and heptadecenoic acids (c17:1) were affected by dietary linseed inclusion, which brought about a significant (p<0.05) reduction of their proportions. conversely, n-3 pufa increased significantly (p<0.01) with linseed dietary inclusion. this confirms the findings of riley et al. (2000). the increase of the proportion of the total n-3 pufa is ascribable to the higher proportions (p<0.01) of α-linolenic (ala, c18:3n-3) and eicosatrienoic (c20:3n-3) acids which trebled and docosapentaenoic acid (dpa, c22:5n-3) ital. j. food sci., vol. 32, 2020 159 that doubled, whereas eicosapentaenoic acid (epa, c20:5n-3) and docosahexaenoic acid (dha, c22:6n-3) remained unchanged. guillevic et al. (2009b) could not find any correlation between ala intake and the proportion of dha in adipose tissues either. riley et al. (2000) hypothesized that the limited accumulation of longer chain pufa in adipose tissues could be due to scant capacity for the synthesis of these products starting from dietary ala. table 3. lipid content (%) and fatty acid profile (% of total fatty acids) of backfat from pigs fed with the experimental diets. items dietary treatment(a) p-value r-mse(b) c le lgse c vs le+lgse/2 le vs lgse (n=4) (n=4) (n=4) lipids 86.14 86.56 87.55 0.752 0.765 4.560 fatty acids: c 10:0 (capric) 0.13 0.10 0.10 0.407 0.988 0.053 c 12:0 (lauric) 0.07 0.05 0.07 0.591 0.328 0.027 c 14:0 (myristic) 1.16 1.15 1.18 0.975 0.659 0.093 c 16:0 (palmitic) 24.66 24.59 24.39 0.735 0.731 0.785 c 17:0 (heptadecanoic) 0.35 0.32 0.30 0.338 0.673 0.055 c 18:0 (stearic) 15.32 14.95 15.47 0.843 0.437 0.896 c 20:0 (eicosanoic) 0.15 0.26 0.22 0.103 0.501 0.076 c 16:1 (palmitoleic) 1.62 1.51 1.57 0.553 0.674 0.208 c 17:1 (heptadecenoic) 0.32 0.26 0.24 0.016 0.517 0.040 c 18:1n-7 (vaccenic) 1.51 1.39 1.65 0.547 0.420 0.281 c 18:1n-9 (oleic) 39.47 38.08 39.30 0.956 0.220 2.040 c 20:1 (eicosenoic) 0.67 0.83 0.81 0.075 0.828 0.126 c 18:2n-6 (linoleic) 12.64 11.60 10.46 0.236 0.458 2.070 c 18:3n-3 (α-linolenic) 0.84 3.26 2.74 0.000 0.103 0.406 c 18:3n-6 (γ-linolenic) 0.09 0.05 0.07 0.026 0.152 0.022 c 20:2n-6 (eicosadienoic) 0.42 0.54 0.50 0.196 0.605 0.116 c 20:3n-3 (eicosatrienoic) 0.12 0.43 0.43 0.000 0.906 0.089 c 20:4n-6 (arachidonic) 0.30 0.25 0.26 0.323 0.859 0.070 c 20:5n-3 (eicosapentaenoic) 0.00 0.15 0.00 0.486 0.254 0.170 c 22:2n-6 (docosadienoic) 0.00 0.00 0.00 0.377 0.466 0.004 c 22:4n-6 (docosatetraenoic) 0.07 0.05 0.04 0.127 0.434 0.027 c 22:5n-3 (docosapentaenoic) 0.09 0.18 0.20 0.000 0.583 0.028 c 22:6n-3 (docosahexaenoic) 0.02 0.02 0.02 0.864 0.510 0.016 total saturated 41.83 41.40 41.72 0.719 0.717 1.185 total monounsaturated 43.58 42.06 43.57 0.587 0.363 2.224 total polyunsaturated 14.60 16.54 14.72 0.523 0.338 2.546 total n-6 13.53 12.49 11.33 0.257 0.470 2.183 total n-3 1.07 4.05 3.39 0.000 0.088 0.484 n-6/n-3 fatty acid ratio 12.64 3.09 3.37 0.000 0.622 0.780 iodine value(c) 63.72 62.98 61.79 0.410 0.520 2.511 (a)c, control (extruded linseed, 0 g kg-1); le, (extruded linseed, 50 g kg-1; vitamin e (α-tocopheryl acetate), 200 mg and se 0.21 mg kg-1); lgse, (extruded linseed, 50 g kg-1; red grape skin extract 3 g kg-1). (b) root mean square error. (c)iv=85.703 + [c14:0] x 2.740 [c16:0] × 1.085 [c18:0] × 0.710 + [c18:2n-6] × 0.986 ital. j. food sci., vol. 32, 2020 160 on the whole, these trends led to a significant (p<0.01) reduction in the n-6/n-3 pufa ratio, as was previously observed also by musella et al. (2009) and riley et al. (2000). in our study, the n-6/n-3 pufa ratio dropped from 12.6 in control pigs to less than 3.5 in linseed fed pigs. thus, the inclusion of 5% extruded linseed in the finishing diet enabled to bring this ratio far below the threshold indicated by simopoulos (2008) to avoid adverse health consequences, without impairing technological parameters of subcutaneous adipose tissue. in fact, most authors (e.g. lebret and mourot, 1998; lo fiego et al., 2005) indicate, as a guarantee of good preservation aptitude, contents of stearic acid (c18:0) and linoleic acid (c18:2n-6) higher than 12 and lower than 15%, respectively and an iodine value minor than 70. as showed in table 3, and as expected, no difference was found between the linseed fed groups (le vs lgse) for any of the parameters taken into account. table 4 shows the fatty acid composition in ltl muscle. in general, the variations observed in fa compositions resembled what already seen in the backfat tissue. in fact, total sfa, mufa and pufa percentages did not differ among dietary treatments (p>0.05). compared to c, linseed groups showed a higher content of lauric acid (c12:0; p<0.01) and lower in vaccenic acid (c18:1n-7; p<0.05). no hypothesis could be put forward to explain these variations that, though trivial, were statistically significant. total n-6 pufa content, as in the backfat depot, although is tendentially higher in c group, was not affected by the dietary treatment and the only significant variation was shown by the docosatetraenoic acid (c22:4n-6; p<0.01), which resulted higher in the c group. the same trend was observed by riley et al. (2000). the total n-3 pufa proportion, as seen in the backfat, was significantly higher (p<0.01) in the linseed groups. in detail, α-linolenic, eicosatrienoic, and eicosapentaenoic acids (p<0.01) more than tripled. thus, epa that did not change with diet in adipose tissue, increased in ltl muscle. this agrees with the results of corino et al. (2008), who observed that epa is preferentially stored in the muscle rather than in the adipose tissue, and riley et al. (2000), who inferred that α-linolenic acid intake elicits eicosapentaenoic acid increments more in muscle than in adipose tissue. hence, also in the muscle, the n-6/n-3 pufa ratio was significantly reduced (p<0.01) to one-third in linseed fed pigs. not even in this tissue, except for the proportion of lauric acid, any variation was found between the two different dietary antioxidants, for any of the parameters taken into account. 3.4. quality characteristics of meat stored in map the analysis of the inner gaseous atmosphere composition of the experimental samples showed that the relative percentages of the gases have not changed throughout the storage time (data not shown). this result is not unexpected, because of the refrigerated (3±1°c) and short storage time length (8 days), the high barrier materials used, which strongly limits the gas transfers in and out the packages, and the bacteriostatic activity of co2 that, slowing the microbial growth, avoids oxygen consumption and as a consequence its decrease. in these conditions, all the modifications registered on the meat samples can be attributed only to the presence or absence of oxygen in the atmosphere surrounding the product and to meat composition. the effects of dietary treatments and of gaseous mixtures in packaging during the 8 days of refrigerated storage on various physico-chemical characteristics of ltl muscle are shown in table 5. the dietary treatments influenced most of the parameters studied. ital. j. food sci., vol. 32, 2020 161 linseed groups exhibited lower weight losses; however, the reduction was significant (p<0.05) only on days 4 and 6. table 4. fatty acid profile (% of total fatty acids) of longissimus thoracis et lumborum muscle from pigs fed with the experimental diets. items dietary treatment(a) p-value r-mse(b) c le lgse c vs le+lgse/2 le vs lgse (n=4) (n=4) (n=4) c 10:0 (capric) 0.11 0.29 0.30 0.355 0.993 0.306 c 12:0 (lauric) 0.05 0.16 0.11 0.000 0.019 0.024 c 14:0 (myristic) 1.07 1.18 1.13 0.343 0.646 0.140 c 16:0 (palmitic) 23.11 23.85 23.48 0.442 0.656 1.117 c 17:0 (heptadecanoic) 0.12 0.14 0.21 0.303 0.185 0.076 c 18:0 (stearic) 13.41 13.50 13.10 0.780 0.381 0.616 c 20:0 (eicosanoic) 0.12 0.12 0.13 0.871 0.822 0.020 c 16:1 (palmitoleic) 2.81 2.99 2.73 0.801 0.261 0.304 c 17:1 (heptadecenoic) 0.07 0.15 0.18 0.062 0.560 0.075 c 18:1n-7 (vaccenic) 3.30 2.84 2.74 0.038 0.686 0.345 c 18:1n-9 (oleic) 39.39 39.79 40.68 0.612 0.646 2.639 c 20:1 (eicosenoic) 0.59 0.52 0.55 0.249 0.618 0.071 c 18:2n-6 (linoleic) 10.52 9.07 9.03 0.276 0.976 2.069 c 18:3n-3 (α-linolenic) 0.40 1.27 1.37 0.000 0.540 0.218 c 18:3n-6 (γ-linolenic) 0.08 0.09 0.13 0.350 0.352 0.057 c 20:2n-6 (eicosadienoic) 0.21 0.19 0.19 0.380 0.971 0.040 c 20:3n-3 (eicosatrienoic) 0.05 0.16 0.18 0.001 0.481 0.043 c 20:4n-6 (arachidonic) 3.53 2.49 2.50 0.093 0.991 0.897 c 20:5n-3 (eicosapentaenoic) 0.07 0.35 0.36 0.004 0.954 0.120 c 22:2n-6 (docosadienoic) 0.01 0.01 0.01 0.167 0.600 0.004 c 22:4n-6 (docosatetraenoic) 0.51 0.19 0.26 0.006 0.497 0.132 c 22:5n-3 (docosapentaenoic) 0.39 0.52 0.60 0.227 0.599 0.214 c 22:6n-3 (docosahexaenoic) 0.08 0.12 0.04 0.992 0.255 0.095 total saturated 38.00 39.23 38.46 0.364 0.469 1.452 total monounsaturated 46.16 46.29 46.88 0.811 0.776 2.824 total polyunsaturated 15.85 14.47 14.67 0.574 0.941 3.574 total n-6 14.85 12.04 12.11 0.173 0.977 3.064 total n-3 1.00 2.43 2.56 0.002 0.755 0.551 n-6/n-3 fatty acid ratio 15.33 4.99 4.76 0.000 0.827 1.438 (a)c, control (extruded linseed, 0 g kg-1); le, (extruded linseed, 50 g kg-1; vitamin e (α-tocopheryl acetate), 200 mg and se 0.21 mg kg-1); lgse, (extruded linseed, 50 g kg-1; red grape skin extract 3 g kg-1). (b) root mean square error. the comparison between the antioxidants added to the linseed diets did not reveal significant differences (p>0.05) in this parameter. in any time-lapse interval considered, the weight loss was unaffected by the packaging atmosphere. the ph of ltl muscle ranged from 5.46 to 5.55 during the 8 days storage. these values are quite common in medium-heavy pigs. dietary linseed inclusion did not affect the ph values, whereas grape skin extract addition yielded higher ph values than synthetic ital. j. food sci., vol. 32, 2020 162 antioxidant at day 6 (p< 0.01). however, in absence of a definite trend, this difference can be attributed to inter-animal variation. our results conflict with the findings of lorenzo et al. (2014), who found that the addition of natural antioxidants, derived from grape seed extract, lowered the value of ph throughout storage. this difference is likely to be ascribable to the different origin of the grape extracts utilized (seed or skin) and, especially, to the fact that in the present work the extract was added to the diet of the pigs and not into the meat. table 5. effect of the diet and packaging on the weight loss (%), ph, tbars values and color variation (∆e), measured on longissimus thoracis et lumborum muscle, refrigerated 8 days (3 ± 1°c). items dietary treatment(a) packaging p-value p-value r-mse(b) c (n=24) le (n=24) lgse (n=24) o2 (n=36) n2 (n=36) o2 vs n2 c vs (le+ lgse/2) le vs lgse day 4 weight loss % 3.67 2.70 2.84 2.86 3.28 0.233 0.021 0.737 0.834 ph 5.52 5.54 5.54 5.55 5.51 0.003 0.116 0.641 0.031 tbars(c) 0.138 0.143 0.131 0.159 0.115 <0.001 0.932 0.186 0.016 δe0_4 (d) 3.58 3.83 3.94 5.30 2.27 0.001 0.724 0.911 1.978 day 6 weight loss % 4.57 3.07 3.87 3.39 4.29 0.076 0.043 0.190 1.178 ph 5.46 5.46 5.50 5.47 5.49 0.059 0.083 0.008 0.026 tbars(c) 0.078 0.032 0.044 0.103 0.001 <0.001 0.015 0.497 0.035 δe0_6(d) 3.81 4.23 3.30 5.12 2.44 <0.001 0.953 0.271 1.644 day 8 weight loss % 5.13 4.22 4.78 4.44 4.98 0.314 0.272 0.396 1.286 ph 5.51 5.51 5.53 5.53 5.50 0.007 0.309 0.230 0.026 tbars(c) 0.157 0.091 0.058 0.177 0.027 0.021 0.208 0.656 0.147 δe0_8(d) 3.88 4.17 3.75 5.33 2.53 0.001 0.922 0.641 1.761 (a)c, control (extruded linseed, 0 g kg-1); le, (extruded linseed, 50 g kg-1; vitamin e (α-tocopheryl acetate), 200 mg and se 0.21 mg kg-1); lgse, (extruded linseed, 50 g kg-1; red grape skin extract 3 g kg-1). (b)root mean square error; (c)tbars (thiobarbituric acid reactive substances) expressed in mg of malondialdehyde per kilogram of muscle. (d)∆ei_j = 𝐿𝑗 − 𝐿𝑖 ! + 𝑎𝑗 − 𝑎𝑖 ! + 𝑏𝑗 − 𝑏𝑖 ! in 02 packaged samples, significant, though negligible, increases (p<0.01) in ph values were recorded at 4 and 8 days of storage. this agrees with muhlisin et al. (2014), who showed that, at increasing oxygen content in the gas mixture, ph of pork was higher than in samples stored at higher levels of nitrogen. however, viana et al. (2005) found that map did not exert a strong effect on ph of fresh pork loin. the color evolution, expressed by ∆e, during refrigerated storage time, did not differ among dietary treatments. the ∆e values were all above the value of 2, which is considered the threshold to appreciate visual changes of the color (lorenzo et al., 2014). with regard to the gas mixtures, the ∆e in the samples stored in oxygen were significantly higher (p<0.01) in any time-lapse interval considered. over storage, the ∆e values remained almost constant in the two gas mixtures, higher than 5 in o2 and roughly half that in n2. ital. j. food sci., vol. 32, 2020 163 fig. 1 reports ltl color parameters evolution. in detail, as shown in fig. 1a, the a* value, which is an index of redness, was consistently higher in high oxygen map over storage, regardless of the dietary treatment. the values observed in n2 map are perceived as grey color, as the values ranged from 3.2 and 4.6 (de santos et al., 2007). the same pattern was observed for the b* value (fig. 1b). figure 1. longissimus thoracis et lumborum muscle color parameters evolution: redness “a*” values (a) and yellowness “b*” values (b) in relation to storage time in map: oxygen (o2) or nitrogen (n2). c, control group (extruded linseed, 0 g kg-1); le group, (extruded linseed, 50 g kg-1; vitamin e (α-tocopheryl acetate), 200 mg and se 0.21 mg kg-1); lgse group, (extruded linseed, 50 g kg-1; red grape skin extract 3.0 g kg-1) ital. j. food sci., vol. 32, 2020 164 as concerns the oxidative stability, in the time-lapses considered, tbars values were unaffected by the dietary treatment on day 4 and 8 (table 5). only on day 6, meat from the control showed a greater oxidation (p<0.05) than in antioxidants groups. no difference was found between le vs lgse. the different modified atmospheres affected the oxidative stability of the muscle, that was always lower in samples packaged in high o2 map, which yielded to a significantly higher tbars values on day 4 and 6 (p<0.01), and on day 8 (p<0.05) of storage. when oxygen is readily available, a substrate such as meat is more prone to oxidation (smiddy et al., 2002) and, in agreement with our results, spanos et al. (2016) observed that samples of ltl muscle stored in map oxygen concentration of 50% or higher showed a significantly lower oxidative stability compared to samples stored under 0% oxygen. however, even the highest determined value of tbars was lower than 0.18 mg mda/kg meat, far below the threshold value of 1.0 mg mda/kg muscle for organoleptic detection of rancidity as suggested by o’grady et al. (2008). considering that tbars values up to 0.6 mg of mda/kg of fresh meat are considered fresh (tarladgis et al., 1960), all our samples, regardless of the different maps, could be classified as fresh meat, through the 8 days of storage. 4. conclusions our results confirm that 5% of dietary extruded linseed included in the pig finishing diet is a suitable means to increase n-3 pufa content and reduce the n-6/n-3 pufa ratio in pig tissues without affecting on live and slaughter performance and impairing technological characteristics of adipose depots. in general, under the point of view of human nutrition, it ameliorates the fatty acid profiles in both backfat and ltl muscle. also, qualitative characteristics and chemical composition of muscle are not affected by dietary linseed inclusion associated with either synthetic or natural antioxidants. in this research linseed feeding, supplemented with supranutritional doses of antioxidants, does not impair oxidative stability compared to a standard diet and reduces weight losses during chilled storage. as expected, high concentration of oxygen in map brings about an increase in oxidative products and yields redder meat, irrespective of the dietary treatment. in linseed fed pigs, dietary red grape skin extract is as effective as synthetic antioxidant in maintaining quality characteristics of pork during storage, even in high oxygen map. acknowledgments this research was funded by regione emilia-romagna por-fesr 2014-2020 grant n. g/2015/730542. “innovating the pork chain through the valorization of vegetal by-products and the use of advanced “omics” and processing technologies, for the sustainable production of fresh and processed pork meat products with a positive impact on human health (green charcuterie)”. the authors thank dr. giacinto della casa (crea-research centre for animal production and aquaculture modena unit – san cesario sul panaro, modena) for taking care of animals, and for formulating and supplying the 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nute g.r., sheard p.r., richardson r.i., hughes s.i. and whittington f.m. 2008. fat deposition, fatty acid composition and meat quality: a review. meat sci. 78:343. paper received may 20, 2019 accepted september 25, 2019 ijfs#165_monteiro_bozza   ital. j. food sci., vol 28, 2016 426 paper functional sausage made from mechanically separated tilapia meat d. peireira bessa1, c.e. teixeira1, r. maia franco1, m. queiroz de freitas1, m.l. guerra monteiro1*, c.a. conte-junior1, l. varon gaze1, f. alves da silva2 and e. teixeira mársico1 1 departament of food technology, federal fluminense university, niterói, rio de janeiro, brazil, 24230-340 2 departament of food engineering, federal de goiás university, rod. goiânia/nova veneza, km 0, goiânia, go, brazil, 74690-900 *corresponding author: tel.: + 55 02126299545; fax: + 55 02126299541 e-mail address: marialuciaguerra@yahoo.com.br abstract physicochemical, bacteriological and sensory parameters of sausages made from waste of nile tilapia with the prebiotic inulin added and reduced sodium were investigated. mechanically separated carcass meat and mechanically separated head meat were processed into sausages with or without inulin and salt replacer. t1 and t3 showed greatest lipid, but lowest carbohydrate levels, a* and b* values (p < 0.05). in general, the inulin formulations showed higher acceptability and purchase intent (p < 0.05). the addition of inulin to low-sodium tilapia sausages is a promising technological strategy to minimize negative effects on the taste and texture from kcl increment. keywords: fish waste, healthy products, inulin, oreochromis niloticus, potassium chloride, salt replacer   ital. j. food sci., vol 28, 2016 427 1. introduction the nile tilapia is the most important species for aquaculture in the world. tilapia is marketed either as whole fish or as fresh or frozen fillets, although consumers prefer fillets. reported fillet yields of nile tilapia vary widely (30-40%), generating a large amount of wastes that are commonly underutilized, used as animal feed, or discarded (monteiro et al., 2012; montanhini neto and ostrensky, 2013). the utilization of fish-processing waste to develop new products provides an important opportunity for the food industry to produce sustainable and value-added products as a source of omega-3 polyunsaturated fatty acids (deckelbaum and torrejon, 2012) and high-value protein. further studies are needed to develop ways to use fish waste to produce functional products with lower costs (monteiro et al., 2014a; palmeira et al., 2014a; palmeira et al., 2014b). some investigators have reported the beneficial effects of including inulin as a fat replacer in chicken and pork sausages, with desirable effects such as improved texture. in addition, inulin has a prebiotic physiological effect, attributed to fructan, which stimulates the growth of beneficial bacteria in the intestine of the host (mendoza et al., 2001; menegas et al., 2013). the reduction of sodium in processed products has constituted a challenge for the food industry, particularly in view of the contemporary consumer’s awareness of the relationship between food and health. the need to reduce salt contents is further reinforced by agreements between the industrial sector and the world health organization (who). the main challenge is to maintain the acceptability as well as physicochemical parameters of the food (desmond, 2006; dotsch et al., 2009; monteiro et al., 2014b), considering microbiological quality standards. however, inulin and sodium replacers in fish products have not been studied. this study evaluated the effect of sodium reduction and inulin addition on physicochemical, bacteriological and sensory parameters of sausages manufactured from mechanically separated meat of nile tilapia. 2. materials and methods 2.1. obtaining mechanically separated tilapia meat the wastes from heads and carcasses of tilapia were obtained from a fish-farming cooperative in rio de janeiro, brazil. the fish heads and carcasses were passed through a deboning machine (kme, são paulo) to remove the muscle from the bones, and were then washed. the mechanically separated carcass meat (mscm) and mechanically separated head meat (mshm) were frozen and packed in 1kg polyethylene packages at -18°c. the samples were transported in cold boxes to keep them frozen during transport to the pilot plant of the centro de tecnologia senai alimentos e bebidas, rio de janeiro, brazil, where they were stored in a freezer (–18°c) until the mshm and mscm were used to produce sausages. four different sausage formulations were prepared: 100% nacl without inulin (t1), 100% nacl + 6% inulin (t2), 50% nacl and 50% kcl + without inulin (t3), and 50% nacl and 50% kcl + 6% inulin (t4). in formulations without inulin, mshm was used as a replacer, and potassium chloride (kcl) was used to reduce the sodium content. all formulations are described in table 1.   ital. j. food sci., vol 28, 2016 428 table 1: formulations of sausages manufactured from mechanically separated tilapia meat. ingredients (%) treatments* t1 t2 t3 t4 mscm** 81.91 81.91 81.91 81.91 sodium chloride 1.3 1.3 0.65 0.65 potassium chloride 0.65 0.65 soybean protein 2.0 2.0 2.0 2.0 mshm** 10.0 4.0 10.0 4.0 inulin 6.0 6.0 polyphosphate 0.5 0.5 0.5 0.5 manioc starch 2.0 2.0 2.0 2.0 sodium erythorbate 0.3 0.3 0.3 0.3 carmine 0.05 0.05 0.05 0.05 collagen 1.0 1.0 1.0 1.0 nitrite 0.25 0.25 0.25 0.25 onion 0.3 0.3 0.3 0.3 garlic 0.2 0.2 0.2 0.2 white pepper 0.1 0.1 0.1 0.1 cilantro 0.05 0.05 0.05 0.05 ginger 0.04 0.04 0.04 0.04 *t1 (100% nacl without inulin), t2 (100% nacl + 6% inulin), t3 (50% nacl and 50% kcl + without inulin), t4 (50% nacl and 50% kcl + 6% inulin). ** mscm mechanically separated carcass meat; and mshm mechanically separated head meat. 2.2. experimental design ten kg and thirty-kg of mshm and mscm were obtained, respectively. for each experimental replication (n = 2), the samples were divided into four groups each with 3.5 kg of mshm (t1 and t3), 1.5 kg of mshm (t2 and t4), and 7.5 kg of mscm. each group was divided at random into 40 g portions, resulting a total of 40 samples per group which were separated in the proposed formulations and analyzed on days 0 and 34 of refrigerated storage, totaling 80 samples units. 2.3. sausage manufacturing to compose the sausage, the ingredients (table 1) were mixed in a cutter. after the emulsion was formed in the cutter, the sausage mass was placed in a manual sausage maker using sausage collagen casings of 21 mm diameter, and separated into sections. the sausage was heat-treated, starting with pre-cooking in a drying oven (incomaf indústria ltda., são paulo, brazil) with circulating hot air at 50°c for 15 min with an open chimney, then raised to 60°c for 30 min with a closed chimney and increased by 5°c every 5 min (steam drying) until the sausage reached 72°c internally, as measured with the aid of a thermocouple. the sausages were then cooled in a water bath to an internal temperature   ital. j. food sci., vol 28, 2016 429 of 40°c. the sausages were stored in a cooling chamber at 5°c until the next day, when the membranes were delaminated and the products were vacuum-packed in polythene bags and stored under refrigeration (5°c). the products were placed in isothermal boxes and transported to the laboratory in rio de janeiro, brazil, where they were kept under refrigeration (4°c) for 34 days until analytical procedures. the analyses on day 0 were performed immediately after the arrival of the samples. 2.4. physicochemical analyses water, ash, protein and lipid contents were evaluated in mshm and mscm on day 0 (aoac, 2012). in the sausages, ph values were determined with a digital ph meter (digimed®dm-22) equipped with a dme-r12 electrode (digimed®) according to conte-júnior et al. (2008). water, ash, protein, lipid and carbohydrate contents were determined by standard methods (aoac, 2012). the carbohydrate content was estimated taking into account the ingredient composition. the energy content (kcal/100g) was estimated according to triki et al. (2013). these analyses were performed immediately after manufacture and repeated after 34 days of storage, to assess the maintenance of quality and identity of the product. in addition, on day 0 the cooking yield was calculated as the cooked weight of the sausages divided by the weight of the pre-cooked sausage, multiplied by 100 (horita et al., 2011). all analyses were performed in quadruplicate for each treatment. the instrumental color parameters were determined using a konica minolta cr-400 colorimeter (konica minolta sensing, osaka, japan) previously calibrated with cie standard illuminant d65, a 8 mm-diameter aperture, and a 2o standard observer (amsa, 2012). samples were macerated to a thickness of 3 cm in a beaker with a diameter of 15 cm. results are expressed in cielab l* (lightness), a* (redness) and b* (yellowness) values. measurements were performed in triplicate for each treatment. 2.5. bacteriological analysis bacteriological analyses of the mshm, mscm and sausages were performed on day 0. total aerobic mesophilic bacteria (tamb), coagulase-positive staphylococcus bacterial counts, and most probable number of thermotolerant coliforms were evaluated. the presence of salmonella spp. was evaluated and the results were expressed as presence or absence in 25 g of sample (apha, 2001). 2.6. sensory evaluation the panelists were recruited from the students, faculty, and staff of the universidade federal fluminense, brazil. sausage samples were analyzed at room temperature (20°c) and were coded with three-digit random numbers. the samples were presented to 100 untrained panelists (38 men and 62 women, 18 to 47 years old) who were instructed to evaluate their overall liking for each sausage sample, using the 9-point hedonic scale (1=dislike extremely, 9=like extremely). the panelists also recorded their purchase intent (5-point scale: 1 = “definitely would not buy”, 5 = “definitely would buy”) (stone and sidel, 1998). these consumer tests were conducted in two stages: with no information (blind – first stage), and with the information “mechanically separated tilapia meat was used to make the sausages, and its use reduces environmental pollution and encourages sustainability” (informed – second stage) (garcia et al., 2009). in the first stage, color, bitter taste, salty taste, succulence, elasticity and softness were also evaluated using a five-point just about right (jar) scale (1 = much too weak, 2 =   ital. j. food sci., vol 28, 2016 430 somewhat weak, 3 = just about right, 4 = somewhat strong, and 5 = much too strong) according to cervantes et al. (2010). an unsalted cracker and a glass of water at 25°c were offered to cleanse the palate between samples. the sensory evaluation was performed two days after the sausage was manufactured, to ensure adequate bacteriological quality. 2.7. statistical analysis data for chemical composition, energy value, ph, overall liking and purchase intent were evaluated separately for treatment and time (days 0 and 34) or stages (blind and informed) by anova. cooking yield, instrumental color parameters, jar data and bacteriological results were analyzed using one-way anova. these data were further analyzed using a tukey test when the means were considered different (p < 0.05). chemical composition of mshm and mscm was evaluated by one-way anova at a 95% confidence interval. in addition, a principal components analysis (pca) was performed to assess the parameters that were influenced by addition of inulin and potassium chloride. partial least squares regression (plsr) was performed to assess if the determinant parameters contributed positively or negatively to the overall liking of the samples. penalty analysis (pa) was used to analyze the jar data in order to identify possible alternatives for product improvement. pearson’s correlation at a 5% significance level (p < 0.05) was performed to correlate the physicochemical and sensory data (color and texture parameters). all statistical analyses were performed using the software xlstat version 2012.6.08 (addinsoft, paris, france). 3. results and discussions 3.1. physicochemical and bacteriological analyses of mshm and mscm mscm and mshm exhibited 0.43% (±0.0006) and 0.73% (± 0.0008) ash, 86.14% (±0.001) and 69.41% (± 0.0069) water, 2.34% (±0.0013) and 18.39% (± 0.0112) lipid, and 4.78% (±0.0056) and 12.62% (± 0.0044) protein, respectively. the mshm had a higher content of lipids, ash and proteins and lower water content than the mscm. monteiro et al. (2012) also observed higher lipid and ash, but lower water content in mshm than in mscm. regarding bacterial quality, salmonella spp. and coagulase-positive staphylococcus were not detected in the samples. the most probable number of thermo-tolerant coliform and tamb counts were within the official limits (icmsf, 1986) which was also observed by monteiro et al. (2012) in mechanically separated tilapia meat. our results suggest that the process of obtaining mechanically separated meat was conducted appropriately, and that the use of tilapia wastes can be a viable alternative for the development of value-added products. 3.2. physicochemical parameters of tilapia sausage water, ash and protein contents did not differ significantly among the treatments (p > 0.05) on both days 0 and 34 (table 2). t1 and t3 showed higher lipid and lower carbohydrate contents than t2 and t4 (p < 0.05). this can be explained by inulin addition, replacing the mshm in t2 and t4. in agreement with our findings, menegas et al. (2013) reported that foods with added carbohydrate (inulin) as a fat replacer showed lower lipid and higher carbohydrate contents.   ital. j. food sci., vol 28, 2016 431 although the lipid content of t1 and t3 differed from t2 and t4, no difference (p > 0.05) was observed in energy value among all treatments, regardless of storage period. thus, in the sausages with lower fat content, the loss of energy value was offset by the increased carbohydrate levels. however, sausages made with mechanically separated tilapia meat residue still have a very low caloric value, similar to low-fat sodium reduced fresh merguez sausage (triki et al., 2013). this is a desirable factor for both fish processors and consumers. the ph values of the sausages did not differ (p > 0.05) among the treatments on days 0 and 34 of refrigerated storage. monteiro et al. (2014c) also observed no difference among ph values of restructured tilapia steaks. the samples were stored under refrigeration, and after 34 days of storage at 4 ± 1°c, the analyses were repeated. no significant differences (p > 0.05) were observed in water, ash, protein, lipid, carbohydrate, energy value and ph values (table 2), which indicates that the identity of the product was maintained after refrigerated storage. table 2: chemical composition, energy value and ph of sausages made from mechanically separated tilapia meat. parameters storage time (days) treatments* t1 t2 t3 t4 water 0 74.05ax±1.76 70.28ax±2.09 73.33ax±3.89 70.22ax±1.50 34 72.86ax±2.69 69.30ax±3.07 73.43ax±2.66 70.02ax±2.53 ash 0 2.99ax±0.51 2.92ax±0.52 3.06ax±0.52 3.02ax±0.38 34 3.05ax±0.51 2.94ax±0.46 3.03ax±0.36 2.93ax±0.42 protein 0 14.26ax±1.51 13.77ax±1.36 14.56ax±1.38 13.71ax±1.00 34 12.61ax±1.37 12.51ax±2.09 14.04ax±0.70 14.33ax±1.29 lipid 0 2.64ax±0.06 0.86bx±0.15 3.16ax±0.09 1.38bx±0.49 34 3.82ax±1.24 1.72bx±1.05 3.02ax±0.39 2.02bx±0.82 carbohydrate 0 6.06bx±2.45 12.18ax±2.70 5.90bx±2.09 11.68ªx±0.82 34 7.66bx±2.33 13.54ax±1.84 6.48bx±2.18 10.71ax±0.82 energy value 0 105.05ax±5.21 111.52ax±6.17 110.24ax±13.14 113.12ax±6.63 34 115.44ax±14.84 119.68ax±15.48 109.28ax±10.80 116.55ax±6.86 ph 0 6.72ax±0.02 6.71ax±0.08 6.75ax±0.04 6.76ax±0.01 34 6.68ax±0.10 6.73ax±0.01 6.72ax±0.06 6.71ax±0.06 * t1 (100% nacl without inulin), t2 (100% nacl + 6% inulin), t3 (50% nacl and 50% kcl + without inulin), t4 (50% nacl and 50% kcl + 6% inulin). water, ash, protein, lipid and carbohydrate expressed in g/100g of sample. energy value expressed in kcal/100g. values are means±sd. a–b means in a row without common superscripts are different (p < 0.05 ); n = 2. x means in a column with common superscripts did not exhibit difference (p > 0.05 ); n = 2. with regard to cooking yield, no differences (p > 0.05) were observed among the treatments (table 3). similarly, horita et al. (2011) and monteiro et al. (2014b) found no significant differences in cooking yield when nacl was replaced with kcl in low-fat   ital. j. food sci., vol 28, 2016 432 bologna and restructured tilapia steaks, respectively. however, triki et al. (2013) reported that replacing sodium chloride with a salt mixture (potassium chloride, calcium chloride and magnesium chloride) increased cooking losses. the direct relationship between fat content and cooking yield is due to the emulsion stability provided by fat. inulin has the ability to form a gel and acts similarly to fat (franck, 2002). in this study, there was no relationship between fat content and cooking yield. similarly, brennan et al. (2004) found no differences in cooking loss (fluid loss) with the inclusion of inulin in spaghetti pasta. however, álvarez and barbut (2013) suggested that inulin powder produces a higher yield compared to inulin gel. felisberto et al. (2015) suggested that significant fluid losses were observed in formulations containing inulin or polydextrose. our results suggest that inulin and kcl addition at the levels of the present study did not affect the product yield. color from tilapia muscle can be strongly affected by meat processing, and consequently influence consumer acceptability. therefore, it is important to study color behavior in fish waste destined for human consumption, to predict changes that may occur in the final product (rawdkuen et al., 2009; monteiro et al., 2014a). t4 exhibited a higher l* value (p < 0.05) than the other treatments (table 3). menegas et al. (2013) observed that the inclusion of inulin increased the lightness of dry-fermented chicken sausage. in addition, monteiro et al. (2014b) observed that the l* value was intensified in restructured tilapia steaks manufactured with 50% nacl and 50% kcl. t2 and t4 presented higher a* values (p < 0.05). these formulations had a lower lipid content (inulin added to replace mshm). candogan and kolsarici (2003) noted that products with reduced lipid tend to be redder, due to concentration of lean meat, which in this study, was represented by the mscm. t3 exhibited lower (p < 0.05) a* and b* values than the other treatments (table 3), suggesting that replacing 50% nacl with kcl can affect color parameters, as also observed by monteiro et al. (2014b) in tilapia products. in contrast to our findings, horita et al. (2011) and canto et al. (2014) found no difference (p > 0.05) in the a* and b* values in reduced-fat mortadella and restructured caiman steaks after replacing 50% nacl with kcl, respectively. nevertheless, it is important to evaluate if the differences detected with instrumental analyses will be perceived by consumers. table 3: physicochemical parameters of sausages manufactured from mechanically separated tilapia meat. physicochemical parameters treatments* t1 t2 t3 t4 cooking yield (%) 78.48a±0.08 82.67a±0.04 77.75a±0.04 77.07a±0.03 l* 63.31b±3.96 63.80b±3.45 63.97b±2.41 67.20ª±4.21 a* 16.57b±0.67 18.15a±0.60 16.13c±0.42 18.53a±0.14 b* 6.85ab±0.15 7.27a±0.70 6.44b±0.08 7.35a±1.23 *t1 (100% nacl without inulin), t2 (100% nacl + 6% inulin), t3 (50% nacl and 50% kcl + without inulin), t4 (50% nacl and 50% kcl + 6% inulin). l* ranges from 0 (black) to 100 (white); a* ranges from red (+a*) to green (−a*); and b* ranges from yellow (+b*) to blue (−b*). values are means±sd. a–c means in a row without common superscripts are different (p < 0.05 ); n = 2. 3.3. bacteriological quality of tilapia sausages no difference (p > 0.05) was observed among treatments. tamb counts were below the limit of 7.0 log cfu/g (icmsf, 1986) in all treatments, ranging from 3.06 to 3.38 log   ital. j. food sci., vol 28, 2016 433 cfu/g. salmonella sp., coagulase-positive staphylococcus and thermo-tolerant coliforms were not detected in any sausage formulation. in the present study, the partial replacement of salts and inulin addition did not compromise the bacteriological quality of the product. desmond (2006) and menegas et al. (2013) reported similar results. nevertheless, the adoption of hygienic procedures before, during, and after processing is essential to obtain a product with optimum quality. our findings suggest that the sausage-making process was conducted appropriately. 3.4. sensory evaluation of tilapia sausages with regard to overall liking, all treatments exhibited mean scores between “like slightly” and “like moderately” (table 4). oliveira filho et al. (2010) found lower overall liking for sausages with different percentages of inclusion of minced tilapia. in this study, differences were observed among the blind samples, where t2 showed higher (p < 0.05) overall liking than t3. after the scorers received information about the products, t3 and t4 exhibited lower (p < 0.05) overall liking than t1 and t2. all formulations received mean scores between “maybe/maybe not” and “probably would buy” (table 4), which indicates the consumer acceptability of this new product. in general, t1 and t2 showed higher scores (p < 0.05) for purchase intent than t3 and t4. the sentence "mechanically separated tilapia meat was used to make the sausages, and its use reduces environmental pollution and encourages sustainability" submitted to the panelists did not affect (p > 0.05) the overall liking and purchase intent of all treatments. however, our results indicate that the inulin formulations had better potential, regardless of any information provided. menegas et al. (2013) reported that acceptability of dry-fermented chicken sausages was not affected by inulin addition. according to franck (2002), when thoroughly mixed with water or another aqueous liquid, inulin forms a particle-gel network, resulting in a white creamy structure with a spreadable texture. this formulation can easily be incorporated into foods to replace up to 100% of the fat. on the other hand, our findings for overall liking and purchase intent indicated that sodium reduction can be still a major challenge for the industry. nevertheless, all formulations exhibited color, taste (bitter and salty), and texture (succulence, elasticity, and softness) attributes close to ideal (2.51 – 3.55) (table 4). color, salty taste, succulence and elasticity did not differ (p > 0.05) among treatments, suggesting that the consumers were unable to differentiate the low-sodium and inulin added in the formulations by these attributes. t3 showed a stronger (p < 0.05) bitter taste than t2, whereas softness was lower (p < 0.05) in t1 compared to the t4 formulation. armenteros et al. (2012) noted that reduction of the salt content by more than 40–50% negatively affected the sensory quality of ham, especially taste, with some bitter and metallic after tastes perceived by consumers. in agreement with our results, palmeira et al. (2014a) observed taste (spicy and bitter) and texture attributes close to ideal in trout meatballs with salt replacement; however, the consumers perceived difference among formulations by these attributes. monteiro et al. (2014b) found close to ideal taste (bitter and salty) and texture attributes in restructured tilapia steaks manufactured with 50% salt replacer (kcl). canto et al. (2014) found a salty taste and texture attributes close to ideal in restructured caiman steaks with 50% kcl. both authors reported that consumers were not able to differentiate among treatments with respect to attributes evaluated.   ital. j. food sci., vol 28, 2016 434 table 4: sensory evaluation of sausages made from mechanically separated tilapia meat. overall liking treatments* t1 t2 t3 t4 blind 6.61abx±1.41 6.79ax±1.51 6.17bx±1.52 6.33abx±1.63 informed 6.93ax±1.35 6.80ax±1.58 6.08bx±1.72 6.20bx1.69 purchase intent t1 t2 t3 t4 blind 3.51abx±0.93 3.57ax±1.15 3.16bx±1.01 3.17bx±1.04 informed 3.68abx±1.00 3.76ax±1.10 3.17cx±1.26 3.28bcx±1.15 jar attributes t1 t2 t3 t4 color 2.74a±0.79 2.55a±0.76 2.77a±0.96 2.51a±0.75 bitter taste 3.10ab±0.54 2.91b±0.60 3.18a±0.66 3.07ab±0.70 salty taste 3.00a±0.55 3.00a±0.65 2.88a±0.73 2.85a±0.69 succulence 2.90a±0.75 2.96a±0.67 2.89a±0.84 2.92a±0.88 elasticity 2.79a±0.74 2.73a±0.83 2.68a±0.91 2.69a±0.90 softness 3.17b±0.83 3.38ab±0.84 3.40ab±0.91 3.55a±0.85 *t1 (100% nacl without inulin), t2 (100% nacl + 6% inulin), t3 (50% nacl and 50% kcl + without inulin), t4 (50% nacl and 50% kcl + 6% inulin). blind with no information; and informed “mechanically separated tilapia meat was used to make the sausages, and its use reduces environmental pollution and encourages sustainability”. values are means±sd. a–c means in a row without common superscripts are different (p < 0.05). x means in a column with common superscripts did not exhibit difference (p > 0.05 ). two principal components (pc1 and pc2) explained 99.73% of total data variance (fig. 1) and separated two groups (t1 and t3; t2 and t4), based mainly on lipid and carbohydrate contents, bitter taste, texture parameters, and a* and b* values. the inulin treatments (t2 and t4) were characterized by greater softness, succulence, carbohydrate, a* and b* values, with a less bitter taste and lower lipid content than t1 and t3. taste and texture attributes were the most important for the salt-replacer formulations. t3 showed a stronger bitter taste and less softness than t4. monteiro et al. (2014b) found that replacing 50% of nacl with kcl slightly increased the bitter taste and negatively influenced the succulence and softness. mendoza et al. (2001) found a softer texture in cooked meat products when the inulin was added. our results suggest that adding inulin can minimize the negative effects of kcl on the sensory parameters of food products. the most important correlations were between softness and succulence (r = 1.00), between softness and elasticity (r = 1.00), between cooking yield and bitter taste (r = 0.99), between cooking yield and salty taste (r = 0.99), between lipid and carbohydrate (r = 0.92) and between lipid and a* values (r = 0.85). these correlations may explain the stronger bitter taste perceived in the formulations with salt replacer (t3 and t4), which was probably influenced by cooking yield. the direct relationship between softness and succulence as well as the inverse relationship between lipid, carbohydrate and a* values may clarify the positive effect of inulin addition on the chemical composition, color and texture parameters observed.   ital. j. food sci., vol 28, 2016 435 t1 (100% nacl without inulin), t2 (100% nacl + 6% inulin), t3 (50% nacl and 50% kcl + without inulin), t4 (50% nacl and 50% kcl + 6% inulin). figure 1: physicochemical and sensory data of sausages made from mechanically separated tilapia meat defined by two principal components. the plsr model (fig. 2) explained 99.4% of consumer acceptance (y-axis) and 93.1% of the untrained-panel sensory scores and physicochemical parameters (x-axis), with a cumulative q2 of 0.925. the x-axis parameters were considered important when their respective ‘variable important to the projection’ was > 1.0 (wold et al., 2001). ash, ph and bitter taste were detrimental to overall liking whereas cooking yield, salty taste, succulence and elasticity positively contributed to overall liking (fig. 3). with regard to cooking yield, t3 and t4 showed similar results, as did t1 and t2. this can be explained by the greater ionic strength of potassium (1.33) than sodium (0.95), which decreases the electrostatic repulsion between the peptide chains and the space between the myofibrils, retaining less water between the myofibrillar spaces (marchese and beveridge, 1984; damodaran et al., 2007). regarding to penalty analysis, major detrimental attributes were those with a > 0.5 penalty score and more that 20% occurrence. t1, t2 and t3 were penalized as having tooweak color (table 5). color intensity (a* and b*) was lower in t1 and t3 than t2 and t4, suggesting that the panelists were unable to differentiate color among treatments. moreover, color was not determinant for acceptability. only formulation t3 was penalized for having a too-bitter taste. the consumers penalized the t3 and t4 formulations as having a too-weak salty taste. this results suggest that use of 50% kcl can decrease the salt perception, which was important for product acceptability. regarding the texture attributes, t1, t3 and t4 were penalized as having too little succulence, and t1 and t3 as having too little elasticity. moreover, only formulation t1 was penalized for being too soft.   ital. j. food sci., vol 28, 2016 436 x axis = physicochemical parameters; y axis = overall liking. t1 (100% nacl without inulin), t2 (100% nacl + 6% inulin), t3 (50% nacl and 50% kcl + without inulin), t4 (50% nacl and 50% kcl + 6% inulin). figure 2: partial least square regression model for sensory attributes and physicochemical parameters of sausages made from mechanically separated tilapia meat. *cy – cooking yield. figure 3: weighted regression coefficients of physicochemical and sensory parameters detrimental to acceptability by partial least squares regression.   ital. j. food sci., vol 28, 2016 437 apparently, kcl affected texture parameters that were determinant for acceptability, whereas inulin addition to foods together with sodium reduction may be an alternative to improve the acceptability of these products, taking into account the enhancement of taste and texture. the effect of fiber addition in products differs, depending on the type and the level of the fiber added, as well as by the presence of other ingredients (jimenez-colmenero et al., 2005). table 5: consumer penalty analysis of the jar diagnostic attributes (percentage of consumers and mean decreases). treatments color bitter taste salty taste too weak too strong too weak too strong too weak too strong t1 35.0* (0.90)# t2 45.0 (1.01) t3 36.0 (0.53) 29.0 (0.83) 27.0 (0.36) t4 29.0 (0.77) treatments succulence elasticity softness too weak too strong too weak too strong too weak too strong t1 27.0 (1.22) 33.0 (0.75) 31.0 (0.57) t2 t3 32.0 (1.04) 42.0 (0.73) t4 34.0 (1.44) t1 (100% nacl without inulin), t2 (100% nacl + 6% inulin), t3 (50% nacl and 50% kcl + without inulin), t4 (50% nacl and 50% kcl + 6% inulin). * the percentage of consumers who found treatments to be too weak or too strong for jar color, bitter taste, salty taste, succulence, elasticity, and softness. # the number in parentheses is the change in mean compared to the consumer response score to overall liking. (-) indicates that less than 20% of consumers choose that jar category. our findings indicate that the level of inulin used was sufficient to maintain the physicochemical and sensory parameters of the tilapia sausages. on the other hand, 50% kcl as a sodium-chloride replacer negatively influenced the sensory attributes. further studies should be performed to evaluate lower levels of kcl and/or the use of other ingredients in this product since herbs and spices is a promising alternative to suppress or decrease the sensory effects caused by the use of kcl (ahn et al., 2004). 4. conclusions sausages manufactured with mechanically separated tilapia meat represent a potential alternative for sustainable use of this waste, with high consumer acceptance. the inclusion of inulin is an option to produce a low-fat food, improve the emulsion stability, and ensure the prebiotic effect of the sausage. replacing sodium with 50% kcl decreased the   ital. j. food sci., vol 28, 2016 438 acceptance and purchase intent of the tilapia sausages; however, the inclusion of inulin in fish products with sodium reduction is a promising technological strategy to solve possible problems with taste and texture due to kcl addition. acknowledgements the authors are grateful for financial support from the fundação carlos chagas filho de amparo à pesquisa do estado do rio de janeiro (faperj), process numbers faperj e-26/112.620/2012, e-26/111.130/2014 and e26/101.403/2014; the conselho nacional de desenvolvimento científico e tecnológico (cnpq), process numbers cnpq 401922/2013-8 and 441987/2014-1; and the coordenação de aperfeiçoamento de pessoal de nível superior (capes)/faperj e-45 papdrj/2013 (e-26/101.403/2014). d.p. bessa was supported by the coordenação de aperfeiçoamento de pessoal de nível superior (capes). the centro de tecnologia (senai) de alimentos e bebidas is gratefully acknowledged for providing the processing-plant facilities. references ahn j., grün i.u. and mustapha a. 2004. antimicrobial and antioxidant activities of natural extracts in vitro and in ground beef. j. food protect. 67:148-155. álvarez d. and barbut s. 2013. effect of inulin, β-glucan and their mixtures on emulsion stability, color and textural parameters of cooked meat batters. meat sci. 94:320-327. amsa. 2012. “meat color measurement guidelines” 1st ed. american meat science association, champaign, usa. aoac. 2012. “official methods for analysis” 19th ed. association of official analytical chemists, gaithersburg, usa. apha. 2001. “compendium of methods for the microbiological examination of foods” 4th ed. american public health association, washington, dc. armenteros m., aristoy m.c., barat j.m. and toldrá f. 2012. biochemical and sensory changes in dry-cured ham salted with partial replacement of sodium by a mixture of potassium, calcium and magnesium. meat sci. 90:361-367. brennan c.s., kuri v. and tudorica c.m. 2004. inulin-enriched pasta: effects on textural properties and starch degradation. food chem. 86:189-193. candogan k. and kolsarici n. 2003. storage stability of low-fat beef frankfurters formulated with carrageenan or carrageenan with pectin. meat sci. 64:207-214. canto a.c.v.c.s., costa lima b.r.c., suman s.p., lazaro c.a., monteiro m.l.g., conte-junior c.a., freitas m.q., cruz a.g., santos e.b. and silva t.j.p. 2014. physico-chemical and sensory attributes of low-sodium restructured caiman steaks containing microbial transglutaminase and salt replacers. meat sci. 96:623-632. cervantes b.g., aoki n.a. and almeida c.p.m. 2010. aceitação sensorial de biscoito de polvilho elaborado com farinha de okara e análise de dados com metodologia de penalty analysis. braz. j. food technol. 1: 3-10. conte-júnior c.a., fernández m. and mano s.b. 2008. use of carbon dioxide to control the microbial spoilage of bullfrog (rana catesbeiana) meat. ch. 3. in “modern multidisciplinary applied microbiology”. a. mendez-vilas (ed.) p. 356-361. wiley-vch verlag gmbh & co. kgaa, germany, de. damodaran s., parkin k.l. and fennema o.r. 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58:109-130. paper received may 19, 2015 accepted august 20, 2015 ijfs#1502_bozza ital. j. food sci., vol. 31, 2019 716 paper variations in nutritive composition of three shellfish species j. pleadina, k. kvrgićb, s. zrnčićc, t. lešića, o. koprivnjakd, a. vulića, n. džafiće, d. oraićc and g. krešić*f acroatian veterinary institute, laboratory for analytical chemistry, savska cesta 143, 10000 zagreb, croatia bcroatian veterinary institute, veterinary institute rijeka, laboratory for analytical chemistry and residues, podmurvice 29, 51000 rijeka, croatia ccroatian veterinary institute, laboratory for fish pathology, savska cesta 143, 10000 zagreb, croatia d school of medicine, department of food technology and control, university of rijeka, braće branchetta 20, 51 000 rijeka, croatia ecroatian veterinary institute, veterinary institute rijeka, laboratory for food and feed microbiology, podmurvice 29, 51000 rijeka, croatia ffaculty of tourism and hospitality management, department of food and nutrition, university of rijeka, primorska 42, 51410 opatija, croatia *corresponding author: tel.: +385 51294714 email address: gretak@fthm.hr abstract nutritive composition, fatty acid profile and health-related lipid indices of natural-born european flat oyster (ostrea edulis), variegated scallop (chlamys varia) and smooth scallop (flexopecten glaber) in 108 samples originating from the adriatic sea, recovered on a monthly basis were investigated. out of three shellfish species, the lowest share of saturated fatty acids, the most favourable ratio of polyunsaturated over saturated fatty acids, the most favourable atherogenic and thrombogenic index, and the most favourable ratio of hypocholesterolaemic over hypercholesterolaemic fatty acids were seen in oysters, sampled during springtime. no statistically significant inter-seasonal differences between basic chemical parameters and fat quality indices were established. keywords: european flat oyster, lipid quality indices, seasonal variations, smooth scallop, variegated scallop ital. j. food sci., vol. 31, 2019 717 1. introduction aquaculture is the fastest-growing food production sector worldwide, shellfish thereby being an important component of global aquatic food supply. the production of marine organisms mainly takes place in sheltered areas or coastal embayments (pogoda, et al., 2011), oyster cultivation thereby being a particularly good example of an extensive production of high value-added products (gibbs, 2004). due to the high nutritional and gastronomic value of these products, consumer demand for cultivated, but also wild shellfish continuously increases. in general, shellfish is a highly nutritious foodstuff, since it contains appreciable quantities of digestible proteins, essential amino acids, bioactive peptides, long-chain polyunsaturated fatty acids, astaxanthin and other carotenoids, vitamin b12 and other vitamins, minerals including copper, zinc, inorganic phosphate, sodium, potassium, selenium, iodine and also other nutrients, which offer a variety of health benefits to consumers (venugopal and gopakumar, 2017). in comparison to other shellfish, european flat oyster (ostrea edulis) represents a product with a higher nutritional value and is hence much higher priced than other shellfish (fao, 2011). literature data have shown that seasonal metabolic activities of shellfish molluscs result from complex interactions between food availability, environmental and growth factors, type of shellfish, but also other parameters (gabbott, 1983). for example, lipid changes seen throughout an annual cycle may be related to the increase in energy and nutritional requirements during gonad development (luzzana et al., 1996), temperature changes (varljen et al., 2004) or diet (henderson et al., 1996). gullian and aguiremacedo (2009) pointed out that, although oysters are tolerant to a broad range of natural variables, this shellfish is susceptible to various forms of physical and chemical disturbances, which cause extreme changes in their metabolism, physiology and defence related-functions, seasonal variations thereby also changing their physiology. variations that occur in different varieties of european shellfish over a 12-month period have not been fully explored yet. additionally, studies of shellfish nutritive composition have been performed only on some species or during certain seasons, mainly on the atlantic oysters (crassostrea gigas) as cultivated shellfish of great economic importance (pazos et al., 1996; soudant et al., 1999; dagorn et al., 2016). on top of that, data on natural-born oysters inhabiting the adriatic sea and other types of shellfish is scarce. investigations into the shellfish composition could provide producers with a useful background information that could also well serve the needs of consumers keen to evaluate health benefits of their shellfish consumption. in view of the above, the aim of this study was to investigate into, and compare, the nutritional properties of european flat oyster (ostrea edulis), variegated scallop (chlamys varia) and smooth scallop (flexopecten glaber) originating from natural beds in the adriatic sea. to the best of our knowledge, this is the first study of basic nutritional composition, fatty acid profile and lipid quality of three natural-born shellfish species populating the adriatic sea and seasonal variations in the above parameters witnessed throughout a year. ital. j. food sci., vol. 31, 2019 718 2. materials and methods 2.1. sampling and sample preparation samples of european flat oyster (ostrea edulis), variegated scallop (chlamys varia) and smooth scallop (flexopecten glaber) were retrieved during 2016 and 2017 from the western coast of the istrian peninsula (fig. 1). this area is extending from the savudrija cape on the north to the border of the territorial sea of the republic of croatia, and from the barbariga cape on the south to the border of the territorial sea of the republic of croatia. this geographic area is highly influenced by strong currents and the vicinity of the mirna river mouth. it hosts natural beds of different species of bivalves like european flat oyster (ostrea edulis), smooth scallop (flexopecten glaber), variegated scallop (chlamys varia), clam (venus verrucosa) and noah’s ark (arca noae). fishermen are collecting mollusks by trawling. variegated scallop samples were collected at the b1 point (45º31’30’’n; 13º27’18’’e), those of smooth scallop at the b2 point (45º13’15,3’’n; 13º30’00’’e) and those of european flat oyster at the b3 point (45º01’14,7’’n; 13º41’46,8’’e) of the area detailed above. figure 1. sampling points at the west coast of the istrian peninsula. ital. j. food sci., vol. 31, 2019 719 shellfish samples were grouped based on the recovery season, that is to say, into the group of samples retrieved during springtime (march, april & may 2016), those retrieved during summertime (june, july & august 2016), those retrieved during autumntime (september, october & november 2016) and those retrieved during wintertime (december 2016 and january & february 2017). each month, samples containing 3 kg of each shellfish under study were sampled from the locations of their growth. in total, 108 shellfish samples (36 oyster, 36 variegated scallop and 36 smooth scallop samples) were analysed within 48 hours after sampling. from a 3 kg-shellfish sample, 300 to 400 g of muscle tissue were obtained and further homogenized using a grindomix gm200 knife mill (retch, germany), so as to obtain a homogeneous sample allowing for the determination of basic chemical composition and fatty acid profile. 2.2. determination of basic chemical composition the moisture content was determined using gravimetric analysis. the samples were dried at 103 ± 2 °c (iso 1442:1997) in an uf75 plus memmert oven (schwabach, germany). the total protein content was determined by virtue of the kjeldahl method (hrn iso 937:1999) using an 8 basic digestion unit (foss, höganäs, sweden) for sample digestion and an automated device for distillation and titration (vapodest 50s, gerhardt, germany). the total fat content was determined using the soxhlet method (hrn iso 1443:1999) that implies the digestion of samples by virtue of acid hydrolysis, followed by the extraction of fats using petroleum ether and a soxtherm 2000 automated device (gerhardt, munich, germany). the ash content was determined according to the iso 936:1998 and made use of a lv9/11/p320 nobertherm furnace (lilienthal, germany). all chemicals used for the analyses were of an analytical grade. carbohydrate content was determined by calculation, based on the determination of water, ash, total protein and fat content. the mean of data obtained from two parallel runs in form of weight percentage (%) and with the accuracy of 0.01% was considered as a result descriptive of a single sample. 2.3. fatty acid profile sample preparation method for the analysis of fatty acid methyl esters was described earlier by pleadin et al. (2015). methyl esters of fatty acids were analysed using gas chromatography (gc) according to the en iso 12966-2:2011 and en iso 12966-4:2015. to the above effect, a 7890ba gas chromatographer equipped with flame ionization detector (fid), a 60-m db-23 capillary column having an internal capillary diameter of 0.25 mm and the stationary phase thickness of 0.25 μm (agilent technologies, santa clara, usa) was used. the components were detected by fid at the temperature of 280 °c, hydrogen flow of 40 ml/min, air flow of 450 ml/min and nitrogen flow of 25 ml/min. the initial column temperature was 130 °c; after a minute, it was increased by 6.5 °c/min until the temperature of 170 °c was reached. the temperature was further increased by 2.75 °c/min until the temperature of 215 °c was attained. the latter temperature was maintained for 12 min and then further increased rate by 40 °c/min until the final column temperature of 230 °c was reached, the latter being maintained for 3 min. one ml of a sample was injected into a split-splitless injector at the temperature of 270 °c and with the partition coefficient of 1:50. the carrier gas was helium (99.9999%), flowing at the constant rate of 43 cm/sec. fatty acid methyl esters were identified by comparing their retention times with those of fatty acid methyl esters contained by the standard mixture, as ital. j. food sci., vol. 31, 2019 720 described earlier by pleadin et al. (2015). the results are expressed as a percentage (%) of a particular fatty acid in total fatty acids, the accuracy thereby being 0.01%. 2.4. nutritional quality of lipids data on fatty acid composition were used for the calculation of the following lipid quality indices: the atherogenic index (ai), the thrombogenic index (ti) and the hypocholesterolaemic/hypercholesterolaemic ratio (hh). the atherogenic index (ai) indicates the relationship between the sum of the main saturates and the sum of the main non-saturates. this parameter was calculated as: ai= [(c12:0 + (4 x c14:0) + c16:0)] / [∑ mufa + pufa n-6+ pufa n-3] (ulbritcth and southgate, 1991). the thrombogenic index (ti) is defined as the relationship between the pro-thrombogenic (saturated) and the anti-thrombogenic fas (mufa, pufa n-6 & pufa n-3). the index was calculated as: ti = (c14:0 + c16:0 + c18:0) / [0.5 x ∑mufa + 0.5 x pufa n-6 + 3 x pufa n-3) + (pufa n-3/pufa n-6)]. the ratio of hypocholesterolaemic over hypercholesterolaemic fatty acids (hh) takes into account well-known effects of certain fatty acids on cholesterol metabolism (santos-silva et al., 2002). it was calculated as: hh = (c18:1n-9 + c18:2n-6 + c20:4n-6 + c18:3n-3 + c20:5n-3 + c22:5n-3 + c22:6n-3) / (c14:0 + c16:0) (ulbritcth and southgate, 1991). 2.5. statistical analysis statistical analysis was performed using the spss statistics software 22.0 (spss statistics, ny ibm, 2013). in order to determine the differences between the sample groups (seasonbased, shellfish type-based), one-way anova and the robust brown-forsythe test were used. the decisions on statistical significance were made at the significance level of p<0.001 and p < 0.05. 3. results and discussion this study provides data on nutritional composition of three natural-born shellfish species originating from the adriatic sea, with a special emphasis on fatty acids and healthrelated lipid indices determined over four seasons of a one-year period. literature data have revealed that habitats, season, feed, species, but also gametogenesis and spawning cycle, can influence the proximate shellfish composition (gabbott, 1983). basic chemical composition of the investigated shellfish determined in this study is shown in table 1. as regards the moisture content, oysters had a significantly lower average value (around 82 g/100 g) as compared to variegated (around 84 g/100 g, p = 0.0028) and smooth scallops (around 87 g/100 g, p < 0.001), representing the shellfish richer in nutrients in comparison to the other two. however, oysters were the only one out of the three shellfish species in which significant seasonal moisture content variations were found (higher moisture content in autumn as compared to spring and summer). moisture contents similar to those we found, with values of 82.1 g/100 g during summer and 81.4 g/100 g during winter, were reported by martino and cruz (2004) for oysters of the crassostrea rhizophorae species. oysters also had significantly higher carbohydrate content (average value around 4.3 g/100 g) as compared to variegated (around 3.2 g/100 g) and smooth scallops (1.4 g/100 ital. j. food sci., vol. 31, 2019 721 g). however, the significant seasonal carbohydrate content variations weren't found for any shellfish (p > 0.05). table 1. basic chemical composition of the analysed shellfish types during a one-year period. shellfish season mean ± sd (g/100 g) moisture protein ash fat carbohydrate european flat oyster (n=36) spring 80.30±0.53d 10.99±0.27d 2.04±0.16 2.37±0.32a,d 4.29±0.63 summer 81.43±0.58d 10.00±0.27 2.14±0.43 2.03±0.25d 4.40±0.07 autumn 83.87±0.61b,c 8.13±1.13b 2.21±0.04 1.20±0.10b,c 4.57±0.77 winter 81.87±1.22 10.27±1.47 2.33±0.20 1.67±0.23b 3.88±0.75 average 81.87±1.50b,c 9.85±1.36 2.18±0.24 1.82±0.50b,c 4.29±0.30b,c variegated scallop (n=36) spring 80.43±7.51 8.88±0.48 3.40±2.42 0.90±0.20 6.39±7.94 summer 85.27±0.40 9.43±0.24 2.56±0.57 0.97±0.23 1.77±0.57 autumn 85.23±1.31 9.90±1.52 1.94±0.20 0.93±0.12 1.99±0.44 winter 85.27±1.25 9.02±2.03 2.06±0.35 0.77±0.15 2.87±1.81 average 84.05±3.95a,c 9.31±1.18 2.49±1.23 0.89±0.17a,c 3.24±1.83a smooth scallop (n=36) spring 86.57±0.35 9.32±1.26 1.70±0.17 1.07±0.31d 1.34±0.29 summer 86.70±0.79 8.71±0.39 2.55±0.61 0.77±0.15 1.28±0.33 autumn 87.63±0.95 8.65±0.88 1.91±0.16 0.30±0.10b 1.51±0.46 winter 87.03±1.42 8.68±0.96 2.27±0.71 0.53±0.23 1.49±0.53 average 86.98±0.92a,b 8.84±0.32 2.11±0.53 0.67±0.35a,b 1.40±0.11a results are expressed as the mean value (mean ± sd) of six results (3 months per season; each month, one sample was taken and analysed in duplicate). statistically significant difference (p< 0.05) within the same column for every shellfish type separately: avs. winter; bvs. spring, cvs. summer, dvs. autumn; avs. european flat oyster, bvs. variegated scallop, cvs. smooth scallop. the average protein content was almost equal in all three studied shellfish species, ranging from 8.84 g/100 g in smooth scallops to 9.85 g/100 g in oysters. the proportion of proteins significantly differed among the shellfish species only in summer. oysters and smooth scallops contained the highest protein levels in spring (10.99 g/100 g and 9.32 g/100 g, respectively) while variegated scallops presented with the highest protein levels in autumn (9.90 g/100 g), although the only statistically significant difference (p = 0.014) was that in the protein content of oysters, which was higher in those collected in spring as compared to those collected in autumn. three shellfish species had quite similar average ash contents, ranging from 2.11 g/100 g in smooth scallop to 2.49 g/100 g in variegated scallop and showing no statistically significant differences, neither across seasons nor across species. based on linear correlation coefficient and slope values from correlation equations related to moisture and fat (y = -0.1818x+16.453; r2 = 0.6275), moisture and protein (y = 0.1153x+19.232; r2 = 0.0885) as well as moisture and ash (y = -0.0679x+7.986; r2 = 0.1681), it is clear that fat content shows the strongest inversely proportional relationship with the moisture content found in the three shellfish. therefore, a decrease in proportion of water is primarily reflected in an increase of fat content, especially in case of oysters. oysters had ital. j. food sci., vol. 31, 2019 722 a significantly higher average fat content (1.82 g/100 g) as compared to variegated (0.89 g/100 g) and smooth scallops (0.67 g/100 g) (p < 0.001), as well as the highest share of fat in winter (p = 0.002), spring (p = 0.001), summer (p = 0.001) and autumn (p < 0.001) in comparison to other shellfish types (data not shown). as regards seasonal influence, both oysters and smooth scallops showed a significantly higher fat content in spring than in autumn (p = 0.005 and p < 0.05, respectively), while in variegated scallops no significant variations were found. since fats have been shown to be involved in spawning-related biochemistry of marine species (ren et al., 2003), the observed variability in fat levels in different sampling times was to be expected. in comparison to the results of lira et al. (2013) that revealed these species to have a higher fat content in winter than in summer, our study failed to confirm such a pattern. however, in the study quoted above, the composition of oysters was analysed using the brazilian cultivated crassostrea rhizophorae oysters sampled in only two seasons winter and summer. nevertheless, our spring sampling could be compared to their winter sampling, confirming the same variability pattern. also, it should be emphasized that the majority of studies were conducted on the pacific oysters (crassostrea gigas) in particular months or seasons, either rendering the inter-comparison impossible or limiting its extent (pazos et al., 1996; soudant et al., 1999; dagorn et al., 2016). in oysters, 27 fatty acids were identified, in all four investigated seasons mostly in the following order of representation: palmitic acid (c16:0), stearic acid (c18:0), oleic acid (c18:1n-9c) and docosahexaenoic acid (dha; c22:6n-3) (table 2). the fatty acid composition and the prevalence of certain fatty acids could be compared to the results of some earlier studies performed on different oyster types (linehan et al., 1999; ezgetabalić et al., 2012; hurtado et al., 2012; lira et al., 2013; pogoda et al., 2013; dagorn et al., 2016). the highest sfa content was determined in summer and autumn, whereas the highest pufa content was determined in spring. it could be assumed that in our samples fatty acid composition of the muscle tissue indicates the differences in selective incorporation of dietary pufas. the study of bivalve food sources populating the adriatic sea (ezgeta-balić et al., 2012) confirmed that bivalves feed on mixed food, the quality of which strongly depends on seasonal changes in food composition. during the period of high phytoplankton presence (spring/summer), bivalve species mainly ingest phytoplankton, but also zooplankton and detritus. during the period of low phytoplankton presence (autumn/winter), bivalves rely on zooplankton and detritus. in line with the findings of ezgeta-balić et al. (2012), we confirmed that oysters accumulate a significant amount of pufas during springtime. as oppose to the results of lira et al. (2013) (although obtained on crassostrea rhizophorae, not ostrea edulis), who determined the dha (c22:6n-3) content to be twice higher in winter than in summer, in our study the highest dha oyster content was observed in spring, with moderately high levels in winter and autumn and the lowest level in summer. the ratios in favour of dha over epa throughout the year confirm the presence of animal component in oyster diets (ezgeta-balić et al., 2012). generally, variegated and smooth scallops were shown to harbour a significantly lower number of fatty acids in comparison to oysters, which could be explained by the fact that food selection is an active process and that different species have different affinities when it comes to food, i.e. various preferences for microalgae (gonzàlez-araya et al., 2012). ital. j. food sci., vol. 31, 2019 723 table 2. fatty acid composition (% of total fatty acids) of european flat oyster (ostrea edulis). season fatty acids spring summer autumn winter c8:0 0.21±0.18 < lod < lod 0.08±0.14 c10:0 0.26±0.23 < lod < lod < lod c12:0 0.06±0.11 < lod < lod 0.60±1.04 c14:0 7.32±0.73 6.51±1.74 5.30±1.28 5.01±1.48 c15:0 1.45±0.00 1.54±0.37 1.43±0.31 1.24±0.45 c16:0 31.57±2.41 39.56±7.40 36.54±3.68 34.90±3.75 c17:0 3.15±0.22 4.42±0.87 4.20±0.67 3.33±1.03 c18:0 6.72±5.88 14.75±1.92 20.27±2.86 14.09±7.13 c20:0 < lod < lod < lod 0.11±0.19 c23:0 0.28±0.48 < lod < lod 0.56±0.49 c14:1 < lod 0.43±0.74 < lod < lod c16:1n-7t 0.50±0.07 < lod 0.47±0.81 0.48±0.48 c16:1n-7c 4.26±0.67 3.14±0.71 2.96±0.50 3.14±0.83 c17:1 0.21±0.18 < lod < lod < lod c18:1n-9c 8.03±0.20 10.18±0.75 9.08±4.91 14.82±9.69 c18:1n-7 2.96±0.24 3.13±0.39 3.12±0.26 2.13±1.85 c20:1n-9 0.60±0.02 1.63±2.12 < lod 0.12±0.20 c24:1n-9 < lod < lod < lod 0.25±0.43 c18:2n-6c 2.72±0.26 2.26±0.44 1.74±1.60 4.31±3.53 c18:3n-6 0.40±0.69 < lod < lod < lod c20:4n-6 0.89±0.19 0.41±0.70 0.37±0.64 0.54±0.66 c18:3n-3 4.35±1.40 1.71±1.72 1.78±1.58 1.11±1.42 c18:4n-3 5.22±0.91 1.69±1.62 1.47±1.41 1.62±2.39 c20:4n-3 0.49±0.43 < lod < lod 0.22±0.38 c20:5n-3 7.31±1.58 3.40±2.96 5.03±1.23 5.15±3.85 c22:5n-3 0.60±0.52 < lod < lod < lod c22:6n-3 10.44±2.05 5.21±4.62 6.25±2.29 6.18±5.28 sfa 51.02±7.19 66.79±11.40 67.74±8.74 59.93±7.99 mufa 16.55±1.12 18.52±2.88 15.63±3.92 20.95±7.68 n-6 4.01±0.88 2.67±1.14 2.11±2.12 4.85±3.41 n-3 28.42±5.67 12.02±10.78 14.52±6.01 14.28±13.01 pufa 32.43±6.46 14.69±11.63 16.64±7.38 19.13±11.66 results are expressed as the mean value (%, mean ± sd) of six results obtained for total fatty acids (3 months per season; each month, one sample was taken and analysed in duplicate); lod (limit of detection) = 0.05%. sfa saturated fatty acids, mufa monounsaturated fatty acids, pufa polyunsaturated fatty acids. raby et al. (1997) found that different species ingest microalgae of different sizes, which is an indicator of their active food selection, the size of microalgae thereby being the major factor influencing the ingestion of food particles. although the three shellfish types were collected from different locations in the adriatic sea (along the coast of the istrian ital. j. food sci., vol. 31, 2019 724 peninsula), the influence of water temperature, salinity and other environmental factors on fatty acid composition should be negligible, due to the small distances between the sampling locations (the same area of the adriatic sea). so, the differences in nutritive composition of three shellfish investigated in this study are probably mainly coming as a result of their different diet preferences. table 3 presents the fatty acid composition of variegated scallop determined in various seasons. same as with oysters, the most dominant fatty acids in variegated scallop were c16:0, c18:0 and c18:1n-9c, whereas dha was not detected. in this shellfish, no statistically significant inter-seasonal differences (p > 0.05) in individual fatty acid, sfa, mufa and pufa contents as were found. however, pufa contents were highly variable within the same annual period, as can be seen from high intra-seasonal standard deviations. higher pufa content was observed in winter in comparison to summertime. table 3. fatty acid composition (% of total fatty acids) of variegated scallop (chlamys varia). season fatty acids spring summer autumn winter c8:0 0.52±0.90 < lod < lod 0.40±0.69 c10:0 0.35±0.61 < lod < lod < lod c12:0 < lod < lod < lod 0.50±0.87 c14:0 8.13±4.77 10.02±1.33 7.18±3.36 3.72±0.96 c16:0 41.20±6.34 44.34±1.10 44.60±1.81 44.56±4.67 c17:0 2.52±4.36 < lod < lod < lod c18:0 31.68±12.47 24.20±4.35 38.73±10.02 35.70±9.55 c16:1n-7 3.67±3.29 3.31±2.95 2.95±2.60 0.70±1.21 c18:1n-9c 7.86±3.61 15.27±2.63 4.48±3.93 12.27±6.89 c18:1n-7 < lod 1.96±1.75 0.78±1.35 < lod c18:2n-6c 1.69±2.93 0.90±1.56 < lod 2.15±3.73 c18:3n-3 2.38±4.13 < lod 1.28±2.21 < lod sfa 84.40±8.64 78.56±4.91 90.51±8.46 84.87±11.83 mufa 11.53±6.32 20.54±5.07 8.21±7.11 12.98±8.10 n-6 1.69±2.63 0.90±1.56 < lod 2.15±3.73 n-3 2.38±4.13 < lod 1.28±2.21 < lod pufa 4.07±7.05 0.90±1.56 1.28±2.21 2.15±3.73 results are expressed as mean value (%, mean ± sd) of six results obtained for total fatty acids (3 months per season; each month, one sample was taken and analysed in duplicate); lod (limit of detection) = 0.05%. sfa saturated fatty acids, mufa monounsaturated fatty acids, pufa polyunsaturated fatty acids. fatty acid composition of smooth scallop seen in various seasons is presented in table 4. same as with variegated scallop, the most dominant fatty acids in smooth scallop were c16:0, c18:0 and c18:1n-9c, while dha presence was not detected. a statistically significant inter-seasonal difference was determined only for c18:0 found in summer (lower value) as compared to that in autumn (higher value) (p = 0.001), which can also be achieved thanks to low intra-seasonal variability witnessed in these two seasons. as for ital. j. food sci., vol. 31, 2019 725 the content of other individual fatty acids, sfa, mufa and pufa, no statistically significant inter-seasonal differences were found (p > 0.05). pufas were not quantified, while a higher sfa content was observed in winter in comparison to summertime. table 4. fatty acid composition (% of total fatty acids) of smooth scallop (flexopecten glaber). fatty acids season spring summer autumn winter c14:0 8.63±3.10 5.91±1.81 1.10±1.90 5.80±3.08 c16:0 44.67±0.89 46.01±3.45 45.29±0.43 44.30±2.45 c17:0 1.88±2.66 < lod < lod < lod c18:0 29.12±11.47 33.47±1.33* 47.22±1.14* 39.49±5.57 c14:1 < lod 1.17±2.02 < lod < lod c16:1n-7 4.78±1.60 1.19±2.06 < lod 2.00±3.46 c18:1n-9c 8.99±0.49 12.25±2.44 6.39±1.31 8.41±3.13 c18:1n-7 1.93±2.73 < lod < lod < lod sfa 84.29±4.82 85.39±5.78 93.61±1.31 89.59±2.11 mufa 15.71±4.82 14.61±5.78 6.39±1.31 10.41±2.11 pufa < lod < lod < lod < lod results are expressed as mean value (%, mean ± sd) of six results obtained for total fatty acids (3 months per season; each month, one sample was taken and analysed in duplicate); lod (limit of detection) = 0.05%. sfa saturated fatty acids, mufa monounsaturated fatty acids, pufa polyunsaturated fatty acids. * statistically significant difference (p < 0.05). the representation of all fatty acid groups (sfa, mufa and pufa) significantly differed (p < 0.05) between the analyzed shell species. oysters contained the smallest proportion of sfas (p < 0.001) and the highest (p < 0.001) share of pufas as compared to other shellfish types. the share of mufa was significantly higher in oysters than in smooth scallop (p = 0.031). it is known that shellfish fatty acid composition usually reflects a fatty acid composition of their diet (phytoplankton or zooplankton), although shellfish have shown a certain ability to elongate (e.g. c16:1 to c18:1, c18:1 to c20:1, c20:5 to c22:5, c20:4 to c22:4) or desaturate (e.g. c20:3 to c20:4) fatty acids (albentosa et al., 1996; delaporte et al., 2005). given that basic shellfish nutrient composition and fatty acid profile are influenced by many parameters, both a correct interpretation of the obtained results and a plausible comparison with the results of other studies require the knowledge on ecological characteristics of areas in which shellfish are cultivated or natural-born. the results pertaining to the nutritional fat quality indices (n-6/n-3, pufa/sfa, ai, ti and hh) determined for each shellfish type in each season and in total, the latter being expressed as the mean value descriptive of the entire one-year study period, are presented in table 5. ratios n-6/n-3 and pufa/sfa are the parameters most commonly used for the assessment of nutritional fat quality. literature has shown that in case of lower n-6/n-3 ratios, the body is more able to make use of n-3 fats (wood et al., 2008). ratio n-6/n-3 has been suggested to be a good tool for comparing relative nutritional values of different species, but this index is of a limited value should the share of individual fatty acids be ital. j. food sci., vol. 31, 2019 726 unknown. due to the fact that fatty acids containing c20 and c22 are more valuable from the nutritional standpoint as compared to fatty acids containing c18, and taking into account their predominance over other n-3 fatty acids, epa and dha are largely responsible for the changes in n-6/n-3 ratio, the latter otherwise being considered as a reliable indicator that enables comparison of relative nutritive lipid values (pleadin et al., 2017). as recently reviewed by weyland et al. (2015), beneficial effects of these fatty acids have been reported for a number of disorders, including cardiovascular, neuropsychiatric and inflammatory diseases, as well as some cancers (mainly colorectal, mammary and prostatic cancer). table 5. nutritional fat quality indices established for the analysed shellfish types during a one-year period. shellfish type season fat quality indices (target values) n-6/n-3 pufa/sfa ai ti hh european flat oyster (n=36) spring 0.14±0.01 0.65±0.21 1.27±0.26 0.46±0.16 0.89±0.17 summer 0.11±0.10 0.24±0.20 2.32±1.53 2.92±3.72 0.54±0.28 autumn 0.13±0.14 0.26±0.13 1.95±0.91 1.30±0.79 0.60±0.28 winter 0.77±1.02 0.34±0.24 1.43±0.32 1.17±0.62 0.81±0.17 average 0.29±0.53 0.37±0.24 1.74±0.89b,c 1.46±1.90 b,c 0.71±0.25b,c variegated scallop (n=36) spring 0.24±0.41 0.05±0.09 6.12±3.82 14.29±15.52 0.25±0.16 summer n.d. 0.01±0.02 4.08±0.96 7.68±2.33 0.30±0.03 autumn n.d. 0.02±0.03 3.80±3.36 6.36±7.32 0.11±0.10 winter n.d. 0.03±0.05 5.68±3.40 16.33±10.03 0.32±0.26 average 0.24±0.41 0.03±0.05 4.92±2.84a 11.17±9.61a 0.23±0.15a smooth scallop (n=36) spring n.d. n.d. 5.15±0.74 11.17±4.38d 0.17±0.00 summer n.d. n.d. 5.48±2.86 13.19±5.81d 0.24±0.07 autumn n.d. n.d. 8.08±2.45 30.12±5.95b,c 0.14±0.03 winter n.d. n.d. 6.65±1.74 17.82±4.54 0.17±0.08 average n.d. n.d. 6.45±2.22a 18.70±8.93a 0.18±0.06a results are expressed as mean value (%, mean ± sd) of six results obtained for total fatty acids (3 months per season; each month, one sample was taken and analysed in duplicate); sfa saturated fatty acids; pufa polyunsaturated fatty acids; hh hypo-/hyper-cholesterolaemic fatty acids ratio = (c18:1n-9+c18:2n-6+c20:4n-6+c18:3n-3+c20:5n3+c22:5n-3+c22:6n-3)/(c14:0+c16:0) ai atherogenic index = [(c12:0+(4xc14:0)+c16:0)]/(∑ mufa+pufa n-6+pufa n-3) ti thrombogenic index = (c14:0+c16:0+c18:0)/[(0.5 x ∑ mufa+ 0.5x pufa n-6+ 3x pufa n-3)+(pufa n3/pufa n-6)] statistically significant difference (p< 0.05) within the same column for every shellfish type separately: avs. winter; bvs. spring, cvs. summer, dvs. autumn; avs. european flat oyster, bvs. variegated scallop, cvs. smooth scallop n.d. (not detected) fatty acids needed for calculation were not detected (< lod). according to health recommendations, n-6/n-3 ratio should be lower than 4, thereby reducing the incidence of chronic food-related illnesses (cordain et al., 2005; simopoulos, 2002). in an annual form, this index was calculable only for oysters, while for variegated scallop it could be provided only for the samples recovered during springtime. in both cases, the determined n-6/n-3 ratios fell within the recommended ital. j. food sci., vol. 31, 2019 727 boundaries, although high intra-seasonal variations were evident. in an annual form, n6/n-3 ratio was not calculable for smooth scallop because of the absence of pufas (values below the lod). pufa/sfa ratio is recommended to be higher than 0.4, so as to reduce the risk of cardiovascular, autoimmune and other chronic diseases (simopoulos, 2002). generally, for two shellfish types in which pufas were present in values above the lod (oyster and variegated scallop), the determined pufa/sfa ratios were significantly lower than the recommended minimum, except in oysters during springtime (0.65±0.21). same as with n6/n-3 ratio, high intra-seasonal variations were noticeable. some authors are of the opinion that an index such as pufa/sfa may prove inadequate for the evaluation of nutritional value of fats, because some sfas do not increase plasma cholesterol. therefore, mensink and katan (1992), and daley et al. (2010), suggested that c12:0 and c14:0 have a more pronounced total cholesterol raising effect than c16:0, whereas c18:0 is neutral when it comes to the concentration of total serum cholesterol, with no apparent impact on either ldl or hdl. myristic acid (c14:0) has a 4-6 times higher potential to increase cholesterol concentrations as compared to c16:0 (ulbritcth and southgate, 1991; bressan et al., 2011). on top of that, pufa/sfa index ignores the effects of mufas, which may have more profound health benefits in terms of coronary disease prevention (orellana et al., 2009). therefore, two additional indices, which take into account different effects that a single fatty acid might have on the incidence of pathogenic phenomena, such as atheroma and/or thrombus formation, i.e. the atherogenic (ai) and the thrombogenic index (ti), were calculated, too. the atherogenic index takes into account the fact that some saturates are considered to be pro-atherogenic (since they facilitate the adhesion of lipids onto the cells the immune and the circulatory system are composed of), while non-saturates are considered to be anti-atherogenic (since that inhibit the formation of plaques and diminish the levels of esterified fatty acids, cholesterol, and phospholipids, therefore preventing microand macro-coronary disease) (ulbritcth and southgate, 1991). the thrombogenic index (ti) shows the tendency towards blood clotting. it is assumed that ais and tis below 1 are beneficial to human health (pleadin et al., 2017). according to the data reported in table 5, only oysters approach the recommended values, while other two shellfish types exceed the maximum limits by far. in order to gain insight into the effect of fatty acids on blood cholesterol, an additional indicator of nutritional quality, i.e. the ratio between hypocholesterolaemic and hypercholesterolaemic fatty acids (hh), was calculated. it is preferable for that index to be higher (santos-silva et al., 2002). the obtained hh values ranged from 0.18±0.06 in smooth scallop and from 0.23±0.15 in variegated scallop, whereas the highest hh index was determined in oysters during springtime (0.89±0.17). in case of oysters and variegated scallop, no statistically significant seasonal differences (p > 0.05) in any of the fat quality indices were determined. in smooth scallop, a significant seasonal difference was determined only for ti (p < 0.05; p = 0.016), with a significantly higher ti in autumn in comparison to spring and summer. with the exception of n-6/n-3 ratio, which was actually determined only in oysters, a significant difference in ti, hh and pufa/sfa indices was determined across the studied shellfish types, while a statistically significant difference in the ai value among the analysed shellfish over different seasons failed to be seen. in spring, oysters had significantly higher pufa/sfa and hh ratios than other shellfish types. in winter and autumn, oysters also showed a significantly higher hh ratio as compared to variegated and smooth scallop. the ti established for smooth scallop in autumn was significantly higher as compared to other ital. j. food sci., vol. 31, 2019 728 shellfish; in winter, the latter index determined for smooth scallop was also higher than that found for oysters. as in the study by lira et al. (2013), fatty acids-related nutritional quality indices were more favourable in winter in comparison to summer period. 4. conclusion the proportion of fat found in all shellfish types under study was low, with the highest average representation in oysters. the proportion of proteins and total minerals in meat of all three shellfish was found to be similar. the representation of saturated fatty acids was generally found to be high, with an unfavourable pufa/sfa ratio that might increase the risk of chronic diseases. out of the three shellfish species under study, the lowest sfa content, the most favourable pufa/sfa ratio and the most favourable ai, ti and hh indices were established in oysters. although oysters harvested in springtime contained the highest proportion of fats and proteins, and therefore presented with the most favourable pufa/sfa, ai, ti and hh indices in that particular season, intra-seasonal variations were huge, so that statistically significant inter-seasonal differences in these parameters in oysters harvested in different times of the year failed to be found. during springtime, smooth scallop also showed the highest representation of fats and proteins, and hence also the most favourable ai and ti indices, but inter-seasonal variations were proven to be either statistically insignificant or significant only in comparison to one out of the three remaining seasons. as for variegated scallop, none of the seasons could be considered as the most favourable when it comes either to fat and protein content or to fat nutritional quality indices. in summary, no statistically significant inter-seasonal differences in basic chemical parameters and fat quality indices descriptive of an edible part of the three shellfish were determined. acknowledgements this study was carried out as a part of the faimmac project (fishery and aquaculture integrated management model along the adriatic coasts) funded from the european maritime and fishery fund 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adriatic sea. j. am. oil chem. soc. 81(8):759-763. doi: doi.org/10.1007/s11746-004-0975-7. venugopal v. and gopakumar k. 2017. shellfish: nutritive value, health benefits and consumer safety. compr. rev. food sci. f. 16(6):1219-1242. doi: doi.org/10.1111/1541-4337.12312. weyland k.h., serini s., chen y.q., su h.-m., lim k., cittadini a. and calviello g. 2015. omega-3 polyunsaturated fatty acids: the way forward in times of mixed evidence. biomed res. int. 2015, id 143109:1-24. doi: doi.org/10.1155/2015/143109. wood j.d., enser m., fisher a.v., nute g.r., sheard p.r., richardson r.i., hughes s.i. and whittington f.m. 2008. fat deposition, fatty acid composition and meat quality: a review. meat sci. 78(4):343-358. doi: doi.org/10.1016/j.meatsci.2007.07.019. iso standard iso 1442:1997. meat and meat products determination of moisture content. international organization for standardization. genève, switzerland. iso standard iso 936:1998. meat and meat products determination of total ash. international organization for standardization. genève, switzerland. iso standard hrn iso 937:1999. meat and meat products determination of nitrogen content (iso 937:1978). international organization for standardization. genève, switzerland. iso standard hrn iso 1443:1999. meat and meat products determination of total fat content (iso 1443:1973). international organization for standardization. genève, switzerland. iso standard en iso 12966-2:2011. animal and vegetables fats and oils gas chromatography of fatty acid methyl esters part 2: preparation of methyl esters of fatty acids. international organization for standardization. genève, switzerland. iso standard en iso 12966-4:2015. animal and vegetables fats and oils gas chromatography of fatty acid methyl esters part 4: determination by capillary gas chromatography. international organization for standardization. genève, switzerland. paper received january 18, 2019 accepted april 18, 2019 ijfs#1566_bozza ital. j. food sci., vol. 32, 2020 399 paper microbiological quality and antimicrobial efficacy of combined oregano essential oil and acetic acid on fresh lettuce oa. ijabadeniyi, a. mbedla and ta. ajayeoba* durban university of technology, durban, 4001, sa *corresponding author: titilayoa@dut.ac.za abstract this study determined the microbiological quality of lettuce purchased at durban markets, and evaluated the antimicrobial effects of oregano essential oil (oeo), acetic acid (aa) and combination (oeo+aa)] on the survival of escherichia coli and l. monocytogenes on lettuce for 6 days. aerobic and anaerobic spore formers, staphylococcus aureus, escherichia coli and l. monocytogenes were microscopically and phenotypically identified from the lettuce. decontamination was higher and significantly different (p>0.05) at 5ºc with combined 0.3% aa+0.1% eoe, and complete inhibition of pathogens was observed on day 2. this formulation can increase antimicrobial efficacy and balance sensory attributes of treated lettuce. keywords: acetic acid, combined treatments, decontamination, oregano essential oil ital. j. food sci., vol. 32, 2020 400 1. introduction there has been a huge increase in the consumption of fresh, minimally processed fruits and vegetables in the last decade. this is due to the minimal labor that is required to prepare these food items and are a great source of a variety of vitamins, minerals and other phytochemicals which are beneficial to health (ramos et al., 2013). the increase of consumption of minimally processed readytoeat vegetables such as lettuce has however led to an increase in the number of reported cases of foodborne outbreaks linked to the consumption of contaminated vegetables (murray et al., 2017). however, information on outbreaks or presence of pathogens in or on fresh produce leading to foodborne outbreaks in south africa is scarce due to the absence of an efficient reporting system (jordaan 2013). most prevalent pathogenic microorganisms reported in contaminated vegetables include bacteria such as e. coli o157:h7, listeria monocytogenes and some salmonella species. these are able to survive under adverse environmental conditions and form biofilms (callejón et al., 2015). decontamination methods used for vegetables in fresh produce industry aim to decrease the microbial populations without necessarily eliminating them (de medeiros barbosa et al., 2016) consumers are aware of the limitations of disinfectants used in fresh-cut produce in terms of taste and freshness (ponce et al., 2004). oregano essential oil (oeo) and acetic acid (aa) have proven to be effective against food borne pathogens such as escherichia coli o157:h7, camphylobacter jejuni, salmonella enterica, and listeria monocytogenes (raeisi et al., 2015). however, the use of these antimicrobials individually requires that food be exposed to large doses for effective inhibition of pathogens. eoe and aa have very strong odors and could impair sensory qualities at high concentrations, which is generally not accepted by customers. therefore, the combination of preservatives serves a promising method to be able to achieve optimum pathogen inhibition without affecting the quality of food (miyague et al., 2015). the combination of technologies with antimicrobial/preservative effects has been used in the food industry to maintain food quality and ascertain that pathogens can be eradicated or controlled (nazer et al., 2005). hence, the objective of this study is to evaluate the effectiveness of individual and combined oregano essential oil and acetic acid on inoculated iceberg lettuce. 2. materials and methods 2.1. collection of samples a total of 60 samples of lettuce and spinach (30 samples each) were purchased from two different retail markets; the open market and fresh retail market (local supermarket). samples were collected in sterile ldpe zip lock bags and stored at 4 ºc until testing. the test pathogens (e. coli o157:h7 atcc 4388 and l. monocytogenes atcc 7644) were collected from the department of biotechnology and food technology, durban university of technology, south africa. eoe and aa were purchased nautica organic’s in durban, south africa. ital. j. food sci., vol. 32, 2020 401 2.2. experimental design for the microbiological quality of leafy vegetables, 60 samples (30 lettuce and 30 spinach samples each purchased equally from retail and open markets) were evaluated for the presence of aerobic and anaerobic spore formers, staphylococcus aureus, e. coli and l. monocytogenes. the assay with oregano essential oil and acetic acid followed a 3x3x2x2 factorial combination. the effect of four factors: type of treatments (oregano eo, acetic acid, oregano eo+ acetic acid); level of concentration (0.05%, 0.1%, 0.3%), contact times (2 min, 5 min) and storage temperatures (5ºc and 20ºc) were evaluated for a duration of 6 days by bioassay. 2.3. microbiological analysis of the samples all experiments were carried out in duplicates. the microbiological testing to isolate and identify aerobic spore formers, anaerobic spore formers, staphylococcus aureus, salmonella spp., listeria monocytogenes and escherichia coli on the leafy vegetables was carried out according to international standard organization protocols as described by ijabadeniyi et al. (2011). 2.3.1. bacterial inoculum preparation a 24 h old culture of l. monocytogenes atcc 7644 and e. coli o157:h7 atcc 4388 were aseptically transferred into 10 ml brain heart infusion broth and tryptic soy broth respectively. the broths were incubated at 37ºc for 24 h and washed by centrifugation (4629xg for 15 min) at 4ºc. serial dilutions of the washed inocula were performed to obtain the desired dilution using absorbance at 600 nm. 2.3.2 assay with essential oil and organic acid treatment suspension preparation suspensions were made by dispersing the treatments into sterile distilled water according to akbas and olmez (2007). the combination treatment was made by mixing the most effective concentrations. fresh lettuce samples that had negative results for the presence of e. coli o157:h7 and l. monocytogenes atcc 7644 were selected for simulative study. lettuce leaves were washed with cold sterile deionized water at 21°c for 2 min. thereafter the leaves were left to dry under a safety hood bio-cabinet and cut into appropriate sizes. leaves were artificially contaminated according to samara et al. (2009). ten grams of the treated samples were immersed into 200 ml of each treatment solution for 2 min and 5 min each, with gentle agitation at room temperature and different concentrations (acetic acid 0.1 and 0.3%; oregano eo0.05 and 0.1%). thereafter, the lettuce was removed from solutions and then placed in 10g samples into polyethylene bags and stored at 5ºc and 22ºc for 6 days. samples were taken for enumeration every 2 days’ interval for a period of 6 days. 2.4. statistical analysis data obtained were subjected to analysis of variance (anova) and t-test using statistical analysis system to determine the significant difference in treatment methods. colony counts were converted into logarithmic values (cfu/g), means and standard deviations were calculated and significance was expressed at p≤0.05. ital. j. food sci., vol. 32, 2020 402 3. results and discussion 3.1. microbiological quality analysis of lettuce and spinach the microbiological quality of leafy vegetables is of great concern as most are usually consumed raw with minimal processing during production. consumer demands for microbiologically safe, fresh vegetables with no chemical preservation have enhanced the need for routine quality analysis of these commodities (carella, 2014). as observed, the total mean log cfu/g of lettuce in open markets (om) were higher and significantly different (p<0.05) than retail markets (rm) while there was no significant difference in spinach samples. all lettuce and spinach samples were positive for s. aureus, aerobic and anaerobic spore formers (table 1). table 1. microbiological quality of spinach and lettuce collected at different retail markets. type of leafy vegetable microorganisms retail market (supermarket) open market lettuce tpc 5.06±0.81c 6.02±0.54b s. aureus 0.37±0.99h 3.28±0.50e asf 1.72±0.95g 3.70±0.21e aasf 1.46±0.71g 2.90±0.73f spinach tpc 7.38±0.08a 7.76±0.39a s. aureus 4.57±0.19d 4.91±0.17d asf 2.33±0.28f 2.61±0.28f aasf 1.94±0.29g 1.83±0.55g results represented as means±standard deviation. means with same superscript letters in the same row are not significantly different (p > 0.05). n= 30 (lettuce), n=30 (spinach). tpc –total plate count, asfaerobic spore formers, aasfanaerobic spore formers. total plate counts had higher values in both samples and markets (lettuce: rm-5.06 log cfu/g; om6.02 log cfu/g; spinach:rm-7.38 log cfu/g; om7.76 log cfu/g) while s. aureus had the least in lettuce (rm-0.37 log cfu/g; om3.28 log cfu/g) and anaerobic spore formers had the least in spinach (rm-1.94 log cfu/g; om1.83 log cfu/g). the high levels of aerobic bacteria found on lettuce and spinach could be due to the large surface area, which allows for easy and fastidious attachment of microorganisms (korir et al., 2016). the difference in s. aureus in the lettuce in both markets could be due to the different environmental conditions handling and cross contamination. improper handling, abuse of temperature, unhygienic practices, un-sanitized contact surfaces that products are exposed to in the open market serve as good sources for the contamination of fresh produce (wiederoder et al., 2012). furthermore, reports have shown that the quality of water used for irrigation during growing seasons, age of leaves and water used for cleaning the leaves before display influences the incidence of bacteria in final produce (merlini et al., 2018). the presence of spore formers may suggest pathogenic bacteria, which may exhibit strong resistance towards chemical and physical sanitizers. contamination of fresh produce by these microorganisms can lead to serious diseases and harm to human health. similar results have been reported by korir et al. (2016) with ital. j. food sci., vol. 32, 2020 403 bacterial counts of 8.02 and 7.49 log cfu/g for spinach and lettuce, respectively. (pingulkar, 2001) also reported aerobic bacterial growth range of 4.3 to 8.9 log cfu/g for fresh-cut vegetables. according to usda regulations, s. aureus observed in open market samples are at unacceptable levels. as observed earlier, the incidence of other pathogens was higher in samples from om than rm. out of the 60 samples, salmonella spp. was detected on 43 [rm: lettuce 6(10.00%), spinach 11(18.33%); om: lettuce 14(23.33%), spinach 12(20.00%)], e. coli was detected on 15 (25.00%)[rm: lettuce 0(0.00%), spinach 2(3.33%); om: lettuce 5(8.33%), spinach 8(13.33%)], while l. monocytogenes was detected on 42 (70.00%) [rm: lettuce 4(6.67%), spinach 10 (16.67%); om: lettuce 12(20.00%), spinach 16(26.67%)]. e. coli, salmonella and listeria all have mechanisms for adherence onto surfaces and all adhere differently onto leaf surfaces (topalić-trivunović et al., 2014). similarly, korir et al. (2016) in their analysis 144 fresh produce samples from retail stores, only four samples were positive for pathogens. e. coli, listeria and salmonella are pathogenic microorganisms that are prominently associated with the diseases/infections caused by the consumption of poor quality contaminated leafy vegetables (singh et al., 2002). environmental sources such as water, soil, air, insects, animals and human activity can cause contamination of leafy vegetables by l. monocytogenes (merlini et al., 2018). the south african guidelines stipulate that these pathogens should not be present in ready-to-eat foods (department of health, 2002; beharielal et al., 2018), therefore, this could represent a public health threat. the low rate of detection of pathogens in samples purchased at retail could be proper handling and storage of the samples. storage temperature and storage period of fresh produce can influence the growth of bacteria (korir et al., 2016). 3.2. antimicrobial effects of aa and oeo on e. coli o157:h7 atcc 4388 at 5 and 22ºc generally, storage at 5ºc was more effective than 22 ºc and eoe showed higher log reductions at both storage temperatures than aa (table 2). however, there was no significant difference in log-reductions with an increase in exposure time from 2 to 5 min. furthermore, there was a complete inhibition of e. coli with 0.1% eoe at day 4. furthermore, the log-reduction increased with increase in storage days. similar result was observed by (poimenidou et al., 2016) who reported a 2.0-2.4 log cfu/g reduction of e. coli o157:h7 on lettuce samples rinsed with acetic acid. 3.3. antimicrobial effects of aa and oeo on l. monocytogenes atcc 7644 at 5 and 22ºc table 3 shows the antimicrobial effects of aa and oeo on l. monocytogenes. similar to the results observed for e. coli reductions, storage at 5ºc was seen to be more effective as compared to storage at 22ºc and a 2 min dip treatment at 0.1% aa yielded 1.54 log cfu/g but an increase in exposure time to 5 min was not significant (1.63 log cfu/g). however, an increase in treatment concentration showed 1.96 log cfu/g reduction at 2 min while 2.17 log cfu/g) was observed at 5 min. (table 4). in addition, there was a progressive logreduction as storage days increased. contrary to the behavior of e. coli, l. monocytogenes showed higher resistance towards treatment with acetic acid, particularly on samples stored at 22ºc. the difference of the pathogens susceptibility could be due to inherent properties of each organism, differences in outer layer (gram positive and negative) and nature of attachment of the pathogen to the lettuce leaf tissues. organic acids generally ital. j. food sci., vol. 32, 2020 404 have a low ph that prevents or inhibit bacterial growth. carella (2014) reported that gram negative bacteria are more susceptible to low ph treatments while essential oils more effective against gram positive bacteria. also, microbial inhibition depends on the concentration of treatment, contact time, mode of application and storage temperature. the limiting factors of effectiveness can be due to the total number and type of microorganisms they are introduced to and how these microbes interact with the acid (samara and koutsoumanis 2009). organic acids have been successfully used for the preservation of fresh fruits and vegetables during pre-harvest and post-harvest operations in the fresh produce industry (hirshfield et al., 2003). they are generally regarded as safe (gras) for use in food production and preservation. antimicrobial activity of organic acids (lactic, citric, acetic, and ascorbic acid) against e. coli and l. monocytogenes was compared on iceberg lettuce and the combination effect of lactic and acetic acid with chlorine to reduce l. monocytogenes on shredded lettuce has been evaluated (park et al., 2011). the individual efficacy of antimicrobials against e.coli o157:h7 and listeria monocytogenes has been reported in numerous studies (akbas and olmez 2007; samara and koutsoumanis 2009; huang and chen 2011; solgi and ghorbanpour 2014; de medeiros barbosa et al., 2016) but higher doses have very strong odours that may negatively affect sensory qualities of food (miyague et al., 2015), hence, the combination of preservatives serves a promising method to be able to achieve optimum pathogen inhibition without affecting the quality of food (nazer et al., 2005; miyague et al., 2015). 3.4. synergistic effect of oeo and aa on e. coli o157:h7 atcc 4388 and l. monocytogenes atcc 7644 table 4 shows the antimicrobial effects of artificially contaminated lettuce in a combined solution of oregano eo and acetic acid (0.1% oregano essential oil + 0.3% acetic acid). at 2 min dip treatment, reductions of 4.86 log cfu/g and 4.95 log cfu/g were observed in e. coli o157:h7 and l. monocytogenes atcc 7644 respectively. at 5 min dip treatment the reductions were not significantly different in e. coli and l. monocytogenes (5.04 log cfu/g, 5.12 log cfu/g). generally, higher log-reduction was observed at 5 ºc than 22 ºc. however, pathogens were not detected on day 4 and 6 storage period. the combined treatment reduced and inhibited the growth of both pathogens. at day 2, storage at temperatures 5 and 22 ºc (table 4), resulted in further reduction of e. coli o157:h7 and l. monocytogenes. the use of aa and oeo lead to a significant reduction of e. coli o157:h7 and l. monocytogenes on lettuce when used individually, more so, the combination of aa and oeo resulted in a further significant reduction of e. coli and o157:h7 l. monocytogenes. combined treatment with oeo and aa completely inhibited the growth of e. coli o157:h7 and l. monocytogenes at day 4. the combination of technologies with antimicrobial/preservative effects is called ‘hurdle technology’ and it has been used in the food industry to maintain food safety (nazer et al., 2005). some studies have reported that the use of essential oils in combination with each other as well as combination with other natural antimicrobials results in enhanced antimicrobial effectiveness as opposed to being used individually (dimitrijević et al., 2007). ital. j. food sci., vol. 32, 2020 405 table 2. comparative antimicrobial effect of oeo and aa on inactivation of e. coli o157:h7 on iceberg lettuce (log cfu/g) over a period of 6 days at 5ºc and 22°c. treatment at 5 ºc type of treatment day 0 day 2 day 4 day 6 2 min 5 min 2 min 5 min 2min 5min 2 min 5 min control 8.13±0.021a 7.27±0.044b 7.04±0.024b 7.25±0.023b aa @ 0.1% 5.39±0.031b 5.20±0.008b 5.11±0.028c 5.01±0.021c 4.36±0.015d 4.07±0.031d 3.98±0.025e 3.78±0.067e aa @ 0.3% 5.07±0.018b 5.02±0.020b 4.42±0.012d 4.19±0.014d 3.98±0.019e 3.71±0.030e 3.64±0.048e 3.40±0.084e oeo @ 0.05% 5.97±0.036b 5.37±0.012b 5.02±0.015c 4.37±0.012d 4.82±0.023d 4.07±0.018d 4.33±0.005d 3.83±0.022 oeo @ 0.1% 3.50±0.057c 3.22±0.056c 3.15±0.044e 2.98±0.082e nd nd nd nd treatment at 22 ºc control 8.13±0.020a 8.26±0.013a 8.46±0.012a 8.91±0.041a aa @ 0.1% 5.39±0.031b 5.20±0.008b 5.46±0.015c 5.41±0.015c 5.60±0.046c 5.51±0.028c 5.63±0.052c 5.47±0.021c aa @ 0.3% 5.07±0.018b 5.02±0.020b 5.04±0024c 4.77±0.047d 4.81±0.03d 4.50±0.057d 4.95±0.041d 4.63±0.057d oeo @ 0.05% 5.97±0.036b 5.37±0.012b 5.45±0.013c 5.09±0.021c 5.39±0.01c 5.06±0.019c 5.47±0.009c 5.23±0.013c oeo @ 0.1% 3.50±0.057c 3.22±0.056c 3.30±0.031e 3.09±0.025e nd nd nd nd means with same superscript letters in the same row are not significantly different (p > 0.05). results represented as means±standard deviation; means (n= 2); ndnot detected, oeooregano essential oil, aaacetic acid. ital. j. food sci., vol. 32, 2020 406 table 3. comparative antimicrobial effect of oeo and aa on inactivation of l. monocytogenes on iceberg lettuce (log cfu/g) over a period of 6 days at 5ºc and 22ºc. treatment at 5 ºc treatment day 0 day 2 day 4 day 6 2 min 5 min 2 min 5 min 2min 5min 2 min 5 min control 8.01±0.033a 7.24±0.021b 6.93±0.05b 7.07±0.018b aa @ 0.1% 6.47±0.013b 6.38±0.027b 6.22±0.022c 6.13±0.014c 6.06±0.037b 6.02±0.029b 6.33±0.011b 6.26±0.023b aa @ 0.3% 6.05±0.008b 5.84±0.031c 5.89±0.016d 5.76±0.022d 5.31±0.006c 5.19±0.01c 5.21±0.021c 4.99±0.031c oeo @ 0.05% 3.63±0.022d 3.25±0.034d 2.84±0.083 nd nd nd nd nd oeo @ 0.1% 3.06±0.027d 2.69±0.124e nd nd nd nd nd nd treatment at 22 ºc control 8.01±0.033a 8.349±0.021a 8.46±0.014a 9.23±0.032a aa @ 0.1% 6.47±0.013b 6.38±0.027b 6.41±0.015c 6.35±0.011c 6.45±0.016b 6.31±0.019b 6.45±0.029b 6.37±0.034b aa @ 0.3% 6.05±0.008b 5.84±0.031c 6.37±0.016c 6.25±0.008c 6.33±0.009b 6.16±0.015b 6.46±0.011b 6.26±0.016b oeo @ 0.05% 3.63±0.022d 3.25±0.034d 3.08±0.051d 2.59±0.15d nd nd nd nd oeo @ 0.1% 3.06±0.027d 2.69±0.124e nd nd nd nd nd nd means with same superscript letters in the same row are not significantly different (p > 0.05). results represented as means±standard deviation; means (n= 2); ndnot detected, oeooregano essential oil, aaacetic acid ital. j. food sci., vol. 32, 2020 407 the mechanism of action of both essential oils and organic acid involves the penetration of bacterial cell membranes and disrupting cytoplasmic functions which eventually lead to cell death, hence the combination of these antimicrobials would results in rapid and increased cell death at low concentrations and contact times (nazer et al., 2005). the addition of small amounts of different natural antimicrobials can play a role in balancing sensory attributes and antimicrobial efficacy of these compounds (zhou et al., 2007). table 4. antimicrobial effect of combined oeo and aa on inactivation of e. coli o157:h7 and l. monocytogenes on lettuce (log cfu/g) over a period of 6 days at 5 and 22ºc. storage day 0 day 2 day 4 day 6 2 min 5 min 2 min 5 min 2min 5min 2 min 5 min e. coli o157:h7 control at 5 ºc 8.13±0.021a 7.28±0.044b 7.00±0.024b 7.25±0.023c sample at 5 ºc 3.27±0.049b 3.09±0.074b 2.54±0.088c 1.00±1.411d nd nd nd nd control at 22 ºc 8.13±0.021a 8.26±0.013a 8.46±0.012a 8.91±0.042b sample at 22 ºc 3.27±0.049b 3.09±0.074b 2.50±0.281c 2.151±0.213c nd nd nd nd l. monocytogenes control at 5 ºc 8.01±0.021a 7.24±0.021b 6.93±0.05c 7.07±0.02c sample at 5 ºc 3.13±0.023b 2.89±0.077b 2.00±0.001c nd nd nd nd nd control at 22 ºc 8.01±0.021a 8.36±0.021a 8.46±0.014a 9.23±0.032a sample at 22 ºc 3.13±0.023b 2.89±0.077b 2.38±0.124c 2.00±0.001c nd nd nd nd results represented as means±standard deviation; means (n= 2); nd not detected. 4. conclusion oregano essential oil and acetic acid, used singly or combined, were effective against e. coli o157:h7 and l. monocytogenes on the fresh leafy vegetable. however, the efficacy of these antimicrobial agents vary with treatment concentration, exposure time and storage temperature. the combined application of 0.1% eoe and 0.3%aa was most effective and achieved a complete inhibition at 4th day at 5ºc storage. this result could serve as preliminary investigation in order to determine the best experimental conditions. research should be done towards evaluating the survival of other pathogens, potential virulence, compatibility to human diet and ready application as sanitizing solutions. acknowledgements this work was based on the research supported in part by the national research foundation (nrf) of south africa for the grant, unique grant no.: 98697. ital. j. food sci., vol. 32, 2020 408 references akbas m.y. and olmez h.o. 2007. inactivation of escherichia coli and listeria monocytogenes on iceberg lettuce by dip wash treatments with organic acids. letters in applied microbiology, 44:619-624. beharielal t., thamaga-chitja j. and schmidt s. 2018. pre-and post-harvest practices of smallholder farmers in rural kwazulu-natal, south africa: microbiological quality and potential market access implications. food control, 92:5362. callejón r.m., rodríguez-naranjo m.i., ubeda c., hornedo-ortega r., garcia-parrilla m.c. and troncoso a.m. 2015. reported foodborne outbreaks due to fresh produce in the united states and european union: trends and causes. foodborne pathogens and disease, 12:32-38. carella l. 2014. use of oregano essential oil and acetic acid to reduce salmonella contamination on romaine lettuce. doctoral dissertation, university of georgia de 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del valle c. and roura s.i. 2004. shelf life of leafy vegetables treated with essential oils. journal of food science, 69. ital. j. food sci., vol. 32, 2020 409 raeisi, m., tajik, h., yarahmadi a. and sanginabadi s. 2015. antimicrobial effect of cinnamon essential oil against escherichia coli and staphylococcus aureus. health scope, 4. samara a. and koutsoumanis k.p. 2009. effect of treating lettuce surfaces with acidulants on the behaivour of listeria monocytogenes during storage at 5ºc and 20 ºc and subsequent exposure to simulated gastric fluid. international journal of food microbiology, 129:1-7. singh n., singh r.k., bhunia a.k. and stroshine r.l. 2002. effect of inoculation and washing methods on the efficacy of different sanitizers against escherichia coli o157:h7 on lettuce. food microbiology, 19:183-193. solgi m. and ghorbanpour m. 2014. application of essential oils and their biological effects on extending the shelflife and quality of horticultural crops. trakia journal of sciences, 12:198-210. topalić-trivunović l., savić a., kovačević j., balešević l., matoš s. and šolaja,m. 2014. the microbiological status of (ready to eat) lettuce before and after standard washing. hemičara, tehnologa i ekologa, 10:51-56. wiederoder m.s., lefcourt a.m., kim m.s. and lo m.y. 2012. detection of freshcut produce processing residues on food contact surface materials using hyperspectral imaging. journal of food measurement and characterization, 6:48-55. zhou f., ji b., zhang h., jiang h., yang z., li j., li j., ren y. and yan w. 2007. synergistic effect of thymol and carvacrol combined with chelators and organic acids against salmonella typhimurium. journal of food protection, 70:1704-1709. paper received april 1, 2019 accepted january 16, 2020 ijfs#1694_bozza ital. j. food sci., vol. 32, 2020 209 paper effect of coconut testa flour on cookie characteristics j.m.n. marikkar*a, r. nagarajab, k.m.s. somawathieb, h.p.t.d. hewapathiranac, c. yalegamac, p. littardid and e. chiavaro*d afood chemistry laboratory, national institute of fundamental studies hanthana road, kandy, sri lanka bdepartment of food science & technology, sabaragamuwa university of sri lanka, belihuloya, sri lanka ccoconut research institute, lunuwila, sri lanka ddepartment of food and drug, parco area delle scienze 47/a, university of parma, 43124 parma, italy *corresponding author: nazrim.ma@nifs.ac.lk; emma.chiavaro@unipr.it abstract a study was conducted to assess the effect of coconut testa flour (ctf) on quality characteristics of cookies by varying the proportion of ctf in wheat flour from 10 to 60% (w/w). cookies samples prepared according to a standard recipe were evaluated by proximate composition, hardness, amylose content, and shelf-life stability. a 30 members sensory panel was employed to determine the critical limit of ctf fortification for acceptable quality cookies. results showed that ctf substitution up to 30% was possible without affecting the overall acceptability of cookies. keeping quality of the cookies remains within the acceptable range throughout three-month storage period. keywords: coconut testa flour, cookies, functional properties, storability, wheat flour ital. j. food sci., vol. 32, 2020 210 1. introduction proper use of agriculture byproducts is an integral part of the sustainable agriculture development. the trend in the world today is to convert byproducts into useful products through the manipulation of microorganisms or recycle them as much as possible. coconut testa, for instance, is a byproduct generated on a daily basis by coconut processing industries such as desiccated coconut, virgin coconut oil and milk powder. previous studies showed that coconut testa constitutes approximately 18% (w/w, wet basis) of the total weight of the whole coconut endosperm (marikkar and madurapperuma, 2012). by using the statistics provided by perera et al (2014), it can be estimated that around 30,000 kg of coconut testa could be generated out of 100,000 nuts of sri lankan tall variety. as the removed testa is either wasted or under-utilized by the processing factories without any value addition, it is timely to undertake research studies to use them in food applications. this might have various direct and indirect environmental and economic impacts reducing the disposal costs and increasing the added value of the final products, giving emphasis to explore their health benefits. recently, adekola et al (2017) suggested that testa of coconut can be used to produce a flour to supplement wheat flour to prepare foods such as cookies with low-glycemic index. according to klunklin and savage (2018), cookies are hardly regarded as healthy snack because of their high levels of rapidly digestible carbohydrate, high fat content, low levels of fiber and only modest amounts of protein. nevertheless, the nutritional value of these product is considered low due to the nutritional composition of refined wheat flour composition. in this background, in recent years, a large variety of vegetable flour products derived from legumes, oilseed, tubers, corn, rice, etc., have been evaluated in cookies formulation in the attempt to produce nutritionally fiber and/or protein enriched products (zucco et al., 2011; chung et al., 2014; paciulli et al., 2018; diaz et al., 2019). as of date, supplementation of wheat flour with defatted coconut testa flour in cookie formulation has been scantily investigated. hence, in this study, the effect of partial substitution of wheat flour by coconut testa flour on the proximate composition, storage stability and consumer acceptance of cookies has been investigated. 2. materials and methods 2.1. materials wheat flour, coconut testa flour, salted butter, icing sugar, fresh milk, potable water and soybean oil, baking powder, distilled water were used for cookie dough preparation. samples of commercial wheat flour were obtained from serendib flour mills (pvt) ltd and samples of partially-defatted coconut testa flour (ctf) of sri lankan commercial variety were supplied as a generous gift by the coconut research institute, lunuwila, sri lanka. other ingredients namely, table salt, fresh milk, soybean oil, and baking powder were obtained from puttlam salt ltd, ambewela milk ltd, lanka soy (pvt) ltd and cargills food city (pvt) ltd, respectively. ital. j. food sci., vol. 32, 2020 211 2.2. chemicals agar culture media, potato dextrose agar were obtained from himedia (pennsylvania, usa); petroleum ether, concentrated sulfuric acid, potato starch, sodium hydroxide , hydrochloric acid, boric acid were purchased from merck (darmstadt, germany); dimethyl sulfoxide, 3,5-dinitrosalicylic acid, sodium potassium tatrate, bromocresol, methyl red, ethanol, methanol, iodine reagent, deionized water, sodium chloride, monosodium phosphate and disodium phosphate were obtained from fluka (buchs, switzerland). 2.3. functional properties of flours 2.3.1 water and oil absorption capacity the water and oil absorption capacities were determined by the method of sosulski et al. (1976). flour samples (one gram, in triplicate) were mixed with 10 ml distilled water and 10 ml of soybean oil separately in 2 centrifuge tubes. samples were kept at room temperature for 30 min and centrifuged with eppendorf 8510r centrifuge (eppendorf, germany) at 3000 rpm for 30min. the volume of free water and free oil were read directly from the centrifuge tube. the water absorption and oil absorption were examined as percent water-bound per gram of flour and percent oil-bound per gram of flour, respectively. 2.3.2 bulk density bulk density of flour sample was examined using the method described by jones et al. (2000) with slight modifications. flour samples (ten grams, in triplicate) were measured in a 50 ml measuring cylinder. the cylinder was tapped on a wooden plank until no visible decrease in volume was noticed and volume of flour sample was recorded. the bulk density was calculated using the following formula: bulk density (g/cc) = weight of sample / volume of sample 2.3.3 swelling capacity the method of okaka and potter (1977) with some modifications was used for determining the swelling capacity. flour samples (one gram, in triplicate) were filled up to 10 ml mark in a 100 ml graduated cylinder was added with water to adjust total volume to 50 ml. the top of the cylinder was tightly covered and mixed by inverting the cylinder. the suspension was inverted again after 2 min and allowed to stand for further 30 min. the volume occupied by the sample was taken after 30 min. 2.3.4 emulsion activity the emulsion activity and stability were followed using the method of yasumatsu et al. (1972). emulsion was prepared in calibrated centrifuged tube by adding flour samples (one gram, in triplicate) into 10 ml distilled water and 10ml of soybean oil. the emulsion was centrifuged at 3000 rpm for 5 min. the emulsion activity was estimated using following equation: emulsion activity (%) = (height of emulsion layer/ total height of mixture) ×100 ital. j. food sci., vol. 32, 2020 212 2.3.5 least gelation concentration least gelation concentration was evaluated using the method of coffman and garcia (1977) with slight modification. flour samples [2 to 30% (w/v), in triplicate] were mixed with 5 ml distilled water and heated at 90°c for 1 h in a hot-water bath. after allowing it to cool under tap water, it was kept at 4 °c for 2 hrs. the least gelation concentration was determined at the concentration when the sample from inverted tube did not slip. 2.4. composite flour preparation composite flour samples were prepared according to the following mixing ratio: t0, 100% wf; t1, wf supplemented with 10% ctf; t2, wf supplemented with 20% ctf; t3, wf supplemented with 30% ctf; t4, wf supplemented with 40% ctf; t5, wf supplemented with 50% ctf; t6, wf supplemented with 60% ctf. 2.5. cookies preparation formulation of cookies was carried out according to a method described in aacc with slight modification (sciarini et al., 2013). cookies were baked in three batches, each with 60 cookies. initially, salted butter (30g) was creamed with icing sugar (40 g), and baking powder (2 g) for 3 min at low speed in a mixer (the bowl will be scraped every minute). water was added thereafter in varying proportions to make the required consistency of dough. mixing was continued using kenwood mixer (model km200, korea) for 2 min at a high speed. finally, 100g of flour was added and mixed for 2 min at low speed while scraping the bowl at every 30 sec. the dough was allowed to rest for 10 min before rollingon and cutting (62 mm diameter, 5.5 mm thick). the cut dough pieces were then baked for 25 min in an oven (biobase forced air drying oven, china) pre-heated at 150 °c. the cooled cookies were then left at 25 °c for 30 min and packed in sealed plastic bags until further analysis. 2.6. sensory evaluation this is a ranking test, which is aimed to rank cookie samples according to the preference of a thirty-member semi-trained panelists. panelists were asked to rank seven coded samples according to the intensity of sensory attributes namely color, smell, taste, texture and overall acceptability of biscuits. a seven-point hedonic scale (1: dislike very much; 2: dislike; 3: dislike slightly; 4: neither like nor dislike; 5: like slightly; 6: like; 7: like very much) was used to evaluate degree of liking for each sensory attribute. 2.7. proximate compositional analysis for sampling under each treatment category, 7 cookies were selected randomly from each of the three batches. moisture, crude fat, crude protein and ash contents of coconut testa flour were determined according to methods described in aoac (2005) manual. the carbohydrate content of the flour was calculated by difference [100(crude protein + crude fat + ash + moisture+ crude fiber)]. ital. j. food sci., vol. 32, 2020 213 2.8. determination of amylose content amylose content was determined according to the iodimetric method described by juliano et al. (1968) with slight modification. iodine reagent was prepared dissolving 1.0 g of iodine and 10 g potassium iodide in water and the volume was made up to 500 ml. a series of standard amylose solutions was prepared dissolving appropriate amounts of amylose in 10 ml of 1.0 n solution of sodium hydroxide and made up to100 ml with water. powdered cookie sample of 100 mg each from t0, t1, t2, and t3 was weighed accurately, and dissolved in 1.0 ml 95% ethanol and 10 ml of 1.0 n sodium hydroxide and heated for 10 min in boiling water bath. the solution was made up to 100 ml in a volumetric flask. an aliquot (2.5 ml) of this solution was then added with 20 ml distilled water and three drops of phenolphthalein. subsequently, 0.1 n hydrochloric was added dropwise until the pink color just disappear. an aliquot (1 ml) of iodine reagent was added into this and the volume was made up to 50 ml. the resulting color was left for 20 min to fully develop before the absorbance reading was monitored at 620 nm with a perkin-elmer lambda 3b double beam uv/visible spectrophotometer. iodine solution of same concentration as above but without amylose was used as reference cell. concentration of amylose in individual cookie sample was calculated using the standard curve. 2.9. hardness test for sampling under each treatment category, three cookies were selected randomly from each of the three batches. hardness of all samples was carried out using brookfield ct3 texture analyzer using the following test criteria was applied in this test. test type: tpa; target: 4.0mm; hold time: 0s; trigger load: 15.0g; test speed: 0.50mm/s; return speed: 0.5mm/s. the value for hardness was derived from the automatic calculation from texture profile analysis curve. 2.10. shelf life studies for storability test, biscuit samples containing 5 cookies were packed under hygienic condition in clean packages and sealed immediately to protect product quality. lowdensity polyethylene (ldpe) bags were used for packaging. during shelf life study, sample bags were kept at room temperature (27°c and rh 60%) until all analyses were performed. the shelf life of cookies was evaluated based on moisture content and the microbial count (total plate count and yeast and mould count) at once in every two weeks’ time continuously for three months. 2.11. microbiological analysis enumeration of aerobic colony count was done by incubating microorganisms in nutrient agar (na) medium under 37°c for 48 hours; the yeast and mould count was done by incubating in potato dextrose agar (pda) medium with 0.01% chloramphenicol held at room temperature (sls 516:1991). 2.12. statistical analysis samples were selected randomly from three batches. all measurements were carried out in triplicate data (n=3). all the results were presented in the form of mean ± standard ital. j. food sci., vol. 32, 2020 214 deviation (sd). data were statistically analyzed by one-way analysis of variance (anova) with using tukey’s test of minitab (version 15) statistical package at 0.05 probability level. results of sensory evaluation results were analyzed using nonparametric friedman rank sum test using the minitab software package version 14.00 (sas 1998). 3. results and discussion 3.1. functional properties of flours the functional properties of cereal and non-cereal flours are known to influence the quality attributes of finished bakery products. a comparative analysis of the physicochemical and functional properties of wheat and coconut testa flours would therefore be beneficial. according to the data presented in table 1, both of these displayed significant (p<0.05) differences in all of the properties studied. the swelling capacity, which is related to the irregular positioning of polymer in granules and small size of starch granules (wong and lelievre, 1982) was higher for ctf (30±0.01) than wf (20±0.01). suresh and samsher (2013) stated that varietal differences, particle size differences, and methods of processing are other factors that may influence the swelling capacity of flour. in an earlier report, chandra et al. (2015) noticed that the swelling capacity of composite flours increased with increasing level of incorporation of rice, green gram and potato flour into wheat flour. from the results of this study, it can be predicted that a composite flour made by mixing wf with ctf would gain a higher swelling capacity with reference to the control. according to table 1, the bulk density of ctf (0.66±0.01) was slightly higher than that of wf (0.49±0.01). the bulk density (g/cm3) of flour is defined as the density measured without the influence of any compression (chandra et al., 2015). according to lam et al. (2008), bulk density of flour samples depends on particle size, density of individual particle, moisture as well as surface characteristics, which are generally influenced by the method of preparation. undoubtedly, the preparation methods of ctf and wf were not similar as one which was cereal based while the other was non-cereal-based. according to suresh and samsher (2013), the high bulk density of flour suggests its suitability for multiple uses in food preparations. water absorption capacity of wf and ctf were 65±0.0% and 75±0.0%, respectively. berton et al. (2002) stated that water absorption capacity is associated with protein and starch contents of flour. however, the particle size reduction and high content of fiber would increase the water absorption capacity of flour (suresh and samsher, 2013; chandrasheker and desikachar, 1984). our preliminary investigation too confirmed that ctf was a rich source of protein and crude fiber, which were relatively higher than those of wheat flour (table 1). as the capacity of protein to enhance the formation and stabilization of emulsions is important for many applications in food products, information on emulsion activity of flour is beneficial (cauvain and young, 2006). in the present study, emulsion activity of ctf was lower (25±0.00) than wf (50±0.00). according to previous researchers, the emulsion activity of flour would increase with higher amounts of soluble proteins (gabra and kaur, 2014; yasumatsu et al., 1972) and reduce with fiber content (yasumatsu et al., 1972). the higher crude fiber content as noticed before in ctf could be a probable reason for lower emulsion activity displayed by ctf. when considering least gelation concentration of the two flour samples, wf showed lower amount (8%, ital. j. food sci., vol. 32, 2020 215 w/v) while ctf displayed a higher value (18%, w/v). this means that wf would form gel quickly at lower concentration than ctf. in an earlier report, akintayo et al. (1999) stated that lowest gelation concentration increased with the gelation of protein. table 1. comparative functional and nutritional properties of coconut testa flour (ctf) and wheat flour (wf). functional/nutritional properties flour ctf wf swelling capacity (ml) 30b±0.01 20a±0.01 water absorption capacity (%) 75b±0.00 65a±0.00 oil absorption capacity (%) 52.7a±2.52 58.50b±1.32 emulsion activity (%) 25a±0.00 50b±0.00 least gelation concentration(%,w/v) 18b±0.00 8a±0.00 bulk density(g/cc) 0.66b±0.01 0.49a±0.01 moisture (%) 2.27±0.42* 14.0±0.03ז ash (%) 4.50±0.14* 1.82±0.29 ז protein (%) 23.82±0.99* 11.68±0.11 ז fat (%) 10.17±1.84* 1.91±0.09 ז total carbohydrate (by difference) 59.24* 70.59 ז each data in the table represents mean of triplicate analysis. means in the same raw bearing different superscripts are significantly different (p<0.05) from each other. *marasinghe et al. (2019) and זmajzoobi et al. (2011). the oil absorption capacity is an indicator of the suitability of the flour in facilitating enhancement of flavor and mouth feel in food preparations. the oil absorption capacity was higher for wf (58.50±1.32) than ctf (52.7±2.52). according to chandra et al. (2015), the presence of high fat content in flours might affect adversely their oil absorption capacity. in this study, ctf having a lower oil absorption capacity was reasonable because our earlier investigation showed that the fat content of ctf was relatively higher (10.17 %) than those of wf (table 1) (maizoobi et al., 2011). suresh and samsher (2013) commented that amino acid composition, protein conformation, surface polarity and hydrophobicity are other factors that may exert influence on the oil-binding capacity of flour. 3.2. sensory attribute evaluation flour is the main ingredient in any biscuit formulations. there have been a number of attempts to improve the nutritional qualities of biscuits by partially substituting wf with non-wheat flour ingredients (klunklin and savage, 2018). replacing wf with other types of flour would most likely affect the nutritional values as well as the organoleptic characteristics of biscuits (hooda and jood, 2005). in order to determine the critical limit of fortification of ctf in biscuit formulation, a proper method of sensory evaluation ital. j. food sci., vol. 32, 2020 216 and statistics are necessary. according to table 2, the preference of the panelists for color attribute sharply dropped after substitution of 10% ctf due to unappealing off-white grainy appearance; but the score values for t2 and above were tended to increase due to brown color development caused by higher amounts of ctf. apart from maillard reaction, the mild brown color of ctf could also be a contributory factor for brown color development in biscuits. this has caused the score values of colour to go up with further substitution. there was, however, no significant difference between the control and the different levels of ctf substitution with respect to smell characteristics (table 2). other attributes such as taste, texture, and overall acceptability were more sensitive to the changing content of ctf in the formulation. with regard to these three sensory attributes, there was no significant difference between the control and the ctf substituted biscuit samples up to the level of 30% substitution (table 2). according to the panelists, a slight bitter taste and undesirable mouth-feel were noticed in biscuit samples exceeding 30% ctf level. therefore, ctf substitution up to 30% would be maximum permissible limit for good quality cookies. hence, the proximate compositional analyses and storage studies were performed only for biscuit samples made out of composite flour containing 0, 10, 20 and 30% ctf supplementation. table 2. results of friedman test along with sum of ranks of sensory attributes of different treatments. treatments color smell taste texture overall acceptability t0 158.0 e 128.0a 152.0c 161.0c 166.5c t1 86.5 a 126.5a 153.5c 156.5c 151.0c t2 103.5 b 107.5a 136.5c 150.0c 148.0c t3 110.0 b 112.0a 141.5c 139.5c 132.5bc t4 123.0 c 116.5a 101.0b 105.5b 111.5b t5 126.5 c 116.5a 78.5a 67.5a 77.0a t6 132.0 d 133.0a 77.0a 60.0a 53.5a cl *** ns *** *** *** means in the same column bearing different superscripts are significantly different from each other. abbreviations: cl, confidence level; ns, not significant; ***p<0.0001. t0, biscuit formulation by wheat flour; t1, biscuit formulation by wheat flour mixed with 10% coconut testa flour; t2, biscuit formulation by wheat flour mixed with 20% coconut testa flour; t3, biscuit formulation by wheat mixed with 30% coconut testa flour; t4, biscuit formulation by wheat flour mixed with 40% coconut testa flour; t5, biscuit formulation by wheat flour mixed with 50% coconut testa flour; t4, biscuit formulation by wheat flour mixed with 60% coconut testa flour. 3.3. proximate composition of biscuits proximate compositions of cookies samples displaying good overall acceptability were compared with those of control sample as shown in table 3. generally, cookies available in the market are generally prepared from wheat flour, which lacks dietary fibre contents and good quality protein due to deficiency in lysine. our preliminary investigation confirmed that composite flour preparation using ctf would be beneficial because ctf ital. j. food sci., vol. 32, 2020 217 was found to be rich in protein (table 1) as well as crude fiber (table 3). in a previous study, appaiah et al. (2014) also indicated that testa of copra was a rich source of protein and crude fiber; it contained about 9.3% of protein and 11.6 % of crude fiber. when compared with the control, cookies prepared with composite flour showed significant increase (p<0.001) on the moisture, protein and crude fiber contents, but with regard to ash and fat contents of cookies, there was no significant (p>0.05) difference among all substitution levels (table 3). this indicated that substitution of wf with ctf did not have any direct impact on both ash and fat contents of the finished products. as previously noticed in table 1, water absorption capacity of ctf was higher than wf due to high fiber content of ctf. this necessitated an extra amount of water to be added to maintain better consistency of the dough at higher level of substitution of ctf. with regard to total carbohydrate content of cookies, there was a significant (p<0.001) decline along with the rising substitution level of ctf. table 3. proximate composition of cookie samples made out of different treatments (g/ 100 g dry matter basis). treatment moisture ash protein fat crude fiber other carbohydrates (by difference) t0 0.79 a±0.04 1.11a±0.15 5.59a±0.50 14.28a±0.41 7.90a±0.14 70.32d± 0.12 t1 0.95 b±0.04 1.17a±0.23 7.43b±0.15 14.59a±0.23 8.95b±0.07 66.91c±0.58 t2 1.24 c±0.02 1.33a±0.00 9.63c±0.18 14.99a±0.47 11.25c±0.5 61.56b±0.75 t3 1.48 d±0.02 1.50a±0.23 11.79d±0.60 15.54a±0.16 15.85d±0.1 53.84a±0.33 cl *** ns *** ns *** *** each data in the table represents mean of triplicate analysis. means in the same column bearing different letters are significantly different from each other. abbreviations: ns, not significant; ***(p<0.0001). for other abbreviations, see table 2. 3.4. amylose content and hardness the data presented in table 4 compares the amylose content of ctf substituted cookie samples with that of the control sample. amylose being a linear polymer contribute important characteristics to starch present in cereals, and hence it can affect the physicochemical properties of the finished products (majzoobi et al., 2011). the control showed the highest amylose content (27.4 %), which fell within the range of amylose content of wheat flour of different cultivars (20 to 30 %) reported in other studies (majzoobi et al., 2011). the amylose contents of t1, t2, and t3 were tended to decline with the decrease of wheat flour component in the composite. in terms of texture, cookies made out of 100% wheat flour was seemed to be the hardest (14.86 n) and the hardness of t1, t2, and t3 were found to decline, with the cookie sample t3 which represented ctf substituted cookies at 30% level displaying the lowest value of 8.27 n. generally, the hardness of cookie is strongly influenced by the development of gluten network whereby gluten promotes the network development by attracting water molecules from the mixture. barak et al. (2013) previously pointed out that wf having a higher gluten ital. j. food sci., vol. 32, 2020 218 content would influence the hardness of cookies. the composite flour made out of ctf substitution would contain lesser amount of gluten, which leads to the decline in the harness of the cookies. table 4. variation of amylose content and harness of cookies1. treatment amylose content (%) hardness (n) t0 27.40 c±0.79 14.86d±0.01 t1 22.77 b±0.50 13.42c±0.02 t2 16.50 a±0.75 11.63b±0.01 t3 15.10 a±0.26 8.27a±0.01 1each data in the table represents mean of triplicate analysis. means in the same column bearing different superscripts are significantly different (p<0.05) from each other. for abbreviations, see table 2. 3.5. shelf-life stability moisture and microbiological stability are important attributes to test the shelf-life stability of products. when considering keeping quality of food materials, the initial moisture content, storage temperature, microbiological quality and packaging materials are important determining factors. in this study, keeping quality of cookies samples are tested in terms of moisture content and microbial growth along the storage period of three months. generally, moisture absorption would not only influence microbiological stability but also textural properties such as hardness. cauvain and young (2006) stated that moisture content of freshly produced cookies should not exceed the limit of 5% while other standards specified that the moisture content of cookies should be less than 6% during storage period (sls 251:1991; bis 1974). according to data presented in table 5, moisture content of samples significantly increased with storage period but none of them exceeded the upper limit of moisture content throughout the three months of storage period. table 5. changes in the moisture content of cookie samples with time. treatment time (in weeks) w0 w2 w4 w6 w8 w10 w12 t0 0.79 a±0.04 0.88a±0.04 1.04b±0.06 1.07b±0.05 1.88c±0.05 2.17d±0.04 2.98e±0.09 t1 0.94 a±0.04 1.22b±0.03 1.35c±0.04 1.49c±0.01 2.07d±0.1 2.59e±0.04 3.20f±0.03 t2 1.25 a±0.03 1.40b±0.02 1.57b±0.01 1.87c±0.05 2.43d±0.08 3.11e±0.09 3.41f±0.07 t3 1.47 a±0.03 1.65b±0.02 1.60b±0.03 2.22c±0.16 2.74d±0.06 3.34e±0.08 3.97f±0.05 each value in the table represents the mean of triplicate analyses. means in the same row bearing different superscripts are significantly (p<0.05) different from each other. abbreviations: w0, week0; w2, week2; w4, week4; w6, week6; w8, week8; w10, week10; w12, week12. for abbreviations, see table 2. ital. j. food sci., vol. 32, 2020 219 the observed gain in moisture content might be due to high water permeability of ldpe as well as the high-humidity conditions of the storage. previously, siripatrawan (2009) stated that there was a moisture migration between the atmosphere and the packaged low‐moisture food product such as rice crackers through packaging material during storage until the food reaches equilibrium with the environment. he noticed that the moisture barrier performance of packaging materials was dependent on permeability coefficient; rice crackers packaged in polypropylene pouches had higher shelf‐life values than those in polyethylene pouches because polypropylene had lower permeability coefficient. in this study, the changes in total plate count and yeast and mold count through the threemonth storage period were compared with initial amount as shown in table 6. according to who standard (who, 1994), total plate count and yeast and mould count of biscuit samples should not exceed 2.0×105 cfu g-1 and 1.0×104 cfu g-1, respectively. although both microbial counts of all samples were found to increase with storage period, none of them exceeded the upper limit along the three-month storage period. this clearly showed that microbiological quality of cookies remains within the safe limit throughout the threemonth storage period. table 6. changes in total plate count, yeast and mold count of cookies samples with time. treatment time (in weeks) w0 w4 w8 w12 total plate count (cfu/g) t0 (1.30 a±0.07)×103 (1.42a±0.09)×103 (2.08b±0.03)×103 (2.53c±0.05)×103 t1 (1.47 a±0.04)×103 (1.57a±0.05)×103 (2.18b±0.03)×103 (2.97c±0.06)×103 t2 (1.57 a±0.05)×103 (1.70a±0.02)×103 (2.46b±0.04)×103 (4.46c±0.15)×103 t3 (1.67 a±0.05)×103 (2.05b±0.04)×103 (2.97c±0.04)×103 (5.53d±0.15)×103 yeast and mould count (cfu/g) t0 (2.23 a±0.2)×102 (2.67ab±0.15)×102 (3.13b±0.12)×102 (6.27c±0.25)×102 t1 (2.30 a±0.26)×102 (3.03b±0.15)×102 (3.50b±0.10)×102 (7.50c±0.30)×102 t2 (2.70 a±0.10)×102 (3.40b±0.10)×102 (4.30c±0.41)×102 (8.30d±0.25)×102 t3 (3.03 a±0.15)×102 (4.03b±0.20)×102 (5.23c±0.20)×102 (9.10d±0.10)×102 each value in the table represents the mean of triplicate analyses. 1means in the same row bearing different superscripts are significantly (p<0.05) different from each other. abbreviations: w0, week0; w4, week4; w8, week8; w12, week12; for other abbreviations see table 2. 4. conclusions this study attempted to evaluate the suitability of ctf as a partial substitute for wf in cookies formulation. sensory evaluation showed that ctf substitution up to 30% was possible without affecting the overall acceptability of cookies. according to proximate analysis, cookies partially substituted with ctf could have enhanced levels of protein and crude fiber contents. the effect of substation on hardness of cookies was positively ital. j. food sci., vol. 32, 2020 220 correlated with amylose content of composite flour; this could also be due to lowering of gluten network formation, which usually brings hardness to cookies. shelf-life studies showed that moisture content and microbiological quality parameters of cookies would remain within the safe limit throughout the three-month storage period. the findings of this study could eventually help enhance the economic value of coconut testa generated as byproduct by the coconut processing industries in sri lanka. acknowledgements partial funding of this study by the national institute of fundamental studies, sri lanka is gratefully acknowledged. references adekola, k.a., salleh, a.b., zaidan, u.h., azlan, a., chiavaro, e., paciulli, m. and marikkar, j.m.n. 2017. total phenolic content, antioxidative and antidiabetic properties of coconut (cocos nucifera l.) testa and selected bean seed coats. ital. j. food sci. 29:741. akintayo, e.t., oshodi, a.a. and esuoso, k.o. 1999. effects of nacl, ionic strength and ph on the foaming and gelation of pigeon pea (cajanus cajan) protein concentrates. food chem. 66:51. aoac international 2005. “official methods of analysis of aoac international”, 18th edn. aoac international, washington dc. appaiah, p., sunil, l., kumar, p.p. and krishna, a.g. 2014. composition of coconut testa, coconut kernel and its oil. j. am. oil chem. soc. 91:917. barak, s., mudgil, d. and singh khatkar, b. 2013. effect of composition of gluten proteins and dough rheological properties on the cookie-making quality. brit. food j. 115:564. berton, b., scher, j., villieras, f. and hardy, j. 2002. measurement of hydration capacity of wheat flour: influence of composition and physical characteristics. powder technol. 128:326. bis 1974. “specification for protein-rich biscuits” is 7487. new delhi, bureau of indian standards. cauvain s. and young l. 2006. ingredients and their influences. in: “baked products: science, technology and practice”. cauvain s. and young l. 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polyphenol profiles and antioxidant activity in italian apples varieties a.m. tarola*1, a.m. girelli2 and f. d’ascenzo3 1laboratory of commodities sciences, department of management, sapienza university of rome, via del castro laurenziano 9, 00161 rome, italy 2department of chemistry, sapienza university of rome, piazzale aldo moro 5, 00185 rome, italy 3department of management, sapienza university of rome, via del castro laurenziano 9, 00161 rome, italy *corresponding author: tel.: +39 0649766312 e-mail address: annamaria.tarola@uniroma1.it abstract in this study ten organic apple varieties grown in italy: renetta osiris, gold rush, braeburn, celato cola, limoncella, cerina, rosada, topaz, jonagored, florina were analysed in order to evaluate some quality parameters. individual phenolics compounds, total phenolics, glucose, fructose and antioxidant activity was determined in pulp and peel extracts. results show that the chlorogenic acid was a predominant component in pulp and peel extracts, with highest value in limoncella and jonagored respectively. a significant correlation between phenolic composition and antioxidant activity was observed. apple varieties renetta osiris and gold rush presented significant values on glucose and fructose in pulp. keywords: apple fruit, dpph, hplc, phytochemicals ital. j. food sci., vol. 31, 2019 244 1. introduction apple (malus domestica) is one of the most ancient fruits. it was born in minor asia, south of black sea and arrived in greece from egypt, where it was grown along nyle valleys (xiii century b.c.). afterwards, it quickly spread all over europe and during the xvi century it appeared in north america (forsline et al., 2003; coart et al., 2006). it is currently the second fruit produced and consumed all over the world (70 million tons). italy produces 1.9 millions tons of apples a year and is the fifth largest producer worldwide and the third largest exporter in the world after china and poland. italy, along with india, is one of the major consumers of apples per capita, each person eats an average of 15 pounds of apples per year (two a week). in germany 10, in france 8, in the united states only 5 (faostat, 2013). apples are grown all over the italian territory, but production is traditionally concentrated in the mountain and foothill regions, particularly in valle d'aosta, piemonte, veneto and trentino-alto adige regions. in the past, the old varieties of fruits locally grown were quickly abandoned accordingly to a higher rationality of production with the choice of more productive varieties. in 1920, about 50 varieties of apples were marketed but they were reduced to 10 today with a loss of 80%. during the twentieth century the development of the sector has grown in connection with the increase of production, using particular attention to the territory and natural cultivation techniques with a low environmental impact. currently, a particular attention is dedicated to the improvement of biodiversity and to the organic production (bertshinger et al., 2004,; raganold et al., 2001). the quality of apple fruits depends on the bioactive compounds: polyphenols, organic acids and carbohydrates. the concentration of the compounds differs with varieties, maturity stage and environmental conditions (eisele and drake, 2005). phenolic substances are considered important in nutrition because of the positive effect on human health as a result of their high antioxidant capacity (serra et al. 2012, eberhard et al., 2000). epidemiologic studies associate the biological activity of these substances to the prevention of cancer and cardiovascular diseases, neuropathies and diabetes (ribeiro et al., 2014; boyer et al., 2004; balasuriya et al., 2012; scalbert et al., 2000). the aim of this study is to evaluate the composition of different apples varieties, produced by organic methods representative of italian biodiversity. the variability on the composition of bioactive compounds: individual phenolic compounds, total phenolics, fructose and glucose in pulp and peel tissue was determined. the influence of the total phenolic compounds on the antioxidant activity was evaluated. 2. material and methods 2.1. standard and reagents the standard compounds gallic acid, (+) catechin, (-) epicatechin, chlorogenic acid, phloretin, rutin, 1,1-diphenyl-2-picryl-hydrazyl (dpph), formic acid, folin-ciocalteau reagent, sodium carbonate, trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2carboxylic acid) were purchased from sigma-aldrich (mo usa). methanol of hplc grade and hydrochloric acid were purchased from merck (germany). the water used was purified in a milli-q system (millipore, bedford, ma, usa). the extracts samples and standard solution were filtered through membrane 0.45 µm (millipore bedford, ma, usa). ital. j. food sci., vol. 31, 2019 245 2.2. apple fruit samples apples samples from the varieties: renetta osiris (trentino alto adige), gold rush (toscana), braeburn (toscana), celato cola (sicilia), limoncella (abruzzo), cerina (lazio), rosada (lazio), topaz (trentino alto adige), jonagored (trentino alto adige), florina (trentino alto adige), were purchased from local organic producers in different italian regions during the period september-october 2015. the analysis was conducted within two days. 2.3. samples preparation and procedure the apple samples of peel and pulp have been separated and the pulp (5 g) and the peel (5 g) fractions were immediately homogenized with methanol containing 1% hydrochloric acid. the samples have been extracted with 10 ml of solvent for 30 min and 10 ml for 30 min using a ultrasonic sonicator at room temperature (25°c). the extract, pulp and peel separately, were combined and filtered through a membrane 0,45 µm pore size prior to injection in hplc for determining the individual phenolic compounds, antioxidant activity and total phenolics. extracts were stored at -4°c for 1 day. later at -20°c for 1 week. the recovery efficiency of phenolic compounds was determined by spiking amounts of standards compounds considered to the fraction pulp and peel of braeburn apple prior to extraction. the recovery study was performed in triplicate. 2.4. apparatus hplc analyses were performed on a shimadzu hplc system, a lc-10at liquid chromatograph equipped with four pumps fcv-10al, a degasser dgu-14a, a rheodyne 7725i injector with a 20 µl sample loop (rheodyne, berkeley, ca, usa) and a photodiode array detector spd-m20a. the eluted compounds were monitored at 280 nm and the adsorption spectra between 250 and 350 nm. spectrophotometric determinations were performed with a shimadzu uv-vis 1800 spectrophotometer. 2.5. identification and quantification of individual phenolic compounds the column used was a c18 supelcosil lc (150 mm x 4.6 mm, 3 µm particle size), and a guard column. the mobile phase was a mixture: formic acid in water (2%, ph 3, solvent b) and formic acid in methanol (2%, ph 3, solvent a). the gradient program was time 0.01 95% b, 15 min 50% b, 20 min 30% b, 25 min 20% b, 30 min 95% b. 5 min of equilibration was required before the next injection. the flow rate was 0.6 ml min and the analyses were conducted at room temperature (25°c). the injected volume was 20 µl. peak identification was performed by comparing the retention times and diode array spectral characteristics with external standards. quantification was performed using calibration curve of standards. data were processed using shimadzu lc solution software. the determination of sugar content, glucose and fructose were determined according method previously described (karkacier et al., 2003). 2.6. total phenolic content assay (tpc) the total phenolics content in the apple extracts was determined using the folinciocalteau procedure according to procedure previously described (singleton et al., 1999) with some slight modifications: 50 µl extract solution was added to 2.5 ml of folin ital. j. food sci., vol. 31, 2019 246 ciocalteu reagent, 2 ml of 20% % na2co3 solution. the mixture was incubated for 30 min at 40°c. absorbance increase was measured at 765 nm. chlorogenic acid was used as standard (0-100 mg l-1), and a calibrate equation was obtained (r2 = 0.9961). total phenolic content is expressed as mg chlorogenic acid g-1. 2.7. antioxidant activity assay the antioxidant activity was estimated using the 1,1-diphenyl-2-picryldydrazyl (dpph). free radical scavenging method according to method previously described with minor adaptation (sharma and bhat, 2009). the dpph reagent 25 mg were dissolved in 1000 ml of methanol(a), and the solution have been diluted 1:10 (b). two ml of solution b were mixed with 100 µl of the extract methanolic solution and kept at 25°c for 30 min in a dark room. a decrease absorbance was determined at 515 nm using spectrophotometer. radical scavenging activity was calculated by the following formula: scavenging % = [abst0min – abs t30min / abs t0min]× 100 where a0 was the absorbance of dpph blank solution, and a was the final absorbance of the tested sample after 30 min of incubation. trolox ((±)-6-hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid) 0.05 mm in methanol was used as a standard to convert the inhibition capacity of the apple extract solution to the trolox equivalent antioxidant activity (r2= 0,9980). 2.8. statistical data analysis and graphic program all results were analyzed by pearson correlation and r2coefficient was determined. these data were elaborated with graph pad prism 5.0 software and statistical significance was estabilished at p<0.05. 3. results and discussion in this study ten apple varieties, by organic production, were selected in order to represent the new orientation of the italian quality organic production aiming to evaluate quality parameters. the apples types considered in the study are usually consumed as fresh fruits in italy: renetta osiris, gold rush, braeburn, celato cola, limoncella, cerina,rosada, topaz, jonagored, florina coming from the organic production of different italian regions: trentino alto adige, toscana, lazio, abruzzo, sicilia. among them: limoncella (abruzzo), cerina (lazio), gelato cola (sicilia) have been produced in small quantities and purchased from selected producers that aim to appraise enhances biodiversity of production territories. 3.1. quantification of individual phenolic compounds the determination of individual phenolic compounds was performed by rp-hplc using the gradient elution method reported in the experimental section. fig. 1 shows the chromatograms of the extracts from the pulp and peel apple of braeburn varieties, obtained at λ 280 nm. ital. j. food sci., vol. 31, 2019 247 pulp peel figure 1. chromatogram of apple braeburn varieties, pulp and peel extracts using rp-hplc, as 280 nm. peaks: 1 gallic acid, 2 (+) catechin, 3 chlorogenic acid, 4 epicatechin, 5 rutin, 6 phloretin. table 1 shows the calibration parameters of the analytical method. concentration of the linear range were determined, for all phenolic compounds, by triplicate analysis of four different standard concentrations. recoveries were determined in both pulp and peel apple extract by spiking apple samples with a solution containing known amounts of all standard mixture compounds prior to the extraction procedure. the study was conducted in triplicate. the obtained values ranged from 92%±2.5 sd 98%±2.2 sd for all analytes. compositions of the individual phenolic compounds: gallic acid, catechin, epicatechin, chlorogenic acid, rutin and phloretin in apples varieties, pulp and peel, are shown in table 2 and table 3. the results on apples pulp show that chlorogenic acid is the major compound present in all examined apples varieties and ranged, in the pulp, between 800.3 µgg-1 limoncella to 52.7 in braeburn, in the peel between 766.6 µgg-1 in jonagored to 187.1 in renetta osiris. ital. j. food sci., vol. 31, 2019 248 table 1. retention time (tr) and calibration parameters of apple phenolics. compounds tr min±sd linear range (µg ml -1) r2 detection limit lod (µg ml-1) gallic acid 7.5±0.04 2-50 0.9958 1.6 (+) catechin 12.4±0.03 2.5-110 0.9902 1.5 chlorogenic acid 13.6±0.06 1.5-200 0.9925 0.7 (-) epicatechin 14.7±0.03 3.0-100 0.9928 0.6 rutin 18.9±0.02 2.5-120 0.9958 0.5 phloretin 19.8±0.03 08-80 0.9991 0.7 mean value (n=3) µg ml-1±sd, l 280 nm. table 2. amounts of phenolics compounds in apples varieties, pulp methanolic extract. apples varieties gallic acid catechin chlorogenic acid epicatechin rutin phloretin renetta osiris 4.7±0.4 34.1±2.2 224.1±2.2 30.3±0.8 35.5±1.3 1.0±1.2 gold rush <1.6 14.9±0.8 243.6±2.0 70.6±1.3 20.1±0.8 3.4±0.8 braeburn <1.6 11.1±2.3 52.7±1.5 170.0±2.1 16.4±0.7 <0.7 gelato cola 4.4±1.7 60.7±2.5 400.1±3.1 89.4±1.7 20.1±0.6 1.7±0.8 limoncella 6.3±1.8 21.2±1.8 800.3±3.5 72.6±1.5 47.2±0.8 <0.7 cerina 5.0±1.0 69.3±1.6 328.5±2.8 79.3±2.1 46.8±1.1 <0.7 rosada 8.3±1.1 110.5±2.8 247.7±1.6 30.0±1.6 13.6±1.1 2.5±1.1 topaz <1.6 44.6±1.6 230.1±2.7 60.5±1.4 18.0±1.2 3.1±1.2 jonagored 7.7±2.1 36.3±1.5 400.2±1.6 18.3±0.8 13.4±0.8 3.2±1.0 florina <1.6 39.2±1.7 180.70±1.5 34.1±1.1 18.8±1.6 3.1±0.8 data are expressed as the mean value ( µg g-1)±sd (n=3). table 3. amounts of phenolics compounds in apples varieties, peel methanolic extract. apples varieties gallic acid catechin chlorogenic acid epicatechin rutin phloretin renetta osiris 4.4±1.2 45.9±0.6 187.1±1.8 65.6±1.0 102.4±2.3 <0.7 gold rush 9.8±1.8 35.9±1.6 400.2±3.2 131.8±0.8 79.4±2.4 4.3±1.3 braeburn 26.7±1.4 77.8±2.8 315.4±3.1 71.2±0.6 131.3±1.8 1.1±1.0 gelato cola 7.2±1.5 100.6±3.1 466.4±3.3 80.1±1.3 128.2±1.6 <0.7 limoncella 12.3±1.6 110.4±3.1 700.2±3.6 100.3±1.7 42.7±2.2 1.2±0.7 cerina 6.3±1.1 120.6±3.3 366.6±2.6 166.8±1.3 71.8±2.1 2.2±0.6 rosada 13.9±1.2 84.7±2.2 500.1±2.7 92.6±0.8 27.7±2.5 <0.7 topaz 2.3±1.6 141.5±2.8 400.3±2.5 140.1±2.1 233.5±1.8 <0.7 jonagored 13.9±1.2 127.2±1.8 766.6±3.1 89.3±2.0 186.4±1.6 10.6±0.5 florina 2.2±2.1 194.3±1.8 203.0±1.8 227.5±2.2 145.8±1.3 19.8±0.7 data are expressed as the mean value ( µg g-1)±sd (n=3). furthermore (−)epicatechin, (+) catechin, rutin, phloretin and gallic acid were minor phenolic constituents. in particular (+) catechin ranged between 11.1 µgg-1in braeburn ital. j. food sci., vol. 31, 2019 249 apple to 110.5 in rosada in pulp extracts, while on the peel between 35.9 µgg-1 gold rush to 141.5 in topaz. epicatechin ranged from 18.3 µgg-1jonagored to 170.0 µgg-1 braeburn in the pulp extracts, while in peel in the range between 65.6-227.5 µgg-1 corresponding to renetta osiris and florina, respectively. rutin content on pulp, ranged from 13.4 µgg-1 jonagored to 47.2 limoncella, on the peel 27.7 µgg-1rosada to 233.5 in topaz. phloretin content ranged from, 0.6 µgg-1on cerina and 3.4 µgg-1 in gold rush on pulp, while on peel 0.6 µgg-1 renetta osiris and 19.8 florina. a poorly significant difference was observed in gallic acid in pulp and peel of all the examined cultivars. however, an higher content of all the examined phenols in peel extracts compared to the one in the pulp one was found with the exception of phloretin in renetta osiris, gelato cola, rosada and topaz. 3.2. total phenolics and antioxidant activity total phenol compounds (tpc) of apple peel extracts are always higher than those in pulp. this is in accordance with the fact that phenolic compounds have the tendency to accumulate in the peel tissues of the plant because of their potential roles in protection against uv radiation and pathogens. in addition, it is clear that total phenol compounds (tpc) of apple peel extracts differed significantly among ten cultivars: tpc was the highest in florina followed, in decreasing order, by jonagored, topaz, rosada, braeburn, limoncella, cerina, gelato cola, rush, renetta osiris. florina was the variety with the highest tpc (9.60±0.8 µg chlorogenic acid g-1) in the peel among all cultivars under study, whereas the lowest value 1.70±0.1 µg chlorogenic acid g-1 was observed in renetta osiris. in pulp, the values ranged between 0.9±0.1 to 4.9±0.5 µg g-1chlorogenic acid; rosada had the lowest values and limoncella, gelato cola and jonagored the highest value. it means that, also in the pulp, tpc is highly related to the specific cultivar. evaluation of antioxidant activity was conducted by dpph method which reveals the colorimetric decrease in absorbance of the radical dpph due to the chemical trapping of the unpaired electron. antioxidants interact with dpph neutralizing its free radical character. table 4 shows the results related to total phenolics and the dpph activity of extracts obtained by using methanol acidified with 1% of hcl. table 4. total phenolics and antioxidant activity in apple varieties, pulp and peel methanolic extract. apples varieties total phenolics* antioxidant activity** (µmol g -1) pulp peel pulp peel renetta osiris 1.0±0.3 1.7±0.1 243.9±0.2 297.2±0.2 gold rush 1.0±0.2 2.3±0.3 236.2±0.2 293.2±0.2 braeburn 1.4±0.2 3.9±0.4 294.8±0.2 363.2±0.3 gelato cola 2.0±0.4 3.0±0.3 319.5±0.6 360.8±0.4 limoncella 2.9±0.5 3.4±0.5 349.4±0.5 307.0±0.4 cerina 1.9±0.2 3.3±0.7 290.7±0.3 348.2±0.4 rosada 0.9±0.1 3.9±0.6 216.6±0.5 351.8±0.3 topaz 4.9±0.5 1.5±0.6 311.1±0.7 397.4±0.4 jonagored 2.0±0.4 6.6±0.6 325.4±0.7 370.9±0.5 florina 1.5±0.2 9.6±0.8 319.8±0.7 377.3±0.6 data espressed* (chlorogenic acid µg g-1)±sd mean value ( n=3). ** data expressed (µmol g -1 trolox)±sd mean value (n=3). ital. j. food sci., vol. 31, 2019 250 in the pulp, the value ranged between 216.6±0.5 to 325.4±0.7 (µmol g-1trolox) respectively for jonagored and rosada varieties, in the peel 293.2±0.2 to 377.3±0.6 (µmol g-1 trolox) for gold rush and florina varieties. the highest values in peel are also evidenced for antioxidant activities. this is may be due to the different distribution of polyphenolics compounds: the peel, in addition to the compounds present in pulp, have additional phenolic compounds not found in the pulp such as anthocyanins and a high amount of quercetin glycosylate (viera et al. 2009). this appears more evident when we compared antioxidant activity in peel extracts between red and yellow apples. the relation between antioxidant activities and polyphenolic concentration is previously reported (van der surs et al, 2001; d’angelo et al., 2007). in this study total phenolic compounds content was positively correlated with antioxidant activity in apple pulp (r2= 0.870) and peel (r2=0.699) respectively. the results suggest that, in apple, phenolic compounds have a significant contribution to the antioxidant capacity according to literature data (lee et al., 2003). 3.3. glucose and fructose content in order to evaluate the sugar content, glucose and fructose have been quantified in apple pulp and peel extracts (figure 2 and figure 3). the fructose content ranged between 1.32 g100g-1 (florina) and 4.45 (gold rush); glucose from 1.25 g100g-1 (florina) to 3.0 (rosada) in the pulp, while in the peel fructose ranged between 0.80 g100g-1 (florina) and 3.40 (renetta osiris), glucose 0.50 g100g-1 (florina) to 2.35 (rosada). the results show the lowest amount in apple topaz, jonagored and florina, whereas these cultivars present significant value in total phenolic content. on the contrary, apple varieties renetta osiris and gold rush, present significant values on glucose and fructose and low content of total phenolic compounds. figure 2. fructose and glucose content in pulp of apples varieties (g 100g-1 ± sd, n=3). 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 fr uc to se -g lu co se g /1 00 g fructose glucose ital. j. food sci., vol. 31, 2019 251 figure 3. fructose and glucose content in peel of apples varieties (g 100g-1 ± sd, n=3). 4. conclusions in conclusion, phenolic compounds, secondary metabolites naturally present in plant material, are important to determine quality characteristics of apple. the bioactive substances quantified show that the commodity is a good source of nutraceutical compounds and denote significant differences in the apples varieties considered. moreover, the apple peel, generally considered as waste, of the examined commodities presents significant contents of individual phenolic compounds, glucose and fructose and, consequently a high antioxidant capacity. acknowledgements the authors acknowledge the funding obtained from sapienza university of rome 2014. references balasuriya n. and rupasingle v.h. 2012. anthypertensive properties of flavonoid-rich apple peel extract. food chem. 135(4):2320. bertschinger, l., mouron p., dolega e. and zürcher m. 2004. ecological apple production: a comparison of organic and integrated apple-growing. acta hortic. 638:321. boyer j. and liu r.h. 2004. apple phytochemicals and their health beneficts. nutritional j. 3:1. coart e., van glabeke s., de loose m., larsen a.s. and ruizi r. 2006. chloroplast diversity in the genus malus: new insights into the relationship between the european wild apple (malus sylvestri smill.) and the domesticated apple (malus domestica borkh). mol. ecol. 15:2171. d’angelo s., cimmino a., raimo m., salvatore a., zappia v. and galletti p. 2007. effect of reddening–ripening on the antioxidant activity of polyphenol extracts from cv. ‘annurca’ apple fruits. j. agric. food chem. 55 (24):9977. eberhardt m.v., lee c.y. and liu r.h. 2000. nutrition-antioxidant activity of fresh apple. nature 405:903. eisele t. and drake s.r. 2005. the partial compositional characteristics of apple juice from 175 apple varieties. j.food comp. anal. 18:213. faostat 2013. world apple production. www.fao.org (accessed 20-02-2013). 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 fr uc to se -g lu co se g /1 00 g fructose glucose ital. j. food sci., vol. 31, 2019 252 forsline p.l., aldwinckle h.s., dickson e.e., luby j.j. and hokanson s.c. 2003. collection, maintenance, characterization, and utilization of wild apples of central asia. hortic. rev. 29:1. lee k, kim y, kim d. and lee c. 2003. major phenolics in apple and their contribution in the total antioxidant capacity. j agric. food chem. 51:6516. karkacier m., erbas m., uslum. k and aksu m. 2003. comparison of different extraction and detection methods for sugar using amino-bonded phase hplc. j. chrom. sci. 41(6):331. raganold j.p., glover j.d., andrews p. k. and hinman h.r. 2001. sustainability of three apple production systems. nature. 410:926. ribeiro f., gomes de mura c., aguiar o., de oliveira f., spadari r., oliveira n., oshima c. and ribeiro d. 2014. the chemopreventive activity of apple against carcinogenesis, antioxidant activity and cell cycle control. eur. j. cancer prev. 23(5):477. scalbert a. and williamson g. 2000. dietary intake and bioavalaibility of polyphenols. jo. nutr. 130:2073. sharma om p. and bhat t. k. 2009. dpph antioxidant assay revisited. food chem. 113: 1202. serra a.t., rocha j., sepododes b., matias a., rodrigo p., agostinho del carvalho f., bronze r., duarte c. & figureira m. 2012. evaluation of cardiovascular protective effect of different varities, correlation of response with composition. food chem. 135 (4):2378. singleton v.l., orthofer r. and lamuela-reventos r.m. 1999. analysis of total phenols and other oxidation substrates and antioxidants by mean of folin-ciocolteu reagent. method enzymol. 299:152. van der sluis a., dekker m.; de jager a. and jongen w.m.f. 2001. activity and concentration of polyphenolic antioxidants in apple:  effect of cultivar, harvest year, and storage conditions. j agric. food chem. 49 (8):3606-3613. vieira fg., borges gs.; copetti c., gonzaga lv., nunes ec. and fett n. 2009. activity and contents of polyphenolic antioxidants in the whole fruit, flesh and peel of three apple cultivars. arch. latinoam. nutri. 59 (1):101. paper received july 24, 2018 accepted september 21, 2018 ijfs#1741_bozza ital. j. food sci., vol. 32, 2020 701 paper the effect of peeling on antioxidant capacity of black radish root e. enkhtuya* and m. tsend department of food engineering, mongolian university of science and technology, baga toiruu 34, ulaanbaatar, mongolia *corresponding author: enkhtsetseg_e@must.edu.mn abstract in this study, freeze-dried peeled and unpeeled root, as well as the juice from peeled and unpeeled root of black radish (raphanus sativus l. var niger) cultivated in mongolia were characterized for their dpph• and abts•+ scavenging activity, reducing power, total phenolics, and flavonoids in order to evaluate the effect of the peel. the juice from the peeled root showed strong antioxidant potential may due to its high phenolic content. however, the ability of the dried unpeeled root extract to quench free radicals and reduce fe3+ was higher than that of the dried peeled root extract. keywords: antioxidant capacity, black radish, peel, phenolic compounds, root ital. j. food sci., vol. 32, 2020 702 1. introduction fruits and vegetables play a vital role in the prevention of degenerative diseases caused by oxidative stress and the improvement of general health as these contain vitamins, minerals, amino acids, dietary fibers, and phenolic compounds. for instance, the prevention of cancer and cardiovascular diseases has been strongly related to the intake of fresh fruits and vegetables rich in natural antioxidants. this suggests that a higher intake of such compounds will lower the risk of mortality from these diseases (willcox et al., 2004). radish (raphanus sativus linn.) is an edible root vegetable of the brassicaceae (cruciferae) family with some other popular vegetables including white and red cabbage, broccoli, brussel sprouts, cauliflower, kohlrabi, rape, and mustard. radish is originally from europe and asia. it grows in temperate climates at altitudes between 190 and 1240 m. it is 30-90 cm high and its roots are thick and of various sizes, forms, and colors (perez gutierrez and perez, 2004). radishes have different skin colors such as red, purple, through pink, black, yellow, and white, while its flesh is typically white (banihani, 2017). the most popular varieties of the radish are red (raphanus sativus l.), white (raphanus sativus l. var white), and black (raphanus sativus l. var niger). among the people of mongolia, black radish is less familiar than red and white radish. a few years ago, some mongolians with diabetes firstly obtained the black radish root from russia due to its positive effect on diabetic conditions. furthermore, local farmers have been cultivating the black radish. it has a rough, black skin with hot-flavored. there are round and elongated varieties. radishes are grown all over the world and mostly eaten raw as a crunchy vegetable, mainly in salads. these can be also brined, fermented (pickled), dried, and cooked in soups or stews. recently, some people prefer to drink its juice due to its certain health benefits such as antioxidant, anti-microbial, anti-viral, anti-inflammatory, antitumorigenic, anti-mutagenic, anti-diabetic, anti-proliferative, hypocholesterolemic, antilithiasic, diuretic, nephroprotective, gastroprotective, and hepatoprotective. it is also very well known for its use in the treatment of bronchitis, diarrhea, gynecological disorders, and jaundice (janjua et al., 2013, agarwal and varma, 2014, banihani, 2017, kumar and patwa, 2018). in general, radish contains carbohydrates, sugars, dietary fibers, protein, various water-soluble vitamins (b1, b2, b3, b5, b6, b9, and c), and minerals (calcium, iron, magnesium, manganese, zinc, potassium, phosphorus, and fluoride). in addition to flavonoids, alkaloids, tannins, and phenolic compounds, the radish was found to have unique bioactive phytochemicals including glucosinolates and isothiocyanates (wang et al., 2010, lugasi and hovari, 2000, banihani, 2017). isothiocyanates are breakdown products resulting from the enzymatic hydrolysis of glucosinolates by the myrosinase. for instance, the most abundant glucosinolates in black radish and its juices are glucoraphasatin and glucoraphanin. these two glucosinolates and their degradation products (raphasatin and sulforaphane) are known for their antioxidant properties, which have been related to cancer and cholesterol gallstones prevention (castro-torres et al., 2014, sarikamis et al., 2015). in addition, radishes have a specific flavor and strong taste due to glucosinolates and their breakdown products. bors et al. (2015) investigated the influence of the variety and vegetative stage on the total phenolic content and antioxidant capacity of radish. significant differences were found in total phenolic content between radish varieties; the highest level was noticed in black radish, followed by red and white radish. black and red radish had similar and significantly higher antioxidant capacity than white radish. regarding the vegetative stage, the highest total phenolic content and antioxidant capacity were found in sprouts, ital. j. food sci., vol. 32, 2020 703 followed by seeds and roots. a positive and highly significant correlation (r2=0.939) was determined between total phenolic content and antioxidant capacity of the radish varieties. lugasi et al. (1998) reported that squeezed juice from black radish root exhibited strong hydrogen-donating ability, reducing power, copper (ii)-chelating property and showed marked free radical (h2o2/•oh) scavenging activity. in their study, only one glucosinolate, namely glucotropaeolin, was detected in the juice by the hplc analysis. in addition, a significant amount of polyphenols was detected in the juice. therefore, the authors supposed that the degradation products of glucosinolates and the polyphenolic compounds are the main biologically active components of the sample. several physiologically active compounds such as isothiocyanates, thiocyanates, indoles, nitriles, epithionitriles, cyano-epithioalkanes, oxazolidine−2−thiones, are released from hydrolysis of glucosinolates by the enzyme myrosinase (lugasi et al., 1998, castrotorres et al., 2014, banihani, 2017). in a study by nikolic et al. (2012), the phenolic compounds content and dpph radical scavenging capacity of black radish depended on root size in such a way that bigger root had a higher content of phenolic compounds and higher scavenging capacity. they also found a positive correlation between the phenolic compound content and radical scavenging capacity. therefore, black radish roots with a weight higher than 100 g are recommended for human nutrition and health benefits. janjua et al. (2013) analyzed five different extracts of black radish root peel for important 14 phytochemicals, namely tannins, saponins, flavonoids, phlobatannins, anthraquinones, carbohydrates, reducing sugars, steroids, phytosterol, alkaloids, amino acids, terpenoids, cardiac glycosides, and chalcones. according to the proximate analysis, the black radish root peel contained 7% moisture and 93% dry matter which was composed of crude protein (28.57%), fats (27.76%) and carbohydrates (39.82%), while fibers were only 1.4% and ash content was 2.43%. the objective of this study was to evaluate antioxidant activity and content of polyphenolic compounds in the black radish root cultivated in mongolia. the antioxidant activities of the black radish root samples were investigated by using three different assays, namely dpph, abts, and frap. total phenolic content and total flavonoid content were also determined by spectrophotometrically. 2. materials and methods 2.1. chemicals and apparatus folin-ciocalteu reagent, 1,1-diphenyl-2-picryl-hydrazyl (dpph), 2,2ʹ-azinobis-3ethylbenzothiazoline-6-sulfonic acid (abts), 2,4,6-tripyridyl-s-triazine (tptz), gallic acid, quercetin, l-ascorbic acid, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) were purchased from sigma-aldrich (mo usa). all other chemicals and reagents were of analytical grade from local suppliers in mongolia. the water used was purified in a milli-q system (millipore, bedford, ma, usa). spectrophotometric determinations were carried out using a shimadzu uv mini 1240 spectrophotometer. ital. j. food sci., vol. 32, 2020 704 2.2. sample collection fresh black radish roots were purchased from a local produce market in ulaanbaatar, mongolia during the period september-october 2018. the radish roots were washed thoroughly under cold running tap water to remove surface impurities as well as to lower microbial load, spread on filter paper, cut-off their crown and tail. afterward, the roots were halved with a stainless steel knife and separated into two parts. one part was peeled off with the knife. the other was processed with peel. 2.3. sample preparation black radish root juice was obtained with the help of a laboratory-scale juice processor and then filtered using a sterilized muslin cloth. the obtained juice was stored at refrigeration temperature (4°c) and analyzed within 2 days. to obtain dried samples, the peeled and unpeeled black radish roots were sliced and freeze-dried for avoiding the loss of their juice rich in biologically active ingredients. the thoroughly dried samples were ground separately to a fine powder in a laboratory mill and then sifted through a mesh 0.5 mm in size. the powdered samples were stored in air-tight containers at 4°c until further use. to assay for antioxidant activity the powdered samples (1 g) were extracted with 50% (v/v) aqueous ethanol (50 ml) on a magnetic stirrer for 120 min at room temperature and centrifuged at 5000 rpm for 10 min at 4°c. the extracts were filtered with a whatman no.5 filter paper and kept in dark at 4°c for further analysis. 2.4. dpph free radical scavenging assay dpph• scavenging ability of the samples was determined as described by adedapo et al. (2009) with a minor modification. briefly, 2 ml of 0.135 mm dpph in ethanol (99.7%) were mixed with 100 µl of the sample solution. after 30 min in the dark, absorbance was measured at 517 nm against a blank (ethanol was used instead of the sample solution). lower absorbance indicates higher scavenging activity. percentage inhibition was calculated by comparing the absorbance of the test sample and the blank. results were also expressed as trolox equivalents (te) by using a calibration curve (r2=0.9973) of trolox (0-300 µm). 2.5. abts radical cation (abts•+) scavenging assay to evaluate abts•+ scavenging ability of the samples, the method of re et al. (1999) was adopted. firstly, to produce a stable abts•+ an aqueous solution of 7 mm abts was oxidized with 2.45 mm potassium persulfate for 12-16 h in the dark at room temperature. before analysis, the abts•+ solution was diluted with distilled water to an absorbance of 0.75±0.05 at 734 nm. a 2 ml of abts•+ solution was mixed with 20 µl of the sample solution and absorbance was read at 734 nm exactly after 7 min. the percentage of abts•+ decolorization was calculated by comparing the absorbance of the test sample and the blank. the blank was prepared by replacing the sample solution with distilled water. the te against abts•+ was also calculated using the calibration curve (r2=0.9993) constructed with the standard trolox (0.1-1.5 mm) under the same experimental conditions. ital. j. food sci., vol. 32, 2020 705 2.6. ferric reducing antioxidant power (frap) assay the frap assay, a method for measuring total reducing power of electron-donating substances, was performed as previously described (benzei and strain, 1996) with a slight modification. frap reagent containing 5 ml of 2,4,6-tripyridyl-s-triazine (tptz, 10 mm) in 40 mm hcl, 5 ml of ferric chloride hexahydrate (20 mm), and 50 ml of acetate buffer (300 mm, ph 3.6) was freshly prepared and warmed at 37°c before use. sample solutions (100 µl) were allowed to react with 3 ml of the frap reagent for 30 min in the dark. absorbance of the colored product was measured at 593 nm. a higher absorbance of the reaction mixture indicates greater reducing power. aqueous solutions of ascorbic acid (0-0.1 mg/ml) were used for calibration (r2=0.9998). the values were expressed as the concentration of ascorbic acid (vitamin c) that is most effective natural antioxidant having the ferric reducing ability. 2.7. determination of total phenolic content the total phenolic content of the black radish root samples was evaluated using the folinciocalteu colorimetric method (singleton et al., 1999). in a test tube, each 20 µl of sample solution was mixed with 1.58 ml of distilled water and 100 µl of 1.8 n folinciocalteu phenol reagent and allowed to stand for 5 min. then 300 µl of 20% aqueous sodium carbonate solution was added and shaken vigorously by a vortex. after incubation at room temperature for 2 h in the dark, the absorbance of the green-blue complex was measured at 765 nm. the results of total phenolic compounds were expressed as gallic acid equivalents (gae), based on a calibration curve (r2 = 0.9996) of gallic acid in the concentration range of 0.1 to 1.0 mg/ml. 2.8. determination of total flavonoid content estimation of the total flavonoids in the black radish root samples was carried out using the procedure reported by adedapo et al. (2009). equal volume of sample solution and 2% anhydrous aluminium chloride in 50% (v/v) ethanol were mixed well and after 1 h at room temperature absorbance was measured at 420 nm. a yellow color indicates the presence of flavonoids. the interference background of the sample solution was corrected by preparing the test without aluminium chloride. the total flavonoid content was estimated from a calibration curve (r2=0.9986) plotted at 420 nm with quercetin (10-50 µg/ml) and expressed as quercetin equivalents (qe). 2.9. statistical analysis all the measurements were taken five times and expressed as mean value ± standard deviation. the data were analyzed using one-way anova for mean differences. statistical significance was declared at p < 0.05. 3. results and discussion bors et al. (2015) investigated the influence of the variety and vegetative stage on the total phenolic content and antioxidant capacity of radish. nikolic et al. (2012) examined the ital. j. food sci., vol. 32, 2020 706 influence of black radish root size on the content and radical scavenging capacity of phenolic compounds. in this study, we evaluated the effect of the peel on the antioxidant activity of the juice and the ethanol extract prepared from black radish root cultivated in mongolia. the juice obtained from black radish root and the ethanol extract of the freezedried root showed potent antioxidant activity in different test systems and contained a significant amount of polyphenols. peeling increased the antioxidant capacity of black radish root juice significantly (p<0.05). on the other hand, the ethanol extract of the dried peeled and unpeeled root exerted statistically similar antioxidant potential. 3.1. dpph• scavenging activity it is well known that the dpph• scavenging ability is attributed to the hydrogen donating ability of phytocompounds. a comparison of dpph• scavenging abilities of the black radish root samples is shown in fig. 1. trolox, a synthetic water-soluble antioxidant analogue of vitamin e, was used as a reference compound, and dpph• scavenging potentials expressed as te of the studied samples were listed in table 1. the black radish root juice were examined for their dpph• scavenging ability after dilution by 10 times with distilled water. the strong dpph• scavenging capacity of 60.9% was detected in the juice from peeled root, while the juice from unpeeled root exerted weak dpph• scavenging capacity of 21.9% (fig. 1), which reflects approximately 2.8-fold difference. however, dpph• scavenging activities of the peeled and unpeeled root extracts were comparable to each other. at a concentration of 50 mg/ml, the percentage scavenging of the peeled root extract was 61.8% whereas that of the unpeeled root extract was 65.7%. the black radish root extracts quenched dpph• with a mean of 414 µmol te/100 g dry weight (table 1). figure 1. dpph• and abts•+ scavenging activity of the black radish root samples (%). values are mean ± standard deviations of five parallel determinations. the vertical bars represent the standard deviations for each data point. values with different superscript letters are significantly different (p < 0.05). *diluted 10 times; **diluted 5 times. ital. j. food sci., vol. 32, 2020 707 there are some reports describing dpph• scavenging activity of black radish root and its juice, but the results varies depending on the experimental condition. for instance, dpph• scavenging capacity of 80% (v/v) ethanol extract from black radish root ranged from 88.3% to 55.6% at a concentration of 5.5 mg/ml depending on the root size and the appropriate ic50 values were 1.59 and 3.54 mg/ml, respectively (nikolic et al., 2012). according to the bors et al. (2015), black radish root showed weak activity (12.23%) to scavenge dpph• at the relatively high concentration (the dry extract prepared from 500 mg freeze-dried powder was recovered in 3 ml methanol). the squeezed juice from black radish has a significant scavenging activity to quench dpph• with an ic50 value of 0.54 ml (lugasi et al., 1998). 3.2. abts•+ scavenging activity the antioxidant capacity of the samples was evaluated by abts•+ assay because proton radical scavenging is an important attribute of antioxidants. abts•+, a protonated radical, is reduced in the presence of hydrogen-donating antioxidants. besides being one of the fastest, abts•+ method also provides good solubility, which allows the analyses of both lipophilic and hydrophilic compounds (re et al., 1999). fig. 1 showed the percentage inhibition of abts•+ by the studied samples, whereas table 1 presented the scavenging abilities expressed as te. the black radish root juice diluted previously 5 times with distilled water was analyzed by abts•+ assay. the juice from the peeled black radish root quenched 76.9% of abts•+ in the reaction mixture (fig. 1), which is about 5.2-fold greater than that found in the juice obtained from the unpeeled root (14.9%). the same as the dpph• scavenging activity, peeled and unpeeled root extracts showed similar ability to scavenge abts+• with a mean of 1510.7 µmol te per 100g dry weight (table 1). at 50 mg/ml, the scavenging percentages were 50.7% and 51.4% for the peeled and unpeeled black radish root extracts, respectively (fig. 1). to our best knowledge, there is almost no data on abts•+ scavenging potential of black radish root samples. 3.3. ferric reducing antioxidant power the reducing power of the black radish root samples was analyzed using the frap assay by comparing with reducing power of ascorbic acid (table 1). the formation of bluecolored tptz−fe2+ complex from colorless oxidized tptz−fe3+ by the action of the electron-donating antioxidants under acidic condition (ph 3.6) was recorded at 593 nm. the reducing ability of the plant samples could be attributed to the number of hydroxyl groups in the phenolic and flavonoid compounds. the studied samples from black radish root intensively reduced fe3+ to fe2+. it was found that 1 g (dry weight) of black radish root and 1 ml of its juice showed reducing power equal to that estimated in 3.5-3.7 and 0.8-1.0 mg of pure ascorbic acid, respectively. in a study by lugasi et al. (1998), the reducing effect of 1 ml of black radish root juice was the same as that of 0.73 µmol ascorbic acid. it is equal to 0.13 mg ascorbic acid per 1 ml of the juice. according to hanlon and barnes, the reducing power of the freeze-dried unpeeled root of black radish (nero tondo variety) was 23.7 µmol ascorbic acid/g which is equal to 4.17 mg ascorbic acid/g. it was comparable to our result. ital. j. food sci., vol. 32, 2020 708 similarly to dpph• and abts•+ scavenging ability, the dried unpeeled root extract and the juice obtained from peeled root showed higher reducing power. however, the margin of difference between reducing power of the juice obtained from the peeled and unpeeled root was narrow. the results indicated that phytoconstituents in black radish are capable of donating electrons that can react with free radicals to convert them into more stable compounds and reduce the oxidized intermediates of lipid peroxidation process. table 1. antioxidant potential of the black radish root samples. sample dpph • scavenging activity abts•+ scavenging activity fe 3+ reducing power dried peeled root 401.47±5.541 1500.45±21.241 349.59±4.243 dried unpeeled root 426.52±4.911 1520.95±109.581 372.23±6.533 juice from peeled root 198.05±0.832 569.66±4.412 100.49±4.954 juice from unpeeled root 71.98±0.432 109.36±1.162 83.90±1.464 1expressed as µmol trolox equivalents/100g. 2expressed as µmol trolox equivalents/100ml. 3expressed as mg ascorbic acid/100g. 4expressed as mg ascorbic acid/100ml. values are mean ± standard deviations of five parallel determinations. 3.4. total phenolic content plant polyphenols are the significant group of compounds acting as free radical scavenging or primary antioxidants; therefore, it is justifiable to determine phenolic content in the plant extract. these can also chelate metal ions that could catalyze the formation of reactive oxygen species, which promotes lipid peroxidation (iqbal et al., 2015). in many studies, phenolic compounds have demonstrated higher antioxidant activity than antioxidant vitamins and carotenoids (velioglu et al., 1998). total phenolic contents (expressed as gae) in the black radish root samples are given in table 2. the amount of total phenolics in the juice ranged from 103 to 146 mg/100ml. lugasi et al. (1998) detected by the folin-denis method relatively low polyphenol content (25.5 mg/100 ml juice) in black radish root juice. in the case of the dried black radish root, total phenolic content was in the range of 750-791 mg/100 g (7.5-7.9 mg/g) dry weight. the lower level of total phenolic content was obtained by bors et al. (2015) in black radish root (4.75 mg of gae/g dry weight) and by hanlon and barnes (2011) in the freeze-dried unpeeled root of the nero tondo black radish (2.4 µg of gae/g). total phenolic content in the unpeeled root was higher than those of the peeled root. these results revealed that black radish root skin contained a considerable amount of phenolic ingredients. however, the juice obtained from the unpeeled root contained lower amount of total phenolics compared to the peeled root juice. it indicated that the phenolic constituents present in the peel could not be extracted into the juice or these substances may be degraded during processing and storage by the action of any factors such as an enzyme. janjua et al. (2013) reported that phytochemicals of black radish root peel were not well dissolved in water as well as methanol. moreover, the lowest yield was of water extract. ital. j. food sci., vol. 32, 2020 709 3.5. total flavonoid content flavonoids exert potent antioxidant activity by several different mechanisms, such as scavenging of free radicals, chelation of metal ions, and inhibition of enzymes responsible for free radical generation. depending on their structure, flavonoids are able to scavenge practically all known reactive oxygen species (panda, 2012). lugasi and hovari (2000) demonstrated that radish contains a significant amount of flavonoids such as kaempferol, quercetin, myricetin, apigenin, and luteolin. before that, some researchers observed 7 mg/kg (0.7 mg/100g) flavonoids including quercetin and myricetin in the radish root (lugasi et al., 1998). the amount of total flavonoid content in the black radish root samples was assessed by the aluminium chloride assay. total flavonoid content in the juice prepared from the peeled root was 0.81 mg qe/100 ml, while total flavonoids were not detected in the juice from unpeeled root (table 2). dried peeled and unpeeled root of black radish contained comparable amounts of total flavonoids with a mean of 15.8 mg qe per 100 g of dry weight. table 2. total phenolics and flavonoids of the black radish root samples. sample total phenolic content total flavonoid content dried peeled root 749.60±14.591 16.00±0.043 dried unpeeled root 791.20±46.711 15.69±0.093 juice from peeled root 146.00±3.422 0.81±0.004 juice from unpeeled root 103.10±2.282 − 1expressed as mg gallic acid equivalents/100g. 2expressed as mg gallic acid equivalents/100ml. 3expressed as mg quercetin equivalents/100g. 4expressed as mg quercetin equivalents/100ml. values are mean ± standard deviations of five parallel determinations. 4. conclusions the juice obtained from the peeled and unpeeled root showed considerable differences in antioxidant activity when it was calculated by the three different methods used in this study. the juice prepared from the peeled root showed strong antioxidant potential may due to its high phenolic content. however, the juice from the unpeeled root contained a relatively low amount of total phenolics and had no flavonoid content. it was supposed that the phenolic constituents including flavonoids present in the peel could not be extracted into the juice or these substances may be degraded. although dried black radish root samples contained significantly different amounts of total phenolics, they showed statistically similar free radical scavenging activities and reducing power. thus, the antioxidant activities of the dried root samples are probably from the combined action of phenolics and other constituents. the results of this study show that the root of black radish cultivated in mongolia contained a significant amount of biologically active phenolics with antioxidant properties. thus, the black radish root may serve as a potential source in functional food development. ital. j. food sci., vol. 32, 2020 710 acknowledgements the authors acknowledge the funding obtained from mongolian foundation for science and technology and mongolian university of science and technology. references adedapo a.a., jimoh f.o., afolayan a.j. and masika p.j. 2009. antioxidant properties of the methanol extracts of the leaves and stems of celtis africana. rec. nat. prod. 3(1): 23. agarwal k. and varma r. 2014. radical scavenging ability and biochemical screening of a common asian vegetable raphanus sativus l. int. j. pharm. sci. rev. res. 27(1):127. banihani s.a. 2017. radish (raphanus sativus) and diabetes. nutrients 9:1014. benzei i.f.f. and strain j.j. 1996. the ferric reducing ability of plasma (frap) as a measure of “antioxidant power”: the frap assay. anal. biochem. 239:70 bors m.d., semeniuc c.a., socaci s. and varva l. 2015. total phenolic content and antioxidant capacity of radish as 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adhikari, 2012). notably, allium plants fortified with se reportedly display higher pharmacological activity than non-fortified ones (ip et al., 2000; adhikari, 2012). moreover, all allium species belong to secondary accumulators of se, even in the seeds (golubkina et al., 2015), with remarkable tolerance to high concentration of this element; accordingly, consumption of se fortified allium parts may give additional benefits to human health, taking also into account that se deficiency has been detected in many countries worldwide (golubkina and papazyan, 2006). much attention is being paid for the last decade to waste valorization technologies of vegetable processing and to search for new natural sources of biologically active compounds. in this respect, the high content of antioxidants and organic sulfur compounds in a. porrum plant parts confer them high protective functions, such as antimicrobial, cardioprotective, hypocholesteremic, hypoglycemic and anticancer activity (radovanović et al., 2015). however, leek has not been deeply investigated as a potential source of pharmacologically beneficial compounds and, in most cases, pseudo-stems are just consumed whereas the leaf blades are discarded. moreover, though this species is a natural source of methylated se-containing amino acids with high anticancer activity, so far only one study concerning soil se biofortification of leek has been carried out (lavu et al., 2012) and no data on leaf se accumulation have been reported. the uneven distribution of elements in the earth crust is the cause of widespread mg, ca, fe, zn, cu, i and se deficiency in quite a few populations (white and broadley, 2009), which leads to numerous attempts to include these elements in bakery products (wimalawansa, 2013; rosell et al. 2015). among the latter, bread is largely consumed in most world countries and this justifies the relevant fortification with different nutrients, in order to produce functional food enriched with natural antioxidants, macro and trace elements (de valença et al., 2017; allen et al., 2006). indeed, the interest in se is due to its powerful antioxidant, immune-modulating, cardio-protective and anti-carcinogenic properties. it is estimated that the mean levels of wheat se are 10550 µg/kg, reaching 3-7 µg/kg in regions with se deficient soils (several provinces of china) and 70 mg/kg in selenosis areas (india) (tamas et al., 2010). with the aim to solve the se-deficiency issue in human organism, the use of se biofortified cereals (lazovélez et al., 2016; bryszewska et al., 2007), se enriched yeast (sсientific opinion, 2008) and bread supplement with se fortified seedlings (bryszewska et al., 2005) have been proposed. the main se chemical form present in the latter products is selenomethionine (tamas et al., 2010), whereas allium plants may provide more powerful anticarcinogen compounds, such as methylated forms of se containing aminoacids (ip et al., 2000; adhikari, 2012). the higher dry matter in leek leaves compared with pseudo-stems simplifies the leaves dehydration process and gives wide chances to leaves powder utilization both as a spice and a supplement in functional food production with remarkable level of se natural antioxidants. ital. j. food sci., vol. 31, 2019 290 indeed, in recent years research has been frequently focused on the use of leaves and peel to produce powders with high antioxidants content and to the utilization of such powders in functional food preparation (ferreira et al., 2015; sonia et al., 2016; odunlade et al., 2017; lakshmi et al., 2017). however, so far leaves powder from se enriched plants has never been used for these purposes, and in the case of allium species this approach can also improve human organism protection against numerous diseases in addition to the increase of se and other antioxidants consumption (gonzález-morales et al., 2017). the aims of the present research were: comparison of leek cultivars in terms of antioxidant and element composition in leaves and pseudo-stems; assessment of the effects of se biofortification on quality characteristics of the most antioxidant-containing cultivar; evaluation of the efficiency of leek leaves powder use in the production of bread fortified with selenium-enriched and non-enriched leek leaves. 2. material and methods 2.1. crop trials research was carried out on leek (a. porrum l.) at the experimental fields of federal scientific center of vegetable production, in odintsovo (moscow, russia, 55°39.51'n, 37°12.23'e) in 2015 and 2016 on a clay-loam soil, with рн 6.8, 2.1 % organic matter, 108 mg kg-1 n, 450 mg kg-1 p2o5, 357 mg kg-1 k2o, exchangeable bases sum as much as 95.2%. mean temperature and relative humidity values from may to october were 13.0, 16.1, 19.8, 18.6, 12.3, 6.4°c and 59.1, 63.8, 69.7, 72.4, 79.1, 81.0 % respectively. the experimental protocol was based on the comparison between nine cultivars (goliath, summer breath, premier, casimir, kalambus, campus, vesta, giraffe, bandit), using a randomized complete block design with three replicates. the sowing was performed on 5 december in 8 x 8 cm trays and the plantlets were transplanted in open field on 14 may, spaced 15 cm along the rows, the latter being 40 cm apart. prior to planting, ploughing at 30 cm depth, hoeing at 15 cm and fertilization with 180 kg ha-1 n, 80 p2o5 and 120 k2o were practiced; during the crops, 40 kg ha-1 n were supplied in three times at two-week intervals and, just in the last n application 7 kg ha-1 of p2o5 and of k2o were also provided. a further experimental trial was carried out in 2016 and 2017, assessing the effects of se biofortification on leek pseudo-stem and leaf quality, by spraying the plants from june to august, once a week, with a total sodium selenate dose of 75 mg m-2; cultivar goliath was used, as it had showed the best quality indicators among the nine varieties compared in the previous year. in either research, commercially ripe plants were harvested at mid-october and on samples taken in each plot the total, pseudo-stem and leaf blade weights were determined. further plant samples were collected, gently washed with water to remove surface contaminants and dried with filter paper. pseudo-stems and leaves were separated, cut with plastic knife, dried to constant weight and homogenized. the resulting powders were subjected to laboratory analysis and further used for bread production. 2.2. bread production trial production of bread was achieved in 2016 and 2017 with three different processing procedures: bread obtained upon addition of leaves powder from leek selenium fortified plants to dough; bread obtained upon addition of leaves powder from leek non-fortified plants; traditionally-made bread. ital. j. food sci., vol. 31, 2019 291 two hundred g of wheat flour with 90 µg selenium /kg d.w., 1.5 g salt and 8 g leek leaves powder were placed in a kenwood dough mixer (model a 907 d) set at highest speed and mixed for 1 min; control samples were not supplemented with leaves powder. then, a suspension of 5 g yeast in 120 ml of water was added and the mixture was further run at high speed for 2 minutes. the dough was later kneaded on the kneading table, rounded into balls by hand and placed in lightly greased fermentation bowl in fermentation cabinet. the dough was then proofed for thirty minutes, baking was done at 250°c for 15 minutes and, next, the baked bread was allowed to cool at room temperature before performing determinations. baking was achieved in triplicate. 2.3. volume, weight and specific volume of loaves produced loaf volume was measured by small seeds displacement method described by khalil et al. (2000). loaf was placed in a container of known volume where onion seeds were run until the container was full. the volume of seeds displaced by the loaf was considered as the loaf volumes, which were measured in a graduated cylinder. the weight of the loaf was determined using a sensitive weighing balance and the specific volume of the loaf was assessed by averaging the loaf volume with loaf weight. the specific volume was calculated according to the equation: specific volume (cm3/g) = loaf volume/loaf weight 2.4. bread porosity bread porosity was determined according to gost procedure (2001). four cylindrical grooves from fresh bread were made with a volume of 27 (±0.5) cm each and weighed simultaneously. the porosity (%) was calculated by the following formula: porosity (%) = [(v-m/1.31):v] x 100 where v is the total volume of bread grooves in cm3; m is the mass of bread grooves in g; 1.31 is the density of non-porous mass of breadcrumb. 2.5. dry matter content the dry matter content in leaves and pseudo-stems of a. porrum as well as in bread samples was assessed after dehydration of the fresh samples in oven at 70°c, until they reached constant weight. 2.6. selenium content se content in leek leaves and pseudo-stems as well as in bread samples was analyzed using the fluorimetric method previously described for tissues and biological fluids (alfthan, 1984). the method includes digestion of dried homogenized samples via heating with a mixture of nitric-chloral acids, subsequent reduction of se+6 to se+4 with a solution of 6 n hcl, and formation of a complex between se+4 and 2,3-diaminonaphtalene. se concentration was assessed in triplicate by recording piazoselenol fluorescence value in hexane at 519 nm λ emission and 376 nm λ excitation. the results precision was checked ital. j. food sci., vol. 31, 2019 292 using a reference standard-lyophilized cabbage at each determination with 150 µg/kg se concentration (institute of nutrition, russia). 2.7. potassium content potassium content in leek pseudo-stems was assessed using aas technique on shimatsu 7000 spectrophotometer (japan) after dry ashing of 2 g leek pseudo-stems and leaves at 420°c, and dissolution of the residues in 15 ml of 3% nitric acid (analytical methods, 1996). 2.8. total soluble solids (tss) and sugars determination of total soluble solids was carried out in water extracts of leek leaves and pseudo-stems using tds-3 conductometer (russia). monosaccharides were determined using ferricyanide colorimetric method, based on the reaction of monosaccharides with potassium ferrycianide (swamy, 2008). total sugars were analogically determined after acidic hydrolysis of water extracts with 20% hydrochloric acid. fructose was used as an external standard. 2.9. polyphenols the concentrations of total phenolics in each sample of leaves, pseudo-stems and bread were determined in 70 % ethanol extract (1 hour at 80 oc) using the folin-ciocalteu colorimetric method, according to golubkina et al. (2017) by unico 2804 uv (usa) spectrophotometer. the phenolic contents were calculated by using a calibration curve of gallic acid constructed with five concentrations of this compound (0-90 μg/ml). phenolic contents were expressed as milligrams of gallic acid equivalents per 100 gram of dry weight (mg gae/100 g d.w.). 2.10. ascorbic acid ascorbic acid content in leek leaves and pseudo-stems was assessed by visual titration of fresh plant extracts in 6% trichloracetic acid with tillmans reagent (aoac, 2012). five grams of fresh leek leaves were homogenized in porcelain mortar with 5 ml of 6% trichloracetic acid and quantitatively transferred to measuring cylinder. the volume was brought to 80 ml using trichloracetic acid, and the mixture was filtered through filter paper 15 min later. the ascorbic acid concentration was determined from the amount of tillmans reagent, which went into titration of the sample. 2.11. antioxidant activity the antioxidant activity of leek leaves and pseudo-stems as well as bread samples was assessed using redox titration method (maximova et al., 2001), via titration of 0.01 n kmno4 solution with ethanolic extracts of leaves, pseudo-stems and bread samples. reduction of kmno4 to colorless mn+2 in this process reflects the amount of antioxidants dissolvable in 70 % ethanol. the values were expressed in mg gae/100 g d.w. the use of kmno4 acidic solution is known to be successfully used for the determination of ocimum basilicum antioxidant potential (srivastava et al., 2015) and antioxidant capacity of serum (zhan et al., 2014). ital. j. food sci., vol. 31, 2019 293 2.12. statistical analysis data were processed by analysis of variance and mean separations were performed through the duncan multiple range test, with reference to 0.05 probability level, using spss software version 21. the data expressed as a percentage were subjected to angular transformation before processing. 3. results and discussion 3.1. leek quality parameters from the evaluation of nutritional indicators of nine leek cultivars, it arose that cultivar goliath had significantly higher content of antioxidants, monosaccharides and potassium compared to the other varieties (table 2). notably, goliath pseudo-stems attained 1.9 to 3.8 fold higher ascorbic acid, 1.6-2.6 fold polyphenols and 1.3-1.8 fold se; leaf polyphenols concentration was 1.3-1.6 times higher. these results suggest that among the nine cultivars examined goliath contains the highest content of polyphenols not only in pseudo-stems but also in leaves which are usually discarded, though their high potential benefits. moreover, a. porrum leaves proved to be better sources of polyphenols than pseudo-stems in all cultivars. conversely, selenium distribution between leaves and pseudo-stems in unfortified plants is less nutritionally important due to the low concentrations of this element. a distinctive feature of cultivar goliath was the high proportion of monosaccharides, accounting for 60.3 % of the total sugar amount in pseudo-stems compared to 18.2-41.9 % in the other varieties (table 1). moreover, goliath pseudo-stems accumulated 2.2 to 11 fold more potassium (fig. 1). interestingly, positive correlations relevant to pseudo-stems were detected between polyphenols and k, se and polyphenols, se and k (r = +0.96; r = +0.97 and r = +0.97 respectively, at p≤0.01) and a negative correlation between leaves and pseudo-stems se content (r = 0.88 at p≤0.01). due to the high nutritional value of cultivar goliath, this variety was chosen in order to assess the effect of se biofortification on leek quality and antioxidant features. ital. j. food sci., vol. 31, 2019 294 table 1. quality and antioxidant indicators of nine leek cultivars. dry matter % plant ascorbic acid mg/100 g polyphenols mg gae/100 g d.w. se µg/kg d.w. sugars % monosaccharides total pseudo-stems pseudo-stems leaves pseudo-stems leaves pseudo-stems pseudo-stems goliath 12.35±0.4e 13.0±0.7a 683±56a 964±72a 107±7a 14±1d 3.8±0.3b 6.3±0.4e premier 15.20±0.5d 8.9±0.5b 432±34b 650±21c 80±5b 65±3b 4.4±0.3a 10.5±0.7c bandit 15.32±0.6d 6.2±0.4c 394±26bc 647±26c 75±5b 48±2c 3.5±0.2c 8.6±0.5d kalambus 17.64±0.6c 4.5±0.4e 319±19de 731±46b 72±4b 74±3b 3.9±0.2b 10.3±0.7c cazimir 18.86±0.6c 5.7±0.5cd 284±20e 728±53b 60±3c 76±4ab 2.8±0.2d 10.7±0.7c giraffe 20.51±0.8b 5.1±0.4d 331±19d 665±41bc 73±4b 49±2c 3.4±0.2c 11.0±0.7c camus 21.36±0.7b 4.5±0.3e 347±21cd 684±43bc 64±3c 81±5ab 2.5±0.2e 12.2±0.8b vesta 23.39±0.8a 4.5±0.3e 301±19de 740±55b 69±3bc 84±5a 2.6±0.2de 14.3±0.8a summer breath 24.28±0.9a 5.4±0.4cd 329±20d 616±42c 72±3b 72±4b 2.8±0.2d 15.1±0.9a m 18.77 6.86 317 686 74,7 62,5 3.3 11.1 sd 3.24 2.69 71 37 8,4 17 0.6 2.1 cv (%) 17.3 39.3 22,4 5,4 11,2 27,2 18.2 18.9 concentration range 4.5-16.9 284-683 616-964 60-107 14-84 2.5-4.4 6.3-15.1 within each column, means followed by different letters are significantly different according to duncan test at p<0.05. ital. j. food sci., vol. 31, 2019 295 figure 1. intervarietal differences in k accumulation by leek pseudo-stems. means followed by different letters are significantly different according to duncan test at p<0.05. 3.2. fortification of leek cultivar goliath with se the data reported in table 2 suggest that the enrichment of leek cultivar goliath with selenium decreased plant weight due to leaf reduction, and significantly increased pseudo-stem occurrence to the total plant weight (+ 49.5%). moreover, the leaves of selenium fortified a. porrum plants contained significantly higher contents of both dry matter and antioxidants compared to pseudo-stems. notably, the contents of all the antioxidants detected were significantly higher in leaves than in pseudo-stems, i.e. 3.86 fold for ascorbic acid, 1.35 fold for polyphenols and 3.2 fold for se. total soluble solids value was also higher in leaves than in pseudo-stems (1.2 fold). table 2. effect of se application on yield, quality and antioxidant indicators of a. porrum cultivar goliath. se fortified plants ratio between fortified plants and non-treated control pseudo-stems leaves pseudo-stems leaves mean weight (g per plant) 196±15a 200±9a 1.1n.s. 0.68* dry matter (%) 11.3±0.3b 13.5±0.3a 1.2* 1.38* ascorbic acid (mg/100 g f.w.) 15.0±0.6b 57.9±0.8a 1.15n.s. 1.04n.s. polyphenols (mg gae/100 g d.w.) 1106±88b 1494±76a 1.62* 1.55* total sugars (%) 6.9±0.4 1.10n.s. monosaccharides (%) 5.9±0.4 1.55* total soluble solids (mg/g) 70.0±2.7b 83.2±3.6a 1.27* 0.72* se content (µg/kg d.w.) 1451±50b 4645±40a 16.1* 332* potassium content (g/kg d.w.) 52.8±5.1a 28.5±2.5 b 1.02n.s. 1.04n.s. n.s. means no statistically significant differences between fortified and control plants; *statistically significant at p≤0.05; within se fortified plants, values along the rows followed by different letters are significantly different at p≤0.05. a b c cd cd cd d e f 0 10 20 30 40 50 60 k c on te nt (g /k g d. w .) ital. j. food sci., vol. 31, 2019 296 according to the described data, leek foliar biofortification with se performed on cultivar goliath is beneficial to leek production, as it led to the increase of pseudo-stem yield as well as polyphenols and selenium concentration. the investigations of lavu (2013) on leek upon soil se supply showed that the lower initial se concentration in non-fortified pseudo-stems the higher the fortification level. the same phenomenon was observed in the present work: the fortification value reached 16.1 in pseudostems with high initial se content (107 µg/kg), whereas it was as much as 332 in leaves with low initial se concentration (14 µg/kg). the uneven distribution of biologically active compounds in a. porrum plants was characterized by higher content of dry matter, ascorbic acid, polyphenols and total soluble solids in leaves, compared to pseudo-stems of selenium enriched plants. similar distribution of these compounds was recorded in control plants, suggesting the nutritional importance of leek leaves, which are unfortunately discarded in the common farming practice. the antioxidant content increase in leaves and pseudostems as a result of selenium fortification is in agreement with the previously reported stimulating effect of selenium absorption on plant antioxidant defense (golubkina, 2016). 3.3. supplementation of bread with leaves powder from se fortified leek plants recent investigations have revealed that 4% supplementation of vegetable leaves power to wheat flour is optimal for producing functional bread (odunlade et al., 2017). physical and biochemical characteristics of bread enriched with a. porrum leaves powder recorded in our research are reported in table 4. the results suggest peculiar changes in bread quality upon supplementation of unfortified or se fortified leaves to flour (table 3). table 3. physical, quality and antioxidant characteristics of bread enriched with a. porrum leaves powder. parameter additives no additive control a. porrum leaves powder se-enriched a. porrum leaves powder dry matter (%) 62.4±1.0ab 60.4±1.0b 64.5±1.1a se content (µg/kg d.w.) 90±1b 90±1b 266±18a total soluble solids (mg/g d.w.) 18.5±0.3c 19.7±0.3b 20.5±0.4a aoa (mg gae/100g d.w.) 3.3±0.2b 5.8±0.2a polyphenols content (mg gae/100g d.w.) 3.2±0.1b 5.6±0.2a specific volume (cm3/g) 2.23±0.08a 2.04±0.07b 1.86±0.08c bread porosity (%) 67.5±0.8a 64.3±0.9b 61.8±0.8c colour white light green light green along each row, values followed by different letters are statistically different according to duncan test at p≤0.05. as far as bread sensory attributes are concerned, the three products did not differ in terms of odor and flavor, whereas the bread supplemented with leek leaves powder showed a light green color, which did not vary between the samples treated with se-fortified or nonfortified leaves. from a practical point of view, this unusual color may be preferred by consumers. ital. j. food sci., vol. 31, 2019 297 notably, bread supplementation with selenium enriched leek leaves powder greatly differs from use of inorganic forms of selenium in bread production. indeed, selenium salts (selenates and selenites) are known to be highly toxic and particularly dangerous upon overdosing. on the other hand, plant treatment with selenium inorganic salts allows to convert the latter to organic selenium derivatives of amino-acids and proteins, and in fact this process is named biofortification. as far as allium species are concerned, such biofortification results in production of methylated forms of selenium-containing aminoacids showing remarkable anticancer activity (ip et al., 2000; adhikari, 2012) which is significantly higher than that associated to the selenomethionine present in selenium enriched yeast (golubkina and papazyan, 2006) notably, the incorporation of leek leaves powder with high se concentration into dough resulted in the highest values of se content, total soluble solids, polyphenols and antioxidant activity of the final product. one hundred grams of such bread contain about 17 µg se, which accounts for 31% of the required selenium consumption (dietary reference intakes, 2000). se losses during bread baking were low and did not exceed 3%, which is consistent with previous investigation results (lyons et al., 2005; rosell et al., 2015; garvin et al., 2011) about the stability of se compounds during baking of bread from se enriched wheat flour. moreover, leek polyphenol stability during baking is higher in se enriched product than under non fortified leaves use (93.7% vs 84.2% respectively). this phenomenon may reflect the well-known antioxidant protective effect of se (golubkina and papazyan, 2006). the results of the present work also prove the effect of se supplementation on bread porosity and specific volume, the latter parameters decreasing according to the following sequence: control > bread with leaves powder from non fortified leek > bread supplemented with se enriched leek leaves powder. the decrease in bread porosity and specific volume was reported in previous studies carried out on bakery products fortified with leaves powder from different plants by odunlade et al. (2017). these authors explained that this phenomenon is the consequence of gluten concentration decrease due to the replacement of the flour portion containing leaves powder. unfortunately, such a statement is not exhaustive for describing the phenomenon relevant to the effect of se enriched leaves on bread porosity and specific volume; it is just indicative that using wheat with high se content in bread production leads to 10% decrease of final product porosity (garvin et al., 2011), which is consistent with the 8.9% decrease relevant to se fortified leek utilization detected in the present study. this peculiarity is presumably associated with se, as the porosity decrease in the case of ordinary leek powder addition results in smaller changes of this parameter (4.7 %). one of the reasons connected to dough rheological properties decrease in case of seenriched leaves powder use, compared to dough with non-fortified leaves and control dough, may be the increased concentration of plant polyphenols under selenium fortification. indeed, despite their high nutritional value, polyphenols are known to cause changes in dough rheology via interaction with proteins, resulting in the decrease of enzymes and yeast activity and thus worsening bread porosity (wang et al., 2007). in particular, gallate and hydroxylate benzol groups of polyphenols form noncovalent bonds with amino-, hydroxyland carboxyl groups of proteins (huang et al., 2004; rosell et al., 2015). another factor possibly affecting dough porosity is the high level of total soluble solids (rosell et al., 2015), which is increased in bread supplemented with a. porrum leaves powder, especially with high selenium content. ital. j. food sci., vol. 31, 2019 298 4. conclusions the experimental investigation carried out on leek in northern europe allows to draw interesting remarks regarding both plant se fortification and bread production using seenriched leaves. in this respect, goliath was identified as the best responsive cultivar to se application for obtaining nutrient-added pseudo-stems and its leaves powder was successfully mixed with dough during the bread making process. the mentioned practice is aimed to valorize both leek crop waste such as leaf blades and a widely consumed daily food such as bread. as arisen from this research, se supply to a. porrum plants entails beneficial effects to human organism as a consequence of the use of either a fresh vegetable or a functional food. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. references aoac. 2012. the official methods of analysis of the association of official analytical chemists international, 2012.22 vitamin c, usa. adhikari p. 2012. biofortification of selenium in broccoli (brassica oleracea l. var. italica) and onion (allium cepa l.). master thesis in plant science. norway. alfthan g. 1984. a micromethod for the determination of selenium in tissues and biological fluids by single-test-tube fluorimetry. anal. chim. acta 65:187. doi: doi.org/10.1016/s0003-2670(00)85199-5 allen l., de benoist b., dary o. and hurrell r. 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10.1155/2014/928595. paper received june 12, 2018 accepted october 23, 2018 ijfs#1523_bozza ital. j. food sci., vol. 31, 2019 808 survey product carbon footprint: still a proper method to start improving the sustainability of food and beverage enterprises a. cimini and m. moresi* department for innovation in the biological, agrofood and forestry systems, university of tuscia, via s. c. de lellis, 01100 viterbo, italy *corresponding author: tel.: +39 0761357497; fax: +39 0761357498 e-mail: mmoresi@unitus.it abstract given the complexity of food production, supply chains and distribution, this paper sustains that the mere assessment of the product carbon footprint might still be regarded as a first trial in the field of improving the sustainability of the food and drink industry. after having reviewed the greenhouse gas (ghg) emissions associated with the agro-food system in industrialized countries, and summarized the main direct environmental impacts of the food industry, the pros and cons of the life cycle assessment (lca) methodology were briefly examined together with the current standard methods used to assess the environmental impact of food and drink products. once a cradle-to-grave product carbon footprint modelling had been developed, some mitigating actions might be tested with the final goal of reducing the ghg emissions associated with the most impacting product life cycle stages. as an example, such a procedure was applied to approximately halve the cradle-to-grave carbon footprint of two cereal-based products (i.e., dry pasta and malt beer). a cost/benefit analysis is required to relate the marginal increase in the product processing costs to each reduction in the product environmental load. keywords: beer, carbon footprint, dry pasta, environmental impact, ghg emissions ital. j. food sci., vol. 31, 2019 809 1. introduction the current food system is regarded as ecologically unsustainable (church, 2005; fooddrinkeurope, 2012; wri, 2013), since fossil fuels are essential requirements for running crop production, animal husbandry, food production and distribution, as well for the construction and maintenance of machinery and processing equipment, transportation vehicles, and infrastructures. the greenhouse gas (ghg) emissions associated with food production and consumption were evaluated to constitute 19-29% of the global ghg emissions (vermeulen et al., 2012). by referring to the major environmental impact categories, such as climate change (cc), ozone depletion (od), photochemical ozone creation (poc), acidification (a), eutrophication (np), resource depletion, human toxicity, and eco-toxicity, the food, drink, tobacco and narcotics area of consumption in the eu-25 was estimated to generate up to 20-30% of the main impact categories (including 22-31% for cc), with the exception of 59% for np (tukker et al., 2006). an increasing number of studies has dealt with the long-term sustainability of the current trends in the production and consumption of food. in particular, the eu standing committee on agriculture research (scar) observed that food production is near to exceed environmental limits; land use change and land degradation, as well as the dependence on non-renewable fossil energy sources, contribute about one-fourth of the ghg emissions; agriculture, including fisheries, is the single largest driver of biodiversity loss (european commission, 2011). the average usa and eu diet, being rich in meat, fat and sugar, is a risk for individual health, social systems and the environment. since the world population is expected to grow from about 7 billion to 9.6 billion people in 2050, as well as the global meat and milk consumption, especially in china and india, the promotion of healthy diets can reduce the environmental footprint of food consumption (european commission, 2011; fao, 2018; moresi and valentini, 2010; wri, 2013). in addition, food processing and retail industries are asked to stimulate the necessary changes in production and consumption patterns (wri, 2013). the food and beverage industry is a major contributor to the eu economy (fooddrinkeurope, 2018), followed by the automotive, machinery and equipment, and chemical industries. as of 2015, it was the major driver of the economy, with turnover of € 1.109 trillion, employment of 4.57 million employees with 294,000 total number of companies. actually, 99.1% (i.e., 280,000) of the companies are small and medium-sized enterprises (smes), these generating 48.1% (i.e., €538 billion) of the overall turnover, 48.4% (i.e., €107 billion) of the value added and 61.3% (i.e., 2.8 million employees) of employments. owing to its environmental and economic importance, an intergovernmental set of 17 sustainable development goals has already been identified in the food sector (fooddrinkeurope, 2019), this being a core part of the 2030 agenda for sustainable development (un, 2015). beyond the general statement of decreasing environmental burdens, such as ghg emissions, waste generation, as well as water and energy consumption, goal 9 aims at building resilient infrastructures, promoting inclusive and sustainable industrialization and fostering innovations. the complex relation between innovation and agro-food sustainability was deeply analyzed by el bilali (2018) in order to identify what type of innovation should be promoted to foster transition towards a more sustainable food system. given the complexity of food production, supply chains and distribution, this paper aimed to present how the mere assessment of the product carbon footprint might effectively help food and drink industries to improve their sustainability. section 2 ital. j. food sci., vol. 31, 2019 810 focused on the ghg emissions associated with the agro-food system in industrialized countries, and especially in italy. section 3 summarized the main direct environmental impacts of the food industry. section 4 briefly reviewed the basic of the life cycle analysis (lca) methodology with the pros and cons of the main standard methods used to assess the food and drink environmental impact. section 5 further discussed if the key elements for sustainable food processing are a priori identifiable or should be considered on a case-by-case basis. finally, the importance of prioritizing the life cycle stages with the highest environmental impact as derived from business-to-consumer lca studies was addressed in section 6. more specifically, by resorting to the cradle-to-grave carbon footprint (cfcg) modelling for two typical cereal-based food and drink products (i.e., dried pasta and malt lager beer), several mitigation options were selected in order to reduce their climate change impact. in spite of assessing the effect of such options on other environmental impact categories, the only estimation of the cfcg was regarded as intrinsically sufficient to promote a first improvement in the sustainability of the great majority of the food and drink enterprises. 2. ghg emissions for the agro-food system in industrialized countries although from the millenary climate observations the warming since the middle of the 20th century might be primarily attributed to natural causes, such as solar activity and random variations (de larminat, 2016), the human contribution cannot be considered negligible (ipcc, 2013). the human population has grown from about 3.0 to 7.7 billion people since 1960 (anonimous, n.d.), and in all probability has exerted a primary impact on the environment. it is, indeed, responsible for the huge release of the so-called greenhouse gases (ghg), namely co2, ch4, n2o, hydrochlorofluorocarbons (hfcs), perfluorinated chemicals (pfcs) and sf6, in the atmosphere. since 1980 the volumetric concentrations of co2, ch4 and n2o in the atmosphere over marine surface sites have definitely increased from about 380 to 405 ppm (noaa, n.d.), 1566 to 1835 ppb and 301 to 328 ppb (eea, 2017), respectively. to allow any person now living on the earth and those expected to live until 2100 the same rights to emit ghgs, the ghg emission space per capita and in a year should be limited to 2400 kg of co2, 59 kg of ch4, and 0.67 kg of n2o, provided the atmospheric concentration of co2 is less than 450 ppm with ch4 and n2o emissions kept at the same levels measured in 1995 (carlsson-kanyama, 1998; ipcc, 1996). thus, the per capita ghg emissions permitted each year within a 20-yr time perspective, as estimated by summing the mass of each ghg times its corresponding global warming potential (ipcc, 2013), would amount to (1x2400+84x59+264x0.67=) 7533 kg co2e yr-1. by referring to the national inventory reports (nir) published by uncc (2018), it is possible to assess whether such permitted ghg emissions are congruent with the ones currently in several countries. in 2007, the direct per capita emissions ranged from 24.0 to 1.6 mg co2e yr-1 for the usa and india, respectively (berners-lee, 2010). as shown in table 1, in 2016 the italian ghg emissions (including those adsorbed by land use, land use change and forestry, lulucf) amounted to circa 398 tg co2e (ispra, 2018), equivalent to the italian per capita cf of about 6.7 mg co2e yr-1. altogether, these emissions were mainly composed of co2, followed by ch4 and n2o, while the contribution of the halogenated compounds (i.e., hfcs, pfcs, nf3, and sf6) was negligible. the main ghg emissions were from the energy sector (347.1 tg co2e), this was followed by the industrial ital. j. food sci., vol. 31, 2019 811 (32.1 tg co2e), agricultural (30.4 tg co2e), and waste (18.3 tg co2e) sectors, while the category lulucf was the main ghg sink (-29.9 tg co2e). more specifically, the agriculture sector mainly emitted ch4 from animal husbandry [i.e., enteric fermentation (14.0 tg co2e) and manure management (3.1 tg co2e)] and rice cultivation (1.7 tg co2e), and n2o from agricultural soils (8.9 tg co2e) and manure management (2.1 tg co2e). the industrial processing ones were mainly due to the iron and steel industry, followed by the chemical, and pulp, paper and print ones. the food processing, beverages and tobacco sector emitted ~3.7 tg co2e (ispra (2018). the contribution of the agro-food sector to the overall direct ghg emissions cannot be directly extracted from any nir. in fact, most of its subsectors (namely, agro-food product transportation; production and transportation of packaging materials; food transport from retailer to consumer’s house; electric energy consumed to preserve foods in the home freezer, fridge, etc.; gas and/or electric energy consumed to cook foods; disposal of food losses or wastes) are aggregated in other sectors. the italian contribution without the consumer and post-consumer phases was found to be about 19% of the overall ghg emissions (moresi, 2014), this falls within the range estimated by tukker et al. (2006). the main direct impacts of food processing are derived from waste generation, water use, and energy use (dieu, 2009). food waste is intense in farms due to spoilage (~21% of supply), but limited to ~7% throughout food processing. food waste may be the loss of inedible materials or rejected products from sorting, grading, peeling, trimming, and squeezing. it may amount to the 50-70% of fresh citrus fruits or crab and shrimp processed (dieu, 2009). packaging materials (i.e., paperand card-board, plastics, glass, metals, and wood) are largely used to protect processed foods not only from deterioration and/or contamination (primary packaging), but also from mechanical damage through the distribution and retailing operations (secondary and tertiary packaging). in food processing large volumes of water are used as the main ingredient, particularly in drink production, as the initial and intermediate cleaning source, transportation conveyor of raw materials, and principal agent used in sanitizing plant areas and machinery (dieu, 2009). the water consumption in fruit and vegetable processing ranges from 4 to 32 m3 per mg of product treated, of which approximately 50% is used just for washing and rinsing. the water used to make beer or milk products may vary from 9 to 18 m3 mg-1. the resulting wastewaters are generally rich in organic matter, and sometimes are contaminated with pesticide residues from raw material treatments. up to 50-60% of the water might be reclaimed and reused after screening, filtering or dilution with fresh water. air emissions during food processing may contain fine particles, combustion products (co, co2, nox), volatile organic compounds, and in the case of fish by-products may contain unpleasant odorous contaminants, such as h2s, and (ch3)3n (dieu, 2009). the energy needs of food industry are of low or medium intensity. some sectors (e.g., wet corn milling, beet sugar, soybean oil mills, malt beverages, meat packaging, canned and frozen fruits and vegetables, bread, and baked products) are however high-energy users (dieu, 2009). the 38% of all the energy consumed by the italian agro-food industry is electric, while the remainder is thermal (mise, n.d.). the total impact of energy use might be lessened by minimizing the energy needs of production, producing energy from waste, and using renewable energy sources. ital. j. food sci., vol. 31, 2019 812 table 1. summary report for the overall italian direct co2 equivalent emissions, including or excluding the net ghg emissions adsorbed from land use, land-use change and forestry (lulucf), as referred to the main ghg sources (i.e., co2, ch4, n2o, and halogenated compounds, hc, as hfcs, pfcs, nf3, and sf6) and sink categories in the year 2016, as extracted from ispra (2018). ghg source co2 ch4 n2o hc subtotal 1 subtotal 2 total sink categories tg co2e 1. energy 334.93 7.66 4.49 0 347.08 a. fuel combustion 332.44 2.93 4.48 0 339.86 1. energy industries 103.79 0.13 0.44 0 104.36 2. manufacturing industries and construction 46.96 0.28 0.71 0 47.94 3. transport 103.38 0.22 0.91 0 104.51 4. other sectors 77.81 2.30 2.41 0 82.52 5. other 0.52 0.00 0.02 0 0.53 b. fugitive emissions from fuels 2.48 4.73 0.01 0 7.22 1. solid fuels 0.00 0.04 0.00 0 0.04 2. oil and natural gas 2.48 4.69 0.01 0 7.18 2. industrial processes and product use 14.76 0.05 0.57 16.72 32.10 32.10 a. mineral industry 10.61 0.00 0.00 0.00 10.61 b. chemical industry 1.46 0.00 0.12 1.49 3.08 c. metal industry 1.71 0.04 0.00 0.01 1.76 d. non-energy products from fuels and solvent use 0.98 0.00 0.00 0.00 0.98 e. electronic industry 0.00 0.00 0.00 0.22 0.22 f. products used as substitutes for ozone-depleting substances 0.00 0.00 0.00 14.66 14.66 g. other product manufacture and use 0.00 0.00 0.46 0.33 0.79 3. agriculture 0.54 18.87 10.98 0.00 30.39 30.39 a. enteric fermentation 14.04 0.00 14.04 b. manure management 3.11 2.12 0.00 5.23 c. rice cultivation 1.71 0.00 1.71 d. agricultural soils 8.86 0.00 8.86 e. prescribed burning of savannas 0.00 0.00 f. field burning of agricultural residues 0.017 0.004 0.00 0.02 g. liming 0.01 0.00 0.01 h. urea application 0.53 0.00 0.53 4. lulucf -31.08 0.40 0.76 0.00 -29.93 -29.93 a. forest land -36.08 0.28 0.001 0.00 -35.80 b. cropland 2.46 0.002 0.03 0.00 2.49 c. grassland -6.64 0.12 0.04 0.00 -6.48 d. wetlands e. settlements 9.01 0.68 0.00 9.69 f. harvested wood products 0.17 0.00 0.17 5. waste 0.09 16.29 1.90 0.00 18.29 18.29 a. solid waste disposal 13.62 0.00 13.62 b. biological treatment of solid waste 0.12 0.53 0.00 0.65 c. incineration and open burning of waste 0.09 0.06 0.02 0.00 0.18 d. waste water treatment and discharge 2.49 1.35 0.00 3.84 total co2 equivalent emissions without lulucf 427.86 total co2 equivalent emissions with lulucf 397.94 ital. j. food sci., vol. 31, 2019 813 3. the environmental impact of food processing the complete supply chain of the food industry from the production of raw materials via food processing to the consumption and disposal by the consumer is quite complex and is schematically sketched in fig. 1. figure 1. simplified flow sheet of the supply chain of the food industry, as adapted from moresi (2014). 4. life-cycle assessment: pros and cons life-cycle assessment (lca) is a technique capable of assessing the environmental impact associated with a product, process or activity during its life cycle from raw material extraction via material processing, packaging, distribution, use, repair and maintenance to the final disposal, that is from cradle to grave (minkov et al., 2016). its procedure is standardized by the international organization for standardization (iso, 2006ab) and is performed in four different phases: i) goal and scope of the study to set the functional unit (i.e., the reference unit), system boundaries, allocation methods and impact categories of choice, as well as the assumptions and limitations used. ii) inventory analysis by constructing a flow chart including all the activities involved in the system boundaries and a flow model to relate all input and output data to and from the environment in order to account for 99% of the mass and energy used in the system under study. iii) impact assessment to convert the inventory analysis results into specific environmental impact categories. these may be also categorized under the development, manufacture, use, and disposal phases of the product examined. iv) interpretation to discuss the outcomes of the above stages, identify the data elements contributing most significantly to each impact category and measure their sensitivity, assess the completeness and consistency of the study, and provide a basis for conclusions and recommendations. several impact categories are used to measure the potential impacts to the natural environment, human health or depletion of natural resources. table 2 lists the main ones together with their characterization models, as derived from manfredi et al. (2012) and morawicki (2012). thus, by summing up any release to air, water or soil yi (expressed in mass, energy, mass-km basis) associated to the system boundaries times its corresponding science-based conversion factor, called characterization factor (fi,j), it is possible to estimate the score of the generic impact category (icj) as: )f (ψic ji, i ij ∑= (1) ital. j. food sci., vol. 31, 2019 814 in particular, the environmental impact of climate change can be directly calculated by using the 100-year time horizon global warming potentials (gwp) relative to the co2 of the ghgs, which were recently reassessed by ipcc (2013). table 2. main impact categories used in several lca standard methods, as extracted from manfredi et al. (2012) and morawicki (2012). impact category category definition indicator unit ref.s climate change (cc) the potential change on the earth climate is due to human activity and ghg release. kg co2e ipcc(2007) ozone depletion (od) the industrial gas concentrations accelerating o3 decomposition in the earth’s stratosphere affect living organisms kg cfc-11e wmo (1999) acidification (a) the release of nox and so2 which combine with water in the atmosphere forms hno3 and h2so3. mol h+e seppälä et al. (2006) eutrophicationaquatic (npa) the release of nand p-rich nutrients in surface waters results in excessive plant growth. fresh water: kg pe; marine water: kg ne struijs et al. (2009) eutrophicationterrestrial (npt) the deposition of n from the emissions released by n-rich nutrients affects terrestrial ecosystems too. mol ne seppälä et al. (2006) photochemical ozone creation (poc) the formation of ground-level o3, as due to the reaction of nox and volatile organic compounds, causes irritation for humans and damage for plants. kg nmvoce van zelm et al. (2008) ecotoxicity-aquatic, freshwater (et) interaction among chemical compounds and organisms in the environment. ctue rosenbaum et al. (2008) human toxicity cancer effects (htc) chemical compounds may cause several types of cancer in humans or ctuh rosenbaum et al. (2008) non-cancer effects (htnc) chronic non-cancer effects including mutagenicity, toxicity, etc. ctuh rosenbaum et al. (2008) particulate matter (pm) particulate matter causes respiratory problems. kg pm2.5e humbert et al.(2011) ionizing radiationhuman health effects (ir) ionizing radiation affects the risk for human cancer incidence and mortality increase. kg u235e dreicer et al. (1995) resource depletion water (rdw) use and depletion of fresh water, minerals and fossil resources impact ecosystems and many species survival. m3 of water related to local water scarcity frischknecht et al. (2008) mineral/fossil (rdmf) kg sbe van oers et al. (2002) land transformation (lt) the extent of changes in land properties and effects on the area affected. kg soil organic matter milà i canals et al. (2007) the environmental performance of food and drink production may be currently assessed by various standard methods, such as those listed in table 3. some of them (i.e., product carbon footprint; pas2050; bilan carbone®, bc; ghg protocol) make use of only the impact category of climate change and give no hint about the overall environmental impact of the products, even if the emissions from direct land-use changes over the previous 20 years are generally included (table 3). other standard methods evaluate from seven (i.e., lca, and environmental product declaration, epd®) to 14 (product environmental footprint, pef) impact categories. their scores are estimated using a series ital. j. food sci., vol. 31, 2019 815 of lca data sources and characterization factors, which obviously are strongly dependent on the lca databases used. there is thus a strong need for reliable databases to achieve a trustworthy assessment of a product life cycle environmental performance, as observed by the food and drink companies involved in several pef pilot tests (fooddrinkeurope, 2017). table 3. brief description of some international standard methods for product and service environmental assessment together with the impact categories (ic) accounted for (same labels as in table 2). standard method description impact categories chosen ref.s life cycle assessment (lca) specifies requirements and provides guidelines for lca studies. cc; od; a; np; poc; rd; lu. iso (2006ab) carbon footprint of product allows the calculation of pcf, based on lca specified in iso (2006ab). cc; luc. iso/ts (2013) pas 2050 provides a standardized guidance for calculating the pcf of goods and services. cc; luc. bsi (2008) bilan carbone® tool developed by the french environment & energy management agency ghg to assess ghg emissions. cc; luc. ademe (2010) environmental product declaration (epd®) tool supported by the swedish government. cc; od; a; np; poc; rd; lu. iso (2006c) ghg protocol defines how measuring, and reporting ghg emissions in the usa. cc bhatia et al. (2011) product environmental footprint (pef) novel european community methodology under development. cc; od; a; npa; npt; poc; et; htc; htnc; pm; ir; rdw; rdmf; lt. manfredi et al. (2012) the greater the number of impact categories accounted for, the more precise the environmental profile of the product under study will be. nevertheless, the estimation with as many as 14 impact categories (table 3) was harshly criticized by numerous stakeholders, such as academia (cimini and moresi, 2018a; finkbeiner, 2014; lehmann et al., 2016), industry (acea, 2013; bdi, 2015), policy-makers (bmub/uba/tub, 2014), and consumer associations (anec, 2012), for being uselessly complex and very expensive. in fact, the federation of german industry (bdi, 2015) estimated an average cost of about 100 k€ for assessing the pef profile of a single product. furthermore, some critical issues were identified to ensure that the lca delivered robust results (notarnicola et al., 2017). in particular, the intrinsic variability of the agricultural system affected the inventory analysis, impact assessment, and interpretation phases. the higher the output per hectare the higher will be the eco-efficiency of the final product. however, long-term sustainability of food production in a given production area is not considered in the current lca method. many lca studies give no details about the soil, climate and weather conditions, timescale adopted, transport distances and modes used to deliver raw materials and final products, as well as the use phase and related wastes. a more meaningful functional unit for food products was also proposed by sonesson et al. (2017) in order to relate the nutritional function of foods to their lca results and account for the sustainable food consumption and food security. how to represent such variability in lca studies without having to collect an enormous number of extra data that would make such studies disproportionately expensive is a primary ital. j. food sci., vol. 31, 2019 816 challenge for lca researchers and practitioners. thus, to allow smalland medium-sized food and drink enterprises to improve their sustainability in the most direct and economical method, the assessment of the product carbon footprint (pcf) appeared to be more useful. in fact, not only was the climate change impact category with the lowest levels of uncertainty (cimini and moresi, 2018a), but also was the major contributor to acidification (r2=0.82), eutrophication (r2=0.66), and photochemical ozone formation (r2=0.86) categories (huijbregts et al., 2006). 5. identification of the key elements for sustainable food processing the food and beverage industry is seeking to improve its environmental performance and identify which actions are suitable for a more sustainable production (moresi, 2014). no food processing nowadays is 100% sustainable owing to the lack of energy, ingredients and packaging materials derived from renewable resources; excessive water use; the inherent ch4 and n2o emissions associated with crop production and animal husbandry; and lack of biodegradable packaging materials (morawicki, 2012). nevertheless, by accounting for only the impact category of climate change, morawicki (2012) suggested a simple and progressive approach to relieve the environmental impact of a food company. first, food processing plant efficiencies for energy, water, and raw and packaging material consumption should be improved and fossil energy usage replaced with renewable one by purchase or self-generation. second, the ghg emissions associated with the transportation of raw materials and final products should be reduced. third, the ghg emissions resulting from the field phase should be minimized. fourth, the impact of the post-consumer disposal of packaging materials, as well as food loss, is to be reduced. despite being firm-oriented, such an approach might result in mitigation actions exerting a minimum reduction in the product carbon footprint. thus, the mitigation opportunities should be prioritized starting from the life cycle stages with the highest contribution to pcf, as previously assessed (cimini and moresi, 2018b). this procedure was specifically applied to improve the sustainability of two typical cereal-based food and drink products, as detailed in the following cases studies. 6. case study no. 1: lager beer production the cradle-to-grave carbon footprint (cfcg) of a malt lager beer was previously estimated (cimini and moresi, 2016, 2018c) by applying the pas 2050 standard method (bsi, 2008). all the aforementioned four lca canonical stages were referred to a functional unit consisting of 1 hl of malt beer, as produced in a large-sized brewery with an annual beer capacity of 3x106 hl and packed in 66-cl glass bottles. the system boundaries for this case study are shown in fig. 2. according to pas 2050 (section 7.2), the geographical and time scopes of this lca study are the western europe and from the years 2006-2016. main process data were of the primary type (cimini and moresi, 2016). by using all the essential data previously given (cimini and moresi, 2018b), the lca model was able to estimate the cfcg as 127 kg co2e per hl of beer. the contribution of the different life cycle stages are shown in fig. 3. ital. j. food sci., vol. 31, 2019 817 figure 2. beer system boundaries, as adapted from cimini and moresi (2018c). the main identification items are listed in the abbreviations and nomenclature section. figure 3. contribution of the different life cycle phases to the cradle-to-grave carbon footprint (cfcg) of 1 hl of beer packed in 66-cl glass bottles in a large-sized brewery, as estimated from the lca model previously developed (cimini and moresi, 2018b), and its cumulative score (see broken line). for the identification items refer to the abbreviations and nomenclature section. ital. j. food sci., vol. 31, 2019 818 the life cycle phases contributing mostly to the cfcg, in descending order, were associated with packaging material manufacture (~56 kg co2e hl-1), overall transportation (~29 kg co2e hl-1), production of malted barley and processing aids (~23 kg co2e hl-1), consumer use (~19 kg co2e hl-1), beer production and packaging (~12 kg co2e hl-1), and waste disposal (1.2 kg co2e hl-1). co2e credits derived from the use of spent grains and surplus yeast as animal feed (2.1 kg co2e hl-1) and from recycling of glass bottles, paper and cardboard wastes (11 kg co2e hl-1). instead of adopting the aforementioned morawicki’s approach to sustainability, a series of improvement opportunities was scheduled to sequentially reduce the contribution of the most impacting life cycle phases of the above reference case. firstly, the replacement of 10% recycled glass bottles with 100% recycled ones reduced the cfcg by about 21 % with respect to the reference case. by shifting the transportation mode from 100% of road freight to 100% of rail freight to manage logistics flows, an additional 10% decrease in cfcg was achieved. the use of organic instead of conventional barley grown locally had the effect of decreasing the cfcg by another 9%. a quasi zero-carbon alternative for electricity generation is solar-photovoltaic electricity. such a shift further lessened the cfcg by 13%. on the contrary, by reducing the delivery distance of malted barley from 500 to 250 km, no significant change was observed in the cfcg; hence, reducing distance had a negligible effect. table 4 shows all the emission factors (efi) that were varied and how the above sequential series of mitigation options practically halved the beer carbon footprint from about 127 to 60 kg co2e hl-1. since the per capita consumption of beer in italy is about 31.8 l yr-1 (assobirra, 2018) and the current italian population is 59,228,336 (worldometers, 2019), the ghg emissions associated with the italian consumption of beer would be reduced from 2.39 to 1.13 tg co2 yr-1. the application of the aforementioned mitigating actions had the effect of limiting the contribution from beer to 0.28% of the overall italian ghg emissions (table 1). table 4. effect of the sequential mitigation strategies used to minimize the cradle-to-grave beer carbon footprint (cfcg) and its cumulative percentage variation with respect to that pertaining to the reference case ( !"#!"# !"!" ∗ ). the sequential stepwise procedure started from the most impacting life cycle phase as resulting from fig. 3. mitigation strategy parameter varied unit cfcg [kg co2e hl -1] ∆𝑪𝑭𝑪𝑮𝒋 𝑪𝑭𝑪𝑮 ∗ [%] beer reference case (*) 127.2 0 100% recycled glass bottles efrb 1.08à0.48 kg co2e kg -1 100.3 -21 malt & beer rail transport efrt 0.168à0.039 kg co2e (mg km) -1 88.2 -31 organic malt efoc 1.143à0.546 kg co2e kg -1 76.6 -40 local malt drm 500à250 km 76.5 -40 photovoltaic electric energy efpee 0.324à0.055 kg co2e kwh -1 60.2 -53 7. case study no. 2: dry pasta production the cradle-to-grave cfcg of an organic durum wheat semolina pasta was previously estimated (cibelli et al., 2017; cimini et al., 2019c) using the pas 2050 standard method (bsi, 2008). all the lca canonical stages were referred to a functional unit consisting of 1 ital. j. food sci., vol. 31, 2019 819 kg of dry pasta, produced in a medium-sized pasta factory with a capacity of approximately 125 gg yr-1 and packed in 0.5-kg polypropylene (pp) bags. the system boundaries for this case study are shown in fig. 4. according to the pas 2050 (section 7.2), the geographical and time scopes of this lca study were the western europe and from the years 2006-2016. finally, the process data were of the primary type, as reported by cimini et al. (2019c). the estimated dry pasta cfcg was about 1.8 kg co2e kg-1, the contribution of all the life cycle phases being plotted in fig. 5. their impacts were therefore ranked as follows: field phase (~0.67 kg co2e kg-1), home pasta cooking (0.65 kg co2e kg-1), pasta production and packaging (~0.20 kg co2e kg-1), transportation (~0.15 kg co2e kg-1), packaging material manufacture (~0.11 kg co2e kg-1), durum wheat milling (~0.05 kg co2e kg-1), end of life of packaging materials (~0.03 kg co2e kg-1) and pasta losses (~0.02 kg co2e kg-1). co2e credits resulted from using wheat milling by-products, and pasta making and packaging wastes for animal feed (~0.07 kg co2e kg-1) as an alternative to soybean meal fodder (cimini et al., 2019c). figure 4. dry pasta system boundaries, as adapted from cimini et al. (2019c). the main identification items are listed in the abbreviations and nomenclature section. figure 5. contribution of the different life cycle stages to the cradle-to-grave carbon footprint (cfcg) of 1 kg of dried organic pasta packed in 0.5-kg pp bags in a medium-sized pasta factory, as estimated from the lca model previously developed (cimini et al., 2019c), and its cumulative score (see broken line). the main identification items are listed in the abbreviations and nomenclature section. 666 52 201 107 12 136 19 -69 651 19 14 -200 100 400 700 1,000 1,300 1,600 1,900 fp mi ppp pam trpaw trfp stpeol bpc cp ps ppeol c f i [g c o 2e k g1 ] cfcg = 1.81 kg co2e kg-1 ital. j. food sci., vol. 31, 2019 820 to improve the sustainability of such product, a series of mitigating actions were programmed to reduce the contribution of the most impacting life cycle phases of the above reference case. in particular, to limit the impact of the primary hotspot (i.e., the consumer and post-consumer ones), the eco-sustainable pasta cooking procedure suggested by cimini et al. (2019ab) was applied by setting the cooking water-to-dry pasta ratio at 2 l kg-1 and the nominal cooking power at 0.4 kw. in this way, the cfcg was cut by 29% with respect to the reference case. use of organic crop rotation enabled the cfcg to be decreased by another 13%. by replacing the methane needed for the steam generating boilers with biogas, the cfcg was further reduced by 7%. use of solar-photovoltaic electricity also lessened the cfcg by an extra 9%. similarly, by shifting from road to rail freight transport, a supplementary 2% reduction in the cfcg was obtained. finally, when the final product or grain delivery distance was shortened from 900 or 150 km to as low as 250 or 50 km, respectively, the cfcg still reduced by 2 or 1%. in total, such a sequential series of mitigating options allowed the dry pasta carbon footprint to be reduced from 1.81 to 0.68 kg co2e kg-1 (table 5). table 5. effect of the sequential mitigation strategies used to minimize the cradle-to-grave dry pasta carbon footprint (cfcg) and its cumulative percentage variation with respect to that pertaining to the reference case ( !!"!"# !"!" ∗ ). the sequential stepwise procedure started from the most impacting life cycle phase as shown in fig. 5. mitigation strategy parameter varied unit cfcg [kg co2e kg -1] 𝜟𝑪𝑭𝑪𝑮𝒋 𝑪𝑭𝑪𝑮 ∗ [%] dry pasta reference case (*) 1.81 0 eco-sustainable cooking pc 2.3à0.4 kwh kg -1 1.28 -29 organic rotation cropping efoc 0.534à0.36 kg co2e kg -1 1.06 -42 thermal energy from biogas efbg 0.231à0.029 kg co2e kwh -1 0.92 -49 photovoltaic electric energy efpee 0.513à0.055 kg co2e kwh -1 0.77 -58 pasta rail transport efrt 0.168à0.047 kg co2e (mg km) -1 0.72 -60 pasta regional distribution dp 900à250 km 0.70 -62 durum wheat local supply drm 150à50 km 0.68 -63 since the per capita consumption of pasta in italy is about 23.5 kg yr-1 (unafpa, 2015), the ghg emissions associated with the italian consumption of dry pasta would reduce from 2.52 to 0.95 tg co2 yr-1. the aforementioned mitigating actions had the effect of reducing the impact of the dry pasta sector to the 0.24% of the overall italian ghg emissions (table 1). 8. conclusions in this work, the main direct environmental impacts of the food industry and ghg emissions for the agro-food system in industrialized countries were analyzed together with the main advantages and disadvantages of the standard methods currently used to assess the food and drink environmental impact. ital. j. food sci., vol. 31, 2019 821 owing to the great deal of money needed to characterize the whole environmental profile of a single product, and the fact that the climate change impact category was by far more reliable than all the other ones used in the epd® and pef standard methods made the assessment of the product carbon footprint a cheaper tool to identify the major hotspots of the food supply chain. thus, it is probably the best method to start improving the sustainability of the 99% of the food and beverage smes. it was used here to select a sequential series of mitigating actions in order to reduce the cradle-to-grave product carbon footprint (cfcg) of 1 hl of beer packed in 66-cl glass bottles from about 127 to 60 kg co2e hl-1, and that of 1 kg of dry organic pasta packed in 0.5-kg pp bags from 1.81 to 0.68 kg co2e kg-1. a cost/benefit analysis might help smes to relate the marginal increase in the overall final product costs to each reduction in the product environmental load. since only the assessment of ghg emissions might result in burden shifting, a further step should investigate the effect of the selected mitigating actions on other environmental impact categories. abbreviations and nomenclature a acidification bc bilan carbone® bpc co2e credits from by-product use as cattle feed; bpp brewing and packaging processing bsg brewer’s spent grain cc climate change cfcg cradle-to-grave product carbon footprint [kg co2e hl-1 or kg-1] cfc trichlorofluoromethane or freon-11 co2e carbon dioxide equivalent cp consumer phase ctue comparative toxic unit for ecosystems ctuh comparative toxic unit for humans de diatomaceous earth dc distribution centers dp distribution distance of packed dry pasta [km] drm supply distance of raw materials [km] ee electric energy efbg emission factor for biogas [kg co2e kwh-1] efoc emission factor for organic crop [kg co2e kg-1] efpee emission factor for photovoltaic electric energy [kg co2e kwh-1] efrb emission factor for 100% recycled glass bottles [kg co2e kg-1] efrt emission factor for rail freight transport [kg co2e (mg km)-1] eol end of life epd environmental product declaration et ecotoxicity – aquatic, freshwater eu european union fij generic i-th characterization factor of the j-th impact category fp field phase ghg greenhouse gas hc halogenated compound hfc hydrochlorofluorocarbon htc human toxicity cancer effects htnc human toxicity non-cancer effects ic impact category ir ionizing radiation – human health effects lca life cycle assessment lt land transformation lulucf land use, land use change and forestry mi milling nir national inventory report nmvoc non-methane volatile organic compound ital. j. food sci., vol. 31, 2019 822 np eutrophication npa eutrophicationaquatic npt eutrophicationterrestrial od ozone depletion pam packaging materials pas publicly available specification pc specific food cooking power [kwh kg-1] pcf product carbon footprint pcwd post-consumer waste disposal pef product environmental footprint pfc perfluorinated chemical pm particulate matter/respiratory inorganics poc photochemical ozone creation pp polypropylene ppeol primary packaging end of life. ppp pasta production and packaging ps pasta scraps pvpp polyvinylpolypyrrolidone q thermal energy rd resource depletion rdmf resource depletion – mineral/fossil rdw resource depletion – water rpm raw and processing materials rr retailer refrigeration r2 coefficient of determination sme smalland medium-sized enterprise stpeol secondary and 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oosterveld et al., 2001; zhao et al., 2008) and also because of its therapeutic effects against cancer, diabetes, cardiovascular diseases (ghatak et al., 2010, di domenico et al., 2009). ferulic acid has been commercially produced from γ-oryzanol in rice bran oil because of easier process. however, the main part of this valuable phenolic acid is presented in plant cell walls cross linked with polysaccharides which may be released by enzymatic or chemical processes (ou et al., 2007). one of these potential sources is sugar beet pulp (sbp), a main byproduct of sugar beet industries. it is a valuable byproduct, but at present it is only used as animal feed. jankovska et al. (2001) set-up a method to determine the ferulic acid content of sugar beet pulp by high-pressure liquid chromatography. a few researches have been previously accomplished to extract ferulic acid from sugar beet pulp by alkaline and enzymatic methods (couteau et al, 1997; kroon et al, 1996; jankovska et al., 2005; donkoh et al., 2012), however, optimization of this process is necessary to understand the interactions between different parameters during extraction, and to minimize the negative effects of chemical processes. the main objective of this research was to compare total phenolic contents of methanol and alkaline sugar beet pulp extracts and optimize alkaline hydrolysis of sugar beet pulp for ferulic acid extraction through response surface methodology via central composite design. finally antioxidant capacity of isolated ferulic acid from sugar beet pulp extract was evaluated. 2. materials and methods 2.1. materials sugar beet pulp (sbp) was provided by isfahan sugar factory (isfahan, iran). trans-ferulic acid as external standard, abts (2,2’-azino-bis(3ethylbenzthiazoline-6-sulphonic acid)) and trolox was purchaced from aldrich chemical co. (milwaukes, w1, usa), sodium hydroxide, ethanol, methanol, kbr, gallic acid, ethyl acetate, potassium persulfate and folinciocalteu reagent were obtained from merck chemical company. 2.2. instrumentation mill type laboratory (panasonic mx -j120-p made in japan). hplc (agilent technologies), equipped with a zorbax c18 column (length 150 mm×4.6mm dpi. 5 μm particle size, 300 å pore size, agilent technologies 1200 series, usa) and coupled online with a uv/vis agilent technologies detector. perkin elmer spectrum 65 ft-ir spectrometer (usa). perkin elmer spectrophotometer, ptfe syring–driven filters (0.22 μm pore size) were provided by biofil (germany). rotary evaporator (heildolph co., germany).   ital. j. food sci., vol 28, 2016 364 2.3. sample preparation sugar beet pulp (sbp) was soaked in water for 3 hours to extract sugar residues and then it was dried in vacuum oven at 40°c for 12 h and ground in a laboratory mill (panasonic mx -j120-p made in japan). the powdered sample was passed through a sieve with mesh size 1 mm was taken for further investigations. 3. preparation of plant extracts 3.1. extraction with methanol methanol was the most commonly used extraction solvent in the assay of phenolic compounds herbs in literatures (dar et al., 2011). in this study, 5 g sbp was mixed in methanol solution (99% v /v) and the extraction of phenolic compounds was performed by reflux for 6 hours at 60°c temperatures. then, the ph of metalonic extracts was adjusted to 2.0, with hcl 6m for lignin precipitation. the mixture was filtered off, and subsequently the filtrate was centrifuged at 9000 rpm for 2 min. the supernatant was filtered and, evaporated to remove excess methanol followed by using a rotary evaporator (heildolph co., germany) under reduced pressure at 40°c. 3.2. extraction with sodium hydroxide in this method, 5 g sbp was placed in an erlenmeyer flask attached to a condenser, and mixed with a 100 ml naoh (1m) solution. it was heated up to 60°c for 6 h and then cooled down to 20° c. once the extraction process was completed, ph was reduced to 2.0, so that the hemicellulose would precipitate. the final mixtures were filtered off, and subsequently 150ml ethyl acetate was added to the filtrates in a 250ml baffled erlenmeyer flask and was shaken in magnet stirrer (300 rpm) at room temperature for15 min to carry out a liquid-liquid extraction (max et al., 2009). the supernatant was vacuum evaporated to remove excess solvent. then the concentrated extract, which contained the phenolic compounds was analyzed for total phenolic compounds. 3.3. determination of total phenolics content the total phenolics content of extracts was determined in accordance with a protocol described by turkmen and velioglu (2005)[15]. 1 ml aliquot of each sample was mixed with 5 ml of folin-ciocalteu reagent (10% in distilled water) in a test tube. after 5 min, 4 ml of sodium carbonate (7.5% in distilled water) were added to each tube, the test tubes were cap-screwed and vortexed. mixtures were kept in dark at ambient conditions for 2 h to complete the reaction. the absorbance was measured at 765 nm with a uv-vis spectrophotometer. gallic acid was used as standard and the analyses were done in triplicate. the results were expressed as mg gallic acid (gae)/g sugar beet pulp extract. 3.4. experimental design response surface methodology (rsm) was used to determine optimum conditions for extraction of ferulic acid from sugar beet pulp. the experiments were designed according to the central composite design (ccd), the most widely used form of rsm. three factors including time (x1), temperature (x2) and sodium hydroxide concentration (x3) were chosen, and ferulic acid concentration (y) were determined using optimization method (table 1).   ital. j. food sci., vol 28, 2016 365 table 1: independent variables and their coded and actual values. parameters symbols coded levels of variables -1.6817(-α) -1 0 1 1.6817(+α) time (hour) x1 2 4.0 7 9.9 12 temperature(°c) x2 30 36.0 45 53.9 60 concentration of naoh(molar) x3 0.5 0.8 1.25 1.69 2 each factor was studied at five different levels (-1.68, -1, 0, 1, 1.68). all variables were taken at a central coded value considered as zero. equation (1) represents the threevariable mathematical model: y= β0+β1x1+β2x2+β3x3+β12x1x2+β13x1x3+β23x2x3+β11x12+β22x22+β33x32 (eq.1) where y is ferulic acid concentration, β0 is the intercept term, β1, β2 and β3 are the linear terms, β11, β22 and β33 are the quadratic terms and β12, β13 and β23 are the interaction terms between three independent variables. the design contains a total of 20 experimental trials. 3.5. determination of ferulic acid concentration the prepared methanolic extracts were passed through 0.22 μm ptfe filters and 10 µl of the filterates were injected into a hplc system, agilent technologies, equipped with a zorbax c18 column (length 150 mm×4.6mm dpi. 5 μm particle size, 300 å pore size, agilent technologies 1200 series, usa) and coupled online with a uv/vis agilent technologies detector. the flow rate was 1.0 ml/min and the oven temperature was 35°c. the mobile phase consisted of methanol and water (1% hac)(65:35, v/v) and the detector was set at 320 nm. all quantitative analyses were made by the external standard method using ferulic acid as an analytical standard. 3.6. conformity tests to declare the mathematical model validity conformity tests were carried out with the same experimental conditions to examine the accuracy of the mathematical model correlations. the error percentage was then calculated according to equation 2 (madadi et al., 2012): error(%) = (actual values − predicted values)/(actual values) (eq.2) 3.7. ferulic acid purification ethanol 95% was added to the brownish extracts to obtain a final concentration of 30% ferulic acid. then, the murky solution was centrifuged at 11,000 g for 20 min (buranov et al., 2009). the supernatant was vacuum evaporated to purify ferulic acid. for further purification of the ferulic acid, it was dissolved in a 6 ml anhydrous ethanol, resulting in a less intense murky solution, and again centrifuged for 20 min at 11,000 g. the supernatant was vacuum evaporated and precipitate was analyzed using fourier transform infra-red (ft-ir) and the methanolic solution of precipitate was injected to high performance liquid chromatography (hplc) (buranov et al., 2009).   ital. j. food sci., vol 28, 2016 366 3.8. ft-ir spectroscopy isolated ferulic acid spectra was recorded on a ft-ir spectrometer in the range of 400-4000 cm-1 using a kbr disc containing 1% finely ground samples (kunst et al., 2003). 3.9. measurement of antioxidant capacity of isolated ferulic acid antioxidant capacity of isolated ferulic acid from sugar beet pulp and pure ferulic acid were performed immediately by abts method. the method used was the abts_+ (radical cation) decolourisation assay (alanon et al., 2011). this assay was based on the ability of different substance to scavange abts cation radical (abts•+(2,2’-azino-bis(3ethylbenzthiazoline-6-sulphonic acid)). the radical cation was prepared by mixing 7mm of abts stock solution with 2.45mm potassium persulfate(1/1,v/v) and leaving the mixture for 4-16h until the reaction was complete and the absorbance was stable. the abts•+ solution was diluted with ethanol to an absorbance of 0.700 ± 0.05 at 734nm for measurement. the photometric assay was conducted on 0.9 ml of abts•+ solution and 0.1 ml of tested sample and mixed for 45 sec; measurements were tacken immediately at 734nm after 15min. the antioxidant activity of the tested sample was calculated by determining the decrease in absorbance by using the following equation: e= ((ac-at)/a)í100 where at and ac are the respective absorbance of tested sample and abts•+ was expressed as µmol. trolox chosen as standard antioxidant and the standard reference curve was constructed by plotting % inhibition value against trolox concentration between 10 and 600 μm. antioxidant activity measurement, expressed as trolox equivalent antioxidant capacity (teac). each sample was measured in triplicate. mean and standard deviation (n = 3) were calculated. 3.10. statistical analysis analysis of variance (anova) followed by duncan’s test was carried out to test for differences between extractants (methanol and alkaline hydrolysis) in the statistical program spss ver. 15.0. significance of differences was defined at the 5% level (p < 0.05). the experimental design and statistical analysis were performed using minitab software (version 16). 4. results and discussion 4.1 total phenolic content significant differences were found in total phenolic contents between two extracts. alkaline extracts contained higher amounts of polyphenols than methanolic extract. the total phenolics content of methanolic and alkaline extract were 121.45 ± 1.32 and 758.638 ± 3.27 mg gae/100 g db, respectively. the results showed that alkaline treatment led to retained higher phenolics, which might be due to an alkaline hydrolysis breaks the ester bond linking phenolic acids to the cell wall and thus is an effective way to release phenolic compounds from polysaccharides. it is clear that chemical processes are more efficient to extract phenolic compounds by hydrolyzing the covalent esteric bonds.   ital. j. food sci., vol 28, 2016 367 in structure of sugar beet pulp cell wall, phenoic compounds such as ferulic and cumaric acids were etherified to lignin and arabinoxylans and forms an alkali-labile cross-link between these two cell wall polymers (torre et al., 2008). such relatively higher content of alkali-labile cross-linkages within the lignin network or between lignin and polysaccharides might explain the fast and easy solubilisation of both phenolic acids by alkaline treatments (noor hasyierah et al., 2011). in other researches, alkaline treatments are commonly used to extract bound phenolic acids and other related compounds from cereal grains, and alkaline hydrolysis totally releases the bound phenolics in a short time at high alkali concentration and temperature (oufnac et al., 2007). phenolic acids such as benzoic and cinnamic acids could not be effectively extracted with pure organic solvents, so mixtures of alcohol-water or alcohol-alkali are recommended. our results are in agreement with the published results (stalikas, 2007; oufnac et al., 2007). 4.2. identification of ferulic acid the intense peak at rt= 3.39 min in the chromatogram of alkaline extracts revealed the presence of ferulic acid in the sugar beet pulp extract. (fig. 1 a and b). figure 1: hplc chromatogram of ferulic acid in c18 reverse phase chromatography at 320 nm. a) standard, b) alkali treated sugar beet pulp extract (peak of ferulic acid = 3.39 min). concentration of ferulic acid were determined according to calibration curve. the concentration of fa in the alkaline hydrolysates obtained in different extraction conditions designed by rsm are presented in table 2. based on the rsm method, result of experimental analysis was presented in table 3. in these results, those effects with calculated p-values less than 0.05 would be significant in the studied range of parameters.   ital. j. food sci., vol 28, 2016 368 table 2: experimental extraction conditions and ferulic acid concentration. run order time (h) t(°c) naoh (m) concentration of ferulic acid (g/100g) x1 x2 x3 1 7.0 45.0 1.25 0.800 2 7.0 45.0 1.25 0.789 3 9.9 53.9 1.69 1.000 4 4.0 53.9 0.80 0.342 5 7.0 45.0 0.50 0.230 6 4.0 53.9 1.69 0.600 7 7.0 45.0 1.25 0.700 8 2.0 45.0 1.25 0.341 9 7.0 60.0 1.25 0.800 10 7.0 45.0 2.00 1.000 11 12.0 45.0 1.25 0.946 12 4.0 36.0 0.80 0.241 13 9.9 53.9 0.80 0.754 14 7.0 45.0 1.25 0.633 15 7.0 45.0 1.25 0.700 16 7.0 45.0 1.25 0.620 17 4.0 36.0 1.69 0.565 18 7.0 30.0 1.25 0.476 19 9.9 36.0 0.80 0.415 20 9.9 36.0 1.69 1.100 based on the results shown in table 3, the extraction of ferulic acid was significantly affected by time (x1), temperature (x2), naoh concentration (x3) and the coupling terms between x2 and x3, while the interaction between terms x1x2 and x1x3, and the second order effect of term x1,x2 and x3 on fa concentration were insignificant (p > 0.05). therefore, after eliminating the statistically insignificant values, the final model is given in eq. (3) as follows: y= -2.10380 + 0.035 x1+0.051x2+ 1.355 x3-0.0158x2x3 (eq.3) the positive and negative signs in front of the terms indicate the synergistic and antagonistic effect of each term on the model response, i.e. fa concentration. the coefficient of determination (r2) for empirical equation from eq. (3) was 0.952.   ital. j. food sci., vol 28, 2016 369 table 3: anova analysis for central composite design. source df seq ss adj ss adj ms f p linear 3 1.11115 1.11115 0.370385 62.14 0.000 a -time 1 0.47184 0.47184 0.471844 79.16 0.000 sign ≤0.05 b-temp 1 0.06196 0.06196 0.061963 10.40 0.009 sign ≤0.05 c concentrati on 1 0.57735 0.57735 0.577348 96.86 0.000 sign ≤0.05 time*time 1 0.00444 0.00733 0.007332 1.23 0.293 temp*temp 1 0.00658 0.00865 0.008651 1.45 0.256 con*con 1 0.01535 0.01535 0.015347 2.57 0.140 interaction time*temp 1 0.00133 0.00133 0.001326 0.22 0.647 time*con 1 0.01523 0.01523 0.015225 2.55 0.141 temp*con 1 0.03188 0.03188 0.031878 5.35 0.043 sign ≤0.05 lack-of-fit 5 0.03109 0.03109 0.006218 1.09 0.463 pure error 5 0.02852 0.02852 0.005703 r2 = 95.21 & α= 0.05 significant parameters (p-value < 0.05). in order to determine the effect of the three independent variables on extraction yield, extraction parameter graph and response surface were generated as a function of two variables, while the third one was held constant at its middle level. the optimum region was determined in terms of maximum concentration of ferulic acid (fig. 2). to assess the quality of the model and to measure how well the suggested model fit the experimental data, the parameters, f-value, lack of fit and r2 were used (montgomery, 2005). in our research f-values were 82.85 and lack of fit of the model was not significant, which implied that the models were significant. the determination coefficients (r2>0.95) were obtained from anova of the quadratic regression models, indicating that less than 4.8% of the total variations was not explained by the suggested model. 4.3. effect of the each process parameter on ferulic acid extraction 4.3.1. temperature as shown in fig. 3, increasing the extraction reaction temperature may cause higher concentration of ferulic acid released, while the other two factors (i.e. time & akali concentration) were constant. it is well known that sugar beet root contains significant quantities of ferulic acid which is etherified to lignin and /or arabinoxylans and through alkali-labile cross-linkages (scalbert, 1986). therefore, cleavage of these bonds at the same time may be possible at higher temperatures. an increase in temperature from 30 to 60°c and time from 2 to 12 hr resulted in an increment of fa from 0.3 to 0.9g/100g when alkali concentration was fixed at the middle level (fig. 2a).   ital. j. food sci., vol 28, 2016 370       figure 2: 3d response surface plot of the interactions. (a) time and concentration when temperature was fixed at 45°c, (b) temperature and time when concentration was fixed at 1.25 m, (c) concentration and temperature when time was fixed at 7 h xu investigations (2005) showed that an increase in alkaline treatment temperature had an important effect on the release of ester-linked p-coumaric and ferulic acids from the cell walls of various cereal straws.   ital. j. food sci., vol 28, 2016 371 figure 3: interactions between extraction parameters naoh concentration and temperature(a), naoh concentration and time(b), time and temperature (c). 4.3.2. naoh concentration as presented in fig. 2b, the ferulic acid concentration significantly increased with naoh concentration. this is because raising the alkaline treatments may dissolve lignin by cleavage of ester linkages in lignin–polysaccharide complexes, which lead to the release and solubilization phenolic acids. different alkaline compounds such as naoh, ca(oh)2, nh4oh, and h2o2 have been previously utilized as catalysts for hydrolysis treatments (rodriguez-vazque et al., 1994). in the present study, naoh was selected because of its more selective action, compared to the other agents, in releasing phenolic compounds such as ferulic acids (torre et al., 2008). it may be obvious that using a too mild alkaline condition may not act effectively for ferulic acid extraction.these results have also been confirmed in previous studies (mussatto et al., 2007; torre et al., 2008). the ffect of time in alkaline hydrolysis on the extracted fa concentration is shown in fig. 2b. by increasing reaction at constant temperature, alkali has more time to release ferulic acid. a positive interaction between the time and naoh concentration and the time and temperature process on the extracted ferulic acid has been observed (figs. 3 b and c), however for higher concentrations of naoh (1.69m), the slope of graph was increased as compared to the low concentrations (0.8 m). at higher concentration of naoh, accelerated solubilisation of ferulic acid has been observed. it may be emphasized that ferulic acid solubilization exhibited a timedependent behaviour and reached a maximum concentration after 12 h of hydrolysis with 2.0n naoh (fig. 3 b). but the main point that should be considered is that severe alkali concentration has a negative dissociation effect on the fa for all temperature / time conditions (fig. 2c and figs. 3 a and b). according to the obtained results, the main key factors affecting the releasing rate of phenolic compounds are alkali concentration and hydrolysis duration. the same result has previously been reported for the effect of these factors on the yield of extraction from the other sources such as paddy straw, sugar cane baggas and agricultural wastes (noor hasyierah et al., 2011; xu et al., 2005; tilay et al., 2008).   ital. j. food sci., vol 28, 2016 372 according to the results in table 3, interaction between temperature and naoh concentration (x2x3) had a statistically significant effect on the extraction yield while the others (i.e: x1x3 and x1x2) were insignificant. increasing the temperature from 36°c to 53°c at a constant time (7 h) and low concentration of alkali (0.8 m) improved the ferulic acid extraction (from 0.32 to 0.54 g/100g fa), but at the same temperature range, using high concentration of naoh caused a reduction of fa concentration (fig. 3a). this is because of the opposite effect of temperature and concentration of naoh on fa extraction, which is shown in eq. (3) by negative sign of the term, x2x3. a clear decrease in ferulic acid concentration took place after the threshold was exceeded and confirmed this negative interaction, so that when temperature was increased from 45 to 60 °c and alkali concentration was raised from 0.5 to 2.0m, an oxidative degradation might happen (fig. 2c). no significant degradation was detected at the lowest alkali level (0.5m). it has also been previously shown that ferulic acid, in its monomeric form, is more resistant to oxidation than its dimeric form during alkaline hydrolysis (bauer et al., 2012). finally, it may be emphasized that the amount of ferulic acid obtained through ccd in the present research was 1.29 g/100 g, which is higher than the maximum amount reported by zhao (2008) in sugar beet pulp (0.800 g/100 g); and this improvement may be mainly due to the optimum condition selected for the extraction process. normal probability plot of residuals produced an approximately straight line which indicated a normal distribution of residuals (the deviation between predicted and actual values) and confirmed the accuracy of the model (noor hasyierah et al., 2011). 4.4. validating the optimal conditions the optimum conditions, as suggested by the software, were 41°c, 2 m naoh, 12 h corresponding to 1.33 g/100 g (predicted value). for verification of these conditions, four replicates were carried out to determine the highest experimental value of ferulic acid. the highest ferulic acid production obtained at these conditions was 1.29 g/100 g. the results of the conformity test on the basis of determining the error percentage demonstrated that process optimization in ccd reliably predicted the fa with acceptable accuracy(3.1%). 4.5. ft-ir spectra and validation of precipitate in the purification stage, the pericipiate that obtained from alkaline extract of 5 g sugar beet pulp was analysed by ft-ir and hplc method. the ft-ir spectrum of the precipitate was compared with the spectrum of the pure ferulic acid to confirm it (fig. 4). the ft-ir spectrum of sample clearly showed the existence of main functional groups in the ferulic acid structure, and the strong and broad band at 3,331.08cm −1 is characteristic of the oh group in phenolic compound. a part from this, c-h stretching of the aromatic ring was at the 2925.82 cm −1 band. the band at 1,649.02 cm-1 corresponds to that of the carbonyl group (c = o). stretching band at 1,328.87 cm −1 is characteristic of c-h vibration on the methyl group, while the vibration for c = c on the aromatic ring is at 692.77 cm−1 band (robert, 1998).   ital. j. food sci., vol 28, 2016 373 figure 4: ft-ir spectra of ferulic acid (isolate and pure). the precipitate was then analyzed using high performance liquid chromatography (hplc) to reaffirm the results as characterized by the ir analysis. 0.15 mg of precipitate was collected and dissolved in 10 ml of methanol solution acting as the solvent before the analysis. finally, the 10 µl of concentrated sample was injected into a hplc machine. the validation was performed with sample of precipitate based on the relative retention times (rrts) and relative peak areas (rpas). the percent of recovery was 64.88% and precision was assessed by analyzing three replicate samples and the relative standard deviation (rsd) was below 2.02% and 4.47% for rrts and rpas, respectively. 4.6. measurement of antioxidant capacity of isolated ferulic acid antioxidant capacity results expressed as µmol of trolox equivalents per milligram of samples. the abts cation radical (abts•+) (pisoschi and negulescu2011) which absorbs at 734 nm (giving a bluish-green colour) is formed by the loss of an electron by the nitrogen atom of abts (2,2’-azino-bis(3ethylbenzthiazoline-6-sulphonic acid)). in the presence of trolox (or of another hydrogen donating antioxidant), the nitrogen atom quenches the hydrogen atom, yielding the solution decolorization. antioxidant capacity for isolated and pure ferulic acid was 0.39±0.01 and 0.55±0.01 respectively. significant differences were found at a significance level of p < 0.05 between isolated and pure samples in the antioxidant capacity values which indicates that precipitates need more purification stage to made pure. result showed that sugar beet pulp is potent source of ferulic acid that can be extracted and use as an antioxidant. 5. conclusions the present study demonstrated that alkaline treatment led to release higher phenolic compounds than methanolic method and the results showed that temperature, time and naoh concentration had significant effects on the ferulic acid solubilization in alkaline media for extraction. the coefficient of determinations (r2) for predicted ferulic acid   ital. j. food sci., vol 28, 2016 374 content showed good correlation with the experimental data at 95% confidence level. the amount of extracted ferulic acid at optimized conditions obtained from the model (i.e: 12 h, 41°c and 2 m) was 1.29 g/100 g. the ft-ir spectrum of isolated sediment clearly showed the existence of main functional groups in the ferulic acid structure. significant differences were found at a significance level of p < 0.05 between isolated and pure ferulic acid in the antioxidant capacity values which indicates that precipitates need more purification stage to made pure. in conclusion suggest that sugar beet pulp is potent source of ferulic acid that can be extracted and use as an antioxidant. references alnon m.e., castro-vazquez l., diaz-maroto m.c., gordon m.h. and pérez-coello m.s. a study of the antioxidant capacity of oak wood used in wine ageing and the correlation with polyphenol composition. 2011food chemistry 128:997-1002. bauer j.l., harbaum-piayda b. and schwarz k. phenolic compounds from hydrolyzed and extracted fiber-rich byproducts. 2012. lwt food science and technology 47:246-254. buranov a.u. and mazza g. extraction and purification of ferulic acid from flax shives, wheat and corn bran by alkaline 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baggasse. 2005. anal. chem. acta 552:207-217. zhao z.h. and moghadasian m.h. chemistry, natural sources, dietary intake and pharmacokinetic properties of ferulic acid: a review. 2008. food chemistry 109: 691-702. paper received may 6, 2015 accepted october 26, 2015 ijfs#1198_bozza ital. j. food sci., vol. 31, 2019 54 paper effects of chia (salvia hispanica l.) seed roasting conditions on quality of cookies h.b. o.1, k.y. song1, k.y. joung1, s.y. shin1 and y.s. kim*1,2 1department of integrated biomedical and life sciences, korea university, 145 anam-ro, seongbuk-gu, seoul 02841, republic of korea 2department of food & nutrition, korea university, 145 anam-ro, seongbuk-gu, seoul 02841, republic of korea *e-mail address: kteresa@korea.ac.kr abstract our aims were to analyze physical changes and antioxidant properties of chia seeds roasted under various conditions (160-200°c, 5-15 min) and to investigate the effects on quality characteristics of cookies. weight loss and water-holding capacity rapidly changed after roasting at 180°c. fatty acid composition showed no significant change, while antioxidant activity of roasted seeds increased. cookies were prepared by replacing 3% of flour with roasted chia seeds (180°c, 0-15 min). baking loss, hardness, and brightness were inversely proportional to roasting time. roasting of chia seeds affected texture and sweetness scores in a consumer preference test. keywords: chia seed, cookie, cooking quality, roasting, sensory evaluation ital. j. food sci., vol. 31, 2019 55 1. introduction cookies are low-moisture, tasty, and crispy baked products comprising three main ingredients: flour, sugar, and butter. cookies are loved by all generations owing to the unique taste and long shelf life. the increase in the awareness about a healthy lifestyle and nutrition among consumers has encouraged many studies on the nutritional ingredients of cookies (jan et al., 2016; park et al., 2015). seeds of chia (salvia hispanica l.), an annual plant originating from central america, were used as a staple food by ancient aztecs in pre-columbian times (valdivia-lópez and tecante, 2015). chia seeds are rich in protein (15-25 g per 100 g), fats (30-33 g per 100 g), dietary fiber (18-30 g per 100 g), and unsaturated fatty acids (17.83 g per 100 g) (álvarez-chávez et al., 2008; martínez-cruz and paredes-lópez, 2014). in addition, chia seeds exert a strong antioxidant effect, owing to the presence of phenol compounds such as quercetin, kaemferol, caffeic acid, and chlorogenic acid (reyescaudillo et al., 2008; taga et al., 1984). for culinary uses, chia seeds are processed into flour, seed oil, or whole seeds. studies on the application of chia seeds to bread, ice-cream, pound cake, and sausage have been carried out (campos et al., 2016; lee, 2013; pizarro et al., 2013; scapin et al., 2015). the ancient aztec roasted chia seeds and used them in the preparation of chiapinolli, a type of flour used in tortillas, tamales, and beverages (cahill, 2003). roasting is a foodprocessing method employed to impart a unique flavor and color to a food. roasting is mainly used for the manufacture of coffee, cocoa, and barley tea. roasting promotes extraction of seed oils and antioxidants owing to modification of the cellular structure of the seed (kim et al., 2002). in addition, roasting is accompanied by a browning reaction, resulting in the production of brown pigments and aroma components. these aminocarbonyl reactants are known to have antioxidant properties and to improve the taste and flavor of the food (dewanto et al., 2002; lin et al., 2016). given the changes in the characteristics of chia seeds after roasting, different qualities of cookies with chia seeds may be obtained by controlling roasting conditions. few studies have shown the changes in roasted chia seeds and their applications in food industry. the aims of this study were to investigate the effects of roasting on physicochemical and antioxidant properties of chia seeds and to find the optimal roasting conditions by evaluation of the quality characteristics of cookies containing roasted chia seeds. 2. materials and methods 2.1. raw materials soft flour (cj cheiljedang co., ltd., incheon, korea), sugar (cj cheiljedang co., ltd.), butter (seoul dairy co., ltd., seoul, korea), and eggs were purchased at a retail market located in seoul to prepare cookies. chia seeds, produced in paraguay in september 2014, were purchased from a supplier (chowonherb, seoul, korea). 2.2. roasting the roasting temperature was set to 160°c, 180°c, or 200°c. chia seeds (10 g) were roasted for 5, 10, or 15 min in an oven (zippel de68-04072d, samsung, seoul, korea). roasted chia seeds were sufficiently cooled at room temperature (25°c) and stored in the freezer (-20°c) until analysis. ital. j. food sci., vol. 31, 2019 56 2.3. physical analysis of chia seeds changes in the mass of chia seeds during roasting were measured using a scale (libror eb-2200hv, shimadzu, kyoto, japan). the water-holding capacity (whc) of the roasted chia seeds was measured by the modified method of alfredo et al. (2009). briefly, 1 g of chia seeds was placed in a flask containing 10 ml of distilled water in a water bath (bs-20, jeio tech, gimpo, gyeonggi) for 24 h incubation at 25°c. the suspension was centrifuged (universal 32 r, hettich, tuttlingen, germany) at 3,000 rpm for 20 min, and the supernatant was weighed. whc was expressed as the weight of water held per gram of the sample. the browning index (bi) was measured by the method of maskan (2001). briefly, 10 g of roasted chia seeds was placed on a petri dish (∅ 90 mm × 15 mm). color values (cie l*, a*, b*, and ∆e) of the chia seeds on the petri dish surface were measured with a colorimeter (cr-400, konica minolta, osaka, japan) in triplicate. chromameter was calibrated with a standard whiteboard (l = 96.90, a = 0.45, b = 1.49). bi was calculated via the following formula: browning index (bi) = [100(𝑥 − 0.31)] 0.17 𝑥 = (𝑎 + 1.75𝐿) (5.645𝐿 + 𝑎 − 3.012𝑏) 2.4. analysis of fatty acids of chia seeds the fatty acid composition of chia seeds was analyzed for fatty acid methyl esters (fames) by gas chromatography with the methods of aocs ce 2-66 and ce 1-62 (aocs, 1998). an agilent technologies 7890n gas chromatograph with a flame ionization detector and a fused silica capillary column (sp™-2560, 100 m × 0.25 mm internal diameter [i.d.] × 0.2 μm film thickness, supelco) was used for the analysis. the operating conditions were as follows: split ratio 200:1, flow rate 1.0 ml he/min, injector temperature 225°c, detector temperature 285°c, initial oven temperature 100°c for 4 min, and endpoint oven temperature 240°c for 17 min (an increase at a rate of 3°c/min). fames were identified by comparing their retention times with those of the standards, and their relative concentrations were calculated as grams per 100 grams of a sample. 2.5. content of total phenols and flavonoids total polyphenol content of roasted chia seeds was analyzed by the folin-denis method (singleton and rossi, 1965). the results were expressed in terms of gallic acid equivalents (mg gae/g). total flavonoid content was measured by the method suggested by davis (1947) and expressed as quercetin equivalents (mg qe/g). 2.6. antioxidant activities of chia seeds dpph (1,1-diphenyl-2-picrylhydrazyl) antioxidant activity was measured by the method of molyneux (2004), and abts (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging activity was measured by the procedure of re et al. (1999). ascorbic acid (sigma aldrich, darmstadt, germany) was used as a reference. the percentage inhibition at various concentrations (100, 50, 33.3, 25, 20, and 16.66 mg/ml) of each ital. j. food sci., vol. 31, 2019 57 sample was calculated using the following formula to estimate the half-inhibitory concentration (ic50; mg/ml) values of dpph and abts: 𝑃𝑒𝑟𝑐𝑒𝑛𝑡𝑎𝑔𝑒 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 𝐴!"#$%"& − 𝐴!"#$%& 𝐴!"#$%"& ×100 where acontrol is the absorbance of 100 µl of ethanol, asample is the absorbance of a 100 µl sample. 2.7. cookie preparation raw chia seeds (group rt0) and chia seeds roasted at 180°c for 5 min (group rt5), 10 min (rt10), and 15 min (rt15) were freeze-dried (fd8508, ilshin biobase co., ltd., gyeonggi, korea). the unroasted and roasted seeds were pulverized by a high-speed grinder (crt04, hungchuan machinery enterprise, taipei, taiwan) and filtered through a 40-mesh sieve. cookies were prepared by the aacc method 10-52 (aacc, 2000) from flour (300 g), butter (180 g), sugar (120 g), and eggs (60 g). each chia seed powder (groups rt0, rt5, rt10, and rt15) was added to cookies via replacement of 3% (9 g) of the flour. butter was creamed by means of a mixer (km400, kenwood, havant, britain) and mixed with sugar and eggs for 5 min. sieved flour and chia seed powder were added to the mixture. the cookie dough was rolled out, cut into a cylindrical shape (∅ 40 mm × 5 mm), and baked for 20 min at 170°c in an oven (zipel de68-04072d, samsung, seoul, korea). the cookies were cooled for 1 h at room temperature (25°c) and then subjected to analysis. 2.8. cookie properties 2.8.1 dough density, baking loss, the spread factor, and ph dough density was measured as an increase in the volume of water. baking loss of cookies was calculated by the comparison between cookie mass and dough mass. the spread factor of cookies was calculated by the procedure of aacc (2000). briefly, six randomly selected cookies were stacked in a line, and their diameter and thickness were measured. the spread factor was calculated by dividing the diameter of a cookie by its thickness. the ph level of dough was measured with a ph meter (sp-701, suntex instruments co., ltd., taipei, taiwan). 2.8.2 quantification of the color of cookies the photographs of cookies with roasted chia seeds were taken by a digital camera (canon ixus 500, tokyo, japan). color values (cie l*, a*, and b*) of six cookies randomly selected from each group were evaluated with a chromameter. the total color difference (∆e) values were calculated as follows: ∆𝐸 = (∆𝐿)! + (∆𝑎)! + (∆𝑏)! where ∆l, ∆a, and ∆b are the difference of l, a, and b value between white board (l: 96.90, a: 0.45, b: 1.49) and sample, respectively. ital. j. food sci., vol. 31, 2019 58 2.8.3 hardness of cookies this parameter was measured repeatedly 15 times for each sample using a rheometer (compac-100ii rheometer sun, sun scientific co., ltd., tokyo, japan) with a no. 5 probe (∅ 5 mm). the operating conditions were as follows: mastication test mode (mode 20), 5 mm distance, and 120 mm/min table speed. 2.9. sensory evaluation a consumer preference test of the cookies was conducted by a panel of 30 people (age 2535 years, 15 males and 15 females). samples were served on a white plate with water. cookies were evaluated for appearance, flavor, texture, an oily taste, sweetness, savory taste, and aftertaste. a method with a 7-point scale, 1 = strongly dislike and 7 = strongly like, was employed to measure the seven parameters. 2.10. statistical analysis all results obtained by measurements were subjected to one-way analysis of variance (anova) in the spss software ver. 23.0 (spss inc., chicago, il, usa). data are presented as mean ± standard deviation (sd). the significance of each experimental value was analyzed by duncan’s multiple-range test (p < 0.05). 3. results and discussion 3.1. physical analysis of chia seeds the mass loss and whc of roasted chia seeds are shown in table 1. at all temperatures, the mass loss increased with roasting time. the mass of roasted chia seeds decreased with an increase in the temperature. in particular, the mass loss of chia seeds roasted at 180°c or 200°c was significantly higher than that of the unroasted samples. wang and lim (2014) described weight loss as a general indicators for determining the roasting degree, and it was divided into two stages: the first stage mainly due to vaporize, and the another stage by formation of co2 and volatiles compounds. since the moisture content of the raw chia seed was 7.00% (data not shown), further mass reduction can be presumed to be due to several volatile compounds such as co2, aldehydes, ketones, alcohols and pyrazines produced by the maillard reaction between sugars and amino acids (xiao et al., 2014). whc of the roasted chia seeds significantly decreased with roasting time. protein denaturation and extraction of seed surface oil during heat treatment may contribute to the rapid decrease in whc. öztürk et al. (2002) reported that whc affects the hardness and spreadability of cookies. thus, the process of roasting of chia seeds was expected to affect the quality of cookies. during the roasting process, the food color gradually darkened due to the formation of a brown pigment from the maillard reaction and caramelization. this change is related to the roasting temperature and time, which are the major parameters that control roasting conditions and processes (kahyaoglu and kaya, 2006). the bi measurement results on chia seeds roasted at 160°c, 180°c, and 200°c are presented in fig. 1. an increase in the bi is an indicator of the nonenzymatic browning process such as the maillard reaction and caramelization (helou et al., 2016). the bi showed no significant change at 160°c but increased at temperature >180°c as a function of roasting time. these results indicated that the maillard reaction proceeded actively in chia seeds above 180°c. ital. j. food sci., vol. 31, 2019 59 table 1. mass loss and water-holding capacity (whc) of chia seeds roasted under. roasting condition mass loss (g / 100 g) whc (g of water retained /g of sample) temperature (℃) time 0 7.67±0.01a 160 5 3.27±0.31f 7.16±0.01b 10 5.93±0.12d 6.71±0.00c 15 6.87±0.50c 3.99±0.00e 180 5 5.53±0.50de 7.69±0.01a 10 6.93±0.50c 4.59±0.00d 15 7.93±0.31b 3.84±0.01e 200 5 5.00±0.72e 7.65±0.01a 10 7.47±0.23bc 3.88±0.01e 15 9.40±0.60a 3.95±0.01e a,b,c,d,e,fmeans with different superscript letters in each column are significantly different according to duncan’s multiple-range test (p < 0.05). figure 1. changes in the browning index (bi) of roasted chia seeds with roasting time. 3.2. fatty acid composition it is known that the fatty acid composition of seed oil determines the physicochemical and nutritional characteristics of seed oil, and can be changed by roasting (hama, 2017). there was no significant difference in the fatty acid composition between raw chia seeds and those roasted under different conditions (table 2). it is expected that there will be no significant change in the quality of seed fat in the temperature and time conditions that we ital. j. food sci., vol. 31, 2019 60 set. yoshida and takagi (1997) reported no significant difference in the quality of sesame oil roasted at temperatures below 200°c. because the roasting conditions in our study were set within the range of normal baking temperatures (160-200°c), the mechanism underlying the change in the fatty acid composition after roasting at higher temperatures and for longer periods remains unclear. yen (1990) reported that the fatty acid composition of sesame, which is similar to that of chia seeds, changed rapidly (to linoleic acid in particular) after roasting at temperatures above 240°c. further studies are needed to evaluate the effect of roasting of chia seeds above 200°c. table 2. fatty acids composition of chia seeds roasted under various temperature and time conditions. roasting condition palmitic acid (g/100 g) stearic acid (g/100 g) oleic acid (g/100 g) linoleic acid (g/100 g) α-linoleic acid (g/100 g) temperature (℃) time 0 6.28±0.04ns 3.26±0.10ns 6.94±0.22ns 18.90±0.99ns 65.22±0.06ns 160 5 6.31±0.19 3.24±0.02 6.86±0.09 18.40±0.00 64.76±0.31 10 6.25±0.03 3.19±0.01 6.74±0.07 18.33±0.16 65.06±0.19 15 6.55±0.48 3.37±0.15 6.70±0.17 18.42±0.18 64.10±0.92 180 5 6.26±0.19 3.23±0.04 6.80±0.10 18.44±0.07 64.85±0.15 10 6.27±0.07 3.26±0.01 6.83±0.07 18.47±0.05 64.90±0.05 15 6.21±0.07 3.22±0.07 6.76±0.09 18.33±0.15 65.06±0.37 200 5 6.33±0.00 3.32±0.03 6.85±0.00 18.78±0.02 64.34±0.00 10 6.18±0.05 3.21±0.01 6.76±0.07 18.43±0.27 64.99±0.36 15 6.49±0.07 3.33±0.06 7.03±0.13 18.74±0.33 64.12±0.68 ns = not significant in each column according to duncan’s multiple-range test (p < 0.05). 3.3. antioxidant activities of chia seeds polyphenol compounds act as antioxidants and can be obtained from fruits, vegetables, and plants. table 3 shows that the total polyphenol and flavonoid content of chia seeds increased with roasting time. these results were similar to those observed for roasted almonds and sesame seeds (jeong et al., 2004; lin et al., 2016). the amino-carbonyl products formed by the maillard reaction act as new antioxidants, thereby enhancing the antioxidant properties (dewanto et al., 2002; nicoli et al., 1999). lee et al. (2013) reported higher total polyphenol and flavonoid contents for green beans as compared to coffee extracts roasted at 190°c. nonetheless, the reverse observation at a high temperature was reported (over 200°c). under all temperature conditions, the ic50 value of dpph and abts decreased with roasting time (table 3). although no significant difference was observed in the ic50 of dpph at 180°c and 200°c, the antioxidant activity tended to increase with an increase in roasting temperature. jeong et al. (2004) mentioned that roasting of sesame seeds at different temperatures and for various periods enhances the antioxidant activities, which positively correlate with the production of melanoidin. durmaz and alpaslan (2007) demonstrated an increase in the antioxidant activity after the maillard reaction. overall, the roasting process was able to enhance the antioxidant activity of chia seeds. ital. j. food sci., vol. 31, 2019 61 table 3. antioxidant activities of chia seeds roasted under various temperature and time conditions. roasting condition total polyphenols (μg gae/ g) total flavonoids (μg qe/ g) dpph ic50 (mg/ml) abts ic50 (mg/ml) temperature (℃) time 0 358.00±5.62f 286.07±2.68e 26.99±9.51a 38.70±0.62a 160 5 369.70±3.12ef 299.21±17.05e 14.94±0.56b 33.96±0.70b 10 438.14±24.01d 359.51±6.50c 14.75±0.79b 28.32±1.28d 15 512.87±1.56ab 394.25±16.34b 11.68±0.63b 23.75±0.46f 180 5 383.21±6.79ef 299.38±5.44e 15.29±1.24b 31.08±2.15c 10 476.85±48.73c 351.49±11.96c 12.50±0.11b 24.20±0.71ef 15 517.37±8.68ab 380.41±19.84b 10.32±0.19b 21.36±1.01g 200 5 393.12±6.80e 323.37±4.76d 13.47±0.19b 25.44±1.15ef 10 493.96±5.63bc 392.93±7.05b 12.47±0.32b 25.94±1.30e 15 538.09±8.11a 421.58±5.20a 11.67±0.29b 21.36±1.01g a,b,c,d,e,f,gmeans with different superscript letters in each column are significantly different according to duncan’s multiple-range test (p < 0.05). 3.4. cookie properties 3.4.1 dough density, baking loss, the spread factor, and ph on the basis of the above results, we roasted chia seeds at 180°c for 5, 10, or 15 min for further experiments. table 4 shows the properties of cookies containing roasted chia seeds. the density and ph of the dough are major indicators of cookie quality, owing to their effects on the hardness, flavor, and color of a cookie (hadinezhad and butler, 2009). no significant difference was observed in dough density (range 1.23-1.26) among the treatment groups. the duration of roasting of chia seeds had no significant effect on ph of the dough; however, ph of the control (6.63) was slightly higher as compared to that of other groups. this observation may be related to the ph difference between the chia seed powder (5.42) and wheat flour (6.82). the baking loss of cookies containing chia seeds, including unroasted seeds, was lower as compared with that of the control (15.82%). these results indicated that the amount of water released during the baking process was smaller because the moisture content (7.00%) of chia seed powder was lower than that of wheat flour. the spread factor determines cookie quality, and high spreadability is indicative of a better cookie (miller and hoseney, 1997). the spread factor was the lowest in group rt0; rt10 and rt15 had a higher spread factor than the control did. cookie spreadability tends to decrease with an increase in the concentration of dietary fiber, owing to the increase in the whc of cookies (mancebo et al., 2015). studies have shown 34.4 g of dietary fiber per 100 g of chia seeds (muñoz et al., 2013), explaining the lower spread factor for rt0 as compared with that of the control. nevertheless, whc significantly decreased after roasting of chia seeds, suggesting that spreadability increased with roasting time. ital. j. food sci., vol. 31, 2019 62 table 4. properties of cookies containing roasted chia seed powders. property density (g/ml) ph baking loss (g/ 100 g) spread factor l a b δe hardness (n) control 1.23±0.02ns 6.63±0.03a 15.82±0.01a 6.15±0.17ab 69.97±1.06a 0.10±1.12b 28.44±0.16a 38.03±0.83d 31.28±3.18b rt0 1.26±0.02 6.41±0.01b 13.03±0.22c 5.65±0.13c 64.57±1.24b 0.60±0.91b 23.33±0.17c 38.98±0.97d 36.19±0.36a rt5 1.24±0.02 6.31±0.02c 13.61±0.00b 5.99±0.07b 62.36±1.23c 1.22±0.25b 23.35±0.37c 40.85±0.86c 32.00±1.14b rt10 1.26±0.02 6.32±0.01c 13.68±0.00b 6.32±0.20a 60.98±0.29c 2.96±0.52a 23.96±0.47b 42.43±0.03b 31.18±1.35b rt15 1.26±0.02 6.33±0.03c 13.63±0.00b 6.22±0.01a 57.74±0.18d 4.11±0.14a 22.64±0.14d 44.66±0.18a 31.54±1.05b control: without added chia seeds, rt0: with chia seeds (raw), rt5: with roasted chia seeds (180°c, 5 min), rt10: with roasted chia seeds (180°c, 10 min), rt15: with roasted chia seeds (180°c, 15 min). a,b,c,dmeans with different superscript letters in each row are significantly different according to duncan’s multiple-range test (p < 0.05). ns = not significant. ital. j. food sci., vol. 31, 2019 63 3.4.2 quantification of the color of cookies a photograph of the cookies is presented in fig. 2. longer roasting time of chia seeds corresponded to darker and larger cookies. the l (lightness) value of the control sample was higher than that of the cookies with chia seeds and tended to decrease with roasting time. the a (redness) value was higher in groups rt10 and rt15 (2.96 and 4.11, respectively). the b (yellowness) value was significantly lower for cookies with chia seeds as compared with that of the control (28.44). ∆e (total color difference) was the lowest (38.03) in the control group and increased with roasting time. the dark color of cookies is attributed to the maillard reaction or caramelization (walker et al., 2012). the dark color of chia seeds affected ∆e of cookies. higher ph of cookies contributes to a better browning reaction (martins et al., 2000). figure 2. photographs of cookies containing roasted chia seed powders. control: without added chia seeds, rt0: with chia seeds (raw), rt5: with roasted chia seeds (180℃, 5 min), rt10: with roasted chia seeds (180℃, 10 min), rt15: with roasted chia seeds (180℃, 15 min). 3.4.3 hardness of cookies this parameter is known to be influenced by moisture content, pore development, and density of cookie dough (chabot, 1979). as illustrated in table 4, the difference in hardness between groups control and rt0 was likely to be associated with the high concentration of dietary fiber (in chia seeds) that increases whc. in comparison to rt0, groups rt5, rt10, and rt15 showed a decreasing trend of hardness; this phenomenon may be due to the inverse relation between hardness and moisture retention. whc of roasted chia seeds decreased with roasting time. sugar loss during the maillard reaction (wong et al., 2008) is reported to affect the hardness of cookies. our results are in line with those reported by vetter et al. (1986) who found a positive correlation between cookie hardness and the amount of added sugar. 3.5. the consumer preference test table 5 shows the results of the survey of consumer preferences regarding the cookies containing chia seeds powder. there were no significant differences in the appearance, flavor, oily taste, sweetness, savory taste, and aftertaste among all the groups. nonetheless, groups rt5, rt10, and rt15 yielded higher texture scores than the control group did, whereas rt0 had the lowest score. hardness was found to be the highest for rt10 (36.19 n). these results are similar to those reported in another study (on cookies containing oak mushroom powder), wherein an inverse relation was observed between mechanical strength and texture preference (jung and joo, 2010). on the other hand, our ital. j. food sci., vol. 31, 2019 64 results contradict the observations reported by joo and choi (2012). as a consequence, rt5 and rt15 showed a high score in overall preference. table 5. sensory preference scores for cookies containing roasted chia seed powders. appearance flavor texture overall preference control 5.25±1.25ns 4.70±1.34ns 4.60±1.35ab 4.66±1.33b rt0 4.75±1.07 4.75±1.45 4.05±1.61b 4.61±1.30b rt5 5.00±1.08 5.05±1.05 5.20±1.24a 5.09±1.10a rt10 4.70±1.17 4.90±1.41 5.45±1.28a 4.97±1.25ab rt15 4.90±1.29 4.95±1.43 5.35±1.27a 5.16±1.27a control: without added chia seeds, rt0: with chia seeds (raw), rt5: with roasted chia seeds (180°c, 5 min), rt10: with roasted chia seeds (180°c, 10 min), rt15: with roasted chia seeds (180°c, 15 min). a,bmeans with different superscript letters in each column are significantly different according to duncan’s multiple-range test (p < 0.05). ns = not significant. 4. conclusions in this study, the effects of roasting conditions on chia seeds and cooking quality of cookies containing chia seeds were investigated. this study confirmed that roasting at 160200°c changes whc, the bi, and color as well as increases the antioxidant activity of chia seeds. cookies with roasted chia seeds, especially rt10 and rt15, were superior in spreadability, hardness and overall preference than control. in conclusion, roasting chia seeds for 10 min at 180°c is preferable for making cookies. references aacc international. 2000. “approved methods of the american association of cereal chemists” 10th ed. aacc international, st. paul, mn, u.s.a. alfredo v.o., gabriel r.r., luis c.g. and david b.a. 2009. physicochemical properties of a fibrous fraction from chia (salvia hispanica l.). lwt-food sci technol. 42:168. álvarez-chávez l.m., valdivia-lópez m.d.l.a., aburto-juarez m.d.l. and tecante a. 2008. chemical characterization of the lipid fraction of mexican chia seed (salvia hispanica l.). int. j. food prop. 11:687. aocs. 1998. “official methods and recommended practices of the aocs” 5th ed. american oil chemists’ society, champaign, il, u.s.a. cahill j.p. 2003. ethnobotany of chia, salvia hispanica l. 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environmental and food sciences, university of molise, via de sanctis, 86100 campobasso, italy e-mail address: antomac@unimol.it abstract the aim of this research is to study the effects of different storage conditions on spanish (alkaline debittering) and natural (directly brined) green olives. laboratory-processed olives were stored in 6% brine or in a vacuum bag without brine, at 6 or 20°c. after 18 months, natural olives showed higher microbial and olive oil stability than naoh-treated olives. the lower ph (<4.80) and higher total phenol content (0.2 g/100 g wet pulp) influenced positively the long shelf life of natural olives. the packaging in 6% nacl brine and in a vacuum bag stored at 20°c gave better performance, while growth of psychrophilic spoilage bacteria occurred at cold temperature. keywords: microbial count, natural green olives, oil oxidation, phenols, sensory profile ital. j. food sci., vol. 30, 2018 415 1. introduction world consumption of table olives (to) continues to increase, passing from 957 to 2,480 thousand tones in the period from 1990/91–2014/15 (iooc, 2017). major producer countries are located in the mediterranean basin (spain, greece, italy, turkey, tunisia, morocco, egypt, and algeria). green spanish-style, natural black greek-style and black ripe californian are the most popular to types; however, many other typologies are produced according to local traditions (ercolini et al., 2006). in fact, to types can vary depending on olive fruit kind (green or black olives), debittering method (chemical or biochemical), fermentation conditions (temperature, levels salt, and type of acid) and finally, canning (sanchez et al., 2006). debittering and fermentation are the main steps of the table olive process. the first is fundamental to remove natural bitterness, while the fermentation is important to prevent microbial spoilage. the typical strong bitter and pungent taste of green olives is due to the high presence of oleuropein, a secoiridoid containing elenolic acid glucoside linked to hydroxytyrosol (ambra et al., 2017; ghanbari et al., 2012). in the chemical process, such as the spanish-style method, olives are debittered in a few hours through a bath in a sodium hydroxide solution that hydrolyzes oleuropein quickly into less bitter compounds (charoenprasert and mitchell, 2012); subsequently, olives are water-washed repeatedly and then brined in 8-10% nacl where a spontaneous lactic acid fermentation occurs in the following months (aponte et al., 2010). olive fermentation is caused by indigenous microbiota, including lactic acid bacteria (lab) and/or yeasts, which transform the sugars in lactic acid with a consequential decrease in ph; in addition, substances inhibiting undesirable microorganisms are produced (bevilacqua et al., 2009). moreover, several microbial strains are able to hydrolyse oleuropein contributing to olive debittering (iorizzo et al., 2016; servili et al., 2006). iooc (2017) indicates the natural olives as “green olives, olives turning colour or black olives placed directly in brine in which they undergo complete or partial fermentation, preserved or not by the addition of acidifying agents”. therefore, raw olives are placed in water or brine as long as required for the spontaneous loss of bitterness. in this case, oleuropeinolytic bacterial/yeast strains degrade oleuropein in synergy with specialized enzymes naturally occurring in the olives (ramirez et al., 2017a; ramirez et al., 2016). however, olive debittering is slow and several months are needed to obtain a satisfactory good-tasting product (sanchez et al., 2006). in recent years, demand of organic and natural foods has become trendy (rozin et al., 2004). in this contest, the demand for natural to risen since they have become popular and appreciated for the nutritional value and distinctive sensory characteristics. several studies on natural to have been carried out focusing on the content of bioactive components (charoenprasert and mitchell, 2012; sakouhi et al., 2008) and microbial characterization (bleve et al., 2015; pereira et al., 2008). moreover, also new systems to accelerate olive debittering with biotechnological approaches as an alternative to chemical treatment have been tried obtaining positive results by using enzymes (de leonardis et al., 2016), selected lab (perricone et al., 2013; hurtado et al., 2010), batches in modified brine (ramirez et al., 2017b) and vacuum impregnation (tamer et al., 2013). in this study, several chemical, microbial and sensory parameters have been tested in green olives laboratory-processed through both the spanish-style and natural methods and stored for a period of time 18 months, under different conditions. specifically, the effects of storing in brine were compared with those in vacuum bags, without brine, in order to evaluate the possibility to reduce the salt, potentially harmful to health. finally, considering that in the supermarkets the packaged olives are frequently placed in ital. j. food sci., vol. 30, 2018 416 refrigerator, the effects of cold temperatures have been studied. the results could be useful to the improvement of natural olives, in olive packaging and conservation systems. 2. materials and methods 2.1. chemicals all reagents were of analytical or hplc grade. gallic acid, hydroxytyrosol and oleuropein were purchased from sigma-aldrich co (st. louis, mo, usa). all the media and the supplements for the microbiological analysis, unless otherwise stated, were from oxoid, basingstoke, hampshire, uk. 2.2. olive processing and storage conditions about 10 kg of green or yellow-green olive fruits were handpicked from ‘cazzarella’ trees, a dual-purpose autochthonous cultivar grown in the molise region (italy). at the laboratory, the olive batch was split and processed as described synthetically in table 1. table 1. olive sample typologies. 1. raw olives: fresh untreated olives. 2. b1-naoh / b1-nat: olives brined in 6% nacl solution (first brining) and stored in the dark under 20 °c constant temperature for 9 months. 3. b2-naoh / b2-nat: olives of point 2 brined in 6% nacl fresh solution (second brining) and stored in the dark under 20 °c constant temperature for other 9 months. 4. c-naoh / c-nat: olives of point 2 packaged without brine in vacuum bags and stored in a refrigerator under 6 °c temperature (c = cold) for other 9 months. 5. t-naoh / t-nat: olives of point 2 packaged without brine in vacuum bags and stored in the dark under 20 °c constant temperature (t = tempered) for other 9 months. -naoh: processed by spanish method; -nat: natural processed. in the preparation of the b1-naoh sample, olives were placed into a 2% naoh solution for 16 hours (ratio olive/lye 1:1.3, w/v); olives were washed repeatedly with water until neutral ph and brined into 6% nacl solution (ratio olive/brine 1:1.3, w/v) in three different closed glass jars. in the case of the b1-nat sample, the olives were immersed directly into 6% nacl solution (ratio olive/brine 1:1.3, w/v); three weeks after (21 days), the brine was renewed with a fresh 6% nacl solution. all jars were stored in the dark under 20°c constant temperature for 9 months. thus, the olives were water-washed repeatedly, analyzed, repackaged and stored for another 9 months as described in table 1 (b2-, c-, t-). 2.3. analytical determinations moisture and total fat were determined on the pulp separated manually from the stone and homogenized. moisture was determined at 105°c on about 5 g of pulp; successively, oil content was measured in dry pulp by using soxhlet extraction with 40-60°c petroleum ether. lactic acid, ph and total phenol were determined on both olive pulp and brine. ital. j. food sci., vol. 30, 2018 417 brine was filtered through a 0.45 μm pvdf syringe-filter before assaying. as for the pulp, about 20 g of fresh pulp was dispersed in 60 ml of distilled water (1:3, w/v) and the flask was put into a sonicator bath for 20 minutes at room temperature by filtering the aqueous phase on paper. the ph measurement was performed through a ph-meter, while the lactic acid was measured by titration with 0.1 m naoh up to ph 7.0. free acidity and fatty acid composition were determined on the oil extracted at cold temperatures from 100 g of olive pulp by following the methods described in pasqualone et al. (2014). the free acidity (expressed as g oleic acid per 100 g pulp olive) was measured following the olive oil european union commission (1991), while the fatty acid composition was determined by gas-chromatographic analysis as described in de leonardis and macciola (2012). p-anisidine value was determined on the cold extracted oil according to aocs (1998). phenol extraction from the olive pulp, determination of total phenols and the hplc analysis with uv detector were carried out in the same conditions described in de leonardis et al. (2016). hydroxytyrosol (hy), dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol (hyeda) and oleuropein (ole) were monitored and each of them was quantified through a hydroxytyrosol calibration curve derived from a plot of area counts versus concentration. hy-compounds were calculated as the sum of hy, hyeda and ole expressed as mg/100 g wet pulp olives as hy equivalent. 2.4. microbial counts microbiological analysis was carried out on the olive pulp according to the method reported in iorizzo et al. (2016) with modifications. for each sample, about 30 g of olives were homogenized in 0.9% nacl (w/v) for 1 min in a stomacher bag (bag mixer-400). pulp homogenates were diluted serially and plated in triplicate by using the following growth media and incubation conditions: plate count agar (pca) at 30°c (72 h) and 15°c (5 days) for total mesophilic and psychrotrophic aerobic bacteria; violet red bile glucose agar (vrbga) at 37°c for 24 h for enterobacteriaceae; mrs agar, added with 0.17 g/l of cycloheximide (sigma-aldrich co, st. louis, mo, usa), at 30°c for 4 days in anaerobiosis (anaerogen kit, oxoid, basingstoke, united kingdom) for lactic acid bacteria (lab); ypd agar (1% yeast extract, 2% peptone, 2% glucose and 2% agar) supplemented with 100 mg/l chloramphenicol at 28°c for 72 h for yeasts; pseudomonas agar base (pab), added with cfc selective supplement, at 30°c for 48 h for pseudomonas spp. 2.5. sensory analysis sensory profile was evaluated according to galán-soldevilla and pérez-cacho (2012), with modifications. the panel group was composed of 15 untrained volunteers selected and led by one olive oil expert taster. training and alignment of panel and sensory profile sheet was developed by using similar commercial products. nine attributes (defects, fruit odor, salty, bitter, acid, firmness, fibrous, crunchy and overall opinion) were selected and evaluated through a seven-point hedonic scale (7: like extremely; 6: like very much; 5: like slightly; 4: neither like nor dislike; 3: dislike slightly; 2: dislike very much; 1 = dislike extremely). value zero was given for absent attribute. during the panel training, panel leader held a discussion on the descriptors and a consensus lexicon was developed about the following nine attributes (galán-soldevilla and pérez-cacho, 2012): a) defects (presence of abnormal negative odor/aroma); b) gustatory sensations (salty: typical taste produced by sodium chloride aqueous solutions; bitter: basic taste produced by diluted aqueous solutions of caffeine: acid: basic taste produced by aqueous solutions of substances like citric acid); d) fruity odor (odor/aroma characteristic of fresh olives); e) kinesthetic sensations (firmness: mechanical property of texture related to the strength ital. j. food sci., vol. 30, 2018 418 required to attain a certain penetration of the olive; fibrous: geometrical property of texture related to the perception of strands oriented in the same direction; crunchy: mechanical property of texture related to the cohesion and strength necessary to break an olive with teeth); f) overall judgment (general grade of appreciation). 2.6. elaboration data each jar was analyzed independently in duplicate (six independent replicates for every sample), by calculating mean and standard deviation. means obtained were compared through anova (duncan's multiple range post hoc tests at p<0.05) by using the spss 13.0 software (spss, inc., chicago, il, usa). 3. results and discussion 3.1. basic olive pulp characteristics processing method strongly affects the final characteristics of table olive (sanchez et al., 2006) and the data reported in table 2 confirm this evidence. olive water content was initially 69.4%. overall, water content in the processed olives was generally higher in the olives treated with naoh than in the natural-processed (nat). b1naoh and b2-naoh samples, stored in the brine, kept the original moisture (69.6 and 72.6%, respectively), while the matching b1-nat and b2-nat samples showed a lower value (63.7 and 67.2%, respectively). however, the observed differences were not statistically significant. conversely, significant moisture reduction was observed in the olives stored in the vacuum bags. specifically, water content was 60.1, 59.0, 53.0 % in the c-nat, t-naoh and t-nat samples, respectively. an increase of lactic acid and a decrease of ph expected as result of the microbial activity. all the naoh-treated olives had about one-higher-point of ph than the natural olives. more specifically, ph values were 5.17, 5.39, 5.56 and 5.73 in b1-naoh, b2-naoh, cnaoh and t-naoh, respectively. these high ph values are potentially harmful being not fit to inhibit growth of spoilage microorganisms. actually, brine acid characteristics of the naoh treated samples (table 2) were below the requirements for trade according to the iooc (2004) that has a set the minimum brine ph value at 4.1; however, addition of acidity regulators is permitted. the ph values of nat olive pulp were 4.62, 4.42, 4.80 and 4.75 in b1-nat, b2-nat, c-nat and t-nat, respectively. the ph value was obviously related to lactic acid, which was found in the nat olives more-than-three-times higher than naoh-treated olives. according to marsilio et al. (2005), lye treatment and subsequent water washing presumably caused sugar and nutrient loss from olive pulp giving rise to insufficient acidification in the naoh-treated samples. in the set group of nat olives, the maximum and minimum values of lactic acid were c-nat (0.24%) and b2-nat (0.11%), respectively. these values are in line with literature (de leonardis et al., 2016; aponte et al., 2012). 3.2. phenolic content many studies have evidenced a close relationship between processing method and content/composition of phenols in table olives (ambra et al., 2017; charoenprasert and mitchell, 2012; blekas et al., 2002). in table 3 phenolic compound evolution is given only for the nat olives due to the phenol content in the naoh-treated olives was negligible. ital. j. food sci., vol. 30, 2018 419 table 2. analytical characteristics of the pulp olive samples and relative canned brine. pulp olive brine moisture % ph lactic acid % ph lactic acid g/l raw olives 69.4(4.3)a,b 5.67(0.06)a nd 7.02(0.06)a n.d. stored in the first brine b1-naoh 69.6(5.2)a,b 5.17(0.38)b,c,d 0.02(0.00)a 5.05(0.27)b 0.4(0.0)a b1-nat 63.7(5.3)b 4.62(0.34)b,c 0.22(0.02)b 4.26(0.16)c 2.5(0.2)b stored in the second brine b2-naoh 72.6(4.2)a,b 5.39(0.41)b,c,d 0.03(0.00)a 5.02(0.25)b 0.5(0.0)a b2-nat 67.2(4.1)a,b 4.42(0.26)b 0.11(0.01)c 4.10(0.22)c 2.5(0.2)b stored in vacuum bags at 6°c c-naoh 69.1(3.8)a,b 5.56(0.29)c,d 0.07(0.03)a c-nat 60.1(3.3)b 4.80(0.31)b,c,d 0.24(0.02)b stored in vacuum bags at 20°c t-naoh 59.0(3.6)b 5.73(0.29)d 0.03(0.00)a t-nat 53.0(3.2)b 4.75(0.26)b,c,d 0.19(0.03)b values are means (standard deviation) of six independent replicates; letters on the column point out significant difference at p<0.05. -naoh: lye processed; -nat: natural processed; n.d. = not detected. table 3. total phenol and hydroxytyrosol (hy) compounds obtained in pulp olives and related brine (natural olives). pulp olives brine total phenols % gae hy-compounds mg/100g as hye total phenols mg/l gae raw olives 1.19(0.08)a 199(13)a b1-nat 0.21(0.01)b 53(4)b 1,791(102)a b2-nat 0.14(0.01)b 25(1)c 1,006(132)b c-nat 0.20(0.01)b 47(3)b t-nat 0.20(0.01)b 62(4)b values are means (standard deviation) of six independent replicates and different letters on the column point out significant difference (p<0.05). specifically, nat olive samples showed total phenol values dropped from 1.19 (raw olives) to about 0.20 g/100g (nat processed olives) as gae (gallic acid equivalent), respectively. although the reduction of the starting phenol content is a coveted result from an organoleptic point of view, presence of residual phenols may be positive from a nutritional point of view, since several phenols are recognized as antioxidant and healthy substances (d’antuono et al., 2016; de leonardis et al., 2013; de leonardis et al., 2008; sanchez et al., 2006). at the end of the first brining (b1-nat), hy-compound content was 53 mg per 100 g olives and insignificant changes were observed in c-nat (47 mg/100 g) and t-nat (62 mg/100g); conversely, hy-compounds halved during the second brining (b2-nat, 25 mg/100g). ital. j. food sci., vol. 30, 2018 420 the european union commission (2012) recently approved a health claim for extra virgin olive oil stating that ‘daily intake of 5 mg of hydroxytyrosol and its derivatives is able to prevent low density lipoprotein (ldl) oxidation’. therefore, nat olives may be claimed as a source of phenols having health-promoting properties. however, the hy-compound values reported in table 3 were certainly underestimated because other hydroxytyrosol linked compounds, known to be present in olives (ambra et al., 2017; charoenprasert and mitchell, 2012), have been omitted in this study for purposes of simplification of the model. according some studies (ramirez et al., 2017a; ramirez et al., 2016; de leonardis et al., 2015), biological and enzymatic oleuropein depletion may occur through a rapid transformation in hy-eda and subsequent hydrolysis with gradual hy release. a similar pathway occurred also in the nat olives, as noted in the graph of fig. 1. figure 1. changes in the table olives of the monitored phenolic compounds, expressed in mg (hy equivalent) per 100 g dry olive pulp. raw olive pulp contained 37, 89 and 158 mg/100 g in dry matter (d.m.) of hy, hy-eda and ole, respectively. after 21 days of brining (when the first brine was replaced, see material and methods), the olive phenol profile was changed in 73, 167 and 22 mg/100 g d.m. of hy, hy-eda and ole, respectively (data not shown). hy-eda is a recognized antimicrobial compound inhibiting the growth of lactic bacteria (medina et al., 2007). after 9 of months brining (b1-nat), ole was completely depleted, while hy-eda was 45 mg/100 g d.m. successively, hy-eda was undetectable in sample b2-nat, c-nat and tnat in which only hy was found in the amounts of 37, 77 and 116 mg/100 g d.m., respectively. 3.3. lipid fraction the characteristics of lipid fraction of the raw olives and samples stored for 18 months are given in table 4. ital. j. food sci., vol. 30, 2018 421 table 4. changes of lipid fraction of olives samples. raw olives naoh-processed olives nat-processed olives b2-naoh c-naoh t-naoh b2-nat c-nat t-nat total oil % w.m. 18.4 (1.1)a 19.5 (1.2)a 19.3 (1.2)a 17.5 (1.1)a 22.1 (1.3)b 22.2 (1.3)b 21.6 (1.3)a,b total oil % d.m. 26.3 (1.6)a 26.8 (1.6)a 28.2 (1.7)a 29.3 (1.8)a 33.0 (2.2)b 36.5 (2.2)c 40.5 (2.5)d free acidity % 0.5 (0.0)a 11.2 (0.7)b 15.3 (0.9)c 13.7 (0.8)d 2.8 (0.2)e 5.5 (0.3)f,a 3.4 (0.2)e p-anisidine value 5.9(0.3)a 33.5(1.7)e 30.7(1.6)d 31.6(1.7)d,e 12.8(0.7)b 29.2(1.5)d 23.1(1.2)c fatty acids % c16:0 16.5 (1.0)a 23.7 (1.4)b,c 24.4 (1.5)b 21.6 (1.3)c,d 17.0 (1.0)a 19.9 (1.2)d 18.2 (1.1)a,d c16:1 1.4 (0.1) 1.4 (0.1) 1.3 (0.1) 1.2 (0.0) 1.3 (0.1) 1.4 (0.2) 1.3 (0.1) c18:0 2.9 (0.2)a 4.2 (0.3)b 4.6 (0.3)b 4.5 (0.3)b 3.4 (0.2)c 3.9 (0.3)b,c 3.8 (0.2)c c18:1 65.5 (4.0)a 60.5 (3.7)a,b 57.1 (3.5)b 60.0 (3.6)a,b 64.5 (3.9)a 63.0 (3.8)a,b 64.3 (2.7)a,b c18:2 11.6 (0.7)a 4.2 (0.3)b 3.2 (0.2)c 5.1 (0.2)d 11.3 (0.5)a 8.2 (0.3)e 9.3 (0.3)f c18:3 0.6 (0.0)a 0.1 (0.0)b 0.1 (0.0)b 0.2 (0.0)b 0.6 (0.1)a 0.3 (0.1)a 0.4 (0.1)a c20:0 0.5 (0.0)a 0.7 (0.0)b 0.8 (0.0)b 0.7 (0.0)b 0.5 (0.0)a 0.6 (0.0)a 0.6 (0.0)a c20:1 0.2 (0.0) 0.3 (0.0) 0.3 (0.0) 0.3 (0.0) 0.3 (0.0) 0.2 (0.0) 0.2 (0.0) c22:0 0.1 (0.0)a 2.9 (0.7)b 4.8 (0.2)b 3.8 (0.6)b 0.3 (0.0)c 1.2 (0.5)c 0.7 (0.3)c c22:1 0.0a 1.5 (0.6)b 2.7 (0.2)b 1.9 (0.8)b 0.1 (0.0)a 0.7 (0.2)c 0.3 (0.3)c values are means (standard deviation) of six independent replicates and different letters on the lines point out a significant difference (p<0.05). a different oil content was observed between naohand nat-processed olives. apparently, total oil content increased in the nat-olives, especially considering total oil on dry matter. tamer et al. (2013) have found that alkali treatment caused oil loss from the olives. moreover, the advanced fermentation status (with large sugar consumption) and the reduced lipolytic activity (with low fat consumption) could explain the apparent increase of oil in the nat-olives (table 4). unfortunately, we did not have enough data to establish a possible link between oil increase and microbial counts and types (table 5). according to what was observed in previous studies (pasqualone et al., 2014; lopez et al., 2011), a higher hydrolytic and oxidative oil degradation was observed in the naoh vs nat-olives. specifically, a considerable increase of free acidity was evident in b2-naoh, c-naoh and t-naoh with values of 11.2, 15.3 and 13.7% on oil extracted, respectively; conversely, free acidity was less than 5.5% for the nat-olives. formation of free fatty acids could be catalysed by the lipases contained in the fruits or synthesized by the environmental microflora. a positive effect of low temperature on the triacylglycerol hydrolysis was evident due to the high free acidity values found in c-naoh (15.3%) and c-nat (5.5%). similarly, oil oxidation was higher in the cold temperature stored olives, as the p-anisidine values show (table 4). as known, p-anisidine assay measures secondary lipid oxidation compounds; the highest p-anisidine values were obtained in the oils extracted from the b2-naoh, c-naoh, t-naoh and c-nat olives. a fatty acid composition of raw olives (table 4) was in agreement with the literature (malheiro et al., 2012). oleic acid was the main fatty acid (65.5%), followed by palmitic acid (16.5%) and linoleic acid (11.6%). in order to make table 4 more readable, the c17:0, ital. j. food sci., vol. 30, 2018 422 c17:1 and c24:0 fatty acids (found in equal or less amount 0.1%) were deliberately omitted; however, these fatty acids did not change during the storage of the olives. significant modifications of fatty acid composition occurred especially in the naoholives, specifically, a reduction of linoleic and linolenic acid was registered in the b2naoh, c-naoh, t-naoh samples; in the same samples a decrease of oleic acid and an increase of palmitic and stearic acid were obtained. it is known that polyunsaturated fatty acids were more affected by oxidation and therefore decreased at a greater extent (caponio et al., 2003; de leonardis and macciola, 2012). in the nat-olives, especially in b2-nat and t-nat, the olive residual phenols have limited oil oxidation, as evidenced by the minor changes of p-anisidine and fatty acid profile. unexpectedly, significant increase of the peaks corresponding to behenic (c22:0) and erucic (c22:1) fatty acids were found in the lipid fraction of stored olives. generally, these long chain fatty acids are present in olive oil in amounts lower than 0.2%, as the data of raw olive oil shows (table 4). in the naoh treated olives, c22:0 and c22:1 were found in amounts higher than 2.9 and 1.5%, respectively. in particular, lipid fraction of the c-naoh sample showed the highest content (c22:0 = 4.8%; c22:1 = 2.7%). conversely, in the nat-olives percentage of c22:0 and c22:1 was lower than naoh olive oil counterpart, but higher than raw olive oil. we have no explanation for this and similar results are missing in literature. perhaps, it is reasonable to hypothesise a microbial origin of c22:0 and c22:1 in the olives, by considering that few oleaginous microorganisms are able to synthesize long chain fatty acids (el bialy et al., 2011; ratledge, 2004). 3.4. sensory analysis graphical presentations of the obtained sensory evaluation (average values) are shown in fig. 2. the panel did not evaluate color; however, naoh-olives were overall lightly yellowgreen, while nat-olives were dark gray-green. in general, sensory profile of naoh-olives was very different from that of nat-olives; these differences were amplified during the second storage. in general, ‘overall opinion’ was more positive for natthan naoholive samples. the b1-naoh obtained an ‘overall opinion’ of 4 points convincing us to continue the storage work for this sample despite its high ph (table 2). however, at the end of study, b2-naoh and t-naoh showed perceptible sensory defects and loss of firmness, fibrous and crunchy, whereas c-naoh was not evaluated due to its pronounced smell of rotten caused by an abnormal fermentation. in addition, salty was the prevailing, if not unique, taste in the tasted naoh olives. conversely, taste of nat-olives was more complex and well-balanced between salty, bitter and acid tastes. salty in the brined b1-nat and b2nat was imperceptible, while bitter taste was perceived clearly in all nat-olives. effectively, bitter taste is a distinctive but pleasant flavor of the natural-style olives (lanza and amoruso, 2016). the intensity of the bitter taste remained essentially unchanged between the b1-nat, c-nat and t-nat samples, while it decreased in b2-nat. finally, a positive fruit odor was perceived clearly up to 18 storage months only in the nat-olives. 3.5. microbiological analysis fermentation of both naohand nat-olives occurred spontaneously without adding any starter culture. the microbial count of raw olives highlighted presence of yeasts (log 7.3 cfu/g), lab (log 2.0 cfu/g) and enterobacteriaceae (log 2.0 cfu/g). heperkan (2013) reports that microbiota of olives include principally yeasts and lactic acid bacteria (lab), members of enterobacteriaceae, clostridium, pseudomonas, staphylococcus, and ital. j. food sci., vol. 30, 2018 423 occasionally moulds. the processing method impacts microbial dynamics affecting greatly quality and shelf life of the to (de angelis et al., 2015). generally, indirectly brined green olives yeasts become the predominant population; however, a correct adding of lab starter may improve lactic fermentation performance (campus et al., 2017; de leonardis et al., 2016; perricone et al., 2013; corsetti et al., 2012). figure 2. sensory profile rings of the table olives. the mean of microbial count and standard deviation of the microorganisms searched in the olive pulp are given in table 5. the most relevant result was absence of detectable microorganisms in b1-nat and b2-nat, apart from yeast cells in b1-nat (7.1 log cfu/g); ital. j. food sci., vol. 30, 2018 424 conversely, in all other samples lab and yeasts coexisted until the end of study (table 5). in addition, neither enterobacteriaceae nor pseudomonas spp. were found in b2-nat, c-nat e t-nat. therefore, low ph (table 2) and high phenol level (table 3) influenced the microbial profile of b1-nat affecting positively the subsequent storage of the nat-olives. generally, the enterobacteriaceae spp., eliminated during fermentation, are not detected at the end of the process (heperkan, 2013). nevertheless, enterobacteriaceae cells were found in all naoh-olives ranging from 3.3 to 4.8 log cfu/g (table 5). certainly, in these samples, high ph values and lack of phenols have favoured the development of enterobacteriaceae and pseudomonas spp., which have contributed to the spoilage of b2naoh, c-naoh and t-naoh samples. moreover, in c-naoh sample, enterobacteriaceae and pseudomonas spp. were not inhibited at cold temperature. indeed, low temperature storage has penalized the populations of yeast and lab favouring the growth of psychrophilic bacteria, which caused lipolysis, oil oxidation (table 4), pectolytic action and, putrefaction (fig. 2). moreover, also for the nat-olives, the conservation at cold temperature was proven to be less effective than that in brine and under temperate conditions. table 5. cell count (log cfu/g) of microorganisms determined on pulp olives. media ypd mrs pca (at 30°c) pca (at 6°c) vrbga pcfc naoh processed olives b1-naoh 6.20 (0.10)a 4.90 (0.10)a 6.10 (0.20)a 6.13 (0.12)a 3.50 (0.20)a 4.10 (0.20)a b2-naoh 5.47 (0.15)c 4.00 (0.20)b 5.00 (0.10)b,c 4.90 (0.20)b 4.80 (0.20)b 0.00 (0.00) c-naoh 6.13 (0.15)a 5.47 (0.15)c 6.10 (0.30)a 5.07 (0.15)b 3.30 (0.20)a 3.80 (0.20)b t-naoh 5.90 (0.20)a 4.67 (0.25)a 5.70 (0.20)d 5.60 (0.20)d 4.03 (0.15)c 4.60 (0.30)c nat processed olives b1-nat 7.10 (0.20)b n.d. n.d. n.d. n.d. n.d. b2-nat n.d. n.d. n.d. n.d. n.d. n.d. c-nat 4.60 (0.10)d 4.83 (0.59)a 4.70 (0.10)c 4.00 (0.10)c n.d. n.d. t-nat 5.17 (0.35)c 4.50 (0.40)a 5.20 (0.40)b n.d. n.d. n.d. values are means (standard deviation) of three independent replicates; letters on the column point out significant difference at p<0.05. n.d. = not detected. 4. conclusions after 18 months of storage, natural green olives showed good nutritional features (hydroxytyrosol, unchanged fatty acid profile), organoleptic identity, microbial safety, low oil hydrolysis and oxidation. conversely, after 9 months of storage, ph values below the requirements for the trade were obtained in the naoh-treated olives. natural olives preserved high total phenols (0.2 g/100 g wet pulp) and a significant level of the hycompounds determined in this study. it is reasonable to suppose that residual phenols have influenced positively polyunsaturated fatty acid preservation and, together with the low ph level, have inhibited the growth of enterobacteriaceae and pseudomonas spp. therefore, the slow olive debittering of natural olives was counterbalanced by a prolonged shelf life. the packaging in 6% nacl renewed brine or in vacuum bag, under a storing ital. j. food sci., vol. 30, 2018 425 temperature of 20°c, gave the best results, while conservation at cold temperature proved to favor the growth of psychrophilic spoilage bacteria. acknowledgements the authors would like to thank margherita di cristofaro for the valuable contribution in editing the manuscript. references ambra r., natella f., bello c., lucchetti s., forte v. and pastore g. 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2006. trends in table olive production. elaboration of table olives. grasas y aceites 57:86-94. servili m., settanni l., veneziani g., esposto s., massitti o., taticchi a., urbani s., montedoro g.f. and corsetti a. 2006. the use of lactobacillus pentosus 1mo to shorten the debittering process time of black table olives (cv. itrana and leccino): a pilot-scale application. journal agricultural food chemistry 54(11):3869-3875. tamer c.e., i̇ncedayı b., yıldız b. and çopur ö.u. 2013. the use of vacuum impregnation for debittering green olives. food and bioprocess technology 6(12):3604-3612. paper received october 20, 2017 accepted february 6, 2018 ijfs#1071_bozza ital. j. food sci., vol. 31, 2019 264 paper comparison of bioactive compounds and sensory evaluation on edible flowers tea infusion n. hussain*a,b, i. ishakb, n. mohd haritha and g. leong pau kuana adepartment of food technology, faculty of food science and technology, universiti putra malaysia, selangor, malaysia bhalal products research institute, universiti putra malaysia, selangor, malaysia *corresponding author: fax: +60389423552 e-mail address: aryatihussain@upm.edu.my abstract france rose buds, jasmine flower, and osmanthus flower are three edible flowers commonly available in malaysia market. composition of these 3 edible flowers is not widely studied. hence, the caffeine, total phenolic content (tpc), volatile compounds, and overall acceptability of tea infusion from france rose buds, jasmine flower, and osmanthus flower were compared. tea infusion from the edible flowers was prepared by boiling it with distilled water. none solvent extraction was carried out to determine the bioactive compounds. tea infusion of osmanthus flower contains the highest caffeine (4.96±1.94 µg/ml), total phenolic content (4.33±0.03 mg gae/g) and overall acceptability (6.16±2.05) compared to france rose buds and jasmine flower. the jasmine flower was found to have the highest number of volatile compounds (13) compared to france rose buds and osmanthus flower. this study indicates that the edible flowers have the potential for application as food ingredient. keywords: france rose buds, jasmine flower, osmanthus flower, caffeine, volatile compounds, sensory evaluation ital. j. food sci., vol. 31, 2019 265 1. introduction edible flowers commonly seen include cauliflower, broccoli, and artichokes (carter et al., 2007). the edible part of cauliflower and broccoli is made of fleshy flower stalks and clusters of flower buds (carter et al., 2007). edible flower petals and flower buds can be eaten raw in salads. refreshing tea infusion can also be made from the flower petals (lim, 2014). in terms of nutritional value of edible flowers; petal of the flowers is a source of vitamins, minerals, and antioxidants thus contribute to an increase interests for consumption (mlcek and rop, 2011). other than as a decoration and culinary purposes, the nutritive and chemoprotective properties of certain edible flowers are welldocumented or under study. edible flowers can also be classified as nutraceutical food (mlcek and rop, 2011). besides the well-recognized wholesome effects of green and black teas prepared from young leaves of camellia sinensis, hot water infusions (teas) of many other plants also may have health benefits. flavonoids and phenolic acids have been used as antioxidants to prevent oxidative damage and control of diseases caused by oxidative stress (na et al., 2014). flower, seeds, and root of osmanthus flower (osmanthus fragrans) is also used as a acesodyne and as a folk medicine for the treatment of liver, stomachache and for other therapeutic purposes such as aerodontalgia, halistosis, rheumatism and physical pain (peng and ji, 2004). the main bioactive components in the extracts of osmanthus fragrans flowers are flavonoids and phenolic acids (xiong et al., 2014), carotenoids, carotenoid-derivatives and volatile constituents (leffingwell, 2002). jasmine flower (trachelospermum jasminoides) is used in the perfume industry, and the scent has been included in at least 55 commercially sold perfumes (basenotes fragrance search, 2014). the inner bark yields a strong fiber that is utilized for making rope, sacks and paper and the stem is used for treating rheumatism and injury in traditional chinese medicine (ill chan noh, 2011; sheu et al., 2009). most publications on jasmine flower are rather concerned about the content of the plant (jing et al., 2012) than about the flowers (joulain, 1987). france rose buds (rosa sp. var. rosa de castillo) are known as edible flowers and have been used for centuries as food components, either in the fresh form or in processed products, such as confectionary and beverages (girard-lagorce et al., 2001). the combination of health benefits with recognized applicability in cuisine raises the possibility of using rose flowers in functional food products. although the health benefits of some well-known edible flowers are commonly studied, the caffeine content, total phenolic content and volatiles compounds in tea infusion from france rose buds, jasmine flower, and osmanthus flower (osmanthus fragrans) still remain unknown. research on sensory evaluation had been conducted by chen et al. (2010) on the attributes of taste, flavor, and overall acceptance on tea infusion from pu-erh teas. zhu et al. (2016) also conducted evaluation of volatile compounds in infusion of oolong tea (fully fermented camellia sinensis l.). instead of using hedonic scale to rate upon answering the questionnaire, 5 aroma terms were used to define the aroma by well-trained panel of ten members: 2-methylpyrazine for ‘‘roast” note, maltol for ‘‘sweet” note, hexanal for ‘‘green and grassy” note, dipropyl disulfide for ‘‘sulphur” note, phenylethyl alcohol for ‘‘floral” note. for the sensory analysis by benvenuti et al. (2016), 5 different organoleptic characteristics of which spiciness, sweetness, softness, scent, and bitterness were included in the evaluation scheme and were expressed in a scale of 1 to 100. in addition, sensory profile of tea infusion of france rose buds, jasmine flower, and osmanthus flower corresponding to the volatile compositions is still insufficient. therefore, this study was presented to compare the caffeine, total phenolic, volatile compounds, and overall ital. j. food sci., vol. 31, 2019 266 acceptability of the 3 types of edible flowers by untrained panelists. the flowers may be further exploit as a source of natural antioxidant for food and nutraceutical applications. 2. material and methods 2.1. sample preparation france rose buds, jasmine flower, and osmanthus flower were purchased from yin onn shd. bhd. (mid valley city) at lower ground floor 035 & 036, mid valley city megamall, mid valley city, lingkaran syed putra,kuala lumpur, malaysia. extraction was prepared using boiled distilled water by 1: 8.82 ratio (ratio of dry mass sample to boil distilled water). the ratio used was modified from bispo et al. (2002). 2.2. chemicals methanols and acetic acid from fisher scientific, uk and caffeine standard from sigmaaldrich, china were used for high performance liquid chromatography (hplc) analysis. folin-ciocalteu and gallic acid from merck, germany and sodium carbonate from fisher scientific, uk were also used for total phenolic content analysis. 2.3. determination of caffeine content using hplc hplc (shimadzu corporation, japan) was carried out to determine and compare the caffeine content of france rose buds, jasmine flower, and osmanthus flower. the experimental procedure was adopted from bispo et al. (2002) with some modifications. the modifications were on the amount of france rose buds, jasmine flower, and osmanthus flower applied in this study. about 17 g of dried edible flowers were used, instead of 4 g as referred to the method bispo et al. (2002). the extraction time was also changed from 3 min to 10 min. aqueous extract was obtained using 150 ml boiled distilled water with 17 g of dried edible flower for 10 min with continuous stirring. the extracts were filtered using syringe filter (chemolab supplies, malaysia) of 0.45 μm twice (double filter) prior injected into hplc. about 20 μl of each sample was injected into hplc. 2.4. total phenolic content (tpc) analysis tpc of france rose buds, jasmine flower, and osmanthus flower were determined and compared using folin-ciocalteu method. the assay was conducted as described by bhebhe et al. (2015). total phenolic content was expressed as gallic acid equivalents (gae). the results were obtained from representative samples prepared from each concentration. calibration and linear curves were determined. 2.5. analysis of volatile compounds using automated gas chromatography-mass spectrometry (gc-ms) the volatile flavor constituents of each sample were analyzed using trace gc ultra gas chromatography system coupled with tsq quantum xls mass spectrometer system (serial no: tqu03227) from thermo fisher scientific (usa) and capillary column of 30 m × 0.25 mm, 0.25 µm film thickness model tg-5ms (thermo scientific, usa). the detail of experimental procedure was adopted from li et al. (2013). ital. j. food sci., vol. 31, 2019 267 2.6. sensory evaluation sensory analysis of france rose buds, jasmine flower, and osmanthus flower were conducted at room temperature (25±2°c) by 50 untrained panelists who need to complete the questionnaire containing 9-point hedonic scales. the evaluation covered the attributes of taste, color, aroma, and overall acceptability where scale 9 represented like extremely and 1 dislike extremely (hajmohammadi et al., 2016; lim, 2011; zheng et al., 2014). 2.7. statistical analysis the experimental design was completely randomized. statistical analysis and comparisons among means were carried out using the statistical package minitab 17. the data collected was analyzed by one-way analysis of variance. the tukey’s post hoc test was applied for comparison of means, and differences are considered significant at the level of p<0.05. linear regression for correlation analysis was performed using microsoft office excel (2007). 3. results and discussion 3.1. caffeine content caffeine content of france rose buds, jasmine flower, and osmanthus flower were determined as shown in table 1 with 17 g of each samples were brewed using boiled distilled water. from the data collected, it shows that osmanthus flower contain significantly (p<0.05) the highest amount of caffeine (4.96±1.94 µg/ml) than france rose buds and jasmine flower, at 0.17±0.00 µg/ml and 0.67±0.03 µg/ml respectively. el-shahawi et al. (2012) reported that caffeine in tea infusion from tea (camellia sinensis l.) is well-known as natural powerful antioxidant and help in the prevention of cardiovascular diseases and cancers. el-shahawi et al. (2012) also revealed that every consumption of 100 ml of brewed tea infusion (camellia sinensis l.), about 1.04 to 212 mg of total catechins and 0.194 to 5.04 mg of caffeine were found in the tea infusion from 29 commercial green tea samples (camellia sinensis l.) in saudi arabia. coffee, tea, and fruits were the most important food sources of total polyphenols. a total of 437 different individual polyphenols were consumed, including 94 of them consumed at a level 1 mg/day (zamora-ros et al., 2015). to date, no specific dietary intake of caffeine in any food product including edible flowers tea infusion related to antioxidant activity has been reported. el-shahawi et al. (2012) reported that twenty-nine green tea samples (camellia sinensis l.) of different origins (china, japan, indonesia, sri lanka and taiwan) from saudi arabian local market have caffeine content ranged from 0.09 to 2.23 mg/g. d’archivio et al. (2016) also reported that average of 2.5±0.2 mg/g of caffeine was detected in tea infusion of oolong tea (semi-fermented camellia sinensis l.). caffeine content in tea infusion of france rose buds, jasmine flower, and osmanthus flower were relatively much lower than both of the green tea and oolong tea infusion as discussed above. ital. j. food sci., vol. 31, 2019 268 table 1. caffeine content of france rose buds, jasmine flower, and osmanthus flower using hplc. type of flower caffeine (µg/ml) france rose buds 0.17±0.00b jasmine flower 0.67±0.03ab osmanthus flower 4.96±1.94a values expressed as mean±standard deviation (n=2). means with different letters within the same column are significantly different at the level of p<0.05. 3.2. total phenolic content (tpc) the total phenolic content (tpc) was determined by folin-ciocalteu assay and the result was shown in table 2.the result was expressed as gallic acid equivalent (gae). table 2 shows that osmanthus flower had significantly the highest tpc value (4.33±0.03 mg gae/g) among the three edible flowers in this study (p<0.05). in addition, the highest caffeine content was also detected in osmanthus flower at 4.96±1.94 µg/ml (table 1) and this could also be related to the highest total phenolic content of osmanthus flower. similarly, na et al. (2014) reported that the total phenolic contents of osmanthus fragrans was higher 16.00±0.57mg gae/g than the other 50 edible types in a range of 0.63±0.03 to 35.84±1.67 mg gae/g wet weight. the tpc value of tea infusion of osmanthus flower in this study was lower than na et al. (2014). the different results obtained may be due to the different parts of plant used, where leaves had higher content of tpc than fruit, stem, or brunches. species type, age, maturity and or environmental stress may also contribute to differences in phenolic content of a plant (watson, 2014). generally, amount of polyphenols in plants is affected by genetics and environment. external environment also triggers the presence and content of polyphenols for the protection of the plant (watson, 2014). the most efficient solvent for polyphenols extraction was 50% dmf for black tea (fully fermented camellia sinensis l.) and 50% acetone for mate tea. higher phenolic content often linked with higher antioxidant activity where it is health benefiting in terms of medicinal properties such as antibiotic, anti-inflammation, anti-cancer and anti-allergic (bhebhe et al., 2015). table 2. total phenolic content of france rose buds, jasmine flower, and osmanthus flower. type of flower total phenolic content (mg gae/g) france rose buds 3.75±0.03b jasmine flower 4.27±0.02a osmanthus flower 4.33±0.03a results expressed in gallic acid equivalent. values are expressed as mean±standard deviation (n=5). means with different letters within the same column are significantly different at the level of p<0.05 3.3. volatile compounds volatile compounds of france rose buds, jasmine flower, and osmanthus flower were determined by automated headspace gcms with the total retention time of 18.30 min. the ital. j. food sci., vol. 31, 2019 269 chromatography of volatile compounds detected in tea infusion of edible flowers and their relative content were summarized in table 3. about 5 volatile compounds were detected in france rose buds by automated headspace gcms. phenylethyl alcohol was the major volatile compound identified from tea infusion of france rose buds at 57.12%, and this agrees with dudareva and pichersky (2006) that phenylethyl alcohol can be detected in france rose buds. phenylethyl alcohol, also known as benzyl carbinol, 2-phenylethanol, and β-phenylethyl alcohol, is a colorless and viscous liquid, rose-like odor, initially a slightly bitter taste then sweet and reminiscent of peach (fenaroli et al., 2000). phenylethyl alcohol has been used as an antimicrobial, antiseptic, disinfectant, fragrance in perfumes and preservatives (burdock, 1997). compound 1-iodo-2-methylundecane (15.75%), tridecane (3.22%), cis-2-methyl-7octadecene (3.18%), and 1-iodo-2-methylnonane (2.67%), classified as hydrocarbon group were detected in tea infusion of france rose buds. antonelli et al. (1997) showed similar findings where the main component in 24 different rose varieties was phenylethanol, but some roses showed unusually high levels of benzyl alcohol. antonelli et al. (1997) also detected 4-vinylphenol, also known as 4-ethenylphenol, p-vinylphenol, phydroxystyrene, and 4-vp, in two samples of rosa gallica out of 24 different rose varieties, but those volatile compounds was not found as analyzed in this study. tridecane detected in this study was similar to lin et al. (2013) where tridecane was extracted by hsspme in a total of 75 oolong tea (fully fermented camellia sinensis l.) at the ranged of 0.30 to 2.50%. benzyl benzoate (6.81%) and 1,6-octadien-3-ol,3,7-dimethyl(3.81%), also known as linalool, were detected in both tea infusion of jasmine flower and osmanthus flower, similarly as lim (2014). the (r)(-)-linalool was found to be the key odorants of jasmine tea flavor. linalool was identified in a high proportion using different solid-phase micro extraction fibres. a total of 13 constituents were identified in the headspace of jasmine flower, with compound linalool (25.01%) was the highest in proportion, followed by benzyl acetate (23.71%) and 3-hexenyl acetate (13.80%) (lim, 2014). pregna-5, 14-diene-3, 20-diol-18-carboxylic acid, 3-acetate-, lactone were the major volatile compound detected in tea infusion of jasmine flower at 31.98%. however, this volatile compound has never been reported in the similar research as reported by lim (2014). this might be due to the different part of the plants used in the experiments as different part consists of different volatile compounds. volatile compositions vary according to genetics, soil, climate, and agricultural practices (toci and farah, 2008). jasmine flower can be further extracted and incorporated as new food ingredient due to its high volatile compounds. 3.4. sensory evaluation sample preparation for sensory evaluation was conducted under the same preparation method for all the above analysis where the tea infusion was prepared by brewing it with boiling water without addition of sugar or honey. the prepared sample was kept at room temperature 25±2°c. about 50 untrained panelists were required to complete the questionnaire to score the attribute of taste, aroma, color, and overall acceptability. the result was recorded in table 4. although jasmine flower and osmanthus flower are originated from the same order and family, france rose buds and osmanthus flowers show significant different (p> 0.05) in sensory attributes, in terms of taste, aroma, color, and overall acceptability. ital. j. food sci., vol. 31, 2019 270 table 3. volatile compounds and their relative contents detected in france rose buds, jasmine flower, and osmanthus flower using hs gcms. no retention time (min) compound name molecular weight (g/mol) france rose buds (%) jasmine flower (%) osmanthus flower (%) • 4.48 cyclohexene,methyl-5-(1-methylethenyl)-, (r) 5.90 2.79 • 4.93 ethyl2-(5-methyl-5vinyltetrahydrofuran-2yl)propan-2-ylcarbonate 13.38 • 5.21 1,6-octadien-3-ol,3,7-dimethyl(linalool) 154.25 3.81 13.04 • 5.49 phenylethyl alcohol 122.16 57.12 • 6.07 2h-pyran-3-ol,6ethenyltetrahydro-2,2,6trimethyl 5.42 • 6.18 heptanediamide,n,n’-di-benzoyloxy 2.07 • 6.79 1,6-octadien-3-ol,3,7dimethyl-,2aminobenzoate 1.68 8.55 • 7.88 megastigma-4,6(e),8(z)-triene 8.05 • 8.44 1h-3a,7methanoazulene,2,3,4,7, 8,8a-hexahydro-3,6,8,8tetramethyl-, 1.97 • 8.95 3-buten-2-one,4-(2,6,6trimethyl-1-cyclohexen1-yl) 3.53 • 9.04 1,3,6,10dodecatetraene,3,7,11trimethyl-, (z,e) 2.21 • 9.68 3-hexen-1-ol benzoate 1.92 • 10.62 tridecane 184.37 3.22 • 11.19 1-[2-o-benzoyl-3,5-odibenzyl-alpha-dribosyl]-5,6dimethylbenzimide 1.07 • 11.44 benzyl benzoate 212.25 6.81 4.74 • 11.77 phenylmalonic acid monobenzyl ester 9.60 6.21 • 12.06 cis-2-methyl-7-octadecene 3.18 • 12.23 1-iodo-2-methylundecane 15.75 • 12.31 ethanone,2,2-dimethoxy-1,2-diphenyl 6.52 3.55 • 13.51 1-iodo-2-methylnonane 2.67 • 13.56 2-[4-methyl-6-(2,6,6trimethylcyclohex-1enyl)hexa-1,3,5trienyl]cyclohex-1-en-1 carboxaldehyde 3.96 • 13.85 cholest-1-eno[2,1a]naphthalene, 3',4'dihydro 2.38 • 14.23 pregna-5,14-diene-3,20diol-18-carboxylic acid,3acetate-, lactone 31.98 –, not found. four constituents selected for analysis are indicated in bold type. ital. j. food sci., vol. 31, 2019 271 table 4. sensory evaluation on attributes including taste, aroma, color, overall acceptability of france rose buds, jasmine flower, and osmanthus flower. type of flower taste aroma color overall acceptability france rose buds 5.48±2.06a 6.24±1.84a 6.56±1.47a 5.90±1.62a jasmine flower 4.34±2.05b 5.24±1.80b 5.60±1.63b 4.96±1.65b osmanthus flower 5.76±2.36a 6.42±2.07a 6.42±1.66a 6.16±2.05a mean±standard deviation (n = 50). values that are followed by different letters within each column are significantly different (p< 0.05). 1 = dislike extremely, 5 = neither like nor dislike, 9 = like extremely. research on sensory evaluation had been conducted by chen et al. (2010) on the attributes of taste, flavor, and overall acceptance on tea infusion from pu-erh teas. zhu et al. (2016) also conducted evaluation of volatile compounds in infusion of oolong tea (fully fermented camellia sinensis l.). instead of using hedonic scale to rate upon answering the questionnaire, 5 aroma terms were used to define the aroma by well-trained panel of ten members: 2-methylpyrazine for ‘‘roast” note, maltol for ‘‘sweet” note, hexanal for ‘‘green and grassy” note, dipropyl disulfide for ‘‘sulphur” note, phenylethyl alcohol for ‘‘floral” note. for the sensory analysis by benvenuti et al. (2016), 5 different organoleptic characteristics of which spiciness, sweetness, softness, scent, and bitterness were included in the evaluation scheme and were expressed in a scale of 1 to 100. table 4 displays a trend where france rose buds and osmanthus flower were scored significantly (p<0.05) higher than jasmine flower in terms of taste, aroma, color, and overall acceptability. osmanthus flower had the most acceptable sensory attributes with the highest score in terms of taste, aroma, and overall acceptability. despite jasmine flower being scored as the lowest in all sensory attributes, it contains the highest number of volatile compounds among the 3 edible flowers. it is believed that the edible flowers serve more than just as decoration and ornamental plants. 4. conclusions this study compared the bioactive components contained in tea infusion of france rose buds, jasmine flower, and osmanthus flower. it was found that tea infusion of osmanthus flower has the highest caffeine 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(1998. inhibitory effect of heliantriol c; a component of edible chrysanthemum, on tumor promotion by 12-o-tetradecanoylphorbol-13acetate in two-stage carcinogenesis in mouse skin. phytomedicine 5(3):215-218. zamaros-ros r., knaze v., rothwell j.a., hémon b., moskal a., overvad k., tjønneland a. et al. 2015. dietary polyphenol intake in europe: the european prospective investigation into cancer and nutrition (epic) study. european journal of nutrition 55(4):1359-1375. zheng x., yu y., xiao g., xu y., wu j., tang d. and zhang y. 2014. comparing product stability of probiotic beverages using litchi juice treated by high hydrostatic pressure and heat as substrates. innovative food science & emerging technologies 23:61-67. zhu j., chen f., wang l., niu y. and xiao z. 2016. evaluation of the synergism among volatile compounds in oolong tea infusion by odour threshold with sensory analysis and e-nose. food chemistry 221:1484-1490. paper received november 3, 2017 accepted october 30, 2018 ijfs#1448_bozza ital. j. food sci., vol. 31, 2019 416 survey household characteristics influencing fish consumption in van province, turkey m. teri̇n* van yuzuncu yil university, faculty of agriculture, department of agricultural economics, 65080 van, turkey *corresponding author: tel.: +905356478210 e-mail address: mustafaterin@yyu.edu.tr abstract this study investigates the relationship between households’ fish consumption frequency and their socio-demographic characteristics and attitudes. using chi-square test of independence, the study compares households’ fish consumption frequencies of never, once a month, twice a month, once a week and more than once a week. the empirical model was estimated using an ordered probit model to obtain the coefficients applied to the calculation of marginal effects and probabilities. the results indicate that, households’ income, children per households, working households’ head, households’ consumption of aquaculture products other than fish and the surveyed being households’ head significantly influence the frequency of fish consumption. keywords: fish consumption, socio-demographic characteristics, ordered probit model, turkey ital. j. food sci., vol. 31, 2019 417 1. introduction in recent years, healthy nutrition has increasingly been encouraged, as a result, different healthy food consumption tendencies are emerged (gilbert, 2000; leek et al., 2000). sea food is an important part of healthy nutrition (trondsen et al., 2003). regular fish consumption reduces the likelihood of many chronic diseases including cardiovascular disease (kornitzer, 2001; pleadin et al., 2017) and contributes significantly to healthy living (verbeke and vackier, 2005). sea food is regarded as one of the most valuable nutrients in terms of the nutrients it contains. sea food products contribute greatly to human nutrition because of high protein ratio, richness in omega-3 fatty acids, and minerals and vitamins they contain (gülyavuz and ünlüsayin, 1999). especially, fish has many benefits to human nutrition. fish meat is easy to digest, contains high protein and is excellent in fat content. in addition, the vitamins and minerals and the low energy of the dietary supplement increase its importance (tatar, 1995; turan et al., 2006; saygi et al., 2015). these factors are main causes that led consumers to change their consumption preferences from red meat to chicken meat and fish meat (rickertsen, 1996; mangen and burrell, 2001). aquaculture plays an important role in ensuring nutritional needs and global food security in both developed and developing countries. in the past 50 years, global average supply of fishery products has increased by 3.2% per year on average, and world population has increased by 1.6%, resulting in an increase in average per capita consumption of aquatic products. in the world, the average per capita annual consumption of aquatic products is estimated to be 20.5 kg in 2017, while it was 9.0 kg, 17.0 kg and 20.2 kg in 1961, 2000 and 2015, respectively. this impressive increase in average fish consumption per capita was mainly due to the increase in production, income, population and urbanization, as well as the development of modern distribution channels (fao, 2018). as a country surrounded by sea, turkey has a significant potential for aquatic products with its lakes, dams, streams and spring waters. fishing in turkey is an important field of activity in terms of being one of the basic livelihood resources in the coastal regions and human nutrition (anonymous, 2014). aquaculture production in turkey has shown significant fluctuations over the years. in 2017, the production of aquaculture products in turkey increased by 7.15% to 630 thousand tons compared to the previous year. out of this production 354 thousand tones (56.2%) was obtained through hunting, and 276 thousand tones (43.8%) were obtained through aquaculture. van province with the biggest lake (van lake) accounts for 23.0% (8310 tons) of inland water fish production in turkey with its pearl mullet (tarek) fish unique for van lake (turkstat, 2018). in addition, there are 24 trout farms in the province and approximately 200 tons of trout is produced yearly (güngör, 2014). in turkey, per capita consumption of aquatic products has ranged from 6.3 to 8.6 kg/year in the last 18 years and has been 5.5 kg/year as of 2017 (turkstat, 2018). the amount of consumption of aquatic products per capita differs significantly between regions in turkey. while per capita consumption was high at regions by seas in giresun and trabzon 28.08 kg/year (aydin and karadurmuş, 2013), in mersin 25.8 kg/year (şen, 2011) in hatay 21.5 kg/year (demirtaş et al., 2014) and in i̇zmir 15 kg/year (çaylak, 2013), domestic, eastern and southeastern regions were below the world average being 13 kg/year in tokat (erdal and esengün, 2008), 12.4 kg/year in isparta (hatirli et al., 2004), 6.5 kg/year in erzurum (uzundumlu, 2017), 4.13 kg/year in kahramanmaraş (ercan and şahin, 2016), 3.8 kg/year in niğde (bashimov, 2017) and 3.4 kg/year in ankara (gül yavuz et al., 2015). ital. j. food sci., vol. 31, 2019 418 there are many factors affecting fish consumption, including socioeconomic structure, general food consumption structure, personal health status and maritime nature of the living area (myrland et al., 2000; trondsen et al., 2004; verbeke and vackier, 2005). but, the most determining factor for purchasing fish is nutrition (adeli et al., 2011). the aim of the study in this context was to determine the socio demographic and behavioral characteristics that affect the frequency of fish consumption of households in urban areas in van. 2. material and methods the main material of the study is the original data collected through questionnaires from 260 households living in the urban area of van. survey was conducted between december 2015 and january 2016. the sample size was determined by ungrouped one stage random likelihood sampling method based on households (collins, 1986; akbay et al., 2007). 𝑛 = 𝑡^2 [1 + (0.02)(𝑏 − 1)] ∗ 𝑝𝑞/𝐸^2 (1) the statistical relationship between the frequency of fish consumption of households and their socio-demographic and behavioral characteristics was estimated using the chi square test. on the other hand, the effects of the socio-demographic and behavioral characteristics of the habits on the fish consumption frequency was estimated using “ordered probit model" method. statistical package for social science (sppss 17.0) and limdep 10 programs were used in the analysis of the data. the ordered probit model is based on the mcfadden (1973) utility maximization theory. the utility function in the research indicates the utility of the consumer in terms of the frequency of fish consumption. however, the level of utility provided here cannot be observed. behind the observable, intermittent and ordered categories (y) in the ordered probit model is assumed to be a continuous, but unobservable, hidden dependent variable. the unobserved, latent dependent variable (y*) is explained by the vector of explanatory variables and the error term. the term error is assumed to have normal distribution (greene, 2012). y* = x'β + ε ε ~ n [0, 1] (2) in the study, households chose one of the five alternatives for fish consumption, the dependent variable was classified according to its size (y = 0, 1, 2, 3, 4). thus, the relationship between the model dependent variable (y) and the unobserved dependent variable (y*) is as follows (chen et al., 2002; greene, 2012). if y*≤0, y=0 if 0<y*≤μ1, y=1 if μ1<y*≤μ2 y=2 if μ2<y*≤μ3 y=3 if μ3 ≤ y* y=4 the dependent variable used in the model is one of the following categories: “y=0”, “y=1”, “y=2”, “y=3” and “y=4” which represents households’ non-fish consumers, households consumes fish once per month, households consume fish once per fifteen days, ital. j. food sci., vol. 31, 2019 419 households consumes fish per week and households consumes fish more than once per week, respectively. in the ordered probit model, the likelihood of the producers selecting one of five alternatives is as follows (greene, 2012). prob (y = 0|x) = φ (− x’β), (3) prob (y = 1|x) = φ (µ1 − x’β) − φ (− x’β), (4) prob (y = 2|x) = φ (µ2 − x’β) − φ (µ1− x’β), (5) prob (y = 3|x) = φ (µ3 − x’β) − φ (µ2− x’β), (6) prob (y = 4|x) = 1 −φ (µ3 − x’β) (7) for all probabilities to be positive, 0<µ1<µ2<...<µj-1 φ shows the cumulative normal distribution function. the solution of the model can be realized by "logarithmic maximum likelihood" method. the marginal effects of the variables are calculated as follows for each probability (greene, 2012). (𝜕𝑃𝑟𝑜𝑏(𝑦 = 0|𝒙))/𝜕𝒙 = −∅(𝒙^′ 𝛽)𝛽, (8) (𝜕𝑃𝑟𝑜𝑏(𝑦 = 1|𝒙))/𝜕𝒙 = [∅(−𝒙^′ 𝛽) − ∅(µ1 − 𝒙^′ 𝛽)]𝛽, (9) (𝜕𝑃𝑟𝑜𝑏(𝑦 = 2|𝒙))/𝜕𝒙 = [∅(µ1 − 𝒙^′ 𝛽) − ∅(µ2 − 𝒙^′ 𝛽)]𝛽, (10) (𝜕𝑃𝑟𝑜𝑏(𝑦 = 3|𝒙))/𝜕𝒙 = [∅(µ2 − 𝒙^′ 𝛽) − ∅(µ3 − 𝒙^′ 𝛽)]𝛽, (11) (𝜕𝑃𝑟𝑜𝑏(𝑦 = 4|𝒙))/𝜕𝒙 = ∅(µ3 − 𝒙^′ 𝛽)]𝛽 (12) 3. results and discussion out of households surveyed 89.2% consumed fish while the remaining 10.8% did not consume fish. in similar studies conducted in van province, 88.2% and 78.53% of the consumers consumed fish, respectively (sari et al., 2000, ceylan, 2006). in some other studies, the fish consumption rate of consumers was 95.8% (gürgün, 2006), 88.3% (orhan and yüksel, 2010), 90.6% (balik et al., 2013), 98.8% (onurlubaş, 2013), 84.0% (olgunoglu et al., 2014), 95.0% (cicek et al., 2014) and 96.52% (djordjevic et al., 2015). out of the households who didn’t consume fish 35.7% stated the reason as fish odor followed by dislike fish, having no habit of fish consumption, and insufficient purchasing power with 25.0 21.4 and 17.9%, respectively. orhan and yüksel (2010) stated that 60.50% of consumers didn’t consume fish due to odor followed by 12.12% having no habit of fish consumption in burdur province, turkey. out of surveyed households 10.8% didn’t consume fish at all. on the other hand, 27.3% of households consumed fish once per two months followed by once per week, once per month and more than once per week with 26.9, 22.7 and 12.3%, respectively (table 1). in the study conducted by güngör (2014), 36.2% of the consumers consumed fish once a month, followed by those who consumed fish once per fifteen days and once per week with 23.0 and 17.8%, respectively. cicek et al. (2014) stated that 28.0% of consumers consumed fish once per fifteen days followed by those who consumed fish once per week and once per month with 25.0 and 23.0%, respectively in elazığ province, turkey. saygi et al. (2015) pointed out that 25% of consumers in i̇zmir, turkey consumed fish once per week while consumers in ankara generally consumed fish once per fifteen days. in a study conducted in serbia more than half of consumer at school age consumed fish once per week (52.24%) and 34.33% once per month (djordjevic et al., 2015). in mexico one third of consumers (32.51%) consumed fish more than once per week followed by 25.62% once per week, 25.62% once per week, 24.24% once per fifteen days and 17.63% once per month (perez-ramirez et al., 2015). hicks et al. (2008) pointed out in american 22.0% of consumers consumed fish more than once, 24.0% of consumers consumed fish once per ital. j. food sci., vol. 31, 2019 420 week, 29.02% of consumers consumed fish once per two-three months and 12.0% of consumers consumed fish once per month. the average monthly income of the households varied between tl 750 and tl 7500 and the monthly average household income was tl 2776.77. while the average monthly income of 7.7% of households was less than 1000 tl, 66.9% of households had a monthly income varied between tl 1001 and tl 3000 and 25.4% of consumers had income above tl 3001. in a study conducted in van province, 14.9% of the households had monthly income between tl 0-1000, 43.8% between tl 1001-3000 and 41.3% had income above tl 3001 (güngör, 2014). the average number of children per household was 2.61 (table 1). out of surveyed households, 26.9% had one child followed by 23.8, 16.9, 12.0 and 11.9% of households with three, one, five and four child, respectively. in a study conducted by ceylan (2006), 55.21% of households in van, turkey had more than one child, 12.5% had one child and 2.08% had no child. gürgün (2006) pointed out that 7.3% of households had no child, 14.6% of households had two and 5.0% had one child in bitlis, turkey. in a study by verbeke and vackier (2005) in belgium, it was found that 57.3% of the households consisted of families with children. table 1. descriptive statistics of the sample. variables values dependent variable fish consumption frequency never y= 0 %10.8 once a month y= 1 %22.7 twice a month y= 2 %27.3 once a week y= 3 %26.9 more than once a week y= 4 %12.3 continuous explanatory variables means income (tl/month) 2776.77 (1453.18) child number 2.61 (1.29) binary explanatory variables values household head (if 1; otherwise 0) %75.8 household head woman (if 1; otherwise 0) %19.6 house wife working (if 1; otherwise 0) %13.8 consumption other seafood except fish (if 1; otherwise 0) %9.6 resides in rental house (if 1; otherwise 0) %33.1 fish prices high (if 1; otherwise 0) %62.3 public spots effect fish consume. positive (if 1; otherwise 0) %65.0 household head working (if 1; otherwise 0) %94.6 standard deviations are given in brackets. the average monthly fish consumption quantity per household and per capita was 6.3 kg and 1.4 kg, respectively. average yearly fish consumption quantity per capita in turkey is 7 kg (turkstat, 2018). in this case, yearly fish consumption quantity per capita in van province is more than that of turkey. pearl mullet (only caught from van lake) consisted 30.0% of the turkey's inland fish production in 2017 (turkstat, 2018). considering ital. j. food sci., vol. 31, 2019 421 factors such as the price of pearl mullet, which is cheaper than the other fish types and being suitable for the taste of the local people, it is expected that the quantity of fish consumption per capita in the province of van is relatively high. this is also confirmed by the research results. the statistical relationship between the socio-demographic and behavioral characteristics of the households and the fish consumption frequency is given in table 2. a statistically significant relationship was found between socio-demographic characteristics such as households’ income, working household head, the households’ consuming aquatic products other than fish, the high prices of fish, positive effect of public spots and fish consumption frequency of households. it can be said that the results obtained are in accordance with the expectations. table 2. socio-demographic characteristics and several attitudes of the sample and fish consumption frequencies. variables never once a month twice a month once a week more than once a week x 2 household head yes 10.2 20.8 26.4 27.9 14.7 5.95 no 12.7 28.6 30.2 23.8 4.8 household head woman yes 13.7 21.6 27.5 25.5 11.8 0.61 no 10.0 23.0 27.3 27.3 12. house wife working yes 2.8 22.2 27.8 33.3 13.9 3.21 no 12.1 22.8 27.2 25.9 12.1 consumption other seafood except fish yes 0.0 16.0 8.0 48.0 28.0 17.40*** no 11.9 23.4 29.4 24.7 10.6 renter yes 10.5 24.4 26.7 27.9 10.5 0.60 no 10.9 21.8 27.6 26.4 13.2 fish prices high yes 17.3 25.3 28.4 19.8 9.3 29.87*** no 0.0 18.4 25.5 38.8 17.3 public spots effects fish consumption positive yes 0.0 24.3 32.5 30.2 13.0 59.47*** no 30.8 19.8 17.6 20.9 11.0 household head working yes 9.3 21.5 27.6 28.5 13.0 17.19*** no 35.7 42.9 21.4 0.0 0.0 income less than 1000 tl 30.0 35.0 30.0 5.0 0.0 23.57*** 1001-3000 tl 10.3 24.1 28.2 27.0 10.3 3001 tl and over 6.1 15.2 24.2 33.3 21.2 ***: 0,01 significant level. ital. j. food sci., vol. 31, 2019 422 the ordered probit model results of the socio-demographic and behavioral characteristics that affect the frequency of fish consumption of the households are given in table 3. the ordered probit model was found to be totally statistically significant using likelihood method (p <0.000). the coefficients of the model were tested using z-rate and standard error. estimated threshold values in the model indicate the numerical relationship between the utility function of the consumer and the consumption frequency (akbay et al., 2007; gunduz and emir, 2010). in view of maddala (1983), the threshold values should be positive and μ1 <μ2 <μ3. the threshold values of the model were positive and statistically significant at 0.01 level. this shows that the consumption frequency categories households are arranged appropriately and the socio-demographic and behavioral characteristics of the households are influential on fish consumption. the marginal effects of the socio-demographic and behavioral characteristics that affect the frequency of fish consumption of the households are given in table 4. while model results are interpreted, marginal effects and coefficients are discussed together. in the study, a positive and statistically significant relationship was found between the household income and the fish consumption frequency (table 3). as a result, the increase in household income will increase the frequency of fish consumption of the households. a tl 1,000 increase in household income will result in reducing the likelihood of no consumption (y=0) and monthly consumption (y=1) by 2.0% and 3.6%, respectively while increase the likelihood of consuming in a weekly (y=3) and more than once a week (y=1), by 3.5% and 2.5%, respectively (table 4). a positive relationship between household income and the frequency and amount of fish consumption has been found in previous studies (akinbode and dipeolu, 2012; can et al., 2015; dauda et al., 2016). in the study, a positive and statistically significant relationship was found between the number of children of households and the frequency of fish consumption (table 3). as a result, one more child in the households will reduce the likelihood of no-consumption (y=0) and consumption per month (y=1) by 1.08 and 1.92%, respectively, while it will increase the likelihood of consumption once per week (y=3) and consumption more than once per week (y=4) by 1.85 and 1.31%, respectively (table 4). in a study by myrland et al. (2000), it was found that the frequency of fish consumption increases by having children between 0-7 years and 8-12 years, having children between 0-7 age group reduce the likelihood of no-consumption and consuming per month by 1.9 and 1.4%, respectively while increasing the likelihood of consuming once per week and once per two weeks by 2.9 and 0.9%. in the study, a positive and statistically significant relationship was found between the heads of households and the frequency of fish consumption (table 3). as a result, the head of households will reduce the likelihood of no-consumption (y=0) and consumption per month(y=1) by 6.36 and 9.26%, respectively, while it will increase the likelihood of consumption once per week (y=3) and consumption more than once per week (y=4) by 9.39 and 5.68%, respectively. a positive and statistically significant relationship was found between the consumption of other aquaculture products except fish and the frequency of fish consumption (table 3). as a result, the households’ consumption of aquaculture products except fish will reduce the likelihood of no-consumption (y=0) and consumption per month (y=1) by 5.55 and 13.58%, respectively, while it will increase the likelihood of consumption once per week (y=3) and consumption more than once per week (y=4) by 11.09 and 13.52%, respectively (table 4). myrland et al. (2000) pointed out that households’ consumption of aquaculture products would increase the fish consumption frequency, namely would decrease the likelihood of non-consumption and consumption per month frequency by 16.2 and 23.7%, respectively while would increase the likelihood of consumption frequency of once per week and twice per week by 32.7 and 24.9%, respectively. ital. j. food sci., vol. 31, 2019 423 in the study, a positive and statistically significant relationship was found between the households’ finding fish prices high and the frequency of fish consumption (table 3). as a result, the households’ considering the fish prices high will reduce the households’ fish consumption frequency. the households’ considering the fish prices high will increase the likelihood of no-consumption (y=0) and consumption per month (y=1) by 6.90 and 13.15%, respectively, while it will reduce the likelihood of consumption once per week (y=3) and consumption more than once per week (y=4) by 12.14 and 10.24%, respectively (table 4). in the study, a positive and statistically significant relationship was found between the households’ thought that public spots had positive effects on fish consumption and the frequency of fish consumption (table 3). the households’ thought that public spots had positive effects on fish consumption will reduce the likelihood of no-consumption (y=0) and consumption per month (y=1) by 9.71 and 13.97%, respectively, while it will increase the likelihood of consumption once per week (y=3) and consumption more than once per week (y=4) by 14.11 and 8.97%, respectively (table 4). a positive and statistically significant relationship was found between the working households’ head and the frequency of fish consumption (table 3). as a result, households’ head working at any place will increase fish consumption frequency. the working households’ head will reduce the likelihood of no-consumption (y=0) and consumption per month (y=1) by 15.28 and 13.84%, respectively, while it will increase the likelihood of consumption once per week (y=3) and consumption more than once per week (y=4) by 16.19 and 7.31%, respectively (table 4). table 3. estimates ordered probit model for fish consumption frequencies. variables coefficient st. error z-statistic p-value constant -0.2056 0.402 -0.51 0.6089 income 0.00016 0.0000546 2.91 0.0036*** child number 0.084 0.043 1.94 0.0528* household head 0.422 0.168 2.51 0.0120*** household head woman -0.158 0.189 -0.83 0.4040 house wife working -0.105 0.233 -0.45 0.6518 other seafood consumption 0.630 0.242 2.61 0.0091** resides in rental house -0.147 0.153 -0.96 0.3352 fish prices high -0.591 0.142 -4.15 0.0000*** public spots 0.648 0.143 4.54 0.0000*** household head working 0.754 0.333 2.26 0.0238** threshold parameters μ (1) 1.031 0.08262 12.48 0.0000*** μ (2) 1.856 0.08267 22.45 0.0000*** μ (3) 2.879 0.11400 25.26 0.0000*** log likelihood = -357.910 restricted log likelihood = -400.952 likelihood ratio statistic (lr) = 86.084 chi-square(10;0.05)=18.307 lr>chi-square=0.000 *:0.1, **:0.05 and ***: 0,01 significant level. ital. j. food sci., vol. 31, 2019 424 table 4. the marginal effects of factors on the probability of relative frequencies for fish consumption. variables y(0) y(1) y(2) y(3) y(4) income -0.000020*** -0.000036*** -0.0000031 0.000035*** 0.000025*** child number -0.01078* -0.01921* -0.00164 0.01850* 0.01313* household head -0.06366** -0.09256*** 0.00552 0.09392** 0.05678*** household head woman 0.2171 0.03566 0.00079 -0.03514 -0.02303 house wife working 0.01426 0.02384 0.00084 -0.02338 -0.01556 other seafood consumption -0.05501*** -0.13582*** -0.05534 0.11094*** 0.13523** resides in rental house 0.01955 0.03337 0.00176 -0.03252 -0.02216 fish prices high 0.06900*** 0.13151*** 0.02331 -0.12144*** -0.10239*** public spots -0.09714*** -0.13973*** 0.00609 0.14111*** 0.08967*** household head working -0.15276 -0.13839*** 0.05612 0.16191** 0.07312*** *:0.1, **:0.05 and ***: 0,01 significant level. 4. conclusions result of this study showed that various socio-economic and demographic factors of households and households’ heads significantly influenced the likelihood of consuming fish. there was a positive relationship between the socio-economic and demographic characteristics of households such as households’ income, children per households, working households’ head, households’ consumption of aquaculture products other than fish, households’ head and the behavioral variables such as households’ thought that public spots affected the fish consumption positively. based on the findings of the study, the following recommendations were made; public or private organization should continue to educate the households’ heads (parents) on the importance of fish on their health. price of fish should be reduced so as to increase the fish consumption in the area since it was observed that price of fish and fish consumption are inversely related. as the income of the 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rickertsen k. 1996. structural change and the demand for meat and fish in norway. european review of agricultural economics 23(3):316.330. sarı m., demirulus h. and söğüt b. 2000. a research on determination of fish meat consumption habits of students in van province. in proceedings of the east anatolian region 4th aquaculture symposium, erzurum, turkey. saygı h., bayhan b. and hekimoğlu m.a. 2015. fishery products consumption in the cities of ankara and i̇zmir in turkey. turkish journal of agriculture-food science and technology 3(5):248-254. şen a. 2011. individuals living in konya and mersin provincial centers comparison of fish consumption on the habits and knowledge level msc. thesis, selçuk university, institute of natural and applied sciences, konya, turkey. tatar o. 1995. nutritional properties of fish and healthy respect. e.u. journal of fisheries & aquatic sciences 12:169-170. trondsen t., braaten t., lund e. and eggen a.e. 2004. health and seafood consumption patterns among women 45-69 years. a norwegian fish consumption study 1996. food quality and preference 15(2):117-128. trondsen t., scholdere j., lund e. and eggen a.e. 2003. perceived barriers to consumption of fish among norwegian women. appetite 41(3):301-314. turan h., kaya y. and sönmez g. 2006. fish meat value and place of human health. e.u. journal of fisheries & aquatic sciences 23:505-508. turkstat 2018. turkish statistical institute. aquaculture statistics. www.tuik.gov.tr/pretablo.do?alt_id=1005 (28.08.2018). uzundumlu a.s. 2017. determining fish consumption behavior among households and the most suitable type of fish in erzurum province. iran j. fish sci. 16(2):684-697. verbeke w. and vackier i. 2005. individual determinants of fish consumption: application of the theory of planned behavior. appetite 44(1):67-82. paper received november 29, 2018 accepted february 24, 2019 ijfs#1221_bozza ital. j. food sci., vol. 31, 2019 19 review edible allium species: chemical composition, biological activity and health effects ž. fredotović and j. puizina* university of split, faculty of science, ruđera boškovića 33, 21 000 split, croatia *corresponding author: tel.: +385 21619290 e-mail address: puizina@pmfst.hr abstract since ancient times edible alliums play an important role in human diet and traditional medicine. the most commonly cultivated allium species are onion (allium cepa l.), garlic (allium sativum l), leek (allium ampeloprasum l.), chive (allium schoenoprasum l.) and welsh or japanese bunching onion (allium fistulosum l.). these species are rich sources of biologically active compounds such as flavonoids, organosulfur compounds and saponins. numerous studies we reviewed in this paper, confirmed their significant antioxidant, antibacterial, anti-inflammatory, antiproliferative and anticancer activities, which makes them an important source of phytonutrients that can contribute to the protection and preservation of human health. keywords: alliums, biological activity, flavonoids, organosulfur compounds ital. j. food sci., vol. 31, 2019 20 1. introduction allium species have been used for centuries in human diet because of their pungent smell and specific taste, but also for medicinal purposes because of their remarkable medicinal properties. the best known and most cultivated species of the genus allium, onion (allium cepa l.) and garlic (allium sativum l.) are widely used as spices and medicinal plants and are therefore the subject of numerous studies. to date, their chemical structure has been thoroughly explored as well as their biological activity. allium species are rich in phytonutrients, mostly flavonoids and organosulfur compounds which exhibit strong antioxidative, antimicrobial, anti-inflammatory and anticarcinogenic activity (ahiabor et al., 2016; albishi et al., 2013; ashraf et al., 2011; benekeblia, 2004; borowska et al., 2013; chang et al., 2013; colina-coca et al., 2017; herman-antosiewicz and singh, 2004; johnson et al., 2016; kaur et al., 2016; khazaei et al., 2017; kim et al., 2013; koca et al., 2016; kumari and ranjan, 2014; kwon et al., 2002; lanzotti et al., 2014; lanzotti, 2006; li et al., 2016; li et al., 2014; min kim et al., 1997; ndoye foe et al., 2016; ortiz, 2015; pan et al., 2018; quintero-fabián et al., 2013; shin et al., 2013; suleiman and abdallah, 2014; thomas et al., 2017; thomson and ali, 2003; yang et al., 2001b). many epidemiological studies showed that regular consumption of allium vegetables can decrease the risk of various diseases such as cardiovascular, respiratory, gastrointestinal diseases and different types of cancer (chen et al., 2009; fleischauer et al., 2000; gonzález et al., 2006; guercio et al., 2014; kim et al., 2018; o’gara et al., 2000; pourzand et al., 2016; turati et al., 2015, 2014; you et al., 1989; zhou et al., 2011). 2. chemical composition the chemical composition of these species is very complex. they contain a variety of different phytochemicals, and the most important constituents are organosulfur compounds. these compounds provide them with characteristic odor and flavor, as well as the majority of biological properties. another important group of chemically active compounds are polyphenols, which include phenolic acids and flavonoids, responsible for the characteristic color of onion bulbs. edible onion parts are rich in carbohydrates, mostly glucose and fructose, while the outer scales of onion bulbs contain significant content of galactose and arabinose. essential amino acids (arginine and glutamic acid), which may be important nitrogen reserves, also contribute to the nutritional value of onion species. they also contain several other complex bioactive components such as saponins, vitamins (a, c, b6, and folate) and minerals (p, k, ca, mg, zn, mn, na, fe, br, j, se, and cu). 2.1. flavonoids flavonoids are the largest group of polyphenolic compounds present in fruit, vegetables, nuts, tea, wine and other food ingredients. common onion (a. cepa l.), garlic (a. sativum l.), and other allium species are the richest sources of dietary flavonoids. the best described property of flavonoids is their antioxidant activity. their structure is essential for their ability to act. the configuration, substitution and number of hydroxyl groups determine their antioxidant activity and ability to scavenge free reactive species. flavonoids have a characteristic structure with two benzene rings (a and b rings, shown in fig. 1) connected with a pyran ring (heterocyclic ring containing oxygen, the c ring, shown in fig. 1). ital. j. food sci., vol. 31, 2019 21 figure 1. the basic structure of flavonoids (pietta, 2000). so far, more than 4000 different kind of flavonoids have been identified. they are divided into groups (fig. 2) by the number and position of hydroxyl groups, level of oxidation and pattern of substitution of the c ring: i) anthocyanins glycosylated derivative of anthocyanidin, present in colorful flowers and fruits; ii) anthoxanthins a group of colorless compounds divided in several categories, including flavones, flavonols, flavanones, flavanols, isoflavones and their glycosides (han et al., 2007). two flavonoid classes are mainly found in onion, flavonols, responsible for yellow and brown skin and anthocyanins which give red to purple color to some onion varieties (rodrigues et al., 2017). figure 2. chemical structure of the main classes of flavonoids (reis giada, 2013). ital. j. food sci., vol. 31, 2019 22 flavonoids are plant pigments, which participate in plant protection against different ecological and physiological stresses such as uv radiation, heat, herbivores and pathogens. flavonoids have been shown to possess a diverse biological property such as antioxidant, anti-inflammatory, antiallergic, antimicrobial, antiviral, anticancer and neuroprotective activity. these properties are structure dependent which makes flavonoids one of the best antioxidants, scavengers of free radicals and a chelator of bivalent cations that cause dna damage which is associated with the development of many diseases (halliwell et al., 2005; panche et al., 2016). anthocyanins and flavonols are the most common subgroups of flavonoids present in allium species. the most abundant flavonols in common onions are two quercetin conjugates, namely quercetin 3,4’-diglucoside and quercetin 4'-monoglucoside (caridi et al., 2007; fredotović et al., 2017; griffiths et al., 2002). these compounds together account for about 80% of total flavonol content with some differences among various allium cultivars. quercetin, myricetin, kaempferol and isorhamnetin glycosides accounted for the remaining 15% total of flavonols in onions. compared to yellow and white onions, red onions showed higher total flavonol content (prakash et al., 2007). also, total flavonol content is higher in the outer layers compared to the inner layers of onion bulb. in addition to flavonoids, onions are rich source of anthocyanins. anthocyanins are class of natural pigments responsible for the color of fruits, vegetables, flowers and grains. they give red or purple color to outer layers of onion bulbs. the most frequent anthocyanins in red onion are cyanidin derivatives (over 50% of all anthocyanins), cyanidin-3-(6"malonylglucoside), cyanidin-3-(6"-malonyl-3"glucosyl-glucoside) and cyanidin-3glucoside. there are also minor amounts of peonidin, petunidin and delphinidin derivatives (ferreres and gil, 1996; fossen et al., 1996; gennaro et al., 2002; rodrigues et al., 2017; slimestad et al., 2007). 2.2. organosulfur compounds allium species are characterized by the rich content of sulfur compounds such as salk(en)yl cysteine sulfoxide (acso), sulfides, alkyl polysulfides and amino acids (who, 1999). first sulfur compounds were isolated from allium species in the middle of the 19th century (semmler, 1892; wertheim, 1844). it has been discovered that volatile sulfur compounds are responsible for the pungent odor of onion species. also, it became clear that these disulfide compounds are not present in intact bulbs, but they are formed by the enzymatic cleavage of precursors upon disruption of the bulb tissue. alliin or (+)-s-allylcysteine sulfoxide, was the first odorless sulfur compound isolated from the cytoplasm of intact garlic cell (stoll and seebeck, 1948). after crushing the onion cell, the enzyme allinase is released from vacuole. the enzyme allinase belongs to the group of c-s lyases and plays a key role in the formation of volatile compounds of the genus allium (keusgen, 2011). acso is decomposed by the enzymatic reaction of allinase to pyruvate, ammonia and sulfenic acid. sulfenic acid immediately produce thiosulfinates by a quick condensation reaction (block et al., 1992). thiosulfinates are very unstable and break down to the mixture of compounds, responsible for specific onion taste: polysulfides, thiosulfinates, capaenes and zwiebelanes (fig. 3). the main thiosulfinate in garlic is diallyl sulfide, derived from allicin, and dipropyl disulfide in common onion, derived from isoalliin (rose et al., 2005). ndoye foe et al. (2016) identified the main components of a. sativum and a. cepa essential oil. a. sativum essential oil was rich in diallyl trisulfide, diallyl disulfide, allyl methyl trisulfide, diallyl sulfide and diallyl tetrasulfide while those from a. cepa was rich in diallyl trisulfide, dipropyl trisulfide, 2-methyl-3,4-dithiaheptane, methyl propyl trisulfide, dipropyl ital. j. food sci., vol. 31, 2019 23 tetrasulfide and 2-propenyl propyl disulfide. these compounds are most likely responsible for their excellent antioxidant and anti-inflammatory activity. figure 3. overview of organosulfide formation from s-alk(en)yl-l-cysteine sulfoxide (acso) in allium species (tocmo et al., 2015). 2.3. other bioactive compounds recent scientific research found several interesting novel compounds isolated from onion such as saponins and peptides. they have been identified so far in over 40 different allium species (sobolewska et al., 2016). saponins are one of the largest classes of surfaceactive secondary metabolites widely distributed in plants, however, their biological functions are not completely understood. they are generally considered to have important roles in defense of plants against pathogens, pests and herbivores and several studies indicated that they can act as natural remedies in treatment of many diseases (augusti, 1990; lanzotti, 2006; morrissey and osbourn, 1999; osbourn et al., 2011; sobolewska et al., 2016; sparg et al., 2004). recent study reported that saponins from allium species possess high cytotoxic activity, which makes them potential candidates for future development as anticancer agents (lanzzoti et al., 2014). 3. biological activity and health effects of edible onions onions are used since ancient times as vegetable and spice because of their special taste and smell. they were also used in folk medicine for treatment of bacterial infections such as dysentery, ulcers, wounds, scars, keloids, and asthma. they have also been used as an ital. j. food sci., vol. 31, 2019 24 adjuvant therapy for diabetes, for prevention of high blood pressure, and loss of appetite (who, 1999). over the last 50 years, an intensive research has been conducted on the evaluation of biological activity of allium plants, their extracts and essential oil. garlic and onion are the best known and two mostly tested allium species. garlic extracts have been reported to possess strong antibacterial (benekeblia, 2004), antidiabetic (ashraf et al., 2011), antiproliferative activity (thomson and ali, 2003; yang et al., 2001a), and ability to inhibit development of cardiovascular diseases (thomson et al., 2006). similar but weaker effects have been proved for onion extracts. 3.1. antioxidant activity allium plants are one of the main food antioxidants. antioxidants prevent cell and dna damage by chelating free oxygen or nitrogen radicals (ros or rns), inhibiting their production, or activating antioxidative enzymes (superoxide dismutase 2sod2, catalase cat and glutathione peroxidasegpx). analysis of the antioxidant activity of allium species is also important because of the proven link between oxidative stress and development of diseases such as atherosclerosis, various forms of cancer and even aging itself. the first study of antioxidant properties of allium plants was performed with crude extracts. investigators have concluded that the organosulfur compounds are primarily responsible for observed antioxidant effects (augusti, 1990; min kim et al., 1997; siegers et al., 1999a). yin and cheng (1998), and benekeblia (2004) concluded that antioxidant activity of onions is not just related with organosulfur compounds but also with phenolic compounds. in vitro and in vivo data reported that onion extracts showed stronger ability to scavenge free radicals, compared to garlic extracts and red onion was more active than yellow onion (gorinstein et al., 2008; nuutila et al., 2003). red onion is the rich source of flavonoids especially quercetin, presented in conjugated form. the dry outer layers of onion, which are wasted during food preparation, contain huge amounts of quercetin and its derivatives (corzo-martínez and corzo, 2007). kaur et al. (2016) proved that methanolic extract of allium cepa have higher antioxidant activity compared to aqueous extract. interestingly, it was observed that aqueous extract had higher phenolic content. kim et al. (2005) investigated antioxidant activity of flavonols isolated from garlic leaves and shoots. they confirmed that quercetin and its compounds possess strong antioxidant activity. kumari and ranjan (2014) study showed that methanolic extract of allium sativum exhibited strong antioxidant activity, which is in correlation with rich phenolic content. fredotović et al. (2017) determined antioxidant potential of two onion methanolic extracts, allium × cornutum and a. cepa. both onions showed strong antioxidant activity. however, a. × cornutum extract showed slightly higher antioxidant activity which was in correlation with his higher total phenolic content. comparison of antioxidant activity of garlic and onion cultivars grown in turkey showed that both species possesses good antioxidant properties which are significantly correlated with their total phenolic content (tpc). among all samples of onion and garlic, red onions had the highest tpc and antioxidant activity (koca et al., 2016). kim et al. (2018) performed a comparative study of bioactive organosulfur compounds and antioxidant activity in three allium species, a. cepa (onion), a. sativum (garlic) and a. ampheloprasum (elephant garlic). results showed that garlic possessed the strongest antioxidant activity followed by elephant garlic and onion. their findings demonstrated a positive correlation between antioxidant activity and organosulfur compounds content. the study of colina-coca et al. (2017) confirmed that feeding rats with high-cholesterol (hc) diet resulted in oxidative stress. hc diet enriched with onion ingredients significantly decreased oxidative ital. j. food sci., vol. 31, 2019 25 stress by activating antioxidant defense system. boyle et al. (2000) performed an in vivo experiment and confirmed the powerful antioxidant effect of onions. they showed that consumption of food rich in flavonoids (in this experiment it was fried red onion) is associated with an increased resistance of human lymphocyte dna to dna strand breakage. different allium species, both cultivated (a. nutans l., a. fistulosum l., a. vineale l., a. pskemense b. fedtsch, a. schoenoprasum l., a. cepa l. and a. sativum l.) and wild (a. flavum l., a. sphaerocephalum l., a. atroviolaceum boiss, a. vineale l., a. ursinum l., a. scorodoprasum l., a. roseum l. and a. subhirsutum l.), were investigated in order to evaluate the antioxidant properties of their bulbs (štajner et al., 2008). the results confirmed that the bulbs and leaves of cultivated allium species possess better antioxidant ability in comparison with other wild species, which makes them promising sources of non-toxic natural antioxidants. therefore, the bulbs and leaves of allium species could be used not only in human diet but also as a source of natural antioxidants and for medical purposes. 3.2. antimicrobial activity in 1858. louis pasteur described the antibacterial effect of garlic for the first time. during world war ii garlic was used as an antiseptic to prevent gangrene (petrovska and cekovska, 2010). recent studies confirmed antibacterial properties of garlic. garlic is effective against gram-positive and gram-negative bacteria, although its extracts were more effective on gram-negative bacteria (dankert et al., 1979; elnima et al., 1983; yoshida et al., 1999a, 1999b). johnson et al. (2016) showed significant antimicrobial potency of aqueous garlic extract against pseudomonas aeruginosa and staphylococcus aureus. aqueous extracts of allium sativum and allium tuberosum were tested against penicillinsensitive s. aureus (pssa) and mrsa. both extracts were able to reduce staphylococcal infection, although only a. sativum showed in vitro anti-staphylococcal activity, but neither of them wasn't effective against mrsa (venâncio et al., 2017). han et al. (1995) showed that biological activity of 1 mg allicin works the same as 15 iu of penicillin. this proved that allicin and other organosulfur compounds present in garlic essential oil are responsible for strong antibacterial activity (ankri and mirelman, 1999; matsuura, 1997). wallock-richards et al. (2014) reported the first evidence for antimicrobial activity of allicin containing garlic extract against bcc (burkholderia cepacia complex), the major bacterial phytopathogen for alliums and intrinsically multiresistant human pathogen. red onion is not so effective against bacteria compared to garlic (bakht et al., 2013; benekeblia, 2004; hughes and lawson, 1991; min kim et al., 1997; santas et al., 2010). onion essential oil showed strong inhibitory effect on growth of gram-positive bacteria. their water extracts showed strong in vitro inhibitory effect on growth of escherichia coli, serratia marcescens, streptococcus sp., lactobacillus odontolyticus, pseudomonas aeruginosa, salmonella typhosa and prevotella intermedia (bakri and douglas, 2005). raw onion extract showed good antimicrobial activity against s. aureus while boiled extract had no activity. in addition, the boiled onion extract showed no antimicrobial activity against both s. aureus and e. coli (ortiz, 2015). ahiabor et al. (2016) confirmed antimicrobial activity of undiluted crude extracts of red and yellow onion (a. cepa l.) and shallot (a. aescalonicum l.) against s. typhi, e. coli and s. aureus. it is interesting that onion and garlic extracts can prevent growth and development of oral bacteria that cause caries (jain et al., 2015; kim, 1997; mishra et al., 2016; thomas et al., 2017). beside antibacterial effect, onion and garlic are also effective against broad spectrum of fungi and yeasts. garlic showed strong inhibitory effect against candida sp., cryptococcus ital. j. food sci., vol. 31, 2019 26 sp., trichophyton sp., epidermophyton sp. and microsporum sp. (ankri and mirelman, 1999; davis et al., 1994; shams-ghahfarokhi et al., 2006; yamada and azuma, 1977) as well as against fungi that produce mycotoxins such as aspergillus parasiticus, a. niger, a. flavus and a. fumigates (benekeblia, 2004; yin and tsao, 1999). recent research showed antifungal effect of garlic essential oil against three trichophyton species (trichophyton rubrum, t. erinacei, t. soudanense) responsible for severe mycoses in humans (pyun and shin, 2006). water extract of red onion was effective against malassezia furfur and candida sp. (shams-ghahfarokhi et al., 2006). the newest studies are consistent with those published research and confirm strong antifungal activity of garlic aqueous and petroleum ether extracts as well as garlic oil tested on candida albicans (li et al., 2016), aspergillus, curvularia and some dermatophyte species (suleiman and abdallah, 2014). allium species stimulate growth of probiotic bacteria (genus lactobacillus and bifidobacterium), which can ferment oligosaccharides, prebiotics that human organism cannot digest on its own. ingestion of probiotic bacteria may reduce the severity and frequency of diarrheal diseases, and development of colon cancer as well as improve lactose digestibility among lactose-intolerant individuals (kaplan and hutkins, 2000). li et al. (2016) investigated the effect of onion juice on milk fermentation by lactobacillus acidophilus. the onion juice stimulated the growth of probiotic bacteria l. acidophilus and maintain their viability. the authors assume that whole constituents of onion juice including polyphenols, sulfur compounds, minerals and fructans together are responsible for stimulation of growth and viability of l. acidophilus. 3.3. anti-inflammatory activity it has been proved that most of the allium members possesses anti-inflammatory effect. mechanism of their anti-inflammatory action can be explained through the interaction of oxidative stress and inflammation. overexpression of pro-inflammatory enzymes such as inos (inducible nitrogen oxide synthetase) and cox-ii (cyclooxygenase) is noticed in some diseases such as atherosclerosis, some types of cancer and inflammatory diseases. their overexpression leads to production of pro-inflammatory mediators such as no (nitrogen oxide) and pg (prostaglandin) which play important role in maintenance of normal blood pressure, inflammatory processes, wound healing and regulation of body temperature (reuter et al., 2010). however, their overexpression can also lead to development of diseases such as colon cancer, atherosclerosis, inflammatory bowel diseases, multiple sclerosis and alzheimer disease. inos and cox-ii enzymes which activate mediators are under control of transcriptional factor nf-κb. nf-κb controls the expression of more than 150 different genes included in regulation of inflammatory processes. recent research has been proved that antioxidants can inhibit activation of nf-κb, and thus reduce the symptoms, and development of mentioned diseases and conditions (devi et al., 2009) quercetin and apigenin are potent inhibitors of nitric oxide (no) and prostaglandin e2 (pge2) production induced by lipopolysaccharide (lps) in the macrophage cell line j774a.1 (raso et al., 2001). they modulate inos and cox-ii enzyme expression. reduced activity of both enzymes by the action of apigenin and quercetin affects the expression of nf-κb (wadsworth and koop, 1999). modulation of inos and cox-ii enzymes by these two flavonoids may be important in the prevention of inflammation and indicate that they might be used as potent anti-inflammatory agents. three active garlic compounds, caffeic acid, s-allyl cysteine and uracil inhibited uvb-induced skin wrinkle formation in mice by decreasing oxidative stress as follows: i) caffeic acid and s-allyl cysteine decrease oxidative stress by direct affecting and modulating nf-κb or ap-1 ital. j. food sci., vol. 31, 2019 27 (activator protein 1), ii) all three active compounds achieve anti-inflammatory effect through the suppression of cox-2 and inos (kim et al., 2013). sac, caffeic acid, uracil, diallyl trisulfide, diallyl sulfide and other garlic compounds can inhibit activity of nf-κb by inhibiting transcription of cytokine genes involved in proinflammatory response. phenolic extract isolated from onion skin showed the potency of inhibition human ldl cholesterol oxidation and cox-2 expression even at concentrations as low as 5 µg/ml (albishi et al., 2013). quintero-fabián et al. (2013) examined the effects of alliin in lipopolysaccharide(lps-) stimulated 3t3-l1 adipocytes by rt-pcr, western blot, and microarrays analysis of 22,000 genes. the phosphorylation of erk1/2, which is involved in lps-induced inflammation in adipocytes, was decreased following alliin treatment. also, the gene expression profile by microarrays showed an upregulation of genes involved in immune response and downregulation of genes related with cancer. their results have shown that alliin is able to suppress the lps inflammatory signals by generating anti-inflammatory gene expression profile and by modifying adipocyte metabolic profile. garlic prevents oxidation of low density lipoprotein (ldl). the oxidation of ldl is under control of lipoxygenase (lox) and inducible no synthase (inos) whose activity is regulated by transcriptional factor nf-κb. oxidized ldl promotes adhesion and platelet aggregation, which stimulates the inflammatory process, resulting in damage of cardiovascular system and development of diseases such as atherosclerosis. garlic water extract and its main component, s-allyl cysteine (sac) inhibits inos in human macrophages and thus reduce oxidation of ldl (geng et al., 1997; ide and lau, 2001). thanks to its strong antioxidative capacity, sac can remove superoxide radical that reacts with no and thus prevent its activity. diallyl-disulfide (dads) also can decrease no production, expression of proinflammatory cytokines and protein expression in raw264.7 murine macrophage cell line (shin et al., 2013) garlic extract and its main compound sac, may be useful for prevention of atherosclerosis (kim et al., 2001). kim et al. (2005) isolated four flavonols from garlic leaves and shoots and checked their antioxidant activity by measuring inhibition of lipoxygenase (lo) and hyaluronidase (hya). quercetin showed the strongest antioxidant activity while its glycosides, isoquercitrin and reynoutrin showed slightly lower activity. allicin, the active substance of garlic, inhibited degradation of iκb (inhibitor of transcriptional factor nf-κb). the degradation of iκb releases active nf-κb, which is then translocated to the nucleus and regulates gene expression. inhibition of nf-κb reduce proinflammatory cytokine expression and synthesis of inflammatory enzymes cox/lox (lang et al., 2004). ali et al. (2000) demonstrate that a. cepa and its thiosulfates can inhibit cox and lox activity, as well as platelet aggregation in the blood. they also confirmed antiasthmatic activity of onion extract and ability to inhibit cancer development. jaiswal and rizvi (2014) explored the effect of onion extract in the regulation of pon1 (paraoxonase 1) expression in male wistar rats subjected to mercuric chloride induced oxidative stress. pon1 is an important enzyme with capability of protection against low-density lipoprotein (ldl) oxidation. onion extract significantly decreased mercuric chloride induced oxidative damage by up-regulating the activity of pon1 enzyme and protected against ldloxidation and lipid peroxidation. 3.4. antiproliferative and anticancer activity antiproliferative effect of allium species have been reported in several studies using different cell cultures. seki et al. (2000) reported about the ability of garlic and onion oil to inhibit proliferation of human promyelocytic leukemia cells. interesting experiment of siegers et al. (1999b) showed that garlic powder and extract alone are unable to inhibit ital. j. food sci., vol. 31, 2019 28 tumor cell growth, but when extract and garlic powder are supplemented simultaneously, there was a significant inhibition of cell proliferation. they suggested that antiproliferative effect of garlic is due to the catalytic break-down of alliin induced by alliinase enzyme. after catalytic breakage of alliin, allicin and polysulfides are synthesized, which are responsible for such a powerful antiproliferative effect. it was also found that onionin a, a natural compound isolated from onion, strongly inhibited ovarian cancer cell proliferation, so it could be used for additional treatment of patients with ovarian cancer (tsuboki et al., 2016). fredotović et al. (2017) demonstrated strong antiproliferative effect of methanolic extracts of two onion species, triploid onion a. × cornutum and a. cepa, on glioblastoma and breast cancer cell lines. the inhibition of glioblastoma cell growth was stronger than the inhibition of breast cancer lines in both onion extracts treatments. as we have already mentioned, allium species are rich sources of flavonoids and organosulfur compounds. the molecular mechanism of antiproliferative action is related with both type of compounds. quercetin glucosides (q 3,4'-diglucoside and q 4'monoglucoside), isolated form four allium species, a. chinese (chinese onion), a. sativum (garlic), a. cepa l. (onion) and a. fistulosum l. (welsh onion) showed to be an effective inhibitor on cell growth of hepg2, pc-3 and ht-29 cells (pan et al., 2018). these results suggest that combination of quercetin glucosides may be responsible for antiproliferative activity of onion extracts on cancer cells (li et al., 2014). chang et al. (2013) demonstrated that among all isolated glucosides from onion extract, q 4'-monoglucoside exhibited the highest antioxidant activity on cancer cells. they also suggested that quercetin glucosides may act as activators of apoptosis in different cancer cell lines. the antiproliferative action of flavonoids may involve the inhibition of the prooxidant process. flavonoids are effective inhibitors of xanthine oxidase (chang et al., 1993), cox and lox (mutoh et al., 2000), and therefore they inhibit tumor cell proliferation. also, they can inhibit polyamine biosynthesis. ornithine decarboxylase is enzyme involved in polyamine biosynthesis and correlated with the rate of dna synthesis and cell proliferation in different tissues. different experiments showed that flavonoids are able to inhibit ornithine decarboxylase and decrease polyamine level leading to inhibition of dna synthesis and cell proliferation (makita et al., 1996; tanaka et al., 1997a, 1997b). the antiproliferative effect of organosulfur compounds seems to be related with their ability to induce apoptosis. the study of sundaram and milner (1996) showed that dads (diallyl disulfide) can reduce the growth of colon, lung and skin tumor cells. dads’ antiproliferative activity depends on the presence of both diallyl and disulfide groups. the antiproliferative mechanism of dads and dats (diallyl trisulfide) is related with the increase of the intracellular free-calcium concentration which may activate calciumdependent endonuclease leading to dna fragmentation and apoptosis (sakamoto et al., 1997; sundaram and milner, 1996). dats possesses anticancer activity both in vitro and in vivo. borowska et al. (2013) showed that dats is more toxic to prostate cancer cells than to noncancerous epithelial cell line pnt1a. cytotoxicity of dats toward pnt1a cell line was reduced which means that pnt1a cells had higher resistance to dats-induced cell death than pc-3 cells. dads also induced apoptosis in hl60, hct-15 and neuroblastoma cells by the production of ros followed by the induction of p53 and activation of caspase-3 which leads to cell death (filomeni et al., 2003; hong et al., 2000; kwon et al., 2002). dads can also affect the cell cycle in human hct-15 cells. it can induce g2/m phase arrest and inhibition of p34 kinase activity because of the decreased p34/cyclin b1 complex formation and subsequent p34 hyperphosphorylation (knowles and milner, 2000). yang et al. (2009) demonstrated that dads induced ros formation and accumulation of ca2+ ions, which induced the apoptosis by promoting caspase-3 activity in colo 205 cells. apoptosis is followed by the increased level of fas, ital. j. food sci., vol. 31, 2019 29 phosphorylation of ask1 and jnk, p53 and apoptotic genes bak and bax leading to the decrease of the antiapoptotic genes bcl-2 and bcl-xl. the flower extract of a. atroviolaceum induced antiproliferative effect against the hella cell line. the mechanism of its action seems to involve the induction of apoptosis through the down regulation of the antiapoptotic bcl-2 gene expression and activation of caspase-9 and caspase-3 mitochondrial death pathway (khazaei et al., 2017). souid et al. (2017) first demonstrated that dried aqueous extract (dae) of a. roseum possesses an excellent antiproliferative effect on chronic myeloid leukemia (clm) k562 cells. the mechanism of dae antiproliferative action was associated with the inhibition of both erk1/2 and pi3k/akt signaling antiapoptotic pathway and induction of apoptotic caspase pathway. furthermore, dae wasn't toxic for normal mouse fibroblast cells. chemical analysis of dae identified a different organosulfur compounds and high amount of allicin, which are known as potent anticancer agents. based on these findings, we can conclude that allium vegetables possess exceptional antiproliferative activity against different cell lines (boivin et al., 2009). this activity is in correlation with their anticancer properties observed in many epidemiological and laboratory studies (fleischauer and arab, 2001; galeone et al., 2006; milner, 2001; talalay and fahey, 2001). these effects are related with both flavonoid and organosulfur compounds. many epidemiological studies have shown that regular consumption of allium vegetables is associated with decreased risk of developing cancer, especially gastrointestinal cancer (bianchini and vainio, 2001; gao et al., 1999; lawson, 1998; witte et al., 1996). two cohort studies and meta-analysis of 19 case-control studies showed that consumption of high levels of allium vegetables reduced risk for gastric cancer development (zhou et al., 2011).the review with summarized findings from epidemiological studies based on allium vegetables intake and gastric cancer risk once again confirmed the beneficial effect of this vegetables (guercio et al., 2014). they concluded that high intakes of alliums, mainly garlic and onion, can prevent gastric cancer development. new epidemiological studies are in line with those of guercio et al. (2014). results from case-control study and meta-analysis confirmed that high intake of alliums, garlic and onion, may reduce gastric cancer risk (turati et al., 2015). a large cohort study carried out in 10 european countries: the european prospective investigation into cancer and nutrition (epic) confirmed the link between increased intake of fruits and vegetables (including allium vegetables) and decreased risk of stomach cancer development (gonzález et al., 2006). in a case-control study conducted in china, it was demonstrated that higher intake of red onion, garlic, chinese onion, welsh onion and leek was correlated with lower risk of esophagus and stomach cancer (gao et al., 1999). the protective role of this vegetables on stomach, esophageal and duodenal cancer development is likely to be associated with their antibacterial activity against helicobacter pylori, a bacterium that plays a key role in the development of these types of cancer (o’gara et al., 2000; you et al., 1989). similar investigation conducted in shanghai confirmed the link between increased intake of food rich in garlic, red onion, chive and leek and decreased risk of prostate cancer development (hsing et al., 2002). although these studies suggested that regular garlic and allium vegetables consumption reduce gastric cancer risk, kim et al. (2018) found no statistically significant association between garlic intake and reduction of gastric cancer risk. it was shown that consumption of red onion and garlic can inhibit colorectal cancer development. six different studies showed that increased intake of raw or cooked garlic reduces the risk of colorectal cancer development from 10 to 50% (fleischauer et al., 2000). in contrast, meta-analysis of eight different cohort studies showed that large intake of allium vegetables does not reduce risk for colorectal cancer (zhu et al., 2014). turati ital. j. food sci., vol. 31, 2019 30 et al. (2014) found that high garlic intake is associated with a 15% reduction in colorectal cancer risk. these case-control studies showed a link between high intake of alliums and a reduction of colorectal adenomatous polyps. yang et al. (2009) found that the intake of raw onion and garlic can be protective against esophageal cancer in taiwanese man. other case-control studies reported that consumption of 7 or more portion of onions per week can be significantly protective against esophageal cell carcinoma (galeone et al., 2006). it has also been confirmed the association between consumption of allium vegetables and lower risk for lung (sankaranarayanan et al., 1994) and brain cancer development (hu et al., 1999). the case-control study was carried out among iranian woman with newly diagnosed breast cancer to investigate the effect of onion, garlic and leek on the breast cancer. the results suggested that the consumption of garlic and leek significantly reduced a risk for breast cancer development, while high consumption of cooked onion may be related with higher risk of breast cancer development (pourzand et al., 2016). several studies have reported that organosulfur compounds as well as flavonoids, such as quercetin 3,4’-diglucoside and quercetin 4'-monoglucoside can protect from cancer development in different tissues. they can activate or inactivate a wide variety of mechanisms to prevent cancer propagation. there are several proposed mechanisms of chemopreventive action of biologically active substances from allium vegetables: inhibition of oxidative damage thanks to their strong antioxidant activity (lawson et al., 1991; nuutila et al., 2003; perchellet et al., 1990; syed et al., 2013), inhibition of cell proliferation and induction of apoptosis (adams-campbell, 2011; altundal et al., 2016; antony and singh, 2011; atashpour et al., 2015; aquilano et al., 2010; chan et al., 2013; chen et al., 2011; chou et al., 2010; duo et al., 2012; hermanantosiewicz and singh, 2004; iciek et al., 2012; kelkel et al., 2012; kim et al, 2013; knowles and milner, 2000; lee yj et al., 2015; lee wj et al., 2015; lee et al., 2010; nagaraj et al., 2010; niu et al., 2011; perchellet et al., 1990; ren et al., 2015; russo et al., 2014; vidya priyadarsini et al., 2010; yi et al., 2010a,b), inhibiting of procarcinogens activation by the their effect on cytochrome p450 (chen et al., 2009; choi et al., 2011; kumar and pandey, 2013; wargovich, 2006; yang et al., 2001b), inhibiting dna damage (anticlastogenic effect) ( fredotović et al., 2014; haza et al, 2011; khanum et al., 2004; lau et al., 1990), inhibition of lipoxygenase and cyclooxygenase activity (anti-inflammatory effect) (adão et al., 2011; ali, 1995; belman et al., 1989;byun et al., 2013; dirsch and vollmar, 2001; chang et al., 2005; elberry et al., 2014; park, 2011; perchellet et al., 1990; prasanna and venkatesh, 2015; rose et al., 2005; wang et al., 2012). additional studies need to be done to confirm the chemopreventive effect of this vegetables as well as the exact mechanism of their action. 4. conclusions for centuries, allium vegetables have been very suitable ingredients in a wide variety of cuisines worldwide. they produce specific chemicals, mostly organosulfur compounds and flavonoids that give them the unique taste and smell but also are responsible for their biological activity. beside these main active compounds, they possess small amounts of saponins which contribute to the health benefits of these vegetables. the antimicrobial activity of allium species has been proved against a wide range of bacteria, fungi and yeasts. numerous studies confirmed them as potent antioxidants capable to catch and inactivate free radicals and therefore prevent oxidative cell damage. strong antioxidant activity was found to be related mainly with sulfur-compounds as well as flavonoids. ital. j. food sci., vol. 31, 2019 31 different studies have indicated that alliums possess anti-inflammatory properties via scavenging reactive oxygen species and through the inhibition of proinflammatory cytokines expression. alliums possess remarkable antiproliferative activities against different cell lines which is directly related with 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gastroenterol. hepatol. 12:1991-2001. paper received april 4, 2018 accepted july 17, 2018 ijfs#1778_bozza ital. j. food sci., vol. 32, 2020 517 review isotope analysis as a means of tracing aquatic products authenticity, source and geographic origins h.t. truonghuynh, g.b. li* and g.k. jaganathan school of medical instrument and food engineering, university of shanghai for science and technology, shanghai 200093, china *corresponding author: lbaoguo@usst.edu.cn abstract aquatic products provide good sources of high-quality protein for humans. tracing the origin of aquatic products is of critical importance both for consumers and suppliers. in recent years, isotope analysis is becoming a key instrument in food products authentication. this work reviews the use of isotope analysis to trace the production sources (wild or farmed) and geographic origins of aquatic products. extensive research has studied the isotope values of freshwater fish to atlantic salmon, sea bass, rainbow trout, and other commercial fish and shellfish. generally, the ratios of carbon (δ13c) and nitrogen (δ15n) stable isotopes were successfully investigated in aquatic products in order to identify the production methods or geographic origins. however, the predictable confidence of isotope analysis can be enhanced in combination with other analytical techniques, such as fatty acids and multi-element profiling. moreover, future research to combine isotope analysis with data fusion and multivariate data evaluation is recommended. keywords: food fraud, provenance, fish, seafood, isotopic abundance ital. j. food sci., vol. 32, 2020 518 1. introduction aquatic products provide good sources of high-quality protein for humans (sapkota et al., 2008). according to the food and agriculture organization’s annual state of world fisheries, total world fish production (capture and aquaculture, excluding aquatic plants) peaked at about 171 million tons in 2016, and is expected to reach 201 million tons in 2030 (fao, 2018). however, the growing number of seafood production and economic globalization have exerted serious pressure on the variety of food products, resulting in food fraud and adulteration (danezis et al., 2016a). in addition, consumers are interested in knowing the origin of aquatic products. numerous cases have been reported with fish products labelled falsely to increase the chances of marketing and sales (fox et al., 2018). in recent years, tracing the origin of fish has become significantly important both for consumers, producers and regulators (posudin et al., 2015). in particular, verifying fish origin and its label description are in compliance is given high importance (danezis et al., 2016b). traditionally, food authentication has been verified using several methods including genomics and proteomics techniques (ceruso et al., 2019, ortea et al., 2016), chromatographic techniques (granato et al., 2018), isotopic and elemental techniques (gopi et al., 2019b), vibrational and fluorescence spectroscopy (cozzolino, 2015, dankowska, 2016), nuclear magnetic resonance spectroscopy (standal et al., 2010), sensory analysis (kiani et al., 2016), immunological techniques (carrera et al., 2014) and others (danezis et al., 2016a, danezis et al., 2016b, gopi et al., 2019a). amongst them, isotope analysis is one of the prominent analytical techniques (danezis et al., 2016b), that has not been commonly used previously but gaining momentum. in particular, isotope analysis could trace the production methods (wild or farmed) and geographic locations of various species; and it is relatively cost-effective (gopi et al., 2019a). during the last several decades, research on food authentication has focused on wine, fruit, vegetables, cereals, meat, dairy products, oils, honey, and eggs (dankowska, 2016). however, there had been little interest in fishery products, but number of studies on fishery authentication are on the rise since 2007 (danezis et al., 2016a). moreover, the investigations on isotope analysis of seafood products have been propelled to the forefront due to its advantages compared with other relevant methods, such as dna, fatty acid and elemental profiling. for instance, the results of dna profiling can be affected by the removal or degradation of dna into small fragments in various treatments (novak et al., 2007, şakalar et al., 2012). fatty acid compositions depend on variability of seasons and diets (grigorakis, 2007), and it is difficult to distinguish the fatty acids profiles of wild and cultured samples (ostermeyer et al., 2014), or between wild and organic samples (molkentin et al., 2015). on the other hand elemental profiling needs more time to prepare samples and large database to discriminate the provenance of each species (gopi et al., 2019a). in this paper, we review isotope analysis used in aquatic products authentication, particularly focusing on the production source (wild or farmed) and geographic origins. our emphasis here is placed on applications, methods, accuracy and productivity of current studies attempting to elucidate the traceability of aquatic food products through isotope analysis. ital. j. food sci., vol. 32, 2020 519 2. isotope ratio analysis isotopes are the atoms of the same element, which have equal numbers of electrons (and protons) but different numbers of neutrons (coplen, 2011, kelly et al., 2005). different isotopes of the same element possess different masses. isotopes have two specific types: stable and unstable (radioactive isotopes). stable isotopes do not decay into other elements. in contrast, radioactive isotopes are unstable and decay into other elements. stable isotopes can be grouped into light and heavy elements isotopes, depending on atomic mass (danezis et al., 2016b). the less abundant stable isotope(s) of an element have one or two additional neutrons than protons, and thus are heavier than the more common stable isotope. the stable isotope abundance of an element is presented in ratio form as the ratio of the heavy-tolight isotopes (e.g. 13c/12c or 15n/14n). since this ratio is small, isotope ratio analysis is normally expressed by the ratio of the heavier and the lighter isotopes to a reference compound of normal isotope ratio, which is reported in standard delta (δ) as parts per thousand (per mil, ‰) (coplen, 2011, kelly et al., 2005) as eq (1): δ!"# = !!"#$!!!"# !!"# (1) where δ!"# is the isotope ratio of the sample expressed in delta units relative to the reference material. r!"#$ and r!"# are the isotope ratios of the sample and reference material, respectively. for bio-elements (2h/1h, 13c/12c, 15n/14n, 18o/16o and 34s/32s) isotope measurements, isotopic ratios are generally investigated by isotope-ratio mass spectrometry (irms) (drivelos and georgiou, 2012). for sr, pb and other heavy isotopes, thermal ionization mass spectrometry (tims), multi-collector-inductively couple plasma mass spectrometry (mcicp-ms), and dynamic reaction cellinductively couple plasma mass spectrometry (mcicp-ms) are used (drivelos and georgiou, 2012). 3. isotope analysis in food authentication the increasing global trade has challenged the guarantee of food safety, transparency and protection of human health (danezis et al., 2016b, kelly et al., 2005). in addition, aquatic food products are highly perishable commodities and traded worldwide, which give certain difficulties for characterizing its provenance (schröder, 2008). therefore, it is important to verify the authenticity of the aquatic products before entering markets. isotopic ratios have been known to be of extreme use in food authentication because food ingredients have variety of isotopes abundance that can reflect its trophic position, food sources, geographic origin, pedology and archaeological sites (danezis et al., 2016b, fuller et al., 2012). stable isotopic ratios of δ13c and δ15n are natural biomarkers to evaluate the effects of different preservation methods on isotopic signatures of fish tissues (arrington and winemiller, 2002, kelly et al., 2006, syväranta et al., 2008), trophodynamics and food sources in time and space (fuller et al., 2012, wyatt et al., 2012). similarly, geographic origin can be recognized by hydrogen, oxygen, sulphur and strontium isotope ratios (kelly et al., 2005). there are various issues concerning the traceability and authentication of fishery and aquatic products. among them, species of origin (fish species), production sources (wild ital. j. food sci., vol. 32, 2020 520 or famed), and geographic origins (locations) are desirable in traceability of fishery and aquatic products (moretti et al., 2003). consequently, isotope ratios can be used in tracing the authenticity of production sources (wild or famed), and geographic origins of aquatic products. 3.1. wild and farmed to date, there are numerous studies focusing to distinguish farmed and wild seafood products, especially since the beginning of the 21st century, due to the increased concerns amongst consumers, who wants to know the origin of fishes (danezis et al., 2016b). the feasibility of using stable isotopes to distinguish recently escaped farmed atlantic salmon (salmo salar) to wild specimen was investigated by dempson and power (2004). their results showed that muscle tissue of wild salmon had significantly enriched nitrogen δ15n but depleted lipid corrected carbon δ13cʹ (the residual δ13c values) than those of escaped farmed salmon. in addition, those authors assumed that the differences of isotope fractionation between farmed and wild fish could be retained depending upon the time of year that farmed fish was escaped relative to the june to august rapid growth period. moreover, their study also supported the fact that adipose tissue can be used as noninvasive utility to determine isotope values in salmonid fishes, as the average δ13cʹ and δ15n of white muscle and adipose fin tissue varied in absolute amount by only 0.5%. the combined measurement of δ13c and δ15n can be useful to differentiate the origin, farmed or wild, brazilian fresh water fish cachara (pseudoplatystoma fasciatum), but seasonal variations need to be concerned (sant’ana et al., 2010). farmed cachara was found to have significantly enriched δ15n in rainy but not dry season, whereas δ13c was found to be enhanced in both seasons. therefore, the authors assumed that δ13c is a better indicator for cachara traceability. the basic of isotope analysis in discrimination of wild (england) and cultured (scotland and greece) sea bass (dicentrarchus labrax) was provided by bell et al. (2007). the isotopic data indicated that δ13c of individual fatty acids 16:0, 18:0, 16:1n-7, 18:1n-9, 18:1n-7, 20:1n-9, and 20:4n-6 were significantly lighter in cultivated sea bass than those of wild specimen. in addition, total flesh oil of farmed sea bass had the lighter δ13c compared to that of the wild specimen, but not for the δ18o of the flesh oil. it was explained by the commercial aquafeed formulations contained more terrestrial-derived raw materials such as wheat, soybean, sunflower, maize, peas and rapeseed meals. although wild bass had a higher choline nitrogen content than cultivated bass, higher δ15n of the flesh lipid total glycerol/choline fraction was observed in cultivated bass than that of wild counterpart. it may be due to the differences in growth rate and maturity of wild (1690 g) and cultivated (386 g) sea bass or seasonal variations of δ15n. nevertheless, those authors stated that because their study only discriminated fish origins from three geographic locations, it warrants further studies combining isotope analysis with other analytical methodologies such as the flesh fatty acids profiles. the study of fasolato et al. (2010) highlighted that the utility of δ13c and δ15n abundance to discriminate the farmed to wild european sea bass. because δ13c abundance can be affected by the variability of tissue lipid content or intramuscular fat (focken and becker, 1998), the δ13c abundance in this study was analyzed from free-fat muscle. similar to previous study of bell et al. (2007), the δ13c values of farmed sea bass were ital. j. food sci., vol. 32, 2020 521 found to be lower than wild specimens. the δ15n abundance showed higher values in wild specimen owing to the higher trophic level of fish feed from the mediterranean sea. in an attempt to extend more heterogeneous range of samples, sea bass (dicentrarchus labrax) from 18 different italian and southern european sources was analyzed for its production sources, wild and cultivated (intensively, semi-intensively, and extensively) by determination of δ13c and δ15n isotopic compositions (farabegoli et al., 2018). however, the results of isotopic abundance were less satisfying as there were merely slight differences in isotopic abundance of δ13c and δ15n between cultivated and wild fish, as well as italian and foreign intensively reared fish. according to the authors, δ13c can be affected by dietary nutrients and habitat shifts; in addition, δ15n can be influenced by the trophic level of fish feed formulations. these may affect results of isotopic abundance to distinguish between wild and cultivated sea bass. it must be noted that the specific choice of isotopic abundance element in particular fatted/defatted samples is important. for example, the sole δ13c abundance in defatted dry matter could not differentiate organic from wild salmon (molkentin et al., 2015). in this case, the combination of δ13c in lipid samples and δ15n in defatted dry matter were needed in the differentiation of organic, conventional and wild fish (molkentin et al., 2015). to search for more authenticated method, wang et al. (2018) suggested to use compound-specific amino acid δ13c fingerprints (δ13caa) on large-numbered of salmon samples, to (1) discriminate organically, conventionally aquaculture to wild fish from pacific to atlantic regions; and (2) detect subtle diet changes by macroalgae or insects with controlled feeding experiments. the bulk isotope values of δ13c and δ15n could be used to trace the salmon origins. those authors found that bulk isotope values resulted in poor discrimination between wild and organic salmon. however, the multivariate analysis of δ13caa data could separate the wild, organic and conventional salmon with high certainty, as well as distinguish diets changes among lab-cultured experimental groups, even between the green (ulva rigida) and red (palmaria) macroalgae inclusion groups. in addition, δ13c of essential amino acids (his, phe, val, ile and leu) in salmon tissue can reflect the dietary sources, therefore they can satisfactorily differentiate fish origins. the research of vasconi et al. (2019) used protein carbon and nitrogen isotope analysis to differentiate the wild and famed european eel (anguilla anguilla) from netherland, denmark and italy; and different farming modes (pond, recirculating aquaculture system, lagoon and wild). multivariate data were performed by principal component analysis and sparse partial least squares discriminant analysis. the results showed that δ13c and δ15n abundance can predictively differentiate lagoon and wild eels, but cannot discriminate male and female eels from netherland and denmark. in addition, the stable isotope analysis results can ratify only the partial of what has been demonstrated by using the fatty acids profile in this study. 3.2. geographic origin in recent years, there has been an increasing interest in isotope analysis to authenticate the geographic origin of fish and shellfish. in particular, the study of ortea and gallardo (2015), using stable isotope ratio and/or multi-element (pb, cd, as, p, s) analyses, has shed some light on not only geographic origin, but also production method, and species authentication of commercially relevant shrimps. the shrimp samples were constituted by 45 individuals of seven different species in nine different geographical ital. j. food sci., vol. 32, 2020 522 origins. multivariate analysis were used for data classification, including principal component analysis, cluster analysis, κ-means hierarchical classification and discriminant analysis (da). the results showed that both stable isotope ratio and multi-element analyses can enhance the prediction capabilities of chemometric technique to discriminate shrimp samples into wild/ farmed, different geographical origins or even biological species, whilst cluster analysis was not appropriate to discern the farm origins. on the contrary, it is of interest to note that kim et al. (2015) stated that isotope analysis is a reliable tool to trace the origin of commercial fish (mackerel, yellow croaker and pollock). biplot of δ13c and δ15n values showed that australian and norwegian mackerel had different spatial and trophic position than those of chinese and korean counterparts (kim et al., 2015). the δ13c of pollock from japan and russia, as well as the δ15n between yellow croaker from korea and china were likely similar because the two areas are close in their geographical distance (kim et al., 2015). those authors also reported that δ13c signature can be more effective in discrimination of geographic origin due to the distinct values of δ13c of the three commercial fish. the geographic origins of commercial hake species (n=60) were evaluated by the isotopic abundance of δ13c and δ15n using bivariate scatter plot, principal component analysis and euclidean hierarchical clustering analysis (carrera and gallardo, 2017). the results facilitated a clear classification of hakes from six geographic coasts: europe, north africa, south africa, north america, south america, and australia. most importantly, the δ13c signature can corroborate the clear discrimination hake species according to latitude. for example, north african and south american hakes were in the adjacent range of δ13c (-14 to -16), whilst australian and north american hakes were in the range of -18 to -20 values of δ13c. to study the carbon cycle at the molecular level, compound-specific isotope analysis is used as the combination of gas chromatography and isotope-ratio mass spectrometry (liu et al., 2017, rieley et al., 1991). compared to the conventional isotope analysis of bulk organic carbon, this technique can reflect the material source more accurately (liu et al., 2017) and understand the carbon fluxes within bio-geochemical systems (rieley et al., 1991). previous studies have applied this technique concerning with source of nutrients in aquatic and terrestrial food webs (larsen et al., 2013) and discrimination of organically, conventionally aquaculture to wild fish (wang et al., 2018). however, few studies have demonstrated the traceability of compound-specific isotope analysis on geographic origins in seafood. one of the first attempts to trace the geographic origin of seafood by this technique was investigated on sea cucumber (apostichopus japonicas) in the coastal area of china (liu et al., 2017). in this study, principal component analysis (pca) and discriminant analysis (da) were used to support the discrimination. although a total of 28 fatty acids was detected in the fatty acid profiles, but stable carbon isotope compositions were only obtained from 26 fatty acids. the δ13c values of fatty acids in both november 2015 and april 2016 were relatively enriched in rushan, wafangdian and pikou, and depleted in the danzi island, the changhai island and muping. principal component analysis (pca) and discriminant analysis (da) allowed researchers to discriminate between different geographic locations of sea cucumber; except for the changhai and zhangzi island in april 2016, because the two islands are both located in the dalian sea and have similar environmental conditions. this study was followed up with the amino acids carbon stable isotope analysis of sea cucumber in an attempt to distinguish the subregions that are close together (zhao et al., 2018). because the δ13caa fingerprint can supply ital. j. food sci., vol. 32, 2020 523 the information of biosynthetic origin and carbon acquisition (scott et al., 2006), as well as the environmental conditions and food sources (gannes et al., 1998, mcmahon et al., 2010); this method resulted in 100% of overall correct classification rate and crossvalidation rate to discriminate 8 locations of wild samples and 3 locations of cultured sea cucumber. especially, the sole δ13c values of gly and ser could similarly discriminate the production method (wild versus cultured) and geographic provinces of sea cucumber, respectively. conservation efforts aimed at tracing the seafood geographic origins also involved the multi-element isotope analysis. for tracing the geographic origins (two northern italian regions and other italian regions) and type of feed (high or low fish content), the relationships between δ13c, δ15n, δ34s, δ2h and δ18o values of proteins and fat fractions of rainbow trout (oncorhynchus mykiss) fillet and those of feed and tank water were evaluated (camin et al., 2018). compared to the δ13c, δ15n and δ34s of feed, those isotopic values of fillet proteins were enriched; and δ13c values of fish fat were depleted. the partial square regression showed that the c, n and s isotopic values of fillet and fish feed were positively correlated within and between material matrixes, and negatively correlated with isotopic values of h and o of feed and h of fillet. whereas, the isotopic signature of environment water δ18o was positively correlated with δ2hprotein and δ18oprotein of fillet. besides that, δ18ofat of fillet was less significant correlated with other isotopic ratios. in addition, the partial least squares-discriminant analysis was applied to check the traceability of two geographic origins and feed type. fish from friuli venezia giulia region was predictably traced by δ15nprotein and δ18oprotein; the trentino fish was marked out by δ2hprotein and δ18oprotein; whilst fish fed with high and low fat-feed were discriminated by δ34sprotein. the discriminant multiclass model reached the average accuracy of 94%. furthermore, the authors suggested that geographical signature is extensively influenced by the local forage of diets. previous studies have also shown that isotope analysis is informative variable to distinguish fish products between different farms (kim et al., 2015, turchini et al., 2008). to be more precise, however, isotope analysis would be a perfect complement to combine with other analytical techniques (carter et al., 2015, gopi et al., 2019b, ortea and gallardo, 2015, turchini et al., 2008). one possibility is the combination with fatty acids profile. in an attempt to distinguishing the geographic traceability of sea cucumber (apostichopus japonicas) in seven locations of northern china sea, the stable isotopes of carbon and nitrogen compositions partially overlapped in some areas, whilst fatty acids profile alone could not discriminate all of the origins (zhang et al., 2017). however, the combination of δ13c and 14:1n-5, or δ15n and 16:0 content could be used to surmount the overlap areas. additionally, the thorough separation of seven sampling locations were achieved when stable isotopes and fatty acid compositions combined with discriminant analysis and the recognition ability was 89.1%. zhang et al. (2019) extended the isotopic analysis of scallops (patinopecten yessoensis, chlamys farreri, and argopecten irradians) of fatty acid δ13c fingerprinting with fatty acid profile in seven sites of china. the results showed that all scallops of 75 samples were discriminated the geographic origins in combination of principal component analysis with the accuracy rate of 100%. to trace the geographic origins of seafood, isotope analysis can be associated with trace metal compositions or elemental profiling (carter et al., 2015, gopi et al., 2019b). for instance, carter et al. (2015) identified that the utility of δ2h and δ13c values in meat component of prawns could distinguish between australian prawns and those imported from neighboring asian countries. in addition, the data of potassium, zinc and arsenic ital. j. food sci., vol. 32, 2020 524 concentrations in prawn meat resembled the results obtained in isotope analysis with minor overlapped areas. therefore, those authors surmised that the association of stable isotope and trace metal analysis would improve the accuracy of classification; however, the discriminant analysis for this combination had not been investigated. to determine the geographic origins and production method (wild or farmed) of asian seabass (lates calcarifer), stable carbon and nitrogen isotope analyses and elemental profiling (31 different elements) were conducted in 38 samples from two australian and one malaysia regions (gopi et al., 2019b). three statistical and ordination methods were used, including univariate (anova) and multivariate (principal component analysis), linear discriminant analysis, and random forest (r package). the accuracy of stable isotope, elemental profiling and the combination of two methods were 84, 72 and 81%, respectively. the incorrect predictions were two, one and none, respectively for three models. it was suggested that the combination of stable isotope and elemental profiling can be accommodated for seafood authentication. however, this study did not cover the seasonal variations of δ15n and had limited sample size and species. to extend the provenance of geographic origins (17 different european areas located in mediterranean sea basin), 144 wild and farmed specimens of european sea bass were analyzed for the carbon and nitrogen isotope and rare earth elements (lanthanum, europium, holmium, erbium, lutetium, and terbium) (varrà et al., 2019). data were anatomized by principal component analysis and orthogonal partial last square discriminant analysis (opls-da). the results showed that the satisfactory classification can be achieved in tracing both for geographical origin and production method by oplsda analysis. in general, isotope analysis has been limited by the absence of reference database on a large scale of different species so that it can be officially applied. while establishing the reliable database, we need to pay attention to some drawbacks of isotope analysis. particularly, isotope fractionations can be influenced by the environmental factors, e.g. growth conditions (liu et al., 2017, zhao et al., 2018), and diet quality, e.g. high versus low dietary protein contents (farabegoli et al., 2018, wang et al., 2018). in addition, the results of isotope analysis can be overlapped due to the seasonal variability (sant’ana et al., 2010), as well as the inappropriate utility of multivariate statistics and chemometrics methods (varrà et al., 2019). 4. conclusions despite the limited studies, this review demonstrated that isotope analysis has been very promising in tracing aquatic food products provenance, especially in production sources and geographic origins. however, considerably more work will need to be done to authenticate the provenance of aquatic products. for example, the studies of isotopic abundance should be extended to various types of fish, seafood and aquatic products. the accuracy in analytical instrumentation and methods need to be firmly established and characterized. in addition, seasonal and environmental effects should be considered in isotopic values of food samples. more broadly, multidisciplinary approach and other analytical techniques, such as chemical characterization, fatty acids profile and multielement profiling, combined with multivariate data evaluation and chemometrics can be further associated with isotopic analysis to improve the level of predictable confidence. ital. j. food sci., vol. 32, 2020 525 references arrington d.a. and winemiller k.o. 2002. preservation effects on stable isotope analysis 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31(4):1029-1044. zhang x., han d., chen x., zhao x., cheng j. and liu y. 2019. combined use of fatty acid profile and fatty acid d13c fingerprinting for origin traceability of scallops (patinopecten yesoensis, chlamys farreri, and argopecten irradians). food chem. 298:124966. zhang x., liu y., li y. and zhao x. 2017. identification of the geographical origins of sea cucumber (apostichopus japonicus) in northern china by using stable isotope ratios and fatty acid profiles. food chem. 218:269-276. zhao x., liu y., li y., zhang x. and qi h. 2018. authentication of the sea cucumber (apostichopus japonicus) using amino acids carbon stable isotope fingerprinting. food control 91:128-137. paper received january 27, 2020 accepted april 28, 2020 ijfs#1207_bozza ital. j. food sci., vol. 31, 2019 1 opinion paper insoluble tomato-fiber effect on wheat dough rheology and cookies' quality m. chouaibi*, l. rezig, a. boussaid and s. hamdi food preservation laboratory, high institute of food industry, 58 alain savary street, elkhadra city, tunis 1003, tunisia *corresponding author: tel./fax: +21671770399/+21671771192 e-mail address: moncef.chouaibi@yahoo.com.au abstract commercial insoluble tomato fiber (itf) was incorporated in wheat-flour dough to prepare cookies at amounts of 2.50, 5, 7.50, and 10 %. it was demonstrated that all wheat dough samples exhibited non-newtonian-thixotropic behaviors at shear rates from 0.001 to 1000 s-1. besides, the oscillatory rheology analysis confirmed that the storage modulus predominated the loss one in the whole frequency range and increased significantly with the increase in the itf concentration. actually, the latter's incorporation from 0 to 10 % increased farinograph water absorption, pasting temperature and peak consistency, and decreased dough stability, amylograph pasting viscosities and fermentation parameters of all tested wheat flours. furthermore, the itf addition was proven to affect the formed cookies, indicating a significant increase in the samples' breaking strength and decrease in their spread ratio. the total polyphenol contents of the formed cookies ranged from 86.98mggae/ 100 g to 376.02 gae/g cookies. the itf incorporation increased the antioxidant activities as measured by dpph, abts and frap scavenging activities. correlations between the analyzed parameters of the cookies' color and ic50 are statistically significant (p< 0.01), suggesting the possible use of itf as an alternative source of bioactive compounds to improve the cookies' quality. keywords: insoluble tomato fiber, wheat flour, rheological properties, cookies, antioxidant activities, quality characteristics ital. j. food sci., vol. 31, 2019 2 1. introduction every year, millions of tons of tomatoes are processed with a residue waste being considered as a good source for food supplements, such as insoluble dietary fibers (idf), fats, proteins and bioactive compounds, namely lycopene and polyphenols (kaur et al., 2008). idf from by-products, whose chemical constituents are chiefly non-starch polysaccharides, namely cellulose, arabinoxylans and β-glucan, is not commonly incorporated in food products due to its adverse effects on food quality, like sensory effects and functionality (ahmed et al., 2013). nowadays, many research works have revealed that idf is beneficial to human health. indeed, the ingestion of insoluble fiber from fruits and vegetables could significantly reduce the plasmatic concentration of cholesterol, implying a decrease in the risk of cardiovascular disease, colon cancer, diabetes and obesity (slavin, 2013). the european prospective investigation into cancer and nutrition (epic) has shown 40% reduction of colorectal cancer risk when consuming more than 30 g of fiber per day (sumczynski et al., 2015). actually, the food and nutrition board recommends 38 g/day for dietary fiber intake (sumczynski et al., 2015). tomato fiber is a by-product of tomato processing industry with high content of idf (71.82 %), soluble dietary fiber (14.33 %), protein (13.30 %), lipid (6.01 %) and ash (3.01) (navarro-gonzalez et al., 2011). some sugars, as glucose, xylose and galactose are present in tomato residue fiber (garcia-herrera et al., 2010). the latter is reported to comprise mainly cellulose (75%), hemicelluloses (15%) and pectin (10%) (hua et al., 2017). since it is rich in bioactive compounds, namely polyphenols and lycopene, it could be used in the development of functional food formulations (navarro-gonzalez et al., 2011). cookies and biscuits can be supplemented with dietary fibers from various sources, wheat bran, inulin carob fiber and many other biopolymers such as galactomannans, pectins and β-glucan (mildner-szkudlarz et al., 2013). the incorporation of dietary fiber into wheat flour interacts directly with the structural elements of three dimensional gluten networks, disrupts the starch gluten matrix and finally changes the mechanical properties of blended dough during mixing, fermentation and baking (liu et al., 2017; martinez et al., 2014; ahmed et al., 2013). since there is no published data on cookies formulations containing itf, to our knowledge, this study is the first to examine the effect of itf incorporation on both physicochemical and rheological wheat dough, and the quality characteristics of the formulated cookies. the obtained results would contribute to better valorize the itf in the cereal-based foods and support product authenticity. 2. materials and methods 2.1. materials the samples of commercial wheat flour produced by “minoterie soukra de tunis”, tunisia” were studied for proximate composition. itf was supplied by “conservas vegetales de extremadura” (conesa), extremadura, spain, and packaged in vacuum bags until the samples were opened for analysis. the itf proximate composition: water content 6.22 g/100 g, protein 0.93 g/100 g, dietary fiber 91.27 g/100 g, lipid 1.01 g/100 g, ash 0.57 g/100 g), itf 79.82 g/100g and soluble dietary fiber 11.45 g/100g. wheat dough samples were packed in low-density polyethylene bags, and then stored for analysis at different itf concentrations. ital. j. food sci., vol. 31, 2019 3 2.2. proximate composition analyses water, protein, lipid and ash contents were determined according to the approved aoac method (aoac, 1990). dietary soluble and insoluble fiber contents were identified according to the method of prosky et al. (1998). total carbohydrate content was estimated by mean-value difference: 100− (% water +% protein+% ash + % lipid+% total fibres). the assessment of wet and dry gluten contents, and gluten index was performed by glutomatic (perten instruments, hägersten, sweden) according to the aacc method (aacc, 2000). wheat flour was substituted by itf at different concentrations (g/100 g) to make a dough blend. both wheat flour and itf was premixed in dry condition using a mixer with a spiral blade, typically used for dough mixing. the wheat dough samples were prepared by mixing different blends in farinograph at a consistency of 500 ub at 30°c. the wheat flour sample without tif was considered as control. 2.3. rheological properties of wheat dough 2.3.1 rheological measurements the rheological properties of prepared dough samples were determined using a strain/stress controlled rheometer (thermo-haake, rheostress 1, germany) equipped with a temperature-control unit (thermo-haake, karlsruhe k15 germany). the rheometer had a cone-plate configuration with a 35-mm cone radius and a 0.14-mm gap between the cone and plate. measurements were conducted in the shear rate range of 0.001 to 1000 s-1 at constant temperature (20°c). twenty-five data points were recorded at 10 s intervals during the shearing. each measurement was replicated seven times on the same sample with two repetitions. experimental data were fitted to herschel-bulkley and power-law (ostwald) equations (1) and (2), respectively: σ = σ! + 𝐾𝛾! (1) σ = kγ! (2) where σ is the shear stress (pa), k is the consistency coefficient (pa.sn), γ is the shear rate (s-1), σ0 is the yield stress and n is the flow behavior index (dimensionless). the thixotropic hysteresis loop area was computed as the difference between the area under the up-flow and the down-flow curve using rheowin v.2.93 (haake, germany) software. 2.3.2 dough viscoelasticity using a parallel plate system (4 cm dia.) at a 500-mm gap, the dynamic rheological measurements were conducted with a rheometer (ar 1000, ta instruments, new castle, de, usa). dynamic shear data were obtained from frequency sweeps over the range of 0.1-100 rad/s. the strain value was obtained in the linear viscoelastic region at 1.5 % strain, and the frequency-sweep tests were performed at 20°c. to obtain the experimental data and to calculate the elastic (g’) and viscous moduli (g’’), data analysis software (version vi. 1.76) was used. aiming to relax the samples before taking the dynamic shear rheological measurements, all samples were allowed a5-min rest at the initial temperatures. these rheological measurements were performed in triplicate. ital. j. food sci., vol. 31, 2019 4 2.3.3 farinograph, alveograph and visco-amylograph tests the itf effect on dough rheology was determined by farinograph alveograph and viscoamylograph tests according to aacc method (aacc, 2000). 2.4. fermentation parameter determination fermentation parameters were assessed by the rheofermentometer f3 (tripette and renaud, france) according to the supplier’s specifications. the fermentation parameters of dough development were determined as follows: maximum dough fermentation height (hm) and the time at which dough reaches maximum height (t1). the measured gas parameters were the volume of gas produced throughout fermentation (vt), the gas retained in the dough at the end of the assay (vr), and the loss volume of gas (vl) in both milliliters (ml). all assays were performed in triplicate, and the average values were adopted. 2.5. formulation of cookies 2.5.1 cookie preparation cookies were prepared from the wheat flour (wf) and other ingredients such as shortenings, sugar, salt and sodium bicarbonate. composite flours, itf and other dry ingredients (sodium bicarbonate, sugar and salt) were mingled together in a bowl and shortenings were added, then mixed in a hobart mixer for 6 min to obtain creamy dough. the specified amount of water was added gradually during continuous mixing until slightly firm dough was obtained. a hundred of baked cookies each weighing approximately 13.4 g were obtained for each recipe. kneaded dough was manually rolled into sheets of required thickness and cut into round shapes, using a 5-cm diameter and 1cm high biscuit cutter. cookies were baked in batches at 195±2.0°c for 20 min. baked cookies were cooled to room temperature (22±1.0°c) and stored in polyethylene bags until analysis. 2.5.2 color determination the color parameters (l*, a* and b*) of the prepared wheat flours and formulated cookies were assessed using a cr-300 colorimeter (konica minolta sensing, inc., osaka, japan). color intensity was measured and expressed using the cie l* a∗ b* coordinate system, where l* represents color lightness, a∗ characterizes red (positive value) and green (negative value) colors. the parameter b∗ indicates yellow (positive value) and blue (negative value) colors. the above analysis was realized in 20 replicates. 2.5.3 physical parameters the cookies' diameter was measured by laying six cookies edge to edge with the help of a scale, rotating them through 90◦, remeasuring them and then taking the average value. the cookies' thickness was measured by stacking five samples one on top of the other and taking the average value. the spread ratio was estimated as diameter/thickness. ital. j. food sci., vol. 31, 2019 5 2.5.4 texture analysis of cookies the cookies texture was assessed using breaking test and whose parameters are breaking strength and fracturability, was determined using a ta.tx. plus texture analyzer (stable micro systems, godalming, surrey, u.k.). the test speed is of 1 mm/s using a knife probe. the peak force from the resulting curve was measured as the breaking strength or breaking force of the cookies. twenty cookies from each formulation were analyzed. 2.5.5 determination of total polyphenol content and antioxidant activities of cookies total polyphenol content and antioxidant activities by dpph and frap of the formulated cookies were determined using the method described by ismail et al. (2014). the total phenol content is expressed as the gallic acid equivalent (mg gae/100 g) of the sample. the cookies antioxidant activities by abts assay were assessed using the method of passos et al. (2017). the antioxidant activities of the formulated cookies are expressed as inhibitory concentration at 50 % (ic50), which in turn, represents the amount of cookies (mg) necessary for 50 % reduction of abts.+, dpph. and frap. ic50 values were calculated according to the non-linear regression algorithm of the plotted inhibition graph percentage compared with the cookie sample concentration. 2.5.6 sensory evaluation of cookies for sensory evaluation, 50 panelists were chosen from the food technology and science department of nutraceutical institute of foods at laval university (25 males and 25 females). the tests were performed under daylight room conditions. sensory analyses were conducted on itf-enriched cookies samples due to their higher physical properties. the order of samples presentation to the panel was randomized. the color, texture, flavor, crispness and overall acceptability of cookies were rated on a 1-9 scale: 1 dislike extremely; 2 dislike very much; 3 dislike moderately; 4 dislike slightly; 5 neither like nor dislike; 6 like slightly; 7 like moderately; 8 like very much; 9 like extremely. 2.6. statistical analysis except for the sensory evaluation that was evaluated in duplicate, all the others were realized in triplicate. the results were statistically analyzed using spss (spss inc., chicago, usa), version 18. while the analysis of variance (anova) was used to identify the significant difference between the results, duncan's test was used to separate the mean with a significance level of 5%. 3. results and discussion 3.1. proximate composition the physicochemical properties of the wheat flour incorporated with itf were examined (table 1), revealing that the itf incorporation levels into wheat flour decreased the water and protein contents from 14.05 to 12.38 % and from 9.58 to 4.33 %, respectively, (p<0.05). the ash content is also known to be another parameter used for the determination of the wheat flour purity. in this study, the ash and fat contents ranged from 0.42 to 0.94 and 0.26 to 1.41 %, respectively, hence the significant variations (p<0.05) between itf-enriched samples and control flour. additionally, the total fiber increased and the carbohydrate ital. j. food sci., vol. 31, 2019 6 contents decreased substantially with the increase in itf from 0 to 10 % (p<0.05) depending on the itf's concentration. likewise, wet and dry gluten contents decreased from 28.20 to 22.56 % and from 12.48 to 9.98 %, respectively, with itf incorporation (p>0.05), which is the same with the gluten index values (p<0.05). furthermore, the results demonstrated that dry gluten content decreased significantly with the incorporation of 2.5 % itf, due to reduction of protein content in the tested wheat flours. the itf incorporation will compete for water during the dough making process, thus making the dough thicker, which in turn may ‘work’ the gluten network more to enable an enhanced water uptake. further itf incorporation would out-compete gluten for the available water and make the gluten network less able to take up water, hence reducing the water binding capacity beyond the itf incorporation in wheat flour. table 1. chemical composition, gluten analysis and color parameters of wheat flour containing insoluble tomato fibre. itf concentration (g/100 g) control 2.5 5 7.5 10 chemical composition water 14.05±0.03a 13.63±0.03b 13.22±0.02c 12.80±0.02d 12.38±0.02e protein 9.58±0.05a 6.77±0.04b 5.95±0.05c 5.14±0.03d 4.33±0.02e ash 0.42±0.02a 0.55±0.01b 0.68±0.01c 0.71±0.01d 0.74±0.01e lipids 0.26±0.01a 0.55±0.01b 0.84±0.01c 0.92±0.01d 0.94±0.01d insoluble fiber 3.54±0.05a 10.21±0.04b 11.87±0.03c 14.54±0.02d 18.20±0.02e soluble fiber 6.75±0.05a 7.08±0.10ab 7.41±0.14bc 7.73±0.20cd 8.06±0.24d total fibers 10.20±0.01a 17.29±0.05b 19.28±0.11bc 22.27±0.17d 26.26±0.23e total carbohydrates 65.40±1.05a 61.21±0.38ab 60.03±0.80bc 58.16±1.24cd 55.35±1.67d gluten analysis wet gluten 28.20±0.03a 26.79±0.02b 25.38±0.02c 23.97±0.02d 22.56±0.03e gluten index 86.86±0.08a 82.51±0.07b 78.17±0.07c 73.84±0.06d 69.48±0.06e dry gluten 12.48±0.02a 11.86±0.02b 11.23±0.01c 10.61±0.03d 9.98±0.02e color parameters l* 95.34±0.04a 93.69±0.03b 92.04±0.03c 90.40±0.04d 88.75±0.03e a* -0.74±0.02a -0.36±0.02b 1.20±0.01c 4.38±0.02d 6.75±0.01e b* 12.78±0.04a 16.27±0.03b 19.67±0.02c 23.27±0.03d 26.76±0.03e chemical composition and gluten analysis were expressed as %. values given are the means of three replicates ± standard deviation. different letters within the same row indicate significant differences (oneway anova and duncan test, p< 0.05). table 1 lists cielab color parameters (l*, a*, and b*) for different wheat flour samples incorporated with itf, indicating the considerable impact of itf incorporation on these parameters. briefly, the lightness value (l*) of the whole-wheat flour was 95.34, which drastically dropped to 88.75 for the highest itf content (10 %). nevertheless, a* value for the whole wheat flour sample was -0.74, which increased significantly, about 4 to 9 times after itf incorporation with the highest value of 6.75 for the sample with the highest itf amount. the yellowness parameter (+b*) increased significantly as itf amount increased. the increase in color values a* and b* in wheat flour samples could be attributed to the ital. j. food sci., vol. 31, 2019 7 creation of more surface areas that possibly increase color intensity as reported by ahmed and al-attar (2015). 3.2. rheological properties of formulated wheat dough 3.2.1 flow behavior all formulated dough samples enriched with itf exhibited non-newtonian behavior at shear rates from 0.001 to 1000 s-1 at 20°c (fig. 1). a b shear rate (1/s) 0 200 400 600 800 1000 1200 s he ar s tre ss (p a) 0 500 1000 1500 2000 2500 10 % itf 7.5 % itf 5 % itf 2.5 % itf control shear rate (1/s) 0 200 400 600 800 1000 1200 sh ea r s tre ss (p a) 0 500 1000 1500 2000 2500 control 10 % itf ital. j. food sci., vol. 31, 2019 8 c d figure 1. shear stress versus shear rate (a). flow curves with a controlled shear rate measured by increasing (forward measurements) and decreasing shear rate (backward measurements) (b). variation of storage (c) and loss modulus (d) with angular frequency (ω) for insoluble tomato fibre-enriched wheat dough at 20°c. there is a nonlinear relationship between shear stress (σ) and shear rate (γ), which is in good agreement with the findings of guadarama-lezam et al. (2016). the power-law and herschel-bulkley models were used to characterize the flow curves of the formulated dough samples. based on the regression coefficient values (r2), the power-law was found to be a better-fit model for flow curves (r2> 0.99), and only the rheological parameters of this model were determined in this study (table 2). furthermore, flow behavior index values (n), at different itf amounts, were in the range of 0.339-0.264 and significant differences between all wheat dough samples (p<0.05) were noted. indeed, the flow plot of the shear stress against shear rate of the investigated dough showed a flow index (n) less than 1 (thinning fluid), indicating that the flow behavior of the examined samples can be described by following a nonnewtonian profile. consequently, all values are less than 1, which further confirms the pseudoplastic behavior of the whole itf-enriched dough. angular frequency (rad/s) 0,1 1 10 100 1000 e la st ic m od ul us g ' ( p a) 100 1000 10000 10 % itf 7.5 % itf 5 % itf 2.5 % itf control angular frequency (rad/s) 0,1 1 10 100 1000 vi sc ou s m od ulu s g ' ( pa ) 100 1000 10000 10 % tif 7.5 % tif 5 % tif 2.5 % tif control ital. j. food sci., vol. 31, 2019 9 the obtained results also showed that the flow index (n) decreased with the increase in itf concentration. actually, the consistency coefficient (k) indicates the viscous nature of a matter. yet, the consistency values (k) of the whole-wheat flour suspension within the studied concentration domain were in the range of 137.22-350.60 pa·sn. the higher consistency values of the formulated dough resulted from the antiplasticizing effect of sugars against water. another reason for the non-newtonian pseudoplastic flow behavior of samples emanates from the presence of high-molecular-weight macromolecules as fibers, gluten, sugars and starch. martinez et al. (2014) have confirmed that the addition of insoluble fibers increases dough consistency due to the insoluble fibers’ effect on the internal structure of wheat doughs. figure 1b shows the flow curves of control and wheat dough at 10 % of itf. shear stress measurements at increasing and decreasing shear rates from 0.001 to 1000 s-1 gave hysteresis loops. the hysteresis area (a) was determined and illustrated in table 2, confirming that its values decreased with the increase in itf concentration. however, significant variations with itf concentration exist in all tested samples (p<0.05). the hysteresis loops indicate a time dependency of the wheat dough rheological properties. this thixotropic behavior is observed with concentrated suspensions and macromolecular solutions due to structural breakdown happening in the specimen during the rheological test. 3.2.2 viscoelastic behavior the oscillatory spectra of the formulated dough samples displayed viscoelastic properties (figs. c-d). the characteristic slopes of the double-logarithmic plots of the storage and loss modulus (g’ and g”) vs angular frequency were quite similar. in this case, the elastic modulus was significantly high and different from the viscous one throughout the covered frequency range. all examined dough samples exhibited higher storage modulus (g’) values than those of g”, thus confirming the elastic behavior of the dough samples and indicating that both moduli values increased with the increase of the angular frequency and itf concentration. these results are comparable with those found by ahmed et al. (2013), guadarramalezama et al. (2016), who have proven that wheat dough was characterized by a solidlike behavior. indeed, they suggested that the increase of mechanical properties is due to limited plasticization effect and the presence of fiber nanoparticles. the experimental data was fitted with power-law models, described as follows: 𝐺!(𝜔) = 𝐾!.𝜔!! (3) 𝐺"(𝜔) = 𝐾".𝜔!" (4) where g’ is an elastic modulus (pa), g” is a loss modulus (pa), ω is an angular frequency (rad/s), and k’, k”, n’ and n” are the experimental constants. table 2 presents the power-law parameters of the elastic and viscous moduli (k’ and k”) of the tested samples undergoing an increase with the increase of itf concentration. in all cases, the k’ values were higher than those of k”, and increased significantly with the rise in the itf level in the wheat dough, (from 850.82 to 1680.92 and from 291.88 to 720.68 pa, respectively). in the same vein, bengtsson et al. (2011) found that the itf increase led to the increase of g’ and g”. thus, the highest k’ and k’’ values were observed for dough sample with 10 % of itf. the exponents n’ and n” were then found to decrease from 0.22 to 0.12 and from 0.23 to 0.181, respectively. besides, no significant variations in exponent values at high levels of itf (p>0.05) were noticed. ital. j. food sci., vol. 31, 2019 10 3.2.3 farinograph properties the use of elevated itf concentration in the wheat flour increased significantly the farinograph water absorption (wa), dough consistency (dc) and dough development time (ddt) (p<0.05) (table 2). while the increase in wa may be due to the presence of protein, fiber and resistant starch contents, the increase in the ddt showed a delay in the hydration and gluten development in the presence of these macromolecules. obviously, variations in wa depend on the protein content, the chemical structure and porosity of itf, the association between molecules and the particle sizes (thebaudin et al., 1997). recently, wang et al. (2002) have demonstrated that the variations in wa is mainly attributed to the greater number of hydroxyl group existing in the fiber structure, allowing more water interaction through hydrogen bonding. these results are in accordance with those found in the literature (mis et al., 2012; ahmed et al., 2013). dough stability time is an indicator of the flour strength, with higher values, indicating stronger doughs, which shows that the different itf amounts modify the ddt (p<0.05). the use of itf improved significantly the dough stability compared with control flour (p< 0.05), which could be explained by higher interactions among itf, water and gluten. furthermore, mixing dough could release water to the dough matrix, causing a quick reduction in dough consistency, and therefore a reduction in ddt (majzoobi et al., 2011). the mixing tolerance index (mti) of itf-enriched dough decreased considerably, with the increase in concentration from 0 to 10 % of itf. table 3 shows that control and wheat dough enriched with 2.5 % of itf has a better dough mixing tolerance with a lower value than the other samples. the increase in mixing tolerance index upon itf addition is probably due to the dilution of gluten protein with the fibers, which may be explicated by the interaction between fiber and gluten influencing the dough mixing properties (ahmed et al., 2013). however, quality index (qi) insignificantly increased from 53.48 to 60.60 bu by the itf addition as a function of increasing rate substitution (p>0.05). ahmed et al. (2013) suggested that the quality index increase emanates from the interaction between gluten and fiber. 3.2.4 alveographic properties table 3 lists the alveographic parameters of dough with and without itf, proving that its incorporation caused an increase in the tenacity (p) and decrease in the extensibility (l), and consequently in the swelling index (g) of the formulated wheat dough (p<0.05). after the itf addition, the configuration curve ratio (p/l) increased and the alveograph strength decreased. this may be due, at least partly, to the reduction in the gluten content caused by the presence of other components, as proteins, fibers, lipids and non-glutenforming proteins, which interfere with gluten formation. it can be concluded that the protein existing in the itf might have instigated partial disruption of the gluten network, causing change in the equilibrium of elasticity and extensibility properties of the whole wheat flour dough. the obtained results also corroborated significant differences between control and itf-enriched dough in term of the deformation energy (w) (p < 0.05). these results are clearly in conformity with those obtained by boubaker et al. (2016). yet, many studies, as wang et al. (2002), reported that commercial fibers' incorporation greatly improved the development of wheat protein behavior. they also stated that the fibres 'incorporation induced a decrease of the proteolytic degradation, being practically neutralized by inulin. ital. j. food sci., vol. 31, 2019 11 3.2.5 visco-amylograph properties to determine the itf incorporation effect on wheat flour-water interaction in cookies dough, a rapid viscoanaylzer (rva) was used, revealing that itf significantly diminished the pasting viscosities (peak, breakdown, final, and setback viscosities) (p<0.05). similarly, the values of the peak paste viscosity of the formulated dough decreased significantly from 1285.56 to 450.44 cp, which was explained probably by their association with high protein amount. thus, the tomato protein is estimated to form complexes with starch granule surface, preventing the release of exudates and lowering the peak viscosity. however, the pasting temperature increased from 86.20 to 90.75°c in flour blends (p<0.05), which, according to martinez et al. (2014), might be related to delay or restricted starch swelling. actually, the amylose leaching and lower pasting viscosities are indications of reduced starch available for gelatinization. therefore, the itf addition is proven to significantly diminish the degree of breakdown from 0.60 (control) to 0.25 (flour at 10 % of tif), showing increased resistance of the swollen starch granule to rupture after cooking as reported by sozer et al. (2014). 3.3. fermentation parameters the fermentation parameters of the itf-enriched wheat dough were assessed by the rheofermentometer, table 3. the maximum dough height (hm) decreased dramatically from 69.17 to 30.84 mm, with the itf addition, regardless of its concentration (p<0.05). however, no significant difference between samples containing 7.5 and 10 % of itf (p> 0.05) was observed. similar results were obtained by martinez et al. (2014) and liu et al. (2017) who discovered that the addition of insoluble fibers led to the decrease of the maximum dough height. nevertheless, the time required to reach the maximum dough height decreased from 2.95 to 1.30 h with the increase in itf concentration (p<0.05), demonstrating substantial differences in the time of reaching the maximum height between wheat flour samples. besides, the co2 production, retention and loss volumes were also determined. indeed, although the itf incorporation decreased the co2 production and retention volumes, it increased its loss volume (p<0.05). these results accord well with those found by martinez et al. (2014), who added different fibers to the wheat flour, proving that the influence differs with the type of fiber used. they suggested that insoluble fibers with larger particles can create points to split the structure, thus enabling gas to escape, affirming the assumption that itf incorporation to wheat flour decreased gas holding capacity. however, correa et al. (2014) have hypothesized that the addition of hydrocolloids could form hydrophilic complexes with protein or starch, which further changed the viscoelasticity and influenced the fermentation character. 3.4. properties of formulated cookies 3.4.1 physical properties table 4 summarizes the physical parameters of the analyzed cookies by substituting wheat flour with itf from 0 to 10% levels. as the itf concentration increased, the diameter of cookies (d) decreased from 53.94 to 51.83 mm, but no significant variations were found between cookies with 5 and 7.5 % of itf. the thickness of cookies (e) increased, confirming the dilution of gluten as reported by ajila et al. (2008). the differences in diameter and thickness are revealed in the spread ratio (d/e), consistently decreasing from 5.17 to 4.02 with itf levels increase (p<0.05). ital. j. food sci., vol. 31, 2019 12 table 2. power-law parameters of insoluble tomato fibre-enriched wheat dough enriched at 20°c. itf (%) 𝛔 = 𝐊𝛄𝐧 𝑮! = 𝑲!.𝝎𝒏! 𝑮" = 𝑲".𝝎𝒏" n k [pa.sn] a [pa.s-1] r2 n’ k'[pa.sn’] r2 n" k"[pa.sn’] r2 control 0.339±0.003a 137.22±9.98a 4.09±0.03a 0.986 0.220±0.002a 850.82±12.00a 0.989 0.230±0.002a 291.88±15.03a 0.994 2.5 0.321±0.002b 153.40±11.80a 3.83±0.02b 0.991 0.194±0.003ab 940.49±14.80b 0.972 0.218±0.003b 370.21±15.21b 0.992 5 0.302±0.002c 191.26±15.74ab 3.51±0.03c 0.978 0.173±0.02ab 1187.66±19.84c 0.988 0.201±0.001c 482.46±12.30c 0.980 7.5 0.284±0.003d 244.19±17.81b 3.33±0.04d 0.983 0.153±0.003bc 1409.37±24.83d 0.991 0.198±0.002c 591.10±12.95d 0.979 10 0.264±0.002e 350.60±19.40c 3.18±0.03e 0.995 0.120±0.003c 1680.92±30.01e 0.998 0.181±0.003d 720.68±15.88e 0.996 a: area of hysteresis loop; n: flow index; k: consistency coefficient; itf: insoluble tomato fibre; r2: regression coefficient; itf: insoluble tomato fibre. values given are the means of three replicates ± standard deviation. different letters within the same row indicate significant differences (oneway anova and duncan test, p< 0.05). table 3. farinograph, alveograph, viscoamylograph and rheofermentation tests of insoluble tomato fibre-enriched wheat flour. test concentration (%, g/100g) parameters control 2.5 5 7.5 10 w (%) 58.80±0.20a 65.70±0.30b 71.42±0.22c 74.04±0.16d 78.68±0.32e dc (bu) 510±3.00a 525±3.00a 565±5.00b 590±10.00c 620±8.00d farinograph ddt (min) 2.00±0.05a 2.30±0.04b 4.02±0.10c 5.10±0.07d 6.30±0.10e dst (min) 7.40±0.10a 7.30±0.05a 7.30±0.08a 6.45±0.10b 5.67±0.13c dmi (bu) 43.20±0.10a 48.55±0.15b 60.40±0.25c 65.23±0.23d 67.78±0.41e qi (bu) 53.48±1.73a 55.00±2.50a 57.09±1.91a 60.20±2.00a 60.60±2.00a p (mmh2o)×10 -4 89.32±2.50a 123.75±0.50b 160.11±1.30c 163.69±2.30c 174.20±2.20d l (mm) 86.56±1.40a 65.44±1.03b 30.19±0.80c 22.77±0.50d 18.82±0.60e alveograph g (mm) 20.65±0.16a 17.96±0.14b 12.20±0.16c 10.59±0.12d 9.63±0.15e p/l ratio 1.03±0.01a 1.89±0.03b 5.30±0.10c 7.19±0.06d 9.26±0.18e wl (j×104) 225.40±5.20a 200.36±3.30b 180.22±1.20c 140.43±2.55d 120.60±3.40e ital. j. food sci., vol. 31, 2019 13 pv (cp) 1285.56±5.70a 1179.25±4.80a 813.60±4.81b 550.02±2.98c 450.44±5.56d hpv (cp) 771.34±3.66a 553.33±4.67b 341.71±2.80c 154.00±3.02d 112.61±1.80e viscoamylograph fv (cp) 1421.63±7.70a 1149.11±9.89b 842.91±6.09c 605.81±5.90d 514.11±3.89e sv (cp) 650.30±4.04a 595.78±5.22b 500.20±3.29c 451.81±2.90d 401.50±5.69e pt (°c) 86.20±0.02a 87.36±0.06b 87.93±0.04c 88.46±0.06d 90.75±0.05e bd 0.60±0.00a 0.49±0.00b 0.42±0.00c 0.28±0.00d 0.25±0.00e hm (mm) 69.17±2.33a 41.23±1.23b 36.89±0.50c 32.76±1.01d 30.84±1.02d t1 (h) 2.95±0.05a 2.52±0.03b 1.83±0.02c 1.47±0.03d 1.30±0.03e rheoermentator vt (ml) 1720.27±19.73a 1680.60±14.40ab 1651.04±14.96bc 1644.3±11.93bc 1610.30±7.85c vr (ml) 1440.06±25.10a 1372.82±18.00ab 1314.10±24.95b 1234.28±15.89c 1144.45±13.70d vl (ml) 280.21±1.27a 307.78±1.40b 336.94±4.95c 410.09±3.97d 465.70±5.85e itf: insoluble tomato fibre; w: water absorption; dc: dough consistency; ddt: dough development time; dst: dough stability time; ds: degree of softening; qi: quality index; p: tenacity (mmh2o); l: dough extensibility; g: swelling index; p/l: configuration curve ratio; wl: deformation energy; pv: pasting viscosity; hpv: hot pasting viscosity; fv: final viscosity; sv: setback viscosity; pt: pasting temperature; db: degree of breakdown; vt: total volume of dioxide carbon; vr: retention volume of carbon dioxide; vl: loss volume of carbon dioxide. values given are the means of three replicates ± standard deviation. different letters within the same row indicate significant differences (oneway anova and duncan test, p< 0.05). table 4. physical, colour, textural and biochemical parameters of insoluble tomato fibre-enriched cookies. itf concentration (g/100 g) parameters control 2.5 5 7.5 10 physical parameters diameter (mm) 53.94±0.06a 53.17±0.03b 52.20±0.10c 52.06±0.06c 51.83±0.03d thickness (mm) 10.43±0.17a 11.24±0.15b 11.67±0.25c 12.22±0.25c 12.91±0.15d spread ratio 5.17±0.08a 4.73±0.06b 4.47±0.10bc 4.26±0.09cd 4.02±0.04d color parameters l* 75.79±0.09a 70.51±0.06b 66.17±0.03c 63.64±0.05d 62.36±0.04e a* 3.22±0.01a 3.94±0.01b 4.17±0.02c 4.91±0.01d 5.43±0.02e b* 27.72±0.03a 31.45±0.05b 35.58±0.02c 38.76±0.03d 39.68±0.01e ital. j. food sci., vol. 31, 2019 14 textural parameters breaking strength (n) 19.88±0.11a 20.94±0.06b 24.47±0.04c 28.06±0.06d 33.47±0.05e fracturability (mm) 0.32±0.02a 0.45±0.02b 0.56±0.02c 0.59±0.02cd 0.63±0.01d biochemical parameters total polyphenol content (mg gae/100) 86.98±2.58a 189.50±7.50b 239.51±3.50c 305.00±10.03d 376.02±6.02e abts (mg/ml) 2.40±0.03a 1.84±0.04b 1.34±0.01c 1.13±0.02d 0.89±0.03e dpph (mg/ml) 181.00±7.00a 91.03±8.04b 14.47±0.07c 9.10±0.10c 3.32±0.05c frap (mg/ml) 35.89±2.34a 16.62±2.12b 6.62±0.03c 1.95±0.05cd 0.78±0.04d abts, dpph, and frap were expressed as inhibitory concentration at 50 % (ic50). values given are the means of three replicates ± standard deviation. different letters within the same row indicate significant differences (oneway anova and duncan test, p< 0.05). ital. j. food sci., vol. 31, 2019 15 however, the decrease in the spread ratio of itf-fortified cookies is probably attributed both to the fact that the composite flour increased water absorption, and the absence of free water in the cookie dough. sharma et al. (2016) mentioned that the decrease in the spread ratio of germinated flour blend cookies may be due to the enzymatic degradation of the starch and protein into smaller sugars and peptides, resulting in the increase of hydrophilic nature within cookies. table 4 shows that cookies became darker (lower l* values), more reddish (higher a* values) and more yellowish (higher b* values) than control cookies with the increase in itf amount. the lightness (l) also decreased proportionally with the increase of the itf amount, reflecting the darkening of the cookies. this color change was confirmed by cookies photos (fig. 2). the decrease of lightness is probably due to the high protein and free sugar contents may accelerate the maillard reaction and therefore change the color of the products. as suggested by mildnerszkudlarz et al. (2013), cookies color depends mainly on their ingredients because the temperature is not high enough for maillard and caramelization reactions. the itf’s color values may affect the color of cookies’ crust. the 10 % level of itf presented more yellowish crust color than control cookies (fig. 2). figure 2. photos of formulated cookies at different levels of insoluble tomato fibre. the breaking strength values of formulated cookies with different itf concentration are illustrated in table 4, which reveals that the breaking force value increased with the increase in itf concentration ranging from 19.88 to 34.47 n. meanwhile, the results showed that there are significant variations in cookies' hardness among all cookies samples (p<0.05) (table 4). the increase in breaking force may be due to the increase in cookies thickness, which gives physical strength to the cookies structure. yet, higher positive correlation was found between itf concentration and breaking strength (r = 0.965, p<0.05), which is in perfect accordance with the study of nasir et al. (2010) who found a positive correlation between the concentration of vegetable flours and cereal foods. besides, the results showed that the itf incorporation decreased the cookies' fracturabilities (p<0.05), revealing a negative correlation between thickness and fracturability of the formulated cookies (r=-0.96, p<0.05). 3.4.2 total polyphenols and antioxidant activities of cookies the results of the total polyphenol content and antioxidant activities are shown in table 4. the total polyphenol content of cookies, with 2.5 to 10 % itf, increased significantly compared to the control cookies, i.e., it increased in parallel with itf supplementation level. while highly negative correlation was found between lightness (l*) parameter and the total polyphenol content (r =-0.977, p< 0.01), positive correlations between a* and b* ital. j. food sci., vol. 31, 2019 16 parameters and total polyphenol content of cookies (r=0.994, p<0.01and r=0.982, p<0.01, respectively). to characterize the antioxidant potential of the formulated itf-enriched cookies, dpph, frap and abts radical scavenging activities were determined, table 4. actually, all ic50values of antioxidant tests decreased with the itf increase, and significant differences between all cookies samples were found (p<0.05). comparing the activities, the highest one was abts followed by those of frap and dpph, whose relationship with the maillard reaction products (e.g., color parameters) was identified by a multivariate analysis to find the possible correlations among measured variables. high correlations were found between the total polyphenol content and antioxidant activities (ic50) (r =-0.982 (abts); r=-0.919 (dpph); r=-0.940 (frap), p< 0.01). correlations between antioxidant activities (ic50) and lightness (l*) (r =-0.998 (abts); r=-0.977 (dpph); r=-0.985 (frap), p< 0.01) were also detected. this implies that the amount and type of polyphenolic substances in cookies are variable, depending mainly on the itf incorporation level. the baking can further increase the antioxidant activities, and the formation of dark color pigment due to maillard browning during baking process can increase the antioxidant properties of the cookies. these findings were confirmed with the high correlations between the lightness and inhibitory concentrations (ic50), which are similar to those obtained by michalska et al. (2008). the latter indicated that maillard reaction products possess cookies' antioxidant activities at elevated temperature, which are in turn in good agreement with the studies of misan et al. (2011) and agila et al. (2008). at a 10 % itf incorporation, the content of polyphenols were 4 times higher than control cookies. 3.4.3 sensory evaluation the score results of sensory evaluation of the tested cookies are illustrated in fig. 3. figure 3. principal component analysis plot of data from sensory evaluation of insoluble tomato fibreenriched cookies. ● itf concentration; ■ sensory parameters control 2.5 % itf 5 % itf 7.5 % itf 10 % itf color flavour texture overall acceptabili ty crispness -3 -2 -1 0 1 2 3 4 -5 -4 -3 -2 -1 0 1 2 3 4 5 f2 (3 6, 47 % ) f1 (60,75 %) biplot (axes f1 and f2: 97,21 %) ital. j. food sci., vol. 31, 2019 17 the first (pc1) and the second (pc2) principal components were 60.75 and 36.47 %, respectively, accounting for 97.21 % of the total variance. the results also revealed that all sensory parameters were positively correlated with both components. indeed, while color, crispness and flavor were positively and highly correlated with pc1, texture and overall acceptability were positively correlated with pc2. the score plot showed that control cookies and those enriched with 7.5 % of tif had high scores of color and overall acceptability. however, compared to control, the sensory parameters of the formulated cookies were found to be different. according to the panels, there are no a significant difference between control and cookies with 7.5 % of itf in term of overall acceptability (fig. 3). it can be concluded that itf can be incorporated into cookies at 7.5 % level. 4. conclusions in this study, the effect of itf on the physicochemical, rheological and antioxidant properties of wheat flour and the formulated cookies were investigated. the results showed that the itf incorporation improved the nutritional and rheological properties of wheat dough and diminished the rheofermentation parameters and pasting properties: higher water absorption, tenacity, stability and peak consistency and smaller extensibility. moreover, itf significantly increased the hardness and the total polyphenol content and enhanced the antioxidant activities of cookies. the cookies enriched with 7.5 % itf had similar overall acceptability with the control cookies. however, compared to the latter, differences on the formulated cookies color and texture were found. therefore, itf might be used for the novel formulation of cookies as an alternative source of dietary fiber and bioactive compounds. acknowledgements thanks are credited to ms. leila mahfoudhi, major teacher of english in the sfax faculty of science, for proofreading and polishing the language of the manuscript. references aacc. 2000. approved methods of the aacc, 10th ed. st. paul, mn: american association of cereal chemists. ahmed j. and al-attar h. 2015. effect of drying method on rheological, thermal, and structural properties of chestnut flour doughs. food hydrocolloid 51:76-87. ahmed j., almusallamb a.s., al-salman f., abdulrahman m.h. and al-salem. e. 2013. rheological properties of water insoluble 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interest. trends food sci. technol. 8(2):41-48. wang j.s., rosell c.m. and de barber c.b. 2002. effect of the addition of different fibers on wheat dough performance and bread quality. food chem. 79(2):221-226. paper received march 26, 2018 accepted august 20, 2018 ijfs#1712_bozza ital. j. food sci., vol. 32, 2020 583 paper a comparative study on the effect of high hydrostatic pressure on ripening of turkish white cheese from different milk species h. yaman*1, e. sarica2 and h. coşkun2 1department of gastronomy and culinary arts, bolu abant izzet baysal university, gölköy campus, bolu, turkey 2department of food engineering, bolu abant izzet baysal university, gölköy campus, bolu, turkey *corresponding author: hulyayaman@ibu.edu.tr abstract high pressure treatment has diverse effect on cheeses depending on their characteristics. in this study, pressure application on turkish white cheese (300 and 450 mpa/5 min) and the changes during ripening were investigated. the 450 mpa pressure process showed an enhanced effect on proteolytic and lipolytic activity of cheeses. besides, 450 mpa pressure treatment was very effective on the microbiological profile, but the other treatment condition exhibited a more moderate antimicrobial effect. although, the total mold-yeast were detected after high-pressure treatment, their existence to a considerable degree was seen at the end of storage. keywords: high pressure, cow cheese, goat cheese, ripening, turkish white cheese ital. j. food sci., vol. 32, 2020 584 1. introduction the ripening of cheese is an expensive process because of the high storage cost, so the reduction in storage cost without affecting the quality of the product would provide significant savings to the cheese industry (el soda and awad, 2011). a reduction in the financial cost of a large quantity of cheese during storage, providing sufficient space for a new product by a fast cycle of stock can be ensured by accelerating ripening. cheese ripening increases by elevated storage temperature, addition of exogenous enzyme or cheese slurries and modified microorganism usage in the dairy industry. recently, the high-pressure treatment on cheese to accelerate ripening has been scientific and of commercial interest to numerous researchers (saldo et al., 2002; o’reilly et al., 2000). it has been reported in previous studies that high hydrostatic pressure (hhp) has been applied to cheese for destroying pathogenic and spoilage bacteria, reducing salting time, allowing the release of starter enzymes and changes in enzyme activity to reduce ripening time without changing cheese quality (saldo et al., 2002). in the event of pressurization process, the breakdown of proteins structure affects textural development, aromatic compound formation and proteolysis by releasing amino acids. in the previous studies, the accelerating effect of hhp treatments on cheese ripening was reported as follows: alteration in enzyme configuration, structural changes in the protein matrix by increasing activity of proteases, as well as bacterial lysis which aid the release of microbial enzymes. there have been various studies on the effect of hhp on cheese such as mozzarella (saldo et al., 2002; o’reilly et al., 2000), gouda (messens et al., 2000), cheddar (rynne et al., 2008), sheep milk cheese (juan et al., 2007) and goat milk cheese (capellas et al., 2001). these researches showed that hhp affects cheese quality and ripening depends on cheese variety, chemical and physicochemical properties of cheeses, pressure level, processing time, and temperature conditions. certain pressure parameters may lead to variable changes in composition, quality properties and texture of different cheeses (o’reilly et al., 2001). although, a good number of researches were published on the cheese ripening properties after applying high hydrostatic pressure, no information has been found on ripening properties of turkish white cheese under pressurization effect. the studies on turkish white cheese have focused on the pressurization effect on microbial inactivation of pathogenic bacteria (l. monocytogenes), total mesophilic aerobic bacteria (tmab), total molds and yeasts, lactococcus, lactobacillus and coliform bacteria (enterobacteriaceae) in cheese (evrendi̇lek et al., 2008), salt distribution of cheese during ripening (koca et al., 2018), as well as textural and microstructural changes after pressure treatment (koca et al., 2011). this study focused on the effect of pressure on ripening properties and hence microbiological changes because the diverse effects of hhp on cheese depend on milk species, composition, structure, and quality. there has been insufficient information on the effect of high pressure on the proteolysis and lipolysis properties of turkish white cheese during ripening. in accordance, it was aimed to determine the effects of moderate level pressure treatment on the chemical, physicochemical, and biochemical changes of turkish white cheese made from goat and cow milk during the ripening period. ital. j. food sci., vol. 32, 2020 585 2. materials and methods 2.1. cheese production full-fat cow and goat milk were obtained from two local dairy farms (i̇kizler süt and bolana, bolu, turkey) and cheeses were produced from 100 liters of pasteurized milk in two different vats. after pasteurizing of raw milk at 65°c for 30 min, the milk was cooled to 32°c. mesophilic starter culture (lactococcus lactis subsp. cremoris and lactococcus lactis subsp. lactis) (dvs-r-704, chr. hansen) at a rate of 0.2% and cacl2 about 0.02% were added into pasteurized milk. then liquid rennet (10g/100l cheese milk (chy-max, chr. hansen) was supplemented as a coagulant when ph reached6.40, and coagulation was completed within 90 min. the coagulum was cut (1 cm cubes) and rested in the whey for 5 min. the curds were transferred into 7x7 cm molds, and the whey was drained spontaneously at room temperature for 8 hours until whey drops were stopped. the cheese blocks were taken in brine (14 g/100g nacl) for 12 h at room temperature. both goat and cow cheese samples were then packaged into airtight and watertight plastic boxes and were separated into three groups depending on the pressure treatment (i) control, (ii) 300 mpa for 5 min and (iii) 450 mpa for 5min for hhp application. after the hhp process, they were stored at +4°c for two months and examined at 0, 30, 60 days of storage. 2.2. high-pressure treatment pilot-scale high-pressure food processor unit (avure technologies, oh, usa) with oneliter pressure chamber was used. vacuum packaged samples were processed with 300 and 450 mpa pressures for 5 min, while process temperature was kept below 6 ºc during the hhp application. control samples without pressure application were packaged the same way as the hhp samples. 2.3. microbiological analyses tmab (iso, 2013), mold and yeast (iso, 2004a), coliform (iso, 2006), mesophilic lactic acid bacteria (iso, 1998) and psychrotrophic bacteria (iso, 2005) were determined in duplicate according to iso (international organization for standardization) standards. 2.4. physicochemical analyses chemical properties of cheese samples were determined with respect to international dairy federation (idf) standards for total solid (iso, 2004b), fat content (idf, 2009), protein content (idf, 2014), salt content (idf, 2006), and titratable acidity (idf, 2012). a ph meter (ph-720, inolab, germany) was used to measure ph value of the homogenate (10 g cheese sample+10 ml distilled water). minolta cr-400 (konica minolta sensing, inc., osaka, japan) equipment was used for color measurements of cheese in d65 mode as l*, a*and b*. 2.5. nitrogen fractionation determination water-soluble nitrogen (wsn) and trichloroacetic acid-soluble nitrogen (tca-sn 12%) fractions were prepared by kuchroo and fox (1982) and polychroniadou et al. ital. j. food sci., vol. 32, 2020 586 (1999), respectively. the nitrogen contents of the soluble extracts were carried out using the kjeldahl method (idf 2014) and the ripening index (ri) was presented as a percentage of wsn to total cheese nitrogen. the acid degree value (adv) of the samples was found in meq koh/100g fat according to case et al. (1985). 2.6. statistical analyses the changes for 2 months-storage were determined by general linear model repeated measures. analysis of variance (anova) and tukey’s multiple comparison tests were used to determine differences between cheese samples in different treatment groups by spss version 23 (spss inc., chicago, usa) package program. simca (soft independent modeling of class analogy) was used for the grouping of cheese samples based on properties under various pressure. the classes were observed to be significantly different from each other when the interclass distances were above 3. 3. results and discussion 3.1. microbiological properties of cheeses the effect of high hydrostatic pressure processing on the microbial properties of cheese samples was shown in table 1. coliform bacteria were not found in both (control and pressurized) cow and goat cheese samples at 1/10 dilution. tmab, lactococci and lactobacilli counts showed a significant decrease in bacterial growth at high pressure treated cheese samples (p<0.05). while aerobic mesophilic bacteria count in cow and goat control cheeses were around 9.13 and 9.6 log cfu/g respectively, average 2-3 log decreasing by 300 mpa and 4-6 log decreasing for 450 mpa pressure were observed in hhp-treated cow and goat cheese samples. the effect of high pressure on lactococci and lactobacilli counts were higher due to elimination of the all starter culture at 450 mpa pressure, processing nearly above the mesophilic bacteria counts. besides, 300 mpa hhptreated cheese samples showed significant decrease in microbial counts of lactic acid bacteria (p<0.05). lactobacilli were affected by hhp more than lactococci in the study. the lactobacilli were the most affected bacteria group from pressurization processing both in cow and goat cheeses. although, total mold/yeast could not be determined for both control and pressure treated samples, their presence increased at the end of the storage and reached about 5.5 log levels at 60 days of storage. growth of yeasts and molds in hhp treated samples compared to the control samples was delayed in goat cheese, but these microorganisms reached about 5 log levels during the storage period. evrendi̇lek et al. (2008) noticed that total yeast and mold after 300-600 mpa high-pressure treatments were below the detection limit, so they could not be detected. similarly, psychrotrophic bacteria in cow cheese were significantly detected at the end of the storage period (p<0.05). however, psychrotrophic bacteria was not found in all goat cheese samples during the storage period daryaei et al. (2008) reported the growth of yeast at 6 weeks of cheese samples treated under similar high-pressure parameters. sublethal injury experiments regarding high pressure applications indicated that high pressure damaged the microorganism population more severely and it took longer to recover (o’reilly et al., 2000). this fact may explain the existence of molds and yeasts counts in cheeses treated at 300 and 450 mpa at 60 days. pressurization processing showed higher microbial reduction ital. j. food sci., vol. 32, 2020 587 effect in goat cheese as compared to cow cheese, this shows that the type of cheese affects the microbial inactivation (o’reilly et al., 2001). table 1. microbiological changes during ripening (at 4ºc) of the cheeses after high pressure treatment at 300 and 450 mpa for 5 min (log cfu/g). cheese type microbial group day treatment control 300 mpa 450 mpa c ow c he es e tmab 1 9.13±0.03a 7.24±0.02b 5.72±0.04c 30 8.60±0.11a 7.07±0.06b 4.00±0.10d 60 8.78±0.15a 7.41±0.61b 4.32±0.62d lactobacilli 1 8.61±0.170a n.d. n.d. 30 4.96±0.08b 3.51±0.11d n.d. 60 4.64±0.11c 3.06±0.44e n.d. lactococci 1 9.00±0.09a 6.82±0.27b n.d. 30 5.00±0.11c 3.79±0.09e n.d. 60 4.78±0.14c 4.28±0.73d n.d. mold/yeast 1 n.d. n.d. n.d. 30 n.d. n.d. n.d. 60 5.32±0.10a 5.58±0.300b 5.41±0.10c psychrotrophic bacteria 1 n.d. n.d. n.d. 30 n.d. n.d. n.d. 60 5.66±0.16a 5.75±0.13a 8.69±0.19b g oa t c he es e tmab 1 9.6±0.07a 5.78±0.16d 3.67±0.09g 30 7.68±0.16b 6.66±0.73c 4.29±0.66f 60 6.04±0.05d 4.84±0.34e 3.30±0.25g lactobacilli 1 9.03±0.11a n.d. n.d. 30 4.91±0.10b 3.20±0.48c n.d. 60 0.50±1.00d 1.00±1.16d n.d. lactococci 1 9.69±0.17a 3.35±0.24d 1.19±1.38e 30 5.67±0.09b 4.64±0.11b n.d. 60 4.27±0.17d 3.47±0.29d n.d. mold/yeast 1 n.d. n.d. n.d. 30 5.34±0.21a 1.46±1.695b n.d. 60 6.00±0.09a 5.67±0.11a 5.28±0.89a psychrotrophic bacteria 1 n.d. n.d. n.d. 30 n.d. n.d. n.d. 60 n.d. n.d. n.d. values represented by mean ± standard deviation. a,b,cdifferent superscript in the same microbial group indicates significant differences (p<0.05). n.d. = not determined in 1/10 dilution. ital. j. food sci., vol. 32, 2020 588 3.2. physicochemical properties of cheeses pressure application did not significantly affect the total solids, protein, and fat contents of both cow and goat cheese samples (fig. 1). the total solid content of the cheeses was found lower than hhp-treated turkish white cheese pressurized up to 400 mpa for 5, 10 and 15 min reported by koca et al. (2011) and evrendi̇lek et al. (2008) due to probably applying different pressing time and load in production. moreover, a significant increase in total solid, protein, and fat contents was found in cow’s milk cheeses on day 60 of ripening (p<0.05), while there were significant decreases in all parameters for goat cheeses (p<0.05). the high beta casein content of goat milk produces a firm and hard structure when processed in turkish white cheese due to the long period of time and spontaneously straining of whey. the differences in structural properties were influenced by high hydrostatic pressure at different levels. the placement of brine into cheese causes an increase in moisture content, in contrast, changes in para casein network due to hhp causes a decrease in moisture content. moreover, saldo et al. (2001) reported the existence of higher amount of bound water in hhp-treated cheese despite presence of same moisture content in control cheese. this phenomenon causes high moisture content in hhp-treated cheese during ripening. this fact also explains the reason for decrease in protein and fat content of goat cheese samples during ripening period. also, the decrease in protein and fat contents may be as a result of the diffusion of proteolysis products and fat from the cheese into brine (hayaloğlu et al., 2002). the goat cheese samples were higher dry solids content as compared to cow cheeses; therefore, goat cheeses resulted in higher moisture content by diffusion of brine into cheese with pressurization. on the other hand, cow cheese was not as firm as goat cheese, and pressure process was affected by the protein networks via breaking down. as a result, the hhp treatment might cause slight water expulsion and result to a decrease in moisture content in the pressure treated cheeses (huppertz et al., 2006). compared to control samples, less water content was reported in la serene cheese treated under 300-400 mpa hhp at 50 days (garde et al., 2007). while the higher water holding capacity was identified in high pressure treated ewes milk cheeses during ripening. none of changes were found for moisture content of goat cheeses (capellas et al., 2001). after the hpp treatment, structure of paracasein matrix of cheese plays an important role in the composition of cheese via acidity development and salt distribution (moschopoulou et al., 2010). the quantity of salt in cheese samples increased both in control and pressurized samples during ripening due to salt diffusion from brine into cheese during storage. koca et al. (2011) reported an increase in the amount of salt in pressurized and unpressurized turkish white cheeses at various pressure levels until the 14th day of ripening. the titratable acidity as lactic acid basis showed a slight reduction by increasing the pressure level, whereas, ph values of the cheese samples increased by pressuring process (table 2). these results were in agreement with results reported by koca et al. (2011) in 50-400 mpa high pressure treated turkish white cheese. the ph-enhancing effect of high pressure on various cheeses was reported by some authors (messens et al., 1998; moschopoulou et al., 2010). the ph value in both cow and goat cheese samples showed a tendency to decrease at the ripening period. the carriage of colloidal calcium phosphate in twosides may create these temporary changes in ph values (moschopoulou et al., 2010). besides, the usage of lactic acid, the formation of alkaline ital. j. food sci., vol. 32, 2020 589 compounds by degradation of big molecules and proteins may be the result of reducing effect (messens et al., 1998). figure 1. effect of hhp treatment (300 and 450 mpa for 5 min) on compositional changes in cow and goat cheese samples during ripening. the color properties (l*, a* b* value) of high hydrostatic pressure treated cheese samples is given in table 2. the high-pressure process did not affect the l* (lightness) and b* (blueyellow) values of the cow cheese samples significantly (p>0.05), whereas, there was a small change in goat cheese (p<0.05). besides, a* (green-red) color parameter tended to increase the greenish color due to release of the whey. the color variations of cheeses are mainly affected by cheese manufacturing techniques and quality properties of the fat phase. as a result of the biochemical reactions during the ripening period, the compositional and structural changes occur and can affect the color changes of various cheeses. the cheese structure is the result of hydrophobic interactions between caseins and the effects of high hydrostatic pressure on the non-covalent bond by their breakdown. therefore, the cheese components produce a new structure of different rheological and color characteristics (saldo et al., 2002). ital. j. food sci., vol. 32, 2020 590 table 2. the physicochemical changes in control and in the pressurized cheeses during ripening. cheese type day control 300 mpa 450 mpa c ow c he es e titratable acidity (% la) 1 0.58±0.02a 0.57±0.00a 0.56±0.03a 30 0.35±0.03b 0.41±0.04c 0.36±0.02b 60 0.38±0.02bc 0.37±0.0bc 0.37±0.02bc ph 1 4.54±0.02ab 4.61±0.00a 4.65±0.01b 30 4.57±0.04ab 4.61±0.04ab 4.59±0.01ab 60 4.52±0.08a 4.56±0.06ab 4.58±0.08ab l* 1 92.38±0.40a 92.26±0.01a 92.79±0.01a 30 92.90±0.14a 92.62±0.15b 93.11±0.81a 60 92.07±0.82a 92.90±0.16c 91.45±1.85a a* 1 -2.46±0.08a -2.38±0.00a -2.89±0.01a 30 -2.37±0.24a -2.51±0.05b -2.81±0.21ab 60 -2.27±0.20a -2.24±0.03c -2.51±0.18b b* 1 16.38±0.64a 16.69±0.04ab 17.65±0.01a 30 16.34±2.18a 17.54±0.00a 16.68±0.26ab 60 16.40±1.57a 16.33±0.44b 16.52±0.74b g oa t c he es e titratable acidity (% la) 1 0.61±0.03a 0.58±0.07a 0.50±0.05b 30 0.38±0.02c 0.35±0.04d 0.38±0.03cd 60 0.34±0.02d 0.39±0.00cd 0.42±0.02c ph 1 4.75±0.10a 4.83±0.08ab 4.92±0.04c 30 4.81±0.07ab 4.87±0.05bc 4.88±0.03bc 60 4.84±0.02b 4.83±0.01ab 4.79±0.01ab l 1 94.27±0.27a 92.82±0.13bc 91.89±0.40d 30 93.16±0.41b 92.37±0.50cd 92.76±0.33bc 60 92.66±0.15bc 94.22±0.40a 93.29±0.37b a* 1 -2.42±0.11ef -3.20±0.21b -3.49±0.14a 30 -2.34±0.05fg -2.54±0.15de -2.68±0.08d 60 -2.22±0.07g -2.70±0.02d -2.96±0.08c b* 1 9.23±0.32a 10.71±0.87b 11.28±0.74b 30 9.55±0.28a 9.04±0.12a 9.09±0.23a 60 9.51±0.27a 9.47±0.16a 9.20±0.19a values represented by mean ± standard deviation. a,b,c different superscript in the same parameter indicates significant differences (p<0.05). 3.3. the changes in nitrogen fractionations the wsn, tca-sn value and ri were determined for the monitoring of proteolysis progress, and acid degree value (adv) for evaluation of lipolysis and the results were given in table 3. no significant difference (p>0.05) was found in wsn or tca-sn levels between hhp-treated cheeses and control cheese for 60 days ripening period in cow cheese. in contrast, both wsn and tca-sn value of goat cheeses were determined by application of 300 mpa pressure compared to 450 mpa in cheeses samples (p<0.05). higher proteolysis was observed in 300 mpa hp treated cheeses compared to control and ital. j. food sci., vol. 32, 2020 591 450 mpa treated samples. the chymosin activity is important for cheese ripening, however, chymosin enzyme has been affected by high hydrostatic pressure depending on pressure force. while most of the peptides were produced rapidly under hp treatment at <300 mpa pressure. the liberation of other peptides was inhibited by pressurization at >300 mpa pressures (scollard et al., 2000). table 3. the effect of high-pressure treatment (300 and 450 mpa for 5 min) on proteolysis and lipolysis of the cheeses during ripening. cheese type control 300 mpa 450 mpa c ow c he es e wsn (%) 1 0.024±0.00a 0.029±0.00ab 0.027±0.00ab 30 0.028±0.00ab 0.032±0.00bc 0.034±0.00cd 60 0.037±0.00de 0.040±0.00e 0.039±0.00e ripening index 1 1.016±0.01a 1.189±0.03abc 1.147±0.00ab 30 1.291±0.09abc 1.376±0.13cd 1.441±0.09de 60 1.670±0.227e 1.662±0.29de 1.588±0.17de tca-sn (%) 1 0.006±0.00a 0.008±0.00ab 0.009±0.01abc 30 0.010±0.00bcd 0.011±0.00bcde 0.009±0.00bc 60 0.013±0.00de 0.014±0.00e 0.012±0.00cde acid degree value 1 0.86±0.08ab 0.56±0.040a 0.67±0.02a 30 1.21±0.39c 1.08±0.09bc 1.02±0.01bc 60 1.16±0.21bc 1.18±0.20bc 1.26±0.22c g oa t c he es e wsn (%) 1 0.025±0.00a 0.030±0.00b 0.027±0.00a 30 0.026±0.00a 0.033±0.00c 0.036±0.00cd 60 0.028±0.00a 0.034±0.00c 0.038±0.00d ripening index 1 0.938±0.02a 1.204±0.06c 1.036±0.11ab 30 1.118±0.09bc 1.421±0.06de 1.512±0.08e 60 1.199±0.06c 1.351±0.09d 1.429±0.03de tca sn (%) 1 0.009±0.00a 0.011±0.00a 0.010±0.00a 30 0.010±0.00a 0.012±0.00a 0.011±0.00a 60 0.461±0.02c 0.345±0.19bc 0.268±0.12b acid degree value 1 0.95±0.01ab 0.99±0.04ab 1.09±0.05b 30 0.80±0.12a 1.15±0.18b 1.09±0.03b 60 1.77±0.29c 1.92±0.14c 1.89±0.03c values represented by mean ± standard deviation. a,b,c different superscript in the same parameter indicates significant differences (p < 0.05). the ripening index value of goat cheese samples was higher than cow cheese samples. the destabilization of casein micelles after high hydrostatic pressure processing may increase as a result of residual coagulant or indigenous milk proteinases activity (huppert et al., 2004) and can cause enhancement effect on their sensibility to proteolytic enzymes. it means that plasmin can play an important role in proteolysis after pressurization. the enhancer effect of high hydrostatic pressure on proteolysis was reported in other cheeses under different pressures (o’reilly et al., 2000; rynne et al., 2008; voigt et al., 2010). ital. j. food sci., vol. 32, 2020 592 similar to proteolytic changes, adv values were affected by high-pressure treatment in goat cheese samples (p<0.05), whereas, there was no significant effect found in cow cheese samples (p>0.05). adv values of all cheese samples showed an increase during storage time. it is obvious that high-pressure treatment influences biochemical reactions during the ripening period of cheese. this phenomenon may occur via the direct effect of pressure on enzyme or reaction and may affect the release of enzymes of lactic acid bacteria. besides the changes during ripening via biochemical reaction on compositional and structural properties of cheeses, hhp produces conformational changes in proteins and may affect enzyme modulation site or active site directly (rovere, 1995). these changes affect proteolysis or lipolysis and show enhancing and reducing effect on the ripening of various cheeses. 3.4. overall composition comparison of cheeses simca of the overall properties of the control and high pressure treated cheeses are presented in fig. 2. it showed the distinctive pattern and 6 well-defined cheese groups. simca is a supervised classification method and provides a model based on the principal component analysis and uses the distance of the mean in each group for discrimination. the distance of mean in each cluster or group are known as interclass distance and if this value is higher than 3, it is significant for identification (kvalheim and karstang, 1992). the interclass distance for all control and pressurized cheeses ranged between 3.74-17.04 (table 4). figure 2. soft independent modeling of class analogy (simca) classification plot of cheese samples treated with high pressure at 300 and 450 mpa for 5 min. the all data were transformed into their centered and normalized mean prior to multiple analysis. ital. j. food sci., vol. 32, 2020 593 table 4. interclass distances between the goat and cow cheese treated with high pressure at 300 and 450 mpa for 5 min based on the simca class projections. groups cow cheese control cow cheese 300 mpa cow cheese 450 mpa goat cheese control goat cheese 300 mpa goat cheese 450 mpa cow cheese control 0.00 cow cheese 300 mpa 3.74 0.00 cow cheese 450 mpa 7.38 6.89 0.00 goat cheese control 6.73 14.33 17.04 0.00 goat cheese 300 mpa 7.35 9.45 10.65 7.48 0.00 goat cheese 450 mpa 8.82 10.04 8.26 11.35 4.68 0.00 interestingly, cow and goat cheese results showed good discrimination, although, both cheese types had similar results. the difference between goat and cow cheeses probably arises from results of the proteolysis and lipolysis test. when wsn and tca-sn values of the cow cheese were not affected by high pressure application, pressurization showed enhancer effect on goat cheese samples, proteolysis and lipolysis properties. the simca pattern showed that control, 300 mpa and 450 mpa pressurized samples of both cow and goat cheeses grouped in the same order and implying increase in pressure created a similar effect on the two cheese species. 4. conclusion the evaluation of the overall results obtained from the study showed that, two different levels of the high-pressure application on white cheeses samples produced from cow and goat milk had varying effects. this method shows the possibility of applying highpressure treatment in the white cheese besides the predetermined reliability. also, the microbial load of lactic acid bacteria is decreased by high-pressure practices. the hpp delayed the growth of yeasts/molds in goat cheese samples compared to the control groups at the end of the ripening period. consequently, increase in the level of hpp provided significant decreasing effect on tmab, lactobacilli and lactococci counts, and increasing effect on proteolysis and lipolysis. the fact that high-pressure has no significant difference in the chemical composition, but have positive results visually indicates the applicability of this technology. acknowledgements this study was supported by bolu abant i̇zzet baysal university, scientific research projects coordination unit (project no: 2015.09.04.958) and was presented at the international conference on agriculture, forest, food sciences, and technologies (icafof 2017 cappadocia, turkey) as an abstract in abstract proceeding book. references capellas m., mor-mur m., sendra e. and guamis b. 2001. effect of high-pressure processing on physico-chemical characteristics of fresh goat’s milk cheese (mato). international dairy journal 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levels of flavour compounds in mature blue-veined cheese. innovative food science emerging technology 11:68-77. doi: doi.org/10.1016/j.ifset.2009.10.009 paper received october 10, 2019 accepted january 31, 2020 ijfs#1141_bozza ital. j. food sci., vol. 31, 2019 87 paper the effect of intramuscular fat content on the meat quality of plw x pl pigs h. jankowiak*, m. bocian and j. barczak department of animal breeding, faculty of animal breeding and biology, utp university of science and technology in bydgoszcz, 85-084 bydgoszcz, poland *e-mail jankowiak@utp.edu.pl abstract this study aimed to determine the effect of intramuscular fat content (imf) on the quality of meat from plw x pl pigs. meat was evaluated in terms of intramuscular fat content (n=80), technological properties, visual and tactile aspects, and colour measurements. it was demonstrated that meat containing more imf was characterised by higher tenderness and marbling, but lower water content and a lower proportion of the yellow b* colour. furthermore, it had a lower c* saturation, a lower h° hue, and lower muscle pigment content. keywords: pigs, intramuscular fat, meat quality ital. j. food sci., vol. 31, 2019 88 1. introduction there are numerous works indicating a significant role of intramuscular fat content (imf) in determining the traits of pork (fernandez et al., 1999a, b; fortin et al., 2005). its content in meat depends on numerous factors such as species, breed and sex (hocquette et al., 2010; wood et al., 2004; bocian et al., 2009; 2012; jankowiak et al., 2010; tyra and żak, 2012; feeding (wood et al., 2004; alonso et al., 2010), method of maintenance and age of slaughtered animals (andrés et al., 2001), body weight at slaughter (latorre et al., 2004), carcass muscling and subcutaneous fat thickness (pietruszka et al., 2015). irrespective of the above factors, intramuscular fat content is intimately related to some quality traits of meat. intramuscular fat (imf) in pork is one of the main factors that influence the sensory quality parameters of meat, especially suitability for processing and cooking (fernandez et al., 1999a; daszkiewicz et al., 2005; czarniecka-skubina et al., 2010). the number and size of intramuscular adipocytes is related to variability in imf content (hocquette et al., 2010). the muscle fibre microstructure and fibre types -affect the accretion rate of intramuscular fat (fiedler et al., 2003; wojtysiak, 2014). according to de vries et al. (2000), consumers mostly prefer pork with an imf content of 2.5-3%. czarniecka-skubina et al. (2007) stated that pork with a higher (>2.51%) imf content, despite having better processing value, tenderness, juiciness, palatability, and marbling, is less accepted by consumers. a minimum imf content of 1.5% was considered necessary to ensure appropriate juiciness, tenderness, and palatability (fortin et al., 2005). reducing imf content may have a negative impact on sensory and processing features of meat (czarniecka-skubina et al., 2007; bocian et al., 2009). according to ellis (2006) an imf content ranging from 1.8% to 2.6% is an indicator of good quality pork. the most popular and common pig breeds in poland are the polish large white (plw), the polish landrace (pl), and their crossbreds (blicharski and snopkiewicz, 2017). the study aimed to determine the effect of imf on some meat quality traits of polish large white x polish landrace (plw x pl) pigs. 2. materials and methods 2.1. animals and sampling the tested meat was obtained from 80 fattening pigs, f1 crossbreds (polish large white x polish landrace), 50% gilts and 50% hogs. the crossbred plw x pl fattening pigs came from and were kept on the same farm under the same environmental conditions, in accordance with welfare requirements. the animals were fed ad libitum with the same complete mixtures, according to standard requirements (grela and skomiał, 2014). the composition and nutritional value of the complete mix are given in table 1. when fattening was complete, the animals were individually weighed and transported to a slaughterhouse about 100 km away. the slaughter was carried out in accordance with the applicable procedures after a 2-hour rest. the average live weight of slaughtered pigs was 106±9.57 kg. ital. j. food sci., vol. 31, 2019 89 table 1. the nutritional value of feed mixtures. fattening period composition of feed mixture 30 to 70 kg 70 to 110 kg ground wheat (%) 20 15 ground barley (%) 25 10 ground triticale (%) 40 60 protein concentratea (%) 15 15 metabolizable energy (mj/kg) 13.20 13.34 crude protein (g/kg) 156 158 acomposition: metabolizable energy, 13.30 mj/kg; crude protein, 37.60%; crude fibre, 2.50%; crude ash, 17,80%; crude fat, 1.20%; ca, 4.40%; p, 1.20%; na, 1.0%; lysine, 4.50%; methionine, 0.56%; tryptophan, 0.50%; threonine, 1.60%; methionine + cystine, 1.40%. vitamin-micromineral per kilogram of complete diet: vitamin a (e 672), 60000 iu; vitamin d3 (e 671), 16600 iu; vitamin e alfa tocopheryl acetate, 716 iu; vitamin k3 as sodium sulfate, menadione 16 mg; thiamine, 12 mg; riboflavin, 24 mg; pyridoxine, 20 mg; cobalamin, 200 mcg; biotin, 400 mcg; niacin, 113 mg; ca-d-pantothenate, 70 mg; betaine, 1080 mg; cu, 160 mg as copper sulfate; fe, 640 mg as iron sulfate monohydrate; mn, 320 mg as manganese oxide; zn, 640 mg as zinc oxi; se, 3.0 mg as sodium selenate; i, 16 mg as anhydrous calcium iodate. on the day following the slaughter, the carcass fat and meat content were determined according to różycki and tyra (2010). the thickness of backfat was determined on the cold right half-carcass at points over the shoulder (at the thickest point), on the back (behind the last thoracic vertebra and the first lumbar vertebra), and at three locations over the loin (cross-section of the gluteal muscle): over the rostral edge of the gluteal muscle, in the middle of the gluteal muscle, and over the caudal edge of the gluteal muscle. the arithmetic mean was calculated from the five measurements of backfat thickness. on a cross-section of the longissimus lumborum muscle taken from the last thoracic vertebra and the first lumbar vertebra, the surface contour was measured (loin eye), and then the determined cross-sectional area was measured using the lucia system (image for image processing and analysis, version 4.82.2004). the research did not require the consent of the local ethical committee. 2.2. meat analysis the acidification of muscle tissue at 45 minutes post slaughter (ph45) and at 48 hours post slaughter (ph48h) was determined using an elmetron cp-401 ph-meter with a blade electrode. the equipment was calibrated using elmetron ph 7.0 and ph 4.0 buffers. the meat quality was evaluated at 48 h post slaughter based on the longissimus lumborum muscle, which were stored at a temperature of 4-6°c. water-holding capacity (whc) was determined using the method developed by grau and hamm (1952) and modified by pohja and niinivaara (1957). a 300 mg sample of minced meat was placed on a whatman 1 filter paper and put between two glass plates; then an even load of 2 kg was applied to it for 5 minutes. the area of juice infiltration was used to calculate the percentage of free water content in the meat, assuming that 1 cm2 of infiltration corresponds to 10 mg of water. the surface of meat juice infiltration was measured using a lucia computer analysis system (system for image processing and analysis, version 4.82.2004). thermal drip was determined at 48 h post slaughter using the method developed by walczak (1959). a 20 g sample of minced meat (20 g) was placed in a hygroscopic gauze and heated in a water bath at a temperature of 85°c for 10 min. after taking the ital. j. food sci., vol. 31, 2019 90 sample out of the water bath, the gauze was removed, then the sample was cooled to a temperature of 4°c and weighed. based on the difference in weight before and after the heat treatment, the percentage weight loss was calculated. shear force was measured using the instron 3342 strength testing equipment with a warner-bratzler attachment (wbsf), in accordance with the methodology provided by szalata et al. (1999). a 120 g meat sample was heated in a water bath until the sample reached a temperature of 70°c on the inside. the heat treatment was performed in a 0.85% nacl solution. then, 10 mm × 10 mm bars were cut along muscle fibres, which were subsequently cut perpendicularly to the muscle fibres. the results were read as maximum shear force expressed in n. the chemical composition of the meat, i.e. water, dry mass, total protein, and intramuscular fat content, was determined in accordance with polish standard pna-82109:2010 with near-infrared transmission spectroscopy (nit) using calibration on artificial neural networks (ann) with the foss foodscan equipment. visual and tactile evaluation was determined 48 h after slaughter on a slice of raw meat weighing 120 g. visual and tactile assessment of the meat was carried out by a trained 10person team. all evaluators had 4 years of experience in assessing pork meat. visual properties of the raw meat were assessed: visual colour intensity according to a 6-grade scale (polish standard pn-iso 4121:1998) on which 1=very light, 6=dark purple; marbling based on canadian and american models on a 10-grade scale (cheng et al., 2015; nppc, 1999) where 1=no intramuscular fat content, 10=very high marbling. tactile evaluation of firmness was on a 7-grade scale (pn-iso 4121:1998) where 1=very firm, 7=very soft. meat colour was also measured on a slice of raw meat at 48 h post slaughter using a minolta cr 310 photocolorimeter (konica minolta, japan) with a measuring port diameter of 50 mm. the equipment was standardized using a cr310 white calibration plate with the following coordinates: y=92.80, x=0.3175 i y=0.3333. colour parameters were determined in the cie system, l*a*b* (l* lightness, a* participation of red, b* participation of yellow) (cie, 1986) using illuminant d 65 and a standard 2° observer. chroma (c*) and hue angle (ho) were calculated according to the formula provided by beattie et al. (1999) and brewer et at. (2001): 𝐶∗ = (𝑎∗)! + (𝑏∗)! , ho=(tan-1 b* / a*) muscle pigment was determined by colorimetry according to the method developed by hornsey (1956). a 40 ml mixture of acetone, water, and concentrated hcl in proportions 40:2:1 was poured over minced meat samples (10 g), which were then extracted for 1 hour. after filtering, the absorbency of the tested solutions was measured using a marcel media spectrophotometer at a wavelength of 640 nm. the optical density value (e) was multiplied by a factor of 680 in order to obtain the proper concentration of hematin expressed as micrograms of hematin per 1 g of meat. 2.3. statistical analysis the results were statistically analysed; the arithmetic mean and the standard deviation for carcass traits, and the standard error (sem) for meat quality traits were calculated. data were verified for homogeneity of variance with the leven’e test; in the absence of homogeneity of variance, statistical significance between groups was calculated using the nonparametric kruskal-wallis test. a probability of p <0.05 was considered statistically significant. ital. j. food sci., vol. 31, 2019 91 the obtained test results were compiled and analysed in three groups that were defined in terms of the intramuscular fat content (imf) of the meat of the plw x pl pigs according to the normal distribution of features (gaussian curve): group i, <1% of imf content; group ii, 1-2.5% of imf content; group iii, >2.5% of imf content. the meats were divided into imf groups to verify the impact on meat quality of increased imf in the meat of plw x pl pigs. pearson’s simple correlation coefficients between the imf content and the meat slaughter traits and quality traits were calculated to numerically summarize the degree of association between any two variables. all calculations were conducted using statistica pl.8.0 data analysis software (statsoft inc. statistica, 2008). 3. results and discussion the data regarding the quantity and weight measurement of warm carcasses, average backfat thickness, and loin eye area are shown in table 2. the obtained values of average backfat thickness indicate a higher fat content of pig carcass than reported in other studies (czarniecka-skubina et al., 2007; tyra and żak, 2012). table 2. mean and standard deviation of carcass characteristics. mean and standard deviation number (n) 80 hot carcass weight (kg) 86.83±8.61 average backfat thickness (mm) 23.23±5.27 loin eye area (cm2) 53.87±7.41 the characteristics of the technological properties of the tested pork are presented in table 3 and analysed according to the intramuscular fat content. the 16 meat samples fell within the first group (lowest fat content), 48 meat samples within the ii group and 16 meat samples within iii group (highest fat content). in numerous tests, the highest percentage of meat samples had up to 2% of imf (daszkiewicz et al., 2005; tyra and żak, 2012). the imf content in the meat studied ranged from 0.79% to 3.20% and significantly differed between all the groups (p<0.01). the acidity of muscle tissue is one of the parameters that determines meat quality and is used to determine the processing and cooking suitability of meat (kajak et al., 2007). meat acidity is measured 45 minutes after slaughter and is a widely recognised criterion that reflects the intensity of post-slaughter changes that lead to meat quality defects such as pse (hofmann, 1994). the ultimate ph is an indicator of meat quality and is associated with water-holding capacity, colour, and tenderness (kajak et al., 2007). in this study, higher ph45 values were observed in group i than in group iii (p<0.05); this can be explained by the fact that the group with the lowest imf content contained carcasses with values from 6.21 to 6.84 ph, which had an impact on higher ph48 values. the meat ph value measured 48 h post slaughter was the highest in the meat with the lowest imf content and differed significantly between group i and group ii and iii (p<0.01). the results of ph45 and ph48 presented in the paper are compatible with the results obtained by jaworska et al. (2007), who showed that with an increase from 1.72% to 2.63% of imf content in meat, the values of ph45 (6.40 to 6.36) and ph48 (5.52 to 5.50) decreased. czarniecka-skubina et al. (2007) demonstrated that pork with the ital. j. food sci., vol. 31, 2019 92 highest imf content (>2.51%) was characterized by a significantly higher final ph (5.63), compared to meat with the lowest (<1.5%) and average (1.51-2.5%) imf content (5.51 and 5.54) (p<0.05); moreover, it was characterized by a darker colour and lower protein content. daszkiewicz et al. (2005) demonstrated that meat with lower imf content (<1.0%) had lower ph45 (6.17) and similar values to obtained in this study at ph48 (5.44). in turn, klont (2005) indicated that a lower final ph causes the meat to have less water retention capacity and to be lighter in colour, while a higher final ph gives it a darker colour, less juice leakage during storage, and positively effects meat quality traits, i.e. succulence, tenderness and taste. the values obtained and presented in this study of ph45 as well as the final ph were typical for meat of good quality in accordance with the assumptions of hofmann (1994) and klont (2005). table 3. characteristics of the technological properties of meat quality (mean value and standard error) in relation to imf content. group imf i ii iii sem p <1% 1-2.5% >2.5% number (n) 16 48 16 number (%) 20.00 60.00 20.00 imf (%) 0.79a 1.59b 3.20c 0.10 0.001 ph45 6.50 a 6.37 6.32b 0.02 0.027 ph48h 5.54 a 5.46b 5.44b 0.01 0.005 whc (% of free water) 19.38a 19.99 21.67b 0.31 0.033 thermal drip (%) 21.20 21.05 21.75 0.24 0.378 wbsf (n) 55.94a 48.38a 37.46b 1.48 0.001 chemical composition of meat water content (%) 73.30a 74.09b 72.73c 0.12 0.001 total protein content (%) 23.09 23.40 23.11 0.07 0.343 (a-c) row means with different superscripts differ significantly at p<0.01. (a-b) row means with different superscripts differ significantly at p<0.05. imf intramuscular fat content; whc water holding capacity; wbsf warner bratzler shear force (n newton); ph45 ph at 45 minutes post slaughter; ph48h ph at 48 hours post slaughter. the meat with the lowest wbsf (shear force) contained the highest amount of imf (group iii) and was more tender than groups i and ii (p<0.01). van laack et al. (2001) evaluated the impact of ultimate muscle tissue acidity (phu) and imf content on tenderness and tenderization of pork. similarly, ramsey et al. (1990) showed that increasing meat imf content decreases its shear force. the studies demonstrated that, along with increasing imf content of the tested meat, the water content decreased and the dry mass content increased (p<0.01). daszkiewicz et al. (2005) observed a similar relationship between the level of imf and the chemical composition of pork. they showed that as the content of imf and marbling increased, the content of dry matter increased and the content of total protein and ash decreased. table 4 contains the results of a visual and tactile evaluation, and an evaluation of colour and muscle pigment of meat. with the increase in imf, marbling in meat increased (p <0.01). ital. j. food sci., vol. 31, 2019 93 higher marbling of pork is associated with a higher imf content (fernandez et al., 1999a; van der wal et al., 1992; van laack et al., 2001). czarniecka-skubina et al. (2007) showed that meat with the highest imf content (≥2.51%) was characterized by a higher marbling (6.06 points) in relation to meat with the lowest (≤1.5%) imf content (3.92 points). also, przybylski et al. (2010) showed more marbling (4.28 points) in meat with 2.27% imf content compared to 1.67 imf meat (2.70 points) (p <0.05). table 4. the results of visual and tactile evaluation of meat colour and muscle pigment content (mean value and standard error) in relation to imf content. group imf i ii iii sem p <1% 1-2.5% >2.5% visual and tactile evaluation visual colour intensity (1-6 scale) 3.7 3.5 3.1 0.08 0.127 marbling (1-10 scale) 1.0a 2.3b 3.9c 0.13 0.001 firmness (1-7 scale) 3.8a 4.2 4.6b 0.08 0.011 colour measurements l*48 53.73 54.78 55.23 0.32 0.560 a*48 16.19 15.78 15.11 0.13 0.037 b*48 6.80 a 4.90ba 3.32bb 0.22 0.001 c*48 17.60 a 16.61a 15.52b 0.16 0.001 ho48 22.63 a 17.02ba 12.36bb 0.70 0.001 muscle pigment (micrograms of hematin per 1 g of meat) 34.27 aa 29.83b 26.99b 0.64 0.002 (a-c) row means with different superscripts differ significantly at p<0.01. l* value represents lightness; a* proportion of red; b* proportion of yellow; c* saturation; ho hue angle. as evaluated tactilely, the highest hardness of meat was observed in samples in group i with a minimum content of imf, compared to group iii which was less hard (p <0.05). meat colour constitutes an important quality indicator (połom and baryłkopikielna, 2004). although lightness l* was highest for meat with the highest fat content, there were no statistically significant differences in terms of imf content. the obtained results were typical of normal meat quality (warris et al., 2006). there were no significant differences in the proportion of red a*, but there was a significant difference in the proportion of yellow b*, which was highest in the group with the lowest imf (p <0.01). quality features of meat colour include colour saturation, colorimetric purity, and dominating light wavelength, referred to as hue. the highest colour saturation c* and h° hue were observed in meat with the lowest (<1%) imf content (p<0.01). similar values of meat colour parameters l*, a*, b*, and saturation c*, and higher values of h° hue than the ones obtained in this study were shown in previous studies (bocian et al., 2015). muscle pigment content is one of the main factors that affect the evaluated meat colour. this study demonstrated significant differences in muscle pigment content. the highest content of muscle pigment was in meat from group i, which also had the lowest imf content compared to group iii, which had higher imf content (p<0.01). these values are similar to the ones obtained previously by bocian et al. (2015) for plw x pl meat. for more detailed interrelations between intramuscular fat content and the characteristics of the tested meat, the simple linear correlations between them were computed. the ital. j. food sci., vol. 31, 2019 94 coefficients of simple correlation between imf and processing properties of meat, subjective visual and tactile evaluation, and its colour are shown in table 5. the correlations between imf and carcass weight, backfat thickness and loin eye area were also computed. the study confirmed a significant negative relationship between imf and meat acidity ph45 (p<0.05) and ph48h, wbsf, water content (p <0.01), visual colour intensity (p <0.05), proportion of red a* and yellow b* colour, its saturation c*, and h° hue, as well as muscle pigment content (p<0.01). table 5. correlation coefficients between intramuscular fat content and other variables in meat. imf ph45 -0.242* ph48h -0.292** whc 0.140 thermal drip 0.032 wbsf -0.493** water content -0.779** total protein content -0.106 visual colour intensity -0.259* marbling 0.908** firmness 0.305** l*48 value represents lightness 0.147 a*48 proportion of red -0.302** b*48 proportion of yellow -0.584** c*48 saturation -0.473** ho48 hue angle -0.560** muscle pigment -0.422** hot carcass weight 0.353** av. backfat thickness 0.195 loin eye area -0.006 (*) statistical significance at p<0.05, (**) statistical significance at p<0.01. intramuscular fat content was not related to carcass fat and meat content; only carcass weight was positively correlated with imf (p<0.01), which may be explained by the impact of age on the higher animal body weight at slaughter. pietruszka et al. (2015) reported a significant positive correlation between imf content and average backfat thickness (r=0.31, p<0.05), and a negative correlation with the loin eye area (r=-0.64, p<0.01); however, in contrast to this study, no significant relationship between imf and ph45 was demonstrated. the high positive correlation between the imf content and marbling (r=0.908) and the negative relationship with the shear force (r=-0.493) (p<0.01) obtained in this study are in line with the findings of li et al. (2013), which also showed lower shear force of meat containing higher imf content. ital. j. food sci., vol. 31, 2019 95 4. conclusions the meat obtained from the most popular polish crossbred pigs contained more intramuscular fat and was characterized by greater shear force, lower ph45 and ph48h, more marbling with lower water content, and a smaller proportion of yellow colour b*. in addition, it was characterized by a lower saturation of colour c*, its tone h°, and lower content of muscle pigment. acknowledgments this study was realized from statutory research funds bs-6/2014 assigned by the polish ministry of science and higher education. references alonso v., del mar campo m., provincial l., roncalés p. and beltrán j.a. 2010. effect of protein level in commercial diets on pork meat quality. meat sci. 85:7-14. andrés i.a., cava r., mayoral a.i., tejeda j.f., morcuende d. and ruiz j. 2001. oxidative stability and fatty acid composition of pig muscles as affected by rearing system, crossbreeding and metabolic type of muscle fibre. meat sci. 59:39-47. beattie v.e., weatherup r.n., moss b.w. and walker n. 1999. the effect of increasing carcass weight of finishing boars and gilts on joint composition and meat quality. meat sci. 52:205-211. blicharski t. and snopkiewicz m. 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fax: +90 2582963262 e-mail address: ezgio@pau.edu.tr abstract in this study, quality properties of six pasta samples, prepared by substituting semolina with smoked trout fillet powder (stfp), were investigated. the addition of stfp to the pasta formulation resulted in an increase in protein, fat and ash content, energy value, antioxidant activity, phenolic content, optimum cooking time, cooking loss, and a* and b* values, but a decrease in carbohydrate content, water absorption capacity, swelling index and l* value. furthermore, sem images revealed that addition of stfp leads to an increase in protein matrix around the starch molecules, and its addition in quantities up to 15% showed acceptable sensory scores. keywords: pasta enrichment, pasta quality, smoked trout fillet powder ital. j. food sci., vol. 31, 2019 111 1. introduction pasta is the most commonly consumed food product after bread among cereal products, and is traditionally produced from wheat semolina and water. the popularity of pasta is a result of its low cost, long shelf life, sensory characteristics and nutritional properties (fradique et al., 2013; kaur et al., 2013; goes et al., 2016). the food and drug administration and the world health organization both recognize pasta as a good vehicle for enrichment with nutrients (beleggia et al., 2011), and it is also known to be a rich source of complex carbohydrates and b vitamins, but as a poor source of protein and essential amino acids. this has led to many studies being carried out to enrich pasta with protein-rich ingredients such as meat, seafood (such as fishes and shrimp), legumes (such as chickpea, split pea, lentil, cowpea, mung bean and faba bean flour) (gallegosinfante et al., 2010; kadam and prabhasankar, 2012; lakshmi devi et al., 2013; liu et al., 2016; petitot et al., 2010; savita et al., 2013; wojtowicz and moscicki, 2014; wood, 2009; zhao et al., 2005). such enrichments can have an effect on certain qualities of pasta, such as color, sensory characteristics, texture and cooking parameters (mercier et al., 2011). fish provide several nutritional benefits, such as high quality proteins that contain essential amino acids, and are a good source of lipids, complex b vitamins and such minerals as phosphorus, magnesium, zinc and iron (goes et al., 2016). fish is usually consumed in its fresh form, but can also be consumed after being dried, salted or smoked. smoking techniques have been used as a preservation method for centuries. besides affecting the characteristic color and flavor of food, smoking processes also has antimicrobial and antioxidative effects that are known to extend the shelf life of foods (lingbeck et al., 2014). before marketing, smoked fish fillet is packed in standard weights. the edges of smoked fillets must be cut to meet a standard weight for packaging. these pieces that showed the same nutritional characteristics as the fish fillets, are discarded as production waste or are processed into low value-added products. such waste can be used in the manufacture of enriched, high value-added food products. the objective of this study is to investigate the chance of utilization of such fillet pieces and to examine the possibility of pasta nutritional value enhancement. it was also aimed to determine the changes in the cooking characteristics, antioxidant activity, microstructural properties, sensory properties and color attributes of pasta samples with the addition of smoked trout (oncorhynchus mykiss) fillet powder (stfp). 2. material and methods 2.1. raw materials smoked trout fillet pieces were dried in a cabinet dryer (yucebas machine analytical equipment industry, izmir, turkey) at 50°c until ˂10% moisture content was achieved. the airflow rate in the cabinet dryer was 0.2 m/s and the relative humidity of the air was in a range of 19–21 %. after drying, the samples were ground into powder of a ˂1000 µm particle size, and the stfp was then stored at -18 °c until use. wheat semolina (particle size ˂450 µm), deionized water and common salt were used in the preparation of each pasta sample. ital. j. food sci., vol. 31, 2019 112 2.2. pasta production the stfp was added to the pasta formulation in quantities of 5, 10, 15, 20 and 25. the formulations of the pasta samples are shown in table 1. the salt content of the stfp was found to be 6.3 %, and so the salt content was adjusted to 1.5 % in the pasta formulations. the ingredients were mixed in the kneading vessel of the laboratory pasta machine (dolly pasta machine, la monferrina, italy) at room temperature for 10 min to make the pasta dough, and the dough was then extruded using the pasta machine. a no. 28 die (6 mm wide, 0.85 mm thick, ptfe) was used to shape the dough, which was then cut into 10 cm lengths. the pasta samples were dried at room temperature until a ˂10% moisture content was reached (~20 h). table 1. formulations of pasta samples. ingredients stfp inclusion level 0% 5% 10% 15% 20% 25% wheat semolina (g) 100 95 90 85 80 75 stfp (g) 0 5 10 15 20 25 deionized water (ml) 34 34 36 39 39 41 salt (g) 1.5 1.2 0.9 0.5 0.2 0 the control sample (unenriched) and the enriched pasta samples were packaged in a moisture-proof material (pet+coex pa) and stored at room temperature for further analyses. 2.3. proximate composition analysis the crude protein content of the samples was determined using the dumas method (shea and watts, 1939) with a dumatherm analyzer (gerhardt gmbh & co. kg, königswinter, germany), while fat content, moisture content and ash content were measured using aoac (1990) methods. carbohydrate content was estimated by subtracting the moisture, protein, fat and ash content from 100% energy values were calculated using the following equation (souci et al., 2000); 𝐸𝑛𝑒𝑟𝑔𝑦 𝑣𝑎𝑙𝑢𝑒(𝑘𝑐𝑎𝑙/𝑔) = 𝐶𝑎𝑟𝑏𝑜ℎ𝑦𝑑𝑟𝑎𝑡𝑒𝑠×4 + 𝑃𝑟𝑜𝑡𝑒𝑖𝑛𝑠×4 + (𝐿𝑖𝑝𝑖𝑑𝑠×9 ) 2.4. total phenolic content and total antioxidant activity of pasta for the extraction of phenolics, 10 ml of aqueous methanol (70:30 v/v) was added to 1 g of the samples. after 10 min of sonication in an ultrasonic bath (e 60 h model, elma co., germany), the mixture was shaken in a mechanical shaker (wiseshake sho-1d, wertheim, germany) for 15 min at room temperature. at the end of the centrifugation (nf 1200 r, nuve, turkey) of mixture at 8500 g at 4°c for 20 min, supernatants were collected. the total phenolic content and antioxidant activity of the extracts were analyzed in duplicate. total phenolic content (tpc) analyses were carried out by the method of singleton et al. (1998), in which 1 ml of extract is poured into a test tube, after which, 5 ml of 10-fold ital. j. food sci., vol. 31, 2019 113 diluted folin-ciocalteu and 4 ml of na2co3 (7.5%) solutions were added. after 2 h incubation in the dark, the absorbance of the mixtures was measured at 760 nm against a reagent blank and standards using a spectrophotometer (pg-80 uv-vis spectrometer, pg instruments, united kingdom). the results were expressed as milligrams gallic acid equivalent (gae)/100 g wet basis. total antioxidant activity (taa) was measured using the 2.2-diphenyl-1-picrylhydrazyl (dpph) method according to thaipong, et al. (2006). twenty four mg of dpph was dissolved in 100 ml of methanol for prepared as a stock solution and then stored at -20 °c until use. the working dpph solution was prepared by mixing the stock solution with methanol to obtain an absorbance value of 1.1±0.02 at 515 nm. the extracts (150µl) were allowed to react with the working solution (2850 µl) for 24 h in the dark, after which the absorbance of the samples was measured at 515nm. the standard calibration curve was linear between 25 and 800 µm trolox, and the results were expressed in µmol trolox equivalent (te)/100 g wet basis. 2.5. pasta cooking tests 2.5.1 optimum cooking time the optimum cooking time (oct) of the samples was determined according to aacc (2000). briefly, 25 g of pasta was broken into 5 cm constant lengths and added into 300 ml of boiling distilled water. every 30 seconds, a piece of pasta was taken out and squeezed between two glass plates. the oct was defined as the time taken until the white center of the sample disappeared. 2.5.2 cooking loss for the determination of cooking loss, 10 g of pasta was cooked for the oct, after which the sample was rinsed with distilled water in a buhner funnel. cooking loss was measured by evaporating the cooking water and rinsing with water in a hot air oven at 105 °c. the residue was weighed and reported as a percentage of the uncooked pasta sample (tudorica et al., 2002). 2.5.3 water absorption capacity after cooking the 10 g pasta samples at oct, the water absorption capacity (wac) was calculated using the following equation (marti et al., 2013): 𝑊𝐴𝐶(%) = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑐𝑜𝑜𝑘𝑒𝑑 𝑝𝑎𝑠𝑡𝑎 − 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑢𝑛𝑐𝑜𝑜𝑘𝑒𝑑 𝑝𝑎𝑠𝑡𝑎 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑢𝑛𝑐𝑜𝑜𝑘𝑒𝑑 𝑝𝑎𝑠𝑡𝑎 ×100 2.5.4 swelling index the swelling index (si) of the samples was determined according to the cleary and brennan (2006). twenty-five g of pasta was cooked in 250 ml of boiling distilled water for oct, and then dried at 105°c. the si was expressed as: ital. j. food sci., vol. 31, 2019 114 𝑆𝐼 = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑐𝑜𝑜𝑘𝑒𝑑 𝑝𝑎𝑠𝑡𝑎 − 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑖𝑒𝑑 𝑝𝑎𝑠𝑡𝑎 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑖𝑒𝑑 𝑝𝑎𝑠𝑡𝑎 2.6. microstructure of raw pasta the pasta samples were freeze-dried (thermo savant modulyod-230, usa) for 8 h, and the freeze-dried samples were coated with gold. the microstructure of the surface of the raw pasta samples was visualized with scanning electron microscopy (sem) (fei quanta 250 feg, usa). 2.7. color measurement the surface color values of the cooked pasta were measured using a hunter lab miniscan xe colorimeter (hunter associates laboratory, reston, va). l*, a* and b* parameters were recorded, and the changes in color resulting from the addition stfp to the pasta formulation were determined according to a color differential index (∆e) that calculated the following equation; ∆𝐸 = ∆𝐿 ! + ∆𝑎 ! + ∆𝑏 ! where: ∆l* was calculated as l* sample-l*control; ∆a was calculated as a* sample-a* control; ∆b was calculated as b*sample-b*control according to the handbook of colour science (yamauchi, 1989), ∆e values describe visual color differences as follows: (0–0.5, trace difference); (0.5–1.5, slightly discernible; hard to detect with the human eye); (1.5–3.0, noticeable; detectable by trained people); (3.06.0, appreciable; detectable by ordinary people); (6.0–12.0, large; large difference in the same color group); (larger than 12; extreme; another color group). 2.8. sensory evaluation the pasta samples were cooked (100 g of pasta in 1 l of boiling water with 10 g salt) for oct and drained. pasta samples were presented individually on plastic trays to each panelist. the sensory evaluation was made by 44 panelists from pamukkale university, department of food engineering (29 females, 15 males; age range 20–50). the panelists scored each sample for color, odor, taste, texture and overall acceptability on a hedonic scale ranging from 1 (dislike extremely) to 7 (like extremely). the samples were labeled with randomly selected three-digit numerical codes. bread and water were given to the panelists for rinse their palates between samples. the final scores were calculated from the average of the 44 scores (aydin and gocmen, 2011; cardenas-hernandez et al., 2016). 2.9. statistical analysis all experiments were performed in duplicate. statistical analyses were made using spss 22.0 (ibm spss inc., chicago, il, usa) software. a duncan’s multiple range test was used and the levels were considered significantly different at p˂0.05. ital. j. food sci., vol. 31, 2019 115 3. results and discussion 3.1. raw materials the moisture, protein, fat, ash and carbohydrate content, and the energy value, taa, tpc and color values of wheat semolina and stfp are shown in table 2. table 2. nutritional and chemical properties of wheat semolina and stfp. wheat semolina stfp moisture (%) 13.41±0.07 7.61±0.45 protein (%) 10.91±0.02 72.49±0.82 fat (%) 1.79±0.37 14.29±0.74 ash (%) 0.90±0.03 5.03±0.22 carbohydrate (%) 73.00±0.40 0.59±0.31 energy value (kcal/100g) 351.69±1.71 420.92±4.57 total antioxidant activity (µmol te/100g) 2.20±0.10 10.06±0.39 total phenolic content (mg gae/100g) 33.30±0.46 51.22±0.91 hunter color values l* 85.61±0.08 53.33±1.06 a* 0.04±0.03 6.27±0.18 b* 18.45±0.08 27.40±0.52 stfp: smoked trout fillet powder. the moisture value of semolina was determined under the maximum limit (14.5%) of the turkish food codex (2002). in addition, the protein content of semolina was detected above the minimum protein content (10.5%) defined in the turkish food codex (2002). the results reveal that while stfp is a remarkable source of protein and fat, semolina is a good source of carbohydrate. it was determined that the use of fish powder in pasta production enhances the nutritional quality of enriched pasta. additionally, the energy values of semolina and stfp were determined as 351.69 kcal/100g and 420.92 kcal/100g, respectively. stfp has a higher energy value than semolina due to its high protein and fat content. furthermore, stfp demonstrates higher total antioxidant activity and total phenolic content than wheat semolina. the high level of antioxidant activity in the stfp can be attributed to the applied smoking process, in that smoke contains various phenolic compounds (goulas and kontominas, 2005; kjallstrand and petersson, 2001). the color measurement indicated that stfp has higher a* and b* values and lower l* values than wheat semolina. 3.2. proximate composition of samples the proximate composition of the control sample and the enriched samples are presented in table 3. moisture content was increased after the addition of stfp, although all results were found to be under the critical moisture value of 13% identified in turkish standard 1620 (2017). the stfp may have increased the moisture content of the samples due to the water holding capacity of stfp proteins during dough preparation (chin et al., 2012; zayas, 1997). ital. j. food sci., vol. 31, 2019 116 table 3. proximate composition of samples. stfp: smoked trout fillet powder. different superscript letters in columns indicate statistical differences (p˂0.05). the protein, fat and ash content of the samples increased with the addition of stfp. in particular, the protein content of the sample with the addition of 25% stfp was detected to be 1.9 times higher than the control sample due to the high protein content (72.49%) of stfp. the fat content of the control sample and 25% stfp-added sample were detected as 1.70% and 7.88% respectively. the control sample was significantly lower than the 10, 15, 20 and, 25 % stfp-added samples (p˂0.05). the ash content analysis revealed 1.33% ash content for the control sample and 2.29% for the 25% stfp-added sample. the ash content of the 25% and 20% stfp added samples were significantly higher than the rest of the groups (p˂0.05). the statistical analysis revealed that the control and stfp 5% samples were similar in this respect (p˃0.05). the carbohydrate ratio decreased significantly with the addition of stfp, while the energy value increased (p˂0.05). although the energy value was higher, it was not regarded as a negative outcome due to the higher fat and protein contents of the enriched samples. the results of the present study concur with those of liu et al. (2016) and chin et al. (2012). 3.3. total phenolic content and antioxidant activity traditionally, meat, fish and some other foods are subjected to smoking not only to give them a special taste, color and odor, but also to improve shelf life, based on the antioxidant and antibacterial properties of smoke (semanova et al., 2016; soares et al., 2016). the taa and tpc of the samples are shown in table 4. table 4. total antioxidant activity and phenolic content of samples sample total antioxidant activity (µm te/100g) total phenolic content (mg gae/100g) control 1.79±0.29b 35.93±1.75c stfp 5% 4.75±0.88a 36.36±1.90bc stfp 10% 4.76±0.92a 38.77±2.24abc stfp 15% 4.99±0.76a 38.83±0.99abc stfp 20% 5.10±1.11a 40.92±2.43ab stfp 25% 5.12±0.41a 42.21±0.30a stfp: smoked trout fillet powder. different superscript letters in columns indicate statistical differences (p˂0.05). sample moisture (%) protein (%) fat (%) ash (%) carbohydrate (%) energy value (kcal/100g) control 9.44±0.15b 13.87±0.28d 1.70±0.19d 1.33±0.03b 73.66±0.59a 365.39±0.47c stfp 5% 10.12±0.06ab 14.29±0.21d 3.61±0.73cd 1.39±0.17b 70.59±0.41a 371.96±4.08bc stfp 10% 10.32±0.26a 17.97±0.18cd 5.52±0.71bc 1.45±0.20b 64.74±0.96b 380.51±3.27ab stfp 15% 10.06±0.55ab 19.72±2.40bc 5.79±1.54ab 1.56±0.08b 62.87±4.41bc 382.48±5.84ab stfp 20% 10.15±0.18ab 23.27±2.93ab 7.22±0.42ab 2.20±0.04a 57.16±2.73cd 386.69±3.00a stfp 25% 10.44±0.45a 25.76±2.51a 7.88±0.88a 2.29±0.11a 53.63±2.19d 388.40±6.66a ital. j. food sci., vol. 31, 2019 117 taa and tpc were both detected in higher values in the stfp than in the semolina (table 2). so enriched samples had higher taa and tpc values than the control samples. the taa values of the samples ranged between 1.79 µm te/100g and 5.12 µm te/100g. the taa of the control sample was significantly lower than stfp-added samples (p˂0.05). the tpc of the samples increased significantly with the addition of stfp to the pasta formulation (p˂0.05), and it was determined that the tpc of the 25% stfp-added sample was 1.2 times higher than the control sample. previous studies have investigated the effects of smoking processes on certain foods, and similar results have been found. shaiban et al. (2006) investigated the effect of different types of woods used for smoking cheese, and found that smoked cheese showed higher total phenolic content and total antioxidant activity than non-smoked cheese. serot et al. (2004) investigated 10 major phenolic compounds in herring fillets smoked using different methods, and found that the sum of the content of 10 phenolic compounds in smoked fillets was strongly affected by the method used. 3.4. pasta cooking tests cooking characteristics are a strong indicator of pasta quality. the cooking characteristics of the pasta samples are shown in table 5. oct ranged from 9.50 to 11.00 minutes, and compared to the control sample, the oct showed an increasing trend with the addition of stfp. higher optimum cooking times have also been reported in meat-based pasta. kadam and prabhasankar (2012) reported that cooking time increased from 8.5 min (control sample) to 14.0 min with the addition of 30% shrimp meat. cooking time is related to the starch gelatinization and water penetration rate. water enter into the starch granule may be restricted by more complex protein networks, which may cause a delay in the start of the gelatinization process (liu et al., 2016). the swelling index of pasta is an indicator of the water absorbed by the proteins during cooking (gopalakrishnan et al., 2011). increasing the stfp ratio in the formulation caused the water absorption capacity and swelling index to decrease significantly (p˂0.05), which could be related to the lower water absorption capacity of the protein network in stfp than in the gluten network. previous studies into bran-enriched and fish powderenriched pasta have reported similar decreases in water absorption capacity and swelling index (aravind et al., 2012; desai et al., 2018; gatta et al., 2017). petitot et al. (2010) determined that pasta samples made with split pea flour and faba bean flour had lower water absorption capacities than the control samples. in contrast, baskaran et al. (2011) reported that pasta enriched with skimmed milk powder and whey protein concentrate had higher swelling index values than the control sample. table 5. cooking characteristics of the control and stfp-enriched pastas. sample optimum cooking time (min) water absorption capacity (%) cooking loss (%) swelling index control 9.50±0.01d 208.32±1.62a 5.87±0.04b 2.74±0.03a stfp 5% 9.75±0.35cd 204.76±3.21ab 7.50±0.01a 2.72±0.05a stfp 10% 10.00±0.01bcd 197.48±5.08bc 7.57±0.23a 2.62±0.07ab stfp 15% 10.50±0.71abc 197.80±2.84bc 7.82±0.69a 2.63±0.01ab stfp 20% 10.75±0.35ab 190.20±1.65cd 8.15±0.50a 2.55±0.04b stfp 25% 11.00±0.01a 182.85±2.53d 8.42±0.15a 2.51±0.08b stfp: smoked trout fillet powder. different superscript letters in columns indicate statistical differences (p˂0.05). ital. j. food sci., vol. 31, 2019 118 cooking loss is defined as the amount of solids lost into the cooking water of a sample cooked for oct (sozer et al., 2007). an analysis identified statistically similar levels of cooking loss in the enriched samples (p˃0.05), while the control sample was significantly different (p˂0.05). increasing the stfp ratio in the pasta formulation increases the level of cooking loss of the samples. the cooking loss values of the samples ranged between 5.87% and 8.42% all of which fall under the acceptable limit of 9% (aacc, 2000). an increase in the proportion of stfp in the formulation results in a decrease in the amount of semolinabased components, such as gluten, in pasta, which leads to a weakening of the gluten network. similarly, some studies on the enrichment of pasta with various ingredients have also shown an increase in the level of cooking loss (aravind et al., 2012; gallegosinfante et al., 2010; islas-rubio et al., 2014; sant’anna et al., 2014). the supplementation of different non-gluten flours in pasta formulation has been reported to dilute the gluten strength and weaken the pasta structure, which may increase the level of dry matter lost into the cooking water (gallegos-infante et al., 2010). in addition, it was stated that pasta had low cooking loss when dried at high temperature. pasqualone et al. (2016) reported that when a high-temperature drying program was adopted, cooking losses were ranged from 3.2 to 4.8 % in control pasta and in pasta samples enriched of lyophilized tomato matrix or with durum wheat bran extracts produced by supercritical carbon dioxide or ultrasound. 3.5. microstructure of raw samples sem images demonstrate the arrangement of the gluten network and starch in pasta (rajeswari et al., 2013). the surface microstructure of raw pasta samples is illustrated in fig 1. images of the control pasta sample show numerous starch granules that appear to vary in both size and shape (fig. 1a). the outer surface of the pasta appears to be coated with a thin protein film (fig. 1a), which has also been reported in previous studies (pasqualone et al., 2016; alireza sadeghi and bhagya, 2008; cunin et al., 1995). as dried pasta has a very limited water system, the starch and protein compete strongly for water during cooking. the less protein surrounding the starch granules, causes the faster starch swelling and gelatinization. (de noni and pagani, 2010).with the addition of stfp to the pasta formulation, the protein matrix around the starch molecules enhances, and the starch molecules are coated with a thicker protein network by the increase of stfp addition ratio (fig. 1b, 1c, 1d, 1e, 1f)., as also observed by alireza sadeghi and bhagya, (2008) and liu et al., (2016).this phenomenon also explained the extension of cooking time by the increasing addition ratio of stfp. 3.6. color measurement the color of pasta is very important quality parameter in terms of consumer preferences (carini et al., 2009). the color parameters of the enriched and control samples are shown in table 6. it can be noted that the l* value (lightness) decreases with the increasing supplementation levels of stfp. the l* values of the control and the 5% stfp-added sample were statically similar (p˃0.05), and significantly higher than the other enriched samples (p˂0.05). pasta samples enriched with stfp had a significantly higher a*(redness) and b*(yellowness) values than the control sample (p˂0.05). overall, the color values indicate that enriched samples have more redness (a*) and more yellowness (b*), but less lightness (l*) values than the control sample. the changes in color were found to be related to the original color of the stfp and the semolina (table 2). ital. j. food sci., vol. 31, 2019 119 a b c d e f figure 1. surface sem images of (a) control pasta, (b) 5% stfp pasta, (c) 10% stfp pasta, (d) 15% stfp pasta, (e) 20% stfp pasta, (f) 25 % stfp pasta. s: starch granules, p: protein network. magnification 2500x. these results were in close agreement with kadam and prabhasankar (2012), aydin and gocmen (2011), alireza sadeghi and bhagya (2008), who reported that samples supplemented with oat flour, shrimp meat and mustard protein isolate had lower l values and higher a and b values than the control sample due to the darker color of the enrichment materials than semolina. p s p s s s p p s p s p s p s p s s p p , p s s p p ital. j. food sci., vol. 31, 2019 120 the color differential index (∆e) was indicated the color changes between the control sample and the enriched samples. the ∆e values of the samples was increased with the addition of stfp to the pasta formulation. according to the handbook of colour science, 10%, 15% and, 20% stfp-added samples can be classified as “large difference in the same color group”, while the classification of the 5% stfp-added sample and the 25% stfpadded sample were “appreciable; detectable by ordinary people” and “extreme, another color” respectively. desai et al., (2018) reported that the incorporation of fish powder into pasta increased ∆e values, while khan et al. (2014) investigated the color changes in sorghum flour-enriched pasta, stating that all the ∆e values of the cooked enriched samples were greater than 12, being classified as “extreme, another color” by yamauchi (1989) in the handbook of colour science. table 6. color parameters of cooked pasta samples. sample l* a* b* ∆e control 68.01±0.45a -3.06±0.02f 14.81±1.10e stfp 5% 67.00±0,34a -2.30±0.10e 18.63±0.01d 4.04±1.05d stfp 10% 65.79±0.47b -1.55±0.10d 20.62±0.33c 6.40±0.66c stfp 15% 64.49±0.52c -0.42±0.27c 22.76±0.64b 9.09±0.30b stfp 20% 62.84±0.59d 0.45±0.13b 24.26±0.95ab 11.32±0.01a stfp 25% 61.87±0.35d 1.24±0.11a 25.25±0.66a 12.85±0.45a stfp: smoked trout fillet powder. different superscript letters in columns indicate statistical differences (p˂0.05). 3.7. sensory evaluation the sensory properties of the samples were evaluated based on color, odor, taste, texture and overall acceptability, and the results are shown in table 7. the control sample and the 5% stfp-enriched samples were found to be statistically similar in all parameters (p˃0.05). table 7. sensory properties of control and smoked trout fillet powder enriched pastas. sample color odor taste texture overall acceptability control 4.84±0.21a 4.86±0.22a 4.78±0.34a 5.01±0.31a 4.90±0.25a stfp 5% 4.98±0.03a 4.88±0.23a 4.77±0.27a 5.00±0.11a 4.86±0.26a stfp 10% 4.75±0.11a 4.50±0.06b 4.36±0.21ab 4.34±0.23ab 4.38±0.23ab stfp 15% 4.84±0.18a 4.17±0.01c 3.94±0.33bc 4.25±0.41ab 4.00±0.30bc stfp 20% 4.77±0.08a 4.19±0.03c 3.46±0.18c 3.84±0.23b 3.63±0.12c stfp 25% 4.48±0.44a 3.90±0.03d 3.48±0.08c 3.73±0.57b 3.56±0.33c stfp: smoked trout fillet powder. different superscript letters in columns indicate statistical differences (p˂0.05). no difference was identified between the color values of all samples (p˃0.05), while odor and taste values decreased significantly with the addition of stfp (p˂0.05). some of the panelists reported an excessive fish odor and taste in the 20% and 25% stfp-added ital. j. food sci., vol. 31, 2019 121 samples, and a decrease in texture values was detected with stfp supplementation. during cooking, it was observed that especially the 20% and 25% stfp-added pastas had a tendency to rupture, and the panelists also pointed out that these pastas were very easily ruptured. the addition of stfp, which causes gluten reduction and a weakening of the gluten network, might reduce the resistance of the pasta. it was determined that the overall acceptability values of all the samples scored above 3.5, which the midpoint in a 7point hedonic scale. this study follows on from the research by chin et al. (2012), who reported that the color, taste and overall acceptability scores of noodles decreased with the addition of surimi powder. on the other hand kadam and prabhasankar (2012), when supplementing pasta with shrimp meat, found from a sensory analyses that the addition of 20% shrimp meat had the best result in the overall score. liu et al. (2016) enriched pasta with beef emulsion, and the overall preference scores showed that a 30% beef emulsion-added sample was statically similar to a commercial pasta sample. 4. conclusions the consumption of fish is known to have many benefits to human health. it can be added to various foods as a good source of enrichment, due mainly to its high protein content. fish meat is considered to be a good source of enrichment for pasta. in the present study, it was determined that the addition of stfp increased the nutritional value of pasta, and also increased the antioxidant activity and phenolic content. the cooking analysis showed that the addition of stfp increased oct and cooking loss, and decreased wac and swelling index. as can be observed from the sem images, the addition of stfp to the pasta formulation leads to an increased protein matrix around the starch molecules. an increased l* value, and decreased a* and b* values have been found in the enriched-stfp samples due to the darker color of stfp than semolina. the sensory analysis revealed overall acceptability scores of all samples above 3.5, although the 20% and 25% stfpadded samples were reported by the panelists to have an excessive taste and odor of fish. the 20% and 25% stfp-added samples were also noted to rupture during cooking. it was determined that the addition of 15% stfp enhanced the nutritional value of pasta, and also had acceptable cooking quality and sensory characteristics. therefore, it can be concluded that fish pieces (production waste material) can be thought as pasta enrichment ingredients in case they are used in the mentioned concentration. acknowledgements this work was funded by pamukkale university, unit of scientific research projects, turkey (project no: 2014fbe052). references aacc. 2000. 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"handbook of colour science". japanese academy of colour science. tokyo, japan. zayas j.f. 1997. "functionality of proteins in food", springer, berlin, heidelberg. zhao y.h., manthey f.a., chang s.k.c., hou h.j. and yuan s.h. 2005. quality characteristics of spaghetti as affected by green and yellow pea , lentil , and chickpea flours. sens. nutr. qual. food 70(6):371-376. doi: doi.org/10.1111/j.1365-2621.2005.tb11458.x. paper received may 11, 2018 accepted august 29, 2018 ijfs#1375_bozza ital. j. food sci., vol. 31, 2019 652 paper new consumer targets towards a traditional spirit: the case of grappa in piedmont (northwest italy) v.m. merlino, s. massaglia*, d. borra and v. mantino department of agricultural, forest and food sciences, university of turin, largo paolo braccini 2, 10095 grugliasco, torino, italy *corresponding author: tel.: +39 0116708622; fax: +39 0112368622 email address: stefano.massaglia@unito.it abstract a choice experiment was conducted in piedmont, italy, to define purchasing preferences and behaviours of grappa consumers. a total of 667 individuals were interviewed at different points of grappa purchase/consumption. the most important attributes considered during grappa purchase by consumer were defined using the best-worst scaling methodology. the latent class analysis was employed to identify consumer clusters characterized by different grappa preferences and consumption styles. for piedmont consumers, grappa choice was related to previous experience, product knowledge and origin. conversely, consumers considered "alcohol content" and "packaging" the two least important factors to be considered during purchase. the lclass analysis allowed the identification of four clusters of grappa consumers that were described in function of socio-demographic variables. keywords: best-worst scaling, cluster analysis, choice factors, italian distillate, socio-demographic variables ital. j. food sci., vol. 31, 2019 653 1. introduction 1.1. grappa history: from illegal product to national symbol european union regulation 110/2008 recognises the intrinsic value of grappa, in particular as a liquor of unique geographical origin and as the only italian product derived from the distillation of pomace, according to the production regulations (ministry of agriculture, food and forestry, decree 5389, 2011). grappa is a traditional and historical italian alcoholic distillate that became a product to drink around 700, although the first methods of distillation date back to the seventh and sixth centuries bc (vaccarini and pillon, 2017; onofri and boatto, 2015). however, a precise date of the first grappa distillation process is still to be defined (antoninetti, 2011). originally, the pomace distillation for liquor production was considered an illegal operation (behrendt and behrendt, 2000) and the distillation process was usable only in medicine and applied sciences. this could explain the mystery that still surrounds the initial date of this liquor. various literature proclaims the official birth of the pomace distillation method for grappa production is attributed to jesuit studies dating back to the mid 1600s, which refined and improved the practice and tools for this production process1. from there on, grappa could be legally consumed as it became a symbol for intellectuals during the italian renaissance (mccracken, 1988), the period when the distillation process received the official academic recognition (antonietti, 2011). today, the social image of this product has evolved over the decades from a product consumed exclusively in local taverns or restaurants, from a defined target of consumers, in a liqueur to be enjoyed on several occasions by all (antonietti, 2011). grappa became a phenomenon linked to different social classes, consolidating its presence in northern italy, but opening up to the channel of mass distribution and advertising. however, grappa has suffered a sharp decrease in consumption in recent decades. in 1974, there were 39 million litres consumed in italy, then dropped to 21 million in 1999. consumption increased to 30 million by 2008 and then plunged back down to 23 million in 2017 (pigozzo, 2018; galletto and rossetto, 2005). in the national context, grappa showed a negative trend in production of 29% from 2006 to 2016, and a drop from 117,000 to 82,000 in anhydrous alcohol (assodistl, 2017). from 2017 grappa production increased recording a significant change in value (from 42.9 to 44.2 million euros, + 5.9%) with recorded quantity from 27,935 to 30,919 anhydrous alcohol (federvini, 2017). nielsen data from 2018 revealed a new positive trend in the italian market (+ 0.8% in volume) (federvini, 2018). the exportation remained however limited: the quantities of product exported in 2017 fell by 12% compared to the previous year (from 28.9 to 25.3 in thousand anhydrous alcohols). the most important markets are germany, which imported bottled grappa for a value of almost 19 million euro, followed by switzerland with 6.4 million euro federvini, 2018). 1.2. research background and aims in several literature researches the consumption occasions, as well as the cultural and socio-demographic variables were analysed as drivers for consumer preferences and behaviour definition regarding agro-food and oenological products (dal vecchio et al., 2018; carsana and jolibert, 2017; dekhili et al., 2011; merlino et al., 2017; mu et al., 2017; schäufele and hamm, 2017; bruwer et al., 2017 borra and ital. j. food sci., vol. 31, 2019 654 tarantola, 2015). even in the case of spirits such as gin, whisky and vodka, researchers have investigated consumer preferences in recent years, including the characteristics and behaviour during purchasing (clarke and koptev, 1992; dubinina and alieva, 2015; guy et al., 1989; gauthier and mazières, 2013). however, in the case of grappa, few studies in literature investigate the consumer preferences and buying behaviours for this product. in general, consumer preferences about liquors, as well as, for grappa, has been evolved meaning that purchases were no longer based exclusively on objective attributes (such as price or economic availability), but also considering emotional and irrational attributes during the product selection (i.e. brand, place of purchase, link with the territory, packaging, certifications, indications or designations of origin) (lock et al., 2006). moreover, unlike food and other beverages, the nutritional and health aspects of alcoholic beverages lose considerable importance for consumer in favour of other more important aspects (aroma, colour, place of production/origin). in addition to preferences, also the characteristics of the typical prototype of grappa consumer, mainly low-middle income and exclusively male, have evolved during the '80s, thanks to the enhancement programs and marketing strategies applied by italian producers, restaurateurs and bars. some quality aspects of grappa have been modified and improved to allow the consumer to choose from a wider range of products. for example, producers improved the alcohol content, which has led to a "softer spirit" with an alcohol content of about 40% instead of the usual 50% or 60% alc. this latter aspect has expanded the opportunities for conventional consumers to enjoy grappa on several occasions (wilson, 2009; antonietti, 2011), transforming this distillate into a more relevant product even for non-experts not used to higher alcohol levels and with a strong flavour. to date, grappa has a new identity and is appreciated by a greater demographic variety, even by young people (demoskopea, 2003). to this end, the aim of our research, conducted in northwest italy, was to investigate on the purchasing preferences and behaviour of grappa's consumers, also to understand if young people were included in the consumers target. at this purpose, the sampling phase was also addressed towards places typically frequented by young individuals. the best-worst scaling (bw) methodology (finn and louviere, 1992; marley and louviere, 2005), already used to analyse consumer preferences in the agri-food sector (lockshin et al., 2015; merlino et al., 2018; girgenti et al., 2016), as well as in other areas (rezaei et al., 2016), was employed in our study. the paper results are structured as follows: firstly, the grappa consumers characteristics and habits are described; then, is defined the importance of 12 quality attributes of grappa expressed by consumers in the decision-making phase; finally, the cluster analysis results and the differences in terms of socio-demographic variables between the consumer targets are analysed. 2. materials and methods a total of 667 individuals were involved in the research to assess grappa's consumer preferences. face-to-face interviews were conducted through a paper questionnaire from march to june 2018 at various grappa purchasing or consumption points (bars, wineries, supermarkets) distributed in cuneo city and in the metropolitan areas of turin (northwest italy) and on the campus of the university of turin. the paper questionnaire consisted of three sections: the first contained questions to investigate the socio-demographic characteristics of respondents; the second part focused ital. j. food sci., vol. 31, 2019 655 on the survey of grappa consumption habits and styles; the third section focused on the analysis of consumer preferences on the twelve attributes of grappa selected for the bestworst scaling application. socio-demographic characteristics of the involved sample are described in table 1. table 1. respondents characteristics in terms of gender, age, number of family components, educational level, annual average income and employment (%). sample (n= 667) gender educational level male 53% primary school 1% female 47% lower secondary school 11% age upper secondary school 71% 18-24 years 40% master's degree 17% 25-35 years 21% 36-50 years 21% annual average income 51-65 years 13% <25,000 €/year 29% over 65 5% 25,000 35, 000 €/year 45% > 35,000 €/year 26% family composition employment one member 11% student 37% two members 13% employed 38% three members 26% self-employed 11% four members 40% retired 7% more than 4 members 10% unemployed 4% housewife 3% 2.1. best-worst scaling methodology and cluster analysis the bw method model helps to identify the most and least relevant attributes for consumers within a designed set of features that describe and characterize a product (seccia et al., 2012). data collection takes place through interviews, during which respondents are asked to indicate between a defined number of attributes organized in sets (from three to five elements), the one that is the most important (best) and the least important (worst) during the purchase and/or selection of the product. the bw method has several advantages compared to traditional methods of discrete choice. firstly, it appears to be easier to understand by consumers and quick to fill out. there is also greater consistency in the options chosen by the consumer, especially when these are the contrary or extreme. finally, the bw method helps to obtain a good amount of information about consumer preferences through a classification of the same (marley and louviere, 2005). using this methodology, respondents evaluate all the attributes present in each set as if they were pairs, consequently choosing the most representative pair for each set that corresponds to the maximum difference pair. for this reason, the bw method is also known as a maximum difference scale (maxdiff) that provides a more efficient evaluation of the coupled data, thereby obliging the respondent to make a discriminating choice ital. j. food sci., vol. 31, 2019 656 between attributes compared to traditional systems of comparison (auger et al, 2007; cohen, 2003; finn and louviere, 1992; marley and louviere, 2005). according to orme (2005) it is advisable to have between four or five attributes per set because a greater number would determine a minimum increase in the information obtained. in our research, 12 grappa attributes have been selected and organized in nine sets of four attributes each, allowing single item to appear three times in the questionnaire. this was feasible by using the appropriate software (sawtooth software v.2.0.2, orem, ut, usa; http://www.sawtoothsoftware.com/). the sawtooth software has also created four different versions of the questionnaire in order to minimize the differences (both subjective and cultural) in the way of personal classification and create greater diversification in the presentation of items. the level of importance for each grappa attribute was evaluated by the average raw bestworst score analysis (casini et al., 2009; cohen, 2003; goodman et al., 2005). this score is a numerical value calculates dividing the bw score (number of best minus number of worst) to the number of respondents and the frequency with which each attribute appears in the set of choices. the confidence limit used for bw score calculation was set equal to 95% and the standard deviation was used to evaluate sample variability. the 12 attributes of grappa selected through a literature research and used in the bw analysis are reported in table 2. table 2. the twelve attributes of grappa selected and used for the best-worst analysis. grappa attributes references taste/flavour diamantidou et al., 2018; louw and lambrechts, 2012; violoni, 2008; finzi, 2007; ubigli, 2001; ubigli and castino, 1992; da porto, 2012; apostolopoulou et al., 2005. packaging/bottle format diamantidou et al., 2018; violoni, 2008; da porto, 2012. brand demoskopea, 2003; prentice and handsjuk, 2016; carsana and jolibert, 2017; louw and lambrechts, 2012. price finzi, 2007; galletto and rossetto, 2005; mu et al., 2017; da porto, 2012. grapevine diamantidou et al., 2018; borsa et al., 2008; galletto and rossetto, 2005; da porto, 2012. aging diamantidou et al., 2018; violoni, 2009; soufleros et al., 2004. information on the label violoni, 2009; da porto, 2012. origin/place of production chandra et al., 2017; schäufele and hamm, 2017; da porto, 2012; louw and lambrechts, 2012. adding aromas/flavour diamantidou et al., 2018; violoni, 2009; asioli et al., 2017; dubinina and alieva, 2015; da porto, 2012; louw and lambrechts, 2012. i know/already tried muller et al., 2010; harrington, 2007. it was recommended to me agnoli et al., 2011; harrington, 2007. alcohol content diamantidou et al., 2018; dubinina and alieva, 2015; pigozzo, 2018; louw and lambrechts, 2012; apostolopoulou et al., 2005. the latent class (lclass) analysis was used to divide the whole sample of individuals into homogeneous groups (clusters) according to their purchasing behaviour and expressed preferences. the sawtooth software automatically created five clusters, each of which is characterized by different values of the following indicators: the akaike constant information criterion (caic), the log-likelihood (ll) and the bayesian information ital. j. food sci., vol. 31, 2019 657 criterion (bic). in our research, the most appropriate segmentation was chosen as the one with the lowest bic value, which, in our case, was corresponding to four clusters, also in accordance with merlino et al. (2018) and dekhili et al. (2011) (table 3). table 3. values of bic of the lclass analysis results: the lowest value was used for clusters number choice. groups replications bic 2 5 5845.89 3 4 5726.20 4 2 5639.28 5 4 5640.28 3. results 3.1. the consumers of grappa: socio-demographic characteristics the 31% of the total sample (n=207 individuals) declared to consume grappa. among those who have declared that they do not consume grappa "non-consumers", the main reason was linked to the organoleptic aspect ("i do not like it"), leaving out other reasons such as health or religious aspects. the sub-sample of grappa consumers was represented mainly by men (77%) with respect to women (23%), and by individuals belonging to the age groups of the youngest (under 35 years), while a minority of over 65 consumed grappa (table 4). the distribution of the genders proportion in the different age groups is described in fig. 1. table 4. age ranges of grappa consumers. age ranges consumer sample (n=207) 18-24 38% 25-35 22% 36-50 21% 51-65 14% over 65 5% from data reported in fig. 1 emerged a majority of men among grappa consumers (77%) compared to women (23%). however, when analysing the distribution between women and men in the different age groups, a majority of women among the youngest consumers emerge, while an evident numerical superiority of men is highlighted in the other considered age ranges. in the over 65 consumers women represented the 40%. grappa consumers differed in level of education and occupational characteristics compared to the whole sample (table 5). regarding the family composition, 43% of consumers represented four-member families, 22% with three members, 16% with more ital. j. food sci., vol. 31, 2019 658 than two children, 13% belonged to families with two members and only 6% to singleparent families. figure 1. genders distribution among the considered age groups and in the sub-sample of grappa consumer. table 5. educational level, family composition, annual average income and employment of grappa consumers. grappa consumers (n = 207) educational level family composition primary school 0% one member 15% lower secondary school 8% two members 14% upper secondary school 68% three members 23% master's degree 23% four members 34% more than 4 members 13% annual average income employment <25,000 €/year 24% student 51% 25,000 35, 000 €/year 44% employed 24% > 35,000 €/year 42% self-employed 14% retired 7% unemployed 7% housewife 0% households with several members (four and five members) had a prevalence of affirmative answers to the question, "someone in the family drinks grappa”. on the contrary, by analysing the family composition of respondents who said they did not consume grappa, the data revealed that 33% belonged to single member families and 20% to two member families. ital. j. food sci., vol. 31, 2019 659 3.2. habits and styles of grappa consumption the majority of grappa consumers involved in this study (69%) stated that they drink grappa occasionally, while the 16% consume grappa once or twice a month and 12% once or more a week. only 2% of consumers drink this liquor every day (table 6). the study found that majority of consumers drink grappa habitually inside their home (35%), followed by 20% of individuals who consumed it in restaurants/pizzerias, special events (17%), in pubs and bars (17%) and at social tastings (10%). only the 2% answered to drank grappa in unspecified occasions. different results emerged from the analysis of answers about the frequency of grappa purchase (the bottle); among consumers, only 21% of those declared to never buy grappa throughout the year, while 42% said they bought it occasionally. the 16% of consumers who bought grappa two to four times a year, while 14% of respondents bought annually. those who bought several times a year represented 8% of the whole sample. about the reasons to purchase grappa, the main expressed by respondents was the convivial consumption with friends (44%), followed by purchase as a gift (31%), and for personal consumption (25%). in the latter case, there was a clear difference in behaviour between products in the case of personal consumption, highlighting a clear prevalence of men in this category. table 6. frequency of grappa consumption and purchase declared by interviewees. 3.3. the importance of grappa attributes the numbers of selected best and worst and the bw average raw score for single grappa attributes for piedmont consumers are reported in table 7. the most important attributes considered during the decision-making process of grappa choice and purchase were "i know it/already tried" with an average raw score of 2.13, followed by "it was recommended to me" (average raw score equal to 1.68), "brand" (raw score equal to 1.71), and "origin/place of production" with a raw score of 1.08. on the contrary, among the attributes that not influence grappa purchase there was the "alcohol content", with the lowest average raw score (-2.30), followed by the "packaging/bottle format" (raw score of -1.71), by "addition of aromas/flavours" and by "information on the label" with average raw score values, respectively, of -1.21 and -0.79. the attributes considered least important by consumer at the time of purchase all present negative raw scores. frequency of grappa consumption frequency of grappa (bottle) purchase everyday 2% several times a year 8% more than 2 times a week 2% 2-4 times a year 16% 1-2 times a week 10% annually 14% 1-2 times monthly 16% occasionally 42% occasionally 69% never 21% ital. j. food sci., vol. 31, 2019 660 3.4. latent class analysis of grappa consumers clusters of consumers defined in function of their expressed preferences for grappa attributes are described in table 8. the same table shows the dimensions of the different clusters, as well as the bw raw score values for each attribute that define their importance within the single consumer group. each of the four clusters has been named according to their expressed preference and perception defined in function of the importance given to the individual factors by consumers. table 7. number of best, number of worst and b-w average raw score for each attributes of grappa. attribute number of selected best number of selected worst b-w average raw score taste/flavour 83 102 -0.40 packaging/bottle format 55 181 -1.71 brand 162 30 1.71 price 75 105 -0.40 grapevine 101 86 0.31 aging 109 87 -0.10 information on the label 66 130 -0.79 origin/place of production 146 48 1.08 adding aromas/flavour 51 150 -1.21 i know/already tried 188 51 2.13 it was recommended to me 163 43 1.69 alcohol content 25 211 -2.30 table 8. average bw raw score for the four clusters representative of considered consumers sample: nonexpert, price sensitive, experts and quality sensitive. clusters nonexpert price sensitive experts quality sensitive cluster dimension 27.4% 15.9% 30.7% 26.0% attributes average raw score taste/flavour 1.37 3.60 -0.01 0.96 packaging/bottle format 0.73 2.84 -0.36 -0.20 make/brand 2.01 2.16 2.70 3.02 price 1.59 3.51 0.15 0.64 grapevine -0.41 3.31 1.87 2.29 aging -0.51 1.72 2.23 2.48 information on the label 1.18 1.19 0.09 1.79 origin/place of production 0.99 3.62 2.21 2.43 adding aromas/flavour 0.83 0.36 0.64 0.97 i know/already tried 2.74 1.68 4.13 1.36 it was recommended to me 2.75 2.85 3.45 0.78 alcohol content 0.00 0.00 0.00 0.00 ital. j. food sci., vol. 31, 2019 661 important differences in attributes preferences evaluation between the four groups of consumers emerged from cluster analysis. the main group (30.7% of the entire sample), called experts, was represented by respondents who considered their consolidated knowledge of grappa, the recommendations on the product, as well as the brand, as the most important factors in the purchasing process. in some aspects, the experts had similarities with the nonexpert group (27.4%). in fact, even for these two types of consumers, the attributes "i know/already tried", "it was recommended to me" and "brand" were the most important factors for the product choice. however, these latter individuals differed from the experts on the least important attributes. for experts consumers, packaging and taste/flavour were irrelevant for grappa selection, whereas the nonexpert considered qualitative aspects such as the grape variety and the aging of the product unimportant factors in the grappa selection. the third group was named of quality sensitive and represented the 26% of the entire sample. respondents that considered discriminant during grappa purchase the grape variety, the product aging and the origin/ place of production, characterized this cluster. among the four groups, quality sensitive was the only one that emphasized the intrinsic qualities of the product, with high raw score values for the information on the label. this group also considered aspects such as price and packaging irrelevant in the decisionmaking process. price sensitive individuals (15.9% of the entire sample) represented the fourth cluster. respondents who considered the price the most important attribute during grappa purchase, followed by the origin/place of production and the taste/flavour, characterized this group. the respondents’ profiles were also analysed considering the consumers sociodemographic characteristics. in particular, if the nonexpert group was characterized by a slight majority of women (58%) compared to men (42%), the price sensitive and expert clusters presented the same distribution with a minority of men (32%) compared to women (68%), while the quality sensitive clusters were represented by 83% of men and only 17% of women. the percentages of individuals divided by age group in the different clusters are shown in table 9. in general, the majority of young people emerge among the expert drinkers of grappa, while the nonexperts were mainly more mature individuals. table 9. age differentiation of respondents belonging to the selected clusters. cluster age ranges (years old) 18-24 25-35 36-50 51-65 >65 price sensitive 41% 0% 27% 23% 9% nonexpert 46% 23% 15% 12% 3% expert 21% 0% 25% 23% 12% quality sensitive 38% 9% 26% 27% 0% ital. j. food sci., vol. 31, 2019 662 figure 2. clusters characterization in function of the annual average income. fig. 2 highlights clusters characterization in function of the annual average income range of respondents. the price sensitive, expert and quality sensitive groups were mostly represented by consumers with a mean yearly income between 25,000 to 40,000 €/year. in general, from our results the intermediate income level emerged as widespread among all the considered clusters, constituting at least one third of each cluster in all cases, excluding the nonexpert group. the clusters composition was then analysed based on the expressed level of grappa knowledge (low, medium, high) declared during the interviews (table 10). the behaviour of the four groups was analysed according to their willingness to spend (in euros) for a bottle of grappa purchase (table 11). table 10. level of grappa knowledge expressed by the four defined clusters of consumers. cluster level of knowledge low medium high nonexpert 89% 11% / quality sensitive 54% 46% / expert 65% 27% 8% price sensitive 70% 21% 9% table 11. willingness to pay (euros ranges) for a bottle of grappa purchase declared by the different clusters of consumers (nonexpert, price sensitive, expert and quality sensitive). cluster price ranges less than 10€ 10-20 € 21-40 € 41-60€ more than € 61 nonexpert 16% 59% 22% 3% 0% price sensitive 0% 33% 48% 10% 5% expert 0% 48% 42% 0% 6% quality sensitive 3% 35% 55% 3% 3% ital. j. food sci., vol. 31, 2019 663 on average, the sample was willing to pay between 21 and 40 euros for grappa purchase. only nonexpert consumers were willing to spend less than 10 euros on grappa, while price sensitive consumers showed a clear price sensitivity by focusing on product value for money. 4. discussion this study analysed grappa consumer characteristics, buying and consumption habits in piedmont. in particular, the preference degree of 12 grappa attributes was measured by dividing the considered sample into four clusters of individuals. subjects with similar behaviours, attitudes and preferences towards grappa product characterized each cluster. the socio-demographic analysis helped with describing the sample of grappa consumers who represented the 31% of the total of interviewed. these individuals were especially men and young subjects under 35 years. this latter result underlines how young people, in particular women, are joining the target group of grappa consumers, and further confirming the evolution of grappa from a product associated with a specific category of consumers to a product for all individuals (antonietti, 2011)2. the probable correlation between the personal consumption of grappa and a more or less habitual drinking within the family emerged from our analysis. although focused only on grappa, this latter result is also confirmed by literary research conducted on the overall consumption of alcohol, confirming the influence of alcohol consumption by family components on the individual behaviour. both scafato et al. (2004) and istat (2016) clearly show how the influence of consumption patterns of parents and, in particular, the head of the family, is a key element in determining behaviour, especially in the younger age group. concerning the consumption habits, the profiled consumer in our study tends to be an occasional consumer who buys grappa infrequently in association with special events, perhaps for convivial consumption with friends or as a gift. these attitudes show differences, however, depending on the gender, revealing a greater propensity of women to buy grappa on specific occasions to taste it in company, in opposition to men respondents that had a greater tendency to buy it for personal consumption. in general, however, while other alcoholic beverages (wine or beer) are consumed more easily due to their alcoholic range, as shown in literature, emerged the tendency of grappa consumers to choose this spirit carefully to taste it on special occasions, without almost never abusing it3. the exploration of grappa consumption habits highlighted how consumers prefer their home as a place to taste this traditional distillate. grappa is often and traditionally drunk after meals, such as lunch or dinner, served as a “digestive” or “to correct a cup of espresso” (antonietti, 2011). these latter results underline that the social factor is extremely related both to the reasons for buying and to the occasions of consumption of grappa. in the case of buying grappa as a gift, the two genders seem to be very similar during purchasing behaviour. the identification of grappa as a gift to give to friends or relatives symbolizes a recognition in this product by the consumer of an added and symbolic value, appreciable as a gift. the results belonging to the best-worst scaling methodology application highlighted as the choice of grappa is driven by the memories arising from a previous tasting experience, a known product and a specific brand, putting the product quality aspects in second place. although grappa producers are focusing their marketing strategies on product enhancing and differentiating through the improvement of aspects such packaging originality, ital. j. food sci., vol. 31, 2019 664 elegance and communicative power4, in our study both packaging and bottle size were among the less important attributes considered during grappa purchase. the lesser importance of the attribute "adding aromas/flavour", on the contrary, is not a surprising result because also literature have confirmed the tendency of grappa consumers to prefer the pure product version, appreciating the taste, aroma, transparency, in particular the “white" colour of grappa (koch, 2008; bellone, 2011; onofri and boatto, 2015, onofri and koch, 2006). clusters analysis allowed the entire sample division into four groups of consumers characterized by homogeneous features and behavioural preferences. the most represented group was that of experts, composed mainly by young men, with a mediumhigh income bracket and a willingness to pay an intermediate price for the purchase of grappa. these consumers reflect the trend of the entire sample by relying on their previous experiences and product image during purchasing. in this case, the experience is accompanied by a greater product knowledge, paying less attention to attributes such as the packaging and the taste/flavour of grappa. the second most represented group was of nonexpert. it is interesting to note that the groups of experts and nonexpert have given importance to the same attributes in the grappa choice in relation to product knowledge and recommendations provided by others. however, the same preferences expressed probably need to be interpreted differently. in fact, for experts, product knowledge and experience give the subject confidence in their own knowledge, which gives them certainty in the choice and reassurance during the purchase phase. the nonexpert group was represented by consumers who rely on their previous experience during the choice, perhaps to make a safe choice of product, and also, not having sufficient knowledge of the product, rely on recommendations for fear of making a mistake in buying. in this group, mainly represented by young people, age has played a key role as it leads to greater inexperience. their willingness to pay for a bottle of grappa was associated with a low-medium price, perhaps in correlation with their lower income bracket. finally, this group was characterized by a higher percentage of women. the group of quality sensitive were mainly mature men who showed greater attention to the intrinsic factors of grappa at the time of purchase, such as aging, the grape variety and geographical origin. probably these consumers are connoisseurs who do not give importance either to price or packaging. quality sensitive probably belong to those expert consumers, enhancers of the gustatory quality of the product that conceive the tasting of grappa as a ritual or a moment to appreciate all the unique connotations conferred by the aging process of a specific producer. these consumers were probably connoisseurs for whom the brand image becomes important again because it is associated with an intrinsic quality of the product, without paying attention to aspects not directly related to the product itself, such as price and packaging. in this category there was a strong prevalence of men, who were willing to pay an average price for the purchase of grappa, as well as characterized by a good knowledge of the product. price sensitive, the last cluster by size, considered price and taste/flavour important attributes during their decision-making process of grappa purchase. their willingness to pay for a bottle of grappa has never fallen below 10 € indicating a lack of confidence in products too cheap and looking for a good value for money. in this case, knowledge plays a fundamental role because no consumer of this group relied on past experience or even on the image of the product because otherwise it doesn’t guarantee quality. in addition, ital. j. food sci., vol. 31, 2019 665 there has been an increase in the average age for this category along with an increase in the middle-income bracket, which is in line with the positive perception of the attribute "price". however, these consumers were looking for the best value for money, also paying attention to the production areas. the combination of price, origin and taste can probably be associated with a consumer who assesses the price as an indicator of superior quality of the grappa product. on the other hand, the information on the label and the addition of flavours/aromas are irrelevant for this category. 5. conclusions this study identified four different profiles of grappa consumers: despite the differentiation in term of preferences and socio-demographical variables, in general, a good part of involved consumers stated to have a medium-low level of knowledge towards grappa, except for a few passionate connoisseurs. the heterogeneity between clusters preferences defines the importance of studying consumer attitudes, especially for products linked to tradition, but whose consumption is limited to special occasions or convivial moments such as grappa. consumer preferences must be interpreted and seen by the producers as a tool and an indicator to deal with marketing and production decisions. grappa has enormous potential; the last twenty years has witnessed the production sector undergoing an evolution that has affected the product, the structure and the organisation of the production chain. the goal of reaching new and younger targets is being realized; however, the intrinsic potential of this product could allow it to expand even further products, opportunities and ways of consumption. the operators of the sector, in collaboration with the points of sale and consumption of grappa, could envisage this objective. a limitation of this research lies in the characteristic of the sample in terms of circumscription in a single geographical area (single region in the northwest of italy), and in the sampling method that could represent a limitation in this type of research. in the future, it could be considered to expand to more areas at the national level and to involve individuals interviewed only in point of grappa consumption. in addition, it would be interesting in future work to assess the level of knowledge of grappa in other areas at the international level and to provide a tool of enhancement to companies with the intention of expanding their market. notes 1www.istitutograppa.org/ita/cosa-e-la-grappa.html/ available at 1/01/2019 2www.istitutograppa.org/ita/stampa/la-grappa-tra-passato-presente-e-futuro-da-vinitaly-buone-prospettive-per-il distillato-di-bandiera.html available at 10/12/2018 3www.grappa.com/ita/grappa_dettaglio.php/titolo=chi_beve_la_grappa/idpagina=10/idnews=1/idsezione=6 available at 10/12/2018 4www.anag.it/premio-design-il-vestito-della-grappa-alla-grappa-clessidra-ma-vince-tutto-il-mondo-della-distillazione/ available at 12/12/2018 ital. j. food sci., vol. 31, 2019 666 references agnoli l., begalli d. and capitello r. 2011. how do values influence the consumer utility for wine and the other alcoholic beverages? a focus on generation y preferences and consumption situations. vineyard data quantification society european association of wine economist, angers france, 18-21 may 2011. apostolopoulou a.a., flouros a. i., demertzis p.g. and akrida-demertzi k., 2005. differences in concentration of principal volatile constituents in traditional greek distillates. food control 16(2):157-164. asioli d., aschemann-witzel j., caputo v., vecchio r., annunziata a., næs t. and varela p. 2017. making sense of the “clean label” trends: a review of consumer food choice behavior and discussion of industry implications. food research international 99:58-71. assodistil. 2017. il settore in cifre. available online at www.assodistil.it/il-mondo-della-distillazione/il-settore-incifre.html. available at 9/10/2018. auger p., devinney t.m. and louviere j.j. 2007. using best–worst scaling methodology to investigate consumer ethical beliefs across countries. journal of business ethics 70(3):299-326. behrendt a. and behrendt b. 2000. grappa. a guide to the best, ny: abbeville press. bellone c. 2011. la grappa nel canale della grande distribuzione in italia. borra d. and tarantola m. 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[accessed 21/11/2018]. paper received october 10, 2018 accepted march 18, 2019 ijfs#1096_bozza ital. j. food sci., vol. 30, 2018 200 review relation between innovation and sustainability in the agro-food system h. el bilali centre for development research (cdr), university of natural resources and life sciences (boku), borkowskigasse 5, 1190 vienna, austria corresponding author: tel: +43 14765416920; fax: +43 14765416909 e-mail address: hamid.elbilali@boku.ac.at abstract this review paper explores the complexity of relation between innovation and sustainability and relates it to the agro-food arena. many scholars argue that meeting the sustainable development goals (sdgs) requires major transformation in modes of innovation. as for the agro-food system, relationship between innovation and sustainability is far from straightforward. innovation (especially technical one) provides a fertile ground for alternative agro-food movements to criticize the over-industrialization of the food system. however, it seems that it is not about questioning innovation tout court, but about what type of innovation (see, sustainable innovation) should be promoted to foster transition towards sustainable food systems. keywords: social innovation, sustainability transitions, sustainable agriculture, sustainable food systems, sustainable innovation, technical innovation ital. j. food sci., vol. 30, 2018 201 1. introduction innovation is rather an ambivalent term and that may explain the existence of different understandings of what innovation means. in fact, innovation has been defined in many different ways to the point that there is somehow a ‘lack of definitional clarity’ (shaver, 2016). oecd and eurostat (2005) describe innovation as “the implementation of a new or significantly improved product (good or service), or process, a new marketing method, or a new organisational method in business practices, workplace organisation or external relations”. innovation refers to a complex phenomenon, involving the production, diffusion and translation of scientific or technical knowledge into new products, techniques, services (menrad and feigl, 2007). according to sterrenberg et al. (2013:62), “innovation is the creation of better or more effective products, processes, services, technologies, or ideas that are accepted by markets, governments, and society. innovation differs from invention in that innovation refers to the use of a new idea or method, whereas invention refers more directly to the creation of the idea or method itself”. this definition clearly shows that there are different types of innovation, with different degrees of application in the agro-food sector. there are also broader understandings of innovation. according to steps centre (2010:1), innovation means “new ways of doing things. this includes not only science and technology, but – crucially – the related array of new ideas, institutions, practices, behaviours and social relations that shape scientific and technological patterns, purposes, applications and outcomes”. innovation concept is strongly linked to that of knowledge, which is fundamental to move towards sustainable practices (grin et al., 2010). knowledge plays an important role in transitions to sustainable food systems. therefore, it is important to pay attention to the different types of knowledge (information, skills, judgment and wisdom) that are needed in different situations (loconto, 2016). however, knowledge, as well as innovation, needed to make transition is often contested and inconclusive (batie, 2008; levin et al., 2012; peters and pierre, 2014). the literature contains many categorisations of innovation along many different dimensions. according to stummer et al. (2010), innovations can be categorized according to innovation type (product, service, process, market), dimension (objective or subjective), scope of change (radical, incremental, reapplied), or how innovation was created (closed or open). the oecd and eurostat (2005) distinguish product, process, marketing and organisational innovations. agricultural innovation as well as innovation in agri-food can be classified using the same categories (avermaete et al., 2004; avolio et al., 2014). schumpeter (1934, 1942) is often identified as the first to feature innovation as a central driver of the economy and to reject neoclassical economics’ idea of a static equilibrium. his idea that the process of innovation “incessantly revolutionises the economic structure from within, incessantly destroying the old one, incessantly creating a new one” (schumpeter, 1942:83) continues to be influential to this day. however, schumpeter and his followers employed the so-called ‘linear model’ in which innovation begins with an invention, is developed into a commercially viable technology in a firm, and is then diffused into the market place (twomey and gaziulusoy, 2014). a consequence of this model was a strong prioritisation of research and development (r&d) and the entrepreneur as the driver of innovation. this is sometimes referred to as the technologyor supply-push perspective of innovation. an alternative perspective put forward in the 1950s and 60s, but still within the linear model approach, was that demand for products and services is more important in stimulating innovation activity and is known as the demand-pull perspective (schmookler, 1966). in the last decades there has been a shift from an innovation concept centred on research to innovation as a result of interactions among several actors that establish diverse ital. j. food sci., vol. 30, 2018 202 networks and linkages (world bank, 2006) in an innovation system. in fact, over the last fifty years, a more nuanced and richer picture of the innovation has emerged, with a wider set of implications for those hoping to assist, shape or direct innovation process. modern innovation theory has moved towards the recognition that innovation is a joint activity involving a large number of actors with different interests, perceptions, capabilities and roles (twomey and gaziulusoy, 2014). it also reshaped the relation between innovation and science (tyfield, 2011). an interesting further development was the recognition of the importance of users (firms and individual consumers) in the innovation process (von hippel, 2005; bogers et al., 2010). according to osburg (2013), innovation theory has seen constant change of its focus over the last decades: concept of newness (1950s), management theory (1960s), demand side (1970s), process innovation (1980s), service innovations (1990s), and, more recently, open innovation (chesbrough, 2003a) and social innovation (van de ven et al., 2008). appreciation of the importance of actor networks is a key idea in modern innovation field. in the mid-1980s the concept of ‘innovation systems’ (freeman, 1995; hekkert et al., 2007; jacobsson and bergek, 2010) was introduced. innovation systems (is) theory is a heuristic framework that starts from the basis that innovation occurs in the context of an entire system. furthermore, there have been efforts towards integrating innovation systems approach and the socio-technical transitions (markard and truffer, 2008). the socio-technical transition approach (kemp, 1994; geels, 2005; rotmans et al., 2000) is an umbrella term that includes, among others, the multi-level perspective on sociotechnical transitions (mlp). the mlp approach differs in focus and scope from the is approach; mlp is conceived in a societal context that is broader than the innovation systems approach (geels, 2005). a central theme of mlp is the recognition of the coevolutionary development of technologies, institutions and social and economic subsystems. mlp is particularly powerful in understanding the complex interplay of different forces at the macro-, mesoand micro-level in creating disruptive change (geels, 2010; geels, 2011). the modern theory of innovation provides a number of concepts and insights similar to that of transition (twomey and gaziulusoy, 2014; tyfield, 2011). the common term ‘transition’ is often used interchangeably with the term ‘systems innovation’, either at the technology system or society-wide level. kemp and rotmans (2005), however, argue that “for the purposes of managing change processes to sustainability it is useful to use the concept of a ‘transition’ rather than system innovation” since it brings into focus the new state, the path towards the end state, the transition problems and the wide range of internal and external developments which shape the outcome. the concept of “transitions” was first coined by alex de tocqueville in the 19th century (coenen-huther, 1996). the term was also utilized in other research areas, such as evolutionary biology, demography, and studies on power relations (marody, 1996). in the 1990s, the ‘transition’ concept was introduced within socio–technical research. in the latter, ‘transitions’ initially referred to large-scale transformations within society or important subsystems, during which the structure of the societal system fundamentally changes (rotmans et al., 2001). more recently, the definition has been refined in such a way by loorbach and rotmans (2010) that the concept now stands for “a fundamental change in structure (e.g. organizations, institutions), culture (e.g. norms, behaviour) and practices (e.g. routines, skills)”. according to sterrenberg et al. (2013:9), radical systems innovations or transitions involve “innovations that are directed to redesigning entire systems of practices and provisions, instead of individual products or processes”. transitions efforts have often borrowed from different strands of research and disciplines, resulting in a myriad of approaches towards understanding and exercising transitions (lachman, ital. j. food sci., vol. 30, 2018 203 2013). overviews of transition theories and approaches can be found in geels (2005), olsthoorn and wieczorek (2006), grin et al. (2010), and markard et al. (2012). the momentum generated by the diffusion of the term ‘sustainable development’ (wced, 1987) spurred an interest in research on transitions towards sustainable futures (markard et al., 2012; lachman, 2013; falcone, 2014). ambiguity, complexity, interconnectedness and multidimensionality of sustainability problems imply that incremental changes are no more sufficient and there is a need for transformative change at the systems level (strn, 2010). embracing the goal of transition towards sustainable systems, the notion of ‘sustainability transition’ was coined (geels, 2011; kemp and van lente, 2011; lachman, 2013). markard et al. (2012:956) defined sustainability transitions as “long-term, multi-dimensional and fundamental transformation processes through which established socio-technical systems shift to more sustainable modes of production and consumption”. sustainability transitions are needed also in the agro-food arena to move towards sustainable food systems. according to the high level panel of experts on food security and nutrition (hlpe, 2014), “a sustainable food system (sfs) is a food system that delivers food security and nutrition for all in such a way that the economic, social and environmental bases to generate food security and nutrition for future generations are not compromised”. this definition clearly shows the strong linkage between food system sustainability and long-term food security. the international panel of experts on sustainable food systems (ipes-food, 2015) pointed out that a multi-directional flow of knowledge between the worlds of science, policy and practice is needed to foster a genuine transformation of food systems, which is necessary to make transition towards sustainability. this is urgently required, among others, because food systems are complex ‘social-ecological’ systems that require different sources of knowledge to be combined. therefore, there is a need for a real food-related knowledge revolution to overcome persistent lock-ins and path dependencies. transition will most likely not depend on one or even a small number of technological innovations, but is likely to arise from a constellation of mutually interacting systems of innovations (twomey and gaziulusoy, 2014). this is particularly true in the case of food system where social innovations seem also important. according to hinrichs (2014), social and organizational innovations are as central to sustainability transitions in food systems as any particular innovative technology. social innovation will likely play an important role in transitions to sustainability in agriculture and food sector that may not be primarily technology-driven (darnhofer, 2015) and the transition to sustainable food systems requires complex and holistic change processes in which social innovation plays as big a role as technological innovation (ipes-food, 2015). the review paper aims to analyse innovation narratives in sustainability literature. in particular, the paper explores the complexity of relation between innovation and sustainability with a particular focus on agro-food systems. the paper is structured as follows. in section 2, i explore the complex relation between innovation and sustainability (cf. sustainable development) by analysing, among others, references to innovation in the outcomes of the main conferences on sustainable development as well as in the sustainable development goals (sdgs). this section also introduces the concept of sustainability-oriented innovation (soi) with particular reference to sustainable innovation and eco-innovation as a way to combine innovation and sustainability. in section 3, i analyse the relation between innovation and sustainability in the agro-food arena and i highlight harmony and conflict areas. sustainable intensification is taken as an example to show diversity of perspectives, agendas and visions regarding sustainable agriculture. the section also analyses attitude of some alternative agro-food movements (e.g. organic agriculture, agro-ecology, food sovereignty, slow food) towards innovation. ital. j. food sci., vol. 30, 2018 204 2. innovation and sustainability: exploring multifaceted linkages the relation between innovation and sustainability can be analysed at least in two different ways: innovation as a driver of sustainability (role of innovation in achieving sustainable development) or sustainability as a driver of innovation (sustainability as a new paradigm and guiding concept for innovation). innovation, science and technology have essential roles to play in meeting the interlinked global environmental, social, and economic challenges of environmental sustainability, poverty reduction, social justice and climate change (steps centre, 2010; un, 2012). in fact, innovation is seen as a route to economic growth and competitive economy as well as to propose effective solutions to real problems such as poverty and environmental challenges (steps centre, 2010). the international assessment of agricultural knowledge, science and technology for development (iaastd, 2009) highlights that knowledge, science and technology (akst) is crucial to address different sustainable development issues such as food insecurity and poverty. it sheds also light on the fact that the scope of agricultural knowledge goes beyond the narrow confines of science and technology (s&t) and encompasses other types of relevant knowledge that is held by agricultural producers, consumers and end-users. the contribution of innovation to sustainability is highlighted in many strategic and policy documents dealing with sustainable development such as the outcomes of the recent world conferences of rio, johannesburg and rio+20 (box 1). innovation was also addressed recently in the context of the 2030 agenda for sustainable development and sustainable development goals (sdgs) (box 2). according to leach et al. (2012:2), delivering sdgs requires a radically new approach to innovation. they add that “what is now needed is nothing short of major transformation—not only in our policies and technologies, but in our modes of innovation themselves—to enable us to navigate turbulence and meet sdgs that respect the safe operating space”. sustainability is considered nowadays a driver for innovation especially in the private sector (e.g. nidumolu et al., 2009). many companies are seizing the strategic opportunities in innovation for sustainability. in fact, sustainability, environmentalism, and corporate social responsibility (csr) have become during the last decades buzzwords among multinational corporations, agribusinesses included, with a risk of ‘green-washing’ (e.g. munshi and kurian, 2005). many private firms see nowadays sustainability as a key factor for their competitiveness and understand that innovation will be crucial factor in developing sustainability. sustainability is often understood as the voluntary integration of environmental, economic and social concerns in firm operations (vilanova and dettoni, 2011). kaskinen et al. (2013) suggest that there are fundamentally three ways of incorporating sustainability into companies; risk aversion strategy (use of certificates and standards as a reaction to an external critique), cost-effectiveness strategy (cost savings through a smart and effective use of resources), and differentiation strategy (using sustainability to distinguish company’s offering on the market). as put by dearing (2000:2), the operational and commercial challenges for companies are “[…] to learn to treat sustainable development as a framework for innovation and to use and extend established management principles to make this framework operational and effective”. ital. j. food sci., vol. 30, 2018 205 box 1. innovation in the outcome documents of world conferences on sustainable development. in ‘our common future’, the final report of the world commission on environment and development (wced, 1987) – that, among others, mainstreamed the concept of sustainable development – there is quite a number of references to innovation. the document is rather critical towards innovation and calls for reorienting technology and managing risk by enhancing capacity for technological innovation in developing countries and adapting recent innovations to their needs. it also calls for broadening the scope of innovation beyond product and process innovations. our common future emphasizes the need to blend traditional and modern technologies and to promote collaborative learning in agriculture: “researchers must learn from and develop the innovations of farmers and not just the reverse” (p. 116). this is a clear stance against linear innovation model. it should be highlighted that much of the focus of our common future is on technological innovation, whereas there is no reference to social innovation; the document even reports that traditional social systems and community control over agricultural practices “may have limited the acceptance and diffusion of technical innovations” (p. 44). interestingly, the first reference to innovation in agenda 21, the rio declaration on environment and development (un, 1992), was in its chapter on changing consumption patterns where it highlighted the multiple sources of innovation “peoples' organizations, women's groups and nongovernmental organizations are important sources of innovation” (p. 15). this broader scope of innovation is confirmed in chapter on conservation of biological diversity that calls for promoting “the wider application of the knowledge, innovations and practices of indigenous and local communities” (p. 150). besides ‘technological innovation’, there is also a clear reference to ‘social innovation’ (p. 129) as well as ‘informal innovations’ (p. 156), and ‘indigenous innovations’ (p. 319). innovation is considered as important to prevent pollution and control environmental degradation. however, agenda 21 also highlighted the importance of assessing the relationship between innovation and development as well as effects of innovation. there is also an entire chapter dedicated to science for sustainable development. in the plan of implementation of the world summit on sustainable development 2002 (un, 2002) as well as in the report of the summit (un, 2002a) there is only one reference to innovation in relation to recognition of the rights of local and indigenous communities as holders of traditional knowledge, innovations and practices regarding biodiversity. the future we want, outcome document of the united nations conference on sustainable development 2012 (un, 2012a), includes also a few references to innovation. in its preamble, it calls for continued and strengthened international cooperation in the area of innovation to achieve sustainable development. it also recognizes the critical role of promoting innovation especially in developing countries and invites governments to create enabling frameworks that foster environmentally sound innovation, including in support of green economy. in its framework for action, it emphasizes the importance of investments in scientific and technological innovation in job creation. once again, the traditional knowledge and innovations of indigenous peoples in relation to biodiversity are recognized. further references to innovation are in the context of education and finance. ital. j. food sci., vol. 30, 2018 206 according to szekely and strebel (2012), ‘strategic innovation for sustainability’ – which focuses on the use of innovation to improve performance in environmental, economic and social dimensions of sustainable development entails improvements that are not only technological/technical but also in business models, thinking, operating procedures and practices, processes, and systems. they consider that types of innovation for sustainability ranges from incremental innovations in products and services or ecodesign (e.g. innovations that focus on improvement in eco-efficiency through reduction in resource inputs such as energy, materials, wastes and emissions), to radical innovations in value chains and processes, to ‘game-changing systemic innovation’. radical transformation of supply chains aims to take more account of the impacts of a company’s products and operations, including environmental (e.g. raw materials sourcing, end-oflife), economic (e.g. competitiveness) and social (e.g. labour conditions) issues. examples of ‘game-changing innovation’ include collaborative consumption (e.g. car sharing and sharing economy in general), that has been enabled by information technology, as well as social entrepreneurship (e.g. micro-credit to the poor). this reminds of the concept of box 2. innovation in the 2030 agenda for development sustainable and sdgs. the general assembly of the united nations adopted on september 25th, 2015, a set of 17 goals (and 169 targets) as part of the 2030 agenda for sustainable development (un, 2015). the only sdg where innovation is explicitly mentioned is sdg9 ‘build resilient infrastructure, promote inclusive and sustainable industrialization and foster innovation’. there are only few references to innovation in the adopted agenda. innovation is considered in the agenda preamble especially regarding its potential in medicine and energy sectors as well as in sustainable urban development; interestingly no reference to agriculture here. moreover, there was no reference to innovation either in relation to the targets of sdg2 ‘end hunger, achieve food security and improved nutrition, and promote sustainable agriculture’. only in the case of sdg8 ‘promote sustained, inclusive and sustainable economic growth, full and productive employment and decent work for all’, technological upgrading and innovation are considered essential to achieve higher levels of economic productivity and there is a call to promote development-oriented policies that support, inter alia, creativity and innovation. innovation is further mentioned two times in targets of sdg9 in reference to upgrading technological capabilities of industrial sectors by, inter alia, encouraging innovation especially in developing countries. nevertheless, the part of the document that abounds with references to innovation is that related to sdg17 “strengthen the means of implementation and revitalize the global partnership for sustainable development” especially in sections dealing with technology (see, access to science, technology and innovation; innovation capacitybuilding mechanism for least developed countries), and means of implementation and the global partnership (see, addis ababa action agenda, which addresses also systemic issues in science, technology and innovation, and established a technology facilitation mechanism tfm; private business activity, investment and innovation). it seems, anyway, that there is a recognition of, and maybe also concerns about, the role of technology and innovation in sustainable development and that may explain the establishment of many mechanisms for follow-up of this issue during the implementation of the 2030 agenda. in fact, tfm that involves representatives of member states, civil society, the private sector and the scientific community – is composed of a united nations inter-agency task team on science, technology and innovation for the sdgs; a collaborative multistakeholder forum on science, technology and innovation for the sdgs; and an online platform to share information on existing science, technology and innovation initiatives. ital. j. food sci., vol. 30, 2018 207 ‘open innovation’ (e.g. chesbrough, 2003; christensen et al., 2005) that calls for a more open approach towards knowledge management and dissemination to assure a wider access to, and consequently use of, knowledge and innovation. open innovation concept stresses innovation and knowledge as public goods (e.g. stiglitz, 2007) to which a wide range of stakeholders should have access. open innovation is the opposite of closed innovation. processes of closed innovation focus on in-house development of innovation before their dissemination to external stakeholders. on the contrary, open innovation focuses on “[...] the use of purposive inflows and outflows of knowledge to accelerate innovation” (chesbrough, 2003). however, finding a balance between reward (for innovation and creativity) and accessibility remains one of the fundamental challenges in science, technology and innovation ecosystems. there is a growing emphasis on the concepts of ‘responsible’, ‘sustainable’, ‘social’ and ‘ecological’ innovation (table 1). table 1. some definitions of concepts of sustainable innovation, eco-innovation and social innovation. concept definition source sustainable innovation sustainable innovation is a process where sustainability considerations (environmental, social, financial) are integrated into company systems from idea generation through to research and development (r&d) and commercialisation. this applies to products, services and technologies, as well as new business and organisation models. charter and clark, 2007 creating new or improved products, services, technologies, processes and management techniques that produce environmental or social benefits along with economic value. chonkova, 2015 sustainability-driven innovation the creation of new market space, products and services or processes driven by social, environmental or sustainability issues. keeble et al., 2005 eco-innovation the process of developing new products, processes or services which provide customer and business value but significantly decrease environmental impact. fussler and james, 1996 eco-innovation is the production, assimilation or exploitation of a product, production process, service or management or business method that is novel to the organisation (developing or adopting it) and which results, throughout its life cycle, in a reduction of environmental risk, pollution and other negative impacts of resources use (including energy use) compared to relevant alternatives. kemp and pearson, 2008 the production, assimilation or exploitation of a novelty in products, production processes, services or in management and business methods, which aims, throughout its lifecycle, to prevent or substantially reduce environmental risk, pollution and other negative impacts of resource use (including energy). ec 2008 in carrillohermosilla et al., 2010 social innovation social innovations ‘are new solutions (products, services, models, markets, processes etc.) that simultaneously meet a social need (more effectively than existing solutions) and lead to new or improved capabilities and relationships and better use of assets and resources. caulier-grice et al., 2012 social innovations are new ideas that meet social needs, create social relationships and form new collaborations. these innovations can be products, services or models addressing unmet needs more effectively. ec, 2017 innovative activities and services that are motivated by the goal of meeting a social need and are predominantly developed and diffused through organizations whose primary purposes are social. mulgan et al., 2007 ital. j. food sci., vol. 30, 2018 208 responsible innovations (ri) address so-called ‘grand challenges’ of our time, such as climate change (ec, 2013), but they are also associated with a range of socio-ethical issues. the network for business sustainability (2012) identified multiple definitions relating to ‘sustainability-oriented innovation’ (soi): eco-innovation, ecological innovation, environmental innovation, frugal innovation, green innovation, green product innovation, inclusive innovation and social innovation. sustainable innovation means paying attention to ecological integrity along social values diversity, promoting fairer und wider distribution of innovation benefits, encouraging plural innovation pathways, fostering inclusive and participatory governance of innovation processes (steps centre, 2010). soi is differentiated from conventional innovation in its purpose and direction as it adds environmental and social consideration to economic profit (bos-brouwers, 2010). key drivers of sustainable innovation include environmental and resource risks, sustainable consumption and production (scp) policies, product environmental regulation and other product policy initiatives as well as market and financial drivers (charter and clark, 2007). also for chonkova (2015), drivers of sustainable innovation include compliance with existing regulation or anticipating future regulations, cost savings by improving resource efficiency as well as social and supply chain pressures. meanwhile, according to nidumolu et al. (2009) to become sustainable, companies should go through five distinct stages of change: viewing compliance as opportunity, making value chains sustainable, designing sustainable products and services, developing new business models and creating next-practice platforms. sustainable innovation is widely recognised as a critical dimension of sustainable development as well as sustainable consumption and production (scp), sustainable food systems included. in fact, the crucial importance of sustainable innovation in these contexts has been recognised since the 1980s and was reinforced since the 1990s not only by united nations (un) but also in the european union (eu). however, sustainable innovation has remained mainly peripheral. nevertheless, the subject is now rapidly moving to centre stage to meet sustainable development challenges of a growing population. in fact, the urgency of adopting sustainable innovation is nowadays recognised as a fundamental step towards a sustainable future (charter and clark, 2007). for instance, the european commission (ec) issued in 2016 two strategic notes dealing with innovation and sustainability namely ‘opportunity now: europe's mission to innovate’ (epsc, 2016) and ‘sustainability now! a european vision for sustainability’ (epsc, 2016a). gerhardt and hubbert (2009) distinguish between conventional innovation (characterised by low sustainability, both environmental and social), green innovation (based on natural resources use and with positive or neutral environmental impact), social innovation (that contributes to social well-being and is accessible by consumers in emerging and developing countries) and sustainable innovation, that’s to say addresses the triple bottom line (elkington, 1997) i.e. is environmentally, socially and economically sustainable. sustainable innovation covers the spectrum of levels of innovation from incremental to radical. stevels (1997) defined four levels of innovation in the context of environmental improvement; from incremental, re-design or green limits, functional or product alternatives, to systems design. according to the network for business sustainability (2012, 2012a), firms can adopt different pathways to become sustainable. these range from ‘operational optimization’ (small incremental changes to improve eco-efficiency) to ‘systems building’ (radical and disruptive changes that have a positive societal impact) through ‘organizational transformation’ (new products, services or business models). a concept similar to green innovation, and to a certain extent also sustainable innovation, is that of eco-innovation (e.g. kemp and foxon, 2007; charter and clark, 2007; ital. j. food sci., vol. 30, 2018 209 reid and miedzinski, 2008; carrillo-hermosilla et al., 2010). however, charter and clark (2007) pointed out that although the two terms, sustainable innovation and eco-innovation, are often used interchangeably, sustainable innovation embraces all dimensions of sustainability (environmental, economic, social/ethical) while eco-innovation addresses mainly environmental and economic dimensions. andersen (2005) distinguishes the following five categories of eco-innovations: add-on innovations, integrated innovations, eco-efficient technological system innovations, eco-efficient organizational system innovations, and general purpose eco-efficient innovations. ecoinnovation can be analysed in terms of its target (product, process, marketing method, organisation, institution), mechanism (modification, re-design, alternatives, creation) and impact (effect on the environment). systemic changes, such as creation and alternatives, generally generate higher environmental benefits (oecd, 2009). the drivers for ecoinnovation include cost reduction, increasing market share, pressure from regulation or communities, improving technical efficiency, green ethos, profits from commercialisation, and improving the company image (kemp and foxon, 2007). many companies as well as a number of governments use eco-innovation to describe their contributions to sustainable development. for instance, eco-innovation is considered to support the lisbon strategy for competitiveness and economic growth in the european union (eu) while preventing or substantially reducing negative impacts on the environment, pollution and improving resource efficiency (oecd, 2009; ec, 2012). the lisbon strategy called for focusing on innovation and sustainable development and was, thus, a clear example of focus on sustainable innovation. this tendency to focus on sustainable innovation in the eu was reaffirmed in the declaration on the ‘strategy for smart, sustainable and inclusive growth’ that should drive the eu until 2020. other key international organisms (e.g. oecd), un agencies (e.g. ilo, wto) as well as the world economic forum advocated similar approaches (vilanova and dettoni, 2011). in fact, according to oecd (2008), eco-innovation is considered to have promise for improving environmental conditions without compromising economic growth. while ecoinnovation has focused mainly on environmental technologies, there is a tendency to broaden the concept scope in order to accommodate more societal concerns (rennings, 2000; meti, 2007). therefore, the overarching concept of eco-innovation is seen as providing vision for pursuing sustainable development. a further concept related to sustainable innovation is that of social innovation (e.g. nicholls and murdock, 2012; caulier-grice et al., 2012; osburg and schmidpeter, 2013), that deals with the integration of environmental and social issues. as put by osburg (2013), social innovation is about adding the social element to innovation or the applied theory of innovation with addition of a relevant and significant social component. according to chonkova (2015), social innovation can be both business oriented (product, process, organisational, marketing) and/or socially oriented (i.e. social innovation). social innovations are considered good not only for the economy but also for society (caulier-grice et al., 2012) as they engage with social problems in a way that is more efficient, just, effective or sustainable than existing solutions (phills et al., 2008). nevertheless, social innovations are not value neutral but rather socially and politically constructed, and context dependent (caulier-grice et al., 2012). according to osburg (2013), open innovation is a must for social innovation, that cannot work with closed innovation processes, as solving current problems in today’s societies requires a constant collaboration across sectors and between different categories of stakeholders. caulier-grice et al. (2012) identified eight common features of social innovation: crosssectoral, open and collaborative, grassroots and bottom-up, pro-sumption (see, productionconsumption) and co-production, mutualism, creating new roles and relationships, better use of assets and resources, and developing assets and capabilities. ital. j. food sci., vol. 30, 2018 210 in a manifesto on innovation, sustainability, development, steps centre (2010) called for a radical shift in how we think about and perform innovation towards a greater respect for and integration of cultural variety, democratic accountability and regional diversity. moving towards innovation for sustainability and sustainable development means nothing less than a radical change in the whole innovation process including agenda setting, capacity building, governance, monitoring, evaluation accountability as well as funding. for that, three arrays of questions related to direction, distribution and diversity, should be addressed (table 2). these three issues are interrelated. for instance, direction matters also because it shapes innovation benefits, risks and costs distribution. meanwhile, the appraisal of innovation directions needs to take into account also benefits distribution and to address social equity and justice issues. furthermore, taking seriously direction and distribution questions, means pursuing deliberately a diversity of innovation pathways to accommodate different needs and aspirations including those of marginalised and poor groups such as small-scale farmers. this, in turn, implies paying attention not only to technical dimension of innovation but also social and organisational ones (steps centre, 2010). these questions are not only still actual but also particularly relevant in agro-food systems. table 2. arrays of questions regarding innovation for sustainable development. issue questions to be addressed technical, social and political directions for change what is innovation for? which kinds of innovation, along which pathways? and towards what goals? distribution who is innovation for? whose innovation counts? who gains and who loses? diversity what and how many kinds of innovation do we need to address any particular challenge? source: steps centre (2010:9). 3. complex and multifaceted relation between innovation and agro-food sustainability innovation has become a key issue in the discussion about the relation between agriculture and sustainability (e.g. fao, 2012; eip-agri, 2013; fao, 2013; ipes-food, 2015; global harvest initiative, 2016). in general, there is a broad consensus on the critical role of innovation to make agriculture not only more competitive but also sustainable. in fact, agricultural innovation is considered vital for meeting the challenges of agriculture development, adapting to climate change and achieving food security (iaastd, 2009; iica, 2014; ec, 2016; unctad, 2017). innovations and modern techniques can strengthen food system resilience, improve resource efficiency in agriculture, and secure social equity thus contributing to the achievement of sustainable food security (hlpe, 2017). the european union has placed emphasis upon innovation as a key element in achieving transformation towards sustainable agriculture (hermans et al., 2010; dwyer, 2013). agricultural research and development (r&d) has been shown to very profitable (alston et al., 2000; rao et al., 2012) and to improve agricultural development, economic growth, and poverty reduction (iaastd, 2009). however, assessing innovation ital. j. food sci., vol. 30, 2018 211 in agriculture only through investment in agricultural r&d shows clearly that the linear model of innovation is still dominant in official arenas and, especially, when it comes to innovation statistics. nevertheless, iaastd (2009) highlights that “there is ample evidence available from the literature that akst investments have contributed significantly to organizational and institutional innovations in the form of methods, tools development, capacity strengthening, and understanding how institutes interact with each other in achieving developmental goals” (p. 516). the high level panel of experts on food security and nutrition (hlpe, 2017) identified in a recent note knowledge and technology as critical emerging issues for food security and nutrition. as there are diverging views on the suitability of different innovations and technologies to improve food security in a sustainable way in different contexts and for different kinds of users – for instance, in the context of small-scale agriculture and agrifood supply chains (e.g. peano et al., 2015; wettasinha, 2016), hlpe (2017) recommended to assess all innovations and technologies against their long-term environmental, economic and social impacts. such an assessment should take into consideration not only technical sustainability and economic profitability but also environmental friendliness and social justice in each use context (e.g. dunmade, 2002; kriesemer and virchow, 2012). the relation between innovation and sustainability (including sustainability transitions) in agro-food system is more complicated than in other systems and sectors. although more recent sustainability transitions research has stressed that important sustainability innovations can be social rather than technological (seyfang and smith, 2007; kirwan et al., 2013), research on food systems change has long favoured a different vocabulary of civic initiatives, community development projects and social movements to reference what sustainability transitions researchers present as manifestations of ‘grassroots innovations’ for sustainable development (hinrichs, 2014). it is widely admitted nowadays that to meet sustainability challenges, more attention should be paid to social innovations, grassroots innovation actors and processes (leach et al., 2012; moulaert, 2013; smith and seyfang, 2013; loconto et al., 2017). similarly, iaastd (2009), suggests that future agricultural innovation needs to address not only simple technological and technical issues but also social ones and to innovate in scales of thinking and action in order to contribute more effectively in addressing pressing challenges such as climate change and food security. likewise, innovation in rural development is widely understood, especially in the european union, in terms of social innovation (i.e. encouraging collective learning cultures, networks, interactions) and cultural innovation (i.e. improving rural context) rather than in the narrow sense of technological innovation (dargan and shucksmith, 2008; dwyer et al., 2012; hermans et al., 2010). this broader understanding of innovation in agriculture is nowadays widespread predominantly in developed countries such as those of the european union, as clearly stated by its standing committee on agricultural research (scar): “innovation is not restricted to a technical or technological dimension. it increasingly concerns strategy, marketing, organization, management and design” (scar-eu, 2012). innovations imply different directions of development, not all of which are sustainable, and which should be subject to democratic debate (strn, 2010). critically inclined research on agricultural and food systems change has generally viewed capital-intensive technologies as contributing to the vast restructuring of food and agriculture and ‘sustaining the unsustainable’ (buttel, 2006), especially referring to genetically modified (gm) crops. because some technologies have abetted industrialization, consolidation, and global neo-liberalization of food and agriculture, technology may be categorically dismissed by some scholars and food system actors as a potentially productive analytical entry point for work on sustainability transitions in food and agriculture (hinrichs, ital. j. food sci., vol. 30, 2018 212 2014). in fact, innovation and technology in agriculture may also negatively affect the environment and rural livelihoods and that may explain increasing mistrust in certain institutionalized forms and fields of science (millstone and van zwanenberg, 2000) such as genetics. management of collective rights and intellectual property rights ipr (cf. fields of big data and genetics) is particularly problematic and challenging in the agricultural sector (hlpe, 2017). in this regard, biotechnologies raise many ethical concerns as highlighted by the european group on ethics in science and new technologies (ege, 2008:59): “the current ipr system (for plant varieties and gm crops) could pave the way for market predominance where a few companies control much of agricultural production, with an impact on innovation and the growth of local economies in developing countries”. the three perspectives on sustainable food security and food system sustainability analysed by garnett (2014) – namely efficiency, demand restraint, food system transformation – also reflect different values and ideologies on the role of technology and innovation in the agro-food arena. for the efficiency perspective, the boundaries of environmental limits can be expanded or overcome by using technology to accommodate humanity. the vision underlying the efficiency perspective is that technology can be used to deliver development goals (e.g. food security, well-being) with less environmental impact. therefore, it can be assumed that advocates of this perspective have a positive attitude towards new technologies and innovation. meanwhile, for the demand restraint perspective, technology is sometimes problematic and can be used by humans to further damage the environment and nature. nevertheless, innovation has always occurred in agriculture as farmers have adapted agricultural practices to changing climate and environment conditions. however, many scholars dealing with agro-food system do not feel comfortable with the current narrow definition of innovation meaning technological and commercialised innovation (levidow, 2015). this narrow ‘technological-deterministic’ understanding of innovation dates back to the early 20th century, when innovation was considered as synonymous of adoption of commercial technological inventions. this innovation model, that emphasises capital-intensive technology, has become profoundly entrenched in research and policy frameworks. the model ignores existing farmers’ knowledge and marginalises their cooperative exchange and learning networks and undervalue their capacity to innovate while favouring a linear knowledge transfer (moschitz et al., 2015) based on the transfer of technology (tot) model (lionberger, 1960; havelock, 1969; chambers and jiggins, 1987). tot model stimulates farmers to capture economies of scale and encourages externalization of significant environmental and social costs (e.g. lal et al., 2005; mukherjee and kathuria, 2006; iaastd, 2009). according to iaastd (2009) report, in general, no recognition is given to farmers’ local and traditional knowledge and their innovation in official systems of agricultural knowledge and science. in fact, the “role of traditional, indigenous knowledge is already being undermined as a result of the growing disconnection with ongoing research activity” (scar-feg, 2007:11). the linear innovation model has privileged laboratory-based and scientific knowledge in research agendas at the expense of farmers’ agro-ecological knowledge (vanloqueren and baret, 2009). this process was seen as causing profound social or cultural changes (godin, 2008, 2015), that are not always positive on farming and rural communities. moreover, inequitable power relationships in agricultural knowledge and information system create barriers to farmers’ innovation (silici, 2014). therefore, in order to contribute more effectively to achieving sustainable food and nutrition security, agricultural research in particular and food-related research in general should adopt a ‘food system approach’ and address at the same time profitability, productivity and ital. j. food sci., vol. 30, 2018 213 sustainability in agricultural and food systems (global panel on agriculture and food systems for nutrition, 2016). multinationals and consulting firms in the agro-food sector seem nowadays more aware about concerns regarding technology and innovation in agriculture. however, they stress the importance of classical technologies (fertilizers, crop protection products) in meeting food security challenge through increase in productivity. for them, the slowing down or plateauing of crop yields, especially cereals, is a big threat to future global food security that requires new breakthrough innovations for the ‘sustainable’ intensification of production. the latter include digital innovation (e.g. soil sensors, drones), biotech innovations (e.g. genetically engineered plants and animals) and process innovation (e.g. vertical farming, hydroponics, aquaponics). however, acceptance of these innovations and technologies is still a problem in agriculture sector: “while these technological innovations have the potential to make a positive impact on agribusiness, the challenge is to find common ground between the significant social, political, and environmental concerns and the business interests surrounding these disruptive changes” (atkearney, 2016:3). in order to gain such an acceptance, the advantages of such technologies for smallholder farmers in developing countries as well as relevance of these breakthrough innovations in reducing the carbon footprint of agriculture and its contribution to climate change are often highlighted. further moves include global transfer of knowledge, also to developing countries, transparency and the democratization of data and better collaboration between agribusiness, government and the civil society (atkearney, 2016). many agro-food companies, including multinationals, are paying more attention to sustainability issues. a clear example of that is publication of periodical sustainability reports (e.g. monsanto, 2017) or dedicating a section of annual reports to sustainability (e.g. bayer, 2017; basf, 2017). in its sustainability report, monsanto makes even a clear reference to sdgs and states that its key principles are: act ethically and responsibly, advocate for biodiversity, advance product stewardship, create a great work environment, drive modern agricultural innovation, engage communities and society, foster collaboration and transparency, improve global food and nutrition security, reduce our environmental impact (monsanto, 2017:5). therefore, there is a clear intention to connect agricultural innovation and technology with societal challenges such as food security, environmental protection, biodiversity as well as ethical concerns. this move of agri-multinationals is often considered as an example of incremental change or even, worse, of ‘greenwashing’ (e.g. scanlan, 2013). however, for instance, szekely and strebel (2012) consider the unilever’s transformation of its tea supply chain for its lipton brand to certified sustainable tea as an example of ‘radical innovation’. in fact, unilever entered into a partnership with the rainforest alliance (rfa) to certify tea supplies focusing on the areas of environmental protection, employee welfare and farm management. this shows clearly the divergences in opinions regarding sustainability transitions in the agro-food arena. in fact, there are several contending paradigms and narratives about sustainable agriculture and way to achieve it (van der ploeg, 2009; levidow, 2011; elzen et al., 2017). a clear example of these contending agendas about agricultural innovation are the ´life sciences & global value chains’ (see, knowledge-based bio-economy) and ‘agroecology & agrofood-energy relocalisation’ in the eu (levidow, 2011). sustainable intensification (e.g. garnett et al., 2013) is a good example to show diversity of visions, agendas and perspectives regarding sustainable agriculture. it clearly shows trade-offs not only between productivity and sustainability aspirations but also between innovation (that ideally should help improving, simultaneously, both productivity and sustainability) and sustainability. sustainable intensification agendas promote a ‘toolkit’ of various options and techniques for reconciling higher productivity with environmental sustainability. the orthodox consensus on ‘technological intensification’ has been ital. j. food sci., vol. 30, 2018 214 challenged by a variety of concerns such as environmental protection, animal welfare, and food quality and safety (loeber and vermeulen, 2012). nevertheless, some agricompanies have seized the ‘sustainable intensification’ momentum to rebrand their products as sustainable intensification tools (constance et al., 2016). in fact, the neoproductivist agenda (e.g. almås and campbell, 2012) has been widely articulated under a sustainable intensification approach that encompasses various agroecological but also even biotechnological methods to increase yield, while also lowering the burdens on the environment (garnett and godfray, 2012). in some contexts, the sustainable intensification toolkit is reduced to only biotechnological solutions such as gm crops (young, 2013). meanwhile, counter-hegemonic global food movements embrace agroecology and community-based food systems. they promote a concept of ‘ecofunctional intensification’ (niggli et al., 2008). however, there are also some attempts to reconcile these two opposed agendas. for instance, a report from the rural investment support for europe (rise) mentioned six systems to achieve sustainable intensification (buckwell et al., 2014): agroecology, biodynamic, organic, integrated, precision farming and conservation agriculture. similarly, in the european union, where mainly capitalintensive technological innovation is emphasized, agroecology has been promoted as a different kind of practice that combines know-how, organizational, social and technological innovation (ifoam eu group et al., 2012). disagreement about model of agriculture that allows reconciling productivity (cf. innovation) and sustainability (especially environmental one) was evident during the setting up of the european innovation partnership (eip) for agricultural productivity and sustainability (eip-agri). in the end, the strategic implementation plan of eip-agri encompassed different approaches such as sustainable intensification, organic farming, low-external input systems (eip-agri, 2013). the attempt to show at least an apparent compatibility and harmony between productivity (see, technological innovation) and sustainability in agriculture is somehow exported to other world regions such as sub-saharan africa. for instance, four different pathways to sustainable intensification of agri-food systems in africa were identified in the prointensafrica project (a horizon 2020 coordination and support action): conventional agriculture pathway, eco-technical pathway, agroecology pathway, and organic agriculture pathway (prointensafrica, 2017). generally speaking, some alternative agro-food movements (e.g. food sovereignty, slow food, agroecology) have a critical attitude towards innovation (especially technical/technological one) while others (e.g. organic agriculture) have evolved towards a more accommodating position. in the manifesto ‘food sovereignty: a future without hunger’, presented by via campesina at the 1996 world food summit (vía campesina, 1996), there is no reference to innovation. this clearly shows a critical attitude towards innovation of this peasant movement. however, access to technology by peasant families, especially women, is stressed. in the declaration of nyéléni (nyéléni, 2007), food sovereignty movement did again no reference to innovation, be it technical or social. however, it made it clear that it is fighting against “technologies and practices that undercut our future food producing capacities, damage the environment and put our health at risk. those include transgenic crops and animals, terminator technology, industrial aquaculture and destructive fishing practices, the so-called white revolution of industrial dairy practices, the socalled ‘old’ and ‘new’ green revolutions, and the “green deserts” of industrial bio-fuel monocultures and other plantations” (p. 3). slow food movement pays a particular attention to the ecological, economic, social and cultural sustainability of the local agro-food systems (e.g. slow food, 2013; peano et al., 2014). it can be argued that the movement has a critical stance with respect to innovation and technology. in fact, references to innovation can be hardly found in the declarations of the movement (e.g. slow food, ital. j. food sci., vol. 30, 2018 215 2008; slow food, 2009). the term innovation has also no place in slow food terminology (slow food, 2015). the organic agriculture movement seems to have nowadays, as it was not always the case, a more accommodating attitude towards innovation. in fact, innovation is even part of the official definition of organic: “organic agriculture is a production system that sustains the health of soils, ecosystems and people. it relies on ecological processes, biodiversity and cycles adapted to local conditions, rather than the use of inputs with adverse effects. organic agriculture combines tradition, innovation and science to benefit the shared environment and promote fair relationships and a good quality of life for all involved” (ifoam, 2010). one of agro-food alternatives is agroecology that has been coined by scientists with the intention to open up scientific preoccupations and to contest technocratic governance of agricultural innovation, oriented towards commercial benefits, agricultural intensification and expansion of global trade (elzen et al., 2017). agroecology is not against innovation in general but certain types of innovation. in fact, as the institute for agriculture and trade policy (iatp, 2013) points out agroecology “is by definition an innovative, creative process of interactions among small-scale producers and their natural environments”. however, agroecology faces the task of challenging the dominant models of innovation in agriculture. beside technological-scientific innovation, it embraces also know-how, social and organisational innovation forms (ifoam eu group et al., 2012). agroecology promotes social and organisational innovation as an alternative strategy across the whole agro-food chain with the aim of strengthening connection between agro-ecological farmers and consumers that support their agro-ecological innovations. these agro-ecological initiatives are variously known as short food supply chains (sfscs) or alternative agrofood networks and they are clear examples of social innovation (galli and brunori, 2013). such new agro-ecologically-inspired local networks and citizen-community alliances can counterweight the dominant agri-food system (fernandez et al., 2013) provided that they professionalise their skills, maintain consumer loyalty, constantly learn and continuously innovate (karner, 2012) thus developing genuine and sustainable local food systems. agro-ecological innovation is key to the transition towards sustainability in the current agro-food system (levidow, 2015). thanks to many social and grassroots movements (e.g. la via campesina), the latin american agroecology agenda (e.g. national plan of agroecological and organic production, planapo, in brazil), inspired transformational strategies elsewhere such as in europe. indeed, according to the eu’s standing committee on agricultural research (scar-feg, 2009), agro-ecological principles should be given priority in agriculture research agendas in the european union. in this context, the european organic sector promotes agro-ecological research with the concept of ‘ecofunctional intensification’ linking farmers’ knowledge and innovation with scientific research (niggli et al., 2008). this new understanding of ‘agro-ecological innovation’ is promoted by a european alliance involving civil society organisations and farmers (arc2020 and friends of the earth europe, 2015). nevertheless, agro-ecological methods, but not necessarily agro-ecological principles, were adopted also by some conventional agriculture actors, such as agrochemical companies and some governments, that incorporated agro-ecological methods into ‘sustainable intensification’ agendas. such a move and process was criticized by many farmers’ organisations, ngos and social movements (levidow et al., 2014; arc2020 and friends of the earth europe, 2015; levidow, 2015a). levidow (2011) explained very nicely the complicated relation between innovation and sustainable agriculture: “nowadays most innovations are promoted under the banner of ‘sustainable development’, but there are different accounts of what is to be sustained. likewise sustainable agriculture has different accounts, so it has become an ambiguous concept – even a ital. j. food sci., vol. 30, 2018 216 contentious one”. accounts of what is to be sustained include agriculture growth, natural resources, current production patterns, livelihoods, ecosystem services, communities, etc. this adds to tensions between and multiple interpretations of environmental, social and economic sustainability in agriculture. 4. conclusions this paper argues that a better understanding of the complex and multifaceted relation between innovation and sustainability in the agro-food arena is crucial to make more effective transition towards sustainable food systems as perception towards innovation in agro-food can be an obstacle to or a lever for the deep and radical change that is needed. it is clear nowadays that innovation is needed to foster sustainability transitions in food system from production to processing, distribution and consumption. while technical innovations are widely used and advocated for a sustainable intensification of food production, social and organisational innovations seem more relevant in the other stages of the food system as they allow improving food chain functioning and governance. however, although innovation and technological progress have had significant benefits in terms of achieving food security, relationship between innovation and sustainability is far from straightforward. in fact, the food system is for sure a contested arena where different worldviews and narratives are confronted and this applies also to innovation. moreover, it should be highlighted that food has also a strong cultural connotation and, for that, all changes in agro-food arena are carefully scrutinised. simply put, while many food system actors emphasize the positive role of innovation and technology in driving progress toward sustainable food systems; innovation (especially technical one) provides fertile ground for alternative agro-food movements (e.g. organic agriculture, slow food, agroecology, food sovereignty) to criticize the over-industrialization of food system. these movements seem more benign towards social innovations, especially grassroots ones, that are seen as a means to bring about the transition towards more sustainable, inclusive and equitable food systems. ultimately, it seems that the issue is not about questioning innovation tout court, but about what type of innovation should be promoted. in other words, it is not about being proor anti-innovation, but about addressing real, essential questions of innovation politics related to direction of change pathways in agro-food systems, distribution and equity, and diversity of options. while moving towards ‘sustainable’ or ‘sustainability-oriented’ innovation seems to be a good compromise and a step in the right direction, thus making transition towards sustainable food systems smoother and more effective, there is no doubt that this does not represent a panacea per se as even the concept of ‘sustainability’ is contested. in fact, one can always ask, a sustainable innovation for whom (as there are winners and losers in any transition or change), where (as sustainability is placeand context-specific), etc. therefore, it can be assumed that innovation approaches need an increased dose of ethics, rules and values when they are applied to food systems and that is also true for all discourses and paradigms regarding sustainability transitions. acknowledgements i would like to thank dr. les levidow (the open university, united kingdom), dr. erik millstone (university of sussex, united kingdom) and dr. allison loconto (fao & inra, france) for constructive comments and valuable suggestions on earlier versions of this paper. ital. j. food sci., vol. 30, 2018 217 references almås r. and campbell h. eds. 2012. rethinking global agricultural regimes: food security, climate change and the future resilience of global 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and food sciences, government college university faisalabad, 38000, pakistan 2national institute of food science and technology, university of agriculture faisalabad, 38000, pakistan 3institute of food science and nutrition, bahauddin zakariya university, multan, 60000, pakistan 4institute of diet and nutrition, university of lahore, 75500, pakistan *corresponding author: drnazirahmad@gcuf.edu.pk abstract this study was planned to develop and characterize extruded multilegume savory bars as a protein supplementary nutrition. legumes were extruded to prepare composite flour. proportions of extruded flour were mixed with whey protein concentrate, honey and palm oil for preparation of protein bar. the product was evaluated for physico-chemical, minerals, calorific value, color, hardness, protein digestibility and sensorial characteristics. in vitro protein digestibility was found from 62.04 to74.98% and in vivo from 65.30 to 84.01%. extrusion process and addition of whey protein concentrates significantly affected the nutritional and sensorial parameters of bars. keywords: legume protein, extrusion, bar, protein digestibility ital. j. food sci., vol. 32, 2020 168 1. introduction malnutrition is an abnormal physiological condition triggered by imbalanced, inadequate or excessive consumption of nutrients (rizwana et al., 2015) while, proteins energy malnutrition (pem) is a change of pathological conditions arises due to deficiency of protein calories (ernest et al., 2013). developing economies are adversely affected by the malnutrition. globally, there are more than 150 million children under the age of five years who are malnourished. the majority of these children are residing in just three countries of the south asia i.e. india, bangladesh and pakistan where almost 54% of child deaths are linked to this menace (unicef, 2016). unhealthy diet, ecological conditions and general living standard have a strong relation with diseases. according to global hunger index (ghi), pakistan is at 11th position from 118 countries with respect to malnourished population (22%), stunted growth (45%), wasting (10.5%) and mortality (8.1%) in children under 5 years of age (ifpri, 2016). likewise, according to national nutrition survey (2011), 58% of the population is facing the food security situation. due to malnutrition, women and children are facing macroand micro-nutrient deficiencies. about 31.5% of the children are underweight, 43.7% are stunted and 15.1% are suffering from wasting. children (39-61%), pregnant women (38-69%) and non-pregnant women (26%-68%) are facing iron, zinc, vitamin a and d deficiencies (gop-pakistan, 2011). pem is one of the important public health issues in developing countries (van der pols-vijlibrief et al., 2014). marasmus, kwashiorkor and marasmic-kwashiorkor are the primary reasons of pem (ernest et al., 2013) that is associated with co-morbidities such as anaemia, tuberculosis diarrhea and malaria (le roux et al., 2010) and these causes may lead to death. several policies have been implemented to overcome the issue of pem that involves different food based strategies such as dietary modifications, food enrichment and supplementation. school health programs are also initiated in various countries to mitigate this situation (onis, 2012). protein as a nutrient is considered a dietary component that evokes the widest array of complex scientific, economic and environmental issues, viewed as the most expensive but essential ingredient forming part of a healthy balanced diet (schönfeldt and hall, 2012). edible legumes belong to the family leguminosae entitled as fabacae. these are termed used for grain legumes which are generally grown up for their edible seeds. legumes also called “a poor man’s meat”. legumes are abundantly cultivated in subtropics and tropics areas of the world. they are the good alternative to animal protein for those people who have limited resources (adebowale and lawal, 2004). they possess amounts of amino acids such as leucine, lysine, aspartic acid, arginine and glutamic acid. they are vital sources for food proteins and also give rational essential amino acids when used with grains or other foods (sarwar et al., 2013). it can be used with other food items to enhance the nutrition. they are also an excellent source of micronutrients as contain riboflavin, thiamin, niacin, selenium, folate, and pyridoxine (usda, agricultural research service 2012). they have good amounts of vitamin a, e and c (raatz; the bean institute 2010). application of extrusion process on legumes modifies the physico-chemical parameters for improving functional properties in target applications (osen et al., 2015). thermal extrusion has advantages as it helps to hinder anti-nutritional factors such as haemagglutinins, trypsin inhibitors, tannins, phytates which inhibit protein functionality and digestibility (alonso et al., 2000). since, legumes have good sources of protein and micronutrients and can be modified into an extruded product. they can be used to prepare protein rich bars, that can diversified the diet with natural approach to enhance the nutritional requirements and reduce the ital. j. food sci., vol. 32, 2020 169 malnutrition of poor regions by using as a cultural food. the present project was planned to prepare extruded multilegume savory bar in order to mitigate pem. according to institute of medicine the recommended daily allowance of protein for adults older than 18 years is 0.8 g/kg/d and the youngsters (under 18 years of age) required 13-52 g of protein per day (nap, 2005). the primary objective of this multilegume product was to provide the enough nutrients that fulfill the daily requirement of protein but will not cross the threshold level for children and adults in the absence of meat products to address pem. 2. materials and methods 2.1. procurement of materials chickpea (cicer arietinum l), mung (vigna radiata), mash (vigna mungo l), soybean (glycine max), whey protein concentrates (wpc 80), palm oil and honey were purchased from the markets of faisalabad, pakistan. 2.2. preparation of extruded multilegume savory bars thermal extrusion has advantages to destroy the anti-nutritional factors such as haemagglutinins, trypsin inhibitors, tannins, phytates, and helps in production of bioactive peptides and improve protein functionality and digestibility. all legumes were soaked for 15 hrs and dried for thermal extrusion. legumes were fed into a twin-screw extruder (dndl 44, bühler ag, uzwil, switzerland) using the method described by tremaine and schoenfuss (2014). optimized extrusion conditions of feed flow rate (60 kg/hr), screw speed (250 rpm), feed moisture content (10%) and barrel exit temperature (160°c) were used for the preparation of extruded powder. extrudates were pelletized and dried in vacuum oven. drying continued at 40°c in oven for 26 hrs. chickpea flour and other legumes (mash, mung and soybean as composite flour) were mixed in proportions and prepared different treatments as described in table 1. whey protein concentrates (3%); honey (3%) and palm oil (3%) were added in each sample for better mixing, sensorial and nutritional properties. purpose of combining proteins from vegan and vegetarian diets is to provide sufficient amounts of some essential amino acids to make complete protein intake. palm oil has good spreading properties, technically useful and economically beneficial as compared to animal fat as well. after mixing, sheeting was done, and cut into bars of 3.5 centimeter (cm) width, 2 cm height, and 9 cm in length. each bar of approximately 50 g was packed individually in aluminum foil. using aacc 2000 methods, moisture content (method no; 44-15.02), crude protein (method no; 990.03), crude fat (method no; 30-10.01), crude fiber (method no; 962.09), ash (method no; 942.05) and nfe content were determined. water activity of prepared bars was determined using a previously described aoac method (aoac, 2012; method no. 978.18). the color values for each treatment were determined through color meter (color test ii, neοhuаus neοtec, germany) by following the method described by hunter (1987). calorific values of bars were determined through oxygen bomb calorimeter (ikаwerke, c2000 basic, gmbh and cο. germany) as described by miller (1959). hardness of bars was measured according to the method of piga et al. (2005). results were obtained on the basis of compression force (kg) used to press the bars. ital. j. food sci., vol. 32, 2020 170 table 1. treatment plan. treatment formulation t0 chickpea flour/composite% as 100/0 t1 chickpea flour/composite% as 70/30 t2 chickpea flour/composite% as 55/45 t3 chickpea flour/composite% as 40/60 t4 chickpea flour/composite% as 25/75 t5 chickpea flour/composite% as 0/100 composite flour contains mung, mash and soybean flours. 2.3. in vitro study for protein digestibility using the method of akeson and stahmann (1964) (with some modifications), in vitro protein digestibility was determined. aliquots of 250 mg of each sample were suspended in 15 ml of 0.1 mol equi/l hcl containing 1.5 mg/ml pepsin (sigma®, st. louis, mo, usa) and incubated for 3 hrs at 37°c in a water bath. hydrolysis from pepsin was stopped after neutralization by adding 7.5 ml of 0.5 mol equi/l of naoh, then pancreatic digestion started by the addition of 10 ml of 0.2 mol/l phosphate buffer (ph 8.0) containing 10 mg of pancreatin (sigma®, st. louis, mo, usa) with 1 ml of 0.005 mol/l sodium azide to hinder microbes growth and incubated at 37°c for 24 hrs. after hydrolysis with pancreatin, 1 ml of 10 g/100 ml of trichloroacetic acid was added and centrifuged at 550×g for 20 min. the supernatant was collected and the total protein content was calculated using kjeldahl (on the basis of nitrogen content) using aoac (2012) method. % digestibility= (ns–nb)/ns×100 ns = nitrogen content in the sample, nb = nitrogen content in the blank. 2.4. in vivo study for true protein digestibility (tpd) male sprague-dawley rats (350±12 g) of 9 weeks old were procured from animal house, national institute of health, islamabad, pakistan and maintained under standard laboratory conditions at 28±2°c with constant light-dark cycle. rats were fed on standardized chow for an acclimation period of 2 days and then 36 rats were divided into groups of 6 rats. rats were fed for 10 days in which 2 days were for acclimation period. rats were weighed on daily basis during study. after 4 days period, spilled food and feces were carefully collected and separated from each rat. the spilled food was dried for 72 hrs in air while collected feces were dried in oven overnight at 100°c, weighed, grinded and analyzed for nitrogen content. weight of spilled food and uneaten food were minus from the total food supplied to rat to determine the nitrogen intake. tpd was calculated as: i– (f–fk) tdp = × 100 i ital. j. food sci., vol. 32, 2020 171 i= intake nitrogen, f= fecal nitrogen, and fk=metabolic or endogenous fecal nitrogen. 2.5. sensory evaluation attributes like color, texture, folding ability, chewаbility, taste and οverаll аcceptаbility of extruded multilegume savory bars were analyzed by а penal of judges using 9point hedonic scale system as described by meilgааrd et аl. (2007). all experiments were conducted in triplicates and average values were considered as mean values. the significance of values was calculated statistically through mean using analysis of variance (anova) at probability of 0.05. 3. resukts 3.1. proximate composition of extruded flour extruded multilegume composite flours for each treatment were prepared by blending various amounts of mung, mash and soybean with chickpea and then analyzed for moisture, crude protein, crude fat, crude fiber, ash, nfe and mineral content. the mean values regarding proximate composition of composite flour is presented in table 2. table 2. proximate composition and mineral profile of flour of chickpea/mung, mash and soybean for savory bar development. chickpea flour/composite flour (%) moisture crude protein2 crude fat crude fiber3 ash nfe t0 3.98±0.18 1 29.26±1.32 3.20±0.05 1.02±0.10 3.03±0.06 57.09±2.04 t1 4.12±0.11 29.89±0.99 3.34±0.03⋆ 1.35±0.13 3.09±0.09 54.17±1.98⋆ t2 4.19±0.24 30.28±2.47 3.78±0.05⋆ 1.98±0.10⋆ 3.43±0.05 53.84±2.61⋆ t3 4.06±0.11 30.49±3.97 3.86±0.03⋆ 2.87±0.13⋆ 3.51±0.09 50.20±1.39⋆ t4 4.29±0.24⋆ 30.58±2.64 4.94±0.05⋆ 3.01±0.10⋆ 4.28±0.05 49.84±2.09⋆ t5 4.48±0.35⋆ 31.43±3.29⋆ 5.53±0.09** 3.30±0.08⋆⋆ 4.34±0.10⋆ 47.50±1.87⋆ mineral profile (mg/100g) na k ca fe zn t0 11.51±0.25 477.00±12.45 81.39±19.10 04.70±0.63 02.54±0.09 t1 13.65±0.29⋆ 614.50±05.23⋆⋆ 113.85±2.62⋆⋆ 06.62±0.25⋆ 03.01±0.09⋆ t2 14.72±0.21⋆ 682.80±09.19⋆⋆ 129.70±5.69⋆⋆ 07.36±0.14⋆ 03.30±0.03⋆ t3 15.86±0.36⋆ 751.87±07.71⋆⋆ 146.55±2.70⋆⋆ 07.75±0.29⋆ 03.45±0.09⋆ t4 16.87±0.49⋆ 820.93±11.40⋆⋆ 155.40±2.22⋆⋆ 07.92±0.23⋆ 03.75±0.08⋆ t5 18.65±0.39⋆ 925.27±11.35⋆⋆ 188.25±4.50⋆⋆⋆ 08.20±0.20⋆ 03.94±0.09⋆ 1mean values (on dry basis) ± standard deviation. different superscripts (⋆) on values in columns show significance difference (p˂ 0.05) within treatment. 2calculated using n x 6.25 for proteins 3calculated by difference of 100 (ash + proteins + fat +starch). ital. j. food sci., vol. 32, 2020 172 the moisture content was ranged from 3.98±0.18 to 4.48±0.35%, crude protein 29.26±1.32 to 31.43±3.29%, crude fat 3.20±0.05 to 5.53±0.09%, crude fiber 1.02±0.10 to 3.30±0.08%, ash 3.03±0.06 to 4.34±0.10% and nfe 47.50±1.87 to 57.09±2.04%. significant (p˂ 0.05) difference in nutritional composition was observed with the increase of multilegume composite flours in formulations. maximum na content was observed in t5 (18.65±0.39 mg/100g) and minimum (11.51±0.25 mg/100g) in t0. k content of formulations was ranged from 477.00±12.45 to 925.27±11.35 mg/100g in which maximum content was observed in t5 and minimum in t0. ca content of formulations was ranged from 81.39±19.10 to 188.25±4.50 mg/100g. the highest ca content was observed in t5 and the lowest ca was noted in t0. maximum values for fe were found in t5 (08.20±0.20 mg/100g) and minimum values in t0 (04.70±0.63 mg/100g). zn content was found lowest in t0 (02.54±0.09 mg/100g) and highest in t5 (03.94±0.09 mg/100g). significant (p˂0.05) difference was found within treatments from control in mineral analysis. 3.2. proximate composition of multilegume savory bars the mean values regarding proximate composition of multіlegume savory bars are shown in table 3. table 3. proximate composition of multіlegume savory bars. savory bar legumes ratio mοіsture crude protein2 crude fat crude fіber3 ash nfe energy4 (calories/100 g) t0 3.99±0.21 1 31.98±0.87 5.03±0.12 0.99±0.26 3.24±0.13 58.03±1.08 418.03±13.04 t1 4.24±0.09 32.76±0.16 5.63±0.24 1.29±0.31 3.45±0.19 56.23±1.98 436.74±12.09⋆ t2 4.35±0.19 33.01±0.34 5.98±0.76 1.92±0.41 3.69±0.23 53.12±2.42⋆ 458.67±10.26⋆ t3 4.40±0.20 33.56±0.23 6.73±0.53⋆ 2.76±0.27⋆ 3.93±0.17 51.89±2.32⋆ 526.18±09.87⋆ t4 4.43±0.29⋆ 33.93±0.49⋆ 7.09±0.49⋆ 2.86±0.65⋆ 4.56±0.32⋆ 50.63±1.59⋆ 530.17±15.76⋆ t5 4.61±0.25⋆ 34.23±0.95⋆ 7.99±1.02* 3.19±0.51⋆ 4.89±0.09⋆ 48.53±0.99⋆⋆ 546.49±19.87⋆⋆ mineral profile (mg/100g) na k ca fe zn t0 11.65±0.12 479.12±4.32 81.29±9.34 04.65±0.43 02.51±0.04 t1 13.71±0.18⋆ 618.32±5.76⋆⋆ 114.31±1.93⋆ 06.68±0.18⋆ 02.97±0.12⋆ t2 14.78±0.31⋆ 685.47±6.20⋆⋆ 130.82±4.32⋆ 07.71±0.07⋆ 03.23±0.31⋆ t3 15.92±0.28⋆ 763.38±8.24⋆⋆ 147.25±3.21⋆ 07.82±0.15⋆ 03.49±0.54⋆ t4 16.93±0.17⋆ 824.52±5.91⋆⋆ 156.32±1.99⋆ 07.89±0.09⋆ 03.87±0.42⋆ t5 18.69±0.16⋆⋆ 927.36±6.32⋆⋆ 189.17±3.35⋆⋆ 08.40±0.87⋆⋆ 04.02±0.41⋆ 1mean values (on dry basis) ± standard deviation. different superscripts (⋆) on values in columns show significance difference (p˂ 0.05) within treatment. 2calculated using n x 6.25 for proteins. 3calculated by difference of 100 (ash + proteins + fat+ starch). 4 caloric values were determined bomb calorimeter. ital. j. food sci., vol. 32, 2020 173 in all treatments, the moisture content ranged from 3.99±0.21 to 4.61±0.25%, crude protein 31.98±0.87 to 34.23±0.95%, crude fat 5.03±0.12 to 7.99±1.02%, crude fiber 0.99±0.26 to 3.19±0.51%, ash 3.24±0.13 to 4.89±0.09% and nfe 48.53±0.99 to 58.03±1.08%. significant (p˂ 0.05) difference was found in all treatments in comparison with control for moisture content, crude protein, crude fat, crude fiber, ash and nfe in bars prepared from multilegumes composite flour. maximum na content was observed in t5 (18.69±0.16 mg/100g) and minimum in t0 (11.65±0.12 mg/100g). k content was ranged from 479.12±4.32 to 927.36±6.32 mg/100g in which maximum content was observed in t5 and minimum in t0. ca content was ranged from 81.29±9.34 to 189.17±3.35 mg/100g. maximum value for fe was found in t5 (08.40±0.87 mg/100g) and minimum in t0 (04.65±0.43 mg/100g). zn content was found highest in t5 (04.02±0.41 mg/100g) and lowest in t0 (02.51±0.04 mg/100g). significant (p<0.05) difference was found for mineral content in bars within treatments in each column. maximum calorific value was noticed in t5 (546.49±19.87 calories/100g) while lowest in t0 (418.03±13.04 calories/100g). 3.3. water activity non-significant (p > 0.05) difference was found for water activities in bar as all values were recorded around 0.50. 3.4. color of extruded multilegume savory bars color reveals the first impression of a food product before consumed. it’s the first score of a like and dislike for food commodity. the mean values for color score of extruded multilegume savory bar are shown in table 4. highest value (58.67±0.14) of l was found in t2 while the lowest value (50.50±0.13) was noticed in t0. maximum color value of a⋆ was 7.85 in t2 while the minimum was 5.30 in t5 that shows coloring trend towards redness. maximum color value for b⋆ was 19.4±0.09 in t5 and lowest was 15.69±0.04 in t3 that shows coloring trend toward yellowness. t1, t2, t4 and t5 were significantly (p<0.05) different from each other while t0 and t3 were non-significantly (p>0.05) different in color value of l*. t0, t2, t3, t4 and t5 were significantly (p<0.05) different from each other while t1 was nonsignificantly (p>0.05) different in color value of a*. t3 and t5 show significant (p<0.05) difference than other treatment in color value of b*. 3.5. hardness of extruded multilegume savory bars hardness is one of the quality attributes, which describe quality of food bars before testing. the mean values of hardness for bars have been listed in table 4. maximum force (kg) was noticed on t5 (8.61±0.76) while minimum on t0 (5.33±0.34). highly significant values were observed for t5, t4 and t3 as compared to others. 3.6. in vitro and in vivo studies for protein digestibility protein digestibility values were calculated for each treatment and results regarding digestibility are shown in figure 1 for both in vitro and in vivo studies. in vitro digestibility was observed between 62.04 to 74.98% while tpd in vivo values ranged from 65.30 to 84.01%. maximum value for tdp was observed in t5 (84.01±3.91) and lowest value in t0 (65.30±3.43). all the treatments were significantly different (p<0.05) from control sample in both studies. ital. j. food sci., vol. 32, 2020 174 table 4. mean values of color and hardness (kg) of multilegume savory bar. color treatments l⋆ a⋆ b⋆ t0 150.50±0.13 6.63⋆ 17.8±0.07 t1 54.81±0.09⋆ 6.33 18.02±0.03 t2 58.67±0.14⋆ 7.85⋆ 16.03±0.02 t3 52.33±0.09 6.08⋆ 15.69±0.04⋆ t4 55.51±0.09⋆ 5.40⋆ 18.80±0.08 t5 57.84±0.12⋆ 5.30⋆ 19.4±0.09⋆ 1mean values of triplicate representations + standard deviation, superscripts (⋆) show the significant difference (p˂0.05) in same column. l⋆ represents the lightness ranging from darkness (0) to lightness (100). a⋆ represents redness varying from greenness (-a⋆) to redness (+a⋆). b⋆ represents the yellowness varying from blue (-b⋆) to yellow (+b⋆). figure 1. the in vivo and in vitro protein digestibility values (%) of different treatments. in vivo protein digestibility was evaluated using rats model and in vitro through pepsin and pancreatin. 3.7. sensory evaluation of extruded multilegume savory bars 0 10 20 30 40 50 60 70 80 90 100 t0 t1 t2 t3 t4 t5 in vivo in vitro treatments % p ro te in d ig es ti bi lit y in vivo and in vitro protein digestibility treatments hardness (kg) t0 15.33±0.34 t1 5.89±0.22 * t2 6.17±0.43 * t3 7.01±0.52 ** t4 7.99±0.56 ** t5 8.61±0.76 *** ital. j. food sci., vol. 32, 2020 175 values regarding aroma, texture and flavor analysis are shown figure 2. 0 1 2 3 4 5 6 7 sweet butter beany spicy herbs a: aroma t1 t2 t3 t4 t5 t0 0 1 2 3 4 5 6 7 8 soft hard crunchy crumbly dense/compact dry b: texture t1 t3 t4 t5 t0 t2 ital. j. food sci., vol. 32, 2020 176 figure 2. mean values were obtained through 3 repetitions by 19 panelists each. (a= aroma, b= texture, c= flavor) score range from 1 = dislike extremely to 9 = like extremely evaluated after 60 days of storage interval. texture observation of bar in t3, t4 and t5 showed significant sweet, butter and bean aroma. texture observation of extruded multilegume savory bars showed that all the treatments had rough texture; t2 showed significant dryness in its texture, t3, t4 and t5 showed crunchy and crumble texture. bars made from t4 showed compact and dense texture whereas bars from t5 were observed significantly soft in texture. flavor analysis showed that t3 has significant flavor of chewy, nutty and grainy, whereas flavor of bitter, burnt and saltish was found in t5. similarly, t4 showed significant saltish flavor. 4. discussion legumes are good source of proteins and transformation of these into bars helps to mitigate the threatening situation of pem. addition of whey protein concentrate, honey and palm oil into bar was also added to improve nutritional and sensorial characteristics. production of protein rich bars lower down pem but also provide various minerals and vitamins to consumer in sufficient quantity to address various body functions. as legumes proportions were increased in treatments, values of protein, ash and energy increased. so, it is clear from results that addition of legumes in composite proportions helps to increase the nutritional status of bars. nutritional value of proteins depends on the availability, digestibility and quantity of essential amino acids present in it. extrusion helps in the inactivation of anti-nutritional factors and improves nutritional values. so, preparation of 0 1 2 3 4 5 6 7 8 bitter burnt chewy nutty grainy saltiness herb/spacy c: flavor t4 t5 t0 t1 t3 t2 ital. j. food sci., vol. 32, 2020 177 bar through extrusion treatment is one of the best method to conserve protein constituents for mitigation of pem. abdel-gawad et al. (2016) prepared composite flours using legumes and wheat and observed protein content between 11.76 to 19.05%, fat content between 1.36 to 3.25%, crude fiber between 0.59 to 1.55% and ash content between 0.63 to 2.40%. all values are slightly different from current study that is due to use of whey protein concentrate and different legume species, as composition varies within species. jahreis et al. (2015) prepared legume flour and found ca content (47-221 mg/100g), k content (1030 to 1760 mg/100g), fe content (4.3 to 7.7 mg/100g) and zn content (2.5 to 4.1 mg/100g); these observations are slightly different from present study. this might be due to addition of different legume species. nadeem et al. (2012) prepared date bars and showed moisture content (15.56 to 18.70%), protein content (7.41 to 14.96%), crude fat (5.55 to 8.37%), crude fibre (3.58 to 3.88%) and ash content (2.30 to 2.91%). as multilegume increases in the bar protein, crude fibre and ash content also increases and improves minerals profile such as na, ca, k, fe, zn and essential amino acids without disturbing the sensorial parameters (bower and whitten 2000). added proteins are functioned to keep the ingredients of food bars intact, maximize the strength, set the structure and contribute to water holding capacity. whey protein possesses viscosity, water holding properties and gel strength that contribute to bar firmness (ortiz et al., 2008). fat content not only provide caloric values but also increases the palatability of bars. additionally, fat possess to act as a binder with sweeteners in agglutination of the ingredient present in bar that helps to impart compactness and firmness to texture of food bars (escobar et al., 1996). color is the first impression of a food product. it’s the first score of a like and dislike food commodity. srebernich et al. (2016) prepared cereal bar with different formulations and found l* value (52.78 to 62.70), a* values (8.39 to 11.93) and b* values (23.53 to 27.90), slight difference was found in our findings in accordance to current findings; this might be due to different composition of bars. rajabi (2017) prepared high protein extruded bars and found hardness values between 6.08 to 9.33 kg; these values are comparable to current findings. increase in harness was observed with the change in composite flour (%) that is might be due to the cumulative effect of different flours. almeida et al. (2015) prepared whey protein supplements and found their protein digestibility between 88.4 to 91.7% in vitro and these values are not comparable to current findings because in this study vegetable protein sources were used that have more antinutritional factors and can form more complex protein structure and may hinder protein digestibility (butts et al., 2012). in vivo true protein digestibility increased as the extruded multilegumes proportions increased in bars. erbersdobler et al. (2017) stated that digestibility of protein is high around 89-96% in weaning foods based on beans and rice and these values have contradictions to current findings and this might be due to use of different legumes and processing conditions. nosworthy et al. (2018) stated that extrusion of beans/legumes ameliorate the protein digestion as compared to other processing conditions. changes in extrusion variables also affect the protein digestibility. with the increase in extrusion temperature, the protein digestibility values are increased as inactivation of protease enzymes occur rapidly as temperature increases. increase in the shear forces increases the protein digestibility, this might be due to denaturation of protein increase with increasing screw speed (bhattacharya and hanna, 1985). in this study screw speed was 250 rpm and barrel exit temperature was 160°c, these conditions might be the reason of increase in protein digestibility of protein bars. ital. j. food sci., vol. 32, 2020 178 extruded multilegume savory bar first time prepared, which has sensorial and nutritional qualities. both nutritional and sensorial qualities of puffed cornmeal were enhanced when blended with milk protein isolates (onwulata, 2010), same is happened in current study. banach et al. (2016) prepared protein bar by using extruded milk protein concentrates that were more cohesive, softer and less textural changes were observed than bars prepared with the spray drying method. hence, extrusion process modifies the physico-chemical parameters of ingredients not only the structural-function relationships of proteins and same findings are observed in the current work. 5. conclusions compositional and sensorial characteristics of multilegume savory bars were assessed to consider the supplementation of protein from plant source (legumes) as alternate of meat protein. proximate analyses showed that proportion of different legumes have significantly increased the nutritional values of savory bar. significant increase in protein and minerals content were observed in bars and they have better protein digestibility in vivo and vitro studies conducted on rats. protein from plant source can be an economical approach to produce these bars to mitigate pem. so, these bars can be 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properties, contents of total phenolics and minerals, and bioaccessibility of antioxidants s. suna, b. incedayi, c.e. tamer, g. ozcan-sinir and o.u. copur* department of food engineering, faculty of agriculture, university of uludag, görükle, 16059, bursa *corresponding author: tel.: +90 22429414 90; fax: +90 2242941402 e-mail address: ucopur@uludag.edu.tr abstract in this study, dried lemon verbena (lippia citriodora kunth) leaves were used for functional beverage production with addition of sucrose and/or sweeteners. carbonated or mineral enriched versions of these beverages were also produced. the highest antioxidant bioaccessibility was obtained from sucrose added and natural mineral water enriched beverage both in frap (47.01 %) and cuprac (11.13 %) assays. in general, all beverages were rich in potassium and the mineral enriched beverages were high in magnesium and calcium. the ascorbic acid value was maximum in carbonated beverages. while energy reduced beverages were rich in total phenolics, sucrose added and natural mineral water enriched beverages showed the highest functionality in terms of bioaccessible antioxidants. keywords: antioxidant capacity, bioaccessibility, herbal tea, lemon verbena, total phenolics ital. j. food sci., vol. 31, 2019 41 1. introduction there has been a growing interest in functional food consumption as a result of an increment in the public awareness on healthy and balanced diet. functional foods are recognized with their health benefits, which related to the high ratios of bioactive components like ascorbic acid, carotenoids, vitamin e and phenolic compounds (miron et al., 2013). these natural antioxidants are widely distributed in several parts of higher plants (bark, flowers, fruits, leaf pods, seeds, stems and wood) and have been investigated worldwide. herbs and spices are among the most important sources of antioxidants and phenolics (yanishlieva et al., 2006). lemon verbena (lippia citriodora), one of these herbal plants, grows spontaneously in south america and is cultivated in north africa and southern europe. it is preferred for refreshing effect, which associated with its lemony flavor since ancient times. fresh leaves are mainly used as a flavoring agent in fish and poultry dishes, vegetable marinades, salad dressings, jams, puddings, and beverages, while dried leaves are mostly used in herbal teas and sorbets (funes et al., 2009). generally, the leaves of this plant are reported to possess digestive, antispasmodic, antipyretic, antioxidant, analgesic, anti-inflammatory, sedative and stomachic properties. in addition, it has been used in infusions for the treatment of asthma, cold, fever, flatulence, colic, diarrhoea and indigestion (ragone et al., 2007). previous studies on lemon verbena mainly concentrated on its chemical characterization and revealed the presence of several phenolic compounds like iridoids, flavonoids, phenolic acids and phenylpropanoids especially verbascoside (funes et al., 2009). it is well known that the health benefits of polyphenols are proportional with the amount of consumption. the bioaccessibility, the amount of an ingested antioxidative compounds that is available for absorption in the gut after digestion (palafox-carlos et al., 2011), should be known, since the phytochemicals must be previously available to exert their biological activities (costa et al., 2014). bioaccessibility of constituents might be changed according to physical properties and chemical composition of the food, its release from the food matrix, possible interactions with other food components, the presence of suppressors or co-factors and individual digestive capacity (parada and aguilera, 2007). there is very limited information about the bioaccessibility of antioxidant capacity of herbs or herbal drinks. today, herbal tea is traditionally prepared by brewing the fresh or dried leaves, stems, roots or seeds with boiled water or using ready to infuse commercial tea bags. however, brewing methods and parameters of plant species differ from one to another. due to erroneous brewing practices, the expected health benefits may be minimized and even adverse effects might seen. the main objective of this study is to produce a new alternative beverage to benefit from the nutritional and functional properties of lemon verbena in different forms. together with physicochemical properties, total phenolic content of beverages, antioxidant capacity and bioaccessibility of antioxidants were investigated. furthermore, the optimization of the process parameters with regard to prevent the mistakes applied in traditional techniques and so producing microbiologically safe, standard and value added beverages were aimed in this research. ital. j. food sci., vol. 31, 2019 42 2. materials and methods 2.1. chemicals all the reagents were analytical grade. tptz (2,4,6-tris(2-pyridyl)-s-triazine) and bile salts were purchased from fluka (switzerland). trolox ((±)-6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid), neocuproine (2,9-dimethyl-1,10-phenanthroline), dpph (2,2-diphenyl-2-picrylhydrazyl), methanol, sodium carbonate, gallic acid, oxalic acid, nitric acid, sodium bicarbonate and sodium hydroxide were purchased from sigma aldrich (germany). pepsin, pancreatin, iron (iii) chloride hexahydrate, folin-ciocalteu reagent, 2,6 dichlorophenol indophenol, copper (ii) chloride, ammonium acetate and hydrochloric acid were supplied from merck (germany). 2.2. materials lemon verbena was purchased from kurtsan food company (bursa, turkey) in the dried form for infusion in the production process of the beverages. natural lemon flavor was obtained from aromsa company (kocaeli, turkey) and natural mineral water was acquired from uludag beverage company (bursa, turkey). 2.3. methods 2.3.1 beverage production the beverages were produced at a pilot scale. a synthetic cloth bag was used as infuser. the plant was infused (1 %) in a boiled water without additional heating. then the extract was cooled down to room temperature and used as the main ingredient of the beverages. afterwards the addition of other ingredients, mixtures were plate filtered (plate filter 60x60 cfp, zambelli enotech, italy). the brix values of the first group of beverages (sucrose added beverages-sb) were adjusted to 8°±0.5 by using sucrose, citric acid, ascorbic acid and natural lemon flavor. the second group (energy reduced beverages-eb) was produced using aspartame and acesulfame-k along others. the amount of substituted aspartame and acesulfame–k in place of sugar were calculated according to the relative sweetness values of these sweeteners. due to the replacement of some sucrose to sweeteners for energy reduction, the brix values of these group beverages were adjusted to 5°±0.5. four different types of beverage were also produced by carbonation-c (ecb, scb) and mineral enrichment-m (emb, smb). eventually, six different beverages were formulated (fig. 1). the carbonation process was applied to improve the refreshing trait of produced beverages. the antimicrobial agents (na-benzoate and k-sorbate) were used in these beverages, so they were not subjected to pasteurization process, since co2 and antimicrobial agents provided for preservation. in the mineral enrichment process, the volume of the tap water was reduced by half and the remaining volume was replaced with natural mineral water. the beverages produced according to these steps mentioned above, were filled into 200 ml glass bottles and pasteurized (except carbonated ones) after capping. the bottles were then cooled and stored at room temperature until analyzed (fig. 1). ital. j. food sci., vol. 31, 2019 43 infusion of dried lemon verbena leaves formulization of beverages from extract filtration filling into bottles and capping pasteurization and cooling (except carbonated beverages) figure 1. flow diagram of beverage production. 2.3.2 evaluation of some physicochemical properties of raw material and beverages the brix (water soluble dry matter) (aoac, 1990), titratable acidity and ph (aoac, 2005), ascorbic acid (simona et al., 2011), color values (l, a, b) (bakker et al., 1986) and turbidity (tajchakavi̇t et al., 2001) analyses were performed on beverages. moisture content, ascorbic acid, and color (l, a, b) were determined in dried lemon verbena leaves to show the physical and chemical properties of raw material. the brix contents of the beverages were analysed using a digital ra-500 model kem refractometer. sevencompact ph/ion mettler toledo ph meter, shimadzu uv 1208 model spectrophotometer, hunterlab colour analyzer (msez4500l; hunterlab, virginia, usa), hach turbidimeter (hach, 2100q) instruments were used for other analyses. all analyses were repeated three times to ensure accuracy of the results. 2.3.3 mineral content to determine the quantity of fe, ca, mg and k in raw material and beverages, nmkl (2007) method was applied using agilent 7500 cx (agilent technologies, usa) model icp-ms. argon (99.9995 % pure, linde, turkey) was used as a carrier gas on icp-ms and ultra-pure water (18 mω·cm at 25 °c resistivity) was generated by purifying distilled water with the new human power i (scholar-uv-pf, 15l/hr) water purification system. standard stock solutions containing 1000 mg l-1 of each element (merck, darmstadt, germany) were used to prepare the calibration standards. standards were prepared in 1 % (v/v) hno3 on a daily basis. approximately 0.5 g of sample was weighed directly in polytetrafluoroethylene (ptfe) flasks after adding 4 ml hno3 (merck suprapur-65 %) and 1 ml h2o2 (merck ultrapur-35 %), then flasks were digested in berghof mws 3+ (germany) microwave digestion system. after cooling down to room temperature, the digested sample was transferred to a 50 ml volumetric flask and diluted with distilled water. all samples were filtered with 0.45 µm filters (hydrophilic pvdf millipore millex-hv) prior to the analysis. the instrumental operating conditions for icp-ms were as follows; plasma parameters; rf-power: 1550 w, sample depth: 8 mm, carrier gas flow: 0.95 l min-1, make up gas flow: 0.15 l min-1, ital. j. food sci., vol. 31, 2019 44 nebuliser pump 0.1 rps, spray chamber temperature: 2 °c, detector parameters; discriminator: 8.0 mv, analog hv: 1710 v, pulse hv: 1490 v. for mineral determination of water used in process, hno3 was directly added to water sample. while k ve ca were analysed by using eppendorf elex 6361 model flame photometer, mg and fe were analysed with perkin elmer optima 2100 dv model icpoes (ayyildiz, 1983). 2.3.4 in vitro digestion procedure to evaluate the functional properties of the beverages, total phenolics, antioxidant capacity and bioaccessibility of antioxidants were investigated. the samples directly taken from each beverage was used for determining the total phenolics and antioxidant capacity. an in vitro digestion enzymatic extraction method, slightly modified version of the one described by vitali et al. (2009) that mimics the conditions in the gastrointestinal tract was used to measure the bioaccessibility of antioxidants. the simulation of gastrointestinal conditions using commercial digestive enzymes (pepsin and pancreatin) is a widely used method for specifying the potential availability of bioactives. briefly, 10 ml of distilled water and 0.5 ml of pepsin (20 g l-1 in 0.1 mol l-1 hcl) were added to 1 ml of sample, ph was adjusted to 2 by using 5 mol l-1 hcl and sample was incubated at 37 °c in a shaking water bath for 1 h. simulation of gastric digestion was stopped by the addition of 1 m nahco3 (to adjust ph to 7.2). 2.5 ml of bile/pancreatin solution (2 g l-1 of pancreatin and 12 g l-1 of bile salt in 0.1 m nahco3) and 2.5 ml of nacl/kcl (120 mmol l-1 nacl and 5 mmol l-1 kcl) were added to the sample and simulation of intestinal digestion was conducted for the following 2 h. samples were centrifuged at 3500 rpm for 10 min and the supernatant was used for the analysis. after gastric and intestinal digestion, digested samples were used to determine the bioaccesibility of antioxidants. bioaccessibility was calculated as the percentage of antioxidant capacity. 2.3.5 total phenolics folin-ciocalteu reagent was used to determine total phenolics as described by spanos and wrolstad (1990). in brief, an aliquot (0.25 ml) of sample, 2.3 ml of deionised water and 0.15 ml of folin-ciocalteu reagent (fc/water, 1:5 v/v) were mixed within 10 ml volumetric flask and vortexed for 15 s at room temperature. after 5 min, 0.3 ml of 35 % na2co3 was added and mixed thoroughly. the absorbance of the mixtures was measured at 725 nm, after incubation for 2 h at room temperature. water was used as the blank, and gallic acid (ga) solution was used for the calibration of the standard curve (r2=0.9835). the phenolic content was expressed as gallic acid equivalents (gae). 2.3.6 antioxidant capacity antioxidant capacity determination methods aim to measure capacity of the antioxidant substances reliably and quickly. so far, various methods were developed, yet only several of them are recommended to be used together to determine the in vitro available antioxidant capacity. 2.3.6.1 dpph (2,2-diphenyl-1-picrylhydrazyl) assay antioxidant capacity of the beverages and digested extracts by using dpph free radical was measured using a modified version of the katalinic et al. (2006). in this method, the antioxidants were allowed to react with the stable radical in methanolic solution. the ital. j. food sci., vol. 31, 2019 45 discoloration of the dpph radicals was monitored through the decrease in absorbance at a characteristic wavelength during the reaction. first, 0.1 ml sample was added to 3.9 ml of 6 x 10-5 m methanolic solution of dpph radical and vortexed (vortex mixer classic, velp scientifica, italia) for 15-30 s. the reaction was allowed to occur in dark at room temperature for 30 mins. a trolox calibration curve (r2=0.9951) was obtained by measuring the reduction in absorbance of the dpph solution in 517 nm in the presence of different concentrations of trolox (10-100 µmol l-1). 2.3.6.2 frap (ferric reducing antioxidant power) assay according to benzie and strain (1996), 3 ml of daily prepared frap reagent (incubated at 37 °c) was mixed with 300 µl of distilled water and 100 µl of the test sample (or extraction solvent for the reagent blank). the test samples, digested extracts and blank were incubated at 37 °c for 60 min. at the end of incubation, absorbance was measured immediately at 595 nm. the frap reagent was prepared by mixing 25 ml of 0.3 mol l-1 acetate buffer (ph 3.6), 2.5 ml of 20 mmol l-1 fecl3 x 6 h2o and 2.5 ml 10 mmol l-1 tptz solution in 40 mmol l-1 hcl. the results were calculated from calibration curve as µmol trolox ml-1 for beverages (r2=0.9975). 2.3.6.3 cuprac (cupric ion reducing antioxidant capacity) assay estimation of cupric ion reducing antioxidant capacity was achieved based on the method of apak et al., (2008). 1 ml 1 x 10-2 m copper (ii) chloride + 1 ml 7.5 x 10-3 m neocuproine + 1 ml 1 m ammonium acetate were added to x ml 10-3 m antioxidant neutral solution + (1x) h2o:vt =4 ml; and the final absorbance was measured at 450 nm after 30 min (r2=0.9947). calculation of antioxidant capacity was done as trolox equivalents (teac values). 2.3.7 sensory analysis the beverages were evaluated based on color, odor, appearance and taste by a panel consisting of 10 trained members using a ranking test (altuğ and elmaci, 2011). according to this test, the panelists ranked the samples from their best favourite one to their least favorite by giving points between 1 and 6. as a result of this statistical test, samples within the range of 22 48 did not show any statistical difference while the samples that ranked below 22 (the mean of all of the panelist’s point values) were preferred and samples ranked above 48 were rejected at the 95 % probability level (p < 0.05). 2.3.8 statistical analysis the experiment was conducted in a completely randomized design with three replications. the results were statistically evaluated by one-way analysis of variance (anova) using the jmp software package version 6.0 (sas institute inc. nc, 27513). when significant differences were found (p < 0.05), the least significant difference (lsd) test was used to determine the differences among means. ital. j. food sci., vol. 31, 2019 46 3. results and discussion 3.1. physico-chemical properties in this study, the physico-chemical properties of dried lemon verbena leaves and beverages were performed in addition to the bioactive content. the moisture content of dried lemon verbena leaves was measured as 7.24±0.04 g 100 g-1. this result was regarded appropriate by the limits turkish standards institute, which was defined as 10 g 100 g-1 max for dried herbs (anonymous, 2014). ebadi et al., (2015) similarly determined 9 g 100 g-1 moisture content in lippia citriodora kunth leaves with different drying methods. physico-chemical properties of lemon verbena beverages are shown in table 1. all data in tables are expressed as means±standard deviations (n = 3). the brix and titratable acidity values of the beverages were adjusted based on the results of market survey on similar beverages conducted prior to the production (ice tea, natural and flavored mineral water, lemonade). the differences between the water soluble dry matter contents of all beverages were found statistically significant (p < 0.05) (table 1). titratable acidity of the beverages was lower than the sum of citric acid and ascorbic acid added in the production. this decrease could be explained by neutralization of the acidity by the hardness of the water used or the buffer salts of the extract (cemeroğlu, 2007). the highest acidity value of ecb could be the result of dissolution of carbondioxide in aqueous medium as carbonic acid (adebayo et al., 2015). the ph values of all beverages were similar with the ph values of some commercial herbal tea beverages determined by phelan and rees (2003). table 1. physico-chemical properties of lemon verbena beverages. analyses sb scb smb eb ecb emb water soluble dry matter (g 100 g-1) 8.10±0.00a 7.50±0.01c 7.80±0.00b 5.20±0.00e 5.50±0.10d 4.90±0.00f titratable acidity (g 100 ml-1)* 0.18±0.00 b 0.17±0.01c 0.15±0.00d 0.18±0.00b 0.19.±0.01a 0.14±0.00e ph 3.32±0.01d 3.62±0.00b 3.79±0.01a 3.33±0.00d 3.54±0.00c 3.79±0.02a ascorbic acid (mg 100 ml-1) 20.48±0.27 bc 28.15±0..30a 20.30±1.60bc 19.36±2.06c 29.57±0.47a 22.19±0.36b color l** 11.63±0.15cd 12.20±0.26ab 12.53±0.31ab 12.03±0.49bc 11.47±0.31d 12.70±0.17a a** -1.63±1.00a -3.73±0.49d -3.07±0.38bcd -2.37±0.46ab -2.60±0.20abc -3.37±0.49cd b** 5.33±0.21bc 6.07±0.11 a 4.90±0.40c 5.70±0.20ab 5.70±0.26ab 5.40±0.17b ntu*** 5.14±0.22b 2.40±0.04f 4.02±0.20c 6.00±0.10a 2.86±0.14e 3.38±0.19d sb: sucrose added beverage; scb: sucrose added and carbonated beverage; smb: sucrose added and mineral enriched beverage. eb: energy reduced beverage; ecb: energy reduced and carbonated beverage; emb: energy reduced and mineral enriched beverage. *: citric acid. ** l means lightness of the beverages, and ranges from black to white (0-100). a negative value of a indicates green, while a positive value indicates red-purple color. positive b indicates yellow and negative blue color. *** nephelometric turbidity unit. (mean values within a column with unlike superscript letters were significantly different (p < 0.05)) ital. j. food sci., vol. 31, 2019 47 the ascorbic acid content of beverages was contributed as both antioxidant source and acidity regulator with citric acid. the highest ascorbic acid contents of scb and ecb samples were related with the production process in which samples were carbonated after addition of antimicrobial agents instead of pasteurization. therefore, the thermal degredation of ascorbic acid was not occured in these beverages. reduction of ascorbic acid in other samples was related with the heat treatment (lešková et al., 2006). the differences between all samples were found to be statistically significant (p < 0.05). the ascorbic acid content of lemon verbena leaves were 5.73±0.27 mg 100 g-1 and the higher ascorbic acid values of beverages compared to the raw-material in this study can be explained by the intentional addition of ascorbic acid during production. costa et al., (2012) reported the ascorbic acid content as 7.20±0.20 mg 100 ml-1 in a beverage prepared with the 0.75 % infusion of rooibos red tea leaves (aspalathus linearis) and 21.40±0.10 mg 100 ml-1 in a beverage produced with 1 % green tea (camellia sinensis) infusion. suna et al., (2016) determined the ascorbic acid content of a mineral enriched erica arborea herbal tea beverage as 28.15±0.30 mg 100 ml-1. the difference between the results might be due to the variety of herb used in the process or its concentration. additionally, tamer et al., (2016) determined ascorbic acid content of linden enriched lemonade as 597.9 mg kg-1 which was found higher than our results as a consequence of high amount of ascorbic acid coming from lemonade. according to the results of the statistical analysis, the overall color parameters for beverages are affected significantly by the different production processes (p < 0.05). as shown in table 1, beverages were in green and yellow colour tones. while emb had the highest l (brightness) value, ecb had the lowest value. the highest turbidity value (6.00±0.1) was measured to be the sample eb, which also had the highest total phenolic content (table 3). it is stated that, chemical turbidity consisted of complexing of some organic compounds like starch, polyphenols, proteins, pectin and minerals like cu and fe (siebert, 1999). accordingly, polyphenols may increase the turbidity by complexing with metals and proteins over time (beveridge, 1997). the color and turbidity values of the products could not be compared due to the absence of similar beverage. 3.2. mineral contents mineral content of raw-material and beverages are given in table 2. it is clear that plants take minerals, which are essential for their life-cycle, from soil. the mineral composition of the plants is also affected from the physical and chemical characteristics of soil, usage of natural or artifical fertilizers, storage conditions, climate, region etc. additionally, the mineral content of the infusions obtained from these plants vary with the mineral amount in leaves and extraction yield (costa et al., 2002). the beverages were rich in k, ca and mg as the raw material used in their production. the same minerals were found in similar values in herbal tea of lippia multiflora in various researches (tettey-larbi, et al. 2015; christine, et al. 2017). fe, ca, mg and k content of water used in our process was 0.03 mg l-1, 13.0 mg l-1, 1.72 mg l-1 and 0.51 mg l-1, respectively. the highest amount of ca and mg were determined in smb and emb whereas ecb had the highest amounts of fe and k. while dried lemon verbena leaves and water used in process were rich in ca, k was higher in beverages. it could be explained with different extraction ratios of minerals. regarding this issue, pytlakowska et al. (2012) studied efficiency of mineral extraction from tea leaves and classified the elements in herb infusions as highly-extractable (>55 %) as k; moderately-extractable (20-55 %) including mg, na, p, b, zn and cu and poorlyextractable (<20 %) comprising al, fe, mn, ba, ca and sr. the researchers also determined the content of some minerals in melissa officinalis infusion (1 %, 10 min brewed) and reported values of 3.90±0.07 µg g-1 (mg kg-1) for fe, 21.0±0.20 mg kg-1 for ca, 198.00±5.00 mg ital. j. food sci., vol. 31, 2019 48 kg-1 for mg, 1449.00±12.00 mg kg-1 for k, respectively. similarly, özcan and akbulut (2008) analysed the mineral content of melissa officinalis infusion (2 %) as 20.11 mg 100ml-1 for ca. while our fe and ca values were higher, mg and k contents were lower than these studies. it could be related to raw material and differences in processing conditions. table 2. mineral content of dried lemon verbena leaves (mg kg-1) and beverage samples (mg l-1). fe ca mg k sb 0.11±0.00c 89.72±2.93d 28.87±0.56d 184.35±4.58c scb 0.26±0.00b 103.58±1.16c 30.26±0.37c 193.48±2.81b smb 0.01±0.00d 122.48±5.17b 43.47±1.29b 121.38±3.37e eb 0.12±0.05c 88.98±0.74d 28.57±0.25d 197.38±1.10b ecb 0.37±0.00a 87.51±0.7d 27.98±0.22d 222.28±1.71a emb 0.01±0.00d 129.85±1.85a 46.21±0.75a 130.78±1.160d raw-material 82.61±1.30 24800±0.02 2814.78±20.00 15600±0.00 *mean values within a column with unlike superscript letters were significantly different (p < 0.05). 3.3. phenolic contents phenolic compounds play an important role regarding in antioxidant effects and defensive action in plants or the human body (boo et al., 2012). total phenolic contents of the beverages were given in table 3. table 3. total phenolic contents of the beverages. sample total phenolics (mg gae 100 ml-1) sb 209.35±3.77d scb 239.33±14.64c smb 232.25±7.67c eb 360.78±14.11a ecb 302.55±11.84b emb 306.80±7.50b mean values within a column with unlike superscript letters were significantly different (p < 0.05). in the average of 2.74-4.71 % of dried lemon verbena polyphenols (7653.46±36.62 mg gae/100 g-1) were transferred to beverages after extraction process. energy reduced beverages had generally higher total phenolic contents than sucrose added beverages. among these eb showed the highest total (360.78±14.11 mg gae 100 ml-1) phenolic value. due to the lack of literature data dealing with phenolic content of lemon verbena beverages, our results were compared with polyphenol content of similar types of samples. for instance, dias et al. (2012) analysed total phenolic content of melissa officinalis infusion (0.50 %) in lyophilized extracts of commercial bag and granulated forms as 959.54±10.02 mg gae ml-1 and 657.06±0.80 mg gae ml-1 respectively, whereas costa et al., (2012) determined green tea infusions’ (1 %) total phenolic content as 29.10±0.50 mg gae 100 ml-1. guimarães et al., (2011) studied infusion and decoction of lemon verbena ital. j. food sci., vol. 31, 2019 49 and fennel mixed herbs and reported phenolics value of decoction and infusion respectively as 389.73±4.00 mg gae g-1 and 438.08±0.19 mg gae g-1. atoui et al., (2005) also determined total phenolic content of 1.25 % infusion of chinese green tea (camellia sinensis) and greek mountain tea (sideritis syriaca) approximately as 507 mg ga 100ml-1 and 37 mg ga 100 ml-1, respectively. our results were different from the literature data owing to the differences in extraction method, concentration and the material. according to velioglu et al., (1998) antioxidant activity and total phenolics were found to be positively and significantly correlated. 3.4. antioxidant capacity and bioaccessibility the antioxidant capacity and the bioaccessibilities of the antioxidant capacity of the beverages are given in table 4. the differences between the antioxidant capacities of the beverages were significant (p < 0.05). the different values obtained from the three assays are due to the different reaction mechanisms or kinetics of the test materials (i.e. dpph, cu2+ and fe3+) quenched/reduced by beverages react according to different mechanism and kinetics (jeszka-skowron et al., 2015). however, the extraction method or differences in concentration make difficult to compare the results. yoo et al., (2008) reported dpph antioxidant capacity of lemon verbena infusion as 86.90±2.20 %. sb, eb and ecb samples analysed with respectively frap, cuprac and dpph assays showed higher antioxidant capacity values than those of others (table 4). table 4. bioaccessibility of antioxidant capacity of the beverages (!mol trolox ml-1). sample dpph (μmol trolox ml-1*) dpph bioaccessibi lity ( %) frap (μmol trolox ml-1*) frap bioaccessi bility ( %) cuprac (μmol trolox ml-1*) cuprac bioaccessibi lity ( %) sb 27.17±0.09a 0.99 32.00±0.78a 19.63 73.35±1.27b 9.26 scb 23.75±0.70b 0.93 19.97±0.25cd 27.84 62.65±6.89c 10.39 smb 24.85±2.64b 0.85 17.55±2.35d 47.01 30.46±6.53e 11.13 eb 27.64±0.08a 0.58 26.60±1.82b 32.07 81.72±2.11a 7.13 ecb 27.78±0.30a 0.50 22.61±1.51c 43.74 69.24±0.84bc 7.47 emb 27.03±0.27a 0.89 19.19±1.72d 37.99 40.83±1.97d 9.65 mean values within a column with unlike superscript letters were significantly different (p < 0.05). the bioaccesibility ratios were higher in order of frap, cuprac and dpph assays. especially smb had the highest bioaccessibility obtained from frap (47.01 %) and cuprac (11.13 %) assays. a possible reason of varying bioaccessibility of antioxidant capacity values could be associated with several factors related to the process conditions, chemical interactions with other phytochemicals, biomolecules present in the food and also the protocols used for the measurements (parada & aguilera, 2007). the bioaccessibility of antioxidant capacity was decrased in this study. in agreement with our data, henning et al., (2014) reported a 21.5 % and 8.1 % decrease of teac (trolox equivalent antioxidant capacity) in green tea and grape seed samples during in vitro simulated digestion. in another study, şahan et al., (2017) reported the bioaccessibility of chicory cultivars with teaccuprac and teacdpph assays between 62.12–73.48 % and 64.6676.21 %, respectively. değirmencioğlu et al., (2016) concluded teaccuprac bioaccessibility of fermented vegetable juices between 16-36 %. although the polyphenols supply major antioxidant potency of the samples, our results displayed that digestion may ital. j. food sci., vol. 31, 2019 50 alter antioxidant properties depending on the variations in polyphenol content (henning et al., 2014). besides, it is known that, structural changes after gi digestion affect both further polyphenol uptake and result in a significant loss of the antioxidant activity (rodríguez-roque et al., 2013). data of this study confirmed that different applications and methods may have an influence on the release of total phenols and their antioxidant capacity, therefore, on the bioaccessible fraction. likewise, phenolic compounds which demonstrate antioxidant capacity would be bioavailable after digestion and might contribute to protection of humans from several diseases (pérez-vicente et al., 2002). 3.5. sensory findings sensory properties of the beverages are shown in fig. 2. for sensorial test, panelists were briefed about the properties of the beverages. the product should have light yellow color related with the extract and should not contain any particles, while the typical odor and taste of the extract should be appreciated. there were no significant differences in color, odor, appearance and taste values between samples and all of the beverages were generally accepted by the panelists (p < 0.05). figure 2. sensory properties of lemon verbena beverages. 4. conclusions overall, the sucrose added and also the sucrose added and mineral enriched beverages were found to be the most nutritive beverages among our products because of the highest bioaccessibility values of antioxidant capacity. in general, energy reduced beverages had higher phenolic content. all samples were preferred as sensorial. as a result of growing market interest in functional drinks, natural herbal extracts became popular due to their high bioactive components. in addition, they are easy to formulate and process. nevertheless, bioaccessibility of a novel herbal tea beverage has not yet been reported in the literature data so far. from this perspective, the bioactive components and their bioaccessibilities in herbal infusions and herbal beverages are needed to be investigated in 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c.h., lee h., moon b. and lee c.y. 2008. relative antioxidant and cytoprotective activities of common herbs. food chem. 106 929-936. paper received february 12, 2018 accepted july 17, 2018 ijfs#1302_bozza ital. j. food sci., vol. 31, 2019 487 paper drying of lime slices by microwave and combined microwave-convective methods n. izli*1, o. taskin1 and g. izli2 1department of biosystems engineering, faculty of agriculture, university of bursa uludag, gorukle campus, 16059, bursa, turkey 2department of food engineering, faculty of natural sciences, architecture and engineering, university of bursa technical, 16190, bursa, turkey *corresponding author: tel.: +902242941604; fax: +902242941402 e-mail address: nazmiizli@gmail.com abstract this research deals with lime samples and aims to investigate the impacts of microwave and combined microwave-convective drying applications. according to the statistical outcomes, models of midilli et al. and page were discovered to give more suitable predictions than the other models. increasing level of both temperature and microwave power, induced a significant reduction in the drying interval while increasing deff values. drying experiment at power 90 w and temperature 55°c ensured the best values in color parameter of a* (greenness) and energy consumption. experiments made with using different techniques will help to select the appropriate drying technique for the relevant sectors. keywords: activation energy, color, drying kinetics, effective moisture diffusivity, specific energy consumption ital. j. food sci., vol. 31, 2019 488 1. introduction the persian lime (citrus latifolia tanaka) belongs to the family of rutaceae (citrus family) and also named as ‘shiraz limoo’, ‘bearss lime’, and ‘tahiti lime’, which is mainly grown in the gulf coast of mexico (lambert et al., 2015; salih, 2015; arredondo et al., 2015). it is an economically significant horticultural crop (kaewsuksaeng et al., 2015). the major flavor components are neural, citral, geranial, α-terpineol, β-bisabolene, βpinene, p-cymene, 1,4-cineole, 1,8-cineole, and 4-terpineol (yadav et al., 2004). it had been used traditionally to treat sinusitis (salih, 2015) and stomachache. also, the cleansing action of mesocarp usage as facial scrub helps in prevention of pimples. besides, the rind is burnt against mosquitoes in some homes (aibinu et al., 2007). human benefit from lime to extract its juice, to prepare beverages, concentrates, squashes and other derivative products such as pectin and citric acid etc. (yadav, 2004). to preserve food, drying is an ancient and prevalent technique. in the food industry, the primary reason to dry food is to reduce packaging, storage, and transportation costs by downsizing end products in terms of both volume and weight. hence, in the food industry, developing economical drying techniques have become a prominent research topic (phoungchandang et al., 2008; tasirin et al., 2014). long drying intervals or high temperatures in conventional air drying may distort the dried product (diaz et al., 2003). many studies have shown that drying with microwave energy usage has many conveniences like shorter drying interval, higher energy efficiency, less space, and faster start-up and shut-down periods (guo and zhu, 2014). however, inhomogeneous distribution in the microwave cavity creates an uneven heating problem. to circumvent a number of the disadvantages of single microwave or hot-air dryers, combine microwaves with hot air one is another methodology (darvishi et al., 2014). microwave and/or microwave–air combined driers were carried out by several researchers to illustrate drying attributes of many agricultural products, for example, parsley (soysal, 2004), peach (wang and sheng, 2006), mint leaves (özbek and dadali, 2007), spinach (karaaslan and tuçer, 2008), tomato pomace (alharahsheh et al., 2009), onion slices (arslan and özcan, 2010), seedless grapes (kassem et al., 2011), sage leaves (esturk, 2012), chili flesh (zhao et al., 2013), okra (kumar et al., 2014) and apple slices (zarein et al., 2015). the aim of conducted research to scrutinize the thin-layer drying behavior of lime slices in both microwave and microwave-convective drying with existing thin-layer drying models. also, effective moisture diffusivity, activation energy, color, energy consumption and specific energy consumption of the lime slices were studied. 2. material and methods 2.1. drying equipment and drying procedure fresh lime samples were obtained from a local grocery one day before the experiments begin and kept at 4±0.5°c temperature. samples had an average diameter of 40±0.08 mm and were sliced to 5±0.03 mm thickness by using a food slicer (nicer dicer, china). in the beginning, the moisture content of the fresh lime samples was identified to be 5.13 (g h2o · g d.m. -1) on a dry basis (d.b.) by using an oven drying at 105°c for 24 hours (ed115 binder, tuttlingen, germany). the drying experiments were conducted at room temperature. a microwave-convective oven (whirlpool amw 545, italy) that works at ∼230 v, 50 hz with 2450 mhz frequency. ital. j. food sci., vol. 31, 2019 489 the system worked in microwave mode at 90 w and 160 w microwave output power levels and in combined microwave-convective mode at 90 w – 55 °c, 90 w – 65 °c, 90 w – 75 °c, 160 w – 55 °c, 160 w – 65 °c, and 160 w – 75 °c microwave output power and temperature combinations with 1 m s-1 air flow. all drying experiments were performed on a 400 mm diameter and 210 x 450 x 420 mm sized glass plate that rotates at the base of the oven. in order to figure out mass, a digital balance (baster, istanbul, turkey) having ±0.01 g accuracy was positioned below the microwave oven device (fig. 1). the loss of the moisture of the lime samples was noted down during drying interval in every 5 minutes. for each lime sample (100 g), these were applied three times and their mean was figured out. figure 1. schematic diagram of experimental microwave-convective drying system (microwave-convective drying chamber (1), rotating glass tray (2) and balance (3)). 2.2. mathematical modelling of drying data the data on moisture content were transformed into the moisture ratio (mr) and adapted by the aid of ten thin-layer drying models (table 1). table 1. selected thin layer drying models used to mathematically model the lime drying kinetics. no model name model references 1 henderson and pabis )exp( ktamr −= bhattacharya et al. (2015). 2 newton )exp( ktmr −= sharma et al. (2005) 3 page )exp( nktmr −= bhattacharya et al. (2015). 4 logarithmic cktamr +−= )exp( unal and sacilik (2011) 5 two term )exp()exp( 10 tkbtkamr −+−= su et al. (2015) 6 two term exponential )exp()1()exp( kataktamr −−+−= sharma et al. (2005) 7 wang and singh 21 btatmr ++= su et al. (2015) 8 diffusion approach )exp()1()exp( kbtaktamr −−+−= unal and sacilik (2011) 9 verma et al. )exp()1()exp( gtaktamr −−+−= omolola et al. (2014) 10 midilli et al. btktamr n +−= )exp( unal and sacilik (2011) ital. j. food sci., vol. 31, 2019 490 the moisture ratio was calculated with the equation presented below: eo et mm mm mr − − = (1) above, mt represents the moisture content (g water g dry matter-1) at a particular time, m0 represents the initial moisture content (g water g dry matter -1), represents the equilibrium moisture content (g h2o · g d. m. -1). me values are proportionately less than mt or m0 values. in consequence, the moisture ratio was made simpler as presented below (arumuganathan et al., 2009): o t m m mr = (2) 2.3. determination of effective moisture diffusivity drying of agricultural products in a falling rate period is embedded into a mass-diffusion equation in accordance with the second law of fick on diffusion is presented in eq. (3) below: 𝜕𝑀 𝜕𝑡 = ∇[𝐷!"" ∇𝑀 ] (3) so as to ascertain the moisture ratio in eq. (4), benefiting from the second law of fick on unsteady state diffusion provided in eq. (3) is possible. crank (1975) put forward the clarification of the diffusion equation for an infinite slab and so uniform moisture distribution at the beginning, negligible shrinkage and external resistance, and constant diffusivity was assumed to be as follows: 𝑀𝑅 = ! !! ! (!!!!)! exp − (!!!!)!!!!!""! !!! ! !!! (4) where: deff symbolizes effective moisture diffusivity in terms of (m2 ·s-1); t symbolizes an interval in terms of (s); l symbolizes half-thickness of samples in terms of (m), and n symbolizes a positive integer. for long drying intervals, only the first term in eq. (4) is significant and the equation is simplified to the one stated below: 𝑀𝑅 = ! !! 𝑒𝑥𝑝 − !!!!""! !!! equation (5) can be articulated in a logarithmic format as can be seen below: ln 𝑀𝑅 = ln ! !! − !!!!""! !!! (5) ital. j. food sci., vol. 31, 2019 491 in eq. (6), effective moisture diffusivity values were identified by plotting experimental drying data in ln (mr) versus drying interval. this plot generates a direct line with a slope calculated below (doymaz et al., 2015): 𝑆𝑙𝑜𝑝𝑒 = ! !!!!! !!! (6) 2.4. computation of activation energy on the grounds that temperature inside the microwave dryer cannot be gauged precisely, modification of the revised arrhenius equation is used to find the activation energy. in this technique, it is deemed that the effective moisture diffusion and the microwave output power ratio are associated with the sample weight (m p-1) rather than air temperature. in this direction, eq. (7) can be utilized in an efficient way, as noted below (özbek and dadali, 2007): 𝐷!"" = 𝐷! − !!! ! (7) above ea symbolizes the activation energy in terms of (w g-1), m symbolizes the mass of raw sample in terms of (g), p symbolizes the microwave power in terms of (w), and lastly, d0 symbolizes the pre-exponential factor in terms of (m2 s-1). 2.5. energy and specific energy consumption the experimental apparatus was plugged into the electricity via a digital electric counter (kaan, type 101, turkey) that has a precision of 0.01 kwh. with the use of the counter, the total energy consumption (et) was measured for whole drying process (kowalski and pawłowski, 2011). energy consumption to dry 100 g fresh lime sample was obtained by applying equation 8. the ekg and w0 symbolize the specific energy needed and the primary mass of the sample, respectively (motevali et al., 2012). 𝐸!" = !! !! (8) 2.6. colour measurement the flesh color of fresh and dried lime samples was determined with hunterlab color analyzer (msez-4500l, reston, virginia, usa) in the l, a, b color scale. color measurements were conveyed in a three-dimensional color space namely l*, a*, and b*, where l* indicates darkness⁄lightness value, a* indicates redness value (if positive) and greenness value (if negative), and b* indicates the yellowness value (if positive) and blueness value (if negative). on the other side l0*, a0* and b0* are color parameters for fresh lime samples. after the calibration of colorimeter against a standard white surface and black one, six replicate measurements were performed for each sample and l*, a*, b*, l0*, a0* and b0* color values were recorded. to illustrate the color changes, chroma (c), hue angle (α) , and total color variance (∆e) values were defined by the following equations (argyropoulos et al., 2011): )( 22 bac += (9) ital. j. food sci., vol. 31, 2019 492 )(tan 1 a b−=α (10) δe = 222 *)*(*)*(*)*( 000 bbaall −+−+− (11) 2.7. statistical analysis this research was realized by the aid of randomized plots factorial design of experimental type. in the course of calculation of the inspected items, three replicates were utilized. while interpreting the outcomes, matlab (mathworks inc., natick, ma) and jmp (version 7.0, sas institute inc., cary, nc, usa) software technologies were employed. significance levels of mean differences were tested and the least significant difference (lsd) test resulted in a 5% significance level. it has been determined that the most convenient model that expresses the drying attributes of lime samples in a thin layer is the one that has lowest reduced chi-squared (χ2) value, lowest root mean square error (rmse) value and the highest coefficient of determination (r2) (arumuganathan et al., 2009). the statistical figures mentioned above are formulated below: nn mrmr n i̇ iprei − − = ∑ =1 2 ,exp, 2 )( χ (12) n mrmr rmse n i̇ iipre∑ = − = 1 exp,, )( (13) where: mrexp,i , symbolizes the experimental moisture ratio for test number i, mrpre,i , symbolizes the estimated moisture ratio for test number i, n symbolizes the observation number, n symbolizes the number of constants in the drying model (doymaz and ismail, 2011). 3. results and discussion 3.1. drying kinetics of dried lime fig. 2 illustrates the drying curves of the lime samples that are dried under distinct microwave and microwave-convective drying settings. drying phase of lime samples took 225, 120, 130, 115, 100, 105, 85 and 75 minutes at 90w, 160w, 90 w – 55°c, 90 w – 65°c, 90 w – 75°c, 160 w – 55°c, 160 w – 65°c, and 160 w – 75°c, respectively. outcomes of the experiments implied that drying intervals of lime samples dried by microwave-convective technique at 90 w – 75°c and 160 w – 75°c reduced 55.6% and 41.7% in comparison to only 90 w and 160 w microwave powers, respectively. the outcomes stated above are in good harmony with former researches. gowen et al. (2008), mohanta et al. (2014), chayjan et al. (2015) found that the use of combined microwave convective technique ensured considerable time savings in drying interval for soybean, ginger and hawthorn ital. j. food sci., vol. 31, 2019 493 samples, respectively. in addition, the average total drying interval at 160 w – 75°c become shorter by 46.2% as against the drying interval at 90 w – 55°c. it can be said that increasing the microwave power level and temperature level ended in a significant reduction in the drying interval of lime samples in combined microwave-convective drying technique. in agreement to our study, karaaslan and tuncer (2010) combined fan-assisted convection (100, 180 and 250°c) and microwave (180 and 540 w) drying and the time to reduce the moisture content of banana slices from the initial 80 % (w.b.) to the final 15 % (w.b.) was highest at 180 w – 100°c and lowest at 540 w – 250°c, respectively. similarly, sadeghi et al. (2013) were measured drying time of lemon slices about 80, 78 and 73 minutes, respectively, when applying 0.97 w g−1 microwave power at 50, 55 and 60°c convective drying. figure 2. a comparison of the experimental and theoretical moisture ratios predicted by the midilli et al. and page models at specific drying times under selected drying conditions (microwave (a) and microwaveconvective (b)). ital. j. food sci., vol. 31, 2019 494 3.2. fitting of drying curves lime table 2 reveals the statistical analysis values obtained from the 10 distinct thin layer drying models, covering the drying model coefficients and the comparison criteria that are benefited from to evaluate the congruous quality, r2, rmse, and χ2. for the statistical parameters, the values varied between 0.9143 and 0.9998 for r2, between 0.0042 and 0.1037, 0.1659 x 10-4 and 106.3810 x 10-4 for rmse and χ2, respectively. in table 3, the page et al. model presented greater r2 and smaller rmse and χ2 values in comparison to other thinlayer drying models for microwave-convective combinations of 90w – 75°c, while the midilli et al. model displayed more suitable statistical values for all other drying settings. in all cases, values of r2, rmse and χ2 in the midilli et al. and page models were ranged from 0.9984 to 0.9998, 0.0042 to 0.0143 and 0.1659 x 10-4 to 1.8507 x 10-4; and 0.9958 to 0.9997, 0.0046 to 0.0211 and 0.1956 x 10-4 to 4.3222 x 10-4, in return. based on the statistical values, the midilli et al. model was the most convenient one for all drying conditions tested, except for drying with the microwave-convective combinations of 90 w – 75°c where the page model is the best one. fig. 2 presents the comparison between the predicted values and experimental ones using the most convenient models with drying interval at chosen drying conditions of lime. as can be seen from these figures, the midilli et al. and page models slightly over-predicted or under-predicted the experimental values, but they are quite close to the experimental results. accordingly, it could be deduced that models of midilli et al. and page sufficiently explained the thin layer drying attributes of lime under the experimental conditions. similar outcomes have been stated by unal et al. (2011), bhattacharya et al. (2015) and su et al. (2015) for midilli et al. and sharma et al. (2005), doymaz and i̇smail (2011), therdthai et al. (2011) for page model. table 2. estimated values of coefficients and statistical analyses obtained from various thin layer drying models for drying of lime using microwave (90 and 160 w) method. no 90 w 160 w model coefficients r2 rmse χ2(10-4) model coefficients r2 rmse χ2(10-4) 1 a=1.164; k=0.01247 0.9632 0.0641 40.4278 a=1.123; k=0.02029 0.9695 0.0566 32.0739 2 k=0.01078 0.9374 0.0836 69.1906 k=0.01803 0.9522 0.0709 50.5494 3 k=0.000555; n=1.641 0.9990 0.0104 1.0139 k=0.002511; n=1.483 0.9958 0.0211 4.3222 4 a=1.403; k=0.00734 c=-0.3089 0.9888 0.0354 12.2592 a=1.505; k=0.01025 c=-0.4523 0.9969 0.0181 3.1788 5 a=-23.08; ko=0.02414 b=24.08; k1=0.02306 0.9955 0.0224 4.8956 a=-14.31; ko=0.03806 b=15.32; k1=0.03578 0.9929 0.0274 7.3938 6 a=0.0000627; k=171.7 0.9360 0.0845 70.7869 a=0.0000608; k=296.6 0.9501 0.0725 52.7683 7 a=-0.00774; b=0.0000141 0.9861 0.0394 15.2627 a=-0.01276; b=0.0000362 0.9954 0.0221 4.8176 8 a=-21.79; k=0.02327 b=0.9567 0.9943 0.0252 6.0009 a=-30.8; k=0.03781 b=0.9704 0.9934 0.0264 6.5723 9 a=-33.45; k=0.02396 g=0.02321 0.9956 0.0221 4.7390 a=-13.74; k=0.03845 g=0.036 0.9934 0.0265 6.8890 10 a=0.9905; k=0.0005298 n=1.645; b=-0.0000446 0.9992 0.00946 0.8400 a=1.016; k=0.005021 n=1.272; b=-0.000886 0.9987 0.0119 1.2692 ital. j. food sci., vol. 31, 2019 495 table 3. estimated values of coefficients and statistical analyses obtained from various thin layer drying models for drying of lime using combined microwaveconvective method. no 90 w – 55oc 90 w – 65oc 90 w – 75oc model coefficients r2 rmse χ2(10-4) model coefficients r2 rmse χ2(10-4) model coefficients r2 rmse χ2(10-4) 1 a=1.135; k=0.01856 0.9609 0.0651 43.1892 a=1.142; k=0.02208 0.9617 0.0659 42.9539 a=1.149; k=0.02614 0.9592 0.0696 49.2828 2 k=0.01632 0.9404 0.0804 66.3224 k=0.01936 0.9402 0.0824 67.7189 k=0.02285 0.9367 0.0867 76.2392 3 k=0.001448; n=1.58 0.9962 0.0202 3.9792 k=0.001695; n=1.606 0.9981 0.0145 1.8601 k=0.001792; n=1.66 0.9996 0.0073 0.6060 4 a=1.637; k=0.008336 c=-0.5794 0.9951 0.0231 5.2789 a=1.511; k=0.01135 c=-0.4383 0.9919 0.0302 8.4956 a=1.461; k=0.01441 c=-0.3763 0.9873 0.0388 15.2013 5 a=-46.82; ko=0.0353 b=47.82; k1=0.03455 0.9919 0.0297 8.8554 a=-32.98; ko=0.04255 b=34; k1=0.04126 0.9943 0.0255 6.1631 a=-35.94; ko=0.05143 b=36.95; k1=0.04992 0.9965 0.0203 4.4728 6 a=0.0000609; k=267.5 0.9379 0.0819 68.9980 a=0.0000627; k=308.7 0.9374 0.0842 70.8216 a=0.0000636; k=359.3 0.9333 0.0889 80.2788 7 a=-0.01129; b=0.0000262 0.9931 0.0274 7.7391 a=-0.01364; b=0.0000412 0.9894 0.0347 11.6260 a=-0.01629; b=0.000061 0.9842 0.0432 18.9157 8 a=-24.67; k=0.03574 b=0.9605 0.9921 0.0293 8.2467 a=-20.28; k=0.0433 b=0.9505 0.9949 0.0241 5.2427 a=-40.28; k=0.05181 b=0.9731 0.9970 0.0189 3.7127 9 a=-43.46; k=0.03522 g=0.03443 0.9921 0.0293 8.6181 a=-42.65; k=0.04245 g=0.04144 0.9949 0.0240 5.5011 a=-15.08; k=0.053 g=0.04934 0.9970 0.0190 3.9366 10 a=1.002; k=0.002416 n=1.42; b=-0.0006716 0.9986 0.0121 1.3012 a=1.003; k=0.002283 n=1.514; b=-0.0003645 0.9989 0.0114 0.9357 a=1.001; k=0.001906 n=1.641; b=-0.0000735 0.9995 0.0074 0.6163 no 160 w – 55oc 160 w – 65oc 160 w – 75oc model coefficients r2 rmse χ2(10-4) model coefficients r2 rmse χ2(10-4) model coefficients r2 rmse χ2(10-4) 1 a=1.151; k=0.02433 0.9590 0.0694 49.2496 a=1.143; k=0.0294 0.9560 0.0728 51.7650 a=1.137; k=0.03362 0.9393 0.0872 74.3240 2 k=0.02119 0.9352 0.0873 78.1182 k=0.02579 0.9346 0.0888 76.5298 k=0.02962 0.9204 0.0999 98.7500 3 k=0.001568; n=1.665 0.9997 0.0046 0.1956 k=0.002196; n=1.661 0.9986 0.0131 1.7386 k=0.001827; n=1.779 0.9972 0.0188 3.1235 4 a=1.498; k=0.01294 c=-0.4127 0.9882 0.0373 14.2787 a=1.568; k=0.01441 c=-0.4931 0.9897 0.0352 11.5807 a=1.89; k=0.01257 c=-0.8272 0.9869 0.0405 16.0290 5 a=-29.68; ko=0.04847 b=30.69; k1=0.04673 0.9971 0.0183 3.4801 a=-18.37; ko=0.05935 b=19.38; k1=0.05599 0.9950 0.0246 5.3362 a=-22.73; ko=0.06944 b=23.74; k1=0.06613 0.9872 0.0400 14.7394 6 a=0.0000597; k=354.9 0.9319 0.0894 82.0501 a=0.0000647; k=398.8 0.9305 0.0915 81.3402 a=0.0000758; k=390.9 0.9143 0.1037 106.3810 7 a=-0.015; b=0.0000502 0.9842 0.0431 19.3033 a=-0.01796; b=0.0000689 0.9864 0.0405 15.1585 a=-0.01974; b=0.000069 0.9844 0.0443 19.6322 8 a=-14.75; k=0.04954 b=0.9292 0.9975 0.0171 2.8964 a=-21.74; k=0.05933 b=0.9512 0.9955 0.0233 5.1105 a=-24.99; k=0.06934 b=0.9558 0.9890 0.0372 11.7403 9 a=-15.14; k=0.04946 g=0.04604 0.9975 0.0171 3.0502 a=-14.52; k=0.06004 g=0.05575 0.9955 0.0233 5.4618 a=-20.43; k=0.0702 g=0.06641 0.9890 0.0372 12.7526 10 a=1.002; k=0.001717 n=1.638; b=-0.0000823 0.9998 0.0042 0.1659 a=1.007; k=0.002971 n=1.564; b=-0.0004321 0.9991 0.0104 1.0207 a=0.9896; k=0.002083 n=1.714; b=-0.0006544 0.9984 0.0143 1.8507 ital. j. food sci., vol. 31, 2019 496 3.3. effective moisture diffusivity effective moisture diffusivity of microwave and microwave-convective dried lime slices ranged from 1.95 x 10-9 and 5.84 x 10-9. it is clear in table 4 that deff values showed an increase with the rise in microwave power and heating temperature. 160 w 75 °c drying setting has the maximum effective moisture diffusivity value and 90 w has the minimum effective moisture diffusivity value. this can be explained by the higher heat caused the higher mass transfer. the diffusivity values derived from the present study were in agreement with values proposed in the literature. doymaz et al. (2015) stated that deff values for dried agricultural products were generally varied between 10–8 and 10–12 m2/s. darvishi et al. (2014) found that deff values of lemon slices dried at 180, 360, 540 and 720 w microwave power level were 1.87, 2.48, 3.29 and 3.95 x 10-8, respectively. similarly, sadeghi et al. (2013) studied on lemon slices dried by using combined microwave convective drying method at 50, 55 and 60°c inlet hot-air temperatures and microwave powers of 185.5 w and 388.5 w. the lowest and highest values of deff are 5.45 × 10−10 (185.5 w 50°c) and 1.25 × 10−9 m2 s−1 (388.5 w – 60°c), respectively. all of the outcomes above verify the rule that the rise in deff leads to decline in the drying interval. 3.4. activation energy the results indicated that ea values of lime are 1.05 w g-1 during microwave drying and 15.99-19.33 w g-1 for microwave-convective drying combinations at 90 w and 160 w, in return. these findings are in congruence with the former researchers. to give an example, zarein et al. (2015) found that the ea of microwave dried apple samples at 200, 400 and 600 w was 12.15 w g-1. the similar trend was determined by amiri chayjan et al. (2015) for hawthorn fruit drying with microwave-convective drying technique. in general, the obtained results of the current study were well consistent with previous high moisture agricultural and food materials studies (ravula et al., 2017). 3.5. energy analysis based on the benchmarking of the energy and specific energy consumption figures obtained during drying experiments of lime samples, it is found that in the 90 w – 55 °c combination required the minimum volume of energy and specific energy, whereas 90 w required the maximum volume of energy and specific energy. out of these drying experiments, the microwave-drying ones consume more energy and they are not costeffective to dry lime samples, whereas the microwave-convective drying combinations are energy-efficient and they consume less energy to dry lime samples. taking into consideration that energy costs high all around the world, a combination of microwaveconvective drying method seems to be an outstanding alternative. according to table 5, the specific energy requirement is a function of air temperature and microwave power, such that when the microwave power level is constant, specific energy requirement to dry lime surges with the rise in air temperature. as drying rate is associated with microwave power and air temperature linearly, it could be detected that the surge in the microwave power and the air temperature consequently end up with the decline in drying interval. similarly, demirel and ismail (2017) stated that total energy consumption depends on the overall drying interval. out of all the drying techniques, the optimal energy consumption arose through microwave drying at 180 w power level with 0.042 kwh energy consumption. the overall energy consumption in all of the combined microwave-convective drying test processes of nectarine was found between 2.03 4.25 ital. j. food sci., vol. 31, 2019 497 kwh. as varith et al. (2007) investigated the peeled longan drying with microwave-hot air combination. the least required specific energy consumption was found 29.7 mj kg-1 water with mw400-300/40-60 and specific energy consumption can reduce the up to 48.2%. table 4. effective moisture diffusivities of dried lime samples. drying method deff (m 2 s-1) 90 w 1.95 x 10-9 90 w 55 °c 3.24 x 10-9 90 w 65 °c 3.89 x 10-9 90 w 75 °c 4.54 x 10-9 160 w 3.24 x 10-9 160 w 55 °c 3.89 x 10-9 160 w 65 °c 5.19 x 10-9 160 w 75 °c 5.84 x 10-9 table 5. energy consumption values of dried lime samples. drying method drying time (min) energy consumption (kwh) specific energy consumption (kwh kg-1) 90 w 269 0.85 10.36 90 w 55 °c 131 0.57 6.93 90 w 65 °c 118 0.60 7.40 90 w 75 °c 97 0.67 8.17 160 w 131 0.57 7 160 w 55 °c 120 0.61 7.48 160 w 65 °c 77 0.62 7.5 160 w 75 °c 33 0.63 7.64 3.6. colour analysis because of to being highly appreciated quality parameter, the effect of drying on the color should be minimized (vega-gálvez et al., 2009). the results concerning the color changes under different experimental conditions are compared to the fresh sample in table 6. as the increase in drying air temperature at constant microwave power lead to decreases in l* values and increases in values of b*. the decrease of l* value implies darker color for all dried lime samples. while negative a* value (greenness) was seen for fresh lime samples (-3.79), it has turn to positive value (redness) in all dried lime samples. besides, the drying at 160 w (28.08) has less negative effect than 90 w (29.21) in color value of b*. according to the ∆e parameter, there were no significant differences except 160 w – 65°c and 160 w – 75°c drying conditions (p<0.05). previous studies such as fadavi and mehrabi (2013), who considered the different temperature treatments (shadow, sun-dried, 40, 105 and 200°c) effect on dried lime and defined that the attractive color is a necessary quality parameter. similar outcomes have been put forward by díaz et al. (2011). ital. j. food sci., vol. 31, 2019 498 table 6. color values of fresh and dried lime samples. drying method color parameters l* a* b* c α° ∆e fresh 62.58±0.06a -3.79±0.08a 26.39±0.05a 26.67±0.06a 98.14±0.17a 90 w 57.52±0.34d 5.14±0.06cd 29.21±0.33cd 29.66±0.33cd 80.06±0.01c 16.84±0.19a 90 w 55 °c 60.78±0.35b 2.90±1.58b 30.81±2.19de 30.96±2.31de 84.80±2.66b 16.64±2.61a 90 w 65 °c 58.88±0.03c 3.60±0.01b 30.10±0.02de 30.31±0.01de 83.23±0.01b 16.56±0.02a 90 w 75 °c 52.78±0.19g 5.62±0.91cd 27.10±0.88ab 27.69±0.67ab 78.29±2.25cd 17.59±0.24a 160 w 54.16±0.23f 4.84±0.49c 28.08±1.53bc 28.50±1.42bc 80.21±1.55c 17.18±1.01a 160 w 55 °c 57.79±0.08d 3.42±0.01b 31.30±0.03e 31.49±0.03e 83.81±0.02b 17.85±0.01a 160 w 65 °c 56.05±0.04e 5.81±0.02cd 29.97±0.01de 30.53±0.01de 79.08±0.01cd 18.26±0.02ab 160 w 75 °c 49.24±0.09h 6.19±0.03d 26.98±0.06ab 27.68±0.06ab 77.12±0.02d 19.95±0.03b a-g mean that different letters in same column with differ significantly (p < 0.05). the rise of drying temperature from 60 to 70 °c without pretreatment resulted color degradation in 3 and 5 mm lime slices. however, the pretreated drying processes end up with fewer color changes and culminate in a product quality analogous to the fresh fruit. 4. conclusions in this research, the influences of microwave and combined microwave-convective drying techniques on the kinetics of drying, color, effective moisture diffusivity, activation energy and energy consumption of lime samples were studied. the obtained outcomes showed that the combined microwave-convective drying technique enabled significantly higher time-saving than microwave drying. the experimental moisture ratio data were tailored to the chosen 10 different thin-layer drying models and the drying attributes are illustrated better in the models of midilli et al. and page. the optimal color parameter of a* (greenness) and energy consumption values are achieved in 90 w – 55°c. alternative techniques should be used to save time or energy in the industrial food process. references aibinu i., adenipekun t., adelowotan t., ogunsanya t. and odugbemi, t. 2007. evaluation of the antimicrobial properties of different parts of citrus aurantifolia (lime fruit) as used locally. afr. j. tradit. complement. altern. med. 4(2):185. al-harahsheh m., ala’a h. and magee t.r.a. 2009. microwave drying kinetics of tomato pomace: effect of osmotic dehydration. chem. eng. process. process intensif. 48(1):524. amiri chayjan r., kaveh m. and khayati, s. 2015. modeling drying characteristics of hawthorn fruit under microwave‐ convective conditions. j. food process. preserv. 39(3):239. argyropoulos d., heindl a. and müller j. 2011. assessment of convection, hot‐air combined with microwave‐vacuum and freeze‐drying methods for mushrooms with regard to product quality. int. j. food sci. technol. 46(2):333. arredondo j., ruiz l., lopez g. and diaz-fleische, f. 2015. determination of the host status of the ‘persian’ lime (citrus latifolia tanaka) for anastrepha ludens (loew) (diptera: tephritidae). j. econ. entomol. 108(1):1. ital. j. food sci., vol. 31, 2019 499 arslan, d. and özcan m.m. 2010. study the effect of sun, oven and microwave drying on quality of onion slices. lwt food sci. technol. 43(7):1121. arumuganathan t., manikantan m.r., rai r.d., anandakumar s. and khare, v. 2009. mathematical modeling of drying kinetics of milky mushroom in a fluidized bed dryer. int. agrophys. 23(1):1. bhattacharya m., srivastav p.p. and mishra, h.n. 2015. thin-layer modeling of convective and microwave-convective drying of oyster mushroom (pleurotus ostreatus). j. food sci. technol. 52(4):2013. darvishi h., khoshtaghaza h.m. and minaei s. 2014. drying kinetics and colour change of lemon slices. int. agrophys. 28:1. demirel f.m. and ismail o. 2017. investigation of the effect of a hybrid drying method on the color quality of nectarine slices and energy consumption. stud. u. babes-bol. che. 62(1):237. díaz d.m., alcalá o.r., avilés c.a., de la cruz medina j. and jimenes, g.r. 2011. drying process design for the thirdqualıty persian lime use from cuitláhuac, veracrúz. méxico. revista iberoamericana de tecnología postcosecha 12(1):69. diaz g.r., martinez-monzo j., fito p. and chiralt a. 2003. modelling of dehydration-rehydration of orange slices in combined microwave-air drying. innov. food sci. emerg. technol. 4:203. doymaz i̇. and i̇smail o. 2011. drying characteristics of sweet cherry. food bioprod. process. 89(1):31. doymaz i., kipcak a.s. and piskin s. 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(sai-namphaung). int. j. food sci. technol. 46(11):2401. unal h.g. and sacilik k. 2011. drying characteristics of hawthorn fruits in a convective hot-air dryer. j. food process. preserv. 35(2):272. varith j., dijkanarukkul p., achariyaviriya a. and achariyaviriya s. 2007. combined microwave-hot air drying of peeled longan. j. food eng. 81(2):454. vega-gálvez a., di scala k., rodríguez k., lemus-mondaca r., miranda m., lópez j., and perez-won m. 2009. effect of air-drying temperature on physico-chemical properties, antioxidant capacity, colour and total phenolic content of red pepper (capsicum annuum, l. var. hungarian). food chem. 117(4):647-653. wang j. and sheng k. 2006. far-infrared and microwave drying of peach. lwt food sci. technol. 39(3):247. yadav a.r., chauhan a.s., rekha m.n., rao l.j.m. and ramteke r.s. 2004. flavour quality of dehydrated lime [citrus aurantifolia (christm.) swingle]. food chem. 85:59. zarein m., samadi s.h. and ghobadian b. 2015. investigation of microwave dryer effect on energy efficiency during drying of apple slices. j. saudi society of agr. sci. 14(1):41. zhao d., zhao c., tao h., an k., ding s. and wang z. 2013. the effect of osmosis pretreatment on hot-air drying and microwave drying characteristics of chili (capsicum annuum l.) flesh. int. j. food sci. technol. 48(8):1589. paper received june 13, 2018 accepted february 1, 2019 ijfs#1654_bozza ital. j. food sci., vol. 32, 2020 292 paper chemical and microbiological characteristics of homogenised ricotta cheese produced from buffalo whey c. tripaldi1, s. rinaldi*1, g. palocci1, s. di giovanni1, m.c. campagna2, c. di russo2 and t. zottola2 1consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca zootecnia e acquacoltura, via salaria 31, monterotondo, rm, italy 2istituto zooprofilattico sperimentale del lazio e toscana, via appia nuova 1411, 00178 roma, italy *corresponding author: simona.rinaldi@crea.gov.it abstract to extend the shelf life of buffalo ricotta cheese, a process was assessed that included a second heat treatment followed by homogenisation and hot packaging. the microbiological and chemical characteristics as well as the oxidation degree of the product were determined over storage for 21 days using total antioxidant activity, redox potential, malondialdehyde content, and protein-bound carbonyl content. homogenised buffalo ricotta cheese has a longer shelf life than traditional ricotta cheese, although the process could be optimised to reduce the total bacterial load during storage. no significant oxidative damage occurred during storage. this innovative process could promote the market expansion of ricotta cheese. keywords: antioxidant activity, carbonyl groups, malondialdehyde, oxidation level, ricotta cheese, shelf life ital. j. food sci., vol. 32, 2020 293 1. introduction ricotta cheese is an italian dairy by-product obtained from whey via the denaturation of whey proteins at 80-85°c. in italy, almost all families purchase ricotta cheese at least once a year (assolatte, 2018). the consumption of ricotta cheese is favoured by its low price. despite its lower commercial value with respect to that of other cheeses, the whey proteins in ricotta cheese have a high biological value owing to their significant content of sulphur-containing amino acids (smithers, 2008). ricotta cheese has a high moisture content and an initial ph above 6.0. its shelf life varies according to the treatment applied after curd floating and the type of packaging (mucchetti and neviani, 2006). fresh buffalo ricotta cheese is marketed locally and mainly consumed within 1-2 days of production. its shelf-life is about 4 days (zottola, personal communication). the fact that the guaranteed shelf life of this product is only a few days hampers its distribution to distant markets. current markets absorb only a small amount of fresh buffalo ricotta cheese in comparison to the actual production potential, indicating that a large proportion of the buffalo whey deriving from mozzarella cheese manufacture is not destined for ricotta cheese but disposed of or destined for non-dairy use. an increase in the shelf life of buffalo ricotta cheese could lead to an increase in the market share; this would in turn lead to a reduction in the quantity of whey to be disposed of. in addition, the recent recognition of the protected designation of origin (pdo) to "ricotta di bufala campana" could increase interest from domestic and foreign markets in a product with a longer shelf life. shelf life can be increased by reducing microbial contamination, improving the hygiene conditions of the cheese plant equipment and environment, and reducing the cooling time (ottogalli et al., 1981; mucchetti and neviani, 2006). furthermore, packaging systems, such as vacuum packaging, can extend the shelf life of ricotta cheese (pintado and malcata, 2000). modified atmosphere (ma) packaging has been used to extend the shelf life of fresh ricotta cheese by up to 14 days (mancuso et al., 2014). a system that ensures a longer shelf life for ricotta cheese is heat packaging. after the whey is drained, ricotta cheese is heat-packaged into suitable containers (mucchetti et al., 2002). additionally, after the whey is drained, ricotta cheese can be subjected to a second thermal treatment and a homogenisation treatment. the homogenisation process occurs at a low pressure and inhibits product syneresis. then, the homogenised ricotta cheese is heat-packaged in sealed plastic containers (mucchetti et al., 2002). the heat packaging is applied to cow ricotta cheese at an industrial level and is less often applied to ricotta cheese from other species, or in smalland medium-sized cheese plants. other product innovations in the food chain have been introduced to meet new consumer needs. these include changes in the manufacturing process, product composition, packaging, and product size and shape, and the introduction of a new method of using the product (lipan et al., 2017). some examples of innovation in ricotta cheese have been described by scarano et al. (2019). to fulfil the needs of the buffalo dairy industry, the present study was designed to improve the technology of small plants to extend the shelf life of buffalo ricotta cheese. a homogenisation of traditional ricotta cheese preceded by heat treatment was suggested. homogenised buffalo ricotta cheese, obtained as described above, differs in sensory properties from traditional buffalo ricotta cheese. homogenisation produces a fine and uniform consistency with consequently greater creaminess (wilbey et al., 2012). ital. j. food sci., vol. 32, 2020 294 there have been many studies regarding the chemical and microbiological characteristics of ricotta cheese (mucchetti and neviani, 2006; mucchetti et al., 2017), but few have focused on its oxidative characteristics (raia et al., 1996). the double heating treatments and homogenisation applied to buffalo ricotta cheese induced us to study the oxidation degree of this type of product at different times of storage. in dairy products, lipids are susceptible to oxidation, a biochemical process that contributes considerably to the degradation of the nutritional and sensory qualities of a product during manufacture and storage; this causes a significant reduction in shelf life (bergamo et al., 1998). this alteration in lipids is determined not only by the absorption of oxygen by both free and esterified unsaturated fatty acids but also by other environmental factors, including exposure to light, high temperatures, and contact with metals (fe, cu, co, ni, and mn) (mortensen et al., 2004). protein oxidation, another type of oxidative damage in dairy products that affects the protein matrix during heat treatment, generally induces changes in amino acid residues and three-dimensional protein structures and may result in the loss of biological functionality (augustyniak et al., 2015; feng et al., 2015). the aim of this study was, therefore, to evaluate the microbiological and chemical characteristics of homogenised buffalo ricotta cheese over storage for 21 days. moreover, the degree of oxidation of the product was investigated by adapting analytical methods to specifically study the ricotta cheese matrix. 2. materials and methods 2.1. process of ricotta cheese the trials were performed in a medium-sized cheese plant located in italy in the lazio region where buffalo mozzarella and traditional ricotta cheese are produced using an artisanal system. the raw material used to produce traditional ricotta cheese utilises the sweet whey drained from mozzarella curd. other important phases of the process are reported in fig. 1. approximately 1.5% (w/v) fresh cream from buffalo whey was added to the sweet whey. then, approximately 0.3% (w/v) nacl was added, and the whey mixture was heated continuously in a large open kettle by direct heating. when the whey grains began to float, sieroinnesto (natural whey cultures obtained from previous cheese making) was added at various amounts according to the extent of the sieroinnesto titratable acidity (2-3% v/v). the heating of the mixed whey was stopped at 85°c, and when the firm curd floated to the surface, it was collected into perforated hoops where the whey was drained. the ricotta cheese was held for one hour at room temperature and then transferred to a cold room at 4°c. while the traditional ricotta cheese was cooling, the temperature, ph, and weight were measured. to produce fresh ricotta cheese with increased shelf life, the following process (fig. 2) was applied. ital. j. food sci., vol. 32, 2020 295 figure 1. flow-chart for the manufacture of traditional buffalo ricotta cheese. figure 2. flow-chart for the manufacture of homogenised buffalo ricotta cheese buffalo whey 1.5% (w/v) fresh cream 0.3% (w/v) nacl heating 2-3% (v/v) sieroinnesto floating of curd (85°c) cooling at 4°c putting in perforated hoops whey drainage at room temperature whey was drained homogenizer-smoother machine pasteurization at 65°c/30 min heat-packaging cooling at 4°c traditional buffalo ricotta cheese homogenization at low pressure thermo-sealing of containers ital. j. food sci., vol. 32, 2020 296 after draining, the traditional ricotta cheese was transferred to a homogeniser-smoother machine. in this machine, which was equipped with a heating system, the ricotta cheese was pasteurised at 65°c for 30 min and then homogenised at 15 bar. preliminary trials were carried out to determine the optimal initial moisture content of the product, pasteurisation parameters, and packaging conditions. the homogenisation process occurred at a low pressure. finally, the product, namely homogenised buffalo ricotta cheese, was immediately heat-packaged in sealed 250 g plastic containers to reduce spoilage risk, and then cooled in a room at 4°c. 2.2. analysed samples sixteen samples were collected per day on three different days during production. hermetically sealed 250 g ricotta cheese packages arrived at the laboratory in refrigerated conditions and were stored at refrigeration temperature (4°c) for a period of 1, 7, 14, and 21 days. microbiological analyses were carried out on the refrigerated samples at the animal prophylaxis research institute for lazio and toscana regions (izslt), latina, italy. chemical and oxidative analyses were carried out on frozen samples at the council for agricultural research and economics, research centre for animal production and aquaculture of monterotondo, rome, italy. for each sample, two ricotta cheese packages were allowed to defrost overnight at 4°c. the sample was adequately homogenised, and the subsamples were taken for analyses. for each analysis, at least two replicates for each sample were performed. 2.3. microbiological analyses bacteria in the fresh cheese samples were detected and enumerated in accordance with international standard methods. an initial suspension was prepared to achieve a uniform distribution of the sample microorganism content. the initial suspension was made by adding 225 ml of diluent to 25 g of sample for bacterial detection, and 90 ml of diluent to 10 g of sample for bacterial enumeration. the samples were subjected to detection methods for salmonella spp. (uni en iso 65791:2017), listeria monocytogenes, and listeria spp. (uni en iso 11290-1:2017). salmonella spp. were detected via pre-enrichment in buffered peptone water (bpw) broth incubated at 36±2°c for 18 h, enrichment in rappaport-vassiliadis soya peptone (rvs) broth and muller-kauffmann tetrathionate-novobiocin (mkttn) broth at 37°c for 24 h, and plating on xylose lysine deoxycholate (xld) agar and salmonella-shigella (ss) agar plates incubated at 37°c and examined after 24 h. listeria monocytogenes and listeria spp. were detected via incubation in half-fraser broth at 30°c for 25±1 h, followed by a second enrichment with fraser broth at 30°c for 25±1 h, which was then plated on listeria agar, according to ottaviani and agosti (aloa), and oxford agar (lsm), and incubated at 37°c for 48 h. β-glucuronidase-positive escherichia coli was detected by incubating samples in tryptonebile-glucuronic medium (tbx) at 44±1°c for 24 h (iso 16649-2: 2001). coagulase-positive staphylococci (staphylococcus aureus and other species) were detected using rabbit plasma fibrinogen agar medium incubated at 37±1°c for 24 h (uni en iso 6888-2: 2004). pseudomonas spp. were detected by culturing the samples on penicillin and pimaricin agar (ppa) at 25°c for 48 h (iso/ts 11059:2009idf/rm 225:2009). ital. j. food sci., vol. 32, 2020 297 enterobacteriaceae was detected by culturing the samples on violet red bile glucose agar (vrbg) at 37°c for 24 h (uni en iso 21528-2: 2017). yeasts and moulds with greater than 0.95 water activity were detected by incubating the samples on dicloran-rose bengal chloramphenicol agar (drbc) at 25±1°c for five days (iso 21527-1: 2008). total mesophilic counts (tmcs) were enumerated by incubating the 3m™ petrifilm™ aerobic count plate at 30±1°c for 72±3 h (afnor 3m 01/1-09/89). total psychrophilic counts (tpcs) were enumerated by incubating the 3m™ petrifilm™ aerobic count plate at 6.5±1°c for ten days (afnor 3m 01/1-09/89; iso 6730: 2005 idf 101). presumed mesophilic and thermophilic cocci were enumerated on m17 agar after aerobic incubation for 48 h at 30°c and at 44°c, respectively. presumed mesophilic lactobacilli were enumerated on deman, rogosa, and sharpe (mrs) agar adjusted to ph 5.4 and incubated anaerobically at 37°c for 72 h. the result of bacteria enumeration was expressed as the number of microorganisms per gram of product. activity water (aw) was determined with an aqualab lite (decagon) principle dew-point measurement (iso18787:2017). 2.4. physical and chemical analyses homogenised ricotta cheese samples were submitted to the following analyses: ph, moisture (idf, 1986), total nitrogen (tn) (fil-idf, 1993), ph 4.6 soluble nitrogen (ph 4.6 sn) (fil-idf, 1991), fat (fil-idf, 2001), ashes (aoac, 2000), and salt (idf, 1988). 2.5. antioxidant activity the dpph method the antioxidant activity of the samples was evaluated using the dpph (1,1-diphenyl-2picryl-hydrazyl radical) method, as reported by unal (2012), for milk and cheese, with some modifications. the purple stable free radical dpph changes to a yellow colour following reduction by antioxidant molecules. this method is commonly used for the evaluation of the antioxidant capacity of plant extracts and food in various contexts (katalinic et al., 2006; nikolova et al., 2011). ricotta cheese samples (2 g) were mixed with 8 ml of 0.11 mm dpph ethanolic solution. the control was prepared by adding 2 ml of ethanol to 8 ml of 0.11 mm dpph ethanolic solution. the mixtures were shaken vigorously and then left standing at room temperature for 20 min in the dark. then, the mixtures were centrifuged for 10 min at 9000 g at 22°c. the absorbance of the supernatant at 517 nm was measured using a double-beam uv-vis spectrophotometer (lambda 25, perkinelmer). absolute ethanol was used as a blank, and analyses were performed in duplicate. the antioxidant activity was expressed as the percentage of inhibition of the dpph radical according to the following equation: % antioxidant activity = (a0 – as) / a0 x 100 where a0 is the absorbance of the control (containing all reagents except ricotta cheese sample), and as is the absorbance of the tested sample. ital. j. food sci., vol. 32, 2020 298 trolox was used as a standard to compare the antioxidant activity of the sample with a reference antioxidant. the antioxidant activity of the samples was expressed in trolox equivalents (mmol trolox eq. /100 g). 2.6. redox potential determination for each sample, the redox potential was determined using a potentiometric method that uses a ph meter (metrohm 827 ph lab) equipped with a platinum electrode (combined pt-ring 6.0451.100) and the potential was expressed in millivolts (mv). 2.7. malondialdehyde analysis the method used for mda determination was based on the detection of mda thiobarbituric acid (tba) fluorescent complexes, in which the mda present in a sample reacts with tba, and the mda-tba complex results in an absorption peak at 532-535 nm (raharjo and sofos, 1993). ricotta cheese samples were prepared according to the methods of raia et al. (1996), with some modifications. a total of 0.4 g of the sample was homogenised in a solution of 3.4 ml of 10% w/v trichloroacetic acid (tca) to precipitate proteins, and 0.20 ml of bht (butylhydroxytoluene) 2.8% w/v ethanolic solution was added as an antioxidant to prevent further lipid peroxidation. the mixture was heated at 90°c for 30 min, quickly cooled in an ice bath for 20 min, and centrifuged at 10,000 g for 10 min to separate the pellet containing the precipitated protein fraction. a supernatant aliquot (300 μl) was added to 700 μl of 0.28% w/v tba and incubated at 90°c for 30 min to induce the formation of the tba-mda complex, and the aliquot was then quickly cooled on ice for 20 min. after centrifugation at 10,000 g for 5 min, the supernatant was collected for the following hplc analysis. hplc analysis was performed using a shimadzu-spd-m10a hplc with a fluorimeter (rf-10a) and zorbax eclipse plus c18 column (4.6 x 250 mm x 5 µm). the isocratic mobile phase consisting of 5 mm sodium phosphate buffer (ph 7.0) and acetonitrile (80:20 v:v) was filtered on whatman 0.2 µm filter paper. aliquots (20 μl) were injected and analysed using a flow rate of 1 ml/min at room temperature. the fluorescent detector was set at an excitation wavelength of 515 nm and emission at 543 nm. the concentration of the mda-tba complex was then calculated based on the calibration curve using a standard solution, and the values obtained were expressed as nmol/g. 2.8. carbonyl determination to evaluate the oxidative damage of proteins in the ricotta cheese samples, the method of reznick and packer (1994) modified by fedele and bergamo (2001) for milk and cheese was used. the method is based on the derivatization of the carbonyl group with dinitrophenol hydrazine (dnph), and the chromophore produced can be detected at 360 nm. in detail, ricotta cheese samples (2 g) were weighed, and 5 ml of 0.2 m sodium citratenaoh (ph 8) at room temperature was added. after vigorous mixing for 5 min, 100 µl aliquots were incubated in the presence of 400 µl of 10 mm dnph in 2.5 m hcl (in duplicate). in total, 400 µl of 2.5 m hcl (in duplicate) was added to the other 100 µl aliquots as a control (blank). ital. j. food sci., vol. 32, 2020 299 after incubation for 30 min in the dark at room temperature, 500 µl of cold 20% w/v tca was added to each sample, and the samples were vigorously shaken. the mixtures were incubated for 20 min on ice and centrifuged for 10 min at 10,000 g at 4°c to cause protein precipitation. the supernatant was removed, and 400 µl of 10% tca was added to the pellet. after incubation for 10 min in the dark, the samples were centrifuged at 10,000 g for 10 min, after which the supernatant was removed. to remove the free dnph, the pellet was washed twice with 1 ml of ethanol/ethyl acetate (1:1 v/v) solution by centrifugation at 10,000 g for 10 min, and the supernatant was removed without disturbing the precipitate. pellets containing protein precipitates were then dissolved in 1 ml of 6 m guanidine hcl in 10 mm phosphate buffer (ph 2.3). the samples were incubated for 30 min in a 60°c water bath, and subsequently the absorbance was measured at 370 nm using a doublebeam uv-vis spectrophotometer (lambda 25, perkinelmer). for the samples treated with 2 m hcl (control without dnph), the protein concentration was determined by measuring the absorbance at 280 nm. the amount of protein was calculated from a bovine serum albumin (bsa) standard curve (0.5-2.5 mg/ml). the net absorbance was calculated as the difference between the absorbance of the sample with dnph and the blank (without dnph). the concentration of protein-bound carbonyls (c carb) was calculated by the molar extinction coefficient of 22,000/m/cm, according to the following formula: c carb (nmol/mg prot) = (δ αbs 360 / 22 mm) * 1000/c prot where δ abs 360 = abs 360 (dnph) abs 360 (blank) is the net measured absorbance, and c prot is the protein content (g/l). the carbonyl concentration, expressed as nmol/mg of protein, was used to provide a global protein oxidation index. 2.9. statistical analysis all microbiological data were log-transformed. the glm procedure of sas software (sas institute inc., 2007) was used for the statistical analysis of the chemical and oxidative analyses of ricotta cheese. a factorial model, including the fixed effect of the storage time of ricotta cheese, was used. the corr procedure of sas software (sas institute inc., 2007) was also used. 3. results and discussion 3.1. process of making traditional buffalo ricotta cheese the initial titratable acidity of the whey transferred to the ricotta cheese kettle ranged from 0.16 to 0.18 g of lactic acid/100 ml. addeo and coppola (1983) found similar values of titratable acidity (0.17 g of lactic acid/100 ml) of starting whey from buffalo mozzarella used for the manufacture of ricotta cheese. the process specifications of pdo “ricotta di bufala campana” require a sweet whey with titratable acidity <0.16 g of lactic acid/100 ml. according to true (1973), in general, the initial titratable acidity of sweet whey destined to produce ricotta cheese, should be less ital. j. food sci., vol. 32, 2020 300 than 0.16 g of lactic acid/100 ml. optimal values are considered 0.13-0.14 g of lactic acid/100 ml. in the traditional process of making buffalo ricotta cheese, approximately 1.5% (w/v) cream was added to the whey. according to “ricotta di bufala campana” specifications, the addition of up to 6% (w/v) milk and 5% (w/v) fresh cream is allowed. the addition of milk or cream enriches the fat content of the whey and improves the sensorial characteristics of the ricotta cheese (shahani, 1979). ricotta cheese manufactured with the addition of milk or cream is softer and creamier and has a delicate texture (pintado et al., 2001). fresh cream made from mozzarella whey transfers a particular flavour to ricotta cheese. to enhance the coagulation and rise of the curd, sieroinnesto was added to hot whey. according to mucchetti et al. (2017), in ovine ricotta cheese production, it is not necessary to reduce the whey ph to favour protein aggregation, while cow and buffalo whey needs to be slightly acidified for better protein aggregation. even the sieroinnesto addition can concur with a particular ricotta flavour. the amount of salt added to the whey (0.3%, w/v) was minimal with respect to the maximum amount allowed by “ricotta di bufala campana” specifications (1%, w/v). nacl dehydrates the whey proteins and has a destabilizing effect on bsa (farkye, 2004). the industrial process of ricotta cheese production (farkye, 2004) involves the stages described below. the whey is first neutralised to ph>6.5 (6.9-7.1) with a naoh solution. manipulation of the ph minimizes protein aggregation and produces a more cohesive coagulum (modler and emmons, 1989). the recommended temperature for milk addition (5-25%) is 65-70°c, while cream is added at 75-80°c. after the addition of cream, nacl (0.5, v/v) is added. then, an acetic or citric acid solution is added for coagulation and curd formation. optimal coagulation and maximum yield occur at ph 5.6-5.8 (weatherup, 1986). in our trials, after three hours of cooling, the temperature of the ricotta cheese decreased on average from 65°c to 17°c. the weight stabilised after three hours starting from the transfer to the basket. weight loss for the traditional ricotta cheese was approximately 15%. the final weight of the shapes was 400-450 g. after three hours, the ph increased from 6.22 to 6.76, while in ricotta cheese from sheep’s milk, the final ph ranged from 6.35 to 6.85 (cherchi et al., 1999; salvatore et al., 2014). 3.2. process of homogenising buffalo ricotta cheese regarding the optimal initial moisture of the product, according to preliminary trials, better results were achieved when the moisture content ranged from 67-70%. the homogeniser-smoother machine used to produce homogenised ricotta cheese was equipped with scraping blades, which stirred the product during heating. if the curd was too dry, the stirring was more difficult, and the curd adhered to the heating surface, reducing the effectiveness of the heat exchange. moreover, the increase in temperature for low-temperature or batch pasteurisation (65°c/30 min) applied to heat the product was very slow. homogenised ricotta cheese was heat-packaged, and to minimise microbiological pollution, the containers were thermo-sealed immediately after being filled. in the preliminary trials, we observed that the application of these conditions contributed to extending the shelf life of the homogenised ricotta cheese. in fact, not sealing the containers immediately after filling them resulted in substantial contamination, particularly with mould and yeasts, after a few days (data not shown). ital. j. food sci., vol. 32, 2020 301 3.3. microbiological characteristics salmonella spp. and listeria monocytogenes, pathogens considered as food safety criteria (reg ce 2073/2005), were not detected (below the detection limit), in accordance with the above regulations. in addition, listeria spp. were not detected. the counts of ß-glucuronidase-positive escherichia coli and coagulase-positive staphylococci (staphylococcus aureus and other species) are considered hygiene markers (reg ce 2073/2005) and were lower than the detection limit of the method <10 colony forming units (cfu)/g (table 1). table 1. microbiological characteristics of homogenised buffalo ricotta cheese during storage. tmc: total mesophilic count; tpc: total psychrophilic count. bacteria values are means±sd of three batches samples, expressed as log 10 cfu/g. additionally, the counts of enterobacteriaceae and pseudomonas spp., which are often associated with changes in the texture and colour of the cheese, were lower than the detection limit (<10 cfu/g). yeasts were <10 cfu/g, while moulds were present only in one sample at day seven and in another at day 14. moulds were within the limits according to iso 21527-1: 2008. penicillium spp. was identified through macroscopic and microscopic traits. these results show that in the homogenised buffalo ricotta cheese made by the process described above, pathogens are undetected, and all microorganisms considered hygiene markers were lower than the detection limit. our results agree with those reported by palmas et al. (1994) during the storage of sheep ricotta cheese subjected to direct hot packaging. storage time day 1 day 7 day 14 day 21 β-glucuronidase-positive escherichia coli (log 10 cfu/g) < 1 < 1 < 1 < 1 coagulase-positive staphylococci (log 10 cfu/g) < 1 < 1 < 1 < 1 enterobacteriaceae (log 10 cfu/g) < 1 < 1 < 1 < 1 pseudomonas spp. (log 10 cfu/g) < 1 < 1 < 1 < 1 yeasts (log 10 cfu/g) < 1 < 1 < 1 < 1 moulds (log 10 cfu/g) < 1 1.30 ± 0.43 2.00 ± 1.41 < 1 tmc (log 10 cfu/g) 4.57 ± 0.41 6.42 ± 0.87 7.37 ± 0.47 8.27 ± 0.20 tpc (log 10 cfu/g) 2.52 ± 2.15 6.88 ± 1.45 7.35 ± 0.44 7.44 ± 0.90 mesophilic lactococci (log 10 cfu/g) 4.34 ± 0.32 4.94 ± 0.30 7.24 ± 0.29 8.56 ± 0.63 thermophilic lactococci (log 10 cfu/g) 4.31 ± 0.01 3.02 ± 2.86 7.20 ± 0.23 8.50 ± 0.71 mesophilic lactobacilli (log 10 cfu/g) 2.72 ± 0.18 < 1 < 1 < 1 aw 0.995 ± 0.001 0.995 ± 0.001 0.994 ± 0.0014 0.993 ± 0.002 ital. j. food sci., vol. 32, 2020 302 the average tmc of our samples increased from 4.57 on day 1 to 8.27 log10 cfu/g on day 21. the same trend was observed for tpc, changing from 2.52 on day one to 7.44 log10 cfu/g on day 21. mesophilic and thermophilic lactococci increased from 4.34 and 4.31 log10 cfu/g on day one to 8.56 and 8.50 log10 cfu/g on day 21, respectively. mesophilic lactobacilli were present only in day one samples, and thermophilic lactobacilli were not detected (table 1). according to mucchetti et al. (2002), industrially produced fresh ricotta cheese, and particularly ricotta cheese subjected to a second heat treatment, had a lower total bacterial count compared to that found in our samples. lactic acid bacteria are the microorganisms most commonly represented in ricotta cheese, and they are an important part of the total bacterial count (mucchetti et al., 2017). the increase during storage in tmc and lab could be due to heat resistant or postcontamination microorganisms, since the high ph and elevated aw do not limit microbial growth. we assume that the microbiological characteristics of the product can be improved by the stricter control of environmental contaminants through the implementation of hygienic conditions during the process. furthermore, to reduce the number of heat-resistant microorganisms, higher temperatures could be used during the second heat treatment. total bacterial count was lower during the storage of the product in industrial ricotta cheese subjected to second heat treatment by 85-95°c (mucchetti et al., 2002). moreover, an essential strategy for the control of mesophilic bacteria, such as sporeforming, is the reduction of cooling times and the strict maintenance of the cold chain during product storage. 3.4. chemical characteristics the chemical characteristics of the homogenised ricotta cheese are reported in table 2. table 2. chemical characteristics of homogenised ricotta cheese from buffalo whey. the mean moisture content of the samples analysed was 65.51%, which was similar to that of the “ricotta di bufala campana” cheese (64.58%) reported previously by several authors (mucchetti and neviani, 2006). the moisture content of industrial ricotta cheese from bovine and sheep whey is higher, 75.72% and 70.03%, respectively (mucchetti et al., 2002), both of which are made in large cheese plants where the sample number moisture (%) dry matter (%) protein (%) soluble protein (%) fat (%) ash (%) 1 67.05 32.95 6.39 0.07 19.42 2.22 2 64.29 35.71 5.41 0.06 24.50 2.07 3 65.18 34.82 5.08 0.09 23.59 1.75 mean 65.51 34.49 5.63 0.07 22.50 2.01 s.d. 1.41 1.41 0.68 0.01 2.71 0.24 ital. j. food sci., vol. 32, 2020 303 industrial process favours better whey recovery in the product than the traditional process (salvadori del prato, 2001; mucchetti et al., 2002). the mean protein and fat contents of homogenised buffalo ricotta cheese were 5.63% and 22.50%, respectively, and the fat: protein ratio was 4.0. the samples analysed had lower protein and higher fat contents than the values found in “ricotta di bufala campana” pdo, which were 9.43% and 19.09%, respectively (mucchetti and neviani, 2006). however, owing to the artisanal processing conditions, the data cited above are very variable: the protein content ranged from 7.10% to 13.46%, and the fat content ranged from 15.87% to 20.78%. the average composition of industrial cow milk ricotta cheese is very different with 9.73% fat content, 9.12% protein content and a fat: protein ratio of 1.07 (mucchetti and neviani, 2006). the mean value of ph 4 soluble protein was 0.07%, similar to that observed by mucchetti et al. (2002) in ricotta cheese from bovine whey at 0.10%. these data indicate the content of undenatured whey protein in ricotta cheese. the use of other techniques such as ultrafiltration can increase the recovery of whey protein in ricotta cheese (maubois and kosikowski, 1978). the mean ash content value was 2.01%, which was higher than that of “ricotta di bufala campana” and cow ricotta cheese (mucchetti and neviani, 2006); their values were 1.23% and 1.16%, respectively. 3.5. physicochemical and oxidative characteristics the physicochemical and oxidative characteristics of the homogenised ricotta cheese from day 1 to 21 are reported in figures 3, 4, 5, and 6. during product storage, the values of all characteristics were not significantly different. the ph values (fig. 3) decreased during storage from 6.90 to 6.55. figure 3. ph of homogenised buffalo ricotta cheese during storage. the increase in lab during product storage could explain the ph decrease (mucchetti et al., 2002). according to hough et al. (1999), the ph of ricotta cheese packaged at temperatures between 65-68°c and stored at 6°c for 20 days decreased to approximately 6.0, and a significant correlation between ph and microbial growth was found. the ph of ital. j. food sci., vol. 32, 2020 304 fresh traditional sheep ricotta cheese packaged in a modified atmosphere decreased from 6.54 on day 1 to 5.97 on day 21 of storage (mancuso et al., 2014). the redox potential (fig. 4) ranged from a minimum of 121 mv on day 1 to a maximum of 134 mv on day 14. figure 4. redox potential of homogenised buffalo ricotta cheese during storage. the values found in industrial cow ricotta cheese were positive (183 mv) (mucchetti et al., 2002). in the samples of homogenised buffalo ricotta cheese not subjected to freezing before analysis, the redox potential was 82.3 mv (data unpublished). redox potential is an important parameter related to food stability. it is affected by many chemical and biological reactions (tango and ghaly, 1999), but it is not routinely measured (caldeo, 2015). the total antioxidant activity (fig. 5) measured as trolox eq. increased slightly from day one (13.69 mmol/100 g or 64.60%) to day 21 (14.05 mmol/100 g or 66.30%). figure 5. antioxidant activity and mda content in homogenised buffalo ricotta cheese during storage. ital. j. food sci., vol. 32, 2020 305 in cheddar cheese, antioxidant activity decreases with an increasing storage period, and in one study was 48% at the beginning of storage and 32% after three weeks of storage (lee et al., 2016). in another study, the antioxidant activity of gouda cheese increased throughout the ripening period from 4.61% on day 0 to 16.38% on day 90 (khan et al., 2018). the authors of the latter study attributed this increase to water-soluble peptides that have antioxidant properties. the total antioxidant capacity measures the activity of many antioxidants that are active (korpela et al., 1995; lindmark-mansson and akesson, 2000), and variations in activity depend on processing conditions. in milk, the changes upon heating were attributed not only to the thermal degradation of naturally occurring antioxidants, such as vitamins and enzymes, but also to the formation of novel oxidative species (calligaris et al., 2004; andrei et al., 2015). in figure 5, the mda values are shown to slightly increase during storage, with a lower content at day seven (1.42 nmol/g or 0.103%) and a higher content at day 21 (1.60 nmol/g or 0.116%). in contrast, one study found that the mda content of buffalo mozzarella cheese significantly increased after four days of storage (taticchi et al., 2017). an increase in mda content was also detected during the storage of high moisture mozzarella from cow’s milk (segat et al., 2013). the average mda content of homogenised ricotta cheese (1.51 nmol/g or 0.11 mg mda/kg) was similar to that of grana padano cheese (1.28 nmol/g) (fedele and bergamo, 2001) and semi-cooked pecorino cheese (0.13 mg/kg) (branciari et al., 2014). in homogenised buffalo ricotta cheese, a negative correlation (-0.76892; p≤0.015) between total antioxidant capacity and mda content was found. other authors have confirmed the active role of antioxidants in preventing or limiting fat oxidation. fedele and bergamo (2001) reported a positive correlation between mda and consumption of αtocopherol. the mda values of homogenised ricotta cheese are far below those of other animal and vegetable foods (papastergiadis et al., 2012), despite the high fat content of this product. in fact, one of the factors that predisposes to lipid oxidation is the high fat content of food (dalsgaard et al., 2010; citta et al., 2017). the carbonyl content is shown in fig. 6 and decreased from 3.93 nmol/mg protein at day one to 3.01 nmol/mg protein at day 21; the average content was 3.52 nmol/mg. figure 6. protein-bound carbonyl content in homogenised buffalo ricotta cheese during storage. ital. j. food sci., vol. 32, 2020 306 in comparison with that of other cheeses (fedele and bergamo, 2001; balestrieri et al., 2002), the carbonyl content of homogenised ricotta cheese is similar to that of cooked cheeses, such as grana padano, caciocavallo, provolone and pecorino romano, and higher than that of fresh cheeses, including buffalo mozzarella cheese. protein-bound carbonyl groups formed during heat treatment are currently used to evaluate the extent of protein oxidation (fedyele and bergamo, 2001; feynaille et al., 2006). moreover, carbonyl content was positively correlated with the temperature of heat treatment (fedeyle and beyrgamo, 2001; scaloyni et al., 2002). 4. conclusions in conclusion, the process adopted to produce homogenised buffalo ricotta cheese ensured that during storage, microorganisms considered hygiene markers were below the detection limit. however, the increase in total mesophilic and lactic bacteria during storage suggests stricter control of environmental contaminants. higher temperatures are also recommended during the second heat treatment to reduce the number of heatresistant microorganisms and to contain the acidification of the product. owing to its oxidative characteristics, homogenised buffalo ricotta cheese is comparable to semi-cooked and cooked cheeses. the low content of malondialdehyde, which remains almost unchanged at the end of storage, confirms the active role of antioxidants present in dairy products in preventing or limiting fat oxidation. the suggested process meets the needs of buffalo dairy producers to obtain a product with a longer shelf life and to increase the distribution to national and international markets away from the production area of origin. acknowledgements we would like to thank regione lazio for the financial support, caseificio casearia casabianca s.r.l. of fondi, latina, italy, for their kind cooperation and for providing buffalo ricotta cheese samples, and sara cirulli who collaborated in the trial. references addeo f. and coppola s. 1983. aspetti 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2019 ijfs#1536_bozza ital. j. food sci., vol. 32, 2020 107 paper textural, physical and retrogradation properties of muffin prepared with kamut (triticum turanicum jakubz) p. lee1, h. oh1, s.y. kim1 and y.s. kim*1,2 1department of integrated biomedical and life sciences, korea university, 145 anam-ro, seongbukgu, seoul 02841, republic of korea 2department of food and nutrition, korea university, 145 anam-ro, seongbukgu, seoul 02841, republic of korea *corresponding author: tel.: +82 29402806, fax: +82 29217207 email: kteresa@korea.ac.kr abstract the effects of kamut flour substitution levels (0%, 25%, 50%, 75%, 100%) on muffin properties were investigated. as kamut flour level increased muffins showed lower height and volume and the l value decreased. the crumb hardness increased with increasing kamut level, and the control showed the lowest value in elasticity, chewiness, and brittleness. the increment of kamut flour level resulted the total flavonoid and polyphenol contents, reducing power, and abts and dpph radical scavenging activities. during storage, the avrami exponent decreased between the control to the sample added with 75% kamut. the crumb air cell number decreased, but the area increased with kamut flour level increment. in sensory evaluation, the samples with kamut level 25% and 50% were acceptable. therefore, muffins with an appropriate level of kamut flour improve the nutritional profile, and quality of baking products. keywords: antioxidant, kamut (khorasan wheat), muffin, physical properties ital. j. food sci., vol. 32, 2020 108 1. introduction in human nutrition, wheat is an important component of cereal-based foods, and it is one of the most consumed food sources on a global scale (sofi et al., 2013). as the awareness of healthy life increases, healthy food products are more in demand. following this trend, various wheat varieties such as whole wheat, organic wheat, and ancient wheat, have emerged in the market (angioloni and collar, 2011; fatma et al., 2017; dinu et al., 2018). among the wheat varieties, kamut (khorasan wheat, triticum turanicum jakubz) is one of the noted ancient grains due to the higher content of selenium content and the protein content. kamut contains 400-1000 ppb of selenium depending on harvesting condition and contains relatively large amounts of 12-18/100 g of protein (wijngaard and arendt, 2006; di loreto et al., 2017). in addition, consuming whole grain and the products from its derivative could be providing antioxidant substances and various cofactors such as copper, iron, zinc and selenium (benedetti et al., 2012). in recent studies (bordoni et al., 2017; carnevali et al., 2014; di loreto et al., 2017), the whole grain kamut was found to contain higher phytochemical contents than common wheat, and it has been attracting attention due to its nutraceutical properties such as high antioxidant, prebiotic activities, and reduction of irritable bowel syndrome symptoms. in addition, in human intervention studies, a volunteer group that consumed kamut products for 8 weeks demonstrated a significant decrease in total and ldl cholesterol and glucose levels while the control group showed no significant changes. the popularity of bakery products, especially with health functionalities, is increasing and as the consumption of cereal-based products increase, these products are important for taking essential nutrients in daily life (alpaslan and hayta, 2006). among the bakery products, muffins are easy to make into various products depending on the ingredients to be added, so the studies on functional muffins such as legume blended muffin, coffee ground residue water extracts muffin, flaxseed muffin and buckwheat muffin (kim et al., 2016; bae and jung, 2013; kaur and kaur, 2018; qian et al., 2017) are briskly. also, muffins have high acceptance for the consumer due to sweet taste and soft texture and are characterized by typical pore formation. previous studies on kamut have been on antioxidant effects of kamut in the rat liver, sourdough bread, flake and muesli, tortillas and cookies (benedetti et al., 2012; carini et al., 2010; chandi et al., 2015; choi et al., 2016; sumczynski et al., 2015). the present study, therefore, focused on antioxidant, baking, rheological, microstructural, storage and quality characteristics of muffins with whole grain kamut and with the aim to find the optimal addition level of kamut for increasing utilization of kamut and development of functional bakery products. 2. materials and methods 2.1. muffin materials kamut (kamut® international, ltd., missoula, usa) cultivated in canada in 2016 was purchased. kamut (khorasan wheat) grains were washed three times and freeze-dried (fd8508, ilshinbiobase co., dongducheon, korea) at -80℃ for 5 days. dried kamut grains were ground (rt-04, wongangbio co., taiwan) for 2min, passed through a 40 mesh sieve twice to obtain kamut flour (kf), and stored at -20℃ until use. a soft wheat flour (wf) (q1, samyang co., asan, korea), salt (chungjungwaon, shinan, korea), sugar (beak-seol, ital. j. food sci., vol. 32, 2020 109 incheon, korea), egg (nature egg, yeo-ju, korea), butter (unsalted pure new zealand butter, fonterra, new zealand), milk (seoul milk, chung-ju, korea), and baking powder (baking soda, gimpo, korea) were acquired at a local market. 2.2 preparation of muffin samples the muffin formulations are presented in table 1. muffins were prepared using a modified method as described by qian et al. (2017). five different muffin samples with various ratio of kf and wf [100 wf (con), 75 wf:25 kf (k25), 50 wf:50 kf(k50), 25 wf:75 kf(k75), 100 kf(k100)] were prepared along with a control sample (100 wf). butter, salt and sugar were whipped with a blender (kmm020, kenwood, havant, england) for 3 min at 40 rpm. the egg was added in two portions and mixed for 5 min at 54 rpm. flour mix and baking powder were added and mixed at 40 rpm for 2 min. finally, milk was blended at 40 rpm for 2 min. the muffins were cooled for 1 h at 25℃ after baking and were used for analyses. table 1. formula for a muffin with different levels of kf. samples con k25 k50 k75 k100 wheat flour (g) 200 50 100 150 0 kamut flour (g) 0 150 100 50 200 salt (g) 2 2 2 2 2 sugar (g) 120 120 120 120 120 egg (g) 70 70 70 70 70 butter (g) 80 80 80 80 80 milk (g) 100 100 100 100 100 baking powder (g) 6 6 6 6 6 con: control. without added kf. k25: 75% wf, 25% kf. k50: 50% wf, 50%kf. k75: 25% wf, 75% kf. k100: 100% kf. 2.3. physicochemical measurement 2.3.1 moisture and brix degree the moisture content was measured at 105℃ in 5.0 g of crumb parts of muffins with a moisture analyzer (mb, ohaus, zurich, switzerland). the sugar content was measured with a digital refractometer (pr-201α, tokyo, japan) having a range of 0-60% by stirring 5.0 g of sample and 50 g of distilled water for 5 min (song et al., 2017). 2.3.2 batter measurement the batter specific gravity was measured at 25℃ by standard methods of analysis (aacc, 2000). the baking loss and baking yield were calculated according to the following formulas using the batter weigh. ital. j. food sci., vol. 32, 2020 110 baking loss (%) = 〔(batter weight – muffin weight)/batter weight〕 × 100 baking yield (%) = (muffin weight/batter weight) × 100 2.3.3 physical properties muffin weight was measured with a digital scale (eb-2200hu, dong-il shimadzu corp., seoul, korea). muffin volume was measured by the method of seed displacement (pyler, 1979). specific volume (ml/g) was determined by dividing the muffin volume (ml) by muffin weight (g). muffin height was measured as the vertical distance from the bottom to the top of the muffin center using vernier calipers. 2.3.4 appearance the color values of both crumb and crust were measured by a spectrophotometer (cr-400, konica minolta co., ltd, tokyo, japan). the color values were shown as lightness (l), redness (a), yellowness (b) and total color difference (△e). the △e was calculated by the following equation. the appearance and cross section of muffins were captured by a digital camera (x-t20, fujifilm, tokyo, japan). ∆𝐸 = (𝐿!"#$%& − 𝐿!"#$%#&%)! + (𝑎!"#$%& − 𝑎!"#$%#&%)! + (𝑏!"#$%& − 𝑏!"#$%#&%)! 2.4. textural analysis of muffin crumb texture profile analysis (tpa) was performed on muffins at a 25℃. the samples (20 mm × 20 mm × 20 mm) were measured by a two-bite compression test using rheometer (compac-100ⅱ, sun scientific, tokyo, japan). in this measurement, the cylindrical probe (20 mm diameter) was mounted and operated at 1.0 mm/s. hardness (n), springiness (%), cohesiveness (%), chewiness (g) and brittleness (g) were determined. hardness refers to the maximum force with the maximum peak of the first compression. springiness is the deformation rate between the first compression and the second compression, defined as the ratio of distances (d1: the maximum distance of the first bite; d2: the distance to the deformed sample surface in the second bite). cohesiveness is the strength of internal bonds and defined as the ratio of area. chewiness is calculated by multiplying the hardness value by the cohesiveness value. brittleness, also called fracturability, is a measure of force at the first peak. springiness (%) = d2/d1 × 100 cohesiveness (%) = a2/a1 × 100 2.5. antioxidant activity properties 2.5.1 antioxidant compound extraction the muffins were freeze-dried at -80℃ for 48 h and ground for 1 min (40 mesh). the muffins were defatted with hexane at a ratio of 1:5 w/v (3 min, 3 times), dried at 45℃ for 5 h, and extracted in a water bath (bs-20, jeio tech, seoul, korea) at 185 rpm for 1 h at 40℃ ital. j. food sci., vol. 32, 2020 111 with 70% methanol at a ratio of 1:10 w/v. the sample extracts were filtered using whatman filter (no. 4) and kept at 4℃ for subsequent experiments. 2.5.2 total polyphenols content total polyphenols content was determined by the folin-ciocalteu method. briefly, 50 µl of 0.9 n folin-ciocalteu’s reagent (merk kgaa, darmstadt, germany) and 150 µl of 20% sodium carbonate solution (merck kgaa, darmstadt, germany) were added sequentially to the extracted samples (20 µl) mixed with distilled water (790 µl). after incubating for 2 h incubation at 25℃, the absorbance of the mixed sample was read at 700 nm using an elisa microplate reader (apollo11lb913, berthhold thechnolosies co., ltd., bad wildbad, germany). the total polyphenol content (㎍ gae/g) was converted to gallic acid equivalents. 2.5.3 total flavonoids content total flavonoids content was examined by the method of zhang et al. (2017). briefly, 150 µl of 5% sodium nitrie (junsei chemistry) was added to 1 ml of samples and incubated in the darkroom at 25℃ for 6 min. subsequently, 0of 10% alcl3 was added to the mixed samples and incubated in the darkroom at 25℃ for 5 min. finally, 1 ml of 1 n naoh was mixed, and the absorbance of the sample mixture was read at 520 nm using an elisa microplate reader (apollo11lb913, berthhold thechnolosies co., ltd., bad wildbad, germany). the total flavonoids content (㎍ qe/mg) was converted to quercetin equivalents. 2.5.4 reducing power reducing power of the extracted samples was determined by a modified oyaizu (1986) method. briefly, 250 µl of 0.2 m phosphate buffer, the mixture of sodium phosphate monobasic solution and sodium phosphate dibasic solution (1:2), and 250 µl of 1% potassium ferricyanide solution were added to 250 µl of samples and incubated for 30 min at 50℃. subsequently, 250 µl of 10% trichloroacetic acid solution was mixed. finally, 500 µl of distilled water and 100 µl of 0.1% fecl3 were added to the sample supernatant (500 µl) and the absorbance of the sample mixtures was read at 700 nm using an elisa microplate reader (apollo11lb913, berthhold thechnolosies co., ltd., bad wildbad, germany). 2.5.5 dpph radical scavenging assay dpph assay refers to the method of joung et al. (2017). the 200 μmol dpph reagent was added to the diluted samples extracts (10, 12.5, 20, 25, 50, 100 mg/ml) and reacted in the darkroom at room temperature for 30 min. the absorbance of the sample mixtures was read at 520 nm using an elisa microplate reader (apollo11lb913, berthhold thechnolosies co., ltd., bad wildbad, germany). ital. j. food sci., vol. 32, 2020 112 2.5.6 abts radical scavenging assay the abts assay was measured with reference to the method of zhang et al. (2017). the abts reagent with the absorbance of 1.5 at 405 nm was added to the diluted samples (10, 12.5, 20, 25, 50, 100 mg/ml) and reacted in the darkroom at room temperature for 60 min. the absorbance of the sample mixtures was read at 405 nm using an elisa microplate reader (apollo11lb913, berthhold thechnolosies co., ltd., bad wildbad, germany). 2.6. air cells determination muffins were cut at the height of muffin mold (2 cm), and images of the bottom half were obtained using a digital camera (x-t20, fujifilm, tokyo, japan). by pore size, air cell number and area were calculated using the imagej software (marcet et al., 2015). 2.7. microstructure of batter and muffins 2.7.1 batter microstructure a drop of batter was placed on a microscope glass slide and covered with a cover glass. the cover glass was used to apply constant force (1 kg) to equalize and thinly spread the batter layer. the batter samples were observed using a microscope (ts100, nikon, tokyo, japan). the infinity capture v6.5.6 for windows software was utilized with the infinity lite camera (lumenera, ottawa, canada). 2.7.2 crumb microstructure scanning electron microscopic (sem) studies were examined using the jsm-6701f (jeol ltd., tokyo, japan). sample preparation for sem was according to the modified method of shin et al. (2018). the crumb samples were freeze-dried (fd8508, ilshinbiobase co.), and pieces of samples (size 2 × 4 mm) were placed separately on aluminum specimen mount using nem tape and conductive graphite (ted pella inc., california, usa). mounted samples were coated with au using the jsm 670-1f (jeol ltd., tokyo, japan) at 10 ma for 2 min. each sample was observed at 10 kv and 1.16 × 10-4 torr vacuum. 2.8. retrogradation kinetics of avrami model retrogradation of the muffin was analyzed with reference to berski et al. (2018). muffin samples were stored at 25℃ for 35 days, and hardness was measured on a rheometer (compac-100ⅱ, sun scientific, tokyo, japan) at the date of production and every week after preparation. according to the avrami equation, the retrogradation status of muffins was measured by analyzing the alteration of hardness with the storage period. avrami equation was as follows: 𝜃 = 𝑒!!"! (1) where θ is the amorphous part remaining after a certain time (t), k is the rate constant, n is the avrami exponent, and t is the storage period. ital. j. food sci., vol. 32, 2020 113 the operating conditions for the rheometer were a tpa test using the cylinder probe no.1 (ф 20 mm), 2 × 2 × 2 cm sample size, 120 mm/min table speed, 66.67% distance, and 10 kg max weight. (el-et) / (el-e0) = 𝑒!!" ! (2) where e0 is the hardness of the initial period (t = 0), et is the hardness after a certain time (t) and el is the greatest hardness value to be reached theoretically (the hardness value of a muffin stored at 25℃ for 35 days). the following equation was obtained by taking a common logarithm of equation (2) as: log〔ln (el-et) / (el-e0)〕 = log k + n log t (3) where n is the avrami exponent (1 ~ 4 values, depending on the crystallization status), and k is rate constant. 2.9. sensorial evaluation the muffin samples were baked 3 h before sensory evaluation. the samples were divided into four parts and supplied with water to each panelist at once in a white plastic plate. the sensory evaluation was completed by 51 panelists from korea university (age range 20-60). the panels evaluated the muffins based on their appearance, flavor, texture, taste and overall acceptability using a hedonic scale of 9 points (9 score is “i like very much” and 1 score is “i dislike very much”). 2.10. statistical analysis the statistical analysis of results was completed using the spss (ibm spss statistics 23, international business machines corporation, new york, usa) program. significant differences were assessed by one-way anova (analysis of variance) followed by duncan’s multiple range test at a 95% significance level (p<0.05). 3. results and discussions 3.1. muffin physicochemical characteristics the physicochemical features of muffins are shown in table 2 (fig. 1.) the results suggest that the addition of kf significantly affected the moisture content and brix degree (p<0.05). muffin crumb moisture content was the highest in k100 (28.18%) and the lowest in con (24.78%). similar to the description by gurpree et al. (2015), this is due to the water absorption of kf. with increasing kf replacement level, muffin moisture content tended to increase. as the result of comparing the water binding capacity of kf and wf, the water binding capacity of kf (225.09%) showed a higher value than that of wf (198.13%) (data not shown). as the water binding capacity results showed, the moisture content of muffins was significantly increased with the addition of kf. the sugar content of muffins was expressed in brix degree, which is the basic criterion to evaluate sugar contents. muffin sugar contents diminished significantly (p<0.05) as the kf substitution ital. j. food sci., vol. 32, 2020 114 increased. due to the maillard reaction, which is the reaction between the reducing sugar and the amino acids. the brix degree represents the content of soluble solids, and the reducing sugar involved in the maillard reaction is also included in the soluble solids. as the kf replacement level increased, the muffin lightness value decreased and became significantly darker, suggesting that the number of soluble solids was reduced by increasing the nonenzymatic browning reaction. according to a study by zhang et al. (2016), the brownish or yellowish color of bakery products with high sugar content were caused by the maillard reaction, which is mainly found in the baking process. the baking loss significantly decreased with increasing kf substitutions (p<0.05). the lowest baking loss was found in k100 (10.67%). the baking yield was the lowest in con (83.43%) and the highest in k100 (89.33%), and it significantly increased as the kf substitution ratio increased. the results are similar to the study of kotoki and daka (2010), suggesting that the water binding capacity affects the water content of muffin crumb, which may affect baking yield and baking loss. the specific gravity of the batter was the lowest in con (0.94), and it significantly increases with the increased in kf substitution levels. the muffin height significantly decreased as the kf substitution rate rose in the batter (p<0.05). the con muffin had the highest volume (121.83 ml) and specific volume (2.89 ml/g), and these were significantly decreased as the kf substitution ratio decreased. while the con muffin showed the lowest weight (42.11 g), it significantly increased with the increasing kf substitution percentage. the lightness (l), yellowness (b), redness (a), and color difference (△e) values of both crust and crumb are shown in table 3. crust l, a and b were the lowest in k100, and the l and b values of crust were the highest in con. the crust color was affected by the maillard or caramelization reaction between proteins and sugar. crumb l and b values decreased significantly, while the a value increased significantly as the kf substitution increased (p<0.05). the crumb color value was affected by the color of the raw material, which was not heated sufficiently to drive the maillard or caramelization reaction (marchetti et al., 2018). the crust △e values of con and k25 were lower than other samples. as the kf proportion increased, the crumb △e values increased significantly (p<0.05). figure 1. photograph of muffins and cross section of muffin crumbs with different levels of kf. ital. j. food sci., vol. 32, 2020 115 table 2. some physicochemical properties of muffin with different levels of kf. samples moisture (%) brix degree baking loss (%) batter yield (%) specific gravity height (mm) volume (ml) specific volume (ml/g) weight (g) con 24.78±0.29 1)d 3.03±0.20a 16.57±0.20a 83.43±0.20d 0.94±0.00e 48.56±0.43a 121.83±2.02a 2.89±0.05a 42.11±0.07d k25 25.95±0.17 c 2.63±0.06b 14.22±0.28b 85.78±0.28c 0.95±0.01d 47.24±0.42b 117.5±1.50b 2.72±0.03b 43.16±0.09c k50 26.92±0.43 b 2.50±0.00bc 11.85±0.03c 88.15±0.03b 0.96±0.00c 47.26±0.47b 114.23±0.76c 2.58±0.02c 44.45±0.02b k75 27.30±0.45 b 2.40±0.00c 11.66±0.13c 88.34±0.13b 1.04±0.00b 46.64±0.21b 107.83±1.26d 2.43±0.03d 44.33±0.10b k100 28.18±0.22 a 2.33±0.06c 10.67±0.09d 89.33±0.09a 1.04±0.00a 45.3±0.33c 90.33±1.26e 2.01±0.03e 45.04±0.05a f-value 46.567*** 23.100*** 591.725*** 591.725*** 616.542*** 28.328*** 226.868*** 317.512*** 782.063*** 1) the data are mean±sd in triplicates. a-edifferent superscripts indicate there are significant differences between values in the same row according to duncan's multiple range test at p<0.05. *p<0.05, ***p<0.001. table 3. color measurement of muffin crust and crumb with different levels of kf. samples crust crumb l2) a b △e l a b △e con 48.51±0.441)a 8.99±0.05b 15.21±0.06a 50.90±0.54b 78.51±0.29a -3.74±0.32d 24.26±0.42a 28.68±0.35e k25 47.78±0.10b 8.75±0.16bc 15.57±0.54a 51.74±0.28b 67.68±0.34b -1.02±0.12c 23.05±0.27b 36.42±0.50d k50 42.87±0.59d 9.64±0.07a 14.17±0.57b 56.11±0.56a 61.45±0.40c -0.79±0.09c 22.02±0.82c 41.63±0.31c k75 44.02±0.30c 7.80±0.18d 13.13±0.32c 55.08±0.99a 55.77±0.16d 0.97±0.11b 20.98±0.12d 45.68±0.20b k100 43.67±0.40c 8.53±0.21c 12.85±0.75c 56.03±0.23a 52.04±0.32e 1.57±0.07a 19.89±0.22e 49.45±0.36a f-value 125.283*** 60.174*** 17.172*** 53.359*** 3370.949*** 455.629*** 44.380*** 1557.270*** 1) the data are mean±sd in triplicates. 2) l: lightness, a: redness, b: yellowness, △e: total color difference. a-e different superscripts indicate there are significant differences between values in the same row according to duncan's multiple range test at p<0.05. *p<0.05, ***p<0.001. ital. j. food sci., vol. 32, 2020 116 3.2. textural properties of muffin crumb the texture of food products is a close factor to the body, such as the feeling of the mouth or fingers. the results of the muffin textural analysis prepared from the various ratio of wf and kf are presented in table 4. the results showed that kf significantly affected the muffin crumb texture (p<0.05). hardness was 1.15 n in con and as the kf ratio increased, the hardness increased significantly (p<0.05), reaching k100 at 3.09 n. the increment in hardness is related to fewer and smaller air cells inside the muffin crumb, and it corresponds to the muffin volume, height, and weight. tess et al. (2015) also mentioned that volume and hardness have an inverse relationship. springiness is related to aeration and freshness of products, and especially in bakery products, high springiness has close relevance to high quality (matos et al., 2014). the k100 muffin showed the highest value in springiness, and an appropriate addition of kamut flour seemed to have a good effect on the muffin textural quality. cohesiveness is the parameter that signifies the perception linked to the energy required to bite the piece of food and sensory brittleness (sanz et al., 2009). in comparison to con, the other samples were significantly (p<0.05) decreased in cohesiveness, which suggests that energy was required for the second compression. the chewiness reflects the parameter associated with the energy required for biting activity from a solid form to a swallowable state (tess et al., 2015). con chewiness showed the lowest value at 0.54 n and it increased significantly (p<0.05) with the increasing kf ratio. k100 chewiness was approximately twice as high as that of con. brittleness is related to the muscle motion of biting food, and it has a correlation with hardness (peng et al., 2002). brittleness in con was 56.57 g, and it significantly (p<0.05) increased, reaching 214.27 g in k100 and following a similar trend as hardness and chewiness. the previous study carried out by pasqualone et al. (2011) demonstrated that the crumb firmness of durum wheat bread was slightly lower than kamut bread, but there was no significant difference. table 4. textural profile analysis of muffin crumbs with different levels of kf. samples hardness (n) springiness (%) cohesiveness (%) chewiness (n) brittleness (g) con 1.15±0.121)c 69.65±3.27c 67.03±2.63a 0.54±0.06b 56.57±9.74e k25 1.40±0.15c 70.25±1.75c 61.56±0.24b 0.61±0.05b 84.04±3.08d k50 1.73±0.25c 75.29±2.11b 59.92±0.92b 0.78±0.10b 110.62±2.52c k75 2.39±0.30b 79.86±1.37a 54.46±1.48c 1.04±0.16a 150.87±3.54b k100 3.09±0.60a 82.32±0.96a 49.17±0.55d 1.25±0.24a 214.27±3.24a f-value 16.882*** 21.252*** 68.269*** 13.309** 426.197*** 1) the data are mean±sd in triplicates. a-edifferent superscripts indicate there are significant differences between values in the same row according to duncan's multiple range test at p<0.05. **p<0.01, ***p<0.001. ital. j. food sci., vol. 32, 2020 117 3.3. antioxidant properties of muffin crumb the kamut flour showed significantly higher antioxidant activities than wheat flour. the total polyphenol content was 23.44 ㎍gae/mg in kamut flour and 17.74 ㎍gae/mg in wheat flour. the total flavonoids content was 27.75 ㎍qe/mg in kamut flour and 8.44 ㎍ qe/mg in wheat flour. in the results of reducing power, kamut flour (0.84) was about four times higher than wheat flour (0.21). dpph and abts results also showed that kamut flour (129.91 ㎍/ml and 130.91 ㎍/ml respectively) had lower ic50 values than wheat flour (162.21 ㎍/ml and 325.79 ㎍/ml respectively) so that kamut flour had higher radical scavenging activities. sofi et al. (2013) reported a comparison of antioxidant activities between kamut flour and wheat flour, and kamut was determined as superior in dpph and fe2+ chelation. in addition, polyphenols and flavonoids were higher in kamut. the antioxidant properties of muffins are presented in table 5. the total polyphenol content and total flavonoid content were determined in terms of gallic acid equivalent and quercetin, respectively. k100 (17.33 ㎍gae/mg) was the richest in polyphenols, which was 2.62 times higher than con. the total flavonoids content was also the highest in k100 (18.54 ㎍qe/mg), and it increased significantly as kf level increased (p<0.05). the replacement of wf with kf showed an increment in reducing power. the reducing power of extracts is regarded as an indicator of antioxidant activities (kaur and kaur, 2018). a significant increment in abts and dpph radical scavenging activities of muffin crumbs was observed as the kf level increased (p<0.05). k100 showed a relatively lower ic50 in comparison to con. table 5. antioxidant activities of muffin crumbs with different levels of kf. con k25 k50 k75 k100 f-value polyphenols (㎍gae/mg) 6.61±0.06 1)e 8.07±0.12d 10.45±0.13c 15.30±0.20b 17.33±0.08a 3953.926*** flavonoids (㎍qe/mg) 9.61±0.25 e 11.77±0.00d 12.80±0.11c 16.75±0.33b 18.54±0.14a 987.048*** reducing power 0.23±0.00 d 0.22±0.00c 0.32±0.00b 0.36±0.00a 0.36±0.00a 9770.3*** dpph (ic50, ㎍/ml) 243.09±3.79d 234.95±1.55c 223.95±.6.67c 224.90±0.87b 213.47±3.48a 25.945*** abts (ic50, ㎍/ml) 263.08±2.90d 237.89±3.58c 223.68±1.91c 219.92±6.32b 207.34±0.22a 104.716*** 1) the data are mean±sd in triplicates. a-e different superscripts indicate there are significant differences between values in the same row according to duncan's multiple range test at p<0.05. *p<0.05, ***p<0.001. 3.4. crumb air cells table 6 presents the number and area of air cells categorized by a particular size range. cross section images of muffin crumbs are shown in fig. 2. during the mixing process, pores are generated in the batter and they grow while baking as co2 production results. ital. j. food sci., vol. 32, 2020 118 table 6. air cell number and area in muffin crumbs with different levels of kf. samples air cells number air cells area <1 pixel2 1-10 pixel2 10-100 pixel2 1001000 pixel2 1000 pixel2 < total 1-10 pixel 2 10-100 pixel2 100-1000 pixel2 1000 pixel2 < total con 61 90 87 81 13 332 4.80±2.71 40.80±27.79 331.22±210.10 2336.69±1194.21 2713.51 k25 52 88 123 66 12 341 4.53±2.55 39.36±23.97 265.05±158.10 2230.58±1445.59 2539.52 k50 98 177 187 59 5 526 4.37±2.28 38.63±24.47 222.32±152.39 1723.6±907.82 1988.92 k75 132 214 257 55 2 660 4.29±2.26 33.77±22.74 224.58±113.84 1412.50±433.46 1675.14 k100 129 218 251 53 3 654 4.28±2.32 34.53±22.09 211.47±110.25 1171.33±152.32 1421.61 figure 2. cellular structure of muffin crumb with different levels of kf. top line: scanned images of the cross section of muffin crumb, bottom line: modified images using imagej. ital. j. food sci., vol. 32, 2020 119 as the substitution ratio of wf to kf increases, the number of pores with the size of 100 pixel2 or less increased, whereas the number of pores decreased over a size of 100 pixel2. the air cells area result showed the opposite tendency to the air cells number. as the kf substitution rate increased, average air cell size increased in all pore size classes. therefore, substitution of wf by kf resulted in a tiny air cell and densely structured crumb. the size of air cells is an important factor affecting the texture of final bakery products (giacomozzi et al., 2018). the air cells growing during baking affects the tender quality, which is related to the crumb hardness. con showed a greater number of large air cells than other samples, which had weak crumb hardness. as mentioned earlier, the hardness increased as the pore size decreased and became dense. 3.5. microstructure of batter and muffins batter microstructure is shown in fig. 3. the micrograph of con batter shows a uniformly spread air bubble and the air bubble size is relatively large and not concentrated in a particular area. in the former study by rajiv et al. (2011), the control muffin batter showed an even and constant size of air bubble distribution. micrograph of k25 batter shows relatively small-sized air bubbles appeared. the air bubbles of k25 batter showed closer formation and density compared to those of the con batter. micrograph of k50 batter exhibits both large-sized and small-sized air bubbles, and it does not form the uniform distribution. compare to con and k25 batter, the smaller air bubbles can be observed to stick together slightly in the k50 batter. the k75 batter micrograph shows unevenly distributed air bubbles, and the small-sized air bubbles are gathered around medium-sized air bubbles. micrograph of the k100 batter shows an air bubble distribution similar to that of the k75 batter is observed, and the air bubbles are denser. fig. 4 presents the scanning electron micrographs. in figs. 4a-1 (con), 4b-1 (k25), 4c-1 (k50), 4d-1 (k75) and 4e-1(k100), the muffin crumbs are magnified 100 times, and the change of the pore size, distribution, and matrix surface can be observed. as the kf level increased, the small-sized pores gradually formed and the granular structure on the matrix surface became larger, such that the matrix appeared to be disconnected. fig. 4a-2 shows the micrograph of the con muffin crumb prepared entirely with wf, which shows gelatinized starch granules buried under the denatured protein matrix. the microstructure of starch granules embedded in a protein matrix is also described by gao et al. (2018). lee et al. (2001) reported that starch granules can be divided into two types, spherical and lenticular-shaped, which were also observed in the blended wheat flour dough. fig. 4b-2 displays the micrograph of the k25 muffin crumb, and it shows fewer starch granules and smoother continuous matrix than that of con. in fig. 4c-2, which is the micrograph of the k50 muffin crumb, a rather rough and ruptured protein matrix is observed. rajiv et al. (2011) reported that disruption of the protein matrix became greater when wheat was replaced by 60% of finger millet. in fig. 4d-2, which exhibits the micrograph of the k75 muffin crumb, the gelatinized starch granules are large, and it appears to be coated. fig. 4e-2 is the micrograph of the k100 muffin crumb, which shows greater and thin starch granules that seem to be entangled in a discontinuous protein matrix. therefore, when the kf level is increased, the starch granules become greater, and continuity of the protein matrix is lost. ital. j. food sci., vol. 32, 2020 120 figure 3. batter microstructure (×100). top line: micrograph of muffin batters con, k25, k50, k75, k100, bottom line: modified images of batter con, k25, k50, k75, k100 using imagej. figure 4. scanning electron micrograph of muffin crumbs. top line: sem micrograph of magnification 100×. bottom line: sem micrograph of magnification 250×. 3.6. retrogradation kinetics the avrami equation describes the retrogradation process kinetics and the result is shown in table 4. starch retrogradation is an important quality determinant in the staling of bakery products. avrami exponent (n) indicates the value of nucleation in crystallization, and it depends on the growth rate of crystallites in short storage periods (collar et al., 1999). the lower the avrami exponent, the slower the crystallization rate, which is more effective for delaying retrogradation (kim et al., 2006; s.-s. kim and chung, 2010; zhang et al., 2017). crystallization is an important factor as it is related to the texture and shelf-life of a bakery product. the avrami exponent showed the highest value in k100, but the value tended to decrease from con (1.5540) to k75 (0.7669). the rate constant (k) refers to the retrogradation time. the reduction in the rate constant describes delays in retrogradation in the presence of carbohydrates. the rate constant tended to decrease with kamut replacement from con (1.8095) to k75 (1.2904), but the k100 (2.1541) showed a sharp increase. according to avrami kinetic results, muffins prepared only with kamut had similar values to the con. carini et al. (2010) reported that whole kamut tortillas were very similar to the control group in textural changes during the storage period. ital. j. food sci., vol. 32, 2020 121 table 7. avrami parameters for muffin crumbs with different levels of kf. avrami avrami exponent (n) rate constant (k) r2 con 1.554 1.8095 0.9988 k25 1.3208 1.4036 0.9998 k50 1.1402 1.3113 0.9996 k75 0.7669 1.2904 0.9995 k100 1.5774 2.1541 1.0000 3.7. sensorial evaluation the sensory evaluation scores for appearance, flavor, texture, sweetness, and overall acceptability of muffins are presented in table 8. the data showed that the sensory score for appearance, flavor, texture, sweetness, and overall acceptability decreased as the kamut flour replacement level was over 50%. in the case of appearance and sweetness, except for con, k25 and k50 scored higher than other samples and became not preferred at levels above 75% kf addition. the lowest appearance score of k100 could be explained due to the dark crumb color by the maillard reaction between sugar and amino acids, increased brittleness, and small muffin volume. the decreased score in sweetness could be related to a decrement in brix degree. regarding flavor and overall acceptability, both k25 and k50 were as high as con. according to statistically analyze result, the con and both k25 and k50 showed no significant difference in flavor and overall acceptability (p>0.05). it showed that the kamut replacement had no significant negative effect on product preference at levels below 50%. the texture is one of the important parameters of sensory evaluation of processed foods (alpaslan and hayta, 2006). the texture was not significantly affected by kamut flour substitution (p>0.05), unlike the mechanical texture results. in previous studies, the partial or complete replacement of wheat flour with kf provided equal or better sensory characteristics (bordoni et al., 2017), and cooked kamut grain ranked highly for sweetness among the various wheat varieties (starr et al., 2015). based on the sensory evaluation results, 25-50% of the kamut added to muffins is considered as a desirable substitute addition level. table 8. sensory evaluation of muffins with different levels of kf. con k25 k50 k75 k100 f-value appearance 7.18±1.80a 6.76±1.45ab 6.27±1.54bc 5.72±1.39cd 5.49±1.55d 10.435*** flavor 6.84±1.49a 6.54±1.43a 6.27±1.61ab 5.84±1.54bc 5.53±1.65c 5.948*** texture 6.10±1.71ns 6.25±1.56 6.11±2.00 5.75±1.62 5.37±1.77 2.161 sweetness 6.94±1.22a 6.25±1.44bc 6.55±1.63ab 5.86±1.46c 6.00±1.93bc 3.985** overall acceptability 6.69±1.69 a 6.37±1.57a 6.45±1.80a 5.73±1.42b 5.47±1.74b 5.009** 1) the data are mean±sd in triplicates. a-e different superscripts indicate there are significant differences between values in the same row according to duncan's multiple range test at p<0.05. **p<0.01, ***p<0.001. ital. j. food sci., vol. 32, 2020 122 4. conclusions replacement of wheat flour by kamut flour in muffins affected the air bubble distribution in batter and this affected on the muffin volume, weight and height. the air cell distribution became denser as kf level increased so that caused increased harness. in addition, with increasing kf level, the greater starch granules and the less protein matrix continuity could be descripted through the microscopic observation. textural properties of crumb changed significantly. addition of kf improved antioxidant capacity. study of muffin firming kinetics revealed avrami exponent value of k100 significantly high, but k75 showed the lowest value. all muffins were considered acceptable and the muffin containing less than 50% of kamut flour level showed a preference score in the sensory evaluation references aacc. 2000. approved methods of the american association of cereal chemists 10th ed. pp.10-91.st. paul, mn:american association of cereal chemists, inc. alpaslan m. and hayta m. 2006. the effects of flaxseed, soy and corn flours on the textural and sensory properties of a bakery product. journal of food quality. 29(6):617-627. angioloni a. and collar c. 2011. nutritional and functional added value of oat, kamut®, spelt, rye and buckwheat versus common wheat in breadmaking. journal of the science of food and agriculture. 91(7):1283-1292. benedetti s., primiterra m., tagliamonte m.c., carnevali a., gianotti a., bordoni a. and canestrari f. 2012. counteraction of oxidative damage in the rat liver by an ancient grain (kamut brand khorasan wheat). nutrition. 28(4):436-441. berski w., ziobro r., witczak m., and gambuś h. 2018. the retrogradation kinetics of starches of different botanical origin in the presence of glucose syrup. international journal of biological macromolecules. 114:1288-1294. bordoni a., danesi f., di nunzio m., taccari a. and valli v. 2017. ancient wheat and health: a legend or the reality? 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63(1):590-598. tess m., bhaduri s., ghatak r.and navder k. p. 2015. physical, textural and sensory characteristics of gluten free muffins prepared with teff flour (eragrostistef (zucc) trotter). journal of food processing and technology. 6(9):490. wijngaard h.h. and arendt e.k. 2006. buckwheat. cereal chemistry. 83(4):391-401. zhang y.y., song k.y., o h., joung k.y., shin s.y.and kim y.s. 2017. effect of pomegranate (punica granatum l.) peel powder on the quality characteristics, retrogradation and antioxidant activities of sponge cake. the korean journal of food and nutrition. 30(3):578-590. zhang l., putranto a., zhou w.b., boom r.m., schutyser m.a. i.and chen x.d. 2016. miniature bread baking as a timesaving research approach and mathematical modeling of browning kinetics. food and bioproducts processing, 100:401–411 paper received february 19, 2019 accepted september 5, 2019 ijfs#1090_bozza ital. j. food sci., vol. 30, 2018 792 paper angiotensin i converting enzyme inhibitory peptides from sword bean y. zhang, p. chen, l. liu, k. li, h. wang and l. wang* school of life science, beijing university of chinese medicine, fangshan district, beijing 102488, china *e-mail address: wanglz@bucm.edu.cn abstract sword bean is a healthy food and herbal medicine in china. in this study, the main components of sword bean were determined. albumin, globulin, prolamin and glutelin were hydrolyzed by pepsin and then the angiotensin i converting enzyme (ace) inhibitory activity was evaluated. our results showed that glutelin peptides manifested the highest ace inhibitory activity with inhibitory ratio of 22.10±1.57% followed by prolamin peptides and albumin peptides of 16.77±0.76% and 16.40±0.42%, respectively, at the final concentration of 0.01 mg/ml. our results strongly suggest that sword bean at some extant have potential to lower blood pressure. keywords: sword bean, main component, angiotensin i converting enzyme, peptides ital. j. food sci., vol. 30, 2018 793 1. introduction hypertension, a major risk factor for cardiovascular and renal diseases, has become the most common serious chronic health problem. the rennin-angiotensin system (ras) is critically involved in the physiological regulation of blood pressure and pathogenesis of hypertension (cat and touyz, 2011). ace, as an essential member of ras, can catalyzes the conversion of angiotensin (ang) i to ang ii by removing a carboxyterminal dipeptide (wysocki et al., 2006). meanwhile, ace metabolizes bradykinin (bk), a vasodilator, to inactive bk-(1-7). therefore, ace inhibitors are effective first-line treatment against essential hypertension (thomas et al., 2004), such as captopril, enalapril and lisinopril. however, these synthetic drugs may also cause obvious side effects including cough, loss to taste, renal impairment, and angioneurotic oedema (antonios et al., 1995). thus, peptides with potent ace inhibitory activity derived from natural food provide an effectively alternative treatment (yu et al., 2006). in recent years, ace inhibitory peptides from natural protein have been successfully isolated, such as corn (yang et al., 2007), soybean (mallikarjun et al., 2006) and coix seed (yuan et al., 2014). recently, the antihypertensive peptides from traditional chinese medicine proteins has drawn considerable attention. sword bean, the seed of the leguminous plant canavalia gladiate, also has been treated as traditional medicine for containing canavanine, hemagglutinin, and concanavalin a (ekanayake et al., 2006). it has been reported that sword bean may exhibit antioxidant activity of eliminating free radicals and against oxidative stress. in addition, it also has strong anti-inflammatory and anticarcinogenic effects. it is reported that soybean paste containing sword bean exhibits higher ace inhibitory effects than other soybean pastes (han et al., 2015). in this study, this medicinal food was chose to prepare the ace inhibitory peptides because of its ace inhibitory activity and rich protein. the aims of this study are: (1) to determine the main components and protein content of sword bean. (2) to obtain peptides with low molecular weight (≤ 3 kd) by hydrolyzing protein with pepsin, and estimate their ace inhibitory activity. (3) to provide some reference for the clinical drug use of sword bean in traditional chinese medicine. 2. materials and methods 2.1. material sword bean was purchased from tongrentang (beijing, china). the voucher specimen (no. 131121003) was deposited at -20°c. pepsin, ace and hippuryl-l-histidyl-l-leucine (hhl) were purchased from sigma-aldrich (st. louis, mo, usa). trifluoroacetic acid (tfa, ms grade) and acetonitrile (hplc grade) were purchased from merck kgaa (darmstadt, germany) and fisher scientific (pittsburgh, pa, usa) respectively. all other chemicals and reagents were analytical grade. 2.2. determination of the proximal compositions protein, fat, moisture and ash content of sword bean were determined according to the chinese pharmacopoeia (commission, 2015). the content of starch was determined by ji (ji et al., 2016). ital. j. food sci., vol. 30, 2018 794 2.3. sequential extraction of seed protein the seeds were ground into powder by a universal high-speed smashing machine, and then defatted with cooled petroleum ether and dried at 40°c overnight. albumin, globulin, prolamin and glutelin were then sequentially extracted with deionized water, 0.5 m nacl, 70% ethanol (containing 0.5% naac, 5% β-mercaptoethanol) and 0.0125 m sodium borate buffer (containing 1% sds, 2% β-mercaptoethanol), respectively. all of the protein solutions were dialysised against deionized water at 4°c for 24 h and then freezedried. the samples were stored at -80°c for further analysis. 2.4. determination of protein molecular weight distribution sds-page was conducted according to the method of krizkova (krizkova et al., 2015) with some modifications to determine the molecular weight distribution of all the protein fractions. all the samples were run for approximately 100 min in 3% stacking gel with a electric current of 10 ma and then for another 100 min in 15% separating gel with 30 ma. after that, the gel was dyed with coomassie brilliant blue overnight and then decolored with bleaching liquid until the strips were seen clearly. 2.5. determination of the amino acid content for determination of amino acid composition, 100 mg samples were subjected to acid hydrolysis with 20 ml of 6 m hcl at 110°c for 24 h. then the lyophilized hydrosate was dissolved in 0.02 m hcl and analyzed by a amino acid analyzer (l-8900; hitachi, tokyo, japan) (wang et al., 2008). 2.6. preparation of enzymatic hydrolysates to produce bioactive peptides, enzymatic hydrolysis method was applied. the protein (2%, w/v) was dissolved in 0.01 m hcl, and pepsin was added with enzyme/substrate ratio of 1/10 (w/w). the mixture was incubated at the temperature of 37°c for 48 h. to terminate the reaction, the mixture was heated 95°c for 5 min. the hydrolysates supernatant was collected after the centrifugation (at 10,000 rpm, 10 min, 4°c). 2.7. ultrafiltration (uf) of protein hydrolysates to produce low molecular weight peptides, the hydrolysates were passed through ultrafiltration membrane(mwco, 3 kd). the peptide concentration of each collected fractions was estimated by the lowry method (sapan, and lundblad, 2015). 2.8. the assay of ace inhibitory activity the ace inhibitory activity was determined according to the method reported by yuan (yuan et al., 2014). briefly, the reaction system contained 10 μl sample, 20 μl ace (2 mu) and 20 μl hhl (2 mm). sample and ace were incubated at 37°c for 10 min prior to adding substrate hhl, and then for an additional incubation for 80 min at the same temperature. to terminate the reaction, 100 μl acetonitrile was added. captopril and borate buffer solution was used as positive and blank control, respectively. ace inhibitory activity was confirmed by monitoring the formation of ha which was generated by hhl under enzymatic hydrolysis. ha was detected by rp-hplc on a c18 column (250 × 4.6 mm, 5 μm, tianhe). the column was eluted by a mobile phase of acetonitrile/water (0.05% ital. j. food sci., vol. 30, 2018 795 tfa) at a volume ratio of 25 : 75 with the flow rate of 1 ml/min. the elution was monitored at 228 nm. the ace inhibitory ratio of each sample was calculated as follows: inhibitory activity (%) = [ (a-b) ⁄ a] × 100% where a was the ha peak area of blank control; b was the ha peak area in the presence of the sample. 2.9. statistical analysis all data were conducted in triplicate and expressed as the mean±sd. the sas 9.3 program was used for multiple comparison, and p < 0.05 were considered to be significant. 3. results 3.1. proximal compositions of sword bean the proximal compositions of sword bean were presented in table 1. the starch content of sword bean ranked first (36.59±2.93%). as the member of leguminosae, the protein content of sword bean accounted for 31.95±0.24%. the moisture and ash contents of this medicinal food all conformed to the requirements of the chinese pharmacopoeia. table 1. proximal compositions (%) of sword bean. protein crude fat moisture ash starch sword bean 31.95 (±0.24) 0.71 (±0.04) 8.33 (±0.01) 3.16 (±0.04) 36.59 (±2.93) results were expressed as the mean±sd (n = 3). 3.2. protein fractions distribution of sword bean protein patterns of sword bean were shown in table 2. considerable variability among albumin, globulin, prolamin and glutelin was observed. albumin had the highest percentage of 70.93±0.25% followed by globulin of 16.75±0.51%. the prolamin and glutelin contents of this leguminous seeds were rather low. insoluble protein in the residue only accounted for 5.83±0.04% indicating effective extraction of protein. table 2. protein pattern of sword bean (% of total protein). albumin globulin prolamin glutelin residual sword bean 70.93a (±0.25) 16.75b (±0.51) 1.48e (±0.02) 7.02c (±0.26) 5.83d (±0.04) results were expressed as the mean±sd (n = 3). different letters of indicated having significantly different (p < 0.05). ital. j. food sci., vol. 30, 2018 796 3.3. sds-page pattern of protein fractions of sword bean the molecular weight (mw) distributions of different protein fractions for sword bean were detected by sds-page, which was shown in fig. 1. albumin and globulin resolved into similar subunits ranging from 97 kd to 19 kd, with the major subunit of 50 kd. the bands of prolamin and glutelin were heterogeneous ranging from 57 to 14.4 kd and 97 to 19 kd, respectively. figure 1. the molecular weight distribution of proteins extracted from sword bean. lane 1, marker; lane 2, albumin; lane 3, globulin; lane 4, prolamin; lane 5, glutelin. 3.4. amino acid composition of seeds flour for sword bean, a total of 13 kinds of amino acid were detected. including almost all the essential amino acids and semi-essential amino acids for human beings. from table 3, it could be seen that phe had the highest percentage of 4.55±0.11 mg/100 mg, while his recorded the lowest (0.50±0.01 mg/100 mg) in sword bean. 3.5. ace inhibitory activity assay the rp-hplc method was utilized to estimate ace inhibitory activity of peptide mixtures (≤ 3 kd). the blank control displayed a strong peak area of ha (fig. 2a), while the positive control (captopril, final concentration of 2×10-9 mol/l) manifested a strong ace inhibition ratio of 91.64±0.07% (fig. 2b). the glutelin peptides (≤ 3 kd) revealed the highest ace inhibitory activity at the final concentration of 0.01 mg/ml with 22.10±1.57 (fig. 2c). all results were showed in table 4. ital. j. food sci., vol. 30, 2018 797 table 3. the amino acid content of sword bean (mg/100 mg). amino acid sword bean (mg/100 mg) asp 2.84±0.03 thra 1.32±0.03 ser 1.49±0.02 glu 3.26±0.07 gly 0.88±0.02 ala 0.68±0.01 cys vala 2.74±0.06 meta ilea 1.07±0.02 leua 1.69±0.05 tyr phea 4.55±0.11 lysa 1.47±0.02 hisa 0.50±0.01 trpa arga 1.56±0.02 pro a(semi-) essential amino acid for human, not detected. the data was expressed as the mean±sd (n = 3). elution time (min) elution time (min) ha hhl b ha hhl a ital. j. food sci., vol. 30, 2018 798 elution time (min) figure 2. rp-hplc chromatograms of (a) blank control, (b) positive control (captopril, final concentration of 2×10-9 mol/l), (c) glutelin peptides (≤ 3 kd) of 0.01 mg/ml. the mobile phase consisted of 25% acetonitrile (containing 0.05% tfa), eluting at a flow rate of 1.0 ml/min and the absorbance of eluent was detected at 228 nm. table 4. ace inhibition rate (%) of peptides (≤ 3 kd) from different protein fractions. peptide ace inhibition rate (%) albumin peptides 16.40±0.42b globulin peptides 12.72±0.29c prolamin peptides 16.77±0.76b glutelin peptides 22.10±1.57a results were expressed as the mean±sd (n = 3). the samples were measured at the final concentration of 0.01 mg/ml. different letters of indicated having significantly different (p < 0.05). 4. discussion food-derived ace inhibitory peptides can provide an effectively alternative treatment for hypertension. there are different methods to produce ace inhitory peptides from precursor proteins, such as enzymatic hydrolysis (chen et al., 2007), microbial fermentation (yamamoto et al., 1994) and chemical synthesis. among these methods, enzymatic hydrolysis is the most commonly used method (yuan et al., 2014). there are a great number of studies have proved that food-derived protein hydrolysates and peptides possess ace inhibitory activity (balti et al., 2010; lassissi et al., 2014; lee et al., 2010). soybean paste containing sword beans exhibits higher ace inhibitory effects (han et al., 2015). research has shown that the presence of hydrophobic amino acids can increase ace inhibitory activity (li et al., 2004). our study showed that sword bean included all the essential amino acids (except met) and semi-essential amino acids for human beings. moreover, sword bean protein may become effective sources for preparation of ace inhibitory peptides because of its high proportion of hydrophobic amino acid and proline, with 44.58% of total amino acid. albumin, globulin, prolamin and glutelin were sequentially extracted, which is of benefit to study different proteins activity. the high levels of protein and starch make sword bean good sources of these nutrients. 5. conclusion our study mainly focused on the ace inhibitory activity of protein hydrolysates. the result showed that glutelin peptides manifested the highest ace inhibitory activity with ha hhl c ital. j. food sci., vol. 30, 2018 799 inhibitory ratio of 22.10±1.57% followed by prolamin peptides and albumin peptides of 16.77±0.76% and 16.40±0.42%, respectively, at the final concentration of 0.01 mg/ml. after further separation, purification and structural identification of hydrolysates (≤ 3 kd), bioactive peptides with better antihypertensive activity might be obtained. our data might contribute to further research into food derived antihypertensive compounds, meanwhile it also provides some reference for the clinical drug use of sword bean in traditional chinese medicine. acknowledgements this study has received financial support from the natural science foundation of china (no. 81872972) 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enzyme inhibitory peptides derived from porcine hemoglobin. peptides 27(11):2950-2956. doi: doi.org/2910.1061/j.peptides.2006.2905.2025. yuan j., liang y., cui s., zhang x., wang l. and qiao y. 2014. angiotensin i converting enzyme inhibitory and antioxidant activity of adlay (coix lacryma-jobi l. var. ma-yuen stapf) glutelin hydrolysate. italian journal of food science 26(3):282-288. paper received november 8, 2018 accepted june 25, 2018 ijfs#1512_bozza ital. j. food sci., vol. 31, 2019 764 paper a clustered-based segmentation of chinese wine consumers by means of kernal fuzzy c-means h. yu and w. ruimei* college of economics and management , china agricultural university, no.17 qinghuadonglu, haidian district, beijing, 100083, p.r. china *corresponding author: tel.: +86-010-62738763 e-mail address: wangruimei@cau.edu.cn abstract the aim of this study is to segment chinese wine consumers based on their preferences, motivations and purchasing behaviors. data from representative 3420 responses were profiled through kernel fuzzy c-means (kfcm), and one-way anova analysis was used to define socio-demographic characteristics of each cluster. this study identified four consumer segments: balanced consumers, credulous consumers, experiential consumers and health sippers. each group showed different demographics, eating and purchase habits. these findings reveal a typology of chinese wine consumers when making the purchase decisions and verify the applicability of kfcm in consumer segmentation. identification of chinese consumer segments provides winemakers a better understanding of the various characteristics of wine consumers and their different purchasing behaviors. this study also provides new contributions to research on the segmentation of the chinese wine market. keywords: segmentation, kernel fuzzy c-means, chinese wine consumer ital. j. food sci., vol. 31, 2019 765 1. introduction with the increased household income and the growing middle class consumers, the level of wine consumption among chinese consumers has significantly increased in recent years. during the past twenty years, china's wine consumption has risen from 10.7 million hl in 1999 to 17.9 million hl in 2017, with an annual growth rate of about 10.9% (oiv, 2018). with a population of 1.39 billion and a gdp of 82.7 trillion cny in 2017 (national bureau of statistics, 2018), china provides a huge market for food and alcohol sales. the rapid growth of wine demand has not only promoted the development of local wine production, but also enabled a large number of foreign wine products enter to the chinese market. for winemakers, china has become a very important target market. as reported by international organization of vine and wine, wine consumption in china shows a positive growth trend. in 2017, wine consumption was 1.79 million kiloliters, accounting for about 7% of global consumption (oiv, 2018). in addition, according to a report by vinexpo, china has become the fifth largest static wine market in the world (vinexpo, 2018). in other words, wine has become one of the most popular products for chinese consumers and is increasingly favored by the younger generation. for chinese wine drinkers, although the per capita alcohol consumption will rise from 1.34 liters/year to 1.53 liters/year, there is still a big gap compared with traditional wine consuming countries – 28 times different from france (iwsr, 2018). despite the increase in the production of domestic wine and the growing demand for imported wines, the market potential of chinese wine consumers is huge. however, the needs and preferences of consumers are heterogeneous which means simple types of wine can’t meet the needs of differentiated markets. because of the competitions in the chinese wine businesses, it is important to segment the market to attract more potential consumers (zeng, 2014). there are very few studies on the segmentation of chinese wine market from the perspective of marketing (capitello et al., 2015; capitello et al., 2014; lee et al., 2015) although market segmentation is an important component and platform for corporate strategic marketing. market segmentation can help companies accurately distinguish valuable targets and select target markets more effectively, find their own position in market competition, and design unique products to meet the needs of consumers in the market. hence, understanding the main drivers and potential motivations for different types of consumers is very important for winemakers. market segmentation also provides a better framework to customize and distribute products to meet different needs of consumers. in addition, it can provide corresponding marketing strategies for marketers. 2. literature review 2.1. understanding customers in the wine industry the different attributes and functions of a product are key factors in determining whether a consumer will purchases it (green et al., 2004). consumers purchase decision can be influenced by intrinsic cues and extrinsic cues as a criterion for judging quality in the process of purchasing products (grunert et al., 2004). intrinsic cues are properties which are inherent to the products, such as ingredients and sensory attributes. extrinsic cues mainly refer to physical attributes such as price, brand, packaging, and origin that are not part of the physical attributes akdeniz et al. (2013). in addition, the marketing ital. j. food sci., vol. 31, 2019 766 strategies of wine shops and the motivation of consumers to purchase are also important factors (atkin et al., 2007; bruwer et al., 2012; bruwer et al., 2011). wine consumption is the specific behavior generated by consumers based on complex psychological activities, including psychological cognition and behavioral awareness (pettigrew and charters, 2010). the cognition includes a variety of psychological activities such as stimulation, impression, thinking and imagination. in general, it is the evaluation of the quality, function and image of a certain wine by the consumers. on the basis of cognition, consumers will have needs of wine, which will form the motivation for wine purchase. when the products meet requirements of consumers, the motivation for wine purchase will gradually be translated into specific wine purchase behavior. many researchers have carried out studies on wine consumption psychology (cognition, motivation, etc.) and behavior, providing an important theoretical reference for people to understand the behavior characteristics and behavior of wine consumers. health benefits, auxiliary dining and interpersonal communication are the main motivations of chinese wine consumers (somogyi et al., 2011). the positive health benefit of wine is that people drink wine in moderation can prevent disease and has anti-aging effect because wine is rich in vitamins and antioxidants (lee, 2009). due to the special color, aroma and taste of wine, it helps to improve the taste of the food when eating with wine (wang, 2016). as a social tool, wine is widely used by chinese consumers to express emotions or enhance friendship (hall et al., 2013). in terms of chinese wine consumer behavior, previous studies have shown that the attribute of product, individual characteristics of consumers and psychological factors of consumers will affect the behavior of chinese wine consumers (pickering and hayes, 2017). various attributes, such as sensory attributes and extrinsic cues including price, brand, and origin are found to be important aspects that influence wine choice (lockshin et al., 2016). when consumers make purchase decisions, the sensory attributes of wine are arguably the most important factor in determining whether people drink or not (charters and pettigrew, 2007; keown and casey, 1995; sarar et al., 2010; thompson and vourvachis, 1995). liu and murphy (2007) have shown that the main reason why chinese wine consumers drink red wines is color, because red symbolizes prosperity and good luck in chinese culture. camillo (2012) also confirmed that sensory attributes such as taste are the main factors affecting wine purchase and consumption. regarding the extrinsic cues of wine, a number of researchers have determined the attributes that have significant effect on wine consumption (xu et al., 2014). they found that the most important factors influencing consumers' choice of wine are previous experiences, quality, brands and recommendations. areta et al. (2018) believe that the quality ratings provided by experts, the vintage and scale of the winery are important in the process of wine consumers’ decision-making. in addition, jovanović et al. (2017) empirically demonstrate socio-demographic factors such as region, place of residence (urban and rural areas), family size, age, income and education; and behavioral-cognitive factors such as the importance of market price, place of purchase and product characteristics play a leading role in wine consumers’ decision-making process. finally, the health function of wine is also an important factor affecting consumer purchases. nowadays, people pay more attention to their health. the healthy function of wine has been gradually recognized and received more attention by chinese consumers. somogyi et al. (2011) found that the perceived health benefits of wine could influence the behavior of wine consumers. pettigrew and charters (2010) also verified that the health benefit is an important reason for wine consumption. ital. j. food sci., vol. 31, 2019 767 2.2. market segmentation theory market segmentation is the process of differentiating consumer groups who have different needs in the market according to certain criteria. market segmentation helps companies meet the needs of each segment more effectively, explore market opportunities, open up new markets, select the target market, and develop effective marketing strategies (marshall and johnston, 2010). raaij and verhallen (1994) showed that the needs of different segments of consumers could offer new opportunity for enterprises. nowadays, researchers mainly segment the wine market from the aspects of personal profile (gender, age, etc.), geographical factors, psychological factors (preference, attitude, etc.) and behavioral factors, like purchase frequency, purchase time, etc. (jain, 2012). spawton (1991) classified consumers into four categories: the wine connoisseur, the aspirational wine drinker, the beverage wine drinker and the new wine drinker based on the consumers’ location, consumers’ attitude towards the brands, and their knowledge and involvement of the wine. a study of chinese wine consumers from a gender perspective was conducted by bretherton and carswell (2013) and the results showed that wine was considered more masculine rather than feminine. it had also verified the results of liu and murphy (2007) and ritchie (2007) at the same time. ogbeide and bruwer (2013) found that older consumers in western countries were more involved in wine related activities from the perspective of age, because they had more time and money to participate in the wine tasting and wine clubs. regarding the places of purchase, hu et al.(2008) showed that 65% of chinese consumers are accustomed to buying wines in supermarkets or restaurants, and the main motivation for drinking is celebration and accompanying meals. more specifically, szolnoki (2018) used two-step clustering to classify tourists in rheingau into four classes: wine and rheingau lovers, wine-oriented tourists, first-time tourists and international tourists based on objective variables. masson et al. (2017) classified wine consumers into six groups from the perspective of drinking occasions. these six groups of consumers were: indifferent occasionals, wine lovers, relaxed amateurs, social networkers, stay-at-home connoisseurs and infrequent moneyminded. malone and lusk (2018) segmented consumers into five clusters: traditional, mavens, uninformed, locavores and premium by k-means cluster analysis, which was based on consumers’ taste perceptions of various brands. most business models are based on pca, factor analysis and k-means method to segment consumers into several groups (díaz et al., 2018; masson et al., 2017; yang et al., 2017). however, the initial point of k-means method is unstable and random, which causes the instability of clustering results and limits the quality of clustering (akbulut et al., 2016; lu and yan, 2015). with the continuous improvement of machine learning methods, cluster analysis based on k-means method are insufficient to meet the requirement of academics and practitioners when segmenting massive data. with introducing fuzzy set theory, ruspini (1969) first adopted the idea of fuzzy in data clustering. data clustering based on fuzzy set theory has become an important step in data mining and machine learning (akbulut et al., 2016). in order to overcome several drawbacks of k-means, kernel fuzzy c-means (kfcm) was proposed. compared with the classical clustering algorithm (k-means and factor analysis), kfcm can better distinguish, extract and amplify useful features, so as to achieve more accurate clustering. at the same time, the convergence speed of the algorithm is faster. when the classical clustering algorithm fails, the kernel clustering algorithm can still get the correct clustering (yang and tsai, 2008; zhang and chen, 2003). the kfcm has been widely used in image ital. j. food sci., vol. 31, 2019 768 segmentation, fault diagnosis and customer segmentation (basu and srinivas, 2014; cai et al., 2009; yang et al., 2011). the purpose of this paper is to use kfcm to segment chinese wine consumers by considering the attitudes, purchase motives and purchasing behavior of chinese wine consumers. the findings will make wine dealers and winemakers better understand the needs of consumers. 3. materials and methods 3.1. questionnaire to achieve the aims of this study, a semi-structured questionnaire was developed and used to classify chinese wine consumers. the questionnaire was based on geographic, demographic, socioeconomic, psychological and behavioral variables (masson and aurier, 2017). it also includes perception of packaging and labeling, consumer motivation, knowledge and involvement and sensory attributes (bruwer et al., 2011) which impact on wine purchase (barber, 2012). the questionnaire is divided into three parts: consumer’ knowledge and his consumption of wine; consumer preferences and motivations for purchasing wine and sociodemographic characteristics. a total of 21 items are divided into six variables: sensory attributes, business marketing, motivation of purchase, consumers’ familiarity, product attributes, and where to buy. in this study, the likert five-point scale method was used to measure the consumers’ attitude. the measurement structure was “very disagree=1, very agree=5”, which were all ordinal measurement, indicating the attitude of consumers to a certain point of view. 3.2. the sample in order to improve the representativeness of the sample and the practical application value of the research conclusions, this study conducted a comprehensive sample survey in 24 provinces, municipalities and autonomous regions in the eastern, central and western regions of china from july to august 2018. the samples covers a wide range of regions, thus could represent the purchasing behavior of wine consumers in different regions of china. before the official investigation, the researchers conducted a centralized and unified training for the investigators and explained the purpose of the survey, the content of the survey, the survey methods and process. the survey was conducted by using convenient sampling because surveys were conducted in shopping malls, streets, residential quarters, parks, farmer's markets, and village markets. the respondents independently filled out the questionnaire according to their actual situation, which would take 20-30 minutes. a total of 4000 questionnaires were collected, of which 2180 were online questionnaires the rest were face-to-face questionnaires. the questionnaires, which had too many missed items and incorrect key information, were removed and finally 3,420 valid questionnaires were actually collected. the sample distribution of the survey was showed in table 1 and the sample coverage is considered comprehensive and representative. ital. j. food sci., vol. 31, 2019 769 table 1. samples distribution of the survey. 3.3. method 3.3.1 confirmatory factor analysis confirmatory factor analysis was used to determine whether items related to wine consumption in literatures could be profiled as general characteristics (hair et al., 2011). for this purpose, confirmatory factor analysis was carried out for 21 selected items, and factors were extracted by maximum rotation of variables. items, which factor loads, were less than 0.5 or items with two or more factor loads greater than 0.5 were excluded from the sampling scale. the results showed that the correlation matrix of the 17 item scales is appropriate (bartlett’s test of sphericity： 2 14653.65χ = ; 435df = ; 0.000;p< the kmo index was 0.861). then, the cronbach's α value was tested to measure the reliability of each factor indicating that the cronbach’s α values of these factors were higher than 0.7, indicating that the internal consistency between the items was better (hair et al., 2011). finally, a six factor solution using varimax rotation procedure was proposed through spss. confirmatory factor analysis showed that 17 variables accounted for 85.65% of the variance and the kmo index was 0.861 (table 2). 3.3.2 cluster analysis kernel fuzzy c-means (kfcm) was used to segment chinese wine consumers based on their purchasing behavior and motivations. the reason for choosing kfcm is that the clustering result of kfcm is more accurate than the general cluster analysis (lu and yan, 2015). we used matlab 2014a software for the analysis. location number of respondents location number of respondents coastal area inland area beijing 240 anhui province 36 hebei province 307 gansu province 100 shandong province 184 liaoning province 147 shanghai 173 ningxia hui autonomous region 94 jiangsu province 316 shanxi province 267 zhejiang province 198 guangxi zhuang autonomous region 41 fujian province 44 henan province 85 guangdong province 253 heilongjiang province 64 tianjin 90 hubei province 70 hunan province 144 jilin province 51 shaanxi province 66 sichuan province 195 yunnan province 147 chongqing 108 total 1805 total 1615 ital. j. food sci., vol. 31, 2019 770 table 2. factor loadings and reliability values. the principle of kfcm algorithm is to map the points of the original space to the feature space by using the kernel function, then directly or indirectly perform algorithm design, analysis and calculation in the feature space, so as to obtain the clustering of the original space. by mapping the kernel function, it can better distinguish, extract and amplify the features that did not appear before, which makes the clustering results more accurate, and also makes the convergence speed of the algorithm improved. for traditional fuzzy clustering analysis, fuzzy c-means clustering is the most widely used method. however, the number of clusters in this type of algorithm is unknown in advance and requires artificially determined determination. in order to better determine the number of clusters in the real structure of the data set and improve the accuracy of cluster analysis, this paper based on the cluster validity function proposed by bezdek et al. (1984) to determine the optimal number of clusters in the data sample set. among them, name items factor loading cronbach’s α intrinsic cues (madeira et al. ,2009) ic1: i usually buy wines with famous brand. 0.792 ic2: the higher the price, the better the quality of the wine. 0.824 0.929 ic3: the quality of wines in well known regions is more reliable. 0.611 ic4: the better the packaging, the higher the grade of the wine. 0.792 ic5: good vintage can produce good wines. 0.755 extrinsic cues (chrea et al., 2011; hall, 2013) ec1: the better the color, the better the wine quality. 0.699 0.845 ec2: the richer the fragrance, the better the quality of the wine. 0.825 ec3: the better the taste, the better the quality of the wine. 0.853 marketing (madeira et al,. 2009) m1: wine advertisement has a big impact on my purchase decision. 0.784 0.932 m2: promotion has a big impact on my purchase decision. 0.744 m3: the location of the store and its interior style impact my choices. 0.607 m4: i trust the recommendation of the sellers. 0.661 reference group (fernandes et al., 2018) rg1: i trust the recommendations of my friends and relatives. 0.755 0.895 rg2: the more rewards offered by the seller, the better the quality of the wine. 0.899 motive (chrea et al., 2011; hall, 2013) mo1: the health benefits of wine affects my choice. 0.678 0.850 knowledge and involvement (brucks, 1985) ki1: the knowledge of wine will influence my choice. 0.711 0.769 ki2: i have very good knowledge about wine. 0.697 ital. j. food sci., vol. 31, 2019 771 the number of clusters that maximize the value of cluster validity index is the optimal number of clusters. 3.3.3 one-way anova analysis descriptive statistical analysis was used to compare the socioeconomic characteristics of each group and wine purchasing and eating habits. in addition, one-way anova was used to compare the mean differences based on age, education and monthly income among the identified groups (lópez-rosas and espinoza-ortega, 2018; skuras and vakrou, 2002). before testing demographic variables and consumption patterns among groups of consumers, homogeneity test of the variance of the population was conducted. the study found that the variances of sociodemographic backgrounds, purchasing and eating habits are the same among different groups of consumers, then one-way anova can be used to analyze these data. the overall one-way anova analysis using f-test were conducted, and when the sig is less than 0.05, it can be considered that there are differences among groups. the results were shown in table 4. 4. results and discussion 4.1. cluster analysis blanchard et al. (2009) argued that the desires and willingness of the consumer can explain consumer’s behavior well and can be used to predict consumers' actual consumption behavior of products. therefore, it is especially important to understand the factors related to consumer behavior. in order to better determine the number of clusters that can reflect the real structure of the data set, it is necessary to calculate the clustering validity function and determine the optimal number of clusters by comparing the index values. the calculation results are shown in figure 1. according to the definition of clustering validity function, the clustering number corresponding to the maximum value is the optimal clustering number of data sets. fig. 1 shows that when the fuzzy weighted index is 1.5, 2, 2.5 and 3, the clustering validity index is the largest when class is 4, so the optimal clustering number is 4. the results of the kfcm revealed four distinct consumer groups that can be named according to the segmenting variables of the consumers (table 3). the scores of the attributes are shown in fig. 2: balanced consumers, credulous consumers, experiential consumers and health sippers. health sippers: this is the largest group of the four consumer groups, with 39.4% of respondents belonging to this group. what makes it different from other groups is that the main motivation for this group to drink wine is for health. they think that drinking wine is good for health and they are more likely to have a high level of health awareness than other groups. it is worth noting that this group scores the highest on knowledge and involvement, which indicates that that they are confident in their knowledge of wine and were care about their health. ital. j. food sci., vol. 31, 2019 772 table 3. categories of wine consumers according to the segmenting variables (a) (b) (c) (d) figure 1. diagram of kfcm validity function's change to data: (a) fuzzy weighted index is 1.5, (b) fuzzy weighted index is 2, (c) fuzzy weighted index is 2.5, (d) fuzzy weighted index is 3. balanced consumers: this group consists of 38.7% of the total respondents. they are "balanced" because consumers in this group consider all aspects of the product in the purchase process, such as the intrinsic and extrinsic cues of wine, the recommendation of family and friends, and the health benefits. the preference scores for marketing strategies such as advertising and shopping guide recommendation are lower compared to the other three groups suggesting that they are more rational. in other words, this group of consumers will evaluate wines by considering the balance of multiple factors during the factor balanced consumers (n=1324) credulous consumers (n=237) experiential consumers (n=515) health sippers (n=1347) reference group 3.7 3.8 1.9 3.1 marketing 3.2 4.2 2.1 2.9 intrinsic cues 3.8 2.9 4.0 3.3 knowledge and involvement 3.3 3.1 2.8 3.4 extrinsic cues 3.5 3.2 3.1 2.8 purchase motive 3.4 3.3 2.1 3.9 2 3 4 5 6 7 8 9 10 1 1.5 2 2.5 3 3.5 4 4.5 5 x 10-3 e ff ec tiv en es s in di ca to r class 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 x 10-3 ef fic ie nc y in de x class 2 3 4 5 6 7 8 9 10 0.5 1 1.5 2 2.5 3 3.5 4 x 10-3 ef fic ie nc y in de x class 2 3 4 5 6 7 8 9 10 0.5 1 1.5 2 2.5 3 3.5 x 10-3 ef fic ie nc y in de x class ital. j. food sci., vol. 31, 2019 773 purchase process. they don’t need to have the highest level of quality for all segmenting variables. experiential consumers: this group of consumers consists of 15.1% of the samples. they show significantly preference for the sensory attributes of wine such as color, taste and aroma. these attributes are usually not confirmed until the product is purchased or used, so consumers can only determine these attributes through "experience." hence, these consumers may not be influenced by their friends' recommendations and the marketing strategies. for this group, only the score of intrinsic cues are high because they just trust their own experiences. they don't drink wines for its health benefit and they don't know much about the knowledge of wine. credulous consumers: these consumers accounts of 6.9% of the respondents. they are more susceptible to extrinsic cues than other groups of consumers, such as corporate promotions, friends' recommendations, etc. they are more likely to be influenced by others during the purchase process and have less knowledge about the wine. they don't care about the quality and health benefits of the wine itself, but the shopping environment and experience can affect their trust and their purchase decision. these results are similar to other researchers on wine consumers. for example, masson et al. (2017) identified six groups of wine consumers using the k-means method. among them, wine lovers have high product involvement in wine. similar to balanced consumers in this study, when choosing wine, they will mainly consider the recommendation of the independent wine experts. like experiential consumers in this study, relaxed amateurs rely on the taste of wines rather than other variables. they don't have much knowledge of wine and only buy what they like. finally, consumers belong to the social network cluster have limited knowledge of wine. they buy wines based on recommendations from others like credulous consumers in this study and are not interested in the intrinsic and extrinsic cues of wines. lópez-rosas and espinoza-ortega (2018) also found that there are four groups of wine consumers in mexico: traditional consumers, consumers in transition, social consumers and consumers linked to the territory. the first group of consumers is similar to balanced consumers because they buy wine based on factors such as origin, brand, awards and quality of wines. they are more rational when make purchase decisions. the second group of consumers is similar to the experiential consumers; they pay more attention to the sensory attributes of wines. then, the third group of consumers are not aware of the attributes of the wine and do not pay attention to the product itself. they often do impulse shopping, just like credulous consumers. for health sippers, annunziata et al. (2016) found that italian consumers who focus on the health warning had a better understanding of the nutritional properties of wines and like wins with detailed nutritional information. these consumers valued wines with health warning followed by nutritional information, when they chose wines. samoggia (2016) studied the effects of wine consumption on health; they found that health-oriented consumers were willing to spend more money on wines, which could improve their health. they are more concerned about the health benefits of the wines. in addition, yoo et al. (2013) have shown that consumers who are more knowledgeable about the health benefit of wines would be willing to spend more money on health-oriented wines. therefore, winemakers and wine marketers should consider health benefits of wines in order to increase the consumers’ desire to buy, especially when developing new wine markets. ital. j. food sci., vol. 31, 2019 774 figure 2. scores of the groups according to the segmenting variables. 4.2. socio-demographic backgrounds, purchasing and eating habits of the groups the analysis of table 4 shows that the proportion of males and females of chinese wine consumers is relatively balanced. the proportion of female consumers is slightly higher than that of men which is in line with masson's (2017) study of chinese consumers. ogbeide and bruwer (2013) pointed out that the age of consumers is related to the level of wine drinking. the results of one-way anova show the age differences among the four groups of consumers. more than half of all respondents are 26 to 45 years old, similar to the findings of china wine barometer – wave 6 (2016). in addition, more than 24% of consumers belonged to balanced consumers. the credulous consumers are more than 45 years old. thus it can be seen that effective marketing can influence purchase decisions of the middle-aged and older consumers. magri et al. (2007) indicated that the main factors affecting consumers' alcohol consumption are consumers’ characteristics and the hedonic motivation. however, other motivations, such as health benefits and appropriate labeling content are also important. the results also show that the health sippers have a higher degree of education. in this study, health sippers have the highest percentage of consumers with college and higher education qualification, which is directly related to their knowledge and involvement score. santos et al. (2006) argued that the difference between wine consumer groups is caused by their level of involvement and knowledge of the product. therefore, the occupation of consumers is also a key factor influencing their purchase. regarding the occupation of consumers, all four groups of consumers have steady occupations, although there is a considerable proportion of students and retired people. it is worth mentioning that the ratio of retired and independents in experiential consumers is higher than in other groups. in this group, wine consumption is related to intrinsic cues, which may be related to their social backgrounds and their belief that drinking wines can to help them to release stress and be more relaxed. ital. j. food sci., vol. 31, 2019 775 table 4. socio-demographic backgrounds, purchasing and eating habits by cluster. variable sample (%) experiential consumers (15.1%) balanced consumers (38.7%) credulous consumers (6.9%) health sippers (39.4%) homogeneity test f-test sig. gender male 48.5 49.5 46.9 47.0 49.5 0.61 0.983 0.400 female 51.5 50.5 53.1 53.0 50.5 age below25 13.6 18.6 14.8 15.0 16.2 0.79 27.309 0.000 26-35 32.8 44.5 25.4 32.0 36.5 36-45 28.2 23.2 31.8 28.2 26.5 46 or above 25.4 13.7 28.0 24.7 20.8 education level secondary/high school or blow 28.7 35.0 18.0 49.0 34.9 0.52 31.297 0.000 college/university 60.4 49.9 70.8 40.4 55.9 graduate level or higher 10.9 15.1 11.2 10.6 9.1 occupation student 13.1 9.7 16.1 14.4 11.2 0.73 1.192 .311 independent 16.4 16.3 12.8 15.7 20.2 retired 18.9 23.5 18.5 17.4 17.8 employed 51.6 50.5 52.6 52.5 50.8 per capita monthly income below 3000 cny 12.7 8.3 15.7 13.1 11.3 0.45 17.247 0.000 3001-5000 cny 13.9 13.6 11.0 26.9 16.3 5001-7000 cny 30.1 29.3 23.2 24.7 36.3 7001-10000 cny 21.3 23.9 20.9 21.6 20.6 above 10000 cny 22.1 24.9 29.3 13.6 15.5 consume bottles of wine in a year? (750ml) 1-2 bottles 25.2 19.2 24.9 28.0 27.3 0.65 3.043 0.028 3-5 bottles 23.0 24.7 22.5 23.3 22.8 6-8 bottles 20.8 24.1 20.3 15.3 21.0 9-11 bottles 12.7 13.6 12.5 13.6 12.4 12-15 bottles 7.9 5.4 9.6 7.6 7.1 16-20 bottles 4.6 5.8 3.9 7.2 4.5 above 20 bottles 5.8 7.2 6.2 5.1 4.9 price of wine (750ml) below 50 cny 1.0 5.1 8.3 5.4 4.9 0.76 45.317 0.000 51-100 cny 13.2 20.1 23.2 27.0 21.7 101-150 cny 29.1 25.7 33.1 33.2 30.0 151-200 cny 23.5 24.7 22.9 18.6 22.0 201-300 cny 16.7 14.7 5.1 9.2 12.2 301-500 cny 9.5 7.8 4.7 4.0 6.3 above 500 cny 7.0 1.9 1.7 2.5 2.9 preferred origin of wine domestic 51.9 39.6 50.6 52.1 57.9 0.82 17.357 0.000 imported 48.1 60.4 49.4 47.9 42.1 dietary preference meat lovers 26.2 23.1 27.6 28.4 25.6 0.25 3.386 .017 vegetarian lovers 21.4 21.0 16.9 30.5 24.3 plasma balance 52.5 55.9 55.5 41.1 50.1 preferred type of wine red wine 65.8 60.8 69.9 44.1 67.4 0.47 3.197 .023 white wine 23.9 24.7 20.3 41.5 24.1 rose wine 10.4 14.6 9.8 14.4 8.5 ital. j. food sci., vol. 31, 2019 776 in terms of monthly income of consumers and the time and price of wine purchased, it can be seen that the proportion of consumers with monthly income of 5001-7000 cny in all groups is quite large. consumers with this range of income prefer to buy wines with prices between 101and 150 cny per bottle. this finding is similar to that of mu et al. (2017). balanced consumers group has the largest proportion of high-income respondents. credulous consumers accounted for the majority of respondents with a monthly income of less than 5,000 cny. this income group’s purchase price was less than 100 cny per bottle. the proportion of this group was much higher than the other three groups. the result analysis suggests that these consumers may be more inclined to promotional information when buying wine because they do not know much about wines and have no interest in the product itself. as for eating habits and frequency of drinking, there is a significant difference in the frequency of consumption by consumers in different groups. credulous consumers drink more frequently than balanced consumers, experiential consumers and health sippers. a large percentage of credulous consumers consume one bottle of wine per month (750ml). the most frequent drinkers are credulous consumers, followed by balanced consumers, experiential consumers, and finally health sippers. in terms of eating habits, most of sampled respondent are consumers with balanced diet. in particular, the proportion of consumers who prefer vegetarian food in credulous consumers is significantly higher than those in other three groups. finally, in terms of the types of wines, the findings show that more than 50% of the respondents prefer domestic red wines. interestingly, more than 60% of experiential consumers prefer imported wines, which may be related to the variety and the richer taste of imported wines. in addition, credulous consumers believe that there is no big difference between drinking red wine and white wine. this, on the other hand, proves that such consumers are relatively ignorant, easy to be affected by extrinsic factors and unable to make their own judgments. from the results of the one-way anova, it can be seen that at the level of 0.05, the four groups of consumers have significant differences in terms of age, monthly income, education level, favorite origin of wines and price of wine purchased regularly. 5. conclusion and implications in the past few decades, the global alcohol market has undergone tremendous changes, and wine is no exception. as a result, winemakers have realized that they need to understand their consumers and markets better. in this research, kfcm was used to study the key factors affecting the purchase behavior of chinese wine consumers. the results show that there are four different groups of wine consumers: experiential consumers, balanced consumers, credulous consumers and health sippers. this market-oriented study provides detailed and practical marketing suggestions for each of the four identified groups. the outcome of this study shows that consumers between the ages of 26 and 45 are the main consumer groups of wine and red wine remains a popular choice of wines. future researches can measure consumer health awareness trends. similarly, future research should expolre how extrinsic attributes (such as nutrition labels, packaging styles) interact with other identified attributes that affect the consumer wine preferences. it should be noted that although this study provides useful findings for consumer segmentation in the chinese wine market, it has certain limitations. for example, the ital. j. food sci., vol. 31, 2019 777 study is based on the participant's self-description. future research should supplement provide additional information by observing the actual purchase behavior of the consumers, such as measuring the real amount of purchased wine in the store, and the consumers who go out to eat together with wines. in addition, since china is a very large country, the regional dietetic culture is quite different. it is necessary to increase the number of consumers in different representative regions in order to improve the representativeness of research in the future. first, for health sippers, wine makers can add detailed nutrition and health information on the labels attached to bottles to improve the possibility of chinese wine consumers’ purchase. in addition, manufactures can increase the promotion of wine through social media and increase the awareness of wine and its health benefits. this will increase the popularity of wines. second, for experiential consumers, according to a study by capitello et al. (2015), chinese consumers tend to try different flavors of wines, therefore the wine makers should conduct more effective market research to ensure the taste and quality of the products and to meet the current changing marketing needs. on the other hand, regular wine tastings and increased communication and understanding of consumers are also effective measures to expand the market and gain more market share. they can also develop and design wines with their own characteristics, create core products and customize different marketing combinations for different products. moreover, through indepth understanding of the characteristics and functions of wines, dealers can establish a multi-category bundle of product combinations. wine stores must have wines of various countries and varieties. only when consumers have more choices can they be more interested in wines and their consumption. third, balanced consumers are rational consumers who have certain knowledge of wine and are not affected by the external influence easily. they like wines and have a relatively high income. therefore, wine makers should provide relevant events to attract these consumers and develop wine tourism projects to increase wine consumption experience to meet the experience needs of wine consumers. by means of drinking cultural propaganda, manufacturers can hold wine related exhibitions of the brand in large and medium-sized cities in china, and cooperate with other media channels such as tv, website, magazines, newspapers and so on to popularize wine drinking experience and culture. through holding different kinds of wine festivals to establish the image of wine brand and promote the customers’ loyalty to increase sales. in terms of publicity and marketing channels, wine producers should also make full use of online sales channels and publicity. under the traditional mode, television, newspapers and magazines are the core channels of advertising, but under the influence of the internet, these traditional media are beginning to be marginalized. social media has gradually become the main channel for people to communicate and communicate. mobile media has begun to occupy people’s lives and become the most influential media channel. finally, in order to cater for the taste of credulous consumers, wine makers should highlight the characteristics of the products, because these consumers have little knowledge of the products. considering that their monthly income is relatively low, lowcost and occasional products discount can be effective for such consumers. in addition, salesmen can be trained to stimulate consumers' interest in products and desire to consume by using different promotion methods, such as free trial drinks, giving gifts and interacting with customers, according to the needs of different sales targets. lastly, advertising through television, radio and social media to increase product awareness is also an effective way of publicity for wine makers and manufacturers (gendall, 2003). ital. j. food sci., vol. 31, 2019 778 appendix questionnaire items acknowledgements this research is supported by chinese agricultural research system (cars-29) and beijing food safety policy& strategy research base. references akbulut y., sengur a., guo y. and polat k. 2016. kncm: kernel neutrosophic c-means clustering. applied soft computing 52:714-724. akdeniz b., calantone r.j. and voorhees c.m. 2013. effectiveness of marketing cues on consumer perceptions of quality: the moderating roles of brand reputation and third-party information. psychology & marketing 30(1):76-89. annunziata a., pomarici e., vecchio r. and mariani a. 2016. nutritional information and health warnings on wine labels: exploring consumer interest and preferences. appetite 106: 58-69. areta á., azcárate i.b. and apezteguía b.i.. 2018. spanish wines in the us market: what attributes do us consumers look for in spanish wines? spanish journal of agricultural research 15(4):1-15. atkin t., nowak l. and garcia r. 2007. women wine consumers: information search and retailing implications. international journal of wine business research 19(4):327-339. barber n. 2012. consumers' intention to purchase environmentally friendly wines: a segmentation approach. international journal of hospitality and tourism administration 13(1):26-47. basu b. and srinivas v.v. 2014. regional flood frequency analysis using kernel‐based fuzzy clustering approach. water resources research 50(4): 3295-3316. name items intrinsic cues ic1: i usually buy wines with famous brand. (madeira et al. ,2009) ic2: the higher the price, the better the quality of the wine. ic3: the quality of wines in wellknown regions is more reliable. ic4: the better the packaging, the higher the grade of the wine. ic5: good vintage can produce good wines. extrinsic cues ec1: the better the color, the better the wine quality. (chrea et al., 2011; hall, 2013) ec2: the richer the fragrance, the better the quality of the wine. ec3: the better the taste, the better the quality of the wine. marketing m1: wine advertisement has a big impact on my purchase decision. (madeira et al,. 2009) m2: promotion has a big impact on my purchase decision. m3: the location of the store and its interior style impact my choices. m4: i trust the recommendation of the sellers. reference group rg1: i trust the recommendations of my friends and relatives. (fernandes et al., 2018) rg2: the more rewards offered by the seller, the better the quality of the wine. motive mo1: the health benefits of wine affects my choice. (chrea et al., 2011; hall, 2013) mo2the auxiliary purpose of wine affects my choice. mo3: the social purpose of wine affects my choice knowledge and involvement ki1: my knowledge of wine influences my choice. 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c-means algorithm. neural processing letters 18(3): 155-162. paper received february 4, 2019 accepted march 15, 2019 ijfs#1422_bozza ital. j. food sci., vol. 31, 2019 573 paper effects of high pressure and marination treatment on texture, myofibrillar protein structure, color and sensory properties of beef loin steaks m. uyarcan* and s. kayaardi department of food engineering, engineering faculty, manisa celal bayar university, manisa, turkey *corresponding author: tel: +902362012273; fax: +902362412143 email: muge.akkara@cbu.edu.tr abstract the influence of high pressure/marination treatment on the texture, myofibrillar protein structure, color and sensory properties of beef loin steaks was studied. combined high pressure and marination treatment at 550 mpa significantly increased beef tenderness, but had a “whitening/brightening” effect on the color of the samples (p<0.05). high-pressure processing caused protein degradation, leading to texture development. furthermore, the panelists gave the highest overall impression score to the 150 mpa pressurized samples. these results show that combined high pressure and marination treatment at 550 mpa can potentially improve the textural properties of beef loin steaks, although it is less favored than pressurization treatment. keywords: beef, high pressure, marination, protein degradation, texture ital. j. food sci., vol. 31, 2019 574 1. introduction high pressure processing has been a hot topic of study among scientists because of its positive effects on the safety, quality and sensory properties of food products (evrendi̇lek et al., 2008). addressing the increasing demand for minimally processed foods with high nutritional and sensory quality, the food industry has employed high pressure processing to develop high quality, fresh, additive-free food products with an extended shelf life. in the european community, high pressurized foods are classified as ‘‘novel foods’’ (campus, 2010). this novel technology also offers an alternative to pasteurization treatment and could have great potential for heat-sensitive foods (duranton et al., 2011). there is particular interest in researching the effects of high pressure on the food matrix (sun and holley, 2010). it has been discovered very recently that short-time high pressure application at lower temperatures develops tenderness in meat and meat products while minimally changing the natural characteristics of the product (bajovic et al., 2012). it has been hypothesized that applying pressure to meat in the postmortem period could cause changes to the enzymes and proteins, especially to the gelatin characteristics of myofibrillar proteins, color, microbial load, ultrastructure and textural properties of meat (schenkova et al., 2007). in research studies, pressure levels applied to post-rigor meat generally ranged from 100 to 600 mpa with short processing times (5-20 min.) at 15-60ºc, according to the purpose of the study (chan et al., 2011; kruk et al., 2011; mcardle et al., 2013; grossi et al., 2014; sikes and tume, 2014; gimenez et al. 2015). researchers have also reported that high pressure technology is a physical additivefree process for meat tenderizing and softening due to its effects on the gel-forming ability of proteins and on texture (buckow et al., 2013). the effective pressure levels have varied from 150 to ≥500 mpa (5 min, 20ºc) for meat tenderization (sun and holley, 2010). furthermore, post-rigor meat tenderization without bleaching of color can be achieved with pressure levels up to 300 mpa for a few minutes at room temperature (campus, 2010). marination treatment using plant additives is another natural way to preserve meat and meat products. in recent years, natural plant extracts with high phenolic contents have been used in meats, due to their safety characteristics and beneficial effects on health, synthetic chemical preservatives. there are numerous studies about plant extracts (such as grape seed, green tea, pomegranate, peanut skin, garlic, rosemary, olive leaf, moringa leaf, nettle, myrtle, and mint leaf extracts) used in meat and meat products (akarpat et al., 2008; alp and aksu, 2010; yu et al., 2010; devatkal et al., 2010; hayes et al., 2010; colindres and brewer, 2011; rababah et al., 2011; biswas et al., 2012; das et al., 2012; özvural and vural, 2012; cao et al., 2013). among these natural extracts, oleoresin rosemary (herbalox®) has been commonly included in food processing as a shelf life extender and flavor developer (ahn et al., 2007). in addition, there are many studies in the literature about the antimicrobial and antioxidant activities of rosemary in different food materials (botsoglou et al., 2007; sasse et al., 2009; nieto et al., 2010; puangsombat and smith, 2010; colindres and brewer, 2011; wojciak et al., 2011; gibis and weiss, 2012; mathenjwa et al., 2012; kim et al., 2013). the literature studies reported that the effective usage level of rosemary extract varied between 0.02-10% in a marinade solution for retarding lipid oxidation and improving sensorial characteristics in meat and meat products (ahn et al., 2007; akarpat et al., 2008; rojas and brewer, 2008; wojciak et al., 2011). ital. j. food sci., vol. 31, 2019 575 the use of a combination of pressure and marination treatment can be an alternative preservation method for meat producers. the combined treatment of pressure and marination can be more efficient at improving meat quality attributes and increasing shelf life. it was reported that the combined treatment of high pressure and natural antioxidants as a multi-hurdle approach can be an alternative treatment in the meat industry (hygreeva and pandey, 2016). however, there are relatively few studies regarding the combined use of high pressure and natural extracts in meat and meat products, and generally chemical preservatives were used for marination in these studies (schenkova et al., 2007; ohnuma et al., 2013; kim et al., 2014; gimenez et al., 2015; rodrigues et al., 2016). in addition, oregano, rosemary, papain plants and carvacrol were used as natural antioxidants for meat and meat products in the studies evaluating a combination of high pressure and marination treatment (bragagnolo et al., 2005; gomez-estaca et al., 2007; de oliveira et al., 2015). although very few studies have been published about the effects of rosemary extract and high pressure treatments on sardines and chicken breast meat, to the best of our knowledge, there has been a lack of information about the effects of combined high pressure and rosemary extract marination on beef quality (bragagnolo et al., 2005; gomez-estaca et al., 2007). the literature studies about combined use of high pressure and natural antioxidants are still in an early stage, and more studies are needed to be conducted (hygreeva and pandey 2016). according to these informations, the main goal of this study was to research the combined effects of high pressure and marination treatment on the textural, color and sensory properties of beef loin steaks and to improve natural new textured meat products. 2. materials and methods 2.1. materials beef loin steaks were supplied by a local retail butcher and cut into 2×10×4 (height×width×length) cm uniform portions weighing an average of 50-70 g before high pressure and marination treatment. oleoresin rosemary extract (herbalox® type w seasoning oil) was supplied by kalsec inc. (kalamazoo, michigan, usa) and used for marination. it is dispersible in water (polar carriers) and oil (nonpolar carriers) with agitation and has a brown, viscous, liquid appearance. eight groups of samples were used in the experiments based on high pressure treatment and high pressure/marination treatment. the samples were divided into (i) 0:control (non-pressurized samples), (ii) 150/0 (150 mpa hpp), (iii) 350/0 (350 mpa hpp), (iv) 550/0 (550 mpa hpp), (v) 1: marinated, non-pressurized sample (vi) 150/1 (150 mpa hpp/marination), (vii) 350/1 (350 mpa hpp/marination) and (viii) 550/1 (550 mpa hpp/marination) groups. all experiments were carried out in triplicate. 2.2. marination treatment marinades were prepared with oleoresin rosemary extract. preliminary experiments were performed to determine the appropriate marinade concentration for preserving and developing meat quality characteristics. each sample was placed in a polyamide/polyethylene bag (apack ambalaj, i̇stanbul, turkey) containing 10 ml of marination solution (including 5% oleoresin rosemary extract) and was kept overnight at 4ºc. on the following day, the marinades were removed from the packages, and all ital. j. food sci., vol. 31, 2019 576 samples were vacuum packaged in double pouches to prevent contamination of the samples by the pressurization medium from bags breaking due to pressurization. 2.3. high-pressure processing high-pressure processing was applied to the non-marinated and marinated samples. as a result of adiabatic heating, pressure treatment increases the temperature of pressuretransmitting fluid and samples, depending on the product composition and initial temperature of the sample (koca et al., 2011). for this reason, the initial temperatures of the samples were adjusted before the high pressure treatment, and the final temperature of the samples after pressurization was monitored with a computer program and found to be approximately 20±2ºc. the high pressure process was carried out in a mse-cip-wb-5500 high pressure food processor (mse teknoloji ltd., gebze, turkey) with a 0.7 l vessel volume. propylene glycol (kimetsan co., ltd., ankara, turkey) was used as the pressure-transmitting fluid. the pressure vessel was surrounded by coils connected to a cooling circulator (model re1050s, lauda dr r. wobser gmbh & co. kg., germany). the temperature of the pressure vessel and the pressure-transmitting fluid inside the pressure vessel were controlled with these coils. the inherent ramp rate was 5 mpa/s, and the pressure was increased to the test pressures of 150 mpa, 350 mpa and 550 mpa within approximately 30 s, 70 s and 110 s, respectively. the samples were held at test pressures for 5 min. after the pressurization, decompression was manually performed in approximately 20 s. during the pressure treatments, the temperature of the pressure-transmitting fluid was monitored with two k-type thermocouples mounted to the center of the top closure of the pressure chamber and positioned close to the sample. in addition, the treatment cycle was controlled by a computer program throughout the pressurization. after pressurization, all samples were stored at 4ºc prior to analysis within 24 h. 2.4. texture profile analysis texture profile analysis (tpa) of samples was carried out with a ta-xt plus texture analyzer (stable micro systems, england). beef loin steaks were cooked in a water bath at 80ºc until reaching an internal temperature of 72ºc and then cooled to room temperature for 45 min before texture analysis. a 5 kg load cell was used in the experiments. the cylindrical samples (1 cm diameter and 2 cm length) were compressed across the fiber direction in two consecutive cycles to 50% of their original height using a cylindrical probe, 38 mm in diameter. the sample was placed under the probe that moved downwards at a constant speed of 2.0 mm s-1 (pre-test), 2.0 mm s-1 (test), and 5.0 mm s-1 (post-test). a time of 5 s was allowed to elapse between the two compression cycles. the tpa parameters (hardness, springiness, cohesiveness, gumminess, chewiness and adhesiveness) were expressed as described by mochizuki (2001). the measurements of each sample were replicated at least six times. all textural analyses were conducted using texture exponent software version 4.0.9.0. (stable microsystems ltd., surrey, england). 2.5. protein solubility myofibrillar proteins were extracted according to the method described by claeys et al. (1995). samples of 2.5 g of minced meat were homogenized with 25 ml of 0.05 m tris, 0.25 m sucrose and 1 mm edta buffer, ph 7.6. homogenate was centrifuged at 1000 × g for 10 ital. j. food sci., vol. 31, 2019 577 min. after centrifugation, the supernatant was removed, and the pellet was suspended in 25 ml of 0.05 m tris, 0.05 m edta buffer, ph 7.6 and sedimented at 1000 × g for 10 min. then, the supernatant was removed again, and the pellet was resuspended in 25 ml of 0.15 m kcl and centrifuged at 1000 × g for 10 min. the same procedure was carried out three times. the myofibril solution was lyophilized and used for further analysis. the lyophilized myofibril extracts were analyzed for protein concentration. the extracts were dissolved in sample buffer (2×laemmli buffer, 2-mercaptoethanol, bromophenol blue, ph 6.8). then, the dissolved extracts were placed in a water bath at 50°c overnight and filtered using whatman no. 1 filter paper. after filtration, the protein concentration of the extracts was determined using the bio-rad quick start bradford assay kit (bio-rad laboratories, hercules, ca, usa) based on the bradford method (bradford, 1976). bovine serum albumin was used as the standard. the myofibrillar protein solubility of the samples was expressed as mg protein/ml extract solution. 2.6. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) sds-page was carried out according to the method of laemmli (1970) using a 12% separation gel and 4.5% stacking gel (bisacrylamide: acrylamide 1:37 [w/w]). the protein concentration of the loaded sample was adjusted to 10 µg for each sample. a protein broad range marker (bio-rad unstained sds-page standards, 161-0317) was used as the molecular weight standard (6.5-200 kda). the electrophoresis run was carried out at 100 v in a mini-protean tetra cell electrophoresis system (bio-rad laboratories, hercules, ca, usa). after the runs, the gels were stained with 0.01 coomassie blue, 50% methanol and 10% acetic acid and then destained in 10% methanol and 7% acetic acid. the gels were visualized, and protein molecular weights were estimated using bio-rad versadoc 4000 mp and quantity one software (bio-rad laboratories, hercules, ca, usa). electrophoresis was carried out in duplicate. 2.7. cooking loss beef loin steaks were placed in plastic bags and cooked in a preheated water bath until the internal temperature of the samples reached 72ºc. then, the samples were taken from the water bath, and excess moisture on the surface of the samples was removed with filter paper. subsequently, the samples were cooled to room temperature and reweighed. the cooking loss (cl) was expressed as a percentage of the weight difference before and after cooking using the following formula described by rodrigues et al. (2016): cl = (initial weight – final weight) / initial weight × 100 2.8. color measurements the color of the samples was measured using a colorimeter (minolta chromameter cr300; minolta camera co., ltd., osaka, japan) with illuminant d65 (light source) and a 10º observation angle. the beef loin steak packages were opened and exposed to air for 10 min prior to analysis. a cielab system was used to determine the color attributes, and the results were expressed as l* (lightness), a* (redness and greenness), and b* (yellowness and blueness). for each sample, five color readings were taken (one at the center and the others from different sides of the sample) at room temperature. the total differences in the color reading values were calculated as described by jung et al. (2003): ital. j. food sci., vol. 31, 2019 578 δ𝐸 = (𝐿∗ − 𝐿!"# ∗ )! + (𝑎∗ − 𝑎!"# ∗ )! + (𝑏∗ − 𝑏!"# ∗ )! the color values of the non-pressurized samples were used as a reference for the sample groups pressurized without marination in calculating ∆𝐸, and the color values of marinated/non-pressurized samples were used as a reference for the marinated pressurized sample groups in calculating ∆𝐸. 2.9. sensory evaluation eight graduate students and lecturers in the department of food engineering at manisa celal bayar university participated in the sensory tests as panelists. the panelists were asked to evaluate the sensory parameters of appearance, color, texture, chewiness, juiciness, flavor and overall acceptability. a hedonic scale of 1-5 was used for each attribute. the 5-point hedonic scale was as follows: like very much (5), like much (4), like (3), like slightly (2) and dislike (1). unsliced raw and cooked samples were presented to the panelists to rate their preferences in terms of appearance, color and texture attributes. in addition, cooked samples were sliced, and a sliced sample from each group was presented to the panelists to rate their preferences in terms of chewiness, juiciness and flavor attributes. the samples were served on plates that were randomly identified with three-digit codes, and a cup of water and bread were given to the panelists to eliminate the residual taste of the samples (djenane et al., 2011). 2.10. statistical analysis all of the experiments were repeated on three separate occasions. the statistical analyses were performed using spss version 25.0 (spss inc., 2017). the experimental data were expressed as the means ± standard deviations. a two-way analysis of variance was conducted to evaluate the effects of high pressure and marination treatment, and the significant differences between pairs of means were tested by duncan’s multiple range test at a confidence level of p<0.05. the results of the sensory analysis using a hedonic scale were evaluated by friedman's (non-parametric) rank test and a wilcoxon test was used to test for pair differences (p<0.05) (meilgaard et al., 2015). 3. results and discussion 3.1. texture profile analysis the textural properties of marinated and marinated pressurized samples are presented in table 1. both pressure and marination treatment had a significant effect on the hardness, gumminess and chewiness of the samples (p<0.05). high pressure treatment alone significantly affected all texture profile parameters, whereas marination treatment was only effective on cohesiveness and adhesiveness (p<0.05). these results suggest that pressure affects the normal texture, marination with rosemary extract partly affects the texture, while the pressure and marination interaction increase the effects on the textural properties of samples. ital. j. food sci., vol. 31, 2019 579 table 1. texture profile parameters of beef loin steaks marinated with rosemary extract and treated with high pressure. *the results are the mean values of three replicates (n=8) ± standard error. means with alphabetical superscripts (a-f) in the same column (within each main effect) are significantly different (p<0.05). **the first number refers to the pressure level, and the second refers to the rosemary extract added (5%). 0: no added extract, 1: added extract. ***l*: lightness; a*: redness and greenness; b*: yellowness and blueness; ∆e: total color difference; sl: significance level; ns: not significant. the combination of marination and high pressure treatment led to an increase in hardness at up to 350 mpa and a slight decrease in hardness at higher pressure values (550 mpa) (p<0.05). high pressure treatment alone also showed a similar trend in the hardness values of the samples, whereas marination treatment alone had no significant effect on hardness (p>0.05). our results were in agreement with those of ma and ledward (2004), who reported that high pressure treatment at or above 200 mpa increased meat hardness. these results could be attributed to the aggregation of pressure-treated myofibrillar proteins at 100-300 mpa, causing increased hardness (ma and ledward, 2004; simonin et al., 2012; rodrigues et al., 2016). the hardness values of all of the samples were significantly decreased at 550 mpa high pressure treatment (p<0.05). in the literature, the decrease in hardness at high-pressure values was explained by the enzymatic hydrolysis of muscle proteins (malinowska-panczyk et al., 2013). furthermore, the lowest hardness values were observed in marinated pressurized (550 hardness (n) springiness cohesiveness gumminess (n) chewiness (n) adhesiveness a: pressure level 0 33.6±0.8c 0.55±0.02a 0.67±0.006a 22.4±0.4c 12.5±0.6c 0.57±0.06c 150 41.0±0.9b 0.60±0.02a 0.62±0.006b 25.4±0.4b 15.1±0.7b 0.35±0.06ab 350 57.9±0.8a 0.55±0.02a 0.59±0.006c 34.2±0.4a 18.7±0.8a 0.47±0.06bc 550 26.2±0.8d 0.48±0.02b 0.62±0.006b 16.4±0.4d 7.7±0.7d 0.18±0.06a sl 0.0 0.04 0.0 0.0 0.0 0.01 b: marination 0 39.2±0.6 0.56±0.02 0.64±0.004a 25.0±0.3 14.0±0.5 0.33±0.04a 1 38.7±0.6 0.52±0.02 0.61±0.004b 24.2±0.3 13.0±0.4 0.46±0.04b sl ns ns 0.0 ns ns 0.04 a×b sl 0.0 ns ns 0.0 0.0 ns samples 150/0** 34.9±1.2d 0.63±0.05 0.64±0.013 22.4±1.2d 14.0±1.2b 0.27±0.03 350/0 61.9±1.3a 0.54±0.04 0.61±0.012 37.7±1.2a 20.3±2.7ab 0.47±0.22 550/0 28.3±3.7e 0.51±0.07 0.65±0.002 18.4±0.9e 9.0±2.1c 0.19±0.12 150/1 47.0±2.0c 0.57±0.03 0.60±0.008 28.3±0.8c 16.2±0.4b 0.43±0.05 350/1 53.9±2.9b 0.55±0.04 0.57±0.004 30.7±0.8b 17.0±1.5ab 0.47±0.07 550/1 24.1±1.8f 0.44±0.03 0.60±0.013 14.4±1.0f 6.4±0.2c 0.17±0.04 ital. j. food sci., vol. 31, 2019 580 mpa) samples. these results showed that pressure treatment of previously marinated meat can be more effective for providing softer texture than pressure treatment alone. the secondary parameters of gumminess and chewiness showed similar changes with hardness. the gumminess and chewiness values of the samples increased with a highpressure treatment up to 350 mpa and decreased significantly at the higher pressure value of 550 mpa. there was also a significant interaction between pressure level and marination on gumminess and chewiness values (p<0.05).the results indicated that the marinated pressurized beef samples were more tender, less gummy and less chewy than the samples that were pressurized alone. it was reported that the loss of myosin structure induced a decrease in gumminess when the texture profile of pressure treated samples was examined with thermograms (angsupanich and ledward, 1998). no significant interaction was found between pressure level and marination for springiness, cohesiveness and adhesiveness of the samples (p>0.05). the springiness, cohesiveness and adhesiveness values of the samples changed variably at different pressure levels. pressure treatment alone had a significant effect on these texture attributes of the samples, whereas marination treatment alone was only effective on cohesiveness and adhesiveness (p<0.05). at the 150 mpa high pressure treatment, the springiness values increased while the cohesiveness and adhesiveness values decreased. a similar relationship was found by angsupanich and ledward (1998) and ashie et al. (1997). on the other hand, some opposing results were found by malinowskapanczyk et al., (2013). at 350 mpa and 550 mpa, the springiness, cohesiveness and adhesiveness decreased. this could be explained by the protective effects of high pressure against heat denaturation of meat proteins (fernandez-martin et al., 1997). in general, fresh meat tenderization depends on resolving two components: actomyosin toughness and background toughness. actomyosin toughness is related to myofibrillar proteins, while background toughness is related to connective tissue and stromal proteins (sun and holley, 2010). the effects of high pressure treatment on meat tenderization can be explained by the changes to myofibrillar protein structure. two possible mechanisms cause myofibrillar protein dissociation and the subsequent decrement in toughness of the meat: thermal degradation of muscle proteins and the enzymatic hydrolysis of proteins (simonin et al., 2012). pressure breaks up the myofibrillar structure and accelerates enzyme activation in meat as mentioned above. in the present study, rosemary extract also showed a positive effect on some textural properties of samples; however, the higher tenderization effect on beef loin steaks was achieved by the combination of high pressure and marination treatment. 3.2. protein solubility protein solubility is one of the most important functional properties of meat proteins (van laack et al., 2000). as a consequence of increased interactions between protein constituents and water, protein solubility can change and cause significant alterations to meat texture (cheftel and culioli, 1997). the effects of high pressure and marination treatment on the solubilization of myofibrillar proteins are shown in fig. 1. according to the results, the high pressure and marination treatment had no effect on myofibrillar protein solubility in the samples. however, the protein solubility of the samples generally decreased with increasing pressure when compared to the untreated group. similar results have been found in previous studies. chapleau et al. (2003) found decreased myofibrillar protein solubility in beef samples subjected to pressure treatment (≤600 mpa) compared to control samples. ital. j. food sci., vol. 31, 2019 581 figure 1. effects of high pressure/marination treatment on the myofibrillar protein solubility of beef loin steaks. furthermore, malinowska-panczyk et al. (2013), souza et al. (2011), grossi et al. (2012) and chan et al. (2011) also reported similar decreases in protein solubility for cod, salmon, pork and beef samples (>60 mpa pressure treatment), pork (215 and 600 mpa pressure treatment) and turkey meat (≤600 mpa pressure treatment), respectively. the literature reports that protein solubility is a good indicator of protein denaturation (van laack et al., 2000). additionally, protein solubility decreases with increasing pressure due to the formation of insoluble protein aggregates that can no longer be extracted (marcos and mullen, 2014). 3.3. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) postmortem degradation of myofibrillar proteins has been reported to be an essential part of postmortem tenderization. the increase in protein degradation reflects lower mechanical tenderness and promotes the development of meat texture (souza et al., 2011). for this reason, it is of great importance to understand the effects of high pressure processing on myofibrillar proteins in considering textural changes in pressurized meat, as described above. fig. 2 shows the sds-page profile of myofibrillar proteins from each of the high-pressure and marination-treated samples. the volume intensity of each protein band is also presented in fig. 3. the protein bands extracted from the samples for myosin heavy chain (mhc) (200 kda), c-protein (135 kda), α-actinin (95 kda), desmin (53 kda), actin (43 kda), tropomyosin (36 kda) and myosin light chain (mlc1, mlc2) (24 kda, 14 kda) were identified on a sds-page gel. similar myofibrillar proteins were also identified by chapleau et al. (2003), chan et al. (2011), omana et al. (2011) and speroni et al. (2014) in beef, turkey, poultry and meatball samples, respectively. in general, increasing the applied pressure reduced the band intensities of the myofibrillar proteins. on the other hand, the sds-page profile of the marinated pressurized samples was similar to that of the samples that were pressurized alone; therefore, we suggest that marination treatment had no effect on myofibrillar protein degradation. the pressuretreated samples had the lowest protein band intensities, and the molecular weights of ital. j. food sci., vol. 31, 2019 582 mainly degraded myofibrillar proteins ranged from 53 kda to 200 kda (mhc, c-protein, α-actinin and desmin). the band intensities of mhc, c-protein, α-actinin and desmin extracted from the pressurized samples were noticeably decreased compared to those of the control samples. the decreased band intensities may have been caused by protein aggregation due to intermolecular disulfide bond formation at the higher pressure levels (angsupanich et al., 1999). myofibrillar proteins were partly degraded with high pressure treatment, and mhc protein was the most degraded protein in the sds-page profile. however, mlc2 protein was found to be unaffected or even decreased in intensity with applied pressure. this may be because myosin aggregation mechanisms involve the dissociation of heavy chains from light chains, so that only myosin heavy chains form aggregates under pressure (speroni et al., 2014). figure 2. sds-page patterns of myofibrillar proteins isolated from beef loin steaks. m: marker, 0: no added extract (control), 150/0, 350/0, 550/0: pressurized, 1: added extract, 150/1, 350/1, 550/1: marinated pressurized ital. j. food sci., vol. 31, 2019 583 figure 3. a: the volume intensity of the different protein bands from sds-page for the high-pressurized sample groups. b: the volume intensity of the different protein bands from sds-page for the marinated high-pressurized sample groups. the changes in band intensities of myofibrillar proteins under pressure are attributed to conformational changes in proteins and thereby decreased solubility due to denaturation following covalent linking or increased solubility due to degradation into lower molecular weight compounds. our results are in accordance with this explanation. the protein band intensities and solubilities decreased in parallel with increasing pressure. 3.4. cooking loss table 2 shows the cooking loss values of the samples. the results indicated that there was no significant interaction between pressure level and marination for cooking loss values of the samples (p>0.05). however, it was found that pressure level and marination separately had a significant effect on cooking loss (p<0.05). a significant difference was observed in all pressure levels compared to the control group (p<0.05). ital. j. food sci., vol. 31, 2019 584 table 2. color and cooking loss values of beef loin steaks marinated with rosemary extract and treated with high pressure. l* a* b* δe cooking loss (%) a: pressure level 0 40.86±0.5c 9.4±0.5b 11.8±0.3d 40.60±0.6a 150 40.69±0.5c 10.0±0.5ab 12.8±0.3c 2.8±0.4c 38.03±0.6b 350 54.41±0.5b 11.3±0.5a 18.6±0.3a 15.4±0.4b 37.55±0.6b 550 57.20±0.5a 8.7±0.5b 17.6±0.3b 17.5±0.4a 39.22±0.6ab sl 0.0 0.01 0.0 0.0 0.0 b: marination 0 46.25±0.3a 10.4±0.3a 14.3±0.2a 12.0±0.4 37.64±0.4a 1 50.33±0.3b 9.3±0.3b 16.1±0.2b 11.8±0.4 40.05±0.4b sl 0.0 0.02 0.0 ns 0.0 a×b sl 0.04 ns ns ns ns samples 150/0** 37.50±0.7e 10.4±1.9 12.1±1.2 3.6±0.9 36.18±0.9 350/0 52.20±0.9c 12.3±0.9 17.3±1.0 14.6±0.9 36.24±2.1 550/0 56.06±1.4b 9.1±1.5 16.7±0.9 17.8±0.9 38.19±0.9 150/1 43.89±1.2d 9.6±0.7 13.6±0.4 2.1±0.8 39.87±0.9 350/1 56.63±0.9ab 10.2±0.3 20.0±0.6 16.2±0.6 38.85±0.4 550/1 58.34±1.8a 8.3±1.2 18.4±0.5 17.2±1.9 40.24±0.7 *the results are the mean values of three replicates (n=8) ± standard error. means with alphabetical superscripts (a-d) in the same column (within each main effect) are significantly different (p<0.05). **the first number refers to the pressure level, and the second refers to the rosemary extract added (5%). 0: no added extract (control), 1: added extract. ***l*: lightness; a*: redness and greenness; b*: yellowness and blueness; ∆e: total color difference; sl: significance level; ns: not significant. both pressure and marination treatment generally resulted in an increase in cooking loss values except for the marinated pressurized (350 mpa) group, but the differences were not significant (p>0.05). the cooking loss values of the samples that were pressurized alone increased with increasing pressure. a similar trend was determined by kim et al. (2014) who reported increased cooking loss values in beef samples pressurized at 300, 450 and 600 mpa compared to the control group. in addition, neto et al. (2015) reported that 100, 200, 300 and 400 mpa high-pressure treatment led to increased cooking loss values in beef samples. these authors also reported that high pressure levels and changes in myofibrillar protein structure at these pressures had a negative effect on the water holding capacity of meat and consequently increased cooking loss. in addition, marcos et al. (2010) explained that sarcoplasmic proteins decreased high-pressure effects on cooking loss but that the increased denaturation of sarcoplasmic proteins induced by pressure had a negative effect on the cooking loss values of meat. the cooking loss values of the marinated pressurized samples decreased at 350 mpa and then increased with increasing pressure. similar results were obtained by barbanti and pasquini (2005) in marinated meat. increasing cooking loss values might be attributed to lower water binding capacity and moisture loss during cooking. ital. j. food sci., vol. 31, 2019 585 3.5. color the color measurements of beef loin steaks marinated with rosemary extract and subjected to high pressure are shown in table 2. statistical analysis showed a two-way interactive effect between pressure level and marination for l* values of the samples (p<0.05). pressure level and marination separately had a significant effect on the a* and b* values of the samples (p<0.05). the l* values showed an increasing trend, while the a* and b* values increased up to 350 mpa and then decreased. marination with rosemary extract also caused a significant increase in l* and b* values and decrease in a* values (p<0.05). pressure level had a significant effect on l* values at pressures above 150 mpa compared to the control group (p<0.05). it was also found that the a* values significantly changed at 150 and 350 mpa pressure levels, whereas the b* values significantly changed at all pressure levels (p<0.05). similar results were also reported by kim et al. (2014), marcos et al. (2010), mcardle et al. (2010), ohnuma et al. (2013) and rodrigues et al. (2016) for beef m. longissimus dorsi, beef supplemented with conjugated linoleic acid, beef longissimus lumborum, beef m. pectoralis profundus and beef treated with sodium hydrogen carbonate, respectively. the highest l* values were determined in marinated pressurized (550 mpa) samples. these increases in l* values caused discoloration of the beef samples, which was attributed to the whitening/brightening effect between the range of 200 to 350 mpa and ferrous (fe2+) myoglobin oxidation to ferric (fe3+) metmyoglobin at pressures above 400 mpa in the literature (simonin et al., 2012; buckow et al., 2013). the whitening/brightening effect occurred due to the following changes: (i) protein coagulation causing loss of sarcoplasmic and myofibrillar protein solubility, affecting their structure and surface properties and (ii) myoglobin denaturation and the displacement or release of the heme group (buckow et al., 2013). mussa (1999) also reported that the lighter color in pressurized meat could be related to alterations in the water content of samples due to drip loss. in the present study, visual color observations of the samples are also shown in fig. 4. figure 4. visual color observation of beef loin steaks. a: control (unpressurized), b: 150 mpa, c: 350 mpa, d: 550 mpa, e: marinatedunpressurized, f: marinated/150 mpa, g: marinated/350 mpa, h: marinated/550 mpa. increasing pressure caused the increase in a* values of samples up to 350 mpa; then, the a* values of the samples decreased at pressures above 350 mpa. jung et al. (2003) found that pressure treatment up to 300 mpa decreased metmyoglobin content and higher pressures led to increased metmyoglobin content in the beef samples. these authors also explained ital. j. food sci., vol. 31, 2019 586 the increases in a* values at pressures up to 300 mpa by the activation of enzymes causing metmyoglobin reduction. our results are in agreement with previous reports for beef samples (jung et al., 2003; marcos et al., 2010; lowder et al., 2014). the b* values represent the intensity of yellowness and blueness in the samples. in the present study, b* values showed a similar trend with a* values. increasing pressure caused by the increase in b* values of samples up to 350 mpa and then the b* values of the samples decreased at pressures above 350 mpa. similar results were found by mcardle et al. (2010), who reported higher b* values in beef samples pressurized at 300 mpa compared to the samples pressurized at 200 mpa and lower b* values in beef samples pressurized at 400 mpa compared to the samples pressurized at 300 mpa. on the other hand, goutefongea et al. (1995) reported an increase in b* values for minced meat samples pressurized at 600 mpa (20ºc for 30 min). these authors related the increase in b* values to the change of the myoglobin chemical state. carlez et al. (1995) also stated that the increase in b* values was due to metmyoglobin formation. the total color difference (∆e) indicates the evaluation of color changes. the results revealed that there was no significant interaction between pressure level and marination for ∆e values of the samples (p>0.05). however, the pressure level had a significant effect on ∆e values (p<0.05). an increase of 10 units in ∆e is thought to significantly change the appearance of meat color (jung et al., 2003). in the present study, an increase of 10 units in ∆e values was found in the samples pressurized at 350 and 550 mpa. 3.6. sensory evaluation the sensory evaluation results of the raw and cooked samples are shown in table 3 and table 4. in general, the addition of rosemary extract (5%) did not positively affect the sensory scores of the samples. it was observed that pressurized samples were evaluated as better than marinated pressurized samples. increasing pressure caused a decrease in the sensory scores of the samples. the panelists showed slightly friedman rank test significant preferences in appearance, color and texture attributes of raw samples and chewiness, juiciness and overall impression attributes of cooked samples (p<0.05). according to the results of the raw samples, the control samples received the highest score for appearance and texture, whereas 150 mpa pressurized samples were rated highest for color. the results of cooked samples also showed that 150 mpa pressurized samples received the highest score for appearance, color, texture, chewiness and overall impression, while 350 mpa pressurized samples were rated highest for juiciness. the panelists did not notice the color difference between the pressurized and nonpressurized cooked samples. it has been reported that high pressure treatments caused visible color changes in raw meat, but the color difference decreased extremely after cooking. our results are in agreement with those of mor-mur and yuste (2003) and simonin et al. (2012). in addition, the panelists recognized the color differences in the raw samples. according to the pair comparisons, there was a significant difference between the control samples and pressurized as well as marinated pressurized samples (p<0.05) in color scores. similarly, the appearance of the raw samples was also significantly influenced by the treatments (p<0.05). these results were also supported by the color measurements shown in table 2. ital. j. food sci., vol. 31, 2019 587 table 3. sensory evaluation of pressurized and marinated pressurized raw samples appearance color texture 0 4.80±0.4 4.70±0.6 4.30±0.7 1 4.10±0.6 4.15±0.7 4.05±0.7 150/0** 4.75±0.4 4.75±0.4 4.20±0.8 350/0 2.60±1.2 2.65±1.2 3.70±1.0 550/0 2.00±1.0 2.00±0.9 3.35±0.8 150/1 3.60±0.8 3.75±0.9 3.80±0.8 350/1 2.15±0.8 2.25±0.9 3.70±1.0 550/1 1.70±0.9 1.90±1.1 3.40±0.9 sl 0.0 0.0 0.0 *the results are the mean values of three replicates (n=8) ± standard error. **the first number refers to the pressure level, and the second refers to the rosemary extract added (5%). 0: no added extract (control), 1: added extract, sl: significance level. the panelists tended to give lower scores for the texture attributes of cooked and raw samples than for the control group. the pair comparisons of texture scores of the control group and the other sample groups were significant except for 150 mpa pressurized groups (150/0, 150/1) and marinated 350 mpa pressurized group (p<0.05). surprisingly, no significant difference was found in the texture attributes of the cooked samples (p>0.05). these results were not in agreement with tpa measurements reported in the previous sections, which were significantly affected by pressure (p<0.05). the contrast between the results could be due to the different preferences reflected by the panelists regarding texture. on the other hand, the panelists gave higher chewiness scores to the pressurized samples (150/0, 350/0) and marinated pressurized samples (350/1) compared to the control group, and the pair significant differences were found between the control group and the 350 mpa pressurized group (350/0, 350/1) (p<0.05). table 4. sensory evaluation of pressurized and marinated pressurized cooked samples appearance color texture chewiness juiciness flavor overall impression 0 3.80±1.0 4.20±0.8 3.95±0.8 3.45±1.2 3.50±1.1 3.80±1.0 3.55±1.1 1 4.00±0.9 4.20±0.7 4.00±0.8 3.55±1.1 3.35±1.1 3.65±1.0 3.55±0.9 150/0** 4.20±0.8 4.25±0.8 4.10±1.1 4.20±0.8 4.00±0.8 4.10±0.9 4.10±1.0 350/0 4.05±0.9 3.05±1.1 3.95±0.8 4.15±0.7 4.25±0.9 4.30±0.8 4.00±0.7 550/0 4.00±0.9 3.05±1.1 3.55±0.9 3.50±1.4 3.50±1.2 3.90±1.0 3.40±1.1 150/1 3.60±1.1 3.60±0.8 3.40±0.9 3.55±0.8 3.40±0.8 3.60±0.7 3.50±0.8 350/1 3.70±1.0 2.90±1.0 3.80±0.8 3.70±1.1 3.65±1.0 3.60±1.0 3.20±1.0 550/1 3.80±1.0 3.15±1.3 3.55±1.1 3.20±1.0 3.15±0.9 3.65±0.8 2.75±0.8 sl ns ns ns 0.0 0.0 ns 0.0 *the results are the mean values of three replicates (n=8) ± standard error. **the first number refers to the pressure level, and the second refers to the rosemary extract added (5%). 0: no added extract (control), 1: added extract, sl: significance level. ital. j. food sci., vol. 31, 2019 588 it was determined that the samples pressurized up to 350 mpa had higher juiciness scores than the control group, while the juiciness scores decreased at 550 mpa pressure levels. macfarlane (1973) stated that decrements in juiciness scores were attributed to increased moisture retention based on the defragmentation of structural proteins into hydrophilic peptides/free amino acids. the panelists gave the highest overall impression scores to the 150 mpa pressurized samples; however, no significant differences were found between the overall impression scores of 150 mpa pressurized samples and the control group (p>0.05). the 350 mpa pressurized samples also received the highest scores for flavor in all sensory characteristics of the cooked samples. according to the sensory evaluation results, the panelists preferred the 150 mpa and 350 mpa pressurized samples most, which were more tender, chewier, juicier and tastier than the control group. in addition, the marinated pressurized samples had lower sensory scores than the samples that were pressurized alone. it was reported that the sensory acceptance among panelists of high pressurized meat products varied and that they generally reported good sensory scores, although with some alterations in aroma and taste components (simonin et al., 2012). 4. conclusion a pressure of 550 mpa improved the tenderness of beef loin steaks but caused a light color in the meat appearance. high pressure processing caused protein degradation, leading to a great change in the protein profile of samples and thereby the development of meat texture. the panelists preferred the samples pressurized at 150 mpa and 350 mpa in the sensory panel. the best overall beef quality was achieved with the combined application of high pressure and marination. high pressure treatment has 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10:175-188. yu j., ahmedna m. and göktepe i. 2010. potential of peanut skin phenolic extract as antioxidative and antibacterial agent in cooked and raw ground beef. j food sci technol. 45:1337-1344. paper received november 4, 2018 accepted april 10, 2019 ijfs#1626_bozza ital. j. food sci., vol. 32, 2020 386 paper effect of extrusion on the total antioxidant capacity and free phenolic compounds of wheat bran by response surface methodology z. jin1, m. wang1, f. wu1, h.y. cai*1,2,3, w.p. jin1,2,3, w. sun1,2,3, x. chen1,2,3, f. li1,2,3, z. wang1,2,3 and w.y. shen1,2,3 1college of food science and engineering ,wuhan polytechnic university, wuhan 430023, hubei, people’s republic of china 2key laboratory of the deep processing of bulk grain and oil authorized by ministry of education, wuhan 430023, hubei, people’s republic of china 3hubei key laboratory for processing and transformation of agricultural products, wuhan 430023, hubei, people’s republic of china *corresponding author: caihongyan1300@163.com abstract there are some antioxidants in wheat bran provides health benefits. but the influence of extrusion pre-treatment on the antioxidant capacity and free phenolic compounds of wheat bran is not clear. herein, it was investigated by response surface methodology (rsm). within the experimental range, free phenolic compounds (fpc) increased gradually with feed moisture and extrusion temperature. and the total antioxidant capacity of extruded wheat bran increased gradually with rising feed moisture and screw speed. the optimized extrusion parameters were extrusion temperature at 86°c, feed moisture at 22% and screw speed at 160 rpm. the total fpa reached 3136.9 μg gae g−1 and the ferulic acid content was 93.4 μg.g−1. extrusion treatment for wheat bran significantly improved the antioxidant properties and increased the concentration of gallic acid and ferulic acid. the effect of extrusion temperature on total free phenol content is extremely significant. keywords: wheat bran, extrusion, response surface methodology, trolox equivalent antioxidant capacity, free phenolic compounds ital. j. food sci., vol. 32, 2020 387 1. introduction wheat bran accounts for approximately 14% of the whole wheat grain. it is consisted of multiple layers, including aleurone layer, the nucellar epidermis, the inner pericarp, and the outer pericarp (from inside to outside) (mateo et al., 2012), (pandey and rizvi, 2009; perales-sánchez et al., 2014). numerous literatures have been found to make a thorough inquiry species of the phenolic compounds exist in wheat, especially in wheat bran fraction (neacsu et al., 2017; rosicka-kaczmarek et al., 2018). in plants, phenolics compounds incorporate a wide variety of compounds including flavonoids, tannins, coumarins, and phenolic acids (hoseney, 2010). what′s more, phenolics compounds including a benzene ring bearing one or lots of hydroxyl groups and phenolic acids that derivatives of either hydroxybenzoic or hydroxycinnamic acid are usually being all cereals (kim et al., 2006; zheng et al., 2015). generally, many beneficial compounds in cereals dedicate to their antioxidant characteristics, such as tocols, carotenoids, polyphenols, flavonoids, anthocyanins, lignans. the comprehensive antioxidant ability of all antioxidants in the cereals is generally represents as total antioxidant capacity (tac) (re et al., 1999; rice-evans et al., 1999). phenolic acids in wheat bran usually can be divided into three type of existing forms: soluble free phenolic acids, conjugated phenolic acids, and insoluble-bound phenolic acids (rosicka-kaczmarek et al., 2018). the free and conjugated phenolic acids make up only a small section, while most of the phenolic acids are bound phenolic compounds by ester and ether linkages with cell wall components such as arabinoxylans and lignin (liu et al., 2016). the aleurone layer is mostly composed of arabinoxylan with a high content of ferulic acid (fa) monomers and low levels of fa dimers (ramos-enríquez et al., 2018a). in fact, different form of phenolic acid worked on various impacts on human health. when the dietary contain bound forms were intake by human, it would be useful in the precaution of colon cancer and other cancers (lei et al., 2012; ramos-enríquez et al., 2018b). however, the intake of soluble free and conjugated forms is attributed to quickly absorption in the stomach and small intestine as well as distribution throughout the body. extrusion technology is a combination of mechanical shearing action, pressure action and thermal energy, which causes the material to be suddenly released from the high temperature and huge pressure state to the normal temperature and pressure and the internal structure and physical and chemical properties of the extruded material would be changed and extrusion technology also is a kind of processing methods to force materials at a predetermined feed rate, to flow through materials and through certain die holes to obtain products of different shapes and properties, and the food is called extruded food (aam et al., 2017). as a high-tech in the field of food processing, extrusion technology opens -up to a new way of production that is simple, mechanized and highly automated for the development of convenience food. besides, it has a high efficiency, and low cost in processing, as well as the products are easy to digest, keep the nutrient at a maximum degree, and is conducive to long-term storage. some evidence has been reported the activities of lipoxygenase and polyphenol oxidase were greatly reduced, and the shelf life was prolonged compared to the wheat bran, which has not been extruded (pilli et al., 2010). what′s more, the content of free phenolic compound in the product was obviously improved, and the antioxidant capacity was also enhanced. therefore, it can be used as a excellent material for making whole wheat food or health-benefit products for improving the nutritional value (brennan et al., 2011). ital. j. food sci., vol. 32, 2020 388 in our previous single factor study, it was found that the antioxidant ability and to phenolic content of extruded wheat bran were higher when the feed moisture was 22-24% and the screw speed was 130-190 rpm at the extrusion temperature of 80-100°c. to improve the antioxidant ability and shelf life of wheat bran, the optimum extrusion condition on the antioxidant capacity and free phenolic compounds of wheat bran was investigated by rsm. and the phenolic compounds extracted from wheat bran before and after extrusion were also identified by ultra-high performance liquid chromatography (uplc). 2. materials and methods 2.1. materials and reagents wheat brans were obtained from hubei sanjie agricultural industrialization co. ltd (hubei province, china); 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (abts) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) were purchased from sigma–aldrich. other chemicals and solvents for chromatographic grade analysis were obtained from merck (darmstadt, germany). all the other chemicals and solvents were of analytical grade. 2.2. sample preparation the wheat brans were milled and sieved to a 60 mesh size. the powder was put in to twoscrew extruder (fmhe36-24, hunan fuma branch food engineering technology co, ltd) and then the extruded wheat brans were dried at 50°c for 18 hours to cut down the moisture content, and then extruded wheat brans were milled and sieved to a 60 mesh size. the powder of extruded wheat bran was kept in a black laboratory bottle, and the bottles were placed at -20°c in refrigerator. 2.3. ultrasound extraction 0.5 g of the dried powder of extruded wheat brans were thoroughly mixed with 10 ml of 60% ethanol and placed in a 50 ml amber laboratory bottle (dhanani et al., 2017). the operating extraction was last for 1.5 hours at 60°c. the supernatants were combined after centrifugation (3622×g, 20 min). after centrifugation the supernatant were evaporated to 2 ml at 45°c and placed in amber laboratory bottle at -20°c until used. 2.4. determination of trolox equivalent antioxidant activity abts assay was implemented the concordat of pellegrini n (re et al., 1999) with a little modifications by condition. abts•+ radical solution was prepared to mix 10 ml of abts stock solution (7mm abts in water) with 176 μl of potassium persulfate (140 mmol/l), which was kept in darkness 12-16 h at 4°c. the abts•+ reagent was diluted with anhydrous ethanol to detect the absorbance of 0.700 ±0.02 at 734 nm (berg et al., 1999). the 0.1 ml of the sample solution and 3.9 ml of diluted abts•+ were thoroughly mixed and placed in a 10 ml amber glass tube and shook at room temperature for 6 minutes. the absorbance of the reaction was measured at 734 nm (a734 = as) using glass cuvettes. then 0.1 ml of sample and 3.9 ml of anhydrous ethanol or 3.9 ml of diluted abts•+ were operated as the above mention and the absorbance a734 remarked as ar and a0 respectively. ital. j. food sci., vol. 32, 2020 389 the calibration curve was set using trolox at the consistence range of 50–1000μmol/l in ultrapure water. the total antioxidant capacity of wheat bran extruded material was expressed as a teac per 1 g of dry matter of a sample (μmol/g). % inhibition rate of abts = 1 − !!!!! !! ×100% (1) 2.5. determination of extruded wheat bran and raw wheat bran total free phenolic content the total free phenolic content was analyzed as determined following described previously (li et al., 2008; vázquez et al., 2015a) with some modification. briefly, a stock solution of gallic acid with pipette gallic acid control solutions was prepared at a concentration of 0.5 mg/ml. with this solution, calibration curve was prepared for the different dilutions. in the dim light, 1 ml of the extract obtained from raw wheat bran and extruded wheat bran were placed in each well of a burette and 9 ml of deionized water, 1 ml of folin-ciocalteu reagent, and 2 ml of sodium carbonate solution (w/w=1/4) were added in orderly. all blanks except the extract was also prepared. then these burettes were put in water bath at 50°c for 0.5 h. secondly, 12 ml of deionized water was added in the burette and put at room temperature for 0.5 h. subsequently, the burettes were read on a spectrophotometer at an absorbance of 745 nm (vázquez et al., 2015a). above all results were represented as gallic acid equivalent (mg gallic acid/g of extruded wheat bran material gae/g of dried sample). 2.6. uplc analysis uplc-pda was used to determine free phenolic compounds extracted from raw and extruded wheat bran. the chromatographic system was made up an acquity uplc (waters, us) equipped with pda. samples were separated using a waters column (acquity uplc@hss t3 1.8 μm, 2.1×150 mm column). the column temperature was maintained at 40°c. methanol-acetonitrile solution (1:1, v/v) and acetic acid (2.50%, v/v) were used as mobile phase a and b, respectively. the gradient program was as follows: 520% a (0-6 min), 20-40% a (6-15 min), 40-70% a (15-18 min), and 70-5% a (18-24 min). phenolic acids were detected at 280 nm. the phenolic acid content was calculated from the peak area according to the calibration curve by using the external standard method and expressed as μg/g dw. 2.7. experimental design the effect of factors such as extrusion temperature (80°c, 90°c, 100°c), screw speed (130 rpm, 160 rpm, 190 rpm), and feed moisture (20%, 22%, 24%) on free phenolic compounds and trolox equivalent antioxidant capacity (teac) were tested. a three-level-three-factor and seven central point factorial design were employed requiring a total of 19 experiments. the bbd was used to determine the optimal extrusion conditions that maximum of teac and fpc of extruded wheat bran. the three independent variables of extrusion temperature (°c, x1), feed moisture (%, x2) and screw speed (rpm, x3) at three levels (-1, 0, +1) were set. the coded and actual values of variables were shown in table 1. ital. j. food sci., vol. 32, 2020 390 table 1. level of coded and real values for factorial design. 2.8. statistical analysis all experiments were carried out in triplicates and results were expressed as means ± standard deviation (n=3). anova was carried out to determine any significant differences (p < 0.05). response surface plots were generated using design-expert 6.0. 3. results 3.1. effect of extrusion condition on teac content of extruded wheat bran the mean values of teac content and the content of fpc each of the 19 treatments at the different extrusion conditions were shown in table 2. the highest of teac content of 14.1143 μmol/g was obtained in experimental run number 7 with an extrusion temperature of 90°c, feed moisture of 22% and a screw speed of 160 rpm. while the lowest teac content of 12.7929 μmol/g was observed in experimental run number 16 with an extrusion temperature of 90°c, feed moisture of 22% and screw speed at 190 rpm. in tables 3 and 4, the estimated regression coefficients and anova of teac of extruded wheat bran was observed. the quadratic regression model was extremely significant (p=0.0004< 0.001) and the lack of fit was not significant (p=0.1609>0.05) at the same time, showing that the model was in good agreement with the experimental data of teac content (bannour et al., 2017; berg et al., 1999; zhen et al., 2016). the regression coefficient (r2 = 0.9264) suggested the experimental and predicted content data had been a good fit in the experiment. the linear and quadratic of feed moisture showed a significant difference, indicating the effect on teac content. moreover, the interactive variables between feed moisture and screw speed showed a significant difference (p=0.0286< 0.05), suggesting the effect on teac content. and then also the interactive variables between extrusion temperature and screw speed showed a significant difference (p=0.0210< 0.05), suggesting the effect on teac content. in order to analyze the effect of interaction of the different variables, the response surface curves were plotted. meanwhile, for the purpose of determining the optimal extrusion condition of the responses of the independent variables with maximized teac content of extruded wheat bran. the concentration of teac content of extruded wheat bran increased with increasing feed moisture and screw speed. however, when the feed moisture was added to nearly 22% and the screw speed added to 160 rpm, the teac content slowly dropped, as described in fig. 1c. teac values also enhanced with the enhanced of extrusion temperature and feed moisture data as shown in fig. 1a. fig. 1c factors level -1 0 1 extrusion temperature (°c) (x1) 80 90 100 feed moisture (%) (x2) 20 22 24 screw speed (rpm) (x3) 130 160 190 1 ital. j. food sci., vol. 32, 2020 391 revealed the increasing teac content with an increase of screw speed and extrusion temperature. table 2. values for teac and fpc of extruded wheat bran extracts under different extrusion conditions. factorial design determination x1 x2 x3 total free phenolic content (mg gae/g) teac (µmol/g) run number extrusion temperature (°c) feed moisture (%) screw speed (rpm) experimental experimental 1 90 24 130 3.04±0.06 13.03±0.01 2 100 22 130 2.83±0.05 13.23±0.01 3 90 22 160 3.14±0.04 13.80±0.01 4 90 20 130 2.94±0.01 13.03±0.05 5 90 24 190 2.78±0.06 13.55±0.02 6 80 24 160 3.04±0.02 13.56±0.02 7 90 22 160 3.18±0.02 14.11±0.01 8 80 22 130 3.14±0.03 13.49±0.01 9 90 22 160 3.17±0.03 13.80±0.01 10 100 22 190 2.99±0.01 13.64±0.03 11 90 22 160 3.15±0.03 13.88±0.04 12 90 22 160 3.21±0.04 13.84±0.01 13 100 20 160 2.88±0.04 13.08±0.01 14 90 22 160 3.21±0.01 13.91±0.07 15 80 20 160 3.07±0.01 13.23±0.01 16 90 20 190 2.92±0.01 12.79±0.01 17 100 24 160 2.90±0.01 13.21±0.03 18 80 22 190 2.92±0.02 13.08±0.05 19 90 22 160 3.13±0.05 13.76±0.02 1 ital. j. food sci., vol. 32, 2020 392 table 3. anova. table 4. anova for quadratic model. response 1: teac of extruded wheat bran. source teac (r2=0.9264) total fpc (r2=0.9579) ss df ms f-value p-value ss df ms f-value p-value model 2.40 9 0.27 12.58 0.0004 0.00 9 0.00 22.74 < 0.0001 lack of fit 0.11 3 0.04 2.45 0.1609 0 3 0.00 2.77 0.1329 pure error 0.09 6 0.01 0 6 0.00 1 source ss df ms f-value p-value model 2.40 9 0.2662 12.58 0.0004 significant a-extrusion temperature 0.0051 1 0.0051 0.2407 0.6354 b-feed moisture 0.1834 1 0.1834 8.67 0.0164 c-screw speed 0.0108 1 0.0108 0.5093 0.4935 ab 0.0107 1 0.0107 0.5035 0.4959 ac 0.1649 1 0.1649 7.79 0.0210 bc 0.1433 1 0.1433 6.77 0.0286 a² 0.1277 1 0.1277 6.04 0.0364 b² 0.8298 1 0.8298 39.21 0.0001 c² 0.5207 1 0.5207 24.60 0.0008 residual 0.1905 9 0.0212 lack of fit 0.1050 3 0.0350 2.45 0.1609 not significant pure error 0.0855 6 0.0143 cor total 2.59 18 1 ital. j. food sci., vol. 32, 2020 393 figure 1. effect of different extrusion conditions on teac and fpc：(a) teac effect of extrusion temperature and feed moisture; (b) teac effect of extrusion temperature and screw speed; (c) teac effect of feed moisture and screw speed; (d) fpc contents effect of extrusion temperature and feed moisture; (e) fpc contents effect of extrusion temperature and screw speed for extrusion; (f) fpc contents effect of screw speed and feed moisture for extrusion. 3.2. effect of extrusion condition on fpc contents of extruded wheat bran table 5 shown that fpc contents of extruded wheat bran quadratic regression model was extremely significant (p< 0.0001). what′s more, the lack of fit had a p-value higher (p=0.1392>0.05). in table 3, the total fpc value of r2 = 0.9579 demonstrated the model to be a well fit for the experimental data of total fpc of extruded wheat bran. individual independent variables extrusion temperature had an extremely significant effect on the total fpc content which was indicated by the linear data model (p=0.0006<0.01), and screw speed had major impact effect on the total phenolic content (p=0.0133<0.05), but feed moisture didn′t show significant effect on the total phenolic content. the interaction between extrusion temperature and feed moisture showed a not significant effect on the total fpc content (p=0.5254> 0.05), and interaction between feed moisture and screw speed also showed a significant effect on the total phenolic content (p=0.0136<0.05). meanwhile, the interplay between extrusion temperature and screw speed also expressed an extremely significant effect on the total fpc content (p=0.001< 0.01). when the extruded temperature was over 100°c, the extruded wheat bran′s antioxidant capacity and total free phenolic contents might decrease, which it may be that the phenolic compounds might be degraded. the results showed that the effect of extrusion temperature on total free phenol content is extremely significant (p=0.0006<0.01). meanwhile, screw speed also indicated an obviously effect on the fpc content (p=0.0133<0.05).the interaction of the three independent variables of extrusion temperature, feed moisture, and screw speed was used to plot the response surface curves for the total phenolic content as shown in fig. 1d, fig. 1e, fig. 1f. increasing the extrusion ital. j. food sci., vol. 32, 2020 394 temperature from 80°c to 100°c at constant feed moisture (fig. 1d) and screw speed (fig. 1e) did change total phenolic content. although, with the extrusion temperature, feed moisture and screw speed increasing, the total fpc contents of extruded wheat bran, as shown in fig. 1d, fig. 1e, fig. 1f. but, when extrusion temperature and screw speed at nearly 100°c and 190 rpm, respectively, a small cut down in total phenolic content was observed (fig. 1e). this expressed that a portion of phenolic compounds would be degraded by over high extrusion temperature and screw speed in extrusion progress. table 5. anova for quadratic model. response 2: fpc of extruded wheat bran. 3.3. verification of predictive optimal extrusion conditions the predicted extrusion conditions of wheat bran extruded material at 85.85°c of extrusion temperature, 22.19% of feed moisture and 154 rpm of screw speed provided the maximum teac content of extruded wheat bran, and the maximum total phenolic content was reached at the predicted conditions of 85.85°c of extrusion temperature, 22.19% of feed moisture and 154 rpm of screw speed. the predicted extruded conditions of wheat bran were the same for the teac content and total phenolic content for convenient operation, and considering the experiment in practice the optimal extraction parameters were adjusted to be 86°c of extrusion temperature, 22% of feed moisture and 160 rpm of screw speed at which that the predicted teac content was 13.8472 μmol g-1 source sum of squares df mean square f-value p-value model 0.0008 9 0.0001 22.74 < 0.0001 significant a-extrusion temperature 0.0001 1 0.0001 26.32 0.0006 b-feed moisture 5.234e-07 1 5.234e-07 0.1360 0.7208 c-screw speed 0.0000 1 0.0000 9.45 0.0133 ab 1.680e-06 1 1.680e-06 0.4365 0.5254 ac 0.0001 1 0.0001 23.14 0.0010 bc 0.0000 1 0.0000 9.36 0.0136 a² 0.0001 1 0.0001 15.42 0.0035 b² 0.0002 1 0.0002 44.71 < 0.0001 c² 0.0002 1 0.0002 44.71 < 0.0001 residual 0.0000 9 3.848e-06 lack of fit 0.0000 3 6.709e-06 2.77 0.1329 not significant pure error 0.0000 6 2.418e-06 cor total 0.0008 18 1 ital. j. food sci., vol. 32, 2020 395 and total phenolic content was 13.19754 mg gae g-1 dw. this strongly suggests that the model is suitable to predict teac content, total phenolic content using extrusion at selected conditions. 3.4 identification and quantification phenolic compounds of extruded wheat bran and raw wheat bran by uplc table 6 showed results about the concentration of each phenolic compound obtained for the extruded wheat bran and raw wheat bran hydroalcoholic extracts. two kinds of phenolic compounds (11.39 μg.g−1 gallic acid and 78.40 μg.g−1 ferulic acid) were identified in raw wheat bran and 4 kinds of phenolic compounds (12.58 μg.g−1 gallic acid, 61.06μg.g−1 caffeic acid, 93.40 μg.g−1 ferulic acid, and 183.64μg.g−1 rutin) in the extruded wheat bran. the compounds caffeic acid and rutin were not identified in the raw wheat bran. so those two phenolic compounds became the significant differences between the raw wheat bran and extruded wheat bran. meanwhile, the raw wheat bran′s teac was 12.38±0.22 μmol.g-1 and extruded wheat bran′s teac was is 13.91±0.04 μmol.g-1. and then the raw wheat bran′s total free phenolic compounds was 2.88±0.09 mg gae.g-1, while extruded wheat bran′s was 3.14±0.07 mg gae.g-1 the extruded wheat bran. above all, the reason why that extruded wheat bran antioxidant capacity and free phenolic compounds significantly higher than the raw wheat bran could be explained by these dates. table 6. characterization of extruded wheat bran extracts. determination this study the raw wheat bran the extruded wheat bran total free phenolic compounds (mg gae.g-1) 2.88±0.09aa 3.14±0.07bb teac (µmol. g-1) 12.38±0.22aa 13.91±0.04bb ferulic acid (µg.g−1) 78.40±0.001aa 93.40±0.000bb gallic acid (µg.g−1) 11.39±0.002aa 12.58±0.001bb caffeic acid (µg.g−1) 0 61.06±0.001 rutin (µg.g−1) 0 183.64±0.001 1 ital. j. food sci., vol. 32, 2020 396 the content of gallic acid (12.58 μg g−1 extruded wheat bran), ferulic acid (93.4 μg g−1 extruded wheat bran) in extruded wheat bran were higher than the raw wheat bran. the extrusion technology helped the wheat bran to break the cell structure of wheat bran and then the phenolic compounds were released by high temperature, strong pressure, and great powerful shear force. phenolic compounds have one or more hydroxyl groups conjugated to an aromatic hydrocarbon group, which characterizes the phenolic structure (chaiyasut et al., 2017; gutiérrez-grijalva et al., 2017; hilbig et al., 2018). the phenolic compounds specially structure bring about these compounds antioxidant activity to a certain degree, which may be higher or lower depending on the position and number of hydroxyls (apeabah et al., 2017; vázquez et al., 2015b). the presence of several phenolic compounds in extruded wheat bran extracts might explicate the antioxidant activity demonstrated for the extruded wheat bran. 4. conclusion in the present work, bbd was successfully carried out to set the extrusion conditions optimized parameters for the antioxidant capacity and free phenolic compounds of extruded wheat bran. in comparison to raw wheat bran, the extruded wheat bran resulted in the higher recoveries of both total free phenolic compounds and trolox equivalent antioxidant capacity, which showed that extrusion could enhance the antioxidant capacity and free phenolic compounds of wheat bran. moreover, both the raw wheat bran and extruded wheat bran, yielded the same phenolic compounds, namely gallic acid，ferulic acid caffeic acid and rutin, were determined by uplc. the study suggested that the use of extrusion pre-treatment in enhancing contents the of desired bioactive components from food industry by-product was a numerous potential extraction technology.from the data of the response surface, the extruded technology is efficient, economic and environmental process technology. because the antioxidant capacity and free phenolic compounds were significantly improved by extrusion treatment. thus, the extruded wheat brans would be a good source of natural antioxidants. acknowledgments the authors gratefully acknowledge the support provide by research and demonstration of key technical equipment for whole wheat flour processing and quality improvement (2018yfd0401002) and the national food industry youth top talent service industry demand independent topic selection project (lq2018203). abbreviations rsm response surface methodology fpc free phenolic compounds gae gallic acid equivalent fa ferulic acid ldl low-density lipoprotein pda photo-diode array uplc ultra performance liquid chromatography teac trolox equivalent antioxidant capacity bbd box-behnken design ital. j. food sci., vol. 32, 2020 397 dw dry 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of murcia, espinardo, 30071 murcia, spain 4department of food engineering, national technological of mexico, tierra blanca campus, 95180 tierra blanca, veracruz, méxico 5licef research center, teluq university, québec, canada *corresponding author: jihen.ben.amor90@gmail.com abstract carrot (daucus carota l.) is the most widely consumed root vegetable since it is an important source of nutritional compounds, mainly antioxidants and sugars. in tunisia, despite the genetic diversity observed in carrot germplasm, including landraces and wild relatives, no research has been conducted on the biochemical composition of carrot. thus, this study aims to analyse carotenoids, soluble sugars, total phenols, total flavonoids and colour properties of 14 carrot landraces, in order to determine the diversity among them and evaluate the relationships among their biochemical contents. the main carotenoids identified were α-carotene, β-carotene and lutein. orange carrots were richer in β-carotene and α-carotene than yellow carrots. the major sugars were sucrose, glucose, fructose and ital. j. food sci., vol. 32, 2020 655 galactose. significant differences were observed among the tunisian carrot landraces with respect to their biochemical composition and colour characteristics. total carotenoids and total sugars ranged from 155.74 to 511.44 μg/g of dw and from 368.77 to 546.79 mg/g of dw, respectively. total phenols and total flavonoids varied from 24.13 to 41.39 mg gae/100 g of dw and from 16.51 to 24.85 µg ce/100 g of dw, respectively. significant, positive and negative correlations were found among the measured parameters. a principal component analysis (pca) and agglomerative hierarchical clustering (ahc) were performed to classify the tunisian carrot landraces on the basis of colour properties and biochemical compounds. the pca divided the landraces into four main groups and ahc classified them into two major clusters. the tunisian carrot landraces were found to be rich in bioactive compounds; they could be good candidates for future breeding programs. keywords: antioxidant compounds, carotenoids, carrot, colour properties, sugars ital. j. food sci., vol. 32, 2020 656 1. introduction carrot (daucus carota l.) is one of the major vegetable crops grown worldwide (rubatzky et al., 1999). this vegetable belongs to the apiaceae family, being the most widely used member. the first carrots, with purple and yellow colours, originated in central asia, asia minor, western europe and england between the 11th and 15th centuries (banga, 1963). the orange carrot was domesticated in europe between the 15th and 16th centuries (banga, 1957; stolarczyk and janick, 2011). due to its nutritional value, moderate price and consumption modes, carrot is the most consumed root vegetable in the world (chaux and foury, 1994). thus, its roots are eaten as crunchy salad; used to prepare juice, sweets and halwa; cooked with mixed vegetables; and preserved by salting, pickling, canning and drying (singh et al., 2018). compared to other vegetables, carrot possesses the highest amount of carotenoids, defined as organic pigments that naturally occur in the chloroplasts and chromoplasts of plants (zielinska and markowski, 2012). these bioactive compounds have an antioxidant activity, protecting plants against oxidative stress, and have been proven to be beneficial to human health, reducing oxidative stress by scavenging free radicals (tan and norhaizan, 2019; stagos, 2020). depending on its carotenoids content, the carrot root can be purple, orange, yellow or white (baranski, 2012). β-carotene constitutes the major portion (60–80%) of the carotenoids, followed by α-carotene (10–40%) and lutein (1–5%), whereas the rest are other minor carotenoids (sun and temelli, 2006). in general, carrot has great acceptability among consumers due to its sweetness, which is governed by its sugars content (simon et al., 1980a). these compounds represent an important sensory indicator for consumers. moreover, sweetness represents one of the most important factors in the acceptance of new commercial vegetable cultivars (nookaraju et al., 2010). the most abundant sugar found in carrot is sucrose, followed by glucose and fructose. the total sugars content in fresh carrot varies from 3% to 10%, whereas soluble sugars can represent up to 70% of the dry carrot (dolores et al., 1999; cazor et al., 2006). besides sugars and carotenoids, which determine carrot sweetness and colour, phenolic compounds also contribute to their organoleptic properties (rubatzky et al., 1999). the major phenolic acids present in carrot are chlorogenic acid, caffeic acid, p-hydroxybenzoic acids, ferulic acid and other isomers of hydroxy cinnamic acid (alasalvar et al., 2001). the content of these compounds varies depending on root colour. chlorogenic acid is the main phenolic acid and represented 52.4, 57.1, 51.4, and 72.5% of the total phenolic compounds in orange, yellow, white, and purple carrots, respectively (sun et al., 2009). the different carrot tissues have similar composition, but the contents of individual phenolic compounds differ and they decrease from the exterior to the interior (gonçalves et al., 2010). phenolic compounds are involved in the resistance to physiological mechanism of plants, and their accumulation in carrots results from exposure to cold, injury or ethylene (rubatzky et al., 1999). flavonoids are represented mainly by anthocyanins, which are richer in purple carrot. they exert beneficial effects on human health, acting as vasodilators (cheng et al., 1993) and platelet disaggregators (gryglewski et al., 1987), and also as efficient antioxidants with free radical scavenging abilities (bahorun et al., 2003). in tunisia, carrots are widely cultivated with an annual production of 201.500 tons, representing 5% of the total vegetable production. they are produced on 5.700 ha of land (~94% as a winter crop and 6% as a summer crop) with an average yield of 35.35 tons/ha ital. j. food sci., vol. 32, 2020 657 (dgpa, 2019). although these locally produced carrots can be yellow or orange, tunisians prefer the latter. tunisia is considered a center of biodiversity for daucus and many other crops because of the diversity of ecosystems and climatic conditions (le floc'h et al., 2010). this diversity has been the subject of several studies in order to identify and enhance this genetic heritage. thus, mezghani et al. (2014; 2017; 2018) studied the genetic diversity of wild carrot, based on morphological and molecular data. this research revealed a great genetic variability among the accessions. moreover, ben amor et al. (2019) characterized tunisian carrot (daucus carota subsp. sativus) landraces collected from different regions by using agro-morphological descriptors related to roots and leaves. the study showed a high phenotypic variability, particularly in root colour, which could be reflected by the biochemical composition and content. thus, the aim of this work was to determine the contents of individual carotenoids and sugars, as well as the total phenols and flavonoids, in different tunisian carrot landraces, and to classify these landraces on the basis of their biochemical compounds and colour properties. 2. materials and methods 2.1. plant material the study material consisted of 14 carrot (daucus carota subsp. sativus) landraces, derived from a collection of 33 carrot landraces originating from the main regions of carrot cultivation in tunisia and conserved at the national gene bank of tunisia. the landraces were selected to maximize the phenotypic diversity, based on our previous study (ben amor et al., 2019). seeds obtained from self-pollinated landraces were sown in the experimental site of the high agronomic institute of chott mariem in tunisia (35.1182 n; 10.7297 e), in november 2016. at the maturity stage, the carrots were manually harvested. uniform roots (five per landrace) were selected, and washed with tap water to remove soil and other dirt. representative samples (500 g) of each set of five roots were taken for subsequent analysis. 2.2. colour measurements the colour measurements were performed on the skin of the carrot roots using a minolta chromameter (cr-410). the measurements obtained are reported in the l*, a*, b* systems. the l* value varies from 0 to 100, representing the darkness or lightness of colour. the a* value ranges from green (-a*) to red (+a*). the b* value varies from blue (-b*) to yellow (+b*). the chroma c* and hue angle h⁰ were also calculated, using the following equations: c*= (a*2 + b*2)1/2; h⁰= tan-1 (b*/a*). the c* value shows the saturation of colour and it is proportional to the colour intensity. the h⁰ is the basic unit of colour and varies from 0⁰ to 90⁰, indicating red and yellow colour, respectively. after the colour measurements, the roots were cut into slices and lyophilized. dry samples were ground and the powders were stored in a refrigerator until analysis. 2.3. extraction and analysis of carotenoids carotenoids were extracted from the lyophilized carrot (0.5 g) using 35 ml of methanol/tetrahydrofuran (1:1, v/v) containing 0.1% butylated hydroxytoluene. the mixture was blended for 5 min and then vacuum filtered through whatman™ no.5 filter ital. j. food sci., vol. 32, 2020 658 paper (whatman, england). the extraction was performed three times, leaving an uncoloured residue. the combined extracts were dried under vacuum at 37 ºc in a rotary evaporator. the residue was re-dissolved in a methanol/tert-butyl methyl ether mixture (1:1, v/v) until the solution reached a final volume of 10 ml. the solution was centrifuged for 10 min at 14 000 rpm (at 4 ºc) and then analyzed (böhm, 2001). the experiment was conducted under dark conditions in order to avoid carotenoids degradation. the quantification of carotenoids was carried out using high-performance liquid chromatography (hplc) with diode array detection (dad). the hplc analysis was performed with methanol (solvent a) and methyl tert-butyl ether (solvent b), using a gradient procedure on a c30 column (250 mm x 4.6 mm, 5 μm, trentec, gerlingen, germany) at 17 ºc and a flow rate of 1.3 ml min-1. carotenoids were quantified at 450 nm and were identified on the basis of the retention time, as described by chen and tang (1998), and according to the dad spectra. standard solutions of the main carotenoids were used to prepare calibration lines, in order to determine the concentrations corresponding to the different peaks of the chromatograms. the concentrations of individual carotenoids were expressed as μg/g of dry weight (μg/g of dw). 2.4. extraction and analysis of soluble sugars the measurement of the soluble sugars content was performed using hplc, according to the method described by nomura et al. (1995). a 0.5 g sample of lyophilized carrot powder was extracted with 5 ml of 80% ethanol (v/v). the mixture was homogenized and then left in an ultrasound bath for 30 min at 60 ºc, before centrifugation at 4 500 rpm for 15 min. the supernatant was recovered and the extraction was repeated twice more. the combined extracts were dried under vacuum at 80 ºc in a rotary evaporator. the residue was re-dissolved in 2 ml of water, filtered and finally analyzed with hplc. the chromatographic separation of the sugars was carried out with a carbo sep cho-682 column (7.8 × 300 mm) held at 80 °c, with distilled water as the mobile phase at a flow rate of 0.4 ml min-1. the temperature for the refractive index detector was set at 45 ºc. standard solutions of sucrose, glucose, fructose and galactose were prepared and calibration lines were made for each one in order to determine the concentrations corresponding to the different peaks in the chromatograms. the concentrations of sugars were expressed as mg/g of dry weight (mg/g of dw). 2.5. extraction and analysis of total phenols and flavonoids 2.5.1 samples preparation total phenols and total flavonoids were extracted from 0.5 g of lyophilized carrots with 4 ml of an acidified methanolic solution (80% metoh + 1% formic acid). the mixture was homogenized, left in an ultrasound bath for 5 min and finally centrifuged for 10 min at 5 000 rpm. the supernatant was collected for the analysis of total phenols and total flavonoids. 2.5.2 analysis of total phenols the quantification of total phenols was performed using a spectrophotometric technique based on the folin-ciocalteu method described by singleton and rossi (1965). in microcuvettes, 1 ml of the sample, 500 μl of diluted folin-ciocalteu reagent (1:10, v/v) and ital. j. food sci., vol. 32, 2020 659 400 μl of na2co3 were mixed. the absorbance was measured at 750 nm after 2 h at room temperature. gallic acid was used as the standard and the data were expressed as mg of gallic acid equivalents/100 g of dry weight (mg gae/100 g of dw). 2.5.3 analysis of total flavonoids the quantification of total flavonoids was conducted spectrophotometric method described by dewanto et al. (2002). an aliquot of 100 μl of each methanolic sample was mixed with 625 μl of distilled water and 37.5 μl of nano2 (5%). after 6 min, 37 μl of alcl3 were added and the mixture was left for 5 min. finally, 250 μl of 1m naoh and 162.5 μl of distilled water were added to the mixture. the absorbance was measured at 510 nm. catechin was used as the standard and the data were expressed as µg of catechin equivalents/100 g of dry weight (µg ce/100 g of dw). 2.6. statistical analysis statistical analyses were performed using appropriate packages in r software, available from the comprehensive r archive network (cran) at http://cran.r-project.org/. for all parameters an analysis of variance (anova) was carried out using the rcmdr package, to determine differences among cultivars. the data are expressed as the mean and standard deviation for each parameter. a pearson correlation analysis was carried out to estimate the relationships among the studied parameters. principal component analysis (pca) and agglomerative hierarchical clustering (ahc) were performed using the factominer package, to determine relationships among the biochemical parameters and to group landraces into homogenous classes. 3. results 3.1. colour parameters the colour characteristics measured showed significant variability among the carrot landraces (table 1). the a* value, indicating the intensity of red colour, ranged from 3.62 for ngbtun560 to 27.73 for ngbtun539. for yellowness b*, ngbtun520 and ngbtun556 showed the highest and lowest values, respectively. all carrot landraces had high values of c* (> 50), which could be due to the high intensity and saturation of the colour. also, all landraces had high values of the lightness parameter l* and the hue angle h⁰; in general, these values were higher in yellow landraces than in orange landraces. 3.2. carotenoids content the analysis of carotenoids with hplc enabled the identification of three major carotenoids: α-carotene, β-carotene (the most abundant one in all samples) and lutein. in addition, the total carotenoids content was estimated as the sum of the individual carotenoids. the anova showed that the individual and total contents of carotenoids depended to a significant extent on the carrot landrace (table 2). the α-carotene content varied from 71.36 μg/g of dw for ngbtun558 to 159.34 μg/g of dw for ngbtun572. the content of β-carotene was highest for ngbtun539 (418.58 μg/g of dw) and lowest for ngbtun560 (71.74 μg/g of dw). for lutein, ngbtun520 had the highest content ital. j. food sci., vol. 32, 2020 660 (68.99 μg/g of dw). the total carotenoids content ranged between 155.74 μg/g of dw (ngbtun560) and 511.44 μg/g of dw (ngbtun539), showing significant and marked differences among landraces. table 1. colour measurements of 14 tunisian carrot landraces1. landrace skin colour a* b* c* h⁰ l* ngbtun512 yellow 11.94±0.38 f 58.97±1.04 e 60.16±1.09 ef 78.55±0.15 d 81.38±0.04 b ngbtun514 yellow 7.14±0.1 gh 54.98±0.32 g 55.44±0.33 g 82.59±0.06 c 74.32±0.59 e ngbtun520 yellow 7.56±0.18 g 66.44±0.32 a 66.86±0.33 a 83.5±0.12 b 83.85±0.15 a ngbtun522 yellow 6.15±0.03 i 60.74±0.17 cd 61.05±0.18 e 84.21±0.01 b 83.99±0.01 a ngbtun523 yellow 6.95±0.5 ghi 62.37±1.44 b 62.75±1.48 d 83.64±0.31 b 80.51±1.04 bc ngbtun527 orange 14.67±0.02 e 51.07±0.09 h 53.13±0.09 h 73.97±0 g 79.36±1.01 c ngbtun539 orange 27.73±0.43 a 59.27±0.39 e 65.43±0.53 b 64.92±0.20 j 74.06±1.17 e ngbtun540 orange 19±0.81 c 61.66±0.68 bc 64.52±0.40 bc 72.86±0.87 h 79.31±1.19 c ngbtun541 orange 16.16±0.75 d 61.38±0.3 bcd 63.47±0.48 cd 75.25±0.58 f 79.52±0.63 c ngbtun556 orange 18.6±1.09 c 50.81±0.66 h 54.1±1.00 h 69.9±0.84 i 76.78±1.15 d ngbtun558 yellow 6.43±0.09 hi 60.48±0.1 d 60.82±0.90 e 83.93±0.09 b 83.73±0.61 a ngbtun560 yellow 3.62±0.51 j 59.1±0.31 e 59.21±0.34 f 86.49±0.47 a 84.84±0.19 a ngbtun567 yellow 14.73±0.09 e 59.19±0.2 e 60.99±0.17 e 76.02±0.13 e 75.87±0.28 d ngbtun572 orange 21.34±0.28 b 56.66±0.4 f 60.54±0.47 e 69.36±0.12 i 76.72±1.22 d p value <0.0001 <0.0001 <0.0001 <0.0001 <0,0001 f value 613.53 157.14 122.88 844.81 62.86 1data are expressed as the mean and standard deviation. a-jdifferent letters in the same column indicate significant differences for p<0.05. 3.3. soluble sugars content the main soluble sugars detected in the tunisian carrot landraces were glucose, fructose, sucrose and galactose, with significant differences among the landraces (table 3). glucose and fructose were the most abundant soluble sugars, followed by sucrose and galactose. in general, the individual sugar contents were: for glucose >96.9 mg/g of dw, for fructose >119 mg/g of dw, for sucrose >46.2 mg/g of dw and for galactose >1.13 mg/g of dw, except for ngbtun558 and ngbtun560 in which galactose was not detected. the total sugars content, represented as the sum of the individual sugars, varied from 368.77 to 546.79 mg/g of dw for ngbtun522 and ngbtun540, respectively. 3.4. total phenols and total flavonoids contents the total phenols and total flavonoids were also analyzed and, as for the carotenoids and sugars, their contents varied significantly among the carrot landraces (table 4). for total phenols, the highest content was recorded for ngbtun520 (41.39 mg gae/100 g of dw), whereas ngbtun527 showed the lowest content (24.13 mg gae/100 g of dw). the total flavonoids content ranged from 16.51 to 24.85 µg ce/100 g of dw for ngbtun539 and ital. j. food sci., vol. 32, 2020 661 ngbtun572, respectively. it is noteworthy that the flavonoids represented a small proportion of the total phenolic compounds in carrots. table 2. content of individual and total carotenoids, expressed as μg/g of dry weight, in 14 tunisian carrot landraces1. landrace α-carotene β-carotene lutein total carotenoids ngbtun512 76.28±0.27 ef 116.77±1.46 f 61.36±1.93 b 254.41±0.19 e ngbtun514 79.00±1.48 e 113.31±1.66 f 50.10±2.66 c 242.41±2.83 e ngbtun520 77.12±0.19 ef 106.77±1.73 fg 68.99±2.94 a 252.88±4.86 e ngbtun522 73.64±0.06 fg 100.32±0.54 g 33.28±0.91 de 207.25±1.52 f ngbtun523 71.49±0.09 g 85.44±1.29 h 29.65±3.51 e 186.58±4.89 g ngbtun527 109.73±2.07 d 235.68±4.31 c 6.07±0.05 i 351.48±6.43 c ngbtun539 79.76±2.06 e 418.58±8.42 a 13.1±0.07 h 511.44±10.55 a ngbtun540 76.94±0.60 ef 210.98±7.33 d 13.17±2.38 h 301.09±10.31 d ngbtun541 122.72±3.21 b 203.29±13.91 d 22.81±8.29 f 348.82±18.99 c ngbtun556 121.72±0.86 b 168.73±5.8 e 5.17±0.84 i 295.62±7.5 d ngbtun558 71.36±0.1 g 74.99±0.39 hi 13.91±2.61 gh 160.26±3.11 h ngbtun560 72.11±0.19 g 71.74±0.44 i 11.89±1.69 h 155.74±2.32 h ngbtun567 115.38±3.78 c 201.31±2.06 d 35.79±1.76 d 352.49±4.08 c ngbtun572 159.34±4.99 a 292.09±17.21 b 18.77±3.67 fg 470.21±25.88 b p value <0.0001 <0.0001 <0.0001 <0.0001 f value 514.05 595.30 129.76 337.71 1data are expressed as the mean and standard deviation. a-idifferent letters in the same column indicate significant differences for p<0.05. 3.5. correlation analysis pearson correlation coefficients (r) were calculated to determine the relationships among the studied parameters. a total of 61 features were correlated at the 0.05 or 0.01 significance level (fig. 1). total carotenoids were significantly and positively correlated with β-carotene (r=0.96) and the redness value a* (r=0.91), but were negatively correlated with the hue angle h⁰ (r=-0.90) and the lightness parameter l* (r=-0.70). the hue angle h⁰ was positively and significantly correlated with the lightness parameter l* (r=0.71), but negatively correlated with the redness value a* (r=-0.98), β-carotene (r=-0.90) and galactose (r=-0.71). significant and positive correlations were also observed between βcarotene and the redness value a* (r=0.93); the yellowness value b* and the chroma c* (r=0.91). the total sugars showed a significant and positive correlation with fructose and glucose (r=0.79 and r=0.73, respectively) whereas glucose had a significant and negative correlation with galactose (r=-0.80). ital. j. food sci., vol. 32, 2020 662 table 3. content of individual and total sugars, expressed as mg/g of dry weight, in 14 tunisian carrot landraces1. landrace sucrose glucose fructose galactose total sugars ngbtun512 85.93±0.31 d 125.19±8.64 ef 200.15±36.94 bc 5.99±1.33 d 417.27±26.66 def ngbtun514 89.57±4.49 d 137.28±1.11de 157.35±18.44 cd 4.88±0.56 e 388.90±13.40 ef ngbtun520 73.73±1.28 e 173.28±2.18 c 183.04±13.93 bc 1.43±0.08 h 431.49±17.48 cde ngbtun522 60.27±1.58 f 133.57±2.18 e 170.38±6.9 bc 4.54±0.18 e 368.77±10.85 f ngbtun523 46.20±3.18 g 188.18±13.96 b 194.43±4.93 bc 1.13±0.10 h 429.95±12.31 cde ngbtun527 137.7±14.29 a 124.02±1.22 ef 119.07±9.00 d 8.04±0.51 c 388.84±18.39 ef ngbtun539 123.95±1.27 b 118.18±5.19 f 156.79±20.15 cd 10.61±0.10 b 409.54±23.98 def ngbtun540 120.66±3.30 b 171.17±1.02 c 252.03±75.64 a 2.92±0.09 g 546.79±73.27 a ngbtun541 85.33±4.15 d 147.07±11.16 d 190.80±17.55 bc 3.47±0.72 fg 426.67±2.96 cde ngbtun556 100.22±8.05 c 96.90±5.84 g 159.55±14.61 cd 14.98±1.47 a 371.65±27.03 f ngbtun558 68.37±3.23 ef 245.47±5.84 a 219.52±17.68 ab 0.00 i 533.36±26.75 a ngbtun560 64.91±0.63 ef 240.00±0.37 a 193.28±22.68 bc 0.00 i 498.19±22.42 ab ngbtun567 121.13±2.25 b 171.63±5.02 c 171.80±18.42 bc 7.47±0.25 c 472.04±15.90 bc ngbtun572 50.49±2.19 g 188.56±15.47 b 206.12±11.71 abc 4.18±0.04 ef 449.35±29.41 bcd p value <0.0001 <0.0001 0.0005 <0.0001 <0.0001 f value 101.53 106.69 4.35 145.43 12.33 1data are expressed as the mean and standard deviation. a-idifferent letters in the same column indicate significant differences for p<0.05. table 4. contents of total phenols and total flavonoids, expressed as mg of gallic acid equivalents/100 g of dry weight and µg of catechin equivalents/100 g of dry weight, respectively, in 14 tunisian carrot landraces1. landrace total phenols total flavonoids ngbtun512 33.29±0.24 cd 20.95±2.19 c ngbtun514 32.38±0.98 d 23.06±0.34 b ngbtun520 41.39±0.65 a 24.57±0.35 a ngbtun522 33.10±0.26 cd 20.56±0.78 cd ngbtun523 27.19±0.64 gh 17.56±0.01fg ngbtun527 24.13±0.34 i 19.36±1.39 cde ngbtun539 36.70±1.55 b 16.51±0.08 g ngbtun540 27.01±0.17 gh 20.79±0.26 c ngbtun541 28.43±0.37 f 19.53±1.55 cde ngbtun556 33.98±0.17 c 22.91±0.30 b ngbtun558 29.70±0.17 e 19.06±0.34 def ngbtun560 24.60±0.83 i 18.41±0.88 ef ngbtun567 26.36±0.30 h 19.64±0.20 cde ngbtun572 28.07±0.08 fg 24.85±0.04 a p value <0.0001 <0.0001 f value 184.39 23.86 1data are expressed as the mean and standard deviation. a-idifferent letters in the same column indicate significant differences for p<0.05. ital. j. food sci., vol. 32, 2020 663 figure 1. pearson correlation coefficients among biochemical and colour parameters of 14 tunisian carrot landraces. the intensity of the pink and blue colours indicates the significance of the correlation existing between each pair of studied parameters. the more the colour turns towards dark pink, the more significant is the positive correlation. the more the colour turns toward dark blue, the more significant is the negative correlation. 3.6. multivariate analysis a principal component analysis (pca) was conducted to determine the differences in the studied parameters among tunisian carrot landraces. this analysis generated 10 axes with distinct percentage contributions to the total variance (fig. 2). the contribution of each parameter to the first five principal components is shown in fig. 3. the first three principal components accounted for 76.1% of the total variance. the first axis explained 42.8% and was associated with the hue angle h⁰, galactose, the redness value a* and total carotenoids. the second axis explained 18.2% and was associated with total sugars, the chroma c* and fructose. the third axis represented 15.1% of the total variance and was correlated with total phenols. the pca scatter plot defined by the two principal components (fig. 4) separated the carrot landraces into four distinct groups. the first group (g1) included ngbtun539, 541, 567 and 572, originated from kairouan, sfax, slimane and siliana, respectively, and having similar contents of α-carotene, β-carotene, total carotenoids and sucrose and similar redness values a*, which were positively ital. j. food sci., vol. 32, 2020 664 correlated among themselves (fig. 1). ngbtun540, cultivated in sfax, diverged from all the other landraces and formed the second group (g2) due to its high contents of total sugars and fructose (table 3). the third group (g3) was formed by ngbtun527 and 556, from sidi-bouzid and gabes, respectively, which had higher contents of galactose than the other landraces (table 3). ngbtun512, 514, 520, 522 and 523, from monastir (moknine, teboulba), and ngbtun558 and 560, from nabeul (manzel-temime), formed the fourth group (g4). these landraces are characterized by a yellow colour, related to the presence of lutein. figure 2. decomposition of the total variation among the components of the pca based on biochemical parameters of tunisian carrot landraces. hierarchical clustering (ahc) was also performed and permitted the grouping of landraces on the basis of similarities (fig. 5). total carotenoids, β-carotene, sucrose and αcarotene were correlated and allowed the grouping of ngbtun527, 539, 541, 556, 567 and 572 into cluster i. these landraces have similar contents of these compounds. total sugars and glucose were strongly correlated, as were fructose and the lightness parameter l*. landraces ngbtun512, 514, 520, 522, 523, 540, 558 and 560 with similar values for these parameters were grouped together into cluster ii. this grouping is in accordance with the pca results. ital. j. food sci., vol. 32, 2020 665 figure 3. contribution of biochemical parameters to the variability on the first five components of the pca. the most significant correlations are indicated by a big, dark-blue circle. figure 4. scatter plot grouping of 14 tunisian carrot landraces based on the first two principal components of the pca. ital. j. food sci., vol. 32, 2020 666 figure 5. dendrogram obtained from the cluster analysis of 14 tunisian carrot landraces. the intensity and variation of colour (from dark red to blue) indicate the correlation existing between the parameters that allowed the discrimination of landraces. 4. discussion in the present study, colour parameters and components of the biochemical composition such as carotenoids, sugars and phenolic compounds were identified in various carrot landraces in order to determine their genetic variability. the results prove that there is a large degree of variation among these landraces with respect to their biochemical composition and its relationship with root colour. food colour is a very important quality attribute influencing the consumer’s choice and preferences (melendez-martinez et al., 2005). it is widely used, together with measurements of pigments contents and flavor, because of its correlation with biochemical properties in vegetables. it is also used to assess food quality in post-harvest and processing conditions (pathare, 2013). our correlation results highlight the association between colour and carotenoids accumulation. thus, the redness value a* was highly correlated with β-carotene, α-carotene and total carotenoids, which give the orange colour to carrot landraces. the yellowness value b* was correlated with lutein and the hue angle h⁰, which had greater values for yellow carrots than for orange ones. the high correlation between the hue angle h⁰ and the lightness parameter l* was accompanied by carotenoids accumulation. the purity of carrot colour for all the landraces was indicated by the high chroma value c*. these results confirm that carotenoids contents can be determined by using a chroma meter (ruiz et al., 2005). the variation of colour in carrot is due mainly to the genotype, development of the plant, temperature and fertilization (bajaj et al., 1980). the most suitable temperature for a better colour, accompanied by the highest content of carotenoids, is considered to be around 16 ºc (banga et al., 1955). the correlation results ital. j. food sci., vol. 32, 2020 667 are consistent with those of a previous study on pumpkin and squash conducted by itle and kabelka (2009), who reported a positive correlation between a* and total carotenoids (r=0.91) and β-carotene (r=0.77); and a negative correlation between total carotenoids and h⁰ (r=-0.83) and l* (r=-0.66), and between h⁰ and β-carotene (r=-0.69). according to reeves (1987), the best parameter to predict the total carotenoid content in pepper is a negative correlation with l*. unlike previous studies (alasalvar et al., 2001), we noted significant correlations between sugars and colour parameters. sucrose was positively correlated with colour parameters, while glucose and fructose were negatively correlated with them. this could be explained by the fact that fructose and glucose accumulation is inversely proportional to sucrose accumulation (suojala, 2000; sekoli et al., 2016). korolev et al. (2000 a, b) confirmed that the fructose and glucose contents increased in carrot root until 50 days after germination, while sucrose increased beyond this date until harvest (90 days). similar to our results, correlations (positive or negative) between individual and total sugars contents have been reported in watermelon genotypes (yoo et al., 2012). glucose and fructose were negatively correlated with the total sugars, while sucrose had a positive correlation. these results show that each sugar contributes to the total sugars content to a different degree and that an increased sucrose content is accompanied by decreased fructose and glucose contents. in the current work, carotenoids were quantified due to their contribution to carrot colour and the general quality of the roots. the type and amount of carotenoids differ among distinct parts of the plant (saini et al., 2015). in carrot, the cortex (flesh) is richer in carotenoids than the core (karabacak and karabacak, 2019). our experiment showed that the carotenoids profile was constituted mainly by β-carotene, α-carotene and lutein, whose contents varied distinctly among the carrot landraces, β-carotene and αcarotene being more highly abundant than lutein in orange coloured carrot . these findings are in conformity with previous studies showing that α-carotene and βcarotene are the major carotenoids in orange carrots, unlike in yellow carrots (nicolle et al., 2004; surles et al. 2004; baranski et al., 2010; jourdan et al., 2015). carrots with orange skin colour have been shown to possess higher amounts of total carotenoids than yellow carrots (alasalvar et al., 2001; grassmann, 2007; sun et al., 2009; singh et al., 2018). these new results indicate that tunisian landraces are richer in carotenoids than ‘’nante’’ hybrids: namely, nante/berlikum (60.21 mg/100 g); nante/maestro (76.47 mg/100 g); nante/forto (72.45 mg/100 g); nante/bolero (72.93 mg/100 g) and nante/champion (79.47 mg/100 g) (rakcejeva et al., 2012). carotenoids biosynthesis depends on the cultivar (nicolle et al., 2004) and is related to genetic factors (rosenfeld et al., 1997). also, these compounds can be affected by the season (horvitz et al., 2004), environmental conditions (simon, 2000), plant maturity (phan and hsu, 1973) and soil (hart and scott, 1995). in fact, high temperatures and dry weather promote an increase in carotenoids contents (fikselová et al. 2010). carotenoids have been found to be more abundant in carrots grown in clay soil than in carrots grown in sandy soil (martín-diana et al., 2007; rico et al., 2007). cultivation practices can affect the content of biochemical compounds. carotenoids accumulation was stimulated by fertilization based on npk (smolen and sady, 2009), while it was inhibited by regular irrigation (fikselová et al., 2010). it has been noted that the post-harvest and storage conditions affect the carotenoids concentration (saini et al., 2015). a temperature of 2-3 °c and 90% relative humidity reduced the carotenoids content by an average of 11% (matějková and petříková, 2010). hence, lyophilization is the most adequate method for better preservation of the nutritional quality of vegetables (saini et al., 2014). the carotenoids and provitamin a ital. j. food sci., vol. 32, 2020 668 contents of carrots are important contributors to human health and the variability of carotenoids (determined using reliable hplc methodology) could be considered as a parameter of selection in breeding programs (simpson, 1983). sugars were also quantified due to their contribution to carrot taste and sweetness. like carotenoids, the sugars content varies among the different parts of the carrot root. for example, total sugars are higher in the crown section (at the shoulders) than in the tip section (sekoli et al., 2016). significant differences were observed in individual sugars as well as total sugars. fructose and glucose were more abundant than sucrose and galactose, the latter being detected in tunisian carrot landraces in lower amounts. galactose has not been determined in similar research related to carrots and other crops. the results seem to be in direct contrast to the findings of dolores et al. (1999), alasalvar et al. (2001) and simon (1985), who showed sucrose to be the major soluble sugar, followed by glucose and fructose. the sugar content in plants, which contributes to sweetness, is controlled by the genotype and the environment (simon et al., 1980.a, b; 1982; 1985). it is a polygenic control with a heritability estimated at 0.45 (baranski et al., 2012). the variation in sugars has been the subject of many studies, but sometimes the results are contradictory. suojala et al. (2000) found low sugar levels in warm seasons, whereas nilsson (1987), hogstad et al. (1997) and rosenfeld et al. (1998) noted the highest sugar levels at high temperatures. fertilization can affect the variation in sugars. in this context, sekoli et al. (2016) found that the sucrose content increased proportionally with the increase in fertilization. according to melo et al. (2006), carrot belongs to the group of vegetables characterized by low phenolic contents since its mean content is <100 mg catechin equivalents/100 g of fresh weight. in our study, the phenolic compounds varied greatly among the carrot landraces. nicolle et al. (2004) found sizable differences in the content of total phenols among different carrot varieties (white, yellow, orange, dark-orange and purple), the values ranging between 4.3 and 4.4 mg gae/g of dry weight for yellow carrot and between 3.3 and 6.0 mg of gae/g of dry weight for orange carrots. however, in our study the carrots showed a lower content of total phenols, varying from 24.6 to 41.4 mg gae/100 g of dry weight for yellow carrots and from 24.1 to 36.7 mg gae/100 g of dry weight for orange carrots. consequently, tunisian carrot landraces appear to have lower contents of total phenols compared with other data of the scientific literature. this difference could be explained by the influence of the genetic factors, environmental conditions and variety (bravo, 1998). in addition, the phenolic compounds concentration depends on the fertilization method (smolen and sady, 2007). for example, the provision of nitrogen permits high concentrations of phenolic compounds (smolen and sady, 2009). also, rozek et al. (2000) noted that the content of phenolic compounds are influenced more by the soil (light, medium or heavy texture) and climatic conditions than by fertilization. the flavonoids are one of the most important groups of phenolic compounds since they exhibit important biochemical and pharmacological effects. indeed, they contribute to the protection against reactive oxygen species (ros) and the inhibition of platelet aggregation. they also have anti-inflammation, anti-atherosclerotic, antitumor, antimicrobial and antiallergic effects (koley et al., 2014). quercetin, luteolin, kaempferol and myricetin are the main types of flavonoids found in carrot (bahorun et al., 2004). however, the information related to the flavonoids content in carrot genotypes is limited. the low values of flavonoids in tunisian carrot landraces are explained by the absence of anthocyanins, which are the most abundant type of flavonoids in the root and are responsible for purple and black colours (singh et al., 2018). different authors (leja et ital. j. food sci., vol. 32, 2020 669 al., 2013; koley et al., 2014; singh et al., 2018) have reported lower amounts of flavonoids in orange and yellow carrots in comparison to purple, rainbow and black carrots. principal component analysis (pca) and clustering (ahc) were performed to classify landraces on the basis of their similarities. information obtained by the pca can help breeders to distinguish between highly differentiated landraces to be used for plant breeding programs. 5. conclusion the contents of sugars and phenolic compounds in the roots varied significantly among 14 tunisian carrot landraces due to agronomic and genetic factors. the colour, an important organoleptic characteristic in carrot, was significantly correlated with the content of carotenoids, oranges landraces having higher contents of α-carotene and β-carotene, whereas yellow landraces had more lutein. considering the high variability of the biochemical parameters and taking 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beres et al., 2017; garcía-lomillo et al., 2017.). possible uses set by the european regulation no. 555/2008 (eu, 2008) consider the exploitation of winery by-products for energy production, animal feeding, soil fertilization or ingredients recovery to be included in pharmaceutical, cosmetic and food processes. in particular, the latter aspect has gained much attention in recent years as many applications have been reported dealing with the enhancement of bioactive molecules from grape pomace for functional food design (galanakis, 2012; yu and ahmedna, 2013; restuccia et al., 2019). polyphenols from winery wastes represent one of the most attractive phytochemicals due to their health-promoting properties including effect on metabolic syndrome (mets) (xia et al., 2010; yu and ahmedna, 2013; xia et al., 2014; teixeira et al., 2014; jara-palacios et al., 2015; chiva-blanch and badimon, 2017). phenolics are usually used for fortification of many food products, including fish and seafood, meat, juices as well as bakery and dairy products (fernàndez-marìn et al., 2013; fontana et al., 2013; beres et al., 2017; garcía-lomillo et al., 2017). mets is characterized by several medical conditions including high blood glucose levels, high serum triglycerides, low value of high-density lipoprotein (hdl), and high blood pressure. mets is associated with several diseases including cardiovascular diseases, heart failure, stroke, hyperuricemia, fatty liver, polycystic ovarian syndrome, erectile dysfunction, and diabetes (mendrick et al., 2018). frequently, patients with mets are overweight or obese. in these patients a progressive reduction in insulin secretion coupled with a progressive rise in insulin resistance are present. moreover, a persistent blood hyperglycaemia determined, at cytosolic and mitochondrial level, an over production of reactive oxygen species (ros) (kaneto et al., 2010). to this regard, a topic of great interest is represented by the diet supplementation with functional foods. in fact, different studies indicated their positive effect on the body weight reduction; their action seemed to be related to the inhibition of the digestive enzymes interfering with the hydrolysis and absorption of lipids and carbohydrates (i.e. α-amylase, α-glucosidase and pancreatic lipase) (tundis et al., 2018; loizzo et al., 2019). recently, akaberi and hosseinzadeh (2016) proposed the use of grapes for mets syndrome treatment. it is well know that grapes are rich in proanthocyanidins that have been used for diverse dedications such as antioxidants, nutritional supplements, preventing atherosclerosis, managing cardiovascular complications, to treat dyslipidaemia, other than as free radical scavenging and lipid lowering. furthermore, several scientific studies and applications, confirmed the absence of toxicity of the tested grape pomace extracts (bladé et al., 2016; costabile et al., 2019; carullo et al., 2019). in this context, the goal of this research was focused on the realization of a new milk kefir, obtained by the addition of a sangiovese pomace extract during the kefir preparation. the supplementation was accomplished in order to improve the antioxidant profile and the impact on metabolism. for this purpose, the grape skin and seeds extracts, obtained by two different procedures, infusion (a classical method) and ultrasound-assisted method (innovative and time-saving), were chemically investigated by hplc analyses and the antioxidant activity was also monitored. skin extract obtained by ultrasound-assisted extraction (uae) resulted the most interesting extract and it was added to kefir grains and skimmed milk. successively, the enriched product was tested for its antioxidant and ital. j. food sci., vol. 32, 2020 369 hypoglycaemic features by in vitro colorimetric assays, α-amylase and α-glucosidase inhibition tests. the hypolipidemic effects by pancreatic lipase inhibition were also assessed. 2. materials and methods 2.1. materials and chemicals sangiovese pomace was kindly given by le moire farm (catanzaro, italy) during september 2019. cell culture and cell culture materials were obtained from sigma-aldrich chemical co. ltd (milan, italy). all chemicals and reagents used in this study were purchased from sigma-aldrich chemical co. ltd (milan, italy) and vwr international (milan, italy). 2.2. preparation of the grape extracts skins and milled seeds (30 g), obtained by manually separation from sangiovese pomace, were added of 200 ml ethanol/water mixture (50:50 v/v) acidified at ph=2 with hcl 37% (v/v) (carrera et al., 2012; gonçalves et al., 2017). the mixture was then subjected to ultrasound assisted extraction (uae) at 30°c for 15 minutes (10 cycles/sec), at an ultrasonic frequency of 40 khz using the ultrasound-bath branson model 3800-cpxh (milan, italy). alternatively, skins and seeds were immersed in a solution ethanol/water (80:20 v/v) and heated at 60°c for 60 minutes. the two procedures were repeated three times. the mixtures were then filtered out and concentrated under reduced pressure using a rotary evaporator buchi rii®. ration extraction between grape sample and solvent mixture was 1:6. all the extracts were stored at -18°c until analyses. the obtained samples were labelled as a (skin extract obtained after uae); b (skin extract obtained after infusion); c (seed extract obtained after uae) and d (seed extract obtained after infusion). 2.3. determination of d-(+)-glucose, d-(−)-fructose and organic acids the sugars level in sangiovese skin and seed extracts was performed using a knauer high liquid chromatography system (asi advanced scientific instruments, berlin, germany) equipped with a knauer hplc pump k-120 (asi advanced scientific instruments, berlin, germany), a rheodyne injection valve with loop of 20 μl and a smartiline ri detector 2300. elution was obtained on a varian meta carb h plus column (300 mm× 7.8 i.d., 5 μm). the column temperature was 55°c and the flow rate was 0.25 ml/min. the mobile phase consisted of 0.01 n h2so4 solution. the hplc analyses of organic acids were performed on a knauer (asi advanced scientific instruments, berlin, germany) system equipped with two pumps smartiline pump 1000, a rheodyne injection valve (20 μl) and a photodiode array detector uv/vis equipped with a semi-microcell. separation was obtained using an acclaim oa column (250 mm×4.0 i.d., 5 μm) at t = 30°c. the mobile phase consisted of 100 mm na2so4 (ph =2.65 with methane sulfonic acid) and the flow rate was 0.6 ml /min. stock solutions of each standard, in different diluted concentration ranging from 0.2-2 g/l, were prepared in ultra-pure water provided by a milli-q system (millipore co., bedford, ma). all solutions were filtered through 0.45 μm glass-microfiber gmf whatman chromatographic filter (hawp millipore co., bedford), before analysis. ital. j. food sci., vol. 32, 2020 370 2.4. phenolic characterization of sangiovese skin and seed extracts high performance liquid chromatography coupled to a diode array detector (hplc-dad) was applied to determine the phenolic profile of sangiovese grape skin and seed extract (kammerer et al., 2004). the analysis was performed on a knauer system (asi advanced scientific instruments, berlin, germany) equipped with two smartiline pump 1000 pumps, a rheodyne injection valve (20 µl) and a uv vis photodiode series detector equipped with a semi-microcell. compounds were separated according the procedure previously described by loizzo et al. (2019) and monitored at 280, 254, 330 and 305 nm. compounds identification and quantification were carried out by comparing the spectra and relative retention times of sangiovese extracts peaks with those obtained by injecting pure standards as selected marker (gallic acid, (+)-catechin and (-)-epicatechin, catechin, caffeic acid, rutin, trans-resveratrol, and quercetin). 2.5. measurement of the total phenolic content (tpc) and total anthocyanins (ta) the total phenolic content (tpc) was detected in the phenolic extracts of grape seeds and skins and kefir or fortified kefir, according to the folin-ciocalteu colorimetric method of restuccia et al. (2011). briefly, a 6.0 ml volume of each properly diluted sample was added with 1.0 ml of folin-ciocalteu reagent and after 3 min with 3.0 ml of 5.0% w/v na2co3. positive control and blank solutions was also prepared by substituting the sample with the same volume of 0.1 (w/v) ascorbic acid and hydro alcoholic solution (50:50 v/v), respectively. after shaking for 2 h, the absorbance value of each mixture was measured at 760 with a jasco v-530 uv/vis spectrometer (jasco, tokyo, japan). the tpc was expressed as mg of gallic acid equivalents (gae) per g of extract for samples a-d and mg gae per l for kefir (m2) and fortified kefir (m2a10). the total anthocyanins (ta) was determined using the differential ph method (loizzo et al., 2019). anthocyanins undergo a reversible modification of the structure with a change in the ph that occurs with a variation in the absorbance spectrum. seven and half mg of extract were added to 5 ml of distilled water. for each sample two dilutions were prepared, one with a 0.025 m hydrochloric acid buffer solution at ph 1, and the other with a 0.4 m sodium acetate buffer solution at ph 4.5, corrected with hydrochloric acid. the solutions were left to equilibrate for 15 min. spectrophotometric reading was per-formed at 510 nm and 700 nm. the results were expressed as cyanidine-3-o-glucoside equivalent (cge) per g of extract. 2.6. determination of total antioxidant capacity (tac) a literature protocol, with few changes was employed to determine total antioxidant capacity (tac) of each extract, kefir or fortified kefir (cirillo et al., 2012). briefly, 0.3 ml of hydro alcoholic solution (50:50 v/v) of sample were added to 1.2 ml of reagent solution (0.6 m h2so4, 28.0 m na3po4, and 4.0 m (nh4)2moo4). the reaction mixture was incubated at 95°c for 150 min and after cooling to room temperature, the absorbance of the mixture was measured at 695 nm. positive control and blank solutions was also prepared by substituting the sample with the same volume of 0.1 (w/v) ascorbic acid and hydro alcoholic solution (50:50 v/v), respectively. the total antioxidant activity of each extract was expressed as mg of catechin equivalent (cte) per g of extract for samples a-d and mg cte per l for kefir (m2) and fortified kefir (m2a10). ital. j. food sci., vol. 32, 2020 371 2.7. determination of scavenging activity on dpph radicals free radical scavenging properties of the extracts were estimated towards dpph (2,2diphenyl-1-picrylhydrazyl)acid)) radical (spizzirri et al., 2011). briefly, in a volumetric flask (10 ml) were placed 1.0 ml of hydro alcoholic solution (50:50 v/v) of each extract, kefir or fortified kefir, 4.0 ml of hydroalcoholic solution (50:50 v/v) and 5.0 ml of ethanol solution of dpph (200 μm), obtaining a solution of dpph with a final concentration of 100 μm. the solution was incubated at 25°c and, after 24 h, the absorbance of the remaining dpph was determined at 517 nm. ascorbic acid 0.1 (w/v) was used as positive control. the scavenging activity of the tested matrices was measured as the decrease in absorbance of the dpph and it was expressed as ic50, defined as the concentration of sample that causes a decrease in the initial dpph concentration by 50%. 2.8. determination of scavenging effect on the abts radical cation free radical scavenging properties of the extracts were estimated in aqueous media towards abts (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) radical (restuccia et al., 2017) abts radical cation (abts.+) solution (7.0 mm) remains in the dark at room temperature for 12-16 h, and then the concentration was adjusted to an absorbance of 0.70±0.02 at 734 nm. in a general procedure, the scavenging effect of the samples was evaluated by adding 500 µl of hydroalcoholic solution (50:50 v/v) on each extract, kefir or fortified kefir at 2.0 ml of the abts.+ radical solution. the mixture was incubated in a water bath at 37°c and protected from light. the decrease of absorbance at 734 nm was measured at the endpoint of 5 min. ascorbic acid 0.1 (w/v) was used as positive control. the antioxidant activity was measured as the decrease in absorbance of the abts and it was expressed as ic50, defined as the concentration of sample that causes a decrease in the initial abts concentration by 50%. 2.9. kefir preparation and enrichment three mg of fresh kefir grains (kefiralia, burumart commerce s.l, spain) were added in a glass flask at room temperature, containing 3 ml, of six type of commercially available milks. the container not-hermetically closed, was posed at 20-25°c for 24 hours with or without the addition of the extract. the six types of milk, purchased in a local market, were labeled ultra high temperature (uht) whole milk (m1), skimmed milk (m2) and partially skimmed milk (m3), pasteurized fresh whole milk (m4), skimmed milk (m5), and partially skimmed milk (m6). the kefir grains obtained by each milk type were finally weighted and the ph value measured by using the ph 211 microprocessor ph meter (hanna instruments italia, milan, italy). the enriched kefir was obtained mixing 1.0, 5.0 and 10.0 mg of the a extract with 10.0 ml of m2 in the same experimental conditions in which ph was measured. 2.10. carbohydrate hydrolysing enzymes inhibition assay the carbohydrate-hydrolyzing enzymes inhibition was detected following the procedure previously described by leporini et al., (2020). α-amylase (0.0253 g in 100 ml of cold water) was mixed with starch solution (0.125 g of potato starch in 25 ml of 20 mm sodium phosphate buffer and 6.7 mm sodium chloride. the mixture was stirred for 15 min at 65°c. sangiovese pomace samples, freeze-dried kefir and enriched kefir at concentrations ital. j. food sci., vol. 32, 2020 372 ranging from 25 to 1000 µg/ml were added to starch solution and left to react with enzyme at 25°c for 5 min. the absorbance was read at 540 nm. in the α-glucosidase assay a maltose solution (12 g of maltose in 300 ml of 50 mm sodium acetate buffer) was mixed with α-glucosidase (1 mg of enzyme in 10 ml of ice cold distilled water) and o-dianisidine (dian) (loizzo et al., 2019). the peroxidase/glucose oxidase (pgo) system-colour reagent solution was obtained by dissolving one capsule in 100 ml of ice-cold distilled water. samples (pomace extracts, freeze-dried kefir and freeze-dried enriched kefir) at concentrations ranging from 25 to 1000 µg/ml were added to 250 µl maltose solution and 5 µl enzyme and left to incubate at 37°c for 30 min. then, perchloric acid was added and the mixture was centrifuged. the supernatant was collected and mixed with dian and pgo, and left to incubate at 37°c for 30 min. the absorbance was read at 500 nm. acarbose was used as positive control in both assays. the ic50 value for each sample, defined as the concentration of sample causing 50% enzyme inhibition was determined from the curves plotted and tabulated. 2.11. pancreatic lipase inhibitory activity pancreatic lipase inhibitory activity was determined by 96-well plate method based on the procedure proposed by el-shiekh et al. (2019). 4-nitrophenyl octanoate (npc), 5 mm in dimethylsulfoxide solution and an aqueous solution of porcine pancreatic lipase enzyme (1 mg/ml), and tris‐hcl buffer (ph 8.5) were prepared. pomace extracts, freeze-dried kefir and freeze-dried enriched kefir at concentrations ranging from 2.5 to 40 mg/ml were added in a well with 6 μl of the enzyme, 6 μl of npc and 279 μl of buffer. the mixture was incubated at 37°c for 30 min. the absorbance was measured at 405 nm. orlistat was used as a positive control. the ic50 value for each sample, defined as the concentration of sample causing 50% enzyme inhibition, was determined from the curves plotted and tabulated. 2.12. statistical analysis all of the data obtained from three replicates (n=3) were presented as mean±standard deviation (sd) were subjected to one-way analysis of variance (anova) using prism graphpad prism version 4.0 for windows, graphpad software (san diego, ca, usa). after that, the tukey’s test was performed to compare all means. differences among means with p<0.05 were accepted as representing statistically significant. dunnett’s test was used to compare each mean of biological data to a positive control mean. differences among means with ****p<0.0001; ***p<0.001; **p<0.01 were accepted as representing statistically significant. 3. results and discussion 3.1. chemical composition of sangiovese pomace skin and seeds extracts grape sugar and acid were monitored by hplc-ri. glucose was particularly abundant in sample a followed by b. differently, fructose was more present in skin extract obtained after infusion (sample b) (13.98 g/100g) (table 1). tartaric acid was the most abundant acid in all samples except seed extract obtained after infusion (sample d). ital. j. food sci., vol. 32, 2020 373 table 1. sugar and acids in sangiovese skins and seeds extracts. glucose (g /100 g) fructose (g/100 g) tartaric acid (g/l) malic acid (g/l) lactic acid (g/l) acetic acid (g/l) fumaric acid (g/l) a 13.79±0.11a 6.01±0.11b 7.81±0.10a 4.86±0.04a 1.82±0.05a 2.68±0.04d 0.005±0.004a b 13.14±0.12b 13.98±0.20a 4.64±0.09c 1.67±0.03d 0.09±0.03d 3.25±0.01b 0.006±0.002a c 0.71±0.07d 0.32±0.07d 6.71±0.14b 2.61±0.04b 0.80±0.04b 4.51±0.04a 0.003±0.001a d 1.17±0.08c 1.19±0.24c 2.74±0.11d 1.78±0.06c 0.46±0.02c 3.05±0.04c 0.003±0.003b a (skin extract obtained after uae); b (skin extract obtained after infusion); c (seed extract obtained after uae) and d (seed extract obtained after infusion). data are expressed as mean ± standard deviation (sd) (n= 3). means within each column and with different lowercase letters are statistically different according to tukey’s test (p<0.05). vitis vinifera genotypes, environmental factors, postharvest treatments and applied extraction procedure for recovery of polyphenolic compounds influenced their amount in the grapes extracts (spigno and de faveri, 2007; bucić-kojić et al., 2009). grape skins and seeds tpc was evaluated by folin-ciocalteu procedure, an analytical methodology based on the electrons transferring from phenolic compounds to the folinciocalteu reagent in an alkaline medium. the tpc values of grape skins and seeds appear strictly related both to raw material (seeds or skins) and extraction procedure (infusion or uae), as reported in the table 2. the tpc in the grape extracts examined in this study ranged from 130.65 to 259.26 mg gae per g of extract. generally, for both raw materials, uae procedure appeared more useful than the infusion technique and higher amounts of tpc were detected in the skins (259.26 mg gae per g of extract, for a), compared with the seed extracts (207.55 mg gae per g of extract, for c). the ta content ranged from 39.31 to 10.24 cge per g of extract for skin extract obtained after uae and seed extract obtained after infusion, respectively. our data are in the same order of magnitude of those reported by mendoza et al. (2012). from hplc analysis gallic acid, (+)-catechin and (-)-epicatechin, catechin, caffeic acid, rutin, trans-resveratrol, and quercetin were identified (table 2). different levels of phenolic compounds could be detected from seed and skin. ital. j. food sci., vol. 32, 2020 374 table 2. tpc and phenolic profile of sangiovese skins and seeds extracts. tpc (mg gae per g extract) ta (cge per g of extract) gallic acid (mg/l) (+)-catechin (mg/l) caffeic acid (mg/l) (-)-epicatechin (mg/l) rutin (mg/l) trans resveratrol (mg/l) quercetin (mg/l) a 259.26±2.12a 39.31±2.89a 2.18±1.04b 147.0±1.15a 4.27±0.07b 76.0±1.15d 13.12±0.14a 21.44±0.18a 247.0±1.39a b 171.48±2.71c 22.48±2.77b 1.64±0.05bc 105.0±1.90c 3.48±0.06c 126.0±1.02c 9.11±0.07ab 14.66±0.11b 210.0±1.30b c 207.55±3.22b 21.65±2.56b 104.0±1.08c 124.0±1.17b 5.87±0.07a 364.0±1.17a 2.17±0.09b n.d. 3.35±1.37c d 130.65±1.10d 10.24±1.13c 88.20±1.16a 122.0±1.12b 2.43±0.06d 232.0±1.12b 2.41±0.05b n.d 1.66±1.24c a (skin extract obtained after uae); b (skin extract obtained after infusion); c (seed extract obtained after uae) and d (seed extract obtained after infusion). n.d.: not detected. data are expressed as mean ± standard deviation (sd) (n= 3). means within each column and with different lowercase letters are statistically different according to tukey’s test (p<0.05). ital. j. food sci., vol. 32, 2020 375 generally, (+)-catechin and (-)-epicatechin represent the main abundant compounds with values in the range105.0-147.0 and 76-364 mg/l, respectively. the applied extraction procedure, influenced a lot the amount of gallic acid. in fact, both c and d are characterized by the highest amount with values of 104.0 and 88.20 mg/l, respectively. a similar consideration could be done for trans-resveratrol and quercetin that are concentrated only in b and a samples (values of 14.66 and 21.44 mg/l, and 210.0 and 247.0 mg/l, respectively). according to our data, both catechin and epicatechin were the main abundant compounds of seeds from v. vinifera cv. frankovka as reported by bucić-kojić et al., 2009. significant amount of gallic acid were also found by doshi et al., (2015) that evaluated the phenolic profile of pusa navarang and merlot seeds and skin. in particular, pusa navarang seed extract showed high amounts of catechin and epicatechin whereas quercetin was abundant in its skin extract. according to our data, transresveratrol was detected only in skin with values of 34.5 and 37.5 mg/l for pusa navarang and merlot varieties, respectively. these values are twofold higher than that found in sangiovese samples. according to godevac et al., (2010) the phenolic composition of grape seed extracts from v. vinifera cv., prokupac, smederevka, italian riesling, traminer, black burgundy, gamay noir, muscat hamburg and gamay bojadiser, showed the flavan-3-ol monomers as the main abundant compounds especially in seeds derived from red wine grapes. regarding rutin, our data showed that this flavonoid glycoside was more concentrated in skin than seeds. however, gengaihi et al., (2014) evidenced a main content of rutin in both red romy and grenache noir seeds. quercetin-3-o-glucoside (39.86 μg/g dry sample), rutin (37.01 μg/g dry sample) and trans-resveratrol (32.88 μg/g dry sample) were the most abundant compounds identified in v. tiliifolia skin (jiménez et al., 2018). the results recorded in the tpc determination are partially in agreement with the concentration of polyphenols recorded by hplc-dad. however, in this comparison it is necessary to consider the contribute of the single class of molecules to tpc value and the contribution of others compounds, such as pigments like anthocyanin, present at high concentration in the grape skins (babbar et al., 2011). furthermore, it should be pointed out that some non-phenolic components also having reducing capacity, such as organic sugars and acids, affect the tpc value, leading to overestimated total phenolic contents. finally, phenolic compounds differently react with the folin-ciocalteu reagent and several flavonoid molecules produce poor responses, leading to an underestimated tpc value (cirillo et al., 2016). 3.2. evaluation of the antioxidant properties of sangiovese skin and seed extracts the results concerning the antioxidant activity expressed as total antioxidant capacity (tac) and radical scavenging activity in aqueous and organic environments appear quite related to tpc values. specifically, tac values, as reported in fig. 1, highlighted as sample a returned the most significant result (326.54 mg cte per g extract), emphasizing the existence of a positive relationship between tpc and tac. some recorded dissimilarities can be explained invoking structural differences in the phenolic molecules that affected antioxidant power of the extracts. literature data indicate that the presence of others interfering molecules, often prevent a linear correlation between tpe and antioxidant activity (fernando et al., 2018). the ability of the extracts as scavenger of lipophilic dpph radical was expressed in terms of ic50 and reported in table 3. ital. j. food sci., vol. 32, 2020 376 figure 1. tac values of sangiovese pomace samples. a (skin extract obtained after uae); b (skin extract obtained after infusion); c (seed extract obtained after uae) and d (seed extract obtained after infusion). a comparison of ic50 values shows that skins extracts (7.6 and 12.0 µg/ml for a and b, respectively) returned 2-times lower values compared to the seeds extracts. a comparison of this data with the hplc-dad analyses displayed as the solubility of recorded polyphenol plays an important role in the lipophilic scavenger activity. in particular, the high concentration of quercetin recorded in the skins extracts (both uae and infusion extraction methodologies) justified the better performances recorded for a and b. moreover, uae process appears in both cases the better technique leading to lower ic50 values. the recorded data showed quite correspondence with the results of tac and tpe. this trend is evident for both the samples and some discrepancies could be justified by considering the different environment (organic and aqueous) in which the assays were performed. the scavenging capacity of the extracts in the aqueous environment against the abts radical was expressed in terms of ic50 value, and table 3 displayed the recorded data. table 3. radical scavenging activity of extracts from skins and seeds of sangiovese cv grape. a (skin extract obtained after uae); b (skin extract obtained after infusion); c (seed extract obtained after uae) and d (seed extract obtained after infusion). data are expressed as mean ± s.d. (n = 3). differences within and between groups were evaluated by one-way analysis of variance test followed by a multicomparison dunnett’s test compared with the positive control (****p<0.0001; ** p<0.01). the analysis of the ic50 values exhibited comparable values for both matrices and the extraction methodologies did not deeply affect the result. however, the scavenging sample dpph ic50 (µg/ml) abts ic50 (µg/ml) a 7.6±0.7** 38.5±0.5**** b 12.0±0.3**** 42.0±1.2**** c 14.8±0.6**** 42.2±2.1**** d 24.5±0.5**** 43.5±1.1**** positive control ascorbic acid 5.0±0.8 1.7±0.3 ital. j. food sci., vol. 32, 2020 377 activity recorded in the aqueous environment appeared more than three times lower compared with the organic one. lack of data concerning extracts antioxidant capacity of the investigated cultivars made very hard to compare the collected values with the literature analyses. in addition, many factors, such as fruit ripening, weather conditions, soil and place of growth, largely affect the distribution of the antioxidants in the vegetable matrix, further complicating whatever qualitative-quantitative comparison (tang et al., 2018). the juxtaposition of these data with the hplc-dad analyses underlined also that the solubility of the recorded polyphenol plays an important role in the lipophilic scavenger activity. in particular, the high concentration of quercetin recorded in the skins extracts (uae or infusion extraction method), justified the better performances recorded for sample b and a. 3.3. kefir enrichment as already stated, six types of milk have been considered for kefir preparation to underline the effect of different raw materials on kefir production (i.e grains growth and ph decrease). as summarized in table 4, after 24 hours, all the considered samples produced a positive variation of the kefir grains mass as well as a decrease in ph values. considering the obtained data (mass and ph) in the same experimental conditions, we decided to enrich only the skimmed milk (m2), which showed the lower kefir grains mass variation, in order to evaluate the improved effect of extract addition. noteworthy, skimmed milk is mostly requested by female population and health fanatics, due to the poor fats content. comparison between m2, m5 and m6, in terms of mass variation and ph decrease, supported the choice to use the former to obtain the functional beverage by adding 1, 5, 10 mg of extract a to 10 ml of m2, before grains addition (dos santos et al., 2017). addition levels have been carefully selected to avoid possible antimicrobial effects exerted by wine polyphenols on kefir microorganisms (garcía-lomillo et al., 2014; katalinić et al., 2010). the highest addition level, in fact corresponded to 0.1% w/w or to a supplementation equivalent to 25.9 mg gae/100g of kefir. table 4. milk type screening. sample mass (g) mass variation ph ph decrease t (h) = 0 t (h) = 24 t (h) = 0 t (h) =24 m1 0.30±0.01 0.37±0.02b 0.07±0.01c 6.98±0.02 4.71±0.01d 2.27±0.01c m2 0.29±0.01 0.31±0.01c 0.02±0.01d 6.99±0.01 4.42±0.01f 2.57±0.01a m3 0.29±0.02 0.44±0.01a 0.15±0.02a 7.01±0.02 5.54±0.02a 1.47±0.02e m4 0.30±0.02 0.44±0.02a 0.14±0.02a 7.02±0.01 4.76±0.02c 2.26±0.01c m5 0.29±0.01 0.39±0.01b 0.10±0.01b 6.97±0.02 4.98±0.01b 1.99±0.01d m6 0.31±0.02 0.41±0.02b 0.10±0.02bc 7.00±0.01 4.65±0.01e 2.36±0.01b data are expressed as mean ± standard deviation (sd) (n= 3). means within each column and with different lowercase letters are statistically different according to tukey’s test (p<0.05). as we recently reported (carullo et al., 2020) for the same kind of samples, the applied fortification level ensured proper activity of lab and yeasts, at the same time, improving antioxidant features and avoiding possible drawbacks. metabolomics analyses of the kefir ital. j. food sci., vol. 32, 2020 378 extracts, in fact, revealed the same compounds produced during fermentation with and without addition, demonstrating that metabolic pathways of lab and yeasts were not influenced by the presence of the wine pomace extracts, at least under the qualitative point of view (carullo et al., 2020). under the technological point of view, tseng and zhao (2013) found that the addition of grape pomace flour to milk at levels higher than 3%, produced an excessive syneresis of yogurt while at levels higher than 5% the coagulation was inhibited. considering sensory features, grape extract at 1% improved the acceptability of yogurt (karaaslan et al., 2011) and only reaching fortification with skin wine pomace at 6% induced decrease in the liking score, especially for the taste and flavor (marchiani et al., 2016). all considered, the supplementation levels employed in this research are far below those generally found in literature and regarded as producing adverse effects. the kefir grains (i.e. m2a1, m2a5 and m2a10) were weighted and their ph was measured after fortification and after 24 h of activity. as reported in table 5, the kefir grains presented different weights, in particular m2a10 resulted the heaviest, although ph values resulted similar among the three kefirs. for this reason, considering that a similar ph value agrees with a similar chemical composition, we decided to investigate only the heaviest kefir (m2a10) for their properties in metabolic syndrome (carullo et al., 2020; settanni et al., 2019). by the comparison between unfortified kefir (m2) with m2a1, m2a5 and m2a10 showed a lower ph decrease probably in relation with high sugars and organic concentration found in the wine pomace extract. more sugars in the environment mean more lactic acid in kefir, thus producing the strong ph decrease. at the same time, wine organic acids can be metabolized and/or partially remain after fermentation. in the latter case, they obviously contribute to the ph decrease. a significant increase in tpc concentrations was observed in m2a10 that showed a tpc of 393.32 mg gae per l in comparison to unfortified kefir (m2) that showed a tpc of 274.74 mg gae per l, as a consequence of the addition of the extract to the unfermented milk before the kefir production. in general, this value appears in accordance with literature data confirming that during the fermentation process, irregular changes in the total phenol content of kefir samples can be observed, due to the biotransformation of phenolic compounds (karaaslan et al., 2011; najgebauer-lejko and sady, 2015; perna et al., 2019; ozcan et al., 2019). table 5. screening of the enriched kefir samples. sample mass (g) mass variation ph ph decrease t (h) = 0 t (h)=24 t (h) = 0 t (h)=24 m2a1 0.29±0.01 0.34±0.01b 0.05±0.01b 7.00±0.01 2.90±0.01c 4.10±0.01a m2a5 0.30±0.01 0.37±0.01b 0.07±0.01b 6.99±0.01 3.00±0.01a 3.99±0.01b m2a10 0.30±0.02 0.59±0.01a 0.29±0.02a 7.01±0.01 2.95±0.02b 4.06±0.02a data are expressed as mean ± standard deviation (sd) (n= 3). means within each column and with different lowercase letters are statistically different according to tukey’s test (p<0.05). ital. j. food sci., vol. 32, 2020 379 3.4. evaluation of the functional properties of kefir fortified with uae-derived sangiovese skin extract polyphenol compounds influence the composition of microbiota by the inhibition of pathogen growth and the stimulation of the commensal bacteria growth and these changes appear deeply related to their chemical structure and concentration (cueva et al., 2010). the action of fermentative enzyme, such as β-glycosidase, produces the hydrolysis of complex phenolic compounds to simpler types producing a significant increase of the total phenolic content (coda et al., 2012). a further increase of the antioxidant activity can be obtained by milk supplementation, before the fermentation process, with a source of bioactive molecules. these compounds can interact with the milk proteins determining changes in the microbiological quality and oxidative stability of the dairy products. the results of antioxidant activity (tac and dpph free radical scavenging assays) showed that enriched kefir had higher antioxidant activity than unfortified kefir (fig. 2 and table 6). table 6. radical scavenging activity of kefir from skins and seeds of sangiovese cv grape. kefir sample dpph ic50 (µg/ml) abts ic50 (µg/ml) m2 107.0± 2.9**** 498.3±8.2**** m2a10 73.4±1.7**** 318.7±7.4**** positive control ascorbic acid 5.0±0.8 1.7±0.3 data are expressed as mean ± sd (n = 3). differences within and between groups were evaluated by oneway analysis of variance test followed by a multicomparison dunnett’s test compared with the positive control (****p<0.0001). figure 2. tac values of kefir (m2) and fortified kefir (m2a10). data clearly showed significant increase of tac value (+47.7% in the fortified kefir respect to the unfortified one), while ic50 value against dpph radical displayed a value that is 14.7% lower compared to control kefir. ital. j. food sci., vol. 32, 2020 380 obtained results highlighted that the fortified kefir displayed improved antioxidant performances, compared to the unfortified sample. 3.5. effect of kefir and its fortified derived products on enzymes linked to mets both kefir and its derived fortified product are able to inhibit the enzymes linked to metabolic syndrome in a concentrationdependent manner. generally, α-amylase resulted more sensible to the action of our investigated samples and the obtained ic50 values are quite similar than that found with the positive control acarbose (table 7). table 7. effect of fortified kefir on enzyme linked to mets. sample α-amylase ic50 (µg/ml) α-glucosidase ic50 (µg/ml) lipase ic50 (µg/ml) m2 71.73±2.5** 86.18±2.8*** 79.15±2.6*** m2a10 69.39±2.2** 84.47±2.7*** 73.01±2.5*** positive control acarbose 50.0±0.9 35.5±1.2 orlistat 37.5±0.92 data are expressed as means ± sd (n= 3). acarbose used as positive control in α-amylase and α-glucosidase tests. orlistat used as positive control in pancreatic lipase test. differences within and between groups were evaluated by one-way anova followed by a multicomparison dunnett’s test compared with the positive control (***p<0.001, **p<0.01). fortified kefir (m2a10) showed an ic50 value of 69.39 mg/ml. value of 84.47 mg/ml was found against α-glucosidase. moreover, m2a10 exerted a promising activity against lipase enzyme (ic50 value of 73.01 mg/ml). previously moreno et al., (2003) demonstrated that grape seed extract (gse) was able to inhibit fat-metabolizing enzyme pancreatic lipase. more recently, hassan et al., (2014) reported the ability of water and ethanol grape seed extracts (wgse and egse) against some pancreatic enzymes including α-amylase and lipase. analysis of data revealed that egse had higher α-amylase inhibitory activity in comparison with wgse. moreover, daily administration of kefir (3.6 ml/200 g) in alloxan-induced diabetes mellitus rats for 20 days, showed a significant reduction in blood glucose, total cholesterol, triglycerides, as well as in low density lipoprotein (ldl) and very low density lipoprotein (vldl) whereas hdl was effectively increased (ghazi et al., 2018). the positive effect of kefir on mets was confirmed, also, by a randomized double-blind placebo-controlled clinical trial on diabetic patients treated with kefir containing lactobacillus casei (600 ml/day) for 8 weeks (ostadrahimi et al., 2015). 4. conclusions the present study assessed the total phenolic and anthocyanin content, hplc-dad phenolic profile, antioxidant activity, α-amylase, α-glucosidase, and lipase inhibitory activities of kefir and sangiovese pomace fortified kefir. between the two extractive methods, uae showed the better performances in the recovery of the bioactive ital. j. food sci., vol. 32, 2020 381 compounds. moreover, uae skin extract (a) characterized by the highest tpc and ta content was selected to be added to skimmed milk (m2) and kefir grains. the amount of sample a added to kefir grains did not inhibit the growth of microorganisms as revealed by the increase weight of fortified kefir grains after 24 hours. fortified kefir showed a better antioxidant activity than unfortified one as evidenced by the comparison of data obtained in all performed assays (tac, dpph, and abts). despite the new beverage demonstrated a good performance in the inhibition of key enzymes linked to metabolic syndrome (α-amylase, α-glucosidase and lipase) this activity is not significantly different from unfortified kefir. in conclusion, our data showed a chemical characterization of winery wastes (sangiovese skins and seeds) also proposing a useful extraction of bioactive molecules to be used as functional ingredients for fermented milk fortification. acknowledgements authors wish to thanks le moire farm (catanzaro, italy) for grape samples supplying. abbreviations abts 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) bht butylated hydroxytoluene cge cyanidine-3-o-glucoside equivalent cte catechin equivalent dian o-dianisidine dm2 diabetes mellitus type 2 dpph 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doi.org/10.3390/ijms11020622 yu j. and ahmedna, m. 2013. functional components of grape pomace: their composition, biological properties and potential applications. int. j. food sci. technol. 48:221-237. doi: doi.org/10.1111/j.1365-2621.2012.03197.x paper received december 20, 2019 accepted february 17, 2020 ijfs#1242_bozza ital. j. food sci., vol. 30, 2018 740 paper composition and thermal properties of quaternary mixtures of palm oil:palm stearin:soybean oil:cocoa butter j.m.n. marikkar*a, n.a.m. yantyb, m. paciullic, m.s. miskandard and e. chiavaro**c afood chemistry laboratory, national institute of fundamental studies, hantana road, kandy, sri lanka bhalal products research institute, universiti putra malaysia, 43400, serdang, selangor, malaysia cdepartment of food and drug, parco area delle scienze 47/a, university of parma, 43124 parma, italy dmalaysian palm oil board, p.o. box 12301, 50774 kuala lumpur, malaysia *,**corresponding author: tel.: +94 812232002; fax: +94 812232131 tel.: +39 0521-905888; fax: +39 0521-906028 e-mail addresses: nazrim@ifs.ac.lk; emma.chiavaro@unipr.it abstract pam oil (po) is a semi-solid substance with potential functional lipid characteristics. a study was carried out to evaluate the effect of addition of soybean oil (sbo), palm stearin (ps) and cocoa butter (cb) on the solidification behavior of po to formulate a mixture to become similar to lard (ld). a total of three mixtures were prepared: po:ps:sbo:cb (38:5:52:5), po:ps:sbo:cb (36:5:54:5) po:ps:sbo:cb (34:5:56:5) (w/w), and identified by the mass ratio of po to ps, cb and sbo. the fat mixtures were compared with lard in terms of the fatty acid and triacylglycerol compositions using gas chromatography and high performance liquid chromatography, thermal properties using differential scanning calorimetry (dsc) and solid fat content (sfc) using p-nuclear magnetic resonance (pnmr). although there were considerable differences between lard and the fat mixtures with regard to fatty acid and triacylglycerol compositions, some similarities were seen on their dsc thermal properties and solid fat content profile. of the fat mixtures, po:ps:sbo:cb (38:5:52:5) displayed closer similarity to lard by having least difference to sfc profile throughout the temperature range and a common dsc thermal transition at around -3.59°c. keywords: palm oil, dsc, lard substitute, cocoa butter, soybean oil, thermal analysis ital. j. food sci., vol. 30, 2018 741 1. introduction animal fats have traditionally been used as medium of frying in many types of foods. they were used in food applications mainly due to economic reasons since voluminous amounts of animal fats are discarded by the industry. flavor imparted on foods by animal fats is an important reason for animal fat use. for instance, in some food cultures, vegetable oils used for frying are blended with small amounts of lard to impart characteristic flavors. although the use of animal fats such as lard, tallow, etc. is already popular, the bad effects of the consumption are also being studied since the consumer perception begins to be negative with regard to the use of animal fat. as a result, there has been a great deal of interest among researchers to investigate various plant-based substitutes as alternative for lard (marikkar and noorziyanna yanty, 2018; hsu and yu, 2002). when exploring these plant-fats, comparing their composition and thermal properties with those of ld was an important aspect of the investigations. for example, floter (2009) highlighted the importance of studying physical properties data in product development. separately, given (1990) also emphasized the influence of fat and oil physicochemical properties on the expression of functionality in baked goods. the special properties of ld are large due to its peculiar nature of tag composition. as reported previously by silva et al. (2009), lpo, opo and spo were the predominating tag molecular species of ld with oleic, palmitic and stearic acids occurring in higher proportions. owing to this reason, ld alternative fats were formulated using plant fat mixtures having these tag molecular species. accordingly, a replacement for ld was investigated using binary fat mixtures of mee fat and palm stearin as well as binary fat mixtures composed of engkabang fat and canola oil (yanty, 2016; nur illiyin et al., 2013). in a separate experiment, yanty (2016) found that ternary blends of avocado (avo) fat, ps and cb also satisfied this requirement. as a notable feature, these fat mixtures displayed solidification behavior closely similar to that of ld. for our knowledge, the compatibility of quaternary fat mixtures comprising palm oil (po), ps, soybean (sbo) and cocoa butter (cb) has not been considered for the said purpose. according to previous studies, po contained approximately 40% oleic, 10% linoleic, 45% palmitic and 5% stearic acids (tan and che man, 2000). as solidification values of po was always higher than that of ld throughout the temperature region, addition of liquid oils such as sbo would be necessary. sbo has very higher amount of oleic and lineic acids, and hence the blending would tend to affect the proportion of palmitic and stearic acid contents. owing to this reason, inclusion of small amounts of ps and cb would be needed to maintain the required proportions of palmitic and stearic acids in the final mixture. in this study, three quaternary plant-based fat blends (po, sbo, ps and cb) were formulated to make comparison to lard with respect to their composition, dsc thermal properties, and solidification profiles. 2. materials and methods 2.1. materials ld was extracted using three batches of adipose tissues of swine collected from local slaughter houses as described in previous reports. samples of po and ps were obtained as generous gift from malaysian palm oil board (mpob). cb and sbo were purchased from malaysian cocoa board and a local supermarket, respectively. all chemicals used in this experiment were of analytical or hplc grade. ital. j. food sci., vol. 30, 2018 742 2.2. preparation of quaternary mixtures the fat samples were melted at 70°c for 1 h before mixing. a total of three fat mixtures were prepared: po:ps:sbo:cb (38:5:52:5), po:ps:sbo:cb (36:5:54:5) po:ps:sbo:cb (34:5:56:5) (w/w), and identified by the mass ratio of po to ps, cb and sbo. all samples were kept under frozen storage at –20°c. prior to analyses, the fat mixtures were removed from frozen storage, and then left static at room temperature for 1 h before being warmed at 70° c until they became completely molten. 2.3. determination of smp and iv smp and iv of the fat samples were determined according to aocs method cc.3.25, and aocs method cd id–92, respectively (aocs, 1999). 2.4. determination of fa composition fatty acid methyl esters were prepared by dissolving 50 mg portion of oil in 0.8 ml of hexane and adding 0.2 ml portion of 1 m solution of sodium methoxide (porim, 1995). the top hexane layer was injected on an agilent 6890n gas chromatograph (agillent technologies, singapore) equipped with a polar capillary column rtx-5 (0.32 mm internal diameter, 30 m length, and 0.25 μm film thickness; restex corp., bellefonte, pa) and a flame ionization detector (fid). split injection was conducted with a split ratio of 58:1, nitrogen was used as a carrier gas at a flow-rate of 1.00 ml/min. the temperature of the column was 50°c (for 1 min), and programmed to increase to 200°c at 8°c/min. the temperatures of the injector and detector were maintained at 200°c. the identification of the peaks of the samples was done with reference to a chromatographic profile containing a set of fatty acid methyl ester standards. the percentage of fatty acid was calculated as the ratio of the partial area to the total area (nur illiyin et al., 2013). 2.5. determination of tag composition the tag compositions of samples were determined according to the method described by yanty (2016) using waters model 510 liquid chromatography equipped with a differential refractometer model 410 as the detector (waters associates, milford, ma). the analysis of tag was performed on a merck lichrosphere rp-18 column (5 μm; 12.5 cm × 4 mm i.d.; merck, darmstadt, germany), which was maintained at 30°. the mobile phase was a mixture of acetone:acetonitrile (63.5:36.5) and the flow rate was 1.5 ml/min. the injector volume was 10 μl of 5% (w/w) oil in chloroform. each sample was chromatographed three times, and the data were reported as peak area percentages. the identification of the peaks of the samples was done using a set of tag standards purchased from sigma-aldrich (deisehofen, germany) as well as the tag profiles of lard (nur illiyin et al., 2013), po (tan and che man, 2000), ps (tan and che man, 2000), cb (segall et al., 2005) and sbo (tan and che man, 2000) reported previously. 2.6. thermal analysis by dsc thermal analysis was carried out on a mettler toledo differential scanning calorimeter (dsc 823 model) equipped with a thermal analysis data station (stare software, version 9.0x, schwerzenbach, switzerland). nitrogen (99.999% purity) was used as the purge gas at a rate of ~20 ml/min. approximately, 4-8 mg of melted sample was placed in a standard dsc aluminum pan and then hermetically sealed. an empty, hermetically sealed ital. j. food sci., vol. 30, 2018 743 dsc aluminum pan was used as the reference. the oil/fat samples were subjected to the following temperature program: 70°c isotherm for 1 min, cooled at 5°c/min to -70°c. the samples were held at -70°c isotherm for 1 min, and heated at 5°c/min to reach 70°c (yanty, 2016) 2.7. determination of sfc sfc was measured according to aocs method cd 16b-93 (aocs, 1999) using a bruker minispec (model mq 20) pulse nuclear magnetic resonance (pnmr) spectrometer (karlsruhe, germany). the sample in the nmr tube was melted at 70°c for 15 min, followed by chilling at 0°c for 60 min, and then held at each measuring temperature for 30 min prior to measurement. melting, chilling, and holding of the samples were carried out in pre-equilibrated thermostatic glycol containing baths, accurate to 0.1°c. sfc measurements were taken at 5°c intervals over the range of 0-70°c. 2.8. statistical analysis all analyses were carried out in triplicate and the results were expressed as mean value ± standard deviation. data were statistically analyzed by one-way analysis of variance (anova), by using tukey’s test of minitab (version 15) statistical package at 0.05 probability level. 3. results and discussion 3.1. fa composition, smp, and iv fa composition of po, ps, sbo, cb, all three quaternary mixtures and ld were presented in table 1. values showed the great variability of the samples, which might influence melting point, iodine values and the shape of dsc thermal curves. according to table 1, the most dominant fa of po was palmitic (43.99%), followed by oleic (39.24%), and linoleic (10.25%) acids. ps, the hard stearin of po contained palmitic (78.9%) as the most dominant fatty acid followed by oleic (10.6%), and stearic (6.32%) acids. in the meantime, cb was found to possess stearic (37.84%) as the predominant fa followed by oleic (32.83%) and palmitic (25.34%). sbo, on the other hand, contained linoleic acid (53.93%) as the most predominant fa, followed by oleic (23.87%), palmitic (11.33%) and linolenic (6.46%) acids. these values were found to be comparable to those reported previously by tan and che man (2000). they found that sbo contained linoleic (53.3%) as the most predominant fa, followed by oleic (23.6%), palmitic (12.6%) and linolenic (6.3%) acids. in fact, additions of ps, sbo and cb into po caused significant (p<0.05) increments in the proportions of stearic, linoleic, and linolenic acids with concurrent decreases in the amounts of palmitic and oleic acids. among these fas, linoleic acid was found to be increased considerably while palmitic and oleic acids were found to decreased. the increment in the proportion of linoleic acid (30.78 to 34.44) of the quaternary mixtures was due to the presence of higher proportion of linoleic acid in sbo. this was far higher than the proportion of linoleic in ld or any other animal fats. however, the proportions of palmitic and oleic in the three mixtures were somewhat lower than the corresponding amounts found in ld. when compared to ld, the palmitic and linoleic acid contents of the quaternary mixtures were significantly (p<0.05) higher while stearic and oleic acid contents were significantly (p<0.05) lower. with the decreasing amount of po in the mixtures, the total sfa content was tended to decrease (from 36.76 to 34.40 %) while the ital. j. food sci., vol. 30, 2018 744 total usfa content was found to increase (from 63.24 to 65.60 %). among the mixture, total usfa (~63%) and sfa (~37%) contents of the po:ps:sbo:cb (38:5:52:5) were found to be roughly similarly to the total usfa (~61%) and sfa (~39%) contents of ld. according to table 1, the smp of po, ps, cb and ld were 30.5, 59.5, 34.25 and 27.5°c, respectively. this measurement was not done for soybean oil because this method does not apply to this type of oil. additions of ps and cb into po were found to cause significant (p<0.05) increases in smp values (from 38.0 to 41.25°c) when compared to that of original po. this could be due to the fact that cb and ps were crystalline solid fats and had comparably higher proportions of palmitic and stearic acids (table 1). with regard to the degree of unsaturation, the iv of po, ps, sbo and cb were 54, 14, 136.85 and 34, respectively. the iv of quaternary mixtures of po:ps:sbo:cb were found to be in the range of 92.26 to 96.95. all fat mixtures of this study displayed significantly higher (p<0.05) iv than either po (54.00) or ld (73.76). based on these results, none of the quaternary mixtures of this study was found to have a smp and iv closely similar to those of ld. the changes in smp and iv of these mixtures as noted before (table 1) could be mainly due to the changes in fa compositions. 3.2. tag composition the tag compositions of po, ps, sbo, cb, quaternary mixtures of po:ps:sbo:cb were compared to that of ld as shown in table 2. po composed of ppo (31.61%), poo (24.76%), ppl (10.19%) and pol (9.96%) as dominant tag molecules. these values were comparably similar to the ranges reported previously (marikkar and ghazali, 2011). on the other hand, ps contained ppp (68.66%), ppo (15.23%), and stop (11.07%) as major tag molecules. the major tag molecules of cb were sop (40.78%), sos (29.35%) and ppo (18.08%). sbo, on the other hand was found to possess lll (23.56%), oll (17.77%), pll (15.82%) and pol (13.69%) as the major tag molecules. after addition of ps, sbo and cb into po, some of the tag molecules were found to increase (e.g. ppl, ool, pol, ppp and sos) while others were tended to decrease (e.g. mmm, mpl, ppl, ooo, poo, ppo, soo, spo and pps). the increments in the proportions of pll, ool and pol could be due to the presence of sbo in the mixture. there were significant increases in the proportions of ppp, and sos as these were major tag species of ps (nor aini and miskandar, 2007) and cb (liu et al., 2007; segall et al., 2005), respectively. tag molecular species namely, llnln, llln, olnln, lll, plln, oll, poln and sss were also found in the mixtures after addition of ps, sbo and cb into po. among the tag molecular species, ppo (ranging from 13.48 to 12.09%) and poo (ranging from 10.87 to 9.70%) were found to reduce dramatically in the mixtures when compared to those of original po (31.61 and 24.76%, respectively). in addition, uuu and ststst tag molecules were found to increase with respect to the original sample of po. for instance, the uuu tag was increased (34.89 to 37.14%) when compared to that of the original sample of po (12.68%). the ststst tag molecules were also found to decrease slightly (24.86 to 22.47%). in the meantime, the amount of ppo in the mixtures was found to be somewhat similar to those of ld. when compared to ld, the amount of uuu and ststst tag of the po:ps:sbo:cb mixtures were found to be higher with concurrent decreases of uust and ustst tag molecules. the increasing proportions of uuu tags in the fat mixtures could have led to the occurrence of greater amounts of oleic and linoleic acids as shown in the overall fa distribution (table 1). among them, ustst tag content of po:ps:sbo:cb (38:5:52:5) was found to be closely (24.86%) comparable to that of ld (26.60%). ital. j. food sci., vol. 30, 2018 745 table 1. basic physico-chemical characteristics and fatty acid composition (%) of po, ps, sbo, cb, quaternary mixtures of po:ps:sbo:cb and ld. po ps sbo cb po:ps:sbo:cb (38:5:52:5) po:ps:sbo:cb (36:5:54:5) po:ps:sbo:cb (34:5:56:5) ld smp 30.50±0.71e 59.50±0.71a nd 34.25±0.35d 41.25±0.35b 39.25±0.35b,c 38.00±0.71c 27.50±0.71f iv 54.00±0.00f 14.00±0.01h 136.85±0.21a 34.00±1.41g 92.26±0.74d 94.36±0.08c 96.95±0.10b 73.76±0.34e fa c12:0 0.33±0.01a 0.20±0.14a,b n.d n.d 0.11±0.01b 0.10±0.01a,b 0.10±0.01b 0.09±0.01b c14:0 1.10±0.00c 1.65±0.07a n.d n.d 0.54±0.01d 0.40±0.01e 0.27±0.02f 1.24±0.01a c16:0 43.99±0.20b 78.90±0.28a 11.33±0.13e 25.34±0.04c 27.88±0.01c 26.69±0.07c 25.72±0.03c 22.68±0.48d c16:1 0.18±0.01b n.d n.d n.d 0.11±0.00b,c 0.08±0.01c,d 0.04±0.01d 1.42±0.05a c18:0 4.36±0.06e 6.32±0.17d 4.42±0.01e 37.84±0.14a 7.83±0.04c 7.86±0.02c 7.87±0.01c 12.70±0.28b c18:1 39.24±0.20 10.90±0.00 23.87±0.00e 32.83±0.26 29.39±0.01b 28.45±0.07c 27.56±0.01d 38.24±0.13a c18:2 10.25±0.06 1.58±0.60 53.93±0.02a 2.92±0.06 30.78±0.05d 32.90±0.10c 34.44±0.09b 20.39±0.04e c18:3 0.19±0.01 n.d 6.46±0.10a n.d 2.96±0.02d 3.28±0.01b 3.56±0.02b 0.98±0.01e c20:0 0.36±0.01c 0.46±0.08c n.d 1.07±0.01a 0.43±0.00c 0.47±0.01c 0.48±0.01c 0.67±0.01b others n.d n.d n.d n.d n.d n.d n.d 1.59 each value in the table represents the mean of three determinations. means within each row bearing different superscripts are significantly (p<0.05) different. abbreviations: po, palm oil: ps, palm stearin: sbo, soybean oil: cb, cocoa butter; ld, lard; fa, fatty acid; smp, slip melting point; iv, iodine value; n.d, not detected. ital. j. food sci., vol. 30, 2018 746 table 2. tag composition of po, ps, sbo, cb, quaternary mixtures of po:ps:sbo:cb and ld. tag po ps sbo cb po:ps:sbo:cb (38:5:52:5) po:ps:sbo:cb (36:5:54:5) po:ps:sbo:cb (34:5:56:5) ld llnln n.d n.d 1.31±0.03a n.d 0.70±0.01b 0.70±0.00b 0.74±0.01b n.d llln n.d n.d 7.66±0.01a n.d 3.93±0.01c 4.10±0.01b,c 4.29±0.01b 1.54±0.21d olnln n.d n.d 0.02±0.00a n.d 0.01±0.00b 0.01±0.00b 0.01±0.00b n.d lll n.d n.d 23.56±0.03a n.d 12.23±0.00d 12.72±0.01c 13.15±0.01b 0.68±0.21e plln n.d n.d 3.64±0.01a n.d 1.90±0.01d 1.97±0.00c 2.05±0.01b n.d oll n.d n.d 17.77±0.01a n.d 9.28±0.01d 9.61±0.00c 9.98±0.01b 4.68±0.08e mmm 0.21±0.01a n.d n.d n.d 0.16±0.01b 0.17±0.00b 0.17±0.01b n.d pll 2.08±0.03f n.d 15.82±0.01a 0.27±0.00g 8.23±0.04d 8.56±0.03c 8.84±0.01b 7.05±0.06e mpl 0.54±0.01a n.d n.d n.d 0.19±0.01b 0.18±0.00b,c 0.17±0.00c n.d poln n.d n.d 0.13±0.01a n.d 0.06±0.00b 0.07±0.01b 0.08±0.01b n.d ool 1.62±0.02d n.d 8.72±0.23a n.d 5.58±0.00c 5.73±0.01c 5.85±0.01c 6.93±0.04b pol 9.96±0.01e 0.32±0.03g 13.69±0.02b 0.85±0.01f 11.31±0.02d 11.86±0.01c 12.15±0.11c 20.00±0.27a ppl 10.19±0.01a 1.03±0.10h 2.25±0.01f 1.55±0.00g 5.02±0.01b 4.73±0.01c 4.52±0.01d 2.62±0.04e ooo 3.97±0.02b 0.13±0.01f 2.02±0.13d 0.69±0.01e 3.14±0.00c 3.13±0.01c 3.12±0.01c 4.33±0.21a poo 24.76±0.01a 2.22±0.02g 1.11±0.02e 2.27±0.02f 10.87±0.04c 10.28±0.07d 9.70±0.04e 20.67±0.11b ppo 31.61±0.01a 15.23±0.03c 0.56±0.03h 18.08±0.01b 13.48±0.20d 12.73±0.05e 12.09±0.02f 10.63±0.01g ppp 4.77±0.03c 68.66±0.13a n.d 0.26±0.01f 4.90±0.04b 4.75±0.02c 4.46±0.01d 0.38±0.00e soo 2.72±0.02c 0.26±0.06g 1.23±0.00f 2.98±0.00b 1.85±0.01e 1.92±0.01e 2.10±0.01d 3.62±0.04a spo 5.65±0.01d 11.07±0.01c 0.54±0.02g 40.78±0.10a 4.58±0.02e 4.35±0.01f 4.15±0.01f 12.52±0.12b pps 0.92±0.01a 0.68±0.04c n.d 0.41±0.01d 0.86±0.01a,b 0.84±0.00a,b 0.82±0.00b 0.81±0.00b sos 0.52±0.01e n.d n.d 29.35±0.01a 1.59±0.01b 1.57±0.01b,c 1.54±0.00c 0.83±0.01d sss n.d 0.41±0.01a n.d 0.40±0.04a 0.02±0.00c 0.02±0.00c 0.02±0.00c 1.31±0.01b others 0.48±0.01 n.d n.d 2.11±0.14 n.d n.d n.d 1.41±0.33 uuu 12.68 0.13 61.06 0.69 34.89 36.00 37.14 18.16 uust 40.06 2.80 35.59 6.37 34.22 34.66 34.92 51.34 ustst 47.97 27.33 3.35 89.76 24.86 23.56 22.47 26.60 ststst 5.90 69.75 n.d 0.66 5.94 5.78 5.47 2.50 each value in the table represents the mean of two determinations. means within each row bearing different superscripts are significantly (p<0.05) different. abbreviations: tag, triacglycerol; po, palm oil; ps, palm stearin; sbo, soybean oil; cb, cocoa butter; ld, lard; o, oleic; p, palmitic; l, linoleic; ln, linolenic; st, stearic; u, unsaturated; s, saturated; n.d, not determined. ital. j. food sci., vol. 30, 2018 747 3.3. thermal characteristics the cooling behaviors of po, ps, sbo, cb, po:ps:sbo:cb mixtures and ld were compared in fig. 1a. the cooling profile of ld (curve a) is characterized by two widely separated transitions: a high (a1, a2) and low (a3) temperature regions. this was roughly similar to the findings reported previously (marikkar and yanty, 2014). according to fig. 1a, the cooling profile of po (curve e) had four cooling transitions; one major sharp peak at 18.8°c (e1) and one broader peak at 5.0°c (e2), with a shoulder peak at -4.2°c (e3) which was in accordance with the previous findings (tan and che man, 2000). in addition to these, a minor peak was also appeared in the lower-temperature region at around -42°c (e4). the cooling thermograms of ps (curve f) and cb (curve h) had one major sharp peak at around 42.8 (f1) and 13.4°c (h2), respectively. in the case of cb, additional small peak (f4) was also found at 30.7°c. as sbo was a liquid oil, its dsc curve (curve g) had three cooling transition at lowtemperature region (below 0°c); the first peak was found at -9.2°c (g1), a broader peak at 37.7°c (g2) and a small peak at -64.5°c (g3). these agreed with the results reported in other studies (tan and che man, 2000). additions of ps, sbo and cb into po brought considerable changes to cooling profiles of the three po:ps:sbo:cb mixtures. only two thermal transitions were displayed by the mixtures; a major sharp peak at around 21°c and a minor peak at around -1°c. with respect to original po curve, the peak-maxima of the thermal transitions of quaternary mixtures were also found to have shifted slightly. these changes in the profiles of mixtures could be attributed to the changing sfa to usfa ratio as noted previously in tables 1 and 2. this has been in accordance with the findings reported by others (nur illyin et al., 2013). figure 1a. dsc cooling thermograms of ld (a), quaternary mixtures of po:ps:sbo:cb (b=38:5:52:5; c= 36:5:54:5; d=34:5:56:5), po (e), ps (f), sbo (g) and cb (h). abbreviations: ld, lard; po, palm oil; ps, palm stearin; sbo, soybean oil; cb, cocoa butter. ital. j. food sci., vol. 30, 2018 748 on the other hand, the major and minor sharp peaks of ld were found at 10.3 and -18.0°c, respectively. the high-melting cooling transitions of po:ps:sbo:cb mixtures were found to be little higher (22.3°c) when compared to that of ld (18°c). these results suggested that none of the quaternary mixtures of po:ps:sbo:cb had thermal transitions exactly matching with the cooling thermogram of ld. however, the peak corresponding to cb 13.4°c (h2) showed a closer similarity to the high melting transition of ld (10.3°c). the melting behaviors of po, ps, sbo, cb, po:ps:sbo:cb mixtures and ld were depicted in fig. 1b. the melting profile of ld (curve a) has five endothermic transitions, which could be classified into two distinct regions namely, low-melting region below 0°c (a1, a2) and highmelting region above 0°c (a3, a4, a5). the native po sample (curve e) had seven endothermic transitions; two major endothermic regions, corresponding to low-melting fraction known as olein and high-melting fraction known as stearin. these were largely confirmatory with the findings reported previously. the high-melting region (above 10°c) consisted of a plateau with a pair of shoulder peaks (c6 and c7), while the low-melting region (below 10°c) contained five overlapping peaks (c1, c2, c3, c4 and c5). ps (curve f) and cb (curve h), on the other hand, had one major sharp peak at 59.0 (f1) and 20.4°c (h2). in addition, cb had one shoulder peak at 14.8°c (h1). in the meantime, sbo (curve g) had four endothermic transitions at lowtemperature region; the profile was comparably similar to that reported previously by tan and che man (2000). this could be due to the fact that sbo was a liquid oil that contained a high amount of usfa (table 1) and uuu tag molecules (table 2). the major peak was found at -27.7 (g2)°c with two shoulder peaks at -20.1 (g3) and -6.5°c (g4). the minor peak was found at -38.2°c (g1). generally, the melting profile of mixtures (curve b, c, d) has six endothermic transitions, which could be classified into three distinct regions namely, lowmelting region below -20°c (peaks at position 1 and 2), middle melting region between -10 to 20°c (peaks at position 3, 4 and 5) and high-melting region at around 40°c (peaks at position 6). figure 1b. dsc melting thermograms of ld (a), quaternary mixtures of po:ps:sbo:cb (b=38:5:52:5; c= 36:5:54:5; d=34:5:56:5) po (e), ps (f), sbo (g) and cb (h). abbreviations: ld, lard; po, palm oil; ps, palm stearin; sbo, soybean oil; cb, cocoa butter. ital. j. food sci., vol. 30, 2018 749 according to fig. 1b, the thermal profiles displayed by the quaternary mixtures were considerably different from the melting profile of original sample of po. the melting profiles of mixtures were found to have one additional minor peak (b2, c2 and d2) with a shoulder peak (b1, c1 and d1) at below -20°c could be attributed to the presence of sbo, which had all of its thermal peaks in the low-melting region. with respect to the original sample, tendset of all quaternary mixtures were found to be shifted to higher temperature region after addition of ps and cb into po. when compared to ld (tendset =37.5°c), all three quaternary mixtures had higher end-set of melting (tendset) (at around 44°c) and lower on-set of melting tonset (at around 45°c). although there were much differences in melting transitions between lard and the mixtures, a closer similarity between them was seen at the peak-maximum of (b3, c3 and d3) and (a2) at -3.59°c. 3.4. solidification behavior a comparison of the sfc profiles of the quaternary fat mixtures and ld was given in fig. 2. the sfc of ld and po at 0 ºc was 30.8 and 68.63%, respectively and tended to decrease gradually until they become 0% at 40 and 55 ºc, respectively. as mentioned previously by yanty (2016), the sfc values of ps and cb were found to drop dramatically at 25 ºc and above 55 ºc, respectively. this unique behaviour of ps and cb was largely in agreement with the observed thermal events in their respective dsc curves where the occurrence of single sharp peak was indicative of the meltdown of the entire tag groups within a narrow temperature range. this rapid meltdown behaviour of these fats was also further discussed in other reports (yanty, 2016). figure 2. solid fat content of po, ps, sbo, cb, quaternary mixtures of po:ps:sbo:cb and ld. abbreviations: po, palm oil; ps, palm stearin; sbo, soybean oil; cb, cocoa butter; ld, lard. ital. j. food sci., vol. 30, 2018 750 however, the sfc value of sbo at 0 ºc was found to be 0.31% and become 0% at 10 ºc. this phenomenon could be due to the presence of high amount of usfas (table 1) and uuu tag molecules (table 2) in sbo. the sfc value of po was found to be higher than that of ld within the temperature range between 0 and 20°c. addition of sbo into po could probably reduce the amount of sfc in this temperature region. however, addition of 50 % of sbo into po was resulted in a big slope (below sfc values of ld) within this temperature ranges and the sfc values tended to be higher than that of ld from 30 to 55°c. although additions of ps into po:sbo mixtures were tended to increase the sfc values at 0 to 20°c, the sfc values were still higher than those of ld at 30 and 35°c. according to another set of sfc data not shown here, addition of cb into po:sbo mixtures did not change the sfc values above 30°c. hence, it was assumed that blending po with an appropriate amount of ps, sbo and cb would help to adjust sfc values of po to become closer to that of lard at almost all temperature regions. the sfc values of three quaternary mixtures in fig. 2 were found to be lower than that of ld in between 0 to 25°c. however, the sfc values of the mixtures were tended to be higher than that of lard above 30°c due to a presence of ps. out of the three quaternary mixtures, po:ps:sbo:cb (38:5:52:5) was found to have sfc value somewhat closer to ld at 0, 5 and 25° c. the calculations presented in table 3 also showed that po:ps:sbo:cb (38:5:52:5) was found to have the least difference to ld in terms of sfc values throughout the temperature range. hence, this mixture was found to be the most compatible to ld in term of solidification behavior. table 3. comparing least difference of sfc values of ld and po:ps:sbo:cb mixtures. temp (°c) po:ps:sbo:cb (38:5:52:5)+/ld po:ps:sbo:cb (36:5:54:5)+/ld po:ps:sbo:cb (34:5:56:5)+/ld 0 0.96 -0.25 -1.13 5 0.17 -0.92 -2.08 10 -2.87 -4.35 -5.68 15 -4.15 -6.04 -7.31 20 -3.59 -5.71 -7.1 25 -1.05 -3.2 -4.58 30 4.63 4.33 3.44 35 4.28 3.69 3.62 40 2.02 1.61 1.29 total 0.4 -10.84 -19.53 abbreviations: po, palm oil; ps, palm stearin: sbo, soybean oil; cb, cocoa butter; temp, temperature. 4. conclusions this study demonstrated the possibility of producing a fat mixture to mimic some of the compositional and thermal properties of ld by blending po with ps, sbo and cb in appropriate ratios. among the three different mixtures formulated, po:ps:sbo:cb (38:5:52:5) was found to have the closest similarity to ld in terms of some dsc parameters and sfc behavior. the sfc values of this mixture were found to display the least difference to those of ld throughout the temperature range. particularly, the closest compatibility in terms of sfc values was seen at 0, 5 and 25°c. in terms of composition, the usfa and sfa contents of this mixture were least difference to those of ld. ital. j. food sci., vol. 30, 2018 751 acknowledgements the authors acknowledge partial funding from a research grant (frgs/2/2013/sg01/upm/02/5) awarded by ministry of higher education malaysia. references aocs. 1999. official methods and recommended practices of the american oil chemists’ society. champaign: american oil chemists’ society. deman j.m. 1999. principles of food chemistry. new york: springer science+business media, inc. floter e. 2009. the role of physical properties data in product development. european j. lipid sci. technol. 111:219. given p.s.j. 1990. influence of fat and oil physicochemical properties on the expression of functionality in baked goods. cere foods world 35:813. hsu s.y. and yu s.h. 2002. comparisons on 11 plant oil fat substitutes for low-fat kung-wans. j. food eng. 51:215. liu k.j., chang h.m. and liu k.m. 2007. enzymatic synthesis of cocoa butter analog through interesterification of lard and triestearin in supercritical carbon dioxide by lipase. food chem. 100:1303. marikkar j.m.n. and yanty n.a.m. 2018. fats, oils and emulsifiers. in: preparation and processing of religious and cultural foods, eakub ali and nina naquiyah (editors). elsevier publishers (in press). marikkar j. m. n. and ghazali h. m. 2011. effect of moringa oleifera oil blending on fractional crystallization behavior of palm oil. intel. j. food prop. 14(5): 1049. nor aini i. and miskandar, m.s. 2007. utilization of palm oil and palm products in shortenings and margarines. euro. j. lipid sci. technol. 109:422. nur illiyin m.r., marikkar j.m.n., shuhaimi m., mahiran b. and miskandar, m.s.a. 2013. comparison of the thermo physical behavior of engkabang (shorea macrophylla) seed fat canola oil blends and lard. j. am. oil chem. soc. 90:1485. segall s.d., artz, w.e., raslan, d.s., ferraz, v.p. and takahashi, j.a. 2005. analysis of triacylglycerol isomers in malaysian cocoa butter using hplc-mass spectroscopy. food res. int. 38:167. silva r.c., cotting l.n., poltronieri t.p., balcao v.m., almeida d.b., goncalves l.a.g., grimaldi r. and gioielli l.a. 2009. the effect of enzymatic interesterification on the physic-chemical properties of blends of lard and soybean oil. lwt–food sci. technol. 42:1275. tan c.p. and che man y.b. 2000. differential scanning calorimetry analysis of edible oils: comparison of thermal properties and chemical composition. j. am. oil chem. soc. 77:143. yanty n.a.m. formulation of fat substitutes using plant-based fats simulating the properties of lard. phd thesis, universiti putra malaysia: selangor d.e., malaysia, 2016. paper received april 26, 2018 accepted june 6, 2018 ijfs#1355_bozza ital. j. food sci., vol. 31, 2019 626 paper characterization of pasta with the addition of cicer arietinum and salvia hispanica flours on quality and antioxidant parameters a.g. cota-gastélum, m.g. salazar-garcía, a. espinoza-lópez, l.m. perez-perez, f.j. cinco-moroyoqui, o. martínez-cruz, f.j. wong-corral and c.l. del-toro-sánchez* department of research and postgraduate in food (dipa), university of sonora. blvd, luis encinas y rosales s/n, colonia centro 83000, hermosillo, sonora, mexico *corresponding author: tel: +526622592208; fax: +526622592208 e-mail address: carmen.deltoro@unison.mx abstract quality parameters, antioxidant properties, in vitro digestion and consumer acceptance were determined in pasta prepared with chickpea and chia flours. pastas fortified with chia and chickpea increased protein, fiber content, total phenols, and antioxidant capacity with respect to the control. more than 85% of the antioxidant capacity and over 90% of the phenolic compounds in cooked pasta were retained after in vitro digestion, which is considered high. pasta prepared with 25% wheat semolina, 10% chia flour, and 65% chickpea flour has high quality parameters, phenol content, antioxidant capacity and consumer acceptance. keywords: cicer arietinum, pasta, phenols, salvia hispanica ital. j. food sci., vol. 31, 2019 627 1. introduction pasta products are basic foodstuffs that are important in human food consumption (gelencsér et al., 2008). the simplicity of pasta production (traditionally manufactured from durum wheat semolina), in addition to its ease-of-handling and storage stability has facilitated its popularity and widespread consumption around the world (chillo et al., 2008). nevertheless, pasta is considered insufficient with regard to its nutritional value, due to a poor source of protein (unless supplemented) and its protein has low amounts of essential amino acids such as lysine. hence, pasta is an excellent vehicle for supplementation with fiber, proteins, and many other valuable healthy components. it could be appropriately designed and would operate as a functional food if healthy components were to be incorporated into its formulation (borneo and aguirre, 2008). some of these ingredients could be chickpea (cicer arietinum) and chia (salvia hispanica) seeds. chickpea is an important grain legume because of its nutritional quality. it is a rich source of complex carbohydrates, protein, vitamins, and minerals (jukanti et al., 2012; hirdyani, 2014). polyphenols are also present in chickpeas. some researches indicate that one of the disadvantages of the presence of phenolic compounds (denominated antinutritionals) is that they bind to proteins through non-covalent interactions (electrostatic and hydrophobic interactions, and hydrogen bonding), which reduces their nutritional availability (mondor et al., 2009). however, these types of compounds are currently of great interest because they are bioactive compounds that can confer significant long-term health benefits (aguilera et al., 2011). polyphenols have been recognized as the most abundant source of antioxidants in our diet due to their activities and beneficial properties such as antihypertensive and antibacterial actions as well as anticarcinogenic properties (yang et al., 2001; thomasset et al., 2007; xu and chang, 2010). in majority of the cases, free radicals are responsible for degenerative diseases such as cancer and diabetes, among others (han et al., 2007), and polyphenols have the ability to scavenge free radicals, thereby preventing associated ailments. the phenolic composition of the chickpea is represented by phenolic acids (p-hydroxybenzoic acid, vanillic acid, ferulic acid, and p-coumaric acid) and flavonols (quercetin, kaempferol, and myricetin), as well as isoflavones (daidzein and genistein) (mondor et al., 2009; sreerama et al., 2010; fares and menga, 2012). one research was found that studied the effects of the toasting of chickpeas on the antioxidant properties of chickpea flour added to durum wheat pasta. in that research, it was concluded that toasting the chickpea increases its phenolic content and antioxidant activity (fares and menga, 2012). however, to the best of our knowledge, there is no information on these compounds in fortified pastas after in vitro digestion. the majority of investigations focused on the health benefits accruing from the digestibility of starch, such as a lower glycemic index for persons with diabetes (goñi and valentín-gamazo, 2003; petitot et al., 2010; flores-silva et al., 2014) and for the prevention of cancer, as well as protection against cardiovascular diseases due to the dietary-fiber content of starch (chillo et al., 2008). the remainder of studies on chickpea-fortified pasta focused on improving its nutritional, cooking, and sensory qualities, in which the authors of these studies concluded that chickpea confers good quality and nutritional properties on pastas (zhao et al., 2005; sabanis et al., 2006; wood, 2009; abou-arab et al., 2010; bashir et al., 2012; padalino et al., 2015). furthermore, chia (salvia hispanica) which is native to southern mexico and northern guatemala has become an important raw material for obtaining functional foods due to its high content of fatty acids, protein, fiber, and secondary metabolites such as phenolic compounds (coates and ayerza, 1996; sandoval-oliveros and paredes ital. j. food sci., vol. 31, 2019 628 lópez, 2013; martínez-cruz and paredes-lópez, 2014). chia seeds are very rich in phenolic compounds and possess a high antioxidant capacity, suggesting that phenolic acids (gallic, caffeic, ferulic, chlorogenic, and rosmarinic), flavonols (quercetin and kaempferol), and isoflovonols (genistein, daidzin, and glycitein) may decrease the invasiveness of cancer cells, remove ros (reactive oxygen species), and improve clinical outcome (sandoval-oliveros and paredes-lópez, 2013; martínez-cruz and paredes-lópez, 2014; reyes-caudillo et al., 2008). according to marineli et al. (2014) and coelho and salas-mellado (2014), the consumption of chia seed can therefore serve as an important alternative for improving consumer health, suggesting its use as a functional food in the human daily diet. previous studies have shown that pasta made with chia flour (7.5% and 10%) had a higher nutritional value and superior technological characteristics (oliveira et al., 2015; menga et al., 2017). due to the many properties that chickpea and chia possess, they are good candidates for use in the fortification of pasta to improve the nutritional quality in pasta formulations. thus, the purpose of this study was to characterize pasta fortified with chickpea flour and chia flour in terms of their quality parameters, antioxidant properties and in vitro digestion. 2. material and methods 2.1. materials commercial wheat (triticum durum) semolina, kabuli-type chickpea (cicer arietinum) and chia (salvia hispanica) were purchased at a local store. chickpea grains were manually cleaned and dried at 60°c for 6 h. to obtain chickpea flour, a perten model lm3100 (perkinelmer, usa) mill was used. the chia grains were ground in an analytical mill (braun aromatic coffee grind ksm2, usa) to obtain chia flour. both flours were then standardized to a particle size of 60 mesh (0.25 mm). all chemical reagents were purchased from local laboratory suppliers and were of analytical grade. 2.2. pasta elaboration each blend was made with different proportions of commercial wheat (t. durum) semolina, chickpea flour, and chia flour, as shown in table 1. chia flour was the same in all formulations (10%) because, in preliminary studies, this amount was optimal for the sensory quality of pasta (data not shown). for its preparation, 100 g of flour was utilized. distilled water was added at 35% absorption (considering the moisture of each mixture, between 35 and 42 ml/100 g of the mixture) and were mixed at room temperature in a mixer machine (professional model 600 hd, usa) at low speed (set 1) for 10 min. afterwards, the dough was allowed to rest for 10 min in a plastic bag at room temperature. first, the proofed dough was laminated in the pasta machine (imperial r 220 model rmn, italy) at setting 3, and finally at setting 1. the pasta was hand-cut into strips approximately 20 cm in length (fresh pasta) using a scissors. the pastas were kept to air-dry for 2 days at ambient temperature according to cleary and brennan (2006). the six pasta samples were placed individually in sealed containers to avoid moisture exchange and were then stored at 5°c. ital. j. food sci., vol. 31, 2019 629 table 1. formulations for pasta made with the wheat semolina, chia flour and chickpea flour. formulations (g/100 g) flour f1 f2 f3 f4 f5 f6 wheat semolina 100 85 65 45 25 0 chia 0 10 10 10 10 10 chickpea 0 5 25 45 65 90 2.3. chemical analysis the testing was performed according to association of official analytical chemists (aoac) official methods. semolina, chia, and chickpea flours and each pasta sample were analyzed for crude protein (method 955.04) and ash (method 920.153) using the aoac (2000) methods. total dietary fiber and fat were determined by methods 985.29 and 920.85, respectively according to aoac (1997). carbohydrates were determined by difference. 2.4. cooking quality determinations the cooking quality of the pasta, such as optimal cooking time, water absorption, weight gain by pasta, and solid loss during cooking, were evaluated using method 66-50 of the american association of cereal chemists (aacc) official methods (aacc, 2000). 2.5. determination of color color was evaluated utilizing the cielab system (hunter lab miniscan ez 45/0-l model, usa). the analyzed parameters included the following: l* (which represents the percentage of brightness, where black is 0% and white is 100%); a* (where +a* is red and a* is green), and b* (where +b* is yellow and -b* is blue). the readings were taken at room temperature on the surface of the pastas, with 10 repetitions for each evaluated sample (oliveira et al., 2015). 2.6. antioxidant activity in determining the antioxidant activity of the different pasta formulations, the extracts were obtained first. all samples (raw and cooked) were adjusted to 0.17 g/ml with 1% hydrochloric acid in methanol (rehman and shah, 2005; li et al., 2007). using an orbital shaker (thermo scientific, usa) for 3 h at room temperature, the super natants of the samples were separated by centrifugation (10,000 rpm, 15 min, and 4°c). dpph [2,2diphenyl-1-picrylhydrazyl] and abts [2,2ʹ-azino-bis-(3 ethylbenzothiazoline-6-sulfonic acid)] assays were employed to quantify the antioxidant activity. additionally, quantification of total phenolic content was determined. 2.6.1 dpph assay the antioxidant properties of the pasta samples using the dpph assay were measured by the method described by molyneux (2004). briefly, a 0.1 ml aliquot of the sample solutions was mixed with 3.9 ml of a free radical dpph solution (6 × 10‒5mol/l). the reaction mixtures were incubated for 30 min in darkness and their absorbance was measured at 515 nm. all analyses were carried out in triplicate and a trolox standard ital. j. food sci., vol. 31, 2019 630 curve was utilized for quantification. the results were reported as micromole of trolox equivalent per gram of sample (µmol te/g). 2.6.2 abts assay the abts assay was performed using the procedure of re et al. (1999). a 2.97 ml of the cation radical solution was combined with 0.03 ml of the extract. the samples were incubated for 30 min at room temperature, and later, absorbance was measured at 734 nm. a control was prepared containing the cation radical solution with no pasta extracts, and another, with the solvent used. all analyses were performed in triplicate and a trolox standard curve was used for quantification. the results were reported as micromole of trolox equivalent per gram of sample (µmol te/g). 2.6.3 total phenolic content quantification of total phenols was assayed by a spectrophotometric method utilizing the folin-ciocalteau reagent (singleton et al., 1999). thereafter, 50 μl of each extract solution was combined with 3 ml of deionized water and 250 μl of 1 n folin-ciocalteau reagent. after 5 min of incubation at room temperature, 750 μl of a 20% sodium carbonate solution and 950 μl of deionized water were added, measuring absorbance at 760 nm. a gallic acid standard calibration curve (0–100 mg/l) was prepared and the results were expressed as mg of gallic acid equivalents per gram of sample (mg gae/g). 2.7. in vitro digestion this assay was conducted according to gil-izquierdo et al. (2002), but with some modifications. the cooked pastas were the samples used in this analysis. a total of 20 g of pasta was cooked in 100 ml of distilled water for 7.5 min. after that, 1 g of cooked pasta was removed, cut into small pieces of approximately 2 mm, and dissolved in distilled water at a material-to-solvent ratio of 1:15. this preparation was mixed in a vortex mixer (daigger, vortex-genie 2) and was adjusted to ph 2.0 with 1 m hcl. a total of 0.5 ml was removed and combined with 0.75 ml of pepsin (315 u/ml prepared in 0.2 m kcl buffer) and 1.75 ml of deionized water. the samples were neutralized with 1.25 m nahco3 after incubation at 37°c in a shaking water bath (wise bath, daihan scientific, wsb-18) at 80 rpm for 2 h. a total of 0.375 ml of pancreatin solution (4 mg/ml, prepared in 0.1 m phosphate buffer saline [pbs]) was added to the samples. afterward, the samples were transferred onto dialysis membranes (12,000 da), placed in an erlenmeyer flask containing 35 ml of 0.1 m pbs solution, and incubated again in the shaking water bath (4 h, 37°c, 80 rpm). antioxidant activity and total phenols were determined before (initial) and after digestion (inside and outside of the membrane). 2.8. consumer acceptance test to evaluate the acceptability of the pasta, a 5-point hedonic scale was used for acceptance testing, in which the upper and lower extremes, respectively, correspond to 5 (liked very much) and 1 (disliked very much). the pasta was placed in boiling water with 5 g of salt and 10 ml of oil, and was subsequently served to tasters. the sensory panel was composed of 100 untrained tasters who were recruited randomly (oliveira et al., 2015). ital. j. food sci., vol. 31, 2019 631 2.9. statistical analysis statistical analysis of the data obtained in all experiments were performed by analysis of variance (anova) using the statgraphics centurion ver. 17.0 xv ver. 15.2.06 statistical software. comparison of means was performed by the tukey least significance test (p<0.05). the experiments were run in triplicate. 3. results and discussion the chemical composition of the samples is presented in table 2. chia and chickpea seeds are rich in protein (27.94 and 22.93%, respectively) and fiber (30.02 and 20.22%, respectively) when compared to other grains such as barley, rice, amaranth, corn, and wheat. several authors found similar results in protein and fiber content in chia and chickpea (mondor et al., 2009; oliveira et al., 2015; sánchez-vioque et al., 1999; muñoz et al., 2012; sargi et al., 2013). despite the fact that chia had the highest quantity of protein, it has not been marketed, to our knowledge, as a source of protein, due to the fact that the profile of amino acids is limiting for schoolchildren (oliveira et al., 2015). however, it can be mixed with other grains to improve protein balance in formulations for adults (ayerza and coates, 2011). this is one of the reasons for the addition of chickpea to the formulation in this study. an increase in protein and fiber content was observed in the pasta formulations by increasing the amount of chickpea, bearing in mind that the amount of chia was the same in all of the formulations. this result is significant because pasta is considered a low nutritional-value product. hence, chickpea considerably fortified the pasta (from f3 to f5) with protein and fiber in comparison with semolina pasta (f1). however, the sample in which semolina was replaced with chia flour and chickpea flour (f6) had the highest amount of protein and fiber, indicating that both seeds are good options for elaborating pasta rich in this type of compound. table 2. chemical properties of chia flour, chickpea flour, and wheat semolina and the different pasta formulations (% dry base). protein (%) fat (%) ash (%) total fiber (%) carbohydrates (%) chia 27.94±0.55a 35.31±0.11a 4.46±0.06a 30.02±0.11b 2.26±0.96e chickpea 22.93±0.25b 8.02±0.31c 2.88±0.02b 20.22±1.12a 45.95±0.58c semolina 14.9±0.37e 1.06±0.03d 0.93±0.03d 4.72±1.41g 78.39±0.34a f1 15.14±0.48e 1.14±0.03d 0.94±0.02d 4.25±1.41g 78.53±0.53a f2 15.01±0.02e 11.31±1.27b 1.06±0.01d 11.42±0.56f 72.62±1.31b f3 17.26±0.44d 12.38±0.42b 1.81±0.02c 14.02±0.63e 68.55±0.68c f4 17.96±0.35d 11.07±0.32b 2.08±0.01b 17.66±1.32d 68.89±0.74c f5 18.92±0.51d 12.35±0.64b 2.65±0.02b 19.29±1.08d 66.08±1.20c f6 24.12±0.38b 10.90±1.42b 3.03±0.10b 21.04±0.99c 61.95±1.81d different letters within the same column indicate statistical differences (p < 0.05). f1: pasta with 100% semolina; f2: pasta with 85% semolina, 10 % chia flour and 5% chickpea flour; f3: pasta with 65% semolina, 10% chia flour and 25% chickpea flour; f4: pasta with 45% semolina, 10% chia flour and 45% chickpea flour; f5: pasta with 25% semolina, 10% chia flour and 65% chickpea flour; f6: pasta with 10% chia flour and 90% chickpea flour. carbohydrates were determined by difference. ital. j. food sci., vol. 31, 2019 632 furthermore, it was observed that fat content for chia-seed is high (35.31%), while fat content (8.02%) for chickpea is low. in comparison with f1, an increase in fat content was observed, from f2 to f6; however, there were no significant differences (p>0.05) between the fat content in these latter samples, indicating that chia could be the principal seed responsible for this increase. alpha-linolenic acid represents 46.72%-62.44% of the total fatty acids in chia seed (peiretti, 2011). it is an omega-3 fatty acid and is popular for preventing and treating diseases of the heart and blood vessels, among other functions (mantzioris et al., 1994; zia-ui-haq et al., 2007). additionally, alpha-linolenic acid is denominated “essential” because it cannot be synthesized by humans, but can only be consumed through foods. therefore, pasta formulations with chia comprise a good option for consuming this compound. on the other hand, ash content in chia is high (4.46%) compared with chickpea (2.88%) and other cereals such as rice, wheat, and sorghum. magnesium, calcium, iron, and phosphorus are the minerals found in abundance in chia seed and are essential for a healthy diet (oliveira et al., 2015; capitani et al., 2012). the quality of many foods depend on the concentration and type of minerals they contain, including their taste, appearance, texture, and stability. in the pasta formulations, the major content of ash was in f5 and f6 (2.65 and 3.03%, respectively), without a significant difference between them (p>0.05). generally, all pastas have a high content of carbohydrates (61.95–78.53%), especially f1, which had the highest content. from f2 to f6, a decrease in these amounts were observed due to the increment of fiber influencing the calculation of carbohydrates, which is determined by difference. similar behavior was obtained by padalino et al. (2014) in pastas enriched with pea flour and by oliveira et al. (2015) with pastas enriched with chia flour. 3.1. cooking quality and color parameters table 3 depicts the results of the cooking quality and color parameters of the pastas. optimal cooking time was the same (7.5 min) for all pasta types. oliveira et al. (2015) reported from 15-16 min cooking time in pastas with chia flour (7.5, 15, and 30%) instead of wheat flour, which is longer than the time frame used in our study. table 3. cooking quality and color parameters for the different pasta formulations. f1 f2 f3 f4 f5 f6 optimum cooking time (min) 7.5a 7.5a 7.5a 7.5a 7.5a 7.5a water absorption (%) 64.1±1.6a 64.5±2.1a 64.7±1.8a 61.1±1.1b 59.0±1.0b 59.6±0.7b percentage weight increase (%) 118.2±4.1a 116.7±7.3a 113.5±5.3b 110.3±9.2b 96.2±1.9c 69.6±3.0d solid loss (%) 4.5±0.5b 4.6±0.3b 4.6±0.2b 4.9±0.0b 4.7±0.4b 6.3±0.9a l* 75.5±0.1a 57.4±1.5b 55.2±1.1b 53.6±0.1bc 51.3±0.7c 48.9±0.6c a 2.9±0.2c 5.0±0.3b 5.4±0.2b 5.9±0.1b 6.5±0.1b 7.1±0.2a b 29.5±0.1a 28.6±0.3a 28.1±0.1a 28.1±0.4a 27.0±2.3a 21.5±0.1b different letters in the same line differ statistically at the 5% level by the tukey’s test. f1: pasta with 100% semolina; f2: pasta with 85% semolina, 10 % chia flour and 5% chickpea flour; f3: pasta with 65% semolina, 10% chia flour and 25% chickpea flour; f4: pasta with 45% semolina, 10% chia flour and 45% chickpea flour; f5: pasta with 25% semolina, 10% chia flour and 65% chickpea flour; f6: pasta with 10% chia flour and 90% chickpea flour. ital. j. food sci., vol. 31, 2019 633 another study with pastas contained 30% of chickpea obtained the cooking time of 10.5 min (zhao et al., 2005). spaghetti with 30% of chickpea flour exhibited an optimal cooking time of 5.75 min (wood, 2009), which is lower than that in our study. thus, our results (7.5 min) are within the average optimal cooking time of pastas fortified with these types of seeds. moreover, water absorption, weight increase, and the loss of soluble solids demonstrated significant differences (p<0.05) across treatments. water absorption was higher, from f1 to f3, without significant differences among them (p >0.05). in this case, the level of substitution did not affect water absorption. several studies indicated that the presence of chia increased water absorption due to the high fiber content of chia flour. the fibers, when mixed with water, form three-dimensional (3-d) networks by means of the mucilaginous compounds that this seed contains (oliveira et al., 2015; muñoz et al., 2012; capitani et al., 2012). however, in our study, water absorption was not affected by the presence of chia. this behavior could be due to the fact that the interactions, mostly with chickpea, possessed fewer free bonds for binding the water molecules, thereby limiting the formation of 3d networks. within this context, the weight increase in the sample is related to the water absorption, demonstrating similar behavior. highest weight increase was obtained for samples with the highest content of semolina (f1 and f2). this fact can be explained by the high interaction with the water of the protein gluten (gliadin and glutenin) and the starch derived from the semolina (feillet and dexter, 1996). similar results were obtained by flores-silva et al. (2014) in spaghetti made with chickpea, unripe plantain, and maize flours. in addition, when semolina was replaced with chia flour and chickpea flour (f6), it exhibited a greater loss of soluble solids, which differed statistically from the rest of the samples (p<0.05). however, there were no significant differences from f1 to f5 (p>0.05), showing that semolina and its interaction with chia and chickpea prevents the loss of soluble solids. hummel (1966) classified pastas according to loss of solids: up to 6% is characteristic of very good quality, up to 8% average quality, and values equal to or greater than 10% are low-quality pastas. hence, according to this classification, pasta enriched with chia and chickpea in the presence of semolina are very good quality pastas. this result can be attributed to the protein contained in the samples, which causes the retention of amylose during cooking (chillo et al., 2008). although, f6 possesses the highest protein content, it is evident that the absence of the semolina proteins increases the loss of soluble solids. oliveira et al. (2015) observed that the addition of chia flour improved the quality of the pastas by reducing the loss of solids. on the contrary, zhao et al. (2005) studied pastas with flours of legumes such as beans and chickpeas, in which the authors observed an increase in loss of solids during cooking. this was attributed to losses in the structural changes of the protein network due to partial substitution of wheat protein by legume protein (torres et al., 2007). however, in our case, the interaction among semolina, chia, and chickpea conferred better quality pastas. the addition of chia and chickpea flour to the pasta showed a reduction in brightness (lower l*), more red (higher a*), and less yellow (lower b*), when compared with semolina pasta (f1), principally when the proportion of chickpea flour was increased (table 3). results of redness (a*) values reached the maximal values (7.1). the greater intensity of the yellow color is a highly desirable feature in pasta products, because it is one of the most influential visual appeals in the acceptance of pastas (chang and flores, 2004). f1 pasta differed statistically from others, featuring greater intensity of the yellow color (b* value), thus favoring acceptability. these results were similar to those of several studies of pasta fortified with chia or chickpea (wood, 2009; abou-arab et al., 2010; oliveira et al., 2015). ital. j. food sci., vol. 31, 2019 634 3.2. antioxidant activity and total phenols dpph, abts, and total phenol determinations of raw and cooked pasta samples are illustrated in fig. 1. antioxidant activity and total phenols content increased when the chickpea concentration increases. similar results were observed by fares and menga (2012) in durum wheat pasta enriched with chickpea flour and by khan et al. (2013) in durum wheat pasta enriched with sorghum. the samples that presented highest antioxidant capacity in both radicals were f5 (dpph 698.54 µmol te/g, abts 1029.16 μmol te/g), and f6 (dpph 756.12 μmol te/g, abts 1063.8 μmol te/g). however, abts exhibits more affinity (fig. 1b) for the pasta compounds than dpph (fig. 1a). this could be explained by the abts reacting more specifically with an h-atom donor than dpph, thereby accomplishing the abts reaction faster (prior et al., 2005; roginsky and lissi, 2005; rosa-alcaraz et al., 2017). additionally, abts can measure hydroand lipophilic compounds (krishnaiah et al., 2011). on the other hand, sharma and bhat (2009) indicated that the dpph radical reacts to a greater degree with lipophilic compounds, but other researches mentioned that this radical does not react mainly with flavonoids, which contain no hydroxyl groups in the b-ring, as well as with aromatic acids containing only one oh-group (von gadow et al., 1997; yokozawa et al., 1998). the phenolic compounds in chia (rosmarinic acid, protocatechuic acid, caffeic acid, and gallic acid, among others) and chickpea (p-hydroxybenzoic acid, vanillic acid, ferulic acid, and pcoumaric acid, among others) comprise hydrophilic compounds to a greater extent (fares and menga, 2012; martínez-cruz and paredes-lópez, 2014; menga et al., 2017). additionally, these compounds have more h-atom donor or aromatic acids containing only one oh-group and, according to what has been previously mentioned, this explains the behavior observed in high affinity for abts rather than for the dpph radical. in our study, antioxidant capacity is higher than in other uncooked pasta (maximum of 1,063.8 µmol te/g); for example, in durum wheat pasta enriched with sorghum that has a maximum of 33.7 μmol te/g (khan et al., 2013). in cooked pasta, the maximal amount obtained in our study was 800.67 μmol te/g, a higher amount compared with pasta supplemented with germinated pigeon pea flour (5.8 μmol te/g) (torres et al., 2007) and in pasta enriched with bean flour (1.26 μmol te/g) (gallegos-infante et al., 2012). temperature affects antioxidant activity, with a loss of approximately 50% in dpph (fig. 1a) and of 30% in abts (fig. 1b). the same behavior was observed in the concentration of phenolic compounds, these decrease is approximately 30% in cooked pasta (fig. 1c). similar results were obtained by khan et al. (2013) in durum wheat pasta enriched with sorghum, where the authors demonstrated a decrease of 20-55% in antioxidant capacity and a decrease of 21-55% of phenolic compounds in cooked pasta. there were no significant differences (p >0.05) between 65% and 90% of chickpea in the formulations in each radical and in the amount of total phenols (14.76 and 16.34 mg gae/g, respectively). these results were higher than those reported by gallegosinfante et al. (2012) in pasta enriched with bean flour (0.49 mg gae/g), by turco et al. (2016) in pasta with faba bean flour (1.85 mg gae/g), and by khan et al. (2013) in wheat pasta with sorghum (1.27-3.22 mg gae/g). the correlation among antioxidant activities with phenols was carried out, and it was observed that this correlation was high (r2 = 0.921). this indicates that phenols were the principal compounds responsible for antioxidant activity in the pasta, while pasta made only with semolina (f1) presented very low antioxidant activity and phenolic content, confirming the effect of the fortification accomplished with chia and chickpea. ital. j. food sci., vol. 31, 2019 635 figure 1. antioxidant activity by dpph (a), abts (b) and total phenols (c) of raw and cooked pastas. columns not sharing a common letter within the same treatment (raw or cooked) of pasta are significantly different (p< 0.05). f1: pasta with 100% semolina; f2: pasta with 85% semolina, 10 % chia flour and 5% chickpea flour; f3: pasta with 65% semolina, 10% chia flour and 25% chickpea flour; f4: pasta with 45% semolina, 10% chia flour and 45% chickpea flour; f5: pasta with 25% semolina, 10% chia flour and 65% chickpea flour; f6: pasta with 10% chia flour and 90% chickpea flour. cooked f1 pasta demonstrated a decrease of approximately 80% when compared with uncooked pasta, indicating that chia-chickpea-fortified pastas have lower cooking losses. 0 150 300 450 600 750 900 1050 1200 f1 f2 f3 f4 f5 f6 a b t s μ m ol t e/ g 0 200 400 600 800 1000 1200 f1 f2 f3 f4 f5 f6 d p p h μ m ol t e/ g raw pasta cooked pasta 0 2 4 6 8 10 12 14 16 18 f1 f2 f3 f4 f5 f6 t ot al p he n ol s m g g a e/ g formulation a a b b b m m n n o a a a b b m m m n n a a b b b m m n n n a ) b ) c ) p c c o c o ital. j. food sci., vol. 31, 2019 636 some authors came to the same conclusion when chickpea-fortified spaghetti and apple by-product-fortified pasta were compared (wood, 2009; lončarić et al., 2014). 3.3. in vitro digestion in vitro digestion was carried out only in cooked pastas using pepsin, pancreatin, and dialysis membranes to simulate the intestine. the most important finding in our study comprised the antioxidant capacity and the amount of phenolic compounds that pass through the membrane, where the outside of the membrane simulates the blood serum. the results revealed major antioxidant capacity in both radicals and the major amount of phenolic compounds outside of the membrane when the amount of semolina is decreased or absent in the formulations (fig. 2). there were no significant differences (p>0.05) in antioxidant capacity between f5 and f6 (figs. 2a and 2b). however, the highest antioxidant activity was detected in abts radical by f5 (655.1 ± 7.56 µmol et/g) and f6 (703.4 ± 14.98 µmol et/g). more than 85% of this antioxidant capacity was maintained outside of the membrane, which is considered high. compared with the f5 and f6 undigested samples in abts radical (781.43 μmol et/g and 800.67 μmol et/g, respectively), there was an approximate decrease of 16% in f5 and 12% in f6 of antioxidant capacity. also, comparison with the f5 and f6 undigested samples (367 µmol et/g and 388.21 µmol et/g, respectively) in the dpph radical revealed a major decrease of approximately 50% in f5 and 45.8% in f6 on the antioxidant capacity. as discussed previously, phenols were the principal compounds responsible for the antioxidant activity in the pasta; thus, if these compounds were affected by the digestive process, consequently, the antioxidant activity was affected. in our case, the phenolic compounds outside of the membrane in f5 (9.82 ± 0.1 mg gae/g) and f6 (10.97 ± 0.3 mg gae/g) exhibited the highest amounts, maintaining approximately more than 90% of these compounds compared with the initial amount (fig. 2c). hence, the differences between antioxidant activities in both radicals could be due to the affinity of the compounds in each sample and/or to their bioaccessibility (fraction of bioactive substance that is released from the food matrix) that consequently affects their bioavailability (amount of phenolic compounds reaching the blood circulation system), (rodríguez-roque et al., 2014). cattaneo et al. (2015) found that after a simulated gastric condition, the antioxidant activity of puffed kernels was high compared to the undigested sample measured by abts and frap methods. rufián-henares and delgado-andrade (2009) obtained similar behavior in flaked wheat-based breakfast cereals measured by abts, dpph and frap methods. in this context, some factors can affect the bioaccessibility and bioavailability of phenolic compounds. studies indicated that in the cell wall of legume/seeds, these compounds could be present in insoluble-bond forms (covalent bonds, 20–60%) (shahidi and yeo, 2016). these insoluble bonds affect the bioaccessibility and consequently, the bioavailability of the phenolic compounds because these bonds need to be cut by specific enzymes that generally are provided by intestinal microbiota (perez-perez et al., 2018). most native polyphenols in foods are in glycoside form, which cannot be absorbed in the intestinal mucosa; therefore, the release of these performed by human and microbial enzymes is a necessary mechanism needed by them to break through the intestinal barrier (valdés et al., 2015). in our study, we focused on the soluble forms (non-covalent bonds) of the phenolic compounds through in vitro digestion without the influence of microbiota. thus, additional research should be carried out on the absorption of phenolics in the human colon. also, hydrophobicity, the membrane-mediated transport, the stability of the compounds, environmental ph, degree of polymerization, interactions with other ital. j. food sci., vol. 31, 2019 637 compounds, molecular mass, and the complexity of the food matrix are some other factors that influence the absorption of phenolic compounds (tarko et al., 2009; rodríguezroque et al., 2014; shahidi and yeo, 2016). in our opinion, the majority of in vitro digestion studies of different fortified pastas have for long focused on starch digestibility. the principal interest of these studies is that sugars are progressively released from pasta during digestion, leading to a standard increase in postprandial blood glucose and insulin response (goñi and valentíngamazo, 2003; petitot et al., 2010; flores-silva et al., 2014;padalino et al., 2015;gelencsér et al., 2008; reyes-caudillo et al., 2008; zheng et al., 2016). our study focused on the bioaccessibility of phenolic compounds and antioxidant capacity after in-vitro digestion in order to provide additional and valuable information on the high effect of chia-chickpea-fortified pasta in the scavenging of free radicals. thus, the antioxidant property of this fortified pasta could be involved in the defense mechanism of the organism against pathologies associated with the attack of free radicals, such as cancer, coronary heart disease, obesity, type 2 diabetes and hypertension, among others (pisoschi and negulescu, 2011). with the results obtained in our research, pasta fortified with chia and chickpea can maintain high levels of phenolic compounds and antioxidant capacity after invitro digestion, therefore, making it capable of providing health benefits. 3.4. consumer acceptance test the results of consumer acceptability are presented in fig. 3. highest overall acceptability by tasters was the pasta with 100% semolina (f1) and samples containing wheat semolina (45% and 25%), chia (10%), and chickpea (45% and 65%) flours (f4 and f5, respectively), which revealed no significant differences (p >0.05). similar results were obtained by zhao et al. (2005) in spaghetti fortified with chickpea. the pasta in which chia and chickpea are utilized instead of wheat semolina (f6) triggered the least acceptance. therefore, the presence of wheat semolina is important for the sensory properties of fortified pastas. additionally, petitot et al. (2010) noted that the addition of a high level of legume flour induced some minor structural changes in pasta. this explains the high acceptability of pastas with a high content of chickpea, containing semolina in their formulation. the addition of chia and chickpea causes inclusion of fibers, dilution of gluten proteins by albumins and globulins, and the largest amount of thin protein films, which may have favored the higher susceptibility of starch to digestive enzymes. consequently, the acceptability of these types of fortified pastas is presently on the increase. if we were required to choose a pasta sample, it would of necessity be the sample that complies with all of the parameters (high quality parameters, phenol content, antioxidant capacity, and consumer acceptance). in accordance with all of the results in this work, this selected sample would be f5 (25% semolina, 10% chia flour, and 65% chickpea flour). ital. j. food sci., vol. 31, 2019 638 figure 2. antioxidant activity measured by dpph (a) and abts (b) and total phenols (c) of digested and undigested (initial) cooked pasta. columns not sharing a common letter within the same status (initial, inside membrane and outside membrane) of pasta are significantly different (p < 0.05). f1: pasta with 100% semolina; f2: pasta with 85% semolina, 10 % chia flour and 5% chickpea flour; f3: pasta with 65% semolina, 10% chia flour and 25% chickpea flour; f4: pasta with 45% semolina, 10% chia flour and 45% chickpea flour; f5: pasta with 25% semolina, 10% chia flour and 65% chickpea flour; f6: pasta with 10% chia flour and 90% chickpea flour. -100 100 300 500 700 900 f1 f2 f3 f4 f5 f6 d p p h μ m ol t e/ g formulation initial inside membrane outside membrane 0 100 200 300 400 500 600 700 800 900 f1 f2 f3 f4 f5 f6 a b t s μ m ol t e/ g formulation 0 2 4 6 8 10 12 14 f1 f2 f3 f4 f5 f6 t ot al p he n ol s m g ea g /g formulation a a ab b b a a b c c a a b b b m w m m n n o n n o p m n n o o w x y z w w x x y w x y z z a) b) c) ital. j. food sci., vol. 31, 2019 639 figure 3. percentage of consumer acceptance of the different pasta formulations. the sensory panel was composed of 100 untrained tasters. identical letters do not differ statistically at the 5% level by the tukey’s test. f1: pasta with 100% semolina; f2: pasta with 85% semolina, 10 % chia flour and 5% chickpea flour; f3: pasta with 65% semolina, 10% chia flour and 25% chickpea flour; f4: pasta with 45% semolina, 10% chia flour and 45% chickpea flour; f5: pasta with 25% semolina, 10% chia flour and 65% chickpea flour; f6: pasta with 10% chia flour and 90% chickpea flour. 4. conclusions in this work, we studied the effect of pasta with the addition of chickpea flour and chia flour in terms of quality parameters, antioxidant properties, and their in vitro digestion. the results revealed that the addition of these flours increases protein and fiber content, indicating that both seeds comprise good options for elaborating pasta rich in these types of compounds. cooking parameters are important for consumers; in our results, the level of substitution of chia and chickpea did not affect water absorption; additionally, the presence of these seeds in the pasta avoided the loss of soluble solids. thus, the interaction among semolina, chia, and chickpea yielded better quality pastas. antioxidant activity and total phenols content increased in the fortified pastas. although, cooking affected the amount of phenols and antioxidant capacity, in vitro digestion studies demonstrated that pasta fortified with chia and chickpea can maintain high levels of phenolic compounds and antioxidant capacity after in-vitro digestion, thus being able to provide health benefits. however, in vivo measurements are needed to confirm any biological effects. chiachickpea-fortified pasta would make an inexpensive, attractive, convenient and healthy food that is acceptable to consumers. therefore, with the results obtained in our research, chia and chickpea can be utilized to develop more value-added products, thus rendering them more economical and affordable for developing countries. acknowledgements this work was supported by grant uso313002193 from the university of sonora, mexico. we are grateful to conacyt-méxico for supporting the phd fellowship, number cvu 590932, granted to liliana maribel perez-perez. 0 5 10 15 20 25 f1 f2 f3 f4 f5 f6 % overall consumer acceptance fo rm ul at io n a a a b b c ital. j. food sci., vol. 31, 2019 640 references aacc. 2000. official methods of the american association of cereal chemists. 10th ed. st.paul, mn, usa. aoac. 1997. official methods of analysis. 17th ed. gaithersburg, md.: assn. of official analytical chemists. aoac. 2000. association of official analytical chemists. official methods of analysis of aoac. 19th ed, 2012. arlington. usa. abou-arab e.a., helmy i.m.f. and bareh g.f. 2010. nutritional evaluation and functional properties of chickpea (cicer arietinum l.) flour and the improvement of spaghetti produced from its. j. am. sci. 6(10):1055. aguilera y., dueñas m., estrella i., hernández t., benitez v., esteban r.m. and martín-cabrejas m.a. 2011. phenolic profile and antioxidant capacity of chickpeas (cicer arietinum l.) as affected by a dehydration process. plant foods hum. nutr. 66:187. ayerza r. and coates w. 2011. protein content, oil content and fatty acid profiles as potential criteria to determine the origin of commercially grown chia (salvia hispanica l.). ind. crops prod. 34(3):1366. bashir k., aeri v. and masoodi l. 2012. physio-chemical and sensory characteristics of pasta fortified with chickpea flour and defatted soy flour. iosr j. environ. sci. toxicol. food technol. 1(5):34.. borneo r. and aguirre a. 2008. chemical composition, cooking quality, and consumer acceptance of pasta made with dried amaranth leaves flour. lwt food sci. technol. 41:1748. capitani m.i., spotorno v., nolasco s.m. and tomás mc. 2012. physicochemical and functional characterization of byproducts from chia (salvia hispanica l.) seeds of argentina. lwt. food sci. technol. 45:94. cattaneo s., hidalgo a., masotti f., stuknyte m., brandolini a. and de noni i. 2015. heat damage and in vitro starch digestibility of puffed wheat kernels. food chem. 188: 286. chang y. and flores h. 2004. qualidade tecnológica de massas alimentícias frescas elaboradas de semolina de trigo durum (t. durum l) e farinha de trigo (t. aestivum l). 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abstract human norovirus has been reported as the major non-bacterial cause of human gastroenteritis due to the consumption of contaminated bivalve mollusks. the european legislation established microbiological criteria only for bacteria (salmonella spp. and escherichia coli), while no viruses have still been considered. in this study, samples of chamelea gallina were harvested along the central adriatic coasts (italy) and artificially contaminated with murine norovirus-1 (mnv-1) up to a final concentration of 103 tcid50/ml in water. they were subject to a depuration process in a closed-circuit system using both ozone and ultraviolet light. four experimental trials (100 specimens/trial) were performed and, at the end of depuration, the digestive glands of mollusks were examined by means of two methods – namely, rt-pcr and tissue culture. the results of rt-pcr ranged from 103.17 to 104.60 tcid50/ml, and the constant presence of mnv-1 was confirmed by the tissue culture as well. in conclusion, no significant viral reduction was obtained, but the contaminated bivalve mollusks remained infectious until the end of the depuration treatment. the proper cooking of live bivalve mollusks could be considered the most important preventive measure against this sanitary risk. keywords: norovirus, clams, depuration, tissue culture, rt-pcr ital. j. food sci., vol. 32, 2020 126 1. introduction norovirus is a non-enveloped, single-stranded positive rna virus, a member of the family caliciviridae, and divided into six genogroups (gi-gvi). however, only the genogroups gi, gii and giv were identified in humans (ilic et al., 2017). moreover, over 40 genotypes based on the capsid were identified (leroux-roels et al., 2017). a novel gii.17 variant emerged in asia (china, japan, korea and taiwan) in 2014 (cheng et al., 2017; suffredini et al., 2017) and was also reported in other countries such as canada, the united states (u.s.), new zealand as well as some european states – i.e. germany, italy, hungary and slovenia (chan et al., 2017). according to the european union rapid alert system for food and feed (eu rasff), the majority of alert notifications involving norovirus in food were reported by denmark, france, italy, the netherlands and norway as notifying countries, and france and serbia as countries of origin, respectively. with regards to border rejection notifications, the main countries of origin were france and serbia, whereas italy and spain submitted to the eu rasff the majority of them (papapanagiotou, 2017). human norovirus (hunov) is reported as the main non-bacterial cause of foodborne outbreaks due to the consumption of live bivalve mollusks. the main clinical symptoms of such an illness, with an incubation period of 10-51 hours, are nausea, sudden onset of vomiting and/or watery non-bloody diarrhea, abdominal or general muscle pain, headache and mild fever (hassard et al., 2017; jeon et al., 2017). in addition, it can lead to more severe conditions, such as dehydratation, hospitalization and potentially death in vulnerable individuals including children and elderly population (trivedi et al., 2013; fusco et al., 2017). bivalve mollusks are filter-feeding organisms that can retain and concentrate in their own body not only nutrients but also suspended viruses or bacteria. however, the european union (eu) legislation (ec, 2004a) established that sanitary controls of live bivalve mollusks must be based only on the detection of escherichia coli used as an indicator of faecal contamination for the classification of production areas, from which they can be collected. moreover, regulation ec no 2073/2005 (ec, 2005) and its amendments reported the absence of salmonella spp. in 25 g of live bivalve mollusks and a range of 230 to 700 mpn/100 g of flesh and intravalvular liquid for e. coli. in the u.s. as well, the standards used for shellfish hygiene controls in both growing areas during primary production and for end-products are represented by faecal or total coliforms (campos et al., 2017). on the contrary, viruses are not investigated as vehicles for foodborne disease transmission according to the above mentioned legislations. generally, viruses show a higher environmental resistance than bacteria, and depuration is poorly effective on decontamination of live bivalve mollusks (varela et al., 2016). the most common depuration systems are based on the use of chlorine, ultraviolet light (uv) and ozone. while chlorine can have organoleptic effects in mollusks and cause the formation of chlorinated by-products, uv and ozone have gained popularity in recent years but both of them can be limited because the first is effective in high flow rates (polo et al., 2014a) and ozone is influenced by some parameters such as temperature, salinity, ph and dissolved oxygen (ilic et al., 2017). the aim of this study is the evaluation of a depuration process in a closed-circuit system using both ozone and uv in clams (chamelea gallina), experimentally contaminated with murine norovirus-1 (mnv-1), because it has genetic and pathological features similar to hunov and therefore it can be used as surrogate (predmore et al., 2015; kim et al., 2017). ital. j. food sci., vol. 32, 2020 127 2. materials and methods 2.1. samples’ collection and depuration process samples of c. gallina (25-32 mm) were harvested along the central adriatic coast of molise region, italy, in 4 different periods (named a to d) of the year 2015 (from january to october) and put into aquariums containing seawater for acclimation and evaluation of their viability. then, they were transferred into tanks filled with artificial marine water (ocean fish, prodac international, padova, italy) and artificially contaminated for 72 hours with the mnv-1 provided by the istituto zooprofilattico sperimentale delle venezie (italy), up to a final concentration of 103 tcid50/ml in water. the depuration process was carried out for 72 hours in a closed-circuit system (tecno impianti international s.r.l., riccione, italy) equipped with uv and ozone. it consisted of 197x72x45 cm tanks with a perlon wool prefilter and hyperactive carbon filter, an active biological filter using lithothamnium calcareum algae and an uv sterilization plant. with regards to the sterilization unit and ozonator, power was 230v and power consumption was 16w and 0.5 a, respectively. aliquots of 100 specimens were analyzed at time 0 as negative control, before being placed in the depuration tanks, and further 3 aliquots were examined at intervals of 24, 48 and 72 hours. the clams were washed, opened and their digestive glands were pooled together and homogenized. then they were analyzed by both tissue culture and rt-pcr according to baert et al. (2008). 2.2. preparation of viral stocks mnv-1 was propagated in monolayers of raw 264.7 (mouse macrophage) purchased from american type culture collection (atcc-lgc standards, milano, italy) and cultured in 75 cm2 tissue culture flasks. the cells were maintaned in dulbecco’s modified eagle medium, dmem (gibco, new york, usa) supplemented with sterile phosphate buffered saline (pbs, ph 7.4), 1% antibiotics (penicillin, nystatin, gentamicin, streptomycin) and 10% fetal bovine serum (merck, darmstadt, germany). they were incubated at 37°c in a humidified atmosphere of 5% co2. for the preparation of viral stocks, the growth medium was removed, the cells were infected with mnv-1 with a virus titer of 103 tcid50/ml and incubated for 48 hours. when significant cytopathic effects were observed, the supernatant was centrifuged at 500 g at 4°c for 30 min. mnv-1 titer was assessed by both rt-pcr assay and traditional virus end point titration according to reed and müench (1938). 2.3. tissue culture assay an aliquot of pooled hepatopancreas (1±0.2 g) was homogenized with sterile quartz sand and diluted 1:10 (w/v) in antibiotics antimycotic solution (100x). the sample was stored at 4°c for 1 hour and centrifuged at 5000 g for 10 min. twenty-four well microplate monolayers of raw 264.7 were infected with 200 !l of the diluted sample (1:10 and 1:100) in dmem and incubated at 37°c in a humidified atmosphere of 5% co2 for 4-6 days. the presence of mnv-1 was evaluated by means of rt-pcr. ital. j. food sci., vol. 32, 2020 128 2.4. rt-pcr assay the glands were pooled and 2±0.2 g were homogenized with 2±0.2 ml of 0.1 mg/ml proteinase k solution (qiagen, hilden, germany), incubated at 37°c with shaking (320 rpm/1 hour) and centrifuged at 3000 g for 5 min. nucleic acids (100 !l of supernatant) were extracted using the biosprint 96 authomatic system (qiagen) with the biosprint 96 one for all vet kit (qiagen) according to the manufacturers’ instructions. ten !l of armored rna west nile virus (hny1999) (asuragen, santa clara, ca, usa) diluted 1:100 were added to each sample as an internal control to check for any rt-pcr inhibition phenomena. monolayers of raw 264.7 infected with10-fold serial dilutions of mnv-1 were used for the devolopment of the rt-pcr assay and the ct value was < 40. the master mix was prepared by using rna ultrasense one-step qrt-pcr system (invitrogen, carlsbad, ca, usa) as reported in table 1. table 1. composition of master mix. reagent c1 a c2 b vol (μl) h2o 5x 1.100 5x ultrasense reaction mix 50x 1x 4.000 rox reference dye (50x) 4.0 μm 1x 0.500 mnv-f 4.0 μm 200 nm 1.000 mnv-r 4.0 μm 200 nm 1.000 mnv-p 20 μm 200 nm 1.000 ns5-2 50 μm 80 nm 0.188 ns5-2f 50 μm 150 nm 0.100 ns5-2r 150 nm 0.100 rna ultrasense enzyme mix 1.000 total 10.0 a c1: initial concentration b c2: final concentration a primer and probe set was selected according to baert et al. (2008). the sequence of primer pairs and probes was as follows: for mnv-1, the probe was 5’fam-cgc ttt gga aca atg-3’mgb (mnv-p), the primer fw was 5’-cac gcc acc gat ctg ttc tg-3’ (mnv-f) and the primer rev was 5’-gcg ctg cgc cat cac tc-3’ (mnv-r); for hny1999, the probe was 5’vic-cca acg cca ttt gct ccg ctg-3’tam (ns5-2), the primer fw was 5’-gaa gag acc tgc ggc tca tg-3’ (ns5-2f) and the primer rev was 5’-cgg tag gga ccc aat tca ca-3’ (ns5-2r). all the primers and probes were purchased from eurofins mwg operon (louisville, usa). ten !l of master mix and 10 !l of viral rna were used for rt-pcr. the assay was performed on a 7900ht fast real-time pcr system (applied biosystems, foster city, ca, usa) at the following thermal conditions: 50°c for 15 min – 95°c for 2 min – 40 cycles (95°c for 15 sec – 60°c for 1 min). the analytical sensitivity of rt-pcr was tested analyzing the serial log10 dilutions of the mnv-1 tissue culture 105.9 tcid50/ml. ital. j. food sci., vol. 32, 2020 129 2.5. statistical analysis the pearson correlation coefficient with confidence intervals of 95% was used to measure the association between time and viral titer. 3. results and discussion the presence of mnv-1 in the artificially contaminated clams was observed by tissue culture assay just after 24 hours of exposure, even if the rt-pcr results showed that an interval of 72 hours was the optimum for the viral contamination because the values increased from 103.60 (24 hours) to 106.60 tcid50/ml (72 hours). the analytical sensitivity of rt-pcr resulted 100.9 tcid50/ml corresponding to 38 ct value (data not shown). the results of the different experiments of the depuration process carried out by the tissue culture assay showed values of 3.98x104 tcid50/ml for trial a and 1.48x104 tcid50/ml for the remaining trials. the viral titer did not vary among the 4 trials, because mnv-1 resulted always vital. the results of rt-pcr ranged from 103.17 to 104.60 tcid50/ml (data not shown). the pearson correlation coefficient was -0.15 (lower limit = -0.55 and upper limit = 0.29) and therefore not significant. a similar study was carried out by polo et al. (2014b) in samples of clams (venerupis philippinarum) and mussels (mytilus galloprovincialis) contaminated with mnv-1 and then depurated for 7 days by means of ozone and uv-c radiation for water sterilization. the average reductions compared with the initial levels of mnv-1 were 60.5% for clams and 91.6% for mussels, but they remained still infectious at the end of the process. polo et al. (2014a) as well found the presence of norovirus in clams and mussels after a depuration process based on water treatment by chlorination. the efficacy of depuration using traditional or closed-circuit system with disinfection by uv was evaluated by savini et al. (2009), which reported no statistically significant differences between depurated and non-depurated samples (i.e. m. galloprovincialis, tapes decussatus and crassostrea gigas) and indicated that the process was not able to remove norovirus. other studies (leal diego et al., 2013; imamura et al., 2016) showed the failure of depuration process applied on oyster samples using a system based on uv. these results demonstrated that the water exchange could be low and the initial contamination was too high (le mennec et al., 2017). therefore, some measures such as the increasing of depuration time and water circulation as well as the continuous exposure to uv treatment could be able to improve the effectiveness of the process. souza et al. (2013) detected mnv-1 in oysters until 96 hours of depuration in a closed system using different uv doses. the presence of mnv-1 after the depuration process described in the present study demonstrated that these viruses can survive and accumulate in live bivalve mollusks, and therefore they represent a source of foodborne disease for consumers. recent studies showed that norovirus strains can selectively accumulate in mollusks due to viral carbohydrate ligands of histo-blood group such as antigens in various tissues of clams, mussels and oysters (polo et al., 2014a; mcleod et al., 2017). therefore, the elimination of norovirus can be difficult using traditional decontamination treatments, because the virus is internalized within the cells of the digestive organs and other tissues of mollusks (kim et al., 2017). ital. j. food sci., vol. 32, 2020 130 according to the report of efsa and ecdc (2017), during 2010-2016 the number of reported foodborne outbreaks linked to calicivirus (including norovirus) was quite stable, with some differences among the eu member states. in particular, a statistically significant increasing trend was described in belgium, france, portugal and the netherlands, while austria, denmark, estonia and hungary reported a decrease. a national information system, called sinzoo was developed in italy aiming at the collection of data regarding food contamination and related zoonoses occurrence (colangeli et al., 2013). such a system highlighted 12 positive out of 176 mollusk samples, even if no outbreak linked to norovirus was described in the year 2017. the monitoring of viral contamination of mollusks represents an important tool for public health, especially because no legislative standards have been established for viruses. however, according to regulation ec no 853/2004 (ec, 2004b), each eu member state may adopt national measures in order to amend non essential elements such as additional health standards for live bivalve mollusks in cooperation with the relevant community reference laboratory, including virus testing procedures and virological standards. the multi-annual regional control plan for both abruzzo and molise regions (ppric 20152018) established sampling of mollusks every 2 months for e. coli and salmonella spp., and every 6 months for viruses. 4. conclusions the consumption of live bivalve mollusks collected from seawater contaminated with sewage pollution, due to malfunctioning of sewerage system, represents an important risk for human health. in this study, no significant viral reduction was observed, the closedcircuit depuration system was not able to reduce the level of mnv-1 and clams remained still infectious until the end of the experimental design. therefore, the ingestion of raw or undercooked mollusks should be avoided expecially by some population categories, such as immunocompromised individuals. acknowledgements the authors thank prof. francesca rosati (sworn translator) for her support in 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noroviruses and progressive emergence of gii.17 in wastewaters in italy (2011-2016) revealed by next-generation and sanger sequencing. food environ. virol. 10(2):141-150. trivedi t.k., desai r., hall a.j., patel m., parashar u.d. and lopman b.a. 2013. clinical characteristics of norovirusassociated deaths: a systematic literature review. am. j. infect. control 41:654-657. varela m.f., polo d. and romalde j.l. 2016. prevalence and genetic diversity of human sapoviruses in shellfish from commercial production areas in galicia, spain. appl. environ. microbiol. 82(4):1167-1172. paper received january 18, 2019 accepted september 19, 2019 ijfs#1068_bozza ital. j. food sci., vol. 31, 2019 75 paper modeling and optimization of process parameters for improving osmotic dehydration of kiwifruit b. liu, w. feng and b. peng* key laboratory of environment correlative dietology, ministry of education, college of food science & technology, huazhong agricultural university, 430070, wuhan, hubei, china *e-mail address: pengbangzhu@163.com abstract osmotic conditions for kiwifruit dehydration were optimized using central composite rotatable design and response surface methodology. the optimal conditions included osmotic time of 4.29 h, sucrose concentration of 70 %, and osmotic temperature of 50 °c. at these optimum values, water loss (wl) exhibited a response value of 45.64 %. the optimized condition was validated and found to be fitted with the experimental values. quadratic regression equations describing the effects of these factors on wl were developed. the osmotic dehydration of kiwifruit was significantly influenced by osmotic temperature, osmotic time, and sucrose concentration. moreover, osmotic process at relatively high temperatures caused a significantly depletion of vc content in kiwifruit. keywords: kiwifruit, osmotic dehydration, optimization, response surface methodology, water loss rate ital. j. food sci., vol. 31, 2019 76 1. introduction kiwifruit is one of the delicious fruits originating from china. although with full of phytochemicals, vitamins, and minerals, it has a relatively short shelf-life due to its highly perishable nature (kaya et al., 2010). the short shelf life of fresh kiwifruit after harvest is becoming one of the main factors that affect the rapid development of kiwifruit processing industry. osmotic dehydration is a potential preservation technique to reduce postharvest losses of fruits and vegetables and produce high-quality intermediate-moisture products (ahmed et al., 2016). it is widely used for partial removal of water from food materials as a pretreatment before further processing to improve texture characteristics, sensory, functional and nutritional properties (chiralt et al., 2001; torreggiani and bertolo, 2001; talens et al., 2002; rastogi and raghavarao, 2004). the use of osmotic dehydration can prolong the shelf life of the kiwifruit, as the water content reduction slows down deteriorative reactions. in osmotic dehydration process, food materials are used to immerse in concentrated solution creating a concentration gradient between the osmotic solution and food materials, the simultaneous mass transfer phenomena mainly include flow of water from the product to the solution, transfer of solute into the product, and leaching of the components of the product. the water is mainly removed by capillary flow and diffusion; meanwhile, leaching and solute uptake occur through diffusion (shi and xue, 2009). the rate of mass transfer during osmotic dehydration can be influenced by many factors, such as type and concentration of osmotic agents, temperature, agitation/circulation of solution, food to solution ratio, food structure, shape and size, thickness of food material, and pre-treatment (da conceicao et al., 2012; akbarian et al., 2013). osmotic temperature and solution concentration are the important factor, which affects osmotic mass transfer (tortoe, 2010). lombard et al. (2008) investigated the influence of the process temperature, pressure and osmotic concentration on the mass transfer process during the osmotic dehydration of south african grown cayenne type pineapple pieces, and the results showed water loss and solids gain increased with temperature and concentration. falade et al. (2007) studied the osmotic mass transfer phenomenon of water melon slabs using three different concentrations of sucrose solution (40, 50 and 60°brix). the water loss and solid gain of the watermelon slabs treated with the higher osmotic solution concentration were found to be higher. cao et al. (2006) found that the optimal conditions for osmotic dehydration of kiwifruit slice were 60% sucrose concentration, 30–40°c osmotic temperature, 150 min osmotic time, and 8 mm slice thickness. meanwhile, the influence of each factor or interactions among the factors should be determined to understand the behavior involved in mass transfer during osmotic dehydration. individual screening of these factors at a time is laborious and requires much experimental work (fernandes et al., 2006). therefore, an optimization technique for osmotic dehydration parameters must be established. response surface methodology (rsm) is an effective mathematical and statistical tool. it not only defines the effect of independent variables but also their interaction effects (myers and montgomery, 1995). the present study aims to determine the optimal osmotic dehydration conditions of independent variables (osmotic temperature, osmotic time, and sucrose concentration) for kiwifruit and validate the optimized conditions based on water loss rate by using rsm coupled with central composite rotatable design. in addition, the effects of different sucrose concentrations on kiwifruit water loss (wl) and solid gain (sg) rates were analyzed. ital. j. food sci., vol. 31, 2019 77 2. materials and methods 2.1. sample preparation and osmotic treatment fresh kiwifruits of xuxiang cultivar were obtained directly from a producer from qinyuan orchard located at mei county (shaanxi, china). the average values of single weight, titratable acidity, and total soluble solid contents in the kiwifruits were 94.2±0.2g, 1.32± 0.34% and 16.2±0.65 brix, respectively. the kiwifruits were washed and cut into cubes (1 cm × 1 cm × 1 cm) to prepare samples. then the cube samples were subjected to osmotic dehydration under different osmotic temperatures, osmotic times, and sucrose concentrations based on the experimental design shown in table 1. the ratio of the sample to the osmotic solution was 1:5 (wt/wt). in order to ensure concentration of the osmotic solution did not change significantly during the experiment, the osmotic system in a vessel was covered with a wrap to prevent evaporation without agitation. the temperature was controlled using a constant temperature water bath. after the osmotic treatment, the samples were removed from the osmotic solution, washed with distilled water, and blotted gently with a tissue paper to remove adhering water for the next analysis (ali et al., 2010; tylewicz et al., 2011). 2.2. central composite rotatable design for optimizing process parameters during kiwifruit osmotic dehydration a central composite rotatable design was used to optimize the conditions for osmotic dehydration of kiwifruit cubes. osmotic temperature (23-57°c), osmotic time (2.3-5.7h), and sucrose concentration (43-77%, w/w) were taken as independent variables to optimize wl rate and determine the efficiency of osmotic dehydration. the experimental data were fitted using multiple linear regression in equation (1) (baş and boyaci, 2007; peng et al., 2015): 𝑌 = 𝑏! + 𝑏!!! ! ! 𝑋! + 𝑏!! ! ! ! ! 𝑋! ! + 𝑏!" ! ! ! ! ! ! 𝑋!𝑋!, ! ! ! ! (1) where y is the wl rate, i and j are the linear and quadratic coefficients, respectively, xi and xj represent the independent variables, and bo, bi, bii, and bij are the regression coefficients. 2.3. mass transfer determination the process kinetic variables of wl and sg rates of the samples were calculated as described by singh et al. (2007) and falade et al. (2007) by using equations (2) and (3) with some minor adjustments: %100% 0 00 x m mmmm wl tt ）（）（ −−− = (2) %100% 0 0 x m mm sg t − = (3) where m0 and m0 are the initial mass weights of the kiwifruit samples and the dry solid mass in the samples (g), respectively; mt and mt are the mass weights of the samples and the dry solids (g) in the samples after the osmotic dehydration time t. ital. j. food sci., vol. 31, 2019 78 2.4. analytical determination moisture content was determined gravimetrically using a vacuum oven by drying to constant weight (aoac, 1997). ascorbic acid (vitamin c, vc) in kiwifruit is the most important vitamin for human nutrition. a standard ascorbic acid solution method was used to determine vc of kiwifruit based on the titration of ascorbic acid with 2,6-dichloroindophenol in acidic solution by the aoac’s official titrimetric method (aoac, 1990). the analysis was done in triplicate, and the result for each sample was averaged. 2.5. statistical analysis all tests were run in triplicate. analysis of variance (anova; origin software, originlab corporation, northampton, ma, usa) was used to indicate significant differences among tests. differences were considered significant at the p≤ 0.01 level. 3. results 3.1. model fitting in this study, central composite rotatable design coupled with rsm was used to optimize osmotic dehydration for kiwifruit cubes. the response of wl rate was selected on the basis that the response directly influenced the following drying efficiency of the product. the three independent variables, namely, osmotic time, sucrose concentration, and osmotic temperature (coded a, b, and c, respectively) were used to optimize the response of wl rate coded y. the experimental design and obtained values are shown in table 1. regression analysis of the response was conducted by fitting a suitable quadratic model in the case of the response variable to assess how well the model represented the data. the results of the analysis of variance (anova) are shown in tables 2 and 3. according to the estimated regression coefficients of the quadratic polynomial model in table 2, nonsignificant factors were removed. the regression model equation in terms of coded value was obtained to express the relationship between the investigated factors and wl rate: y=37.32 + 2.54 × a + 2.86 × b + 8.24 × c 0.21 × a × b 0.98 × × a × c 0.34 × b × c 2.31 × a2 0.82 × b2 1.82 × c2 (4) the regression model was a function of changes in sucrose concentration, osmotic temperature and time. the f-value of 38.80 implied that the model was very significant (p < 0.01) and accurately predicted the wl rate of the samples. moreover, the r2 of 0.7884 for the model is in reasonable agreement with the adjusted r2 of 0.9471, and the adequate precision of 21.532 indicates that the model has an adequate signal to noise ratio (table 3). as shown in table 2, osmotic time, sucrose concentration, and osmotic temperature significantly affected the kiwifruit dehydration rate (p < 0.01); the model of prob > f and less than 0.01 indicated that the regression equation exhibited high significance and reliability. meanwhile, the r2 of the regression model was found to be 0.972, greater than 90%, indicating the significant relationship between the independent variable and the response value (table 3). ital. j. food sci., vol. 31, 2019 79 table 1. experimental design and measured values of wl rate for osmotic dehydration of kiwifruit. no. a-osmotic time /h b-sucrose concentration/% c-osmotic temperature/℃ y-water loss rate*/% 1 4.00(0) 76.82(+1.68) 40.00(0) 41.20±1.84 2 4.00(0) 60.00(0) 40.00(0) 37.31±1.53 3 5.00(+1) 50.00(-1) 50.00(+1) 40.44±1.97 4 3.00(-1) 50.00(-1) 50.00(+1) 38.41±1.25 5 4.00(0) 60.00(0) 23.18(-1.68) 20.65±1.13 6 5.00(+1) 50.00(-1) 30.00(-1) 23.23±0.89 7 2.32(-1.68) 60.00(0) 40.00(0) 24.64±1.54 8 4.00(0) 43.18(-1.68) 40.00(0) 29.09±1.32 9 4.00(0) 60.00(0) 56.82(+1.68) 44.02±1.28 10 4.00(0) 60.00(0) 40.00(0) 37.31±1.05 11 5.68(+1.68) 60.00(0) 40.00(0) 37.26±1.57 12 4.00(0) 60.00(0) 40.00(0) 37.31±1.44 13 5.00(+1) 70.00(+1) 50.00(+1) 43.81±1.58 14 3.00(-1) 70.00(+1) 30.00(-1) 23.23±1.88 15 3.00(-1) 50.00(-1) 30.00(-1) 17.67±1.05 16 4.00(0) 60.00(0) 40.00(0) 37.31±2.15 17 3.00(-1) 70.00(+1) 50.00(+1) 43.01±1.86 18 4.00(0) 60.00(0) 40.00(0) 37.31±1.43 19 5.00(+1) 70.00(+1) 30.00(-1) 28.34±1.62 20 4.00(0) 60.00(0) 40.00(0) 37.31±1.45 ( ) coded levels for actual values of different parameters during osmotic dehydration of kiwifruit. *each combination with triplicate and water loss rate expressed by average value ± standard deviation. table 2. anova of wl rate regression model for osmotic dehydration of kiwifruit. source sum of squares degree of freedom mean square f value p-value* model 1251.66 9 139.07 38.80 <0.0001 a 88.29 1 88.29 24.63 0.0006 b 111.41 1 111.41 31.08 0.0002 c 926.79 1 926.79 258.54 <0.0001 ab 0.35 1 0.35 0.098 0.7602 ac 7.68 1 7.68 2.14 0.1739 bc 0.91 1 0.91 0.25 0.6251 a2 76.60 1 76.60 21.37 0.0009 b2 9.75 1 9.75 2.72 0.1302 c2 47.52 1 47.52 13.26 0.0045 residual 35.85 10 3.58 lack of fit 35.85 5 7.17 pure error 0.000 5 0.000 total 1287.51 19 a: osmotic time (h); b: sucrose concentration (%); c: osmotic temperature (℃). *p-values less than 0.01 indicate model terms are significant, and values greater than 0.1 indicate the model terms are not significant. ital. j. food sci., vol. 31, 2019 80 table 3. anova for response surface quadratic model. terms values standard deviation 1.89 mean 33.94 coefficient of variation (%) 5.58 r2 0.9722 adjusted r2 0.9471 predicted r2 0.7884 adequate precision* 21.532 *adequate precision measures the signal to noise ratio. a ration greater 4 is desirable. 3.2. linear effect of osmotic variables on wl rate for kiwifruit dehydration osmotic time, sucrose concentration, and osmotic temperature significantly affected (p < 0.01) the wl rate of the samples at the linear level (table 2). the coefficients of linear terms in the regression equation (equation 4) indicated that the wl rate of the samples was mainly influenced by osmotic temperature (p ≤ 0.01), followed by sucrose concentration (p ≤ 0.01) and osmotic time (p ≤ 0.01). in addition, the quadratic terms of osmotic temperature and time (p < 0.05) had significant effects, while the interaction of factors had no significant effect (p＞0.05) on wl rate within the investigated range (table 2). 3.3. interactive effect of osmotic variables on wl rate for kiwifruit dehydration considering the interactive effect of osmotic variables, fig.1 shows the response surface plot and contour plot of kiwifruit wl rate under the effects of input parameters of osmotic time, sucrose concentration, and osmotic temperature. some profiles for the quadratic response surface plot in the optimization of the two parameters were obtained by keeping the other parameter at zero levels for wl rate. as shown in fig.1a, the wl rate first gradually increases with increasing osmotic time and sucrose concentration and subsequently maintains a steady state. this trend may be rationalized by considering that the intracellular free water movement speed in kiwifruit accelerates with increasing sucrose concentration. the wl rate will gradually decrease with decreasing amount of free water. when the osmotic pressure between the solution and the internal kiwifruit cells reach the equilibrium, the wl rate will not change. fig. 1b and 1c demonstrate the same trends that the wl rate first increases and subsequently maintains a steady state under the interaction between two parameters. at lower sucrose concentration with increasing osmotic temperature, the wl rate increases gradually, but as the sucrose concentration increases, the wl rate increases rapidly with increasing osmotic temperature (fig.1b). similarly, the interaction between osmotic temperature and time showed similar positive correlation (fig.1c).this might be due to higher temperature led to swelling and plasticizing cellular membrane and rapider release of moisture from the kiwifruit cells, and viscosity of the sucrose solution was lower at higher temperature, which improved water loss from common surface of kiwifruit and osmotic solution. ital. j. food sci., vol. 31, 2019 81 a b c figure 1. response surface and contour plots for response of kiwifruit water loss rate during osmotic dehydration (a: the interaction between the osmotic time and sucrose concentration; b: the interaction between the sucrose concentration and the osmotic temperature; and c: the interaction between the osmotic time and temperature). 3.4. determination and experimental validation of optimal conditions process parameters can be optimized by finding the stationary point of the model equation in the ranges of tested independent parameters (peng et al., 2015). the optimal conditions were determined by maximizing the desirability of the response using design expert ital. j. food sci., vol. 31, 2019 82 software (version 6.0.4 by stat-ease, inc., mn, usa). the optimal conditions included osmotic time of 4.29 h, sucrose concentration of 70 % , and osmotic temperature of 50 ℃ with a predicted response value of 45.64 % for wl rate. a confirmation test was conducted using the optimum parameters identified by rsm to verify the adequacy of the regression models. the fitted values predicted by the models were compared with the experimental data. under these optimal conditions, the experimental value of wl rate is consistent with the predicted value with 3.89 % standard deviation (table 4). these values did not show any significant difference (p > 0.05). response surface method is reasonable and effective for optimization of wl rate of kiwifruit. table 4. optimal conditions and validation. osmotic time (h) osmotic temperature (℃) sucrose concentration (%) predicted water loss rate (%) experimental water loss rate (%)* standard deviation (%) 4.29 50 70 45.64 43.81 3.89 *experimental water loss rate expressed by average value with triplicate to eliminate the errors. 3.5. effect of sucrose concentration on wl and sg rates change in wl and sg rates for osmotic dehydration of kiwifruit under different sucrose concentrations was showed in fig. 2. the results showed that the wl and sg rates have similar trends in 50%, 60%, and 70% sucrose concentrations. osmotic time had a substantial effect on mass transfer kinetics. increasing the time increased the percentage of water loss and solid gain. from fig. 2a, wl rate rapidly increased in the first 5 h of osmosis, then increasing slowly. this phenomenon is due to the largest pressure difference between the kiwifruit cells and the surrounding hypertonic solution, thereby promoting the osmotic dehydration of kiwifruit in the initial stage of the penetration process and inducing rapid diffusion of the water molecules. as osmotic time continues, the pressure difference gradually decreases and the structural changes in kiwifruit tissues gradually occur, the mass transfer tends to reach the dynamic equilibrium state. the wl rate increases with increasing sucrose concentration, and higher concentrations of osmotic solution could facilitate the removal of moisture from the texture of food product and resulted in lower moisture contents and higher wl rate from the texture, consistent with some other reports. lenart (1992) reported that increasing the concentration of an osmotic solution led to high wl rate until the equilibrium level was achieved; by contrast, low-concentrated sucrose solution led to small wl and sg rates (tortoe, 2010). similarly, ramaswamy (2005) studied the effect of osmotic time on mass transfer, and the results showed that mass exchange occurred at a faster rate within the initial 2h followed by a reduction in drying rate during further processing time. the kiwifruit sg rate showed similar trends in 40%, 60%, and 80% sucrose concentrations (fig. 2b). the sg rate increased continuously throughout the osmotic time in the test range, and the increase in the sucrose concentration could raise the sg rate. high concentration promotes sucrose mass transfer from the solution to the kiwifruit cells. the concentration of an osmotic agent affects the mass transfer kinetics during osmotic dehydration (herman-lara et al., 2013). the difference in osmotic potential between the solution and the fruit sample resulted in a high diffusion rate of the solute and water (azoubel and murr, 2004; phisut, 2012). similarly, lazarides (1994) reported that apple processed at a temperature of 30 and 50°c resulted in higher sugar gain (up to 55%) ital. j. food sci., vol. 31, 2019 83 compared to room temperature condition. it is due to the swelling of membrane and plasticizing effect, which enhances the permeability of the membrane. a b figure 2. variation of wl and sg rates with time during kiwifruit osmotic dehydration under different sucrose concentrations at 50℃. 3.6. change of vc content in untreated and osmotic treated kiwifruits comparison of ascorbic acid (vc) content in untreated and osmotic treated kiwifruit samples at different osmotic temperatures was showed in fig. 3. the vc content of kiwifruit was significantly decreased by osmotic dehydration. it may be that vc is transferred from the kiwifruit to the osmotic solution with the water molecule moving from the inside of the kiwifruit during the osmotic process. moreover, the vc content of osmotic treated kiwifruit was decreased significantly with increasing the osmotic temperature, this may be because the internal molecular movement in kiwifruit osmotic system at high temperature were much faster, which accelerated the loss of internal vc molecules. this result is in agreement with the report by cao et al.(2006), which osmotic temperature was the most significant factor affecting the ascorbic acid loss. however, chakraborty and samanta (2016) found that the optimally dehydrated kiwifruit ital. j. food sci., vol. 31, 2019 84 demonstrated a significant increase in the ascorbic acid content by simultaneous osmotic dehydration using fructose as osmotic solution and vacuum drying under far‐infrared radiation. figure 3. comparison of vc content in untreated and osmotic treated kiwifruit samples at different osmotic temperatures. bars with different capital letters at each osmotic temperature are significantly different at p< 0.05. bars with different small letters at treated samples are significantly different at p< 0.05. 4. conclusions the optimization of the osmotic conditions for kiwifruit dehydration was successfully examined using the rsm. the optimal conditions comprised osmotic time of 4.29 h, sucrose concentration of 70 %, and osmotic temperature of 50 ℃ with a response value of 45.64 % for the wl rate. the wl rate of the kiwifruit cubes was mainly influenced by osmotic temperature (p ≤ 0.01), followed by sucrose concentration (p ≤ 0.01) and osmotic time (p ≤ 0.01). moreover, the vc content was decreased significantly with increasing the osmotic temperature. the optimized condition was validated and found to be fitted with the experimental values. therefore, osmotic dehydration of kiwifruit highly depends on osmotic temperature, osmotic time, and solvent concentration. the predicted model for wl rate established by the response surface quadratic regression provided an adequate mathematical description of kiwifruit osmotic dehydration. acknowledgements this research was partially supported by the fundamental research funds for the central universities (2662016py100). the authors acknowledge the anonymous reviewers very much for their valuable comments and improvements. references aoac. 1990. official methods of analysis of the association of official analytical chemists, 15th ed. arlington va: association of official analytical chemists. aoac. 1997. official methods of analysis of the association of official analytical chemists, 16th ed. arlington va: association of official analytical chemists. 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process by systematic approach systems. journal of food engineering 104(3):438-444. yetenayet b. and hosahalli r. 2010. going beyond conventional osmotic dehydration for quality advantage and energy savings. ethiopian journal of applied sciences and technology 1:1-15. paper received november 8, 2017 accepted june 25, 2018 ijfs#1597_bozza ital. j. food sci., vol. 32, 2020 4 opinion paper prebiotic effects of xylanase modification of β-glucan from oat bran on bifidobacterium bifidum x.j. qiua,b, w.x. zhenga,b, l. zhanga,b, y.l. shia,b, j.h. hua,b, y.l. lia,b, z.y. liua,b and m.d. zhu*a,b ainner mongolia university of technology, hohhot, inner mongolia, china binner mongolia energy conservation and emission reduction engineering technology research center for fermentation industry, hohhot, inner mongolia, china *corresponding author: tel.: 15904710399 email: zhumingda2003@163.com abstract oat β-glucan (bg) was isolated from oat bran, and the xylanase treatment was conducted to obtain modification of β-glucan (mbg). the relative molecular weight (mw) of bg and mbg was determined by gel permeation chromatography (gpc). results demonstrated that the mw of bg was reduced from 1.66×104 to 5.43×103. we assessed the prebiotic effect of bg and mbg on human colon bifidobacterium bifidum（b. bifidum). our findings suggest that the addition of bg and mbg resulted in a lower ph of the fermentation broths. both lactic and acetic acid production increased in the fermentation broths. while bg was found to significantly promote the proliferation of b. bifidum , mbg had a greater effect on b. bifidum. keywords: β-glucan, bifidobacterium bifidum, modification, prebiotic, xylanase ital. j. food sci., vol. 32, 2020 5 1. introduction oat bran is a by-product of the oatmeal production, produced during the milling process. it is a mixture that is mainly composed of seed coat, the aleurone layer, and oat germ, which accounts for 8%~12% of the total mass of oat seeds. it contains a variety of nutrients, such as dietary fibre, fat, protein, and minerals, and a large amount of dietary fibre represents a valuable renewable resource (zheng et al., 2017). oat bg is a soluble dietary fibre (sdf) present in the oatmeal grain endosperm and aleurone cell wall. its main components are (1-3) and (1-4)-β-d-glucan. after oatmeal processing, the bg is enriched in wheat bran. following claims made by the us food and drug administration (fda), many researchers have demonstrated an association between oat bg and a reduction in the risk factors of cardio-vascular disease, in particular by lowering the blood cholesterol and glucose levels (vitaglione et al., 2008; yan et al., 2017), and at the same time regulating the immune system and strengthening resistance (li et al., 2018). oat bg has also been found to relieve the immunosuppression of tumour cells and to have a good therapeutic effect on patients with early-stage cancer (mei et al., 2018). the enzymatic method involves the use of enzymes to enzymatically decompose a raw material and to remove surface impurities to obtain insoluble dietary fibre, which is then further enzymatically modified to obtain a water-soluble dietary fibre. studies have shown that the enzyme treatment of bran can effectively change the functional properties of dietary fibre by changing the structure or molecular rearrangement of polysaccharides (santala et al., 2014). the modification of bran by xylanase and cellulase can increase the soluble dietary fibre content of oat bran and reduce its water binding ability (lebesi et al., 2012). laccase assisted by high hydrostatic pressure and cellulose bran can increase the content of soluble dietary fibre, alter the honeycomb structure of dietary fibre, and produce new polysaccharides (ma et al., 2016). after the treatment of rice bran with xylanases, including amylase, glucoamylase, protease, and cellulase, the total phenolic, flavonoid, iron reducing antioxidant capacity, and oxygen free radical absorption capacity of modified dietary fibre has been found to significantly improve (liu et al., 2017). however, the precise role of bg in the enzymatic treatment of the human requires further elucidation. the definition of prebiotics has been suggested by gibson et al. (2017) as “a substrate that is selectively utilized by host microorganisms conferring a health benefit”. the catabolism of prebiotic carbohydrates by metabolic activity of the gut microbiota primarily produces short chain fatty acids (scfa). the most abundant scfa in the colon is acetate, which in general represents more than half of the scfa content detected in feces (louis et al., 2007). prebiotic substrates can selectively promote the growth of beneficial microorganisms and induce changes in the levels of these scfa in healthy individuals (lecerf et al., 2012). thus, scfa levels represent an indirect measure of the level of beneficial microorganisms in the gut and their impact on human health. in this study, inner mongolia oat bran was used as raw material to extract oat bg by xylanase enzymatic hydrolysis to explore the prebiotic effects on bifidobacterium bifidum before and after enzymatic hydrolysis. ital. j. food sci., vol. 32, 2020 6 2. materials and methods 2.1. chemicals and media the xylanases (1.67 millikatal (mkat)/g) used in this study were purchased from yuanye biotechnology co., ltd. (shanghai, china). all other chemicals and media were of analytical grade and obtained from haibo biotechnology co., ltd. (qingdao, china). b. bifidum (strain number cicc 6168) was purchased from china center of industrial culture collection. hplc-grade water was prepared using a milli-qplus purification system (millipore corp., bedford, ma, usa). oat bran was produced in the inner mongolia autonomous region (hohhot, china). 2.2. crude bg extraction the isolation of bg was performed as follows: fat was removed from oat bran using a 60mesh screen with a soxhlet extractor. the defatted oat bran (50 g) was then soaked in distilled water (0.5 l), then gelatinized at 100°c for 15 min, and finally incubated with heat-resistant a-amylase at 95°c for 1.5 h to remove starch, followed by centrifugation (2000 ×g, 15 min). insoluble dietary fibre (idf) was the resulting precipitation. the supernatant was isoelectrically deproteinized in a naoh solution (0.75 mol/l, 50°c, 2 h). the ph was adjusted to 6.5 to remove protein. after centrifuging at 2000 ×g for 5 min, 200 ml of 950 g/kg ethanol solution was added to the crude bg precipitate (60°c, 2 h). the precipitation (crude bg) was then dried at 60°c for 12 h in an air-drying oven. 2.3. enzymatic hydrolysis of bg the enzymatic treatment was conducted as follows: the precipitation (idf) was dried at 60°c for 12 h using an air-drying oven, then grinded and sieved through a 250 mm mesh. the crude bg and idf were combined and treated with xylanase. the mixture (30 g) was soaked in hot water (500 ml) at 50°c for 0.5 h. then, xylanase was added at 667 nanokatal/g (mixture) and the slurry was incubated for 2 h (50°c, ph 5.0). the treated slurry was centrifuged at 2000 ×g for 5 min after the inactivation of the enzyme in a boiling water bath for 10 min. the resulting insoluble material was washed twice with hot water (pre-heated to 50°c) and centrifuged (2000 ×g, 5 min) again. then, 150 ml of 950 g/kg ethanol solution was added to the precipitated crude modification of β-glucan (mbg), and the residue was dried at 60°c for 12 h using a hot-air oven. 2.4. bg and mbg purification bg crude powder (0.1 g) and mbg crude powder (0.1 g) were dissolved in 5 ml of water, respectively. after full dissolution, the solution was used for anion exchange column chromatography, using water as the eluent, at a flow rate 30 ml/h. using 10 ml per tube, the eluent was collected from each tube. the polysaccharide distribution was determined using the phenol-sulfuric acid method, and the protein content was determined by 280 nm colorimetry. a single peak of polysaccharide was combined. the bg solution and mbg solution obtained were concentrated to 5 ml under reduced pressure, respectively, and the supernatant was centrifuged for use. the bg and mbg concentrates treated by deae sepharose cl-6b anion exchange column chromatography was respectively used for gel permeation column chromatography using ital. j. food sci., vol. 32, 2020 7 water as an eluent at a flow rate of 30 ml/h. the eluate was collected from each tube. polysaccharide distribution and protein content were detected by the phenol-sulfuric acid method and 280 nm colorimetry, respectively. a single peak of polysaccharide was combined. bg and mbg were concentrated under reduced pressure before freeze drying. 2.5. determination of average molecular weight (mw) of bg and mbg the mw of bg and mbg was determined by gpc. the purified bg and mbg (1 mg) were analysed using a pl aquagel-oh mixed chromatographic column at 45°c. the mobile phase consisted of 0.1 mol/l nano3 at a flow rate of 0.9 ml/min. the quantification was performed using a vex differential refractive index detector (pl gpc-220, agilent technologies inc. california, usa). 2.6. the prebiotic effect of bg and mbg on b. bifidum 2.6.1. strains activation b. bifidum freeze-dried strains were used, such that the strains were activated and cultured before carrying out the experiments. the formulation of medium was provided by the china center of industrial culture collection, and is provided in table 1. here, 30 ml of bbl medium was placed in 100 ml vial, followed by vacuum pumping and sterilization at 121°c for 30 min. the inoculation operation was carried out in an anaerobic incubator. for this, b. bifidum lyophilized powder was fully dissolved and inoculated into 1 ml of sterile medium. the culture period of bacteria is normally 1~2 days, however the first-generation revival culture needed to be extended appropriately. as such, the duration of this experiment was 3 days. the cells were statically cultured at 37°c in an anaerobic culture incubator. the first-generation revival cultured cells that survived were sub-cultured for two generations with 10% inoculation, with a culturing time per generation of 2 days. table 1. bifidobacterium bifidum medium (bbl) formula (per litre, /l). name dose name dose yeast extract 3 g beef extract 10 g peptone 10 g soluble starch 1 g glucose 5 g l-cysteine hydrochloride 0.5 g sodium chloride 3 g sodium acetate 3 g resazurin 3 mg ph 6.8 2.6.2 effect of bg and xylanase mbg on the growth of b. bifidum to investigate the effect of bg and mbg on the growth of b. bifidum, bg and mbg were used as the sole carbon source. bg and mbg were substituted for the glucose in the bbl medium. here, 0.5 g of bg and mbg were used to replace glucose, and 100 ml of bbl medium was prepared with an inoculation amount of 5%. after inoculation, the solution was placed in a 37°c anaerobic culture incubator for 24 h. the od value was measured using an ultraviolet-visible spectrophotometer at a wavelength of 600 nm after 24 h. the ital. j. food sci., vol. 32, 2020 8 bbl medium not inoculated with bacteria was used to adjust the reading to zero. as can be seen in table 1, 15 g of agar was added to bbl medium to prepare the bbl agar medium. the fermentation broth was coated and inoculated into bbl medium before culturing in an anaerobic incubator at 37° c for 24 h. 2.6.3 b. bifidum fermentation broth ph determination the initial ph of the b. bifidum fermentation broth was measured before fermentation, and a sample was measured once every 6 h. for the measurement, 5 ml of the fermentation broth was sampled, centrifuged at 2000 ×g for 10 min, and the resulting supernatant was measured using a ph meter. 2.6.4 effect of bg and mbg on the concentration of scfa here, 0.5 g of bg and mbg were added to 100 ml of bbl medium with glucose. bbl medium with 5 g of glucose per litre was used as a negative control. the inoculation amount was 5%. after the inoculation was completed, it was incubated at 37°c in an anaerobic culture incubator for 24 h. the bacteria solution was filtered through a 0.22 μm filter membrane before performing high performance liquid chromatography after 24 h. the three groups of b. bifidum solutions were analysed using a hpx-87h chromatographic column at 50°c. the mobile phase consisted of 0.005 mol/l sulphuric acid at a flow rate of 0.5 ml/min. the quantification was performed by high performance liquid chromatography (waters 2695, waters technology co., ltd. milford, usa). this quantification was also carried out on a bacterial solution cultured for 0 h, that is immediately after inoculation, to determine the increased in lactic acid and acetic acid production and used as the control group. 2.7. statistical analysis all measurements were carried out at least in triplicate. the results presented are the mean±standard deviation (sd) of each treatment (n=3). the differences were considered significant when p<0.05. 3. results and discussion 3.1. determination of mw of bg and mbg the mw distribution of untreated bg and mbg is shown in fig.1. the mw represents the statistical average molecular weight of the polysaccharide compared to the average weight of different molecular weights, and the average molecular weight (mn) represents the statistical average molecular weight of the molecules in the polysaccharide with different molecular weights. according to fig. 1(a) and table 2, the mw of the bg which was not subjected to the enzyme treatment was 1.49×104. the mw/mn value was 1.03, which was close to 1, indicating that the mw distribution of bg was uniform, and the distribution was concentrated around the average molecular weight. as shown in fig. 1(b) and table 2, the mbg exhibited four mw segments, 5.98×105, 2.68×104, 1.66×104, and 5.43×103, respectively. the mw/mn values were 1.10, 1.01, 1.02, and 1.23, respectively, that is close to 1, indicating that the mw distribution of mbg was uniform and that the distribution was concentrated ital. j. food sci., vol. 32, 2020 9 on the average of the four molecular weight segments. among them, the mbg of two mw fractions, 5.98×105 and 2.68×104, accounted for 2.20% and 8.78% of the total content, respectively, and the content was low. after xylanase enzymatically cleaves the bran xylan, the macromolecular bg originally linked to xylan may be isolated. its high molecular weight (hmw) of 1.66×104 is similar to that of non-enzymatically-treated bg, and should be the same type of bg. the mbg with a low molecular weight (lmw) of 5.43×103 could be the result of enzymatic hydrolysis of xylanase. xylanase destroys the β-1,4glycosidic linkage of connecting bg, thereby decreasing the mw of bg, with a resulting molecular weight of 1.66×104. among these, mbg with molecular weight of 5.43×103 accounted for 86.28% of the total content. the use of xylanase can be also a smart strategy bg to convert lmw, the mw of mbg showed that the mw of bg was reduced from 1.66×104 to 5.43×103. the mw of bg plays an important role in determining the physiological efficacy of bg in terms of health benefits. incorporating lmw bg may influence the palatability of food and has been shown to lower cholesterol in men (pins et al., 2005) and animals (wilson et al., 2004). a b figure 1. mw distribution of bg and mbg. ital. j. food sci., vol. 32, 2020 10 table 2. mw of each component of bg and mbg. peak number mp mn mw mz area (mv.secs) % area b1 12237 14501 14938 15420 777.567 100 m1 578382 544525 598568 660548 94.3102 2.75 m2 25501 26587 26868 27167 75.6232 2.20 m3 15732 16311 16600 16899 300.825 8.78 m4 5024 4415 5435 6492 2959.55 86.28 furthermore, the action of xylanase on idf leads to the formation of two mw fractions, 5.98×105 and 2.68×104, which may be water soluble. as illustrated in fig. 2, this conversion of highly polymerized idf into sdf was achieved by performing a tailored enzymatic treatment. polysaccharides and other polymers are cross-linked to the cell wall of the cereals with other components to form a structural network. b-(1-3,1-4)-d-glucans and arabinoxylans are the major cell wall polysaccharides in oat bran and are composed of a backbone of β-(1,4) linked d-xylopyranosyl residues. moreover, a-l-arabinofuranoside can be present at the c (o)-3 and/or the c (o)-2 positions of the xylose moieties, and arabinoxylans can be cross-linked to ferulic acid at the c (o)-5 positions via ester linkages. many of these polysaccharides would require enzymatic hydrolysis to be removed from the structure, formed by covalent and non-covalent cross-linking. for example, cellulose and hemicelluloses treatments can be used to improve the quality of fiber-enriched oat bran by using xylanase treatment on the fibre fraction. enzymatic methods have been used as means for modification to improve the extractability of polysaccharides and increase yields (laurikainen et al., 1998). similar studies have reported changes in the chemical bonds of the polysaccharide and bran dietary fibre molecular structures via various enzymatic treatments (saulnier et al., 2009; ya et al., 2017). figure 2. schematic representation of the effects of xylanase on bg. ital. j. food sci., vol. 32, 2020 11 3.2. the prebiotic effect of bg and mbg on b. bifidum 3.2.1 effect of bg and mbg on the growth of b. bifidum according to the experimental methods, in which the od600 of the bacterial solution of b. bifidum was measured in vitro after 24 h of anaerobic fermentation, the od value of the bacterial solution was 0.041 cultured in bbl medium of 5 g/l glucose at 37 °c for 24 h, while the od value of the bacterial solution were 0.089 and 0.244 cultured in bbl medium of 5 g/l bg and 5 g/l mbg, respectively. the od value of the bacterial solution increased significantly after the addition of bg. the od value of the bg bacterial solution then increases again after xylanase treatment. the positive correlation between the od value of the bacterial solution and the number of bacteria suggests that bg can promote the proliferation of b. bifidum. however, the promotion of proliferation was greater after the addition of mbg. this may be due to b. bifidum being more easily oxidized in the lmw segment than in the hmw segment of bg during the oxidation of sugar. non-digestible polysaccharides cannot be degraded by mammalian enzymes. therefore, following ingestion, these glycopolymers are delivered intact to the large intestine, where they may influence the growth or metabolic activity of members of the gut microbiota. in this context, there is growing scientific evidence of the possible prebiotic effects elicited by non-digestible polysaccharides towards various microorganisms of the mammalian gut (vitaglione et al., 2008; tan et al., 2006). after bg enters the large intestine as a soluble dietary fibre, probiotics such as b. bifidum pass through the extracellular glycosidase to promote bg degradation and utilization, thereby promoting the proliferative metabolism of probiotics. 3.2.3 change in ph of b. bifidum fermentation broth the decrease in the ph of the fermentation broth was mainly the result of organic acid production during fermentation. as can be seen in fig. 3, compared to the negative control, the ph of the fermentation broth decreased rapidly after the addition of untreated bg and mbg. the environment changed from alkaline to acidic after 24 h of fermentation. the addition of mgb resulted in a more rapid decrease of the fermentation broth’s ph compared to bg. metabolically produced organic acid reduced the intestinal ph environment, resulting in an acidic environment for the intestines, thereby inhibiting the growth of harmful bacteria and promoting to intestinal health. there is also considerable evidence that supports the role of fibre in the promotion of health by its ability to modulate gut microbiota composition and metabolism (slavin et al., 2013). the proposed benefits of fibre on the intestinal microbiota are associated with their uptake and utilization by putative health-promoting bacteria species and the subsequent cross-species metabolism of fermentation by-products (holscher et al., 2015; verbeke et al., 2015; tap et al., 2015). ital. j. food sci., vol. 32, 2020 12 figure 3. the effects of time on ph in fermented broth. note: b1: bbl medium of 10 g/l glucose; b2: 5 g/l bg plus 5 g/l glucose bbl medium; b3: 5 g/l mbg plus 5 g/l glucose bbl medium. 3.2.4 effect of bg and mbg on the concentration of scfa the formula of the medium (table 1) was obtained via an optimization study based on strong metabolic and molecular studies by the china center of industrial culture collection. the production of lactic acid in the bacterial solution was measured after culturing the cells, as described in the methods. the effect of bg and mbg on the concentration of lactic acid and acetic acid in the fermentation broth is shown in figure 4. b. bifidum was cultured at 37℃ for 24 h. the yields of lactic acid and acetic acid in the control (b1, 10 g/l glucose bbl medium ) were 0.5316±0.0033 g/l and 0.3927±0.0043 g/l, whereas the fermentation of b. bifidum with 5 g/l bg plus 5 g/l glucose bbl medium and 5 g/l mbg plus 5 g/l glucose bbl medium, respectively, as a carbon source resulted in greater yields of lactic acid and acetic acid than the control. an analysis of the metabolic activity in both cultures showed that lactic acid production increased 56% during bg treatment (0.8316±0.0265 g/l) and by 184% during mbg treatment (1.5091±0.0151 g/l; p<0.05) compared to the control. the acetic acid production increased 12% during bg treatment (0.4388±0.0033 g/l) and by 30% during mbg treatment (0.5117±0.0046 g/l; p<0.05) compared to the control (figu. 4). these results demonstrated that the addition of bg and mbg to b. bifidum during fermentation with glucose as a substrate can significantly promote the production of metabolic acid, since the increase in acid production of mbg was greater. the overall beneficial effects produced by mbg were higher than those induced by bg. the addition of mbg and bg to the cultures beneficially influenced the fermentation patterns of b. bifidum, demonstrate by the higher scfa production and remarkably higher levels of lactic acid. ital. j. food sci., vol. 32, 2020 13 figure 4. effects of bg on the lactic acid and acetic acid concentration in fermentation solution (g/l). b1: bbl medium of 10 g/l glucose; b2: 5 g/l bg plus 5 g/l glucose bbl medium; b3: 5 g/l mbg plus 5 g/l glucose bbl medium. the scfa microbial metabolites are of particular interest, and have been suggested to promote health by regulating hormone release in the gut, as well as the cholesterol synthesis/metabolism to enhance satiety, also exerting anticancer and anti-inflammatory effects (vinolo et al., 2011; wong et al., 2006). the experiments showed that b. bifidum could produce acid by glycolysis with glucose as the carbon source. the mbg of the small molecular segment was more easily utilized by b. bifidum than the bg of the large molecular segment. the promotion of lactic acid production by probiotics was also more evident. the fermentation properties of b. bifidum are strongly influenced by the degree of polymerization of non-digestible polysaccharides. the increased scfa production can be explained by the additional b. bifidum biomass resulting from the prebiotic effect of bg and mbg. 4. conclusions oat bran dietary fibre was treated with xylanase to obtain mbg, which varied in its structure and properties compared to the original bg. we found that the mw of mbg was reduced from 1.66×104 to 5.43×103, as determined by gel permeation chromatography. the addition of bg and mbg to the fermentation broth of b. bifidum significantly promoted the proliferation of b. bifidum, and the proliferation of b. bifidum in the lmw segment of mbg was greater. the b. bifidum bacteria metabolites, lactic acid and acetic acid, were detected. moreover, with glucose and bg as the carbon sources, the acid production of b. bifidum increased significantly. the production of lactic acid production in mbg increased significantly as a result. ital. j. food sci., vol. 32, 2020 14 acknowledgments the project was partially supported by technology cooperation project for industryuniversity-research of hohhot (grant no. 2015150103000137) and fundamental research funds for mongolia industrial university (grant no. zy 201803). references 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zhaolin*2, 3 1college of biological sciences and biotechnology, beijing forestry university, beijing 100083, p.r. china 2analysis and testing center, beijing forestry university, beijing 100083, p.r. china 3department of beijing key laboratory of forest food process and safety, beijing forestry university, beijing 100083, p.r. china *corresponding author: zhaolinlv@126.com abstract the volatile composition of five blueberry varieties from two different regions was analysed by dynamic headspace (purge and trap, p&t) coupled to gas chromatographymass spectrometry (gc-ms). under the optimized conditions, the p&t method was successfully validated, showing good linearity, high accuracy, good reproducibility and a low limit of detection. a total of 80 volatiles were identified, including 19 esters, 30 alcohols, 18 aldehydes, 7 ketones and 6 other compounds. furthermore, a spider web diagram was constructed to compare the flavour profiles of these blueberries, and the obtained results demonstrated that blueberries from different locations have different flavour profiles. keywords: aroma active compounds, blueberry, gc-ms, p&t, volatile compounds ital. j. food sci., vol. 32, 2020 483 1. introduction blueberries have been recognized by the scientific community and consumers for their health-promoting potential (silva et al., 2017). the history of blueberry cultivation in china is approximately 20 years old, and chinese blueberries were mainly introduced from the united states and japan. there are three main types of blueberries: highbush (vaccinium corymbosum), lowbush (vaccinium angustifolium), and rabbiteye (vaccinium virgatum). highbush blueberries can be further divided into northern highbush and southern highbush blueberries (du and rouseff, 2014). northern highbush and lowbush blueberries are the predominant varieties in the greater khingan range. southern highbush and rabbiteye blueberries are generally grown to the south of the yangtze river (he and wu, 2010). in addition to being rich in vitamins and anthocyanins, blueberries are rich in volatile compounds such as ethyl acetate, butyl acetate, and 1-nonanal. flavour and aroma are two of the most important fruit quality characteristics and ultimately determine consumer acceptability and purchase decisions (du and rouseff, 2014). volatile compounds are important contributors to fruit aroma, which is one of the main characteristics that determine blueberry organoleptic quality and style (sun et al., 2013). different proportions of volatile components determine the overall aromatic properties (lv and lin, 2015). people realized the importance of volatile compounds with regards to aroma approximately 50 years ago. however, due to equipment being less advanced, studies of blueberry aroma are still very limited. the volatile compounds of highbush blueberries were analysed by parliament and kolor in 1975, and 18 individual components were identified by mass spectrometry, infrared analysis and gas chromatographic retention times (parllment and kolor, 1975). hall et al. (1970) used gas-liquid chromatography (glc) to examine the aromatic composition of lowbush blueberries. acetaldehyde, methyl acetate, ethyl acetate and ethyl alcohol were reported as the major aromatic compounds. currently, with the emergence of detection techniques with high sensitivity and accuracy, such as gas chromatography-olfactometry (gc-o), gas chromatography-mass spectrometry (gc-ms) and liquid chromatography-mass spectrometry (lc-ms), more volatile compounds at relatively low concentrations and thresholds are expected to be detected. purge and trap (p&t), also known as dynamic headspace, has been widely used for the preconcentration of volatile compounds (larreta et al., 2008). with p&t, an inert gas is purged throughout the sample in the same way as which we breathe, making this technique suitable for correlation with organoleptic studies (aznar and arroyo, 2007). it can be applied to solid or liquid matrices (murat et al., 2012). compared to spme, the high recovery of very volatile compounds and the low dispersion associated with the use of a totally automated system are the main advantages of p&t-gc-ms-based methods (soria et al., 2009). in this study, five blueberry varieties from two major blueberry production areas were identified (i) by purge and trap coupled to gas chromatography-mass spectrometry (p&tgc-ms). to provide a representative analysis of the blueberry volatiles, we first (ii) optimized this method and evaluate its correctness and then (iii) drew a spider web diagram to compare the flavour profiles of these blueberries. ital. j. food sci., vol. 32, 2020 484 2. materials and methods 2.1. plant materials all the samples were purchased from the hulun buiroroqen pristine production co. ltd. blueberries were squeezed into juice, diluted three-fold, and filtered for analysis. in this work, a total of five blueberry taxa were used to study volatiles. these taxa included two wild blueberries and three cultivated blueberries (table 1). table 1. blueberry taxa in this study*. taxa characters origin population wild blueberry around the humus mohe area of greater khingan range wh-m wild blueberry around the stones mohe area of greater khingan range ws-m cultivated blueberry bluecrop greater khingan range cb-g cultivated blueberry powderblue greater khingan range cp-g cultivated blueberry britewell yangzhou cb-y *cb-g is northern highbush blueberry; cp-g and cb-y are rabbiteye blueberries. 2.2. chemicals nacl and n-alkanes (c6-c22) were purchased from beijing chemical reagents co. ltd. (beijing, china). analytical grade 2-methylbutyraldehyde, ethyl acetate, 2-nonanone, linalool, and ethyl caprylate were purchased from sigma-aldrich co. ltd. (shanghai, china). 2.3. volatile compounds extracted by purge and trap p&t was performed by an eclipse 4660 purge and trap sample concentrator with a 4551a autosampler (oi analytical company, usa) and a #10 trap. three millilitres of each juice sample was placed in a 5 ml purge tube. nitrogen gas was utilized as a purge at 10 psi at 25°c. the other analytical conditions were as follows: trap temperature: purge, 30°c; desorption, 190°c; transfer line, 110°c; and valve oven, 110°c. time: purge 11 min; desorption 1 min. 2.4. gc-ms conditions chromatographic analysis was performed in a gc-ms (qp2010 ultra, shimadzu corporation, japan) system equipped with a rtx-5ms capillary column (0.25 mm×30 m×0.25 μm) (restek, usa). helium was used as the carrier gas at a linear velocity of 1.0 ml/min. the column temperature was held at 50°c for 5 min, increased to 180°c at a rate of 10°c/min, increased to 210°c for 5 min at a rate of 5°c/min, and finally increased to 280°c at a rate of 20°c/min. the mass selective was operated in the electron ionization mode at 70 ev and a scan range m/z of 45-400. ital. j. food sci., vol. 32, 2020 485 2.5. identification of volatile compounds volatile compounds were identified by matching their mass spectra with those of the known compounds from the nist 11/11s edition library. the relative odour activity value (roav) was calculated to measure the contribution of each volatile compound towards the whole aroma profile and was calculated using the following equation (zhuang et al., 2008; gu et al., 2012). roavs were calculated by using eq. (1): roavi = !"% !"#$%% × !"#$% !" ×100 (1) where “stan” is the volatile compound that has the highest relative contents; roavi is the odour activity value of the compound in sample i; ci is its content; and ti is its odour threshold concentration. compounds with a roav ≥1 significantly contribute to the aroma. (zhuang et al.,2016). 2.6. statistical analysis significant differences in the volatile compounds of the five blueberry varieties obtained from duplicate analysis were determined by one-way anova with spss 17.0 for windows (spss inc., chicago, il). statistically significant differences were determined at p<0.05. the originpro system (v8.5 sr6, originlab corporation, northampton, ma, usa) was used for statistical analysis. 3. results and discussion 3.1. optimization of the p&t-gc-ms method this study optimized the following p&t extraction parameters: sample volume, purge temperature and purge time. ethyl acetate, ethyl caprylate, 2-methylbutyraldehyde, 2-nonanone and linalool were used as standard compounds for optimization of the p&t-gc-ms method. as shown in fig. 1, varying volumes of blueberry juice (3, 4, and 5 ml) were placed in the trapping apparatus flask and purged for 11 min at 25°c. for ethyl octanoate, ethyl acetate, and linalool, there was a considerable difference between the various sample volumes (p<0.05). for 2methylbutyraldehyde and 2-nonanone, the relative percentages of these standard compounds in the 3 ml groups increased compared with the high sample quality group, but there was no significant difference (p>0.05). this study also showed that the number of volatile substances obtained from 3, 4, and 5 ml was 52, 50, and 49, respectively. the reason for this result may be that a high liquid level is too close to the top of the purge trap, so when a large amount of n2 purifies the liquid, extra water could be purged into the trap, which can shorten the trap life in the same way as a longer purge time (deng et al., 2011). ital. j. food sci., vol. 32, 2020 486 figure 1. effect of sample volume on the extraction efficiency; purge temperature = 25°c, purge time = 11 min. figure 2. effect of purge temperature on the extraction efficiency. sample volume = 3 ml, purge time = 11 min. ital. j. food sci., vol. 32, 2020 487 fig. 2 showed the effect of purge temperature on the extraction efficiency. the purge temperature varied between 25-60°c with 3 ml of sample volume for 11 min. the amount of total volatiles detected in blueberries gradually decreased as the purge temperature increased from 25°c to 60°c, probably due to the amount of water that reached the trap and decreased the sensitivity; therefore, ambient temperature was maintained in all the experiments (campillo et al., 2004). the effect of purge time on the sensitivity is shown in fig. 3. the purge time was varied between 8 and 14 min with 3 ml of sample volume at 25°c. finally, a value of 11 min was chosen as the optimal time, since 8 and 14 min led to a slight decrease in the peak area and total number. eight minutes is too short mainly because the volatile substances are not fully blown out. indeed, 14 min decreased the signals because a flow of n2 that was too long could move the volatiles from the trap before desorption and reduce the final signal (campillo et al., 2004). figure 3. effect of purge time on the extraction efficiency. sample volume = 3 ml, purge temperature = 25°c. verification and quantitative analysis (of p&t-gc-ms method) once the final purge conditions were selected, these five aroma standards were detected. table 2 shows the results from method validation: linearity, recovery, reproducibility, lod and loq. linearity the linearity of the method was evaluated by analysing a series of aromatic standards. linearity was found in the concentration range between 5 and 160 µg/l, with high ital. j. food sci., vol. 32, 2020 488 reproducibility and accuracy. regression analysis of the experimental data points showed a linear relationship with excellent regression coefficients (r2>99%) for 2-methylbutyraldehyde, ethyl acetate, 2-nonanone, linalool, and ethyl caprylate. table 2. performance parameters of the p&t method for the volatile compounds in blueberry. compounds linearity (r2) recovery (%) c.v. (%) lod (μg/l） loq (μg/l) 2-methylbutyraldehyde 0.9982 117.01 4.3418 0.78 2.60 ethyl acetate 0.9971 91.52 1.7276 0.90 3.00 2-nonanone 0.9957 103.47 2.8291 1.02 3.40 linalool 0.9933 99.33 5.4841 1.06 3.50 ethyl caprylate 0.9964 106.18 7.1420 1.29 4.30 recovery recoveries ranged between 96% and 120%, indicating that the accuracy of the method meets the experimental requirements and that the results are reliable. reproducibility reproducibility was evaluated by using the coefficient of variation (cv%) for replicate analyses. the cv% values obtained are shown in table 3. cv% values were found to be <8% in the case of relative proportions (hakala et al., 2002). the smallest cv% was found for ethyl acetate (1.73%), and the largest was found for ethyl octanoate (7.14%). in the range of esters, as the carbon number increases, the coefficient of variation also increased. as shown above, the p&t-gc-ms technique was reproducible enough to allow for comparative comparison studies of the volatiles of different varieties (hakala et al., 2002). determination of the limit of detection (lod) and the limit of quantification (loq). the lod was calculated as the concentration required to obtain a signal that was three times higher than that of the baseline signal (pino and oueris, 2010). detection limits were below 1.29 µg/l for all volatiles. the loq can also be estimated as the concentration of analyte producing a signal that is 10 times that of the noise (s/n = 10) (pino and oueris, 2010). from the above results, good linearity, high accuracy, very good repeatability and a low limit of detection were achieved (deng et al., 2011). there were also good recoveries and reproducibility. this method can be applied for research on the volatiles in blueberries. in conclusion, 3 ml of sample purged at 25°c for 11 min were selected as the best extraction conditions for the p&t methodology developed in this study. ital. j. food sci., vol. 32, 2020 489 table 3. analysis of volatile compounds from different blueberry varieties. no. ri tr (min) compounds relative content (%) wh-m ws-m cb-g cp-g cb-y esters 1 487 1.603 methyl acetate 0.14±0.01 2 584 1.644 ethyl formate 9.9±0.50a 8.81±0.43a 9.54±0.51 a 3 586 1.891 ethyl acetate 60.94±3.05a 66.21±3.24a 4.89±0.22c 1.58±0.09c 17.36±0.95b 4 686 2.518 ethyl propionate 0.48±0.02a 0.11±0.01b 5 686 2.540 propyl acetate 0.14±0.01a 0.10±0.01a 6 686 2.620 methyl butyrate 0.03±0.00b 0.84±0.03 a 7 778 2.796 isopentyl formate 0.24±0.02c 0.60±0.03b 1.00±0.04 a 8 785 3.053 ethyl butyrate 0.04±0.00a 0.04±0.00a 9 785 3.247 butyl acetate 9.80±0.50a 9.30±0.42a 5.09±0.27 b 10 820 4.680 ethyl isovalerate 1.78±0.10b 1.32±0.08b 12.66±0.68 a 11 864 5.193 amyl acetate 3.97±0.21a 3.80±0.24a 12 869 6.330 prenylacetate 0.02±0.00b 0.24±0.01 a 13 869 6.414 ethyl 3,3-dimethylacrylate 0.02±0.00 14 983 7.614 ethyl 2-hydroxy-3-methylbutanoate 0.04±0.00 15 1029 8.875 hexenyl acetate 0.16±0.01a 0.06±0.00b 16 1043 8.900 butyl pentanoate 0.01±0.00b 0.02±0.00b 0.17±0.01 a 17 1047 9.117 hexyl acetate 0.02±0.00a 0.01±0.00a 18 1277 18.500 l-bornyl acetate 0.10±0.01 19 1294 19.949 2-methylpropyl benzoate 0.07±0.00c 0.06±0.00c 0.40±0.02a 0.27±0.01b alcohols 20 662 2.156 n-butyl alcohol 0.10±0.00b 16.41±0.76 a 21 788 2.275 cyclopentanol 0.57±0.03 22 700 2.817 2-methyl-1-butanol 0.12±0.01b 0.32±0.02a 23 769 2.982 2-penten-1-ol 0.12±0.01 ital. j. food sci., vol. 32, 2020 490 24 995 4.659 6-methyl-heptanol 1.85±0.08a 0.26±0.01 b 25 858 4.934 2-hexen-1-ol 0.05±0.00d 0.17±0.01cd 0.51±0.03b 1.19±0.07a 0.26±0.01c 26 860 5.020 hexyl alcohol 0.51±0.03b 0.67±0.03b 2.08±0.12b 3.77±0.20a 2.07±0.98b 27 960 7.780 n-heptanol 0.10±0.00 28 969 8.092 1-octen-3-ol 0.07±0.00c 0.06±0.00c 0.35±0.02b 0.38±0.02b 0.94±0.05a 29 969 8.835 citronellol 1.49±0.06 30 971 9.401 3-ethyl-4-methyl-1-pentanol 0.12±0.01 31 1042 9.535 4-isopropyltoluene 0.08±0.00 b 0.31±0.02a 32 1055 9.664 2-ethylhexanol 1.84±0.11c 1.16±0.01c 11.2±0.52a 9.17±0.43b 10.61±0.59ab 33 1059 9.795 eucalyptol 0.07±0.00bc 0.10±0.00b 0.39±0.02a 0.10±0.01d 0.06±0.00c 34 1060 11.095 1-octanol 0.13±0.01b 0.09±0.00b 0.92±0.05a 0.87±0.03ab 0.33±0.02ab 35 1063 11.157 dihydromyrcenol 1.88±0.10 36 1082 12.093 linalool 0.05±0.00c 0.03±0.00c 0.88±0.04b 0.93±0.04b 2.68±0.14a 37 1138 12.753 fenchyl alcohol 0.10±0.01b 0.53±0.03 a 0.18±0.01b 38 1153 14.249 menthol 0.22±0.02 39 1158 14.598 borneol 0.96±0.05 40 1159 14.622 1-nonanol 0.08±0.00c 0.05±0.00c 0.44±0.02b 0.75±0.03a 41 1164 14.817 dl-menthol 0.68±0.03b 0.85±0.04a 0.65±0.03b 0.47±0.02c 42 1187 14.914 4-terpineol 0.15±0.01c 0.12±0.01c 0.80±0.03a 0.27±0.01b 43 1198 15.422 (-)-α-terpineol 0.07±0.00c 0.05±0.00c 0.51±0.02a 0.38±0.02b 0.06±0.00c 44 1228 17.356 geraniol 0.99±0.05 b 13.28±0.57a 45 1258 18.133 1-decanol 0.06±0.00b 1.50±0.07 a 46 1200 18.536 cis-anethol 0.06±0.00b 0.04±0.00b 0.17±0.02a 47 1262 18.978 thymol 0.10±0.00 48 1457 24.504 1-dodecanol 0.37±0.15 49 1543 28.659 cedrol 0.07±0.00 aldehydes and ketones 50 508 1.589 propionaldehyde 2.93±0.14 b 2.78±0.12b 3.66±0.20a 51 543 1.725 isobutyraldehyde 0.16±0.01 b 0.53±0.02 a 52 555 1.815 2-butanone 0.92±0.04 ital. j. food sci., vol. 32, 2020 491 53 643 2.008 2-methylbutyraldehyde 1.57±0.04 a 1.22±0.07b 54 644 2.279 1-penten-3-one 0.04±0.00b 0.65±0.03 a 55 654 2.384 3-pentanone 0.17±0.01 b 0.84±0.05 a 56 715 2.982 2-pentenal 0.08±0.00 57 791 3.613 4-methyl-3-pentene-1-one 0.07±0.00 c 22.98±1.13a 21.9±0.99a 8.49±0.37b 58 806 3.654 hexanal 0.88±0.04b 16.59±0.96 a 17.3±0.87a 59 831 4.225 furfural 0.14±0.01b 0.12±0.01b 0.12±0.01b 4.13±0.23a 60 853 5.500 2-heptanone 0.01±0.00c 0.22±0.01 b 1.68±0.07a 61 841 5.816 4-methylhexanal 0.02±0.00b 0.17±0.01 b 2.07±0.11a 62 913 7.358 2-heptenal 0.04±0.00 63 982 7.509 benzaldehyde 0.07±0.00d 0.04±0.00d 0.65±0.03b 0.92±0.06a 0.21±0.01c 64 1005 8.823 octanal 0.03±0.00d 0.17±0.01 b 0.21±0.01a 0.13±0.00c 65 1013 10.619 2-octenal 0.04±0.00 66 1052 11.762 2-nonanone 0.03±0.00b 0.03±0.00b 0.22±0.01 a 67 1104 12.251 nonanal 1.38±0.06c 1.39±0.07c 7.01±0.42b 9.22±0.51a 2.18±0.14c 68 1112 14.194 (2e)-nonenal 0.18±0.02 69 1151 15.300 2-decanone 0.02±0.00 70 1204 15.807 decanal 1.84±0.01a 0.50±0.03bc 0.58±0.02b 0.47±0.03c 71 1208 16.063 2,4-dimethylbenzaldehyde 0.16±0.01c 0.10±0.01c 0.57±0.03a 0.45±0.02b 72 1263 16.358 5-hydroxymethylfurfural 4.46±0.29 73 1402 22.595 dodecyl aldehyde 0.01±0.00b 0.14±0.01 a 74 1420 23.826 (z)-geranyl acetone 0.36±0.02d 0.58±0.03c 0.74±0.04b 1.19±0.06a others 75 877 4.514 3,7-dimethyl-1-octene 0.02±0.00b 0.61±0.03 a 76 883 5.574 phenylethylene 0.05±0.00c 0.04±0.00c 0.18±0.01b 0.27±0.01a 77 1029 10.851 acetophenone 0.04±0.00b 0.03±0.00b 0.14±0.01b 3.37±0.20a 78 1231 14.992 naphthalene 0.48±0.02c 0.46±0.02c 1.63±0.08b 2.35±0.13a 0.45±0.02c 79 1407 22.876 cedarene 0.84±0.05 80 1668 25.545 butylated hydroxytoluene 0.77±0.04c 1.17±0.06b 2.53±0.11a ital. j. food sci., vol. 32, 2020 492 3.2. identification of the volatile compounds in five blueberry varieties as shown in table 3, the volatile compounds in the five blueberry varieties were identified. a total of 80 volatiles were identified, including 19 esters, 30 alcohols, 18 aldehydes, 7 ketones and 6 other compounds. the number of identified volatile compounds in each blueberry variety ranged from 30 to 53. wh-m and cb-g had the highest (53) and the second highest number (47) of volatile compounds, respectively, while cb-y had the smallest number (30) of volatile compounds. esters are considered to be contributors to fruity and floral notes (wang et al., 2009). a total of 21 ester compounds were detected in the five blueberry varieties. ethyl acetate is a common compound that has a strong fruity aroma. among the 5 groups, the sum of the esters was higher in ws-m and wh-m blueberries than in the other cultivated groups. esters were abundant in wild blueberries, contributing 87.66-90.44% of the total volatiles (table 4). although 13 esters in total were found in wild blueberries in this study, ethyl acetate, ethyl formate, butyl acetate and amyl acetate accounted for more than 80% of the total esters in wh-m and ws-m. the unique esters of wh-m were methyl butyrate, prenylacetate, ethyl 3-methyl-2-butenoate, ethyl 3,3-dimethylacrylate, and ethyl 2hydroxy-3-methylbutanoate, with the latter two in agreement with previous results (beaulieu et al., 2014). l-bornyl acetate was only detected in cb-g. 2-methylpropyl benzoate was detected in all varieties except cb-y. esters were not considered to be as important as aldehydes to the aroma in highbush blueberries, while they have been identified as important volatiles in some rabbiteye blueberries, which is consistent with previous results (du and rouseff, 2014). the total content of alcohols accounted for 4.7-35.98% of the total volatiles (table 3). the content of alcohols was significantly higher in cultivated blueberries than in wild blueberries. of the 30 alcohols identified in this study, 8 were identified in all five varieties: 2-hexen-1-ol, hexyl alcohol, 1-octene-3-ol, eucalyptol, 1-octanol, linalool, 2ethylhexanol, and (-)-α-terpineol. among them, 2-ethylhexanol was dominant, with relative contents ranging from 1.16% to 11.20% (table 4). 2-methyl-1-butanol was detected in ws-m and wh-m. 2-penten-1-ol, 3-ethyl-4-methyl-1-pentanol, borneol and menthol were only detected in cb-g. the unique alcohols in cp-g and cb-y were citronellol and cyclopentanol, respectively. a total of 25 different aldehydes and ketones in blueberry juice were identified, accounting for 3.16%-68.67% of the total volatiles (table 4). the sum of the aldehydes and ketones in cp-g was significantly higher than that in other varieties, and it was also significantly higher in cultivated blueberries than in wild blueberries. nonanal and benzaldehyde were the predominant aldehydes found in the five blueberry varieties. whm had a significantly higher decanal content than that of the other aldehydes. in all cultivated groups, 4-methyl-3-pentene-1-one was the major component, accounting for more than 20% of the total aldehydes in cb-g and cp-g. 2-pentenal, 2-heptenal, and 2octenal were only detected in cb-g. additionally, (2e)-nonenal, 5-hydroxymethylfurfural and dodecyl aldehyde could be used to distinguish cp-g from the other varieties. 2butanone was only detected in cb-y. ital. j. food sci., vol. 32, 2020 493 table 4. the aroma-active compounds (roav > 1) in different blueberries*. no. volatile threshold （μg/l） sensory attributes aroma classificatio n roav wh-m ws-m cb-g cp-g cb-y 1 ethyl formate 150 fruity 1 0.54 ±0.03b 0.44± 0.02b 1.83 ±0.10a 2 ethyl acetate 5 fruity 1 100.00±5.00a 100.00±4.89a 13.95±0.63b 3.43±0.20b 100.00 ±5.47a 3 butyl acetate 66 sweet, banana, 1,3 1.22±0.06b 1.06 ±0.05b 2.22 ±0.12a 4 1-octene-3-ol 1 mushroom 4 0.57 ±0.00c 0.45 ±0.00c 4.99±0.29b 4.12 ±0.22b 27.07±1.44a 5 linalool 6 sweet lemon 1 0.07 ±0.00c 0.04 ±0.00c 2.09 ±0.10b 1.68 ±0.07b 12.86 ±0.67a 6 geraniol 40 rose 2 0.19 ±0.01b 4.74 ±0.20a 7 2-methylbutyraldehyde 1 stimulating, coffee, sweet 1,3,6 17.03 ±0.43 b 35.14±2.02a 8 hexanal 5 fragrant, grassy 5 1.44 ±0.07c 47.33 ±2.74a 37.53±1.89b 9 4-methylhexanal 3 fruity, rose 1,2 0.05 ±0.00b 0.81±0.05b 7.48±0.40a 10 octanal 0.7 rose, orange 1,2,3 0.35 ±0.00c 3.46 ±0.20b 3.25 ±0.15b 5.35 ±0.00a 11 nonanal 1 floral, citrus, slightly spicy 1,2,6 11.32 ±0.49c 10.50 ±0.53c 100.00±5.99a 100.00±5.53a 62.79 ±4.03b 12 decanal 3 fruity 1 5.03 ±0.03b 1.26±0.08d 2.76±0.10a 1.70±0.11c ∗intensity: 1-fruity, 2-floral, 3-sweet, 4-fatty, 5-fragrant, 6-stimulating ital. j. food sci., vol. 32, 2020 494 3.3. determination of the aroma active compounds in different blueberries considering that volatile compounds have different thresholds and people have different sensitivities to them, the relative content cannot reflect the true contribution that every volatile compound makes to the whole aroma profile. therefore, we used roavs to detect the contribution of volatile compounds to the whole aroma profile (yi et al., 2016). fourteen aroma active compounds were selected from five blueberry varieties, which are shown in table 4. there were four aroma active compounds (ethyl acetate, 1-octene-3-ol, linalool, nonanal) with higher roavs in five varieties. ethyl acetate and nonanal possessed the highest roavs in wild blueberries and cultivated blueberries, respectively. cb-y had the highest roav summations, which was significantly higher than the other four varieties. aldehydes were the most abundant chemical group, with aromatic activity found in five blueberry varieties. 2-methylbutyraldehyde, hexanal, 4-methylhexanal, 1-octanal, nonanal and decanal contributed to stimulating, fragrant, fruity, rose, floral and fruity aroma notes, respectively. 2-methylbutyraldehyde was observed only in cp-g. 2-methylbutyraldehyde has stimulating, coffee, and sweet aroma notes, with a very low threshold (1 !g/l) in cpg. aldehydes made a major contribution to blueberry aromas, which is in agreement with previous results (du and rouseff, 2014; horvat and senter, 1985). alcohols were the next most abundant group, including 1-octene-3-ol, linalool, and geraniol, contributing mushroom, lemon and rose aroma notes. 1-octene-3-ol and linalool were identified in the five blueberry varieties. three esters, including methyl acetate, ethyl acetate and butyl acetate, were aroma active. ethyl formate had a high threshold value (150 !g/l) and a high relative content. however, its roavs were low (0.32-1.53). ethyl acetate contributed a fruity aroma to the five varieties and possessed the highest roav in wild blueberries. butyl acetate contributed sweet and banana aroma notes. however, it has not been previously reported as contributing to blueberry aroma. although wild blueberries had higher contents of volatile compounds, their characteristic aroma notes were less than those of cultivated blueberries. the reason may be that the aroma of fruit is not completely dependent on the concentration of the volatile compound but it is closely related to its threshold. the threshold of volatile compounds found differed greatly among the varieties studied. for example, the relative contents of ethyl formate in the two wild blueberries were higher than those in cultivated blueberries, but the roavs were lower because the threshold value of methyl acetate was high (150 !g/l). six descriptors (fruity, floral, sweet, fatty, fragrant, and stimulating) were used to provide an assessment of the five blueberries. to reflect the difference in aroma among different blueberry varieties, the roav of each blueberry aroma component was taken as the logarithm base 10, and the aromatic series of the five blueberry juices on the spider web diagram are shown in fig. 4. ital. j. food sci., vol. 32, 2020 495 figure 4. aromatic series in blueberries based on aroma activity values. the analysis showed that wh-m and ws-m could mostly be described as having fruity and floral notes due to the higher roavs of ethyl acetate and nonanal in the samples. cbg and cp-g had higher values for the attributes fragrant and floral due to their large quantities of hexanal and nonanal. the difference between cb-g and cp-g lies in the fact that cb-g exhibited a greater sweet component. the roav of 1-octene-3-ol was higher in cb-y; thus, cb-y was perceived to have a fatty aroma. considering the volatile composition of these blueberries, samples had higher values for the attributes fruity and fragrant due to their large quantities of aldehydes and alcohols. 4. conclusions and future work the p&t extraction method coupled to gc-ms analysis was a quick and efficient method for the evaluation of blueberry volatiles, and the results demonstrated that 3 ml of sample volume purged at 25°c for 11 min were the best extraction conditions. a total of 80 volatiles were identified in five blueberry varieties using the p&t-gc-ms technique. the volatiles of blueberries were composed of mainly aldehydes, alcohols, esters, and terpenes. among the identified compounds, 12 compounds (roav>1), including ethyl formate, ethyl acetate, butyl acetate, 1-octene-3-ol, linalool, geraniol, 2-methylbutyraldehyde, hexanal, 4-methylhexanal, octanal, nonanal and decanal, were considered aroma active. the spider web diagram showed that the sensory characterization of the five varieties was distinct due to the different quantities of volatile compounds. acknowledgements this work was supported by water and soil conservation development and management center, ministry of water resources. (project no.2020 swxyjskf001). -1,5 -1 -0,5 0 0,5 1 1,5 2 2,5 fruity floral sweet fatty fragrant stimulating wh-m ws-m cb-g cp-g cb-w ital. j. food sci., vol. 32, 2020 496 references aznar m. and arroyo t. 2007. analysis of wine volatile profile by purge-and-trap–gas chromatography–mass spectrometry. j. chromatogr. a. 1165:151-157. beaulieu j.c., stein-chisholm r.e. and boykin d.l. 2014. qualitative analysis of volatiles in rabbiteye blueberry cultivars at various maturities using rapid solid-phase microextraction. j. amer. soc. hort. sci. 139:167-177. campillo n., viñas p., lópez-garcı ́a i., aguinaga n. and hernández-córdoba m. 2004. purge-and-trap capillary gas chromatography with atomic emission detection for volatile halogenated organic compounds determination in waters and beverages. j. chromatogr. a. 1035:1-8. deng x., liang g., chen j., qi m. and xie p. 2011. simultaneous determination of eight common odors in natural water body using automatic purge and trap coupled to gas 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liu, t. 2016. comparison of different extraction methods in the analysis of volatile compounds in pomegranate juice. food anal method. 9:2364-2373. zhuang k., wu n., wang x., wu x., wang s., long x. and wei x. 2008. “roav” method: a new method for determining key odor compounds of rugao ham. food sci. 29:370-374. paper received august 28, 2019 accepted december 20, 2019 ijfs#1131_bozza ital. j. food sci., vol. 30, 2018 809 survey consumer perspective regarding dried tropical fruits in turkey g. cinar department of agricultural economics, faculty of agriculture, aydın adnan menderes university, turkey e-mail address: gokhan.cinar@adu.edu.tr abstract the purposes of this study are to evaluate the tropical fruit (banana, kiwi, pineapple) preferences of consumers in turkey and their willingness to pay and to assess the factors that affect this willingness to pay. in this context, tropical fruit products were presented in packages of 50 grams to 386 individuals who had never tasted these products before, and after the products had been tasted, surveys were administered. the findings revealed that dried banana has sensory issues related to hardness and taste and that dried kiwi has sensory issues related to taste and odour. the results show that improving the taste characteristics and increasing the emphasis on health while promoting the products could have a positive impact on increasing the demand for these products in turkey. keywords: consumers, dried fruits, sensory analysis, willingness to pay ital. j. food sci., vol. 30, 2018 810 1. introduction in turkey, fruit is a widely cultivated product. however, demand for banana, pineapple and kiwi, which are tropical fruits, is met through imports because of climatic conditions and land constraints in turkey. in 2015, turkey had a total of 5,896,156 usd of pineapple imports, 108,334,990 usd of banana imports and 2,945,173 usd of kiwi imports. many of these imports are from countries such as costa rica, ecuador, chile, guatemala and panama (anonymous, 2016). importing from other countries increases the risk that these products will be decayed. the protections applied to remove this risk, however, make it difficult to maintain quality standards that differ from country to country. these issues can be resolved by drying the products. drying essentially means removing the moisture from the product with the help of the sun or mechanical devices (chang et al., 2016). this process reduces packaging, shipping and transportation costs for the product due to the decrease in its weight and mass and reduces the measures needed for product protection (omolola et al., 2017). on the other hand, dried fruits contain more antioxidants, fibre and vitamins than do fresh fruits (bennett et al., 2011). this information shows that dried fruit consumed regularly and in the correct amount can reduce risk for various health factors such as glycaemia and cardiovascular diseases (jeszka-skowron et al., 2017). although dried fruits have advantages related to both consumer health and commercial risk, few studies have focused on consumer preferences for these products. accordingly, jesionkowska et al. (2009) determined the factors affecting the consumption preferences of dutch, french and polish consumers who expressed that they consumed dried fruit and fruit products at least once a month. similar studies have been conducted on dutch, french (sijtsema et al., 2012) and chinese (wu, 2017) consumers. in these studies, it was argued that emphasis on health can play an especially important role in increasing the consumption of these products. alphonce et al., (2015) assessed consumer preference in europe and the willingness of consumers to pay extra for tropical dried fruit produced in africa. the organic status of dried fruit and fair trade factors have been shown to influence consumer willingness to pay extra for dried fruit. various fruits such as dried apricots, figs and grapes, which are extensively cultivated in turkey, are consumed frequently and are well recognized by consumers. therefore, the turkish consumer is familiar with dried fruit products and the process of their production. however, in turkey, dried kiwi, banana and tropical fruits such as pineapple have recently started to be sold. currently, these products are occasionally sold in private stores, in a limited number of supermarkets and online. therefore, consumers in turkey do not know much about these tropical dried fruits, and many of them have never tasted these products. specifically, approximately 75-90% of the food products that are newly introduced into a market may fail in the first few years after being launched (talavera et al., 2017). to understand and improve market success for new products, consumer analysis is needed (chen et al., 2013; de andrade et al., 2017). however, consumers’ decision to eat fruits and derivative products is the result of the interaction between a large variety of factors (sabbe et al., 2009). studies show that the foremost factor governing consumer preferences is the fruit’s internal features such as taste, odour, colour, and firmness (pollard et al., 2002; harker et al., 2008). another important factor is the importance that the consumer gives to health and their lifestyle (pollard et al., 2001). considering health, it is indicated that the probability of purchasing fruit is affected positively when health-related information or the possible benefits of the product are noted on the label (silvestri et al., 2018). according to sabbe et al., (2008), consumers’ socio-demographic structures such as gender and habitat are ital. j. food sci., vol. 30, 2018 811 related to their willingness to purchase tropical fruits. in addition, while consumers can be affected by a wide range of external product features such as brand, price, convenience, availability, and packaging in food selection, they pay more attention to price, especially when buying fruit (sabbe et al., 2009). overall, apart from consumers’ socio-demographic features, the price of the product and the health-consciousness and lifestyle of the consumer are important considerations in fruit consumption. for this reason, these matters were the primary focus of this study. realizing consumers’ sentimental expectations, leads to consumer satisfaction and increases the marketing of the product (grunert, 2002). halagarda and suvala (2018) argue that referring to consumers’ experiences on the current products increases the level of significance of the research outcomes. migliore et al., (2017) has emphasized that not recognizing and being unfamiliar with tropical fruits is one obstacle to purchasing. a product overview was written and a taste test administered in order to overcome these problems as well as to appraise these products, which are new to the market from the consumers’ point of view. the fundamental hypothesis underlying this study is that the willingness to buy a new fruit product can change depending on an individual’s sensitivity towards health and the internal features of the product, as well as the demographic and socio-economic features of consumers. that information contributes to the choice of the target market. in addition, another hypothesis formed claims that sensory evaluation tests presented to the consumers helps them to evaluate the characteristics of the products. that information, if exists, contributes to the development of the sensual features of the product. the last hypothesis indicates that a new product has a perceptual position compared to the various derivative products. that information helps to determining possible competitor products. in this context, the main purpose of this study is to determine the willingness of consumers who have never tasted dried tropical fruits (kiwi, banana, pineapple) to pay for these products. in addition to this objective, other objectives of my study are as follows: • determining the amount of money that consumers, who want to buy dried tropical fruits, are willing to pay; • revealing the factors that affect willingness to pay for dried tropical fruits; • analysing the degree of preference for and deficiencies of dried tropical fruits depending on their sensory properties; and • determining the consumption preference hierarchy for dried tropical fruits compared to their other derivative products. given the assumption that dried tropical fruit products are quite new for many consumers, it is important to understand the different factors that affect consumers’ willingness to purchase and consumption behaviour in terms of developing promotion policies. few studies have examined consumers’ preferences for dried fruits, and none of these studies focused on the turkish market. through an extensive literature search and application of the different methods used in this study, better marketing strategies can be developed for the decision makers and operators in the dried tropical fruit industry. ital. j. food sci., vol. 30, 2018 812 2. materials and methods 2.1. overview this study was conducted in izmir, which is located in the western part of turkey and is the biggest city in the area. participants who had not tasted dried tropical fruits before were given banana, kiwi, and pineapple in packs of 50 grams to taste free of charge. the participants were then asked to rate the taste, colour, appearance, hardness, and odour of these products in points. then, a questionnaire was administered to the participants, and the consumers' willingness to purchase these products and the factors that influence this willingness were determined. finally, the participants' preference for different types of these products (fresh fruit, mineral water, fruit milk, fruit yogurt) and the ranking of the derivative products in the consumer mind were determined based on the information gathered from these same questionnaires. spss 21 (statistical package for social sciences) and r package programs were used for the statistical analysis in this study. the materials and methods used in this study at presented in detail below. 2.2. sample selection the location of the survey was i̇zmir province, as it contains the largest city in the aegean region and the third largest city in turkey. the total population of i̇zmir province is 4,223,545. this province, according to code tr31, represents a region of its own. in the study, the number of consumers surveyed was determined by the following sample formula (newbold, 1995): n = np(1− p) (n −1)σp 2 + p(1− p) where n is the sample size, n is the population size (4,223,545), and p is the prediction rate (0.5 for the maximum sample size) and the probability level confidence interval (95% confidence interval, 𝜎p: 0.02551 for 0.05 margin of error from the equation of 1.96𝜎p: 0.05). accordingly, the number of consumers randomly selected for the face-to-face survey was 385. questionnaires were administered to a total of 386 consumers, of which there were 193 females and 193 males; thus, the numbers of male and female consumers were equal. the main constraint of this study is about the sample collected from a limited part of turkish society. the sample in this study may not be a representative of all segments of the turkey population. the research can be expanded with sample size acquired from different regions and cultures. 2.3. sensory evaluations in this study, dried tropical fruits (kiwi, banana, pineapple) were evaluated by consumers who had not previously tasted such products before. participants were selected on a volunteer basis. the products were presented to the participants free of charge as dried kiwi, banana, and pineapple in packs of 50 grams. a little time was given, and water was provided in order to minimize residual effects between tasting the different products. the participants were then asked to rate the taste, odour, appearance, firmness, and colour of each product objectively on a 9-point likert-type scale (1. dislike extremely, 5. neither like ital. j. food sci., vol. 30, 2018 813 nor dislike, 9. like extremely). on this scale, for example, for firmness, 1 defines the expression “the product is extremely firm”, while 5 defines the expression “the product is neither firm nor soft”, and 9 defines the expression “the product is an ideal firmness”. although this evaluation method, which makes use of previous studies (zhao et al., 2007; barrett et al., 2010), is generally used by expert panellists, it can also be used by consumers (worch et al., 2010). 2.4. conditional valuation method and lower bound meaning for payment in this study, the conditional evaluation method was applied to determine consumer willingness to pay for dried tropical fruits. for this method, a hypothetical market is created for any goods or services that cannot be sold on the market, the benefits that can be gained from such goods or services are explained, and how much consumers are willing to pay for the benefits that they receive in consuming these goods or services is determined (carson, 2000; uzmay and cinar, 2017). in this research, dried kiwi, banana and pineapple were first tasted by consumers in complementary packages of 50 grams. these consumers had not previously tasted these products before. after this step, consumers were informed about the products, specifically, the products' health advantages. then, consumers were asked separately about kiwi, banana and pineapple and whether they would like to buy these products. if the response was positive, the final price that they were willing to pay was determined. the prices obtained from each consumer were converted to the consumer's general willingness to pay based on the lower bound method presented below (blaine et al., 2003). lower bound method (lbm) = (p00∏ )+ (pii∏ i=1 k ∑ −pi−1) where π0 is the cumulative percentage of willingness to pay, p0 is the lowest payment boundary, and k is the number of boundaries. 2.5. logistic regression in this study, the dual logistic regression method is used to determine the factors that affect consumer willingness to pay for dried fruit. in the logistic regression model, the dependent variable is discrete, and the estimated probability values range from 0 to 1. a general logistic regression model is expressed below (gujarati, 1995). pi = f(zi)= f(α +βxi)= )()( 1 1 1 1 ixzi ee βα+−− + = + where pi=i is the probability that the ith individual chooses a specific option, f is the cumulative probability function, α is the constant coefficient, β is the estimation parameter for each independent variable, and x is the independent variable. in this study, consumer dried fruit consumption status (no/yes) was the dependent variable in the logistic regression model. models are designed separately for the three separate products. in the models, the dependent variables are consumers who are not willing to pay for the dried products. the independent variables are education level of the consumers, understanding of a healthy life, gender, income, age and body mass index. the response obtained from the expression "i think i am a consumer who understands a healthy life" represented the consumers' understanding that a healthy life is included in the response model. for this, ital. j. food sci., vol. 30, 2018 814 before receiving their answer, it was expressed to the consumers that health-consciousness involves living a healthy life, eating healthy, exercising, sleeping well, and being in touch with nature frequently. accordingly, the consumers were asked to evaluate themselves objectively. in addition to this process, the study also used hypothesis tests such as mannwhitney u, friedman, and kendall's w. 2.6. fuzzy pairwise comparison method and multidimensional scaling analysis in this study, after the products were tasted by the consumers, questionnaires were administered. the sections included in the questionnaire were in the order of the consumption preferences among fresh fruit, fruit yoghurt, plain fruit, fruit milk, fruit soda and dried tropical fruits. in this section, the fuzzy pairwise comparison method was used. the steps of the method can be summarized as follows (tanaka, 1997). pairwise comparisons were presented to indicate individual preferences. the total distance in a comparison was equal to 1. if gkh=0.5, then k≈h; if gkh>0.5, then k>h; and if gkh<0.5, then k<h. the number of paired comparisons of the objectives (c) was determined as c=[(z.(z-1))/2]. z referred to the preferred number of objectives in the formula. in this study, 10 comparisons of five different products were presented to everyone. for each pairwise comparison, the gcr preference was obtained. measurement of the preference degree of r according to c can be expressed as gcr=1-grc. then, the fuzzy preference matrix was generated. gcr = 0 𝑖𝑓 𝑐 = 𝑟 ∀ 𝑐,𝑟 = 1,… .𝑛 𝑔!" 𝑖𝑓 𝑐 ≠ 𝑟 ∀ 𝑐,𝑟 = 1,… . .𝑛 in this study, a 5×5 fuzzy preference matrix was created for each individual as follows: g = the separately preferred density of each objective (µj) was obtained using the following equation: µj = 1 − 𝐺!"!!!!! /(𝑛 − 1) !/! the value of µj ranged between 0 and 1. this information provides a more efficient structure than traditional sorting methods (gunden and terrence, 2012). the objective hierarchy obtained by fuzzy pairwise comparisons was converted to perceptual maps using multidimensional scaling. this analysis obtained the projection of the objects in a kdimensional space based on the determined distance between n objects according to p argument. this projection used the distance between units. this analysis used kruskal's stress to determine concordance with distances between estimated distances. stress = dij −kıj( ) 2 ∑∑ (d ij 2)∑ 0 g12 g13 . g1r g21 0 . . . g31 g32 0 . . . . . 0 . gc1 . . . 0 ital. j. food sci., vol. 30, 2018 815 3. results 3.1. consumer profile in table 1, various demographics such as gender, age and educational level of the participants are presented. according to the regional results of the turkish statistical institute's research on income and living conditions for 2014, the average annual household income was 32,639 tl. the monthly average annual income is 2719 tl. in this study, the average household income was 2904 tl. according to turkstat, the average height of turkish citizens is 167.2 centimetres, and the average weight is 71.5 kilograms (anonymous, 2003a). the weight and height ratios of the participants were obtained with the aim of establishing the body mass index variable to use in the model at a subsequent stage. in the present study, the average weight of the participants was 70 kilograms, and the average height was 168 centimetres. participants with a primary education accounted for 51.3% of the total participants, 32.4% of the participants were high school graduates, and 16.4% of the participants were university graduates. this information agrees with the educational characterization of i̇zmir province. approximately 49.6% of the izmir population is female, and 51.4% is male. based on these data, the numbers of male and female participants selected for the sample group were equal. the household size of the participants was 4. in general, the demographic findings corresponded to the turkish consumer profile. table 1. participants' socioeconomic characteristics*. demographic variable minimum maximum mean weight (kilograms) 50 130 70.292 size (centimetres) 150 190 168.450 age (year) 18 75 41.233 number of people in the household 1 10 4.256 income level of the household (turkish lira) 1400 11000 2904.330 educational status primary/secondary school 198 51.3% high school 125 32.4% college/faculty/postgraduate 63 16.4% gender female 193 50% male 193 50% *at the time of the survey, 1 usd=2.72 tl. as previously mentioned, consumers that had not consumed dried tropical fruits (banana, kiwi, pineapple) prior to the study were selected. in addition, consumers with no chronic illnesses caused by fruits and who generally consumed fruit fresh, especially banana, kiwi and pineapple, were also selected. the consumers were asked open-ended questions about the most consumed, the most liked and the most disliked fruit types that they had consumed in the previous year. the responses obtained are presented in table 2. according to the responses, the most consumed fruits in the last year were apples, followed by oranges. these findings are consistent with the turkish statistical institute's data on the consumption of food items. accordingly, apple is the most consumed fresh ital. j. food sci., vol. 30, 2018 816 fruit in turkey, with an annual consumption of 67,634,642 kilograms (anonymous, 2003b). in general, it can be stated that the fruit consumption characteristics of the consumers participating in the survey were well matched with the turkish consumer. specifically, the fact that these fruits can be produced in turkey, can easily be accessed, and are cheaper are the most basic reasons for them being consumed the most. in addition, some products (watermelon, melon) are consumed by consumers as fruit although they are regarded as plants in the literature. on the other hand, the most popular fruit is banana, and the least popular fruit is grapefruit. additionally, 62.2% of the consumers preferred to buy fresh fruits from the bazaar, 20.2% from the supermarkets and 10.6% from grocery stores. for these selections, product type, cost and availability are the primary factors affecting their selection. 3.2. sensory data analysis in this research, sensory evaluation by consumers was performed on dried kiwi, banana and pineapple. the evaluated sensory criteria were hardness, smell, taste, appearance and colour. the highest score for these criteria was 9, while the lowest score was 1. when evaluated in terms of overall score, the highest score was for dried pineapple (7.14). this score was described as a "good at a moderate level" on the sensory evaluations scale. in addition, the second-best score was for kiwi (5.96). this score was described as "neither good nor bad". the lowest score belonged to dried banana, with an average of 4.54. this score was described as "slightly bad". figure 1 presents the sensory evaluation data for the products. according to these data, the average hardness scores were 1.6 for banana, 7.4 for kiwi and 7.5 for pineapple. the taste score averages were 2.1 for banana, 5.1 for kiwi and 6.7 for pineapple. the colour score averages were 6.8 for banana, 7.5 for kiwi and 7.2 for pineapple. the average point of appearance was 5.8 for banana, 6.6 for kiwi and 7.1 for pineapple. the average scores for smell were 6.4 for banana, 3.2 for kiwi and 7.2 for pineapple. in summary, banana had a low score in terms of hardness and taste, and kiwi had a low score in terms of taste and smell. pineapple also had a higher value than the other products in five criteria. 3.3. willingness to pay table 3 presents consumer willingness to pay for dried products. according to the table, 62.2% of consumers were reluctant to buy dried banana. of these consumers, 78.3% attributed the reason for not buying this product to the problems with taste and hardness, and 92.1% attributed the reason to other sensory attributes. on the other hand, the lowest price given for this product was 0.10 tl and the highest price was 10 tl by consumers who wanted to buy the product. however, findings from the lower bound of the payment methods showed that the general consumer group tended to be willing to pay 0.91 tl for a 50 gram package of dried banana. when the dried kiwi findings were examined, it could be observed that 52.2% of the consumers wanted to buy dried kiwi, while 47.8% did not want to buy it. among the consumers who were reluctant to purchase this product, 78.7% had reasons relating to product odour and taste, and 86.4% had reasons relating to other sensory properties. on the other hand, for the consumers who wanted to buy the product, the lowest price for this product was 0.10 tl, and the highest price was 13 tl. however, the findings from the lower bound method show that the general consumer group tended to be willing to pay 1.30 tl for a 50 gram package of this product. ital. j. food sci., vol. 30, 2018 817 table 2. consumer preferences for fresh fruit consumption. the fruit that i consume the most my favourite fruit the fruit that i never eat frequency % frequency % frequency % apple 118 30.570 banana 119 30.829 none 100 25.907 orange 77 19.948 strawberry 46 11.917 grapefruit 51 13.212 banana 61 15.803 plum 45 11.658 medlar 27 6.995 tangerines 34 8.808 apple 41 10.622 quince 25 6.477 plum 28 7.254 kiwi 29 7.513 grape 23 5.959 strawberry 15 3.886 peach 13 3.368 alligator pear 18 4.663 watermelon 13 3.368 watermelon 12 3.109 apple 16 4.145 kiwi 11 2.850 cherry 12 3.109 plum 11 2.850 pear 5 1.295 tangerines 12 3.109 orange 11 2.850 melon 4 1.036 pineapple 10 2.591 diospyros kaki 10 2.591 pomegranate 4 1.036 orange 9 2.332 eriobotrya japonica 10 2.591 pineapple 3 0.777 grape 7 1.813 apricot 10 2.591 apricot 3 0.777 unripe almond 6 1.554 pear 9 2.332 peach 3 0.777 pear 5 1.295 pomegranate 9 2.332 grape 3 0.777 melon 4 1.036 peach 8 2.073 ages 2 0.518 apricot 4 1.036 strawberry 7 1.813 quince 1 0.259 pomegranate 3 0.777 mulberry 5 1.295 cherry 1 0.259 other 9 2.331 other 36 9.324 total 386 100 386 100 386 100 ital. j. food sci., vol. 30, 2018 818 figure 1. sensory evaluation for dried banana, pineapple and kiwi. while 60.1% of the consumers were willing to pay for the dried pineapple, 39.1% did not want to buy this product. accordingly, dried pineapple was the product with the highest willingness to pay. of the consumers, 71.2% that were reluctant to purchase had issues relating to taste, and 89.8% of the consumers indicated that they had issues with other sensory characteristics. however, among the consumers who wanted to buy the product, the lowest price given for this product was 0.25 tl, and the highest price was 10 tl. however, the findings from the lower bound method showed that the general consumer group tended to be willing to pay 1.97 tl for a 50 gram package of this product. in general, the highest willingness to pay was for dried pineapple. the second highest willingness to pay was for dried kiwi, followed by dried banana. 3.4. factors influencing consumer preferences in this part of the study, factors affecting consumer willingness to buy dried banana, kiwi and pineapple have been revealed. for this, a logistic regression model was used, and each product was analysed separately (table 4). consumers who wanted to purchase products were the dependent variables in the model, and the independent variables were their socio-demographic characteristics. the data obtained indicated that the models were significant (p<0.05). the correct estimation rate of the dependent variable was 71.2 for dried banana, 70.7 for dried kiwi and 69.7 for dried pineapple. the explanatory power of the dependent variables (r2) for the independent variables was 16.3% for dried banana, 24.7% for dried kiwi and 24.0% for dried pineapple. in table 4, the direction of the b coefficient represented the odds ratio (probability of occurring) of the exp (b) coefficient. the findings showed that gender affected the consumption of dried fruit. female participants wanted to pay more for dried products than did the male participants. while this willingness to pay was significant for dried pineapple and kiwi, it was not statistically significant for banana. according to these results, females tended to buy 6.99 (1/0.143) times more dried pineapple than males and 4.83 (1/0.207) times more dried kiwi than males (table 4). 1 2 3 4 5 6 7 8 9 hard taste color view smell banana kiwi pineapple ital. j. food sci., vol. 30, 2018 819 table 3. willingness of consumers to pay for dried banana, pineapple, and kiwi. dried kiwi (tl/package) dried pineapple (tl/package) dried banana (tl/package) wtp(tl) frequency % c% wtp(tl) frequency % c% wtp(tl) frequency % c% 13 1 0.26 0.26 10 12 3.11 3.11 10 3 0.78 0.78 10 7 1.81 2.07 8 1 0.26 3.37 5 21 5.44 6.22 7 1 0.26 2.33 7 4 1.04 4.40 4 3 0.78 7.00 6 1 0.26 2.59 6 3 0.78 5.18 3 22 5.70 12.70 5 30 7.77 10.36 5 51 13.21 18.39 2.5 4 1.04 13.73 4 8 2.07 12.44 4 11 2.85 21.24 2 40 10.36 24.10 3.5 1 0.26 12.69 3 43 11.14 32.38 1.5 8 2.07 26.17 3 28 7.25 19.95 2.5 5 1.30 33.68 1 33 8.55 34.72 2.75 2 0.52 20.47 2 47 12.18 45.85 0.75 2 0.52 35.24 2.5 2 0.52 20.98 1.5 6 1.55 47.41 0.5 8 2.07 37.31 2.25 1 0.26 21.24 1 38 9.84 57.25 0.25 1 0.26 37.57 2 38 9.84 31.09 0.75 1 0.26 57.51 0.1 1 0.26 37.83 1.5 7 1.81 32.90 0.5 12 3.11 60.62 0 240 62.18 100.00 1 35 9.07 41.97 0.25 1 0.26 60.88 total 386 100.00 0.75 1 0.26 42.23 0 151 39.12 100.00 0.5 8 2.07 44.30 total 386 100.00 0.25 1 0.26 44.56 0.1 1 0.26 44.82 0 213 55.18 100.00 total 386 100.00 lbm=1.30 tl/package (26.0 tl/kg) lbm=1.97 tl/package (39.4 tl/kg) lbm=0.91 tl/package (18.2 tl/kg) ital. j. food sci., vol. 30, 2018 820 table 4. results of the logistic regression model. independent variable description wtp dried banana wtp dried kiwi wtp dried pineapple b exp(b) s.e. b exp(b) s.e. b exp(b) s.e. educational status college/faculty high 0.025 1.025 0.256 -0.281 0.326 0.745 -0.617 0.539 0.329 primary/secondary -0.189 0.828 0.324 -0.580 0.560* 0.260 -0.671 0.511* 0.258 phla no 1.270 3.262* 0.233 1.662 5.271* 0.238 1.635 5.130* 0.267 yes gender women -0.552 0.576 0.621 -1.575 0.207* 0.752 -1.946 0.143* 0.687 male income scale 0.001 1.001* 0.001 0.001 1.001* 0.001 0.001 1.001* 0.001 age scale -0.007 1.007 0.020 0.006 0.994 0.021 0.015 0.985 0.022 bmi scale 0.019 1.020 0.011 -0.016 0.984 0.011 -0.017 0.983 0.011 constant -4.296 0.014* 0.951 0.186 1.205 0.870 1.396 4.037 0.880 model details model coefficients -0.497 0.608* 0.105 -0.208 0.812* 0.102 0.442 1.556* 0.104 x2 49.230* 78.612* 75.257* r2 16.3 24.7 24.0 log likelihood 462.757 452.345 441.426 predicted 71.2 70.7 69.7 *p<0.05. ital. j. food sci., vol. 30, 2018 821 on the other hand, the tendency to consume products was not statistically significant in terms of education, except for pineapple. consumers with a primary/secondary school level of education were willing to buy fewer products than were those with a college/faculty level of education. for dried kiwi and pineapple, this information was statistically significant. those with education at the college/faculty level tended to purchase 1.95 (1/0.511) times more dried pineapple and 1.78 (1/0.560) times more dried kiwi than did those with a primary/secondary level of education. in addition, an increase in income significantly increased the willingness to pay for all three products (p<0.05). however, there was no significant relationship of consumer age or body mass index with the desire to consume these products. finally, a positive relationship was found between consumers being health-conscious consumers and their willingness to purchase products (phla). according to this result, those who expressed that they were health-conscious consumers tended to buy 3.26 times more dried banana, 5.27 times more dried kiwi and 5.13 times more pineapple than did those who did not pay attention to health (p<0.05). in general, income, health consciousness and gender were influential factors on willingness to purchase. 3.5. the hierarchical selection of consumers and perceptual mapping of products in table 5, the fuzzy comparison results are presented. this method has been applied to determine the hierarchy of consumer preference for banana, pineapple and kiwi products as fresh, in mineral water, in fruit milk, in fruit yoghurt and as dried fruit. the validity of the fuzzy comparison method was tested by the friedman test and kendall's w test. the friedman test determines whether consumers are behaving differently in at least one product selection. accordingly, the h0 hypothesis was rejected, and at least one ranking was found to be different from the others (p<0.01). this method was carried out after consumers tasted the dried tropical fruit products. the findings indicated that fresh banana, kiwi and pineapple were most preferred by participants (0.651). specifically, the preference to consume fresh product was statistically significantly higher in males than in females (p<0.05). the second highest preference for consumers was to consume these products in mineral water (0.438). there was no significant difference between males and females in the consumption of the product in mineral water (p>0.05). the third highest preference for consumers was to consume the products as fruit milk (0.401). the preference for consuming the product as fruit milk was statistically significantly higher in females than in males (p<0.05). the fourth highest preference for consumers was to consume the products as fruit yoghurt (0.366). the preference for consuming the product as fruit yoghurt was statistically significantly higher in females than in males (p<0.05). the lowest preference for consumers was to consume the products dried (0.203). the preference for consuming the product as dried was statistically significantly higher for females than for males (p<0.05). however, in table 5, consumption preference order by gender is also presented. when the table is examined, the preference order of the male's products was as follows: fresh (0.715), in mineral water (0.467), in fruit milk (0.350), in fruit yoghurt (0.310) and dried (0.178). for females, the order of preferences was as follows: fresh (0.587), in fruit milk (0.452), in fruit yoghurt (0.423), in mineral water (0.410) and dried (0.228). on the other hand, males preferred fruit yoghurt and fruit milk, while females were more indifferent about making similar selections as mineral water, fruit yoghurt and fruit milk. ital. j. food sci., vol. 30, 2018 822 table 5. fuzzy pairwise comparison findings. variable gender mean preference mann-whitney u asymp. sig. general mean** std. deviation general preference fresh fruits woman 0.587 1 13924.00 0.000 0.651 0.285 1 male 0.715 1 mineral water woman 0.410 4 12943.00 0.080 0.438 0.235 2 male 0.467 2 fruit milk woman 0.452 2 14235.00 0.000 0.401 0.253 3 male 0.350 3 fruit yoghurt woman 0.423 3 13502.00 0.000 0.366 0.211 4 male 0.310 4 dried fruits woman 0.228 5 16046.00 0.018 0.203 0.200 5 male 0.178 5 **significant by friedman test for p<0.01; kendall’s w = 0.267. ital. j. food sci., vol. 30, 2018 823 in this research, multi-dimensional scaling analysis was used to create a perception map for consumers for different types of tropical fruits (kiwi, banana, pineapple). this analysis included products seen as substitutes in the market and their differentiation from one another (kinnear and taylor, 1996). according to multidimensional scale findings, the stress value was 0.01786, and the r2 value was 0.99863. these values indicated that the findings could be interpreted. figure 2 presents a consumer perception map generated from fuzzy comparison data. accordingly, consumer perceptions differed according to the different selling processes of the fruits. only fruit milk and fruit yoghurt were given the same weight by the consumers, while fresh fruit was placed in another dimension with dried fruit and with mineral water. according to the findings of the positioning matrix, the most distant perceived products were fruit soda and dried fruit, with a distance of 3.970, and the closest perceived products were fruit yoghurt and fruit milk, with a distance of 0.787. dried fruit was the closest product to the fresh fruit, with a distance of 2.206. however, the distance between dried fruit and fruit milk was 2.841, and the distance between dried fruit and fruit yoghurt was 2.844. hence, since fresh fruit was the closest product to dried fruit, it may be a rival for it. however, dimensional distinctions and overall distance values indicated that consumers perceived dried tropical fruits in a different position compared to other products. figure 2. two-dimensional positioning of the general perception for the products. 4. discussion and conclusions this study was conducted to evaluate the factors affecting turkish consumer preferences for dried tropical fruits (banana, kiwi, pineapple) and the willingness of these consumers to pay for these products. significant results have emerged from this research. ital. j. food sci., vol. 30, 2018 824 first, the results of the sensory evaluation method showed that there were issues with dried bananas related to taste and hardness and that the issues for dried kiwi were related to odour and taste. the sensory properties of pineapple were better than those of the other products. in alphonce et al., (2015)’s study of european consumers, the average firmness of dried banana was defined as being below average, with 4 points, while its taste was defined as being average, with approximately 5 points. both the firmness and taste scores of pineapple were just above average (5 points). in this study of turkish consumers, however, the firmness and taste scores of dried banana were less than those in alphonce et al., (2015)’s study, with 1.6 and 2.1 points, respectively. the average taste score given by turkish consumers to pineapple was 6.7, and the average firmness score was 7.5. these scores were above average. european consumers gave the highest scores to dried mango, pineapple, and banana, in order. in this study, turkish consumers preferred dried pineapple, kiwi, and banana, in order. in previous studies, dried kiwi was not evaluated sensually by consumers. generally, the dried banana taste and hardness issues and the average scores for dried pineapple agree with the study of european consumers (alphonce et al., 2015). the maturity of the fruit before drying is the most important factor affecting fruit taste and flavour because the maturity level of the fruit is related to the sugar level. very mature fruits contain high levels of concentrated sugar. during the drying process, this sugar becomes more apparent, and the dried fruit can be very sweet. conversely, this highly concentrated sugar will lead to a worse taste after drying. on the other hand, excessive drying can harden the fruit. the presence of protections against decay may cause odour problems. therefore, it is important to determine the level of moisture that does not disturb the drying style or the product taste (manzungu and machiridza, 2001). the products had equal and acceptable features in terms of appearance (shape and size) and colour. this information suggests that suppliers should focus on improving poor sensory features rather than the appearance of the products. the second important result of the survey is related to the willingness to pay for the products. it has been emphasized in previous studies that sensory attributes of food products are effective in procurement decisions (topcu et al., 2015). specifically, taste is one of the most important factors affecting the purchase preferences for both dried products (endiyani and salima, 2017) and fruits (kamenidou et al., 2002, panico et al., 2011). the results of this study also confirmed these data. in this study, 37.8% of the consumers did not want to buy dried banana, 44.8% did not want to buy dried kiwi, and 38.8% did not want to buy dried pineapple. many of the consumers who did not want to buy these products cited sensory characteristics, especially taste. in addition, the research results showed that the willingness to pay was 18.2 kg/tl for dried banana, 26.0 kg/tl for dried kiwi and 39.4 kg/tl for dried pineapple. the lowest willingness to pay was for banana. the market sales price for these products in turkey is 36 kg/tl. accordingly, consumers were willing to purchase only dried pineapple for a price above market value. therefore, the market prices and sensory characteristics of dried banana and kiwi show that they are very unlikely to be consumed frequently by consumers. the third important result of the study relates to the socio-demographic characteristics of the consumers, which affect the willingness to pay for products. the results showed that income had a positive effect on the willingness to pay for these products. these data overlap with a study conducted on consumers in zimbabwe relating their preferences for dried fruit (manzungu and machiridza, 2001). in addition, health consciousness has a significant impact on the willingness to consume a product. this result agrees with research on chinese consumer preferences for dried mango (wu, 2017). additionally, female consumers were more willing to purchase these products than were males. the education level was particularly influential on the consumption of kiwi and pineapple. studies examining consumption habits of turkish consumers have indicated that gender, ital. j. food sci., vol. 30, 2018 825 education level and income level are influential in food purchasing decisions. in addition, high-income, highly educated female consumers are more sensitive to food safety (goktolga et al, 2006). recently, increasing income levels resulting from economic development in turkey and increasing education levels, combined with the need for a balanced diet, have created a new consumer market. in this context, the sociodemographic characteristics of the consumer group that wants to purchase dried tropical fruits may facilitate the desire of the target consumer for these products. the fourth important result of the study is the creation of a hierarchy of preferences for different states of the products. different studies on dried products have revealed that products are preferred in their fresh state as opposed to their dried state (sijtsema et al, 2012, owureku-asare et al, 2017). the belief that fresh fruit has more vitamins causes french consumers to consume more of the product when it is fresh (jesionkowska et al, 2007). however, this situation may differ from country to country (jesionkowska et al, 2008). the results of this study support the idea that consumers prefer fresh fruit, as turkish consumers preferred fresh products from among all the different types of fruit products. however, consumers primarily preferred products in mineral water, then fruit milk and then fruit yoghurt. the differences in the consumption preferences and rankings of these products according to gender have been determined. specifically, females were more indifferent about whether the product was in mineral water, fruit yoghurt or fruit milk. the averages for dried tropical fruits differed from the other products. in addition, consumers positioned dried tropical fruits differently than other forms of the same fruits. this result suggests that tropical dried fruit is unique. therefore, the consumer may not make comparisons with other products while purchasing these products. this information can be used for developing advertising strategies. in general, the results indicate that sensory issues related to these products need to be addressed. in addition, when determining the target market, more attention 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science 29(2):209-221. worch t., lê s. and punter p. 2010. how reliable are the consumers? comparison of sensory profiles from consumers and experts. food quality and preference 21(3):309-318. wu d. 2017. consumer preference on intrinsic quality attributes of dried mango among chinese consumers. d. thesis. food technology, china. zhao x., chambers e., matta z., loughin t.m. and carey e.e. 2007. consumer sensory analysis of organically and conventionally grown vegetables. journal of food science 72(2):87-91. paper received january 17, 2018 accepted june 25, 2018 ijfs#1529_bozza ital. j. food sci., vol. 31, 2019 749 paper immobilization and characterization of β-glucosidase from gemlik olive (olea europea l.) responsible for hydrolization of oleuropein s. onat and e. savaş* department of food engineering, faculty of engineering, university of balikesir, cagis, 10145 balikesir, turkey *corresponding author: tel: +900266 6121194-95, fax: +900266 6121257 e-mail address: esavas@balikesir.edu.tr abstract the β-glucosidase (β-d-glucoside glucohydrolase, ec 3.2.1.21) enzyme was purified from gemlik variety olive (olea europea l.). the purified enzyme was immobilised onto supermagnetic nanoparticles in order to stabilise the enzymatic efficiency and increase usage in the food industry. the purified and immobilised enzyme was characterised by molecular weight, kinetic parameters and optimum ph and temperature values comparatively. the enzyme is a monomer with a mass of approximately 40 kda. the kinetic values of the immobilised and purified enzymes were 1.34 mm and 384.61 u/mg; 0.37 mm as km and 370.37 u/mg as vmax respectively. keywords: β-glucosidase, olea europea l., pnpg, purification, spmn ital. j. food sci., vol. 31, 2019 750 1. introduction olive is a fruit which consists a lot of biotransformal compounds. there is a wide variety of phenolic compounds in olea europaea l. which are important for sensorial properties. they also have substantial effects on human health such as nutritional, physiological and pharmaceutical effects. unripe olive fruit has important phenolic secoiridoids causing bitter taste. these polyphenolic substances called oleuropeins have many aldehydic or dialdehydic forms such as hydroxytyrosol and tyrosol, transformed by β-glucosidase as a part of the defence mechanism in the plant tissue (garcia-rodriquez et al., 2011; de leonardis et al., 2015). as a result of this system, many oleuropein-related compounds are also available from olives, rearranged via aglycon by the elenolic acid ring. the quality and quantity of these substances change by variety, tissue by tissue (leave, fruit, etc.) and in terms of ripening stage (bianchi, 2003). olives gradually get rid of their bitterness at the ripening stage. this occurs by β-glucosidase gradual hydrolyzation of oleuropein and leads to changes in taste (guirimand et al., 2010). β-glucosidases are biologically active enzymes that hydrolyse 1.4 β-glycoside bonds between carbohydrate molecules (ünal and sener, 2017). olive β-glucosidases (de leonardis et al., 2015) (β-d-glucoside glucohydrolases, ec 3.2.1.21) show high substrate specificity (mazzuca et al., 2006) as in other plant species (savas et al., 2018). they hydrolyse the β-glycosidic bonds in oligosaccharides or other glucose moieties and ester bonds in oleuropein, which is responsible for bitterness (velẩzquez-palmero et al., 2017). after the reaction, β-glucosidases lose their catalytic activities like other enzymes. some techniques may be used in food processing systems (debittering, flavour enrichment etc.) to stabilise enzyme usage. enzyme immobilisation by covalent bonding is used for stability and reuse of enzymes. for this purpose, different bulk or magnetic materials could be used as a matrix. superparamagnetic iron oxide nanoparticles (spmn) that are used for high colloidal stability, magnetism and be biocompatible materials are preferred as immobilisation matrices (ma et al., 2009). these are small synthetic ϒ-fe2o3 or fe3o4 particles with a core size of < 10 nm and well dispersed in a liquid for biomedical applications. they may be removed easily with simple magnetism from the reaction medium. gemlik variety olives are from the north-west turkey (uylaşer and şahin, 2004). this variety, which are processed olives for direct consumption has high oil content. considering the rare reports on the topic indicates the catalytic activity of olive βglucosidases. the objective of this study is to show that gemlik variety olive fruits could be used as a one β-glucosidase source for biotechnological application in the food industry. in this context, purified and immobilised forms of β-glucosidases (β-d-glucoside glucohydrolase) were investigated as a naturally occurring enzyme involved in the biotransformation of oleuropein. 2. material and methods 2.1. materials gemlik variety olives that were used in our study were obtained from balikesir at their black maturity stage. they were brought to the laboratory in cold storage conditions ital. j. food sci., vol. 31, 2019 751 (+4oc). after the washing and sorting steps, they were used for obtaining acetone powder as the enzyme source. all chemicals that were used in our study were supplied from sigma-aldrich (st. louis, mo, usa), and protein molecular weight markers were supplied from thermo scientific (waltham, ma, usa). they were of the highest grade available. 2.2. preparation of acetone powder acetone powder was used as the enzyme source in this study (savas et al., 2018). 100 g of olive pulp was homogenised for 2 min in 750 ml of cold acetone (-20°c) using a homogenizer for preparation. the homogenate was filtered by whatmann no 1 filter paper, and retentate was extracted three times with 500 ml of acetone (−20°c) to remove oil residues. reddish purple residues on the filter were air-dried at room conditions on blotting papers and held at -20oc for enzyme assays. 2 g of the acetone powder was homogenised in 100 ml of a cold extraction buffer (4°c) (ph 9.5) using an ultra torrax homogenizer. the mixtures were centrifuged at 15,000 rpm for 30 min at 4°c, and crude extracts were obtained from the supernatant. 2.3. chromatographic study the further step of enzyme purification was carried out based on the method by kara et al. (2011) by hydrophobic interaction chromatography. as defined in the method, the solid ammonium sulphate in concentrations from 0 to 50% was added to the crude extract at +4°c for ammonium sulphate precipitation. the reaction mixture was centrifuged at 15000 rpm for 30 min (+4oc), the sediment was dissolved in 50 mm of the sodium phosphate buffer (ph 6.8), and the final saline concentration of the mixture was set to 1m ammonium sulphate. the hydrophobic column was synthesized using 10% cnbr in a 1:1 solution of sepharose 4b and distilled water for the second step of the purification process. the ph of the mixture was stabilised at 11 for 8–10 min. the gel obtained was filtrated and washed with a cold 0.1m nahco3 buffer (ph 10). after the reaction mixture was combined with the saturated l-tyrosine, the solution was stirred for 90 min. after the gel, washing and diazotization of 1-naphthylamine in this complex was fixtured to the sepharose-4b-ltyrosine. the further steps were carried out as described in the method by 3 ml of enzyme solution loaded onto the hydrophobic column. 1 ml fractions were gathered at a flow rate of 30 ml/h in a linear gradient. the fractions were collected with the highest protein content and used in next studies as the purified enzyme. 2.4. immobilisation superparamagnetic nanoparticles (spmn) were synthesized specifically for the use of enzyme immobilization (kockar et al., 2010). 20-100 mg of fe+2/+3 superparamagnetic nanoparticles (spmn) was placed into 2 ml of a 0.003 m phosphate buffer (ph 6) with 0.1 m of nacl, and 0.5 ml of carbodiimide solution (0.025 g/ml in buffer) was placed into the reaction medium. the reaction medium was sonicated for 10 minutes. 2 ml of purified βglucosidase enzyme was added and sonicated for 30 minutes. ital. j. food sci., vol. 31, 2019 752 2.5. characterisation assays and protein determination in all steps for olive β-glucosidase extraction, purification and further studies, activity of the enzyme was measured at 410 nm against para-nitrophenyl-β-d-glucopyranosides (pnpg) as substrate (lowry et al., 1951). 70 μl of enzyme solution in 50 mm sodium acetate (ph 5.5) and 70 µl of substrate were added in a 96 well plate. incubation of the well content was facilitated at 37◦c for 30 min in triplicates. 70μl of 0.5m na2co3 was added into the medium for stopping the reaction, and the absorbance values were determined by spectrophotometry. enzyme activity was expressed as μmol p-nitrophenol composed per minute in the reaction medium under these terms. molecular mass values of protein were estimated using a commonly used standard (bovine serum albumin bsa). sds polyacrylamide gel electrophoresis (sds-page) was carried out to estimate the molecular weight of olive β-glucosidase according to the method reported by li et al. (1997) using a minigel system (bio-rad laboratories, usa). after gel colorization with coomassie brilliant blue r-250 and decolorization with 7.5% acetic acid in 5% methanol to detect protein bands, the gel was photographed with uv light. 25 mm sodium acetate (2.0–10.0) buffers were used for the ph optima assays (kara et al., 2011). activity measurements were achieved by using 5 mm of substrate (ph 5.5) at the temperature range of 25 to 65ºc for 30 min to determine the optimum temperature. the thermal stability was determined by incubating at 70 for 30 min and then cooling down to 4 °c. p-npg (concentration range from 0.12-2.38 mm) and oleuropein were studied for determining km and vmax values. glucose, citric acid, lactic acid, sodium hydroxide and sodium chloride were studied as potential inhibitors. activity measurements of the samples were performed, and 1.75 mm of pnpg and 1 mm of ni+1, zn+1, cr+1, mn+2, mg+2 and co+1 were used in the reaction medium. the effect of various concentrations (0.0029-0.0297 mm) of deltamethrin, chlorpyrifos and alphacypermethrin as widely used pesticides against olive insects on olive β-glucosidase were also studied using 1.25 mm of pnpg as the substrate. results are given as relative activity, which the enzyme activity in the noninhibitor medium is considered 100. the inhibitor concentration, which reduces the enzymatic activity by 50% (ic50 values), was determined by the relative plots. a fourier transform-infrared spectroscopy (ft-ir) analysis was performed to prove the correctness of the enzyme immobilisation after the purification step. ir spectra of fe3o4 superparamagnetic nanoparticles, β-glucosidase and immobilised β-glucosidase on the superparamagnetic nanoparticles were obtained by using the kbr pellets preparation technique in atr cells (600-4000cm-1) with a perkin elmer-1600 series device. 3. results and discussion 3.1. enzyme extraction and purification protein assays were achieved by using acetone powder as previously reported (koudounas et al., 2015). although it is known that the use of acetone leads to mutual effects, thanks to usage of acetone, it is possible to obtain concentrated proteins without pigments derived from fresh fruits (romero-segura et al., 2009). furthermore, acetone powder usage also makes it possible to use as stock enzyme source in the absence of the olive fruit. ital. j. food sci., vol. 31, 2019 753 hydrophobic interaction chromatography (savas et al., 2018) was used by precipitation with ammonium sulphate to separate β-glucosidase from the olive acetone powder. after precipitation of the β-glucosidase active fractions with ammonium sulphate, 97% of the activity was measured (table 1). in this part of the process, great proteins except βglucosidase were removed, and the quantity of the protein was reduced from 297 to 23 mg. the elution pattern of enzyme activity and total protein concentrations for all fractions that were collected on the hydrophobic column are shown in fig. 1. the fractions that had the highest enzyme activity were pooled. the enzyme was purified 163-fold from the remaining particles with clear homogeneity with an overall enzyme yield of 9.90% and a specific activity of 6291.7777 u/mg (table 1). the purification yield values were higher than those previously reported for olive (li et al., 2005; mazzuca et al., 2006) and several sources (cameron et al., 2001; li et al., 2005; verma et al., 2011). minimal sequential steps, matrix and ligand characteristics (hydrophobic structure of 1-napthylamine, sepharose-4b gel matrix and l-tyrosine arm) led to increased purification factors. more purification steps could result in better purification rates. however, more steps cause a dramatic decrease in enzyme activity and protein amounts. table 1. purification of β-glucosidase from olive (olea europaea cv. gemlik). purification steps volume (ml) total protein (mg) total activity (u) specific activity (u/mg) yield (%) crude extract ammonium sulphate hydrophobic chromatography 40 10 2 297.27 23.19 0.18 11430.88 11123.31 1132.52 38.4528 479.6597 6291.7777 100 97.30 9.90 figure 1. purification of olive fruit β-glucosidase by hydrophobic interaction chromatography. the enzyme activity and total protein concentrations were determined from all fractions that were collected, as described in section 2. the enzyme activity was expressed as µmol of p-/o-nitrophenol liberated per minute in the reaction. a single band with an apparent molecular mass of ca. 42 kda was seen by standard methods of sds-page electrophoresis (fig. 2). the β-glucosidase of the olive is a ital. j. food sci., vol. 31, 2019 754 monomer like other plant sources e.g. tea, citrus. there are many β-glucosidase results reported as monomer and oligomer from different plant sources e.g. 68 kda from rauvolfia serpentina, 92 kda (verma et al., 2011), 37 kda from tea leaves (li et al., 2005), 65 kda from almond (he and withers, 1997), from sweet cherry fruit (prunus avium l.) (gerardi et al., 2001) and 55 kda from citrus sinen-sisvar. valencia (kaya, 2014). estimated molecular mass of β-glucosidase from olive was reported previously as 55-65.5 kda (romero-segura et al., 2009; kara et al., 2011; kaya, 2014). these different results indicate the molecular mass of β-glucosidases from olives depending on variety. figure 2. sds-page analysis of the β-glucosidase purified from olive (olea europaea cv gemlik) fruit. the enzyme was electrophoresed at ph 8.3 on a 12% polyacrylamide gel and stained with coomassie brilliant blue r-250. lane 1: molecular weight standards (β-galactosidase, 116kda; bovine serum albumin, 66.2kda; egg albumin, 45kda; lactate dehydrogenase, 35kda; rease bsp981 (e. coli), 25kda; β-lactoglobulin,18.4kda; lysozyme, 14.4kda); lane 2: purified β -glucosidase. 3.2. characterisation of enzyme sources the activities of purified and immobilised olive β-glucosidase in different ph are shown in fig. 3. the optimal ph was found at 5.5 for both enzymes. the purified enzyme showed higher activity relatively in the range of ph 4.5-6 with any activity at ph 2 and 8. the optimal ph values of β-glucosidases that were determined in previous studies from olive fruit (romero-segura et al., 2009; kara et al., 2011; koudounas et al., 2015), from citrus (cameron et al., 2001), from wheat (sue et al., 2000a) and rye (sue et al., 2000b) were similar and higher than β-glucosidases from rice (ph 4.5) (akiyama et al., 1998), soybean (ph 4.5) (masaru et al., 1995), barley (ph 5.0) (leah et al., 1995) and lower than vanilla bean (ph 6.5) (odoux et al., 2003) and maize (ph 5.8) (cuevas et al., 1992). ital. j. food sci., vol. 31, 2019 755 figure 3. effect of ph on activity of purified olive (olea europaea cv gemlik) fruit β-glucosidase. ph optima of β-glucosidase purified from olive (olea europea l.) and immobilised form. the optimum temperature was determined in the purified and immobilised enzyme sources respectively as 50 and 55oc by using p-npg as a substrate (fig. 4a). temperature optima of the immobilised enzyme were higher than the free enzyme by 5oc like other immobilised enzymes (singh et al., 2011). the enzyme became more stable after the immobilization process by means of the vineyard structures. the enzyme purified from olive fruit showed maximum activity at 50oc and 35oc with p-npg and oleuropein as substrates, respectively (fig. 4b). the purified enzyme lost its catalytic activity at the end of the 30th min at 70oc with p-npg as a substrate in the 50 mm acetate buffer (fig. 4c). it was reported that plant β-glucosidase showed maximal hydrolytic activity towards pnpg at 40–45°c in citrus sinensis var. valencia fruit (cameron et al., 2001), 25–30°c in rye (sue et al., 2000b), 45°c in soybean (masaru et al., 2005), 50°c in rice (akiyama et al., 1998), 60°c in barley (leah et al, 1995) and 40°c in vanilla bean (odoux et al., 2003). the temperature optima of our enzyme were similar to that for β-glucosidases taken from tea (li et al., 2005), rice (akiyama et al., 1998) and maize (cuevas et al., 1992) using pnpg as a substrate. the enzyme was still active by 33% at 4°c and 11% of the initial level at 25°c after 8 weeks (data not shown). this temperature-sensitive enzyme was more stable at cold conditions. 3.3. f-tir analysis the f-tir charts indicated patterns of purified and immobilised olive β-glucosidase on to spions, fe3o4 superparamagnetic nanoparticles (spmn) and processed spions (fig. 5). the peak of the enzyme at 1400cm-1 was lost on the spmn activated by carbodiimide (without enzyme) and enzyme-bond spions, while the new peak occurred in the range of 1000-1100 cm-1. it was concluded according to the ir spectrum that, the new bond indicated spmn-carbodiimide activation. in the enzyme bond spmn pattern, the characteristic peaks revealed that bonding of the enzyme onto nanoparticles took place. ital. j. food sci., vol. 31, 2019 756 0 20 40 60 80 100 120 25 30 35 40 45 50 55 60 65 re la ti ve a ct iv it y (% ) temperature (oc) i̇mmobilised purifieda 0 20 40 60 80 100 120 25 30 35 40 45 50 55 60 65 re la ti ve a ct iv it y % temperature (oc) oleuropein pnpgb figure 4. effect of temperature on olive β-glucosidase activity. temperature optima of a) β-glucosidase purified from olive (olea europea l.) and immobilised form using p-npg as the substrate, and b) purified enzyme using different substrates. c) thermal stability of purified olive β-glucosidase by using p-npg as the substrate. ital. j. food sci., vol. 31, 2019 757 figure 5. ft-ir spectra of immobilization support material, pure enzyme and enzyme bound nanoparticle. 3.4. substrate specificity enzyme kinetics were studied using p-nitrophenyl-β-d-glucopyranosides (p-npg) and oleuropein as substrates. the β-glucosidase from olive tissues activates oleuropein by converting the secoiridoid glucoside moiety of the oleuropein when the tissues are damaged by insects or herbivores (konno et al., 1999). olive fruit β-glucosidases that were able to hydrolyse olive glucosides exhibited high substrate specificity to oleuropein (romero-segura et al., 2009). the enzyme was effectively active on p-npg and oleuropein with the km values of 0.37 and 1.7 mm and the vmax values of 370.3 and 1000 u/mg, respectively (table 2). although the affinity of the olive β-glucosidase for p-npg was considerably higher than for oleuropein, the activity was lower. the higher βglucosidase affinity for p-npg was reported on sorghum (cicek and esen, 1998), tea leaves (li et al., 2005), orange (cameron et al., 2001) and olive (kara et al., 2011). table 2. kinetic parameters of purified and immobilised β-glucosidase from olive with different substrates. β-glucosidase substrates vmax(u) km vmax/km purified p-npg 370.37 0.37 1001 oleuropein 1000 1.7 588.23 immobilised p-npg 384.61 1.34 287.02 oleuropein 2000 6 333.3 ital. j. food sci., vol. 31, 2019 758 3.5. effects of inhibitors β-glucosidases have been researched with reversible and irreversible inactivators (he and withers, 1997; rempel and withers, 2008). the purified enzyme was incubated in the presence of glucose, lactic acid, sodium hydroxide, sodium chloride, citric acid, some pesticides and mg2+, mn2+, ni1+, co1+, cr1+, zn1+ ions using p-npg as the substrate (1.66 mm) to determine the inhibition kinetics. although a strong inhibitory effect could not be detected at the studied concentrations, citric acid was more effective with ic50 of 2.38 mm than glucose with ic50 of 23.33 mm and lactic acid with ic50 of 145.7 mm (fig. 6a-f). figure 6. inhibition of the β-glucosidase purified from olive (olea europaea l.) fruit by p-npg activity (%) curve in the presence of different a) glucose, b) lactic acid, c) citric acid, d) nacl and e) naoh concentrations, f) lineweaver–burk plot with various concentrations of p-npg for ic50 . it was reported that citric acid is the main organic acid followed by succinic acid in turkish olive varieties (aslan and ozcan, 2011). it is the most commonly used agent in ital. j. food sci., vol. 31, 2019 759 olive fermentation for initial acidity in the fermentation medium. it is contemplated that the concentration of the citric acid in the reaction medium will result in a lower ph level than the ph optima of the enzyme. glucose is the main compound for enzymatic saccharification of cellulolytic substrates and presents as fermentable sugar in the reaction medium. similar inhibitory effects of glucose have been reported for β-glucosidase in olive in a previous study (kara et al., 2011; savas et al., 2018), whereas in another study, it was stated that no inhibitory effect of glucose was determined (romero-segura et al., 2009). differences in the sequence of amino acids found in the structure of plant β-glucosidases reflect observed quaternary structures and change in the properties of the active site (yu et al., 2007). it is thought that, as a result of this, the kinetic parameters will be changed. these differences in olive βglucosidases, whose molecular weights and other kinetic parameters are different from each other, are due to the differences in protein structures depending on the variety. it is necessary to study the kinetic parameters of olive β-glucosidases of different varieties and at different maturity levels for future studies. generally, plant origin β-glucosidases were reported to be resistant to high glucose concentrations (ramani et al., 2012). unlike other chemicals that are used, the observed slight activation by nacl and naoh may be explained by the ions’ effect of na+ on enzyme structure stabilization. β-glucosidases were reported as metalloprotein and required metal ions for action (ramani et al., 2012). the inhibition kinetics were determined in the presence of mg+, mn+, ni+, co+, cr+ and zn+ to verify the effects of several metal ions on olive β-glucosidase activity (fig. 7). figure 7. the effects of some heavy metal ions on the activity of β-glucosidase purified from olea europea l. the crude enzyme activity was indicated as the control. the activity of the control was accepted as 100%. trials were achieved by three replicates. metal ion concentration in this study was in the range from 1 to 1.75 mm. the relative enzyme activity was 67.36, 76.46, 74.85, 65.75, 59.33 and 91.4 presence of mg2+, mn2+, ni1+, co1+, cr1+ and zn1+ ions, respectively. the results showed that all metal ions, which were 0 10 20 30 40 50 60 70 80 90 100 110 control mg mn ni co cr zn re la ti ve a ct iv it y (% ) heavy metals (1mm) ital. j. food sci., vol. 31, 2019 760 used showed successful inhibitory effects on olive β-glucosidase. especially cr+ and mg+ were more effective ions as the inhibitor onto olive β-glucosidase, while ni1+ was mentioned in the literature as an inhibitor of olive β glucosidase like cd 2+ pb 2+ cu 2+ and ag. 1-10 mm of mg2+ and mn2+ were reported as strong activators for fungal βglucosidases (ma et al., 2011; chen et al., 2012; bai et al., 2013) except for neocallimastix patriciarum w5 (chen et al., 2012). our findings were mostly incompatible with those obtained by other studies. this is thought to be caused by the diversity of the enzyme sources. the metal ions that are mentioned above are found in the chemicals mostly used as antifungal agents. the ic50 values of deltamethrin, chlorpyrifos and alphacypermethrin as the most commonly used pesticides in olive farming were 0.0323, 1.25 and 13.29 mg/l respectively. deltamethrin is used as insecticide in olive trees. as the reported optimum concentration that was used was 0.31 for olive tree, the obtained ic50 value was sufficient for inhibition of olive β-glucosidase. 4. conclusions the olive β-glucosidase was purified from gemlik variety turkish olives, and the free enzyme was immobilised onto fe+2/+3 superparamagnetic nanoparticles to increase the stability and reusability for industrial applications. this is the main enzyme the is responsible for oleuropein hydrolysis during the maturation period. at the same time, there is also industrial use of it for aroma formation and debittering step in table olive production. in previous studies, basic characterisation of olive β -glucosidase was investigated. however, this is the first time where this enzyme was immobilised onto superparamagnetic nanoparticles and characterization of the immobilised enzyme was studied comparatively. in the study, the potential activator and inhibitor effects of the substances under the process conditions in table olive production were determined for the purpose of developing materials which can be used in the debittering process of table olive production. because of the nanomaterial that was used is biocompatible and riskfree, it may be safely used in food production. after immobilization, the enzyme became more stable under environmental conditions. acknowledgements this study was funded by the research fund of the university of balikesir (bap.3.2016.0001), and turkish research council project grant no 110o778, turkey and patented under number of pt2013-01335. abbreviations kda kilodalton hic hydrophobic interaction chromatography spmn superparamagnetic nanoparticles eu/mg enzyme unit/milligram sds-page sodium dodecyl sulphate polyacrylamide gel electrophoresis dna deoxyribonucleic acid rna ribonucleic acid uv ultra violet ir infra-red temed n,n,n’,n’,tetramethylenediamine ital. j. food sci., vol. 31, 2019 761 pnpg p-nitrophenyl-alpha-d-galactopyranoside onpg ortho-nitrophenyl-galactopyranoside pnpgal p-nitrophenyl a-dgalactopyranoside onpgal orto-nitrophenyl-β-d-galactopyranoside pmsf phenylmethylsulfonyl fluoride edta ethylenediaminetetraacetic acid dtt dithiothreitol bsa bovine serum albumin dmpd n, n-dimethyl-p-phenylenediamine dpph di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium h2o2 hydrogen peroxide hydrogen peroxide tris tri hydroxymethyl)aminomethane ftir fourier transform infrared spectroscopy kbr potassium bromide pe purified enzyme ie immobilised 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by harvest year lwt food sci. and technol. 79:190-198. verma o.p., singh a., singh n.and chaudhary o.2011. isolation, purification and characterization of β-glucosidase from rauvolfia serpentina j. chem. eng. process. technol. 2:119-123. velázquez-palmero d., romero-segura, c., garcía-rodríguez,r. hernández, m.l., vaistij, f.e., graham i.a., pérez, a.g., and martínez-rivas j.m. 2017. an oleuropein β-glucosidase from olive fruit is involved in determining the phenolic composition of virgin olive oil. front plant sci. 8:1902. yu h.l., xu j.h., lu w.y. and lin g.q. 2007 identification, purificationand characterization of b-glucosidase from apple seed as a novel catalyst for synthesis of o-glucosides. enzyme microb. technol. 40:354-361. paper received february 15, 2019 accepted april 15, 2019 ijfs#1343_bozza ital. j. food sci., vol. 31, 2019 253 paper changes in the antioxidant capacity and polyphenols content of rye-buckwheat cakes fortified with spices during their long-term storage m. starowicz* and h. zieliński department of chemistry and biodynamic of food, division of food science, institute of animal reproduction and food research of the polish academy of sciences, tuwima 10, 10-748 olsztyn, poland *corresponding author: tel.: +48509078453; e-mail address: m.przygodzka@pan.olsztyn.pl abstract the long-term storage of cakes may affect their functional and physicochemical properties. in this study, we investigated changes in the antioxidant capacity and polyphenols content of rye-buckwheat cakes with selected spices, e.g. cloves, nutmeg, cinnamon, vanilla, allspice and a commercial blend of spices, during their 18-month storage at 23°c, 60% humidity. measurements were performed after each 6 months. during storage, chemical markers decreased considerably. the observed decrease in the antioxidant capacity was highly correlated with contents of total phenols, flavonoids and rutin. the use of cloves, cinnamon and a spice mix in rye-buckwheat recipe offered a higher functionality of cakes. keywords: antioxidant capacity, flavonoids, rye-buckwheat cakes, storage, total phenolics, spices ital. j. food sci., vol. 31, 2019 254 1. introduction according to the report of the polish agriculture market agency, the consumption of cookies and cakes had been increasing till 2008 and became stable for many years (agriculture market agency, 2013). producers develop new recipes to attract consumers with new flavors and enrich confectionery products with healthy ingredients. spices are well-known sources of natural antioxidants, which might be used as preservatives or flavor enhancers in cookies and biscuits (embuscado, 2015). a wide range of compounds with different molecular structures has been suggested to be responsible for the high antioxidant status of spices (yashin et al., 2017). therefore, they might significantly influence the quality and sensory parameters of confectionery products, but also increase their functional properties like e.g. their antioxidant activity (nanditha and prabhasankar, 2009). pasqualone et al. (2014) described the positive influence of grape marc (by-product with high antioxidant potential) on biscuits’ flavor and sensorial parameters, e.g. by more intense color and fruity odor formation. powders are the main form in which spices are incorporated into confectionery products. therefore, bajej et al. (2006) demonstrated that better bakery parameters as well as consumer acceptability were achieved for biscuits incorporating mint powder than for biscuits with either mint extract or menthol. in some cases, however, essential oils of spices – which are generally acceptable by consumers – are added to provide the antioxidant capacity of confectionary goods (basuny et al., 2012). cinnamon, mint, nutmeg and cloves were indicated as the most commonly used spices and herbs in confectionary (nanditha and prabhasankar, 2009). garlic, ginger, basil, turmeric or coriander powders were also used in some studies with wheat bread (dziki et al., 2014). the addition of spices, as the main sources of antioxidants, is expected to prolong storage stability of confectionery products, ensure consumer acceptability and provide healthy properties. according to many reports, consumer acceptability may be affected by the formation of oxidative compounds, which might create an unpleasant aroma and/or rancid and bitter taste in stored products. oxidation may be effectively inhibited by antioxidants, therefore recipes of confectionery products were modified to increase antioxidants content in many ways. for example, watanabe et al. (2014) reported that the addition of quinoa flour to wheat cookies increased their antioxidant capacity and then improved their oxidative stability during 50-day storage period (lower peroxide value of cookies incorporating 7.5% of quinoa flour). zieliński et al. (2012) demonstrated a greater increase in the antioxidant capacity of ginger cakes made of rye flour, instead of wheat flour, after long-time storage. they speculated this might also be due to the formation of some of the maillard reaction products, e.g. melanoidins, which was indicated by the higher values of the browning index. in turn, jensen et al. (2011) reported no significant changes in the oxidative status during storage of wheat bread incorporated with a rosemary extract or an α-tocopherol solution. however, enhanced hydroperoxide formation was determined in the samples prepared with α-tocopherol, which means that the rosemary extract protected food from oxidation. in studies of ning et al. (2017) the addition of green tea powder to wheat bread significantly increased its overall antioxidant capacity and inhibited the formation of peroxides during 8-day storage (which was described as lower peroxide values, even at only 1% of green tea addition) as compared to wheat bread. also interesting results were obtained by dar et al. (2016), who developed rice snacks with a combination of cereal brans. according to their results, the antioxidant capacity of these snacks decreased significantly during 6 months of their storage, i.e. about 25 up to 28%. they also noticed the negative effect of storage on total phenolic contents, which might be due to lowstability of molecules in the matrix and advanced degradation process of phenolic ital. j. food sci., vol. 31, 2019 255 compounds. the reason of these changes was explained as “dilution of antioxidant components by increased moisture” or oxidation of antioxidant molecules during longterm storage, but these are only speculations as causes these changes are still unknown. little literature focused on changes in the antioxidant capacity and phenolic content in cereal products is available today. therefore, the aim of this study was to determine changes in total phenolic and flavonoid contents during 18-month storage of rye-buckwheat cakes fortified with selected spices. the spices used in rye-buckwheat cakes formula were: cloves, nutmeg, cinnamon, vanilla, allspice and a commercial blend of spices for cakes. the content of rutin, as the main polyphenol occurring in buckwheat flour and honey, was determined to control its changes during storage period. furthermore, antioxidant capacity was evaluated by measurements of the scavenging ability against dpph• and abts+• radicals. this study focused also on the antioxidant stability of rye-buckwheat cakes during storage. moreover, spices with the best impact on storage stability of the analyzed cakes were selected. the measurements were performed every 6 month till the 18th month of storage period and compared with results obtained immediately after baking. 2. material and methods 2.1. chemicals standards of rutin (quercetin-3-rutinoside), gallic acid and catechin as well as chemicals used for antioxidant capacity determination: 2,2-azinobis(3-ethylbenzothiazoline-6sulfonic acid) diammonium salt (abts), 2,2-diphenyl-1-picrylhydrazyl (dpph), 6hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox), sodium nitrite, aluminum chloride, and sodium carbonate were purchased from sigma (usa). ethanol, acetonitrile and formic acid were obtained from merck (germany). folin-ciocalteu reagent was provided by poch (poland). for hplc analysis, water was purified with the mili-qsystem (milipore, usa). the spices used in rye-buckwheat cakes formula as: cloves (syzygium aromaticum l.), nutmeg (myristica fragrans h.), cinnamon (cinnamomum verum j.), vanilla (vanilla mill.), allspice (pimenta dioica l.) and a commercial blend of spices for cakes, were provided from local market (kotányi, poland). according to the producer‘s declaration, commercial spice mix contains cinnamon, pepper, clove, anise, coriander, fennel and nutmeg. tpc values of each spice were previously calculated in our studies (przygodzka et al., 2014): clove 156.67±3.52 (mg gae g-1), nutmeg 11.80±0.55, cinnamon 180.59±14.23, vanilla 8.08±0.49, allspice 183.92±1.26, and spice mix 101.33±5.66. also tf of selected spices was measured (mg rutin g-1): clove 19.78±0.54, nutmeg 10.21±0.17, cinnamon 96.81±1.95, vanilla 3.97±0.37, allspice 16.82±0.07, and spice mix 64.21±1.82. 2.2. preparation of rye-buckwheat cakes and their storage conditions preparation of rye-buckwheat cakes with selected spices was described in detail in our previous work (przygodzka et al., 2016).the composition of ingredients used is listed in table 1. the rye-buckwheat cakes fortified with cloves, nutmeg, cinnamon, vanilla, allspice, and a spice mix were prepared while rye-buckwheat cakes without spices served as a control sample. after baking, the cakes were cooled down and part of them were freeze-dried, ground into powder and then used directly for analyses (“0”), whereas the others were packed into plastics bags with clips, stored at room temperature, without light ital. j. food sci., vol. 31, 2019 256 for 6, 12 and 18 months, and afterwards defrosted and ground into powder to prepare cake extracts. table 1. composition of rye-buckwheat cakes with selected spices. ingredients (g) c cakes with spices rye flour 70 70 light buckwheat flour 30 30 buckwheat honey 50 50 sugar 20 20 baking powder 3 3 butter 25 25 cloves/ nutmeg/ cinnamon/ vanilla/ allspice or spice mix 0 2 2.3. rye-buckwheat cake extracts preparation about 100 mg of powdered rye-buckwheat cakes were placed in 2-ml eppendorf flasks, to which 1 ml of an extraction solvent (ethanol/water 1:1, v/v, the solvent was selected according to our previous study (przygodzka et al., 2014) was added. the mixture was vortexed for 1 min and then extracted with ultrasonic waves for 30 sec. then, it was centrifuged at the maximum speed and a temperature of 4ºc for 15 min. the supernatant was then transferred to a 5-ml volumetric flask and the residues were re-suspended in another 1-ml portion of the ethanol/water solution. this step was repeated until 5 ml of the extract had been obtained. these extracts were used for further analyses of rutin, total phenols and flavonoids contents, and for antioxidant capacity determination with abts and dpph assays. 2.4. determination of rutin, total phenolics and flavonoids contents the content of rutin in rye-buckwheat cakes was determined with the hplc system (shimadzu, japan) with a uv detector (spd-10a) set up at 330 nm according to the procedure presented by zielińska et al. (2010). five concentrations of a rutin standard were prepared in triplicate in the range of 1.0-40 μm, then solutions were filtered through nylon filter membranes, with pore size of 0.45 µm, before injection. the results were expressed in μg g-1 of dry weight. the tpc and tf contents were determined as previously described by przygodzka et al. (2014). tpc content was expressed in terms of mg gallic acid equivalents (gae) g-1 of dry weight, whereas tf content was expressed as mg of catechin (ca) g-1 of dry weight. 2.5. determination of antioxidant capacity (ac) by abts and dpph assays the antioxidant capacity of rye-buckwheat cakes enriched with spices was determined by abts assay as described by przygodzka et al. (2014). also scavenging ability against dpph radicals was measured according to przygodzka et al. (2015). the results provided by both abts and dpph assays were expressed as μmol of trolox equivalents (te) g-1of dry weight. ital. j. food sci., vol. 31, 2019 257 2.6. statistical analysis the results of the analyses are given as the means and the standard deviation of three independent extractions (mean±sd). statistical one-way analysis of variance (anova) using fischer test was performed. the significance level was set at p< 0.01. pearson correlation coefficients were calculated to enable correlations analysis of the results of rutin, total phenolic and flavonoids contents, and results of abts and dpph assays. the correlations were calculated for each type of rye-buckwheat cakes fortified with selected spices over 18-month storage period. 3. results and discussion 3.1. changes of rutin, total phenols and flavonoids contents of rye-buckwheat cakes during storage changes in rutin content in cakes made of rye and buckwheat flour mixtures with selected spices during 18-month storage are illustrated in fig. 1. generally, a progressive reduction of rutin content was observed in all samples. it the control samples, it decreased significantly after 6 months of storage (almost 45%), to reach finally 86% decrease after 18 months of storage. in the case of cakes with spices addition, after 6 months the lowest decrease was noticed in cakes with allspice and cloves, from 173 to 89 µg g-1 and 320 to 190 µg g-1, respectively. figure 1. the influence of storage time on rutin content in rye-buckwheat cakes fortified with selected spices. sd were not higher than 10%. a further decrease in rutin content was observed after 12 months, and after 18 months the lowest rutin content was determined in the cakes with cinnamon and the highest one in the cakes with the spice mix and allspice. the final rutin content was the highest in the cakes fortified with the spice mix, cloves and vanilla. our observations are in an agreement with findings reported by sakač et al. (2016), who also observed a decrease of rutin content in rice-buckwheat cookies (80:20, v/v) during 16-month storage. they noticed the difference between rutin content in unpacked and packed cookies after the 0 50 100 150 200 250 300 350 0 6 12 18 r ut in c on te nt (µ g/ g) storage time (months) c1 with cloves with nutmeg with cinnamon with vanilla with allspice with spice mix ital. j. food sci., vol. 31, 2019 258 first 6 months of storage, while afterwards no difference was demonstrated. in this case, it might be hypothesized that the lower rutin content was associated with the formation of new molecules with another compound like lysine, as it was described by zhang et al. (2016), or that rutin might be degraded to quercetin during the baking process (luškič et al, 2016). moreover, dadáková et al. (2011) noticed that 12month storage of black elderflowers at 22ºc impacted a decrease of rutin amount, even much more than of chlorogenic acid. however, storage of asparagus at 4ºc or as freeze-dried flour had no significant influence on rutin content (stoffyn et al., 2012). moreover, rutin content decrease could be ascribed to the oxidation processes. in model studies of buchner et al.(2006), a decrease in rutin concentration was observed in solutions with rutin kept under air perfusion. this effect might be reduced with vacuum packaging of cakes. not many studies have been focused on changes in contents of individual polyphenols in bakery and/or confectionery products during storage, whereas a high number of research works is available on the monitoring of changes of total phenolic compounds. therefore, the total phenolic compounds (tpc) content was monitored every 6 months in rye-buckwheat cakes fortified with selected spices (table 2). its decrease was observed in all cakes over the entire period of storage. after 6-month storage it decreased by 6% in the control cakes and by 2% and 24% in the samples with spice mix and nutmeg, respectively. after another 6 months of storage, a significant decrease of tpc content was noticed in almost all cakes samples except for control and with allspice. then, after 18 months the greatest loss of tpc was determined in the cakes with nutmeg (33%) and the lowest one in the cakes with the spice mix (9%). a significant decrease in tpc content, by 73 and 58%, was also observed in the cakes with cloves and spice mix. despite the fact that the tpc content decreased by more than a half, still a very high tpc content was noted in the cakes with cloves. moreover, the highest tpc content was reached in the cakes made of light buckwheat flour and fortified with spice mix, cloves, cinnamon and then allspice. the changes in tpc contents were higher compared to these presented previously by zieliński et al. (2012), which might be a consequence of rapid rutin degradation in cakes made of buckwheat flours. therefore, the higher tpc content in rye-buckwheat cakes before the storage might be due to higher amounts of honey and ginger in their recipe. our results are in accordance with findings reported by sakač et al. (2016), who observed that tpc decreased along the prolonged storage period. after 6-month storage, the tpc content in rice-buckwheat cakes decreased by about 25%, then after 12 months by up to 55%. moreover, these authors found no difference between tpc contents in the samples stored at 23 and 40°c, whereas slightly higher tpc values were observed in unpacked than in packed cakes. table 2. content of total phenolic compounds (tpc) and flavonoids (tf) in rye-buckwheat cakes fortified with selected spices: cloves, nutmeg, cinnamon, vanilla, allspice and spice mix. tpc [mg gae g-1] storage time spices 0 m 6 m 0-6 m 12 m 0-12 m 18 m 0-18 m c (without spices) 1.12±0.03ga 1.05±0.07eab ↓ 6% 1.04±0.01fb ↓ 7% 0.85±0.03ec ↓ 24% cloves 2.11±0.04ca 2.04±0.06ba ↓ 3% 1.73±0.06bcb ↓ 18% 1.68±0.03bb ↓ 20% nutmeg 1.56±0.10ea 1.18±0.04db ↓ 24% 1.09±0.01ec ↓ 30% 1.04±0.01dd ↓ 33% cinnamon 2.28±0.05ba 2.01±0.06bb ↓ 12% 1.80±0.06bc ↓ 21% 1.66±0.02bd ↓ 27% vanilla 1.32±0.08fa 1.25±0.07db ↓ 5% 1.14±0.01dc ↓ 14% 1.01±0.04dd ↓ 23% allspice 1.84±0.07da 1.63±0.05cb ↓ 11% 1.62±0.05cb ↓ 12% 1.57±0.02cb ↓ 15% spice mix 2.70±0.09aa 2.64±0.03aa ↓ 2% 2.53±0.05ab ↓ 6% 2.46±0.03ab ↓ 9% ital. j. food sci., vol. 31, 2019 259 tf [mg ca g-1] storage time spices 0 m 6 m 0-6 m 12 m 0-12 m 18 m 0-18 m c (without spices) 2.02±0.01fa 1.59±0.02eb ↓ 21% 1.37±0.07gc ↓ 32% 1.24±0.01ed ↓ 38% cloves 3.45±0.01aa 3.11±0.06ab ↓ 10% 3.01±0.07ab ↓ 13% 2.86±0.05ac ↓ 17% nutmeg 2.20±0.01ea 2.13±0.02db ↓ 3% 1.98±0.01fc ↓ 10% 1.63±0.02dc ↓ 26% cinnamon 2.95±0.05ba 2.81±0.05bb ↓ 5% 2.46±0.05bc ↓ 16% 2.00±0.02bd ↓ 32% vanilla 2.47±0.02dca 2.35±0.02cb ↓ 5% 2.18±0.01dc ↓ 12% 1.65±0.03dd ↓ 33% allspice 2.21±0.02ea 2.15±0.05dab ↓ 3% 2.10±0.02eb ↓ 5% 1.83 ±0.04cc ↓ 17% spice mix 2.96±0.03ba 2.79±0.08ba ↓ 6% 2.54±0.04cb ↓ 14% 1.94±0.05bc ↓ 34% a, bmeans in the same column with different letters as superscripts are significantly different (p <0.001). a, bmeans in the same row with different letters as superscripts are significantly different (p<0.001). in our case, however, the changes in tpc contents were smaller. dar, sharma and nayik (2016) also found a smaller decrease of tpc content (at about 17%) in snacks prepared from rice flour and a blend of three brans: wheat, oat and rice (2:1.5:1.5), during storage at room temperature. also liang and were (2018) reported on the loss of phenols in butter cookies with different addition of sweeteners. furthermore, the greatest decrease in tpc contents after 24-hour storage was observed in the cookies with honey and the lowest one in these with xylitol. this might be due to interactions between sugars (glucose, fructose etc.) and formation of maillard reaction compounds. the loss of tpc was also explained by the formation of favorable polyphenol-sugar adducts which were further rearranged to brown pigments (melanoidins) (zhang, chen and wang, 2013). the pearson coefficient of correlation between tpc and rutin content (table 4) was very high for almost all samples of cakes fortified with cinnamon (0.987), allspice (0.985), spice mix (0.982), nutmeg (0.974), and vanilla (0.937). the main flavonoids of common buckwheat kernels are flavonol glycosides: rutin, quercetin, and kaempferol-3-rutinoside (tian, li and patil, 2002). total flavonoid (tf) content was also monitored during rye-buckwheat cakes storage and its significant changes were determined. in the control samples, tf values decreased from 2.02 for “0” time, through 1.59 after 6 months and 1.37 after 12 months and finally to 1.24 mg g-1 after 18 months. furthermore, after 6 months no significant decrease was observed in the cakes with addition of nutmeg, and allspice, whereas 5% decrease was noticed in cinnamon and vanilla, and 6% decrease of tf was noticed in spice mix. then, 12-month storage caused a further decrease in tf content in the range from 5 to 16% in the cakes with allspice and cinnamon, respectively. after 18 months, the least changes of tf contents were observed in the cakes fortified with cloves and allspice. therefore, the highest tf content was determined in the cakes with cloves (2.86 mg g-1), cinnamon (2.00 mg g-1), and spice mix (1.94 mg g-1). the decrease in tpc and tf contents in all samples of cakes was observed, however their values in the samples fortified with spices were still higher than in the control sample. according to results of tpc and tf content determination, after long-time storage of the spices-fortified cakes, the cloves, spice mix, and cinnamon might be recommended as good additives for confectionery goods. according to values of correlation coefficients (table 4), it can be concluded that in the control samples and in the cakes with cloves and spice mix, rutin might have the highest contribution to tf levels in these samples. whereas, a correlation between tpc and tf contents was the highest in the samples with vanilla (0.970), spice mix (0.950), and cinnamon (0.936). ital. j. food sci., vol. 31, 2019 260 3.2. changes of the antioxidant capacity of rye-buckwheat cakes during storage in the next step of our study, antioxidant capacity was monitored in rye-buckwheat cakes for 18 months of storage based on the scavenging ability of abts and dpph radicals. results of these determinations are presented in table 3. in control cakes, the abts values decreased only by 1 and 4% after 6 and 12 months of storage, respectively. then, a high decrease (27%) was noticed after 18 months. in ryebuckwheat cakes fortified with spices and stored for 6, 12 and 18 months, the least changes in the antioxidant capacity were observed in the cakes with the spice mix (from 1 to 12%) and vanilla (from 4 to 14%). in turn, the greatest decrease in the antioxidant capacity was noticed in the cakes with cinnamon (17%) and allspice (14%) after 6 months, and in the cakes fortified with cinnamon (2527%) after 12 and 18 months. in the samples with nutmeg no change was observed after 6 months, then after 12 months the antioxidant capacity dropped drastically to 21% and finally after 18 months to 25%. a decrease of the antioxidant capacity of snacks enriched with bran was also described by dar, sharma and nayik (2016). they reported that after 6 months of storage, abts values decreased by about 17-18% (conventional and microwave-assisted extraction, respectively). in turn, sakač et al. (2016) demonstrated that in packed samples of cookies formulated from 80% of rice and 20% of buckwheat, the antioxidant capacity increased in the first months of storage but after 10 months started to decrease. whereas from the beginning of storage period, a decreasing tendency was noticed in the packed samples, which were stored at higher than room temperature. table 3. antioxidant capacity determined with abts and dpph assays in rye-buckwheat cakes fortified with selected spices: cloves, nutmeg, cinnamon, vanilla, allspice and spice mix. abts [μmol te g-1] storage time spices 0 m* 6 m 0-6 m 12 m 0-12 m 18 m 0-18 m c (without spices) 21.13±0.88fa 20.94±0.32fab <1% 20.36±0.94fb ↓ 4% 15.36±0.15gc ↓ 27% cloves 55.52±2.73ba 47.91±0.40bb ↓ 14% 45.62±1.16bb ↓ 18% 43.18±0.40bc ↓ 22% nutmeg 30.49±0.84ea 30.47±0.28ea <1% 24.16±0.17eb ↓ 21% 22.98±0.36ec ↓ 25% cinnamon 49.38±0.19ca 41.10±0.14cb ↓ 17% 37.24±0.41cc ↓ 25% 36.04±0.29cd ↓ 27% vanilla 21.87±1.21fa 20.91±0.78fab ↓ 4% 19.83±0.76gb ↓ 9% 17.12±0.25fd ↓ 14% allspice 40.86±2.28da 35.98±0.75db ↓ 12% 34.09±0.63db ↓ 17% 32.55±0.87dc ↓ 20% spice mix 63.24±1.31aa 62.61±0.57aa <1% 57.03±0.13ab ↓ 10% 55.23±0.72ac ↓ 12% dpph [μmol te g-1] storage time spices 0 m* 6 m 0-6 m 12 m 0-12 m 18 m 0-18 m c (without spices) 17.48±0.57ca 16.94±0.67cab ↓ 3% 16.71±0.10bb ↓ 4% 16.30±0.80bb ↓ 7% cloves 22.35±0.70aa 21.96±0.88aa ↓ 2% 21.99±0.68aa ↓ 2% 21.56±0.04aa ↓ 4% nutmeg 9.99±0.16ga 9.96±0.49ga ↓ <1% 9.57±0.35ea ↓ 4% 9.08±0.06fb ↓ 10% cinnamon 21.39±0.61ba 19.62±0.16bb ↓ 8% 16.38±0.27bc ↓ 23% 16.76±0.65bc ↓ 22% vanilla 12.26±0.72fa 12.29±0.53fa ↑ <1% 12.11±0.09da ↓ 2% 11.08±0.02eb ↓ 10% allspice 15.96±0.60da 13.78±0.26eb ↓ 12% 13.88±0.08cb ↓ 13% 12.07±0.26dc ↓ 24% spice mix 15.12±0.19ea 15.23±0.54da ↑ <1% 13.83±0.25cc ↓ 9% 14.71±0.04cb ↓ 3% a, bmeans in the same column with different letters as superscripts are significantly different (p <0.001). a, bmeans in the same row with different letters as superscripts are significantly different (p<0.001). ital. j. food sci., vol. 31, 2019 261 table 4. coefficients of correlations between rutin, tp and tf contents and antioxidant capacity provided by abts and dpph assays for 18-month stored of rye-buckwheat cakes fortified with selected spices. type of cakes rutin vs tpc rutin vs tf tpc vs tf rutin vs abts tpc vs abts tf vs abts rutin vs dpph tpc vs dpph tf vs dpph abts vs dpph c (without spices) 0.842 0.999 0.821 0.734 0.983 0.707 0.987 0.713 0.833 0.831 cloves 0.863 0.981 0.881 0.966 0.875 0.998 0.989 0.751 0.742 0.926 nutmeg 0.974 0.859 0.735 0.872 0.754 0.881 0.855 0.914 0.975 0.932 cinnamon 0.987 0.877 0.936 0.995 0.978 0.842 0.931 0.958 0.964 0.948 vanilla 0.937 0.848 0.970 0.890 0.986 0.996 0.703 0.971 0.997 0.950 allspice 0.985 0.695 0.717 0.988 0.977 0.798 0.908 0.946 1.000 0.950 spice mix 0.982 0.885 0.950 0.955 0.982 0.916 0.666 0.947 0.987 0.698 the antioxidant capacity was also measured using the dpph method (table 3) and a decreasing tendency was observed in dpph values of the rye-buckwheat cakes. this result is in agreement with study of rinaldi et al. (2014), who observed dpph decrease in chestnutwheat bread even after 3 days of storage. the ability of control cake to scavenge dpph• radicals mildly decreased from 3 to 7% during 18-month storage period. the 6-month storage had no significant influence on dpph value decrease of ginger cakes with nutmeg, spice mix (both around 1%) and cloves (2%), whereas in the cakes with vanilla addition a slightly increase of dpph value was observed (less than 1%). with time of storage, successive reduction was observed in the scavenging ability against dpph radicals. in the cakes fortified with cinnamon, the dpph value decreased even by 23%, whereas in the samples with cloves addition no significant change was noted. after 18 months, the highest reduction in dpph values was determined in the cakes with cinnamon (22%) and allspice (24%) addition. in contrast, the least changes in dpph values were observed in the cakes with spice mix (3%) and cloves (4%). our observations are in accordance with findings reported by sakač et al. (2016), and by dar, sharma and nayik (2016), who observed that dpph values increased by about 20% and 17% during storage of cereal products, respectively. it may be suggested that not only storage conditions but also the type and amount of flours used in formulae of cookies can influence the antioxidant capacity during storage. however more studies are needed in this respect because in other studies a decrease of the antioxidant capacity was also observed after 1 and 2 days of storage of barley bread, but it was not recognized as a significant change (holtekjɵlen et al., 2008). according to abts and dpph methods, the highest values of antioxidant activity were found in the buckwheat cakes with cloves, cinnamon and spice mix. moreover, other methods could be used to evaluate the antioxidant activity to ensure better verification of tendencies in antioxidant activity changes. the highest coefficients of correlation (table 4) between rutin content and abts values were calculated for the samples with cinnamon (0.995), allspice (0.988), cloves (0.966), and spice mix (0.955). furthermore, high values of tpc vs. abts correlation were noted for the samples with vanilla (0.986), spice mix (0.982), cinnamon (0.978), and allspice (0.977). the high coefficients of correlation between tf and abts were calculated for the samples with cloves (0.998), vanilla (0.996), and spice mix (0.916). whereas the dpph radical method demonstrated a high correlation between rutin content and dpph value in the buckwheat cakes with cloves (0.989), cinnamon (0.931), and allspice (0.908); as well as between tpc and dpph value in the samples with addition of vanilla (0.971), cinnamon (0.958), spice mix (0.947), allspice (0.946), and nutmeg (0.914). similar pearson coefficients were calculated for the correlation between tf content dpph value. it can be concluded ital. j. food sci., vol. 31, 2019 262 that the antioxidant capacity of rye-buckwheat cakes fortified with spices was highly related to bioactive compounds composition. the neo-formed antioxidants during baking or storage period have no significant contribution to the final antioxidant potential. 4. conclusions in this study, polyphenols, flavonoids and rutin contents, and antioxidant capacity of ryebuckwheat cakes enriched with cloves, nutmeg, cinnamon, spice mix, allspice or vanilla were monitored during 18-month storage. the loss of rutin, flavonoids and polyphenols, and antioxidant capacity was observed in both non-enriched and spices-fortified cakes. however, in the rye-buckwheat cakes fortified with selected spices all parameters were still higher as compared to the non-fortified ones. it was shown that the rye-buckwheat cakes fortified with cloves, cinnamon and spice mix maintained the highest functional parameters as tpc, tf, and rutin contents and antioxidant capacity after 18-month storage. moreover, the loss of antioxidant capacity of the cakes fortified with spices was highly correlated with phenolics content. therefore, the cloves, cinnamon and spice mix may be recommended to improve the quality of rye-buckwheat cakes. in future studies, it would also be advisable to develop cake formulas with other types of flour originating from tartary buckwheat or tartary buckwheat sprouts (being reach sources of antioxidants), whereas amounts of these ingredients need to be carefully studied to achieve also high consumer acceptance and mask their bitter taste and potential a stringent after taste in the finished product. therefore, except for polyphenol-rich ingredients, a vacuum packaging or more appropriate bags should be used to prevent rapid degradation of polyphenols and/or formation of oxidation products. references agriculture market agency. 2013. the cereal market in poland. 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author: olfajamel@yahoo.fr abstract therapeutic levels of lactulose were used with commercial starters (yoflex 801, yoflex 901 and yomix 486) in yoghurt. in fact, yoflex 801 was supplemented with 1.5% lactulose resulting in minor yoghurt quality alterations. this co-culture was retained to study the influence of lactulose levels (0, 4, 6, and 8 %) on yoghurt quality for 28 days at 4°c. kinetic parameters, syneresis, proteolysis degree, and sensory characteristics were improved by increasing lactulose dose; thus, thixotropic and pseudoplastic gel was shown. accordingly, functional yoghurt fermented with yoflex 801, containing 4 to 6 % of lactulose, proved to be the most adequate choice. keywords: dose, lactulose, prebiotic, starter, yoghurt ital. j. food sci., vol. 31, 2019 783 1. introduction yoghurt is one of the most popular fermented dairy products, widely consumed all over the world, owing to its nutritional and sensory characteristics solicited by consumers (lovedayet al., 2013). it is produced by lactic fermentation of two specific strains: streptococcus thermophilus and lactobacillus delbruekii subsp. bulgaricus (codex stan 2432003). yoghurt has nutritional and health benefits, such as improving digestibility and lactose utilization. it promotes gut health and has a hypocholesterolemic action (adolfsson et al., 2004; weerathilake et al., 2014). bioactive compounds such as probiotics and prebiotics are usually added in yoghurts to enhance its functionality, quality and therapeutic properties (özer et al., 2005; cruz et al., 2013a). prebiotics are substrates that are selectively utilized by host microorganisms conferring a health benefit (gibson et al., 2017). prebiotics cannot be digested by the enzymes of the human gastrointestinal tract, however, they are fermented in the large intestine by colonic microflora, producing lactic acid, short chain fatty acids (acetic, propionic and butyric) and gases (garcia et al., 2008; nicholson et al., 2012). therefore, intestinal ph is reduced and harmful and pathogenic microorganisms proliferation are inhibited (rolim, 2015; wang, 2009). also, prebiotics prevent diarrhea and other diseases like colon cancer (mann et al., 2007). besides, they act in the absorption of calcium and establish favorable mechanisms to immunomodulation as well as beneficial effects on lipid metabolism and various cardiovascular risk factors (delgado et al., 2011). prebiotics including lactulose, inulin and oligofructose are considered as bifidogenic factors (roberfroid, 2000; rafter et al., 2007). thus, they are used in the formulation of dairy products, such as fermented milk (özer et al., 2005), italian cheese (ferrão et al., 2016; belsito et al., 2017; ferrão et al., 2018), whey beverage (guimaraes et al., 2018) and ice cream (balthazar et al., 2017) in order to add a functional value to these products and improve their technological characteristics. lactulose is a prebiotic (fric, 2007) used as a drug to treat illnesses, particularly chronic constipation (aider and de halleux, 2007; lee‐robichaud et al., 2010). moreover, it stimulates the growth of bifidobacteria (pharm and shah, 2008; olano and corzo, 2009). in this regard, lactulose effects are dose dependent (bothe et al., 2017), for instance, 2 g of administrated lactulose would increase the short-chain fatty acid levels of the intestinal content (mizota et al., 2002). besides, bifidogenic effects of lactulose are acquired when 5 g of lactulose are consumed every day. therefore, when bacterial counts of bifidobacterium, lactobacillus and anaerostipes increase, subsequently, acetate, butyrate and lactate increase with a decrease of branched-chain fatty acids. likewise, 7.5 g dose of lactulose, daily, allows decreasing ammonia levels (aguirre et al., 2014). accordingly, lactulose appears as an important food ingredient that might be further explored for the production of new functional foods, and thus its future large scale production for food and nutraceutical purposes is anticipated. for the best of our knowledge, there are few researches about lactulose effects on technological properties of yoghurt starters as well as on yoghurt characteristics. in this connection, with the present study we intend to formulate new functional yoghurt and explore the possible application of lactulose as a prebiotic agent in this product when varying his concentration. then, the first aim of this study is to chiefly evaluate the effect of lactulose on the acidification kinetics and post-acidification, syneresis, proteolysis degree and growth of three different commercial yoghurt starters during storage, in order to select yoghurt ital. j. food sci., vol. 31, 2019 784 cultures, possessing a low affinity to lactulose, and thus yielding functional yoghurt similar to the conventional one, as requested by consumers. the second aim of this work is to evaluate the effect of the incorporation of lactulose at different doses on the quality of new developed yoghurt inoculated with the selected starter. the lactulose dose effect was determined on biochemical, microbiological, rheological, and sensory yoghurt properties when compared with control that lead to choose the most adequate concentration having the least effects during refrigerated storage. 2. materials and methods 2.1. yoghurt manufacture and study design for yoghurt production, 5% of skim milk powder was added to skimmed milk (not fat solid =10%). thus, enriched milk was homogenized and heated to 95°c for 3 min. the pasteurized milk was then rapidly cooled down to 43 +/1°c and divided into six batches. three control batches were inoculated with three combinations of frozen starters composed of two strains, streptococcus thermophilus and lactobacillus delbrueckii subsp. bulgaricus, at a concentration of 20 ml/l, which corresponds to an initial count ofabout 8 log cfu/ml. these technological starters named, respectively, yoflex 901, yoflex 801 and yomix 486 were purchased from chr. hansen’s dairy cultures (hoersholm, denmark). they are the most commonly used ones by dairy industries in tunisia in terms of yoghurt manufacture. thereafter, 1.5% of lactulose was added to each sample of the remaining batches (chimica mugello society, italy), before inoculation with one of the three cocultures. subsequently, milk was distributed in sterilized sealed containers, incubated at 43°c until ph reached 4.6 and acidity reached 75 °d, and then cooled and stored at 4 °c. finally, dornic acidity, total solids, proteolysis degree, syneresis and lactic acid bacteria counts were determined after 24 h of production each week, during 4 weeks of refrigeration storage. after highlighting the best co-culture for yoghurt with lactulose, in the first part of this study, we focused, in the second part, on the effect of lactulose dose. accordingly, for yoghurt preparation, the same steps were applied as described above. in fact, four batches were prepared and supplemented with 0, 4, 6 and 8% of lactulose. each sample was then inoculated with the selected starter. all previous analyses and viscosity measurements were performed on the obtained yoghurt samples. sensory evaluation was also carried out at days 1, 14 and 28, during refrigeration storage. 2.2. total solids, ph and dornic acidity total solids, ph values and dornic acidity (expressed as degree dornic) were measured according to aoac (1995) and afnor (1980), respectively. kinetic parameters were also considered in this study: (i) the maximum acidification rate (vmax), expressed in 10-3ph units/ min, (ii) the time to reach the maximum acidification rate (tvmax), and (iii) the time to complete the fermentation (tph4.6), expressed in hours. ital. j. food sci., vol. 31, 2019 785 2.3. syneresis the gel was stirred at 4°c for 60 s and centrifuged for 20 min at 12,075 g in an ultracentrifuge (beckman usa) (rinaldoni et al., 2009). syneresis (%) was calculated as mass of the separated serum from the gel after centrifugation, relating to the total mass of gel that was centrifuged. 2.4. bacterial enumerations the enumeration of lactic acid bacteria was performed by using de man rogosa and sharpe agar (ph=6.2±0.2; oxoid, france) after the incubation of plates at 45°c for 48h (guiraud, 1998). 2.5. evaluation of the proteolysis for proteolysis determination, the fractional precipitation method, described by hassouna et al. (1999), was used. total nitrogen (nt) and soluble nitrogen at ph 4.6 (ns) were assayed after mineralization of organic nitrogen followed by distillation according to the kjeldahl reference method (aoac, 1990). the protein content of yoghurt samples was calculated as follows: tn (g of nitrogen ⁄ 100 ml of yoghurt) x 6.38 (duncan et al., 2008). proteolysis degree = 100 x [ns (g of nitrogen⁄100 ml of yoghurt) ⁄tn (g of nitrogen ⁄ 100 ml of yoghurt)] as described by boulares et al. (2011). 2.6. rheological measurements the rheological properties were determined according to the method described by nguyen et al. (2015) with a slight modification. briefly, flow curves of yoghurt samples were analyzed with a rotary viscometer rheometric rm180 (rheomat, caluire, france), equipped with coaxial cylinders’ geometry. the bob and the cup used had 15.18 (r1) and 21 mm (r2) radius, respectively, giving a ratio r1/r2=0.72. viscosity measurements at increasing and decreasing strain rates were conducted between 0.01 and 500 s−1. the viscometer was controlled by rsi orchestrator v6.5.8 software. flow properties were assessed at temperature 4°c. the regulation of temperature during the rheological measurements was obtained using a circulator bath (julabo gmbh, germany). the area of thixotropic hysteresis loop was determined using rsi orchestrator v 6.5.8 software, which calculates the difference between the area under the up-flow curve and the down-flow curve. 2.7. sensory evaluation throughout the storage period at 4°c (1st , 14th and 28th day), the sensory properties of experimental yoghurts were evaluated by a jury of panelists consisting of 20 trained members (8 male and 12 female, aged between 24 and 45 years). the trained panelists were students from the tunisian higher institute of food industry and the training was conducted according to the method described by hootman (1992) and meilgaard et al. (2006). the test was performed inside a uniformly illuminated room, at approximately 25 °c. the obtained yoghurts were coded with a random six-digit number and served to panelists in a randomized order. the main descriptors, used to evaluate appearance, taste and texture, were sweet taste, bitter taste, mouth feel, granular texture, whey exudation, ital. j. food sci., vol. 31, 2019 786 white color and overall acceptance, consisted in a 9-point scale (dantas et al., 2016; silva et al., 2018). 2.8. statistical analysis the obtained data were statistically evaluated using a one-way analysis of variance (anova) with ducan’s test for mean comparison to highlight significant differences (p < 0.05) among yoghurt samples. all the experiments were carried out in triplicate. 3. results and discussion 3.1. effect of lactic starter variation and lactulose addition on yoghurt quality 3.1.1 effects on yoghurt fermentation changes in kinetic parameters of acidification during the fermentation of control and lactulose incorporated yoghurts, using three different commercial starters, are shown in table 1. table 1. kinetic parameters of acidification of yoghurt using different starter co-cultures with and without lactulose at 1.5%. yoghurt starters lactulose vmax (10-3phunits/min) tmax (hours) tph4.5 (hours) yoflex 801 control 19.33±0.02 3.5±0.19 5±0.20 with lactulose 19.33±0.01 2.5±0.17 4±0.22 yoflex 901 control 16.61±0.02 3.5±0.22 4.5±0.19 with lactulose 19.67±0.03 3±0.25 4±0.27 yomix 486 control 20.00±0.01 3±0.24 4.5±0.21 with lactulose 23.33±0.02 2±0.23 3.5±0.22 as expected, on the basis of chemical acidification reaction that underlies the fermentation process, ph dropped during 3.5–5 h (tph4.6) to values of 4.6 in all experiments. yomix 486 exhibited the fastest acidifying kinetics (vmax = 20±0.01 x 10-3 ph units /min; tmax = 3 h); followed by yoflex 801. in fact, according to almeida et al. (2009), different acidification profiles of labs depend on their peculiar capacity to use nutritive compounds of milk, which could account for the differences in the kinetic parameters observed amongst the various yoghurts. thus, labs capacity to produce lactic acid, which is the main product of the metabolic activity of starter cultures, depends on the strains and their associations (béal et al., 1999). indeed, it is known that a synergic proto-cooperation between streptococcus thermophilus and lactobacillus bulgaricus takes place during yoghurt fermentation (lourens-hattingh and viljoen, 2001). furthermore, vmax increased by the incorporation of lactulose (1.5%) (except for yoflex 801), reaching 19.67±0.03 and 23.33±0.02 x 10-3 ph units/min, in fermented yoghurt, respectively, with yoflex 901 and yomix 486. the time for reaching maximum acidification rate (t max) was reduced by 1h, 0.5 h and 1 h, respectively, for yoflex 801, yoflex 901 and yomix 486. besides, the time for ital. j. food sci., vol. 31, 2019 787 reaching tph4.6 was reduced by 1h for yoflex 801 and 0.5h for both yoflex 901 and yomix 486. these differences could be attributed to the high rate of l. bulgaricus in yomix 486 starter and/or the differences between strains and species of labs for lactulose metabolism. 3.1.2 effects on yoghurt quality during storage quality parameters of yoghurts fermented by each of the three different lactic starters with or without lactulose at 1.5%, over 28 days of refrigeration storage, are illustrated in table 2. total solids content of yoghurts were evaluated during refrigeration storage. initial total solids levels were between 98±0.38 and 102±0.95 g/l for control yoghurts. however, after lactulose incorporation, these values reached 111±0.98 and 114±1.24 g/l. hence, the presence of prebiotics increased the total solids content of milk bases. these results were in accordance with other studies reporting that the addition of prebiotics in mix increase total solids content (aryana and mc grew, 2007). however, no significant differences (p > 0.05) between total solids values during storage period were noted. postacidification of the yoghurts displayed an increase over the storage period. dornic acidity values growths were 13.5, 18 and 19°d for control fermented yoghurt, respectively, with yoflex 801, yoflex 901 and yomix 486. the values changed to be 14.5, 20 and 26.5°d, when lactulose was added. indeed, acid-production trend during storage was similar to other research studies (çelik, 2007). the lowest postacidification was obtained with yoflex 801, against the highest one noted for yomix 486 starter, especially in yoghurt, wherein lactulose (1.5%) was added. these findings suggest that l. bulgaricus strain of yomix 486 was able to assimilate more lactulose than the other co-culture strains. moreover, l. bulgaricus produces more lactic acid when lactulose is available (hernandezhernandez et al., 2012). for syneresis (table 2), a steady increase in all tested samples was recorded, with the progress of storage time until the 21st day. syneresis levels increased from 62% to 73% and from 60% to 77%, respectively, for yoflex 901 and yomix 486. the use of yoflex 801 culture was associated with weak whey separation, compared to the other starters, during all storage time. syneresis values rose from 58% to 69%. these findings could be attributed to the capacity of each strain to produce exopolysaccharides. in fact, amatayakul et al. (2006) reported that syneresis could be reduced by starters producing exopolysaccharides. besides, there were conflicting findings about fermentation parameters effects on molecular characteristics of exopolysaccharides (mende et al., 2016). furthermore, ibrahim (2015) noted that frail gel was obtained when the fermentation time of camel milk was long. the main reasons for syneresis might be ascribed to the structural rearrangements in casein micelles in the gel network and the rate of solubilization of colloidal calcium particles. in this study, a longer fermentation period was achieved by yoflex 801 culture. however, higher syneresis was observed in yoghurt yoflex 901 or yomix 486 (table 2). therefore, the primary reason for higher syneresis was considered to be the type of strains in each co-culture. as indicated in table 2, at the 28th day, syneresis percentage exhibited little decrease. these results were in accordance with akgun et al. (2017) findings, pertaining to probiotic yoghurts. as determined by mende et al. (2016), medium acidity was linked to the interaction between polysaccharides molecules and protein network. indeed, acidity affects protein network charges, and consequently their joining with polysaccharides would be modified as well. ital. j. food sci., vol. 31, 2019 788 table 2. variations in total solids, postacidification, syneresis, proteolysis degree and lactic acid bacteria counts of yoghurt fermented using three different starters, with or without lactulose at 1.5%, for 28 days of storage at 4°c. parameters starters lactulose (1.5%) storage period (days) 1 7 14 21 28 total solids yoflex 801 control 98±0.38 98±0.88 99±0.84 99.4±0.85 99.18±1.22 with lactulose 111±0.98** 111.5±0.97** 112±0.94** 113±1.24** 112±0.85** yoflex 901 control 99±0.99 99.9±0.56 101±0.85 100±0.76 101.1±0.97 with lactulose 113±0.74** 113.5±0.54** 112±0.65** 112±0.85** 114±1.14** yomix 486 control 99±1.12 102±0.54 101±0.68 100±0.75 102±0.95 with lactulose 111±0.98** 112±0.65** 113±0.94** 114±1.24** 113.2±0.85** dornic acidity yoflex 801 control 78±0.43 82±0.72* 85±0.45* 89±0.66* 92.5±0.34* with lactulose 81.5±0.28** 84±0.63* 90±0.52*,** 95±0.38*,** 96±0.71** yoflex 901 control 76±0.50 84±0.52* 86±0.34* 87±0.35* 94±0.38* with lactulose 76.5±0.91 92±0.45*,** 93±0.34** 94±0.92** 96.5±0.26** yomix 486 control 79±0.5 90±0.52* 94±0.81* 93±0.73 98±0.90* with lactulose 81±0.38** 91±0.27* 96±0.63* 101±0.63*,** 107.5±0.38*,** syneresis (%) yoflex 801 control 58±0.05 60±0.02* 67±0.1* 67±0.01 66±0.4* with lactulose 63±0.01** 63±0.01** 67±0.12* 69±0.02*,** 68±0.3*,** yoflex 901 control 62±0.03 67±0.01* 69±0.1* 73±0.01* 70±0.2* with lactulose 63±0.02** 68±0.02*,** 70±0.08*,** 74±0.02*,** 72±0.1*,** yomix 486 control 60±0.01 65±0.1* 70±0.01* 77±0.3* 74±0.5* with lactulose 61±0.01** 66±0.01*,** 75±0.5*,** 79±0.1*,** 75±0.7* proteolysis degree (%) yoflex 801 control 33±0.71 36±0.57* 39.5±0.42* 43±0.57* 44.5±0.71* with lactulose 40±0.57 ** 42.6±0.42*,** 43.75±0.35*,** 46±0.71*,** 49.5±0.71*,** yoflex 901 control 39±0.28 41±0.99 43.9±0.28 46.8±0.58* 49.2±0.14* with lactulose 44±0.71** 45±0.2 49.2±0.14*,** 50.49±0.58** 52.99±0.56*,** yomix 486 control 28±0.42 32±0.56* 38±0.35* 43.9±0.57* 49±0.71* with lactulose 30±0.42** 38±0.35*,** 46±0.5*,** 48±0.71*,** 56±0.56*,** lab counts (log cfu/ml) yoflex 801 control 8.70±0.3 8.75±0.1 9.08±0.1* 9.39±0.3* 9.60±0.1 with lactulose 8.85±0.1 9.22±0.24*,** 9.32±0.2** 9.55±0.2* 9.85±0.1*,** yoflex 901 control 9.05±0.1 9.15±0.1* 9.45±0.17* 9.65±0.34 9.80±0.22 with lactulose 9.15±0.2 9.45±0.3 9.75±0.12** 9.90±0.19 10.10±0.13*,** yomix 486 control 9.01±0.2 9.32±0.2* 9.84±0.1* 9.94±0.2 10.4±0.1* with lactulose 9.22±0.2 9.58±0.14*,** 10.1±0.12*,** 10.12±0.24 10.9±0.16*,** data are presented as the mean±sd of three separate experiments. *, significant differences between storage period (p< 0.05); **, significant differences between control and supplemented yoghurt at the same storage time (p< 0.05). ital. j. food sci., vol. 31, 2019 789 otherwise, yoghurts incorporating 1.5% lactulose had higher syneresis values at each storage period. however, these differences were not significant (p > 0.05) compared to control, except for yomix 486 samples, which recorded the highest syneresis percentage. this might be assigned to lower ph obtained when lactulose was added, which caused an unstable gel network with a continuous changing arrangement, thus, resulting in disturbed protein micells as described by donkor et al. (2007) and möller and vrese (2004). yoghurt protein content was 4.6%.this value is in compliance with the standard (codex stan 243-2003), which requires content in minimal equal protein of 2.7 %. table 2 presents proteolysis degree obtained in yoghurt supplemented with 1.5% and fermented with yoflex 801, yoflex 901 and yomix 486. as expected, proteolysis degrees increased for all yoghurt samples, for 28 days of refrigeration storage. these results induced extracellular proteases activity of lactic acid bacteria through the storage period (yuksel and erdem, 2010). besides, nielsen et al. (2009) proved that proteases are active during refrigeration storage. further, proteolysis degrees were higher, at each storage period, when 1.5% of lactulose was added. similar results were obtained by yuksel and erdem (2010) and donkor et al. (2007). in fact, they also demonstrated that proteolysis levels depend on the nutrients available to proteolytic microorganisms. lactic acid bacteria counts were converted to log scale and reported in table 2. even with the addition of lactulose, lab count was maintained over108 cfu/ml. this result was in good agreement with the codex alimentarius commission (codex stan 243-2003), which established that the counting of lactic acid bacteria must be over 107 cfu/ml. it is concluded that lab counts become higher in yoghurt samples incorporating lactulose. indeed, over the storage period, their counts increased by 0.9, 0.75 and 1.39 log cfu/ml, in control fermented yoghurts, respectively, with yoflex 801, yoflex 901 and yomix 486. these increases became 1, 0.95 and 1.68 in yoghurts, wherein lactulose (1.5%) was added. these results were consistent with tabatabaie and mortazavi (2008) who reported that in yoghurt containing lactulose (1 and 3%) during 5 weeks of cold storage, the survival of l. rhamnosus lba and b. bifidum cect considerably improved. rastall and maitin (2002) found that the highest count of bifidobacteria was noted when adding xyloligosaccharide and lactulose, however, the largest increase in lactobacilli was obtained when adding foss. thus, generally, lactulose was a more effective growth promoter for lactic strains compared to inulin. on the other hand, differences were not significant at each storage period. indeed, probiotic bacteria metabolise prebiotics more than yoghurt starters. furthermore, when lactulose was incorporated, the lowest lab counts changes were obtained in yoghurts with yoflex 801. however, greater proliferations were noted for yoflex 901 and yomix 486 starters. therefore, it can be concluded that lactulose had high prebiotic effect on yoflex 901 and yomix 486, followed by yoflex 801. indeed, it seems that the stimulatory impacts of prebiotics on lactic acid bacteria viability depends on several factors such as strain type and final ph. hence, low stimulation of starter bacteria, low postacidification, low proteolysis and low syneresis were sought to obtain functional food, having similar characteristics to the conventional food. hereinafter, yoflex 801 is chosen to be used in the rest of the study. ital. j. food sci., vol. 31, 2019 790 3.2. characteristics of yoghurt added with different dose of lactulose 3.2.1 effects of lactulose dose on yoghurt fermentation and post-acidification concerning acidification kinetics, shown in table 3, it was observed that lactulose yoghurts exhibited higher vmax than the control; values obtained ranged from 19.33±0.02 to 25±0.02 103ph units/min, respectively, for control fermentation milk and samples added with 8% of lactulose. however, the time (tmax) to reach vmax was 3.5 h for control, 3 h for both 4% and 6% of lactulose and 2.5 h for 8%. moreover, the time to reach ph= 4.6 was 5 h for control, 4 h for both 4% and 6% of lactulose and 3.5 h for 8%. these findings were in accordance with those of özer et al. (2005), revealing that inulin and lactulose addition at different concentrations reduced the incubation period of yoghurt. in this regard, lactic acidity values increased significantly (p < 0.05) during refrigeration storage in all yoghurt samples (table 4). indeed, metabolism of yoghurt bacteria continued during the 28 days of storage at 4°c, as shown previously in the first part of the study. moreover, when lactulose was supplemented, overall postacidification increased weakly (1 to 3°d). this data was in agreement with cruz et al. (2013b) results, reporting that the supplementation of different doses (2, 4, 6 and 8%) of oligofructose as prebiotic has no significant effect on post acidification. these findings are desirable in modern yoghurt industry, and endorse the choice of yoflex 801 as starter in this study. however, oliveira et al. (2011) proved that the addition of lactulose in skim fermented milk by probiotic lab in coculture with s. thermophilus decreased ph at the final period of storage, indicating a bifidogenic effect for bifidobacterium lactis. table 3. kinetic parameters of acidification of yoghurt fermented with yoflex 801 and added with lactulose at different doses (0, 4, 6 and 8%). lactulose dose (%) vmax (10-3phunits/min) tmax (hours) tph4.5 (hours) control 19.33±0.02 3.5±0.19 5±0.2 4 20±0.01 3±0.17 4±0.22 6 21.6±0.03 3±0.21 4±0.21 8 25±0.02 2±0.23 3.5±0.19 3.2.2 effects of lactulose dose on yoghurt quality during storage the parameters of control yoghurts and those obtained at different lactulose concentrations (4, 6 and 8%) fermented with selected yoflex 801 starter, for 28 days at 4°c, are presented in table 4. total solids content of the four obtained yoghurts displayed an increase (p <0.05) when lactulose concentrations rose. values varied from 97.5±0.9 g/l (control sample) to 178±0.9 g/l (8% lactulose). indeed, de castro et al. (2008) reported that the addition of prebiotic was associated with a total dry extract increase. moreover, these findings outlined that lactulose was still in yoghurts and would be available for consumers as prebiotic, in order to improve health. ital. j. food sci., vol. 31, 2019 791 table 4. variations in post-acidification, total solids, syneresis, proteolysis degree and lactic acid bacteria counts of yoghurt containing various doses of lactulose (0, 4, 6 and 8%) and fermented with yoflex 801, for 28 days of storage at 4 °c. storage period (days) lactulose dose (%) dornic acidity total solids (g/l) syneresis (%) proteolysis degree (%) lab counts (log cfu/ml) 1 control 79±0.13 97.5±0.9 61±0.12 32.38±0.71 8.35±0.14 4 80.33±0.2* 125.33±1.2* 58±0.5* 37.1±0.56* 8.56±0.22* 6 81±0.2* 155.5±0.8* 56±0.1* 42±0.42* 8.77±0.5 8 82±0.2* 178±0.9* 53±0.18* 45.3±0.14* 8.86±0.12 7 control 83.66±0.12** 98±0.85 62±0.16** 35.5±0.28** 8.48±0.2 4 86.33±0.49*,** 127±1.24* 60±0.2*,** 38.3±0.78* 8.79±0.18* 6 88.66±0.23*,** 155.66±1.22* 59±0.5*,** 44±0.59*,** 9.07±0.1* 8 90.33±0.45*,** 168±0.85* 55±0.8*,** 47.5±0.71*,** 9.13±0.1** 14 control 86±0.39** 98±0.97 65±0.12 37.8±0.35** 8.86±0.14 4 88.33±0.23*,** 123±1.24 * 62±0.3*,** 40.4±0.58* 9.08±0.22*,** 6 89±0.25*,** 155±1.9* 62±0.4** 45±0.28* 9.3±0.2** 8 90±0.13*,** 170±1.74* 57±0.18*,** 49.5±0.5* 9.38±0.1** 21 control 90.66±0.1** 99±0.95 68±0.1 41±0.35** 9±0.1** 4 93.33±0.2*,** 124±0.99* 63±0.24*,** 42.6±0.42*,** 9.11±0.12 6 94.6±0.12*,** 153±1.14* 64±0.1*,** 47±0.14*,** 9.31±0.13*,** 8 94.33±0.17*,** 171±1.41* 59±0.18*,** 50.6±0.58* 9.56±0.1** 28 control 94.66±0.39 98.8±0.85 70±0.09 43.8±0.28 9.66±0.14** 4 97±0.25*,** 125±1.9* 65±0.02*,** 45±0.42* 9.87±0.2** 6 97.33±0.1*,** 152±1.37* 65±0.01** 48.5±0.71* 10.01±0.2** 8 100.66±0.49*,** 169±1.25* 60±0.03*,** 52.49±0.35*,** 10.16±0.24** data are presented as the mean±sd of three separate experiments. *, significant differences between lactulose dose at the same storage time (p< 0.05); **, significant differences between the same dose of lactulose at different storage periods (p< 0.05). ital. j. food sci., vol. 31, 2019 792 furthermore, initial syneresis values varied from 53±0.18 % to 61±0.12 %. besides, whey separation increased significantly (p <0.05) during storage in all samples, and decreased with lactulose dose increase. syneresis reached 60±0.03 % to 70±0.09 at the28th day of cold storage (table 4). these results could be elucidated by the effective role of prebiotics in increasing water-holding capacity in the texture (reid et al., 2003). moreover, some studies revealed that using prebiotic compounds, such as inulin and lactulose at optimum concentrations, might reduce the percentage of syneresis. in addition, these findings could be related to the total solids. in fact, when dry extracts increased, syneresis decreased (estevez et al., 2009). thus, lactulose levels would improve yoghurt quality by reducing syneresis, which is not sought by dairy industry. on the other hand, during storage, proteolysis degrees increased significantly (p < 0.05). these findings are in line with our previous results and suggest that although lactulose dose did not affect lab growth, it was involved into their proteolytic activity, as reported by özer et al. (2005). however, it is noteworthy that proteolysis would generate free amino-acids, which improve the sensory properties of dairy fermented products. further, lab counts over the storage period (table 4), increased in all samples. correspondingly, lactulose dose weakly affected lab growth. these results were in good agreement with özer et al. (2005) findings, who did not note any significant effect of lactulose (2.5%) on the growth of yoghurt starter bacteria. likewise, those data asserted previous results when different starters were used with 1.5% of lactulose. 3.3. rheological properties variation in this study, as shown in table 5, the results revealed that the increase of lactulose concentration and storage period give rise to the increase of the yield stress values (0.11±0.020.44±0.01 pa), consistency coefficients (1.96±0.04 3.62±0.03 pa.sn) and hysteresis area, and the slow decrease of flow index values. this can be explained by the breakdown of the yoghurt structure during storage after shear. indeed, the increase of consistency values of the formulated samples could be assigned to the increase of the total solid content in lactulose yoghurts, especially when lactulose dose ranged from 4% to 8 % (p < 0.05). therefore, an increase in lactulose concentration was accompanied with an increase in pseudoplasticity. moroever, lactulose contributed in forming the best structural arrangement in the enriched yoghurts. thus, its addition increased the rates of aggregation and curd firming reactions in the casein gels, which was in line with the result reported in previous work (arango et al., 2013). the two-way anova test was made to ascertain the effects of the storage period, lactulose concentration as well as the interaction between the storage period and lactulose concentration on rheological parameters (yield stress, flow index, consistency coefficient and hysteresis area). the influence of both factors on each variable tested was clear with p values < 0.05, except for flow index, which had no significant p values (p > 0.05) in terms of storage period and interaction between the storage period and lactulose concentration. on the other hand, flow curves of control and 8% lactulose enriched yoghurts, shown in fig. 1, yield hysteresis loops. all samples exhibited thixotropic behavior as illustrated in other studies, covering set yoghurts (ciron et al., 2012; espírito-santo et al., 2013 and ilicic et al., 2014). ital. j. food sci., vol. 31, 2019 793 table 5. variations in rheological parameters of yoghurt containing various doses of lactulose, for 28 days of storage at 4 °c. storage time (days) lactulose dose (%) yield stress σ0 (pa) flow index n consistency coefficient k (pa.sn) hysteresis area a (pa/s) r 2 control 0.11±0.02a 0.65±0.02a 2.05±0.04a 1170.05±30.10a 0.993 1 4 0.15±0.02ab 0.63±0.02a 3.13±0.03b 1256.45±24.20b 0.987 6 0.18±0.01b 0.61±0.01a 3.27±0.07bc 1290±20.02b 0.996 8 0.21±0.01b 0.60±0.01a 3.41±0.05c 1319.30±26.22b 0.975 control 0.12±0.01a 0.66±0.02a 2.13±0.05a 1193.06±40.46a 0.966 7 4 0.19±0.02b 0.61±0.02ab 3.22±0.03b 1268.21±35.87a 0.973 6 0.26±0.01c 0.58±0.02ab 3.45±0.05c 1277.09±34.91a 0.992 8 0.32±0.01d 0.57±0.02b 3.52±0.04c 1394.20±30.33b 0.991 control 0.14±0.02a 0.64±0.03a 2.21±0.05a 1292.50±13.70a 0.989 14 4 0.25±0.02b 0.61±0.01ab 3.38±0.06b 1289.68±19.88a 0.995 6 0.31±0.03bc 0.56±0.01ab 3.48±0.05bc 1314.59±16.64a 0.970 8 0.40±0.03c 0.54±0.02b 3.64±0.06c 1349.70±15.50b 0.989 control 0.15±0.0a 0.64±0.02a 2.10±0.04a 1291.18±15.72a 0.964 21 4 0.28±0.02b 0.60±0.01ab 3.40±0.03b 1296.26±15.93ab 0.989 6 0.34±0.02b 0.57±0.01bc 3.48±0.02b 1302.53±113.08ab 0.976 8 0.44±0.01c 0.53±0.01c 3.62±0.03c 1303.46±12.66b 0.985 control 0.13±0.01a 0.68±0.03a 1.96±0.04a 1287.73±14.28a 0.989 28 4 0.30±0.01b 0.61±0.01ab 3.38±0.04b 1288.34±15.01a 0.974 6 0.36±0.01b 0.55±0.03b 3.50±0.05bc 1299.87±13.53ab 0.997 8 0.43±0.03c 0.52±0.02b 3.64±0.04c 1312.22±14.13b 0.966 data are presented as the mean±sd of three separate experiments. different superscript letters indicate statistically significant (p<.05) differences in a column at the same storage time. figure 1. hysteresis loops of set yoghurts (control and 8 % of lactulose) after 1 day of storage at 4°c. 0 100 200 300 400 500 600 0 20 40 60 80 100 120 140 160 shea rate γ (1/s) sh ea r s tr es s σ (p a) control 8% ital. j. food sci., vol. 31, 2019 794 steffe (1996) reported that thixotropic property is observed particularly in fragile structures and the three-dimensional network formed is completely destroyed as in the case of set yoghurts. accordingly, it is clear that the sample enriched with lactulose has shear stress values, higher than those found in the control. in fact, yoghurts viscosities increased with the increase of lactulose concentration. a non-linear relationship was detected between shear stress (σ) and shear rate (𝛾 ̇). these findings were in accordance with those of sahet al. (2016), cuiet al. (2014) and ciron et al. (2012). based on the values of r2 coefficient, the herschel-bulkley model was found to be a better-fit model for flow curves (r2> 0.96) and only rheological parameters of this model are presented in this study (table 5). the obtained data were fitted to herschel–bulkley model according to: σ = σ! + 𝐾𝛾! where 𝜎 𝜎represents the shear stress (pa), k is the consistency coefficient (pa.sn), γ is the shear rate (s-1), 𝜎! is the yield stress and n is the flow behavior index (dimensionless). moreover, the plot of the shear stress against shear rate of the yoghurt samples under investigation yielded a flow index n of less than 1 (thinning fluid) (0.53±0.01 0.68±0.03), indicating that their flow behavior had a non-newtonian profile. 3.4. sensory evaluation table 6 represents comparative sensory analysis among yoghurts supplemented with different doses of lactulose (0, 4, 6 and 8%) using scoring methodology, after storage for days 1, 14 and 28. table 6. variations in sensory evaluation of yoghurt containing various doses of lactulose (0, 4, 6 and 8 %) for 28 days of storage at 4°c. *: significant differences between lactulose doses at the same storage time (p< 0.05). storage period (days) lactulose dose (%) sweet taste bitter taste mouth feel granular texture whey exsudation white color overall acceptance 1 control 1.9±0.4 2.28±0.7 5.11±0.5 5.18±0.75 3.9±0.8 2.95±0.8 3±0.35 4 2.78±0.3* 2.63±0.5 3.15±0.6* 4±0.65 4.56±0.4 3.72±0.6 3,65±0.25* 6 4.95±0.5* 2.85±0.35 4.88±0.7 2.65±0.75 4±0.3* 5.44±0.5 4,43±0.6 8 6.84±0.6* 2.48±0.2 6.58±0.25 1.79±0.8 2.9±0.38* 5.31±0.3 6,2±0.39 14 control 2.45±0.6 3.22±0.3 3.58±0.25 4.2±0.24 4,78±0.32 3.3±0.2 3,11±0.32 4 5±0.35 3.25±0.25 3±0.7 3.9±0.9 3,75±0.35* 3.25±0.7 3.65±0.62 6 6±0.5 2,5±0.24* 3.65±0.4 4.45±0.75 4.56±0.56 3.6±0.56 3.4±0.23* 8 6±0.7 2.99±0.3* 5.15±0.8* 1.72±0.65 3.14±0.38 2.91±0.34 5.65±0.39* 28 control 2.1±0.2 2,5±0.3 4.29±0.65 4.09±0.9 4.72±0.42 3.34±0.85 3.11±0.8 4 3±0.35* 2.78±0.24 4.5±0.45 3.5±0.3 3.5±0.6 4.28±0.77 3.5±0.77 6 3.78±0.4 2.3±0.35 5±0.35* 3.4±0.6 4.52±0.9 4.27±0.36 4±0.65 8 5.3±0.3* 2.85±0.7 7.1±0.5* 2.9±0.8 4.4±0.42 4.1±0.39 4.34±0.55 ital. j. food sci., vol. 31, 2019 795 the panelists could identify differences (p < 0.05) in the sweet taste during storage. moreover, when lactulose dose increased, sweet taste score increased, being from 1.9±0.4 to 6.84±0.6 at the first storage day, respectively, for control yoghurt and when 8 % of lactulose was added. indeed, lactulose had a considerable sweetness power (westhoff et al. 2000). the bitter taste and color scores of the yoghurt samples were not affected by lactulose addition. otherwise, lower score of granular texture and whey exudation were obtained in yoghurt with higher lactulose dose. besides, mouth feel was better, when lactose dose or storage period increased, especially for 6 and 8% of lactulose. the overall appreciation increased when lactulose dose increased. scores reached, at the first day, 6.2±0.39 for yoghurt with 8% of lactulose against 3±0.35 for control yoghurt. this is probably ascribed to the sweetness power of lactulose. literatures about the effects of prebiotics on sensory attributes of fermented milk products are rather conflicting. seydin et al. (2005) found that yoghurts containing inulin had good flavor and smooth texture. further, haydari et al. (2011) reported that increasing the concentration of prebiotics led to a weaker sensorial gel firmness and scoopability probably ascribed to depletion flocculation of milk proteins during fermentation. except for inulin, increasing the concentration of prebiotics resulted in less smooth oral texture, and, likewise, higher concentration of prebiotics possessed less flavor acceptability and total acceptability. 4. conclusions starter type had significant effects on kinetic parameters, postacidification, syneresis and proteolysis degree of yoghurt containing 1.5% of lactulose. thus, yoflex 801 was the adequate co-culture to use in prebiotic yoghurt supplemented with lactulose. with respect to lactulose therapeutic dose, increased levels (4, 6 and 8%) reduced syneresis and improved sensory characteristics. however, concerning rheological characteristics, yoghurts supplemented with lactulose had a weak gel, with a thixotropic and pseudoplastic behavior, peculiarly 8% of lactulose. hence, 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weerathilake w.a.d.v., rasika d.m.d., ruwanmali j.k.u. and munasinghe m.a.d.d. 2014. the evolution, processing, varieties and health benefits of yogurt. international journal of scientific and research publications 4:1-10. westhoff g.m., kuster b.f.m., heslinga m.c., pluim h. and verhage m. 2000. lactose and derivatives. inullmann's encyclopedia of industrial chemistry. doi:doi.org/10 1002/14356007.a15-107. yüksel z. and erdem y.k. 2010.the influence of transglutaminase treatment on functional properties of set yogurt. international journal of dairy technology 63:86-97. paper received january 4, 2019 accepted april 11, 2019 ijfs#1218_bozza ital. j. food sci., vol. 31, 2019 205 paper effects of ultrasound treatment on structural, chemical and functional properties of protein hydrolysate of rainbow trout (oncorhynchus mykiss) by-products g.b. misira and s. koral*b acentral fisheries research institute, vali adil yazar street, no:14, kasustu, trabzon, turkey bkatip çelebi university, faculty of fisheries, fish processing technology department, çiğli, i̇zmir, turkey *corresponding author: serkan.koral@ikc.edu.tr abstract in this study, the effects of ultrasound treatment on biochemical, physical, structural and functional properties of fish protein hydrolysate of rainbow trout (oncorhynchus mykiss) by-products were investigated. enzymatic hydrolysis was conducted by alcalase 2.4 l, ph 8, 1 h at 60°c, and enzyme/substrate ratio at 0.5%. a probe-type ultrasound was used for ultrasound assisted hydrolysis (uh) process. higher protein recovery was obtained in uh than in the conventional enzymatic hydrolysis (ch). the highest foaming capacities of ch and uh were measured as 137.5% and 152.5%, respectively (p<0.05). overall, our data suggest that ultrasound treatment helps to improve the functional properties such as foaming capacity and stability. keywords: by-products, fish protein hydrolysate, oncorhynchus mykiss, rainbow trout, ultrasound hydrolysis ital. j. food sci., vol. 31, 2019 206 1. introduction seafood processing industry generates high amounts of by-products during the processing steps. these by-products include backbone, head, tail, viscera, blood and cut-offs. pertaining to species and the process applied, the volumes of these by-products vary from 20 to 70% of the whole raw material. such amount of by-products brings about pollution and create severe problems at disposal points. based on the present industrial practice, most of the by-products are either discarded or used for various feed applications by processing them into fish silage, fishmeal and oil (hsu, 2010). the frame has a high-value biochemical composition with potential for higher-value food applications (arason et al., 2009; klomklao and benjakul, 2018). an increasing trend in industrial applications for the utilization of fishery byproducts, is the manufacture of water-soluble fish protein hydrolysates (fph). this will give an increased yield of solubilized proteins due to reduced molecular weight and an increase in the number of ionizable groups (kristinsson and rasco, 2000). besides utilization in the replacement of animal meals from other sources (li et al., 2018), protein hydrolysates can be used as functional additives in the food processing industry with many functional properties, such as water holding, gelling and foaming capacities, fat absorption, emulsifying and also antioxidant and antimicrobial activities. however, alternative methodologies have become necessary for improving yield, functional properties and bioactivity in protein hydrolysates. the quality and functional characteristics of the obtained products vary with the usage of different enzymes and production conditions. it is therefore, necessary to test alternative innovative technologies that will enhance product quality. these technologies need to be safe, cheap and easy to apply. it should also have no toxic and side effects. power ultrasound is an emerging and promising technology that has been applied in a variety of fields (arvanitoyannis et al., 2015). recently, enhancement in peptide production by ultrasound has become a focus of research in the food industry. with ultrasonic pretreatment of substrates, the enzymatic hydrolysis of wheat germ protein can be significantly stimulated (zang et al., 2015). in addition, hydrolysis of food protein can also be enhanced using sonicated enzymes (kadam et al., 2015). the ultrasound treatment seems to be useful in accelerating the release of peptides. ultrasound treatment is regarded as safe, non-toxic and environmentally friendly. it is also considered to be more advantageous to other technologies and is covered by "green technologies" (kentish and ashakkumar, 2011). food technologists focus on protein production using high-intensity ultrasound application to support enzymatic hydrolysis, and produce high-throughput peptides. in this study, it was aimed to determine the effects of ultrasound treatment on biochemical, physical, structural, antioxidant and functional properties of the fish protein hydrolysate that was produced from rainbow trout by-products. 2. material and methods 2.1. materials a total of 45 (weighing 12 kg) individual fresh rainbow trout (with average length and weight being 29.36±1.72 cm and 264.83±53.42 g, respectively), obtained from a local fish farming company, were transferred to the laboratory in styrofoam box with ice. after evisceration, the by-products (head, backbone, fins, tail and skin) were separated by hand and used as raw material; total weights of by-products are presented in table 1. to ital. j. food sci., vol. 31, 2019 207 minimize microbial contamination and internal enzyme activity, viscera was excluded. the food-grade alcalase 2.4 l (au/kg sigma aldrich, novozymes, bagswaerd, denmark) was used. all chemical reagents used for the experimental analysis were of analytic grade. the hydrolysation process was done on the same day the raw material reached the laboratory. table 1. total weights of by-products of rainbow trout. type total weight (g) head 2074,10 tail and backbone 1226,11 fins and skin 1298,93 total by-products used as raw material 4599,14 viscera (not used) 2194,51 2.2. methods 2.2.1 preparation of raw material by-products were chopped with mincing machine (super meat grinder, 5 mm; pore size), mixed with distilled water (1:1 w/w) and homogenized (200 rpm for 2 minutes) using wisetis˓hg-15d (daihan, seoul, korea). 2.2.2 preparation of protein hydrolysis protein hydrolysates were prepared using the ph-stat method according to sathivel et al. (2005) and kangsanant et al. (2004), with slight modifications. for maximum activity and stability of the enzyme, all reactions were conducted at ph 8 (adjusted with 1n naoh) and a temperature of 60°c. the prepared homogenate was used as raw material, divided into two equal aliquots and then placed in glass examination vessels. experiments were carried out in the shaking water bath (wisebath, wertheim, germany) agitating at 200 rpm for ch (conventional enzymatic hydrolysis) and uh (ultrasonicassisted enzymatic hydrolysis) processes, using alkalase (0.5% by weight of raw material). for ultrasound assisted system, a probe type ultrasound equipment (sonics vibra cell, usa, tapered micro tip, 142 x 6 mm) was used and the probe was immersed into the experimental vessel with 40% ultrasonic amplitude, pulse duration of 10 s ontime; 20 s off-time. in both vessels, the temperature was increased to 60°c for enzyme activation and kept constant during the experiment. hydrolysis was initiated by addition of enzyme and terminated after 60 min, the enzyme was inactivated by increasing the temperature to 90 ºc for 10 min. coarse filtration was applied to heated suspensions using glass cotton and filter paper. thereafter, filtrates (6000 g) were centrifuged in a refrigerated centrifuge (universal 320 r, hettich, germany) at 4°c for 35 min. after centrifugation, 3 separate phases occurred in the separation funnel; bottom phase: insoluble protein, middle phase: soluble protein heavily liquid, and upper phase: lipid fraction light liquid. the middle layer was collected. the supernatants were stored in a freezer at -80ºc and dried in a freeze-dryer (labconco freezone 2.5 benchtop freeze dryer, usa) for 48 hours. the resulting powdery hydrolysates were vacuum packed and stored in a freezer at -80°c until analysis. the hydrolysis process of ch and uh groups were done in duplicate. all the analysis for the ch and uh groups were performed in three parallels. ital. j. food sci., vol. 31, 2019 208 2.2.2.1 yield of fph fph yield was calculated following the method used by ilhan and gülyavuz, 2003. yield of fph (%) = [weight of fph (g)/weight of by-products (g)] x100 (1) yield of protein (%) = [(wf × pf)/ (wi × pi)] x100 (2) where wf is the weight in grams of fph, pf is the protein content (%) of fph, wi is the weight of by-products in grams and pi is the protein content (%) of by-products (pires et al., 2012). 2.2.3 determination of the degree of hydrolysis (dh) dh was analyzed with ph-stat method described by wrolstad et al., 2005. about 10 g of freeze-dried sample was weighted; hydrolysis conditions of fish by-products were applied. the solution was stirred with the magnetic stirrer (ika, rct basic, germany) and ph was adjusted to 8.0 with 0,1 n naoh for 60 min. naoh consumption was reported every 5 min. results were given as a percentage. the equation used in the calculation is given below; dh (%) = b × nb × 1/α × 1/mp × 1/htot × 100 (3) b: amount of alkali consumed (ml) nb: normality of the alkali; 0.5 n (= 0.5 mmol/ml) mp: the mass of substrate (protein (g), %n ×6.25) 1/α: the calibration factors for ph-stat htot: the content of peptide bonds. adler-nissen (1986) assumed htot as 8.6 mmolg-1 of protein and α as 1 for fish. 2.2.4 determination of biochemical composition total crude lipid content was determined by soxhlet extraction method and crude protein content was analyzed by kjeldahl method. the total protein content was calculated as %n using the standard conversion factor of 6.25. (aoac, 1990, method 2.507); moisture and ash contents were determined using aoac 1990 method 985.14 and method 7.009, respectively. 2.2.5 amino acid analysis total amino acid analyzes were carried out in kazlıçeşme r and d test laboratory (ab0513-t), an accredited laboratory in istanbul, turkey. after pre-column derivatization with hplc (agilent 1260 infinity), agilent eclipse aaa method was modified using fld/dad detectors and determined by an in-house laboratory method. a 0.2 g sample was weighed and mixed with 5 ml of 6 n hcl and stored in the condenser for 24 h. depending on the amount of amino acid, 0.6 g to 2 g of sample was transferred to 100 ml balloon flask, after addition of 5 ml norvaline standard, the flask volume was completed to 100 ml. thereafter, 0.5 μl of the filtered sample was injected into the device and analyzed. opa (ortho phthalaldehyde), fmoc (fluorenylmethoxy chloroformate) and borate was used as the derivatizing agent. ital. j. food sci., vol. 31, 2019 209 2.2.5.1 hplc conditions mobile phase a; 40 mn na2hpo4 (ph 7.8) and mobile phase b; asentonitrile/ methanol/water (45/45/10), a flow rate of 2 ml/min. zorbax eclipse-aaa 4.6 * 150 mm (3.5 μm) was used as the column. the column temperature was set at 40°c. the injection volume of sample was 0.5 μl. dad detector wave lengths were 338nm, 10nm bw; ref: 390 nm, 20 nm bw (for opa-amino acid) and 262 nm, 16 nm bw; ref: 324 nm, 8 nm bw (for fmoc-amino acid). 2.2.6 measurement of the color the color was measured using a color meter (konica minolta (specktropen cr10 japan)). three measurements were taken from the samples of ch and uh. l * (brightness), a * (redness), b * (jaundice), w (whiteness), chroma and h hue angle /saturation degree; w = 100 –[(100-l *) 2 + a * 2 + b * 2)]1/2 (5) chroma = (a * 2 + b * 2) ½ (6) h= b */ a * (7) (pires et al. (2012) 2.2.7 determination of functional properties 2.2.7.1 protein solubility protein solubilities of ch and uh were determined as reported by american oil chemists society (aocs) (1989). fph’s were dispersed in the water (10 g/l); ph of solutions were adjusted to 3, 5, 7 and 9 with 0.5 n naoh or 0.5 n hcl for 45 min with constant stirring. the solutions were then centrifuged for 30 min at 2.800 g. n contents in 15 ml of supernatants were determined according to the kjeldahl method; protein solubility (%) = protein content of the supernatant/total protein content. 2.2.7.2 foaming capacity and foaming stability foaming capacity (fc) and foaming stability (fs) were performed according to wilde and clark, 1996 and shahidi et al., 1995, with slight modifications. three g of fph was mixed with 100 ml of distilled water, then transferred into a 250ml graduated cylinder. the mixture was homogenized at 11000 rpm for 1 min at room temperature. the total volume was measured at 0, 1st, 5th, 10th, 40th and 60th min. fc was expressed as foam expansion at 0 min, while fs was expressed as foam expansion at 60 min. 2.2.7.3 oil binding capacity and water holding capacity oil binding capacity was determined by the protocol of shahidi et al., 1995. five hundred mg hydrolysate was put in a centrifuge tube and 10 ml of sunflower oil was added. after being thoroughly vortexed for 1 minute, it was centrifuged (hettich universal 320 r refrigerated centrifuge) at 4500 g for 30 min at a temperature of 4°c, thereafter the unconnected oil was discharged. the oil binding capacity was expressed as weight of fat (g) absorbed per gram of sample. water holding capacity was analyzed following the centrifuge method described by cobb and hyder (1972), with slight modifications. five hundred mg of fph was weighted into a centrifuge tube and 20 ml distilled water was added. the mixture was vortexed for 30 s, ital. j. food sci., vol. 31, 2019 210 then put in a dark place at room temperature for 6 h. thereafter, the tube at 2800 g was placed into the centrifuge for 30 min. obtained supernatant was filtered from whatman paper no: 1 and the volume of liquid was weighted. the water holding capacity calculation was done by dividing the volume of the filtrate obtained from the initially used water volume by the amount of sample. the results are expressed as ml/g. 2.2.8 scanning electron microscopy (sem) the surface morphology of ch and uh thin films was investigated using a jsm-6610 (jeol) scanning electron microscope (sem) equipped with an energy dispersive x-ray (edx) analyzer operated at 20 kv acceleration voltages. prior to the observation, the investigated specimens were coated with about 250 angstroms of gold by quorumsc7620 sputter coater. 2.2.9 antioxidant activity assay to observe antioxidant capacities of the groups, copper (ii) ion reducing antioxidant capacity (cuprac) and fe (iii) ion reducing antioxidant power methods were used. radical scavenging activity was determined by abts+•2,2’-azinobis-(3-etilbenzotiazolin-6sülphonic acid) radical scavenging method. 2.2.9.1 copper (ii) ion reducing antioxidant capacity assay (cuprac) the method is based on the reduction of copper (ii)-neokuproine to copper (i)neokuproine after addition of antioxidant solution to the medium (apak et al., 2004; mentese et al., 2015). a total of 10 mm cu(ii) chlorure (sigma chemical co, usa), 7.5 mmneokuproine (sigma chemical co, usa), and 1 m ammonium acetate tampon solution at ph 7.0 (one ml each) were pipetted into the test tubes. about 20 µl sample solutions were added to the medium and vortexed. final volume was completed to 4.1 ml and 1080 µl distilled water was added and again vortexed. the same procedure was applied for trolox® standard. after incubation at room temperature for 50 min, absorbance was read at 450 nm (1601uv-shimadzu, australia). using trolox® curve (8 4 2 1 0.5 0.25 0.125 0.0625 mm trolox®, (r2=0.999)), trolox® equivalent antioxidant capacity (mg teac/mg substance) per mg substance was calculated for each substance. 2.2.9.2 iron (iii) ion reducing antioxidant capacity assay (frap) the method is based on the measurement of the absorbance of the complex, fe2+ tptz complex at 593 nm (benzie and strain, 1999; can and baltas, 2016). firstly, 300 mm acetate buffer at ph 3.6 was dissolved in 40 mm hcl, and 10 mm tptz (2,4,6-tris (2pyridyl)-s-triazine and 20 mm of fecl3.6h2o solution were prepared. freshly prepared solutions were mixed in a ratio of 10: 1: 1 and frap reactive was obtained. a total of 100 ml aliquots of samples and 3000 µl frap reactive were transferred to each sample tube and vortexed. the reaction mixture was incubated for 5 min at room temperature, and absorbance was read at 593 nm. the same treatments were carried out for feso4.7h2o standard (r2 = 0.999) prepared at concentrations of 15.63 31.25 62.50 125 250 500 1000 μm, respectively. the absorbance of the test tubes, which were allowed to incubate for 5 min at room temperature, was measured at 593 nm (1601uv-shimadzu, australia) and the standard feso4.7h2o curve was used to calculate the equivalent antioxidant capacity (mm feso4.7h2o/mg substances). ital. j. food sci., vol. 31, 2019 211 2.2.9.3 abts•+cationic radical scavenging method the radical scavenging activity of the abts•+ [2,2'-azino-bis (3-ethylbenzothiazoline-6sulfonic acid)] groups were studied according to re et al., 1999; yilmaz et al., 2017. a 7 mm solution of abts in water was prepared and 10 ml of this solution was mixed with 2.45 mm and 5 ml potassium persulphate solution and allowed to incubate at room temperature for 18 hours to enable the formation of abts•+ cationic radical. the resulting radical solution was diluted with phosphate buffer (pbs) at ph 7.4, to give an absorbance of 0.700 ± 0.020 at 734 nm. 200 μl of the test compound (dissolved in dmso) was added to 1800 μl of the radical solution, vortexed, and after 5 min, the absorbance was read on the uv-visible spectrophotometer (1601 uv-shimadzu, australia) at a wavelength of 734 nm. the radical scavenge value of the groups was calculated from the following formula. the study consisted of three replications for each substance and standard. radical scavenging (%) = [(odcontrol-odtest)/( odcontrol)x100] 2.2.10 statistical analysis the obtained data were analyzed by analysis of variance (one way anova) and when significant differences were found, comparisons among means were carried out using the tukey and mann whitney u test (data not provided in the normality of assumptions) under the program called jmp 5.0.1 (sas institute. inc. usa) and spss 18.0 (spss inc., chicago, il) (sokal and rohlf, 1987). a significance level of 95% (p<0.05) was used throughout the analysis. 3. results and discussion 3.1. yields of by-products, fph’s (ch and uh) and the protein of fph the yield of by-product was calculated as 38.32 % using the data shown in table 1. the yields of ch and uh were calculated as 9.82% and 10.54%, respectively. ultrasound application may have increased the yield because ultrasound have a positive effect on alcalase activity due to the ability to break down the molecular aggregates, giving enzymes the opportunity to yield higher accessibility for reaction and increasing activity, (mcclements, 1995). ma et al. (2011) studied the mechanism of ultrasonic impact on protease activity and their results showed that ultrasound had an effect on the activity of alcalase. protein yields of fph’s were also calculated as 57.13% for ch and 61.76% for uh. liaset et. al. (2000) produced protein hydrolysate from atlantic salmon frames without heads. they used alcalase for conventional enzymatic hydrolysis and fph protein recovery was observed to be 61.8%. the higher protein recovery of the present study may be affected by different hydrolysis conditions. 3.2. biochemical composition of by-products from rainbow trout biochemical composition of raw material (trout by-products), ch and uh, are shown in table 2. ital. j. food sci., vol. 31, 2019 212 table 2. proximate composition of by-products, fish protein hydrolysates (ch) and (uh) protein(%) lipid(%) moisture(%) ash(%) by-products 14.82±0.18a 6.45±0.48a 72.19±1.38a 3.54±0.24a (ch) 86.40±0.32b 0.05±0.01b 1.36±0.08b 6.25±0.40b (uh) 86.75±0.28b 0.05±0.01b 2.10±0.18c 5.95±0.32b ch: conventional enzymatic hydrolysis, uh: ultrasonic-assisted enzymatic hydrolysis, ± sd: n: 3. the different superscript lowercase letters (a,b) represent statistical differences amongst the groups (p<0.05). protein content in by-products was very low (14.82%). the ch and uh samples have high and similar protein contents. the high protein content of fph is due to the characteristics of the hydrolysis process. during this process, proteins are solubilized and insoluble materials are removed by centrifugation (chalamaiah et al., 2010). lipid contents were the same for ch and uh and are very low (0.05%). this also may be as a result of characteristics of the hydrolysis process; the membranes tended to round up and form insoluble bubbles, which could cause the removal of membrane structural lipids. during the centrifugation stage after hydrolysis, these lipids separated into different layers and were removed from the medium. it is a desired feature for protein hydrolysates to have low lipid content (shahidi et al., 1995). as estimated, the moisture content of byproducts was high. whereas, it was very low in ch and uh because fphs were freezedried at the end of the hydrolysis process. the difference between ch and uh was significant (p<0.05). since by-products have skin and bone parts, ash content was higher than trout flesh (average 1.21%) (turcomp, 2014), but it was higher in ch and uh than the by-products; this may be due to the increased salts by addition of alkali into the medium to adjust the ph 8 during the hydrolysis process (benjakul and morrissey, 1997). there was no significant difference in the ash contents of ch and uh. 3.3. amino acids analysis the functional differences in hydrolysates are closely related to the amino acid groups present in the structures. table 3 shows the total amino acid contents in by-products, ch and uh, respectively. accordingly, values for by-products of amino acids were lower than all amino acid values of fph (p<0.05). amino acid contents of ch and uh were higher and close to each other. total amino acid contents were calculated as 2.01g/100g for by-products, 80.54g/100g for ch and 82.65g/100g for uh. generally, ultrasound application helps to open the surface of the substrate and increases the enzyme activity. as a result, it supports hydrolysis process. glycine was the highest in all groups. among the hydrophobic amino acids, (valine, methionine, leucine, isoleucine, alanine, tryptophan, phenylalanine and tyrosine), the value of leucine was the maximum and methionine was the minimum. indicating the increased antioxidant activity, the sum of the hydrophobic amino acids was calculated as 22.98g/100g for ch and 23.72g/100g for uh. differences for all amino acids of ch and uh were not significant, except valine (p<0.05). rajapakse et al., 2005 stated that hydrophobic amino acids, such as phenylalanine and glycine are highly soluble in lipids. soluble amino acids have more capability to gain closer access to the radicals than neutral or hydrophilic amino acids. ital. j. food sci., vol. 31, 2019 213 table 3. total amino acid contents of by-products, ch and uh(g/100 g). amino acid by-product ch uh cysteine 0.00±0.00a 1.81±0.25b 1.94±0.01b aspartate 0.12±0.01a 6.80±0.06b 7.10±0.13b glutamate 0.09±0.01a 10.98±0.11b 11.45±0.28b aspargine nd nd nd serine 0.11±0.01a 3.19±0.01b 3.24±0.17b glutamine nd nd nd histidine 0.06±0.01a 1.93±0.00b 1.90±0.04b glycine 0.36±0.01a 12.05±0.16b 12.06±0.38b threonine 0.11±0.01a 3.00±0.01b 3.08±0.15b arginine 0.09±0.01a 5.59±0.06b 5.75±0.20b alanine 0.14±0.01a 5.70±0.06b 5.88±0.17b tyrosine 0.12±0.01a 1.95±0.05b 2.08±0.06b valine 0.14±0.01a 3.06±0.02b 3.21±0.03c methionine 0.11±0.01a 1.65±0.04b 1.74±0.02b norvaline 0.02±0.00a 0.01±0.01a 0.02±0.01a tryptophane 0.06±0.01a 0.33±0.06b 0.34±0.06b phenylalanine 0.08±0.00a 3.84±0.05b 3.99±0.11b isoleucine 0.07±0.01a 1.91±0.04b 2.05±0.08b leucine 0.07±0.01a 4.84±0.04b 5.13±0.11b lysine 0.09±0.01a 5.83±0.13b 5.89±0.08b hydroxyproline 0.13±0.01a 2.28±0.08b 2.07±0.16b sarcosine nd nd nd proline 0.11±0.01a 3.89±0.05b 3.77±0.08b total 2,01±0,04a 80,54±0,75b 82,65±0,60b ch: conventional enzymatic hydrolysis, uh: ultrasonic-assisted enzymatic hydrolysis, ± sd: n: 3. the different superscript lowercase letters (a,b,c..) represent statistical differences amongst the groups (p<0.05). 3.4. measurement of the color at the end of the hydrolysis, yellowish brown liquid mixtures were obtained in both vessels (ch and uh). the liquid mixtures had three layers; the bottom, including bones brown in color, the middle; a dark yellowish brown clear liquid, and the top dense brown liquid. after centrifugation, collected liquids (ch and uh) were bright dark yellow. the colors of freeze-dried powders of ch and uh were creamy yellow and l*, a*, and b* values are presented in table 4. table 4. l*, a* and b* and w, c, h values of ch and uh. l* a* b* w c h ch 85.80±0.84a 2.90±0.51a 22.60±1.35a 73.15±0.78a 22.79±0.48a 7.80±0.46a uh 83.90±0.60a 3.50±0.30a 24.30±0.30a 71.10±0.56a 24.10±0.30a 6.80±0.34a ch: conventional enzymatic hydrolysis, uh: ultrasonic-assisted enzymatic hydrolysis, ± sd: n: 10. the different superscript lowercase letters (a,b,c..) represent statistical differences amongst the groups (p<0.05). ital. j. food sci., vol. 31, 2019 214 l*, a*, and b* values observed in ch were similar with uh. using l*, a*, b* values whiteness, chroma and h values were calculated (table 4). 3.5. dh fig. 1 illustrates the variation in the dh of ch and uh under experimental conditions, which showed a rapid increase in both groups due to many peptide bonds cleaved up to around 20th min regardless of ultrasound application. after this time, as fewer peptide bonds were available for cleavage, the reaction rate reduced for ch and uh. after about 35-40 min, a small decrease occurred in the dh of uh, which was significantly lower than dh of ch (p<0.05). this observation was not in accordance with the theory that ultrasound application would yield a higher dh than the conventional hydrolysis. kangsanant et al. (2014) produced enzymatic hydrolysate from nile tilapia assisted by continuous ultrasound with 40w. the researchers observed that ultrasound assisted hydrolysis provoked a decrease in dh, but this decrease was not significant compared to other researches which focused on different food items. this may be due to the low intensity of ultrasound applied in the present study (huang et. al., 2015; zhang et. al., 2015). figure 1. evolution of dh during the hydrolysis of ch and uh. ch: conventional enzymatic hydrolysis, uh: ultrasonic-assisted enzymatic hydrolysis, ± sd: n: 3. the different letters (a,b) represent statistical differences amongst the groups (p<0.05). 3.6. sem analysis to find out the structural effect of ultrasonic treatment on fph, the microstructure of lyophilized ch and uh were observed by sem under different magnifications. fig. 2 illustrates the sem images of ch and uh. as shown in fig. 2, different microstructures were obtained in ch and uh. uh had larger aggregates plate-shaped morphology and a smooth surface structure, whereas ch had smaller aggregates both in the form of round structures and plate-shaped morphology. the differences might be due to the changes in application of ultrasound that led to the unfolding of uh molecules. as a result, higher hydrophobic groups might occur at the surface of the molecules and interaction of these reaction time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 d h (% ) 0 5 10 15 20 25 30 ch uh a b a a a a a a a a a a a a a b b a a b b a b a b a b a ital. j. food sci., vol. 31, 2019 215 groups with each other formed larger structures. hu et al. (2013) found that treatments of different frequencies and times of ultrasound were effected on the structure of soy protein isolate dispersions. in their study, after ultrasound treatment, samples had larger and more heterogeneous structures. also, they observed that longer ultrasound application might result in larger structure size. zhou et al. (2016) investigated the effects of heat, ultrasound and combinations of heat/ultrasound and ultrasound/heat on corn gluten meal hydrolysate. researchers used 40 khz frequency, on-time 10 s and off time 3 s, 40 min duration at 20°c. they observed that the control was in the form of massive texture, but the surface became incompact and porous after ultrasound pretreatment. figure 2. sem analysis of ch (a,b,c) and uh (d,e,f). a d b e f c ital. j. food sci., vol. 31, 2019 216 in the present study, results are inconsistent with those emphasized studies. different shapes may be due to the different ultrasonic conditions (pretreatment, ultrasonic-assisted hydrolysis and time, temperature, etc.) and the raw material used in the study. 3.7. functional properties in the food industry, proteins have a special attribution in food products due to their several significant functional characteristics. among the functional properties, emulsifying, foaming, thickening and gelling capacities are often affected by their solubility (damodaran, 1997). soluble peptides obtained from enzymatic hydrolysis of proteins, can contribute to improving the emulsion and the foaming characteristics (raymundo et al., 2000). ultrasound applications led to an improvement in the functional properties of different food items (bryant and mcclements, 1999). but ultrasonic treatment conditions and variation in the rheological and thermos-physical properties of protein sources are considered effective on the functional properties of protein hydrolysates (avad et al., 2012). 3.7.1 protein solubility protein solubility of ch and uh are shown in fig. 3. it was low at acidic ph and gradually increased with increasing the ph, after neutral ph, it decreased again to ph 9. both groups have the highest solubility at ph 7, and the lowest at ph 3. the differences between groups were significant, except ph 3 (p<0.05). as shown in fig. 3, a parallel trend in ch and uh, ultrasound treatment was shown to improve the protein solubility. figure 3. protein solubility of ch and uh. ultrasound changes the conformation and structure of protein and hydrophilic amino acid residues directed towards water (arzeni et al., 2011). this situation explains the case of higher solubility of uh than ch. protein solubility is one of the most important representative factors in protein functionality. in the food industry, improvement in solubility led to a potential improvement in the functional properties of proteins (pelegrine and gasparetto, 2005). ph values 3 5 7 9 p ro te in s ol ub ili ty (% ) 90 92 94 96 98 100 102 uh ch a a a b a b b a ital. j. food sci., vol. 31, 2019 217 3.7.2 foaming capacity (fc) and foaming stability (fs) fc and fs of ch and uh are shown in fig. 4 a, b. the fc of uh in each duration was significantly higher than that of ch. the highest values for fc of ch and uh were measured accordingly as 137.5% and 152.5%, respectively. at the end of 60 min, it decreased to 11.0% in ch and 20.0% in uh. diffusion of soluble proteins, rapid conformational change and reorganization of molecules at air-water interface are needed in the protein-based foam formation (nalinanon et al., 2011). parallel to fc, fs of uh was also significantly higher than ch, this difference was significant (p<0.05) after the 5th to 60th min (fig. 4b). figure 4. foaming capacity (a) and stability (b) of ch and uh. ch: conventional enzymatic hydrolysis, uh: ultrasonic-assisted enzymatic hydrolysis, ± sd: n: 3. the different letters (a,b) represent statistical differences amongst the groups (p<0.05). it was reported that protein solubility has an important effect on functional properties of protein hydrolysates. in the present study, the results of fc and fs were in accordance with the solubility values. as the solubility increased with the ultrasound treatment, fc and fs increased. researchers also reported that higher solubility results in higher foaming characteristics from different protein sources (soria-hernández et al. 2015). jambrak et al., (2009) illustrated that ultrasound application is an effective way to improve the physical properties of soy proteins. anon, 2008 reported “the degree of hydrolyzation determines the functionality of the end products. low degree of hydrolyzation results in highly functional foaming agents and high degree of hydrolyzation results in hydrolysed vegetable protein (hvp) which are used in soups and sauces as flavor enhancers”. 3.7.3 oil binding capacity (obc) and water holding capacity (whc) obc shows a major functionality of ingredients in the food industry. kristinsson and rasco (2000) stated oil binding capacity ranged from 2.86 to 7.07 ml of oil/g of protein for atlantic salmon protein hydrolysates. the bulk density of the protein, the degree of hydrolysis and enzyme used in hydrolysis process affect this functionality. water holding capacity is another important factor. it especially improves the textural properties of duration (min) 0 1 5 10 40 60 f o a m in g c a p a ci ty ( % ) 0 20 40 60 80 100 120 140 160 ch uh a b a b a b a b a a bb duration (min) 0 1 5 10 40 60 f o a m in g s ta b ili ty ( % ) 0 20 40 60 80 100 ch uh a b a a a a a b a b ital. j. food sci., vol. 31, 2019 218 foods. different ingredients derived from proteins are used in muscle foods to improve water holding functions. data on obc and whc of ch and uh are presented in table 5. uh has a better obc than ch (p<0.05). on the contrary, whc was lower in uh than ch, but this difference was not significant (p>0.05). table 5. oil absorption and water holding capacity of ch and uh. ch uh oil absorption capacity (g/g oil) 4.47±0.23a 6,36±0.40b water holding capacity (ml/g) 5.40±0.57a 4,70±0.14a ch: conventional enzymatic hydrolysis, uh: ultrasonic-assisted enzymatic hydrolysis, ± sd: n: 3. the different superscript lowercase letters (a, b, c..) represent statistical differences amongst the groups (p<0.05). 3.8. antioxidant activity different measurement methods are used for antioxidant capacity determination. since only one experiment cannot give reasonable results, it was observed that the item act as antioxidant. accordingly, antioxidant activities of ch and uh were measured using the methods; cuprac, frap and abts•+ radical scavenging activities. many studies have shown that all protein hydrolysates consist of peptides or smaller protein fractions that are hydrogen donor and could react with radicals to convert them to more steady products, thereby finalizing the radical reaction (kittiphattanabawon et al., 2012). 3.8.1 cuprac and frap antioxidant activity cuprac method is easily used to measure total antioxidant capacities of both hydrophilic and lipophilic antioxidants (yavaşer, 2011). the results of the antioxidant activity obtained using the cuprac and the frap methods of the uh and ch are given in table 6. trolox equivalent antioxidant capacity (teac) values of the groups (according to the cuprac method) were calculated on the trolox® standard and feso4.7h2o standard. table 6. antioxidant activities of uh and ch, (cuprac (mm trolox/mg compound) and frap (mm feso4.7h2o/mg compound) methods). compounds teac values (µm trolox®/mg mixture) frap values (µm feso4.7h2o/mg mixture) uh 244.89±0.020a 13.175±0.009a ch 230.23±0.017b 12.161±0.003b ch: conventional enzymatic hydrolysis, uh: ultrasonic-assisted enzymatic hydrolysis, ± sd: n: 3. the different superscript lowercase letters (a,b,c..) represent statistical differences amongst the groups (p<0.05). teac method is based on electron transfer such as trolox equivalent antioxidant capacity (sarmadi and ismail, 2010). teac values for uh were significantly higher than ch (p<0.05). reduction activities of uh and ch to iron (iii) and iron (ii) were calculated according to frap method. frap values of uh were higher than ch (p<0.05) (table 6). in ital. j. food sci., vol. 31, 2019 219 both methods, higher antioxidant activities of uh samples might be due to change in the structures of fractions as the effect of ultrasound. jiang et al. (2014) stated that ultrasonic treatment causes higher interactions of protein hydrophobic sites exposed to the surface of the molecules and buried inside the molecules. 3.8.2 abts•+ radical scavenging activity the total radical scavenging capacities of ch and uh were determined using the abts•+ radical scavenging assay. abts•+ is generated by oxidation of abts with potassium per sulfate and is reduced in the presence of such as hydrogen or an electron donating antioxidant (binsan et al., 2008). the sc50 values for abts•+ radical scavenging activities of the ch and uh were presented in table 6. the ch exhibited efficient radical scavenging activity when compared to uh, at the all final concentration (table 7). increased compound concentrations caused an increase in radical scavenging ability. abts scavenging activity increased with increasing concentrations and it was stated that some amino acids like histidine, methionine, cysteine, phenylalanine and tyrosine might be effective in increasing the abts+ radicals scavenging activities (chalamaiah et al. 2010). aromatic amino acids in hydrolysates are capable of stabilizing free radicals by donating an electron. in the present study, total amounts of these amino acids were similar in ch and uh (11.18g/100g and 11.65g/100g, for ch and uh, respectively). histidine shows capabilities of stabilizing free radicals by donating an electron and inhibiting lipid oxidation through chelating and lipid trapping of the imidazole ring. in the present study, histidine was higher in ch than uh. lower sc50 values of ch display a higher radical scavenging effectiveness. the sc50 values for abts•+ method of ch and uh were found as 160.0 and 180.10 µg/ml, respectively (table 7). in a study, abts scavenging activities were similar for control and ultrasound pretreated (91.2% and 92.7%) bighead carp hydrolysate, at a hydrolysate concentration of 30% (yang et al., 2016). ital. j. food sci., vol. 31, 2019 220 table 7. abts•+ radical scavenging activities at various final concentrations (%) and sc50 values of the uh and ch. compounds abts•+ method radical scavenging (%) sc50 values (µg/ml) 1000 µg/ml 500 µg/ml 250 µg/ml 125 µg/ml 62.5 µg/ml 31.25 µg/ml uh 86.15±1.12a 77.54±0.72a 61.85±0.48a 36.62±0.30a 19.62±0.22a 6.92±0.12a 180.10±0.68a ch 87.08±0.90a 79.08±0.50a 64.31±0.56a 42.58±0.42b 24.46±0.28b 9.85±0.08b 160.00±0.45b ch: conventional enzymatic hydrolysis, uh: ultrasonic-assisted enzymatic hydrolysis, ± sd: n: 3. the different superscript lowercase letters (a,b,c..) represent statistical differences amongst the groups (p<0.05). ital. j. food sci., vol. 31, 2019 221 4. conclusion this research shows that fph derived from trout by-products may have a potential utilization as a functional and nutritional ingredient in food systems with desirable properties. ultrasound application improves protein solubility and it affects especially foaming capacity and stability, as well as oil absorption capacity of fph. there 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(2016). effect of high intensity ultrasound on physicochemical and functional properties of soybean glycinin at different ionic strengths. innovative food science and emerging technologies 34:205-213. paper received july 23, 2018 accepted september october 30, 2018 ijfs#1702_bozza ital. j. food sci., vol. 32, 2020 540 paper quality and mycobiota composition of stored eggs i. regecová, m. pipová*, p. jevinová, s. demjanová and b. semjon department of food hygiene and technology, university of veterinary medicine and pharmacy, komenského 73, 041 81 košice, slovak republic *corresponding author: monika.pipova@uvlf.sk abstract the aim of this study was to monitor the quality and mycobiota composition of table eggs during storage period. the most significant changes in the egg weight and water activity were observed on day 7. to identify the mycobiota present on the eggshell by pcr method, a newly designed procedure for the extraction of fungal dna based on a combination of commercial isolation kit, proteinase k and ultrasound was implemented. identified mold genera included penicillium spp., cladosporium spp., fusarium spp. and alternaria alternata group. their ratio varied considerably during storage with the dominance of penicillium spp. on day 14. keywords: egg quality, haugh units, micromycetes, pcr, storage, table eggs ital. j. food sci., vol. 32, 2020 541 1. introduction egg is one of the most nutritious low-energy foods possessing all the proteins, vitamins and minerals needed for human health (tolik et al., 2014). because of its beneficial composition, it may also be used as nutraceuticals and protein ingredients for food applications (zambrowicz et al., 2015). however, during egg collection, there is significant risk of contamination of the shell surface with microscopic filamentous fungi. by laying out, an egg gets to the external environment, which becomes its source of contamination. the majority of egg microbiological contamination studies deal primarily with microorganisms of bacterial origin (bahobail et al., 2012; jones et al., 2015; erkmen and bozoglu, 2016; karimiazar et al., 2019), secondarily with fungal contamination (tomczyk et al., 2019). micromycetes are able to grow under conditions which are unsuitable for bacteria (extreme ph, low aw, wide range of enzymatic activity). spores of various mold genera are regularly found on the shell surface and can furher penetrate into the egg contents (tomczyk et al., 2018). contamination of the eggshell may be initiated by ambient conditions during egg collection and storage, which may be as a result of the quality of litter and feed, the presence of dust, air temperature and humidity. therefore, the presence of numerous fungal genera (aspergillus, cladosporium, drechslera, penicillium, stemphylium and fusarium) on the eggshell has been reported in many studies (kokkonen et al., 2010; rohweder et al., 2011; tomczyk et al., 2019). fungi not only degrade the substrate on which they occur, they also cause numerous diseases due to the presence of spores in the air (skóra et al., 2015). mycotoxins, which are secondary metabolites produced by some fungal strains, are of the greatest risk for consumers (sypecka et al., 2004). freshness, the characteristic most commonly related to egg quality, declines with time and temperature after laying (hidalgo et al., 2006). several chemical and physical changes occur during egg storage such as increase in albumen ph, thinning of the thick albumen, water evaporation through the shell (lucisano et al., 1996), enlargement of air cell, development of maillard reaction (hidalgo et al., 2006). the most common indices used to evaluated egg freshness are air cell height (eu, 2007) and thick albumen height, expressed as haugh units (usda, 1995). storage conditions (especially the temperature) also have a great impact on mycological contamination and the production of fungal exoproducts on the shell surface. temperatures below 5°c slow the aging process, but primarily reduce the development of microorganisms. if the refrigerator temperature decreases below ‒2°c, the egg contents would start to freeze. according to the european legislation (ec, 2008), shelleggs must not be exposed to refrigeration at temperatures lower than 5°c. another significant parameter in egg storage is the relative air humidity which is adjusted to reduce as much as possible the losses caused by water evaporation. this results in a weight decrease and changes in individual egg quality indicators. in general, the lower the storage temperature, the higher the relative humidity (steinhauserová et al., 2003). within the storage period, diversified mycoflora, including potentially toxinogenic and toxinogenic species of micromycetes, is found in the eggs. therefore, the identification of micromycetes is necessary (thrane et al., 2004). current routine methods used for the detection and identification of microscopic filamentous fungi often require culture isolation and further morphological and physiological characterisation (simmons, 2007). however, it does not provide sufficient distinction among species. the limitation of these techniques is unstable micromycete cell morphology, which may vary within the species and also dependent on ambient conditions (rainieri et al., 2003). the development of molecular techniques has ital. j. food sci., vol. 32, 2020 542 enabled the identification of not only the genus, but also the individual species of micromycetes. currently, numerous identification methods are generally in use. dna reassociation techniques are used to detect dna complementarity. the disavantage of these methods is inability to distinguish closely related species. gene sequence comparison no longer has this limitation. however, detection of rrna genes, such as the internal transcriptional region (its), is the preferred method (kurtzman et al., 2011). for proper identification of micromycetes by pcr methods, sufficient amount and high purity of the dna are necessary. the aim of this study was to detect changes in quality of table eggs during the storage period of 28 days and to identify mold genera present on the eggshell using the pcr method. for correct and accurate identification it is necessary to obtain dna which is often very complex in micromycetes. to extract the fungal dna, the cell wall, cytoplasm, and nuclear membrane must be first effectively lysed. however, the structure of fungal cell wall itself prevents lysis and sufficient nucleic acid isolation (čmoková et al., 2014). therefore, a new effective procedure for lysing fungal cells and further dna extraction has been designed and implemented in this study. 2. materials and methods micromycetes were isolated from 120 brown-shell table eggs, laid by the lohmann brown crossbreed laying hens in cages, weight category m (ec, 2008). the eggs were graded, labeled and stored in the breeding stock at an average temperature of 14.3°c and a relative humidity of 61%. sets of 30 sample units were taken every 7 days. among them, water activity (aw) of the eggshell was determined in 10 eggs, another 10 eggs were checked for selected egg quality indicators and the enumeration of microscopic filamentous fungi according to stn iso 21527-1 (2010) and stn iso 21527-2 (2010) was carried out using the last 10 eggs. since molds are aerobic organisms and colonize mainly the shell, enumeration of molds was focused on the eggshell surface. 2.1. determination of water activity (aw) of eggshell and egg quality indicators water activity of the eggshell was determined by the labmaster-aw (novasina ag, lachen, switzerland) at a constant temperature (25°c) for 20 min±2.48 min. measurements were repeated three times for individual egg. eggshell breaking force was measured in accordance with the manufacturer's instructions by the egg force reader (orka food technology ltd., ramat hasharon, israel) a compact system for automatic measuring of eggshell rupture point. the unit of strength measurement (kilogram-force, kgf) is calculated as gently applied force on the eggshell until it cracks. egg analyzer™ (orka food technology ltd., ramat hasharon, israel) was used to determine the egg weight, haugh units (hu), quality grade and yolk color. the device is able to measure egg weight (g), height of the thick albumen (mm), and color of the yolk. the first two measurements were used for calculation of haugh units that indicate egg quality. the equation for working out the rating is shown below: hu = 100 log (h − 1.7w0.37 + 7.6), ital. j. food sci., vol. 32, 2020 543 where: hu = egg quality in haugh units w = egg weight in grams h = height of the thick albumen in mm (nagy et al., 2011) yolk color intensity was compared to the roche yolk color fan (rcf) ranging from 1 to 15 (1 means pale yellow and 15 dark yellow). after evaluation, the egg contents were transferred to large petri dishes with diameter of 200 mm each, they were then placed on a dark flat surface and both the height and the width of the egg yolk as well as those of the thick albumen were measured using a micrometer gauger and a ruler. subsequently, the yolk index (yi) and albumen index (ai) were calculated according to nagy et al. (2011) as follows: albumen index = height of the thick albumen (mm) x 100/ [length of the thick albumen (mm) + width of the thick albumen (mm)/2] yolk index = height of the yolk (mm)/yolk diameter (mm) x 100 2.2. fungal strains and culture condition microscopic filamentous fungi were removed from the eggshell surface as described by cupáková et al. (2010). for this purpose, ten eggs of each experimental group were tested every week. individual egg was transferred aseptically into a sterile plastic bag and sterile peptone water (0.1 wt%) in a volume of 100 ml was added. the sample was then shaken for 15 minutes using the orbi-shakertmjr (benchmark scientific inc., sayreville, usa). further decimal dilutions were prepared in accordance with stn en iso 6887-1 (2017). as described by the standard procedure, the first two decimal dilutions were spread in parallel on the surface of dichloran glycerol agar medium (dg-18; oxoid, basingstoke, uk) and dichloran-rose bengal chloramphenicol agar medium (drbc; oxoid, basingstoke, uk) in a volume of 0.1 ml and incubated for 5 days at 25°c (stn iso 21527-1 and stn iso 21527-2, 2010). colonies were further subcultured individually on the surface of czapek agar medium (oxoid, basingstoke, uk) and incubated at 25°c for another 7 days. after incubation, 1 to 5 colonies were subjected to macroscopic and microscopic identification. macroscopic evaluation included the growth rate, color, texture and topography of fungal colonies. microscopic study of different mold genera was carried out by preparing slides stained with lacto phenol cotton blue and observed under light microscope. 2.3. dna isolation because the cell wall of micromycetes is difficult to break, there was a challenge to obtain pure and concentrated fungal dna. therefore, the following three pre-isolation techniques were designed: 1. mycelium in a quantity of 10-50 mg was taken with a sterile scalpel from the surface of czapek agar medium, transferred into 1.5 ml eppendorf, exposed to liquid nitrogen for 5 min and then heated to 95°c for 5 min. freezing and heating was repeated three times. the dna isolation was then performed with the help of commercially available e.z.n.a.® fungal dna mini kit (omega bio-tek, norcross, usa). ital. j. food sci., vol. 32, 2020 544 2. proteinase k (macherey-nagel, düren, germany) in a volume of 10 µl was added to eppendorfs with 10-50 mg mycelium and incubated for 30 min at 37°c. after that, the dna isolation procedure was carried out in accordance with instructions of the commercially available e.z.n.a.® fungal dna mini kit manufacturer (omega bio-tek, norcross, usa). 3. both zircon and glass beads (1:1) in a volume of 0.2 ml and proteinase k (machereynagel, düren, germany) in a volume of 10 μl were added to fungal mycelium (10-50 mg) in the eppendorf. samples were incubated at 37°c for 30 min. after that, 800 μl of lysing solution fg1, which was a part of the e.z.n.a.® fungal dna mini kit (omega bio-tek, norcross, usa) was added. samples were then incubated at 65°c for 10 min with ultrasound waves of 500 hz. further dna isolation was performed according to recommended procedure for the commercially available e.z.n.a.® fungal dna mini kit (omega bio-tek, usa, norcross, usa). the purity and concentration of dna was measured using spectrophotometer (shimadzu, wien, austria). the obtained supernatant was used as a source of dna in pcr reactions. 2.4. primer design the forward primer its 212d and the reverse primer its 549 amplified a specific dna fragment for penicillium spp. (336 bp; table 1). table 1. dna sequences of primers used in this study. primer name primer sequence (5′-3′) annealing temperature (°c) pcr product genbank-embl accession number reference penicillium spp. its 212d aaatataaattatttaaaactttc 46 336 bp lc 317718.1 pedersen et al., 1997 its 549 ctggataaaaatttgggttg a. alternata group 18s‒f aggatccattggagggcaagt 61 99 bp ay 563301 pavón et al., 2010 18s‒r tccaactacgagctttttaactgca altsp‒f gagaacagcttcatggacttctcttt 61 195 bp altsp‒r cgcggcagtagttgggaa alt a–f cgcatcctgccctgtca 60 118 bp alt a–r gttggtagccttgatgttgaagc cladosporium spp. ms1 cagcagtcaagaatattagtcaatg 51 370 bp ay 291273 zeng et al., 2006 ms2 gcggattatcgaattaaataac clado‒pf tactccaatggttctaatattttcctctc 51 87 bp clado‒pr gggtacctagacagtatttctagcct fusarium spp. its‒fu‒f caactcccaaacccctgtga 55 410 mk 156681.1 abdelsalam et al., 2003 its‒fu‒r gcgacgattaccagtaacga ital. j. food sci., vol. 32, 2020 545 these primers were designed based on its region and 5.8s rrna sequences from penicillium spp. available in the genbank-european molecular biology laboratory database (genbank-embl; pedersen et al., 1997). alternaria-specific and alternaria alternata-group-specific primer pairs were designed based on alt a 1 gene sequences from alternaria spp. available in the genbank-embl database (table 1; pavón et al., 2010). both the forward primer altspf and the reverse primer altspr amplified a dna fragment of 195 bp. in all alternaria spp. the primer pair altaf and altar amplified a specific dna fragment (118 bp) for alternaria alternata group. finaly, the primer pair 18sf and 18sr, based on conserved 18s rrna gene, was used as positive amplification control of the assay (table 1; martín et al., 2009; pavón et al., 2010). mitochondrial (mt) small subunit rrna (ssu rrna) of cladosporium was amplified by pcr using the universal fungal mitochondrial primers ms1 and ms2. two specific primers clado‒pf and clado‒pr were designed for multiplex pcr assay (table 1). the expected amplicon size for primer pair clado-pf/r was 87 bp (zeng et al., 2006). two primers were designed based on sequence information obtained to amplify specific fragments within the its regions of fusarium spp. the initial tests for specificity revealed that the primer pair its-fu-f and its-fu-r were highly specific for fusarium genus (table 1; abd-elsalam et al., 2003). all the primers were synthesized by the same commercial company (ecoli s.r.o., bratislava, slovakia). 2.5. pcr amplifcation the amplification was done in a volume of 20 μl containing 1 ng to 10 ng of dna, 0.5 µl of each primer (concentration 10 pmol/µl), 4.0 μl hot firepol® blend master mix (solis biodyne, tartu, estonia) in the thermal cycler (tc-512, techne uk, staffordshire, united kingdom) using the same cycling conditions for each primer sets, with an initial cycling step at 95°c for 12 min, followed by dna denaturation at 95°c for 20 s, annealing at diferent temperatures depending on the type of primer used (table 1) for 60 s, and elongation at 72°c for 2 min. the amplification was terminated by cooling to 6°c. the following reference strains were used as positive controls in this study: penicillium chrysogenum ccm f-362, fusarium sporotrichioides ccm f-352, alternaria alternata ccm f397, cladosporium cladosporioides ccm f-348 (czech collection of microorganisms, brno, czech republic). 2.6. sensitivity of specific detection assays to determine the minimum detectable amount of fungal dna in three established pcr assays, variable quantities of fungal genomic dna ranging from 10 ng to 100 ng were used as dna template. the pcr products were size fractionated in agarose gels (1.5%) and visualized using gelred™nucleic acid gel stain (biotium inc., fremont, usa). amplicons were visualized by uv transillumination using the mini bis pro® (dnr bioimaging systems ltd., neve yamin, izrael). the identity of pcr products with the selected primers was confirmed by a commercial company (gatc biotech ag, cologne, germany). the dna sequences obtained from fungal strains were searched for homology to those available at the genbank-embl database using the blast program (ncbi software package). ital. j. food sci., vol. 32, 2020 546 2.7. statisical analysis two-way analysis of variance (anova) and tukey test for multiple comparison of means with a confidence interval set at 95% was conducted with r statistics software (r core team, 2018). the storage period was set as the main factor. multiple factor analysis was conducted in r statistics software (r core team, 2018) with “factominer” (sebastien et al., 2008) and “factoextra” package (kassambara and mundt, 2007) according to semjon et al. (2018). 3. results and discussion pre-market table eggs must be sorted, packed and stored under appropriate conditions to maintain temporary shelf life. according to commission regulation (ec) no. 589/2008, the minimum shelf life of fresh eggs is 28 days from the day it is laid. storage conditions are limited by storage temperature where the lower limit is 5°c. however, the upper limit is not defined, it only requires maintaining the optimum egg quality. 3.1. changes in egg quality indicators quality parameters of table eggs change during storage. within the entire storage period, the average values of egg weight ranged from 60.47±2.838 to 57.83±3.368. the most significant egg weight loss (2.64 g±0.87) in this study was recorded on day 7. the decrease in egg weight was accompanied by a decrease in water activity (aw) in the eggshell. similarly, the most significant decrease in water activity was recorded on day 7. on the same day, the average shell firmness achieved its maximum value. shell firmness is related to water evaporation and subsequent drying of the eggs. the rate of water evaporation is influenced by the permeability of the shell and the number of pores in the shell (simeonovová and míková, 2003). as already reported, the above-mentioned three egg indicators are interrelated. due to egg drying, the increased water evaporation has a negative effect on egg weight (matušovičová et al., 1986), which has also been confirmed in this study. similar results were published by de leo et al. (2018). the authors reported an average weight loss of 1.67% after 35 days of egg torage. according to simeonovová et al. (1999), evaporation of water from egg contents depends on the environment, in particular temperature and humidity, as well as on the storage period. alleoni and antunes (2004) also reported a reduction in egg weight during storage. the hu indicates egg quality and the calculation is based on both the height of the thick albumen and the egg weight (caner and yüceer, 2015). the initial value of hu represents the main marker for evaluating egg quality, and its expression provides an indication of the egg shelf-life as well as the storage conditions (figueiredo et al., 2014). as seen in table 2, mean haugh unit values ranged between 53.74 and 59.80 during egg storage in this study. de leo et al. (2018) also confirmed a hu decrease during the storage period of 42 days, which corresponds to similar earlier studies (caner, 2005; caner and yüceer, 2015; morsy et al., 2015). in general, haugh units decrease during egg storage. this can be explained by changes in protein structure resulting in albumen thinning (simeonovová et al., 1999). results of jin et al. (2011) were consistent with those obtained in this study. in contrast, lower values ital. j. food sci., vol. 32, 2020 547 within the entire storage period were reported by samli et al. (2005) and akyurek and okur (2009). all of the cited authors stated that haugh units were decreasing over time. as also reported by tomczyk et al. (2019), longer egg storage period resulted in a more noticeable decline in haugh unit values. a similar decrease was observed in the albumen index, which is based on both the width and height of the thick albumen. the values ranged from 1.370 to 10.500, depending on the storage period. albumen index was decreasing with prolonged egg storage. the most significant decline occurred on day 7, when the maximum weight loss and shell firmness, as well as minimum eggshell breaking point, water activity and haugh units were all determined. similarly, lazar (1990) also reported a decrease in albumen index value with increase in storage time and míková and davídek (2000) ranked the albumen index among the main indicators of egg freshness. table 2. changes in physico-chemical parameters during egg storage. storage period (days) 1 7 14 21 28 water activity (aw) 0.91±0.00 a 0.91±0.00a 0.91±0.01a 0.91±0.00a 0.91±0.00a weight (g) 60.47±2.84a 58.20±3.71a 58.23±2.60a 59.00±2.14a 58.23±2.26a color 8.33±1.63a 9.17±2.23a 9.50±1.64a 9.33±0.52a 9.00±1.26a haugh units 59.80±23.03a 57.85±11.91a 58.33±14.28a 56.15±13.83a 54.57±9.76a albumen index (%) 10.50±1.30a 6.86±3.49b 2.25±1.44c 1.23±0.26c 1.42±0.77c yolk index (%) 36.70±2.90a 35.51±2.59a 32.92±2.63ab 34.85±1.67ab 31.02±2.99b eggshell breaking force (kgf) 3.55±1.06 a 4.97±1.08a 4.69±0.73a 4.21±1.25a 3.75±1.53a egg quality grade aa‒ba aa‒ba aa‒ba aa‒ba ba a-c different superscripts in each row indicate significant differences between the mean values (tukey´s, p<0.05). the yolk index is also an excellent indicator of egg freshness, which is based on the measurements of the yolk height and width (yüceer and caner, 2014). yolk index ranged from 30.74% to 36.90% depending on the length of storage. yolk width increased during storage as the vitelline membrane lost its elasticity due to aging. similarly, in a study by de leo et al. (2018), the yolk index ranged from 30.99% to 39.96% during 42 days of egg storage. as reported by nedomová (2012), the yolk index decreases with prolonged egg storage. during storage, the structure of vitelline membrane is changed, its strenght decreases, which may, in a combination with water gained from the egg albumen, lead to the enlargement and deformation of the yolk (simeonovová et al.,1999). similar decrease in yolk index during egg storage was also observed in other studies (samli et al., 2005; akyurek and okurk, 2009; nedomová and simeonovová, 2010), where the storage period was identified as the limiting factor. another indicator of egg quality is the yolk color. the yolk color is a consumer factor without any nutritional significance. in this study, the yolk color ranged from 8 to 10 la roche scale and became more intense as the egg aged. similar to other studies (míková and davídek, 2000; jinn et al., 2011), it becomes more intense during egg storage. ital. j. food sci., vol. 32, 2020 548 changes in egg quality parameters during storage may also be accompanied by egg contamination with microscopic filamentous fungi. therefore, not only the temperature, but also the relative humidity is of great importance during egg storage. microscopic filamentous fungi need sufficient moisture to grow. thus, eggs are more frequently contaminated in high relative humidity environments. de reu et al. (2006) and messens et al. (2007) reported that a higher level of eggshell contamination led to a better possibility for penetration of micromycetes into the egg contents. shell pores allow a potential route of entry for fungi and this can lead to both health hazards and a foul smell and taste of an egg. therefore, detecting egg contamination is an important aspect of public health concern. in addition, volatile organic compounds (vocs) are produced by micromycetes as they proliferate.these chemicals are emitted back into the environment through the eggshell. currently, 69 fungi are known as volatile emitters (lemfack et al., 2014). the fact that table eggs are often contaminated with fungal spores was also confirmed by tomczyk et al. (2019) during three weeks of egg storage under various storage conditions. neamatallah et al. (2009) also reported the presence of microscopic filamentous fungi in 38% of eggs in their study with an average count of 3.53 log cfu/ml. the authors have identified the following mold genera: aspergillus (14%), penicillium (9%), fusarium (1%), mucor (6%), rhizopus (4%), and cladosporium (5%). in this study, micromycetes were isolated from the shell surface using dg−18 and drbc selective culture media. according to stn iso 21527-1, drbc culture medium is used for enumeration of yeasts and molds in food products with water activity greater than 0,95 (e. i. egg contents). however, the measurements in this study gave a value of aw ≤ 0.95 in the eggshell which requires the use of dg−18 medium (stn iso 21527-2). some mold genera and species are xerophilic and need lower aw to grow. therefore, mycological examination of the eggshell was performed using both the above-mentioned culture media in this study. for genus identification, macroscopic evaluation of colony size and characteristics on special culture media is of great importance. czapek agar medium often provides useful information with regards to growth rates on low water activity media. however, experience has shown that the brand of agar medium used may affect the appearance of colonies that grow on it (samson and pitt, 2000). therefore, microscopic evaluation of fungal structure is used for more accurate genus identification. phenotypic key identification is difficult, because these characteristics are unstable and micromycetes sometimes appear to be atypical with slow spore formation and aberrant production of conidiophores (chełkowski and visconti, 1992). as seen in table 3, fungal isolates in this study belonged to penicilium spp., fusarium spp., alternanria spp. and cladosporium spp. after inclusion of mold isolates into individual genera, the ability of both culture media to capture these genera was evaluated (tables 2 and 3). despite the fact that stn iso 21527 recommends the use of two selective culture media depending on water activity of particular food samples, different capture ability has been confirmed for both media. dg-18 medium was more suitable for isolation of cladosporium spp. and alternaria spp. on the contrary, capture of fusarium spp. and to a lesser extent penicillium spp., was significantly higher on drbc medium. similar results have been published by weidenborner et al. (1995) and pereira et al. (2010). the authors reported that drbc medium permited the isolation of a wider range of fungal genera/species, regardless of the type of food under study. in this study, the counts and composition of mycoflora on the eggshell varied during the storage period. similar to changes in egg quality indicators, changes in numbers of micromycetes were also confirmed between 7 and 28 days of egg storage. however, these ital. j. food sci., vol. 32, 2020 549 changes did not show any statistical significance (table 2). with the use of drbc medium, significant changes in the composition of mycoflora have been noticed on day 21, when cladosporium spp. was no more detected and a significant increase in fusarium isolates (57%) has been observed. on day 28, cladosporium accounted for only 16% of the total mycological representation. regarding the dg-18 medium, changes in the genus composition were first noticed on day 14 with the dominance of penicillium spp. (table 3). the presence of alternaria spp. (14%) was confirmed on day 21 of egg storage. table 3. mycological contamination of eggshells during the entire storage period. day log cfu/ml percentage of isolates drbc 1 1.95±1.98 cladosporium spp. (50%) penicillium spp. (25%) fusarium spp. (25%) 7 2.23±2.88 cladosporium spp. (50%) penicillium spp. (33%) fusarium spp. (17%) 14 1.85±1.65 cladosporium spp. (42%) penicillium spp. (16%) fusarium spp. (42%) 21 1.85±1.76 penicillium spp. (43%) fusarium spp. (57%) 28 2.15±1.73 cladosporium spp. (16%) penicillium spp. (42%) fusarium spp. (42%) dg‒18 1 1.95±1.87 cladosporium spp. (50%) penicillium spp. (40%) fusarium spp. (10%) 7 1.90±1.96 cladosporium spp. (86%) penicillium spp. (14%) 14 1.90±1.88 cladosporium spp. (20%) penicillium spp. (60%) fusarium spp. (20%) 21 1.48±1.43 cladosporium spp. (58%) penicillium spp. (14%) fusarium spp. (14%) alternaria spp. (14%) 28 1.95±1.85 cladosporium spp. (56%) penicillium spp. (44%) the results are expressed as the (average±standard deviation) of six independent measurements. all the physico-chemical and mycological parameters of table eggs during the storage period of 28 days were further analyzed statistically using the method of multiple factor analysis (mfa) where the storage period was the main qualitative factor. the results of mfa showed four selected components that explained 63.39% of the total variation in the results of experimental eggs. the first dimension (dim1) explained 18.66% of variation, dimension 2 (dim2) 17.22%, dimension 3 (dim3) 15.20%, and dimension 4 (dim4) 12.92%. contribution of the analyzed data in dim1 was mainly related to storage period (51.36%, r=0.95) and physicochemical variables (46.70%, r=0.94). the first two dimensions explained 35.88% of variance (fig. 1). the highest contribution in dim 1 included albumen index (r=0.92), yolk index (r=0.62) and egg weight (r=0.41). dim2 was characterized by the contribution of the effect of storage period (48.78%, r=0.89) and microbial parameters (37.25%, r=0.80) on analyzed variables. microbiota count analyzed on drbc agar (r=0.78) with water activity (r=-0.49) were correlated in dim2. in the first two dimensions, these variables were correlated on statistical significant level of alfa<0.05 (fig. 2). dim3 was mostly related with the storage period, as well as with combination of physicochemical and microbial parameters of experimental eggs. correlation coefficients for microbial variables in dim3 were determined as follows: cfu analyzed on dg‒18 agar (r=0.57), water activity (r=0.45), yolk index (r=-0.39) and eggshell breaking force (r=-0.47). ital. j. food sci., vol. 32, 2020 550 the highest contribution on dim4 was related with physicochemical variables (43.38%). however in dim4 only the correlated cfu analysed on dg‒18 agar (r=0.39) was statistically significant. figure 1. plot of individuals in the first and second extracted dimensions during egg storage period. from the results obtained by the mfa method, it can be concluded that the effect of storage conditions on experimental eggs was significant. the mfa method showed differences between experimental egg groups during the storage period. analyzed variables of experimental eggs were more similar to each other on days 14, 21 and 28 than on other days during the storage period. on experimental days 1 and 7, the eggs were significantly different from each other as well as from experimental eggs analyzed on days 14, 21 and 28 in all monitored variables. 3.2. isolation of fungal dna to overcome the poor diagnostic sensitivities and long turnaround times associated with the detection and identification of fungal pathogens in samples by cultivation, noncultivation methods including the polymerase chain reaction (pcr) are increasingly being used for exact confirmation and more accurate identification of micromycetes. the ital. j. food sci., vol. 32, 2020 551 ultimate sensitivity of any pcr assay for the detection of fungal pathogens depends on the efficient lysis of fungal cells in the tissue sample and the purification of dna that is free of pcr inhibitors. fungi have cell walls that impede lysis and the recovery of nucleic acids. furthermore, highly sensitive and specific nucleic acid-based methods for the detection of fungi necessitate the use of dna extraction reagents that are free of contaminating fungal nucleic acids (fredricks et al., 2005). figure 2. correlation plot of variables in the first and second extracted dimensions during egg storage period. in the case of the direct detection of molds in food samples (especially eggshell), dna yield and purity depends primarily on the quantity and quality of the material taken. to isolate fungal dna, it is first necessary to effectively disrupt the cell wall, lyse the cytoplasm and nucleus membrane, precipitate the proteins and remove the dna from a number of inhibitors that can reduce the effectiveness of pcr. the individual steps can be executed using chemicals by known methodological procedures, or using commercial kits to facilitate isolation. in this case, however, it may be a problem to optimize the methodological process, because some chemicals in diagnostic kits are subject to corporate secrecy (čmoková et al., 2014). the cell walls can be disrupted by homogenising of frozen sample in mortar using liquid nitrogen (garg et al., 2009; uchida et al., 2009), by ital. j. food sci., vol. 32, 2020 552 vortexing with glass or zircon beads (ebihara et al., 2009; sato et al., 2010), or by repeated freezing (litz and cavagnolo, 2010). membrane lysis is made with detergents (e.g. triton x-100, sodium laurysulfate) followed by nucleic acids release into a buffered solution which contains chelators, most commonly edta and bonding calcium cations serving as a nucleases cofactor to prevent cleavage of the released dna. to increase the nucleic acid purity, proteinase k is sometimes added into the lysis solution to cleave proteins including dna-bound histones. a commonly used method is the incubation with proteinase k and subsequent completion of isolation using a commercial kit (bergmans et al., 2010; alexander et al., 2011; beifuss et al., 2011; wisselink et al., 2011). in this study, three isolation procedures of fungal dna were used and compared: 1. combination of liquid nitrogen and heat 95°c; 2. proteinase k and fg1 lysis solution; and 3. zircon and glass bead isolation with simultaneous effects of proteinase k and ultrasonication of 50 hz. measurements of dna concentration (table 4) provided useful information on which of the three test procedures is the most effective for cell wall lysis and dna extraction. dna samples with the highest concentration and purity were used for further analysis. after pcr identification, the effectiveness of isolation procedures in relation to individual mold genera was re-evaluated. table 4. comparison of dna concentrations (ng/µl) yielded by three extraction procedures (average±sd). procedure 1 procedure 2 procedure 3 one-way anova p value penicillium spp. 36.372±17.272c 49.146±26.657b 97.669±12.225a <0.001 cladosporium spp. 12.462±4.807b 35.585±11.433a 38.175±20.410a <0.001 fusarium spp. 22.335±9.134c 64.222±18.859b 71.554±17.674a <0.001 alternaria alternata group 10.867±0.103c 29.017±0.618b 49.650±0.812a <0.001 a-c different superscripts in each row indicate significant differences between the mean values (tukey´s, p<0.05). as can be seen in table 4, the lowest average values for all identified genes were obtained by isolation procedure using a combination of thermal shock and liquid nitrogen. among four mold genera, this procedure was most effective for penicilium spp. isolates, where the average dna concentration achieved a value of (36.372±17.272 ng/μl). the first procedure proved to be the worst for isolates of the genus cladosporium. the second isolation procedure involving the use of proteinase k and a lysing solution fg1 appeared to be sufficient for isolates of the genus fusarium (average dna concentration was 64.222±18.859 ng/μl). the third method of isolation with a combination of zirconium and glass beads, proteinase k and ultrasonic waves, appeared to be the most effective for all four mold genera tested (figs. 3-5). however, the highest average dna concentration was recorded in penicillium spp. (97.669±12.225 ng/μl). the last procedure was also sufficient for cladosporium spp. (38.175±20.410 ng/μl) where the other isolation methods did not yield satisfactory results. in general, the purity of dna obtained by all the three isolation procedures was very high (1.75-1.91). ital. j. food sci., vol. 32, 2020 553 a b figure 3. identification of penicillium spp. (a) and fusarium spp. (b) comparison of three dna isolation procedures (1 3). figure a: line 1 100 bp ladder; lines 4,7,10 reference strain penicillium chrysogenum ccm f-362; lines 2,3,5,6,8,9 isolates of penicillium spp. (336 bp). lines 2,3,4 procedure 1; lines 5,6,7 procedure 2; lines 8,9,10 procedure 3. figure b: line 1 100 bp ladder; lines 2,4,6 isolates of fusarium spp.; lines 3,5,7 reference strain fusarium sporotrichioides ccm f-352 (410 bp). lines 2,3 procedure 1; lines 4,5 procedure 2; lines 6,7 procedure 3. a b figure 4. identification of alternaria alternata comparison of three dna isolation procedures (1 3). figure a: line 1 100 bp ladder; lines 2,5 reference strain alternaria alternata ccm f-397; lines 3,4,6,7 isolates of alternaria alternata (118 bp). lines 2,3,4 procedure 2; lines 5,6,7 procedure 1. figure b: line 1 100 bp ladder; line 2 negative control; lines 3,4,5 isolates of alternaria alternata; line 5 reference strain alternaria alternata ccm f-397 (118 bp). lines 3,4,5 -procedure 3. statistically significant differences (p<0.001) in the dna concentrations obtained by three isolation procedures were observed in penicillium spp., fusarium spp. and alternaria alternata group (table 4). in cladosporium spp., statistically significant difference was only recorded for the first isolation procedure where the minimum average concentration of dna was obtained. the effectiveness of fungal dna isolation using a combination of mechanical and chemical actions was also confirmed by liu et al. (2000). however, other authors have reported various methods of cell wall destruction. in the most common ital. j. food sci., vol. 32, 2020 554 method, fungal mycelium is milled with liquid nitrogen or glass beads (lee et al., 1988, wu et al., 2001). some researchers also used dry ice (griffin et al., 2002), glass or magnetic beads (faggi et al., 2005), enzymatic cleavage (li et al., 2002), or benzyl chloride (xue et al., 2006). although these techniques generally provide dna of satisfactory quantity and quality, the greater problem arises with the cladosporium dna. melanized cell walls contain complex of polysaccharides and various secondary metabolites, including complex phenolic compounds, which hamper successful isolation of dna (adams et al., 1994). due to the complexity of the fungal cell wall, conventional methods are often not appropriate for dna extraction (karakousis et al., 2006) and, compared to mammalian cells (wong et al., 2007), the use of rigorous techniques is required (yano et al., 2003). figure 5. isolation of cladosporium spp. comparison of three dna isolation procedures (1 3). line 1 100 bp ladder; lines 2,3,4,5,7,8,9,10,12,13,14,15,16 isolates of cladosporium spp.; lines 6,11,17 reference strain cladosporium cladosporioides ccm f-348 (87 bp). lines 2,3,4,5,6 procedure 1; lines 7,8,9,10,11 procedure 3; lines 12,13,14,15,16 procedure 2. in this study, the combination of glass and zircon beads, proteinase k, ultrasound, and a commercially distributed insulating kit, yielded dna in sufficient concentration and quality to identify the genus cladosporium by pcr method. the pcr products obtained in this study were further separated by agarose gel electrophoresis. this procedure verified the dna integrity required for reliable identification of fungal isolates at the genus level by the pcr method. 3.3. identification of micromycetes identification of fungal isolates from table eggs was performed with the help of pcr method. the main advantage of this method lies in its high sensitivity and detection rate (mehlig et al., 2014) ranging from several hours to 2-3 days. methods that allow the detection of genus without futher specification (pan-dermatophyte pcr) are focused on conservative dna segments (kano et al., 2003). most current methods used to identify major fungal species are targeted at the ribosomal dna domains (including 18s, its1, 5,8s, its2 and 28s). specifically, the its region provides sufficient differentation among micromycete species. methods based on classical pcr are most frequently used to detect fungal genus as a whole without more precise determination (brillowska et al., 2007, ital. j. food sci., vol. 32, 2020 555 2010a,b; kondori et al., 2010; kim et al., 2011). by this method, 30 isolates of fusarium spp. (fig. 6a) and 27 isolates of penicillium spp. (fig. 6b) were identified in this study, where the its region and 5.8s rrna were used as the target site. a b figure 6. identification of fusarium spp. (a) and penicilium spp. (b) by pcr method. figure a: line 1 100 bp ladder; lines 2,3,4,5,6,7,8,9 isolates of fusarium spp. (410 bp). figure b: line 1 100 bp ladder; lines 2,3,4,5,6,7 isolates of penicillium spp. (336 bp). unlike classical pcr, multiplex pcr offers identification of major mold species (li et al., 2002). multiplex pcr was used to identify the prevalent species within alternaria alternata group and cladosporium spp. (figs. 7a and 7b). alt a 1 gene sequence was selected as the target site for identification of alternaria alternata group. strains of cladosporium spp. (35 isolates) were identified by specific sequence in mitochondrial (mt) small subunit rrna (zeng et al., 2006). a b figure 7. identification of alternaria alternata group (a) and cladosporium spp. (b) by mutliplex pcr method. figure a: line 1 50 bp ladder; lines 2,3 isolates of alternaria alternata group (400 bp, 195 bp, 118 bp), lines 4,5 unidentified isolates. figure b: line 1 100 bp ladder; lines 2,3,4,5,6,7,8,9,10 isolates of cladosporium spp. (370 bp, 87 bp). ital. j. food sci., vol. 32, 2020 556 the pcr products obtained in this study were further sequenced and the homologues of the amplified dna sequences were searched for in the genbank-embl database. pcr genus identification correlated perfectly with the results of both macroscopic and microscopic identifications. based on these results, fungal isolates from the eggshells were identified as cladosporium spp. (35 isolates), fusarium spp. (30 isolates), penicilium spp. (27 isolates), and alternaria alternata group (1 isolate). with the exception of cladosporium spp., the remaining three fungal genera are potentially toxinogenic common pathogens of food crops. their increasing occurrence in table eggs was reported by neamatallah et. al. (2009). the authors found that 38% of the examined eggs were contaminated by potentially toxinogenic micromycetes of the genus aspergillus (14%), penicillium (9%), fusarium (1%), mucor (6%), rhizopus (4%) and cladosporium (5%). gilardi et al. (2015) confirmed the presence of the same species on the eggshell as found in this study (alternaria alternata group), which was manifested as black spots on the inner shell membrane. based on these findings, there is a high probability that the contaminating toxinogenic micromycetes on the eggshell may serve as a potential source of mycotoxins found in the egg contents. this theory has already been confirmed by el malt (2015). mycological analysis of stored eggs also indicated the presence of micromycetes on the shell surface in other studies. in nigeria, micromycetes of the genus penicillium were found in 82.5% of the examined egg samples (obi and igbokwe, 2009). greco et al. (2014) reported that penicillium is the second most common genus of microscopic filamentous fungi that contributes to the contamination of eggs in buenos aires, la pampa and the province of rio negro. this genus was also placed at the forefront by el malt (2015). the prevalence of penicillium spp. has also been demonstrated in this study. problems with the occurrence of microscopic filamentous fungi in stored eggs concern the entire world, but especially african countries which account for around 5% of the world egg production (mottet and tempio, 2017). in these countries, environmental conditions are combined with very poor hygiene, resulting in the survival and proliferation of microorganisms (salihu, 2015). 4. conclusion the results of this study confirmed that storage period has significant impact on egg quality. therefore, commercial freshness of table eggs can be extended by maintaining appropriate storage period and adequate storage conditions. in this study, changes in egg quality and the counts of micromycetes were observed during egg storage at an average temperature of 14.3°c and a relative humidity of 61%. the composition of eggshell mycoflora indicates a risk araising from the presence of potentially toxinogenic micromycetes belonging to fusarium spp., penicillium spp. and alternaria alternata group. rapid and reliable identification of mold genera by specific pcr methods requires high quality and purity of fungal dna. for that purpose, a new effective method of dna extraction from fungal cells based on the combination of a commercial isolation kit, proteinase k and ultrasound was designed and implemented in this study. acknowledgements this work was supported by iga uvlf 01/2017 „identification of toxigenic species micromycetes isolated from eggs“, scientific grant agency of the ministry of education, science, research and sport of the slovak republic and the slovak academy of sciences (vega 1/0705/16) and the slovak research and development agency (apvv-18-0039). ital. j. food sci., vol. 32, 2020 557 references abd-elsalam k.a., aly i.n., abdel-satar m.a., khalil, m.s. and verreet j.a. 2003. pcr identification of fusarium genus based on nuclear ribosomal-dna sequence data. african j. biotechnol. 2(4):82. adams r.p., miller j.s., golenberg e.m. and adams j.e. 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serdang, selangor, malaysia 2halal products research institute, putra infoport, 43400, upm, serdang, selangor, malaysia *corresponding author: najjahazharna@gmail.com abstract this study addressed the recent interest in utilizing waste as source of natural food product to build a safer environment. the sample used was watermelon rind, a byproduct of an edible fruit, often ignored for its bland taste. thermosonication is a method of treating food beverage to improve product quality, but its effects, specifically on rind beverage has not been extensively studied. this study determined the effects of temperature and time (optimization using response surface methodology) on the physicochemical, vitamin c content and microbial load of rind beverage stored under different temperatures for a week. thermosonication at 65 ºc for 60 min significantly affected juice separation (stored under 4 ºc in a week) (11.3%), tss (12.9 °brix), the total color difference value (1.7) and microbial load (6.2 log cfu/ml). themosonicated rind juice can be stored longer at temperature below 4ºc, which is beneficial to both the consumers and the country at large. keywords: physicochemical, storage, thermosonication, vitamin c, waste, watermelon rind ital. j. food sci., vol. 31, 2019 632 1. introduction the evaluation on fruit waste or fruit by-product has become a subject of interest in the food industry. the aim is to promote the use of natural waste in food application for a safer cause and a cleaner environment. fruits such as watermelon, banana and papaya are examples of functional food with high fiber and nutrition value. their intake helps to reduce the risk of cardiovascular disease such as coronary heart disease and stroke (wu et al., 2015). previous studies have reported that nutrient contents are higher in fruit peels and seeds than in the fruit pulp (morais et al., 2015; moo-huchin et al., 2015; santos et al., 2014). the unappealing taste of watermelon rind has been the main reason for discard despite its edibility and health-promoting content (al-sayed and ahmed, 2013). recent studies claimed watermelon rind act as a good thickening, foaming and emulsifying agent, as it contains pectin and large quantity of antihypertensive and antioxidant properties due to its polysaccharides content (petkowitcz et al., 2017; romdhane et al., 2017). watermelon rind was also used as wheat flour substitute in cake, pan bread, cookies and yellow noodles (al-sayed and ahmed, 2013; el-badry et al., 2014; naknaen et al., 2016; ho and darci, 2016). a mixture of 5% flour and 10% fat with watermelon rind powder slowed down staling and inhibited lipid oxidation and free fatty acids formation during storage (al-sayed and ahmed, 2013). the substitution had positive effects on human health including antioxidant activity that scavenges free-radicals (leong and shui, 2002; lewinsohn et al., 2005), converts citrulline to arginine for boosting the immune system, circulatory system and heart, as well as relaxing blood vessels in cases of cardiovascular diseases and cancer (rimando and perkins-veazie, 2005). however, research on the application of watermelon rind as juice beverage seems limited. honey was chosen as sweetener for the rind juice because it has better antibacterial and medicinal properties compared to sugar (mandal and mandal, 2011) as well as its common use as preservative (bogdanov et al., 2008). a milk sample that contains honey lasted longer and inhibited bacterial growth better than the sample without honey at 4ºc storage (krushna et al., 2006). the efficient antibacterial activity of honey in food was due to its hydrogen peroxide and polyphenol contents (white, 1978; snowdon and cliver, 1996). besides, high sugar-sweetened beverage intake contributes to heart complication, rise in blood pressure, weight-gain and cavities (corliss, 2016). in beverage production, heat treatment is applied to maintain food stability and sensory quality. pasteurization is the conventional method used to extend the shelf-life of fruit juices by applying heat that kills or inactivates certain enzymes and microorganisms (yeasts, molds and bacteria), which contribute to the juice’s spoilage (polydera et al., 2003). however, the heating effect of pasteurization was found to detract the natural quality of fruit juices, resulting in flavor loss and other changes. the thermal application also decreases the product’s physicochemical and nutritional values like vitamin c and e contents, polyphenols, ph and color (dubrovic et al., 2011; giner et al., 2013; santhirasegaram et al., 2013). a study of the pasteurization effect on physicochemical properties of physalis (physalisperuviana l.) juice reported that pasteurization at 90ºc for 2 min significantly improved the juice’s organoleptic characteristics and reduced the ascorbic acid content from 38.90 to 30.20mg/100g during storage. the ascorbic acid content of the pasteurized juice was lower than the ascorbic acid content preserved in the fresh physalis juice during storage (rabie et al., 2015). thus, alternative technologies such as ultrasound and high hydrostatic pressure were developed to reduce the effects on product quality. ultrasound is sound waves having frequency that ital. j. food sci., vol. 31, 2019 633 exceeds 20 khz, which can affect the physical, mechanical and chemical properties of food (awad et al., 2012). the application of ultrasound in food processing and food preservation can improve the food texture and flavor by enhancing heat and mass transfer processes. the treatment also serves as assistance to the existing thermal treatments to compensate for the loss of nutritional values caused by heat (knorr et al., 2011). previously, low power ultrasound had been used in treating vegetables and fruits in pre and postharvest applications (awad et al., 2012). ultrasound treatment alone gives low germicidal effect since the process greatly depends on microorganisms’ type, processing parameters and sonication medium in microbial destruction (cheng et al., 2007). the combination of ultrasound and moderate heating, known as thermosonication, inactivates heat-resistant enzymes and kills microorganisms at lower temperature within a shorter period (abdullah and chin, 2014; abid et al., 2014). a study of the thermosonication effect on tomato showed that the treatment was effective in inactivating the enzyme pectin methylesterase, which degrades pectin and reduces the viscosity of tomato juice (eskin, 1979). there are also studies, which reported that thermosonication effectively inactivated various enzymes including polyphenoloxidase (cheng et al., 2007), peroxidase (ercan and soysal, 2010; gamboa et al., 2012) and polygalacturonase (terefe et a.l, 2009). instead of relying on sound waves alone to initiate bubble cavitation, thermosonication involves both sound waves and temperature, where the temperature is controlled to produce maximum cavitation bubbles for juice extraction (patist and bates, 2008; holtung et al., 2011). thus, more polyphenols can be pertained (abid et al., 2014). thermosonication treatment also increases microbial inactivation rate in fruit juice. the killing mechanism involved is caused by thinning of cell membranes, increasing localized heat and pressure as well as increasing the production of free radicals. the treatment with combination of heat and ultrasound resulted in extensive cell damage and breakage on e. coli k12 cells (lee et al., 2009). examples of fruit and vegetable juices that have been studied using combined treatment include kasturi, lime juice (bhat et al., 2011), apple juice (abid et al., 2013), soursop juice (dias et al., 2015), grape juice (aadil et al., 2015) and carrot juice (zou and jiang, 2016). they produced better results than the juice subjected to only heat treatment. although,several studies have been done on the effect of ultrasound on fruit juices, none has been done on the optimization of thermosonication condition on watermelon rindhoney beverage. the application of thermosonication treatment with a short processing time in juice preparation has categorized it as requiring minimal processing for freshness and health purposes. however, the study on thermosonication in improving the quality of fruit waste juice beverage is yet to be extensively conducted. the objective of this study is to determine the effect of different thermosonication condition (temperature and time) on watermelon rind beverage containing honey by evaluating the physicochemical, vitamin c content and microbiological properties of the beverage stored under different conditions for a week. 2. materials and methods 2.1. watermelon sample and honey approximately 2.5 kg of red seedless watermelon kept as a whole fruit at room temperature and honey were purchased from the local market in sri serdang, selangor. ital. j. food sci., vol. 31, 2019 634 both watermelon and honey were stored in a chiller of 4ºc for 2 days and a week, respectively, before analysis. 2.2. proximate analysis of raw watermelon rind the proximate analysis was carried out on seeded red watermelon rind (srwr) and seedless red watermelon rind (slrwr) to determine which watermelon rind is more suitable in terms of high fiber content for the development of watermelon rind beverage containing honey. the proximate compositions include, crude fiber, crude fat, and ash content, crude protein, carbohydrate and moisture content (aoac, 2006). 2.3. preliminary experiment a preliminary experiment was done to determine the acceptable sweetness of honey in watermelon rind (v/v %). a 12 ml honey was mixed in 100 ml watermelon rind juice to get a 12% watermelon rindhoney juice. a range of 12% to 17% of honey to watermelon juice mixture was prepared and distributed to untrained panelists. the result showed that more than 80% of participants selected 13% (v/v) as the most acceptable sweetness. 2.4. preparation of watermelon rind juice the watermelon was separated into respective parts of rind, flesh and skin using a stainless-steel knife and the rind was further cut into cubes. the rind was cleaned and washed before being put into the juice blender mix-898m (panasonic, japan). after blending, the rind puree was obtained. the rind puree was then transferred to a cloth sieve and squeezed to obtain the juice. for control sample, 100 ml of watermelon rind juice was weighed using a weighing balance (scaltec, uk) and was transferred into glass bottles with metal caps sterilized in boiling water (100ºc). such method was used for the control sample since watermelon rind juice is consumed without any heat treatment and there has been no prior study on the development of watermelon rind juice. as for sample undergoing treatment, watermelon rind juice and honey were weighed before honey was mixed manually into watermelon rind juice using a spoon with 13% (v/v) in 100 ml watermelon rind juice. once completely mixed, the juice was transferred into sterilized glass bottles sealed with metal caps prior to thermosonication treatment. analysis was conducted for a week but signs of mold growth was visible on the surface of the control sample on day 4 of storage. 2.5. thermosonication process thermosonication treatment was applied at three different temperatures, 25, 45 and 65ºc for 10, 35 and 60 min respectively, using an ultrasonic cleaner bath (dc150h; delta, china). thermosonication treatment is regarded as a better alternative method because it has better nutrient retention capacity than the conventional method (pasteurization at 71 to 82 ºc) of treating orange and lime juice without altering or degrading the nutritional contents of the beverages (khandpur and gogate, 2016). the ultrasonic cleaner bath is a rectangular container (300 x 160 x 150 mm) with the maximal tank capacity of 7.5 l with frequency of 40 khz and power w. these parameters 150 were chosen based on the study of thermosonication of grapefruit juice (aadil et al., ital. j. food sci., vol. 31, 2019 635 2015), which also used ultrasonic cleaner bath to carry out ultrasonic treatment. six liters of distilled water was poured into the bath as transmission medium. the ultrasound treatment sends acoustic waves that creates bubbles, whereby these bubbles induce either stable cavitation or transient cavitation phenomena (chowdhury and viraraghavan, 2009; thangavadivel et al, 2012). watermelon rind beverage was kept sterilized glass bottles with metal caps at room temperature (25ºc) and chill temperature (4ºc) for further analysis. 2.6. experimental design response surface methodology (rsm) was used to determine the effect of two independent variables; temperature (25 to 65ºc) and duration (10 to 60 min) of ultrasound. these variables were chosen based on parameters of ultrasound on fruit and vegetable juices (bhat et al., 2011; aadil et al., 2015; zou and jiang, 2016). in this experiment, 14 runs (table 2) were generated from the central composite design (ccd) with two independent variables, each with three levels of low, center and high (table 1). table 1. level of independent variable using ccd. independent variable independent variable level low (-1) centre (0) high (+1) temperature (ºc) 25 45 65 time (min) 10 35 60 table 2. generated experimental runs with variable combination obtained from ccd. experimental run blocks independent variable temperature (ºc) time (min) 1 1 25 10 2* 1 45 35 3 1 65 60 4* 1 45 35 5 1 65 10 6* 1 45 35 7 1 25 60 8 2 45 10 9 2 25 35 10* 2 45 35 11 2 45 60 12* 2 45 35 13 2 65 35 14* 2 45 35 *centre point. ital. j. food sci., vol. 31, 2019 636 2.7. ph the rind beverage ph was determined using calibrated ph meter model jenwah 305, (keison, uk). approximately 50 ml of beverage was placed in a 200 ml beaker and stirred continuously while inserting the meter rode into the beaker (aoac, 2006). the ph value was taken and comparison was done between day 1, 4 and 7 of storage. 2.8. separation (%) the rind beverage separation was determined by thoroughly mixing the beverage and then transferring 100 ml of beverage into 100 ml graduated measuring cylinder. it was left to stand for 30 min at room temperature. then, the volume of the top clear beverage serum was recorded by reading the level of measuring cylinder. the readings of separation were recorded and comparison was done between day 1, 4 and 7 of storage. the determination of separation was done using foodtech method 17 (fmc, 2005). 2.9. total soluble solid (°brix) the total soluble solid of watermelon beverage was evaluated using handheld refractometer (0-32°brix). a drop of watermelon beverage was placed and spread on the refractometer window and the °brix value was determined. the total soluble solid values were taken and compared between day 1, and 7 of storage. the determination of total soluble solid was done according to aoac (2006). 2.10. color the color of watermelon beverage containing honey was determined using hunter lab ultrascan pro colorimeter (hunter associate laboratory, international commission, reston, usa) with easymatch qc software. the measurement l* (lightness), a* (redness) and b* (yellowness) color scale was taken with regular transmission (rtran) mode. the samples were placed in a transparent rectangular transmission cell unit until full to avoid air space inside the transmission cell that will interfere with the reading of color measurement. analysis was done in triplicates to obtain accurate data analysis (pathare et al., 2013; assawarachan and noomhorn, 2010). total color difference (tcd) value was also calculated using the following equation: √(𝐿∗−𝐿˳∗)2+ (𝑎∗−𝑎˳∗)2+ (𝑏∗−𝑏˳∗)2 (1) where; l* = l* for sample lo* = l* for control a* = a* for sample ao* = a* for control b* = b* for sample bo* = b* for control ital. j. food sci., vol. 31, 2019 637 2.11. vitamin c content (ascorbic acid) the analysis was conducted to estimate and compare the content of ascorbic acid between day 1, 4 and 7 of storage using the aoac international method 967.21 (horwitz and latimer, 2006). metaphosphoric acid-acetic acid solution was prepared by adding 100 ml deionized distilled water with 20 ml of acetic acid (emsure, germany). then, 7.5 g of metaphosphroic acid (fisher scientific, uk) was added and stirred. mixture was diluted to 250 ml with distilled water. the mixture was filtered into an amber bottle with lid using a filter paper and refrigerated until further use. ascorbic acid standard solution was prepared by weighing approximately 50 mg of ascorbic acid (emsure, china). thereafter, it was diluted to 50 ml with prepared metaphosphoric acid-acetic acid immediately before use. in preparing indophenol solution-dye, 50 ml deionized water and 42 mg sodium carbonate were added in a 150 ml beaker and stirred. then, 50 mg 2,6 dichloroindophenol sodium salt (camlab, uk) was added. mixture was diluted to 200 ml with deionized distilled water. the mixture was filtered into an amber bottle with lid and was refrigerated until further use. two milliliters of standard ascorbic acid solution in 5 ml metapshosphoric acidacetic acid solution was titrated against the dye solution until a light yet distinct rose pink color was obtained. the initial and final burette reading was recorded. five milliliters metaphosphoric acidacetic acid solution and 2 ml of watermelon rind beverage was pipetted into 50 ml erlenmeyer flask and it was titrated against the dye solution until a light yet distinct rose pink color was obtained. the initial and final burette reading was recorded. the amount of ascorbic acid was estimated using the following equation: mg of ascorbic acid/ g or ml of sample = (x-b) x (f/e) x (v/y) (2) where; x = average ml for test solution titration b = average ml for test blank titration f = mg ascorbic acid equivalents to 1.0 ml indophenol standard solution e = sample weight (g) or volume (ml) v = volume of initial test solution y = volume of test solution titrated 2.12. microbiological analysis total plate count, yeast and mold count were determined to compare results between day 1, 4 and 7 of storage using the spread plate method. for total plate count, plate count agar (pca) was used. for yeast and mold count, potato dextrose agar (pda) was used. a series of dilutions (10-1 to 10-5) were made from watermelon rind beverage sample in 0.1% peptone water. then, 0.1 ml of dilution from the appropriate desired dilution series (10-3 to 10-5) were pipetted onto the center of the agar plate surface. l-shaped glass spreader or hockey stick was dipped into alcohol and flamed over a bunsen burner. the plates were incubated at 37ºc for 24 h. the log cfu/ml value of the sample was calculated using eq. (a.3). the determination of pca and pda was achieved 21 using fda’s standard method of bacteriological analytical manual (fda, 2001). ital. j. food sci., vol. 31, 2019 638 cfu/ml = log [(no. of colonies x dilution factor of plate)/ aliquot plated] (3) 3. results and discussion 3.1. proximate composition of srwr and slrwr researches on nutritional content of watermelon flesh have been done widely (sabeetha et al., 2017; nonga et al., 2014; tlili et al., 2011; yau et al., 2010), however, very few research has been done on the watermelon rind. the knowledge of nutritional content of watermelon rind can reassure consumers to accept the rind as a potential food product instead of it being considered as waste (fila et al., 2013). the proximate analysis for srwr and slrwr was conducted to investigate which type of rind is more suitable to be applied in this study. table 3 shows the result of proximate analysis for srwr and slrwr. table 3. proximate analysis of srwr and slrwr. proximate composition (%) red watermelon rind seeded (srwr) seedless (slrwr) moisture 90.69a±1.31 87.42b±0.49 ash 5.03a±0.78 5.00a±0.40 crude protein 0.39a±0.09 0.57a±0.10 crude fat 0.49a±0.29 0.70a±0.26 crude fiber 2.10b±0.76 4.48a±0.65 carbohydrate 1.30a±0.31 1.83a±0.50 data are mean±sd of three replicates. values with the same letter within a row is not significantly different at 5% level (p<0.05). as shown in table 3, there were no significant difference in ash, crude protein, crude fat and carbohydrate between srwr and slrwr. however, the moisture content in srwr (90.69±1.31) was significantly higher than in slrwr (87.42±0.49). also, the crude fiber content in slrwr (4.48±0.65) was significantly higher than in srwr (2.10±0.76). soluble fiber intake from juice makes it easier for absorption of vitamins, minerals and important phytonutrients (ruiz-gutiérrez et al., 2014). when compared with other studies, it was observed that the rind (0.30g/100g) does have higher content of fiber compared to the flesh (0.19g/100g) (fila et al., 2013). therefore, slrwr was selected to be used in the development of watermelon rind beverage containing honey since it has a significantly higher crude fiber content than srwr. 3.2. fitting the rsm to significant independent variable the effects of temperature and time of thermosonication on the watermelon rind beverage containing honey was studied using central composite design (ccd). the response variables are ph and separation. table 4 shows the experimental data obtained for both response variables. the linear, quadratic and interaction effects of time (x1) and ital. j. food sci., vol. 31, 2019 639 temperature (x2) on each response variables (yi) of watermelon rind beverage containing honey are shown in table 4. table 4. the matrix of central composite design (ccd) and experimental data obtained for the response variables analysed; ph (y1) and separation (y2) (mean±sd). independent variables response variables run order blocks temperature (°c) time (min) ph (y1) separation (y2) 1 1 25 10 5.6±0.0 36.0±1.0 2* 1 45 35 5.7±0.0 29.0±6.0 3 1 65 60 5.9±0.0 5.0±0.0 4* 1 45 35 5.7±0.1 28.5±5.5 5 1 65 10 5.7±0.1 34.0±1.0 6* 1 45 35 5.7±0.1 35.0±0.0 7 1 25 60 5.6±0.0 29.5±6.5 8 2 45 10 5.7±0.1 37.0±0.7 9 2 25 35 5.6±0.0 35.0±0.7 10* 2 45 35 5.7±0.1 27.0±8.0 11 2 45 60 5.7±0.1 13.0±0.7 12* 2 45 35 5.6±0.1 36.0±2.0 13 2 65 35 5.9±0.1 17.5±5.5 14* 2 45 35 5.6±0.1 30.0±0.7 *centre point. the estimated regression coefficients for the response variables with their corresponding r1, r2 (adj), f-value and p-value of lack of fits are also included in table 5. table 5. regression coefficient, r2, adjusted r2, probability values and lack of fit for the final reduced models. regression coefficient ph (y1) separation (y2) b0 5.7 30.2 b1 0.1* -7.3* b2 0.1* -9.9* b21 0.0 -1.9 b22 0.0 -3.2 b12 0.1* -5.6* r2 0.9 0.9 r2 (adj) 0.8 0.9 *significant level at p < 0.05 bi: the estimated regression coefficient for the main linear effects. bii: the estimated regression coefficient for the quadratic effects. bii: the estimated regression coefficient for the interaction effects. x1= time, x2= temperature ital. j. food sci., vol. 31, 2019 640 table 5 shows the estimated regression coefficients of the final reduced model. the linear, quadratic and interaction effects of time (x1) and temperature (x2) on ph (y1) and separation (y2) were studied. the results showed that a high coefficient of determination (r2 > 0.8) was achieved for all regression models. hence, it is concluded that more than 80% of the variation of the response can be accurately explained as a function of time and temperature of thermosonication treatment. the experimental and predicted values for both response variables at optimum and least optimum conditions showed no significant difference (p > 0.05). hence, rsm was effective in estimating the effects of thermosonication conditions on ph and separation of watermelon rind beverage containing honey. this was seen in another study of ultrasonic treatment of soursop juice, where rsm was demonstrated to be an effective technique for investigating the effects of ultrasound intensity and processing time on polyphenol oxidase residual activity, temperature increase, phenolic compounds, ascorbic acid content and total color difference (tcd) on soursop juice. the predicted and experimental values showed no significant differences (dias et al., 2015). similar result was observed in another study of enzymatic inactivation and antioxidant properties of blackberry juice using thermoultrasound, where time and temperature showed high correlation coefficients with the mathematical model of rsm. the predicted and experimental values for pectin methylesterase residual activity, polyphenol oxidase residual activity, diammonium salt, ascorbic acid, total phenolic compounds and anthocyanins of blackberry juice were not significantly different (cervantes-elizarraras et al., 2017). 3.3. thermosonication treatment conditions in thermosonication for juice treatment, temperature and time are examples of factors that influence the fruit juice quality. this experiment employed preliminary treatment time from a previous study of ultrasound effect on grapefruit juice, with treatment time ranging from 0 to 90 min at 20ºc to 60ºc (aadil et al., 2015). the result showed that grapefruit juice sonicated for 90 min had significantly higher total carotenoids, lycopene, sugar contents and phenolic compounds with significant decrease in viscosity and microorganisms load (aadil et al, 2015). from the rsm model, the optimum condition was determined based on the ph and lowest separation (%) of watermelon rind juice. from tables 6 and 7, the experimental and predicted values for both response variables at optimum and least optimum conditions showed no significant difference. hence, rsm is effective in estimating the effects of thermosonication conditions on ph and separation of watermelon rind beverage containing honey. 3.4. thermosonication effect on physicochemical and microbiological properties of control and treated rind beverage acidity is usually correlated with the measure of ph (yau et al., 2010). the ph value is usually used to determine the processing requirements and for regulatory purposes (liu et al., 2012). as shown in table 8, no significant difference was observed between the ph values of control and treated beverages, indicating that thermosonication does not alter the ph of beverages. a study of ultrasound done on blackberry juice also showed no significant effect on the juice’s ph value and the treatment was able to maintain the juice’s ph value at 3.27 to 3.83 (ramírez-moreno et al., 2017). the accepted range of ph for ital. j. food sci., vol. 31, 2019 641 commercial non-dairy beverages is from 2.1 (lime juice concentrate) to 7.4 (spring water) (seow and thong, 2005). the acidity of beverage has to be controlled as it is one of the major causes of dental erosion amongst consumers (reddy et al., 2016). table 6. values of response variables at good conditions (n=3). response watermelon rind beverage containing honey predicted experimental ph 5.9a 5.0a separation (%) 5.2b 5.3b factors optimum condition time (min) 60 temperature (ºc) 65 values with different letters within a row is significantly different at 5% level (p < 0.05). table 7. values of response variables at the least good conditions (n=3). response watermelon rind beverage containing honey predicted experimental ph 5.4a 5.2a separation (%) 39.6b 40.5b factors least optimum condition time (min) 10 temperature (ºc) 25 values with different letters within a row is significantly different at 5% level (p < 0.05). separation is an important factor for consumer preference as it contributes to the physical appearance of juice. it measures the suspension degree of existing solid particles in the fruit juice to reduce layering. a comparison between juice separation (%) for treated and non-treated watermelon rind juice was done. the results in table 8 shows that the juice separation (%) for control, optimum and least optimum are all significantly different from one another, showing temperature and time did influence juice separation (%). watermelon rind juice treated under elevated temperature caused a significant decrease in juice separation (%). high temperature increases the kinetic movement of juice molecules which make it to collide with one another more rapidly. the mechanical forces developed from bubble cavitation disintegrate larger molecules into smaller molecules, hence, enhancing the solubility of juice molecules (demirdoven and baysal, 2008; santhirasegaram, 2013). as thermosonication variables (time and temperature) increases, size of the particles in cantaloupe and grapefruit juice reduces, thereby providing better uniformity and stability which lead to reduced juice separation (aadil et al., 2015; fonteles et al., 2012). separation is one of the most common problems in fruit juice production, which makes it difficult to maintain the solids in suspension or dispersion in the beverage over a long period of time (de-leon and boak, 1984). it was shown that as temperature increases, ital. j. food sci., vol. 31, 2019 642 the cloudiness of juices also increases (cervantes-elizarraras et al., 2017). however, another study stated that ultrasonic helps to increase clarity in juices. ultrasonic has been shown to activate pectinesterase, which is capable of destabilizing colloidal pectin molecules suspended in a juice, making the juice clearer (cheng et al., 2007). the total soluble solid (tss) of fruits depends mainly on the type of cultivar, fruit species, maturity status, agricultural practices and seasonal changes (nayak et al., 2017; tasnim et al., 2010). as shown in table 8, tss of control is significantly lower than beverages treated under optimum and least optimum conditions. the significantly different tss value may be due to the addition of honey in the treated beverages. however, there is no significant difference between the tss value of beverage thermosonicated at optimum condition and beverage thermosonicated at least optimum condition. this indicates that thermosonication did not significantly affect the tss of beverage. a study done on purple cactus pear juice using thermosonication reported that the treatment had minimum effect on the juice tss (cruz-casino et al., 2015). a study carried out by abid et al. (2014) using thermosonication on apple juice also reported insignificant difference in the tss of treated and non-treated apple juice. a combined treatment of thermosonication and pulse electric field on orange juice also resulted in no significant changes in tss (walkingribeiro et al., 2009). for color evaluation, there is no significant difference for l* value between control sample and sample thermosonicated at the least optimum conditions, but there is a significant difference for a* and b* values. the comparison between control sample and sample treated at optimum conditions showed no significant difference for b* value, whereas, there is a significant difference in l* and a* values. these results indicate that thermosonication greatly influenced the beverage color (lightness and redness). the study shows that there is no significant difference (p > 0.05) in vitamin c content of beverage between control and the treated sample at the least optimum condition, while there is a significant difference (p < 0.05) between the vitamin c content of beverage treated under optimum condition with control beverage and beverage treated under least optimum condition. the statistic indicates that beverage treated with thermosonication at optimum conditions have lower vitamin c compared to untreated and treated beverages at least optimum conditions of thermosonication. vitamin c (ascorbic acid) is an important antioxidant that possess protective properties against some types of cancers (adekunte et al., 2010). as shown in table 8, there was no significant difference (p > 0.05) between control and thermosonicated sample at the least optimum condition. this result is similar with results obtained from studies on thermosonication of apple, watermelon and tomato juice (abid et al., 2013; rawson et al., 2011; walking-ribeiro et al., 2009). however, there was significant difference (p < 0.05) between control and thermosonicated sample at optimum condition similar to studies on thermosonication of orange and strawberry juice (tiwari et al., 2008; hart and henglein, 1985). sample that was thermosonicated at optimum condition and stored at 25ºc experienced complete loss of vitamin c compared to other treatment methods and storage conditions. microbiological analysis showed that the log cfu/ml of tpc and pda for both sonicated samples reduced significantly compared to the control sample. this result is similar with the sonication of orange, strawberry, carrot and mango juice (zou and jiang, 2016; santhirasegaram et al., 2013; tiwari et al., 2008; tomadoni et al., 2017). it was concluded that with the increase in ultrasound treatment time, destruction of microbes was greater (zou and jiang, 2016). this was similar with the storage study of strawberry juice (tomadoni, et al., 2017). ultrasound has also shown to have destructive ital. j. food sci., vol. 31, 2019 643 effect on microorganisms in honey (puttongsiri and haruenkit, 2010). reduction in microbial rate is a probable effect from total polyphenols increment, contributed by the release of free amino acids through the decomposing of cell structure, caused by ultrasound and thermal treatment (d’arcy, 2017). the microbial inactivation by power ultrasound occurred due to cavitation, localised heating and free radical formation. free radicals were formed during the application of ultrasound to liquids due to sonolysis of water, and these free radicals had a bactericidal effect. table 8. effect of thermosonication conditions on physicochemical and microbiological properties of control beverage compared with treated beverage. properties treatment control optimum condition least optimum condition physicochemical ph 5.47a±0.07 5.24a±0.01 5.49a±0.02 separation (%) 51.0a±1.0 12.0c±2.7 32.7b±2.5 total soluble solid (°brix) 4.0b±0.0 13.0a±0.0 13.0a±0.0 color values l* 69. 0b±2.0 72.7a±0.6 71.5ab±1.0 a* 2.1b±0.2 7.2a±0.1 7.0a±0.6 b* 30.1a±0.5 29.8ab±0.5 28.2b±1.0 tcd 6.5a±1.0 6.3a±0.9 vitamin c content (mg/100 ml) 0.70a±0.00 0.00b±0.00 0.70a±0.10 microbiological tpc (log cfu/ml) 7.2a±0.3 4.6b±0.7 5.3b±0.5 pda (log cfu/ml) 6.5a±0.3 4.1b±0.1 4.2b±0.1 data are mean±sd of three replicates values with different letters within a row is significantly different at 5% level (p < 0.05). 3.5. thermosonication effect on physicochemical and microbiological properties of control and treated rind beverage stored at 25ºc and 4ºc for a week as shown in table 9, all sonicated samples treated at optimum and the least optimum condition stored at 25ºc and 4ºc showed significant decrease in ph within a week of storage. this is due to the increase in lactic acid bacteria and the possible formation of hydroxyl methyl furfural throughout the storage period as well as the increase in titrable acidity (bhardwaj et al, 2005; khandpur & gogate, 2016; wang et al., 2005). the values of ph for stored watermelon rind beverage containing honey after being treated with thermosonication ranges from 3.77 to 5.49 and it has already been stated that most beverages or juice have their ph ranges between 3.5 and 5.5 during storage (pearson, 1995). therefore, from the results, the ph of sonicated watermelon rind beverage containing honey is still in the acceptable range even for a week storage. a comparison for juice separation (%) between juices stored under 25 and 4°c for 7 days was done. the separation (%) of rind juices thermosonicated at 65°c for 60 min showed significant gradual increase from day 1 to day 7 when stored under 25°c. fruit juices have the same taste, color and aroma as their whole fruit because mechanical separation exerts minimal effect on the juices’ physicochemical properties. thus, they still contain colloids ital. j. food sci., vol. 31, 2019 644 (pectin, proteins and polyphenols) and fibers, contributing to the cloudy appearance of juice. presence of pectin hinders the formation of aggregation of juice particles and prevents floating substances from settling (hui et al., 2006). during storage under ambient temperature, the increase in separation may be due to the breaking down of pectin by pectinase into simple sugars. hence, the juice separation is harder to maintain when stored under ambient temperature. at storage temperature of 4°c, the low temperature and high relative humidity inhibits enzyme activities, thereby, minimizing the increase of separation in juice (singh and mathur, 1983). the separation (%)of rind juice thermosonicated at optimum condition and rind juice thermosonicated at least optimum condition differed significantly, however, the increase in separation (%) through day 1 storage until day 7 storage is still temperature dependent. the tss of beverages for both treatment showed no significant decrease throughout the storage period under both storage conditions. a similar result was obtained from storage study of strawberry ultrasonicated at 40 khz for 10 and 30 min, where control (untreated juice), thermally treated juice (pasteurization at 90ºc for 60 s) and ultrasonicated juice showed no significant difference in tss after 10 days of storage at 5°c (tomadoni et al., 2017). for color analysis, the a* value significantly decreased from day 1 to day 7 for all sonicated samples. the beverage thermosonicated at optimum conditions and stored at 25 and 4oc showed decreasing tcd value during a week of storage, while beverage thermosonicated at least optimum condition and stored at 25 and 4oc showed inconsistent change in tcd value during a week of storage. this shows that low temperature and short duration of sonication and storage at 25oc has the highest negative impact on the beverage color. among the primary factors of consumers’ evaluation on the freshness of fruits and vegetable products is the color attribute. better color characteristics will give better appeal to the consumers (kays, 1999). the increase in l* was similar with studies of ultrasonic treatment for orange, strawberry, soursop, watermelon and mango juice (dias et al., 2015; rawson et al., 2011; santhirasegaram et al., 2013; tiwari et al., 2008; tomadoni et al., 2017). the tcd value of beverage was thermosonicated at optimum and the least optimum condition was more than 2, which indicated that there were visually noticeable differences in the sonicated samples compared to control. however, on the seventh day of storage, the color difference between the beverage thermosonicated at optimum condition and the control cannot be visually perceived. a study done on purple cactus pear showed that the color of juice thermoultrasonicated at 80% for 25 min was not visually different from control at the first day of storage only, but afterwards, the color of thermosonicated juices were visually different from control (cruz-casino et al., 2015). the cavitation caused by ultrasonic may accelerate chemical reactions, increasing diffusion rate, dispersion, aggregates formation and particles breakdowns which leads to color changes in treated beverages (sala et al, 1995). increase in l* value was also observed in sonicated honey, which were due to the reduction in crystal size (quintero-lira et al., 2017). the l* value of sample sonicated at the least optimum condition and stored at 25ºc had significantly increased, similar to the result of sonicated orange juice after storage (tiwari et al., 2008), whereas the sample sonicated at least optimum temperature and stored at 4ºc showed a decrease in day 4 and an increase in day 7. studies showed that an increase in l* value was due to the partial precipitation of unstable suspended particles, whereas, a decrease is due to oxidative darkening (genovese et al., 2006). this result is similar with thermosonicated strawberry juice (herceg et al., 2013). studies show that a decrease in a* and b* values might be attributed to the development of ital. j. food sci., vol. 31, 2019 645 browning degree in the beverage (ibarz et al., 2005). the results show that low temperature and short duration of sonication and storage at 25ºc has highest negative impact on color. based on the study of thermosonication on purple cactus pear juice, changes in color may be due to acceleration of chemical reactions by the increase in temperature, dispersion, particles breakdown and formation of aggregates by cavitation of thermosonication (cruz-casino et al., 2015). the degradation of vitamin c as shown in table 9 could be due to the formation of free radical by sonication reaction, associated with the oxidative process (aguilar et al., 2017). table 9. effect of thermosonication conditions on physicochemical properties of beverage stored at 25ºc and 4ºc for a week. physicochemical properties storage (day(s)) storage condition 25ºc 4ºc treatment optimum least optimum optimum least optimum ph 1 5.24aa±0.01 5.49aa±0.02 5.24aa±0.01 5.15aa±0.02 4 4.39bb±0.01 4.30bb±0.01 4.91bb±0.01 4.98bb±0.01 7 3.77cc±0.01 3.78cc±0.01 4.14cc±0.01 4.32cc±0.01 separation (%) 1 21cc±2 36ca±1 7bd±1 31bb±1 4 53bb±1 61ba±1 10ad±2 36ac±2 7 84ab±1 91aa±1 11ad±1 39ac±1 total soluble solid (°brix) 1 13.0aa±0.0 13.0aa±0.0 13.0aa±0.0 13.0aa±0.0 4 13.0aa±0.1 13.0aa±0.1 13.0aa±0.1 13.0aa±0.1 7 12.9aa±0.1 12.9aa±0.1 12.9aa±0.1 12.9aa±0.1 color l* 1 73.0aa±0.5 72.8ba±2.5 72.4aa±0.4 73.4ba±0.1 4 69.9bc±0.2 78.1aba±2.2 72.0ab±0.2 71.1cb±0.8 7 69.0bc±0.7 79.0aa±1.9 68.9bc±0.1 75.6ab±0.6 a* 1 8.2aab±0.1 6.4ab±2.0 8.0aab±0.0 9.2aa±0.0 4 4.9ba±0.1 5.2aa±0.2 6.3ba±1.1 5.7ba±0.2 7 3.1ca±0.1 0.3bb±0.2 3.1ca±0.1 0.1cb±0.1 b* 1 30.1bab±0.1 27.4ab±2.3 30.7ca±0.1 30.4ca±0.1 4 23.3cc±0.2 20.0bd±0.1 32.9ab±0.1 33.2aa±0.1 7 31.7aa±0.2 28.3ab±0.8 31.3ba±0.2 32.3ba±0.1 tcd 1 7.9aa±0.9 6.9ba±1.3 6.8aba±0.2 8.4aa±0.0 4 7.5ab±0.2 13.9aa±1.4 5.9abc±0.8 5.3cc±0.2 7 2.0bc±0.3 10.3aba±1.9 1.7bc±0.2 7.3bb±0.6 vitamin c (mg/100 ml) 1 0.00ab±0.06 0.67aa±0.02 0.67aa±0.02 0.67aa±0.02 4 0.00aa±0.00 0.00ba±0.00 0.00ba±0.00 0.00ba±0.00 7 0.00aa±0.00 0.00ba±0.00 0.00ba±0.00 0.00ba±0.00 data are mean±sd of three replicates. values with different lower case letters within a column is significantly different at 5% level (p<0.05) between storage day(s). values with different capital letters within a row is significantly different at 5% level (p<0.05) between storage conditions. ital. j. food sci., vol. 31, 2019 646 the vitamin c content for beverage treated at least optimum condition were retained for at least a day of storage under both temperatures (25°c and 4°c). studies also showed that the degradation of vitamin c in juices is influenced greatly by the presence of air; therefore, juice should be degassed prior to treatment for better retention of ascorbic acid (oms-oliu et al., 2009). in some cases, degradation of vitamin c occur in order to protect other compounds such as carotenoids, phenolic compounds or anthocyanins (tiwari et al., 2008). this shows that ascorbic acid is an unstable compound, making it very easy to degrade. therefore, for better retention of vitamin c, milder processing conditions should be used (basmaci, 2017). as shown in table 10, all sonicated samples showed an increase in log cfu/ml of tpc and pda within a week of storage. for all samples, on pda, there is only growth of yeast with no growth of fungi. from this experiment, the control sample would not be compatible for human consumption starting from day 0, since the maximum limit of microorganisms in minimally processed food is 7 log cfu/ml (tomadoni et al., 2017). as for sample sonicated at optimum condition, stored at both 25 and 4ºc, the watermelon rind beverage containing honey were still fit for consumption even after reaching the 7th day of storage. the application of ultrasound treatment at high temperature can regulate microbial growth in watermelon juice containing honey at a level whereby the juice shelf life could be prolonged. table 10. effects of optimum and least optimum sonication conditions on microbiological properties of sample stored at 25 and 4 ºc for a week. microbiological properties storage (day(s)) storage condition 25ºc 4ºc treatment optimum least optimum optimum least optimum tpc (log cfu/ml) 1 4.6ba±0.5 5.3ba±0.7 4.6ba±0.4 5.3ba±0.4 4 5.5ba±0.5 6.2aba±0.3 5.0aba±0.6 6.2aba±0.8 7 6.8aa±0.3 7.4aa±0.5 6.3aa±0.6 7.0aa±0.2 pda (log cfu/ml) 1 4.2ba±0.5 5.2ba±0.3 4.1ba±0.3 5.2ba±0.6 4 5.1ba±0.4 6.1ba±0.2 5.0aba±0.6 6.1aba±0.7 7 6.8aa±0.7 7.2aa±0.5 6.2aa±0.5 7.2aa±0.4 data are mean±sd of three replicates. values with different lower case letters within a column is significantly different at 5% level (p < 0.05) between storage day(s). values with different capital letters within a row is significantly different at 5% level (p < 0.05) between storage conditions. 4. conclusions this study shows that the optimum condition for thermosonication process for watermelon rind-honey beverage is at 65°c for 60 min. thermosonication has no significant effect on the ph and tss of beverage. however, throughout the storage period, the ph of beverage decreased, depending on the storage temperature but it is still safe for consumption. the % separation, color, vitamin c content and microbial load of beverage were all significantly affected by thermosonication. the changes in beverage ph, % separation and color during storage period were temperature dependent. all the results ital. j. food sci., vol. 31, 2019 647 showed that the beverage treated with thermosonication can be preserved all through the storage period of a week. therefore, it can be concluded that watermelon rind beverage containing honey can be better treated using thermosonication method rather than thermal treatment alone to obtain longer beverage shelf life. however, further enhancement study should be done for better vitamin c retention in fruit juices using thermosonication method. acknowledgments this research was supported by faculty of food science and technology, universiti putra malaysia. references aadil, r.m., zeng, x.a., zhang, z.h., wang, m.s., han, z., jing, h. and jabbar, s. 2015. 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ijfs1760_bozza_new ital. j. food sci., vol. 32, 2020 1 letter from the italian journal of food science editor-in-chief p. fantozzi dipartimento di scienze agrarie, alimentari ed ambientali, università di perugia, via s. costanzo, 06126 perugia, italy corresponding author: tel.: +39 075 5857910 telefax +39 075 5857939-5857943 email: paolo.fantozzi@ijfs.eu ital. j. food sci., vol. 32, 2020 2 dear readers and authors, we are starting our thirty-second year of ijfs, which will be an important anniversary for all of us. the journal has substantially improved his if and citation index (0.736 published in 2018 journal of citation reports, scopus citescore 2019: 1.11, citescore tracker2019: 1.00) i consider these values very important and rewarding the journal inside the list of peer reviewed international journals. everyone of us may locate easily our journal in the 2018 official jcr list for food science and technology. during the last five years (2015-2019) we published 316 papers of 1736 received, with an acceptation rate of 18,2%. accepted paper from abroad raised steadily during the years, reaching 79,4 % during 2019. their acceptation rate (20,1%) is also consistent with the general value. since 1989, when the university of perugia founded the journal, i was appointed as the editor-in-chief (eic). starting 2009 i took over the journal, still leaving, as before, the editorial responsibility to chiriotti editori. in october 16, 2019, in consideration of the importance of the requesting company and of the official scientific guaranties to the preservation of the existing evaluation system, i decided to transfer the journal headline to codon publications, brisbane, australia, but maintaining for myself the permanent appointment as eic. codon publication agreed also to continue the journal publication 1) under the seal of the university of perugia , appointing the journal “under the aeges of the university of perugia” and 2) under the seal of the italian society of food science (sistal), enforcing, as before, the existing papers evaluation system managed by the co-editors of the sistal. i would like in this occasion express my gratitude to the following professors who acted in the past as unique journal sistal co.editors and accepted to continue their work even in the future: chiavaro emma università degli studi di parma del caro alessandra università degli studi di sassari de noni ivano università degli studi di milano hidalgo alyssa università degli studi di milano loizzo monica rosa università della calabria rantsiou kalliopi università degli studi di torino rolle luca giorgio carlo università degli studi di torino vincenzi simone università degli studi di padova vittadini elena giovanna università di camerino practically, all future papers submitted to ijfs until 30th of june 2020 will be processed, as before, inside the existing web platform of chiriotti editori. starting 1st of july 2020, this open access platform will not accept anymore papers. ital. j. food sci., vol. 32, 2020 3 all submitting authors will be asked to redirect and submit their papers to the new codon publication wopen access web platform. you will find proper and sound information during the next months in the section “announcement” of the ijfs chiriotti platform. in looking forward to continue our important collaboration, i wish all of you a fruitful 2020. paolo fantozzi #109_cansian_bozza ! ital. j. food sci., vol 28, 2016 733 paper listeria monocytogenes adhesion to food processing surfaces (boning knives) and the removal efficacy of different sanitizers j. barbosa1, v. grzybowski1, m. cuppini1, j. flach1, c. steffens*1, g. toniazzo1 and r. l. cansian1 1 department of food engineering, uri erechim, av. 7 de setembro, 1621, cep 99700-000, erechim rs, brazil *corresponding author. tel.: +55 5435209000; fax: +55 5435209090 e-mail address: claristeffens@yahoo.com.br abstract the adhesion and biofilm formation of listeria monocytogenes on new and used boning knives (handle and blade) were observed. the number of pathogens on both surfaces increased with the contact time, forming a biofilm after 2 h. peracetic acid or hot water effectively removed adhered l. monocytogenes at each of the contact times and concentrations evaluated. biguanide showed lower removal efficacy on used surfaces, but had increased effectiveness at concentrations of 2.0%. the new knife blades had a lower roughness and flattened morphology in comparison with used surface keywords: bacterial adhesion, sanitizer, listeria monocytogenes, nactivation, boning knives ! ital. j. food sci., vol 28, 2016 734 1. introduction food contact surfaces constantly must pass a microbiological evaluation for efficiency control and sanitizing procedures, in order to avoid contamination of the food produced (pinheiro et al., 2010; almeida et al., 2013). the exposure of surfaces to pathogens may take place either by direct contact with contaminated objects or indirectly through airborne particles. according to kusumaningrum et al. (2003), many bacteria, including l. monocytogenes may survive on utensils for hours or days after the initial contamination and can persist on industrial equipment and installations with high potential for adhesion and biofilm formation at low temperatures (giaouris et al., 2014; carpentier and cerf, 2011). cell surface hydrophobicity and production of extracellular polymeric substances influence the rate and extent of attachment of microbial cells. another factor that can also influence bacterial adhesion is the surface roughness (rodriguez et al., 2008; skovager et al., 2013). bacterial adhesion to stainless steel, glass, rubber and polypropylene surfaces is a potential source of contamination that may lead to disease transmission (chavant et al., 2007). the adhered cells are highly resistant to acids present in sanitizers, to desiccation and to heat. tolerance to increased sub-lethal concentrations of disinfectants or resistance to lethal concentrations are documented, because sessile cells produce exopolysaccharides that protect against chemical agents (joseph et al., 2001; carpentier and cerf 2011; silva et al., 2010). several sanitizers are available with different uses, but they do not always eliminate bacteria to the expected level (beltrame et al., 2012). in this way, it is very important to know the factors involved in the adherence of biofilms to surfaces, which could be useful to improve methods for disinfection of food processing surfaces (boning knives). therefore, the purpose of this study was to investigate adhesion and biofilm formation of l. monocytogenes on the handle and blade of new and used boning knives. this study also evaluated the removal of bacteria on the surface of the knife using different contact times and concentrations of chemical sanitizers (peracetic acid and biguanide) or hot water. 2. materials and methods in this study, bacterial adhesion and inactivation were evaluated on new and used boning knives using gram-positive l. monocytogenes (atcc 7644). the bacterial culture was purchased from the instituto oswaldo cruz, mantained at -80°c and revitalized in luria bertani broth (lb merck, darmstadt, germany) at 30°c, 24 hours before the experiments. 2.1. bacterial adhesion and quantification of adhered cells in order to assess bacterial adhesion, the polypropylene (pp) handle and stainless steel (ss) blade of new boning knives (professional line mundial® model 5315-6) and the same model of used knives (use in a cattle slaughterhouse for 45 to 60 days) were studied. the entire knife surface (handle and blade) was prepared according to the follows steps: cleaning by manual rubbing with water and neutral liquid detergent, rinsing with water and then by sterile distilled water and air drying. for sanitization, the surfaces were exposed to ultraviolet light (254 nm) for one hour. after cleaning, the entire knife surface was swabbed in order to confirm the absence of initial contamination (negative control), before carrying out the experiments. ! ital. j. food sci., vol 28, 2016 735 the bacteria were incubated in lb broth for 24 h at 35-37°c. next, 10 ml of inoculum (3 log cfu/ml) was inoculated into 1.5 l lb broth that had been previously poured into a polyvinylchloride (pvc) tube with a 10 cm diameter to accommodate the entire knife, and the samples were incubated for 24 h at 35-37°c. afterward, the knives (new or used) were removed from the pvc tube with sterile forceps and rinsed with water to remove the planktonic cells. the entire surface of the knife handle and knife blade were swabbed separately to assess contamination. dilutions were performed in peptone water, plated onto lb agar, and incubated at 35-37°c for 24 h. the number of adherent cells was assessed in intervals of 0.1, 0.5, 1, 2, 6, 24 and 48 h at 35°c. these times were selected in order to simulate the factory production time, which is the period that knives remain in contact with products without sanitization. 2.2. characterization of the boning knife surface by contact angle the hydrophobicity and hydrophilicity of the new and used blades with and without l. monocytogenes adhesion for 6 h, were determined by contact angle with a drop of water using a contact angle metre (ksv instruments, helsinki, finland). the measurements were performed at 25°c and 45% humidity, by depositing 4.0 !l of water with a hamilton syringe. the handles were not evaluated in this analysis because the texture of the handle produces differences in surface that did not permit the stable formation of a drop on the surface. 2.3. characterization of the boning knife surface by atomic force microscopy (afm) the morphology and average roughness (ra) of the new and used blades with and without l. monocytogenes adhesion for 6 h were analyzed with a dimension v (veeco instruments inc.) afm, using a silicon nitride tip, with a spring constant of 42 n/m and resonance frequency of 285 khz. all images were obtained in tappingtm mode at a scan rate of 1 hz. the images were processed with the aid of gwydion© 2.1 data analysis software. the handles were not evaluated due to the characteristics of the non-slip coating that had very large differences in height, which did not permit a surface scan. the ra value (arithmetic mean deviation of the profile) is the most common measure used to define the surface roughness (verran et al., 1991). afm scans were performed in 500 x 500 nm2 areas on each surface. the roughness was calculated from three scans in different areas. 2.4. l. monocytogenes inactivation in this study, peracetic acid (pluron 461 ap®) and biguanide (pluron 463 ap®) sanitizers were studied; they were prepared in sterilized water immediately prior to testing, according to the supplier’s instructions. in inactivation experiments, the entire knife was analyzed at intervals of 1, 2 and 6 h, simulating industrial conditions. after rinsing with deionized water, the knives were immersed in beakers containing 500 ml of the respective sanitizer solution at concentrations of 0, 0.2, 0.5, 1.0 or 2.0% (v/v) for 10 min at 25°c, to evaluate their efficacy against cell attachment. the hot water treatment was performed by immersing the knife surface in a hot water bath (82.2°c) for 15 s, according to 175/2005/mapa method 2 (brasil 2005). bacterial presence was quantified by the enumeration method as previously described (kim et al., 2008) using swabs of the knives. ! ital. j. food sci., vol 28, 2016 736 2.5. statistical analysis the results of l. monocytogenes counts were converted to decimal logarithmic values (log cfu/cm2) and subjected to tukey's test at a 5% significance level using statistica 8.0 software (statsoftinc®, usa). all experiments were run in triplicate and repeated with three separate knives. 3. results and discussions 3.1. listeria monocytogenes adhesion the adhesion of l. monocytogenes on new and used boning knives (handles and blades) is shown in fig. 1. a rapid bacterial growth was observed for the first 6 h, with a tendency to stabilize at 48 h. adherence occurred on handles and blades and the adhesion velocities were similar for used and new materials. however, statistical analysis showed a significant difference in adherence to new and used polyethylene handles between 6 and 48 h (fig. 1a). the knives a non-slip coating on the surface of the handle, which conveys firmness and grip during handling, however, this feature promoted easy adhesion of bacteria on used surfaces. figure 1: adhesion kinetics of l. monocytogeneson(a) new polyethylene handles (npp) and usedpolyethylene handles (upp) and (b) newstainless steel blades (nss) and usedstainless steel blades(uss). biofilm formation with 3.0 log cfu/cm2. means (± standard deviations), for each time tested, who having the * symbol are significantly different (p<0.05). ! ital. j. food sci., vol 28, 2016 737 l. monocytogenes showed a greater capacity for adhesion onto ss than to pp (fig. 1) from 6 h in new, and 24 h in used knives. these results confirm those by teixeira et al. (2008), who observed greater adhesion on ss as compared with the extent of adhesion on pp (cutting board). ronner and wong (1993) defined biofilm formation as a recovery of greater than 3.0 log cfu/cm2 adhered cells. thus, according to this criterion, l. monocytogenes biofilm growth occurred after 1 h of contact on the handles (new and used), and from 30 min (new) to 1 h (used) on the ss surface. 3.2. characterization of the surface of the boning knife by contact angle water contact angle is a quantitative measurement of surface wettability, and also can be used to evaluate the cleanliness of the material surface. throughout this study, a contact angle of 70±1.0 and 82±1.0 degrees was obtained for new stainless steel (nss) and used stainless steel (uss) surfaces without microbial adhesion, respectively. these results agree with those found by bernardes et al. (2010), who found a value of 70±7.9 on ss surfaces. after 6 h of bacteria exposure, contact angles of 19±1.4 and 30±2.1 were obtained on nss and uss respectively, showing that the surfaces became more wettable after microbial adhesion. these results corroborate with those of chavant et al. (2007), who observed better adhesion and biofilm formation of l. monocytogenes on hydrophilic (ss) rather than on hydrophobic (pp) surfaces. these decreases in the value of contact angle from 70 down to 19 (nss) and 80 down to 30 (uss) may be attributed to the l. monocytogenes surface composition (molecules such as proteins, teichoic and lipoteichoic acids) (bereksi et al., 2002). 3.3. morphological and roughness characterization the morphology of new and used blades was analyzed through use of afm before and after l. monocytogenes adhesion (fig. 2). roughness values (ra) of 5.1±2.3, 14.3±1.7, 17.4±2.4 and 28.8±2.1 nm were obtained from new and used blades without and with l. monocytogenes adhesion, respectively. new blades had a low roughness and flattening morphology compared with used surfaces, which is very important to note because studies have shown that an increase in ra value will cause a corresponding increase in microbial adhesion on surfaces (whitehead et al., 2004). this increase may be due to protective cells present in microscopic niches. roughness values of 800 nm or less are generally used to describe a hygienic surface (flint et al., 1997). these values were found for all surfaces evaluated in this work (fig. 2). teixeira et al. (2008), observed that ss is a material with high surface roughness, similar to what was measured in this study for the surface of the knife blade (mundial®). taylor et al. (1998) observed a significant increase in the attachment of pseudomonas aeruginosa and staphylococcus epidermidis on polymethyl methacrylate when the surfaces had a small increase in roughness (40-1240 nm). bacterial adhesion has been found to increase numerically with surface roughness, by comparing bacterial adhesion on used versus abraded ss and for ra value increases from 602 to 706 nm or from 484 to 698 nm (holah and thorpe, 1990). thus, it was found that the roughness data obtained after the adherence of l. monocytogenes are in agreement with those from the literature, but no correlation was demonstrated between ra and the number of adhering cells. from this evaluation of the surface characteristics of boning knives, it can be observed that with an increase of the surface roughness, there was a decrease in the contact angle. ! ital. j. food sci., vol 28, 2016 738 figure 2: roughness values on nss and uss with and withoutl. monocytogenes adhesion. mean values and standard deviations (error bars) are indicated. observations correspond to on 500 x 500 nm scan area. 3.4. efficacy of different sanitizers on food processing surfaces to evaluate their ability to sanitize new and used knives, biguanide and peracetic acid sanitizers were used at different concentrations (0.2, 0.5, 1.0 and 2.0% v/v) for 10 min, or hot water (82.2°c) was used for 15 s. the boning knives remained in contact with l. monocytogenes for different contact times (1, 2 and 6 h) to simulate industrial conditions for adhesion. l. monocytogenes was resistant to biguanide sanitizer under the different contact times studied (table 1). similar efficacy levels have been obtained by other researchers. martín-espada et al. (2014) observed that 1.61% peracetic acid was effective against p. aeruginosa biofilms formed on polystyrene surfaces, inhibiting almost 100% of the microbial population. similarly, beltrame et al. (2015) evaluated the efficacy of 0.5% peracetic acid at inactivating l. monocytogenes cells adhered to cutting boards and observed that it was able to reduce the amount of adhered cells by 100%, after 3 h of contact time with bacteria. cabeça et al. (2012) observed adhesion of l. monocytogenes on ss surfaces and studied the inactivation after treatment with 0.5% biguanide and 0.5% peracetic acid. the authors verified an initial count of 6.2 log cfu/cm2 with reduction to 2.9 log cfu/cm2 and 1.1 log cfu/cm2 using biguanide and peracetic acid, respectively, showing that peracetic acid was more effective against l. monocytogenes cells. for the sanitizer evaluated, the suppliers recommend a maximum disinfection concentration of 0.5% for both biguanide and peracetic acid. thus, there was a variation in the effectiveness of the procedures, based on decimal reductions, in microbial effect conveyed by the different sanitization process investigated, as demonstrated in table 2. in the maximum contact time (6h) between the knife and the microorganism, the low concentration of peracetic acid (0.2%), lower than that recommended by the supplier, showed reliable results from the point of microbiology safety. similar results were observed for hot water, where the presence of surviving l. monocytogenes was not observed. on the other hand, the maximum concentration of biguanide recommended by ! ital. j. food sci., vol 28, 2016 739 the supplier was not able to remove adherent cells, with maximum efficacy of only 50% on npp (table 2). table 1: reduction of the l. monocytogenes count (log cfu/cm2) as a function of contact time (1, 2 and 6 h) and the biguanide sanitizer concentration (0.2, 0.5, 1.0 and 2.0%).results in log cfu/cm2. biguanide concentration (%) contact time boning knives initial count r* 0.2% % r* 0.5% % r* 1.0% % r* 2.0% % 1h npp 3.5 2.0±0.2c 57,1 2.7±0.1b 77.1 3.3±0.1a 94,3 3.5±0.3a 100 upp 3.7 0.5±0.1c 14.3 1.7±0.1b 45,9 1.8±0.2b 48.6 3.0±0.1a 81.1 nss 3.4 0.6±0.1c 17.6 1.3±0.1b 38.2 3.2±0.1a 94.1 3.4±0.1a 100 uss 4.1 0.2±0.1d 4.9 1.7±0.1c 41.5 2.8±0.1b 68.3 4.0±0.1a 97.6 2h npp 3.7 1.7±0.1b 4.6 2.4±0.2ab 64.9 2.8±0.2a 75.7 2.7±0.1a 73.0 upp 3.8 0.8±0.1c 21.0 1.8±0.1b 47.4 1.8±0.1b 47.4 2.7±0.1a 71.0 nss 3.5 0.2±0.1d 5.7 0.9±0.1c 25.7 1.9±0.1b 54.3 3.3±0.1a 94.3 uss 4.1 0.6±0.1d 14.6 1.6±0.1c 39.0 2.1±0.1b 51.2 3.3±0.1a 80.5 6h npp 4.2 1.1±0.1c 26.2 2.1±0.1b 50.0 2.5±0.2a 59.5 2.8±0.1a 66.7 upp 4.8 0.1±0.1c 2.1 2.3±0.2b 47.9 2.7±0.2ab 56.2 3.1±0.2a 64.6 nss 4.7 1.0±0.1d 21.3 2.0±0.1c 42.6 2.7±0.1b 57.4 3.9±0.1a 83.0 uss 4.8 0.7±0.1c 14.6 0.5±0.1c 10.4 2.4±0.1b 50.0 3.7±0.1a 77.1 npp: new handles; upp: used handles, nss: new blades; uss: used blades. *r decimal reduction (log cfu initial population-log cfu end population submitted to the sanitizers application). values followed for the same letters in the lines do not differ statistically according to thetukey test, with 95% confidence range. table 2: reduction of the listeria monocytogenes count (log cfu/cm2) in boning knives after hot water and sanitizer treatments (6 h of contact time, sanitizer concentrations 0.5% biguanide and 0.2% peracetic acid). boning knives initial count biguanide r* % peracetic acid r* % hot water r* % npp 4.2 2.1±0.1 50.0 4.2±0 100 4.1±0.1 100 upp 4.8 2.3±0.2 47.9 4.8±0 100 4.8±0 100 nss 4.7 2.0±0.1 42.6 4.7±0 100 4.7±0 100 uss 4.8 0.5±0.1 10.4 4.8±0 100 4.8±0 100 npp: new handles; upp: used handles; nss: new blades; uss: used blades. * decimal reduction (initial population log cfu end population log cfu). thus, this work confirms that the most effective treatments were the hot water and peracetic acid sanitizer, whereas biguanide showed lower performance. additional studies should be performed to improve the action of biguanide on removal of l. monocytogenes from food preparation surfaces. the treatments with hot water and peracetic acid were shown to be effective for the inactivation of bacteria that had initially adhered. this result can be associated to the disinfectant activity of peracetic acid based on the release of active oxygen, which may ! ital. j. food sci., vol 28, 2016 740 disrupt the chemiosmotic function of the lipoprotein cytoplasmic membrane and transport through the dislocation or rupture of cell walls. this can also be effective against outer membrane lipoproteins, facilitating its action against gram-negative cells. the intracellular peracetic acid may also oxidize essential enzymes, which may damage the bases of dna molecules (kitis, 2004). moreover this sanitizing agent has low environmental hazards and does not produce toxic compounds upon reaction with organic materials. likewise, the application of hot water was also an effective means of bacterial inactivation. the mechanism of action of hot water treatment is multifactorial. the exposure to this high temperature is likely to affect most components of the bacterial cell including the cell wall, cell membrane, enzymes and proteins, dna and rna (phua et al., 2014). differences between the bactericidal effect of biguanide solutions and peracetic acid were observed, corroborating data obtained by silva et al. (2010), who showed that peracetic acid was a more effective bactericidal agent than the quaternary ammonium compound (whose mechanism of action is similar to biguanide). (a) (b) figure 3: afm characterization of the knives surface in 3d (a) new without bacterial adhesion, (b) used without bacterial adhesion, (c) new with bacterial adhesion and (d) used with bacterial adhesion. ! ital. j. food sci., vol 28, 2016 741 sodium dichloroisocyanurate, hydrogen peroxide and peracetic acid have been evaluated for their abilities to inactivate the biofilm formed by s. aureus on ss and glass surfaces; peracetic acid showed a significant difference (p < 0.05) compared with the three disinfectants used (marques et al., 2007). the higher efficacy of peracetic acid was explained by its high capacity to oxidize cellular molecules. the survival of bacteria after cleaning and sanitizing is a potential danger to the food industry and the consumer (rodgers et al., 2001). this study demonstrates the need for specific tests in order to select products to be used to clean surfaces that come into contact with food. furthermore, bacteria can obtain high resistance through adaptation, genetic elements, stress response and biofilm formation (bridier et al., 2011). it should be noted that an appropriate and effective cleaning process is extremely important, since the american public health association (apha) recommends a maximum tolerated limit of 2 cfu/cm2 in order to consider a food contact surface appropriate (vanderzant and splittsstoesser, 1992), whereas the world health organization (who) suggests limits of 30 cfu/cm2. based on the obtained results, this study confirms that hot water and peracetic acid was fully effective in removing l. monocytogenes cells, which had adhered onto new and used boning knives, whereas biguanide was not efficient in removing the bacterial cells. 4. conclusions adherence of l. monocytogenes occurred on handles and blades, and the adhesion velocities were similar between the used and new materials. regarding the morphology and contact angle of the surfaces, an increase of the wettability and roughness on the used stainless steel surface in relation to the new stainless steelsurface was observed. disinfection with peracetic acid was effective at all contact times and concentrations evaluated; no surviving bacteria was found after sanitizer application in all conditions investigated. the hot water treatment (82.2ºc for 15 s) also was effective in reducing l. monocytogenes adhesion on the surfaces tested. biguanide showed lower efficacy on new and used handles and blades, but had increased effectiveness at concentrations of 2.0% with 1 h of contact time. acknowledgements the authors thank cnpq, capes, fapergs, science and technology secretary rs and uri-erechim for the financial support for this research. references almeida g. et al. 2013. foci of contamination of listeria monocytogenes in different cheese processing plants. int. j. food microbiol. 167:303-309 doi:http://dx.doi.org/10.1016/j.ijfoodmicro.2013.09.006 beltrame c.a. et al. 2012. influence of different sanitizers on food contaminant bacteria: effect of exposure temperature, contact time, and product concentration ciência tecnol. alim. 32:228-232. beltrame c.a. et al. 2015. adhesion of listeria monocytogenes to cutting board surfaces and removal by different sanitizers. j. verbr. lebensm. 10:41-47 doi:10.1007/s00003-014-0923-7 bereksi n., gavini f., benezech t. and faille c. 2002. growth, morphology and surface properties of listeria monocytogenes scott a and lo28 under saline and acid environments. j. appl. microbiol. 92:556-565. ! 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fax: +38521329461 e-mail address: tea@ktf-split.hr abstract in this study, the gastrointestinal stability of carotenoids (lycopene and β-carotene) extracted from raw and freeze-dried samples of tomato, carrot and red pepper was investigated. extracted carotenoids fractions (lycopene and β-carotene) were submitted to a two-phase in vitro digestion process using human gastrointestinal enzymes. the use of freeze-drying has a strong effect on the enhancement of the gastrointestinal stability of carotenoid, especially after simulated intestinal phase. in addition, the effect of ph on carotenoid stability is much lower in freeze-dried plant material than in raw samples of tomato, carrot and red pepper. the food matrix also plays important role in carotenoids gastrointestinal stability rate, which was found to be the most stable in red pepper. keywords: carotenoids, in vitro digestion, lycopene, β-carotene ital. j. food sci., vol. 31, 2019 173 1. introduction environmental influences such as contamination, ultraviolet (uv) radiation, smoking, stress and improper diet may result in cell damage caused by free radicals, believed to be the cause of many degenerative diseases, such as certain types of cancer, cardiovascular disease, type 2 diabetes, age-related macular degeneration (amd) and cataracts, among others, and even mortality caused by some of these serious diseases (maiani et al., 2009; fiedor and burda, 2014; meyers et al., 2015; nolan et al., 2015; sluijs et al., 2015). in order to protect itself, the body uses antioxidants and neutralizers of the free radicals, which are commonly provided by the diet. carotenoids, which are considered as the most widely distributed pigments in nature (schwartz et al., 2008), are known to be very efficient scavengers of singlet oxygen (1o2), as well as other reactive oxygen species (ros). carotenoids are responsible for the attractive, yellow to red colour of fruit and vegetables, which is the first attribute that consumers evaluate. although more than 700 different carotenoids have been identified so far, just six of them are commonly analysed in foods and blood: three hydrocarbon compounds carotenes (β-carotene, α-carotene, lycopene) and three oxygenated forms xanthophylls (β-cryptoxanthin, lutein, zeaxanthin) (olmedilla-alonso, 2017). the composition and the content of carotenoids in foods are dependent on different factors e.g. variety and maturity of species, cultivation practices and methods of food processing. several reviews and databases on food sources of carotenoids, intake, stability and bioavailability have been published (heinonen et al., 1989; hart and scott, 1995; leth et al., 2000; murkovic et al., 2000; o’neill et al., 2001; kim et al., 2007; fernández-garcía et al., 2012; nagao, 2014; meyers et al., 2015; olmedillaalonso, 2017). therefore, in order to understand the relationship between nutrition and health in humans, it is important to know not only the amounts of consumed carotenoids but to what extent they are absorbed from the different dietary sources, their bioavailability, respectively (olmedilla-alonso 2017). in general, stability under environmental conditions, gastrointestinal stability, the bioaccessibility as well as the bioavailability of functional food ingredients represent main factors affecting usefulness of the intake of certain foodstuffs (nagao, 2014). carotenoids have very low bioavailability because they are quite susceptible to conditions found in the digestive tract (temperature, ph). likewise, they are less bioavailable due to extreme hydrophobicity, and it also depends on other factors: release from the food matrix, solubilization in the digestive tract, absorption in intestinal epithelia, and metabolism (nagao 2014). also, it may be due to the fact that they can be bound in carotenoproteins; in green leafy vegetables carotenoids are found bound in chloroplasts and in carrot root, αand β-carotene are largely in crystal forms. therefore, the carotenoids are not easily solubilized out of these tissues by the digestive process (institute of medicine 2000), mostly due to rigid cell walls. they are more readily released in ripe fruit as well as processed vegetables then in fresh one, which substantially improves their bioavailability (nagao 2014). it is known that the application of a thermal treatment and/or mechanical homogenization, as well as addition of fats and oils in diet, are all techniques that enhanced the bioavailability of dietary carotenoids (fernández-garcía et al., 2012), just because of increased bioaccessibility by dispersing them in digestive tract. on the other hand, dietary fibres have been thought to decrease bioaccessibility by binding bile acids (nagao, 2014). following digestive release in stomach and upper intestine, the hydrophobic components aggregate in lipid emulsion droplets, which partitioned into mixed micelles in the small intestine. the formation of micelles allows carotenoids to be soluble in the hydrophobic interior and carotenoids, which are not in that form are not typically bioaccessible and remain unabsorbed (fernández-garcía et al., 2012; neilson et al., 2017). ital. j. food sci., vol. 31, 2019 174 in order to study the rate of gastrointestinal stability of biologically active components, the use of in vitro digestion models procedures represents effective tool due to its simplicity, low cost and putative production of many digesta fractions. a large number of in vitro studies have been carried out on the bioavailability of carotenoids and their assimilation during the digestive process and a number of models are suggested to mimic in vivo digestion over the years (ferruzzi et al., 2006; granado-lorencio et al., 2007; failla et al., 2008; courraud et al., 2013; kopec et al., 2017), but there is a lack of studies on the stability of carotenoids under gastrointestinal conditions using human gastrointestinal juices which comprise a complex mixture of enzymes present in multiple isoforms, enzyme inhibitors and bile salts that are important for the digestion process. the aim of this study was to determine carotenoid stability after simulated gastric and duodenal phases of simulated digestion process. raw and freeze-dried samples of tomato, carrot and red pepper, vegetables rich in carotenoids, were used for their extraction. the stability rate of carotenoids (β-carotene and lycopene) in relation to digestion enzymes and to the effect of ph after each simulated digestion phase was determined spectrophotometrically. 2. material and methods 2.1. chemicals lycopene, β-carotene and bht (butylhydroxy toluene) were purchased from sigma – aldrich (basel, switzerland). all solvents (hexane, acetone, ethanol) were of pro analysis purity and were purchased from kemika (zagreb, croatia). 2.2. samples raw samples of carrot, tomato and red pepper were purchased from green local market. raw samples were processed immediately after the purchase. samples were firstly cut in small pieces and immediately homogenized in the blender to obtain a pulp. then, the pulp was treated with argon in order to avoid rapid oxidation process. the pulp was not stored, because for repetition of experiments only fresh samples were used. immediately after purchasing, one part of samples was freeze-dried using freeze-dry system (labconco, usa) at the temperature of –50°c and the pressure of 0.2 mb, during the period of 3 days. after freeze-drying, samples were grinding into a spice grinder and immediately used for further analyses. second part of samples were fresh (raw) and were immediately prepared for extraction in the manner that samples were cut into approximately 2 to 4 cm cubes, and then homogenized using a hand blender (bosch maxomixx, germany). 2.3. extraction of carotenoids the extraction of carotenoids from freeze-dried and raw samples of carrot, tomato and red pepper was done using two procedures described by alda et al. (2009) and fish et al. (2002). for extraction procedure, the following solvents were used: acetone, hexane and ethanol (2:1:1). sample from homogenized raw plant material or freeze-dried plant material (1 g) was mixed with 25 ml of solvent mixture, under subdued lighting at room temperature and the bottle was treated with argon in order to protect lycopene from degradation. after solvent addition, the samples were shacked during the period of 30 min (180 rpm, room temperature). after that, 1 ml of deionized water was added and the samples were left for approximately 5 min in order to obtain two separate layers. upper ital. j. food sci., vol. 31, 2019 175 layer (the fraction with lycopene) was separate and stored in glass, dark flasks, treated with inert gas (argon) and stored at – 20°c until analysis. 2.4. extraction of β-carotene extraction of β-carotene was done according to procedure described by davis et al. (2008). for extraction procedure, the following solvents were used: bht in acetone (0.05%), ethanol and hexane (1:1:2). homogenized raw plant material or freeze-dried plant material (0.6 g) was mixed with 15 ml of solvent mixture under subdued lighting at room temperature and the bottle was treated with argon to protect β-carotene from degradation. after solvent addition, the samples were shacked during the period of 10 min (180 rpm, room temperature). after that, 3 ml of deionized water was added and the samples were left for approximately 5 min in order to obtain two separate layers. upper layer (the fraction with β-carotene) was separate and stored in glass, dark flasks, treated with inert gas (argon) and stored at –20°c until analysis. 2.5. spectrophotometric measurement of lycopene and β-carotene spectrophotometric measurements were performed on uv/vis spectrophotometer specord 200 spectrometer (analytik jena gmbh, germany) and iraffinity-1 fourier transform infrared (ftir) spectrometer (shimadzu, japan). ir spectra were recorded by using kbr transmision cell, in the spectral area 4000-400 cm-1 and with resolution 4 cm-1. abbreviations used are for streching (n), deformation (d). for measurement of lycopene and β-carotene, the calibration curve was done using different concentrations of lycopene and β-carotene. a 0.01 g of β-carotene and lycopene was dissolved in hexane (100 ml) to obtain the concentration of 100 μg/ml (stock solution). after that, the working solution was done (20 μg/ml) and the following concentrations were prepared: 7, 6, 5, 4, 3, 2, 1 μg/ml. the concentration of lycopene in prepared samples was determined by uv/vis spectrophotometer at 503 nm. hexane was used for detection of zero. concentration of lycopene in raw and freeze-dried samples of carrot, tomato and red pepper was determined according to following formula (fish et al., 2002): (a503 × 31.2) / g of sample absorbance of β-carotene was determined using uv/vis spectrophotometer at 450 nm. 2.6. isolation of human juices human gastric and duodenal juices were collected from four donors (two males and two female) without known gastrointestinal pathology, and who were not taking acid secretion inhibitors or antibiotics. gastric and duodenal juices were aspirated through the endoscope. eight hours before the procedure, all liquid or food intake was ceased. for each patient, 3 ml of initially aspired juice were discarded and the remaining amount was collected in a sterile tube, which was centrifuged to remove mucus and cell debris. in order to reduce inter-individual variations, batches of pooled gastric and intestinal juices were prepared and then stored at –20°c until use. the approval for the collection of digestive juices was obtained from the ethics committee of the university hospital centre split, croatia. ital. j. food sci., vol. 31, 2019 176 2.7. enzymatic activity of juices the procedure described by almaas et al. (2006) was used to determine enzymatic activity of the prepared pooled human gastric juice samples. pepsin activity was measured using 2.5% solution of bovine haemoglobin. the solution was prepared in 0.2 mol/l phosphate buffer (ph=7.6) and then acidified (to ph=3) using h2so4. in order to determine the human duodenal juice activity, casein solution (1%) dissolved in 0.2 mol/l phosphate buffer (ph=7.6) was used. a volume of 500 µl of prepared protein solutions was incubated with 5, 20 or 50 µl of gastrointestinal juice. the digestion reactions were stopped with the addition of 1 ml of 10% trichloroacetic acid (tca). samples were measured spectrophotometrically at 280 nm. one unit of enzyme activity (u) is defined as the amount of enzyme that causes the absorbance change of 1 between the blank and the sample, after 20 min at 37°c. 2.8. in vitro digestion a two-phase digestion procedure was performed according to the method described by furlund et al. (2013). gastric and intestinal digestion phases were performed at 37°c, in horizontal shaking bath (180 rpm). the volume of digestive juice corresponding to 1 u of enzymatic activity was 20 µl of human gastric juice and 25 µl of human duodenal juice. the ph of the samples was adjusted to ph=2.5 using 1 mol/l hcl for gastric phase, and to ph=7.5 using 2 mol/l naoh for intestinal phase. the concentration of human juices used for this assay was 20 u per g of plant material for gastric and 62.4 u per g of plant material for intestinal phase. a 0.6 g of both fresh and freeze-dried samples were used for digestion process. the incubation period of gastric phase was 30 min, while digested intestinal samples were collected after 120 min of intestinal phase. before spectrophotometric analyses, the digested samples were centrifuged before spectrophtometrical analyses by microcentrifuges myspin 12 (thermo scientific, usa) at room temperature, during 10 min at 9000 rpm. enzymatic reactions were stopped on ice and the samples were stored at –20°c until analyses. all digestion processes were run in duplicate. stability rate of lycopene and β-carotene represents the ratio of their concentrations before in vitro digestion and after gastric or intestinal digestion phases. samples were dissolved in n-hexane and according to uddin et al. (2009) and poojari et al. (2009) digestive enzymes retained their stability in non-polar solvents such as n-hexane. the gastrointestinal stability rate (%) of lycopene and β-carotene was calculated according to the following formula: (sample concentration after digestion/sample concentration before digestion) x 100 2.9. statistics statistical analysis was performed using graphpad instat3 software (graphpad software inc., san diego, ca, usa). the relationship between the obtained parameters was described using pearson’s correlation coefficient r. differences at p<0.05 were considered to be statistically significant ital. j. food sci., vol. 31, 2019 177 3. results and discussion the influence of digestion process on the carotenoids stability is not completely explored. in this study we explored the stability rate of carotenoids (lycopene and β-carotene) from raw and freeze-dried red carrot (daucus carota), tomato (solanum lycopersicum) and red pepper (capsicum annuum) after gastric and duodenal simulated digestive phase using human digestive juices. the analysis of the uv/vis spectrum of the obtained extracts showed strong peaks at 444, 471 and 502 nm. ftir spectral peaks, in the range of 3082 2835 cm-1 indicate the presence only of c-h bonds, ie. 3082 3011 cm-1 which correspond to c(sp2)-h bonds and 2965 2835 cm-1 to c(sp3)-h bonds stretching. peaks are observed at 1643 n (c=c, alkene), 1435 and 1375 d (ch2, ch3, bend). the quantities of carotenoids in the extracted fractions of raw freeze-dried samples were determined spectrophotometrically by previously described methods. although not the absorbance peak of greatest magnitude in hexane, the absorbance peak at 503 nm was used for lycopene determination in order to minimize interference from other carotenoids. if generally accepted, nominal carotenoid contents of red-fleshed watermelon, fresh red tomato, and pink grapefruit are utilized (holden et al., 1999) together with molar extinction coefficients at 503 nm in hexane for those carotenoids (zechmeister et al., 1943; zechmeister et al., 1943a), the potential error can be estimated if absorbance contributions by other carotenoids are ignored. such a calculation suggests that constituent carotenoids other than lycopene will contribute to the absorbance at 503 nm 0.2% for red-fleshed watermelon, 0.4% for fresh red tomatoes, and 0.6% for pink grapefruit (fish et al., 2002). previous reports of the major carotenoids detected in the investigated material showed that the carrot is a significant source of β carotene (bystricka et al., 2015), s. lycopersicum of lycopene (baranska et al., 2006), while the unique keto carotenoids of red pepper capsanthin, capsorubin and cryptocapsin impart brilliant red colour to ripen chilly pods, while the yellow orange colour is from β carotene, zeaxanthin, violaxanthin and β-cryptoxanthin (arimboor et al. 2015). results showed that the concentration of carotenoids (lycopene) in raw and freeze-dried samples ranged from 37.73 to 53.93 mg/kg and 61.09 to 61.92 mg/kg, respectively, with the highest one in red pepper extracts. on the other hand, the absorbance peak at 450 nm was used for β-carotene estimation in the investigated samples. the results showed that the carotenoid (β-carotene) concentration in raw and freeze-dried samples ranged from 15.14 to 27.92 mg/l, and 21.43 to 56.17 mg/l, respectively. the difference in the stability of carotenoids (lycopene and β-carotene) between raw and freeze-dried plant material as well as the difference in their gastrointestinal stability in relation to different plant matrix were detected. the high stability of carotenoids from freeze dried food rich with carotenoids is already reported by several authors (cinar, 2005; chen et al., 2007; vasque-caicedo et al., 2007). however, this is the first report on the gastrointestinal stability of carotenoids from fresh and dried samples. gastrointestinal stability of β-carotene and lycopene was evaluated using gastric and duodenal human juices. some authors reported that colonic microbiota can maximize the bio-accessibility of carotenoids by digestion of plant cell walls (djuric et al., 2017). results presented in table 1 show that the stability of carotenoids (lycopene) were significantly higher in red pepper than in carrot and tomato after simulated gastric phase. generally, lycopene stability after simulated gastric digestion was much higher in freezedried plant material than in raw plant material. the difference in lycopene gastric stability was lower in freeze-dried plant matrix (carrot, tomato and red pepper). interestingly, the stability rate of lycopene after simulated gastric digestion was extremely high in freezedried red pepper and tomato (96.04 and 92.09%, respectively). lycopene was not stable after simulated duodenal digestive phase in carrot and in tomato, or its stability ital. j. food sci., vol. 31, 2019 178 was very low (27.98% in raw red pepper). the use of freeze-drying significantly improved its duodenal stability. generally, lycopene stability in raw and in freeze-dried plant material was much lower after simulated duodenal phase in comparison with its stability after simulated gastric phase as shown in table 2. courraud et al. (2013) also reported high stability of carotenoids after simulated gastric incubation (in their study they used commercial digestive enzymes). table 1. stability rate of carotenoids (lycopene) from a) raw, and b) freeze-dried samples of carrot, tomato and red pepper after simulated gastric phase. sample carrot (daucus carota l.) tomato (solanum lycopersicum l.) red pepper (capsicum annum l.) a) concentration in raw sample [mg/kg] 37.73±0.12 45.48±0.20 53.93±0.77 concentration after gastric phase [mg/kg] 16.35±0.84 20.12±0.94 32.80±0.19 stability [%] 43.00±0.23 44.14±0.47 60.81±0.45 b) concentration in freeze-dried sample [mg/kg] 61.09±0.24 60.45±0.55 61.92±0.41 concentration after gastric phase [mg/kg] 52.26±0.12 52.54±0.90 59.47±0.34 stability [%] 86.91±0.87 92.09±0.55 96.04±0.63 table 2. stability rate of carotenoids (lycopene) from a) raw, and b) freeze-dried samples of carrot, tomato and red pepper after simulated duodenal phase. sample carrot (daucus carota l.) tomato (solanum lycopersicum l.) red pepper (capsicu annum l.) a) concentration in raw sample [mg/kg] 37.73±0.12 45.48±0.20 53.93±0.77 concentration after duodenal phase [mg/kg] / / 15.09±0.56 stability [%] / / 27.98±0.36 b) concentration in freeze-dried sample [mg/kg] 61.09±0.24 60.45±0.55 61.92±0.41 concentration after duodenal phase [mg/kg] 38.08±0.78 36.12±0.24 42.16±0.20 stability [%] 62.33±0.11 59.75±0.22 68.08±0.14 according to results presented in tables 3 and 4 the gastrointestinal stability of carotenoids (β-carotene) differs from that of lycopene. after duodenal digestive phase β carotene was not the most stable in red pepper, as it was the case for lycopene. in comparison with lycopene, β-carotene showed moderate stability rate after duodenal phase in both, raw and freeze-dried plant material, while lycopene was not stable in raw carrot and tomato after duodenal phase. interestingly, the stability rate of β-carotene significantly decreased after duodenal digestive phase in freeze-dried red pepper (table 4). ital. j. food sci., vol. 31, 2019 179 table 3. stability rate of carotenoids (β-carotene) from a) raw, and b) freeze-dried samples of carrot, tomato and red pepper after simulated gastric phase. sample carrot (daucus carota l.) tomato (solanum lycopersicum l.) red pepper (capsicum annum l.) a) concentration in raw sample [mg/kg] 15.14±0.35 21.54±0.40 27.92±0.24 concentration after gastric phase [mg/kg] 3.57±0.26 10.47±0.34 20.41±0.25 stability [%] 23.59±0.17 48.64±0.32 73.13±0.30 b) concentration in freeze-dried sample [mg/kg] 21.43±0.29 33.28±0.35 56.17±0.27 concentration after gastric phase [mg/kg] 6.42±0.45 18.89±0.29 51.84±0.18 stability [%] 30.00±0.28 56.77±0.38 92.30±0.45 table 4. stability rate of carotenoids (β-carotene) from a) raw, and b) freeze-dried samples of carrot, tomato and red pepper after simulated duodenal phase. sample carrot (daucus carota l.) tomato (solanum lycopersicum l.) red pepper (capsicum annum l.) a) concentration in raw sample [mg/kg] 15.14±0.35 21.54±0.40 27.92±0.24 concentration after duodenal phase [mg/kg] 5.38±0.36 12.80±0.29 13.54±0.19 stability [%] 35.59±0.22 59.45±0.26 48.50±0.34 b) concentration in freeze-dried sample [mg/kg] 21.43±0.29 33.28±0.35 56.17±0.27 concentration after duodenal phase [mg/kg] 11.42±0.26 22.93±0.40 11.38±0.24 stability [%] 53.33±0.20 68.93±0.24 20.26±0.36 in this study the influence of ph on carotenoids (lycopene and β-carotene) stability was explored (tables 5 and 6). as it is shown in table 5 the influence of ph on lycopene stability was stronger in raw than in freeze-dried plant material. generally, it can be seen that carotenoids (lycopene and β-carotene) were more stable at ph 2.5 than at ph 8.0. correlations between the stability of carotenoids in acidic and alkaline conditions were found to be significant (r=0.6926, p=0.0125) and extremely significant (r=0.9170, p<0.0001) for tables 5 and 6, respectively. freeze-drying technique significantly improve lycopene stability at low ph. interestingly, concerning the stability of β-carotene there is no significant difference between raw and freeze-dried plant material. ital. j. food sci., vol. 31, 2019 180 table 5. the influence of ph (gastric and duodenal) on the stability rate of carotenoids (lycopene) from a) raw, and b) freeze-dried samples of carrot, tomato and red pepper. sample carrot (daucus carota l.) tomato (solanum lycopersicum l.) red pepper (capsicum annum l.) a) concentration in raw sample [mg/kg] 37.73±0.12 45.48±0.20 53.93±0.77 concentration at ph 2.5 [mg/kg] 16.06±0.89 23.76±0.22 37.61±0.87 stability [%] 42.56±0.78 52.12±0.47 69.73±0.87 concentration at ph 8 [mg/kg] 10.70±0.78 8.51±0.32 31.32±0.56 stability [%] 27.21±0.51 18.67±0.23 58.07±0.45 b) concentration in freeze-dried sample [mg/kg] 61.09±0.24 60.45±0.55 61.92±0.41 concentration at ph 2.5 [mg/kg] 55.03±0.44 52.47±0.70 57.30±0.66 stability [%] 90.65±0.54 86.79±0.10 92.53±0.53 concentration at ph 8 [mg/kg] 8.44±0.20 39.30±0.24 38.52±0.30 stability [%] 13.81±0.12 65.01±0.17 62.20±0.78 table 6. the influence of ph (gastric and duodenal) on the stability rate of carotenoids (β-carotene) from a) raw, and b) freeze-dried samples of carrot, tomato and red pepper. sample carrot (daucus carota l.) tomato (solanum lycopersicum l.) red pepper (capsicum annum l.) a) concentration in raw sample [mg/kg] 15.14±0.35 21.54±0.40 27.92±0.24 concentration at ph 2.5 [mg/kg] 6.80±0.12 12.06±0.26 17.58±0.35 stability [%] 44.97±0.54 56.02±0.22 62.98±0.26 concentration at ph 8 [mg/kg] 4.41±0.29 4.54±0.30 16.75±0.28 stability [%] 29.13±0.18 21.12±0.22 60.02±0.56 b) concentration in freeze-dried sample [mg/kg] 21.43±0.29 33.28±0.35 56.17±0.27 concentration at ph 2.5 [mg/kg] 12.18±0.15 21.04±0.29 46.22±0.52 stability [%] 56.87±0.45 63.23±0.17 82.29±0.13 concentration at ph 8 [mg/kg] 5.38±0.18 7.74±0.42 33.93±0.25 stability [%] 25.12±0.20 23.27±0.09 60.42±0.15 4. conclusions results of this study showed that the use of freeze-drying greatly improved gastrointestinal stability of carotenoids (lycopene and β-carotene) from carrot, red pepper and tomato in comparison with raw plant material, especially after intestinal digestive phase. in addition, the effect of ph on the stability of carotenoids is lower in freeze-dried plant material. also, carotenoids stability depends on the food matrix (carotenoids were the most stable in red pepper). ital. j. food sci., vol. 31, 2019 181 this article contains a study with human digestive juices. the approval for the collection of digestive juices was obtained from the ethics committee of the university hospital centre split (11/09/2014). references alda l.m., gogoaşă i., bordean d-m. and niţă l. 2009. lycopene content of tomatoes and tomato products. j. agroaliment. proc. technol. 15(4):540-542. doi: doi.org/10.1016/s0963-9969(99)00053-8 almaas h., cases a.l., devold t.g., holm h. langsrud t., aabakken l., aadnoey t. and vegarud g.e. 2006. in vitro digestion of bovine and caprine milk 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lähteenmäki, 2013; perito et al., 2019). in particular, consumers seem more interested in environmental aspects associated with organic production, that have more direct benefits on health than other environmental issues (for example, the use in the production process of less water, fuel, workers’ rights, etc.,) (defrancesco et al., 2017; lähteenmäki, 2013). though in recent decades sustainable food consumption has been extensively studied (verain et al., 2012), it is no easy matter to assess the consumer preferences for environmental concerns, combined with other aspects (e.g., brand, origin, price, frequency of consumption, etc.). van dam and van trijp (2011) pointed out sustainability as an abstract verbal construct with no objective meaning. according to szolnoki (2013), it is still very difficult to define the term sustainability because not only each country, but also each entrepreneur and every individual has a different understanding of sustainability, especially in the wine sector. however, even if it is not feasible to have a unanimously accepted definition of sustainability, a general consensus is focused on a consumption pattern that covers a wide variety of aspects (e.g. environment, health and safety of the food products, rural and local development, …) (verain et al., 2012). taking in to consideration the wine sector, consumer perception of sustainable wines seems generally associated to the terms such as organic and local (zucca et al., 2009; cbi, 2016). the sustainable wine is traditionally considered as superior quality wine, due to the production without pesticides and fertilizers (considered harmful to health) in the vineyard (berghoef and dodds, 2013) and consumers are not willing to trade off this specific quality of a wine for only environmental/social features (lockshin and corsi, 2012). moreover, consumers pay attention to different issues, such as price, origin, and production process (gallenti et al., 2019). they seem interested in environmentally friendly matters, but only with organic labels (sogari et al., 2013; grankvist and biel, 2001; magnusson et al., 2003; defrancesco et al., 2017) and geographical indication labels (boncinelli et al., 2019; sáenz-navajas et al., 2013). as a matter of fact, sustainability aspects are credence attributes because consumers cannot determine by themselves if a wine is produced following sustainable practices (fernqvist and ekelund, 2014; ginon et al., 2014; schaufele and hamm, 2017) and the expectations that credence attributes have an effect on consumers’ perceived quality (fernqvist and ekelund, 2014; schaufele and hamm, 2017). wine is one of the most differentiated products on the market (schaufele and hamm, 2017) and the market relies strongly on how consumers perceive wine quality (loureiro, 2003). sustainability in the wine sector could play an important role in the wine business and it could be a way to both differentiate wines, to deal with food safety constraints and to guarantee the future development of the sector (sellers-rubio and nicolaugonzalbez, 2016; nait mohand et al., 2017). moreover, eco-labels may offer new attributes for consumers’ choices (delmas and lessem, 2017; klohr et al., 2013; schaufele and hamm, 2017). as empirical researches pointed out, consumers see vintages, wine producers and the region of origin as signs of quality (de francesco et al., 2012; gallenti et al., 2019; jaeger et al., 2013; yang and paladino, 2015; schaufele and hamm, 2017). in ital. j. food sci., vol. 32, 2020 224 particular, origin plays an important role in the european wine sector (loureiro, 2003; veale and quester, 2008). according to perrouty et al. (2006), geographical indication label gives added value to the product and affects both perception of wine quality and its process of purchase (pucci et al., 2016). italy is the first wine producer (european leader of wines certified with geographical indication or organic label1) and the second largest consumer country worldwide after france (nomisma, 2017). nevertheless, the italian wine market has been affected by a deep transformation due to new competitors on the world market (argentina, australia, chile, new zealand, the united states and south africa), a constant decrease in the quantities consumed, a lower willingness to pay due to the general economic crisis and a demand for new quality attributes (mainly sustainable and local products) by wine consumers (crescimanno and galati, 2014). in this framework, despite researches on wine appears to be copious in current literature (see e.g., dal vecchio et al., 2018; d’amico et al., 2016; di vita et al., 2014; fabbrizzi et al., 2017; marinelli et al., 2014; marone et al., 2017; sellers-rubio and nicolau-gonzalbez, 2016), to our knowledge, much is still to be studied about on drivers that can push consumers towards both local and sustainable attributes. specifically, this paper tries to fill a gap in the literature, with the purpose of understanding the consumers’ willingness to consume sustainable and local wine. specifically, we sought to answer three research questions: what interest do italian consumers have in sustainable wines? what interest do italian consumers have in local wines? which variables are important predictors of consumers’ willingness to buy these wines? to this purpose, the paper is articulated in three sections: sampling procedure and methodological approach, results, discussion and final remarks. 2. materials and methods 2.1. the questionnaire in order to explore the drivers of the consumers’ inclination to consume sustainable and local wine, a structured survey was developed. consumers were recruited through invitations to participate in the online survey (performed by google drive) via social networks (twitter, facebook and whatsapp), university students’ networks and email (see e.g. sogari et al., 2015). snowball sampling recruitment was also adopted, using interpersonal relations and connections among university students to reach a large number of participants. data were collected using an online survey between january and july 2015. following general studies on food science (e.g. verbeke, 2015), in the questionnaire the meaning of sustainable and organic wine used in our survey was explained to respondents. the online questionnaire2 consisted of two separate sections. the first section collected basic socio-demographic information: gender, age, area and region of residence and education level of respondents. the second section gathered information on consumption frequencies of wine, type of wine preferred and consumption frequencies of wine out of home. to understand the feelings regarding sustainable wine, according to the literature (d’amico et al., 2016; sogari et al., 2016; lockshin and corsi, 2012; forbes et al., 2009) the respondents ital. j. food sci., vol. 32, 2020 225 were asked if an organic label is a guarantee of sustainability of higher quality product, naturalness and food safety with respects the environment. moreover, it has been asked the willingness to consume sustainable and local wine and the attention paid to wine cellar brand. in addition, to understand the respondent’s perceptions concerning local origin of wine, we asked if they thought local origin of wine is a guarantee of territoriality and origin of raw materials (perito et al., 2019). the last questions collected information on consumer interest towards producer name and nutrition label. it is important to underline that following pomarici and vecchio (2014), most questions of the survey were based on the formula yes/no. 2.2. the sample and data analysis we surveyed a sample of 1,099 wine consumers in italy. the questionnaire was validated in a pilot study on a sample of 100 consumers. this pre-test was developed exclusively to discover any possible misinterpretation, error or duplication in the questionnaire. to explore the drivers of people’s inclination to consume sustainable and local wine, and following the current literature (e.g. pomarici and vecchio, 2014; sellers, 2016; verbeke, 2015) a binary logistic regression model was used. the binary dependent variable yi takes the values “yes” and “no” and the probability of success p(y= yes|x) represents the probability that an individual is willing to consume sustainable and local wine conditioned by variables of the questionnaire. in a first analysis, all potential explanatory variables were included in the logistic model. many estimated coefficients are associated to non-significant p-values and the relative variables are excluded from the final model. the final model parameterization is selected on the basis of the model aic, using a mixed “backward” and “forward” stepwise selection strategy. in formula, the final model is: p(y=yes׀x) =β0 + β1wine + β2 wine_out + β3 lab_org + β4 wine_company_name + β5 local + β8 market_name + β9 nutr (1) among the parameters of the model, we looked at the β values and at the significance of each factor. the factors were then interpreted according to the odds’ ratio, which shows the probability increase/decrease of the wiliness to consume sustainable and local wine when the considered variable increases or decreases. all computations are carried out using r version 3.5.1 (r development core team, 2018). 3. results and discussion 3.1. descriptive results the sample consists of 564 females and 535 males and 74.4% of people belong to age range 18-45 while, 46.2% of respondents are young (18-35 years) (table 1). it is important to highlight that the sample analysed in this study is not representative of the whole national population. about 85% of the respondents come from urban area and 50.41% has a high level of education (i.e. university college or postgraduate degrees). ital. j. food sci., vol. 32, 2020 226 table 1. socio-demographic information of sample (n = 1,099). variable label % gender male gen_m 48.69 female gen_f 51.31 total 100.00 age ranges 18-25 years 12.83 26-35 years 33.39 36-45 years 29.21 46-55 years 16.20 56-65 years 6.92 65 + 1.46 total 100.00 area of residence urban area urb 84.99 rural area rural 15.01 total 100.00 education level primary or secondary school low 49.59 university or postgraduate degree high 50.41 total 100.00 *source: our elaboration on data survey. the findings show that 88.44% of the sample is inclined to consume organic and local wine and 43.13% of respondents consume wine 2 o 3 times a week. 56.96% prefer red wine and 55.14% of our sample think that the organic label (lab_org) is a guarantee of sustainability and a higher quality product. . moreover, 51.41% of respondents do not think that the local origin of wine (local) is a guarantee of territoriality and origin of raw materials. most respondents do not pay attention to neither wine cellar brand or to producer name. about 51.0% pay attention to nutrition labels when buying wine. descriptive statistics of the sample are presented in table 2. table 2. descriptive statistics of sample (n = 1,099). variable label % readiness to consume sustainable and local wine no y 11.56 yes 88.44 total 100.00 frequency of consumption of wine in terms of times a week or a month wine never 0.00 ital. j. food sci., vol. 32, 2020 227 sometimes a year 1.82 once a month 4.19 2 o 3 times a month 11.46 once a week 35.67 2 o 3 times a week 43.13 every day 3.73 total 100.00 kind of wine red 56.96 white 38.31 rosè 4.73 total 100.00 frequency of consumption of wine in restaurants, wine bar, etc, in terms of times a week or a month wine_out never 4.73 sometimes a year 18.38 once a month 17.93 2 o 3 times a month 19.65 once a week 18.56 2 o 3 times a week; 18.38 every day 2.37 total 100.00 do you think that sustainable/organic label is a guarantee of high quality product, naturalness of food, food safety respecting at the same time the environment? lab_org no 44.86 yes 55.14 total 100.00 when you buy wine, do you pay attention to wine cellar brand? wine_company_ name no 53.96 yes 46.04 total 100.00 do you think that local origin of wine is a guarantee of the origin of raw materials supporting at the same time local economies and the culture of landscape? local no 51.41 yes 48.59 total 100.00 when you buy wine, do you pay attention to producer name? maker name no 67.88 yes 32.12 total 100.00 when you buy wine, do you pay attention to nutrition label? nutr no 49.04 yes 50.96 total 100.00 source: own elaboration on survey data. ital. j. food sci., vol. 32, 2020 228 3.2. model results table 3 shows the results of the binary logistic regression model with the estimated coefficients (β), their standard errors (s.e.), wald x2-statistics, significance levels, odds ratios (exp(β)) and goodness-of-fit statistics. the goodness of fit as measured by mcfadden’s pseudo-r² is equal to 0.32. relatively low value of r2 is expected for models based on samples with large number of observations (gujarati, 2004), as it is happened in our case (1,099 observations). multi-collinearity was not a major issue in the model as it was tested through variance inflation factors (vif) (the highest value is equal to 1.19). table 3. parameters of the logistic regression (n = 1,099). variable β s.e. wald sig. exp (β) intercept -3.097 0.585 27.977 1.23 e-07 0.045 wine 0.542 0.109 24.596 7.07 e-07 1.719 wine_out 0.306 0.085 12.825 0.000342 1.358 lab_org 2.709 0.295 84.221 2 e-16 15.018 wine_company_name 1.883 0.264 50.554 1.16 e-12 6.576 local 0.971 0.248 15.295 9.19 e-05 2.641 maker name 0.430 0.271 2.509 0.113 2.509 nutr -0.381 0.231 2.705 0.099 2.705 aic: 553.24 pseudo-r2 = 0.32 -2log likelihood statistic = 533.24 source: our elaboration on survey data. our findings show that consumers who drink wine frequently (wine) and consume it even outside home (wine_out) are more likely to consume sustainable and local wine, 1.71 times and 1.35 times (respectively), than other people. therefore, the “wine consume frequency” exerted a positive effect on willingness to consume sustainable and local wine. moreover, the lab_org variable is the most influential factor in the model (wald x2 = 84.2) followed by wine_company_name variable with wald x2=50.5. this last variable (wine_company_name) is referred to the attention that respondent could have to winery’s brand when he/she buys wine. participants who indicated organic label (lab_org) as guarantee of sustainable product and other aspects (high quality product, naturalness of food, food safety and environmental feeling) are 15.0 times more likely to consume sustainable and local wine than other consumers. in our sample people that pay attention to wine company name (wine_company_name) are 6.57 times more likely to be ready to consume sustainable and local wine than other consumers. it is generally well known by the literature that producer and brand name should interest more consumers when they buy a bottle of wine (pomarici et al., 2017). in addition, participants who indicated local product (local) as guarantee of quality are 2.6 times more likely to be ready to consume sustainable wine than other consumers. the results are similar to veale and quester’s (2008) study where origin of product influences consumers’ wine quality assessment. ital. j. food sci., vol. 32, 2020 229 3.3. discussion our results showed a strong interest in respondents towards sustainable and local wine and highlight some important marketing implications for the italian wine companies. in fact, the findings suggest that producing and marketing wine with sustainability and local characteristics are promising strategies for quality differentiation (schaufele and hamm, 2017) and that could allow catching new niches of italian wine market. another interesting result that emerges from current study is that organic label and local origin variables go towards in the same direction in our model, confirming that, in according to loureiro (2003), if the perceived quality of region (local) is negative, wine organic label is not a useful marketing strategy and the people are unwillingness to consume sustainable and local wine. in accordance with sogari et al. (2015) our results suggest that consumers who perceive sustainable certification as a guarantee of high quality standards have a more positive attitude towards such wine. in fact, consumers may interpret an organic label as a mark of quality (boncinelli et al., 2019) because an organic certification is indicative of positive health effects (mann et al., 2012), environmental benefits (mueller loose and remaud, 2013), and great taste (boncinelli et al., 2019; kim and bonn, 2015). sustainable labelling certification could be an effective strategy to provide consumers with accurate, understandable and trustworthy information to encourage them to buy sustainable wines (ginon et al., 2014). moreover, it is interesting underline that the consumers of our sample inferred that a product is environmentally sustainable in the presence of logos (golan et al., 2001). in fact, organic labels are the most influential factor in our model, followed by winery’s brand and local label. also local labels play an important role in differentiating wines because, when the consumers do not know the winery’s brand, they base their choice on labelling of origin as a quality standard (loureiro, 2003). in this situation, the consumer relies on the image of the region (negative or positive) that guarantees and promotes that particular label. (loureiro, 2003) our results emphasized the need to create eco-labels that communicate clearly both the environmental attributes of wine (delmas and lessem, 2017) and the benefits associated with them. even if our sample is not representative of the whole italian population, the findings could be useful points on which to reflect if we consider that recently the wine market in italy has been affected by a deep transformation due to both growing international competition and changing of the internal demand. this last aspect is due to new orientation of consumers towards attributes of quality wines (crescimanno and galati, 2014). for these reasons, our findings could be useful for italian wine producers that want to enter in the sustainable wine market niche. in particular, italian wine producers could address their marketing strategies towards an adult who drinks wine frequently and preferably in restaurants, pubs and bars, he/she is environmentally friendly and more interested in the local origin of his/her food choices than other consumers. in this context, wine producers should focus on the communication of their environmental commitment through appropriate marketing tools (pomarici et al., 2016) that help consumers to identify sustainable products (pomarici and vecchio, 2014). in particular, marketers should focus on wine label information (i.e. organic, local and brand) available when consumers inspect the bottle in order to drive the consumers’ inclination to consume sustainable and local wine. ital. j. food sci., vol. 32, 2020 230 4. conclusions this paper has to be considered as an attempt to contribute to the current literature on the willingness to consume sustainable and local wine by italian consumers and on the main factors that might affect the readiness of consumers. profiling consumers who are willing to consume sustainable and local wine could be a first step towards a better understanding of consumers’ decisions on sustainable and local food. our findings indicated that the large majority of the interviewed italian consumers are willing to consume sustainable and local wine. the sustainable and local wine consumer of our survey could be described as an adult, who regularly consumes wine, even outside home, more label-conscious and environmentally friendly. our findings are even more important if we consider that the italian wine market (as world market one) has been affected by a deep transformation due to also new orientation of consumers towards attributes of quality wines. in particular, our results emphasize the need to have sustainable labelling that communicates clearly both the environmental attributes of wine and the benefits associated with them. moreover, our results might be useful to drive marketing strategies because wineries could benefit by promoting these wines as organic and local. our findings suggest that wine label information (organic, local and brand) might be a useful tool to promote sustainable and local wine and at the same time help to give a positive perception of the whole italian wine sector. however, the present study shows some limitations that could be considered and addressed in future research. first of all, the sample is not representative of the italian population and it suffers from a smaller degree of self-selection. in particular, the questionnaire, to reach the broadest population, was not directed towards specific targets (e.g., university students) and the survey itself is not based on explicit selection mechanism. an additional limitation is that we opted for verbal descriptors to identify the wine attributes, which might mimic a real market in a less realistic way. to avoid this limitation, further studies should simulate real shopping environments where the choice sets are designed with visual labelling elements, such as images or symbols to increase the accuracy of the results. notes 1italy is the largest producer of wine with protected designation of origin (pdo) 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enological eligibility of grape clones based on the simca method: the case of the sangiovese cultivar from tuscany v. canuti*1, s. puccioni2, p. storchi2, b. zanoni1, m. picchi1, and m. bertuccioli1 1gesaaf, department of agriculture, food and forestry systems, university of florence, via donizetti 6, 50144, florence, italy 2crea, council for research in agriculture and agricultural economy analysis, viale santa margherita 80, 52100, arezzo, italy *corresponding author. tel.: +39 0552755517, fax +39 0552755500 e-mail address: valentina.canuti@unifi.it abstract sangiovese is the most widespread italian red grape cultivar and it constitutes the basis of internationally known wines. it has a large diversity of clones whose performances vary with environmental conditions due to interaction with the weather and soil. in this study, the performance of grapes from seven sangiovese clones was evaluated by analyzing grapes from four vineyards in the chianti classico region in tuscany over the ripening period. in order to assess the enological eligibility of grape clones, a grape reference model was developed using chemical parameters from commercially available sangiovese wines, by performing a soft independent modeling of class analogy (simca). keywords: anthocyanin profile, clone plasticity, ripening, sangiovese, simca, wine quality ital. j. food sci., vol. 30, 2018 185 1. introduction sangiovese is the most widespread italian red cultivar and, according to the last agricultural census of the ministry of agricultural, food, and forestry policies (http://catalogoviti.politicheagricole.it), the total area planted with sangiovese was 69787 ha, equivalent to 10.3% of the total area of vineyards in italy. 47% of these vineyards are in tuscany, producing 92.5% of the sangiovese world output. sangiovese constitutes the basis of internationally known wines such as chianti, brunello di montalcino, nobile di montepulciano, and, furthermore, its use is allowed in the production of 11 docg (denominazione di origine controllata e garantita), 103 doc (denominazione di origine controllata) and 99 igt (indicazione geografica tipica) wines all over italy. the selective pressure carried out by mankind over the centuries in different growing conditions has induced the diversification of sangiovese into many clones (campostrini et al., 1995). nowadays, 116 clones of sangiovese are listed in the italian national registry of grapevine varieties. clone performances vary with environmental conditions according to the interaction between the grapevine, weather and soil (barbeau et al., 1999) and they can influence wine quality and typicality. some authors (tonietto and carbonneau, 2004) consider that climate is the most dominant factor in determining grape quality and that is responsible for the terroir effect. a few studies have been conducted on the physiological and productive response of sangiovese clones to environmental and pedoclimatic factors. on the other hand, while some authors have focused on the relationship between the sangiovese grape composition and different grapevine growth conditions, they only monitored standard parameters such as ph, titratable acidity, and sugar content (di collalto et al., 2000). however, some studies on other cultivars, such as tempranillo, pinot noir, merlot, and cabernet sauvignon, have shown that, within the same grape variety, different clones can be distinguished by comparing their field performances including yield components (number and weight of clusters and berries, pruning weight) and the chemical compositions of grapes including anthocyanin content (revilla et al., 2009, fidelibus et al., 2006, fidelibus et al., 2007, castagnoli et al., 2006, arozarena et al., 2002, ranković-vasić et al., 2015) and anthocyanin profiles (guidoni et al. 2002, ryan and revilla, 2003, downey et al., 2006, gonzález-neves et al., 2004, ortegaregules et al., 2006). these studies demonstrated that climate and soil are crucial factors in determining the final composition of grapes. however, it is not easy to predict how these changes could affect the final characteristics of wines. integrated approaches can identify the relationship between grape chemical features, wine chemical composition, and wine sensory attributes, in order to predict wine flavor from grape composition and provide a practical tool for guiding the winemaking process (zanoni et al., 2010, forde et al., 2011). many studies (cadot et al., 2012, kontoudakis et al., 2011, koundouras et al., 2006, ristic et al., 2010) have proposed different methodological approaches to measure the qualitative characteristics of grapes and the related wines, but a complete methodological approach to assess the eligibility of grapes in order to obtain a wine with well-defined characteristics has not been taken into account by these authors. a recent study proposed a multivariate statistical approach to relate grape features, and enological and agronomical practices with the composition of wines, in order to provide a practical tool to manage the vineyard and the winemaking process and preserve or enhance wine typicality (canuti et al., 2017). the aim of this study was to create a tool capable of discriminating grapes, depending on their chemical composition, according to a given enological objective. for this purpose, a ital. j. food sci., vol. 30, 2018 186 grape eligibility model (gem) was developed by computing the chemical parameters of commercially available sangiovese wines, grapes, and the relevant wines from previous experiments by performing a soft independent modeling of class analogy (simca). the model was then applied to assess the enological eligibility of seven sangiovese clones grown in four vineyards in different growing areas of the chianti classico region in tuscany. 2. materials and methods 2.1. grape samples the present study was carried out using seven vitis vinifera cv. sangiovese clones listed in the italian national registry of grapevine varieties as reported in table 1. table 1. sangiovese clones studied for performance evaluation. registered clones code area of origin ss-f9-a5-48 f9 lamole (florence tuscany, italy) rauscedo 24 r24 predappio (forlì cesena emilia romagna, italy) montalcino 42 m42 montalcino (siena tuscany, italy) ap-sg-1 ap1 cossignano (ascoli piceno marche, italy) peccioli 1 pec peccioli (pisa tuscany, italy) rauscedo 10 r10 lamole (florence tuscany, italy) sg-12t 12t predappio (forlì cesena emilia romagna, italy) the vineyards were located in four different estates in the chianti classico docg area in tuscany. these were situated in the provinces of florence (panzano and greve in chianti) and siena (castellina in chianti and castelnuovo in berardenga) and coded with the letters a, b, c, and d respectively. each vineyard was planted in 1990 and has an average surface area of 1.7 hectares. the vines were spaced 2.8 m (between rows) × 1.0 m (between vines in the row), resulting in a density of 3571 vines/ha, in a randomized block design with a minimum of three replicates. each clone was grafted on to 420 a (vitis berlandieri x vitis riparia) rootstock. the vines, with a permanent unilateral cordon, were spur-pruned and trained to a vertical shoot-position. the same soil management techniques were applied in all the vineyards, in which the rows were alternately covered with a spontaneous permanent grass and tilled. no irrigation system was installed. the meteorological indices, based on the data recorded by the nearest climate stations to the experimental vineyards and the characteristics of the soils, are available at the reader's request. grapes were harvested for sampling at three different ripening stages during the 2005 vintage (september 10th, 20th and 30th). in 2005, rain events were quite intense at the beginning of september, as indicated by the data collected at the local meteorological stations. on each sampling date, 2 kg-samples of grapes were collected from portions of different clusters picked randomly from all the randomized blocks and analyzed. ital. j. food sci., vol. 30, 2018 187 2.2. chemicals the acetonitrile and o-phosphoric acid (hplc grade) were purchased from panreac (barcelona, spain). the malvidin-3-monoglucoside (m3mg) (hplc grade) was purchased from extrasynthèse (genay, france). all of the other chemicals were of the highest purity available and were purchased from sigma-aldrich (milan, italy). 2.3. instrumentation the hplc analyses were carried out on a perkin-elmer 200 lc series equipped with an autosampler and a diode-array detector (perkin elmer, shelton, ct, usa). the ultravioletvisible (uv/vis) absorbance of the samples was measured on a perkin elmer lambda 35 uv/vis spectrophotometer (perkin elmer, shelton, ct, usa). 2.4. grape analysis the technological ripeness of the grapes was measured following official oiv methods (compendium of international methods of analysis – oiv – oeno 21/2004). two hundred berries were pressed to extract their juice. the juice sugar contents (brix), titratable acidity (g/l), and ph were measured after centrifugation of the juice at 3000 rpm for 10 min. the berry weight was determined as the ratio between the total weight and the number of berries. phenolic maturity was measured as described by saint-criq et al. (1998). the anthocyanin profiles (expressed as relative abundance of the different anthocyanins) of the same grape extracts at ph 3.2 were determined by hplc, according to a previously published method (peng et al., 2002) used to determine the phenolic maturity by acquiring a chromatogram at 520 nm. at the same time, the tannin contents (expressed as peak height) were determined by acquiring a chromatogram at 280 nm, as described in canuti et al. (2012). chromatograms were acquired, recorded, and processed using total chrome navigator software (perkin elmer). 2.5. analysis of commercial and experimental wines standard parameters were measured in the wines following official eu methods (official methods of wine analysis, reg. 440/2003). color intensity (ci) and hue (hue) were measured according to glories (1984) and the total phenols index (tpi) was measured as described by ribereau-gayon (1970). monomer anthocyanin contents, expressed as mg/l of malvidin-3-monoglucoside (m3mg), colored polymeric pigments (cpp) expressed as mg/l of m3mg, and tannins (expressed as peak height) were determined by hplc (peng et al., 2002). chromatograms were acquired at 520 and 280 nm respectively, using the same hplc parameters reported in the grape analysis section. 2.6. statistical analysis the analysis of variance (anova) was performed using statgraphics centurion (ver. xv, statpoint technologies, warrenton, va), considering clones, growing area, and sampling date as factors. principal component analysis (pca) and the soft independent modelling of class analogy (simca) were performed using unscrambler (v10.3, camo process as, oslo, norway). the simca analysis enables the assessment of which factors are decisive in determining the classification. indeed, in this classification method, each class is described by an ital. j. food sci., vol. 30, 2018 188 independent principal component analysis model. new samples are classified on the basis of their fit with the different pca models. the optimal number of pcs for each model is chosen independently since the classes may exhibit different shapes and structures. for new samples the residuals and scores are calculated for each pca model. the residuals provide information on the ability of each model to describe the new data, like a sort of object-to-model distance, while the scores can be combined to measure the distance between the object and the model center. 2.7. grape quality model and evaluation of clone performance a model evaluating the grape quality, and the consequent performance of the clones, was built in order to provide an objective tool to assess the grapes’ eligibility for winemaking. the classification method (simca) consisted of describing each class of samples (wines and grapes), identified by their chemical composition, in independent principal component analysis (pca) models. grape and wine samples were classified on the basis of their membership limit within the different pca models (wold and sjostrom, 1977). in particular, the methodology to build the model and evaluate the clone performances consisted of three phases as follows: phase 1 – sangiovese wine eligibility model (wem) in phase 1, a sangiovese wine eligibility model was built which described the chianti classico region wines. the aim of this phase was to establish a definition of sangiovese wines from the region. for this purpose, 37 commercial chianti classico docg wines (coded with numbers from 101 to 137) were analyzed to determine their alcohol contents, titratable acidity, ph, total phenol index, total anthocyanins, tannins, color intensity, and hue. later, a global pca was run considering all the parameters for the 37 commercial wines. all the wines used to build the wem were produced with 100% sangiovese grapes without oak contact and analyzed one year after the harvest. phase 2 – sangiovese grape eligibility model (gem) the aim of phase 2 was to create a grape eligibility model (gem) starting from the wem built in phase 1. for this purpose, 30 sangiovese grape samples from the chianti classico region were analyzed to determine their sugar content, ph, titratable acidity, total and extractable anthocyanins, cellular maturity index, phenolic richness, and tannins. the grapes were then vinified, on an industrial scale, to obtain 30 experimental sangiovese wines (coded with numbers from 1 to 30) that were analyzed, after aging for one year, to determine their alcohol contents, titratable acidity, ph, total phenol index, total anthocyanins, tannins, color intensity, and hue. finally, the chemical composition parameters of 30 sangiovese grape samples and the related experimental wines were statistically analyzed in order to correlate the grapes to wine composition. a global pca of the experimental wines was performed and the resulting model was compared with a simca analysis to assess which wines fitted the wem. the grape samples whose wines fitted the wem obtained in phase 1 were used to create the grape eligibility model (gem) by running a global pca which considered all of the grape parameters. ital. j. food sci., vol. 30, 2018 189 phase 3 – evaluation of clone performance in order to evaluate the clones’ performances, the grape samples were classified according to their composition using a simca analysis to assess which grapes fitted the gem. outliers were detected during the exploratory analysis by calculating the hotelling’s t2 distance diagnostic tool for each class of samples. 3. results 3.1. grape clone characteristics the data collected from grape sample analyses at three different ripening stages were statistically analyzed and the results are reported in table 2. table 2. f-values and interactions for chemical parameters analyzed in the grape samples. variable sd c ga c x ga ga x sd c x sd sugar 13.97*** 2.86* 70.43*** 4.46*** 4.25*** 3.79*** ph 15.45*** 5.65*** 21.08*** 1.23 ns 1.48 ns 1.53 ns titratable acidity 65.61*** 14.35*** 112.52*** 6.43*** 4.11*** 9.24*** berry weight 4.02* 13.78*** 93.69*** 11.52*** 5.76*** 5.01*** total potential in anthocyanins 14.37*** 23.41*** 54.05*** 13.06*** 13.57*** 5.00*** extractable anthocyanins 10.46*** 20.47*** 70.42*** 10.50*** 9.89*** 5.21*** cellular maturity index 22.66*** 6.09*** 18.27*** 7.10*** 6.65*** 6.26*** phenolic richness 6.48** 5.15*** 7.67*** 2.87*** 2.58* 2.08* di/tri substituted anthocyanins ratio 21.82*** 15.93*** 92.03*** 27.48*** 7.90*** 2.07* tannins 5.05* 46.30*** 186.47*** 46.18*** 4.48*** 5.39*** *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.001; ns, not significant; c, clone; ga, growing area; sd, sampling date. all the variables measured have been significantly affected by the factors clone (c), growing area (ga), sampling date (sd) factors and their interactions, with the exception of ph (table 2). furthermore, the anova results indicate that ga was the most influential factor; in fact, the f-values of ga were the highest for all the considered grape parameters. the c factor was found to be a determining factor for the concentrations of phenolic compounds (tannins, potential and total anthocyanins). as expected, the sd factor did not only influence the cellular maturity index but also the quality of the anthocyanins, modifying the di-substituted and tri-substituted pigment ratios. with regard to the interactions between the various factors, the results suggest that the interaction between clone and growing area (c x ga) was the most significant, in relation to tannin contents (f = 46.18) and the di/tri index (f = 27.48). the mean plots for the standard and phenolic parameters of the grape samples are shown in figs. 1 and 2, respectively. the berry weight (bw) provides a qualitative and quantitative indication about the berry size and the skin to pulp ratio. the most important factor that influenced bw (table 2) was ga, which showed the highest f value (93.69). according to fig. 1, bw remained fairly constant during the ripening stage, indicating that the sd factor had less influence on bw (f – value = 4.02) if compared to the ga and c factors which had a higher statistically ital. j. food sci., vol. 30, 2018 190 significant influence (table 2). in particular, clone f09 showed a higher bw in comparison to clone 12t, which produced smaller berries. growing areas a and d seemed to stimulate the growth of the berry more than growing area b. clone r24 had the highest berry weight variability, producing the lightest berries (1.24 g) in area b and the heaviest (2.37 g) in d. the clone pec stood out in area c with an average weight of 2.35 g. clones f09, ap1, and r10 produced grapes with a more constant berry weight in all the growing areas. figure 1. mean plots of standard parameters of grape samples (sugar, ph, titratable acidity, berry weight, and cellular maturity index); significance at 95% confidence level according to fisher’s least significant difference (lsd) procedure. bars represent lsd; total number of grape samples for each plot = 252. all of the factors had a statistically significant influence in relation to the grapes’ sugar content, and ga was the one with the greatest effect (f-value = 70.43). there were also significant effects when considering the interactions between factors. for example, fig. 1 ital. j. food sci., vol. 30, 2018 191 shows that the sugar content increased, as expected, during ripening, with clone r24 having the highest sugar content, and growing area b promoting the accumulation of sugars. the sugar content of the grapes produced by clones 12t and ap1 showed the most unstable and extreme values, which varied between 25.0 brix in b and 20.5 in the other zones. r10 showed constant concentrations (21.3 brix) in all the growing areas. figure 2. mean plots of phenolic parameters of grape samples (total potential in anthocyanins, extractable anthocyanins, phenolic richness, di/tri substituted anthocyanins ratio, and tannins); significance at 95% confidence level according to fisher’s least significant difference (lsd) procedure. bars represent lsd; total number of grape samples for each plot = 252. concerning the titratable acidity and ph (table 2), the most determining factor was again ga (f value = 112.52 and 21.58 respectively). moreover, the titratable acidity decreased as the ph increased at the three different sampling dates (f = 65.61). clone ap1 showed the highest ph and clones 12t and r10 the highest titratable acidity (fig. 1). ital. j. food sci., vol. 30, 2018 192 growing area b produced grapes with the highest ph and the lowest titratable acidity in comparison to the other gas. the grapes grown in area a showed the highest titratable acidity and the lowest ph. the titratable acidity was similar for all the clones in growing area b, while the results showed a high variability in the other zones. f09 reached the lowest value of titratable acidity (6.65 g/l) in area d. clone r10 displayed the highest values in zones a (8.59 g/l) and c (8.08 g/l). moreover, in growing area a, clones 12t (8.72 g/l) and r24 (8.57 g/l) stood out for their high titratable acidity. the cellular maturity index (ea%) measures the berries’ ability to release anthocyanins: the lower the ea index value, the higher the extractable potential of the anthocyanins (saint criq et al., 1998). the evolution of this factor at the different sds (fig. 1) highlighted a progressive cellular maturity that should result in a better release of the phenolic compounds during winemaking. statistical analysis (table 2) pointed out that ea% mostly depended on the sd (f = 22.66) and ga factors (f = 18.27) and that growing areas a and b promoted the ripening of the grape skin cells the most. moreover, clone 12t showed the highest ea% (fig. 1). the cellular maturity index was stable for clone pec (49) while it was inconstant for f09 (38 in b and 51 in c) and m42 (40 in a and 56 in c). the ea% was the highest for all the clones in growing area d. during the ripening of the grapes, there was a constant and significant increase in phenolic richness and total potential in anthocyanins from the first to the second sd and a significant decrease at the third sd (fig. 2). instead, the extractable anthocyanins increased over time, reaching the maximum at the second sampling date and remaining constant between the second and third sds. the tannin contents remained constant throughout the ripening stages; this result could indicate that the increase in the total amount of phenolic compounds was due to the accumulation of monomeric phenols (fig. 2). however, clones pec and r24 showed the highest tannin content, and there were significant differences induced by the ga factor, with growing areas a and d having the lowest values and areas b and c showing the highest value for tannin contents. the largest differences in phenolic composition between the grapes emerged when considering ga as the main factor. in fact, the content of total potential (f = 54.05), extractable anthocyanins (f = 70.42) and tannins (f = 186.47) was higher in zone b and lower in all the other growing areas. moreover, the differences in the levels of phenolic richness were less remarkable but still significant for growing areas b, c, and d, which showed higher values than area a. upon examining the chemical profile of the different sangiovese clones, it is clear that there were differences among them due to the content of total anthocyanins but not at the level of phenolic richness. clone r24 showed a different behavior when compared to all the other clones, resulting in significantly higher contents of total potential and extractable anthocyanins and tannins. regarding the clone x growing area interactions, clone f09 always showed similar levels of phenolic richness (54), while r24, with values that ranged between 47 in a and 56 in d, was the clone most influenced by the ga factor. the tannin content was similar for all the clones in areas a and b; a higher variability was observed in the other zones where clones pec, r24 (in b), and f09 (in c) stood out with higher values. the anthocyanin content, both potential and extractable, showed the highest variability in zone b where clones 12t (1879 and 946 mg/kg) and r24 (2011 and 983 mg/kg) stood out with the highest values. in area b, the values were higher for all the clones with the exception of f09 (1043 and 642 mg/kg) which, on the contrary, was the richest in anthocyanins in growing area c (1609 and 744 mg/kg). different considerations should be made regarding the ratio between the diand trisubstituted anthocyanins (di/tri) and the anthocyanin profiles of the different clones. ital. j. food sci., vol. 30, 2018 193 clone r24 showed the highest di/tri value and growing areas a and b showed larger levels of di/tri in comparison to areas c and d (fig. 2). regarding the relative abundance of anthocyanins, it was seen that the pigments were mainly represented by delphinidin-3-o-glucoside (del), cyanidin-3-o-glucoside (cya), peonidin-3-o-glucoside (peo), petunidin-3-o-glucoside (pet), and malvidin-3-oglucoside (mal). in agreement with results reported elsewhere (mattivi et al., 2006, arapitsas et al., 2012), the acylated anthocyanins were found in very low amounts (sum of total relative area below 2% of the total anthocyanin content, data not shown). due to the low amounts of acylated pigments in the sangiovese cultivar, only glucoside anthocyanins were taken into consideration for the statistical analysis (del, cya, peo, pet and mal). the variability in di/tri levels, according to the multifactor anova, was affected by all the factors taken into consideration (table 2). all the clones had similar di/tri values with the exception of clone r24 (f = 15.93), which showed a higher value (fig. 2). small variations occurred during the ripening period (f = 21.82). in this case too, the growing area was the most influential factor on this parameter (f = 92.03), with areas a and b showing the highest values of the index. the di/tri ratio was similar for all the clones in growing areas a and c while in area b the values varied between 1.37 for clone r24 and 0.49 for r10. upon analyzing the anthocyanin profile (table 3), mal resulted the major component of the anthocyanin pool present in the sangiovese grapes from all the different clones; while del was the smallest component. table 3. grape clone anthocyanin percentages and significance1. anthocyanins factors del cya pet peo mal σ tri-sub clone 12t 9.5c 20.5a 13.3cd 16.9b 39.7bc 62.5ab ap1 9.1b 19.5a 12.8b 19.8d 38.7b 60.6ab f09 6.8a 19.9a 10.4a 18.6c 44.1d 61.3ab m42 9.7c 19.5a 13.4d 16.5b 41.0c 64.1b pec 9.4c 23.7b 13.7d 14.8a 38.1b 61.2ab r10 10.2d 19.9a 14.1c 14.3a 41.2c 65.5b r24 10.3d 24.5a 12.9bc 17.4b 34.6a 57.8a growing area a 9.9c 24.2c 13.7b 16.2b 35.8a 59.4a b 8.9b 24.7c 12.3a 18.7d 35.2a 56.4a c 9.9c 16.6a 13.8b 15.2a 44.4b 68.1c d 8.5a 18.9b 12.0a 17.5c 43.0b 63.5b sampling date 9/10 9.4a 19.6a 13.0a 16.5a 41.3c 63.7b 9/20 9.3a 22.8a 12.8a 17.3b 37.6a 59.7a 9/30 9.3a 20.8a 12.9a 16.9ab 39.9b 62.1ab 1different letters within the same row mean significant differences (significance at 95% confidence level according to fisher’s least significant difference (lsd) procedure). del: delphinidin-3-o-glucoside; cya: cyanidin-3-o-glucoside; peo: peonidin-3-o-glucoside; pet: petunidin-3-o-glucoside; mal: malvidin-3-oglucoside; σ tri-sub: sum of tri-substituted anthocyanins. ital. j. food sci., vol. 30, 2018 194 cya was the second most abundant anthocyanin in sangiovese grapes. the sum of the trisubstituted anthocyanins (del, pet and mal) ranged between 57.8-65.5%. the results showed that the average anthocyanin profile of sangiovese corresponded to the one described in the literature (mattivi et al., 2006, arapitsas et al., 2012). however, significant differences in the anthocyanin composition of the grapes were related to c, ga, and sd (table 3). 3.2. grape quality model and evaluation of clone performance phase 1 – sangiovese wine eligibility model (wem) the model was built by running a pca which considered the chemical parameters of 37 commercial sangiovese wines from the chianti classico region. the scores of the wines relative to the two first pcs and the hotelling t2 ellipse (95% confidence level) are represented in fig. 3. figure 3. principal component analysis (pca) and hotelling t2 ellipse of the commercial wines, numbered 101 to 137. by encircling all the samples, the hotelling statistic evidenced the absence of outliers among the wines. the statistical analysis confirmed, moreover, a homogeneity between the samples, stating that they reasonably belong to the same class. for the construction of the model, the number of pcs was chosen to reach a level of explained variance between 80 and 90%. for the wem, three principal components that accounted for 84.11% of the total variance were considered adequate. phase 2 – sangiovese grape eligibility model (gem) the wem built in phase 1 was used to classify 30 experimental wines according to their analytical profiles. the simca analysis allowed the identification of 23 wines that fitted the model criteria, while rejecting wine samples 6, 13, 20, 22, 23, 27, and 28 (table 4). the grapes used in the experimental wines that resulted eligible by applying the wem were considered suitable for the construction of a grape eligibility model (gem). the ital. j. food sci., vol. 30, 2018 195 analytical parameters of the 23 grape samples were then used to calculate a pca in order to set up the gem. table 4. classification of the experimental wines (simca). numbers 1 to 30 represent the wine samples. (●) wine which fitted the wem, (-) wine which did not fit the wem. experimental wine experimental wine 1 ! 16 ! 2 ! 17 ! 3 ! 18 ! 4 ! 19 ! 5 ! 20 6 21 ! 7 ! 22 8 ! 23 9 ! 24 ! 10 ! 25 ! 11 ! 26 ! 12 ! 27 13 28 14 ! 29 ! 15 ! 30 ! fig. 4 reports the pca performed with the chemical parameters of the eligible grapes previously selected using the simca analysis. in this case too, no outliers were found according to the hotelling t2 test, confirming the homogeneity among the samples and indicating that they reasonably belong to the same class. figure 4. principal component analysis (pca) and hotelling t2 ellipse of the eligible grapes. the numbers inside the ellipse represent the grape samples whose wines fitted the wem. ital. j. food sci., vol. 30, 2018 196 similarly, as was done for the construction of the wem, to build the gem the number of pcs was chosen to reach a level of explained variance between 80 and 90%. for the purpose of this study, four pcs, which accounted for 81.72% of the total variance, were considered adequate. the simca analysis also allowed us to assess which factors are decisive in determining the classification of wines. the modeling power represents the contribution of each factor to building the model expressed as the variance of each variable. any variable having a modeling power higher than 0.3 was considered relevant in the model. in our study all the considered variables were relevant and in particular, hue (0.709), color intensity (0.704), monomer anthocyanins (0.699), and titratable acidity (0.628) were the variables with the greatest modeling power for the wines, followed by total phenol index (0.584), alcohol content (0.554), tannins (0.504), and ph (0.498). phase 3 – evaluation of clone performance lastly, the chemical profile of the grape clones was compared with the gem to classify their performance as a function of the clone, the sampling date, and the different growing areas. the results indicating which grapes had the chemical characteristics to fit the gem are reported in table 5. table 5. classification of the grapes (simca) as a function of the clone (12t, ap1, f09, m42, pec, r10, r24), the sampling date (9/10, 9/20, 9/30) and the different growing area (a, b, c, d); (●) grapes fitting the gem, (-) grapes not fitting the gem. a b c d 9/10 9/20 9/30 9/10 9/20 9/30 9/10 9/20 9/30 9/10 9/20 9/30 12t ! ! ! ap1 ! ! ! ! ! f09 ! ! ! ! ! m42 ! ! ! pec ! ! ! ! r10 ! ! ! ! r24 ! ! ! ! the clones showed a different performance according to the different growing areas and their ripening stage. considering the growing area as a factor, it can be seen that in zone a the ideal ripeness was reached in only four cases. in zone a, furthermore, only three clones were in accord with the parameters defined by the model: 12t, f09, and pec. area c, on the contrary, showed the highest number of clones that produced grapes with the desired characteristics. with regard to the performances of the individual clones, f09 resulted the only clone able to fit the model in all the growing areas on at least one of the sampling dates. the grapes from clones 12t, m42 and r10 fitted the model in two distinct zones: clone 12t in areas a and c; clone m42 in areas b and c; and clone r10 in areas b and d; the other clones (ap1 and r24) reached the maturity required by the model in three growing areas (areas b, c and d for clones ap1 and r24, and areas a, c, and d for clone pec). total potential in anthocyanins (0.797), extractable anthocyanins (0.699), and tannins (0.684) were the variables with the greatest modeling power for the grapes (expressed as ital. j. food sci., vol. 30, 2018 197 the variance of each variable) followed by cellular maturity index ea% (0.674), phenolic richness (0.607), titratable acidity (0.562), ph (0.505), and sugar (0.497). these results show the strong influence of the soil and climate on the ripeness parameters of sangiovese grapes. in growing area b five clones already fitted the model in the first sampling period, producing the fastest ripening. in this case, the grapes tended to exit the eligibility model over time. this highlights how the gem determined the unsuitability of grapes due to over-ripening, and therefore identified precise periods within which the quality of the grapes reaches its peak in relation to a particular enological target (wem). table 5 also shows how the model determined not only thresholds of acceptability but a real eligibility criterion. indeed, when all these variables are taken into account together, throughout the model, it creates a “space of acceptability where grapes transit over time,” with some of the grapes entering the space as they ripen while others, which were initially acceptable, are later expelled from the space of suitability by the model, because of overripening. it should be emphasized that the study was carried out in a single year, in order to propose a methodological approach. on the other hand, an adequate number of replicates over the years, taking into account the seasonal trends, should be analyzed when studying the performance of clones in a given area. 4. conclusions in the present study, grapes from seven sangiovese grape clones cultivated in the chianti classico region in tuscany were chemically characterized and compared, over the ripening period, to understand if non-genetic factors could affect the performances of different clones. in the first part of the study, the results showed that grape characteristics were influenced by all the factors considered: clone, growing area, and sampling date. the largest differences between the grapes, according to the phenolic composition, emerged when considering the growing area as a factor and the total anthocyanin content as a variable. the anthocyanin profiles were also affected by the different growing conditions and clones; the most abundant anthocyanins were malvidin-3-o-glucoside and cyanidin-3-oglucoside while acylated anthocyanins were detected in a very low amount (less than 2%), confirming the results for sangiovese wines reported elsewhere. in the second part of the study, a statistical model was developed to evaluate the impact of sangiovese variability on grape eligibility referring to a given enological target. for this purpose, a chianti classico sangiovese wine reference model (wem) was developed with the chemical characteristics of commercial wines from the same area. by comparing the composition of the experimental wines that fitted the wem with the relevant grapes, a model (gem) was defined that allowed us to discriminate the sangiovese grapes on the basis of their suitability to produce wines with the desired properties. the model could be expanded by inserting additional features such as aroma compounds or by using quick analysis methods such as ft-nir that can easily predict some chemical grape and wine parameters. with an adequate number of replicates, the proposed approach could be useful in zoning studies or in determining the performance of different varieties or clones with the goal of producing a typical sangiovese wine of the chianti classico region. moreover, it could be implemented as a more rational use of available analytical data both to monitor grape maturation trends and to improve the management of winemaking processes by transforming chemical analysis databases into active decision-making tools. ital. j. food sci., vol. 30, 2018 198 acknowledgements thanks to julian herszage for his help with the revision of the manuscript. references arapitsas p., perenzoni d., nicolini g. and mattivi f. 2012. study of sangiovese wines pigment profile by uhplcms/ms. j. agric. food chem. 60:10461-10471. arozarena i., ayestarán b., cantalejo m.j., navarro m., vera m., abril i. and casp a. 2002. anthocyanin composition of tempranillo, garnacha and cabernet sauvignon grapes from highand low-quality vineyards over two years. eur. food res. tech. 214:303-309. barbeau g., cousin m., blin 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revilla e. 2003. anthocyanin composition of cabernet sauvignon and tempranillo grapes at different stages of ripening. j. agric. food chem. 51:3372-3378. saint-criq de gaulejac n., vivas n. and glories y. 1998. maturité phénolique: definition et control. rev. française d’œnologie 173:14-21. tonietto j. and carbonneau a. 2004. a multicriteria climatic classification system for grape-growing regions worldwide. agric. forest meteorol. 124:81-97. wold s. and sjostrom m. 1977. simca: a method for analyzing chemical data in terms of similarity and analogy. chemometr theory appl 52:243-282. zanoni b., siliani s., canuti v., rosi i. and bertuccioli m. 2010. a kinetic study on extraction and transformation phenomena of phenolic compounds during red wine fermentation. int. j. food sci. & tech. 45(10):2080-2088. paper received september 14, 2017 accepted november 17, 2017 ijfs#1005_bozza ital. j. food sci., vol. 30, 2018 289 paper beverages based on ricotta cheese whey and fruit juices a. rizzolo and g. cortellino* council for agriculture research and economics, research centre for engineering and agro­food processing (crea­it), department of milan, via venezian 26, 20133 milan, italy *e-mail address: giovanna.cortellino@crea.gov.it abstract for studying antioxidants, sugars and organic acids compositions and their impact on sourness index (si) and total sweetness index (tsi), beverages were produced by blending ricotta-cheese whey (rcw), by-product of ricotta cheese production, with fruit juices. pear-rcw had higher malic acid and sorbitol, blueberry-rcw higher citric acid, total phenolics and anthocyanin pigments, apple-rcw higher sucrose and fructose, and strawberry-rcw higher glucose. blueberry-rcw had the highest si and the lowest tsi, while apple-rcw had the lowest si and the highest tsi. higher quality beverages may be obtained by using apple juice (‘yellow’ type) and the apple:blueberry (50:50) blend (‘red’ type). keywords: monomeric anthocyanin pigment, percent polymeric color, sourness index, total phenolic compounds, total sweetness index ital. j. food sci., vol. 30, 2018 290 1. introduction the whey-based fruit juice drinks could be considered novel functional beverages in which the nutraceutical components coming from fruit are combined with those of whey, so strengthening the functional value of the resulting product (hözer and kirmaci, 2010). by changing the type of fruit juice used in the formulation, the functional properties of the beverage can be modulated. the presence of anthocyanins, flavonols, catechins and phenolic acids in blueberries and strawberries has been related to the prevention of oxidative stress by scavenging reactive oxygen species and free radicals (brambilla et al., 2008; zafra-stone et al., 2007; granato et al., 2016), and risks reduction of several diseases, such as cardiovascular diseases and cancer (navindra, 2010). pear juice, being rich in hydroxycinnamic acids, glycosides of the flavonols quercetin and isorhamnetin, catechins and procyanidin polymeric units with chain lengths of up to 25 units, has a potent antioxidant power, and its singlet oxygen quenching abilities have been linked to its protective activities against both human erythrocyte lyses and protein oxidation (ferreira wessel et al., 2016). apple juice, containing polyphenols, including flavonoids (quercetin glycosides, procyanidins, epicatechins, chlorogenic acids, and phloretin glycosides) and phenolic acids, and apple fruit may reduce the risk of chronic disease by various mechanisms, including antioxidant, antiproliferative, and cell signaling effects (hyson, 2011). several researches have focused on the development of whey-based fruit blends with various formulations exploiting acid whey and sweet rennet-based whey in order to select the optimal mixture based on sensory perception (djurić et al., 2004; goudarzi et al., 2015; sakhale et al., 2012), as the poor sensory profile of whey protein beverages still remains a challenge to consumer acceptance. rizzolo and cortellino (2017) replaced whey with ricotta-cheese whey (rcw, scotta), the main by-product of ricotta cheese production obtained after flocculation of whey proteins induced by thermal treatment of cheese whey at 85-90°c for about 20 min, and produced ‘yellow’ and ‘red’ fruit-rcw-based beverages (juice/rcw ratio: 80/20, 14% soluble solids content), using apple, pear, blueberry and strawberry pectin-free juices. furthermore, cortellino et al. (2015) showed that the type of fruit juice used in the rcw-based beverage impacted on sensory perception, and suggested blending the blueberry juice with the apple ones in order to buffer the high sourness of the blueberrybased drink, which on the other hand was preferred for color, and at the same time to enrich the apple-based drink, which was preferred for taste, with anthocyanin compounds from blueberry. in this study, we investigated how antioxidants, sugars and organic acids compositions of the rcw-fruit juice beverages could be modulated by changing the type of fruit juice used in the formulation, considering pear-rcw and apple-rcw beverages for the ‘yellow’ type and blueberry-rcw, strawberry-rcw, apple:blueberry 50:50-rcw; apple:blueberry 75:25rcw beverages for the ‘red’ ones. 2. materials and methods 2.1. preparation of rcw-fruit juice beverages the rcw-fruit juice beverages have been prepared from a frozen cow rcw (dairy industry of lombardy region) and four types of frozen concentrated clear fruit juices without any preservers and colorants addition (g. mariani & c. s.p.a., brescia, italy) according to the process flow diagram described by rizzolo and cortellino (2017). ital. j. food sci., vol. 30, 2018 291 chemical composition, nitrogen distribution and mineral composition of rcw were reported in table 1 (monti et al., 2015). before blending, rcw was thawed at 2°c for 24 h and filtered in order to remove the majority of fat, whereas the concentrated clear fruit juices (65 % pectin-free strawberry, 65 % pectin-free blueberry, 70 % pear and 70 % apple) were thawed and diluted to 16.6 % soluble solids content for the preparation of the following beverages: apple-rcw, pearrcw, strawberry-rcw, blueberry-rcw, apple:blueberry (50:50, v/v)-rcw and apple:blueberry (75:25, v/v)-rcw drinks. hereafter the two formulations containing a blend of apple and blueberry juices are referred to as rcw-mix50:50 for apple:blueberry (50:50, v/v)-rcw drink and rcw-mix75:25 for apple:blueberry (75:25, v/v)-rcw beverage. table 1. chemical composition, nitrogen distribution and mineral composition of rcw. adapted from monti et al. 2015. chemical composition (g/100 g) mineral composition (mg/kg) ph 6.06 lactose 2.72 k 1023 p 218.00 total solids 4.16 glucose 0.01 ca 267.00 mg 158.00 ash 0.45 galactose 0.10 fe 0.48 cu 0.07 fat 0.05 citric acid 0.14 mn < 0.01 se 0.008 total proteins 0.39 lactic acid 0.13 na 881.00 zn 0.40 nitrogen distribution % g/100 g g/100 g % npn/nt true protein (nt-npn)*6.38 denatured protein (nt-ncn)*6.38 α-la β-lg α-la+β-lg cmp peptides 45.9 0.21 0.05 0.02 0.046 37.7 24.0 38.2 nt = total nitrogen; npn = non-protein-nitrogen; ncn = non-casein-nitrogen; α-la = α-lactalbumin; β-lg = β-lactoglobulin; cmp = casein macropeptide. beverages were packed in 125 ml uncolored glass bottles capped with twist-off lid. in order to obtain beverages standardized at about 14 % of soluble solids content, 16.6 % fruit juices and rcw were blended in 80:20 (v/v) ratios separately for every bottle. six bottles per juice type were divided into two lots and each lot (3 bottles/lot) was separately pasteurized by autoclaving (ghizzoni, parma, italy). pasteurization temperatures were detected in the autoclave and at the core of the bottle by means of flexible thermocouple probes and monitored with e-val 2.10 software system; for strawberry, apple and blueberry based beverages, characterized by ph<4.2, the lethal rate f_100^10 value was 11 while for pear beverage, having ph>4.2, an f_100^10=16 value was adopted. after pasteurization bottles were kept for 15 days on open shelves at ambient temperature (20°c); then 50 ml aliquots from every bottle were frozen at −20°c till analysis. for each beverage, 3 bottles per pasteurization lot (6 replicates/beverage) were analyzed for physical-chemical parameters, (ph, titratable acidity, soluble solids content, color) soon after the 15 days of shelf life at 20°c, and for soluble sugar and organic acid compositions, phenolics, anthocyanins, polymeric color and total antioxidant capacity after overnight thawing at 2°c of the frozen samples. each bottle was analyzed separately (1 bottle=1 replicate). ital. j. food sci., vol. 30, 2018 292 2.2. physical-chemical analyses the ph values and titratable acidity (ta) were determined with a titroprocessor (model 682, metrohm ag, switzerland) by titrating with 0.1 mol/l naoh to ph=8.2. color parameters were measured by means of a spectrophotometer cm-2600 (minolta, japan) equipped with a sample holder for 10 mm-plastic cells suited for liquid analysis, using the primary illuminant d65 (cie, 2006) and 2° observer in the l*, a*, b* color space. specular component included (sci) mode was selected and a white calibration tile was used as background. from l*, a*, b* values, chroma (c*) and hue (h°) were computed according to: 𝐶∗ = 𝑎∗! + 𝑏∗! (1) h°=arctangent (b*/a*) × 360/ (2×3.14) (2) with h°=90° corresponding to the yellow color and h°= 360° to the violet one. 2.3. determination of total phenolic content (tpc) and total anthocyanin pigments (map) total phenolic content (tpc) was measured spectrophotometrically by the folinciocalteau assay (singleton and rossi, 1965) and was expressed as gallic acid equivalents (mg gae/100 ml). total monomeric anthocyanin pigment (map) was estimated spectrophotometrically by the ph-differential method (giusti and wrolstad, 2001) and was expressed as cyanidin 3-glucoside equivalents (mg c3ge/100 ml). color density (cd) and percent polymeric color (ppc) were measured according the bisulphite bleaching method described by giusti and wrolstad (2001). 2.4. determination of antioxidant capacity the antioxidant activity (antox) was measured spectrophotometrically by using the stabilized artificial free radical 2,2-diphenyl-1-picrylhydrazyl (dpph) according to the method described by lo scalzo et al. (2004) and was expressed as gallic acid equivalents (mg gae/100 ml). 2.5. soluble sugar and organic acid compositions soluble sugars were quantified by hplc (forni et al., 1992) using a rezex™ rpmmonosaccharide pb+2 (8%) (300x7.8 mm, phenomenex) column. organic acids were analyzed on a hypersil gold (5 µm, 4.6x250 mm, thermo scientific) column kept at 30°c with a variable wavelength detector (uv-1575) set at 210 nm, using as mobile phase 0.02 m orthophosphoric acid at the flow rate of 0.7 ml/min. data of sugar composition were expressed as g/100 ml of beverage, whereas those of organic acids as mg/100 ml of beverage. 2.6. sourness index and total sweetness index the sour taste of each beverage was estimated by means of sourness index, taking into account the fact that, within the same ph level, sourness increases with increasing acid concentration (sowalski and noble, 1998). ital. j. food sci., vol. 30, 2018 293 hence, sourness index was computed according to the following equation: 𝑆𝑜𝑢𝑟𝑛𝑒𝑠𝑠 𝐼𝑛𝑑𝑒𝑥 = 𝐶!"#×𝑎 + 𝐶!"#×𝑏 + 𝐶!"#×𝑐 + 𝐶!"#×𝑑 (3) where: cmal, ccit, cfum and clac are the amounts of malic, citric, fumaric and lactic acids, respectively, expressed in g/100 ml, whereas a, b, c and d are the estimated sourness units (table 2) of malic, citric, fumaric and lactic acids, respectively, at the ph value of each beverage. table 2. estimated sourness units of malic acid (a), citric acid (b), fumaric acid (c) and lactic acid (d ) at the ph value of each beverage used for computation of sourness index. the values are taken from the plot estimated sourness in units from ratio scales of moskowits (1971) vs ph in the 3.0-4.4 range reported by sortwell (2005). a b c d apple 3.2 3.3 1.8 2.5 pear 2 2 1 1 strawberry 4.1 3.6 2.5 4 blueberry 7 5.3 4 8 mix 50:50 5.3 4.7 3 6 mix 75:25 5 4.1 2.8 5 total sweetness index (tsi) was estimated by multiplying the amounts of each sugar by its relative sweetness respect to sucrose according to the following equation: 𝑇𝑆𝐼 = 𝐶!"#×1.00 + 𝐶!"#×0.45 + 𝐶!"#×0.91 + 𝐶!"#$×0.37 + 𝐶!"#$×0.22 (4) where: csuc, cglu, cfru, csorb and clact are the average amounts of sucrose, glucose, fructose, sorbitol and lactose, respectively, expressed in g/100 ml, multiplied for the relative sweetness values respect to sucrose as determined by moskowitz (1970) from sweetness power functions related to the weight of solute in solution with the exponent fixed at 1.3. 2.7. statistical analysis the statgraphics v.5.2 (manugistic inc., rockville, md, usa) software package was used. data were submitted to one-way analysis of variance (anova) and means were compared by tukey’s test (p<0.05). 3. results and discussion 3.1. physical-chemical analyses table 3 shows the physical-chemical parameters of the six types of rcw-fruit juice beverages. as expected, a clear influence of the type of fruit juice used as ingredient in the beverage formulation was found for ph, ta and color parameters. the two ‘yellow’ formulations had higher ph and lower ta than the four ‘red’ drinks, with the apple-rcw drink having lower ph and higher ta than the pear-rcw ones. strawberry-rcw ital. j. food sci., vol. 30, 2018 294 beverage had higher ph than all the three formulations based on blueberry juice, and a ta amount intermediate between that of blueberry-rcw drink and the two apple:blueberrybased beverages. considering the formulations based on blueberry juice, ph increased and ta decreased as the proportion of apple juice added increased (table 3). color parameters indicated that ‘yellow’ beverages had lighter and more vivid color (higher l* and c*) than the four ‘red’ beverages. the pear-rcw drink had a yellower (hue value more close to 90°) and lighter (higher l*) color than apple-rcw drink, while all the ‘red’ beverages had a very dark and grayish color (l*<25 and c*<2.5), with blueberry-rcw and rcw-mix50:50 drinks having lower l* and c* than rcw-mix75:25 beverage. the ‘red’ beverages, in addition, differed for hue, which indicated a violet-red color for strawberryrcw drink (h ≈ +15°) and a red-violet one for blueberry-rcw beverage (h ≈ −9°). the blending of blueberry juice with apple juice provoked a shift of hue towards the violet-red color, with the higher change at the higher proportion of apple juice (apple:blueberry 50:50, h ≈−2°; apple:blueberry 75:25: h ≈ +5°). a similar scenario on the relationship between proportion of apple juice added to a fruit juice containing anthocyanins and shift of hue from red violet to violet red has already been reported also for blends of clear apple juice with clear sour cherry juice (nani et al., 1991) and blackcurrant juice (nani et al., 1993). table 3. physical-chemical parameters (ta, titratable acidity; l* lightness; c* chroma; h°, hue angle) of rcw-fruit juices beverages. data are mean±standard error (n=6). means within columns having different letters are significantly different (tukey’s test); p-value, significance of f ratio. ph ta (meq/100 ml) l* c* h° apple 3.80±0.01 b 6.77±0.03 e 44.4±0.07 b 25.1±0.05 b 76.6±0.1 e pear 4.49±0.05 a 3.34±0.05 f 52.2±0.08 a 25.8±0.05 a 85.2±0.0 f strawberry 3.61±0 c 15.80±0.14 b 25.2±0.03 c 2.2±0.05 c 15.6±0.4 d blueberry 3.19±0.04 e 19.23±0.10 a 24.6±0.05 e 1.4±0.05 d 351.3±1.4 c mix 50:50 3.35±0.01 d 13.28±0.04 c 24.5±0.07 e 1.4±0.02 d 357.9±2.3 b mix 75:25 3.45±0.02 d 11.02±0.04 d 24.8±0.01 d 2.4±0.18 c 364.7±0.3 a p-value < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 3.2. sugar and organic acid compositions both sugar and organic acid compositions (tables 4 and 5) were clearly influenced by the ingredients. the sugar and organic acid compositions of the rcw were analyzed according to the methods described in section 2.5 suitable to quantify components from fruit juices. the following data were obtained (mean±standard error, n=3): lactose, 2.08±0.07 g/100 ml; lactic acid, 0.41±0.03 g/100 ml; acetic acid, 0.13±0.002 g/100 ml; citric acid, 0.11±0.005 g/100 ml; fumaric acid, 0.53±0.02 mg/100 ml, whereas glucose and galactose were present in trace levels, below the limit of quantification of the method. in agreement with these data, and taking into account the proportion of rcw used in all the formulations (20 %), in all the beverages it was not possible to quantify galactose and acetic acid from rcw, and, as expected, the amounts of lactose (approx. 416.7±26.4 mg/100 ml) and lactic acid (approx. 66.8±2.9 mg/100 ml) did not differ among formulations (tables 4 and 5). ital. j. food sci., vol. 30, 2018 295 table 4. amounts of organic acids (mg/100 ml) of rcw-fruit juices beverages. data are mean±standard error (n=6). means within columns having different letters are significantly different (tukey’s test); p-value, significance of f ratio. malic acid lactic acid citric acid fumaric acid apple 483.5±6.8 b 69.0±2.9 a 29.5±0.4 e 0.34±0.01 b pear 1,004.0±24.5 a 63.8±2.4 a 28.3±1.7 e 0.41±0.05 b strawberry 375.9±9.6 de 66.3±2.2 a 792.6±16.8 b 1.62±0.18 a blueberry 351.4±8.3 e 69.9±1.8 a 938.8±9.3 a 1.60±0.36 a mix 50:50 425.5±0.8 cd 69.0±3.2 a 424.1±4.5 c 0.41±0.01 b mix 75:25 442.3±3.1 bc 63.0±3.4 a 278.4±4.9 d 0.23±0.01 b p-value < 0.001 ns < 0.001 < 0.001 table 5. amounts of sugars (mg/100 ml) of rcw-fruit juices beverages. data are mean±standard error (n=6). means within columns having different letters are significantly different (tukey’s test); p-value, significance of f ratio. sucrose glucose fructose sorbitol lactose apple 1,219±10 a 3,362±40 c 7,206±76 a 362±8 b 392±15 a pear 1,039±12 b 2,941±30 d 5,880±65 d 3,286±40 a 433±12 a strawberry 235±11 e 4,484±116 a 6,581±176 bc 247±10 c 402±16 a blueberry 4±2 f 4,505±119 a 6,022±168 cd 37±2 d 396±13 a mix 50:50 489±16 d 4,184±99 ab 7,016±150 ab 209±4 c 461±15 a mix 75:25 717±11 c 3,928±72 b 7,172±127 b 266±8 c 416±13 a p-value < 0.001 < 0.001 < 0.001 < 0.001 ns as for the other organic acids (table 4) and sugars (table 5), the amounts and proportions were in agreement with those from the literature for the four fruit species (eisele and drake, 2005 for apple; sha et al., 2011 and viera arroyo et al., 2013 for pear; kallio et al. 2000 for strawberry; rizzolo et al., 2002 and bett garber et al., 2005 for blueberry) and, hence, mirrored the characteristic of the type of fruit juice used in the formulation. our results on organic acid compositions showed that apple-rcw and pearrcw beverages had higher malic acid content than strawberry-rcw and blueberry-rcw ones, with pear-rcw showing the highest amount (approx. 1 g/100 ml). blueberry-rcw beverage had the highest amount, and strawberry-rcw one the second highest amount of citric acid and both these red beverages were characterized by the highest fumaric acid contents (approx. 1.6 mg/100 ml). the blending of blueberry juice with apple juice induced an increase in malic acid and a decrease in citric acid contents, with the major changes observed in the rcw-mix75:25 drink, but not significant changes in fumaric acid amounts. considering the sugar composition (table 5), apple-rcw beverage had the highest amounts of sucrose and fructose, while pear-rcw drink the second highest amount of sucrose, about a tenfold quantity of sorbitol than all the other formulations, and the lowest amounts of glucose and fructose; strawberry-rcw drink was characterized by the highest amount of glucose and about one fifth of sucrose concentration of apple-rcw and pear-rcw formulations. in contrast, blueberry-rcw beverage had very low amounts of sucrose (<10 mg/100 ml) and sorbitol (<40 mg/100 ml) and glucose as much as strawberry-rcw drink. the blending of blueberry juice with apple juice induced an increase of sucrose, fructose and sorbitol and a decrease in glucose, with the major changes observed in the rcw-mix75:25 drink. ital. j. food sci., vol. 30, 2018 296 the different organic acid composition coupled to a different ph value and the different sugar composition exerted an influence on sour taste intensity and total sweetness of beverages. in fact it has been reported that, within the same concentration level, sourness intensity increases with decreasing ph, as well as that, within the same ph level, sourness increases with increasing acid concentration (sowalsky and noble, 1998); furthermore, da conceicao neta et al. (2007) found that sour taste intensity was linearly related to the molar concentration of hydrogen ions and to the molar concentration of all organic acid species that have at least one protonated carboxyl group, and, hence, that weaker organic acids were more sour at a given ph than a stronger organic acid because a larger fraction of the carboxyl groups were protonated. on these bases, in order to have a rough estimation of sourness for each rcw-fruit beverage, the sourness index, computed according to the eq. (3) reported in section 2.6, was used (fig. 1, top). basing on this index rcw-fruit beverages can be ordered from the most to the less sour as following: blueberry > strawberry = apple:blueberry 50:50 > apple:blueberry 75:25 > pear >apple. as for sugars, in order to have an estimate of the sweetness for each rcwfruit formulation, the total sweetness index, calculated according to the eq. (4) reported in section 2.6, was used (fig. 1, bottom). figure 1. sourness index (top) and total sweetness index (bottom) of rcw-fruit juices beverages. bars refer to standard error (n=6). sample abbreviations: mix 50:50, rcw-apple:blueberry (50:50 v/v); mix 75:25, rcw-apple:blueberry (75:25 v/v). means having different letters are significantly different (tukey’s test). ital. j. food sci., vol. 30, 2018 297 considering the tsi, apple-rcw beverage was characterized by the highest total sweetness, and the blueberry-rcw drink by the lowest; strawberry-rcw had lower tsi than apple-rcw, but higher than blueberry-rcw, while pear-rcw, rcw-mix50:50 and rcw-mix75:25 drinks had tsi values not different from apple-rcw and strawberry-rcw ones. even if sourness index and tsi are an approximation of the more complex scenario which actually occurs, as they do not consider the suppression effect of sugars on sourness intensity (savant and mcdaniel, 2004), they could have a practical use in evaluating the sensory impact of a product starting from the compositions in the principal organic acids and sugars. in fact, sourness index and tsi results are in agreement with the scores of sensory taste pleasantness reported by cortellino et al. (2015), which gave to blueberry-rcw beverage the lowest score, being judged too sour and astringent, and to apple-rcw drink the highest. 3.3. total monomeric anthocyanins and color indices the mean concentration of total monomeric anthocyanins was highest in blueberry-rcw drink, decreased with blending of blueberry juice with apple juice (approx. −53 % for rcw-mix50:50; approx. −73 % for rcw-mix75:25) and was lowest in strawberry-rcw beverage (table 5). our results for blueberry-rcw drink are in line with previous reports on anthocyanins content of blueberry juices, which varied according to the cultivar and juice production conditions from 8 to 130 mg/100 g (lee et al., 2002; müller et al. 2012; rizzolo et al., 2002; rossi et al., 2003), whereas the map content of strawberry-rcw drink was low if compared to the 36.2-38 mg/100 ml range found by odriozolaserrano et al. (2008) for a 7 % clear strawberry juice after heat treatments at 90°c. the color indices were measured exploiting the ability of bisulphite to form colorless adducts with monomeric anthocyanin compounds but not with the polymeric pigments (giusti and wrolstad, 2001) which are formed by polymerization reaction mostly occurring by binding monomeric anthocyanins with other phenolics compounds, such as phenolic acid and condensed tannins (türkyilmaz and özkan, 2012). cd (monomeric anthocyanin compounds plus polymeric pigments) of blueberry-rcw drink was twice as much than cd of the other three red formulations, while pc (polymeric pigments) was highest in blueberry-rcw drink and with blending of blueberry juice with apple juice it decreased approx. by 49 % for rcw-mix50:50 and approx. 61 % for rcwmix75:25 beverages. as a consequence, the three formulations based on blueberry juice had ppc values of approx. 50 % independently from the proportion of blueberry juice used in the beverage (table 6). this value is in line with lee et al. (2002) results on 14-15 % pasteurized blueberry juices, for which ppc ranged from 40.7 to 50.4 %. a different scenario was observed for strawberry-rcw drink; notwithstanding cd was not different from the value observed for the two beverages prepared using blueberry juice blended with apple juice, ppc was 30 % higher than the ppc values found for the three blueberry based formulations. as a matter of fact, rizzolo and cortellino (2017) found for the strawberry-rcw beverage already before the pasteurization step an high ppc value (approx. 71 %) coupled to low map (approx. 46 mg c3ge/100 ml) and suggested that likely anthocynanins had undergone some degradation already at the factory during the production of the 65 % pectin-free strawberry juice used for the preparation of the drink. beverage contained 20% of rcw and consequently very small quantity of total protein (0.08 g/100g), which only partially can be considered as true protein (0.05 g/100g). rcw brought α-lactalbumin, β-lactoglobulin, casein macropeptide and peptides to the drink; these compounds may interact with anthocyanins increasing their stability as reported in the literature. ital. j. food sci., vol. 30, 2018 298 anthocyanins are susceptible to chemical degradation and consequently to color fading in the presence of vitamin c. chung et al. (2015) demonstrated that the addition of biopolymers significantly enhanced the color stability of purple carrot anthocyanin in model beverage during storage. the best stability was obtained by adding heat denatured whey protein isolate, which through complexation with anthocyanin reduced its degradation due to ascorbic acid. a fluorescence quenching study showed that the anthocyanin formed stronger interactions with the protein through hydrogen bonding than with the ascorbic acid. moreover chung et al. (2017) suggested that also the addition of three amino acid (l-phenylalanine, l-tyrosine and l-tryptophan) and a polypeptide (ɛ-poly-l-lysine) may prolong the color stability of the same beverage. the most significant improvement was observed for l-tryptophan, which interacted with anthocyanins mainly through hydrogen bonding but also by some hydrophobic interaction. table 6. monomeric anthocyanin pigments (map), color density (cd), polymeric color (pc) and percent polymeric color (ppc) of ‘red’ rcw-fruit juice beverages. data are mean±standard error (n=6). means within columns having different letters are significantly different (tukey’s test); p-value, significance of f ratio. map (mg c3ge/100 ml) cd pc ppc strawberry 24.34±4.11 c 5.15±1.20 b 3.76±0.65 b 80.0±6.1 a blueberry 119.04±4.28 a 12.01±0.12 a 5.87±0.10 a 48.9±0.6 b mix 50:50 56.03±0.98 b 5.86±0.02 b 3.00±0.02 bc 51.2±0.4 b mix 75:25 33.22±0.30 c 4.11±0.11 b 2.29±0.05 c 55.7±0.5 b p-value < 0.001 < 0.001 < 0.001 < 0.001 3.4. total phenolic content and antioxidant capacity total phenolic content and antioxidant capacity of rcw-fruit based beverages as expected greatly depended on the type of fruit juice used for the preparation of the beverage (table 7). the mean amount of tpc was highest in blueberry-rcw drink and decreased with blending of blueberry juice with apple juice [−50 % for apple:blueberry (50:50); approx. −52 % for apple:blueberry (75:25)]. strawberry-rcw beverage had the second highest tpc amounts, which was higher than the values found for both apple:blueberry blends. strawberry-rcw drink had higher antioxidant capacity than blueberry-rcw beverage, even if the difference between the mean values was very low (approx. 1.8 mg gae/100 ml). similarly to what found for tpc, antioxidant capacity of apple:blueberry blends was approx. 50 % (rcw-mix50:50) and 47 % (rcw-mix75:25) of that found for blueberry-rcw drink. apple-rcw and pear-rcw drinks were characterized by the lowest tpc amounts along with the lowest antioxidant capacity. the higher values of antioxidant capacity found for the four ‘red’ beverages may be related to the presence of anthocyanins coupled with to higher tpc content compared to the two ‘yellow’ drinks, compounds which have demonstrated powerful in vitro antioxidant capacity at various tests (tabart et al., 2009). our results on tpc agree well with those in previous studies on blueberry (casati et al, 2012; granato et al., 2015; rizzolo et al., 2002), apple (granato et al., 2015), pear (saeeduddin et al., 2015) and strawberry (cao et al., 2012; hartmann et al., 2008) juices, while data of antioxidant capacity cannot be directly compared with those reported in the literature for apple (granato et al., 2015; tabart et al., 2009), pear (saeeduddin et al., 2015), strawberry (cao et al., 2012; hartmann et al., 2008; ital. j. food sci., vol. 30, 2018 299 odriozola-serrano et al., 2008) and blueberry (granato et al., 2015; casati et al., 2012) juices due to either differences in the methodology adopted for the dpph reaction with the samples or to the use of a different type of test used to estimate the antioxidant capacity of juice samples. the fact that strawberry-rcw beverage showed similar antioxidant capacity to that of blueberry-rcw drink, notwithstanding the lower map and tpc amounts, could be due to the higher proportion of anthocyanin polymers (higher value of ppc), whose antioxidant capacity likely compensates for the loss of antioxidant capacity due to monomeric anthocyanin degradation, as well as to the formation of maillard reaction products in response to thermal treatment which exert antioxidant capacity (yilmaz and toledo, 2005). table 7. total phenolics content (tpc) and antioxidant capacity (antox) of rcw-fruit juices beverages. data are mean±standard error (n=6). means within columns having different letters are significantly different (tukey’s test); p-value, significance of f ratio. tpc (mg gae/100 ml) antox (mg gae/100 ml) apple 47.3±0.5 d 3.26±0.02 d pear 38.5±1.0 d 1.86±0.03 d strawberry 216.9±2.5 b 34.03±0.78 a blueberry 325.4±3.3 a 32.22±0.42 b mix 50:50 162.7±1.8 c 16.20±0.38 c mix 75:25 156.0±2.3 c 15.41±0.252 c p-value < 0.001 < 0.001 4. conclusions consumer interest in “healthy” foods and beverages has increased over the last decade, and rcw-fruit juice beverages may be considered novel functional drinks in which the functional status of the product could be modulated by changing the type of fruit juice used in the formulation. considering the interplay among antioxidant capacity, phytonutrient content and beverage sourness and sweetness indices, it could be concluded that higher quality fruit juice-rcw beverages may be obtained by using apple juice for the ‘yellow’ type and the apple:blueberry (50:50) blend for the ‘red’ ones. specifically, apple juice was preferred to the pear one as, being equal for total sweetness and sourness index, it was richer in total phenolic content and antioxidant capacity. as for the apple:blueberry (50:50) blend, it represented a good compromise having an acceptable sourness index along with a remarkable source of phenolic compounds and monomeric anthocyanin pigments. acknowledgements the authors would like to thank nicola luccini for the contribution to preparation and analyses of rcw-juice drinks. this research was carried out within the “twinning italy-canada activities in research and innovation in the agro-food area – canadair” project funded by the italian ministry of agriculture (ministry decree 27240/7303/2011). ital. j. food sci., vol. 30, 2018 300 references bett-garber k.l., lea j.m., watson m.a., grimm c.c., lloyd s.w., beaulieu j.c., stein-chrisholm r.e., andrzejewski b.p. and marshall d.a. 2015. flavor of fresh blueberry juice and the comparison to amounts of sugars, acids, anthocyanidins, and physicochemical measurements. j. food sci. 80:s818-s827. brambilla a., lo scalzo r., bertolo g. and torreggiani d. 2008. steam blanched highbush blueberry (vaccinium corymbosum l.) juice: phenolic profile and antioxidant capacity in relation to cultivar selection. j. agric. food chem. 56:2643-2648. cao x., bi x., huang w., wu j., hu x. and liao x. 2012. changes of quality of high hydrostatic pressure processed cloudy and clear strawberry juices during storage. innov. food sci. emerg. tech. 16:181-190. casati c.b., sánchez v., baeza r., magnani n., evelson p. and zamora m.c. 2012. relationships between colour parameters, phenolic content and sensory changes of processed blueberry, elderberry and blackcurrant commercial juices. int. j. food sci. technol. 47:1728-1736. chung c., rojanasasithara t., mutilangi w. and mcclements d.j. 2015. enhanced stability of anthocyanin-based color in model beverage systems through whey protein isolate complexation. food res. int. 76:761-768. chung c., rojanasasithara t., mutilangi w. and mcclements d.j. 2017. stability improvement of natural food colors: impact of amino acid and peptide addition on anthocyanin stability in model beverages. food chem. 218:277-284. cie, 2006. colorimetry part 2: cie standard illuminants. 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j. and winefield c. 2013. genetic parameters for sugar content in an interspecific pear population. eur. j. hortic. sci. 78:56-66. yilmaz y. and toledo r. 2005. antioxidant activity of water-soluble maillard reaction products. food chem. 93:273-278. zafra-stone s., yasmin t., bagchi m., chatterjee a., vinson j.a. and bagchi d. 2007. berry anthocyanins as novel antioxidants in human health and disease prevention. mol. nutr. food res. 51:675-683. paper received august 30, 2017 accepted november 13, 2017 ijfs#1313_bozza ital. j. food sci., vol. 31, 2019 401 paper imaging, sensory properties and fatty acid composition of parma ham and “nero di parma ham” g. pastorelli*1, s. panseri1, g. curone1, a. zanon3 and m. di giancamillo1 1department of veterinary medicine, università di milano, italy 2department of veterinary sciences and technologies for food safety, università di milano, italy 3dvm freelancer *corresponding author: grazia.pastorelli@unimi.it abstract this paper provides preliminary results on “nero di parma” ham analysing fatty acid composition from fat and muscle tissues in three locations. computerized tomography (ct) and sensory characteristics were also performed. the same measurements were investigated in parma ham too. significant differences in terms of saturated and monounsaturated fatty acids composition were observed between hams resulting lower and higher in “nero di parma” ham than parma ham respectively. results detected by ct showed an inverse ratio of fat and muscle between the hams. “nero di parma” ham highlighted some descriptors such as oiliness, brightness, redness significantly different from parma useful to define the sensory profile of a typical product, which had never been tested. keywords: fatty acids, “nero di parma ham”, parma ham, sensory profile ital. j. food sci., vol. 31, 2019 402 1. introduction dry curing is a common way to preserve pork in some mediterranean countries. in italy, parma ham is the main consortium for the production of dry cured ham labeled with protected designation of origin (pdo), processing over 8 million thighs (data referred to 2017). parma ham represents a product of great economic value covering about half of the trading value of carcasses from italian heavy pigs. in addition, new interest is now addressed to the health promoting characteristics of parma ham and therefore, the fatty acid composition of its lipids is important to relate the profile to medical guidelines. alongside the intensive pig farming conducted according to criteria of industrialization, an attractive production related to tradition is emerging. it concerns native breeds that are characterised by peculiar traits low input rearing conditions and suitable high quality food products. in the present work “nero di parma” breed has been considered. it is a modern recreation of the ancient nera parmigiana breed, which significantly declined almost to the point of extinction. however, selection programs are being designed to reintroduce the “nero di parma” pig breeding tradition; with the dm 11781 of 20 may 2016 the “nero di parma” pig obtained the recognition of the breed. animals are fed fresh grass, corn, barley and wheat, as well as broad beans, berries, roots and acorns. the characteristics of the meat of “nero di parma” are linked to the presence of intramuscular fat due to the ability to accumulate subcutaneous fat between the muscle fibers compared to the white breeds such as the large white and landrace and their crossings. fat plays an important role in the development of the chemical and sensorial characteristics of cured meat products (ventanas et al., 2007; jimenez-colmenero et al., 2010). moreover, much attention is being paid to fatty acid profile in animal products because of its effects on consumers’ health (wood and enser, 1997). scientific evidence (who, 2003) and nutritional guidelines recommend a reduction in total fat intake, particularly of saturated fatty acids (sfa), which are associated with an increased risk of obesity, hypercholesterolaemia and some cancers (wood et al., 2004), while a high intake of monounsaturated and n-3 polyunsaturated fatty acids has been shown to have an inverse effect (ganji et al., 2003). however, there are few data on “nero di parma” pigs in terms of meat quality and those related to seasoned ham to the best of our knowledge are missing. the aim of paper is to provide preliminary results on some qualitative parameters of the “nero di parma” ham by evaluating fatty acid composition in fat and muscle in three locations (initial part, central and the final part of the ham), the structure of the ham through the computerized tomography (ct), sensory characteristics and measuring the same parameters in parma ham. 2. material and methods 2.1. sampling a total of 6 raw cured hams, 3 “nero di parma” hams and 3 parma (pdo) were provided by a local producer, within the same factory in order to ensure the same production technology. the two types of hams had different ripening maturation times: 30 and 24 months respectively. the choice depended on the high fat (either fat coverage either as intermuscular fat) of “nero di parma” that requires longer times of ripening. therefore “nero di parma” ham is marketed at 30 months, and parma ham at 24 months. before starting chemical analysis, on the whole ham the spiral multi slice ct was carried out; subsequently the hams were sliced and used for sensory analysis. during the sensory ital. j. food sci., vol. 31, 2019 403 analysis whole slices (including muscle and external fat) at three different locations (initial, central and final part) were sampled according to fig. 1. samples were vacuum-packaged and stored at -20ºc for 2 weeks until subsequent chemical analysis. figure 1. sampling location. section 1 corresponds to initial part; section 2 corresponds to central part; section 3: corresponds to final part 2.2. fatty acid analysis lipid extraction was conducted using chloroform: methanol (2:1) according to the method of folch et al. (1957). lipids were extracted from each sample of the muscle, and from each sample of trimmed adipose tissue. the extracts were dried under vacuum on a rotary evaporator (laborota 4000, heidolph instruments, milan, italy). fatty acid composition was measured after methylation of the samples. fatty acid methyl esters were prepared with boron trifluoride methanol according to procedures developed by morrison and smith (1964). the methyl esters were separated on a carlo erba instruments chromatograph (gc 6000 vega series 2) equipped with fused silica gel capillary column (0.25 mm i.d.25 m) having a 0.2-mm internal coating of cyanopropyl siloxane (cp-sil 88, chrompack). the furnace temperature was 180°c, and injector and detector temperatures were 240°c. for all samples, retention times and peak areas were determined by chromatography (nelson analytical, manchester, nh). the identities of the peaks were verified by comparison with the retention times of standard fatty acid methyl esters. the results were expressed as the percentage of the total fatty acid composition. all the analyses were carried out in duplicate. atherogenicity (ai) and thrombogenicity (ti) indices (ulbricht and southgate 1991), which characterize the potential proneness to atherosclerosis and thrombosis in humans were used to assess dietetic values of ham. in detail ia indicates the relationship between the sum of the main saturated fatty acids and that of the main classes of unsaturated; the former being considered pro-atherogenic (favoring the adhesion of lipids to cells of the immunological and circulatory system), and the latter anti atherogenic (inhibiting the aggregation of plaque and diminishing the levels ital. j. food sci., vol. 31, 2019 404 of esterified fatty acid, cholesterol, and phospholipids, by preventing the appearance of micro and macro coronary diseases). index of thrombogenicity is defined as the relationship between the pro-thrombogenetic (saturated) and the anti-thrombogenetic fatty acids (mufas, pufas – n6 and pufas – n3). 2.3. sensory analysis eleven trained assessors belonging to onas (national organization salumi taster) undertook the sensory analysis on 2-mm slices of ham. the choice of descriptors was decided by open discussion in two preliminary sessions. twenty sensory descriptors covering appearance (redness, whiteness, marbling, oiliness, brightness), odor (aged, fresh pork meat, rancid, mold), taste (sweetness, saltiness, bitter), flavor (aged, fresh pork meat, rancid, butter, fresh fat), texture (fibrousness, dryness, firmness) were chosen. a 9-point scale was used, where 0 meant absence and 9 meant maximum intensity of the descriptor. the sensory evaluation was repeated in three sessions carried out on three different days (en iso 13299, 2010). each member of the panel assessed two types of hams in each session. slices of ham samples were coded with three random numbers and were presented to the members according to macfie et al. (1989). 2.4. computed axial tomography hams were submitted to 16-slices ct scanner (brightspeed® ge medical systems italia s.p.a., milan, italy) applying the following protocol: tube voltage 110 kvp, x-ray tube current 200 ma, revolution time 1, slice thickness 1.25 mm, spiral pitch factors 0.9375, convolution kernel standard, rows and columns 512x512. all images were transferred to the picture archiving and communication system (mypacs – md saronno italy), processed with a certified medical software (osirixpro® 64 bit, aycan medical systems) and reconstructed using smoothing and edge enhancement algorithms in all the three spatial transverse planes. the program can differentiate the fat from the lean as a consequence of the significant difference in hounsfield units (hu) existing between the two tissues (negative values for fat and positive for lean). hounsfield units (also named ct numbers) express the x-ray attenuation of the tissues, which is a measure of its density in a given region of interest (roi). in each slice, a roi based on the density of the tissues examined (fat or lean) was selected in order to obtain the hu surface area values. therefore, the “compute volume” function of the software was used to calculate the volume. 2.5. statistical analysis the ham was the experimental unit for statistical analysis. data were analyzed by means of variance analysis including typology (parma and “nero di parma”), tissue (muscle and fat) and location (initial, central and distal part), as main effect. newman-keuls test was used to assess differences between means. the sensory data for each attribute were submitted to three-way anova with typology, judges, replicates and their interaction as effects. the significance of these effects was tested with the f-test and the comparison between mean values was tested with t student. differences with probability levels of p<0.05 were considered significant. effects were deemed significant at p<0.05, and a trend was noted when p<0.10. statistical analyses of the data were performed using spss software (spss inc., chicago, il) ital. j. food sci., vol. 31, 2019 405 3. results and discussion pig farming plays a key role within animal production, particularly in north italy where about 70% of the italian pig production is located. the main strength factor of the italian pig industry is represented by the top quality of its production labeled with the pdo (protected designation of origin) or pgi (protected geographical indication) marks. they represent an important market value and must continuously be monitored from a qualitative point of view. fat and fatty acids, whether in adipose tissue or muscle, contribute importantly to various aspects of meat quality and are central to meat nutritional value (wood et al., 2008). it is generally assumed that intramuscular fat (imf) content positively influences sensory quality traits, including flavor, juiciness and tenderness of meat, whereas a low amount of fat results in a less tasty meat (tous et al., 2013). it is well known that traditional breeds are fattier than industrial ones. generally, they have more adipose tissue thickness and more intramuscular fat (gandemer, 2009). drycured ham from conventionally reared modern breeds of pigs compared to dry-cured ham from iberian pigs had lower levels of saturated fatty acids (sfa) and polyunsaturated fatty acids (pufa) (jiménez-colmenero et al., 2010). the fa composition of dry-cured hams is due to many factors, such as genetic features and differences in the rearing and feeding systems. results of fernández et al. (2015) for fatty acid composition of analyzed samples of spanish dry cured ham showed approximate mean percentages of sfa and monounsaturated fatty acid (mufa) of about 43% of the total fa, and 13.87% of pufa. in a study conducted by bermúdez et al. (2012) on the effect of the inclusion of chestnut in the finishing diet (from three different diets: concentrate, mixed and chestnut) on fatty acid profile of dry-cured celta the percentage of sfa (35 %) pufa (13%) and mufa (51%) in mixed diet were similar to those found herein. in the present study the most abundant fatty acids in both types were monounsaturated fatty acids (mufa) (mainly c18:1 c-n9) followed by sfa and finally polyunsaturated fatty acids. the different typology caused some changes in the fatty acid composition of dry-cured ham (table 1). in particular, “nero di parma” ham showed a significant lower sfa content and higher mufa than parma ham as a direct consequence of the high oleic acid content of acorns as reported in iberian pigs (ruiz et al., 1998; ruiz-carrascal et al., 2000). numerically, the values of oleic acid in “nero di parma” agree with those found in spanish ham (perez-palacios, 2010). moreover, campo and sierra (2011) monitored different varieties of spanish dry cured ham produced from the same company in two years (19952007). the oleic acid (c18: 1n9) levels found overlap those found in “nero di parma” ham in the present work. the levels found in this study are lower but not too far from those of fernández et al. (2007) who found approximately 49% of c18:1 n-9, considered a healthy product in a diet. moreover, the oleic acid content of “nero di parma” ham agrees with values coming from pigs of parma dop circuit fed with 6% sunflower oil in which a significant increase of oleic acid in the treated group was found (bosi et al., 2000). “nero di parma” ham compared to parma showed a significantly lower content of both stearic acid (c18: 0) and myristic acid (c14: 0). moreover, the content of total n-3 pufa and particularly α-linolenic acid (ala, c18:3n-3) was greater (p<0.05) in “nero di parma” than parma dry cured ham. this is likely due to the different rearing system, which characterize the two typologies. according to muriel et al. (2002), free-range rearing and feeding pigs on pasture increase the levels of long chain n 3 fatty acids in porcine muscles which is in agreement with the present results. ital. j. food sci., vol. 31, 2019 406 table 1. effects of typology (parma ham and “nero di parma ham”) on fatty acid composition of muscle and adipose tissue of dry-cured ham. muscle tissue fat tissue p value1 parma “nero di parma parma “nero di parma” t2 typ3 tex x typ c14:0 1.07±0.17 0.87±0.2 1.17±0.36 0.92±0.3 ns * ns c16:0 24.00±0.87 23.40±0.86 24.01±0.85 24.03±2.4 ns ns ns c16:1n7 2.42±0.81 2.37±0.77 2.00±0.81 2.24±0.59 ns ns ns c17:0 0.16±0.53 0.15±0.48 0.15±0.44 0.17±0.39 ns ns ns c17:1 0.12±0.04 0.15±0.03 0.13±0.04 0.15±0.02 ns ns ns c18:0 11.00±0.95 9.15±1.26 11.23±1.40 9.53±1.37 ns *** ns c18:1n9 44.40±2.01 47.40±2.87 44.96±2.43 46.5±4.02 ns *** ns c18:2n6 10.38±0.83 9.90±2.27 11.11±1.12 10.74±1.40 ns ns ns c20:0 0.08±0.01 0. 08±0.03 0.07±0.04 0.10±0.02 ns ns ns c18:3n3 0.32±0.10 0.42±0.10 0.38±0.08 0.48±0.06 ** *** ns c20:1n9 0.64±0.10 0.71±0.15 0.60±0.12 0.68±0.20 ns ns ns c20:2n6 0.37±0.06 0.34±0.06 0.39±0.10 0.35±0.10 ns ns ns c20:3n3 0.02±0.02 0.03±0.01 0.03±0.03 0.04±0.03 ns ns ns c20:3n6 0.07±0.06 0.06±0.03 0.05±0.08 0.03±0.02 ns ns ns c20:4n6 0.48±0.25 0.58±0.44 0.10±0.17 0.11±0.07 *** ns ns c23:0 0.23±0.21 0.15±0.14 0.04±0.05 0.13±0.30 ns ns ns sfa 36.58±1.34 33.82±1.78 36.74±1.52 34.89±3.74 ns *** ns mufa 51.77±1.56 54.85±2.94 51.18±2.03 53.83±3.75 ** *** ns pufa 11.65±0.95 11.32±2.77 12.07±1.39 11.77±1.55 ns ns ns n-6 pufa 10.94±0.93 10.53±2.65 11.27±1.30 10.89±1.47 ns ns ns n-3 pufa 0.35±0.11 0.44±0.11 0.41±0.10 0.52±0.07 † *** ns 2t= tissue; 3 typ: tipology; probability (*) statistical significance at p<0.05, (**) statistical significance at p<0.01, (***) statistical significance at p<0.001, (†) = p<0.10 previous studies conducted in heavy pigs (pastorelli et al., 2003) (160±10 kg of bw) and in light pigs (santos et al., 2008) showed that the fatty acid composition of dry cured ham depends on the dietary fatty acid composition. both studies observed an increase in ala content in dry-cured ham obtained from pigs fed diets enriched in n-3 pufa. the increase of linolenic acid and monounsaturated fatty acids content, in “nero di parma ham” is certainly positive considering the indications provided by human diet for the important role of monounsaturated fatty acids and fatty acids of the omega 3 series. a trend was noted for n-3 pufa in the two tissues analysed (table 1). as far as linolenic acid is concerned, the content is higher in adipose tissue than in muscle (0.43 vs 0.37), according to musella et al. (2009) and as expected. in contrast, eicosapentaenoic acid (epa) and docosapentaenoic acid (dpa) are preferentially stored in the organs or muscle rather than in the adipose tissue. in the present work, these fatty acids have not been quantified. no significant effect has been found for tissue x typology. typology did not affect (p>0.05) linoleic acid (c18:2n6) in any of analysed tissue. regarding the curing process of meat and in order to obtain adequate fat quality, adipose tissue should contain no more than 12% of c18:2n-6 and exceed 12% of c18:0 (mourot and hermier, 2001). adipose tissues studied in the present research were within those ranges. ital. j. food sci., vol. 31, 2019 407 the nature and proportion of individual fatty acids, especially sfa to pufa, is important in assessing the quality and nutritional value of fats. the proportions of these fatty acid groups have been used to calculate the very important risk factors for atherogenic index (ai) and thrombogenic index (ti). a lower ai value indicates a lower proportion of saturated to unsaturated acids, which reduces ability of endothelium of blood vessels to attach lipids and plaque formation. the ti at its lower index indicates a lower risk of occurrence of disturbance of the blood coagulation process and blood clotting. both these indices in the present study were at an appropriate low level in both typologies of ham, close to that observed in wild boar meat (razmaite et al., 2012). both ai (0.42 vs 0.45) and ti (1.00 vs 1.20) indices were significantly lower in “nero di parma” ham than parma ham. our results are consistent with those obtained by cebulska et al. (2018), who reported an average value of 0.40 and 1.07 for ai and ti respectively in pork meat originating from pigs of polish native pure breeds. a significant reduction of the nutritional indexes has been reported in cow’s milk (caroprese et al., 2010), ewe’s milk (cieslak et al., 2010; caroprese et al., 2011), and rabbit meat (peiretti and meineri, 2010) after linseed feeding. cieslak et al. (2010) showed reduction of the atherogenic index from 1.4 to 0.5 and thrombogenic index from 0.8 to 0.4. okrouhlá et al. (2013) reported a thrombogenic index (1.33 and 1.28 vs. 0.80 and 0.85) in meat of barrows and gilts. according to previous studies (lorenzo et al., 2012, franco et al., 2006) that found the effect of carcass location on the fatty acid profile, we investigated the effect of location in three different locations of the ham. fatty acid composition differences in relation to location were found. in particular, they were identified for the following fatty acids: c18:0, c18: 1n9, c20: 0, c20: 3n3, c20: 4n6 with consequent effect on sfa (p = 0.007), mufa (p<0.001) and n-6 pufa that showed just a trend (p = 0.075). in particular, it is noted that sfa exhibit a higher concentration in the initial part (36.73), the mufas in the final part (54.99) and the pufa in the central part (12.49) of the sampled hams (fig. 2). figure 2. effect of sampling location on saturated fatty acids (sfa), polyunsaturated fatty acids (pufa) and monounsaturated fatty acids (mufa) content. ital. j. food sci., vol. 31, 2019 408 the highest content of mufa in the final part agrees with results of cava et al. (2000) who found higher percentages of oleic acid and mufa in a deeper muscle than in superficial one. sfa are mainly located in the initial part corresponding to a location with higher adipose accumulation. the nutritional value of adipose tissues is related to high values of pufa/sfa, mufa/ sfa ratios and low values for n-6/n-3 ratio. in the present study all cited ratios were significantly improved in “nero di parma” ham. 3.1. sensorial analysis there are various kinds of dry-cured hams in mediterranean countries, distinguished by the origin and the quality of raw material. dry-cured hams can be categorized into two types: (1) originating from traditional breeds, usually accompanied by extensive outdoor rearing systems and (2) originating from modern lean breeds, raised under intensive systems (čandek-potokar and škrlep, 2012). consumer demand regarding the sensory quality of dry cured ham varies according to the region and local habits. many highly regarded traditional products are based on the exploitation of natural resources like acorn pastures in the current case or in the case of the iberian pig. taste is the key factor affecting consumer satisfaction with dry-cured ham (resano et al., 2011). however, familiarity with a product affects quality expectations and perceptions of consumers, which explains why different dry-cured ham characteristics are appreciated in different countries. the least square means of the different attributes for the samples of dry cured ham are reported in the spider plot (fig. 3). the panelist effect was significant (p<0.01) for all descriptors; this statement is very common in sensory evaluation because of a different use of scores and disagreement within the assessment of sample (lea et al., 1997). no effect for replicates and no interaction between panelists × replicates and samples × replicates were found except brightness (p=0.018) and sweetness taste (p = 0.024). these results underline the excellent reproducibility of scores given by the panelists and excellent homogeneity of samples during repetitions. typology x replication interaction was not significant; this result would therefore seem to indicate that the mean scores for each sample given by the panelists for each descriptor are satisfactory estimates of the sensory profile of parma and “nero di parma” ham. anova of the sensory data showed a significant effect in the following descriptors: redness, marbling, oily, brightness, aged odor, rancid both in odor and flavor, sweetness, bitter and dryness. numerical values of the considered descriptors of parma ham are comparable with literature (laureati et al., 2014). redness descriptor was higher in “nero di parma” (7.4 vs 5.88) than parma ham. this result is consistent with those previously reported by (estévez et al., 2003, 2006) who described a higher a* value, chroma and iron content in muscles from rustic pig breeds than in those from selected ones. in addition to the breed effect, the characteristics of the iberian pigs’ livestock farming could have influenced since the pigments and iron concentrations and the red colour of the muscles increase with the animal age and the physical exercise (lawrie, 1998). this descriptor is linked to the sensory perception of meat color: consumers consider bright red meat as fresh and are adverse to brown meat. in addition, the highest oiliness appearance of “nero di parma” agrees with values of montanera ham (jurado et al., 2007) due to a high fat content of “ham as underlined by ct. the highest value of oiliness produced the highest brightness value in “nero di parma” (5.27 vs 4,22). ital. j. food sci., vol. 31, 2019 409 figure 3. sensory analysis results: mean values for each sensory descriptor by parma and “nero di parma” ham. for each descriptor the relevant significance is reported. (***significant at p< 0.001, **significant at p<0.01, *significant at p<0.05). in addition, the fat unsaturation affects dryness of ham meat resulting higher in parma ham according to results of ruiz et al. (2002) obtained in meat. however, an high fat content is associated with the phenomena of oxidation; the panel, both aroma and odor evaluation, found a greater value of rancid in the “nero di parma” ham (p <0.001 and p <0.003) than parma ham. this attribute is often present in longmatured products, such as ham, and it is also reported in the iberian typology (garciagonzalez et al., 2006; ruiz et al., 2002). rancidity is considered as a defective note of the product if present in high density. however, in contrast to the negative effect of lipid oxidation in pork, the formation of volatile lipid oxidation components during ripening/fermentation is important for the oxidative flavour note of parma ham. it is well known that the typical ham aroma is related to intramuscular lipid composition and to the extent of lipolysis and oxidation of lipids during processing (berdaguè, et al., 1993; buscailhon et al., 1994). in fact, muscle and adipose tissue lipids are subject to intense lipolysis by the action of lipases, generating free fatty acids that, at a second stage, are transformed to volatiles as a result of oxidation. sensory profiles of dry cured ham are strongly affected by these enzymatic reactions (toldra et al., 1997). “nero di parma” also showed the most intense aged odor according to the prolonged seasoning period (30 vs 24 months). as for marbling, a significant difference was obtained between the two types, higher in parma than in “nero di parma” ham (5.13 vs 4.53). it must be emphasized that marbling score is not only affected by the total fat area of fat ital. j. food sci., vol. 31, 2019 410 flecks, but also by the distribution of fat (cheng et al., 2015). the total lipid content of muscle was equal to an average value of 7.1% (s.e. 0.88) and 10.2% (s.e. 1.26) (p = 0.65) in parma ham and “nero di parma ham” respectively. this result agrees with ct analysis as described below. taste descriptor related to sweetness, a typical characteristic of parma ham, highlighted in parma ham higher values (3.93 vs 3.43) than “nero di parma ham”. in contrast, “nero di parma”, characterized by a lower sweetness, resulted more bitter (3.18 vs 2.28); this attribute could be caused by a greater presence of myoglobin. higher myoglobin levels in bull meat have been indicated as leading to greater sensations of metallic, liver, and bitter flavors. as recently reported, native pig breeds are characterized by the highest redness and the higher content of heme pigments (cebulska et al., 2018). in order to satisfy the requirements of the modern meat industry, it is important to develop some non-destructive, accurate, and rapid techniques for assessing meat quality and safety as recently reviewed (xiong et al., 2017). computerized tomography has been used mostly in medicine for diagnostic purposes. nevertheless, it has also been proven to be useful in other fields and its application has been extended to palaeontology, geology and also to food technology and the meat science field. application of computed tomography (ct) in meat science is based on the different x-ray attenuations that different tissues produce. it has been demonstrated to be useful in the estimation of body composition in animals and to measuring lean percentage in pig carcasses (picouet et al., 2010). in the present paper hounsfield units (hu), transformed by the software as cm3, were then expressed as a percentage; the different distribution of lean and fat can be appreciated from the images reported in fig. 4. figure 4. distribution of fat and lean tissue in parma ham and “nero di parma ham”. ital. j. food sci., vol. 31, 2019 411 as graphically presented in fig. 5 the opposite content of lean and fat component in the two types of analyzed hams (“nero di parma” 39% and 55%, parma: 59% and 34%) could be noted respectively. surprisingly, connective content is similar between the two types. monziols et al. (2006) evaluating by magnetic resonance imaging the proportion of different carcass cuts reported a value of 62% (st. dev. 7.3) and 20.2% (st. dev. 6.3) in muscle and fat of ham respectively. the percentage of muscle overlaps the values found in parma ham. the highest fat content of “nero di parma ham” is due to an adaptive mechanism to the environment, which is known as thrifty genotype and which was firstly described in humans (neel, 1962) and also in iberian pigs (garcia-contreras et al., 2018). the thrifty genotype facilitates accommodation to seasonal cycles of feasting and famine because the ability to store fat in excess during food abundance enables survival during periods of scarcity. an higher fat content implicates a longer ripening process from which depends all chemical and physical changes affecting volatile compounds during processing. figure 5. the lean, fat, and connective tissue surface ratio in “nero di parma” ham and parma ham. the connective tissue content of meat varies with species, chronological age, state of nutrition of the animal and muscle fiber characteristics (klont et al., 1998). meat texture is directly related to the size of muscle fiber and the amount of connective tissue (joo et al., 2013). moreover, connective tissue also undergoes morphological changes during meat-aging (bailey and light 1989; nishimura 2015). the results of ct are consistent with the evaluation of the sensory analysis carried out on the ham; in this study no difference was found in texture also in accordance with the study of nishimura (2010). 4. conclusions in conclusion, the results of the quality tests conducted on raw cured ham from the “nero di parma” pig emphasize high processing value and nutritional value of the meat of these animals. the profile of fatty acids and the proportions of individual fatty acids expressed in the form of disease risk indices (ai, ti) allow us to characterize the “nero di parma” ital. j. food sci., vol. 31, 2019 412 ham as a product with health value. “nero di parma” ham with the high monounsaturated and omega-3 fatty acids content, could play an important role for human health; moreover, the omega6/omega3 ratio was improved in “nero di parma” ham more than in parma ham. the present study provides additional data on parma pdo ham with regard to the fatty acid composition, and sensory analysis, confirming literature and the positive evaluation of this product. the sensorial profile of a typical product is the starting point for a strategy to enhance the typicality. the results of this preliminary study indicated that “nero di parma” ham, for which sensory investigations are not known, highlighted some descriptors such as oiliness, brightness, redness significantly different from parma ham, and useful to define the sensorial profile of a typical product. this study is only a first approach from which further investigations can emerge. the numbers will have to be expanded in order to reach a definitive 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deposition and eating quality in pig. meat sci. 67:651. xiong z., sun d.w., pu h., gao w. and dai q. 2017. applications of emerging imaging techniques for meat quality and safety detection and evaluation: a review. crit. rev. food sci. nutr. 57:755. paper received june 21, 2018 accepted october 15, 2018 ijfs#1252_bozza ital. j. food sci., vol. 30, 2018 752 paper gc-c-irms characterization of synthetic bis(methyl-thio)methane in truffle flavorings m. bononi*1, f. tateo1, f. benevelli2, a. pennetta3 and g. de benedetto3 1department of agricultural and environmental sciences, university of milan, via celoria 2, 20133 milan, italy 2bruker italia s.r.l., viale lancetti, 43, 20158 milan, italy 3department of cultural heritage, university of salento, s.p. 6, monteroni, lecce, italy *corresponding author: tel.: +39 0250316538 e-mail address: monica.bononi@unimi.it abstract bis(methyl-thio)methane (bmtm), the molecule which provides “white truffle-like” flavor was characterized by physico-chemical methods. analysis by gc-c-irms of eight samples of synthetic bmtm from various raw material suppliers allowed the investigation of the δ13c values. more, ten samples purchased on the italian flavoring market, declared as synthetic bmtm principal component diluted in olive oil were analyzed by gc-c-irms. the results of both sample groups allowed us to define the range of δ13c values of synthetic bmtm. we verified if the simple proposed extraction method allows to measure the δ13c value of bmtm also identified in seasonings of the italian market declared on label as “white truffle flavored olive oil”. in all twenty samples purchased on the market, the data strictly corresponded with synthetic bmtm as the principal component. measurements by 1h nmr made on synthetic bmtm and bmtm extracted from “white truffle-like flavor” confirmed that the adopted extraction method using methanol-d4 determined the isotopic distribution of 13c/12c ratio in two characteristic sites of this molecule. keywords: white truffle flavoring, bmtm, gc-c-irms, 1h nmr ital. j. food sci., vol. 30, 2018 753 1. introduction tuber magnatum (white truffle), tuber aestivum (summer truffle), and tuber melanosporum (black truffle) are the most well-known species belonging to the genus tuber f.h. these species have been the object of numerous studies using various analytical systems for the identification of the compounds that provide the distinctive aroma of these fungi (bellesia et al., 1996; díaz et al., 2002; díaz et al., 2003; talou et al., 1989). several reports have considered the constituents responsible for the typical aroma and have also studied the quantitative and qualitative fluctuations of these compounds, depending upon truffle type and geographical origin (costa et al., 2015; fiecchi et al., 1967; gioacchini et al., 2008; mauriello et al., 2004; pelusio et al., 1995). presently, pure natural bmtm derived from truffles is not available on the raw material flavor market because the levels of bmtm in truffles are too low for satisfactory isolation of a significant quantity of this molecule (schmidberger and schieberle, 2017). also, the commercial cost of the natural bmtm from the white truffle would be unsustainable because the raw material is very expensive (borsino del tartufo, 2018). therefore, due to the non-feasibility of bmtm isolation from the natural matrix, no measurements using gas chromatography-combustion-isotope ratio mass spectrometry (gc-c-irms) have been previously carried on. the european union (eu) regulation 1334/2008 allows the citation of the flavoring source (in our case “white truffle”) only if the flavoring components are obtained exclusively or by at least 95% (w/w) from the source material referred to and the other maximum 5% can derive only from other natural sources. therefore, the citation in the label “natural flavoring”, without the citation of a defined natural source, may only be used if all the flavoring components derive from natural sources. if one or more synthetic compounds are present in a flavoring formulation, the label must report only the term “flavor” without any reference to a food, food category or a vegetable or animal flavoring source (reg. eu 1334, 2008). the corresponding naturally-occurring identical compound, easily synthesized by the oil industry and supported in olive oil (pacioni et al., 2014), is used as a flavoring agent for truffle flavored food products. among the “white truffle-like” flavored foods, extra virgin olive oil, flavored with bis(methyl-thio)methane (bmtm), used as a seasoning, occupies the most important position in the market. using a simple extraction method and the gc-c-irms analytical technique, we aimed to ascertain the reliability of characterizations of the synthetic bmtm molecule present in “white truffle-like” flavors and seasonings. following the eu regulation 1334/2008 before cited, the identification of synthetic bmtm in the formulation of flavors and seasonings does not allow the use on the label the term “natural white truffle flavor”, or “natural flavor” or “white truffle flavor”, but only the term “flavor”. synthetic bmtm characterization by gc-c-irms, confirmed by 1h-nmr, allowed us to demonstrate the identity of the bmtm molecule extracted from “white truffle-like” flavors purchased on the italian flavoring market and from olive oil seasonings available on the italian consumer market. 2. materials and methods 2.1. solvents methanol (anhydrous) (99.8) and methanol d-4 were purchased from sigma aldrich (milan, italy). ital. j. food sci., vol. 30, 2018 754 2.2. samples analyzed by gc-c-irms eight samples of bmtm (c2h8s2) declared to be synthetically derived were obtained. the first purchased was a standard (> 99%) from sigma aldrich (italy) and the others were purchased on the flavor raw material market (fci, frutarom, moellhausen, penta, treatt, sterling, sigma aldrich). ten samples of “white truffle-like” flavors purchased on the italian flavorings market and declared as synthetic bmtm diluted in olive oil. twenty “white truffle-like” seasonings purchased on the italian market and consisting of flavored extra virgin olive oil, or olive oil, or sunflower oil. all the seasonings were declared to contain olive oil or extra-virgin olive oil except the seasoning n. 4, which contains sunflower oil. the product n. 8 reported on the label “natural flavor”. the product n. 13 reported on the label “white truffle natural flavor”. the products n. 6, 11, 16, and 19 reported on the label “white truffle flavor”. the other products reported on the label “flavor”. 2.3. samples analysis by 1h nmr two samples were analyzed, one corresponding to the standard bmtm (sample n.1 in table 1) and another corresponding to a “white truffle-like” flavoring (sample n.1 in table 2). 2.4. samples preparation for gc-c-irms analysis, samples of synthetic bmtm (table 1) were dissolved in anhydrous methanol (99.8) at a concentration of 60 µl ml-1. the mix was vortexed for 1 min. for samples of “white truffle-like” flavoring (table 2) an aliquot of 3 ml was vortexed with 1 ml of methanol for 5 min and then centrifuged at 5000 rpm for 5 min. the methanol layer was isolated by a pasteur pipette and utilized for the analysis. for samples of seasonings, an aliquot of 5-10 ml was vortexed with 1 ml of methanol for 5 min and then centrifuged at 5000 rpm for 5 min. the methanol layer was isolated by a pasteur pipette and utilized for the analysis. for nmr analysis, a sample of standard bmtm (sample n.1 in table 1) was dissolved in methanol d-4 at a concentration of 60 µl ml-1. the mix was vortexed for 1 min. for sample of a “white truffle-like” flavoring (sample n.1 in table 2), an aliquot of 3 ml was vortexed with 1 ml of methanol d-4 for 5 min and then centrifuged at 5000 rpm for 5 min. the methanol layer, isolated by a pasteur pipette, was utilized for the analysis. 2.5. gc-c-irms analysis the system consisted of an agilent technology 7890a gas chromatograph equipped with a g4513a autosampler (agilent technology, germany) and coupled to an isoprime stable irms gc5 (isoprime, cheadle, uk) via a combustion interface under a continuous flow of helium. the combustion interface consisted of a ceramic furnace with a copper oxide and platinum catalyst at 850°c. the carbon stable isotope ratio was determined by referring to the international standard vienna peedee belemnite (δ13cvpdb) with a defined 13c content. the crm used for the gc-c-irms multipoint calibration were n-undecane (δ13c: -26.11‰, chiron c0414.11-150-cy), n-pentadecane (δ13c: -30.22‰, chiron c0418.15-150-cy) and nhexadecane (δ13c: -34.87‰). the hexadecane delta value was obtained by ea-irms using the following primary standards: glucose (δ13c: -10.76‰, sigma aldrich bcr657), ital. j. food sci., vol. 30, 2018 755 polyethylene (δ13c: -32.15‰, iaea iaea-ch-7) and lithium carbonate (δ13c: -46.60‰, nist rm 8545). isotope ratios were expressed as values (‰) and calculated on the basis of the following equation: (i r sa – i r ref) δi e = i r ref where “i” is the mass number of the heavier isotope of element e (in this case 13c), r sa is the respective isotope ratio of the sample and rref is the relevant internationally recognized reference material. the delta values were multiplied by 1000 and expressed in units “per mill” (‰) (coplen, 2011). the gc was operated using a hp-5 capillary column, 30 m x 0.32 mm i.d., 0.25 µm film thickness (agilent – italy). helium was used as the carrier gas at a flow rate of 1.2 ml. the oven temperature program was initially 50°c (held for 1 min), then increased to 150°c at a rate of 5°c min-1, then increased to 250°c at a rate of 20°c min-1 (held for 1 min). the injector temperature was 220°c and the injection volume was 1 ml (split 1:10). data were collected in triplicate. 2.6. nmr analysis the 1h nmr spectra were measured on a bruker spectrometer aviii400 equipped with a samplexpress sample changer and using a bbiz probe. the spectra were acquired using a 30° excitation pulse width of 2.66 µs, relaxation delay of 40 sec, and acquisition time of 5 s at 298 k. t1 relaxation times of the quantified peaks were measured with inversionrecovery experiments to assure that no bias from a short d1 would arise. the longest t1 measured for the standard bmtm (sample n.1 in table 1) was 6.96 s, found for the ch2 peak, while for the “white truffle-like” flavoring (sample n.1 in table 2) we observed 6.02 sec for the same peak as the longest t1. thus, the chosen d1 was sufficient to ensure a complete relaxation of the magnetization and to give fully quantitative results. the spectral width was 40 ppm centered at 6.5 ppm to ensure that the baseline was perfectly flat, as this is a precondition to correct integration and quantification of the peaks. for each spectrum, 64 scans were summed. the acquisitions were performed in a fully automated way. the spectra were processed with 260 k points and an exponential multiplication of 0.3 hz. the data were acquired, processed, and analyzed using the software program topspin 3.5 from bruker biospin. phasing and baseline correction were performed manually. in the case of the standard bmtm (sample n.1 in table 1), the two main peaks of the h1-c12 were considered together with both satellites for each peak. however, in the case of the “white truffle-like” flavoring (sample n.1 in table 2), only one satellite per peak was considered due to the superposition with other signals in the matrix. the intensity of the peak was doubled for the calculation of the isotopic ratio. 3. results 3.1. gc-c-irms the samples of commercially available synthetic bmtm could be readily prepared for gcc-irms analysis by simple dilution in anhydrous methanol (table 1). ital. j. food sci., vol. 30, 2018 756 similarly, the extraction method adopted for samples reported in tables 2 and 3 allowed us to isolate bmtm as a methanolic extract for use in gc-c-irms measurements. for samples reported in tables 1 and 2, corresponding to bmtm declared from synthesis and “white truffle-like” flavors purchased on the italian flavorings market, respectively, the δ13c values varied in the tight range of -42.24 and -43.40‰. the above cited range, deduced from analysis of a sufficiently large number of samples (standards and flavorings) produced with “bmtm” certified from synthesis, was very useful for characterization of the “synthetic” authenticity of the molecule subjected to δ13c measure. in addition, the same data, as expected, was not consistent with the carbon isotope ratio natural abundance ranges, as documented in literature data (van leeuwen et al., 2014). data obtained for seasonings of table 3 are also included in the range between -42.34 and -43.26‰, and was not significantly different from the values corresponding to the synthetic samples cited above. table 1. gc-c-irms data produced by eight samples declared as bmtm from synthesis and available on the flavoring market. sample δ13c ‰ (mean) s 1 -43.35 0.43 2 -42.30 0.35 3 -43.05 0.34 4 -42.47 0.32 5 -42.24 0.35 6 -43.40 0.34 7 -42.26 0.34 8 -43.14 0.41 table 2. gc-c-irms data produced by ten samples, all declared as “white truffle-like” flavors, purchased on the italian flavorings market and consisting of synthetic bmtm as the principal component diluted in olive oil. sample d13c ‰ (mean) s 1 -42.50 0.58 2 -42.43 0.53 3 -43.12 0.41 4 -42.40 0.34 5 -42.55 0.38 6 -42.64 0.47 7 -43.18 0.52 8 -42.38 0.45 9 -42.46 0.37 10 -42.52 0.46 ital. j. food sci., vol. 30, 2018 757 table 3. gc-c-irms data produced by twenty samples of white truffle flavored oils purchased on the italian market (glass pack 40-250 ml). seasoning δ13c ‰ (mean) s 1 -42.34 0.42 2 43.08 0.34 3 -42.81 0.52 4 -42.63 0.36 5 -43.16 0.38 6 -42.86 0.48 7 -43.02 0.35 8 n.d. --- 9 -42.53 0.46 10 -43.12 0.43 11 -43.06 0.37 12 -43.18 0.51 13 -42.68 0.45 14 -42.48 0.39 15 -43.04 0.37 16 -43.10 0.41 17 -42.66 0.56 18 -42.74 0.46 19 -43.26 0.38 20 -42.45 0.42 3.2. 1h nmr characterization of bmtm the aim of our study was to develop an approach using nmr methodology to characterize the bmtm synthetic molecule of a standard sample and to verify the possibility of using the extraction method referred in “2.4 sample preparation” to characterize the synthetic bmtm present in a “white truffle-like” flavor. we measured the values 13c/12c for the two sites corresponding to ch2 and ch3 in the bmtm molecule extracted from the two samples. our data showed the structural isotopic distribution of 13c/12c in the characteristic sites of these two molecules. 3.2.1 bmtm from synthesis (sample 1, table 1) the 1h nmr spectra of the synthetic standard bmtm were acquired using two aliquots of the same sample diluted in methanol-d4 and in replicates to evaluate the experimental cv% (coefficient of variation %) from 8 trials. the bmtm 1h nmr spectrum in methanol-d4 displayed two singlets, resonating at 3.68 and at 2.15 ppm. the signals are attributed to the ch2 (3.68 ppm) and the ch3 (2.15 ppm). the 13c satellites of the two peaks were easily recognizable and they appeared as doublets with a heteronuclear j coupling of 149.9 hz for the ch2 and of 138.5 hz for the ch3. the doublets were centered at the isotropic resonances of the peaks. as expected, the intensities of the satellites were significantly lower than those of the main peaks, which were attributed to the 1h attached to the 12c. ital. j. food sci., vol. 30, 2018 758 the ratio between the intensities of satellites due to the 1h 13c heteronuclear coupling and that of the main peak allowed us to extract the integral ratio between 13c and 12c in a site specific manner. the 1h nmr spectrum (fig. 1) and the results (table 4) indicated that the ratio of 13c/12c gave the same value for both sites (ch2 and ch3). this value is in agreement with the expected 13c/12c global ratio. figure 1. 1h spectrum of the bmtm from synthesis, available on the flavor raw material market (sample 1, table 1). the asterisk indicates the 13c satellites. the dotted lines indicate the integral regions. table 4. data %13c measured by 1h nmr spectra in replicates of two aliquots for each sample: synthetic bmtm standard (sample 1 in table 1), synthetic bmtm from “white truffle-like” flavor (sample 1 in table 2). % 13ch bmtm synth. bmtm synth. std. flav. replicates -ch2-ch3 -ch2-ch3 1 1.02 1.06 1.01 0.99 2 1.03 1.02 1.07 0.99 3 1.05 1.05 1.03 1.06 1 1.05 1.05 0.99 1.02 2 1.02 1.00 0.99 0.99 3 1.00 1.01 0.99 1.04 4 1.01 1.00 0.99 1.02 5 1.03 0.99 1.03 1.05 mean 1.03 1.02 1.01 1.02 cv% 0.02 0.03 0.03 0.03 al iq uo t 1 al iq uo t 2 ital. j. food sci., vol. 30, 2018 759 3.2.2 bmtm extracted from synthetic white truffle-like flavoring (sample1, table 2) the same approach described for the synthetic bmtm standard was applied to the synthetic bmtm in “white truffle-like” flavoring. as in the case of the molecule bmtm from synthesis, the spectrum was acquired in 8 replicates derived from two extracts produced from two aliquots of synthetic bmtm from “white truffle-like” flavoring. the 1h spectra of the two aliquots extracted with methanol-d4 showed similar features to that of the synthetic bmtm standard, with the two singlets in the same position (3.67 ppm for ch2 and 2.14 ppm for ch3) and the same heteronuclear j couplings (149.9 hz for the ch2 and of 138.5 hz for the ch3). the 0.01 ppm shift is attributable to a matrix effect and it is reproducible in all the replicates. the spectrum acquired is reported in fig. 2 and also in this case, as reported in table 4, the ratio of 13c/12c gave the same value for both sites (ch2 and ch3). as shown in fig. 2, the presence of a potentially interfering substance often found in “white truffle” flavor (identifiable by gc/ms as methylsulfinyl(methylthio)-methane) did not hinder the identification of 13c satellites and the % 13c values were strictly comparable to data obtained for synthetic bmtm standard. to clarify, the 1h nmr spectrum of the sulfoxide is easily recognizable by these features: one singlet at 2.71 ppm attributable to one terminal methyl; one singlet at 2.33 ppm attributable to the second terminal methyl; two doublets centered at 3.98 ppm and at 3.83 ppm with a j=13.8 hz attributable to the ch2 protons that became diastereotopic upon asymmetric oxidation. figure 2. 1h spectrum of the “white truffle” flavor purchased on the italian flavor market and consisting of synthetic bmtm as the principal component diluted in olive oil. the asterisk indicates the 13c satellites. the dotted lines indicate the integral regions. the inset shows the expansion of the 4.05-3.75. these peaks are attributed to the ch2 protons in a molecule identified as methylsulfinyl(methylthio)-methane. ital. j. food sci., vol. 30, 2018 760 4. discussion the simple extraction method, adopting methanol as the solvent, is useful for application to provide data by gc-c-irms of a synthetic bmtm standard, in flavoring from the raw material market (see table 1), and in italian flavoring from the commercial market (see table 2). the δ13c‰ data collected for all these matrices ranged between -42.24 (σ 0.35) and -43.40‰ (σ 0.34). clearly, the range is external to the natural abundance of the carbon isotope ratio, and allows one to identify the synthetic origin of bmtm if this molecule is used to produce seasonings made by the addition to olive oil or other vegetable oils. the δ13c‰ data obtained with the same simple extraction method on twenty seasonings purchased on the italian market allowed us to deduce that the synthetic bmtm molecule is clearly identifiable. in fact, the δ13c‰ values reported in table 3, except for the sample n. 8, which does not contain bmtm, exhibited data in the range -42.34 and -43.26‰, identified as characteristic of synthetic bmtm. most seasoning samples – representing about 75% of the total seasoning samples examined (namely, n. 1-5, 7, 9, 10, 12, 14-15, 17-18 and 20 in table 3) are compliant with the eu regulation 1334/2008 because they contain synthetic bmtm and on the label only the term “flavor” is correctly used. samples n. 6, 11, 13, 16, and 19 in table 3 are not compliant with the eu regulation 1334/2008 because they contain synthetic bmtm and report on label the reference to the term “truffle”. specifically, the term “white truffle flavor” was used for samples 6, 11, 16, 19, while “natural white truffle flavor” for sample 13. for the sample n. 8 in table 3, the only one resulting as not containing bmtm, and reported on label as produced with “natural flavor”, the judgment of compliance or otherwise with the eu regulation 1334/2008 does not depend from the bmtm identification (natural or synthetic), but from the origin of all the molecules constituting the flavor. in fact, a natural flavor used for a seasoning can be realized totally with natural molecules. in this case, the final judgment is not linked to the bmtm identification, but from the naturalness of all the components of the flavor used. there was no evidence of data that did not fall within the range characteristic of synthetic bmtm, and it is justified by being anyhow unavoidable due to the lack of commercial availability of natural bmtm in the raw material flavor market. adopting the same extraction method, but using methanol d-4 as the solvent, we demonstrated the feasibility of 1h nmr measures to calculate % 13c/12c in two characteristic sites -ch2and -ch3. this investigation, applied to a declared synthetic bmtm standard (sample 1 in table 1) characterizes the integral ratio between 13c and 12c in a site specific manner. in fact, the 1h nmr spectrum (fig. 1) and the results (table 4) indicated that the ratio of 13c/12c gave the same value for both sites (ch2 and ch3), in agreement with the expected 13c/12c global ratio. data produced from a synthetic bmtm standard were not statistically different from the corresponding data produced for a “white truffle-like” flavor, as shown in table 4. 5. conclusions the data reported in this paper are the first gc-c-irms and 1h nmr contributions to the characterization of synthetic bmtm available on the flavoring market and in some “white truffle-like” flavors. all the analyzed seasoning (except one sample not containing bmtm) produced gc-c-irms data of bmtm in agreement with the values of the synthetic molecule. ital. j. food sci., vol. 30, 2018 761 references bellesia f., pinetti a., bianchi a. and tirillini b. 1996.volatile compounds of the white truffle (tuber magnatum pico) from middle italy. flavour frag. j. 11:239-243 borsino del tartufo, 2018. www.tuber.it/en/borsino-del-tartufo.php. (most recent access date 12 april 2018). coplen t.b. 2011. guidelines and recommended terms for expression of stable-isotope-ratio and gas-ratio measurement results. rapid commun. mass spectrom. 25:2538-2560. costa r., fanali c., pennazza g., tedone l., dugo l., santonico m., sciarrone d., cacciola f., cucchiarini l., dachà m. and mondello l. 2015. screening of volatile compounds composition of white truffle during storage by gcxgc(fid/ms) and gas sensor array analyses. lwt food sci. technol. 60:905-913. díaz p., señoráns f.j., reglero g. and ibáñez e. 2002. truffle aroma analysis by headspace solid phase micoextraction. j. agr. food chem. 50:6468-6472. díaz p., ibáñez e., señoráns f.j. and reglero g. 2003. truffle aroma characterization by headspace solid-phase microextraction. j. chromatogr. a 1017:207-214. european union (eu) regulation (ec) no 1334/2008 of the european parliament and of the council of 16 december 2008 on flavourings and certain food ingredients with flavouring properties for use in and on foods and amending council regulation (eec) no 1601/91, regulations (ec) no 2232/96 and (ec) no 110/2008. off. j. eur. union, l: legis. 2008, 51, 34-50. fiecchi a., galli kienle m., scala a. and cabella p. 1967. bis-methylthiomethane, an odorous substance from white truffle, tuber magnatum pico. tetrahedron lett. 8:1681-1682. gioacchini a.m., menotta m., guescini m., saltarelli r., ceccaroli p., amicucci a., barbieri e., giomaro g. and stocchi v. 2008. geographical traceability of italian white truffle (tuber magnatum pico) by the analysis of volatile organic compounds. rapid commun. mass sp. 22:3147-3153. mauriello g., marino r., d’auria m., cerone g. and g.l. rana. 2004. determination of volatile organic compounds from truffle via spme-gc-ms. j. chromatogr. sci. 42:299-305. pacioni g., cerretani l., procida g. and cichelli a. 2014. composition of commercial truffle flavores oils with gc-ms analysis and discrimination with an electronic nose. food chem. 146:30-35. pelusio f., nilsson t., montanarella l., tilio r., larsen b., facchetti s. and madsen j. 1995. headspace solid-phase microextraction analysis of volatile organic sulfur compounds in black and white truffle aroma. j. agr. food chem. 43:2138-2143. schmidberger p.c. and schieberle p. 2017. characterization of the key aroma compounds in white alba truffle (tuber magnatum pico) and burgundy truffle (tuber uncinatum) by means of the sensomics approach. j. agr. food chem. 65:9287-9296. talou t., delmas m. and gaset a. 1989. the volatile components of tinned black perigord truffles tuber melanosporum vitt. flavour frag. j. 4:109-112. van leeuwen k.a., prenzler p.d., ryan d. and camin f. 2014. gas chromatography-combustion-isotope ratio mass spectrometry for traceability and authenticity in foods and beverages. comprehensive rev. food sci. f.13:814-83. paper received april 30, 2018 accepted july 25, 2018 ijfs#1655_bozza ital. j. food sci., vol. 32, 2020 622 paper development of a dynamic model to predict the fate of pathogenic escherichia coli in diced cucumber under changing temperatures j. ha1, e. park2, j.s. kim3,4, j. lee1, s. lee1, s. kim1, y. choi2, h. oh2, y. kim2, y. lee2, y. seo2, j. kang2 and y. yoon*1,2 1risk analysis research center, sookmyung women’s university, seoul 04310, korea 2department of food and nutrition, sookmyung women’s university, seoul 04310, korea 3research division of strategic food technology, korea food research institute, jeollabuk-do, 55365, korea 4department of food biotechnology, korea university of science and technology, daejeon 34113, korea *corresponding author: yyoon@sm.ac.kr abstract escherichia coli has been detected in a variety of foods, particularly in salad vegetables, such as diced cucumbers. however, it is difficult to control this pathogen in salad vegetables, because they are consumed without additional preparation or cooking. thus, the objective of this study was to develop dynamic models to describe the kinetic behavior of e. coli in diced cucumber. the diced cucumber was inoculated with e. coli, and stored at 10°c, 20°c, 25°c, and 30°c; cells counts were then performed using petrifilm™ plates. the baranyi model was used to calculate lag phase duration (lpd; h) and maximum specific growth rate (µmax.; log cfu/g/h). these parameters were then fitted to a polynomial model, as a function of temperature, and a subsequent dynamic model was developed in accordance with these primary and secondary models. the performance of the model was evaluated by comparing predicted data with observed data to calculate the root mean square error (rmse). as temperature increased, lpd decreased, but µmax increased. the secondary model effectively described the temperature effect on lpd and µmax, where r2 equaled 0.972-0.983. in the validation stage, rmse value of 0.272 suggested that model performance was appropriate to predict cell counts in diced cucumber, and these predictions remained appropriate under changing temperatures. these results indicate that e. coli can grow rapidly in diced cucumber at high storage temperatures, and present a useful dynamic model for describing the kinetic behavior of e. coli in this vegetable. keywords: escherichia coli, cucumber, mathematical model, dynamic model ital. j. food sci., vol. 32, 2020 623 1. introduction interest in health and diet has led to an increase in the production and consumption of fresh vegetables in recent years (wirsenius et al., 2010; vereecken et al., 2015). a survey conducted by nguyen et al. (2015) reported that 26% of foodborne illness is due to consumption of contaminated fruits and vegetables. although consumers usually think that fruit and vegetable salads are microbiologically safe, foodborne pathogens can survive and replicate in fresh vegetables (callejón et al., 2015; bennett et al., 2018). in addition, fresh vegetables are eaten raw and can, therefore, be more dangerous than other food products, following exposure to foodborne bacteria. specifically, there have been many cases of food poisoning due to contamination of sliced cucumber (decraene et al., 2012; angelo et al., 2015). escherichia coli is a facultative anaerobic, gram-negative bacillus that belongs to the enterobacteriaceae family (paterson, 2006; stepien-pysniak, 2010). e. coli is considered an indicator organism for contamination (choi et al., 2018) and major cause of foodborne illness, particularly via contamination of fresh vegetables by enteroinvasive e. coli (eiec), enterohemorrhagic e. coli (ehec), enteropathogenic e. coli (epec), enterotoxigenic e. coli (etec), and enteroaggregative e. coli (eaec) (nataro and kaper 1998; olsen et al., 2000; jang et al., 2017). among these pathotypes, etec is the most frequent cause of food poisoning and outbreaks caused by ehec have been emerging in recent years (gould et al., 2013; catford et al., 2014). predictive microbiology is a strategy that employs mathematical models to estimate the kinetic parameters of foodborne pathogens, and is aimed at securing food safety via the prevention of potential risks or hazards (whiting and buchanan, 1997; yoon, 2010). most predictive models are developed using a constant temperature (ha et al., 2019; lee et al., 2019); however, a number of variables, such as temperature and humidity, change during food storage and distribution. for this reason, a dynamic model should be used to describe the fate of foodborne pathogens under these changing conditions (ha et al., 2015; choi et al., 2016). therefore, the objective of this study was to develop a dynamic model to describe the kinetic behavior of e. coli in diced cucumber at a range of temperatures. 2. materials and methods 2.1. e. coli prevalence in cucumbers to evaluate e. coli contamination levels, 24 cucumbers were purchased from conventional markets or grocery stores in korea. twenty five-gram portions of cucumber were placed into sterile filter bags (3m, st. paul, mn, usa), and 225 ml 0.1% buffered peptone water (bpw; becton dickinson and company, bd, franklin lakes, nj, usa) was added prior to homogenization for 60 sec. for quantitative analysis of e. coli, 1 ml of the homogenate was dispensed into a petrifilm™ e. coli/coliform count plate (3m, usa), which was then incubated at 37°c for 24 h. any blue colonies with associated gas bubbles were identified and counted. for qualitative analysis of e. coli, 1 ml of the homogenate was added to e. coli (ec) broth (bd, usa) containing a durham tube and cultured at 44.5°c for 24-48 h. the aliquot in the gas-producing tube was then streaked on eosin methylene blue (emb) agar (bd, usa) and incubated at 37°c for 24-48 h. the typical colonies representing e. coli, with a green color and metallic sheen, were counted. finally, the 16s rrna gene was ital. j. food sci., vol. 32, 2020 624 amplified using the following primers: 27f (5'-aga gtt tga tcm tgg ctc ag-3') and 1492r (5'-tac ggy tac ctt gtt acg act t-3') and sequenced for identification. to determine if the e. coli isolates were pathogenic, genomic dna was extracted using a dneasy blood and tissue kit (qiagen, hilden, germany) and amplified by pcr, using a powerchek™ diarrheal e. coli 8-plex detection kit (kogene biotech, seoul, korea), according to the manufacturers’ protocols. the following pcr conditions were used: initial denaturation at 95°c for 12 min, followed by 32 cycles of 95°c for 30 sec, 60°c for 45 sec, 72°c for 60 sec, and a final extension at 72°c for 10 min. the characteristics of the genes used in pcr are shown in table 1. to confirm amplification of the target gene, the pcr product was resolved on a 1.5% agarose gel in 1x tae buffer (biosesang, seongnamsi, korea). 2.2. preparation of inocula e. coli (nccp14038, nccp14039, nccp15661, and nccp11142) were cultured in 10 ml tryptic soy broth (tsb; bd, usa) at 37°c for 24 h. one milliliter of culture was transferred into 10 ml fresh tsb and subcultures were incubated at 37°c for 24 h. the subcultured strains were mixed into a tube, and then centrifuged at 1,912 ×g at 4°c for 15 min. the supernatants were discarded, and the cell pellets were washed twice with phosphatebuffered saline (pbs: ph 7.4; 0.2 g kh2po4, 1.5 g na2hpo4·7h2o, 8.0 g nacl, and 0.2 g kcl in 1 l distilled water). the cell pellets were then resuspended in pbs, and further diluted with pbs to a final concentration of 5 log cfu/ml for inoculation. 2.3. development of predictive models cucumbers were diced into 25-g portions, then dipped into the pathogenic e. coli inocula for 3 min, drained for 10 min, and then placed in a sterile bag. the samples were stored at 10°c, 20°c, 25°c, and 30°c up to 96 h, depending on storage temperature. to enumerate e. coli in the cucumbers, the samples were aseptically transferred to sample bags (3m, usa) containing 225 ml bpw and homogenized using a pummeler (bagmixer; interscience, st. nom, france). the homogenates were serially diluted in bpw and 1 ml of each dilution was transferred to a petrifilm™ plate. the plates were incubated at 37°c for 24 h and the colonies were then manually counted. the experiment was repeated three times for each temperature. the primary model was developed by fitting the cell count data to the baranyi model, using dmfit curve fitting software (institute of food research, norwich, uk) to calculate lag phase duration (lpd; h) and maximum specific growth rate (µmax; log cfu/g/h). the equation was as follows: 𝑁! = 𝑁! + 𝜇!"#×𝐴! − ln 1 + !"# !!"#×!! !! !"#(!!"#!!!) eq. 1 where nt is the bacterial cell count at time t, and n0 and nmax are the initial and final bacterial cell counts in a growth curve, respectively. at is the adjustment function, which denotes the physiological status of bacterial cells when defining the lpd (baranyi and roberts 1994). lpd and µmax values were further-analyzed using a polynomial model as a function of temperature to develop a secondary model as follows: lpd or µmax = 𝑎! + 𝑎!𝑇 + 𝑎!𝑇! eq. 2 ital. j. food sci., vol. 32, 2020 625 where ai are the coefficient values and t is the storage temperature (°c). also h0 values were calculated for describing the initial physiological status of bacterial cells. 2.4. validation to evaluate model performance, additional experiments were performed at 15°c and 23°c. during storage, “observed data” for e. coli cell counts were obtained as described above. these observed data were then compared to the predicted data, calculated using the model. table 1. target genes for escherichia coli pathogen type with pcr. pathogen target gene size (bp) eaec1) aggr 757 ehec2) vt1 (stx1) 637 etec3) lt 530 epec4) bfpa 400 ehec vt2 (stx2) 297 epec eaea 231 etec st (sth/stp) 167 eiec5) ipah 141 1)enteroaggregative e. coli. 2) enterohemorrhagic e. coli. 3)enterotoxigenic e. coli. 4)enteropathogenic e. coli. 5)enteroinvasive e. coli. the differences between the observed and predicted data were quantified by calculating the root mean square error (rmse), bias factor (b factor) and accuracy factor (a factor) as follows: rmse = 1/n× observed data − predicticed data ! eq. 3 𝐵 factor = 10[ !"# (!"#$%&'%(# !"#$%& !"#$%&$' !"#$%& /!] eq. 4 𝐴 factor = 10[ !"# !"#$%&'%(# !"#$%& !"#$%&$' !"#$%& /!] eq. 5 where n represents the number of data points. 2.5. development of a dynamic model to describe the e. coli growth in cucumbers at changing temperatures, a dynamic model was developed with the equation suggested by baranyi and roberts (1994), in accordance with primary and secondary models detailed above. to evaluate the performance of the dynamic model, e. coli-inoculated cucumber samples were stored at fluctuating temperatures (10°c-28°c), and cell counts were performed as described above. ital. j. food sci., vol. 32, 2020 626 these cell counts were then compared with the predicted cell counts generated using the dynamic model. 2.6. statistical analysis lpd and µmax data were analyzed with a general linear model using sas® software version 9.4 (sas institute, inc., cary, nc, usa). the mean comparisons among storage temperature were performed using a pairwise t-test at α = 0.05. 3. results and conclusions e. coli and coliform bacteria were not detected in the quantitative analysis of cucumber samples, although e. coli was detected in one sample by qualitative analysis. this organism was identified as non-pathogenic e. coli via 16s rrna gene sequencing (fig. 1). e. coli has been detected in salad vegetables in previous studies (viswanathan and kaur, 2001; rahman and noor, 2012), and specifically, food poisoning associated with cucumbers has been reported in canada and sweden (decraene et al., 2012; kozak et al., 2013). thus, e. coli can be considered as an important risk in cucumbers and the behavior of this bacteria in cucumbers should be investigated. figure 1. multiplex pcr results for escherichia coli pathogen type isolated from cucumbers using primers targeting aggr, vt1, lt, bfpa, vt2, eaea, st, and ipah genes. lane 1: 100 bp-marker; lane 2: negative control; lane 3: positive control; lane 4: e. coli isolated from cucumbers. cell counts increased gradually when cucumber samples were stored at 10°c; however the counts increased rapidly in cucumbers stored at 20°c-30°c, reaching stationary phase within 12-24 h, depending on storage temperature (fig. 2). in addition, lpd (1.73-5.00 h) was very short at this temperature range (1.73-5.00 h) (table 2). similarly, µmax was measured as 0.01 log cfu/g/h in samples stored at 10°c, and increased values (0.29-0.42 log cfu/g/h) were recorded from samples stored at 20°c-30°c (table 2). these results indicate that if cucumbers are contaminated with e. coli, the bacteria can replicate and cross contamination can occur during cutting. e. coli can grow very quickly in diced cucumber during preparation, indicated by low lpd values of 3.10 and 1.73 h for storage at 25°c and 30°c, respectively. ital. j. food sci., vol. 32, 2020 627 a b c d figure 2. bacterial populations of pathogenic escherichia coli in cucumbers during storage at 10°c (a), 20°c (b), 25°c (c), and 30°c (d) for 96, 48, 48, and 48 h respectively; • observed value, fitted line. table 2. the parameters calculated by the baranyi model for pathogenic escherichia coli growth in cucumber. storage temperature (oc) lpd 1) (h) μmax 2) (log cfu/g/h) h0 3) n0 4) (log cfu/g) nmax 5) (log cfu/g) r 2 10 11.15±1.35a 0.01±0.00d 0.10 2.85±0.16 3.47±0.24 0.648 20 5.00±0.64b 0.29±0.03c 1.45 2.98±0.17 6.96±0.21 0.989 25 3.10±0.03c 0.37±0.03b 1.15 3.06±0.20 7.44±0.49 0.983 30 1.73±0.12c 0.42±0.02a 0.73 3.01±0.20 7.48±0.52 0.975 1)lag phase duration. 2)maximum specific growth rate. 3)parameter specifying the initial physiological state of cells. 4)initial cell concentration. 5)maximum cell concentration. a-dmeans within the same column with different superscript letters are significantly different (p<0.05). these results are consistent with those observed in a study by abdul-raouf et al. (1993). the h0 (which is a value multiplied by lpd and µmax) is the value obtained by quantifying the initial physiological status (baranyi and roberts, 1994; grijspeerdt and vanrolleghem, 1999; mckellar, 2001); this measure was higher at temperatures above 20°c (0.73-1.45) (table 2). this indicates that cells grown at temperatures over 20°c can adapt to the actual environment more quickly, and therefore, storing contaminated cucumbers above 20°c may increase the risk. ital. j. food sci., vol. 32, 2020 628 to evaluate the effect of the temperature on the kinetic parameters (lpd and µmax), a secondary model was developed in which r2 was calculated to be 0.972-0.983 (fig. 3), indicating that this model was appropriate to describe the effect of temperature on kinetic parameters. the rmse value was calculated to evaluate model performance, where a value close to zero indicates that the predicted values are the same as the observed values (kim et al., 2018). in this study, rmse was calculated as 0.272, indicating that the developed models were appropriate for describing the kinetic behavior of e. coli in cucumbers. also, b factor and a factor were 0.98 and 1.04, respectively. rose (1999) showed that the developed model was suitable if the b factor was 0.9 to 1.05 and the a factor was below 1.15. thus, the developed models in our study were appropriate. using the model, e. coli cell counts were predicted at changing temperatures (10°c-28°c), simulating storage, distribution, and preparation, and these predicted values were similar to the observed cell counts (fig. 4). a b figure 3. secondary predictive model for lag phase duration (lpd) (a) and maximum specific growth rate (!max) (b) of escherichia coli in cucumbers, as a function of temperature. figure 4. dynamic model for escherichia coli in cucumbers (symbol: observed cell counts, line: predicted cell counts). ital. j. food sci., vol. 32, 2020 629 this result indicates that this dynamic model is appropriate for 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needed for global food production under scenarios of dietary changes and livestock productivity increases in 2030. agric. syst. 103:621-638. yoon y., 2010. principal theory and application of predictive microbiology. food sci. indust. 43:70-74. paper received july 2, 2019 accepted february 28, 2020 ijfs#1440_bozza ital. j. food sci., vol. 31, 2019 591 paper effect of ultrasound treatment on physicochemical, functional and nutritional properties of a safflower (carthamus tinctorius l.) protein isolate m.r. zuñiga-salcedoa, j.a. ulloa*a,b, p.u. bautista-rosalesa,b, p. rosas-ulloab, j.c. ramírez-ramírezc, y. silva-carrillod, r. gutiérrez-leyvac and c. hernándeze aprograma de maestría en ciencias biológico agropecuarias. unidad académica de agricultura, universidad autónoma de nayarit, carretera tepic-compostela km 9, 63780 xalisco, nayarit, méxico bcentro de tecnología de alimentos, universidad autónoma de nayarit. ciudad de la cultura amado nervo, 63155 tepic, nayarit. méxico cunidad académica de medicina veterinaria y zootecnia, universidad autónoma de nayarit, carretera compostela-chapalilla km 3.5, 63700 compostela, nayarit, méxico dunidad académica de agricultura, universidad autónoma de nayarit, carretera tepic-compostela km 9, 63780 xalisco, nayarit, méxico ecentro de investigación en alimentación y desarrollo unidad mazatlán, av. sábalo cerritos s/n, 82100 mazatlán, sinaloa, méxico *corresponding author: tel.: +523112118851; fax +523112118861 e-mail address: arulloa5@gmail.com abstract the effect of ultrasound exposure time (15 and 30 min) at 130 w and 40 khz on the physicochemical, functional, and nutritional properties of a safflower protein isolate (spi) was evaluated. the moisture content, bulk density, and aw of the spi were significantly (p < 0.05) decreased by ultrasound compared to the untreated control. in contrast to the control, the spi exposed to ultrasound for 30 min had increased protein solubility (by 9.7% and 3.7% at ph 6 and 7, respectively), least gelation concentration (by 2.0% at ph 6), and oil absorption capacity (by 3.0%). no significant differences (p < 0.05) were observed in amino acid composition, chemical score, or predicted protein efficiency ratio of the control and the spi exposed to ultrasound. ultrasound treatment would benefit the application of spi in the food industry for ground meat formulations, meat substitutes and extenders, doughnuts, baked goods, and soups. keywords: ultrasound treatment, safflower protein isolate, physicochemical properties, functional properties, nutritional properties ital. j. food sci., vol. 31, 2019 592 1. introduction safflower (carthamus tinctorius l.) is one of the oldest cultivated crops and usually is grown for flowers that are used for coloring, flavoring foods, dyes, medicinal properties, and livestock feed (peiretti, 2017). safflower is gaining attention due to its oil yield potential and the ability to grow under high temperatures, drought, and high salinity (hussain et al., 2016). safflower is produced in over 20 countries, but in 2016, the russian federation, kazakhstan, mexico, and the usa were the main producers, producing about 71% of the 948,516 t of safflower produced worldwide (fao, 2018). oil and meal are the two main products that come from safflower production. oil is the primary product and has both food and industrial uses. the seed oil content of safflower ranges from 30 to 45% (liu et al., 2016). safflower meal, a by-product of the safflower-oil industry, contains approximately 20-25% protein, and is currently marketed as animal feed (tiril and kerim, 2015). however, according to some studies, a protein isolate from safflower meal could represent an opportunity to recover proteins for human consumption (ulloa et al., 2011; paredes-lópez and ordorica-falomir, 1986). on the other hand, the application of ultrasonic technology in the food industry is currently attracting much attention (hu et al., 2013). ultrasound is an acoustic wave with a frequency >20 khz (o’sullivan et al., 2016a). high intensity ultrasound (hius, 20100khz) might have a wide variety of applications in the food industry (zhang et al., 2014) because it can alter the physicochemical properties and/or structure and functional properties due to cavitation effects on vegetable proteins such as soy protein isolate (jambrak et al., 2009), black bean protein isolate (jiang et al., 2014), and jackfruit seed protein isolate (resendiz-vazquez et al., 2017). a recent study by xiong et al. (2018) showed that ultrasonic treatment caused partial unfolding of the proteins of pea protein isolate, which improved foam ability and stability. according to malik et al. (2017), solubility, emulsifying capacity, emulsion stability, foaming capacity, foam stability, and oil binding capacity of a sunflower protein isolate were improved significantly after application of hius. zhang et al. (2018) found that the hius treatment modified the protein structure and significantly enhanced the solubility of rice proteins, as well as emulsifying properties and foaming properties. however, the results of the application of ultrasound treatment on protein properties depend on some conditions such as frequency, amplitude, time, temperature, ionic strength, ph, and concentration, as well as intrinsic characteristics of the protein source (higuerabarraza et al., 2016). to our knowledge, no studies have focused on the potential effects of hius application on safflower protein isolate (spi). therefore, the objective of this study was to evaluate the effect of hius on the physicochemical, functional, and nutritive properties of a protein isolate obtained from safflower meal. 2. material and methods 2.1. materials and chemicals the safflower meal (23% protein, moisture 8%, 5.4% ash, 1.4% fat, and total carbohydrates 62.2%) used in this study was purchased from aceitera la junta, s.a. de c.v. (limited company of variable capital) (guadalajara, jalisco, mexico). all chemical reagents were j.t. baker analytical grade and purchased from diseño tecnológico en laboratorios, s. a. de c.v. (guadalajara, jalisco, méxico). ital. j. food sci., vol. 31, 2019 593 2.2. preparation of protein isolate the spi was prepared according to the method reported by ulloa et al. (2011). briefly, the protein was recovered in batches of safflower meal suspension (120 l) by adding one part safflower meal to 30 parts filtered tap water, which was then mixed for 45 min at room temperature (25°c). the ph of the suspension was adjusted to 9.0 with diluted naoh during mixing. the insoluble residue of the protein suspension was separated by continuous centrifugation. the protein extract was concentrated using a pilot-scale ultrafiltration unit (osmonic inc., minnetonka, usa) equipped with a polysulfonate membrane cartridge with a molecular weight cut-off of 100 kda. extracts were concentrated to one-fifth of their original volume, then diluted with filtered tap water and concentrated by diafiltration for further purification of protein retentate. dilution consisted of adding an amount of water equal to four times the retentate volume after the first concentration. three cycles of diafiltration were required to produce a protein isolate with a protein content ≥90.0% dry weight. finally, the diafiltrated protein extract was spray dried in a model tower no. 1 drier (niro atomizer, sa, monterrey, nuevo león, mexico) to obtain the powder of protein isolate. 2.3. ultrasound treatment aqueous suspensions of spi containing 10% of protein (w/v) were prepared in 100-ml beakers by magnetic stirring for 15 min. ultrasound treatment was performed in an ultrasound bath branson model mth-3510 (130 w and 40 khz; a tank capacity of 5 l; internal dimensions of 290 × 150 × 150 mm; an acoustic energy density of 0.026 w cm-3). beakers containing spi suspension were placed directly into the ultrasound bath to receive the treatment for 15 or 30 min, while the control spi suspension was placed into a water bath at 25°c; the temperature during ultrasound treatment increased by <2°c. the ultrasonic intensity introduced in the system measured by calorimetry according to jambrak et al. (2014) was 1 wcm-2. afterward the suspensions of spi exposed to ultrasound and the control were lyophilized in a freezone freeze-dry system (labconco, kansas city, mo, usa) and stored at room temperature in sealed containers for further analysis. 2.4. physicochemical and microstructure characteristics moisture, protein (n × 6.25), and ash contents were determined according to aoac methods (1995). water activity (aw) was measured at 25°c using an aqualab 4tev (decagon devices inc., pullman, washington, usa), on coarse powder samples (3 g). prior to testing samples, the water activity meter was turned on and allowed to warm up for 30 min and calibrated by filling a plastic disposable cup half filled with a saturated sodium chloride solution. the accuracy of water activity values was ±0.003. the color was determined with a minolta cr-400 color meter (konica minolta sensing ltd, inc., tokyo, japan). the measured values were expressed according to the cielab color scale l* (lightness), a* (redness-greenness), and b* (yellowness–blueness). the 𝐿!∗ , 𝑎!∗, and 𝑏!∗ values of the white standard tile used as reference were 94.44, -0.23 and 3.89, respectively. total color difference (∆e) was calculated as: ∆𝐸∗ = 𝐿!∗ − 𝐿∗ ! + 𝑎!∗ − 𝑎∗ ! + 𝑏! ∗− 𝑏∗ ! !/! (1) the bulk density was determined in triplicate using the method described by piornos et al. (2015) and expressed as g ml-1. ital. j. food sci., vol. 31, 2019 594 the microstructure of the freeze-dried spi samples was observed with a scanning electron microscope (sem; sec, mini-sem sne-3200m, south korea) at an accelerating voltage of 20 kv. before using the sem, the samples were coated with gold using an ion sputter coater (mcm-100, sec). 2.5. functional properties protein solubility profile of the spi as a function of ph was determined according to the method reported by piornos et al. (2015). the water absorption capacity (wac) and oil absorption capacity (oac) were measured according to the method described by ulloa et al. (2011) and expressed as g water or oil absorbed per g protein. soy oil (fábrica de aceites la central, s. a. de c.v., guadalajara, jalisco, méxico) was used in the determination of oac. the emulsifying activity (ea) and emulsion stability (es) were determined according to the method reported by deng et al. (2011) using soy oil. the least gelation concentration (lgc) at ph 2, 4, 6, 8, and 10 was determined according to the method reported by benelhadj et al. (2016). foaming capacity (fc) and foam stability (fs) were measured using the method described by stone et al. (2015). 2.6. amino acid composition and protein nutritive quality hydrolysis and quantification of amino acids were performed according to the methods described by vázquez-ortíz et al. (1995) using a pro star-210 varian high-performance liquid chromatographic system (varian associates, inc. usa). amino acids were expressed on a protein basis, equivalent to g per 16 g of protein. the tryptophan content was not determined. the nutritive quality of proteins was estimated by determination of chemical score (cs) and predicted protein efficiency ratio (per) according to the procedure described previously by ulloa et al. (2015). 2.7. statistical analysis analyses of samples were done in triplicate and data were analyzed using one-way anova. significant differences (p < 0.05) between samples were determined from a tukey’s test using spss statistics version 20 (ibm corporation, new york, usa). 3. results and discussion 3.1. physicochemical and microstructure characteristics the effect of ultrasound exposure time on the physicochemical characteristics of spi is shown in table 1. the protein and ash contents of the spi exposed to ultrasound for 15 and 30 min were not significantly (p > 0.05) different from the control (table 1). however, the moisture content of spi was significantly (p < 0.05) reduced from 5.08% (control) to 4.01% and 4.53% when samples were exposed to ultrasound for 15 and 30 min, respectively. the lower moisture content of spi treated with ultrasound was due to a higher effect of compressions and expansions induced by the sound waves passing through the food medium, which make moisture removal easier (awad et al., 2012). bulk density is a property used to characterize powder products. it is of great importance for economical and functional reasons, for example, for reducing packaging costs, which depends on the combined effects of interrelated factors, like particle size, number of ital. j. food sci., vol. 31, 2019 595 contact points, and intensity of attractive inter-particle forces (piornos et al., 2015). as shown in table 1, the exposure to ultrasound of spi significantly decreased the bulk density 4.9–6.5% as compared to the control. such a decrease in the bulk density was due to the samples being treated with ultrasound and freeze drying, as these samples had larger and more heterogeneous structures in protein isolates in comparison with the control (hu et al., 2013; resendiz-vazquez et al., 2017). the aw of a food system is a thermodynamic property, which is defined as the ratio of water vapor pressure of food to the saturated water vapor pressure at a given temperature. it is considered to be a good quality indicator for the safety and stability of foods with respect to microbial growth and biochemical reactions (tadapaneni et al., 2017). the aw of spi exposed for 15 and 30 min to ultrasound was lower than that of the control (table 1). as discussed previously, sound waves passing through the food medium make moisture removal easier, which also influenced the reduction in aw of spi as was observed in this study. however, both the control and the spi exposed to ultrasound had values of aw below a limiting level to ensure microbial stability, because it is generally accepted that no microbial growth will occur at aw < 0.66 (ulloa et al., 2015). table 1. effect of ultrasound exposure time on the physicochemical properties of safflower protein isolate. property ultrasound exposure time (min) 0 (control) 15 30 protein content (%) 87.54±0.52 88.17±0.13 87.03±1.01 ash content (%) 6.92±0.11 6.88±0.12 6.81±0.13 moisture content (%) 5.08a±0.13 4.01c±0.12 4.53b±0.11 bulk density (g ml-1) 0.61a±0.1 0.57b±0.1 0.58b±0.1 water activity 0.341a±0.002 0.286c±0.007 0.300b±0.004 color l* (lightness) 60.62±0.11 60.35±0.19 60.19±0.21 a* (redness-greenness) 4.21±0.06 4.23±0.03 4.18±0.08 b* (yellowness-blueness) 17.45±0.29 17.41±0.17 17.77±0.25 δe (color difference) 36.70±0.17 36.94±0.21 37.22±0.27 values are mean±standard deviation of three determinations. values followed by different superscript letters in the same line are significantly different (p < 0.05). the color characteristics of spi exposed to ultrasound for 15 and 30 min in comparison with the control treatment are shown in table 1. there were not significant (p > 0.05) differences in l*, a*, b*, and ∆e values among samples of control spi and those exposed to ultrasound. the effect of ultrasound on color in food depends on intrinsic characteristics of food and ultrasound conditions (bi et al., 2015). adekunte et al. (2010) reported a decrease in l*, a* and b* values of tomato juice after sonication due to the degradation of lycopene. cheng et al. (2007) found that guava juice treated by ultrasound for 30 min showed a significant change in the ∆e value due to a decrease in l* value and an increase in a* and b* values. valero et al. (2007) found that ultrasound treatments had no significant effect on color scoring in orange juice. the degradation of color might be due to the effect of cavitation of ultrasound that could induce both chemical (e.g., by generation of radicals) and mechanical degradation of biomolecules (adekunte et al., 2010); however, such a phenomenon was not observed in the spi exposed to ultrasound in this study. in general, a similar behavior on the effect of ultrasound exposure time on the ital. j. food sci., vol. 31, 2019 596 physicochemical properties of safflower protein isolate of this study was reported by flores-jiménez et al. (2019) for canola protein isolate. fig. 1 shows a set of sem images of control spi (a) and spi exposed to ultrasound for 15 (b) and 30 min (c). it was observed that samples b and c obtained after ultrasonic treatments and freeze-drying had larger and more heterogeneous structures than sample a (untreated spi). these results might have been caused by ultrasound treatment inducing the unfolding of proteins and increasing the surface hydrophobic groups of the spi molecules (hu et al., 2013; jiang et al., 2014). figure 1. effect of ultrasound exposure time (control = a, 15 min = b and 30 min = c) on the microstructure of safflower protein isolate. 3.2. functional properties solubility is one of the most important functional attributes of proteins because it affects the texture, color, and the sensory properties of products containing them. it is closely associated with water retention capacity and other physicochemical and functional properties. solubility of proteins depends on the composition, molecular weight, and surface characteristics of constituent amino acids and environmental factors such as ph, temperature, and ionic strength, and it can be affected by some chemical and physical treatments (timilsena et al., 2016). table 2 shows the effect ultrasound exposure time on the protein solubility of spi at different ph values. only at ph 6 and ph 7, did the spi exposed to ultrasound for 30 min have a beneficial effect on protein solubility, increasing 9.7% and 3.7%, respectively, in comparison to the control. this increase in protein solubility may be due to the conformational change during ultrasonic treatment and formation of soluble protein aggregates from insoluble protein (hu et al., 2013). however, such an increase in protein solubility depends on ultrasonic conditions (jiang et al., 2014), as well as the ph at which the proteins are solubilized, as was observed in this study. besides, for safflower proteins, the solubility values were minimum at the ph value of 5.0, which is the isoelectric point of safflower proteins, for ultrasound treatments and control treatment. the results of wac and oac by effect of ultrasound exposure time of spi are shown in table 3. the wac decreased from an initial value of 2.14 g h2o g-1 protein to 1.94 g h2o g-1 protein after ultrasound treatment for 30 min. according to resendiz-vazquez et al. (2017), the ultrasound treatment might denature the molecular structure of proteins and cause an increase in the hydrophobic surface, which can lead to low values of wac, as was observed in proteins from jackfruit seeds. in contrast, the oac of samples of spi exposed to ultrasound for 15 and 30 min were significantly (p < 0.05) higher compared to the control spi. the oac increased from an initial value of 0.99 g oil g-1 protein to 1.19 g oil g-1 protein and 1.30 g oil g-1 after exposure to ultrasound for 15 and 30 min, respectively. the ital. j. food sci., vol. 31, 2019 597 increase of oac in proteins might be attributed to the exposure of hydrophobic groups after ultrasound treatment (higuera-barraza et al., 2016; zhou et al., 2016), which allowed the physical entrapment of oil (meinlschmidt et al., 2016). table 2. effect of ultrasound exposure time at different ph on protein solubility (%) of safflower protein isolate. ph ultrasound exposure time (min) 0 (control) 15 30 2 90.95±0.68 87.22±1.43 88.10±0.06 3 81.06a±0.68 65.32b±0.68 67.16b±0.69 4 17.98a±0.68 12.59b±0.01 13.73b±0.01 5 14.57a±0.01 12.11b±0.66 13.20ab±0.69 6 49.61b±1.37 50.32b±0.01 54.41a±0.61 7 61.16b±0.01 57.94c±1.28 63.44a±0.04 8 89.75a±0.74 71.88b±0.68 73.49b±1.43 9 92.32a±0.12 86.34b±1.31 88.32ab±1.38 10 95.72a±0.68 90.26b±1.37 93.00ab±0.06 values are mean±standard deviation of three determinations. values followed by different superscript letters in the same line are significantly different (p < 0.05). table 3. effect of ultrasound exposure time on water and oil absorption capacity of safflower protein isolate. property ultrasound exposure time (min) 0 (control) 15 30 water absorption capacity (g h2o g -1 protein) 2.14a±0.03 2.15a±0.03 1.94b±0.02 oil absorption capacity (g oil g-1 protein) 0.99c±0.01 1.19b±0.02 1.30a±0.01 values are mean±standard deviation of three determinations. values followed by different superscript letters in the same line are significantly different (p < 0.05). proteins are of particular interest as emulsifying agents in food formulations such as frozen desserts, salad dressings, comminuted meats, mayonnaise, cake batters, milks, and coffee whiteners, due to their ability to adsorb and form viscoelastic films at oil-water interfaces (o’sullivan et al., 2016b). according to the results presented in fig. 2, ultrasound treatment had no effect on ea and es of spi. yanjun et al. (2014) reported that ultrasound pretreatment increased the emulsifying activity index (eai) and es index of milk proteins. zhou et al. (2016) found that the ultrasound treatment increased or decreased eai of soybean glycinin depending on the ionic strength. in another study, the ea of defatted rice bran protein concentrate was higher (p < 0.05) than that of the ultrasound treatment, but the es was not significantly different (p > 0.05) from the control (chittapalo and noomhorm, 2009). the emulsifying properties of food proteins depend upon the solubility, molecular flexibility, surface hydrophobicity, and stability of the protein structure (zhou et al., 2016), which can be modified or not when are exposed to different ultrasound conditions as was observed in this study. ital. j. food sci., vol. 31, 2019 598 figure 2. effect of ultrasound exposure time on the emulsifying activity (ea) and stability (es) of safflower protein isolate. gel properties are important functional characteristics of protein isolates, which are widely used as gelling agents to improve the texture and water holding capacity of meat products. gelation is often an aggregation of denatured proteins, which involves the formation of a network, retaining significant amounts of water and transforming liquid sample to solid, which exhibits a certain degree of order where both covalent and noncovalent interactions are involved (chen et al., 2016). lgc indicates the gelling capacity of protein. fig. 3 shows that the spi exposed to ultrasound for 15 and 30 min at ph 2-10 had no effect on lgc, except for the ultrasound treatment of 30 min at ph 6, where the lgc increased from 6.0% (w/w) to 8.0% (w/w), which implies a reduction of the gelling capacity. the behavior of the lgc of spi by effect of ph in this study was similar to that showed for lupin (piornos et al., 2015), jackfruit (resendiz-vazquez et al., 2017), and cashew nut shell (yuliana et al., 2014) protein isolates. figure 3. effect of ultrasound exposure time on the last gelation concentration (lgc) of safflower protein isolate. ital. j. food sci., vol. 31, 2019 599 the fc and fs of protein isolates are functional properties that determine their application in food systems, where aeration and overrun is required (e.g. baked foods, whipped toppings and ice cream mixes) (shevkani et al., 2015). because of surface-active properties, the proteins form foam when they are whipped (malik et al., 2017). according to the results obtained in this study, the exposure to ultrasound for 15 and 30 min did not modify the fc and fs of spi with respect to the control (fig. 4). figure 4. effect of ultrasound exposure time on the foaming capacity (fc) and foam stability (fs) of safflower protein isolate. some reports showed that fc and fe of protein isolates from whey (jambrak et al., 2008) and soy (zhang et al., 2014) were improved after ultrasound treatment for both 20 khz and 40 khz treatments, but no effect in foaming at 500 khz treatment was observed, as occurred for spi in this study. the improvement of the foaming properties in protein suspensions by ultrasound may be due to protein partial denaturation, which causes a higher air-water diffusion interface due to an increase in cohesiveness and flexibility of the foams (higuera-barraza et al., 2016). 3.3. amino acid composition and protein nutritive quality amino acid composition and nutritive quality of proteins in terms of cs and per of spi exposed to the ultrasound treatments in comparison with the control (table 4) were not significantly different (p < 0.05). of all the amino acids present in spi, half corresponded to essential amino acids. on the other hand, glutamic acid was the amino acid with higher concentration in the spi, as well as in the hempseed, soy (wang et al., 2008), and pennycress protein isolates (hojilla-evangelista et al., 2014). according to amino acid requirements for adults (fao/who, 1991), the first limiting amino acid of spi was lysine. therefore lysine was considered for calculating cs values for the spi exposed to ultrasound for 15 min, 30 min, and the control, which were 48.4, 48.2, and 48.9 respectively, and were not significantly different (p > 0.05) from one another (table 4). ital. j. food sci., vol. 31, 2019 600 table 4. effect of ultrasound exposure time on the composition of amino acid and protein quality of safflower protein isolate. parameter amino acid composition (g/16 g n) reference for adults (fao/who, 1991) ultrasound exposure time (min) 0 (control) 15 30 essential amino acid lysine 2.69±0.08 2.66±0.15 2.65±0.05 5.5 threonine 5.08±0.25 4.78±0.21 4.31±0.57 4.0 valine 2.93±0.09 3.01±0.44 2.70±0.14 5.0 methionine+cysteine 2.13±0.01 1.93±0.37 2.21±0.14 3.5 cysteine isoleucine 2.25±0.46 2.36±0.10 2.20±0.20 4.0 leucine 6.39±0.94 7.06±0.36 6.57±0.14 7.0 phenylalanine+tyrosine 4.10±0.94 4.16±1.12 3.56±0.60 6.0 tyrosine 2.58±0.10 2.97±0.73 2.40±0.20 tryptophan nd nd nd total essential amino acids 25.58±0.70 25.96±1.10 24.19±0.80 no essential amino acid histidine 3.84±0.05 3.42±0.32 4.09±0.46 arginine 18.65±1.08 18.57±0.74 19.08±0.56 aspartic acid 6.78±0.57 6.73±0.41 6.60±0.48 serine 8.25±0.58 7.20±0.51 7.99±0.60 glutamic acid 20.96±0.86 22.28±0.73 21.17±0.83 glycine 8.83±0.52 9.09±0.58 9.61±0.26 alanine 7.11±0.37 6.75±0.55 7.27±0.86 total non-essential amino acid 74.42±0.81 74.04±0.75 75.81±0.90 nutritive quality chemical score 48.9±1.53 48.4±2.7 48.2±0.83 per 2.16±0.22 2.42±0.20 2.26±0.04 values are mean±standard deviation of three determinations. values followed by different superscript letters in the same line are significantly different (p < 0.05). nd = not determined. per = predicted protein efficiency ratio. with respect to the per, the values obtained for the spi exposed to ultrasound for 15 min, 30 min, and the control were not significantly different (p < 0.05) from one another and had values of 2.42, 2.26, and 2.16, respectively, which were higher than the values 2.14 and 2.04 for desi chickpea and soy protein isolates, respectively (wang et al., 2010). 4. conclusions application of ultrasound at 130 w and 40 khz 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zooprofilattico sperimentale della puglia e della basilicata (izs pb), via manfredonia 20, 71121 foggia, italy 2istituto zooprofilattico sperimentale della sardegna (izs sa)“g. pegreffi”, via vienna 2, 07100 sassari, italy *corresponding author: giovanna.lasalandra@izspb.it abstract this study aimed to estimate the prevalence of methicillin-resistant staphylococcus aureus (mrsa) from 500 retail meat products in south italy from june 2016 to june 2018, including 150 raw bovine, 120 pork, 150 chicken and 80 horse meat. after bacteriological analysis, 12 (2.4%) samples tested positive for mrsa. isolates were characterized by antimicrobial susceptibility, spa typing and mlst. mrsa were also investigated by pcr for the presence of enterotoxins, lukf-pv-luks-pv and icaa-icad genes. antimicrobial susceptibility testing showed that mrsa isolates were multidrug resistant. one strain harboured pvl-encoding genes (8.3%). seven mrsa isolates of 12 (58.3%) carried seh enterotoxin encoding gene. the icaa and icad genes were both present in 10 isolates (83.3%). mrsa isolates in retail meat may serve as a potential source of exposure to mrsa for humans and monitoring of food-producing animals and hygiene standards should be strictly and carefully considered throughout the entire meat chain to ensure food safety. keywords: biofilm, food safety, methicillin-resistant staphylococcus aureus, panton valentine leukocidin, retail meat ital. j. food sci., vol. 32, 2020 411 1. introduction staphylococcus aureus is considered as one of the major foodborne pathogens and is responsible for a wide spectrum of infections worldwide (wu et al., 2018). methicillinresistant s. aureus (mrsa) poses a public health issue because of its multiple antimicrobial resistance and data on the occurrence of mrsa in food-producing animals and food is underestimated as the report is currently voluntary (efsa and ecdc, 2019). traditionally, mrsa strains are distinguished into two distinct epidemiological groups, hospital-associated mrsa (ha-mrsa) and community-associated mrsa (ca-mrsa) (tang et al., 2017). ca-mrsa frequently harbour staphylococcal cassette chromosome mec (sccmec) types iv or v and the genes luks-pv-lukf-pv encoding the subunits of the panton-valentine leucocidin, a cytotoxin that causes leukocyte lysis or apoptosis via pore formation (boyle-vavra and daum, 2007). ha-mrsa typically possess larger-size sccmec types i, ii or iii and often exhibit resistance to multiple classes of antimicrobial agents strains (shore et al., 2014). a third group of mrsa, known as livestock-associated mrsa (la-mrsa), has recently been identified and it infects livestock and companion animals, as well as some other farm animal species and wild animals. la-mrsa have mainly sccmec types iva or v, although, non-typeable cassettes and sccmec type xi have also been found (butaye et al., 2016). methicillin resistance is primarily attributed to the altered penicillin binding protein (pbp2a), encoded in the meca gene, which has a reduced affinity for β-lactam antibiotics. recently, a homolog of the meca, mecc (mecalga251) was identified in mrsa strains from humans and livestock that were phenotypically resistant to methicillin, but tested negative for the meca gene. the mecc gene shares about 70% nucleotide homology with meca and is located in sccmec xi (velasco et al., 2015). the nuc gene is considered a marker for the detection of s. aureus and encodes for a thermostable nuclease (costa et al., 2005). s. aureus has the ability to form biofilms on various materials and surfaces. biofilms in the food industry can cause serious hygienic problems as the bacteria could adhere to the food contact surfaces and contaminate foodstuffs (rode et al., 2007). the mechanism of biofilm formation is promoted by ica locus containing four genes, namely icaa, icab, icac, icad. the product of ica locus is the polysaccharide intracellular adhesin (pia), that mediates intercellular aggregation of bacterial cells. pia was found to be the main exopolysaccharide component of the staphylococcal biofilm (arciola et al., 2015). the icaa gene encodes for a transmembrane enzyme, nacetylglucosaminyltransferase that contribute to the synthesis of the poly-nacetylglucosamine polymer and requires icad for full functioning (ciftci et al., 2009). in recent years, mrsa isolation from fresh retail meat has been reported in u.s.a., saudi arabia, korea, denmark, finland, germany, spain and switzerland (efsa and ecdc, 2019; ge et al., 2017; kim et al., 2015; tang et al., 2017), suggesting that these products may pose a potential risk for mrsa transmission to humans (buyukcangaz et al., 2013). to the best of our knowledge, there is little data available on the prevalence of mrsa contamination in fresh meats sold at retail prices in italy. genotyping of s. aureus isolated from retail meat is an important tool in epidemiological studies of infection and contributes to better understanding of the pathogen’s dissemination. several molecular methods have been developed for typing s. aureus isolates, such as pulsed field gel electrophoresis (pfge), multilocus sequence typing ital. j. food sci., vol. 32, 2020 412 (mlst) and spa typing (strommenger et al., 2006; enright et al., 2000; bannerman et al., 1995). the aims of this study were to evaluate the prevalence of mrsa in fresh meat samples sold at retail prices in southern italy and investigate the molecular characteristics of mrsa isolates as regards some virulence-associated genes, and antimicrobial resistance profiling for epidemiological studies and risk assessment purposes in the “one health” perspective. 2. materials and methods 2.1. isolation and identification of mrsa a total of 500 fresh meat samples, over a two-year period (june 2016-june 2018), were collected at retail markets by local health officials, and transported to the laboratories of the istituto zooprofilattico sperimentale della puglia e della basilicata (izs pb) and analysed for the detection of s. aureus. these samples comprised 150 raw bovine, 120 pork, 150 chicken, and 80 horse meat samples. isolation and identification of s. aureus were performed according to en iso 6888 1-2 1999. presumptive s. aureus colonies (black colonies with a zone of clearing of the medium) were identified by conventional biochemical methods and plated onto blood agar. after 18-24 h of incubation at 37°c, s. aureus isolates were subcultured on chromagar™ mrsa (chromagar, paris, france). dna was extracted from an isolated bacterial colony using the instagene matrix™ (bio-rad, segrate (mi), italy), following the manufacturer's instructions. all s. aureus isolates were screened by multiplex pcr for 16s rrna (monday and bohach, 1999), nuc (costa et al., 2005) and meca/mecc (garcía-álvarez et al., 2011) genes in order to confirm s. aureus species and to detect methicillin resistance. confirmed mrsa isolates (one strain per sample) were further characterized and tested for antimicrobial susceptibility. 2.2. in vitro antimicrobial susceptibility antimicrobial susceptibility of mrsa isolates was determined by disc diffusion method according to the guidelines of clinical laboratory standards institute (2013). a total of eleven antibiotics were included: penicillin (10 units), oxacillin (1 µg), cefoxitin (30 µg), cephalothin (30 µg), gentamicin (10 µg), kanamycin (30 µg), erythromycin (15 µg), tetracycline (30 µg), trimethoprim-sulfamethoxazole (25 µg), chloramphenicol (30 µg), enrofloxacin (5 µg). the mic of teicoplanin of the mrsa isolates was determined by etest® (biomérieux italia spa, bagno a ripoli (fi) italy), following clsi interpretative breakpoints (2017). s. aureus atcc 25923 was included for quality control. 2.3. genotyping the polymorphic x region of the protein a gene (spa typing) was amplified according to a published protocol (strommenger et al., 2006). amplification of seven housekeeping genes (arcc, aroe, glpf, gmk, pta, tpi, and yqil) by multilocus sequence typing (mlst) was performed as described by enright (2000). the dna sequences were submitted to the staphylococcus mlst database (http://saureus.mlst.net/) to obtain the allelic profiles of mrsa strains. ital. j. food sci., vol. 32, 2020 413 2.4. sccmec typing and detection of enterotoxins, lukf-pv-luks-pv and icaa-icad genes sccmec elements were typed by multiplex pcr as previously described (garcíaálvarez et al., 2011; kondo et al., 2007). the sccmec type iv was subtyped according to zhang (2012). mrsa isolates were screened by three multiplex pcr for the detection of 12 genes encoding staphylococcal enterotoxins (for sea to see, for seg to sej, for sem to seo), using twelve specific primer sets as previously described (boerema et al., 2006; jarraud et al., 2002; løvseth et al., 2004; monday and bohach, 1999; rosec and gigaud, 2002). all mrsa strains were subjected to a pcr assay to test the presence of lukf-pvluks-pv and icaa-icad genes encoding respectively, panton-valentine leukocidin (pvl) and polysaccharide intercellular adhesin (pia), as described elsewhere (hesje et al., 2011; zmantar et al., 2008). furthermore, the ability of the mrsa strains to form biofilm was tested using a semi-quantitative adherence assay by microtiter plate (mtp) according to zmantar (2010). the assay recorded the optical density (od) at 570nm of adherent biofilm after incubation for 24 h at 37°c. biofilm formation was classified as highly positive (od570 ≥ 1), low grade positive (0.1 ≤ od570< 1), or negative (od570< 0.1). 3. results and discussion methicillin-resistant staphylococcus aureus is a public health concern and food contaminated by mrsa may serve as a potential vehicle for transmission to humans (efsa, 2009). this study reports on the prevalence of mrsa isolated from fresh meat and sold at retail prices as well as the characterization of some virulence-associated genes and antimicrobial resistance profiling. in this survey, among 500 retail fresh meat samples subjected to bacteriological analysis, 72 (14.4%; 72/500) tested positive for s. aureus and 12 (2.4%; 12/500) for mrsa. total s. aureus counts performed using standard microbiological procedures were below 103 colony forming units per gram (cfu/g) in all tested samples. all mrsa isolates carried the meca gene and none carried the mecc gene. several surveys have estimated the prevalence of mrsa in retail meat worldwide. in a study from u.s.a., ge et al. (2017) found that 27.9% of the retail meats examined was contaminated by s. aureus and 1.9% tested positive for mrsa. another study from u.k. recovered mrsa from 7.3% of retail meat samples (fox et al., 2016). previous studies conducted in italy reported a contamination rate of meat product samples of 10% and 0.5%, but no mrsa strain was found among the isolates (normanno et al., 2007a; normanno et al., 2007b; traversa et al., 2015). probably, these differences in the prevalence of mrsa in retail meats could be due to the different geographical area, sampling and collection period. in this report, most mrsa isolates (5/12; 41.7%) were t127/st1/sccmec type iva, seh positive and pvl-negative. the 16.7% (2/12) of the isolates was t174/st1/sccmec type iva, ses and negative pvl genes. other recovered mrsa strains were: t386/st1/sccmec type iva (1/12; 8.3%) and t599/st1/sccmec type iva (1/12; 8.3%), both seh positive and pvl-negative, t044/st80/sccmectype ivc (1/12; 8.3%), ses negative and pvl positive, st97 t1236/st97 (1/12; 8.3%) and t899/st398 sccmec type v, both ses and pvl-negative (1/12; 8.3%) (table 1). st1 is a clone frequently implicated in human infections and spatype 127 is the prevalent clone involved in cases of invasive mrsa infections in europe (monaco et al., 2013). mrsa t127/st1 was also found in cows, sheep, goats and pigs in ital. j. food sci., vol. 32, 2020 414 italy and other european countries (agersø et al., 2012; alba et al., 2015; papadopoulos et al., 2018). mrsa t127/st1 clone is often detected in italian pig industry and the presence of a pig reservoir from this lineage has been hypothesized (alba et al., 2015; franco et al., 2011). moreover, seh gene is considered to be constitutive of st1, independently from the host origin (monecke et al., 2011). mrsa with genotype t044/st80/sccmectype ivc pvl-positive belongs to cc80 and lacks enterotoxin genes. first recognized in denmark in 1993, now it is widely spread throughout europe, north africa, sub-saharan africa and the middle east (monecke et al., 2011). it is mainly associated with skin infections in the community, but rarely causes invasive infections (david et al., 2010). the presence of this clone in retail horse meat underlies the spread of this mrsa-st80 clone. indeed, there are studies from various countries about nosocomial infections in horses due to mrsa causing a variety of infections (cuny et al., 2017; islam et al., 2017; steinman et al., 2015). in this survey, only one isolate (1/12; 8.3%) harboured pvl-encoding genes. the finding of mrsa st80/t044 pvl-positive suggests human handlers as potential source of contamination of meat, with pvl being a marker of ca-mrsa. another genotype recovered in this study from bovine retail meat was mrsa t1236/st97. in this isolate, sccmec was not detected. other studies reported this genotype to be associated with sheep, goats and cows as methicillin-susceptible s. aureus (feltrin et al., 2016; porrero et al., 2012). st97 (cc97) is generally responsible for bovine mastitis. less commonly, it was found in small ruminants, pigs, and humans. this clonal complex is the second most prevalent mrsa lineage in pig finishing holdings in italy and one of the s. aureus lineages associated with cattle, particularly with bovine mastitis (feltrin et al., 2016). the finding of mrsa strains in raw fresh meat should be considered carefully since meat may expose humans to this microorganism. it would be desirable monitoring health status of animals and implement control measures for breeding and slaughtering to avoid contaminations of their meat by s. aureus. mrsa t899/st398 sccmec type v pvl-negative is considered an important livestock-associated (la)-mrsa present in pigs, poultry, calves, companion animals, horses and other farm animal species in many countries. this clone was found in retail chicken meat in england (fox et al., 2016), in germany (kraushaar et al., 2017) and china (wang et al., 2014). la-mrsa may also pose an occupational risk for those people in close contact with livestock and their derived carcasses, especially pig farmers, cattle farmers, poultry farmers, slaughterhouse workers and veterinarians (hadjirin et al., 2015). these persons are more likely to be colonized with mrsa and spread the microorganism in the community. hence, retail meat may be a route for transmission of ca-mrsa (e.g. mrsa st1-t127) and la-mrsa to humans. several foods are implicated in staphylococcal food poisoning (sfp) such as raw meat, sausages, raw milk and raw milk cheese, in which contamination could be due to animal carriage or to infections of animal origin. colonized food handlers, rather than animals, are likely sources of contamination after heat treatment of the food (basanisi et al., 2017). the emetic activity of enterotoxins has been demonstrated only for sea, seb, sec, sed, and see (johler et al., 2015). the 58.3% (7/12) of the mrsa isolated in this study harboured seh gene, but in literature, there is little data available on the prevalence of mrsa in sfp (table 1) (jørgensen et al. 2005; and ostyn et al. 2012). nevertheless, the risk of human infection cannot be ignored. consequently, more attention should be paid during food handling and storage in order to reduce the potential role of food in the dissemination of successful mrsa lineages. ital. j. food sci., vol. 32, 2020 415 biofilm matrix is considered to be a significant virulence factor because when growing in this mode of life, microorganisms become more tolerant to antimicrobial agents and extremely difficult to eradicate. in this survey, the 83.3% (10/12) of mrsa isolates carried both the icaa and icad genes; the16.7% (2/12) of the isolates harboured only icad gene (table 1). as regard the ability to form biofilm, the mrsa strains were biofilm producers with mtp method, although, production level varied. among these, 83.3% of isolates (10/12) were low grade biofilm positive and 16.7% (2/12) were strongly biofilm producers (table 1). these strains could be of concern for the meat industry since bacteria in biofilms can be resistant to the normal disinfection and prophylaxis methods. special attention should be paid to hygiene procedures in farms and food facility. table 1. antimicrobial resistance profiles, genotypic characteristics and virulence-associated genes of the 12 mrsa isolates analyzed in this study. n° source of meat resistance to *: sccmec spa type mlst ses lukfpv/luks-pv icaa icad #od570 1 horse p, ox, fox, kf, k, e, te iva t174 st1 + ++ 2 horse p, ox, fox, kf, k, e, te iva t174 st1 + ++ 3 horse p, ox, fox, kf, cn, k, e, enr ivc t044 st80 + + + ++++ 4 bovine p, ox, fox, kf, e, te nd t1236 st97 + + ++++ 5 bovine p, ox, fox, k, te, kf, stx iva t127 st1 seh + + ++ 6 bovine p, ox, fox, k, te, kf, stx iva t386 st1 seh + + ++ 7 pork p, ox, fox, cn, k, e, kf, te iva t127 st1 seh + + ++ 8 pork p, ox, fox, cn, k, e, kf, te iva t127 st1 seh + + ++ 9 pork p, ox, fox, cn, k, e, kf, te iva t127 st1 seh + + ++ 10 pork p, ox, fox, cn, k, e, kf, te iva t127 st1 seh + + ++ 11 pork p, ox, fox, k, e, te, kf iva t599 st1 seh + + ++ 12 chicken p, ox, fox, te, kf, stx v t899 st398 + + ++ *antibiotic abbreviations: p, penicillin; ox, oxacillin; fox, cefoxitin; kf cephalothin; cn, gentamicin; k, kanamycin; e, erythromycin; te, tetracicline; enr, enrofloxacin; stx, trimethoprimsulfamethoxazole.#strongly biofilm positive (++++), low grade biofilm positive (++). global consumption of antimicrobials has increased worldwide causing the development of resistance to several antimicrobial agents in bacteria and this might result to serious problems. in this study, mrsa isolates were resistant to penicillin, oxacillin, cefoxitin, cephalothin followed by tetracycline (11/12; 91.7%), kanamycin (10/12; 83.3%), erythromycin (8/12; 66.7%), gentamicin (5/12; 41.7%), trimethoprim-sulfamethoxazole (3/12; 25%), enrofloxacin (1/12; 8.3%). the mic value of teicoplanin was ≤ 1.5 µg/ml for all mrsa isolates (table 1). multidrug resistance in retail meat was also observed in other studies (jackson et al., 2013; tang et al., 2017). aminoglicosides, macrolides, ital. j. food sci., vol. 32, 2020 416 penicillins, and tetracyclines are some of the classes of antimicrobial agents extremely important for veterinary medicine considering the wide range of applications and diseases to be treated (wendlandt et al., 2015). therefore, controlling the use of antibiotics in farming could limit the risk of transmission of multidrug resistant pathogens among animals and potentially to humans through the food chain. 4. conclusions in this study, raw bovine, pork, chicken and horse meat samples were positive for mrsa, although, the level of prevalence was low and varied between meats of different origin. the data obtained from this survey suggest that the presence of mrsa in fresh retail meats could be the result of human contamination due to colonized food handlers or cross-contamination of carcasses during 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bakhrouf a. 2008. detection by pcr of adhesins genes and slime production in clinical staphylococcus aureus. journal of basic microbiology, 48:308-314. doi: doi.org/10.1002/jobm.200700289. paper received july 3, 2019 accepted february 14, 2020 ijfs#1208_bozza ital. j. food sci., vol. 31, 2019 150 paper effect of the production process on the content of anthocyanins in dried red-fleshed potato cubes e. rytel*1, a. tajner-czopek1, a. kita1, a. sokół-łętowska2, a.z. kucharska2 and k. hamouz3 1department of food storage and technology, wrocław university of environmental and life sciences, 37/41 chełmońskiego st., 51-630 wrocław, poland 2department of fruit, vegetable and plant nutraceutical technology, wrocław university of environmental and life sciences, 37/41 chełmońskiego st., 51-630 wrocław, poland 3department of plant production, czech university of life sciences in prague, 129 kamýcká st., 165 00 praha 6, suchdol, czech republic *e-mail address: elzbieta.rytel@upwr.edu.pl abstract this study aimed at determining the effect of particular stages in the laboratory manufacture of dehydrated potato cubes on the stability of anthocyanin content in redfleshed potato varieties. the raw material used in the study was potatoes of the following three red-fleshed varieties: rosemarie, herbie 26, and rote emma. the analysed potato varieties differed in their respective content of anthocyanins and polyphenols. a higher content of these compounds was found in potatoes of rote emma cv. (216 mg/100 dm polyphenols and 37.3 mg/100 g anthocyanins). the greatest losses of anthocyanins were noted after peeling and pre-drying and those of total polyphenols were noticed after blanching, pre-drying and drying. in comparison to the raw material, only ca. 25% of anthocyanins and ca. 31% of total polyphenols remained in the finished product. among the analysed varieties, rote emma might be recommended for the production of dried potato cubes. this is because the highest content of biologically active compounds was present in potatoes of this variety after the production process. keywords: colour-fleshed potatoes, anthocyanins, dehydrated potato cubes ital. j. food sci., vol. 31, 2019 151 1. introduction in comparison to other plant materials (fruits or vegetables), potatoes are convenient study materials, owing to their wide applicability, high availability, high consumption across the world and very good adaptation capabilities. they are one of the few plant materials that produce high crop yields in different climatic zones and under various soil conditions. although potatoes are materials that are well known to consumers, many scientists worldwide are still undertaking analyses of their chemical composition. such a high interest in this raw material results from its varietal diversity and, therefore, from its rich chemical composition (brown et al., 2008; lisińska et al., 2009; rytel et al., 2014). recent studies have addressed biologically active compounds of the potatoes varieties that have intensively coloured flesh, containing anthocyanins, which are known for their antioxidative properties (lachman and hamouz, 2005; furrer et al., 2017; valiñas et al., 2017). anthocyanins constitute a large group of plant pigments included in the natural phytonutrients that are soluble in water and occur in almost all parts of a plant (bridle and timberlake, 1997; rodriguez-saona et al., 1998; castañeda-ovando et al., 2009, piątkowska et al., 2011). in cells, they occur in vacuoles in the form of granules of various sizes. a few hundred natural pigments and over 100 chemically synthesised ones are known today. anthocyanins are widely applied in the food industry as colourants due to their intensive and attractive colour. in addition, their therapeutic properties have been used in folk medicine for years. today, they are being increasingly used in the cosmetic and pharmaceutical industries (wrolstad, 2000; eichorn and winterhalter, 2005). anthocyanins are unstable compounds. they undergo various transformations in the water environment depending on ph value, which, in turn, might contribute to a change in the colour of the products that contain them. few scientific reports are available on the effect of processing conditions on anthocyanins in redor purple-fleshed potato varieties (perla et al., 2012; lachman et al., 2013; kita et al., 2015). anthocyanins of potatoes are acylated derivatives of cyanidin, and their colour differs depending on the medium ph. potatoes contain anthocyanins, which are stable not only in an acidic medium, such as in the form of pigments isolated from fruits, but also in neutral and slightly basic media (eichorn and winterhalter, 2005; friedman and levin, 2009; chung et al., 2017). a dynamic increase has recently been observed in the manufacture of potato products, the main ones including french fries, chips and dehydrated potato products. production of the latter is successively increasing in response to the needs of the market. today, consumers look for ‘convenient’ foods that not only enable the fast preparation of meals in households or catering facilities but which are also characterised by high organoleptic and nutritional values. the drying process facilitates the possibility of manufacturing a wide array of preserved semi-products or potato products that meet these criteria. a new and interesting solution is the use of red-fleshed and purple-fleshed potatoes to manufacture such products. this study aimed at determining the effect of particular stages in the laboratory manufacture of dehydrated potato cubes on the stability of anthocyanin content in redfleshed potato varieties. ital. j. food sci., vol. 31, 2019 152 2. material and methods 2.1. material the raw material used in the study was potatoes of the following three red-fleshed varieties: rosemarie, herbie 26 and rote emma, all of which were sourced from the plantations of the czech university. the study was conducted in the growing season from 2015 to 2016 in three technological replications. the effect of particular stages in the laboratory manufacture of dehydrated potato products on changes in the content of anthocyanins in the raw material, semi-products and finished products that were made from colour-fleshed potatoes was investigated. the method of dehydrated dice production in laboratory conditions was as follows: the potatoes were washed, peeled (1.5 mm) using a laboratory carborundum peeler, diced into 10×10×10 mm cubes by a manual cutting device in the laboratory and rinsed with distilled water at a temperature of 20°c. subsequently, the potato cubes were blanched in water at 75°c for 5 min and pre-dried in a laboratory oven at 120°c for 1 hour. afterward, the temperature of drying was decreased between 55 and 60°c to obtain a final moisture content of about 12% (about 8 hours). a total of 1 kg samples of potato were taken during each stage of laboratory processing (lisińska and leszczyński, 1989; rytel, 2012; rytel et al., 2014; rytel et al., 2017). the raw material (unpeeled potatoes) was determined for the following proximate chemical composition: dry matter, starch, total and reducing sugars. wet samples, which included unpeeled potatoes, peeled potatoes, skins, potato after blanching and pre-drying, were frozen and lyophilised by using a freeze dryer (temperature -35°c, pressure 5 pa, time 12 h) (edwards, england). all raw materials, semi-products and finished products obtained during laboratory production were ground in a laboratory mill. the prepared samples were examined for determining the content of anthocyanins. 2.2. extraction of anthocyanins the samples were prepared according to the method described by nemś et al. (2015). the freeze-dried raw materials, semi-products and finished products were extracted with 70% aqueous acetone (0.1% acetic acid) in a graduated tube. the mixture was homogenised using a vortex and allowed to stand for 2 h at room temperature. the acetone-water solution was partitioned with chloroform to remove lipophilic compounds. next, the acetone-water fraction was collected and put into a büchi rotary evaporator (merck, darmstadt, germany) until all residual acetone evaporated. the remaining extract was brought to a known volume with 50% methanol and stored at 20°c until it was analysed. the samples were filtered with 0.45 µm and 0.22 µm filters before hplc-pda and uplcms/ms analyses. 2.3. quantification of anthocyanins by hplc-pda the content of anthocyanins was determined according to kucharska et al. (2017) by using a dionex (usa) hplc system equipped with an ultimate 3000 model of a diode array detector, an lpg-3400a quaternary pump, an ewps-3000si autosampler and a tcc3000sd thermostated column compartment, all of which were controlled by the chromeleon v. 6.8 software. the cadenza imtakt column c5-c18 (75 × 4.6 mm, 5 µm) (portland, usa) was used. the following solvents constituted the mobile phase: 4.5% formic acid (solvent a) and 100% acetonitrile (solvent b). the following elution conditions were applied: 0-1 min 5% b in a; 1-20 min 25% b in a; 20-27 min 100% b in a; and 27-30 ital. j. food sci., vol. 31, 2019 153 min 5% b in a. the flow rate was 1 ml/min, and the injection volume was 40 μl. the column was operated at 30°c. anthocyanins were monitored at 520 nm, and their content was expressed in cyanidin 3-o-glucoside equivalents (cyge)/100 g dm. 2.4. identification of anthocyanins by uplc-qtof-ms/ms the method for anthocyanin identification was previously described by mizgier et al. (2016). anthocyanins were identified on acquity ultra-performance liquid chromatography (uplc) system coupled with a quadrupole-time of flight (q-tof) ms instrument (uplc/synapt q-tof ms, waters corp., milford, ma, usa) with an electrospray ionisation (esi) source. they were separated on an acquity tm beh c18 column (100 mm × 2.1 mm i.d., 1.7 μm; waters) (merck, darmstadt, germany). the detection wavelength was set at 520 nm. the mobile phase was a mixture of 4.5% formic acid (solvent a) and 100% acetonitrile (solvent b). the gradient program was as follows: initial conditions 99% (a), 12 min 75% (a), 12.5 min 100% (b) and 13.5 min 99% (a). the flow rate was 0.45 ml/min, and the injection volume was 5 μl. the column was operated at 30°c. the major operating parameters for the q-tof ms were set as follows: capillary voltage, 2.0 kv; cone voltage, 40 v; cone gas flow, 11 l/h; collision energy, 28-30 ev; source temperature, 100°c; dissolution temperature, 250°c; collision gas, argon; dissolution gas, nitrogen; flow rate, 600 l/h; data acquisition range, m/z 100-2000 da; and ionisation mode, positive. the data were collected by the mass-lynx tm v 4.1. software. 2.5. analytical methods the dry matter content of fresh potato samples and freeze-dried materials was determined by the reduced weight after drying at 105°c until a constant weight was achieved (horwitz and latimer, 2005). the contents of total and reducing sugars were determined by the colorimetric method with dns (horwitz and latimer, 2005). the starch content was determined in raw potato tubers by measuring their specific gravity while the quantity of anthocyanins was analysed by hplc-pda (kucharska et al., 2017), and their profile by uplc-qtof-ms/ms (mizgier et al., 2016) of samples that were prepared as described in the work of nemś et al. (2015). the polyphenol content was determined using the folin-ciocalteu colorimetric method, as described by singleton et al. (1999) and kita et al. (2015). all analyses were carried out in triplicate. 2.6. statistical analysis the study’s results were subjected to statistical calculations by using the statistica 13.1 software (statsoft polska sp. z o.o., kraków, poland). the significance of the differences between mean values was determined by conducting a multi-way analysis of variance and duncan’s test (p≤0.05). all experiments were performed in three technological replications within two years of investigation, and the present results show the mean values of all data in a combined way. 3. results and discussion potatoes of red-fleshed and purple-fleshed varieties are rarely used to manufacture fried or dried food products. this is because of their lesser popularity among producers and consumers and, consequently, their lower availability in the market. in addition, potatoes ital. j. food sci., vol. 31, 2019 154 of colour-fleshed varieties usually have a higher content of total sugars and reducing sugars in comparison to those of the common yellow-fleshed or white-fleshed varieties. these compounds determine the colour of the finished product (kita et al., 2015). dehydrated potato products should be manufactured from potatoes that have a high content of dry matter (from 21 to 25%) and starch (from 15 to 19%) and those in which the content of reducing sugars is below 0.5% (lisińska et al., 2009). in the potatoes of redfleshed varieties that were analysed in our study, contents of dry matter, starch and reducing sugars met the above requirements in tubers of herbie 26 and rote emma var. potatoes of rosemarie var. had a lower content of dry matter and starch and over 0.6% of reducing sugars (table 1). table 1. chemical composition of raw potatoes. chemical compounds (g/100 g fresh matter, fm) potato variety herbie 26 rosemarie rote emma dry matter 22.8±0.12c 17.9±0.09a 20.9±0.10b starch 15.4±0.11b 14.0±0.11a 15.4±0.12b total sugar reducing sugar 0.71±0.09a 0.51±0.08a 0.86±0.07b 0.60±0.09b 0.69±0.10a 0.69±0.10a a, b, c different letters indicate significant differences among the varieties following the lsd test (p>0.05), ± sd (standard deviation); n = 6. taking into consideration the attractive colour of the flesh and the higher content of biologically active compounds, potatoes of redand purple-fleshed varieties might be an interesting alternative to traditional light-fleshed potato varieties and might be recommended for the manufacture of potato products. tables 2 and 3 present the contents of anthocyanins and total polyphenols determined at particular stages in the laboratory manufacture of dehydrated potato cubes. ital. j. food sci., vol. 31, 2019 155 table 2. contents of pigments (mg/100 g dry matter, dm) and total polyphenols (mg/100 g dm) in red-fleshed potatoes and skins. pigments potato variety herbie 26 rosemarie rote emma unpeeled potatoes skins unpeeled potatoes skins unpeeled potatoes skins pelargonidin-3-rutinoside-5-glucoside 3.20±0.12b 6.81±0.11a 9.46±0.13b 10.5±0.12a pelargonidin-3-rutoside 2.04±0.11b 4.35±0.10a 2.90±0.12a 1.36±0.07c 2.33±0.10a 6.32±0.10b pelargonidin-3-caffeoylrutinoside-5glucoside 4.85±0.20 a 0.74±0.08a 4.94±0.09a 2.98±0.08b 8.48±0.11b 15.5±0.14c pelargonidin-3-pcoumaorylrutinoside-5glucoside 2.95±0.10 b 0.40±0.06a 1.81±0.11a 2.20±0.10b 10.5±0.12c pelargonidin-3-feruloylrutinoside-5glucoside 13.1±0.11 c 6.38±0.11b 5.10±0.13a 6.31±0.12b 6.52±0.11b 0.43±0.03a sum of analysed anthocyanins 26.1±0.13b 18.7±0.10b 14.7±0.12a 12.8±0.11a 37.3±0.22c 32.8±0.17c total polyphenols 188±4.53a 196±5.13b 186±3.99a 94±4.01a 216±5.32b 204±4.89c a, b, c different letters indicate significant differences among varieties following the lsd test (p>0.05), ± sd; n = 6. table 3. content of pigments (mg/100 g dm) and total polyphenols (mg/100 g dm) in potatoes after particular technological stages. potato variety technological stage pigments total polyphenols pelargonidin-3rutinoside-5glucoside pelargonidin-3rutoside pelargonidin3-caffeoylrutinoside-5glucoside pelargonidin-3-pcoumarylrutinoside5-glucoside pelargonidin-3feruloylrutinoside5-glucoside sum of analysed anthocyanins h er bi e 26 potato after peeling 2.54±0.10c 1.62±0.09b 0.52±0.07b 0.07±0.06c 9.39±0.12d 14.1±0.12d 185±3.71c potato after blanching 2.22±0.11b 0.15±0.04a 0.54±0.05b 0.04±0.008b 5.51±0.10c 8.46±0.11c 179±2.55b potato after pre-drying 0.60±0.04a 0.10±0.01a 0.50±0.06b 0.01±0.007a 4.30±0.09b 5.51±0.10b 172±4.01b potato after drying 0.18±0.08a 2.10±0.08a 2.28±0.09a 141±1.98a r os em ar ie potato after peeling 1.03±0.09c 3.51±0.10d 1.22±0.09c 2.49±0.11c 8.25±0.12d 181±4.02d potato after blanching 0.39±0.01b 3.45±0.11c 0.96±0.08b 2.09±0.08b 6.89±0.11c 168±3.25c potato after pre-drying 0.29±0.03b 2.79±0.12b 0.41±0.06a 1.81±0.10a 5.30±0.12b 151±3.44b potato after drying 0.20±0.02a 2.47±0.10a 1.80±0.11a 4.47±0.09a 146±2.56a r ot e e m m a potato after peeling 1.32±0.09c 0.94±0.07b 3.32±0.14c 10.7±0.14d 5.90±0.12b 22.2±0.17c 194±2.07d potato after blanching 1.02±0.08b 0.72±0.05a 2.41±0.10b 7.18±0.12c 6.02±0.12b 17.3±0.18b 152±2.67c potato after pre-drying 0.81±0.03a 1.10±0.09a 4.18±0.10a 4.99±0.11a 11.1±0.10a 128±1.98b potato after drying 1.09±0.08a 5.42±0.11cb 4.88±0.10a 11.4±0.11a 112±1.91a a, b, c, d different letters indicate significant differences among varieties following the lsd test (p>0.05), ± sd; n = 6. ital. j. food sci., vol. 31, 2019 156 the mean content of total polyphenols in potatoes of the analysed varieties was 197 mg/100 g dm. the highest content was found in both skins and whole tubers of rote emma potatoes, whereas the lowest was found in rosemarie potatoes (table 2). the content of anthocyanins in the analysed potatoes ranged from 14.7 mg/100 g dm (rosemarie var.) to 37.3 mg/100 g dm (rote emma var.). their contents were lower in skins and were on average 21.4 mg/100 g dm (table 2). however, the lowest anthocyanin content was determined in skins of rosemarie var. according to other authors (fossen et al., 2003; friedman and levin, 2009; furrer et al., 2017), the content of anthocyanins in potatoes might vary greatly from a few to a few dozen mg per 100 g dm. as reported by hamouz et al. (2011), the anthocyanin content in potatoes of purple-fleshed varieties ranged from 6.88 mg/100 g-1 dm (valfi var.) to 57.3 mg/100 g dm (violette var.) and in potatoes of red-fleshed varieties from 13.5 mg/100 g dm (rosalinde var.) to 21.2 mg/100 g dm (highland burgundy red var.). in turn, according to kita et al. (2013), purple-fleshed potatoes contain these compounds in a range from 40.2 to 184.7 mg/100 g dm. the quantitative and qualitative composition of anthocyanins in potatoes is highly diverse and depends, primarily, on the potatoes’ variety, cultivation site and weather and climatic conditions (lachman et al., 2009; hamouz et al., 2011). according to sulc et al. (2017), the red-fleshed potato varieties might also contain other anthocyanin glucosides, e.g. peonidin, apart from pelargonidin. in contrast, as claimed by other authors (lachman et al., 2009; nemś et al., 2015), both the flesh and skins of potatoes contain the same anthocyanins. however, potatoes of the red-fleshed varieties that were analysed in the present study differed in both the composition and content of acylated compounds (table 2). in rote emma var. potatoes, the majorly identified anthocyanin glucoside was pelargonidin-3-p-cumaorylrutinoside-5glucoside, whereas, in potatoes of herbie 26 and rosemarie varieties, it was pelargonidin3 feruloylrutinos-5-glucoside (table 2). pelargonidin 3-rutinoside-5-glucoside was also found to be a predominating glucoside in skins of herbie 26 and rote emma var. potatoes. in contrast, the skins of potatoes of rote emma var. differed in the composition of glucosides. they did not contain pelargonidin-3-p-cumaorylrutinoside-5-glucoside, and the majorly identified compound in it was pelargonidin-3-feruloylrutinoside-5-glucoside (table 2). according to valiñas et al. (2017), the flesh of potatoes differs in the composition and contents of individual anthocyanins. this is probably due to the migration and transport of metabolites between flesh and skin or vice versa. as of now, however, no research works have addressed this issue. the first technological stage of processing potatoes into most dried products is peeling. in this study, the potatoes were peeled manually, so the depth of peeling might be greater and exceed 1.5 mm. there were no differences in the composition of anthocyanin glucosides in the peeled potatoes, but their losses were observed. after peeling, the total content of anthocyanins decreased by 43% on average (fig. 1). according to furrer et al. (2017), peeled potatoes contain ca. 7% fewer anthocyanins in comparison to the non-peeled ones. the great differences in the loss of anthocyanins, which was observed after potato peeling, might have resulted from the manner and depth of skin removal. in the study conducted by furrer et al. (2017), the potatoes were industrially peeled; therefore, their peeling depth could be significantly less than after manual peeling. during deeper manual peeling, the skin was removed along with the layer of flesh underneath. according to lachman et al. (2013), manual peeling of potatoes up to a depth of ca. 1-2 mm does not affect anthocyanin loss; however, the lack of such an effect depends on the variety. in potatoes of the purple-fleshed variety, namely, violette, the content of anthocyanins decreased by 41% after peeling, whereas it increased by 127 to 286% in potatoes of other colour-fleshed varieties (lachman et al., 2013). anthocyanins occur in higher amounts in the flesh of potatoes rather than in the skin. ital. j. food sci., vol. 31, 2019 157 therefore, during the shallower peeling of tubers, the percentage content of these compounds in dry matter increases. figure 1. anthocyanin residue (%) in potatoes after technological stages of dehydrated dice processing (mean of years). another stage of processing involves blanching potato cubes. the extensive disintegration of the raw material (cubes) and its exposure to a temperature of 75°c for 15 min caused successive loss of anthocyanins (table 3, fig. 1). after blanching, their total content of 60 46 30 30 0 20 40 60 80 100 potato after peeling potato after blanching potato after pre-drying potato after drying rote emma var. 54 32 21 9 0 20 40 60 80 100 potato after peeling potato after blanching potato after pre-drying potato after drying herbie 26 var. 56 47 36 30 0 20 40 60 80 100 potato after peeling potato after blanching potato after pre-drying potato after drying rosemarie var. ital. j. food sci., vol. 31, 2019 158 anthocyanins decreased by 19% on average in potatoes of the rote emma and rosemarie varieties, and by 40% in those of herbie 26 var. compared to the peeled potatoes (table 3). as reported by other authors (mulinnaci et al., 2008; lachman et al., 2013), losses of anthocyanins after blanching might range from 16 to 29%. the extent of these losses can be determined by the ph value of the blanching bath, the degree of raw material disintegration, temperature and the time during which the product is exposed to it (mulinnaci et al., 2008). according to furrer et al. (2017), the stability of anthocyanins in potatoes depends on their variety and the type of heat treatment, specifically, after thermal processing (i.e. blanching, freezing, roasting and frying), the content of anthocyanins decreased by 3 to 29% on average in purple-fleshed potatoes. however, it increased by a few percentage points in red-fleshed potatoes. as demonstrated by lachman et al. (2013), processes, such as roasting, microwaving and steaming, prevent anthocyanin losses. after such their processes were conducted, these authors reported a significant increase in total anthocyanins in purple-fleshed and red-fleshed potatoes, even though the above processes were applied to whole tubers with skins (non-peeled and nondisintegrated). probably, this method of material preparation has a protective effect on the anthocyanin content in potato tubers. as reported by brown et al. (2008), microwaving and cooking cause smaller changes in anthocyanin content than frying or roasting. in our study, blanched potato cubes were pre-dried at 120°c for 1 hour. the impact of high temperature on the material contributed to the successive loss of anthocyanins. after this stage, their content decreased by 23% (rosemarie var.) to 35% (herbie 26 var.) compared to the blanched potatoes; however, no changes were found in the composition of the analysed anthocyanin glycosides (table 3). further drying of potato cubes at 50°c for 8 hours caused changes in the composition of the studied compounds. in the case of herbie 26 var., the potato cubes that were dried to a moisture content of ca. 12% (finished product) did not contain pelargonidin-3-rutinoside-5-glucoside, pelargonidin-rutinoside5-glucoside or pelargonidin-3-p-cumaorylrutinoside-5-glucoside and were characterised by the lowest total content of anthocyanins (2.28 mg/100 g) in comparison to the potatoes of the other varieties (table 3). according to other authors (kita et al., 2013; nemś et al., 2015), high-temperature processes (over 100°c), such as frying or extrusion, not only cause greater losses of anthocyanins that range from 50 to 80% but also changes in the composition of anthocyanin glycosides. according to castaneda-ovando et al. (2009), anthocyanins are highly unstable and susceptible to degradation. their stability depends on multiple factors, e.g., ph, storage temperature, chemical structure, their content, exposure to light and oxygen, solvent and presence of enzymes, flavonoids, proteins or metal ions. in our study, the greatest losses of anthocyanins in the production process of dehydrated potato cubes were attributed to the processes of peeling (43% on average) and pre-drying (31% on average), whereas blanching caused their content to decrease by only 26% on average (fig.1). this was despite the considerable disintegration of the material. changes in the content of anthocyanins varied depending on the variety, and the greatest losses were found upon processing potatoes of herbie 26 var. the production process of dry potato cubes also caused losses in total polyphenols (table 3). the greatest loss of these compounds was noted after the following thermal processes: blanching, pre-drying and drying. according to kita et al. (2015), the heat processes used in potato production not only cause the degradation of phenolic compounds but also the transformation of different groups of polyphenols. the content of anthocyanins in the finished product was ca. 23% on average (fig. 1) and that of polyphenols was ca. 31% of their initial content in the raw material (tables 2 and 3). despite high losses of total polyphenols and anthocyanins during the manufacture of dehydrated products, potatoes of red-fleshed and purple-fleshed varieties might be an alternative to the potatoes with yellow or white flesh. literature data support the ital. j. food sci., vol. 31, 2019 159 conclusion that smaller losses of anthocyanin compounds occur upon processing potatoes with skin, and, perhaps, the use of this type of material should be recommended. 4. conclusions the analysed potato varieties met the established requirements for tubers intended for the manufacture of dehydrated potato products. however, only potatoes of rosemarie var. had lower than recommended content of dry matter and starch and over 0.6% of reducing sugars. potatoes of rote emma variety might be recommended for the production of dried potato cubes, as they met all the requirements and had the highest content of total polyphenols and anthocyanins. in addition, dried potato cubes made of this variety preserved the highest content of biologically active compounds. the process of laboratory production of dehydrated potato cubes caused losses of anthocyanins and total polyphenols in semi-products and finished products. the greatest losses of anthocyanins were noted after peeling, and pre-drying and those of total polyphenols were noticed after blanching, pre-drying and drying. in comparison to the raw material, only ca. 23% of anthocyanins and ca. 31% of total polyphenols remained in the finished product. acknowledgements this publication was supported by the wroclaw centre of biotechnology programme at the leading national research centre (know) for the years 2014-2018. references bridle p. and timberlake c.f. 1997. anthocyanins as natural food colours selected aspects. food chem. 58:103-109. brown c.r., durst r.w., wrolstad, r. and de jong w. 2008. variability of phytonutrient content of potato in relation to growing location and cooking method. potato res. 51: 259-270. castañeda-ovando a., pacheco-hernández ma., páez-hernández m.e., rodríguez j.a. and galán-vidal c.a. 2009. chemical studies of anthocyanins: a review. food chem. 113:859-871. chung ch., rojanasasithara t., mutilangi w. and mcclements d.j. 2017. stability improvement of natural food colors: impact of amino acid and peptide addition on anthocyanin stability in model beverages. food chem. 218:277-284. eichorn s. and winterhalter p. 2005. anthocyanins from pigmented potato (solanum tuberosum l.) varieties. food res. inter. 38:943-948. fossen t., ovstedal d.o., slimestad r. and andersen o.m. 2003. anthocyanins from a norwegian potato cultivar. food chem. 81:433-437. friedman m. and levin c.e. 2009. analysis and biological activities of potato glycoalkaloids, calystegine alkaloids, phenolic compounds, and anthocyanins. 1st ed. in ‘advances in potato chemistry and technology’. j. singh and l. kaur (eds.). pp. 127-161. elsevier, oxford, uk. furrer a., cladis d.p., kurlich a., manoharan r. and feruzzi m.g. 2017. changes in phenolic content on commercial potato varieties through industrial processing and fresh preparation. food chem. 218:47-55. hamouz k., lachman j., pazderů k., tomášek j., hejtmánková k. and pivec v. 2011. differences in anthocyanin content and antioxidant activity of potato tubers with different flesh colour. plant soil environ. 57(10):478-485. horwitz w. and latimer g. 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china 3key laboratory of viticulture and enology, ministry of agriculture *corresponding author: tel.: +86 106273671717 e-mail address: wsmu@cau.edu.cn abstract this study investigates the importance rating of influencing factors in driving wine consumption under four specific situations, that is, gift, banquet, party, and self-drinking, and thus achieves consumer segmentation. the affecting factors containing wine quality and socio-demographic variables are measured on a national representative sample (n=609) in china. lasso method is used to select the factors, and a binary classifier v-twin support vector machine (v-tsvm) is extended to a multi-classification case by using a “one-versus-one” approach, which predicts the purchasing behavior of consumers. the monthly income, occupation, and knowledge of a consumer toward wine, the origin of wine, the vintage, and advertisement, are critical factors in driving consumption. wine color and packing emerge as leading factors when consumer purchase wine for gift and banquet. promotion significantly contributes to wine price selection for banquet, party, and self-drinking. results show that the importance ranking of determinants varies under different purchasing motivations. in addition, the recognition accuracy can be considerably increased with prior knowledge of the consumption purpose. the nonlinear classifier is recommended for application because this classifier performs better than the linear one. this paper offers a fresh perspective on wine consumption behavior in china by applying two machine learning methods to identify and quantify determinants in specific situations. the results significantly assist wine managers to provide informed decisions with regard to wine production and marketing. keywords: wine motives, personal traits, wine price, influential factor, consumers’ purchasing behavior ital. j. food sci., vol. 30, 2018 776 1. introduction the chinese wine market has been flourishing in recent years with the improvement of the living standards of people and influence of affluent western lifestyles. in 2015, china has produced and consumed 11.5 million and 16 million hectoliters of wine, respectively, and ranked sixth in the global wine production (oiv, 2016). the prediction of the purchase behavior of consumers has been recognized as a significant research topic over the past decades. the accurate prediction of purchasing behavior enables wine dealers to accurately locate consumers’ demands, formulate appropriate marketing strategies, and achieve consumer segmentation. a study on the influence of purchasing motivation will aid in understanding the progress of consumer decision-making. previous studies have shown that a wine consumer exhibits different purchasing motivations under various consumption scenarios in a descriptive way (geraghty and torres, 2009). the main motivations of chinese wine consumers include health care, auxiliary dining, and social contact (li, 2014). people perceive wine as a healthy and nutritional product that can be recommended for regular intake to prevent diseases because wine contains many kinds of organic acids, minerals, and vitamins (tang, 2008; mu, et al., 2016). consumers have different preferences for wine attributes, when they drink at home, drink with friends and give as gifts (quester and smart, 1998; li, 2014; chen, 2014). drinking with friends at parties is casual and relaxed, whereas the major function of wine is to please other people in a business banquet (hall et al., 2001). currently, an increasing number of chinese aim to give red wine as a gift to display affection or enhance friendship, especially during festivals. wine consumption is influenced by many interrelating factors, such as wine product properties, lifestyle and situations of an individual, and psychological factors of consumers (pickering and hayes, 2017; schmitt, 1997). various attributes, such as taste, color, aroma, brand, production, and label information, are found to be important aspects that determine wine choice (thorpe, 2009). lockshin et al. (2017) summarized several methods used in marketing in combination with sensory science techniques to understand the changing consumer preferences in china. the consumption behavior of chinese are highly related to the educational background of consumers, wine-related activities, wine taste, country of origin, quality, and price (balestrini and gamble, 2006; camillo, 2012). most business models are based on a linear equation to estimate the weight of such factors when measuring the response of purchase intention to the contextual factors. the commonly used linear models are linear discriminant and logistic regression analyses (culbert et al., 2017; honoré-chedozeau et al., 2017; li, 2014; yormirzoev, 2016). the prediction models for purchase behavior are over-concentrated and over-reliant on these linear models compared with other research fields. in addition, principal component analysis (pca) is also combined with the linear models to reduce the dimensionality of factors (jolliffe, 2002; chang, et al., 2015; tsourgiannis et al., 2015). however, using pca to extract the component feature may lose certain important information. the meaning of comprehensive evaluation function is unclear when the labels of load factor in the principal component are positive and negative; thus, this function is sensitive to the relative scaling of the original variables and has low variable interpretation. moreover, we can collect additional consumer data information with the development of communication technologies. analyses based on traditional linear models are insufficient in achieving the requirement of academics and practitioners (daykin and moffatt, 2002; thong and solgaard, 2017). in recent decades, increasing machine learning approaches have emerged. the least absolute shrinkage and selection operator (lasso) is recognized for its capability to exploit ital. j. food sci., vol. 30, 2018 777 information from ordinary data and flexibility to capture different effects of explanatory variables (tibshirani, 1996). the lasso method can continuously shrink certain coefficients to zero and automatically select a subset of variables. in addition, the lasso method has better variable interpretability than other feature selection methods, such as principal component regression and least squares regression (tian et al., 2015). the support vector machine (svm) has been considered an effective and promising binary classifier for its unique advantages (vapnik, 1995). the introduction of kernel function maps training variables into a high-dimensional space, thereby successfully solving the nonlinear svm. many variants of svm have been proposed since then, and several binary svms have been successfully extended to multi-class scenarios by applying “one-versusone” (ovo) and “one-versus-all” (ova) strategies (tomar and agarwal, 2015; wang and zhou, 2017). the svms have been widely applied in various aspects that range from disease diagnosis and bankruptcy prediction to consumption behavior prediction (e.g., electricity, health product, and building energy) (bahamonde et al., 2007; guo, 2013; kavaklioglu, 2011). this study aims to use two representative machine learning methods, that is, lasso and ovo v-tsvm, to investigate the determinants on the wine price selection under free and four purpose-based choices, that is, gift, banquet, party, and self-drinking, so as to predict the price of wine purchased by a consumer and estimate the effects of major factors selected through the lasso method simultaneously. 2. materials and methods 2.1. conceptual framework numerous researches discipline including economics, marketing, psychology, and products, have a shared interest in consumers’ behavior. more and more researchers have increasingly concentrated on consumers’ attitudes, motivation, perceptions and preferences for wine. previous studies show that the motivation for purchasing wine varies under different purchasing situations (barreiro et al., 2008). moreover, goodman (2009) found that previous tasting experience and opinion of other people significantly influence wine purchasing behavior. the knowledge of consumers toward wine positively and notably affects the wine purchasing behavior of these consumers (hussain et al., 2007). consumers with higher production involvement are less sensitive to wine price, whereas consumers with lower production involvement focus more on price discounts (jaege et al., 2009). furthermore, many researches have shown that consumers’ purchase choices are well related with age and education in wine consumption. based on the previous studies and combining with characteristics of wine consumption, the factors affecting wine consumption were summarizes in fig. 1. it covers a range of purchasing motivations, reference group factor, marking factors, wine quality factors, the knowledge level towards wine and characteristics of consumers. ital. j. food sci., vol. 30, 2018 778 figure 1. conceptual framework of consumer’s purchasing behavior for wine. 2.2. questionnaire the questionnaire of chinese consumers’ decision making behavior towards wine (it is shown in the appendix) was designed which consisted of 30 questions. this questionnaire includes the following contents: (1) questions regarding the purchasing behaviors of consumers (the frequency of purchasing and drinking). (2) questions investigating the price of wine that consumers frequently purchase. the consumers selected seven kinds of wine price, that is, 1=“$0-7.5,” 2=“$7.6-15.1,” 3=“$15.222.6,” 4=“$ 22.7-30.1,” 5=“$ 30.2-45.2,” 6=“$ 45.3-75.3,” and 7=“$75.4 and above.” based on the literature review, four usually types of motivation (gift, banquet, party, and selfdrinking) for wine consumption were extracted and described in the questionnaire. besides, the consumers were asked to choose the price of wine that they purchase for the specific purpose; (3) questions that belong to multi-item scales, which measure factors that influence consumer purchasing, such as influence of others, quality of wine, enterprise marketing factors, knowledge of consumers. this study investigates the 10 items of wine quality factors, namely, the origin of wine and vintage, effects, packing, brand, label information, color, aroma, taste, and awards. the enterprise marketing factors contains 4 items, i.e. advertisement, promotion, service and attitude of the salesperson, and store location and environment. the 16-item scale was collected using a 5-point likert scale from 1=“strongly disagree” to 5=“strongly agree.” (4) consumers’ socio-demographic characteristics: gender, age, marital status, monthly income, education background, and occupation. all the six features use the numbers “1, 2, 3, …” to assign the variable level from low to high. ital. j. food sci., vol. 30, 2018 779 2.3. survey considering the sampling frame and economic development level in different regions, we hired and trained several undergraduate students from china agricultural university to answer the survey. we realized that young people are the main force in wine consumption and many wine tasting groups are found on the internet. the survey was conducted in 2016 and lasted for five months. a total of 1600 questionnaires were distributed in many provinces of china, and 995 questionnaires were returned. in the returned questionnaires, the respondents were instructed to evaluate the statement “in the past year, how often did you purchase wine?” the data were “cleaned” by removing responses of “never bought wine.” therefore, the respondents in this study are consumers who, on one occasion, purchased wine. finally, 609 questionnaires were used for final analysis. 2.4. methods the analysis of the data consisted of two steps. first, the lasso method was conducted to select the determinants. in theory, the discrimination ability we can obtain is robust when we use considerable features. however, an excessive number of features may increase the learning speed and lead to “overfitting” problem. the accurate selection of features is a prerequisite for a high prediction accuracy. the lasso method penalizes the regression coefficients with an l1 penalty, shrinking many of the features to zero. any features with non-zero coefficients are “selected” through the lasso method, which indicates that these selected features contribute most to the wine purchasing behavior of consumers. second, the ovo mv-tsvm method was used to predict the behavior of chinese wine consumers. to the best of our knowledge, the v-tsvm (peng, 2010) was initially proposed for binary problems. owing to the k-class scenario, we use the ith class as the positive and jth as the negative to construct a binary v-tsvm classifier. the ovo mv-tsvm method need to construct k(k−1)/2 binary v-tsvm classifiers. for a new testing point, we obtain the vote for each class and assign its label with a maximum vote. for the nonlinear case, we used the gaussian kernel function ker(xi ,x j )=e − xi −x j 2 /2r2 and grid research to find the optimal parameter. all algorithms were written and operated in matlab 2014a, and all statistical analyses were conducted using the spss version 20 and microsoft office excel version 2013 software. 3. results the whole cronbach’s of the questionnaire is 0.776, f=334.221, sig=0.00, thereby indicating that the survey has a high internal consistency. the response rate of questionnaire is 62.19%. a majority of the respondents (63.71%) would purchase wine once or twice a year, and 81.94% would drink two or more bottles of wine in a year. the 609 samples were collected from 21 provinces, cities, and autonomous regions in china. we inquired the per capita monthly income of the above areas from the china statistical yearbook 2016, on which we calculated the global per capita monthly income as a standard, and the value is 780.06$. the provinces where the samples were collected are located in eastern china, and most of these samples were relatively advanced in the ital. j. food sci., vol. 30, 2018 780 economic area. a total of 9.69% participants would purchase wine as a gift, 21.18% for banquet, 30.05% for parties, and 39.08% for self-drinking. the results of wine price that the consumers purchased are listed in table 1. based on these samples, 65.51% would purchase wine in the price range of 7.6-30.1$ with free choice. the average price is 30.20$ (sd=0.83), with a 95% confidence interval of (28.58, 31.85). for the purpose of gift, 55.83% would select the wine price above 30.2$, and the average price is 40.64$ (sd=0.91). for the purpose of banquet, 64.20% would select the wine price in the range of 15.2-45.2$, and the average price is 30.71$ (sd=0.74). for the purpose of party, 64.86% would select the wine price in the range of 7.6-30.1$, and the average price is 27.97$ (sd=0.69). for the purpose of self-drinking, 65.19% would select the wine price in the range of 7.6-30.1$, and the average price is 28.11$ (sd=0.75). table 1. statistical results of consumer's purchased wine price. wine price ($) free-choice (％) gift-based (％) banquet-based (％) party-based (％) self-drinking-based (％) 0-7.5 2.63 1.15 1.64 3.28 4.43 7.6-15.1 22.99 12.15 17.24 20.69 22.33 15.2-22.6 20.85 12.15 21.02 21.35 22.50 22.7-30.1 21.67 18.72 23.15 22.82 20.36 30.2-45.2 12.32 19.70 20.03 19.05 14.45 45.3-75.3 9.85 17.41 10.84 8.87 10.84 above 75.3 9.69 18.72 6.08 3.94 5.09 mean* 30.20 40.64 30.71 27.97 28.11 sd.* 0.83 0.91 0.74 0.69 0.75 95%confidence interval* (28.58, 31.85) (38.89, 21.82) (29.27, 32.17) (26.65, 29.35) (26.61, 29.59) note: *are the results of 10000 times bootstrap resampling results. the characteristics of the sample’s demographics are detailed in table 2. the average age is 35.18 years (sd=0.42). the average monthly income is 774.67$ in 10000 times bootstrap estimation, which is nearly the same as the standard 780.06$. the respondents are 52.71% male and 47.29% female; a total of 32.35% are single, and 67.65% are married. a majority of the respondents who attained a college degree were 76.52%, 18.56% are senior high or in a special school, and only 4.93% are in primary or junior high school. the respondents vary in careers, 8.21% are students, 2.30% are peasantry, 25.94% are freelance, 2.96% are unemployed or retired, 11.99% are staffs of state-owned companies, 13.30% are staffs of foreign or private enterprises, 15.60% work as party and government officers, 9.36% work in education and scientific research units, and 10.34% work in other fields. inspired by forleo et al. (2017), we lists the associations of wine consumption prices with demographics in table 3. it is obvious that monthly income and occupation are significant no matter in what purpose-based. there are about 10% high-income and 3~4% lowincome consumers choose high-priced wine. male and female showed differences in the wine purchasing for free-choice, gift-giving and banquet-based purpose. there are 17.73% male and 14.12% female consumers choose wine price above 30.2$. the gender difference is not obvious in party-based and self-drinking based wine purchasing. there are only 8% elder people (above 46 years) choose high-priced wine (above 30.2$), and the percentage increased to 12% for gifted purpose. the single consumer and married consumer acted ital. j. food sci., vol. 30, 2018 781 different in wine price-choosing for party-based and self-drinking based purpose. statistically significant differences between education and wine-price choosing for gifted and banquet-based purpose were identified. about 30% highly educated consumers choose high-priced wine, and only 1% consumers with primary or junior high school background chose high-priced wine. fig. 2 illustrates the results of statistical affecting factors, where the mean of wine knowledge is the highest at 4.04, and the mean of advertisement is the lowest at 3.18. table 2. statistical features of respondents. demographic characteristics category percentage sample population(n) gender male 52.71 321 female 47.29 288 age 18-25 22.33 136 26-35 31.86 194 36-45 24.14 147 46-55 17.24 105 above 55 4.43 27 mean/sd.* 35.18 0.42 95%confidence interval* (34.35, 36.01) marital status single 32.35 197 married 67.65 412 per capita monthly income ($) 0-301.2 13.46 82 301.3-451.8 14.29 87 451.9-753.0 32.35 197 753.1-1054.2 20.69 126 1054.3-1506.0 9.52 58 1506.1-2259.0 4.76 29 above 2259.0 4.93 30 mean/sd.* 774.67 21.81 95%confidence interval* (732.09, 818.99) educational background primary or junior high school 4.93 30 senior high or special school 18.56 113 junior college or undergraduate 62.73 382 postgraduate and above 13.79 84 job students 8.21 50 peasantry 2.30 14 freelance 25.94 158 unemployed/retired 2.96 18 staffs of state-owned companies 11.99 73 staffs of foreign or private enterprises 13.30 81 party and government officers 15.60 95 education and scientific research units 9.36 57 else 10.34 63 note: *the bootstrap estimate was calculated as the mid-value of the range. ital. j. food sci., vol. 30, 2018 782 table 3. association of wine consumption prices with demographics. items-prices gender age marital status monthly income education occupation free-choice 0.004* 0.111 0.097 0.000*** 0.095 0.001*** gift-based 0.014* 0.021* 0.369 0.000*** 0.002** 0.000*** banquet-based 0.017* 0.000*** 0.215 0.000*** 0.010** 0.000*** party-based 0.160 0.000*** 0.012* 0.000*** 0.136 0.000*** self-drinking based 0.230 0.001*** 0.024* 0.000*** 0.110 0.000*** note: *0.01<p<=0.05; ** 0.001<p<=0.01; *** p<=0.001. figure 2. statistical results of affecting factors. the trace plot of coefficient through the lasso method and the corresponding crossvalidated mse under free choice are depicted in fig. 3, where different color lines represent various affecting factors (b1 to b23) in fig. 3(a). the vertical bold blue and green dashed lines stand for the parameter under minmse and 1se, respectively, and we only record the weights of factors under the minimum and 1se in table 4. from left to right in fig. 3(a), the factors gradually shrink to zero, and the last factor that became zero represents the most important determinants. in the minmse position, 16 factors are selected. the factor selected through the lasso method significantly affects the wine consumption behavior under free choice, which maintains a null hypothesis. in the index1se position, only five factors are left; these factors are monthly income of consumers, the vintage, the origin of wine, purchasing motivation, and the service and attitudes of salesmen. we can achieve a prediction accuracy of 62.56% and 63.01% by using the first five important features for prediction in linear and nonlinear ovo mvtsvm, respectively. the algorithm relies considerably on the parameter, and the process of grid research is presented in fig. 3(c). ital. j. food sci., vol. 30, 2018 783 (a) (b) 232220191716135 4 3 1 df 10 -4 10 -3 10 -2 10 -1 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 lambda trace plot of coefficients fit by lasso lambdaminmse lambda1se b1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14 b15 b16 b17 b18 b19 b20 b21 b22 b23 10 -4 10 -3 10 -2 10 -1 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 lambda m se cross-validated mse of lasso fit ital. j. food sci., vol. 30, 2018 784 (c) figure 3. (a) the trace plot of coefficients by lasso, (b) cross-validated mse of lasso fit under free-choice, (c) the influence of the parameter v, kernel parameter r and prediction accuracy for nonlinear ovo mv-tsvm with free-choice. #b1: purchasing motivation, b2: others' influence, b3: advertisement, b4:promotion, b5: service and attitudes of salesman, b6:store location and environment, b7: the origin of wine, b8: the vintage, b9:effect, b10: packing, b11:brand, b12: label information, b13: color, b14: aroma, b15: taste, b16:awards, b17: the wine knowledge, b18: gender, b19: age, b20: marital status, b21:monthly income, b22: education, b23: job. similarly, the parts of experimental results under four purposes, that is, gift, banquet, party, and self-drinking, are also summarized in table 4. for the gift purpose, the first five important affecting factors are as follows: monthly income, occupation, wine knowledge of consumers, wine color, and production origin. for the banquet purpose, the first three determinants are monthly income, the origin of wine, the vintage. for the party purpose, the first five important affecting factors are as follows: monthly income, the origin of wine, wine knowledge of consumers, the vintage, and occupation. for the self-drinking purpose, the first five determinants are as follows: monthly income, the origin of wine, wine knowledge of consumers, occupation, and the vintage. the accuracy of nonlinear ovo mv-tsvm can achieve 80.12%, 72.46%, 63.49%, and 74.69% by using the selected factors. 0 0.2 0.4 0.6 0.8 1 -10 -5 0 5 10 40 45 50 55 60 65 vr a cc ur ac y 0.6, 28, 63.01% ital. j. food sci., vol. 30, 2018 785 table 4. part of the lasso results and prediction results using ovo mv-tsvm. metrics free-choice gift-based banquet-based party-based self-drinking based coef. coef coef. coef coef. coef coef. coef coef. coef min λ index1se min λ index1se min λ index1se min λ index1se min λ index1se purchasing motivation -0.227 0 0.067 0 0.050 0 0.078 0 0.064 0 others' influence -0.021 0 0.112 0 -0.017 0 -0.087 0 -0.134 0 advertisement 0.007 0 -0.083 0 -0.119 0 -0.025 0 -0.100 0 promotion -0.058 0 0.008 0 -0.041 0 -0.102 0 -0.102 0 the salesman' service and attitudes 0.079 0 -0.105 0 0.005 0 0.002 0 0.015 0 store location and environment 0.049 0 0.040 0 0.017 0 0.050 0 0.076 0 the origin of wine 0.350 0.014 0.098 0.014 0.195 0.070 0.174 0.088 0.231 0.140 the vintage 0.256 0 0.248 0 0.217 0.007 0.164 0.053 0.152 0.002 effect 0.027 0 -0.043 0 0.049 0 0.092 0 0.045 0 packing 0.019 0 -0.112 0 -0.022 0 -0.047 0 -0.035 0 brand -0.217 0 -0.111 0 -0.125 0 -0.132 0 -0.108 0 label information -0.146 0 -0.026 0 -0.009 0 -0.019 0 -0.015 0 color 0.059 -0.059 -0.301 -0.059 -0.134 0 -0.058 0 0.036 0 aroma 0.101 0 0.022 0 0.100 0 0.038 0 0.095 0 taste -0.127 0 0.070 0 0.016 0 -0.050 0 -0.093 0 awards 0.047 0 -0.011 0 -0.006 0 0.065 0 -0.024 0.000 the wine knowledge 0.076 0.149 0.328 0.149 0.183 0 0.265 0.098 0.275 0.07 gender 0.136 0 0.240 0 0.163 0 0.041 0 0.195 0 age -0.116 0 0.016 0 -0.020 0 -0.036 0 -0.026 0 marital status -0.015 0 0.025 0 -0.007 0 0.103 0 0.051 0 monthly income 0.434 0.109 0.174 0.109 0.247 0.136 0.257 0.202 0.237 0.163 education -0.160 0 0.093 0 0.098 0 0.000 0 0.047 0 occupation -0.038 0.057 0.089 0.057 0.043 0 0.055 0.021 0.055 0.016 intercept 2.172 3.578 1.918 3.578 1.041 3.248 0.897 2.049 0.692 2.240 ital. j. food sci., vol. 30, 2018 786 df 23 5 23 5 23 3 23 5 23 5 mse 2.250 2.573 2.542 2.573 2.035 2.068 1.938 1.938 2.233 2.249 se 0.060 0.103 0.110 0.103 0.105 0.127 0.061 0.062 0.077 0.070 λ 6.26e-05 0.168 3.89e-05 0.168 4.62e-05 0.241 4.83e-05 0.131 4.51e-05 0.162 (i)percentage correct prediction 62.56 79.22 65.22 49.21 61.45 (ii)percentage correct prediction 63.01 80.12 72.46 63.49 74.69 #i: linear ovo mv-tsvm; ii: nonlinear ovo mv-tsvm. ital. j. food sci., vol. 30, 2018 787 4. discussion this study provides a fresh perspective on the wine consumer segmentation based on importance ratings of factors by applying two popular machine learning methods. in addition, these segments are predictive in specific purchasing situations. the results provide valuable information for policymakers and marketing managers. first, the consumers would pay a high wine price for gift giving, and a relatively low wine price for self-drinking. the importance rankings of determinants in wine consumption vary under different purchasing situations. these rankings will guide a salesperson in utilizing the key points when recommending wine products to customers. for example, a salesperson should primarily realize the monthly income, occupation, degree of wine knowledge of a consumer, wine color, the origin of wine, if the wine is purchased as a gift. the ovo mv-tsvm can aid in predicting the type of consumers when the information is acquired. the retailer or salesman can then recommend wine with the corresponding price. if the purchasing motivation is for a banquet, then the retailer should primarily focus on the income of the consumer, the origin of wine, and the vintage. this result is our most important discovery. furthermore, using the lasso method can reduce the complexity before prediction because the relevant features can be selected, whereas the irrelevant features become zero under a fixed value. second, the nonlinear ovo mv-tsvm behaves better than the linear ovo mvtsvm. these results indicate that the data we collected are linearly inseparable, thereby leading to poor testing accuracy with linear models. the kernel trick in the ovo mv-tsvm maps the data into a high-dimensional space, hence successfully making the data linear separable in the projected space (shawe-taylor and cristianini, 2004). accordingly, the nonlinear model is suggested in wine consumption prediction area. under the free choice, the nonlinear ovo mv-tsvm can only achieve 63.01%, whereas this strategy obtains high-prediction accuracies under the four purchasing situations. the pre-knowledge of the purchasing situation of a consumer would help marketers provide an accurate recommendation to wine consumers. in terms of prediction accuracy, the case of purchasing under the gift purpose can explain the purchase behavior because its accuracy achieves 80.12%, followed by self-drinking, banquet, and party. in addition, the prediction accuracy of the ovo mv-tsvm is significantly affected by the choice of parameters. the prediction accuracy must select the appropriate parameters beforehand in practical applications. third, most personal traits are the important determinants of wine consumption (pickering and hayes, 2017). the monthly income and occupation of consumers are the leading positive influential factors in wine consumption regardless of the purchasing conditions, hence implying that consumers with high monthly income and a favorable job prefer wine with a high price. occupational difference reflects the social status of consumers to a certain extent, liu and murphy (2007) found that wine was seen as a symbol of one’s social status and sophistication. gender positively influence the high priced wine purchasing, especially in free choice, giftgiving and banquet purpose. male consumer would prefer higher-priced of wine than female consumer. education positively influences the high priced wine purchasing when the purposes are gift and banquet, whereas education is an insignificant factor for party. age of consumers is found to be sensitive and negative to wine purchasing for banquet, party, and self-drinking purpose, thereby implying that young chinese consumers prefer higher priced wine than elder persons who are ital. j. food sci., vol. 30, 2018 788 more thrifty. marital status is not a leading factor for wine purchasing, while it emerges as a relatively significant factor for party-based wine purchasing. fourth, the knowledge of consumers about wine is a major positive driver for wine consumption (barber et al., 2008). in consumption decision-making, the consumers rely on their professional knowledge, especially on their subjective knowledge. consumers will behave confidently and rely on their judgment when these consumers assume that they have professional knowledge, and vice versa. these results imply that consumers will spend more money on wine if they absorbed the wine culture. fifth, the vintage and origin of wine are found to be the more important determinants for prediction than wine taste and awards. the weights of the vintage and oration of wine are positive, which means consumers would pay more if they value the wine vintage and origin. for banquet, wine taste and aroma have a positive effect on wine price selection, which indicates that consumers would pay more money to receive a favorable quality of the wine. wine color and packing are important factors when consumers purchase wine as a gift, whereas these factors are insignificant for self-drinking, which provides valuable suggestions for wine sellers and producers to sell or produce wine. for party, banquet, and self-drinking, the effect of wine emerges as a significant positive factor in high-priced wine selection. this result provides a hint to the winemakers to produce banquet and self-drinking wine with favorable efficacy to attract consumers, thus increasing the returns. consumers attach wine aroma when purchasing wine for self-drinking and banquet compared with the purposes of gift and party. furthermore, consumers are nonsensitive to wine brand and label information. our findings are meaningful for and can be implemented by winemakers and suppliers to formulate appropriate strategies in accordance with the preference and purchasing purpose of consumers. sixth, this study demonstrates that purchasing motivation is a positive driver for wine consumption. previous studies have shown that people trust their families, friends, colleagues, or acquaintances; recommendations of other people have a significant influence on the purchasing behavior of consumers (goodman, 2009). moreover, consumers are especially sensitive to opinions of other people when these consumers purchase wine for self-drinking and party, and the effect is negative to high-priced wine selection. by contrast, the influence is positive for gifts. the results suggest that wine dealers can invite wine critics to recommend gifting wine on tv or take measures for expanding the influence of friends. lastly, advertisement is a driver for purchasing wine regardless of the purpose, and the influence is negative for high-priced wine, which warns the wine dealers to invest reasonably in advertisements because excessive advertisement expenditure can conversely affect the sale of wine. the promotion activities of enterprises can inspire the latent purchasing behavior of consumers (pohjanheimo et al., 2010). consumers are more influenced by the service and attitude of the salesperson than the promotion, store location, and environment when these consumers purchase wine as a gift. managers are encouraged to improve the service and skills of their sales personnel to improve their sales of wine as a gift. promotion becomes a significant factor when consumers purchase wine for banquet, party, or selfdrinking, and the influence is negative because promotions result in an increased price of wine, thereby reminding managers to create suitable promotional activities. the store location and environment emerge as a driver when consumers purchase wine for self-drinking, and the influence is positive for selling high-priced wine, thereby indicating that consumers would pay a relatively high price for self-drinking if the store location is near their home or the environment is comfortable and clean. ital. j. food sci., vol. 30, 2018 789 5. conclusion and future work the present study explored 23 factors that are associated with wine consumption under four specific purchasing situations based on a surveyed sample of the chinese population. the lasso method was used as a primary empirical tool for selecting the most affecting features, and the ovo mv-tsvm method was used to predict the purchasing behavior of consumers. the findings can be applied in various commercial fields. limitations should be noted that may aid in drawing avenues for future research, although our study exhibits interesting results in terms of using the machine learning methods. first, the samples collected in the study were from 21 provinces in central and eastern china. although the respondents who completed the survey were rewarded with a monetary deposit into their alipay and wechat accounts as an incentive, the recovery rate of the questionnaire is not high enough. owing to the limited time and resources, we were unable to collect data from western and southwestern china, such as xinjiang, xizang, and yunnan provinces. the samples used in this study might not be representative of the whole county. second, information on consumer perception for a specific wine product, such as claret, white wine, or sweet red wine can be collected. culbert et al. (2017) investigated the sensory profiles and consumer acceptance of different styles of australian moscato and led us to focus on the determinants of a specific wine product in china. this area helped us predict the purchasing behavior of chinese consumers, adjust models, and provide useful suggestions for marketers and companies to create reasonable and timely adjustments. third, the list of factors in this study was not intended to be exhaustive, and other factors could be incorporated. for example, we only set one item to investigate the knowledge of consumers toward wine consumption. to the best of our knowledge, familiarity with wine involves various aspects, such as grape varieties, viticulture, wine process, wine tasting, and wine storage management. further, the questions on the consumption of wine and human health (tamburro et al., 2017) can be considered in further research. the emergence of machine learning methods leads to new research topics on wine consumption. the other 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gallina artificially contaminated with vibrio parahaemolyticus and vibrio vulnificus. contamination experiments were used to assess the accumulation capacity of clams in poorly, moderate and highly contaminated waters (vibrio spp103/ml, 104/ml, 106/ml). all bivalves were exposed to contamination for 72 h and 8 experiments were performed. accumulation capacity varied with respect to initial level. comparing experiments dataset with two vibrios species, clams showed a different specific accumulation: in particular, molluscs evidenced a scarce accumulation capacity of v.vulnificus. depuration trials were performed in close-circuit seawater-disinfection system that uses filtration, ultra violet (uv) and ozone. bivalves samples were collected every 12h until 3 days of depuration. most of the depuration trials with v.parahaemolyticus showed a decrease in initial bacterial loads (4times lower values) after36-48 hours, but in subsequent periods, the trend remained stationary. in v. vulnificus tests, clams showed a scarce depuration capacity instead. future studies are still required to assess the efficacy of the depuration process in reducing pathogenic vibrio strain naturally accumulated in clams and to prevent significant economic losses to stakeholders due to long depuration periods. keywords: food safety, depuration, clams, v. parahaemolyticus,v.vulnificus ital. j. food sci., vol. 30, 2018 603 1. introduction the exploitation of bivalve molluscan shellfish is of great social and economic importance in the coastal ecosystems of southern europe (berthou et al., 2005). in particular, the landing of venus gallina play an important economic role in the central and northern adriatic coasts of italy, where it has considerably increased in the last decades with the introduction of the hydraulic dredges (morello et al., 2005; moschino and marin, 2006). the venerid bivalve, venus gallina, is a mollusc distributed throughout the mediterranean and black sea (moschino and marin, 2006). the interest towards this clam increased also in relation to its nutritional characteristics. in fact, it has interesting dietetic properties due to the low lipid and cholesterol contents, presence of phytosterols and the high percentages of healthy polyunsaturated fatty acids (orbanet al., 2006). since bivalves are filter-feeding organisms, they can accumulate pathogenic microorganisms (for example, bacteria, human viruses and microalgae), having a significant health risk if consumed raw or lightly cooked (cook, 1991; lees et al., 2010). generally, bivalves’ bacterial composition is dominated by gram-negative bacteria like vibrionaceae and enterobacteriaceae (cao et al., 2009). among the indigenous microbiota of coastal environments, the family vibrionaceae, particularly v. parahaemolyticus, v. vulnificus and v. cholerae, is targeted as a causative agent of human disease due to the consumption of shellfish (butt et al., 2004; normanno et al., 2006; ripabelli et al., 1999). the occurrence of potentially pathogenic vibrio spp. in coastal waters and shellfish of european countries has already been documented in italy, spain and france (barbieri et al., 1999; hervio-heath et al., 2002; martinez-urtaza et al., 2008, roque et al., 2009). the number of vibrio spp. in shellfish varies widely and depends on the geographical area, environmental conditions and local parameters. salinity and temperature are in fact important parameters in the dynamics of vibrios in marine systems (hsieh et al., 2008; blackwell and oliver, 2008; depaola, 2003; pfeffer et al., 2003; randa et al., 2004). some studies have shown that v. parahaemolyticus, v. vulnificus and v. cholerae enter a viable but non-culturable state when water temperature average is below 15°c (colwell and grimes, 2000; roszak and colwell, 1987). on the other hand, temperatures above 20°c favor the growth of vibrio spp. in seawater (blackwell and oliver, 2008; depaola et al., 2003). in order to reduce shellfish contamination and to minimize the inherent risks of shellfish consumption, legislation sets requirements for sample collection, wet storage, bivalve selfpurification by depuration and/or relaying (tank construction and operation, packaging, labelling), shellfish processing, laboratory analytical methodologies and product distribution (reg. n.853/2004/ec, reg. n.854/2004/ec). the legislation employs a classification to the seafood harvesting areas according to bacterial indicators of sanitary quality (e. coli). this classification determines whether shellfish can be sent for direct consumption or must be treated prior to commercialisation (lees, 2000). furthermore, the current european legislation does not specify limits for vibrio spp. (ec, 2001). therefore, preventive measures should be implemented for vibrio spp., taking into account that they are naturally found in seawater and normal constituents of mollusc flora (barile et al., 2009) and some strains (such as v. parahaemolyticus) are the major cause of epidemics associated with the consumption of bivalves (mead et al., 1999; wittmann and flick, 1995). relaying and depuration are common approaches to reducing bacterial loads in shellfish. in the relaying process, shellfish is transferred before harvest from polluted areas to an unpolluted waterway for natural biological purification. depuration consists of a flowthrough or recirculation system of chemically (chlorine, ozone, iodophores, and activated oxygen) or physically (uv irradiation) disinfected water to allow purification under ital. j. food sci., vol. 30, 2018 604 controlled conditions (lees, 2000). effectiveness of the depuration process depends on the diversity and physiology of shellfishes, initial loads of bacterial strains, environmental conditions (temperature, salinity, ph and so on.) and purification system (joven et al., 2011; scheneider et al., 2009). several studies have been carried out to evaluate the effect of depuration in physiological and microbiological aspects of some clams species, such as ruditapes decussatus, venerupis senegalensis, venus gallina and mercenaria mercenaria (el-schenawy, 2004; howard et al., 2003; maffei et al., 2009). in general, the depuration process is particularly efficient in the reduction of total viable counts and e. coli levels (anacleto et al., 2013), but not with vibrio spp. in fact, vibrio spp. requires longer depuration periods than e. coli to become effective (colakolu et al., 2014; cozzi et al., 2009; croci et al., 2002; lopez-joven et al., 2011). croci et al. (2002) indicated that 44 h of depuration process led to a decline in vibrio by a factor of only 10, whereas, cozzi et al. (2009) describes that 72 h reduced vibrio contamination to a level close or below the detection limit of the methods. also, lopez-joven et al. (2011) reported that at least 10 days depuration at 20°c is effective in reducing vibrio load. in this context, the main objective of this study was to investigate the depuration capacity of venus gallina artificially contaminated for three days with vibrio parahaemolyticus and vibrio vulnificus running tests. 2. materials and methods 2.1. collection of samples and treatments venus gallina specimens were taken at about 250-500 m from molise coastline in classified areas. clams were harvested with hydraulic dredges. the specimens (mean size: 25-32 mm) were taken to the laboratory in refrigerated containers (4°c) within 2 hours. dead or damaged specimens were discarded and the remainder was divided into seven aliquots of 250 clams. before the trial, specimens were acclimatised for 72h in aquariums with 100 litres of recirculated and artificial seawater (instant ocean aquarium system salinity: 35‰). temperature was fixed at 18°c. after acclimatization, ten specimens for each aliquots underwent bacteriological analysis (v. parahaemolyticus, v. vulnificus) to evaluate the sanitary conditions and the initial load of each pathogen for determining the contamination levels. 2.2. contamination experiment vibrio parahaemolyticus atcc 17802 and vibrio vulnificus atcc 27562 were used for trials. bacterial contamination was conducted in 8 l (0.12 m3) tanks filled with artificial seawater (ocean fish marine salts-prodac). in this study, contamination experiments were conducted to assess the accumulation capacity of clams in poorly, moderate and highly contaminated waters (103/ml, 104/ml, 106/ml). contamination levels were chosen from results of previous studies (barile et al. 2009; barile et al. unpublished). details of each contamination trial are reported in table 1. to test the influence of temperature on vibrio vulnificus accumulation in clams, two trials were conducted at different artificial conditions: 35‰salinity and 22°c temperature (test vv2), 30‰salinity and 25°c temperature (test vv3). parameters were established considering results of previous studies (colwell and grimes, 2000; blackwell and oliver, 2008). ital. j. food sci., vol. 30, 2018 605 all bivalves were exposed to contamination for 72 h. after contamination period, ten replicates for each test were analyzed to evaluate the variability of their accumulation capacity. table 1. details of contamination trials. species bacterial load temperature salinity test vibrio parahaemolyticus 2.30*103 22°c 35‰ vp1 vibrio parahaemolyticus 3.75*104 22°c 35‰ vp2 vibrio parahaemolyticus 9.37*104 22°c 35‰ vp3 vibrio parahaemolyticus 1.12*106 22°c 35‰ vp4 vibrio vulnificus 2.30*103 22°c 35‰ vv1 vibrio vulnificus 7.5*104 22°c 35‰ vv2 vibrio vulnificus 7.5*104 25°c 30‰ vv3 vibrio vulnificus 1.12*106 22°c 35‰ vv4 2.3. depuration experiment at the end of contamination, all clams were transferred to depuration system (fig. 1), except for vv3 test. specimens were placed in a single layer on a plastic grill to avoid contact with the bottom of the tank and thus, minimise recontamination. figure 1. trial depuration system. depuration systems consisted of tanks fitted with a wool/perlon prefilter and hyperactive carbon filter, an active biological filter using lithothamnium calcareum algae and a uv sterilisation plant (fig. 2), and filled with artificial seawater (ocean fish marine saltsprodac). the pump takes water from the tank containing the biological filter (l. calcareum ital. j. food sci., vol. 30, 2018 606 algae) and pumps it into the uv sterilisation unit and then on, through the bubbler to reach the tank. it then crosses to the chamber fitted with the wool/perlon and the carbon filters, through the serpentine cooling unit (in which ozonation also takes place) and back to the tank containing the biological filter. the water is then taken back up by the pump and recirculated. the water temperature is controlled by a thermostat (18°c). the chemical, physical and microbiological parameters of water (temperature, salinity, dissolved oxygen, vibrios detection) and clams vitality were checked daily to control proper functioning of aquarium. bivalves samples were collected every 12h for 3 days. samples were immediately processed using the methods reported below. figure 2. depuration system:(a) pump;b) uv sterilisation unit;c) tank;d) bubbler;e) wool/perlon filter;f) carbon filter;g) serpentine cooling unit;h) biological filter;i) ozonation air pump. 2.4. enumeration of vibrio parahaemolyticus and vibrio vulnificus for quantification of vibrio parahaemolyticus, a mpn modified method was used. clam meat was added to alkaline peptone water (apw). serial ten-fold dilutions were prepared in apw, incubated at 37°c for 16-18h and subsequently plated on thiosulphate citrate bile salt sucrose agar (tcbs) and incubated at 37°c for 18-24h. biochemical confirmation was performed with individual and miniaturised tests (api 20e: 4154106 for v.parahaemolyticus, 5346007 for v. vulnificus; api 20ne: 7077444 for v. parahaemolyticus, 7432055 for v.vulnificus). for the quantification of vibrio vulnificus, analyses were conducted as described above, using the selective medium m-cpc (incubation at 40°c for 18-24h). results are expressed in mpn/100g (most probable number). 3. results and discussion 3.1. clams mortality during trials mortality ranged from zero to five specimens during all trials. it should be stressed that, in this study, depuration experiments were carried out in a closed system, with a periodic control of water parameters. in addition, clams were placed in depuration systems at a ital. j. food sci., vol. 30, 2018 607 lower density than those commonly contained in bins of depuration centers. these excellent conditions could influence positively clams mortality rates. 3.2. depuration system parameters chemical and physical parameters of water showed minimal variations (lower than 5%). no pathogen was detected in water samples. 3.3. contamination experiment vibrio parahaemolyticus accumulation values were in one lower order of magnitude than water concentrations. in fact, specimens of venus gallina showed vibrios median values of 430mpn/100g, 9200 mpn/100g, 1100mpn/100g and15000mpn/100g respectively, at water levels of 2,3*103/ml; 3,75 *104/ml; 9,37 *104/ml; and 1,12 *106/ml. considering experimental results, microbial loads changed markedly among different replicates in each trial (table 2). table 2. vibrio parahaemolyticus values after 72h contamination at controlled conditions: temperature 22°c and salinity 35%. test vp1 test vp2 test vp3 test vp4 mpn/100 g mpn/100 g mpn/100 g mpn/100 g min 92 6.800 740 1.500 max 930 17.000 2.200 360.000 average value 435 10.200 1.237 51.680 median value 430 9.200 1.100 15.000 vibrio vulnificus accumulation values did not show a clear pattern regarding water concentrations (table 3). this data was particularly evidenced in test vv4, where high contamination levels in water corresponded to a very low vibriovulnificus loads in clams. moreover, tests comparison between two vibrios species showed that shellfish accumulated fewer vibrio vulnificus than vibrio parahaemolyticus. table 3. vibrio vulnificus values after 72h contamination at controlled conditions: temperature 22°c and salinity 35%. test vv1 test vv2 test vv4 mpn/100 g mpn/100 g mpn/100 g min 36 74 74 max 230 15000 430 average value 125 4110 222 median value 92 1215 230 besides, bivalve’s interand intra-specie variability is well known in studies on microbiological contamination. in fact, the amount of water filtered is between twenty and one hundred liters per day, independent of the environmental conditions (richards, ital. j. food sci., vol. 30, 2018 608 1988; robertson, 2007). bivalve molluscs feeding physiology determine the accumulation of pathogenic microorganisms filtered from the overlaying water (burkhardt and calci, 2000; ho and tam, 2000). these phenomena may also partially explain seasonal and geographical differences in microbial content of bivalves (hernroth et al., 2002). in contamination experiments with vibrio vulnificus conducted under different environmental conditions (test vv2 and test vv3), values were not comparable (table 4). in particular, at salinity of 30‰ and temperature of 25°c, most of the recorded values ranged from<30 to 350mpn/100g: it may indicate a lower accumulation capacity of clams oralower capacity of pathogen to survive and multiply under these conditions. these facts supported reports from other studies. kaspar and tamplin (1993) described that the greatest accumulation of microorganisms in hard-shelled clams occurred during certain periods in the spring, at temperatures ranging from 11.5 to 21.5°c. burkhardt et al. (1992) showed that temperatures outside the range of 13-22°c and salinities greater than 25 ppt reduced the survival of v. vulnificus in seawater. moreover, oliveira et al. (2011) reported that annual variation in water temperature and salinity influence shellfish’s physiological state and therefore, affects the capacity of siphoning and accumulate microbial species. table 4. vibrio vulnificus values after 72h contamination levels under different conditions: salinity of 35‰ and temperatures of 22°c (test vv2), salinity of 30% and temperatures of 25°c (test vv3). test vv2 test vv3 mpn/100 g mpn/100 g min 74 36 max 15000 9200 average value 4110 1437 median value 1215 92 at the end, contamination experiment conducted in this study evidenced a variability infiltration capacity; the presence of such inhomogeneous results provide useful suggestions for planning future tests in terms of the number of organisms to be tested and number of organisms and replicas to be examined at different times. the remark of this variability, although, it led to greater difficulty in data processing, was a crucial and essential factor in the discussion of depuration tests. 3.4. depuration experiment in depuration trials, vp1-vp3 (fig. 3), bacterial load showed a decrease after 48 hours with values four times lower than the initial contamination levels. instead, in tests vp2 and vp4, after 36 hours of depuration, recorded values were four times lower than those at the start. observed decreases remained fairly constant in the subsequent hours of depuration during all tests. this finding suggests that the initial phase of pathogen’s elimination was followed by a "plateau" phase. concerning v.vulnificus levels during the first depuration trial (vv1), a not clear trend was found over time (fig. 4). after 48 hours of depuration, lower values were recorded with respect to the initial load, and at the end of experiment (after 72 hours of depuration), values were in one lower order of magnitude with respect to the initial load. ital. j. food sci., vol. 30, 2018 609 figure 3. temporal changes in vibrio parahaemolyticus levels during depuration tests. in tests vv2 and vv3 (fig. 4), with low initial values (92 and 230mpn/100g), depuration levels lower than 30mpn/100g occurred after 36 hours. in test 2, this trend has remained constant, while in test 3, higher values (92mpn/100g) were detected after 48 hours. in all depuration treatments with v. vulnificus, recorded values were within the range defined by the initial contamination levels, then the apparent decrease may be associated with the variability in contaminated organisms and not to an effective depuration treatment. figure 4. temporal changes in vibrio vulnificus levels during depuration tests. in this study, although, it is known that the initial contamination levels affect the depuration efficiency and higher contaminated shellfish require longer purification periods, comparing trials with medium-high initial loads (103 to 105mpn/100g) showed similar trends. this evidence could be explained by a different response to purification processes by vibrios species. on the other hand, the presence of different pathogens could ital. j. food sci., vol. 30, 2018 610 affect clams filtration capacity differently. such observations are in fact recorded for other bivalves: crassostrea virginica oysters showed longer depuration times for vibrio spp. than e. coli and salmonella tallahassee (murphree and tamplin, 1991). results of this research are in agreement with a previous study on depuration of venus gallina by e. coli, salmonella tiphymuriumand vibrio parahaemolyticus (barile et al., 2009). in addition, in regard to purification methods, previous studies reported that v. parahaemolyticus is sensitive to ultraviolet irradiation and chlorine dioxide (hamamoto et al., 2007, wang et al., 2010). ren and su (2006) examined the effects of electrolyzed oxidizing (eo) water depuration in reducing v. parahaemolyticus and v. vulnificus in laboratory-contaminated oysters and found that both species could only be reduced by approximately 1.0-log unit after 8h at room temperature. considering that adopted purification system was fitted with a uv sterilisation and ozonation plant, the data obtained confirmed those reported in literature. concerning depuration experiments involving v. vulnificus, clams showed a scarce depuration capacity, although, further studies would be needed to give more strength to this hypothesis. clams purification capacity may have been influencedby temperature and salinityconditions of depuration processes (18°cand 35‰).some studies indicated the effects of water temperature on depuration of vibrio spp. in other molluscan species, especially oysters. limited reductions in v. parahaemolyticus (1.2 log mpn/g) and v. vulnificus (2.0 log mpn/g) were observed in oysters after depuration with a uv sterilizer at 22°c for 48 h (chae et al., 2009). on the contrary, tamplin and capers (1992) reported that levels of v. vulnificus accumulated naturally in gulf oysters increased by 5 log mpn/g after depuration in uv-sterilized water at 23°c for 48 h. in this study, at temperatures of 18°c, reductions in one order of magnitude were recorded in only two tests with v. parahaemolyticus. specific studies are required to determine the optimal conditions for shellfish microbial depuration. 4. conclusions shellfish production is done globally and their nutritional and economic value is wellknown. however, filter-feeding bivalves is an efficient transmitter of seafood-born disease. in fact, the emergence of vibrio spp as human pathogen is of particular concern to shellfish producers. very few data are available on the number of pathogenic vibrios and more information is needed to improve the quantitative risk assessment concerning the presence of vibrios in shellfish (cantet et al., 2013; who, 2011). bacterial indicators used for shellfish health evaluation were reported as inadequate predictors of the presence of autochthonous bacterial human enteric viruses. over a long period of time, the high-risk nature of this product and underestimation factors have been well documented in many investigative reports and international agencies. this study emphasizes a limited capacity of venus gallina to release vibrios, in fact, clams with v. parahaemolyticus showed a depuration capacity only in initial phases, while clams with v. vulnificus showed a scarce depuration capacity. bivalves quality is reduced with time due to lack of feed in the depuration process. stakeholders experience significant economic losses when depuration periods above 48h are implemented. more sensitive and reliable depuration procedures must be developed and a better knowledge of the parameters affecting the kinetics of the processes of depuration is still needed. ital. j. food sci., vol. 30, 2018 611 references anonymous. 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(eds). shellfish safety and quality. woodhead pub lmt, cambridge, pp. 509-541. tamplin m.l. and capers g.m. 1992. persistence of vibrio vulnificus in tissues of gulf coast oysters, crassostrea virginica, exposed to seawater disinfected with uv light. 1992. appl environ microbiol. 58(5):1506 -1510. wang d., zhang d., chen w., yu s. and shi x. 2010. retention of vibrio parahaemolyticus in oyster tissues after chlorine dioxide treatment. int j food microbiol 137:76-80. wittman r.j. and flick g.j. 1995. microbial contamination of shellfish: prevalence, risk to human health, and control strategies. ann rev pub health 16:123-40. who world health organization, 2011. risk assessment of vibrio parahaemolyticus in seafood: interpretative summary and technical report, p. 200. http://www.fao.org/docrep/014/i2225e/i2225e00.pdf paper received october 17, 2017 accepted may 5, 2018 ijfs#1599_bozza ital. j. food sci., vol. 32, 2020 645 paper fatty acid profile and sensory properties of roe deer meat after modified atmosphere storage t. daszkiewicz* and j. kondratowicz department of commodity science and animal raw material processing, university of warmia and mazury in olsztyn, oczapowskiego 5, 10-957 olsztyn, poland *corresponding author: tomasz.daszkiewicz@uwm.edu.pl abstract the aim of this study was to determine the effect of cold storage (2°c, 7 and 21 days) under vacuum and modified atmosphere (ma) conditions on the fatty acid (fa) profile and sensory properties of the longissimus thoracis et lumborum from male roe deer. the total content of polyunsaturated fa tended to decrease during storage. storage conditions had a limited (p>0.05) influence on the fa composition. vacuumand ma-packaged meat was characterized by high sensory quality during storage. however, samples packaged in ma composed of 40% co2+60% n2 had a tendency to higher average scores for taste desirability after the third week of storage. keywords: game, cold storage, meat quality ital. j. food sci., vol. 32, 2020 646 1. introduction the consumption of game meat is relatively low despite its high nutritional and culinary value (schulp et al., 2014). there are various reasons for the above, and one of them is a low variety of venison preservation techniques on the consumer market. in retail, game meat is available mostly in the form of vacuum-packaged and deep frozen products. vacuum packaging inhibits the growth of aerobic microorganisms, limits lipid oxidation and prolongs the product's shelf life (stella et al., 2018), but it also has several drawbacks, such as the dark color of meat and considerable drip loss, which compromises consumer acceptance (sakowska et al., 2016). the meat of wild animals is vacuum packaged on account of its unique quality attributes. game meat is characterized by a high content of unsaturated phospholipids (valencak et al., 2015) and heme iron in myoglobin (wiklund et al., 2006), and it is susceptible to auto-oxidation, which compromises its quality. to preserve the freshness of venison, oxygen is removed from the packaging during the vacuum packaging process. freezing decreases water activity, slows down chemical and biochemical processes, inhibits microbial growth and extends the product's shelf life (zhou et al., 2010). however, freezing, frozen storage and thawing lead to adverse changes in meat quality. modified atmosphere (ma) packaging preserves the attributes of fresh meat (djenane and roncalés, 2018). the gas composition of ma is selected for a given type of meat to prevent undesirable changes in quality caused by microbial growth and oxidation or to preserve its attractive color (beef) (zhang et al., 2015). modified atmosphere packaging eliminates the drawbacks of vacuum packaging, such as considerable drip loss as well as packaging and product deformation, through the application of reduced pressure that increases consumer acceptance (ščetar et al., 2010). the effects of storage conditions (ma composition) and cold storage time on the quality of meat from farm animals, including poultry, and fish have been extensively researched (zhang et al., 2015). however, their influence on the quality of venison, in particular meat from wild-living animals, remains insufficiently investigated. therefore, the objective of this study was to determine the effect of vacuum packaging and ma packaging on the fatty acid profile and sensory properties of meat from male roe deer (capreolus capreolus l.). 2. materials and methods 2.1. sampling, packaging and storage the experimental materials comprised samples of the longissimus thoracis et lumborum (ltl) muscle collected from the carcasses of 16 male roe deer aged 3 to 5 years, hunterharvested in the forests of north-eastern poland (sępopol plain, region of warmia and mazury) in june and july during one hunting season. during carcass dressing in a meat processing plant (within 48-54 h of harvest), right and left ltl muscles were cut out, placed in polyethylene bags on ice, and transported to the laboratory. each muscle was divided into samples of similar weight, which were allocated to groups a, b, c and d. samples a were immediately subjected to laboratory analyses, samples b were vacuumpackaged, samples c and d were packaged in ma containing 40% co2+60% n2 and 60% co2+40% n2, respectively. the samples were packaged in barrier bags made of ethylenevinyl alcohol (evoh) copolymer with the following gas permeability: o2=1 ml/ m2/24 h/bar/23°c, n2<0.1 ml/m2/24 h/bar/23°c, co2=1.6 ml/m2/24 h/bar/23°c, h2o=3 ital. j. food sci., vol. 32, 2020 647 g/m2/24 h/23ºc using the pp-5mg (015) vacuum packaging machine (tepro s.a., koszalin, poland). the samples were stored in a cooling chamber without forced air-flow at a temperature of 2ºc for 7 and 21 days. meat quality was determined based on an analysis of the fatty acid profile of intramuscular fat (imf), an evaluation of the sensory properties of meat, and shear force measurements. 2.2. analytical procedures the sensory properties of meat were evaluated after cooking in 0.6% nacl solution at 96ºc (±2°c) until internal temperature reached 80ºc, as described by daszkiewicz et al. (2012). five panelists on a 5-point scale (5 points most desirable, 1 point least desirable) evaluated meat quality. prior to the evaluation, the panelists had been trained in the sensory properties of cooked venison based on cooked beef loin as the reference standard. the panelists assessed encoded samples composed of 1 cm x 1 cm x 1 cm meat cubes, cut from the center of each cooked sample, cooled to room temperature. redistilled water was made available to the panelists for mouth cleansing between samples. the sensory properties (aroma, taste, juiciness, tenderness) of up to 5 meat samples were assessed per session. the maximum shear force required to cut meat samples (5 cylinder-shaped samples, 1.27 cm in diameter, 2 cm in height) across the grain was measured using a warner-bratzler head (500 n, speed 100 mm/min) attached to the instron 5542 universal testing machine (instron, canton, massachusetts, usa). the samples were prepared as described by honikel (1998). intramuscular fat (imf) was extracted by soxhlet extraction (aoac, 1990) with diethyl ether as the solvent in the soxtec™ 2050 auto fat extraction system (foss analytical, hilleroed, denmark). the fatty acid profile of imf was determined by gas chromatography using a unicam pu-4600 gas chromatograph with a flame ionization detector (fid) on a glass capillary column (length: 2.10 m, inner diameter: 4.0 mm); detector temperature 250ºc, injector temperature 225ºc, column temperature 200ºc, carrier gas argon, carrier gas flow rate 50 ml/min. fatty acid methyl esters were prepared by the modified peisker method with chloroform: methanol: sulphuric acid (100:100:1 v/v) (żegarska et al., 1991). 2.3. statistical analysis the results were processed statistically by one-way anova with the use of statistica ver. 13.3 software (tibco software inc.). the statistical significance of differences between mean values in groups was determined using the bonferroni correction at p≤0.05. 3. results and discussion 3.1. sensory properties of meat a sensory evaluation confirmed high quality of meat from male roe deer irrespective of storage conditions (table 1). stored meat samples were characterized by lower aroma intensity than non-stored samples, and the differences were higher (p≤0.05) in mapackaged meat. stored samples received similar scores for aroma intensity, only vacuumpackaged samples stored for 21 days scored higher (p≤0.05) than samples packaged in ma ital. j. food sci., vol. 32, 2020 648 containing 60% co2 and 40% n2. storage time had no influence (p>0.05) on aroma desirability but after 21 days, ma-packaged meat received higher (p≤0.05) scores for this attribute than vacuum-packaged samples. table 1. sensory properties (points) and shear force values (n) of meat from male roe deer stored in a modified atmosphere (arithmetic means±sd). parameter storage (days) vacuum modified atmosphere 40% co2+60% n2 60% co2+40% n2 aroma intensity 0 4.13±0.85 4.13±0.85a 4.13±0.85a 7 3.72±0.68 3.38±0.59b 3.28±0.55b 21 3.75±0.86x 3.34±0.54bxy 3.22±0.45by aroma desirability 0 5.00±0.00 5.00±0.00 5.00±0.00 7 4.88±0.22 4.94±0.17 4.97±0.13 21 4.87±0.22x 5.00±0.00y 5.00±0.00y taste intensity 0 4.09±0.52 4.09±0.52a 4.09±0.52a 7 3.91±0.55 3.78±0.45b 3.75±0.37b 21 4.19±0.31x 4.00±0.00aby 4.06±0.17axy taste desirability 0 5.00±0.00a 5.00±0.00a 5.00±0.00a 7 4.84±0.24a 4.84±0.24ab 4.88±0.22a 21 4.28±0.82bx 4.78±0.36by 4.38±0.62bxy juiciness 0 3.59±0.38 3.59±0.38 3.59±0.38ab 7 3.44±0.54 3.66±0.44 3.69±0.40a 21 3.38±0.43 3.38±0.43 3.38±0.47b tenderness 0 4.44±0.48a 4.44±0.48a 4.44±0.48 7 4.56±0.57a 4.63±0.50ab 4.72±0.41 21 4.97±0.12bx 4.88±0.29bxy 4.75±0.37y shear force 0 21.27±4.42a 21.27±4.42a 21.27±4.42a 7 17.98±3.39b 16.94±2.76b 18.86±4.46ab 21 16.24±2.20b 17.14±2.48b 16.72±2.68b values within a row with different superscript letters (x-y) are significantly different (p≤0.05). values within a column with different superscript letters (a-b) are significantly different (p≤0.05). during storage, taste intensity decreased in samples stored for 7 days compared with nonstored samples and samples stored for 21 days. this trend was more pronounced (p≤0.05) in ma-packaged meat than in vacuum-packaged meat. an analysis of the effect of storage conditions on taste intensity revealed that after 21 days, vacuum-packaged samples scored higher (p≤0.05) than samples packaged in ma composed of 40% co2 and 60% n2. roe deer meat stored for 21 days was characterized by lower (p≤0.05) taste desirability than non-stored meat and meat stored for 7 days. taste deteriorated at the slowest rate in meat packaged in ma composed of 40% co2 and 60% n2, and at the fastest rate in vacuum-packaged meat and meat packaged in ma containing 60% co2 and 40% n2. similar results were reported by seman et al. (1989) who observed a decrease in the flavor desirability of meat from farm-raised male red deer. in their study, vacuumpackaged samples and samples packaged in ma consisting of 100% co2 were stored for 6, ital. j. food sci., vol. 32, 2020 649 12 and 18 weeks at -1ºc. the undesirable changes in palatability most likely resulted from enzymatic and chemical reactions, and the accumulation of microbial metabolites. when analyzing the effects of chemical processes on the sensory properties of meat, attention should be paid to peroxidation of lipids, in particular polyunsaturated fatty acids (pufas) (papuc et al., 2017). meat juiciness was comparable (p>0.05) in non-stored samples and ma-packaged samples stored for 7 days. the value of this attribute decreased after 21 days of storage. storage conditions had no effect (p>0.05) on meat juiciness. however, ma-packaged samples stored for 7 days received somewhat higher average scores for juiciness than vacuumpackaged samples. no differences (p>0.05) in juiciness were reported by hur et al. (2013) in beef, piaskowska et al. (2016) in fallow deer meat and by seman et al. (1989) in red deer meat packaged in ma consisting of co2 and n2 or 100% co2 vs. vacuum-packaged samples. orkusz (2018) and clausen et al. (2009) observed a decrease in the juiciness of samples (goose meat and beef, respectively) stored in ma containing 50-80% o2. the analyzed meat was characterized by desirable tenderness, which further improved during storage, and differences (p≤0.05) were found for vacuum-packaged samples and samples packaged in ma consisting of 40% co2+60% n2. after 21 days of storage, vacuum-packaged meat had the highest tenderness scores and meat packaged in ma composed of 60% co2+40% n2 had the lowest tenderness scores (p≤0.05). the tenderness of samples packaged in ma consisting of 40% co2 and 60% n2 was at an average level. the results of tenderness evaluation were reflected in shear force values, which were lower (p≤0.05) in stored meat than in non-stored samples. no differences (p>0.05) were found between the average shear force values of meat stored for 7 and 21 days. storage conditions had no effect (p>0.05) on shear force values. the increase in tenderness of roe deer meat and the decrease in shear force values after cold storage, observed in our study, were related to the activity of endogenous and bacterial proteolytic enzymes. according to wiklund et al. (2014), meat from selected cervid species does not require aging due to its high tenderness, which probably would not show a further improvement during the process. paulsen et al. (2005) found no considerable differences in the average values of shear force between samples of roe deer meat stored in vacuum (3.5ºc, 132 h) and samples collected from roe deer carcasses on day 5 post mortem. seman et al. (1989) noted no differences (p>0.05) in the shear force values of meat from male red deer stored in vacuum and ma (100% co2) for 18 weeks. 3.2. fatty acid composition of intramuscular fat storage time and conditions had no effect (p>0.05) on the content of saturated fatty acids (sfas) in imf (table 2). nevertheless, an increase in c 16:0 and c 18:0 content (in particular c 18:0) during storage contributed to an increase (p>0.05) in the total sfa pool. similarly to sfas, storage time and conditions had a limited influence on the content of ufas (table 3). the differences between mean values in groups were small and statistically significant only for margoleic acid (c 17:1) and gadoleic acid (c 20:1). in vacuum-packaged meat, the content of c 20:1 was lower (p≤0.05) in non-stored samples and samples stored for 21 days compared with those stored for 7 days. after 7 days of storage, the content of c 17:1 and c 20:1 was higher (p≤0.05) in vacuum-packaged samples than in samples packaged in ma composed of 60% co2+40% n2 and 40% co2+60% n2, respectively. ital. j. food sci., vol. 32, 2020 650 table 2. percentage of saturated fatty acids in total fatty acids in the intramuscular fat of meat from male roe deer stored under vacuum and modified atmosphere conditions (arithmetic means ± sd). parameter storage (days) vacuum modified atmosphere 40% co2+60% n2 60% co2+40% n2 c 12:0 0 1.06±0.61 1.06±0.61 1.06±0.61 7 1.02±0.49 1.09±0.71 0.92±0.35 21 0.86±0.55 0.98±0.64 0.81±0.49 c 14:0 0 1.96±0.50 1.96±0.50 1.96±0.50 7 1.90±0.27 2.05±0.66 1.81±0.34 21 1.88±0.41 1.97±0.86 1.97±0.45 c 15:0 0 0.57±0.15 0.57±0.15 0.57±0.15 7 0.56±0.18 0.59±0.19 0.53±0.11 21 0.64±0.45 0.59±0.20 0.58±0.14 c 16:0 0 23.72±2.64 23.72±2.64 23.72±2.64 7 23.59±1.47 23.86±1.84 23.58±2.16 21 24.18±1.66 24.39±2.98 24.69±2.84 c 17:0 0 1.37±0.15 1.37±0.15 1.37±0.15 7 1.37±0.15 1.40±0.23 1.35±0.19 21 1.37±0.19 1.39±0.18 1.38±0.19 c 18:0 0 23.91±1.85 23.91±1.85 23.91±1.85 7 24.22±3.14 24.56±2.18 24.03±1.85 21 25.27±2.78 24.63±2.86 25.24±2.32 c 20:0 0 0.44±0.19 0.44±0.19 0.44±0.19 7 0.45±0.17 0.49±0.30 0.39±0.10 21 0.51±0.38 0.41±0.18 0.46±0.26 sfas 0 53.04±4.13 53.04±4.13 53.04±4.13 7 53.11±3.25 54.04±4.07 52.62±3.48 21 54.72±4.09 54.35±3.78 55.13±3.70 sfas saturated fatty acids. storage time had no effect (p>0.05) on total content of ufas, but the total content of pufas was lower in stored samples, particularly (p≤0.05) those vacuum-packaged and packaged in ma containing 40% co2 and 60% n2, than in non-stored samples (table 3). as a result, the pufas/sfas ratio was slightly lower in stored meat (table 4). the total content of monounsaturated fatty acids (mufas) increased (p>0.05) in meat stored for 7 days and decreased (p>0.05) in meat stored for 21 days (table 3). increased pufa content is desirable in view of the nutritional value and health-promoting properties of meat but they can also lead to undesirable changes in the product’s quality during storage. auto-oxidation of pufas contributes to undesirable changes in the aroma and taste of meat as well as to the formation of compounds that decrease the nutritional value of meat, and toxic compounds (papuc et al., 2017). therefore, meat packaging must protect the product against the adverse effects of oxygen. this applies also to the meat of wild animals, which is rich in both pufas and heme iron that catalyzes auto-oxidation. in practice, vacuum and ma packaging are used to prevent ital. j. food sci., vol. 32, 2020 651 oxygen from entering the package oxygen is replaced with appropriately selected gas mixtures. table 3. percentage of unsaturated fatty acids in total fatty acids in the intramuscular fat of meat from male roe deer stored under vacuum and modified atmosphere conditions (arithmetic means ± sd). parameter storage (days) vacuum modified atmosphere 40% co2+60% n2 60% co2+40% n2 c 14:1 0 0.15±0.08 0.15±0.08 0.15±0.08 7 0.17±0.07 1.12±0.06 0.15±0.07 21 0.41±0.72 0.27±0.42 0.13±0.07 c 16:1 0 2.22±0.20 2.22±0.20 2.22±0.20 7 2.38±0.43 2.50±0.45 2.36±0.38 21 2.28±0.66 2.34±0.38 2.43±0.47 c 17:1 0 0.24±0.04 0.24±0.04 0.24±0.04 7 0.29±0.04x 0.26±0.05xy 0.23±0.02y 21 0.29±0.12 0.27±0.05 0.27±0.10 c 18:1 0 26.28±4.46 26.28±4.46 26.28±4.46 7 27.53±4.38 27.12±4.42 26.54±4.75 21 26.79±3.92 26.02±4.89 25.88±2.99 c 18:2 0 12.07±2.41 12.07±2.41 12.07±2.41 7 11.07±1.95 10.78±1.92 11.94±3.60 21 10.75±2.46 11.39±2.81 11.22±3.25 c 18:3 0 2.19±0.47 2.19±0.47 2.19±0.47 7 1.85±0.53 1.93±0.57 1.98±0.62 21 1.80±0.60 1.92±0.71 1.75±0.59 c 20:1 0 0.39±0.13a 0.39±0.13 0.39±0.13 7 0.55±0.09bx 0.44±0.10y 0.48±0.10xy 21 0.40±0.05a 0.41±0.12 0.42±0.15 c 20:4 0 4,80±4.73 4.80±4.73 4.80±4.73 7 3.06±0.65 2.81±0.77 3.69±1.81 21 2.57±1.25 3.03±1.00 2.77±1.07 mufas 0 29.28±4.31 29.28±4.31 29.28±4.31 7 30.92±4.36 30.44±4.23 29.77±4.83 21 30.16±3.46 29.31±4.98 29.13±2.97 pufas 0 19.07±4.89a 19.07±4.89a 19.07±4.89 7 15.97±2.74b 15.53±2.83b 17.61±5.80 21 15.12±3.62b 16.35±4.14ab 15.74±4.66 ufas 0 46.96±4.13 46.96±4.13 46.96±4.13 7 46.89±3.25 45.96±4.07 47.38±3.48 21 45.28±4.09 45.65±3.78 44.87±3.70 mufas monounsaturated fatty acids; pufas polyunsaturated fatty acids; ufas unsaturated fatty acids (mufas + pufas). values within a row with different superscript letters (x-y) are significantly different (p≤0.05). values within a column with different superscript letters (a-b) are significantly different (p≤0.05). ital. j. food sci., vol. 32, 2020 652 table 4. the ratio of unsaturated fatty acids to saturated fatty acids in the intramuscular fat of meat from male roe deer stored under vacuum and modified atmosphere conditions (arithmetic means ± sd). parameter storage (days) vacuum modified atmosphere 40% co2+60% n2 60% co2+40% n2 ufas/sfas 0 0.90±0.14 0.90±0.14 0.90±0.14 7 0.89±0.11 0.86±0.13 0.91±0.12 21 0.84±0.13 0.85±0.13 0.82±0.12 mufas/sfas 0 0.56±0.11 0.56±0.11 0.56±0.11 7 0.59±0.11 0.57±0.11 0.57±0.11 21 0.56±0.09 0.55±0.12 0.53±0.07 pufas/sfas 0 0.36±0.11a 0.36±0.11a 0.36±0.11 7 0.30±0.05ab 0.29±0.06b 0.34±0.12 21 0.28±0.08b 0.30±0.08ab 0.29±0.10 mufas monounsaturated fatty acids; pufas polyunsaturated fatty acids; ufas unsaturated fatty acids (mufas + pufas). values within a column with different superscript letters (a-b) are significantly different (p≤0.05). the beneficial effect of oxygen-free packaging that limits lipid peroxidation in game meat was reported by farouk and freke (2008). they noted a lower content of malondialdehyde (an indicator of auto-oxidative changes) in samples of the semimembranosus muscle of red deer, which were vacuum-packaged and stored for 9 months, in comparison with samples that were placed in bags that permitted free exchange of air between the inside and outside of the packaging. unfortunately, vacuum and ma packaging with oxygen-free gas mixtures cannot completely inhibit undesirable changes in the quality of cold-stored meat products. under industrial conditions, o2 cannot be entirely removed from the vacuum or ma packaging. according to berruga et al. (2005), the presence of “residual oxygen”, less than 2% is sufficient to promote lipid peroxidation. in the present study, this could be the reason for a decrease in pufas content in the imf of roe deer after storage. 4. conclusions due to the absence of significant changes in sensory properties and fatty acid profile between vacuum-packaged and ma-packaged samples of roe deer meat during storage, the suitability of those packaging methods and the composition of gas mixtures should be analyzed in view of the values of other attributes that are important from the consumer’s perspective (color, drip loss, microbial counts). references aoac. 1990. “official methods of analysis” 15th ed. association of official analytical chemists, washington, dc. baryłko-pikielna n., kossakowska t. and baldwin z. 1964. the selection of optimal method to prepare beef and pork for the sensoric evaluation. roczniki instytutu przemysłu mięsnego 1:111-132. ital. j. food sci., vol. 32, 2020 653 berruga m.i., vergara h. and galleo l. 2005. influence of packaging conditions on microbial and lipid oxidation in lamb meat. small ruminant res. 57:257-264. clausen i., jakobsen m., ertbjerg p. and madsen n.t. 2009. modified atmosphere packaging affects lipid oxidation, myofibrillar fragmentation index and eating quality of beef. packag. technol. sci. 22:85-96. daszkiewicz t., kubiak d., winarski r. and koba-kowalczyk m. 2012. the effect of gender on the quality of roe deer (capreolus capreolus l.) meat. small ruminant res. 103:169-175. farouk m.m. and freke c. 2008. packaging and storage effects on the functional properties of frozen venison. j. muscle foods 19:275-287. djenane d., roncalés p. 2018. carbon monoxide in meat and fish packaging: advantages and limits. foods 7, 12. honikel k.o. 1998. reference methods for the assessment of physical characteristics of meat. meat sci. 49:447-457. hur s.j, jin s.k., park j.h., jung s.w. and lyu h.j. 2013. effect of modified atmosphere packaging and vacuum packaging on quality characteristics of low grade beef during cold storage. asian-australas. j. anim. sci. 26:17811789. orkusz a. 2018. effects of packaging conditions on some functional and sensory attributes of goose meat. poultry sci. 97:2988-2993. papuc c., goran g.v., predescu c.n. and nicorescu v. 2017. mechanisms of oxidative processes in meat and toxicity induced by postprandial degradation products. compr. rev. food sci. f. 16:96-123. paulsen p., bajer f., winkelmayer r., smulders f.j.m. and hofbauer p. 2005. zu qualitätsparametern von vakuumverpacktem rehfleisch. gewonnen durch zerlegung von rehen 12 bzw. 24 h nach dem erlegen. fleischwirtschaft 11:14-17. piaskowska n., daszkiewicz t., kubiak d. and zapotoczny p. 2016. quality of meat (longissimus dorsi) from male fallow deer (dama dama l.) packaged and stored under vacuum and modified atmosphere conditions. asian austral. j. anim. 29: 1782-1789. sakowska a., guzek d. and wierzbicka a. 2016. effects of carbon monoxide treatment before vacuum packaging on the physical parameters and consumer evaluations of raw beef. food sci. technol. (campinas) 36:485-492. ščetar m., kurek m. and galič k. 2010. trends in meat and meat products packaging a review. croat. j. food sci. technol. 2:32-48. schulp c.j.e., thuiller w. and verburg p.h. 2014. wild food in europe: a synthesis of knowledge and data of terrestrial wild food as an ecosystem service. ecol. econ.105:292-305. seman d.l., drew k.r. and littlejjohn r.p. 1989. packaging venison for extended chilled storage: comparison of vacuum and modified atmosphere packaging containing 100% carbon dioxide. j. food protec. 52:886-893. stella s., bernardi c., tirloni e. 2018. influence of skin packaging on raw beef quality: a review. j. food qual. volume 2018, article id 7464578. valencak t.g., gamsjäger l., ohrnberger s., culbert n.j. and ruf t. 2015. healthy n‑6/n‑3 fatty acid composition from five european game meat species remains after cooking. bmc res. notes 8:273-278. wiklund e., sampels s., manley t.r., pickova j. and littlejohn r.p. 2006. effects of feeding regimen and chilled storage on water-holding capacity, colour stability, pigment content and oxidation in red deer (cervus elaphus) meat. j. sci. food agric. 86:98-106. wiklund e., farouk m. and finstad g. 2014. venison: meat from red deer (cervus elaphus) and reindeer (rangifer tarandus tarandus). animal front. 4:55-61. żegarska z., jaworski j. and borejszo z. 1991. evaluation of the peisker modified method for extracting methyl esters from fatty acids. acta acad. agricult. tech. olst. technol. aliment. 24:25-33. zhang m., meng x., bhandari b., fang z. and chen h. 2015. recent application of modified atmosphere packaging (map) in fresh and fresh-cut foods. food rev. int. 31:172-193. paper received may 28, 2019 accepted april 21, 2020 ijfs#1532_bozza ital. j. food sci., vol. 31, 2019 565 short communication influence of tomato powder on comminuted meat product quality m. modzelewska-kapituła and a. więk* department of meat technology and chemistry, faculty of food sciences, university of warmia and mazury in olsztyn, plac cieszyński 1, 10-719 olsztyn, poland *corresponding author: tel.: +48 895233207 e-mail address: adam.wiek@uwm.edu.pl abstract this study investigated the influence of tomato powder on the quality attributes (ph, colour, cooking loss, oxidative stability, sensory quality) of comminuted meat products. the addition of 2.0% and 2.5% of tomato powder significantly decreased lightness, increased redness and yellowness and delayed oxidation processes in the products. tomato powder lowered the ph values of batters, but had no impact on ph or cooking losses of the products. products enriched with tomato powder were more acceptable than the control sample. the results indicate the beneficial impact of tomato powder on the quality of comminuted meat products. keywords: functional foods, lipid oxidation, lycopene, meat product, sensory quality, tomato ital. j. food sci., vol. 31, 2019 566 1. introduction tomatoes are valuable components of the human diet due to a high content of carotenoids, vitamins (c, k, e, b1, b2, b5, b6, pp, h) and mineral compounds (k, na, p, mg, ca, fe, cu, zn, mn). among the carotenoids, the most abundant is lycopene (fernandes et al., 2016). although fresh tomatoes are available on the market throughout the year, there are also many different processed tomato products offered to consumers, including ketchup, tomato puree (passata), concentrate, whole and cut canned tomatoes, sun-dried tomatoes and tomato powder. although the processing of fruit and vegetables might reduce the concentration of valuable bioactive compounds, in the case of lycopene, thermal treatment of tomatoes increases lycopene concentration in the products from up to 4.2 mg/100 g in fresh tomatoes to up to 265 mg/100 g in tomato powder (skiepko et al., 2015). tomato powder (tp) is produced from tomatoes subjected to drying and grinding, while lycopene itself might be obtained from waste material, such as tomato peel or seeds, or using chemical and microbial syntheses (hernandez-almanza et al., 2016). a beneficial impact on human health has been attributed to lycopene consumption, due to its potential in alleviating chronic diseases, including cancer and coronary heart disease. lycopene is recognized as a reactive oxygen species (ros) scavenger and may therefore control ros-mediated cell growth (hernandez-almanza et al., 2016; palozza et al., 2011). it has also been demonstrated that it induces cell-to-cell communication and improves the functioning of immune and hormone systems and may prevent osteoporosis (hernandez-almanza et al., 2016; sołtysiak and folwarczna, 2015). in order to induce a beneficial impact of lycopene on the human body, such as combating oxidative stress and adverting chronic diseases, from 5 to 10 mg of lycopene should be consumed daily (rao and shen, 2002). the main sources of lycopene in the diet in poland are fresh and processed tomatoes, tropical fruit and watermelons. the average daily lycopene intake in poland is estimated at 7 to 7.5 mg, which is similar to levels noted for inhabitants of california (6.6 mg) and canada (6.4 mg) (sołtysiak and folwarczna, 2015). although the intake of lycopene is higher than the minimum recommended level, an increase in its consumption (e.g. in meat products containing processed tomatoes) would be beneficial. incorporating lycopene into meat products has gained much interest among food scientists and in the meat industry. numerous papers concerning the effect of lycopene addition on the quality of meat products have been published (calvo et al., 2008; eylier and oztan, 2011; garcia et al., 2009; hayes et al., 2013). however, to the authors’ knowledge there are few products with tomato powder available on the market. although the influence of lycopene on meat product colour is well-documented, the sensory quality of the products depends strongly on the amount and the source of lycopene, the nature of the food matrix and its composition. the influence of lycopene on lipid oxidation in meat products during storage is not fully recognized due to various factors affecting the process, related to raw materials (composition, physical state, comminution degree), methods used in meat processing, storage conditions, the length of storage, etc. in a previous study by the authors, the effect of adding from 0.2 to 1.0% tomato powder to comminuted meat products stored under vacuum up to 14 days was investigated (modzelewska-kapituła, 2012). all products showed good sensory quality, but the addition of tomato powder did not retard lipid oxidation in meatloaves stored in vacuum packages. since higher amounts of tomato powder were not used, the present study was undertaken to investigate the effect of adding 2.0% and 2.5% of tomato powder on the quality of comminuted meat products. moreover, in contrast to the ital. j. food sci., vol. 31, 2019 567 previous study, the products were stored under aerobic conditions in a refrigerator for up to seven days, which resembles the conditions under which the products might be stored in households after opening a vacuum package in which products might be distributed and the influence of tomato powder on fat oxidation was studied. therefore, the aim of the present study was to investigate the effect of an increased amount of tomato powder (up to 2.5%) on comminuted meat product quality, including ph values, thiobarbituric acid reactive substances (tbars), sensory quality and colour. 2. material and methods 2.1. materials and production the meatloaves were produced from pork neck, which was ground twice through a size 5 mesh and mixed manually in a bowl with ice water (10%), bread crumbs (4%), fried onion (3%), an egg, salt (1.5%), pepper (0.1%), nutmeg (0.1%) and fresh garlic (0.07%). the amount of additives was calculated in respect to the mass of comminuted meat. three treatments were produced: control – with no addition of tomato powder (tp) and with 2.0% and 2.5% of tp (altoma 9010, diana naturals, antrain, france, kindly provided by kaczmarek-komponenty, mrowino, poland). tp was added to a batter in the required amount and mixed manually to obtain an even distribution within the product. the batters (ca. 300 g) were placed in the individual aluminium forms and heated at 180°c in dry air to 72°c in a geometric centre. after thermal processing, products were cooled to 4°c±1°c and stored in darkness under aerobic conditions at 4°c±1°c until the 7th day after production. three independent batches of meatloaves were produced on three different occasions. according to the technical data sheet provided by the producer, the tp contained dehydrated tomato (lycopersicum esculentum l., min. 99% dry matter) and anti-caking agent: silicon dioxide (e551). it was a powder (100% < 1 mm) showing a medium solubility in water and red colour. total carotenoid content, expressed as lycopene equivalent (ec dir 95/45: od 472 nm e 1%, 1 cm = 3,450, hexane) > 850 mg/kg, fresh tomato ratio: 17:1 (w/w). indicative nutritional data for 100 g of tp were as follows: carbohydrates from 45.0 g to 75.0 g, fat up to 1.0 g, proteins (n × 6.25) from 5 g to 15 g, minerals from 2.0 g to 4.0 g, energy from 200 kcal to 370 kcal. 2.2. methods the following analyses were conducted: sensory evaluation (at day 1, the next day after production), ph measurements (day 1, 3, 7), colour evaluation (day 1) and tbars (day 1, 3, 7). cooking loss was calculated based on the differences in the mass of the products before and after thermal treatment. acidity (ph) values were determined in batters and products (day 1, 3, 7) in homogenates prepared with 10 g of a batter or a product and 10 g of distilled water (ph-meter hi 8314c, hanna instruments polska, olsztyn, poland). three homogenates were prepared for each sample. the 2-thiobarbituric acid reactive substances (tbars), as an indicator of fat oxidation in products, were determined according to the modified salih method (pikul et al., 1993; ital. j. food sci., vol. 31, 2019 568 described in detail in modzelewska-kapituła, 2012) at day 1, 3 and 7. two replicates from each sample were prepared. the colour (cie lab) of the meatloaves was determined at day 1, using miniscan xe plus (hunterlab, reston, usa) in three different positions on the surface and the cross-section of the products. sensory evaluation was conducted by six panellists trained and experienced in sensory evaluation, using difference and preference tests (scoring and ranking methods, respectively). the following attributes of the products were scored on a 6-point scale: overall appearance (1 – irregular shape, surface extremely dry or wet; 6 – regular shape, surface dry and clean), colour on the cross-section (1 – extremely pale, atypical, uneven; 6 – uniform, desirable), consistency (1 – extremely greasy, crumbling; 6 – compact, elastic), taste (1 – bland or extremely intensive, atypical; 6 – perceptible, typical), aroma (1 – bland or extremely intensive, atypical; 6 – perceptible, typical). the panellists were then asked to rank the products according to their preferences from the most desirable (value of 1) to the least desirable (value of 3). the samples for evaluation were served sliced (5 mm thick) at a temperature of approx. 10°c, randomly presented on white plates, coded with two-digit, random numbers. water at room temperature and bread were provided for cleansing the palate between samples. the evaluation was carried out at room temperature (approx. 20°c) under fluorescent lighting. in total, three sensory analysis sessions were performed, during which three meat product samples were assessed per session. 2.3. statistical analysis the results were analysed using statistica 12 (statsoft. inc., tulsa, usa) at a significance level p < 0.05. the influence of tomato powder addition on colour, cooking loss and batter ph were evaluated using one-way variance analysis, as well as the influence of storage time on ph and tbars. the results of sensory evaluations were analysed using a nonparametric kruskal-wallis test, whereas the results of a preference test were analysed using χ2 pearson’s test. 3. results and discussion the influence of tp addition on ph and cooking loss of products is presented in table 1. the addition of tp significantly lowered the ph values of the batters, which was caused by acidic ph of tomatoes. tp had no impact on the ph of final products or cooking losses. the ph values of products with and without tp addition did not change during 7day storage in a refrigerator (p˂0.05). reduction in ph values as a result of tomato products, such as dried tomato peel, sundried tomatoes and tomato paste, when added to meat products was reported also by other authors (condogan, 2002; garcía et al., 2009; østerlie and lerfall, 2005). reduced ph values of meat batters might increase cooking losses due to reduced water holding capacity of meat proteins (huff-lonergan and lonergan, 2005). in the present study, the decrease in ph values was apparently too low to exert an adverse effect on cooking loss. ital. j. food sci., vol. 31, 2019 569 table 1. the influence of tomato powder (tp) on ph and cooking loss of comminuted meat products. attribute product 0% tp 2.0% tp 2.5% tp ph: batter 6.1±0.1 a 5.9±0.1 b 5.7±0.1 b ph: product day 1 6.8±0.2 aa 6.6±0.2 aa 6.5±0.2 aa ph: product day 3 6.8±0.2 aa 6.6±0.2 aa 6.5±0.2 aa ph: product day 7 6.8±0.1 aa 6.6±0.1 aa 6.5±0.1 aa cooking loss (%) 8.0±0.1 a 8.5±0.1 a 8.9±0.1 a a,b means in rows with different letters differ significantly at p < 0.05; a means in columns with the same letter do not differ significantly at p < 0.05. the colour of the surface and the cross-section of the products containing tp and those without the ingredient differed significantly and, moreover, the amount of tp affected the intensity of the cross-section colour (table 2). the surface of the control samples (0% tp) had higher l* and lower a* and b* values than tp-containing products (2.0% tp and 2.5% tp). a cross-section of the control sample also had higher l* values than both tpcontaining samples. the values of a* and b* increased with the increase in tp addition, which indicates that when more tp was added to the batter, the cross-section colour of the products became more intense and the proportion of red and yellow hues was higher and, thus, the colour turned toward orange. these results resemble those presented by garcía et al. (2009), eyiler and oztan (2011), hayes et al. (2013). table 2. the influence of tomato powder (tp) on the colour and sensory attributes of comminuted meat products. attribute product 0% tp 2.0% tp 2.5% tp colour: surface l* 43.5±5.3 a 23.6±1.9 b 35.2±4.5 b a* 7.4±1.5 b 15.8±0.8 a 15.7±1.5 a b* 19.6±3.4 b 23.4±1.7 a 22.2±2.5 a colour: cross-section l* 59.6±5.4 a 53.5±3.8 b 53.5±2.8 b a* 5.4±0.8 c 13.8±2.3 b 16.7±1.3 a b* 17.8±0.7 c 23.6±1.7 b 26.7±0.7 a sensory quality overall appearance 3.9±1.1 a 4.4±1.1 a 4.4±0.9 a colour of the cross-section 4.0±1.2 a 4.3±1.0 a 3.8±1.0 a consistency 4.6±0.9 a 4.5±1.1 a 4.3±1.0 a taste 4.4±1.1 a 4.8±1.2 a 4.7±1.2 a aroma 4.5±1.2 a 4.3±0.9 a 4.2±1.0 a a,b,c means in rows with different letters differ significantly at p < 0.05. ital. j. food sci., vol. 31, 2019 570 sensory analysis comprised the evaluation of particular attributes of products (table 2) and the indication of the most preferred product. all products were scored between 3.8 and 4.8, which indicated their good sensorial quality and there were no significant differences between the control and tp containing products. these results are in line with those of garcía et al. (2009), who used dry tomato peel (dtp) in hamburgers and noted no significant differences between the control sample and those produced with a 3% addition of dtp in odour and texture. calvo et al. (2008) also reported no differences in a hedonic test of dry fermented sausages produced with dry tomato peel up to 1.2% (w/w). panellists more often chose products with tp (2.0% and 2.5%) as more preferred than the control (0% tp) (p<0.05). the results indicate that a tp addition in the range from 2.0% to 2.5% positively affected the sensory quality of comminuted meat products. calvo et al. (2008) found that the colour of meat products (sausages) influenced the preferences of consumers, although in their study the addition of dry tomato peel (0.9 and 1.2%) to sausages lowered their acceptability. thus, it might also be concluded that the acceptability of meat products with tomato powder depends on the product type and its characteristics. tomato powder affected tbars in the products during 7-day cold storage in aerobic conditions (fig. 1). in the control sample, which did not contain tp, tbars increased in storage time and significant differences between day 1, day 3 and day 7 were noted (p<0.05). in the product, which contained 2.0%, tbars increased between day 1 and 3 and remained unchanged between day 3 and 7, whereas in the product (which contained 2.5% tp) tbars did not change during the storage period. figure 1. the influence of storage time on tbars in meatloaves produced with the addition of tomato powder (2.0% tp, 2.5% tp) and without the tomato powder (0% tp). a,b,c means within each treatment with different letters differ significantly at p < 0.05. ital. j. food sci., vol. 31, 2019 571 these findings suggest that tomato powder has the potential to inhibit oxidation in meat products stored under aerobic conditions and its effectiveness depends on the amount of tomato powder. the anti-oxidative effect of lycopene added to paprika salami was also reported by rohlík et al. (2013), which supports the results of the present study. on the other hand, it was also noted that tbars values at day 1 were the highest for 2.5% tp, whereas they were the lowest for 0% tp products. the results indicate that the method used for tbars determination, which is suitable for evaluation of the oxidation process in meat and meat products, might give misleading results, probably due to the elution of carotenoids from tp-containing samples, which contributes to higher absorbance values. that is why the results of tbars were not compared between samples containing different amounts of tp, but analysed taking into consideration only the storage time. on the other hand, the increase in tbars along with the increased amount of tomato products (powder, paste) in meat products was also noted by eyiler and oztan (2011), deda et al. (2007) and hayes et al. (2013), who attributed it to the pro-oxidative effect of lycopene used in higher concentrations. tbars is used as a lipid oxidation indictor in meat and processed meat products. a threshold of 2 mg malondialdehyde/kg sample is regarded as the minimum tbars value that causes the off-flavour in meat and meat products (eyiler and oztan, 2011). no samples investigated in this study exceeded 2 mg ma/kg during seven days of storage under aerobic conditions, which indicated that their quality was not reduced by excessive lipid oxidation. similar values were noted in the previous studies, in which meat products with lycopene addition were stored vacuum-packed (modzelewska-kapituła, 2012; eyiler and oztan, 2011). however, in the previous studies, the antioxidant properties of lycopene in cooked meat products, stored in vacuum-packages were not proven, probably due to evacuation of the oxygen from vacuum packages. 4. conclusions in conclusion, tomato powder added in amounts of 2.0% and 2.5% to a comminuted meat product increased its sensory acceptability and oxidative stability, changed the colour towards more orange and decreased ph, although it had no influence on cooking losses. thus, the meat products obtained using 2.5% of tomato powder might be a healthier alternative to the ready-to-eat meat products currently available on the market. however, before launching them on the market, the results of the present study should be complemented with an evaluation of consumer acceptability of the products and determination of microbial quality, due to the lack of preservatives such as sodium nitrite. lycopene content in the products should also be examined, since the storage of tomato products (e.g. tomato puree) might reduce lycopene content (marković et al., 2007). the application of tomato powder as a source of lycopene and the lack of food additives provides the opportunity to introduce “clean label” products with nutritional benefits for the consumer. acknowledgements the authors would like to thank the technical support of natalia wdowiak. the study was financed from the statutory funds of the faculty of food sciences, university of warmia and mazury in olsztyn. project financially supported by minister of science and higher education in the range of the program entitled "regional initiative of excellence" for the years 2019-2022, project no. 010/rid/2018/19, amount of funding 12.000.000 pln. ital. j. food sci., vol. 31, 2019 572 references calvo m.m., garcía m.l. and selgas m.d. 2008. dry fermented sausages enriched with lycopene from tomato peel. meat sci. 80:167-172. condogan k. 2002. the effect of tomato paste on some quality characteristics of beef patties during refrigerated storage. eur. food res. technol. 17:125-133. deda m.s., bloukas j.g. and fista g.a. 2007. effect of tomato paste and nitrite level on processing and quality characteristics of frankfurters. meat sci. 76:501-508. eyiler e. and oztan a. 2011. production of frankfurters with tomato powder as a natural additive. lwt-food sci. technol. 44:307-311. fernandes f.a.n., rodrigues s., garcía-pérez j.v. and cárcel j.a. 2016. effects of ultrasound-assisted air-drying on vitamins and carotenoids of cherry tomatoes. drying technol. 34:986-996. garcía m.l., calvo m.m. and selgas m.d. 2009. beef hamburgers enriched in lycopene using dry tomato peel as an ingredient. meat sci. 83:45-49. hayes j.e., canonico i. and allen p. 2013. effects of organic tomato pulp powder and nitrite level on the physicochemical, textural and sensory properties of pork luncheon roll. meat sci. 95:755-762. hernández-almanza a., montañez j., martínez g., aguilar-jiménez a., contreras-esquivel j.c. and aguilar c.n. 2016. lycopene: progress in microbial production. trends food sci. technol. 56:142-148. marković k., hruškar m. and vahčić n. 2007. stability of lycopene in tomato purée during storage. acta aliment. 36:8998. modzelewska-kapituła m. 2012. effects of tomato powder on color, lipid 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properties of lycopene and utilizing it to produce functional foods. żywność. nauka. technologia. jakość. 103: 20-32. sołtysiak p. and folwarczna j. 2015. effects of lycopene on the skeletal system. post. hig. med. dośw. 69:243-251. paper received february 19, 2019 accepted may 20, 2019 ijfs#1663_bozza ital. j. food sci., vol. 32, 2020 420 paper effects of pectin-based edible coatings containing a bacteriocin of bacillus methylotrophicus bm47 on the quality and storage life of fresh blackberries y. tumbarski*1, n. petkova2, m. todorova2, i. ivanov2, i. deseva3, d. mihaylova4 and s.a. ibrahim5 1department of microbiology, university of food technologies, 26, maritsa blvd., 4002 plovdiv, bulgaria 2department of organic chemistry and inorganic chemistry, university of food technologies, 26 maritsa blvd., 4002 plovdiv, bulgaria 3department of analytical chemistry and physicochemistry, university of food technologies, 26 maritsa blvd., 4002 plovdiv, bulgaria 4department of biotechnology, university of food technologies, 26, maritsa blvd., 4002 plovdiv, bulgaria 5food microbiology and biotechnology laboratory, food and nutritional sciences program, north carolina a & t state university, greensboro, nc 27411-1064, usa *corresponding author: tumbarski@abv.bg abstract the aim of the current research is to investigate the effects of edible coatings based on celery pectin singly and in combination with a bacteriocin of bacillus methylotrophicus bm47 on the quality and storage life of fresh blackberries under refrigeration conditions. in this study three experimental groups were prepared: uncoated blackberries as a control, blackberries with 1% pectin coatings and blackberries with 1% pectin coatings+bacteriocin of b. methylotrophicus bm47. during the storage at 4°c and 75% rh for 16 days, the weight loss, decay percentage, total soluble solids (tss), titratable acidity (ta), ph, organic acids, sugars, total phenolic content, total anthocyanins and antioxidant activity were analyzed. ital. j. food sci., vol. 32, 2020 421 the results showed that the application of pectin and pectin+bacteriocin coatings led to a reduction in weight loss with 6.3% and 6.7% compared to the control fruit on the 16-th day of storage. a decrease in decay percentage was also noticed, which was most pronounced in the pectin+bacteriocin coated fruit compared to the pectin coatings and control. the pectin and pectin+bacteriocin coatings reduced tss levels with 0.4% and 0.6%, respectively compared to the control on the 16-th day of the storage, but did not affect ta and ph values. the pectin and pectin+bacteriocin coatings had no effect on decreasing total phenolic and anthocyanin contents or the concentration of sugars (glucose and fructose) in both treatments and the control fruit. the pectin and pectin+bacteriocin edible coatings exhibited a protective effect on the ascorbic acid content, maintaining concentrations of 57.5 mg/100 g of fw and 58.8 mg/100 g of fw (day 16), which were close to the initial values. the pectin and pectin+bacteriocin treatments had also a positive impact on antioxidant activity in the coated blackberries. both edible coatings effectively inhibited its decrease with the prolongation of the storage time and kept antioxidant levels of 231.8 te/100 g of fw and 232.4 te/100 g of fw (day 16) that were close to the initial values. keywords: bacillus methylotrophicus, bacteriocin, biopreservation, blackberry, edible coatings ital. j. food sci., vol. 32, 2020 422 1. introduction blackberry (rubus fruticosus) is a perennial plant in the family rosaceae whose native populations grow primarily in the mediterranean region of europe. the cultivated blackberry is grown all over the world, and in recent years, worldwide production and consumption of blackberries continue to increase (strik et al., 2007). the blackberry fruit is an excellent source of antioxidants, including various phenolic compounds (phenolic acids, tannins, stilbenes, flavonoids and anthocyanins) and ascorbic acid as well as large amounts of other compounds such as vitamins, minerals, fibers and volatiles (de souza et al., 2014). the rich phytochemical composition and nutritional characteristics of blackberry fruit have been shown to exert a positive impact on human health. for example, the high levels of antioxidants in blackberries have been found to exhibit antiinflammatory, anticarcinogenic, antimutagenic and antimicrobial properties (gyawali and ibrahim, 2014). the bioactive components in blackberries also reduce cholesterol levels, cause an analgesic effect and help to strengthen blood vessels (nile and park, 2014). some recent studies have shown that blackberry polyphenols exhibit a neuroprotective effect by delaying degenerative brain processes (tavares et al., 2012). on the other hand, blackberry is a highly perishable fruit with a very short market life. the storage time of blackberries after harvesting is limited due to their high susceptibility to physical injuries, desiccation and fungal spoilage caused by botrytis cinerea, rhizopus sp. and mucor sp. which results in a rapid loss of commercially acceptable appearance. these post-harvest changes can be suppressed by storage at low temperatures or by creating modified atmospheres (lower oxygen and increased carbon dioxide levels) that delay tissue senescence. advanced technologies such as ozone treatment and gamma radiation can also be used to decrease microbial decay and prolong the storage life of blackberries (maftoonazad et al., 2007; oliveira et al., 2013). the conservation, distribution and marketing of blackberries can also be significantly improved through some non-conventional methods for extending the storage time such as the application of thin layers of biopolymers known as edible coatings, which protect the fruit from physical injuries, chemical and microbiological activities (falguera et al., 2011). edible coatings and films act as barriers against moisture loss, oxygen, carbon dioxide and lipid transfer, thus preventing dehydration, desiccation and deterioration of the fruit quality (cui et al., 2016). edible coatings provide additional benefits in perishable fruits by reducing respiration rate, improving textural quality, and helping to retain natural color and volatiles, thereby protecting the fruit’s nutritional value (corbo et al., 2015). the nature of the edible coating material is essential for the coating’s effectiveness. existing data in the literature show that hydrocolloids (polysaccharides and proteins) and lipids are among the most suitable structural matrices for edible coatings production. most edible coatings include cellulose and its derivatives, starch, alginate, chitosan and pectin. numerous studies have described and evaluated the protective effects of cellulose derivatives, starch, alginate and chitosan, but research on the application of pectin-based edible coatings is very limited (mahajan et al., 2018). pectins are biopolymers comprised of (1→4) α-d-galactopyranosyluronic acid units naturally esterified with methanol. based on the content of methyl esters or the degree of esterification (de) that has a decisive effect on solubility and gel formation properties, pectins are divided into two groups – high-methoxylated (de>50%) and lowmethoxylated (de<50%) pectins (corbo et al., 2015). pectins are also polysaccharides that are commonly used as gelling agents in the food industry. as such, pectins are of great ital. j. food sci., vol. 32, 2020 423 interest as edible coating agents in fruit biopreservation due to their unique colloidal characteristics, strong gel formation properties and ability to provide an excellent oxygen barrier and aroma preservation. however, the primary disadvantage of all polysaccharide-based coatings is that they do not ensure a good moisture barrier due to their hydrophilic nature (oms-oliu et al., 2008; valdés et al., 2015). berries contain high levels of sugars, other nutrients and water that make them an excellent substrate for microbial growth. in addition, the low ph of berry fruits is a prerequisite for fungal spoilage, which affects product quality, storage and distribution, resulting in significant economic losses (tournas and katsoudas, 2005). the resistance of some fungi to conventional fungicides and the proven negative effects of chemical treatments on human health have led to many limitations in the use of such chemicals. consequently, research efforts have become more focused on the development of safer preservation methods that employ non-toxic biologically active compounds. thus, the biological control of spoilage by antimicrobial substances such as bacteriocins synthesized by bacillus sp. and lactic acid bacteria (lab) are a promising alternative to the traditional fungicide application. bacteriocins are safe; they do not alter the organoleptic characteristics of food and can be applied directly or indirectly by in situ production. some lab bacteriocins have been tested and have already shown promising potential against microbial spoilage of fruits and fruit products (barbosa et al., 2017). other bacteriocins with strong antifungal activity such as those produced by bacillus methylotrophicus bm47 might also be prospective candidates for solving the problems associated with fungal decay (tumbarski et al., 2018). the current study thus aimed to examine the application of edible coatings based on celery pectin alone and in combination with a bacteriocin synthesized by bacillus methylotrophicus bm47 on the quality and extension of the storage life of fresh blackberry fruit. 2. materials and methods 2.1. materials 2.1.1 fruit fresh blackberries (rubus fruticosus) were purchased from the local fruit market in plovdiv, bulgaria. the fruit was selected based on size, shape, color and absence of physical injuries. the blackberries were placed in brown paper bags and then immediately transferred into a fridge bag (7°c) to the laboratory to conduct the experiments. 2.1.2 pectin low-methoxylated pectin was extracted from celery tubers (apium graveolens var. rapaceum d.c.) purchased from the local fruit market in plovdiv, bulgaria. the tubers were washed with tap water, checked for impurities, and then peeled, coarsely chopped and dried at 40°с. before extraction, the tubers were finely ground in a laboratory homogenizer and pre-washed with 70% ethanol acidified with 2% hydrochloric acid in order to obtain alcohol insoluble solids. pectin was extracted by ultrasound-assisted extraction with 2% aqueous ammonium oxalate using the method of petrova et al. (2017). the degree of esterification (de) and anhydrouronic acid content (auac) were determined by titration ital. j. food sci., vol. 32, 2020 424 method (petrova et al., 2017). the degree of acetylation (da) was measured by the hydroxamic acid reaction method with β-d-glucose pentaacetate as a standard. all chemicals were purchased from sigma-aldrich, merck (st. louis, mo, usa). the molecular mass (mm) was assessed by high-performance size-exclusion chromatography (hpsec) analysis. separation was conducted using an elite lachrom hplc system (vwr™ hitachi, tokyo, japan) coupled with a column shodex oh-pack 806m (8 mm × 300 mm) and a refractive index detector chromaster 5450 with an aqueous 0.1 m sodium nitrate solution at a flow rate of 0.8 ml/min (ognyanov et al., 2018). the obtained celery pectin was characterized as low-esterified pectin with de 46%, da 2%, auac 70% and average mm of 912694 g/mol. 2.1.3 bacteriocin a bacteriocin synthesized by the strain bacillus methylotrophicus bm47 (previously isolated from a natural thermal spring in the haskovo region of bulgaria) was used in the experiment. the bacteriocin, purified by fast protein liquid chromatography (fplc), contained an antimicrobial peptide with mm of 19578 da as characterized in an earlier research (tumbarski et al., 2018). 2.2. methods 2.2.1 experiment design the edible coating solution (1%) was prepared by dissolving 4 g of celery pectin in 400 ml of distilled water at 45°c in a magnetic stirrer ika® rct classic (ika®-werke gmbh & co. kg, staufen, germany) at 800 rpm for 30 min. next, 0.5% glycerol monostearate cutina® gms (henkel, düsseldorf, germany) was added as a plasticizer, and the solution was stirred under identical conditions as outlined above for 15 min. the pectin solution was then divided into two equal parts of 200 ml each. after cooling to room temperature, 100 au/ml (0.15 mg/ml) of the purified bacteriocin of b. methylotrophicus bm47 was added to the second part and stirred without heating for 15 min (tumbarski et al., 2019). the blackberries were disinfected by being dipped in 1% sodium hypochlorite (sigmaaldrich, merck, germany) for 3 min, then washed three times with tap water and dried at 25°c for 2 h in a forced air mlw drying chamber (labexchange, burladingen, germany). after drying, the blackberries were separated into three experimental groups of 30 berries each as follows: uncoated blackberries as a control (group 1), blackberries with pectin coatings (group 2) and blackberries with pectin coatings + bacteriocin (group 3). the blackberries from group 2 and group 3 were immersed in the relevant coating solutions for 2-3 min. the treated fruits were then dried at 25°c for 2 h in a drying chamber with forced air mlw (labexchange), after which all groups were placed in plastic boxes and stored under refrigeration conditions (4°c and 75% rh) for 16 days (tumbarski et al., 2019). 2.2.2 visual observation all groups were examined at the beginning of the experiment (i.e. 0 day) and on the 4-th, 8-th, 12-th and 16-th day of storage. during the storage period, an observation of the morphological changes and fungal growth was made, and samples for analyses were taken. ital. j. food sci., vol. 32, 2020 425 2.2.2.1 weight loss percentage for determination of weight loss (wl) and decay percentage, three separate groups of 10 blackberries each with the same treatments control, pectin and pectin + bacteriocin were prepared and stored under identical conditions. to measure wl, each group was weighed at the beginning of the experiment (i.e. 0 day) and on the 4-th, 8-th, 12-th and 16-th day of storage. wl was defined as the difference between the initial weight of each experimental group and the weight of the same group determined on the relevant monitoring day. the results were calculated as a percentage loss of the initial weight (tumbarski et al., 2019). 2.2.2.2 decay percentage the decay percentage was determined as follows: the number of berries with visible decay or morphological changes was expressed as a percentage of the initial number of all berries in the relevant experimental group (tumbarski et al., 2019). 2.2.3 physico-chemical parameters 2.2.3.1 total soluble solids, titratable acidity and ph the total soluble solids (tss) content was determined by a portable abbe refractrometer (officine galileo, campi bisenzio, italy). the samples were preliminary homogenized with a special device polytron (kinematica ag, luzern, switzerland), a few drops of blackberry juice were put on the prism glass, and the tss value was immediately read and recorded. the titratable acidity (ta) was measured by titration of 2 ml of blackberry juice with 0.1 n naoh (sigma-aldrich, merck) using phenolphthalein (sigma-aldrich, merck) as an indicator until the appearance of a pale pink color persisted for over 1 min. the results were calculated as the mean value of three successive experiments and expressed as the percent of malic acid. the ph values for each experimental group were measured by a ph-meter wtw ph 7110 (wtw, weilheim, germany) at 23°c (tumbarski et al., 2019). 2.2.3.2 total phenolic content the total phenolic content (tpc) was measured using a folin-ciocalteu reagent (sigmaaldrich, merck) by the method of stintzing et al. (2005). to conduct the analysis, 1 ml of folin-ciocalteu reagent was mixed with 0.2 ml blackberry juice and 0.8 ml 7.5% na2co3 (sigma-aldrich, merck). the reaction was performed in darkness at room temperature for 20 min. the absorbance was measured by uv/vis spectrophotometer camspec m107 (spectronic-camspec ltd., leeds, uk) against a blank at λ=765 nm, and the results were expressed as mg equivalent of gallic acid (gae)/100 g of fresh weight (fw) according to a calibration curve (ivanov et al., 2014). 2.2.3.3 total anthocyanins content the total anthocyanins content (tac) was estimated using the ph differential method (lee et al., 2005). the samples of blackberry juice (0.2 ml) were mixed with buffers (sigma-aldrich, merck) at ph 1.0 and ph 4.5 (1.8 ml), and the absorbance was measured by uv/vis spectrophotometer camspec m107 (spectronic-camspec ltd.) against a blank ital. j. food sci., vol. 32, 2020 426 at λ=510 nm and λ=700 nm. the results were expressed as mg cyanidin-3-glycoside equivalents/100 g of fw. 2.2.3.4 antioxidant activity the antioxidant activity was determined by the 2,2-diphenyl-1-picrylhydrazyl (dpph) free radical-scavenging method. to perform this assay, 0.15 ml of blackberry juice was mixed with 2.85 ml of a freshly prepared 0.1 mm methanol solution of dpph (sigmaaldrich, merck). the samples were incubated in darkness for 15 min at 37°c. the reduction of absorbance was measured at λ=517 nm by uv/vis spectrophotometer camspec m107 (spectronic-camspec ltd.) against a blank prepared with methanol (sigma-aldrich, merck), and the percentage of inhibition was calculated (ivanov et al., 2014). 2.2.3.5 high pressure liquid chromatographic (hplc) analysis of organic acids the content of organic acids was determined by an elite lachrom hplc-dad system (vwr™ hitachi) according to ivanov et al. (2017). the separation was conducted on a discovery® hs c18 column (5 µm, 25 cm × 4.6 mm) at 30°с. the isocratic elution was conducted with 25 mm кн2ро4 (ph 2.4) as a mobile phase at a flow rate of 0.5 ml/min. l(+)-ascorbic acid was detected at λ=244 nm and l-malic acid at λ=210 nm. the obtained results were expressed as mg/100 g of fw. 2.2.3.6 hplc analysis of monoand disaccharides chromatographic separation and measurement of sugars were performed on an elite lachrom hplc system coupled with a refractive index detector chromaster 5450 (vwr™ hitachi). the separation was performed on a shodex® sugar sp0810 column (300 mm × 8.0 mm) with pb2+ and a guard column shodex sp-g (5 μm, 6 mm × 50 mm) operating at 85°c mobile phase d.h2o with a flow rate of 1.0 ml/min and injection volume of 20 μl. the results were expressed as g/100 g of fw (petkova et al., 2014). 2.2.4 statistical analysis each analysis was independently replicated three times, the data were presented as mean value, and the standard deviation (±sd) was calculated (tumbarski et al., 2019). 3. results and discussion 3.1. visual observation during the first four days of storage at 4°c and 75% rh, a slight increase in weight loss (wl) in all experimental groups was observed, which was greater in the control blackberries than in the pectin and pectin+bacteriocin coated fruit. during this time no morphological changes in any experimental group were observed (figs. 1 and 2). on the 8th day of the refrigerated storage, the first visible signs of dehydration in the control blackberries appeared, while the coated fruit remained unaffected (fig. 3). the wl of the control blackberries was greater with 1.8% compared to that of the treated fruit. on the 12 ital. j. food sci., vol. 32, 2020 427 th day of the storage period, the first visible signs of fungal growth in the control and pectin-coated fruit appeared, while the bacteriocin-containing coatings prevented effectively the blackberries from decay and no spoilage changes were observed (fig. 4). the wl of the control blackberries was greater with 2.1% (pectin) to 2.4% (pectin+bacteriocin) compared to the coated fruit. at the end of the observation period (day 16), the dehydration in the control group continued to increase, and the difference in the wl between the uncoated blackberries and the coated fruit reached 6.3% (pectin) and 6.7% (pectin+bacteriocin), respectively. the fungal spoilage process was most pronounced in the control and pectin-containing coatings, while the blackberries whose coatings contained a bacteriocin remained unaffected (fig. 5). as seen from the results summarized in table 1, the pectin-based edible coatings singly or in combination with a bacteriocin of b. methylotrophicus bm47 effectively protected the treated fruit from moisture/weight loss and desiccation during the entire storage period, which helped to extend their storage life and to improve their commercial appearance. the protective effect of the bacteriocin of b. methylotrophicus bm47 and the attendant decrease in the decay percentage were related to the bacteriocin’s strong antifungal properties as described in our previous research (tumbarski et al., 2018). a similar tendency for weight loss during storage at low temperature was reported by guerreiro et al. (2015), who used citrus pectin and alginate as edible coatings for red raspberry fruit. in addition, it was demonstrated that pectin active coatings improved the physico-chemical, microbiological and sensory quality of strawberries from 6 (control) to 15 days at 4°c and 90% rh (valdés et al., 2015). figure 1. effects of pectin-based and pectin-based edible coatings with bacteriocin of b. methylotrophicus bm47 on the appearance and morphology of fresh blackberries at the beginning of the experiment (day 0). c control; p pectin (1%); p+b pectin (1%) + bacteriocin. ital. j. food sci., vol. 32, 2020 428 figure 2. effects of pectin-based and pectin-based edible coatings with bacteriocin of b. methylotrophicus bm47 on the appearance and morphology of fresh blackberries at day 4 during refrigerated storage (4°c and 75% rh). c control; p pectin (1%); p+b pectin (1%) + bacteriocin. figure 3. effects of pectin-based and pectin-based edible coatings with bacteriocin of b. methylotrophicus bm47 on the appearance and morphology of fresh blackberries at day 8 during refrigerated storage (4°c and 75% rh). c – control; p pectin (1%); p+b – pectin (1%) + bacteriocin. figure 4. effects of pectin-based and pectin-based edible coatings with bacteriocin of b. methylotrophicus bm47 on the appearance and morphology of fresh blackberries at day 12 during refrigerated storage (4°c and 75% rh). c control; p pectin (1%); p+b pectin (1%) + bacteriocin. ital. j. food sci., vol. 32, 2020 429 figure 5. effects of pectin-based and pectin-based edible coatings with bacteriocin of b. methylotrophicus bm47 on the appearance and morphology of fresh blackberries at day 16 during refrigerated storage (4°c and 75% rh). c control; p pectin (1%); p+b pectin (1%) + bacteriocin. 3.2. changes in total soluble solids, titratable acidity and ph the total soluble solids maintained a constant level of 14.1% in all experimental groups during the first four days of storage. on the 8-th day, a slight increase in tss in the control group and pectin-coated blackberries was detected, while pectin+bacteriocin coatings maintained tss levels equal to the initial (14.1%). the tss levels continued to rise gradually over the storage period due to water migration in the environment and fruit desiccation. by the end of the observation period (days 12 and 16) the treated blackberries had lower tss values (with 0.2% to 0.4% for pectin-coated fruit and 0.4% to 0.6% for pectin+bacteriocin-coated fruit) compared to the uncoated blackberries (table 1). these results demonstrated that pectin-based edible coatings, especially those with the addition of bacteriocin, provided a protective barrier against moisture loss and consequently a reduced attenuation of the fruit quality. a slight increase in ta and a decrease in ph values in all groups were observed, both of which are normally associated with post-harvest changes of fruits. the results presented in table 1 demonstrate that pectin and pectin+bacteriocin coatings did not consistently influence these two parameters, which remained similar to those observed in the uncoated blackberries until the end of the storage period. tosun et al. (2008), who studied the behaviour of ph during blackberry ripening, observed average values of 3.20 in unripe fruits, 2.64 in red fruits and 3.14 in mature fruits. according to antunes et al. (2003), the initial values of ph 3.59 and 3.39 reached 3.94 and 4.09 after 12 days of storage, respectively; there was an increase in ph of 0.40 and 0.70. however, in our case, a decrease in ph values of 0.30 was observed at the end of 16 days. in our study, an increase in titrable acidity was observed during the storage (table 1) from 1.09 to 1.70 %. our valuеs were higher than reported from meneghel et al. (2008), who showed constant values for acidity during 18 days of storage, with average values of 0.86, 0.89 and 0.85 g of citric acid per 100 g for coated blackberry cv. comanche. the similar observation of increase in titrable acidity was reported by oliveira et al. (2012) in blackberry (rubus spp.) conservation with edible coating. ital. j. food sci., vol. 32, 2020 430 table 1. effects of pectin-based and pectin-based edible coatings with bacteriocin of b. methylotrophicus bm47 on the physicochemical characteristics of fresh blackberries during refrigerated storage. day coating parameter wl(%) decay(%) tss(%) ta(%) ph 0 c n.a. n.a. 14.1 1.09±0.06* 3.72±0.03 p n.a. n.a. 14.1 1.08±0.03 3.71±0.06 p+b n.a. n.a. 14.1 1.09±0.07 3.72±0.08 4 c 7.6 0 14.1 1.16±0.02 3.63±0.04 p 6.7 0 14.1 1.19±0.04 3.62±0.02 p+b 6.7 0 14.1 1.20±0.03 3.54±0.06 8 c 12.0 0 14.5 1.30±0.05 3.60±0.04 p 10.2 0 14.3 1.41±0.01 3.56±0.03 p+b 10.2 0 14.1 1.55±0.06 3.52±0.01 12 c 17.7 20.0 14.7 1.57±0.03 3.57±0.02 p 15.6 10.0 14.5 1.61±0.01 3.45±0.04 p+b 15.3 0 14.3 1.67±0.04 3.42±0.05 16 c 23.7 40.0 15.2 1.70±0.06 3.42±0.01 p 17.4 20.0 14.8 1.72±0.02 3.46±0.05 p+b 17.0 0 14.6 1.74±0.01 3.44±0.03 c control; p pectin (1%); b bacteriocin; wl weight loss; tss total soluble solids; ta titratable acidity; n.a. not analyzed; * ±standard deviation (±sd). 3.3. changes in organic acid content the results showed that the content of ascorbic acid in blackberries declined in all treatments during the first 4 days of storage (table 2). this finding was most likely associated with a stress reaction resulting from the effects of low temperatures on fruit metabolism. thereafter, ascorbic acid concentrations gradually increased in all treatments relative to the storage period. the application of pectin-based coatings singly and in combination with bacteriocin of b. methylotrophicus bm47 effectively maintained higher levels of ascorbic acid in the treated fruit compared to the control fruit during the entire monitoring period. by the end of the storage period (16-th day), ascorbic acid levels had reached 56.8 mg/100 g of fw (control), 57.5 mg/100 g of fw (pectin) and 58.8 mg/100 g of fw (pectin+bacteriocin). these levels were close to the value noted at the beginning of the experiment (62.3 mg/100 g of fw). our values for ascorbic acid content in fresh and coated blackberries were higher than reported values for different fresh blackberry cultivars (12.3–16.4 mg/100 g of fw) (deighton et al. 2000). the detected levels of ascorbic acid content were near to cornelaian cherry cultivars (48.4-73.1 mg/100 g of fw) and higher than its level in other berries as raspberry (21.231.1 mg/100 g of fw) and strawberries (46 mg/100 g of fw) (pantelidis et al. 2007). the concentration of malic acid in both the control and coated blackberries progressively increased, with storage time, reaching levels of 56.9 mg/100 g of fw (control), 58.3 mg/100 g of fw (pectin) and 61.3 mg/100 g of fw (pectin+bacteriocin) by the 16-th day of storage as compared to the initial level of 31.3 mg/100 g of fw. our values for malic acid were more than twenty times higher than reported content in raspberry cultivars (zorenc et al. ital. j. food sci., vol. 32, 2020 431 2017) and wild growing blackberries (mikulic-petkovsek et al. 2012). neither type of coating had an inhibitory effect on the increasing concentration of malic acid that is normally associated with post-harvest changes in fruits (table 2). these results correlated with the increasing levels of titratable acidity and decreasing ph values in all experimental groups during the storage period (table 1). this could be explained with by increased metabolism of fruits after harvesting, in which a greater consumption of organic acids is needed as substrates for the respiratory process. as a result of this, there is a greater conversion into simple sugars during maturation (oliveira et al. 2012). table 2. effects of pectin-based and pectin-based edible coatings with bacteriocin of b. methylotrophicus bm47 on organic acids content and sugar content of fresh blackberries during refrigerated storage. day coating organic acid (mg/100 g) sugar (g/100 g) ascorbic malic glucose fructose 0 c 62.3±0.14* 31.3±0.22 3.0±0.21 3.4±0.22 p 62.2±0.15 31.1±0.21 3.0±0.24 3.4±0.19 p+b 62.1±0.13 31.2±0.17 3.0±0.18 3.4±0.23 4 c 47.5±0.16 31.3±0.14 n.a. n.a. p 49.8±0.23 33.1±0.15 n.a. n.a. p+b 51.8±0.21 31.3±0.23 n.a. n.a. 8 c 53.4±0.19 33.2±0.24 3.0±0.29 3.4±0.27 p 54.9±0.22 45.6±0.16 2.8±0.23 3.3±0.21 p+b 55.2±0.18 46.2±0.13 2.7±0.25 3.4±0.28 12 c 56.0±0.15 47.7±0.15 n.a. n.a. p 56.4±0.17 56.1±0.18 n.a. n.a. p+b 58.2±0.21 56.2±0.22 n.a. n.a. 16 c 56.8±0.24 56.9±0.21 2.8±0.25 3.2±0.28 p 57.5±0.15 58.3±0.19 2.5±0.22 3.1±0.24 p+b 58.8±0.21 61.3±0.15 2.3±0.29 3.2±0.23 c control; p pectin (1%); b – bacteriocin; n.a. not analyzed; * ±standard deviation (±sd). 3.4. changes in sugar content the values of glucose and fructose in blackberries were measured at the beginning (day 0), middle (day 8) and end (day 16) of the storage period (table 2). the detected sugars in all samples were only glucose and fructose as their content was closer to reported values for wild grown blackberry 35g/kg of fw (mikulic-petkovsek et al. 2012). however, in our study sucrose was not found in blackberry samples. the results showed that on the 8-th day of the experiment, the concentration of glucose in the control fruit matched the initial level of 3.0 g/100 g of fw, while the glucose level in the treated fruit decreased slightly to 2.8 g/100 g of fw (pectin) and 2.7 g/100 g of fw (pectin+bacteriocin). thereafter, the concentration of glucose decreased in all treatments and reached levels of 2.8 g/100 g of fw (control), 2.5 g/100 g of fw (pectin) and 2.3 g/100 g of fw (pectin+bacteriocin) by the 16-th day of storage. fructose levels remained almost constant despite prolongation of the storage time. during the first 8 days of storage, all treatments maintained their initial level ital. j. food sci., vol. 32, 2020 432 of 3.4 g/100 g of fw, except the pectin-coated fruits, which showed a concentration of 3.3 g/100 g of fw. at the end of the storage period (day 16), the concentration of fructose had decreased slightly in all experimental groups, reaching levels of 3.2 g/100 g of fw (control), 3.1 g/100 g of fw (pectin) and 3.2 g/100 g of fw (pectin+bacteriocin) (table 2). these results demonstrated that pectin-based edible coatings singly or in combination with bacteriocin did not protect the sugar content in coated blackberries under refrigerated storage conditions. 3.5. changes in total anthocyanins content as seen in fig. 6, the initial level of total anthocyanins of 109.2 mg/100 g of fw was in accordance with that reported in the literature (104 – 198 mg/100 g of fw) (pantelidis et al., 2007), but gradually decreased in all treatments. this trend was recorded through the end of the observation period. by the 16-th day of storage, total anthocyanins reached concentrations of 100 mg/100 g of fw (control), 94.2 mg/100 g of fw (pectin) and 92.3 mg/100 g of fw (pectin+bacteriocin), indicating that the application of both coatings failed to delay the reduction of anthocyanins in blackberries during refrigerated storage. the negative effect of low temperatures on anthocyanin concentration during the storage of fresh berries was previously reported by kalt et al. (1999). lowered anthocyanins and ascorbic acid content during cold storage of strawberries was also confirmed by cordenunsi et al. (2005). however, the authors observed a positive impact on other parameters such as sugars, while levels of flavonols, ellagic acid, tpc and antioxidant activity remained almost the same or even decreased at all tested temperatures (6, 16 and 25°c). figure 6. effects of pectin-based and pectin-based edible coatings with bacteriocin of b. methylotrophicus bm47 on the total anthocyanins content of fresh blackberries during refrigerated storage. c control; p pectin (1%); p+b pectin (1%) + bacteriocin. ital. j. food sci., vol. 32, 2020 433 3.6. changes in total polyphenols content during the first four days of refrigerated storage, the tpc in both the control and treated blackberries maintained relatively high levels that were close to the initial concentration of polyphenols of 186 mg gae/100 g of fw (0 day) (fig. 7). however, by the end of the observation period (16-th day), the tpc declined for all experimental groups to concentrations of 174.2 mg gae/100 g of fw (control), 173.8 mg gae/100 g of fw (pectin) and 173.1 mg gae/100 g of fw (pectin+bacteriocin). these results demonstrated that the coatings did not effectively protect the polyphenolic content, and this progressive decrease was most likely associated with the breakdown of cell structures that is caused by natural senescence processes in the fruits (tumbarski et al., 2019). figure 7. effects of pectin-based and pectin-based edible coatings with bacteriocin of b. methylotrophicus bm47 on the total phenolic content of fresh blackberries during refrigerated storage. c control; p pectin (1%); p+b pectin (1%) + bacteriocin. 3.7. changes in antioxidant activity the results obtained by the dpph method showed that the antioxidant activity of control blackberries gradually decreased throughout the monitoring period, reaching its lowest value of 222.2 mmol te/100 g of fw on the 16-th day. as seen from the results presented in fig. 8, the pectin and pectin+bacteriocin coatings effectively inhibited this decrease and maintained antioxidant levels in the treated blackberries that were higher than those in the uncoated ones, keeping the antioxidant levels close to the initial value of 233.9 mmol te/100 g of fw (0 day). the positive impact of pectin and pectin+bacteriocin coatings on antioxidant activity could be associated with a reduction in the respiration rates and the resultant protective effect on other biologically active compounds that possess strong antioxidant activity such as phenolic acids, hydrolysable tannins, vitamin e, carotenoids, minerals and enzymes. further exploration of these antioxidant compounds is warranted in a future study. ital. j. food sci., vol. 32, 2020 434 figure 8. effects of pectin-based and pectin-based edible coatings with bacteriocin of b. methylotrophicus bm47 on the antioxidant activity of fresh blackberries during refrigerated storage. c control; p pectin (1%); p+b – pectin (1%) + bacteriocin. the biopreservation of blackberry fruit using different edible coatings has also been investigated by other authors. for example, oliveira et al. (2012) reported the effects of three types of edible coatings chitosan (1.5%), cassava starch (2.5%) and kefir grains in water (20%) on the storage life of the blackberry cultivar tupy stored at temperatures of 0°c and 10°c. the authors stated that during the 18 days of storage, all coatings effectively reduced the loss of weight at 0°c, but cassava starch and kefir grains-based coatings were not efficient at 10°c. during storage, a significant increase in ph was observed in all treatments at both 0°c and 10°c, with the control fruit showing the lowest ph values. the authors also reported that at 10°c, the anthocyanins levels in all treatments progressively decreased until the 12-th day but thereafter increased significantly in the control fruit and in blackberries with chitosan coatings. at 0°c, the same trend of lowered anthocyanins concentrations in the control and chitosan coatings was observed, while cassava starch and kefir grains-based edible coatings maintained elevated and relatively stable anthocyanins levels. pérez-gallardo et al. (2014) developed more hydrophobic edible coatings based on modified tapioca starch added to 0.5 and 1.0% beeswax microparticles and then examined the quality of freshly harvested blackberries during storage at 4°c for 16 days. this study showed that the coated fruit exhibited greater weight loss than uncoated fruit, but the increase in concentration of beeswax particles from 0.5 to 1% led to a decrease in weight loss of 11.55±0.71 and 9.72±0.42%, respectively, compared to the control fruit (7.6±0.13%) at the end of the experiment. uncoated blackberries showed higher anthocyanins content than coated ones. the coated blackberries with 0.5% beeswax microparticles exhibited low but similar tac, while those coated with 1% beeswax microparticles revealed higher values and a slight, but significant decrease at the end of the storage period. by the end of the storage period, the tpc was significantly higher in uncoated blackberries than in coated fruit. ital. j. food sci., vol. 32, 2020 435 gol et al. (2015) evaluated the effects of edible coatings comprised of chitosan, alginate and carboxymethyl cellulose on the improvement in the quality and storage life of indian blackberry or jamun fruit (syzygium cumini l.). the results from this study showed that fruit treated with these three coatings at concentrations of 1% and 1.5% exhibited a significant delay in wl along with a reduction in decay percentage as well as positive effects on tss, ph, ta and sugars in comparison to the uncoated fruit. these coatings also had a positive impact on maintaining a higher concentration of antioxidants during the 16 days of storage and had a positive effect on the inhibition of cell wall-degrading enzyme activities. 4. conclusions the present study demonstrated that the application of celery pectin-based edible coatings singly and in combination with a bacteriocin of bacillus methylotrophicus bm47 represents a promising approach for extending storage life in processed blackberries. the bacteriocincontaining edible coatings effectively inhibited the fungal growth, significantly reduced the decay incidence, delayed the increase in tss and helped to preserve some of the health benefitial properties of the fresh fruits, specifically the ascorbic acid content and antioxidant activity. our results suggested that the bacteriocin synthesized by b. methylotrophicus bm47 in the composition of edible coatings could find a successful application as a biopreservative for improving product quality, storage life and the safety of blackberry fruit. acknowledgements this work was financed by fund ’science‘ of the university of food technologies, plovdiv, bulgaria (grant 01/19-h). the authors declare no conflict of interest. references antunes l.e.c., duarte-filho j. and souza c.m. 2003. conservação pós-colheita de frutos de amoreira-preta. pesq. agrop. brasileira. 38:413. barbosa a.a., mantovani h.c. and jain s. 2017. bacteriocins from lactic acid bacteria and their potential in the 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and lante a. 2019. biopreservation of fresh strawberries by carboxymethyl cellulose edible coatings enriched with a bacteriocin of bacillus methylotrophicus bm47. food technol. biotechnol. 57(2):230. valdés a., burgos n., jiménez a. and garrigós m.c. 2015. natural pectin polysaccharides as edible coatings. coatings. 5(4):865. zorenc z., veberic r., koron d. and mikulic-petkovsek m. 2017. impact of raspberry (rubus idaeus l.) primocane tipping on fruit yield and quality, not. bot. horti. agrobo. 45(2):417. paper received august 15, 2019 accepted january 31, 2020 ijfs#1697_bozza ital. j. food sci., vol. 32, 2020 251 paper drying characteristics of ‘ankara’ pear slices y.b. öztekin*1 and k. sacilik2 1department of agricultural machinery and technologies engineering, faculty of agriculture, ondokuz mayis university, 55139, samsun, turkey 2department of agricultural machinery and technologies engineering, faculty of agriculture, ankara university, 06130, ankara, turkey *corresponding author: tel.: +90 3623121919; fax: +90 362 4576034 email: yurtlu@omu.edu.tr abstract this study evaluated the effects of drying temperature and pre-treatment on the rehydration capacity and color parameters of sliced pears (cv. ankara). drying trials were conducted at 55, 65, and 75°c. pre-treatment consisted of immersion of pear slices in a citric-acid solution or blanching in hot water. pre-treatment was found to have a significant effect on both rehydration capacity and color, with higher temperatures and pre-treatment resulting in decreases in drying time and increases in rehydration capacity. effective diffusivity values ranged between 1.12×10-10 and 2.94×10-10 m2/s. blanched pear slices had the lowest ea values (15.51 kj/mol), followed by the samples immersed in citric acid (28.03 kj/mol) and the untreated samples (33.48 kj/mol). the midilli et al. model displayed the best fit to the drying data of five models tested based on the statistical criteria evaluated. natural color of fresh pear was best preserved with lower drying temperatures and pre-treatment with citric acid. keywords: color properties, convective drying, moisture content, rehydration capacity ital. j. food sci., vol. 32, 2020 252 1. introduction pear is one of the most important fruits in turkey and around the world. in 2017, turkey accounted for approximately 503,004 tons of the 24.17 million tons of pears produced world-wide, making it the 5th largest pear producer behind china (16.53 million tons), argentina (930,340 tons), the united states (677,891 tons) and italy 772,577 tons) (faostat, 2019). turkey is also a center of genetic diversity, with over 600 of the more than 5,000 varieties found throughout the world (karadeni̇z, 1999). one of the most important pear varieties in turkey is the ‘ankara’ pear, which originated in ankara and is grown mainly in turkey’s central anatolia region, especially in the province of ankara (erdoğan et al., 2007). ‘ankara’ pear trees produce medium-sized, green fruit with smooth surfaces, thin skins, short, thick stalks and juicy, fragrant flesh that melts in the mouth. the fruit are also easy to store (dumanoğlu et al., 2006; erdoğan et al., 2007). vegetables and fruits contain basic nutrients that are important for human health. because fruits and vegetables are cultivated on a seasonal basis and have a high-water content that makes them easily perishable, various preservation techniques have been developed so that fruits and vegetables can be consumed throughout the year (quiles et al., 2005). dehydration, although a highly complicated product-processing technique (maskan, 2000), is the basic method used for reducing moisture levels in order to minimize on-going microbial reactions, prevent deterioration (krokida and marinos-kouris, 2003), and increase the shelf life (das et al., 2001) of agricultural products. of the many drying methods available, convective drying, which represents one of the most common of all postharvest technologies, allows for high-quality products that preserve close to their original color (doymaz, 2004). pears are consumed in various forms, both fresh and dried. dried pears are consumed directly as snacks and are also widely used as inputs in the food industry. the design, operation, and maintenance of fruit-drying systems require a good understanding of drying characteristics. studies have evaluated drying characteristics of different varieties of pears, such as ‘d’anjou’ (park et al., 2002) and ‘deveci’ (doymaz, 2013), as well as different techniques, including convective drying (gonzález-martínez, 2006) airdrying (doymaz, 2013; doymaz and i̇smail, 2012), osmo-vacuum drying (amiripour et al., 2015), mid-infrared-freeze drying (antal et al., 2017), and microwave-vacuum drying (taskin et al., 2019). however, the literature includes no data on the drying behavior of the ‘ankara’ pear variety, whose texture varies greatly from that of other varieties, especially the ‘deveci’ pear. thus, this study was carried out to examine how drying temperature and pre-treatment by either immersion in a citric acid solution or blanching in hot water affect the drying characteristics and quality parameters (i.e. moisture content, rehydration capacity, color) of ‘ankara’ pear. 2. materials and methods 2.1. material the pears used in this study (cv. ankara) were obtained from a local market in ankara, turkey. pears were kept refrigerated at 5ºc and removed 12 hours prior to the trials to obtain equilibrium. pears were then sliced into sound, homogenous samples of 5±0.5 mm thickness and randomly distributed among 3 groups according to pre-treatment, as follows: citric acid: pear slices were immersed in a citric-acid solution (5 g/l) for 3 min at ital. j. food sci., vol. 32, 2020 253 room temperature; blanching: pear slices were blanched in 85°c water for 3 min and then rinsed with running water; untreated: pear slices received no pre-treatment. 2.2. drying pears were dried according to sacilik et al. (2010) using a convective hot-air dryer (57 x 68 x 57 cm) comprised of a perforated basket (576 cm2 x 12 cm), an adjustable fan, an electric heater, and a load-cell system attached to a pc (fig. 1). drying runs were carried out at 55, 65 and 75 °c, with a constant air velocity of 1 m/s (izli et al., 2019). a minimum of 250 g of pear slices was used for each run. pear slices were dried with tissue paper and then placed uniformly into the basket, which was positioned in the drying system after it had been allowed to idle for 20 min to reach thermal stabilization. initial moisture content of pears was measured at 120 ºc using an hb43-s halogen moisture analyzer (mettler toledo, switzerland) and recorded as 572.04% d.b. (85.12% w.b.). during the drying process, moisture loss from samples in the drying basket was measured using a load cell and continuously recorded using specially developed software connected to a pc. once moisture-loss measurements were completed, dried samples were evaluated for rehydration capacity and color. 2.3. effective diffusivity and activation energy a falling-rate drying period can be observed in drying pear slices, with moisture and/or vapor migration controlled by diffusion. in this case, fick’s second law can be derived as follows (crank, 1975; sacilik and unal, 2005): )( leff l md t m ∇∇= ∂ ∂ (1) figure. 1. the diagram of drying system. where ml is the local moisture content in % d.b., t is the drying time in min, and deff is the effective diffusivity in m2/s. assuming moisture migration to be realized through diffusion, shrinkage to be negligible, and diffusion coefficients and temperatures to be constant (crank, 1975) yields the following equation: ital. j. food sci., vol. 32, 2020 254 ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ +− + = − − = ∑ ∞ = 2 22 0 22 0 4 π)12( exp )12( 1 π 8 h tdn nmm mm m eff ne e r (2) for long drying periods, by considering only the first term in the series and, given the relatively small size of me as compared to m and m0, reducing moisture ratio (mr) to m/m0 equation 2 can be simplified to yield equation 3: ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ −= 2 2 2 0 4 π π 8 lnln h td m m eff (3) where mr is the dimensionless moisture ratio, m is the moisture content at any time in % d.b., me is the equilibrium moisture content in % d.b., m0 is the initial moisture content in % d.b., h is the half-thickness of the slab in sample in m, and n is a positive integer. effective diffusivity and drying air temperature are correlated using the arrhenius equation (equation 4): ⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ −= a a eff rt e dd exp0 (4) where d0 is the pre-exponential factor of the arrhenius equation in m2/s, ea is the activation energy in kj/mol, r is the universal gas constant in kj/mol.k, and ta is the absolute air temperature in k. 2.4. modelling of drying data drying data were fitted to five selected models (table 1). moisture ratios were determined using the following equation: e e r mm mm m − − = 0 (5) where mr is the moisture ratio, m, me and mo are, respectively, the moisture content at any time, the equilibrium moisture content, and the initial moisture content in % d.b. mr was further reduced to m/m0 , given the continuous fluctuation of relative humidity during the drying processes, (diamente and munro, 1993). data were analyzed by using statistica 6.1 (statsoft inc., usa) software package. drying rate constants and model coefficients were calculated according to levenberg-marguardt, and the statistical validity of the selected drying models was assessed according to the criteria put forth in equations 6, 7 and 8 (sacilik et al., 2010; yurtlu, 2011): ∑ = − = n i ir ipreriexr m mm n p 1 exp,, ,,,,100 (6) ital. j. food sci., vol. 32, 2020 255 ( ) 2/1 1 2 ,,,, 1 ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ −= ∑ = n i ipreriexr mmn rmse (7) zn mm n i ipreriexr − − = ∑ =1 2 ,,,, 2 )( χ (8) where mr,ex,i is the ith experimental moisture ratio, mr,pre,i is the ith predicted moisture ratio, n is the number of observations, and z is the number of constants. r2 was used as the primary comparison criteria. goodness of fit was also examined based on p, rmse and χ2 (yurtlu, 2011). table 1. selected drying models. model no model name model references 1 page mr = exp(-kt m) agrawal and singh (1977) 2 logarithmic mr = a exp(-kt) + c yagcioglu et al. (1999) 3 two-term mr = a exp(-kt) + b exp(-k0t) henderson (1974) 4 approximation of diffusion mr = a exp(-kt) + (1 a) exp(-kbt) yaldiz and ertekin (2001) 5 midilli et al. mr = a exp(-kt m) + bt midilli et al. (2002) 2.5. rehydration capacity and color parameters of pear slices rehydration capacity is of paramount importance for dried products. in this study, rehydration capacity was determined by immersing 10 g of dried pear slices into 85 °c water for 3 min, drying the pear surfaces with paper towels, and measuring the mass of the rehydrated sample using an electronic digital scale (±0.001 g), with rehydration capacity expressed as the ratio of the mass of the rehydrated sample to the mass of the dried sample (prakash et al., 2004). color properties are also among the important quality parameters of dried fruits (elicin and sacilik, 2005). in this study, color measurements were obtained from 5 points on the surface of each pear sample using a minolta cr-300 chromameter, and the average measurement was calculated. hue angles and color differences between raw and dried samples were calculated with the help of equation 9 and equation 10 (sacilik and unal, 2005): )(tan * * 1 a b h −= (9) 20 2 0 2 0 )()()( fff bbaalle −+−+−=δ (10) where h is the hue angle°, δe is the color difference, l0, a0 and b0 are the color lightness, green-red and blue-yellow values of raw pear slices, and lf, af and bf are the color lightness, green-red and blue-yellow values of dried pear slices. ital. j. food sci., vol. 32, 2020 256 3. results and discussion 3.1 hot-air drying curves of pears pear (cv. ankara) drying characteristics are presented in figs. 2, 3 and 4 according to drying temperature and pre-treatment procedures. as the figs show, pear moisture content was observed to decrease continuously over time from 572.04% d.b. to between 4.43% d.b. and 19.22% d.b. moisture content was significantly affected by drying temperature, citric-acid treatment, and blanching. untreated pears required drying times of 1,560, 1,080 and 900 min at 55, 65 and 75 ºc, respectively, to reach their final moisture content, as compared to 1,140, 900 and 660 min for pear samples pre-treated with citric acid and 840, 720 and 600 min for samples blanched in hot water. these figs. – representing decreases in drying time of 46% at 55ºc and 33% at 65ºc and 75ºc for blanched pears as compared to untreated pears – demonstrate that water diffusion increases with pre-treatment. similar results have been reported by doymaz (2010) for amasya red apples, by doymaz (2013) for pear, by vardin and yilmaz (2018) for pomegranate arils, and by pandey et al. (2019) for green peas. figure 2. drying curves for ‘ankara’ pear at 55°c. 0 100 200 300 400 500 600 0 5 10 15 20 25 30 m oi st ur e co nt en t (% d .b .) drying time (h) untreated blanch citric acid ital. j. food sci., vol. 32, 2020 257 figure 3. drying curves for ‘ankara’ pear at 65°c. figure 4. drying curves for ‘ankara’ pear at 75°c. 3.2. effective diffusivity and activation energy from equation 3, a plot of ln(mr) vs. the time provides a straight line with a slope s of: 2 2 4 π h d s eff= (11) the highest deff values were obtained for the blanched pear samples, followed by the citricacid treated and the untreated samples (table 2). deff values were observed to increase with increases in air temperature due to accelerated moisture diffusion, which could be due to an increase in water permeability caused by cracks in the sample surfaces. the deff values obtained for ‘ankara’ pear slices in the present study are comparable to values ranging from 1.59×10−10 to 7.64×10−10 m2/s obtained for ‘d’anjou’ pear at 40 ºc 80 ºc (park et al., 2002), from 2.27×10-10 to 4.97×10-10 m2/s for “organic apple” at 40 ºc 60 ºc (sacilik and 0 100 200 300 400 500 600 0 5 10 15 20 m oi st ur e co nt en t (% d .b .) drying time (h) untreated blanch citric acid 0 100 200 300 400 500 600 0 5 10 15 20 m oi st ur e co nt en t (% d .b .) drying time (h) untreated blanch citric acid ital. j. food sci., vol. 32, 2020 258 elicin, 2006), from 2.66×10-10 to 4.56×10-10 m2/s for üryani plum at 50ºc 70ºc (sacilik et al., 2006), from 0.85×10−10 to 2.18×10−10 m2/s for pear slices at 55ºc 75ºc (doymaz, 2012), and from 8.56×10-11 to 2.25×0-10 m2/s for ‘deveci’ pear slices at 50ºc 71ºc (doymaz, 2013). activation energy values were obtained by plotting ln(deff) vs. 1/t (fig. 5), which yielded a straight line indicating an arrhenius dependence on temperature. using equation 4, activation energy values for untreated pear samples, pear samples treated with citric acid, and blanched pear samples were obtained using equations 12, 13 and 14, respectively, as follows: table 2. effective diffusivity for ‘ankara’ pear at various air temperatures. air temperature, °c deff x10 10, m2 /s r2 untreated 55 1.12 0.9747 65 1.56 0.9878 75 2.26 0.9713 citric acid 55 1.45 0.9951 65 1.90 0.9839 75 2.61 0.9821 blanched 55 2.12 0.9709 65 2.44 0.9756 75 2.94 0.9825 figure 5. arrhenius-type relationship between deff and ta. untreated samples: ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ −×= − t deff 23.4026 exp1036.2 5 (12) (r2=0.9979), citric acid-treated samples: -23,0 -22,6 -22,2 -21,8 0,0028 0,0029 0,003 0,0031 ln d ef f 1/ta, (k-1) untreated blanch citric acid ital. j. food sci., vol. 32, 2020 259 ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ −×= − t deff 19.3371 exp1015.4 6 (13) (r2=0.9965), blanched samples: ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ −×= − t deff 72.1865 exp1019.6 8 (14) (r2=0.9916). the highest value of activation energy was obtained for the untreated samples (ea=33.48 kj/mol), followed by the citric-acid treated samples (ea=28.03 kj/mol) and blanched samples (ea=15.51 kj/mol). these values are in line with the range (15-40 kj/mol) specified by rizvi (1986) for various foods. 3.3. parameter estimation estimated values of drying models and comparison criteria (r2, p, rmse and χ2) are given in table 3. selected models offered a good fit to data. of the 5 models examined, the midilli et al. had highest r2 and lowest p, rmse and χ2 values, indicating it to be the best model in terms of fitness to data. comparisons of the experimental data and the predicted moisture ratios obtained using the midilli et al. model for ‘ankara’ pear slices at 55, 65 and 75°c are presented in fig 6. as the fig. show, there is very good conformity between the actual and the predicated data, confirming the goodness of fit of the midilli et al. model. 3.4. quality parameters (rehydration and color retention) air temperature as well as pre-treatment, either with a citric-acid solution or by blanching, significantly affected the rehydration capacity of ‘ankara’ pears (table 4). the highest rehydration values were observed for the blanched pear slices dried at 75ºc. at every temperature examined, the blanched pear slices showed the greatest rehydration capacity, followed by the samples treated with citric acid and the untreated samples. increases in air temperatures during drying resulted in increases in rehydration capacity, with increases of 5.43%, 4.64% and 10.54%, respectively, for untreated samples, samples treated with citric acid, and blanched samples when temperatures were increased from 55 to 75 ºc. this finding can be explained by an increase in the rate of moisture removal with increases in air temperature, which leads to less shrinkage and thus an accelerated rate of rehydration. similar results have been reported by amiripour et al. (2015), hebda et al. (2019) and singh et al. (2006). table 5 shows the hunter color values for pears by air temperature and pre-treatment procedures. the lowest a* values and the highest l* and h values were observed at 55°c regardless of pretreatment. h and l* values decreased with increases in temperature, whereas a* values increased with increases in temperature, demonstrating that browning occurred as a result of temperature increases. similar results were reported by wang and chao (2003), elicin and sacilik (2005) and sacilik and elicin (2006). ital. j. food sci., vol. 32, 2020 260 table 3. statistical criteria of the models for ‘ankara’ pear. °c model no model coefficients r2 p (%) rmse χ2 untreated 55 1 k=0.0985; m=1.2518 0.9974 25.54 1.84x10-2 3.65x10-4 2 a=1.0627; k=0.164; c=-0.0126 0.9932 23.20 3.06x10-2 10.15x10-4 3 a=-0.0405; k0=0.5464; b=1.4754; k1=0.2142 0.9980 21.22 1.69x10-2 3.11x10-4 4 a=6.901; k=0.113; b=0.9935 0.9915 28.90 3.42x10-2 12.67x10-4 5 a=0.99; k=0.082; m=1.3664; b=0.0015 0.9993 9.65 1.02x10-2 1.14x10-4 65 1 k=0.0982; m=1.3124 0.9988 10.84 1.33x10-2 1.97x10-4 2 a=1.12; k=0.1527; c=-0.0858 0.9942 26.81 2.98x10-2 9.92x10-4 3 a=05257.; k0=0.1863; b=0.5265; k1=0.1863 0.9894 29.24 4.14x10 -2 19.24x10-4 4 a=-7.4478; k=0.3419; b=0.908 0.9920 10.02 1.34x10-2 1.99x10-4 5 a=0.9795; k=0.0836; m=1.395; b=0.00061 0.9992 7.24 1.15x10-2 1.49x10-4 75 1 k=0.156; m=1.3316 0.9962 44.90 2.24x10-2 5.47x10-4 2 a=1.0545; k=0.273; c=0.0065 0.9904 35.97 3.65x10-2 14.54x10-4 3 a=2.2259; k0=0.3951; b=-1.2366; k1=0.6568 0.9965 42.11 2.24x10-2 5.51x10-4 4 a=9.9874; k=0.1899; b=0.9696 0.9878 50.74 4.11x10-2 18.46x10-4 5 a=0.9826; k=0.1336; m=1.4489; b=0.00165 0.9994 9.72 0.96x10-2 1.03x10-4 citric acid 55 1 k=0.1352; m=1.1923 0.9991 12.76 1.09x10-2 1.32x10-4 2 a=1.0648; k=0.1897; c=-0.0213 0.9970 13.48 2.08x10-2 4.83x10-4 3 a=0.5246; k0=0.2011; b=0.5246; k1=0.2009 0.9965 8.83 2.31x10 -2 5.99x10-4 4 a=-5.7317; k=0.121; b=1.0676 0.9955 17.59 2.57x10-2 7.36x10-4 5 a=1.0034; k=0.131; m=1.2332; b=0.0012 0.9997 3.73 0.64x10-2 0.45x10-4 65 1 k=0.1254; m=1.3216 0.9987 12.84 1.40x10-2 2.23x10-4 2 a=1.1207; k=0.1869; c=-0.0836 0.9937 29.66 3.20x10-2 11.74x10-4 3 a=0.4054; k0=0.2271; b=0.6489; k1=0.2271 0.9891 30.74 4.35x10 -2 21.85x10-4 4 a=-6.8642; k=0.4215; b=0.899 0.9987 11.83 1.44x10-2 2.39x10-4 5 a=0.9798; k=0.1081; m=1.4065; b=0.0007 0.9991 8.66 1.24x10-2 1.78x10-4 75 1 k=0.198; m=1.3054 0.9982 13.01 1.69x10-2 3.41x10-4 2 a=1.1148; k=0.2576; c=-0.0823 0.9931 30.98 3.49x10-2 14.66x10-4 3 a=0.5247; k0=0.3119; b=0.5247; k1=0.3119 0.9886 31.51 4.67x10 -2 26.72x10-4 4 a=-5.384; k=0.147; b=1.1161 0.9929 32.32 3.53x10-2 14.97x10-4 5 a=0.9738; k=0.1699; m=1.4089; b=0.0011 0.9988 8.65 1.48x10-2 2.69x10-4 blanched 55 1 k=0.1757; m=1.2674 0.9987 17.28 1.41x10-2 2.29x10-4 2 a=1.0863; k=0.2434; c=-0.0475 0.9944 32.44 3.03x10-2 10.62x10-4 3 a=-0.0407; k0=0.2745; b=1.01; k1=0.2746 0.9922 26.57 3.71x10 -2 16.11x10-4 4 a=-5.789; k=0.4965; b=0.8918 0.9988 16.57 1.41x10-2 2.29x10-4 5 a=0.9843; k=0.1584; m=1.3390; b=0.00098 0.9990 9.79 1.32x10-2 2.03x10-4 65 1 k=0.2161; m=1.2946 0.9987 17.90 1.44x10-2 2.45x10-4 2 a=1.0827; k=0.2986; c=-0.0391 0.9927 28.95 3.56x10-2 15.06x10-4 3 a=-0.4799; k0=0.3298; b=0.5734; k1=0.3298 0.9911 21.92 4.11x10-2 20.21x10-4 4 a=-4.5469; k=0.6298; b=0.859 0.9989 16.06 1.37x10-2 2.21x10-4 5 a=0.9813; k=0.1921; m=1.3988; b=0.0021 0.9995 2.65 9.61x10-2 1.11x10-4 75 1 k=0.281; m=1.2235 0.9996 11.58 0.83x10-2 0.82x10-4 2 a=1.08; k=0.3377; c=-0.0442 0.9963 24.31 2.55x10-2 7.96x10-4 3 a=-2.7851; k0=0.7071; b=3.7845; k1=0.5749 0.9996 11.08 0.86x10-2 0.93x10-4 4 a=0.1349; k=0.3568; b=0.9989 0.9914 28.21 3.91x10-2 18.68x10-4 5 a=0.9936; k=0.2729; m=1.257; b=0.00103 0.9996 7.70 0.78x10-2 0.75x10-4 ital. j. food sci., vol. 32, 2020 261 (a) (b) (c) figure 6. conformity of the midilli et al. for ‘ankara’ pear at 55°c (a), at 65°c (b) and at 75°c (c). 0,0 0,2 0,4 0,6 0,8 1,0 0 5 10 15 20 25 30 m r drying time (h) untreated, experimental citric acid, experimental blanch, experimental midilli et al. model 0,0 0,2 0,4 0,6 0,8 1,0 0 5 10 15 20 m r drying time (h) untreated, experimental citric acid, experimental blanch, experimental midilli et al. model 0,0 0,2 0,4 0,6 0,8 1,0 0 5 10 15 20 m r drying time (h) untreated, experimental citric acid, experimental blanch, experimental midilli et al. model ital. j. food sci., vol. 32, 2020 262 table 4. rehydration capacity for ‘ankara’ pear at various temperatures. air temperature, °c rehydration capacity untreated 55 3.31 65 3.35 75 3.49 citric acid 55 3.45 65 3.54 75 3.61 blanched 55 3.51 65 3.69 75 3.88 table 5. color values for ‘ankara’ pear at various temperatures. air temperature hunter color values °c l* a* b* δe h° untreated 55 72.69 5.04 33.27 10.09 81.40 65 68.04 4.46 26.33 14.62 80.13 75 66.38 6.57 29.22 15.43 77.50 citric acid 55 78.16 2.91 34.03 8.97 85.25 65 75.69 5.79 34.96 9.41 80.58 75 67.71 5.20 31.98 12.85 80.56 blanched 55 69.45 6.26 30.73 12.54 78.43 65 64.72 6.99 30.77 16.61 77.65 75 63.98 7.69 34.37 16.29 77.60 in terms of pre-treatment, the present study found samples pre-treated with citric acid had higher h and l* values as compared to blanched and untreated samples at each air temperature tested. moreover, the samples treated with citric acid had smaller ∆e values than both the blanched and untreated samples, indicating that pre-treatment with citric acid helped to preserve the original color of pear slices. overall, the natural color of fresh pear was best preserved when slices were pre-treated with a citric-acid solution and dried at the lowest air temperature (55°c). 4. conclusions in conclusion, drying temperature and pre-treatment with either a citric-acid solution or by blanching in hot water significantly affected the moisture content, rehydration capacity and color parameters of ‘ankara’ pear slices. blanched pear slices required shorter drying times than samples treated with citric acid as well as untreated samples. when compared to untreated pears, blanched pear slices required 46% less time for drying at 55ºc and 33% less time at 65ºc and at 75ºc. deff values were observed to decrease with decreases in ital. j. food sci., vol. 32, 2020 263 temperature and were lower for untreated pears than for pre-treated pears. ea values were highest for untreated samples (33.48 kj/mol), followed by citric acid-treated (28.03 kj/mol) and blanched samples (15.51 kj/mol). based on the evaluated statistical criteria, the midilli et al. model showed the best fit to the drying data of all the models tested. rehydration capacity of pear slices was seen to decrease with decreases in drying temperature. the natural color of fresh pear slices was best retained when the samples were pre-treated with citric acid and dried at the lowest air temperature. references agrawal y.c. and singh r.d. 1977. thin layer drying studies on short grain rice. asae paper no: 3531 asae, st. joseph, mi. amiripour m., habibi-najafi m.b., mohebbi m. and emadi b. 2015. 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30(1):156-169. wang j. and chao y. 2003. effect of 60co irradiation on drying characteristics of apple. j. food eng. 56(4):347-351. yurtlu y.b. 2011. drying characteristics of bay laurel (laurus nobilis l.) fruits in a convective hot-air dryer. afr. j. biotechnol. 10(47):9593-9599. yagcioglu a., degirmencioglu a. and cagatay f. 1999. drying characteristics of laurel leaves under different drying conditions. in proceedings of the 7th international congress on agricultural mechanization and energy, pp. 565-569, adana, turkey, may 26-27. yaldız o. and ertekin c. 2001. thin layer solar drying some different vegetables. drying technol. 19(3):583-596. paper received september 24, 2019 accepted november 6, 2019 ijfs#1150_bozza ital. j. food sci., vol. 30, 2018 650 paper myosin heavy chain isoforms, fatty acid composition, sensory evaluation and quality of cinta senese pig meat s. velotto1, m. rabie ashkezary2, s. de camillis1, v. alfeo2 and a. todaro*2 1department of promotion of human sciences and the quality of life, university of study of roma san raffaele, via val di val cannuta 247, roma, italy 2department of agricultural, food and forest sciences, università degli studi di palermo, viale delle scienze ed.4, 90128 palermo, italy *corresponding author. tel. +39 3451228409 e-mail address: aldo.todaro@unipa.it abstract the aims of this study were to examine the effects of myosin heavy chain (mhc) isoforms on cinta senese meat and sensory quality. the research was carried out on 65 pigs and muscle samples characteristics such as mhc isoform, meat quality, fatty acid composition, and sensory were evaluated. the results demonstrated that mhc slow isoform content was significantly correlated with ph24h (r=0.25, p<0.05) and drip loss (r=-0.31, p<0.005), whereas the content of mhc isoforms was only weakly correlated with fatty acids. sensory evaluation was done by a trained panel test and the results shown that the mhc fast/slow ratio was correlated with the juiciness (r=-0.32, p<0.005), off-flavor (r=0.33, p<0.01), and tenderness attributes (r=-0.42 to -0.46). we therefore conclude that the content of mhc isoforms can be one of the most important factors for examination of overall quality of cinta senese pigs. keywords: fatty acid, fiber, myosin, quality, tenderness ital. j. food sci., vol. 30, 2018 651 1. introduction to date, studies have demonstrated that muscle fibre composition plays an important role in meat quality traits (dai, 2009). skeletal muscle is composed of various types of fibers. muscle fiber types have different biochemical characteristics, including oxidative and glycolytic capacities, contraction speed, fiber size, myoglobin, and glycogen content (schiaffino and reggiani, 1996). muscle fiber type i has slow-twitch, oxidative metabolic characteristics, and a low glycogen content. type iia is a fast oxidativeglycolytic fiber. on the other hand, type iib has fast-twitch, glycolytic metabolic characteristics, and high glycogen content (velotto, 2012). another study has indicated that during postmortem period, some glycolysis enzymes might be the candidate predictors for meat discoloration (wu et al. 2015). therefore, during the postmortem period, muscle fiber type composition may impress metabolite content (fernandez, 1995). the composition of type iib fiber has a positive correlation with meat lightness and drip loss (ryu and kim, 2006), whereas type iib fiber has a negative correlation with juiciness and flavor (taylor, 2004). there is a clear relationship between meat quality to consumer satisfaction and flavor, texture, juiciness, tenderness and meat palatability (behrends, 2005; calkins and hodgen, 2007). moisture content in fresh and cooked meat influence on the juiciness of meat and also intramuscular fat (imf) content contributes to the perceived juiciness (fortin, 2005). glycolytic rate during the postmortem period is related to myosin heavy chain (mhc) isoforms content, therefore mhc isoforms content can influence ultimate meat quality traits (gil, 2003). our objective was to investigate the effects of mhc isoforms to postmortem meat quality traits, fatty acid composition, and sensory evaluation in cinta senese pigs. the cinta senese pig has existed for much longer than any of the other white breeds in northern europe: the large white, the yorkshire and the landrace. the renewed interest in the breeding of the cinta senese is quite recent. the battle to safeguard the breed is in progress and is succeeding. following the line of these perspectives, we aimed the examination of the effects of mhc isoforms on cinta senese meat and sensory quality. 2. materials and methods 2.1. preparation of muscle samples 65 cinta senese pigs, one year old, (25 gilts and 40 castrated male pigs) were investigated. in order to follow the recommendation of the national research council (nrc, 1998), we fed the pigs with a commercial diet. animals were treated according to the guidelines of the european community on the treatment of experimental animals (reg. ec 1/2005). the slaughterhouse had eec mark with reference to rules 852/853/854/2004; 2076/2005. pigs were anesthetized by an intramuscular injection of ketamine and sumianxin (rules120/2008/cee). samples of longissimus dorsi muscles were recovered from the carcasses at the 7-8th thoracic vertebra, 48 h after slaughter and were prepared for analysis. the slaughtering process was based on a traditional mechanized slaughter line. this method has the processes of vat scalding (62°c), dehairing, singeing/flaming, and polishing. the muscle samples were cut into 0.4×0.4×1.5 cm pieces, frozen in liquid nitrogen and stored at -80°c until analysis for mhc isoforms. the pork loins (the 10-13th thoracic vertebrae) were taken for meat quality assessment after 24 h of chilling at 4°c and then instantly stored at -20°c without chopping until measurements of imf content, fatty acid composition, and sensory quality were made. ital. j. food sci., vol. 30, 2018 652 2.2. myosin heavy chain isoform content eight frozen sections of each muscle sample were prepared and placed in 1.5-ml tubes. myofibrils were prepared according to the proposed method of the talmadge and roy (1993), and bradford's method (bradford, 1976) was used to determination of the protein concentration of each sample. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) was used to analyze mhcs (talmadge and roy, 1993) and separated into slow and fast isoforms. the mhc bands were identified by coomassie brilliant blue staining. these findings were evaluated using an image analysis system for quantitative measures (leica application suite interactive measurement). the percentage of each mhc isoforms was taken from the ratio of the density of each mhc band to the all mhc band densities (slow and fast), and the mhc fast/slow ratio was obtained from the ratio of the density of fast mhc bands to the density of slow mhc bands within each sample (choi, 2010). 2.3. meat quality measurements muscle ph was obtained at 24 h postmortem (ph24h) using a spear-type portable ph meter (hanna instruments, ph 210). meat color parameters was assessed after exposing the surface to air at 4°c for 30 min by the lightness (l*), redness (a*), and yellowness (b*) system (c.i.e., 1978) using a minolta chromameter (cr-300, minolta camera co., japan). the mean value of three replicates conducted on each sample and also color (1=pale pink is gray to white; 6=dark purplish-red) and marbling (1=1.0% imf; 10=10% imf) were assessed visually (nppc, 2000). three parameters including, drip loss, filter-paper fluid uptake (ffu), and cooking loss were used to evaluate the water holding capacity (whc). according to honikel (1987), driploss was calculated after weighing (70 gr). the sample was placed in a net surrounded and then hung in an inflated plastic bag for 48 h at 4°c. the sample weighed again after 48 h and drip loss was calculated as percent change in weight. in order to measurement of ffu (kauffman, 1986), filter paper (whatman #2, 42.5 mm in diameter) was weighed, then putted on the surface of the sample to absorb fluids (<2s) and then was re-weighed. ffu was expressed as milligrams of exudates absorbed by the filter-paper (eikelenboom et al., 1996). this was how we measured the cooking loss. following 24 h of chilling, loin sections were placed in thin-walled polyethylene bags and were then putted in a continuously heated water bath (80°c). samples were cooked to 71°c internal temperature that measured using a thermometer with a handheld probe; (tes-1300, tes electrical electronic co., taiwan) and were then held in ice water for 15 min. finally, the samples were removed from ice water and therefore were taken from the polyethylene bag, blotted dry, and weighed. cooking loss was expressed as a percentage of the initial sample weight (honikel, 1987). for measurement of warner-bratzler shear force (wbs), loin sections were cut into 2 cm thick chops and therefore, cooked meat samples for wbs were prepared as described for cooking loss samples. after cooking we took eight to ten cores (1.25 cm diameter) from the steak parallel to the longitudinal orientation of muscle fibers. wbs was examined by an instron universal testing machine (model 1011, instron corp., usa) equipped with a warner-bratzler shearing device using a crosshead speed of 200 mm/min and a load capacity of 10 kn. samples were sheared perpendicular to the long axis of the cores. 2.4. intramuscular fat content and fatty acid composition imf content was analysed using the soxhlet method with a solvent extraction system (aoac, 2000). for fatty acid composition analysis, the fat was extracted following the ital. j. food sci., vol. 30, 2018 653 procedure described by folch (1957). homogenized meat (1.5 gr) was blended with an extraction solvent of chloroform/methanol (2:1, v/v) twice, filtered, and then placed in a separator funnel and mixed with saline solution (0.9% nacl). after phase separation, chloroform lipid fraction was washed using extraction solvent, whereas the aqueous methanol fraction was discarded. lipid extracts were concentrated using a rotary evaporator and were then placed in test tubes for subsequent gas chromatographic analysis. before the gas chromatograph analysis, methylation of lipids was performed by adding 2 ml of sodium methoxide, distilled water, and heptane. gas chromatograph analysis was carried out using a gas chromatography-mass selective detector (gc, agilent 7890n, usa; msd, agilent 5975a, usa) equipped with a hp-innowax column (length of 30m, internal diameter of 0.25mm, film thickness of 0.25 μm). operating conditions included a helium flow rate of 0.7 ml/ min, a fid setting of 260°c, a splitsplitless injector setting of 220°c with an injection rate of 120 ml/min, and an injection volume of 1 μl. the temperature program was composed of an initial hold at 140°c for 4 min, ramping to 220°c at 4°c/min. retention time and area of each peak were computed using agilent software. individual fatty acid peaks were identified by comparing the retention times with those of known mixtures of standard fatty acids (fame, sigmaaldrich co, usa). fatty acid composition was expressed as the percentage of total methylated fatty acid. data were initially recorded and listed as the percentage of individual fatty acids in each sample. total saturated fatty acid (sfa) was computed as the sum of c12:0, c14:0, c16:0, c17:0, and c18:0. monounsaturated fatty acid (mufa) included c16:1, c18:1n-9, c18:1n-7, and c20:1n-9, whereas total polyunsaturated fatty acid (pufa) was calculated as the sum of c18:2n-6, c18:3n-6, c18:3n-3, c20:2n-6, c20:3n-3, c20:4n-6, c20:5n-3, and c22:6n-3. the ratios of mufa to sfa and pufa to sfa were then calculated. 2.5. sensory evaluation eleven panelists were selected by standing external descriptive panel. 65 pork samples were evaluated in three replications. the sensory analysis took place in 6 weeks sessions of up to 1.5 h each. according to the american meat science association (amsa, 1995) and published procedures (meilgaard et al., 1991), we performed formal trainings for the panelists. samples were thawed overnight at 4°c, and then cooked without salt or spice in a humid heat oven (mcs312cf4, electrolux, sweden) set at 180°c until they reached an internal temperature of 70°c, which was measured using a thermometer (tes-1300, tes electrical electronic co., taiwan). they were then immediately sliced into 1.3×1.3×1.3 cm3 pieces. samples were held in a water bath (54°c) until presented to the panelists simultaneously in a compartmented plate and three-digit codes were used to name them and served one at a time in random order. in order to eliminate the taste from the previous sample, the evaluators were served distilled water (30°c) and about 3 to 5 min elapsed before evaluation of the next samples. sensory properties including softness, initial tenderness, juiciness, flavor intensity, off-flavor intensity, chewiness, rate of breakdown and amount of perceptible residue were evaluated. 2.6. statistical analysis sas pc software (sas institute, 2004) was used to analysis the content of mhc isoforms in terms of means and standard deviations. means, standard deviations, and overall ranges are presented as results. correlations among data obtained were calculated using pearson’s correlation coefficient (r). ital. j. food sci., vol. 30, 2018 654 3. results and discussion 3.1. myosin heavy chain isoform content in pig muscle table 1 illustrates the results of means and standard deviations for mhc isoform contents of the longissimus dorsi muscle in cinta senese pigs. mean values of the mhc slow and fast isoforms were 4.88% and 84.02%, respectively, and the fast/slow ratio was 23.21. according to information obtained, differences in the mhc isoform composition were observed among muscles. table 1. content of the myosin heavy chain (mhc) isoforms of the longissimus dorsi muscle in cinta senese pigs. parameter μ sd mhc slow isoform (%) 4.88 2.28 mhc fast isoform (%) 84.02 2.28 mhc fast/slow ratio (%) 23.21 20.30 3.2. meat quality table 2 shows the correlations between the content of mhc isoforms and meat quality measurements. this experiment found a significant correlation between muscle ph24h and content of mhc slow (r=0.25, p<0.05), fast (r=-0.25, p<0.05) isoforms and fast/ slow ratios (r=-0.30, p<0.01). the results showed that the mhc fast/slow ratio indicated a positive correlation with drip loss (r=0.36, p<0.001) and ffu (r=0.24, p<0.05). whereas mhc slow isoforms content had an opposite tendency. table 2. correlation coefficients between the content of myosin heavy chain (mhc) isoforms and meat quality measurements of the longissimus dorsi muscle in cinta senese pigs. mhc isoform parameter slow isoform fast isoform fast/slow ratio muscle ph24h 0.25 1 -0.251 -0.302 lightness (l*) 0.04 -0.04 -0.05 redness (a*) 0.12 -0.12 -0.10 yellowness (b*) 0.241 -0.241 -0.17 drip loss -0.312 0.312 0.363 filter-paper fluid uptake -0.221 0.221 0.241 cooking loss 0.12 -0.12 -0.9 warner-bratzler shear force 0.03 -0.03 -0.03 color 0.231 -0.231 -0.12 marbling 0.005 -0.005 -0.02 statistically different values (1p<0.05; 2p<0.01; 3p<0.001). ital. j. food sci., vol. 30, 2018 655 3.3. fat content table 3 shows the correlation between content of mhc isoforms and fatty acid composition. the results revealed that the mhc isoform contents were not related to lipid content and marbling score. in the present study, limited relationships emerged from the content of mhc isoforms with individual fatty acids (data not shown) as well as sfa, mufa, pufa, and ratio of pufa and sfa. table 3. correlation coefficients between the content of myosin heavy chain (mhc) isoforms, intramuscular fat (imf) and fatty acid composition of the longissimus dorsi muscle in cinta senese pigs. mhc isoform parameter slow isoform fast isoform fast/slow ratio imf -0.079 0.079 0.11 sfa 0.17 -0.17 -0.9 mufa 0.03 -0.03 0.04 pufa -0.10 0.10 0.005 mufa+pufa -0.17 0.17 0.9 mufa: sfa -0.12 0.12 0.16 pufa: sfa -0.12 0.12 0.03 3.4. sensorial features analyzing all quality parameters panelists were asked to leave comments for each attribute if they felt the need. particularly they found pig meat good for softness and initial tenderness but they didn’t consider it excellent. table 4 illustrates a correlation between the content of mhc isoforms and the sensory quality of cooked pork. table 4. correlation coefficients between the content of myosin heavy chain (mhc) isoforms and sensory evaluation of cooked meat of the longissimus dorsi muscle in cinta senese pigs. mhc isoform parameter slow isoform fast isoform fast/slow isoform softness 0.251 -0.251 -0.463 initial tenderness 0.251 -0.251 -0.433 juiciness 0.18 -0.18 -0.322 flavor intensity 0.231 -0.231 -0.19 off-flavor intensity -0.251 0.251 0.332 chewiness 0.211 -0.211 -0.423 rate of breakdown 0.231 -0.231 -0.443 mouth coating -0.02 0.02 0.17 amount of perceptible residue 0.261 -0.261 -0.423 score distributions: softness, soft to hard; initial tenderness, tender to tough; chewiness, tender to chewy; rate of breakdown, fast to slow; juiciness, not juicy to extremely juicy; flavor intensity, no pork flavor to full pork flavor; off-flavor intensity, none to strong off-flavor; mouth coating, none to very much; amount of perceptible residues, none to abundant. .statistically different values (1p<0.05; 2p<0.01; 3p<0.001). ital. j. food sci., vol. 30, 2018 656 in this study, a significant correlation was found between content of mhc isoforms and tenderness attributes including softness, initial tenderness, chewiness, rate of breakdown, and amount of perceptible residue. a positive correlation was shown between the content of mhc slow isoform and tenderness characteristics, whereas a negative correlation was found between the fast/slow ratio and softness (r=-0.46, p<0.001), initial tenderness (r=0.43, p<0.001), chewiness (r= -0.42, p<0.001), rate of breakdown (r=-0.44, p<0.001), and amount of perceptible residue (r=-0.42, p<0.001). the results showed that, there was no significant correlation between juiciness and content of mhc slow and fast isoforms, whereas there was a negative correlation between juiciness and the fast/slow ratio (r=0.32, p<0.01). we observed that there are positive and negative correlations between flavor intensity and mhc slow isoform content (r=0.23, p<0.05) and mhc fast isoform (r= -0.23, p<0.05), respectively, even if there weren’t great differences. on the other hand, off-flavor intensity had inverse relation to flavor and there were positive and negative correlations between off-flavor intensity and mhc fast isoform (r= 0.25, p<0.05) and mhc slow isoform content (r= -0.25, p<0.05), respectively. 4. discussion the aim of this study was to investigate the effects of mhc isoforms to postmortem meat quality traits, fatty acid composition, and sensory evaluation in cinta senese pigs. according to information obtained, differences in the mhc isoform composition were observed among muscles. scientists (sazili et al., 2005) detected a higher mhc fast isoform content in the longissimus dorsi and tensor fasciae latae muscles in comparison with the supraspinatus, semitendinosus, and trapezius muscles. however, in crossbred pigs (yorkshire x landrace x duroc), the mhc slow and fast isoform contents of the longissimus dorsi muscle were 6.61% and 93.38%, according to choi et al. (2006). differences seen in glycogen content and enzyme activities between muscles may be related to fiber type composition and the physical activity level of the muscle (granlund, 2011). choi and kim (2009) reported that the mhc fast isoform have a fast atpase and high anaerobic capacity, whereas the mhc slow isoform have a slow atpase and a high aerobic capacity in single muscle fibers. in contrast with our results mhc 1 isoform content was negatively correlated with the lightness and glycolytic enzyme activity, and was positively correlated with oxidative enzyme activity (choi, 2009). these demonstrated how the composition of mhc isoforms leads to a decline in the rate and extent of ph caused by lactate overproduction. our results show a significant correlation between muscle ph24h and content of mhc slow, fast isoforms and fast/ slow ratios. according to ryu and kim (2006), muscles harboring a higher percentage of mhc fast isoform tend to show a more rapid ph decline than muscles harboring a higher percentage of mhc slow isoform. genetic and pre-slaughter factors that influence postmortem rate of glycolysis and ph decline are used to determine the occurrence of pse (pale, soft, exudative) meat (kazemi, 2011). protein denaturation occurs when in high temperature of the muscle, rapid rate of postmortem glycolysis leads to a rapid ph fall (bendall and swatland, 1988; kauffman et al., 1998). the denaturation of myofibrillar proteins and particularly myosin is related to the low water holding capacity of pse meat (offer and knight, 1988; offer, 1991). muscles with a higher extent of protein denaturation show a higher percentage of mhc fast isoforms and higher degrees of fluid loss by exudation than muscles with a lower extent of protein denaturation (choi, 2010). our results showed that the mhc fast/slow ratio indicated a positive correlation with drip loss. ital. j. food sci., vol. 30, 2018 657 according to peter et al (1972), type i fiber contains greater amount of lipid, some of which presumably serves as a source of aerobic metabolic fuel; in contrast, type iib contains greater amounts of glycogen and glucose (hintz et al., 1984), and also glucose uses as fuel (choi, 2009). our results however revealed that the mhc isoform contents were not related to lipid content and marbling score. lipid composition is one of the main characteristics related to meat quality affected by muscle fiber type. according to leseigneur-meynier and gandemer (1991), total phospholipids (pls) and pufa of pls in pork are influenced by muscle fiber type. variations in the imf content are mainly due to changes in the triglyceride content, therefore, high imf contents imply a high level of triglycerides (ruiz-carrascal, 2000). in this study, a significant correlation was found between content of mhc isoforms and tenderness attributes including softness, initial tenderness, chewiness, rate of breakdown, and amount of perceptible residue. a positive correlation was shown between the content of mhc slow isoform and tenderness characteristics, whereas a negative correlation was found between the fast/slow ratio and softness, initial tenderness chewiness, rate of breakdown, and amount of perceptible residue. proteins and structures that bind and entrap water, specifically the myofibrillar are responsible for the mechanism of waterholding capacity (whc). it was earlier demonstrated that there is a direct effect of ph, ionic strength, and oxidation on the ability of myofibrillar protein and myofibrils and muscle cells to entrap water (huff-lonergan, 2005). glycolysis causes a more rapid ph decline, and muscle contraction, thereby, resulting in a high drip loss (zelechowska, 2012). it has also been declared that ph can affect meat tenderness (zheng et al., 2017). it is generally accepted that after cooking, muscles with a higher whc are more tender and juicy meat than muscles with a lower whc (jeong, 2010; kang, 2011). fiber type composition affects the sensory quality of cooked (kang, 2011; nam, 2009). in the present study, a positive correlation exists between content of mhc slow isoform and two parameters including ph and whc. moreover a high ratio mhc fast/slow isoforms and a high content of the mhc slow isoform are associated with tough and tender meat, respectively. according to oury (2009), a positive correlation exists between the content of the mhc slow isoform and the initial tenderness of rectus abdominis muscle from charolais heifers. in our study we noticed, as previously asserted, that there was a major percentage of fast-twitch glycolytic fibers, so that the whc was low, consequently cinta senese pig had high drip loss and low cooking loss. probably this feature was associated with the muscle chosen. there is a positive correlation between the proportions of type i fibers and sensory assessed tenderness in beef (maltin et al., 1998) and pork (ockerman et al., 1984) has been found. it was earlier demonstrated that effect of muscle fibre traits on meat tenderness are not uniform (orzechowska, 2008). according to totland (1988), the superficial regions in bovine semitendinosus muscle contained a high percentage of type iib fibers and were more tender than the deeper muscle layers, with a high percentage of type i fibers. there is a negative correlation between the percentage of type iib fibers and toughness in pigs (karlsson, 1993), whereas there is a positive correlation between the percentages of type i fibers and meat toughness in bulls. strydom (2000) reported that sensory tenderness shows a positive and negative relationship with the percentage of type i and iib fibers, respectively. on the other hand, we demonstrated that, there was no significant correlation between juiciness and content of mhc slow and fast isoforms, whereas there was a negative correlation between juiciness and the fast/slow ratio. according to huff-lonergan (2002), lipid content can influence the sensory features including texture, tenderness, flavor, and juiciness. also dall aaslyng (2008) reported that water holding capacity (whc) of the meat might can influence the juiciness. in this study, the higher whc in muscles with a lower fast/slow ratio compared to muscles with a higher fast/slow ratio ital. j. food sci., vol. 30, 2018 658 probably explains the relationship between juiciness and content of mhc. taste and aroma were defined as flavor (moody, 1983), which is based on taste-active compounds, flavor enhancers and aroma components with over 880 compounds presently identified in cooked beef alone (macleod, 1998). tenderness, juiciness and flavor are as components of the palatability of meat (muchenje, 2008). fatty components cause different flavors among beef, pork, chicken, turkey, and lamb and also fatty tissues give them specific flavor profiles. when the fat acts as one of the flavor agent heated, they are combined with amino acids from proteins and other components and therefore, they release (dinh tran nhat thu, 2006). there is about twice as much phospholipid than glycolytic ones in oxidative muscles. muscle fiber type composition may influence flavor through the phospholipids (lefaucheur, 2006) as major determinants of cooked meat flavor (meynier and gandemer, 1994). particularly phospholipids have an important role in the development of flavor intensity (mottram and edwards, 1983). high content of type i fiber is related to juiciness and flavor, whereas high type iib fiber content tends to be associated with tougher meat (choi and kim, 2009). according to our results, the offflavor intensity had inverse relation to flavor and there were positive and negative correlations between off-flavor intensity and mhc fast isoform and also with the mhc slow isoform content respectively. 5. conclusions the present study revealed that the composition of mhc isoforms influences the sensory quality of cooked pork, including tenderness features. a notable point that should be 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thickness determination: comparison between histological observation and texture analysis determination f. battista1, d. tomasi1, d. porro2, f. caicci3, s. giacosa4 and l. rolle4,* 1cra, centro di ricerca per la viticoltura, consiglio per la ricerca e la sperimentazione in agricoltura, 31015 conegliano, italy 2fondazione edmund mach, centro di trasferimento tecnologico, via mach 1, s. michele a/a (tn), italy 3dept. of biology, university of padova, via u. bassi 58/b, 35121 padova, italy 4università degli studi di torino, dipartimento di scienze agrarie, forestali e alimentari, largo paolo braccini 2, 10095 grugliasco (to), italy *corresponding author: tel. +39 011 6708558, fax +39 011 6708549, email: luca.rolle@unito.it abstract we analyzed the relation between the assessment of grape berry skin thickness by means of histology sections and instrumental mechanical properties measurements. berry skin of vitis vinifera l. cultivar corvina vineyards from valpolicella valpantena zone (verona, italy) were tested, evidencing a strong correlation between the two thickness determination methods. the middle or equatorial berry skin portion was found to be the less variable in instrumental skin thickness determination. in addition, unlike other studies, no correlation between the skin thickness and cell layers number was found. mailto:luca.rolle@unito.it ital. j. food sci., vol. 27 2015 137 introduction the skin or exocarp forms the grape’s dermal system: depending on thickness and berry size, it accounts for between 5 and 18 % of the fresh weight of ripe berries (ojeda et al., 2002). the skin is composed of epidermis, covered with a waxy cuticle, and a underlying outer hypodermis (considine and knox, 1979). during grape berry development from fecundation to ripening the skin thickness varies consistently (coombe and mccarthy, 2000). immediately after the fruit set until the véraison, when berry weight increases, epidermis and hypodermis cells expands in the tangential direction, increasing their area by 15% and 33%, respectively. after the véraison, the mesocarp cells continue their expansion, and the hypodermal and epidermal cells lose size, acting inversely in comparison with mesocarp cells (schlosser et al., 2008). thus, the skin become thinner in the berry during ripening: the relative thickness of the skin decreases from one-eighth to one-hundredth of the total berry diameter between fruit set and maturity stages (keller, 2010). the skin thickness is one of the most important grape skin morphological characteristics affecting the gas exchange regulation, the berry susceptibility to fungal diseases and the resistance to mechanical injuries (rosenquist and morrison, 1989; kök and çelik, 2004). the skin thickness varies depending on variety (muganu et al., 2011; giacosa et al., 2012) and clone (rolle et al., 2012a), confirming that this parameter is genetically influenced: this could be useful to further understand some different varietal characteristics, such as the susceptibility to fungal diseases or the aptitude to the post-harvest dehydration process. furthermore, the skin thickness seems to be related with the environmental conditions: in the alpine area cv. nebbiolo berries with similar sugar content showed a generally thickest skin than in the hill side (rolle et al., 2012b). this highlights that the skin thickness is very sensitive to the climate and the bunch microclimate conditions (porro et al., 2008; muganu et al., 2011), although a direct relation with water regimes in cv. muscat blanc in open field conditions was not found (giordano et al., 2013). since epidermis and hypodermis cells contain chloroplasts and phenolic-rich vacuoles (keller, 2010), skin berry properties (break force and thickness) can aid in the assessment of the phenolic content during the ripening. in particular, the skin thickness represents a useful indicator to predict anthocyanin extractability, and thinner skins seems to be characterized by higher anthocyanin extractability (río segade et al., 2011a). so, thickness can be useful to support the choice of the harvest data and to rationalize maceration and winemaking processes, thus allowing winemakers to best exploit the grape potential reached in the vineyard. berry skin thickness assessment can be obtained with histological observation or instrumental methods, i.e. texture analysis (letaief et al., 2008a; rolle et al., 2012c). the instrumental skin thickness measurement permits to minimize the sample treatment without using reagents or special procedures, speeding up the analysis process. the aim of this study was to compare the two cited skin thickness measurement methods (histology and texture analysis) among several vineyards, in order to assess differences between the two techniques and also to investigate the relationship between the thickness and the cell layer number of the analyzed samples. a preliminary test on the influence of the sampling berry skin portion on the instrumental skin thickness determination was also carried out. materials and methods grape samples grapes were collected from four vineyards located in the “valpolicella valpantena” denomination of origin, just to the north of verona, italy (45°29’22”n, 11°0’49”e). the vineyards were fifteen years old, planted with corvina (clone isv13) grafted on kober 5bb. the vines were trained with simple guyot and the rows were oriented north to south. the number of the vineyards analyzed was considered to be sufficient for this kind of study, following previous studies involving berry skin thickness variation analysis which considered from 3 to 7 vineyards (río segade et al. 2011b; rolle et al., 2012b). each vineyard was analyzed in duplicate (two subsamples) using this random sampling schema: each subsample was obtained by sampling fifteen bunches (one per chosen grapevine). from each bunch, twenty intact berries were selected, then the 300 resulting berries were used for the following analysis. histology for the histological characterization, ten berries from each subsample were randomly chosen, and the protocol by bozzola and russell (1998) for histological observation followed. a berry section was cut from each berry and immediately fixed in 2.5% glutaraldehyde (ted pella inc., redding, ca, usa) diluted with 0.1 mol/l sodium cacodylate buffer solution at ph  7.4 overnight at 4  °c. then, they were incubated for 1 hour at 4 °c in osmium tetroxide 1 % in 0.1 mol/l sodium cacodylate buffer, and then water washed three times. after that, the samples were dehydrated in a graded ethanol series and embedded in an epoxy resin (sigma-aldrich, st. louis, mo, usa). semithin  sections (1 µm) were obtained with an ultrotome v (lkb) 138 ital. j. food sci., vol. 27 2015 ultramicrotome, counterstained with toluidine blue 1  %  and observed with a leica dmr  optical microscope equipped with a camera (leica dfc 480). measurements of skin thickness and cell skin number were made using an imaging software (imagej 1.38; wayne rasband; national institutes of health, usa) considering the epidermis and hypodermis. instrumental skin thickness a ta.xtplus universal testing machine (stable micro systems, godalming, uk) was used, operating in the following conditions (letaief et al., 2008a): 5 kg load cell, p/2 2-mm cylindrical flat probe, hdp/90 platform, test speed 0.2 mm/s, data rate 500 points per second, data acquisition and integration using the exponent software from the same manufacturer. all the analysis were done at 20±2 °c. the probe was calibrated by force and distance before each session, the latter to define the starting point 1 mm above the platform. a pulp-free clean portion of the peeled skin sample was then placed on the hdp/90 platform base, letting it adhere on the platform surface, and a 0.2 mm/s compression movement was applied by the probe. the berry skin thickness (sp sk ) instrumental parameter was defined as the distance (in µm) between the point when the probe touched the skin sample and the platform base. in correctly defining the touch point, it was necessary to include a 0.05 n instrumental trigger to avoid the “tail” effect (letaief et al., 2008a), as described in fig. 1. in order to assess the variability of the instrumental measurements influenced by the analysis position and intra-berry, a preliminary test was carried out. ten berries were randomly taken from all the previously-formed subsamples: these berries were analyzed in fifteen different spots each, equally distributed in the top (close to the peduncle), equatorial (middle), and bottom side of the berry. instrumental berry skin thickness (sp sk ) values were then calculated, the results normalized based on the equatorial position, and the relative standard deviation (%rsd) calculated for the three spots and for the intra-berry measurements variation in the same spot. for the instrumental skin thickness determination, ten berries from each subsample were randomly chosen. the berries were singularly treated, they were peeled and a skin portion from the equatorial berry position analyzed using the aforementioned method, with the thickness calculated as sp sk (µm). statistical analysis statistical analysis was performed using the statistical software package ibm spss statistics (ibm corporation, armonk, ny, us). the tukey-b test at p < 0.05 was used in order to establish statistical differences by one-way analysis of variance (anova). results and conclusions the instrumental skin thickness preliminary test results were shown in table 1. given 100 the equatorial position skin thickness average, fig. 1 typical force-distance curve of berry skin thickness mechanical test, magnified in the y-axis in order to highlight the “tail” effect influence and the need of a trigger threshold. table 1 comparison between instrumental mechanical skin thickness evaluation on top, equatorial and bottom positions of the berry, and mean variation of intra-berry measurements. berry analysis position normalized sp sk a %rsd average intra-berry %rsd top 78.3a 20.1 14.84 equatorial 100.0b 9.9 9.09 bottom 102.3b 17.2 14.07 instrumental berry skin thickness (sp sk ) data is expressed as normalized result (n = 50) with respect to the equatorial side (given as 100). for each measurement the relative standard deviation (%rsd) is reported. intra-berry %rsd calculated as average of the ten %rsd values found analyzing each berry in 5 different spots. asp sk normalized result values are significantly different at p <0.001. different letters in sp sk normalized results mean significance at p < 0.05 (tukey-b test) among berry analysis position. ital. j. food sci., vol. 27 2015 139 the other measures were normalized accordingly, and the relative standard deviation (%rsd) calculated. the bottom section gave similar results compared to the equatorial one, however the relative standard deviation measured is about 75 % higher than that of the latter position. the berry top skin section (close to the peduncle) showed lower sp sk values in relation to the other two sections considered, but the higher relative standard deviation of the group. regarding the intra-berry variation, the equatorial position was found the less variable, that means several measurements of the skin thickness on the same berry gave the more similar results when done on the equatorial position, with respect on measurements done only on the top position, or only on the bottom position. the difference in the berry skin mechanical behavior induced by the analysis position was also found by letaief et al. (2008b), which found significantly different berry skin break force and energy values depending on the puncture position when testing berries of cabernet sauvignon, pinot noir and nebbiolo cultivars. the berry skin break force and energy values in the top position (labeled a3) were found the lower ones in most cases, as found for the instrumental thickness parameter in the present study. this can lead to the hypothesis of a link between these skins mechanical parameters, however no evidence of a meaningful correlation between skin break force and thickness analyzed on the same position (berry lateral side) was found in a previous study conducted on grapes from several cv. mencía vineyards (río segade et al., 2011a). fig. 2 shows a picture of the berry skin section taken with the optical microscope: in the picture there were reported the different tissues that form the skin, epidermis and hypodermis. the outermost two-three cell layers were considered to be the epidermis, while the seven to nine cell layers immediately below the epidermis were considered to be the hypodermis (hardie et al., 2008). immediately below to these layers there were polygonal shape cells that were considered to be the mesocarp (schlosser et al., 2008). the data collected with the histological method is reported in table 2. the measured skin cell layers number ranged between 8 to 11, with the average vineyard values resulted from 8.87 to 9.65, in agreement with the values reported for other varieties (considine and knox, 1979; muganu et al., 2011). the cell layers number showed significant differences between vineyards: a and c showed smaller values (8.87 and 8.95 respectively) compared with b (9.65), while d showed an intermediate average cell number (9.23). also the skin thickness measured from the histological samples showed significant differences between vineyards. the vineyard a showed the thickest skin (173 µm) while the vineyard d showed the thinnest one (152 µm), with b fig. 2 picture of traverse section of corvina berry skin at maturity taken with leica dmr optical microscope. in the picture there are indicated the epidermis (ep), hypodermis (hy), and mesocarp (me). 140 ital. j. food sci., vol. 27 2015 table 2 skin cell layers number and thickness evaluated by histology, and skin thickness by instrumental mechanical properties technique, of the analyzed vineyards (two groups per vineyard). vineyard cell layers number thickness thickness signb [by histology] [by histology, µm] [mechanical as sp sk , μm] a 8.87±0.69a 173±5c 176±8c ns b 9.65±0.65b 163±5b 164±8b ns c 8.95±0.72a 161±5b 160±7b ns d 9.23±0.92ab 152±5a 153±5a ns signa ** *** *** data is expressed as average±standard deviation (n = 20). different letters means significance at p < 0.05 (tukey-b test) among vineyards. a: ** and *** means significance among vineyards at p < 0.01 and 0.001, respectively. b: ns means not significant differences between thickness determinations (in a same vineyard sample). and c showing intermediate values (163 and 161 µm, respectively). the skin thickness estimated with the texture analysis equipment (as sp sk ) showed similar values with respect to those measured on the histological sections. indeed, there was not statistical difference between the values measured using the two different techniques. this was confirmed by the high correlation (r2 = 0.9242) found between the values recorded with the two methods, as shown in the correlation graph in fig. 3a. the obtained values highlighted that there was no correlation (r2  =  0.1391) between the skin thickness and the cell layers number, both analyzed by histology (fig. 3b). this means that the different skin thickness recorded between cv. corvina vineyards was due essentially to the different epidermis and hypodermis cells size. some authors reported that the skin thickness is strictly dependent on the different number of cell layers (considine and knox, 1979; roudot, 2006; hardie et al., 2008; muganu et al., 2011) but they based their observation on different grape varieties with respect to the present study. we can conclude that the comparison between histological observation and texture analysis determination confirmed that the instrumental skin thickness technique by texture analysis give similar values of skin thickness in relation to those obtained by histology. the use of the texture analysis method can speed up the analysis process, minimizing the sample treatment. fig. 3 correlation between skin thickness measured by means of instrumental mechanical properties technique and histology, and between histological cell layers number and skin thickness. each point represents a berry group (n = 10, two groups per vineyard). ital. j. food sci., vol. 27 2015 141 references bozzola j.j. and russell l.d. 1998. electron microscopy: principles and techniques for biologists (second edition). jones and bartlett publishers, sudbary, ma 01776, usa. considine j. and knox r. 1979. 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f., giacosa s., río segade s., cagnasso e. and gerbi v. 2012b. assessment of physicochemical differences in nebbiolo grape berries from different production areas and sorted by flotation. am. j. enol. vitic. 63: 195-204. rolle l., siret r., río segade s., maury c., gerbi v. and jourjon f. 2012c. instrumental texture analysis parameters as markers of table-grape and winegrape quality: a review. am. j. enol. vitic. 63: 11-28. rosenquist j.k. and morrison j.c. 1989. some factors affecting cuticle and wax accumulation on grape berries. am. j. enol. vitic. 40: 241-244. roudot a.c. 2006. some considerations for a theory of plant tissue mechanics. sci. aliments 26: 409-426. schlosser j., olsson n., weis m., reid k., peng f., lund s. and bowen p. 2008. cellular expansion and gene expression in the developing grape (vitis vinifera l.). protoplasma 232: 255-265. paper received july 30, 2014 accepted november 12, 2014 ijfs#1318_bozza ital. j. food sci., vol. 31, 2019 604 paper the impact of lemon juice on the marination of anchovy (engraulis encrasicolus): chemical, microbiological and sensory changes v. šimat*1, a. mićunović1, t. bogdanović2, i. listeš2, i. generalić mekinić3, i. hamed1 and d. skroza3 1university department of marine studies, university of split, r. boškovića 37, 21000 split, croatia 2regional veterinary institute split, croatian veterinary institute, poljička cesta 33, 21000 split, croatia 3department of food technology and biotechnology faculty of chemistry and technology, university of split, r. boškovića 35, 21000 split, croatia *corresponding author: tel.: +38521510192 e-mail address: vida@unist.hr abstract this paper investigates the qualitative changes (physical, biochemical, microbiological and sensory) in anchovy fillets during the maturation, in a marinade made from fresh lemon juice and olive oil with the addition of acetic acid. marination was carried out at 4°c and it required 7 days until sensory acceptability of the product was achieved. at marination end point, a ph value of 4.2, aw of 0.85 and nacl content of 4% were measured for the products. volatile and non-volatile amines were extracted from the marinated anchovy fillets, and the detected concentration implies that the accumulation of these components during processing was weak (<14 mg tvb-n/100 g, <0.5 mg tma/100 g, <30 mg biogenic amines/kg). the lemon juice showed good preservative effect, it reduced the bacterial count in the products, while the addition of 0.5% acetic acid improved the sensory attributes of the product. keywords: marination, anchovies, quality control, tvb-n, tma, biogenic amines, lemon juice ital. j. food sci., vol. 31, 2019 605 1. introduction marinating is one of the oldest chemical preservation methods used for fish. during the process, the preservative effect is achieved by different concentrations of organic acids and salt. this effect is based on reducing the product’s ph value and water activity, and increasing the salt content, resulting in a retardation of bacterial activity and enzymes. marination promotes the structural characteristics of fish flesh, giving the product a characteristic flavour and aroma, and tender texture, but a limited shelf life of 1 to 6 months in refrigerated storage. cold marinades achieve fish ripening in acetic acid and salt solution without any heat treatment. after maturation has been completed, the product is packaged in oil, sauces or a new salt/acid solution with the addition of spices (mclay, 1972). the quality of the final product is directly influenced by the initial quality of the raw material. in promoting the mediterranean diet as a healthy way of living, minimally processed seafood products have become extremely popular. thus, there is a growing interest in marinated fish products, and especially traditional carpaccio-like products (cold-marinated fish in fruit juice citric acid and salt). so far, the time required for the maturation of fish fillets in citric juice has been estimated empirically. depending on the thickness of the raw material used for marination, it is generally considered that thin slices of bigger fishes (such as tuna) are ready for consumption after two hours, while the fillets of smaller fish (anchovy or sardine) need at least two days of maturation in a lemon juice-oil mixture. there is a real lack of information on how the properties of fillets change during maturation in citric juice, which would be useful for the industrial production of carpaccio-like products. in food preparation processes, citric acid has shown a positive effect on the texture and colour of raw materials (topuz et al., 2016). fresh lemon juice appears to be a good natural choice for marinating processes. it contains about 3% sugars, ascorbic acid, minerals such as potassium and phenolics, all of which have health-promoting properties and antioxidant effects (gonzález-molina et al., 2009). the only european representative of the engraulidae family, the anchovy (engraulis encrasicolus, l.), belongs to the group of small pelagic fish caught along the mediterranean shores and widely distributed in the adriatic sea. they are characterized by a gentler texture and lower fat content than sardines (šimat and bogdanović, 2012), which makes them a good raw material for minimally processed fish products. on the other hand, anchovies present a risk of infestation by anisakis spp. (mladineo et al., 2012; šimat et al., 2015); thus, they must be frozen prior to processing. the marinating of thawed fish presents a challenge with respect to fillet texture, juiciness and colour, since the freezing process and storage in a freezer result in denaturation of muscle proteins. anchovy carpaccio is a minimally processed, natural fish product traditionally prepared from fish fillets, marinated in lemon or lime juice and salt, with the addition of oil and spices. during maturation, fillets become glossy white, juicy and tender with a gentle acidic taste. the blood stains on the belly part of the fillet appear on the fillet as a result of the freezing process and ruin the appearance and overall acceptability of the product; therefore, the quality of the raw material is of key importance for the production of high quality products. since lightly preserved fish products, in general, have a low salt content (<6 % nacl w/w) and their ph value usually exceeds 5.0, carpaccio-like products are on the very edge of that definition; they have a low salt content and a ph value below 5. after processing, these products are stored at refrigerated temperatures (4-6°c) and consumed without prior heating. consequently, this research was conducted in order to investigate the optimal duration of maturation as well as qualitative changes (physical, chemical, microbiological and sensory) in anchovy fillets during the maturation process in a marinade made from fresh lemon juice and olive oil. additionally, the effect of the addition of acetic acid (final ital. j. food sci., vol. 31, 2019 606 concentration of 0.5% and 1% in the marinating bath) to the lemon juice and olive oil marinade was investigated. the aim was to determine the time required for completion of the maturation process, and the physical, chemical and microbiological changes underlying the maturation process. the objectives of the maturation process were to obtain sensory acceptability of the product, glassy-white colour and soft fillet texture, characteristic flavour, satisfactory salt content and decrease in the ph and water activity values, and to investigate the preservative effect of lemon juice. 2. material and methods 2.1. raw material preparation and marinating anchovies used for marinating were caught in southern part of the adriatic sea (fao fishing area 37.2.1.), placed on ice onboard, transported to a local factory where they were frozen in a brine-air blast freezer and stored at -20°c for four months. the traditional method of preparing carpaccio marinade was somewhat modified to attain a better quality product. the fish were thawed, beheaded, gutted and filleted, and the fillets transferred to a nacl (200 g/l) solution (ratio 1:1) for 22 minutes to remove excess blood, intestines or scales. the fillets were then washed in icy water and drained. marination was carried out in round polypropylene containers, in three lots. the first lot, 140 g of brined filets, were packed with 70 ml fresh lemon juice and 70 ml olive oil per container. lots two and three were processed in the same way, with the addition of acetic acid to reach a final concentration of 0.5% and 1% in the marinating bath, respectively. the containers were closed, stored at 4±2°c and shaken several times before the first sampling for analyses. preliminary investigation results were used for preparation of the sampling plan; thus, five randomly chosen containers were sampled on days 2 and 7 in order to investigate physical, chemical, microbiological and sensory qualitative changes. before the analyses, the liquid was drained from all the containers and the fillets were homogenized using a laboratory homogenizer (kinematica microtron mb 550, switzerland). the homogenized mass of three containers was used for physical and chemical analyses. the remaining two containers were used for microbiological analysis and sensory assessment. 2.2. chemical composition analyses the chemical composition of anchovy fillets was determined as moisture content by gravimetric method, crude protein (aoac, 2000), crude lipid (bligh and dyer, 1959) and crude ash (aoac, 2000). all analyses were repeated six times and the results were presented on a wet weight basis (percentage). 2.3. physical and chemical analysis for the ph measurements, 10 g of fillets were homogenized with 10 ml distilled water. the ph value of the fish homogenate was measured using a digital iskra ph-meter ma 5705 with a combined glass electrode (iskra model 0101, slovenia). water activity (aw) was determined at room temperature using an aw meter (rotronic ag, hygropalm aw1-set 40, rotronicinstrument corp., basserdorf, germany). total volatile basic nitrogen (tvb-n, mg/100 g) and trimethylamine (tma, mg/100 g) were analysed according to šimat et al. (2009) using a kjeldahl distillation unit (model b-324, büchi, switzerland) and automatic titration (methrom 702 set ⁄ met titrino). the 2-thiobarbituric acid (tba, mg ital. j. food sci., vol. 31, 2019 607 malonaldehyde/kg) assay, as an index of lipid oxidation, was performed according to the procedure of vyncke (1970) and lemon (1975). the total of eight biogenic amines, namely β-phenyletylamine, cadaverine, histamine, putrescine, spermidine, spermine, tryptamine and tyramine, were determined in samples using high-performance liquid chromatography (hplc), according to the method described by eerola et al. (1993) and modified for smaller particle size column (šimat and dalgaard, 2011). the standards of biogenic amines that were used were obtained from sigma-aldrich (st. louis, mo, usa). the hplc analyses were carried out using a classical agilent 1200 series lc system (agilent technologies inc., waldbronn, germany) and the separation was performed on a c18 agilent column (zorbax eclipse xdb 50 × 4.6 mm id, 1.8 μm particle size). all analyses were done in triplicate and the results expressed as mg/kg of the sample. 2.4. microbiological analysis samples and decimal dilutions were prepared according to the method described by özogul et al. (2006). for all microbial counts, 25 g of anchovy fillet was taken and homogenized with 225 ml of buffered peptone water (bpw, biolife italiana). a series of decimal dilutions were obtained and inoculated on appropriate media. total viable count was enumerated on plate count agar (pca, biolife italiana) at 30°c for 24-48 h using the pour plate method. psychrotrophic microorganisms were also enumerated using pca as the medium. plates were incubated at 7°c for 10 days. the lactic acid bacteria count was determined using the pour plate method on de man, rogosa and sharpe agar (biolife, italiana) after incubation at 30°c for 72 hours. enterobacteriaceae were enumerated from violet red bile glucose agar (vrbga, biolife italiana) after incubation at 37°c for 48 hours. the number of colony forming units (cfu) per gram of sample was calculated from the number of colonies obtained on selected plates retained from three successive dilutions and the results were presented as log cfu/g. each dilution was poured in triplicate. 2.5. sensory analysis the sensory attributes of the marinated fish fillets were evaluated by a panel of 8 trained panellists according to the method described by sallam et al. (2007). the panellists, composed of msc students, university staff and fish industry technologists, were educated on specific positive and negative sensory aspects of cold marinated fish. every day, during seven evaluation days, fillet samples of two different products were served to each panellist in covered porcelain dishes coded with 3-digit randomly chosen numbers. an eight-point hedonic scoring scale was used for evaluation of the appearance (8 = extremely acceptable to 1 = extremely unacceptable), colour (8 = faint pink to 1 = deep brown), texture (8 = firm and consistent to 1 = extremely soft or extremely hard), odour (8 = characteristic saury odour to 1 = extreme off-odour), rancidity (8 = no rancidity to 1 = extremely rancid), juiciness and tenderness (8 = extremely juicy/tender to 1 = extremely dry/tough), sour and salty taste (8 = not sour/salty to 1 = extremely sour/salty), as well as flavour and aftertaste (8 = characteristic saury flavour to 1 = extreme off-flavour). a ninepoint hedonic scale (9 = like extremely; 8 = like very much; 7 = like moderately; 6 = like slightly; 5 = neither like nor dislike; 4 = dislike slightly; 3 = dislike moderately; 2 = dislike very much; 1 = dislike extremely) was used for evaluation of overall acceptability. the maturation process was performed at refrigerated temperature (4±2°c) and it was considered complete when sensory acceptability of the product was satisfactory (glassywhite colour, soft fillet texture, favourable effect in terms of odour and flavour). this was ital. j. food sci., vol. 31, 2019 608 achieved after seven days of maturation (preliminary study, data not shown). thus, the sensory analysis 2-day data (generally accepted end of maturation) and 7-day data (end of maturation based on preliminary study) are presented in this paper. 2.6. statistical analyses in order to establish significant differences between data obtained for different marinating baths (with and without acetic acid), one-way variance analysis (anova) was performed using the statgraphics centurion xvi 16.1 (statpoint technologies, inc.) statistical package, and fisher's least significant difference (lsd) procedure at p value of <0.05. 3. results and discussion taking into account the economic value of anchovy and increasing production of lightly preserved and uncooked fish products, knowledge on quality changes during processing will ensure standardization of the production processes as well as general safety requirements for consumers. in marinated products, a synergic effect of salt and acid content ensures specific product characteristics, such as taste, appearance or texture, but also environmental conditions that delay the growth of microorganisms causing spoilage. the addition of 0.3% acetic acid is considered to have a bactericidal effect, especially against gram-negative bacteria (ray and bhunia, 2008). carpaccio products are prepared from raw fish fillets, thinly sliced and marinated in lemon juice and olive oil. anchovy is a small pelagic fish whose fillets are thin and suitable for this type of product; however, the european anchovy in the adriatic sea represents a moderately highlyinfected paratenic host of anisakis pegreffii (mladineo et al., 2012; šimat et al., 2015) and, therefore, freezing of the fish before the cold marinade processing is obligatory. during freezing and storage, fish changes such as protein denaturation and lipid oxidation may influence the marinating process, which means that the overall acceptability of such products is usually lower, the colour and texture being less appealing to consumers. table 1 shows the moisture, protein, lipid and ash content of thawed, brined and marinated anchovy fillets after two and seven days of maturation at 4±2°c. the moisture content in thawed fillet was lower than in raw fish (0.78%, data not shown) due to the combined brine/air blast system used for freezing the fish. it is an individually quick freezing technology that uses a brine solution chilled at -21°c for freezing and air blast freezer for glaze fixation and final freezing of the product. small pelagic fish such as anchovy reach a temperature of -18°c in 8-9 minutes but this results in lower moisture and higher nacl content (0.23% in raw material) than in fresh samples. the lipid content of anchovies used in this study was lower than in previous reports for this species (duyar and eke, 2009; kocatepe and turan, 2012; özogul et al., 2017). atlantic or black sea representatives of this species, in general, have different proximate composition and higher lipid content than anchovies caught in the adriatic sea (šimat and bogdanović, 2012). the brining of the fillets also decreased moisture content and this remained relatively constant during the marinating process. the statistical analysis attempted to determine the interaction of the investigated parameters, i.e. whether the chemical composition was dependent on the particular stage of production as well as the acetic acid addition at the beginning (day 2) and at the end of the maturation process (day 7). the brining of the fish before the marinating process had a statistically significant effect (p<0.05) on the chemical composition of the fillets (table 1). both brining and marinating significantly affected the lipid content (table 1) when compared to thawed fillets. according to the statistical analysis, the difference between three groups of marinated ital. j. food sci., vol. 31, 2019 609 products was found to be significant (p < 0.05). the protein and ash content was higher in marinated samples, but did not change significantly during the marinating process. for fillet brining, strong brine (200 g/l) was used to avoid swelling of the flesh (weak brine) or surface crystallisation of filets (if saturated brines are used). the marinating process also significantly influenced the changes of all the investigated chemical parameters (table 2). table 2 shows changes in the physical and chemical parameters of anchovy fillets during marination in lemon juice, and lemon juice with added acetic acid (0.5 and 1%). the results allow concluding that the brining step ensured satisfactory content of nacl in the fillets (4.12%) while lemon juice ensured ph values of 4.2 in all marinated products. when the brining step (dipping the fillets in nacl solution for 22 minutes) was applied to raw fresh fillets, the salt content was higher than 6% (data not shown), which makes the fillets too salty in sensory terms. the brining step made the thawed fillets whiter, hardened the consistency of the fillets by drawing some water out of them and achieved appropriate salinity of the product (tables 1, 2 and 4). the addition of acetic acid did not affect the nacl content at the end of maturation (7 days). in the literature, significantly higher salt contents were found in marinated fish products. kilinc and cakli (2005) marinated sardine (sardina pilchardus) in a solution containing 7% acetic acid and 14% salt. marination was preceded by brining of the fish in 10% aqueous salt solution. the authors reported a statistically significant change in the salt content during marination and storage of marinated sardines. on the first day of maturation, the salt content was 4.93% and after 22 days 7.12%. the marinating process used for engraulis anchoitae (brined in 10% nacl, marinated in a solution containing 3% acetic acid and 10% salt) resulted in 4.97% nacl in the fish meat on the first day of storage (yeannes and casales, 2008). citric acid has shown to be an excellent choice of natural additive for fish maturation and preservation as it controls the acidity of the product, softens the consistency of muscle, gives it a lighter colour and sour taste, and enhances the impact of conservation. the ph value of the prepared lemon juice marinade was 2.35±0.15. the addition of acetic acid in the proposed concentrations did not have any effect on the ph of the marinade or the fillets during maturation. the combined effect of aw (< 0.9) and ph (4.2) in marinated anchovies places these products in the inhibition zone of growth of pathogenic bacteria and many of the deteriorant microorganisms (cabrer et al. 2002). low ph values of fish marinated in acetic acid and salt solutions are recorded in the scientific literature. the ph values of 3.9 were recorded in anchovies marinated with acetic acid (concentration of 50100 g/l) in a study by poligne and colligan (2000). on the 20th day of storage, the ph value reached its maximum (4.21) and thereafter remained constant for 60 days. gökoğlu et al. (2009) recorded decreasing ph in fresh anchovy, from 6.3 to 4.57, during marination in 30 g acetic acid/l and 150 g nacl/l. kilnic and cakli (2004) marinated sardine (ph = 6.72) in a solution with a ph value of 3.86, while in the marinated fillet they achieved a ph value of 4.23 at the beginning and 4.11 at the end of maturation (22 days). during 7 days of maturation in a bath prepared from 30 g acetic acid/l, fuselli et al. (1994) obtained a ph value of 4.2 for e. anchoita fillets. freshness, deterioration of freshness, and spoilage of seafood products can be monitored over time through the dynamics and speed of changes in biochemical parameters (ólafsdóttir et al., 1997) by numerous biochemical tests that have been developed. tba has been used as an indicator of lipid oxidation of fish through the determination of secondary lipid oxidation products generated by hydroperoxide decomposition, while tvb-n and tma values are widely used as criteria of freshness and degree of spoilage of fish muscle (ólafsdóttir et al., 1997). ital. j. food sci., vol. 31, 2019 610 table 1. changes of proximate composition of anchovy (engraulis encrasicolus) fillet during marination in a lemon juice/olive oil mixture, and a lemon juice/olive oil mixture with the addition of acetic acid to reach a final concentration of 0.5% and 1% in the marinating bath (n=6). thawed fillet brined fillet marinated fillet (day 2) marinated fillet (day 7) marinated fillet + 0.5% acetic acid (day 2) marinated fillet + 0.5% acetic acid (day 7) marinated fillet + 1% acetic acid (day 2) marinated fillet + 1% acetic acid (day 7) % moisture 74.88±0.26 a 70.05±0.34 b 70.07±0.34 b 69.68±0.24 c 70.75±0.18 b 69.19±0.36 c 70.75±0.11 b 69.42±0.14 c % protein 20.11±0.11 a 20.89±0.12 a 23.32±0.06 b 23.14±0.10 b 23.47±0.12 b 23.41±0.21 b 21.47±0.12 c 21.49±0.18 c % lipid 2.79±0.14 a 2.32±0.12 b 1.68±0.10 c 1.69±0.06 c 1.54±0.05d 1.52±0.06 d 1.74±0.05 e 1.71±0.06 e % ash 2.31±0.01 a 4.62±0.06 b 4.07±0.03 c 4.24±0.02 d 3.94±0.03 c 4.18±0.02 d 3.35 ±0.03 e 4.47±0.02 b a-emean value±standard deviation in the same raw followed by different superscript are significantly different (p<0.05). table 2. changes in the physical and chemical parameters of anchovy (engraulis encrasicolus) fillet during marination in a lemon juice/olive oil mixture, and a lemon juice/olive oil mixture with the addition of acetic acid to reach a final concentration of 0.5% and 1% in the marinating bath (n=6). chemical analyses thawed fillet brined fillet marinated fillet (day 2) marinated fillet (day 7) marinated fillet + 0.5% acetic acid (day 2) marinated fillet + 0.5% acetic acid (day 7) marinated fillet + 1% acetic acid (day 2) marinated fillet + 1% acetic acid (day 7) ph 6.22±0.02 a 5.67±0.03 b 4.23±0.04 c 4.22±0.11 c 4.24±0.04 c 4.21±0.06 c 4.21±0.04 c 4.23±0.08 c aw 0.88±0.01 a 0.77±0.01 b 0.85±0.04 c 0.85±0.07 c 0.85±0.08 c 0.86±0.06 c 0.84±0.04 c 0.86±0.02 c nacl (%) 1.34±0.12 a 4.12±0.21 b 4.02±0.2 c 4.10±0.14 b 3.87±0.12 d 4.02±0.14 c 3.61±0.12 e 4.27±0.26 f tba (mg malonaldehyde/kg) 2.04±0.14 a 2.31±0.18 b 3.67±0.21 c 3.37±0.17 d 4.06±0.21 e 3.65±0.12 c 2.94±0.14 e 2.10±0.19 a tvb-n (mg/100 g) 12.41±0.21 a 14.04±0.36 b 10.35±0.32 c 10.47±0.24 c 8.05±0.26 d 9.59±0.32 e 8.24±0.16 d 8.75±0.12 d tma(mg/100 g) 0.36±0.04 a 0.32±0.04 a 0.29±0.02 b 0.27±0.04 b 0.25±0.08 c 0.22±0.05 d 0.24±0.08 c 0.21±0.05 d a-f mean value±standard deviation in the same raw followed by different superscript are significantly different (p<0.05). ital. j. food sci., vol. 31, 2019 611 changes in biochemical parameters of anchovy fillet during marination in lemon juice, and lemon juice with the addition of 0.5 and 1% acetic acid are shown in table 2. regarding the tba test, quality fish products are limited to 5 mg malonaldehyde (mda)/kg, while the fish is considered safe for consummation up to a maximum level of 8 mg mda/kg (sallam et al., 2007). the tba values of thawed and brined fillets were low, <2.5 mg mda/kg indicating good quality of the raw material. both brining and marination processes significantly (p<0.05) increased tba (>3 mg mda/kg) values, while over 7 days of maturation these values were slightly lower. the marination bath was prepared using fresh lemon juice and olive oil, which might influence the tba values in marinated fillets. the data suggest that the tba values of marinated fish are within the good quality limits after 7 days of maturation. the tvb-n value is related to species, catching season and region, age and sex of the fish (kilinc and cakli, 2004). according to huss (1988), the tvb-n value in fresh fish is typically between 5 and 20 mg/100 g of flesh, whereas 30 to 35 mg/100 g is considered a limit of acceptability. in our study, the tvb-n value of thawed anchovies was 12.41 mg/100 g, while data reported in the literature ranged from 7.32 to 12.77 mg/100 g (pons-sanchez-cascado et al., 2005b; olgunoğlu et al., 2009; özogul et al., 2010; topuz et al., 2016). after the brining step, the tvb-n value increased to 14.04 mg/100 g; however, two days in the lemon juice/olive oil mixture resulted in a significant reduction (p<0.05) of tvb-n values. reduction of tvb-n through the marinating process has been reported in the literature and can be attributed to the action of acid and salt whereby the tvb-n components are extracted from marinated filets. kilinc and cakli (2004) reported a decrease in the amount of tvb, from 10.24 to 6.53 mg/100 g, during marination of sardine (s. pilchardus) in 7% acetic acid and 14% salt bath. aksu et al. (1997) report the same for anchovy; and the value of tvb decreased from 8.7 in the raw material to 7.41 mg/100 g in the final product (6% oacetic acid, 14% nacl). pons-sánchezcascado et al. (2005a) detected a decrease of tvb-n, from 7.32 to 6.04 mg/100 g, during marination of anchovy in a wine vinegar marinade. in this study, lemon juice appeared to have a strong extractive effect on tvb-n components, which can be attributed to the presence of citric acid. during a shrimp marination process, a marination bath containing citric acid (2%) and salt (4%) resulted in a lower tvb-n value, from the initial 27.5 to as low as 7 mg/100 g shrimp flesh (cadun et al., 2008). a similar lowering effect of lemon juice was observed for tma, a non-protein fraction found in marine fish produced from the reduction of trimethylamine oxide by bacterial activity. the tma values observed in thawed and brined fillet (0.36 and 0.32 mg/100 g) were significantly reduced during maturation (table 2). since tma values in fresh fish were around 1 mg/100 g, and the limit of acceptability was found to be 5 mg/100 g (huss, 1988), the quality of the investigated fillets, in terms of tma, was good and remained good during the entire maturation period. the range of tma for small pelagic fish ranges from 0.49 to 3.21 mg/100 g (pons-sánchez-cascado et al., 2005b; kilinc and cakli, 2005; gökoğlu et al., 2009). the content of tvb-n and tma were not analysed in the marinade bath during maturation, but based on previous research conducted by ponssánchez-cascado et al. (2005a). it can be assumed that lemon juice marinade has a strong extraction effect on the tvb-n and tma components of anchovy fillets and, considering the low initial values, high levels should not be expected in such products. during the storage of marinated sardines in marinating baths with the addition of 2 and 4% acetic acid and 10% nacl at 4°c, gökoğlu et al. (2004) reported a statistically significant increase in the amount of tma in the flesh of sardines; however, in both types of baths, this amount did not exceed 1.7 mg/100 g after 150 days of storage. similarly, tma did not exceed 2 mg/100 g in marinated anchovies (30 g/l acetic acid and 150 g/l salt) after several months of storage of the marinated anchovies in oil and tomato sauce at ital. j. food sci., vol. 31, 2019 612 a temperature of 4°c (gökoğlu et al., 2009). kilinc and cakli (2004) recorded values lower than 2 mg tma/100 g in marinated sardine flesh (7% acetic acid and 14% salt) during the marinating process (22 days). pons-sánchez-cascado et al. (2005a) recorded a slight increase in the amount of tma after 3 months of storage of cold marinated anchovy in wine vinegar, but these quantities did not exceed 1 mg tma/100 g of flesh. the presence of biogenic amines (bas) in seafood is involved in various toxicological reactions and can constitute a risk to consumer health. the accumulation of high levels of bas is associated with a relevant growth (>7 log cfu/g) of decarboxylating microorganisms (gardini et al., 2016) and indicates a need for better hygiene and process control during production. the decarboxylation process can be catalyzed by naturally occurring endogenous amino acid decarboxylases present in animal or plant cells and by exogenous enzymes produced by various microorganisms under favourable conditions (halász et al., 1994). there is no legal regulation on the ba content of foods (besides histamine); however, a profile of ba in foods provides a better overview of the quality of certain products. the accumulation of bas during brining and marination of anchovy fillets is shown in fig. 1. out of eight bas (β-phenyletylamine, cadaverine, histamine, putrescine, spermidine, spermine, tryptamine and tyramine) determined in this work, only cadaverine, spermine and tyramine were detected in thawed and marinated anchovy fillets. a total of 20.62 mg ba/kg was observed in thawed fillets, which corresponds to the quantities of bas found in fresh/thawed anchovy reported in the literature (veciana-nogués et al., 1996; pons-sánchez-cascado et al., 2005a, özogul et al., 2017). after the brining process, the cadaverine and spermine values remained unchanged while tyramine concentration increased from 4.32 to 14.86 mg/kg. this resulted in significantly higher (p<0.05) ba content (30.58 mg/kg). the lowest content of total ba during the marinating process was observed for the lemon juice/olive oil mixture, with 0.5% acetic acid, and was 15.22 mg/kg. spermine was detected in small amounts (4-6 mg/kg) and its concentration remained unchanged during maturation. together with spermidine (undetected in marinated filets), spermine is not related to bacterial spoilage but rather a physiological ba needed for cell growth (veciananogués et al., 1996). the increased levels of tyramine in anchovies during the marination process suggest that some enzymatic processes occur during lemon juice/olive oil marination that supports ba formation. in marinades, amino acid decarboxylation by bacterial enzymes is limited by the ph value, nacl content and aw value, which directly affect the growth of bacteria (pons-snáchez-cascado et al., 2005b). furthermore, acidic conditions increase the activity of cathepsin, which results in an increase of free amino acids, the precursors for ba formation (meyer, 1965; pons-sánchezcascado et al., 2005b). tyramine is often reported as dominant ba in anchovy products, both marinated and salt-ripened (veciana-nogués et al., 1996; pons-sánchezcascado et al., 2005a) but also in fresh anchovies during storage (özogul et al., 2017). tyramine production is generally related to lactic acid bacteria and orally administered tyramine (concentrations over 125 mg/kg) can be potentially toxic (ladero et al., 2010). no other bas were detected in marinated fillets; however, the ba content associated with a lemon juice/olive oil marinating mixture was not analysed. during production of vinegar-marinated anchovies, pons-sánchez-cascado et al. (2005a) recorded a total of 9.80 mg/kg of bas per kilogram of raw material (not including agmatine), and 35.44 mg/kg after 3 months of storage. the authors did not find any β-phenylethylamine or tryptamine in the raw material or marinated product. they concluded that vinegar acts as an extractive solvent of biogenic amines from fish, and that the levels of some bas (cadaverine and putrescine) decreased in fish flesh during the marinating process in tandem with their increased levels in vinegar. according to veciana-nogués et al. ital. j. food sci., vol. 31, 2019 613 (1996), β-phenylethylamine and tryptamine are present only in fish with a high content of other bas. in general, enterobacteriaceae and lactic acid bacteria are associated with the accumulation of the main biogenic amines: histamine, cadaverine, putrescine and tyramine in seafood products (özogul et al., 2017). the low tma values and ba accumulation data obtained through microbiological analyses are given in table 3. the lemon juice/olive oil mixture had an inhibitory effect on total viable bacteria count, psychrophilic bacteria count and lactic acid bacteria count. the addition of acetic acid inhibited the growth of total viable count and psychrophilic bacteria at first two days of maturation, but by the end of maturation the investigated bacteria adapted to the new conditions and their count increased. no enterobacteriaceae, which are primarily responsible for cadaverine production (marino et al., 2000; özogul et al., 2017), were detected in the carpaccio products, which corresponds to low ba production in marinated anchovy fillets. total or partial inhibition of microorganisms by the application of a marinating process has been recorded previously by fuselli et al. (1994) in marinated anchovy (e. anchoita), with 3% acetic acid and 10% salt; aksu et al. (1997) in marinated anchovy e. encrasicolus (6% acetic acid and 16% salt); kilnic and cakli (2004) in sardine marinade (7% acetic acid and 14% salt); olgunoğlu et al. (2009) in anchovy e. encrasicolus (4.5% alcoholic vinegar, 10% salt and 0.2% citric acid). figure 1. biogenic amine formation in anchovy (engraulis encrasicolus) fillet during marination in a lemon juice/olive oil mixture, and a lemon juice/olive oil mixture with the addition of acetic acid to reach a final concentration of 0.5% and 1% in the marinating bath (n=3). in general, the marinating process does not completely inactivate bacteria. they are able to continue to grow, faster or slower depending on their ability to adapt to the new media surrounding them (fuselli et al., 1994). resistance of microorganisms to the acidic environment is variable and depends on their ability to adapt to new conditions. according to ray and bhunia (2008), brief exposure of microorganisms under suboptimal conditions triggers their cellular mechanisms and allows them to be more resistant and better adapted to harsh environments. the authors state that acid tolerant bacteria may, by exposure to slightly acidic conditions (ph 5.0-5.8), exhibit resistance to survival at ph 2.4-4.0. the results of sensory evaluation of marinated anchovies are given in table 4. ital. j. food sci., vol. 31, 2019 614 table 3. changes in the microbiological count of anchovy (engraulis encrasicolus) fillet during marination in a lemon juice/olive oil mixture, and a lemon juice/olive oil mixture with the addition of acetic acid to reach a final concentration of 0.5% and 1% in the marinating bath (n=3). microbiological analyses (log cfu/g) thawed fillet brined fillet marinated fillet (day 2) marinated fillet (day 7) marinated fillet + 0.5% acetic acid (day 2) marinated fillet + 0.5% acetic acid (day 7) marinated fillet + 1% acetic acid (day 2) marinated fillet + 1% acetic acid (day 7) total viable count 1.13±0.62 nd 0.91±0.24 0.26±0.14 0.34±0.11 0.42±0.18 <10-1 0.28±0.09 psychrophilic bacteria 1.16±0.14 nd 0.56±0.11 <10-1 <10-1 0.94±0.09 <10-1 0.56±0.12 lactic acid bacteria 0.56±0.02 nd 0.65±0.28 <10-1 0.62±0.12 <10-1 0.73±0.12 <10-1 enterobacteriaceae <10-1 nd <10-1 <10-1 <10-1 <10-1 <10-1 <10-1 mean value±standard deviation, nd – not determined. table 4. results of the sensory analysis of anchovy (engraulis encrasicolus) fillet during marination in a lemon juice/olive oil mixture, and a lemon juice/olive oil mixture with the addition of 0.5 and 1% acetic acid (% on a wet weight basis) (n=6). sensory attribute marinated fillet (day 2) marinated fillet (day 7) marinated fillet + 0.5% acetic acid (day 2) marinated fillet + 0.5% acetic acid (day 7) marinated fillet + 1% acetic acid (day 2) marinated fillet + 1% acetic acid (day 7) appearance 7.87±0.92 a 7.90±0.46 a 7.90±0.62 a 7.98±0.08 b 7.76±0.91 c 7.90±0.85 a colour 6.90±1.08 a 7.47±0.88 b 6.73±0.81 c 7.93±0.17 d 6.87±0.74 a 7.83±0.65 d texture 7.92±0.94 a 7.93±0.68 a 7.53±0.99 b 8.00±0.08 c 8.00±0.02 c 7.47±0.62 b odour 7.41±0.94 a 7.94±0.68 b 7.62±0.74 c 7.81±0.56 d 7.96±0.99 b 7.38±0.87 a rancidity 6.97±1.12 a 7.42±0.74 b 7.24±0.42 b 7.45±0.99 b 7.79±1.21 c 7.45±0.91 b juiciness 7.92±0.81 a 7.93±1.18 a 7.53±0.60 b 7.98±0.22 c 8.00±0.69 c 7.47±0.88 b sour and salty taste 6.85±0.99 a 7.38±1.14 b 6.91±0.62 a 7.63±0.48 c 7.85±0.87 d 6.35±0.63 e flavour and aftertaste 5.61±0.88 a 6.27±0.97 b 5.45±0.71 c 7.81±0.87 d 7.72±0.49 d 6.48±0.47 a overall acceptability (scale from 1-9) 7.31±1.12 a 8.54±1.12 b 7.24±0.94 a 8.94±0.66 c 7.90±0.78 d 7.35±0.49 a a-emean value±standard deviation in the same raw followed by different superscript are significantly different (p<0.05). ital. j. food sci., vol. 31, 2019 615 based on the results of sensory analysis, the time required for maturation of anchovy fillets in the lemon/olive oil mixture was 7 days at 4°c. depending on the acid and nacl content, maturation temperature, bath:fish ratio etc., time needed for complete maturation of anchovy fillets ranges from 22 days at 4°c (pons-sánchez-cascado et al. (2005a) to 9 days at 20°c (cabrer et al., 2002). in general, better quality of fish is achieved when maturation takes place at lower temperatures over a longer period. results for texture, juiciness and odour obtained on the second day of maturation in the lemon/olive oil mixture were high (7.9) and did not change during 7 days. however, products containing acetic acid showed improvement of these attributes over maturation. on day 2, all samples had a fishy (raw) flavour and a strong aftertaste and thus obtained lower grades from the panellists. also, the colour was not glossy white, but yellowish and so colour grades were lower as well. after 7 days, the sensory acceptability of the product was satisfactory and grades were high for all investigated attributes. glassy-white opaque colour, soft fillet texture, favourable effect in terms of odour and flavour were obtained. at this point, all assessed attributes (with the exception of odour) and overall acceptability of the product were the highest for the product in the lemon/olive oil mixture, with 0.5% acetic acid. the addition of 1% acetic acid resulted in an acidic aftertaste that covered the flavour and good aroma, thus changing the odour of the product and resulting in lower grades. to the best to our knowledge, this paper presents the first results on the quality changes (physical, chemical, microbiological and sensory) of anchovy fillets during maturation in a lemon juice and olive oil marinade. the described process, consisting in the brining of fillets in a 200 g/l nacl solution and marination of fillets in a lemon juice and olive oil mixture (50:50) over 7 days, achieved glassy-white colour and soft fillet texture, characteristic flavour and satisfactory salt content. the process resulted in a decrease of ph and water activity and good chemical characteristics of the final product. low amine accumulation led us to assume that lemon juice has an extracting effect on volatile and non-volatile amines from anchovy fillets. therefore, their role as an index of freshness/quality for carpaccio-like products is questionable. further studies should include an analysis of the marination bath. moreover, marination in lemon juice showed good preservative effects (total viable bacteria, psychrophilic bacteria and lactic acid bacteria count were reduced). the addition of 0.5% acetic acid improved the overall acceptability of the product. the results of this study could be of interest to the fish industry and contribute and enrich the offer of minimally processed seafood on the market. acknowledgments this work was fully supported by the croatian science foundation, under project ip-2014-09-6897. the authors wish to thank canicula d.o.o., gizdavac, croatia for their support during this investigation. references aksu h. erkan n. colak h. varlik c. gokoglu n. and ugur m. 1997. some changes in anchovy marinades during prodution in different acid – salt concentration and determination of shelf life. 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martínez-cerezo et al., 2005a; santos et al., 2007; teixeira et al., 2005). the suckling lamb meat produced under selected conditions in castilla y león region (spain) was granted by the eu’s protected geographical indication (pgi) “lechazo de castilla y león” (commission regulation eec no. 2107/99). the pgi lambs must belong to one of the authorized breeds – churra, castellana or ojalada – or their crosses, have to be fed on maternal milk and slaughtered under 35 d old. in 2017 the pgi-protected lambs were approximately 300 thousand, being churra the predominant breed (sánchez, 2018). suckling lamb meat is usually stored under aerobic refrigerated conditions in retail meat premises during a short period of time before being sold to the consumers (marm, 2010). the pgi appellation allows aerobic refrigerated storage for a maximum of 8 d, as well as other storage methods in order to extend the meat’s shelf-life as long as storage does not negatively affect the meat colour and edible quality. during aerobic refrigerated storage of carcasses or meat joints ageing takes place resulting in more tenderness, although meat becomes unacceptable to the consumer over time due to microbial spoilage (bellés et al., 2017). edible quality changes in suckling lamb joints during aerobic refrigerated storage have been studied (martínez-cerezo et al., 2005a, 2005b; vieira and fernández, 2014). these studies reported that 4-5-d storage periods resulted in a decrease in meat hardness and fibrosity and that 4 d could be enough time to obtain a high-quality meat. retail premises are increasingly offering vacuum packaged suckling lamb joints, or chops obtained from previously vacuum packaged joints. vacuum packaging provides an anoxic environment between the meat surface and the packaging retarding microbial spoilage. rubio et al. (2016) studied the shelf-life of vacuum packaged suckling lamb forelegs stored at 4 ºc and found a suitable storage period of up to 16 d. ageing is not stopped by vacuum storage during the retail period (bellés et al., 2017). freezing prolongs meat shelf-life; however, freezing and thawing can affect juiciness, flavour and colour to a level that depends on the meat characteristics and the freezing, frozen storage and thawing conditions (leygonie et al., 2012). no studies have addressed the effect of frozen storage on the quality of suckling lamb meat after thawing; however, there are a number of studies on its effect on the meat of older lambs (3-4 months old), concluding that thawed meat showed small differences in quality as compared to fresh meat, e.g. lower juiciness (bueno et al., 2013; muela et al., 2016, 2012). the storage-related changes in suckling lamb meat have been scarcely studied. the aim of the present study is thus to explore the effect of three different storage procedures (7-d aerobic refrigerated storage, 3-week refrigerated storage under vacuum, or 3-month frozen storage) on the colour and water holding capacity of suckling lamb meat, as well as on the texture and oxidative stability of this meat after cooking. 2. material and methods 2.1. meat samples and storage thirty-two legs from churra pgi “lechazo de castilla y león” lambs were sampled. according to the pgi specifications, the lambs were fed on ewe`s milk, slaughtered between 20 and 35 d of age, and their carcasses weighed between 4.5 to 7.0 kg. legs were purchased from the local market on 8 different days, up to 6 legs per day, during a 1 ital. j. food sci., vol. 31, 2019 429 month period. all the sampled legs had to come from a different lamb and the postmortem time at purchasing had to be one day. twenty-four legs had to come from males and eight from females in order to resemble the distribution of gender in the market. after sampling all legs were carried to the lab at 0-6 ºc, wrapped with a cling film (polyvinyl chloride, pvc; oxygen permeability of 580 ml m−2 h−1), and stored at 3 ºc until the following day. afterwards, the covering film, the tail, and the epiploic fat adhered on the leg surface were removed and the legs were weighed. the legs, weighing 785 g±184 g (standard deviation) were randomly assigned to one of the following storage groups, (i) non-stored (ns), with no further storage; (ii) aerobic refrigerated storage (ars) at 3 ºc for 7 d with the legs wrapped with pvc cling film; (iii) vacuum-packaged refrigerated storage (vrs) at 3 ºc for 21 d using a 150-µm film (polyamide/polyethylene 30/120; oxygen permeability of 1.25 ml m-2 h-1); and (iv) frozen storage (fs), freezing the legs wrapped with pvc cling film in a freezing tunnel (-25 ºc for 60 min) and then stored at -18 ºc for 90 d followed by a 12-h (3 ºc) thawing period. the proportion of male-female samples in the assignment was balanced to adjust for the potential confounding effect of sex (8 legs per group with 2 out of them being from female lambs), which could be expected to be of little or no significant (miguélez et al., 2008). the ars, vrs and fs groups would be representative, respectively, for whole lamb joints stored in a cool room, packaged under vacuum and frozen, for extending shelf-life. the ns group would serve as a control to compare the quality changes due to storage method. 2.2. sample preparation and meat analysis 2.2.1 raw meat after the storage, the covering film was removed from the legs, the legs were weighed, and ph was measured in the semimembranosus muscle using a 52-32 puncture ph electrode (crison instruments, barcelona, spain). colour was measured at three different points on the external surface of gracilis muscle where it showed the thinnest epimysium and lack of visible fat, using a spectrophotometer cm-700d (konica minolta sensing inc., osaka, japan) operating in triplicated with a d65 illuminant, sci mode, 11 mm aperture for illumination and 8 mm for measurement, and 10° visual angle. the results were expressed according to the cie l*a*b* system and the ratio of reflectance at 630 nm and at 580 nm (r630/r580) was calculated as discoloration index in cut meat surfaces exposed to oxygen due to storage, i.e. the lower the ratio, the higher the proportion of metmyoglobin relative to oxymyoglobin plus deoxymyoglobin (amsa, 1995). the top-round-cap muscle group – semimembranosus, adductor and gracilis muscles – was then separated from the ns legs on the following day of purchasing or from the rest of the legs after the storage. this muscle group was sliced transversely into two equal-sized parts and 1 h after slicing the water holding capacity (whc) as assessed in duplicate determining the percentage of expressible juice released by a 300 mg (290-310 mg) meat sample placed on filter paper after a 5 min compression under a 1-kg weight (grau and hamm, 1957), and the colour of the cut surface following the above-mentioned procedure were determined. chroma (the squared root of a*2+b*2) was calculated to better describe the vividness of colour in the meat surface (young et al., 1999). 2.2.2 cooked meat the legs, without the top-round-caps, were roasted (180 ºc) in a forced-air oven rotating them every 15 min until the leg core temperature reached 80 ºc, and then tempered at ital. j. food sci., vol. 31, 2019 430 20 ºc for one h. the biceps femoris and semitendinosus muscles were separated, individually vacuum packaged and frozen at -25 ºc for up to 15 d until further instrumental texture and oxidative stability analyses, which were carried out after a 12-h 3 ºc thawing period. the texture profile analysis (tpa), as predictor for sensory texture (ruiz de huidobro et al., 2005), was determined in three 1.0-cm thick cubes cut from the long head of the biceps femoris muscles after being tempered at 21 °c for 1 h using a ta-xt2i analyser. the operating conditions used were a 25-kg load cell, 0.5 mm/s test speed, 10 mm/s preand post-test speeds, compression percentage of the initial height of 80%, perpendicularly to the muscle fibre, and a 10-s elapse between the 2 compression cycles. oxidative stability was determined in duplicate using the thiobarbituric acid reactive substances (tbars) test (nam and ahn, 2003). for this purpose, the semitendinosus muscle was transversely cut in two similar parts. one was used for immediate analysis and the other was wrapped with pvc cling film, stored for two days at 3 ºc under darkness and then analyzed. results were expressed as the increment in tbars during storage, i.e the difference between tbars before and after the two days storage. 2.3. statistical analysis the experimental data were analyzed using a univariate analysis of variance (anova) with storage method as fixed factor – to assess weight losses the ns group was excluded of the model. when the fixed factor showed significance (p < 0.05) the anova was followed by the least square difference (lsd) test. the spss statistics software (version 23; ibm, somers, ny, usa) was used. 3. results and discussion 3.1. raw meat the mean colour values (± standard deviation) of the gracillis muscle for the 32 legs before storage were as follows: l*, 44.0±2.4; a*, 6.6±1.4; b*, 11.6±2.3, and the mean ph was 5.74±0.08. no differences were found among the legs assigned to the different storage methods (p > 0.05). the highest weight losses due to storage were found in the vrs (table 1). the ars weight losses were comparable to those found in legs submitted to frozen storage and thawing (fs) suggesting the suckling lamb meat to have a good ability to reabsorb water during thawing. in contrast, fs lamb showed the highest expressible juice percentage (table 1), indicating the lowest whc. decreases in meat whc due to frozen storage have been widely recognized and related to disruption of the muscle fibre structure and denaturation of muscle proteins (leygonie et al., 2012). freezing meat at slow freezing rates, i.e. using air temperatures between -20 and -33 ºc (as done in the present study) results in significant water diffusion from the muscle fibres into the intercellular spaces, fibre separation and myofibril damage (grujić et al., 1993). the ph of ns, ars and fs meat did not differ significantly among them; however, a higher ph was found in the vrs meat (table 1). the ph increment during refrigerated storage has been attributed to a switch from a glycolytic to an amino acid-degrading microbial metabolism (nychas et al., 2008). in contrast to the present results, callejascardenas et al. (2014) found no significant change in lamb ph after a similar vacuum storage period. the discrepancy among studies could be attributed to the differences between the lamb ages at slaughter, i.e. 3-months vs 3-weeks in the former and present study, respectively, and explained by a faster microbial growth and/or lower muscle ital. j. food sci., vol. 31, 2019 431 glycogen content in the meat from the younger lambs. in spite of that the ph of thawed meat tends to be lower than prior to freezing (leygonie et al., 2012), as seen in this study and in previous studies on light lamb (muela et al., 2010), frozen storage showed no effect on ph. table 1. effect of storage method on leg weight loss, expressible juice of adductor muscle, ph and colour characteristics# of gracilis muscle surface and adductor muscle cut surface (after 1 h of slicing) in suckling lamb legs. ns (n=8) ars (n=8) vrs (n=8) fs (n=8) sem p-level weight loss (%) 0.85b 1.29a 0.88b 0.136 * expressible juice (%) 19.9b 17.6b 21.4b 26.7a 1.462 ** ph 5.70b 5.75b 5.96a 5.79b 0.017 *** colour of muscle surface l* 42.88a 43.11a 45.23a 39.98b 0.990 ** a* 7.34 8.02 6.76 6.22 0.630 n.s. b* 11.60 10.70 8.76 8.80 1.004 n.s. r630/r580 2.52 a 2.05b 2.43a 1.99b 0.111 ** colour of muscle cut surface l* 40.28a 37.54a 39.06a 33.32b 1.264 ** a* 7.62 9.37 9.20 8.01 0.724 n.s. b* 15.12 17.42 16.05 16.63 0.611 # chroma 16.09 19.81 18.53 18.59 0.785 # #: l*, lightness; a*, redness: b*, yellowness; r630/r580, wavelength reflectance ratio (630 nm and 580 nm wavelengths). ns: non-stored, 2-day post-mortem refrigerated legs. ars: refrigerated legs (2-day post-mortem) wrapped with air-permeable cling film and stored for 7 days at 3 ºc. vrs: refrigerated legs (2-day post-mortem) stored under vacuum for 21 days at 3 ºc. fs: refrigerated legs (2-day post-mortem) wrapped with airpermeable cling film, frozen and stored for 3 months at -18 ºc and then thawed at 3 ºc overnight. sem: standard error of the mean. p-level: n.s.: not significant; *: p < 0.05; **: p < 0.01; ***: p < 0.001. ab: means in the same row showing different superscripts are significantly different (p < 0.05; least significant difference test). colour in the muscle leg surface was affected by storage (table 1). fs resulted in a significant lower l* value as compared to ns, ars and vrs. accordingly, other studies found frozen storage to decrease l* in comparison with not-frozen meat (moore and young, 1991; muela et al., 2010). this difference should be attributed to changes affecting light scattering on the meat surface and could negatively affect the colour preference by lamb consumers, which has been strongly related to l* (callejascárdenas et al., 2014; hopkins, 1996). it might specially affect suckling lamb meat, characterized by a bright white to light pink colour (erasmus et al., 2017). as another adverse effect on leg appearance, freezing tended to produce a more pronounced redblood colour into the superficial large blood vessels visible on the leg internal face (fig. 1). the differences found for r630/r580 ratio suggest that meat discoloration occurred during those storage methods allowing the higher exposure of meat to oxygen, i.e. ars and fs, since the r630/r580 ratio is inversely related to metmyoglobin formation (amsa, 1995) during aerobic storage (mancini and hunt, 2005). discoloration due to oxygen exposure is ital. j. food sci., vol. 31, 2019 432 considered to be a regular phenomenon (mckenna et al., 2005; moore, 1990). a decrease in that ratio was previously observed during a 10-d aerobic refrigerated storage period in suckling lamb chops (mateo et al., 2018). muela et al. (2016) also reported discoloration in the meat of 3-month age lambs after one month of frozen (-18 ºc) storage as detected by a trained sensory panel. figure 1. pairs of photographs from four of the legs used in this study (a to d) just before being freezing (top photo of the pair) and just after thawing (bottom); an arrow and ellipse have been drawn in each photo to locate and highlight the main difference in leg appearance due to frozen storage. the effect of the storage on the colour of cut meat after the 1-hour blooming period was significant for l* and near-to-significant for b* and chroma (table 1). l* value was the lowest in the fs meat confirming the results obtained for the colour in the whole leg muscle surface and suggesting a lower consumer colour acceptance. furthermore, a nearto-significant differences suggest b* higher chrome values in ars as compared to ns meat, which should not be considered an advantage for ars meat because suckling lamb meat is not valuated by a more vivid colour (more chroma) but by a white pale pink colour (erasmus et al., 2017). other studies have reported an increase in b* due to ageing as the most relevant change in the colour of sliced ruminant meat (boakye and mittal, 1996; vieira and fernández, 2014). the latter of them, studying specifically suckling lamb, reported a significant increase in the b* of cut meat surface due to a 5-d dry ageing period. however, none of those studies explained this effect. 3.2. cooked meat table 2 shows the effect of storage on the tpa of cooked lamb. hardness was significantly affected (p < 0.05) by storage, masticability was near-to-significantly affected (p < 0.1) and ital. j. food sci., vol. 31, 2019 433 no effect was detected on elasticity and cohesiveness. significant differences in hardness were found between ns exhibiting the highest values and vrs lamb with the lowest. there is a general agreement that refrigerated aerobic storage tends to decrease the instrumental hardness of lamb (martínez-cerezo et al., 2005b; starkey et al., 2015), with this decrement depending on storage length. the effect of refrigerated storage on suckling lamb hardness has been however scarcely studied. vieira and fernández (2014) reported a decrease in instrumental and sensory hardness in cooked suckling lamb due to a 5-d storage period of carcasses when carcasses were chilled under a conventional regimen (2 ºc for 24 h); however, hardness did not decrease with storage when carcasses were slowly chilled (12 ºc for 7 h and then 2 ºc for 22 h). table 2. effect of storage method on the texture profile analysis (tpa) in suckling lamb cooked meat and the lipid oxidation calculated as increment in thiobarbituric-acid reactive substances during a two-day aerobic refrigerated storage period. ns (n=8) ars (n=8) vrs (n=8) fs (n=8) sem p-level tpa hardness (n) 18.90a 17.06ab 15.35b 16.45ab 8.814 * elasticity 0.45 0.43 0.43 0.43 0.013 n.s. cohesiveness 0.45 0.44 0.43 0.44 0.009 n.s. masticability (n) 3.87 3.24 2.88 3.10 0.264 # lipid oxidation due to storage (∆ mg malonaldehyde/kg of meat) 4.15 3.98 5.48 3.60 0.522 # ns: non-stored, 2-day post-mortem refrigerated legs. ars: refrigerated legs (2-day post-mortem) wrapped with air-permeable cling film and stored for 7 days at 3 ºc. vrs: refrigerated legs (2-day post-mortem) stored under vacuum for 21 days at 3 ºc. fs: refrigerated legs (2-day post-mortem) wrapped with airpermeable cling film, frozen and stored for 3 months at -18 ºc and then thawed at 3 ºc overnight. sem: standard error of the mean. p-level: n.s.: not significant; # p < 0.1; *: p < 0.05. ab: means in the same row showing different superscripts are significantly different (p < 0.05; least significant difference test). results from previous studies seem to agree with the decrease in instrumental hardness resulting from vrs. martínez-cerezo et al. (2005b) found in suckling lamb meat from three different breeds, one of them churra, that a 16-d vacuum storage period significantly decreased the tpa-20%-compression hardness as assessed in raw meat and associated this effect to degradation of muscle structure. furthermore, using the same meat samples, martínez-cerezo et al. (2005a) reported a clear and steady tenderization effect as assessed in cooked meat by a sensory panel. bórnez et al. (2010) also evaluated the effect of a 21-d refrigerated storage of suckling lamb joints under modified atmospheres on the hardness of meat after cooking and observed a decrease in the shear-force. the meat of 1month age suckling lambs, as that in this study, is considered to be tenderer and easier to swallow as compared with meat from weaned older lambs, i.e. 2-month age lambs (gorraiz et al., 2000), and tenderness in suckling lamb seems to be highly valued by consumers (sañudo et al., 2007; vieira and fernández, 2014). notwithstanding, an excess in tenderness might not be desirable. studies are needed to order to establish a lowvalue tenderness threshold for consumers. ital. j. food sci., vol. 31, 2019 434 in partial agreement with our results, no significant effect of a 3-month -18 ºc storage was found on the sensory tenderness of meat from lambs weighing twice as much as those of this study (bueno et al., 2013; muela et al., 2012). however, frozen meat, in contrast with non-frozen meat, can show similar or lower instrumental hardness and, at the same time, higher toughness as perceived by consumer panels (leygonie et al., 2012). this discrepancy between instrumental and sensory results has been attributed to the lower juiciness in thawed meat after cooking together with the positive relationship between sensory juiciness and tenderness. the effect of frozen storage on the sensory tenderness of suckling lamb meat deserves further study. the increment in tbars value during the 2-d aerobic storage period in cooked meat was near-to-significantly affected by the previous storage (p < 0.1; table 2). the mean tbars values just after cooking were 1.6±0.9 mg of malonaldehyde/kg of cooked meat, with no differences between treatments (p = 0.610). the value for the tbars increment in vrs cooked meat was higher than those from the other storage methods. no studies have been found on the oxidative stability of cooked suckling lamb to compare with our results. in light lamb raw meat, neither an up-to-6-month frozen-storage of meat from 3-4-month age lambs (muela et al., 2010) nor a 18-d vacuum refrigerated storage of suckling lamb (rubio et al., 2016) resulted in increased tbars values. a tbars value of 2 mg of malonaldehyde/kg was suggested as the threshold for raw beef, just before being cooked, over, which rancidity flavour can be perceived by consumers (campo et al., 2006). however, the application of this threshold to the present study should be not reliable because the tbars thresholds for warmer-over-flavour detection appears to depend on different factors such as species, animal age, or whether the meat is raw or cooked (fernández et al., 1997). studies should be done to determine the tbars levels in cooked suckling lamb above which oxidized flavours are detectable. 4. conclusions the use of three commonly used methods for increasing the shelf-life of churra suckling lamb joints could diminish the meat acceptability with regard to non-stored meat. aerobic storage for 7 d can result in discoloration at the joint muscle surface and increased colour intensity in the surface of fresh cut chops; therefore, it seems to be not advisable to use longer aerobic storage periods to that already established in the pgi’s standard (8 d). vacuum storage (21 d) would result in increased meat ph and softer meat, and frozen storage would give muscle surface discoloration in thawed meat joint and lower water holding capacity. sensory studies are required for evaluating the association between l* value and chroma in suckling lamb chops and consumer purchasing intent, and that of tenderness, whc and tbars on the meat edible quality. acknowledgements authors are grateful to the asociación de ganaderos de ganado ovino de raza churra for its support in sampling. they are also grateful for a doctoral grant from conacyt (mex/ref. 288189) and to the university of león who provided general support to this study. references amsa. 1995. research guidelines 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and collagen characteristics. meat sci. 105:32-37. teixeira a., batista s., delfa r. and cadavez v. 2005. lamb meat quality of two breeds with protected origin designation. influence of breed, sex and live weight. meat sci. 71:530-536. vieira c. and fernández a.m. 2014. effect of ageing time on suckling lamb meat quality resulting from different carcass chilling regimes. meat sci. 96:682-687. young o.a., priolo a., simmons n.j. and west j. 1999. effects of rigor attainment temperature on meat blooming and colour on display. meat sci. 52:47-56. paper received january18, 2019 accepted march 12, 2019 ijfs#1519_bozza ital. j. food sci., vol. 31, 2019 705 paper liking and sensory description of protein substitutes in phenylketonuria subjects: a case-study in northern and southern italy c. proserpio*1, e. verduci2, i. scala3, p. strisciuglio3, j. zuvadelli2 and e. pagliarini1 1department of food, environmental and nutritional sciences (defens), university of milan, milan, italy 2department of paediatrics, san paolo hospital, department of health sciences, university of milan, italy 3 department of translational science – pediatric section, federico ii university hospital, naples, italy *corresponding author: cristina.proserpio@unimi.it abstract nowadays, it is important to make effort to develop new formulations for subjects affected by rare diseases who need to follow a lifetime diet to maintain a good health. the purpose of the study was to evaluate the acceptability and to obtain a sensory descriptive analysis of protein substitutes (glycomacropeptide gmp formulas vs l-amino acid formulas) involving subjects affected by phenylketonuria in northern and southern italy. results demonstrated in both groups of subjects a greater acceptability of gmp samples, characterized by sweet and mild taste, mild odor, and natural color, compared with amino acid formulations. these sensory attributes should be considered during product development as a key factor influencing subjects’ satisfaction. keywords: acceptability, cata, food development, food formulations, odor, taste ital. j. food sci., vol. 31, 2019 706 1. introduction sensory perception and food preferences play a key role in determining food choice and, thus, directly influence the diet quality (cox et al., 2015). the study of the influence of sensory and hedonic individual differences in products acceptability is extremely important in modern food product development, especially for the ones meant to satisfy the needs of specific target populations (e.g, diabetics, elderly, obese etc). in this context, it is also important to make effort to develop new formulations for subjects affected by rare diseases who need to follow a lifetime diet to maintain a good state of health. between rare diseases, phenylketonuria (pku) is an inborn metabolism error, which, if untreated, could lead to severe brain damage (giovannini et al., 2012). the main purpose of pku patients’ treatment is to control the blood phenylalanine (phe) concentration to prevent severe health consequences (blau, 2016). the treatment for pku is mainly a lifetime diet based on low-protein foods in combination with amino acid supplements. these supplements without phe, are enriched with vitamins and minerals, and some products are also added with fat and carbohydrates to ensure normal growth and good health through adulthood (van spronsen et al., 2017). compliance with this strict diet becomes a challenge over time, especially in adolescence, and this is primarily due to the unpleasant taste of the amino acid supplements and the variety of available formulations (macdonald et al., 2010, aguiar et al., 2015). in order to achieve better compliance with diet through the lifespan, protein supplements should satisfy the need for better taste and easier management (van spronsen et al., 2008). the number of available protein substitutes for pku subjects is increasing over time (feillet and agostoni, 2010). in this context, beside the alternatives to traditional substitutes, the casein glycomacropeptide (gmp) is a 64-amino acid peptide from cheese whey which is rich in specific essential amino acids and is the only known natural protein source free from phe (ney et al., 2009; solverson et al., 2012). the lack of empirical studies aimed at evaluating subjects’ satisfaction for these substitutes reinforce the need to evaluate pku subjects’ perception regarding which sensory characteristics should have low-phe formulations to be more appreciated. only one recent study (proserpio et a., 2018) has been conducted in an ambulatory context involving pku subjects leading to define the sensory drivers of liking of protein substitutes in northern italy. however, intracultural differences between the northern and southern regions of italy are well recognized, not only referred to industrialization and economic prosperity, but also to cultural values and social structures (ruggiero et al., 2000). these differences are also clearly reflected in different dietary patterns, which lead to a higher percentage of overweight and obese subjects in the southern areas. in this context, some national data collected from the survey “indagine multiscopo dell’istituto nazionale di statistica (istat, 2015)” showed that, in the southern regions, the consumption of food rich in carbohydrates prevails while healthy and low energy dense food consumption characterized the north west area. due of the above, the aim of the present study was to evaluate the acceptability of new gmp formulas compared with the more traditional amino-acid mixtures involving subjects affected by phenylketonuria in northern and southern italy. a sensory descriptive analysis was also conducted in order to better understand which are the sensory characteristics that are related to the acceptance of these formulations that should be considered during the products development. ital. j. food sci., vol. 31, 2019 707 2. materials and methods 2.1. subjects a total of sixty-six subjects (mean age: 25.6±5.9 years, 34 women and 32 men) gave informed consent and completed the study. thirty-three subjects who were admitted to department of pediatrics, san paolo hospital (milan, italy), and thirty-three subjects referred to the department of pediatrics, federico ii university hospital (naples, italy) were recruited. all participants were following a low-phenylalanine diet (metabolic control phe (umol/l): milan= 642.1±309.2; naples= 720.9±295.2). the exclusion criteria were: pregnancy, food allergies to whey proteins, severe neurological and functional disorders. the present study was performed according to the principles established by the declaration of helsinki after the protocol was approved by the institutional ethics committee (protocol approval n°210). 2.2. samples 4 l-amino acid formulas (aa) and 4 glycomacropeptide (gmp) formulas, flavored with neutral, chocolate, strawberry and tomato aromas were prepared as reported in proserpio et al., (2018). the gmp flavored formulas (gmp_strawberry and gmp_tomato) were prepared by adding 2g of flavoring powder (aromaxx erdbeere; aromaxx tomate-basilikum, metax istitut fur diatetikmamoxi, torino, italy) to the neutral formulation (gmp_neutral) which consisted of 100 ml of glytactin rtdtm (cambrooke-quaris, roma, italy). the gmp chocolate flavored sample (gmp_chocolate) consisted of 100 ml of glytactin rtdtm chocolate (cambrooke-quaris, roma, italy). the aa neutral formula (aa_neutral) was prepared by mixing 16.5 g of powder high in lamino acid (xphe energy kid neutral, metax istitut fur diatetikmamoxi, torino, italy) and water to reach a final volume of 100 ml. the aa flavored formulas (aa_tomato; aa_chocolate) were prepared by adding 2g of flavoring powder (aromaxx tomatebasilikum; aromaxx schoko, metax istitut fur diatetikmamoxi, torino, italy). the lamino acid strawberry flavored samples (aa_strawberry) were prepared using 16.5 g of xphe energy kid erdbeere (metax istitut fur diatetikmamoxi, torino, italy) and water. each of these samples provides 5 g/100 ml protein equivalents. all the formulas were provided by mamoxi (torino, italy) and cambrooke-quaris (roma, italy). 30 ml of each sample were presented monadically, in a randomized and balanced order, to each participant in plastic cups labelled with three-digit codes. water was available for rinsing the palate between the samples. 2.3. experimental procedure sessions were conducted between 10:30-12:30 in quiet rooms under similar light conditions in both group of subjects (milan and naples). they were asked to refrain from consuming anything but water for 2 hours before the test (hungry state). subjects started by filling in the questionnaire on general appetite. subsequently, they had to score their overall liking and, after a rest of 5 min, to made a sensory descriptive evaluation (‘check-all-that-apply’ questionnaire: cata; varela and ares, 2012) for each sample. all samples were prepared on the same day of the session and were presented at room temperature (20-22 °c). ital. j. food sci., vol. 31, 2019 708 2.4. general appetite to ensure that both subjects from milan and naples were in a similar hunger state, they were asked to rate their appetite at the beginning of the session by filling out a questionnaire on general appetite (hunger, fullness, desire to eat, and thirst) all measured on 100mm visual analogue scales (vas, ‘not at all’: scored 0; ‘very’: scored 10). 2.5. overall liking evaluation subjects were asked to taste the samples monadically and to express their liking scores on a 100mm vas anchored by the extremes ‘‘extremely disliked” (rated 0) and ‘‘extremely liked” (rated 10). the experimenters provided instructions for the use of the scale prior to tasting (lawless et al., 2010). 2.6. sensory descriptive evaluation the sensory descriptive evaluation was performed using the ‘check-all-that-apply’ (cata) questionnaire. a separate group of 12 untrained subjects (age range: 20-40 years) attended a pilot test to define the suitable terms to describe the samples using a free listing questionnaire (varela and ares, 2012). the eight low protein samples were provided to the subjects and they were asked to evaluate the sensory characteristics and to write all attributes for describing their color, appearance, odor, taste, flavor and texture. the individual development of the attributes was followed by an open discussion. subsequently, the experimenters finalized the list of terms, selecting the most mentioned and the most common words in order to avoid synonymous (jaeger et al., 2015). finally, the questionnaire consisted of a list of 27 sensory attributes: 10 for the appearance (light brown, dark brown, light yellow, dark yellow, light pink, dark pink, natural color, artificial color, brightness and opaque), 6 for the odor (natural odor, artificial odor, mild odor, strong odor, milk odor and vanilla odor), 8 for the taste/flavor (sweet, bitter, salty, sour, mild taste, strong taste, milk flavor and vanilla flavor) and 3 for the texture (thin, thick and floury). subjects who took part to the experimental sessions were asked to check from the list all the terms that they considered appropriate to describe each sample. the position of attributes was randomized. 2.7. data analysis a paired samples t-test was performed to compare the general appetite ratings (100mm vas: hunger, fullness, satiety, desire to eat, and thirst) in the northern and southern groups before the samples evaluation. a linear mixed model procedure was carried out on overall liking scores considering ‘samples’, ‘gender’ (women and men), ‘city’ (northern and southern) and their two-way interactions as fixed factors. subjects were added as random effect while age as covariate. when a significant difference (p<0.05) was found, least significant difference (lsd) post hoc test was used. these statistical analyses were performed using ibm spss statistics for windows, version 24.0 (ibm corp., armonk ny). for the sensory descriptive evaluation, a data set was generated as 0/1 matrix, that is, “1” if the term was selected by the subjects and “0” if term was not selected. a frequency table was made from the total count of each term for each sample. cochran’s q test was carried ital. j. food sci., vol. 31, 2019 709 out for each of the 27 terms to detect differences in participants’ perception of the evaluated samples. correspondence analysis (ca) was used to obtain a bi-dimensional representation of the samples and to show relationship between samples and terms from the cata questionnaire. since the results provided by northern and southern subjects were similar to each other, the ca results are reported showing all the 66 subjects. penalty-lift analysis (plaehn, 2012; meyners et al., 2013) was also performed in order to study which cata terms were positively or negatively related with liking scores. these statistical analyses were performed using xlstat-sensory® software for windows, version 2015.6.01 (addinsoft™, france). a p-value of <0.05 was considered significant. results 3.1. general appetite the baseline general appetite ratings (table 1) confirmed that feelings of hunger, fullness, desire to eat, and thirst were not significantly different in the two groups of subjects (northern and southern). table 1. general appetite ratings (means±sem), as measured on 100 mm vas, provided by northern and southern subjects. general appetite northern southern t p hunger 4.47±0.45 4.53±0.46 0.08 0.94 fullness 3.43±0.43 3.53±0.49 1.69 0.09 desire to eat 4.99±0.48 4.45±0.45 0.82 0.41 thirsty 4.77±0.44 5.86±0.37 1.90 0.07 3.2. overall liking evaluation the main factor ‘samples’ was found to have a significant effect on liking scores (f(7;441)= 58,75; p<0.001). generally, gmp samples obtained significant higher liking scores (m=4.3±0.1) compared with the aa formulas (m=2.7±0.2). a significant effect of the interaction ‘samples’ * ‘city’ on liking scores was also found (f(7;441)=2.95, p<0.01). the mean liking scores by samples in subjects from milan and naples are provided in table 2. looking at the results gave by northern subjects, the highest liking scores were obtained by the gmp_chocolate and gmp_strawberry, which were comparable to each other. the aa_strawberry samples obtained the highest score between the aa formulas, even if all aa samples were not acceptable (score <5). similarly, southern subjects preferred the gmp and aa samples flavored with strawberry aroma. contrariwise, the tomato flavored samples, both gmp and aa formulas, obtained the lowest hedonic ratings in both subjects from milan and naples. ital. j. food sci., vol. 31, 2019 710 table 2. liking scores (means±sem) for each samples provided by northern and southern subjects. different letters (in column) indicate significant differences according to post hoc test within each group of subjects. samples liking scores northern southern p gmp tomato 1.10a±0.38 1.23a±0.39 0.78 n.s neutral 4.48c±0.37 4.45cd±0.39 0.89 n.s chocolate 5.64d±0.37 4.84d±0.38 0.13 n.s strawberry 6.45d±0.38 6.13e±0.40 0.57 n.s aa tomato 1.17a±0.38 0.94a±0.38 0.72 n.s neutral 1.23a±0.37 3.10b±0.39 0.00 *** chocolate 3.00b±0.38 3.33bc±0.39 0.55 n.s strawberry 4.05c±0.37 5.17de±0.38 0.03* *p<0.05; *** p<0.001. comparing the results obtained in the two groups of subjects no significant differences have been highlighted between the gmp samples, while significant differences have been found in aa samples. in particular, significant higher scores were provided to aa_neutral and aa_strawberry by southern subjects. the main factor ‘gender’ and the interactions ‘sample’ * ‘gender’; ‘city’ * ‘gender’ were not significant (f(1,64)= 0.015, p=0.90; f(7,441)= 0.70, p=0.80; f(1,64)=0.004, p=0.95, respectively). 3.3. sensory descriptive evaluation the contingency table below (table 3) shows the frequency of terms checked by northern and southern subjects to describe the eight samples. significant differences (p<0.001) were found in the frequency for all the 27 terms within the five sensory attributes categories evaluated, suggesting that the pku subjects involved perceived differences between samples in terms of their sensory characteristics. the correspondence analysis, used to obtain a bi-dimensional representation of the samples and the relationship between samples and terms from the cata questionnaire, resulted in two dimensions accounting for 60.62% of variance in the data. as shown from fig. 1, samples were clearly discriminated by subjects according to their aroma. indeed, samples flavored with strawberry aroma (aa_strawberry and gmp_strawberry) are positioned in the upper left side of the map while the samples added with tomato aroma (aa_tomato and gmp_tomato) are situated in the upper right side of the map. in the lower part of the map, gmp samples without aroma (gmp_neutral) and the chocolate one (gmp_chocolate) are well distinguished from the l-amino acid formulas with the same aromas (aa_neutral and aa_chocolate). ital. j. food sci., vol. 31, 2019 711 table 3. contingency table for the sensory descriptive analysis evaluation. sensory attributes aa samples gmp samples chocolate strawberry neutral tomato chocolate strawberry neutral tomato appearance artificial color 9 28 12 20 10 24 14 26 natural color 17 7 24 6 19 7 25 7 light yellow 0 0 5 1 12 0 19 1 dark yellow 0 0 0 3 12 0 1 7 brightness 2 18 15 4 11 5 12 5 light brown 5 0 0 45 25 0 3 35 dark brown 60 0 0 7 4 0 0 4 opaque 19 3 6 18 13 13 16 23 light pink 0 58 0 0 0 15 0 4 dark pink 0 2 0 6 0 47 0 9 odor artificial odor 21 25 32 34 12 23 10 35 mild odor 15 38 19 2 26 24 27 6 milk odor 4 4 8 0 14 6 32 2 vanilla odor 1 7 13 1 13 4 11 2 strong odor 21 5 11 43 10 19 7 40 natural odor 20 14 8 3 20 9 18 3 taste sweet 14 39 6 1 41 51 33 5 sour 10 12 17 26 2 3 6 23 salty 12 6 15 38 3 0 5 31 bitter 27 9 28 24 8 1 6 18 mild taste 11 27 18 2 31 27 32 5 strong taste 35 20 28 51 7 23 9 45 flavor milk flavor 6 9 2 0 24 10 35 1 vanilla flavor 1 4 8 0 16 3 8 2 texture thin 18 44 39 29 24 25 32 25 thick 32 0 17 16 18 21 20 25 floury 21 2 14 12 11 8 6 8 3.4. relating sensory profiling with liking a penalty-lift analysis was carried out to understand which sensory attributes were mainly associated to the overall liking of the samples. the analysis showed to which extent liking increased or decreased when the subjects related a certain cata terms to the samples. as inferred from fig. 2 liking scores were significantly positively associated with the sensory attributes: ‘sweet’ (p<0.0001), ‘mild taste’ (p<0.0001), ‘mild odor’ (p<0.0001) and ital. j. food sci., vol. 31, 2019 712 ‘natural color’ (p<0.0001). contrariwise, the terms that significantly decreased the samples’ acceptability were: ‘salty’ (p<0.0001); ‘strong taste’ (p<0.0001); ‘bitter’ (p<0.0001); ‘strong odor’(p<0.0001); ‘light brown’(p<0.0001); ‘artificial odor’(p<0.0001), ‘opaque’(p<0.001) and ‘thick’(p<0.05). figure 1. attributes and products plot obtained from cata total frequency counts. figure 2. penalty-lift analysis of sensory attributes across all samples. ital. j. food sci., vol. 31, 2019 713 4. discussion the purpose of the present study was to deepen the evaluation of the acceptability of protein substitutes (gmp formulas vs l-amino acid formulas) involving subjects affected by phenylketonuria in northern and southern italy. a sensory descriptive evaluation was performed to define which sensory characteristics are mainly related to the acceptance or to the refusal of these products that should be taken into account in developing new formulations for specific target populations. overall, the present results demonstrated in both subjects from milan and naples a greater acceptability of gmp samples compared with the more traditional amino acid formulations, besides the reported differences in dietary pattern among the regions of italy (ruggiero et al., 2000; istat, 2015). in particular, gmp samples flavored with chocolate and strawberry, were the most appreciated by the northern group, while the sample flavored with strawberry was the most appreciated by the southern group. these results confirmed our previous findings with a group of northern subjects (proserpio et al., 2018) in which a greater acceptability was depicted for gmp beverages flavored with chocolate and strawberry compared with the l-amino acid formulations flavored with the same aromas. according to the present results lim and collaborators (2007) showed that a gmp chocolate beverage was significantly more liked compared with the same flavored amino acid beverage. both groups of subjects from milan and naples gave to the tomato flavored samples the lowest liking scores. even if the gmp samples received generally average higher liking scores (m=4.3±0.1) compared with the aa formulas (m=2.7±0.2), it is important to consider that the type of added aroma was a key driver of the acceptance. indeed, the gmp sample, as well as aa formulation, flavored with the tomato aroma were evaluated as not acceptable by both group of subjects. these samples were not appreciated since they were perceived as salty, sour and characterized by strong odor and taste. the tomato flavored formulations maybe were perceived as unpleasant since tomato aroma, signaling savory product, is not usually used as flavoring in low-phenylalanine products, especially as beverages. thus, subjects maybe have perceived tomato flavored samples as really far from their usual food habits. indeed, it is well known that food habits and also consumers’ expectations could influence the sensory perception, the liking, and consequently the actual food consumption (köster, 2009). confirming this hypothesis, two different studies demonstrated that cracker samples with gmp, a product expected to be characterized by salty taste, obtained higher liking scores compared with the low-protein crackers (lim et al., 2007; van calcar et al., 2012). considering the sensory attributes mainly associated to the overall liking of the samples, the present study demonstrated that the acceptability increased when the samples were characterized by the attributes: ‘sweet’, ‘mild taste’, ‘mild odor’ and ‘natural color’. the information achieved by the present results could be useful to understand which sensory characteristic should have a low-phenylalanine product to be more accepted by the subjects. consequently, more appreciated products could facilitate dietary compliance that is a challenge over time, especially in adolescence (macdonald et al., 2010, aguiar et al., 2015). moreover, it has also been demonstrated that, besides the higher acceptance of gmp formulations compared the more traditional ones, gmp could also be considered as a more physiological source of dietary protein and promote higher satiety compared with synthetic amino acids (van calcar et al., 2012). ital. j. food sci., vol. 31, 2019 714 as future perspective, it should be useful to evaluate other gmp base products using validated sensory approaches, as it has been performed in the present study, besides considering mainly the nutritional composition of these products. in conclusion, the present results suggest that, in order to improve the protein substitutes’ sensory quality, these formulations should be characterized by a sweet and mild taste, a mild odor, and a natural color. indeed, it is important to consider the pku patients’ satisfaction as a key factor during the product development in order to improve the diet quality through the lifespan. acknowledgments we would like to express our thanks to mamoxi (torino, italy) and cambrooke-quaris (roma, italy) for supplying the samples. references aguiar a., ahring k., almeida m.f., assoun m., quintana a.b., bigot s., caris a. et al. 2015. practices in prescribing protein substitutes for pku in europe: no uniformity of approach. mol. genet. metab. 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of food choice: a psychological perspective. food qual. prefer. 20(2):7082. lawless h.t., popper r. and kroll bj. 2010. a comparison of the labeled magnitude (lam) scale, an 11-point category scale and the traditional 9-point hedonic scale. food qual. prefer. 21(1):4-12. lim k., van calcar s.c., nelson k.l., gleason s.t. and ney d.m. 2007. acceptable low-phenylalanine foods and beverages can be made with glycomacropeptide from cheese whey for individuals with pku. mol. genet. metab. 92(1):176-178. macdonald a., gokmen-ozel h., van rijn m. and burgard p. 2010. the reality of dietary compliance in the management of phenylketonuria. j. inherit. metab. dis. 33(6):665-670. ney d.m., gleason s.t., van calcar s.c., macleod e.l., nelson k.l., etzel, m.r., wolff j. et al. a. 2009. nutritional management of pku with glycomacropeptide from cheese whey. j. inherit. metab. dis. 32(1):32-39. meyners m., castura j.c. and carr b.t. 2013. existing and new approaches for the analysis of cata data. food qual. prefer. 30(2):309-319. plaehn d. 2012. cata penalty/reward. food qual. and prefer. 24:141-152. ital. j. food sci., vol. 31, 2019 715 proserpio c., pagliarini e., zuvadelli j., paci s., re dionigi a., banderali g., verduci e. et al. 2018. exploring drivers of liking of low-phenylalanine products in subjects with phenyilketonuria using check-all-that-apply method. nutrients. 10(9):1179. ruggiero g.m., hannöver w., mantero m. and papa r. 2000. body acceptance and culture: a study in northern and southern italy. eur. eat. disorders rev. 8(1):40-50. solverson p., murali s.g., brinkman a.s., nelson d.w., clayton m.k., yen c.l.e. and ney d.m. 2012. glycomacropeptide, a low-phenylalanine protein isolated from cheese whey, supports growth and attenuates metabolic stress in the murine model of phenylketonuria. am. j. physiol. endocrinol. metab. 302(7):885-895. van calcar s.c. and ney d.m. 2012. food products made with glycomacropeptide, a low-phenylalanine whey protein, provide a new alternative to amino acid–based medical foods for nutrition management of phenylketonuria. j. acad. nutr. diet. 112(8):1201-1210. van spronsen f.j. and burgard p. 2008. the truth of treating patients with phenylketonuria after childhood: the need for a new guideline. j. inherit. metab dis. 31(6):673-679. van spronsen f.j., van wegberg a.m., ahring k., bélanger-quintana a., blau n., bosch a.m., huijbregts s.c. et al. 2017. key european guidelines for the diagnosis and management of patients with phenylketonuria. lancet. diabetes endocrinol. 5:743-756. varela p. and ares g. 2012. sensory profiling, the blurred line between sensory and consumer science. a review of novel methods for product characterization. food res. int. 48(2):893-908. paper received february 5, 2019 accepted june 20, 2019 ijfs#1706_bozza ital. j. food sci., vol. 32, 2020 596 paper bromatological composition and effect of temperature on the rheology of eggplant pulp r. marsiglia1, l. mieles-gómez, s. lastra, s.e. quintana and l.a. garcía-zapateiro* faculty of engineering, food engineering program, research group in complex fluids engineering and food rheology (ifcra), university of cartagena, 130015 cartagena, colombia *corresponding author: lgarciaz@unicartagena.edu.co abstract this study aimed to determine the bromatological composition and the behavior of rheological parameters on the pulp eggplant (solanum melongena). bromatological analyzes were performed according to the reference methods, in which a percentage of moisture 90.98%, total carbohydrates 6.86%, crude fiber 1.94%, crude protein 1.19%, fat 0.31% and 0.49% of ash has been obtained. viscous flow curves were calculated in the steady state over a temperature range of 10-80ºc, and the rheological properties of the pulp were evaluated as a function of temperature. the pulp showed pseudoplastic behavior (shear thinning type) at all temperatures, and the relationship between viscosity and the carreau-yasuda model (r2>0.99). the arrhenius equation was fitted to the data for the apparent viscosity of the pulp with respect to temperature, with activation energy e𝑎=1081.61 j/mol. the results provide information on bromatological composition and the rheological behavior of eggplant pulp and may have applications in the design of processes using this raw material. keywords: carreau-yasuda model, eggplant (solanum melongena), bromatological analysis, pulp, rheological behavior, shear thinning ital. j. food sci., vol. 32, 2020 597 1. introduction eggplant or aubergine (solanum melongena) is an economically important crop that is widely cultivated in tropical and subtropical areas of the world, and its cultivars produce a wide variety of fruits with different shapes, sizes and colors (salunkhe and desai, 1984; san josé et al., 2013; niño-medina et al., 2017). the global consumption of eggplant has increased in recent years, due to the numerous benefits of this crop, such as the presence of metabolites that contribute significantly to a healthy diet (sun et al., 2015). it has also been found that extracts of eggplant can successfully suppress the development and growth of tumors, metastasis, inflammation and heart disease (nisha et al., 2009). eggplant is one of the vegetables on which great importance is placed in the colombian caribbean, due to its strong suitability for export, the aforementioned health benefits, its high content of phenols and antioxidant activity, and its contribution to the diet in terms of low calorie content, very low sodium content and high fiber content (aramendiztatis et al., 2010). according to food and agriculture organization fao (2015) data from 2015, eggplants used for consumption were fresh (more common), or in industrial processes, mainly frozen. in industrial processes, this vegetable can be used as cubes or dice, such as in slices, jams and jellies. the products of fruits and vegetables contain pulps as basic raw materials, which in most cases are transported via pipes and tanks and are agitated and mixed with other raw materials, pasteurized and evaporated in heat exchangers and continuous evaporators. in addition, other operations are applied such as sieving, snubbing, mixing and various thermal treatments. in order for these operations to be technically and economically feasible, it is important to have a knowledge of the properties of the pulp (ibarz et al., 1996; ortega-quintana et al., 2015). the rheological behavior is one of the most important properties in the development of new products, and is very useful in the design of unit operations, guarantees of highquality food and beverages, and process optimization. in addition, rheological approaches are essential tools for food engineering, since rheology is linked to the processing and stability of food as well as its sensory qualities. physical properties such as density, specific heat and thermal conductivity are of great importance for food and beverages, as they are closely related to their sensory and rheological characteristics (augusto et al., 2012; de castilhos et al., 2017). several factors affect the rheological behavior of fruit and vegetable pulps, and temperature is the most likely to affect the viscosity of the pulp (holdsworth, 1971). an understanding of the influence of temperature on viscosity is therefore fundamental in obtaining better knowledge of the rheological behavior of fruit and vegetable derivatives during processing at high temperatures (ibarz et al., 1996). various studies have been carried out on the rheological behavior of pulps from different botanical sources; most of these do not comply with newton's law of viscosity, and it is said that they behave like non-newtonian fluids. their behavior can be described by a power law (holdsworth, 1971) or by the herschel-bulkley model in the case where they have a non-zero yield stress (steffe, 1996). the main objective of this research is to determine the effect of temperature on the behavior of the rheological parameters of eggplant pulp. ital. j. food sci., vol. 32, 2020 598 2. materials and methods the eggplants used were acquired from a food supply center in cartagena, bolivarcolombia, and were in a state of commercial maturity and free from mechanical damage. 2.1. pulp extraction the selected fruits were weighed, washed and blanched at 80ºc for five minutes. pulp was obtained using a refining 1.5 mm opening mesh, giving a pulp to facilitate rheological measurements. this was then packed in hermetic bags and stored in refrigeration at 4ºc for 24 hours for further rheological analysis. 2.2. bromatological evaluation bromatological analyses were performed on the eggplant pulp. moisture content, ash, protein, fat, total carbohydrates and raw fiber were determined using the methods described by the association of official analytical chemists (aoac) (2000): moisture: dehydration in an oven at 105ºc aoac 33.7.03 method 926.08; ash: y combustion at 450ºc for 12 hours aoac 33.7.07 method 935.42; crude protein: using the method of macro kjeldahl, aoac 33.7.12 method 926.123; fat: aoac 972.28 ethereal extraction method; total carbohydrates: aoac 923.03; crude fiber: a.o.a.c. 985.29. 2.4. rheological evaluation steady flow tests were carried out to give viscosity curves at temperatures of 10ºc, 20ºc, 25ºc, 40ºc, 60ºc and 80ºc for samples without a previous history of shear, using a controlled-stress rehometer (modular advanced rheometer system mars 60, haake, thermo-scientific, germany), equipped with peltier temperature control and measuring system, using a stainless steel plate-plate geometry (rough surface ø 35 mm) with 1 mm gap, over a range of shear rates between 0.001 and 1000 s-1 (franco et al., 1998). prior to measurement, all samples were left at rest for 600 s to allow for relaxation, and the temperature of the samples was kept constant at 20±0.1°c using a peltier system, following the methodology used by quintana et al. (2017). 2.4. statistical analysis the data were analyzed with a unidirectional anova using spss software (version 17.0 for windows) in order to determine statistically significant differences (p < 0.05) between the samples. all tests were performed in triplicate. 3. results and discussion 3.1. bromatological analysis table 1 shows the average values for moisture, total carbohydrates, total fiber, protein, fat and ash, obtained in triplicate. the results show that the eggplants used in this study mostly contained water at a percentage of 90.98±0.016%, a carbohydrate content of ital. j. food sci., vol. 32, 2020 599 6.86±0.17% and a fiber content of 1.94±0.07%, fairly close to the values reported by garcía et al. (2003), moreiras et al. (2013) and icbf (2015). table 1. bromatological analysis. sample (%) moisture total carbohydrates crude fiber crude protein fat ash eggplant 90.98±0.16 6.86±0.17 1.94±0.07 1.19 ±0.04 0.31±0.02 0.49±0.02 eggplant represents an important source of dietary fiber, and therefore has health benefits in terms of its effects on digestive regularity and the prevention of diseases such as constipation, hypercholesterolemia, hyperglycemia and obesity, which are partly related to intake of fiber (alves dos santos et al., 2002). its beneficial physiological effects lie in its texture and consistency, since it acts as a sponge that binds to foods rich in cholesterol, releasing accumulated wastes from the intestinal wall, which would otherwise be difficult to expel. it also increases the fecal mass, which results in a reduction in carcinogenic risk and faster elimination from the body (cañas-ángel et al 2011.) 3.2. rheological evaluation the variation of the viscosity with the deformation speed was observed using steady flow tests at different temperatures. fig. 1 shows the viscous flow curves for the eggplant pulp as a function of the deformation speed, at temperatures of 10°c, 20°c, 25°c, 40°c, 60°c and 80°c. these curves show the characteristics of a non-newtonian fluid of rheofluidifying (shear thinning) type, since they combine the characteristic properties of elastic and both solid-like and liquid-like properties, characterized by a potential decrease in viscosity with respect to the shear rate (muller, 1973). on the other hand, a constant viscosity value 𝜂! and at high deformation 𝜂! velocities are observed at low deformation speeds. this behavior can be explained by the breaking of the reticular structure of polysaccharide molecules during shearing, as explained by bhandari et al. (2002). in a reticular system, the speed at which existing molecular interactions break becomes higher than the speed at which they reform, with increasing shear rate. the result is a lower intermolecular resistance to the flow, and hence a lower viscosity (díaz-ocampo et al., 2012). similar behaviors have been observed in several fruit pulps, such as squash pulp (cucurbita moschata) (quintanaa et al., 2018); papaya pulp (carica papaya) (quintana et al., 2017); borojó pulp (borojoa patinoi cuatrec) (díaz-ocampo et al., 2012); nispero pulp (achras sapota l.) (andrade et al., 2009) and mango, papaya and peach purees (guerrero and alzamora, 1998). among the models most often used to describe the rheological behavior of fruit pulps is the ostwald de waele power law (toralles et al., 2006). numerous authors have successfully described the flow behavior of various pulps using this model, for example, peach pulp (muñoz et al., 2012) and mango pulp of different varieties (vidal et al.,2004; ortega-quintana et al., 2015; figueroa-flórez et al., 2017). in this case, the experimental data for the viscosity and shear rate were fitted to the carreau-yasuda model (carreau, 1972), which gave us the best statistical parameters including a minimum correlation coefficient r2>0.99282: ital. j. food sci., vol. 32, 2020 600 𝜂 = 𝜂! + 𝜂! − 𝜂! 1 + (𝜆!γ̇)! !!! ! (1) this model represents a fluid that follows newton's law of viscosity at low deformation speeds and obeys a power law at high shear rates (méndez-sánchez et al., 2010). it uses five parameters: 𝜂! corresponds to the newtonian viscosity at low shear rates; 𝜂! is the newtonian viscosity at high deformation speeds; 𝜆! is the carreau time constant; 𝑎 is the transition control factor, which is a dimensionless constant; and 𝑛 is the parameter of the power law model. in the case where n=1, the model is reduced to the linear newtonian model, for example, the navier-stokes equations. for fluidifying liquids (n<1), the viscosity decreases with an increase in the shear rate. 10-5 104 10-3 10-2 10 -1 100 10 1 102 103 104 10-2 10-1 100 101 102 103 104 105 106 eggplant 10°c eggplant 20°c eggplant 25°c eggplant 40°c eggplant 60°c eggplant 80°c carreau mod el η (p a. s) γ (1/s)• figure 1. viscous flow curves for s. melongena pulp at different temperatures (10ºc, 20ºc, 25ºc, 40ºc, 60ºc, 80ºc) fitted to the carreau-yasuda model. fig. 1 shows the experimental data (viscosity vs. shear rate) fitted to the carreau-yasuda model. at the initial points, the apparent viscosity tends to be constant, and is represented as the null viscosity 𝜂𝑜. it subsequently begins to decrease, and the viscosity curve enters a logarithmic drop phase, where at high deformation speeds it tends to behave like a newtonian fluid, following the behavior of the carreau model. this decrease in viscosity with an increase in shear rate is called shear thinning, and similar results have been obtained in rheological studies of papaya pulp (quintana et al., 2017). the fitting parameters of the carreau-yasuda model are shown in table 2. the results demonstrate rheofluidifying (shear-thinning) behavior, with values for the flow behavior index of less than unity (n<1) for the different conditions of temperature. the viscosity decreases with the shear rate, and hence it is not considered an increase or decrease in the ital. j. food sci., vol. 32, 2020 601 fluidifying character of the pulps. however, changes in temperature affect the viscosity, since an increase in temperature produces greater intermolecular interaction in the aqueous phase of the pulp, causing repulsion between the suspended particles, lower resistance to flow, and consequently a decrease in the apparent viscosity (figueroaflórez et al., 2017). this effect was also found by quintana (2016), who showed that the results depended strongly on the heat treatment applied to the pulp before rheological analyses were carried out. table 2. rheological parameters of the carreau-yasuda model for the viscosity of eggplant pulp with variation in shear rate, at temperatures of 10-80°c. temp. 𝜼𝟎 𝜼! 𝝀𝒄 𝒂 𝒏 r 2 10°c 70390.99±1495.38 4.31e-47±0.01 2129.66±136.20 1.50±0 0.10±0.02 0.99 20°c 48987.00±751.80 0.02±0.01 882.40±23.87 2.15±0.14 0.06±0.01 0.99 25°c 96193.78±583.63 5.14e-37±0 980.64±0 5.00±0.33 0.02±0.01 0.99 40°c 23964.83±654.76 0.08±0 643.23±10.68 17.61±5.43 0.08±0.01 0.99 60°c 83051.70±5478.35 0.13±0.3 1222.66±48.54 1.25±0.13 0.05±0.02 0.99 80°c 164922.94±5552.34 0.05±0.01 39.73±8.43 2.09±0.21 0.16±0.01 0.99 the effect of temperature on the apparent viscosity of fluid foods (at constant shear rate) can be explained by the arrhenius equation (dak et al., 2007; rao and tattiyakul, 1999), expressed as: 𝜂 = 𝐴𝑒𝑥𝑝 !! !" (2) where a is the pre-exponential factor; 𝐸𝑎 is the activation energy, a parameter used to evaluate the thermal dependence (j/mol); r is the gas constant (8.314 j/mol k); and t is the temperature absolute (k). in this model, the apparent viscosity decreases in an exponential way with temperature. a shear rate of 15 s-1 was selected, since operations such as flow in pipes, mixing and agitation involve a range of shear speed of 10–1000 s-1 (steffe, 1996). the experimental data for η can be well modeled by the arrhenius equation, as shown in fig. 2, and the values for r2 are found to be greater than 0.9387. the effect of temperature on apparent viscosity (at a constant shear rate) can also be observed in fig. 2. in this case, the activation energy e𝑎=1081.61894 j/mol was lower than the values reported for squash pulp (𝐸𝑎 = 1229.46 j/mol) (quintana et al., 2018); acerola pulp (𝐸𝑎=12637.8914292.45 j/mol) (pereira et al., 2014); sapodilla pulp (𝐸𝑎=12637.89 j/mol) (andradepizarro et al., 2010) and tomato paste (𝐸𝑎=8600-13000 j/mol) (dak et al., 2008), indicating that eggplant pulp has a higher sensitivity to changes in temperature in a food transpiring process; in other words, the internal structure of eggplant pulp is more affected by temperature than the other pulps or food products mentioned above. the activation energy is a very important parameter in terms of the movement of the molecules, as it influences when the temperature increases in the liquids, allowing them flow more easily, due to a high activation energy at high temperatures (memnune et al., 2005). in this case, an increase in temperature causes a decrease in the viscosity of the ital. j. food sci., vol. 32, 2020 602 liquid phase, thus increasing the movement of the suspended particles and causing a decrease in the viscosity of the pulp (pelegrine et al., 2002). 0,0028 0,0029 0,0030 0,0031 0,0032 0,0033 0,0034 0,0035 4 6 8 10 viscosity arrhenius model η ( p a. s) temperature (1/k) τ figure 2. variation in the values of η obtained at 15 s-1 as a function of temperature and fit to the arrhenius model. 4. conclusions based on the results obtained in this investigation, the eggplant pulp have a the mayor content expressed in percentage for moisture and carbohydrates, 90.98 and 6.86±0.17 respectively. the rheological properties of the pulp behave like a non-newtonian fluid of rheofluidifying type (shear thinning). all samples showed a decrease in viscosity with shear rate, which was fitted to the carreau-yasuda model with r2>0.99. the influence of temperature on the behavior of the pulp was observed, and the eggplant pulp was found to lose pseudoplasticity and become less consistent as the temperature increases and its rheological parameters are affected. the relationship between the temperature and the apparent viscosity of the pulp can be represented by the arrhenius equation, where an increase in temperature causes a decrease in viscosity. finally, by examining the rheological and flow 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vendruscolo j. and vendruscolo c. 2006. reológia de purê homogeneizado de pêssego: efeito da temperatura e concentração. braz. j. food technol. 9(1):1. vidal j., pelegrine d. and gasparetto c. 2004. efeito da temperatura no comportamento reológico da polpa de manga (mangífera indica l. keitt). ciênc. tecnol. aliment. 24(1):039. doi: doi.org/10.1590/s010120612004000100008 paper received september 15, 2019 accepted february 14, 2020 ijfs#1510_bozza ital. j. food sci., vol. 31, 2019 617 paper antioxidant activity as well as vitamin c and polyphenol content in the diet for athletes b. frączek*1, m. morawska2, m. gacek3 and k. pogoń4 1department of sports medicine and human nutrition, university school of physical education in cracow, al. jana pawla ii 78, 31-571 cracow, poland 2department of sports medicine and human nutrition, university school of physical education in cracow, al. jana pawla ii 78, 31-571 cracow, poland 3department of sports medicine and human nutrition, university school of physical education in cracow, al. jana pawla ii 78, 31-571 cracow, poland 4department of fruit, vegetable and mushroom technology, university of agriculture in cracow, poland *corresponding author: tel.: +48 126831002; fax: +48 126831223 e-mail address: barbara.fraczek@awf.krakow.pl abstract the aim of the study was to analytically evaluate the total content of vitamin c and polyphenols as well as the antioxidant potential of daily food rations planned for athletes. chemical analyses showed that an average food ration for women (2,120.1 kcal, 90.8 g protein, 53.1 g fat and 354.0 g carbohydrates) contained 5.5±2.6 mg vitamin c and 20.1±4.1 mg polyphenols in 100 g fresh mass. an average food ration for men (2,648.8 kcal, 112.5 g protein, 63.1 g fat and 447.4 g carbohydrates) contained 5.6±1.4 mg vitamin c and 22.9±8.1 mg polyphenols in 100 g fresh mass. the antioxidant potential of an average ration for women expressed as reducing power (frap index) in 100 g fresh mass was 8.2±0.7 mmol fe+2, and for men, 8.9 ±0.9 mmol fe+2. the antioxidant potential of an average ration prepared for women and men expressed as antiradical activity against dpph in 100 g fresh mass was respectively: 2.7±02 mmol and 2.7±0.4 mmol trolox equivalent. balanced food rations rich in products with high nutrient density can ensure the appropriate intake of vitamin c and polyphenols and high antioxidant potential of the diet. keywords: vitamin c, polyphenols, antioxidant potential, diet, athletes, chemical analyses ital. j. food sci., vol. 31, 2019 618 1. introduction the situation of intensive physical exercise leads to the disruption of body homeostasis, also in terms of prooxidant-antioxidant equilibrium (oxidative stress) as a result of intense metabolic processes and the influence of psychological and environmental factors. intensive physical exercise induces the overproduction of reactive oxygen species, which cause oxidative damage to tissues as a result of peroxidation of lipids, proteins and dna. by reducing the skeletal muscle contraction strength, speeding up fatigue and lowering immunity, they reduce athletes' performance (sung et al., 2016; yavari et al., 2015). high oxidative stress occurring in athletes generates increased demand for antioxidant vitamins and polyphenols (heaton et al., 2017; morillas-ruiz et al., 2006; orlando et al., 2018; schneider et al., 2018; yavari et al., 2015). a rich source of dietary antioxidants is fruit and vegetables with high contents of bioactive substances, including vitamin c, carotenoids and polyphenols (phenolic acids and flavonoids with anthocyans) (naderi et al., 2018; sikora et al., 2008). the swiss food pyramid for athletes recommends daily consumption of 5 portions of fruit and vegetables in all colors, ensuring a wide range of bioactive substances (walter et al., 2007). in this context, planning balanced food rations, rich in products with high nutrient density, such as fruit and vegetables and other products with high nutrient density, is of special importance in athletes' diet (yavari et al., 2015). literature review shows that many works present the nutritional value, including the content of antioxidant substances and antioxidant activity of selected products and dishes, such as e.g., honey (cianciosi et al., 2018), fruit (aires et al., 2017; naderi et al., 2018; shin et al., 2018; zenteno-rramírez et al., 2018), vegetables (jaiswal et al., 2012; sotiroudis et al., 2010), legumes (durazzo et al., 2013), whole grains (durazzo et al. 2015), several varieties of wheat and black barley (siebenhandl et al., 2007), different cereal grain species (van hung, 2016), sour cherry juice (ferretti et al., 2010; mccormick et al., 2015), sprouts (hotnog et al., 2017), as well as grilled chicken salad and spaghetti with tomatoes and parmesan cheese (frączek and gacek, 2013; gacek et al. 2012). fewer works describe the nutritional value and antioxidant properties of daily food rations (aliakbarlu et al., 2014; bedogni et al., 1999; koréissi-dembélé et al., 2017; marconi et al., 2018; zloch et al., 2018). evaluation of antioxidant potential of different products and dishes is one aspect of the innovative approach to research on food (durazzo, 2017). therefore, this study focused on vitamin c and polyphenol content as well as antioxidant properties of complete daily food rations planned for athletes with consideration of products and dishes they prefer, in accordance with qualitative and quantitative recommendations. the study aimed to answer the question whether it is possible to prepare food rations that meet the qualitative and quantitative recommendations for athletes (balanced in terms of energy and macroelements intake) and at the same time are rich in dietary antioxidants and have high antioxidant properties. the innovative approach to the issue is the verification through chemical analyses of the nutritional value of food rations based on theoretical databases. in this context, the aim of the study was to analytically evaluate the total content of vitamin c and polyphenols as well as the antioxidant properties of daily food rations planned for athletes (women and men) of disciplines that require them to maintain low body mass. ital. j. food sci., vol. 31, 2019 619 2. materials and methods 2.1. material daily food rations were prepared on the basis of dietary databases for polish athletes (women and men) professionally doing disciplines that require them to maintain low body mass, with consideration of their dietary preferences. the criterion for the open selection of participants was doing the sport professionally for at least 3 years. the explored group of athletes for whom the menus were prepared included people aged 1830 (22±3.8), representing the following disciplines: long-distance running, middle-distance running, triple jump, race walking, ballet, artistic gymnastics, rhythmic gymnastics, ski jumping, nordic combined, synchronised swimming and dancing. the age and somatic indices of the participating athletes are presented in table 1. body composition (total fat mass tbf and total body water tbw) was measured using bioimpedance testing method (body comp mf from akern). table 1. statistical characteristics of anthropometric indices of the study subjects. group statistics age (years) body weight (kg) body height (cm) tbf (%) tbw (%) bmi (kg/ m2) overall mean 22.0 58.4 171.5 11.7 64.6 19.8 sd 3.8 6.9 7.1 4.6 3.4 1.6 men mean 21.3 62.6 175.3 9.1 66.5 20.0 sd 3.7 5.3 6.4 3.3 2.4 1.5 women mean 22.9 53.2 166.8 15.1 62.2 19.1 sd 3.8 4.9 4.7 3.8 2.9 1.4 the athletes' dietary preferences were determined for 185 products and dishes using a 5point hedonic scale (5 – like very much, 4 – like, 3 – neither like nor dislike, 2 – dislike, 1 – dislike very much). the proposed menus included products and dishes for which a high level of preference was obtained (mean values in the 4.64-4.00 range for 41 products, such as vegetables, fruit, cereal products, flour products, poultry, eggs and egg dishes). products that were disliked were excluded from the diet (mean preference indices in the 2.50-2.99 range, such as pumpernickel, soy products, margarines, fish in cream sauce and in oil). based on the analysis of dietary preferences, two weekly menus were prepared – 14 daily food rations (7 for women: w1-w7 and 7 for men: m1-m7). the menus were prepared in accordance with qualitative (walter et al., 2007) and quantitative recommendations for athletes (kreider et al., 2010; potgieter, 2013) on the basis of data concerning products' and dishes' nutritional value in polish “tables of food ingredients and nutritional value” (kunachowicz et al., 2005). the quantitative assumptions for the planned food rations are presented in table 2. the nutritional value of dishes was calculated on the basis of the adopted recipes, taking into consideration raw products (and values for edible parts). ital. j. food sci., vol. 31, 2019 620 table 2. detailed assumptions for intended diet plans for women and men (crp – daily food serving). energy/nutrient women men energy (kcal) 2162.0 2779.0 protein (g/kg b.w.) 1.7 1.7 protein (g) 90.4 106.4 protein (% of energy) 16.7 15.3 fat (g/kg b.w.) 1.0 1.0 fat (g) 53.2 62.6 fat (% of energy) 22.1 20.3 carbohydrates (g/kg b.w.) 6.5 7.4 total carbohydrates (g) 350.4 470 indigestible carbohydrates (g) 315.4 430 carbohydrates (% of energy) 61.2 64.4 dietary fiber (g) 25-40 25-40 the planned daily food rations were prepared in a food laboratory and then sent for chemical analyses to the malopolska centre of food monitoring at the university of agriculture in krakow. each food ration was prepared and analyzed twice in 2 samples (each ration was evaluated in 4 iterations). tables 3 and 4 present the basic list of products and dishes in the weekly menus for athletes (women and men). table 3. weekly menu prepared for women (the list of products/dishes). breakfast lunch dinner supper w1 strawberry porridge chocolate pudding and a nectarine tomato soup with pasta, grilled chicken breast with rice and grilled vegetables rye bread with fish spread and vegetable salad w2 rye bread with honey, white cheese and vegetables banana shake (with buttermilk) tomato cream soup, beef chops with barley groats, raw salad (carrot, apple) vegetable salad with toasts w3 muesli with natural yoghurt, rye bread with white cheese and vegetables fruit salad with natural yoghurt cucumber soup with rice, tagliatelle with shrimp layer salad (vegetables, boiled eggs) with yoghurt sauce and white rye bread w4 scrambled eggs with rye bread, an orange steamed dumplings with shake (buttermilk, strawberries) vegetable soup with potatoes, spaghetti bolognese rye bread with, white cheese and vegetables, fruit yoghurt w5 porridge with natural yoghurt, rye bread with chicken ham and vegetables yoghurt ice-cream zucchini cream soup, cod fillet with rice and boiled vegetables pancakes with strawberry jam, rye bread with cheese and tomato w6 rye bread with egg spread, natural yoghurt, lettuce, a nectarine pancakes with roasted apple beetroot soup with potatoes, farfalle with chicken and broccoli rye bread with white cheese spread and tomatoes w7 rye bread with strawberry jam and mozarella, tomato, natural yoghurt white rice with strawberry mousse tomato cream cheese, beef steak with pearl barley and lettuce lecsó with white rye bread ital. j. food sci., vol. 31, 2019 621 table 4. weekly menu prepared for men (the list of products/dishes). breakfast lunch dinner supper m1 scrambled eggs with rye bread, an orange steamed dumplings with strawberry shake (with buttermilk) vegetable soup, spaghetti bolognese rye bread with, white cheese and vegetables, fruit yoghurt m2 porridge with natural yoghurt, rye bread with chicken ham and vegetables yoghurt ice-cream, sponge cake zucchini cream soup, roasted cod fillet with potatoes and vegetables pancakes with strawberry jam, rye bread with cheese and tomato m3 rye bread with egg spread and lettuce, a nectarine pancakes with roasted apple beetroot soup with potatoes, farfalle with chicken and broccoli rye bread with white cheese spread and tomato, cherry yoghurt with muesli m4 porridge with raspberry, a roll with strawberry jam banana shake (with buttermilk) tomato soup with pasta, chicken breast with rice and cabbage salad nice-style salad with rye bread m5 muesli with natural yoghurt, rye bread with gouda cheese and vegetables, a banana strawberry dumplings tomato cream soup with toasts, risotto with vegetables rye bread with fish spread and vegetable salad m6 muesli with natural yoghurt, rye bread with white cheese, vegetables and strawberry jam, an apple yoghurt ice-cream, sponge cake leek cream soup with a baguette, beef steak with rice and rocket and tomato salad rye bread with chicken ham, egg and vegetables m7 muesli with natural yoghurt, rye bread with mozarella cheese and tomato fruit salad with fruit yoghurt cucumber soup with rice, chicken pizza rye bread with white cheese spread and smoked salmon with vegetables the mean mass of complete food rations for women was: 2,108.5 g (w1), 2,046.5 g (w2), 2,266.5 g (w3), 2,073.0 g (w4), 2,034.0 g (w5), 2,014.0 g (w6) and 2,085.5 g (w7), and for men: 2,359.5 g (m1), 2,572.5 g (m2), 2,454.5 g (m3), 2,824.0 g (m4), 2,566.5 g (m5), 2,507.0 g (m6) and 2,349.5 g (m7). the mean mass of an average food ration prepared for women (w1-w7) was: 2,090.42±92.29 g, and for men (m1-m7): 2,519.07±161.56 g. 2.2. preparation of materials the rations were homogenized and air-frozen using a freezing chamber (feutron-type 3626-51, germany) within 90 minutes in order to reach a temperature of -30°c in the thermal centre. the frozen material was then dried. drying parameters: initial product temperature: -30°c, condenser temperature: -52°c, shelf temperature: 20°c; drying: 6 hours of total drying time at shelf temperature of 30°c. drying was carried out for 24 hours. 2.3. chemical analyses homogenates were used to determine dry mass content, and lyophilized samples were used to test the other indices. dry mass content was determined in accordance with the aoac procedure (2005, no. 930.04) with the weighing method, by drying up to solid mass at a temperature of 105 °c. total nitrogen was determined in accordance with the aoac ital. j. food sci., vol. 31, 2019 622 procedure (2005, no 978.04) with the kjeldahl method using distillation unit b-324 (büchi, switzerland). protein content was determined using the 6.25 rate of conversion, and fat content in accordance with the aoac procedure (2005, no. 920.39) with the soxhlet method using diethyl ether-based extraction. total ash content was determined in accordance with the aoac procedure (2005, no. 920.05) by incinetaring the material at 485°c, and dietary fiber content in accordance with the aoac procedure (2005, no. 991.43) enzymatic fermentation and drying the remnants at 105 °c. total carbohydrates content and the energy value of the rations were determined in accordance with the guidelines of fao (2003). the vitamin c content in food rations was tested via the hplc method according to en 14130 (2003). the analysis was performed on a thermo scientific dionex ultimate 3000 uplc chromatograph with a dad detector. the extract was injected onto an onyx monolithic c 18 column (100 x 4.6 mm). elution was carried out using 0.1 m metaphoric acid at a flow rate of 1 ml/min. absorbance measurement was carried out at a wavelength of λ = 254 nm. the sum of l-ascorbic acid and dehydroascorbic acid was determined after reduction with l-cysteine according to en 14130 (2003). to determine antioxidant properties, 80% methanol extracts were made from lyophilized food rations using sonification. the total polyphenol content was determined using the method described by singleton et al. (1999). the appropriate amount of extracts from lyophilized food rations was collected and a reaction was carried out with the folin-ciocalteu reagent in the presence of na2co3. after 60 minutes, absorbance was read on a hitachi uv-vis spectrometer, type u-2900 (hitachi, japan) compared to the blind sample at λ = 675 nm. the results were read on the basis of a standard curve prepared for gallic acid. antioxidant activity against dpph radical (1,1-diphenyl-2-pictyhydrazole) was determined using the method described by pekkarinen et al. (1999). extracts from lyophilized food rations were mixed with a dpph radical solution, and after 10 minutes of reaction, the absorbance was measured on a hitachi uv-vis u-2900 (hitachi, japan) uvvis spectrophotometer at a wavelength of λ = 516 nm. the percentage of radical scavenging level (% rsa) was determined by referring the absorbance of extracts from lyophilized food rations to the absorbance of the blind sample. the value of antioxidant activity against dpph radical is expressed in trolox millimoles (water-soluble αtocopherol analogue – 2-carboxyl-6-hydroxyl-2,5,7,8-tetramethylchromate). antioxidant activity using the frap method was determined according to the procedure described by benzie and strain (1996). extracts from lyophilized food rations were mixed with a tptz solution (2,4,6-tripyridyl-s-triazine) and fecl3 in an acetate buffer. after 10 minutes of incubation at 37°c, absorbance was measured on a hitachi uv-vis u2900 (hitachi, japan) uv-vis spectrophotometer at a wavelength of λ = 595 nm against a blind sample. the value of antioxidant activity determined by the frap method was expressed in millimoles of fe2+ ions. 3. results the analyses showed that an average food ration prepared for women contained: 2,120.1 kcal, 90.8 g protein, 53.1 g fat and 354.0 g carbohydrates, and for men: 2,648.8 kcal, 112.5 g protein, 63.1 g fat and 447.4 g carbohydrates (tables 5 and 6). ital. j. food sci., vol. 31, 2019 623 table 5. nutritional value of food rations planned for women (m±sd). w1 w2 w3 w4 w5 w6 w7 w1-w7 energy value (kcal) 2066.0 2228.8 1969.8 2121.2 2135.0 2117.2 2203.0 2120.1±86.1 protein (g) 86.8 88.5 84.7 93.3 92.0 95.3 94.9 90.8±4.1 fat (g) 51.1 58.4 44.4 52.9 52.9 54.0 58.0 53.1±4.7 sfas (g) 12.1 18.0 12.6 13.5 14.6 13.5 19.2 14.8±2.7 mufas (g) 27.5 29.1 22.6 27.6 28.1 28.9 27.2 27.3±2.2 pufas (g) 11.4 11.3 9.1 11.8 10.1 11.7 11.6 11±1.0 carbohydrates (g) 346.6 380.9 336.7 349.3 351.4 349.2 363.8 354±14.3 dietary fiber (g) 31.9 43.7 28.8 31.3 28.6 36.8 38.5 34.2±5.6 table 6. nutritional value of food rations planned for men (m±sd). m1 m2 m3 m4 m5 m6 m7 m1-m7 energy value (kcal) 2585.7 2754.9 2578.9 2683.6 2714.8 2667.2 2556.2 2648.8±76.0 protein (g) 116.7 114.4 114.5 100.4 110.7 112.5 118.7 112.5±6.0 fat (g) 65.5 68.7 62.0 60.7 63.7 61.4 59.7 63.1±3.1 sfas (g) 15.2 18.4 15.6 15.1 21.0 19.4 21.2 18±2.7 mufas (g) 34.9 38.2 32.7 32.9 28.0 29.5 30.1 32.3±3.5 pufas (g) 15.4 12.1 13.7 12.7 14.6 12.5 8.3 12.8±2.3 carbohydrates (g) 420.7 460.0 426.9 484.3 466.5 452.9 420.4 447.4±25.1 dietary fiber (g) 38.3 40.2 36.2 50.4 41.7 36.9 34.4 39.7±5.3 w1/m1 first daily food serving, w2/m2 second daily food serving, w3/m3 third daily food serving, w4/m4 fourth daily food serving, w5/m5 fifth daily food serving, w6/m6 sixth daily food serving, w7/m7 seventh daily food serving the chemical analyses showed that 100 g fresh mass of an average food ration prepared for women contained: 5.5±2.6 mg vitamin c and 20.1±4.1 mg polyphenols, and for men, respectively: 5.6±1.4 mg and 22.9±8.1 mg. the antioxidant properties of an average ration for women expressed as reducing power (frap index) in 100 g fresh mass was found to be 8.2±0.7 mmol fe+2, and for men 8.9±0.9 mmol fe+2. the antioxidant properties of an average ration developed for women and men expressed as antiradical activity against dpph in 100 g fresh mass was respectively: 2.7±02 mmol and 2.7±0.4 mmol trolox equivalent. the respective values for dry mass were higher (tables 7 and 8). ital. j. food sci., vol. 31, 2019 624 table 7. vitamin c and polyphenol content as well as antioxidant potential of food rations planned for women (per 100 g fresh and dry mass) (m±sd). evaluated indices w1 w2 w3 w4 w5 w6 w7 w1-w7 100g fm vitamin c (mg) 5.0±0.3 3.5±0.2 5.3±0.2 3.6±0.2 5.0±0.2 11.1±0.4 5.0±0.2 5.5±2.6 polyphenols as gallic acid (mg) 16.4±5.4 23.1±3.4 23.2±2.0 21.2±5.6 12.60.9 21.6±1.2 22.9±0.8 20.1±4.1 reducing activity (the frap method) (mmol fe+2) 7.5±0.4 9.2±0.4 7.6±0.4 7.9±0.2 7.9±0.8 7.9±0.4 9.1±0.8 8.2±0.7 antioxidant activity against dpph (mmol te – trolox equivalent) 2.3±0.6 2.8±0.2 2.8±0.1 3.0±0.4 2.7±0.2 2.8±0.2 2.8±0.3 2.7±0.2 100g dm vitamin c (mg) 21.0±1.0 13.2±0.6 24.3±1.2 14.3±0.7 19.8±0.9 43.3±2.1 19.6±0.9 22.2±10.1 polyphenols (mg) as gallic acid 68.3±21.5 86.1±13.9 106.8±9.3 84.0±21.9 49.6±4.2 83.9±6.5 88.9±2.8 81.1±17.9 reducing activity (the frap method) (mmol fe+2) 31.3±1.1 34.4±1.4 35.1±2.1 31.6±0.6 31.1±3.8 31.0±1.8 35.5±3.7 32.9±2.0 antioxidant activity against dpph (mmol te – trolox equivalent) 9.4±2.3 10.5±0.6 12.9±0.4 12.0±1.5 10.5±0.8 10.9±0.8 10.9±1.1 11.0±1.1 antioxidant activity against dpph (%rsa for extract: 20 mg lyophilized food ration/g) 13.1±3.1 14.4±0.9 17.8±0.5 16.6±2.0 14.5±1.2 15.0±1.1 15.1±1.5 15.2±1.6 table 8. vitamin c and polyphenol content as well as antioxidant potential of food rations planned for men (per 100 g fresh and dry mass) (m±sd). evaluated indices m1 m2 m3 m4 m5 m6 m7 m1-m7 100g fm vitamin c (mg) 4.9±0.3 5.9±0.3 4.2±0.3 4.8±0.3 5.1±0.3 8.6±0.5 5.8±0.3 5.6±1.4 polyphenols (mg) as gallic acid 26.2±1.0 19.4±0.8 20.1±0.2 18.7±1.3 16.9±2.8 19.4±1.7 40.2±6.2 22.9±8.1 reducing activity (the frap method) (mmol fe+2) 9.1±0.1 7.4±0.2 8.5±0.4 8.2±0.4 9.0±0.5 9.6±0.7 10.4±0.2 8.9±0.9 antioxidant activity against dpph (mmol te – trolox equivalent) 2.8±0.1 2.4±0.2 2.5±0.2 2.6±0.1 2.8±0.9 2.3±1.2 3.6±0.3 2.7±0.4 ital. j. food sci., vol. 31, 2019 625 100g dm vitamin c (mg) 18.4±1.2 22.8±1.4 16.4±1.0 20.2±1.3 19.8±1.2 33.1±2.1 21.8±1.4 21.8±5.4 polyphenols (mg) as gallic acid 97.7±3.4 74.8±3.5 78.8±1.9 78.8±5.7 65.2±11.1 75.1±8.2 151.4±25.2 88.8±29.2 reducing activity (the frap method) (mmol fe+2) 33.8±0.3 28.4±0.7 33.3±1.2 34.8±1.8 34.7±2.1 37.2±2.8 39.1±1.1 34.5±3.4 antioxidant activity against dpph (mmol te – trolox equivalent) 10.3±0.1 9.4±0.8 9.7±0.9 11.0±0.5 10.8±3.8 8.9±4.7 13.6±1.5 10.5±1.6 antioxidant activity against dpph (%rsa for extract: 20 mg lyophilized food ration/g) 14.2±0.2 13.0±1.0 13.4±1.3 15.2±0.6 15.0±5.2 12.3±6.4 18.8±2.1 14.6±2.1 4. discussion different groups of sports disciplines have different dietary requirements, connected with the kind of exercise and the dominant energy pathways, development of specific motor properties and the rigor of maintaining body mass and composition. one of special groups is athletes who train disciplines that require maintaining low body mass and low fat content, which is connected with planning a balanced diet with relatively lower energy intake but high nutrient density (rich in i.a., vegetables, fruit, wholegrain cereal products, legumes, fish and nuts) (thomas et al., 2016). the presented original study showed that food rations prepared for athletes (women and men) of disciplines that require maintaining low body mass, in accordance with qualitative and quantitative recommendations, balanced in terms of energy and basic nutrients intake, rich in fruit and vegetables and other products with high nutrient value and with high content of dietary antioxidants, including vitamin c and polyphenols, ensuring high antioxidant properties (with the energy intake of 2,120 kcal in female and 2,649 kcal in male athletes). high vitamin c and polyphenol content results from including in the prepared food rations an appropriate number of portions of products being natural sources of dietary antioxidants (i.a., fruit and/or vegetables in each meal). the mean content of vitamin c found in the prepared food rations, i.e., 5.5 mg/100 g fresh mass (women) and 5.6 mg/100 g fresh mass (men), with the assumption of their average weight of 2,090.42 g for women and 2,519.07 g for men, corresponds to vitamin c content of 114.97 mg (women) and 141.07 mg (men). comparing the content of vitamin c in an average (within a week) food ration to the norms of physiological demand for vitamin c (rda: 75 mg/day for women and 90 mg/day for men) (kreider et al., 2010), additionally increased in athletes (potgieter, 2013), shows that this diet can ensure its normative intake and the demand can be covered, also in the conditions of physical exercise. the described mean content of vitamin c in an average food ration corresponds to satisfying the rda demand of 153.29% (in women) and 156.74% (in men). high vitamin c content in the prepared food rations results from high vitamin c content in products and dishes included in them, such as raw fruit and vegetables (kunachowicz et al., 2005). extremely high content of vitamin c occurs in red peppers, but it is also high in tomatoes and lettuce (kunachowicz et al., 2005), which are the ingredients of many salads and (cream) soups planned in the analyzed food rations for athletes. ital. j. food sci., vol. 31, 2019 626 the total content of polyphenols in the analyzed food rations is also the product of their high content in the ingredients. the mean content of polyphenols found in the prepared food rations, i.e., 20.1 mg/100 g fresh mass (women) and 22.9 mg/100 g fresh mass (men), with the assumption of their average weight of 2,090.42 g for women and 2,519.07 g for men, corresponds to polyphenol content of 420.17 mg (women) and 576.87 mg (men). the recommended intake of polyphenols, promoting good functioning of the body, is estimated at 250-500 mg/day (sikora et al., 2008). an average food ration, supplying this amount of polyphenols, covers the recommended intake. a study aimed to estimate the intake of phenolic compounds with the diet showed that a statistical pole consumes approx. 440 mg polyphenols/day, and important sources of these antioxidants are vegetables (31%) and fruit (23%) (sikora et al., 2008). it is estimated that western societies consume on average 50-800 mg, and eastern up to 2 g flavonoids/day. an average mediterranean diet provides 100-1000 mg flavonoids/day (sikora et al., 2008). in the czech population the average intake of plant polyphenols was 426.6 mg/day (less than in other european as well as non-european, countries, including spain, france, ireland, brazil etc.) (zloch et al., 2018). a population of elderly japanese (mostly men) consumed 1492 mg/day of polyphenols on average, and coffee and green tea were the largest sources of polyphenols in their daily life (taguchi et al., 2015). total polyphenol content in fruit and vegetables varies. in vegetables the content is the highest in: broccoli (290 mg/100 g), carrots (156 mg/100 g) and onions (150 mg/100 g) (cieślik et al., 2006). in apples, peaches and mandarines the content of extracted polyphenols was between 18.8 and 28.0 mg/100 g fresh fruit (arranz et al., 2009). out of the vegetables used to prepare the dishes in the analyzed food rations, the richest in polyphenols are red peppers (68.50 mg/100 g), onions (45.81 mg/100 g) and garlic (36.10 mg/100 g) (cieślik et al., 2006). foods' antioxidant properties are correlated with the content of substances with antioxidant properties, including vitamin c and polyphenols. the antioxidant potential of an average food ration determined in this study, expressed as reducing power (frap index) was: 8.2 mmol fe+2/100 g ration fresh mass (for women) and 8.9 mmol fe+2/100 g ration (for men), and expressed as antiradical activity against dpph was: 2.7 mmol/100 g ration fresh mass (for women and men). antioxidant properties of food rations prepared for athletes is the product of content of bioactive substances with antioxidant properties and antioxidant potential of individual products/dishes and results from including the recommended number of portions of fruit and vegetables with high antioxidant activity, including honey and wholegrain cereal products. it is worth pointing out that antioxidant capacity of vegetables is usually lower than that of fruit, especially berries, and higher than that of cereal products. antioxidant activity of fruit varies from 1.02 (pears) to 3.91 mmol/100 g (strawberries), and of vegetables, mushrooms and legumes from 0.27 (cucumbers) to 6.91 mmol/100 g (beans). high potential (>2 mmol/100 g) also occurs in peas, dill, dock, red cabbage and beetroot. especially high antioxidant potential expressed as dpph scavenging activity has been described for brussels sprouts and red peppers (ec50 7.8 mg and 11.9 mg, respectively) (cieślik et al., 2006; sikora et al., 2008). significant antioxidant properties of fruit and vegetable snacks have also been reported (gramza-michałowska and człapka-matyasik, 2011). with reference to antioxidant properties of wholegrain cereal products, studies have shown that the antioxidant potential of boiled wholegrain pasta (expressed as frap index) is from 3.26±0.08 to 19.52±1.28 μmol/g dry mass (durazzo et al., 2014). another study concerning dishes preferred by athletes showed high antioxidant properties of chicken salad (0.29 mmol/100 g) and tomato spaghetti (0.35 mmol/100 g) (gacek et al., 2012). ital. j. food sci., vol. 31, 2019 627 antioxidant activity of the prepared and analyzed food rations, being the product of content of antioxidant substances, including vitamin c and polyphenols, proves they are useful in a rational diet, also for people engaging in intensive physical exercise, who need more antioxidants. exogenous antioxidants have an impact on the total antioxidant capacity and physical fitness in athletes (morillas-ruiz et al., 2006; schneider et al., 2018), so an important aspect of a rational diet is the appropriate intake of vegetable bioactive substances. research has confirmed the importance of diet with high antioxidant properties for the restoration of athletes' antioxidant status. in this respect, it has been shown that antioxidant-rich diet improved the redox status of triathlon athletes (schneider et al., 2018), and high consumption of flavones from cocoa improved the total antioxidant capacity during workout and regeneration in professional cyclists (decroix et al., 2017). it has been confirmed beyond doubt that satisfying higher nutritional demand, also in terms of vitamin c and other antioxidants, promotes the implementation of dietary strategies connected with muscle regeneration, glycogen replenishment, preventing fatigue, the improvement of immunity and preparation for further training and contests (heaton et al., 2017; myburgh, 2014). it is recommended to apply dietary strategies that improve diet antioxidant potential (so-called antioxidant food ration) (yavari et al., 2015). when discussing the importance of antioxidants in the diet of physically active people, we may refer to a study that showed a positive influence of 6-week nordic walking training on the improvement of blood antioxidant protection system in women over age 55 (cebula et al., 2017). due to some cases of nutritional deficiencies and because of the importance of antioxidants for increasing antioxidant activity and protecting skeletal muscles from oxidation damage caused by physical exercise, researchers attempt to study the supplementation of athletes' diet with antioxidant substances, including melatonin (leonardo-mendonca et al., 2017), coenzyme q (orlando et al., 2018), vitamin c (yavari et al., 2015), and l-carnitine (sung et al., 2016) and whey protein (xu et al., 2011). some studies have also shown the risk of negative impact of supplementing high doses of vitamin c (1g/day) and e (≥260 iu/day) on disorders in skeletal muscles adjusted to long training sessions (mason et al., 2016). functional drinks based on almonds and olive oil enriched with α-tocopherol and docosahexaenoic acid (dha) can also be used to modulate oxidative stress and improve effort tolerance of athletes. they also help improve blood polyphenol concentration in older athletes and increase the expression of antioxidant enzyme genes in peripheral blood cells after exercise in young athletes (capo et al., 2016). supplementation with purple grape juice displayed ergogenic activity (by delaying exhaustion) and led to increasing antioxidant activity and decreasing the concentration of inflammation markers in recreational runners (toscano et al., 2015). literature also includes other examples of supplementing athletes' diet with antioxidant substances (yavari et al., 2015). the presented results can be useful in planning diet and dietary strategies improving the antioxidant properties of the diet of people with high physical activity and increased nutritional needs. regular consumption of fresh fruit and vegetables, whole grains, legumes and beans, sprouts and seeds is an effective and safe way to cover the antioxidant needs of physically active people. ital. j. food sci., vol. 31, 2019 628 5. conclusions balanced food rations prepared for athletes (with the mean energy value of approx. 2,120 kcal for women and approx. 2,649 kcal for men included the normative amount of vitamin c (114.97 mg – women and 141.07 mg – men) and polyphenols (420.17 mg – women and 576.87 mg – men). balanced food rations for athletes, including fruit and/or vegetables in each meal and the normative amount of vitamin c and polyphenols, have high antioxidant properties. preparing balanced food rations for athletes, providing not only appropriate amounts of energy and macroelements but also bioactive substances (vitamin c and polyphenols) as well as high antioxidant properties is possible, though not easy. chemical analyses confirmed the nutritional value of food rations planned on the basis of tables of products' and dishes' nutritional value. reserch funding the study was supported from the european union funds within the framework of the european social fund “doctus lesser poland fund for grants for phd students”. references aires a., carvalho r., matos m., carnide v., silva a.p. and goncalves b. 2017. variation of chemical constituents, antioxidant activity, and endogenous plant hormones throughout different ripening stages of highbush blueberry (vaccinium corymbosum l.) cultivars produced in centre of portugal. journal of food biochemistry 41:416. aliakbarlu j., 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*corresponding author: tel.: +390116708558 e-mail address: luca.rolle@unito.it abstract this study aimed to investigate the physico-chemical characteristics of ‘‘freisa’’ red winegrapes from five growing areas at three ripeness degrees. wines were produced with grapes from each growing area. results highlighted that “freisa” grapes have relatively hard skins (break force>0.8 n), medium anthocyanin content (>800 mg/kg grapes), and high flavanol content (pro>2500, fva>1200 mg/kg grapes). skin thickness, total anthocyanin and flavanol contents were significantly influenced by environmental conditions and ripeness. grapes from monferrato area showed the thickest skins and highest contents of anthocyanins but also of seed flavanols. coherently, wine color characteristics and phenolic composition depended on growing area. keywords: phenolic composition, texture parameters, “freisa” grapes, growing areas, ripeness, minor varieties ital. j. food sci., vol. 31, 2019 686 1. introduction vitis vinifera l. cv. “freisa” is an italian native and historic red grape variety included in the national register of vine varieties from 1970 (gu 149-17/06/1970). this variety grows mainly in the central part of the piedmont hills (north-west italy), in the municipalities of albugnano (at), castelnuovo don bosco (at), pino d’asti (at), and moncucco torinese (at) (ajassa et al., 2015), although vineyards of this grape variety are present also in other zones of the piedmont region. the first citation of “freisa” grapes is dated in 1517 in a customs document of pancalieri town (to), while the first vine-plantation was near neive (cn) in 1692 (mainardi, 2003). since then, in the centuries “freisa” grapevines spread throughout all piedmont because of its good yield, resistance to late frosts and diseases, plasmopara viticola in particular (pecile et al., 2018). outside piedmont, this variety has a little spread in other three regions of italy (valle d’aosta, lombardia, and veneto), while abroad some vines are planted in argentina and in the usa state of california (schneider et al., 2013). the italian national institute of statistics (istat) in 1970 reported in italy 7,410 hectares planted with “freisa” variety, while the last census in 2010 recorded a decrease of planted surface to 1,041 hectares. of those, 80% of “freisa” planted area is located in the piedmont region, and about 420 hectares in the province of asti (regione piemonte, 2018). nowadays, the italian nursery production of “freisa” vines is active with a production over 150,000 vine plants in 2017 vintage (pecile et al., 2018). the strong linkage between this variety and its piedmont growing area is evidenced by five designations of controlled origin (doc, denominazioni di origine controllata): freisa d’asti doc, freisa di chieri doc, colli tortonesi doc freisa, monferrato doc freisa, and langhe doc freisa (rossi, 2012). furthermore, from 2014 three of these designations (freisa d’asti doc, langhe doc, and monferrato doc) are included in a major wine-producing area of piedmont that became a unesco world heritage site (unesco italia, 2019). over the years, some investigations on “freisa” variety were done. regarding viticultural aspects, lisa et al. (2005) showed that the most suitable training system for “freisa” variety is the lateral cordon trellis system, able to satisfy quality and consistent yields. moreover, astegiano and ciolfi (1974), cravero and di stefano (1992), gerbi et al. (2005), and rolle et al. (2008a) studied some aspects of the phenolic composition of “freisa” grapes and showed that the content of tannins was generally high (> 3500 mg/kg) and of anthocyanins in the skins was satisfactory (> 800 mg/kg). rolle et al. (2008a) also observed a difficult extraction of anthocyanins from the skin into the must and, on the contrary, a relevant contribution of tannins from the seeds. furthermore, gerbi et al. (2005) and rolle and guidoni (2007) showed that the anthocyanin profile of “freisa” grapes was characterized by preponderance of di-substituted forms, cyanidin3-o-glucoside (about 20%) and peonidin-3-o-glucoside (about 50%), similarly to “nebbiolo” grape variety. indeed, schneider et al. (2004) and lacombe et al. (2013) studied the genetic profile of “freisa” variety and established that it is an offspring of the italian red grape varieties “nebbiolo” and “avanà”. furthermore, schneider et al. (2004) found that there is a genetic relationship between “freisa” variety and the white grape variety “viognier”. wines produced from “freisa” grapes have had supporters and critics in the past. generally, the wine made from ripe “freisa” grapes has smooth and lightly astringent tannins. however, when the grapes do not reach the optimal ripeness degree, the resulting wines have a high level of acidity and tannicity, leading to sensory perceptions of ital. j. food sci., vol. 31, 2019 687 excessive astringency and bitterness (schneider et al., 2013). in order to mitigate this fact and to produce quality wines, both for wines to be consumed young or after an ageing period, the winemaking of “freisa” grapes often involves the partial removal of the seeds from the fermenting must after at least 48 hours from the beginning of alcoholic fermentation (rolle et al., 2008a). although some technical information on “freisa” grapes is already present in scientific literature, to our knowledge, no comprehensive physico-chemical characterization of grapes and wines has already been reported. therefore, the main aim of this work was to investigate the mechanical properties of berry skin and the total extractable phenolic composition of berry skins and seeds from “freisa” grapes, taking into consideration five different growing areas in the south-east of piedmont region and three different grape ripeness levels. indeed, the study aimed to assess the real impact of the production area and/or ripeness degree on grape characteristics. finally, monovarietal wines were produced from “freisa” grapes of each production area in order to propose appropriate enological techniques according to the grape features. 2. materials and methods 2.1. vineyards grape samples of vitis vinifera l. cv. “freisa” were collected from different vineyards located in five south-east piedmont growing areas (north-west italy): astigiano (san paolo solbrito, at), collina torinese (chieri, to), langhe (barolo, cn), tortonese (monleale, al), and monferrato (casorzo, at). the production zone was delimited by the following geographical coordinates: 44.374−45.023 n latitude and 7.500–8.974 e longitude, including the five designations of controlled origin linked to “freisa” wines: freisa d’asti doc, freisa di chieri doc, langhe doc freisa, colli tortonesi doc freisa, and monferrato doc freisa, respectively. the five mentioned vineyards, chosen each as representative of the respective growing location, were planted for commercial use and met the designation rules: the vines were at least 10 years old, planted on medium slope hills with exposure south (astigiano, collina torinese, and langhe locations), east (tortonese location) or south-east (monferrato location), vertical growth by lateral cordon trellis system, and guyot pruned. from each production area, “freisa” grapes were harvested in plastic boxes (maximum capacity of 20 kg to avoid grape crushing during transport) and about 20 kg were randomly selected and transported to the laboratory, while 150 kg were brought to the experimental cellar of the university of turin for the winemaking process. 2.2. grape samples and density class selection once in the laboratory, the berries were manually separated from the stalk with harvest shears and then placed on paper trays. about 200 berries were randomly taken for the determination of standard chemical parameters and other 200 berries were used for the evaluation of phenolic extractability indices. all the other berries were classified according to their density (i.e., total soluble solid content) by flotation as described by fournand et al. (2006) and rolle et al. (2011b), in order to improve the physiological homogeneity inside the density class, to permit the comparative evaluation between ripeness levels at harvest, and to assess growing area effects. ital. j. food sci., vol. 31, 2019 688 in brief, for each growing area, the berries were floated and separated using saline solutions ranging from 130 to 190 g/l of sodium chloride, with densities spread between 1088 and 1125 kg/m3. after flotation, all berries were washed with water and dried using absorbent paper. the three most represented density classes (by weight) were chosen: 1100 kg/m3 (lower), 1107 kg/m3 (middle), and 1115 kg/m3 (higher). for each one, 20 berries were randomly selected for the determination of skin mechanical properties, and three sub-samples of 10 berries were used for the determination of skin and seed phenolic composition. 2.3. mechanical properties of grapes for the texture analysis test, a universal testing machine (utm) taxt2i texture analyzer (stable micro systems, godalming, surrey, uk), equipped with a hdp/90 platform and a 5 kg load cell, was used. the determination of the skin hardness parameters was carried out, on the whole berries placed in a horizontal plane on the metal plate of the utm, by a puncture test using a sms p/2 n needle probe (letaief et al., 2008). berry skin break force (n, as fsk), berry skin break energy (mj, as wsk), and berry skin young’s modulus (n/mm, as esk) were measured. then, berry skin thickness (µm, as spsk) was determined, on a portion of skin (ca. 0.25 cm2) removed by a razor blade from the lateral side of each berry, by a compression test using a 2-mm sms p/2 flat cylindral probe (río segade et al., 2011a). for each sample, 20 berries were individually analyzed for each test. 2.4. phenolic compounds extractability trials for each replicate (n = 3), 10 berries were weighed and peeled. the skins and the seeds were manually removed from the pulp using a laboratory spatula, counted, weighed, and quickly immersed separately in 50 ml of a hydroalcoholic buffer solution at ph 3.20 containing 5 g/l of tartaric acid, 2 g/l of na2s2o5, and 12% v/v of ethanol. the skin and seed samples were then placed in an oven at 25°c for 12 h and one week, respectively (di stefano and cravero, 1991; río segade et al., 2014). the skins into the buffer solution were homogenized at 8000 rpm for 1 min with an ultra-turrax t25 high-speed homogenizer (ika labortechnik, staufen, germany), centrifuged for 5 min at 3500 × g at 20°c using a pk 131 centrifuge (alc international, milano, italy), and the supernatant was collected for analysis. in the case of seeds, they were removed from the buffer solution while the extract was used for the determination of the seed phenolic fraction. 2.5. wine production in brief, the “freisa” grapes harvested in each location were separately destemmed, crushed, and the mash was added with 40 mg/l of potassium metabisulfite. after about 8 h, selected yeasts (lalvin brl97, lallemand inc., montreal, canada) were inoculated at a dose of 20 g/hl. alcoholic and malolactic fermentation were carried out at controlled temperature of 27±2°c and 19±1°c, respectively. at the end of the fermentations, 60 mg/l of potassium metabisulfite were added and wines were cold-stabilized at 0 ºc for 2 weeks, filtered (seitz k300 grade filter sheets, pall corporation, port washington, ny, usa), and then bottled in glass bottles of 0.75 l with cork stoppers. ital. j. food sci., vol. 31, 2019 689 2.6. chemical analysis 2.6.1 reagents and standards solvents of hplc-gradient grade and all other chemicals of analytical-reagent grade were purchased from sigma-aldrich (milan, italy). the solutions were prepared in deionized water produced by a milli-q system (merck millipore, darmstadt, de). chemical standards of malvidin-3-o-glucoside chloride and cyanidin chloride were supplied by extrasynthèse (genay, france), whereas (+)-catechin was purchased from sigma-aldrich. 2.6.2 standard chemical parameters of grapes and wines standard chemical parameters of grape musts, obtained by manual crushing and centrifugation, and wines were determined according to oiv (2016) methods. in particular, the following parameters were determined: grape potential alcohol degree (% v/v; oiv-oeno 466:r2012), must ph (oiv-ma-as313-15:r2011), total acidity (g/l as tartaric acid; oiv-ma-as313-01:r2015), wine alcohol content (% v/v; oiv-ma-as31201a:r2016), and wine dry net extract (g/l; oiv-ma-as2-03b:r2012 and oiv-ma-as31102:r2009). the contents (g/l) of reducing sugars (as sum of glucose and fructose), tartaric acid and malic acid in grape musts were determined by hplc (torchio et al., 2010). 2.6.3 phenolic extractability indices and phenolic composition of grapes and wines phenolic extractability indices in grape berries were assessed for each sample in accordance with the procedure described by glories and augustin (1993), modified by cagnasso et al. (2008). the extractant solution at ph 1 was prepared just before use by mixing equal volumes of 1.0 m of hydrochloric acid and 2 g/l of potassium metabisulfite, while the extraction at ph 3.2 was carried out using a buffer solution containing 5 g/l of tartaric acid. the parameters obtained at ph 1 and ph 3.2, namely total phenolic content (a280) and total anthocyanin content (ta1 and ta3.2), were used for the determination of the following extractability indexes: cellular maturity (ea%) and seed maturity (mp%). the latter index was assessed by taking into consideration the average ratio (tar) between the skin contents of total phenols (a280) and total anthocyanins (expressed as g/l), equal to the value of 40 (cagnasso et al., 2008). the ea% and mp% indexes were calculated as follows: ea% = [(ta1 − ta3.2)/ta1] × 100; mp% = {[a280 − ((ta3.2/1000) × tar)] /a280} × 100). on berry skin and seed extracts, and on resulting wines, spectrophotometric assessments were done in order to evaluate their phenolic composition. total anthocyanins index (mg malvidin-3-o-glucoside chloride/kg grape or l wine, as ta) was evaluated in berry skin extracts and wines (di stefano and cravero, 1991; torchio et al., 2010), while monomeric anthocyanins index (mg malvidin-3-o-glucoside chloride/l wine, as ma) was determined only in wines previous isolation on polyvinylpolypyrrolidone (pvpp) and elution with an ethanol:water:hcl 37% (70:30:1) solution (di stefano et al., 1989). for both skin and seed extracts, and wines, total flavonoids index (mg (+)-catechin/kg grape or l wine, as tf), flavanols vanillin assay (mg (+)-catechin/kg grape or l wine, as fva), and proanthocyanidins assay based on bate-smith reaction (mg cyanidin chloride/ kg grape or l wine, as pro) were evaluated (di stefano and cravero, 1991; torchio et al., 2010). a uv-1800 spectrophotometer (shimadzu corporation, kyoto, japan) was used for all analysis. the skin anthocyanin profile was determined by hplc ital. j. food sci., vol. 31, 2019 690 dad after purification on a 1-g sep-pak c18 spe cartridge (waters corporation, milford, ma, usa) according to the protocol described by río segade et al. (2014). the chromatographic separation was performed on a lichrocart analytical column (25 cm × 0.4 cm i.d.) purchased from merck (darmstadt, germany) and packed with lichrospher 100 rp-18 (5 μm) particles supplied by alltech (deerfield, il, usa), using formic acid/water (10:90, v/v) and formic acid/methanol/water (10:50:40, v/v) as mobile phases. the different individual anthocyanin forms were expressed as area percentage of total forms. 2.6.4 wine color parameters wine color was evaluated by the ciel*a*b* parameters including lightness (l*), red/green color coordinate (a*), and yellow/blue color coordinate (b*) according to the method oivma-as2-11 (oiv, 2016). furthermore, the color intensity (on 10 mm optical path) and the color hue were calculated using the method oiv-ma-as2-07b (oiv, 2016). a uv-1800 spectrophotometer (shimazdu corporation) was used with 2-mm path length cuvettes. 2.7. statistical analysis statistical analyses were carried out using r statistics software version 3.4.0 (r core team, 2017). levene’s and shapiro-wilk’s tests were used for assessing the homogeneity of variance and analysis of variance (anova) residuals normality, respectively. in case of heteroscedasticity, we used the anova with welch’s correction, followed by tamhane’s t2 post-hoc test when null hypothesis was rejected. in the case of homoscedasticity, we used one-way anova and the tukey hsd test for p < 0.05 to assess significant differences between groups. 3. results and discussion 3.1. grape chemical composition at harvest and berry sorting in all five “freisa” grape samples harvested from the representative vineyards of each growing area studied, the content of sugars at harvest was suitable to meet the minimum potential alcohol degree [% v/v] indicated in the disciplinary rules of each designation of controlled origin (table 1). regarding the phenolic maturity of unsorted grapes, the cellular maturity index (ea%) and the seed maturity index (mp%) varied between 35-45% and 77-82%, respectively, depending on the growing area. these values agreed with those previously reported by rolle et al. (2008a) and confirmed particular varietal characteristics, such as the difficulty for releasing anthocyanins from the skin during maceration (ea% >30) and a high contribution of tannins from the seeds (mp% >30) (ribéreau-gayon et al., 2006). moreover, in the present work, the astigiano and tortonese growing areas showed the lowest contents not only of potential anthocyanins (extracted at ph 1) but also of extractable anthocyanins (extracted at ph 3.2), the grapes from tortonese area also showing the lowest anthocyanin extractability (ea% =45). ital. j. food sci., vol. 31, 2019 691 table 1. grape must standard chemical parameters and phenolic extractability indices of “freisa” grapes harvested in five piedmont growing locations. location parameter astigiano collina torinese langhe tortonese monferrato designation of origin freisa d’asti doc freisa di chieri doc langhe doc freisa colli tortonesi doc freisa monferrato doc freisa minimum potential alcohol content [% v/v] 10.5 10.5 10.5 11.0 10.0 density at 20°c 1.102 1.106 1.097 1.110 1.106 sugars content [g/l] 239 249 227 258 247 potential alcohol degree [% v/v] 14.2 14.8 13.5 15.3 14.6 ph 3.57 3.44 3.40 3.61 3.59 total acidity [g/l as tartaric acid] 5.51 6.30 6.65 6.27 6.43 tartaric acid [g/l] 5.54 6.81 6.61 6.76 5.18 malic acid [g/l] 2.33 2.00 2.35 2.23 3.31 ea% 37 35 40 45 40 mp% 82 78 77 82 77 ta3.2 [mg/kg malvidin-3-o-glucoside chloride] 605 762 758 571 766 a280 on extract at ph 3.2 132 137 130 125 135 ta1 [mg/kg malvidin-3-o-glucoside chloride] 959 1169 1269 1032 1273 ea% = cellular maturity index; mp% = seed maturity index; ta3.2 = total anthocyanins extracted at ph 3.2; a280 = absorbance at 280 nm; ta1 = total anthocyanins extracted at ph 1. the distribution percentages of all “freisa” grape berries in different density classes at harvest were determined for the five vineyard locations. in agreement with data reported in scientific literature (kontoudakis et al., 2011; rolle et al., 2012), bell-shaped distributions were found (data not shown). in general, as already reported by fournand et al. (2006), the difference in the total sugar content of the berries belonging to two consecutive density classes was ~17 g/l (i.e. 1% v/v potential alcohol). for all the growing areas, the three more representative density classes were 1100 kg/m3, 1107 kg/m3, and 1115 kg/m3. more than 80% of the berries were belonging to these three density groups. in table 2 some berry physical traits are shown for the three considered density groups. in order to evaluate the real impact of the growing area and ripeness degree, two comparisons were done by statistical analysis: the first among the five locations considering the same density class, the second among the three density classes inside each growing area. the first comparison showed that the grapes from astigiano location presented, in two density classes out of three (the middle and the higher), the highest values of both berry and skin weight while, on the contrary, the grapes from collina torinese had the lowest berry weight values. for these two parameters, the second comparison showed that the ratio of skin and berry weight increases with increasing the sugar contents. this ratio ranged between 7.3 and 9.1% in the low density class, 8.2 and 8.9% in the middle density class, and 9.6 and 12.2% in the high density class. ital. j. food sci., vol. 31, 2019 692 table 2. physical parameters of “freisa” grapes harvested in five piedmont growing locations and sorted according to density. density class location berry weight (g) skin weight (g) seeds (n) single seed weight (g) 1100 kg/m3 astigiano 2.21ab,α 0.17ab,α 2.0a 0.04ab collina torinese 1.93a,αβ 0.14a 2.5ab,αβ 0.04a langhe 2.18ab 0.18bc 2.3ab 0.04ab tortonese 2.18ab 0.18ab,α 2.1a,β 0.05b monferrato 2.41b,β 0.22c,β 2.9b,β 0.04ab sign (1) * *** ** * 1107 kg/m3 astigiano 2.44b,β 0.20b,β 2.4b 0.04 collina torinese 2.03a,β 0.18ab 2.7b,β 0.04 langhe 2.19ab 0.19ab 2.5b 0.04 tortonese 1.97a 0.17a,α 1.7a,α 0.05 monferrato 2.10a,α 0.18ab,α 2.6b,αβ 0.04 sign (1) ** * ** ns 1115 kg/m3 astigiano 2.29c,α 0.24c,γ 2.6 0.04 collina torinese 1.78a,α 0.17a 2.1α 0.04 langhe 1.90ab 0.21ab 2.1 0.04 tortonese 1.97ab 0.24bc,β 2.0αβ 0.04 monferrato 2.03b,α 0.21bc,β 2.4α 0.04 sign (1) *** *** ns ns sign (2) **, *, ns, ns, * ***, ns, ns, **, ** ns, *, ns, *, * ns, ns, ns, ns, ns values are expressed as average (n = 3). different latin letters within the same column indicate significant differences (1) among zones for the same berry density (tukey hsd test; p < 0.05). different greek letters within the same column indicate significant differences (2) among the three density classes for the same zone (tukey hsd test; p < 0.05). sign: *, **, ***, and "ns" indicate significance at p < 0.05, 0.01, 0.001, and not significant, respectively. regarding seeds, although their weight was similar among the locations and the density classes, the variability of their number influenced the ratio between seeds and berry weight, the highest percentage value (5.4%) being in the grapes from collina torinese location belonging to the middle density class and the lowest value (3.6%) being in the grapes from astigiano and tortonese location belonging to the lower and the middle density class. grape berries of the same diameter and/or fresh weight have different total soluble solid concentrations as a consequence of the functional relationship among berry sugar accumulation, transpiration, and water accumulation (šuklje et al., 2012). indeed, they may belong to different density classes. this aspect could be related not only to the environmental conditions of the vineyard but also to the position of a specific berry in a bunch and the relative position of a bunch in the vine. all these factors are of great importance because they influence the relative skin and seed weight and therefore berry phenolic composition (roby et al., 2004). ital. j. food sci., vol. 31, 2019 693 3.2. berry skin mechanical properties the berry skin mechanical parameters measured on “freisa” grapes from the five different locations and sorted by density are available in table 3. the data showed high values of skin break force (fsk ≥ 0.824 n) and skin break energy (wsk ≥ 0.782 mj), in agreement with letaief et al. (2008) who reported higher values of skin hardness parameters for “freisa” grapes with relation to other six grapevine varieties, including the genetically related “nebbiolo” grape variety. although “freisa” can be considered a grape variety with a ‘hard’ skin, when compared with other varieties growing in the same vineyard in a single vintage (i.e. same bioclimatic indexes), the skin hardness parameters (fsk and wsk) for “freisa” berries did not show the highest values (rolle et al., 2011a). in this sense, “becuet” (variety grown in mountain environment) and some varieties of “teinturier” (cultivar with coloured pulp) were characterized by harder skins. table 3. berry skin mechanical properties of “freisa” grapes harvested in five piedmont growing locations and sorted according to density. values are expressed as average ± standard deviation (n = 20). different latin letters within the same column indicate significant differences (1) among zone for the same berry density (tukey hsd test; p < 0.05). different greek letters within the same column indicate significant differences (2) among the three density classes for the same zone (tukey hsd test; p < 0.05). sign: *, **, ***, and "ns" indicate significance at p < 0.05, 0.01, 0.001, and not significant, respectively. density class location berry skin break force [fsk. n] berry skin break energy [wsk. mj] berry skin young’s modulus [esk. n/mm] berry skin thickness [spsk. µm] 1100 kg/m3 astigiano 0.855±0.182 0.862±0.278 0.386±0.082ab 190±35a collina torinese 0.907±0.121 0.817±0.178 0.440±0.058b 192±34a langhe 0.892±0.120 0.868±0.173 0.400±0.055b.β 188±29a.α tortonese 0.869±0.141 0.884±0.256 0.386±0.057ab.β 215±25a monferrato 0.879±0.201 0.893±0.272 0.342±0.061a 244±35b sign (1) ns ns *** *** 1107 kg/m3 astigiano 0.875±0.175 0.896±0.268 0.380±0.074 199±29a collina torinese 0.892±0.148 0.880±0.236 0.391±0.062 213±24ab langhe 0.897±0.137 0.906±0.256 0.380±0.050αβ 218±27ab.β tortonese 0.838±0.128 0.877±0.211 0.332±0.035α 230±23b monferrato 0.835±0.229 0.822±0.280 0.354±0.099 225±34b sign (1) ns ns ns ** 1115 kg/m3 astigiano 0.879±0.130 0.934±0.217 0.340±0.038a 209±22 collina torinese 0.870±0.162 0.782±0.217 0.417±0.090b 211±28 langhe 0.857±0.156 0.860±0.223 0.356±0.064a.α 224±31β tortonese 0.824±0.160 0.872±0.247 0.339±0.058a.α 231±17 monferrato 0.945±0.137 0.953±0.219 0.387±0.066ab 223±43 sign (1) ns ns *** ns sign (2) ns, ns, ns, ns, ns ns, ns, ns, ns, ns ns, ns, *, **, ns ns, ns, ***, ns, ns ital. j. food sci., vol. 31, 2019 694 no significant differences in fsk and wsk were observed among density classes and growing areas. although it is normal to observe high coefficients of variation for these parameters in berry skin analysis, this behaviour may highlight the assumption that the berry skin hardness traits (fsk and wsk) are firstly variety dependent and not markedly influenced by the ripeness degree and the growing area. in fact, rolle et al. (2008b) have observed similar values of fsk and wsk at the last weeks during ripening of “nebbiolo” variety. these skin mechanical parameters give high resistance to “freisa” grapes against fungal pathogens (rosenquist and morrison, 1988) and to handling injury during harvest, transport, and postharvest treatments (kök and çelik, 2004). from a technological point of view, a higher skin hardness is generally associated to a slower anthocyanin release into the must-wine during maceration-fermentation but, with a longer maceration, the anthocyanin extraction yield is higher (rolle et al., 2008b). this aspect is particularly important and favorable for “freisa” grapes and for all the wine grapes varieties rich in 3’-hydroxylated anthocyanins because these pigments are extracted preferentially during the initial phase of maceration and may be easily oxidized by the enzymes present in the juice (gonzález-neves et al., 2008). regarding other berry skin hardness-derived parameter, significant differences were found for young’s modulus (esk) that measures the rigidity or stiffness of tissues. inside each density class, zone effects were observed. particularly, the grapes of collina torinese location had the highest values of esk (> 0.390 n/mm). furthermore, ripeness effects were evidenced in some growing areas (langhe and tortonese), with a tendency to decrease esk values when increasing the density class, as observed by torchio et al. (2010) on “barbera” grapes. for skin thickness (spsk, table 3), the values obtained in the present study agreed with the reported by letaief et al. (2008) for “freisa” grapes. in this study, some significant differences were found among growing areas and density classes. monferrato location showed a significantly higher value of skin thickness with respect to other locations when considering the grapes belonging to the lowest density class considered (244 µm). furthermore, also in the other density groups this location evidenced high skin thickness values (225 and 223 µm, respectively, in the middle and in the higher density class). on the other hand, astigiano location was characterized by thinner skins (190-209 µm). previous studies have highlighted that precipitation indices, which could strongly influence berry water availability in the last ripening weeks, are responsible for differences in skin mechanical parameters among production areas (rolle et al., 2011a). moreover, the influence of rain on skin thickness has been already reported for mondeuse grapes during on-vine withering (rolle et al., 2009). significant differences in this skin mechanical parameter among the density groups inside each location were found only for langhe samples (p < 0.001), with an increasing trend from 1100 kg/m3 class to the higher density classes. a slight increasing tendency was found also for other locations and similarly observed by torchio et al. (2010) on “barbera” grapes, by río segade et al. (2011b) on galician grapes, and by rolle et al. (2012) on “nebbiolo”. moreover, rolle et al. (2011b) showed on “nebbiolo” grapes that stiffer and thicker berry skins allowed respectively the higher accumulation and extraction of proanthocyanidins, while harder skins provided higher concentration and extraction of oligomeric flavanols. villangó et al. (2015) evidenced that thicker skins had the highest content of anthocyanins in “syrah”, while río segade et al. (2011a) found on “mencía” grapes that thinner skins were characterized by a higher release of anthocyanins. therefore, although the changes in the skin mechanical properties during ripening are generally limited, the preliminary knowledge of these parameters could help the planning ital. j. food sci., vol. 31, 2019 695 of the harvest time (i.e. selection among different vineyards) and the strategy of maceration process, in order to guarantee the rapid degradation of the grape skin and an improved extraction of its components (río segade et al., 2014, río segade et al., 2015). 3.3. skin anthocyanin content and profile the skin total anthocyanin content (task) and profile of “freisa” samples are shown in table 4. the first comparison shows that there were differences in total anthocyanin content among growing areas for all the density classes considered (p < 0.05 for density class of 1100 kg/m3, and p < 0.001 for 1107 and 1115 kg/m3). this behaviour highlighted a zone effect on grape total anthocyanin content because climate indices, sunlight and soil conditions are factors of great relevance on anthocyanin biosynthesis (spayd et al., 2002). the lowest and highest contents of anthocyanins were found in the grapes from astigiano and monferrato locations, respectively, belonging to the three density classes, even though the differences between the two locations were more relevant for the grapes belonging to the highest density classes (156, 418, and 475 mg/kg for 1100, 1107, and 1115 kg/m3, respectively). although probably there is not an only influential factor, the south-east exposure of the monferrato vineyard could favour the biosynthesis of anthocyanins or reduce their degradation, with respect to the south orientation of the astigiano vineyard, as a consequence of temperature and/or sunlight effects. these same samples had also the lowest and highest values of spsk, respectively, in accordance with villangó et al. (2015) that observed on “syrah” grapes a higher concentration of anthocyanins in the thicker skins. the second comparison showed that total extractable concentrations of anthocyanins almost always increased with increasing berry density, with significance at p < 0.05 in three cases (collina torinese, langhe, and tortonese locations) and at p < 0.01 in the case of monferrato location. a density effect on anthocyanins concentration is evident and confirms the assumptions of torchio et al. (2010), who previously observed this behaviour on “barbera” grapes, and of rolle et al. (2011b) on “nebbiolo” grapes. in this study, “freisa” grapes were characterized by a high average percentage of simple anthocyanin glucosides (95.3% of total forms on average), with a higher content of free disubstituted anthocyanins (66.0% on average) with respect to free tri-substituted free forms (29.3% on average), as already reported in literature (rolle and guidoni, 2007; ferrandino et al., 2012). in the present study, peonidin-3-o-glucoside was the major anthocyanin compound found, with an average content of 48.1%, followed by malvidin-3o-glucoside and cyanidin-3-o-glucoside with an average percentage of 19.2 and 17.8% of total forms, respectively. a similar anthocyanin profile was evidenced for “nebbiolo” grapes and for other minor, ancient grape varieties diffused in north-west italy close to the alps such as “avanà”, “doux d’henry”, “grignolino”, “neretto di bairo”, “pelaverga piccolo”, and “rastajola”, which showed a percentage of peonidin-3-o-glucoside of about 45-55% on total anthocyanin forms (gerbi et al., 2005; ferrandino et al., 2012). some differences in the anthocyanin profile among locations and density classes were observed, even if the variations are not easily attributable to environmental or ripeness factors, as already discussed by ortega-regules et al. (2006). in the lower density class, the grapes from collina torinese location had the lowest content of di-substituted anthocyanins (cyanidinand peonidin-3-o-glucoside, 56.1%) and the highest content of tri-substituted ones (delphinidin-, petunidin-, and malvidin-3-o-glucoside, 38.8%). ital. j. food sci., vol. 31, 2019 696 table 4. total extractable anthocyanins content and relative profile of “freisa” grapes harvested in five piedmont growing locations and sorted according to density. density class location total anthocyanins task delphinidin-3o-glucoside [%] cyanidin-3-oglucoside [%] petunidin-3-oglucoside [%] peonidin-3-oglucoside [%] malvidin-3-oglucoside [%] acetyl-3-oglucosides [%] cinnamoyl-3o-glucosides [%] 1100 kg/m3 astigiano 834±53ab 3.93±0.11a 19.43±0.61b.α 5.44±0.06a 49.17±1.02b.α 17.06±0.36a.β 1.28±0.13b 3.69±0.05ab.β collina torinese 961±69ab.αβ 5.23±0.30b.β 14.02±0.93a 7.09±0.29b.β 42.06±2.27a.α 26.52±2.18b.β 0.31±0.05b 3.77±0.39b langhe 924±76ab.α 3.54±0.36a 20.00±1.81b 4.93±0.18a.α 50.99±1.36b 16.60±0.69a 0.95±0.03a.α 2.99±0.31a.α tortonese 777±16a.α 3.24±0.65a.α 17.57±0.66ab 4.57±0.55a.α 53.17±3.34b.β 16.45±1.39a.α 1.32±0.04b.α 3.69±0.22ab monferrato 990±100b.α 3.17±0.52a.α 18.51±2.21b 4.79±0.62a.α 51.63±1.78b.β 17.24±2.58a 1.34±0.11b 3.32±0.24ab sign (1) * *** ** *** *** *** *** * 1107 kg/m3 astigiano 773±75a 3.53±0.96 21.35±0.60b.β 5.05±0.95 50.47±5.09αβ 14.96±2.70αβ 1.28±0.13 3.36±0.69αβ collina torinese 869±52a.α 3.99±0.59α 15.67±2.09a 5.69±0.35α 50.52±2.59β 19.76±1.41α 1.14±0.09 3.23±0.23 langhe 1122±44b.β 4.67±0.42 18.89±2.07ab 6.16±0.40β 44.81±3.49 20.78±3.71 1.30±0.31αβ 3.39±0.66αβ tortonese 847±45a.αβ 4.22±0.38αβ 17.65±1.09ab 5.57±0.44αβ 48.67±1.77αβ 19.00±2.00αβ 1.32±0.11α 3.56±0.30 monferrato 1191±41b.β 4.87±0.66β 15.47±0.91b 6.64±0.69β 47.75±2.57αβ 20.88±1.76 1.35±0.13 3.34±0.24 sign (1) *** ns ** ns ns ns ns ns 1115 kg/m3 astigiano 785±50a 3.25±0.14a 22.42±0.24b.β 4.57±0.09a 54.07±0.86b.β 11.90±0.34a.α 1.05±0.02a 2.74±0.20a.α collina torinese 1050±50b.β 4.88±0.42b.αβ 15.97±0.90a 6.61±0.31b.β 46.77±2.09ab.αβ 21.23±0.68b.α 1.11±0.15a 3.43±0.31b langhe 1042±74b.αβ 4.94±0.88b 17.65±2.19ab 6.51±0.90ab.β 43.25±4.87a 22.13±4.97ab 1.52±0.16b.β 4.01±0.14c.β tortonese 936±77ab.β 5.40±0.76b.β 16.31±0.18a 6.87±0.70ab.β 43.67±3.85a.α 22.24±2.36b.β 1.54±0.06b.β 3.97±0.27c monferrato 1260±29c.β 5.28±0.31b.β 16.28±0.96a 7.13±0.50b.β 45.45±2.06a.α 21.13±1.87b 1.41±0.08b 3.33±0.17b sign (1) *** ** *** ** ** *** ** ** sign (2) ns,*,*,*,** ns,*,ns,*,** **,ns,ns,ns,ns ns,**,*,**,** **,*,ns,*,* ***,**,ns,*,ns ns,ns,*,*,ns *,ns,*,ns,ns values are expressed as average ± standard deviation (n = 3). different latin letters within the same column indicate significant differences (1) among zone for the same berry density (tukey hsd test; p < 0.05). different greek letters within the same column indicate significant differences (2) among the three density classes for the same zone (tukey hsd test; p < 0.05). sign: *, **, ***, and "ns" indicate significance at p < 0.05, 0.01, 0.001, and not significantly respectively. task = total anthocyanins (mg malvidin-3-o-glucoside chloride/kg berries). ital. j. food sci., vol. 31, 2019 697 on the contrary, in the higher density class, the grapes from astigiano location had the highest content of di-substituted anthocyanins (76.5%) and the lowest content of trisubstituted forms (19.7%). when the ripeness effect was evaluated for each location, in the grapes from astigiano (and partly collina torinese) location there was a relative increase of di-substituted anthocyanins and a decrease of tri-substituted ones when increasing the density class considered, while in the grapes from langhe, tortonese, and monferrato locations the opposite effect was observed. as previously commented for total anthocyanins, the south orientation of the astigiano vineyard seems to influence negatively the percentage of the most stable forms, namely acylatedand malvidin-derivatives. it also can be hypothesized that higher rainfall and lower temperatures during the pre-veraison period could probably affect positively the f3’h activity, resulting in a higher di-substituted flavonoid accumulation, such as peonidin and cyanidin-3-o-glucoside, and negatively the f3’5’h activity with a lower trisubstituted flavonoid accumulation, such as delphinidin-, petunidinand malvidin-3-oglucoside (ferrandino and guidoni, 2010). 3.4. total flavonoids and flavanol composition of skins and seeds the skin and seeds flavonoid content and flavanol composition of “freisa” grapes harvested in the five different locations and sorted by flotation are shown in table 5. regarding grape skins, significantly higher contents of total flavonoids (tfsk) were found in the grapes from langhe and monferrato locations when belonging to the middle and higher density classes (p < 0.001). however, an increase of tfsk was observed with the increase of sugar level for all locations with exception of astigiano, as also occurred for task content. differently, few differences were found in skin proanthocyanidin and oligomeric flavanol contents (represented by prosk and fvask parameters, respectively) among locations for each density class and no differences were observed among the berries belonging to the different density classes for the same growing area. a similar behaviour of prosk and fvask was previously observed in “nebbiolo” grapes with berry density (rolle et al., 2012). particularly, in this study, the highest contents of both larger and smaller molecular mass flavanols were found in the grapes skins from astigiano, especially in the middle density class [prosk, 2849 mg cyanidin chloride/kg berries; fvask, 1122 mg (+)–catechin/kg berries]. this location had also the highest values of fvask/prosk ratio (0.36 and 0.39, respectively, in the lower and in the middle density class), thus evidencing an important contribution of the oligomeric flavanol fraction assessed by fvask parameter. regarding seeds, a remarkable aspect of “freisa” grapes is the high content of flavanols reactive to vanillin (fvas) with respect to proanthocyanidins (pros) and, as a consequence, the high fvas/pros ratio (> 0.57). rolle et al. (2008a) have also observed a similar relationship between oligomeric and polymeric flavanols in “freisa” grapes and therefore a varietal behaviour is evident. this could represent an issue when seeds flavanol extraction into the must during the maceration is high, with the risk of producing unbalanced wines for bitterness and astringency. in fact, flavanols vanillin assay (fva) quantifies monomeric and oligomeric flavanols, which are more bitter than the polymeric ones, while the astringency is positively correlated to mean degree of polymerization (mdp) and galloylation degree (vidal et al., 2003, cheynier et al., 2006). ital. j. food sci., vol. 31, 2019 698 table 5. skin and seeds total extractable phenolic composition of “freisa” grapes harvested in five piedmont growing locations and sorted according to density. density class location skins seeds total flavonoids [tfsk] proanthocyanidins [prosk] vanillin assay [fvask] fvask/prosk ratio total flavonoids [tfs] proanthocyani dins [pros] vanillin assay [fvas] fvas/pros ratio 1100 kg/m3 astigiano 3770±154 2800±88 999±117b 0.36±0.03b 1310±115bc 1476±5b 847±69a 0.57±0.03a collina torinese 3802±29 α 2204±253 649±138a 0.29±0.03ab 860±49a 961±22a 621±70a 0.65±0.06a langhe 3816±160α 2305±161 750±18ab 0.33±0.03ab 1011±137ab 981±126a 758±125a 0.77±0.03b tortonese 3482±70α 2217±181 691±147ab 0.31±0.04ab 780±154a 986±145a 593±115a 0.60±0.03a monferrato 3710±210α 2501±380 648±122a 0.26±0.01a 1425±69c.β 1684±87c.β 1327±88b.β 0.79±0.02b sign (1) ns ns * * *** *** *** *** 1107 kg/m3 astigiano 3481±104a 2849±140b 1122±206b 0.39±0.06b 1205±142cd 1278±163c 942±131cd 0.74±0.05b collina torinese 3736±149 a.α 2054±183a 584±39a 0.28±0.01ab 1059±125bc 1187±224bc 771±96bc 0.65±0.04ab langhe 4249±162b.β 2787±73b 878±242ab 0.32±0.09ab 898±25b 903±49ab 663±21ab 0.74±0.06b tortonese 3813±24a.β 2516±211ab 722±135ab 0.29±0.04ab 642±111a 800±89a 470±89a 0.58±0.04a monferrato 4241±212b.β 2809±372b 650±76a 0.23±0.04a 1384±49d.β 1644±112d.β 1137±116d.β 0.69±0.08ab sign (1) *** ** * * *** *** *** * 1115 kg/m3 astigiano 3541±210a 2651±82 796±207 0.30±0.08 979±100b 1075±134 693±78ab 0.64±0.01 collina torinese 4326±180 bc.β 2317±255 605±35 0.26±0.03 868±39ab 1014±86 575±42ab 0.57±0.01 langhe 4204±53bc.β 2727±328 778±83 0.29±0.04 947±102b 1042±126 698±53ab 0.67±0.03 tortonese 3863±263ab.β 2287±442 672±118 0.30±0.03 708±53a 861±18 520±57a 0.60±0.06 monferrato 4567±125c.β 2885±141 819±53 0.28±0.03 949±111b.α 1111±160α 707±94b.α 0.64±0.07 sign (1) *** ns ns ns * ns * ns sign (2) ns,**,*,*,** ns,ns,ns,ns,ns ns,ns,ns,ns,ns ns,ns,ns,ns,ns ns,ns,ns,ns,*** ns,ns,ns,ns,** ns,ns,ns,ns,*** ns,ns,ns,ns,ns values are expressed as average ± standard deviation (n = 3). different latin letters within the same column indicate significant differences (1) among zone for the same berry density (tukey hsd test, p < 0,05). different greek letters within the same column indicate significant differences (2) among the three density classes for the same zone (tukey hsd test; p < 0.05). sign: *, **, ***, and "ns" indicate significance at p < 0.05, 0.01, 0.001, and not significant, respectively. tfsk,s = total flavonoids (mg (+) – catechin/kg berries); prosk,s = proanthocyanidins (mg cyanidin chloride/kg berries); fvask,s = flavanols vanillin assay (mg (+) – catechin/kg berries). ital. j. food sci., vol. 31, 2019 699 the content of flavanols in “freisa” seeds showed differences mainly induced by the growing area, with the highest content of tfs, fvas and pros in the grapes from monferrato location and belonging to the lower and middle density classes [pros, 1684 and 1644 mg cyanidin chloride/kg berries; fvas, 1327 and 1137 mg (+)–catechin/kg berries, respectively]. among density classes, a decreasing tendency of flavanols content from the lower to higher density class was observed, only being significantly relevant in the grapes from monferrato location. these results agreed with the higher seed number found in the berries from the monferrato vineyard, particularly in those belonging to the lower density class. indeed, it is widely recognised that during the berry development the concentration of monomeric flavanols decreases rapidly (gonzález-manzano et al., 2004) and that the histological and histochemical modifications (i.e. solidification of the cells rich in tannins) occurring during grape ripeness influence negatively the aptitude for the extraction of these compounds (cadot et al., 2006). 3.5. “freisa” experimental wines assessment monovarietal wines were produced for each single “freisa” designation of origin considered in this study, and then analysed according to their general chemical composition, chromatic characteristics, and phenolic content (table 6). as expected, the alcohol content in all five “freisa” wines were higher than the minimum limits imposed by disciplinary rules and always higher than 13% v/v. in the same way, also total acidity and dry net extract were satisfactory with values higher than 5.27 g/l as tartaric acid and 25.8 g/l, respectively. the wines produced by grapes from astigiano and collina torinese locations had the highest values of total acidity (6.26 and 6.27 g/l as tartaric acid, respectively), with the latter presenting also the highest alcohol content (14.6% v/v). wines produced by grapes from tortonese location showed the highest value of dry net extract (28.1 g/l). regarding colour characteristics, the highest values of lightness (l*), red/green colour coordinate (a*), and yellow/blue colour coordinate (b*), related to the lowest value of colour intensity, were found in the wine from astigiano location that also had the lowest content of total anthocyanins (task). as opposed, the wine from monferrato location showed the lowest values of l* and of the coordinates a* and b*, related to the highest value of colour intensity, and a high value of colour hue (more red-orange nuances). the anthocyanin content of “freisa” wines ranged between 198 and 303 mg malvidin-3o-glucoside chloride/l. it is well known that anthocyanins are strongly related to wine chromatic characteristics, but paissoni et al. (2018) demonstrated that they also can contribute to in-mouth properties in function of their content and acylation. these authors estimated the perception threshold of total anthocyanins at 255 mg/l, and evidenced that acetylated and cinnamoylated anthocyanins contribute to in-mouth sensory properties more than glucoside forms. regarding “freisa” wines analysed in the present study, only in two wines out of five (langhe doc freisa and monferrato doc freisa) the content of total anthocyanins was higher than the proposed threshold. taking into account that “freisa” grapes had a low amount of acylated forms (table 4), it may be hypothesized that the wine contains limited acylated anthocyanins and, therefore, presents a very small contribution to in-mouth sensory traits. for these reasons, the contribution of anthocyanins to in-mouth sensory properties of “freisa” wines can be excluded in most cases. ital. j. food sci., vol. 31, 2019 700 table 6. compositional characteristics of wines produced from “freisa” grapes harvested in five piedmont growing locations. location wine parameter astigiano collina torinese langhe tortonese monferrato designation of origin freisa d'asti doc freisa di chieri doc langhe doc freisa colli tortonesi doc freisa monferrato doc freisa minimum alcohol content [% v/v] 11.0 11.0 11.0 11.0 11.0 minimum total acidity content [g/l as tartaric acid] 5.50 4.50 4.50 4.50 5.00 minimum dry net extract [g/l] 19.0 19.0 19.0 20.0 20.0 alcohol content [% v/v] 13.7 14.6 14.1 14.1 14.1 total acidity [g/l as tartaric acid] 6.26 6.27 5.85 5.27 5.48 dry net extract [g/l] 25.8 26.7 27.0 28.1 27.3 l* 20.7 17.7 19.1 18.8 16.9 a* 55.1 52.4 54.0 52.7 51.1 b* 51.3 49.6 51.7 49.9 49.4 color intensity [a.u., 10 mm optical path] 17.6 18.2 18.1 18.5 19.6 color hue 0.87 0.79 0.78 0.92 0.90 taw [mg malvidin-3-o-glucoside chloride/l] 198 238 303 237 276 maw [mg malvidin-3-o-glucoside chloride/l] 64 83 135 85 107 maw/taw [%] 32 35 45 36 39 tfw [mg (+)-catechin/l] 2217 2050 2048 2449 2155 fvaw [mg (+)-catechin/l] 2136 1679 1580 2209 1665 prow [mg cyanidin chloride/l] 3654 2875 3038 4044 3213 fvaw/prow 0.58 0.58 0.52 0.55 0.52 the values in the first three numerical rows are the levels to achieve in the wines for each appellation, in the latter three are the levels achieved in the produced wines. l* = lightness; a* = red/green color coordinate; b* = yellow/blue color coordinate; taw = total anthocyanins; maw = monomeric anthocyanins; tfw = total flavonoids; prow = proanthocyanidins; fvaw = flavanols vanillin assay. the highest percentage of monomeric anthocyanins on the total content (45%) was found in langhe doc freisa wine, which had also the highest content of total anthocyanins (303 mg malvidin-3-o-glucoside chloride/l). on the contrary, the lowest monomeric/total anthocyanin ratio (32%) and total anthocyanin content (198 mg malvidin-3-o-glucoside chloride/l) were found in freisa d’asti doc wine. in both wines the data confirmed the trends found in the grape anthocyanins (table 1). about wine flavanols, the two highest contents of smaller molecular mass tannins [fvaw, 2209 and 2136 mg (+)-catechin/l] were found in the wines from tortonese and astigiano locations, respectively. these two wines were produced from the grapes with high values of seed maturity index (mp%=82, table 1). as previously mentioned, smaller molecular mass tannins (flavanol monomers and oligomers) are perceived as more bitter, and therefore high contents of fvaw in wines could be involved in high bitter sensations. for this purpose, some production strategies could be useful: for instance, when both technological and phenolic maturity are not satisfactory, the grape ital. j. food sci., vol. 31, 2019 701 dehydration could aid to reach a better sugars/acids ratio and a higher level of seeds lignification with a lower flavanols release. furthermore, when technological maturity is satisfactory, the removal of the seeds from the must 48-96 h after the beginning of the fermentation process could limit the extraction of flavanols during macerationfermentation (rolle et al., 2008a). 4. conclusions the physico-chemical characteristics of the piedmont minor variety vitis vinifera l. cv. “freisa” were comprehensively studied, considering grapes from five different growing areas and at three ripeness levels defined by density sorting. mechanical properties and phenolic composition of “freisa” grapes were differently affected by these factors, and some parameters showed a strong varietal character. particularly, berry skin hardness parameters (break force and break energy), which influence extraction kinetics of phenolic compounds, were slightly affected by environmental conditions and ripeness degree. instead, the other two mechanical parameters, namely skin young’s modulus and thickness, varied among the locations and berry density classes considered. the first one decreased with increasing the ripeness degree while, on the contrary, the second one showed an increasing tendency that affects positively the skin extractable content of phenolic compounds as follows. berry skin total anthocyanin content increased significantly with increasing the density class, in agreement with higher values of skin thickness, and also was significantly affected by the growing area. nevertheless, berry skin flavanol contents (monomeric oligomeric and polymeric) varied only among locations, while seed flavanol contents varied among the locations and the density classes with a tendency to decrease from the less ripe to the ripest grapes berries considered. grapes from monferrato growing area showed the thickest skins, and coherently the highest contents of total anthocyanins but also of seed flavanols. the crucial point of “freisa” grapes has been confirmed to be the high release of flavanols from the seeds during winemaking. this aspect highlights the importance of a careful management of the maceration process in cellar and the possibility of using 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161 paper corn grain brushing for deoxynivalenol reduction r. čolović1, đ. vukmirović1, l. pezo*2, j. kos1, d. čolović1, f. bagi3 and n. memiši4 1institute of food technology (fins), university of novi sad, car lazar boulevard 1, 21000 novi sad, serbia 2institute of general and physical chemistry, university of belgrade, studentski trg 12-16, 11000 belgrade, serbia 3faculty of agriculture, university of novi sad, trg dositeja obradovića 8, 21000 novi sad, serbia 4ad imlek tolminska 10, 24000 subotica, serbia *e-mail address: latopezo@yahoo.co.uk abstract for the purpose of deoxynivalenol (don) reduction in corn samples, a laboratory brusher was developed. the brusher had two main parts, motionless screen and fast rotating brush. corn kernels were placed on the motionless screen of the brusher, and fast rotating round shaped polypropylene bristle brush was put into the motionless screen in order to clean the surface of the kernels. the experiment consisted of 27 brushing trials with variations in process parameters (don concentration, brushing time, and speed of rotating brush). brushing of corn that is don infected resulted in the reduction of don concentration in all samples. the maximal limit of don reduction at optimal conditions was 83.6%. thus, the presented process can be considered as highly efficient. the proposed technology did not cause any changes in the physical appearance of kernel, nor were damages observed on kernel surface during the brushing process. also, there were no whole grain losses detected in any of the process parameter combination. keywords: brushing, corn, decontamination, deoxynivalenol ital. j. food sci., vol. 31, 2019 162 1. introduction well-known and frequently mentioned secondary metabolites of fungi, mycotoxins, commonly occurred in cereal grains and other food and feedstuffs. they belong to the most toxic contaminants among a wide range of food commodities (council for agricultural science and technology, 2003; kourousekos and theodosiadou, 2018). these highly harmful compounds are extremely thermostabile; for instance, pure aflatoxin b1 can be destroyed only at temperatures above 160°c (karlovsky et al., 2016). during “field to fork” chain, contaminated food materials are not exposed to temperature, which can lead to mycotoxin decontamination, or if it is exposed, the process does not last long enough to be decontaminated. moreover, conditions on the field as well as conditions during further manipulation with cereal products oftentimes promote fungal growth and mycotoxin production. exposure to the mycotoxins is somehow unavoidable (magan and olsen, 2006). chronic consumption of contaminated goods can adversely affect human and animal health and can be lethal to some animal species. main toxic effects of mycotoxins are teratotoxicity, carcinogenicity, hepatotoxicity, nephrotoxicity, embryotoxicity and immunosuppression (pestka, 2010; anfossi et al., 2010). one of the best ways to manage contamination is to apply pre-harvest and post-harvest preventive strategies, that is by avoiding the emergence of mycotoxins. however, when the material is already contaminated, mycotoxins should be inactivated, destroyed, or removed from the commodity. besides, the nutritive value and acceptability of the products should be preserved, and technological properties of the product should be retained (avantaggiato, 2012). nowadays, various procedures are applied for mycotoxin decontamination, which can be divided into three main groups: chemical, biological, and physical. chemicals, such as oxidizing agents, chlorinating agents, acids, and alkali, have been used for the deactivation of mycotoxins. despite the fact that some of these agents were found to be effective, the chemical procedures are not widely used due to their negative effect on product palatability and nutritive value, potential toxicity, high operational costs, and long operational time (amézqueta et al. 2009; zaki et al., 2012). biological detoxification, which is based on biotransformation and/or biodegradation principles, is widely used in the deactivation of mycotoxins contaminated feedstuffs, as well as for food and beverages. enzymes, microorganisms or a specific organic compound derived from microorganisms interact with mycotoxins in the gastrointestinal tract of animals to form a non-toxic stable complex. to enable sufficient contact surface between contaminated substrate and biological additive, grain materials are usually milled before the addition of bio-binder. therefore, when biological detoxification is performed, the grains could not be preserved in a whole kernel form, nor the effects of the binders observed before grains are consumed by animals (kolosova and stroka, 2011; karlovsky et al., 2011). physical treatment of contaminated materials includes, sorting, screening, milling, washing, irradiation, thermal treatments, adsorbing, etc. some of these procedures can be found in conventional grain storage and/or processing technologies, with the main purpose of improving the nutritional quality of grains, and not to remove mycotoxins (trenholm et al., 1991). taking into account that most of these operations do not destroy mycotoxins, the effect of physical procedures is often weak to moderate (kabak, 2009; manafi and khosravinia, 2012; marin et al., 2013). generally, the selected method for mycotoxin decontamination should be efficient, relatively simple, inexpensive, and not time-consuming. ital. j. food sci., vol. 31, 2019 163 for the purpose of mycotoxin decontamination in grain, a team of scientists at the institute of food technology, university of novi sad, serbia, developed an intensive laboratory grain brusher. preliminary trials have shown a significant effect of the brushing process on the aflatoxin (af) removal (čolović et al., 2013). although there is a considerable number of papers referring to the effects of the scouring process on mycotoxin reduction, the literature on the effects of cleaning grain surface without damage of pericarp is limited (schaarschmidt and fauhl‐hassek, 2018). considering all the aforementioned facts, the main objective of this study was to present a new method of physical detoxification, primarily. furthermore, we wanted to investigate the influence of processing parameters (processing time (t), and rotational speed (v)) on reduction rate of don in maize samples naturally contaminated with this mycotoxin in three different concentrations and to determine the optimal conditions of brush procedure applied. response surface methodology (rsm) was used since it was proven to be an effective tool for optimizing a wide variety of processes (liaudanskas et al., 2018). experimental results were subjected to analysis of variance (anova) to show the relationship between applied assays. 2. materials and methods 2.1. material samples of mycotoxin contaminated corn were collected from commercial warehouses within the serbian northern province of vojvodina. sampling of three corn samples was performed in accordance with commission regulation 401/2006 (european commission, 2006). the total amount of aggregate sample (10 kg) was homogenized using nauta mixer, model 19387 (nauta patenten, netherlands). after homogenization, the aggregate sample was quartered to get 3000 g of the representative sample. obtained representative corn samples were again homogenized using rotation drum mixer (model syth0,05, muyang group, china) and quartered to get sub-samples of 100 g per contaminated sample (27 sub samples of each contaminated sample). initial don concentrations in naturally contaminated samples were 7.5 mg/kg (don1), 10.6 mg/kg (don2), and 14.8 mg/kg (don3). 2.2. chemicals and reagents acetonitrile used for hplc analysis (all hplc grade, purity ≥ 99.9%) was purchased from merck (darmstadt, germany). ultra-pure water was produced by milli-q purification system (milli-q from millipore, usa). don (concentration of 100 µg/ml) standard was purchased from sigma-aldrich (steinhem, germany). standard solutions were prepared in acetonitrile and stored at –10 °c. those solutions were used for solvent based calibration and for fortification of blank corn samples. 2.3. sample preparation sub-samples of 100 g were ground to a 1 mm particle size using laboratory mill (knifetectm 1095 mill, foss, hoganas, sweden), and additionally quartered to obtain subsamples of 25 g. the sub-samples were further extracted with 100 ml of acetonitrile:water (84:16, v/v) and shaken vigorously for 30 minutes on a laboratory griffin flask shaker (griffin and george, wembley, england). the extract was filtered through no. 4 filter paper (whatman, maidstone, uk). three ml of extract was cleaned up on mycosep® 225 ital. j. food sci., vol. 31, 2019 164 trich for don determination (romer labs, inc., union, mo). the cleaned-up extract was evaporated to dryness (reacti-therm ™ manifold, thermo fisher scientific, inc., usa), and reconstituted in 300 ml of mobile phase. 2.4. hplc analysis of don and validation procedure agilent 1200 (agilent technologies inc., usa) hplc instrument system equipped with diode array detector (dad) was used for determination of don. the detection was performed at 220 nm. agilent column eclipse xdb-c18, 1.8 µm, 4.6 x 50 mm was used. the mobile phase consisted of an isocratic mixture of water-acetonitrile (80:20, v/v), with a flow rate of 0.25 ml/min. each sample was analysed in duplicate. before application in the experiment shown, hplc/dad method was developed and validated for don determination. the validation parameters were determined and calculated according to eu commission decision procedure (2002/657/ec) by analysing certified reference material as well as spiked corn samples at three different levels. crm with certified don content of 900±100 µg/kg (d-w-164) was supplied by trilogy analytical laboratory (trilogy® reference material, washington, usa). the results of methodology validation are shown in table 1. as can be seen, the obtained validation parameters were in compliance with the recommendations given in regulation 2006/401/ec (ec, 2006). table 1. validation parameters for don determination. validation parameters crm spiked concentration (µg/kg) 1000 5000 10000 rsdr 6.56 7.12 8.15 5.94 rsdr 8.81 9.22 10.1 6.56 recovery 96.7 94.5 92.5 95.1 crm certified reference material (naturally contaminated corn, d-w-164, trilogy® reference material, washington, usa). rsdrrelative standard deviation calculated under repeatability conditions (%). rsdrrelative standard deviation calculated under reproducibility conditions (%). 2.5. processing contaminated corn, in a kernel form, was subjected to the brushing process. for this purpose, laboratory brusher developed at the institute of food technology, university of novi sad, serbia, was used (fig. 1). the brusher had two main parts, fast rotating brush (a) and motionless screen (b). corn kernels were placed onto the motionless screen to cover the surface (approximately 100 g) of the brusher (laboratory test sieve was used for this purpose), and fast rotating round shaped polypropylene bristle brush was put down into the motionless screen. the purpose of the corn kernel brushing was to remove dust from the surface of whole kernels, and to brush it out together with broken kernels through the openings in the motionless screen. aspiration of the dust is provided from the bottom side of the screen by connecting it to the central aspiration system, in order to facilitate separation, and to prevent excessive dusting, as well as inhalation of toxic substances. brushing time was set at 30, 60, and 90 s, respectively, while speed of rotating brush was set at 400, 800, and 1200 rpm, respectively. ital. j. food sci., vol. 31, 2019 165 figure 1. laboratory grain brusher. a fast rotating brush, b motionless screen. extent of don reduction (edon (%)) was calculated using the following equation: red.1 3 1 3 1 100%− − ⎛ ⎞ = − ⋅⎜ ⎟⎜ ⎟ ⎝ ⎠ don don don c e c (equation 1) 2.6. experimental design and statistical analysis the experimental data used for the study of experimental results were obtained using a 32 full factorial experimental design; each of the 3 specific don contaminated samples were processed at 2 parameters and at 3 levels. independent experimental factors for each of the samples are shown in table 2. table 2. independent experimental factors and their levels. experimental factor symbol coded factor’s level -1 (low) 0 (centre) +1 (high) t – brushing time (s) x1 30 60 90 v – speed of rotating brush (rpm) x2 400 800 1200 descriptive statistical analyses of all the obtained results were expressed by means, for each treatment. collected data were subjected to anova to explore the effects of process variables. the evaluation of rsm and anova of the obtained results was performed using statistica software version 12 (statsoft inc. 2013, usa)®. ital. j. food sci., vol. 31, 2019 166 the experimental data used for the analysis were derived according to rsm. the main advantage of rsm is a reduced number of experimental runs that provide sufficient information for statistically valid results. the rsm equations describe the effects of the test variables on the observed responses, determine test variable interrelationships and represent the combined effect of all test variables in the observed responses, enabling the experimenter to make efficient exploration of the process (čolović et al., 2016, brlek et al., 2013). the following second order polynomial (sop) model was fitted to the experimental data. six models of the following form were developed to relate six responses (y) and three process variables (x), for each of the different mixtures. 2 2 2 0 12 1 2 1 1 ,k k ki i kii i k i i y x x x xβ β β β = = = + ⋅ + ⋅ + ⋅ ⋅∑ ∑ (equation 2) where: 0kβ , kiβ , kiiβ , 12kβ are constant regression coefficients; ky , deoxynivalenol concentration after reduction (cdonred), for three initial concentrations of deoxynivalenol (don1-3), while x1 brushing time (t); x2-speed of rotating brush (v). 3. results and discussion the results for the effects of brushing process on don reduction have been shown in fig. 2 from the least to the most contaminated samples, respectively. the brushing of infected corn resulted in the reduction of concentration of don in all samples, matter on don concentration, brushing time or speed of rotating brush. the best results of brushing for don concentration of 7.5 mg/kg (don1) and 14.8 mg/kg (don3) were recorded at the highest levels of rotating speed and the longest time of brushing, respectively. the samples before and after brushing for rotating speed of 1200 rpm and brushing time of 90 s, as well as tailings collected from the aspiration system has been shown in fig. 3. however, samples contaminated with concentration of 10.6 mg/kg (don2) have shown the highest level of decontamination at rotating speed of 1200 rpm and a duration of 60 s. looking at fig. 2, it is clear that the results of decontamination level were unpredictable for rotation speed of 400 and 800 rpm. the reason for that is probably due to the insufficient rotating speed of brush, so decontamination level is rather accidental and not dependent on brushing time. at rotation speed of 1200 rpm, decontamination level increased with the increase in processing time. a similar conclusion can be made for don2 concentration. yet, decontamination level for don2 samples differ insignificantly for rotation speed of 1200 rpm and processing time of 60 and 90 s (approx. 81% and 78% respectively). it seems that for this don concentration, sufficient time of brushing for maximum decontamination is 60s at a brushing speed of 1200 rpm. an average mycotoxin reduction of 27 samples was 50.9%, showing that the grain brushing process was very effective in the removal of don. also, only five of 27 samples had don reduction less than 40%, while only two of those had don reduction less than 20%. generally, for a short brushing time and low speed of rotating brush, don reduction was lower. visconti et al. (2004) showed that concentration of don is the highest in the outer layers of grains such as bran. this also explains why intensive cleaning of the outer layers of corn applied in our study reduced don concentration. ital. j. food sci., vol. 31, 2019 167 figure 2. extent of don reduction (%) by application of grain brushing process. a-g different letters within the same set of samples show significantly different means of observed data at p<0.05 level. figure 3. the infected sample, contaminated with heavy don concentration (a), the sample after brushing treatment (for rotating speed of 1200 rpm and brushing time of 90 s) (b), and the brushed tailings collected from the aspiration system (c). physical dehulling of the outer layer of maize can reduce af decontamination by 92% (siwela et al., 2005). however, this method removes natural protection of kernel and it does not preserve its integrity. for any combination of process parameters, changes in physical integrity of kernel or damages to the kernel surface were not observed during the brushing process. since there is no damage of the grain, kernels could preserve its biological function and its natural protection from microbial contamination. therefore, it is 0 10 20 30 40 50 60 70 80 90 30 s 60 s 90 s 400 rpm 800 rpm 1200 rpm 30 s 60 s 90 s 30 s 60 s 90 s samples mean±2.sd don1 don2 don3e d o n (% ) a b b c d d d e f a a b c c d e fg a b ccdd d e f g ital. j. food sci., vol. 31, 2019 168 reasonable to expect that kernels decontaminated by brushing have a longer shelf life than kernels decontaminated with similar mechanical treatments, which include surface breakage of kernels. it cannot be neglected that, by this method, none of the total mass of treated kernels was removed and as a result, in that way, there were no material losses. as wu and munkvold (2008) stated in their paper that deals with the economic costs of mycotoxin’s presence in feed, removing of screenings and broken kernels from maize after sieving reduced don contamination by 73%, however, mass loses were extensively high and accounted for approx. 69%. removal of contaminated maize can also be performed by flotation and density segregation (grenier et al., 2014). fungal damaged kernels are mostly, also, mycotoxin-contaminated. since they have different physical properties than non-infected kernels, they can be separated by density segregation or by fractionation on so-called gravity tables. however, these methods are not mycotoxin specific, so kernels contaminated with fungi, but without presence of mycotoxins are also removed. a group of italian authors showed in their paper an electronic optical technique of sorting infected and healthy apricot kernels based on the discoloration of contaminated kernels (zivoli et al., 2016). although, they succeeded in removing up to 59% of aflatoxin accumulated in naturally-contaminated samples, the obtained results were highly variated, such that the proposed method cannot be considered as reliable and need to be improved. it may be less effective in comparison with the manual method of sorting, such as removal of contaminated grains, which is, on the other hand, highly time demanding.kushiro (2008) in his review paper showed that don concentration could be decreased by 86% when infested wheat kernels are removed. on the other hand, park (2002) combined removal of extensive mould growth kernels with the cleaning of maize kernels for reduction of af content, and obtained a reduction of 40% to 80%. that is similar to the maximal mycotoxin reduction in our study, where extensive mould growth kernels were not removed. however, the maize was not infected with the same mycotoxin. same author also used dry milling process for fractionation of af b1 content. highest levels of mycotoxin were found in the germ and hull fractions. grits, low-fat meal and low fat flour contained only 6 to 10% of af. since a prerequisite for fractionation is to comminute the kernel, authors of present study changed the physical structure of corn kernels. also, fractionation resulted in the concentration of toxin in separate fractions, without removal of mycotoxins from the material. washing grains with tap water significantly reduced the mycotoxin level and this can be applied to food and feeds (fandohan et al., 2005). yet, costs of drying grains are too high, so it is reasonable to use this method only prior to wet milling or ethanol fermentation. the same goes for rinsing and flotation techniques. most of the physical methods of decontamination can be generally considered as efficient, but the major problems occur with implementation of these processes on a commercial scale. as earlier mentioned, milling, combined with sieving causes significant mass losses, and most of other established methods are time consuming, which cannot be said for the presented method of kernel brushing. laboratory grain brusher presented in this study has been shown to be generally efficient on a small scale level. meanwhile, development of a continuous semi-industrial scale brusher is in progress with the accent on maintaining same efficacy as it was on a lab scale device. anova shows the significant effects of independent variables to the responses (table 3). the sop models for all variables were found to be statistically significant and the response surfaces were fitted to these models. the linear term of v was the most influential in the don1 reduction calculation (statistically significant, at p < 0.10 level). the prediction of don reduction was influenced by a linear term of v and the quadratic term of t (both ital. j. food sci., vol. 31, 2019 169 statistically significant at p < 0.10 level). the linear term of v was very influential for don reduction calculation (p < 0.05), for don3. all sop models had an insignificant lack of fit tests, which means that all the models represented the data satisfactorily (table 3). a high r2 is indicative that the variation was accounted and that the data fitted satisfactorily to the proposed model (sop in this case). the coefficients of determination for don reduction prediction were very good and showed the good fit of the model to experimental results. table 3. anova calculation for don reduction during grain brushing process. df cdonred 1 cdonred 2 cdonred 3 t 1 124.519 32.491 1082.945 t2 1 1.660 947.083** 42.844 v 1 407.825** 1350.000** 2764.210* v2 1 135.942 279.609 78.972 t × v 1 108.160 462.656 1.097 error 3 161.312 442.659 757.681 r2 0.828 0.874 0.840 abbreviations: t brushing time, v speed of rotating brush, df degrees of freedom. + significant at p<0.01 level, *significant at p<0.05 level, **significant at p<0.10 level, error terms have been found to be statistically insignificant. using these models, the contour plots of the extent of don reduction (edon) were plotted and superimposed to ascertain the optimum processing conditions (fig. 4). figure 4. optimum region obtained after superimposing the contour plots of the system response. ital. j. food sci., vol. 31, 2019 170 optimization of the process is performed to ensure rapid processing conditions with high don reduction. optimum operating conditions were derived with a few iterative steps in finding processing parameters that gave the highest reduction of don. contour plots of the extent of don reduction showed that maximum reduction was obtained at higher initial concentration and higher speed of rotating brush, as was expected. the optimal conditions for don reduction were t = 90 s, v = 1200 rpm, which consistent with experimental results. the maximal extents of don reduction at optimal conditions were: 75.3%, 78.2% and 83.6%, for the initial don concentration of 7.5 mg/kg, 10.6 mg/kg and 14.8 mg/kg, respectively. 4. conclusions this study presented fast technological process which successfully detoxified corn without causing any changes in the physical integrity of the kernel, nor damages of kernel surface. therefore, the presented laboratory brusher could easily grow into its industrial version, especially due to its simple construction. the higher reduction of don was obtained for the higher initial concentration, longer polishing time, and higher speed of rotating brush. further, brushing of corn infected with don resulted in reduction of concentration in all processed samples. it is important to note that the proposed process did not lead to whole grain loss, but only fine particles, unlike other effective physical decontamination methods, such as sieving or fractionation. acknowledgements this study has been supported by the provincial secretariat for higher education and scientific research of the autonomous province of vojvodina through the project “application of novel and conventional processes for removal of most common contaminants, mycotoxins and salmonella, in order to produce safe animal feed in the territory of ap vojvodina”, project no. 114-451-2505/2016-01. references amézqueta s., 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accepted august 14, 2018 ijfs#1376_bozza ital. j. food sci., vol. 31, 2019 470 paper development of fruit leather from actinidia arguta by-product: quality assessment and shelf life studies g. giacalone, t.m. da silva, c. peano and n.r. giuggioli* department of agricultural, forest and food sciences (disafa), università degli studi di torino, largo paolo braccini 2, 10095 grugliasco, torino, italy *corresponding author: tel.: +390116708646; fax: +390116708658 e-mail address: nicole.giuggioli@unito.it abstract strategies are needed to reduce the substantial fruit losses of actinidia arguta (speciality fruits) caused by the high perishability of the edible skin. fruit leathers are considered a valuable opportunity to meet the requirements of today’s consumers searching for healthy and time-saving products. this work aimed to (a) develop a novel baby kiwifruit leather product (four formulations) and (b) evaluate the best formulation under different shelf life conditions. storage at 25°c and polyethylene samples presented the best colour and received acceptable scores for sensory evaluation at the end of the shelf life. keywords: actinidia arguta, formulations, leather, shelf life, storage ital. j. food sci., vol. 31, 2019 471 1. introduction in 2014, the high level panel of experts (hlpe) on food security and nutrition of the united nations food and agriculture organisation (fao) affirmed that ‘a sustainable food system is a food system that ensures food security and nutrition for all in such a way that the economic, social and environmental bases to generate food security and nutrition of future generations are not compromised’. today, the topic of the agro-food system sustainability transitions remains high on the agenda of many countries of the european union and international organisations (international energy agency, organisation for economic co-operation and development, and the world bank). the scientific community that works with the agro-food industry agrees with solutions about waste and food losses prevention, minimisation and valorisation (porat et al., 2018). in the fruit and vegetable sector, this issue represents a critical point for products that belong to speciality foods, such as berry fruits (murphy et al., 2002; girgenti et al., 2016), which need to be marked with high qualitative standards, and small aesthetic defects are enough to compromise the competitivity of these products in the market chain. although several studies have reported about the opportunity to valorise fruit waste (ma et al., 1993; laufenberg et al., 2003; federici et al., 2009; pathak et al., 2017) and suggested approaches to reduce the fresh fruit losses in the supply chain, there are currently limited research publications about the fate of specialities fruits that do not meet market specifications. a related option could be represented by the creation of new processed products with a high nutrient profile to fulfil the increasing demand for innovative and time-saving products (as well as the needs of consumers, who are looking for healthy products to prevent health issues, such as diabetes, obesity and cardiac diseases). earlier investigations evaluated the various effects of the drying (kaya et al., 2010), pasteurisation (lespinard et al., 2012), freezing (cano and marín, 1992; park et al., 2016) and canning (cano and marín, 1992) of processed fruits. processing of new fruit puree formulations revealed to be a well-performing strategy to prolong shelf life and diversify the market, by creating novel products, which may retain or even improve the nutraceutical profile (simal et al., 2005; torres et al., 2013). among these strategies, fruit leathers seem to be innovative dried products with high levels of convenience, as they represent a suitable choice for consumers searching for nutritious snacks (sharma et al., 2013). fruit leather processing is characterised by fruit puree dehydration, which is necessary to obtain a solid product. apple, mango and a range of tropical fruit, for example, can be used to produce fruit leathers. however, the formulation is highly dependent on the type of fruit that is incorporated into the puree mix because of variations in the acid, sugar and pectin contents. thus, the formulation may contain different types of sugars and, sometimes, additives, such as preservatives (vatthanakul et al., 2010; quintero et al., 2012; diamante et al., 2014) and hydrocolloids (vatthanakul et al., 2010; alhinai et al., 2012), which are occasionally included to improve the rheological characteristics or to retain the fruit’s natural colour (quintero et al., 2012; diamante et al., 2014). processed fruit products are overall healthy since they are less caloric than traditional snacks and are also an important source of fibre that promotes a sense of satiety (diamante et al., 2014), so many feasibility studies have been developed to assess fruit leather production (concha-meyer et al., 2016). given that baby kiwifruit (actinidia arguta) suppliers (due to the high perishability of the edible skin of the berries) have not yet found specific strategies for fruit non-compliant with the fresh market standards, and no prior literature has assessed the shelf life stability of baby kiwifruit leather, this work ital. j. food sci., vol. 31, 2019 472 had two goals. the first was to develop a novel baby kiwifruit leather product (four formulations) presenting a composition as close as possible to the fresh fruit profile, namely, with low sugar and an attractive bright green appearance, and second, to evaluate the physicochemical quality of the developed product during storage. 2. material and methods 2.1. materials frozen baby kiwifruit (a. arguta) puree added with 10% sucrose was provided by far, a fruit processing industry located in cuneo (italy). fresh apples (malus domestica, cv granny smith), glucose syrup 33 dextrose equivalent (de) (selca srl, italy), sucrose (reire, italy), invert sugar (zucchero & c., italy), low-methylated pectin powder (reire), citric acid powder (reire), fructose powder (reire) and fresh lemon (citrus limon) were provided by the department of agricultural, forest and food sciences (disafa department). polyamide/polyethylene (pa/pe) pouches, with a thickness of 0.09 mm, a water vapour rate of 5 g m-2 d-1 (at 23°c, 85% relative humidity), and an oxygen permeability of than 65 cc m2 d-1 atm-1 (at 23°c, 75%), were used for shelf life studies. 2.2. formulation development preliminary trials were conducted to select the best formulation. the first trial involved thawing 4 kg of frozen puree that was then weighed and combined with varying proportions of the ingredients mentioned above (section 2.1) to generate four different formulations (table 1). whole apples were cut and blended into a puree before being added to all formulations, to improve the texture uniformity of the final product. direct heat treatment with an open copper pan (100°c, for about 30 min) was applied to allow proper mixing of the product during the concentration process. all formulations were concentrated until the soluble solids content (scc) reached 70% brix. afterwards, the formulations were transferred to four separate trays, covered with baking paper to avoid external contamination, and left at 25°c for almost 24 h, to allow gelatinisation. the definitive formulation was chosen by a sensory evaluation panel of ten semi-trained judges, using a 5-point hedonic scale to describe the overall acceptance (1 = “dislike extremely”, 5 = “like extremely”). table 1. formulations 1, 2, 3, 4 produced on first trial. formulation 1 formulation 2 formulation 3 formulation 4 39% baby kiwi puree 8% granny smith apple 33% sucrose 14% invert sugar 3% water 2% low methylated pectin powder 1% citric acid powder 38% baby kiwi puree 58% sucrose 2% low methylated pectin powder 1% citric acid powder 1% water 71% baby kiwi puree 22% granny smith apple 5% glucose syrup 33 de 1% citric acid powder 1% water 43% baby kiwi puree 28% granny smith apple 28% glucose syrup 33 de 1% water ital. j. food sci., vol. 31, 2019 473 2.3. shelf life test and fruit quality assessments the best solid formulation (formulation 3a) was cut into 36 leathers (5.0 cm × 5.0 cm × 0.5 cm) that were divided into four samples: two samples were stored at 25°c (room temperature) and the remaining two samples maintained under an accelerated shelf life condition (37°c) in a static air oven for 21 days, respectively. for both shelf life tests, one of the two samples was packaged in awith polyethylene (hdpa/pe) foils, giving rise to four definitive leather samples: environment (e), environment pe (epe), oven (o) and oven pe (ope). pa/pe packaging was selected due, owing to its transparent surface, in order to that, alloweding the consumers to appreciate the product’s appearancespect. at the start of storage (day 0), baby baby kiwifruit raw puree (raw puree), raw formulation and the leather samples were analysed for scc, colour, texture parameters, total phenolic content (tpc), antioxidant capacity (ac) and sensory attributes. leather samples were assessed every 7 days for 21 days, using the same criteria as applied at the start of storage (0 day) plus weight loss. 2.3.1 weight loss determination leather samples were weighed on an electronic balance (se622, wvr science education) (±0.001 g) at day 0 and every 7th day. the average of triplicate measurements was calculated, and the percentage weight loss was computed as follows: weight loss (%) = (!!!) ! × 100 (1) where a is the initial weight (day 0), and b is the weight recorded every 7 days for 21 days. 2.3.2 soluble solids content (scc) the ssc was determined using an atago pocket pad-1 digital refractometer for raw puree, raw formulation and leather at day 0, and on every 7th day for the leather sample (aoac 932.12). ten grams of each leather sample was homogenised, and the scc (%) of the filtered pulp was measured in triplicate. 2.3.3 colour and browning index (bi) colour was measured in triplicate at day 0 and every 7th day for leather samples, using a minolta cr-400 chromameter (konica minolta sensing, inc., osaka, japan) pre-calibrated against a standard white tile. parameters l*, a* and b* were recorded at three different points on each sample, and browning index (bi), defined as brown-colour purity, which is usually used as an indicator of the extent of browning in sugar-containing food products (perez-gago et al., 2005) was also calculated (quintero ruiz et al., 2012): bi = (! ! !.!") !.!"# × 100 (2) where x is the chromaticity coordinate calculated from the x, y, z tristimulus values, according to the following formulae: 𝑥 = ! (! ! ! ! !) (3) ital. j. food sci., vol. 31, 2019 474 x = 𝑋𝑛× !∗ !"" + (!∗!!") !!" ! (4) y = 𝑌𝑛×((!∗!!") !!" )! (5) z = 𝑍𝑛×[ !!∗ !"" + (!∗!!") !!" ]! (6) where xn, yn and zn correspond to white reference tristimulus values of xn = 91.97, yn = 93.8 and zn = 107.98. 2.3.4 texture parameters a texture profile analysis (tpa), acquired using a ta.xtplus texture analyser (stable micro systems, usa) (30 kg load cell), was evaluated to determine some of the most important tpa parameters. a compression test was conducted, using a 75-mm aluminium flat probe to a 55% strain, with a pre-test, test and post-test speed of 5, 1 and 5 mm s-1, respectively, and 5 g trigger force. 2.3.5 total phenolic content (tpc) determination and antioxidant capacity (ac) the folin–ciocalteu (singleton and rossi, 1965) and ferric reducing/antioxidant power (frap) tests (benzie and strain, 1996) were undertaken to assess the tpc and ac, respectively. for both analyses, 10 g of raw puree, 10 g of raw formulation and 10 g of leather, were each extracted with solvent (500 ml methanol, 1.4 ml concentrated hcl and 23.8 ml of deionised water [dw]) for 60 min under reduced light conditions, followed by homogenisation (15 min) to obtain sample extracts. for the tpc test, the supernatant of each sample was mixed with 30 ml dw and 2.5 ml folin–ciocalteu reagent. after 8 min incubation, the mixture was combined with 10 ml of sodium carbonate solution (15% w/v) and incubated at room temperature (25°c) for 2 h. absorbance was measured at 765 nm (uv-1600 pc spectrophotometer, vwr), and the results expressed as milligrams of chlorogenic acid equivalents (cae) per 100 g dw (torres et al., 2015). the ac assay was carried out using stock solutions (frap) containing 0.3 m acetate buffer (3.1 g sodium acetate, 1 l dw and 16 ml acetic acid), 10 mm 2,4,6-tripyridyl-s-triazine solution in 40 mm hcl, and 20 mm fecl3 solution. readings of the coloured product (ferrous–tripyridyltriazine complex) were recorded at 595 nm, and the results expressed as mmol fe2+ kg-1. triplicate measurements were acquired for both assays. 2.3.6 sensory analysis a sensory evaluation was undertaken on leather samples at day 0 and 21, by a panel of ten semi-trained judges (aged 25-65 years) from the disafa department. leathers were brought to 25°c and presented to panellists coded, who appraised the samples for hardness, sweetness, sourness, flavour and overall acceptance, using a 5-point hedonic scale. panellists’ average responses were considered for each attribute. ital. j. food sci., vol. 31, 2019 475 2.4. statistical analysis all the statistical analyses were computed using spss 20 for windows (spss, inc., chicago, il, usa). analysis of variance (anova) was conducted, using the software ibm-spss 22 (2015), followed by tukey’s honestly significance difference test (p<0.05). 3. results and discussion 3.1. formulation development at the end of the first trial, all formulations differed by colour, texture and taste. formulations 1 and 2 presented the desired firmness and flexibility, due to lowmethylation pectin powder addition and its gelling property, which promotes hydrogen bonds between carboxyl groups and water, and ionic bonds with calcium (ca2+) and other divalent metals (valenzuela and aguilera, 2015). although actinidia species are known to have low-pectin content (reddy et al., 2015), formulation 3 with no added pectin powder presented an acceptable texture, afforded by the endogenous pectic compounds of the granny smith apples that comprised 22% of the recipe (table 1) and are known to have a high pectin content (quintero ruiz et al., 2012). furthermore, the extremely low amounts of glucose syrup (5%) and fructose (5%) in this formulation favoured concentration and the outcome of a desirable texture. the addition of sugar compounds strengthens pectic gels up to a maximum point, after which, such property may reduce (orego et al., 2014). this behaviour is explained by the presence of low molecular weight compounds, such as glucose, fructose, sucrose and organic acids, which have a low glass transition temperature. these molecules may affect the product texture because they are highly hygroscopic in their amorphous state and show high molecular mobility at temperatures above the glass transition temperature leading to a less stable, sticky and rubbery leather (dirim et al., 2015). in this work, this undesirable texture was observed only in formulation 4, which had the greatest glucose syrup content (28 %), in the absence of pectin powder. besides texture, sourness was also an important factor in determining the overall acceptance of baby kiwifruit leathers. while formulations 1, 2 and 4 presented a sugary and well-balanced taste, respectively, formulation 3 was considered too sour by the panellists. a second trial was, therefore, necessary to optimise baby kiwifruit formulations. given the aim was to develop a formulation that produced a leather presenting a composition as similar to that of the fresh fruit, only formulations 3 and 4 were modified in the second trial, generating formulations 3a and 4a, respectively. these formulations were preservative-free, with a relatively less sour taste (created by replacing the citric acid powder with fresh lemon juice) and an appropriate texture (table 2). formulation 4a had less glucose syrup than formulation 4, to reduce the low molecular weight compounds while the glucose syrup content of formulation 3a was higher than formulation 3, to improve taste. fructose power was added to both formulations as a sweetener. finally, formulation 4a incorporated green apple to enhance the gel flavour. the same concentration process and sensory analysis of the original formulations were applied to formulations 3a and 4a. although both formulations presented a desirable texture and taste, formulation 4a did not keep a desirable colour, due to its high sugar content, so only formulation 3a was assessed for shelf life quality. ital. j. food sci., vol. 31, 2019 476 table 2. formulations 3a and 4a, produced during second trial obtained by optimization of formulations 3 and 4. formulation 3a formulation 4a 66% baby kiwi frozen puree 47% baby kiwi frozen puree 22% apple granny smith 31% apple granny smith 5% glucose syrup 33 de 10% glucose syrup 33 de 5% fructose powder 10% fructose powder 1% lemon juice 1% lemon juice 1% water 1% water 3.2. shelf life test and fruit quality assessments 3.2.1 weight loss, soluble solids and acidity weight loss was statistically different among all leather samples and did not alter during shelf life (fig. 1), except sample ope, which demonstrated a slight increment at 21 days. figure 1. weight losses (%) of environment (e), environment pe (epe), oven (o) and oven pe (ope) samples during storage. 1different capital letters (a–b) show significant differences (p≤ 0.05) within treatment. 2different lower-case letters (a-d) show significant differences among treatments (p ≤ 0.05) for each storage time. major weight loss was observed for ope and o samples. in this work, even though moisture content was not determined, weight loss might be related to the moisture loss during shelf life, as demonstrated in previous trials (diamante et al., 2014). enhancement of this parameter might indicate a poor shelf life stability of samples since this trend could change the texture, colour and nutrient composition. lower weight loss was noted for the pa/pe packaged samples than the unpackaged ones, under both storage conditions, suggesting that pa/pe packaging was efficient to avoid or delay weight loss in ope, e and pe samples when compared with their unpackaged ital. j. food sci., vol. 31, 2019 477 counterparts, stored under the same temperature condition. conversely, the pa/pe packaging was insufficient to circumvent the augmented weight loss trend of ope sample during simulated long-term shelf life (i.e., the accelerated storage condition). 3.2.2 soluble solids content (ssc) figure 2 shows the ssc of raw puree, raw formulation and leather samples. during leather manufacturing, the addition of glucose syrup increased the ssc from 16% (raw puree) to 25% (raw formulation). owing to the concentration process, leather samples at day 0 reached 75%, which was higher than the 65% scc needed for sugar-acid-high-methoxyl pectin gelation (quintero et al., 2012). an increase in the ssc is mainly a result of the concentration of the sugars and is important to the gel flavour, as the hydrogen bonded sugar–water molecules will be entrapped in the pectic gel. this phenomenon is possible only at a high sugar concentration of 55 g 100 g-1 (torres et al., 2013). differences between samples were observed, especially at 14 and 21 days. oven samples registered significantly higher ssc values than environment samples, irrespective of the storage time. this finding was consistent with the literature that indicates an increase of ssc during storage is a consequence of dehydration and possible conversion of complex carbohydrates into sugars and other soluble components (kuchi et al., 2014). in this work, carbohydrates were probably represented by the starch content since frozen puree was produced from unripe fruit, not suitable for the fresh market, though further analysis of those components should have been done, to provide a better understanding of the product composition. the ssc trend demonstrated in this work also occurred in the simulated long-term storage products (o/ope samples), corroborating the work of chavan and shaik (2015). differences between packaged and unpackaged samples were also noticed: those with pe packaging presented lower ssc values at the end of shelf life when compared with the unpackaged samples under the same temperature condition. although, from day 7, the brix values within samples fluctuated and, at the end of the shelf life, were higher than the initial values (0 day), demonstrating poor stability of the samples, with and without packaging. these results are different from quintero et al. (2012), wherein apple leathers packaged with a metalised-plastic material did not show any modifications because of the superior protective property of the packaging material. as far as we know, there are no other shelf life studies of pa/pe packaged fruit leather. although, the high water vapour transmission rate (5 g m-2 d-1) of pa/pe packaging means it is not as efficient at preserving the product’s quality as other packaging materials, such as polyester/aluminium/polyamide/polyethylene, which has a water vapour transmission rate of 0.1 g m-2 d-1 (fu et al., 2018). 3.2.3 colour and browning index (bi) the colour degradation of leather samples was highly correlated with the evolutions of l* and bi during shelf life, so only those two parameters are presented in this work. at the end of shelf life, l* values decreased significantly for o, ope and e samples, indicating a darkening of the colour. only epe samples retained initial l* values (table 3), which demonstrates a protective effect of pa/pe packaging but exclusively in simulated shortterm shelf life. parameter l* expresses the degree of brightness, which can be related to browning reactions and production of dark pigments, such as melanoidins (quintero et al., 2012; patel et al., 2013), caused by oxygen exposure, moisture and an extended shelf life (patel et al., 2013). thus, the trend found for ope when simulating a longer shelf life, illustrated the inadequacy of pa/pe in preventing the quality loss of this sample, as its l* values were even higher than those of o samples. ital. j. food sci., vol. 31, 2019 478 figure 2. soluble solids content of raw puree, raw formulation and leather samples at day 0; and brix of environment (e), environment pe (epe), oven (o) and oven pe (ope) samples during storage. 1different capital letters (a-c show significant differences (p ≤ 0.05) within treatment. 2different lower-case letters (a-d) show significant differences among treatments (p ≤ 0.05) for each storage time. table 3. l* evolution of environment (e), environment pe (epe), oven (o) and oven pe (ope) samples during storage. samples l* day 0 day 7 day 14 day 21 e 26,71 a1 n.s.2 26,04 ab bc 25,85 ab a 25,13 b a epe 26,71 n.s. n.s. 26,76 n.s. ab 25,62 n.s. a 25,81 n.s. a o 26,71 ab n.s. 28,03 a a 25,88 b a 25,68 b a ope 26,71 a n.s. 24,78 b c 22,40 c b 23,24 c b 1different capital letters (a-c) in the same row show significant differences (p ≤ 0.05) within treatment. 2different lower-case letters (a-b) in the same column show significant differences among treatments (p ≤ 0.05) for each storage time. the bi (fig. 3), which is a combination of the l*, a* and b* values, and used as a colour darkening indicator of leathers (quintero ruiz et al., 2012; torres et al., 2013), contrasted with the l* values and demonstrated colour quality loss only for o samples. for these samples, the bi increased from 54.11 to 89.68. conversely, e and epe samples were stable, and ope displayed only a slight decrease at 14 days of shelf life, probably related to the decline in b*, from 6.25 to 2.46, as the b* values of the other samples increased from 6.22 to 4.32 (e), 4.90 (epe) and 7.52 (o), thereby affecting the estimation of the bi of ope throughout the shelf life. for this reason, the l* values were a better indicator of colour quality loss during shelf life. the browning reactions were probably caused by the degradation of ascorbic acid, present at high amounts in the raw material (mcneilage et al., 2001; mcneilage et al., 2004) and probably initially reduced by the ital. j. food sci., vol. 31, 2019 479 concentration process and preparation of leather samples, but degraded further during shelf life studies. ascorbic acid is strongly affected by temperature but may incur browning reactions, even at 25°c (patel et al., 2013). the formation of browning compounds during storage could also be due to the maillard reaction initiated by the interaction between reducing sugars and amino groups, and ultimately generates brown pigment precursors, such as hydroxymethylfurfural (maskan, 2001; lespinard et al., 2012). figure 3. browning index evolution for environment (e), environment pe (epe), oven (o) and oven pe (ope) samples during storage. 1different capital letters (a-b) show significant differences (p ≤ 0.05) within treatment. 2different lower-case letters (a-b) show significant differences among treatments (p ≤ 0.05) for each storage time. 3.2.4 texture parameters adhesiveness and springiness express the rheological and mechanical properties of organic materials (arana, 2012) and might indicate the efficiency of the packaging material adopted for stabilising the leather texture, as well as how the endogenous pectin and low molecular weight compounds might affect texture during the concentration process and shelf life. a limited amount of literature has demonstrated the tpa measurements of leather (orego et al., 2014). adhesiveness is an important parameter of dried fruit products since it could infer the stickiness of the products (nishinari and fang, 2018). the adhesiveness values (table 4) of samples enhanced to become closer to 0, indicating a lower sensation of stickiness of samples at the end of shelf life. samples o and ope had significantly different values from the other samples during shelf life since they were the least sticky. according to valenzuela and aguilera (2015), high adhesiveness values of leather at day 0 (-237.68) could be related to the presence of low molecular weight compounds in the leather formulation. it means the high adhesiveness levels found in the current work are probably explained by the use of fructose and glucose syrup 33 de in the leather formulations. these components contribute a large number of monomers relative to the other ingredients and with lower de values, lowering the glass transition temperature, which is the temperature at which products may become increasingly sticky. in contrast, glucose syrup 17 de and 19 de, used by valenzuela and aguilera (2015), contribute to relatively high molecular weight polymer ital. j. food sci., vol. 31, 2019 480 compounds, which can compete with sugars for hydrogen bonding with water, thereby avoiding the product stickiness. during shelf life, the water loss of samples (suggested by the weight loss and ssc concentration) might have reduced the adhesiveness, a phenomenon most frequently apparent for o samples, attributed to the higher temperature condition of 37 versus 25°c. similarly, al hinai et al. (2013) verified that adhesiveness was also influenced by the moisture content of leather samples. springiness values (table 5) fluctuated during shelf life. pectin was found to be the most important factor influencing (increasing) springiness of fruit leather (orego et al., 2014). consequently, the decrease in springiness is possibly explained by endogenous pectin gel degradation, leading to the formation of new compounds, such as pectic acids and uronic acid (kuchi et al., 2014) that might have occurred during shelf life. the absence of significant differences between packaged and unpackaged samples for both texture parameters at the end of shelf life reaffirmed the pe packaging was inadequate in maintaining the texture stability of the leathers. 3.2.5 total phenolic content (tpc) determination and antioxidant capacity (ac) during leather production, the tpc of the raw puree decreased in the raw formulation but then increased in the leather samples at day 0. the tpc values did not differ significantly among the leather samples or during shelf life (fig. 4). figure 4. total polyphenol content of raw puree, raw formulation and leather samples at day start, and total polyphenol content evolution for environment (e), environment pe (epe), oven (o) and oven pe (ope) samples during storage. 1capital letters (n.s) show absence of significant differences(p ≤ 0.05) within treatment. 2lower-case letters (n.s.) show absence of significant differences among treatments (p ≤ 0.05) for each storage time. the major polyphenol compounds in baby kiwifruit are phenolic acids, tannins and flavanols (leontowicz et al., 2016). in our work, the tpc of raw puree (334.79 mg gae 100 g-1) was very similar to fresh fruit, as also shown by zou et al. (2012). baby kiwifruit is currently marked in europe as berries, due to its size, commercial availability and ital. j. food sci., vol. 31, 2019 481 nutraceutical quality. however, even though the tpc values are higher than raw apple puree (landl et al., 2010), they are still lower than strawberry and blueberry puree (patras et al., 2009). moreover, the addition of apples to the raw puree during the formulation process further lowered the tpc amounts in the raw formulation, thereby deviating the product from the berries nutraceutical profile. leather samples at day 0 presented an increased tpc (563.93 gae 100 g-1), as a result of the concentration process, wherein water loss meant all the compounds present were concentrated, in concurrence with observations by suna et al. (2014), lutz et al. (2015) and concha-meyer et al. (2016). in this work, the ssc increased almost three-fold while the tpc values only doubled. thus, on a dry weight basis, it is suggested that heat treatment would have led to a loss of phenolic compounds, despite the absolute increased values. no changes within and between leather samples during shelf life affirmed that shelf life and packaging conditions did not affect the tpc, in agreement with quintero et al. (2012) and torres et al. (2013). although, fig. 5 emphasises the large variability in the data, especially among o and ope samples. considering the substantial fluctuations in the data of almost all analysis, blending of the ingredients must be further improved to ensure homogeneity of the samples. figure 5. antioxidant capacity of raw puree, raw formulation and leather samples at day 0. antioxidant capacity of environment (e), environment pe (epe), oven (o) and oven pe (ope) samples during storage. 1capital letters (n.s).show absence of significant differences (p ≤ 0.05) within treatment. 2lower-case letters (n.s.) show absence of significant differences among treatments in(p ≤ 0.05) for each storage time. the ac of raw puree, raw formulation and leather samples at day 0 were similar to each other. this trend was not expected since tpc has been well correlated to ac in fresh, pureed and dried fruits, respectively (karadag et al., 2009; orego et al., 2014). the ital. j. food sci., vol. 31, 2019 482 absence of significant differences within and among samples highlights that pe packaging and storage time did not affect the ac of leathers (fig. 5). the ac stability after day 7 well correlated with the tpc trend. this pattern was also observed by sacchetti et al. (2008) and concha-meyer et al. (2016). 3.2.6 sensory evaluation figure 6 illustrates the scores obtained for each leather sample at days 0 and 21. overall, at day 0 the descriptors hardness (3.2), sweetness (3.2), flavour (4.2) and overall liking (3.5) scored highest while sourness (2.2) scored lowest, for all samples. at the end of storage, flavour decreased, and sourness increased, in all samples. sweetness decreased for o and ope samples while hardness remained stable for e and epe. these results contrasted with the ssc analysis, which showed an increase in ssc content of o and ope samples, suggesting an increased sweetness. probably, the ssc increase was accompanied by a similar trend in the acidity content, which was not assessed in this work. both o and ope exhibited particularly enhanced hardness values, arising from the intense dehydration of the leathers. overall acceptance remained stable during the shelf life of e and epe samples while for o and ope samples, this descriptor drastically decreased, as a result of hardening. table 4. adhesiveness parameter of tpa for environment (e), environment pe (epe), oven (o) and oven pe (ope) samples during storage. samples adhesiveness (n*s) day 0 day 7 day 14 day 21 e -237,68 b1 n.s.2 -69,91 aab -58,02 aab -47,98 aab epe -237,68 b. n.s. -115,64 ab -67,71 ab -71,10 ab o -237,68 b n.s. -1,01 ab -21,64 aa -1,08 aa ope -237,68 b n.s. -70,92 aab -59,98 aab -17,95 aa 1different capital letters (a–c) in the same row show significant differences (p ≤ 0.05) within treatment. 2different lower-case letters (a–b) in the same column show significant differences among treatments (p ≤ 0.05) for each storage time. table 5. springiness parameter of tpa for environment (e), environment pe (epe), oven (o) and oven pe (ope) samples during storage. samples springness (s) day 0 day 7 day 14 day 21 e 0,49 n.s.1n.s.2 0,63 n.s. b 0,71 n.s.ab 0,88 n.s.n.s. epe 0,49 b n.s. 0,69 a b 0,45 b b 0,72 a n.s. o 0,49 c n.s. 0,56 b a 0,90 a a 0,88 a n.s. ope 0,49 b n.s. 0,56 ab b 0,71 a ab 0,77 a n.s. 1different capital letters (a–c) in the same row show significant differences (p ≤ 0.05) within treatment. 2different lower-case letters (a–b) in the same column show significant differences among treatments (p ≤ 0.05) for each storage time. ital. j. food sci., vol. 31, 2019 483 figure 6. sensory analysis scores of environment (e), environment pe (epe), oven (o) and oven pe (ope) samples at day 0 and after day 21. 4. conclusions the development of leather formulations could be a promising opportunity to improve the baby kiwifruit supply chain, solving the a. arguta losses and offering a processed product that meets the requirements of current consumers seeking healthy and convenient food. baby kiwifruit formulations considered for obtaining a healthy product with low sugar and a bright green appearance were achieved by adjusting the recipe to offer a balanced content of fructose and glucose syrup. in turn, it reduced the maillard reaction and favoured formation of a desirable texture, generating a flexible but solid product after the concentration process. in this investigation, preparing baby kiwifruit leather with high fruit content allowed shelf life extension and improved the nutritional properties of the fresh raw material by the concentration-induced reduction in the water content and, consequently, enhanced sugar and polyphenol concentrations. however, the accelerated shelf life experiment (simulating long-term conservation) exposed baby kiwifruit leathers to intense browning reactions and dehydration, leading to the darkening of o and ope samples and hardening of the texture up to a level that highly compromised the overall acceptance. pa/pe packaging was inconsistent at maintaining leather quality by the end of the shelf life experiment, regardless of a shelf life extension. additional research is needed to identify new material packaging that is suitable to prevent quality loss during shelf life, and further analysis should be done to 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quince leathers made without preservatives and with enhanced antioxidant activity. lwt food science and technology 62(2):996-1003. ital. j. food sci., vol. 31, 2019 486 valenzuela c. and aguilera j. m. 2015. effects of different factors on stickiness of apple leathers. journal of food engineering 149:51. vatthanakul, s., jangchud, a., jangchud, k., therdthai, n. and wilkinson, b. 2010. gold kiwifruit leather product development using quality function deployment approach. food quality and preference 21(3):339-345. zou l., wang z., fan z., tian s. and liu j. 2012. evaluation of antioxidant and antiproliferative properties of three actinidia (actinidia kolomikta, actinidia arguta, actinidia chinensis) extracts in vitro. international journal of molecular science (13):5506. paper received september 10, 2018 accepted february 25, 2019 ital. j. food sci., vol. 30, 2018 553 paper development of chicken roll recipe using response surface methodology a.y. aydar*, b. gürel and s. kayaardi department of food engineering, faculty of engineering, manisa celal bayar university. muradiye, manisa 45140 turkey *e-mail address: alevyuksel.aydar@cbu.edu.tr abstract the purpose of this study was to determine the optimum quantities of ingredients to yield a chicken roll product with desirable textural properties and coloring and a minimum cooking loss. response surface methodology (rsm), a statistical technique, was applied for optimization. the optimum quantities for chicken roll production were found to be 8.66 g, 75.00 g and 53.83 g for wheat flour, distilled water, and minced chicken, respectively. the lowest cooking loss was achieved by a recipe with high wheat flour and distilled water content, whereas the highest cooking loss was observed in the recipe with the lowest wheat flour content and the highest minced chicken content. keywords: chicken, poultry, optimization, texture profile analysis, response surface methodology ital. j. food sci., vol. 30, 2018 554 1. introduction the consumption of poultry meat is gradually increasing around the world. because of its high-quality protein and relatively low fat content, poultry meat, especially chicken meat, plays an important role in the human diet. in addition to this high nutritional value, the relatively low cost and great variety of chicken meat products make chicken meat a widely favored food (chouliara et al., 2008; mok et al., 2017). in recent years, ready-to-eat (rte) foods have become highly preferred food products. some of the leading causes of the rising consumer demand for rte foods include: changes in consumers’ lifestyles, households in which both parents work long hours, the convenience of consumption, the minimal time required for rte meal preparation, and the charm of flavorful products (bae et al., 2010; jiang and xiong, 2015). the consumption of rte foods has risen almost 20% from 2007 to 2012; during these years, european consumers’ demand for rte meat and poultry products (e.g. meat balls, burger patties, sausages) has increased in accordance with this trend (ferreira et al., 2016). in australia, the consumption of rte meat products has recently increased from 20% to 50% (jiang and xiong, 2015). according to a global report on the rte food market, rte food consumption is expected to rise 21.8% between 2018 and 2023 (global ready to eat food market report, 2017). hydrocolloids (e.g. pectin, xanthan, starch, guar gum, alginate) are frequently used in various food products as thickeners, gelling agents, emulsifiers, stabilizers, fat replacers, clarifying agents, flocculating agents, clouding agents, and whipping agents (li and nie, 2014; viebke et al., 2014). guar gum, commonly used as a hydrocolloid, is a good stabilizer and water-binder, and it provides desirable structure and a slick fat-like mouthfeel for food products (andrés et al., 2006). starches occupy an important role in meat recipes (feng et al., 2013). due to its unique white color, excellent mouthfeel properties, bland taste, and relatively small granules (2-7 µm), rice starch has become an alternative fat replacer that provides good textural properties for food products (park et al., 2007; resconi et al., 2015; tomaschunas et al., 2013; wani et al., 2012). because gum increases the viscosity of starch and affects gelatinization, the utilization of gum and starch in food systems has been researched in many studies (kim and yoo, 2006; yoo et al., 2005). the interactions between starches and gums enhance the rheological properties of starch, improve overall product quality, and reduce the cost of the products (kim and yoo, 2006; shi and bemiller, 2002). it was reported that guar gum and xanthan gum increased the viscosity of cationic tapioca starch suspensions (chaisawang and suphantharika, 2005). feng et al. (2013) studied the physicochemical properties, texture, and sensory evaluation of chinese cantonese-style sausage prepared from the mesona blumes gum-rice starch mixture; they reported that this gel could be used as a fat substitute in sausage (feng et al., 2013). alginate, a polysaccharide obtained from brown seaweed, is used as a thickening, filmforming, gel-producing, and emulsion-stabilizing agent in the food industry; this is due to its high water-solubility, high biodegradability, and low price, relative to natural casings (comaposada et al., 2015; marcos et al., 2016; nakauma et al., 2016). mostly used in meat products, sodium alginate can be combined with calcium ions at room temperature to create a uniform, transparent, water-insoluble, and thermo-irreversible gel (boles and shand, 1998; comaposada et al., 2015; leon et al., 2016). calcium chloride solutions are most commonly used to provide the calcium ions necessary to achieve gelatinization (comaposada et al., 2015; hassan and ramaswamy, 2011). the us food and drug administration applies the label ‘‘generally recognized as safe (gras)’’ to citric acid, which is commonly used in meat and poultry processing for its antimicrobial and tenderizing effects (khare et al., 2016). in some studies, combining ital. j. food sci., vol. 30, 2018 555 citric acid and lactic acid reduced the microbial load of chicken drum sticks, while negatively affecting the sensory parameters (zhu et al., 2016). coating chicken fillets with carrageenan, cinnamon oil, and citric acid extends the shelf life of chicken meat, under chilled conditions (khare et al., 2016). in previous studies, chicken rolls were produced and analyzed with different formulations and methods (breclaw and damson, 1970; du et al., 2003; furumoto and stadelman, 1980; gillett and carpenter, 1992; wang and chen, 1987; xiao et al., 2011; yim et al., 2015). however, to the best of our knowledge, there is no published study addressing the optimization of chicken roll formulation based on some preferred properties. the aim of this study was to determine the optimum quantities of ingredients for a chicken roll product of a desirable color, with textural properties such as low hardness and high resilience, and a minimum cooking loss. 2. materials and methods 2.1. experimental design rsm was employed to determine the optimum values of three independent variables and evaluate the combined effects of those parameters. as the purpose of this study was to evaluate the interaction effects between food hydrocolloids and other ingredients, the water used in the experiments needed to be free of salt or any other impurities. for example, common drinking water contains salts that can change the effects of calcium chloride and sodium alginate; thus, distilled water was used. the wheat flour (5-35 g), distilled water (35-75 g), and minced chicken (20-60 g) were the parameters and levels employed. the coded values and the original values of the independent parameters are shown in table 1. for the purpose of evaluating the pure error and the curvature of the complete design, a three-level three-factor box-behnken design (bbd) was implemented, which indicated the need to compose a total of 17 experiments with 5 replicates of the center point. table 1. variables and their levels in response surface design. coded levels independent variables symbols -1 0 1 wheat flour (g) a 5 20 35 distilled water (g) b 35 55 75 minced chicken (g) c 20 40 60 2.2. sample preparation fresh skinless chicken breasts were obtained from keskinoğlu poultry and hatchery ind. inc., manisa, turkey. different amounts of wheat flour (5-35 g), distilled water (35-75 g), and minced chicken (20-60 g) were added to obtain chicken rolls according to the experimental design. the other ingredients, which are sodium alginate, rice starch, guar gum, citric acid, wheat flour, and calcium chloride, were obtained from smart chemistry inc., i̇zmir, turkey. before preparing the chicken meat batter, breast meat was ground through the mincer. sodium alginate (2.4 g), rice starch (1.7 g), guar gum (0.85 g), citric acid (0.2 g), and wheat flour were added to 60±5˚c distilled water, and blended for 1 minute. the minced chicken was added to this mixture, which was then blended for 1 ital. j. food sci., vol. 30, 2018 556 minute. the chicken meat batter was thinned to 2 mm and dipped in a 125 ml 7% (w/v) calcium chloride (cacl2) solution for 1 minute. then the batter was molded into round shapes (each with an internal diameter of 10 cm), sealed into plastic bags, and cooked in a 90˚c water bath for 5 minutes (until the contents of the bags reached an internal temperature of 72˚c). 2.3. color measurement a chroma meter (konika minolta cr-5, konika minolta, inc., japan) was used to measure the surface color of chicken rolls based on the hunter lab system. hunter lab values were expressed as l* (lightness), a* (redness), and b* (yellowness). color measurements were taken from both sides of each chicken roll and the mean color values of three replicates were calculated. 2.4. cooking loss determination the chicken rolls were individually weighed before and after cooking. the cooking loss was calculated using the following formula, and expressed as a percentage. cooking loss (%) =[(𝑅𝑎𝑤 𝑤𝑒𝑖𝑔ℎ𝑡 −𝐶𝑜𝑜𝑘𝑒𝑑 𝑤𝑒𝑖𝑔ℎ𝑡)/𝑅𝑎𝑤 𝑤𝑒𝑖𝑔ℎ𝑡]×100 2.5. texture profile analysis (tpa) the tpa of the chicken rolls was performed using ta.xt plus texture analyzer (stable micro systems, uk). four uniform test samples, each with a 25 mm diameter, were cut from each chicken roll for the tpa, and the test samples were compressed twice, to 50% of their original height, using a 36 mm cylindrical probe (p/36r) at a test speed of 10.02 cm/min. force-time deformation curves were obtained, using a 50 kg load cell. the curves were calculated using texture exponent 2.0.6.0 software (stable micro systems). texture profile parameters were defined as below: hardness (n) = maximum peak force for the first compression cycle. adhesiveness (n.min) = negative force area for the first bite to pull away the compressing probe. springiness (ratio) = ability of the sample to recover its original shape between the end of the first bite and the start of the second bite. cohesiveness (ratio) = ratio of the positive areas during the second compression area to the first compression area. gumminess (n) = hardness × cohesiveness. chewiness (n) = springiness × gumminess. resilience (ratio) = ratio of the area during the withdrawal of the first compression to the area of the first compression. 2.6. statistical analysis for optimization, the design-expert version 7.0.0 (state-ease inc., minneapolis, mn, usa) was used to evaluate the experimental design, statistical analysis, and regression models. linear, quadratic, cubic or 2fi models were obtained according to experimental data. constant terms a, b, and c (linear coefficients for wheat flour, distilled water, and minced chicken, respectively), ab, ac and bc (interactive term coefficients), a2, b2, and c2 (quadratic term coefficients) were the coefficients of the model. to evaluate the fitness of the model, correlation coefficient (r2), adjusted determination coefficient (adj-r2) and adequate precisions were used. the model was determined to fit when its p value ˂ 0.05, ital. j. food sci., vol. 30, 2018 557 lack of fit p value ˃ 0.05, and adeq. precision > 4. for statistical significance, when a level was set at p < 0.05, an analysis of variance (anova) was used to examine differences between means. 3. results and discussion 3.1. color measurement l*, a*, and b* values of samples are shown in table 2. l* values were between 68.140 and 76.540. the coefficients of the variables in the regression models and their significance are shown in table 3. table 2. design and results of box-behnken experiments (color values and cooking loss). runs a b c l* a* b* cooking loss (%) 1 0 0 0 72.839 -0.113 11.793 -0.50 2 0 1 -1 71.186 -0.824 9.724 -0.98 3 -1 1 0 75.268 -1.059 10.623 0.68 4 0 0 0 73.402 -0.334 11.738 -0.34 5 0 1 1 75.321 -0.018 11.871 2.12 6 1 0 1 70.067 0.474 12.479 2.27 7 0 0 0 73.433 0.013 12.385 3.76 8 1 -1 0 68.140 1.648 14.702 1.88 9 -1 0 1 76.540 0.373 11.067 8.82 10 0 -1 1 74.013 0.587 11.819 1.83 11 1 1 0 72.434 0.216 12.236 -1.41 12 0 -1 -1 69.260 0.593 13.353 2.63 13 -1 -1 0 75.288 -0.120 11.450 5.26 14 1 0 -1 68.195 0.558 13.003 2.50 15 -1 0 -1 72.358 -0.988 10.490 5.11 16 0 0 0 73.357 -0.194 12.120 2.05 17 0 0 0 72.897 -0.249 11.282 0.69 a: wheat flour; b: distilled water; c: minced chicken. effects on the l* value of the chicken rolls that were statistically significant for the model (p< 0.05) included: the linear effects of wheat flour (a), distilled water (b), and minced chicken (c); the interaction effects of wheat flour and distilled water (ab), wheat flour and minced chicken (ac); and the quadratic effect of minced chicken (c2) (table 3). the l* value decreased when the amount of distilled water and wheat flour increased. however, it increased when the amount of minced chicken increased. previous studies reported a high correlation between moisture and l* value for meat products (costa-corredor et al., 2009; garcía-esteban et al., 2003; sanabria et al., 2004). devatkal et al. (2011) determined that the addition of 10% sorghum flour into the chicken nugget formulation tended to lower the l* value of the products. because meat muscles contain a high amount of water, increasing the meat content in the product increases the l* value (devatkal et al., 2011). in our study, increasing minced chicken content yielded greater lightness of the chicken rolls, but increasing the water content decreased the l* value of the samples. it is suggested that the cooking loss of the products increased in accordance with increased levels of water content. thus, an increasing concentration of pigments (e.g., myoglobin) during the cooking process produces a darker coloration in the chicken rolls. ital. j. food sci., vol. 30, 2018 558 as we observed in our results, garcía-esteban et al. (2003) also found that ham semimembranosus (sm) muscle has a lower l* value, depending on dehydration (garcíaesteban et al., 2003). the quadratic regression model for l* value is as follows: l* = +73.192.58a + 0.94b + 1.87c +1.08ab0.58ac0.87c2 a* values of the chicken rolls were between-1.059 and 1.648. the linear effects of wheat flour (a), distilled water (b), minced chicken (c), and the interaction effect of wheat flour minced chicken (ac) were significant (p< 0.05). the 2fl regression model for a* value results is shown below: a* = +0.033 + 0.59a0.55b + 0.26c0.36ac a* values of the chicken rolls increased as distilled water content decreased, according to the regression model. as increased water content has a diluting effect on the myoglobin responsible for the color pigment of the meat, increasing the water content in the chicken roll resulted in a lower a* value. khare et al. (2015) also reported that decreasing the meat level in chicken noodles reduced the redness caused by the concentration of pigments (khare et al., 2015). the b* values of samples ranged from 9.724 to 14.702. wheat flour (a) and distilled water (b) had a significant linear effect on b* value (p< 0.05). thus, the decreased quantity of wheat flour yielded low b* values. the interaction effect of distilled water--minced chicken (bc) was statistically significant for the model (p< 0.05). it has been reported that the addition of 7.5% and 10.0% bean flour to beef sausages increases the yellowness of samples, depending on the dilution of the myoglobin in the meat and, to some extent, the color of the flour additives (dzudie et al., 2002). the 2fl regression model for b* value is as follows: b* = +11.89 + 1.10a 0.86b + 0.92bc although the results of l* value are similar to some studies previously conducted on different poultry meat products, b* values were relatively higher than the values reported by previous studies (heaton et al., 2000; tang and cronin, 2007; tang et al., 2005). also, a* values were relatively lower than the values reported by tang and cronin (2007) and tang et al. (2005). differences for a* and b* values could be related to the amounts of wheat flour and rice starch used in this study or to the different types of poultry meat used in previous studies. analysis of variance (anova) of the regression models for all responses is shown in table 4. ital. j. food sci., vol. 30, 2018 559 table 3. the coefficients of the variables in the regression models and their significance. constant a b c ab ac bc a2 b2 c2 model hardness 89.05 17.02* -16.33* 1.49 _ _ _ _ _ _ linear adhesiveness -9.97e-003 -2.39e-003* -3.65e-003* +1.35e-003 -1.65e-004 +3.42e-003* -2.52e-003 _ _ _ 2fl gumminess 69.68 12.40* -13.65* -0.21 _ _ _ _ _ _ linear chewiness 59.79 9.57* -13.04* 0.39 _ _ _ _ _ _ linear resilience 0.38 -6.24e-003 -0.019* -0.015* _ _ _ _ _ _ linear l* 73.19 -2.58* 0.94* 1.87* 1.08* -0.58* -0.15 -0.53 0.13 -0.87* quadratic a* 0.033 0.59* -0.55* 0.26* -0.12 -0.36* 0.20 _ _ _ 2fl b* 11.89 1.10* -0.86* 0.083 -0.41 -0.27 0.92* _ _ _ 2fl cooking loss 1.13 -1.83* -1.40* 0.72 0.32 -0.98 0.97 1.87* -1.40 1.67 quadratic *significant at 5% level, a: wheat flour; b: distilled water; c: minced chicken. table 4. anova for examination of every regression model adequac. model lack of fit responses f value p value r2 adj-r2 adeq. precision ss p value hardness 23.51 <0.0001* 0.8443 0.8084 17.274 753.45 0.0735** adhesiveness 6.89 0.0041* 0.8051 0.6882 9.504 3.409e-005 0.5450** springiness 2.87 0.0771** 0.3985 0.2596 5.511 4.281e-003 0.9260** cohesiveness 3.36 0.0520** 0.4367 0.3067 5.834 4.169e-003 0.1790** gumminess 24.04 <0.0001* 0.8473 0.8120 17.486 432.80 0.1292** chewiness 17.34 <0.0001* 0.8001 0.7539 14.692 471.02 0.0980** resilience 4.92 0.0169* 0.5316 0.4235 7.626 2.698e-003 0.7154** l* 50.07 <0.0001* 0.9847 0.9650 24.761 1.19 0.0854** a* 15.77 0.0001* 0.9044 0.8471 14.717 0.61 0.0558** b* 20.65 <0.0001* 0.9253 0.8805 16.417 0.91 0.5800** cooking loss 4.00 0.0405* 0.8374 0.6282 8.227 4.46 0.7217** adj-r2.adjusted determination coefficient; ss. sum of square; *significant; ** not significant. ital. j. food sci., vol. 30, 2018 560 high f values (50.07, 15.77 and 20.65, for l*, a*, b*, respectively) and low p values (<0.0001, 0.0001, and <0.0001, for l*, a*, b*, respectively), indicate that the models were significant for l*, a* and b* values. the r2 of predicted models for l*, a*, and b* values were 0.9847, 0.9044, and 0.9253, respectively. the adj.r2 value indicated a degree of linear fit between the predicted and experimental values, which were 0.9650 for l* value, 0.8471 for a* value, and 0.8805 for b* value. the p value of the lack-of-fit test was 0.0854 for l* value, 0.0558 for a* value and 0.5800 for b* value; these values indicate that the model fit the experimental data. based on these results, the model of color parameters was adequate for predicting within the range of the variables employed. 3.2. cooking loss determination the cooking loss values of samples ranged from-1.41% to 8.82%, as shown in table 2. the linear effects of wheat flour (a) and distilled water (b) and the quadratic effect of wheat flour (a2) on the cooking loss of the chicken rolls were statistically significant for the model (p< 0.05) (table 3). the negativity in cooking loss value can be explained by the interaction between the water in the water bath and in the chicken roll recipe during the cooking process. guar gum is widely used in the food industry for its thickening properties, which operate by its interaction with water and stabilizing effect on food matrices, and by its ability to favorably interact with gluten proteins and increase dough stability (linlaud et al., 2009; sandhu et al., 2015). in our study, the thickening effect of guar gum on higher levels of water resulted in lower amounts of cooking loss. therefore, increased quantities of distilled water produced low cooking loss values. wang and chen (1987) also found that the cooking yields of poultry meats were higher when more water was added (wang and chen, 1987). the quadratic regression model for cooking loss results is shown below: cooking loss = +1.131.83a1.40b + 1.87a2 the model was significant for cooking loss, with regard to f value (4.00) and p value (0.0405). the r2 of the predicted model for cooking loss was 0.8374, indicating that more than 83% of the variability in cooking loss could be explained by the model. the regression model shows that cooking loss could be predicted within the range of the variables employed. 3.3. texture profile analysis (tpa) the results of the tpa are shown in table 5. the hardness values of the chicken rolls were between 57.410 n and 123.069 n. wheat flour (a) and distilled water (b) had a significant effect on the hardness of the samples (p< 0.05) (table 3). the hardness of the chicken rolls rose when large amounts of flour were added to the formulation, but it fell when large amounts of water were added. this can be explained by the high water-and fat-absorption properties of flour and the softening effects of water. as we observed in our results, devatkal et al. (2011) reported that the addition of 10% sorghum flour into chicken nuggets’ formulation significantly increased the nuggets’ hardness (p<0.05). by contrast, dzudie et al. (2002) found that the shear force and hardness of beef sausages decreased with the addition of common bean flour. ital. j. food sci., vol. 30, 2018 561 table 5. design and results of box-behnken experiments (tpa). runs a b c hardness (n) adhesiveness (n.min) springiness cohesiveness gumminess (n) chewiness (n) resilience 1 0 0 0 82.077 -0.007 0.874 0.776 63.918 56.298 0.362 2 0 1 -1 72.848 -0.015 0.803 0.767 55.889 45.065 0.351 3 -1 1 0 67.193 -0.009 0.883 0.774 51.914 46.218 0.370 4 0 0 0 85.947 -0.013 0.889 0.808 69.472 58.818 0.372 5 0 1 1 78.032 -0.018 0.845 0.739 57.384 49.187 0.328 6 1 0 1 110.629 -0.007 0.859 0.762 84.605 73.447 0.361 7 0 0 0 81.591 -0.008 0.796 0.786 63.968 51.408 0.383 8 1 -1 0 123.069 -0.010 0.840 0.753 92.152 77.474 0.368 9 -1 0 1 78.479 -0.009 0.878 0.773 60.679 53.317 0.374 10 0 -1 1 103.109 -0.003 0.900 0.786 80.905 72.685 0.392 11 1 1 0 83.436 -0.015 0.814 0.768 63.492 51.823 0.375 12 0 -1 -1 119.455 -0.010 0.877 0.786 94.217 83.165 0.408 13 -1 -1 0 86.515 -0.005 0.895 0.818 70.623 63.327 0.410 14 1 0 -1 108.615 -0.018 0.860 0.818 88.664 76.981 0.409 15 -1 0 -1 57.410 -0.006 0.848 0.812 46.509 40.315 0.409 16 0 0 0 91.872 -0.008 0.832 0.798 72.836 61.076 0.394 17 0 0 0 83.586 -0.008 0.821 0.805 67.320 55.864 0.417 a: wheat flour; b: distilled water; c: minced chicken. ital. j. food sci., vol. 30, 2018 562 this can be explained by the fact that the beef sausage formulation contained meat in large quantities and lacked any hydrocolloid that would have increased the hardness (devatkal et al., 2011). the linear regression model for hardness is as follows: hardness = + 89.05 + 17.02a16.33b adhesiveness results of the samples ranged from-0.018 n.min to-0.003 n.min. the linear effects of wheat flour (a) and distilled water (b) and the interaction effect of of flour-minced chicken (ac) were statistically significant (p< 0.05) for the adhesiveness of chicken rolls (table 3). verma et al. (2015) also reported that incorporating 8% pea hull flour into low-fat low-salt chicken nuggets resulted in a decrease in the adhesiveness value of the samples (verma et al., 2015). the 2fl regression model for adhesiveness is shown below: adhesiveness =-9.967e-0032.393e-003a3.654e-003b+ 3.420e-003ac springiness values of the chicken rolls were between 0.796 and 0.900. cohesiveness results of the samples ranged from 0.739 to 0.818. the high p values for springiness and cohesiveness indicated that none of the regression models were a fit for these textural parameters. gumminess values of the chicken rolls ranged from 46.509 n to 94.217 n. the linear effects of wheat flour (a) and distilled water (b) were significant for the gumminess of the chicken rolls. these effects showed that decreasing the quantity of wheat flour or increasing the quantity of distilled water yielded less gummy chicken rolls. devatkal et al., (2011) found similar results with an addition of 10% sorghum flour, which enhanced the gumminess of the chicken nuggets (devatkal et al., 2011). furthermore, it was reported that 10% sorghum flour or 10% finger millet flour significantly increased the gumminess of chicken patties (p<0.05)(das et al., 2015). the linear regression model for gumminess is as follows: gumminess = +69.68 + 12.40a13.685b chewiness values of the samples ranged from 40.315 n to 83.165 n. as they were for gumminess, the linear effects of wheat flour (a) and distilled water (b) were statistically significant for the chewiness of chicken rolls. the chewiness value decreased as the quantity of distilled water increased, while chewiness increased as the quantity of wheat flour increased. it has been determined that incorporating 8% pea hull flour into low-fat low-salt chicken nuggets increases the chewiness of the samples. adding 10% sorghum flour to the recipes for chicken nuggets and for chicken patties recipes resulted in significantly higher chewiness values (p<0.05)(das et al., 2015; devatkal et al., 2011; verma et al., 2015). the linear regression model for chewiness is shown below: chewiness = +59.79 + 9.57a13.04b resilience values of the chicken rolls were between 0.328 and 0.417. the distilled water (b) and the minced chicken (c) each had an individual linear effect that was statistically significant for the model of resilience. thus, the resilience value of the chicken rolls increased when the amount of either the distilled water or the minced chicken was decreased. the linear regression model for resilience is as follows: resilience = +0.380.019b0.015c ital. j. food sci., vol. 30, 2018 563 while the values of hardness are similar to those of the turkey rolls reported by tang et al., 2005, the values of cohesiveness and gumminess are relatively higher than those of the same turkey rolls (tang et al., 2005). these differences with regard to cohesiveness and gumminess (between our chicken rolls and the previously studied turkey rolls) could be related to the cooking methods and food additives used in this study. the p value, f value, r2, adj-r2, and adeq. precision estimates of the adequacy of each model are shown in table 4. the models are significant for hardness, adhesiveness, gumminess, chewiness, and resilience; this is reflected by high f values (23.51, 6.89, 24.04, 17.34 and, 4.92, respectively) and low p values (<0.0001, 0.0041, <0.0001, <0.0001, and 0.0169, respectively). as springiness and cohesiveness had low f values (2.87 and 3.36, respectively) and high p values (0.0771 and 0.0520, respectively), the models are not significant for springiness and cohesiveness. the r2 of the predicted models are 0.8443 for hardness, 0.8051 for adhesiveness, 0.8473 for gumminess, 0.8001 for chewiness, and 0.5316 for resilience; however the r2 for springiness and cohesiveness are relatively low (0.3985 and 0.4367, respectively). the adj.r2 values are 0.8084 for hardness, 0.6882 for adhesiveness, 0.8120 for gumminess, 0.7539 for chewiness and 0.4235 for resilience. the low adj.r2 values for springiness and cohesiveness (0.2596 and 0.3067, respectively) indicate quite a low degree of linear fit between the predicted and experimental values for these models. based on these results, the models for hardness, adhesiveness, gumminess, chewiness, and resilience are adequate for predicting within the range of the variables employed, but the models for springiness and cohesiveness are not adequate for this purpose. 3.4. optimization of quantities of ingredients and the validation of the model to optimize textural properties, color, and cooking loss values, numerical optimizations were applied. the aim of this study was to provide the formulation of the chicken roll resulting in the highest values of l*, a*, springiness, resilience and the lowest values of b*, cooking loss, hardness, adhesiveness, gumminess, and chewiness. as optimization criterion, all relevant product characteristics of chicken rolls were selected on the basis of maximum yield and quality variables, as determined by previous studies (andrès et al., 2006; andrés et al., 2006; du et al., 2003; somboonpanyakul et al., 2007).the effects of wheat flour, distilled water, and minced chicken contents on l* value (figs. 1a-c) and cooking loss (figs. 1d-f) are shown in 3-d surface plots. the wheat flour of 8.66 g, the distilled water of 75.00 g, and the minced chicken of 53.83 g are identified as the predicted optimum quantities of ingredients for the chicken roll. based on these optimized quantities, the predicted values are 76.153 for l*, -0.3576 for a*, 11.3484 for b*, 3.02492 for cooking loss, 60.8873 for hardness, -0.01428 for adhesiveness, 46.508 for gumminess, 39.7841 for chewiness and 0.3564 for resilience. the desirability value of these predicted values was 0.703. to verify the model’s adequacy, all experiments were performed with optimum formulations. the experimental values of each response were compared with the predicted values, and the calculated error rates are shown in table 6. the error rates of all responses are lower than 10%. thus, a high level of agreement was observed between the experimental values and the predicted values. ital. j. food sci., vol. 30, 2018 564 a) d) b) e) c) f) figure 1. response surface plots for l* value (a,b,c) and cooking loss (d,e,f). ital. j. food sci., vol. 30, 2018 565 4. conclusions the interest in poultry and poultry products has grown over the last decade. to meet this increasing demand in today’s living conditions, different fast food products need to be developed. a limited number of studies have been conducted on the optimization of new product development. in this study, the most suitable recipe for the chicken roll, a fast and healthy product with desirable textural and physical properties, has been determined. according to the results of this study, adding wheat flour to the chicken roll recipe increases the hardness and gumminess of the product. however, increasing the water content in the formulation decreases the hardness, gumminess and chewiness of the product. it was observed that the l* values of the chicken rolls made with a low wheat flour content and a high minced chicken content were relatively higher than those in the other experiments. as a result of this study, an alternative poultry product that is readyto-eat and rich in desired textural and quality characteristics has been developed; future studies should aim to determine the product’s sensorial and microbiological properties. acknowledgements the keskinoğlu poultry company (akhisar-manisa, turkey) is gratefully acknowledged. references andrès s., zaritzky n. and califano a. 2006. the effect of whey protein concentrates and hydrocolloids on the texture and colour characteristics of chicken sausages. int. j. food sci. technol. 41(8):954-61. andrés s.c., garcía m.e., zaritzky n.e. and califano a. n. 2006. storage stability of low-fat chicken sausages. j. food eng. 72(4):311-19. bae h-j., 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affected by dietary oxidation levels and packaging. j. food sci. 76(4):612-17. yim d-g., ahn d.u. and nam k-c. 2015. effect of packaging and antioxidant combinations on physicochemical properties of irradiated restructured chicken rolls. korean j. food sci. anim. resour. 35(2):248-57. yoo d., kim c. and yoo b. 2005. steady and dynamic shear rheology of rice starch-galactomannan mixtures. starch/staerke. 57(7):310-18 zhu y., xia x., liu a., zou l. and zhou k. 2016. effects of combined organic acid treatments during the cutting process on the natural microflora and quality of chicken drumsticks. food control. 67:1-8. paper received january 20, 2018 accepted march 27, 2018 ijfs#1107_bozza ital. j. food sci., vol. 31, 2019 195 paper presence of destruxin a and beauvericin in cereals e. dreassi*1,, c. zamperini1, a. cito2, v. francardi2 and m. botta1 1department of biotechnology, chemistry and pharmacy, siena university, via a. moro 53100, siena, italy 2consiglio per la ricerca in agricoltura e analisi dell’economia agraria, research center for plant protection and certification, via di lanciola 12/a, 50125 florence, italy *corresponding author: tel.: +39 0577234321; e-mail address: elena.dreassi@unisi.it abstract a lc-ms/ms method for the detection of destruxin a (dtx a) and beauvericin (bea) in cereals was developed, validated and applied to commercial products collected in italian markets in the years 2015-2016. results showed that bea contaminated 59 % of the samples even if only 15 of them (34%) showed quantifiable residues (comprised between 0.11 and 7.51 ng/g). the sample of red rice contaminated with the highest bea level was also contaminated with dtx a (0.28 ng/g). finally, no significant differences were detected between contaminated samples based on the production year and the agronomic technology used (organic or conventional farming). keywords: lc-ms/ms, mycotoxins, organic, conventional ital. j. food sci., vol. 31, 2019 196 1. introduction cereals supply and demand have been steadily increasing in recent years (usda, 2017), however these products are exposed to pre-/post-harvest fungal infections potentially dangerous for humans and animals and responsible for economic losses (who-iarc, 2002; peraica et al., 1999; zain, 2011). fungal toxicity is mainly due to the production of mycotoxins. these secondary metabolites, produced by molds as natural protection, are generally thermostable and resistant to food transformation processes (karlowsky et al., 2016). for these reasons, mycotoxins are considered the main chronic dietary risk factors and therefore a correct evaluation of the real contamination and co-occurrence of these products is required. among all mycotoxins, european union has set a maximum level in food only for aflatoxins, ochratoxin a, patulin, deoxynivalenol, zearalenone, fumonisins, t-2 toxin and ht-2 toxin (commission regulation 1881/2006; commission recommendation 2013/165/eu). recently, a particular attention was paid in ec to enniatins and beauvericin (bea). efsa’s panel on contaminants has reported the occurrence of enniatins and bea in european foods and feeds in 2014 in the food chain (efsa, 2014). even if no concerns for human health have been related to the acute exposure to these mycotoxins, given the lack of relevant in vivo toxicity data, no reliable conclusions can be drawn on chronic exposure to these compounds (contam, 2014). many fungi, such as beauveria bassiana and fusarium spp., produce bea and, in the last period, b. bassiana is widely used as entomopathogenic mycoinsecticide alone or in combination with metarhizium anisopliae (wang and xu, 2012). the metarhizium spp. and other ubiquitous soil fungi, produce a family of cyclic peptide toxins termed destruxins (dtxs). to date, a number of dtxs have been identified and placed in five major subgroups (a-e) with dtx a, b and e as the most predominant one (hsiao and ko, 2001; wang et al., 2009; ibraim and asker, 2012). they are known to possess cytotoxic and cytostatic effects on mammalian and insect cells with dtx a and e being the most toxic (skrobeck and butt, 2015). for some authors, mycotoxins from mycoinsecticides have limited ways to enter in environment and the risks of contaminating foods are negligible (hu and zhang, 2016). differently from bea, no analytical data are currently available on the occurrence of dtxs in food chain and the present work aimed to investigate of the real occurrence of dtx a, along with bea, in cereals purchased from the italian market in 2015-2016 period. based on our previous experience with these two analytes and on the extraction solvents reviewed in literature (hsiao and ko, 2001; wang et al., 2009; cito et al., 2014 and 2016; butt et al., 2009; taibon et al., 2015; blesa et al., 2012; sørensen et al., 2008), a validated lc-ms/ms method was optimized in order to determine simultaneously both analytes in commercial organic or conventional farming cereals samples. 2. materials and methods 2.1. chemicals the standards of dtx a and bea were obtained from sigma-aldrich s.r.l (milano, italia). all reagents were obtained by sigma unless stated otherwise. acetonitrile, dichloromethane and ethyl acetate, used for the mycotoxins extractions, were of analytical grade while acetonitrile used for chromatographic analysis was of hplc grade. milli-q quality water (millipore, milford, ma, usa) was used. ital. j. food sci., vol. 31, 2019 197 2.2. standard solutions standard solutions were prepared by dissolving each compound with methanol in a volumetric flask and then diluted with methanol to make the working solutions. 2.3. sample extraction the cereals samples (maize, barley, oat, rice, red rice, amaranth, millet, wheat and spelt) were purchased in local supermarkets. in the first step, a representative portion of the cereal samples (100 g) was mixed well with a food chopper. an accurately weighed portion of all the samples (10 g) was placed in a centrifuge tube and 25 ml of acetonitrile or dichloromethane: ethyl acetate (1:1, v/v) was added. the extractions were carried out using an ika labortecnhik homogenizer model t25 basic (ika werke gmbh & co., staufen, germany) for 5 min at 13500 rpm. the supernatant was transferred after centrifugation, and another aliquot of extraction solvent was added to the residue and homogenized. the organic fractions were pooled and evaporated to dryness under vacuum by rotary evaporation (temperature of the bath, 20°c), and the residue redissolved in 500 µl of acetonitrile. the sample was filtered with 0.45 µm minisart srp 4 (sartorius: goettingen, germany) and used for the lc/ms-ms analysis. 2.4. quantification and recovery the quantitative analysis of bea and dtx a was based on calibration curves obtained analysing spiked samples (10 g of equimolar mixture of barley, oat, maize, rice, wheat and spelt) at different concentrations ranged between 0.1 and 100 ng/g. for the equations, six points with different concentrations were used. extraction recoveries were determined by spiking untreated powdered equimolar mixture of cereals with standard solutions to obtain tree different final concentrations (0.1, 10 and 100 ng/g for each investigated compound). after the solvent evaporated, the samples were extracted, as reported above. recovery values were calculated as the ratio of the peak area obtained from the extraction of the fortified samples to the corresponding peak area determined by a single-point calibration standard. 2.5. lc–esi-tandem ms analysis chromatography-mass spectrometry system consisted of a varian apparatus (varian inc.) including a vacuum solvent degassing 20 unit, two pumps (212-lc), a triple quadrupole msd (mod. 320-lc) mass spectrometer with esi interface and varian ms workstation system control ver. 6.9 software. the chromatographic separation was performed by using a kinetex 2.6µm c18 100å column (phenomenex) (100 mm×4.6 mm). the sample was injected (5 µl) after filtration. chromatographic analysis was carried out by using acetonitrile and aqueous solution of formic acid (0.05%) (3:97 v/v). the flow rate was 0.1 ml/min. the instrument operated in positive mode and esi parameters were: detector voltage 1250 v, drying gas pressure 18.0 psi, desolvation temperature 300.0°c, nebulizer gas 42.0 psi, needle voltage 6000 v and shield voltage 250 v. nitrogen was used as nebulizer and drying gas. collision induced dissociation was performed using argon as the collision gas at a pressure of 1.8 mtorr in the collision cell. the selected reaction monitoring (srm) transitions as well as the capillary voltage and the collision energy are summarized in table 1. quantitative analysis was performed in srm to maximize sensitivity. for each investigated compound the [m+h]+ species were selected as precursor ions. two srm ital. j. food sci., vol. 31, 2019 198 transitions (table 1), the first one for quantification and the second one for confirmation purpose, were acquired by using the experimental conditions described above. table 1. chromatographic and selected reaction monitoring (srm) parameters used in the analysis (retention time (tr), quantification and confirmation transitions, collision energy and capillary voltage). compound tr (min) quantification transition (m/z) collision energy (ev) confirmatory transition (m/z) collision energy (ev) capillary voltage (v) dtx a 3.52±0.08 578.1→465.1 -28.5 578.1→436.8 -22.5 86.29 bea 4.32±0.06 784.2→244.0 -25.0 784.2→262.0 -24.5 140.00 2.6. validation procedure and evaluation of the matrix effect the specificity of the method was assessed by analysing blank samples (one sample for each analysed cereals) and blank samples spiked with the investigated compounds, according to the procedure reported above. assay selectivity was defined by evidence of non-interference at retention times and ion channels identical to those of bea and dtx a in the blank samples. in order to determine the linearity of the method, calibration curves (obtained from five replicate experiments) were constructed by analysing spiked cereal samples (10 g of equimolar mixture of barley, oat, maize, rice, wheat and spelt fortified before extraction) ranged between 0.1 and 100 ng/g. the linearity was evaluated by linear least-squares regression analysis. the detection limit (lod) was defined as the concentration for which a signal-to-noise ratio equal to 3 was obtained. the quantification limit (loq) was defined as the lowest concentration for which an accuracy between 80% and 120% and a precision with a coefficient of variation of±20% or less was obtained over six measurements, with a signalto-noise ratio superior or equal to 10. assay precision was determined by repeatability (intra-day) and intermediate precision (inter-day). intra-day precision was evaluated by assaying added blank cereal samples, six replicates set at the same concentration (0.1, 10 and 100 ng/g), during the same day. the between-day precision was studied by assaying added blank cereals samples, six replicates set at the same concentration (0.1, 10 and 100 ng/g), on different days (5 days). the accuracy of the method was also evaluated at the same concentration levels and expressed as relative error % (re). the recovery data were determined by spiking blank cereal samples with standard solutions (three concentrations analysed in triplicate). after spiking, the samples were extracted as previously described. recovery values were calculated by comparing the analytical results of the samples through overall extraction procedure with those obtained from blank samples fortified after extraction. in order to study the matrix effect (me), blank samples were processed and spiked later to obtain three final concentration levels (set b: six samples with final concentrations of 0.1, 10 and 100 ng/g). the response (peak area) was compared with directly injected standard solutions (set a: six samples prepared in methanol at the same concentration levels). the matrix effect (me) was evaluated by comparing the mean peak area of the spiked samples (post-extraction addition) with corresponding standard solutions at equivalent concentrations prepared in methanol. the me values were then calculated as follows: me (%) = a/b×100 (matuszewski et al., 2003). ital. j. food sci., vol. 31, 2019 199 3. results and discussion 3.1. lc–esi-tandem-ms optimization the selected reaction monitoring (srm) was performed to enhance sensitivity and specificity of the analysis. the ms/ms dissociation study was optimized, for each single standard compound, by varying the cone voltage and collision energy, using the flow injection analysis (fia) of working standard solutions at a flow rate of 0.01 ml/min directly through the electrospray probe. [m+h]+ ions were found to be the most abundant ones and selected as precursor ions for the target compounds. the ms–ms breakdown for dtx a showed a fragmentation pattern similar to that reported by other authors (wang et al., 2009; butt et al., 2009). as expected the dtx a ion [m+h]+ at 578.1 m/z represented the most abundant ion without any adduct. the collision-induced dissociation (cid) experiments showed common losses of amino acids following ring opening. as previously described by other authors, bea tends to be readily ionized via esi to form [m+h]+ ion. as determined in this work, [m+h]+ ion was found to be the most abundant one and selected for bea analysis. the ms/ms tuning experiments displayed product ions scan spectra of bea in accordance with those reported in literature (sørensen et al., 2008; song et al., 2009). most intense fragments were selected for dtx a and bea quantification and confirmatory purposes (table 1). 3.2. method optimization and validation procedure the method was validated for accuracy, precision, specificity, linearity and sensitivity. in order to control for variability in recovery from biological samples and factors that can affect the instrumental response, various cereals samples were assayed. cereals used for calibration and for recovery studies were analysed to verify the absence of each investigated compound before performing the analysis. the analysis of blank samples showed the absence of interfering endogenous compound peaks at the same ion channel or retention time of dtx a or bea. two extraction methods were tested in order to identify a unique system to quantify contemporarily both mycotoxins. satisfactory mean recoveries for bea (89 and 72 % respectively) and only with binary mixture for dtx a (56 %) were obtained by using both selected extraction solvents (acetonitrile and dichloromethane: ethyl acetate 1:1 v/v) (table 2). table 2. mean extraction recoveries obtained with the two extraction procedures (experiments conducted on equimolar mixture of barley, maize, oat, rice, spelt and wheat) (n=3). spiked concentration (ng/g) extraction with acetonitrile ch2cl2:ethyl acetate (1:1 v/v) recovery (mean±sd) rsd% a recovery (mean±sd) rsd% a dtx a 0.1 10 100 38.6±7.8 43.1±6.4 39.2±5.8 20.3 14.8 14.9 66.7±6.66 68.2±5.52 79.9±3.46 10.4 8.10 4.3 bea 0.1 10 100 88.4±5.0 88.7±3.1 89.4±2.9 5.9 3.4 3.2 55.8±6.7 54.9±6.2 57.5±4.7 12.0 11.2 8.1 arelative standard deviation. ital. j. food sci., vol. 31, 2019 200 the matrix-induced effects, such as signal enhancement or suppression, were also evaluated according to matuszewski et al. (2003), and the results obtained in presence of an extract of equimolar mixture of barley, maize, oat, rice, spelt and wheat are summarized in table 3. an enhancement of the absolute response was observed for both analytes with both the extraction systems. very close results were obtained for all matrices when tested separately (results not shown), therefore, calibration curves were generated from blank constituted of an equimolar-mixed cereals sample (barley, oat, maize, rice, wheat and spelt) spiked before extraction to avoid and minimize any uncertainty related to the matrix-induced effects. table 3. matrix effects obtained with the two extraction procedures (experiments conducted on equimolar mixture of barley, maize, oat, rice, spelt and wheat) (n=3). spiked concentration (ng/g) matrix effect (mean±sd) acetonitrile ch2cl2:ethyl acetate (1:1 v/v) dtx a 0.1 10 100 146.1±11.3 137.8±9.9 136.9±7.9 120.6±10.6 130.6±9.6 124.8±9.0 bea 0.1 10 100 147.9±8.5 148.7±4.7 143.1±8.5 156.6±10.7 137.2±8.7 140.8±8.5 based on the results obtained in these preliminary stages, the binary extraction mixture was selected for the continuation of the work, ensuring satisfactory recovery values for both analytes. calibration curves (five replicate experiments) were constructed and the method was found to be linear within the range 0.1–100 ng/g with correlation coefficient above 0.9994. the equations of the curves, obtained by a least squares fit, are reported in table 4. table 4. regression plot parameters for dtx a and bea quantification in mixed cereals (experiments conducted on equimolar mixture of barley, maize, oat, rice, spelt and wheat). range (ng/g) regression plots parameters r2 loq a (ng/g) lodb (ng/g) dtx a 0.1-100 y = 24423x+6127 0.9994 0.1 0.03 bea 0.1-100 y = 46020x+7695 0.9997 0.1 0.03 aquantification limit. bdetection limit. the selected method was validated in term of precision and accuracy (results are reported in table 5). intra-day and inter-day precision, expressed as the relative standard deviation (rsd %) values, were always less than 15 % (n = 6) for both the analytes. the relative errors (re %) ranged from −12.00 % for bea to +14.00 % for dtx a obtained at loq levels. the reported results indicated that the developed method is precise, accurate, reproducible and utilizable for determination of the two compounds in cereal-based foods. compared to the loq present in literature our values are comparable for both analytes (taibon et al., 2015; blesa et al., 2012; sørensen et al., 2008; tolosa et al., 2017; malachová et al., 2014). ital. j. food sci., vol. 31, 2019 201 table 5. the intra-day and inter-day precision and accuracy of the method (n=6). compound analysis type spiked concentration (ng/g) measured concentration (media±sd) rsd%a accuracy (relative error %) b dtx a intra-day 0.1 10 100 0.11±0.01 10.52±1.12 101.03±3.40 9.09 10.65 3.40 +8.20 +5.20 +1.00 inter-day 0.1 10 100 0.12±0.01 10.54±0.84 103.06±2.35 11.67 7.97 2.28 +14.00 +5.40 +3.06 bea intra-day 0.1 10 100 0.09±0.01 9.90±1.24 101.20±4.23 14.44 12.53 4.18 -12.00 -1.00 +1.20 inter-day 0.1 10 100 0.09±0.01 9.95±0.90 109.20±3.68 13.33 9.35 3.37 -9.40 -0.50 +9.20 arelative standard deviation. baccuracy = relative error % = (measured-spiked)/spiked x 100. 3.3. analysis of dtx a and bea content in cereals the validated method (extraction accomplished with dichloromethane: ethyl acetate, 1:1 v/v) was successfully applied to quantify dtx a and bea levels in 44 commercial products collected in the years 2015-2016 (table 6). results showed that bea contaminated 59 % of the sample even if in only 15 samples (34%) quantities were higher than loq (included in the range 0.11 and 7.51 ng/g). contamination data are in accord with bea occurrence reported by various authors and collected in the efsa contam panel report (2014). the high number of positive samples is due to the good lod values obtained with our method (0.03 ng/g for both compounds). either no significant differences were detected between percentages of contaminated samples based on the year of production or the agronomic technology used (organic or conventional farming). only one sample (red rice presenting the highest bea levels) resulted contaminated by dtx a (0.28 ng/g). although the number of analyzed samples is limited, the low levels of bea and the substantial absence of dtx a confirm that acute exposure to these toxins could do not indicate concern for human health. however, careful monitoring in foods is essential in order to provide a correct estimate of chronic exposure to these toxins. ital. j. food sci., vol. 31, 2019 202 table 6. occurrence and content of dtx a and bea in commercial products (44 cereals samples) collected in the years 2015-2016 (each sample was analysed in triplicate). year bea content (ng/g±sd) amaranth barley oat oat flakes maize millet red rice rice spelt wheat 2015 ---a ---a 0.18±0.01 0.17±0.02a 0.19±0.02a 0.58±0.05a 7.51±1.16b 1.28±0.16 0.53±0.07a 3.92±0.13 0.27±0.03a ---a ---2.48±0.48 --- 0.98±0.11a ------ >lod --- >lod --- 2016 ---a >lod >lod ---a 0.21±0.03 >lod a --->lod a >lod a >lod --->lod 1.54±0.18 >lod ---a ------ --- 0.11±0.01 5.12±0.59 >lod --- aorganic product. bred rice sample containing also dtx a (0.28±0.04 ng/g). ital. j. food sci., vol. 31, 2019 203 acknowledgments we thank iuni margaret laura trist for helping with the manuscript writing. references blesa j., marín r., lino c.m. and mañes j. 2012. evaluation of enniatins a, a1, b, b1 and beauvericin in portuguese cereal-based foods. food additives and contaminants a 29:1727-1735. butt t.m., ben el hadj n., skrobek a., ravensberg w.j., wang c., lange c.m., vey a., shah u.k. and dudley e. 2009. mass spectrometry as a tool for the selective profiling of destruxins, their first identification in lecanicillium longisporum. rapid communication in mass spectrometry 23:1426-1434. cito a., mazza g., strangi a., benvenuti c., barzanti g.p., dreassi e., turchetti t., francardi v. 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hutwimmer s., strasser h. and burgstaller w. 2009. destruxin production of metarhizium anisopliae under carbon and nitrogen exhaustion. journal of basic microbiology 49:404-411. who-iarc, evaluation of carcinogenic risks to humans, iarc monographs vol. 82, iarc press, lyon, france, 2002. zain m.e. 2011. impact of mycotoxins on human and animals. journal of saudi chemical society 15:129-144. paper received december 17, 2017 accepted august 30, 2018 ijfs#1163_bozza ital. j. food sci., vol. 30, 2018 673 paper pasta-making properties of the new durum wheat variety biensur suitable for the northern mediterranean environment g. visioli*1, t. vamerali2, c. dal cortivo2, s. trevisan2, b. simonato3 and g. pasini2 1department of chemistry, life sciences and environmental sustainability, university of parma, parco area delle scienze 11/a, 43124 parma, italy 2department of agronomy, food, natural resources, animals and the environment, university of padua, viale dell'università 16, 35020 legnaro-padua, italy 3department of biotechnology, university of verona, strada le grazie 15, 37134 verona, italy *e-mail address: giovanna.visioli@unipr.it abstract industrial pasta is commonly made from mixtures of semolina from different durum wheat varieties, and there is a very low market presence of mono-varietal pasta from local, short supply chains. in this work, dough rheological properties and pasta quality traits of the new durum wheat cv. biensur, which has a high hmw/lmw-gs ratio, were evaluated with a view to developing short-chain, mono-varietal pasta production in ne italy. chemical and sensory analyses on short-cut pasta, viz. tubetti, made with semolina from cv. biensur at two drying temperatures revealed that it has good technological characteristics and stability, excellent cooking and sensory properties, and is comparable to the high-quality commercial reference cv. aureo. we conclude that biensur provides farmers and traders with new market opportunities and offers improvements to the environmental and economic sustainability of the durum wheat chain. keywords: mono-varietal pasta, short-chain production, pasta texture, hmw and lmw gs, gliadins, technological characteristics ital. j. food sci., vol. 30, 2018 674 1. introduction durum wheat (triticum durum desf.) is one of the most important crops worldwide with an average annual production of 32.6 million tons (mmt), and is the most widely grown crop in the mediterranean area with an annual production of about 18 mmt (international grain council, 2015). italy is a major producer of durum wheat in the mediterranean, with an average production of about 4.0 mmt/year, about 67% of which is grown in the south of italy and used mainly for pasta production (d’egidio, 2007). pasta is made from the kneaded dough of semolina flour and water, which is shaped by a press and stabilised by drying. the pasta-making industry generally uses mixtures of different durum wheat varieties as blending yields semolina with high technological properties. as a consequence, there is very little mono-varietal pasta currently on the market, therefore it would be worthwhile studying the effects of individual cultivars on pasta quality (padalino et al., 2014). protein content and gluten characteristics are the main factors influencing pasta quality (sissons et al., 2005; cubadda et al., 2007), but their relative importance depends on many factors, including genotype, and environmental and processing conditions, such as drying temperature (d’egidio et al., 1990; novaro et al., 1993). the performance of durum wheat in pasta making is related, in particular, to the storage proteins of grains, i.e., glutenins and gliadins, which influence dough strength, extensibility and stability (sissons, 2008). glutenins are large aggregates of sub-units of either low molecular weight (lmw; 31–51 kda) or high molecular weight (hmw; 80-140 kda) joined by disulphide bonds (varzakas et al., 2014). gliadins are alcohol-soluble proteins, which fall into four groups, ω, γ and α/β, based on their electrophoretic mobility and molecular weight (45-25 kda). glutenins are mainly polymeric proteins responsible for dough elasticity, whereas gliadins are monomeric and determine characteristics related to extensibility. aside from the amounts of proteins and types of gluten proteins, the glutenin to gliadin and the hmw-gs to lmw-gs ratios are also directly related to the balance between dough strength and extensibility (samaan et al., 2006; sissons, 2008). drying temperature, one of the most important factors in the pasta production process, gives rise to highly aggregated proteins that are cross-linked via covalent bonds and disulphide bonds. a higher drying temperature intensifies polymerisation of the proteins into a protein network, which entraps the starch granules thereby preventing starch leaching during cooking and increasing the pasta’s sensory properties and cooking quality (zweifel et al., 2003; padalino et al., 2016). in recent years, the environmental impact of the entire pasta production cycle, from the cropping system in the field to semolina production techniques and packaging, has been reviewed (bevilacqua et al., 2007). moreover, under a recent italian decree (legislative decree, 2017), it is now obligatory to declare the origin of the durum wheat grains used in pasta production. in light of this, an advantageous strategy would be to promote local, short food supply chains in order to improve environmental and economic sustainability. in this regard, a recent study assessed the new cv. biensur and found it offered a suitable combination of high grain yield [7.31 t/ha, 132% higher than the italian mean (3.15 t/ha) and 37% higher than the veneto regional mean (5.32 t/ha) (source: istat, averages of the 12-year period 2006-2017)] and high quality semolina when grown under sustainable agronomic management on the edge of the cultivation area of this species in the mediterranean (visioli et al., 2018). the aims of this research, therefore, were: a) to evaluate the dough rheological properties and the quality of the pasta obtained from triticum durum cv. biensur cultivated in ital. j. food sci., vol. 30, 2018 675 northern italy with a view to its possible use in the production of mono-varietal pasta; b) to evaluate the effects on pasta quality of two different drying temperatures during industrial processing. comparative analyses were made against a high-quality standard represented by cv. aureo. 2. materials and methods 2.1. field experiment and grain production a field experiment was carried out in a sandy loam soil at the miana serraglia farm (mira, venice, italy), located close to the venetian lagoon, during the 2012-2013 growing season. the durum wheat cv. biensur (apsovsementi, voghera, italy) was grown in a 13.6 ha field. in accordance with local recommendations, 215 kg/ha of nitrogen fertilizer was applied: 200 kg n/ha to the soil as ammonium nitrate and 15 kg n/ha (uan, urea-ammoniumnitrate) by foliar spraying at the flowering stage. the wheat was sown late in october and harvested early in july. grain samples of 100 kg were collected to carry out dough tests and manufacture the pasta. 2.2. gluten protein quantification and chemical composition grains of the cv. biensur (triticum durum desf.) were ground in an experimental laboratory mill (buhler mlu202 roller mill; braunschweig, germany) at the scientific technology park of the molise region (campobasso, italy) in order to obtain fine semolina with a particle size similar to that of the reference control (200 to 350 µm). the reference control was a high-quality commercial semolina (cv. aureo) used in industrial, mono-varietal pasta production. protein, starch, fat, total fibre and ash contents were quantified in the semolina samples. total protein content was quantified by the kjeldahl 2001.11 method (aoac, 2000). in addition, relative quantification of the gliadin and the high-molecular-weight (hmw) and low-molecular-weight (lmw) glutenin (gs) fractions in the semolina of both biensur and aureo was carried out using the protein sequential extraction procedure (visioli et al., 2016) followed by quantification by bradford assay (bradford, 1976). three technical replicates were performed for each variety. sds-page was performed on a miniprotean tetra cell (bio-rad) on 8%, 12% and 15% acrylamide gel for the hmw-gs, lmw-gs and gliadin fractions, respectively, as previously described (visioli et al., 2016). following gel staining and image acquisition, protein molecular weights (mw) were identified and relative quantification of the gliadin, lmw-gs and hmw-gs in each gel was carried out using the image lab 4.5.1 (bio-rad) software. the total starch content in the semolina samples was determined according to the 996.11 method (aoac, 2000), while the fat content was estimated according to the 2003.05 method (aoac, 2000). total fibre content was measured according to the official 991.43 method (aoac, 2000), and ash content according to the 942.05 method (aoac, 2000). chemical analyses were performed in triplicate and the results expressed on a dry matter basis. short-cut pasta (tubetti) made from both the biensur and reference (aureo) semolina samples (fig. 1) was processed using a pilot system at the pavan-map impianti factory (galliera veneta, padua, italy). briefly, pasta samples were prepared in accordance with italian legislation (presidential decree n°187, 2001) by mixing water and semolina to form a dough with a 30% moisture content. the dough was driven through a vacuum system then extruded to mould the pasta. samples of biensur and aureo pasta were dried ital. j. food sci., vol. 30, 2018 676 at two different temperatures, low (maximum temperature 60°c, for 9 h) or high (maximum temperature 85°c, for 5 h), to obtain a final moisture content of 11% dw. the pasta samples from the two wheat varieties dried at the low and high temperatures are henceforth referred to as biensur lt, biensur ht, aureo lt and aureo ht. figure 1. appearance of the “tubetti” pasta made from mono-varietal semolina of cv. biensur compared with the reference cv. aureo at two drying temperatures, 60°c for 9 h (lt) or 85°c for 5 h (ht). 2.3. farinographic evaluation the properties of the semolina samples were measured with a farinograph (t6, promylograph, max egger, austria) according to the approved 54-21 method (aacc, 2000). farinograph tests allowed us to determine: (i) the water absorption (g water per 100 g of semolina) required to reach a dough consistency of 500 pu (promylograph units); (ii) dough stability, defined as the length of time the dough maintains its maximum consistency; (iii) dough weakening, defined as the reduction in dough consistency (as pu) after 20 minutes of mixing. the analyses were performed in triplicate. 2.4. cooking properties 2.4.1 determination of optimal cooking time pasta samples (50 g) were cooked in deionised boiling water (500 ml). optimal cooking time (oct), defined as “al dente”, was determined by pressing the pasta between two glass slides at different times during cooking and observing the time it took the starchy white core of the pasta to disappear (abécassis et al., 1994). ital. j. food sci., vol. 30, 2018 677 2.4.2 cooking loss and water absorption the pasta samples were drained immediately at the oct to halt the cooking process. cooking loss was defined as the amount of solids lost in the cooking water (d’egidio et al., 1990). in brief, the cooking water was collected in a beaker, placed in an air oven at 110°c and evaporated until dry. cooking loss was the weight of the residue expressed as a percentage of the initial weight of the pasta (g solids per 100 g of dry pasta). water absorption was measured as the increase in the weight of the pasta after cooking and expressed as a percentage of the weight of the uncooked pasta. cooking losses and water absorption were determined with 3 individual measurements (replicates). 2.4.3 texture analyses and colour determination pasta firmness, viz. the resistance to a bite with the incisors through the cooked pasta, and stickiness, i.e., the material adhering to the surface of the cooked pasta, were determined using a ta.xt plus texture analyser (stable micro systems, uk) equipped with a 5 kg load cell. the firmness test was performed according to the aacc 16-50 method. a single tubetto (12 mm thick) was oriented perpendicularly to a knife probe, cut, then compressed at a speed of 0.5 mm/s. firmness was measured as the maximum peak force curve (n) required to compress the pasta sample. for the stickiness test, the pasta was oriented perpendicularly, as described above, to a rectangular probe. excess water was removed before testing. the probe applied a compression force to the pasta sample (1 kg), was held in contact for 2 seconds then withdrawn at the same speed as above (0.5 mm/s). stickiness was measured as the maximum peak force curve (g/s) required to withdraw the probe from the surface of the sample. the average value of five replicates was reported for each test. the colour of cooked pasta was determined using a reflectance colorimeter (minolta®, cr300, japan) following the cie-l*a*b* colour system, where the l* value (brightness) ranges from black (0) to white (100), the chroma a* value ranges from green (-60) to red (+60), and the chroma b* value ranges from blue (-60) to yellow (+60) (minolta, 1993). each colour data represents the mean of three measurements on different pasta samples. 2.4.4 sensory evaluation of pasta to assess acceptability of the pasta made from cv. biensur, a sensory evaluation was carried out by 15 panel members (9 women, 6 men; ages ranging from 22 to 40 years) with experience in general food evaluation. the four pasta samples (biensur ht and lt, and aureo ht and lt) were cooked “al dente” without the addition of salt, drained and kept warm until serving in randomised order on plastic plates labelled with random 2-digit codes. panellists were asked to evaluate colour, flavour and texture properties (firmness, stickiness) on a five-point scale from 1, low intensity, to 5, high intensity. they were also asked to score the overall quality of the product based on these same attributes using the same five-point scale. the attribute scores for each sample and panel member were subjected to a one-way analysis of variance (anova) to obtain mean sensory scores for each of the 15 panel members. 2.5. statistical analysis statistical analysis of the data was performed with the statgraphics centurion xiv software (statpoint technologies, inc., warrenton, va, usa) and the results compared by ital. j. food sci., vol. 30, 2018 678 one-way anova. significant differences between treatments were determined by tukey’s test. 3. results and discussion 3.1. chemical composition of the semolina and rheological properties of the dough table 1 shows a comparison of the compositions of the refined semolina from cv. biensur and the commercial high-quality semolina from cv. aureo, which is already used in italy to produce the mono-varietal pasta voiello® (naples, italy). although biensur had a lower protein content (138.8 vs. 146.8 mg/g dw), the levels were high in both compared with the minimum levels (105 mg/g dw) required by italian legislation (presidential decree n° 187, 2001) and were commercially acceptable. in pasta making, the quantity and quality of wheat storage proteins is important in determining essential dough properties, such as stability and firmness (samaan et al., 2006; sissons, 2008). table 1. chemical and gluten protein composition of semolina samples (mg/g of dw) of cv. biensur compared with the commercial reference cv. aureo. total protein1 gli 2 hmw-gs2 lmw-gs2 gs/gli hmw/lmw-gs moisture total fibre starch ash lipids biensur 138.8b 64 a 12 a 24 b 0.56 b 0.51 a 14.11a 3.17a 74.9a 0.79a 1.73a aureo 146.8a 58 b 12 a 30 a 0.72 a 0.42 b 13.65a 3.10a 73.2b 0.80a 1.80a within each parameter, different letters indicate significant differences (tukey test, p ≤ 0.05; n = 5). 1kjeldahl method. 2percentage of total gluten proteins, which were: biensur 22.3±0.33, aureo 24.2±0.06 mg/g semolina. biensur has been recently recognised as a high-yielding variety, with higher gs/gliadin and hmw/lmw-gs ratios than other italian cultivars, and an optimal allelic gs configuration (bx7 and by8) (fig. 2), suggesting that high productivity can be combined with good quality through suitable breeding programmes (visioli et al., 2018). the hmw-gs configuration is indicated as bx7 and by8 for cv. biensur and as bx6 and by8 for cv. aureo. the lmw-gs pattern in the two varieties is indicated as the lmw-2 protein group, which, in the modern cultivars, replaced the low quality lmw-1 protein configuration (d’ovidio and masci, 2004). the gliadin fractions ω, γ, α/β were indicated according to the molecular weight range in relation to molecular weight markers. besides protein content, the types of gluten proteins, and the ratios between glutenins and gliadins, and between hmw-gs and lmw-gs are known to be directly related to the balance between dough strength and extensibility (samaan et al., 2006; sissons, 2008). biensur semolina had a lower total gluten protein content than aureo (22.3±0.33 vs. 24.2±0.06 mg/g flour; p ≤ 0.05), a lower percentage of the lmw-gs fraction and a higher gliadin fraction with respect to total gluten proteins (table 1). however, our results confirm biensur as having a higher hmw/lmw-gs ratio than aureo (0.51 vs. 0.42) and an acceptable gs/gli ratio (0.56), which are important parameters for gluten technological quality (table 1). we also found differences between the varieties in the abundances of the hmw-gs x-type and y-type sub-units and the most common lmw-gs (42 and 37 kda), as ital. j. food sci., vol. 30, 2018 679 previously reported (visioli et al., 2018). biensur had higher proportions of x-type hmw sub-units and 37 kda lmw-gs than aureo (fig. 2). regarding the gliadin fractions, biensur had a greater amount of α/β gliadins (rich in cys residues) and a lower fraction of ωgliadins (poor in cys residues) than aureo, although they had similar amounts of γgliadins, which are very rich in cys residues. gluten composition and the relative amounts of sub-units are known to contribute to the technological quality of semolina. biensur also had more starch than aureo (749 vs. 732 mg/g dw). figure 2. sds-page of hmw-gs, lmw-gs and gliadin sub-units extracted from cv. aureo and biensur semolina (a), and their relative abundances (%) obtained by densitometric analysis (b). we compared samples of biensur and aureo for dough stability and weakening using a farinograph (table 2) and found the two varieties to have very similar levels of dough stability, while cv. aureo had better indices of dough weakening and water absorption. although there were no significant differences between the two varieties in dough stability, after 20 minutes of mixing the biensur dough was found to have a higher dough weakening index, meaning a lower tolerance to mechanical mixing. ital. j. food sci., vol. 30, 2018 680 table 2. farinographic indices (means; n = 3) of dough samples of cv. biensur compared with the commercial reference cv. aureo. water absorption (%) dough stability (min) dough weakening (pu) biensur 52.4b 11.5a 35a aureo 55.7a 12.0a 25b within each parameter, different letters indicate significant differences (tukey test, p ≤ 0.05). 3.2. physical and sensory characteristics of pasta we looked at the most important quality indicators to properly compare the pasta obtained from the two varieties. good quality pasta should meet the criteria of high water absorption, low cooking losses and good texture (cubadda et al., 2007; bruneel et al., 2010). after cooking, it should be firm enough to resist surface disintegration and have no excessive stickiness. the data regarding water absorption, cooking loss, firmness and stickiness of all pasta samples at the optimal cooking time are similar for the two durum wheat varieties (table 3). table 3. cooking properties [optimal cooking time (oct), water absorption and cooking loss (n = 3)], firmness and stickiness measured by texture analyzer (n = 3), and colour indices of pasta samples of cv. biensur compared with the commercial reference cv. aureo dried at different temperatures (lt = 60°c for 9 h; ht = 85°c for 5 h). cooking quality colour oct (min.sec) water absorption cooking loss firmness stickiness l* a* b* (%) (%) (n) (g/s) biensur ht 9.0 111.90a 2.96a 5.70a 81.6a 57.10a -1.15a 17.6a biensur lt 8.3 111.58a 3.00a 5.65a 82.3a 58.67a -2.4b 18.0a aureo ht 9.0 111.90a 2.96a 5.80a 80.1a 60.57a -1.9ab 18.4a aureo lt 8.3 111.70 a 2.97a 5.71a 81.1a 59.40a -2.41b 17.7a within the same parameter, values with the same letter are not significantly different from each other (tukey test, p ≤ 0.05). analyses of variance for these parameters did not reveal any significant differences between biensur and aureo pasta dried at the same temperature. this provides confirmation that biensur, despite having a lower protein content than aureo, has good gluten quality and is therefore suitable for high quality pasta production. there were also no significant differences between biensur and aureo pasta dried at different temperatures: in all cases the gluten network seems to provide similar shear resistance and equally restricts starch swelling and leaching. this was probably because the gluten quality of cv. biensur has a better hmw-gs configuration (bx7-by8) and a higher hmw/lmw-gs ratio than aureo, as well as an acceptable gs/gli ratio, as previously described (table 1), which plays an important role in the formation of a strong protein network. moreover, we consider that the difference between the lt and ht drying temperatures is not so great as to affect the pasta structure. indeed, only large increases in drying temperature would modify the pasta structure, with positive effects on the sensory ital. j. food sci., vol. 30, 2018 681 properties and cooking quality (pasini et al., 2015; padalino et al., 2016), especially when total protein content is low (cubadda et al., 2007). high drying temperatures, particularly >70 °c, are also known to lower protein digestibility (petitot et al., 2009; stuknyte et al., 2014). in this trial, we detected slight improvements to pasta firmness and lower cooking losses in both varieties dried at the high temperature compared with the lower (85 °c vs. 60 °c), but they were not statistically significant. however, biensur was slightly stickier than aureo, probably due to its higher starch content, and the higher drying temperature seems to be effective in slightly reducing this effect. the sensory properties of the cooked pasta, such as colour, flavour and texture (firmness and stickiness), play an essential role in determining consumer acceptability of the product, especially in traditional pasta-consuming countries (d’egidio and nardi, 1998). sensory evaluation of pasta made from the cv. biensur and from the reference cv. aureo showed there to be no significant differences between them for any of the parameters tested (table 4), which is consistent with the texture analysis (table 3) and hence shows good overall acceptability. texture and flavour appear to play a major role in sensory evaluation, but the initial impact is also highly influenced by colour. similar brightness (l*) and b* values were observed for all cooked pasta samples. differences between the ht and lt pasta samples were found, as indicated by a significant increase in the a* value (redness) under the higher drying temperature (table 3), which is known to be correlated with non-enzymatic browning (anese et al., 1999). although it is difficult to compare results from different studies because of the different drying cycles and raw materials used, our results are in accordance with those of other authors who investigated the effects of drying temperatures and the role of gluten content in pasta quality (cubadda et al., 2007; padalino et al., 2016). table 4. summary of the sensory properties (n = 15) of pasta samples of cv. biensur compared with the commercial reference cv. aureo dried at different temperatures (lt = 60°c for 9 h; ht = 85°c for 5 h). colour flavour firmness stickiness overall acceptability biensur ht 2.7a 3.5a 4.4a 1.7a 3.0a biensur lt 2.5 a 3.5 a 4.1a 1.9 a 2.8 a aureo ht 3.0a 3.8a 4.8a 1.5a 3.5a aureo lt 2.6a 3.7a 4.5a 1.4a 3.4a each attribute was assessed on a 5-point scale from 1, low intensity, to 5, high intensity 4. conclusions to cultivate durum wheat in the northern latitudes of the mediterranean region, greater attention needs to be paid to varietal choice and crop management, particularly nitrogen nutrition and pathogen control, as the climatic conditions are extreme for this species (lower temperatures, higher humidity). currently, high quality semolina is mainly associated with high-protein cultivars, such as aureo, the reference in our trial, although this variety commonly fails to reach high grain yield targets and may not be economically sustainable for farmers in the potentially high-yield, fertile soils of ne italy. one of many wheat cultivars, biensur grown at the extreme northern edge of the mediterranean region has been recently found to have high yield, appreciable protein ital. j. food sci., vol. 30, 2018 682 contents and a good gluten sub-unit configuration for pasta making (visioli et al., 2018). we therefore felt there was a need to assess whether the characteristics of this cultivar meet the requirements for producing high-quality pasta from large field cultivations, and whether the drying temperature may mitigate possible weaknesses during processing. this study suggests that cv. biensur is a good candidate to increase italy’s production of mono-varietal pasta by extending cultivation of it to the more fertile soils of the po plain, given that the dough has high technological characteristics and the pasta very good sensory properties, comparable to well-established commercial mono-varietal semolinas. the effect of drying temperature (high or low) was minimal, suggesting that the intrinsic characteristics of individual varieties are of central importance, as reported by padalino et al. (2014), and that the most energy/economically sustainable technological processes can be selected without compromising pasta quality. there is currently rising market demand for mono-varietal brands, a situation that could stimulate local cultivation of specific durum wheat cultivars to supply short-chain pasta production, thereby offering new market opportunities for farmers and traders, especially in light of the recent italian decree (legislative decree, 2017) requiring the origin of the wheat to be indicated on the label. as all the steps in this project (cultivation, milling, pasta-making) were carried out on a large scale, we are confident that the results will be useful for future development of the chain in ne italy. furthermore, having demonstrated that essential sensory (mechanical) properties, such as firmness and stickiness, can be faithfully measured by a texture analyser and panellists’ judgements, we are sure that consumers will find the quality of pasta made from cv. biensur acceptable. acknowledgements the work was carried out with financial support awarded to prof. giuliano mosca, university of padua, and prof. nelson marmiroli, university of parma, by the ager project grant 2010-0278 “environmental and economic sustainability for yield and quality production of durum wheat supply chain”. the authors thank pavan s.p.a. (galliera veneta, padua, italy) for manufacturing the pasta, and dr. claudio pollini for technical assistance. the authors wish to thank tessa say for revising the english text. references abécassis j., abbou r., chaurand m., morel m.h. and vernoux p. 1994. influence of extrusion conditions on extrusion speed, temperature, and pressure in the extruder and on pasta quality. cereal chem. 71:247. aacc. 2000. “approved methods”10th ed. american association of cereal chemists, s. paul, mn, usa. anese m., nicoli m.c., massini r. and lerici, c.r. 1999. effects of drying processing on the maillard reaction in pasta. food res. int. 32:193. aoac. 2000. “official methods of analysis” 17th ed. association of official analytical chemists, washington, dc, usa. bevilacqua m., braglia m., carmignani g. and zammori f.a. 2007. life cycle assessment of pasta production in italy. j. food qual. 30:932. bradford m.m. 1976. a rapid and sensitive method for the quantitation of microgram quantities of protein 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1universidade estadual de maringá, center of agricultural sciences, department of animal science, av. colombo, 5790, 87020-900, maringá, paraná, brasil 2universidade de são paulo, faculty of animal science and food engineering usp, avenida duque de caxias norte, 225, 13635-900, pirassununga, são paulo, brasil 3universidade estadual paulista, faculty of agricultural and veterinary sciences of jaboticabal, via de acesso prof. paulo donato castellane s/n, 14884-900, jaboticabal, são paulo, brasil 4department of agriculture, food, environment and forestry, università degli studi di firenze unifi, firenze, italy 5universidade federal da grande dourados, agricultural sciences faculty, rodovia dourados itahum km 12, unidade 2, 79.804-970, dourados, mato grosso do sul, brasil *corresponding author: mlrsouza@uem.br abstract aiming evaluate the effects of smoking techniques on the quality of tilapia fillets, a 2x2 factorial scheme experiment was conducted comprising two smoking techniques (hot and cold) and two pigmentation (with and without). cold smoked fillets and fillets with pigmentation demonstrated a greater yield. hot smoked fillets were tenderer and presented lower moisture and greater ash and protein contents. the pigmentation did not influence the smoked fillet composition, but ash content was greater in fillets without pigmentation. the sensory acceptance of hot smoked fillets was better. the pigmentation influenced the color and appearance; however, fillets without pigmentation gave better flavor. keywords: benzo(a)pyrene, chemical composition, oreochromis niloticus, organoleptic aspects, smoked yield ital. j. food sci., vol. 32, 2020 451 1. introduction smoking is a technique that has been used since antiquity in order to preserve food from the effects of natural degradation and oxidation (varlet et al., 2007). the degree of conservation of the fish depends on the synergistic actions between the addition of salt, the preservative effects from smoke compounds (phenols, aldehydes, and organic acids), and the dehydration that occurs during the smoking process (fuentes et al., 2010). however, smoking is currently more utilized for its organoleptic qualities, as it is a process that provides fewer preservative benefits but more sensory qualities such as aroma, flavor, color, and it also adds value to the product (cardinal et al., 2006). smoked fish is a highly nutritional food that contains polyunsaturated fatty acids, fatsoluble vitamins, essential minerals, and essential amino acids for humans (bilgin et al., 2008). traditionally, there are two methods of smoking: hot and cold; these differences are obtained by temperature changes in the smoking chamber. cold smoking is done at 33°c so that the intense thermal treatment is avoided and the nutrient structure is preserved (arvanitoyannis and kotsanopoulos, 2012). as a result, cold smoking does not offer adequate protection against harmful microorganisms, thus decreasing the shelf-life of the cold smoked fish. in the hot smoking process, the temperature ranges from 70 to 80°c, which results in baking of the meat (arvanitoyannis and kotsanopoulos, 2012). the heat and dehydration reduce the water activity of the fish, thus limiting the growth of microorganisms and increasing the shelf-life (abolagba and osifo, 2004). the smoking process occurs in three steps: salting, heating, and smoking. salt is used to preserve and to enhance the flavor of the smoked fish (guizani et al., 2014), to help in the dehydration process, inhibit microorganism growth, and extract the salt soluble protein (cheng et al., 2007). the appearance is the first factor that influences the consumer who is buying smoked products. to get a better color in smoked fish, artificial coloring can be added, or the smoking time can be extended, the latter of which would lead to more weight loss in the fish and possible economic losses (beraquet and mori, 1984). in the smoking process, color can be added to either intensify or subdue the golden red color. natural dyes in foods have been utilized to give or to intensify color as well as to restore the color in products after the smoking process. the artificial pigmentation can ensure greater color uniformity, and the product becomes more attractive, which can significantly influence its acceptability with consumers. nile tilapia (oreochromis niloticus) is the fourth most farmed fish worldwide (fao, 2018) and the most widely farmed in brazil (peixebr, 2019). its meat has a high nutritional value, featuring good taste and texture, and its fillet provides a good acceptance (souza et al., 2015). therefore, an experiment was conducted to evaluate the effects of hot and cold smoking techniques on the quality, yield, and organoleptic characteristics in fillets of in natura nile tilapia (oreochromis niloticus). ital. j. food sci., vol. 32, 2020 452 2. materials and methods 2.1. animals and experimental procedures method was carried out in accordance with the guidelines of the brazilian college for animal experimentation (cobea). we used 250 nile tilapia (oreochromis niloticus) and submitted them to depuration for 48 hours in tanks with running water and without feed. after the depuration, animals were euthanized by severing the spinal cord followed by hand filleting. the fillets were vacuum-packed and kept cooled for 12 hours until the smoking time. from the 500 fillets that were obtained, half were assigned to the cold smoking treatment while the other half was assigned to hot smoking. the two fillets from each fish were randomly distributed to be pigmented or not. the smoking of the fillets was achieved by using an industrial smoker (model arprojet, arprotec, valinhos, brazil), with smoke produced via wood friction (wooden rafter from pink eucalyptus). for the smoking process, the fillets (with and without pigmentation) were immersed in a 20% brine solution at a 2:1 ratio of brine solution (weight/volume) for 30 minutes. after this period, the fillets were washed in running water and were placed in the screen of the smoking cart. for the pigmentation process, the fillets were submerged in a water solution (1 kg fillets/1 liter of solution) with annatto extract (6 ml/l) for 15 minutes. the pigmented and unpigmented fillets were taken to the cold chamber (0 to 1oc), where they remained for 7 hours to remove superficial water (drainage). afterwards, the fillets were placed in the smoking chamber to achieve partial drying (cold smoking = 30ºc and hot smoking = 50ºc) for 60 minutes. after, smoke was added to the process; the temperatures in the hot smoking treatment ranged from 50ºc to 80ºc for 3 hours, while in the cold smoking treatment the temperatures ranged from 30ºc to 40ºc for 5 hours. at the end of the smoking process, the fillets were taken to a cold chamber (0° to 1ºc), and after the cooling process, they were vacuum packed and individually labeled. 2.2. scanning electron microscopy three samples from the dorsal part of the smoked fillets were removed for scanning electron microscopy analysis; they were fixed in 2.5% buffered glutaraldehyde and then received 1% osmium tetroxide for 2 hours. afterwards, the samples were washed in phosphate buffer, dehydrated in ethanol, and dried at a critical point with co2. the specimens were metallized with gold-palladium ions and electron-micrographed with jeol jsm-5410. 2.3. fillet yields and areas in order to determine the yield, 90 fillets per treatment were utilized, and each weight was multiplied by 2 (number of fillets per fish). all yield data was calculated in relation to the total animal weight. fillet yields were analyzed for in natura and smoked treatments, and calculations were made to determine the losses that occurred during the smoking process (in natura fillet yield minus smoked fillet yield). to determine the fillet area, the fillets were placed on parchment paper and were circumscribed by using a pencil. after, a geographic information system was utilized with ital. j. food sci., vol. 32, 2020 453 the geocoded information processing system – spring program (inpe, 1999), which was developed by the national institute of spatial research – inpe in order to complete the calculation of the fillet areas. 2.4. quality parameters in smoked fillets compared with in natura fillets 2.4.1 chemical composition analysis four fillets per treatment collected before and after the smoking process were used for the chemical composition analysis. these samples were packed in plastic bags, identified, and stored at -18ºc until the analyses. the samples of in natura and smoked fillets were ground using a multiprocessor to obtain a homogeneous sample. aliquots from this sample were used for the chemical composition determinations (4 replications; moisture, ash, and crude protein) according to the official methodology of aoac (2005), while total lipid determination was achieved based on the methodology described by bligh and dyer (1959). 2.5 color, texture, and ph ten smoked fillets per treatment and three in natura fillets (control) were used for the evaluation of the color. the fillet color was determined using a portable colorimeter (miniscan xe, hunterlab, reston, va, usa) with a d65 light source (6500ºkelvin), observation angle of 10º, and a 30 mm opening measurement cell while using the scale l*, a*, and b* of the cielab system that was developed by judd and hunter (hunter, 1975). the shear force was determined in 10 smoked fillets per treatment and 3 in natura fillets (control). the fillets were sheared with a warner bratzler catheter at 500 mm/min by using a texturometer (ta.xt2i, sms, surrey, england). the force as a function of the deformation was calculated using the average of 10 measures in different positions per fillet sample, which was expressed in kg using the program texture expert v.1.15 (sms, surrey, england). the ph of the smoked fillets was determined using a portable ph meter (dm2, digimed, são paulo, brazil) in 10 fillets per treatment after the end of the smoking process. 2.6. organoleptic characteristics for the sensory analysis, the method utilized to evaluate the acceptability of the products was the preference test, which represents the sum of all sensory perceptions and considers the opinions of the consumers. this test measures consumer preference in order to predict the acceptability of a product. thus, the acceptance attributes test was conducted by using a 9-point hedonic scale varying from “extremely disliked” (1 point) to “extremely liked” (9 points), as according to dutcosky (2007). forty non trained tasters were used for the sensory evaluation. within 36 hours after the smoking process, the fillets were cut and the samples were standardized in terms of weight and portion of the fillet (25 g). then, the samples were packed in aluminum papers and identified. the tasters randomly received the samples on plates coded with three random numbers, and a sheet for sensory analysis was provided to evaluate flavor, internal color, aroma, texture, salt content, and general acceptance. also, entire fillets per treatment were evaluated in terms of appearance attributes and the superficial fillet color. ital. j. food sci., vol. 32, 2020 454 2.7. benzo(a)pyrene production aliquots (n = 4) from samples of in natura and smoked fillets that were utilized in the chemical composition were also used to determine the benzo(a)pyrene content. the samples were subjected to saponification with methanol koh, liquid-liquid extraction with cyclohexane and dimethylformamide water (9:1, v/v), and cleaning via column chromatography of silica gel. the determination of benzo(a)pyrene was performed using high performance liquid chromatography with fluorescence detection (hplc). 2.8. experimental design the experiment was completely randomized in a 2 × 2 factorial design consisting of two smoking techniques (hs = hot smoking and cc = cold smoking) and two pigmentation treatments (fwp = fillet with pigmentation and fop = fillet without pigmentation) with 90 replications per treatment to determine yield analysis and losses during processing. for the chemical composition (n = 4), color determination (n = 10) and texture (n = 10) characteristics of smoked fillets, in natura fillets (control) were added in the design. three in natura replications were used for color and texture, while for the other measurements we used the same number of replications as the smoked fillets (n = 4). for the determination of fillet area (n = 20) the type of process was included in the analysis (in natura and smoked). for the determination of benzo(a)pyrene (n = 3), we compared the hs and cc fillets with the in natura fillets (control). the fillet was considered the experimental unit. the results of the analyzed variables were submitted to variance analysis by glm procedure from the statistical computer program statistical analysis system (sas, 2005). and the means were compared using the tukey test with a 5% probability level. for the sensory analysis, the friedman test (chi-square test) was utilized with the nonparameterized tukey test (α = 0.05). 3. results 3.1. yield, area, and area losses due to processing in natura fillets presented with a similar weight, while smoked fillets presented with an average weight of 73.77 g/fillet. the smoking and the pigmentation processes affected the smoked fillet weight (table 1). the hot smoked fillets presented with a lower weight (p<0.01) than cold smoked fillets, while the pigmented fillets were heavier (p<0.01) than those without pigmentation. a significant difference was not observed in the fillet yields. a significant interaction was observed (p<0.01) in the smoked fillet yield and the smoked fillet losses (table 1, fig. 1). we observed that the cold smoking technique demonstrated a greater yield independent of fillet pigmentation (fillets with pigmentation = 24.90%) and lack of pigmentation (fillets without pigmentation = 25.03%) (fig. 1a). considering hot smoked fillets, the fillets with pigmentation presented with a greater yield (23.85%) than fillets without pigmentation (22.20%). regarding the losses that occurred during the smoking process, an interaction was observed between smoking techniques and pigmentation (table 1, fig. 1b). cold smoked fillets (fillets with pigmentation = 11.54% and fillets without pigmentation = 11.52%) ital. j. food sci., vol. 32, 2020 455 presented with lower losses during the smoking process than hot smoked fillets (fillets with pigmentation = 12.51% and fillets without pigmentation = 14.43%). table 1. means of weight, yield, losses and areas that occurred during the processing of nile tilapia fillets (oreochromis niloticus) that were submitted to cold and hot smoking techniques, with and without pigmentation. factors of variation fillet weight (g) yield (%) smoked losses (%) in natura smoked filleting smoked smoking technique (t) cold (cc) 112.32±17.25 a 76.94±13.35 a 36.51±1.12 a 24.97±1.68 11.53±1.52 hot (hs) 112.43±16.10 a 70.81±11.56 b 36.52±0.90 a 22.98±1.96 13.51±1.97 pigmentation (p) with (fwp) 113.37±16.87 a 75.82±12.67 a 36.40±0.95 a 24.37±2.19 12.03±2.08 without (fop) 111.43±16.42 a 71.82±12.68 b 36.59±1.05 a 23.54±1.89 13.05±1.84 f test technique (t) 0.01ns 14.67** 0.00 ns 77.42** 87.04** pigmentation (p) 0.83ns 5.84* 2.25 ns 11.71** 20.81** interaction t × p 0.02ns 3.55ns 0.36 ns 16.20** 21.63** means in the same column that are followed by different letters differed by tukey test (p<0.05) ** significant (p<0.01); nsnon-significant (p>0.05). (*) means with the same lowercase letter for smoking technique within the kind of pigmentation, while uppercase letters for pigmentation within smoking technique did not differ by tukey test (p>0.05). data expressed as mean ± standard deviation. smoking technique significantly affected (p<0.01) the fillet area. the hot smoked fillets had a greater superficial area (41.92 cm2) than cold smoked fillets (37.17 cm2). the use of pigments did not affect the fillets' area, but a significant reduction (p<0.01) in area was observed when the fillet processing technique was evaluated (in natura = 41.99 cm2 and smoked = 37.11 cm2). the smoking process decreased the fillet area by 11.62% (4.88 cm2) when compared to in natura fillets. the pigmentation did not interfere (p>0.05) in the loss of fillet area. figure 1. (a) smoked yeld (%); (b) smoked losses (%), unfolding of the interaction between smoking technique x pigmentation in fillets of nile tilapia (oreochromis niloticus). (*) means followed by the same lowercase to the factor smoking technique and uppercase to pigmentation does not differ by tukey test (p>0.05). vertical bars represent the standard deviation of the mean. ital. j. food sci., vol. 32, 2020 456 3.2. chemical composition variation occurred in the chemical composition of in natura fillets when compared to the final product (table 2). hot smoked fillets had a lower moisture content (68.90%) than cold smoked fillets (72.24%), while crude protein and ash contents were significantly greater (25.20 and 3.85% respectively) in hot smoking. the smoking technique did not influence total lipid content (table 2). it was observed that fillet pigmentation affected the ash and energy content. the pigmented fillets presented with lower ash contents (2.84%) than those without pigmentation (4.37%). table 2. means of chemical composition(*) of nile tilapia fillets (oreochromis niloticus) submitted to cold and hot smoking techniques with and without pigmentation. factors of variation moisture (%) protein (%) lipids (%) ash (%) smoking technique (t) cold (cc) 72.24±2.37 a(1) 22.50±2.30 b 2.10±0.25 a 3.19±0.87 b hot (hs) 68.90±2.29 b 25.20±1.71 a 2.08±0.99 a 3.85±0.91 a pigmentation (p) with (fwp) 71.45±2.60 a 23.60±1.98 a 2.19±0.98 a 2.84±0.43 b without (fop) 69.67±2,92 a 24.07±2.87 a 2.04±0.26 a 4.37±0.58 a control = in natura 80.83±0.58 17.48±0.13 1.18±0.07 1.00±0.01 f test control vs fatorial 102.62** 45.36** 5.74* 227.61** smoking technique (t) 13.46** 10.31** 0.004 ns 23.59** pigmentation (p) 3.74 ns 0.45 ns 0.26 ns 103.60** interaction t x p 0.09 ns 0.31 ns 0.47 ns 0.32 ns (1)for each factor, means of the same factor in a column that are followed by the same letter did not differ by tukey test (p >0.05). ns non-significant (p>0.05) *significant (p<0.05) **significant (p<0.01) (*) 2 replications per sample were used for protein, lipids, and ash content, while 3 replications were used for moisture content. moisture content is showed in franco et al. (2013). data expressed as mean ± standard deviation. 3.3. color, texture, and ph of the fillets when analyzing the fillet color (figure 2), we found that unpigmented in natura fillets had an average chroma value of a* and b* of -1.5 and 9.54, respectively. the values of a* and b* increased in function of the smoking technique and pigmentation. pigmented fillets had values for a* and b* that were significantly greater than those without pigmentation, while the hot smoking process yielded greater chroma values than cold smoked fillets. the unpigmented hot smoked fillets presented with a lightness (66.04) that was significantly greater (p <0.05) than pigmented fillets (62.45). cold smoked fillets with pigmentation (48.74) did not differ from those cold smoked fillets with pigmentation (49.46) in terms of lightness. ital. j. food sci., vol. 32, 2020 457 figure 2. means of color system (cielab) for smoked and in natura fillets with interactions between smoking technique and fillet pigmentation. means following the same uppercase letter (lowercase) for the factor pigmentation within each smoked technique (smoked technique within each pigmentation) did not differ by tukey test at 5% of probability level. *indicates that for each smoking technique and pigmentation treatment, the mean differs from control (in natura). vertical bars represent the standard deviation of the mean. there was an interaction between smoking techniques and pigmentation for shear force of the fillets (fig. 3a). cold smoked fillet shear force values (with pigmentation = 6.40 kg and without pigmentation = 6.11 kg) were greater (p <0.05) than hot smoked fillets values (with pigmentation = 2.71 kg and without pigmentation = 3.09 kg). there was a significant difference between smoking techniques and pigmentation in fillets in comparison to in natura fillets. through scanning electron microscopy, we can observe the film surrounding the fillets (fig. 3b). in cold smoked fillets, collagen fibers were observed (fig. 3c). cold smoked fillets presented with a lower (p <0.01) ph value (6.43) than hs fillets (6.94). the fillets without pigmentation had a lower ph (6.59) in relation to fillets with pigmentation (6.78) due to dehydration in fillets without pigmentation. the ph changes that occurred in cold versus hot techniques are due to water loss during the process, with more water loss occurring in hot smoked fillets (ph = 6.94). the greatest smoking time decreased the ph values in cold smoked fillets (ph = 6.43). 3.4. benzo(a)pyrene production when analyzing the benzo(a)pyrene content in cold smoked fillets (0.45 µg/kg) and hot smoked fillets (0.49 µg/kg) and comparing them to in natura fillets (0.26 µg/kg), we observed that the smoking process affected the benzo(a)pyrene content, wich was significantly higher (p <0,05) in the fillets subjected to smoking (regardless of type, hot or cold) compared to fillets in natura. ital. j. food sci., vol. 32, 2020 458 figure 3. (a) means of shear force (kg) of interaction when compared between smoking technique (cold and hot) and pigmentation (with = fwp and without = fop). means following the same uppercase (lowercase) letter for the factor pigmentation within of each smoking technique (smoking technique within each pigmentation) did not differ by tukey test at a 5% probability level. * indicates that for each smoking technique and pigmentation treatment, the mean following the * differs from control (in natura). vertical bars represent the standard deviation of the mean. (b) electron-micrograph of smoked fillets depicting the film that is formed by protein denaturation and leaching of fat, which is associated with the compound action generated by organic matter pyrolysis. (c) presence of collagen fiber bundles among muscle tissue of cold smoked fillets. 3.5. organoleptic characteristics there was a significant effect (p <0.05) of smoking technique on flavor, aroma, texture, salt content, superficial and internal color, and general acceptance. hot smoked fillets were the best evaluated by tasters. the pigmentation had a significant effect on attributes such as appearance, flavor, superficial color, and salt content in smoked fillets. fillets with pigmentation received greater scores than fillets without pigmentation for appearance, superficial and internal color, and flavor (table 3). ital. j. food sci., vol. 32, 2020 459 there was a significant interaction (p<0.05) between smoking technique and pigmentation on salt content. hot smoked fillets presented with greater scores by tasters when compared to pigmented and unpigmented cold smoked fillets in terms of salt content. table 3. probability value for the friedman chi-square test, means and non-parameterized tukey multiple comparison test values (α = 0.05) based on the scores assigned by tasters for nile tilapia smoked fillets. entire fillet portion fillet appearance(1) color internal color aroma flavor texture salt content general acceptance smoking technique (t) cold (cc) 6 a 6 b 5 b 5 b 4 b 4 b 5,5 b 4 b hot (hs) 6 a 7 a 8 a 7 a 8 a 8 a 8 a 7 a pigmentation (p) with (fwp) 7 a 7 a 6 a 6 a 6 b 6 a 6 b 6 a without (fop) 5 b 6 b 6 a 6 a 7 a 6 a 7 a 7 a f test smoking technique (t) 0.6985 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 pigmentation (p) 0.0001 0.0001 0.3897 0.3481 0.0445 0.4320 0.0183 0.2893 interaction t×p 0.8575 0.4652 0.4497 0.7237 0.1573 0.0947 0.0112 0.0679 coeficiente of variation (%) 3.85 1.89 3.22 2.89 1.66 2.52 3.01 2.99 (1)for each factor, means in the same column that have the same letter did not differ significantly by the nonparameterized tukey multiple comparison test (α = 0.05). 4. discussion in the present study, the losses that occurred during the smoking process were greatest for hot smoked fillets (13.51%). according to sigurgisladottir et al. (2000), fillet weight losses between 10 and 25% during the smoking process, due to dehydration and fat leaching in muscle tissues, is commonly observed. a significant difference was not observed in the fillet yields. thus, we can infer that fish weights were homogeneous, and that we used one filleting method and the same person to perform the filleting. the cold smoking technique demonstrated a greater yield independent of fillet pigmentation and lack of pigmentation. considering hot smoked fillets, the fillets with pigmentation presented with a greater yield than fillets without pigmentation. these results were also observed by franco et al. (2010), who reported that matrinxa fillets with skin (brycon cephalus) had a lower hot smoked fillets yield (33.79%) when compared with cold smoking yields (34.46%). regarding the losses that occurred during the smoking process, cold smoked fillets presented with lower losses during the smoking process than hot smoked fillets. although pigmentation did not interfere in the losses for cold smoked fillets, it did affect hot smoked fillets. this fact is due to the dehydration that occurred in the fillets, as hot ital. j. food sci., vol. 32, 2020 460 smoked fillets presented with lower moisture contents; despite the fact that there was no significant difference observed, the fillets with pigmentation had the lowest moisture content. the smoking process decreased the fillet area by 11.62% when compared to in natura fillets. thus, the losses that occurred during the smoking process are related to the weight, thickness, and area of the fillets. the hot smoked fillets demonstrated lower losses in fillet area compared to cold smoked fillets; the temperature used in the process led to greater losses in the product due to the greater reduction in moisture content of the fillet. in the chemical composition, the greater values for crude protein and total lipids observed in smoked fillets in relation to in natura fillets are due to the effects of dehydration. these results were also observed by vasiliadou et al. (2005) for hot smoked dourade (sparus aurata); the total lipid concentration increased from 7.55% (in natura fish) to 12.92% (smoked fish), crude protein content ranged from 20.65% (in natura) to 25.67% (smoked), while moisture content decreased from 69.96 % to 57.45% after the smoking process. the greater ash content in the in natura fillets compared to the smoked fillets may be due to sodium chloride absorption in the muscle tissue during the brining process of the smoked fillets. this result may also be due to the varied nutrient concentration, which is a side effect of moisture losses during dehydration, which takes place during the smoking process. the pigmented fillets presented with lower ash contents, and this can be related to the loss of salt in the fillets due to the annatto extract solution that was used in the fillet pigmentation process. when analyzing the fillet color, the values of a* and b* increased in function of the smoking technique and pigmentation. the processing technique (time x temperature) that was used influenced the chroma a* and b* and led to lower chroma values in cold smoked fillets. cardinal et al. (2001), when studying atlantic salmon (salmo salar), relayed that the temperature did not affect these two color parameters. beraquet and mori (1984) observed that smoking time contributed intensively to color formation in smoked fishes (mackerel scomber japonicus); in other words, cold smoked fishes (8 hours at temperature below 35ºc) presented with a golden yellow color that was more intense than hot smoked fishes due to the long exposure time of the fillets to smoke. it was theorized that reactions between carbon compounds and proteins are responsible for color formation in the smoked fish surface, while absorbed phenolic compounds are deeply related to the flavor and aroma of the smoked product (huda et al., 2010). there was an interaction between lightness (l*) and chroma a* and b*. pigmented fillets had values for a* and b* that were significantly greater than those without pigmentation, while the hot smoking process yielded greater chroma values than cold smoked fillets. the smoking process results in water losses of the meat, which decreases the fillet lightness (fuentes et al., 2012). however, for hot smoked fillets in the present study, the increase in processing temperature led to a greater release of lipids, which resulted in brighter fillet surfaces independent of the pigmentation treatment, and consequently, caused an increase in the lightness. bixin, natural pigment annatto seed (bixa orellana l.), is the compound responsible for food pigmentation, and it has a good thermal stability below 100ºc (giridhar et al., 2014). in both cold and hot smoking processes, the smoking chamber temperature did not exceed 100ºc, which therefore did not alter the fillet color. this can be demonstrated by the homogeneity of the results for chroma a* and b*. the unpigmented hot smoked fillets presented with a lightness that was significantly greater than pigmented fillets. cold smoked fillets with pigmentation did not differ from ital. j. food sci., vol. 32, 2020 461 those cold smoked fillets with pigmentation in terms of lightness. the lightness increased due to steam because actomyosin is denatured by heat. however, if the smoking process is independent of temperature, the lightness decreases once the smoking process deposits chemical compounds, which are produced naturally via wood pyrolysis, into the smoked product. hot smoked unpigmented fillets had greater values for a* and b* and yielded a golden red color; these values were greater than those of pigmented cold smoked fillets, whose red tone was also significantly less. the greater smoking time results in blackened fillets, so the dehydration and fillet color should be controlled to obtain a more acceptable final product (hassan, 1988). lysine participates with the ε-amina group in the initial steps of the maillard reaction (siskos et al., 2005), as seen in the active reaction of aldehyde (formaldehyde, glyoxal, furfural, coniferaldehyde, and sinapaldehyde) from the smoke with the amina group of the lysine. the smoking process increases the lysine content in the fish due to the maillard reactions (akintola, 2015). thus, this can justify the greater values (a* and b*) observed in hot smoked fillets when compared to cold smoked fillets. despite the shorter smoking time in the hot smoking process (3h), the temperature provides greater amounts of dehydration and more reactions between lysine and smoke compounds, thereby obtaining a more intense coloration. when we evaluated the shear force of the fillets, cold smoked fillet shear force values were greater than hot smoked fillets values. the salt that was used in the smoking process can affect the final texture of the smoked fillet, which can be seen in terms of water retention capacity, isoelectric point, and protein functionality. thus, greater salt concentrations are responsible for a firmer texture (gallart-jornet et al., 2007), which then results in a better flavor and greater stability during storage. the production of a superficial film in smoked meat is due to the protein denaturation that occurs as a result of dehydration, which is associated with salt and heating (hassan, 1988). this film on the surface may prevent excessive leaching of fat or evaporation (sigurgisladottir et al., 2000). through scanning electron microscopy, we can observe the film surrounding the fillets. in hot smoked fillets, this was very important, because they were tenderer than cold smoked fillets. thus, they needed a more consistent structure to avoid disruption via touch or light pressure. this is important mainly for fillets or fish that are kept hanging inside of the smoking chamber in order to avoid falling during the smoking process. collagen is the largest component of the intramuscular connective tissue of fish, and it has an important role in maintaining fillet integrity and muscle cohesiveness (aussanasuwannakul et al., 2012) since it contributes to meat stability and firmness. during the smoking process, the activity of endogenous proteases increases, and these enzymes hydrolyze muscle proteins, thereby breaking down the connective tissue (hultmann et al., 2004) and altering the texture of the smoked fillet. collagen contributes to the texture of in natura fishes, but it is not important in the texture of baked fish (haard, 1992). the textural resistance of baked muscle decreases with increasing moisture contents up to 79% (lee and toledo, 1976). above 79% of moisture, the resistance decreases, thus reflecting the effects of shear force and compression. morris et al., (2004) reported that hot smoking results in protein denaturation via heat, but there are no theoretical explanations for the phenomenon relating to the thermoviscoelastic properties of the muscle tissue. in hot smoked fillets, despite the lower moisture content (68.90%), a temperature between 50 and 80ºc was enough to alter the structure of collagen fibers and increase tenderness of the fillets, while the film on the fillet surface was responsible for keeping the surface intact until the end of the process. in cold smoked ital. j. food sci., vol. 32, 2020 462 fillets, collagen fibers were observed; therefore, the temperature utilized was not severe enough to denature the collagen fibers. during the smoking process, polycyclic aromatic hydrocarbons (pah) can be formed by organic matter from the wood (visciano et al., 2008). polycyclic aromatic hydrocarbons are a compound group that consists of three or more condensed aromatic rings, which are produced during the incomplete combustion process involving wood, coal, or oil, of which the benzo[a] pyrene is the most studied because it is highly carcinogenic (wretling et al., 2010). regarding the ph of the fillets, cold smoked fillets presented with a lower ph value than hot smoked fillets, and the fillets without pigmentation had a lower ph in relation to fillets with pigmentation, due to dehydration in fillets without pigmentation. the ph changes that occurred in cold versus hot techniques are due to water loss during the process, with more water loss occurring in hot smoked fillets. the greatest smoking time decreased the ph values in cold smoked fillets, and these changes are due to acid absorption from the smoke, moisture loss, as well as the reaction between phenol or polyphenol and carbonyls with proteins and amino groups, respectively (hassan, 1988). when analyzing the benzo(a)pyrene content in cold smoked fillets and hot smoked fillets and comparing them to in natura fillets, we observed that the smoking process affected the benzo(a)pyrene content, wich was significantly higher in the fillets subjected to smoking (regardless of type, hot or cold) compared to fillets in natura. however, the mean values that we observed are considered low. the temperature influences the amount of benzo(a)pyrene, as does the exposure time of the product to the smoke compound in pyrolysis. the smoked fish, in adequate conditions, normally present with a low amount of benzo(a)pyrenes, and the maximum level for benzo[a]pyrene in smoked fish and smoked fishery products is 5.0 μg/kg (wretling et al., 2010); the values in this study are within the range reported. in sensory analysis, hot smoked fillets were the best evaluated by tasters. fillets with pigmentation received greater scores than fillets without pigmentation for appearance, superficial and internal color, and flavor. during the smoking process, the phenols contained in the smoke are responsible for conferring the desirable sensory properties as well as important antioxidants (stołyhwo and sikorski, 2005). the lipids in the fish absorb the aromatic substances present in the smoke. we observed that cold smoked fillets had lower scores for flavor and aroma, which can be associated with the fat content in the fillets and the temperature used in the process. when the temperature is high (above 35ºc), the fat in the muscle moves to the surface; this improves the appearance and retention of aromatic substances in the fillets, which therefore creates a better aroma and flavor (beraquet and mori, 1984). hot smoked fillets presented with greater scores by tasters when compared to pigmented and unpigmented cold smoked fillets in terms of salt content. this is due to the smoking process since moisture losses occurred in the hot smoked fillets. in the fillets without pigmentation, there was a consequent increase in the salt concentration in the smoked fillets, thus providing a tasty fillet in terms of salt content. in temperatures above 30ºc, the fish acquires better flavor and coloration when compared to cold smoked products from the same time period (beraquet and mori, 1984). this finding confirms the observations in the present study, where the flavor was significantly greater for hot smoked fillets than cold smoked fillets. the same phenomenon occurred in terms of superficial and internal color of the fillets, because the tasters attributed the greatest scores to hot smoked fillets when compared to cold smoked fillets. ital. j. food sci., vol. 32, 2020 463 hot smoked matrinxa fillets presented with the best results in terms of flavor, salt content, and acceptance, while cold smoked fillets had the best results for color and appearance (franco et al., 2010). according to these authors, aroma and texture were not influenced by the smoking techniques. the tasters believed that hot smoked fillets had a better texture (score 8) when compared to cold smoked fillets (score 4). this fact is due to the tenderness of the hot smoked fillets, as these fillets had a lower shear force. the tasters did not observe any difference in the texture of pigmented and unpigmented fillets, and when this characteristic was analyzed in terms of shear force, no difference was observed. 5. conclusions time and temperature affected the fillet quality in terms of composition, sensory attributes, yield, area, salt content, color, and ph. the pigmented hot smoked fillets presented with the best results regarding quality (chemical composition, salt content, color, texture, water activity, and ph) and organoleptic characteristics; however, they presented with the lowest yield in terms of processing, and consequently, had greater weight losses. the smoking process altered the chemical composition of the smoked fillets by reducing moisture and providing a greater concentration of crude protein, total lipids, and ash. the hot smoked fillets presented with a lower moisture content, but had a greater crude protein and ash content. the pigmentation interfered with ash content. the pigmentation improved the color and general acceptance of smoked fillets, and was also associated with a greater yield for hot smoked fillets. the reddish color (a*) and the yellow color (b*) produced a golden red fillet that was more pigmented in hot smoked fillets than the others; these fillets were considered to be the most adequate by tasters. in terms of public health (or food 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cross-linked with smoke condensate and glutaraldehyde s. barbut* and m. ioi university of guelph, department of food science, guelph, ontario, canada, n1g 2w1 *corresponding author: tel.: +15198244120 e-mail address: sbarbut@uoguelph.ca abstract collagen films were produced from five commercial collagen dispersions used for in-line sausage co-extrusion production. films were prepared by partially dehydrating in a salt solution (30%) and crossed linked with smoke condensate (15%) or glutaraldehyde (ga; 01%). both treatments increased the tensile strength (0.32 to 0.91 mpa) and reduced % elongation while differences among the dispersions were observed. overall, % elongation generally decreased with a higher degree of cross linking. transmission electron micrographs revealed that collagen fibers were swollen to varying degrees, likely influencing the mechanical behaviours. protein concentration affected the transparency of the films with a difference of 100% in light transmission between the clearest and most opaque film studied. cross-linking with ga appeared to thermally stabilize films up to 80˚c. aldehydes in the smoke condensate were identified by gas chromatography showing highest concentration of benzaldehyde, 4-hydroxy-3,5-dimethoxy. keywords: casings, coating, collagen, cross-linking, glutaraldehyde, smoke condensate ital. j. food sci., vol. 31, 2019 645 1. introduction sausage products rely on casings to contain, form and bind the raw ground/emulsified meat placed inside them. after cooking (heat induced gelation) of the meat proteins, the casings can be removed from the product by the food processor (e.g., frankfurters produced in cellulose casings), by the consumer (salami produced in plastic casings) or consumed with the product (pepperettes produced in natural casings). prior to the early 20th century almost all casings were derived from animal intestines. since then, advancements in casing technology and manufacturing have led to several types of synthetic casings (e.g., cellulose, plastic) that can be tailored to a given process and product. with increasing sophistication, the production of manufactured casings was moved to designated plants, where collagen derived from hides is extracted, formed into tubes and then crosslinked with a strong crosslinking agent such as glutaraldehyde. when co-extrusion was more recently commercialized, on a large industrial scale (savic and savic, 2016), direct casing application was moved to the meat processing facilities. however, the issue of finding an acceptable mild crosslinking agent, that will also enable operating at a high speed production line, is still a challenge today (i.e., glutaraldehyde is not permitted to be used in food production areas, and in the off-premises casing manufacturing plants it is thoroughly washed away prior to shipping) and hence the need for the current investigation. co-extrusion is the process of extruding a cylindrical core of sausage meat, while simultaneously extruding an outer layer of casing material directly on to the meat’s surface. subsequent processes include: brining, smoking, drying and cooking. they are needed to stabilize and harden the casing material by osmotically removing water, and later chemically cross-linking the building blocks of the casing material (e.g., collagen, alginate). stabilizing the casing is essential, as the casings require strength and elasticity to undergo linking, packaging, storage and reheating. since casings are formed directly on the meat’s surface, co-extrusion systems must use a suitable brining (e.g., concentrated salt solution) and cross-linking agents that will set the casings fairly quickly (morgan et al., 1998). smoke condensates are commonly used as chemical cross-linking agents, as their aldehydes form covalent linkages between collagen molecules and fibers. exposure to smoke condensates enhances the mechanical properties of the casing, and also provides the sausage with colour, flavour and some antimicrobial properties. in co-extrusion, smoke condensate is favoured over traditional smoking (burnt wood shavings) because of the ability to work with higher concentrations, product’s uniformity, and the intense colour they can provide during a short exposure time (morgan et al., 1998; bontjer et al., 2011). smoke condensates are produced by combustion of hard woods (e.g., cherry, oak), while the smoke is moved through a shower of water droplets that collect the aldehydes, ketones, furans, phenols, and acids (toledo, 2007). when dry regenerated collagen casings are produced (in special plants), glutaraldehyde (ga) is used as a cross-linking agent, and is later thoroughly washed off the casings before the drying process (avery and bailey, 2008; morgan et al., 1998; savic and savic, 2016). glutaraldehyde is also used as a leather tanning agent, production of medical constructs made from collagen, and tissue fixative (covington, 1997; cheung and nimni, 1982). it is able to form stable bonds among proteins and increase the mechanical properties of collagen fibers. the mechanism of glutaraldehyde’s reactions has been studied quite a lot in order to help improve the modification of collagenous materials (cheung and nimni, 1982; barbut, 2015). overall, it has been shown that ga reacts with amine groups, specifically lysine, to form heterocyclic compounds. subsequent oxidation reactions produce pyridine rings (englert et al., 2007). although ga is a ital. j. food sci., vol. 31, 2019 646 highly functional cross-linking agent, cytotoxic effects have limited its direct food applications, including in semi-liquid casing application as used in co-extrusion. however, as mentioned above in designated casing producing plants it is used as part of the manufacturing of regenerated collagen casings that subsequently go through an extensive washing process to remove all the ga residues. overall, there has been a substantial effort to understand the properties of collagenous materials and the effects of different cross-linking agents (o’sullivan et al., 2006; wolf et al., 2006; tomihata et al., 1994; olde damik et al., 1995). however, little has been published about the effectiveness of cross-linking agents on the performance of commercial co-extrusion collagen products. this study was designed to address this area in two steps. first, the effects of smoke condensate (currently used in meat processing plants for the wet co-extrusion process), and glutaraldehyde (used in special plants to make regenerated dry manufactured collagen casing) concentration and contact time were examined by evaluating the mechanical properties of collagen films. this was done to provide an insight into potential manipulation of the mechanical properties of coextrusion collagen casings. the second study examined the mechanical, microstructural and thermal properties of cross-linked films to compare the similarities and differences in functionality of different commercial collagen dispersions. 2. material and methods 2.1. study i. manipulation of cross-linking conditions 2.1.1 preparation of films five commercial collagen dispersions used for sausage extrusion processes were obtained from major manufacturers. the dispersions were labeled as: collagen 1 through 5 (c1, c2, c3, c4, c5). protein content was determined by the dumas method (leco fp528, st joseph, mi, usa) using a nitrogen factor of 6.25. moisture content was determined by drying triplicate samples (5 g) at 105°c for 24 h. the method of film formation was adapted from the work of harper et al. (2013) with alginate solutions and was performed at 4˚c to reduce adhesion during film formation. briefly, the samples were degassed using a vacuum packager (multivac canada inc., woodbridge, on, can) at 7.3 kpa for 25 s, then again at 7.3 kpa for 50 s and 75 s (settings 4, 6 and 8, respectively) to remove gas bubbles that were incorporated during processing, as they can create weak spots in the films. while still in the vacuum pack bags, the dispersions were mixed by rolling the dispersions 10 times in adjacent directions. later, approximately 3 g portions were rolled on a stainless steel board between two layers of plastic film, with a stainless steel roller. the roller had a recess of 0.50 mm in order to achieve uniform film thickness. the plastic sheet on the roller side of the film was removed and the remaining plastic sheet with the collagen film on it was then placed in a 30 wt.% sodium chloride (prepared in deionized water) for 5 min, in order to dehydrate the film. the nacl treatment mirrors the industrial practice of dehydrating the film (avery and bailey, 2008). after 5 min, the film was strong enough to hold together when removed from the plastic sheet. the plastic sheet was folded onto the formed film to prevent further dehydration of the film before it was moved to the cross-linking solution. the goal of the first study was to determine the best exposure time and cross-linking agent concentration to obtain a good quality film/casing. for this part we used collagen 2, which is one of the most common dispersions currently used by the meat industry. the partially dehydrated films (i.e., by the salt solution) were cross-linked with either liquid ital. j. food sci., vol. 31, 2019 647 smoke condensate (charsol select 24p liquid smoke, red arrow products, manitowoc, wi, usa) or glutaraldhyde (em grade, canemco, canton de gore, qc, can). the application of liquid smoke is a standard treatment used by the meat industry, while glutaraldehyde was used here as a control treatment with a known aldehyde (crosslinker) concentration. the films were immersed in a 15 vol.% smoke condensate in deionized water, i.e., based on industry recommendation/procedure. films were held in the diluted smoke condensate bath for 10, 20, 40 or 80 s. following cross-linking, films were covered with a plastic sheet to avoid drying before testing. as indicated above, glutaraldehyde (ga) was used because its mechanism of cross-linking is better understood, thus its effects could be used as a reference/standard. films were cross-linked in solutions of 0.1, 0.5 and 1.0 vol.% ga in 1m hepes buffer at ph 7.4 (fisher scientific, ottawa, on, can). the partially dehydrated films were immersed in the ga solution for 5, 10 or 20 min intervals (note: longer times were used because of the low ga concentrations), followed by a 5 min rinse in water to remove ga residues. similar to cross-linking with smoke condensate, the films were later covered with plastic to avoid dying before testing. 2.1.2 mechanical properties the standard tensile test (astm, 2010) was used, by employing a texture analyzer (taxt2i, texture technologies corporation, scarsdale, ny, usa) with a gripper distance set at 50 mm, trigger force at 5 g, test speed at 2 mm/s and test distance of 25 mm. six measurements were made to calculate an average thickness, using a digital micrometer (testing machines inc., islandia, ny, usa), of each film in each of the three trials. films were cut into 75 mm × 25 mm strips. the average thickness and width of the films were used for the tensile stress calculations. tensile strength (maximum stress the film endured prior to breaking) and the percent elongation (maximum elongation the film reached prior to breaking) were determined from the generated stress–strain curve. puncture force was also examined using the texture analyzer. in this test, a 5 mm stainless steel ball probe was used to puncture films mounted in an extensibility fixture with 10 mm diameter round openings (ta-108s5, texture technologies, corporation, scarsdale, ny, usa). the test speed was 1 mm/s and the trigger force was 5 g. the distance to puncture and work of puncture were determined from the force–distance curve. a total of eighteen measurements were used for each of the treatments (six measurements per trial). 2.1.3 experimental design and statistical analysis the experiment was designed as a randomized complete block with 3 independent trials. each trial consisted of evaluating six samples per treatment. the statistical analysis was performed using sas version 9.2 (sas inst., cary, nc, usa). a general linear model was used for the analysis of variance (anova). the film type means and interactions were compared by using tukey's multiple comparison analysis with a p-value ≤ 0.05, which was used to detect statistical significance. 2.2. study ii. evaluation of cross-linked collagen dispersions 2.2.1 preparation of films collagen films were prepared from the five dispersions, as described before. in this part they were cross-linked with either 15 vol.% smoke condensate in deionized water, for 40 s, or with 0.5 vol.% ga in 1m hepes buffer at ph 7.4 for 5 min, followed by a 5 min rinse. ital. j. food sci., vol. 31, 2019 648 2.2.2 characterization of smoke condensate the smoke condensate was diluted in methanol and injected directly to a gas chromatograph (gc-ms 1200, varian, palo alto, ca, usa) to determine both volatile and semi-volatile compounds. compounds were tentatively identified based on the nist library provided with the instrument. 2.2.3 transmission electron microscopy (tem) tem was performed on collagen films, raw collagen and cellulose fibers as controls. the films were fixed in 2.0% glutaraldehyde, buffered with hepes (ph 7.4) for 90 min. this was followed by fixing in osmium tetroxide and dehydration through a series of graded ethanol solutions (50%, 70%, 80%, 90%, and 100%), each for 10 min. once the films were dehydrated, they were run through a series of propylene oxide and spurr’s resin solutions (1:0, 3:1, 1:1, 1:3 and 0:1) to ensure that the resin was thoroughly incorporated, prior to embedding. the resin was polymerized for 24 h at 60˚c. samples were then cut into 70 to 90 nm sections on a microtome (reichert ultracut s, leica microsystems inc., concord, on, can), fixed on grids, stained with saturated uranium acetate and lead citrate (hayat, 2000). negative staining was used to prepare raw collagen and cellulose fibers as controls. the collagen control was prepared by hydrating dried bovine hide sample (provided by the meat laboratory at the university of guelph) for 24 h at 23˚c. the ph was then lowered to 2 and blended to aid in dispersion and fiber swelling. the cellulose control was a 0.1 wt.% solution of powdered cellulose (arbocel, jrs, schoolcraft, mi, usa). one drop of the control solution was placed on a formvar-coated grid and the excess solution was removed with a filter paper. a drop of 2% uranium acetate was then applied to stain the sample. after 30 s, the excess uranium acetate was removed with a filter paper and the grids were dried, on the bench top. samples were examined by a tem (philips cm 10, philips scientifics, eindhoven, nb, nl) and photographed (olympus morada camera, olympus soft imaging system, berlin, ger) using item imaging software (item software, whiteley, hph, uk). 2.2.4 optical property the light transmission (380–780 nm) of the films was evaluated using a single beam spectrophotometer (usb 2000, ocean optics inc., dunedin, fl, usa). the following settings were used: integration time: 100 ms: scans to average 2; and boxcar width 4. the light transmission was measured on twelve films per collagen sample. 2.2.5 mechanical properties of thermally treated films cross-linked films from collagen 2 (c2) were mounted in the puncture fixture and placed in a plastic bag. the fixture and bag were lowered into a water bath at 40, 50, 60, 70 or 80˚c and held for 15 min. the puncture test was performed immediately after thermal treatment. the puncture test was performed as previously described. once again, a total of eighteen measurements were used for each of the thermal treatments (six measurements per trial). ital. j. food sci., vol. 31, 2019 649 2.2.6 experimental design and statistical analysis the experiment was designed as a completely randomized block with 3 independent trials. each trial consisted of evaluating six sub-samples per treatment. the statistical analysis was performed as indicated above. 3. results and discussion 3.1. study i. manipulation of cross-linking conditions 3.1.1 mechanical properties the goal of the first study was to determine the best exposure time to obtain a good selfsupporting collagen film, and for this we focused on collagen 2, which is currently the most commonly used preparation. the results of the tensile test (fig. 1) demonstrate that there were no significant differences (p > 0.05) in the tensile strength and % elongation when films were exposed to smoke condensate for 10 to 80 s. the results were obtained by using collagen 2, which is one of the most commonly used collagen dispersions in the industry. the ph of the dispersion was 2.21 and is in line with the values of the other dispersions (c1=2.06; c3=2.01; c4=2.67; c5=2.04). according to manufacturers, the swelling agent for c3 was hcl, for c4 it was hcl/acetic acid; this information was not provided for c1, 2 and 5. overall, the results reveal that after 10 s exposure the film gains a certain degree of cross-linking, and further exposure does not have a significant effect. it should be pointed out that after 10 s the film is removed from the solution but not rinsed so the amount of liquid smoke picked up by the film keeps on working. overall, crosslinking, by intramolecular bonds, are formed between collagen molecules and fibers. the formation of covalent cross-links prevents collagen fibers from sliding past each other, thus reducing the film’s ability to undergo deformation (rault et al., 1996; paul and bailey, 2003). from a practical point of view, the results indicate that the meat processor would not be able to significantly modify the tensile properties of their casings by increasing the contact time, within this range (10 to 80 s). as noted in the method section, the concentration of smoke condensate used here was based on industry practices/recommendations. figure 1. mechanical properties of collagen films produced with increasing exposure times to smoke condensate (15 vol.% in deionized water). samples made from collagen 2. means with the same letter are not significantly different (p > 0.05). ital. j. food sci., vol. 31, 2019 650 this study employed a commonly used liquid smoke condensate, currently used by various meat processors for co-extrusion applications. overall, it is known that the composition of smoke condensates can vary, because of variations in the proportion of compounds such as aldehydes and phenols in the hard wood used to produce the condensates (guillén and manzanos, 1999; montazeri et al., 2013). since aldehyde compounds are linked to the smoke’s cross-linking ability, variations in their concentration could potentially produce different results related to tensile strength. in order to address this point, laboratory grade glutaraldehyde (ga) with a known aldehyde concentration was also used in our study. the mechanical properties of the glutaraldehyde-treated films (table 1) show that the tensile strength and % elongation were basically in the same range as obtained by the smoke condensate. when using ga there were no significant interactions between the ga concentration and exposure time. also, the results indicate that there was no significant difference (p > 0.05) in the percent elongation between 5 and 20 min exposure (fig. 2). thus, exposure time appears to have a greater effect on tensile strength when cross-linking with ga. this difference might be attributed to the concentration of the cross-linking agent in the ga solution. the results also suggest that manipulating the concentration of ga will have significant differences (p < 0.05) on the tensile and puncture properties of the collagen films. the most notable effect of increasing ga concentration (0.1 to 1.0 vol.%) was the significant decrease in percent elongation and distance to break (fig. 2). these observations are consistent with those discussed by avery and bailey (2008). overall, it appears that over cross-linking with ga would make the films less stretchable. table 1. mechanical properties of collagen films produced with increasing exposure time and concentration of glutaraldehyde; samples made from collagen 2. concentration vol.% time min tensile strength mpa percent elongation % distance to break mm work to break n*mm 5 0.564±0.14 22.475±2.11 3.012±0.26 1.537±0.56 0.1 10 0.765±0.09 23.213±1.72 2.911±0.31 1.590±0.28 20 0.833±0.12 20.741±0.96 2.787±0.35 1.566±0.31 5 0.502±0.06 20.137±1.21 2.517±0.24 1.068±0.05 0.5 10 0.664±0.02 20.381±1.43 2.338±0.44 1.058±0.37 20 0.577±0.05 19.661±3.28 1.952±0.48 0.672±0.19 5 0.708±0.08 20.921±2.96 2.071±0.63 1.630±0.90 1.0 10 0.680±0.15 15.877±2.80 1.784±0.72 1.276±0.61 20 0.764±0.16 17.591±3.68 1.634±0.59 1.135±0.69 3.2. study ii. evaluation of cross-linked collagen dispersions 3.2.1 mechanical properties the mechanical properties of casings play a crucial role in sausage production. they affect sausages’ structural integrity, shape, volumetric changes, textural properties, and behavior during processing (savic and savic, 2016). for example, during traditional production, casings must withstand tensile stresses during stuffing, and hanging in the smokehouse. in addition, casings must provide compressive strength during meat protein gelation (heating) and exhibit elasticity during cooling the product (savic and savic, 2016). ital. j. food sci., vol. 31, 2019 651 examining properties such as percent elongation, and tensile strength, can help to predict the success of a casing. figure 2. mechanical properties of collagen films produced with increasing contact times to glutaraldehyde and concentration of glutaraldehyde. samples made from collagen 2. the contact time means were averaged across all concentration (0.1, 0.5, 1.0 vol.%) and the concentration means were averaged across all contact times (5, 10 and 20 min). means in the same graph with same letter are not significantly different (p > 0.05). in the second study, the five different collagen dispersions were compared. overall, they all had a protein content or 4.3±0.8% (c1=5.1; c2=3.6; c3=3.7; c4=4.4; c5=4.8%) and moisture content of 94.5±1.0% (c1=93.5; c2=95.5; c3=93.5; c4=94.0; c5=93.7%). after partially drying the casings, by exposing them to a salt solution (30% salt as is also done by the industry), all the films showed a pretty similar moisture content (76.0±1.5%, where ital. j. food sci., vol. 31, 2019 652 c1=74.5; c2=77.5; c3=74.5; c4=75.0; c5=77.0%). tensile strength and percent elongation results (table 2) showed that there were some significant differences among the various collagen films. the film produced with collagen 4 (c4) had the lowest tensile strength (0.32 mpa) when cross-linked with smoke condensate, and c3 (0.41 mpa) when crosslinked with ga. in any case, both c3 and c4 showed low tensile strength under both cross-linking treatments. savic and savic (2016) indicated that a greater degree of native and intact fibrillar structures produce higher strength and elasticity in collagen casings. the c3 and c4 collagen dispersions may have undergone further degradation during processing, resulting in their lower tensile properties. for instance, excessive mechanical or alkaline modification, during corium separation, can result in greater degradation of the native collagen fibers (savic and savic, 2016). the influence of protein concentration on the mechanical properties was also investigated (table 3). when comparing adjusted mechanical properties (i.e., per % protein), again c3 and c4 were found to result in low tensile strength values when cross-linked with smoke condensate. when cross-linked with ga, c3 showed the lowest value (0.077 mpa/% protein). when looking at % elongation, the samples that showed the lowest values when tested as received (i.e., their original protein content) also showed the lowest values when compared on a % protein basis (sample c5 treated with smoke condensate, and sample c3 treated with ga; tables 2 and 3). these results indicate that the mechanical properties of films may be more dependent on the quality of protein, rather than on protein content. another factor that may have led to differences in mechanical properties was the concentration of cellulose fibers within the partially dried films tested here. mathew et al. (2012) observed that interactions between cellulose nanofibers and collagen significantly increased the strength and break strain of fully dry collagen films. note: see below additional discussion and micrographs concerning cellulose fibers. it is important to note that cross-linking has a dramatic effect on the mechanical properties of partially dehydrated films (i.e., with salt solution). cross-linking with smoke condensate or ga appeared to increase the tensile strength of collagen films by approximately 100% and 200% (respectively) over the values obtained after salt solution partial drying (barbut, unpublished data), which were in the range of 0.2 to 0.3 mpa. in addition, the distance to break was reduced by 30% and 60%, when cross-linked with smoke condensate and ga, respectively. as suggested by others (avery and bailey, 2008; covington, 1997; paul and bailey, 2003) cross-links increase the mechanical strength by forming bonds among the collagen molecules/fibers and preventing them from sliding past each other, creating a stiff but brittle network within the films. overall, there were some differences in the mechanical properties between films crosslinked with smoke condensate and ga. in general, films cross-linked with ga showed greater tensile strength than those cross-linked with liquid smoke (table 2). smoke condensates typically contain over 300 compounds (toledo, 2007) therefore, the differences observed in tensile strength can likely be attributed to the purity of the crosslinking agent. the smoke condensate was analyzed to determine the type and concentration of its aldehyde compounds (table 4). it was observed that the aldehyde compounds were all mono-functional aldehydes. the effect of various monoand dialdehydes (formaldehyde, gluteraldehyde, crotanaldehyde and glyoxal) on the thermal and conformational stability of type i collagen has been investigated by fathima et al. (2004). their work suggested that the aldehydes differ in their ability to improve the thermal stability of collagen. it was observed that the increase in thermal stability followed the order: formaldehyde > gluteraldehyde > glyoxal > crotanaldehyde, thus demonstrating that di-aldehydes are not necessarily more functional than monoaldehydes. ital. j. food sci., vol. 31, 2019 653 table 2. mechanical properties of cross-linked films: c1 (collagen 1), c2 (collagen 2), c3 (collagen 3), c4 (collagen 4) and c5 (collagen 5). collagen cross-linker1 tensile strength 2 mpa percent elongation2 % distance at break3 mm work to break3 nmm thickness mm c1 sc 0.67±0.04a 24.80±1.23ab 3.85±0.22a 2.75±0.48a 0.35±0.01ab c2 sc 0.53±0.02ab 26.32±1.18a 3.39±0.18a 1.52±0.14a 0.30±0.01b c3 sc 0.38±0.05b 22.41±0.78abc 3.21±0.30a 1.24±0.34a 0.34±0.01ab c4 sc 0.32±0.07b 21.37±0.99bc 3.27±0.35a 1.23±0.31a 0.36±0.02a c5 sc 0.39±0.16b 18.81±2.08c 2.59±0.04a 0.85±0.10a 0.38±0.03a c1 ga 0.91±0.17d 26.26±4.65d 2.77±0.52d 1.79±0.57d 0.36±0.03d c2 ga 0.66±0.02e 20.38±1.43d 2.34±0.44d 1.06±0.37d 0.45±0.01d c3 ga 0.41±0.13f 18.95±2.56d 2.66±0.39d 1.35±0.27d 0.38±0.05d c4 ga 0.61±0.20ef 24.18±3.72d 2.74±0.29d 1.52±0.11d 0.38±0.04d c5 ga 0.60±0.18ef 22.04±2.60d 2.87±0.60d 1.91±0.76d 0.39±0.05d 1smoke condensate (sc), glutaraldehyde (ga). 2tensile test. 3puncture test. 4means in columns with same letter are not significantly different p > 0.05: letters a-crefer to smoke condensate treated films; d-eglutaraldehyde treated films. table 3. protein adjusted mechanical properties of cross-linked films: c1 (collagen 1), c2 (collagen 2), c3 (collagen 3), c4 (collagen 4) and c5 (collagen 5). collagen cross-linker1 tensile strength 2 mpa / %protein percent elongation2 % / %protein distance at break3 mm / %protein work to break3 n*mm / %protein c1 sc 0.092±0.01ab 3.36±0.17bc 0.38±0.07a 0.24±0.08a c2 sc 0.101±0.00a 4.98±0.23a 0.45±0.08a 0.20±0.07a c3 sc 0.062±0.01b 3.67±0.13b 0.44±0.06a 0.22±0.04a c4 sc 0.056±0.01b 3.68±0.17b 0.47±0.05a 0.26±0.02a c5 sc 0.058±0.02b 2.77±0.31c 0.42±0.09a 0.28±0.11a c1 ga 0.140±0.03d 4.05±0.72d 0.43±0.08d 0.28±0.09d c2 ga 0.131±0.00d 4.02±0.28d 0.46±0.09d 0.21±0.07d c3 ga 0.077±0.03e 3.59±0.48d 0.50±0.07d 0.26±0.05d c4 ga 0.120±0.04de 4.77±0.73d 0.54±0.06d 0.30±0.02d c5 ga 0.118±0.04de 4.34±0.51d 0.57±0.12d 0.38±0.15d 1smoke condensate (sc), glutaraldehyde (ga). 2tensile test. 3puncture test. 4means in columns with same letter are not significantly different p > 0.05: letters a-crefer to smoke condensate treated films; d-eglutaraldehyde treated films. ital. j. food sci., vol. 31, 2019 654 it was suggested that the number of cross-links formed influence the stability of the crosslinks, therefore the reactivity of the aldehyde is of greater importance. the reactivity of mono and di-aldehydes is thought to stem from sterochemical factors and ph (increase uptake at higher ph) as was indicated by bowes and cater (1968). although according to our calculation the smoke condensate had a higher percentage of different aldehydes (0.95%; table 4), compared to the 0.5% glutaraldehyde used in the experiment, it appears that glutaraldehyde is a more reactive cross-linker that overall provided higher tensile strength values to the films (tables 2 and 3). it should be mentioned that the ph of the cross-linking solution may have also affected the mechanical properties of the films. morgan et al. (1998) suggested that neutralizing the acidic collagen dispersion would result in water loss. similar to dehydrating films with a salt brine, the removal of water will result in shorter distances between the collagen molecules and hence improve the stability of the collagen structure. the smoke condensate was used as is (i.e., not buffered; at ph 3.5) while the ga was buffered (ph 7.4). thus the buffered solution could have increased the neutralization of the collagen gels, and in turn, improving stabilization. table 4. tentative identification of aldehyde compounds found in the commercial smoke condensate, the relative concentration (%) and approximate concentration in the cross-linking bath. tentative id relative % cross-linking bath1 % 2,4-diethoxybenzaldehyde 0.47 0.07 4-methyl 2,5-dimethoxybenzaldehyde 1.24 0.19 benzaldehyde, 4-hydroxy-3,5-dimethoxy 3.22 0.48 2-oxa-6-azatricyclo[3.3.1.1(3,7)]decane-6-carboxaldehyde 0.47 0.07 3,5-dimethoxy-4-hydroxycinnamaldehyde 0.92 0.14 (aldehyde in smoke condensate) (6.32 0.95) 1 the original smoke condensate received was diluted to 15 vol.% using deionized water. 3.2.2 electron microscope imaging the collagen fibers appear to have varying degrees of swelling or hydration (fig. 3). overall in natural tendon tissue, collagen fibrils are long, slender and show cylindrical structures, but can differ in length, diameter, uniformity, and telopeptide size, as a result of collagen type and interactions (wess, 2008; cameron et al., 2002). the alignment of collagen molecules in a staggered array conformation develops areas of overlap and gaps. the overlap and gap regions of the parallel arrays give collagen fibrils their distinctive striated pattern with a periodicity of 640-700 å (wess, 2008). the control collagen sample (obtained by us from beef hide) was imaged to compare the likeness of the banded fibers (fig. 4a). it appeared that, at this magnification, the banded structures were indicative of collagen fibers. in addition, the bulk material, presented in this micrograph, shares similar characteristics to the collagen tissue studies by meyer et al. (2005). although some collagen fibers display a banding pattern or cross-striations (fig. 3), the majority of the fibers presented in the current work appear to be swollen. since commercial collagen dispersions are extracted from hides that have varying degree of natural cross-linking, the degree of cross-links varies depending on animal age, growing conditions, overall muscle activity, etc. overall, there is an increase in natural cross-links, ital. j. food sci., vol. 31, 2019 655 with increasing age and therefore the solubility also decreases (meyer et al., 2005). in addition, the connective tissue extraction process would be expected to result in varying degree of fiber degradation. figure 3. transmission electron micrographs of collagen film: collagen c1 (a), collagen c2 (b), collagen c3 (c), collagen c4 (d), collagen c5 (e). black bar represents 1 !m. the final collagen structure supports the mechanical properties of the film because a more intact fibrillar structure produces higher strength and elasticity in collagen casings (savic and savic, 2016). the c4 appeared to have the most swollen or hydrated fibers, with little visible banded fibrils remaining (fig. 3d). since c4 generally revealed lower tensile strength and percent elongation (table 2), one may presume that greater swelling and hydration during collagen extraction, with hcl, and acetic acid resulted in lower mechanical properties. however, definite conclusions cannot be drawn as the orientation and plane at which the fibrils are viewed is not known with certainty. ital. j. food sci., vol. 31, 2019 656 figure 4. transmission electron micrographs of a collagen control obtained from beef hide (a), a cellulose control (b) and a cellulose fiber embedded in a collagen film network (c). black bar represents 1 !m in micrograph (a) and 5 !m in micrographs (b) and (c). another interesting observation was that there appears to be differences in the linearity of fibers. the fibers in c1 appear to be arranged in a less linear way compared to the other treatments. c1 also appears to have a higher concentration of more condensed fibers, which may have hindered alignment of the fibers during film preparation by rolling. it should be pointed out that during the formation of commercial co-extruded casings, a counter rotating extrusion head applies shear forces, which orientates and elongates the collagen fibers (hoogenkamp et al., 2015). orienting the fibers improves the mechanical properties of casing by reducing splitting (bontjer et al., 2011). overall, it would be interesting to further investigate this topic to further assist the industry. microstructure imaging the films did not suggest significant differences in the concentration of cellulose in the five dispersions. as previously mentioned, cellulose fibers are commonly added to modify the mechanical properties and porosity of collagen casings (savic and savic, 2016; barbut, 2010). to verify that the larger fiber structures were derived from cellulose, a cellulose control (fig. 4b) was imaged, demonstrating the difference in structure and magnitude. an example of a cellulose fiber embedded in the collagen network of one of the dispersions can be seen in fig. 4c. overall, cellulose fibers can be distinguished based on their larger size, lack of striation and electron density. 3.2.3 optical properties of films the degree of light transmission through casings affects consumer’s ability to evaluate the meat product inside (savic and savic, 2016). light transmission measurements were ital. j. food sci., vol. 31, 2019 657 taken to evaluate the transparency of cross-linked collagen films (fig. 5). among the collagen films, c5 was the least transparent and c2 was the most. simple visual inspection also confirmed that c5 was more opaque than the other samples. the micrograph of the c5 films also showed high concentration of individual fibrils and sub-fibrils, which could result in higher light scattering. light transmission is also affected by the composition of each casing (savic and savic, 2016). c2 was on the lower end of protein content (3.6%), which may have contributed to it being the most transparent. figure 5. the light transmission (380 to 780 nm) through cross-linked collagen films; c1 (collagen 1), c2 (collagen 2), c3 (collagen 3), c4 (collagen 4) and c5 (collagen 5). a films cross-linked with liquid smoke; b films cross-linked with glutaraldehyde. films treated with liquid smoke tended to have lower transmission than films treated with glutaraldehyde (fig. 5). this observation is likely a result of some dark smoke components that became deposited on the films. there was no difference in the ranking of transparency between the liquid smoke and glutaraldehyde treated films. this observation is somewhat inconsistent with tanaka et al. (2011), who observed that increased crosslinking results in lower transmission of wet collagen films cross-linked with 1-ethyl-3-(3dimethylaminopropyl) carbodimide and n-hydroxysuccimide. 0 20 40 60 80 100 380 420 460 500 540 580 620 660 700 740 780 li gh t t ra ns m is si on (% ) wavelength (nm) c1 c2 c3 c4 c5 a 0 20 40 60 80 100 380 420 460 500 540 580 620 660 700 740 780 li gh t t ra ns m is si on (% ) wavelength (nm) c1 c2 c3 c4 c5 b ital. j. food sci., vol. 31, 2019 658 3.2.4 mechanical properties of thermally treated films table 5 shows the puncture distance and work to puncture as an effect of heating temperature. in general, as heating increases, puncture distance significantly decreased and work to puncture showed a trend of decreasing. this is in line with the expectation that collagen tissue, in muscle foods, softens as temperature goes above the collagen denaturation temperature of 60˚c (miles et al., 2005). overall, there were no measurements recorded from testing the films cross-linked with smoke condensate because the films became too fragile to be evaluated by the texture analyzer. as previously suggested, the collagen in films cross-linked with smoke condensate may have remained more swollen and had higher water content. the higher water content may have resulted in lower thermal stability of these films. miles et al. (2005) suggested that the increased thermal stability is a result of the water content of the fibers. they observed that crosslinking reduced the axial separation between molecules, thus reducing the amount of entrapped water. avery and bailey (2008) observed that cross-linking agents, having different cross-link length, had no effect on denaturation temperature when hydration was done in the same way. table 5. mechanical properties (puncture distance and work to puncture) of collagen films cross-linked with glutaraldehyde and thermally treated. films produced with collagen 2. thermal treatment puncture distance mm work of puncture mj no treatment 3.781±0.75a 2.576±0.82a 40°c 3.180±0.17ab 2.053±0.16a 50°c 3.151±0.57ab 2.584±0.45a 60°c 2.838±0.25ab 1.844±0.63a 70°c 2.423±0.13b 1.265±0.30a 80°c 2.578±0.23b 1.518±0.40a 1means in columns with a similar letter are not significantly different (p > 0.05). in the present study, collagen may have not undergone full thermal denaturation by 80˚c when films were cross-linked with ga (avery and bailey, 2008). cross-linking with ga has been observed to increase the denaturation temperature of collagen to approximately 100˚c (miles et al., 2005). our results may suggest that the thermal stability of cross-linked casings depend more on moisture content than cross-links. 4. conclusions it was observed that manipulating the smoke condensate contact time (10, 20, 40, 80 s) did not result in significant differences (p > 0.05) in the mechanical properties of collagen films. similarly, the ga contact time (5, 10, 20 min) did not result in significant differences in mechanical properties, when averaged across the ga concentrations employed. although contact time had little effect, increasing the concentration of cross-linking with ga (0.1, 0.5 1.0%) significantly decreased the % elongation (22 to 17%) and distance to break (3.0 to 1.6 mm). this suggests that increasing the ga concentration increases crosslinking and brittleness of the film. ital. j. food sci., vol. 31, 2019 659 there were some significant differences between the mechanical properties of the different cross-linked films. it was suggested that fiber structure, cellulose content and ph of the cross-linking agent could influence the mechanical properties. therefore manufacturers of regenerated collagen casings and producers of co-extruded sausages should consider these differences when selecting a source of collagen for their casings. in addition, meat processors should monitor the concentration and ph of their crosslinking solution as solutions from a continuous liquid smoke drip/spray application are known to become diluted over time. as discussed in the paper, these changes may significantly affect the mechanical properties of the collagen films. overall, proper understanding is required when selecting the raw collagen dispersion and the conditions for the co-extrusion process. furthermore, if products are going to be cooked, the casings may require further heating and dehydration, which will also change their mechanical properties. acknowledgements the authors would like to thank the ontario ministry of agriculture, food and rural affairs for financial support. references astm. 2010. standard test method for tensile properties of this plastic sheeting. method d882-10, philadelphia, pa. avery n. and bailey a. 2008. restraining cross-links responsible for the mechanical properties of collagen fibers: natural and artificial. in “collagen structure and mechanics”, p. fratzl (ed.), p. 81. springer science, new york, ny. barbut, s. 2015. casings. in: “the science of poultry and meat processing”, p.13-54 and 62. isbn-978-088955-625-6. free download: www.poultryandmeatprocessing.com. barbut s. 2010. microstructure of natural, extruded and co-extruded collagen casings before and after heating. ital. j. food sci. 22:126. bontjer m.b.h., kuijpers m.w.j.t. and van den berg k.w. 2011. method for preparing food products by co-extrusion, in 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(eds.), p. 275. american chemical society, washington, usa. wess t.j. 2008. collagen fibrillar structure and hierarchies. in: “collagen structure and mechanics”. p. fratzl (ed.), p.55. springer science, new york, ny. wolf k.l., sobral, p.j.a. and telis v.r.n. 2006. characterization of collagen fibers for biodegradable films production. iufost 13:801. paper received june 3, 2018 accepted february 13, 2019 ijfs#1727_bozza ital. j. food sci., vol. 32, 2020 275 paper isolation of baby lima bean (phaseolus lunatus) proteins fractions and evaluation of their antioxidant activity m. zambrano1, g. vásquez2, d. morales2, r. vilcacundo2 and w. carrillo*3 1espol polytechnic university. faculty of mechanical engineering and production sciences, campus gustavo galindo, km 30.5 vía perimetral, guayaquil, guayas, p.o. box 09-01-5863, ecuador 2laboratory of functional foods, faculty of foods sciences and engineering in food and biotechnology, technical university of ambato, av. los chasquis y rio payamino, campus huachi, ambato, tungurahua 1801334, ecuador 3department of research, faculty of health sciences, technical university of babahoyo, av. universitaria km 21/2 av. montalvo. babahoyo, los rios, 120301, ecuador *corresponding author: wi.carrillo@uta.edu.ec abstract the aim of this work was to obtain fractions of baby lima bean protein concentrate (lbpc) from (phaseolus lunatus) and to evaluate their antioxidant activity. lbpc was prepared by alkaline extraction and isoelectric precipitation at ph 4.5. lbpc was subject to gastrointestinal digestion simulation. lbpc was fractionated using a deae affi-gel blue gel column. lbpc presented a protein content of 77.20% with a protein solubility capacity ranging between 37.34% to 99.98%. the antioxidant activity was evaluated using the frap, dpph and orac methods. lbpc cationic fractions presented a high antioxidant activity when using the orac method with values ranging between 0.47 to 3.37 µmol te/µmol of sample. keywords: baby lima bean, phaseolus lunatus, simulated gastrointestinal digestion, protein concentrate, fractions, antioxidant activity ital. j. food sci., vol. 32, 2020 276 1. introduction soybean protein isolate (spi) are produced with defatted soybean flour. spi is used in the food industry as a food ingredient for different purposes due to its functional properties such as a high solubility, a high foaming capacity and a high protein content. in ecuador, soybeans and spi imports for food purposes are high. the government has implemented measures to promote the search for new native matrices that allow obtaining protein concentrates and isolates. baby lima bean (phaseolus lunatus) is a legume belonging to the fabaceae family. this crop has been described for the exceptional potential in adaptation to lowland tropical conditions and potentially important as a food legume (drago et al., 2016; wu et al., 2016). animal proteins, such as meat, milk, fish and eggs, are generally expensive and relatively difficult to acquire, which has led to a worldwide increase in research into vegetable protein sources. for their high protein content, legumes have formed an important part of this search being a cheaper, an alternative protein source and an important crop nitrogen fixative (sathe and salunkhe 1981). legumes are a source of quality protein in developing countries of south america such as ecuador, colombia, peru and venezuela. phaseolus lunatus seeds have a high protein content with 26% of protein content (betancur-arcona et al., 2009). the protein isolate obtained from phaseolus lunatus presents a protein content of 71.13%-73.75%. this protein content in lima bean makes this plant to be an excellent protein source for food industry applications (chel-guerrero et al., 2002). phaseolus lunatus can be used to manufacture concentrate and isolate protein with excellent functionals properties. the easiest way to obtain proteins from animal and plant sources is through alkaline extraction followed by isoelectric precipitation. for the characterization of the proteins present in protein isolates, different analytical techniques such as chromatography methods have been used. many ion exchange columns (iec) have been used to fraction different protein concentrates and isolate them to evaluate their biological activities and their possible use as functional ingredients in the food industry (carrillo et al., 2016a; 2016b; 2018; de castro and cason 2017; gaspard, 2017; tabtabaei et al., 2017). phaseolus angularis (red bean) globulins have been fractionated using an iec of deaesepharose. native and heated fractions were characterized using the electrophoresis technique. phaseolus vulgaris, phaseolus lunatus, canavalia ensiformis and mucuna pruriens legume plants belonging to the fabaceae family have been hydrolyzed and fractionated with different methods such as ultrafiltration with membrane. total hydrolysates and fractions have been described with different biological activities such as antioxidant, antibacterial, antihypertensive and antihyperglycemic activities using in vivo and in vitro models for their evaluation (cárdenas et al., 2018; cunsolo et al., 2007; luna-vital et al., 2015; magaña et al., 2015; mamilla and mishra 2017; yoshida 1989). different antioxidant activity methods are used to evaluate the potential of different compounds to determine the quality of foods of animal and vegetal sources. these antioxidant activity methods allow to enhance the nutritional and biological value of these components. these methods allow a biological characterization of bioactive compounds as polyphenols, proteins and peptides. among the most used methods to evaluate antioxidant activity are the orac and frap methods. there is a high interest in finding new natural antioxidant compounds. vegetal proteins can be a source of compounds with antioxidant abilities such as polyphenols, lipids, and proteins. proteins can be hydrolyzed with different enzymes, being possible to simulate human condition of gastric digestion and duodenal digestion (hernández-ledesma et al., 2004; orsini delgado et al., 2011; vilcacundo et al., 2018a;). ital. j. food sci., vol. 32, 2020 277 protein concentrates and protein isolates with antioxidant activities have been reported by different authors (je et al., 2005; olagunju et al., 2018; orsini-delgado et al., 2016; pownall et al., 2010). for example, quinoa protein concentrate (qpc) chenopodium quinoa and amaranth protein concentrate (apc) amaranthus caudatus have been described with antioxidant activity and the capacity to inhibit lipid peroxidation in the zebrafish model (vilcacundo et al., 2017; 2018b). the aim of this study was to obtain baby lima bean (phaseolus lunatus), protein concentrate, and determine its in vitro digestibility. protein concentrate was fractionated using an exchange column, and the antioxidant activity was evaluated using the orac, frap and dpph methods. 2. materials and methods 2.1. reagents porcine gastric mucosa pepsin (4500 u/mg), porcine pancreas pancreatin (10000 u/mg), 2,2’-azobis (methylpropionamide)-dihidrochloride (aaph), 2, 4, 6-tripyridyl-s-triazine (tptz), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox), 2-diphenyl-1picrylhydrazyl (dpph), fluorescein disodium (fl) and trifluoracetic acid (tfa) were obtained from sigma-aldrich (st. louis, mo, usa). the other reagents used in this study were of analytical grade. 2.2. material vegetal baby lima bean (phaseolus lunatus) samples were obtained from a native crop of the recinto la sequita of manabí region (manabí-ecuador). the seeds of baby lima bean were collected in july,2016. the samples are registered in the espol sample collection. the humidity of beans was determined with a value <10%. then, the beans were ground using a perten laboratory mill 3100 and sifted in a sieve advantech durataptm dt168 with mesh # 70 (0.210mm). the obtained flour bean was vacuum packed and stored at laboratory temperature. 2.3. proximate analysis proximal flour bean analysis and lbpc was performed, according to the official methods of the official association of analytical chemists (aoac 1997). moisture content was determined by the aoac 925.10 method (aoac 1997), protein by the aoac 2001.11 kjeldhal method (factor 6.25) (aoac 2001), fat by the aoac 922.06 method (aoac 1997), and ash by the aoac 923.03 method(aoac 1997). 2.4. preparation of lbpc lbpc was prepared according to poveda et al. (2016). the bean flour was defatted with hexane (1:10 w/v) for 24 h. then, 100 g of defatted flour was resuspended in milli-q water (1:10, w:v) at ph 9.5 for 15, 30, 45 and 60 min at 40°c. then, the solution obtained was centrifuged at 5000 x g for 30 min at 25°c. the supernatant solution was adjusted to ph 4.5 using 1 n hcl and centrifuged for 20 min at 8000 x g at 25°c. the precipitated obtained was stored and then adjusted at ph 7.0 with 0.1 m naoh. then, the neutralized precipitated was lyophilized and frozen at -80°c. ital. j. food sci., vol. 32, 2020 278 2.5. protein solubility capacity lbpc were dissolved in distilled water at a concentration of 0.2% (w:v) and the ph of the suspension was adjusted to ph 2.0 – ph 10.0 using solutions of 0.001n hcl and 2n naoh. the suspensions were shaken for 1 h and centrifuged at 10000 rpm for 10 min in a sorvall legend micro 17 centrifuge (thermofhiser scientific, germany). the content of protein in the supernatant was analyzed with the bca protein method. the content of soluble protein was expressed as the percentage of the content of protein present in the sample (pazmiño et al., 2018). 2.6. fractionation of lbpc with an anion-exchange column 20 mg/ml of lbpc was centrifuged at 10000 x g for 20 min and 1 ml of supernatant was injected in the column of anionic exchange chromatography deae affi-gel® blue gel (bio-rad, hercules, ca, usa). then, 10 ml of buffer tris-hcl 50 mm at ph 7.0 was loaded in the column to elute proteins not adhered in the column as proteins with charge positive. then, 10 fractions of 1 ml each, were collected, then 10 ml of nacl 500 mm were loaded in the column to elute the retained proteins (negative proteins) (qi et al., 2001). 10 fractions of 1 ml each, were collected to be analyzed using the sds-page electrophoresis method. protein contents of fractions were determined using the bca method. 2.7. gastric and duodenal digestion of lbpc lbpc (10 mg/ml) was subject to simulated gastric digestion using pepsin enzyme (2000 u/ml) at ph 3.0 for 2 h at 37°c with agitation. the pepsin enzyme was inactivated by heating at 80°c for 10 min. one milliliter of gastric digestion was mixed with one milliliter of pancreatin enzymes (100 u/ml) at ph 7.0 for 2 h at 37°c. the enzymatic reaction was stopped by heating at 90°c for 10 min (piñuel et al., 2019). 2.8. % degree of hydrolysis by the orthophthalaldehyde (opa) assay the hydrolysis degree (%dh) of gastric and duodenal digest from lbpc was determined using the opa method described by piñuel et al. (2019). opa solution: 25 ml of sodium tetraborate (100 mmol/l) was mixed with 2.5 ml of 20% (w/v) sodium dodecyl sulfate, 40 mg of opa was dissolved in 1.0 ml of methanol and 100 µl of 2-mercaptoethanol. the final volume was of 50 ml. derivatization opa: 10 µl of the sample was mixed with 3.4 ml of the opa solution and the mixture was stored at 25°c for 2 min. the absorbance was measured at 340 nm. %dh was determinate with the equation: %dh = abs × 1,934 × d/c where abs is the sample absorbance, d is the factor of dilution and c is the concentration of protein in lbpc (mg/ml). 2.9. sds-page electrophoresis analysis of lbpc and their fractions lbpc and lbpc fractions were analyzed using the sds-page electrophoresis method. the samples were analyzed using 15% polyacrylamide gels in a mini-protean ital. j. food sci., vol. 32, 2020 279 electrophoresis system (bio-rad, hercules, ca, usa). the standard protein (precision plus proteintm unstained standard, bio-rad) with a range of 10 kda-250 kda was used to determine the molecular weight of proteins present in the samples (quinteros et al., 2016; toapanta et al., 2016). 2.10. rp-uhplc analysis of lbpc fractions lpbc and lbpc fractions were analyzed by rp-uhplc technique using the agilent 1200 infinity series uhplc (agilent technologies, waldbron, germany). the signal of the chromatograms was registered at the wavelength of 280 nm. the separation of the samples was made with the help of a zorbax ec c18 agilent poroshell 120 (4.6 mm x 50 mm x 2.7 µm) analytical column. the solvents used were solvent a [milli-q water + tfa 0.37%] and solvent b [acetonitrile + tfa, 0.270%]. samples were eluted at 1.0 ml/min with a lineal gradient from 0% to 70% of solvent b for 15 min. before analysis all samples were filtered with a membrane filter of 0.45 µm and then were centrifuged for 2 min at 12000 rpm at 4°c. samples were injected for three times (lara et al., 2017). 2.11. antioxidant activity of lbpc and their fractions the colorimetric assays ferric ion reducing antioxidant power (frap) and 2, 2-diphenyl-1picrylhydrazyl (dpph) was performed following the procedure described by benzie and strain (1996) and piñuel et al. (2019) respectively. the antioxidant capacity of lbpc and lbpc fractions was evaluated using an frap assay. the solutions used were 300 mm acetate buffer at ph 3.6, 10 mm tptz solution in 40 mm hcl, and 20 mm fecl3 6h2o solution. a new working solution was prepared by a mixture of 25 ml acetate buffer with a 2.5 ml tptz solution, and 2.5 ml fecl3 6h2o solution and then incubated at 37°c before use. lbpc and fractions (150 ml) were mixed with 2850 ml of the frap solution for 30 min in darkness. readings of the colored product were measured at a wavelength at 593 nm. the standard trolox linear curve was used as control (20 to 1200 mm). all results were expressed as mm of trolox equivalents (te) per g sample. all experiments were made in triplicate. lbpc and fractions were used to evaluate their antioxidant activity using the dpph method. the ability to capture free radicals by antioxidants was analyzed using the radical species dpph, measuring the decrease of the absorbance at 517 nm spectrophotometrically (spectrum sp-2100uv/sp spectrophotometer, china). each assay was made five times, the value of antioxidant activity being expressed as mg of te/100 g sample. the oxygen radical absorbance capacity fluorescein (orac-fl) assay was made according to moore et al (2005). a synergy 2 multi-mode microplate reader (biotek, winooski, vt, usa) was used. samples and trolox standards were prepared with 50% acetone. all other reagents were prepared in a 75 mmol/l phosphate buffer (ph 7.4). each well in a 96-well plate contained 30 μl of 20 μmol/mg sample or 50% acetone for blank and 225 μl fluorescein (81.63 nmol/l). the plate with a cover was incubated for 20 min in 37°c, and then 25 μl aaph (0.36 mol/l) were added to each well to start reaction, resulting in a final total volume of 280 μl. the fluorescence was recorded every minute for 2 h at 37°c, where excitation and emission of wavelengths were 485 and 528 nm. trolox was used as standard (0-200 µmol) with a standard curve (y=0.034x+0.6068), r2=0.999. standards and samples were performed in triplicate. results were expressed as µmol te/µmol of sample. ital. j. food sci., vol. 32, 2020 280 2.12. statistical analysis results are presented as means±standard deviation from three replicates of each experiment. differences between mean values were determined by the analysis of variance (anova). the post hoc analysis was made by the tukey and dunnet test. all tests were considered significant at p <0.05 using the software graphpad prism 4. 3. results and discussion 3.1. analysis proximal of flour bean and lbpc the table 1 shows the proximate analysis of flour bean and lbpc. flour bean present moisture with a value of 8.95% and lbpc present a value of 9.37%. flour bean present a protein content of 20.48% and lbpc present a protein content of 77.20%. lbpc present an increase of protein content. the carbohydrates content of flour bean was 65.42%. lbpc present a decrease with a value of 10.63%. chel-guerrero et al. (2002); gallegostintoré, et al. (2004); torruco-uco et al. (2009); betancur-ancona et al. (2009); guzmán-méndez et al. (2014) and drago et al. (2016) report protein contents from lima bean with values of 71.13%, 71.13%, 71.80%, 69.90%, 71.88% y 74.06%. the protein content in this study is higher compared to the values reported. these differences are may be due to the varieties used in each study. table 1. proximate analysis of flour bean and lbpc. analysis flour bean (%) lbpc (%) moisture 8.95%±0.14 9.37%±0.26 protein 20.48%±1.17 77.20%±0.02 fat 1.57%±0.10 0.56%±0.03 ashes 3.58%±1.23 2.24%±0.30 carbohydrates 65.42±2.25 10.63±3.10 results are expressed as mean±standard deviation (n=3) 3.2. protein solubility capacity protein solubility is considered one of the most important functional properties in proteins. when a food protein has high solubility, this ingredient can be used for many industrial proposes, a food protein with low solubility decreases the industrial possibilities when used as an ingredient. solubility capacity can affect other protein functional properties. the flour bean solubility curve is shown in fig. 1a. proteins are more soluble in acidic regions (ph 2.0) and in alkaline regions (ph 12) with a value of 100%. between ph 3.0 to ph 5.0 the solubility is reduced. between ph 6.0 to ph 10.0 the solubility of proteins is relatively good. lbpc proteins solubility profile presents the typical u-shape of the legume extracts, with a minimum solubility at the isoelectric point and a greater solubility at low acidic ph and high alkaline ph (pazmiño et al., 2018). at ph 2.0 and ph 12.0, ital. j. food sci., vol. 32, 2020 281 lbpc present the highest percentage of solubility with values of 99.98±0.04% and 98.82±0.05%. at ph 6.0, these proteins present solubility values of 45.71±0.02% and at ph 8.0 these proteins present solubility values 57.95%. the lowest solubility was reached at ph 4.0 with a value of 37.34% (fig. 1b). seidu et al. (2015) reported protein solubility of protein from skin lima bean with percentage between 25% to 90%. betancur-ancona et al., 2009 reported protein solubility capacity of protein isolate obtained of lima bean with values of 15% to 70%. chel-guerrero et al., 2002 reported protein solubility of protein isolate from lima bean with values between 5% to 70%. figure 1. percentage of solubility of flour and lbpc at different phs. a) flour bean b) lbpc. 3.3. lbpc protein profile and their digestibility lbpc was obtained by alkaline extraction at ph 9.5 for 15-30-45 and 60 min of agitation at 40°c, followed by isoelectric precipitation at ph 4.5 and then analyzed by sds-page electrophoresis. fig. 2a shows the lbpc proteins profile with bands between 10 kda to 100 kda. the bands with the higher intensity present molecular weights of 22 kda, 25 kda and 30 kda. the bands with the lower intensity were the bands with low molecular weights with 10 kda, 15 kda, 20 kda, 55 kda and 60 kda. the protein profile is composed as follows: one band of 100 kda, one band of 70 kda, doublet with 55 kda and 60 kda, triplet with 40 kda, 35 kda and 30 kda, triplet of 20 kda, 22 kda and 25 kda. sparvoli et al. (1996) reported a similar protein profile identified as phaseolin from lima bean (phaseolus lunatus) with molecular weights of one band (70 kda), doublet (54 kda 58 kda), triplet (32 kda, 35 kda and 38.5 kda) and doublet (21 kda 25 kda). these samples were analyzed for reaction of western blot against phaseolin (phaseolus vulgaris). the lbpc protein profile was the same at the different times alkaline extraction was assayed (15-30-45-60 min). flour of phaseolus lunatus presents a 20.48% of protein content. lbpc present a higher protein content with a value of 77.20% (table 1). ital. j. food sci., vol. 32, 2020 282 figure 2. sds-page electrophoresis analysis of lbpc and gastric and duodenal digest of lbpc. a) profile protein of lbpc obtained at different times at 40°c and ph 4.5. mw (molecular weight). b) lbpc control (without hydrolysis). lane 1: gastric digest of lbpc, lane 2: duodenal digest of lbpc. in common bean (phaseolus vulgaris) phaseolin protein content represents around 50% of the total seed protein (boschin et al., 2014). it is made up of a small number of polypeptides and presents an extensive variation in the electrophoretic pattern, mostly observed among wild-growing beans. comparison of electrophoretic patterns of total seed proteins of cultivated species belonging to the genus phaseolus showed that the phaseolus lunatus pattern is quite different from the others, lacking major polypeptides with molecular mass similar to those of phaseolin (lioi, 1987). different authors have reported 7s globulin (vicilin) from phaseolus vulgaris. with molecular weights between 40 kda to 54 kda. montoya et al. (2008; 2010) reported the protein profile of a different variety of phaseolus. they reported a high content of globulins and reported 2 to 6 bands between 40 kda to 54 kda. they identified these bands as vicilin (7s globulin). carrasco-castilla et al. (2012) reported phaseolus vulgaris protein profiles. they identified ten protein bands with molecular weights (mw) ranging from 15 to 200 kda in the protein isolate. the 41 kda and 46 kda bands, correspond to the phaseolin subunits and the most abundant proteins. the 15 kda, 18 kda, 25 kda and 32 kda, bands correspond to proteins belonging to the lectin-family. garcía-mora et al. (2015) reported a protein profile from phaseolus vulgaris. var. pinto, with bands between 10 kda to 97 kda. bands with molecular weights of 25 kda, 45 kda and 50 kda were identified as phaseolin. phytohemagglutinins (32 kda), α-amylase inhibitor (18 kda) and α-amylase β subunit (15 kda) were identified in the pinto bean protein concentrate. phaseolin band in phaseolus vulgaris present high intensity and represent about 50% of total protein content. phaseolus lunatus presents an absence of this high intensity band. phaseolin protein of phaseolus lunatus corresponds to another band with a different molecular weight. lbpc and their fractions were subject to an in vitro gastrointestinal digestion simulation using pepsin enzyme for the gastric phase and a pepsin/pancreatin enzymes mix for the duodenal phase. in the gastric phase, bands with molecular weights of 20 kda, 22 kda and 25 kda present resistance to hydrolysis with pepsin. we found bands with molecular 1 2 mw 250 150 100 75 50 37 25 20 15 10 5 2 kda lbpc mw 250 150 100 75 50 37 25 20 15 10 5 kda a b 15 min 30 min 45 min 60 min time of extraction control digests ital. j. food sci., vol. 32, 2020 283 weights of 50 kda and under 10 kda. phaseolin protein presents resistance to hydrolysis with pepsin/pancreatin (fig. 2b). in the duodenal phase, the protein profile of hydrolysate present bands with molecular weights between 20 kda to 50 kda. bands with low molecular weight were partially hydrolyzed with pepsin/pancreatin. bands of 20 kda, 22 kda and 25 kda present resistance to duodenal hydrolysis. phaseolus lunatus phaseolin protein presents resistance to hydrolysis with pepsin and pepsin/pancreatin. phaseolin from different phaseolus species has been reported to be resistant to enzymatic hydrolysis with pepsin and pepsin/pancreatin. lbpc gastric digest present 9.3% dh and lbpc duodenal digest present 16.2% dh. these results are in accordance with the resistance at the hydrolysis observed in the electrophoresis analysis. betancur-ancona et al. (2009) reported protein isolate (phaseolus lunatus) hydrolysis using an enzymatic (trypsin, chymotrypsin and peptidase) mix, with a hydrolysis degree of 79.8% of hd. different in vitro hydrolysis methods have been used to evaluate in vitro hydrolysis of common bean seeds. there are differences in the %hd reported in these studies. these differences must be due to the type and variety of seeds, geographic position of the cultivar and the differences in the method of hydrolysis and enzymes used, time of incubation, phs of simulation, temperature, proportion of enzymes and combination of enzymes. for example, montoya et al. (2008; 2010) reported hydrolysis of isolated phaseolin, treated and not treated thermally, of 43 varieties hydrolyzed with pepsin at ph 2.0 and hydrolyzed with pepsin and pancreatin dissolved at ph 7.5. in the gastric phase, at 120 min of incubation with pepsin the %hd was 5.2% in the unheated sample and 7.5% in the heated sample. in the duodenal phase, at 360 min of incubation with pepsin and pancreatin, the %hd was 11% to 27% for the unheated sample and 57% to 96% for the heated sample. the gastrointestinal digest presented a high % hd but the results were different depending on the variety of phaseolus vulgaris used. torruco-uco et al. (2009) reported hydrolysates of phaseolus vulgaris from mexico obtained with alcalase and flavourzyme for 30 min. they found a %dh of 49.48% and 26.05% respectively. 3.4. characterization of fractions of lbpc by sds-page electrophoresis lbpc fractions were obtained using a deae affi-gel blue gel chromatographic column. ten cationic and anionic fractions were obtained to be analyzed with rp-uhplc and sds-page electrophoresis. the anionic fractions numbered as fraction 1, f1, fraction 2, f2, fraction 3, f3 and fraction 4, f4 were chosen for their high protein content. the cationic fractions numbered as fraction 5, f5, fraction 6, f6, fraction 7, f7 and fraction 8, f8 were chosen for their high protein content. fig. 3 shows the protein profile of cationic and anionic lbpc fractions. all fractions present an identical profile of proteins with molecular weights between 20 kda to 50 kda. triplet with bands of 20 kda, 22 kda and 25 kda were the bands with higher intensity. these bands correspond to the phaseolin protein. in the cationic fractions, f5 and f6 present a higher protein content than f3 and f4. in the anionic fractions, f3 and f4 present a higher protein content than f5 and f6. the cationic fractions present a higher protein content than the anionic protein content. sparvoli et al. (1996) reported cationic fractions from phaseolus lunatus obtained by an ion exchange chromatography with a mono-q hr5/5column coupled to the fplc system. they reported nine cationic fractions with presence of triplet bands with molecular weights of 32 kda, 35 kda and 38.5 kda in all fractions. these bands correspond to the ital. j. food sci., vol. 32, 2020 284 phaseolin protein. in this study, all fractions present triplet of bands but with lower molecular weights (20 kda, 22 kda and 25 kda). the protein content fractions were determined using the bca method. the anionic fractions, f1 (10.4%), f2 (11.5%), f3 (17.4%) and f4 (49.4%) have the percentage of protein content in brackets. these previous results show a correlation of the protein content with the intensity of the band in the gels of polyacrylamide. cationic fractions f5 and f6 present a higher protein content with values of 77.9% and 77.2% of protein respectively. figure 3. protein profile of lbpc and cationic and anionic fractions using sds-page electrophoresis analysis. 3.5. characterization of lbpc fractions by uhplc analysis cationic and anionic fractions of lbpc were characterized using the uhplc technique. the cationic fractions f5, f6, f7 and f8 have the same profile of proteins with four peaks in the chromatogram. f8 present higher intensity in the absorbance at 280 nm. peak number one shows the highest intensity with a value of 20 au. these results show a correlation with the intensity of the bands in the gel sds-page and the percentage of protein determined by the bca method. these results suggest that this fraction present a higher protein content. these proteins are rich in tryptophan, this amino acid is absorbed at 280 nm (fig 4). lbpc anionic fractions show three peaks. f1 and f2 present minor absorbance at 280 nm. these results show a correlation with the sds-page electrophoresis results of these fractions. f3 and f4 present major absorbance at 280 nm and in the gel sds-page these fractions present a higher protein content because the staining is strong. peak number one of fractions f3 and f4 shows a high content of tryptophan amino acid in these sequences, if we look at the polyacrylamide gel and the percentage of protein calculated by the bca method (fig. 5). ital. j. food sci., vol. 32, 2020 285 figure 4. rp-uhplc analysis of anionic fractions of lbpc. a) anionic fraction f1, b) anionic fraction f2, c) anionic fraction f3 (and d) anionic fraction f4. figure 5. rp-uhplc analysis of cationic fractions of lbpc. a) cationic fraction f5, b) cationic fraction f6, c) cationic fraction f7 and d) cationic fraction f8. 3.6. antioxidant activity of lbpc and fractions of lbpc fig. 6 shows the results of lbpc and their fractions antioxidant activity, using the frap method. all samples assayed present antioxidant activity. lbpc present a value of 1.67±0.14 mg te/g of sample. this is the highest value. the positive fractions present the ital. j. food sci., vol. 32, 2020 286 higher activities, f5 with value of 1.26±0.06 mg te/g of sample, f6 present a value of 1.61±0.37 mg te/g of sample, f7 present a value of 1.15±0.05 mg te/g of sample, f8 present a value of 1.53±0.26 mg te/g of sample. lbpc negative fractions were active with values between 1.15±0.05 to 1.23±0.18 mg te/g of sample. f8 present the highest value with 1.23±0.18 mg te/g of sample. positive fractions were more active than negative fractions but the lbpc sample was more active than the negative fractions. this situation shows the correlation with the orac activity. in the orac activity, positive fractions were more active than negative fractions. figure 6. antioxidant activity of lbpc and their fractions using the frap method. different letters represent significant differences between lbpc vs sample as p <0.05 (n=3). antioxidant activity of lbpc fractions also was evaluated using the dpph method. lbpc cationic fractions were more active than lbpc negative fractions. fig. 7 shows the antioxidant activity of lbcp fractions. f5 present a value of dpph of 189.38 µmol te/g of fraction, f8 present a value of dpph of 191.33 µmol te/g of fraction, f7 show a value antioxidant of 196.17 µmol te/g of fraction and f8 show a value of 205.19 µmol te/g of fraction. this sample present higher antioxidant activity. lbpc negative fractions present value of dpph between 36.79 to 48.96 µmol te/g of fraction. lbpc control present a value of 84.08 µmol te/g of lbpc. different works have been reported fractions obtained of food proteins using different methods of isolation or separation with biological properties. for example, rodriguez saint-jean et al. (2013) have described fractions isolated of αs2 casein bovine with strong antiviral activity against the infectious hematopoietic necrosis virus of salmonid fish. the fractions were isolated using ionexchange chromatography. fig. 8 shows the lbpc antioxidant activity results and their fractions using the orac method. 0.0 0.5 1.0 1.5 2.0 2.5 f1 (-) f2 (-) f3 (-) f4 (-) f5 (+) f6 (+) f7 (+) f8 (+) lbpc µm ol t e/ g sa m pl e a a a a a a b b b ital. j. food sci., vol. 32, 2020 287 figure 7. antioxidant activity of lbpc and their fractions using the dpph method. different letters represent significant differences between lbpc vs sample as p <0.05 (n=3). figure 8. antioxidant activity of lbpc and their fractions using the orac method. a) anionic fractions, b) cationic fractions. different letters represent significant differences between lbpc vs sample as p <0.05 (n=3). 0 50 100 150 200 250 f3 (+) f4 (+) f5 (+) f6 (+) f5 (-) f6 (-) f7 (-) f8 (-) lbpc µ m ol t e/ g s am pl e a a a a b b b b c 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 5 µmol 10 µmol 20 µmol 25 µmol 50 µmol 100 µmol 200 µmol µm ol t e /µ m ol sa m pl e b) lbpc f5 (+) f6 (+) f7 (+) f8 (+) d d d d d d d c c c c c c c a a a a a a a b b b b b b b b b b b b b b 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 5 µmol 10 µmol 20 µmol 25 µmol 50 µmol 100 µmol 200 µmol µ m o l t e/ µ m o l sa m p le a) lbpc f1 (-) f2 (-) f3 (-) f4 (-) d d d d d d d c c c c c c c e e e e e e e a a a a a a a b b b b b b b ital. j. food sci., vol. 32, 2020 288 non-digested lbpc showed an orac value between 0.64±0.06 to 0.81±0.06 µmol te/ µmol of sample. vilcacundo et al. (2018) reported quinoa protein concentrates with values of 0.42±0.03 µmol te/µmol of protein. nongonierma et al. (2015) reported quinoa proteins with orac values of 0.26±0.07 µmol te/mg of sample. lbpc cationic fractions (f5 and f6) present lower antioxidant activity than lbpc with orac values between 0.47±0.09 to 0.56±0.09 µmol te/µmol of sample and 0.48±0.03 to 0.61±0.03 µmol te/µmol of sample respectively. the cationic fractions f7 and f8 present higher antioxidant activity than the f5 and f6 and anionic fractions (f1, f2, f3 and f4) with orac values between 1.75 to 2.24±0.03 µmol te/µmol of sample and 2.64±0.05 to 3.37±0.05 µmol te/µmol of sample respectively. the anionic fraction f1 present an absence of antioxidant activity using the orac method, f3 and f4 present higher orac values (1.03±0.06 to 1.31±0.06 µmol te/µmol of sample) and (1.52±0.06 to 1.94±0.06 µmol te/µmol of sample). fraction f4 present orac values between 0.83±0.08 to 1.06±0.08 µmol te/µmol of sample. carrillo et al. (2016a; 2016b) have described hydrolysates and fractions isolate of lysozyme protein with antioxidant activity and capacity to inhibit lipid peroxidation in zebrafish larvae, the fractions from lysozyme were isolated using the iec technique. peptides were identified by hplc-esi-ms-ms. peptides presented antioxidant activity using the orac method. carrillo et al. (2018) have described fractions isolated of lysozyme protein with antibacterial activity against escherichia coli and staphylococcus carnosus. fractions were separated using a membrane of cationic exchange chromatography technique. piñuel et al. (2019) have reported antioxidant fractions obtained of digest of phaseolus vulgaris separated using ultrafiltration membrane of 3 kda and 10 kda. fractions with biological activities can be isolated of food proteins of animal or vegetal sources and can be used for different proposes. when the proteins are subject to enzymatic hydrolysis the peptides can be identified and synthetized to be studied. 4. conclusions baby lima bean (phaseolus lunatus) seeds from ecuador used in this study present a high protein content. these seeds can be a new source of vegetable protein and used for different purposes in the food industry. in this study, lbpc was obtained using an alkaline extraction, followed by an isoelectric precipitation, lbpc present a complex proteins profile. phaseolin is the most abundant protein in the phaseolus lunatus protein concentrate. phaseolin protein presents resistance to hydrolysis with pepsin in the gastric phase and to pepsin/pancreatin in the duodenal phase. lbpc 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marikkar et al., 2011). the quality and nutritional characteristics of chicken nugget significantly affected by factors such as processing treatments, raw material and additives (yogesh et al., 2013). supplementation of meat products with protein sources, legumes, and oilseed products has the ability to reduce the cost of products (asgar et al., 2010), and to improve nutritional, functional and sensory characteristics of these product. in recent times, some efforts were conducted to formulate modified meat products by changing in the contents of fat and fatty acids, and/or by inclusion of functional ingredients into meat products or by removing or reducing the substrates that are considered as a hazard to the human health (jimenezcolmenero et al., 2001; fernandez-lopez et al., 2005). vegetables and fruit contain more than one type of bioactive compounds. diets with high levels of vegetables and fruit have a good protection against cancers (valcke et al., 2017) and cardiovascular disease (dos santos et al., 2017). cauliflower (brassica oleracea var. botrytis l.) is one of the most popular vegetables grown all over the world and has a wide diversity of uses as a dish or as an ingredient in soups or salads. cauliflower is a good source of vitamins, folic acid as, dietary fibers, proteins, mineral elements, eg, phosphorus, magnesium manganese, potassium, and iron (ahmed and ali, 2013; kapusta-duch et al., 2017). the main objective of the current study was to evaluate the effect of the incorporation of different levels of cauliflower on the nutritional and sensory characteristics of chicken nuggets. 2. materials and methods fresh, boneless, skinless, chicken breast fillets and chicken skin were obtained from poultry slaughterhouse, giza, egypt. cauliflower, freshly harvested, free from insect and mechanical damage was purchased from the local market in giza governorate, egypt (january, 2018). sodium tripolyphosphate was obtained from el-gomhouria company for trading chemicals and medical appliances (building 23, el-sawah street, al ameria, cairo, egypt. dried bread crumbs (6.8% moisture, 12.59% protein, 3.94% fat, 0.96% ash, 0.82 fiber and 81.69% carbohydrates,) were purchased from modern bakeries (rich bake) company, 6th of october city, giza, egypt. refined salt, white pepper powder, fresh garlic paste and fresh onion were obtained from local market, giza, egypt. 2.1. preparation of frozen cauliflower the cauliflower florets were cut into small bite-sized pieces (about five cm in diameter and five cm in length), blanched for three min in boiling water containing 0.5% sodium acid pyrophosphate. blanched cauliflower samples were drained on a stainless sieve until cold, packed in polyethylene bags, and stored at -25°c for further uses. ital. j. food sci., vol. 32, 2020 48 2.2. chicken nugget formulations chicken nugget samples were prepared according to the previous procedures described by arshad et al., 2017, using the ingredients listed in table 1. chicken breasts were cut into smaller chunks (2 cm height × 2 cm width × 2 cm length). chicken breast chunks were ground twice in meat grinder (moulinex model me605131). frozen cauliflower and seasonings (refined salt, white pepper powder, fresh garlic paste and fresh onion) were added into the formulations (table 1). the mixture was minced twice in the abovementioned grinder. the mixture was weighed and formed into nugget pieces (25 g weight, 1.5 cm thick, 8 cm length, and 3.5 cm width). the formulated nuggets were pre-dusted (with wheat flour), batter-coated and breaded (with dried bread crumbs). the breaded nuggets were pre-fried in sunflower oil at 175±5°c for 59 seconds. fried nuggets were drained on absorbent paper towels, and cooled to room temperature (25°c). the samples were packed in polyethylene bags, and stored at -25°c. hygienic practices were applied during the preparation, packaging and storage processes of the chicken nugget products. table 1. chicken nugget formulations. ingredients (%) control formula 1 formula 2 formula 3 formula 4 chicken breast 70 70 70 70 70 chicken skin 20 15 10 5 frozen cauliflower 5 10 15 20 crushed ice 5 5 5 5 5 refined salt 1.5 1.5 1.5 1.5 1.5 fresh garlic paste 0.5 0.5 0.5 0.5 0.5 fresh onion 2.5 2.5 2.5 2.5 2.5 white pepper powder 0.30 0.30 0.30 0.30 0.30 sodium tripolyphosphate 0.20 0.20 0.20 0.20 0.20 2.3. frying (cooking) process two kilograms of sunflower oil were placed in frying pan, preheated and maintained at 180±5°c for 5 minutes before frying process. frozen chicken nuggets were fried for 5 minutes. fried nuggets were drained on absorbent paper towels, and cooled to 50°c for sensory evaluation. other portions of samples were allowed to cool to room temperature (25°c) before further tests. 2.4. total phenolics, total flavonoids and antioxidant activity of cauliflower extract 2.4.1 preparation of crude extract of frozen white cauliflower frozen white cauliflower was subjected to water extraction.10 g of frozen white cauliflower was extracted using 100 ml deionized water for 5 h at ambient temperature (25°c). the mixture was mixed and homogenized in stainless steel blender. the resulting mixture was filtered through whatman no 40 paper. the collected filtrates were packed in teflon tubes and kept at -25°c for further use. total phenolic content, total flavonoids ital. j. food sci., vol. 32, 2020 49 content and antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (dpph) assay of cauliflower extract were determined according to the procedures described by ahmed and ali (2013). the content of phenolics was expressed as milligram of gallic acid equivalents per g of extract. flavonoid contents were expressed as mg of catechin equivalents per g of extract. antioxidant activity was expressed as the inhibition (%) of the absorbance at 515nm using a uv-vis spectrophotometer (lambda 9; perkin elmer, shelton, ct, usa). 2.5. physicochemical properties and nutritive value of formulated nuggets 2.5.1 determination of chemical composition the content of moisture, ash, fiber, protein and fat were assayed according to the official procedure described by aoac (2005). total carbohydrate percentage was calculated by difference. total caloric (kcal/100g sample) were calculated according to choi et al, 2010 as follow, for fat (9 kcal g-1), protein (4.02 kcal g-1), and carbohydrates (3.87 kcal g-1). 2.5.2 determination of cholesterol cholesterol content was determined according to the previous procedures described by turhan et al. (2007). the concentration of cholesterol was expressed as mg/100 g, dry weight basis of nugget samples. 2.5.3 ph the ph value was determined by mixing a 10 g of nugget sample with 100 ml of deionized water. the mixture was filtered through whatman filter paper number 1, and the ph of the filtrate was measured at room temperature (yogesh et al., 2013) using a ph meter (shanghai second analytical instrument factory, shanghai, china). 2.5.4 colour properties nugget's colour was measured using a minolta colourimeter (model cr-400, minolta, japan, calibrated using a standard white porcelain plate l*=97.75, a*=-0.48,b*=+2.31), with a measuring area of 8 mm diameter). the nugget samples were placed in a transparent plate and placed directly on the path of light to determine the colour parameter values of l*, a* and b*(ngadi et al., 2007) 2.6. cooking properties 2.6.1 cooking yield the percentage of cooking yield was calculated according to the following equation (yogesh et al., 2013). cooking yield percentage = (weight of fried (cooked)/ weight of raw (uncooked) nugget) x 100 ital. j. food sci., vol. 32, 2020 50 2.7. sensory characteristics sensory evaluation of fried chicken nuggets was carried out by a fifteen of trained judges from who are belonging to food technology research institute, agriculture research center, giza, egypt). all judges are knowledgeable about the properties of chicken nuggets and familiar with chicken and meat products. the panelists were seated in individual cabins in a temperature-controlled room at 25°c lighted by daylight fluorescent lights. rectangular strips approximately 2×2× 4 cm3 were served to the panelists. chicken nugget samples were evaluated for appearance, juiciness, texture, flavour and overall acceptability by using 9-point hedonic scale, 9 = like extremely, 8 = like very much, 7 = like moderately, 6 = like slightly, 5 = neither like nor dislike, 4 = dislike slightly, 3 = dislike moderately, 2 = dislike very much and 1 = dislike extremely (yogesh et al., 2013). cups of drinking water were provided for judges to clean their mouth between samples. 2.8. statistical analysis results are expressed as mean±sd. data were statistically analyzed according to the procedures described by (gomez and gomez, 1984). spss version 18.0 (spss inc., chicago, il, usa) was used to analyze data. duncan’s multiple range mean separation was performed where the anova procedure showed significance. 3. results and discussions 3.1. sensory characteristics of chicken nuggets formulated with different levels of frozen cauliflower in order to determine the acceptability of the chicken nugget formulations, sensory evaluation tests were carried out. the transformed data of appearance, juiciness, texture, flavor and overall acceptability of cooked (fried) chicken nuggets formulated with different levels of cauliflower are presented in table 2. the appearance scores of cooked chicken nuggets ranged from 7.50 to 8.19. nugget samples incorporated with 20% of frozen cauliflower showed the lowest (p ≤ 0.05) scores of appearance. however, no significant differences were observed in appearance values between control samples and those nugget samples formulated with 5, 10 and 15% of frozen cauliflower. no significant (p ≥ 0.05) variations were detected in juiciness value between control samples formulated with 20% of chicken skin and those batches formulated with 5, 10 and 15%of frozen cauliflower as fat replacer. on the contrary, the lowest juiciness value was recorded for those samples containing 20% of frozen cauliflower. this findings attributed to the effect of frying process on reduction of the moisture content of fried food and increase the crispy and hardness of fried vegetables. a critical aspect of battered and breaded chicken products is the contrast between the crispy and oily outer layer and the soft cooked interior (dobraszczyk et al., 2006). crispy texture of fried food products determines the consumer acceptability. the texture of cooked chicken nugget significantly affected by the addition of cauliflower as fat replacer. chicken nuggets formulated with various levels of cauliflower had significantly (p ≤ 0.05) higher texture scores than control samples without cauliflower addition. the highest (p ≤ 0.05) scores of texture were recorded for chicken nuggets incorporated with 15 and 20% cauliflower as fat replacer followed by those samples containing 10 and 5% cauliflower. on the other hand, control samples ital. j. food sci., vol. 32, 2020 51 formulated with chicken skin had significantly (p ≤ 0.05) the lowest score of texture (7.81). additions of carrot and sweet potato were found to be effective in improving texture scores (bhosale et al., 2011). the highest flavour scores (8.22 and 8.14) were recorded for control sample and those samples incorporated with 5% frozen cauliflower. the perception of fat in food is a complex process involving many sensory modalities (texture, aroma and flavour) (feron and poette, 2013). no significant variations were detected in flavor scores among nugget batches formulated with 10, 15 and 20%of frozen cauliflower as fat replacer. the increasing of consumer awareness about healthy foods led the efforts made by the food manufacturers to develop new items of food with positive properties. some efforts have been made to produce alternative meat products by changing in the amounts of lipid contents, by inserting of with bioactive compounds or plant-based phytonutrients into meat products (jimenez-colmenero et al., 2001; fernandez-lopez et al., 2005). no significant variations (p ≥ 0.05) were recorded in the overall acceptability scores among chicken nugget batches. generally speaking, the incorporation of frozen cauliflower as fat replacer had positive effects on the acceptability and overall quality of chicken nuggets. table 2. sensory characteristics of cooked (fried) chicken nuggets formulated with different levels of cauliflower (n= 15). parameters control formula 1 formula 2 formula 3 formula 4 lsd at 0.05 appearance 8.19a±0.2 8.15a±0.1 8.05a±0.1 8.00a±0.2 7.50b±0.3 0.30 juiciness 8.17a±0.14 8.09a±0.18 8.04a±0.24 7.90a±0.10 7.45b±0.15 0.28 texture 7.81b±0.18 8.10ab±0.13 8.15ab±0.25 8.20a±0.14 8.20a±0.20 0.27 flavor 8.22a±0.10 8.14a±0.06 7.80b±0.12 7.69b±0.15 7.56b±0.18 0.25 overall acceptability 8.14 a±0.16 8.12a±0.14 7.98a±0.10 7.92a±0.09 7.51a±0.14 0.33 means in a line with different letters are significantly different (p ≤ 0.05). lsd, least significant difference at p ≤.05 according to duncan’s multiple-range test. 3.2. total phenolics, total flavonoid and antioxidant activity by dpph of crude extract of frozen white cauliflower the nutraceutical quality depends on the proximate composition, particularly, the quantity of the phenols and flavonoids. table 3 shows total phenolics, total flavonoid and antioxidant activity by dpph of crude extract of frozen white cauliflower. the amount of total phenolics found in crude extract of frozen white cauliflower was 6.76 mg / g. studies found that phenolics are strictly responsible for antioxidant activity of vegetarian sources. these compounds can contribute candidly to scavenge free radicals (ahmed and ali, 2013; arshad et al., 2017). the total concentration of flavonoids for crude extract of cauliflower was 1.65 mg/g. it has been proven that flavonoid compounds possess antioxidant, antimicrobial, free radicals scavenging and antitumor activities (fernandez-lopez et al., 2005). the crude extract of white cauliflower exhibited remarkable free radicals scavenging activity was 54.9%. the high level of phenolic compounds in white cauliflower makes it as a potential ingredient for meat and chicken products to improve their nutritive value and enhance their ability to scavenge free radicals. ital. j. food sci., vol. 32, 2020 52 table 3. total phenolics, total flavonoid and antioxidant activity by dpph of crude extract of frozen white cauliflower. data are expressed as the mean±standard deviation. 3.3. chemical compositions, energy content and cholesterol content of raw and cooked chicken nuggets formulated with different levels of cauliflower the proximate composition of raw and cooked chicken nuggets incorporated with different levels of cauliflower is shown in tables 4. moisture content of nugget samples was significantly affected by addition of various levels of frozen cauliflower into chicken nugget formulas. moisture content of raw chicken nuggets ranged from 61.73 to 66.08%. significant differences (p ≤ 0.05) were observed in the moisture content between nugget samples formulated with cauliflower and those control samples (without cauliflower addition). nugget samples formulated with frozen cauliflower had significantly (p ≤ 0.05) higher moisture content than control samples without cauliflower. the highest (p ≤ 0.05) moisture content (66.08%) was recorded for chicken nuggets incorporated with 20% cauliflower, followed by nuggets samples containing 15% cauliflower (65.76%). the lowest content of moisture was observed for control samples (61.73%). at the same time, no significant differences (p ≥ 0.05) in moisture content were observed among chicken nuggets incorporated with 20, 15 and 10% of frozen cauliflower as fat replacer. the increases in moisture content in nuggets incorporated with cauliflower may be attributed to the ability of cauliflower fiber to hold water. pectic-polysaccharide-rich fiber of cauliflower has been used as a water holding agent for enhancing the quality properties of model foods (mckee, and latner, 2000). fat content of raw chicken nuggets ranged from 0.31 to 13.20%. fat content of chicken nugget samples was markedly affected by the addition of frozen cauliflower as fat replacer. substitution of chicken skin with different levels of frozen cauliflower caused significant decreases in fat content of nugget samples. fat content of chicken nuggets incorporated with 20, 15, 10, and 5% of cauliflower as fat replacer were about 42.58, 3.91, 1.96 and 1.31 times as low as in control samples which containing 20% chicken skin as source of fat, respectively. the amount of fat recovered from chicken skin ranged from 22.6 to 38.9% of the initial weight of skin, according to the extraction conditions (piette et al., 2001). these levels of fat representing 51.5 to 89.6% of the fat initially contained in chicken skin. blanched cauliflower contains low levels of fat ranged from 1.93 to 2.20% of the dry weight. (ahmed and ali, 2013), this low level of fat in cauliflower contributes well in reducing fat content of chicken nuggets formulated with different levels of cauliflower. no significant differences (p ≥ 0.05) in protein content were observed among chicken nuggets formulated with different levels of cauliflower and control samples without cauliflower addition. properties white cauliflower extract total phenolics (mg/g) 6.76±0.39 flavonoids (mg/g) 1.65±0.08 free radical scavenging (%) 54.9±1.08 ital. j. food sci., vol. 32, 2020 53 table 4. chemical compositions, energy content and cholesterol content of raw and cooked chicken nuggets formulated with different levels of frozen cauliflower. adata are expressed as the mean±standard deviation. values followed by the same letter (a-e) are not significantly different (p≤0.05). bby difference. clsd, least significant difference. dnd, refers to not detected. generally, control samples contain considerably lower ash content than those samples formulated with frozen cauliflower (p ≤ 0.05). ash content of formulated chicken nuggets ranged from 1.68 to 2.17%. the highest (p ≤ 0.05) ash content (2.17%) was recorded for chicken nuggets incorporated with 20% cauliflower. however the lowest content (1.68%) was recorded for control sample without cauliflower addition. ash content of chicken nuggets formulated with 20, 15 and 10% of cauliflower as fat replacer were about 1.29, 1.16 and 1.11 times as great as in control samples which containing 20% chicken skin as source of fat, respectively. fiber content of formulated chicken nuggets ranged from 0.92 to 3.78%. nuggets formulated with different levels of cauliflower had higher levels of fiber than control samples. control samples without cauliflower addition had significantly (p ≤ 0.05) the lowest level (0.92%) of fiber content. the highest (p ≤ 0.05) content of fiber (3.78%) was recorded for chicken nuggets formulated with 20% cauliflower followed by those samples formulated with 15% cauliflower (3.09%). this finding attributed to the fact chemical composition of raw chicken nuggets (n= 5) parameters control formula 1 formula 2 formula 3 formula 4 lsd at 0.05 moisture (%) 61.73c±0.63 63.31b±0.80 65.08a±0.92 65.76a±1.01 66.08a±0.79 1.514 fat (%) 13.20a±0.72 10.01b±0.11 6.71c±0.31 3.37d±0.29 0.31e±0.08 0.688 protein (%) 17.85a±1.03 17.90a±2.02 17.95a±3.01 18.09a±1.08 18.23a±1.03 3.30 ash (%) 1.68b±0.21 1.76b±0.17 1.88ab±0.22 1.95ab±0.09 2.17a±0.13 0.307 crude fiber (%) 0.92e±0.07 1.81d±0.13 2.47±c0.02 3.09b±0.19 3.78a±0.24 0.322 carbohydrates (%)b 4.62 e±0.28 5.21d±0.36 5.91c±0.13 7.74b±0.21 9.43a±0.32 0.497 energy content (kcal per 100 g) 208.42 a±0.71 182.20b±0.38 155.41c±0.85 133.00d±0.77 112.56e±0.32 1.16 cholesterol(mg/ 100g) 113.89 a±1.98 86.79b±2.08 63.42c±1.31 29.11d±0.94 nde 2.65 chemical composition of cooked (fried) chicken nuggets (n= 5) moisture (%) 51.74c±0.94 53.70b±1.08 55.42ab±0.87 56.24ab±0.66 56.26a±0.82 1.80 fat (%) 20.31a±0.17 16.53b±0.31 12.81c±0.33 9.97d±0.69 6.85e±0.43 0.76 protein (%) 19.59a±1.19 19.60a±1.68 19.92 a±2.02 20.13a±0.69 20.16a±1.87 2.85 ash (%) 1.79b±0.16 2.15a±0.23 2.28a±0.08 2.46a±0.17 2.51a±0.12 0.292 crude fiber (%) 1.08e±0.09 1.76d±0.11 2.61c±0.06 3.07b±0.10 3.84a±0.14 0.18 carbohydrates (%)b 5.49 e±0.41 6.27d±0.37 6.96c±0.19 8.13b±0.32 10.38a±0.09 0.53 energy content (kcal per 100 g) 282.78 a±0.69 251.82b±0.47 222.29c±0.76 202.11d±0.47 182.86e±0.39 1.04 cholesterol (mg) 125.50 a±1.07 94.86b±0.67 68.12c±2.54 32.58d±1.46 nd e 2.59 ital. j. food sci., vol. 32, 2020 54 that cauliflower is a good source of dietary fiber. in this regard, ahmed and ali, 2013 reported that blanched cauliflower florets contain 11.52 -12.74% crude fiber on a dry weight basis. carbohydrate content of nugget samples varied from 4.62 to 9.43%. control sample had lower levels of carbohydrates than those samples formulated with different levels of cauliflower. carbohydrate content of chicken nuggets incorporated with 20, 15, 10, and 5% of frozen cauliflower as fat replacer were about 2.04, 1.67, 1.29 and 1.12 times as high as in control samples containing 20% chicken skin as source of fat, respectively. carbohydrate content of blanched cauliflower florets was estimated to be about 50% of the total dry weight (ahmed and ali, 2013). energy content (kcal) of formulated chicken nuggets ranged from 112.56 to 208.42 per 100g. the highest content of energy (208.42 kcal) was recorded for control sample formulated with 20% of chicken skin as source of fat. the lower values of calories were recorded for those samples formulated with different levels of cauliflower. calories (kcal) of chicken nuggets incorporated with 20, 15, 10, and 5% of cauliflower as fat replacer were about 1.85,1.65, 1.34 and 1.14 times as low as in control samples which containing 20% chicken skin as source of fat, respectively. foods with a lower level of fat provide fewer calories than foods with a higher level of fat. these findings attributed to the fact that one gram of lipids provides 9 kcal, while one gram of protein or carbohydrates provides 4 kcal. cholesterol content (mg/100 g) of formulated chicken nuggets ranged from not-detectable level to 113.89. control samples formulated with chicken skin contain the highest levels (113.89 mg/100 g) of cholesterol. skin of poultry has the greatest cholesterol level compared with poultry meat or poultry fat. in this regard, mendez-lagunas et al., 2015 reported that the cholesterol content of chicken skin was 131 mg/100 g of raw wet tissue. bragagnolo (2009) reported also that raw poultry meat has approximately 27 to 90 mg cholesterol/100 g, while cooked poultry meat has around 59 to 154 mg/100 g. samples formulated with various levels of cauliflower had significantly (p ≤ 0.05) lower levels of cholesterol than control samples without cauliflower addition. cholesterol level of the formulated chicken nuggets can be arranged in the decreasing order as follows: control samples > nuggets with 5% cauliflower > nuggets with 10% cauliflower > nuggets with 15% cauliflower > nuggets with 20% cauliflower. as cauliflower florets are vegetarian diets, they are eaten as cholesterol free diets. several studies proved that incorporation of vegetarian based foods into diets could promote health and reduce the risk of cholesterol and heart disease (sadler, 2004). moisture content of cooked nuggets was considerably lower than that of raw (un-cooked) for all the nugget samples. this decrease in water content may be attributed to the fact that frying process resulted in water expulsion from chicken samples where the frying temperature is higher than the boiling temperature of water. moisture content of cooked chicken nuggets ranged from 51.74 to 56.24%. the lowest moisture content was observed for cooked control nuggets formulated without cauliflower addition. cooked nuggets formulated with different levels of cauliflower contain higher level of moisture than control samples. this finding attributed to the capacity of cauliflower fibers to hold water during frying process. fat content of cooked-fried chicken nuggets ranged from 6.85 to 20.31%. cooked control samples contain significantly (p ≤ 0.05) the highest level of fat (20.31%). fried items uptake fat during frying process, which is a health concern as excessive lipid consumption can contribute to obesity and heart disease (wolfram 2003). cooked nuggets formulated with different levels of frozen white cauliflower have lower amounts of fat than control samples formulated with 20% chicken skin. fat content of cooked chicken nuggets formulated with 5, 10, 15 and 20% of frozen white cauliflower were about 1.22, 1.58, 2.03 and 2.96 times as low as in control samples formulated with 20% chicken skin as source of fat, respectively. this finding attributed to the fact that certain cruciferous ital. j. food sci., vol. 32, 2020 55 vegetables of the genus brassica including cauliflower, contain little fat and energy (mukherjee et al., 2008). a critical element of deep-fat fried food is the high level of fat that is absorbed during the frying process, reaching in some cases up to 40% of the total weight of fried product. several investigations have reported that excess consumption of fat is a key dietary contributor to coronary heart disease and perhaps cancer of the breast, colon, and prostate (koene et al., 2016). no significant differences (p ≥ 0.05) in protein content were observed among cooked chicken nuggets. ash content of cooked nuggets varied from 1.79 to 2.51%. increased ash content was noticed in all the cooked nuggets when compared to raw chicken nuggets. losses of moisture, occurring during frying process resulted in higher ash content in cooked nuggets as compared to the uncooked nuggets cooked nuggets formulated with different levels frozen white cauliflower contain greater levels of ash than control samples without cauliflower addition. no significant (p ≥ 0.05) differences in ash content were observed among cooked samples formulated with various amounts of cauliflower. fiber content of cooked nuggets ranged from 1.08 to 3.84%. as expected nuggets formulated with frozen white cauliflower had higher levels of fibers than control sample without cauliflower addition. fiber content of cooked nuggets formulated with 20, 15, 10, and 5% of frozen white cauliflower were about 3.55, 2.84, 2.41 and 1.62 times as high as in control samples containing 20% chicken skin as source of fat, respectively. incorporation of dietary fiber into foods may be an effective power for enhancing functional and nutritional properties as reported earlier, there is a dramatic rise in the demand of food items with high levels of fiber and low levels of lipids as they are very efficient in lowering of fat absorption by the product, particularly fatty acids and cholesterol (borderias et al., 2005), that could be useful in reducing obesity. cooked chicken nuggets formulated with frozen white cauliflower had significantly (p ≤ 0.05) higher values of carbohydrates than control samples without cauliflower addition. the highest value (10.38%) of carbohydrates was recorded for nugget samples incorporated with 20% of frozen cauliflower. energy content (kcal) of cooked chicken nuggets ranged from 182.86to 282.78. as expected, control samples are higher in energy content than those samples formulated with frozen white cauliflower. in the current study, reducing the content of chicken skin from 20% to 0% and replacing it with frozen-blanched cauliflower caused significant (p ≤0.05) decreases in energy content of cooked nuggets. at a 25% chicken skin replacement, the energy content was reduced by 10.94%, while the nuggets sample in which 100% chicken skin was replaced, the energy content was reduced by 35.33%. the inverse relation between fat content and energy content was observed in different types of meat products formulated with dietary fiber (méndez-zamora et al., 2015; keenan et al., 2014). cholesterol content (mg/100 g) of cooked nuggets varied from not-detectable concentration to 125.50. frying process caused significant (p ≤ 0.05) increases in cholesterol content of formulated chicken nuggets. the cholesterol content in the control (cooked) sample was the highest, amounting 125.50 mg / 100 g. cholesterol levels were higher in fried chicken nuggets compared to un-fried samples. these increases in cholesterol content may attributed to the loss of moisture content during frying process, which leading to changes in cholesterol levels (padiani et al., 2002; turhan et al., 2007). in this regards, medina et al. (2015) reported that frying process caused significant increases in cholesterol content of the refrigerated nuggets; they added also that such changes in cholesterol as a consequence of frying could also be related to the slight dehydration caused by the heat treatment. the cholesterol content of chicken nuggets decreased with respect to the incremental addition of frozen cauliflower as a chicken skin substitute (p ≤ 0.05), cholesterol content of cooked chicken nuggets supplemented with 5, 10 and 15% of frozen white cauliflower were about 1.32, 1.84 and 3.85 times as low as in ital. j. food sci., vol. 32, 2020 56 control samples incorporated with 20% chicken skin as source of fat, respectively. cholesterol has not been detected in nugget samples formulated with 20% frozen white cauliflower as fat replacer. incorporation of hydrated potato flakes as a type of carbohydrates-based fat replacers caused significant reduction in cholesterol content of beef patties (ali et al., 2011). 3.4. physicochemical characteristics of cooked chicken nuggets formulated with different levels of frozen cauliflower 3.4.1 ph no significant differences in ph values between control samples and those samples incorporated with different levels of frozen cauliflower. this means that the reduction in chicken skin content and the addition of frozen white cauliflower did not affect (p ≤ 0.05) the ph of formulated chicken nuggets (table 5). kim et al., 2015 showed that the ph has not been significantly altered for chicken nugget formulated with various contents of chicken skin and wheat fiber mixture. the value of ph affects the ability of meat and meat products to retain moisture. water-holding capacity (whc) of meat reaches a minimum when the ph value is at the isoelectric point of meat proteins. whc is an important factor for meat products as it affects both the yield and the quality attributes of the end product (yogesh et al., 2013). table 5. ph, cooking yield and color measurements of cooked chicken nuggets formulated with different levels of frozen cauliflower (n= 5) parameter control formula 1 formula 2 formula 3 formula 4 lsd at 0.05 ph 6.1a±0.01 6.1a±0.01 6.1a±0.03 6.1a±0.02 6.1a±0.04 0.029 cooking yield 93.44b±1.02 94.28ab±0.90 95.60a±0.51 96.4a±0.38 96.64a±0.28 1.24 l (lightness) 69.55a±1.2 67.10ab±1.8 66.60ab±0.90 64.90b±2.2 64.80b±0.60 2.67 a (redness) 4.50b±0.1 4.61b±0.3 4.61b±0.4 4.73b±0.2 7.74a±0.30 0.50 b (yellowness) 29.50a±0.9 29.50a±1.10 30.00a±0.75 30.05a±1.3 30.10a±0.80 1.80 data are expressed as the mean±standard deviation. values followed by the same letter(a-e)are not significantly different (p≤0.05). lsd, least significant difference. 3.4.2 cooking yield table 5 illustrates the impact of substitution of chicken skin with different proportions of frozen white cauliflower on the cooking yield of chicken nuggets. cooking yield of formulated chicken ranged from 93.44 to 96.64%. generally, these high values of cooking yield may be attributed to the fact that significant amounts of fat were absorbed from frying media during cooking (frying) process (mellema, 2003). cooked chicken nuggets formulated with frozen white cauliflower had significantly (p ≤ 0.05) higher cooking yield values than control samples without cauliflower addition. the lowest value (93.44%) of cooking yield was recorded for control samples. the addition of frozen cauliflower had ital. j. food sci., vol. 32, 2020 57 positive effects for the cooking yields of the nuggets (table 5). these increases in cooking yield may be attributed to the ability of cauliflower florets fibers to hold moisture and lipids during frying process. it was proved that the yield percentages of cooking depends on cooking temperature, time of cooking, the amounts and type of additive, as well as the type of fat and dietary fiber in the meat products (choi et al., 2014; keenan et al., 2014; méndez-zamora et al., 2015). 3.4.3 color measurement of chicken nuggets formulated with different levels of frozen cauliflower measurement of texture and colour using instrumental methods can provide quantified indicators for determining processing operations to improve and enhance quality properties of finished products (ngadi et al., 2007). the surface color is one of the major physical attributes that determine consumer’s acceptability of finished products (syuhairah et al., 2016). the l* values of formulated cooked nuggets ranged from 64.80 to 69.55. the l* value of control chicken nuggets was higher (p ≤ 0.05) than chicken nuggets formulated with different levels of frozen cauliflower (table 5). the lightness values of nuggets were clearly affected (p ≤ 0.05) by the percentage of incorporated fats. colour lightness of nuggets significantly (p ≤ 0.05) reduced with decreasing the content of chicken skin (table 5). the results showed also that the increase in level of cauliflower incorporation results in linear decrease of the lightness values of nugget samples. the lower values were recorded for nugget samples incorporated with 15 and 20% of frozen white cauliflower. however, those samples formulated with 5 and 10% of frozen white cauliflower as fat replacer showed slight decreases in lightness values,which might be due to the presence of moderate amount ( 15 and 10%) of chicken skin. the a* value of formulated cooked nuggets ranged from 4.50 to 7.74. the addition of frozen cauliflower at 5, 10 and 15% as fat replacer had no significant effect on the a* value of the formulated samples. nuggets samples formulated with 20% cauliflower as fat replacer had significantly the highest value of redness (a* value). redness may not be a desirable colour in fried food products in general (krokida et al., 2001). however redness in cooked meat products is a desirable factor for consumer preferences (yogesh et al., 2013). no significant (p ≥ 0.05) differences in b* value (yellowness) were found among formulated chicken nugget samples (table 5). 4. conclusion in conclusion, sensory evaluation results revealed that chicken nuggets supplemented with different levels of cauliflower had overall palatability that was similar to control samples with 20% chicken skin. ash, fiber and carbohydrates contents in chicken nuggets formulated by incorporating 20% of frozen cauliflower were higher compared to control samples without cauliflower addition. it was observed also that the addition of cauliflower reduced the fat content of reformulated nuggets. the highest cooking yield was found in the sample containing 20% of cauliflower as fat replacer. references ahmed f. a. and ali rehab f. 2013. bioactive compounds and antioxidant activity of fresh and processed white cauliflower. biomed. res int. 367819:1-9. ital. j. food sci., vol. 32, 2020 58 ali rehabf m., el-anany a.m. and gaafar a.m. 2011. effect of potato flakes as fat replacer on the quality attributes of low-fat beef patties. advance journal of food science and technology 3(3):173-180. aoac. 2005. official methods of analysis. 18th edn. association of official analytical chemists; 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in yellow, light yellow, and cream-coloured potato tubers after short-term storage and boiling m. grudzińska*1 and d. mańkowski2 1plant breeding and acclimatization institute national research institute, poland 2laboratory of seed production and plant breeding economics, plant breeding and acclimatization institute, national research institute in radzików, poland *corresponding author: mag.g1@wp.pl abstract this study measured the changes in bioactive compounds [l-ascorbic acid (aa) and total phenolic (tp) compounds] and antioxidant activity (measured in trolox equivalents, te) in six potato (solanum tuberosum l.) varieties with yellow, light-yellow, and creamcoloured flesh after several different treatments. the experimental materials included raw tubers and both peeled and unpeeled tubers that had been boiled. analyses were conducted immediately after harvest and after 3 months of storage at 5°c and 8°c. flesh colour significantly affected the aa and tp contents in tubers. the difference in aa content was 0.195 mg·g-1 dm between creamand yellow-coloured tubers and 0.086 mg·g-1 dm between cream and light-yellow tubers. differences in tp contents between tubers with different flesh colours did not exceed 33%. significant losses in aa were detected in yellowand light-yellow-fleshed tubers that had been peeled and cooked after harvest (44 and 46%, respectively). cooking peeled tubers significantly decreased the antioxidant activity in potatoes regardless of flesh colour and storage treatment. unpeeled cooked tubers had significantly higher antioxidant activity than raw tubers after harvest. irrespective of flesh colour, high linear correlations were found between (aa)×(te) for cooked peeled tubers. a significant determination coefficient (r2) was observed between (tps)×(te) for raw and cooked unpeeled light-yellow and yellow-coloured tubers. the linear relationship between tps and te after cooking was significant for unpeeled tubers. the greatest matching of the model characteristics of the interdependence of features (φ2) was 75% for (aa) × (te) and 80% for (tps) × (te). keywords: antioxidant activity, ascorbic acid, phenolics, boiling, potato, storage ital. j. food sci., vol. 32, 2020 779 1. introduction potatoes (solanum tuberosum l.) are a rich source of nutrients, particularly complex carbohydrates (starch), phenolic compounds (hejtmànkova et al., 2009, lachman et al., 2012, navarre et al., 2010, rumboa et al., 2009, teow et al., 2007), and vitamin c (l-ascorbic acid, aa), with aa levels ranging from 14 to 25 mg·100-1 g fresh matter (fm) depending on the variety (burgos et al., 2009, grudzińska and zgórska, 2011, han et al., 2004, valcarcel et al., 2015). the importance of these compounds in the human diet has been emphasized by recent studies on their health-promoting properties (welch et al., 2005, cahill et al., 2009). until recently, it was thought that the processing of potatoes, such as cooking, degrades antioxidants and reduces their activity. however, the impact of processing on antioxidant activity is not always straightforward. for example, rumboa et al. (2009) found that a reduction in the natural antioxidant content in a food product (potato extracts) may be accompanied by an increase in antioxidant activity. research on the bioactive compounds in potato (lachman and hamouz, 2005, reyes et al., 2005, lachman et al., 2012, hejtmànkova et al., 2009, bellumori et al., 2017) has focused on variations in potatoes with different flesh colours (e.g., white, red, pink, and purple), growing locations (jansen and flamme, 2008, lachman et al., 2008, valcarcel et al., 2015, perla et al., 2012, silveira et al., 2016), and cultivation systems (brazinskiene et al., 2014, grudzińska et al., 2016) and the effects of cooking (mulinacci et al., 2008, lachman et al., 2012, burgos et al., 2013, bellumori et al., 2017) and blanching conditions (marangoni et al., 2019). however, these studies have not unequivocally demonstrated a correlation between changes in the levels of bioactive compounds and antioxidant activity in potato tubers after storage or non-peeled tubers after cooking using a best-fit model. in particular, such studies have not been performed in potato varieties with cream, light yellow, and yellow flesh, the most popular flesh colours in europe. the aim of this study was to determine the changes in bioactive compound levels and antioxidant activity in raw potatoes and in peeled and unpeeled boiled potato tubers with yellow, light-yellow, and cream-coloured flesh after short-term storage at 5°c and 8°c. we also developed a model describing the association of these changes. 2. materials and methods 2.1. chemicals all reagents used in this study, including 2,6-dichloroindophenol (puriss p.a 97.0%), oxalic acid (puriss p.a ≥99.0%), acetone (puriss p.a 99.5%), l-ascorbic acid (l-aa) standard solution (puriss p.a ≥90%), trolox ((±)-6-hydroxy 2,5,7,8-tetramethylchroman-2-carboxylic acid (97%), 2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) (abts) (activity 90– 110%), potassium persulfate (puriss p.a 98%), ethanol (puriss p.a 96%), folin–ciocalteu reagent, chlorogenic acid (puriss p.a ≥98.0%), and sodium carbonate (puriss p.a 99%) were purchased from sigma aldrich, fluk, poch, or linegal chemicals. ital. j. food sci., vol. 32, 2020 780 2.2. plant materials the experimental materials were six varieties of potato (solanum tuberosum l.). the varieties and their characteristics are shown in table 1. table 1. characteristics of the potato varieties examined in this study. variety origin of seed tubers skin colour flesh colour average yield [t ha-1]1 cooking type ametyst poland yellow cream 64.4 medium meal aruba poland yellow cream 34.4 slightly floury ingrid netherlands yellow light yellow 41.2 slightly floury flaming poland red light yellow 33.3 slightly floury altesse france yellow yellow 44.9 intermediate type elanda poland yellow yellow 44.0 slightly floury 1characteristics from the national register of varieties of potato ed. xv potatoes were grown in experimental fields at the experimental station of the plant breeding and acclimatization institute, research division jadwisin, poland. agronomic inputs used in conventional systems are shown in table 2. table 2. agronomic inputs used in conventional systems. crop production practice conventional system fertilisation 4–5 t ploughed rye straw + 1 kg mineral nitrogen per 100 kg straw + mustard as a catch crop; n: 100 kg.ha-1, p: 53 kg.ha-1, k: 150 kg.ha-1 weed control mechanical tillage + herbicides: 2012: afalon-1.9 l.ha -1, titus+trend (60 g.ha-1 + 0.5 l.ha-1) 2013: linurex-1.8 l.ha-1, titus + trend (60 g.ha-1 + 0.5 l.ha-1) colorado potato beetle control chemical insecticides: 2012: actara -60 g.ha-1 2013: actara 2 times per season -70 g.ha-1, apacz-40 g.ha-1 late blight control chemical fungicides 2012: ridomil-2 l.ha -1, revus-0.6 l.ha-1, ranman-0.2 l.ha-1, altima0.4 l.ha-1, ranman-0.2 l.ha-1, 2013: revus-0.6 l.ha-1 after harvest, the potato tubers (whole crop) were placed in an experimental storage room under the following conditions: 1) during the preparatory period for the first two weeks after harvest, the temperature was maintained at 15°c with 95±2% relative humidity; 2) subsequently, over a two-week period, the temperature was gradually lowered to 8°c (chamber i) or 5°c (chamber ii) while maintaining the same relative humidity (95%). post-harvest potato samples were collected for analysis immediately after harvest (the third set of ten days [decade] of september). post-storage samples were collected after 3 months of storage at 5°c and 8°c (the third decade of january). in each test, the samples were collected at random times (each laboratory sample consisted of ca. 50 tubers ~40 mm in size). ital. j. food sci., vol. 32, 2020 781 2.3. sample preparation the samples were prepared for analysis in the following manner: 1) tubers were left raw, unpeeled, and uncut; 2) uncut tubers were peeled (cut slit width 1.33 mm) and boiled in water in a beaker (standard proportions of 0.5 kg of potatoes and 0.7 dm3 of boiling water without added salt) for approximately 15±2 min (beginning when the tubers were placed into boiling water); and 3) tubers were left unpeeled and boiled. 2.4. measurement of dry matter the dry matter content was determined using a two-stage drying-weighing method involving drying at 60°c for 12 hours, followed by 105°c until the sample maintained a constant weight. 2.5. extraction of hydrophilic fractions freeze-dried samples were ground into a fine powder with a freezer mill 6770. five grams of freeze-dried powder was vortexed for 2 min in 25 ml hexane, and the mixture was filtered through a buchner funnel. the hexane extraction step was repeated twice, and the combined lipophilic extracts were evaporated to dryness at 50°c in a vacuum evaporator. the residue produced after hexane extraction was extracted twice in 25 ml of acidified methanol (7% acetic acid in 80% methanol) to obtain the hydrophilic fraction. the final volume of the hydrophilic fraction was adjusted to 50 ml with acidified methanol. 2.6. measurement of abts radical-scavenging activity the abts radical-scavenging activity of the hydrophilic fractions was determined as described by rice-evans et al. (1997) using the modifications described by re et al. (1999). the abts_+ solution consisted of 7 mm abts salt and 2.45 mm potassium persulfate (final concentration) in 25 ml of distilled water. the mixture was allowed to stand in the dark at room temperature for 12–16 h before use. the abts_+ solution was diluted with 95% ethanol (approximately 600 µl abts to 40 ml 95% ethanol) to obtain an absorbance of approximately 0.7 (±0.02) at 734 nm. fresh abts_+ solution was prepared for each analysis. antioxidant or standard solutions (20 µl) were mixed with 1 ml of diluted abts_+ solution and incubated at 30°c. the absorbance at 734 nm was read every minute for 30 min. ethanol (95%) was used as a blank. trolox (0 to 500 µm) was used as a standard. free radical scavenging activity was expressed as µmoles of trolox per 100 grams of sample (µmol te·100 g-1). 2.7. measurement of total phenolics total phenolic contents were measured by the folin-ciocalteu method using the modifications described by singleton et al. (1999). the hydrophilic extract (0.5 ml) was diluted with distilled water to 5 ml, to which 0.5 ml folin-ciocalteu reagent was added and allowed to react at room temperature for 3 min. after the addition of 1 ml of 1 n sodium carbonate, the mixture was incubated at room temperature for 1.5 h. the absorbance was measured at 725 nm using a spectrophotometer (t70+ uv/vis) with distilled water as a blank. chlorogenic acid was used as a standard. total phenolic contents were reported as milligrams per gram dry matter (mg tps·g-1 dm). ital. j. food sci., vol. 32, 2020 782 2.8. measurement of l-ascorbic acid l-ascorbic acid (aa) concentrations were measured using a standard spectrophotometric method (polish standard pn-a04019) based on the ability of aa to reduce the dye 2,6dichloroindophenol. briefly, a 10-g sample of potato tuber was extracted in 0.4% oxalic acid by homogenizing the sample in an ultra turrax t25 for 3 min at 13,500 rpm. the extract was filtered through filter paper under a vacuum and adjusted to 100 ml with the same extraction solution. next, 5 ml of the extract was reacted with 2 ml of 2,6dichloroindophenol (1.6%) for 2 min. the absorbance was measured at 500 nm using a spectrophotometer (t70+ uv/vis) with oxalic acid and 2 ml of 2,6-dichloroindophenol (1.6%) as a blank. the aa concentration was quantified via comparison with a standard curve of l-aa. the aa content was reported as milligrams per gram dry matter (mg aa·g-1 dm). 2.9. statistical analysis two and three-way analyses of variance (anovas) based on fixed model and multiple regression analysis were conducted to determine if the studied factors significantly differed from the analysed features. significant differences between means for the objects (after confirming the existence of these differences using f-test in analysis of variance) were determined using tukey’s multiple comparison procedure with p≤0.05. relationships (after confirming the existence of these relationships using multiple regression model analysis) were described using determination coefficients (r2) and convergence coefficients (ϕ2). calculations were performed using statistica software (v.12). 3. results and discussion 3.1. ascorbic acid (aa) according to burgos et al. (2009), murniece et al. (2011), hamouz et al. (2008), and valcalcer et al. (2015), the aa content in potato tubers after harvest is dependent on the variety, place of cultivation, and environmental conditions during growth. according to hamouz et al. (2008), the aa content in raw potatoes can vary from 14 to 1.093 mg·g-1 dm, while according to burgos et al. (2009), it can vary from 295 to 1.677 mg·g-1 dm in the current study, the highest aa content was detected in potato tubers after harvest (light yellow-fleshed potato, 1.142 mg·g-1 dm) (table 3). flesh colour significantly affected aa content in tubers. we observed significant differences in aa content between tubers with yellow-coloured flesh (1.100 mg·g-1 dm) and cream-coloured flesh (0.952 mg·g-1 dm). by contrast, hejtmánkova et al. (2009) showed that white and yellow potatoes did not differ in terms of aa content, whereas hamouz et al. (2008) determined that aa content was 2.9-times higher in red and purple potatoes than in potatoes with yellow and white flesh. valcalcer et al. (2015) studied 5 potato varieties with white flesh, 7 with yellow flesh, 20 with light-yellow flesh, and 25 with cream-coloured flesh and found that the aa content in tubers is determined by both environmental conditions (location, climate) and variety. in the current study, the highest aa content was detected in raw tubers of the altesse variety (yellow flesh) and the lowest was detected in potatoes of the ametyst variety (cream-coloured flesh). the difference in aa content between these varieties reached 26%. ital. j. food sci., vol. 32, 2020 783 short-term storage at both 5°c and 8°c significantly affected the aa content in raw potatoes. potato tubers contained approximately 40% more aa immediately after harvest than potatoes stored for 3 months. consistent with this finding, keijbets and ebbenhorst-seller (1990) recorded aa losses of 20–60% in potatoes during the first 4 months of storage. such patterns were also observed by abong et al. (2001), who showed that the losses of aa in raw potatoes were much higher during the first months of storage than during the final storage period. similar studies were carried out by grudzińska and zgórska (2011), who showed that in potato tubers stored through autumn (up to 90 days), aa losses reached 10%, while in potatoes stored through winter (up to 150 days), aa losses reached 22%. rivero et al. (2003) showed that the reductions of aa levels after 20 weeks of storage (140 days) reached 50% in some varieties. we found that aa contents were significantly lower in tubers stored at 5°c (0.702 mg·g-1 dm) vs. 8°c (0.967 mg·g-1 dm). similar associations were observed by külen et al. (2013) who recorded a loss of aa in raw potato tubers in the first months of storage at 4°c. these observations are consistent with the conclusions of sapei and hwe (2014), whose study on the kinetics of vitamin c degradation revealed that losses of vitamin c in products stored at lower temperatures could be reduced by the simultaneous presence of sucrose. potatoes stored at lower temperatures accumulate this sugar grudzińska at el. (2016). no interactions between flesh colour and the time of tuber storage in relation to storage temperature have been demonstrated. statistical analysis of our results revealed that boiling had a significant effect on aa contents in potato tubers (table 4). the greatest changes in aa content were observed in boiled potatoes directly after harvest (~40% loss of aa), while the smallest changes were observed in boiled potatoes after 3 months of storage at 5°c. in addition, peeling tubers before thermal processing increased the losses of aa by approximately 7%. peeling had a significant effect on the aa content in boiled tubers with cream-coloured flesh after harvest (peeled 0.715 mg·g-1 dm; unpeeled 0.466 mg·g-1 dm) and in yellow-coloured potatoes (peeled 0.607 mg·g-1 dm; unpeeled 1.009 mg·g-1 dm). these differences were not observed in tubers that were boiled after storage. similar association were noted by rytel and lisińska (2007). lachman et al. (2013) detected significant losses (up to 69%) of aa in boiled peeled potatoes compared to raw tubers. in the current study, such large differences were not observed. the greatest differences between aa losses were recorded after harvest in potatoes with yellow and light-yellow flesh that were peeled and boiled (44% and 46%, respectively). statistical analysis of the results revealed significant interactions between flesh colour, cooking, and storage (table 4). the lowest aa content was detected in unpeeled boiled potatoes with cream-coloured flesh after harvest (0.466 mg·g-1 dm), while the highest aa content was detected in unpeeled boiled yellow-coloured potatoes after harvest (1.009 mg·g-1 dm) and in peeled boiled yellow-coloured potatoes after storage at 8°c (0.955 mg·g-1 dm). ital. j. food sci., vol. 32, 2020 784 table 3. aa content [mg g -1 dm] in potato tubers under different treatments. variety after harvest after storage 5°c 8°c r aw after cooking m ea n r aw after cooking m ea n r aw after cooking m ea n u np ee le d p ee le d u np ee le d p ee le d u np ee le d p ee le d cream-coloured flesh ametyst 0.889 0.455 0.685 0.676ab 0.695 0.540 0.615 0.616a 0.840 0.800 0.740 0.793bc aruba 1.016 0.518 0.746 0.760b 0.655 0.524 0.592 0.590a 0.885 0.592 0.743 0.740b mean 0.952 0.466a 0.715c 0.711 0.675 0.532b 0.603bc 0.603 0.862 0.696c 0.741c 0.767 light yellow flesh ingrid 1.344 0.786 0.585 0.905cd 0.798 0.559 0.560 0.639a 0.986 0.740 0.659 0.795bc flaming 0.941 0.482 0.647 0.690ab 0.468 0.603 0.533 0.534a 0.821 0.389 0.879 0.696ab mean 1.142 0.634bc 0.616bc 0.797 0.633 0.581b 0.546b 0.587 0.903 0.564b 0.769c 0.746 yellow flesh altesse 1.147 0.830 0.614 0.863c 1.014 0.705 0.624 0.781bc 1.132 0.816 0.860 0.936d elanda 1.054 1.188 0.601 0.947d 0.582 0.823 0.750 0.718b 1.136 0.920 1.051 1.035e mean 1.100 1.009e 0.607bc 0.906 0.798 0.764c 0.687c 0.750 1.134 0.868d 0.955e 0.986 mean 1.065 0.703 0.646 0.702 0.626 0.612 0.967 0.709 0.822 mean of cooked 0.674a 0.619 a 0.765 b homogenous groups are denoted by letters (a, b, c and a, b, c). means with different letters are significantly different. a, b, c means with different letters are significantly different between varieties after harvest and storage. a, b, c means with different letters are significantly different between varieties with different flesh colours after harvest and storage. ital. j. food sci., vol. 32, 2020 785 table 4. sources of variation and anova results for aa content in potato tubers under different treatments (statistical analyses of the data shown in table 3). sources of variation anova results sum of the squares degrees of freedom mean square f statistic p-value significance variety (v) 0.965 5 0.193 16.363 0.0001 *** storage temperature (s) 0.603 2 0.302 25.562 0.0001 *** boiling (b) 0.920 2 0.460 38.982 0.0025 ** (v) × (s) 0.382 10 0.038 3.237 0.0001 *** (v) × (b) 0.558 10 0.056 4.733 0.0001 *** (s) × (b) 0.544 4 0.136 11.518 0.0001 *** (v) × (s) × (b) 0.887 20 0.044 3.759 0.0001 *** flesh colour (fc) 0.769 2 0.384 21.372 0.0001 *** (fc) × (s) 0.076 4 0.019 1.051 0.3861 n.s (fc) × (b) 0.233 4 0.058 3.238 0.0162 * (fc) × (s) × (b) 0.332 8 0.041 2.307 0.0279 * n.s. not significant; *, significant at α=0.05; **, significant at α=0.01; ***, significant at α=0.001. 3.2. total phenolics (tps) table 5 shows the tp contents in the potato tubers under different treatments. flesh colour had a significant effect on tp content. the highest tp content was found in tubers with yellow flesh both after harvest and after storage (3.766 mg·g-1 dm after harvest to 6.350 mg·g-1 dm after storage). the tp content was significantly lower in tubers with cream-coloured flesh (f2.633 to 3.831 mg·g-1 dm) and light-yellow flesh (3.363 to 4.335 mg·g-1 dm). similar results were obtained by valcarcel et al. (2015) and tierno et al. (2015). the differences in tp contents between raw tubers with yellowand cream-coloured flesh were 1.125 mg·g-1 dm for tubers after harvest, 1.268 mg·g-1 dm for tubers stored at 5°c, and 2.520 mg·g-1 dm for tubers stored at 8°c. for raw tubers with light-yellow and yellow flesh, the differences in tp contents were much lower, ranging from 0.395 mg·g-1 dm after harvest to 2.016 mg·g-1 dm after storage at 8°c. differences in tp contents between tubers with different flesh colours (yellow, light yellow, and cream) were no greater than 33%. lachman et al. (2008) observed much greater differences in tp contents (58%) between potato tubers with purple and yellow flesh. storage temperature significantly affects tp contents in tubers (grudzińska and zgórska, 2011, külen et al., 2013). we found that the tp content was significantly higher in tubers stored at the higher temperature (8°c) (3.830 to 6.350 mg·g-1 d.m) than at 5°c (2.982 to 4.250 mg·g-1 d.m). kumar and ezekiel (2009), grudzińska and zgórska (2011), and grudzińska and barbaś (2017) found that at high storage temperatures, tubers germinate more frequently and lose turgor. al weshahy et al. (2013) found that the tp content in tubers significantly decreased during the first 4 weeks of storage (regardless of temperature) and significantly increased after 8 weeks of storage. at the higher storage temperature (8°c), we observed significant variation in the ital. j. food sci., vol. 32, 2020 786 differences in tp content, with the greatest differences observed in raw tubers with lightyellow flesh (ingrid and flaming) and yellow flesh (altesse and elanda). table 5. tp contents [mg g -1 dm] in potato tubers under different treatments. variety after harvest after storage 5°c 8°c r aw after cooking m ea n r aw after cooking m ea n r aw after cooking m ea n u n p e el e d p e el e d u n p e el e d p e el e d u n p e el e d p e el e d cream-coloured flesh ametyst 2.433 3.043 2.511 2.662a 2.907 5.414 4.194 4.171de 3.455 4.125 3.875 3.818cd aruba 2.834 3.242 2.461 2.845ab 3.058 6.105 4.134 4.432ef 4.208 4.963 3.316 4.162de mean 2.633 3.142 2.486 2.754b 2.982 5.760 4.164 4.302cd 3.831 4.544 3.595 3.990c light yellow flesh ingrid 3.547 4.029 2.916 3.497bc 3.980 6.869 4.726 5.191ef 5.106 6.138 4.400 5.214ef flaming 3.180 2.786 1.978 2.648a 2.127 5.102 2.802 3.343bc 3.565 4.276 2.643 3.494bc mean 3.363 3.407 2.447 3.072bc 3.053 5.985 3.764 4.267cd 4.335c 5.207 3.521 4.354cd yellow flesh altesse 3.510 3.870 3.847 3.742bc 4.316 7.067 5.904 5.762fg 5.343 6.276 4.790 5.469ef elanda 4.007 4.075 3.257 3.779bc 4.184 7.747 5.118 5.683f 7.363 8.938 5.047 7.116g mean 3.758 3.973 3.552 3.761c 4.250 7.407 5.511 5.722d 6.35l 7.607 4.918 6.292d mean 3.251 3.507 2.828 3.428 6.384 4.479 4.840 5.786 4.011 mean of cooked 3.167 a 5.431 b 4.898 ab homogenous groups are denoted by letters (a, b, c and a, b, c). means with different letters are significantly different. a, b, c means with different letters are significantly different between varieties after harvest and storage. a, b, c means with different letters are significantly different between varieties with different flesh colours after harvest and storage. in unpeeled tubers, tp contents were significantly higher in cooked vs. raw potatoes, regardless of flesh colour and whether the tubers were stored or analysed after harvest. similar results were obtained by navarre et al. (2010) and burgos et al. (2013), who reported an increase in the contents of phenolic compounds in potato tubers after cooking, which varied depending on the method of cooking (boiling, steaming, baking). according to blessington et al. (2010), the increase in tp contents in potato tubers after cooking may be related to the higher extractability of these compounds from the cooked tuber cell matrix compared to the uncooked matrix. in the current study, the greatest difference in tp content was detected between raw and unpeeled cooked tubers after storage at 5°c (2.778 to 3.157 mg·g-1 dm, respectively) regardless of flesh colour, and the smallest difference was detected in tubers after harvest (0.044 to 0.509 mg·g-1, respectively). higher tp levels were observed in unpeeled boiled tubers regardless of whether they were stored at either temperature or measured immediately after harvest. tp contents were significantly lower in peeled vs. unpeeled potatoes. similar results were obtained by lechmana et al. (2008), who measured 30.4–38.7% differences in phenolic compound ital. j. food sci., vol. 32, 2020 787 contents as a result of peeling. similarly, dao and fridman (1992) detected substantial amounts of phenolic compounds just below the skin to ~2 mm into the flesh of potato tubers. tierno et al. (2015) reported that peeling allows for the migration and degradation of phenolic compounds during cooking. therefore, cooking potatoes without peeling them is an effective method for reducing the loss of phenolic compounds. statistical analysis of our results showed no significant interaction between the factors flesh colour, cooking, and storage (table 6). table 6. sources of variation and anova results for tp content in potato tubers under different treatments (statistical analysis of the data shown in table 5). source of variation anova results sum of the squares degrees of freedom mean square f statistic p-value significance variety (v) 73.607 5 14.721 167.538 0.0001 *** storage (s) 60.932 2 30.466 346.723 0.0001 *** boiling (b) 51.210 2 25.605 291.401 0.0001 *** (v) × (s) 14.851 10 1.485 16.901 0.0001 *** (v) × (b) 7.792 10 0.779 8.867 0.0001 *** (s) × (b) 32.893 4 8.223 93.587 0.0001 *** (v) × (s) × (b) 5.495 20 0.275 3.127 0.0050 *** flesh colour (fc) 52.628 2 26.314 55.612 0.0001 *** (fc) × (s) 7.068 4 1.767 3.734 0.0077 ** (fc) × (b) 1.796 4 0.449 0.949 0.4401 n.s (fc) × (s) × (b) 2.821 8 0.353 0.745 0.6514 n.s n.s., not significant; *, significant at α=0.05; **, significant at α=0.01; ***, significant at α=0.001. 3.3. antioxidant activity table 7 shows changes in antioxidant activity in potato tubers under different treatments. flesh colour had no significant effect on the antioxidant activity of raw tubers. higher antioxidant activities were observed in potatoes with light-yellow flesh after harvest (0.589 µmol te·g-1), and lower antioxidant activities were observed in potatoes with creamcoloured flesh (0.460 µmol te·g-1), but these differences were not significant (tables 7 and 8). after harvest, the antioxidant activity ranged from 0.460 µmol te·g-1 in cream-coloured tubers to 0.554 µmol te·g-1 in tubers with yellow flesh. after 3 months of storage at both temperatures, the antioxidant activity of tubers significantly decreased by 0.264 µmol te·g1 for light-yellow tubers and 0.168 µmol te·g-1 for cream-coloured tubers. similar observations were made by rosenthal and jansky (2008), who detected higher antioxidant activity in potato tubers directly after harvest than after storage. according to their study, antioxidant activity did not changed after up to 135 days of storage. in the current study, significant changes in antioxidant activity were observed in tubers after 90 days of storage at 5°c. at the higher storage temperature (8°c), the antioxidant activity in potatoes was similar to that after harvest. ital. j. food sci., vol. 32, 2020 788 table 7. antioxidant activity [µmol te·g-1] in potato tubers under different treatments. variety after harvest after storage 5°c 8°c r aw after cooking m ea n r aw after cooking m ea n r aw after cooking m ea n u np ee le d p ee le d u np ee le d p ee le d u np ee le d p ee le d cream-coloured flesh ametyst 0.553 0.348 0.442 0.447 bc 0.145 0.354 0.267 0.255 a 0.557 0.388 0.496 0.480 c aruba 0.429 0.718 0.294 0.480 c 0.240 0.354 0.395 0.329 a 0.496 0.685 0.321 0.500 cd mean 0.481c 0.533d 0.368b 0.460 0.192a 0.354b 0.331b 0.292 0.526cd 0.538d 0.408bc 0.490 light yellow flesh ingrid 0.678 0.990 0.348 0.672 d 0.297 0.111 0.476 0.294 a 0.462 0.449 0.233 0.380 b flaming 0.456 0.641 0.405 0.500 cd 0.432 0.294 0.347 0.357 ab 0.307 0.429 0.503 0.413 bc mean 0.567d 0.815e 0.376b 0.589 0.362b 0.202a 0.411bc 0.325 0.384cd 0.439d 0.368bc 0.397 yellow flesh altesse 0.440 0.523 0.532 0.498 c 0.294 0.381 0.321 0.332 ab 0.350 0.510 0.489 0.449 bc elanda 0.746 0.577 0.510 0.611 d 0.415 0.361 0.422 0.399 b 0.658 0.617 0.368 0.547 cd mean 0.593d 0.550d 0.521cd 0.554 0.354b 0.371b 0.371b 0.365 0.504cd 0.563d 0.428c 0.498 mean 0.550 b 0.632 c 0.421 ab 0.303 a 0.309 a 0.371 a 0.471b 0.513b 0.401ab mean of cooked 0.527 c 0.340 a 0.457 b homogenous groups are denoted by letters (a, b, c and a, b, c). means with different letters are significantly different. a, b, c means with different letters are significantly different between varieties after harvest and storage. a, b, c means with different letters are significantly different between tubers with different flesh colours after harvest and storage. ital. j. food sci., vol. 32, 2020 789 table 8. sources of variation and anova results for antioxidant activity in potato tubers under different treatments (statistical analysis of the data shown in table 7). sources of variation anova results sum of the squares degrees of freedom mean square f statistic p-value significance variety (v) 0.164 5 0.033 156.747 0.0001 *** storage (s) 0.788 2 0.394 1876.919 0.0001 *** boiling (b) 0.135 2 0.068 322.795 0.0001 *** (v) × (s) 0.252 10 0.025 120.186 0.0001 *** (v) × (b) 0.309 10 0.031 147.103 0.0001 *** (s) × (b) 0.245 4 0.061 291.976 0.0001 *** (v) × (s) × (b) 0.705 20 0.035 168.099 0.0001 *** flesh colour (fc) 0.063 2 0.031 2.778 0.6810 n.s (fc) × (s) 0.149 4 0.037 3.303 0.0147 ** (fc) × (b) 0.016 4 0.345 0.345 0.8471 n.s (fc) × (s) × (b) 0.304 8 3.379 3.379 0.0022 ** n.s., not significant; *, significant at α=0.05; **, significant at α=0.01; ***, significant at α=0.001. the cooking of peeled tubers led to a decrease in antioxidant activity regardless of flesh colour and storage condition. the antioxidant activity was significantly higher in cooked unpeeled tubers (0.632 µmol te·g-1) than in raw potatoes after harvest (0.550 µmol te·g-1) and after storage at 8°c (0.512 µmol te·g-1). according to blessington et al. (2010), perli et al. (2012), and lachman et al. (2013), cooking changes the antioxidant activity in tubers. these changes depend on the cooking method and pre-treatment of potatoes (peeling). as a result of peeling, the contents of the phenolic compounds flavonoids, flavones, anthocyanins, and lutein in potato tubers decreased by approximately 46–54%, leading to a significant reduction in antioxidant activity in tubers after cooking (perla et al., 2012). nara et al. (2006) indicated that potato tuber peels have high antioxidant activity, suggesting they could be used as a new source of natural antioxidants. statistical analysis of our results revealed a significant effect of variety on antioxidant activity (table 8). the highest antioxidant activity was detected in raw tubers of the elanda variety directly after harvest (0.748 µmol te·g-1) and in unpeeled cooked tubers of the aruba variety (0.718 µmol te·g-1), while the lowest antioxidant activity was detected in raw tubers of the ametyst variety (0.145 µmol te·g-1) after storage at 5°c and in unpeeled cooked tubers of the ingrid variety (0.111 µmol te·g-1). 3.4. correlation analysis the correlation between phenolic compound content and antioxidant activity is widely known (lachman et al., 2008, reyes et al., 2005, rumboa et al., 2009, teow et al., 2007, nzaramba et al., 2013). the correlation coefficients between the parameters examined in the above-mentioned studies ranged from 0.430 (teow et al., 2007, alweshahy et al., 2013) to 0.930 (reddivari et al., 2007), with the size of the coefficient ital. j. food sci., vol. 32, 2020 790 depending on the research material, crop location, and climatic conditions during the growing season. table 9 shows the coefficient of determination and convergence coefficient between antioxidant activity and the aa and tp contents depending on flesh colour. the coefficient of determination between (aa) × (te) in raw potatoes ranged from r2 = 0.563 for light-yellow tubers to r2 = 0.737 for yellow tubers. table 9. determination and convergence coefficients between antioxidant activity (te) and l-ascorbic acid (aa) and total phenolic compounds (tp) contents in raw, unpeeled, and peeled potatoes after cooking depending on flesh colour. flesh colour (aa)×(te) (tps)×(te) raw unpeeled peeled raw unpeeled peeled determination coefficient (r2) cream 0.688*** 0.758*** 0.743*** 0.109n.s 0.019n.s 0.182n.s light yellow 0.563** 0.277n.s 0.702*** 0.646** 0.781*** 0.266n.s yellow 0.737*** 0.197n.s 0.613** 0.793*** 0.669*** 0.399 n.s convergence coefficient (φ2) cream 31.2 24.2 25.7 89.1 98.1 81.8 light yellow 43.7 72.3 29.8 35.4 21.9 73.4 yellow 26.3 80.3 38.7 20.7 33.1 60.1 n.s., not significant; *, significant at α=0.05; **, significant at α=0.01; ***, significant at α=0.001. in unpeeled cooked tubers, significant dependencies (r2 = 0.758) were observed only in tubers with cream-coloured flesh. in potatoes with yellow and light-yellow flesh, such relationships were not observed (r2 = 0.197; r2 = 0.277, respectively). other dependencies were observed for peeled cooked potatoes: regardless of flesh colour, the determination coefficients were statistically significant (0.613 to 0.743). the highest convergence of features was observed for peeled and unpeeled cooked tubers with cream-coloured flesh (24.2 and 25.7%, respectively). different relationships were obtained for the (tps) × (te) model. for cream-coloured tubers, the determination coefficient (r2) was not statistically significant (0.019 for unpeeled cooked tubers to 0.182 for peeled cooked tubers). the convergence coefficient ranged from 98 to 81%, indicating that the antioxidant activity in cream-coloured tubers was shaped by other factors and not by tp content. non-significant relationships (0.182 to 0.399) were also obtained for peeled cooked tubers irrespective of flesh colour. these results are consistent with the finding of dao and fridman (1992) that significant amounts of phenolic compounds are present just below the peel to ~2 mm of the potato tuber. seijo-rodríguez et al. (2018) found that due to the high correlation between tp content and antioxidant activity, tp content could be used as an indicator of the antioxidant activity of a tuber. our findings do not fully confirm this notion, as this indicator could be determined only in raw and unpeeled cooked tubers but would be difficult to determine using peeled cooked tubers due to the different level of phenolic compound accumulation under the peel. ital. j. food sci., vol. 32, 2020 791 fig. 1 shows the relationship between the contents of aa and tps and antioxidant activity in raw tubers and in unpeeled and peeled cooked tubers regardless of flesh colour. the higher the aa content in boiled peeled potatoes, the higher the antioxidant activity (fig. 1). figure 1. the relationship between antioxidant activity and the contents of l-ascorbic acid and total phenolic compounds in raw potatoes and in unpeeled and peeled potatoes after cooking. the correlation coefficient between antioxidant activity and aa content for peeled boiled potatoes was r2 = 0.411. hejtmánková et al. (2009) did not detect a significant correlation between aa content and antioxidant activity (r2 = 0.08) in unpeeled raw potatoes. we obtained similar results, but our results were for unpeeled boiled potatoes (r2 y = 0,4196x + 0,5111 r² = 0,1697 y = 0,7739x + 0,3885 r² = 0,4112 y = 1,1335x + 0,472 r² = 0,7963 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 0 0,2 0,4 0,6 0,8 1 1,2 l-ascorbic acid [mg g-1 d.m.] antioxidat activity [µmol te·g-1 f.m] non-peeled potatoes peeled potatoes raw potato liniowy ( non-peeled potatoes) liniowy ( peeled potatoes) liniowy (raw potato) y = -3,8566x + 6,7608 r² = 0,284 y = -5,2317x + 6,4 r² = 0,4095 y = 7,805x + 1,33 r² = 0,6761 0 1 2 3 4 5 6 7 8 9 10 0 0,2 0,4 0,6 0,8 1 1,2 phenolic compouds [mg·g-1 d.m] antioxidant activity [µmol te·g-1] non-peeled potatoes peeled potatoes raw potato liniowy (non-peeled potatoes) liniowy ( peeled potatoes) liniowy (raw potato) ital. j. food sci., vol. 32, 2020 792 = 0.169). the highest correlation for aa content and antioxidant activity was obtained for raw tubers (r2 = 0.796). in the current study, the correlation between the content of tp compounds and antioxidant activity was inversely proportional in boiled potato tubers (fig. 1): the lower the tp content, the higher the antioxidant activity. a similar relationship was detected by rumboa et al. (2009) (r2 = 0.56). such irregularities might indicate that the antioxidant activity in tubers after boiling is also shaped by other bioactive compounds. there was a significant dependence between tp content and antioxidant activity in potatoes after boiling for unpeeled cooked tubers. in peeled cooked tubers, such relationships were not observed (r2 = 0.023) (fig. 1b). al-weshahy and rao (2009) showed that phenolic compound levels are often higher just below the peel of potato tubers than deeper inside the tuber, but this depends on the variety and the colour of the peel itself. however, according to nara et al. (2006), phenolic compounds found in the peel (in both free and bound form) show high antioxidant activity, whereas those in the flesh show low antioxidant activity. 4. conclusions here we demonstrated that flesh colour has a significant effect on aa and tp contents in tubers. the difference between the aa contents was 12.5% in creamvs. yellow-coloured tubers and 16.5% between creamand light-yellow-coloured tubers. differences in tp contents between potatoes with different flesh colours did not exceed 33%. significant aa loses were found in peeled boiled yellow and light-yellow tubers after harvest (44 and 46%, respectively). significant interactions were observed between aa contents and flesh colour, boiling, and storage. both storage treatments significantly decreased the antioxidant activity of tubers irrespective of flesh colour. boiling significantly decreased antioxidant activity in peeled tubers regardless of flesh colour and storage treatment. unpeeled boiled tubers had significantly higher antioxidant activity than raw tubers after harvest. regardless of flesh colour, high correlations were observed between (aa) × (te) for peeled boiled tubers. significant r2 values were observed between (tps) × (te) for raw and unpeeled boiled light-yellow and yellow tubers. the relationship between tp content and te in potatoes after boiling was significant for unpeeled tubers. the greatest matching of the model characteristics of the interdependence of features (φ2) was 75% for the model (aa) × (te) and 80% for the model (tps) × (te). abbreviations aa ascorbic acid. dm dry matter. tps total phenolics. te antioxidant activity, trolox equivalents. references abong g.o., m.w. okoth j.k. and imungi kabira j.n. 2001. losses of ascorbic acid during storage of fresh tubers, frying, packaging and storage of potato crisps from four kenyan potato cultivars. am. j. food technol. 6:772-780. ital. j. food sci., vol. 32, 2020 793 al-weshahy a. and venket rao a. 2009. isolation 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journal of food composition and analysis. 41:58-65. teow ch.c., truong van-den, mcfeeters r.f., thompson r.l., pecota k.v. and yencho g.c. 2007. antioxidant activities, phenolic and ß-carotene contents of sweet potato genotypes with varying flesh colours. food chem. 103:829-838. welch r.w., price r.k., lee a.m. and strain j.j. 2005. uptake and antioxidant activity of oat and wheat phenolics in humans. w: icc (eds) cereals the future challenge. book of abstracts 15-15. valcarcel j., reilly k., gaffney m. and nora o’brien n. 2015. total carotenoids and l-ascorbic acid content in 60 varieties of potato (solanum tuberosum l.) grown in ireland potato research. 58:29-41. doi: doi.org/10.1007/s11540-0149270-4 paper received november 30, 2019 accepted september 11, 2020 ijfs#1077_bozza ital. j. food sci., vol. 30, 2018 362 paper the eco-efficiency of the dairy cheese chain: an italian case study m.b. forleo*a, n. palmieria and e. salimeib adepartment of economics, university of molise, via f. de sanctis, 86100 campobasso, italy bdepartment of agriculture, environment and food sciences, university of molise, via f. de sanctis, 86100 campobasso, italy *corresponding author: tel. +39 0874404454 *e-mail address: forleo@unimol.it abstract the eco-efficiency of mozzarella cheese production was investigated in two dairy chains that differ in liquid whey recycling, with whey recycling (b) and without whey recycling (a), in cow diets. the total eco-efficiency (total gva/total gwp) for 1 kg of mozzarella cheese ranged from € 0.19 (b) to € 0.16 per kg co2-eq (a). the cheese-making phase of each diet accounted for about 3% of gwp total emissions. the mozzarella cheese making phase had the highest eco-efficiency ratio, while the milk production phase showed the lowest economic value and the highest impact. findings suggest improvements in reducing the environmental burden of the primary phase while increasing its economic value. keywords: carbon footprint, cheese whey recycling, eco-efficiency ratio; economic value added, mozzarella cheese production ital. j. food sci., vol. 30, 2018 363 1. introduction food supply chains are increasingly associated with environmental impacts, and this has brought global attention to the sustainability of the agri-food systems (fantozzi et al., 2015). dairy products have a great impact, especially in terms of resource depletion and greenhouse gas emissions (gonzález-garcía et al., 2013). furthermore, the dairy industry is considered responsible for a significant impact due to the characteristics of its wastewaters and effluents (mirabella et al., 2014). solid waste treatment and wastewater treatment along the dairy chain affect several environmental indicators. cheese whey is the main pollutant generated from cheese production that can cause several environmental impacts (prazeres et al., 2012). thus, cheese whey cannot be discharged directly into the environment without appropriate treatment. according to some authors (succi et al., 1986), apart from potential environmental benefits, liquid whey is also an interesting animal diet ingredient from an economic point of view, especially when distances from the cheese industry are short and costs of handling and transportation are high. in the framework of a circular economy approach, the reuse of whey in dairy cows' diet may minimize resources use and waste production from cheese making. in this regard, the european commission has recently adopted an action plan on the circular economy where the value of products, materials, and resources is maintained in the economy as long as possible, and the generation of waste is minimizedto develop a sustainable and competitive economy with low carbon content and efficient resource use. assessing the environmental performance of dairy chains can reduce their impacts and improve the efficiency of resource use (mu et al., 2017). life cycle assessment (lca) is a methodology widely used to investigate the environmental impact of food production. sala et al. (2017) underlined the importance of the environmental and socio-economic impacts associated with the food supply chains and indicated life cycle thinking and assessment as key elements in identifying more sustainable solutions for global food challenges. furthermore, notarnicola et al. (2015) deepened the issue of lca in the agri-food sector with case studies, methodological issues and best practices. existing literature reports several studies that addressed different topics related to the lca of cheese production. kim et al. (2013) conducted a us-based lca to determine the environmental impacts of cheddar, mozzarella cheese and dry whey from cradle-to-grave. gonzález-garcía et al. (2013) studied the life cycle of mature cheese production in portugal from a cradle-to-gate perspective and identified the environmental hotspots. palmieri et al. (2017) applied an lca approach to assess the impacts of mozzarella cheese production and evaluate the contribution of different strategies in a traditional dairy chain. global warming potential is one of the most studied impacts of dairy products. rotz (2018) reviewed the models for evaluating ghg emission from dairy farms —along a continuum from relatively simple models for single ghg emission sources to very detailed simulations over the whole farm production system— and concluded that lca is a comprehensive method for quantifying and evaluating the different sources of emissions over the full cycle. colombini et al. (2015) applied an lca cradle-to-farm-gate to assess the global warming potential of milk production in three forage systems scenarios and lactating cow diets. hawkins et al. (2015) estimated how the formulation of the ration and the associated land allocation decisions, contribute to reductions in ghg emissions of the intensive dairy production systems in ontario. van middelaar et al. (2013) studied the environmental effect of replacing grass silage with maize silage in a feeding strategy ital. j. food sci., vol. 30, 2018 364 and applied a life cycle assessment to predict ghg emissions at chain level. finally, finnegan et al. (2015) measured the global warming potential associated with the processing of raw milk into 11 dairy products in the republic of ireland following a cradle-to-processing factory gate boundary. a general result from literature suggested that raw milk production is the most impactful phase along the chain due to feed production and animal emissions. few studies dealt specifically with the environmental impact of mozzarella cheese production. two studies investigated the impact of american and canadian mozzarella cheese production (kim et al., 2013; vergé et al., 2013) by considering several impact categories. concerning the italian mozzarella product, a study (dalla riva et al., 2017) investigated a cradle-to-processing-gate lca of two types of mozzarella (the traditional one produced from raw milk, and the mozzarella obtained from curd) focusing mainly on transformation and consumption of mozzarella cheese, also dealing with different environmental impacts. a study by palmieri et al. (2017) focused on several impact categories of both farm and factory phases based on some study cases of the mozzarella production in italy. helmes et al. (2016) assessed the carbon footprint of an italian mozzarella facility dealing with the sensitivity of lca results according to different allocation choices. finally, falcone et al. (2017) applied the lca approach to assess the environmental effect of a shelf life extension technique in the lacto fermented italian mozzarella cheese production. under a wider sustainable perspective, the assessment of a dairy product should be extended beyond environmental impacts by considering its profitability and economic performance. recent studies started focused on the economic and environmental assessment of dairy products by using different approaches and focusing on minimising costs and/or on maximising profits. soteriades et al. (2016) proposed to combine the lca approach with the data envelopment analysis (dea) method in order to holistically assess dairy farm ecoefficiency by maximising output per unit of environmental impacts. kirilova and vaklieva-bancheva (2017) designed an optimal “green” portfolio for curd production in bulgaria to demonstrate the role of the environmental impacts measured in terms of wastewater and co2 emissionswithin a profit maximization function that includes the costs of the above impacts. murphy et al. (2017) compared male dairy calf-to-beef production systems based on different animal performance and applied economic profitability and ghg emissions models to highlight the best performing system per each perspective. hawkins et al. (2015) used an optimization model of ration formulation to determine how specific ghg targets can be reached while maximising net returns to an intensive dairy farming system. wettemann and latacz-lohmann (2017) estimated the potential costs and ghg emissions savings for a sample of 216 dairy farms in northern germany using an inputoriented data envelopment analysis and showed that cost and ghg emission reductions are complementary across a wide range. an economic approach focused on costs is also followed by huysveld et al. (2017) that analysed a sample of 103 specialized dairy farms in flanders (belgium) and showed potential simultaneous savings in costs and overall natural resource demand (up to 48%). falcone et al. (2017) applied a life cycle assessment and life cycle costing methods in order to assess the environmental and economic impacts of innovations in the lacto-fermented mozzarella cheese production in calabria region. finally, hessle et al. (2017) studied different production scenarios of the dairy chain in sweden by performing a life cycle method to assess the best environmental performance and by quantifying the costs in the primary production of dairy and beef to find out the most cost-efficient production models. ital. j. food sci., vol. 30, 2018 365 another approach that integrates economic and environmental assessment is based on the eco-efficiency ratio (saling, 2016). eco-efficiency is defined as economic efficiency combined with environmental benefits and deals with three main goals: the reduction of resource consumption, the reduction of environmental impacts, and the increase of product value. the concept of eco-efficiency has been applied to several agricultural products to estimate the value added per kg of ghg emitted into the atmosphere for each system studied. in the dairy sector, basset-mens et al. (2009) applied an eco-efficiency analysis of milk production in flanders. meul et al. (2007) studied the eco-efficiency of milk production in some flemish dairy farms, but the authors intended eco-efficiency in terms of ecologic and not economic terms and measured an indicator based on nitrogen and energy use efficiency. to the best of our knowledge, few studies considered the eco-efficiency of the dairy chain. a study measured the economic performance of the cheese production chain by calculating the gross value added (gva) of stages along the chain (van middelaar et al., 2011). another study (sanjuan et al.. 2011) measured the economic added value and the net income of mahon-menorca cheese production under different scenarios regarding technical and cleaner production criteria. however, that study included the assessment of the cheese production phase and excluded the milk production phase. a different approach to eco-efficiency was applied in a study that related the environmental performances with the economic efficiency in the use of dairy farms inputs (iribarren et al., 2011). this study aims to contribute to the literature on the environmental and economic performances of the mozzarella cheese production by measuring its eco-efficiency ratio based on an italian case study. the study answers the question, "how much value is added per kg of ghg emitted to the atmosphere?". firstly, the environmental and economic assessments were implemented; subsequently, the two perspectives were combined within an eco-efficiency analysis. in an earlier study (palmieri et al., 2017) an environmental analysis was performed according to a global approach. the present study goes further by focusing on the carbon footprint assessment and adding the analysis of the economic performance of mozzarella cheese production. 2. materials and methods 2.1. the environmental assessment 2.1.1 goal and scope definition the main purpose of the study was to calculate the eco-efficiency ratio of mozzarella cheese production based on raw milk produced following different feeding strategies. the environmental impact of the dairy cheese chain was based on ghg emissions, and the economic performances considered the gva of the dairy cheese chain. the value added per ghg emission of one kg of mozzarella cheese produced was finally measured. the carbon footprint (cf), an important index of the climate change impacts of food production within the whole supply chain (roma et al., 2015), was measured by an attributional life cycle assessment methodology (baitz, 2017; iso 14040, 2006; iso 14044, 2006). the cf of 1 kg of mozzarella cheese is defined as the sum of all ghgs emitted along the production cycle (röös et al., 2014). gwp is expressed in co2 equivalent (co2-eq) using weights of 1,28 and 265 for carbon dioxide (co2), methane (ch4) and nitrous oxide (n2o), respectively (assuming 100 years lifespan; ipcc, 2015). ital. j. food sci., vol. 30, 2018 366 furthermore, an economic analysis considered the added economic value of the dairy cheese chain as the difference between total revenues and total costs for intermediate consumption (van middelaar et al., 2011). intermediate consumption costs measure the value of goods and services consumed, including raw materials, services, and other operating expenses, other than fixed assets. the gva does not include labor costs, depreciation, nor interest loan payment; when considering the depreciation of fixed capital, a net value added is obtained. the gva indicator was chosen because it is frequently used to measure the economic sustainability of agricultural systems (van middelaar et al., 2011). the final goal of jointly assessing the environmental and economic performances in the case study was pursued by measuring the eco-efficiency ratio (gva/gwp) of mozzarella cheese production based on milk produced following different feeding strategies. 2.1.2 functional unit and system boundary the functional unit (fu) of the environmental and economic analysis was expressed per 1 kg of mozzarella cheese produced from 8.11 l of cow milk. the lca system boundary (fig. 1) refers to the first two phases of a dairy chain, namely the dairy farming and the cheese-making phases. the boundary considers: the dairy farm including the agricultural processes of feedstuffs and the whole life cycle of cows -; and the cheese factory -including all the activities that take place for the mozzarella cheese making, from the milk reception to the mozzarella production and the whole liquid whey disposal (the wastewater treatment plant or recycled into the cow diets). two dairy diets that differ in the usage/non-usage of liquid whey were assessed. in relation to the different disposal of the liquid whey, along with the two diets (a and b diets), two different chains are considered. in a chain, the whole amount of liquid cheese whey is mixed with the wastewater effluent from the mill and delivered to a municipal wastewater treatment plant (fig. 1). in the b chain, the whole amount of liquid whey produced at cheese-making level is delivered to the farm where it is used, after microbial stabilization, in animal feeding as partial substitute of drinking water. the physical allocation method was used in the baseline scenario to share the environmental burden between milk and meat at the farm level, while the environmental burden of the mozzarella production was totally allocated to curd (gonzález-garcía et al., 2013). the percentages of physical allocation at case farm level were 88% to milk and 12% to meat (as live weight cow and calf) (idf, 2015). the manure/slurry allocation was not necessary because farmyard manure was recycled as fertilizer in the feed cultivation. ital. j. food sci., vol. 30, 2018 367 figure 1. system boundaries: dairy farm and cheese factory. abbreviations: see table 1. table 1. list of abbreviations and acronyms. alca attributional life cycle assessment cf carbon footprint co2 carbon dioxide cu cereal unit allocation method ch4 methane fpcm fat and protein corrected milk fu functional unit gva gross value added ghg greenhouse gas emissions gwp global warming potential ipcc intergovernmental panel on climate lca life cycle assessment n2o nitrous oxide wwtp wastewater treatment plant ital. j. food sci., vol. 30, 2018 368 2.1.3 life cycle inventory data for the life cycle inventory analysis partly comes from the inlatte project (tables 2-4) and were collected through a questionnaire drawn according to the guidelines for the application of lca to food and agricultural products (neri, 2009). secondary data (table 5) were taken from both the ecoinvent database v. 3.0 (weidema et al., 2013) and literature (franchini and neri, 2004; neri and borsari, 2005; kim et al., 2013). primary data were collected from two firms (a dairy farm and a cheese factory) located in molise region (it). data from the case farm reported the milk quantity and quality, the italian friesian cow rations and water consumption, and the manure/slurry produced. the case farm experimented two different dietary strategies: a diet including ensiled forages and no liquid whey usage (a diet) and a diet including both silages and liquid whey (b diet). data reported in table 2 summarise the management of animals in the case farm. for the present study, 36 lactating cows were divided into two groups of 18 cows each which were homogeneous and comparable in terms of milk yield and days of lactation and parity. the average fat and protein corrected milk (fpcm) yield has been calculated on a 305 days basis for each experimental group and used in the lca study. the fpcm yield was calculated according to finnegan et al. (2015). table 3 shows the composition of the diets. in this regard, it is worth noting that feedstuffs were offered as total mixed rations, except for the microbiologically stabilized liquid cheese whey offered to b diet cows as partial substitute of drinking water. water consumption in b diet was, therefore, lower than that in the a diet. primary data from the cheese factory have been recorded throughout the experiment and summarised in table 4. mozzarella cheese for fresh consumption traditionally obtained directly and solely from liquid milk is the dairy product considered in the study. table 2. case farm characteristics. case farm data cow breed holstein friesian number of lactating cows 36 number of dry cows 9 dairy replacement calves and heifers, n. 32 number of calves (male) 18 days of production/year (lactating cows) 305 males raised as beef cattle, age (days) calves: 20 milk production diets milk yield – fpcm (kg/per yr) a 8,332 b 8,039 % fat a 4.03 b 3.99 % true protein a 3.68 b 3.60 ital. j. food sci., vol. 30, 2018 369 table 3. water consumption and characteristics of diets on a dry matter (dm) basis. diets calves diet a b water consumption (l/day) 10 10 liquid whey (kg/day) total dm intake (kg/ day) 1.96 1.96 heifers diet a b water consumption (l/day) 35 25 liquid whey (kg/day) 0.57 total dm intake (kg/ day) 4.53 5.10 lactating cow diet a b water consumption (l/day) 80 50 liquid whey (kg/day) 1.48 total dm intake (kg/ day) 20.06 21.54 dry cow diet a b water consumption (l/day) 40 40 liquid whey (kg/day) total dm intake (kg/ day) 13.08 13.08 when real data were not available, inventory data were collected from literature and ecoinvent database (v. 3.0) (weidema et al., 2013), as reported in table 5. emissions considered in the study were drawn from literature (table 6). data for the raw milk and whey transportation and for the wastewater treatment plant for whey disposal came from ecoinvent database. table 4. cheese factory data. products data kg of mozzarella produced by 8.11 l of milk 1 kg of whey produced by 1 kg of mozzarella 0,89 fat in mozzarella (g/kg of product) 185 protein in mozzarella (g/kg of product) 154 fat in whey (g/kg of product) 2 protein in whey (g/kg of product) 7 resources consumption electricity consumption (kwh/ kg of mozzarella) 0,20 heat consumption (mj/kg of mozzarella) 0,11 water consumption (l/kg of mozzarella) 18,08 data source: inlatte project. ital. j. food sci., vol. 30, 2018 370 table 5. secondary data considered in the study. source feed cultivation and processing barley ecoinvent database (v. 3.0) maize meadow hay milk powdered franchini and neri (2004); ecoinvent database (v. 3.0) mixed feed ecoinvent database (v. 3.0) mineral feed sugar beet pulp soybean meal 44% triticale silage mozzarella production milk reception ecoinvent database (v. 3.0); pasteurisation franchini and neri (2004); ecoinvent database (v. 3.0) heating, inoculation and coagulation ecoinvent database (v. 3.0) curd cutting curd transfer and ripening spinning and molding hardening and salting raw milk transportation ecoinvent database (v. 3.0) for diesel track of 16 t capacity. real distance from the dairy farm to the factory 10 km wastewater treatment ecoinvent database (v. 3.0); moderately large municipal wastewater treatment plant with a three-stage process (mechanical, biological and chemical) table 6. emissions considered in the study. emissions source enteric and animal housing emissions ch4 emissions and the ammonia emissions battini et al. (2016); emep/eea (2009) nitrous oxide (n2o) emissions from animal housing not considered according to battini et al. (2016) storage emissions emissions of methane (ch4) and nitrous oxide (n2o) dalla riva et al. (2014); ipcc (2006) (tier 2); using ispra (2008) methods ammonia (nh3) emissions due to manure/slurry storage falconi et al. (2011) using ispra (2008) method nitrogen oxides (nox) emissions battini et al. (2016) using the factor by ipcc (2006) emissions related to manure/slurry spreading n2o, nh3, nox and nitrate leaching battini et al. (2016) using ipcc (2006) the p leaching run-off emissions battini et al. (2016) emission factor of potassium, copper and zinc neri and borsari (2005) 2.2. economic assessment and eco-efficiency ratio of the dairy chain the eco-efficiency indicator is based on data from both environmental and economic accounting systems. the higher the indicator value, the higher the economic performance per unit of environmental burden. since ecological and economic data need to be derived ital. j. food sci., vol. 30, 2018 371 from the same data set (muller et al., 2015), we collected information based on the annual budget of the considered dairy farm and the cheese factory. the economic data for both stages, milk production and mozzarella cheese making, are shown in table 7. the b dairy chain had lower total costs than a chain due to both the elimination of treatment costs of whey in the wwtp and saved transportation costs of whey from the cheese factory to the dairy farm. the factory and the farm agreed to equally share the costs of both whey transportation (from the cheese factory to the dairy farm) and whey management at firm’s level. finally, the lower costs of b chain were due to the reduction of water consumption in the diet. the eco-efficiency analysis was applied to the two stages of mozzarella cheese production (i.e., milk production and mozzarella cheese-making phases). the eco-efficiency of each stage was computed by dividing its economic value added by its ecological impact (van middelaar et al., 2011). table 7. cheese factory and dairy farm economic data. economic data units cheese factory (€/kg of mozzarella) dairy farm (€/8,11 l of milk) a chain b chain a chain b chain gross revenue €/kg 6.10 6.10 4.00 4.00 variable and fixed costs €/kg 5.10 4.90 3.44 3.37 economic value added €/kg 1.00 1.20 0.56 0.63 source: data came from the dairy farm and cheese factory case studies. 2.3. sensitivity analysis: allocation method and variability of gva the choice of the allocation procedure for agricultural co-products may affect the results of lca study as discussed in flysjo et al. (2012) and helmes et al. (2016). both studies compared the dry matter and the economic allocation methods for assessing the impact of dairy industry and underlined the need for testing results against different approaches. for this reason, a sensitivity analysis for environmental impacts was performed by changing the allocation method of milk according to a cereal unit (cu) method (brankatschk and finkbeiner, 2014). this sensitivity analysis involved only the case farm level, as in many reported studies (fantin et al., 2012; gonzález-garcía et al., 2013; kim et al., 2013; van middelaar et al., 2011), because milk production is more impactful than cheese-making. the cu allocation method is based on the metabolizable energy content of product and co-product for feed purpose so that it allows considering agricultural products and co-products used in different sectors. the environmental burden was allocated 86.6% to milk, 6.8% to live-weight dairy cow and 6.6% to live-weight fattening male calf (brankatschk and finkbeiner, 2014). furthermore, if the economic dataset was based on the annual reports of the dairy farm and the cheese factory -and therefore are real and accurate-, a further sensitivity analysis was performed to estimate the effect a ±10% change of gva of the two stages for each dairy chain. ital. j. food sci., vol. 30, 2018 372 3. results and discussion 3.1. the carbon footprint of 1 kg of mozzarella: baseline allocation results of the environmental impact of 1 kg of mozzarella cheese showed that raw milk production was the most impactful phase along the considered supply chain, irrespective of the diet followed at the farm level (fig. 2). figure 2. carbon footprint of 1 kg of mozzarella cheese in a supply chain (on the left side) and b chain (on the right side): milk and mozzarella production (physical allocation). note: transport refers both to the milk delivered to the dairy factory (supply chain a and b) and to the liquid whey delivered to the dairy factory (b supply chain) or the wastewater treatment plant (wwtp; a supply chain). milk production was the most critical phase along the dairy chain, with contributions of 96% (a diet) and 97% (b diet) of the global warming potential (gwp). the high contribution of milk production phase to the environmental impact of the mozzarella dairy chain observed is consistent with the study of dalla riva et al. (2017), even considering the farm gate-to grave perspective followed by the authors. a similar conclusion was in the study of finnegan et al. (2015) that, although was based on different cheese product and fluid milk, showed that milk production contributes to gwp within 81% 97% range (depending on the amount of raw milk per kg of the six cheese products considered in the study). the remainder contribution being mainly due to the processing phase. the environmental impacts of milk production phase were due to emissions of both methane from the enteric fermentation process and dinitrogen monoxide and carbon dioxide from manure management and spreading, confirming the study of gonzálezgarcía et al. (2013) which referred to the cheese chain in portugal. methane from enteric fermentation and manure management was also the main ghg emission source in other studies dealing with cheese (kim et al., 2013) and milk production (vida and tedesco, 2017). in the studies of van middelaar et al. (2011) and santos et al. (2016), the enteric fermentation was the main emission source affecting gwp. according to van middelaar et al. (2011), the stage that contributed most to total global warming potential along the production chain of dutch semi-hard cheese was on-farm milk production (65%), mainly due to enteric fermentation. in a study by santos et al. (2016) about the cheese production in a small-sized dairy industry in brazil, the contributions of the raw milk production ranged from 70 to 98% depending on the different midpoint impact categories. ital. j. food sci., vol. 30, 2018 373 the cheese-making phase of each diet accounted for about 3% of gwp total emissions. mozzarella production phase showed impacts due to carbon dioxide from heat consumption during the cheese making process. this result confirms van middelaar et al. (2011) findings that measured the contribution of semi-hard cheese-making and packaging phases in about 3% 4% of gwp emissions, each. even in the study of helmes et al. (2016), the contribution from the processing step of mozzarella production was quite limited compared to raw milk and transport impacts. furthermore, in our study, impacts of transportation of both milk —from dairy farm to cheese factory— and whey, either from factory to the wastewater plant or from factory to the dairy farmwere negligible due to the close distance between the locations of the two firms involved, the farm and the factory. a similar result was reported in the study of finnegan et al. (2015) where liquid milk transportation contributed for less than 0.5%, whichever dairy products considered in the assessment. the relative burden of the wastewater treatment (in a diet) along the whole dairy chain was also considered insignificant. comparing impacts between the chains, results based on a cradle-to-processing-gate boundary showed that the b dairy chain had a cf 1% higher than the a chain per unit of product. the carbon footprint of mozzarella cheese in a chain was 9.65 kg co2-eq/kg mozzarella cheese, while it was 9.81 kg co2-eq /kg mozzarella cheese in b chain. the b dairy chain, although with the liquid whey usage, appeared to be a slightly worse solution due to a lower milk yield (8,039 kg fpcm) compared with a chain (8,332 kg), confirming that the environmental impact increases at decreasing milk yields (nemecek et al., 2011). study findings were similar to those reported in kim et al. (2013) where the carbon footprint of us mozzarella cheese was 9.30 kg co2-eq/kg. furthermore, the results of our study are consistent with the study of helmes et al. (2016), even if these authors considered different scenarios (mozzarella with ricotta or mozzarella with whey powder) from that of the present study. according to santos et al. (2016), gwp emissions of cheese production were 14.44 kg co2-eq/kg of product, while in vergé et al. (2013) the carbon footprint of canadian dairy products was significantly lower than the one assessed in this analysis. however, both studies cannot be directly compared to the present findings due to several differences related to the final cheese products, to the production process and different methodological choices. in our study, gwp emissions of mozzarella cheese-making phase were 0.32 kg co2-eq with a diet and 0.29 with b diet. these findings are quite in line with the study of finnegan et al. (2015) that calculated the gwp emission of six groups of dairy products (not mozzarella cheese) and showed that gwp emissions from the dairy processing phase ranged 0.11-2.5 kg co2-eq/kg according to the different groups of studied products. in conclusion, despite different environmental assessment methods used in literature, the milk production is the process that mostly contributed to the environmental impact. improvement alternatives at the dairy-farm level are therefore required, and they involve many aspects, among which is the use of fertilizers for feedstuffs cultivation. in this regard, koesling et al. (2017) assessed the variations in nitrogen utilisation of conventional and organic dairy farms in norway. these researchers concluded that, for both a dairy farm and system area, n-surpluses increased with increasing use of fertilizer n per hectare, biological n-fixation, and imported concentrates and roughages, while they decreased with higher production per area. pagani et al. (2016) investigated direct and indirect energy inputs in a sample of dairy farms -either grain-based, forage-based or organicand demonstrated that potential reduction in the overall energy input could be achieved by shifting to organic farming, switching to forage-based farming, and by promoting reduced use of fertilizers. both studies highlighted the importance of good agronomy that utilizes available nitrogen and reduces energy inputs properly. ital. j. food sci., vol. 30, 2018 374 other studies focused on improvements in the composition of dairy ration to mitigate the environmental impact. hawkins et al. (2015) suggested that feeding decisions have important implications for ghg emissions from intensive dairy production due to the wide variation in emissions from alternative crops that can be used in the ration. patra et al. (2011) reviewed several potential methane mitigation options such as animal interventions (i.e., number and productivity of animals or genetic selection), dietary interventions, suppression of rumen methanogens, and new potential technologies, by underlying areas worthy of investigation for ch4 mitigation and improvements most likely to be adopted by farmers. finally, white (2016) proposed a farm-scale diet optimization model to reduce land use, water use, and ghg emissions within dairy production systems and assessed how improved energy and protein use efficiency reduces the environmental impacts of dairy production systems. finally, improvements in the environmental profile of cheese production should also be directed at the dairy factory level, mainly due to a high-energy consumption of machinery used during the production process. however, according to van middelaar et al. (2013), mitigation strategies may be case-specific and must consider the level of the analysis –at animal, farm and chain level-. to achieve a sustainable mozzarella cheese production chain, not only its environmental impact must be considered and minimized, but also the economic value that is added along the chain. 3.2. the eco-efficiency of the dairy chain the total eco-efficiency (total gva/total gwp) of 1 kg of mozzarella cheese accounted for € 0.19 per kg co2-eq in the b supply chain and € 0.16 per kg co2-eq in the a supply chain (table 8). findings showed that dairy chain in case of b diet had a better eco-efficiency ratio per unit of ghg emitted to the atmosphere. table 8. carbon footprint and gross value added (gva) per functional unit (fu=1 kg mozzarella cheese), and eco-efficiency of the two stages in the dairy chain (physical allocation). stage gwp (kg co2-eq/fu) economic performance gva/fu (€) eco-efficiency total gva/ total gwp a chain b chain a chain b chain a chain b chain milk production 9.33 9.52 0.56 0.63 0.06 0.07 mozzarella cheesemaking 0.32 0.29 1.00 1.20 3.12 4.13 total 9.65 9.81 1.56 1.83 0.16 0.19 under the economic viewpoint, the b dairy chain had lower total costs than the a chain due to: 1) the elimination of treatment costs of whey in the wwtp at cheese factory level; 2) the reduction of water consumption due to whey usage in b diet; 3) finally, to lower transportation costs. the total value added for 1 kg of mozzarella cheese was € 1.56 for the a dairy chain and € 1.83 for the b chain. when considering the distribution of total gva along the chain, milk production accounted for a lower economic weigh (36 % in a chain and 34 % in b chain) compared to the value contribution of the cheese making process. for the above reasons, mozzarella cheese making had the highest eco-efficiency ratio for each dairy chain (€ 3.12 ital. j. food sci., vol. 30, 2018 375 in a chain and € 4.13 in b chain) and added the highest economic value per unit of environmental impact. the average gva per 1 kg of fat and protein correct milk (fpcm) for the milk production phase was € 0.56 (per 8.11 kg fpcm to produce 1 kg of mozzarella) for the a dairy chain and € 0.63 per (8.11 kg fpcm to produce 1 kg of mozzarella) for the b chain. our results were consistent with the van middelaar et al. (2011) study that calculated the economic performances of a cheese chain as defined in this study (i.e. gross value added per environmental impact of stages along a production chain) and showed that the milk production contributed 34% to the total gva of mozzarella cheese production. furthermore, the economic performance of mozzarella production phase accounted for € 1.00 for the a chain and for € 1.20 in the b supply chain, confirming the van middelaar et al. (2011) results that showed a gva of € 1.04 for the cheese-making phase. the above differences, while negligible, were likely due to both different local markets, products, and manufacturing costs and prices. for this reason, two sensitivity analyses were carried out to test eco-efficiency results against changes in the economic indicator and to test environmental results against an allocation method different from the one applied in the baseline analysis. 3.3. sensitivity analysis results results of the sensitivity analysis confirm previous results about the eco-efficiency of mozzarella cheese production. the first sensitivity analysis (table 9) showed that results from the cu allocation were lower than results achieved through a physical allocation for each dairy chain, but the differences in the value of the carbon footprint were negligible (around 1% for each chain). furthermore, comparing findings based on cu allocation for the two dairy chains, results were consistent with those presented in fig. 2 based on the physical allocation method (data are available on request). the b dairy chain confirmed its lower environmental performance. table 9. sensitivity results of the carbon footprint to the allocation method (physical and cu allocation). stage gwp (kg co2-eq/fu) physical allocation cu allocation a chain b chain a chain b chain milk production 9.33 9.52 9.18 9.37 mozzarella cheesemaking 0.32 0.29 0.32 0.29 total 9.65 9.81 9.50 9.66 fu=1 kg mozzarella cheese the second sensitivity analysis (table 10) was performed to estimate the effect of ±10% change of gva for each stage, for each dairy chain and each allocation method on the ecoefficiency ratio. compared with the baseline scenario, the ±10% change of gva modified the eco-efficiency scores in the range ±0.04 €/kg co2-eq, (e.g., from a score of 0.14 to 0.18 and from a score of 0.16 to 0.20 €/kg co2-eq, respectively in the a and b chains under the physical allocation method). finally, findings showed higher eco-efficiency values with a cu allocation than a physical allocation method. even in this case, results reported small changes in the absolute values of the eco-efficiency per 1 kg of mozzarella cheese and ital. j. food sci., vol. 30, 2018 376 showed that the best-performing dairy chains did not change. therefore, the dairy chain in case of b diet had the best eco-efficiency ratio per unit of ghg emitted to the atmosphere. from the two sensitivity analysis, it is possible to affirm that study results are not very much influenced by the choice between the two considered allocation methods, nor by the change in the economic value added. table 10. sensitivity results of the economic performance (±10% change of gva) and of the eco-efficiency scores in the two dairy chains (physical versus cu allocation and ±10% change of gva). change stage economic* performance gva/fu (€) eco-efficiency* scores gwa/gwp (€/kg co2-eq) physical allocation cu allocation a chain b chain a chain b chain a chain b chain +10% of gva milk production 0.62 0.69 0.06 0.07 0.07 0.08 mozzarella cheese-making 1.10 1.32 3.44 4.55 3.44 4.55 total 1.72 2.01 0.18 0.20 0.19 0.21 baseline scenario milk production 0.56 0.63 0.06 0.07 0.06 0.07 mozzarella cheese-making 1.00 1.20 3.12 4.13 3.13 4.14 total 1.56 1.83 0.16 0.19 0.17 0.20 10% of gva milk production 0.50 0.57 0.05 0.06 0.05 0.06 mozzarella cheese-making 0.90 1.08 2.81 3.72 2.81 3.72 total 1.40 1.65 0.14 0.16 0.15 0.17 *the different allocation method (physical or cu allocation) does not imply any variation in the economic performance (gva), while it influences the environmental assessment (gwp, as reported in table 9) and the eco-efficiency results (because the eco-efficiency is the ratio between total gva/total gwp). 4. conclusions in this paper, the eco-efficiency ratio of mozzarella cheese production is assessed in an italian case study according to the handmade cheese making system considering two different diets at the farm level, including or not including liquid cheese whey in cows’ diet. from an environmental point of view, one of the main findings of the study was that the primary phase had the highest impact within the mozzarella cheese supply chain. for the phases along the dairy chain, the mozzarella cheese making had the highest ecoefficiency ratio for each dairy chain and produced the highest economic value per unit of environmental impact. the milk production phase added the lowest value of total gva in both dairy chains while showing the highest environmental impact in ghg terms. to reduce the environmental impact of the dairy chain and the wastage of a mozzarella cheese co-product, we assessed the carbon footprint of two dairy chains changing the diet composition at case farm level and using the liquid whey in cows' diet. the study hypothesis was that the use of the by-product of mozzarella cheese production within the local dairy chain would provide benefits under both environmental and economic perspectives. from the environmental point of view, the b supply chain with the whey showed an environmental performance per unit of mozzarella cheese lower than that of the a chain, although in a negligible measure, due to the effect of the milk yield in the primary phase. however, when considering the economic assessment of the two diets, the comparison of the eco-efficiency indicator evidenced a better performance of the b chain whose value per unit of impact was higher thanks to the liquid whey recycling. ital. j. food sci., vol. 30, 2018 377 study findings lead to certain conclusions on the need of improving both sides of sustainability. on the economic side, improvements are needed in the market mechanisms to set costs and revenues that increase the value added along the dairy chain, mainly at the farm level. under an environmental perspective, based on the carbon footprint assessment, improvements in the milk production should provide practices and alternatives that can further reduce the primary phase emissions up to the limit allowed by the ruminant physiology. finally, the circularity in nature and economic cycles should be further analysed to improve the performances of both sides of sustainability. by recycling the liquid whey and strengthening the relation between dairy farms and cheese factories at a local level, some economic benefits (the cost of whey transportation and the disposal costs of liquid whey) emerged, while the environmental burden of whey treatment is avoided. the best scenario satisfying both environmental and economic goals would realise a reduction in costs related to efficiency improvements in the usage of natural resources and dairy chain by-products, and a lower environmental burden associated with production processes. concerning the revenues, the best scenario would be related to the attainment of a price premium for the environmental performances of the dairy products. for example by leveraging on marketing tools, such as environmental 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this identification has unlocked new possibilities for their application to wine quality evaluation. here, cyclic and non-cyclic b-type proanthocyanidins, along with other phenolic compounds as well as sensory and oenological parameters, were characterized in eleven pinot noir wines. the wines were produced from grapes harvested in different vineyards and under different winemaking conditions. with principal component analysis (pca) based on the cyclic proanthocyanidins or their relative proportions, it was possible to differentiate the wines according to specific winemaking conditions. moreover, cyclic proanthocyanidins were related to the overall sensory quality of pinot noir wines. keywords: pinot noir, cyclic proanthocyanidins, winemaking, phenolic profile, high-resolution massspectrometry, sensory analysis ital. j. food sci., vol. 32, 2020 338 1. introduction red pinot noir wine is a light-to-medium-bodied wine with a complex aroma profile (casassa et al., 2018). it is produced in several viticultural areas as well as in south tyrol (italy). several commercial frauds involving the marketing of pinot noir have been recorded. for instance, some producers were convicted in 2010 of mislabeling 13.5 million l of pinot noir wine that was replaced with cheaper wines made with merlot and syrah grape varieties (takeoka et al., 2011). for this reason, assessing the commercial quality of pinot noir wines and investigating a wider selection of authenticity markers became advisable. several studies have been proposed for comparative authenticity assessments of pinot noir and other wines. for example, south tyrolean pinot noir wines were differentiated from cabernet sauvignon using proton-transfer mass spectrometry analysis (spitaler et al., 2007). furthermore, the polyphenol content and antioxidant activity of nouveau wines made from pinot noir and other grape varieties (pellegrini et al., 2000) were studied. in addition, the comparison of the phenolic and sensory profiles of organic wines made from pinot noir grapes and other varieties was performed (lante et al., 2004). pinot noir showed a content of phenolic compounds (including phenolic acids) comparable to cabernet sauvignon and cabernet franc (van leeuw et al., 2014). however, pinot noir wines are lighter in color compared to other wines because of a lower total anthocyanin content (peterlunger et al., 2002). also, the content of tannins in pinot noir grapes is lower compared to other red wines (casassa et al., 2018; harbertson et al., 2008). phenolic compounds can be used to differentiate wines according to the winemaking technique (baiano et al., 2009, siren et al., 2015; zhang et al., 2018), grape variety (boselli et al., 2004; perestrelo et al., 2018; van leeuw et al., 2014), vintage (bellomarino et al., 2010; geana et al., 2016; giacosa et al., 2019), and geographical origin (granato et al., 2011; rocchetti et al., 2018; stockham et al., 2013). the anthocyanin profile is currently one of the most employed parameters for authenticity assessment studies (oiv, 2007; villano et al., 2017). however, anthocyanins as chemical markers have a limited application for several reasons: they can be applied only to red wines, and furthermore, during the aging of wine, anthocyanins are oxidized or transformed into oligomeric and polymeric pigments through condensation reactions with flavanols (he et al., 2012; zhang et al., 2018). thus, the anthocyanin content decreases in aged wines, and the assessment of the grape varieties used to make red wine may be difficult. for this reason, more stable chemical markers should be identified and investigated for authenticity purposes with respect to the grape variety. a recent study highlighted the presence of an unconventional cyclic b-type tetrameric procyanidin (also known as ‘crown’ procyanidin) in cabernet sauvignon, providing also its full structural characterization (zeng et al., 2019). several studies have also identified the profiles of cyclic b-type tetrameric, pentameric, and hexameric procyanidins and prodelphinidins in red and white wines (longo et al., 2018a,b,c; longo et al., 2019; merkyte et al., 2020), including pinot noir. the role of proanthocyanidins (pac) as chemical markers to evaluate wine quality and authenticity is promising, as their profile and the relative proportions of the different congeners were preliminarily found to be dependent on the grape variety used for winemaking (longo et al., 2018c; longo et al., 2019). besides, cyclic proanthocyanidins (c-pac) showed greater stability towards strongly acidic and depolymerising conditions in comparison to (conventional) non-cyclic proanthocyanidins (nc-pac) (zeng et al., 2019). these c-pac compounds showed also ital. j. food sci., vol. 32, 2020 339 more resistance than their nc-pac analogues towards fragmentation during mass spectrometric analysis (longo et al., 2018a). in this report, the profile of c-pac was studied in eleven pinot noir wines from the same winery but produced with different winemaking practices. the aim of this study was to investigate the profile of pac in these wines in relation to specific winemaking factors, such as the use of raisins or undesired stuck fermentations and the location of the vineyards. in addition, other phenolics and the sensory profiles were discussed. the results shed light on the possible role of c-pac in relation to the effects of specific winemaking practices or geographical location of the vineyards. 2. materials and methods 2.1. wine samples, chemicals, and materials eleven red dry wines obtained from 100% pinot noir grapes were produced and donated by a local winery (franz haas, montagna, bz, italy). the grapes were harvested in 2016 in different vineyards located between 350 and 800 m a.s.l. in trentino-south tyrol (italy). the mass of grapes obtained for each vinification was 3.5 t. the maceration lasted eight days at a constant fermentation temperature of 26°c. the samples differed for aspects such as the altitude, location, and orientation of the vineyards and for the winemaking practices as described in table 1. table 1. description of the eleven pinot noir wines in terms of vineyard, altitude, location, orientation, and winemaking techniques. wine vineyard altitude (a.s.l./m) location (orientation) winemaking technique 1 a 400 pinzano (bz) (south west) grape mass 3.5 t; 8 days maceration, 25-26°c fermentation temperature 2 a 400 pinzano (bz) (south west) as wine 1, but a thermal maceration at 42°c was applied for 8 hours prior to alcoholic fermentation held at 20°c 3 a 400 pinzano (bz) (south west) as wine 1, but it underwent a stuck fermentation followed by a second inoculation with supplementary addition of so2 4 b 780 trentino (south east) as wine 1 5 c 750-800 aldino (bz) (south) as wine 1 (grapes have been treated with a leaf fertilizer) 6 c 750-800 aldino (bz) (south) as wine 1 7 d 650 gleno (bz) (south west) as wine 1 8 e 350 mazzon (bz) (north west) as wine 1 9 e 350 mazzon (bz) (north west) as wine 1 10 e 350 mazzon (bz) (north west) as wine 1, but with 20% of non-destemmed grapes 11 e 350 mazzon (bz) (north west) as wine 1, but using 100% raisins ital. j. food sci., vol. 32, 2020 340 2.2. hplc-dad-hrms/ms analysis solvents and standard compounds for the hplc-hrms/ms analysis were purchased from sigma-aldrich ltd. all chemicals were lc-ms grade. the preparation of wine samples and the hplc-hrms/ms analysis were performed according to the procedure reported by longo et al., 2018a with slight modifications. briefly, 20 ml of each wine were concentrated under low pressure (11 mbar) at 40°c. then, a gentle n2 flux was applied for 30 min and the samples were re-dissolved (with a sonication for 5 min) to a final concentration 10 times higher. finally, all samples were filtered (0.2 µm) before hplc injection. a q-exactive hrms instrument (thermo fisher scientific, rodano, milano, italy) was coupled to an agilent 1260 hplc (agilent technologies italia s.p.a., cernusco sul naviglio, milano, italy) with a 16 channel dad detector. the chromatographic separation was carried out using an ods hypersyl c18 lc column (125 mm × 4.6 mm i.d., 5 μm, thermo fisher scientific), which was protected with a hplc pre-column filter (ods hypersil, 5 µm pore size, 10 x 4 mm drop-in guards, thermo fisher scientific) at a flow rate of 1 ml.min-1. the mobile phase consisted of solvent a (0.1% v/v formic acid in 0.02 mol.l-1 ammonium formate in water) and solvent b (0.1% v/v formic acid in saturated ammonium formate acetonitrile). the gradient program of solvent b was as follows: from 0 to 21 min 5%, 21 to 22 min 25%, 22 to 27 min 95%, 27 to 28 min 5%, followed by a reequilibration step (5% b) from 28 to 35 min. the dad spectra were recorded from 210 to 600 nm and provided real-time monitoring at 280 nm, 320 nm, 365 nm, 420 nm and 520 nm (+/4 nm). a post-column flow splitter valve (upchurch scientific) was used to feed both analyzers in parallel (dad and hrms) at a fixed ratio. for the full ms analysis, the hesi source was operated in positive ionization mode for the analysis of proanthocyanidins and in negative ionization mode during the analysis of the phenolic profile. the following conditions were used: sheath gas at 20 (arbitrary units), auxiliary gas at 5 (arbitrary units), auxiliary gas temperature at 250°c, spray voltage at +3,500 kv, capillary temperature at 320°c and rf s-lens at 70 (arbitrary units). the mass range was from m/z 500 to 2,000 with the full ms set resolution of 70,000 (@200 m/z), agc target at 3.106, max injection time of 300. full ms parameters were: ms/ms agc target 106, max. injection time 300, ft-ms set resolution 35,000, loop count 5, isolation window 2 or 3 m/z with 1 m/z offset, normalized collision energy 15 ev (positive mode) and from 30 to 60 ev (negative mode). for data-dependent settings: minimum agc target 3.103, apex trigger from 2 to 8 sec, charge exclusion from 3 to 8 and higher, dynamic exclusion 3 sec, “if idle” tool set to “pick others.” lock masses were constantly employed to correct mass deviations across the full ms acquisition range throughout the experiments. the hplc-dad data were collected and analyzed by the openlab software while the hplc-ms data were collected and analyzed with xcalibur 3.1 software and compound discoverer 2.0 (thermo fisher scientific). simple phenolic compounds quantitation was achieved at hplc-dad with external calibration and with injection of standard compounds (peaks integration at 280 nm). 2.3. standard oenological characterization acetic acid, glucose and fructose, free and total so2 were measured using an automatic multi-parametric analyzer – miura one (exacta+optech labcenter s.p.a., san prospero, italy). all samples were filtered (0.2 µm, cellulose acetate filter) before the analysis without any specific sample preparation. reagents for the enzymatic analysis of wines were ital. j. food sci., vol. 32, 2020 341 purchased from exacta+optech labcenter s.p.a. (san prospero, italy). the total acidity was measured according to oiv (oiv, 2015a). the alcohol content was measured with a malligand ebulliometer. 2.4. sensory evaluation a group of eight trained panelists (4 females and 4 males) aged from 30 to 50 years were recruited at free university of bozen-bolzano, faculty of science and technology. an initial qualitative analysis phase consisted in presenting the wine samples in order to define a common vocabulary of the sensory descriptors for pinot noir wines. then, nineteen sensory descriptors were identified and evaluated with the procedure of the round table (yasar et al., 2018). the visual descriptors were clarity, hue, and color intensity. the olfactory descriptors were olfactory intensity, floral, fruity, herbaceous, spicy, liquor, maderized, caramelized aromas, and solvent. the gustatory descriptors were alcoholic, softness, sweetness, acidity, sapidity, tannicity, and balance. each descriptor was evaluated using a 10-point scale (1 = no perception, 10 = high intensity). the bottles were opened just before each sensory session and 30 ml of wine were offered randomly to the panelists in iso glasses codified with 3-digit number at around 18°c. the presentation order of the samples was counterbalanced between and within participants. the participants were provided with mineral water to rinse their mouths between samples. at the end of the session, an overall quality judgment was also requested. 2.5. statistical analysis principal component analysis was performed using xlstat (version 2019.2.2.59417, addinsoft, paris, france). nipals (non-linear iterative partial least squares) algorithm was preliminary applied to account for sparse missing values in the chemical datasets (wold et al., 1984). the relative abundances of non-cyclic and cyclic proanthocyanidins and their relative ratios were auto-scaled (mean-centered followed by division of each column i.e. variable by the standard deviation of that column). the average ratings of each sensory descriptor were instead only mean-centred as they all shared the same 10-point scale for the evaluation. ‘overall judgment’ was used as supplementary variable (non-active) in the sensory analysis. 3. results and discussion in table 1, the information on each analyzed pinot noir sample is reported. samples 1, 4, 5, 6, 7, 8, and 9 were produced with the same winemaking procedure (mass of 3.5 t for each sample; 8 d maceration, 25-26°c fermentation temperature). the main differences among the cited samples were the altitude and the geographical orientation of the vineyards. samples 1, 2, and 3 differed for the winemaking practice used: to produce wine 2, a thermal maceration at 42°c was applied for 8 h before the alcoholic fermentation; wine 3 instead underwent an unwanted stuck fermentation; thus, it was re-inoculated with selected yeast and then added with supplementary so2 to prevent off-fermentations (di mattia et al., 2015). wine 11 was obtained from grapes harvested in the same vineyard (e) of wines 8, 9, and 10, but using 100% raisin grapes obtained by cutting some vine shoots and leaving the clusters hanging on the plants for a few days. wine 10 was made ital. j. food sci., vol. 32, 2020 342 with 20% of whole clusters (non-destemmed and uncrushed) that were left in the must during maceration/fermentation. 3.1. oenological parameters the standard oenological results are presented in table 2. the alcohol content in pinot noir wines ranged from 12.8% (sample 4) to 15.4% (sample 11). as expected, wines 4, 5, and 6 obtained from the vineyards located in the highest sites showed the lowest alcohol content due to the lowest degree of grape ripeness whereas wines 1-3 and 8-11 showed the highest alcohol content since the grapes were cultivated in lower vineyards (table 2). the highest alcohol content of sample 11 compared to the other pinot noir wines could be expected since this wine was made with 100% raisins (with higher sugar content). the ph ranged from 3.2 (sample 4) to 3.5 (sample 6). the first four wines had lower ph compared to the others. the ph fitted the usual ph range of red wines (3.0 – 4.0) (jacobson, 2006). the total acidity measured in samples 1-3, 5, and 79 was 5.6 g.l-1 tartaric acid. samples 4, 6, 10, and 11 had a higher total acidity (6.2 – 6.8 g.l-1 tartaric acid). all pinot noir wines had low acetic acid content (within the legal threshold of 1.2 g.l-1 acetic acid equivalents, oiv, 2015b and oiv, 2012). all the wines were dry and most of them showed a residual sugar content ranging from 0.06 g.l-1 (wines 3 and 6) to 0.44 g.l-1 (wine 7) (fernandeznovales et al., 2009). wine 11 (made with 100% raisin grapes) contained the highest residual sugar content (1.63 g.l-1). interestingly, wines 5 and 6 had the lowest glucosefructose levels (0.07 and 0.06 g.l-1, respectively). the free so2 levels were relatively low (12 – 18 mg.l-1) and the total so2 (73 – 108 mg.l-1) was within the legal limits (oiv, 2012). table 2. oenological parameters of the eleven pinot noir wines. wine 1abv (%) ph 2total acidity (g.l-1) acetic acid (g.l-1) 3gl-fr (g.l-1) 4fso2 (mg.l-1) 5tso2 (mg.l-1) 1 14.4 3.38 5.6 0.21 0.19 14 107 2 14.4 3.33 5.6 0.24 0.17 14 93 3 13.7 3.25 5.6 0.41 0.06 13 108 4 12.8 3.21 6.8 0.40 0.14 12 88 5 13.1 3.48 5.6 0.25 0.07 14 79 6 13.4 3.54 6.2 0.32 0.06 13 82 7 14.7 3.42 5.6 0.36 0.44 12 83 8 14.8 3.41 5.6 0.31 0.31 15 73 9 14 3.48 5.6 0.30 0.22 14 90 10 14.5 3.46 6.5 0.39 0.30 18 91 11 15.4 3.50 6.8 0.40 1.63 18 90 1abv: alcohol by volume (% v/v); 2g/l tartaric acid; 3gl-fr: glucose-fructose (g.l-1); 4fso2: free sulphur dioxide (mg.l-1); 5tso2: total sulphur dioxide (mg.l-1). ital. j. food sci., vol. 32, 2020 343 3.2. profiles of proanthocyanidins the proanthocyanidins (pac) profile was analyzed by means of hplc-hrms and the results are reported in table 3. both non-cyclic procyanidins (nc-pc) and cyclic procyanidins (c-pc) were found in higher concentrations in pinot noir samples, compared to prodelphinidins (pd). all wines except sample 3 had a high content of dimeric procyanidins (nc-2 pcs). the abundances of nc-pc decreased at a higher degree of polymerization (dp). the highest amount of nc-6 pc (non-cyclic hexameric procyanidin) was present in wine 11. wine 3 had instead the lowest amount of c-pac. also, wines 10 and 11 stood out with a higher content of c-6 pc (cyclic hexameric procyanidin) with respect to other samples. furthermore, wine 11 had almost twice as much of c-5 pd (cyclic pentameric prodelphinidin) compared to wines 7 and 8. principal component analysis was performed using auto-scaled pac variables, to highlight trends within the dataset that may suggest relationships between the pac profiles and the different factors involved. in previous studies on the distribution of procyanidins (longo et al., 2019) and prodelphinidins (longo et al., 2018c) in wines, the relative (%) ratios were applied: these showed clear dependency upon the grape variety, but no study has yet addressed their relationship with the winemaking practices or the geographical origin. these ratios correspond to the proportions (%) of any cyclic congener over the total amount of cyclic + non-cyclic congeners by number and composition of monomers as reported in previous reports (longo et al., 2018c; longo et al., 2019). the pca bi-plot of these ratios is shown in fig. 1. the total variance explained by the first two principal components is 84.0% (pc1: 69.6% + pc2: 14.4%). all variables are in positive correlation with the first principal component, except for the ratio of c-pd (cyclic prodelphinidins) with one and three (epi)gallocatechin units (indicated as %c-4-1-oh and %c-4-3-oh respectively). all %c-pc (relative (%) ratios of procyanidins) showed strong correlations among each other and also with most of the pd. wine 3 is well separated from the other wines, which are clustered in the central area of the bi-plot. this is probably caused by the occurrence of a stuck fermentation: namely, as the fermentation halted prematurely, the extraction of the polyphenols from the berry skins was hampered, since the reached concentration of ethanol was lower in comparison to the other samples. after that event, sample 3 was racked before being reinoculated with the yeast. removing the skins at an early stage of maceration presumably prevented the completion of the extraction of polyphenols. however, this also slowed down the extraction of the non-cyclic congeners, since these are less polar compounds than the cyclic ones and require higher percentages of ethanol for their extraction. instead, the cyclic compounds were still extracted in higher proportions (as evidenced in fig. 1). hence, the relative ratios (%) of cyclic congeners were “over-expressed” in sample 3. notably, these percentages do not represent absolute concentrations, but instead they are just the relative proportions (%) of c-pac over c-pac plus nc-pac (by dp and composition). indeed, the data in table 3 show that the peak areas in sample 3 are lower for all compounds than in the other samples. notably, a recent study on the kinetics of skin extraction for c-pc in cabernet sauvignon showed that these compounds are extracted almost completely at the beginning of maceration (jouin et al., 2019), while ncpc are only extracted over time with the increasing formation of ethanol. ital. j. food sci., vol. 32, 2020 344 table 3. relative abundances (integrated total ion current) of non-cyclic and cyclic proanthocyanidins in the eleven pinot noir wines. pac nc-2 pc i nc-2 pc ii nc-2 pc iii nc-2 pc iv nc-2 pc v nc-3 pc nc-4 pc c-4 pc nc-5 pc m/z 579.1497 579.1497 579.1497 579.1497 579.1497 867.2124 1155.2760 1153.2604 1443.3392 w in e sa m pl es 1 22274956 22306888 22323575 22374250 22325218 7750638 2404025 162851 480128 2 37425812 37336457 37336457 37354174 37354706 19392028 7135274 183528 1925495 3 639543 639543 630124 639543 639543 79649 3475 25201 3698 4 33026883 33012707 33004346 33012578 32999883 15520019 5166134 84876 1522658 5 49790473 49787516 49753715 49788501 49788582 23452844 8328293 157309 2525998 6 34515047 34495827 34503463 34515042 34499696 11871865 4004473 104562 967950 7 58252407 58023701 58083779 58145139 58145139 26156359 7954429 316117 1873445 8 45458622 45411956 45413310 45358853 45368291 25007492 8781802 301711 2467726 9 12463012 12487106 12463722 12467415 12462662 4193665 1141355 131150 242130 10 26662439 26665699 26664558 26668416 26646611 11066048 4442351 174339 1332144 11 29720456 29692215 29720858 29720262 29720035 20466723 9604520 67151 3235409 pac c-5 pc nc-6 pc c-6 pc nc-2 pd 1-galloc nc-3 pd 1-galloc nc-3 pd 2-galloc nc-3 pd 3-galloc nc-4 pd 1-galloc m/z 1441.3213 1731.4010 1729.3870 595.1446 883.2072 899.2021 915.1970 1171.2710 w in e sa m pl es 1 168545 28905 8308 0 103469 12973 787 638805 2 145329 348388 16802 565 71849 7113 0 1081774 3 22871 1632 990 0 427 0 230 221433 4 114903 251288 24875 0 80485 7434 0 1301338 5 174167 467959 35912 575 231898 30436 514 2028349 6 118992 155456 9765 0 157400 25428 5041 1023981 7 263641 272430 13032 308 268426 45048 1024 1960693 8 365229 446402 43924 0 164426 19409 1344 2336156 9 117526 18048 4672 0 47356 6993 349 432706 10 211433 307633 36639 0 107349 26470 1365 2719 11 111594 784272 38554 0 39650 3466 0 1874952 ital. j. food sci., vol. 32, 2020 345 pac nc-4 pd 2-galloc nc-4 pd 3-galloc nc-4 pd 4-galloc c-4 pd 1-galloc c-4 pd 2-galloc c-4 pd 3-galloc c-4 pd 4-galloc nc-5 pd 1-galloc m/z 1187.2660 1203.2605 1219.2550 1169.2557 1185.2507 1201.2456 1217.2405 1459.3343 w in e sa m pl es 1 40168 7470 1317 34465 2686 3725 0 60521 2 45131 2996 735 60435 3717 14213 0 238163 3 0 289 308 1277 198 0 0 0 4 73318 10593 3446 61911 4280 21048 1941 369331 5 331689 34976 4516 88724 8139 6740 1307 632792 6 160249 20578 1964 42105 6604 5050 10487 232513 7 225155 29024 3917 109277 22825 14161 3913 383685 8 226537 17780 4891 99430 18169 4590 482 618359 9 17701 10183 3886 21521 1577 18515 397 27818 10 109507 17819 2251 49828 11919 17315 448 295178 11 183308 4883 1081 210398 23102 3371 0 756973 pac nc-5 pd 2-galloc nc-5 pd 3-galloc nc-5 pd 4-galloc c-5 pd 1-galloc c-5 pd 2-galloc c-5 pd 3-galloc c-5 pd 4-galloc c-5 pd 5-galloc m/z 1475.3291 1491.3240 1507.3189 1457.3191 1473.3140 1489.3090 1505.3039 1521.2988 w in e sa m pl es 1 7874 0 0 49574 10550 1964 565 0 2 9260 790 0 53667 5422 1422 294 0 3 676 306 0 3030 886 0 0 0 4 23878 3065 0 45778 5108 1889 0 0 5 106745 16166 0 75154 16454 1808 0 0 6 36370 7013 601 43342 8661 4357 0 0 7 44404 3795 0 95602 26777 5586 295 0 8 73686 4936 0 123419 30556 3078 0 257 9 2255 1900 0 36030 6991 1400 0 0 10 31967 4855 0 68776 19911 4301 0 0 11 88643 7073 0 123989 13498 0 0 0 abbreviations: nc – non-cyclic; c – cyclic; numbers after nc or c indicates the number of monomer units of catechin or epicatechin (e.g. nc-2 is non-cyclic dimer); pc – procyanidins, pd – prodelphinidin; the last number in prodelphinidins indicates the number of gallocatechins in the oligomeric chain. ital. j. food sci., vol. 32, 2020 346 figure 1. pca bi-plot of the relative ratios of proanthocyanidins (%) of the eleven pinot noir wines. the vectors of the ratios of procyanidins are dashed, whereas the vectors for prodelphinidins are full. the first number in the abbreviations indicates the number of the monomers forming the proanthocyanidin. the second number shows the number of gallocatechin units in the oligomeric chain of prodelphinidins. f1 and f2, principal components. %c-n-m-x: ratio of relative abundance of a cyclic oligomer over the sum of relative abundances for cyclic and non-cyclic, considering the same relative compositions in (epi)catechins and (epi)gallocatechins and number of composing monomeric units. in the formula: c = cyclic, n = number of monomeric units, m = number of (epi)gallocatechins in the structure, x = -oh if the compound is a prodelphinidin or empty if it is a procyanidin. in fig. 2, the pca models, which were elaborated over the relative abundances of ncpac (2a) and c-pac (2b) are shown separately. the lack of nc-pac in wine 3 is again confirmed in fig. 2a (84.4% of total variance), where wine 3 is situated on the opposite side of pc1 with respect to all the variables. wine 11 had higher concentrations of ncpac and the highest concentrations of residual sugars and alcohol (table 2). in fact, the grapes used for winemaking of sample 11 had been cut and left to dry hanging on the vine before the harvest, which had the effect of concentrating even further the polyphenols besides the sugars. notably, in figs. 2a and 2b the values used represent absolute abundances, as they are integrated peak values obtained with the hplc-hrms analysis (table 3). wines 3 and 11 are clearly separated from the others in 2a and 2b respectively, and the trends for the variables are shown: wine 3 was on the opposite part of most descriptors, while wine 11 was driven by the c-pd with one or two (epi)gallocatechin units. ital. j. food sci., vol. 32, 2020 347 figure 2. pca bi-plots of non-cyclic proanthocyanidins (a) and cyclic proanthocyanidins (b) in pinot noir wines. nc non-cyclic, c cyclic. the vectors of procyanidins are dashed, whereas the vectors for prodelphinidins are full. the first number in the abbreviations indicates the number of the monomers. the second number shows the number of gallocatechin units in the oligomeric chain of prodelphinidins. f1 and f2, principal components. 3.3. profiles of simple phenolics overall, none of the evaluated simple phenolic variables could distinguish significantly groups of samples; therefore, they were not included in the previous statistical analysis (data not shown). instead, they are just mentioned qualitatively. seven monomeric phenolic compounds (gallic acid, protocatechuic acid, 4hydroxybenzoic acid, vanillic acid, catechin, caffeic acid, ferulic acid) were identified (table 4), and concentrations were evaluated by standard injection according to longo et al. (2017) for phenolic compounds. gallic, vanillic and caffeic acids were present in all samples. the highest amount of gallic acid was shown in wine 10, vanillic acid in wine 11, and caffeic acid in wines 7, 10 and 11. wine 1 showed a higher content of protocatechuic acid; wine 3 was higher in ferulic acid; wines 10 and 11 in 4-hydroxybenzoic acid; wines 7 and 8 in catechin. 3.4. sensory evaluation of pinot noir wines fig. 3 shows the pca bi-plot for the sensory data. the first two components explained 46.2% of the total variance. the first principal component (26.6% of the total variance) was correlated with wine balance and the overall judgment on wine quality. besides, pc1 was correlated with softness, sweetness, herbaceous, floral and fruity aromas. the second principal component (19.6%) was correlated with clarity, tannicity (astringency), and caramelized descriptors, which were inversely correlated with a maderized descriptor. as shown in fig. 3, wines 1, 2, and 3 (vineyard a) were clustered on the left part of the graph. samples 1 and 2 showed a very similar trend; thus the thermal maceration of wine 2 did not remarkably affect the sensory properties. however, wine 3 was characterized more by alcoholic, liquor, and maderized variables, and it was lacking in tannicity. wines 5 and 6 (vineyard c) were situated in the center of the plot. wines 8, 9, and 10 (vineyard e) were ital. j. food sci., vol. 32, 2020 348 situated on the same side as wine 11 (vineyard e). the wines 9, 10, and 11 were the most balanced and with a high overall judgment assigned by the panelists. finally, the other two wines – 4 (vineyard b) and 7 (vineyard d) – were well separated from the other samples. table 4. concentration of simple phenolic compounds in the eleven pinot noir wines evaluated by hplcdad (280 nm) standard injections. calibration curves with r2 = 0.999 for evaluated compounds. wine gallic acid (µm) protocatechuic acid (µm) p-hydroxybenzoic acid (µm) vanillic acid (µm) (+)-catechin (µm) caffeic acid (µm) ferulic acid (µm) 1 171 672 0 206 5 14 2 2 229 1 0 440 2 24 1 3 225 63 0 304 5 20 4 4 203 56 2 300 3 22 0 5 220 40 0 319 2 21 0 6 213 47 0 308 1 20 0 7 221 1 2 348 21 30 0 8 153 1 0 404 31 29 0 9 253 1 4 256 3 16 0 10 311 1 6 314 0 31 1 11 180 1 6 482 0 30 1 figure 3. pca bi-plot of the sensory data across the eleven pinot noir wines. overall judgment was used as a supplementary variable. f1 and f2, principal components. ital. j. food sci., vol. 32, 2020 349 4. conclusion using the profile of cyclic and non-cyclic proanthocyanidins, the separation of the most different samples of pinot noir wines, such as sample 3 (that had experienced a stuck fermentation) and sample 11 (that was produced using raisin grapes) was similar to that achieved with sensory analysis. sample 3, with low proanthocyanidins concentration (including the cyclic ones), was described by the panel as highly maderized and lacking in tannins. conversely, wine 11 (made with raisin grapes) contained the highest amount of cyclic tetrameric prodelphinidins and it was described as a balanced wine with a high overall quality judgment by the panel. the ratios between cyclic and non-cyclic proanthocyanidins confirmed the different solubility and extractability of these compounds and did reflect the occurrence of a stuck fermentation followed by racking and re-inoculation. thus, the profile of cyclic and non-cyclic proanthocyanidins was affected by specific factors, such as the stuck fermentation or the use of 100% raisins. both of these factors were related to the sensory quality judgement of pinot noir wines. acknowledgements the authors would like to thank the winery franz haas srl (montagna, south tyrol, italy) for supplying the pinot noir wines. this work was supported financially by the provincia di bolzano (italy) (beschluss der landesregierung nr. 1472, 07.10.2013) and is part of the wineid (wine identity card) interdisciplinary project of unibz (no. 3666). 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january 13, 2020 ital. j. food sci., vol. 30, 2018 522 paper trimethylamine as a freshness indicator for seafood stored in ice: analysis by gc-fid of four species caught in the tyrrhenian sea t. nevigato*, m. masci, i. casini, r. caproni and e. orban consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria (crea) research centre for food and nutrition, via ardeatina 546, rome 00178, italy *corresponding author: tel.: +39 0651494658 e-mail address: teresina.nevigato@crea.gov.it abstract in seafood products, trimethylamine (tma) is an indicator of the conservation status. it is almost absent in freshly caught samples, and its content increases during spoilage. in the present work, a new simple gc-fid method that uses a commercial capillary column, specifically designed, was applied. tma was measured at increasing time intervals in four marine species caught in the tyrrhenian sea and stored in ice; 852 individuals were analyzed. an assessment of the maximum allowable time of storage in ice was made for each species. existing guidelines for the level of trimethylamine are reviewed and discussed. keywords: trimethylamine (tma), seafood, freshness, shelf life, storage in ice, gas chromatography-flame ionization detector (gc-fid) ital. j. food sci., vol. 30, 2018 523 1. introduction fish, mollusks, crustaceans, and other marine species are among the most perishable food products (bourigua et al., 2011; dimogianopoulos and grigorakis, 2014; sterniša et al., 2016), and their spoilage leads to the formation of some substances that may cause intoxication when ingested (biji et al., 2016). seafood spoilage leads also to the formation of low-molecular-weight volatile amines, which results in off-flavors. the main compound responsible for the typical smell of spoiled fish is trimethylamine (tma). tma is a good chemical marker of freshness (popelka et al., 2014): its concentration increases with spoilage by bacterial degradation of tmao (trimethylamine n-oxide), an important osmoregulatory organic molecule that is commonly found in the muscle of marine fish (treberg and driedzic, 2002). a bad conservation status of seafood represents a topic of safety concern. ideally, seafood should be stored under conditions such that bacteria cannot grow at all: when preserved in ice, the product is safe only within a limited period. as such, an indicator of freshness may be very useful. tma is actually used as an indicator of seafood conservation status in ice (bourigua et al., 2011; toniolo et al., 2014; baliño-zuazo and barranco, 2016). the various analytical techniques for the determination of tma include colorimetric assays (penapereira et al., 2010), flow injection analyses (ruiz-capillas and horner, 1999), biosensor analysis (bourigua et al., 2011), capillary electrophoresis (timm and jørgensen, 2002), and hplc utilizing derivatization (baliño-zuazo and barranco, 2016). for gas chromatography, there are no simple instrumental methods available that allow a direct split/splitless injection into a capillary column. packed columns were used for this purpose, especially in the past (veciana-nogues et al., 1996). alternative methods use complex hyphenated techniques such as headspace-gas chromatography and solid phase microextraction-gas chromatography (krzymien and elias, 1990; dehaut et al., 2016). in the present work, an innovative instrumental method was applied. thanks to a capillary column specifically designed for the volatile amines and coated with a proprietary phase, simple instrumental analysis using gas chromatography with a flame ionization detector (gc-fid) was possible. this was done by injecting the liquid sample in split mode. 2. materials and methods 2.1. chemicals and reagents trimethylamine hydrochloride (tma·hcl) and n-propylamine hydrochloride (n-pa·hcl) were purchased from sigma aldrich® (st. louis, mo, usa). toluene, potassium hydroxide (koh), and trichloroacetic acid (tca) were from carlo erba reagents® (milan, italy). testmix cp0043 was from varian® (walnut creek, ca, usa). standard amine solutions were prepared by dissolving tma·hcl and n-pa·hcl in distilled water in order to obtain the desired concentrations expressed as free bases (tma and n-pa). 2.2. seafood samples red mullet (mullus barbatus), european anchovy (engraulis encrasicolus), and deep-water rose shrimp (parapenaeus longirostris) were caught by professional fishermen during the months of june, july and september for the summer campaign and during the months of ital. j. food sci., vol. 30, 2018 524 february, march and april for the winter campaign. atlantic mackerel (scomber scombrus) was caught in the summer campaign only. for red mullet two different sizes were selected and analyzed in order to verify whether spoilage is influenced by this parameter. in some cases, the study had to take into account the amount of sample available at the moment. fishing was done by trawling along the coast of the tyrrhenian sea, near civitavecchia located in the region of latium in central italy. the collected seafood samples were selected directly on board and separated by species and size, and then were placed in polystyrene boxes and covered with ice. the polystyrene boxes were stored in a refrigerated cell at 0-1°c until the day of the analyses that started on day 1 and continued on days 3, 6, 8, 10, and 13. the size and number of the individuals collected are reported in table 1. table 1. size of the seafood species collected (minimum – maximum) and total number of individuals analysed. weight (g) length (cm) number of individuals summer winter summer winter summer winter red mullet, big size (mullus barbatus) 52-276 23-99 16-27 13-20 17 42 red mullet, small size (mullus barbatus) 19-61 7-33 12-18 9-15 48 173 european anchovy (engraulis encrasicolus) 10-18 9-23 12-14 12-16 120 138 atlantic mackerel (scomber scombrus) 51-135 19-26 25 deep-water rose shrimp (parapenaeus longirostris) 8-25 6-24 10-16 10-14 109 180 2.3. sample preparation 2.3.1 filleting and homogenization on the day of the analysis, a minimum of 2-4 and a maximum of 10-30 individuals of each species, based on the number available, were pooled. this was followed by gutting, skinning, and filleting. subsequently, homogenization was carried out for 30 seconds at a low speed with a waring blender (model 8010e, waring® products division, new hartford, ct, usa) and by using a previously cooled stainless-steel cup. the resulting homogenate was ready for tma extraction. 2.3.2 tma extraction analyses were performed in duplicate. sample preparation followed the methods of perez martin et al. (1987) and veciana-nogues et al. (1996) with minor modifications. approximately 10 g of homogenized product was weighed into 250 ml plastic bottles. to the weighed sample, 40 ml of tca 6% (w/w) solution was added, and then a second homogenization was carried out for 1 min at 11000 rpm with an ultra turrax homogenizer (model t25b, ika®, staufen, germany); the plastic bottle was kept immersed in ice to prevent any heating. ital. j. food sci., vol. 30, 2018 525 subsequently, centrifugation was carried out for 10 min at 4°c (12000 rpm, 22214g) using a beckman coulter avantitm j25 ultracentrifuge. the supernatant was collected in a 100 ml volumetric flask by using a funnel with an inserted whatman n° 2 filter paper 15 cm in diameter. the filter, residue pulp, and plastic bottle were washed with distilled water to ensure that all of the tma extracted was quantitatively collected. finally, the solution was brought to volume with distilled water. the solution (tca extract) was transferred into 10 ml tubes and kept at -30°c until the day of gas chromatographic analysis. 2.3.3 sample preparation for gc injection on the day of the gas chromatographic analysis, the tca extract from the previous step was left to thaw at room temperature. to 7 ml of the tca extract in a glass tube, the internal standard (is) n-pa·hcl was added. the added amount corresponded to a concentration of 19.04 mg/l of n-pa as free base. subsequently, 2 ml of toluene and 10 ml of koh 65% (w/v) solution were added, and the tube was shaken at 1500 rpm for 1 min by means of a vortex mixer (heidolph®, schwabach, germany). one microliter of the toluenic upper layer was injected in gc-fid. 2.4. instrumental analysis the apparatus used was a 6890 agilent gas chromatograph with a flame ionization detector (gc-fid), equipped with a cp-volamine fused silica capillary column (60 m × 0.32mm i.d., 0.45 mm o.d., 5 µm film thickness, from varian®). helium as the carrier gas was used in constant flow mode at 0.9 ml/min. the operating conditions were as follows. the initial oven temperature was 45°c, which was held for 10 min and then increased to 250°c at a rate of 20°c/min. this final temperature was maintained for 10 min. the fid temperature was 300°c, while the injector temperature was 270°c. injections were made in split mode (5:1) with an injection volume of 1 µl. fig. 1 shows two gas chromatograms for a shrimp sample and a standard solution of tma. figure 1. gc-fid chromatograms, peak of tma. standard solution of tma and a shrimp sample. chromatograms are overlaid. ital. j. food sci., vol. 30, 2018 526 2.5. analytical quality control instrumental performance was investigated by using the testmix cp0043 provided by varian®. the composition of the testmix was 0.1% of each component, including tma, in isopropanol. for the quantitative analysis of tma, an appropriate calibration curve was constructed (fig. 2). figure 2. calibration curve used to quantify tma in the seafood samples. solutions of known composition for the points of the calibration curve were prepared in tca by using pure tma·hcl. pure n-pa·hcl was used as is. the real samples that were observed to have very high levels of tma were again prepared and appropriately diluted before the gc injection to fall within the linear dynamic range of the calibration curve. the limit of quantitation (loq) is the amount of tma in the seafood sample that is easily quantifiable because of the good signal-to-noise ratio of the final chromatographic peak. the limit of detection (lod) is the amount of tma with a signal-to-noise ratio that is sufficient only for the detection of its presence without the possibility of a reliable integration. for the present method, we measured a loq of 3 mg of tma per kilogram of seafood sample and a lod of 2 mg/kg (corresponding to a loq of 0.07 mg n/100 g and to a lod of 0.05 mg n/100 g when tma is expressed in mg n/100 g of sample). blanks and recovery measurements were carried out to ensure that no false positives nor false negatives were produced. for the recoveries, the tca extract (section 2.3.2) coming from a red mullet sample was spiked with tma at three different concentration levels, as reported in table 2. recovery was 90.2±8.0%; this is similar to that reported by other researchers (perez-martin et al., 1987). ital. j. food sci., vol. 30, 2018 527 table 2. tma recovery measurements. sample measured concentration of tma in the native tca extract (mg/l) expected concentration of tma in the spiked tca extract (mg/l) measured concentration of tma in the spiked tca extract (mg/l) recovery (%) red mullet, big size winter campaign (t2) no adding 2.11 level 1, adding 3.20 2.70 84.4 level 2, adding 4.29 4.26 99.3 level 3, adding 5.37 4.66 86.8 after each addition of tma, the extract was normally processed and injected in gc-fid. 3. results and discussion 3.1. guidelines for tma concentration tma is considered a good indicator of fish spoilage when the product is preserved in ice; however, there is no regulation for tma levels (macé et al., 2012). the european community regulation cites that when the organoleptic examination reveals any doubt as to the freshness of the fishery products, samples may be taken and subjected to laboratory tests to determine the levels of tma (reg. ce no 854/2004); surprisingly, the regulation itself does not provide any tolerance or reference value. there are, however, generally accepted values or values recommended by international organizations. tma is sometimes reported in milligrams of tma per kilogram of fish; in other cases, it is reported in milligrams of tma-n (milligrams of nitrogen) per 100 g of fish, a situation that can be potentially confusing. for clarification, we report the equation for converting tma into tma-n and vice versa: )100/(0237288.0)/( gmgntmakgmgtma −=× (1) the food and agriculture organization of the united nations (fao, 1988) reports that good-quality cold-water fish contains less than 63 mg/kg of tma. el marrakchi et al. (1990) investigated the freshness of marine fish (sardina pilchardus) both by tma determination and by sensory evaluations by two official veterinary inspectors. in their study the product was judged as fresh up to a tma level of 50 mg/kg (1.19 mg/100g of tma-n). this is in good agreement with what has been reported by fao, and is the same as the maximum reference tma content cited by the italian istituto zooprofilattico sperimentale (izsum, ministry of health) for the freshness of marine fish (haouet, 2001). veciana-nogues et al. (1996) reported that hake can be graded as excellent quality when its tma level is lower than 42 mg/kg (1 mg/ 100 g of tma-n). we see that recommendations by national and international organizations, as well as experimental works of researchers, agree that the range 42-63 mg/kg of tma (1-1.5 mg/100g of tma-n) in marine fish is the limit below which the freshness status is at an optimum. ital. j. food sci., vol. 30, 2018 528 3.2. deep-water rose shrimp as table 3 shows, shrimps exhibited a tma content suggestive of a bad freshness state after three days of storage in ice (151.7 mg/kg in the summer campaign). we must emphasize that on day 3 the colour of the shrimps had in fact turned black from the original pink. it can be concluded that the shelf life of deep-water rose shrimp in ice is very limited when the product does not undergo to any conservative treatment, as is generally done (zhang et al., 2015). another work (laghmari and el marrakchi, 2005) confirms the short shelf life (3-5 days) of p. longirostris stored in ice. as it can be seen for shrimps in table 3 after many days of storage in ice the tma content in some marine species could also stabilize or even decrease: this is a known phenomenon because the content of the precursor tmao runs out, but that does not mean, of course, an improvement in the conservation status. 3.3. red mullet red mullet (m. barbatus) stored in ice showed a status of good freshness until 8 days, when the tma level remained constantly below 65 mg/kg for both sizes and campaigns studied (table 3). it appears that even over 8 days the product in some cases is in an acceptable status of conservation. at long storage times in ice (i.e. 8 days and 10 days) the bigger size tends to release a lower quantity of tma than the smaller size. this means that the larger size is more resistant to degradation than is the smaller one. it was already observed in other fish species a better resistance to degradation for bigger sizes (orban et al., 2011). a similar shelf life was obtained in a study in which sensory and microbiological analyses were carried out on red mullet (özyurt et al., 2009). 3.4. atlantic mackerel atlantic mackerel (s. scombrus) was caught in the summer campaign only. s. scombrus is a species of great commercial importance that is mainly distributed as a canned product. a bad conservation process of the product can lead to high levels of histamine in mackerel during storage, which may cause health problems for consumers (scombroid fish poisoning). this is due to the relatively high content of free histidine in this species, which during bad storage is converted to histamine (bennour et al., 1991). therefore an indicator of freshness is extremely useful. from table 3 it can be deduced that at 8 days in ice, the rejection status was reached being 170.2 mg/kg the tma content; up to six days, the product can be considered in good state. a similar shelf life for mackerel stored in ice has been reported (bennour et al., 1991). in the study by bennour, the histamine concentration was also measured. it was concluded that even when mackerel is allowed to spoil in ice until it becomes unfit to eat (over eight days), the level of histamine does not rise much above 5 mg/100 g of flesh, the level established by the united states food and drug administration as a guidance value (fda, 2011). 3.5. european anchovy from the tma content in table 3 we can be deduce that the maximum allowable time of storage in ice for anchovies is 5-6 days in summer. in fact, the tma concentration at day 6 is 68.1 mg/kg in the summer campaign. this result is perfectly in agreement with a study on e. encrasicolus collected in the mediterranean area during summer (pons-sánchez-cascado et al., 2006). such ital. j. food sci., vol. 30, 2018 529 work performed microbiological and sensory assays on anchovies stored in ice. a team of eight panel members trained in fish freshness assessment developed the schemes proposed for raw anchovies, and the final judgement was that the limit of acceptability for anchovies is reached after 5 days of storage in ice. the mean size of the individuals was practically the same in the work of pons-sánchez-cascado et al. and in the present one. the winter campaign seems to indicate a possible slightly longer time of storage in ice. table 3. concentrations of tma measured for different seafood species at different days of storage in ice. days of storage in ice species tma (mg/kg) tma-n (mg/100g) summer winter summer winter 1 day (t0) red mullet, big size n.d. n.d. n.d. n.d. red mullet, small size 5.4±0.2 < 3 0.13±0.00 < 0.07 european anchovy 28.8±2.6 16.2±0.3 0.68±0.06 0.38±0.01 atlantic mackerel 18.2±0.6 0.43±0.01 deep-water rose shrimp 40.3±0.3 18.3±15.8 0.96±0.01 0.43±0.37 3 days (t1) red mullet, big size 11.2±0.2 < 3 0.27±0.00 < 0.07 red mullet, small size 7.8±0.1 5.2±0.4 0.18±0.00 0.12±0.01 european anchovy 32.6±0.1 21.7±0.1 0.77±0.00 0.51±0.00 atlantic mackerel 48.1±1.6 1.14±0.04 deep-water rose shrimp 151.7±2.3 76.8±4.8 3.60±0.06 1.82±0.11 6 days (t2) red mullet, big size 24.9±0.2 18.3±0.0 0.59±0.00 0.43±0.00 red mullet, small size 23.6±0.1 20.0±0.1 0.56±0.00 0.47±0.00 european anchovy 68.1±0.7 49.6±0.0 1.62±0.02 1.18±0.00 atlantic mackerel 80.2±0.0 1.90±0.00 deep-water rose shrimp 283.4±11.1 623.9±98.9 6.72±0.26 14.81±2.35 8 days (t3) red mullet, big size 33.8±1.8 24.5±6.8 0.80±0.04 0.58±0.16 red mullet, small size 46.6±0.0 64.8±1.1 1.10±0.00 1.54±0.03 european anchovy 153.4±0.5 57.0±8.4 3.64±0.01 1.35±0.20 atlantic mackerel 170.2±0.3 4.04±0.01 deep-water rose shrimp 548.4±8.6 1041.0±12.6 13.01±0.20 24.70±0.30 10 days (t4) red mullet, big size 98.4±0.8 2.33±0.02 red mullet, small size 46.3±0.1 142.5±14.0 1.10±0.00 3.38±0.33 european anchovy 192.9±0.4 71.6±1.7 4.58±0.01 1.70±0.04 atlantic mackerel 187.3±0.6 4.44±0.02 deep-water rose shrimp 638.6±1.1 679.2±40.3 15.15±0.03 16.12±0.96 13 days (t5) atlantic mackerel 261.6±2.2 6.21±0.05 deep-water rose shrimp 1229.1±257.7 1089.0±87.2 29.16±6.11 25.84±2.07 results are reported as mean±standard deviation (n = 2) and are expressed both in mg/kg of tma and in mg/100g of tma-n (see equation 1, section 3.1). n.d. = not detected (below the lod) ital. j. food sci., vol. 30, 2018 530 3.6. comparison among species figures 3 and 4 plot the tma content measured in all species as a function of the days of storage in ice. it is evident that shrimps constantly released a much higher amount of tma when compared with fish species: this resulted in a much lower shelf life. figure 3. tma content of the different species as a function of the days of storage in ice (summer campaign). figure 4. tma content of the different species as a function of the days of storage in ice (winter campaign). ital. j. food sci., vol. 30, 2018 531 3.7. method validation in order to assess the reliability of the method presented here, a very different approach for evaluating the freshness status was applied. the species investigated for the tma content (red mullet big size, red mullet small size, european anchovy, atlantic mackerel) were also analyzed for their total volatile basic nitrogen (tvb-n) content, a widely accepted spoilage indicator that was established by the european community (comm. reg. ec no 2074/2005). measurements were carried out according to the ec official method (decision 95/149/ec). they involved acid extraction followed by alkalinization, distillation, and titration. comm. reg. ec no 2074/2005 stipulates that fish is unfit for human consumption when the tvb-n content is above 25-35 mg/100 g. on the other hand, the value 1.5 mg/100g of tma-n is the limit above which the fish begins to lose the optimum state of freshness (section 3.1). we can easily see in table 4 that the two indicators are in perfect agreement. table 4. maximum allowable time of storage in ice as indicated both by tvb-n and tma-n content (mg/100g) for the summer campaign. day 1 day 3 day 6 day 8 day 10 tvb-n red mullet, big size 13.66±0.29 15.50±0.15 15.78±0.17 17.44±0.27 not analyzed red mullet, small size 12.11±0.52 14.86±0.04 16.40±1.22 18.74±0.17 19.83±0.56 european anchovy 14.49±0.54 16.80±0.08 23.04±0.81 30.43±0.14 37.97±0.97 atlantic mackerel 19.30±0.27 26.54±0.23 25.72±1.24 35.81a 57.94±1.04 tma-n red mullet, big size n.d. 0.27±0.00 0.59±0.00 0.80±0.04 not analyzed red mullet, small size 0.13±0.00 0.18±0.00 0.56±0.00 1.10±0.00 1.10±0.00 european anchovy 0.68±0.06 0.77±0.00 1.62±0.02 3.64±0.01 4.58±0.01 atlantic mackerel 0.43±0.01 1.14±0.04 1.90±0.00 4.04±0.01 4.44±0.02 asingle measure results are reported as mean±standard deviation (n = 2). n.d. = not detected analyses of tvb-n were performed on another aliquot of the same homogenate that was processed for tma from the tvb-n content we can conclude that red mullet is in a good conservation status up to 8 days (10 days for the small size), since the concentration remains always below 20 mg/100 g. an identical situation is indicated by tma, which never exceeded 1.10 mg/100 g in red mullets. for european anchovy and atlantic mackerel, the tvb-n level is acceptable up to day 6 ( ≤ 25 mg/100 g), and it begins to exceed 30-35 mg/100 g on day 8, when degradation starts. the same is valid for the tma content, which shows that on day 8 degradation is starting (3.64 and 4.04 mg/100 g). from the above it may be concluded that tma, as measured in the present study using the developed gc-fid method, is a reliable freshness indicator. ital. j. food sci., vol. 30, 2018 532 4. conclusions a quick and easy gas chromatographic method for the analysis of tma in seafood was developed and validated. a capillary column and a classical split injection were used so greatly simplifying the procedure. it was applied to different seafood species caught in the tyrrhenian sea that had been stored in ice. thanks to its simplicity, the method appears very suitable for routine controls. by comparison with fish species, crustaceans such as shrimps exhibited a much greater release of tma, which resulted in a much shorter shelf life (1-2 days). fish species maintained a good freshness state for almost a week, up to 8-10 days for red mullet, with the bigger sizes being more resistant to degradation than the smaller ones. in the present study, the existing guidelines for the tma content are reviewed and discussed. not always there is full clarity on this topic in the literature. the generally accepted criterion for an “optimum freshness state” is a tma content below 42-63 mg/kg (1.0-1.5 mg/100g of tma-n). observations made in the present research fully confirm this limit, at least for the marine species here investigated. acknowledgements research funded by the italian ministry of agricultural food and forestry policies. references bennour m., el marrakchi a., bouchritf n., hamama a. and el ouadaa m. 1991. chemical and microbiological assessments of mackerel (scomber scombrus) stored in ice. j. food protect. 54:784. 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281667860@qq.com abstract the composition of intramuscular phospholipids fatty acids in longissimus dorsimuscle (ld), left-hind leg muscle (ll) and abdominal muscle (am) of inra rabbit slaughtered between 35 to 90 days old were investigated. significant decreasing of intramuscular phospholipids (% total intramuscular lipids) was observed in three muscles as age increased (p < 0.05). the highest phospholipids content was found in ll in both male and female rabbits during the growth period, and the phospholipids content in three muscles of the males were higher than that of the females. abundant amount of unsaturated fatty acids (ufa), especially polyunsaturated fatty acids (pufa) characterised the fatty acid composition of the intramuscular phospholipids (32.94-55.79%), and the percentage of pufa in the muscles were all significantly decreased during the growth of male and female inra rabbit (p < 0.05). in addition, a significant reduction of pufa/sfa ratio and a significant increase of sfa + mufa were observed (p < 0.05). major fatty acids, such as palmitic acid (c16:0), stearic acid (c18:0), oleic acid (c18:1n-9), linoleic acid (c18:2n-6) and arachidonic acid (c20:4) changed more obviously than other fatty acids. the analysis of partial least square regression (plsr) showed that the composition of phospholipid fatty acids varied in age, muscle and gender, and the nutritional value of the phospholipid fatty acids decreased with age distinctly, the am had better nutritional value of phospholipids. keywords: rabbit, fatty acids, intramuscular phospholipids, composition ! ital. j. food sci., vol 28, 2016 684 1. introduction functional foods are a tool that can be easily used in reducing public health costs. compared to meats of other animal species, rabbit meat is characterized by high levels of polyunsaturated fatty acids (pufa) and n-3 fatty acids, high levels of protein with essential amino acids, high digestibility value, lower cholesterol contents and significant source of vitamin b family (vitamins b2, b5, b6, b3, b12) etc. (dalle zotte and szendrö, 2011). moreover, rabbit meat consumption could become a good way of providing bioactive compounds to human consumers, since the rabbit meat fatty acids profile may be favorably modified by the inclusion of raw materials rich in unsaturated fatty acids (ufa) in the diet (dal bosco et al., 2004; hernández, 2008; kouba et al., 2008). rabbit meat is considered as dietetically healthy, relatively rich in n-3 pufas and with a lower n-6 to n-3 ratio (7�12) than pork, veal or chicken meats (dalle zotte, 2002; hernández and gondret, 2006). unlike pork or beef meat, it contains two important metabolites from α-linolenic acid (ala), docosahexaenoic acid (dha, c22:6n-3) and eicosapentaenoic acid (epa, c20:5n-3) in detectable levels (combes and dalle zotte, 2005; eiben et al., 2010). inra rabbit is imported from france and have high breeding efficiency. in recent years, interests have focused not only on the amount of intramuscular phospholipids but also on the composition of fatty acids. the intramuscular fat (imf), which characterises the amount of fat, is one of the major factors affecting the palatability of meat (hocquette et al., 2010). muscle lipids are composed of polar lipids, mainly phospholipids (rich in pufa) located in the cell membranes, and triacylglycerols (high levels of saturated fatty acids (sfa) and monounsaturated fatty acids (mufa)) along the muscle fibres (de smet et al., 2004). in the imf, pufa are restricted almost exclusively to the phospholipids fraction (wood et al., 2003). thus, the amount of intramuscular phospholipids in the meat is an important factor (gray et al., 1996). phospholipids consist of long-chain fatty acids attached to a phosphoryl group. since the fatty acids chains can vary in length and degree of saturation, each phospholipid class possesses numerous molecular species with different chemical and biological properties (marco et al., 2004; wang et al., 2009). cambero et al. (1991) first analysed the phospholipid content and classes in rabbits and they provide important information on phospholipid prevalence according to breed and feeding, specifying that phospholipid differs also according to age and gender. generally, the analysis of phospholipids is based the determination of the phospholipid classes (phosphatidyleth-anolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, sphingomyelin, lysophosphatidylcholine etc.) with high performance liquid chromatography (alasnier and gandemer, 1998; peterson and cummings, 2005; boselli et al., 2008). however, practically limited literature data are available on the determination of the fatty acids in intramuscular phospholipids by gas chromatography after purification of the polar lipid fraction, especially the intramuscular phospholipids from inra rabbit. hence, in order to provide a database for the characterization of nutritional quality of inra rabbit meat, the composition of intramuscular phospholipids fatty acids and the effect of ages, genders and muscles on the composition and nutritional value of fatty acids were investigated by gas chromatography. 2. materials and methods 2.1. sampling of inra rabbit meat ! ital. j. food sci., vol 28, 2016 685 a total of 200 35 days old weaned inra rabbits (20 males+20 females per age) were provided by college of animal science and technology, southwest university. the ingredients and proximate chemical composition of the diet were shown in the table 1. table 1: the ingredients and proximate chemical composition of the diet. item diets ingredients proportion(%) corn 24.2 wheat bran 19 soybean meal 10.82 alfalfa meal 36 corn germ cake 4 rapeseed 3 powder 0.5 dicalcium 0.8 lysine 0.07 methionine 0.11 salt 0.5 premixa 1 nutrition proportion (%) dry matter 89.8% crude protein 16.0% fat 3.3% lysine 0.7 methionine 0.6 calcium 0.95 phosphorus 0.59 acid detergent fiber 33.2% neutral detergent fiber 21.4% digestible energyb 10.5 mj/kg a the premix contains (per kg of diet): vitamin a, 10000 iu; vitamin d3, 1000 iu; vitamin e, 30 mg; vitamin k, 1 mg; vitamin b1, 1 mg; vitamin b2, 3.5 mg; vitamin b6, 2 mg; vitamin b12, 0.01 mg; niacin, 50 mg; folic acid, 0.3 mg; choline, 1000 mg; zn, 30 mg; cu, 5 mg; mn, 15 mg; fe, 30 mg; i, 1 mg. b digestible energy (kcal/kg dm) = tdn×4400 (nrc, 1985). they were maintained in a closed building under natural environmental conditions in individual wire mesh cages, equipped with metal troughs and automatic nipple drinkers. the rabbits had free access to feed and water. the rabbits were bred under similar production system and slaughtered at the age of 35, 45, 60, 75, and 90 d in a local commercial slaughterhouse. the facilities of the slaughterhouse met the requirements of the institute of animal care and use committee (iacuc), which is funded by the united states national institutes of health. after 24 h post-mortem, the longissimus dorsi muscle (ld), left-hind leg muscle (ll), and abdominal muscle (am) (ventral musculus) of the carcass were removed and immediately vacuumpacked and frozen at -20°c until analyzed. ! ital. j. food sci., vol 28, 2016 686 2.2. intramuscular lipid content and fatty acid composition analysis intramuscular lipids were extracted according to folch et al. (1957). total lipid content was measured by weighing after solvent evaporation. the content of imf was expressed as percent of the muscle weight. fractions of intramuscular phospholipids were prepared with silica cartridges (sep-pack, waters, milford, ma, usa) by the method of juaneda and rocquelin (1985). phospholipids were quantified by phosphorous determination (bartlett, 1959). the relative content of phospholpids was expressed as percent of the imf weight, while the absolute content was expressed as percent of the muscle weight. the phospholipids were methylated with boron fluoride-methanol (sigma aldrich) according to morrison and smith (1964). the fatty acids methyl esters were analyzed by a qp-2010 gas chromatograph (shimadzu, kyoto, japan) equipped with a flame ionization detector and a split injector. one microliter of fa methyl esters was injected in split mode (5:1) onto a rtx-wax capillary column (restek, bellefonte, pa, usa; 30 m × 0.25 mm id × 0.25 µm film thickness). the temperature of the column was programmed as follows: 1 min at 140°c, increments of 8°c/min to 180°c and held at 180°c for 2 min, increments of 3°c/min to 210°c then increments of 5°c /min to 230°c and held at 230°c for 10 min. the temperature of the injector and the detector both were 250°c. the flow rate of the carrier gas (n2) was 1.5 ml/min. identification of fatty acids was performed by comparison of the retention times with those of standards (sigma). the results were expressed as percent of the total fatty acids methyl esters present. 2.3. statistical analysis the statistical analysis system (1996) was used to determine means, standard errors and analysis of variance. duncan’s multiple range test was used to compare differences among means. an alpha level of p < 0.05 was considered significant. the effect of ages, muscles and genders on the composition of intramuscular phospholipid fatty acids were performed by anova-partial least squares regression (a-plsr). ten 0/1 indicators variables (35, 45, 60, 75, 90 d, female, male, ld, ll, am), sfa+mufa, and pufa/sfa in the x-matrix and 21 kinds of fatty acids (c12:0 c22: 6n-3 were represented by the number 1-21) in the y-matrix. ellipses represent r2=0.5 (50%) and 1.0 (100%). a plsr was performed using the unscrambler software, version 9.7 (camo asa, trondheim, norway). all data was centered and standardized before analysis. 3. results and discussions 3.1. variation of content of total intramuscular lipids and phospholipids of inra rabbits 3.1.1. variation of intramuscular lipid content the intramuscular lipid content (% muscle weight) of ld, ll and am from male and female inra rabbits were significantly increased (p <0.05) with age (table 2). the intramuscular lipid content of the three muscles of male and female rabbits was all increased. during the growth of inra rabbits, the am showed the highest content of intramuscular lipid, followed by ll and ld. hernàndez and dalle zotte (2010) reported that the leanest cut of meat in the rabbit carcass was the loin and the hindleg was the quantitatively important cut because of its low lipid content compared to the other meats, which was consistent with our investigation (male: 0.77-1.21%, female: 0.79-1.33%). ! ital. j. food sci., vol 28, 2016 687 hence, the lipid content depended greatly on the age, gender and muscle. during the growth period from 35 d to 90 d, the deposition degree of total intramuscular lipid of the females in am (2.88% to 5.42%) was significantly higher than that in the ll (1.19% to 1.78%) and ld (0.79% to 1.33%). table 2: comparison of intramuscular lipid content of inra rabbit at different agesa. ld a ll am male female male female male female 35 dbc 0.77±0.02d 0.79±0.08c 1.16±0.12d 1.19±0.15c 2.41±0.16d 2.88±0.12e 45 d 0.80±0.08cd 0.82±0.06c 1.20±0.06d 1.24±0.12c 2.67±0.09c 3.18±0.08d 60 d 0.96±0.11bc 1.01±0.10bc 1.30±0.03c 1.45±0.13b 2.74±0.01c 3.58±0.20c 75 d 1.15±0.13ab 1.25±0.24ab 1.49±0.12b 1.64±0.07ab 4.60±0.03b 4.83±0.05b 90 d 1.21±0.13a 1.33±0.21a 1.66±0.02a 1.78±0.06a 5.16±0.20a 5.42±0.17a a ld, longissimus dorsimuscle; ll, left-hind leg muscle; am, abdominal muscle. b results were expressed as means ± se, data were means of three replicates. c values in the same column with different letters were significantly different (p<0.05), male: a-d, female: ae. 3.1.2. variation of intramuscular phospholipids content the percentage of phospholipids in the total intramuscular lipids of both male and female inra rabbits was significantly decreased at the three muscles with age (p <0.05) (table 3). a higher percentage of phospholipids characterized the total lipids (22.35-53.81%), however, the phospholipid contents in the meat of both new zealand white and the commercial hybrid ranged from 9% to 19% total lipid (cambero et al., 1991). table 3: comparison of intramuscular phospholipids content (intramuscular lipid weight %) of inra rabbit at different agesa. lda ll am male female male female male female 35 dbc 43.47±1.33a 42.11±1.96a 53.81±2.82a 51.03±2.22a 42.21±2.36a 37.01±1.98a 45 d 35.02±1.88b 33.64±1.53b 38.98±1.48b 36.78±1.65b 34.94±2.88b 31.99±1.68b 60 d 32.28±1.02b 31.14±1.92b 36.35±1.44b 33.87±1.60b 28.93±1.22c 25.31±1.16c 75 d 27.93±1.58c 24.13±0.17c 30.21±2.14c 27.84±2.39c 27.87±1.91c 23.36±2.96c 90 d 26.84±1.72c 23.52±0.86c 28.89±2.18c 26.78±1.16c 25.40±1.32c 22.35±1.29c a ld, longissimus dorsimuscle; ll, left-hind leg muscle; am, abdominal muscle. b results were expressed as means ± se, data were means of three replicates. c values in the same column with different letters were significantly different (p<0.05), male: a-c, female: ac. the highest relative phospholipids content was found in the ll in both male and female inra rabbits at different ages. in addition, the relative content of phospholipids in inra rabbit abdomen showed significant gender differences, whereas that in legs and back phospholipids content appeared more pronounced gender differences after the age of 60 d. however, the absolute percentage of intramuscular phospholipids in the three muscles ! ital. j. food sci., vol 28, 2016 688 did not vary obviously (p > 0.05) (fig. 1). among the three muscles, the am showed the maximum absolute percentage of intramuscular phospholipids, followed by ll and ld, and the absolute percentage of intramuscular phospholipids in males were higher than that in females. according to wood et al (2008), phospholipids remained constant or increase little, as muscle fatness increases, whilst triacylglycerols increased to a higher extent, which may explain this phenomenon. figure 1: comparison of intramuscular phospholipids content (muscle weight %) of inra rabbit at different ages (a: male rabbits, b: female rabbits). 3.2. effect of age, muscle and gender on composition of intramuscular phospholipids the comparative intramuscular phospholipids fatty acids in ld, ll and am of both male and female inra rabbits at 35, 45, 60, 75, 90 d were shown in table 4 (ld), table 5 (ll), and table 6 (am), respectively. high levels of ufa (the sum of pufa and mufa), especially the abundance of pufa, including the long chain (c20-22) pufa in muscle, were observed in all samples. in muscle, significant percentage is phospholipids, which has a much higher pufa content in order to perform its function as a constituent of cellular membranes (wood et al., 2008). the ratio of sfa and mufa in ld, ll and am increased significantly with age in both gender rabbits (p < 0.05), whereas the ratio of pufa among the muscles were all significantly decreased (p < 0.05). in addition, a significant reduction of pufa/sfa ratio and a significant increase of sfa + mufa were observed (p < 0.05). during the growth, the phospholipids pufa percentage (% intramuscular fatty acids) was significantly higher in the ll than that in the ld and am of male rabbits, corresponding to the lowest mufa percentage in male ll. the phospholipids pufa percentage in female ld was the lowest, while the mufa in female legs was the minimum. compared to the ld and ll, the am existed more obvious gender differences in sfa and ufa percentage of phospholipids. ! ital. j. food sci., vol 28, 2016 689 table 4: composition of the fatty acids of intramuscular phospholipids (%) in longissimus dorsi of inra rabbit at different agesa. 35d 45d 60d 75d 90d male female male female male female male female male female c12:0b 0.10±0.02a 0.03±0.01c 0.05±0.01b 0.08±0.02b 0.12±0.03a 0.17±0.02a -d c14:0 0.26±0.05e 0.28±0.05b 0.47±0.01d 0.52±0.15a 0.60±0.17c 0.57±0.07a 0.79±0.05b 0.58±0.05a 0.83±0.05a 0.53±0.04a c14:1 0.36±0.02b 0.41±0.02a 0.26±0.02c 0.46±0.04a 0.11±0.02d 0.41±0.06a 0.34±0.03b 0.46±0.11a 0.50±0.02a 0.46±0.02a c15:0 1.19±0.11d 1.46±0.22e 1.65±0.16c 1.57±0.10d 1.81±0.22b 2.47±0.31c 2.02±0.18a 3.20±0.32a 1.10±0.03d 2.79±0.13b c16:0 20.03±0.03e 17.16±0.19e 21.64±0.38d 20.30±0.05d 22.37±0.11c 21.25±0.09c 23.71±0.14b 23.34±0.12b 25.11±0.30a 28.22±0.02a c16:1n-7 0.24±0.03c 0.29±0.03d 0.31±0.02c 0.48±0.06a 0.34±0.03c 0.39±0.02c 0.52±0.02b 0.44±0.04b 1.75±0.24a 0.44±0.01b c17:0 0.50±0.02a 0.48±0.02a 0.42±0.03b 0.47±0.04a 0.38±0.02b 0.39±0.02b 0.48±0.02a 0.40±0.01b 0.41±0.01b 0.50±0.02a c17:1 0.56±0.04d 0.72±0.09e 0.82±0.05a 1.34±0.07a 0.92±0.07b 1.15±0.07b 0.66±0.05c 0.93±0.05d 0.41±0.01e 1.01±0.03c c18:0 11.04±0.03e 12.58±0.40d 11.72±0.07d 13.75±0.10c 12.56±0.13c 13.82±0.03c 13.11±0.02b 14.31±0.24b 13.50±0.03a 14.45±0.04a c18:1n-9 13.34±0.02e 13.54±0.15e 13.99±0.53d 13.69±0.15d 15.01±0.03c 14.43±0.03c 15.80±0.04b 14.61±0.06b 17.89±0.02a 17.75±0.07a c18:1n-7 1.63±0.03a 1.64±0.21a 1.52±0.31a 0.88±0.12d 1.00±0.03b 1.06±0.01c 0.86±0.02b 1.15±0.02b 0.65±0.02c 0.65±0.02e c18:2n-6 25.91±0.07a 25.00±0.23a 24.29±0.28b 20.24±0.13b 22.67±0.02c 19.14±0.10c 20.77±0.02d 17.65±0.11d 20.21±0.07e 15.09±0.08e c18:3n-3 0.27±0.03d 0.35±0.03b 0.41±0.02b 0.58±0.02a 0.48±0.02c 0.58±0.03a 0.78±0.06a 0.56±0.02a 0.37±0.02b 0.38±0.12b c20:0 0.05±0.01a 0.06±0.01b 0.08±0.03a 0.17±0.01a 0.11±0.03a 0.17±0.02a 0.09±0.02a 0.17±0.01a 0.13±0.02a 0.16±0.06a c20:1n-9 0.18±0.01ab 0.17±0.02b 0.11±0.03b 0.11±0.03c 0.18±0.06ab 0.22±0.02a 0.37±0.03a 0.14±0.01bc 0.16±0.02ab 0.10±0.02c c20:2n-6 0.68±0.03d 0.73±0.03e 0.71±0.03d 0.83±0.06d 1.19±0.02c 0.90±0.03c 1.65±0.03a 1.15±0.02b 1.74±0.03b 1.23±0.01a c20:3n-6 0.63±0.02e 0.68±0.04c 0.91±0.02d 1.21±0.07a 1.26±0.03c 1.20±0.06a 1.54±0.02b 1.21±0.04a 1.88±0.03a 0.82±0.05b c20:4n-6 13.80±0.08a 14.58±0.05a 11.78±0.08b 14.01±0.13b 9.72±0.14c 13.64±0.15c 9.47±0.04d 12.99±0.11d 8.81±0.05e 10.36±0.10e c20:5n-3 5.01±0.04a 5.53±0.03a 4.84±0.02b 5.03±0.12b 4.60±0.04c 4.60±0.03c 3.91±0.03d 4.04±0.06d 2.77±0.10e 3.40±0.02e c22:5n-3 2.58±0.02a 2.49±0.03a 2.47±0.01b 2.33±0.08b 2.34±0.02c 2.21±0.04c 1.84±0.01d 1.62±0.04d 1.12±0.03e 1.11±0.03e c22:6n-3 1.65±0.02a 1.83±0.02a 1.56±0.02b 1.41±0.04b 1.40±0.03c 1.23±0.01c 1.29±0.02d 1.03±0.01d 0.69±0.02e 0.56±0.03e sfac 33.16±0.12e 32.06±0.06e 36.03±0.69d 36.86±0.78d 37.96±0.11c 38.83±0.34c 40.19±0.13b 41.99±0.26b 41.07±0.31a 46.65±0.07a pufa 50.54±0.17a 51.18±0.24a 46.96±0.34b 45.64±0.07b 43.68±0.17c 43.51±0.35c 41.26±0.07d 40.27±0.30d 37.56±0.09e 32.94±0.09e mufa 16.30±0.07e 16.76±0.28d 17.02±0.77d 17.50±0.85c 18.36±0.18c 17.66±0.06bc 18.55±0.19b 17.74±0.04b 21.37±0.22a 20.41±0.02a a results were expressed as means ± se, data were means of three replicates. b values in the same column with different letters were significantly different (p<0.05), male: a-e, female: a-e. c sfa, total saturated fatty acids; mufa, total monounsaturated fatty acids; pufa, total polyunsaturated fatty acids. d ”-” : undetected. ! ital. j. food sci., vol 28, 2016 690 table 5: composition of the fatty acids of intramuscular phospholipids (%) in left-hind leg muscle of inra rabbit at different agesa. 35d 45d 60d 75d 90d male female male female male female male female male female c12:0b 0.05±0.01b 0.04±0.01b 0.04±0.01b 0.09±0.01a 0.27±0.02a 0.05±0.02b -d c14:0 0.16±0.02b 0.23±0.06d 0.22±0.07b 0.41±0.11c 0.34±0.09b 0.54±0.13bc 0.77±0.16a 0.95±0.11a 0.26±0.01b 0.68±0.02b c14:1 0.35±0.03b 0.37±0.02b 0.22±0.02c 0.14±0.02c 0.14±0.01d 0.51±0.03a 0.23±0.04c 0.32±0.01b 0.52±0.01a 0.52±0.03a c15:0 1.12±0.25d 1.55±0.09b 1.46±0.35c 1.60±0.09b 1.51±0.36bc 2.39±0.47a 1.53±0.03b 1.77±0.13b 1.66±0.01a 1.84±0.07b c16:0 19.57±0.05e 16.98±0.13d 20.65±0.08d 20.48±0.07c 21.55±0.11c 21.95±0.06b 22.63±0.13b 22.01±0.22b 24.08±0.05a 24.66±0.20a c16:1n-7 0.34±0.02c 0.37±0.02d 0.32±0.02c 0.39±0.02cd 0.36±0.02c 0.41±0.02c 0.51±0.18b 0.75±0.02a 1.94±0.03a 0.48±0.01b c17:0 0.40±0.03c 0.43±0.01b 0.44±0.01b 0.36±0.01c 0.38±0.02c 0.50±0.02a 0.55±0.03a 0.40±0.03b 0.40±0.02c 0.49±0.01a c17:1 0.51±0.09bc 0.67±0.02b 0.94±0.12a 2.08±0.29a 0.86±0.13a 0.89±0.11b 0.57±0.01b 0.54±0.05b 0.35±0.02c 0.78±0.02b c18:0 11.79±0.06e 13.12±0.06e 13.15±0.04d 14.78±0.10d 14.61±0.03c 15.29±0.10c 15.97±0.04b 15.91±0.03b 16.12±0.02a 16.32±0.03a c18:1n-9 11.78±0.05e 12.09±0.12e 12.48±0.02d 12.34±0.06d 13.24±0.16c 13.60±0.12c 14.83±0.02b 13.94±0.15b 15.10±0.02a 15.75±0.07a c18:1n-7 1.48±0.06ab 1.39±0.03a 1.48±0.02ab 0.85±0.01c 1.41±0.10a 0.99±0.05b 1.53±0.02b 1.00±0.08b 0.61±0.01c 0.70±0.03d c18:2n-6 29.86±0.11a 26.34±0.02a 26.58±0.07b 20.62±0.25b 24.59±0.01c 18.77±0.72c 21.37±0.05d 18.21±0.02d 20.84±0.02e 15.45±0.05e c18:3n-3 0.37±0.02c 0.37±0.02cd 0.37±0.02c 0.72±0.06a 0.54±0.01ab 0.40±0.02c 0.83±0.37a 0.56±0.01b 0.50±0.03c 0.35±0.04d c20:0 0.05±0.01b 0.07±0.01a 0.06±0.01b 0.17±0.03a 0.13±0.03a 0.17±0.18a 0.09±0.02b 0.12±0.03a 0.08±0.01b 0.13±0.01a c20:1n-9 0.18±0.02b 0.18±0.02bc 0.17±0.02b 0.11±0.02d 0.27±0.02a 0.20±0.02b 0.28±0.03a 0.28±0.01a 0.27±0.01a 0.15±0.02c c20:2n-6 0.83±0.02d 0.83±0.02e 0.87±0.02c 0.93±0.01d 1.11±0.02b 1.03±0.02c 1.15±0.01b 1.41±0.02b 1.29±0.02a 1.53±0.02a c20:3n-6 0.83±0.02d 0.78±0.02e 0.93±0.02c 1.08±0.02d 1.08±0.04b 1.04±0.01c 1.18±0.02a 1.10±0.02b 0.95±0.02c 1.16±0.01a c20:4n-6 12.33±0.10a 15.29±0.07a 11.95±0.18b 14.42±0.07b 10.29±0.02c 13.94±0.05c 9.95±0.05d 13.56±0.09c 9.84±0.02d 12.59±0.07d c20:5n-3 3.77±0.06a 4.93±0.02a 3.58±0.03b 4.73±0.05b 3.34±0.03c 4.45±0.11c 3.12±0.01d 4.21±0.03d 3.09±0.02e 4.17±0.05d c22:5n-3 2.40±0.04a 2.33±0.02a 2.34±0.02b 2.31±0.03b 2.33±0.02b 2.06±0.04c 1.87±0.02c 1.79±0.03d 1.34±0.04d 1.59±0.08e c22:6n-3 1.82±0.15a 1.63±0.02a 1.75±0.03a 1.37±0.02b 1.66±0.03b 1.27±0.28c 1.05±0.01c 1.17±0.02d 0.83±0.02d 0.67±0.04e sfac 33.14±0.29e 32.44±0.06d 36.02±0.56d 37.90±0.01c 38.77±0.11c 40.89±0.33b 41.53±0.23b 41.16±0.04b 42.60±0.07a 44.13±0.11a pufa 52.22±0.45a 52.50±0.10a 48.37±0.26b 46.19±0.22b 41.95±0.07c 42.51±0.39c 40.51±0.35d 42.01±0.19d 38.67±0.03e 37.50±0.12e mufa 14.64±0.16e 15.07±0.14d 15.61±0.09d 15.91±0.21c 16.28±0.14c 16.61±0.07b 17.96±0.13b 16.83±0.19b 18.72±0.08a 18.37±0.08a a results were expressed as means ± se, data were means of three replicates. b values in the same column with different letters were significantly different (p<0.05), male: a-e, female: a-e. c sfa, total saturated fatty acids; mufa, total monounsaturated fatty acids; pufa, total polyunsaturated fatty acids. d ”-” : undetected. ! ital. j. food sci., vol 28, 2016 691 table 6: composition of the fatty acids of intramuscular phospholipids (%) in abdominal muscle of inra rabbit at different agesa. 35d 45d 60d 75d 90d male female male female male female male female male female c12:0b 0.17±0.01a 0.07±0.02a 0.07±0.02b 0.04±0.01b 0.07±0.02b 0.06±0.01ab -d c14:0 0.53±0.16a 0.39±0.05d 0.38±0.07ab 0.65±0.19a 0.30±0.02b 0.41±0.06c 0.44±0.14ab 0.59±0.04b 0.27±0.02b 0.69±0.03a c14:1 0.25±0.02b 0.15±0.02d 0.21±0.02b 0.18±0.01c 0.60±0.35a 0.27±0.01b 0.24±0.02b 0.28±0.02b 0.47±0.05ab 0.56±0.19a c15:0 1.82±0.56a 1.36±0.02d 2.53±0.76a 1.68±0.39c 2.34±0.79a 1.80±0.57b 2.88±0.52a 1.83±0.51b 1.98±0.13a 1.94±0.04a c16:0 21.22±0.07c 12.65±0.01d 22.17±0.08bc 12.90±0.26d 22.67±0.31b 14.06±0.13c 22.97±0.06b 17.23±0.06b 23.87±1.09a 21.37±0.11a c16:1n-7 0.34±0.03d 0.34±0.02e 0.30±0.02d 0.48±0.02d 1.02±0.32b 0.65±0.02c 2.02±0.24a 1.05±0.02b 2.04±0.06a 1.56±0.12a c17:0 0.43±0.02bc 0.44±0.02a 0.44±0.01b 0.39±0.02b 0.41±0.03c 0.33±0.01c 0.52±0.01a 0.38±0.03b 0.37±0.02d 0.47±0.01a c17:1 0.82±0.23ab 0.61±0.11bc 0.93±0.28ab 1.27±0.14a 1.16±0.25a 0.74±0.19b 0.92±0.18ab 0.46±0.11c 0.65±0.03b 0.47±0.01c c18:0 10.00±0.08e 13.24±0.03e 10.58±0.02d 15.50±0.06d 10.95±0.28c 17.04±0.11c 11.42±0.34b 17.39±0.05b 13.48±1.35a 17.67±0.40a c18:1n-9 13.93±0.03b 13.22±0.02d 14.21±0.78b 13.65±0.50cd 14.66±0.24b 14.10±0.07c 16.42±0.28a 14.65±0.06b 16.89±0.85a 15.40±0.38a c18:1n-7 1.20±0.04a 1.42±0.03a 1.12±0.27a 0.90±0.19c 0.65±0.03b 0.71±0.07d 0.50±0.04b 1.13±0.04b 0.99±0.38ab 1.34±0.07a c18:2n-6 24.23±0.07a 25.26±0.10a 23.91±0.03b 22.09±0.09b 23.48±0.58b 20.80±0.19c 22.78±0.29bc 19.73±0.13d 22.56±0.99c 17.21±0.21e c18:3n-3 0.35±0.01c 0.35±0.02e 0.31±0.02d 0.60±0.02b 0.56±0.03b 0.51±0.02c 0.85±0.02a 0.67±0.02a 0.54±0.01b 0.45±0.02d c20:0 0.13±0.03a 0.10±0.02ab 0.07±0.01b 0.08±0.01b 0.11±0.01ab 0.11±0.02a 0.09±0.01b 0.12±0.01a 0.07±0.01b 0.13±0.01a c20:1n-9 0.26±0.01b 0.22±0.02b 0.17±0.02b 0.23±0.02b 0.30±0.02ab 0.27±0.02a 0.22±0.02b 0.27±0.02a 0.37±0.12a 0.20±0.03c c20:2n-6 0.96±0.02c 0.76±0.04e 1.11±0.03d 0.98±0.02d 1.14±0.03b 1.14±0.02b 1.23±0.07a 1.07±0.02c 1.08±0.05b 1.24±0.02a c20:3n-6 0.95±0.01d 0.83±0.02e 1.00±0.03c 1.13±0.02d 1.28±0.03b 1.43±0.01c 1.38±0.07a 1.78±0.05b 0.99±0.05cb 1.96±0.02a c20:4n-6 12.93±0.14a 17.88±0.08a 11.71±0.03b 17.40±0.15b 10.72±0.41c 16.08±0.12c 8.92±0.40d 13.64±0.10d 8.67±0.41d 11.22±0.17e c20:5n-3 5.65±0.06a 5.84±0.07a 5.23±0.02b 5.48±0.07b 4.44±0.15c 5.16±0.02b 3.48±0.10d 4.59±0.04c 2.88±0.14e 3.59±0.09d c22:5n-3 2.24±0.03a 2.96±0.03a 2.12±0.02b 2.73±0.02b 1.76±0.07d 2.72±0.02b 1.87±0.08c 1.91±0.07c 1.08±0.01e 1.56±0.03d c22:6n-3 1.60±0.14a 1.92±0.03a 1.45±0.01b 1.61±0.06b 1.37±0.08c 1.59±0.03b 1.08±0.04d 1.24±0.01c 0.76±0.01e 1.08±0.02d sfac 34.30±0.30d 28.25±0.27e 36.22±0.98c 31.25±0.95d 36.84±0.73c 33.82±0.28c 38.31±0.35b 37.54±0.43b 40.03±0.50a 42.26±0.36a pufa 48.91±0.45a 55.79±0.37a 46.84±0.14b 52.03±0.22b 44.75±0.21c 49.43±0.34c 41.59±0.82d 44.63±0.12d 38.56±1.65e 38.31±0.53e mufa 16.79±0.16a 15.96±0.09b 16.94±0.77a 16.72±0.56c 18.41±0.48b 16.75±0.07d 20.10±0.49c 17.83±0.06b 21.41±1.28d 19.43±0.21a a results were expressed as means ± se, data were means of three replicates. b values in the same column with different letters were significantly different (p<0.05), male: a-e, female: a-e. c sfa, total saturated fatty acids; mufa, total monounsaturated fatty acids; pufa, total polyunsaturated fatty acids. d ”-” : undetected. ! ital. j. food sci., vol 28, 2016 692 in terms of fatty acids composition of intramuscular phospholipids of inra rabbit, sfa among the muscles were mainly composed of palmitic (c16:0) and stearic (c18:0), mufa were mainly represented by oleic (c18:1), whereas pufa consisted of linoleic (c18:2) and arachidonic acid (c20:4). according to cambero et al. (1991), the c16:0, c18:0, c18:1 and c18:2 were together representing more than 70% of the total fatty acids. a higher percentage of pufa characterized the fatty acids composition of phospholipids (kanatt et al., 2006). in our study, the percentage of pufa in males and females were accounted for 37.56-52.22% and 32.94-55.79% respectively in the intramuscular phospholipids during growth period from 35 d to 90 d. long chain n-3 and n-6 pufa were mainly found in phospholipids (enser et al., 2000; cooper et al., 2004), which was also in good agreement with our investigation. however, the fatty acids composition was rarely detected in different rabbit muscles during different feeding days, especially on a particular rabbit species. comparing the variety of intramuscular phospholipids from ld, ll and am during the growth stages from 35 d to 90 d, c16:0, c18:0, palmitoleic acid methyl ester (c16:1n-7), c18:1n-9, cis-11,14-eicosadienoic acid methyl ester (c20:2n-6) and cis-8,11,14-eicosatrienoic acid methyl ester (c20:3n-6) increased significantly (p < 0.05) in both genders, whereas c18:2n-6, c20:4n-6, c20:5n-3, c22:5n-3 and c22:6n-3 decreased significantly (p < 0.05). the percentage of c16:0 and c18:0 in female-ld significantly increased (p < 0.05), and both c18:2n-6 and c20:4n-6 in female-ll significantly decreased (p < 0.05). moreover, the percentage of c20:4n-6 in female-am decreased faster than other samples during the test days. however, other fatty acids did not showed apparent changes. according to alasnier and gandemer (1998), the fatty acid composition of individual phospholipid classes was related to metabolic type of fibre in the rabbit, and the differences in fatty acid composition of phosphatidyl ethanolamine, phosphatidyl choline and cardiolipin explained a large part of the differences in fatty acid compositions of the total phospholipids of glycolytic and oxidative muscles. as the major ingredient of feeds for all species, the incorporation of c18:2n-6 into the muscles, in relation to the amount in the diet, was greatest among other fatty acids. c18:2n-6 was deposited in muscle phospholipids at a high level where it and its long chain products c20:4n-6 competed well for insertion into phospholipids molecules (wood et al., 2008). comparing the changes of fatty acids in the ld, ll and am, the deposition rate of c16:0 was faster in the ld of inra rabbits (both males and females) than that in the ll and am. however, the c16:0 had lowest percentage and slowest deposition rate in am. in terms of c18:0, ll was sequentially higher than ld and am in male rabbits, while am was higher than ll and ld in female rabbits. meanwhile, the percentage and deposition rate of c18:0 in am were also higher in females than that in males. for the c18:1n-9 percentage, ld showed the highest and fastest deposition rate in both genders. the percentage of c18:2n-6 in ll was the highest, and decreased in the maximum levels with age. according to wood et al (2008), the higher percentage of c18:2n-6 in phospholipids compared with neutral lipids in all species mean that muscle from lean animals has relatively higher percentages of this major pufa. in addition, during the growth period from 35 d to 90 d, the initial content of c20:4n-6 in ld was higher compared with other muscles, and showed the fastest reducing rate. no significantly variation was found among other fatty acids components. during the growth period of inra rabbit, the n-6/n-3 values for ld, ll, and am ranged from 4.02 to 6.61, 4.71 to 5.72 and 3.97 to 6.33 in males, and 3.88 to 5.05, 4.05 to 4.67 and 3.95 to 4.73 in females, respectively. there is an increasing recognition of the health benefits of pufa in general, and of n-3 pufas in particular, because these fatty acids are essential for humans (alessandri et al., 1998; conquer et al., 2011). nutritional value is determined primarily by the ratio between sfa and pufa in meat and the balance between fatty acids of the n-6 and n-3 series. unfortunately, western diet is very high in n! ital. j. food sci., vol 28, 2016 693 6 fatty acids relative to n-3 fatty acids (enser et al., 2000; hargis and van elswyk, 1993). nutritionist recommendations are for a ratio of n-6/n-3 pufa of less than 5 (wood et al., 2003; kouba et al., 2003), and a ratio of n-6/n-3 below about 4.0 is required in the diet to combat various ‘‘lifestyle diseases’’ such as coronary heart disease and cancers (simopoulos, 2004; williams, 2000). according to fao/who, the recommended dose of essential pufa in a healthy daily diet is 5/1 to 10/1 (n-6/n-3) (dalle zotte and szendrö, 2011), and a lower ratio is more desirable in reducing the risk of many of chronic diseases, even if the optimal ratio may vary depending on the disease under consideration (simopoulos, 2002). therefore, it can be suggested that the intramuscular phospholipids of inra rabbits is recommended. alasnier and gandemer (1998) reported that the phosphatidyl ethanolamine of oxidative muscles contains less 18:2n-6 and more 18:0 and long chain pufa of the n-6 and n-3 series than that of glycolytic ones; phosphatidyl choline of oxidative muscles contains more 18:0 and less 16:0 and 18:2n-6 than that of glycolytic ones; cardiolipin of the oxidative muscles contains less 18:2 n-6 than those of the glycolytic ones. in addition, they suggested that a part of the composition difference could be related to high mitochondria content of the oxidative muscles compared to the glycolytic ones. to check this hypothesis, further investigations are required to determinate the fatty acid composition of individual phospholipid classes of both mitochondria and microsomes in rabbit muscles. 3.3. analysis of plsr on composition of intramuscular phospholipids the analysis of plsr showed that the first and second main ingredients explained 43% and 33% y variables, respectively. from the nutritional point of view, the pufa/sfa value is often used to evaluate the nutritional value of the meat, and higher value represents better nutritional value. however, the higher sfa + mufa value of the meat are, the tenderness juiciness and the better flavor are (cameron and enser, 1991). on the contrary, if the content of pufa is too high, the tenderness, flavor and juiciness of meat are poor. hence, the sfa + mufa value can be used to measure the quality indicators of samples after processing. the sfa + mufa value of intramuscular phospholipid fatty acids of inra rabbit was located in the bottom right renderings, indicating the higher the nutritional value of the sample in the bottom right, and the pufa/sfa values were located in the top left of the renderings (fig. 2). thus, the better flavor of the sample after processing is closer to the top left. on the first principal component, the composition of phospholipid fatty acids showed obviously different in ages, genders and muscles. the ld was closer to the sfa + mufa, indicating the better phospholipid processed-flavor after processing of ld. however, the am was closer to pufa/sfa, indicating the better phospholipid nutritional value of am. on the second principal component, the composition of phospholipid fatty acid of the raw material showed obviously different in ages and genders. the nutritional value of the total lipid decreased with age. in addition, the 35 d-feedstock located in the top left oval, closely to 12 (c18: 2n-6), 18 (c20: 4n-6), 19 (c20: 5n-3), 20 (c22: 5n-3), 21 (c22: 6n-3) and other pufas, while the 90 dfeedstock located in the bottom right of the ellipse, closely to 6 (c16:1n-7,), 2 (c14:0), 3(c14:1n-6), 5 (c16:0), 10 (c18:1n-9) and some other saturated and monounsaturated fatty acids. moreover, the intramuscular phospholipids of male rabbits was closely related to the 5 (c16:0) and 12 (c18:2n-6), that is the c16:0 and c18:2n-6 percentage in muscle was higher in male rabbits than that in the females. the intramuscular phospholipid of female rabbits was closely related to the percentage of 9 (c18:0)�14 (c20:0) and 18 (c20:4n-6), that is the c18:0, c20:0 and c20:4n-6 levels in muscle were higher in female rabbits than that in the males. among the three sections, the am had better phospholipid nutritional ! ital. j. food sci., vol 28, 2016 694 value, which may be due to the higher percentage of pufa, and the ld and ll showed better phospholipid flavor after processing, which may be due to the higher percentage of 13 (c18:3n-3) and 7 (c17:0), 15 (c20:1n-9), respectively. figure 2: a plsr correlation loadings plot for first 2 principal components (pcs). ten 0/1 indicators variables (35 d, 45 d, 60 d, 75 d, 90 d, female, male, ld, ll, am), sfa+mufa, and pufa/sfa in the x-matrix and 21 kinds of fatty acids (c12:0 c22:6n-3 were represented by the number 1-21) in the y-matrix. ellipses represent r2=0.5 (50%) and 1.0 (100%). overall, the composition of phospholipid fatty acids at different ages, genders and muscles showed significant difference. the effects of age, gender and muscle on the composition of phospholipid fatty acids mainly reflected on the first principal component. however, on the second principal component, only ages and genders showed the obvious difference of phospholipid fatty acids composition. 4. conclusions inra rabbits are a meat source of nutritious quality, containing low content of intramuscular lipids, low ratio of n-6/n-3, whilst high content intramuscular phospholipids (% lipid). a higher content of pufa characterised the fatty acids composition of the phospholipids, and the significantly decrease of pufa in intramuscular phospholipids during growth were observed. among the fatty acids from intramuscular phospholipids, sfa mainly consists of c16:0 and c18:0, mufa consist of c18:1, and pufa consist of c18:2 and c20:4. there is a wide variation of total lipids content and fatty acid composition at different ages, genders and muscles in inra rabbit. by the analysis of plsr, the nutritional value of the phospholipid fatty acids decreased with age, and the am showed better nutritional value than ld and ll. the absolute data analysis is important for recommendations and suggestion of the consumption of dietary ! ital. j. food sci., vol 28, 2016 695 phospholipids from animal sources. further investigation is necessary to explore the properties of processing and nutritional characteristics of different sections from inra rabbit meat. acknowledgements the authors are grateful to college of animal science and technology, southwest university for the help in the meat sample supply. this study was funded by the special public welfare industry (agriculture) research program of china (grant no. 201303144), the national rabbit industry technology system program (grant no. cars-44d-1), the doctoral scientific research foundation of minnan normal university (grant no. 2006l21513), and the program of education and scientific research for young and middle-aged teachers in fujian province (grant no. jat160296). references alasnier c., gandemer g. 1998. fatty acid and aldehyde composition of individual phospholipid classes of rabbit skeletal muscles is related to the metabolic type of the fibre. meat sci. 48:225-235. alessandri j.m., goustard b., guesnet p., durand, a. 1998. docosahexaenoic acid concentrations in retinal phospholipids of piglets fed an infant formula enriched with long-chain polyunsaturated fatty acids: effects of egg phospholipids and fish oils with different ratios of eicosapentaenoic acid to docosahexaenoic acid. am. j. clin. nutr., 67:377-385. bartlett g.r. 1959. phosphorus assay in column chromatography. j. biol. chem., 234:466-468. boselli e., pacetti d., curzi f., frega n.g. 2008. determination of phospholipid molecular species in pork meat by high performance liquid chromatography-tandem mass spectrometry and evaporative light scattering detection. meat sci. 78:305-313. cambero m.i., de la hoz l., sanz b., ordóñez j.a. 1991. lipid and fatty acid composition of rabbit meat: part 2.phospholipids. meat sci. 29:167-176. cameron n.d., enser m.b. 1991. fatty acid composition of lipid in longissimus dorsi muscle of duroc and british landrace pigs and its relationship with eating quality. meat sci. 29:295-307. combes s., dalle zotte a. 2005. rabbit meat: dietetic properties and processing characteristics. 11émes journées de la recherche cunicole, paris france, pp. 167-180. conquer j.a., holub b.j. 1998. effect of supplementation with different doses of dha on the levels of circulating dha as non-esterified fatty acid in subjects of asian indian background. j. lipid res. 39:286-292. cooper s.l., sinclair l.a., wilkinson, r. g., hallett, k. g., enser, m., wood, j. d. 2004. manipulation of the n-3 polyunsaturated acid content of muscle and adipose tissue in lambs. j. anim. sci. 82:1461-1470. dal bosco a., castellini c., bianchi l., mugnai c. 2004. effect of dietary α-linolenic acid and vitamin e on the fatty acid composition, storage stability and sensory traits of rabbit meat. meat sci. 66:407-413. dalle zotte a. 2002. perception of rabbit meat quality and major factors influencing the rabbit carcass and meat quality. livest. prod. sci. 75:11-32. dalle zotte a., szendrö z. 2011. the role of rabbit meat as functional food. a review. meat sci. 88:319-331. eiben cs., végi b., virág gy., gódor-surmann k., maró a., odermatt m., zsédely e., tóth t., schmidt j. 2010. effect of different dietary ratios of sunflower and linseed oils on growth and carcass traits of rabbits. livest. sci. 131:15-22. enser m., richardson r.i., wood j.d., gill b.p., sheard, p. r. 2000. feeding linseed to increase the n-3 pufa of pork: fatty acid composition of muscle, adipose tissue, liver and sausages. meat sci. 55:201-212. folch j., lees m., stanley g.h.s. 1957. a simple method for the isolation and purification of total lipids from animal tissues. j. biol chem., 226, 497-509. gray j.i., gomaa e.a., buckley d.j. 1996. oxidative quality and shelf life of meats. meat sci., 43, s111-s123. hargis p.s., van elswyk m.e. 1993. manipulating the fatty acid composition of poultry meat and eggs for the health conscious consumer. world's poult. sci. j. 49:251-264. ! 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simopoulos a.p. 2004. omega-3 fatty acids and antioxidants in edible wild plants. biol res. 37:263-277. simopoulos a.p. 2001. n-3 fatty acids and human health: defining strategies for public policy. lipids 3:s83-s89. simopoulos a.p. 2002. the importance of the ratio of omega-6/omega-3 essential fatty acids. biomed. pharmacother., 56, 365−379. wang d., xu w., xu x., zhou g., zhu y., li c., yang m. 2009. determination of intramuscular phospholipid classes and molecular species in gaoyou duck. food chem. 112:150-155. williams c.m. 2000. dietary fatty acids and human health. ann. zootech. 49:165-180. wood j. d., enser m., fisher a.v., nute g.r., sheard p.r., richardson r.i., hughes s.i., whittington f.m. 2008. fat deposition, fatty acid composition and meat quality: a review. meat sci. 78:343-358. wood j.d., richardson r.i., nute g.r., fisher a.v., campo m.m., kasapidou e. 2003. effects of fatty acids on meat quality: a review. meat sci. 66:21-32. paper received december 11, 2014 accepted september 10, 2015 ijfs#1403_bozza ital. j. food sci., vol. 32, 2020 16 paper beef traditional food: consumer before purchase preferences based on quality c. silvestri*, b. aquilani, m. piccarozzi and a. ruggieri department of economy, engineering, society and business, university of tuscia, viterbo, italy *corresponding author: c.silvestri@unitus.it abstract the aim of the paper is to study beef quality cues and attributes in italy, comparing regions where beef is considered traditional food and regions where it is not. a quantitative research has been conducted; both a factor analysis and a cluster analysis were performed. quality cues and/(or) attributes distinguish consumers when before purchase preferences are considered. traceability and safety issues have become crucial in the before purchase phase. the paper suggests enhancing knowledge about contextual factors, besides quality cues and attributes, able to shape consumer preferencesand before purchase expectations to create new value offering to satisfy consumers’ changing expectations concerning beef. keywords: beef quality, consumer perception, intrinsic quality cues, extrinsic quality cues, expected quality, traditional food ital. j. food sci., vol. 32, 2020 17 1. introduction food quality has always beenand is still today, one of the most interesting topics not only for academics, but above all for consumers. the meat quality issue has been in the spot light since 1995 (e.g., cardello, 1995; grunert, 1995; moskowitz, 1995), when beef attracted much attention just after the emergence of bse (bovine spongiform encephalopathy). however, over the last 22 years a number of quality standards, regulations and safety programs have been introduced, not only at a national level. the most recent one, at the european level, was introduced in 2014 focusing on traceability; an issue that had already emerged in literature (e.g., banović et al., 2012) together with beef safety (e.g., de barcellos et al., 2010; van wezemael et al., 2010). given that beef is an experience product, consumers shape their before purchase expectations, building on extrinsic and intrinsic cues and when faced with choosing unbranded beef they almost always rely on price (banović et al., 2012), even if the relationship between price and quality has not always been demonstrated (solomon et al., 2007). besides visual impressions understood as extrinsic and intrinsic cues (e.g., bello acebròn and calvo dorpico, 2000; grunert et al., 2004), also sensory impressions e.g. quality attributes like taste – affect beef purchase choice as demonstrated through studies performed after consumption (e.g., bello acebròn and calvo dorpico, 2000). however, to the best of the authors’ knowledge, none of the papers on beef have considered that past sensory impressions could play a role together with extrinsic and intrinsic cues in consumer preferences and purchase choice, before purchase. therefore, the paper aims at studying the impact of past sensory impressions as well as extrinsic and intrinsic quality cues in consumer before purchase preferences, paying attention to traceability and safety issues still not studied at length. therefore, two initial research questions emerge: • what is the role of extrinsic and intrinsic cues and of sensory impressions, based on past experience at the moment of purchase? • in this context how do traceability and safety issues affect consumer preferences and choices? these two research questions could also be affected by other elements, like familiarity, which has already been studied by banović et al. (2010, 2012). the third research question can be put forward as: • do consumers in regions where beef is a traditional food consider the impact of traceability and safety issues differently at the moment of purchase? to perform the study the authors chose two different regions: tuscany where beef is considered a traditional food and latium, one of the nearest regions to tuscany, but where beef is not a traditional food and the most famous pdo is “abbacchio romano”, a type of lamb. protected designation of origins (pdos) and protected geographical indications (pgis), have been defined by the european union in the domain of geographic indications (european regulation 1151/2012). the european union supports traditional quality products and the way they are produced, highlighting that "for a product name to ital. j. food sci., vol. 32, 2020 18 be protected as a pdo there must be an objective and exclusive link between the features of the product and its geographical origin" (london economics, 2008, p. 6). indeed, the paper also responds to the recent call for country-specific research into the beef domain, given that preferences for this food vary across different countries (ardeshiri and rose, 2017). the paper is structured as follows. firstly, there is a literature review focusing on papers that discuss various issues concerning food quality, meat quality and then beef quality. after the methodology section, results are illustrated and discussed. the paper ends with conclusions in which limitations, future steps of further research, as well as theoretical and managerial implications, are presented. 1.1. literature review 1.1.1 an overview of quality types in food and meat studies although there are several definitions of food quality in literature, according to grunert (2005), literature agrees that “quality has an objective and a subjective dimension”, (p. 371). objective quality refers to technical and physical characteristics necessary to have quality food, while subjective quality is about consumer perception of quality (grunert, 1995; 2005). steenkamp (1990) elaborated this concept perceived qualityas the match between product characteristics and consumer preferences. cardello (1995) suggested that “food quality is a complex concept” (p. 163) where various factors converge and should be measured by both objective indices (e.g., nutritional or physicochemical characteristics) and subjective indices linked to person, place and time. oude ophuis and van trijp (1995) as well as moskowitz (1995) stated that “food quality is a multi-faceted concept” (p. 157) and has a very subjective nature because it changes from person to person. following this through, food quality must be understood as a “human perceptual/evaluative construct” (moskowitz, 1995, p.167), therefore only consumer judgment can establish the quality of food. according to tolosa et al. (2005) quality is a “multidimensional phenomenon” (p. 419) and it can be described as a “set of attributes that must be perceived by the consumer” (p. 419). for these authors, subjective characteristics influence food quality more than objective features. in particular, grunert (1995), proposed three distinct types of food quality: (1) product-oriented quality to be understood as all physical characteristics of food which can be objectively measured; (2) process-oriented quality, namely all characteristics of the food production process and (3) user-oriented quality, referring to consumer subjective quality perception. brunsø et al. (2005), building on this classification, introduced a fourth quality type, namely “quality control”, defined as “the standards a product has to meet in order to be approved for a specific quality class” (p. 84), e.g. iso 9001 or specific standard quality beef. focusing on subjective quality, literature agrees to distinguish between multidimensional and hierarchical approaches (brunsø et al., 2005). according to the multidimensional approach, the combination of a number of quality dimensions or attributes determines the quality perception of a product (e.g. food) (verdù jover et al., 2004; brunsø et al., 2005). the two most important classifications in this approach are, on the one hand, the one regarding search, experience and credence characteristics (darby and karni, 1973; ital. j. food sci., vol. 32, 2020 19 nelson, 1970, 1974) and on the other, the one proposing the separation of intrinsic quality cues from extrinsic quality cues (olson and jacoby, 1972; olson, 1977). according to the economic theory, search and experience are evaluated at a different time from the moment in which the consumer carries out his purchase the first, before purchase, for example, refers to price or color; the second, after buying for example, refers to taste. credence, instead, cannot be established either before or after purchase, because it is based on trust and faith in the product information provided e.g. exclusiveness (oude ophuis and van trijp, 1995; grunert et al., 2004; brunsø et al., 2005; fandos and flavián, 2006). the second classification is part of the psychological theory and distinguishes between intrinsic and extrinsic quality cues. intrinsic quality cues, according to grunert et al. (2004), brunsø et al. (2005), tolosana et al. (2005) and espejel et al. (2007) can be understood as “part of the physical characteristics of the product”; they are “related to technical specifications, which also involve physiological characteristics” (bello acebrón and calvo dopico, 2000, p. 230), while extrinsic quality cues refer to characteristics “related to the product, but are not physically part of it” (oude ophuis and van trijp, 1995, p. 178). quality cues can, therefore, be evaluated only prior to consumption. quality attributes, on the other hand, can only be ascertained through consumption, namely when the consumer eats the prepared meat (steenkamp, 1990; ophuis and van trijp, 1995). indeed, bello acebrón and calvo dopico (2000) defined quality attributes as “functional and psychological benefits or consequences provided by the product and they are unobservable prior to consumption” (p. 231). therefore, when purchasing, consumers base their choices on quality cues (steenkamp, 1989, 1990), while hoping quality attributes will meet their expectations. casweell (2000) maintained that quality perception depends on both intrinsic/extrinsic quality cues and “information environment”, that is search, experience and credence quality, which are “vertically/horizontally differentiated” (p. 225). in his model casweell integrates the two classifications of quality dimensions. this point of view is shared by burnués et al. (2003), who proposed a model integrating intrinsic and extrinsic quality cues with search, experience and credence quality, in order to analyze the extrinsic quality cues of beef perceived as indicators of quality in europe. the hierarchical approach focuses on the association “between product attributes and more abstract, more central cognitive categories such as values, which can motivate behavior and create interest for product attributes” (brunsø et al., 2005, p. 85). the frameworks on which the hierarchical approaches are based are the “means-end chain models” (olson and reynolds, 1983; gutman, 1991), which link product characteristics to deeper purchasing motivation. to clarify the distinction between multidimensional and hierarchical approaches, it is important to understand subjective quality perception. indeed, these two approaches have played a key role in developing the total food quality model (tfqm) proposed by grunert et al. (1997). tfqm integrates several approaches to consumer quality perceptions (darby and karni, 1973; fishbein and ajzen, 1975; gutman, 1982) and tries to explain, on the one hand, which factors are able to influence consumer purchase intentionand on the other, the concept of customer satisfaction as the gap between expected and experienced ital. j. food sci., vol. 32, 2020 20 quality (oliver, 1990; grunert et al., 2004; vimiso et al., 2012). in doing this the authors distinguished 'before' from 'after' purchase evaluation. tfqm shows how quality expectations, in the 'before purchase' phase, come from the evaluation of available quality cues. according to steenkamp, (1990), consumers use 'cues' to determine the value of the product. therefore, it is necessary to consider them together with quality attributes. for this reason, the authors proposed a more complex model than those used in the past, one where the distinction between quality cues and attributes is considered. an overview of food quality types identified by the abovementioned studies is presented in fig. 1. figure 1. an overview of food quality types from the literature review. source: our elaboration. 1.2. quality perception in beef consumption over the years, various studies have considered meat quality and especially beef quality issues. grunert (1997) analyzed how consumers evaluate the quality of beef, developing research in four european countries: france, germany, spain and the uk. through focus groups, the author identified the intrinsic quality cues (cut, color and fat), the extrinsic quality cues (price, origin and information on animal production) and quality ital. j. food sci., vol. 32, 2020 21 attributes (taste, tenderness, juiciness, freshness, leanness, wholesomeness, nutrition). in this study, grunert demonstrated that some quality cues were crucial to consumer perception, even if their effect could be positive on some (e.g., on lean meat) and negative on others (e.g., price). moreover, he observed that all quality attributes have an important impact on purchase choice and should be considered as a uni-dimensional quality concept. all the above-mentioned quality dimensions were used by the same author some years later (grunert et al., 2004) in order to understand how to use the feedback obtained from consumers on subjective quality perception dimensions, to develop new products in the meat sector deemed to better suit desires. focusing on intrinsic quality cues, color/appearance, fat and cut are the three quality dimensions most analyzed by various authors starting with grunert (1997). in the same vein, mcilveen and buchanan (2001) used these quality dimensions of intrinsic quality cues, to analyze the factors, which influence beef consumer choices. these authors demonstrated that expectations about quality play a crucial role in evaluating beef quality and that consumers combine sensory (intrinsic) properties – colour, cut and fat in this study-, with extrinsic factors like place of purchase, country of origin, price, brandand quality attributes like appearance, texture, flavour and leanness, to predict and evaluate beef quality. brunsø et al. (2005) also used visual stimuli colour, fat and cut in order to understand danish consumer meat quality perception, demonstrating that consumers are very sensitive to visual stimuli even if this might involve dissatisfaction at the consumption moment. for this reason, brunsø et al., (2005) also stressed the need to educate the consumer in order to improve his consumption experience. for this sensory analysis, the following quality factors were used: cut, fat and colour (three intrinsic quality cues) and tenderness, juiciness, good taste, wholesomeness, nutritional value, freshness, leanness (the latter being quality attribute expectations). the same quality dimensions (intrinsic quality cues and quality attributes) with the addition of safety were used by banović et al. (2009) in order to study how portuguese consumers perceive beef quality. however, in their research, authors also focused on extrinsic quality cues. they also studied the relationship between intrinsic and extrinsic quality cues (price, origin and brand) and how these features were used by consumers to shape their quality perception at the moment of purchase. results showed that brand is the predominant extrinsic quality cueand that experienced eating quality has a crucial role in future purchase intentions. "differences in the consumers’ quality perception of national branded, national store brandedand imported store branded beef" were studied by banović et al. (2010, p. 54). they observed that consumers perceived the national branded beef as better under all quality cues and aspects in respect to all other branded beef. the same authors in 2012, published another paper focusing on how intrinsic and extrinsic cues affected beef quality consumer perception, also considering different levels of consumer familiarity with a particular beef product. results demonstrated that color is the intrinsic quality cue most used to evaluate quality when there is high-familiarity with beef. on the contrary, for consumers not familiar with beef, brand plays a crucial role. borgogno et al. (2015) also focused on this topic; they compared "consumer’s liking and perception of meat quality attributes as a function of their familiarity and involvement with fresh meat" (p. 139) and results showed that, regardless of familiarity level, consumers assign great importance to the visual appearance of meat. brand in the beef sector is very important because in this ital. j. food sci., vol. 32, 2020 22 domain meat is mostly sold unbranded. for this reason, according to bredahl (2003), analyzing "consumers’ quality perception is particularly difficult" (p. 65). the author proposed further research be developed on this topic in order to improve knowledge about the formation of perceived quality and to understand how consumers use and combine quality cues, focusing on brand information. this author demonstrated that brand, as an extrinsic quality cue, is the basis for evaluating both expected eating quality and expected health quality. intrinsic quality cues identified by bredahl (2003) were fat, color, meat juice and cut, while extrinsic quality cues were “brand name, price, cardboard tray, product label, package sleeve, information leaflet, recipes, promotion boards and the information scanner” (p. 69). finally, quality attributes studied “nutritional value, healthiness, freshness, leanness, tenderness, taste and juiciness” (bredahl, 2003, p. 69). research on the role of the brand in consumer quality perception, also demonstrated that consumers associate safety (quality attributes) with brand, in particular when there is no familiarity with beef. concerning the safety topic, de carlos et al. (2005) performed a qualitative study on the perception of beef in spain. they observed that the most significant factors affecting quality perception were color, fat content intrinsic quality cues and price extrinsic quality cue among others (table 1 and 2). quite surprisingly, the study highlighted that spanish consumers, even if aware of the controls carried out by various beef authorities, prefer not to rely on them. according to bernuéns et al. (2003), for some consumer groups, an indicator of safety and nutritious/healthy meat is animal feed and not origin. in their research, the authors focused on different extrinsic quality cues (origin/region of production, animal breed, environmentally friendly, processing/packaging, animal welfare storage, animal feeding) in order to study the role of this extrinsic quality cue on the willingness of consumers to pay for beef, developing their research over five european regions. they conclude identifying clusters of consumers according to the importance of extrinsic quality cues. the high level of importance given to animal welfare by consumers, as a dimension of extrinsic quality, has also been demonstrated by lagerkvist et al. (2014). the authors analyzed how food labels and packaging information on place of origin influence consumer purchasing decisions. lagerkvist et al. (2014) studied the price-quality tradeoffs issue, highlighting that consumers base their decisions on price when they lack information about intrinsic quality cues. also merlino et al. (2018) proved that price, for italian consumers, is the most important factor in meat purchasing. however, results showed that italian consumers are also sensitive to “animal welfare” which plays an important role in the choice of buying meat. according to verbeke and ward (2006), information cues on labels in the beef sector are very important because they help consumers orient their purchasing choices. in particular, the authors developed a study in belgium, in order to understand which information cues on beef labels greater influenced consumers and to evaluate the impact of a campaign aimed at informing consumers about beef traceability. in this case verbeke and ward (2006) focused only on extrinsic quality cues, without deepening the role of intrinsic quality cues or quality attributes on consumer purchase decisions, unlike bello acebrón and calvo dopico (2000) who developed a study in spain demonstrating that consumers shape their expectations about beef quality building on both intrinsic cues (e.g., color and fat) and extrinsic cues (e.g., price and origin of animal). these authors also observed that quality attributes, evaluated during consumption, are: taste, tenderness and juiciness. in particular, these authors studied the relationship ital. j. food sci., vol. 32, 2020 23 between expected quality and perceived quality at the moment of cooking. resano et al., (2018) focused on consumer preferences of veal attributes; authors proved that regional origin and health information play a stronger role than guaranteed tenderness at the moment of purchasing. to analyze consumer meat quality perceptions several authors used the tfqm model. in particular vimiso et al. (2012) applied the tfqm model in order to compare rural consumer meat quality perceptions, measured through intrinsic and extrinsic quality cues, with meat trader quality perceptions. quality dimensions used in this research were color and fat intrinsic quality cues-and place of slaughter, packaging, beef class and price extrinsic quality cues. quality attributes considered were: juiciness, tenderness, freshness, leanness. saeed (2013) and saeed and grunert (2014), through the application of the tfqm model, focused on beef production processes. saeed (2013) used the tfqm in order to analyze the change in consumer quality perception concerning four new processed beef products, both in the pre and post consumption phase. quality cues selected for this study were: beef color, fat, appearance, cut, trim and ingredients. taste, freshness, nutrition, juiciness, wholesomeness were considered among the quality attributes and evaluated at the point of beef consumption, in order to study consumer perceptions. saeed and grunert (2014), using tfqm, focused on four different new beef product processes and underlined that cue evaluations as well as “expected/experienced quality and purchase motive fulfillment” affect purchase intention but act differently before and after trial (p. 451). they investigated quality cues before and after trial like appearance, color, fat, etc.; expected quality and experienced quality like taste, freshness, juiciness, etc.; purchase motives before and after trial and, finally, purchase intention before and after purchase. the studies of beef product processes are very important because according to resurreccion (2003) “the development of low-fat products is another strategy to increase the consumption of beef” (p. 13). indeed, the author studied factors influencing consumer purchase behavior, suggesting that changes in consumer preferences depend on factors such as health concerns, change in demographics, need for convenience, changes in the distribution of meat, as well as price. colle et al., (2016) developed a technical study to determine the influence of postfabrication ageing on beef quality characteristics and consumer sensory perceptions of biceps femoris and semi-membranous steaks. quality attributes selected for this study were: tenderness, juiciness and flavor. based on previous research of consumer decision-making about red meat, from which the amount and type of visual fat emerged as a major factor in consumer choice (i.e., banović et al., 2012, banović et al., 2009, banović et al., 2010, brunsø et al., 2005), banović et al. (2016) focused on the effect of fat content on visual attention and on the choice of red meat, as well as on gender differences, developing a study conducted on 105 portuguese meat consumers. results show that consumers pay more attention and more often choose meat products with lower fat content, particularly if they are female. the relationship between meat color and fat and consumer perception was also studied by ristić et al. (2017) who develop a sensorial analysis in order to evaluate consumer attitudes towards sensory properties of chicken, royal and beef salami, all meat products from zlatiborac meat company. the authors proved that consumers pay great attention to these intrinsic quality cues; especially older consumers, perhaps because they are more aware of health aspects related to the food products they purchase. according to ital. j. food sci., vol. 32, 2020 24 subbaraj et al. (2016), meat color is one of the cues available for consumers to gauge overall meat quality and wholesomeness; the authors, performing a technical study based on hydrophilic interaction liquid chromatography–mass spectrometry (hilic–ms), were able to state that “colour stability of meat is determined by several factors both inherent to the animal and post-slaughter conditions, including ageing, storage/packaging and display times" (subbaraj et al., 2016, p. 163). finally, henchion et al., (2017) developed a systematic review in order to determine the relative importance of beef quality attributes from a consumer perspective, considering search, experience and credence quality attributes. the aim of the study was to provide relevant information that may be considered in future iterations of quality assurance schemes, to increase consumer satisfaction and, potentially, to increase returns to industry. tables 1-3 show quality dimensions studied by the above-mentioned authors in order to analyze and understand consumer perception of beef. table 1. intrinsic quality cues. author type of meat analyzed country intrinsic quality cues colour/appea rance f at c ut meat juice trimm ing marbli ng grunert, (1997) beef france, germany, spain, uk x x x acebroen & calvo dopico (2000) beef spain x x mcilveen and buchanan, (2001) beef ireland x x x bredahl (2003) beef denmark x x x x grunert et al., (2004) beef and pork france, germany, spain, uk x x x resurreccion, (2004) beef france, germany, spain, uk and usa x x brunsø et al., (2005) beef danish x x x de carlos et al., (2005) beef spain x x x banović et al., (2009) beef portugal x x x banović et al., (2010) beef portugal, brazil x x x banović et al., (2012) beef portugal x x x vimiso et al., (2012) beef south africa x x saeed et al., (2013) beef denmark x x x borgogno et al., (2014) beef italy x x x saeed and grunert (2014) beef denmark x x banović et al., (2016) beef portugal x colle et al., (2016) beef idaho usa x subbaraj et al., (2016) beef southland, new zealand x henchion et al., (2017) beef x x merlino et al., (2018) beef italy x source: our elaboration. ital. j. food sci., vol. 32, 2020 25 table 2. extrinsic quality cues. author type of meat analyzed country extrinsic quality cues price origin/quality certification promotion label information/ information on animal production place of purchase brand butcher recommendation beef class store image storage package/ presentation animal welfare recipes grunert, (1997) beef france, germany, spain, uk x x x acebroen & calvo dopico (2000) beef spain x x x x x mcilveen and buchanan, (2001) beef ireland x x x x bernués et al., (2003) beef england, italy, france, scotland and spain x x x x x bredahl (2003) beef denmark x x x x x x* x grunert et al., (2004) beef and pork france, germany, spain, uk x x x resurreccio n, (2004) beef france, germany, spain, uk and usa x de carlos et al., (2005) beef spain x x x x x x ital. j. food sci., vol. 32, 2020 26 verbeke and ward (2006) beef belgium x x banović et al., (2009) beef portugal x x x banović et al., (2010) beef portugal, brazil x x x x x banović et al., (2012) beef portugal x x x borgogno et al., (2014) beef italy x x x x x x x x vimiso et al., (2012) beef south africa x x x x lagerkvist et al. (2014) beef swedish x x x x henchion et al. (2017) beef x x x x x x merlino et al., (2018) beef italy x x x x x x resano et al., (2018) beef spain x x x source: our elaboration. * cardboard tray, package sleeve. table 3. quality attributes expectations/experience. author type of meat analyzed country quality attributes taste/flavour tenderness juiciness wholesomeness/healthiness nutrition value leanness safety freshness smell grunert, (1997) beef france, germany, spain, uk x x x x x x x acebroen & calvo dopico (2000) beef spain x x x mcilveen and buchanan, (2001) beef ireland x x x x ital. j. food sci., vol. 32, 2020 27 bredahl (2003) beef denmark x x x x x x x grunert et al., (2004) beef and pork france, germany, spain, uk x x x x x x x resurreccion, (2004) beef france, germany, spain, uk and usa x x x x x x brunsø et al., (2005) beef danish x x x x x x x banović et al., (2009) beef portugal x x x x x x x x banović et al., (2010) beef portugal, brazil x x x x x x banović et al., (2012) beef portugal x x x x x x vimiso et al., (2012) beef south africa x x x x x saeed et al., (2013) beef denmark x x x x x borgogno et al., (2014) beef italy x x saeed and grunert (2014) beef denmark x x x x x henchion et al., (2017) beef x x x x x* x* merlino et al., (2018) beef italy x x x x resano et al., (2018) beef spain x source: our elaboration. note: * henchion et al., (2017) classify nutrition value and safety as credence attributes together with origin, animal welfare, production system/feeding, environmental issues, traceability, processing technologies (ageing, irradiation, halal/kosher) and breed. ital. j. food sci., vol. 32, 2020 28 2. material and methods 2.1. questionnaire and data collection based on the study of espejel et al. (2007), a questionnaire was prepared to investigate the relationship between intrinsic quality cues, extrinsic quality cues, expected quality of beef and customer behavior. the questionnaire was divided into three different areas of analysis: (i) perceived quality cues (extrinsic and intrinsic) (table 4), (ii) evaluation of expected quality (table 4), (iii) customer profile: containing information on socio-demographic features (table 9). dimensions of quality cues and expected quality attributes were drawn from the literature review. the likert measurement scale was used to measure consumer perception, with a score assigned to the respondents between 1 and 6, ranging from ‘strongly disagree’ (scoring value 1) to ‘strongly agree’ (scoring value 6); an even scale was chosen in order to avoid central tendency bias of the responses (likert, 1932; matell and jacoby, 1971; bernués et al., 2012; silvestri et al., 2018), to measure customer before purchase preferences and expectations, three types of questions were formulated: two single choice questions, three dichotomic questions and two questions measured on a likert scale 1-6. as the aim of the research was also to understand how the perception of beef quality changes from one region to another and therefore if the traditional food issue could affect consumer preferences and purchase choices, the study was performed in two central italian regions that have the closest percentage of beef production: latium 35,9% and tuscany 32,2% (ismea, 2016). tuscany was selected as it is the only italian region where beef is part of the traditional cuisine (miele and murdoch, 2002). in particular, from this region grosseto and orbetello were selected, both pertaining to grosseto province, which is the administrative center where beef livestock is the most important of all central italian provinces (istat, 2010; ismea, 2016). in latium, viterbo was selected as the nearest province to tuscany and the province where beef livestock is less important than in other provinces in latiumand rome where beef livestock is the most important in the region, but the pdo is “abbacchio romano” (istat, 2010; http://ec.europa.eu/). the data collection was performed thus: viterbo (latium) june, 17-19, 2016; grosseto (tuscany) june, 24-26, 2016; rome (latium) july, 1-3, 2016; orbetello (tuscany) july, 8-10, 2016. to ensure both the homogeneity of data collection conditions within four hypermarkets and the possibility of contacting the most heterogeneous consumers – also working people and families questionnaires were collected at weekends. consumers were interviewed at the meat counter of the hypermarket once they had picked up a beef package. the difficulty in identifying the meat consumers led, as it usually does in market research activities, to the adoption of a no probabilistic model, in particular of a random sampling (bracalente et al., 2009, saeed et al., 2013). the sample analyzed was composed of 447 individuals. the data collected was analyzed using the statistic program "stata 12 data analysis and statistical software" (www.stata.com). ital. j. food sci., vol. 32, 2020 29 2.2. factor analysis and cluster analysis data presented in table 4 shows that all quality dimensions significantly influence preferences and beef purchase decisions. in particular, among the intrinsic quality cues, the most important attribute is color (average value of 5.40); the extrinsic quality cues are affected by price (average value of 5.85) and quality certification (average value of 5.52). expected quality is homogeneously affected by all attributes. safety and juiciness are the only quality attributes that present a lower average value (safety average value of 3.99; juiciness average value of 4.64) cronbach α was used to test internal consistency for all items under respective variables (namukasa, 2013). following hair at al. (2006) who stated that cronbach α coefficient over 0.6 is adequate for basic research, it is possible to argue that the sample of this study shows good internal consistency. also performing the kaiser-meyer-olkin (kmo) test whose result must exceed the 0.5 limit (kaiser, 1974; hair et al., 2006; santouridis and trivellas, 2010), the sample was found appropriate to perform the factor analysis. finally, the correlation test was used to verify whether or not the observed variables contain misleading redundancies or make the results insignificant. table 4. descriptive statistics of quality dimensions. measures items variable obs mean std. dev. min ma x alpha kmo intrinsic quality cues cut iq1 447 4.67 1.47 1 6 0.696 0.780 color iq2 447 5.40 1.06 1 6 0.811 fat iq3 447 4.79 1.33 1 6 0.823 extrinsic quality cues origin eq1 447 4.67 1.48 1 6 0.667 0.744 price eq2 447 5.85 0.53 2 6 0.623 quality certification eq3 447 5.52 0.89 1 6 0.681 brand eq4 447 5.44 1.03 1 6 0.641 expected quality attributes nutritional value exq1 447 5.57 1.01 1 6 0.612 0.844 freshness exq2 447 5.23 1.08 1 6 0.845 taste exq3 447 5.53 0.85 1 6 0.882 tenderness exq5 447 5.23 1.34 1 6 0.806 smell exq6 447 5.66 0.92 1 6 0.833 juiciness exq7 447 4.64 1.36 1 6 0.789 wholesomeness/healthine ss exq8 447 5.57 1.01 1 6 0.782 safety exq9 447 3.99 1.75 1 6 0.716 overall 0.746 0.790 source: our elaboration on the data set. ital. j. food sci., vol. 32, 2020 30 in order to determine the number of the most important factors, the screen plots tool introduced by cattell (1966) was used. fig. 2 shows that the first four factors are the only ones with eigenvalues greater than 1. figure 2. screen plot of eigenvalues after factor analysis. source: our elaboration. table 5 shows orthogonal varimax rotation of the factors where the first four have eigenvalues greater than 1 and also encompass 51.58% of the information contained in the original data set. table 5. rotation: orthogonal varimax (kaiser off). factor variance difference proportion cumulative factor 1 2.5322 0.4394 0.1688 0.1688 factor 2 2.0928 0.2172 0.1395 0.3083 factor 3 1.8756 0.6393 0.1250 0.4334 factor 4 1.2363 0.0824 0.5158 source: our elaboration on the data set; number of obs 447; retained factors 4. from the results obtained from the joint use of the two above illustrated analytical tools, the first four factors were considered to identify the new variables. ital. j. food sci., vol. 32, 2020 31 factor interpretation was achieved by considering the so-called saturation matrix (table 6) where correlation between original variables and factors were identified. table 6. saturation matrix (factor loadings). new variables measures items variable factor 1 factor 2 factor 3 factor 4 uniqueness beef quality features – fa1 intrinsic quality cues color iq2 0.6457 0.2420 -0.0077 0.1189 0.5104 fat iq3 0.5069 0.1431 -0.0077 0.1674 0.6945 quality expected attributes freshness exq2 0.7152 0.0449 0.1304 0.0066 0.4694 taste exq3 0.6020 0.2464 0.0132 0.0346 0.5755 tenderness exq5 0.5764 0.2474 0.1252 -0.0191 0.5906 smell exq6 0.6871 0.1779 0.0312 0.0066 0.4952 flavor & healthiness – fa2 intrinsic quality cues cut iq1 0.0792 0.5961 0.1815 0.2020 0.5646 quality expected attributes nutritional value exq1 0.0093 0.5929 0.2193 0.1985 0.5609 juiciness exq7 0.2954 0.7237 -0.0277 -0.1143 0.3751 wholesome ness/healthi ness exq8 0.2429 0.7203 0.1169 -0.0723 0.4033 safety & traceability – fa3 extrinsic quality cues origin eq1 0.0775 0.2822 0.7074 -0.0060 0.4140 quality certification eq3 0.0565 0.0700 0.8034 0.0198 0.3460 quality expected attributes safety exq9 -0.0003 -0.0473 0.6513 -0.0325 0.5725 price & brand – fa4 extrinsic quality cues price eq2 -0.0108 0.0749 -0.1864 0.7960 0.3259 brand eq4 0.1347 -0.1279 0.3760 0.6774 0.3653 source: our elaboration on the data set. table 6 shows that factor1 synthesizes the variables related to the attributes of intrinsic quality cues (like color and fat) and expected quality (like freshness, taste, tenderness and smell). factor 2 synthesizes the variables related to the attributes of intrinsic quality cues (like cut) and expected quality (like nutritional value, juiciness, wholesomeness/healthiness). factor 3 synthesizes the variables related to the attributes of extrinsic quality cues (like origin and quality certification) and expected quality (like safety) and finally factor 4 synthesizes the variables related to the attributes of extrinsic quality cues (like price and brand). through factor analysis, the number of variables was reduced from 15 to 4. this result highlights that consumers do not have a clear idea of how literature classifies the different quality dimensions of meat. for research purposes the hierarchical method of ward (fabbris, 1997; dahl and næs, 2004; annunziata and vecchio, 2013) was used and the number of groups was determined by inspecting the dendrogram. ital. j. food sci., vol. 32, 2020 32 using the information derived by the calinski/harabasz indicator (table 7) together with the dendrogram analysis, four groups were identified. table 7. calinski/harabasz indicator. number of clusters calinski/ harabasz 2 76.06 3 86.52 4 98.83 5 94.23 6 91.79 7 88.22 source: our elaboration on direct survey table 8 shows the four meat consumer groups related to the new variables of quality dimensions. on the basis of the correlation link intensity it is possible to define the characteristics of the four clusters. table 8. cluster analysis in relation to new factors of quality – correlation link intensity. cluster fa1 fa2 fa3 fa4 cluster 1 -1.971 -0.409 -0.392 0.184 cluster 2 0.577 0.038 -1.763 0.335 cluster 3 0.189 0.284 0.323 -1.184 cluster 4 0.231 -0.045 0.390 0.420 total -1.57e-10 5.82e-10 -1.87e-09 -1.42e-09 source: our elaboration on direct survey. cluster 1 seems to be indifferent to all studied quality dimensions of beef, unlike the other three clusters. indeed cluster 2 is characterized by consumers focused on beef quality features (fa1), cut, nutritional value, juiciness, wholesomeness/healthiness (fa2) are essential for cluster 3, while safety and traceability (fa3) and price and brand (fa4) are fundamental to cluster 4. in order to validate the segmentation into 4 clusters, confirmatory analysis was developed. the statistical significance of socio-demographic variables (categorical variables) was validated through the test study of pearson chi-square (adanacioglu and albayram, 2012), while the statistical significance of numeric variables was validated through the study of variance (vermeir and verbeke, 2008; yadavalli and jones, 2014). ital. j. food sci., vol. 32, 2020 33 the largest group is cluster 4, 51.23% followed by 23.71% of cluster 3, while cluster 1 and cluster 2 are the smallest ones (cluster 1 represents 12.08% of the sample and cluster 2 12.98%). cluster 1 is mainly composed of young men, aged between 20-29 and 30-39. they are students, workers, entrepreneurs and teachers, residing mostly in tuscany (grosseto) and latium (viterbo province). they purchase beef every day or one day a weekand they do not read the traceability label because they stated they don’t understand its meaning. for this reason, most cluster 1 consumers are not willing to pay a higher price for a better beef quality system. those who are ready to pay more declared that they would be ready to pay up to a 10% increase on the market price to have a better beef quality system. for cluster 1 consumers, quality certification is synonyms with safety (scores assigned on the likert-scale from 3 to 5) and media information affects their perception of beef quality (scores assigned on the likert-scale from 4 to 6). cluster 2 consumers were focused on beef quality features (fa1). this represents 12.98% of the sample and it consists mainly of young men aged between 20-29 and 30-39. they are students, employees, freelancers and artisans, resident in tuscany (grosseto) and latium (viterbo). they purchase beef two or three times of week. like cluster 1 consumers, they do not read the traceability label because they stated they don’t understand it. they are not willing to pay a higher price for a better beef quality system. those who are ready to pay more declared that they are ready to pay up to a 10% increase on the market price to have a better beef quality system. for cluster 2 consumers quality certification is not synonyms with safety (scores assigned on the likert-scale from 1 to 2) and media information affects their perception of beef quality (scores assigned on the likert-scale from 3 to 5). cluster 3 is composed of women and men aged between 20-29, 40-49 and 50-59 years, students, entrepreneurs, freelancers and unemployed resident in tuscany (grosseto province) and latium (roma). consumers of cluster 3 focus their attention on the traceability label and quality certification too and are willing to pay up to 10% more than the beef market price in order to have a better quality system. some cluster 3 consumers consider beef quality certifications as synonymous with safety (scores assigned on the likert-scale 4) while others do not (scores assigned on the likert-scale 1). some cluster 3 consumers claimed to be greatly influenced by media information (scores assigned on the likert-scale 6) while others said the opposite (scores assigned on the likert-scale). finally, they buy beef two or three times a week, more than once a month or less than once a month. finally, cluster 4 is the largest group (51.23% of simple) and it is composed of women aged between 50-59 and over 60, predominantly housewives, teachers and pensioners living in tuscany (grosseto province). for them the traceability label and quality certification of beef are essential factors in making their purchase decision. however, they are willing to pay up to 5% more than the current beef market price in order to have a better quality system. finally, they consider beef quality certifications as synonymous with safety (scores assigned on the likert-scale from 5 to 6) and they buy beef every day and once a week. some of them are greatly influenced by media information (scores assigned on the likert-scale 5) while others stated that they are not influenced by such information at all (scores assigned on the likert-scale from 1 to 2) (table 9). ital. j. food sci., vol. 32, 2020 34 table 9. socio-demographic characteristics and purchase intention of the meat consumers of the four clusters. sociodemographic behavioral variables sample (n) cluster 1 (n= 54; 12.08% ) cluster 2 (n= 58;12.98% ) cluster 3 (n= 106; 23.71% ) cluster 4 (n= 229, 51.23%) f % f % f % f % f % gender male 142 31.77 23 42.59 21 36.21 34 32.08 64 27.95 female 305 68.23 31 57.41 37 63.79 72 67.92 165 72.05 total 447 100 54 100 58 100 106 100 229 100 age group 20-29 68 15.21 14 25.93 11 18.97 17 16.04 26 11.35 30-39 51 11.41 11 20.37 12 20.69 6 5.66 22 9.61 40-49 63 14.09 4 7.41 8 13.79 23 21.7 28 12.23 50-59 107 23.94 7 12.96 9 15.52 32 30.19 59 25.76 ≥60 158 35.35 18 33.33 18 31.03 28 26.42 94 41.05 total 447 100 54 100 58 100 106 100 229 100 professional category student 43 9.62 7 12.96 9 15.52 12 11.32 15 6.55 employee 82 18.34 9 16.67 16 27.59 19 17.92 38 16.59 worker 32 7.16 6 11.11 4 6.9 7 6.6 15 6.55 housewife 69 15.44 6 11.11 7 12.07 14 13.21 42 18.34 entrepreneur 16 3.58 4 7.41 2 3.45 6 5.66 4 1.75 freelance 41 9.17 4 7.41 6 10.34 18 16.98 13 5.68 teacher 23 5.15 3 5.56 2 3.45 5 4.72 13 5.68 pensioner 104 23.27 12 22.22 8 13.79 20 18.87 64 27.95 artisan 4 0.89 0 0 2 3.45 1 0.94 1 0.44 unemployed 12 2.68 1 1.85 1 1.72 3 2.83 7 3.06 other 21 4.7 2 3.7 1 1.72 1 0.94 17 7.42 total 447 100 54 100 58 100 106 100 229 100 residence viterbo 70 15.66 7 12.96 18 31.03 14 13.21 31 13.54 province of viterbo 70 15.66 13 24.07 8 13.79 14 13.21 35 15.28 civitavecchia 62 13.87 5 9.26 8 13.79 17 16.04 32 13.97 grosseto 101 22.6 13 24.07 14 24.14 22 20.75 52 22.71 province of grosseto 39 8.72 4 7.41 5 8.62 14 13.21 16 6.99 orbetello 43 9.62 5 9.26 1 1.72 6 5.66 31 13.54 other provinces of tuscany 62 13.87 7 12.96 4 6.9 19 17.92 32 13.97 total 447 100 54 100 58 100 106 100 229 100 ital. j. food sci., vol. 32, 2020 35 purchase frequency everyday 22 4.93 6 11.32 0 0 4 3.81 12 5.29 2-3 times a week 220 48.43 25 45.28 32 54.39 54 50.48 109 47.14 1 time per week 163 37.22 20 37.74 20 35.09 36 34.29 87 38.33 2-3 times a month 38 8.52 3 5.66 5 8.77 10 9.52 20 8.81 less than once a month 4 0.9 0 0 1 1.75 2 1.9 1 0.44 total 447 100 54 100 58 100 106 100 229 100 knowledge the traceability label yes 368 82.33 42 77.78 22 55.17 96 85.85 209 88.65 no 79 17.67 12 22.22 36 44.83 10 14.15 19 11.35 total 447 100 54 100 58 100 106 100 228 100 read the traceability label yes 370 82.74 42 77.78 22 37.93 96 90.57 210 91.67 no 77 17.26 12 22.22 36 62.07 10 9.43 19 8.33 total 447 100 54 100 58 100 106 100 229 100 willingness to pay a higher price for a better meat quality system yes 392 87.7 42 77.78 43 74.14 102 96.23 205 89.52 no 55 12.3 12 22.22 15 25.86 4 3.77 24 10.48 total 447 100 54 100 58 100 106 100 229 100 how much more 5% more than the current market price 191 48.6 21 48.84 21 48.84 40 39.22 109 53.17 up to 10% more than the current market price 130 33.08 16 37.21 15 34.88 32 31.37 67 32.68 over 10% more than the current market price 72 18.32 6 13.95 7 16.28 30 29.41 29 14.15 total 393 100 43 100 43 100 102 100 205 100 how much quality labels are safety synonyms for consumers 1= in no way 52 11.63 2 3.7 10 17.24 17 16.04 23 10.04 2 23 5.15 4 7.41 7 12.07 6 5.66 6 2.62 3 48 10.74 10 18.52 8 13.79 12 11.32 18 7.86 4 105 23.49 16 29.63 13 22.41 29 27.36 47 20.52 5 146 32.66 18 33.33 15 25.86 28 26.42 85 37.12 6=very much 73 16.33 4 7.41 5 8.62 14 13.21 50 21.83 total 447 100 54 100 58 100 106 100 229 100 ital. j. food sci., vol. 32, 2020 36 how much media information influences their meat purchasing choices 1= in no way 227 50.78 22 40.74 26 43.86 62 58.1 121 52.42 2 32 7.16 4 7.41 4 7.02 5 4.76 19 8.37 3 44 9.84 4 7.41 8 14.04 9 8.57 22 9.69 4 57 12.75 11 20.37 11 19.3 11 10.48 22 9.69 5 52 11.63 8 14.81 7 12.28 8 7.62 28 12.33 6=very much 35 7.83 5 9.26 2 3.51 11 10.48 17 7.49 total 447 100 54 100 58 100 106 100 229 100 source: our elaboration. 3. discussion the above results help to answer the research questions on which the paper is built. factor analysis helps understand what the relationships are between extrinsic cues, intrinsic cues and expected quality attributes – sensory impressions based on past experience-, therefore answer the first research question: • what is the role of extrinsic and intrinsic cues as well as sensory impressions based on past experience at the moment of purchase? from table 6 it is clear that extrinsic quality cues are linked to safety which is an expected quality attribute, while intrinsic quality cues are linked to all other expected quality attributes, namely freshness, taste, tenderness, smell. these results are in line with previous studies. in particular, origin, safety and quality certifications – e.g. quality labels – (cluster 4) have already been considered as quality cues important to determine consumer preferences and choices before beef is purchased (e.g. grunert, 2005). brunsø et al. (2005) also highlight the importance of quality controls, stating that this is the third dimension of quality. instead, grunert (2005) states that information available about “breed, age of animaland slaughtering date are predictive” of taste and tenderness, but “few consumers feel confident in using them” (p. 376). cluster 4 represents 51.23% of the entire sample. it is made up of older women, aged from 50 to over 60. consumers grouped in cluster 4 consider beef traceability as well as quality certifications of paramount importance and predictive of beef safety. moreover, for these consumers price and brand are the most important features to signal quality as also suggested by grunert et al. (2004). price has long been studied in beef quality literature, almost together with brand (bello acebrón and calvo dopico, 2000; bredahl, 2003; grunert et al., 2004; grunert, 2005; tolosana et al., 2005; banović et al l., 2010; banović et al., 2012). indeed, for grunert et al. (2004) brand if combined with quality and reliability built over time, can be considered the most important extrinsic quality cue when purchasing beef for consumers not so aware of beef features and therefore struggling to formulate their expectations about beef quality cues. in this same vein bredahl (2003) and banović et al. (2010; 2012) demonstrated that consumers focus on brand when they are not so familiar with beef products, which leads to hesitation at the moment of purchase. besides brand, price is also used by hesitant consumers as predictive of beef quality (bello acebrón and calvo dopico, 2000; tolosana et al., 2005; merlino et al. 2018). cluster 4 consumers are also ready to pay more than the ital. j. food sci., vol. 32, 2020 37 average beef market price (maximum +5%) to rely on a better quality system as already stated by bello acebrón and calvo dopico (2000) and grunert et al. (2004). on the contrary bredahl (2003), carrying out a study on the danish beef market, found that price is not considered such an important extrinsic quality cue for danish consumers. our insights about cluster 4 are in line with past studies concerning consumer behavior, stating that older women pay more attention seeking information about product safety and quality. (e.g. richardson et al., 1996; roszkowska-hołysz, 2013). from our study, media information seems decisive in determining older women’s purchasing choices. in this domain, kuo et al. (2011) found that in general all women adopt a more “protective behavior”(p. 5) than men, in that they are more aware of food risks and the importance of safety issues. finally, results are in line with the study conducted by banterle and stranieri (2008), which showed that, among european consumers, italians are more sensitive to the issue of safety and food certification. the research shows that italians make extensive use of information reported on labels, such as information on certification and meat origins. intrinsic quality cues and part of the expected quality attributes, apart from safety, are of paramount importance when the consumer is aware of the product and its special quality features. in this respect bredhal et al. (1998), for example, pointed out that making the relationship clear between expected and organoleptic characteristics e.g. intrinsic quality cues – is important to understand how consumers shape their expectations about beef. this study confirms that these characteristics are important at least for cluster 2, which represents 12.98% of the entire sample. cluster 2 is made up of young men who are mostly unaware of traceability labels and don’t read them. they are willing to pay over 10% of the current beef market price to have better quality beef and their beef consumption is on average once a week. these consumers seem to pay great attention to intrinsic quality cues and results are in line with several studies conducted in the literature. in primis, the male gender, whose result is discriminating for cluster (2) and cluster (1), confirms the study of several authors like e.g. sobal (2005), cavazza et al. (2015), basfirinci and cilingir uk (2017), according to whom the consumption of red meat is an expression of virility and strength and is more associated with the male identity. indeed, the female is associated with sweet foods (lupton, 1996), fruit (o'doherty and holm, 1999) and dietetic products (mooney and lorenz, 1997; basfirinci and cilingir uk, 2017). finally, lennernäs et al. (1997), biloukha and utermohlen (2001) and piggford et al. (2008) showed that "sensory appeal" (piggford et al., 2008, p.19) (including smell, appearance, palatability and pleasure), represent the factors that influence the male purchases. according to peng et al. (2005), in fact, male consumers pay more attention to product quality and the purchasing environment, than do female consumers. cluster 1, representing 12.08% of the sample is made up of young men, mostly students, employees and laborers. they frequently consume beef, but seem not to be affected by any quality cue and/or attribute at the moment of purchase. these results are not surprising in that, in general, men have less shopping experience and pay less attention to information about safety and quality than women (e.g. tzimitra-kalogianni et al., 2003; kuo et al., 2011). cluster 3 individuals (23.71% of sample) give a component as the visual aspect of the meat (cut) an attribute (succulence) that can be evaluated through taste and two other attributes (nutritional values and wholesomeness) that cannot be measured because they are part of ital. j. food sci., vol. 32, 2020 38 the beliefs, which can be found in the purchase psychological factors (font-i-furnols and guerrero, 2014). the cut is linked to these attributes, because the amount of fat in the meat varies according to the cut and, as stated by shan et al. (2016), consumers are very attentive to these aspects. in particular, young italians, who among various purchasing factors also consider livestock feeding, since there is a relationship between this and nutritional value and healthiness (banterle and stranieri, 2008). moreover, while several studies claimed that women are more attentive to factors such as nutritional value and healthiness (for both health and body care reasons) compared to men (drewnowski and hann, 1999; holm, 2003; shan et al., 2016), this distinction does not emerge from the results of the present study. cluster 3 is composed of both men and women. the results show that even men are becoming more sensitive to these issues nowadays. traceability and safety issues emerged to a certain extent in the previous discussion when we analyzed the identified cluster characteristics, but our study also focuses specifically on this issue with the second research question. • how traceability and safety issues affect consumer preferences and choices? traceability labels were found important for cluster 4. in particular, consumers in this cluster are aware of traceability labels and read them. also, it can be observed that cluster 4 consumers are also ready to pay a higher price than the actual average beef price for a better quality system. to understand why, we considered the traditional food issueand found that consumers falling in this cluster ready to pay a higher price to have a better quality system are 205 out of 229 representing 89.52% of cluster 4 and are mostly resident in tuscany (43.24%) – where beef is a traditional food. the importance of safety issues as a whole has already been highlighted in literature, above all after the emergence of bse (e.g. brunsø et al. 2005; grunert, 2005), but the results of this study seem to suggest that consumers today are more aware of beef quality related issues for health in general and especially when this food is known and frequently purchased, these features become of paramount importance. the third research question introduced the traditional food issue, not yet considered in literature, which seems to play a role in beef purchase choice. • do consumers in regions where beef is a traditional food, consider the impact of traceability and safety issues differently at the moment of purchase? cluster 1, 2 and 3 consumers are mostly resident in latium (45%, 58.61% and 42.46% of the sample) where beef is not a traditional food and they seem not to be affected by traceability and safety issues at the moment of purchase. on the contrary, consumers in cluster 4 are aware of traceability and safety issues and are mostly resident in tuscany (43.24% of the sample) where beef is a traditional food (miele and murdoch, 2002). in this sense, it seems that residence – e.g. traditional food could be considered a discriminating factor affecting evaluation linked to traceability and safety issues before beef purchase. 4. conclusion this paper adds some insights into beef meat consumer preferences before purchase: (a) quality cues and/or attributes diversely affect consumers with various socio-demographic characteristics; (b) being a traditional food can affect consumer choices; (c) traceability and ital. j. food sci., vol. 32, 2020 39 safety have become crucial in shaping before purchase consumer preferences, especially after the emergence of bse some years ago. this is also because national and international bodies have focused their attention on these issues, obtaining feedback in terms of the importance of these issues recognized by some consumers. the paper also has some limitations, which could be of help to identify future avenues of research. principally: (a) the number of questionnaires and the limited places in which they were collected; future studies should consider other italian regions but also other countries, verifying the role of the traditional food issue in a more focused way; (b) the study just considers quality cues and attributes before purchasing and does not compare them with the after purchase experience; this could be another future avenue of research. among theoretical implications, the most important refers to the attempt to widen the perspective used to study beef quality and its cues and attributes to better understand consumer preferences and purchasing choices. even if familiarity with beef products has been studied (banović et al., 2010; 2012), other contextual factors could play a role and they should be understood better to paint the “full picture” in this domain. adopting the managerial perspective, it becomes clear that it is crucial to firms operating in this industry to know which quality cues and attributes are important in shaping different consumer cluster expectations and preferences. in particular, new value offerings could be shaped ad hoc for different and above all emerging clusters considering, besides beef quality cues and attributes, socio-demographic 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determination of microbial contamination, ph and temperature changes in sheep and cattle carcasses during the slaughter and pre-cooling processes in konya, turkey ü. gürbüz1,2, a.e. telli*1, h.a. kahraman3, d. balpetekkülcü4 and s. yalçın1 1selçuk university, faculty of veterinary medicine, department of food hygiene and technology, konya, turkey 2kyrgyz -turkish manas university, faculty ofveterinary medicine, department of food hygiene and technology, bishkek, kyrgyzstan 3mehmet akif ersoyuniversity, faculty of veterinary medicine, food hygiene and technology department, burdur, turkey 4giresun university, engineering faculty, foodengineering department, giresun, turkey *corresponding author: tel.: +90 5334698994; fax: 03322410063 e-mail address: ezgiyilmaz@selcuk.edu.tr abstract this study was conducted to determine microbial contamination, ph and internal temperature changes in sheep and cattle carcasses during slaughter and chilling stages. samples were analysed for the presence of salmonella spp. and enterobacteriaceae and aerobic colony counts (acc) were performed. air sampling was also performed in slaughtering areas and chilling rooms. mean values of accs were between 2.57±0.61 and 4.71±0.24 log cfu/cm2 and between 3.51±0.48 and 5.19±0.28 log cfu/cm2, whereas enterobacteriaceae counts were between 0.89±0.46 and 2.61±0.10 log cfu/cm2 and between 0.55±0.37 and 3.63±0.39 log cfu/cm2 in cattle and sheep carcasses, respectively. enterobacteriaceae contamination in the shoulder region of cattle carcasses after washing, enterobacteriaceae contamination in all regions in sheep carcasses after chilling and acc in the shoulder region of sheep carcasses after chilling all exceeded the limits of ec regulation (ec no 2073/2005). keywords: air sampling, enterobacteriaceae, salmonella spp., slaughtering stages, acc ital. j. food sci., vol. 30, 2018 829 1. introduction it is desirable to keep the initial microorganism load in meat as low as possible and to observe hygiene rules during slaughtering. therefore, it is very crucial that slaughtered animals are cut according to hygiene rules in slaughterhouses. carcasses naturally have a low level of microbial flora and can be regarded to be sterile immediately after slaughtering. microbial contamination can occur in slaughtered animals in most of the stages throughout the slaughtering process. the slaughtering steps are basically followed by bleeding, dressing, evisceration, washing and finally storage. the hygienic bleeding and dressing system described by fao is to allow the animal to move up from the back leg to an upright position to allow the bleeding to continue until the blood flow reaches negligible level. besides, inadequate hygiene conditions along the dressing cause the bacteria to spread from the carcass to the knives and to the hands of the operators. contamination of carcasses may also occur through direct contact with equipment or hands of the personnel or may also occur indirectly through microorganisms in the air of the slaughterhouse following slaughter (unterman et al., 1997, gill and baker, 1998, burfoot et al., 2006). the european union legislation has declared that the enterobacteriaceae content and aerobic colony count (acc) should be used as hygiene criteria throughout the slaughtering process and that measures should be taken if the values increase above the criteria for slaughtered animals during the slaughtering process (barco et al., 2015). the legislation required monitoring of the above bacterial groups as process hygiene criteria for cattle, sheep and other slaughtered animals and were declared to be hazard analysis and critical control point (haccp) indicators for an acceptable food processing system (ec no 2073/2005; barco et al., 2015). according to the legislation, the acc and enterobacteriaceae limits for carcasses of cattle and sheep were declared as minimum (m) and maximum (m) values were 3.5 log cfu/cm2 5.0 log cfu/cm2 and 1.5 log cfu/cm2 2.5 log cfu/cm2, respectively. the european food safety authority (efsa) panel on biological hazards (biohaz) has also introduced the current requirement that the interior temperature of the carcass should not higher than 7 °c immediately after the post-mortem examination, before transporting. a panel of researchers has stated that temperature-time profiles can be applied to obtain similar or reduced levels of carcass contamination and that contamination levels at this temperature range are typically related to the initial level of contamination. in most studies on carcass surface microbiology, non-invasive methods are used, such as the swab method (mcevoya, 2004). the swab method is the preferred method for carcass sampling according to haccp requirements for european union slaughterhouses (pepperell et al., 2005). there are a number of published studies in which swab sampling methods have been utilised (anderson et al., 2005; blagojevic, 2012; barco et al., 2015; petruzzelli et al., 2016; alonso-calleja et al., 2017). carcass samples used in this study were slaughtered using procedures based on ‘good hygiene practices’ and ‘haccp’ principles, related to european union regulation 852/2004. the main purpose of the present study was to determine whether the turkish food codex hygiene criteria and commission regulation (ec) no 2073/2005, are met in cattle and sheep slaughterhouses in the konya province, which is the biggest producer of red meat in turkey. in addition, we aimed to detect the airborne contamination in slaughtering area and cold storage rooms, to identify the sources of contamination during slaughtering and to detect the incidence (presence or absence) of salmonella spp. contamination in sheep and cattle carcasses. ital. j. food sci., vol. 30, 2018 830 2. materials and methods 2.1. sample collection in this study, changes in microbial flora, ph and temperature in cattle and sheep carcasses during different slaughter stages were investigated in three different large-scale slaughterhouses (with a daily cutting capacity of at least 40 cattle, according to the classification of turkish slaughterhouses) between december 2013 and april 2016 in konya, turkey. swab samples moistened with sterile buffered peptone water (bpw) were collected using the swab technique consisting of 5 vertical and horizontal passes described by usda with slight modification. we swabbed an area of 10 × 10 cm2 from five randomly chosen regions of the carcasses, including two shoulders, two rumps and briskets, after three different cutting stages (dressing, evisceration and washing) and after storage of the same carcass in chilling rooms for 24 h. a total of 480 samples from sheep (n = 240) and cattle (n = 240) were collected from the carcasses. samples were cold chain transported to the laboratory, and microbiological analyses were performed within 3 h of sampling. samples were cold chain transported to the laboratory 2.2. microbiological analysis acc were performed as follows: 1 ml from a 1:10 diluted swab sample was poured onto plate count agar (pca, merck 105463) plates. incubation was performed under aerobic conditions at 37°c for 24 h. the total number of enterobacteriaceae was determined according to the international organization for standardization (iso) 21528–2:2004. the procedure was as follows: 1 ml of serial dilutions in buffered peptone water (bpw, merck, germany), were poured onto violet red bile glucose agar (vrbg, merck, germany) and incubated at 37°c for 24 h. typical colonies grown on plates were quantified after incubation. isolation and identification of salmonella spp. was performed using the method recommended by iso 6579:2002 + a1:2007 with slight modifications. accordingly, swab samples in bpw were incubated overnight at 37°c for pre-enrichment. for selective enrichment, 0.1 ml of the pre-enriched culture was added to 10 ml of modified rappaportvassiliadis broth (mrvb, merck, germany) and incubated at 41.5°c for 24-48 h. subsequently, 0.1 ml from the enriched culture was streaked onto xylose lactose tergitol 4 (xlt4, merck, germany) agar supplemented with xlt4 selective supplement (merck, germany). these plates were incubated at 37°c for 24 h. dna isolation was performed from five selected black or black-centred colonies on each plate which were considered to be ‘presumptive salmonella colonies’ grown on xlt4 agar. 2.3. conventional m-pcr for detecting salmonella spp. dna isolation from suspicious salmonella colonies was performed using the boiling method. following optimisation of pcr conditions, conventional multiplex-pcr (m-pcr) was performed. gene primers used for salmonella spp. are shown on table 1. the m-pcr master mix comprised 1 u taq polymerase and taq buffer (5 mm kcl and 0 mm tris-hcl), 1.5 mm mgcl2, 0.025 mm of each primer, 0.9 µm inv-a primers and 0.4 µm ie1 and flic-c primers in a 20-µl reaction volume. the m-pcr protocol comprised an initial denaturation step for 5 min at 95°c followed by 30 cycles of 1 min at 95°c, 1 min at 58°c and 30 s at 72°c, with a final extension step of 7 min at 72°c (paiao et al., 2013). ital. j. food sci., vol. 30, 2018 831 table 1. the primer pairs used in this study. primers product length reference salmonella spp. (inv-a) f:gtgaaattatcgccacgttcgggcaa r:tcatcgcaccgtcaaaggaacc 284 bp rahn et al., 1992 s. enteritidis (ie-1) f:agtgccatactt ttaatgac r:actatgtcgatacggtggg 316 bp wang and yeh, 2002 s. typhimurium (flic-c) f:cccgcttacaggtggactac r:agcgggttttcggtggttgt 432 bp paiao et al., 2013 2.4. air sampling air sampling was employed to determine the number of acc and fungal counts in slaughter operation and cold storage rooms during processing. pca (merck, germmany) was used for acc and potato dextrose agar (merck, germany) supplemented with 10% tartaric acid solution was used for fungal counts in the air sampler (air ideal 3p, biomerieux, france). the air sampler device was placed 1-1,5 m above the floor along the slaughter line and chilling rooms. in the sampling areas, 190 l of air was vacuumed by placing the petri dishes on the vacuum surface of the device. the plates were incubated before determining microbial counts. the samples were taken six independent times on different sampling days. 2.5. determination of ph and temperature ph and temperature values of the carcass were measured during different sampling stages using a portable ph and temperature probe (testo 205, germany). changes in ph and temperature of sheep and cattle carcasses were also determined during the following slaughtering steps: dressing, evisceration, washing and chilling. 2.6. statistical analysis data obtained from the study was analysed using spss software package 21.00. data was subjected to variance analysis (one-way anova) and two sample t-tests in accordance with the experimental design. significant differences (p < 0.05) were identified using multicomparisons of the means, with duncan’s test, within the variance analysis. means and standard errors of the means were reported. 3. results and discussion in our study, acc in cattle carcasses were observed between 2.57±0.61 and 4.71±0.24 log cfu/cm2 and enterobacteriaceae counts were observed between 0.89±0.46 and 2.61±0.10 log cfu/cm2. further, contamination with enterobacteriaceae in the shoulder region after washing was found to exceed the limits of ec regulation for cattle carcasses. our highest acc in cattle carcass were observed in shoulders and rumps after dressing and in briskets after washing (fig. 1). contamination levels were not found to be statistically different (p > 0.05) for acc in shoulders, rumps and brisket regions at different stages of cattle slaughtering. ital. j. food sci., vol. 30, 2018 832 figure 1. acc at different slaughtering stages of cattle and sheep carcasses (log10cfu/cm2) data are presented as mean±standart error. data with different superscript (x,y; a,b; α,β) indicate significant difference (p<0.05). although there were no statistically significant differences in the total number of enterobacteriaceae in cattle carcasses (p > 0.05) in shoulders, rumps and brisket regions at all stages of slaughtering (p > 0.05; fig. 2), we observed that enterobacteriaceae contamination was the highest after the washing stage. this can be explained by partial contamination originating from faeces or internal organs that are spread to other regions during washing. in sheep carcass samples, the highest level of enterobacteriaceae contamination was observed at the chilling stage, and this was statistically significant (p < 0.05; fig 2). in sheep carcasses, mean values of acc were between 3.51±0.48 and 5.19±0.28 log cfu/cm2, whereas enterobacteriaceae counts were between 0.55±0.37 and 3.63±0.39 log cfu/cm2. further, acc in the shoulder region and enterobacteriaceae contamination in all regions after the chilling stage exceeded the limits of ec regulation. the highest acc were found in all sampled parts after the chilling stage (fig 1) and this was statistically significant (p < 0.05). in a similar study, zweifel et al. (2014) obtained similar results in cattle carcasses and they determined that chilling was the most important stage for preventing contamination. cattle and sheep carcasses were compared in terms of slaughtering steps and contamination levels in different sampling regions. statistical graphs and a comparison table of acc and enterobacteriaceae count from both sheep and cattle carcasses at different slaughtering stages are given below (p < 0.05, table 2). acc were higher in the shoulder region of cattle carcasses than in that of sheep carcasses (p < 0.05, table 2). similar differences were observed in acc in rump area after washing and that in brisket after the dressing process in the two carcasses (p < 0.05). further, acc in the rump area of sheep carcasses had higher contamination than that in the rump area of cattle carcasses during the dressing stage. after evisceration, contamination in the brisket of sheep carcasses was higher than that in the brisket of cattle carcasses (p < 0.05, fig. 3.). ital. j. food sci., vol. 30, 2018 833 figure 2. enterobacteriaceae counts at different slaughtering stages of cattle and sheep carcasses (log10cfu/cm2). data are presented as mean±standart error. data with different superscript (x,y; a,b; α,β ) above each bar indicate significant difference (p<0.05). table 2. comparison the acc and enterobacteriaceae contamination levels of cattle and sheep carcasses. shoulder rump brisket processing steps x± sx x± sx x± sx p acc (log cfu/cm2) after dressing cattle 4,71±0,24a 2,99±0,69b 4,01±0,21ab * sheep 4,55±0,17 4,70±0,28 3,51±0,48 after evisceration cattle 4,46±0,23 4,08±0,47 2,93±0,68b sheep 4,66±0,18 4,45±0,26 4,48±0,21 after washing cattle 4,46±0,26a 4,22±0,20a 2,57±0,61b ** sheep 4,22±0,21 4,19±0,19 3,79±0,48 after chilling cattle 4,44±0,24 3,54±0,26 2,92±0,68 sheep 5,19±0,28 5,44±0,24 4,60±0,59 enterobacteriaceae (log cfu/cm2) after dressing cattle 2,14±0,16a 2,06±0,22a 1,14±0,40b * sheep 0,83±0,42b 2,30±0,45a 0,55±0,37b * after evisceration cattle 2,45±0,33 2,13±0,50 1,77±0,52 sheep 1,09±0,47 1,31±0,54 1,82±0,51 after washing cattle 2,61±0,10a 2,28±0,30a 1,77±0,48b * sheep 1,12±0,38 1,16±0,39 1,55±0,44 after chilling cattle 1,69±0,50 1,41±0,50 0,89±0,46 sheep 3,63±0,39 3,52±0,49 3,15±0,59 x,y: values within a row with different letters are significantly different (p<0.05), x: mean, sx: standard error of mean *: p<0.05;**: p<0.01. ital. j. food sci., vol. 30, 2018 834 figure 3. comparison the acc levels of cattle and sheep carcasses. data are presented as mean±standart error. data with different superscript (x,y; a,b; α,β) above each bar indicate significant difference (p<0.05). the shoulder region of cattle carcasses after dressing and in the shoulder and rump regions after washing had higher enterobacteriaceae contamination values than those of sheep carcasses (p < 0.05, fig 4.), this implied that the washing process in cattle was inadequate. figure 4. comparison enterobacteriaceae spp. contamination levels of cattle and sheep carcasses.data are presented as mean±standart error. data with different superscript (x,y; a,b; α,β) above each bar indicate significant difference (p<0.05). other previous studies (barboza de martinez et al., 2002; mies et al., 2004) have supported these findings and stated that the washing process during slaughtering may be ineffective in decreasing the rate of bacterial contamination. therefore, many investigators suggest that washing should be done with effective disinfectants and that more strict preventive measures should be taken. a similar study (barboza de martinez et al., 2002) investigated the effect of nisin and lactic acid against bacterial loads, including acc, total coliforms and e. coli in cattle carcasses. researchers stated that washing alone did not reduce the bacterial load and that spraying with a mixture of lactic acid and nisin provided the highest reduction in all bacterial groups tested. in another study (mies et al., 2004), researchers assessed the effect of washing cattle carcasses with or without ital. j. food sci., vol. 30, 2018 835 disinfectant solutions prior to slaughtering on aerobic plate, coliform, e. coli and salmonella counts. the greatest reductions were observed in mean logs of bacterial counts in groups sprayed with 4%-6% ethanol and lactic acid. our microbial counts are comparable with those obtained in other surveys at the national and international level using similar techniques (swabbing or sponging) (gill and baker, 1998; duffy et al., 2001; yalçın et al., 2004; pearce et al., 2005; salmela et al., 2013; petruzelli et al., 2016). gill and baker (1998) examined aerobic counts and coliform and e. coli contamination rates on randomly selected sheep carcass surfaces using the swab method in canada. the highest e. coli load was detected on shoulder and rump regions after the dressing stage and that the acc load was the highest after the evisceration stage. duffy et al. in 2001 investigated lamb carcasses in the united states using sponge sampling to detect the presence of salmonella spp and e. coli and determine acc and total coliform loads after the chilling process. salmonella spp. was detected in 1.5% samples, whereas acc, total coliform and e. coli counts were observed at 4.42, 1.18 and 0.70 log cfu/ cm2, respectively. a similar study in turkey by yalçın et al. (2004) stated that acc in sheep carcasses were 2.96, 3.10, 2.81 and 1.69 log cfu/ cm2 after dressing, evisceration, washing and chilling steps, respectively. moreover, in ireland pearce et al. (2005) reported that acc in sheep carcasses, as tested by the excision method, in the thorax, shoulder-neck, chest-brisket, and flank areas were 3.4, 3.6, 3.5 and 2.4 log cfu/cm2, respectively, whereas measurements using the polyurethane sponge method indicated 2.9, 2.8, 3.3 and 2.5 log cfu/cm2 and that using the cellulose acetate sponge method indicated 2.7, 2.5, 2.7 and 2.3 log cfu/cm2 in the same stages, respectively. salmela et al. (2013) also assayed acc of carcasses sampled by excision and swab methods in finland; results were 3.77 and 3.16 log cfu/cm2, respectively. researchers reported that acc and enterobacteriaceae and e. coli counts were higher in the excision method than the swab method. the authors reported that enterobacteriaceae was detected using the excision and swab methods in 72% and 76% carcasses, respectively, whereas e. coli was detected in 48% and 61% carcasses, respectively. similar to our findings, petruzzelli et al. (2016) investigated acc and enterobacteriaceae contaminations in ovine, bovine and swine carcasses in italy using the sponge method following ec regulations. they found that contamination levels in bovine, ovine and swine carcasses, when measuring acc were 1.96, 2.27 and 2.27 log cfu/cm2, respectively, whereas enterobacteriaceae counts were 0.01, 0.27 and 0.20, respectively. they also stated that cattle carcasses had significantly lower levels of acc and enterobacteriaceae counts than swine and ovine carcasses. alonso-calleja et al. (2017) also studied lamb carcasses in two slaughterhouses in spain. they stated that total viable counts (tvc, 2.74 log cfu/cm2) were higher than enterobacteriaceae (2.21 log cfu/cm2) counts and that there was a high correlation between them. they also stated that 0% and 30.8% of the samples in abattoir a and 10% and 40% of the samples in abattoir b exceeded ec regulations for tvc and enterobacteriaceae counts, respectively. an overall evaluation demonstrated that as the samples progressed through the steps of the slaughtering process, an increase in the total number of microorganisms in the shoulders, rump and brisket regions was observed after dressing in both cattle and sheep carcasses. in this context, gill et al. (2003) argued that despite the use of decontamination methods, such as pasteurisation, hot-water washing or lactic acid spraying, after the evisceration step, the increase in bacterial counts after chilling of carcasses was related to the proliferation of initial microorganisms rather than subsequent contaminations. however, it has been determined that accs, as one of the determinative criteria of slaughtering hygiene, are also in conformity with the turkish food codex regulations on microbiological criteria of meat and meat products and commission, except in the shoulder region of sheep carcasses after the chilling stage. in addition, enterobacteriaceae ital. j. food sci., vol. 30, 2018 836 limits were exceeded in shoulder, brisket and rump regions of sheep carcasses after the chilling process and in the shoulder region of cattle carcasses after washing. thus, it is very important to take necessary precautions to prevent contamination, particularly during the process of washing and evisceration during slaughtering. likewise, it is thought that the chilling process should be performed at maximum performance and speed and that carcass decontamination methods should be applied, if necessary, to minimise the growth of pathogenic and spoilage microorganisms. the present study aimed to determine the presence of salmonella spp. in sheep and cattle slaughtering process. however, no salmonella spp. were detected in the 480 samples analysed, which is highly satisfactory in terms of slaughtering hygiene and public health. salmela et al. (2013) did not detect any salmonella spp. by swab and excision methods in carcasses in any of the samples, similar to that observed our present study. nevertheless, madden et al. (2001) found that three of the 200 samples from cattle carcasses were contaminated with salmonella spp. chavez et al. (2015) used the sponge sampling technique to collect samples (n = 142) after the washing step and before the chilling step and determined that 18% cattle carcasses were contaminated with salmonella spp. at the three inspected abattoirs. similarly, hald et al. (2003), collected pig carcass samples from 12 slaughterhouses in five countries of europe. the researchers stated no salmonella was found in one country while 5.3 % of 3485 samples found to be positive in the other four countries. in a recent study in ethiopia, muluneh and kibret (2015) found 7.6 % of the beef carcasses collected from an abattoir positive for salmonella spp. we observed that as the sheep and cattle slaughtering process progressed, the temperature progressively decreased in all carcass regions and that this was statistically significant (p < 0.05; table 3). the temperature observed after storage did not reach the desired low temperature (4°c-6°c), indicating that cooling is insufficient. this demonstrates the necessity for systematically controlling the size of carcasses, internal temperature, air flow and humidity of the chilling rooms. it was observed that inner temperatures of sheep carcasses after chilling storage were lower than cattle. nevertheless, it has been observed that this decrease in temperature does not provide any advantage in reducing the microbial load. as a matter of fact, according to the efsa panel on biohaz (2014a,b), it has been declared that it is important to go to the transport stage while the chilling process is performed on the carcasses so that the number of spoilage bacteria cannot reach an unsatisfactory level. table 3. ph and temperature (°c) values of the shoulder and rump regions at different slaughtering stages. sheep cattle shoulder rump shoulder rump processing steps x±sx x±sx x±sx x±sx ph after dressing 6.40±0.09a 6.35±0.05a 6,40a±0,09 6,35a±0,05 after evisceration 6.48±0.12a 6.14±0.05ab 6,48a±0,12 6,14ab±0,05 after washing 6.33±0.23a 6.15±0.01ab 6,33a±0,23 6,15ab±0,01 after chilling 5.61±0.28b 5.28±0.04c 5,61b±0,28 5,28c±0,04 p* *** *** *** *** °c after dressing 38.66±0.22a 38.48±0.38a 32,77ab±1,54 34,47b±1,93 after evisceration 38.12±0.26a 37.46±0.47a 33,85a±2,82 37,50a±0,35 after washing 36.27±0.68b 37.22±0.92a 28,23b±3,95 37,05ab±0,85 after chilling 7.72±0.77c 8.13±0.85b 11,43c±1,18 13,36c±1,24 p* *** *** *** *** ital. j. food sci., vol. 30, 2018 837 air sampling results for acc and fungal counts were found to be lower in the chilling rooms than in the slaughtering area (table 4; p < 0.05). these values indicated that the hygiene in chilling rooms is satisfactory. this can explain why microbial counts were reduced in the chilling rooms compared with those in the slaughtering area and increasing general hygienic conditions is critical for reduction of carcass microbial growth. in a similar study, prendergast et al. (2004) also investigated the relation between airborne bacterial counts and carcass contamination in two abattoirs in ireland. the acc were found to be between 1.79-3.49 log10cfu/m3 at different stages of slaughtering. they also stated the clean areas of slaughtering process had a lower microbial load than the dirty areas. researchers found the correlations between carcass contamination and aerial load was low. similarly, burfoot et al. (2006) noted that airborne contamination of cattle and sheep carcasses during the evisceration phase is less of a concern than other contamination sources. table 4. ac, yeast and molds counts in the slaughtering room and cold storage room (log10 cfu/m3). areas acc (x±sx) yeast and molds (x±sx) slaughtering room 2.54±0.01a 1.23±0.12 cold storage room 1.98±0.03b 1.04±0.01 p *** a, b, c: the differences between different letter values in the same column are significant (p <0.05). n: number of samples. x: mean value. sx: standard error of mean, *: p<0,05;**: p<0,01;***: p<0,001. 4. conclusion in the present study, enterobacteriaceae limits were exceeded in shoulder, brisket and rump regions of sheep carcasses after the chilling process and in the shoulder region of cattle carcasses after washing. further, acc in the shoulder region and enterobacteriaceae contamination in all regions after the chilling stage exceeded the limits of ec regulation. the highest acc were found in all sampled parts after the chilling stage. this is thought to be due to the provision of a suitable environment as a result of poorly cooled chilled rooms to gradually increase microbial load, which is relatively low in cutting stages. furthermore, it is satisfactory that salmonella spp. is not detected in the present study, but it should be considered that this result may be due to possible insufficiency of the swabbing method compared with the excision. in conclusion, the microbial quality of meat for consumption is closely related to public health. thus, it is critical to understand specific microbial risks of each work step, from slaughtering to chilling of carcasses. microbiological analyzes which are carried out only at the end of the process, may not provide realistic information on the main causes of the microbial contamination. therefore, the microbiological criteria which are ‘process-based’ related to measurements including different methods at various stages of the process should be 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polymerase chain reaction primers for the detection of salmonella enteritidis in foodsand faecal samples. lett. app. microbiol. 32:422-427.3. yalçın s., nizamlıoğlu m. and gurbuz u. 2004 microbiological conditions of sheep carcasses during the slaughtering process. journal of food safety 24(2):87-93. doi: doi.org/10.1111/j.1745-4565.2004.tb00377.x. zweifel c., capek m. and stephan r. 2014. microbiological contamination of cattle carcasses at different stages of slaughter in two abattoirs. meat science 98:198-202. doi: doi.org/10.1016/j.meatsci.2014.05.029. paper received april 12, 2018 accepted june 14, 2018 ijfs#1787_bozza ital. j. food sci., vol. 32, 2020 893 paper mango-seed extract and sulphites as promoters of color and bioactive compounds retention during tray drying of mango slices a. jiménez-durán1, n.f. santos-sánchez1, b. hernández-carlos, h.r. juliani2 and r. salas-coronado*1 1 instituto de agroindustrias, universidad tecnológica de la mixteca. carretera huajuapan-acatlima km 2.5, 69000 huajuapan de león oaxaca, méxico 2 department of plant biology and pathology, rutgers, the state university of new jersey, foran hall/ cook campus, 59 dudley rd, new brunswick, 08901 new jersey, united states *corresponding author: rsalas@mixteco.utm.mx abstract the study objective was to evaluate effect of mango-seed extract alone or in combination with sodium metabisulphite on the content of vitamin c, free phenols, six phenols compounds, and total carotenes, and color in mango slices dried at 60ºc until 15% moisture. from drying curves were calculated effective diffusivities [1.17-1.35x10-9 m2•s-1] and drying rate constants [1.53±0.90x10-3-2.27±0.80x10-3] using midilli’s model. results showed that combination of mango-seed extract with sodium metabisulphite has an important role in retention of vitamin c and carotenes, and an enrichment of phenolic compounds was found in dried mango slices. keywords: dried mango; mango-seed extract, sodium metabisulphite, free phenols; total carotenes; vitamin c ital. j. food sci., vol. 32, 2020 894 1. introduction mango (mangifera indica l.) is one of the most important fruits grown commercially in tropical and subtropical regions in the world. since mango fruit is highly perishable, it is generally transformed to a dried product (lin et al., 2016) for prolonged shelf life. fresh mango is characterized by its high content of phenolic compounds, vitamin c and carotenes. since these compounds are antioxidants, they are able to impart beneficial properties for the consumer health (siddiq et al. 2013). mangoes have phenolic compounds derived from phenolic acids (schieber et al., 2000) such as gallic acid, caffeic acid, p-coumaric acid, etc. gallic acid has antioxidant, antiinflammatory, and anticarcinogenic activity (velderrain-rodríguez et al., 2018). schieber et al. (2000) reported that mango pulp has 5.9 mg gallic acid•(100 g dry mass)-1. other phenolic acids found in mangoes are caffeic acid [6.6 mg•(100 g dry mass)-1] and ferulic acid [8.9 mg•(100 g dry mass)-1] (abdalla et al., 2007). in addition, methyl gallate is a potent cell protector against oxidative stress, reduces lipid peroxidation (lpo) and reactive oxygen species (ros) (whang et al., 2005). lee et al. (2010) reported that methyl gallate suppresses t regulatory cells (treg) in mice’s malignant tumors. mangiferin is a cglucosyl-xanthone found in some mango varieties, such as tommy atkins, haden and ubi. this compound has a wide range of biological properties, because it is gastroprotective, analgesic, antibacterial and cytoprotective (masibo and he, 2008). schieber et al. (2000) reported that mango pulp has 3.8 mg mangiferin•(100 g dry mass)1. vitamin c is one of the most abundant compounds in mango fruit and its concentration varies with the fruit maturity, as well as with the post-harvest handling and processing methods. rocha-ribeiro et al. (2007) reported a concentration of 94.0 mg aae•(100 g dry mass)-1 for vitamin c in fresh mango var tommy atkins grown in brazil. vitamin c decreases from thermal effects and exposure to air and light (liu et al., 2014). mango is also a good source of carotenes. β-carotene is the most abundant in mango fruits (varakumar et al., 2011). β-carotene content is often used as an indicator of damage extent to mangoes during processing and storage (tharanathan et al., 2006). manthey and perkins-veazie (2009) reported a concentration of 32.9 to 59.1 mg of carotenes•(100 g dry mass)-1 in mango tommy atkins mexican mangoes. during the mango convective drying process, considerable degradation of these bioactive compounds occurs (méndez-calderón et al., 2018). hence, to minimize bioactive compound degradation it may be beneficial to carry out mango pretreatments prior to the drying process (yao et al., 2020). the pretreatment choice depends on the drying method and characteristics of desired product. additionally, pretreatments may improve product quality by retaining color of fresh mango and reducing product darkening. guiamba et al. (2016) evaluated the retention of β-carotene and vitamin c (dehydroascorbic and ascorbic acids) in osmotically dried mango pretreated with calcium chloride or ascorbic acid. this study consisted in an initial osmotic dehydration using 45º brix sucrose solutions added with 1% calcium chloride or 1% ascorbic acid. then samples were dried in an air convection oven at 50ºc or 70ºc. the authors reported that both pretreatments significantly improved retention of vitamin c and all-trans-β-carotene in dried products. in other study, chen et al. (2007) performed mango drying experiments using hot drying air and freeze-drying in presence of 1% sodium hydrogen sulphite or 1% ascorbic acid. the authors reported that use of pretreatments during drying mangoes reduced carotenes degradation. this behavior also was observed by jiménezhernández et al. (2017) when studied effect osmotic dehydration of mango slices in an emulsion based on inulin and piquin-pepper oleoresin. the results showed a retention of ital. j. food sci., vol. 32, 2020 895 68.8% of ascorbic acid and 95.5% of β-carotene at 30ºc. also, this study found a strong decrease in retention of ascorbic acid (43.6%) when the process was carried out at 50ºc. while retention of β-carotene was 83.0%. recently, dereje et al. (2020) dried mango slices using four pretreatments (lemon juice, salt solution dips, hot water blanching and control) and four drying methods (solar, tray, freeze and fluidized bed drying) to assess effect of pretreatments and drying methods on qualities of dried mango slices. the results showed that pretreatments and drying methods had significant effects on color antioxidants of the dried mango slices. the ascorbic acid and total phenol contents were affected by drying methods and had respective values of 33.18-41.24 mg aae•(100 g dry mass)-1 and 131.13251.12 mg of gallic acid equivalents (gae)•(100 g dry mass)-1. on the other hand, it has been reported that mango-seed extracts contain a significant amount of free phenols, 27.7±0.1 g gae•(100 g dry mass)-1 (bernal-mercado et al., 2018). for this reason, this same study used mango-seed extract as antioxidant agent of fresh-cut mango. the results showed that a solution mango-seed extract at 0.63% (m/v) contributed to preservation of fresh mangoes cubes due to increasing free phenols from 306.0 to 364.9 mg gae•(100 g dry mass)-1. additionally, in this study found that mango-seed extract contains 60 mg of gallic acid•(100 g dry mass)-1, 42.0 mg of mangiferin•(100 g dry mass)-1, 77 mg of caffeic acid•(100 g dry mass)-1 and 12.6 mg of p-coumaric acid•(100 g dry mass)-1. also, mango-seed extracts have been used to develop antioxidants films (adilah et al., 2018). to the best of our knowledge, there are no reports about use of mango-seed extracts as a fruit drying pretreatment. considering the above, aim of study was to evaluate effect of different pretreatments on retention of color and antioxidant compounds (free phenols, vitamin c and total carotenes) during tray drying of tommy atkins mango slices. three pretreatments were used for tray drying of tommy atkins mango slices: 0.5% sodium metabisulphite (pt1), 1.44% mango-seed extract (pt2) and 0.5%/1.44% (w/v) sodium metabisulphite/mangoseed extract (pt3). hplc was used to quantify the main phenolic compounds present in the dried products. 2. materials and methods 2.1. sampling procedure and sample preparation tommy atkins mangoes (mangifera indica l.) were obtained from porfirio díaz market located in huajuapan de león city, oaxaca, méxico. mango fruits were selected based on fruit size and pulp color as measured by cielab b* parameters, which defines yellow color. it was also verified that the fruits had no physical damage. mangoes were peeled with a home peeler and slices of 6.0 ± 0.1 cm x 4.0 ± 0.1 cm (length x width) and 3.8 ± 0.4 mm thickness were obtained with a cutter. 2.2. determination of mango physicochemical parameters the physicochemical characteristics of fresh mangoes were evaluated from soluble solids content, moisture percentage, titratable acidity and ph, which are briefly described below. soluble solids content (aoac 932.12). a drop of fresh mango juice was placed in abbe refractometer and °brix of sample was measured. moisture percentage (wrolstad et al., 2005). a known amount of mango pulp (3 g) was weighed into pre-weighed and dried crucibles. the samples were then placed in an oven ital. j. food sci., vol. 32, 2020 896 at 105°c for 24 h. after that, dried samples were placed in a desiccator for 30 min at room temperature and anhydrous conditions and were finally weighed. titratable acidity and ph (dea et al., 2013). mango pulp sample was blended for 2 min to homogenize the sample and the blend was filtered through a cotton plug. a potentiometer previously calibrated with standard solutions (ph 4 and 7) was used to measure sample´s titratable acidity. for titration 10 g of sample was used. a 0.1 n sodium hydroxide solution was continuously added, while the ph was measured until sample reached a ph of 8.3. 2.3. preparation of the mango-seed extract tommy atkins mango seeds were cut into small pieces, ground in a blender for 2 min and sieved through a #40 mesh to obtain a fine powder. subsequently, 25 g of powder was weighed and mixed with 500 ml of 99.5% methanol and sonicated for 30 min at room temperature. mixture was allowed to stand until a phase separation was observed. supernatant was decanted and filtered through a whatman filter paper #1. pellet was treated with same extraction procedure three times. following this, filtrates were collected, combined and evaporated on a rotary vacuum evaporator at 40°c until 11 g of solvent-free extract was obtained as a viscous reddish-brown liquid. extract was dissolved in water to make a final volume of 100 ml in a volumetric flask. finally, extract solution was stored at -20°c for preservation until further use. 2.4. pretreatment of mango slices 700 g of mango slices were immersed for 3 min in 1.4 l of pretreatment solutions at 25ºc. solutions were used only once, after they were discarded. solutions used as pretreatment were 0.5% sodium metabisulphite solution (pt1), 1.44% mango-seed extract solution (pt2) and a combination of 0.5% sodium metabisulphite and 1.44% mango-seed extract solutions (pt3). following this, slices were drained for 1 min. mango slices without pretreatment was used as control. 2.5. dryer and drying conditions a tray dryer (santos-sánchez et al., 2012,) equipped with a tray rotating mechanism and a heating-air flow control, was used in this work to perform drying of 700 ± 8 g of mango slices. mango slices were dried at 60°c, air velocity of 1.2 m•s-1 and 20 rpm tray rotation velocity. moisture loss was quantified by weighing slices every 15 min until moisture content was about 15%. drying time was around 110 min. for each determination, mass of three samples was measured. 2.6. determination of effective diffusivity (deff) and drying rate constant (k) drying curves (moisture ratio versus drying time) were used to calculate effective diffusivity (deff) and drying rate constant (k). drying curves were performed in triplicate. moisture ratio (mr) of food slices can be predicted from sherwood and newman model, equation 1. this equation relates mr with drying time (t), thickness of food slice (l) and effective diffusivity (deff). deff is related with fourier number (f) through equation 2. in this study, deff was calculated from curve slope expressed by simplified sherwood and ital. j. food sci., vol. 32, 2020 897 newman model, equation 3 (ashraf et al., 2012). the k values were calculated from nonlinear regressions of midilli equation (midilli et al. 2002). (1) (2) where mrt = moisture ratio at time t, mro = initial moisture ratio, mr* = equilibrium moisture ratio and f = fourier number. compared to values of mrt and mr0, the value of mre is relatively smaller, so the mr can be reduced to mr = mrt/mr0 (mewa et al., 2018). considering that for this study, n = 0 and diffusion occurs through two faces of mango slice (trays used for drying are perforated), mr can be expressed from equation 3. deff values were obtained from slope of time versus ln mr curves. (3) equation 4 was used to estimate k as well as n, a and b values, nonlinear regressions were applied to equation of midilli et al. (2002), where mr is moisture ratio, t is the drying time, k is drying rate constant and a, b and n are the model constants. the regression was performed with interreg 2014 (kroll-software). (4) 2.7. free phenols quantification of mango-seed extract and the mango slices for free phenols determination, 0.2 g of mango seed was mixer with 3.1 ml of 60:40 % (v/v) ethanol:water or 2 g of mango pulp was mixed with 25 ml of 60:40 % (v/v) ethanol:water. the mixture was milled in a home blender for 1 min and subsequently filtered through cotton wool. extracts were obtained in triplicate. free phenols quantification was performed in a biotek elx-808 microplate reader using modified folinciocalteu method described by ochoa-velasco et al. (2016). extract or standard (40 µl) was pour in a microplate well with 40 µl of 0.1 m folin-ciocalteu reagent. the reaction mixture was allowed to stand for 3 min in microplate reader, and stirred for 15 s at low velocity. subsequently, 40 µl of 0.5% sodium carbonate (w/v) was added and mixed by suction with a multichannel pipette. mixture was allowed to stand for 30 min at 40°c, after which it was stirred at medium speed in microplate reader. finally, sample absorbance was read at 765 nm. a calibration curve was prepared using gallic acid solutions with known concentrations. free phenols content in mango-seed extract and mango samples was determined using calibration curve and was expressed in mg of gae•(100 g dry mass)-1. all measurements were made in triplicate. € mr = mrt −mr * mro −mr * = 8 π2(2n +1)n=0 a ∑ exp −(2n +1)2 π2 4 f ⎡ ⎣ ⎢ ⎤ ⎦ ⎥ € f = deff *t l2 € lnmr = ln 8 π2 − π2deff 4l2 ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ t € mr = aexp −ktn( ) + bt ital. j. food sci., vol. 32, 2020 898 2.8. vitamin c quantification the quantification of vitamin c was performed following procedure described by ochoa-velasco et al. (2016). briefly, 0.05 g of dried mango (0.3 g of fresh mango) was mixed with 1.00 ml of 1% metaphosphoric acid solution. subsequently, mixture was sonicated in an 8510 sonicator (branson ultrasonics co., usa) for 15 min at room temperature. then mixture was centrifuged for 15 min at 900 g. supernatant containing vitamin c was used for discoloration reaction of 2,6-dichloroindophenol sodium salt (dcip) in a 96 well microplate plate. to carry out this reaction, 70 µl of extract was mixed with 70 µl of a solution of 30 ppm dcip and allowed to stand for 1 min at room temperature in absence of light. finally, mixture was stirred for 15 s and then absorbance at 515 nm was measured in a biotek elx-808 microplate reader. vitamin c content in samples was determined using calibration curve and expressed in mg of ascorbic acid equivalents (aae)•(100 g dry mass)-1. all measurements were made in triplicate. 2.9. phenolic compounds quantification by hplc a mixture of 0.2 g of dried mango and 2.5 ml of a 70:30% (v/v) methanol:water solution with 0.1% ch3cooh was vortexed for 5 min. subsequently, mixture was sonicated for 10 min at room temperature, followed by centrifugation for 10 min at 900 g. supernatant was then removed and the pellet was re-extracted for a second time. supernatants were pooled and dried on a rotary vacuum evaporator at 40°c. extracts were obtained in triplicate. dried extracts were redissolved in 500 ml of water and solution was cleaned up by eluting it through c18 spe cartridges of 2.8 ml (alltech, usa). the clean-up procedure consisted first in elution of 1 ml of deionized water through c18 spe cartridge. subsequently, extract solution was loaded into cartridge and eluted with 5 ml of water, followed by 1 ml of a 1:1 (v/v) methanol:water solution, and finally with 1 ml of methanol. fractions were collected in hplc vials. phenolic compounds quantification was performed in an hplc instrument equipped with a photodiode array detector (water, alliance 2695, usa) and a c18 column of 4.6 mm x 250 and 5 µm inner diameter (phenomex, usa). the following phenolic measuring standards were employed: mangiferin, methyl gallate, ferulic acid, gallic acid, caffeic acid and p-coumaric acid. a calibration curve was performed for each standard. a 10 μl injection volume and a 1 ml•min-1 flow was used. a binary mobile phase was used (a = 0.1% formic acid in water, b = 0.1% formic acid in acetonitrile) with the following gradient program: 0 min 10% b, 5 min 10% b, 15 min 80% b and 30 min 100% b, 35 min 10% b. the mangiferin was monitored at 365 nm and phenolic acids and methyl gallate were monitored at 280 nm for (rocha-ribeiro et al., 2008). all measurements were made in triplicate. 2.10. total carotenes quantification a modification of method described by wrolstad et al. (2005) was used for total carotene quantification. briefly, 0.3 g of dried mango was weighed and ground in a mortar with 3 ml of water. for fresh mango samples, 1.0 g pulp sample was homogenized and mixed with 2 ml of water. mixture was placed in a 10 ml amber vial with subsequent addition of 4 ml of 95% ethanol was added, followed by vortexing for 4 min. this procedure was performed in triplicate. then the mixture was vacuum filtered through filter paper. supernatant was then poured through a 25 ml filtration funnel with 10 ml of hexane, and then manually stirred. filtration funnel was allowed to stand for 2 min. ital. j. food sci., vol. 32, 2020 899 subsequently, ethanolic phase was removed. absorbance of hexane phase was measured at 450 nm in a lambda 32 uv-vis spectrophotometer (perkin elmer, usa). sample measurements were performed in triplicate. equation 5 was used for the calculation of total carotenes of sample expressed as mg of β-carotene•(100 g dry mass)-1. all measurements were made in triplicate. [total carotenes] = !!"#•!"#!"#$%&"'(!") !"#.!" !"•!"!!• !"#$%& !"## (!) • 100 (5) 2.11. sulphites quantification sulphites quantification in dried mango slices obtained from pt1 and pt3 pretreatments was carried out analogously to reported by li and zhao (2006). briefly, solutions used to prepare samples and standards were 0.1 mm ethylenediaminetetraacetic disodium salt (edta) solution, 1000 ppm tris(hydroxymethyl)aminomethane (tris) buffer solution at ph 8 and 0.3 mm 5,5-dithio-bis-(2-nitrobenzoic acid) [dtnb, also called ellman's reagent]. 0.1 mm edta and tris buffer solutions were prepared with deionized and degassed water for 25 min at 25ºc. subsequently, edta 0.1 mm solution was used to prepare 1000 ppm sodium metabisulphite solution. tris buffer solution was used to prepare 0.3 mm dtnb solution. before being employed, all solutions were degassed for 15 min at 25ºc. on the other hand, around 1 g of dried mango slices (pt2 and pt3), were drymilled in a mortar to form a homogeneous paste. 20 mg of this paste was mixed with 1 ml of 0.1 mm edta. later, samples were shaken in a vortex for 2 min and degassed for 15 min at 25ºc on ultrasound, followed by a centrifugation for 5 min at 900 g. supernatants were separated and then used to carry out colorimetric reaction with 0.3 mm dtnb. this consisted of a mix 60 µl of sample or standard with 60 µl of 0.3 mm dtnb in a 96-well plate. also, two reaction blanks were prepared, the first was prepared by mixing 60 µl of sample with 60 µl of 0.1 mm edta, while the second blank was prepared by mixing 60 µl of 0.3 mm dtnb with 60 µl of 0.1 mm edta. reaction mixtures were allowed to stand for 5 min at 25ºc, then stirred for 30 s in a biotek® model elx808 microplate reader and absorbance at 405 nm was measured. calibration curve was built with five sulphites standards, which were prepared at concentrations in interval of 6 to 20 ppm from 1000 ppm sodium metabisulphite solution. all measurements were made in triplicate. 2.12. color determination a hunterlab ultra scanvis (usa) spectrophotometric colorimeter was used to determine cielab color parameters of mango samples. d65 illuminant was used with an observation angle of 10° and a 0.9525 cm slit. for each drying data point, the cielab color parameters (l*, a* and b*) of mango slices were measured at ten different points to obtain an average. also, angle hue* was calculated with equation 6. three mango slices were measured for every drying data point. 𝐻𝑢𝑒∗ = 𝑎𝑟𝑐 𝑡𝑎𝑛 ! ∗ !∗ (6) ital. j. food sci., vol. 32, 2020 900 2.13 statistical analysis a randomized experimental design was applied in this study, one-factor anova tests and duncan´s means comparison method were used with a significance level α = 0.05 between treatments and variables. design-expert® v.6.0 software was employed for these analyses. 3. results and discussion 3.1. physicochemical parameters of the mango pulp soluble sugars content and titratable acidity are related to fruit ripeness stage. the determinations of these parameters for fresh mango pulp are listed in table 1 and are similar to values reported by siddiq et al. (2013) for ripe mango samples. visual color scale reported by brecht (2010) for tommy atkins mango was used to report state of mango ripeness in this study. according to this scale, fresh mango fruits ripeness was 5. the l* value was 63.56±4.73, accounting for a low degree of darkness, while b* parameter (57.81±4.22) indicates an intense yellow color, which was greater than that reported by rocha-ribeiro et al. (2008) (table 1). table 1. physicochemical properties of fresh tommy atkins mango. parameter this study literature mango weight (g) 671.46±80.47 soluble solids (°brix) 16.22±1.62 14-16b moisture (%) 88.30±1.09 ph 3.78±0.15 3.4±0.1a acidity % 0.47±0.07 0.9±0.0a color l* 63.56±4.73 55.0-61.1b a* 11.45±1.95 11.5-14.4b b* 57.81±4.22 40.0-50.0b mean ± standard deviation, n = 3. asiddiq et al. (2013). brocha-ribeiro et al. (2008). 3.2. physicochemical, chemical and antioxidant properties of mango seed tommy atkins mango seeds of 24.6±7.5 g weight and a 6.5±0.5 cm length used for study had a 37.7±0.3% moisture content. extraction yield was 12.2±0.3 g of extract•(100 g of seed mango)-1, which was comparable with yields reported by dorta et al. (2012) for mangoseed extracts var. keitt using 50% aqueous acetone solvent and 60 min ultrasound, 12.0±1.0 g of extract•(100 g of seed mango) -1. concentration of feee phenols in mango seed extracts was 23.9±0.0 g gae•(100 g dry mass)-1. this concentration was superior to that reported by sogi et al. (2013), for mango var. tommy atkins from usa, 20.03-11.23 g gae•(100 g dry mass)-1, which was previously dried using different drying methods ital. j. food sci., vol. 32, 2020 901 (freeze drying, tray drying, vacuum drying and infrared drying) to extraction. in this study, freeze drying allowed highest retention of phenols compounds in mango seed extract. on the other hand, bernal-mercado et al. (2018) obtained a total phenol content of 27.7±0.1 g gae•(100 g dry mass)-1 from mango var. haden. this last extract was obtained from a maceration at 25ºc for 10 days in darkness. this indicates that extraction assisted by ultrasound, in addition to being fast, allows a high retention of phenolic compounds in extracts from mango seeds. additionally, it is important to avoid drying mango-seed samples with hot air. 3.3. drying curves during the drying time from 0 to 15 min, free water heating and evaporation occur slowly for pt1 pretreatment. all drying curves presented a significant moisture reduction in range from 15 to 75 min, fig. 1. this behavior can be explained considering that during this drying period mango slice surface is wet, thus forming a continuous free water film. consequently, there is no resistance to water transfer from solid surface to surrounding air. on the other hand, drying rate decreased after 75 min of drying for all pretreatments and control, indicating start of a decreasing drying rate period. 3.4. effective diffusivities and constant drying rate effective diffusivity, deff,, for different drying pretreatments and control (data no showed) lied in range of 1.17-1.35 x 10-9 m2•s-1. these values are similar to those reported by dissa et al. (2008) for 5 mm-thick mango slices, dried at 60°c. a comparative analysis of deff means attested that there are not significant differences between mango pretreatments. midilli’s n, k and a constants were calculated with equation 4. the determination coefficients (r2) for all pretreatments were higher than 0.99. the b constant for this study was zero, and n and a constants were found in ranges of 1.42±0.09-1.54±0.13 and 7.16±0.047.75±0.90, respectively. constant k is considered a measure of water evaporation rate from the mango slice. the k values for pretreatments and control were not significantly different as compared by the duncan’s mean comparison test (p<0.05) and were found in range of 1.53±0.90x10-3-2.27±0.80 x 10-3. the k values obtained in this study are similar to that reported by murthy and manohar (2014) for slices of dried mango at 60ºc and air velocity of 2.25 m•s-1: k = 0.054, n = 1.022 and r2 = 0.969. while n value, which corresponds to drying kinetics order, is higher in present study than in that reported by murthy and manohar (2014). it should be noted that an order of drying kinetics close to unity is indicative that drying process depends almost exclusively on temperature and as n value increases, it is indicative that other variables are also contributing significantly to food drying process. these variables are drying air velocity, mango slices thickness, concentrations and types of pretreatments, among others. ital. j. food sci., vol. 32, 2020 902 figure 1. drying curves for mango slices with different pretreatments. control = mango dried without pretreatment, pt1 = 0.5% (w/v) sodium metabisulphite, pt2 = 1.44% (w/v) seed extract mango, pt3 = seed extract mango/sodium metabisulphite [1.44%/0.5% (w/v)]. each curve is mean of three drying replicates. 3.5. free phenols fig. 2 shows free phenols content for dried and fresh mango slices. free phenols content of fresh mango was 195.28±5.48 mg gae•(100 g dry mass)-1, which is within range reported by manthey and perkins-veazie (2009) of 171.8-257.3 mg gae•(100 g dry mass)-1, for a mexican tommy atkins variety. from duncan test comparisons, it can be implied that since control and pt1 pretreated mango slices (0.5% metabisulphite) are not statistically different (p<0.05), pt1 pretreatment did not have a significant effect on phenol retention (about 46%). this retention amount is similar to that reported by chong et al. (2013) (50.4%) who performed dried of mango slices using cold/hot air treatment. treatment used by authors consisted of applying a flow of cold air (11.54±0.26ºc) either at beginning or during dehydration process with hot air at 53.95±0.03ºc. on the other hand, dried samples which were pretreated with mango-seed extract (pt2 and pt3) had a much greater phenol content than corresponding values of both control and fresh mango samples (346.96±19.69, 368.00±11.84 mg gae•(100 g dry mass)-1, fig. 2). this implies that, as opposed to bisulphites-only treatment (pt1), addition of mango-seed extract not only aided in retention of phenolic compounds but also in the increase of their concentration to 77.8 and 88.4% in pt2 and pt3 pretreatments, respectively. this effect appears to be due to the diffusion of phenolic compounds from pretreatment extract to the mango slices during the immersion period. it is also remarkable that in the case of pt3 pretreatment, addition of sodium metabisulphite caused a greater increase in total phenol content than that caused by use of the mango-seed extract alone (pt2) (fig. 2). this seems ital. j. food sci., vol. 32, 2020 903 to indicate that sodium metabisulphite had a synergistic effect on retention and fortification of phenolic compounds during mango drying. figure 2. free phenols content [mg gallic acid equivalents gae•(100 g dry mass)-1] in fresh mango and pretreated dried. mean ± standard deviation, n = 3. control = mango dried without pretreatment, pt1 = 0.5% (w/v) sodium metabisulphite, pt2 = 1.44% (w/v) seed extract mango, pt3 = seed extract mango/sodium metabisulphite [1.44%/0.5% (w/v)]. superscripts a-d showed a significant difference (α = 0.05) according to the duncan test. 3.6. profile of main phenolic compound in dried mango samples the phenolic compound concentration profile of different pretreated samples was measured by hplc for gallic acid, methyl gallate, mangiferin, caffeic acid, ferulic acid and p-coumaric acid (table 2). mangiferin concentrations were not affected by sample pretreatments with respect to control. mango slices dried without pretreatment (control) had a similar behavior than mango sliced pretreated with metabisulphite (pt1), except for gallic acid. the pt1 pretreatment (0.5% sulphites only) caused a significant increase in the retention of gallic acid only (33.0%). on the other hand, pt2 pretreatment (1.44% mangoseed extract) significantly increased content of methyl gallate (27.4%), caffeic acid (70.9%), ferulic acid (244.4%), and p-coumaric acid (87%) with respect to control. these results are also in agreement with total phenol assays in which pt2 samples had a higher phenolic content than both control and fresh samples, which confirms that pt2 dried products were enriched with phenolic compounds of mango-seed extract. this phenolic enrichment can be explained by considering that mango-seed extract solutions had a 10-fold higher concentration of free phenols than that present in fresh mango pulp, so molecular diffusion occurs from the extract to pulp by a concentration driving force. in general, pt3 pretreatment samples (0.5% sulphites and 1.44% mango-seed extract) displayed greater increments in phenolic compounds concentration than those observed in pt2 pretreatment, with exception of methyl gallate, which is statistically equal in both treatments. in addition, pt3 samples presented a sharp increase in concentrations of gallic acid and caffeic acid with respect to pt2 pretreatment. this result agrees with free phenol ital. j. food sci., vol. 32, 2020 904 observations for pt2 and pt3 pretreatments, thus confirming synergic effect of bisulphites and mango extract on phenol enrichment of mango dried products. table 2. phenolic compounds content in dried slices of tommy atkins mango quantified by hplc. pretreatment compound [mg•(100 g dry mass)-1] gallic acid methyl gallate mangiferin caffeic acid ferulic acid p-coumaric acid control 13.49±0.02ª 7.26±0.15ª 5.31±0.45a 6.16±0.26a 10.86±0.52a 8.24±0.45a pt1 17.98±0.29b 6.83±0.31ª 4.75±0.70a 5.52±0.53a 11.61±0.57a 7.99±0.28a pt2 14.71±0.18c 9.25±0.49b 5.02±0.17a 10.53±0.66b 37.40±0.47b 15.41±0.67b pt3 26.42±0.16d 10.08±0.28b 5.65±0.50a 26.10±0.71c 45.61±0.31c 16.30±0.55c control = mango dried without pretreatment, pt1 = 0.5% (w/v) sodium metabisulfite, pt2 = 1.44% (w/v) seed extract mango, pt3 = seed extract mango/sodium metabisulfite [1.44%/0.5% (w/v)]. mean ± standard deviation, n = 3. superscripts a-d = mean difference significant in columns (α = 0.05) by duncan test. in the calibration equation y = area and x = concentration of corresponding phenol compound. 3.7. vitamin c content fresh mango samples had a vitamin c content of 135.59±3.40 aae•(100 g dry mass)-1 which is similar to that reported by rocha-ribeiro et al. (2007) (94.0 mg aae•(100 g dry mass)-1). vitamin c contents in dried samples (fig. 3) were greatly superior to those described by ndawula et al. (2004), who dried mango slices of 3-5 mm thickness in an open solar dryer. these authors reported a vitamin c content of 25.4 mg aae•(100 g dry mass) -1 and a 15.5% vitamin c retention in dried slices. vitamin c content in pt2 (32.15±1.21 aae•(100 g dry mass)-1) pretreated samples (mango-seed extract only), was not significantly different than control (32.34±1.58 aae•(100 g dry mass)-1, p<0.05), thus indicating that pt2 pretreatment did not have a significant effect on retention of vitamin c in dried product. on the other hand, sulphites-added samples (pt1 and pt3) presented a higher vitamin c content than control samples, which indicates that bisulphites pretreatment efficiently promoted retention of vitamin c in dried mango (fig. 3). finally, pt3 pretreatment (combined sulphites and mango-seed extract) presented a 3-fold vitamin c content with respect to the control (96.52±5.09 aae•(100 g dry mass)-1, fig. 3). thus, similarly to phenol results, combined pretreatments of sulphites and mango-seed extract yielded an improved vitamin c retention in dried product (71.2%) as compared with individual pretreatments alone, which indicates a synergistic effect of such pretreatments on retention of antioxidant compounds during mango drying. however, in contrast to phenol results, sulphites pretreatment (pt1) was the most effective at maintaining ascorbic acid content of dried product. ital. j. food sci., vol. 32, 2020 905 figure 3. content of vitamin c [mg ascorbic acid equivalents aae•(100 g dry mass)-1] in samples of fresh mango and pretreated dried. mean ± standard deviation, n = 3. control = without pretreatment, pt1 = 0.5 % (w/v) sodium metabisulphite, pt2 = 1.44% (w/v) mango seed extract, pt3 = [1.44%/0.5% (w/v)] mango seed extract/sodium metabisulphite. superscripts a-d showed a significant difference (α = 0.05) according to the duncan test. 3.8. total carotenes content in dried products the total carotenes content in fresh mango pulp was 25.54±0.81 mg of β-carotene•(100 g dry mass)-1, fig. 4, was within range reported by manthey and perkins-veazie (2009), who reported concentrations from 32.9 to 59.1 mg of carotenes•(100 g dry mass)-1 in mexican tommy atkins mangoes. total carotenes content in sulphites-pretreated samples was 1.8 times higher than those reported by chen et al. (2007) for mango slices of 3 x 9 cm pretreated with 1% sodium bisulphite and dried with hot air at 60ºc. the variation in these results is probably due to different drying times and slice thickness. guarte et al. (2005) reported a carotene content of 6.80 mg•(100 g dry mass)-1 for pulp mango, a very similar value to those measured for control and the pt2 samples in the present study. from total carotene quantification of dried samples (fig. 4), it was observed that mangoseed extract pretreatment (pt2) did not prevent carotene degradation since total carotene content with this pretreatment was similar than that of control [6.99±0.33 mg of βcarotene•(100 g dry mass)-1, 7.06±0.321 mg of β-carotene•(100 g dry mass)-1, respectively, fig. 4]. sulphites pretreatment (pt1) caused only a slight increase in total carotenes concentration with respect to control, which accounted for 29.3% of carotenes retention. on the other hand, pt3 combined pretreatment (sulphites and mango-seed extract) caused a remarkable increase in total carotenes content of dried sample with a 40.2% carotenes retention. despite the fact that in this pretreatment carotenes retention was lower than 50%, total carotenes concentrations were 2.4 times higher than those obtained by chen et al. (2007) using a 1% sodium bisulphite treatment. in agreement with the previous results in this work, retention of carotenes was influenced synergistically by pretreatment with both the sulphites and mango-seed extract. furthermore, the synergistic effect of combined pretreatments on carotene retention was more pronounced than with the other compounds, considering that in this case the individual pretreatments alone had little or no effect. a very high pearson’s correlation of total carotenes with vitamin c (r = 0.9580, ital. j. food sci., vol. 32, 2020 906 p = 0.0420) showed total carotenes are directly related with vitamin c content in mango slice during drying process. figure 4. content of total carotenes [mg β-carotene•(100 g dry mass)-1] in samples of fresh mango and pretreated dried. mean ± standard deviation, n = 3. control = without pretreatment, pt1 = 0.5 % (w/v) sodium metabisulphite, pt2 = 1.44% (w/v) mango seed extract, pt3 = [1.44%/0.5% (w/v)] mango seed extract/sodium metabisulphite. superscripts a-d showed a significant difference (α = 0.05) according to the duncan test. these results also could be ascribed to diffusion of antioxidant compounds from mangoseed extract/sodium metabisulphite solution to mango slice during pretreatment. this diffusion of antioxidant compounds could have contributed to maintain the solid structure of mango slices and thus reduce damage of mango pulp cells during drying process. the mango cells integrity during drying possibly reduced antioxidants compounds diffusion, including carotenes, from cell inside to mango slice surface. adiletta et al. (2016) provided evidence related to effect of pretreatments on solid structure preservation of foods during drying process. this study consisted in evaluating the pretreatment effect based nacl 0.5% and trehalose 0.5% on eggplants drying. they observed by scanning electron microscopy (sem) an increase in dried samples porosity, preserving its solid structure, while samples without pretreatment showed collapse and shrinkage phenomena. it is suggested that sodium metabisulphite behaves analogously to nacl, promoting pores on the surface of mango slices during drying process, facilitating diffusion phenomena. also, the combined effect of sodium metabisulphite with mangoseed extract, potency the decrease of antioxidant compounds degradation in mango slices. 3.9. sulphites content in dried mango slices from pt1 and pt3 sulphites are utilized as antioxidant additives for preventing oxidation, kept flavour and color, inhibit the growth of microorganisms that promote food spoilage, and also are anti ital. j. food sci., vol. 32, 2020 907 browning agents for controlling enzymatic and non-enzymatic (maillard) reactions (lou et al., 2017). otherwise, sulphites are allergenic components that can cause allergic reactions in asthma patients and people with diminished sulphite oxidase activity (soubra et al., 2007). additionally, these compounds can cause skin reactions (vally et al., 2009) and dna damage (meng et al., 2005). hence, food safety organizations have considered an acceptable limit for sulphites in foods. sulphites concentrations in mango slices pretreated from pt1 and pt3 were 820.10±11.45 and 900.28±43.97 mg sulphites•(kg dry mass)-1, respectively. duncan's test showed a significant difference (α = 0.05) between samples pt1 and pt3. the latter showed approximately 9% more sulphites than pt1. result indicates that mango-seed extract promoted an increase in sulphites diffusion towards the mango slice during immersion. also, it is important to mention that sulphites concentrations in mango dried slices pt1 y pt3 are within the limit established by the codex alimentarius commission, created by the world health organization and the united nation’s food and agriculture organization (fao), which supports a general maximum of 1250 mg•(kg so3–2)-1 in dried fruits (liao et al., 2013). 3.10. product color the color parameters cie l*a*b* and hueº were determined for fresh and dried slices mango, table 3. table 3. color parameters cie l*a*b* and hue angle in dried slices and fresh mango. pretreatment l* a* b* hue angle control 65.61±3.00b 16.97±1.37c 66.74±4.33b 75.72±1.00a pt1 60.50±4.20ª,b 14.14±1.87b 58.81±5.01a 76.39±2.17a pt2 66.83±3.97b 12.33±1.53a,b 63.44±4.64a,b 78.96±1.59b pt3 71.38±3.39b 10.88±1.88a 69.35±2.37b 81.08±1.61b fresh 63.56±4.73b 11.45±1.95a 57.81±4.22a 78.84±1.43b control = mango dehydrated without pretreatment, pt1 = 0.5% (w/v) sodium metabisulfite, pt2 = 1.44% (w/v) seed extract mango, pt3 = seed extract mango/sodium metabisulfite [1.44%/0.5% (w/v)]. mean ± standard deviation, n = 3. the letters a-c showed a significant difference in columns (α = 0.05) with duncan test. the values for l*, a*, b* and hueº were found in ranges 60.50-71.38, 10.88-16.97, 57.8169.35 and 75.72-81.08, respectively. parameter l* indicates brightness degree of the sample on a scale from 0 (black) to 100 (white). the l* values allow affirm that pretreatment of mango slices with sulphites help to avoid the darkening of dried mango, as it expected. however, when sulphites are combined with extract of mango-seed, the effect is lost. this last behavior is attributed to the phenoloxidase enzymes present in seed mango extract that degrade the phenolic compounds to melanines. these compounds are responsible of darkening of mango slices during drying process. the temperature and sonication time used to obtain mango-seed extract, according to cheng et al. (2013) do not inactivate phenoloxidase enzymes. these enzymes are inactivated only at temperatures above 62°c and ultrasound frequencies above 20 khz. ital. j. food sci., vol. 32, 2020 908 parameter a* indicates sample redness degree and as sample color is redder, a* has a bigger positive magnitude. results obtained for a* show drying process induces an increase of redness in mango slices without mango-seed extract (control and pt1), table 3. parameter b* with positive values indicates yellowness degree of sample. dried sample with 0.5% sodium metabisulphite and seed mango extract (pt3) show the higher valor of b*, 69.35±2.37. this result is concordant with carotenes content and indicates a combined effect protective of sodium metabisulphite and phenol compounds present in pretreatment (pt3). hue angle is one color property, defined as the degree to which a stimulus can be related with red, orange, green, yellow, green, blue and violet. a hue° value of 90 represents a yellow tone. therefore, from the data in table 3 it can be affirm that samples pretreated with 0.5% sodium metabisulphite (pt2 and pt3) have a yellower tone than control and pt1 samples. 4. conclusions midilli’s model fitted very well to experimental data of mango slices dried without pretreatment and with the three pretreatments (pt1, pt2 and pt3) used in the present study. comparative analysis of midilli’s constants, k, a and b using duncan’s means test showed pretreatments did not influence drying process of mango slices. also, the results of antioxidant compounds quantification showed (1.44%/0.5%) mango-seed extract/sodium metabisulphite used as a pretreatment (pt3) has an important role on retention of vitamin c and carotenes in dried mango slices. the free phenols content was quadrupled compared to dried slices without pretreatment (control) and nearly were doubled compared to fresh mango. mango slices pretreated with mango-seed extract were strongly enriched with gallic acid, caffeic acid, ferulic acid and p-coumaric acid, also with, methyl gallate to a lesser extent in relation to mango slices that did not receive a pretreatment (control). furthermore, sulphites content of this dried product is within limit established by the codex alimentarius of fao. finally, this is the first study reporting combined use of mango-seed extract with sulphites as a pretreatment for drying mango pulp. mango seed, which is generally an agro-industrial residue, used to obtain extracts rich in phenolic compounds and using these as a pretreatment for drying mango pulp and other foods could become commercially feasible in the proximate future. acknowledgments the authors thank carol ann hayenga for her english assistance in the preparation of this manuscript and the reviewers for supplying suggestions and recommendations to accomplish standards of journal. analleli jiménez-durán thanks the consejo nacional de ciencia y tecnología (conacyt) from the mexican government for its financial support 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in press. doi: doi.org/10.1016/j.foodchem.2019.12547 paper received february 1, 2020 accepted july 4, 2020 ijfs#1368_bozza ital. j. food sci., vol. 31, 2019 311 paper turkish-coffee enriched with rose: a promising combination e. karabudak1, e. aksoydan2, d. ağagündüz*1 and m. ergül3 1gazi university, faculty of health sciences, department of nutrition and dietetics, emniyet mahallesi, muammer yaşar bostancı caddesi 16, 06500 beşevler, ankara, turkey 2başkent university, faculty of health sciences, department of nutrition and dietetics, bağlıca kampüsü fatih sultan mahallesi, 06790 etimesgut, ankara, turkey 3san francisco state university, college of business, hospitality and tourism management department, usa *corresponding author: tel.: +903122162601; fax: +903122162636 e-mail address: duygu_turkozu@ymail.com abstract the purpose of this study was to develop a new & healthy version of turkish-coffee enriched with rose. conventionally roasted coffee arabica l. beans for turkish-coffee & dried-rosa damascene mill.[7/0,7/0.5,7/1.5,7/2g(w/w)] was grinded. total-phenolic contents (tpc), total-antioxidant (tas) & oxidant-status (tos) were measured and oxidatif stress index (osi) was calculated. consumer panel testing was done. tpc of the coffee samples with 1.5 & 2g rose was different according to plain coffee (p<0.05). tas value (mmol/l) of plain-coffee was 2.2±0.11 while the values of the coffees including 0.5g,1.5g,2g rose were 2.4±0.09,2.4±0.05,2.7±0.02, respectively. tos value (µmol/l) of plain-coffee was 17.6±0.24, while the values of the coffees including 0.5 g, 1.5 g and 2 g rose were 13.0±1.00, 9.4±1.30, 9.4±0.31, respectively (p<0.05). osis of coffee samples including 0, 0.5, 1.5, 2 g rose were found to be 7.7, 5.2, 3.6, 2.7 respectively (p<0.05). the coffees including 1.5 & 2 g rose had the highest sensory-scores. turkish-coffee including rose will strengthen already existing phenolic & antioxidant features of coffee, and thus contribute the improvement of health & taste. keywords: antioxidants, coffee, oxidants, polyphenols, rose, turkish coffee ital. j. food sci., vol. 31, 2019 312 1. introduction recent studies in nutrition & health have focused on more detailed research on the effects of foods on health (kri̇s-etherton et al., 2002; kri̇s-etherton et al., 2004; mrc, 2017). these studies are mostly conducted on the components of foods, processing techniques and alternative development (bier et al., 2015; poti̇ et al., 2015). the food industry has embarked on a consumer-oriented mission with new product development or product modifications matching with scientific nutrition and health recommendations (bier et al., 2015). in particular, many herbal foods have become important raw materials for many fields due to their bioactive compounds, especially phenolic compounds (el 2008). the bioactive compounds found and defined in foods vary according to their numbers, chemical structures and functions (fi̇lho et al., 2007; kri̇s-etherton et al., 2002; kri̇s-etherton et al., 2004). coffee is a globally consumed beverage and recent studies showed that consuming coffee in acceptable amounts have potential health benefits (li̇ang et al., 2016). it’s known to be a natural antioxidant and recent studies comment on antioxidant effects of coffee along with other benefits and linking them to prevent various common diseases (agui̇ar et al., 2016). turkish coffee, a traditional delicacy for the turks with its unique flavor and aroma is becoming a popular beverage globally (özgür, 2012). coffee arabica l., which is the most used bean type for turkish coffee, is widely used in pharmacology, homeopathy, therapeutics and gastronomy due to its health benefits (capek et al., 2014). rosa damascena is a herb with economical value. turkey is a leading manufacturer of rose and rose products especially around the city of isparta (anon 2003). in addition to its economical value, incorporation of “rosa damascena” into pharmacology, homeopathy, therapeutics and gastronomy demonstrates a broad range of its uses and health benefits (boskabady et al., 2011; mahboubi̇, 2016). researchers indicate that rose products are also natural antioxidants, and thus use of rose as nutraceutical foods is useful for both health aspects and adding aesthetic value and taste to make it appealing to consumers (mlcek and rop 2011; kovatcheva-apostolova et al., 2008). additionally, there are studies regarding its anelgesic, antimicrobial, antioxidant, anti-inflammatory, antidiabetic and antidepressant features fields (boskabady et al., 2011; mahboubi̇, 2016). it is considered that antioxidant and antimicrobial effects of rosa damascena originate from its phenolic content and essential fat composition (özkan et al., 2004). citronellol and geraniol are the two main compounds found in the essential oil of rosa damascena and responsible for its pharmacological activities (mahboubi̇, 2016). moreover, quarcetin, kaempferol and their glycosides are the flavonol glycosides, which are responsible for the high antioxidant activity of rosa damascena flowers, petals and extract (boskabady et al., 2011). however, it is reported that further research is required on the use of rosa damascena plant in preclinical and clinical investigations (mahboubi̇, 2016). the purpose of this study is to develop a new and healthy version turkish coffee with rose through preserving its traditional and nutritional value while investigating consumers’ liking and preferences. besides contributing the efforts for improvement of health and strengthening already existing phenolic content and antioxidant features of coffee which is a widely consumed and traditional beverage in turkey, this study will ensure that a turkish-origin healthy beverage will be introduced as an innovative design to the world. ital. j. food sci., vol. 31, 2019 313 2. material and methods this study was conducted on two stages. the first stage consists of the provision of sample, preparing the samples for chemical analysis and the measurements of phenolic content and antioxidant capacity. the second stage consists of the preparation of turkish coffees and tasting the coffee samples prepared by using coffee and spent rose, which are mixed in certain amounts [7/0, 7/0.5, 7/1.5, 7/2 g (w/w)]. 2.1. preparation of rose and coffee samples fresh (unfaded) petals of unprocessed rosa damascena, which were harvested in isparta, were dried at room temperature. before having been ground, rose petals and coffee beans were stored within closed dark glass jars at room temperature until analysis and/or tasting panel day so that they preserved their compound and freshness. the most consumed coffee in the world (85-90%), coffee arabica l. beans were selected for the study. raw coffee beans were roasted in the coffee shop for 4 minutes at 220 0c. the samples were prepared according to the traditional turkish coffee standard published by the turkish coffee culture and research association (özgür, 2012). coffee beans and rose petals were fine ground with a grinding machine (krupps f203 electric grinder®) on the analysis day. fine ground 7 g coffee was prepared with 70 ml water (traditional coffee cup measure). to ensure the volume of coffee cup, the samples of turkish coffee cups were also collected from coffee manufacturers and coffee shops, and their average service volumes were calculated. instead of steel/copper coffee pot, which is used for the preparation of traditional turkish coffee, an electricity coffee pot (arçelik k® 3300) was used to prepare multiple samples and ensure standardization. coffee samples were prepared in avarege 1.15 minutes. fine ground coffee and rose petals were taken in certain amounts [7/0, 7/0.5, 7/1.5, 7/2 g (w/w)] to prepare four different samples as described above. samples were taken from only the drinkable part of the coffee to eppendorf tubes one minute after the rose petal coffee was ready. three samples of each experimental coffee were prepared and all samples were analyzed for three times. 2.2. measurement of total phenolic content (tpc) the amount of phenolic compounds in the spent rose (rosa damascene mill.), turkish coffee beans and all coffee samples with spent roses were determined by folin-ciocalteu colorimetric method (si̇ngleton et al., 1999). for sample extraction, spent rose and coffee beans were dissolved and homogenized in 80% ethanol and heated for 5 minutes. extracts filtered with whatman filter paper (number 4). then, 80% ethanol was added to residue after filtration and heated for 10 minutes and filtered again up to ensure extraction. extracts of dry coffee-rose samples and drinkable part of the coffee samples with rose in eppendorf tubes dilueted and analysed using a folin-ciocalteu reagent. tpcs were expressed as mg/l gallic acid equivalents (gae) extract. 2.3. measurement of total antioxidant status (tas) tas levels were measured using commercially available kits (relassay, turkey). the novel automated method is based on the bleaching of characteristic color of a more stable abts (2,2′ azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) radical cation by antioxidants. the assay has excellent precision values, which are lower than 3%. the results were expressed as mmol trolox equivalent/l (erel, 2004). ital. j. food sci., vol. 31, 2019 314 2.4. measurement of total oxidant status (tos) tos levels were measured using commercially available kits (relassay, turkey). in the new method, oxidants present in the sample oxidized the ferrous ion-o-dianisidine complex to ferric ion. the oxidation reaction was enhanced by glycerol molecules abundantly present in the reaction medium. the ferric ion produced a colored complex with xylenol orange in an acidic medium. the color intensity, which could be measured spectrophotometrically, was related to the total amount of oxidant molecules present in the sample. the assay was calibrated with hydrogen peroxide and the results were expressed in terms of micromolar hydrogen peroxide equivalent per liter (μmol h2o2 equivalent/l) (yumru et al., 2009). 2.5. calculation of the oxidative stress index (osi) the ratio of tos to tas was accepted as the oxidative stress index (osi). for calculation, the resulting unit of tas was converted to μmol/l, and the osi value was calculated according to the following formula (yumru et al., 2009): osi (arbitrary unit) =tos (μmol h2o2 equivalent/l)/tac (μmol trolox equivalent/l) 2.6. sensory evaluation the samples prepared in the same order as the coffees prepared for chemical measurements. sugar was not added to coffee samples. fine ground coffee and rose petals were taken in certain amounts [7/0, 7/0.5, 7/1.5, 7/2 g (w/w)] to prepare traditional turkish coffee samples as described above. panelists were chosen among the people (20-64 years) who consume turkish coffee regularly, accepted participating in the study voluntarily, do not smoke and do not have any mouth and dental (tooth decay etc.) and chronic disease that may affect their palate. each group is expected to consist of 10-15 individuals in such studies considering the fact that there is not any data in the literature with regard to the appropriate number of panelists and qualitative research aspect of the study (çokluk et al., 2011). therefore, 15 panelists participated in the study. panelists were chosen in “food preparation laboratory” and they were seated in a way that they could not communicate with each other during the tasting stage. each panelist was also given a number randomly. the panelists were informed for approximately fifteen minutes about the procedures of evaluation and the points to be taken into account before coffee presentation. the coffees prepared by the researchers with rose petals in four different amounts were presented in turkish coffee cups, which had the same volume and design (70 ml). in order that panelists could differentiate the tastes of coffee samples, they were presented every ten minutes and participants were asked to eat a fat-free salt cracker and gargle with water at the intervals. after each coffee taste, the panelists wrote down their rating points form for each coffee sample. the evaluation was made from the lowest point (0 points) towards the highest point (10 points). panelist evaluation scores were assessed separately for each sample under the following criteria: • smell (the harmony of coffee and rose smell, dominant one etc.) • aroma (harmony and range of aroma component) • taste (the taste and aroma combination experienced when the coffee sample was tasted) ital. j. food sci., vol. 31, 2019 315 • aftertaste (the duration of feeling the taste at the back of palate after swallowing the sample) • acceptability • preference (consumption preference) • general impression (panelist’s own remarks) this study was approved by baskent university institutional review board (project no: ka17/83) and supported by baskent university research fund. clear explanations were provided for the individuals with regard to the purpose of the study, after which written informed consent was obtained from all participants in accordance with the declaration of helsinki (world medical association). 2.7. statistical analysis all statistical analyses were performed using spss (the statistical package for social sciences) version 20.0 (spss inc., chicago, il, usa). percentage, mean ± standard deviation (sd) values were taken for the evaluation of the data. in addition, a kolmogorov-smirnov test was used to determine whether the panelist evaluation ratings had a normal distribution. panelists' evaluation ratings for the coffee samples were shown as mean ± sd and median (minimum-maximum). kruskal wallis and mann-whitney-u tests were used to compare the means of tpc, tas, tos and osis of coffee samples prepared with rose in four different amounts. differences among means with p < 0.05 were accepted as representing statistically significant differences. 3. results and discussion 3.1. evaluation of the total phenolic content and antioxidant/oxidant status table 1 shows the tpc (mg/l gae), tas (mmol/l), tos (μmol/l) and osis of coffee samples prepared with raw and spent rose and coffee samples in different amounts. accordingly, the tpc of raw dry coffee and rose was 611.3±1.19 mg/l gae and 605.2±0.64 mg/l gae respectively. there was no statistically difference between the tpc of plain coffee sample (445.7±6.66 mg/l gae) and that of the samples prepared with 0.5g rose (458.4±11.75) (p>0.05). however, tpc of the coffee samples prepared with 1.5 and 2g rose (463.1±6.42 and 479.3±12.56 mg/l gae) was statistically different compared to that of plain coffee sample. in addition, tpc of the coffee samples with 1.5 and 2g rose was not different from that of the sample with 0.5g rose (p>0.05). tpc of the coffee samples with 1.5 and 2 g rose was similar to each other (p>0.05). the evaluation of tas of the samples showed that the tas of raw/dry coffee and rose samples was 3.1±0.00 mmol/l and 2.3±0.02 µmol/l respectively. the evaluation of tas of the coffees prepared with rose in different amounts and without any rose showed that the tas value of plain coffee was 2.2±0.011 mmol/l, while the values of the coffees including 0.5g, 1.5g and 2g rose were 2.4±0.09, 2.4±0.05 and 2.7±0.02 mmol/l respectively, and thus all tas values of coffee samples with rose were different from each other (p<0.05). the evaluation of tos of the samples showed that the tos values of raw/dry coffee and the spent rose were 9.5±0.02 µmol/l and 16.8±0.06 µmol/l respectively (table 1). in addition, the evaluation of tos of coffee samples showed that the tos value of plain coffee was 17.6±0.24, while the values of the coffees including 0.5 g, 1.5g and 2g rose were 13.0±1.00 µmol/l, 9.4±1.30 µmol/l and 7.5±0.31 µmol/l respectively. the difference between the tos values of all coffee samples with rose was important (p<0.05). ital. j. food sci., vol. 31, 2019 316 the evaluation of osis of all samples showed that the osis of raw/dry coffee and rose samples were 3.0 and 7.2 respectively. osis of coffee samples including 0, 0.5, 1.5, 2 g rose were found to be 7.7, 5.2, 3.6 and 2.7 respectively (p<0.05) (table 1). table 1. the total phenolic content (gae/g), total antioxidant status (mmol/l), total oxidant status (μmol/l) and oxidative stress indexes of coffee samples including rose in different amounts*. sample tpc (mg/l gae) tas (mmol/l) tos (µmol/l) osi powder and raw dry coffee 611.3±1.19 3.1±0.00 9.5±0.02 3.0 spent rose 605.2±0.64 2.3±0.02 16.8±0.06 7.2 boiled (per turkish coffee cup) (w/w) 7 g coffee 445.7±6.66 a 2.2±0.11a 17.6±0.24a 7.7a 7 g coffee + 0.5 g rose 458.4±11.75a 2.4±0.09a 13.0±1.00b 5.2b 7 g coffee + 1.5 g rose 463.1±6.42 b 2.6±0.05b 9.4±1.30c 3.6 c 7 g coffee + 2 g rose 479.3±12.56 b 2.7±0.02c 7.5±0.31d 2.7d *tpc: total phenolic content, tas: total antioxidant status, tos: total oxidant status, osi: oxidative stress index. a-dvalues are the mean ± sd of three replicates. values with different letters in the same column are statistically different (p<0.05). 3.2. sensory evaluation in this experiment, plain and coffee with rose (with different concentrations) were tasted in the panel and 66.7% of the panelists were female, while 33.3% of them were male. the mean age of the panelists was 43.8 ±7.5 years. 46.7% of the panelists were drinking turkish coffee every day, 46.7% several times a week and 6.6% once a week. 66.7% of the participants were drinking sugar-free turkish coffee, 26.7% of them preferred coffee with little sugar and 6.6% consumed coffee with sugar. table 2 shows the evaluation scores (smell, aroma, taste, aftertaste, acceptability, preference and general impression) of the panelists about the coffee samples with rose in different amounts. the sensory evaluation scores given to coffee samples with rose in different amounts show that panelists gave more points to the coffee samples containing 1.5 g and 2 g rose than plain coffee sample (6.2±2.2 vs. 6.1±2.3, respectively) (table 2). in addition to this, the sensory evaluation scores of the coffee samples containing 0.5g rose are similar to or less than that of plain coffee (table 2). when general impression scores of the coffee samples as a result of the panel test and total antioxidant and total oxidant status of coffee samples are evaluated, the interpretation of consumer preference and both tas and tos of coffee samples showed that the coffee sample containing 2 g rose both had relatively the highest tas and low tos and got the highest score from the panelists. additionally, the coffee sample containing 1.5 g rose content had relatively higher tas and lower tos than plain coffee and received higher general impression score from the panelists. ital. j. food sci., vol. 31, 2019 317 table 2. panelist evaluation scores of coffee samples with rose in different concentrations*. sample smell aroma taste aftertaste acceptability preference general impression 7 g coffee 5.7±2.8 5 (1-9) 5.8±1.7 6 (3-8) 6.3±2.4 7 (3-9) 6.8±1.6 7 (4-9) 6.1±2.3 6 (3-9) 5.8±2.3 6 (3-9) 5.8±1.77 6(4-9) 7 g coffee+0.5 g rose 6.6±1.5 6 (4-8) 5.7±1.8 6 (3-8) 6.4±1.7 7 (4-8) 6.8±0.7 7 (6-8) 5.3±1.34 6 (3-7) 5.6±1.9 6 (3-9) 5.4±2.09 6(3-9) 7 g coffee+1.5 g rose 7.3±1.4 8 (5-9) 7.0±1.7 8 (4-9) 6.5±1.8 6(4-9) 7.9±0.7 8(7-9) 6.4±1.7 6 (4-9) 6.1±1.7 6 (4-9) 6.2±2.2 6(4-9) 7 g coffee+2 g rose 7.0±1.6 8 (5-9) 6.3±1.9 6 (3-9) 6.4±1.9 6 (4-9) 7.4±1.8 8 (5-10) 6.0±2.3 5 (3-9) 5.7±2.2 5 (3-8) 6.1±2.3 5(3-9) *scores were shown as mean ± sd and median (minimum-maximum points). 4. discussion coffee is consumed worldwide and one of the most popular beverages. a number of epidemiologic and clinic studies proved that coffee consumption may prevent several chronic and degenerative diseases, such as cancer, cardiovascular disorders, diabetes, and parkinson's disease (ludwig et al., 2014). within this scope, this study was conducted to strenghten potential health effects and antioxidant feature of turkish coffee and improve health condition by adding rose petals to the traditional beverage turkish coffee which is widely consumed in turkey. it was found out in the study that tpc of the raw and dry coffee beans of coffee arabica l roasted for 4 minutes at 2200 c was 611.3±1.19 mg/l gae, while the value decreased to 451.4±1.20 mg/l gae after the coffee was boiled. moreover, tas value decreased while tos values and osis increased. an important family of phenolic compounds, chlorogenic acids are green coffee compounds which are formed by the esterification of caffeic, ferulic, and p-coumaric trans-cinnamic acids with (−)-quinic acid and associated with hepatoprotective, hypoglycemic and antiviral activities because of their antioxidant effects (farah and donangelo, 2006). chlorogenic acids (cga) and related compounds (caffeoylquinic acids, dicaffeoylquinic acids, feruloylquinic acids, p-coumaroylquinic acids and mixed diesters of caffeic and ferulic acids with quinic acid and their isomers) constitutes the most important phenolic fraction of green coffee bean (clifford, 2000) and its content reaches up to 14% in dry substance (farah and donangelo, 2006). the chlorogenic acid content of a 200 ml (7-oz) cup of coffee has been reported to range from 70-350 mg (cli̇fford, 1999). during the roasting, cga can turn into isomerized, hydrolyzed or degraded lower molecular weighted materials. roasted beans of coffee arabica l. contain 1.9-2.5 g/100g cga in average, while the roasted beans of coffee canephora contain 3.3-3.8 g/100 g in average and these average values change depending on the type of coffee beans (farah, 2012). especially roasting coffee beans at high temperatures (230°c) turns a part of cga into quinolactons and melanoidins, and thus cga content decreases after roasting (farah 2006; farah, 2012). a study showed that roasting coffee beans causes 23% cga loss because of degredation and constitutes condensed form (42-62 mmol/100 g) and esterlinked melanoiding forms (1.1-1.6 mmol/100 g) (coelho et al., 2014). it was considered after this study that the decrease in the tpc after the roasted dry coffee was boiled in water at high temperature was the result of the transformation and condensation of the free forms of cga into quinolactons and melanoidins through various mechanisms due to degredation. moreover, the decrease in tpc also caused decrease in total antioxidant capacity and increase in oxidant capacity and osi value. ital. j. food sci., vol. 31, 2019 318 the determination of tpc, tas, tos and osi values of a coffee prepared in turkish coffee preparation method (7 g/70 ml) for the first time was one of the important findings of this study. coffees are prepared in different methods such as drip or filter, plunger or cafetière, espresso, cappuccino, moka-napoletana, percolator, soluble or instant and flavored (karabudak et al., 2015). preparation method of turkish coffee is different from other coffee types (özgür, 2012). for the traditional preparation of turkish coffee, fine ground powder coffee is added to cold water and the coffee is boiled. this produces a strong coffee with a layer of foam on the surface and sediment (not meant for drinking) that settles on the bottom of the cup. ludwi̇g et al. (2012) indicated that brewing time and preparation method affected the antioxidant amount of coffee (ludwi̇g et al., 2012). it is generally reported that the total antioxidant capacity of 7-10 g of coffee is 150-300 mg/g (yashi̇n et al., 2013). in a comprehensive evaluation made by using different methods, ferric reducing antioxidant power (frap) values of espresso, instant coffee, decaffeinated espresso coffee were 129.4, 108.6, 93.0 mol fe+2/l respectively (pellegrini et al., 2003). additionally, it was found out that total radical trapping antioxidant power (trap) of espresso was 66 mol trolox/l, that of instant coffee 52.4 trolox/l and that of decaffeinated espresso 45.8 mol trolox/l. trolox equivalent antioxidant capacity (teac) values for espresso, instant coffee and decaffeinated espresso were found to be 36.5, 32.5 and 27.0 mol trolox/l respectively (pellegri̇ni̇ et al., 2003). therefore, the presence and amount of bioactive compounds have important roles in health effects depending on the preparation method of coffee (peters, 1991). however, the unstandardized methods and amounts (water and coffee amounts) and different unit of measurement (w/w, w/v, w/dose, and w/cup) make it difficult to compare the results of studies with other studies and the coffees prepared with different methods (capri̇oli̇ et al., 2015). this is one of the most important limitations in the literature. rosa damascena mill's antimicrobial, anti-inflammatory, anticancer effects and protective effects against neurological, cardiovascular and liver diseases were proved in a number of in vitro and animal studies (nayebi̇ et al., 2017). the flowers, petals and hips of rosa damascena contain terpenes, glycosides, flavonoids and anthocyanins. in addition, this plant contains carboxylic acids, myrcene, vitamin c, kaempferol, quarcetin, tannin and essential oils and organic acids (boskabady et al., 2011). a study showed that three flavonol glycosides, specifically quercetin-3-o-glucoside, kaempferol-3-o rhamnoside and kaempferol-3-o-arabinoside, contained in rosa damascena, are responsible for antioxidant activity. in our study, the tpc and tas of spent rose leaf powder were found to be quite high (605.2±0.64 mg/l gae and 2.3±0.02 mmol/l, respectively). in another study conducted by using rosa damascena extracts grown in the same region of turkey, tpc of fresh leaf extract of rosa damascena was 276.0±2.93 mg/l gae, while that of spent flower was found to be 248.9±2.96 mg/l gae. antiradical activities of fresh and spent leaf were determined, through α-diphenylα-picrylhydrazyl (dpph), to be %74.51±1.65 and %75.94±1.72 at 100 ppm respectively. additionally, determined by the method depending on phosphomolybdenum complex formation, the antioxidant activity of fresh leaf extract (372.2±0.96 mg/g) was higher than that of spent leaf extract 351.3±0.84 mg/g (özkan et al., 2004). the reason of the difference between the results of our study and above mentioned study may be a number of factors such as seasons and the difference in analysis techniques, land and improvement methods although rosa damascenas of the same region were used. the aim of this study was to increase existing tpc and tas value of turkish coffee by adding spent rose flower grown in isparta region of turkey. as a result, tpc and tas values increased and tos and total osi values decreased after 0.5 g, 1.5 g and 2.0 g rose aroma were added to plain coffee. the coffee sample containing 2g rose (the highest amount) had the highest tpc and tas values and lowest oxidant content and osi values. ital. j. food sci., vol. 31, 2019 319 some studies suggest that rosa damascena plant may be used as a medical source in the prevention and treatment of many diseases caused by free radicals (boskabady et al., 2011). it is considered that it would be beneficial to carry out preclinical and clinical evaluations of above mentioned rose coffee samples in future studies. oxidative stress is associated with the excessive increase in oxidant levels and/or antioxidant capacity. the atoms or molecules, which contain one or more unpaired electron(s), are called oxidants or free radicals in biological system. oxidants deteriorate cell structure and extracellular matrix and cause damages in genetic structure by distorting dnas. therefore, free radicals have a role in the pathogenesis of various diseases such as atherosclerosis, neurodegenerative diseases, cancer, allergies, diabetes and cataract (yumru et al., 2009). this study evaluated the osis of the coffee samples containing rose in different amounts and revealed that osi values of coffee samples decreased as the amount of rose increased. in another study, rats were given 50, 75, 100 and 200 mg/kg/day of ethanol extract produced from rosa damascena petals for 10 days orally and it was found out that all doses of rosa damascena prevented lipid peroxidation and the highest antioxidant activity was observed after the consumption of 200 mg/kg (shahri̇ari̇ et al., 2007). therefore, coffee with rose consumption may be considered to support the antioxidant defense system in order to prevent free radical formation and prevent biological damage. coffee is one of the most widely consumed beverages throughout the world because of its unique sensory properties (kreuml et al., 2013). so, aromatic components are very important in coffee beverages, because they are the principal constituents of sensory experience for coffee consumers (jaimes et al., 2015). the aroma of the coffee comes from caffeine and trigonelline alkoloid, chlorogenic acid, kahweol and cafestol and melanoidin, which is a maillard reaction products (ludwig et al., 2014). one of the way for strenghten aroma profile of coffee is inserting a herb with rich source of aroma such as rose. aroma compounds of rose vary according to parts of herb and its recovering periods (feng et al., 2008; zhao et al., 2016). at the full opening stage of rose, β-citronellol citronellol acetate, phenethyl alcohol, geranyl acetate, geraniol, phenethyl acetate, nerol, nhexyl acetate and α-myrcene, and alcohols are the major constituents of aroma (zhao et al., 2016). consumer appreciation/evaluation plays an important role in the innovation studies of food and various drinks. therefore, this study includes panelist evaluation tests conducted on the coffee samples containing different amounts of rose aroma. in these evaluations, panelists ranked coffees in terms of their smell, aroma, taste, acceptability, preference and general impression and the coffee samples containing 1.5 g and 2 g rose aroma received relatively highest scores generally. these coffee samples had the highest phenolic content and antioxidant status, but lowest oxidant status and osi values. coffee consumers prefer products, not only good flavour and taste, but also good for health (jaimes et al., 2015). so, through this study, it was to develop a new and relatively healthy version turkish coffee with rose through preserving its traditional and nutritional value while investigating consumers’ liking and preferences. 5. conclusions it is considered to strenghten existing phenolic content and antioxidant features of turkish coffee especially with turkish coffees containing 1.5 and 2 g rose as an innovative design. moreover, these coffee samples are considered as the antioxidant & healthy products enriched with a different aroma and appreciated by consumers. nonetheless, it is necessary to conduct a further study for more detailed investigation of the consumption ital. j. food sci., vol. 31, 2019 320 doses, possible potential short and long term health effects and risks of these promising combinations. as far as is known, this is the first study that a promising combination of coffee and rosa damascena is shown as an innovative design. this study has a number of limitations. first of all, total antioxidant and oxidant profile were focused in this study. so, each phenolic composition of rose and coffee samples related to the antioxidant and oxidant activity were not determined. nevertheless, it is considered that this study will light the way for other studies. furthermore, this study focused on turkish coffee, which was a particular kind of brewed coffee. so, the results could not be generalized to the effects of all coffee types. finally, when comparing to other studies, our number of panelists might be relatively less or not because of rigid inclusion criteria. further sensory evaluation may conduct with a larger sample size, including different age groups and populations. it is believed that taking into consideration these situations would be useful in future studies. the author(s) received no financial support for the research, authorship, and/or publication of this article. references aguiar j., estevinho b.n. and santos l. 2016. microencapsulation of natural antioxidants for food application-the specific case of coffee antioxidants-a review. trends food sci. 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reagent. methods enzymol 299:152-178. yashin a., yashin y., wang y.g. and nemzer b. 2013. antioxidant and antiradical activity of coffee. antioxidants (basels) 2(4):230-245. ital. j. food sci., vol. 31, 2019 322 yumru m., savas h.a., kalenderoglu a., bulut m., celik h. and erel o. 2009. oxidative imbalance in bipolar disorder subtypes:a comparative study. prog. neuropsychopharmacol. biol. psychiatry 330(6):1070-1074. zhao c.y., xue j., cai x.d., guo j., li b. and wu s. 2016. assessment of the key aroma compounds in rose-based products. j. food drug anal. 24(3):471-476. paper received august 28, 2018 accepted november 15, 2018 ital. j. food sci., vol. 30, 2018 497 paper comparative 1dand 2d-electrophoretic protein profiles of ancestral and modern buckwheat seeds grown in the italian alpine region j. capraro*1, c. magni1, a. giorgi2, m. duranti1 and a. scarafoni1 1department of food, environmental and nutritional sciences (defens), faculty of food and agriculture, università degli studi di milano, via celoria 2, milan, italy 2department of agricultural and environmental sciences, unimont, via morino 8, 25048 edolo, brescia, italy *corresponding author: tel.: +39 0250316823; fax: +39 0250316800 e-mail address: jessica.capraro@unimi.it abstract buckwheat is an old crop whose seeds are under-utilized. the protein composition of these seeds, however, makes them suitable as much needed ingredients for the production of gluten-free products. several buckwheat species and local cultivars are known worldwide. in this work, 1d and 2d electrophoresis were used to characterize and compare the seed protein profiles of two buckwheat species (fagopyrum esculentum and fagopyrum tataricum). the two analyzed cultivars of f. esculentum represent authentic landraces of an italian alpine valley, named valtellina. the protein profiles of f. tataricum and the two f. esculentum cultivars did not show major differences. however, narrow but significant differences were present between these two landraces, allowing their discrimination at protein level. this work represents a molecular-based approach to the designation of origin and authenticity of local buckwheat varieties and their tracing in flours for human food. keywords: buckwheat, electrophoresis, fagopyrum spp., historical landraces, proteome, traditional cultivation ital. j. food sci., vol. 30, 2018 498 1. introduction buckwheat is a pseudo-cereal seed of the class dicotyledoneae, genus fagopyrum, and smartweed family. it originated from and was domesticated in eastern himalaya regions and can be cultivated in flat and mountainous regions, as long as the climate is cold. buckwheat came to europe in the late middle age (ohnishi, 1993) and, early traces of its cultivation in italy were found in property documents of a family in teglio, valtellina, dating back to the middle of the sixteenth century (ferranti et al., 2002). after a long history of growth and food use and a remarkable decline in the last decades, there has been a renewed interest in the crop. among the reasons for this recent reappraisal are the growing worldwide need for sustainable nutrient sources (duranti and scarafoni, 2015) and the claimed health benefits of various buckwheat components (li et al., 2008; izydorczyk et al., 2014; zhou et al., 2015), as it occurs for many other seeds (scarafoni et al., 2007). buckwheat seeds (fagopyrum spp.) display a high fiber content, ranging from 12 to 18% with very low lipid levels (about 3%) and relatively low carbohydrate levels, around 50% (eggum et al., 1980). the presence of biologically-relevant compounds, such as flavonoids, flavones, phytosterols, thiamin-binding proteins (li and zhang, 2001) and rutin, with antioxidant properties (kreft et al., 2002), all add nutritional value to this seed. the protein content is similar to or greater than that of wheat, from 12% d.w. upward. the peculiar composition of the protein fraction makes these seeds and their proteins suitable as main ingredients for many food applications, including foods for coeliac patients. phylogenetic relationships and polymorphisms of buckwheat have been studied at both genetic and protein levels (li et al., 2008; ohnishi and matsuoka, 1996; yasui and ohnishi, 1998; du et al., 2004; zeller et al., 2004; rout and chrungoo; 2007). wide margins for implementing tailored and finalized applications exist, because not all molecular and compositional features of these seeds have been thoroughly investigated for their optimal exploitation. modern analytical approaches allow the use of molecular-based strategies for the comparison, selection and improvement of crops and these activities are becoming crucial for mankind in the near future. electrophoretic techniques applied to the protein fraction may represent a complementary and effective approach to the genetic/genomic analysis of plants (gorinstein et al., 2005; capraro et al., 2008). in this work, we applied a fast and reliable methodology based on electrophoretic analyses of seed proteins to identify candidate quality marker to be used to test and guarantee the designation of origin and authenticity of local buckwheat varieties. we focused our attention on two remnant cultivars of fagopyrum esculentum, l.; one which is locally named ‘nustran’ and the other called ‘furest’ or ‘francese’ or else ‘curunin’ which has recently been identified as an authentic valtellina landrace by genetic analyses (barcaccia et al., 2016). in this work we will refer to this latter cultivar as ‘furest’. it is still cultivated in teglio (valtellina), an italian central alpine valley village. the first landrace, ‘nustran’, is an original teglio ecotype, whereas ‘furest’ has been cultivated in teglio only since the beginning of the twentieth century. in appearance, the two cultivar seeds show only slight morphological differences (barcaccia et al., 2016). in addition, a modern variety of fagopyrum tataricum has also been included in our comparative work. ital. j. food sci., vol. 30, 2018 499 2. materials and methods 2.1. materials buckwheat seeds were kindly supplied by raetia biodiversità alpine, teglio, valtellina, italy. fagopyrum esculentum, l. was of the varieties nustran and furest; fagopyrum tataricumwas of variety n’zibaria. 2.2. methods 2.2.1 protein extraction dry seed kernels were manually ground to a meal in a mortar. the protein fractions of the resulting flours were extracted under stirring at room temperature for two hours following two procedures: a non-denaturing one, consisting of a solution containing 50 mmtris-hcl and 0.5 m nacl at ph 7.5, and a denaturing one (redry solution), consisting of a solution containing 8 m urea, 2 m thiourea, 20 mg/ml3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate(chaps) and 65 mm 1,4-dithiothreitol (dtt) at ph 8.5. the ratios, 1/20 (w/v) in non-denaturing conditions and 1/40 (w/v) in the denaturing buffer, were used. the slurries were centrifuged at 12,000g at room temperature for 20 min, and the extracted proteins were conserved at -20°c until used. two experimental replicas for each condition were carried out. 2.2.2 electrophoretic techniques isoelectric focusing (ief) was performed on 7 cm ph 3–10 linear ipg strips (amersham biosciences) as described by capraro et al. (2008). sds-page for either 1d and 2d analyses was carried out according to laemmli (1970) on 12% polyacrylamide gel using a mini-protean ii cell (bio-rad). the gels were stained with coomassie blue. protein extracts were analyzed in triplicate. 3. results and discussion buckwheat seed proteins include either albumins and globulins, typical of the legume grains, and prolamins at high and low molecular weight (hmw and lmw) (naiłęcz et al., 2009), typical of cereals seeds. for this reason, the performances of two extraction protocols were assessed. the extraction of proteins under non-denaturing conditions using a saline alkaline buffer (fig. 1, lanes marked with nd, namely tnd, nnd and fnd) resulted in the prevalent solubilization of buckwheat globulins, as shown by 1d sdspage analysis. the presence of two main bands at about 40 and 25 kda (la and lb, respectively in fig. 1) is typical of the reduced 13s globulin family of most species (casey et al., 1985) and the greater mrminor band components around 60 kda likely correspond to the 7s globulin subunits (v in fig. 1). under these conditions, a lower number of high mr bands with respect to the samples extracted under denaturing conditions was visible. according to naiłęcz et al. (2009), these new bands likely correspond to the prolamin family, which are insoluble, unless denatured and reduced. ital. j. food sci., vol. 30, 2018 500 figure 1. sds-page under reducing conditions of proteins from seeds of fagopyrum species, namely tataricum (t), and esculentum landraces, ‘nustran’ (n) and ‘furest’ (f) extracted under non-denaturing (nd) and denaturing extraction conditions (d). la and lb stand for 13s globulin acidic and basic subunits, respectively; v stands for 7s globulin family. see text for further details. in the 1d separation of fig. 1, a clearly distinct pattern of f. tataricum protein profile from those of the two f. esculentum cultivars was visible in the range 40-70 kda. in particular, in samples td, three distinct bands were visible in the range 50 66 kda while only two polypetides were detectable in the other two samples. overall, the 1d protein profiles of f. esculentum ‘nustran’ and ‘forest’ were more similar to each other than that of f. tataricum. the greater resolution of 2d electrophoretic analysis was used to get a more detailed comparison of the three protein patterns (fig. 2). based on the results described above, the 2d ief/sds-page analyses were carried out under denaturing and reducing extraction conditions to get the most complete picture of the respective proteomes. indeed, the 2d maps of the analyzed fagopyrum spp. allowed the identification of some of the seed’s main protein components. the spot groups marked with la and lb display intensities and positions which definitely identifies these spots as acidic and basic subunits of the 13s globulin, respectively. the ‘train spot’ group is typical of the high mr 7s globulin chains with their peculiar pi isoforms (radović et al., 1996; magni et al., 2007). in this latter case, however, closely migrating hmw prolamins with variable pi and mr around 50 kda were likely present too, as detailed by naiłęcz et al. (2009). prolamins of intermediate and low mr, though barely recognizable, seemingly spread in the map at more acidic pis and with greater migration coefficients, according to naiłęcz et al. (2009). the 2d electrophoretic maps confirm and detail the 1d profiles by showing a greater similarity between the two f. esculentum cultivars and their difference with f. tataricum spot pattern. indeed, the region of the 13s globulin acidic subunits appears quite different and some major spots, which are circled in the panel t of fig. 2, contributed to significantly diversify the map of f. tataricum with respect to those of f. esculentum. however, differences between the two f. esculentum landraces were also detectable. quantitative differences among common spots, could be noted too. most of these differences were found in the low mr, neutral ph region of the 2d maps. the observed intervarietal differences cannot be attributed to climatic, pedological or edaphological causes, since the seeds arise from same cultivation area and grower. likely, these ital. j. food sci., vol. 30, 2018 501 differences represent authentic biodiversity source. in an extension of this work, it would be interesting to identify those differing spots, which are unlikely related to classical storage proteins because of their low mr and clear-cut shape of the spots, and to associate them with peculiar phenotypic or nutritional features of the two cultivars. figure 2. two-dimension electrophoretic maps of proteins extracted from seeds of fagopyrum species, namely tataricum (t) and esculentum landraces: ‘nustran’ (n) and ‘furest’ (f), all extracted under denaturing conditions (see details under methods). v: 7s globulin chains; p: prolamins chains; la: 13s globulin acidic subunits; lb: 13s globulin basic subunits. 4. conclusions in the perspective of future uses of landraces and their derivatives in foods suitable for people with coeliac disease or in nutraceutical formulations, the development of an agile ital. j. food sci., vol. 30, 2018 502 methodology to identify landraces components is needed. this work represents a first step in this direction, making available specific 2d electrophoretic maps for given landraces and thus helping in their identification and tracing in flours for human food. it is worthy of note that the two cultivar seeds are very similar in appearance, with ‘furest’ being smaller in size and of lighter grey color (barcaccia et al., 2016). these minimal differences make the need for molecular fingerprinting more compelling. this work, by revealing even minor interspecific and intervarietal differences in the protein expression patterns of buckwheat seeds, may open the gateway to the identification of hidden useful properties, such as resistance to adverse environmental conditions and seed quality characteristics, and to the valorization of these crop populations. references barcaccia g., volpato m., gentili r., abeli t., galla g., orsenigo s., citterio s., sgorbati s. and rossi g. 2016. genetic identity of common buckwheat (fagopyrum esculentum moench) landraces locally cultivated in the alps. genet. resour. crop evol. 63: 639. capraro 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research: 195. zhou x., wen l., li z., zhou y., chen y. and lu y. 2015.advances on the benefits of bioactive peptides from buckwheat. phytochem. rev. 14: 381. paper received december 13, 2017 accepted january 28, 2018 ital. j. food sci., vol. 30, 2018 504 paper the effect of temperature and method of drying on isot (urfa pepper) and its vitamin c degradation kinetics ş. dağhan1, a. yildirim*1, f. mehmet yilmaz2, h. vardin1 and m. karaaslan1 1harran university, faculty of engineering, food engineering department, sanliurfa, turkey 2adnan menderes university, engineering faculty, food engineering department, aydın turkey *corresponding author: tel. +90 5068430765 e-mail address: ayildirim10@gmail.com abstract this study investigated drying and vitamin c degradation kinetics in isot (red peppers) at different temperatures (55, 65 and 75°c) and conditions (vacuum and hot air drying). the drying temperature and method had a significant effect on the loss of vitamin c. vacuum dried samples at 55°c retained the highest quantity of vitamin c, while the samples dried at 75°c in a cabinet dryer lost the highest amount of vitamin c. the results showed that vitamin c is especially sensitive to the presence of oxygen and higher temperatures. the weibull model was found to provide the mathematical equation best describing the ascorbic acid degradation kinetics in red peppers, while the page model best reflected the drying kinetics. keywords: drying, ascorbic acid, isot, degradation kinetics, mathematical modeling ital. j. food sci., vol. 30, 2018 505 1. introduction red pepper (capsicum annuum l.) is native to american tropical zones and largely grown in relatively moderate regions as well. red pepper is widely consumed as a fresh vegetable crop or is processed into various food products like roasted, dehydrated, flaked or powdered pepper. one of the oldest and most common processing methods is drying, and it is utilized to enhance the shelf life of food crops. lowering water activity during drying prevents microbial and biochemical decay of food (kamiloglu et al., 2014). dried red peppers are used in the production of ready to eat soups, ketchup, snacks, potato chips, dressings and sauces. peppers are not just used as a colorant, or flavoring, and/or as a source of pungency, but pepper is a good source of vitamin c, antioxidants and bioactive compounds. among the antioxidant phytochemicals, polyphenols deserve a special mention due to their free radical scavenging properties. the red pepper, especially, has a significantly higher total phenolic content than green pepper. red pepper contains a higher level of β-carotene (5.4 μg/g), capsanthin (8.0 μg/g), quercetin (34.0 μg/g) and luteolin (11.0 μg/g) (nadeem et al., 2011). however, the drying process may negatively affect food quality parameters such as nutritional value, bioactive-compound content, color and texture (fazaeli et al., 2011) non-enzymatic browning reactions, loss of pungency and degradation of bioactive compounds are the major changes occurring in red peppers during drying. red peppers are also a good source of lycopene, β-cryptoxanthin, fiber and an array of vitamins such as a, k and c. vitamin c is one of the numerous bio-functional compounds found in red peppers. several studies reported on the ascorbic acid content of red peppers (lee et al., 1995; kumar and sape, 2009). even though there are large differences in varieties kumar and sape, 2009), red peppers are generally recognized as a good source of vitamin c, and its vitamin content can be as high as 186 mg/100 g of fresh weight. vitamin c has been shown to be associated with alleviation of cardiovascular disease and high blood pressure, with increased immune function, increased iron absorption and the promotion of high iron stores. retention of vitamin c in dried foods indicates that processing conditions were not harsh and thus the other micro-nutrients present in the food matrix were most likely retained (hiwilepo-van hal et al., 2012). as vitamin c is a hydrophilic, heat-sensitive vitamin that is especially prone to both chemical and enzymatic oxidation, its concentration in food systems can be considered as a quality factor in plant-based foods. the use of dried food crops is mainly to enhance the quality, flavor and acceptability of the prepared dishes. to minimize the loss or destruction of a food component such as vitamin c or its color during processing, kinetic models that describe destruction rates and their dependence on such factors as temperature, moisture content and water activity must be determined. the study of drying behaviour of different materials has been a subject of interest for various researchers on both theoretical and application grounds for the past 16 years. recently, there have been many studies on the drying behaviour of various vegetables and fruits, such as mushrooms and pollen (midilli et al., 1999), potato (gogus and maskan, 1998), onion (sarsavadia et al., 1999), green beans and pumpkin (yaldiz and ertekin, 2001), grapes (yaldiz et al., 2001), pistachios (midilli, 2001) and peppers (di scala and crapiste, 2008; veras et al., 2012; darvishi et al., 2014). mathematical equations and kinetic models are required for the design of optimal procedures for food-processing steps such as drying and storage (kaymak-ertekin and gedik, 2005). this study was carried out to report on the degradation kinetics of ascorbic acid and the drying kinetics of red peppers processed under different drying conditions at various temperatures. the loss of nutritional quality during food processing ital. j. food sci., vol. 30, 2018 506 has drawn more and more attention in recent years as nutrient deterioration can be the limiting factor determining the consumer demand, especially for vegetable-fruit-based foodstuffs. a review of the degradation kinetics of vitamins in fruits, vegetables and cereals during thermal processing was published by villota and hawkes (1986). thus, food processors are concerned in protecting nutrient quality and developing the technological capability to predict nutrient losses during handling and processing. this requires the identification and understanding of the processing parameters responsible for nutrient degradation. considering the rare reports available on ascorbic acid degradation kinetics in dried fruit or vegetables, and the growing interest in many bio-functional compounds including ascorbic acid in recent years, it is important to study the degradation kinetics of these compounds. the degradation kinetics of vitamin c gives a better insight into corresponding food processing conditions, and thus, helps to adapt the best processing methods to preserve the nutritional content of the products. this study was designed to determine the optimum drying conditions for preserving the micro-nutrients of urfa pepper (i̇sot) that has a large trading potential in urfa province. therefore, we studied the drying of red peppers using different drying techniques at different temperatures and established the vitamin c degradation equations. 2. materials and methods 2.1. materials red pepper (capsicum annuum l.) cultivated in şanlıurfa province was purchased from local markets and used for dried red pepper production. peppers were sliced by hand before the drying processes. the samples obtained either during or at the end of the drying process were stored in a freezer (-20°c) until the analyses were carried out. 2.2. chemicals meta-phosphoric acid (hpo3) was obtained from merck (germany), ascorbic acid standard with 99% purity and methanol of hplc grade were both obtained from sigma-aldrich company (germany). 2.3. drying equipment the drying processes were carried out in vacuum and cabinet (hot air) dryers. a vacuum dryer (wiseven, wov-70, witeg, germany) was used to dehydrate red peppers under vacuum conditions. the dryer has three metal shelves. split red peppers were laid on each shelf in a density of 1600 g/m2. red peppers were dried under the conditions of -0.1 mpa atmospheric pressure and at temperature levels of 55, 65 and 75°c. a cabinet dryer (elektro-mag, m7040-r, turkey) was used for drying red pepper using hot air with a velocity of (1.2 m/s). the dryer has three grill shelves, a fan and ventilation hole. split red peppers were laid on each shelf in density of 1600 g/m2 and dried at three different temperatures (55, 65 and 75°c). the temperature values were determined according to the results of preliminary experiments. all drying processes were carried out in triplicate. ital. j. food sci., vol. 30, 2018 507 2.4. calculations the drying rate (dr) of peppers was calculated using eq. 1, where mt and mt+dt are the moisture content (kg of water per kg of dry solid) at t and t +dt, where t is the drying time in minutes. dr = mt+dt − mt dt (1) for drying model selection, drying curves were fitted to 5 well known drying models, which are given in table. 1. the moisture ratio (mr) of pepper during the drying experiments was calculated using the following equation: mr = mt − me mo − me (2) where mt, mo, and me are the moisture content at any drying time (in minutes), and the initial and equilibrium moisture content (%, d.b.), respectively. the values of me are relatively small compared to those of mt or mo, hence the error involved in the following simplification is negligible (aghbashlo et al., 2008) and accordingly we can write: mr = mt mo (3) table 1. models used to fit the drying data. model equation reference eq. no. page mr =exp(−ktn ) diamonte and munro (1993) (4) modified page mr =exp[(−(kt)n )] white et al. (1981) (5) newton mr =exp(−kt) henderson (1974) (6) henderson and pabis mr = aexp(−kt) zhang and litchfield (1991) (7) wang and singh mr =1+at +bt2 wang and singh (1978) (8) for the effect of drying temperature on the rate constant (k) for eq. 4, page’s model was used with arrhenius’s equation (eq. 9): ln(k)= ln(ko)− ea rt (4) where, t, ea, r and ko are the drying temperature in k, the activation energy in kj mol−1, the ideal gas constant of 8.314×10−3 mol−1 k−1 and the pre-exponential factor, respectively. all calculations were done in triplicate. 2.5. sample preparation for hplc analysis firstly, dried and frozen red pepper samples were blended (yazıcılar, g1, turkey) to make fine powder. approximately 2 g of dried red pepper was weighed into a centrifuge tube ital. j. food sci., vol. 30, 2018 508 (50 ml) and it was combined with 50 ml of 3% metaphosphoric acid and shaken for 5 minutes and then centrifuged at 4000 rpm for 10 minutes. the supernatant was then transferred to a 100 ml volumetric flask with 3% metaphosphoric acid. each sample was filtered into vials before injection into an hplc column. 2.6. ascorbic acid analysis by hplc the detection and quantifying of l-ascorbic acid levels in the samples was carried out using hplc equipped with a uv-dad detector (shimadzu), according to puwastein et al. (2011) and stefanelli et al. (2014). the hplc equipped with an lc-20ad pump, autosampler, and an ods c18 column (250 mm×4.6 mm×5 µm) was used, and the uvdad detector was set to a wavelength of 254 nm. the isocratic mobile phase was methanol: water (5:95, v/v) at a ph of 3 fixed by h3po4, and the flow rate was 1ml/min with an injection volume of 20 µl. various concentrations of standard ascorbic acid was used to obtain a calibration curve, and the peak areas were used to calculate the ascorbic acid content. results were expressed as mg/100g of dry matter. all hplc measurements were carried out in triplicate. 2.7. statistical analyses one-way analysis of variance (anova) was carried out with spss 16.0 to determine the main influence of vacuum and hot air drying techniques on the drying and ascorbic acid degradation parameters. the duncan multiple range test was employed to compare the differences among groups. the non-linear regression analysis was done using a sigma plot (version 10) software package. the correlation coefficient (r2) was one of the main criteria for selecting the best model. in addition to the coefficient of correlation, the goodness of fit was determined by root mean square error (rmse) values, residual-predicted plot and experimental-predicted plot. for a quality fit, the r2 values should be close to 1 and the rmse values should be lower. 3. results and discussion 3.1. change of moisture ratio and drying rate during hot air and vacuum drying drying of the red peppers by both hot air and vacuum dryers started with an initial moisture content of around 91.26% (w.b.) and continued until a final moisture content of 12.50% (w.b.) was reached. the variations of moisture ratio with time at 55, 65 and 75°c for both hot air and vacuum dryers are given in fig. 1. as expected, an increase in drying temperature resulted in a decrease in the drying time for both hot air and vacuum drying of red peppers at different temperatures (55, 65 and 75°c). the times to reach 12.50% (w.b.) moisture content from the initial moisture content of red peppers at 55, 65 and 75°c were found to be 960, 742 and 594 minutes for hot air drying, and 247, 231 and 195 minutes for vacuum drying, respectively. based on the final moisture content (12.50% w.b.), drying time decreased from 960 minutes to 247 minutes at 55oc when comparing hot air drying to vacuum drying. similarly at 65oc, drying time decreased from 742 minutes to 231 minutes, and at 75°c drying time decreased from 594 minutes to 195 minutes. the vacuum drying was able to reduce the drying time of peppers by 74.1% at 55oc, 69.3% at 65°c and 67.0% at 75oc, compared with hot air drying (fig. 1). these results are in good agreement with previous observations for ital. j. food sci., vol. 30, 2018 509 mint leaves (giri and prasad, 2007) and for mushrooms (therdthai and zhou, 2009). the changes in the drying rates (dr, % d.b min−1) versus both drying time (minimum) and moisture content (%, d.b) for both hot air and vacuum dryers are shown in fig. 2. it is apparent that the drying rate decreased with drying time for both hot air and vacuum drying. the drying rate also decreased continuously with decreasing moisture content or for increasing drying times. these findings are in the agreement with previous studies (togrul and pehlivan, 2002; yaldiz and ertekin, 2001; yaldiz et al., 2001). there is no constant-rate drying period in these drying rate curves, and all the drying operations are seen to occur over the falling rate period. hot air time (min) 0 200 400 600 800 1000 1200 m oi st ur e r at io (m o/m t) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 55 oc 65 oc 75 oc page model vacuum time (min) 0 50 100 150 200 250 300 350 m oi st ur e ra tio (m t/m o) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 55 oc 65 oc 75 oc page model 55 oc time (min) 0 100 200 300 400 500 600 700 800 900 1000 1100 m oi st ur e r at io (m t/m o) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 hot air vacuum page model 65 oc time (min) 0 100 200 300 400 500 600 700 800 900 1000 1100 m oi st ur e r at io (m t/m o) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 hot air vacuum page model 75 oc time (min) 0 100 200 300 400 500 600 700 800 900 1000 1100 m oi st ur e r at io (m t/m o) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 hot air vacuum page model figure 1. fitting of the average experimental and simulated data of hot air and vacuum drying to the page model at 55, 65 and 75°c temperatures. ital. j. food sci., vol. 30, 2018 510 hot air time (min) 0 200 400 600 800 1000 1200 d ry in g ra te (k g w at er /k g dr y so lid .m in ) 0 1 2 3 4 5 55 oc 65 oc 75 oc vacuum time (min) 0 50 100 150 200 250 300 350 d ry in g ra te (k g w at er /k g dr y so lid .m in ) 0 5 10 15 20 25 30 55 oc 65 oc 75 oc 55 oc time (min) 0 100 200 300 400 500 600 700 800 900 1000 1100 d ry in g ra te (k g w at er /k g dr y so lid .m in ) 0 2 4 6 8 10 12 14 16 18 hot air vacuum 65 oc time (min) 0 100 200 300 400 500 600 700 800 900 1000 1100 d ry in g ra te (k g w at er /k g dr y so lid .m in ) 0 5 10 15 20 25 hot air vacuum 75 oc time (min) 0 100 200 300 400 500 600 700 800 900 1000 1100 d ry in g ra te (k g w at er /k g dr y so lid .m in ) 0 5 10 15 20 25 30 hot air vacuum hot air moisture content (%, d.b.) 0 200 400 600 800 1000 d ry in g ra te (k g w at er /k g dr y so lid .m in ) 0 1 2 3 4 5 55 oc 65 oc 75 oc vacuum moisture content (%, d.b.) 0 100 200 300 400 500 600 d ry in g ra te (k g w at er /k g dr y so lid .m in ) 0 5 10 15 20 25 30 55 oc 65 oc 75 oc 55 oc moisture content (%, d.b.) 0 100 200 300 400 500 600 700 800 900 1000 1100 d ry in g ra te (k g w at er /k g dr y so lid .m in ) 0 2 4 6 8 10 12 14 16 18 hot air vacuum ital. j. food sci., vol. 30, 2018 511 65 oc moisture content (%, d.b.) 0 100 200 300 400 500 600 700 800 900 d ry in g ra te (k g w at er /k g dr y so lid .m in ) 0 5 10 15 20 25 hot air vacuum 75 oc moisture content (%, d.b.) 0 100 200 300 400 500 600 700 800 d ry in g ra te (k g w at er /k g dr y so lid .m in ) 0 5 10 15 20 25 30 hot air vacuum figure 2. drying rate versus time and moisture content at different temperatures for hot air and vacuum drying of peppers. increases in the drying temperature of red peppers also increased the drying rate and decreased the drying time (fig. 2). the drying time is shorter when the temperature is higher, such as 75oc, and can be explained by the increase in the drying rate due to the increased heat transfer potential between the air and the peppers, therefore favoring the evaporation of water from the peppers. the effect of temperature on the drying rate has been also studied and reported on by some researchers (henderson and pabis, 1961; akpinar et al., 2003). the vacuum drying was able to increase the drying rate of peppers by 76.6% at 55°c, 73.6% at 65°c and 70.2% at 75°c, compared to hot air drying (fig. 2), respectively. increases in the drying rates confirmed the decrease in drying times when the peppers were dried with a vacuum dryer. these results are in good agreement with the study by giri and prasad (2007) for mushroom vacuum drying with a drying time reduction range of 70 to 90%, and earlier observations (kaymak-ertekin, 2002; akpinar et al., 2003). 3.2. the drying kinetics of peppers 3.2.1 modeling of pepper moisture ratio as a function of drying time fig. 1 shows the change in moisture ratio of pepper samples with time at 55, 65 and 75°c for hot air and vacuum drying. it can be seen that the moisture ratio decreases continuously with drying time for both hot air and vacuum drying. the mass transfer within the samples was more rapid during higher temperatures because more heat generation within the sample created a large vapor pressure difference. the advantage of vacuum drying is to accelerate the drying process, to increase the mass transfer through an increased pressure gradient between the inner and outer layers, and to maintain the drying process at lower temperatures (pere and rodier, 2002). the statistical and regression results from the 5 different models are given in table 2. the r2 and rmse values for models were found to be in the range of 0.8596 to 0.9966 and 0.13 to 0.02, respectively. based on the criteria of the highest r2 (0.9996 to 0.9697) and the lowest rmse (0.02 to 0.06), page’s model was selected as the most suitable model to represent the hot air and vacuum drying behavior for red peppers. the model parameters were significant with a 95% confidence interval, and the predictive curves arising from the page model (fig. 1) adequately described the experimental data. the residual-predicted plot for the page ital. j. food sci., vol. 30, 2018 512 model regressed on the data is displayed in fig. 3 and the residual points seem to be randomly distributed, with most residuals lying within two standard deviations. table 2. statistical parameters of the drying models for different conditions. process temp. (oc ) model a b k (min-1) n r 2 rmse page 8.73x10-7(±0.03)a, x 2.16(±0.01)a,x 0.9966 0.02 modified page 1.57 x10-3 2.16 0.9966 0.02 55 newton 1.42 x10-3 0.8596 0.12 hend.andpabis 1.17 1.72 x10-3 0.9056 0.10 wang and sing -6.92x10-4 -3.40x10-7 0.9691 0.05 page 1.18x10-6(±0.06)b, x 2.15(±0.02)a,x 0.9841 0.04 hot air modified page 1.86x10-3 2.15 0.9841 0.04 65 newton 1.77x10-3 0.8616 0.13 hend.andpabis 1.18 2.12 x10-3 0.9033 0.11 wang and sing -1.14x10-3 1.00x10-7 0.9308 0.09 page 2.76x10-6(±0.03)c, x 2.14(±0.03)a,x 0.9697 0.06 modified page 2.53 x10-3 2.14 0.9697 0.06 75 newton 2.49 x10-3 0.8867 0.12 hend.andpabis 1.17 2.90 x10-3 0.9174 0.10 wang and sing -1.96x10-3 1.01x10-6 0.9229 0.10 page 7.31x10-4(±0.02)a, y 1.71(±0.02)a,y 0.9950 0.02 modified page 5.90 x10-3 1.71 0.9950 0.02 55 newton 5.77 x10-3 0.9254 0.09 hend.andpabis 1.11 6.49 x10-3 0.9465 0.08 wang and sing -4.05x10-3 2.95 x10-6 0.9722 0.05 page 9.34 x10-4(±0.03)b, y 1.28(±0.01)a,y 0.9858 0.04 modified page 7.25 x10-3 1.28 0.9858 0.04 65 newton 7.41 x10 -3 0.9692 0.06 vacuum hend.andpabis 1.05 7.80 x10-3 0.9734 0.05 wang and sing -5.51x10-3 7.64 x10-6 0.9902 0.03 page 1.57 x10-3(±0.08)c, y 1.09(±0.02)a,y 0.9875 0.03 modified page 9.32 x10-3 1.09 0.9875 0.03 75 newton 9.45 x10-3 0.9857 0.04 hend.andpabis 1.02 9.59 x10-3 0.9860 0.04 wang and sing -7.04x10-3 1.30x10-5 0.9839 0.04 means in the same column with different superscript letters are significantly different, a to c (temperature), x to y (dryer), and p ≤ 0.05. values in parentheses are standard deviations. furthermore, the experimental-predicted plot was close to linear, indicating a good model fit to the page and weibull models in describing the drying characteristics of red peppers (fig. 3). from the page model, n was found to be greater than 1.0 (the lowest value was 1.09), which means that the relationship between the moisture ratio and time was unlikely to be first order kinetics. thus, page’s model offered improved predictability of drying kinetics ital. j. food sci., vol. 30, 2018 513 over other models. for page’s model, k for the pepper samples increased from 8.73×10−7 min−1 to 2.76×10−6 min−1 (a 68.37% increase) when drying temperature changed from 55°c to 75°c for hot air drying. also, a 97.52% increase was obtained in the value of k for vacuum drying within the same temperature range. when comparing hot air and vacuum drying of peppers at 55oc temperature, the k value for the page model increased from 8.73×10−7 min−1 to 7.31×10−4 min−1, a 99.88% increase. vacuum drying produced a similar percentage increase in the k value at 65°c and 75°c (99.87 and 99.82%). these results are supported with results in section 3.1, which is related to the increased drying rate and decreased drying time with vacuum drying. moisture ratios predicted 0.0 0.2 0.4 0.6 0.8 1.0 1.2 r es id ua ls -0.15 -0.10 -0.05 0.00 0.05 0.10 0.15 hot air drying 55 oc hot air drying 65 oc hot air drying 75 oc vacuum drying 55 oc vacuum drying 65 oc vacuum drying 75 oc ascorbic acid degredation predicted 0.0 0.2 0.4 0.6 0.8 1.0 1.2 r es id ua ls -0.06 -0.04 -0.02 0.00 0.02 0.04 0.06 hot air drying 55 oc hot air drying 65 oc hot air drying 75 oc vacuum drying 55 oc vacuum drying 65 oc vacuum drying 75 oc moisture ratios predicted 0.0 0.2 0.4 0.6 0.8 1.0 1.2 ex pe rim en ta l 0.0 0.2 0.4 0.6 0.8 1.0 1.2 hot air drying 55 oc hot air drying 65 oc hot air drying 75 oc vacuum drying 55 oc vacuum drying 65 oc vacuum drying 75 oc ascorbic acid degredation predicted 0.0 0.2 0.4 0.6 0.8 1.0 1.2 e xp er im en ta l 0.0 0.2 0.4 0.6 0.8 1.0 1.2 hot air drying 55 oc hot air drying 65 oc hot air drying 75 oc vacuum drying 55 oc vacuum drying 65 oc vacuum drying 75 oc figure 3. residual-predicted and experimental-predicted plots for page and weibull models at different temperatures (55, 65 and 75°c) for hot air and vacuum drying 3.2.2 a general model to describe the moisture ratio (mt/mo) as a function of drying time and temperature arrhenius plots of the natural logarithm of the rate constant (k) versus the inverse of t (k) (eq. 9) for both hot air and vacuum drying of peppers are superposed in fig. 4. the activation energy, ea, is related to the slope of this graph, and shows that the temperature dependence of the drying rate constant (k) was fitted to a linear model. the computed values of ea were found to be 54.4 kj mol−1 for hot air and 36.0 kj mol−1 for vacuum drying of peppers, respectively. compared to the hot air drying, the activation energy decreased when the vacuum drying was applied for drying red pepper. this corresponds to the increase of the drying rate and decrease of the drying time. a similar comparable result was obtained by therdthai and zhou (2009) for mint leaves for both hot air and vacuum drying. also, the activation energy is shown to be sensitive to the drying rate constant against temperature. the greater activation energy value, the higher sensitivity of ital. j. food sci., vol. 30, 2018 514 the k value of pepper to the temperature. in general, activation energy values for food and agricultural crops lie in the range of 12.7 to 110.0 kj mol−1 (aghbashlo et al., 2008). 1/t (1(k) 0,00290 0,00295 0,00300 0,00305 ln (k ) -16 -14 -12 -10 -8 -6 -4 hot air vacuum fitted t (k) 325 330 335 340 345 350 n 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 hot air vacuum fiited 1/t (1/k) 0.00290 0.00295 0.00300 0.00305 ln (1 /α ) -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 hot air vacuum fitted t (k) 325 330 335 340 345 350 β 0.3 0.4 0.5 0.6 0.7 0.8 0.9 hot air vacuum fitted figure 4. arrhenius plot of the page (drying rate constant, k and n) and weibull (kinetic reaction rate constant for ascorbic acid, 1/α and β) models for temperatures of 55, 65 and 75°c for hot air and vacuum drying of peppers. the activation energy of pepper for the present study compares satisfactorily with similar data for red pepper reported in published literature by turhan et al. (1997), kaymakertekin (2002) and di scala and crapiste (2008), at 28.4, 42.8 and 33.8 kj mol−1, respectively. the parameter values of page’s model obtained for k and n by the least square fit of experimental points are given below for both hot air and vacuum drying: hot air drying ln(k)= 5.8966− 6540.9601 t (r2=0.9211) (10) n = 2.488−0.001*t (r2=0.9999) (11) vacuum drying ln(k)= 5.9359− 4331.2057 t (r2=0.9524) (12) n =11.838−0.031*t (r2=0.9524) (13) combining page’s model with equations 10 to 13, the following general models can be derived to describe the drying kinetics of pepper as a function of time and temperature: ital. j. food sci., vol. 30, 2018 515 mr =exp[−363.798*exp(−6540.9601 1 t )*t2.488−0.0010*t ] (for hot air drying) (14) mr =exp[−378.380*exp(−4331.2057 1 t )*t11.838−0.031*t ] (for vacuum drying) (15) these expressions can be used to estimate the moisture ratio of red pepper and for temperature at any time with great accuracy during the hot air and vacuum drying processes. similar expressions were also obtained by several researchers for different agricultural products (akpinar et al., 2003; kaymak-ertekin, 2002; gowen et al., 2007; yildirim et al., 2011; chayjan et al., 2011). 3.3.1 ascorbic acid degradation kinetics for pepper during drying ascorbic acid degradation was modeled using first order and weibull models (eq. 16 and 17). the weibull model is flexible owing to the inclusion of a shape constant in addition to the rate constant and has been employed to describe microbial, enzymatic and chemical degradation kinetics (cunha et al., 1998; manso et al., 2001). ct co =exp(−k1 *t) (16) ct co =exp(−( t α )β) (17) where ct is the ascorbic acid concentration at a time t, co is the initial ascorbic acid concentration, ct/co is the ascorbic acid degradation ratio at any time, k1 is the rate constant, β (dimensionless) is the shape constant (first order kinetics model is applicable when β=1) and α is the scale parameter in minutes. in general, according to the literature, the average vitamin c content for red peppers is around 23.84-78.67 mg/100g (andrews, 1995; teodoro et al., 2013). the vitamin c content for the red peppers used in this study was 54.35 mg/100 g. red pepper is known to be one of the richest sources of vitamin c compared with other fruits and vegetables (marin et al., 2004; sarker and gohda, 2013), thus vitamin c was selected as the quality parameter for this study. a graphical representation of the experimental and predicted vitamin c degradation in the pepper samples during vacuum and hot air drying at 55, 65 and 75°c is shown in fig. 5. as seen in the figure, at any time during drying, the loss of vitamin c increased with increasing temperature (p<0.05) in both drying conditions due to the heat-sensitive nature of vitamin c. the heated air inherently exposes the products to oxidation, reducing their ascorbic acid content (vega-galvez et al., 2008). the degradation kinetics of vitamin c was assessed with first order and weibull models. the weibull distribution function has an interesting potential for describing microbial, enzymatic and chemical degradation kinetics (cunha et al., 1998; oms-oliu et al., 2009). table 3 shows the results of fitting vitamin c retention in red pepper to the first order model and weibull model distribution. the weibull model (eq. 17) yielded a good fit to the vitamin c experimental data, and the weibull distribution seemed to be suitable considering the high determination coefficients (r2 = 0.9904 to 0.9975) and the low rmses (0.01 to 0.03). the values of kinetic constants (α) and (β) of the weibull model were obtained by fitting eq. 17 to the experimental data. the α and β values obtained from the weibull model were directly affected by drying temperature. the β values for hot air drying were 0.59, 0.55 and 0.38 at 55, 65 and 75oc, respectively, and 0.79, 0.59 and 0.44 for vacuum drying at the same temperature levels. the constant β represents a behavior ital. j. food sci., vol. 30, 2018 516 index, and if β < 1 the reaction rate decreases with time and a degradation rate higher than the exponential is observed at the beginning of the process (cunha et al., 1998; marfil et al., 2008). fig. 5 supports this idea as there was an initial high rate of vitamin c loss at relatively higher moisture contents, followed by a period of less rapid degradation as the moisture content decreased. similar tendencies in vitamin c degradation during drying were also observed in other studies (di scala and crapiste 2008; erenturk et al., 2005). therefore, retention of vitamin c is not only dependent on drying conditions but also on the sample moisture content. the α values were 241.83, 168.08 and 56.07 at 55, 65 and 75oc, respectively for hot air drying, and 383.05, 235.79 and 87.45 for vacuum drying at the same temperature levels (table 3). higher α values indicate lower degradation rates or, in other words, a longer time before nutrient collapse (marfil et al., 2008). the parameter α was dependent on both temperature and dryer type in this study. the degradation rate was less in vacuum drying and for lower drying temperatures. an oxygen-deficient medium and less drying time could be reasons for the lower degradation rate of the vitamin c in vacuum drying (yilmaz et al., 2017). hot air time (min) 0 100 200 300 400 500 600 700 800 900 1000 1100 a sc or bi c ac id d eg ra da tio n (c t/c o) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 55 oc 65 oc 75 oc weibull model vacuum time (min) 0 50 100 150 200 250 300 350 a sc or bi c ac id d eg ra da tio n (c t/c o) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 55 oc 65 o c 75 oc weibull model 55 oc time (min) 0 100 200 300 400 500 600 700 800 900 1000 1100 a sc or bi c ac id d eg ra da tio n (c t/c o) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 hot air vacuum weibull model 65 oc time (min) 0 200 400 600 800 1000 1200 a sc or bi c ac id d eg ra da tio n (c t/c o) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 hot air vacuum weibull model 75 oc (e) time (min) 0 100 200 300 400 500 600 700 800 900 1000 1100 a sc or bi c ac id d eg ra da tio n (c t/c o) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 hot air vacuum weibull model figure 5. ascorbic acid degradation curves for pepper samples using the weibull model for hot air and vacuum drying at 55, 65 and 75°c. ital. j. food sci., vol. 30, 2018 517 the final vitamin c contents of the vacuum dried peppers were 45, 43 and 28% of the initial value at 55, 65 and 75°c respectively, as the drying operation was terminated at 12.50% moisture content (w.b.). vitamin c retention for hot air drying was 20, 19 and 15% at 55, 65 and 75°c, respectively. as seen in these results, the degradation rate was higher for hot air drying and elevated temperatures. vega-galvez et al. (2008) reported the ratio of final to initial vitamin c was in the range 0.18, 0.17, 0.15 and 0.16 for the lamuyo variety red pepper dried at 50, 60, 70 and 80°c, respectively. sigge et al. (1999) reported 25 to 40% retention of vitamin c during dehydration of green peppers processed between 55 and 75°c. indeed, drying at 55 and 65°c in this study produced similar vitamin c degradation. the results from di scala and crapiste (2008) also showed the same nutritional degradation level for red peppers from the ascorbic acid retention standpoint at 60 and 70°c. vitamin c degradation rates during drying depended mostly on the combination of temperature and time parameters. in addition to that, air composition, sample shape and also food composition (aw, enzymes, ph) are directly related to vitamin c retention (santos and silva, 2008). table 3. first order and weibull model parameters for drying pepper samples at different temperatures during hot air and vacuum drying. process temp. (oc ) model α (1/α) β k1 r 2 rmse 55 first order 4.13x10 -3 0.9024 0.08 weibull 241.83(±1.35) c,x 4.14x103(±0.04)a,y 0.59(±0.01)c,x 0.9883 0.03 hot air 65 first order 5.39x10 -3 0.9048 0.08 weibull 168.08(±0.87) b,x 5.95x103(±0.02)b,y 0.55(±0.02)b,x 0.9975 0.01 75 first order 10.80x10 -3 0.8926 0.08 weibull 56.07(±2.22) a,x 17.83x103(±0.05)c,y 0.38(±0.01)a,x 0.9955 0.02 55 first order 3.00x10 -3 0.9790 0.03 weibull 383.05(±3.84) c,y 2.61x10-3(±0.03)a,x 0.79(±0.04)c,y 0.9972 0.01 vacuum 65 first order 4.73x10 -3 0.9226 0.06 weibull 235.79(±2.45) b,y 4.24x10-3(±0.03)b,x 0.59(±0.02)b,y 0.9940 0.02 75 first order 9.02x10 -3 0.8897 0.09 weibull 87.45(±3.42) a,y 11.43x103(±0.05)c,x 0.44(±0.01)a,y 0.9904 0.03 means in the same column with different superscript letters are significantly different, a to c (temperature), x to y (dryer), and p ≤ 0.05. values in parentheses are standard deviations. 3.2.3 a general model to describe ascorbic acid degradation as a function of drying time and temperature the temperature dependence of ascorbic acid degradation on the basis of weibull’s model can be described by an arrhenius type equation where plots of ln (1/α) eq. (18) versus the reciprocal of drying temperature in absolute degrees resulted in straight lines (fig. 4). ln(1/α)= ln(1/αo)− ea rt (18) ital. j. food sci., vol. 30, 2018 518 where, t, ea and r are the drying temperature in k, the activation energy in kj mol−1 and the ideal gas constant of 8.314×10−3 mol−1 k−1, respectively. the parameter values obtained by weibull’s model (α and β) by least square fit of the experimental points are given below for both hot air and vacuum drying: hot air drying ln(1α)=19.68− 8298.00 t (r2=0.9131) (19) β = 4.06−0.0105*t (r2=0.8867) (20) vacuum drying ln(1α)=19.57− 8398.94 t (r2=0.9555) (21) β = 6.52−0.0175*t (r 2=0.9932) (22) the high activation energies for both hot air (68.99 kj mol-1) and vacuum drying (69.83 kj mol-1) obtained in this study based on weibull’s model suggest that vitamin c is highly susceptible to temperature changes (oms-oliu et al., 2009; marfil et al., 2008; karaaslan et al., 2014). the degradation rates of ascorbic acid during vacuum drying was lower than that of hot air drying, but its activation energy was higher. these results indicate that vacuum drying was more stable than hot air drying. from the regression of linear fit of arrhenius curves for weibull’s model, the following general model can be derived to describe the ascorbic acid degradation kinetics of pepper during drying as a function of time and temperature: hot air: ct co =exp[−(3.54x108 *exp(−8298 1 t )*t)4.0557−0.0105*t ] (23) vacuum: ct co =exp[−(3.15x108 *exp(−8398.9383 1 t )*t)6.5217−0.0175*t ] (24) with the models developed (eqs. 23 to 24), the time and temperature dependent (instantaneous) ascorbic acid content can be estimated for both the dryer types. this study revealed the effects of dryer type, drying temperature, drying time and moisture removal on the vitamin c content of the red pepper. also, our findings demonstrated that the weibull distribution is likely to be a useful tool for describing vitamin c changes in red pepper under different drying conditions. 4. conclusions the drying rate of peppers increased under vacuum drying by 76.6% at 55°c, 73.6% at 65°c and 70.2% at 75°c, respectively. vacuum drying could reduce the drying time of peppers by 74.1% at 55oc, 69.3% at 65°c and 67.0% at 75°c, compared to hot air drying. the drying time is shorter when the temperature is higher due to the increase of the drying rate, which means an enhanced heat transfer potential between the air and the peppers, therefore favoring the evaporation of water from the peppers. the page model provided the best fit of five drying kinetic models with the highest r2 values (0.9697 to 0.9996) and the lowest rmse values (0.06 to 0.02). weibull’s model yielded a good fit with ascorbic acid degradation data, and the model distribution seemed to be suitable considering the high determination coefficients (r2 = 0.9904 to 0.9975) and low rmse values (0.01 to 0.03). increases in drying temperature increased the percentage of the k ital. j. food sci., vol. 30, 2018 519 value in page’s model to 68.37% for hot air drying and 97.52% for vacuum drying. vacuum drying increased the k value of the page model with a 99.88% increase at 55°c, 99.87% at 65°c and 99.82% at 75°c. the activation energy of peppers based on drying kinetics decreased from 54.4 kj mol−1 to 36.0 kj mol−1 with vacuum drying. this confirms the increase in drying rate and decrease in drying time. on the other hand, high activation energies for both hot air (68.99 kj mol−1) and vacuum drying (69.83 kj mol−1), based on ascorbic acid degradation that were obtained in this study, suggest that vitamin c is highly susceptible to temperature changes. the degradation rates of ascorbic acid during vacuum drying were lower than that of hot air drying, while its activation energy was higher. these results indicate that vacuum drying was more stable than hot air drying. new general expressions obtained for both drying and ascorbic acid degradation kinetics can be used to estimate the moisture ratio and the ascorbic acid degradation ratio of red pepper at any time and temperature with a great accuracy during the hot air and vacuum drying processes. we concluded that vacuum drying can be more advantagoeus over hot air drying with respect to drying time and ascorbic acid degradation. acknowledgements the authors would like to thank harran university scientific research council for financial support, and uğur keskin for technical assistance. abbreviations co initial ascorbic acid concentration in mg 100g-1 ct ascorbic acid concentration at any time in mg 100g−1 dad diode array detector d.b. dry base dr drying rates ea activation energy in kj mol−1 hmf hydroxymethylfurfural k drying temperature in k (kelvin) ko pre-exponential factor k, n parameter values of page’s model m.c. moisture content mr moisture ratio mt moisture content at any time mo initial moisture content me equilibrium moisture content r2 coefficient of determination rmse root mean square 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96010-900, pelotas, rs, brazil bembrapa clima temperado, rodovia br 392, km 78, 9º distrito, monte bonito, caixa postal 403, cep 96010971, pelotas, rs, brazil *corresponding author: tel.: +555332758105; fax: +555332758221 e-mail address: jardelaraujoribeiro@gmail.com abstract this study aimed to evaluate the effect of erythorbic acid (ea), sodium erythorbate (se) and kojic acid (ka) to control the pulp browning of minimally processed (mp) ‘royal gala’ apples. physicochemical and sensorial properties of mp apples were evaluated during a shelf life testing. se and ea resulted in the highest levels of phenolic compounds, antioxidant activity and ppo and pod inhibition. sensorial analysis results revealed that se and ea treatments preserved the flavor, odor, color, succulence, firmness and overall quality of mp apples for up to 9 days. in conclusion, both se and ea are suitable antibrowning agents for mp ‘royal gala’ apples. keywords: antioxidant activity, enzymes, minimal processing, phenolic compounds, sensorial analyses ital. j. food sci., vol. 31, 2019 574 1. introduction apple (malus domestica borkh) is a widely consumed fruit in many westernized diets due to its year-round market availability (vieira et al., 2011). there is also a large retail demand for minimally processed (mp) apples due to practicality during consumption, transportation, and health benefits for those who consume. nevertheless, apples are very sensitive to enzymatic browning, either by the high content of phenolic compounds and/or the activity of oxidative enzymes such as polyphenoloxidase (holderbaum et al., 2010) and peroxidase (rojas-graue et al., 2008). enzymatic browning in mp apple is a major problem for the food processing industry, and must be controlled to prevent undesirable changes in color, flavor and nutritional value (toivonen and brummell, 2008; ioannou and ghoul, 2013). an alternative to avoid the browning effect on minimally processed fruit pulps is the use of antioxidants (galgano et al., 2015). lcysteine, erythorbic acid, sodium erythorbate, and kojic acid, used in combination with calcium chloride, are examples of antioxidants agents to preserve mp products quality. lcysteine, commonly used to control/avoid pulp browning in mp products (ali et al., 2016; cabezas-serrano et al., 2013; ghidelli et al., 2014), has been reported to have different mechanisms of action: a) inhibition of ppo (ni eidhin et al., 2006); b) adduct formation with o-quinones resulting in non-colored compounds during oxidation (richard-forget et al., 1992); and c) reduction of o-quinones to their polyphenols precursors (cilliers and singleton, 1990). erythorbic acid (d-isoascorbic acid) (kall and andersen, 1999), has also been used in mp products such as potatoes (cacace et al., 2002), canned applesauce and beer (andersen, 1999). this antioxidant has been reported with similar antioxidants properties of its stereoisomer (ascorbic acid), however, it does not have the vitamin c activity, but it is at least five times cheaper (martin-belloso and soliva-fortuny, 2011). sodium erythorbate is a reducing agent (buta et al., 1999) commonly used in the food industry to reduce the superficial oxidation of mp fruits (gross et al., 2016; sapers and miller, 1998) and meat products (sommers et al., 2002). kojic acid has been used in japan as food additive due to its inhibitory properties over different oxidase enzymes. this antioxidant has been reported in several food matrices such as syrup, flour, meet, flavoring and vegetables (scpp, 2008). in addition, it has been reported that kojic acid significantly reduced the browning in liberty apple slices (son et al., 2001), amasya apple juice (iyidogan and bayindirli, 2004) and litchi pericarp (shah et al., 2017). calcium chloride, when used in association with antioxidants, is responsible for the reduction of physiological imbalances and maintenance of color. in addition, it is recommended due to its firming action on a wide range of fruits (techakanon and barrett, 2017). despite the use of different antioxidants in mp apples, erythorbic acid, sodium erythorbate and kojic acid have never been tested as antibrowning agents in fresh-cut ‘royal gala’ apple, as far as we are concerned. in this context, the objective of this study was to evaluate the sensorial characteristics and physicochemical variables of mp ‘royal gala’ apples treated with erythorbic acid, sodium erythorbate and kojic acid associated with calcium chloride, under cold storage for 9 d, to simulate shelf-life conditions. 2. material and methods 2.1. harvesting and storage of fruits royal gala apples (malus domestica borkh), at commercial maturity stage, were harvested from a commercial orchard located in the vacaria city (28º 30' 44" s, 50º 56' 02" w), rio ital. j. food sci., vol. 31, 2019 575 grande do sul state, brazil. the apples had the following average characteristics: starch content of 4.80, measured according to streif (1984); pulp firmness of 12.84 n; concentration of total soluble solids of 12.51ºbrix and titratable acidity of 0.23 g of malic acid 100 g-1 fw. the apples were selected by size (approximately 110 g) and for the absence of visible mechanical damage and rot and stored at 1.0°c ± 0.5°c and relative humidity of 90.0 % ± 5.0 %, at the postharvest physiology laboratory from embrapa clima temperado. 2.2. treatments fruits were sanitized by dipping into a sodium hypochlorite solution (100 ppm, ph 6.5, at 6.5±1.5ºc) for 10 min, cut into eight wedge shape longitudinal slices with approximately the same size, with the removal of the central core and seeds, preserving the epidermis. the experimental treatments consisted of dipping the apple slices into different solutions for 1 min, as described below: treatments* distilled water (w) l-cysteine chloride 0.60 % (lc) sodium erythorbate 5.00 % (se) erythorbic acid 3.00 % (ea) kojic acid 0.07 % (ka) cacl2 1.00 % (cc) w+cc (negative control) x x lc+cc (positive control) x x se+cc x x ea+cc x x ka+cc x x *the treatments w+cc (negative control) and lc+cc (positive control) were designed to be ineffective and highly effective against browning, respectively. after dipping, the apple slices were drained for 5 min and placed onto expanded polystyrene trays, covered with pvc film (9 μm thickness) and stored for four different periods (0 d, 3 d, 6 d and 9 d), at 4±1.0°c and rh of 90.0±5.0 %, to simulate the shelf life of mp apples. the samples analyzed at 0 d had been exposed to each treatment for 8±1 h. 2.3. reagents reagents were purchased from different suppliers: calcium chloride (≥ 99 %) from synth (diadema, sp, brazil), l-cysteine (≥ 98 %) from vetec (duque de caxias, rj, brazil), sodium erythorbate (≥ 98 %) and erythorbic acid (≥ 99 %) from daxia doce aroma ind. com. ltd. (são paulo, sp, brazil) and kojic acid (≥ 99 %) from chengdu jinkay biology engineering co., ltd. (são paulo, sp, brazil). 2.4. physical and chemical analysis 2.4.1 total soluble solids (tss) tss was determined using a portable refractometer (atago, model pal-1) at 20ºc and the results were expressed in ºbrix. ital. j. food sci., vol. 31, 2019 576 2.4.2 titratable acidity (ta) ta was determined on apple pulp juice. the titration endpoint was determined with a ph meter (quimis model q400a). briefly, 10 ml of pulp juice (diluted in 90 ml distilled water) was titrated with 0.1 m naoh to a ph 8.1 endpoint. the results were expressed as grams (g) of malic acid per 100 g-1 of fresh weight (fw). 2.4.3 tss/ta ratio tss/ta ratio was calculated by dividing the tss values by ta values. 2.4.4 loss of mass mass loss was calculated by the equation 1. 𝐿𝑜𝑠𝑠 𝑜𝑓 𝑚𝑎𝑠𝑠 % = !"#$#%& !"##!!"#$% !"## !"#$% !"## 𝑥 100 equation 1 2.4.5 pulp firmness measured according to melo et al. (2009) using a texture analyser (ta-xt plus 40855, stable microsystems, england) with a 2 mm diameter probe, penetration depth of 5 mm, pre-test velocity of 1.0 mm s-1; 2.0 mm s-1 test; post-test of 10.0 mm s-1 and force of 5 kg. the readings were performed in the middle portion of the pieces and the results were expressed in newton (n). 2.4.6 color apple color of equatorial region of slices was measured using a minolta cr-400 colorimeter with a cie l*a*b* reading system, proposed by the comission internacionale de i’eclairage (cie). the browning index was calculated from l*, a* and b* values according to palou et al. (1999) and hue or chromatic hue, represented by the hue (hº) angle, was calculated as the tangent arc of b*/a* quotient. the result was expressed in degrees. 2.4.7 total phenolic compounds measured according to the folin-ciocalteu method adapted from swain and hillis (1959). briefly, 250 μl aliquot of the extracts (the same used for dpph• analysis) was combined with 250 μl of 0.25 m folin-ciocalteu reagent and 4000 μl ultrapure water. after 3 min of reaction, 500 μl of 0.5 m na2co3 was added, following incubation for 2 h at room temperature and absorbance reading at 725 nm. the results were expressed as grams of chlorogenic acid equivalents (cae) per 100 g-1 of fw. chlorogenic acid standard curve (0.0 mg ml-1 to 0.5 mg ml-1) was used. 2.4.8 antioxidant activity (dpph) the antioxidant activity was evaluated using the method described by brandwilliams et al. (1995) with some modifications. first, 10 ml of methanol was added to 2.5 g of fresh apple and homogenized during 1 min (ultra-turrax homogenizer, ika, artur nogueira, sp). extracts were centrifuged (eppendorf centrifuge 5810 r) at 3050 g for 30 min at 1.0ºc. the supernatant was collected and stored at −18ºc until analysis. apple extracts (100 μl) were added to 3900 μl dpph solution (in methanol), and the reaction ital. j. food sci., vol. 31, 2019 577 mixture was kept in the dark for 24 h. after this period, the absorbances were spectrophotometrically read at 515 nm, and results expressed as mg of trolox equivalent per 100 g-1 of fw. 2.4.9 peroxidase (pod) enzyme activity the pod enzyme activity was determined according to the methodology described by coelho and salas-mellado (2014), adapted for micro quantities. five grams (5.0 g) of apple pulp were homogenized (ultra-turrax homogenizer, ika, artur nogueira, sp) with 0.2 g of polyvinylpyrrolidone (diluted in 20 ml of 0.2 m phosphate buffer ph 6.0) for 3 min. the homogenate was centrifuged (eppendorf centrifuge 5810 r) at 3050 g, for 30 min at 1ºc. the supernatant (apple extract) was collected and maintained at ±4ºc. an aliquot of 45 μl of apple extract was mixed with 230 μl of a mixture of phosphate buffer (1.5 ml, ph 6.5, 0.05 m), distilled water (2.0 ml), hydrogen peroxide (1.0 ml, 0.08 %, v/v) and guaiacol (0.5 ml, 1 %). samples were incubated at 37°c with readings (molecular devices spectramax 190 spectrophotometer) at 470 nm at time zero and after 15 minutes of reaction. the enzymatic activity was calculated based on the amount of protein, where a unit of enzyme activity was defined as the amount of enzyme causing the increase in absorbance of 0.01per min-1. the results were expressed in units of enzyme/μg of protein. 2.4.10 polyphenoloxidase (ppo) enzyme activity ppo enzyme activity was measured by following the methodology described by rai et al. (2011), with some adaptation. briefly, 55 μl of the above apple pulp extract was added to 220 μl of the mixture consisting of phosphate buffer (1.5 ml, ph 6.5, 0.05 m), catechol (0.5 ml, 0.05 m) and water (2.0 ml). the reaction occurred at 37°c with reading (molecular devices spectramax 190 spectrophotometer) at 425 nm at time zero and 30 min. the enzymatic activity was calculated based on the amount of protein where a unit of enzyme activity was defined as the amount of enzyme, which causes an increase in absorbance of 0.01 per min-1. the results were expressed in units of enzyme / μg of protein. 2.4.11 protein protein concentration was determined according to bradford (1976), using the reagents from sigma-aldrich® (bradford reagent, #b6916 and bsa protein standard, #p0834) and the corresponding protocol to assay the protein samples on 96 well plate. the absorbance was read at 595 nm in a spectrophotometer (molecular devices spectramax 190), and the results were expressed in µg of protein per µl. 2.5. respiratory gas analysis the o2 and co2 concentration in the headspace of the hermetically sealed packages were determined every 3 d using oxybaby 6.0 witt-gasetechnik gauge analyzer. the results were expressed as a percentage (%). 2.6. sensory analysis of minimally processed apple slices a set of possible sensory judges, comprising male and female participants (between 20 and 40 years old) who consumed apple frequently, were trained for the evaluation of apple attributes under analysis during 30 days (2 weekly meetings of 15 minutes). the top 17 judges, those who showed better results, were selected. for each period of storage (0 d, ital. j. food sci., vol. 31, 2019 578 3 d, 6 d and 9 d), five samples (12.0±1.0 g each) were identified with three-digit code and randomly provided to the judges. a glass of water was provided for cleaning the palate between one sample and another. flavor, odor, darkening, succulence, firmness and overall quality acceptability were the parameters evaluated using structured scales of 9 cm labelled "bad" to "excellent". this information was converted into scores from 0 to 9, respectively. the evaluation was performed at room temperature and in uniform laboratory conditions. the analysis was submitted to the evaluation by the research ethics committee of the faculty of medicine of the federal university of pelotas and approved for being in accordance with resolution 196/96 of the national health council (caae 48625015.1.0000.5317). 2.7. statistical analysis the experimental design was completely randomized, in a 5 x 4 two-factorial scheme (five treatments x four storage periods) with three replicates. data were analyzed for variance (p≤0.05) using the statistica 7.0 program. for significant results, the means were compared by the lsd least significant difference (p ≤ 0.05) test. the multivariate factorial analysis technique was also applied, using the varimax rotation to improve the interpretation of the factors, using statgraphics centurion xvii software. 3. results and discussion it is important to notice that the samples analyzed at 0 d had been exposed to the corresponding treatment for about eight hours before analysis, causing the treatments to affect the results even at 0 d. changes in the ratio (tss/ta), commonly used to evaluate the fruit quality, can impact apple flavor (piagentini and pirovani, 2017). treatments with w+cc, lc+cc, se+cc and ka+cc resulted in a ratio increase (fig. 1a) over the 9 days of storage. this ratio increase can be explained by the conversion of organic acids in non-acid molecules during the respiratory metabolism (pech et al., 2008), or due to the sugar accumulation by loss of humidity. apples treated with ea+cc showed a decrease on ratio, probably due to the higher acidity (1.18 g of citric acid/100 ml, ph 2.39) of this antioxidant agent. mass loss can compromise the shelf-life of mp products (sanchis et al., 2016). minimal processing exposes fruits and vegetables tissues to a lower water pressure environment and can cause substantial loss of mass. w+cc and lc+cc were the treatments with higher mass loss (fig. 1b). however, these values were lower than 1 %, not enough to change the fruit quality. in a similar study, pizato et al. (2013) and qi et al. (2011) obtained higher values for mass loss. significant mass loss has physiological effect and can compromise fruits and vegetables appearance, texture and nutritional quality (sanchis et al., 2016). results for pulp firmness (fig. 1c) were not affected by the loss of mass, although slight variations occurred over the 9 days of storage. according to gang et al. (2015), the use of cacl2 has an essential role in the maintenance of pulp integrity and firmness due to its action as bio-membrane stabilizer, supporting the integrity of the membrane cell wall. besides, appropriated processing and storage conditions and use of antioxidants contribute to prevent pulp softening. ital. j. food sci., vol. 31, 2019 579 figure 1. effect of sodium erythorbate 5.0 % (se) + calcium chloride 1.0 % (cc), erythorbic acid 3.0 % (ea) + cc and kojic acid 0.07 % (ka) + cc on physicochemical variables: mean values for ratio (a), mass loss (b), pulp firmness (c) and pulp color variables: a* (d), hue angle (e) and browning index (f) for minimally processed ‘royal gala’ apple recorded at 0 d, 3 d, 6 d and 9 d, at 4.0ºc. negative control (nc) consisted of distilled water (w) + cc, and positive control (pc) consisted of l-cysteine chloride 0.6 % (lc) + cc. the vertical bar indicates the least significant difference (lsd) with (p≤0.05). color change in the pulp of the mp apples is associated with the oxidative processes due to the rupture of the cell membranes during product preparation. green-red chromatic coordinate (a*) is an important parameter for the study of browning since brown color represents a combination between green (-a *) and red (+a *) (kim and lee, 2008). the green-red chromatic coordinate (a*) values were high for w+cc treatment in all periods of storage (fig. 1d), corresponding to a more reddish apple pulp. lower values of a* coordinate (yellow to green fruits) were observed for treatments lc+cc, se+cc, ea+cc and ka+cc until day 6. however, the result of a* coordinate value for ka+cc treatment increased between day 6 and day 9. this increase on reddish color of apples treated with ka+cc can be due to the reversible linkage of kojic acid and ppo enzyme (chen et al., 1991). the process of oxidative coloration is triggered by the cell membrane rupture and 35 45 55 65 75 85 r at io (s st /t itr at ab le a ci di ty ) w+cc (nc) lc+cc (pc) se+cc ea+cc ka+cc lsd = 1.79(a ) 2.5 3.0 3.5 4.0 4.5 pu lp f ir m ne ss (n ) lsd = 0.11(c) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 m as s lo ss (% ) lsd = 0.02(b) -5 -4 -3 -2 -1 0 pu lp c ol ou r a* lsd = 0.29(d) 90 95 100 105 110 0 3 6 9 pu lp c ol ou r hu e an gl e (º h) period of storage (days) lsd = 0.99(e) 15 25 35 45 55 65 0 3 6 9 pu lp c ol ou r br ow ni ng in de x period of storage (days) lsd = 3.32(f) ital. j. food sci., vol. 31, 2019 580 the resulting mixture of polyphenol substrates with ppo (toivonen and brummell, 2008). ppo enzyme when in contact with oxygen catalyzes two reactions: (1) hydroxylation of monophenols in diphenols and (2) oxidation of diphenols in quinones. the first is relatively slow and results in colorless products. the second one is relatively fast, and the resulting quinones are colored. subsequent reactions of the quinones lead to the accumulation of melanoidin, which is the brown pigment (cortellino et al., 2015). fig. 1e displays the hue angle values obtained for the different antioxidant treatments. the treatments lc+cc, se+cc, ea+cc and ka+cc resulted in a yellow-green hue throughout the nine days of storage. on the other hand, apples treated only with water and calcium chloride (w+cc), showed a tendency to brown, from the beginning of storage period, intensifying slightly over the 9 days of storage. these antagonistic results point to the importance of the antioxidant to inhibit the darkening of the pulp. calcium chloride, besides maintaining pulp firmness, can also be responsible for the maintenance of color. this effect is related to its action on the prevention of the cellular membrane degradation, with consequent reduction in substrates release for ppo activity (toivonen and brummell, 2008), protecting, therefore, the color of mp products (perez-cabrera et al., 2011). regarding the browning index (fig. 1f), the treatments lc+cc, se+cc, ea+cc and ka+cc maintained the pulp color, without statistical differences on the 6-day and 9-day of evaluation. overall results for the color measurements revealed that all treatments maintained the apple pulp color satisfactorily, despite the differences between the mechanism of action of the antioxidants. l-cysteine has the ability to inhibit the enzymatic browning caused by ppo, through a competitive mechanism that captures o-quinones through the formation of cysteinyl adducts (richard-forget et al., 1992). the subgroup erythorbates comprise two compounds, erythorbic acid and sodium erythorbate. erythorbic acid is a stereoisomer of ascorbic acid, with strong reducing properties, differing only from the relative position of the hydroxyl groups and of the hydrogen in the fifth carbon atom in the molecule (carocho et al., 2018). this acid reacts with oxygen and can thus remove it from a closed system (lee et al., 2012). sodium erythorbate antioxidant activity is due to the quenching of singlet oxygen, hydrogen donation and as a reducing agent (carocho et al., 2018). erythorbates have essentially the same function (fidler et al., 2004) and are widely used as alternatives to ascorbic acid and its salts in products that the action of vitamin c is not necessary (martin-belloso and soliva-fortuny, 2011). the antioxidant properties of kojic acid can be attributed to its ability to inhibit the thyroxinase and polyphenoloxidase enzymes, and its chelating activity (mitani et al., 2001). the mechanisms of action of kojic acid are similar to those of l-cysteine and ascorbic acid since they occur through interference with the absorption of o2 required for the enzymatic reaction, reduction of the quinones to diphenols, or the combination of both (chen et al., 1991). the antioxidant activity (dpph scavenging) of se+cc treatment was higher than all the other treatments during the entire period of storage (fig. 2a), with significant decrease (24.3 %) from day 0 to day 9. this result can be attributed to sodium erythorbate ability to prevent oxidation (figueiredo et al., 2014; reische et al., 2002); or capture of the oxygen present in the medium through stable chemical reactions, preventing this oxygen of acting as propagator of auto-oxidation or as synergist in the regeneration of primary antioxidants (ramalho and jorge, 2006). on the other hand, the ea+cc treatment showed a very similar activity to se+cc treatment during the first six days of storage. however, a marked drop of dpph activity (35.8 %) was observed between day 6 and day 9. this decrease can be attributed to the non-enzymatic degradation of erythorbic acid in aerobic conditions (corzo‐martínez et al., 2012; cropotova et al., 2016). other treatments, w+cc, lc+cc and ka+cc, although oscillating and presenting statistical ital. j. food sci., vol. 31, 2019 581 difference between them, maintained a mean dpph activity of 91.9 mg trolox per 100 g of fw. ‘royal gala’ apple treated with se+cc and ea+cc showed the highest values of total phenolic content (fig. 2b), which was maintained throughout the storage period. other treatments (w+cc, lc+cc and ka+cc) showed total phenolic compounds contents within the values reported in the literature for apples, 50 mg per 100 g fw to 380 mg per 100 g fw (ceymann et al., 2012). sodium erythorbate was reported to preserve the phenolic compounds in mp oyster mushroom (ventura-aguilar et al., 2017). ascorbic acid is a well-known antioxidant that prevents the polyphenol degradation due to its reducing action (gil et al., 1998). thus, it can be inferred that its stereoisomer, erythorbic acid, has a similar capacity, effectively preserving the polyphenols content as observed in this experiment. results of phenolic compounds and antioxidant activity in apples were correlated. treatments that preserved the largest amount of phenolics were the same ones that maintained the highest antioxidant activity. besides the antioxidant capacity, phenolic compounds are also responsible for apples flavor (zhang et al., 2017). in addition, they have biological functions such as immunomodulatory and antiinflammatory properties in humans and animals (zhang et al., 2017). therefore, the preservation of these properties is important to maintain the quality of the fruit and, consequently, the health benefit for consumers. figure 2. effect of sodium erythorbate 5.0 % (se) + calcium chloride 1.0 % (cc), erythorbic acid 3.0 % (ea) + cc and kojic acid 0.07 % (ka) + cc on dpph activity (a), total phenolic content (b), ppo (c) and pod (d) activities for minimally processed ‘royal gala’ apple recorded at 0 d, 3 d, 6 d and 9 d, at 4.0ºc. negative control (nc) consisted of distilled water (w) + cc, and positive control (pc) consisted of l-cysteine chloride 0.6 % (lc) + cc. the vertical bar indicates the least significant difference (lsd) with (p≤0.05). 0 100 200 300 400 500 d pp h (m g t ro lo x pe r 1 00 g -1 fw ) w+cc (nc) lc+cc (pc) se+cc ea+cc ka+cc (a ) lsd = 5.58 75 100 125 150 175 200 t ot al p he no ls (m g c a e p er 1 00 g -1 fw ) (b) lsd = 3.66 0 5 10 15 20 0 3 6 9 pp o (u / µ g of p ro te in ) period of storage (days) (c) lsd = 0.59 0 10 20 30 40 0 3 6 9 po d (u / µ g of p ro te in ) period of storage (days) (d) lsd = 1.24 ital. j. food sci., vol. 31, 2019 582 regarding the enzymatic activity, changes in ppo activity (fig. 2c) were treatmentdependent. apples treated with se+cc, ea+cc and lc+cc showed the lowest enzymatic activity. this result was reflected in the preservation of total phenolics, antioxidant activity, and maintenance of mp fruit color. inactivation of ppo by ea+cc and lc+cc can also be attributed to the low ph of these solutions (2.22 and 1.62 respectively). as the ideal ph for ppo activity is 7.0, by reducing the ph its activity decreases rapidly, suggesting that the effect of ph is very important for most solutions that aim to inhibit the browning of the mp pulp. similar results were obtained by tsouvaltzis and brecht (2017) studying “russet burbank” potatoes where the immersion of mp tubers in h2so4 (< 0,04 %), ph 2.39, reduced the ppo activity in comparison to control samples. they attributed the reduction of ppo activity to the deviation from the ideal ph (5 to 7). another mechanism for browning inhibition of lcysteine is attributed to its conjugation with o-quinones, forming colorless compounds, or by reducing o-quinones to the precursor phenolic compounds (cilliers and singleton, 1990). according to richard-forget et al. (1992), these conjugated compounds may act as competitive inhibitors of ppo. however, they advert that in the presence of an excess of quinones, after all cysteine is consumed, the quinones can react with the cysteine-quinone addition compounds, forming violet pigments (richardforget et al., 1992), which were not observed in our experiment. nevertheless, se+cc (ph 7.0) inactivated almost completely the enzymatic activity regardless of the ph value of the antioxidant solution. this antioxidant agent has no direct effect on ppo activity, but rather on browning since this compound acts as a free radical scavenger and changes the redox potential, reducing o-quinones to diphenols (mosneaguta et al., 2012). ka+cc treatment did not completely inhibit the enzymatic activity, probably because this acid could not act satisfactorily in one of these mechanisms: prevent the absorption of the o2 required for the enzymatic reaction; to reduce the quinones to diphenols; or to the combination of the two previously mentioned (chen et al., 1991). besides the positive effect provided by antioxidants, calcium chloride, which has similar properties to sodium chloride, generates chlorine dioxide under acidic conditions, which is involved in the ppo inhibition (gomes et al., 2014). this observation suggests that the enzymatic inactivation results from synergism between antioxidants agents and cacl2. although ppo is recognized as the main enzyme related to enzymatic browning in apples, it is also necessary to study the changes in pod enzymes activity, since these enzymes may also contribute to change the color of this fruit (jang and moon, 2011). results obtained for pod activity (fig. 2d) and ppo activity (fig. 2c) were similar. apples treated with lc+cc showed the highest enzymatic activity at day zero, which decreased at day 3 and day 6, increasing at day 9. pod activity was close to zero for se+cc and ea+cc treatments. since these antioxidants are structurally similar to ascorbic acid (aa), this effect is in accordance with those of jang and moon (2011), who reported that the presence of aa effectively reduced pod activity in mp apples. reduction of pod activity in fruits treated with aa could be the result of lower oxidative stress on their surface due to the antioxidant nature of aa or the pod-enzyme-hydrogen-donor complex formation (saba and sogvar, 2016). these results suggest that treatments containing sodium erythorbate and erythorbic acid may be an efficient way to maintain the quality of mp apples without pulp browning during storage. concerning the respiratory gases, oxygen (o2) and carbon dioxide (co2) in the headspace of the packages, all the treatments showed a similar pattern, with a decrease in o2 (fig. 3a) and increase in co2 (fig. 3b) over the 9 d of storage. the o2 concentration for all the treatments (lc+cc, se+cc, ea+cc, ka+cc) remained lower than the control treatment (w+cc), possibly due to the higher metabolism of apples not treated with antioxidants. ital. j. food sci., vol. 31, 2019 583 figure 3. respiratory rate of minimally processed ‘royal gala’ apples treated with sodium erythorbate 5.0 % (se) + calcium chloride 1.0 % (cc), erythorbic acid 3.0 % (ea) + cc and kojic acid 0.07 % (ka) + cc based on concentration of o2 (a) and concentration of co2 (b) in the headspace of the pack, recorded at 0 d, 3 d, 6 d and 9 d, at 4.0ºc. negative control (nc) consisted of distilled water (w) + cc and positive control (pc) consisted of l-cysteine chloride 0.6 % (lc) + cc. the vertical bar indicates the least significant difference (lsd) with (p≤0.05). the respiratory process consumes o2 and releases co2 because of the oxidation of carbohydrates, lipids and proteins, which are present in the product. in addition, higher respiration rates indicate faster overall metabolism and deterioration (venturaaguilar et al., 2017). according to manurakchinakorn et al. (2012), the reduction of o2 and the increase of co2, a consequence of the respiratory process, is detectable in mp fruits even if stored in closed containers permeable for gas exchange. interestingly, for unknown reasons, apples treated with lc+cc accumulated in the packs an average of 26.9 % more co2 than the apples treated with other antioxidants. sensory analysis showed that apples treated with the antioxidants lc+cc, se+cc and ea+cc had no noticeable darkening in the pulp (fig. 4a), being classified as very good and excellent. on the other hand, apples treated with w+cc presented a considerable increase in browning during the nine days of evaluation, reaching the marketing limit from the sixth day. the ka+cc also showed increased browning, however with lower intensity. flavor parameter (fig. 4b) was only affected for apples treated with lc+cc. acceptability of apples treated with lc+cc was lower than for other treatments, already on the first day of evaluation. this behavior was intensified along the storage periods to the point of being between the marketing and inedible limits. the presence of calcium chloride did not significantly modify the flavor of the samples, which corroborates that this salt does not modify the flavor of the fruits. due to the high acidity of the ea+cc treatment, it was necessary to carefully reduce the droplets of this antioxidant on the epidermis of the apple slices, since when this happened, the evaluators noticed and reported the unpleasant sensation caused by this antioxidant solution. this peculiarity makes this treatment a secondary option of using this product. considering the odor attribute, apples treated with lc+cc also showed low acceptability (fig. 4c). evaluators were able to perceive the sulfur-containing compounds in the samples containing lcysteine, which agrees with the results obtained by perez-gago et al. (2006). other treatments, although with lower intensity, also had a decrease in the odor attribute along the storage. this decrease can be attributed to the volatilization of compounds responsible for the aroma of the fruit due to damages caused in the pulp during the minimal processing. the attributes succulence and firmness (data not presented) did not present 18 19 20 21 22 23 0 3 6 9 r es pi ra to ry ra te o 2 (% ) period of storage (days) w+cc (nc) lc+cc (pc) se+cc ea+cc ka+cc (a ) lsd = 0.079 0.5 1.0 1.5 2.0 0 3 6 9 r es pi ra to ry ra te c o 2 (% ) period of storage (days) (b) lsd = 0.032 ital. j. food sci., vol. 31, 2019 584 statistical difference between the treatments, being in the range between very good and excellent. when including all evaluated attributes and score the overall quality, apples treated with se+cc, ea+cc and ka+cc presented high acceptability since all evaluated attributes were satisfactory (fig. 4d). figure 4. sensorial characteristics of minimally processed ‘royal gala’ apple treated with sodium erythorbate 5.0 % (se) + calcium chloride 1.0 % (cc), erythorbic acid 3.0 % (ea) + cc and kojic acid 0.07 % (ka) + cc, and recorded at 0 d, 3 d, 6 d and 9 d; at 4.0ºc: pulp browning (a); characteristic flavor (b), characteristic odor (c) and overall quality (d). negative control (nc) consisted of distilled water (w) + cc, and positive control (pc) consisted of l-cysteine chloride 0.6 % (lc) + cc. the vertical bar indicates the least significant difference (lsd) with (p≤0.05). opposite situation occurred with w+cc treatment, mainly due to the appearance of undesirable brown pigments on the surface of the slices due to the oxidation processes. lc+cc treatment was able to inhibit pulp browning completely, however, the apple taste and odor significantly changed, leading to low acceptability of apples treated with this antioxidant. the changes caused by l-cysteine in the odor are attributed to enzymatic reactions, mainly the degradation of methionine, cysteine and derivatives. these enzymatic degradations lead to the formation of volatile compounds containing aliphatic sulfur, such as thiols and polysulfides (varlet and fernandez, 2010). in view of these results, the application of sodium erythorbate and erythorbic acid proved to be viable alternatives for the maintenance of the sensorial and physical-chemical attributes of 0 2 4 6 8 w+cc (nc) lc+cc (pc) se+cc ea+cc ka+cc pu lp b ro w ni ng 0 storage days 3 storage days 6 storage days 9 storage days poor: inedible fair: limit of usability good: limit of marketability very good excellent (a ) lsd = 0.19 1 3 5 7 9 w+cc (nc) lc+cc (pc) se+cc ea+cc ka+cc c ha ra ct er is tic o do r antioxidants excellent very good good: limit of marketability fair: limit of usability poor: inedible (c) lsd = 0.28 1 3 5 7 9 w+cc (nc) lc+cc (pc) se+cc ea+cc ka+cc c ha ra ct er is tic f la vo r excellent very good good: limit of marketability fair: limit of usability poor: inedible (b) lsd = 0.18 1 3 5 7 9 w+cc (nc) lc+cc (pc) se+cc ea+cc ka+cc o ve ra ll qu al ity antioxidants excellent very good good: limit of marketability fair: limit of usability poor: inedible (d) lsd = 0.28 ital. j. food sci., vol. 31, 2019 585 apples evaluated in this study. in addition, erythorbic acid has a relatively low cost because calcium 2-ketogluconate is easily converted during glucose fermentation (lee et al., 2012). this antioxidant is also reported to potentiate the non-heme iron uptake (fidler et al., 2004), helping to provide the nutritional needs of iron (singh et al., 2016). according to halagarda and suwala (2018), the importance of sensory analysis at every stage of the product development or improvement process is due to significant cost and failure reduction after product launch. moreover, the fulfilment of expectations, especially the sensorial ones, leads to consumer satisfaction, increasing the likelihood of commercialization of the product (grunert, 2002). the factorial analysis allowed us to verify the most important factors for fruit quality. in this analysis, five factors explained 70.86 % of the total variability of the experiment (data not shown). the factor 1 (termed pulp color) explained 26.25 % of total variability (fig. 5a), and showed higher correlation coefficients with the variables index of darkening (0.963), b* (0.946), chroma (0.942) and ºhue (-0.875). on the positive axis of the abscissa are the variables with the higher positive correlation coefficients values (index of darkening, b*, sensorial darkening and chroma) and on the negative axis is the variable with the higher negative correlation coefficient value (hue). it appears that these variables evolved together, being classified in the same factor, which indicates that the differences were detected by the sensory panel of judges. regarding the distribution of the treatments in the factors, it can be observed that the treatment w+cc (negative control), as expected, resulted in slices of apples with higher levels of darkening, being grouped in the positive axis of the abscissa on the graph. the other treatments, with a low level of darkening, were grouped predominantly on the negative axis of the abscissa. the factor 2 (termed sensorial quality), which explains 20.55 % of total variability, showed higher correlation coefficients with the variables odor (0.909), taste characteristics (0.882) and overall quality (0.865). all of them were located on the positive axis of the ordinate. it can be observed that the values of sensory quality in apples treated with se+cc, ea+cc and ka+cc, resulted in a good level of acceptability by the sensory panel of judges. on the other hand, the application of lc+cc presented lower sensory quality. in relation to the storage periods (0 d, 3 d, 6 d and 9 d) it can be observed (fig. 5b) that periods in which the mp fruit presented the best quality are located on the positive axis of the ordinate axis, and mp fruits of inferior quality are located on the negative axis of the ordinate. -18 -15 -12 -9 -6 -3 0 3 6 9 12 -12 -9 -6 -3 0 3 6 9 12 15 18 21 fa ct or 2 (2 0. 55 % ): se ns or ia l qu al ity factor 1 (26.25 %): pulp color w+cc (nc) lc+cc (pc) se+cc ea+cc ka+cc (a) ital. j. food sci., vol. 31, 2019 586 figure 5. factorial analysis of minimally processed ‘royal gala’ apples treated with sodium erythorbate 5.0 % (se) + calcium chloride 1.0 % (cc), erythorbic acid 3.0 % (ea) + cc and kojic acid 0.07 % (ka) + cc (a) recorded at 0 d, 3 d, 6 d and 9 d (b); at 4.0ºc. factor 1: color and factor 2: sensorial quality after varimax orthogonal rotation. negative control (nc) consisted of distilled water (w) + cc, and positive control (pc) consisted of l-cysteine chloride 0.6 % (lc) + cc. the vertical bar indicates the least significant difference (lsd) with (p≤0.05). this result indicates that mp apples slices lose quality as the refrigerated storage period increased. however, this reduction in quality is a normal process, as many compounds are metabolized, resulting in senescence and cell death. 4. conclusions apple browning is of high concern for the industry of minimally processed fruits since it has a high impact on color, flavor and nutritional value. our results showed that treatment with either 5 % sodium erythorbate or 3 % erythorbic acid, associated with 1 % calcium chloride, controlled the apple pulp browning and preserved the phenolic compounds and the antioxidant activity for up to 9 days under cold storage. besides, both antioxidants significantly inhibited the peroxidase and polyphenoloxidase enzymes and preserved the sensorial quality, which was found to be equivalent to the fresh fruit. thus, the treatment of mp ‘royal gala’ apples with 5 % sodium erythorbate or 3 % erythorbic acid, associated with 1 % calcium chloride, can be suitable to extend the product shelf-life. acknowledgements the authors acknowledge the brazilian government (embrapa clima temperado, capes and cnpq) for funding this study, including a ph.d. scholarship, and rasip agro pastoril s/a for providing logistic support and experimental material. -18 -15 -12 -9 -6 -3 0 3 6 9 12 -12 -9 -6 -3 0 3 6 9 12 15 18 21 fa ct or 2 (2 0. 55 % ): s en so ria l q ua lit y factor 1 (26.25 %): pulp color 0 da ys of stora ge 3 da ys of stora ge 6 da ys of stora ge 9 da ys of stora ge (b) ital. j. food sci., vol. 31, 2019 587 references ali s., khan a.s. and malik a.u. 2016. postharvest l-cysteine application delayed pericarp browning, suppressed lipid peroxidation and maintained antioxidative activities of litchi fruit. postharvest. biol. technol. 121:135 doi: doi.org/10.1016/j.postharvbio.2016.07.015 andersen f.a. 1999. final report on the safety assessment of ascorbyl palmitate, ascorbyl dipalmitate, ascorbyl stearate, erythorbic acid, and sodium erythorbate. int. j. toxicol. 18(3_suppl):1. doi: doi.org/10.1177/109158189901800303 bradford m.m. 1976. a rapid and sensitive method for the quantitation of 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abbey et al., 2014). dairy products, cereals, candies, jellies, ice-cream, soft drinks, yogurts, fillings, liqueurs, and powdered juices are the most common food items in which this dye is added (meinicke and jorge, 2008; yuan et al., 2016). overuse of synthetic colorants is a major source of food intoxication (koutsogeorgopoulou et al., 1998). there are various adverse health effects associated with ingesting excess amounts of synthetic colorants, such as cancer, genetic diseases, etc. (tsuda et al., 2001; das and mukherjee, 2004), asthma, abortions, weakened immune systems, and allergic reactions (geoffrey and felix, 1991; hinton, 2000; bhattacharjee, 2014). additionally, synthetic colorants have been associated with behavioral effects in children, such as hyperactivity, decreased iq scores, and boosted aggression (geoffrey and felix, 1991; hinton, 2000; bhattacharjee, 2014). synthetic sy specifically has been linked with anaphylactic reactions and cardiovascular complications (angioedema, vasculitis, and thromboxane synthesis inhibition) in individuals who present a sensitivity to the compound (sardi et al., 2010). studies have shown that most synthetic colorants bind directly to dna and cause structural and numerical incongruities (hamdy et al., 2000; mpountoukas et al., 2010). in addition, it has been found that semi-toxic doses of sunset yellow leads to changes in total lipid storage of the body when exposed to animal models. as lipids have structural functions in biological membranes of the body, this might stimulate their metabolism and may cause potential liver injuries such as necrosis (mathur et al., 2005). similar to most other developing countries, it is a common scenario in bangladesh that industrial and non-industrial sectors are involved in food production and processing activities. food industry uses various synthetic colorants, which are the most interesting groups of food additives as the colorful food products attract consumers (kucharska and grabka, 2010). however, their range of use and amounts are restricted across the world (sun et al., 2013). the non-industrial sector produces twoto three-times the amount of food items compared to the industrial sector. unfortunately, quality control systems are lacking in the non-industrial sectors, leading to high production of substandard food products potentially compromising the health of consumers and thus, indirectly leading to a financial burden for the nation. based on the results of the international research and recommendation of codex committee on food additives and contaminants (ccfac), the acceptable daily intake (adi) value of food colorants is set across the world (bessonov et al., 2011; ganesan et al., 2011). the adi of permitted food colorants varies from 0.1 mg/kg body weight (erythrosine red) to 25mg/kg body weight (fast green fcf) (swaroop et al., 2011). however, there is warranted concern over the amount of synthetic colorants in food products as certain companies exceed the upper limit recommended by the adi. therefore, it is necessary to monitor the total daily intake of all food colorants to ensure the adi is not exceeded (joint, on food additives, organization and others, 1965, 1991). due to the wide industrial use of food dyes to color foods it is important to determine the amounts of synthetic colorants commonly added to foods in bangladesh. current analytical detection methods are costly and require substantial resources that are not available in bangladesh. therefore, the present study was conducted to determine the color range of sy in different brands of orange jellies collected from different shops of tangail city, bangladesh, using a simple and cost effective high-performance liquid chromatography (hplc) method. the analytical determination of this particular color ital. j. food sci., vol. 31, 2019 186 may aid in establishing a method for detecting synthetic colorants and contribute to overall quality assurance and consumer safety. 2. material and methods 2.1. chemicals and reagents sodium acetate 97%, hplc-grade chloroform, n-hexane, and acetonitrile were obtained from merck (darmstadt, germany). glacial acetic acid was procured from sigma chemical co. (darmstadt, germany). diethyl acetate from rci lifescan ltd. was used. deionised water (18.2 mω) used for chromatography processing was procured from a barnstead nanopure water purification system (barnstead, usa). sunset yellow fcf (e110) was purchased from rayner, co. ltd. (london, england). 2.2. samples a total of 54 samples from six brands of orange jellies (research raw material) were purchased from three stores in tangail city, tangail, bangladesh after getting verbal consent from the shopkeepers. all samples were considered valid based on their expiry dates. the weights of the samples were 200 gm to 500 gm. the collected samples were preserved in refrigerator at 4 °c in the laboratory of the department of food technology and nutritional science, mawlana bhashani science and technology university (mbstu). ethical approval was taken from the research cell of mbstu. 2.3. mobile phase the mobile phase was prepared using the modified method of pylypiw and grether (2000). it consisted of a mixture of 3.0 mm acetate buffer (ph ~4.00) and hplc-grade acetonitrile with a ratio of 17:3. acetate buffer was prepared by mixing of 1.0 ml of glacial acetic acid and 1.0 g of sodium acetate trihydrate in 1.0 l de-ionized water and mixed well. the mixture was filtered with a filter membrane (pore size 0.2 µm). 2.4. preparation of standard solution approximately100 mg of anhydrous sunset yellow was taken in a 25 ml volumetric flask. ten ml 85% aqueous acetonitrile was added to the volumetric flask and was shaken well. finally, 85% aqueous acetonitrile was added up to mark. the solution was filtered with syringe filter. the standard stock solution-1 was labeled as 4 mg/ml. approximately 5 ml of stock solution-1 was taken in 50 ml volumetric flask and mobile phase was added up to the mark. the standard solution-2 was labeled as 0.4 mg/ml standard solution. from the stock solutions, working standard solutions 0.0, 1.0, 5.0, 10.0, 20.0, 40.0 µg/ml were prepared by dilution of aliquots. the solution was filtered through sample filters (pore size 0.2 µm) prior to inject into the column. 2.5. preparation of sample solution approximately 1.0 gm of orange jelly was weighted accurately and placed in a conical flask and it was made to 10 ml by adding aqueous 85% acetonitrile solution and mixed well by vigorous shaking for 10 minutes. about 2.0 ml of the solution was filtered through sample filter (pore size 0.2 µm) and the filtrate was then diluted 5 times and placed in an ital. j. food sci., vol. 31, 2019 187 eppendorf tube. finally, 20 µl was injected into the hplc column. the concentration of injected sample solution was 20 µg/ml. calculation of sample concentration: ! !"×!"" !" !" !"×!""" !" = 0.02 mg/ml=20µg/ml 2.6. sample solution preparation for spiked/recovery assay approximately 2.0 gm of orange jelly and 1.0 mg of sy was weighted accurately and placed in a conical flask and 85% aqueous acetonitrile solution was added to it to make 20 ml solution and mixed well by vigorous shaking for 10 minutes. about 2.0 ml of the solution was filtered through sample filter (pore size 0.2 µm) and the filtrate was then diluted 5 times and placed in an eppendorf tube. finally, 20 µl was injected into the hplc column. the concentration of injected sample solution was 20 µg/ml. 2.7. chromatographic analysis the chromatographic system consisted of a shimadzu isocratic pump, a degasser, column, oven, a uv-vis detector, and a lc workstation class-vp for data acquisition and analysis. each of orange jelly samples of 1.0 g was diluted 1:10 with mobile phase and then the sample was again diluted 1:5 with mobile phase. after that the solution was transferred into dry eppendorf tube. the clear aqueous solution was filtered through a ptfe syringe filter. then the solution was transferred to the dry hplc vials. 20 µl of the sample was injected into the injector. for the chromatographic analysis, a luna 5µ c18 (2) 100a column (250 × 4.6 mm) was used and the column temperature was set at 33 °c. the sunset yellow analysis was performed with isocratic solvent system using sodium acetate and acetic acid buffer (ph ~4.0)/acetonitrile17:3 with a flow rate of 1.0 ml/min. 2.8. sy identification and quantification optimum absorption wavelength for sy color was evaluated before and using standard solutions with uv-spectrophotometer. the determined wavelength for the analysis of sy was 480 nm. several runs were made to determine the retention time for the analysis. the retention time for this color was used for the identification of the color present in different brands of orange jellies. quantification of the studied colors was done by external standard calibration. five level analytical curves (0.00, 1.0, 5.0, 10.0, 20.0, 40.0 µg/ml) were used and the mean of 3 injections of each standard was used to represent each calibration point. by plotting analytes (y) against the concentration (µg/ml) of the color the peak areas were measured. to determine the slope, y-intercept and the correlation coefficients of the standards plots, least square linear regression analysis was used. limit of detection (dl) and limit of quantification (ql) were determined by considering 3 and 10 times the signal to noise ratios respectively estimated by the regression lines as mentioned in the previous report (macdougall et al. 1980). for hplc method validation the performance parameters, i.e., precision, linearity, limit of detection, limit of quantification, the expanded uncertainty were calculated. by spiking known amounts of the studied colors to the unprocessed sample and comparing the output with the same sample without spiking, recovery evaluations were carried out. ital. j. food sci., vol. 31, 2019 188 recoveries were calculated by differences of concentrations and were expressed as percentages. 2.9. statistical analysis each test was performed in triplicate. the descriptive analyses (means, median, standard errors coefficient of variation) were summarized. data were expressed as mean±standard error (se). one-way analysis of variance (anova) was carried out using spss software version 20 at a significance level of 5%. the least significant difference (lsd) test was used to detect differences in means. 3. results and discussion 3.1. analysis of chromatogram numerous analytical methods have been developed for analyzing synthetic colorants in response to the concern regarding their adverse effects. the most preferred technique for quantitative determination of the colorants is using hplc, as it is relatively simple, cost effective, and has less environmental impact due to fewer hazardous chemicals used. in this study, an efficient and accurate hplc analytical method was used for the determination of sunset yellow in different brands of orange jellies collected from local markets of tangail city, bangladesh. this method was simple to use, had good operational stability, and gave reliable and reproducible results. the developed hplc method was applied to analyze the sy color range in orange jellies. the retention time of the sunset yellow standard was 5.62±0.2 minutes and approximate time was set at 10 minutes. (figs. 1 and 2). the calibration curve (fig. 3) for sy was obtained by plotting the peak areas of different concentrations of the working standard solutions (0.0, 1, 5, 10, 20 and 40 µg/ml). the recovery range was 96-131%. table 1 describes the analytical characteristics of the hplc method. there was a strong linear relationship (r2 = 0.999) obtained between the concentration of sy and the peak area within the hplc chromatogram at 480 nm (fig. 3). the detection and quantification limits were calculated as 1.14 mg/100 ml and 3.46 mg/100 ml, respectively. figure 1. hplc chromatogram of 10 µg/ml sunset yellow standard solution with a retention time of 5.625 min. minutes 0 1 2 3 4 5 6 7 8 9 10 vo lts -0.002 0.000 0.002 0.004 vo lts -0.002 0.000 0.002 0.004 1. 21 7 3. 00 0 3. 22 5 5. 62 5 6. 02 5 6. 09 2 6. 35 0 6. 65 8 9. 91 7 detector a (480nm) sunset yellow std 10ugper ml 090715 sunset yellow std 10ugper ml retention time ital. j. food sci., vol. 31, 2019 189 figure 2. hplc chromatogram of sunset yellow present in orange jelly brand-1 (obtained from shop 1) with a retention time of 5.433 min. figure 3. calibration curve for the sunset yellow standard. table 1. analytical characteristics of hplc method. parameter value accuracy 107±14.3 slope 4020 intercept 0 linearity range 1.32 µg/ml to 40 µg/ml correlation coefficient 0.999 steyx 1390 lod 1.14 mg/100 ml loq 3.46 mg/100 ml minutes 0 1 2 3 4 5 6 7 8 9 10 vo lts -0.005 0.000 0.005 vo lts -0.005 0.000 0.005 0. 15 8 0. 32 5 0. 51 7 0. 71 7 4. 65 0 4. 80 0 5. 01 7 5 .4 33 6. 15 0 6. 53 3 7. 20 0 7. 70 0 7. 88 3 8. 07 5 8. 64 2 8. 85 0 detector a (480nm) sunset yellow p3 080715 sunset yellow p3 01 retention time y = 4,0206x r² = 0,99949 0 20 40 60 80 100 120 140 160 180 0 5 10 15 20 25 30 35 40 45 pe ak a re a × 1 03 concentration of std solution in µg/ml ital. j. food sci., vol. 31, 2019 190 3.2. calculation of % recovery of sy in spiked samples approximate 500 µg of sy was added to the 1.0 g of unspiked sample containing sy 4049.41 µg/g. the observed value of sy of the spiked sample was calculated 4560.60 µg/g. so the recovered added sy was 511.19 µg. finally, the % recovery was 102.23%. 3.3. determination of sy in samples the results in tables 2 and 3 show the levels of the sy from the six brands of orange jellies collected from three different shops with different batches. after hplc analysis, majority of the orange jellies contained the same synthetic color as was mentioned on their respective product labels. chromatogram’s peak from five out of the six orange jelly brands, were identified as sy matched with the sy standard. no peak in the chromatogram of the samples of brand 6 was matched to the peak of sy standard. table 2. level of sy (mg/100 g) in different brands of orange jellies. orange jelly concentration of sunset yellow (mg/100 g) mean±sd (n = 3) p-value shop sample 1 sample 2 sample 3 brand 1 1 25.10 27.40 28.70 27.07±1.49 0.118 2 17.30 15.20 25.80 19.43±4.58 3 25.70 28.20 17.90 23.93±4.39 brand 2 1 42.50 40.40 42.00 41.63±0.90 0.351 2 37.33 44.30 39.20 40.28±2.95 3 39.20 40.50 37.90 39.20±1.06 brand 3 1 10.10 8.70 8.89 9.23±0.62 0.833 2 8.89 10.79 7.49 9.06±1.35 3 9.02 9.25 8.07 8.78±0.51 brand 4 1 19.40 18.50 17.40 18.43±0.82 0.109 2 13.8 17.00 18.70 16.50±2.03 3 18.00 20.3 19.70 19.33±0.97 brand 5 1 12.90 13.60 13.30 13.27±0.29 0.003 2 13.10 11.30 12.10 12.17±0.74 3 9.95 11.20 10.10 10.42±0.59 brand 6 1 nd nd nd nd 2 nd nd nd nd 3 nd nd nd nd *nd = not detected. the concentrations of sy in the studied orange jellies varied between 8.78 to 41.63 mg/100 g, depending on the brand in table 2. among the collected samples of brand 1, sy concentration (mean ± sd) of two samples were higher than the bsti value as a whole, but the difference was not statistically significant (p = 0.118). sy values in all samples of brand 1 exceeded the recommended value of eu. when compared to the sy concentration of brand 2, all values exceeded the bsti and eu recommended values. no samples of brand 3 exceeded the eu and bsti values. on the other hand, all samples of brand 4 exceeded ital. j. food sci., vol. 31, 2019 191 the eu recommended value, but did not exceed the bsti value. the sy values in brand 5 sample were within the bsti value, but only one was within the eu value. however, brand 6 showed no trace of sy. a discrepant finding compared to the product label from brand 6 was observed. added synthetic color can be reduced during manufacturing process due to formation of inorganic salts as byproducts e.g. nacl (kirschbaum et al., 2003). orange jellies from brand 2 contained the highest amount (41.63 mg/100 g) of sy in table 3, which was 2 times more than the maximum value of bsti and 4 times from the eu value (fig. 4). again, when comparing among different brands, sy showed significant difference among the brands (p = 0.003) in table 3. among the statistical parameters, the mean value, standard errors, and coefficient of variance were 19.5 mg/100 g, 13.0 and 76% respectively. table 3. summary of concentration of sy (mg/100 g) in selected brands of orange jellies. sample concentration of sunset yellow (mg/100 g) mean ± sd (mg/100 g) (n = 9) p-value shop 1 shop 2 shop 3 brand 1 27.07 19.43 23.93 23.48±3.14 pb = 0.079, pe < 0.001 brand 2 41.63 40.28 39.20 40.37±0.99 pb <0.001, pe <0.001 brand 3 9.23 9.06 8.78 9.02±0.19 pb < 0.001, pe = 0.018 brand 4 18.43 16.50 19.33 18.09±1.18 pb = 0.018, pe <0.001 brand 5 13.27 12.17 10.42 11.95±1.17 pb < 0.001, pe = 0.003 brand 6 nd nd nd nd *nd= not detected; pb – value compared with bsti; pe – value compared with eu. figure 4. comparison of sy concentration in different brands of orange jelly with bsti and eu standard range. alves et al. (2008) showed that the concentration of sy in one brand of mango juice powder was significantly higher compared to the maximum regulated value of 10 mg/100 g. the intakes of colors such as tartrazine, erythrocine and sy were higher in children due to the ingestion of foods containing high concentrations of colors (9.45 and 4.0 mg) (rao and sudershan, 2008). another study showed that the consumption of chewing gum 20 10 23,48 40,37 9,02 18,09 11,95 0 0 10 20 30 40 50 bsti eu brand 1 brand 2 brand 3 brand 4 brand 5 brand 6 c on c. o f s un se t y el lo w (m g/ 10 0g ) brands ital. j. food sci., vol. 31, 2019 192 contains the greatest amount of tartrazine (e102), and jellies contain quinoline yellow (e104), ponceau 4r (e124) and allura red (e129) (malczyk et al., 2015). on the other hand, the consumption of colored beverages significantly increases the adoption of sy (e110) and azorubine (e122) (malczyk et al., 2015). the amount of tartrazine that is not secreted through urine, widely metabolized by intestinal microflora in which some metabolites are absorbed through the intestine ( khera et al., 1979; watabe et al., 1980; elhkim et al., 2007). bento et al. (2015) found the highest concentration containing 75.30±3.85 mgl-1 of ins102 colorant in one sample of milk drink among 15 samples of yogurt and milk drink. they also mentioned that ins122 was the most commonly used dye which was 33% of yogurt and milk drink samples ranging from 1.43 to 11.75 mgl-1. 4. conclusions all but two of the samples tested in this experiment contained sunset yellow in accordance with the standard range accepted by the bsti. sunset yellow was absent in one brand, while the other brand substantially exceeded the amount of sunset yellow compared to the standard range accepted by the bsti and eu. the differences in sunset yellow concentrations across the six brands of orange jellies highlight the need for improved product labeling. by providing this information to consumers and manufacturers would not only be aiding the health of consumers but they would also be assuaging the food adulteration reputation that currently taints the food product system. according to the bangladesh pure food ordinance (2005), there is prohibition of use of intoxicated food color in food. this is considered as a frightening issue which might be hazardous for public health. therefore, further research is required to evaluate different category food products with this method to see the reproducibility of the results described here. acknowledgements authors thank the ministry of science and technology, bangladesh for financial support through grant number 39.009.006.01.00.049.2013-2014/bs-154/154/1-4. s.h.k. designed the study, and a.i. and m.s. collected data from the experiments. a. i. and l. b. contributed to data analysis and manuscript preparation. additional editing was performed by m.i.h. and m.z.a. references were obtained by m.a.z. all authors carefully read and approved the manuscript. references abbey j., fields b., o’mullane m. and tomaska l. d. 2014. food additives: colorants. encycl food saf. 2:459. alves s.p., brum, d.m., branco de andrade, é.c. and 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toxicological assessment, intolerance reactions and maximum theoretical daily intake in france. regul toxicol. and pharmacol. 47(3):308. ganesan l., margolles-clark e., song y. and buchwald p. 2011. the food colorant erythrosine is a promiscuous proteinprotein interaction inhibitor. biochem. pharmacol. 81(6):810. geoffrey b. a. and felix m. b. 1991. canthaxanthin and the eye: a critical ocular toxicologic assessment. j. toxicol. cutan ocul. toxicol. 10(1-2):115. hamdy a., mekkawy a.a., massoud a. and el-zawahry m. 2000. mutagenic effects of the food colour erythrosine in rats. probl. forensic sci. 43:184. hinton, d.m. 2000. us fda“redbook ii” immunotoxicity testing guidelines and research in immunotoxicity evaluations of food chemicals and new food proteins. toxicol pathol. 28(3):467. joint f.a.o., on food additives w.h.o.e.c., organization w.h. and others 1965. specifications for the identity and purity of food additives and their toxicological evaluation: food colours and some 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induced by red food dyes orally administered to pregnant and male mice. toxicol. sci. 61(1):92. watabe t., ozawa n., kobayashi f. and kurata h. 1980. reduction of sulphonated water-soluble azo dyes by microorganisms from human faeces. food cosmet toxicol. 18(4):349. yuan y., zhao x., qiao m., zhu j., liu s., yang j. and hu x. 2016. determination of sunset yellow in soft drinks based on fluorescence quenching of carbon dots. spectrochim acta a mol biomol. spectrosc 167:106. paper received august 8, 2018 accepted september 9, 2018 ijfs#1652_bozza ital. j. food sci., vol. 32, 2020 195 paper influence of relative humidity on dried ca++-alginate films and composites made with soy and pectin s. barbut* and a. harper food science department, university of guelph, canada, n1g 2w1 *corresponding author: sbarbut@uoguelph.ca abstract dried ca-alginate films were manufactured±pectin or soy protein isolate (spi) as well dried un-gelled (no ca2+) pure alginate films: conditioned at either 57% or 100% relative humidity (rh). with the exception of the un-gelled alginate films, all films conditioned at 100% rh were more transparent than at 57% rh. high rh films resulted in higher % elongation at break, distance and work to puncture values than their corresponding films conditioned at 57% rh. atr-ftir scans showed several peak shifts when the film forming solutions were gelled with ca2+ and when the ‘wet’ films were dried. keywords: alginate, film, pectin, relative humidity, soy protein isolate, tensile strength ital. j. food sci., vol. 32, 2020 196 1. introduction global production of packaging materials is increasing and was estimated to exceed 180 million tons per year (cutter, 2006; nura, 2018). food packaging accounts for a large portion of this material and is estimated to attribute to roughly 70% of the $100 billion packaging market in the u.s. alone (cutter, 2006). much of this packaging material is made from synthetic plastics. however, mounting environmental concerns about plastic use and the rising cost of petroleum has led to an increased interest in natural food packaging material such as polysaccharides, proteins and/or lipids (rhim, 2004; cutter, 2006; da silva et al., 2009; janjarasskul and krochta, 2010). however, edible packaging materials are not normally meant to entirely replace conventional packaging. rather, the efficiency of food preservation can be improved by using primary edible packing together with less nonedible packaging. a good example is replacing plastic or cellulose casings used for frankfurters with manufactured co-extruded (in line) collagen casings, alginate or their combination (barbut, 2015). the frankfurters are then packaged in a strong oxygen barrier plastic pouch, which can be used as a barrier during cooking and/or distribution. a popular example of a polysaccharide that has been used in edible films is alginate. it is used as food wraps (e.g., spring rolls) and for small diameter sausage casings. one of the more desirable properties of alginate is its ability to very quickly form cold-set gels in the presence of calcium (ouwerx et al., 1998; mørch et al., 2006). alginates are derived from marine brown algae (phaeophyceae) and also produced by soil bacteria (stephen et al., 2006). chemically, alginate is made up of (1→4) β-d-mannuronic (m) and α-lguluronic (g) acid. regions in alginate made up of solely m or g residues are referred to as m or g blocks and these areas are interspersed with mg alternating blocks (fang et al., 2007; lee et al., 2018). several factors influence the use of edible films in food applications including: availability, cost, functional attributes, mechanical properties, optical quality, barrier properties, structural resistance to water and sensory acceptability (da silva et al., 2009; janjarasskul and krochta, 2010). typically protein and polysaccharide films are fairly good oxygen barriers at low to medium rh and have good mechanical properties but, due to their hydrophilic nature, are poor water vapour barriers (rhim and ng, 2007). today, several large and medium size sausage manufacturers are using co-extrusion technology. unofficial estimates put this number at 30% of the us market. this allows sausage manufacturers to move from a batch to a continuous process and thus increase production volumes (barbut, 2015). additionally, fewer people handle the product and thus the risk of microbial contamination of fresh sausages is reduced (anonymous, 2012). some manufacturers are also using cook in the bag technology, and therefore further reduce the risk of re-contamination (after cooking) with pathogens such as listeria. however, there are several challenges of casings manufactured from solely alginate or collagen. researchers have reported that alginate casings are prone to calcium migration (e.g., casings dissolve over time), while collagen casings may shrink more during frying (visser, 2012). one solution is producing composite co-extruded casings/films, which take advantage of synergistic interactions between individual components. the industry has already adopted this approach by adding ingredients such as starch and cellulose to co-extruded alginate casings, as well as producing co-extruded alginate-collagen hybrid casings. although these composite alginate casings are used commercially, there is little or no scientific literature regarding the properties of such ‘wet’ films. in fact, most of the literature reports on ‘dry’ alginate films for use after they have been cast. for example, ital. j. food sci., vol. 32, 2020 197 researchers have created composite ‘dry’ alginate films with proteins such as, whey, soy, and whey (shih, 1994; villagomez-zavala et al., 2008; wang et al., 2010), or polysaccharides such as pectin, kappa-carrageenan, pullulan, sago starch, and carboxymethyl cellulose (xu et al., 2003; tong et al., 2008; da silva et al., 2009; fazilah et al., 2011; gohil, 2011; bierhalz et al., 2012; paşcalău et al., 2012; xiao et al., 2012; galus and lenart, 2013). in certain cases, synergism was seen when ‘dry’ composite films made from 50:50 alginate and low methoxyl pectin showed higher tensile strength and elongation than either pure alginate or pectin films (galus and lenart, 2013). harper et al. (2013) started publishing on the ‘wet’ film area (90-95% water content), which recently became popular with the use of co-extruded sausage casings. they examined how various protein (whey, soy, and gelatin) and carbohydrate (pectin, cellulose, starch, carrageenan, and gellan gum) additives influence the mechanical and microstructural properties of composite films/casings. however, so far very few comparisons have been made between the physical properties of ‘wet’ and dried alginate films. understanding the role water plays in these films is important for future development of this area. therefore, the objective of this work was to explore how drying ‘wet’ alginate and alginate composite films affect these physical properties. additionally, the effect of rehydration of the dried films (i.e., exposing the dried films to high humidity) on their physical properties has been studied. for this work, composite alginate films were manufactured with either soy protein isolate (spi) or low-methoxylated pectin and compared to pure alginate films. 2. materials and methods 2.1. film preparation the ‘wet’ alginate films were produced according to the methods described by harper et al. (2013). briefly, 5% (w/w) alginate (grindsted® alginate fd 6965, danisco usa inc., rochester, ny, usa) solution was prepared. later, 1.5 g portions were rolled onto a plastic covered stainless steel board using a stainless steel roller with a recess of 0.34 mm. the roller, with the film on it, was placed into a 5% (w/w) cacl2 (fisher scientific, fair lawn, nj, usa) bath for 1 min to gel the film. soy protein isolate (spi; soy protein supro 515, protein 90%, supplied by hela spice canada, inc., uxbridge, on, canada) was dissolved in 230c double deionized water to form a 1% wt. protein solution. a 0.25% wt. pectin solution was made by dissolving low methoxyl pectin (lmp; genu® pectin type lm-104 as-z, cp kelco, lille skensved, denmark) into 700c double deionized water. 5% wt. alginate was also mixed by hand (total mixing time of 15 min) into both the protein and pectin solutions. additionally, a control sample of 5% wt. alginate with no protein or pectin was made. to remove some of the air incorporated into the solutions during mixing, the solutions were degassed by using a vacuum packager (multivac canada inc., woodbridge, on, canada) at 7.3 kpa for 25, 50 and 75 sec. the solutions were allowed to hydrate for a minimum of 2 hr at 23°c prior to film formation. the ‘wet’ films were produced using a stainless-steel roller and then placed into the calcium bath. alginate films were also produced without calcium as a gelling agent. in this case, alginate solution was rolled between two sheets of plastic on the stainless-steel board. the top layer of plastic was carefully peeled away from the film, leaving the film on the bottom sheet of plastic for drying. ital. j. food sci., vol. 32, 2020 198 2.2. drying and conditioning of the films the ‘wet’ alginate and alginate composite films were pinned to a plastic covered smooth piece of wood (to prevent curling) and left to dry overnight at room conditions (~230c and ~52% rh). the alginate films without calcium were simply left on the bottom sheet of plastic to dry. the following day the films were removed from the plastic and placed in jars with either 57% rh (nabr controlled) or 100% rh, and left to condition overnight. prior to conditioning, the films that would later be used for tensile testing were cut into 75 mm x 25 mm strips using a razor blade, and their thickness measured. three thickness measurements (top, centre and bottom) were taken on each film using a digital micrometer (testing machines inc., islandia, ny, usa) and the average thickness of each film was used for the tensile stress calculations. 2.3. mechanical properties the puncture and tensile properties of the films were evaluated using a texture analyzer (ta.xt icon. texture technologies corp., hamilton, ma/stable micro systems, godalming, surrey, uk). the films were kept in their rh respective jars right until the testing. for the puncture test a 5 mm ball probe was used to puncture the uncut films. the trigger force was set at 5 g and the test speed was 10 mm/s. from the generated forcedistance graph, the force (n), distance (mm) and work (n mm) to puncture the films were determined. tensile testing was conducted according to the astm-d882 standard tensile testing procedure (astm, 2010). the grippers were spaced 50 mm apart and the test distance was set at 75 mm. the trigger force for testing was 8 g and the test speed was 2 mm/s. the tensile strength (mpa), % elongation at break and the young’s modulus of the films were determined from the generated stress-strain graphs. 2.4. optical properties the visible light transmission (400-780 nm) of the films was measured using a single beam spectrophotometer (usb 2000, ocean optics inc., dunedin, fl, usa). the integration time was 25 ms with 2 scans to average and a boxcar width of 4. in total eighteen films per treatment were scanned (six per trial). the average light transmission of the eighteen films was calculated for each of the treatments. 2.5. atr-ftir analysis spectra were acquired using an ftir spectrometer (irprestige-21, shimadzu corporation, tokyo, japan) equipped with a total attenuated reflection press (miracle™, pike technologies, madison, wi, usa). scans were taken of the alginate and alginate-low methoxyl pectin film forming solutions, gelled ‘wet’ films prior to drying and the gelled dried films conditioned at 57% rh. additionally, the alginate and low methoxyl pectin powders used to make the films were scanned. all of the powders were conditioned at 57% rh overnight. for consistency, all of the films were scanned with the side of the film that was touching the roller, during gelling in the calcium bath, face up. all samples were tested at 23°c. the data were collected from 600-4000 cm-1 with an average of 30 scans per sample and a resolution of 4 cm-1. the second derivatives of the original spectrums were ital. j. food sci., vol. 32, 2020 199 taken using grams-32 spectral analysis software (galactic industries corp., salem, nh, usa) to determine the wavenumber of the peaks. sampling was performed in triplicate. 2.6. statistical analysis the experimental design was a completely randomized block with three independent trials. for both the puncture and tensile tests six films per treatment were tested in each of the three trials. the average of the six measurements was taken for each treatment in each trial and used for statistical analysis (sas version 9.2, sas inst., cary, nc, usa). a general linear model was used for analysis of variance (anova) and a tukey’s multiple comparison analysis (p-value ≤ 0.05) was used to detect statistical significance between film type means. 3. results and discussion 3.1. mechanical properties of formed films the purpose of this work was to compare the physical properties of dried alginate and alginate composite films to their ‘wet’ film counterparts. in our previous work, when low methoxyl pectin (lmp) or soy protein isolate (spi) were added to ‘wet’ alginate films, the composite films exhibited different mechanical properties from the pure ‘wet’ alginate films (harper et al., 2013; harper et al., 2015). for this reason, pectin and spi were chosen as ingredients for the alginate composite films manufactured in this study. in order to be able to directly compare the dried films in this study to the ‘wet’ films produced in earlier work, the films were manufactured in the same way. in the present study, the films were dried and then conditioned at 57% and 100% relative humidity in order to also evaluate how rehydration influences the films’ physical properties. overall, all of the dried films (conditioned at 57% or 100% rh) showed higher puncture force and tensile strength but lower puncture distance and elongation values compared to their corresponding ‘wet’ films. for example, the percent elongation at break was 140% for the ‘wet’ alginate-pectin films (harper et al., 2015) and 4.6% and 12.3% for the dried alginate-pectin films conditioned at 57% and 100% rh, respectively (table 1). the control alginate film gelled with ca had an elongation value of 88% while after drying and conditioned it to 57% or 100% rh the values were only 5.8% and 12.0%, respectively. therefore, it is important to emphasize that the rehydrated dried films (i.e., 100% rh films) did not have the same mechanical properties as their original ‘wet’ films, thus demonstrating that drying caused irreversible changes in the alginate film structure. all the dried and reconditioned films could be tested after conditioning at 57% and 100% rh, with the exception of the alginate films that were not gelled with calcium. after conditioning at 100% rh, the un-gelled alginate films were very sticky and unable to hold their shape for testing. for this reason, no mechanical or optical tests were performed on these films. as expected, differences in puncture force were seen between the films conditioned at 57% and 100% rh (fig. 1). interestingly, the trend was not consistent for all treatments. in the case of the gelled alginate and alginate-pectin films, the films conditioned at 100% rh required less force to puncture than those conditioned at 57% rh, while the opposite was true for the alginate-spi films. remunan-lopez and bodmeier (1997) also explored the differences in puncture strength between ‘dry’ and ‘wet’ alginate films. they found that ‘wet’ films had lower puncture strength than ‘dry’ ital. j. food sci., vol. 32, 2020 200 films, which they attributed to the plasticizing effect of water. a similar trend was expected in this work. it should be noted that the ‘wet’ films in their study were immersed in water as opposed to exposed to humid air, which may have influenced the uptake of water by the films and thus their resulting mechanical properties. table 1. mechanical properties of dried calcium-alginate (alg) films with and without low methoxyl pectin (lmp) or soy protein isolate (spi) conditioned at 57% and 100% relative humidity. treatment distance to puncture (mm) work to puncture (n mm) tensile strength (mpa) elongation at break (%) young’s modulus (mpa) thickness (um) alg (no ca) 57 3.2±0.3c 7.3±0.4c 90.9±32.8a 3.7±1.3b 2729.2±298.4a 0.007±0.002c alg 57 6.6±0.5b 29.4±1.9b 30.0±5.2b 5.8±1.3b 867.2±294.0b 0.018±0.002b alg 100 11.3±0.5a 39.5±4.3a 9.9±0.3b 12.0±2.9a 179.7±15.5c 0.020±0.001b alg lmp 57 5.7±0.3b 27.8±3.5b 38.8±5.7b 4.6±0.6b 1190.9±228.1b 0.018±0.002b alg lmp 100 11.9±0.1a 41.0±1.8a 9.9±1.5b 12.3±2.4a 178.7±45.2c 0.018±0.002b alg spi 57 2.7±0.4c 4.8±1.1c 24.5±3.5b 3.2±0.3b 989.6±265.6b 0.035±0.002a alg spi 100 11.6±0.3a 31.6±2.4b 4.4±0.1b 16.1±3.2a 64.5±4.4c 0.034±0.001a means±standard deviation, a-c show significant differences (p<0.05) between means (n=18). figure 1. puncture force of dried calcium-alginate (alg) films with and without low methoxyl pectin (lmp) or soy protein isolate (spi) conditioned at 57% and 100% relative humidity (rh). note: alg with no ca does not form a film and hence could not be evaluated. a-e show significant differences (p<0.05) between means (n=18). d,e a a e b b,c c,d 0 2 4 6 8 10 12 14 16 alg (no ca) alg alg lmp alg spi p un ct ur e fo rc e (n ) treatment 57% rh 100% rh ital. j. food sci., vol. 32, 2020 201 the puncture force trends between treatments are similar to what we have previously reported for the ‘wet’ alginate films (harper et al., 2015). in both the dried (57 & 100% rh) and ‘wet’ alginate films, it took significantly less force to puncture the alginate-spi films (3.8n) than the pure alginate films (6.3n). this may be a result of the soy proteins interrupting the alginate film network. no differences in puncture force existed between the alginate-pectin (5.8n) and pure alginate films (5.3n) for the ‘wet’ or dried films (harper et al., 2015). as expected, the un-gelled pure alginate films had significantly lower puncture force than the gelled pure alginate films. similarly, it has been reported that ‘dry’ alginate films gelled with calcium have increased tensile strength over their ungelled counterparts (fazilah et al., 2011). differences in the puncture distance were also observed between the dried films conditioned at 57% and 100% rh (table 1). in all cases, the films conditioned at 100% rh showed a greater distance to puncture than the films conditioned at 57% rh. again, this is likely due to the plasticizing effect of water in the 100% rh conditioned films. although no differences were observed between the three 100% rh treatments, the alginate and alginate-pectin films conditioned at 57% rh showed significantly higher puncture distance values than the alginate-spi and un-gelled alginate films conditioned at 57% rh. similar trends were observed for ‘wet’ alginate films where the pure ‘wet’ alginate films had significantly higher puncture distance values (17mm) than ‘wet’ alginate-spi films (13mm) (harper et al., 2013), but not significantly different values from the ‘wet’ alginate-pectin films (17mm) (harper et al., 2015). the work to puncture results followed a similar pattern as the puncture distance, with the films conditioned at 100% rh having significantly higher work values than those conditioned at 57% rh (table 1). both the alginate-spi and un-gelled alginate films conditioned at 57% rh required less work to puncture than the pure alginate films conditioned at 57% rh. no difference in the work to puncture existed between the alginate-pectin and pure alginate films conditioned at either relative humidity. tensile testing is another method used to measure the strength and elasticity of biopolymer films. fewer differences were seen in the tensile strengths of the dried films gelled with calcium and then reconditioned at either 57% or 100% rh (table 1). our previous work indicated that, ‘wet’ alginate pectin films had significantly higher tensile strength than the pure ‘wet’ alginate films. this was also the case here when the films conditioned at 57% rh were compared. in general, it was expected that films conditioned at 57% rh would have higher tensile strength than those conditioned at 100% rh. other work has shown that increasing the relative humidity of ‘dry’ calcium-alginate films decreases their tensile strength (olivas and barbosa-cánovas, 2008). however, the authors also reported that this effect was less pronounced when plasticizers such as glycerol, sorbitol, fructose or peg-8000 were absent from the film formulations. they argued that the non-plasticized films had a reduced capacity to absorb water and thus their mechanical properties were less influenced by changes in relative humidity. in the current work, plasticizers were not used. this may explain why more differences between the 57% and 100% rh films tensile strengths were not observed. the tensile strengths of the ca-alginate films in the present study were lower than some of the values reported in the literature for ‘dry’ alginate films. tensile strengths of 85.9, 64.7, and 24.1 mpa were reported for dried glycerol-plasticized calcium-alginate films conditioned at 50%, 56%, and 98% rh, respectively (rhim, 2004; olivas and barbosacánovas, 2008). in each of these cases, the alginate films were dried prior to being immersed into cacl2. alternatively, the films in the present study were gelled with cacl2 prior to drying, which may account for some of the differences observed between the ital. j. food sci., vol. 32, 2020 202 studies. the percent elongation at break (% eab) values in the present study (table 1) were similar to those reported for other ‘dry’ alginate and alginate composite films (rhim, 2004; olivas and barbosa-cánovas, 2008; fazilah et al., 2011; gohil, 2011; galus and lenart, 2013). similar to the puncture distance, the % eab was significantly higher for films conditioned at 100% rh than films conditioned at 57% rh (table 1). unlike the puncture distance, no significant differences were found between the various films conditioned at 57% rh. contrarily, galus and lenart (2013) reported higher elasticity values for blended ‘dry’ alginate-pectin films than pure alginate or pure pectin ‘dry’ films. gohil (2011) also reported that adding up to 60% low methoxyl pectin improved the elasticity of ‘dry’ alginate films. additionally our previous work on ‘wet’ alginate films showed that alginate-pectin films had significantly higher % eab than pure alginate films (harper et al., 2015). therefore, it appears that any benefit low methoxyl pectin imparted on the elasticity of ‘wet’ alginate films was lost when the films were dried. on the other hand, adding soy protein isolate to the alginate films did not influence the % eab of either the ‘wet’ (harper et al., 2013) or dried (57% 100% rh) films. the young’s modulus is a measurement of the film’s stiffness. similar to the tensile strength, the un-gelled alginate film had the highest young’s modulus of all of the films (table 1). no differences in modulus were seen between the gelled alginate, alginatepectin and alginate-spi films conditioned at the same relative humidity, but in all three cases the films conditioned at 57% rh had higher stiffness values than those conditioned at 100% rh. the alginate-spi films were significantly thicker than the alginate-pectin and gelled alginate films while the un-gelled alginate films were the thinnest (table 1). since the un-gelled films were rolled between two sheets of plastic (with a roller), and not submerged in a cacl2 bath, these films were thinner than the gelled films prior to drying and thus, it is no surprise that the dried un-gelled films were thinner than the dried gelled films. all the dried films (conditioned at both rh) in the present study had higher puncture force and tensile strength values than their corresponding ‘wet’ films produced in earlier work. for instance, the ‘wet’ alginate-spi films had a tensile strength of 1.6 mpa (harper et al., 2013), while the dried alginate-spi films had tensile strengths of 24.5 and 4.4 mpa for the films conditioned at 57 and 100% rh, respectively. on the other hand, all the ‘wet’ films had higher puncture distance and % eab than their corresponding dried (57 & 100% rh) films. the % eab values in the present study ranged from ~3 to 16% whereas those previously reported for ‘wet’ alginate films were ~80 to 140%. therefore, it can be concluded that drying the films improved their puncture and tensile strength but decreased their elasticity. as mentioned before, even after rehydration the films did not have the same properties as the original ‘wet’ films. 3.2. films optical properties with the exception of the un-gelled alginate films, all of the films conditioned at 100% rh were more transparent than the films conditioned at 57% rh (fig. 2). visually, the alginate-pectin, alginate-spi and pure alginate films conditioned at 57% rh were a whitish colour. on the other hand, the pure alginate and alginate-pectin films conditioned at 100% rh were clear and transparent (upper lines in fig. 2), while the alginate-spi films conditioned at 100% rh had a slight yellowish-brown tint to them. it is believed that the whitish colour of the films conditioned at 57% rh was a result of salt crystals on the surface of the film. this hypothesis was supported by sem images which showed very ital. j. food sci., vol. 32, 2020 203 small salt crystals on the surface of the alginate-spi, alginate-pectin and pure alginate films conditioned at 57% rh (images not shown). it would also explain why the un-gelled alginate films conditioned at 57% rh were much more transparent than all of the other films conditioned at 57% rh. at both relative humidity conditions, the alginate-spi films appeared to be least transparent of the films, although a greater difference was seen for the films conditioned at 100% rh. the alginate-pectin films conditioned at 57% rh also appeared to be slightly less transparent than the pure alginate films. this is in agreement with other researchers who have reported ‘dry’ alginate films to be more transparent than ‘dry’ pectin films (parris et al., 1995; da silva et al., 2009; galus and lenart, 2013). figure 2. visible light transmission (n=18) of dried calcium-alginate (alg) films with and without low methoxyl pectin (lmp) or soy protein isolate (spi) conditioned at 57% and 100% relative humidity. 3.3. ftir the atr-ftir spectra of the alginate, and alginate-pectin film forming solutions as well as the ‘wet’ and dried (57% rh) films are shown in fig. 3. in addition, the alginate and pectin dry powders that were used to make the films were scanned (fig. 3). table 2 summarizes the main vibrational peaks of the various spectra in the fingerprint region (1750-800 cm-1). assignments of the peaks was based on literature (sartori et al., 1997; kacurakova et al., 2000; pereira et al., 2009; papageorgiou et al., 2010). calcium is known to interact with the carboxylate groups in the alginate. the two absorbance bands associated with the symmetric and asymmetric stretching vibration of the coogroups of alginate occurred at 1426-1414 cm-1 and 1597-1591 cm-1 for the films and film forming solutions. this is in agreement with other studies that reported these two ital. j. food sci., vol. 32, 2020 204 peaks to occur in the 1422-1404 cm-1 and 1622-1596 cm-1 ranges (sarmento et al., 2006; mohamadnia et al. 2007; jaya et al., 2009; papageorgiou et al., 2010; paşcalău et al., 2012). it should be noted that there were actually two peaks detected in the 1600 cm-1 region in the present study; however, the peak at 1635-1631 cm-1 has been attributed to the water in the films as it was not present in the alginate powder spectra (fig. 3). figure 3. atr-ftir spectra in the 4000-675 cm-1 range of alginate (a), and alginate-low methoxyl pectin (b). film forming solutions (a, e); ‘wet’ films (b, f); ‘dry’ films (c, g); and alginate (d), and low methoxyl pectin (h) powders. table 2. assignment of the main vibrational peaks for the atr-ftir spectra in the 1750-800 cm-1 range. ital. j. food sci., vol. 32, 2020 205 vibrationa alginate alginate-low methoxyl pectin film forming solution ‘wet’ film ‘dry’ film alginate powder film forming solution ‘wet’ film ‘dry’ film pectin powder c=o stretching 1743 1676 1635 1632 1633s 1633 1632 1631s asymmetric coo stretching 1597s 1591s 1596 1594 1596s 1594s 1595 1589 symmetric coo stretching 1414 1418 1426 1410 1414 1418 1422 1441s 1408 1326 c-o stretching 1312 1318 1312 s=o stretching 1224 c-o, c-c & c-o-c stretching 1126 1102 1032 1125 1085 1028 1117 1080 1028 1124 1088 1027 1126 1101 1032 1124 1086 1028 1117 1081 1028 1141 1100 1072 1047 999 992 1010 989 995s 1000 993 1010 989 1013 c-o stretching 950 945 941 944 949 944 939 947 c-o of 3,6anhydrogalactose 886 c-o-so3 on c4 of galactose 830 mannuronic acid residues? 824 811 822 asartori et al., 1997; kacurakova et al., 2000; pereira et al., 2009; papageorgiou et al., 2010. s shoulder peak. for both treatments, the peak associated with the symmetric stretching vibration of the cooshifted to a higher wavenumber (1414-1415 cm-1 to 1418-1420 cm-1) when the film forming solution was gelled with calcium. paşcalău et al. (2012) reported a similar shift when ‘dry’ alginate-kappa-carrageenan films were gelled with calcium. sartori et al. (1997) also observed an increase in wavenumber of the symmetric stretching vibration of the coowhen sodium alginate was gelled with calcium. they stated that a peak shift should be expected as the environment around the carboxyl group changes when na+ ions are replaced by ca2+ ions, since the two ions have different charge densities, radii and atomic weights. a second shift of the peak associated with the symmetric stretching vibration of cooto higher energy (wavenumber) was observed when the gelled ‘wet’ films were dried. in this case, the peak shifted from 1418-1420 cm-1 to 1422-1426 cm-1. shifts in the peak associated with the asymmetric stretching vibration of the cooon the alginate were less defined. several other peak shifts occurred when the alginate and composite solutions were gelled with calcium into ‘wet’ films. these included shifts from 1102-1101 cm-1 to 1086-1085 cm-1, 1032 cm-1 to 1028 cm-1, and 1000-999 cm-1 to 993-992 cm-1. sartori et al. (1997) also reported shifts towards lower wavenumbers for several peaks in the 1150-1000 cm-1 region when sodium alginate was gelled with calcium. they suggested that the shift to lower frequencies was caused by weakening in the c-c and c-o bonds, likely due to these ital. j. food sci., vol. 32, 2020 206 bonds being shared with calcium ions. they also found a new peak at 1010 cm-1 when sodium alginate was gelled with calcium. while this peak was not observed in the ‘wet’ films in the current work, it was present in the spectra of the dried films. other peaks that were only observed in the dried film spectrum include the peaks at 1312 cm-1 and 824-820 cm-1. it is suspected that these peaks were masked by the large percentage of water (~95%) in the ‘wet’ films. drying the ‘wet’ films also caused shifts towards lower frequencies of several peaks. these included shifts from 1125-1124 cm-1 to 1117 cm-1, 1086-1085 cm-1 to 1081-1080 cm-1, and 993-992 cm-1 to 989 cm-1. overall, there were no differences observed between the alginate, and alginate-pectin treatments in either the film or the film forming solutions’ spectrum. it was expected that a peak around 1760-1740 cm-1 would be observed in the alginate-pectin treatments due to the esterified carboxyl groups of the pectin (ismail et al., 2012). it is thought that because the amount of pectin added to the alginate was low (0.25%), the intensity of these peaks was too low to be detected in the analysis of the composite film and film forming solution spectra. 4. conclusions the results demonstrate that water plays a critical role in determining the mechanical properties of alginate films. dried alginate films conditioned at 57% rh had different mechanical and optical properties than their corresponding dried alginate films conditioned at 100% rh. while drying gelled ‘wet’ alginate films appeared to increase their puncture and tensile strength, it also decreased their elasticity. rehydrated dried films did not have the same properties as the original ‘wet’ films, suggesting that drying caused irreversible changes in the alginate film structure. dried alginate-pectin films did not show significantly different puncture or tensile properties compared to pure ca-alginate films at either 57% or 100% rh. this was contrary to some of our earlier work on ‘wet’ films which showed ‘wet’ alginate-pectin films to have higher tensile stress, % eab, and puncture work than pure ‘wet’ ca-alginate films (harper et al., 2015). therefore, any benefits low methoxyl pectin imparted on ‘wet’ alginate films, appeared to be lost once the films were dried. on the other hand, adding spi to both the dried and reconditioned films (57% & 100% rh), as well to ‘wet’ alginate films, decreased their puncture force and work but did not affect the films’ tensile properties. adding low methoxyl pectin did not cause any detectable differences in the ftir spectra of the films or film forming solutions in the 1750-800 cm-1 region. however, for 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"food polysaccharides and their applications" (2nd ed). crc press, new york, ny. tong q., xiao q. and lim l.t. 2008. preparation and properties of pullulan-alginate-carboxymethylcellulose blend films. food res. int. 41(10):1007-1014. villagómez-zavala d.l., gómez-corona c., martínez e.s.m., pérez-orozco j.p., vernon-carter e. j. and pedroza-islas r. 2008. comparative study of the mechanical properties of edible films made from single and blended hydrophilic biopolymer matrices. rev. mex. ing. quím. 7(3):263-273. visser p.r. 2012. casings for foodstuffs u.s. patent application us 2012/0114807 a1. wang l., auty m.a.e. and kerry j.p. 2010. physical assessment of composite biodegradable films manufactured using whey protein isolate, gelatin and sodium alginate. j. food eng. 96(2):199-207. xiao q., lim l.t. and tong q. 2012. properties of pullulan-based blend films as affected by alginate content and relative humidity. carbohydr. polym. 87(1):227-234. xu j.b., bartley j.p. and johnson r.a. 2003. preparation and characterization of alginate-carrageenan hydrogel films crosslinked using a water-soluble carbodiimide (wsc). j. membrane sci. 218(1-2):131-146. paper received july 26, 2019 accepted october 12, 2019 ijfs#1851_bozza ital. j. food sci., vol. 32, 2020 691 paper evaluation of enzymatic hydrolysis applied to fish by-product oil through chemical parameters c. moroni silvaa, f. gerald limaa, k. furtado da costaa, v. amaral ribeiroa, r.s. ribeiro leiteb, m.e. petenucic, j.m.l. neves gelinskid, g. graciano fonseca*c,d and c. prenticea alaboratory of food technology, school of chemistry and food, federal university of rio grande, rio grande, rs, brazil blaboratory of enzymology and fermentation processes, faculty of biological and environmental sciences, federal university of grande dourados, dourados, ms, brazil claboratory of bioengineering, faculty of biological and environmental sciences, federal university of grande dourados, dourados, ms, brazil dcenter of biotechnology, postgraduate program in science and biotechnology, university of west of santa catarina (unoesc), videira, sc, brazil *corresponding author: ggf@ufgd.edu.br abstract large amounts of fish waste are produced by the fish processing plants. this waste could be used to obtain high value-added products, such as long chain polyunsaturated fatty acids. thus, the aim of this work was to evaluate the enzymatic hydrolysis of low commercial value crude fish oil through chemical parameters. crude fish oil was obtained from mixed fishmeal production and was chemically refined. candida rugosa lipase ay "amano" 30 was utilized to catalyze the enzymatic hydrolysis. the acidity index, iodine value, saponification index and peroxide value were used to characterize the samples studied. the chemical refining process yielded 62.75% and improved the quality of crude fish oil by reducing the acidity index around 91%. the best results of hydrolysis degree (23.45%) and iodine value (120 g i2 g-1) were obtained at 45ºc after 6 h of lipase action. the iodine value of crude oil was not affected by processing, indicating that the nutritional quality was preserved in the refined oil. despite the hydrolysis process showed good results, it was not sufficient to concentrate the unsaturated fatty acids of refined oil, as indicated by the iodine value. keywords: acidity index, candida rugosa, iodine value, lipase, refined fish oil ital. j. food sci., vol. 32, 2020 692 1. introduction the fish processing plants produce around 50% of waste from the total processed fish (arruda et al., 2006). this waste is rich in organic and inorganic compounds, and its improper disposal causes negative environmental impacts, particularly on the margins of water bodies or in unlicensed landfills (feltes et al., 2010). fish waste could be utilized to obtain high value-added products, since fish oil can be produced from whole fish, roe and by-products from the processing of fish. fish oil is widely used in aquafeeds as supply of long chain omega-3s fatty acids for aquaculture, especially for carnivorous fish e.g. salmonids. the direct use of fish oil in human foods and capsules is an increasingly significant outlet the so-called, “nutraceuticals”, which use has been increasing even more rapidly than that in aquaculture, at around 15% per annum (pike and jackson, 2010). fish oil is an important source of long chain polyunsaturated fatty acids (lc-pufa), mainly the eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) (monroig et al., 2018). recent studies revealed that lc-pufas prevented short and long term memory impairment induced by chronic sleep deprivation (azoulbi et al., 2019). there is also abundant evidence that increasing the intake of omega-3s fatty acids can soften the symptoms of neurodegenerative and neurological diseases, improving memory and cognitive function (cutuli et al., 2014; zhou et al., 2018). however, crude fish oil presents impurities e.g. free fatty acids, monoand diglycerides, phosphatides, steroids, vitamins, hydrocarbons, pigments, carbohydrates, proteins and their degradation products and colloidal materials (menegazzo et al., 2014). the production of high quality oils requires the largest possible removal of non-triglyceride components (prior et al., 1991). the refining process aims precisely to remove these non-triglyceride components, conferring the oil best features. one of the most promising techniques to fish oil processing is the use of lipase-catalyze enzymatic hydrolysis. this process favors the release of lipids from a protein matrix while preserving the nutritional value of fats for their application in food industry. the high specificity of lipases in relation to triacylglycerol substrate has suggested a large number of applications in the pharmaceutical and food areas, being used mainly for the production of particular fatty acids with low energetic consumption (padilha and augusto-ruiz, 2007; ferreira-dias et al., 2013). concentrates of epa and dha may be prepared by selective hydrolysis of fish oils using lipases or by selective esterification of dha and other free fatty acids (ranjanmoharana et al., 2016). the modification of lipids from oils usually involves a process catalyzed by lipase for fat hydrolysis, modification of triacylglycerol, and synthesis of esters. the process is attractive, since the lipase shows a high efficiency, using little amounts, especially in immobilization process (ferreira-dias et al., 2013; rajendran et al., 2009). thus, this work aimed at evaluating the effect of enzymatic hydrolysis of low commercial value crude fish oil submitted to chemical refining. to this aim, samples submitted to chemical refining for increasing time and at different temperatures were characterized for chemical parameters. ital. j. food sci., vol. 32, 2020 693 2. materials and methods 2.1. crude fish oil and enzyme crude fish oil was obtained as a by-product of the mixed fishmeal production from the torquato pontes fisheries industry, located in rio grande, rs, brazil. the crude oil was kept under refrigeration. the enzyme utilized was candida rugosa lipase ay "amano" 30, supplied by amano enzymes (usa). 2.2. chemical refining of crude fish oil the chemical refining of crude fish oil was realized according to the methodology described elsewhere (morais et al., 2001; menegazzo et al., 2014). fig. 1a shows all the process steps. 2.3. enzyme application in the crude fish oil the enzyme application in crude fish oil was adapted from sun et al. (2002) and is shown in fig. 1b. the chemically refined fish oil was hydrolyzed with candida rugosa lipase ay "amano" 30 by adding phosphate buffer solution (ph 7.0) with the enzyme, and 100 μmol of cacl2 solution. figure 1. chemical refining (a) and concentration (b) of fish oil. ital. j. food sci., vol. 32, 2020 694 the samples were placed in a reactor with stirrer and temperature-controlled bath. aliquots were collected at defined intervals to determine the acidity index and the degree of hydrolysis (enzymatic hydrolysis). after the reaction, lipases were inactivated with methanol (lipase inactivation) and the free acids were neutralized with a naoh solution 20% (neutralization). the separation of glycerides from the fatty acids was performed by using a separator funnel. the oily mixture was added of 100 ml of hexane and 50 ml of distilled water (separation of glycerides). the lower aqueous layer was separated and discarded, while the upper layer containing glycerides was washed twice with 50 ml of distilled water (washing) and the remaining water was removed by a layer of anhydrous sodium sulfate (drying/filtration). the glycerides were recovered after removal of solvent on a rotary evaporator (evaporation). then, unsaturated fatty acid was obtained as final product. 2.4. chemical characterization of samples the characterization of the crude, refined fish oils and the unsaturated fatty acids was carried out according to aocs (2004) methods: acidity index (ai) (cd 3d-63), iodine value (iv) (cd 1b-87), peroxide value (pv) (cd 8-53), and saponification index (si) (cd 3-25). all analyses were performed in triplicate. 2.5. determination of lipase activity the lipase activity was performed using the method described by sugihara et al. (1990) an olive oil emulsion was prepared and added of 50 mm acetate buffer (ph 5.6) and 100mm cacl2. the enzyme solution added varied from 5 to 50 μl. the reaction was maintained at 37°c under stirring of 500 rpm for 30 min. after that, the reaction was inactivated with ethanol, then titrated with koh 50mm, to determine the amount of fatty acids released by enzymatic reaction.19 the lipase activity was calculated using equation 1: activity = ! mol ! ×10! !mol mol × ! 1000ml ×!volumenaoh ml time(min) (1) 2.6. hydrolysis degree the hydrolysis degree of oils after the enzymic treatment was calculated according to equation 2: hydrolysis(%) = ai(hydrolyzedoil!ai(non!hydrolyzedoil) si(non!hydrolyzedoil)!ai(non!hydrolyzedoil) 𝑥100 (2) where: si is the saponification index, and ai is the acidity index. 2.7. statistical analysis a factorial experimental design 22, with three central points, was applied to obtain statistical models for the parameters studied (hydrolysis time and temperature) in function of the considered responses (hydrolysis degree and iodine value). the studied factors with the respective real and coded levels are shown in table 1. ital. j. food sci., vol. 32, 2020 695 table 1. variable levels and limits for the 22 experimental factorial design. independent variable level -1 0 1 hydrolysis time (h) 2 4 6 hydrolysis temperature (ºc) 45 50 55 all statistical analysis was performed using the statistica 6.0 software and the validity of quadratic model was performed by variance analysis (anova) at 5% probability by fisher test. 3. results and discussion 3.1. characterization of fish oils table 2 shows the yield of each step of chemical refining. the refined fish oil yielded 62.75%, which is acceptable, since refining stages causes many losses during process, as occur in neutralization step with naoh. this alkali was more effective in bleaching, even if it caused saponification of a small part of neutral oil in the same time it promotes the neutralization of free fatty acids. the washing was essential to remove all naoh residue. notwithstanding, the importance of neutral oil drying lies in that the moisture, during the oil storage, can cause hydrolysis and increase the acidity, as well as oxidation of the heated oil. at the filtering stage, the yield dropped from 67.74% to 62.75%. this loss could be associated to the use of adsorbents in higher amounts than the minimum required, resulting in a greater loss of oil. table 2. yield of chemical refining process. stage of the process yield (%) crude fish oil 100.00 degumming 91.85 neutralization 81.65 washing 74.82 decantation 70.12 drying 67.74 filtration 62.75 refined fish oil 62.75 the chemical characterization of crude fish oil and refined fish oil is shown in table 3. ital. j. food sci., vol. 32, 2020 696 table 3. chemical characterization of crude fish oil and refined fish oil. parameter crude fish oil refined fish oil acidity index (ai) (mg naoh g-1) 5.05±0.02a 0.45±0.04b iodine value (iv) (g i2 g-1) 121±0.13a 121±0.11a peroxide value (pv) (meq. kg-1) n.d* n.d* saponification index (ai) (mg koh g-1) 182±0.25a 182±0.19a *not detectable. **different letters in the same line indicated a significant difference (p<0.05) by the tukey test. a significant difference (p<0.05) was observed between the ai of crude fish oil and refined fish oil, showing that the chemical refining was effective in reducing that value. free fatty acids contents are usually associated with undesirable flavour and textural changes (visentainer et al., 2015). the iv, which is associated with the degree of unsaturated fatty acids of oil (crexi et al., 2010), showed no significant difference (p>0.05) between the samples studied. the si also presented no significant difference (p>0.05) between results (table 3). however, they are in accordance with the values reported for oils from marine animals (160-196 mg koh g-1) (araújo, 2001). the peroxide value was not detected due to the addition of antioxidant, which inhibited the oxidation-reduction reactions used to determine this parameter. in general, the recommendation of peroxide value for human consumption is of 8 meq kg-1 oil (boran et al., 2006). oliveira et al. (2016) also observed similar results for ai of crude and refined tuna byproduct oils. these authors observed a reduction of 85% in ai, whereas a higher reduction of 91% was obtained in the present study (table 2). moreover, the ai of refined fish oil is in accordance to other fish oils, e.g. nile tilapia (0.08 mg naoh g-1) and hybrid sorubim (0.03 mg naoh g-1) (menegazzo et al., 2014). regarding iv results, they indicated that the unsaturated compounds were preserved during the process, which was also observed in other study with nile tilapia and hybrid sorubim oils (menegazzo et al., 2014). in general, iv of both samples are in accordance to the value reported by ackman (1966) for oils from marine animals, varying from 110 to 193 g i2 g-1. menegazzo et al. (2014) 3.2. determination of the lipase activity the lipase activity for the enzyme candida rugosa amano ay was 1.78 u ml-1 (35.7 u g-1) according to sugihara et al. (1990). two experiments were carried out to verify the lipolytic activity. the relation among the enzyme concentration and the lipolytic activity was similar to the value supplied by the manufacturer, which is 30,000 u g-1 at the enzyme concentration of 2.5 x 10-4 g ml-1. 3.3. enzymatic hydrolysis the results of enzymatic hydrolysis according to the experimental design are showed in table 4. a decrease in the iv was observed with the temperature increased from 50 to 55ºc. this behavior is associated to the oxidation of unsaturated fatty acids present in the sample, reducing the iv. ital. j. food sci., vol. 32, 2020 697 table 4. factorial experimental design matrix and results for hydrolysis degree and iodine value for the hydrolyzed oil. experiment temperature (°c) time (h) hydrolysis degree (%) iodine value (g i2 g -1) 1 -1 (45) -1 (2) 22.79±0.12 b 121±0.19 b 2 -1 (45) +1 (6) 23.45±0.15 a 120±0.20 c 3 +1 (55) -1 (2) 14.65±0.10 f 179±0.13 a 4 +1 (55) +1 (6) 20.4±0.09 c 107±0.17 g 5 0 (50) 0 (4) 22.63±0.18 b 117±0.12 d 6 0 (50) 0 (4) 18.90±0.11 e 113±0.22 f 7 0 (50) 0 (4) 21.00±0.12 d 114±0.15 e *different letters in the same line indicated a significant difference (p<0.05) by the tukey test. the experiment 2 showed the best results with hydrolysis degree (23.45%) and iv (120 g i2 g-1), whereas the other experiments showed minor values. according to those results, it was noted that the optimal temperature and hydrolysis time with candida rugosa amanoay was at 45ºc and 6 h. therefore, another experiment was realized to correlate hydrolysis degree and acidity index. fig. 2 shows the hydrolysis results obtained with the optimal conditions (45°c and ph 7.0) for the enzyme candida rugosa ay "amano" 30 after 12 h, according to sugihara et al. (1990). according to fig. 2, a sharp increase was observed in hydrolysis at the first 0.5 h (up to 14.1%). the maximal hydrolysis degree was obtained at 8 h (27.8%). in this meantime, the hydrolysis degree augmented at a relatively constant rate. after that, the values oscillated without great variability, showing that was not necessary to prolong the period of hydrolysis for a long time and that acidity index showed similar behavior, as expected. figure 2. kinetics of hydrolysis degree (■) (%) and acidity index (●) (mg koh g-1 oil) of refined fish oil. 0 10 20 30 40 50 0 10 20 30 40 50 60 0 2 4 6 8 10 12 14 h yd ro ly si s de gr ee (% ) a i ( m g k o h g -1 ) time (h) ital. j. food sci., vol. 32, 2020 698 other researchers also studied the enzymatic hydrolysis for extraction of unsaturated fatty acids from fish oil (iberahim et al., 2018; babajafari et al., 2017). babajafari et al. (2017) compared enzymatic hydrolysis and chemical methods of oil extraction from rainbow trout. the chemical methods showed higher yield (16.58%) values than enzymatic hydrolysis (13.65% 150ppm concentrated protease). however, the fatty acids composition showed that enzymatic hydrolysis concentrated the lc-pufa (epa and dha), whereas the chemical methods concentrated mufa and pufa (linoleic acid and α-linolenic acid). moreover, enzymatic hydrolysis is a safety method about food quality and environmentfriendly extraction. iberahim et al. (2018) observed that catfish oil submitted to enzymatic hydrolysis showed no significant difference (p<0.05) in their fatty acids content, it means, mufa content before hydrolysis was 48.41% and after 47.99%; pufa content decreased from 20.32% to 19.32% after enzymatic hydrolysis by lipase. this behavior could be associated to oil auto-oxidation and photo-oxidation, since pufa are more likely to undergo oxidation. pv and ai increased during the enzymatic hydrolysis, indicating the oil oxidation. those results are similar to obtained in this study (tables 3 and 4), since ai increased during the enzymatic hydrolysis process and iv indicated no change in fatty acids chains. despite results showed that hydrolysis of refine oil was affected by temperature and time (table 4), no significant difference was observed between the iv of refined oil (table 3) and hydrolyzed oil (table 4). an increase in iv was expected with the hydrolysis process, since the enzyme has the ability to concentrate the unsaturated fatty acids. so, those results indicated that the hydrolysis process was not efficient. thus, other process such as winterization (amorim et al., 2015) and ultrafiltration processes (koris et al., 2006) might be also evaluated to this kind of raw material to improve its nutritional quality, increasing the unsaturated fatty acids content. 4. conclusions the chemical refining yielded 62.75% and was efficient at removing undesirable compounds from crude fish oil, since the acidity index decreased by around 91% during the processing. the iodine value and saponification number of refined oil were not affected, ensuring its nutritional quality as a food ingredient. the best results of hydrolysis degree (23.45%) and iodine value (120 g i2 g-1) were obtained at 45ºc after 6 h of lipase action. however, this hydrolysis process was not efficient in concentrating the unsaturated fatty acids of the refined oil since no significant difference (p>0.05) was observed between the iodine value of the refined oil and the hydrolyzed oil. thus, further research using this kind of residue is necessary in order to obtain isolated fatty acids (omega-3) or increasing the content of health beneficial fatty acids. acknowledgments the authors are indebted to the brazilian research funding agencies cnpq, capes and fundect for their financial support. ital. j. food sci., vol. 32, 2020 699 references ackman r.g. 1966. empirical relationships between iodine value and polyunsaturated fatty acid content in marine oils and lipids. j. am. oil chem. soc. 43:385-389. doi: doi.org/10.1007/bf02646795. aidos i., schelvus-smit r., veldnan m.b., luten j., padt a.v.d. and boom r.m. 2003. chemical and sensory evaluation of crude oil extracted from herring byproducts from different processing operations. j. agric. food chem. 50:1897-1903. doi: doi.org/10.1021/jf020684p. amorim c.d., camilo a.g., oliveira c., petenucci m.e. and fonseca g.g. 2015. turning pork processing waste into value-added chemicals for the food industry. sustain. mat. technol. 6:9-13. doi: doi.org/10.1016/j.susmat.2015.09.001. aocs. 2004. american oil chemists society: official methods and recommended practices of the american oil chemists’ society. champaign: aocs press. araújo j.m.a. 2001. food chemistry theory and practice. 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of two molecular forms of geotrichum candidum lipase. j. biochem. 107:426-430. doi: doi.org/10.1093/oxfordjournals.jbchem.a123061. visentainer j. v., petenuci m.e., biondo p.f., montanher p.f., santos junior o.o. and claus t. canola: a química analítica do processamento aos compostos bioativos. curitiba: appris, 2015. zhou m.m., che h.x., huang j.q., zhang t.t., xu j., xue c.h. and wang y.m. 2018. comparative study of different polar groups of epa-enriched phospholipids on ameliorating memory loss and cognitive deficiency in aged samp8 mice. mol. nutr. food res. 62:1-13. doi: doi.org/10.1002/mnfr.201700637. paper received march 27, 2020 accepted june 5, 2020 ijfs#1805_bozza ital. j. food sci., vol. 32, 2020 605 paper quality of istrian and slavonian dry-fermented sausages j. pleadin*1, t. lešić1, g. krešić2, t. bogdanović3, m. malenica4, i. kos5, b.s. pulić6, s. petričević3, g. kušec7 and n. vahčić8 1croatian veterinary institute, laboratory for analytical chemistry, savska cesta 143, 10000 zagreb, croatia 2faculty of tourism and hospitality management, department of food and nutrition, university of rijeka, primorska 42, 51410 opatija, croatia 3croatian veterinary institute, regional veterinary institute split, poljička cesta 33, 21000 split, croatia 4department of biotechnology university of rijeka, radmile matejčić 2, 51000 rijeka, croatia 5faculty of agriculture university of zagreb, department of animal science and technology, svetošimunska cesta 25, 10000 zagreb, croatia 6administrative department of agriculture, forestry, hunting, fishery and water management, šetalište pazinske gimnazije 1, 52000 pazin, croatia 7faculty of agrobiotechnical sciences osijek, josip juraj strossmayer university of osijek, vladimira preloga 1, 31 000 osijek, croatia 8faculty of food technology and biotechnology, university of zagreb, pierottijeva 6, 10000 zagreb, croatia *corresponding author: pleadin@veinst.hr abstract the aim of this study was to investigate nutritive and sensorial quality of istrian and slavonian pork-meat dry-fermented sausages. sensorial analysis resulted in statistically significant differences (p<0.05) between these products in 11 sensorial parameters analysed. significantly higher protein content was determined in istrian in comparison with slavonian dry-fermented sausage, although the latter content significantly varied across the producing households. no significant differences in particular fatty acid esters and saturated, monounsaturated and polyunsaturated fatty acids were obtained. although some of the nutritional quality indices failed to meet health recommendations, the obtained values are consistent with data published for pork meat products. keywords: croatian households, dry-fermented sausages, nutritive properties, sensory properties, traditional pork meat sausages ital. j. food sci., vol. 32, 2020 606 1. introduction a long tradition of dry-cured meat production in rural households exists all over the world, especially in the mediterranean countries. a very important group of such products are dry-fermented sausages, whose properties widely vary due to various processing procedures often lacking uniformity (talon et al., 2007; garcíagonzález et al., 2013). suitable chemical and sensory markers enable better linkage between raw matter and processing parameters, and thus result in higher uniformity and consistency of the products (chizzolini et al., 1996; virgili and schivazappa, 2002; zanardi et al., 2010). a wide variety of dry-fermented sausages is characterized by specific flavour investigated into by a number of studies focused on clarifying the control mechanisms affecting the flavour development (toldrá, 1998; garcía-gonzález et al., 2013). the highest impact on sensory properties of these products is that of smoking and ripening operations (jerković et al., 2010; kovačević, 2014). of particular significance are the processes of fat lipolysis, free fatty acids’ formation, and degradation and oxidation of short-chain fatty acids, since these key reactions, taking place during ripening, affect the formation of specific odour and taste of the final product (kozačinski et al., 2006; miličević et al., 2014; marušić et al., 2014). raw material quality is influenced by farm animals’ genotype, manners of their keeping and feeding, procedures applied before slaughtering, and post-slaughtering conditions. technological processes, such as conditioning, fermentation, drying, smoking and ripening, as well as different technological parameters, such as temperature, relative humidity and air/smoke velocity, influence the properties of the final product, too. due to the application of various technological processes, the activity of technological microflora, especially during the fermentation process and long-term maturation of sausage stuffing during production, complex microbiological, physicochemical and biochemical changes take place in fundamental building materials (fats, proteins and carbohydrates), resulting in water loss and increase in dry matter weight (kovačević, 2014). during the recent decades, studies devoted to fermented meat products have mainly focused on evaluation of physicochemical, microbiological and sensory properties (comi et al., 2005, rantsiou et al., 2005, di cagno et al., 2008) in order to contribute to the better characterization of final products, the definition of unique quality markers and the improvement of product specification protocols essential when dealing with products of the protected designation of origin (pdo) or geographical indication (pgi) (bogdanović et al., 2016). however, it is known that significant differences exist among traditional dry-fermented sausages produced in different countries or even in the same country (kos et al., 2009; zanardi et al., 2010). as for traditional dry-fermented sausages produced in croatia, slavonian sausage is produced in the eastern croatia and represents a very important croatian pgi food brand. the same applies to istrian sausage produced in the western croatia, which is in the process of becoming a protected product. even though both sausages are produced from pork meat, istrian sausages contain less fat tissue and are spiced with pepper and garlic. the ingredient that makes these sausages specific is the local wine (malvasia). unlike these, slavonian sausages are spiced with garlic and red pepper and contain in general a larger amount of fat tissue. the production of slavonian sausage calls for the minimum of 70% of the secondand the third-category pork meat and the minimum of 30% of solid fat tissue, while the production of istrian sausage makes use of the meat of the same categories, but of not more than 6% of bacon. mincing (6-8 mm hole for slavonian and at least 10 mm for istrian ital. j. food sci., vol. 32, 2020 607 sausage) is followed by the addition of salt and spices; in case of slavonian sausage, sweet and hot red pepper and garlic are added, while in case of istrian sausage pepper and malvasia enriched with garlic extract and prepared according to a specific procedure, are added. in both cases, a thoroughly mixed mixture of the above-detailed ingredients is stuffed in pork small intestine used as casing, which should be at least 70 cm long when it comes to slavonian, and not longer than 50 cm when it comes to istrian sausage. in the further course of slavonian sausage production, the product is conditioned for a day, coldsmoked for 14 days tops, and ripened and dried in chambers for at least 45 days at temperatures not higher than 16°c, relative humidity thereby being kept at 70-85%. since istrian sausages should not be smoked, the subsequent course of their production upon stuffing involves ripening in chambers at temperatures of 9-16°c and with relative humidity of 65 to 85%. istrian sausages can be released to the market 30 days after the production commencement, while the entire production of slavonian sausages takes 60 days at least. as for their physicochemical properties, slavonian sausage is allowed to contain no more than 40% of fat, while water activity should be kept below 0.90; on the other hand, istrian sausage should contain 16% of proteins at the minimum and 40% of water at the maximum. according to specification, slavonian sausage has an elongated cylinder shape; one sausage pair should be at least 35 cm long and measure 2-3 cm in cross-section (ma, 2019). the casing is reddish/brown and should be free of smudges, folders, cracklings and surface moulds. the texture is expected to be solid and elastic, but not rubberish; the sausage should be easy-to-cut, not prone to crumbling, and easy-to-chew. the stuffing should be dark red in its cross-section, except for the fat tissue, which is coloured either white or orange; the stuffing resembles a mosaic, should be well entangled and free of holes, lacking a dark margin under the casing. prior to cutting, slavonian sausage has a smoky smell of an ash tree, hornbeam or beech; once the sausage is cut, the smell of fermented meat and garlic is released. istrian sausage assumes the shape of an elongated cylinder, measuring at least 50 cm in length and having a rounded diameter. the casing should not be damaged and should tightly adhere to the stuffing. in its cross-section, the sausage is solid, mosaic-like, the meat thereby being coloured red and the fat tissue being coloured white. the sausage should be compact and holes-free, while its stuffing should not get separated during cutting. given the fact that among complex factors that drive consumer dry-fermented sausages’ choice, nutritive and sensory properties are the major criteria, the aim of this study was to investigate into these qualities of the most important types of traditional croatian dryfermented sausages. the study was performed on istrian and slavonian sausages produced in different households situated in two croatian main fermented meat production regions. based on fatty acid content, nutritional quality indices were also assessed for both types of products. 2. materials and methods 2.1. sampling the study involved 40 samples of traditional croatian dry-fermented sausages named istrian (n=20) and slavonian sausage (n=20). sausages were collected randomly during a two-year period (2018-2019) from markets, fairs and producing households. the products originated from two croatian regions: the western region (istra and primorje) and the ital. j. food sci., vol. 32, 2020 608 eastern region (slavonia and baranja). pursuant to the ordinance on meat products (og 62/18) adopted by the republic of croatia, these sausages belong to the group of drycured sausages falling into the category of non-thermally processed meat products. the sausages were produced according to traditional recipes using traditional technologies observed by the producing rural household. to the end of their production, pork meat of the first, the second and the third category was used, together with fat and various amounts of salt and spices, depending on the sausage type (kovačević, 2017). 2.2. sensory characteristics sensory analysis was carried out by a trained panel (of 9 assessors). the assessors were selected and generically trained according to the iso standard (iso 11132:2012). sensory analysis was carried out in the sensory laboratory of the faculty of food technology and biotechnology according to iso 8589:2007. sensory assessment made use of a quantitative descriptive analysis (qda) and a unipolar numerical intensity scale was developed in collaboration with the centro studi assaggiatori (brescia, italy). the intensity of each sensorial property was assessed using an ascending left-to-right numerical scale, “zero” thereby representing the absence of a given sensorial property and “nine” its highest intensity. at each panel session, a repeated sample of a given product group was available; for each assessor, a number of statistical parameters descriptive of his/her efficacy was calculated. plates containing single-coded samples were served into sensory compartments, together with all materials required for sensory assessment. each assessor carried out a sensory assessment of the intensity of objective visual food item properties (colour of the minced meat, colour uniformity, fat content, cohesiveness), smell-related properties (favourable smell, unfavourable smell, smoky smell), taste-related properties (tenderness, juiciness, saltiness, sweetness, sourness, bitterness, spiciness) and aroma (coming from aromatic herbs, spice herbs, ripe meat, biochemical properties, fresh pork meat, moulds). the above was followed by the assessment of subjective properties (cross-section attractiveness, smell attractiveness, consistency attractiveness, maturity, richness of appealing aromas, steadiness of appealing aromas, overall attractiveness), which made use of the same numerical, intensity-measuring scale. 2.3. physicochemical analysis samples were cut into small pieces and homogenized for 15 sec at 6,000 rpm using a grindomix gm 200 (retsch, haam, germany). sample preparation was completed in full line with iso 3100-1:1991. all samples were analysed for physicochemical parameters within the next 48 hours upon arrival into the laboratory. the extracted fat was stored in a refrigerator at -18°c pending fatty acid composition analysis carried out within the next 48 hours. all chemicals used for analyses were of an analytical grade. the ph value was determined in a homogenate diluted with distilled water (1:10, p/v) using a ph/ion 510 – bench ph/ion/mv meter (eutech instruments pte ltd/ oakton instruments, usa) according to the ph/ion 510 instruction manual. water activity (aw) was measured at the room temperature (20±2ºc) using a rotronic hygrolab 3 (rotronic ag, bassersdorf, switzerland). the ph-value and water activity given for each sample represent the mean value of two independent measurements. the water content was determined gravimetrically (iso 1442:1997) at 103°c in an oven (uf75 plus, memmert, schwabach, germany), while the ash content was established according to iso 936:1998 by ital. j. food sci., vol. 32, 2020 609 virtue of burning the samples in a furnace at 550°c (lv9/11/p320 nobertherm, lilienthal, germany). the total fat content was determined using the soxhlet method (iso 1443:1973), which involves digestion of a sample in an acidic environment followed by fat extraction with petroleum ether using a soxtherm 2000 automated device (gerhardt, munich, germany). the total protein content was determined using the kjeldahl method (iso 937:1978) that employed a unit 8 basic digestion block (foss, höganäs, sweden) and an automated distillation & titration device (vapodest 50s, gerhardt, munich, germany). the salt content determination made use of the multiple standard addition potentiometric technique that employs an ion-selective electrode and a na easyplustm analyser (mettler toledo, germany). based on the established sodium content, the representation of sodium chloride (salt) was determined stoichiometrically. the carbohydrate content was calculated based on the water, ash, total protein and fat content. each sausage sample was analysed in duplicate and the results were expressed in form of weight percentage (%) with the accuracy of 0.01%. quality control of analytical methods used was performed using the reference material (rm) tet003rm (fapas, york, england). 2.4. fatty acid profile sample preparation for the analysis of fatty acid methyl esters was described earlier by pleadin et al. (2019). fatty acid methyl esters were analysed using gas chromatography (gc) according to iso 12966-4:2015 and en iso 12966-4:2015. to the above effect, a 7890ba gas chromatographer equipped with flame ionization detector (fid), a 60-m db23 capillary column having an internal capillary diameter of 0.25 mm and the stationary phase thickness of 0.25 μm (agilent technologies, santa clara, usa) was used. the components were detected by fid at the temperature of 280°c, hydrogen flow rate of 40 ml/min, air flow rate of 450 ml/min and nitrogen flow rate of 25 ml/min. the initial column temperature was 130°c; after a minute, it was increased by 6.5°c/min until the temperature of 170°c was reached. the temperature was further increased by 2.75°c/min until the temperature of 215°c was attained. the latter temperature was maintained for 12 min and then further increased rate by 40°c/min until the final column temperature of 230°c was reached, the latter being maintained for 3 min. one ml of a sample was injected into a split-splitless injector at the temperature of 270°c and with the partition coefficient of 1:50. the carrier gas was helium (99.9999%), flowing at the constant rate of 43 cm/sec. fatty acid methyl esters were identified by comparing their retention times with those of fatty acid methyl esters contained by the standard mixture. the results were expressed as a percent-share (%) of an individual fatty acid in total fatty acids, the accuracy thereby being 0.01%. each sausage sample was analysed in duplicate. the material used for quality control was crm bcr 163 (institute for reference materials and measurements, geel, belgium) that has a specified content of seven individual fatty acids. 2.5. nutritional quality of lipids data on fatty acid composition in terms of the mean values obtained by the analysis of two replicates, were used for the calculation of the following lipid quality indices: the atherogenic index (ai), the thrombogenic index (ti) and the hypocholesterolaemic/hypercholesterolaemic ratio (hh). the atherogenic index (ai) ital. j. food sci., vol. 32, 2020 610 indicates the relationship between the sum of the main saturates and the sum of the main non-saturates. this parameter was calculated as follows: ai = [(c12:0 + (4 x c14:0) + c16:0)] / [∑ mufa + pufa n-6+ pufa n-3] (ulbritcth and southgate, 1991) the thrombogenic index (ti) is defined as the relationship between the pro-thrombogenic (saturated) and the anti-thrombogenic fas (mufa, pufa n-6 & pufa n-3). the index was calculated as follows: ti = (c14:0 + c16:0 + c18:0) / [0.5 x ∑mufa + 0.5 x pufa n-6 + 3 x pufa n-3) + + (pufa n-3/pufa n-6)]. the ratio of hypocholesterolaemic over hypercholesterolaemic fatty acids (hh) takes into account well-known effects of certain fatty acids on cholesterol metabolism (santossilva et al., 2002). it was calculated as follows: hh = (c18:1n-9 + c18:2n-6 + c20:4n-6 + c18:3n-3 + c20:5n-3 + c22:5n-3 + c22:6n-3) / (c14:0 + c16:0) (ulbritcth and southgate, 1991). 2.6. data analysis statistical analysis was performed using the spss statistics software 22.0 (spss statistics, ny ibm, 2013) and the big sensory soft (centro studi assaggiatori, brescia, italy, 2005). in order to determine the differences between istrian and slavonian dry-fermented sausage in terms of physicochemical properties, fatty acid composition and sensory parameters, the independent sample t-test was used. the decisions on statistical significance were made at the significance level of p≤0.05. 3. results and discussion 3.1. sensory characteristics sensory qualities of fermented meat products are adjudicated based on their aroma, appearance, flavour, texture, aftertaste and sound properties (flores, 2011). it is important to point out that contrary to some foods, sensory assessment of fermented meat products, including fermented sausages, is not standardized, since no consensus on sensory attributes that should be evaluated hasn’t been reached yet. in several papers, these attributes have been selected and assessed through complex procedures involving sensory vocabulary generation so as to be able to compile a lexicon to be used to describe a sensory profile of a given fermented meat product, as well as through quantitativedescriptive analysis (ruiz-pérez-cacho et al., 2005; garcía-gonzález et al., 2006; garcía-gonzález et al., 2008; benedini et al., 2012). as for croatian householdproduced dry-fermented sausages, it has already been concluded that variations in their overall quality, especially sensory characteristics, represent a huge problem (frece et al., 2014). ital. j. food sci., vol. 32, 2020 611 the results of sensory evaluation of both istrian and slavonian dry-fermented sausage using quantitative descriptive analysis, carried out within this study frame, are shown in fig. 1. figure 1. descriptive sensory profile of objective characteristics (appearance, odour, texture, taste and aroma) of istrian and slavonian dry-fermented sausage. sensorial analysis resulted in statistically significant differences (p<0.05) between these products in 11 out of the total of 20 sensory characteristics analysed, including cohesiveness (p=0.001), smoky odour (p<0.001), tenderness (p<0.001), juiciness (p=0.008), sweetness (p<0.001), sourness (p<0.001), spiciness (p<0.001), aroma generated by the presence of aromatic herbs (p<0.001), ripe meat quality (p=0.011), biochemical parameters (p=0.016) and mould presence (p<0.001). these significant differences can be attributed to the differences in processing and to the use of different ingredients and spices during the production of the two. the cohesiveness is stronger in istrian in comparison with slavonian sausage. slavonian sausage scored higher for its smoky odour than did istrian sausage, whose score in this regard was around 0. the latter was to be expected given that slavonian sausage is smoked with cold smoke, while istrian sausage should not be smoked at all. slavonian sausage scored higher for tenderness and juiciness. it is known that these parameters are in correlation with moisture and fat content. istrian sausage was shown to be sweeter, as oppose to slavonian sausage, which was proven sourer, but they turned out to be equally salty. istrian sausage must have a delicate taste (never sour) and should be moderately salty, while the taste of slavonian dry-fermented sausage, according to its specifications (ma, 2019), should be mildly hot but never bitter, coming as a result of the combination of fermented meat, garlic, salt and red pepper used in its production. slavonian dry ital. j. food sci., vol. 32, 2020 612 fermented sausage is spicier as compared to istrian one due to the presence of red pepper spiking, while istrian sausage scores higher when it comes to aromatic herbs, which can also be attributed to spices added, i.e. garlic and local wine malvasia. the principal intensity scales applied in sensory profiling of similar dry-fermented sausages varied in terms of the different point intensity scales as well as the unstructured line scale (flores, 2011). the lack of consensus regarding the application of sensory scales sometimes makes the comparison and discussion of the results of similar studies quite difficult. however, the results pertaining to the texture descriptors (tenderness, juiciness), obtained in this study, are in agreement with those reported by kos et al. (2015) for dry sausages made from domestic pig and wild boar meat. in general, studies that employed sensorial analysis in order to compare dry-ripened sausages with similar fat levels reported higher sensory scores for texture attributes (fonseca et al., 2015). moreover, the amount of fat affects the colour of smoked sausages and is responsible for a high score obtained on the sensory attributes’ assessment scale (ahmad and amer, 2012), as was the case in this study, too. the smell and the taste of smoked sausages come as the result of decomposition of carbohydrates, lipids and proteins mediated by enzymes, as well as the due to the spices used in the production and the production process itself (kaban and kaya, 2009). the results descriptive of smelland taste-related attributes of istrian and slavonian sausages, obtained in this study, can also be compared to those obtained by kos et al. (2015), who also claimed that dry sausages made from domestic pig and wild boar meat have an intense aroma, higher salinity, more pronounced spiciness, pronounced herbal and ripe meat aroma, and more stable flavour. the intensity of subjective characteristics of istrian and slavonian dry-fermented sausage, assessed as a part of descriptive sensory analysis, is shown in fig. 2. figure 2. descriptive sensory profile of subjective characteristics of istrian and slavonian dry-fermented sausage ital. j. food sci., vol. 32, 2020 613 no significant differences (p>0.05) in any of the assessed attributes were established, showing a good acceptability of both types of dry-fermented sausages. 3.2. physicochemical properties unlike industrial production, household technologies applied during the production of fermented sausages are not regulated, so that a number of production-related factors, such as uneven weight and quality of raw materials and differences in production technologies, result in the diversity of composition of the finished products. therefore, physicochemical properties of dry-fermented sausages show huge variability across individual producers and production periods (kozačinski et al., 2008). industrial production of traditional dry sausages leans on traditional recipes also used in rural households, but, as opposed to seasonal household production, is carried out under controlled processing conditions and is not seasonal in nature, thus allowing for a continuous market supply throughout a year. recent studies have shown that, due to healthy trends in meat products’ consumption in terms of low-fat and low-salt products’ preference, meat producers are facing a new challenge that comes down to attaining fat and salt reduction without any loss in sensory qualities (jiménez-colmenero et al., 2009, flores et al., 2013). physicochemical parameters of istrian and slavonian dry-fermented sausage determined in this study are shown in table 1. the results pertinent to chemical parameters indicate a similar nutritional composition of these two products, with statistically significant differences (p<0.05) in ph value, protein and ash content. table 1. physicochemical parameters of istrian and slavonian dry-fermented sausages. parameter ph water (% w/w) protein (% w/w) fat (% w/w) ash (% w/w) salt (% w/w) ch (% w/w) istrian dry-fermented sausage 5.72±0.59 24.99±5.79 30.66±5.05 38.76±8.44 5.00±0.72 5.51±0.96 0.59±0.43 slavonian dry-fermented sausage 5.12±0.27 28.10±4.28 26.68±3.57 39.48±8.58 4.54±0.59 4.16±0.67 1.20±0.92 p value 0.000 0.085 0.013 0.801 0.049 0.234 0.490 ch carbohydrates. results are expressed as the mean value (%, mean±sd); one sausage sample was taken and analysed in duplicate. the ph value is an indicator of fermentation and ripening of a meat product (salgado et al., 2005) and is commonly used for the assessment of their shelf-life. in dry-fermented sausages, the ph value spans from 4.7 to 6.3 (dellaglio et al., 1996; zanardi et al., 2010; demeyer et al., 2000; moretti et al., 2012; pleadin et al., 2014). in this study, the mean ph values equalled to 5.11 for slavonian sausage and 5.72 for istrian sausage, i.e. were within the acceptable range specified above. literature data suggest that, due to the longer drying and ripening of dry-fermented sausages and a high share of lean meat used in the preparation of their stuffing, water and protein content in finished ripened sausages are on an equal level (about 30-40% w/w), ital. j. food sci., vol. 32, 2020 614 suggesting a high nutritional value of the finished product (pleadin et al., 2014). in dryfermented sausages, the water content mostly raises up to 40% w/w (vignolo et al., 2010). the ratio of water over proteins of 1.2 to 1.3 is typical of dry sausages (incze, 2007). in this study, a higher protein as compared to water content was determined in istrian dry-fermented sausage, while in slavonian dry-fermented sausage the ratio of water over proteins was determined to be 1.05. when analysing dry sausages produced in croatian households, water over protein ratio ranged from 1.0 to 2.1, showing that these products are of a high nutritional value (kozačinski et al., 2008). the amount of total fat present in dry-fermented sausages generally varies widely (21.70 to 55.40% w/w) depending on the recipe and the producing household, but also on the origin of raw materials. such variations can be attributed to the differences in the amount of added fatback and the choice of more or less fatty meat made by individual manufacturers (pleadin et al., 2014). the ash content of meat products generally ranges from 3.52% to 6.06% w/w (ockerman and basu, 2007; jiménez-colmenero et al., 2010; karolyi and čurić, 2012). salt (sodium chloride) is an essential ingredient of fermented sausages, so that meat products are one of the richest sodium chloride food sources contributing to the increased water and fat binding capacity, the formation of colour, taste and texture of the product and its microbiological safety. the salinity of a product depends on the amount of added salt and the duration of drying and ripening of the product (wirth, 1986), and has a significant impact on hardness and elasticity of meat products and their resistance to chewing (kovačević et al., 2010). in fermented sausages’ stuffing, an average share of salt ranges from 2.0% to 2.6%, whereas during the drying process the value keeps growing up to its final level found in the finished product (ockerman and basu, 2007; stahnke and tjener, 2007). jiménez-colmenero et al. (2001) demonstrated that larger amounts of salt exceeding the value of 4-6% have been established in numerous fermented meats studies, which was also the case in both sausages under this study (4.165.51%). since the glucose content in meat is too low or much too variable, different carbohydrates like glucose, sucrose, lactose, maltodextrin, corn syrups, starches and sorbitol are added into fermented sausages’ stuffing (mastanjević et al., 2017) so as to enhance the growth of technological microflora (lucke, 1994). sugars, mostly glucose, facilitate dry sausage fermentation process, since they pose as a substrate for the lactic acid production and contribute to the specific aroma development. sugars added into fermented sausage stuffing in the maximal percent-share of 2% (usually 0.3-0.8% w/w) ensure the ph decrease from the initial 5.8-6.0 down to 4.8-5.4 (lucke, 2000). the carbohydrate content established in sausages under this study (0.59% in istrian and 1.20% in slavonian dryfermented sausage) can be explained in view of the above. 3.3. fatty acid profile table 2 shows the fatty acid composition and fat quality indices of istrian and slavonian dry-fermented sausage. statistical analysis of the fatty acid composition-related data did not show any significant differences between these two types of sausages (p>0.05). no significant differences in fatty acid esters and saturated (sfa), monounsaturated (mufa) and polyunsaturated (pufa) fatty acids were obtained, revealing these sausages to have the fatty acid profile of a typical pork meat product, with small composition variations attributable to the differences in the amount of added fatback and the stuffing fatness. the proportions of fatty acid groups found in both analysed sausages were as ital. j. food sci., vol. 32, 2020 615 follows (in descending order): mufa (46.66-46.83%) > sfa (42.91-43.00%) > pufa (10.2610.33%). this trend has also been confirmed in earlier studies of fatty acid profile of meat and meat products (marušić radovčić et al., 2014; woods and fearon, 2009; pleadin et al., 2014). table 2. fatty acid composition (% of total fatty acids) of istrian and slavonian dry-fermented sausage. fatty acid istrian dry-fermented sausage slavonian dry-fermented sausage p value c10:0 0.09±0.01 0.09±0.01 0.565 c12:0 0.08±0.01 0.08±0.01 0.968 c14:0 1.41±0.11 1.43±0.15 0.714 c16:0 25.39±0.30 25.92±1.22 0.274 c17:0 0.39±0.09 0.40±0.12 0.779 c18:0 14.31±1.12 14.21±1.13 0.817 c20:0 0.43±0.23 0.43±0.19 0.961 c21:0 0.46±0.18 0.44±0.10 0.805 sfa 42.91±1.98 43.00±2.22 0.921 c16:1 2.31±0.34 2.56±0.29 0.087 c18:1t 0.19±0.04 0.20±0.03 0.749 c18:1c 44.18±2.50 43.74 ±2.99 0.689 c22:1 0.14±0.09 0.17±0.07 0.386 mufa 46.83±2.65 46.66±3.13 0.885 c18:2n-6 8.97±3.17 9.13±2.64 0.586 c18:3n-6 0.23±0.02 0.24±0.03 0.469 c18:3n-3 1.01±0.16 0.95±0.20 0.365 pufa 10.26±3.25 10.33±2.59 0.805 n-6 9.22±3.19 9.37±2.64 0.902 n-3 1.04±0.15 0.96±0.20 0.246 results are expressed as the mean value (%, mean±sd); one sausage sample was taken and analysed in duplicate. lod (limit of detection)=0.05%. sfa saturated fatty acids, mufa monounsaturated fatty acids, pufa polyunsaturated fatty acids. as proven earlier, fatty acids most represented in different pork meat sausages are oleic (18:1n-9c), palmitic (16:0), stearic (18:0) and linoleic (c18:2n-6c) acid (lešić et al., 2017). the same was established in the study of fatty acid composition of industrial slavonian kulen (pleadin et al., 2014). oleic acid, as the predominant fatty acid in meat and meat products, ranged from 43.74±2.99% in slavonian sausage to the similar 44.18±2.50% in istrian dry-fermented sausage, and accounts for roughly 94% of all mufas in both products. among sfas, palmitic and stearic acid were shown to be dominating and accounted for roughly 93% of all sfas in both types of sausages. linoleic acid, as the dominating pufa, accounted for roughly 87-88% of all pufas in both sausages. ital. j. food sci., vol. 32, 2020 616 3.4. nutritional quality indices pufa/sfa and n-6/n-3 ratios are the most common parameters used to evaluate the nutritional quality of fat (jimenez-colmenero et al., 2010). additional indices, which take into account different effects that a single fatty acid might have on the incidence of pathogenic phenomena, such as atheroma and/or thrombus formation, i.e. the atherogenic (ai) and the thrombogenic index (ti), were also calculated. in order to gain insight into the effect of fatty acids on blood cholesterol, the ratio of hypocholesterolaemic over hypercholesterolaemic fatty acids (h/h) was determined, too. nutritional fat quality indices determined in this study for istrian and slavonian dryfermented sausage are shown in table 3. no statistically significant differences (p>0.05) in any of the indices were determined between the two. table 3. nutritional fat quality indices calculated for istrian and slavonian dry-fermented sausage. nutritional quality indices recommended value istrian dryfermented sausage slavonian dryfermented sausage p value n-6/n-3 < 4 8.90±3.07 10.28±4.18 0.340 pufa/sfa < 0.4 0.24±0.08 0.24±0.06 0.971 ai < 1 0.55±0.05 0.56±0.05 0.791 ti < 1 1.45±0.13 1.46±0.13 0.879 h/h as higher 2.01±0.17 1.98±0.17 0.676 h/h hypo-/hypercholesterolaemic fatty acids ratio; ai atherogenic index; ti thrombogenic index. results are expressed as the mean value (%, mean±sd); one sausage sample was taken and analysed in duplicate. literature has revealed that consumption of animal fats is related to an excessive intake of sfas and an increased proportion of n-6 pufas (n-6/n-3 ratio). it has been shown that in the current diet of consumers from western countries, the n-6/n-3 ratio is roughly 15-20+, while according to health recommendations it should be less than 4 if the incidence of chronic diet-related diseases is to be reduced (simopoulos, 2002; cordain et al., 2005). in order to meet health recommendations or reduce the risk of cardiovascular, autoimmune and other chronic diseases, the pufa/sfa ratio should be higher than 0.4 (simopoulos, 2002). in this study, the pufa/sfa ratio was in line with health recommendations, whereas n-6/n-3 ratio was approximately two to three times higher than the maximal recommended value. the mean n-6/n-3 ratios obtained in this study for istrian and slavonian sausage are not extremely high (about 10), however, still not in accordance with health recommendations; at least, they are consistent with the results of previous studies of similar pork meat products (lešić et al., 2017). the ai is considered to be a particularly useful index because, in addition to describing mufa content, it places the emphasis on myristic acid (c14: 0), which is believed to have the most harmful cardiovascular effects (higgs, 2002). the ai index takes into account the fact that some saturates, primarily myristic and palmitic acid, are considered to be proatherogenic (since they facilitate the adhesion of lipids onto the cells the immune and the circulatory system are composed of), while non-saturates are considered to be antiatherogenic (since they inhibit the formation of plaques and diminish the levels of ital. j. food sci., vol. 32, 2020 617 esterified fatty acids, cholesterol and phospholipids, therefore preventing microand macro-coronary disease) (ulbritcth and southgate, 1991). it is assumed that ais below 1 are beneficial to human health (pleadin et al., 2019). for both types of sausages under this study, the ais having the mean values of about 0.55 were in line with the recommendation, and similar to those found in other studies of different types of sausages (del nobile et al., 2009; stajić et al., 2011; razmaite and švirmickas, 2012; romero et al., 2013). the ti indicates the risk of blood clotting and represents the ratio of pro-thrombogenic (certain saturated) and anti-thrombogenic (unsaturated) fatty acids. for both sausages analysed within this study, the tis were about 1.5 times higher than the recommended values, which is consistent with literature data on similar types of sausages (del nobile et al., 2009; romero et al., 2013). recent studies show that fatty acids with an even number of c atoms (lauric, myristic and palmitic acid) increase the concentration of total and ldl cholesterol, as well as promote not only coagulation, but also inflammatory processes and insulin resistance (calder, 2015). the h/h index takes into account known effects of certain fatty acids (especially oleic and linoleic acid) involved in cholesterol metabolism. the higher value of this index shows better effects for human health (santos-silva et al., 2002). oleic acid, cis-mufa fatty acids in general and linoleic acid can reduce both total and ldl cholesterol, thereby reducing the risk of cardiovascular disease (calder, 2015). due to the wide range of potential beneficial biological effects (cell membrane functionality, gene expression and lipid metabolism), n-3 pufas play a role in the prevention and treatment of inflammatory processes, thus reducing the risk of cardiovascular disease and some cancers (arterburn et al., 2006). in this study, the h/h values approximated to 2 for both types of sausages; such a value is typical of meat, especially pork meat (santos-silva et al., 2002; razmaite and švirmickas, 2012). 4. conclusions despite of the fact that istrian and slavonian dry-fermented sausages are produced in two climatically different regions, they are nutritionally related. significant differences exist in their sensory properties due to the differences in traditional recipes used in their production. however, from the sensory evaluation standpoint, the existing sensory characteristics are highly accepted by consumers. since this research showed that certain nutritional indices, especially n-6 over n-3 ratio, are not within the desirable limits and in line with health recommendations, modifications to the fatty acid composition of these sausages, to be attained primarily 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707 paper comparison of the fatty acid profile in the meat of pigs and wild boars k. stasiak*a, a. roślewskaa, m. staneka, h. jankowiakb, d. cygan-szczegielniaka and m. bocianb adivision of biochemistry and toxicology, faculty of animal breeding and biology, utp university of science and technology, mazowiecka 28, 85-084 bydgoszcz, poland bdepartment of pig breeding and horses, department of animal breeding and biology, utp university of science and technology, mazowiecka 28, 85-084 bydgoszcz, poland *correspomding author: tel.: +48 523749729, fax: +48 523228158 e-mail address: stasiak@utp.edu.pl abstract the aim of the study was to compare the fatty acid profile of the longissimus lumborum muscle from organically raised pigs: 20 samples from złotnicka spotted pigs, 20 samples from f1 crossbred pigs (polish large white x polish landrace) and 16 samples from wild boar. the content of saturated fatty acids in the meat of animals from all the groups was similar. statistically significant differences were calculated for monounsaturated fatty acids and polyunsaturated fatty acids. the meat of wild boar had the highest content of arachidonic acid but the lowest content of palmitoleic acid, oleic acid and α-linolenic acid. keywords: fatty acid profile, meat, pigs, wild boar ital. j. food sci., vol. 30, 2018 708 1. introduction due to its content of many nutrients, meat plays an important role in the human diet. meat provides our bodies with high value protein, essential amino acids, as well as trace elements, b-group and antioxidant vitamins and fatty acids (fa). the composition of fatty acids, especially the ratio of polyunsaturated fatty acids (pufa) to saturated fatty acids (sfa), is significant for human health (strazdina et al., 2013). according to serrano et al. (2007), saturated fatty acids are regarded as the cause of cardiovascular diseases, as they increase blood pressure and the concentration of the ldl fraction of cholesterol, while monounsaturated fatty acids (mufa) and polyunsaturated fatty acids (pufa) decrease the concentration of bad cholesterol (ldl) and increase the concentration of the good cholesterol (hdl), which results in reducing the risk of heart diseases and atherosclerosis (garcia rebollo et al., 1998; gerhard et al. ,2004). compared to beef, pork is characterised by a favourable fatty acid profile, i.e. lower sfa content and higher pufa content. in comparison with poultry meat, pork (despite a lower total pufa content) shows a much more beneficial n-6 to n-3 fatty acids ratio (blicharski et al., 2013; ivanović et al., 2013). recently, more and more consumers have been paying attention not only to the quality and safety of food, but also to the environmental aspects of its production. the main regulations regarding organic food production are included in the following legal acts: council regulation (ec) no. 834/2007 and regulation (ec) no. 889/2008. according to these regulations, livestock should be fed with plant feeds and feed produced in accordance with the principles of organic farming. at the same time, the laws take into account the possibility of using additives containing some trace elements, vitamins and minerals (sundrum et al., 2000). in organic farms, plant protection products or veterinary medicines should not be used. organically raised animals are fed diets without synthetic additives and gmo preparations. these requirements undoubtedly affect the quality of the product obtained. the meat quality of domestic animals, which were bred in natural conditions means greater food safety for the consumer (skobrák et al., 2011). according to grela and kowalczuk (2009), organic meat products derived from fattening pigs are characterised by a higher content of nutrients. pork obtained from pigs reared in the organic system contains higher amounts of intramuscular fat and more unsaturated fatty acids (angood et al., 2008; sundrum et al., 2000; hansen et al., 2006). in turn, the main source of feed for wild boar living in their natural habitats is plants (grasses, leaves, roots, shrubs, seeds, forest fruits) and, less frequently, avian eggs, snails, insects, earthworms, larvae and beetles (skobrák et al., 2011; rozmaite et al., 2012). due to the natural environment in which wild animals live, venison is often defined as an organic food. the aim of the study was to compare the fatty acid profile of the longissimus lumborum muscle from organically raised złotnicka spotted and f1 crossbred pigs (polish large white × polish landrace) and from wild boar. 2. materials and methods 2.1. animals the study used 20 złotnicka spotted pigs, 20 f1 crossbred pigs (polish large white × polish landrace) and 16 wild boar (hogs and gilts in equal amounts). in each group, the gender ratio of the tested animals was close to 1:1. the pigs originated from an organic ital. j. food sci., vol. 30, 2018 709 farm in the kujawsko-pomorskie province, where they were fed a diet containing 12.6 mj metabolisable energy and 156 g total protein. the feed was comprised of 25% triticale, 20% rye, 10% barley, 10% wheat, 10% oats, 10% lupin, 5% pea, 5% rapeseed and 5% vitaminmineral mixture. the piggery met all the conditions of welfare defined by polish law (regulation of the minister of agriculture and rural development, 2010). at the end of fattening, when złotnicka spotted pigs reached 106.2±3.66 kg of body weight (aged 5.5-6 month) and f1 crosses reached 114.25±2.59 kg (aged 6.5-7 month), the animals were slaughtered under uniform standard production conditions in accordance with polish law and standards in place. from the right halves of the carcasses, samples of the longissimus lumborum muscle were collected (between the 1st and the 4th lumbar vertebra). in turn, two-year-old wild boar (weighing 38-60 kg on average) were shot in the podkarpackie province during the hunting season by different hunters, in accordance with regulation no. 45 and no. 48 of the minister of the environment. the samples were removed from the longissimus lumborum approximately at the level of the 1-2 lumbar vertebra of the carcasses. local hunters provided all samples within 24 hours after shooting. 2.2. samples and fatty acid analysis for further analyses, samples were placed in sterile, tightly sealed bags, chilled to 4°c and transported to a laboratory. after freeze-drying (lyovac gt2, finn-aqua), the samples were analysed for fatty acids through extraction with a chloroform and methanol solution in accordance with the method described by folch et al. (1956). the fatty acid profile of methyl esters was determined by gas chromatography (varian 3800 gc, usa) with a flame ionisation detector, using a supelcowax 10 column (30 m × 0.32 mm × 0.25 μm). the temperature of the injector was 230°c, and that of the detector was 250°c. the volume of the injected sample was 1µl (split 1:50). the carrier gas was helium at a flow rate of 1.5 cm3min-1. the analyses were performed at a temperature range of 90 to 225°c (11°c min-1), 225°c for 6 min, and then an increase from 225 to 240°c (6°c min-1) and 240°c for 19 min. the fatty acid methyl esters were identified with supelco pufa-2 animal source and supelco 37 component fame mix standards (supelco, usa). the composition of fatty acids was expressed as a percentage of total fatty acids. in addition, the indices of fatty acid metabolism were specified. the elongase index was calculated as the ratio of c18:0 to c16:0. the thioesterase index was calculated as the ratio of c16:0 to myristic acid (c14:0). the 9-desaturase index was calculated as 100 times the ratio of the palmitoleic acid (c16:1) percentage to the sum of acids: c16:1 and c16:0. the 9desaturase index was calculated as 100 times the ratio of oleic acid (c18:1) to the sum of acids: c18:1 and c18:0 (zhang et al., 2007). 2.3. statistical analysis the results were statistically analysed with statistica 8.0, and the means and standard deviations are provided in the table. one-way analysis of variance (anova) and the posthoc scheffe test were used to compare the proportion of different fatty acids in the longissimus lumborum muscle of the animals. the normality of data distribution and the homogeneity of variance were tested with the shapiro-wilk and levene tests, respectively. the correlations between the analysed fatty acids were determined based on the coefficients of pearson’s correlation. differences were considered significant at p < 0.05. ital. j. food sci., vol. 30, 2018 710 3. results and discussion the percentage of different fatty acids in the longissimus lumborum muscle of the animals, depending on genetic type and species, is presented in table 1. table 1. fatty acid profile (% of total fa) of fat from the m. longissimus lumborum of the studied animals. zs plw x pl wild boar n=20 n=20 n=16 c14:0 1.02±0.21a 1.26±0.27a 1.80±0.39b c16:0 26.88±1.48a,b 28.22±0.91a 25.14±4.21b c18:0 14.91±2.82a 13.29±1.60a 15.36±0.85a c16:1 n7 3.05±0.45a 3.64±0.74c 1.30±0.68b c18:1 n9 41.65±2.58a 40.76±3.38a 31.59±6.17b c18:3 n3 2.09±0.66a 2.03±0.59a 0.79±0.16b c20:4 n6 10.40±1.87a 10.80±2.99a 24.01±3.15b sfa 42.81±3.50a 42.78±0.95a 42.31±3.93a mufa 44.70±2.81a 44.40±3.90a 32.90±5.77b pufa 12.48±2.34a 12.83±3.36a 24.80±3.10b n3/n6 0.20±0.05a 0.19±0.05a 0.03±0.01b n6/n3 5.34±1.46a 5.62±1.87a 32.03±9.89b thioesterase index1 27.07±4.35a 23.23±4.63a 14.88±5.24b elongase index2 0.55±0.10a,b 0.47±0.07a 0.63±0.12a ʌ9 desaturase (c16) index3 10.10±1.35a 11.35±1.88a 4.74±1.95b ʌ9 desaturase (c18) index4 73.69±4.48a 75.32±3.52a 66.79±4.27b results were expressed as means±sd. values marked in the same row with different letters (a,b) are statistical significantly different at p < 0.05. sfa, mufa, pufa, n6, n3 = sum of all saturated (sfa), monounsaturated (mufa), polyunsaturated (pufa), n6 and n3 fatty acids, zs złotnicka spotted pigs, plw x pl crossbred pigs (polish large white × polish landrace), 1calculated as 16:0/14:0, 2calculated as 18:0/16:0, 3calculated as 100 × [16:1n-9/(16:1n-9 + 16:0)], 4calculated as 100 × [18:1n-9/(18:1n-9 + 18:0)] among saturated fatty acids (sfa), the presence of myristic (c14:0), palmitic (c16:0) and stearic acids (c18:0) was found. compared to pig muscle, the wild boar muscle had a significantly highest content of c14:0 (approx. 1.8%). in turn, the muscle of plw x pl crossbreeds contained statistically more c16:0 acid than wild boar muscle (approx. 3.08%). unlike jankowiak et al. (2010), the present statistical analysis showed no significant differences in the content of palmitic acid between the meat of złotnicka spotted pigs and f1 crossbreeds (plw × pl). despite the differences in the content of individual fatty acids, the total sfa in the meat of the animals in each group did not exceed 43%. the obtained result was confirmed in the studies of other authors (petrović et al., 2014; grela and kowalczuk, 2009). while sfa content remained at the same level in all the groups, considerable differences occurred in the group of monounsaturated fatty acids (mufa). the lowest concentration of mufa was observed in wild boar muscle, in which the content of palmitoleic (c16:1 n7) and oleic acids (c18:1 n9) differed significantly from that determined for the pig muscle from both study groups. compared to other authors, the content of acids (c16:1 n7 and ital. j. food sci., vol. 30, 2018 711 c18:1 n9) determined in the present study was very similar, although the content of mufa was lower by 4.81% for organically raised pigs (grela and kowalczuk, 2009) and by 3.9% for wild boar (ivanović et al., 2013). this difference is caused by the presence of additional acids (c18:1 n7). the highest total pufa (24.8% of all fatty acids) was determined in wild boar muscle. this value was confirmed in the research of other authors (sales and kotrba, 2008). the high level of these acids translates into an appropriate pufa/sfa ratio, which, according to wood et al. (2003), should exceed 0.4. for wild boar muscle, this ratio is 0.5861. the present level was slightly lower than published by dannenberger et al. (2013) for wild boar living in the northern part of germany (0.65÷1.05). in our study, the content of pufa in wild boar meat was twice as high as the values obtained for the muscles of the pigs from both study groups. their pufa level was similar to the values published by cebulska et al. (2018), for złotnicka spotted pigs, and by grześkowiak et al. (2010), who determined the fatty acid profile of the muscle of polish landrace × polish large white pigs. in the present study, arachidonic acid (c20:4 n6) showed the highest percentage among pufa. likely, the high content of c20:4 n6 is due to the absence of c18: 2 n6. this was significantly higher in wild boar muscle compared to pig muscle. statistical analysis did not reveal significant differences in the content of this acid between the meat from złotnicka spotted and plw × pl pigs. the second determined pufa acid was αlinolenic acid (c18:3 n3; ala). the muscles of pigs from both study groups contained similar amounts of this acid, which formed approx. 2% of total fatty acids. in contrast, the wild boar meat was less abundant in this acid (only 0.79%). in comparison with other authors, the ala acid content determined in our own research was higher. according to pedrazzoli et al. (2017), meat of wild boars up to 2 years should contain on average 1.47% of this acid (0.62% more than in the muscle of pigs), while the meat of older wild boars only 0.99% (0.2% more than in the present study). the ala (α-linolenic acid, c18: 3 n3) and la (linoleic; c18: 2 n6) acids supplied with food may undergo enzymatic transformation. elongase enzymes lengthen carbon chains, and desaturases produce additional double bonds, resulting in the formation of polyunsaturated fatty acids with lengths of at least 20 c atoms (achremowicz and szary-sworst, 2005). in fatty acid synthesis, thioesterase is responsible for terminating the reaction and releasing the newly synthesised fatty acid. the thioesterase index (c16:0/c14:0), which indicates a catalysis of palmitic acid synthesis from miristic, was higher (p < 0.05) in pig muscle than in wild boar, while the elongase index, as an indicator of c18:0 synthesis from c16:0 (c18:0/c16:0), remained at the same level (0.47-0.63) in all the groups. ʌ9-desaturase catalyses the conversion of c16:0 and c18:0 to c16:1 and c18:1, the 2 major mufa of pork lipids. greater index values mean greater desaturase activity. the highest ʌ9 -desaturase (c16) and (c18) indexes were found in pig muscle. these results agree with those obtained by daza et.al. (2017) and zhang et al. (2007). between the analysed fatty acids, 11 correlations were found for the meat of zs pigs and 16 correlations for the meat of plw × pl pigs. statistically significant relationships recurred between myristic acid and palmitic, palmitoleic and arachidonic acids, between palmitoleic acid and stearic and oleic acids, and between stearic acid and oleic and arachidonic acids (tables 2-3). ital. j. food sci., vol. 30, 2018 712 table 2. coefficients of correlation (rxy) between fatty acids determined in the m. longissimus lumborum of zs pigs. c16:0 0.81* c16:1 0.56* 0.18 c18:0 –0.22 0.21 –0.57* c18:1 –0.10 –0.45* 0.43* –0.68* c18:3 0.05 –0.16 –0.02 –0.40* –0.19 c20:4 –0.44* –0.56* –0.19 –0.43* –0.02 0.64* c14:0 c16:0 c16:1 c18:0 c18:1 c18:3 * significant at p < 0.05 table 3. coefficients of correlation (rxy) between fatty acids determined in the m. longissimus lumborum of f1 crossbred pigs (plw × pl). c16:0 0.77* c16:1 0.82* 0.66* c18:0 –0.81* –0.77* –0.95* c18:1 0.63* 0.38 0.65* –0.67* c18:3 –0.87* –0.50 –0.58* 0.57 –0.61* c20:4 –0.63* –0.45 –0.63* 0.64* –0.98* 0.56 c14:0 c16:0 c16:1 c18:0 c18:1 c18:3 * significant at p < 0.05 contrary to pig meat, only 5 correlations were found for wild boar meat (table 4). the only correlation shared by the analysed fatty acids for all study groups was a negative correlation between palmitoleic acid and stearic acid (r= -0.57 for zs; r= -0.95 for plw × pl; r= -0.76 for wild boar; p < 0.05). table 4. coefficients of correlation (rxy) between fatty acids determined in the m. longissimus lumborum of wild boar. c16:0 –0.15 c16:1 0.10 0.73* c18:0 –0.27 –0.34 –0.76* c18:1 0.23 –0.90* –0.63* 0.29 c18:3 0.05 0.02 –0.01 0.25 0.10 c20:4 –0.33 0.38 0.24 –0.20 –0.74* –0.35 c14:0 c16:0 c16:1 c18:0 c18:1 c18:3 * significant at p < 0.05 to ensure normal function of the human body, it is particularly important to maintain the proper pufa n6 to pufa n3 ratio, which should range between 1:1 and 4:1 (simopoulos, 2002). in both pig groups under study, this ratio was slightly higher (5.3:1 ital. j. food sci., vol. 30, 2018 713 and 5.6:1), while in the case of wild boar muscle, it was by the highest (32.03:1). according to marciniak-łukasik (2011), excess n6 fatty acids in the diet inhibits the metabolism of n3 fatty acids, which disrupts the physiological balance of the biologically active compounds obtained from them. 4. conclusions wild boar meat does not differ significantly in sfa content from the meat of organically raised pigs of the zs breed and f1 crossbreds (plw × pl). statistically significant differences were noted in the mufa and pufa content. wild boar muscle proved the richest in polyunsaturated fatty acids. the appropriate amounts of individual fatty acids determined in the pig muscles translate into a more health-promoting ratio of n6 to n3 acids. the n6 to n3 fatty acids ratio determined in wild boar muscle was the highest, but the least desirable. references achremowicz k. and szary-sworst k. 2005. polyunsaturated 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stalder k.j., goodwin r.n., lonergan s.m. and beitz d.c. 2007. effects of breed, sex, and halothane genotype on fatty acid composition of pork longissimus muscle. j. anim. sci. 85:583-591 doi: doi.org/10.2527/jas.2006-239 paper received march 6, 2018 accepted june 5, 2018 paper ital. j. food sci., vol. 27 2015 191 keywords: bactoscan fc, conversion line, cow milk, total bacterial count new national conversion line for bactoscan fc in italy: a step forward g. bolzoni1*, a. marcolini1, g. delle donne1, b. appicciafuoco† and a.m. ferrini † 1national reference centre for bovine milk quality, izsler, brescia, italy †national reference laboratory for milk and milk products, istituto superiore di sanità, roma, italy *corresponding author: tel. +39 030 2290541, fax +39 030 2290537, email: giuseppe.bolzoni@izsler.it abstract to improve the reproducibility of flow cytometry technique for total bacterial count in milk, a conversion from instrumental results (impulses/μl) to the reference method resultes (cfu/ml) is needed. in 2008 in italy, a project for a common conversion line for bactoscan fc was initiated. in this paper we report on the second phase of the project focusing on the statistical procedure used to evaluate the validity of the data. the new conversion line, representative of national milk (2,732 valid samples from 29 labs) obtained from both rounds of the study is: log 10 (cfu ml-1) = log 10 (ibc µl-1) x 0.939 + 2.559, with s y:x = 0.282 with an application range up to 70,000 ibc µl-1. mailto:giuseppe.bolzoni%40izsler.it?subject= 192 ital. j. food sci., vol. 27 2015 introduction regulation (ec) 1664 (ec 1664:2006) established that the reference method for determining total bacterial count at 30°c in raw milk is en iso 4833 (iso 4833:2003), however the use of alternative methods is acceptable when they are validated against the reference method in accordance with the protocol set out in en/iso standard 16140 (iso 16140:2003) or other similar internationally-accepted protocols. in the case of milk, iso 21187 (iso 21187:2004) and iso 16297 (iso 16297:2013) are examples of other such protocols. en iso 4833 instructs that colonies grown in defined conditions must be counted after 72 h of incubation at 30°c whereas flow cytometry instruments count free cells independently from their physiological status or their capability to develop into a colony. the counts are obtained from electrical impulses (derived by the fluorescence of bacterial dna and rna stained by fluorochrome ethidium bromide) and must be converted into cfu ml-1 equivalents, as this is the regulatory unit of measure. this conversion (when calculated by a single laboratory) is the main reason for the low reproducibility of the alternative method in spite of its otherwise better repeatability, rapidity and cost effectiveness compared to the reference method and this could have major consequences both from economic and food safety points of view. currently the flow-cell automatic instruments for total bacterial count are indispensable to the centralized and specialized laboratories in charge of large numbers of milk samples per day. for this reason, at the end of 2008, the reference centre for bovine milk quality of izsler launched a project for a “common conversion line” for bactoscan fc (foss, dk), the most commonly used instrument in italy. the result of that study (bolzoni and marcolini, 2010) was adopted on a voluntary basis by several laboratories in our country in the last few years. in 2012, with the coordination of the italian national reference laboratory (nrl) for milk (istituto superiore di sanità), a second round of the project was developed with the objectives to: verify the results of the first round of the project; study a wider range of milk contamination levels; derive a conversion formula that is more tailored to italian milk, meaning a single, mandatory conversion formula to be applied at the national level; propose a statistical model to evaluate the reliability of the raw data. materials and methods the study involved 29 laboratories from all over italy. the number of samples analyzed from each laboratory and for the different levels of contamination was determined on the basis of their previous participation or not in the first round of the project in 2008 (table 1). the protocol adopted in 2008 (bolzoni and marcolini, 2010) was adopted again in 2012 with the intention of producing comparable data. participating laboratories, during the period from january to june 2012, selected samples of cow bulk tank milk (refrigerated and without preservatives) from those submitted for daily analytical activity. the instrument’s calibration status was checked through an inter-laboratory trial using lyophilized milk samples at 3 different contamination levels that were shipped to participants (data not shown). considering that iso/ts 19036 (iso 19036, 2006) estimates that the standard deviation for aerobic mesophilic flora in milk (s r = 0.12) is affected more by operative conditions (s cond = 0.09) than by the initial suspension (s is = 0.04), it was decided that the reference method would be performed using 2 plates per dilution with one series of dilutions. in each laboratory, immediately before analysis, each sample was mixed as stated in iso 6887-5 (iso 6887-5, 2010), tested in duplicate by the bactoscan fc and immediately analyzed by the reference method. a single series of at least 3 decimal dilutions was pretable 1 selection of samples – percentage of samples and respective ranges of impulses required from each lab. range impulses % samples analyzed % samples analyzed (ibc µl-1) (from 10 to 50 samples)a (from 50 to 100 samples)b 0-20 3 3 21-100 30 10 100-1,000 30 10 1,000-5,000 25 10 5,000-10,000 4 30 10,000-50,000 4 27 50,000-99,999 4 10 a: laboratories with participation in the project prior to 2009. b: laboratories without participation in the project prior to 2009. ital. j. food sci., vol. 27 2015 193 pared with quarter-strength ringer’s solution (the level of dilution was established on the basis of the previous instrumental results); 1 ml of each dilution was dispensed in each of 2 plates of milk-pca medium and then incubated at 30°c ± 1°c for 3 days. each participating lab contributed their data on the bactoscan fc double counts in “impulses” (ibc µl-1) and colonies counts from the two plates of each dilution to a database. after the relevant controls of raw data (see: point “d” in the “selection of results” section below) as indicated by iso 7218 (iso 7218:2010; iso 144612:2005) and the additional controls (see: points “e” and “f”), the linear mixed effect model (lme) was applied to produce the regression line of the data from the “valid samples”. the statistical evaluation of the results is described in the following section. the software “procedure r 2.15” and excel 2010 (microsoft corp., redmond, wa) were used. results and discussion range of measurement and linearity the ratio between observed values (o.v.) and expected values (e.v.) in impulses µl-1 (ibc µl-1) from serial dilutions of ad hoc heavily contaminated milk samples was taken as an indicator of linearity of the instrumental signal response. the ratio o.v./e.v. ~ 1 (fig. 1) suggests the acceptable instrumental linearity continues up to 50,000 ibc µl-1, which is well above the producer’s declared limit of 30,000 ibc µl-1 and confirms our previous evaluation (bolzoni et al. 2000, bolzoni et al., 2001). since one of the aims of the work was to evaluate whether a broader range of instrumental measures could be accepted without affecting the conversion line, values > 30,000 ibc µl-1 were also considered. ratio o.v./e.v. = 0.9 was adopted as an arbitrary lower limit of acceptability of the linearity indicator (equivalent to 3 standard deviations from the mean of the ratios obtained). these considerations allowed us to accept 70,000 ibc µl-1 as the upper limit for the range of application of the conversion line. we would like to note that samples with ibc µl-1 > 30,000 (approximately > 4,000,000 cfu ml-1) are rather unusual in italy. selection of results of the 1,827 total milk samples analyzed, which is equivalent to more than 10,000 analytical results produced by 29 participating laboratories, the selection process for valid data led to the rejection of 499 (27%) samples due to the following factors: a) unreliability – 19 samples were eliminated for absence of correspondence between the instrumental results and the reference method results or errors in the report transmission results. b) out of range of measurement – 65 samples were eliminated because their values were outside the established range of linearity (12 samples lower than 10 ibc µl-1 l and 53 higher than 70,000 ibc µl-1). c) instrumental repeatability – 31 samples were eliminated because the difference between replicates exceeded the repeatability limit of the bactoscan fc: critical log difference between replicates > 2.83 s r (p 95%). additionally 12 samples were eliminated because they exceeded the instrumental reproducibility limit (s r ). d) maximum minimum numbers of colonies on the plates and proportionality between dilutions – plates outside the range 10 324 colonies were not considered for the count (iso 7218:2007). the g2 factor test, which compares the relationship between pairs of plates and dilutions, led to the elimination of 179 samples. e) sub-dispersion of reference method results no laboratories were eliminated on this basis (which compares the relationship between observed and expected values on plates) but the frequency of sub-dispersed samples was one criterion used for the selection of laboratories described in point f. f) single laboratory performance evaluation – the effect of each individual laboratory on the extrapolation of the final regression line was considered on the basis of the following factors: – excessive or insufficient dispersion of the individual lab’s regression line; – high frequency of sub-dispersed results from the reference method; – high frequency of eliminated results from the g2 factor test. the dispersion of data around single-lab regression lines is reported in table 2 as s y:x . givfig. 1 bactoscan fc linearity: the relationship between the observed values and the ratio of the observed value to the expected value. 194 ital. j. food sci., vol. 27 2015 table 2 dispersion of the conversion line for individual laboratories (s y:x). lab code samples (n) intercept slope sy:x 40 50 2.1184 1.0309 0.0139 27 98 2.9025 0.7797 0.0930 14 42 2.4432 0.9911 0.1455 38 36 2.2563 1.0279 0.1577 31 93 2.3363 1.0408 0.2517 35 52 2.1976 1.0711 0.2556 41 16 2.1718 1.0859 0.2594 1 40 2.1280 0.9966 0.2676 39 88 2.6219 0.8914 0.2766 15 26 2.5538 1.0119 0.3086 11 26 2.5408 0.9711 0.3118 23 98 2.6394 0.9257 0.3223 24 50 2.4829 0.9593 0.3291 6 68 2.2774 1.0927 0.3365 28 54 3.5260 0.6413 0.3546 37 79 3.6620 0.5508 0.3707 22 76 2.7561 0.8592 0.3756 26 55 2.1806 0.9484 0.3766 7 22 2.4747 1.0033 0.3830 29 24 2.7238 0.9293 0.3893 33 89 3.0733 0.6950 0.4104 34 103 2.1782 1.2029 0.4145 25 97 2.8959 0.7690 0.4286 30 36 2.8759 0.7964 0.4379 8 34 2.6250 0.9531 0.4410 32 29 3.1796 0.6974 0.4567 9 30 3.0099 0.8643 0.6225 36 32 2.5325 0.6897 0.6386 21 110 3.1939 0.8881 0.8504 en s y:x < 0.40 is a criterion for acceptability (listed as a “tentative value” in iso 16297:2013), nine of twenty-nine labs were over range. of the nine, six were considered borderline and only laboratories 21, 36 and 9 were eliminated for over-dispersion. furthermore laboratory 40 was eliminated for sub-dispersion, which suggested their results were not completely reliable. two laboratories exhibited a high frequency of eliminated samples by the g2 factor test (> 50% of samples); in the first case we decided to eliminate all results (lab 36, which had already been eliminated for high dispersion as mentioned above), whereas in the second case (lab 28) we decided to preserve the remaining “valid results” considering the very low value of dispersion of its regression line (0.3546 s y:x ). evaluation of the regression line the lme model was applied to produce the regression line of the selected 1,388 valid samples. multi-step selection of outliers (residual standard deviation > 2.58) was preliminarily applied (iso 21187:2004). in synthesis, after a 3-step sequential elaboration, 65 outliers were eliminated, narrowing the number of valid results to 1,323 and improving the s y:x value from 0.3547 to 0.2781. after the third step, no significant improvement in the level of estimation could be obtained so no further elimination of data was considered appropriate. the following conversion equation was calculated from the 1,323 residual samples (characteristics of the conversion equation are reported in table 3): log 10 (cfu ml-1) = log 10 (ibc µl-1) x 0.946 + 2.569 fig. 2 shows the conversion line from 2012 alongside the conversion line from 2009 (black dashed line) (6), calculated by: log 10 (cfu ml-1) = log 10 (ibc µl-1) x 0.911 + 2.599 the conversion line from 2012 is very similar to the line from 2009 although differences are seen at high and very high contaminatable 3 characterization of the conversion line from 2012. parameters coefficient st. error t sig low high intercept 2.569 0.038 67.57 0.000 2.493 2.645 slope 0.946 0.009 106.91 0.000 0.928 0.964 number of samples = 1,323; s y:x = 0.278. fig. 2 distribution of data from the 2012 conversion line compared with the 2009 conversion line. ital. j. food sci., vol. 27 2015 195 tion levels as a consequence of the extension of the measurement field in the second round of the project. in figs. 3 and 4, the distribution of random effects in the lme for data from individual laboratories is presented. statistically four labs were found to be apparently different from the others: numbers 6 and 34 overestimated their counts while numbers 1 and 26 underestimated their counts. no factors affecting this distribution could be identified (e.g. bacterial flora, sample characteristics, or systematic bias in reference method execution), so the data from these labs were kept in the regression line calculation. new national conversion line considering that the same procedure and the same statistical evaluation were used in both rounds of the project, we considered it not only possible but also appropriate to pool the valid results from 2009 and 2012 and to run a new mixed statistical evaluation. taking a step back before the respective outliers were excluded, a new multi-step selection was performed on the 1,474 valid results from 2009 combined with the 1,388 from 2012. the total elimination of 130 samples at the third step of selection led to no further increase in estimation (table 4). the final regression line was computed from 2,732 samples and it is represented by the equation: log 10 (cfu ml-1) = log 10 (ibc µl-1) x 0.939 + 2.559 the characteristics of the combined regression line are reported in table 5 and fig. 5. fig. 4 distribution of random effect coefficients from the labs compared with a normal distribution (2012 conversion line). fig. 3 q-q plot of random effects from each laboratory in the linear mixed effect model table 4 multi-step selection of outliers on 2009 and 2012 aggregated data. step no. samples (n) s y:x intercept slope min std max std residual residual 1 2,862 0.3533 2.591 0.921 4.503 -5.798 2 2,793 0.3048 2.575 0.931 2.989 -3.103 3 2,752 0.2886 2.565 0.937 2.707 -2.691 4 2,732 0.2821 2.559 0.939 2.651 -2.645 5 2,724 0.2796 2.558 0.939 2.660 -2.597 6 2,718 0.2778 2.557 0.939 2.620 -2.590 table 5 characterization of the new national conversion line (2009 and 2012 pooled results). parameters coefficient st. error t sig low high intercept 2.559 0.032 80.77 0.000 2.496 2.622 slope 0.939 0.006 150.38 0.000 0.927 0.952 number of samples = 2,732; s y:x = 0.282 196 ital. j. food sci., vol. 27 2015 alyzed and their results should be entered into the geometric mean of the last three months, as per the calculation system defined by reg. ec 853:2004. the present project led to the creation of a conversion relationship between impulse µl-1 and cfu ml-1for the enumeration of the total bacterial counts in italian raw cow milk using a bactoscan fc. in summary the conversion line incorporates the following points: – the conversion relationship was constructed according to iso 21187:2004; – the level of accuracy obtained was satisfactory (s y:x = 0.282 log 10 ); – the number of samples was representative of italian milk production variability; – 80% of all italian laboratories involved in milk control by routine method joined the project. the new conversion line appeared robust and representative of milk quality and variety in italy, with a range of application up to 70,000 ibc µl-1. it was ultimately validated and adopted as the national conversion line in italy. this is an important advance for both the industry and public hygiene because the use of a unique conversion line should significantly improve the reproducibility of the bacterial count results obtained by bactoscan fc in italy. in addition, the use of the conversion line for highly-contaminated samples is a further contribution to improve analytical harmonization. data quality control was focused on the evaluation of data entry quality and consequently the accuracy and robustness of the elaborated conversion line. this was done by checking the raw data (agreement between pairs of plates, and proportionality between successive dilutions). references bolzoni g., marcolini a. and varisco g. 2000. evaluation of the bactoscan fc. 1. accuracy, comparison with bactoscan 8000 and somatic cells effect. milchwissenschaft(2), 67-70. bolzoni g., marcolini a. and varisco g. 2001. evaluation of bactoscan fc. second part: stability,linearity, repeteability and carry-over. milchwissenschaft, 56(6), 318-321. bolzoni g. and marcolini a. 2010. bactoscan fc project for unified conversion line in italy. milchwissenshaft, 65(3), 309-310. international organization for standardization. 2003. en iso 4833:2003 microbiology of food and animal feeding stuffs -horizontal method for the enumeration of microorganisms -colony-count technique at 30 degrees c. international organization for standardization. 2003. iso 16140: 2003 microbiology of food and animal feeding stuffs -protocol for the validation of alternative methods. international organization for standardization. 2005. iso 14461-2:2005 (idf 169-2: 2005) milk and milk products -quality control in microbiological laboratories -part 2: determination of the reliability of colony counts of parallel plates and subsequent dilution steps. international organization for standardization. 2006. iso/ ts 19036:2006 microbiology of food and animal feeding stuffs guidelines for the estimation of measurement uncertainty for quantitative determinations. fig. 6 distribution of random effect coefficients from labs compared with a normal distribution (new conversion line). fig. 5 the new national conversion line (computed from 2009 and 2012 aggregated data). the distribution of random effects in the lme for individual laboratory data is presented in fig. 6. conclusions during the first round, in 2008, the main focus was on milk samples with total bacterial counts around 100,000 cfu ml-1, the european legal limit for compliance (reg. ec 853:2004). contrastingly, during the second round, laboratories were invited to include milk samples with high to very high levels of bacterial contamination in order to test the instrumental response to bacteria levels outside of the linear range indicated by the bactoscan fc producers. in routine situations the submission of very highly contaminated samples is a rare occurrence, however they should nonetheless be anital. j. food sci., vol. 27 2015 197 international organization for standardization. 2006. uni en iso 21187:2004 milk -quantitative determination of bacteriological quality -guidance for establishing and verifying a conversion relationship between routine method results and anchor method results. international organization for standardization. 2007. uni en iso 7218: 2007 microbiology of food and animal feeding stuffs – general requirements and guidance for microbiological examination. international organization for standardization. 2010. iso 6887-5: 2010 microbiology of food and animal feeding stuffs -preparation of test samples, initial suspension and decimal dilutions for microbiological examination part 5: specific rules for the preparation of milk and milk products. paper received april 10, 2014 accepted august 6, 2014 international organization for standardization. 2013. iso 16297/ idf 161: 2013 milk bacterial count protocol for the evaluation of alternative methods. the european parliament and the council. 2004. regulation (ec) no 853/2004 of the european parliament and of the council of 29 april 2004 laying down specific hygiene rules for food of animal origin. oj l 139, 30/04/2004, 55-205. the european parliament and the council. 2006. commission regulation (ec) no 1664/2006 of 6 november 2006 amending regulation (ec) no 2074/2005 as regards implementing measures for certain products of animal origin intended for human consumption and repealing certain implementing measures. oj l 320, 18/11/2006, p. 13-45. ijfs#120_limonta_bozza   ital. j. food sci., vol 28, 2016 440 paper pest detected in packed food: ten years of analysis l. limonta*, s. savoldelli, l. süss§ and d. p. locatelli defens, department of food, environmental and nutritional sciences, università degli studi di milano, via celoria 2, 20133 milan, italy §via valle aurina 7, 20152 milan, italy *corresponding author. tel.: +39 0250316753; fax: +39 0250316748 e-mail address: lidia.limonta@unimi.it abstract more than one hundred food complaints, coming from food industries, food stores, and customers, were analyzed over a ten-year period (2004-2013). in the samples of plant products and animal products, the prevalent pests were insects and rodents while in animal products, mites were also found. the highest percentages of stored products’ pests in plant products were represented by coleoptera (62.1) and lepidoptera (48.2), while diptera were mainly crop pests (12.5) or species of hygienic concern (33.3). in animal products, the highest number of complaints concerned milk and dairy products, and the contaminations were caused by insects, mites, and mice. keywords: pests, infestation, products contamination   ital. j. food sci., vol 28, 2016 441 1. introduction different types of foreign matter are reported in food, and insects are considered one of the most important foreign matter problems (lewis, 1993; edwards and stringer, 2007). as stated by the fda defect levels handbook (2014), an infestation is: “the presence of any live or dead life cycle stages of insects in a host product, …; or evidence of their presence …; or the establishment of an active breeding population …”. an inaccurate use of integrated pest management and of hazard analysis and critical control points in food processing and retailing can facilitate the occurrence of pests; the detection of extraneous materials in processed food causes the significant loss of revenue and image to the companies involved. animal contaminations can derive not only from crops, food industries, and stores but also from dwellings when food is improperly conserved (trematerra and fleuratlessard, 2015). the presence of insect in food repulses customers, and moreover, the presence of pests can cause hygienic problems, e.g. cockroaches, domestic flies and rodents can contaminate food with pathogens (gorham, 1991; macovei et al., 2008; sulaiman et al., 2011; pava-ripoll et al., 2012; wasala et al., 2013). complaints about cereal products were considered in a previous paper, and the pests most frequently associated with contamination were flying insects. the moth plodia interpunctella (hbn.) (lepidoptera, pyralidae) and the beetle sitophilus oryzae (l.) (coleoptera, curculionidae) were the pests that were most commonly responsible for food contamination, and rodent droppings were found in a few cases (süss et al., 2014). pasta was more commonly infested by insects because the strip of cardboard packaging is not always well-glued, and one or two series of aeration holes in flexible packaging allow insect entry (gorham, 1991; locatelli and gambaro, 1999; süss et al., 2014; trematerra and savoldelli, 2014). p. interpunctella contaminated confectionery products, made with ingredients that are susceptible to attack, namely, flour, cocoa, nuts, and dried fruits (süss et al., 2014). in the present study complaints about extraneous materials, such as insects, rodent droppings, and hairs, visible to the naked eye, in plant and animal food source were analyzed. 2. materials and methods samples, coming from food industries, food stores, and customers, were analyzed in the entomological laboratory of university of milan from 2004 to 2013. in the present study, we analyzed food related complaints of samples belonging to plant products (88), and animal products (16), in a total of 104 samples. samples were delivered in original packages, unwrapped packages, or without packages. in some cases, the samples were delivered frozen or cooled. samples were stored at the temperature of the retail store and analysed within 48 hours. when the sample was delivered in the original package, the first step in the analysis was a visual inspection to ensure the integrity of the package and the presence of any obvious sign of infestation. the presence of mechanical-related holes, holes due to the activity of insects, or sealing defects, was verified before opening packages. airtight packages were then verified by immersion in water. holes were scrutinized under a stereo-microscope to verify if they were of mechanical origin or due to the insect activity (riudavets et al., 2007). where insects were found, developmental stage and larval age were noted, and we also noted if the insects were dead or alive (süss et al., 2014).   ital. j. food sci., vol 28, 2016 442 for each category, we reported the number and/or the percentage of samples contaminated with insects, rodents or other animals. insects were classified and divided into two different categories: crop pests and stored product pests. 3. results in the samples of plant products and animal products, the most represented pests were insects and rodents (table 1), while mites were also found in animal products. among insects (table 2), coleoptera (40.3%) and lepidoptera (37.5%) were the most represented in plant products, while diptera (33.3%) were in animal products. only 15.2% of plant product complaints concerned food in opened packages, while animal product complaints mainly concerned unwrapped packages. table 1: percentages of pests in samples of plant products (88) and animal products (16). pests plant products animal products insects 81.8 70.5 mites 11.8 anellida 1.1 rodents 11.4 11.8 other vertebrates 1.1 other contaminants* 4.6 5.9 *plastic fragments, feather, seed and vegetable debris. table 2: percentages of insect orders present in samples of plant products and animal products. insect plant products animal products coleoptera 40.3 25.0 lepidoptera 37.5 16.8 diptera 12.5 33.3 hymenoptera 8.3 thysanura 1.4 orthoptera 1.4 dermaptera 1.4 8.3 dictyoptera 1.4 hemiptera 1.4 8.3 psocoptera 2.7   ital. j. food sci., vol 28, 2016 443 3.1. plant products in complaints about plant products, pests of stored products represented the highest percentages in coleoptera (62.1) and lepidoptera (48.2), while diptera were mainly crop pests or species of hygienic concern (table 3). fruits and vegetables (21.6%), canned vegetables (18.2%), and cocoa (15.9%) were the foods most susceptible to complaints (table 4) followed by frozen vegetables (11.4%), mushrooms (9.1%), ready to eat fresh vegetables (7.9%), and fresh vegetables (4.5%). table 3: complaints about plant products: relative values of lepidoptera, diptera and coleoptera (each order was considered 100) distributed according to origin of pests (in the case of “other”, lepidoptera pests were unidentified, in the case of diptera pests were of hygienic relevance). origin of pests lepidoptera diptera coleoptera crop 37.0 66.7 37.9 stored products 48.2 62.1 other 14.8 33.3 table 4: number and percentage of complaints about different food plant products. food complaints no. % canned vegetables 16 18.2 cocoa 14 15.9 dried fruits and vegetables 19 21.6 frozen vegetables 10 11.4 fruit juice 2 2.3 grinded coffee 1 1.1 mushrooms 8 9.1 olive oil 2 2.3 ready meals 1 1.1 ready to eat fresh vegetables 7 7.9 sugar 2 2.3 vegetable stock cube 2 2.3 vegetables 4 4.5 among crop pests, noctuid moths larvae or locusts were found in salads and spinach, acanthoscelides obtectus say, callosobruchus maculatus (f.) and zabrotes subfasciatus (bohemann) (coleoptera, chrysomelidae) infested dried and canned pulses, tuta absoluta (meyrick) (lepidoptera, gelechiidae) and elaterid larvae were detected in tomatoes, noctuid larvae were found also in canned artichokes and tomatoes, and elaterids in canned jam. ostrinia nubilalis (hübner) (lepidoptera, crambidae) contaminated grilled   ital. j. food sci., vol 28, 2016 444 peppers, while carabidae, ground beetles, were detected in spinach and also in chamomile. alive stored product pests such as lasioderma serricorne (f.) (coleoptera, anobiidae) developed in chamomile, spices and herbal tea, plodia interpunctella infested cocoa products, and nuts and dead larvae of p. interpunctella were found in coffee, vegetable stock cube, and instant mashed potatoes. in cocoa products, ephestia spp. (lepidoptera, pyralidae) were also detected, and accidental contamination by blow flies, forficula auricularia l. (dermaptera, forficulidae), and attagenus sp. (coleoptera, dermestidae) was observed; the presence of ahasverus advena (waltl) (coleoptera, silvanidae) and carpophilus sp. (coleoptera, nitidulidae) in cocoa beans revealed the presence of molds. one adult psocid (psocoptera), probably present in the cupboard, contaminated an open sugar bag while another was detected in a bottle of olive oil. sometimes, species not directly linked to the food were found, but their presence seriously increases the risk of contamination, particularly with regard to insects of hygienic relevance. examples were one larva of musca domestica l. (diptera, muscidae) and one adult of muscina sp. (diptera, muscidae) in tomato sauce and a periplaneta americana (l.) (dictyoptera, blattidae) nymph in dried mushrooms. occasional infestations caused by larvae of attagenus sp. (coleoptera, dermestidae) in an orange soft drink and in fruit juice, blow fly in cocoa, and by parts of other animals, such as one lizard tail, one earthworm, and one feather, were also detected. mus domesticus schwartz & schwartz (rodentia, muridae) contaminated different foods: dead mice were found in the cocoa bean, in frozen vegetables, in tomatoes and potatoes. legs of mice and one apodemus sp. (rodentia, muridae) were also present in spinach. also, droppings of mouse were detected in sugar, oil, and sesame seeds, while mouse hairs were found in dates. 3.2. animal products in the case of food complaints in animal products the packages were already unwrapped, except for eggs. the highest percentage of complaints concerned milk and dairy products (62.5%) and the contaminations were caused by insects, mites, and mice. in detail, one adult of diptera sciaridae contaminated a milk bottle, while different insects were detected in dry milk: larvae of dermestid and anobid beetles, and the thorax of an adult stink bug, forficula auricularia (dermaptera, forficulidae), was found in fruit yoghurt. cheese was contaminated with tyrophagous putrescentiae (schrank) (acarididae, acaridae) mites, but also with plastic debris. in dry milk, an adult of m. domesticus was found, while droppings were detected in cheese. diptera were also found in salami, one blowfly larva, and one muscid pupa in an egg package. only in one case was a larva of p. interpunctella recorded in a meat product: this involved a breadcrumbed chicken breast and the infestation derived from the breadcrumb. as far as fish products are concerned, one larva of tenebrio molitor l. (coleoptera, tenebrionidae) was in canned tuna, one larva of m. domestica was in canned octopus, and one larva of an unidentified moth was in fish baby food. 4. comments and conclusion food infestation mainly concerned insects and rodents to a lesser extent. crop insects were frequently detected in frozen and ready to eat vegetables, as the pest hides in the vegetable and leaves are sometimes difficult to wash due to their conformation.   ital. j. food sci., vol 28, 2016 445 vegetable food samples included wrapped and unwrapped packages while complaints about animal products were always about already unwrapped packages. this case makes it difficult to exactly state the origin of contamination, but it should frequently be ascribed to improper conservation after purchase. plodia interpunctella was the most frequent pest; it infested cocoa products, dried and dehydrated vegetables, dried beans, chamomile, spices and herbal tea, nuts, coffee, vegetable stock cubes, instant mashed potatoes, and was also recorded in breadcrumbed meat. ready to eat products with dried mushrooms were infested by dead larvae of diptera mycetophilidae. fungus gnats avoid already senescent or decaying mushroom, usually spoiled by diptera phoridae, sciaridae, and calliphoridae (locatelli et al., 2006). therefore, the presence of mycetophilidae indicated that the mushroom was fresh. ahasverus advena and carpophilus sp., which infest nuts and dried fruits that are incorrectly stored (woodroffe, 1962), were detected in cocoa beans; their presence reveals the development of molds (sinha, 1974; pierce et al., 1991) that can produce mycotoxins (david et al., 1974). also, cockroaches and flies in food cause concern as they are linked to the transmission of pathogens (sasaki et al., 2000; de jesu´s et al., 2004; talley et al., 2009). in a few cases, the infestation was caused by different live insect stages; often, only one dead insect was detected in the samples. in a previous paper on cereal products (süss et al., 2014), live insects in different stages were detected. this time, few live insects were in dried food such as chamomile, cocoa, nuts and herbal tea. rodent droppings and hair were found both in plant and animal food. rodent contamination is unacceptable for health reasons (meerburg and kijlstra, 2007) and indicates negligence and laxity in applying prevention measures during production and storage in warehouses and dwellings. sometimes, customers confused soil and pieces of plastic with droppings. some pests not typically associated with the products were also found. larvae of p. interpunctella often nestled under lids and in jars. in these cases, the insects used the packaging as a shelter, but the effect on the customer was nevertheless extremely negative. in other cases, the insect was embedded in the multilayer film, as sometimes packaging industries overlook insect prevention in processing departments (riudavets et al., 2007). an integrated approach of controlling food safety throughout the entire food production chain has become an important issue in attaining a greater food safety level (valeeva et al., 2004; trematerra, 2013; trematerra and fleurat-lessard, 2015). quality assurance of food industries asks entomologists about the identification of pests’ species, and information on biology and ethology of pests in order to establish the weak point of production processes. in the majority of our cases, it was not possible to trace the origin of infestation. consumers frequently reported contamination several days or weeks after purchase, i.e. packages have already been opened (sometimes a part of the package was missing) and part of the food has been consumed. as turner and ali (1996) reported, often products which are left, partially used and open, in a cupboard will absorb water vapor and possibly become attractive to psocids in the kitchen. in many cases, no information was available about storage conditions after the packages were opened. references david m.h., mills r.b. and sauer d.b. 1974. development and oviposition of ahasverus advena (waltl) (coleoptera, silvanidae) on seven species of fungi. j. stored prod. research 10 (1):17-22. de jesu´s a.j., olsen a.r., bryce j.r. and whiting r.c. 2004. quantitative contamination and transfer of escherichia coli from foods by houseflies, musca domestica l. (diptera: muscidae). int. j. food microbiol. 93:259-262.   ital. j. food sci., vol 28, 2016 446 edwards m.c. and stringer m.f. 2007. the breakdowns in food safety group observations on patterns in foreign material investigations. food control 18:773-782. food and drug administration. 2014. http://www.fda.gov/food/guidanceregulation/guidancedocumentsregulatoryinformation/sanitationtransportatio n/ucm056174.htm. gorham j.r. 1991. food pests as disease vectors. in: “ecology and management of food-industry pests”. j.r. gorham (ed.), pp. 477-482. the association of official analytical chemists. lewis d.f. 1993. a tutorial and comprehensive bibliography on the identification of foreign-bodies found in food. food struct. 12 (3):365-378. locatelli d.p. and gambaro j. 1999. susceptibility of pasta package to the attack of some infesting insects. foreign title: suscettibilità di confezioni di pasta alimentare all'attacco di alcuni insetti infestanti. tecnica molitoria 50 (5):525-530. locatelli d.p., süss l. and panizzolo f. 2006. localization of diptera larvae in dried mushrooms belonging to boletus spp. foreign title: indagine sulla localizzazione di larve di ditteri in funghi essiccati. industrie alimentari 45 (462):1018-1024. macovei l., miles b. and zureki l. 2008. potential of houseflies to contaminate ready-to-eat food with anti biotic-resistant enterococci. j. food prot. 71 (2):435-439. meerburg b.g. and kijlstra a. 2007. role of rodents in transmission of salmonella and campylobacter. j. sci. food agric. 87:2774-2781. pava-ripoll m., goeriz pearson r.e., miller a.k. and ziobro g.c. 2012. prevalence and relative risk of cronobacter spp., salmonella spp., and listeria monocytogenes associated with the body surfaces and guts of individual filth flies. appl. environ. microbiol. 78 (22):7891-7902. pierce a.m., pierce jr. h.d., borden j.h. and oehlschlager a.c. 1991. fungal volatiles: semiochemicals for storedproduct beetles. j. chem. ecol. 17:581-597. riudavets j., salas i. and pons m.j. 2007. damage characteristics produced by insect pests in packaging film. j. stored prod. res. 43:564-570. sasaki, t., kobayashi m. and agui n. 2000. epidemiological potential of excretion and regurgitation by musca domestica (diptera: muscidae) in the dissemination of escherichia coli o157:h7 to food. j. med. entomol. 37:945-949. sinha r.n. 1974. seasonal abundance of insects and mites in small farm granaries. env. ent. 3:854-862. sulaiman i.m., anderson m., khristova m., tang k., sulaiman n., phifer e., simpson s. and kerdahi k. 2011. development of a pcr-restriction fragment length polymorphism protocol for rapid detection and differentiation of four cockroach vectors (group i "dirty 22" species) responsible for food contamination and spreading of foodborne pathogens: public health importance. j. food prot. 74 (11):1883-1890. süss l., savoldelli s., limonta l. and locatelli d.p. 2014. ten years of food complaints about cereal products. integrated protection of stored products iobc-wprs bulletin 98:43-48. talley j.l., wayadande a.c., wasala l.p., gerry a.c., fletcher j., desilva u. and gilliland s.e. 2009. association of escherichia coli o157:h7 with filth flies (muscidae and calliphoridae) captured in leafy greens fields and experimental transmission of e. coli o157:h7 to spinach leaves by house flies (diptera: muscidae). j. food prot. 72 (7):1547-1552. trematerra p. 2013. aspects related to decision support tools and integrated pest management in food chains. food control 34:733-742. trematerra p. and savoldelli s. 2014. pasta preference and ability to penetrate through packaging of sitophilus zeamais motschulsky (coleoptera: dryophthoridae). j. stored prod. res. 59: 126-132. trematerra p. and fleurat-lessard f. 2015. food industry practices affecting pest management. stewart postharvest review 12: 1-7. turner b. and ali n. 1996. the pest status of psocids in the uk. proceedings of the second international conference on urban pests. k.b. wildey (ed.):515-523. valeeva n.i., meuwissen m.p.m. and huirne r.b.m. 2004. economics of food safety in chains: a review of general principles. njas 51-4:369-390.   ital. j. food sci., vol 28, 2016 447 wasala l., talley j.l., desilva u., fletcher j. and astri w. 2013. transfer of escherichia coli o157:h7 to spinach by house flies, musca domestica (diptera: muscidae). phytopathology 103 (4):373-380. woodroffe g.e. 1962. the status of the foreign grain beetle, ahasverus advena (waltl) (col., silvanidae), as a pest of stored products. bull. entomol. res. 53 (3):537-540. paper received april 12, 2015 accepted december 20, 2016 ijfs#1765_bozza ital. j. food sci., vol. 32, 2020 858 paper trend of polychlorinated dibenzo-p-dioxins and dibenzofurans (pcdd/pcdfs) in beehive matrices s.m.r. tulinia*, r.m. specchiab , o.r. laib, c. muccioloc, m. amorenaa and g. crescenzob adepartment of bioscience and agro-food and environmental technology, teramo university, località piano d’accio, 64100 teramo, italy bdepartment of veterinary medicine, university of bari aldo moro, 70121 bari, italy csalerno’s a.s.l., department of prevention food hygiene service, 84135 salerno, italy *corresponding author: tel. +390861266988, fax: +390861266987 email address: stulini@unite.it abstract polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (pcdd/pcdfs) are well-known persistent organic pollutants (pops) with highly toxic potential. these compounds are released in the environment as a complex mixture of various congeners which shown significant physico-chemical differences, as well as different environmental fates. pcdd/pcdf mixtures change spatially and temporally in the environment and biota, complicating the risk assessment and regulatory control for human and animal exposure. considering the well-known role of honeybees as bioindicators for pesticides, heavy metals and other chemicals, the present study has been developed to assess the use of honeybees and honeybee products in biomonitoring projects about pcdd/pcdfs. three dadant-blatt type beehives, located since march 2017 in the headquarter of ducati motor holding s.p.a. (borgo panigale, bologna, italy) have been used as monitoring stations. honeybees, honey and beeswax have been sampled and analyzed for pcdd/pcdfs detection in june and in september of the same year. among the analyzed ital. j. food sci., vol. 32, 2020 859 matrices, beeswax has shown the highest who-teq values, probably due to its lipidic nature capable of accumulating fat-soluble, non-volatile, persistent organic pollutants. hexaclorodibenzo-p-dioxin (hxcdd), usually measured in vegetables and fruits, has been detected only in honey samples. maximum levels of pcdd/pcdfs are settled by commission regulation (ec) no 1259/2011 of 2 december 2011, but only on animalderived products. considering the role of dietary-model adopted by the consumers on toxic substances dietary intake and associated exposure risks, limits on botanical derived products are needed. but more controls about bee-products are advisable also in order to reduce the exposure risk for bees and for protecting biodiversity. keywords: pops, pcdd/pcdfs, honeybees, bio-indicators, environment, health ital. j. food sci., vol. 32, 2020 860 1. introduction various attributes make the honeybee (apis mellifera) the “ideal bioindicator” (tong et al., 1975; stöcker, 1980; wallwork-barber, 1982; raes et al., 1992; leita et al., 1996; zhelyazkova, 2012). due to the intense forager activity and the high sensitivity of bees toward toxic substances, the hives can give informations about environmental pollution via health-status and high mortality of bees or via the residues detection in honey, pollen, propolis, beeswax, royaljelly, larvae and bees (conti and botrè, 2001; crane, 1984; bogdanov, 2006; chauzat et al., 2011; perugini et al., 2017). as far as the biomonitoring of environmental pollution is concerned, honeybees have been used in a lot of investigations to evaluate different type of contaminants (kirkham and corey, 1977; bromenshenk et al., 1985; tonelli et al., 1990; franco et al., 1997; franco et al., 1998; celli and maccagnani, 2003; balayiannis and balayiannis, 2008; porrini et al., 2014). however, only few studies have tried to evaluate the possible application of honeybees as bioindicators for dioxin and furan detection (porrini et al., 2014; özkök et al., 2018). polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (pcdd/pcdfs) are well-known persistent organic pollutants (pops), with highly toxic potential (semanainen et al., 2002; birnbaum et al., 2003). these compounds are released in the environment as a complex mixture of various congeners, produced primarily as by-products of chemical manufacturing activities and during the combustion of municipal and chemical waste (hutzinger et al., 1985; meneses, 2004). atmospheric transport and deposition processes lead to the dispersion of these compounds into soils, plant surfaces, bodies of water and sediments (van den berg et al., 1994; lohmann and jones, 1998). due to the significant differences in physico-chemical properties (solubilities, volatilities, rates of degradation/metabolism, exc.) of each congener, the complex mixtures of pcdd/pcdfs change spatially and temporally in the environment and in animal tissues (schrenk et al., 1991; wegiel et al., 2018; zheng et al., 2008). due to their lipophilic properties, pcdd/pcdfs may concentrate in fatty tissues and bioaccumulate through the food chain (traag et al., 2006). considering the well-known role of honeybees as bioindicators, the present study has been developed to evaluate the distribution of pcdd/pcdfs in the hive. due to their wide use, honey and beeswax contamination could also represent an important safety concern. nevertheless, this investigation has the main purpose of improving data about the possible application of honeybees as bioindicators for monitoring environmental pollution and human exposure risks. 2. materials and methods 2.1. beehives location monitoring station in the headquarter of ducati motor holding s.p.a. (borgo panigale, bologna, italy), was placed in an important italian industrial area, at less than 7 km from bologna central station. bologna city represents one of the most populated cities in italy (2783 p/km2) and its province extends for about 3702 km2 representing in italy, the most productive industrial area for metalworking and engine sector. the monitoring station, consisting of three beehives (bh1, bh2, bh3), dadant-blatt type with 12 frames have been ital. j. food sci., vol. 32, 2020 861 located in the headquarter of ducati motor holding s.p.a. (borgo panigale, bologna, italy) at the end of march 2017. the hives, homogeneous in colony strength (colonies bring up on 10 frames) were placed into a wooden gazebo open frontally and laterally, with roof on top, at the distance of 40 cm between them. as suggested by porrini et al, (2014), all the beehives have been provided with cages for dead bees sampling (under-basket type) and were periodically monitored. 2.2. sampling honeybees, honey and beeswax were sampled from each beehive in june and in september 2017. bees have been collected alive in airtight container directly from the combs, taking care to not involve the queen. honey and wax have been collected as two honeycomb centrifuged for honey extraction. stored at −20°c for 24 hours, each sample (50g) was homogenized with liquid nitrogen by a crushing mill (ika, wilmington, nc), then analyzed for pcdd/pcdfs detection. the analyzed congeners have been reported in table 1. 2.3. chemical analysis determination of pcdd/fs were performed following analytical methods based on international norms for dioxin analysis, such as epa 1613 (epa, 1994), and following the requirements of european directives related to this subject. extraction of the fat fraction, including the compounds of interest, was performed in soxhlet apparatus with solvents (hexane/dichlormethane or hexane/diethyl ether). beeswax samples were directly dissolved in 20 ml of hexane. sample extracts were purified in a 4 cm diameter multilayer column, containing (top to bottom) na2so4 , 44% h2so4/silica, 22 %h2so4/silica, naoh/silica and agno3/silica. pcdd/fs were eluted with hexane. the purified extracts were fractionated in spe pre-packed carbon tubes (supelclean envi-carb), from supelco (bellefonte, pa, usa). the obtained pcdd/f fractions were evaporated to 15 µl under nitrogen stream and corresponding pcdd/f 13c syringe standards (1,2,3,4-tecdd and 1,2,3,7,8,9-hxcdd) were added. samples were analysed in a 6890n gas chromatograph (agilent, santa clara, ca, usa), coupled to an autospec ultima high resolution mass spectrometer (micromass, manchester, uk), operating in electronic impact ionization mode and at 10,000 resolving power. for the pcdd/f analysis, samples were injected (2 µl) on splitless mode (1 min) into the injector at 280 ºc. the chromatograph was fitted with a rtx-5ms column (60 m x 0.25 mm i.d., 0.25 µm) from restek (bellefonte, pa, usa). carrier gas was helium at 250 kpa constant pressure mode. the temperature program was 150 ºc (held for 1 min), increased at 30 min-1 to 200 ºc, increased at 3 ºc min-1 to 235 ºc (held for 10 min) and increased at 6 min-1 to 300 ºc (held 17 min). monitored masses were those proposed by epa 1613 method (epa, 1994). samples were quantified according to the isotopic dilution method, with the use of 13c12-labelled pcdd/f as internal standards. among 200 pcdds and 70 pcdfs, 17 congeners considered dangerous from a toxicological point of view (council regulation (eu) 1259/2011), have been evaluated for the present investigation (table 1). ital. j. food sci., vol. 32, 2020 862 table 1. loq, lod and who-tefs (van den berg et al., 2006) of researched pcdd/pcdf congeners. loq (pg/g) lod (pg/g) who 2005 tefs 2, 3, 7, 8 tetrachlorodibenzo-p-dioxin (tcdd) 0.04 0.02 1 1, 2, 3, 7, 8 pentachlorodibenzo-p-dioxin (pecdd) 0.05 0.025 1 1, 2, 3, 4, 7, 8 hexachlorodibenzo-p-dioxin (hxcdd) 0.10 0.05 0.1 1, 2, 3, 6, 7, 8 hexachlorodibenzo-p-dioxin (hxcdd) 0.10 0.05 0.1 1, 2, 3, 7, 8, 9 hexachlorodibenzo-p-dioxin (hxcdd) 0.10 0.05 0.1 1, 2, 3, 4, 6, 7, 8 heptachlorodibenzo-p-dioxin (hpcdd) 0.25 0.13 0.01 1, 2, 3, 4, 6, 7, 8, 9 octachlorodibenzo-p-dioxin (ocdd) 0.50 0.25 0.0003 2, 3, 7, 8 tetrachlorodibenzofuran (tcdf) 0.04 0.02 0.1 1, 2, 3, 7, 8 pentachlorodibenzofuran (pecdf) 0.05 0.025 0.03 2, 3, 4, 7, 8 pentachlorodibenzofuran (pecdf) 0.05 0.025 0.3 1, 2, 3, 4, 7, 8 hexachlorodibenzofuran (hxcdf) 0.10 0.05 0.1 1, 2, 3, 6, 7, 8 hexachlorodibenzofuran (hxcdf) 0.10 0.05 0.1 2, 3, 4, 6, 7, 8 hexachlorodibenzofuran (hxcdf) 0.10 0.05 0.1 1, 2, 3, 7, 8, 9 hexachlorodibenzofuran (hxcdf) 0.10 0.05 0.1 1, 2, 3, 4, 6, 7, 8 heptachlorodibenzofuran (hpcdf) 0.25 0.13 0.01 1, 2, 3, 4, 7, 8, 9 heptachlorodibenzofuran (hpcdf) 0.25 0.13 0.01 1, 2, 3, 4, 6, 7, 8, 9 octachlorodibenzofuran (ocdf) 0.50 0.25 0.0003 2.4. who-tef and who-teq the concepts of toxic equivalency factor (tef) and total toxic equivalent (teq) have been developed and introduced by the world health organization (who) to facilitate risk assessment and regulatory control of exposure to pcdd/pcdfs mixtures. the who-tef estimates the toxic potential of each congener comparing its affinity for a cytosolic receptor protein (aryl hydrocarbon receptor – ahr) with the highest affinity associated to 2,3,7,8-tetrachlorodibenzo-p-dioxin (tcdd) (table 1). the who-teq is operationally defined by the sum of each compound concentration multiplied by its tef value and represents an evaluation of the total 2,3,7,8-tcdd–like activity of the pcdd/pcdfs mixture, as well as of their total potential toxicity (van den berg et al., 1998, van den berg et al., 2006; van den berg et al., 2013). two different methods can be used for who-teq evaluation. usually, it is calculated as lower-bound for environmental matrices, considering the undetectable concentrations equal to zero. instead, for high-lipid-content food products it is calculated with the upper-bound method, considering the undetectable concentrations equal to the detection limit of each congener (lod) (commission regulation (eu) no 589/2014). for the present study the who-teqs were calculated on honeybees, honey and beeswax pcdd/pcdfs concentrations with both lower-bound and upper-bound methods (table 2). 3. results and discussion ingestion of contaminated food is the principal way of human exposure to pcdd/pcdfs, accounting for 90% if compared to other ways such as inhalation and dermal contact (sweetman et al., 2000). this concern about the human health impact and continuous ital. j. food sci., vol. 32, 2020 863 encouragement from scientific committees to monitor food samples across europe, have led to numerous international and local studies on the concentration of dioxins in particular food items or on the estimation of the daily intake from food (european commission, brusseles, june 2002; focant et al. 2002; karl et al., 2002). seafood represents the most contaminated foodstuff and the congeners most frequently detected in all type of analyzed foodstuff were ocdd and hpcdd, as well as pecdd. regarding dietary intake evaluation on human health it was carry out combining data on consumption habits with the different concentrations of pcdd/pcdfs expressed in who-teq found in food samples (bordajandi et al., 2004). honey and other bee-products are included in nutritional habits of a lot of country and currently are widely used also as dietary supplements for health purposes. however these product were never been taking into account for human dietary exposure calculation and then never analysed for pcdd/pcdfs evaluation. this study has been performed for improving this lack o data profile on food pcdd/pcdfs concentration and to evaluate a possible application of honeybee as bio-indicators for pcdd/pcdfs monitoring in the environment. results are showed in tables 2 and 3. octachlorodibenzo-p-dioxin (ocdd), as well as being reported in other studies regarding pcdd/pcdfs, is the congener most frequently detected during the present investigation (bordajandi et al., 2004; domingo et al., 1999). it has been quantified in all the analysed matrices (figure 1 and figure 2). aside for one honeybee sample collected in september (bh1), that reported 0.07 pg/g of 2, 3, 7, 8 – tetraclorodibenzofuran (tcdf), ocdd was the only congener detected in honeybee samples. this trend is confirmed also for honey samples. tcdf has been quantified only in one sample of honey collected in september from bh1 (0.07 pg/g), while interesting concentrations of ocdd have been detected in all honey samples collected in june and in september (figs. 1 and 2). detectable concentrations of 1, 2, 3, 4, 7, 8 hexaclorodibenzo-p-dioxin and 1, 2, 3, 6, 7, 8 hexaclorodibenzo-p-dioxin (hxcdd), usually measured in vegetables and fruits (domingo et al., 1999), have been detected, in june and in september, only in honey samples (figs. 1 and 2). honey is a natural product that honeybees make from blossom’s nectar or from secretions coming from living parts of plants (özkök et al., 2017), but özkök et al. (2018) monitoring pcdd/pcdfs in honeybee pollen (honey component) had encountered different results. 2, 3, 7, 8 – tcdf, 1, 2, 3, 7, 8 – pecdd, 2, 3, 4, 7, 8 – pecdf, showed the higher concentrations with both analytical methods employed for the study. however, mentioned studies confirm with present data, that maximum levels should be established also for cereals, vegetables and bee-products, in which not negligible concentrations have been reported (özkök et al., 2018). moreover, vegetables represent the most frequent consumed food for a healthy diet and dietary-model adopted by the consumers should be considered important for assessing daily pollutants intake for humans (domingo et al., 1999; schecter et al., 2006). the most toxic pcdd/pcdf congeners are 2,3,7,8-substituted tetra-, penta-, and hexachloro compounds that, in addition to ocdd, have the greatest tendency to bioaccumulate (cohen et al., 2002; bocio and domingo, 2005). nevertheless, the highest concentrations registered during the present investigation and associated to heptaclorodibenzo-p-dioxin (hpcdd) and octachlorodibenzo-p-dioxin (ocdd), have been detected in beeswax samples collected in june (fig. 1). ital. j. food sci., vol. 32, 2020 864 table 2. concentrations detected in june 2017. bees honey wax analyzed congeners bh1* (pg/g) bh2* (pg/g) bh3* (pg/g) average* (pg/g) sd bh1* (pg/g) bh2* (pg/g) bh3* (pg/g) average* (pg/g) sd bh1* (pg/g) bh2* (pg/g) bh3* (pg/g) average* (pg/g) sd 2,3,7,8 tetraclorodibenzop-diossina (tcdd) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd 1,2,3,7,8 pentaclorodiben zo-p-diossina (pecdd) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd 1,2,3,4,7,8 esaclorodibenzo-pdiossina (excdd) <lod <lod <lod nd nd 0,11 0,11 0,15 0,12 0,02 <lod <lod <lod nd nd 1,2,3,6,7,8 esaclorodibenzo-pdiossina (excdd) <lod <lod <lod nd nd <lod 0,12 0,12 0,08 0,07 <lod <lod <lod nd nd 1,2,3,7,8,9 esaclorodibenzo-pdiossina (excdd) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd 1,2,3,4,6,7,8 eptaclorodibenzo-pdiossina (epcdd) <lod <lod <lod nd nd <lod <lod <lod nd nd 2,01 1,39 1,73 1,71 0,31 octaclorodibenzo-pdiossina (ocdd) 0,52 0,56 <lod 0,36 0,31 0,54 0,52 0,51 0,52 0,02 12,05 9,08 9,97 10,37 1,52 2,3,7,8 tetraclorodibenzofu rano (tcdf) <lod <lod <lod nd nd <lod <lod <lod nd nd 0,09 0,07 0,08 0,08 0,01 1,2,3,7,8 pentaclorodiben zofurano (pecdf) <lod <lod <lod nd nd <lod <lod <lod nd nd 0,07 0,07 0,08 0,07 0,01 2,3,4,7,8 pentaclorodiben zofurano (pecdf) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd 1,2,3,4,7,8 esaclorodibenzo furano (excdf) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd ital. j. food sci., vol. 32, 2020 865 1,2,3,6,7,8 esaclorodibenzo furano (excdf) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd 2,3,4,6,7,8 esaclorodibenzo furano (excdf) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd 1,2,3,7,8,9 esaclorodibenzo furano (excdf) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd 1,2,3,4,6,7,8 eptaclorodibenzo furano (epcdf) <lod <lod <lod nd nd <lod <lod <lod nd nd 0,41 1,20 0,86 0,82 0,40 1,2,3,4,7,8,9 eptaclorodibenzo furano (epcdf) <lod <lod <lod nd nd <lod <lod <lod nd nd 0,25 <lod <lod 0,08 nd octaclorodibenzofu rano (ocdf) <lod <lod <lod nd nd <lod <lod <lod nd nd 0,84 0,66 0,88 0,79 0,12 table 3. concentrations detected in september 2017. bees honey wax analyzed congeners bh1* (pg/g) bh2* (pg/g) bh3* (pg/g) average* (pg/g) sd bh1* (pg/g) bh2* (pg/g) bh3* (pg/g) average* (pg/g) sd bh1* (pg/g) bh2* (pg/g) bh3* (pg/g) average* (pg/g) sd 2,3,7,8 tetraclorodibenzo-pdiossina (tcdd) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd 1,2,3,7,8 pentaclorodibenzo-pdiossina (pecdd) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd 1,2,3,4,7,8 – esaclorodibenzop-diossina (excdd) <lod <lod <lod nd nd 0,11 0,16 0,15 0,14 0,03 <lod <lod <lod nd nd 1,2,3,6,7,8 – esaclorodibenzop-diossina (excdd) <lod <lod <lod nd nd 0,11 0,18 0,12 0,14 0,04 <lod <lod <lod nd nd 1,2,3,7,8,9 esaclorodibenzo-pdiossina (excdd) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd 1,2,3,4,6,7,8 eptaclorodibenzo-p-diossina (epcdd) <lod <lod <lod nd nd <lod <lod <lod nd nd 0,58 1,36 1,36 1,10 0,45 octaclorodibenzo-p-diossina (ocdd) 0,77 0,90 0,79 0,82 0,07 0,52 0,59 0,52 0,54 0,04 5,68 8,42 10,04 8,05 2,20 2,3,7,8 tetraclorodibenzofurano (tcdf) <lod <lod 0,07 0,07 0,00 <lod <lod 0,07 0,07 0,00 0,14 0,15 0,16 0,15 0,01 ital. j. food sci., vol. 32, 2020 866 1,2,3,7,8 pentaclorodibenzofurano (pecdf) <lod <lod <lod nd nd <lod <lod <lod nd nd 0,05 0,08 0,05 0,06 0,02 2,3,4,7,8 pentaclorodibenzofurano (pecdf) <lod <lod <lod nd nd <lod <lod <lod nd nd 0,09 0,08 0,05 0,07 0,02 1,2,3,4,7,8 esaclorodibenzofurano (excdf) <lod <lod <lod nd nd <lod <lod <lod nd nd 0,12 0,10 0,10 0,11 0,01 1,2,3,6,7,8 esaclorodibenzofurano (excdf) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd 2,3,4,6,7,8 esaclorodibenzofurano (excdf) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd 1,2,3,7,8,9 esaclorodibenzofurano (excdf) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd 1,2,3,4,6,7,8 eptaclorodibenzofurano (epcdf) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd 1,2,3,4,7,8,9 eptaclorodibenzofurano (epcdf) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod <lod nd nd octaclorodibenzofurano (ocdf) <lod <lod <lod nd nd <lod <lod <lod nd nd <lod <lod 1,31 1,31 0,00 ital. j. food sci., vol. 32, 2020 867 figure 1. average concentrations of pcdd/pcdfs detected in hive matrices in june 2017. figure 2. average concentrations of pcdd/pcdfs detected in hive matrices in september 2017. 0 2 4 6 8 10 12 2, 3 , 7 , 8 tc d d 1, 2 , 3 , 7 , 8 pe cd d 1, 2 , 3 , 4 , 7 , 8 h xc d d 1, 2 , 3 , 6 , 7 , 8 h xc d d 1, 2 , 3 , 7 , 8 , 9 h xc d d 1, 2 , 3 , 4 , 6 , 7 , 8 h pc d d 1, 2 , 3 , 4 , 6 , 7 , 8 , 9 o cd d 2, 3 , 7 , 8 tc d f 1, 2 , 3 , 7 , 8 pe cd f 2, 3 , 4 , 7 , 8 pe cd f 1, 2 , 3 , 4 , 7 , 8 h xc d f 1, 2 , 3 , 6 , 7 , 8 h xc d f 2, 3 , 4 , 6 , 7 , 8 h xc d f 1, 2 , 3 , 7 , 8 , 9 h xc d f 1, 2 , 3 , 4 , 6 , 7 , 8 h pc d f 1, 2 , 3 , 4 , 7 , 8 , 9 h pc d f 1, 2 , 3 , 4 , 6 , 7 , 8 , 9 o cd f co n ce n tr at io n p g/ g bees honey beeswax 0 1 2 3 4 5 6 7 8 9 2, 3 , 7 , 8 tc d d 1, 2 , 3 , 7 , 8 pe cd d 1, 2 , 3 , 4 , 7 , 8 h xc d d 1, 2 , 3 , 6 , 7 , 8 h xc d d 1, 2 , 3 , 7 , 8 , 9 h xc d d 1, 2 , 3 , 4 , 6 , 7 , 8 h pc d d 1, 2 , 3 , 4 , 6 , 7 , 8 , 9 o cd d 2, 3 , 7 , 8 tc d f 1, 2 , 3 , 7 , 8 pe cd f 2, 3 , 4 , 7 , 8 pe cd f 1, 2 , 3 , 4 , 7 , 8 h xc d f 1, 2 , 3 , 6 , 7 , 8 h xc d f 2, 3 , 4 , 6 , 7 , 8 h xc d f 1, 2 , 3 , 7 , 8 , 9 h xc d f 1, 2 , 3 , 4 , 6 , 7 , 8 h pc d f 1, 2 , 3 , 4 , 7 , 8 , 9 h pc d f 1, 2 , 3 , 4 , 6 , 7 , 8 , 9 o cd f co n ce n tr at io n p g/ g bees honey beeswax ital. j. food sci., vol. 32, 2020 868 among the analysed matrices, beeswax has shown the highest number of detectable congeners. hpcdd, ocdd, tcdf, pentachlorodibenzofuran (pecdf), heptachlorodibenzofuran (hpcdf) and octachlorodibenzofuran (ocdf) have all been detected in beeswax (figs. 1 and 2). a larger number of congeners have been quantified in beeswax samples collected in september as compared to those collected in june (fig. 2) and indeed, the highest who-teq values calculated for the present investigation have been associated to them (table 4). table 4. average who-teq lowerand upper-bound values, calculated on the average pcdd/pcdf concentrations measured in honeybee, honey. june 2017 september 2017 honeybees honey beeswax honeybees honey beeswax who-teq lower bound 0.0001 pg/whoteq/g 0.0088 pg/whoteq/g 0.0382 pg/whoteq/g 0.0025 pg/whoteq/g 0.0164 pg/whoteq/g 0.0630 pg/whoteq/g who-teq upper bound 0.1884 pg/whoteq/g 0.1913 pg/whoteq/g 0.2159 pg/whoteq/g 0.1894 pg/whoteq/g 0.1971 pg/whoteq/g 0.2181 pg/whoteq/g higher values of who-teq lowerand upper-bound have been registered in september then in june for all the analyzed matrices (table 2). the highest amounts of who-teq lowerand upper-bound have been associated to the beeswax samples in june and in september (table 2). beeswax composition, consisting in a mixture of fatty acids, fatty alcohols, paraffinic hydrocarbons and free fatty acids, is capable of accumulating of fat soluble, non-volatile and persistent pollutants (tulloch, 1980; johnson et al., 2010; serra-bonvehí and orantes-bermejo, 2010; ravoet et al., 2015; perugini et al., 2017). however, the mechanisms of beeswax contamination have not been well investigated yet. beeswax is made by young worker bees who have never been out of the hive and its contamination could be the result of chemicals transmigration between different matrices, as well as the result of degradation/metabolism processes allowed by the bees consuming contaminated pollen and nectar. currently, regarding pcdd/pcdfs, a possible bioaccumulation phenomenon in the hive cannot be excluded. similarly to animal’s fat-tissues, beeswax is the main reservoir for pcdd/pcdfs mixtures that change in the “hive tissues” during the exposure time according to specific degradation/metabolism processes. although further studies would be advisable, honeybees, honey and beeswax data suggest the possible use of them as indicators for pcdd/pcdfs distribution in the environment (bees), in vegetable foodstuffs (honey) and in animal fat-tissues (beeswax). 4. conclusions “honeybees monitoring stations” have been confirmed as an effective and inexpensive method for controlling the levels of pcdd/pcdfs, as well as other pollutants, in the environment. nevertheless, honey and beeswax contamination also represents an important concern for beekeeping practices and for honeybee products consumer health. ital. j. food sci., vol. 32, 2020 869 honey is an important food product in many countries and beeswax finds important applications in food, cosmetic and pharmaceutical industries, representing possible sources of exposure for humans (perugini et al., 2017). considering the beekeeping common practice to recycle not controlled beeswax for wax foundation sheets production, it can become a source of subsequent recirculation of pollutants in the hive, with serious risks for honeybee health and for biodiversity protection (mullin et al., 2010; wu et al., 2011; wu et al., 2012). based on who-tef and who-teq concepts, the commission regulation (ec) no 1259/2011 of 2 december 2011 set pcdd/pcdfs maximum levels modifying those established by the commission regulation (ec) no 1881/2006 of 19 december 2006. these limits are settled mainly for animal-derived products and expressed as pg whopcdd/f-teq/g, but currently, no maximum levels are applied to cereals, fruit and vegetables, or to honey and other honeybee products. according to many studies this lack should be revised in order to guarantee risk assessment and regulatory control of exposure to pcdd/pcdfs mixtures, taking into account real nutritional habit in different countries. cereals, vegetables and bee-products have long been considered with a “low-impact” for humans daily intake, but recent studies (özkök et al., 2018) have demonstrated that interesting concentrations can also be found in honeybee pollen (honey component) as well in cereals and vegetables (bordajandi et al., 2004; domingo et al., 1999; focant et al., 2002; karl et al., 2002; schecter et al., 2006). acknowledgements this work was made possible thanks to the commitment of ducati motor holding s.p.a. 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while red chilli variety had the lowest antioxidant activities (abts was 3.89 μmol te/g fw, dpph was 2.82 μmol te/g fw and frap was 16.95 μmol te/g fw). the methanolic extracts of different peppers showed strong but different antiinflammatory activity values (8.22 μg/ml 9.52 μg). yellow bell, red and green chilli had the highest anti-inflammatory activity followed by green and red bell extracts, respectively. the results suggest that these varieties of pepper could contribute as sources of important antioxidant and anti-inflammatory related to the oxidative stress and inflammation prevention. keywords: pepper, antioxidant, anti-inflammatory, cell line ital. j. food sci., vol. 32, 2020 266 1. introduction the interest in natural food full of antioxidants and their therapeutic properties have recently increased dynamically. pepper fruit belongs to the genus capsicum, solanaceae family with of more than 200 varieties (zimmer et al., 2012; allemand et al., 2016). pepper has spread of names reckoning on location and type; and the most common pepper names are chili, bell, green and red or just pepper (sung et al., 2016). pepper is considered an excellent source of bioactive nutrients (aminifard et al., 2012). the nutritive composition of peppers depends mainly on the several factors, including cultivar, agricultural practice (organic or conventional), maturity and storage conditions (nimmakayala et al., 2016). pepper fruits are popular due to their characteristic as flavor, texture, firmness and bright colors (silva et al., 2014a). in addition, peppers consumption is recommended due to the positive bioactive compounds impact on health, such as minerals, vitamins and antioxidant compounds (silva et al., 2013; silva et al., 2014b). the phytochemicals in pepper fruits have been reported to possess many pharmacological and biochemical properties, such as anti-allergic, anti-carcinogenic, antioxidant and anti-inflammatory activities (rokayya et al., 2019). they can be eaten fresh, pickled, smoked, dried, or in sauces (alvarez-parrilla et al., 2012). in fact, in chinese medicine, pepper has been used for of stomach aches, arthritis, rheumatism, skin rashes, dog/snake bites and flesh wounds treatments (kim et al., 2016). the antioxidant properties were tested in several studies by using different approaches (lonkar and dedon, et al., 2011). free radical scavenging activity (dpph), ferric reducing antioxidant power (frap), and trolox equivalent antioxidant capacity (abts) assays are the three most frequently used for measuring the antioxidant activities (magalhaes et al., 2011). oxidative stress plays an essential role in cardiovascular diseases and pathogenesis cancer (montecucco et al., 2011). the redox stress triggers the activation of immune cells which release pro-inflammatory cytokines, reactive nitrogen and oxygen species causing pathological pathways and physiological imbalances (lonkar et al., 2011). the present study, therefore aims to determine the total antioxidant, flavonoid and phenol, measure the bioactive activities such as (frap), (abts), (dpph), no production and cell viability (mtt). 2. materials and methods 2.1. chemicals and cells abts (2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), dpph (2,2-diphenyl-1picrylhydrazyl), frap, quercetin, folin-ciocalteau, trolox reagents and ascorbic acid, were from (sigma, louis, mo, usa). dulbecco’s modified eagle medium (dmem) and lps were purchased from sigma inc. (st. louis, mo, usa). the murine machrophage cell line, raw 264.7 was purchased institute of biological sciences (shanghai, china). 2.2. sample preparation five different pepper varieties purchased from shenyang city, china: yellow bell, red bell, green bell, red chilli and green chilli, respectively. bell type (yellow, red and green) and chilli type (red and green). yellow, red and green are bell type from flowering plants genus in the nightshade family (solanaceae); pepper with thick skin fruits ital. j. food sci., vol. 32, 2020 267 (approximately 112–217 g in weight); red and green chilli are the fruits of genus capsicum plants which are nightshade family (solanaceae) members. chilli peppers are varieties with cone shape and medium size (61-91 g in weight). peppers were purchased in a local supermarket at commercial maturity. all the pepper varieties were cleaned and cut tissues into cubic of 10 * 10 * 10 mm3 before processing. freeze-dried (fd) treatment was operated 2h at -80°c then put in freeze drying machine (alpha 1-4 lsc, germany) at 50°c and 0.04 atm for 48h. tissues were grounded to powder, packed in n2-vacuumed amber bottles and stored at -80°c until use. 2.3. antioxidant extraction pepper powder (1.5 g) of each sample was extracted with 10 ml of 80% methanol, by stirring and sonicating for 20 min. the extracts were centrifuged at ~3000g for 20 min, the supernatant was stored at 4°c. 2.3.1. total antioxidant, phenolic and flavonoid content determination the total antioxidant capacity was determined by using ascorbic acid as a standard. the results were expressed as μg of ascorbic acid equivalent (aae) per mg (parasad et al., 2013). the flavonoid content was determined on triplicate aliquots of the homogenous pepper extract (ilahy et al., 2011). thirty-microliter aliquots of the extract were used for flavonoid determination. samples were diluted with 90 μl methanol, 6 μl of 10% aluminum chloride, 6 μl of 1mol/l potassium acetate were added and finally 170 μl of methanol was added. the absorbance was done at 415 nm after 30 min. quercetin was used as a standard for calculating the flavonoid content (mg qe/g of fw). 2.3.2. antioxidant activity determination (abts), (dpph) and (frap) assay antioxidant activity was measured using the abts+ decoloration method using radical abts+ (2,2-azino-di-(3-ethylbenzothialozine-sulphonic acid) (kaur et al., 2013). dpph assay was to evaluate ability of antioxidants toward the stable radical dpph. a 0.2 ml aliquot of a 0.0062 mm of dpph solution, in 20 ml methanol (95%) was added to 0.04 ml of each extract and shaken vigorously (shumaila et al., 2013). frap was operated according to the procedure (young et al., 2013). the frap reagent included 300 (mm) acetate buffer, ph 3.6, 10 (mm) tptz in 40 (mm) hcl and 20 (mm) fecl3 in the ratio 10:1:1. reduction of the ferric-tripyridyltriazine to the ferrous complex, which was measured at 593 nm. results were expressed at μmol te/g fw. 2.4. anti-inflammatory activity 2.4.1. anti-inflammatory extraction extracts were prepared by homogenization of 4 g of freeze-dried sample in 10 ml of 80% methanol, using an ultra turrax digital homogeniser t-25 (ika werke gmbh & co., staufen, germany). further, supernatants were combined and filtered using whatman no. 1 paper. the concentrate was evaporated under vacuum to dryness. finally, the extract residue was dissolved in (dmso) dimethyl sulfoxide solution to give a final concentration of 20 mg/ml. ital. j. food sci., vol. 32, 2020 268 2.4.2. cell viability assay mitochondrial respiration was determined by a mitochondrial-dependent reduction of 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (mtt) assay. briefly, treated cells (1x105 cells/ml) were incubated with 5 mg/ml (mtt) in 96-well plates for 4h and solubilized in dmso (150 μl/well). the extent of the reduction of mtt within the cells were measured at 490 nm (alet et al., 2015). 2.4.3. no production measurement for no production determination, the amount of no2 in the supernatant of the media was measured by the griess method (suresh and priya, et al., 2015). cells were incubated for 24h, after which the cell culture medium (0.2 ml) were added to aqueous extract of pepper varieties containing the griess reagents (1%) sulfanilamide, (0.1%) naphthyl ethylene diamine dihydrochloride in (5%) h3po4. the no production was then determined based on the absorbance at 540 nm. 2.5. statistical analysis data from replications of all varieties were subjected to a variance analysis (anova) using spss (16.0.). significant difference between the means was determined by duncan’s new multiple range test (p<0.05). the correlation between the studied parameters was determined by (pca) using xlstat software. 3. results and discussion 3.1. total antioxidant, phenol and flavonoid contents in our study, all the extracts exhibited high total antioxidant activity values from 9.52 μg aae/mg fw in green chilli to 12.70 μg aae/mg fw in green bell (table 1). table 1. total antioxidant, phenol and flavonoid contents of selected pepper tissue varieties. total antioxidant total phenol total flavonid µg aae/mg mm (teac) mg qe/g yellow bell 12.68±0.15a 20.53±0.64a 89.85±7.45d red bell 11.57±0.60b 21.09±0.93a 141.67±10.12b green bell 12.70±0.57a 19.91±2.14a 113.60±7.96c red chilli 10.21±0.18c 20.45±1.71a 75.85±4.20d green chilli 9.52±0.10c 20.67±1.63a 163.95±8.35a values are the average of three individual samples each analyzed in triplicate ±standard deviation. different uppercase superscript letters respectively indicate significant difference (p < 0.05) analyzed by duncan’s multiple-range test. ital. j. food sci., vol. 32, 2020 269 the antioxidant activity can be attributed to flavonoids and polyphenolic compounds found in it (kim et al., 2016). total phenol contents ranged from 19.91 mm (teac) in green bell to 21.09 mm (teac) in red bell. red and green chilli total phenol had similar values, 20.45 mm (teac) and 20.67 mm (teac), respectively. flavonoid contents ranged from 75.85 mg qe/g fw to 163.95 mg qe/g fw, green chilli peppers had the highest flavonoid contents followed by red bell and green bell. red chilli and yellow bell varieties had lower flavonoid contents 75.85 mg qe/g fw and 89.85 mg qe/g fw, respectively than the other varieties (113.60 163.95 mg qe/g fw). 3.2. antioxidant activity (abts, dpph and frap) antioxidant activity results of pepper varieties were expressed as μmol te/g fw in (fig. 1). the antioxidant activity measured by abts+ assay was between 3.89 μmol te/g fw for red chilli and 5.18 μmol te/g fw for yellow bell. the results obtained by dpph assay were between 2.82 μmol te/g fw for red chilli and 43.29 μmol te/g fw for green chilli. the values of pepper varieties obtained by frap assay were between 16.15 μmol te/ g fw for red chilli and 46.36 μmol te/ g fw for green bell. similar results of antioxidant activity using dpph and frap assays have been reported (rahim and mat, 2012). however, the antioxidant capacity also depends on many other factors including environmental conditions, genetics, post-harvest storage conditions, production techniques used, ext. (anna et al., 2014). frap dpph abts 5 10 15 20 25 30 35 40 45 50 55 abbbba d e a c b b cc a µ m ol t e /g fw yellow bell red bell green bell red chilli green chilli b figure 1. antioxidant activities (abts, dpph and frap) contents were expressed as (μmol te/g fw). 3.3. anti-inflammatory activity the cytotoxicities of pepper extracts in lps-induced macrophages were evaluated in a range of 0–200 μg extract/ml using mtt reduction assay after 24h (fig. 2). therefore, results indicated that the different concentration ranges of used in this study to treat the cells did not exert any cytotoxic effect. analysis of no production revealed that placing unstimulated raw 264.7 cells in culture medium for 24h produced a basal amount of nitrite. when the cells were incubated with extracts from these varieties after treatment ital. j. food sci., vol. 32, 2020 270 with lps for 24h, the medium concentration of nitrite increased markedly. excessive production of no in macrophages represents a probably noxious result, if not counteracted will cause the onset or progression of the many sickness pathologies (hamidreza et al., 2017). significant concentration dependent inhibition was detected when cells were cotreated with lps and various concentrations of the five variety extracts (fig. 3). yellow bell red bell green bell red chilli green chilli 0 25 50 75 100 125 c a b bb b aaa c bc aabab c aaa ab a a abab b c el l v ia bi lit y (% ) 0 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml 200 µg/ml figure 2. cell viability. yellow bell red bell green bell red chilli green chilli 0 2 4 6 8 10 12 14 d d dddd e d b b b b b a a aaa c c cc c a c n o (µ g/ m l) control 0 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml figure 3. no production by raw 264.7 cells. ital. j. food sci., vol. 32, 2020 271 pepper extracts induced a significant (p<0.05) dose-dependent suppression of no production. all tested extracts showed high anti-inflammatory value in a range 0-100 μg concentration where red, green chilli and yellow bell extracts were the best varieties followed by green and red bell varieties. these results indicated that pepper had a noticeable effect on scavenging free radicals. no production value grew equally in a dose dependant manner in all varieties except in red bell variety. a different reaction course was found for red bell. red bell showed an inverse relationship between the antiinflammatory and the dose dependant manner at 25 μg/ml. 3.4. correlation between antioxidant and anti-inflammatory activities the analysis expressed that green chili produced the lowest antioxidant level 9.52 μg aae/mg fw with a little high anti-inflammatory activity 8.22 μg/ml (fig. 4). yellow bell 12.68 μg aae/mg fw and green bell 12.70 μg aae/mg fw produced the highest antioxidant and high anti-inflammatory activity values 8.51 μg/ml and 8.23 μg/ml fw, respectively. yellow bell red bell green bell red chilli green chilli 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 µ g/ m l µ g a a e /m g anti-inflammatory total antioxidant figure 4. correlation between antioxidant and anti-inflammatory activities. the highest inflammatory activity level in red bell 7.25 μg/ml produced high antioxidant level 11.57 μg aae/mg fw. red chilli produced a little high antioxidant and antiinflammatory activity level 10.21 μg aae/mg fw and 8.99 μg/ml, respectively. 3.5. principal component analysis antioxidant and anti-inflammatory activities measurements had been submitted to (pca) to presence of five subspecies of peppers, as seen in table 2. the structuring accessions showed 70.38% of total variation. axes were retained because they expressed 36.44% (axes 1), 33.94% (axes 2). axes 2 was made positively by (dpph), no production, total phenol and flavonoid. the inertia was made negative by cell viability. data projection on plans as ital. j. food sci., vol. 32, 2020 272 outlined by inertia axes of pca from pepper samples showed vital significant variations between varieties. the (figs. 5 and 6) present the plots of the scores/the correlation loadings respectively. in fact, once applying principal component analysis, it appeared that there was a discriminate structure. yellow bell and green bell were grouped together. as for red bell, red and green chilli were individualized. table 2. discriminate variables factors of principal components analysis. f1 f2 proper value 2.92 2.72 variability (%) 36.44 33.94 cumulative (%) 36.44 70.38 total antioxidant +29.57 total phenol +8.95 total flavonoid +33.62 frap +31.95 dpph +23.61 abts +16.15 cell viability -13.22 no production +11.56 figure 5. plots of the scores for antioxidant and anti-inflammatory activities content. total antioxidant total phenol total flavonid frap dpph abts cell viability no production -1 -0,75 -0,5 -0,25 0 0,25 0,5 0,75 1 -1 -0,75 -0,5 -0,25 0 0,25 0,5 0,75 1 f2 (3 3. 94 % ) f1 (36.44 %) variables (axes f1 and f2: 70.38 %) ital. j. food sci., vol. 32, 2020 273 figure 6. plots of the xloadings antioxidant and anti-inflammatory activities content. 4. conclusion the results of this study offer an experimental basis for the event of recent ways to provide knowledge of many pharmacological and biochemical properties. although these results warrant further in-vivo studies, the presented in-vitro data suggest the potential of pepper to attenuate inflammation and oxidative stresses. acknowledgments this work is financially supported by national student innovation and entrepreneurship project, no 201910166090 to declare. references alet v.t., annie m.j. and duncan c.a. 2015. limitations of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2h-tetrazolium bromide (mtt) assay when compared to three commonly used cell enumeration assays. bmc res notes. 8(47):1-10. allemand a., leonardi b.f., zimmer a.r., moreno s., romão p.r. and gosmann g. 2016. red pepper (capsicum baccatum) extracts present anti-inflammatory effects in vivo and inhibit the production of tnf-α and no in vitro. journal of medicinal food 19(8):759-767. 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siró et al., 2008; camilo, 2003; smith et al., 2020). oral nutritional supplements (onss) represent a specific product category, which includes beverages highly dense in macroand micro-nutrients that are thermally treated to provide a 12 month shelf-life (mcclements, 2015a). from a colloidal perspective, onss are oil-in-water emulsions in which the lipid phase, containing a mixture of vegetable oils (e.g., sunflower and soy) and lipophilic bioactive compounds, is dispersed into a water phase enriched in water-soluble vitamins, salts, polysaccharides, in addition to proteins. the proteins are derived from dairy or plant sources, and it is common that mixtures of both are used. surface-active molecules such as proteins, polysaccharides and phospholipids are usually added to create a protective layer at the oil-water interface and prevent the oil droplets from coalescing. it is well known that oil-in-water emulsions display an inherent tendency to destabilise through a variety of physicochemical mechanisms, including gravitational separation and droplet aggregation (mcclements, 2015a, mcclements, 2020). the ingredients selected in the formulation of onss ultimately determine their sensory properties, such as appearance, aroma and texture, which contribute to consumer liking. previous studies have reported that onss possess poor sensory properties and low consumer acceptability which, in turn, can negatively affect consumption rates (gosney, 2003; thomas et al., 2018; regan et al., 2019). given that these beverages may function as a sole source of nutrition, this is highly problematic. considering the importance of sensory properties and consumer acceptability for onss, tribology can serve as a useful instrumental means of understanding how these products are perceived during consumption in the oral cavity. tribological parameters (i.e., coefficient of friction at the boundary and mixed regimes) have been linked to important sensory attributes, such as astringency and creaminess (malone et al., 2003; de hoog et al., 2006; dresselhuis et al., 2008; vardhanabhuti et al., 2011; campbell et al., 2017; priyanka et al., 2020). vardhanabhuti et al. (2011) found an increase in both astringency perception and coefficient of friction in dairy protein suspensions with increasing protein content from 0.5 to 10% w/w at acidic ph. moreover, a strong correlation between perceived creaminess and tribological parameters as a function of fat content was identified in several studies of emulsion systems (malone et al., 2003; de wijk and prinz, 2005). onss, due to their high nutrient density, and diverse ingredient combinations, represent a highly complex food matrix and to the best of our knowledge, information on ingredient interactions, colloidal properties, physical stability and consumer acceptability of onss are not widely available. therefore, the aim of this work was to undertake a comprehensive assessment of the physicochemical and sensory properties of onss with protein, fat and carbohydrate contents ranging from 4.0-10.0%, 2.6-6.7%, 8.7-20.4%, respectively. the information gathered will help underpin the formulation of onss with high physical stability and sensory acceptability. ital. j. food sci., vol. 32, 2020 958 2. materials and methods 2.1. oral nutritional supplements forty commercial onss in different flavours (e.g., vanilla, strawberry, chocolate) were procured from six different suppliers and reviewed. in order to have a broad overview of the physicochemical properties of ons, six samples at low ( ̴ 4%), medium ( ̴ 6%) and high ( ̴10%) protein content were selected for further analysis. the ons samples were obtained from three different suppliers and all samples had a common flavour (i.e., vanilla). the onss were packaged in 250 ml plastic bottles, with the exception of sample a, which was packaged in a 250 ml metallic can. the samples were stored at 22±1°c prior to testing and were analysed within 4 months of their manufacture, according to the supplier information. table 1 shows the macro-nutrient composition of the samples along with their ingredient lists. table 1. macronutrient content (% w/v of product) and ingredient declaration for protein, carbohydrate and fat for the oral nutritional supplements studied a f. macronutrient a b c d e f protein (%) 4.0 4.3 6.4 6.8 10.0 10.0 ingredients milk proteins, soy protein isolate milk proteins, soy protein isolate milk proteins, soy protein isolate milk proteins milk proteins milk proteins, soy protein isolate carbohydrate (%) 13.5 8.7 20.4 18.8 12.4 15.0 ingredients sucrose, maltodextrin sucromalt, maltodextrin, fructo oligosaccharide sucrose, hydrolysed corn starch, fructo oligosaccharide, oat fibre, gum arabic, carboxymethyl cellulose, soy polysaccharide sucrose, hydrolysed corn starch, gum arabic, soy poly saccharide sucrose, maltodextrin glucose syrup, maltodextrin fat (%) 3.3 3.5 5.1 2.6 6.7 5.6 ingredients canola oil, high oleic sunflower oil canola oil, high oleic sunflower oil canola oil, high oleic sunflower oil, corn oil medium chain triacylglycerol from palm kernel, canola and soy rapeseed oil, sunflower oil vegetable oils 2.2. analysis 2.2.1 colour colour analysis was carried out using a tristimulus colorimeter (chromameter-2 reflectance, minolta, osaka, japan) equipped with a cr-300 measuring head. the ital. j. food sci., vol. 32, 2020 959 instrument was standardised against a white tile before measurements. colour was expressed in l* (luminosity), a* (green to red components for negative and positive values, respectively) and b* (blue to yellow components for negative and positive values, respectively) parameters. 2.2.2 particle size distribution particle size distribution of the onss was determined using a laser light diffraction unit (mastersizer 3000, malvern instruments ltd, worcestershire, uk) equipped with a 300 rf (reverse fourier) lens and he-ne laser (λ of 633 nm). sample was introduced to the mixing chamber and dispersed in ultrapure water until a laser obscuration of 12% (± 0.5%) was reached. the water refractive index was set at 1.33, while that of the onss was measured using a refractometer (atago™ r–5000 hand-held refractometer, atago co., ltd) and ranged between 1.34 and 1.38 at 20°c. data were presented as volume-based particle size distribution together with d4,3 values, which represent the volume mean diameter. 2.2.3 rheological properties the rheological properties of the selected onss were determined using a controlled-stress rheometer (ta discovery hybrid rheometer, ta instruments, crawley, west sussex, uk) equipped with a concentric cylinder geometry. viscosity was measured as a function of shear rate in the range 1-300 s− 1 at 22°c. the power law (eq.1) was applied to the data obtained: 𝜏 = 𝐾𝛾! (eq. 1) where τ is shear stress (pa), k is consistency coefficient (pa·sn), γ̇ is shear rate (s-1) and n is flow behaviour index (anema et al., 2014). all the curves showed an r2 ≥0.99. the results are reported as apparent viscosity at 50 s-1, with this shear rate previously demonstrated to relate best to thickness perception during sensory assessments (ross et al., 2019). 2.2.4 accelerated physical stability an analytical centrifuge (lumisizer®, l.u.m. gmbh, berlin, germany), which measures the intensity of transmitted near infra-red (nir) light as a function of centrifugation time and position over the length of a cell held horizontally over the light path, was used to measure the rate and the extent of separation in onss. polycarbonate cells (2 mm light path) were filled with 400 μl of onss with a wide-bore needle. measurements were performed at 25°c and 2200g for 60 min. for calculating the change in transmission over time, integration limits were set at 109 and 130 mm. data were reported as integral transmission (%) as a function of the running time (crowley et al., 2016) and the creaming rate was calculated by linear regression analysis. 2.2.5 tribology tribological assessment was conducted on the selected onss to determine variations in friction and lubrication properties using a method as described by batchelor et al. (2015). tribological measurements were conducted using a mini traction machine (mtm2 pcs instruments, london, uk) consisting of a 19.05 mm stainless steel ball loaded onto a 46 ital. j. food sci., vol. 32, 2020 960 mm diameter silicone elastomer disc, both of which are independently driven, allowing for different motion between the two, and the temperature of measurement was maintained at 20°c. this temperature was chosen since the products are retained in the mouth for less than 20 s and therefore, it is a more representative temperature than body temperature (i.e., 37°c) (batchelor et al., 2015). stribeck curves were constructed for all of the investigated systems by measuring traction in the range 1-200 mm/s with a normal force of 2 n, which is the normal force typically applied to dairy-based products during oral processing (hori et al., 2009; mills et al., 2013; batchelor et al., 2015; nguyen et al., 2016). 2.2.6 sensory analysis the sensory analysis was conducted using untrained assessors (n=25) recruited from university college cork (ireland) in the range 21–50 years. selection criteria for assessors were availability for testing and motivation to participate on all days of the experiment and the assessors also needed to be regular dairy beverage consumers. onss were coded with a randomly selected 3-digit code and presented in duplicate (stone et al., 2012). consumers evaluated both intensity attributes and hedonic in the same session, but separated by an interval to allow training and descriptor explanation with reference to a table of descriptions provided (fellendorf et al., 2017). the assessors were asked to rate the samples on a continuous line scale from 1 to 10 cm, in which 1 corresponded to ‘extremely low descriptor intensity’, and 10 to ‘extremely high descriptor intensity’ (ranking descriptive analysis, rda) (richter et al., 2010). additionally, each assessor was asked to indicate their degree of liking on a 10-cm line scale ranging from 0 (dislike extremely) at the left to 10 (like extremely). sessions were carried out at 22°c under white light and sensory evaluators were instructed to use still water provided to cleanse their palates between tastings. 2.3. statistical data analysis the results presented are the average of at least three measurements and are reported as mean value±standard deviation. statistical analysis was performed using r v.2.15.0 (the r foundation for statistical computing). bartlett’s test was used to check the homogeneity of variance, one-way anova was carried out and the tukey test was used to determine statistically significant differences among means (p<0.05). linear regression analysis by least squares minimisation was performed using microsoft excel 2007 (microsoft corporation, redmond, wa, usa). the goodness of fit was evaluated based on correlation coefficients (r2) and p values. correlation analysis between instrumental measurements and sensory attributes was carried out using statistica (statistica for windows v. 10, statsoft, inc.). ital. j. food sci., vol. 32, 2020 961 3. results and discussion 3.1. colloidal properties of oral nutritional supplements 3.1.1 colour selected onss were firstly characterized for their colour coordinates (table 2). high luminosity and yellowness values, indicated by l* and b* co-ordinates, with values ranging between 62.6 to 74.3 and 14.7 to 26.2, respectively, were recorded for ons samples. low red point values, indicated by a*, with values ranging between -0.1 and 2.8 were displayed in the samples. similar results have been reported for nutritional beverages enriched in dairy protein and carbohydrates and have been attributed to brown melanoidin-based pigments produced in the latter stages of the maillard reaction upon sterilisation (van boekel, 1998; liu and zhong, 2015; chen and o’mahony, 2016; drapala et al., 2017). liu and zhong (2015) reported l*, a* and b* values ranging between 73.0 and 66.3, -2.9 and 1.2, 2.8 and 37.0, respectively, in mixtures of whey protein isolate, maltodextrin and lactose at ph 3.0-7.0 thermally treated at 130°c for 30 min. similar results have been observed in nutritional beverages containing 8.5% milk protein isolate and 5% carbohydrate (i.e., maltodextrin, corn syrup or glucose) at near-neutral ph (ph 6.48-6.78) subjected to thermal treatment at 121°c for 15 min (chen and o’mahony, 2016). table 2. chromaticity co-ordinates (l*, a*, b*), particle size mean diameter (d4,3), rheological properties and traction coefficient for the oral nutritional supplements studied a f. sample chromaticity co-ordinates particle size distribution parameter rheological properties traction coefficient (-) l* a* b* d4,3 (µm) flow behaviour index (-) viscosity at 50 s-1 (mpa·s) boundary regime (2.5 mm s-1) mixed regime (25 mm s-1) (-) a 72.1±0.0d 0.6±0.0d 14.7±0.0f 0.30±0.00c 0.954±0.021ab 7.2±0.0e 0.239±0.021a 0.128±0.005c b 71.4±0.0c 0.6±0.0d 18.5±0.0d 0.31±0.00e 0.801±0.001d 17.3±0.0d 0.221±0.025b 0.143±0.014a c 68.1±0.0f 0.8±0.0b 22.8±0.0b 8.16±0.05a 0.948±0.005bc 43.4±1.0b 0.217±0.018b 0.093±0.019d d 62.6±0.0e -0.1±0.0e 26.2±0.0a 2.05±0.02b 0.891±0.003c 19.3±0.1dc 0.243±0.033a 0.131±0.013a e 74.3±0.0a 2.8±0.0a 15.8±0.0e 0.64±0.00c 0.968±0.021a 20.6±0.1c 0.231±0.021a 0.115±0.013ab f 72.7±0.0b 0.7±0.0c 19.7±0.0 c 0.37±0.00d 0.927±0.003cd 56.0±0.5a 0.204±0.024c 0.092±0.008d a, b, c, d: means with different letters in the same column are significantly different (p<0.05). n.a.: not applicable. 3.1.2 particle size distribution samples a, b, e and f displayed particle size distributions with a dominant peak in the nano-sized region (0.01-1 µm) and d4,3 ranged between 0.30 to 0.64 µm (fig. 1a, b, e, f and table 2). a bimodal distribution, with two distinct peaks in the nano-sized region and in the micron-sized region (1-100 μm) was observed for both samples c and d; d4,3 values for ital. j. food sci., vol. 32, 2020 962 both samples c and d were 8.16 and 2.05 µm respectively (fig. 1c, d; table 2). the presence of a micron-sized peak may be attributed to hydrocolloids (i.e., including gum arabic, oat fibre and carboxy-methyl cellulose) present in the formulations, in agreement with the work of huang et al. (2001), who reported a particle size distribution similar to those in this study for emulsions containing hydrocolloids (0.5%) including methylcellulose and gum arabic. the results indicated that dairy and soy protein (i.e., a, b, e and f) aided the formation of nano-sized particle distributions, perhaps due to synergistic emulsifying properties of dairy and plant proteins when blended (ho et al., 2018). figure 1. particle size distribution data for the oral nutritional supplements studied a f. 3.1.3 rheological properties the rheological properties, including flow behaviour index and apparent viscosity at 50 s-1 of the onss, are reported in table 2. all the samples displayed shear thinning behaviour with flow behaviour index (n) ranging between 0.801 and 0.968. these values are typical of protein suspensions, which form weak structures that are disrupted upon application of shear stress (walstra et al., 2006). the viscosity ranged between 7.2 and 56.0 mpa·s across all the onss, with the samples ranked from lowest to highest as follows: a < b < d < e < c < f (table 2). samples a and b, having the lowest protein content (4%), displayed the lowest viscosity (7.2 and 17.3 mpa·s), whereas in samples d and e higher viscosity was observed with values ranging between 19.3 and 20.6 mpa·s. the highest viscosity values were recorded in ons c and f, with values of 43.0 and 56.0 mpa·s, respectively, and may be attributed to the high proportions of carbohydrates (greater than 15%), proteins (greater than 6%) and fat (greater than 5%) in these samples (dickinson, 2003; pitkowski et al., 2009; huppertz et al., 2017; quinzio et al., 2018). ital. j. food sci., vol. 32, 2020 963 3.1.4 physical stability the physical stability of onss was investigated by measuring the changes in light transmission during centrifugation of the samples. the integral transmission as a function of centrifugation time, together with the clarification rate are shown in fig. 2. an increase in integral transmission with progressive centrifugation time was observed, indicative of phase separation. samples a and b displayed clarification rates of 0.41 and 0.124 integral transmission·min-1 (it%·min-1), respectively, while in sample c, a slight change was observed (0.068 it%·min-1). on the other hand, no changes in the integral transmission were observed in samples d, e and f, thus indicating high stability of the onss (fig. 2). the highest clarification rate (i.e., 0.41 it%·min-1), and therefore the lowest physical stability observed in sample a may be ascribed to its low viscosity (i.e., 7 mpa·s), whereas, the relatively high stability of samples c, d, e and f may be attributed to viscosity values higher than 18 mpa·s, associated with the presence of hydrocolloids (e.g., hydrolysed corn starch, oat fibre, gum arabic) in samples c and d. it is well known that hydrocolloids are able to improve the physical stability of emulsion-based systems by increasing the viscosity (mcclements, 2015b). it has been demonstrated that the addition of guar gum from 0 to 0.5% (w/w) in an oil-in-water emulsion containing 10% oil resulted in reduction in the creaming rate from 10 to 100 min (vélez et al., 2003; yousefi and jafari, 2019). sample physical stability (integral transmission·min-1) r2 a 0.410±0.003 0.993 b 0.124±0.003 0.999 c 0.068±0.003 0.995 d n.a. n.a. e n.a. n.a. f n.a. n.a. figure 2. changes in transmission profile during centrifugation at 2200g for 60 min at 20°c for the oral nutritional supplements studied a ( ), b (l), c ( ), d (×), e ( ), f (o). shown inset is the separation rate expressed as integral transmission·min-1 and r2 computed from the slopes of the linear regression at 2200g for the oral nutritional supplements studied a f. 3.2. tribology assessment tribology has previously been used to model the friction and lubricity of onss that would occur between oral surfaces in the mouth (laiho et al., 2017). a stribeck curve (data not shown), which represents the traction coefficient as a function of speed motion, can be typically divided into the boundary regime (< 10 mm/s) and the mixed regime, (10-100 ital. j. food sci., vol. 32, 2020 964 mm/s) which are usually associated with astringency (rossetti et al., 2009) and creamy textures (chojnicka-paszun et al., 2012), respectively. within the boundary regime (<10 mm/s), systems d, a and e exhibited the greatest values of friction coefficient, with values ranging between 0.231 and 0.243, respectively, whereas samples b, c and f displayed the lowest values for friction coefficient with values between 0.217 and 0.204, respectively. according to the results, the ranking of the investigated systems from highest to lowest value at the boundary regimes was as follows: d > a > e > b > c > f (table 2). this trend can be ascribed to different, interlinked factors, including particle size distribution, viscosity and protein type (i.e., casein and whey) as well as sources (i.e., dairy and plant) (ley, 2008; hughes et al., 2011; zhao et al., 2016, vardhanabhuti et al., 2011; malone et al., 2003; de wijk and prinz, 2005). conversely, in the mixed regimes (10-100 mm·s-1), lower friction coefficients indicate a creamier texture, which can be associated with sensory attributes such as thickness, smoothness, slipperiness and softness (akhtar et al., 2006; chojnicka-paszun et al., 2012; batchelor et al., 2015). the ranking in terms of creamy texture, from the lowest to the highest friction coefficient at the boundary regimes, corresponding to the most to least creamy, respectively, was as follows: f > c > e > a > d > b (table 2). this trend for texture is consistent with the composition of the investigated systems (table 1) and was predominately ascribed to the fat content, whereby the creamier systems (i.e., f, c and e) had fat content >5% (w/w), and the less creamy systems (i.e., b, d and a) had a fat content <4% (w/w). the work of akhtar et al. (2005) showed that higher levels of fat in oil-in-water emulsions stabilized with sodium caseinate contributed to an enhanced perception of creaminess. furthermore, the trend in friction coefficient is aligned with the apparent viscosity at 50 s-1 (table 2), whereby measurement of viscosity at this shear rate is typically related to the perception of thickness (akhtar et al., 2006; stokes et al., 2013; dickinson, 2018). 3.3. sensory evaluation the sensory profiles of the selected onss were mapped by ranking descriptive analysis, which included intensity of beige colour, vanilla aroma, cooked flavour, thickness and astringent after-taste (table 3). a wide range of beige colour intensity, with values ranging between 3.7 and 7.7 was recorded for the ons samples and the following rank, from the lowest to the highest, a < e < b < f < c < d was observed. the vanilla aroma intensity ranged between 3.8 and 7.5, with a lowest to highest ranking of b < a < e < d < f < c across the samples. cooked flavour, which originates from sulphur compounds in thermally processed protein-based beverages, was mildly (3.3-5.1) perceived and the onss displayed the following rank, from lowest to highest: b < a < e < d < f < c. a positive correlation (0.847, p<0.05) between cooked flavour and the proportion of carbohydrate, one of the prerequisite substrates for the maillard reaction, was found (mellema and bot, 2009). a wide range of thickness values, from 2.5 to 7.0 was recorded and the lowest thickness perception was perceived in onss having the lowest protein content (4.0 and 4.3% for a and b, respectively). the highest thickness values were recorded in samples c, d and f, which contained relatively high levels of protein (>6%) and carbohydrate (>15%), such as guar gum and carboxy-methyl cellulose. the results displayed a strong positive correlation between thickness perception and instrumental viscosity (0.869, p<0.05) as well as thickness perception and mixed regime values (-0.772, p<0.05), in agreement with the literature (malone et al., 2003; dresselhuis et al., 2008; ross et al., 2019). ital. j. food sci., vol. 32, 2020 965 table 3. data for ranking descriptive analysis and sensory hedonic evaluation of the oral nutritional supplements studied a f. a, b, c: means with different letters in the same column are significantly different (p<0.05). 1 corresponds to extremely low descriptor intensity; 10 corresponds to extremely high descriptor intensity. 2 corresponds to dislike extremely; 10 corresponds to dislike extremely. sample ranking descriptive analysis1 hedonic evaluation2 colour (degree of beige) vanilla aroma cooked flavour thickness sweet taste after-taste liking of appearance liking of aroma liking of flavour liking of texture overall acceptability a 3.7±0.1b 3.8±0.2c 3.9±0.7a 2.5±0.2c 4.3±0.3b 3.4±0.1a 4.9±0.0b 5.5±0.3ab 4.3±0.3b 5.2±0.3b 4.5±0.0 b b 5.4±0.4ab 5.4±0.5b 3.3±0.1a 4.2±0.1bc 7.0±0.0a 2.9±0.1a 6.1±0.0a 6.2±0.2a 5.9±0.2a 6.2±0.1b 5.7±0.1a c 6.5±0.4ab 4.5±0.3bc 5.1±0.1a 7.0±0.2a 4.9±0.3b 3.8±0.2a 4.7±0.1b 5.3±0.1ab 4.1±0.3b 5.1±0.1b 4.0±0.2b d 7.7±0.4ab 7.5±0.1a 4.9±0.3a 5.5±0.2ab 7.4±0.8a 3.0±0.5a 3.6±0.0c 4.9±0.1bc 4.2±0.1b 5.7±0.2ab 4.0±0.2b e 4.0±0.4b 4.4±0.2bc 4.2±0.0a 5.1±0.7ab 5.6±0.0ab 3.1±0.2a 5.9±0.2a 5.2±0.1ab 4.9±0.1ab 5.9±0.1ab 5.2±0.1a f 5.5±0.1ab 4.1±0.1bc 5.0±0.2a 6.9±0.4a 6.2±0.3ab 3.7±0.5a 5.7±0.2a 4.1±0.2c 4.2±0.1b 5.3±0.0ab 4.0±0.1b ital. j. food sci., vol. 32, 2020 966 medium sweet intensity, with values ranging between 4.3 and 7.0 was assessed across onss and the following order, from highest to lowest sweet intensity, a < c < e < f < b < d was observed. across the samples, the intensity of after-taste, which is typically perceived in protein-based beverages, ranged between 2 and 3.5 and no significant (p > 0.05) differences were observed, probably due to the tailored ingredients balance in the formulations (de wijk and prinz, 2005; vardhanabhuti et al., 2011). the consumer preferences for ons products were assessed by hedonic testing, which encompasses liking of appearance, aroma, flavour, texture and overall acceptability (table 3). onss displayed a medium liking score in appearance, with values ranging between 4.7 and 6.1. sample d displayed the lowest liking in appearance (i.e., 3.6), whereas samples b, e and f showed the highest liking scores with values of 6.1, 5.7 and 5.9, respectively. significant differences (p<0.05) in liking in aroma were observed within the selected onss and samples f and d obtained the lowest scores (i.e., 4.1 and 4.9), while b displayed the highest liking in aroma (i.e., 6.2). for liking of flavour, significant differences (p<0.05) across the samples were found. the liking of texture scores ranged between 5.1 and 6.2 and samples ordered, from lowest to highest as c < a < f < d < e < b. the overall acceptability reported low-medium scores, with values ranging between 4 and 5.7, in agreement with the literature for protein-based beverages (ye et al., 2011; thomas et al,. 2016; withers et al., 2014). a negative correlation between overall acceptability and cooked flavour (-0.879, p<0.05) or astringent after-taste (-0.902, p<0.05) was observed. the panellists identified samples, namely b and e, as the most preferred products, with 48% and 23% of the preferences, respectively (table 3). the high preference for samples b and e may be ascribed to the high sweet intensity, vanilla aroma and thickness values together with low cooked flavour and after-taste intensity recorded in the aforementioned samples. 4. conclusions in this study, colloidal, tribological and sensory properties were investigated in order to better understand the physical and sensory quality attributes of onss. samples characterized by high viscosity and small particle size possessed the highest physical stability. onss containing dairy and soy protein blends with lipid content higher that 5% had low coefficient of friction values in both boundary and mixed regimes, which are usually associated with astringent after-taste and creamy texture, respectively. lowmedium liking scores were displayed across all the samples and the highest preference was recorded for samples with high sweet intensity, vanilla aroma, thickness and low values in cooked flavour and after-taste. the detailed information on rheological, colloidal, physical stability, tribological and sensory properties reported in this study will support the development of new onss with high physical stability and enhanced sensory acceptability. acknowledgments this work was funded by the food institutional research measure (firm) administered by the irish department of agriculture, food and the marine under the project on “dehydration/rehydration dynamics for development of ‘smart” dairy ingredients (grant number 11/f/061). ital. j. food sci., vol. 32, 2020 967 references akhtar m., stenzel j., murray b.s. and dickinson e. 2005. factors affecting the perception of creaminess of 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fermentations a review. food res. int. 89:39-47. paper received march 6, 2020 accepted july 15, 2020 ijfs 1331_bozza ital. j. food sci., vol. 31, 2019 618 paper coagulase positive staphylococci enumeration and enterotoxins detection in milk and dairy products from central italy l. iannetti1, p. visciano2, c. marfoglia1, g. iannitto1, g. parisciani1, m. sericola1, d. petrone1, m.s. mangieri2, f. pomilio1 and m. schirone*2 1istituto zooprofilattico sperimentale dell’abruzzo e del molise “g. caporale”, via campo boario, 64100 teramo, italy 2faculty of bioscience and technology for food, agriculture and environment, university of teramo, via r. balzarini 1, 64100 teramo, italy *corresponding author: tel.: +390861266911 e-mail address: mschirone@unite.it abstract this study aims at enumerating coagulase positive staphylococci (cps) in 404 samples of milk and dairy products collected in own-checks or during the official controls from different dairy industries located in central italy. these microorganisms were enumerated using iso 6888-2:1999/amd. 1:2003 and only when they exceeded 105 cfu/g, the presence of any of the seven more common staphylococcal enterotoxins (sea, seb, sec1, sec2, sec3, sed and see) was also investigated. own-checks samples resulted always below the detection limit, whereas among those collected by the competent authorities in the framework of official controls, provola (100%) and mozzarella (22.9%) samples were positive to cps, with mean values of 1.8x102 and 2.8x105 cfu/g respectively. such values exceeded the maximum limits set by commission regulation (ec) no. 2073/2005, resulting in a request of hygiene improvements in the first case; in the second case, the presence of staphylococcal enterotoxins in 8 (2.7%) mozzarella samples out of 298 investigated cheese products was also observed, resulting in their withdrawn from the market. therefore, this study aims at highlighting that monitoring of cps incidence in dairy products and subsequent testing of cheeses for enterotoxins when appropriate represent an important tool for public health in order to avoid the occurrence of foodborne outbreaks. keywords: coagulase positive staphylococci, milk, dairy products, process-hygiene criteria, staphylococcal enterotoxins ital. j. food sci., vol. 31, 2019 619 1. introduction italian cheese production boasts a great variety of dairy products ranging from fresh to ripened cheeses such as mozzarella, pecorino and parmigiano reggiano. their quality is closely associated with parameters such as the territory of production, pedoclimatic characteristics, and anthropic components (schirone et al., 2012). abruzzo and molise regions located in central italy have no cheeses with protected designation of origin (pdo) or protected geographical indication (pgi), except for caciocavallo silano and mozzarella di bufala campana for molise region, as reported in the publicly-accessible updated register of pdo and pgi of regulation (eu) no. 1151/2012 (eu, 2012). in contrast, according to the italian decree no. 350 dated september 8, 1999 (mipaaf decree, 1999), 14 and 12 traditional cheeses are reported as traditional food products made in abruzzo and molise regions respectively. as par as such traditional cheeses are concerned, raw milk microbiota rather than microorganisms deriving from the dairy environment or equipment can affect the safety of the end product (sánchez-gamboa et al., 2018; schirone et al., 2018). staphylococci are widespread in nature, being found on the skin and mucous membranes of animals and humans, but also in the environment (ruiz et al., 2016). coagulase positive staphylococci (cps) are the most common causative agents of subclinical mastitis in dairy cows (rajic-savic et al., 2015). several studies (jorgersen et al., 2005; neder et al., 2011) reported that some strains isolated from bulk milk can produce enterotoxins and, as such, they are considered biological hazards for the dairy industry. among them, staphylococcus aureus is one of the most important disease-causing agents of foodborne intoxication in humans caused by the consumption of dairy products but, since it is also found in 30-80% of human population, the personnel working in a food industry can contaminate food with unhygienic food handling and manufacturing practices (soltan dallal et al., 2016). according to commission regulation (ec) no. 2073/2005 (ec, 2005) and further amendments, cps should be investigated in different categories of cheese, both ripened an unripened, made from raw or pasteurized milk, or after a stronger heat treatment, for process control purpose. the process-hygiene criteria established by the above quoted regulation differ on the basis of cheese categories and can be applied at the end of the manufacturing process or at the time when the number of staphylococci is expected to be the highest. the current legislation on microbiological criteria requires that only when such values exceed 105 cfu/g the cheese batch must be analyzed for staphylococcal enterotoxins, even if some authors reported that levels of s. aureus ranging from not detectable to 1.5x1010 cfu/g, with a median value of 3x107 cfu/g, could cause foodborne outbreaks (wallin-carlquist et al., 2010). their formation is influenced by some parameters, such as temperature, ph, water activity, redox potential and bacteria antagonism (schelin et al., 2011). the most common detected staphylococcal enterotoxins named as classical (sea, seb, sec, sed and see) can cause several symptoms at rapid onset (2-8 h), including hypersalivation, nausea, vomiting, abdominal cramping and diarrhoea, after the ingestion of contaminated food (martins et al., 2014; mehli et al., 2017). staphylococcal food poisoning usually resolves within 24-48 h with no specific treatment; but it can be more severe or even fatal when occurring in infants, elderly or immune-compromised people (rola et al., 2016). the aim of the present investigation was the enumeration of cps in milk and dairy products produced in dairy industries located in abruzzo and molise regions, central italy, in order to check the process-hygiene criteria established by regulation (ec) no. 2073/2005 and to detect staphylococcal enterotoxins only in not compliant samples. ital. j. food sci., vol. 31, 2019 620 2. material and methods a total of 404 samples of milk and dairy products were obtained from different dairy industries located in abruzzo and molise regions (central italy) in the framework of ownchecks (n=118) and official controls (n=286). the milk samples (n=91) were differentiated into raw milk (n=14), bulk milk (n=28), whole milk (n=21), low-fat milk (n=19) and skimmed pasteurized milk (n=9). among the dairy products, butter (n=9) and cream (n=6) were sampled. all the other remaining dairy products belonged to different categories of cheese (n=298) and could be grouped according to the process-hygiene criteria of commission regulation (ec) no. 2073/2005 − namely raw milk cheese (n=18), ripened cheese from pasteurized milk (n=36) and unripened cheese from pasteurized milk (n=244). among the latter, only caciotta samples were produced from raw cow’ or sheep’s milk; while most of cheese samples were represented by unripened cheeses, made from pasteurized cow’ or water buffaloes’ milk, usually “pasta filata” fresh cheeses (i.e. burrata, mozzarella, provola, scamorza and fiordilatte), semi-soft cheeses and ricotta. ripened cheeses were also from pasteurized cow’ or sheep’s milk and included semi-hard cheeses and grated cheese. all samples were analyzed at the istituto zooprofilattico sperimentale dell’abruzzo e del molise − that is, the laboratory in charge for official controls in those regions, which also tested samples taken in the framework of own-checks from some local dairy industries. samples collected over a 18-month period, including the whole year 2017 and the first 6 months of the year 2018, were involved in the present study. they were investigated for cps enumeration by the analytical reference method iso 6888-2:1999/amd. 1:2003. such a method is based on the use of rabbit plasma fibrinogen agar that allows the easy differentiation of coagulase positive and negative colonies with no need of confirmatory steps (viçosa et al., 2009). briefly speaking, duplicate poured plates of the quoted medium were prepared and inoculated using decimal dilutions of the test sample (or initial suspension, if solid) and then incubated at 37°c for 18-24 h or more, if needed. the calculation of cps per g of a sample was made counting typical colonies on petri dishes. according to prescriptions from commission regulation (ec) no. 2073/2005, samples with cps counts exceeding 105 cfu/g were also analyzed for the detection of staphylococcal enterotoxins using the vidas® staph enterotoxin ii (set2) kit (biomerieux, france) according to the fda bacteriological analytical manual (bam), 8th edition, chapter 13a (revision march 2011). such a method allows the detection of any of the seven more common staphylococcal enterotoxins (sea, seb, sec1, sec2, sec3, sed, see). in a few words, toxins were extracted from the test samples (25 g or ml) adding extraction buffer, blending and centrifuging according to the manufacturer’s instructions. the supernatant was pumped through moistened absorbent cotton placed in a syringe, using the plunger; then 500 μl of the filtrate was placed in the sample well of the vidas set2 reagent strip and the assay was finally performed with a mini vidas® instrument (biomerieux, france). 3. results and discussion most dairy products, including semi-hard and grated cheese, resulted below the detection limit of cps (<1 cfu/ml or <10 cfu/g). table 1 shows the results reported in the different cheese types, in relation to the presence of unsatisfactory results of cps enumeration according to the regulation (ec) no. 2073/2005. ital. j. food sci., vol. 31, 2019 621 table 1. number of cheese samples (n) and unsatisfactory cps enumeration for each cheese type according to the category of the regulation (ec) no. 2073/2005. limits are expressed in parentheses; the lowest is allowed only for 2 samples out of 5, the highest is never allowed. samples raw milk cheeses (104-105cfu/g) ripened cheeses* (102-103 cfu/g) unripened cheeses* (10-102 cfu/g) n unsatisfactory n unsatisfactory n unsatisfactory caciotta 18 0** ricotta 10 0 soft cheese 10 0 semi-soft cheese 5 0 semi-hard cheese 27 0 grated cheese 8 0 burrata 8 0 mozzarella 35 8 provola 5 5 scamorza 73 0 other “pasta filata” cheeses 99 0 *made from milk or whey that has undergone pasteurization or a stronger heat treatment; **cps detected below regulatory limits coagulase positive staphylococci were only found in samples from official controls, while own-checks were always below the detection limit. the results of cps in milk samples were below the detection limit (<1 cfu/ml) except for 5 samples of raw milk collected in the framework of official controls from a small dairy industry in molise region, that showed values ranging from 2.9x103 to 3.5x103 cfu/ml with a mean value of 3.5 log cfu/ml (fig. 1). the microbial contamination of raw milk may originate from animals, due to endogenous or udder infection, or from faeces and skin, and/or from the environment during milking or storage (verraes et al., 2014). only some categories of unripened cheese resulted positive to cps − namely caciotta, provola and mozzarella samples. in particular, the values of cps ranged from 4.8x103 to 5.4x103 cfu/g in 5 (33.3%) caciotta samples with a mean value of 3.7 log cfu/g. however, they did not exceed the limits (104-105 cfu/g) of process-hygiene criteria established by commission regulation (ec) no. 2073/2005 for cheeses made from raw milk. all (100%) provola samples resulted positive to cps, ranging from 1.2x102 to 2.2x102 cfu/g with a mean value of 2.3 log cfu/g (fig. 1). these values exceeded the limits (10102 cfu/g) for unripened cheeses made from pasteurized milk and therefore process hygiene improvements were required to the involved dairy industry. in addition, 8 (22.9%) samples of mozzarella out of 35 exceeded the regulatory limits up to a maximum value of 3.5x105 cfu/g, with a mean value of 5.4 log cfu/g (fig. 2). in such last case, as values over 105 cfu/g of cps were detected, staphylococcal enterotoxins were also investigated according to commission regulation (ec) no 2073/2005 and their presence was always confirmed in 25 g (100%). ital. j. food sci., vol. 31, 2019 622 figure 1. coagulase positive staphylococci counts (cfu/ml or g) in milk and cheese samples. figure 2. coagulase positive staphylococci counts (cfu/g) in mozzarella samples exceeding the regulatory limit. the results always below the detection limit for samples made in own-checks could be due to sampling made not at random, at least by some dairy industries – rather they aimed at selecting less manipulated products or those perceived to be safer, in order to reduce the possibility of encountering samples positive to cps. on the contrary, some samples taken in the framework of official controls showed high values of cps, stressing the importance of controls directly carried out by competent authorities. however, they usually did not exceed the process hygiene criteria, except for provola and mozzarella samples. these results could suggest the importance of good hygiene and manufacturing practices during cheese production in the first case, whereas the presence of staphylococcal enterotoxins in mozzarella samples with high values of cps (> 105 cfu/g) confirmed a potential serious risk for consumers. all positive samples belonged to the same batch produced in a small 0 1 2 3 4 milk caciotta provola l og c f u /m l o r g samples 4 5 6 1 2 3 4 5 6 7 8 l og c f u /g samples ital. j. food sci., vol. 31, 2019 623 dairy industry in molise region and were withdrawn or recalled in accordance with article 19 of regulation (ec) no. 178/2002 (ec, 2002). that batch was probably contaminated, even though it remains unknown if such contamination could be due to raw materials, lack of good hygiene practices or even to the handlers themselves, representing healthy carriers of cps. however, some studies reported that pasteurized milk cheese caused outbreaks of foodborne diseases also at a higher incidence rate than those linked to raw milk cheese consumption (yoon et al., 2016). moreover, failure of thermal processing, contamination of the products after pasteurization or abuse of storage temperature could allow the proliferation of cps in dairy products as well, after heat treatment. some bad hygiene habits such as coughing or sneezing and not washing hands when handling cheese production equipment could be a source of contamination of the end products (rosengren et al., 2010). other authors (delbes et al., 2006) reported lower cps values than our results in raw cow’s milk collected from france, ranging from undetectable to about 1.1x103 cfu/ml. as par as cheeses are concerned, rosengren et al. (2010) detected cps in 69% of raw milk cheeses and 6% of cheeses made from pasteurized milk, even if staphylococcal enterotoxins were never detected. according to the results of our investigation, mean values of cps (2.6x103 cfu/g) were detected in raw milk cheese produced in a south tyrolean malga and the presence of staphylococcal enterotoxins was also confirmed, causing symptoms referable to staphylococcal food poisoning (armani et al., 2016). according to the european union summary report on trends and sources of zoonoses in 2016 (efsa and ecdc, 2017), a monitoring study on milk samples from different animal species carried out in italy reported that 110 and 55 out of 165 samples were positive for s. aureus and staphylococcus spp. respectively. another italian survey on 6,482 milk samples described in the above quoted report showed that 451 were positive for s. aureus, 54 for staphylococcus intermedius and 628 for staphylococcus spp. johler et al. (2018) found 80% of artisan raw milk cheese samples positive to s. aureus from 25 dairies located in italy. some isolates exhibited at least one gene encoding some of staphylococcal enterotoxins. another italian study (cremonesi et al., 2007) on raw milk cheese samples (fresh, soft, semi-hard and hard cheeses) reported that no enterotoxins were detected in samples positive to high concentrations of s. aureus. 4. conclusions the results of the present study have highlighted the presence of cps in some raw milk and unripened cheese, as well as the detection of staphylococcal enterotoxins in eight samples of mozzarella. therefore, the monitoring of cps incidence in milk and dairy products represents an important tool for public health, especially when high values are detected. the ubiquity of such microorganisms and the lack of good manufacturing practices in milk and cheese production represent a risk factor to consumers, so that the improvement of hygiene procedures such as regular 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expression and production od staphylococcus aureus enterotoxin a in processed pork meat. int. j. food microbiol. 141:s69-s74. paper received july 17, 2018 accepted december 14, 2018 ijfs#1433_bozza ital. j. food sci., vol. 31, 2019 593 paper influence of fertilisation type on the quality of virgin rapeseed oil m. kachela, a. matwijczukb, a. niemczynowiczc, m. koszela and a. przywaraa adepartment of machinery exploitation and management of production processes, głęboka 28, 20-612 lublin, poland bdepartment of biophysics, university of life sciences in lublin, akademicka 13, 20-950 lublin, poland cdepartment of relativity physics, university of warmia and mazury in olsztyn, słoneczna 54, 20-710 olsztyn, poland *corresponding author: tel.: +48 815317131 email address: magdalena.kachel@up.lublin.pl abstract the aim of the study was to determine whether, and to what the type of fertilisation employed can affect the quality of the harvested spring rape and the oil extracted therefrom through virgin oil pressing. the article presents the results of research conducted with the use of atr-ftir absorption spectroscopy of selected cold pressed vegetable oils. the study included three types of rapeseed oil from spring plants grown with traditional npk mixture fertilisation and digestate. the oxidative stability of the pressed oil was also determined and soil parameters such as: content of absorbable nutrients and acidity in the experimental plots. keywords: art-ftir spectroscopy, cold pressed oils, fat, fatty acids, fertiliser, oxidative stability ital. j. food sci., vol. 31, 2019 594 1. introduction with the view of increasing rapeseed yields, numerous artificial fertilisers have been used in recent years to modify the soil content of micro-organisms and drastically alter the general environmental conditions of the soil. this situation has a negative impact on both the sustainable development of soil fertility and, above all, quality of the natural environment (ramirez et al., 2012). health and environmental concerns force societies to support a revival of traditional methods of food production that are consistent with the guidelines for sustainable agriculture (cetin and sevik, 2016). the trend led to the development of the concept of the so-called eco-efficiency, which entails the use of natural fertilisers produced through natural processing of agricultural waste in agricultural biogas plants (czekala et al., 2012; dennehy et al., 2016). as observed by möller and müller (2012), the use of digestate containing large amounts of macroand micronutrients normally found in the natural environment provides a viable alternative to mineral fertilisers, capable of improving soil fertility and quality of farm produce by providing plants with easily absorbable elements (such as nitrogen, phosphorus and potassium). depending on the quality and nutritional status, digestate application has been shown to be advantageous in terms of crop yield, plant nutrient uptake (terhoeven-urselmans et al., 2009; andruschkewitsch et al., 2013) soil health (vaneeckhaute et al., 2013), and enhancement observed in dehydrogenase activity (garcía-sanchez et al., 2015). the technological and nutritional value of rapeseeds is determined by their chemical composition, in particular the content of nutrients and anti-nutrients, which in turn is closely related to the qualities of the plant species itself and primarily defined by genetic factors. under specific conditions, the nutrient content can, however, vary significantly depending on the cultivar, soil type, fertilisation, weather conditions, and technological processing procedures employed (heating, steaming, autoclaving, etc.) (krasucki et al., 2001). low quality raw material – containing immature seeds, contaminated, damaged, excessively moist, affected by the processes of fat hydrolysis and oxidation – will produce low quality oil characterised by short shelf life. consequently, the most valuable oils are those extracted from the freshest, highest quality material in which the oxidative processes are still relatively unadvanced. the process of fat oxidation is the primary cause of diminished quality manifested in the loss of oil’s organoleptic and nutritional value (ziemlański and budzyńska-topolowska, 1991; poyato et al., 2014). the oxidation rate is conditioned by a number of factors, including the content of fatty acids, presence of proand antioxidants and, particularly in the case of cold pressed oils, the storage conditions (wroniak and łukasik, 2007). the growing interest in natural and safe food, including oils and fats, is a testament to its value as a rich source of healthy nutrients, whose consumption facilitates prevention of numerous, particularly digestive diseases and is beneficial to the overall health of organisms (harel et al., 2002). the seeds of both spring and winter rape are used in the production of cooking oil and provide a key raw material in the production of (increasingly popular and sought-after) biofuels for compression-ignition engines, particularly in europe (van duren et al., 2015, atabani et al., 2013) and certain countries in africa and asia, including china (wu and leung, 2011; shin et al., 2012). it can be safely assumed that europe is currently the leading global producer of rapeseed (usda, 2016). compared to other oils such as sunflower oil, corn oil, sesame oil or olive oil, rapeseed oil is characterised by a particularly rich chemical composition. the high content of fat and protein as well a wide range of fatty acids, including large quantities of ital. j. food sci., vol. 31, 2019 595 n-3 fatty acids such as α-linolenic acid (c18:3), palmitic acid (c16:0), palmitoleic acid (c16:1), stearic acid (c18:0), oleic acid (c18:1), linoleic acid (c18:2), linolenic acid, as well as its n-6/n-3 ratio, make it an attractive offering for various consumer groups. said acids are very beneficial from the perspective of human health. however, high content of αlinolenic acid, often exceeding 10%, increases the rapeseed oil’s susceptibility to autooxidation, thus negatively affecting its oxidative stability (shahidi, 2005; parry et al., 2005). the presence of lipids, carbohydrates, sterols, aliphatic alcohols, tocopherols and a combination of pigments ensures antioxidative activity and high quality, both in terms of nutritional and sensory value (damerau, 2015; tsaknis et al., 1997; szydłowskaczerniak et al., 2013). notably, as reported in a number of studies comparing cold pressing to other methods of oil extraction, due to the presence of anti-oxidants (carotenoids, tocopherols), as well as other phenolic compounds whose small quantities are introduced into the oil in the process of pressing the seeds, cold pressed oils are characterised by one of the highest values of oxidative stability (cichosz and czeczot, 2011). it is noteworthy that the final quality of both the seeds themselves and the oil extracted therefrom is dependent on a variety of diverse factors, including climate, type of plantation, methods of harvest, storage, preliminary processing and, above all, the process of cold pressing itself. the increased awareness of the value of oil consumption stimulated the development of better quality products and renewed interest in methods of extracting vegetable oil without the use of chemical reagents, or at least with minimum use thereof. due to the above, in recent years, we have observed a turn back to the methods of oil extraction involving cold pressing and virgin oil pressing (szydłowska-czerniak et al., 2013) under which, due to the low temperatures under which the process takes place, the extent of degradation of bioactive compounds can be substantially reduced (ramadan, 2013). the simplicity of the process of extracting the oils selected for the present study simultaneously allows them to preserve much of their original colour, high nutritional value, high bioactive compounds content, as well as the characteristic taste and smell. the aim of the study was to: 1. determine and compare selected growing conditions based on soil analysis after fertilisation with a mineral npk mixture and digestate. 2. analyse the quality of seeds in terms of fat and protein content in spring rape seeds after using fertilisers containing different amounts of nitrogen. 3. analyse selected quality parameters of seeds intended for oil extraction and the quality of the thus obtained virgin rapeseed oil. the scope of the study included: determination of the content of moisture, fats, fatty acids, and protein in the respective seeds, an analysis of the oil extracted therefrom and determination of its oxidative stability. a comparative analysis of atr-ftir spectra was employed for the purposes of identifying/highlighting differences in the chemical makeup of the respective oils. 2. materials and methods the research material comprised three spring rape cultivars: ‘markus’ – ‘m’, ‘bios’ – ‘b’ and ‘feliks’ – ‘f’ obtained from hodowla roślin strzelce sp. z o.o. the aforementioned rapeseed cultivars were grown in two combinations on experimental plots of 27 m2 located in the lublin voivodship (51o16’19.8”n 22o24’34.0”e), poland (fig. 1). in the first version of the experiment, natural fertiliser (digestate) obtained from the agricultural biogas farm wikana bioenergia sp. z o.o. in piaski, lublin voivodship, was used, while in the second ital. j. food sci., vol. 31, 2019 596 version, the traditional multi-compound fertilizer yara npk 5-14-28 was applied (ammonium nitrogen 5 %, p14% k 28 %, so3 – 12.5%, cao 3%). the digestate in the amount of 97 l was distributed in a plot of 27 m2 (36,000 l/ha). the ph of the digestate used in the cultivation of spring rape was 8.73. the cultivars grown in the control plot – c did not receive any of the aforementioned fertilisers. the products included in the study were obtained from a two-year experimental plot cultivation (2015 and 2016). after harvest, the seeds were cleaned and stored in laboratory conditions for a period of one month in 20oc ambient temperature and 70% ambient humidity to equalise the moisture content. subsequently, the seeds were cold pressed to extract oil. the oil was then refrigerated for a period of one week in 4oc, in dark bottles, to separate the oil from impurities. calculations were conducted for triplicate mean values. all the results obtained in the course of the two-year experiment were averaged and tabularised (see tables below). figure 1. distribution of experimental spring rape plots. weather information relating to the period from march to september 2015 and 2016 was obtained from the institute of meteorology and water management national research institute imgw-pib as recorded at the weather station lublin-radawiec. the measurements of soil microelement content prior to the application of the selected fertilisers and after the harvest were conducted at the regional chemical-agricultural station in lublin. soil samples were tested for the available nutrient content, in particular phosphorus and potassium, in accordance with the applicable norms with respect to those nutrients, i.e. pn-r-04024:1997, as well as magnesium in mg per 100g od soil, in accordance with pn-iso 10390:1997. soil acidity, ph (kcl) and potential liming requirements were also considered. the digestate content of major nutrients and heavy metals was determined. the laboratory tests were conducted at the regional chemical-agricultural station in lublin in accordance with kq/pb-17-76-77 rev. 04 of 02.07.12. the biogas plant feedstock used in the production of biogas and digestate consisted of: corn silage, whey and plant waste. rape seeds were analysed in terms of fat content. the fat content was determined using soxhlet method in accordance with pnen 1163:1999, i.e. by multiple, continuous extraction from pulverised and pre-dried product using an organic extraction solvent, followed by removal of the solvent and determination of the fat content by weight. the ital. j. food sci., vol. 31, 2019 597 tests were conducted at the central agro-energy laboratory of the university of life sciences in lublin. rapeseeds were tested for fatty acids content in accordance with pm-en iso 5509:2001. “vegetable and animal fats and oils” – the analysis of fatty acid methyl esters was performed by way of gas chromatography at the central agro-energy laboratory of the university of life sciences in lublin. rape seeds were analysed in terms of protein content. the protein content was determined using the kjeldahl method, in accordance with pn – a – 04018/a z 3:2002, i.e. by way of sulphation of organic nitrogen compounds using concentrated sulphuric acid in the presence of a catalyst, and subsequent alkalization of the solution, distillation and titration with sulphuric acid of ammonia bound by boronic acid. the oil was extracted using a continuous action screw press with exchangeable nozzles, 8 mm in diameter with a set of microscopic sieves, manufactured by farmet duo. before activation, the press was preheated to 60oc ±10oc. once the press was active and the pressing temperature equalised, the pressing process itself was initiated. stabilisation was achieved after pressing oil from approximately 1kg of seeds, with the temperature stabilised at approximately 70oc. the pressing temperature was measured using an amadigit thermometer. after the extraction, the oil was stored in dark-glass bottles at 5oc to achieve natural decantation over a period of 6 days. afterwards, the oil was ready for laboratory analyses. the oxidative stability was measured using a rancimat 670 apparatus (metrohm ag, herisau, switzerland). oil samples (2.5 g) were weighed into reaction vessels and heated to 1200c under a dry air flow of 20 l h-1. the volatile compounds released during oxidation were collected into a cell containing distilled water, and the increasing water conductivity was continually measured. the time taken to reach the conductivity inflection point was recorded as the induction period (it) expressed in hours. all determinations were carried out in triplicate. the normal time was calculated using stabnet software, which controlled the rancimat apparatus. infrared absorption spectra were recorded with a vertex 70 ftir (bruker) spectrometer. the attenuated total reflection (atr) configuration was used with 20 internal reflections of the znse crystal plate (45° cut). typically, 16 scans were collected, fourier-transformed, and averaged for each measurement. absorption spectra at a resolution of one data point per 1 cm-1 were obtained in the region between 4000 and 600 cm-1. the instrument was continuously purged with n2 for 40 min. before and during measurements. the znse crystal plate was cleaned with ultra-pure organic solvents from sigma-aldrich. the spectral analysis was performed with grams/ai software from thermogalactic industries (usa). all experiments were carried out at 25°c. in the recent years, chemometric methods have played an important role in studies on food quality as well as the identification and quantification of major constituents of foods. the use of chemometric methods combined with spectroscopic techniques has been analysed in previous research. the literature is extensive and details the theoretical aspects of applying such methods as e.g. principal component analysis (pca), hierarchical cluster analysis (hca) or partial last squares regression (pls). examples are the analysis pork fat or lard as a whole of matter extracted from pork (rohman et al., 2011), the determination of fat in milk samples (inon et al., 2004), analysis of extra virgin olive oil adulterated with palm oil (rohman and che mana, 2010). principal component analysis (pca) is the most popular mathematical method of reducing data dimensionality with a minimum loss of information. pca allows us to visualize and interpret data. another multivariate method with which we can hierarchize the data is ital. j. food sci., vol. 31, 2019 598 hierarchical cluster analysis (hca), which searches for objects, which are close together in the variable space. hca and pca methods combined with spectroscopic techniques have been successfully used for the purposes of determining food quality as well as identifying and quantifying major constituents of foods. the objective of the present study was to overview the similarities and differences observed between oil samples extracted through cold pressing and also involved the content of fatty acid composition in rapeseeds. the results obtained for the selected oil products were analysed statistically by way of mean value and ± standard deviation determination, as wells as post–hoc tukey's test, with the level of significance of p < 0.05, using statistica 10 software. furthermore, correlations between the respective variables were evaluated using pca and hca. pca transforms the original, measured variables into new uncorrelated variables. hca was applied to the data in order to identify similarities between different oils samples. hca calculates the distances (or correlations) between all oils samples based on euclidean distance. 3. results and discussion table 1 presents the relevant weather conditions including atmospheric pressure, air temperature and rainfall in the years of cultivation, 2015 and 2016, for the period from march (sowing) to august when the crop was collected by way of two-stage harvest. as shown in the relevant data, 2015 was characterised by lower rainfall (10.08 mm h2o) relative to 2016, when the total rainfall in the analysed period was (12.59 mm h2o). the mean daily air temperature in 2015 was higher than in 2016, with the exception of april and may, respectively 7.75 and 12.37°c. table 1. weather conditions in 2015 and 2016. month year 2015 year 2016 pressure (hpa) temperature ( oc) rainfall (mm h2o) pressure (hpa) temperature (oc) rainfall (mm h2o) 3 991.41 4.70 1.31 985.26 3.46 2.04 4 986.86 7.75 1.20 983.69 8.91 1.20 5 986.75 12.37 2.20 985.82 14.44 1.09 6 989.60 26.63 0.63 986.30 18.34 1.78 7 986.81 21.60 1.42 987.12 18.91 4.49 8 990.38 25.15 0.30 990.73 18.07 1.52 9 989.19 14.63 3.02 990.73 15.15 0.47 an important gap given that a significant popularisation of inorganic n fertilizers in the coming decades is expected to occur in developing countries as a consequence of population growth and increasing food demands (farnworth et al., 2017). oil and nitrogen (n) content in soil and rapeseed is an important quality indicator affecting oil yield and protein content of the meal (grant et al., 2011). nitrogen content has a significant impact on protein synthesis and must be supplied in adequate amounts to ital. j. food sci., vol. 31, 2019 599 ensure optimum crop yield. too much n in the soil can lead to a nutrient imbalance that can restrict protein synthesis and reduce rapeseed growth and seed yield (grant et al., 2012; lemke et al., 2009). according to rathke et al. (2005), the yield of oilseed rape involves balancing the synthesis of oil and crude protein in the seeds, as well as the energy and carbon dioxide (co2) budget of the photosynthetic pool. table 2 presents the results of the analysis of major nutrient and heavy metal content in the digestate used for the spring rape cultivation. the same confirm the presence of numerous major nutrients. however, one should not that with respect to primary major nutrients such as: nitrogen, phosphorus and potassium, the levels observed in the applied digestate (respectively: 0.119; 0.12; 5.37 g/l) were significantly lower than those present in industrial fertilisers. odlare et al. (2008) reported in his research high content of mineral nutrients (nitrogen, phosphorus, potassium) in fermentation wastewater, while rehl (2011) observed that in terms of response time digestate was in fact comparable to mineral fertilizers due to that fact that the n, p, and k nutrients were more readily accessible and available for the plants. based on the subsequent analyses of the obtained results it can be reported that the tested digestate samples contained virtually no heavy metals, while the overall high major nutrient content supported the usability of digestate as a potential alternative fertiliser. furthermore, comparetti (2013) and kouřimská et al. (2012) observed that digestate contains organic matter that has a significant positive influence on the physicochemical quality of the fertilised soil. table 2. content of major nutrients and selected heavy metals in digestate used for cultivation of spring rape. element unit content nitrogen (g/l) 0.119 phosphorus (g/l) 0.12 potassium (g/l) 5.37 calcium (g/l) 0.28 magnesium (g/l) 0.07 cadmium (mg/l) <0.43 lead (mg/l) <0.43 nickel (mg/l) <0.43 chromium (mg/l) <0.43 copper (mg/l) 0.43 zinc (mg/l) 2.00 manganese (mg/l) 2.26 iron (mg/l) 70.82 table 3 presents the averaged results obtained in the course of the two-year study in terms of major nutrient content in the soil prior to an after the application of selected fertilisers for the purposes of spring rape cultivation. based on the obtained, data it was observed that the ph of the soil in both cases remained relatively stable and did not change significantly after the sowing. with respect to nutrient content in plants relative to the levels or phosphorus, potassium and magnesium, the values varied significantly in the samples selected for the study. in all cases, the content of said nutrients was significantly ital. j. food sci., vol. 31, 2019 600 higher in the fertilised plots when compared to the control plots, both prior to sowing and after the harvest. the analysis of soil content after the harvest revealed similar values for both experimental variants, which in both cases remained higher than those observed for the control, and were e.g.: 64.5 mg /100g of phosphorus in the plot fertilised with digestate compared to 25.1 mg /100g in the control plot. in the case of potassium, its concentration in the control plot was 20.04 mg/100g, while in the digestate plot it was 60.05 mg/100g. data related to magnesium content analyses returned very similar results, i.e. 9.6 mg/100g in control plots and 21.8 mg/100g in lots fertilised with digestate. given the above, the primary agricultural use of digestate, given its physical and chemical properties, is biofertilisation. such usability of fermentation wastewater was also suggested, based on obtained research results, by kouřimská et al., (2012), eickenscheidt et al., (2014), vázquez-rowe et al., (2015). kouřimská et al., (2012) reported that digestate used in fertilisation improved soil fertility, as well as quality of the crop and the plants’ resistance to biotic and abiotic factors. the statistical analysis using the post-hoc tukey test at the significance level of p<0.05 also revealed statistically significant differences in the content of the respective nutrients between the control plots and the experimental fertilised plots. statistically significant differences were also observed between the content of the analysed nutrients in the soil prior to sowing and after the harvest, with the exception of control plots relate to the use of traditional npk fertilisation. table 3. content of available nutrients and soil acidity. experimental variant plot type acidity liming needed content of available nutrients (mg 100/g) ph in kcl reaction phosphorus p2o3 potassium k2o magnesium mg prior to sowing digestate control 6.97 neutral no 23.8a 16.3a 10.3a exp. plot 7.22 alkaline no 42.17b 53.17b 13.75b npk fertilizer control 7.06 alkaline no 21.1c 15.7a 9.5a exp. plot 5.51 acidic yes 23.57a 20.55c 14.05b after harvest digestate control 6,99 neutral no 25.1d 20.04c 9.6a exp. plot 7.52 alkaline no 64.5e 60.5e 21.8c npk fertilizer control 7.06 alkaline no 21.6c 16.7a 9.55a exp. plot 5.37 acidic yes 26.5d 23.3d 17.2b a, b, c, d, e – statistically significant cultivar differences relative to the control; mean values marked with the same letter are not statistically significantly different (p > 0.05). the yield obtained from spring rape plants in 2015/2016 was below expectations and the unfavourable weather conditions observed in the summer in both years resulted in yield discrepancies. it is assumed that the correct moisture content of rapeseed suitable for further storage ought to be between 5 and 9%, with 10% being within the admissible range in some countries, e.g. canada (gawrysiak-witulska et al., 2012). table 4 presents ital. j. food sci., vol. 31, 2019 601 the values of initial moisture during harvest, moisture reported during analyses, as well as fat and protein content of the rapeseeds. the initial moisture content during harvest was between 8.3 and 10% dry mass, respectively for all the samples. during storage, the value dropped on average by 1% relative to the initial moisture content, which rendered it acceptable by oil and fat industry standards, i.e. the harvested seeds would be accepted for further potential storage. the tests conducted with the aim of determining the fat content in seeds revealed that this particular characteristic was primarily dependent on genetic conditions. seeds of spring rape are characterised by slightly lower fat and protein content (respectively 48.6% and 24%) compared to winter rapeseed (warmiński et al., 2001). in our study, the protein concentration wasn’t strongly affected by the application of n, regardless of the fertilizer used. since n is a major structural component of protein, increasing n supply frequently leads to an increase in protein concentration (malhi and gill, 2007). in both experimental cases, the fat content turned out to be fairly similar, varying between 40.23 and 42.57% for digestate cultivations, with the lowest value recorded for ‘feliks’ control, and between 41.33 and 42.53% for ‘feliks’ control cultivar on traditional npk fertiliser. the protein content is undoubtedly an important parameter influencing the usefulness of various oils, which is why its measurement is necessary when determining the potential usability of the fat-free leftover material found in rapeseeds as e.g. raw material in the production of dietary protein supplements (gawrysiak-witulska et al., 2012). the analysis of protein content revealed that the same was slightly higher in seeds cultivated using natural fertiliser (digestate) and was 23.30% (for ‘bios’ cultivar) and between 23.23% and 22.47% (for ‘feliks’ cultivar). we concluded that the application of digestate had a subtle but noticeable influence on yield and quality compared to mineral fertilization. the obtained results in terms of fat and protein content were consistent with values reported by other authors (murawa and warmiński, 2005). in the research of rathke et al. (2005) analyzing the effect of applying cattle manure, it was observed that seed yield of winter oilseed rape tended to increase with increasing n fertilization rate, while the oil content in the seeds declined. cheema et al., (2001) and mason and brennan, (1998) reported the negative influence of n fertilization on the oil (fat) content in seeds. the statistical post-hoc tukey test conducted at the significance level of p<0.05 revealed no statistically significant differences in terms of the protein content between the respective rapeseed cultivation variants. at the same time, such significant differences were observed for the fat content. mensink and katan (1990) say that high fatty acids content in oils is desirable because of their health benefits. the fatty acid content varies significantly between vegetable oils obtained from different sources, particularly depending on the actual plant cultivar used and plant maturity. other factors with significant influence include: the region in which the plants are grown and specific climatic conditions (murkovic et al., 1996). given the above, table 4 presents the primary fatty acids content in spring rapeseeds during the first stage of storage treated as the point of reference for the subsequent extracted oil measurements. the fatty acid content in oils was determined for specific rape cultivars (as reflected by the obtained measurement results) and returned the approximate percentages of 60% oleic acid, 21% linoleic acid, and 10% linolenic acid (krygier, 1997). comparable results were obtained in the cultivation and harvest of winter rape, where the highest reported content of mufas and pufas was respectively 68.33% and 34.55% (tys et al., 2006). in terms of the analysed seeds, the fatty acid content was within the range approved by the codex alimentarius (2011). ital. j. food sci., vol. 31, 2019 602 table 4. moisture, fat and protein content in seeds. experiment cultivar initial moisture (%) moisture content (%) fat content (% dry mass) protein content (% dry mass) control ‘bios’ 10.00±0.06aa 9.93±0.06aa 42.57±0.15aa 23.35±0.35aa ‘feliks’ 9.50±0.06aa 8.80±0.10bb 40.23±0.85cb 22.47±0.21aa ‘markus’ 9.90±0.08aa 8.93±0.21ba 40.73±0.64cb 23.37±0.15ab digestate ‘bios’ 9.20±0.05baa 8,17±0.06aaa 41.37±0.12baa 23.30±0.10aaa ‘feliks’ 8.30±0.06baa 7.27±0.06aaa 41.67±0.38baa 23.23±0.06aaa ‘markus’ 9.03±0.07baa 7.97±0.12aaa 41.97±0.06baa 22.83±0.21aaa npk fertilizer ‘bios’ 8.75±0.05baa 7.97±0.06aaa 41.80±0.10aaa 22.73±0.06aaa ‘feliks’ 8.60±0.05baa 7.57±0.06aaa 42.00±0.44baa 23.57±0.31aaa ‘markus’ 8.95±0.09baa 7.67±0.06aaa 41.33±0.06baa 22.37±0.40aaa a, b, c statistically significant cultivar differences relative to the control; a, b statistically significant differences between cultivars under the same field experiment; a, b, statistically significant differences between the respective fertilizer type experiments relative to the same parameters; mean values marked with the same letter are not statistically significantly different (p > 0.05). based on the obtained results (table 5) it can be concluded that in the case of seeds grown with the use of digestate, the content of oleic acid (c18:1) and linoleic acid (α-c18:3) was lower in the control than in the “ecologically” grown samples of ‘bios’, ‘markus’ and ‘feliks’ cultivars. an analogous situation was also observed in seeds cultivated using traditional npk fertilisation. the statistical tuckey’s post-hoc analysis revealed statistically significant differences in terms of acid content between the cultivars and their respective control, in terms of c18:1n9c for ‘bios’ and ‘feliks’, in terms of c18:2n6c (only for ‘feliks’); and in terms of c18:3n3 again for ‘bios’ and ‘feliks’, where the experimental plots were cultivated on digestate. statistical differences were observed in terms of the c18:1n9c content between particular seed cultivars from the cultivation fertilized traditionally with npk, specifically between ‘bios’ seeds on the one hand, and ‘feliks’ and ‘markus’ seeds on the other. differences were also observed in terms of c18:3n3 acid between seed cultivars grown on digestate, specifically between bios’ seeds on the one hand, and ‘feliks’ and ‘markus’ seeds on the other. in terms of sfas in the ‘bios’ cultivar, the highest values were observed in the control, where it reached 7.85%, compared to 7.53% for traditionally fertilised crops. in terms of the other two cultivars, the highest saturated fatty acids content was observed in the digestate cultivations 7.99% followed by the control 7.76% (‘feliks’) and 7.76% for traditional fertilisation (‘markus’). the lowest content of those fatty acids was observed in ‘markus’ seeds grown on digestate: 7.55%. a very similar situation could be observed with respect to monounsaturated fatty acids. after analysing the results obtained for omega 3 acids, it can be concluded that the same fluctuated between 6.22 and 7.64%, with the lowest values observed for feliks seeds both in the control (6.90%) and the experimental cultivations, respectively 6.22% for digestate and 6.31% for npk. the highest content of the same was observed for ‘bios’ seeds cultivated using the biofertilizer (7.64%). the content of omega 6 acids in the seeds of analysed cultivars varied between 17.90 and 20.98%. the highest values were observed for the control ‘feliks’ cultivar (20.48%) and the lowest for ‘markus’ at 17.90% (on traditional fertiliser). ital. j. food sci., vol. 31, 2019 603 the statistical analysis using tukey’s post-hoc test at the level of significance of p<0.05 revealed clear statistical difference in the content of mufas between the control plots and plots fertilised with digestate for the ‘bios’ cultivar, as well as between the control plots and experimental plots fertilised with either digestate of npk mixture for the ‘feliks’ cultivar. the statistical analysis also revealed significant differences in terms of the content of mufa acids in the analysed seeds, specifically for ‘bios’ vs. ‘feliks’ and ‘markus’ grown on digestate, and between ‘bios’ and ‘markus’ in the npk cultivation. in terms of pufas, statistical differences were also observed for the ‘bios’ and ‘feliks’ cultivars relative to seeds obtained from their respective control plots. the statistical analysis revealed the existence of certain differences between the respective analysed cultivar types. significant differences in the content of pufas were observed in the control cultivation between ‘bios’ and ‘markus’ seeds on the one hand and ‘feliks’ seeds on the other. moreover, discrepancies were observed between the three cultivars grown on digestate as well as between ‘bios’ on the one hand and ‘markus’ and ‘feliks’ on the other in the npk cultivation. statistical differences were also observed in the content of omega 6 acids for the ‘feliks’ cultivar when comparing the control plots to the experimental plots, regardless of the fertilisation method. furthermore, significant differences in terms of their levels were noted in the control between ‘bios’ and ‘markus’ seeds on the one hand and ‘feliks’ seeds on the other. simultaneously, the statistical analysis revealed no statistical differences in terms of the content of omega 3 acids between the experimental cultivations and the control. differences of borderline statistical significance were observed between particular cultivars fertilized with digestate, specifically between ‘bios’ seeds on the one hand and ‘feliks’ and ‘markus’ on the other. according to jeleń et al. (2000) major components of vegetable oils are fatty acids, both saturated and unsaturated, mainly bound to glycerol as triacylglycerols. the allyl groups in unsaturated fatty acids are highly susceptible to free-radical reactions. de leonardis and macciola (2012) reported that the influence of fatty acid composition on oil stability has been varied between respective cases. in general, oils that are more unsaturated oxidise more readily than their less unsaturated counterparts. in the presence of oxygen, unsaturated fatty acids undergo decomposition even at low temperatures. the process of lipid oxidation results in deterioration of food quality, which can manifest itself through unpleasant odour leading consumers to reject the product (shahidi, 2005). it is the decomposition of hydroperoxides that constitute the main non-volatile compounds directly involved in the decomposition of volatile compounds, such as alkenes, aldehydes, alcohols, esters, acids and carbohydrates, that deteriorates the taste of food and is responsible for the rancid smell of soap. moreover, the oxidative degradation of lipids can damage biological membranes and protein, and as such can pose a direct threat to human life (malheiro et al., 2013). ital. j. food sci., vol. 31, 2019 604 table 5. fatty acid content of the rapeseeds. a, b statistically significant cultivar differences relative to the control; a, b statistically significant differences between cultivars under the same field experiment; a, b, statistically significant differences between the respective fertilizer type experiments relative to the same parameters; mean values marked with the same letter are not statistically significantly different (p > 0.05). specification (%) rapeseeds control ,b’ ‘b’digestate ‘b’ – npk fertilizer control ‘f’ ,f’ digestate ‘f’ – npk fertilizer control ‘m’ ‘m’ digestate ‘m’ – npk fertilizer c14:0 0.07±0.02 aaa 0.08±0.02aaa 0.07±0.02aaa 0.09±0.02aaa 0.08±0.02aaa 0.09±0.02aaa 0.09±0.02aaa 0.08±0.02aaa 0.07±0.02aaa c16:0 4.47±0.71aaa 4.24±0.68aaa 4.26±0.68aaa 4.74±0.76aaa 4.66±0.74aaa 4.52±0.72aaa 4.39±0.70aaa 4.46±0.71aaa 4.39±0.70aaa c16:1 0.27±0.01aaa 0.25aaa 0.28aaa 0.31±0.01aaa 0.30±0.01aaa 0.28aaa 0.27±0.01aaa 0.27aaa 0.26aaa c17:0 0.05±0.01aaa 0.05±0.01aaa 0.05±0.01aaa c17:1 0.07±0,01aaa 0.10±0.01aaa 0.08aaa 0.08±0.01aaa 0.08±0.01aaa 0.08aaa 0.09±0.01aaa 0.07aaa 0.07±0.01aaa c18:0 2.05±0.21aaa 1.99±0.21aaa 2.01±0.21aaa 1.71±0.18aaa 2.04±0.21aaa 2.08±0.22aaa 1.91±0.20aaa 1.87±0.19aaa 2.11±0.22aaa c18:1n9c +c18:1n9t 65.01±0.60aaa 63.81±0.48bba 64.24±0.52aaa 62.41±0.33cb b 65.76±0.67aca 65.56±0.65aca 64.60±0.56aaa 64.69±0.57aba 65.24±0.62aca c18:2n6c+c18:2n6t 18.19±0.57aaa 18.74±0.74aaa 18.52±0.67aaa 20.48±0.27ab b 17.99±0.51aaa 18.04±0.53aaa 18.49±0.66aaa 18.78±0.75aaa 17.90±0.48aaa c18:3n6 (gamma) c18:3n3 (alpha) 6.72±0.19aaa 7.64±0.22aaa 7.14±0.20aaa 6.90±0.20aaa 6.22±0.18aba 6.31±0.18aaa 7.17±0.20aaa 6.38±0.18aba 6.50±0.18aaa c20:0 0.69±0.06aaa 0.68±0.06aaa 0.67±0.06aaa 0.63±0.06aaa 0.69±0.06aaa 0.72±0.06aaa 0.64±0.06aaa 0.65±0.06aaa 0.72±0.06aaa c20:5 c20:1 1.50±0.23aaa 1.54±0.23aaa 1.65±0.25aaa 1.66±0.25aaa 1.38±0.21aaa 1.44±0.22aaa 1.45±0.22aaa 1.65±0.25aaa 1.77±0.27aaa c22:0 0.35±0.03aaa 0.34±0.03aaa 0.35±0.03aaa 0.37±0.03aaa 0.36±0.03aaa 0.37±0.03aaa 0.33±0.03aaa 0.35±0.03aaa 0.33±0.03aaa c22:2 c24:1 0.17±0.03aaa 0.14aaa 0.15aaa 0.19±0.01aaa 0.14aaa 0.14aaa 0.14±0.03aaa 0.16±0.03aaa 0.10aaa sfa 7.85aaa 7.49aaa 7.52aaa 7.76aaa 7.99aaa 7.95aaa 7.56aaa 7.55aaa 7.76aaa mufa 67.13aaa 65.98baa 66.68aaa 64.71bbb 67.66aba 67.49aaa 66.67aaa 67.15aba 67.74aba pufa 25.10aaa 26.60baa 26.03baa 27.53abb 24.27bba 24.41bba 25.87aaa 25.53aca 24.70aba omega 3 6.72aaa 7.64aaa 7.14aaa 6.90aaa 6.22aba 6.31aba 7.17aaa 6.38aba 6.50aaa omega 6 18.19aaa 18.74aaa 18.52aaa 20.48bbb 17.99aaa 18.04aaa 18.49aaa 18.78aaa 17.90aaa ital. j. food sci., vol. 31, 2019 605 the oxidative stability of the analysed oil samples was determined using the rancimat method. said stability is affected by a number of factors, including above all: the content of fatty acids, antioxidants and a whole range of other minor compounds. table 6 presents the results of analyses performed in relation to the oxidative stability of the studied oils. in the case of oils extracted from plants cultivated on digestate, the shortest induction time was observed for the ‘bios’ cultivar and was 5.21 h, while in the case of the respective control the same was three hours longer (8.31 h). the longest period of oxidative stability was recorded for the ‘feliks’ oil, specifically 8.17 h, the same result as that observed in the respective control – 8.17 h. the results of measurements conducted for oil extracted from seeds cultivated using npk and digestate fertilisation revealed relatively high oxidative stability compared to that reported by other authors. in a study by cichosz and czeczot (2011), the induction time of oxidative processes (rancimat test) in pressed and refined rapeseed oil was respectively: 4.5 and 4.7 h. results obtained by wroniak and łukasik (2007) with respect to the oxidative stability of cold-pressed rapeseed oils with various antioxidative additives varied between 3.76 and 4.38 h. in the case of oils obtained from traditionally fertilised cultivations, the longest induction time was observed for control ‘markus’ oil – 9.30 h, and the shortest for ‘bios’ oil – 8.41. in terms of oxidative stability of oils analysed relative to normal time, a discrepancy was observed for ‘bios’ oil obtained from seeds grown on digestate, npk mixture and the respective control, where it was 24.4 h (control), 20.96 h (digestate) and 22.02 h (npk). a similar situation was recorded for ‘markus’ oil (tale 6). the conducted statistical analysis using tukey’s post-hoc test for the significance level of p<0.05 also revealed slight statistical induction time differences between ‘bios’ grown on digestate and the respective control plants. in the case of the ‘markus’ cultivar, slight statistical differences were observed between the control and oil from the npk plots. the results of the statistical analysis also indicated the presence of significant differences between the respective cultivars from particular field experiments. seeds grown on digestate showed differences in terms of the time of oil induction, specifically between ‘bios’ seeds on the one hand and ‘markus’ and ‘feliks’ seeds on the other. significant differences were also observed between the fertilizer types for the respective rape cultivars. specifically, for ‘bios’ seeds in the case of the control and ‘bios’ and ‘markus’ seeds grown on digestate and npk. in the discussed case it should be noted that the seeds were subjected to no heating processes prior to extraction. the obtained oxidative stability values were considerably higher than those reported in other studies (4.35 or 4.39h) (kondratowiczpietruszka, 2014; ciemniewska-żytkiewicz et al., 2014). fig. 2 presents the atr-ftir spectra for selected oil samples. for easier interpretation of the results, table 7 provides a breakdown of all characteristic bands (maximums) for selected samples together with the corresponding vibration of the respective functional groups. samples were placed on a znse crystal under n2 atmosphere (as described above in materials and methods). spectra in the infrared region (atr-ftir) have well resolved bands that can be assigned to functional groups of particular components of food and biodiesel. edible fats and oils as well as some biodiesel substances consist basically of triglycerides groups with different substitution patterns, which are mainly differentiated by the degree and form of unsaturation of the acyl groups and their length (guillen and cobo, 1997; hong et al., 2005; scholze and meier, 2005; rohman and che man, 2010; van de voort, 1992). fig. 2 shows the infrared spectrum of selected oil samples chosen for the spectroscopic research. ital. j. food sci., vol. 31, 2019 606 table 6. analysis of the induction time for oils. parameter experiment cultivar measurements induction time control ‘bios’ 8.31±0.02aaa normal time 24.40±0.53aaa oxidative stability (h) induction time ‘markus’ 8.58±0.55aaa normal time 21.39±0.46aba induction time ‘feliks’ 8.17±0.04aaa normal time 21.11±0.93aba induction time digestate ‘bios’ 5.21±0.13bab normal time 20.96±0.02bab oxidative stability (h) induction time ‘markus’ 7.71±0.21aba normal time 21.42±0.11aaa induction time ‘feliks’ 8.17±0.05aba normal time 21.66±0.38 aaa induction time npk fertilizer ‘bios’ 8.41±0.07aaa normal time 22.02±0.23bac oxidative stability (h) induction time ‘markus’ 9.30±0.08cab normal time 23.89±0.65aba induction time ‘feliks’ 8.51±0.18aaa normal time 21.77±0.77aba a, b, c statistically significant cultivar differences relative to the control; a, b statistically significant differences between cultivars under the same field experiment; a, b, c, statistically significant differences between the respective fertilizer type experiments relative to the same parameters; mean values marked with the same letter are not statistically significantly different (p > 0.05). figure 2. atr-ftir spectra for selected rapeseed oil samples for the following cultivars: ‘bios’, ‘feliks’ and ‘markus’, respectively, ‘bios’ npk– (7) solid black line, ‘bios’ digestate – (6) solid grey line, ‘feliks’ npk – (5) solid blue line, feliks digestate – (4) solid light-blue line and ‘markus’ npk – (3) solid red line, ‘markus’ digestate – (2) solid brown line. the spectra are presented in the spectral range of 600 – 3800/cm. ital. j. food sci., vol. 31, 2019 607 although some authors have suggested specific assignments of spectral bands in oils and fats (guillen and cobo, 1997; li et al., 2013; qian et al., 2007), there are some, which are notoriously difficult to decidedly assign to a specific group. table 7 shows the frequencies of the characteristic bands or shoulders in the spectra of 7 oil samples (guillen and cobo, 1997), as well as their assignment to respective functional groups (guillen and cobo, 1997; yang et al., 2005; hong et al., 2005; scholze and meier, 2005; rohman and che man, 2010), their vibration mode and intensity in spectra typical for the ir region. it should be noted that the assignment of bands corresponding to the stretching vibration in ftir modes is usually easier than the assignment of bands corresponding to the bending vibration modes due to overlap or vibration of this group. thus, in the absorption spectra of our oil samples, we can observe that some bending vibrations of the methylene group are produced between 1350 and 1150/ cm. this stretching vibration originates from –c-h vibrations in –ch3 (~1350/cm) groups and deformation vibrations in this group (~1150 cm-1). on the other hand, stretching vibrations of the c-o bond of esters are composed of two coupled asymmetric vibrations, c-c(=o)-o and o-c-c, the first being more important (guillen and cabo, 1997; hong et al., 2005; rohman and che man, 2010); these bands occur in the region between 1300 (some c-c(=o)-o) and 1000/cm (of this combination’s group). the c-c(=o)-o band of saturated esters appears between 1240 and 1163/cm, and in unsaturated esters the vibration is produced at lower frequencies. on the other hand, the o-c-o band of esters derived from primary alcohols appears in the zone between 1064 and 1031/cm, while for those derived from secondary alcohols the band appears approximately at 1100/cm (rohman and che man, 2010; li et al., 2013). both kinds of esters are present in triglyceride molecules. however, some authors assign the band at about 1238/7 /cm exclusively to out-of-plane bending vibrations of the methylene group (guillen and cabo, 1997; li et al., 2013; qian et al., 2007). two bands in table 7 have been assigned in a different way: the bands at approximately 1400/cm and 1319/cm. the first band, at about 1400/cm, has been assigned by some authors to terminal methyl groups on the aliphatic chains of oil components (gurdeniz and ozen, 2009). the second band, at about 1319/cm, was observed in all samples where absorption reached between 967 and 914/cm. in this context, it must be pointed out that the band at about 914/cm, which appears in all oil samples, has been related to the bending vibration of cis disubstituted olefinic groups (guillen and cabo, 1997; qian et al., 2007) and to vinyl groups. although the oil spectra examined in this articles seem to be similar, they show differences in the intensity of their bands as well as in the exact frequency at which the maximum absorbance is produced in each case, due to the different nature and composition of the respective oil samples under study. further characteristic areas of vibration are found in the bands with the maximum of approximately 1740/cm, typically associated with vibrations of the c=o carbonyl group in esters (hong et al., 2005; scholze and meier, 2005). at the same time, a very weak band, or more specifically extension of the band with the maximum at approximately 1705/cm, reveals very weak vibrations of the carbonyl group in acids. in turn, bands with the maximum at approximately 1650/cm correspond to vibrations originating from stretching vibrations of the –c=cgroup (due to cistransformation). a characteristic area is also found in bands with the maximum of approximately 1460/cm originating from deformation vibrations in the -c-h groups of ch2 and ch3 (bending (scissoring)). the subsequent area of bands from 890 to 660/cm represents the characteristic deformation vibrations of -hc=chgroups (cisconformation, extra plain) and ring vibrations of the groups (δ(-(ch2)nand – ital. j. food sci., vol. 31, 2019 608 hc=ch(cis-)) (guillen and cabo, 1997; scholze and meier, 2005; li et al., 2013; qian et al., 2007). as we progress to vibrations within higher wavenumber ranges, one should mention the significant stretching vibrations =c-h (trans-) with the maximum at approximately 3060/cm which originate from the vibrations of triglyceride fractions. a very characteristic feature of stretching =c-h vibrations in the cisconfiguration are the relatively intensive vibrations at approximately 3006/7 /cm. next, a number of vibrations with the maxima at approximately 2955, 2920 and 2855/cm originate from the stretching vibrations of–c-h in the respective -ch3, ch2 groups belonging to the aliphatic groups in triglycerides (hong et al., 2005; scholze and meier, 2005; qian et al., 2007). characteristic vibrations associated with specific functional groups were described in detail for all studied samples in table 7. furthermore, it is noteworthy that in the spectra of the studied samples of oil extracted from rape grown on digestate and traditional fertilizer mixture (fig. 2), there are clearly visible differences in terms of the shape of spectra in the 1770 – 1670/cm area. in most of the studied samples there is apparent, slight amplification of the band at 1742/3/cm (responsible for vibrations of the c=o group) on the side of lower wave numbers, with the clearly defined extreme at approximately 1710/cm (with the relatively lowest intensity in the control spectrum), which can be associated with the formation of a hydrogen bond between the c=o…h-o-h groups in oil samples selected for the study. simultaneously to the appearance of the band at 1710/cm (under both fertilisation regimens), there is a clear increase in the intensity of bands at approximately 1360, 700/cm, which is associated to stretching vibrations in c-o and c-c groups (as described above). the area between 1100 – 1300/cm is also responsible for the stretching vibrations of the co group, but the same varied only slightly between the studied oil samples, regardless of their origin (i.e. cultivation with the use of digestate or traditional fertiliser) (li et al., 2013; qian et al., 2007). with the reducing affinity of the constituent molecules for the formation of the hydrogen bond between c=o…h-o-h, the bands show a slight increase in intensity. a similar correlation can be observed both in rapeseed oil samples originating from traditionally fertilised plots and from plots fertilised with digestate. moreover, it would be prudent to once again mention bands from the range of 2960 to 2840/cm, which are responsible for the symmetrical and asymmetrical vibrations of ch2 and ch3 groups in the aliphatic sections of triglycerides. their rates vary significantly depending on the origin of the respective samples, i.e. from rapeseed grown in plots fertilised traditionally or with the use of digestate. the variations observed in this area are very well correlated with variations in the fatty acids profile presented in table 5. the aforementioned spectral changes also confirm the slight discrepancies in the overall fat content in the studied oil samples, as presented in table 4 above. one should also point out the clear discrepancies in terms of band intensity between the particular rapeseed cultivations, relative to the method of fertilisation. in all spectrum-wide band intensity measurements the same was clearly lower in in the case of plots fertilised with digestate as compared to traditional fertilisation. with regard to a comparison between specific cultivars, the most intensive bands were observed for ‘bios’, followed by ‘markus’, with the lowest values observed for ‘feliks’. the described differences correspond to the respective overall nutrient content in the studied samples of rapeseed oil. the main band differences for all samples, relative to the method of fertilisation, can be observed in bands with the respective maxima at 1745, 1709, 1368, 1220, 1154 and 1146/cm, which confirms the discrepancies in the overall fatty acids profile of the analysed samples. ital. j. food sci., vol. 31, 2019 609 table 7. position of the maxima of absorption spectra in relation to the respective vibrations for selected spring rapeseeds grown with the use of traditional and digestate fertilisation, within the spectral range of 3800-550/cm. ftir types and origin of vibration position of the maximum (/cm) npk fertilizer digestate control ’b’ ’f’ ’m’ ’b’ ’f’ ’m’ ’b’ ’f’ ’m’ – 3463 3476 3476 3474 3476 3474 3476 3474 -c=ow (overtone) and ν(=c-hvw, trans-) 3005 3066 3064 3063 3061 3066 3067 3181 – – 3006 3007 3007 3007 3007 3006 3059/3007 3062/ 3007 ν(=c-hm, cis-) – 2969 2954 2954 2953 2955 2954 – – νas(-c-hm, -ch3) – 2955 2923 2922 2921 2922 2922 2953 2956 νas(-c-hvst, -cha) and νs(-c-hvst, -cha) (aliphatic groups in triglycerides) 2922 2923 – – – – – 2922 2923 2854 2871/2853 2853 2853/ 2872 2853/ 2870 2853/ 2872 2852/ 2872 2854 2852 2731 2731 – 2731 2730 – 2729 2730 2729 -c=ovw fermi resonance 2676 2677 2676 2635/2680 2675 2674 2678 2678 2679 – 2584 – – – – 2335/2361 – 2339/23 60 2360/ 2336 2362/ 2337 2361 – – 2362 – – – 1743 1743 1744 1743 1743 1743 1743 1743 1744 ν(-c=ovst) in ester 1704 1704 1704 1705 1705 1705 1704 1702 1705 ν(-c=ovw) in acid – 1654 1654/ 1559 1654 1654 1654 1653/ 1558 1653 1653 νvw(-c=c-, cis-) 1462 1462 1462 1462 1462 1462 1463 1462 1462 δvw(-c-h) in ch2 and ch3 group, bending (scissoring) 1417 1441/1418 1440/ 1417 1416 – 1418 1440/ 1418 1439/ 1418 1440/ 1418 νvw(-c-h, cis-) bending (rocking) – 1401 1400 1400 – 1400 1400 1400 1401 νw, m, vw(-c-h, -ch3), bending 1377 1376 1377 1377 1377 1351/ 1377 1377/ 1350 1376/ 1349 1377 1367 – – – – – – 1365 1349 1353 1350 1349 1350 1318 1316 1317 1351 1317 1318 1318 1317 1318/ 1302 1301 1300 1300 1300 1300 1301 1299 1279 1277 1278 1278 1277 1279 νm(-c-o) or δm(-ch2-) 1278 1278 1277 – – – – – – – 1265 1262 1264 1261 1262 – 1237 1229 1237 1237 1237 1236 1237 1237 1234 1161 1160 1160/ 1141 1160 1143/ 1159 1141/ 1160 1161 1142/ 1160 1160/ 1143 νst(-c-o) or δst(-ch2-) 1120 1119 1119 1119 1119 1119 1120 1120 1119 νm(-c-o) 1097 1097 1096 1097 1096 1096 1097 1097 1097 νm,vw(-c-o) 1066 1060 1061 1062 1062 1059 1060 1063 1060 1030 1030 1030 1030 1031 1030 1030 1030 1031 966 965 967 966 914/965 913/966 913/ 967 912/ 965 913/969 /949 δw(-hc=ch-, trans-) bending out of plane ital. j. food sci., vol. 31, 2019 610 913/ 867/ 851 911 912/ 867/ 849 848/ 868/ 913 – 870 689/ 867 694/ 869 791 δvw(-hc=ch-, cis-) bending out of plane 766 767 765 765 – 766 770 765 765 δ(-(ch2)nand –hc=ch (cis-) bending (rocking) 723 722 722 721 – 722 722 722 722 – 689 691 694 – 687 662 664 690 ν – stretching, δ – deformation, s – symmetrical, as – asymmetrical, st – strong, vst – very strong, w – weak pca is a mathematical tool, which performs a reduction in data dimensionality and allows the visualization of underlying structure in experimental data and relationships between data and samples. ftir spectra data of 7 oil samples were used in principal component analysis (pca). pca of ftir spectra using absorbencies at 16 wavenumber regions was carried out using statistica 13 advanced statistics software. the pca score plot (fig. 3) was obtained from the correlation matrix of peak absorbencies at 16 frequency regions, namely 3007, 2921, 2853, 1744, 1654, 1461, 1418, 1373, 1235, 1161, 1119, 1096, 1031, 966, 913, 724/cm. in pca, the first principal component (pc1) and the second principal component (pc2) account the largest and the next largest of variable variation. pc 1 explain 99,32% variance, meanwhile pc 2 accounted 0,62%; therefore the variance can be described by the first two pcs. figure 3. projection of cases on the pc 1 and pc 2 plane. ital. j. food sci., vol. 31, 2019 611 hierarchical cluster analysis (hca) was performed in order to observe similarities or dissimilarities between the ‘bios’, ‘markus’ and ‘feliks’ oil samples from spring plants grown with traditional npk mixture fertilisation and digestate (d). the dissimilarity of different clusters was defined by euclidean distance and calculated by single linkeage method. the results obtained are presented in the dendrogram structure, showing the different groups. on the fig. 4 we present the plot obtained from the 7 oil samples of ‘bios’, ‘markus’ and ‘feliks’. considering the cut-off of 0.1 dissimilarity units we can distinguish three clusters (in red, green and blue colour, respectively). the first cluster, green coloured, is a small cluster, aggregated on the far left arm of the dendrogram. this cluster is formed by f-npk oil of ‘feliks’ sample. the second cluster, red coloured, is composed of all ‘eco-digestate’ oil samples including ‘bios’, ‘markus’ and ‘feliks’ samples and samples of control and of m-npk. this result suggests that these oils have a physicalchemical properties more similar than the others. the sample of b-npk oil is aggregated in third cluster (blue coloured) on the right arm of dendrogram. figure 4. dendrogram analysis (hca) of all samples in the 3800-600/cm spectral region. the pca analysis was applied to fatty acid data. the obtained results are presented in figs. 5-6. the greatest effect on pc 1 in pca is positively correlated with the content of c18:1n9c +c18:1n9t (0.017), c16:0 (0.0127) and is negatively correlated with the content of pufa (-0.0471). in turn, pc2 is negatively correlated with c18:2n6c+c18:2n6t and omega 6 (-0,0091) and positively correlated with the content of c18:3n3 (alpha) and omega 3 (0,0096). ital. j. food sci., vol. 31, 2019 612 figure 5. the plot of two first principal components after pca analysis of fatty acid compositions. projection of the cases on the pc 1 and pc 2 plane. figure 6. the plot of two first principal components after pca analysis of fatty acid compositions. projection of the variables on the pc 1 and pc 2 plane. ital. j. food sci., vol. 31, 2019 613 4. conclusions the obtained results reveal a correlation between the employed method of fertilisation, using digestate or a traditional npk mixture, and the soil condition as well as the quality of the products, i.e. spring rapeseed and the oil cold-pressed therefrom. based on said results it can be concluded that statistically significant differences can be observed in terms of the content of nutrients available to plants between the control and fertilised plots. statistically significant differences were also observed between the content of the analysed nutrients in the soil prior to sowing and after the harvest, with the exception of control plots related to the use of npk fertiliser. analyses of the oil in terms of its oxidative stability revealed no direct correlation between the qualities of particular oils and the method of cultivating the seeds from which they were extracted, although the statistical analysis did reveal significant differences in terms of oil obtained from the ‘bios’ cultivar, which was characterised by the lowest stability relative to digestate fertilisation. it is noteworthy that clear, although not particularly big, differences in terms of band intensity in the general cross-section of the ftir spectrum were observed for samples fertilised with the mineral mixture, as compared to digestate. 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faisalabad, pakistan 2center of excellence for olive research and training, barani agricultural research institute, 48800 chakwal, pakistan 3department of food science, lyallpur institute of advanced studies, 38000 faisalabad, pakistan 4department of clinical nutrition, nur international university, 54000 lahore, pakistan 5riphah college of rehabilitation and allied health sciences, riphah international university, 38000 faisalabad, pakistan 6department of food science and technology, khwaja fareed university of engineering and information technology, 64200 rahim yar khan, pakistan *corresponding author: iqrayasmin8@gmail.com abstract current study was designed to explore the nutritional and antioxidant potential of pakistani apricot’s varieties (marghulam, halman, kakas and shakanda). the highest values of moisture, crude fat, crude protein, fiber and ash (7.95±0.05%, 2.29±0.07%, 3.22±0.06%, 3.82±0.91% and 4.26±0.08%) were found in marhulam. the maximum value of total phenolic contents (tpc) 30.75±0.09mg/100ggae and dpph 40.64±0.04% was determined in marghulam and halman respectively. cookies were prepared with incorporation of apricot powder (10%) and evaluated for compositional, mineral, tpc, antioxidant activity, physical parameters, color, texture and sensory attributes. based on sensory evaluation, cookies supplemented with 10% marghulam flour got the highest score. keywords: antioxidants, apricots, cookies, organolyptic characteristics ital. j. food sci., vol. 32, 2020 832 1. introduction fruits and vegetables are at the base of the food pyramid and rich source of nutrients, bioactive compounds i.e. phytochemicals (flavonoids, phenolics, and carotenoids), minerals, vitamins and fibers (liu 2013). vitamins a, b and c along with magnesium, potassium, calcium and iron are present in fruits like papaya, mango, pineapple, lemon, guava, jackfruit. globally, people consume fruits as a staple food. for example, banana is eaten by south americans as the main course of their meal. our food shortages can be minimized to a considerable extent by banana, jackfruit, guava and pineapple (yahia et al., 2019). apricot (prunus armenia l.) is one of the nutritious fruit and a member of the rosaece family. “apricot” is a derivative of the latin term praecocia, meaning “early maturing” or “precocious”. china started its farming, three thousand years before christ (karaca et al., 2019). the total area destined to apricot’s farming in pakistan accounts for 31256 hectares. moreover, in punjab, the apricot cultivation area is 42 hectares. furthermore, the highest production of 220276 tons of apricot is in baluchistan, with a total area of 28901 hectares (kousar et al., 2019). apricots are greatly valued both fresh and dried. a dried fruit delivers a sufficient amount of energy and nutrients, which is particularly important for the population of the mountainous karakoram zone (chen et al., 2020). apricot cultivation significantly contributes to the agricultural income of the pakistani province of gilgit-baltistan, where the local varieties marghulam, kakas, halman and shakanda are mostly cultivated. apricot has a distinctive nutritive value amongst stone fruits. it has a high content of proteins (8%), crude fiber (11.50%), fat (2%), minerals (4%), sugars (>60%), vitamins (a, b complexes, c and k) and organic acids (malic and citric acid), as well as a substantial amount of flavonoids and phenolics (kafkaletou et al., 2019). dried apricots such as marghulam and halman are rich source of nutrients, minerals and vitamin c. dried apricots can also be used for the preparation of different value-added food products (kiralan et al., 2019). previous efforts were made to increase the nutritional and organoleptic characteristics of cookies through supplementation. uchoa et al. (2009) supplemented apple and guava pomace (dehydrated fruit powders) at different level. the cookies prepared with 15% and 20% substitution showed the best results in sensory evaluation. similarly, tańska et al. (2016) incorporated fruit pomace (rosehip, rowan, blackcurrant and elderberry) and studied the effect of antioxidants on the properties of shortbread cookies. likewise, pasqualone et al. (2019) studied the acorn flour substitution effect on the physicochemical and sensory properties of biscuits. various authors (cheng and bhat 2016; ertaş and aslan 2020; kapoor and ranote 2016; lepionka et al., 2019; yousaf et al., 2013; zucco et al., 2011) supplemented legume, pulses, seeds, cereals and evaluated the effect of substitution on quality of cookies. functional food can be described as the food that can deliver consumers with a health benefit beyond their basic nutrients. functional bakery products are gaining much importance to improve the health and wellbeing of consumers. different functional beverages, functional bakery products, functional cereal-based food and dairy-based foods are available in the market (ninfali et al., 2019). among bakery products, cookies are full of carbohydrates and fats, and have low levels of fiber, vitamins and minerals, but are largely consumed all around the world. cookies are considered the most common and consumable bakery items (curutchet et al., 2019). to improve the nutritional profile of cookies, the present study is designed to develop potentially functional cookies by ital. j. food sci., vol. 32, 2020 833 supplementing them with dried powdered apricots after that, the quality testing of prepared cookies was carried out. 2. materials and methods the present study was carried out in the food and nutrition laboratory, government college women university, faisalabad, pakistan. however, some of the product’s analyses were carried out at the university of agriculture, faisalabad, pakistan. 2.1. raw materials apricots varieties kakas, marghulam, halman and shakanda were acquired from mountain agriculture research centre (marc) located in jaglot, in the gilgit baltistan province, pakistan. the selected trees were seven-year-old and were subjected to the same agricultural practices. three healthy plants of the same age of each variety were selected during the years 2018 and 2019. apricots were harvested healthy and at full ripening stage (deep yellow color with red blush). all varieties were harvested on the same day to provide fruits with uniform maturity, color, size and firm texture. particulates of herbicide, insecticide and dust present on fruit exterior were removed by washing. the commercial straight grade flour (sgf) and remaining ingredients for cookies formulation were purchased from the local market of faisalabad, pakistan. all reagents, chemicals and standards were bought from sigma-aldrich (sigma-aldrich tokyo, japan) and merck (kgaa, merck darmstadt, germany). 2.2. preparation of apricot powder apricots were dehydrated by the sun-drying method. initially, the apricots were washed, pitted, cut into small pieces (1.5 cm x 1.5 cm) and placed on wire mesh tray covered with cloth. the apricots were dried under direct sunlight with an overall maximum day time temperature (37°c) and a minimum night temperature (29°c) for 48 h until the final moisture (6.0%) was reached. then the dried apricots were ground by using an electric grinder. the dry apricot powder was sieved, to ensure a homogeneous particle size, through a mesh sieve of 250 µm (retsch test sieve, germany). the powder was stored in sealed polyethylene bags at 4°c for further analyses. 2.3. proximate and antioxidant analysis of apricot powder apricot powder of all varieties was analyzed individually for proximate composition and antioxidant potential. the proximate analyses (moisture, crude fat, crude protein, crude fiber, ash and nfe) were performed following the corresponding methods as stated in aoac (2006). folin-ciocalteu’s test was used to measure the total phenolic content and antioxidant activity (%) was assessed based on free radical scavenging action through 2, 2diphenyl-l-picrylhydrazyl (dpph) assay (idris, 2019). 2.4. mineral analysis of apricot powder the mineral content of apricot powders was determined by the ashing method (aoac, 2006). the acquired ash was digested with 5 ml of 6 m hcl in a water bath. ca, fe, mn, ital. j. food sci., vol. 32, 2020 834 cu, zn and mg of apricot powders were determined in an atomic absorption spectrophotometer whereas na and k were determined by flame photometer and p by using uv-spectrophotometer. 2.5. β-carotene content of apricot powder β-carotene was isolated through column chromatography by using aluminum oxide with an absorbent column length of 10 cm. β-carotene moves down through the absorbent column afore all other pigments and is collected till the chosen pigment/s moved through the column and eluent becomes colorless. the color intensity of the eluent with known volume was measured with a spectrophotometer at 452 nm while acetone in petroleum ether (3 % v/v) used as blank (girón et al., 2019). 2.6. preparation of cookies cookies were prepared from composite blends by using the method of han et al. (2004). the standardized cookie recipe was followed that includes wheat flour 40 g, butter 26 g, sugar 18 g, egg 9, milk 5 ml and yeast 1.6 g. apricot powder and wheat flour (10:30) ratio was set based on different experimental trails while remaining ingredients remained the same. all the ingredients were mixed properly to make the dough. then, the dough was rolled into cookies sheets. the cookies were baked at a temperature of 220 °c for 8 min. after baking, the cookies were cooled at room temperature and stored in polyethylene bags until further analyses. 2.6.1. proximate, mineral, antioxidant and β-carotene of apricot supplemented cookies (asc) proximate composition (moisture, fat, protein, fiber, ash and nfe) of apricot supplemented cookies was determined according to the standard procedures of aoac (2006). the mineral content of apricot supplemented cookies was also determined (aoac, 2006). total phenolic contents were determined through folin-ciocalteu’s method and antioxidant activity (%) was assessed through 2, 2-diphenyl-l-picrylhydrazyl (dpph) assay (idris 2019). β-carotene was estimated by the method described by girón et al. (2019). 2.6.2. physical analysis of apricot supplemented cookies thickness and diameter of apricot supplemented cookies were measured with vernier caliper at two different positions for each cookie and the average was calculated. the spread ratio was calculated using the formula: diameter of cookies/height of cookies. the bake loss of cookies was calculated by weighing five cookies before and after baking. the average difference in weight was noted and represented as percent bake loss (chauhan et al., 2016). 2.6.3. color of apricot supplemented cookies the colorimeter was used to measure the color of cookies based on l, a*, b* value. l* values range (0-100) and represents black to white, a* values represent the red color, and b* values show yellow color (chauhan et al., 2016). ital. j. food sci., vol. 32, 2020 835 2.6.4. texture analysis of apricot supplemented cookies the texture was determined by texture profile analysis (tpa) by using a texture analyzer (brookfield, middleboro, usa). the probe was p/75 aluminum, 75 mm diameter and the test speed was 0.5 mm/s. sample cookies were always placed with the top upward. texture profile analysis includes several parameters i.e. hardness, springiness, cohesiveness and chewiness (gupta et al., 2011). 2.6.5. sensory evaluation of apricot supplemented cookies sensory evaluation of cookies was performed by a group of ten male and female semitrained panelists (20-35 years of age). the assessment of the overall sensory value of cookies was experienced by the sense of sight, taste, and touch. the 9 points hedonic scale rating test was used to assess the degree of acceptance. the panelists were offered with an assessment method, which described several sensory criteria and score choices with number rankings. when all the assessment forms were completed, the data was analyzed. the cookies were evaluated for different sensory attributes like taste, texture, color, appearance, and overall acceptability (ganorkar and jain, 2014). 2.7. statistical analysis the obtained data were evaluated by one-way analysis of variance (anova) with the help of statistix 8.1 (florida, usa). the level of significance (α 0.05%) was determined through the analysis of variance (anova) method under the principles explained by montgomery et al. (2008). 3. results and discussion 3.1. proximate composition of apricot powder the proximate analysis of different apricot varieties is presented in table 1. the highest moisture content was observed in v1 (7.95%) followed by v2 (7.39%) while the lowest moisture content was observed in v4 (6.09%). the present results are contradictory to the outcomes of ashraf et al. (2018), they reported that the apricot powder had moisture content (6.85%). the difference might be due to different varieties used in both studies. storage stability, processing and value of food are linked with its low moisture content (hussain et al., 2006). due to the nutritional differences, marghulam had the highest moisture content as compared to other apricot varieties. the highest value of fat content was reported in v1 (2.29) followed by v2 (2.15%). the present outcomes on fat contents are in line with the finding of hussain et al. (2010), they reported that the apricot powder contains 2.31% fat content. similarly, another researcher arshad et al. (2010), reported that the fat content of three different varieties of apricot marghulam, halman and shakanda was 3.10%, 2.2% and 4.56%, respectively. the compositional differences may be due to genetic and environmental variation (hacıseferoğulları et al., 2007; özcan and hacıseferoğulları, 2007). the ash content is considered as an indicator of minerals present in any commodity. the highest value of ash content was observed in v1 (4.26%) followed by v2 (4.21%). the results ital. j. food sci., vol. 32, 2020 836 of present study were according to the results of akin et al. (2008). the highest value of fiber content was observed in v1 (3.82%) followed by v2 (3.62%) and the lowest value was observed in v4 (3.23%). ali et al. (2014b) reported that the fiber content is 3.85% in apricot powder. crude fiber is also a significant part of fruits and important in sustaining health through improving bowel movement and taking extra fat from the blood. marghulam powder has the highest amount of fiber as compared to other apricot varieties. this compositional difference is common among varieties due to their differences in genotypes, geography and agricultural practices (ali et al., 2014a). table 1. proximate, antioxidant, β-carotene and mineral of apricot powder. apricot powder v1 v2 v3 v4 moisture (%) 7.95±0.05a 7.39±0.04b 6.75±0.03c 6.09±0.02d fat (%) 2.29±0.07a 2.19±0.06b 2.15±0.05c 2.12±0.04d protein (%) 3.22±0.06a 3.18±0.05b 3.15±0.04c 3.12±0.03d fiber (%) 3.82±0.09a 3.62±0.08b 3.42±0.07c 3.23±0.06d ash (%) 4.26±0.08a 4.21±0.07b 4.19±0.06a 4.12±0.04a nfe (%) 78.50±0.09d 79.50±0.10c 80.02±0.11b 80.06±0.012d dpph (%) 32.77±0.07b 40.64±0.06a 30.54±0.05c 28.43±0.03d tpc(mg/100ggae) 30.75±0.09a 29.64±0.04b 28.54±0.02c 27.43±0.03d ß-carotene (mg/100g) 46.12±1.00a 40.07±0.02b 35.41±0.07c 30.74±0.04d na (mg/100gdw) 22.05±1.30a 18.71±0.60b 15.79±0.23c 14.30±0.60d k (mg/100gdw) 2240±38.00a 1868±29.67b 1730±30.00c 1620±25.00d ca (mg/100gdw) 111.30±3.20a 105±2.40b 98.40±2.28c 76.71±0.72d mg (mg/100gdw) 144.89±6.18a 139.90±6.20b 132.70±3.50c 118. 90±4.30d zin (mg/100gdw) 3.28±0.21a 2.70±0.18b 1.43±0.16c 108±0.11d fe (mg/100gdw) 9.96±0.50a 6.34±0.41b 4.04±0.31c 3.90±0.24d cu (mg/100gdw) 0.50±0.05a 0.40±0.04b 0.32±0.01c 0.26±0.02d p (mg/100gdw) 250.80±7.24a 247.90±5.30b 212.40±3.48c 190.60±3.10d mean carrying same letters are not significantly different from each other. values are expressed as mean ±sd (n=3). whereas, v1: marghulam, v2: halman, v3: shakanda, v4: kakas. 3.2. total phenolic content (tpc) and antioxidant activity of apricot powder the highest value of tpc was observed in v1 (30.75 mg/100g ga) followed by v2 (29.64 mg/100g gae) and v3 (28.54 mg/100g gae) while the lowest tpc value was found in v4 (27.43%) as presented in table 1. the current outcomes of tpc are similar to the findings of (wani et al., 2017), they reported that tpc contents of different varieties of apricot harcot, rival and cuminis haley were 25.44 mg/100g gae, 32.86 mg/100g gae and 24.87 mg/100g gae, respectively. dpph value was highest in halman while the maximum tpc content was found in marghulam. the highest antioxidant value was observed in v2 (40.64%) followed by v1 (32.77%) whilst the lowest in v4 (28.43 %). the current outcomes of dpph are in coherence ital. j. food sci., vol. 32, 2020 837 with the results of wani et al. (2015) who reported that dpph of three varieties of apricot harcot, rival and cuminis haley was 21.68%, 34.16% and 44.62% respectively. the difference in phenolic content and antioxidant might be due to varietal, genetic and geographical difference. 3.3. mineral content of apricot powder the potassium concentration was found to be highest in v1 (2240mg\100gdw) followed by v2 (1868mg\100gdw) and v3 (1730mg\100gdw). the highest sodium content was found in v1 (22.05 mg\100gdw) followed by v2 (18.71 mg\100gdw). the highest calcium content was present in v1 (111.30 mg\100gdw) followed by v2 (105.18 mg\100gdw) and v3 (98.40mg\100gdw). the maximum iron content was in v1 (9.96mg\100gdw) followed by v2 (6.34 mg\100gdw) and v3 (4.04mg\100gdw). the present results of different apricot varieties are following the outcomes of ali et al. (2014b). mineral composition showed that apricot varieties contain considerable amounts of minerals. the mineral composition showed that marghulam variety has high mineral content followed by halman shakanda and khakhas. these conclusions are in line with the previous study on turkish apricots (hacıseferoğulları et al., 2007). however, differences in data might be due to the genotype, geographical origin, environment and agronomic practices (chen et al., 2020). 3.4. β-carotene content of apricot powder the β-carotene were found to be highest in v1 (46.12mg/100g) followed by v2 (40.07mg/100g) and v3 (35.41mg/100g). the present results of apricot cookies are consistent with the outcomes of ali et al. (2014), who reported that marghulam β-carotene (50.12±1.00 mg/100g) followed by shakanda (46.07±0.88 mg/100g), jahangir (40.54±0.78 mg/100g), shirini (39.45±0.740 mg/100g), yagoo (36.41±0.700 mg/100g) and halman (30.61±0.50 mg/100g). bioactive compounds i.e. phenolics and carotenoids are important quality index parameters of fruits and vegetables (girón et al. 2019). 3.5. proximate composition of apricot supplemented cookies the proximate composition of apricot supplemented cookies varied significantly among different treatments (table 2). the highest moisture content was found in t1 (7.85%) followed by t2 (7.80%) and the lowest moisture content was found in t4 (6.24%). the present results of different apricot cookies are in accordance with the results of mundeja and hirdyani (2014), they reported 6.38% moisture content in apricot cookies. similarly, another research work of peter ikechukwu et al. (2017), found that the moisture content of whole wheat flour cookies was 7.70%. the reduction in crude fat content might be due to the lipolytic activity of the enzymes i.e., lipase and lipooxidase (varshney et al., 2008). the protein content reduction in different cookies might be due to the hydrolysis of the peptide bond with the help of protease enzyme that results in the splitting of protein molecules (wani et al., 2015). the difference in fiber content may be due to the deprivation of hemicelluloses and other structural polysaccharides. the highest ash content was found in t1 (1.36%) followed by t2 (1.30%). similar results were reported by other researchers (gayas et al., 2012; pratyush et al., 2015). ital. j. food sci., vol. 32, 2020 838 3.6. total phenolic content and antioxidant activity of apricot supplemented cookies the total phenolic content and antioxidant activity varied significantly for treatments (table 2). total phenolic content and the antioxidant value decreased in apricot cookies as compared to apricot powder due to the processing loss during baking. the maximum tpc value was found in t1 (25.69 mg/100g gae) followed by t2 (24.60 mg/100g gae). the highest dpph value was found in halman and the highest tpc value was found in t1. dpph is a free radical compound that has been widely used to estimate the free radical scavenging activity (amarowicz et al., 2004). the highest value of dpph was found in t2 (38.52%) followed by t1 (31.60%) and t3 (28.39%) while the lowest dpph value was found in t4 (25.30%). 3.7. mineral content of apricot supplemented cookies the mineral contents of different apricot cookies also varied significantly (p<0.05) among all treatments. potassium, sodium, calcium and iron concentration was found to be highest in t1, these values for these minerals were 1940 mg\100g, 20.05 mg\100g, 108.29 mg\100g and 8.90 mg\100g on dry weight basis respectively. the present results of various apricot cookies are consistent with the outcomes of i̇ncedayi et al. (2016). similarly, akin et al. (2008) determined the k, mg, ca and zn contents of eleven apricot varieties similar to the present study as 1227-3455 mg/100 g, 110.4-284.4 mg/100 g, 87-240.5 mg/100 g and 1.384.24 mg/100 g based on dry matter, respectively. the data about mineral contents (mg/100 g) revealed k as the predominant element. these mineral compounds are present in any part of the plant and their concentration differs concerning crop, variety, growth condition, irrigation, genetics, temperature fluctuation, seasons, pre-harvest and postharvest treatments. k, ca and mg are considered major minerals of the apricot fruit (drogoudi et al., 2008). for the human body, minerals are vital for a diversity of basic physiological and metabolic processes that include enzyme activation, muscle contraction, bone health, conduction of nerve impulse, transport of oxygen, antioxidant activity, acid and immune function of blood. an adult requires almost 350 mg mg, 3000 mg k and 1000 mg ca intake per day (williams, 2005). 3.8. β-carotene content of apricot supplemented cookies maximum ß-carotene content was found in t1 (43.79 mg\100g) followed by t2 (36.99 mg\100g) and t3 (32.88 mg\100g). the present results of various apricot cookies are consistent with the results of igual et al. (2012). the carotenoids content is lesser than that stated for fresh apricot, which can be elucidated by the detail that drying and baking may lead to degradation of carotenoids due to oxidation and high temperature (dragovic-uzelac et al., 2007). in addition to β-carotene, apricot fruit and its products contain smaller amounts of α-carotene, γ-carotene, zeaxanthin and lutein (fraser and bramley 2004). 3.9. physical analysis of apricot supplemented cookies physical properties (thickness, diameter, spread ratio, and bake loss) of cookies are presented in table 3. results revealed that there was a significant difference (p<0.05) among each treatment in terms of thickness, diameter, spread ratio and bake loss. the highest diameter was noted in t1 (53.99 mm) followed by t2 (53.77 mm), t3 (52.96 mm). ital. j. food sci., vol. 32, 2020 839 table 2. proximate, antioxidant, β-carotene and mineral of apricot supplemented cookies. apricot cookies t0 t1 t2 t3 t4 moisture (%) 5.85±0.06e 7.85±0.11a 7.80±0.10b 6.84±0.09c 6.24±0.08d fat (%) 7.68±0.01e 9.77±0.05a 9.26±0.04b 8.27±0.03c 8.07±0.02d protein (%) 5.15±0.01e 5.75±0.05a 5.48±0.04b 5.38±0.03c 5.24±0.02d fiber (%) 0.72±0.01e 1.45±0.05a 1.33±0.04b 1.19±0.03c 1.14±0.02d ash (%) 1.17±0.01e 1.36±0.05a 1.30±0.04b 1.25±0.03c 1.22±0.02d nfe (%) 79.04±0.09a 77.35±0.08b 75.41±0.07c 73.68±0.06d 63.78±0.05e dpph (%) 20.6±0.09 e 31.60±0.04b 38.52±0.02a 28.39±0.03c 25.30±0.02d tpc(mg/100ggae) 15.69±0.12e 25.69±0.06a 24.60±0.04b 23.37±0.15c 22.26±0.03d ß-carotene (mg/100g) 15.61±0.30e 43.99±0.05a 36.99±0.03b 32.88±0.02c 27.50±0.04d na (mg/100gdw) 8.40±0.40e 20.05±1.28a 16.71±0.58b 12.79±0.20c 10.30±0.57d k (mg/100gdw) 1250±20.00e 1940±36.00a 1762±27.60b 1690±24.00c 1599±20.00d ca (mg/100gdw) 50.92±0.60e 108.29±3.10a 102.16±2.38b 94.40±2.25c 72.71±0.69d mg (mg/100gdw) 110±4.27e 140.89±6.15a 136.88±6.20b 128.68±3.48c 116.90±4.27d zn (mg/100gdw) 0.99±0.07e 2.99±0.17a 1.69±0.16b 1.40±0.14c 1.06±0.09d fe (mg/100gdw) 1.99±0.20e 8.90±0.48a 5.32±0.38b 3.02±0.29c 2.90±0.22d cu (mg/100gdw) 170±3.06e 240.79±6.24a 236.77±4.28b 208.38±2.45c 188.58±2.09d p (mg/100gdw) 0.18±0.01e 0.48±0.04a 0.38±0.03b 0.28±0.02c 0.24±0.02d mean carrying same letters are not significantly different from each other. values are expressed as mean ±sd (n=3). whereas, t0: cookies prepared with straight grade flour, t1: cookies prepared with straight grade flour and marghulam (90:10), t2: cookies prepared with straight grade flour and halman (90:10), t3: cookies prepared with straight grade flour and shakanda (90:10), t4: cookies prepared with straight grade flour and kakas (90:10). table 3. physical analysis of apricot supplemented cookies. treatments weight (g) thickness (mm) diameter (mm) spread ratio bake loss (g/100g) t0 12.16±0.10 e 9.78±0.41a 51.11±0.04e 5.60±0.03e 16.99±14.8aa t1 15.47±0.20a 8.75±0.11 b 53.99±0.17a 6.99±0.09a 16.36±14.87b t2 15.38±0.21b 8.48±0.09 c 53.77±0.15b 6.70±0.06b 14.99±14.87c t3 14.35±018 c 8.35±0.07d 52.96±0.12c 6.55±0.05c 14.50±14.87d t4 13.66±0.15 d 8.20±0.06e 52.09±0.09d 6.40±0.04d 14.25±14.87e mean carrying same letters are not significantly different from each other. values are expressed as mean ±sd (n=3). whereas, t0: cookies prepared with straight grade flour, t1: cookies prepared with straight grade flour and marghulam (90:10), t2: cookies prepared with straight grade flour and halman (90:10), t3: cookies prepared with straight grade flour and shakanda (90:10), t4: cookies prepared with straight grade flour and kakas (90:10). the present results for diameter are coherent with the results of sharif et al. (2009) who reported that the diameter of the cookies increases as the concentration of defatted rice bran increases. the current findings are contradictory to the work of kamaljit et al. ital. j. food sci., vol. 32, 2020 840 (2010), they reported that the pea flour combination reduces the diameter of the cookie. the highest weight was observed in t1 (15.47g) followed by t2 (15.38) and t3 (14.48g). the present results are coherent with the results of chauhan et al. (2016). baking loss of treatment cookies was decreased compared to control. this is due to the water holding capacity of apricot powder compared to wheat flour due to its high-protein content. 3.10. color analysis of apricot supplemented cookies the color values varied significantly (p<0.05) among the treatments (table 4). l* a* and b* values were highest in t1 65.58, 24.27 and 47.76 respectively. the current results are in line with the findings of i̇ncedayi et al. (2016). hegedú´ s et al. (2010) described hue angle values of 62.63-84.63, l∗ values of 60.15-72.43 and chroma values of 51.66-68.48 of fruit flesh of selected apricot cultivars. particularly, l* values were similar to the results of kamaljit et al. (2010). many scientists have reported that the l* value is a measure of a browning index in fruits and fruit products. the difference in color might be due to the moisture level of the apricot supplemented cookies or variation in processing conditions. color is a key factor for assessing the baking performance of cookies, which not only provides adequacy to the raw materials but also provides information regarding baking excellence (pasha et al., 2011). it is one of the demanding characteristics to determine the quality of a product. variation of color in cookies during baking is a dynamic process in which color changes as the baking proceeds (mepba et al., 2007). table 4. color of apricot supplemented cookies. treatments l* a* b* t0 50.79±0.22 e 9.69±0.99e 27.70±0.04e t1 65.58±0.16 a 24.27±0.88a 47.76±0.09a t2 64.72 c±0.17b 18.15±0.87b 38.76±0.07b t3 61.74±0.12 c 15.20±0.80c 35.71d±0.08c t4 57.79±0.14 d 12.48±0.82d 31.72±0.06d mean carrying same letters are not significantly different from each other. values are expressed as mean ±sd (n=3). whereas, t0: cookies prepared with straight grade flour, t1: cookies prepared with straight grade flour and marghulam (90:10), t2: cookies prepared with straight grade flour and halman (90:10), t3: cookies prepared with straight grade flour and shakanda (90:10), t4: cookies prepared with straight grade flour and kakas (90:10). 3.11. texture profile of apricot supplemented cookies the maximum hardness was found in t1 (150.74n) followed by t2 (145.66n) and t3 (135.54n) (table 5). hardness is the textural property, which is one of the most important assessment parameters of baked food items. maximum springiness was found in t1 (74.55%) followed by t2 (62.22%) and t3 (57.66%). the lowest springiness was recorded in t4 (50.66%). the current findings are comparable with pereira et al. (2013), they reported that hardness, springiness, cohesiveness and chewiness of maria type cookies was 158.74 n, 72.22%, 0.78 and 93.02 n, respectively. these differences might be due to varietal, genetic and geographical differences. these outcomes might be due to other reasons such ital. j. food sci., vol. 32, 2020 841 as protein denaturation, irregular structure, water holding capacity, moisture content, solubilization of proteins, protein coagulation and fat content. table 5. texture analysis of apricot supplemented cookies. treatments hardness (n) springiness (%) cohesiveness chewiness(n) t0 120.55±20.78 e 40.22±8.79e 0.40±0.01d 35.63±6.53d t1 150.74±37.79 a 74.55±11.79a 0.70±0.09a 90.02±23.71a t2 145.66±23.80 b 62.22±10.22b 0.60±0.07ab 69.10±21.54abc t3 135.54±14.87 c 57.66±7.92c 0.55±0.06c 51.64±21.80bc t4 130.88±20.80 d 50.66±6.22d 0.50±0.04cd 39.47±15.20c mean carrying same letters are not significantly different from each other. values are expressed as mean ±sd (n=3). whereas, t0: cookies prepared with straight grade flour, t1: cookies prepared with straight grade flour and marghulam (90:10), t2: cookies prepared with straight grade flour and halman (90:10), t3: cookies prepared with straight grade flour and shakanda (90:10), t4: cookies prepared with straight grade flour and kakas (90:10). 3.12. sensory evaluation of apricot supplemented cookies sensory evaluation is important for quality assessment and commonly done by the panelist’s judgment. sensory evaluation of apricot supplemented cookies depicted significant effect (p<0.05) of apricot powder on different sensory parameters (color, flavor, taste, texture and overall acceptability). after control, t1 got a maximum score for all the sensory parameters (fig. 1). the color acts as an indicator of the final baked items and is linked to differences in taste and smell. the decreasing tendency in texture scores was probably owing to humidity from the atmosphere (sharif et al., 2009). figure 1. sensory evaluation of apricot supplemented cookies. t0: cookies prepared with straight grade flour, t1: cookies prepared with straight grade flour and marghulam (90:10), t2: cookies prepared with straight grade flour and halman (90:10), t3: cookies prepared with straight grade flour and shakanda (90:10), t4: cookies prepared with straight grade flour and kakas (90:10) 0 2 4 6 8 10 color flavor taste texture overall acceptability t0 t1 t2 t3 t4 ital. j. food sci., vol. 32, 2020 842 4. conclusions apricot is one of the nutritious fruit, which is consumed either fresh or dried all around the globe. a blend of straight grade flour and apricot powder could make excellent baked product with improved economic worth. it not only improves the overall acceptability but also improves the nutritive value of the products without adding much to the cost of the product. this shows the effective utilization of apricot powder in baked goods. the result of the study revealed that the marghulam variety has high antioxidant activity as compared to other varieties. the cookies made out of straight grade flour supplemented with 10% marghulam apricot powder were satisfactory in terms of functional, nutritional and organoleptic characteristics as compared to control and other treatments. the results of the present study could be beneficial for the development of functional cookies. acknowledgments the authors are thankful to the department of food science and technology, government college women university faisalabad and faculty of food, nutrition and home sciences, university of agriculture, faisalabad, pakistan for research facilities and technical support. authors are really grateful to dr. nazia khalid for proofreading. references akin e.b., karabulut i. and topcu a. 2008. some compositional properties of main malatya apricot (prunus armeniaca l.) varieties. food chemistry 107(2):939-948. ali s., masud t., abbasi k.s., ahmad a., mahmood t. and ali a. 2014a. biochemical attributes of 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20, 2020 accepted august 20, 2020 ijfs#1657_bozza ital. j. food sci., vol. 32, 2020 466 paper food service operators behavior and knowledge on gluten-free meals and requirements of public canteens m. tamburro1, m.l. sammarco1, l. di eleonora2 and g. ripabelli*1 1department of medicine and health sciences “vincenzo tiberio”, university of molise, campobasso, italy 2food and nutrition hygiene unit, local health institution umbria n. 2, umbria region, terni, italy *corresponding author: ripab@unimol.it abstract this study provides a summary of celiac disease (cd) epidemiology in italy, canteens distribution and training courses for food service operators (fsos) analyzing 2007-2017 data. furthermore, behavior and knowledge of fsos and organizational requirements for gluten-free (gf) meals in school and hospital canteens in central italy were investigated. in parallel to an increased cd occurrence at national level, gf foods demand is significantly growing. our survey in 20 inspected canteens revealed important knowledge and awareness gaps among fsos, as well as lack of procedures and structural requirements for safely providing gf meals, underlining the need to improve education on topic. keywords: celiac people, central italy, gluten contamination, public food supply, food preparation, training ital. j. food sci., vol. 32, 2020 467 1. introduction celiac disease (cd) is a chronic small intestinal immune-mediated enteropathy with a spectrum of disorders related to the trigger of gluten ingestion in susceptible individuals (gulino et al., 2016; degeorge et al., 2017; lebwohl et al., 2018). generally, cd clinical presentation encompasses classic gastrointestinal or malabsorption symptoms, such as chronic diarrhea, weight loss, bloating, flatulence, and abdominal pain (ludvigsson et al., 2013; downey et al., 2015; elli et al., 2015). because of the most recent epidemiological shift, non-classical symptoms are becoming frequent either in childhood or in adulthood, and commonly include iron deficiency anemia, abnormal liver functions, bone disease, and skin disorders (rubio-tapia et al., 2013; lebwohl et al., 2018). during the latest 10 years, cd has become a significant public health concern globally (sapone et al., 2012; singh et al., 2018), primarily involving the areas of the world characterized by high wheat consumption. a recent meta-analysis estimated a pooled global cd seroprevalence and biopsy-confirmed cd prevalence of 1.4% and 0.7%, respectively, with the highest prevalence in europe (0.8%) and oceania (0.8%), and the least in south america (0.4%) (singh et al., 2018). furthermore, biopsy-confirmed cd prevalence was reported to be 1.5 times more common in females than in males, and approximately twice more common in children than in adults (singh et al., 2018). the high cd prevalence has been mostly associated to the widespread consumption of gluten containing cereals, and to occurrence of human leucocyte antigen (hla) dq2 and hla dq8 predisposing alleles in the global population, ranging 0-28% and 1-9%, respectively (lionetti and catassi, 2014). although both required, these factors are not sufficient for clinical cd development, since only 30% of subjects eventually develop the disease (lionetti et al., 2015). cd almost affects about 1% of the european population (rubio-tapia et al., 2012; choung et al., 2017), even though a large proportion of undiagnosed and consequently untreated subjects has been estimated (elli et al., 2015). furthermore, there are huge differences in cd prevalence that could be not explained only by genetic and environmental risk factors. for example, germany has the lowest cd prevalence than other european countries, whilst sweden and finland the highest (mustalahti et al., 2010). in italy, the revision of the “essential levels of care” (italian ministerial decree 12/01/2017) involved the shift of cd and its clinical variant of dermatitis herpetiformis in the list of chronic disabling diseases. the transition was considered as necessary because both clinical forms could not be longer included within the prevalence limits of the rare diseases established below 5/10,000 inhabitants at european level. furthermore, the italian law n. 123/2005 on “rules for the protection of cd people” has recognized cd as a social disease and promoted adoption of measures for these patients (legge 4 luglio 2005, n. 123; capuozzo et al., 2013). indeed, the national government assigns annually dedicated resources to the regions, and authorizes public money spending for gluten-free (gf) meals distribution in the school, hospital and public canteens, as well as for professional training for food service operators (fsos). in particular, according to the law 123/2005, school (public and private) and hospital (public and private hospitals, hospices and public nursing homes, institutional care and private nursing homes) canteens, as well as those attached to public facilities (public institutes and administrations) must guarantee gf meals for cd people who request it. at present, a balanced gf diet based on the combination of naturally and certified processed gf food represents the only effective treatment for celiac people (la vieille et ital. j. food sci., vol. 32, 2020 468 al., 2014; gulino et al., 2016; bascuñán et al., 2017), providing a rapid improvement and resolution of symptoms. although the effectiveness of gluten sources elimination in an absolute and permanent way is proved, numerous difficulties could affect a complete adherence to gf diet, including costs of gf food, variable quality of information regarding the status of ingredients, and potential gluten exposure when travelling or eating out (barratt et al., 2011; rubio-tapia et al., 2013). the threshold below which gluten is safe is unknown; however, the available evidences advised that exposure to less than 10 mg/day is unlikely to cause histological changes to the intestinal mucosa for cd individuals (bold and rostami, 2011; la vieille et al., 2014, 2016). indeed, according to the international food standards codex alimentarius, gluten level in gf food may not exceed 20 parts per million (ppm), which corresponds to 20 milligrams of gluten per kilogram, or per liter of product (rubio-tapia et al., 2013; farage et al., 2017a; standards codex alimentarius fao-who; eu law-eur-lex). of concern, it should be considered that cross-contamination could frequently occur when gf food come in contact with glutencontaining grains and their derivatives during any stage of food preparation process, or if the same equipment/utensils handling food products or surfaces are used (koerner et al., 2011; farage et al., 2017a; rostami et al., 2017; verma et al., 2017). therefore, avoiding cross-contamination during meals preparation, especially in restaurants and public canteens, is the key factor to ensure the quality of gf food for cd people. however, having a meal out could represent a serious risk concern when there is a lack of knowledge regarding gf food by the staff involved (farage et al., 2014). in order to contribute to a better quality of life for cd patients, it is important to establish effective strategies to prevent contamination and enable safe production of gf food (farage et al., 2017a). these perspectives clearly include the development of appropriate instruments or approaches for verification in loco of non-conformities related to the potential crosscontamination in the production process (farage et al., 2017b), and the improvement of professional training, being awareness of cd related issues among chefs and cooks highly dependent by the education level (schultz et al., 2017). this study aims to describe the state of the art and the epidemiological background regarding cd occurrence in italy, as well as an overview of the public canteens administrating gf meals and training on this issue by examining all available data at national level. furthermore, the results of a survey conducted among fsos of hospital and school canteens in terni province, umbria region, central italy, are reported. in particular, the investigation explored about fsos knowledge and behaviors related to cd issues, as well as on the structural and organizational requirements for gf meals preparation and administration, and measures developed and in place established to minimize the likelihood of gluten contamination. 2. methods 2.1. epidemiological data on cd, canteens mapping and training in italy, the general directorate for hygiene and food safety and nutrition of the ministry of health releases an annual census to the parliament on cd (www.salute.gov.it/portale/documentazione/). by searching in the thematic area of “nutrition” and the publication years, annual reports to the parliament on cd items were available since 2007. indeed, data on cd prevalence, distribution of public canteens ital. j. food sci., vol. 32, 2020 469 involved in gf meals administration, and specific training for fsos in relation to 20072017 reference period were pooled and analyzed (guidarelli et al., 2008, 2009, 2010; de stefano and silano, 2011, 2012, 2016, 2018, 2019; de stefano et al., 2015, 2016). 2.2. setting of the survey involving fsos the survey involved 20 public canteens located in terni province, umbria region, central italy. particularly, 14 nursery school, 4 primary school and 2 hospital canteens were included, being involved in the production and/or administration and/or sale of gf products to consumers at time of the study. the canteens were inspected by technicians of the prevention in the environment and the workplace of the food and nutrition hygiene unit, department of prevention of umbria region, local health institution n. 2. 2.3. survey tools for data collection data were collected during the regular inspection activities using two different approaches, an interview and a checklist, which were both administered to 20 fsos (one for each inspected canteen) in charge of the entire food processing. hence, fsos were faceto-face interviewed by technicians to ascertain their knowledge about cd and glutenrelated aspects and their behavior for the control of gluten contamination risk. similarly, a checklist was completed by fsos to verify the structural and organizational requirements for gf food preparation and administration. in details, the interview was structured in two sections concerning knowledge and behavior. the knowledge section was composed of five questions concerning general and theoretical aspects on cd, particularly referring to food permitted and forbidden (list of 20 food types) in the diet for celiacs, and specific precautions. the section referred to behavior was structured in seven questions in order to investigate about their attitudes in the management and control of the entire gf food processing. in particular, the checklist was structured into six areas, reflecting different stages of gf food production, preparation and administration to verify the compliance with procedural, structural and organizational requirements for control of gluten contamination risk. hence, the checklist included 24 questions concerning hazard analysis and critical control points (haccp) plan; purchase and storage of raw food materials and available equipment (control procedures for raw ingredients; storage and equipment/utensils for food preparation and manipulation); food preparation and cooking (areas, practices, equipment); food administration; staff hygiene and procedures (clothing); personnel training (food hygiene and cd issues). 2.4. data analysis cd prevalence, public canteens involved in gf meals administration, and specific courses for fsos obtained by reports to italian parliament from 2007 to 2017 were analyzed in terms of temporal trend, and through descriptive statistics (mean ± standard deviation and median encompassing all the reference period). the overall data from the interview and checklist were collected and analyzed using statistical package for social science (spss) software version 25.0. to each item from both the interview and the checklist, 1-point level was assigned when a correct answer was provided, whilst 0-point if fsos erroneously or not answered. ital. j. food sci., vol. 32, 2020 470 therefore, results from the interview were evaluated compared to the maximum achievable score of 31, comprised of 24 plus 7-points of knowledge and behavior questionnaire sections, respectively, while the score for checklist varied between 0 and 23points. 3. results 3.1. cd prevalence, gf meals canteens and training courses, italy, 2007-2017 in italy, according to the last epidemiological mapping and 2017 annual census on cd by ministry of health, 206,561 celiacs have been diagnosed, for a mean prevalence of 0.34% (de stefano and silano, 2019), with a percentage increment of 4.1% compared to the previous year (fig. 1). from 2007 to 2017, a steady increase of the number of diagnosed cases was observed (mean number of cd subjects=144,389±46,330 (sd), median=148,662), which along the whole period corresponded to +220.8% increase of diagnoses, ranging from 64,398 in 2007 to 206,561 cases in 2017 (fig. 1). during 10 years, the highest variation occurred between 2007 and 2008 (+27.2%), and between 2008 and 2009 (+34.9%), while lowest variations were found in the most recent years (fig. 1), although with a high number of new diagnoses. additionally, two third of cd population were females (145,759 vs 60,802 males) with a 1m:2f mean proportion (de stefano and silano, 2019), and this trend has been constantly observed in the last decade (fig. 2). during 20072017 period, cd cases were on average 98,849±35,771 (median=104,334 cases; range: 35,017-145,759) among females, and 42,017±14,479 (median=44,253 cases; range: 16,23960,802) among males, respectively. the age group in which cd was prevalently registered corresponded to 19-40 years with 71,371 individuals (34.5%) (de stefano and silano, 2019). figure 1. number of cd cases, italy, 2007-2017. ital. j. food sci., vol. 32, 2020 471 figure 2. number of cd cases stratified by gender, italy, 2007-2017. in 2017, the national health service has provided an amount of 320,111.59€ to the italian regions in order to guarantee gf food and 534,427.43€ for training courses to fsos (de stefano and silano, 2019). to support gf diet, almost 250 million euros have been spent for gf products, with an annual national average of 1,200.00€ per capita (de stefano and silano, 2019). data from the regional registry offices in 2017 reported that 42,814 canteens covered the scope of law 123/2005: 28,718 (67%) were school, 7,997 (19%) hospital and 6,099 (14%) of public organizations. during 2007-2017, a significant increase of the number of canteens was observed (mean±sd=39,761±3,040; median=39,110), with the highest prevalence of school canteens in the national territory (fig. 3) (de stefano and silano, 2019). in the overall period, school, public and hospital canteens were 28,103±1,704 (median=28,248; range: 24,693-30,810), 7,029±1,778 (median=6,410; range: 6,032-9,301), and 4,506±2,031 (median=3,823; range: 4,320-9,301), respectively. from 2017 data, 755 specific training courses with an average of 5 hours for each course were activated (fig. 4) in the national territory, involving 19,068 fsos. since 2017, the number of courses was increased compared to the latest two years. in the whole period, on average, 669±362 (median=645) specific courses were managed, involving 15,120±5,403 (median=15,968) fsos, with high participation since 2010. 3.2. interview to fsos among various listed food, all fsos demonstrated to know that bread, pasta and cutlet are forbidden food in the diet for cd individuals, whilst 15% (n=3) and 20% (n=4) erroneously answered that cookies and barley coffee can be consumed, respectively (fig. 5). other permitted food categories in the diet for celiacs were correctly identified, such as cooked vegetables, olive oil, tomato paste, eggs, meat, fish, sugar, honey, and coffee. conversely, only 80% (n=16), 85% (n=17), and 55% (n=11) knew that consumption of milk, rice and strawberries are allowed in gf diet, respectively. in addition, raw ham was ital. j. food sci., vol. 32, 2020 472 improperly considered as forbidden in cd diet by 25% (n=5), as well as natural yoghurt (without cereals) and butter by 15% (n=3) and 10% (n=2), respectively (fig. 5). figure 3. number of school, hospital and public canteens administering gf food, italy, 2007-2017. figure 4. courses on cd and gf food for fsos, italy, 2007-2017. furthermore, 30% (n=6) erroneously stated that cd patients can introduce small amounts of gluten with diet, and only 75% (n=15) proved to be aware that gluten is not removed by cooking foods (table 1). about management and control of gluten contamination risk in food preparation and administration, 20% (n=4) of fsos did not know that gf food should be stored in clearly identified and separated areas. furthermore, gf food preparation should occur in a well-identified area and separated kitchen for 30% (n=6). only 25% (n=5) of fsos knew that equipment (i.e. oven, deep fryer, plates, etc.) and utensils (i.e. cookware, tableware, etc.) should be used exclusively for gf food preparation (table 1). ital. j. food sci., vol. 32, 2020 473 figure 5. are the following food categories permitted in the diet for cd individuals? all fsos demonstrated to know that, if lacking a double equipment for celiac and nonceliac people, it is a prerequisite to separate their use in time, ensuring satisfactory cleaning conditions prior to use. all fsos proved to be aware that hand washing and control of clothing are measures highly required in the meal service, as well as the proper identification of dishes for cd consumers to avoid a promiscuous service. however, a special coat when preparing gf meals should be used by 30% (n=6) of fsos (table 1). based on the answers provided, only 6 (30%) fsos scored 24-points being the maximum level of knowledge, while the remaining scored 18-23-points. regarding skills related to gf meals, the maximum level was reached only for two fsos, while the remaining ranged as 4-6, and more often (65%) with a 5-points score. largely, the inadequate response was related to the equipment dedicated for gf food because considered unnecessary. by pooling data, none of the fsos had the ideal level (maximum score=31) of knowledge and behavior, and only eight showed a quasi-optimal score reaching 28-30-points, while the remaining scored in the intermediate range of 23-26-points. 3.3. requirements for gf meals processing resulting from checklist with respect to haccp plan implemented in the inspected canteens, there was a specific section on gluten and management of critical control points (ccps) to minimize and control the risk of accidental and/or cross-contamination only for 40% (n=8) (table 2). furthermore, there was no opportunity to appreciate the presence, in paper form or online, of the handbook of the italian association for celiacs in 55% (n=11) of canteens. before storage, control procedure for raw materials, semi-finished and finished food products at the reception were not available in 45% (n=9), and no specific procedure for nonconforming food was found in 60% (n=12) (table 2). ital. j. food sci., vol. 32, 2020 474 table 1. knowledge on cd and gf food as reported by 20 fsos through the interview. items correct answer yes n (%) no n (%) • cd subjects could introduce small amounts of gluten with diet no 6 (30.0) 14 (70.0) • gluten is removed by cooking foods no 5 (25.0) 15 (75.0) • gf food must be provided by the consumers no 4 (20.0) 16 (80.0) • gf food should be stored in a separate and well identified area yes 16 (80.0) 4 (20.0) • preparation of gf meals must take place in a separate and well identified area yes 6 (30.0) 14 (70.0) • equipment and utensils must be dedicated for gf food preparation yes 5 (25.0) 15 (75.0) • it is necessary prior sanitation and time differentiation when a double equipment is not available yes 20 (100) 0 • a dedicated coat should be used when preparing for cd subjects yes 14 (70.0) 6 (30.0) • hand washing and control of uniform must be regularly performed in food administration yes 20 (100) 0 • gf meals must be clearly identified to avoid promiscuous service yes 20 (100) 0 ital. j. food sci., vol. 32, 2020 475 table 2. checklist on procedural, structural and hygienic requirements in gf meals preparation and administration. items correct answer yes n (%) no n (%) • a section for gf products is included in the haccp plan yes 8 (40.0) 12 (60.0) • in place availability of the italian booklet of association for celiacs yes 9 (45.0) 11 (55.0) • the control of any raw materials, semi-finished and finished products is performed before storage yes 11 (55.0) 9 (45.0) • there is a procedure for discarding non-conforming products yes 8 (40.0) 12 (60.0) • gf raw materials are stored in separate areas and in identifiable shelves yes 20 (100) 0 • gf food are stored in closed and identifiable containers yes 20 (100) 0 • gf food are identified and placed in separate containers in the fridge/freezer yes 13 (65.0) 7 (35.0) • the storage of gf products takes place in a dedicated fridge/freezer yes 4 (20.0) 16 (80.0) • there is a separate room/area for preparing gf food yes 4 (20.0) 16 (80.0) • a temporal differentiation with previous sanitization is performed if a separate zone for gf food is not available yes 16 (80.0) 4 (20.0) • a sanitation areas procedure is available yes 5 (25.0) 15 (75.0) • the registration of the sanitization control is carried out yes 5 (25.0) 15 (75.0) • there are equipment, tools and utensils dedicated for meals for cd people yes 4 (20.0) 16 (80.0) • a temporal differentiation is performed with previous sanitation if utensils are used for gluten-containing foods yes 4 (20.0) 16 (80.0) • there is a procedure for the sanitization of equipment and tools yes 5 (25.0) 15 (75.0) • presence of washbasins yes 7 (35.0) 13 (65.0) • hand washing and clothing control are regularly carried out yes 16 (80.0) 4 (20.0) • the identification of the dishes for cd people and non-promiscuous service are always performed yes 19 (95.0) 1 (5.0) • there is dedicated clothing that is placed in exclusive rooms yes 2 (10.0) 18 (90.0) • a clothing and hygiene procedure for personnel is available yes 12 (60.0) 8 (40.0) • a specific training on cd and related issues is regularly performed yes 11 (55.0) 9 (45.0) ital. j. food sci., vol. 32, 2020 476 storage activities of gf raw materials were carried out in separate areas and in special shelves, as well as gf food preserved in closed and identifiable containers. however, welldefined local or area for preparing gf food, or utensils/equipment dedicated were not available for 80% (n=16) of the canteens, although a time differentiation for their use was performed, with prior sanitization (table 2). similarly, a fridge or freezer exclusively dedicated to store perishable gf food was not found in 80% (n=16) of canteens, although a separation from gluten-containing products was ensured in the 65% (n=13) of cases. when locals and equipment/tools were shared and used for both gf and non-gf foods, a formal registration of the sanitation procedure before use was not present for 75% (n=15) of the facilities. furthermore, 20% (n=4) of fsos declared to do not regularly practice hand washing and control of the uniform in food administration, and 5% (n=1) stated to do not identify meals/dishes intended or not intended for cd consumers (table 2). assessing hygienic practices and training, 90% (n=18) did not comply with the requirements of clothes dedicated for gf food and retained in distinct cabinets. furthermore, 60% (n=12) did not have a formalized procedure on management of staff clothing, and washbasins were present in the 35% (n=7). at time of the inspection, 45% (n=9) of fsos have not attended a certified training course on cd and issues related to gf preparation and administration (table 2). based on the responses provided for the checklist, the score ranged between 6 and 19 (ideal score=23), and the majority scored 17 (n=4, 20%), followed by 10 and 7 (each n=3, 15%). 4. conclusion health protection of cd individuals is an important goal, especially for most sensitive categories, such as children, patients admitted at hospitals, and people of school age who represent a vulnerable young-adult group (forleo et al., 2017; tamburro et al., 2017). in this study, results from 20 fsos revealed knowledge gaps on the basic and general theories about cd, as well as the lack of best practices for control risk of gluten contamination. part of fsos was not aware that consumption of some products could lead to high risk, while other naturally gf food were erroneously considered as forbidden. similar findings were found in other studies (karajeh et al., 2005; young and thaivalappil, 2018), reporting that food service personnel were less knowledgeable on cd than general population, underlining a paucity of tailored education that should be addressed through specific training. furthermore, concerns emerged about the beliefs of some fsos that cd subjects can introduce small amounts of gluten with diet that, however, it should not exceed 20 mg/kg (regulation (eu) no 609/2013). moreover, the protracted intake of products contaminated with gluten traces may cause persistent intestinal damage and symptoms in non-responsive cd patients, who fail to ever respond to gf diet, or have recurrence/relapse of symptoms despite the gf diet (hollon et al., 2013). while fsos were aware of the importance to use equipment and utensils in different time for gf and gluten-containing food, inappropriate behaviors were observed for special coat in gf meals preparation. in fact, although kitchen equipment and utensils used for glutencontaining foods may not pose a high risk for cd patients, cross-contamination during food preparation or cooking can occur, and it should be avoided cleaning utensils, and washing hands/surfaces regularly (studerus et al., 2018). ital. j. food sci., vol. 32, 2020 477 the complete removal of gluten from diet is difficult, being a pervasive nutrient that may contaminate gf products along the production chain (lee et al., 2014). recently, a systematic review on cross-contamination from gluten higher than 20 ppm in gf food in europe (falcomer et al., 2018) has revealed percentages of contamination ranging from 10%-13% in the united kingdom and sweden (storsrud et al., 2003; mcintosh et al., 2011) to 56%-70% in finland and spain (collin et al., 2004; hernando et al., 2008). one study conducted in italy did not report gluten contamination among gf foods tested (manfredi et al., 2015). quantification of gluten is difficult, being a combination of different components (microheterogeneity) classified as gliadins, glutenins, globulins and albumins, whose measure is unpractical (verma et al., 2017), especially in processed foods (diaz-amigo and popping, 2012). nonetheless, several approaches for detecting gluten proteins are available, such as immunological tests mainly through elisa kits, proteomic analysis with mass spectrometry, and dna-based methods applying polymerase chain reaction (haraszi et al., 2011). however, none of these methods is considered universally acceptable for a high sensitive detection, showing each technology advantages and disadvantages (slot et al., 2016). our results from checklist showed a non-optimal compliance with structural and procedural requirements for warranting food safety value to cd subjects, as well as a lack of adherence to hygiene principles and in relation to a continuing education on gf diet, indicating that targeted and effective interventions are necessary. like our survey, a retrospective study analyzing characteristics of the gf food chain in a school service in an italian region (bioletti et al., 2016) reported that 71% of sampled schools were inadequate for at least one of the production stages. certainly, food separation for cd people has to be handled in order to reduce the chance of cross-contamination. at present, it is managed as strictly as for allergic subjects, and whether this approach is necessary remains to be tested in prospective studies. the same oven for gluten-containing and gf food can be used, as long as the two types are not baked at the same time (bianchi et al., 2018); when specific requirements are complied, also the simultaneous cooking can be a safe procedure (vincentini et al., 2016). nearly half of recruited fsos in our survey has not attended a training course on cd and related issues, similarly to another study reporting that half of chefs had not received formal training (schultz et al., 2017). indeed, a comprehensive and specialized training has been recommended for fsos (lee and xu, 2015; shafie and azman, 2015; radke et al., 2016), and interventions to improve knowledge and practice of food service personnel should be implemented (young and thaivalappil, 2018). moreover, to meet gf food quality specifications, compliance with basic food safety concepts at all stages of product life cycle is decisive for ensuring harmless foods for celiacs (bioletti et al., 2016), as well as appropriateness of a haccp plan (petruzzelli et al., 2014). in conclusion, our study remark the need to provide effective education and proper resources for food service establishment personnel to gain knowledge and strengthen awareness on gf foods, and improve or enhance practical skills, attending courses conceived to assess their ability for safely serving celiac consumers. in addition, good manufacturing practices have a positive effect on gf foods production, and are useful to identify priority areas for improving comprehension of cd issues and practices among fsos, to prevent accidental gluten 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m., ripabelli g., forleo m.b. and sammarco m.l. 2017. dietary behaviours and awareness of seasonal food among college students in central italy. ital. j. food sci. 29:667‒680. verma a.k., gatti s., galeazzi t., monachesi c., padella l., baldo g.d., annibali r., lionetti e. and catassi c. 2017. gluten contamination in naturally or labeled gluten-free products marketed in italy. nutrients. 9:e115. vincentini o., izzo m., maialetti f., gonnelli e., neuhold s. and silano m. 2016. risk of cross-contact for gluten-free pizzas in shared-production restaurants in relation to oven cooking procedures. j. food prot. 79:1642-1646. young i. and thaivalappil a. 2018. a systematic review and meta-regression of the knowledge, practices, and training of restaurant and food service personnel toward food allergies and celiac disease. plos one. 13:e0203496. paper received august 7, 2019 accepted february 5, 2020 ijfs#1666_bozza ital. j. food sci., vol. 32, 2020 528 paper engineering properties of two hazelnuts varieties and its kernel relation to harvest and threshing k.ç. selvi*, e. yesiloğlu cevher and h. sauk ondokuz mayıs university, faculty of agriculture, agricultural machinery and technologies department, 55139 samsun, turkey email: kcselvi74@gmail.com abstract in this research, several engineering properties of two hazelnut cultivars (palaz and çakıldak varieties) were determined and compared in terms of linear dimensions, mass, sphericity, surface area, projected area, true and bulk densities, porosity, repose angle, shell ratio, terminal velocity, rupture force, energy, deformation and drag coefficient. these properties are necessary for the design of much equipment for harvesting, processing, and transportation, sorting, separating and packing. also, rupture force and deformations were determined which are the most discriminant parameters that can be used to describe the behavior under compression. in both cultivars, these values were also determined within the kernels. keywords: hazelnut, pomological properties, strength properties ital. j. food sci., vol. 32, 2020 529 1. introduction hazelnut is the nut of the hazel and therefore includes any of the nuts deriving from species of the genus corylus, especially the nuts of the species corylus avellana. it is also known as cobnut or filbert nut according to species (martin et al., 2014). a cob is roughly spherical to oval, about 15-25 millimeters (0.59-0.98 in) long and 10-15 millimeters (0.39-0.59 in) in diameter, with an outer fibrous husk surrounding a smooth shell (anonymous, 2016a) it has been mentioned in historical documents that hazelnut was produced 2300 years ago in the black sea coast on the north of turkey and it is known that hazelnut has been exported from turkey to other countries for the last 6 centuries. turkey, which is one of the few countries in the world with favorable weather conditions for hazelnut production, accounts for 75% of the global production and 70-75% of the exportation (anonymous, 2016b). the main hazelnut producing countries in the world are turkey, italy, spain, usa and greece. although hazelnut is also produced in the former soviet union, iran, romania and france, these countries do not have a major input in the world hazelnut trade. turkey has an average production of 550,000 tons of shelled nuts in recent years. on the other hand, the production of italy and georgia, additional important producing countries, varies between 100,000-125,000 tons and 40,000-80,000 tons respectively (anonymous, 2018). the following hazelnut varieties are cultivated in turkey: tombul, palaz, cakildak, kara, fosa, min-cane, uzunmusa, kan, kargalak, cavcava, sivri, aci, kus, yuvarlak badem and yassi badem. they differ from each other in some properties (ozdemir and akinci, 2004). a specific know-ledge of some hazelnut engineering properties such as linear dimensions, shapes, porosity, volume, density, terminal velocity, rupture force, etc. and the variation between the hazelnut varieties is required to design of hazelnut processing instrument. the running of many types of machines is influenced accurately by the size and shape of the fruit enterer, and so in order to study a given process should be described accurately. for example, sphericity is one of the most important characteristics as it greatly affects the processability of hazelnuts for the food industry. for this reason, hazelnut varieties with better sphericity need to be grown more (mohsenin, 1980). the volume and density of agro-food products play an important role in applications such as design of silos, drying, mechanical compaction, stability of pellets and wafers, seperation and grading, evaluation of maturity, or quality evaluation (glinski et al., 2011). terminal velocity and drag coefficient plays also a significant assignment for the separation, the pneumatic conveying off goods and the cleaning foreign materials (guner, 2007). similarly, the rupture force is also important and indicates failure over a significant. in mechanical processing of the fruits, most of the damage occurs in the harvesting and threshing as well as mechanical conveying and other equipment (ozdemir and akinci, 2004). for example; dynamic forces during fruit transport and handling cause by far the most bruise damage (zeebroeck et al., 2007). the evaluation of mechanical properties of hazelnuts (whole fruit, shell and kernel) has been developed over the past years with the objectives to obtain industrial processes and improve the use of hazelnuts as food ingredient. the experimental characterization of shells and kernels is a challenging topic to improve the quality of the final product. many literature papers describe procedures to find the mechanical properties of raw and roasted kernels (braga et al., 1999; aydin, 2002; alasalvar et al., 2003; demir and cronin, 2004; özdemir and akinci 2004; ghirardello et al., 2009; delprete and sesana 2014) and the experimental testing on hazelnuts generally relies on compressive testing of kernel and shell by means of ital. j. food sci., vol. 32, 2020 530 universal testing machines. in addition to these studies, some papers (e.g. güner et al., 2003; koyuncu et al., 2004; vursavuş and özgüven, 2005; valentini et al., 2006,) have experimental values of the compressive load needed to crack the shell of hazelnuts, walnuts and pine nuts. compressive force-crosshead displacement curves are widely used to measure textural properties in food products (carcel et al., 2012): initial slope, maximum force, energy until failure and other curve-related parameters have been described and correlated with textural parameters of hazelnuts. the physical characteristics of the hazelnut kernel have an important role on the crispness and crunchiness sensory parameters especially on the roasted nuts (saklar et al., 1999) and the water activities have direct effects on mechanical characteristic (borges and peleg, 1997). in a test campaign (demir and cronin, 2004), a small rectangular prismatic specimen, including the inner cavity present in the core of each hazelnut, was cut from the whole kernel to simplify the calculation of stress and elastic modulus when a compressive axial force loads the specimen section. again the specimen geometry affected the results and it did not allow obtaining material properties. di matteo et al., (2012) evaluated also some mechanical properties of chemical-peeled hazelnut kernels, such as firmness and rigidity, to study an original industrial process to improve the kernel pellicle removal. a mechanical characterization of whole nut, kernel and shell was conducted (delprete and sesana, 2014) in order to aid the design and construction of selecting machines. these mechanical properties are affected by numerous factors, such as the moisture content and loading direction (chengmao et al., 2017). also, nut shell characteristics, such as hardness and thickness, were measured and correlated to the biological cycle of the nut weevil of curculio nucum (coleoptera: curculionidae) pest and to the damage by its larvae (guidone et al., 2007) stress the importance of physical properties evaluation. there were three aims of this study. the first was to investigate the some pomological (physical) properties of two hazel nut varieties and its kernel widely grown in the turkey. the second aim was to determine of strength properties of nuts and kernels and last aim was to determine of some frictional and aerodynamic properties of nuts and its kernel. 2. materials and methods 2.1. sample preparation and material testing system in this study, two hazelnut varieties (palaz and çakıldak) that chosen randomly were used for all the experiments. the 30 nuts and kernels in four replicates of each variety were tested. samples were supplied from the different hazelnut growers (2015 harvest season; samsun, turkey). the experiments performed as soon as possible after hazelnuts purchased. samples were kept in a refrigerator until analyses were performed. the hazelnuts were cleaned manually to remove all foreign matter, immature, broken or spoilt nuts. these experiments were carried out in the laboratory of the department of agricultural engineering, ondokuz mayıs univesity, samsun. the mechanical properties of hazelnuts under compression load were measured by a lloyd instrument universal testing machine (lloyd instrument lrx plus, lloyd instruments ltd, an amatek company). the device has three main parts: moving head, driving unit and data acquisition system (load cell, computer and connections and nexygen plus software) (fig. 1). ital. j. food sci., vol. 32, 2020 531 figure 1. lloyd instrument universal testing machine. 2.2. determination of some pomological (physical) properties of nuts and kernels the initial moisture content of hazelnut varieties (palaz and çakıldak) were determined by using a standard method and were found to vary between 6.38-7.38% and 6.52-7.71% db (db = dry basis) respectively (usda, 1970). to arrange the average linear dimensions (length (l), width (w) and thickness (t)) of the hazelnuts cultivars, a sample of 30 hazelnuts were randomly selected and the dimensions and mass of each hazel nuts used were determined. the dimensions of the hazelnuts were measured with a digital caliper, which had an accuracy of 0.01 mm. the geometric mean diameter, surface area and the sphericity of the hazelnuts were calculated by using the following relationships (mohsenin, 1980): dg =(lwt)1/3 (1) ɸ=dg/l*100 (2) s=π.dg2 (3) where, dg is the geometric mean diameter in mm; ɸ is the sphericity in %; s is surface area in mm2 and l is the length (mm), w is the width (mm) and t is the thickness (mm) (fig. 2). ital. j. food sci., vol. 32, 2020 532 figure 2. three dimensions of nut. sample mass (m) and thousand mass (m1000) were measured by using a digital balance with a sensitivity of 0.001 g. the fruit mass was determined on 30 randomly selected hazelnuts and kernels and converted to a thousand mass. also, shell ratio (rs) was calculated by the measurement of nut mass (m) and shell mass (ms) (özdemir and akinci, 2004). projected area (p) (y axes) was determined from the pictures of hazelnuts and kernels which were taken by a digital camera (canon 600 d), in comparison with the reference area to the sample area by using the sigma scan pro 5 program. the true density (ρk), were determined using the liquid displacement method and the bulk density (ρb) was determined with a weight per hectoliters tester which has calibrated in kilogram per hectoliters (deshpande and ojha, 1993; aydin, 2002; demir et al., 2002). the porosity was determined by the following equation ε=1-(ρb-ρk) (4) where (ρb) is bulk density and (ρk), is true density in kgm-3 (mohsenin, 1980; sitkei, 1986). 2.3. determination of strength properties of nuts and kernels to determine the strength a property of hazelnuts and kernels, biological material test device (lloyd instrument universal testing machine) was used (fig. 1). in this study, hazelnuts and kernels were compressed between two parallel plates at a constant rate of 10 mmmin-1 based on the preliminary tests. rupture force and deformation were determined from the force-deformation curve, where there is a sudden drop in force. to arrange the effect of loading positions on strength properties a coordinate system describing the three main compression positions of hazelnut and kernels is shown in fig. 3. the energy absorbed in rupture point for was determined from the diagram by measuring the area under the force-deformation curves. ital. j. food sci., vol. 32, 2020 533 figure 3. applied force axis of nuts. 2.4. determination of some frictional and aerodynamic properties nuts and its kernels the determination of the angle of repose (φ) of nuts and kernels was used a funnel tube (smallest diameter 50 mm, biggest diameter 150 mm and height 300 mm) and a bow with discharge gate at the bottom. after filling the box with sample, the gate was quickly removed. the height of fruit pile above the floor (h) and the diameter of the heap of sample (r) was measured and used to determine the angle of repose. the angle of repose was calculated with the measurement of the height (h) of conical shape at the center and radius (r) of the free samples over the surface (ertekin et al., 2006) φ=tan-1(h/r) (5) terminal velocity was determined using a wind tunnel. for each test, a sample (nut, kernel) was dropped into the air stream from the top of the wind tunnel, and air was blown up the column to suspend the material in the air stream. the air velocity near the location of the sample suspension was measured by a digital hot-wire anemometer with an accuracy of 0.1 ms-1. in addition, the drag coefficient was calculated as following equation (mohsenin 1980) 𝐶𝑑 = 2𝑚𝑔'𝜌𝑝 − 𝜌𝑓, 𝜌𝑝𝜌𝑓𝐴𝑝𝑉𝑡 2 (6) where: ap is projected area of the particle (m2), cd is drag coefficient (-), g is acceleration due to gravity (9.81 ms-2), m is mass of samples (kg), vt is terminal velocity (ms-1), ρp is density of samples (kgm-3) and ρf is density of air (1.206 kgm-3) 3. results and discussion frequency distributions of the dimensional properties of two hazelnut cultivars are given in table 1. ital. j. food sci., vol. 32, 2020 534 table 1. frequency distributions of the dimensional properties of two hazelnut cultivars. palaz çakıldak dimensions frequency nuts kernels nuts kernels length (mm) %86.7 16.60-18.27 12.37-16.26 17.25-19.64 13.36-17.59 width (mm) 19.14-20.94 13.12-16.78 15.42-18.66 11.09-14.78 thickness (mm) 16.35-17.96 11.98-15.27 14.38-16.40 9.89-13.49 according to the results of frequency distributions of the dimensional properties of two hazelnut and kernels cultivars; about 86.7% of the hazelnut were between 16.60 and 19.64 mm in length, 15.42 and 20.94 mm in width and 14.38 and 17.96 mm in thickness for both cultivars. some physical properties of the palaz and çakıldak nuts and kernels are given in tables 2 and 3. the moisture contents were 6.63% and 6.96% for palaz and çakıldak nuts and 4.56% 4.50% for kernel respectively. while mean nut length was 17.73 mm, nut width was 19.89 mm and thickness was 17.08 mm and kernel length was 14.21 mm, kernel width was 14.94 mm and thickness was 13.36 mm for palaz. these values were 18.44 mm, 16.85 mm, 15.20 mm and 14.70 mm, 12.75 mm, 11.82 mm for nuts and kernel of çakıldak, respectively. all sizes of palaz except length are bigger than those of çakıldak. shell ratio (46.72 %) and thickness (1.21 mm) of the palaz were higher than the çakıldak (45.90 %) and (1.10 mm). for the palaz, mean mass of nut and 1000 nuts mass was 1.58 g and 1591.98 g for nuts and 0.79 g and 793.61 g for kernels. the same values of çakıldak were 1.59 g and 1612.18 g for nuts and 0.80 g and 792.23 g for kernels. when the nut mass in this study was compared with previous studies, the mean mass of the fruit was with in normal limits (güner et al., 2003). the average value of the geometric mean diameter was calculated as 18.05 mm for palaz and 16.76 mm for çakıldak nuts, respectively. the same values were 14.12 mm and 13.00 mm for palaz and çakıldak kernels. sphericity is an expression of a shape of a solid relative to that of a sphere of the same volume while the aspect ratio relates the width to the length of the fruit which is an indicative of its tendency toward being oblong in shape (ertekin et al., 2006). these values were 1.042 and 0.91 for nuts and 1.37 and 0.89 for palaz and çakıldak, respectively. bulk densities for nuts and kernels of palaz were between 392 kg/m3 and 450 kg/m3 and 482 kg/m3 and 578 kg/m3. the same values were between 406 kg/m3 and 468 kg/m3 and 483 kg/m3 and 522 kg/m3 for çakıldak. the porosity ranged between 53.50% and 61.30% for palaz nuts and 54.70% and 62.70% for çakıldak nuts. the same values were between 58.70% and 64.30% for palaz and 54.70% and 62.70% for çakıldak for kernels. bulk densities and porosities values were similar with the literature values (ozdemir and akinci, 2004). the mean surface and the projected area of the palaz nut were found 889.17 mm2 and 221.762 mm2. for çakıldak variety the same values were calculated as an 883.92 mm2 and 211.740 mm2 respectively. when the same properties were examined for the kernel of the palaz and çakıldak, the surface and the projected area were found 663.21 mm2 and 167.23 mm2, 533.03 mm2 and 132.20 mm2, respectively. the mean and sd values of some mechanical properties of the two nut cultivars obtained from the measurements and calculations at moisture contents of 6.63% and 6.96% (w.b.) are shown in table 4. ital. j. food sci., vol. 32, 2020 535 table 2. some physical properties of nuts. cultivar palaz çakıldak min max mean s.d min max mean s.d length (mm) 15.53 18.43 17.33 0.024 15.06 20.01 18.44 0.045 width (mm) 18.71 21.40 19.89 0.025 15.10 18.74 16.85 0.035 thickness (mm) 16.21 18.21 17.08 0.021 13.92 16.42 15.20 0.024 shell ratio (%) 44.33 48.71 46.72 0.230 44.69 46.78 45.90 0.215 shell thickness (mm) 1.02 1.66 1.213 0.005 0.90 1.39 1.10 0.004 geo. mean dia. (mm) 16.82 18.72 18.05 0.017 15.73 17.99 16.76 0.022 sphericity 0.99 1.109 1.042 0.001 0.86 1.062 0.91 0.002 surface area (mm2) 889.17 1100.34 1024.65 1.957 777.66 1016.50 883.92 2.315 projected area (mm2) 221.76 298.84 257.23 0.785 201.73 247.13 211.74 0.598 true density (kgm-3) 714.23 801.42 748.65 1.235 705.39 753.54 721.98 1.383 bulk density(kgm-3) 392.64 450.32 412.42 0.983 406.43 468.24 436.71 0.871 porosity 0.53 0.613 0.587 0.005 0.54 0.62 0.59 0.004 mass (g) 1.58 1.753 1.627 0.010 1.57 1.63 1.58 0.011 1000 mass (g) 1591.98 1739.86 1669.25 12.26 1568.42 1634.83 1612.18 10.54 table 3. some physical properties of kernels. cultivar palaz çakıldak min max mean s.d min max mean s.d lenght (mm) 12.08 17.010 14.21 0.057 10.16 18.87 14.70 0.060 width (mm) 12.57 17.22 14.94 0.046 9.31 15.00 12.75 0.046 thickness (mm) 11.26 15.79 13.36 0.037 8.82 13.87 11.82 0.046 geo. mean dia. (mm) 12.88 15.52 14.12 0.025 10.83 14.78 13.00 0.033 sphericity 0.84 1.15 1.37 0.004 0.69 1.18 0.89 0.004 surface area (mm2) 521.30 756.30 663.21 2.213 368.77 686.45 533.03 2.676 projected area (mm2) 140.79 198.50 167.23 0.898 112.64 178.06 132.20 0.785 true density (kgm-3) 817.12 1020.12 984.42 3.452 824.56 978.76 845.31 4.349 bulk density (kgm-3) 482.17 578.67 523.23 1.924 483.33 522.35 489.60 1.345 porosity 0.58 0.64 0.61 0.003 0.54 0.62 0.59 0.005 mass (g) 0.78 0.92 0.842 0.012 0.72 0.84 0.79 0.015 1000 mass (g) 793.61 934.51 869.73 10.25 735.25 865.79 792.23 10.743 the force values required to initiate nut rupture were obtained from the same experiments. from table 4, it is seen that the rupture force was higher along the y-axis compared with the xand z-axes for both cultivars. this could be because the area of contact between the nut pit and compression plates was larger along the y-axis than those along the xand z-axes. this is in agreement with the finding of aktaş et al. (2007). ital. j. food sci., vol. 32, 2020 536 table 4. some mechanical properties of nuts. palaz çakıldak min max mean s.d min max mean s.d z axes rupture force (n) 186.94 266.79 217.96 2.742 105.61 289.50 211.24 6.733 deformation (mm) 0.70 1.19 1.01 0.018 0.59 1.45 0.97 0.029 energy (j) 0.07 0.16 0.12 0.013 0.03 0.21 0.11 0.006 hardness (n/mm) 166.39 265.12 220.02 3.52 175.59 265.08 216.96 3.24 x axes rupture force (n) 191.53 312.91 251.04 3.86 129.77 313.34 222.68 6.449 deformation(mm) 0.57 0.96 0.78 0.013 0.53 1.22 0.88 0.023 energy (j) 0.055 0.160 0.100 0.004 0.034 0.189 0.10 0.005 hardness (n/mm) 286.54 381.08 324.43 2.89 184.27 328.93 257.28 4.988 y axes rupture force (n) 191.81 339.39 274.88 5.341 155.61 396.46 239.57 7.248 deformation(mm) 0.56 1.32 0.97 0.023 0.45 2.77 1.02 0.067 energy (j) 0.051 0.23 0.13 0.005 0.04 0.27 0.10 0.007 hardness (n/mm) 244.47 342.78 288.25 3.442 95.32 408.73 273.16 8.272 in addition to rupture force, energy values, deformation and hardness were calculated as some mechanical properties. maximum and minimum energy values except min energy for çakıldak cultivar (0.034 j-x axes) were found along the y axes as 0.23 j for the palaz and 0.27 j for the çakıldak and 0.05 j for the palaz. depending on rupture force, maximum deformation was obtained on the y axes as a 1.32 mm and 2.77 mm for the palaz and çakıldak, respectively. the response of hardness to loading position in the x axes was higher than the hardness in the z and y axes loading position for the palaz, while hardness value in the y axes was max for çakıldak. this difference among the two type nut cultivars may be due to the shell properties of the varieties. terminal velocity, drag coefficient, repose angel and static and dynamic coefficient of the two hazelnut varieties and their kernels were given in table 5. table 5. terminal velocity, drag coefficient and repose angel of the palaz and the çakıldak. cultivar palaz çakıldak min max mean s.d min max mean s.d n ut terminal velocity (m/s) 11.26 13.89 13.15 0.744 11.84 13.67 13.30 0.544 drag coefficient 0.351 0.389 0.362 0.012 0.358 0.391 0.367 0.011 repose angel (°) 22.615 23.12 22.814 0.175 22.578 22.845 22.751 0.090 k er ne l terminal velocity (m/s) 11.77 14.05 13.66 0.672 12.73 13.84 13.70 0.309 drag coefficient 0.363 0.377 0.369 0.005 0.370 0.388 0.377 0.005 repose angel (°) 24.231 25.813 24.934 0.464 23.887 24.867 24.53 0.389 the terminal velocity of hazelnuts and kernels, the values ranged from 13.26 to 13.89 m/s and from 11.77 to 14.05 m/s, respectively. the terminal velocity of kernels is higher than ital. j. food sci., vol. 32, 2020 537 that of hazelnuts. these differences in results can be attributed to the increase in mass of the individual nut or the kernel per unit when their frontal areas were presented to the air stream to suspend the material. the repose angle with hazelnuts and kernels were varying from 22.58° to 23.12° and from 23.89° to 25.81° respectively. this is due to the higher sphericity of hazelnuts and kernels which results from allowing them to slide and roll on each other. 4. conclusion several physical and mechanical properties of the two (the palaz and the çakıldak) hazelnut varieties were described in order to design a specific machine for harvesting, threshing, conveying, cleaning, separating, storing, etc. according to these results. some remarkable points for the study can be summarised as follows. 1. moisture content was 6.63% for hazelnut and 4.56% for kernel of the palaz cultivar and 6.96% for hazelnut and 4.50% for kernel of the çakıldak cultivar. the average hazelnut length, width, thickness and geometric diameter were 17.34 mm, 19.90 mm, 17.09 mm and 18.06 mm for hazelnut and 14.21 mm, 14.94 mm, 13.36 mm and 14.13 mm for kernels of the palaz, respectively. the average hazelnut length, width, thickness and geometric diameter were 18.45 mm, 19.85 mm, 15.21 mm and 16.77 mm for hazelnut and 14.71 mm, 12.75 mm, 11.82 mm and 13.00 mm for kernels of the çakıldak, respectively. the average 1000 mass for hazelnut and kernels were 1669.25 g and 869.74 g for the palaz and 1612.18 g, 792.24 g for the çakıldak, respectively. 2. the mean true and bulk density, angle of repose and terminal velocity of the palaz hazelnut and kernel were 748 kg/m3 984 kg/m3, 412 kg/m3 523 kg/m3, 22.81° 24.93° and 13.15 m/s 13.66 m/s, respectively. the same values were 721 kg/m3 845 kg/m3, 436 kg/m3 489 kg/m3, 22.75° 24.53°and 13.30 m/s 13.70 m/s, respectively. 3. the sphericity, surface area, projected area and porosity of the hazelnut and kernels were 1.04 % 1.37 %, 1025 mm2 663 mm2, 257 mm2 167 mm2 and 0.59 0.61 for the palaz cultivar, respectively. the same values were 0.91 % 0.89 %, 883 mm2 533 mm2, 211 mm2 132 mm2 and 0.59 0.59 for the çakıldak cultivar, respectively. 4. the rupture force was higher along the y-axis compared with the xand z-axes for both cultivars. the biggest mean deformation value was 1.01 mm on z axes for the palaz and 1.023 mm on y 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publishers. mohsenin n.n. 1970. physical properties of plant and animal materials. gordon and breach, new york. özdemir f. and akıncı i̇. 2004. physical and nutritional properties of four major commercial turkish hazelnut varieties. journal of food engineering 63:341-347. saklar s., ungan s. and katnas s. 1999. instrumental crispness and crunchiness of roasted hazelnuts and correlations with sensory assessment. journal of food science and technology 64:101-1019. ital. j. food sci., vol. 32, 2020 539 usda. 1970. official grain standards of the united states. us department of agricultural consumer and marketing service, grain division, revised. valentini n., rolle l., st evigny c. and zeppa g. 2006. mechanical behaviour of hazelnuts used for table consumption under compression loading. journal of the science of food and agriculture 86:1257-1262. vursavuş k. and özgüven f. 2005. fracture resistance of pine nut to compressive loading. biosystems engineering 90:185-191. zeebroeck m.v., linden v.v., ramon h., baerdemaeker j.d., nicolai b.m. and tijskens e. 2007. impact damage of apples during transport and handling. postharvest biology and technology 45:157-167. paper received august 14, 2019 accepted march 13, 2020 paper ital. j. food sci., vol. 27 2015 181 keywords: fresh red meat consumption, principal component, k-means cluster and multiple regression analyses, sensory and hedonic quality attributes how sensory and hedonic quality attributes affect fresh red meat consumption decision of turkish consumers? y. topcu*, a.s. uzundumlu and d. baran department of agricultural economics, college of agriculture, ataturk university, 25240-erzurum, turkey *corresponding author: tel. +90 442 2311393, yavuztopcu@atauni.edu.tr; ytopcu25@hotmail.com, ytopcu025@gmail.com abstract the aim of the study is to explore how the sensory and hedonic quality attributes of the fresh red meat affect its consumption preference and amounts of turkish consumers. the data obtained from 385 households in erzurum were used for principal component (pca), k-means cluster and multiple regression/correlation (mrc) analyses. the results of the study highlighted considerably that the sensory quality attributes on their consumption preference had a much bigger effect than the hedonic ones for each cluster. however, its price and their income accepted as the important indicators of the hedonic ones, but a much lower impact on all clusters. mailto:yavuztopcu@atauni.edu.tr mailto:ytopcu25@hotmail.com mailto:ytopcu025@gmail.com 182 ital. j. food sci., vol. 27 2015 introduction in recent years, the increases of the red meat prices due to the contractions in the meat supply covering the production, processing and marketing of the meat and the meat products obliged the political units of the government to import live red meat materials, and thus they have provided stabilization at some levels for the meat supply and price (yavuz et al., 2013). however, the origins of the meat sources among the consumers consuming the fresh red meat have been considered as an important decision factor. a few portion of them, therefore, exhibited the buying attitude and behaviors within a moderate consumption trend of those preferred the meat sources imported while the others formed the purchase models with a positive motivation in the red meat consumption preferences based on the national meat sources. there was a string relationship between the origin of the red meat sources as an indicator of the consumers’ purchase decisions and both the sensory quality including in its intrinsic quality attributes and the hedonic quality consisting of its extrinsic quality attributes (bernues et al., 2003). the strong motivational effects of their individual, demographic, socioeconomic, psychological and health characteristics, on the other hand, are among the primary preference factors, as well (topcu, 2012). turkish consumers’ purchase decisions based on the food safety and quality of the fresh red meat have changed considerably due to its confidential and sensory quality attributes affected directly by microbiological insecurity (dioxin effect, bse and salmonella diseases) of foreignorigined red meat sources, and by the contamination risks with various pollutants (antibiotic and hormones) in the last years (topcu, 2015; topcu and uzundumlu, 2012). they have also concerned about deterioration of their health conditions with the meat consumption of the imported animals being exposed to genetic manipulations, and then about inheriting to the future generations of the negative phenomenon (topcu, 2012). in addition to imported live animal sources, presence of unhealthy fresh red meats penetrating to domestic markets uncontrollably and illegally, unknown origin, exposing to some additives at levels threatening human health, and the pollution, contamination and microbiological insecurity of the manufacturing meats in the livestock farms and meat processing facilities under unhygienic conditions have caused the consumers to exhibit more sensitive purchase attitude and behaviours by considering the sensory quality attributes (yavuz et al., 2013). on the other hand, not only digestive and coronary heart diseases caused by obesity based on the passive lifestyles revealed the communication and information age but also chronic illnesses resulting from nutrition with the intensive protein diets consisting of the red meats and the impacts of the life circle of the mature people have gradually reduced the consumers’ red meat consumption tendencies, and thus nowadays their purchase decisions towards the red meats have been changing habitually (realini et al., 2013; topcu, 2012). the analyses of the customers reviews/assessments about the intrinsic quality attributes having a direct relationship between the sensory quality and confidential attributes (food quality measured by the chemical and microbiological tests) of the red meats such as the structural and visual characteristics (realini et al., 2013; mccarty et al., 2003) are of a much more importance. on the other hand, they also focus on the extrinsic quality attributes correlating with the hedonic quality attributes such as the actual product image, the cost to customer, the origin of the meat source, disposable income (troy and kerry, 2010; burnues et al., 2003; mccarty et al., 2003), and reflecting the consumers’ individual characteristics and determining their purchase decisions about the fresh red meat. therefore, eliminating the factors affecting negatively their consumption satisfaction and loyalty accepted at the focal point of the production, consumption and marketing activities, determining the main attributes accelerating the consumption trends of the homogenous target consumers masses, and then designing the marketing tactic and strategies for them could provide the important advantages in terms of the efficiency of the production resources and the maximization of the expected utilities on all market dynamics. the diet meeting the main requirements of the people by providing the metabolic energy needed for biological organisms consists of the plant and animal foods, and thus they must be consumed to sustain a healthy and balanced life of the people at an adequate level (topcu and uzundumlu, 2012). in particular, the animal-derived proteins such as the meat, milk, eggs, cheese being of a sufficient level for the essential amino acids are also suitable in terms of digestion. about 75-80% of high-quality animal-derived proteins are transformed into the body proteins. in contrast, plant-derived proteins classified as lowquality protein have an insufficient level of some essential amino acids, and it is benefited from only 40% of their proteins since their digestion is fairly difficult (topcu, 2012). a person must consume 70 gr proteins per day for an adequate and balanced diet; therefore, it must be at least half of animal origin protein (seker et al., 2011). the consumption amounts of the animal origin foodstuffs per capita are very low in turkey, but that of the plant-derived foods is greater than that of the developed countries (karkacier, 2000). if people fed with cereal-based food products give more weight into ital. j. food sci., vol. 27 2015 183 the animal origin ones in turkey, and they gain the proper eating habits, it could be contributed significantly to the improvement of the life quality of the community. today, the consumption levels of the animal-origin products are considered as a development indicator of the countries, and thus as their socioeconomic structures improves, the consumption amount of the protein foods increases in contrast to reduce that of the carbohydrate foods (yaylak et al., 2010). as a result of all this, the meat includes an important part of people’s daily diets in usa, eu and other developed countries, and it meets 15%, 40% and 20% of the energy, protein and fat requirements per day, respectively, but its consumption fluctuates significantly in various regions of the world (daniel et al., 2011). for example, while the annual meat consumption amounts per capita in 2011 were calculated as 142, 125, 82 and 80 kg in austria, usa, germany and uk, representing the leader countries in the meat production and consumption, respectively, it was only 12 kg in turkey. according to the data obtained from the research area (erzurum), on the other hand, the annual meat consumption amount per capita calculated as 17.8 kg in 2008, 12.5 and 11.5 kg in 2011 and 2012 were found (topcu and uzundumlu, 2012). the substitution of the imported red meats instead of the red meat derived from the animal husbandry based on a quality pastures and a wide variety flora having a significant impact on the red meat quality caused the consumers’ preferences shift to other meat groups in the research area, and their red meat consumption trends decreased significantly by influencing negatively its sensory and hedonic quality attributes (topcu and uzundumlu, 2012). as stated above, the annual red meat consumption amount per capita in the research area and turkey was about 5-8 times less than that in developed countries, but this difference continued to increase steadily, in the last five years. especially, there has been an increasing trend towards the poultry meat consumption due to the dissatisfactions of not only the sensory quality attributes at the meat production, manufacture and retail levels based on the imported meat materials but also the hedonic quality attributes pointing the actual product images. however, it has been experienced the significant changes such as the region origin and the types of the red meat sources in the consumption preferences of the consumers obtained from the red meats about 58, 34 and 36% of the total meat consumption in the developed countries, turkey and the research area, respectively (mcafee et al., 2010; lichtenstein et al., 2006). the red meat consumption amounts at much low levels in turkey and the study area could result from not only the sensory and hedonic quality attributes of the fresh red meat but also the socioeconomic and individual attitudes of the consumers and the confidence to the manufacturer, marketer and retailers (realini et al., 2013; troy and kerry, 2010; mccarty et al., 2003). however, the supply amount of the fresh red meat in the research area is higher than its demand amount due to its high sensory quality attributes resulting from the advantages of the organic cattle fattening farming based on the quality pastures and rich flora varieties under the agroecological and topographical properties at the high mountainous areas of the region (topcu and uzundumlu, 2012). the consumers, therefore, considering the advantages of both the hedonic quality attributes reflecting the images of the core and actual products focused on the numerous factors of the marketing mix and higher sensory quality attributes have preferred and consumed intensively them. as a result, the consumers residing in the research region and migrating from the research area to the different regions of turkey have not only maintained their demands for the red meats with the region of the origin to provide much higher main utility but also contributed to the regional/rural development through the improvement of the farmer families’ life qualities. on the other hand, the inefficient policies implemented to meet the fresh meat deficit without effect on the rural/regional developments and the sensory quality attributes affecting negatively the consumer satisfaction with the penetration of the imported red meat to domestic markets caused by the supply contraction of the red meat have led the consumers with dissatisfactions to reflect the different purchase decisions, in the last decade. all these individual responses of the consumers about the fresh red meat and their purchase decisions have emerged as the acceptable results of the relationships between the sensory and hedonic quality attributes. this study, therefore, was designed to reach all the objectives mentioned above. in this scope, the main aims of the study are to determine how the sensory and hedonic quality attributes affect the fresh red meat consumption decisions of turkish consumers; and then to construct the target homogenous consumer segments based on their consumption frequencies, and finally to analyze the effectiveness of the factors effecting on their consumption amounts. material and methods material the preliminary data used in this study were obtained from a survey consisting of turkish consumers’ attitude and behaviors with respect to the fresh red meat consumption de184 ital. j. food sci., vol. 27 2015 cisions conducted in erzurum,1 turkey. in order to determine the sample size (selected statistically from the local household consumers through simple random sampling method) population minimizing sample bias and representing the main population correctly, the city center was divided into three districts covering aziziye, palandoken and yakutiye districts with 6.562, 30.022 and 44.075 households at the west, south and north-east parts of erzurum, respectively (anonymous, 2013). methods method used in determination of the sample size in order to calculate the sample size for each district, the following formula was used (topcu et al., 2010). 385 )1(** 2 2 = − = c ppz n where, n = sample size, z = z value (1.96 for 95% confidence level), p = percentage making a choice (0.5 used for sample size needed), c = confidence interval (used 0.05 = ±5). then, based on the population of each district, the weighted sample size and distribution of the surveys for each district were determined proportionally. a total of 385 surveys were distributed with 52, 136 and 197 questionnaires allocated to the aziziye, palandoken and yakutiye districts, respectively. methods used in the preparation of the questionnaires participants of the survey were asked to respond to each statement indicating the significance level of the fresh red meat attributes for them. a liker-type scale was used (where 1 refers to the least important and 5 refer to the most important attribute). table 1 reported that 35 attributes of the fresh red meat consisted of 17 sensory quality attributes and 18 hedonic quality ones (topcu, 2015; topcu and uzundumlu, 2012). of five numeric variables referring to the consumers’ socioeconomic characteristics, on the other hand, two referred to their monthly fresh red meat consumption amount and purchase frequency and three included in their monthly income ($), the price per kg of the fresh red meat ($/kg), and the share of its expenditure within total food one. methods used in the statistics analyses after editing and coding, the primary data were first used in principal component analy1 erzurum (39°45’n latitude and 41°15’e longitude) is located in the north-east region of turkey and is on the silk road. erzurum is one of the biggest provinces in the region with a total population 778.195, and the city centre population is 509.474. 2 a factor extraction method used to form uncorrelated linear combinations of the observed variables. the first component has the maximum variance. successive components explain progressively smaller portions of the variance and are all uncorrelated with each other. pca is used to obtain the initial factor solution. it can be used when there is a single correlation matrix. 3 this method is an orthogonal rotation method that minimizes the number of variables that have high loading on each factor. it simplifies the interpretation of the factors. sis (pca)2 to determine the main factors related to the product attitudes influencing on the consumers’ fresh red meat purchase patterns. pca is a data reduction technique that reduces the number of variables used in an analysis by creating new variables (called factors) that combine redundancy in the data (spss 15.0, 2006). the first step in pca is to determine the number of relevant factors. this was conducted by pca using the varimax rotation method (vrm)3. pca was used initially to identify underlying aspects explaining a correlation among a set of the food product attributes. the purpose of pca was to identify those attributes accounting for a relatively large proportion of the variance in the sample. in the second and final step of the statistical analyses, the main factors obtained from pca were used for k-means cluster and multiple regression/correlation mrc analyses, respectively. in the second step, according to the fresh red meat consumption frequencies, therefore, the target consumers were separated to three homogeneous clusters including in light users (2-3 times per month), medium users (2-3 times per 15 days) and heavy users (2-3 times per week) (topcu, 2012), and then the main factors were allocated to the homogeneous consumer clusters based on the meat consumption frequencies of the target consumers by k-means cluster analysis. in the final step, mrc analysis was used for the measurements of the efficiency of the main factors and socioeconomic variables (independent variables) effecting on the consumers’ meat consumption amounts (dependent variable). in the mrc model taken into consideration the variables related to the main factors obtained from the pca and some socioeconomic characteristics of the consumers, therefore, it was tried to measure the relationships between monthly consumption amount of the fresh red meat per household as a dependent variable and the main factors including in their sensory and hedonic quality attributes as long with the consumers’ socioeconomic characteristics called as the independent variables. in order to test whether or not the normal distribution of the main factors’ group scores deital. j. food sci., vol. 27 2015 185 rived from pca and socioeconomic variables collected from the consumers exhibited, was applied the various transformations techniques, and finally logarithmic transformation one providing the closest distribution to the normal was chosen. on the other hand, the parameter coefficients were estimated by using ordinary least squares (ols). individual and group significance of these coefficients were tested using t and f tests, respectively. in order to evaluate whether to be any econometrical problem among the variables, it was tested the overall multicollinarity and auto-correlation problems by considering the variance-inflating factor (vif) and durbinwatson d statistics, respectively. multicollinearity among variables was detected by calculating (vif) (gujarati, 2005; spss 15.0, 2006). mrc model could be written as following equation: ),,,,,,,,,,,,( idisincmeprcfororgorglocmesdurcosconnutvalimagvisqlycrerismicsafaromaffmcons e= dependent variable fmcons : monthly fresh red meat consumption amount (kg) independent variables aroma : aroma of the fresh red meat (sensory quality attribute) micsaf : microbial safety (associated with sensory quality attributes) creris : chemical residue-related risk (associated with sensory quality attributes) visqly : visible quality (sensory quality attribute) imag : accual fresh meat image (hedonic quality attribute) nutval : nutritional value (associated with sensory quality attributes) coscon : cost to the consumer (hedonic quality attribute) mesdur : meat sources and its durability (associated with hedonic quality attribute) orgloc : local-origined organic meat willingness (hedonic quality attribute) fororg : foreign-origined meat willingness (hedonic quality attribute) meprc : the price of the meat ($) (hedonic quality attribute) disinc : the disposable income (hedonic quality attribute) all analyses were run in sequence with the spss 15.0 statistical software. these techniques were used widely to analyze the meat attributes playing the important roles on the consumers’ consumption preference and amounts in marketing studies (topcu and uzundumlu, 2012; krystallis et al., 2007; diez et al., 2006; oliver et al., 2006; verbeke and vackier, 2004; bernues et al., 2003). results and discussion results of pca related to the sensory and hedonic quality attributes of fresh red meat kaiser normalization (kmo) which compares partial correlation coefficients with observed ones including in the sensory and hedonic quality attributes effecting on the fresh red meat consumption preferences of the turkish consumers was calculated as 0.85, and this means that the data set for the pca was at a perfect level since the test score was greater than 0.5 (table 1). on the other hand, the chi-square value calculated for bartlett’s test of sphericity statistics of their main factors with respect to the consumers’ meat consumption preferences was calculated as 5060.68 (p: 0.000), and thus the unit matrix hypothesis was rejected (p<0.01). the pca using varimax rotation method grouped the thirtyseven variables related to the sensory and hedonic quality attributes for fresh red meat into the ten main factors with eigen-values greater than 1, and the main factors explained the 71% of the total variance (table 1). as shown in table 1, the results of the study showed that aroma (f1) and visible quality (f4) affecting the fresh red meat consumption preferences of turkish consumers by explaining about 29.6 and 5.3% of total variance constituted directly the first and second sensory quality factors, respectively. by having an important impact on the quality attributes of f1 and f4; moreover, microbial safety (f2) and chemical residue-related risk (f3) associated much closely with the sensory quality attributes of the fresh red meat were interpreted as the second and third sensory quality factors indicating about 7.5 and 7.1% of total variance. the same main factors for the sensory quality attributes based on the various similar variables taken into consideration by topcu and uzundumlu (2012), kergoat et al. (2010), troy and kerry (2010), aaslyng et al. (2007), krystallis et al. (2007) were reported in their previous researches, as well. on the other hand, the results of the present study indicated that actual product image (f5); nutritional value (f6), cost to the consumer (f7) and meat sources and durability (f8) of the fresh meat; local-origined organic meat willingness (f9) 186 ital. j. food sci., vol. 27 2015 table 1 the measurement results of pca with regard to the sensory and hedonic quality attributes based on the fresh red meats consumption decisions of turkish consumers. factos interpretions and the variables factor loadings* f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 aroma (f1: aroma) flavour 0.841 0.078 0.121 0.127 0.032 0.127 0.102 0.039 0.015 0.029 succulence 0.831 0.076 0.061 0.138 0.047 0.076 0.014 -0.007 -0.035 -0.014 smell/odour 0.750 0.083 0.115 0.083 0.205 0.051 0.026 0.165 -0.035 -0.038 juiciness 0.705 -0.003 0.086 0.146 0.017 0.178 0.054 0.120 0.099 0.277 visible fat 0.530 0.251 -0.021 0.070 -0.014 0.246 0.217 0.068 0.355 -0.333 microbial safety (f2: micsaf) dioxin scare 0.079 0.880 0.192 0.088 0.103 0.071 0.037 0.008 0.112 -0.019 salmonella disease 0.088 0.875 0.096 0.120 0.096 0.162 0.053 0.040 0.049 0.006 bse 0.081 0.802 0.133 0.151 -0.004 0.130 0.119 0.050 0.044 0.011 trustworthiness 0.090 0.666 0.073 0.119 0.209 0.262 0.163 0.122 -0.188 -0.242 chemical residue-related risk (f3: creris) meat manufacturing methods 0.139 0.161 0.698 0.090 0.178 0.108 0.087 0.086 0.059 -0.013 antibiotic residues 0.128 0.352 0.687 0.022 0.220 0.172 0.010 0.064 -0.111 0.193 hormone residues 0.017 0.449 0.603 0.153 0.092 0.148 0.065 0.156 -0.163 0.122 visible quality (f4: visqly) freshness of the meat 0.346 0.164 0.010 0.789 0.028 0.155 0.085 -0.038 0.032 0.039 meat part obtained from the animal 0.038 -0.007 0.077 0.772 0.084 0.091 0.098 0.211 0.013 0.080 natural structure of the meat 0.197 0.103 0.138 0.762 0.161 0.164 0.074 -0.015 0.064 0.065 the meat colour 0.104 0.234 0.093 0.740 0.149 0.121 0.119 0.154 0.001 -0.078 tenderness 0.140 0.053 0.342 0.527 0.077 -0.012 0.210 0.231 0.237 0.244 accual fresh meat image (f5: imag) brand name 0.179 -0.020 0.239 0.113 0.769 -0.006 0.041 0.021 0.145 0.016 package 0.029 0.142 0.012 0.130 0.672 0.133 0.306 0.274 -0.006 0.080 advertisement -0.066 0.002 0.141 0.181 0.647 0.055 0.152 0.251 0.398 -0.114 free of additives/natural product 0.264 0.144 0.190 0.091 0.627 0.193 0.173 -0.044 -0.030 0.005 labelling 0.134 0.134 0.156 0.068 0.605 0.151 0.474 0.131 -0.151 0.055 nutritional value (f6: nutval) brand name 0.112 0.111 0.200 0.216 0.153 0.832 0.000 0.091 0.070 0.072 package 0.206 0.153 0.230 0.183 0.115 0.820 0.056 0.097 0.114 0.109 advertisement 0.248 0.254 0.147 0.139 0.072 0.764 0.168 0.018 -0.064 0.021 organic product 0.172 0.029 0.188 0.100 0.175 0.754 0.171 0.095 0.064 -0.044 cost to the consumers (f7: coscon) discounts in the meat prices -0.019 0.136 0.220 0.158 0.143 -0.031 0.707 0.065 0.295 0.006 the initial price of the meat 0.120 0.114 0.040 0.069 0.253 0.098 0.702 -0.030 -0.103 -0.029 outlets/sales points of the meat 0.187 -0.034 0.097 0.146 0.154 0.087 0.693 0.238 0.196 0.041 willingness to buy meat sources and its durability (f8: mesdur) beef 0.180 0.068 0.144 0.212 0.002 0.064 0.113 0.777 0.030 -0.037 sheep and goad meats 0.052 0.024 0.036 0.026 0.253 0.046 -0.017 0.747 0.221 0.049 the shelf life of the meats 0.330 0.050 0.245 0.063 0.105 0.104 0.327 0.551 -0.128 -0.024 local-origined organic meat (f9: orgloc) willingness to buy local-orig. meat 0.301 -0.055 0.159 0.124 0.094 0.309 0.020 -0.007 0.593 0.127 willingness to buy organic meat 0.068 0.192 0.076 0.222 0.136 0.239 0.137 0.235 0.582 0.130 willingness to buy foreign-origined meat (f10: fororg) willingness to buy foreign-orig. meat -0.055 0.145 -0.019 0.029 0.107 0.063 0.118 0.113 0.062 0.803 eiqen-values 10.655 2.689 2.554 1.914 1.545 1.452 1.233 1.173 1.119 1.034 share of explained variance (%) 29.598 7.468 7.095 5.318 4.292 4.033 3.426 3.263 3.109 2.872 cumulative share of that (%) 29.598 37.066 44.160 49.478 53.763 57.803 61.229 64.492 67.600 70.472 kmo (kaiser-meyer-olkin) statistic 0.848 bartlett’s test of sphericity [chi square (2, df:630):5060.68](p:0.000) * bold numbers indicated the largest loading for each variable. ital. j. food sci., vol. 27 2015 187 and foreign-origined meat willingness (f10) constructing the hedonic quality attributes of the fresh red meat consisted of about 4.3, 4.0, 3.4, 3.3, 3.1 and 2.9% of total variance, respectively (table 1). when the obtained main factors related to the hedonic quality attributes in the present study were compared with the others presented by the results of the previous researches; while the f5, f6 and f7 factors with respect to the hedonic quality were supported directly with the results of the studies conducted by furnols et al. (2011), krystallis et al. (2007), audebert et al. (2006), bernues et al. (2003) and verbeke and vackier (2004), the f8, f9 and f10 factors focused on the attributes of the hedonic quality were of the strong relationships with the results of those presented by furnols et al. (2011), napolitano et al. (2010), bernues et al. (2003), mccarty et al. (2003). as a result of all these, the sensory quality attributes explaining about 70% of the main factors effecting on the fresh red meat consumption preferences of turkish consumers were of a much bigger influence than the hedonic quality ones. results of cluster analysis related to the sensory and hedonic quality attributes of fresh red meat the sensory and hedonic quality attributes derived from the pca and effecting on the fresh red meat purchasing patterns of turkish consumers were separated into three homogeneous consumers segments through k-means cluster according to their red meat purchasing frequencies including the light, medium and heavy users. the main factors reflecting turkish consumers’ attitude and behaviors impacting on each homogeneous consumers segment were given in table 2. the results of the study showed that the heavy users of the fresh red meat (c1) preferred its consumption according to the aroma and visible quality being the main components of the sensory quality by taking into consideration the local-origined organic meat willingness accepted as an indicator of the hedonic quality attributes. therefore, the homogeneous consumers in the c1 determined their purchase attitude and behaviors by considering the sensory quality attributes of the red meat delivered from the animal materials fattened under the advantages of the superior agroecological characteristics of the study area. the fresh red meat presentations positioned at the retailer shelves by highlighting their core benefits, and causing no changes on the sensory quality attributes of them with the designation of the local-origined meat under the generic brands for the consumers in c1 could have an important impact on their purchase patterns. the results of the study also indicated that the medium users of the fresh red meat (c2) focused on the purchase patterns constructed by a combination of the factors affecting the sensory quality including in the microbial safety and chemical residue-related risk associated with willingness to obtain the salutary meats minimizing the negative effects on human healthy at the feeding and nourishment levels of the livestocks and the manufacture levels of the red meats. they, moreover, paid a major attention to the hedonic quality related to the nutritional value and the cost of the ownership minimizing their expenditures by being provided easily and table 2 final cluster centres and the number of cases in each cluster. main factors clusters* heavy users (c1)** medium users (c2)** light users (c3)** aroma (f1) 0.465 -0.122 -0.127 micsaf (f2) -0.751 0.423 -0.174 creris (f3) -0.641 0.342 0.073 visqly (f4) 0.156 -0.241 0.076 imag (f5) -0.263 -0.034 0.333 nutval (f6) -0.955 0.328 0.215 coscon (f7) -0.042 0.079 -0.009 mesdur (f8) 0.117 -0.890 0.473 orgloc (f9) 0.330 -0.423 0.108 fororg (f10) -0.078 -0.320 0.222 disposable income (disinc) 3.570 3.280 2.980 the prices of the meats (meprc) 1.340 1.330 1.330 number of total cases in each cluster *** 81 193 111 % of total cases in each cluster 21% 50% 29% * bold numbers indicate the largest final cluster centre scores for each factor. ** according to f statistics, the final cluster center scors were found very importance (p<0.01). *** the total number of the cases (n): 385. 188 ital. j. food sci., vol. 27 2015 comfortably to the consumer of c2. the implementations of differentiated actual product strategies based on the price discriminations under own brands of the manufacturers of the red meat with food safety that there was no negative impact on their health, therefore, could also affect positively their purchasing motivation by emphasizing the core benefits of the red meat for c2. the results of the study, furthermore, explained that the light users of the fresh red meat (c3) tended to buy the fresh red meat with the hedonic quality attributes positioned to the focus point of the purchase attitude psychology alienated under the actual fresh meat image with the foreign-origined meat willingness, its resources and durability. they, thus, preferred the national or global brands exhibiting the actual fresh red meat image by turning to durable foreign-origined meat sources with long shelf life. it could be implemented the marketing tactic and strategies focused on the designs of the actual fresh red meat image under the name of the national and global brands for the consumers in c3. results of mrc analysis related to the sensory and hedonic quality attributes of fresh red meat the results of the statistical tests in table 3 reported that the vif values with 1.06 and 2.47 indicating the scores between 1.00 and 2.50 determining the acceptable reference range and 1.85 durbin-watson d statistics positioned between d u (1.96) and 4-d u (1.68) referred that there were no the econometrics problems for multicollinarity and auto-correlation in the mrc model (gujarati, 2005; kalayci, 2005). according to the results diagnosing the econometrics problems, the data sets could be used directly for the mrc model. the determination statistics, ols estimates of the parameter coefficients and other statistic measurements such as f and t, collinearity and correlation matrix scores were given in table 3. the results of the mrc analysis indicated that the determination coefficient (r2) and adjusted (adj.) r2 was calculated as 0.51 and 0.50 in the mrc model, and thus all the independent variables explained the least 50% of the dependent variable. rejecting the null hypothesis, furthermore, referring that there was no the relationships between the dependent variable and all the independent variables by making the parameter coefficients of them equal to zero, f-statistic was calculated as 6898 (p:0.000). on the other hand, the partial regression coefficients of all the independent variables taking into consideration t-statistics, except for those that of the microbial safety (micsaf), the cost to the consumers (coscon), the meat sources and its durability (mesdur) and the foreign-origined meat willingness (fororg), were statistically found to be meaningful (t c(df:12;0.01/0.05) ); and then the signs of their coefficients including in the chemical residue-related safety (creris) and the price of the meats (meprc) with negative and the others with positive ones at the levels (p:0.01 and 0.05) were also found consistent with the economic theories. the results of the mrc analysis also highlighted that the visible quality (visqly), the aroma (aroma), the nutritional value (nutval), the accual fresh meat image (imag), meprc, the local-origined organic meat willingness (orgloc), creris, the disposable income (disinc) were of very important impacts on the fresh red meat table 3 the measurement results of the mrc analysis and some statistic tests. n: 385 r2: 0.51 adj.r2: 0.50 f c(12;361) : 6.898* dw d c : 1.85 variables mrc model collinearity statistics correlations β:coefficientsa std. error t c -value p-value tolerance vif zero-order partial part constant 1.478 0.563 2.623 0.009* logaroma 0.484 0.189 2.556 0.011* 0.374 2.472 0.027 0.133 0.121 logmicsaf 0.023 0.093 0.244 0.807 0.550 1.818 0.037 0.013 0.012 logcreris -0.161 0.086 -1.868 0.053** 0.559 1.788 0.102 -0.098 -0.088 logvisqly 0.553 0.194 2.844 0.005* 0.487 2.483 0.093 0.148 0.135 logimaj 0.245 0.086 2.845 0.005* 0.461 2.167 0.115 0.148 0.135 lognutval 0.370 0.136 2.716 0.007* 0.413 2.418 0.035 0.142 0.129 logcoscon -0.107 0.084 -1.277 0.202 0.603 1.658 0.016 -0.067 -0.060 logmesdur 0.008 0.081 0.095 0.924 0.682 1.465 0.022 0.005 0.005 logorgloc 0.210 0.109 1.920 0.056** 0.428 2.338 0.078 0.101 0.091 logfororg 0.004 0.026 0.156 0.876 0.778 1.285 -0.019 0.008 0.007 logmeprc -0.240 0.091 -2.633 0.009* 0.947 1.056 -0.140 -0.137 -0.125 logdisinc 0.106 0.072 3.791 0.001* 0.886 1.129 0.133 0.078 0.070 a the coefficients consisted of the standardized coefficients. * (p<0.01) **(p<0.05). ital. j. food sci., vol. 27 2015 189 consumption amounts of turkish consumers (p<0.01 and 0.05). in relation to the profiles of the cluster segments based on the underlying dimensions of the sensory and hedonic quality attributes emerged from pca, the satisfaction of visqly and aroma identifying the sensory quality attributes of the fresh red meat supported by orgloc of turkish consumers in c1 increased considerably very high effects on their meat consumption amounts with 0.55 (p<0.01), 0.48 (p<0.01) and 0.21 (p<0.01) rates, respectively. when considered the credence quality characteristics assigned to the sensory quality attributes affecting the fresh red meat consumption amounts of turkish consumers in c2, there was an adverse relationship between creris (β: -0.16 p<0.05) and its consumption amounts, but not nutval (β: 0.37 p<0.01). as minimized its creris, and improved its nutval, therefore, it could be increased those at a lower level than all the others. imag (β: 0.24 p<0.01) standing for the hedonic quality attributes and nutval (β: 0.37 p<0.01) symbozing the sensory ones, on the other hand, had a moderate impact on those of them in c3. overall, the average agreement of all the cluster members in relation to the price of the meat (meprc) exhibiting an adverse relation with its consumption amounts occurred at a moderate level (β: -0.24 p<0.01), but the disposable income (disinc) having a linear relation with those substantiated at lower levels (β: 0.11 p<0.01) than the others. the results of the present study confirmed briefly that the sensory quality attributes, especially visqly, aroma, nutval, on the fresh red meat consumption amounts had a much higher impact than the hedonic quality attributes such as imag, meprc, disinc. in particular, the results emphasized that the pleasure of turkish consumers provided from its consumption were under not only the effects of the numerous postpurchasing criterion (aroma, nutritional value, microbial safety, etc.) but also the impacts of the various pre-purchasing processes based on the visual quality cues (colour, freshness, natural structure, specific animal parts and meat outlets, etc.) impacting on their purchase decisions. the results of the present study with regard to the sensory and hedonic quality attributes of the fresh red meat are in line with the results of the previous studies reported that the consumers paid particular much more attention to the sensory quality attributes of the red meat inspected visually in a pre-purchasing process (colour, freshness, natural structure, fat content, specific animal parts and meat outlets) (realini et al., 2013; troy and kerry, 2010; aaslyng et al., 2007; verbeke and vackier, 2004), and then to its credence attributes perceived such as nutritional value (vitamin and protein content), microbial freeness (salmonella, dioxins, bse) (krystallis et al., 2007; verbeke and vackier, 2004) and chemical freeness (hormones, antibiotics) (krystallis et al., 2007; verbeke and vackier, 2004) rather than the hedonic ones (meat image, price, income, country of origin) (furnols et al., 2011; napolitano et al., 2010; krystallis et al., 2007; audebert et al., 2006; oliver et al., 2006; verbeke and vackier, 2004; bernues et al., 2003). conclusions the results of the study confirmed clearly that the heavy users in c1 attributed a much more importance to the sensory quality attributes based on willingness to buy the local-origined organic meat endorsing the aroma as an indicator of the post-purchasing satisfaction followed by the fresh red meat consumption and the visual quality considered in the pre-purchasing process of it than the others. on the other hand, the medium and light users in c2 and c3 paid also a more attention to the sensory quality attributes including in the credence attributes such as the microbial and chemical freeness and the nutritional value of it than the members of c1 as considered the efficiency scores of the sensory and hedonic quality attribute variables effecting on its consumption amounts of turkish consumers in mrc model. consequently, the sensory quality attributes had a much bigger impact on its consumption decision and amounts of turkish consumers than the hedonic quality attributes in the overall agreements with regard to the importance of all the factors. therefore, the fresh red meat consumption amount in turkey maintaining at a very low level of the annual fresh red meat consumption amount per capita (about 12 kg) when compared with the leading countries in the meat consumption could be increased meaningfully by the innovative red meat and meat products designed through the product and price differentiations under the production, manufacturing and marketing tactic and strategies taking into account first its core benefit based on the sensory quality attributes such as the visible quality, aroma and credence attribute, and then its actual benefit focused on the hedonic quality attributes (the country of the origin, its actual image and price and their disposable 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odemiş, izmir. j. animal prod. 51(1): 21-30. paper received february 14, 2014 accepted august 6, 2014 paper 198 ital. j. food sci., vol. 27 2015 keywords: food safety, listeria monocytogenes, fresh produce, food borne illnesses, tomatoes, food borne pathogens inactivation of listeria monocytogenes atcc 7644 on tomatoes using sodium dodecyl sulphate, levulinic acid and sodium hypochlorite solution e. mnyandu*, o.a. ijabadeniyi and s. singh department of biotechnology and food technology, durban university of technology, durban 4001, south africa *corresponding author: tel. 002778 0248617 email: mketiwae@yahoo.com abstract the human pathogen listeria monocytogenes poses a serious threat to public health. a study was carried out to evaluate the effectiveness of four sanitizers, used individually or combined, against l. monocytogenes atcc 7644. the contact times for bacteria and sanitizer were varied to 1, 3 and 5 minutes. levulinic acid, sodium dodecyl sulphate (sds), sodium hypochlorite solution (chlorine) and a combination of sds and levulinic acid (mixture) were tested. results revealed that 0.5% levulinic acid, when used individually, is capable of reducing the surviving colonies by 3.63 log cfu/ml, 4.05 log cfu/ml, 6.71 log cfu/ml after exposure for 1, 3 and 5 minutes respectively. sds resulted in an 8 log cfu/ml reduction after 1, 3 and 5 minutes. a combination of 0.5% levulinic acid and 0.05% sds caused a 3.69 log cfu /ml reduction, 4.4 log cfu/ml reduction, 7.97 log cfu/ml reduction for 1, 3 and 5 minutes respectively. chlorine was the least effective with 2.93 log cfu/ml reduction, 3.16 log cfu/ ml reduction and 4.53 log cfu/ ml reduction respectively. when stored for up to 72 hours at 4°c, the surviving colonies remained viable and decreased in number significantly p < 0.05 = 0.001. the titratable acidity of samples treated with levulinic acid and samples treated with sds/lev mixture was lowered significantly compared to the control sample. no significant differences were noted in these same parameters for samples treated with chlorine or sds. the application of sds in the fresh produce industry as a sanitizing agent may be successful in eradicating or reducing the viability of l. monocytogenes on fresh produce, thereby replacing the routine chlorine washing. mailto:mketiwae%40yahoo.com?subject= ital. j. food sci., vol. 27 2015 199 introduction the increase in fresh produce consumption has caused a rapid evolution in the fresh produce industry (johnston et al., 2005). this, coupled with recommendations to eat minimally processed foods, has led to an increase in the consumption of fresh fruits and vegetables among consumers (berger et al., 2010). the consumption of minimally processed foods and fresh produce has also been encouraged among the immune compromised populations, such as those affected by hiv/aids, children and pregnant women (berger et al., 2010; gandhi and chikindas, 2007). consumer demands and habits have also shifted, with many consumers in the busy world preferring to eat ready-to-eat foods and eating from salad bars (oms-oliu et al., 2010; berdegué et al., 2005). a variety of fresh produce such as lettuce cantaloupes, peppers, tomatoes, herbs and green leafy vegetables, among others, have been linked to food borne illnesses associated with either salmonella, escherichia coli o157:h7 or listeria monocytogenes contamination (tauxe et al., 2010). contamination of fresh produce by these pathogens occurs by various means. ijabadeniyi et al. (2011a) cited irrigation water as major pre-harvest source of contamination of fresh produce. other factors as cited by johnston et al. (2005) include use of biocides as fertilizer, poor worker hygiene and poor sanitation. l. monocytogenes among other food borne pathogens have been implicated as a public health threat (velusamy et al., 2010) and are estimated to cause about 1,600 incidents of illness, more than 1400 hospitalisations and about 250 deaths per year in united states (kyle, 2012). these pathogens are responsible for food borne listeriosis. they can grow in the soil, drains and on food preparation surfaces (gálvez et al., 2010; pan et al., 2006; djordjevic et al., 2002). they have been largely associated with dairy products, but recent research has also shown their increasing association with fresh produce (gandhi and chikindas, 2007) including tomatoes. tomatoes are widely consumed and can be eaten raw, partially cooked or can be processed into other products. they are a very rich source of carotenoids, folate, vitamin c, mineral elements and phenolic compounds (frusciante et al., 2007). of major importance are the antioxidants (carotenoids). epidemiological research has shown that the antioxidants are capable of preventing chances of cancers and cardio vascular diseases (leonardi et al., 2000). tomatoes also provide a dietary source of soluble and insoluble fibres such as pectin, hemicellulose, and cellulose. due to their nutritional value, they form an important part of the human diet. the elimination of food borne pathogens that can contaminate tomatoes is essential for preventing food borne illnesses that may be associated with the consumption of tomatoes. many methods are being used to try and eliminate the food borne pathogens. use of phage or phage products in food production has been considered as a novel method for bio-control of pathogens in fresh and ready-to-eat food products (hagens and loessner, 2010), but the cost associated with their use is very high. other methods include bacteriocin-activated films high-hydrostatic pressure, high-pressure homogenization, in-package pasteurization, food irradiation, pulsed electric fields, or pulsed light and electrolyzed water (gálvez et al., 2010). sanitizers such as carvacrol, vanillin, peroxyacetic acid, hydrogen peroxide, n-acetyl-l-cysteine and citrox among others have also been tried (abadias et al., 2011). sanitizers affect cell components, for example proteins, dna, rna and cell wall constituents through physicochemical interactions or chemical reactions. they cause irreversible damage to these structures and a loss of cell contents, thereby rendering the bacteria inactive or dead (cerf et al., 2010). the action of sanitizers is governed by contact time (exposure time), ph and temperature, among other factors. some researchers conclude that sanitizers are not effective in eradicating food borne pathogens when used individually, although a combination of agents increases the sanitizer ability (sagong et al., 2011; zhao et al., 2009). recent studies have also shown that if not used properly, sanitizers can be detrimental to the quality of fresh produce (salgado et al., 2013; guan et al., 2010). with regard to tomatoes, ph and acidity are the most important determinants of tomato quality (anthon et al., 2011), hence the interaction of tomatoes with sanitizers during washing should be monitored. the study was performed to evaluate the effectiveness of sds, chlorine and levulinic acid in reducing the viability of l. monocytogenes on tomatoes and the effect of these sanitizers on ph, titratable acidity and total soluble solids. materials and methods fresh produce tomatoes were purchased from a local supermarket on three separate occasions in durban, south africa. on the day of purchase the tomatoes were washed in running water. the tomatoes were then washed in 70% alcohol (ijabadeniyi et al., 2011a). prior to subjection to different sanitizer treatments, the tomatoes were tested for the presence of l. monocytogenes. bacterial strains listeria monocytogenes atcc 7644 (mer ck, south africa) was used for this study. the 200 ital. j. food sci., vol. 27 2015 strain was cultured in fraser broth for 24 hours at 37ºc and stored at 4ºc (ijabadeniyi et al., 2011a). prior to each experiment, a fresh culture was prepared from the stock culture by sub-culturing in fraser broth for 24 hours at 37ºc, an 8 log cfu/ml culture of l. monocytogenes, using mcfarland standards (ji et al., 2010). chemicals and chemical treatments sodium dodecyl sulphate (sds), levulinic acid, sodium hypochlorite solution, all purchased from merck, south africa, were tested, individually or combined with varying contact times (1, 3 and 5 minutes) for their effect on l. monocytogenes atcc 7644 in tomatoes. the chemicals were used as follows; 1% sds individually 0.5% levulinic acid individually 200 ppm sodium hypochlorite solution individually and 0.5% levulinic acid/0.05% sds combined and termed mixture. inoculation of bacterial strains into tomatoes the method of zhao et al. (2009) was followed. a 25 g sample of tomatoes was cut into approximately 5 cm long pieces in the lamina flow hood. the samples were submerged into bacterial suspension (108 cfu/ml, 50 ml of bacterial solution into 950 ml of distilled water) for 60 seconds and then air dried for 20 minutes in the lamina flow hood. the samples were then suspended into 500 ml test solution and agitated by a magnetic stirrer at 100 rpm for 1, 3 and 5 minutes. following treatment, the individual samples were placed in double zipper bags containing 25 ml of phosphate buffered saline and pummelled for one minute. the suspension was serially diluted (1:10) in 0.1% buffered peptone water and enumerated for l. monocytogenes atcc 7644. enumeration of l. monocytogenes a method by taormina and beuchat (2001) was followed. populations of l. monocytogenes atcc 7644 were determined by surface plating serially diluted samples; 0.1 ml in duplicates on listeria selective agar (oxford formulation; oxoid ltd, wade road, basingstoke, hants uk). plates were incubated for 24 hours at 37ºc, after which colonies were counted. preparation of samples for scanning electron microscopy untreated samples and samples subjected to chlorine, levulinic and sds/lev were used for sem viewing. a method used by ijabadeniyi et al. (2011b) was followed with a few modifications, according to the requirements of university of kwazulu natal microscopy unit. pieces of tomatoes inoculated with l. monocytogenes atcc 7644 and subjected to different treatments were cut into small pieces of 2 x 2 mm using a sterile blade. primary fixation was carried out in 2.5% glutaraldehyde for 12 hours, followed by rinsing three times in phosphate buffer (0.1 m, ph 7.0). post fixation was done using 0.5% osmium tetroxide for one hour. fixed samples were dehydrated in graded ethanol (30%, 50%, 75% and 100%) each for 5 minutes. the samples were then dried in a critical point dryer with carbon dioxide as a transition gas. the samples were mounted on specimen stubs and coated with gold palladium. the samples were then analysed using desmond clarence scanning electron microscopy. analysis of tomato physicochemical properties preparation of samples: the method of zhao et al. (2009) was followed for sample preparation, except that the tomato was further homogenised into slurry. a 25 g sample of tomatoes was cut into approximately 5 cm long pieces. the samples were then suspended into 500 ml test solutions as follows: 25 grams of tomatoes + 500 ml de-ionised water (control) 25 grams of tomatoes + 500 ml 1% sds 25 grams of tomatoes + 500 ml of 0.5% levulinic acid 25 grams of tomatoes + 500 ml of 200 ppm sodium hypochlorite solution 25 grams of tomatoes + 500 ml of 0.5% levulinic acid/0.05% sds (mixture) the samples were agitated by a magnetic stirrer at 100 rpm for 1, 3 and 5 minutes (contact times). after each contact time was achieved, samples were immediately drained and the tomatoes were homogenized to form a slurry using waring commercial laboratory blender. the slurry was used to test for ph, titratable acidity and total soluble solids immediately. determination of ph the determination of ph was done on freshly made tomato paste using the thermo scientific orion 2star ph meter. the electrodes were rinsed with distilled water in between samples. determination of titratable acidity for estimating titratable acidity, the slurry was filtered using whatman syringe filters. a 100 ml of the filtrate was titrated by adding 0.1n sodium hydroxide until a ph of 8.1 was attained. the volume of the sodium hydroxide added to the solution was multiplied by a correction factor of 0.064 to estimate titratable acidity as a ital. j. food sci., vol. 27 2015 201 percentage of citric acid (cheema et al., 2014; turhan and seniz, 2009). determination of soluble solids content tss is an index of soluble solids concentration in fruit. for an estimation of soluble solids content, 1.5 ml tomato slurry was centrifuged at 10,000 rpm (15 min, 25°c), and the supernatant was filtered through whatman nonsterile syringe filters (0.45 μm). the filtered tomato serum (40 μl) was measured using a digital refractometer atago (atago, usa inc. kirkland, wa, usa). measurements were taken once for each sample, and 70% ethanol was used to clean in between samples. the refraction index was expressed as percent soluble solids in°brix (wilkerson et al., 2013; javanmardi and kubota, 2006). data analysis three trials were conducted for each experiment. analysis of the data was performed using spss version 21 (ibm statistics). analysis of variance was conducted with repeated measures and greenhouse geisser correction to study the effect of contact time on the survival of l. monocytogenes, atcc 7644 and the effect of each sanitizer on the survival of l. monocytogenes atcc 7644 at varied time intervals (0, 24, 48 and 72 hours). the number of surviving colonies was plotted against contact time (1, 3 and 5 minutes) and also against time interval (0, 24, 48 and 72 hours). log reductions for each contact time and sanitizer were also calculated and is presented in a table. pair wise comparison with bonferroni adjustment was used to determine any significant difference between subjects. to analyse results for physicochemical properties, anova was used to assess if there was a significant difference in ph, total soluble solids and titratable acidity of treated and untreated tomato samples. results effect of storage time, sanitizer treatments and contact time on the survival of l. monocytogenes atcc 7644 the treatment of l. monocytogenes with sanitizers resulted in a decrease in the populations of bacteria. all the sanitizers tested had the ability to reduce the surviving colonies, with varying degree of effectiveness. among the sanitizers tested, sodium hypochlorite solution was the least effective, with the highest counts of surviving colonies. the next in the list is levulinic acid, then a mixture of sds and levulinic (termed mixture), with sds the most effective of them all. the results of repeated measures (castro et al.) with greenhouse-geisser correction showed that there was a significant difference at 5% level between effectiveness of sanitizers used, [f(3, 9) = 63.00; p< 0.05 = 0.01]. the surviving colonies were reduced progressively as storage time increased from 0 hours to 72 hours. the means of surviving colonies are shown in table 1. marginal means for each sanitizer’s contact time were also plotted in fig. 1 for 1, 3 and 5 minutes. as shown in the figure, sodium hytable 1 mean 1 count of l. monocytogenes atcc 7644 after treatment with different sanitizers at different contact times and storage times. contact times time intervals 0 hours 24 hours 48 hours 72 hours 1 minute a 5.36±0.02 a 5.14±0.03 a 5.02±0.03 a 4.75±0.04 chlorine 3 minutes a 5.06±0.03 a 5.06±0.03 a 4.78±0.05 a 4.45±0.04 5 minutes b 4.17±0.09 b 3.77±0.09 b 3.33±0.10 b 2.60±0.09 1 minute c 4.60±0.01 c 4.59 ±0.02 c 4.27±0.08 c 4.01±0.06 sds/lev 3 minutes c 4.35±0.05 c 4.24 ±0.06 c 3.39±0.36 c 2.53±0.08 5 minutes d 1.33±0.15 d 1.40±0.03 d 0.56±0.09 d 0.00 1 minute e 4.68±0.03 e 4.60±0.02 e 4.15±0.14 e 4.06±0.11 levulinic 3 minutes e 4.68±0.03 e 4.34±0.09 e 4.12±0.10 e 2.60±0.30 5 minutes f 3.17±0.07 f 2.06±0.04 f 1.50±0.10 f 0.43±0.20 1 minute g 0.00 g 0.00 g 0.00 g 0.00 sds 3 minutes g 0.00 g 0.00 g 0.00 g 0.00 5 minutes g 0.00 g 0.00 g 0.00 g 0.00 mean counts ±standard deviation (log 10 cfu /ml). 1means followed by different letters in the same column are significantly different. 202 ital. j. food sci., vol. 27 2015 fig. 1 means of surviving colonies of l. monocytogenes atcc 7644; based on marginal means. the highest means associated with chlorine show that it was least effective. pochlorite has the highest mean values, meaning that the highest number of surviving colonies was observed after exposure to this sanitizer compared to other solutions. sodium chloride was thus not very efficient in reducing survival of the pathogen in this particular study. increasing the contact time (1, 3 and 5 minutes) significantly reduced the surviving colonies for all sanitizers tested at 5% level; [f (3, 180) = 30.70; p< 0.001]. however, the results of anova with green house geisser correction showed that the reduction for 1 minute and 3 minutes of treatment were not significantly different (p = 0.16). this shows that increasing the contact time of each of the sanitizer to 3 minutes did not make much difference to the surviving colonies. overall log reductions when exposed for 1 minute to 200 ppm chlorine, l. monocytogenes were inactivated by 2.93 log cfu/ml. a log reduction of 3.16 log cfu/ ml and 4.53 log cfu/ml was achieved after increasing contact time to 3 minutes and 5 minutes respectively. a mixture of 0.5% levulinic acid and 0.05% sds (mixture) reduced the surviving colonies to 3.69 log cfu/ml, 4.4 log cfu/ml and log 7.97 cfu/ml after exposure for 1 minute, 3 minutes and 5 minutes, respectively. using 0.5% levulinic acid resulted in log reductions of 3.63 log cfu/ml, 4.05 log cfu/ ml and 6.71 cfu/ml after exposure for 1 minute, 3 minutes and 5 minutes. the overall log reductions are presented in table 2. observations of specimens using a scanning electron microscope samples of tomatoes treated with sodium hypochlorite solution, levulinic acid and mixture were viewed under sem to verify the existence of colonies even after exposure to sanitizers. the results for this current work showed that there were surviving colonies after exposure to sodium chlorite solution, levulinic acid and a mixture. however, viewing samples treated with the above sanitizers did not clearly show the remains of surviving colonies. the sem images did not show the presence of an abundance of bacteria on the surfaces. it is possible that the bacteria that were inoculated on the surfaces could have been washed out during the sample preparation procedure. the procedure used for sample preparation might not be suitable in this specific case. bacteria might also have migrated into other hidden sections of the pictures due to the irregularities of the topography. table 2 log reductions (cfu/ ml) for chlorine, mixture, levulinic acid and sds at 1, 3 and 5 minutes. overall log reduction sanitizer 1 minute 3 minutes 5 minutes chlorine 2.93 3.16 4.53 mixture 3.69 4.40 7.17 levulinic 3.63 4.05 6.71 sds 8.00 8.00 8.00 ital. j. food sci., vol. 27 2015 203 titratable acidity, ph and total soluble solids of tomato samples table 3 presents the results of the ta, ph and tss. the ta of tomato samples treated with levulinic acid and sds /lev mixture was significantly different from the control (p< 0.05). the ta for levulinic acid treated tomatoes was 2.78%, 2.81%, and 2.81%; while the ta for mixture treated tomatoes was 3.81%, 3.73%, 3.74% for 1, 3 and 5 minutes respectively. the ph for levulinic acid and mixture treated tomato samples was relatively lower than the ph of the control sample, as shown in the table. there was no significant difference between the ta and ph of the mixture and levulinic acid treated samples. the ta for tomato samples treated with sds was 0.16 and for samples treated with chlorine was 0.15%, 0.14% and 0.14%. these results did not vary significantly from the control. the ph for the sds and chlorine treated samples were also slightly different from the control sample, as shown in the table. tss for levulinic acid treated samples were reduced significantly to 3.20% brix for 1, 3 and 5 minutes, while the tss for mixture treated samples was reduced to 3.24, 3.26, 3.24% brix respectively. though the tss for sds treated and chlorine treated samples were reduced, the effect was not significant according to the findings of this study. contact time was varied from 1 minute to 5 minutes; but there were no significant changes in theses parameters from 1 minute to 5 minutes. discussion the food manufacturing industry depends on the use of sanitizers for reducing the risk associated with food borne pathogens. many sanitizers have been tried, but to date food borne pathogens are still a problem in the food and fresh produce industry. some researchers have suggested that this is due to development of resistance by the bacteria with repeated exposure to sanitizers (mani-lópez et al., 2012; riazi and matthews, 2011). most fruits and vegetable units resort to chlorine based sanitizers because they are cheaper and have a long standing credibility with reducing surviving bacteria. however, this is proved not to be the case in this current research, as well as other previous research. findings from this study show that though chlorine has been widely used for washing produce and sanitising food surfaces, it is not really capable of killing all food borne pathogens. this is shown by high mean counts associated with chlorine as presented in the results above. chlorine washing has also been tried on escherichia coli o157:h7 and salmonella, but the reports on that work also shows that chlorine is not effective against food borne pathogens (keskinen et al., 2009b). other researchers also agree that chlorine cannot reduce food borne pathogens effectively (ijabadeniyi et al., 2011b; allende et al., 2009; mahmoud et al., 2007). several research projects are under way to try table 3 effects of levulinic acid, chlorine, sds/lev mixture and sds on physicochemical properties of tomatoes. tomato treatment contact ph of sample titratable acidity total soluble times (% citric acid) solids (%brix) 1 minute 4.77 a 0.16 a 4.90 a distilled water 3 minutes 4.78 a 0.14 a 4.90 a 5 minutes 4.78 a 0.16 a 4.90 a levulinic acid 1 minute 3.61 b 2.78 b 3.20 b 3 minutes 3.67 b 2.81 b 3.20 b 5 minutes 3.69 b 2.81 b 3.20 b mixture (sds/lev) 1 minute 3.81 b 2.76 b 3.24 b 3 minutes 3.73 b 2.78 b 3.26 b 5 minutes 3.74 b 2.78 b 3.24 b chlorine 1 minute 5.09 a 0.15 a 4.60 a 3 minutes 5.17 a 0.14 a 4.63 a 5 minutes 5.20 a 0.14 a 4.61 a sds 1 minute 4.68 a 0.16 b 4.65 b 3 minutes 4.88 a 0.16 b 4.61 b 5 minutes 4.87 a 0.16 b 4.63 b each value represents the mean of three trials. for each parameter, the values significantly different at p ≤ 0.05 are indicated by different letters. samples treated in distilled water were used as control. chlorine = sodium hypochlorite solution. 204 ital. j. food sci., vol. 27 2015 to find other alternative sanitizers because of the challenges that are associated with chlorine (keskinen et al., 2009a). some researchers point out that its ph sensitivity affects its effectiveness (zhao et al., 2009). another challenge is that it diminishes quickly upon contact with organic matter and hence leads to reduced effectiveness (neal et al., 2012). other concerns raised include the environmental and health risks associated with the formation of carcinogenic halogenated disinfection by-products such as trihalomethanes (gil et al., 2009; kim et al., 2009). for these reasons chlorine has not been gainfully useful in the fresh produce industry in recent years. though it has been a long standing sanitizer in the food industry, other sanitizers that have been shown to be more effective than chlorine; through this research and previous research can be employed for the betterment of microbiologically quality of fresh produce. levulinic acid is applied in the food manufacturing industry as a food additive. it has been designated as a generally safe additive to food by the food and drug administration (fda)(zhao et al., 2009). levulinic acid disrupts the membrane structure of bacteria due to its polarity, thereby exposing cell constituencies and lethality (thompson, 2007). levulinic acid can be used over a wide ph and temperature range (sagong et al., 2011). in this particular study, levulinic acid showed mean counts that were much lower than those of chlorine. with these findings, it can be concluded that levulinic acid at 0.5% can perform better than a 200ppm sodium hypochlorite solution against l. monocytogenes atcc 7644. other researchers also tried levulinic acid in their work with related findings. thompson et al. (2008) concluded that it was effective in inhibiting outgrowth of l. monocytogenes in ready-to-eat meat products. other studies using lactic acid, acetic acid and levulinic acid on meat revealed that though levulinic acid is effective, it does not provide as effective decontamination as lactic acid, nor as much residual protection as acetic acid (carpenter et al., 2011). levulinic acid shows potential in the fresh produce industry, and therefore further research can be pursued on the most usable concentrations and most applicable pathogens. its detrimental effects on quality should be taken into consideration as well. sodium dodecyl sulphate is generally regarded as a safe (gras) food additive (lu and wu, 2012). in this study, using 1% sds alone resulted in 8 log cfu/ml reduction of l. monocytogenes. sds has amphilic properties (12 carbon chain attached to sulphate group) and its anti-microbial effectiveness increases when ph is decreased, it has the ability to denature cell proteins and damage cells membranes irreversibly (zhao et al., 2009). the action of sds was much better than that of levulinic acid in this particular study when they were used individually. this is because levulinic acid has a shorter carbon chain (5 carbons and a hydroxyl group), which makes it a weak acid, therefore its effectiveness is less than sds. extra care must be taken if sds is employed in fresh produce as it was established during this study that very low concentrations of 1% can have a very large impact on survival of pathogens. a combination of 0.05% sds and 0.5% levulinic acid was also used in this study. findings show that this mixture achieved better results as compared to levulinic acid alone. many researchers have reported on the advantages of mixing sds and levulinic acid. the findings of zhao et al. (2009) show an increased antimicrobial activity by the combination of sds and levulinic acid against salmonella and e. coli o157: h7. gurtler and jin (2012) found that a combination of 2% acetic acid, lactic acid and levulinic acid reduce salmonella on tomatoes. ortega et al. (2011) reported that a combination of levulinic acid and sds was highly effective against e. coli when exposure times were increased to 30 and 60 minutes. on the contrary, guan et al. (2010) reported that a combination of these had no commercial value as they have detrimental effects on the quality of fresh produce. combining sanitizers has shown to have a positive contribution in the food market. this has potential for implementation in the fresh produce industry. the implementation of a combination of sanitizers can be tried together with an assessment of their effects on sensory qualities. increasing exposure time significantly decreased the surviving colonies of l. monocytogenes. in this particular study, a greater fall in surviving colonies was achieved at 5 minutes exposure time. this is evidence that the longer the bacteria are exposed to chemicals, the greater the chances of reducing their survival. park et al. (2011) also reported that log reductions increase with increasing contact times. other writers indicate that an exposure time of 3 minutes is effective against food pathogens (mattson et al., 2011). ding et al. (2011) and møretrø et al. (2012) also reported that the effectiveness of a sanitizer depends on treatment time. other writers also note that a significant decrease in the bacterial counts occurs in the first minute and the subsequent decrease after one minute is not significant (stebbins et al., 2011; tirpanalan et al., 2011). from the reports written by other writers and from this research, it can be said that contact time is one of the factors that should be monitored when using sanitizers. insufficient contact time will lead to high survival after treatment, while extended contact time may lead to damage in the sensory qualities of fresh produce. the bacteria where further stored for a period of 72 hours at 4 0 c. during this storage period l. monocytogenes survived up to 72 hours after being treated with sanitizers, except in ital. j. food sci., vol. 27 2015 205 sds. survival of pathogens after a storage period of 72 hours is also reported by ijabadeniyi et al. (2011b). sufficient exposure of pathogens to sanitizers is paramount to reduce surviving colonies, as some have the ability to recover even after being treated with sanitizer. for sanitizers to be effectively used on tomatoes, they should cause negligible changes to ph and titratable acidity of the tomatoes, the major determinants of tomato quality (anthon et al., 2011). in this research it was revealed that sanitizers can alter the physicochemical attributes of fresh tomatoes if the sanitizers come into contact with the sub surfaces, thereby affecting the final sensory quality of tomatoes. other recent studies also point out that sanitizers can affect sensory qualities of fresh produce to some extent (pérez-gregorio et al., 2011). in this study, major effects were noted on the ph, ta and tss with levulinic acid and mixture. changes effected by sds and chlorine were not significant. these findings pronounce sds as a better sanitizer to replace the routine chlorine washing as it causes minimal changes to quality. in previous studies, sds was tested together with organic acids and hydrogen peroxide on blue berries and no significant difference was detected in ph and total anthocyanin value between untreated and treated blueberries (li and wu, 2013). in another study using iceberg and romane lettuce, chlorine had high quality scores for romane lettuce, but caused quality deterioration on iceberg lettuce. a combination of sds and tsunami did not show any effect on sensory attributes of iceberg lettuce either (salgado et al., 2013). though levulinic acid did not have favourable results in this particular study, previous studies report that using levulinic acid caused no sensory changes in turkey meat and pork sausages (vasavada et al., 2003). a combination of sds and levulinic did not give favourable results in this study. other studies also report that sds used in combination with other sanitizers such as levulinic acid are of low commercial value compared to chlorine, since they cause detrimental effects to sensory attributes (guan et al., 2010). total soluble solids were reduced for all treatments with levulinic acid having the highest reductions followed by sds/lev mixture. this could have been attributed to leaching of contents into treatment solutions as a larger surface area of the subsurface area of tomatoes was exposed. leaching of materials is also reported by alegria et al. (2009). though previous studies also report that longer contact times result in deterioration of sensory characteristics (rico et al., 2007), there was no significant difference for all attributes tested in relation to contact time in this particular study. contact of sanitizers with sub-surfaces of fresh produce should be minimised to prevent unnecessary damage to sensory quality attributes. acknowledgements the authors would like to extend their sincere gratitude to the durban university of technology, faculty of applied sciences, for the financial support towards this study. the authors also want to thank the university of kwazulu natal microscopy unit for their contributions towards this study. conclusion this works confirms that use of sanitizers in food processing at shorter contact time of 1 minute may not eradicate food borne pathogens. sds alone is capable of destroying l. monocytogenes, causing no detrimental effect to sensory attributes of tomatoes. it is also important to 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sci., vol. 30, 2018 628 paper effect of chitosan coating enriched with cumin (cuminum cyminum l.) essential oil on the quality of refrigerated turkey breast meat t. taheri*a, a. fazlaraa, l. roomianib and s. taheria adepartment of food science and technology, ahvaz branch, islamic azad university, ahvaz, iran bdepartment of fisheries, ahvaz branch, islamic azad university, ahvaz, iran e-mail address: tinataheri18@yahoo.com abstract a solution containing 2% chitosan, 1% cumin seeds' essential oils and 1% acetic acid was applied for the coating preparation. in all of the treatments the total viable counts and applied psychrophilic bacteria decreased significantly compared to the control through the storage time. the ph, total volatile basic nitrogen (tvb-n), peroxide value (pv) and sensory attributes in all the treatments were significantly detected lower than the same parameters of the controls. the results of our investigation revealed that chitosan+cumin and ascorbic acid retarded spoilage and oxidative changes in refrigerated turkey breast meat. keywords: antibacterial properties, lipid oxidation, meat products, natural antioxidant ital. j. food sci., vol. 30, 2018 629 1. introduction products produced by poultry meats are considered as the food products with considerable growing interest in market in many parts of the world due to their low production cost compared to further meat products including beef, lamb and pork meats. they contain low fat, high nutritional value as well as distinct interesting taste and flavor. however, poultry meats due to their nature and composition, (i.e high moisture and protein contents) as well as high ph value (low acidity value), they have ideal and appropriate environments for pathogenic microorganisms' growth (latou et al., 2014; vaithiyanathan et al., 2011). the approach to extend and increase the shelf life of raw poultry meats and its products may present a challenge for meat and poultry industries not only in iran but also in further areas. microbial growth as well as lipid oxidation reactions may lead to undesirable and unwanted organoleptic alterations through the storage period in meat products (ouattara et al., 2000). one of the most commonly used methods to preserve food products safe and increase their shelf life is the addition of natural compounds with antibacterial and antioxidant properties in food products. since synthetic antioxidants have been reported to behave as carcinogens and mutating agents, more attention has been directed to the employment of natural antioxidants (taheri et al., 2012; wannes et al., 2010). several studies have been performed in this area including application of essential oils obtained from plant sources (raeisi et al., 2015; chen et al., 2014; taheri et al., 2013, allahghadri et al., 2010), chitosan obtained from natural origins (ojagh et al., 2010; no et al., 2007), and organic acids of natural resources (shaltout et al., 2014; bin jasass, 2008) with the aim of retardation of microbial deteriorations and oxidation reactions (latou et al., 2014). chitosan is categorized as a modified and natural carbohydrate polymer derived from deacetylation of chitin [poly-β-(1 → 4)-n-acetyl -d-glucosamine), and is a major component of the shells of crustaceans including crab, shrimp, and crawfish and is ranked second in the abundance among further natural biopolymers (after cellulose) (nowzari et al., 2013; no et al., 2007). due to the chitosan’s intrinsic antimicrobial and anti-oxidative properties and having appropriate characteristics in film preparation, as well as its biocompatibility and biodegradability properties, chitosan has attracted much attention as a natural additive (preservative) in not only nutraceutical but also in pharmaceutical and cosmetic industries (yuan et al., 2016; kanatt et al., 2013; fan et al., 2009). several studies have been performed on the antibacterial, antioxidant, and potential health benefit properties of the essential oils extracted from spices and herbs particularly from apiacea family (allahghadri et al., 2010; zhang et al., 2009). one of these valuable plants is cumin (cuminum cyminum l.), named “zira” growing in middle east particularly in iran. this plant species is also commonly grown in cyprus, lebanon, morocco, malta, turkey, spain, russia and india as well as china (erdeni et al., 2013). antioxidant and antibacterial activities are detected as one of the most considerable functional properties of cumin seeds. cumin seeds may have considerable potential to be used as an antioxidant agent in food products; the achieved results of several performed studies have shown that γ-terpinene is a predominant detected compound with potential health benefits in cumin seeds (raeisi et al., 2015; chen et al., 2014; iacobellis et al., 2005). application of a biodegradable film or coating is considered as a novel approach to protect not only meat products but also further food products against deteriorating agents (nowzari et al., 2013). a combination of chitosan and cumin essential oils may be formulated to prepare biodegradable films with considerable protective effects. the aim of the present study is to evaluate and assess the effect of chitosan coating enriched by cumin essential oil in combine of acetic acid to be used in enhancements of the shelf life and quality of turkey fillet stored at (4±1°c). ital. j. food sci., vol. 30, 2018 630 2. materials and methods 2.1. turkey breast preparation fresh turkey breast samples (18 kg) were purchased from a local market in ahvaz (khuzestan province, south iran). the average weight of each breast piece was set on 3 kg. the turkey breast samples were placed and sealed in an ice box with ice (refrigeration temperature) and transferred to food and drug administration laboratory at jundishapur university within 30 minutes. turkey breast samples were filleted manually and carefully washed with cold water. the weight of each fillet was set on 110±5 g. the prepared fillets were used for the selected experiments. 2.2. cumin seeds cumin seeds' essential oil (cuminum cyminum l.) and the required chemicals and reagents for gc-ms analysis (gas chromatography mass spectrometry) were provided from barij essence company in iran. 2.3. preparation of the coating film and treatment of the fillets a chitosan solution was prepared with 2% (w/v) chitosan (sigma chemical co, medium molecular weight, viscosity 200-800 cp) in 1% v/v acetic acid. 20 g of chitosan solution was mixed well with 900 ml of distilled water and the obtained mixture was stirred for 10 min, afterward 10 ml of glacial acetic acid was added to the mixture and stirred at room temperature by achieving a smooth solution. the solution was reached and diluted up to 1000 ml by the addition of distilled water. glycerol was added as a plasticizer to the achieved solution with the concentration of 0.75 ml. g-1 and was stirred for 10 min (nowzari et al., 2013; ojagh et al., 2010). thereafter the cumin essential oil (ceo), (mixed with tween 80 (aldrich chemical co., steinheim, germany), was added to the final prepared solution (bazargani glilani et al., 2015; ojagh et al., 2010) to comfort distribution and complete incorporation of cumin seeds' oil in the final solution. the final coating solution consisting of 2% chitosan, 1% acetic acid, 0.75 % glycerol and 0.2 % twen 80 as well as cumin seeds' essential oil 1% was homogenized under aseptic conditions for 1 min. the fillet samples were divided in 3 separated groups. samples of the first group were left untreated (control, sterile distilled water), and two other groups were treated by the following solution: chitosan 2% ceo 1% and acetic acid 1%. each sample was immersed for 30 min in the solution (yuan et al., 2016). then the prepared meat samples were removed from the solution and allowed to drain for 30 min before packaging (coating) (kanatt et al., 2013). after that, all of the fillet samples were individually packed in sterile ldpe containers; all of the containers were kept in a refrigerator at the temperature of 4±1°c. fillet samples were experimented on days 0, 3, 6, 9, 12 and 15 after packaging and storage, and analyzed for chemical and microbial tests as well as sensory alterations analysis. each experiment was done in triplicate. 2.4. microbial analysis 10 g of each sample was mixed with 90 ml of sterile saline (0.85 % nacl) solution in a sterile stomacher bag and was stomached continuously for 1 min. other decimal dilutions were prepared from the achieved uniform solution or dilution of stomacher and cultivated in an appropriate microbial medium regarding the type of the microbial test. the total viable count (tvc) was determined by pour plate medium approach with the use of plate ital. j. food sci., vol. 30, 2018 631 count agar (pca) (merck, germany). the inoculated plates were incubated at 37 ֯◌c for 48 h for total viable count, and at 10 ֯◌c for 7 days for psychrophilic count. the achieved results were expressed as log 10 cfu. g-1 of samples (nowzari et al., 2013). 2.5. ph value determination for determination of ph value, 5 g of turkey breast samples (of each treatment) were homogenized for 1 min with 45 ml of distilled water. the ph value was measured using a standardized portable ph meter (toa, kobe, japan; taheri et al., 2013). 2.6. determination of peroxide value (pv) peroxide value (pv) was determined in the lipid extract according to the method described previously by (aoac, 2000). the achieved results were expressed as milliequivalents peroxide value per each kg of lipid (meq o2/kg lipid). 2.7. determination of total volatile basic nitrogen (tvb-n) total volatile basic-nitrogen (tvb-n) values were detected by the direct distillation approach according to (goudlas and kontoinas, 2005) method. the micro diffusion method was determined by distillation after the addition of mgo to the homogenized turkey breast samples. the tvb-n value (mg nitrogen. 100 g-1 breast sample) was determined regarding the consumed volume of sulphuric acid reagent. 2.8. sensory evaluation and analysis sensory analyses were performed by an instructed taste panel consisting of five experienced judges, according to the guidelines presented in table 1 (octavian and octavian, 2010). three different categories were ranked in the prepared questioners: 3 scores for excellent, 2 scores for acceptable, and 1 for unacceptable rates. the assessed and studied parameters in sensory assessment were detected as the following: appearance, odor, color, consistency and elasticity. table 1. sensorial attributes and quality of refrigerated turkey breast. attribute excellent acceptable unacceptable appearance without slime present on surface slime present in some part of the surface slime present on the entire surface muscular elasticity fast return slow return no return odor characteristic off odors (slight sulphurous or ammoniacal) foreign (rancid, acid, putrid) color pink dark pink pale pink 2.9. statistical analysis spss software version 22 was applied for data analysis in the present study. all of the selected experiments for each sample were performed in triplicate. nonparametric statistics were used to analyze the achieved data. the analysis of variance (anova) test ital. j. food sci., vol. 30, 2018 632 was applied to detect the significant difference among the obtained data in the confidence level of 0.05. duncan’s multiple range tests were applied to compare the achieved means in the confidence level of 0.05. 3. results and discussion 3.1. microbial analysis it has been well known that through the refrigeration storage time of poultry meat, an extensive range of bacterial species may be characterized (vasilatos and savvaidis, 2013). changes in the value of total viable counts (tvc) in turkey breast fillet during the refrigerated storage are presented in (fig. 1). the initial tvc (log10 cfu. g-1) of the control, acetic acid and chitosan-cumin treatment were detected 4.77, 4.62, and 3.90 (log10 cfu. g-1), respectively. according to the recommended and standard limits (7 log10 cfu. g-1) for fresh meat (raeisi et al., 2015), the samples have shown acceptable quality. these achieved results were in solid agreement of those from bazargani-gilani et al. (2015) and latou et al. (2014) for fresh chicken meat, and raeisi et al. (2015) in term of fish fillet. all of the studied samples expressed an increased tvc value with enhancements in storage time (p<0.05). in 6 days after storage, the obtained mean of tvc of control samples was detected 6.54, which was close to the maximum allowed limit of tvc (7 log10 cfu. g-1) for raw meats, indicating the shelf life confined 5 6 days. this phenomenon (6 days shelf life) may be attributed to the longer acidic environment started after the bleeding of slaughtered animals (shaltout et al., 2014). for untreated samples (control), the tvc presented a value rather than the acceptable healthy limit in 9 days after storage time (8.76 log10 cfu. g-1). in contrast, the tvc values for the treated samples with acetic acid (1%) and chitosan (2%) + cumin (1%) was determined lower than the proposed standard value by the end of day 15th of storage period. reduction in microbial count by floating of the samples in the prepared solution mixed of chitosan, cumin and acetic acid, have been reported previously for chicken meat by (bin jasass, 2008), turkey breast by (vasilatos and savvaidis, 2013) and shrimp by (yuan et al., 2016). in the present study, the chitosan + cumin 1% treatment was the most effective formulation against tvc. gram-negative psychotrophic bacterial species (ptc) are detected as the major group of microorganisms leading to spoilage of aerobically stored fresh meats at chilled temperatures (nowzari et al., 2013; ojagh et al., 2010). in the current study, the initial ptc (day 0) in the control sample was detected 3.55 (log10 cfu. g-1), it was determined 4.04 and 3.194 (log10 cfu. g-1), in meat samples coated by aa and ch + c, respectively. furthermore, the growth pattern on tvc and ptc expressed an increasing rate through storage period (fig. 2). for all of the treatments, storage time had significant (p < 0.05) effects on the ptc value (log10 cfu. g-1), on day 6th of storage time, the mean value of ptc of the control samples increased up to 7.57 and spoilage started to appear as a slight foul smell. a significant reduction (p<0.05) of ptc value was detected in the samples treated by aa compared to the control ones. the effectiveness of acetic acid against microorganisms may be attributed to a decrease in ph and metabolic inhibition by the undissociated acid molecules as detected and reported by bin jasass (2008). in control samples, the ptc was detected rather than the acceptable limit on day 6th of storage time (7.57 log10 cfu. g-1). in contrast, the tvc values for the treated samples with chitosan + cumin (5.78 log10 cfu. g-1) remained lower than the proposed standard value by the end of day 15th of storage period. the antimicrobial activity of cumin essential oil might be attributed to the phenolic compounds (bazargani-gilani et al., 2015; raeisi et al., ital. j. food sci., vol. 30, 2018 633 2015; allahghadri et al., 2010). chitosan has been reported to be effective as an antimicrobial agent (shahidi et al., 1999; fan et al., 2009; kanatt et al., 2013). chitosan may act on the cells of the microorganisms and pathogens, therefore by alterations in the permeability of the cytoplasmatic membranes, may lead to the leakage of intracellular electrolytes and protein compounds out, as a result, it may lead to the death of the cells (yuan et al., 2016; bazargani-gilani et al., 2015). the mechanism of action of chitosan appears to be associated with the disruption of the lipopolysaccharide layer of the outer membrane of gram-negative bacteria (nowzari et al., 2013; pereda et al., 2011), as well as its function as a barrier against oxygen penetration (ojagh et al., 2010; jeon et al., 2002). figure 1. changes in total viable count (tvc) of turkey breast samples during refrigerated storage. figure 2. changes in psychrotrophic counts (ptc) of turkey breast samples during refrigerated storage. 0 1 2 3 4 5 6 7 8 9 10 0 3 6 9 12 15 t v c (l og 1 0 cf u/ g) storage time (days) control acetic acid chitosan+cumin 0 2 4 6 8 10 12 0 3 6 9 12 15 p t c (l og 10 c fu /g ) storage time (days) control acetic acid chitosan+cumin ital. j. food sci., vol. 30, 2018 634 3.2. ph value the ph and its alteration during storage time (0, 3, 6, 9, 12 and 15 storage days) for turkey breast in the control and two other treatments (aa and ch+c) have been shown in (fig. 3). an increase in ph from 5.86 to 7.07, 5.32 to 5.87, and 5.14 to 5.20 in the control, samples treated by aa 1%, and ch 2 % + c 1% was observed respectively, during 15 days storage time. for all of the treatments, storage time showed a significant (p < 0.05) effect on the ph values.. the initial ph value of the control sample was significantly (p < 0.05) higher than those from all of the treated samples. in all of the turkey breast samples, the values of ph showed a decrease by the day 6th of storage time, and then increased significantly. decrease of ph may be attributed to increasing of solubility of co2 at storage time, affecting growth of aerobic microflora (taheri et al., 2013), while an increase in ph of the control sample may be due to an increase in volatile compound contents (e.g. ammonia and trimethylamine), produced by either endogenous or microbial enzymes through storage time (fan et al., 2009; bazargani–gilani et al., 2015). no significant difference was observed between the ph values of treatment groups (aa and ch+c) through 15 days of storage time (p>0.05), with exceptions in samples stored for 3 and 15 days (p< 0.05). samples treated by acetic acid, chitosan and cumin essential oils expressed a gradual increase throughout storage period, probably due to the presence of the acidified antimicrobial agents (bazargani–gilani et al., 2015) and phenolic compounds (ibrahim and el-sherif, 2008). the achieved results of the present study are in solid agreement of those for chicken breast (containing pomegranate juice and chitosan and essential oil) (bazargani–gilani et al., 2015), beef meat (containing organic acid) (shaltout et al., 2014) and marinated chicken thigh (sodium lactate and lactic acid) (smaoui et al., 2012). figure 3. changes in ph values of turkey breast samples during refrigerated storage. 0 1 2 3 4 5 6 7 8 9 0 3 6 9 12 15 ph storage time (days) control acetic acid chitosan+cumin ital. j. food sci., vol. 30, 2018 635 3.3. total volatile basic nitrogen (tvb-n) total volatile basic nitrogen (tvb-n) mainly composed of ammonia and primary, secondary and tertiary amines, is extensively used as an indicator for meat deterioration determination. an increase in this parameter may be attributed to the activity of bacterial species and endogenous enzymes (fan et al., 2009; kyrana et al., 1997; nowzari et al., 2013; duan et al., 2010). the tvb-n values for turkey breast samples through storage period have been presented in (fig.4) as well. figure 4. changes in tvb-n values of turkey breast samples during refrigerated storage. an increase in tvb-n value was observed from 14.30 to 37.16, 14.23 to 32.40, and 14.23 to 28.06 mg n. 100 g-1, in control, samples treated by aa 1%, and ch+c 1%, respectively, through storage time for 15 days. according to (balamatsia et al., 2006), the value of 28-29 mg n. 100 g-1 is proposed as the highest acceptable level in poultry meat products. in the present study, the tvb-n values were detected higher than the acceptable limit by 12 and 15 days of storage time for the untreated samples (control) and the treated ones by acetic acid (1%), respectively. however, tvb-n values in treated samples by chitosan + cumin remained lower than the limit of acceptable index throughout the entire storage time. higher microbial counts resulted in a significant increase in the basic nitrogen fraction for the untreated samples compared to chitosan treated samples (nowzari et al., 2013; mohan et al., 2012). the tvb-n level presented enhancements gradually along with the time of storage for the control and both of the treated samples (p<0.05). by 6 days after storage, the tvb-n value of the control samples increased significantly compared to the treated samples, (p<0.05). at the end of the storage time, the tvb-n value of the control sample was detected significantly rather than the studied treatments (p<0.05). these achieved results are in line with those of previous studies that reported, the tvb-n value of fish and chicken meats treated by acetic acid, chitosan, and cumin essential oil showed reductions significantly (p<0.05) (raeisi et al., 2015; smaoui et al., 2012). an increase rate in tvb-n value of turkey breast treated by chitosan coating was also 0 10 20 30 40 50 0 3 6 9 12 15 t v b -n ( m g n /1 00 g) storage time (days) control acetic acid chitosan+cumin ital. j. food sci., vol. 30, 2018 636 inhibited compared to the control, that is in agreement with the achieved results of (yuan et al., 2016) and (ojagh et al., 2010). in addition, an increase in tvb-n value of turkey breast treated by chitosan + cumin coating was significantly lower than the samples treated by acetic acid by 15 days after storage period, presenting the synergism effect of chitosan coating on tvb-n value once used in combination with cumin seeds’ essential oils. 3.4. peroxide value (pv) determination lipid oxidation value was detected regarding the pv formation (primary oxidation compounds). the peroxide value of the studied samples indicates the concentrations of peroxide and hydroperoxide compounds produced during early stage lipid oxidation process. the peroxide value is commonly determined for a sample, and a sharp increase indicates the end of the shelf-life for the studied samples (taheri et al., 2013). alterations in pv values of the control sample and both treatments (aa and ch + c) during 15 days of storage time at the temperature of 4±1°c have been presented in (fig. 5). figure 5. changes in pv values of turkey breast samples during refrigerated storage. initial pv values of control, samples treated by acetic acid, and chitosan + cumin were determined as the following: 3.41, 3.21, and 2.10 meq o2. kg-1 followed by enhancements up to 13.83, 11.66, and 10.33, respectively. all of the samples expressed an increased pv value in turkey breast fillets once the frozen storage time increased (p<0.05). in control samples, pv values were detected higher than other samples in the current study during the storage time. the differences among detected peroxide values of the treated and untreated samples (control) may be associated with different bioactivity properties of the materials with natural origins. plant phenols and flavonoids are known to inhibit lipid peroxidation by quenching lipid peroxy radicals and reduce and/or chelate iron ions in lipoxygenase enzymes, and as a result of it, may prevent the initiation of lipid peroxidation reactions (allahghadri et al., 2010). the antioxidant property of cumin 0 2 4 6 8 10 12 14 16 0 3 6 9 12 15 p v ( m eq o 2/ k g) storage time (days) control acetic acid chitosan+cumin ital. j. food sci., vol. 30, 2018 637 seeds’ essential oil may be associated to phenolic and γ-terpinene compounds as well as monoterpenes (chen et al., 2014; raeisi et al., 2015). according to the achieved results, it is concluded that cumin seeds’ essential oils may have a significant effect on lipid oxidation retardation. similar results were reported by further scientists (raeisi et al., 2015; allahghadri et al., 2010). the results of the present study indicate that chitosan coating is effective in retarding the production of pv in turkey breast fillets stored by refrigeration. jeon et al. (2002) demonstrated that chitosan may be considered as a potential natural antioxidant for stabilizing shelf life enhancements in food products containing lipid. these results are in solid agreement with those of (hu et al., 2002), who reported that chitosan loaded by cinnamon essential oils was effective against lipid oxidation in pork meats stored at 4±1°c. the achieved results of the present research confirmed the obtained results by (bazargani-gilani et al., 2015), who reported that chitosan coating was effective in retarding of lipid oxidation reactions in chicken breast fillets stored at 4±1°c. moreover (nowzari et al., 2013) reported, that chitosan coating is effective in retarding of pov in trout fillets stored at 4±1°c. 3.5. sensory evaluation the sensory qualities of turkey breast samples were assessed in terms of appearance, color, odor, meat consistency and elasticity, with the use of a three-point hedonic scale (1 representing unacceptable and 3 indicating excellent). the turkey breast samples were considered acceptable for customers by 2, suggested by (octavian and octavian, 2010). the obtained results of the sensory evaluation of turkey breast samples have been shown in (table 2). table 2. changes in sensory attributes score of turkey breast samples stored at 4±°c. sensory attributes treatment 0 3 storage period (days) 6 9 12 15 appearance control aa ch+c 3.0±0.00aa 3.0±0.00 aa 3.0±0.00 aa 3.0±0.00 aa 3.0±0.00 aa 3.0±0.00 aa 2.6±0.54bab 3.0±0.00 aa 3.0±0.00 aa 1.2±0.44 bb 2.6±0.54aab 2.8±0.44aab 1.0±0.00 cb 2.0±0.70 bb 2.4±0.54 ab 1.0±0.00 bb 1.8±0.44 ab 2.0±0.00 ac meat elasticity control aa ch+c 3.0±0.00 aa 3.0±0.00 aa 3.0±0.00 aa 2.8±0.44 aa 3.0±0.00 aa 3.0±0.00 aa 2.8±0.44 aa 3.0±0.00 aa 3±0.00 aa 1.8±0.44 bb 2.6±0.54 ab 2.8±0.44aab 1.0±0.00 cc 1.8±0.83 bc 2.4±0.54 ab 1.0±0.00 cc 1.6±0.54 bc 2.0±0.00 ac odor control aa ch+c 3.0±0.00 aa 3.0±0.00 aa 3.0±0.00 aa 2.6±0.54 bb 3.0±0.00 aa 3.0±0.00 aa 2.0±0.00 bc 2.6±0.54 abb 3.0±0.00 aa 1.6±0.54 bd 2.2±0.44 abc 2.8±0.44aab 1.0±0.00 ce 1.6±0.54 bd 2.4±0.54 ab 1.0±0.00 be 1.6±0.54 abd 2.0±0.00 ac color control aa ch+c 3.0±0.00 aa 3.0±0.00 aa 3.0±0.00 aa 2.8±0.44 aa 3.0±0.00 aa 3.0±0.00 aa 2.8±0.44 aa 2.8±0.44aab 3.0±0.00 aa 1.6±0.54 cb 2.4±0.54bbc 3.0±0.00 aa 1.0±0.00 bc 2.2±0.44 ac 2.2±0.44 ab 1.0±0.00 bc 1.4±0.54 bd 2.0±0.00 ab means in column with different small letters indicate significant differences (p<0.05) among treatments, and means in row with different capital letters indicate significant differences (p<0.05) as result of refrigerated storage time. at the beginning of the evaluations, the appearance, odor, color, and consistency of breast fillets were fresh. as expected, progressive quality deteriorations of samples were observed, as a result of an increase in storage time (p<0.05). the sensory evaluation result is associated with the microbial and chemical properties. due to a high lipid oxidation and microbial growth, the control samples (untreated) of turkey breast fillet showed deterioration, appearing as off-odor, and slimy as well as discoloration after 6 days of ital. j. food sci., vol. 30, 2018 638 storage period. in comparison, chitosan + cumin coated samples showed an acceptable sensory score up to 15 days of storage time. thus, antioxidant and antimicrobial effects of chitosan + cumin coating may minimize the oxidative reactions, and as a result extending the products’ shelf life while maintaining their quality as well (ojagh et al., 2010). the achieved results indicated that chitosan + cumin treatment led to the highest score among other treatments in all of the studied sensory properties through storage time. this phenomenon may be attributed to the unique flavor of cumin seeds’ essential oil. addition of cumin seeds’ essential oil to chitosan coating enhanced the beneficial effects on color, odor, and overall acceptability of turkey breast fillets significantly (p<0.05) in the final days of storage periods. considerable correlations among the microbial and chemical qualities as well as sensory attributes were found by (bazargani-gilani et al., 2015; kanatt et al., 2013; shaltout et al., 2014; vasilatos and savvaidis, 2013; raeisi et al., 2015; ojagh et al., 2010) where were in solid agreement of the achieved results of the current study. 4. conclusions fresh meats and their products are very susceptible to deterioration by microbial growth and oxidative reactions. the shelf life of refrigerated turkey meat is normally short due to the chemical and microbial activities in it, leading to quality loss and spoilage. the achieved results of microbial (tvc and ptc), physicochemical (ph, tvn-b and pv) and sensory evaluation analyses indicated that acetic acid and ch + c (chitosan and cumin oil) coating on turkey breast fillets may lead to the maintenance of qualitative characteristics, improvement of microbial safety and extension of the shelf life of meat products through the chilled storage period. chitosan + cumin treatment could maintain turkey breast fillet shelf life till the end of the storage period (day 15), while acetic acid treatment and the control sample had a shelf life just for 9 and 6 days, respectively. therefore, natural preservatives such as chitosan + cumin oil combination may be used as a safe preservation approach to extend the shelf life of chilled turkey meat and its products. acknowledgements the authors appreciate jundishapur university and chamran university (ahvaz, iran) for their collaboration and facilities provided during the current study. references allahghadri t., rasooli i., owlia p., nadooshan m.j ., ghanfari t., taghizadeh m. and astaneh s.d. 2010. antimicrobial property, antioxidant capacity, and cytotoxicity of essential oil from cumin produced in iran. journal of food science 75:h54-h61. aoac. 2000.“official methods of analysis” 17th edition. association of official analytical chemists, arlington, washington (usa). balamatsia c.c., rogga k., badeka a., 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63(1):519-526. iacobellis n.s., lo cantore p., capasso f. and senatore f. 2005. antibacterial activity of cuminum cyminum l. and carum carvi l. essential oils. journal of agriculture food chemistry 53(1):57-61. ibrahim s.m. and el-sherif s.a. 2008. effect of some plant extracts on quality aspects of frozen tilapia (oreochromis niloticus l.) fillets. global veterinaria 2(2):62-66. jeon y.j., kamil j. y.v. a. and shahidi f. 2002. chitosan as an edible invisible film for quality preservation of herring and atlantic cod. journal of agriculture food chemistry 20:5167-5178. kanatt s.r., rao m.s., chawla s.p. and sharma a. 2013. effects of chitosan coating on shelf-life of ready-to-cook meat products during chilled storage. food science and technology 53:321-326. kyrana v.r., lougovois v.p. and valsamis d.s. 1997.assessment of shelf life of maricultured gilthead sea bream (sparus aurata) stored in ice. international journal of food science and technology 32:339-347. latou e., mexis s.f., badeka a.v., kontakos s. and kontominas m.g. 2014. combined effect of chitosan and modified atmosphere packaging for shelf life extension of chicken breast fillets. lwtfood science and technology 55:263-268. mohan c.o., ravishankar c.n., lalitha k.v. and srinivasa gopal t.k. 2012. effect of chitosan edible coating on the quality of double filleted indian oil sardine (sardinella longiceps) during chilled storage. food hydrocolloids 26:167-174. no h.k., meyers s.p., prinyawiwatkul w. and xu z. 2007. applications of chitosan for improvement of quality and shelf life of foods. journal of food science.72:r87-r100. nowzari f., shabanpour b. and ojagh s.m. 2013. comparison of chitosan–gelatin composite and bilayer coating and film effect on the quality of refrigerated rainbow trout. food chemistry 141:1667-1672. octavian b. and octavian b. 2010. raw chicken leg and breast sensory evaluation. food science and technology 11(1):2530. ojagh s.m., rezaei m., razavi s.h. and hosseini s.m.h. 2010. effect of chitosan coatings enriched with cinnamon oil on the quality of refrigerated rainbow trout. food chemistry 120:193-198. ouattara b., simard r.e., piette g., begin a. and holley r.a. 2000. inhibition of surface spoilage bacteria in processed meats by application of antimicrobial films prepared with chitosan. international journal of food microbiology 62:139148. pereda m., ponce a.g., marcovich n.e., ruseckaite r.a. and martucci j.f. 2011. chitosan–gelatin composites and bilayer films with potential antimicrobial activity. food hydrocolloids 25:1372-1381. raeisi s., young quek s., ojagh s. m. and alishahi a.r. 2015. effect of cumin (cuminum cyminum l.) seed and wild mint (mentha longifolia l.) leaf extract on the shelf life and quality of rainbow trout ( oncorhynchus mykiss) fillets stored at 4 ͦc± 1. journal of food safety 1-11. shahidi f., arachchi j.k.v. and jeon y.j. 1999. food applications of chitin and chitosans. trends in food science and technology 10(2): 37-51. ital. j. food sci., vol. 30, 2018 640 shaltout f.a., gerges m.t. and shewail a. a. 2014. impact of organic acids and their salts on microbial quality and shelf life of beef meat. food safety science 2:360-370. sharifi-rad j., hoseini-alfatemi s.m., sharifi-rad m. and teixeira da silva j.a. 2015. antibacterial, antioxidant, antifungal and anti-inflammatory activities of crude extract from nitraria schoberi fruits. 3 biotech 5(5): 677-684. smaoui s., ben hlima h. and ghorbel r. 2012. the effect of sodium lactate and lactic acid combinations on the microbial, sensory, and chemical attributes of marinated chicken thigh. poultry science 91:1473-1481. taheri s., motallebi a.a., fazlara a. and aghababyan a. 2013. effect of zataria multiflora boiss (avishan shirazi)essential oil on oxidative progress in frozen cobia fish fillets during storage. journal of aqua food product technology 22:310321. taheri s., motallebi a. a., fazlara a., aftabsavar y. and aubourg s.p. 2012. effect of previous ascorbic acid treatment on the fatty acid profile of cobia (rachycentron canadum) fillets during frozen storage. international journal of fats and oils 63(1):70-78. vaithiyanathan s., naveena b., muthukumar m., girish p. and kondaiah n. 2011. effect ofdipping in pomegranate (punica granatum) fruit juice phenolic solution on the shelf life of chicken meat under refrigerated storage (4° c). meat science 88(3):409-414. vasilatos g.c. and savvaidis i.n. 2013. chitosan or rosemary oil treatments, singly or combined to increase turkey meat shelf-life. international journal of food microbiology 166:54-58. wannes w. a., mhamdi b., stiti j., jemia m. b., ouchikh o., hamdaoui g., kchouk m.e. and marzouk b. 2010. antioxidant activities of the essential oils and methanol extracts from myrtle (myrtus communis var. italica l.) leaf, stem and flower. food chemical toxicology 48:1362-1370. yuan g., lv h., tang w., zhang x. and sun h. 2016. effect of chitosan coating combined with pomegranate peel extract on the quality of pacific white shrimp during iced storage. food control 59:818-823. zhang h., kong b., xiong y.l. and sun x. 2009. antimicrobial activities of spice extracts against pathogenic and spoilage bacteria in modified atmosphere packaged fresh pork and vacuum packaged ham slices stored at 4 ͦ c. meat science 81:686-692. paper received february 2, 2018 accepted may 7, 2018 ijfs#1917_bozza ital. j. food sci., vol. 32, 2020 743 paper lactic acid bacteria microbiota of “pirot`s kashkaval” d. bojana*1, m. nebojša2, c. dragoljub3 and s. dragiša1 1faculty of technology, university of niš, bulevar oslobođenja 124, 16 000 leskovac, serbia 2college of agriculture and food technology, ćirila i metodija 1, 18 400 prokuplje, serbia 3faculty of technology, university of novi sad, bulevar cara lazara 1, 21 000 novi sad, serbia *corresponding author: bojana.danilovic@junis.ni.ac.rs abstract “pirot`s kashkaval” is an autochthonous dairy product belonging to pasta-filata cheeses. in order to determine the changes in the lactic acid bacteria microbiota during 60 days of ripening, 315 lactic acid bacteria strains were isolated from ewe`s and cow`s milk cheese and identified as enterococcus faecium, pediococcus acidilactici, pd. pentosaceus, lactobacillus casei/rhamnosus, lb. plantaum, lb. casei, lb. fermentum, lb. paracesei, lb. rhamnosus and streptococcus macedonicus. enterococci were the most dominant isolated strains. in the final stages of ripening, the increase of the population of lb. casei and pd. acidilactici was observed for ewe`s and cow`s milk cheese, respectively. keywords: cow`s milk cheese, ewe`s milk cheese, molecular identification, lactic acid bacteria, “pirot`s kashkaval”, ital. j. food sci., vol. 32, 2020 744 1. introduction “pirot`s kashkaval” cheese is an autochthonous dairy product produced in pirot, republic of serbia and its surroundings by a specific technology. the production process, which differs this type of cheese from other produced in serbia includes the cooking of partly fermented curd prior to ripening. this type of cheese has light to intensively yellow color, monolith, partially layered and elastically-plastic structure. the combination of cow’s and ewe`s milk is, usually, being used for the production. the taste of “pirot`s kashkaval” is mildly sour and piquant, specific and depends on the type of used milk (ostojić et al., 2012). the production is characteristic for the process of cooking of the sliced fermented curd for 5-8 min. at temperature of 72-75°c. the cheese curd is then salted, mixed, molded and additionally ripened for a few months (mančić and mančić, 2005). the cooking of curd in hot water has the significant influence on its microbiota. thermal treatment of fermented curd is a way of pasteurization of cheese dough, which leads to certain biochemical and microbiological processes. during this process the significant drop in bacterial and yeast number occurs, so it can be very important in the cases when it’s not possible to get good quality milk and when milk pasteurization is not applied (alrubai, 1979). the formation of sensory profile of cheeses is the result of metabolic activity of the present microbiota (benito de cardenas, et al., 1990; mustafa, 2006, duan et al., 2008). products of glicolysis, proteolysis of casein and lipolysis of fats, as the main metabolic functions of lactic acid bacteria (lab), have a great influence on cheese flavor. since starter cultures are not added during the production of “pirot`s kashkaval”, cheese flavor originates from metabolic products of autochthonous microbiota. the advantages of autochthonous microbiota are fast growth and development and production of specific sensory characteristics of the products. furthermore, determination of the microbiota composition is an important step in analyzing traditional fermented dairy products and eventual devinition of autochthonous starter cultures. the aim of this work was the isolation and characterization of lab present during the process of ripening of “pirot`s kashkaval” unique in serbia for the production process. for that purpose, 10 samples of cheese produced from cow`s and ewe`s milk were collected during 60 days of ripening and analysed. 2. materials and methods 2.1. cheese samples “pirot`s kashkaval” was produced by a traditional process from cow’s and ewe`s milk. after the addition of rennet, the whey was separated, and the fresh curd was fermented for a few days. the fermented curd was sliced and cooked in hot water (72-75°c). after that, the curd was salted, molded and ripened in a ripening chamber. sampling was performed during the ripening process of 10-15 days at 25°c and until the end of the two months ripening period at 10°c. samples of cow`s milk cheese (cc) and ewe`s milk cheese (ec), were collected during the process of ripening after 1 (cc1 and ec1), 5 (cc5 and ec5), 20 (cc20 and ec20), 30 (cc30 and ec30) and 60 (cc60 and ec60) days of ripening. the samples were then packed in ital. j. food sci., vol. 32, 2020 745 vacuum, transported and kept up to 3 days at + 5°c to the moment of microbiological analyses. 2.2. isolation and determination of lab number cheese samples (10 g) were transferred aseptically to 90 ml of 2% (w/v) sodium citrate solution (t=45°c) and homogenized for 30 minutes. the isolation of lab was performed by serial dilution method. a volume of 1 ml of appropriate dilution was transferred into a petri dishes and layered with mrs (torlak, belgrade, serbia), m17 (merck, darmstadt, germany) and mse agar (tripton 10 g l-1, gelatine 2.5 g l-1, yeast extract 5 g l-1, sucrose 100 g l-1, glucose 5 g l-1, sodium citrate 1 g l-1, sodium azide 0.075 g l-1 and agar 13 g l-1) for determination of presumptive lactobacilli, lactococci and leuconostocs, respectively. after solidification, second layer of medium was poured in order to achieve micro-aerophilic conditions preferable for the growth of lab. plates with mrs and m17 agar were incubated for 48 h at 30 and 45°c in order to determine both mesophilic and thermophilic lab strains, while the mse agar plates were incubated 48h at 30°c. determination of total number of mesophilic bacteria was performed on nutrition agar (na) plates (torlak, belgrade, serbia) (48 h, 30°c). the determination of number of bacteria was performed in triplicate and the values are presented as the mean value. after the incubation and enumeration, at least 30 lab colonies were selected from each sample and purified. preliminary characterization of the isolates was done by gram staining and catalase test. bacterial cultures were stocked in liquid medium (mrs and m17 broth), with addition of 20% (v/v) glycerol at the temperature of -20°c until further analysis. 2.3. molecular identification of isolates the extraction of total dna, pcr amplification with (gtg)5 primer and electrophoresis were done by already described method (nikolić et al., 2008). sequencing of 16s rrna genes was performed by multiplying of fragments by u968 (5′aacgcgaagaaccttac-3′) and l1401 (5′-aacgcgaagaaccttac-3′) primers (randazzo et al., 2002) and using taq dna polymerases. reaction was carried out in pcr system 2700 (applied biosystems) with the following parameters: starting denaturation of dna during 5 min at 94°c, 30 cycles of 30 s at 94°c, 30 s at 55°c, 30 s at 72°c and the extension of incomplete products for 7 min at 72°c. electrophoresis of multiplied products was done at 1% (w/v) agar gel with the addition of ethidium bromide. multiplied fragments were purified using qiaquick pcr purification kit/250 (qiagen gmbh, hilden, germany), while their sequencing was done in macrogen in seoul, south korea. blast algorithm (www.ncbi.nlm.nih.gov/blast) was used for determining the most similar sequence from ncbi base. 3. results and discussion 3.1. determination of number of bacteria the changes of total mesophilic bacteria and lab number determined at mrs, m17 and mse agar plates in analyzed ewe`s and cow`s milk cheese samples are shown in figs. 1 and 2, respectively. during the first 5 days of ripening, a slight increase of total number of ital. j. food sci., vol. 32, 2020 746 mesophilic lab determined at mrs agar plates was noticed, from 7.7 to 7.8 log cfu g-1, after which the number decreases to 6.4 log cfu g-1 (fig. 1). figure 1. the change of number of bacteria during ripening of pirot cheese produced from ewe`s milk at мrs (■), m17 (▲), мse (♦) and na (●) plates incubated at 30 (full symbols) and 45 °c (empty symbols). figure 2. change of the number of bacteria during ripening of pirot cheese produced from cow`s milk at мrs (■), m17 (▲), мse (♦) and na (●) plates, incubated at 30°c (full symbols) and 45°c (empty symbols). the number of microorganisms on m17, mse and nutrition agar plates decreased in the first 5 days of ripening. thus, number of mesophilic lab at m17 agar plates decreased from 8.3 to 6.9 log cfu g-1 during the first 5 days, after which it was constant with the value of 7.1 log cfu g-1. the number of thermophilic lab was lower compared to the number of mesophilic lab, regardless the used medium. also, number of thermophilic bacteria decreased continually after the 30th day of ripening. the total number of mesophilic bacteria incubated at nutrition agar plates in first 5 days decreased from 7.9 to ital. j. food sci., vol. 32, 2020 747 7.2 log cfu g-1, and that value maintained to the end of ripening. the number of mesophilic lab determined on mse agar plates decreased in first 5 days from 7.5 to 5.2 log cfu g-1 and after that it increased to 6.8 log cfu g-1 (fig. 1). the number of bacteria in cow`s milk cheese samples is shown in fig. 2. the increase of the lab number at the beginning of fermentation (to 5th day) was only noticed on m17 agar plates for both mesophilic (from 7.5 to 7.7 log cfu g-1) and thermophilic bacteria (from 7.3 to 7.8 log cfu g-1). in the following 15 days, number of mesophilic lab decreased to 6.9 log cfu g-1, and thermophilic to 7.2 log cfug-1. at the end of fermentation, the number of lab on m17 agar plates was 7.3 log cfu g-1. the number of lab on mrs agar in the first 20 days decreased for both bacterial groups reaching the value of cca 6.0 log cfu g-1. mesophilic and thermophilic lab on mrs agar plates reached maximum on the 30th day of ripening (7.7 log cfu g-1), afterwards a slight decrease was observed (fig. 2). high initial number of lab in both cheese types can be explained by the process of fermentation, which took place before curd cooking. the fermented curd was thermally processed by immersion in hot water before sampling, but it can be assumed that heat stressed cells remained viable. the decrease of the lab number was observed, regardless the type of milk used for making cheese, in the period from the 1st to the 5th day of ripening due to the adaptation of the microbiota to the ripening conditions. this decrease is in accordance with the literature results (alrubai, 1979; gobbetti et al., 1997; baruzzi et al., 2002), which indicate that cooking of curd has a great influence on the microbiota and lead to a significant decrease of total number of bacteria in the first stage of ripening. the differences in the lab number of cow`s and ewe`s milk cheese are probably the result of the different microbiota composition in these two cheese types. the stagnation of lab number during the ripening of cow`s milk cheese lasts longer (30 days, fig. 1) in relation to the samples of ewe`s milk cheese (20 days, fig. 2). after that, the constant number of bacteria was achieved in the range from 6.5 to 7.4 log cfu g-1, for both type of cheese. at the end of fermentation, the lowest number was observed for thermophilic lab on mrs agar plates. this can be explained by the ripening conditions (temperature 10°c) which favors the growth of mesophilic bacteria. the domination of mesophilic lab in the later stages of pasta filata cheese ripening has already been reported by succi et al. (2016). determined lab number in analyzed samples is in accordance with earlier researches for “pirot`s kashkaval” (mijačević et al., 2005a; mijačević et al., 2005), italian cheese pugliese, silano and molise, produced from pasteurized cow milk (gobbetti et al., 2002; coppola et al., 2003; piraino et al., 2005), mozzarella from cow milk (de candia et al., 2007) and taleggio cheese from lombardi in italy (gobbetti et al., 1997). 3.2. identification of lactic acid bacteria isolates from 5 different samples of pirot`s ewe`s milk cheese 173 lab isolates were obtained. the identification was performed by (gtg)5-pcr fingerprinting and 16s rrna gene sequencing. according to the (gtg)5-pcr fingerprinting low level of diversity was observed among the isolates and representative fingerprints are shown in fig. 3. most of the isolates belong to cocci (130), while 43 belong to bacilli. isolates were identified as the representatives of enterococcus faecium, pediococcus acidilactici, pediococcus pentosaceus, lactobacillus casei/rhamnosus, lactobacillus plantaum, lactobacillus casei, lactobacillus fermentum, lactobacillus paracesei and streptococcus macedonicus. among them, the most dominant were en. faecium (50%) and pd. acidilactici (16%), while the least isolated strains were lb. fermentum (1%) and lb. paracasei (0.5%). during 60 days of ripening of pirot`s ital. j. food sci., vol. 32, 2020 748 cow`s milk cheese 142 lab isolates were isolated and identified as en. faecium (44.4% of isolates), pd. acidilactici (50%), pd. pentosaceus (0.7%), lb. plantarum (1.4%), lactobacillus rhamnosus (0.7%) and lb. casei (2.8%). the presence of different lab strains is important in cheese ripening due to their unique metabolism products (cogan, 2000). dominancy of particular lab strain is directly dependent of milk type, animal breeding, and way of feeding, type and quality of pasture, altitude, and production process, cheese storage conditions and many other (topisirović et al., 2006). the presence and prevalence of concrete lab strains in analysed cheese samples is mainly affected by lab heat resistance during the curd cooking. among lab strains isolated from pirot`s cheese samples, the representatives of genera enterococcus and pediococcus dominate, while in a smaller percentage lactobacillus spp. and streptococcus sp. were present. the obtained results are in accordance with literature data according to which lab isolated from cheese belong to genera enterococcus, pediococcus, lactobacillus, streptococcus and lactococcus (fleet, 1999; poznanski et al., 2004; meng et al., 2018; vandera et al., 2019). figure 3. representative (gtg)5-pcr fingerprints of lab strains isolated from pirot cow`s and ewe`s milk cheese: en. faecium (1,2), pd. acidilactici (3), pd. pentosaceus (4), lb.plantarum (5), lb. casei (6,7,8), lb. fermentum (9), lb. paracesei (10,11), lb. rhamnosus (12), lb. casei/rhamnosus (13) and st. macedonicus (14). 3.3. microbiota dynamics in “pirot`s kashkaval” the results of monitoring changes in microbiota during ripening of cheese made from ewe`s milk are presented on fig. 4. after 24 hours (sample ec1), the most frequently isolated was en. faecium (60%), while st. macedonicus (36%) and pd. acidilactici (4%) were isolated as well. in sample after 5 days of fermentation (ec5) the population of en. faecium increased (85%), as well as pediococci (9%), while streptococci were not isolated. the percentage of enterococci decreased significantly after 20 days (ec20), while the percentage of pediococci (pd. acidilactici – 47% and pd. pentosaceus – 18%) and lactobacilli increased (lb. casei/rhamnosus 6% and lb. plantarum 9%). in the sample after 60 days of fermentation (ec60) en. faecium (55%) was dominant, while lb. casei (20%), pd. acidilactici (14%) and lb. casei/rhamnosus (3%) were also isolated. lb. fermentum (5%) and lb. paracasei (3%) were identified only in this sample. the change of microbiota during 60 days cow`s milk cheese ripening is shown in fig. 5. ital. j. food sci., vol. 32, 2020 749 figure 4. frequency of isolation of lab strains after 1 (ec1), 5 (ec5), 20 (ec20), 30 (ec30) and 60 (ec60) days of ripening of pirot ewe`s milk cheese. figure 5. frequency of isolation of lab strains after 1 (cc1), 5 (cc5), 20 (cc20), 30 (cc30) and 60 (cc60) days of ripening of pirot cow`s milk cheese. at the beginning of ripening, in the sample cc1, two strains of lab where isolated: en. faecium with 91% and pd. acidilactici, 9%. the population of en. faecium decreased (66%), while the population of pd. acidilactici (33%) increased in the sample cc5. after 20 days of ripening (cc20) pd. acidilactici was isolated in the frequency of 39%. on the other hand, the presence of en. faecium was on the previous level, 47%. in this sample, lb. casei was isolated with 13% of population. to the end of ripening, pd. acidilactici was dominant with 97% in the sample cc30 and 85% in the sample cc60. also, another representative of ital. j. food sci., vol. 32, 2020 750 pediococci, pd. pentosaceus, was isolated, with 4% of total population of cc60. lactobacilli were isolated in low frequency in the samples after 30 days (lb. casei 3%) and 60 days (lb. rhamnosus 4% and lb. plantarum 7%) of ripening. generally, enterococci were the most numerous genera in pirot`s cheese, thus en. faecium made 50% of total identified microbiota in ewe`s milk cheese and 44% in cow`s milk cheese. this strain was identified in all phases of ripening in ewe`s milk cheese, and the highest number was observed at the beginning of ripening. in the cow`s milk cheese samples, en. faecium dominated in the first stage of ripening, while it was not isolated after 30 days. enterococci often present the considerable part of lab microbiota of many traditionally produced types of cheese (fontecha et al., 1990; cogan et al., 1997; morea et al., 1999; domingos-lopes et al., 2017; vandera et al., 2019). the presence of enterococci (especially en. faecalis and en. faecium) is characteristic for cheese made of ewe`s or goat’s milk. as well, these two species have a very important role in lipolysis, proteolysis and production of diacetyl in cheese (giraffa, 2002). the prevalence of enterococci in analysed samples are probably the result of the use of raw milk for the cheese production. the number of enterococci can be up to 60000 times higher in cheese produced from raw milk than from pasteurized milk (pappa et al., 2019). pediococci often take part in autochthonous microbiota of cheese and have a significant impact on ripening of many cheese types (bhowmik and mart, 1990). pd. acidilactici was isolated from all analyzed cheese samples. in ewe`s milk cheese the highest percent of isolation was in the sample after 20 days of ripening, while in cow`s milk cheese it was dominant in the final stage of ripening. pd. acidilactici and pd. pentocaseus were identified in cheese produced in the mediterranean (poznanski et al., 2004; morea et al., 2007; aydemir et al., 2015; de pasquale et al., 2019), while in ripe italian cheese parmigiano reggiano they are predominant (gobbetti et al., 2002). on the other hand, in the analysis of pirot`s cow`s milk cheese (ostojić, 2012), this bacterium was not identified. the other lab strains identified in pirot`s cheese belonged to lactobacilli (lb. plantarum, lb. casei/rhamnosus, lb. rhamnosus, lb. casei and lb. paracasei) and streptococci (st. macedonicus). lactobacilli represent dominant population of many types of cheese where they have a significant effect on formation of aroma compounds (beresford et al., 2001; wouters et al., 2002; de pasquale et al., 2019). lb. plantarum was isolated from italian and argentinean cheese (zago et al., 2011) and lb. fermentum was isolated from caciocavallo pugliese (morea et al., 2007). lb. rhamnosus was identified in cheese parmigiano reggiano (coppola et al., 2005; succi et al., 2005; de dea lindner et al., 2008; bove et al., 2011) and irish cheddar cheese (mlalazi et al., 2011). lb. casei and lb. paracasei were isolated and identified in spanish cheese cabrales (belén-flórez et al., 2006), turkish cheese kasar (aydemir et al., 2015) and italian cheese montasio (marino et al., 2003). these lactobacilli can have good probiotic potential and possibility to produce different bacteriocins (mlalazi et al., 2011; zago et al., 2011; milićević et al., 2014). the largest diversity of lab strains in the analysed samples was observed after two months ripening period. high diversity of lab strains and significant changes in microbiota composition has been reported in various types of pasta filata cheeses as the result of different processing (curd cooking and stretching) and ripening conditions (gobbetti et al., 2018). additionally, greater diversity of lactobacillus strains has been noticed in the later stage of ripening, in correlation with the results of sant`anna et al. (2019). in pirot`s ewe`s milk cheese the population of st. macedonicus was also detected. this bacteria was first identified in greek cheese kasseri (tsakalidou et al., 1998; georgalaki et al., 2000) and was also isolated from italian types of cheese from raw ital. j. food sci., vol. 32, 2020 751 milk: asiago, montasio, monte veronese, morlacco, spressa, fontina, ragusano, mozzarella (lombardi et al., 2004), nostrano di primiero (poznanski et al., 2004), toma piemontese (zeppa et al., 2004) as well as from pirot cow`s milk cheese (ostojić et al., 2012). this streptococcus has good acidification, proteolytic and bacteriocin characteristics, thus some authors claim it as multi-functional and very suitable for the production of dairy products (de vuyst and tsakalidou, 2008). microbiological profile of lab population similar to lab population of pirot`s cheese was reported for traditionally made types of cheese in the region of the mediterranean: caciovallo molise (coppola et al., 2003), caciocavallo pugliese (morea et al., 2007), montasio (marino et al., 2003), toma piemontese (zeppa et al., 2004), nostrano si primiero (poznanski et al., 2004) and kasar (aydemir et al., 2015). 4. conclusions “pirot`s kashkaval” is an autochthonous product traditionally made in serbia. in order to continue and promote the production of this cheese, is very important to understand the composition and the changes in microbiota which occurs during ripening. since lab are being recognized as the most important in cheese ripening, the isolation and identification of lab from “pirot`s kashkaval” is of primary importance. lab microbiota isolated during ripening of “pirot`s kashkaval” made of cow`s and ewe`s milk was constituted of the genera enterococcus, pediococcus, lactobacillus and streptococcus. the most dominant species, in both types of cheese, was en. faecium. during the ripening of cow`s milk cheese, the population and diversity of lactobacilli increased. on the other hand, ripening of ewe`s milk cheese was characterized by the increase of pediococci population. understanding of the microbiological changes which occurred during ripening of “pirot`s kashkaval” can contribute to the definition of starter cultures suitable for industrial production of “pirot`s kashkaval”. aknoledgments this work was performed at the university of nis, faculty of technology in leskovac. references alrubai a. 1979. protein changes during the ripening of cheese produced with the addition of different proteolytic enzymes, phd theses, university of belgrade aydemir o., harth h., weckx s., dervişoğlu m. and de vuyst l. 2015. microbial communities involved in kaşar cheese ripening. food microbiol. 46:587-595 baruzzi f., matarante a., morea m. and cocconcelli p.s. 2002. microbial community dynamics during the scamorza altamurana cheese natural fermentation. j. dairy sci. 85:1390-1397. belén-flórez a., lópez-díaz t.m., álvarez-martín p. and mayo b. 2006. microbial characterisation of the 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and manachini p.l. 2004. characterization of autochthonous lactic acid bacteria from an artisanal italian cheese. acta agriculturae slovenica. 84:3-9. paper received june 10, 2020 accepted august 6, 2020 ijfs#1269_bozza ital. j. food sci., vol. 30, 2018 715 paper effect of heating on chemical parameters of extra virgin olive oil, pomace olive oil, soybean oil and palm oil a.m. giuffrè*, m. caracciolo, c. zappia, m. capocasale and m. poiana dipartimento di agraria, università degli studi mediterranea di reggio calabria, contrada melissari, 89124 reggio calabria, italy *corresponding author: tel.: +39 9651694362 e-mail address: amgiuffre@unirc.it abstract this work studied the oxidative stress on the chemical properties of extra virgin olive oil, pomace olive oil, soybean oil and palm oil during heating. the highest relative increase in free acidity was found in pomace olive oil. peroxide value as an absolute value (meq o2/kg) was lowest in palm oil 1.4 (unheated), 4.0 (180°c 120 min), 6.4 (220°c 120 min). extra virgin olive oil had lower spectrophotometric indices (k232, k270 and ∆k) compared to the solvent extracted oils. total phenols were highest in the extra virgin olive oil (196.8 mg/kg) and decreased to 59.8 and to 66.8 mg/kg after 120 min of heating at 180°c and 220°c respectively. a decreasing trend was also found in the tocopherol content with the highest % reduction (-79.5%) in evoo heated at 220°c for 120 min. this was in agreement with the antioxidant activity trend measured with the abts assay (2,2’azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) (155.9 μm te/100 g) and with the oxygen radical absorbance capacity (orac) assay (316.1 μm te/100 g) in the unheated extra virgin olive oil. keywords: abts, dpph●, evoo, heating, orac, oxidation ital. j. food sci., vol. 30, 2018 716 1. introduction lipids play a key role in human health. in the human diet, they are an important and essential part along with carbohydrates and proteins, representing not only a source of energy and protection for the organs and the body thanks to their functional properties, but also taking part in the metabolic processes, being components of bio-membranes and serving as carriers of biologically active substances (vaskova and buckova, 2015). edible oils are chemically unstable and susceptible to oxidation processes, especially if they are in the presence of oxygen, light, moisture, heat (calvo et al., 2012), enzymes and traces of metals (vaskova and buckova, 2015). oxidation is the main cause of deterioration of oils and fats, which besides reducing shelflife, sensorial characteristics and nutritional value, also produces toxic compounds (poyato et al., 2014). more than 400 chemical components have been detected in deteriorated fried edible vegetable oils (paul and mittal, 1996). introducing lipid oxidation products into the diet may lead to an increased risk of cardiovascular disorders, such as the formation of atherosclerotic plaques (halliwell and chirico, 1993). oxidative stress seems to be linked to many multi-factorial diseases, especially cancers, cardiovascular diseases and inflammatory disorders. oxidation alters physiologically important molecules, including proteins, lipids, carbohydrates and nucleic acids, together with the modulation of gene expression and the inflammatory response (laguerre et al., 2007). the endogenous antioxidants of vegetable oils provide a natural resistance to oxidative stress. among all categories of olive oil, extra virgin olive oil has gained significant importance from the gastronomical, nutritional, therapeutic and economic point of view. it is considered the best olive oil for its organoleptic characteristics, stability and chemical composition (calvo et al., 2012). the biological activities associated with the consumption of extra virgin olive oil (antioxidant, anti-inflammatory, chemo-preventive and anti-cancer) have promoted the use of this oil, not only as food but also as an ingredient in a wide range of industrial food products (calvo et al., 2012). virgin olive oil is fundamental in the mediterranean diet and contributes to its health benefits (boskou, 2015) and it is widely used in the countries of the mediterranean basin, such as italy (proto and zimbalatti, 2015). pomace olive oil is a secondary product in the olive oil industry, but it is important because otherwise olive pomace would be considered a waste. although, palm oil is the most widely consumed edible vegetable oil in the world, with a production of 60.96 million metric tons in 2015-2016 (statista.com, 2017), controversial results have been found in relation to its use and human health (mancini et al., 2015). soybean oil is the second most widely consumed edible vegetable oil in the world, with a production of 51.45 million metric tons in 2015-2016 (statista.com, 2017). after oil extraction, the residue is widely used as animal feed. this work has been based on these premises and aims to study the chemical property variations during heating of the three most popular edible vegetable oils (extra virgin olive oil, soybean oil and palm oil), together with pomace olive oil, which could become more important in the edible vegetable oil market. the aim of this work was to study the variation in the chemical properties of the three most popular edible vegetable oils during heating at 180 and 220°c and for 30, 60 and 120 min. ital. j. food sci., vol. 30, 2018 717 2. materials and methods 2.1. vegetable oils four vegetable oils were used in this experiment: extra virgin olive oil (evoo) and pomace olive oil (po) were produced in the harvest year 2016-2017 in the region of calabria (south italy) and bought directly from the producer, palm oil (p) and soybean oil (so) were purchased in a supermarket. the oils were analysed before heating and after heating at two different temperatures (180°c and 220°c) for 30, 60 and 120 minutes for each temperature. a 100 g aliquot of each oil was placed in a glass pyrex container which was heated in an oven. nine glass pyrex containers for each oil and for each temperature (180 and 220°c) were prepared, three of them for each temperature were taken out of the oven after each time established by the experimental design (30, 60 and 120 min) to conduct analyses in triplicate. 2.2. reagents diethyl ether, ethyl alcohol, sodium hydroxide, phenolphtalein, chloroform, acetic acid, potassium iodide, soluble starch, cyclohexane, p-anisidine, methanol, folin-ciocalteu reagent, sodium carbonate, gallic acid, 1,1-diphenyl-2-picrylhydrazyl radical (dpph●), 2,2΄-azobis(2-methylpropionamide) dihydrochloride (aaph), trolox, 2,2-azinobis-(3ethylbensothiazoline)-6-sulfonic acid (abts), potassium peroxodisulphate, ethanol were from sigma-aldrich (st. louis, mo, usa), fluorescein, sodium thiosulphate, buffer phosphate were from carlo erba, (milan, italy). 2.3. free acidity (fa) fa analysis was conducted according to annex ii of the consleg (2015) for olive oil analyses. a 5 g aliquot of each oil was dissolved in 25 ml solution of diethyl ether/ethylic alcohol (1:1, v/v) and titrated with a 0.1 n naoh aqueous solution using 1% phenolphthalein in ethanol as an indicator. results are expressed as g oleic acid/100 g. 2.4. peroxide value (pv) determination of pv was performed according to annex iii of the consleg (2015) for olive oil analyses. a 2 g aliquot of each oil was dissolved in a 25 ml solution of acetic acid/chloroform (3:2, v/v) and 1 ml of a saturated aqueous solution of potassium iodide was added. the mixture was shaken for 1 minute before being placed in the dark for five minutes. after this time, it was titrated with a 0.01 n sodium thiosulphate solution using a 1% starch soluble solution as an indicator. results are expressed as meq o2/kg. 2.5. p-anisidine value (p-anv) the p-anv analysis was conducted as described by the norme grassi e derivati method ngd c 36-79 (ngd 1979). each sample was diluted 1:100 (m/v), with isooctane (for spectrophotometry type), after which it was allowed to react with p-anisidine. the optical density of the solution was measured at 350 nm in a uv/vis spectrometer model lambda 2, perkin elmer, waltham, massachusetts usa. ital. j. food sci., vol. 30, 2018 718 2.6. totox this index is given as the sum of 2pv and p-anv. 2.7. spectrophotometric indices spectrophotometric indices were determined as described in annex ix of the european regulation (consleg, 2015). each sample was diluted 1:100 (m/v) with cyclohexane and the specific extinctions were measured at 232, 266, 270 and 274 nm against a blank (only cyclohexane). an uv/vis spectrometer model lambda 2, perkin elmer (waltham, ma, usa), was used. 2.8. antioxidant extract (ae) ae was obtained with the method proposed by goldsmith et al. (2014) modified as follows: 5 g of each sample was mixed for extraction with a 5 ml of methanol/water solution (80:20, v/v). the mixture was vigorously shaken with a vortex for 1 min and then centrifuged at 5000 rpm for 7 min. the supernatant containing the antioxidants was kept. the operation was repeated one more time and the two extracts were mixed together to obtain the first ae. after this, two more ae were prepared to obtain three different ae from the same oil and to analyse each oil in triplicate. 2.9. total phenolic content total phenolic content of the ae was determined using the folin-ciocalteu assay (singleton et al., 1999; giuffrè et al., 2017a). two ml of ae, 10 ml of bi-deionised water, 2.5 ml of folin-ciocalteu reagent and 10 ml of a 7% sodium carbonate in bideionised water solution were placed in a 50 ml glass flask. at this point the volume was made up to 50 ml with bi-deionised water. a blank was prepared substituting ae with bideionised water. after 1 hour in the dark, the absorbance was measured at 765 nm in an agilent 8453 spectrophotometer (santa clara, ca, usa). the total phenolic content was calculated on the basis of a calibration curve. data were expressed as mg gallic acid/kg. 2.10. antioxidant activity (dpph assay) hydrophilic the dpph assay measures the radical scavenging activity of a vegetable extract, in this case from a vegetable oil. the dpph assay method was developed by brand-williams et al. (1995) and it was adapted to an olive oil application (kalantzakis et al., 2006). it is spectrophotometrically determined by measuring the disappearance of the 1,1-diphenyl-2picrylhydrazyl radical (dpph●) at 515 nm. a 0.10 ml aliquot of ae was added to 2.40 ml of a 60 µm dpph methanolic solution. the mixture was shaken for five minutes in the dark. after this, the decrease in absorbance was measured at 515 nm in an agilent 8453 spectrophotometer (santa clara, ca, usa). results were expressed as % inhibition (mean ± s.d.) using the following formula: % inhibition = [(t0 – t5)/t0] x 100. 2.11. antioxidant activity (dpph assay) oil the dpph assay measures the radical scavenging activity of a vegetable oil. it was conducted in an uv/vis spectrometer model lambda 2, perkin elmer (waltham, ma, usa), using the method proposed by kalantzakis et al. (2006), modified as follows. firstly, the oil was diluted with ethyl acetate (1:10, v/v). secondly, 500 µl of diluted oil ital. j. food sci., vol. 30, 2018 719 were added to 2 ml of a 10-4 m dpph● solution, previously prepared with ethyl acetate and, thirdly, the absorbance of the mixture was measured immediately at 515 nm (t0) and after 30 minutes of incubation (t30). the results were calculated with the following formula: % inhibition = [(t0 – t30)/t0] x 100 and they were expressed as % inhibition. 2.12. antioxidant activity (abts assay) the abts assay determines the radical scavenging activity of an extract using an ethanol solution of 2,2-azinobis-(3-ethylbensothiazoline)-6-sulfonic acid (abts) and potassium peroxydisulphate. for its determination the method proposed by re et al. (1999) was applied, with the following modifications. a 0.050 ml aliquot of ae was added to 2.450 ml of a 7mm abts ethanolic solution and was vigorously shaken in the dark for 6 minutes. after this, the decrease in absorbance was measured at 734 nm in an agilent spectrophotometer, model 8453 (santa clara, ca, usa). results were expressed as % inhibition using the following formula: % inhibition = [(t0 – t6)/t0] x 100. 2.13. antioxidant activity (orac assay) ae the orac assay was proposed by cao et al. (1993). the orac assay measures the antioxidant activity of an oil extract and is determined on the basis of the oxidative damage to the fluorescent protein (ninfali et al., 2001). aaph was used as the generating species of peroxyl radicals, and trolox as an antioxidant standard. a 150 µl aliquot of fluorescein solution (96 nm in a 7.4 ph buffer phosphate solution) and 30 µl of aaph (133 mm in a 7.4 ph buffer phosphate solution) were added to 20 µl of ae previously diluted 1:30 (v/v) in a 7.4 ph buffer phosphate solution. the fluorescence decrease was measured using a perkin elmer victor x2 (waltham, ma, u.s.a.). the final reaction tested and the concentrations of the different reagents were determined following fernández-pachon et al. (2005). results were expressed as µmol trolox/100 g. 2.14. statistical analysis analyses of samples were conducted in triplicate and mean and standard deviation were calculated by the excel 2010 version software. analysis of variance (one-way anova) was performed by spss software version 17.0 for windows (spss inc., chicago, il, u.s.a.), using the tukey test and the significance level was set at p < 0.05. the effect of temperature and heating duration were analysed by a two-way anova by spss software version 17.0 for windows (spss inc., chicago, il, u.s.a.). 3. results and discussion 3.1. free acidity a vegetable oil is mainly composed of tryglycerides. each vegetable oil has a specific triglyceride composition: olive oil contains mainly triolein (giuffrè, 2013; 2014); palm oil contains mainly dioleylpalmitoylglycerol (21-25%) and dipalmitoyloleylglycerol (30-34%) (endo et al., 2011); soybean oil contains mainly trilinolein (21-22%) and dilinoleolein (1516%) (sudar et al., 2003). the hydrolysis of tryglycerides produces free fatty acids as the main degradation products. when the oil temperature reaches 150°c a part of the glycerol evaporates and ital. j. food sci., vol. 30, 2018 720 the remaining glycerol promotes the production of free fatty acids by hydrolysis (naz et al., 2005). this process is accelerated when a food containing water is added to the oil. the higher the water quantity, the higher the oil degradation. in our work, evoo had the highest initial fa but it showed the lowest percentage increase with temperature and with time. when the oils were heated at 180°c for 30, 60 and 120 min, the highest percentage increase in fa was in p: 30, 40 and 50% respectively. when the oils were heated at 220°c for 120 min, the highest percentage increase in fa was in po: 146.7% (table 1). if the absolute fa data are considered, the lowest values were found in p and in so oils in which 0.18 g/100 g and 0.17 g/100 g (as oleic acid) were found after the most drastic treatment (220°c for 120 min). azimah et al. (2017) used palm oil to fry potatoes at 175°c for 0, 10 and 20 times and found an initial increase in free acidity from 0.18 % (0 times, i.e. no fried oil) to 0.27% (10 times fried) whereas no increase was found from 10 to 20 times of frying. bulut and yilmaz (2010) used a refined pomace olive oil to fry 35 g patties whose dough contained flour (56%), water (42%) dry yeast (0.5%), baking soda (0.5%) and salt (0.5%) and found an increase from 0.27% to 0.28%, 0.34%, 0.43%, 0.52% and 0.59% in fried oil after respectively 0, 1, 2, 3, 4 and 5 days. 3.2. peroxide value the pv analysis is based on the quantification of the primary oxidation products, mainly hydroperoxides (saad et al. 2007). during peroxidation, unsaturated fatty acids are oxidized by o2. this is an auto-catalytic reaction which induces the formation of free radicals from fatty acids and starts the oxidation of the remaining non-oxidized fatty acids. in evoo, the initial pv was 8.1 meq o2/kg, i.e. well below the maximum (20 meq o2/kg) stated by both the consleg (2015) and the ioc (2015). after 120 min heating, pv increased in evoo up to 19.4 meq o2/kg (+139.5%) at 180°c and 20.9 meq o2/kg (+158.0%) at 220°c. under the worst conditions (220°c and 120 min heating), po and so were 11.8 and 9.5 meq o2/kg respectively and p showed at the same time both the lowest absolute pv and the highest percentage increase (357.1%), this was due to the very low initial pv (1.4 meq o2/kg) which is well below the 10 meq o2/kg required by the codex stan (2013) for a refined edible vegetable oil. after 120 min of p heating, pv increased to 4.0 meq o2/kg (+185.7%) at 180°c and to 6.4 meq o2/kg (+357.1%) at 220°c. after 60 min heating at 180°c, the pv was 9.9, 4.92, 5.7 and 3.9 meq o2/kg for evoo, po, so and p respectively (table 2), i.e. always below the maximum of 10 meq o2/kg, stated by the codex stan (2013). p showed the lowest increase in absolute value: 6.4 meq o2/kg after 120 min at 220°c. gharby et al. (2016) heated extra virgin olive oil and refined olive oil from morocco (cv picholine) at 100°c for 120 h and found a variation from 2.30 to 32.43 meq o2/kg oil in the former and a variation from 0.60 to 375.10 meq o2/kg oil in the latter. jaarin and kamisah (2002) fried sweet potatoes in palm oil and soy oil for 10 min at 180°c and used the same oil five times with an interval of at least five hours between each heating and found an increase from 2 (fresh oil) to more than 9 meq o2/kg oil in the palm oil and an increase from 5 (fresh oil) to 11 meq o2/kg oil in the soy oil. 3.3. p-anisidine value p-anv is an appropriate method for evaluating the secondary products of lipid oxidation (qing et al., 2016) and it is related to the formation of non-volatile aldehydes (2-alkenals) and ketones which are responsible of the rancid odour and taste in a fat. ital. j. food sci., vol. 30, 2018 721 table 1. free acidity (g oleic acid/100 g). at the top of the table, one-way anova experiment where unheated and heated oils are considered: means followed by different letters in the same column are significantly different according to tukey’s test (**, p < 0.01; ***, p < 0.001). at the bottom of the table, two-way anova experiment where only heated oils are considered: temperature, time, temperature x time (n.s., p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001). difference (%) is calculated on the unheated oil. sign. *** *** ** *** evoo difference % pomace difference % soybean difference % palm difference % unheated oil 0.50±0.02 b - 0.15±0.01 c - 0.12±0.01 c - 0.10±0.01 c - 180°c/30min 0.51±0.01 b 2.0 0.17±0.01 c 13.3 0.14±0 bc 16.7 0.13±0.01 bc 30.0 180°c/60min 0.53±0.02 b 6.0 0.18±0.01 bc 20.0 0.14±0.01 abc 16.7 0.14±0 b 40.0 180°c/120min 0.53±0.01 b 6.0 0.19±0.01 bc 26.7 0.16±0.02 ab 33.3 0.15±0.01 ab 50.0 220°c/30min 0.51±0.01 b 2.0 0.17±0.01 c 13.3 0.14±0.01 abc 16.7 0.14±0 b 40.0 220°c/60min 0.52±0.01 b 4.0 0.21±0.01 b 40.0 0.16±0.01 ab 33.3 0.16±0.01 ab 60.0 220°c/120min 0.61±0.02 a 22.0 0.37±0 a 146.7 0.17±0.01 a 41.7 0.18±0.01 a 80.0 temperature n.s. n.s. n.s. n.s. time n.s. ** * n.s. temperature x time *** n.s. n.s. n.s. table 2. peroxide value (meq o2/kg). at the top of the table, one-way anova experiment where unheated and heated oils are considered: means followed by different letters in the same column are significantly different according to tukey’s test (***, p < 0.001). at the bottom of the table, two-way anova experiment where only heated oils are considered: temperature, time, temperature x time (n.s., p > 0.05; *, p < 0.05; ***, p < 0.001). difference (%) is calculated on the unheated oil. sign. *** *** *** *** evoo difference % pomace difference % soybean difference % palm difference % unheated oil 8.1±0.3 f -3.9±0.01 f - 2.4±0.03 g - 1.4±0.05 f - 180°c/30min 9.4±0.1 e 16.1 4.4±0.04 e 12.8 4.3±0.10 f 79.2 3.7±0.06 e 164.3 180°c/60min 9.9±0.2 e 22.2 4.9±0.07 c 25.6 5.7±0.01 d 137.5 3.9±0.04 d 178.6 180°c/120min 19.4±0.5 b 139.5 5.9±0.11 b 51.3 7.3±0.04 b 204.2 4.0±0.05 d 185.7 220°c/30min 11.6±0.6 d 43.2 4.7±0.01 d 20.5 4.6±0.05 e 91.7 4.2±0.04 c 200.0 220°c/60min 14.8±0.2 c 82.7 5.9±0.08 b 51.3 7.1±0.06 c 195.8 5.5±0 b 292.9 220°c/120min 20.9±0.2 a 158.0 11.8±0.03 a 202.6 9.5±0.04 a 295.8 6.4±0.04 a 357.1 temperature n.s. n.s. n.s. n.s. time * n.s. * n.s. temperature x time *** *** *** *** ital. j. food sci., vol. 30, 2018 722 the importance of p-anv, mainly in a rectified oil, is due to the scarce effect on removing the secondary oxidisation products by deodorisation which, instead, diminishes the pv. for this reason, if before its rectification, an edible vegetable oil has suffered a heavy and continuous oxidative damage, this can be revealed by the p-anv analysis. after 120 min heating at 220°c, p showed the lowest initial p-anv (31.4) of the studied oils, whereas so showed the highest panv (94.4) and the highest percentage increase (1715.4%) compared to the unheated oils (table 3). considering values after heating at 180°c for 120 min, the p-anv was 2.6 (evoo), 3.1 (po), 2.8 (so) and 2.0 (p) times lower than p-anv found at 220°c heating for 120 min. if values at 220°c are considered, it is worthy of note that p-anv after 120 min heating is almost double in po and p and more than double in evoo compared to p-anv found after 60 min heating, whereas a very little increase was observed at 180°c between 60 and 120 min heated oils. xu et al. (2015) studied the p-anv variation in palm oil used to fry potatoes at 170°c for 75 frying batches conducted over 3 days and found a constant increase with 85 as the final value. ahmad tarmizi and ismail (2014) used refined, bleached, and deodorized palm olein to fry potatoes at 180°c for a 56 h period and found an initial p-anv increase from 0.8 (0 h – fresh palm olein) to 37.0 (32 h heating), thereafter they measured a constant decrease until 31.4 (48 h) and a final increase 35.6 (56 h). they also mixed palm olein with sunflower oil, canola oil and cotton seed oil and in all cases the p-anv during heating was higher than in palm olein. 3.4. totox this is an indicator of the overall oxidation state and quality of the oil (saad et al., 2007). the higher the totox the higher the oil’s oxidisation. totox values are listed in table 4. the initial lowest totox value was found in p (4.5) followed by so (10.1), po (16.0) and evoo (20.6). if totox is considered after 120 min heating at 180°c, the initial classification varies from p (23.6), po (35.7), so (48.3) to evoo (59.9). if totox is considered after 120 min heating at 220°c, the initial value increases to 44.3 in p, 96.5 in evoo, 97.6 in po and to 113.4 in so. this was due to the different antioxidant content and to the different fatty acid composition of each oil. evoo had the highest total phenolic content (table 8) and p had the highest saturated fatty acid content (data not published). as a consequence, even if evoo had the highest initial totox, the heating treatment caused the lowest percentage increase after 120 min at 220°c (368.4%): this was due to the highest phenolic content (table 8). another low increase in terms of totox was found in p (44.3 after 120 min at 220°c), because of the lowest unsaturated fatty acid content (less than 60%) and the lowest polyunsaturated fatty acid content (less than 13%) of this oil (data not published). xu et al. (2015) studied palm oil during the frying of potatoes at 170°c and found a constant increase from 10 (fresh oil) to 115 after 75 frying cycles (5 min each one). srivastava and semwal (2015), in coconut oil heated at 180°c for 8 h found a continuous and significant increase in totox value from 8.91 (fresh oil/0 h) to 33.95 after 8 h heating. 3.5. k232, k270 and ∆k thermal oxidation causes the isomerisation of double bonds contained in the unsaturated fatty acids and the formation of trans isomers (marinova et al., 2012). the absorbance at 232 nm gives information about the presence of diene conjugates, which are formed during the oil rectification. also, the oxidation products present in an edible vegetable oil vary the spectrum of uv absorption and increase values read on the spectrophotometer. the lower this value, the better the oil quality. ital. j. food sci., vol. 30, 2018 723 table 3. p-anisidine value. at the top of the table, one-way anova experiment where unheated and heated oils are considered: means followed by different letters in the same column are significantly different according to tukey’s test (***, p < 0.001). at the bottom of the table, two-way anova experiment where only heated oils are considered: temperature, time, temperature x time (n.s., p > 0.05; ***, p < 0.001). difference (%) is calculated on the unheated oil. sign. *** *** *** *** evoo difference % pomace difference % soybean difference % palm difference % unheated oil 4.4±0.1 f -8.3±0.07 f -5.2±0.12 f -1.8±0.10 e - 180°c/30min 7.4±0.6 e 68.2 9.6±0.08 e 15.7 5.6±0.04 f 7.7 2.0±0.07 e 11.1 180°c/60min 17.4±0.5 c 295.5 21.2±0.13 d 155.4 20.2±0.69 e 288.5 11.9±0.04 d 561.1 180°c/120min 21.0±0.1 b 377.3 24.0±0.39 c 189.2 33.6±0.83 c 546.2 15.5±0.33 c 761.1 220°c/30min 13.4±0.6 d 204.5 21.4±0.11 d 157.8 27.2±0.64 d 423.1 2.4±0.15 e 33.3 220°c/60min 20.8±0.1 b 372.8 40.5±0.08 b 388.0 47.6±1.06 b 815.4 17.5±0.36 b 872.2 220°c/120min 54.6±1.2 a 1140.9 73.9±0.01 a 790.4 94.4±1.13 a 1715.4 31.4±0.33 a 1644.4 temperature n.s. n.s. n.s. n.s. time n.s. n.s. n.s. n.s. temperature x time *** *** *** *** table 4. totox. at the top of the table, one-way anova experiment where unheated and heated oils are considered: means followed by different letters in the same column are significantly different according to tukey’s test (***, p < 0.001). at the bottom of the table, two-way anova experiment where only heated oils are considered: temperature, time, temperature x time (n.s., p > 0.05; ***, p < 0.001). difference (%) is calculated on the unheated oil. sign. *** *** *** *** evoo difference % pomace difference % soybean difference % palm difference % unheated oil 20.6±0.7 f -16.0±0.1 f -10.1±0.2 g -4.5±0.2 g - 180°c/30min 26.1±0.8 d 26.7 18.5±0.01 e 15.6 14.2±0.2 f 40.6 9.5±0.1 f 111.1 180°c/60min 37.3±0.3 d 81.1 31.1±0.3 d 94.4 31.6±0.7 e 212.9 19.8±0.1 d 340.0 180°c/120min 59.9±1.0 b 190.8 35.7±0.6 c 123.1 48.3±0.8 c 378.2 23.6±0.4 c 424.4 220°c/30min 36.6±0.6 d 77.7 30.8±0.1 d 92.5 36.4±0.6 d 260.4 10.9±0.2 e 142.2 220°c/60min 50.4±0.5 c 144.7 52.3±0.2 b 226.9 61.7±1.2 b 510.9 28.5±0.4 b 533.3 220°c/120min 96.5±1.2 a 368.4 97.6±0.1 a 510.0 113.4±1.2 a 1022.8 44.3±0.4 a 884.4 temperature n.s. n.s. n.s. n.s. time n.s. n.s. n.s. n.s. temperature x time *** *** *** *** ital. j. food sci., vol. 30, 2018 724 the best findings were revealed in evoo (1.686 for unheated evoo and 2.663 for h-evoo at 220°c for 120 min). p showed the second lowest values at 180°c and at 220°c after 30 min heating. in p a slight reduction in k232 was found at both 180 and 220°c from 60 and 120 min heating (table 5). po showed the second lowest findings at 220°c and 60-120 min heating. the absorbance at 270 nm gives information about the presence of triene conjugates. linoleate oxidation products or degradation of hydroxylinoleate produce conjugated trienes absorbing at 270 nm (marinova et al., 2012). findings are listed in table 6. so showed the initial absolute highest value and this negative condition was found after all the studied treatments, whereas it showed the lowest relative increase during heating (68.9% after 120 min at 220°c). evoo always showed both the absolute lowest value and the highest relative increase with time and with temperature. the lowest evoo values were due to its being the only non-rectified vegetable oil in this study. p showed the second lowest findings after evoo. ∆k is a spectrophotometric index indicating the maximum absorbance at 270 nm. in the case of a rectified oil, and mainly in the case of po, the absorbance value in this zone increases and the spectrophotometric profile has a characteristic trend with three maximum levels due to the presence of trienes. of the three peaks, the most pronounced is the central one at 270 nm. to judge an edible vegetable oil it is also important to take into account the absorbance of the two lateral peaks at 266 nm and 274 nm. evoo showed the lowest ∆k before and after heating, this was because evoo is not rectified and because of the low ∆k value before heating (0.001), which was reversed in the subsequent steps during heating (table 7). it is noteworthy that the ∆k value observed in evoo after 120 min of heating at 220°c was lower than the ∆k value observed in the three other oils before heating; this demonstrates, if necessary, the better food properties of evoo if compared to other edible vegetable oils. so always presented the highest values and from the ∆k point of view this is the worst oil. gharby et al. (2017) treated edible oils at 100°c for 120 h and found an increase from 1.57 (fresh oil) to 2.59 (after 120 h heating) in an evoo and a progress from 1.78 to 2.79 in a refined olive oil under the same thermal and time conditions. in the same study k270 was found to increase from 0.13 to 0.33 in evoo and from 0.55 to 1.99 in a refined olive oil. azimah et al. (2017) measured the presence of conjugated dienes at 234 nm in palm oil used to fry potatoes at 175°c, they read 5.36 as a specific extinction in the fresh oil and 5.40 and 5.21 after 10 and 20 frying cycles respectively. 3.6. total phenolic content vegetable oils contain many biologically active components, which exert antioxidant activity, evoo is consumed unrefined, differently from other edible vegetable oils. this implies that evoo contains many minor bioactive compounds such as phenols whose content was found to decrease in evoo during olive fruit ripening (sicari et al., 2009; sicari et al., 2010). table 8 describes the total phenolic content evolution of the four studied oils during heating treatments. the highest total phenolic content was found in evoo (196.8 mg/kg) whereas the lowest content was found in so (15.0 mg gallic acid/kg). heating lowered the total phenolic content and a continuous decrease was measured with heating and with time in all the studied oils. the lowest total phenolic content was found in the samples heated for 120 min at 220°c. the highest loss in total phenolic content (as a percentage) was in evoo because it had (ab origine) the highest total phenolic content, thus, the highest total phenolic content to be lost during heating. santos et al. (2018) studied evoo during the frying of white potatoes at 175°c and found a loss in total phenolic content from fresh oil (564 mg/kg) to the 28 hrs fried oil (171 mg/kg), with the minimum content (114 mg/kg) after 16 hrs frying. ital. j. food sci., vol. 30, 2018 725 table 5. k 232. at the top of the table, one-way anova experiment where unheated and heated oils are considered: means followed by different letters in the same column are significantly different according to tukey’s test (*, p < 0.05; ***, p < 0.001). at the bottom of the table, two-way anova experiment where only heated oils are considered: temperature, time, temperature x time (n.s., p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001). difference (%) is calculated on the unheated oil. sign. *** * *** *** evoo difference % pomace difference % soybean differenc % palm difference % unheated oil 1.686±0.01 - 2.858±0.02 - 2.967±0.04 -2.287±0.13 - 180°c/30min 1.802±0.02 6.9 2.898±0.07 1.4 3.012±0.08 1.5 2.568±0.06 12.3 180°c/60min 1.931±0.02 14.5 2.927±0.02 2.4 3.099±0.06 4.4 2.742±0.01 19.9 180°c/120min 2.297±0.01 36.2 2.987±0.12 4.5 3.113±0.01 4.9 2.722±0.01 19.0 220°c/30min 1.820±0.02 7.9 2.922±0 2.2 3.053±0.06 2.9 2.827±0.03 23.6 220°c/60min 2.594±0.01 53.9 2.934±0.01 2.7 3.104±0 4.6 3.518±0.05 53.8 220°c/120min 2.663±0.03 57.9 3.039±0.05 6.3 3.143±0.01 5.9 3.212±0.06 40.4 temperature n.s. n.s. n.s. n.s. time n.s. * ** n.s. temperature x time *** n.s. n.s. *** table 6. k270. at the top of the table, one-way anova experiment where unheated and heated oils are considered: means followed by different letters in the same column are significantly different according to tukey’s test (***, p < 0.001). at the bottom of the table, two-way anova experiment where only heated oils are considered: temperature, time, temperature x time (n.s., p > 0.05; ***, p < 0.001). difference (%) is calculated on the unheated oil. sign. *** *** *** *** evoo difference % pomace difference % soybean difference % palm difference % unheated oil 0.113±0.01 g -1.203±0.01 g -2.174±0.05 e - 0.971±0.03 f - 180°c/30min 0.166±0.01 f 46.9 1.466±0.01 f 21.9 2.308±0.10 d 6.2 1.054±0 e 8.5 180°c/60min 0.407±0.01 d 260.2 1.713±0.02 d 42.4 2.550±0.09 d 17.3 1.211±0.02 d 24.7 180°c/120min 0.545±0 b 382.3 1.871±0.02 c 55.5 3.237±0.18 b 48.9 1.555±0 c 60.1 220°c/30min 0.350±0 e 209.7 1.639±0.01 e 36.2 2.841±0.01 c 30.7 1.227±0.02 d 26.4 220°c/60min 0.511±0 c 352.2 2.279±0.01 b 89.4 3.664±0.03 a 68.5 1.639±0.04 b 68.8 220°c/120min 0.890±0.01 a 687.6 2.729±0.03 a 126.8 3.672±0.04 a 68.9 2.364±0.01 a 143.5 temperature n.s. n.s. n.s. n.s. time n.s. n.s. n.s. n.s. temperature x time *** *** *** *** ital. j. food sci., vol. 30, 2018 726 table 7. ∆k. at the top of the table, one-way anova experiment where unheated and heated oils are considered: means followed by different letters in the same column are significantly different according to tukey’s test (***, p < 0.001). at the bottom of the table, two-way anova experiment where only heated oils are considered: temperature, time, temperature x time (n.s., p > 0.05; *, p < 0.05; ***, p < 0.001). difference (%) is calculated on the unheated oil. sign. *** *** *** *** evoo difference % pomace difference % soybean difference % palm difference % unheated oil 0.001±0 g -0.071±0 c -0.194±0.03 d -0.082±0 e - 180°c/30min 0.005±0 f 400.0 0.077±0 c 8.5 0.234±0.04 d 20.6 0.086±0 e 4.9 180°c/60min 0.025±0 d 2400.0 0.104±0 bc 46.5 0.249±0.03 cd 28.4 0.105±0 d 28.0 180°c/120min 0.048±0.02 b 4700.0 0.106±0 bc 49.3 0.599±0.19 ab 208.8 0.152±0 b 85.4 220°c/30min 0.019±0 e 1800.0 0.109±0.01 bc 52.5 0.460±0.02 bc 137.1 0.116±0 c 41.5 220°c/60min 0.031±0 c 3000.0 0.136±0.01 d 91.5 0.650±0.04 ab 235.0 0.155±0.01 b 89.0 220°c/120min 0.061±0 a 6000.0 0.172±0.03 a 142.3 0.684±0.01 a 252.6 0.447±0 a 445.1 temperature * n.s. n.s. n.s. time * n.s. n.s. n.s. temperature x time n.s. n.s. * *** table 8. total phenolic content (mg gallic acid/kg). at the top of the table, one-way anova experiment where unheated and heated oils are considered: means followed by different letters in the same column are significantly different according to tukey’s test (***, p < 0.001). at the bottom of the table, two-way anova experiment where only heated oils are considered: temperature, time, temperature x time (n.s., p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001). difference (%) is calculated on the unheated oil. sign. *** *** *** *** evoo difference % pomace difference % soybean difference % palm difference % unheated oil 196.8±5.1 a -40.0±0.2 a -15.0±0.6 e -43.0±0.6 a - 180°c/30min 155.5±3.0 b -21.0 35.5±3.0 b -11.3 12.2±0.3 b -18.7 39.5±1.6 b -8.1 180°c/60min 95.1±4.0 c -51.7 29.6±0.3 c -26.0 10.5±0.4 c -30.0 33.3±0.9 c -22.6 180°c/120min 59.8±2.2 d -69.6 27.6±1.1 cd -31.0 10.1±0.3 c -32.6 26.5±2.0 d -38.4 220°c/30min 91.5±2.9 c -53.5 30.0±0.7 c -25.0 9.6±0.5 c -36.0 33.1±0.6 c -23.0 220°c/60min 63.8±1.8 d -67.6 24.3±0.2 de -39.3 7.1±0.5 d -52.7 27.2±0.9 d -36.7 220°c/120min 66.8±1.2 d -66.1 22.5±1.3 e -43.8 5.2±0.4 e -65.3 21.8±0.6 e -49.3 temperature n.s. *** * ** time n.s. *** n.s. ** temperature x time *** n.s. *** n.s. ital. j. food sci., vol. 30, 2018 727 3.7. total tocopherol content tocopherols are important components of vitamin e which was found to prevent the risk of prostate cancer (coc, 2015), to maintain the integrity of long-chain pufas in the membranes of cells and thus maintain their bioactivity (traber and atkinson, 2007), to have beneficial effects as an antioxidant against reproductive disorders, thus it is recommended for women of reproductive age (mutalip et al., 2018), and to exert an antioxidant activity during a vegetable oil’s shelf-life (evans et al., 2002). the initial tocopherol content depends on many factors such as cultivar and extraction procedure, for this reason different tocopherol contents are present in the literature in the unheated oils. the following contents have been reported: for evoo 125-214 mg/kg (ninfali et al., 2002); for po: 300 mg/kg (pignitter et al., 2016); for s: 1030 mg/kg (evans et al., 2002), 340 mg/kg (grilo et al., 2014); for p: 201 mg/kg (xu et al., 2015), 500 mg/kg (kouski et al., 2015). in the oils studied in our work, p contained the highest initial total tocopherol content (249 α-tocopherol mg/kg) and showed the lowest percentage decreasing trend at 180°c and at 220°c. so was found to have the second highest tocopherol content (199.6 mg/kg), whereas po had the lowest content i.e. 68.6 mg/kg in the unheated po and 37.3 mg/kg after 120 min heating at 180°c and 18.8 mg/kg after 120 min at 220°c heating (table 9). our results are confirmed by other authors who always found a decreasing trend in tocopherol content during heating even if with a different rate depending on the type of oil, cooking system and applied temperature (hassanein et al., 2003; hamid et al., 2014; javidipour et al., 2017). 3.8. antioxidant activity (abts assay) the aa is partially a consequence of total phenolic content and, more generally, it is a consequence of the physico-chemical properties of each oil studied in this work. evoo showed the highest abts-aa at the start of the experiment (155.9 μm te/100 g) and in all the six applied treatments. of the refined oils, p and so showed the lowest percentage difference in all the six treatments, i.e. the lowest decrease in terms of percentage (table 10). aydeniz and yilmaz (2016) studied a refined winterized peanut oil and found a decreasing trend in antioxidant activity during frying of patties at 180°c for four consecutive days (5-5.5 hrs per day): the oil showed 3.1 mm teac/100 g oil at 0 day and 1.8 (day 1), 1.2 (day 2), 0.8 (day 3), 0.5 (day 4); in this experiment the antioxidant activity was lower than in our experiment but the peanut oil had a very low initial total phenolic content (0.013 g/kg oil). 3.9. antioxidant activity (dpph hydrophilic assay) p showed the highest aa-dpph-hydro in the hydrophilic extract after each treatment (51.8-38.9 μm te/100 g), whereas so always showed the lowest aa-dpph-hydro (6.6-3.7 μm te/100 g). evoo showed the second highest aa-dpph-hydro. this was probably due to the high phenolic content in evoo (table 8) and to the high tocopherol content in p, in accordance with findings of antioxidant content reported by other authors (hamid et al., 2014). in all our studied oils a constant decrease in terms of aa was revealed (table 11). 3.10. antioxidant activity (dpph oil assay) the highest aa-dpph-oil was found in unheated so (117.0 μm te/100 g) and p (115.7 μm te/100 g), whereas in unheated evoo the antioxidant activity was 73.5 μmol te/100 g (table 12). according to kalantzakis et al. (2006) who compared virgin olive oil with so during 10 hours heating and found so to have a higher aa-dpph-oil before and after heating, this was probably due to the higher presence of tocopherol content in so. ital. j. food sci., vol. 30, 2018 728 table 9. total tocopherol content (mg α-tocopherol/kg). at the top of the table, one-way anova experiment where unheated and heated oils are considered: means followed by different letters in the same column are significantly different according to tukey’s test (***, p < 0.001). at the bottom of the table, two-way anova experiment where only heated oils are considered: temperature, time, temperature x time (n.s., p > 0.05; *, p < 0.05; ***, p < 0.001). difference (%) is calculated on the unheated oil. sign. *** *** *** *** evoo difference % pomace difference % soybean difference % palm difference % unheated oil 169.2±2.7 a -68.6±0.3 a -199.6±0.7 a -249.1±1.2 a - 180°c/30min 105.8±2.1 b -37.5 51.3±0.8 b -25.2 169.5±0.9 b -15.1 213.0±0.4 b -14.5 180°c/60min 77.2±1.5 c -54.4 46.0±0.8 c -32.9 138.0±0.3 d -30.8 179.1±0.7 d -28.1 180°c/120min 44.4±1.4 e -73.8 37.3±0.6 d -45.6 86.4±0.3 f -56.7 126.5±0.3 f -49.2 220°c/30min 59.4±3.9 d -64.9 44.8±0.5 c -34.7 158.1±0.6 c -20.8 201.5±0.3 c -19.1 220°c/60min 48.4±0.7 e -71.4 25.7±0.6 e -62.5 116.6±0.4 e -41.6 156.3±0.4 e -37.2 220°c/120min 34.7±1.6 f -79.5 18.8±0.4 f -72.5 53.7±0.5 g -73.1 91.2±0.8 g -63.4 temperature n.s. n.s. n.s. n.s. time n.s. n.s. * * temperature x time *** *** *** *** table 10. antioxidant activity, abts assay (μm te/100g). at the top of the table, one-way anova experiment where unheated and heated oils are considered: means followed by different letters in the same column are significantly different according to tukey’s test (***, p < 0.001). at the bottom of the table, two-way anova experiment where only heated oils are considered: temperature, time, temperature x time (n.s., p > 0.05; *, p < 0.05; **, p < 0.01). difference (%) is calculated on the unheated oil. sign. *** *** *** *** evoo difference % pomace difference % soybean difference % palm difference % unheated oil 155.9±3.83 a -62.5±2.47 a -51.8±0.22 a -51.8±0.36 a - 180°c/30min 145.6±1.28 b -6.6 56.3±1.32 ab -9.9 37.9±0.22 b -26.9 49.1±2.12 a -5.3 180°c/60min 131.3±1.71 c -15.8 52.8±4.56 bc -15.5 36.4±0.27 bc -29.9 44.8±1.20 b -13.5 180°c/120min 112.3±0.31 d -27.9 45.5±1.55 cd -27.1 30.7±0.79 de -40.8 43.1±0.21 b -16.9 220°c/30min 124.6±1.17 c -20.1 44.9±5.51 cd -28.0 32.4±3.27 cd -37.6 44.5±2.14 b -14.2 220°c/60min 111.1±3.69 d -28.7 42.9±3.80 d -31.3 33.8±1.24 bcd -34.8 39.1±0.25 c -24.6 220°c/120min 100.5±3.29 e -35.5 29.1±2.01 e -53.4 27.2±1.58 e -47.5 38.9±0.44 c -24.9 temperature * * * * time * n.s. * * temperature x time ** n.s. n.s. n.s. ital. j. food sci., vol. 30, 2018 729 in all the oils studied in our work, the aa-dpph-oil assay showed higher values if compared to aa-dpph-hydro (tables 11-12). this was in accordance with the findings of espín et al. (2000) who analysed the aa-dpph of untreated edible vegetable oils (total fats or ft), of its methanolic extracts (mf) and of the oil after methanolic extraction (lf) and found the sum mf + lf always quantitatively comparable with tf value. the decreasing trend of the aa-dpphoil was demonstrated by other authors in different heating conditions. gomez-alonso et al. (2003) studied evoo used for french fries at 180°c and found a decrease in the aa of the oil from 740 μmol te/kg (fresh oil) to less than 250 μmol te/kg during a total of 2 h frying over 6 days. a reduction in radical scavenging activity (dpph assay) was also found during deepfrying in palm oil and rice bran oil (hamid et al., 2014). kobyliński et al (2016) studied the effect of specific oil surface in rapeseed oil during heating at 180°c and found 459.5 μmol te/100g as an aa-dpph value in the fresh oil and an aa-dpph value ranging from 3.3 μmol te/100g to 72.65 μmol te/100g when the level of oil in pan-five different oil layer heights was changed from 0.5 cm to 2.5 cm; this was expected considering that during heating of a thin oil layer there is a greater oxygen absorption per unit oil than during heating in a larger amount of oil. 3.11. antioxidant activity (orac assay) the orac assay confirmed results obtained by abts assay with evoo showing the highest orac-aa. before heating evoo had the highest orac-aa value (316.1 μm te/100 g), after 30 min heating it decreased to 220.9 μm te/100 g (-30.1%) and to 142.6 μm te/100 g (-54.9%) at 180°c and 220°c respectively (table 13). p showed the second highest orac-aa (65.4 μm te/100 g) and it decreased to 40.7 μm te/100 g (after 30 min heating) and to 32.5 μm te/100 g (after 120 min heating) at 180°c. when p was heated at 220°c, the orac-aa was 27.1 μm te/100 g (-58.6%) and 15.9 (-75.7%) respectively after 30 and 120 min heating. it is worthy of note that oils from different cultivars could have a different orac even if they have the same total phenolic content, in fact zullo and ciafardini (2008) studied some single components of the phenolic fraction in an evoo and found gallic acid to have a greater influence on orac than caffeic acid and oleuropein. 3.12. one-way anova, two-way anova, correlation matrix 3.12.1 free acidity one-way anova showed high significant differences in so and showed very high significant differences in every other oil (p < 0.001), (table 1). two-way anova analysis showed that temperature had no significant effect on the fa variation in the four oils analysed in this study (table 1). the same was for heating duration on evoo and p, whereas the heating duration influenced significantly the fa variation in so (p < 0.05) and highly significantly in po (p < 0.01). fa showed a very good positive correlation with p-anv especially in evoo (0.9175), in po (0.9567) and in p (0.8193), a good correlation was found in so (0.7634), (tables 14-17). fa was negatively very well correlated with aa-dpph-oil in p (-0.8569), in so (-0.8372), in evoo (-0.8067) and a good correlation was found in po (-0.7820), (tables 14-17). similar studies have been conducted on other edible vegetable oils. giuffrè et al. (2017b), studied the influence of high temperature and time of heating on sunflower seed oil at 180-210-240°c for 15-30-60-120 min and found a constant slight increase in fa which was influenced by time of heating (p < 0.01) and by the interaction between temperature and time of heating (p < 0.001). ital. j. food sci., vol. 30, 2018 730 table 11. antioxidant activity, dpph hydrophilic assay (!m te/100g). at the top of the table, one-way anova experiment where unheated and heated oils are considered: means followed by different letters in the same column are significantly different according to tukey’s test (**, p < 0.01; ***, p < 0.001). at the bottom of the table, two-way anova experiment where only heated oils are considered: temperature, time, temperature x time (n.s., p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001). difference (%) is calculated on the unheated oil. sign. *** ** *** *** evoo difference % pomace difference % soybean difference % palm difference % unheated oil 47.0±3.37 a -10.7±0.44 a -6.6±0.30 a -51.8±0.36 a - 180°c/30min 43.6±0.88 a -7.3 8.3±2.82 b -22.9 6.4±0.14 a -3.9 49.1±2.12 b -13.4 180°c/60min 29.6±1.71 b -37.1 7.8±0.41 b -27.4 6.3±0.17 a -5.5 44.8±1.20 bc -21.5 180°c/120min 19.9±0.35 c -57.7 6.8±1.05 b -37.0 3.8±0.45 b -42.9 43.1±0.21 bc -24.9 220°c/30min 28.8±2.05 b -38.7 7.5±0.06 b -29.8 4.5±0.25 b -32.7 44.5±2.14 b -14.9 220°c/60min 19.6±0.46 c -58.3 6.6±0.11 b -38.7 3.8±0.45 b -42.6 39.1±0.25 cd -32.1 220°c/120min 12.7±2.87 d -73.0 5.0±1.78 b -53.6 3.7±0.24 b -43.7 38.9±0.44 d -43.3 temperature * n.s. n.s. n.s. time * n.s. n.s. n.s. temperature x time ** n.s. *** * table 12. antioxidant activity, dpph oil assay (!m te/100g). at the top of the table, one-way anova experiment where unheated and heated oils are considered: means followed by different letters in the same column are significantly different according to tukey’s test (***, p < 0.001). at the bottom of the table, two-way anova experiment where only heated oils are considered: temperature, time, temperature x time (n.s., p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001). difference (%) is calculated on the unheated oil. sign. *** *** *** *** evoo difference % pomace difference % soybean difference % palm difference % unheated oil 73.5±2.02 a -69.8±0.14 a -117.0±0.21 a -115.7±1.97 a - 180°c/30min 71.7±1.84 a -2.3 68.0±0.19 b -2.6 107.4±0.46 b -8.2 109.9±1.14 ab -5.0 180°c/60min 56.7±1.59 c -22.8 66.2±0.19 c -5.2 105.6±1.06 c -9.8 106.8±0.46 bc -7.7 180°c/120min 43.4±1.82 e -40.9 61.4±0.55 d -12.0 101.2±0.49 e -13.5 101.2±0.34 cd -12.6 220°c/30min 64.5±1.14 b -12.1 49.9±0.19 e -28.5 105.2±0.60 c -10.1 107.4±4.24 bc -7.1 220°c/60min 50.3±1.97 d -31.5 48.5±0.25 f -30.6 103.3±0.42 d -11.7 95.9±2.44 d -17.1 220°c/120min 31.3±0.92 f -57.3 39.0±0.56 g -44.2 96.5±0.28 f -17.5 76.9±3.59 e -33.5 temperature * ** n.s. n.s. time * n.s. * n.s. temperature x time * *** ** *** ital. j. food sci., vol. 30, 2018 731 table 13. antioxidant activity, orac assay (!m te/100 g). at the top of the table, one-way anova experiment where unheated and heated oils are considered: means followed by different letters in the same column are significantly different according to tukey’s test (***, p < 0.001). at the bottom of the table, two-way anova experiment where only heated oils are considered: temperature, time, temperature x time (n.s., p > 0.05; *, p < 0.05; ***, p < 0.001). difference (%) is calculated on the unheated oil. sign. *** *** *** *** evoo difference % pomace difference % soybean difference % palm difference % unheated oil 316.1±44. 9 a -18.5±0.4 a -9.1±0.2 a -65.4±0.5 a - 180°c/30min 220.9±9.1 b -30.1 17.5±0.2 b -5.4 6.0±0.3 b -34.1 40.7±0.5 b -37.8 180°c/60min 164.8±15.5 c -47.9 14.9±0.5 d -19.5 5.4±0.3 b -40.7 33.0±1.2 c -49.5 180°c/120min 117.2±3.6 de -62.9 10.4±0.5 f -43.8 1.9±0.4 e -79.1 32.5±1.7 c -50.3 220°c/30min 142.6±4.8 cd -54.9 15.8±0.1 c -14.6 5.6±0.3 b -38.5 27.1±1.7 d -58.6 220°c/60min 114.6±6.2 de -63.8 11.4±0.2 e -38.4 3.7±0.4 c -59.3 25.7±1.6 d -60.7 220°c/120min 93.2±3.5 e -70.5 8.3±0.6 g -55.1 2.0±0.4 e -78.0 15.9±0.3 e -75.7 temperature n.s. * n.s. * time n.s. * n.s. n.s. temperature x time *** *** *** *** table 14. correlation matrix between chemical properties of extra virgin olive oil before and after heating. free acidity peroxide value p-anisidine value totox k232 k270 δk total phenolic content total tocopherol content abts assay dpph oil assay dpph hydro assay orac assay free acidity 1 peroxide value 0.7212 1 p-anisidine value 0.9175 0.8327 1 totox 0.8782 0.9339 0.9756 1 k232 0.6966 0.8660 0.8148 0.8692 1 k270 0.8467 0.9097 0.9605 0.9806 0.8886 1 δk 0.7308 0.9246 0.8663 0.9257 0.8266 0.9370 1 total phenolic content -0.4955 -0.7815 -0.6420 -0.7242 -0.7787 -0.8193 -0.8114 1 total tocopherol content -0.5516 -0.7862 -0.6825 -0.7522 -0.7590 -0.8258 -0.8059 0.9712 1 abts assay -0.6686 -0.9000 -0.8187 -0.8852 -0.8964 -0.9337 -0.8946 0.9420 0.9358 1 dpph oil assay -0.8067 -0.9410 -0.9067 -0.9583 -0.9017 -0.9751 -0.9476 0.8231 0.8071 0.9180 1 dpph hydro assay -0.6740 -0.8832 -0.8162 -0.8770 -0.8895 -0.9377 -0.8989 0.9457 0.9151 0.9824 0.9277 1 orac assay -0.5367 -0.7590 -0.6743 -0.7362 -0.7544 -0.8144 -0.7819 0.9658 0.9909 0.9246 0.7899 0.9115 1 ital. j. food sci., vol. 30, 2018 732 table 15. correlation matrix between chemical properties of pomace olive oil before and after heating. free acidity peroxide value p-anisidine value totox k232 k270 δk total phenolic content total tocopher ol content abts assay dpph oil assay dpph hydro assay orac assay free acidity 1 peroxide value 0.9822 1 p-anisidine value 0.9567 0.9594 1 totox 0.9677 0.9733 0.9985 1 k232 0.6195 0.6929 0.6657 0.6752 1 k270 0.8903 0.8958 0.9677 0.9600 0.6691 1 δk 0.8319 0.8192 0.9000 0.8901 0.4356 0.9147 1 total phenolic content -0.6913 -0.7133 -0.8265 -0.8099 -0.7159 -0.9166 -0.8354 1 total tocopherol content -0.7774 -0.7884 0.8825 -0.8699 -0.6649 -0.9669 -0.8702 0.9557 1 abts assay -0.8324 -0.8515 -0.8992 -0.8957 -0.6938 -0.9081 -0.8359 0.8692 0.9038 1 dpph oil assay -0.7820 -0.7803 -0.8851 -0.8705 -0.5545 -0.8693 -0.8576 0.8128 0.8508 0.9036 1 dpph hydro assay -0.6438 -0.6561 -0.7034 -0.6987 -0.5697 -0.7818 -0.7970 0.8631 0.8084 0.7472 0.6855 1 orac assay -0.7645 -0.8156 -0.8592 -0.8563 -0.6961 -0.9253 -0.8343 0.9020 0.9210 0.8563 0.7379 0.7645 1 table 16. correlation matrix between chemical properties of soybean oil before and after heating. free acidity peroxid e value panisidine value totox k232 k270 δk total phenolic content total tocophero l content abts assay dpph oil assay dpph hydro assay orac assay free acidity 1 peroxide value 0.8486 1 p-anisidine value 0.7634 0.9021 1 totox 0.7834 0.9254 0.9984 1 k232 0.7388 0.8092 0.6786 0.7037 1 k270 0.7403 0.8763 0.8545 0.8672 0.6972 1 δk 0.6422 0.8081 0.7969 0.8075 0.6311 0.9588 1 total phenolic content -0.7696 -0.9082 -0.9027 -0.9137 -0.7510 -0.9094 -0.8266 1 total tocopherol content -0.8382 -0.9883 -0.8815 -0.9057 -0.7950 -0.8478 -0.8057 0.8532 1 abts assay -0.7822 -0.8473 -0.7025 -0.7297 -0.7304 -0.7681 -0.7315 0.8641 0.8297 1 dpph oil assay -0.8372 -0.9456 -0.8172 -0.8436 -0.7957 -0.8273 -0.7721 0.9095 0.9295 0.9649 1 dpph hydro assay -0.7477 -0.7865 -0.7539 -0.7668 -0.6029 -0.9041 -0.8835 0.8095 0.7913 0.7824 0.7881 1 orac assay -0.8181 -0.9422 -0.7523 -0.7861 -0.7668 -0.8531 -0.8294 0.8282 0.9519 0.9050 0.9489 0.8302 1 ital. j. food sci., vol. 30, 2018 733 table 17. correlation matrix between chemical properties of palm oil before and after heating. free acidity peroxid e value panisidine value totox k232 k270 δk total phenolic content total tocopherol content abts assay dpph oil assay dpph hydro assay orac assay free acidity 1 peroxide value 0.8886 1 p-anisidine value 0.8193 0.8009 1 totox 0.8656 0.8769 0.9901 1 k232 0.7557 0.8806 0.7031 0.7704 1 k270 0.8501 0.8485 0.9605 0.9695 0.7227 1 δk 0.7400 0.7391 0.8894 0.8869 0.5450 0.9507 1 total phenolic content -0.8787 -0.8762 -0.8904 -0.9198 -0.7836 -0.9019 -0.7545 1 total tocopherol content -0.8819 -0.8447 -0.9343 -0.9476 -0.6783 -0.9243 -0.8087 0.9611 1 abts assay -0.8357 -0.9083 -0.8202 -0.8709 -0.9062 -0.8264 -0.6571 0.9163 0.8552 1 dpph oil assay -0.8569 -0.8765 -0.9411 -0.9605 -0.7405 -0.9787 -0.9373 0.8628 0.8971 0.8137 1 dpph hydro assay -0.8470 -0.9208 -0.9016 -0.9392 -0.8087 -0.8897 -0.7764 0.8892 0.9081 0.9053 0.9203 1 orac assay -0.8753 -0.9559 -0.7051 -0.7897 -0.7985 -0.7587 -0.6362 0.8734 0.8246 0.8823 0.7829 0.8540 1 ital. j. food sci., vol. 30, 2018 734 3.12.2 peroxide value the two-way anova analysis showed that in all the studied oils, the pv increase was not significantly influenced by temperature but was always very highly significantly influenced by the interaction between temperature and time (table 2). pv was found to be very good and positively correlated with k270: 0.9097 evoo, 0.8958 po, 0.8763 so and 0.8485 p, whereas a very high and negative correlation was found with aa-abts: -0.9000 evoo, -0.8515 po, -0.8473 so and -0.9083 p (tables 14-17). 3.12.3 p-anisidine value the one-way anova analysis showed that very highly significant differences exist in all the studied oils between treatments. the two-way anova analysis showed that in all the studied oils, the p-anv increase was not significantly influenced by temperature or by time of heating but it was always very highly significantly influenced by the interaction between temperature and time (table 3). in the three rectified oils, p-anv was found to have negative and good or very good correlation with the parameters indicating antioxidant activity. in po, p-anv was found to be correlated with pv (0.9594), with totox (0.9985) and with k270 0.9677). in so, p-anv was found to be correlated with pv (0.9021), with totox (0.9984) and with total phenolic content (0.9027). in p, p-anv was found to be correlated with totox (0.9901), with k270 (0.9605) and with aa-dpph oil (0.9411). in evoo, a negative and very good correlation was found with abts assay (0.8187), aa-dpph-oil (-0.9067), aa-dpph hydro (-0.8162), (tables 14-17). 3.12.4 totox the one-way anova analysis showed very high significant differences between treatments. the two-way anova analysis showed that in all the studied oils, the totox variation was not significantly influenced by temperature or by time of heating but it was always very highly significantly influenced by the interaction between temperature and time (table 4), according to two-way anova results of p-anv (table 3). totox showed a very good positive correlation with k270: 0.9806 in evoo, 0.9600 in po, 0.8672 in so and 0.9695 in p (tables 14-17). 3.12.5 k232 the two-way anova demonstrated that k232 was not significantly affected by temperature in all oils, whereas the interaction between temperature and heating duration influenced very highly significantly (p < 0.001) evoo and p (table 5). a low correlation with ∆k in all the rectified oils was found in k232: 0.4356 in po, 0.6311 in so and 0.5450 in p whereas k232 had a good correlation with ∆k in evoo (tables 1417). 3.12.6 k270 two-way anova demonstrated that k270 was not influenced by temperature and heating duration in all the four studied oils, whereas a very high significant influence was found in the interaction between the two variables (table 6). both k270 and p-anv are used as indices to indicate a prolonged oxidation. k270 was found to be very well correlated with p-anv in evoo (0.9605), in po (0.9677), in so (0.8545) and in p (0.9605), (tables 14-17). ital. j. food sci., vol. 30, 2018 735 3.12.7 ∆k the one-way anova analysis showed very high significant differences between treatments in all the four studied oils (p < 0.001). the two-way anova analysis showed evoo as the most influenced by heating duration and by the temperatures p < 0.05 for both the applied variables, whereas p was very highly significantly influenced (p < 0.001) by the interaction between the two treatments (table 7). ∆k was found to have a very good correlation with k270 and the highest correlations were in the two seed oils: 0.9588 in so and 0.9507 in p (tables 14-17). 3.12.8 total phenolic content the one-way anova analysis showed very highly significant differences between treatments. the two-way anova analysis showed a different situation for each oil. evoo was not influenced by temperature and by time, but it was very highly significantly influenced by their interaction. po was very highly significantly influenced by temperature and by time (p < 0.001), but their interaction was not significant. the total phenolic content in so was not influenced by time but was significantly influenced by temperature (p < 0.05) and very highly significantly influenced by their interaction (p < 0.001). in p, the interaction between time and temperature had no significant effect whereas temperature and time caused a highly significant effect (p < 0.01). total phenolic content showed a negative very good correlation with k270 in evoo (-0.8193), in po (0.9166), in so (-0.9094) and in p (-0.9019), (tables 14-17). 3.12.9 total tocopherol content one-way anova analysis showed a very high significant lowering (p < 0.001) of the total tocopherol content during heating both at 180 and 220°c. the greatest lowering effect was produced at 220°c with a reduction accounting for -79.5% (evoo), -72.5% (po), -73.1% (s) and -63.4% (p) after 120 min of heating treatment (table 9). the two-way anova experiment demonstrated very high significant differences by the combined effects of temperature x time in all the four studied oils and not significant differences if only the temperature effect was considered (table 9). a very good positive correlation (minimum 0.8532 in s) was always found with the total phenolic content and with the orac assay (minimum 0.8246 in p), (tables 14-17). 3.12.10 antioxidant activity (abts assay) the one-way anova analysis showed very highly significant differences between treatments (p < 0.001). the two-way anova analysis showed that in all the studied oils, the temperature significantly influenced the abts-aa (p < 0.05). time of heating significantly influenced the abts-aa in all oils except in po in which the significance was 0.057. the interaction between the two studied variables was not significant in all the studied oils except in evoo in which a highly significant effect (p < 0.01) was found (table 10). abts-aa showed a negative very good correlation with pv in evoo (-0.9000), in po (0.8515), in so (-0.8473) and in p (-0.9083) and a positive very good correlation with total phenolic content: 0.9420 in evoo, 0.8692 in po, 0.8641 in so and 0.9163 in p (tables 1417). ital. j. food sci., vol. 30, 2018 736 3.12.11 antioxidant activity (dpph hydrophilic assay) the one-way anova analysis showed very highly significant differences between treatments (p < 0.001) in all oils except in po in which a highly significant difference was found (p < 0.01). the two-way anova analysis showed that temperature caused no significant difference in so and p, in po the significance was 0.053 and in evoo the temperature caused significant differences (p < 0.05). the interaction between temperature and time caused no significant differences in the treatments of po, whereas in the other oils the significance was p < 0.05 for p, p < 0.01 for evoo and p < 0.001 for so (table 11). 3.12.12 antioxidant activity (dpph oil assay) one-way anova evidenced very highly significant differences between samples (p < 0.001), (table 12). two-way anova showed that the interaction between time and temperature influenced the aa-dpph-oil as follows: evoo (p < 0.05), so (p < 0.01), po and p (p < 0.001). no significant effect resulted from the temperature treatment in s and p, the same was for the time treatment in po (table 13). 3.12.13 antioxidant activity (orac assay) the one-way anova analysis showed very highly significant differences between treatments (p < 0.001) in all oils. the two-way anova analysis showed that temperature caused no significant effect in evoo and so, whereas in po and p a significant effect was found (p< 0.05). the time of heating showed no significant effect on evoo, so and p. the interaction between temperature and time of heating caused very highly significant effects (p < 0.001) in all the studied oils (table 13). orac assay showed the highest positive correlation with total phenolic content in evoo (0.9658) and in po (0.9020), (tables 14-15). in so the highest positive correlation was found with aa-dpph-oil (0.9489), whereas abts was the highest correlated in p (0.8823), (tables 16-17). the correlation between orac values and phenolic content was also studied by ninfali et al. (2001) who found a positive correlation (r = 0.78925) by analyzing commercially available evoos. 4. conclusions the findings of this work suggest how to manage temperatures and heating duration during cooking with an extra virgin olive oil, a pomace olive oil, a soybean oil and a palm oil. the four studied vegetable oils showed four different behaviours in relation to temperature and heating duration. extra virgin olive oil and palm oil showed the best performances in term of resistance to oxidation. all the heated oils showed a reduction in antioxidant activity when compared to control (unheated oil). a lower antioxidant activity was found in the heated oils because phenols and, in general, the antioxidants are destroyed during heating. the best cooking temperature was found to be at 180°c, which caused the lowest oil deterioration, as well as being less expensive if compared to 220°c. when extra virgin olive oil, pomace olive oil, soybean oil and palm oil are heated at 180°c they can 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1478. zullo b.a. and ciafardini g. 2008. the olive oil oxygen radical absorbance capacity (dpph assay) as a quality indicator. eur. j. lipid sci. technol. 110:428. paper received may 21, 2018 accepted may 25, 2018 ijfs#1393_bozza ital. j. food sci., vol. 31, 2019 451 paper the effect of an increase in paste temperature between malaxation and centrifugation on olive oil quality and yield: preliminary results l. guerrini*1, p. masella1, g. angeloni1, b. zanoni1, c. breschi1, l. calamai2,3 and a. parenti1 1dipartimento di gestione dei sistemi agrari, alimentari e forestali (gesaaf), università degli studi di firenze, piazzale delle cascine 15, 50144 florence, italy 2dipartimento di scienze delle produzioni agroalimentari e dell’ambiente (dispaa), università degli studi di firenze, piazzale delle cascine 18, 50144 florence, italy 3istituto bioscienze e biorisorse, (ibbr) cnr, florence, via madonna del piano 10, sesto fiorentino, italy *e-mail address: lorenzo.guerrini@unifi.it abstract olive oil extraction conditions are usually a compromise between quality and yield. enhancing yield decreases quality, and vice versa. we aim to understand if a change in temperature between malaxation and centrifugation can result in a better compromise. thus, malaxation is carried out at 20°c and oil is extracted at 20°c, 27°c, and 35°c. the results show that a moderate increase in temperature between malaxation and centrifugation (i.e. from 20°c to 27°c) increases both yield and quality of the oil. conversely, an excessive temperature increase (i.e. from 20°c to 35°c) leads to the production of rancid-related compounds. keywords: olive oil, phenols, quality, temperature, yield ital. j. food sci., vol. 31, 2019 452 1. introduction the extra virgin olive oil extraction involves several subsequent steps. briefly, olives are washed and crushed to obtain a paste. then, the olive paste is kneaded (i.e. malaxation phase) and centrifuged to separate the olive oil from water and exhaust solids (i.e. pomace) (guerrini et al., 2017a). temperature control during extraction is a key issue in extra virgin olive oil (evoo) production, since it affects both yield and quality (boselli et al., 2009). the effects of temperature on yield are mainly related to the malaxation and centrifugation steps. during malaxation, temperature influences the physic, chemical and enzymatic activity of olive paste. on the one hand, higher temperatures enhance the coalescence of oil droplets, decrease viscosity, and accelerate the diffusion of substances from the aqueous to the oily phase. usually, higher temperatures increase yield (trapani et al., 2017a). during malaxation, the slow and continuous kneading of the paste merges oil small droplets into larger ones, creating a continuous liquid phase that can easily be separated mechanically (kalua et al., 2006). drops with a diameter >30 µm are considered to be separated more easily with the decanter centrifuge (martinez moreno et al., 1957). furthermore, the viscosity of olive oil and the consistency of the olive paste decrease with an increase in temperature, facilitating evoo extraction in the decanter (guerrini et al., 2017b). the temperature during the extraction process has a marked effect on some evoo quality parameters. high temperatures enhance enzymatic reactions involving lipolytic, hydrolytic and oxidative degradation, affecting both phenols and aroma. furthermore, higher temperatures increase the vapor pressure of volatile substances, and may causes aroma losses (tamborrino, 2014). phenols are considered indicators of evoo quality for their functional, nutritional, and sensory properties (rodis et al., 2002). their recovery into the evoo is strongly affected by temperature. high temperatures enhance phenol solubility in the oily phase (rodis et al., 2002) and their release from olive tissues on the one hand, while, on the other hand, they improve the rate of enzymatic activity involved in phenol consumption and molecular weight reduction (trapani et al., 2017b). evoo flavor is mainly described by fruity and defective attributes. the european directive ec 1019/2002 on marketing standards for olive oil states that the fruity attribute must be perceivable, while no defective attributes should be present. the fruity attribute has been related to lipoxygenase (lox) pathway compounds (angerosa et al., 2001). lox activity is significantly affected by temperature. in the same way, temperature affects the appearance in the oil of all enzymatic-related defects (zhu et al., 2014). current practice is to select the malaxation temperature to maximize desired effects (i.e. higher yield, phenolic content and lox-related compounds) and minimize undesirable ones (i.e. phenol degradation and off-flavor biosynthesis). recently, heat exchangers have provided a valuable technological solution to improve the thermal control of olive paste. the first study of heat exchangers in olive mills was conducted by amirante et al. (2006). destoned olive paste was heated between the crusher and the malaxer to obtain the same yield for stoned and destoned paste (otherwise yield is lower for the latter). on the other hand, in two studies, veneziani et al. (2015, 2017) cooled the olive paste between the crusher and malaxer, and found an increase in evoo quality without a decrease in yield. these studies highlight that malaxation temperature plays a central role for the evoo quality. however, we want to test if a fast temperature increase after the malaxation can improve yield without decrease the oil quality. in fact, in a theoretical instantaneous temperature increase, we can reasonable suppose that only the physical effects (change in olive oil/paste viscosity, and change in solubility) occurs. on ital. j. food sci., vol. 31, 2019 453 the contrary, the chemical and enzymatic reactions occur during the time required for the temperature increase. hence, given the improved ability to control temperature during the process, in this work we test, the effect of heating olive paste between the malaxer and the centrifuge. trials explore the possibility of conducting malaxation at one temperature and extraction at another. the aim was to test if such heating has advantages in terms of both evoo yield and quality. 2. material and methods 2.1. trials olives (cv. frantoio) were manually harvested in tavarnelle val di pesa (italy) at the beginning of december 2017. the following day they were washed and crushed. processing was designed to reduce oxidative impact and limit any damage to oil quality due to oxygen. hence, 1.2 kg of the obtained olive paste was weighed and malaxed in sealed conditions for 30 min at 20°c. the result was divided into three portions of 400 g each. each portion was put in a sealed jar and the temperature was bring at 20°c, 27°c, or 35°c in one of three thermostatic baths. the time required for the temperature increase was 7 min. preliminary trials established the water temperature needed in each of the thermostatic baths to obtain these three temperatures in 7 min. then the paste was centrifuged for 2 min at 2000 rpm (trapani et al., 2017a) to separate olive oil from other phases. three replicates were conducted. 2.2. yield, water and oil content the oil extraction yields were calculated both as the actual yield (oy) and as an extractability index (ei): 𝑂𝑌 = !"! !"! ∙ 100 (1) 𝐸𝐼 = !"! !"!" ∙ 100 (2) where: oex was the measured extracted olive oil (g); olm was the measured olive paste (g); ocom was the oil content of the olive paste (g), which was determined from the oil content of the olive fruits (%). water content was measured using the gravimetric method. total oil content was determined in olive paste and pomace with hexane in an automatic randall extractor (mod. 148, velp scientifica, milan, italy), following the analytical technique described in cherubini et al. (2009). results were expressed as the percentage of total weight. 2.3. chemical analysis olive oils were tested for free fatty acid content, peroxide value, k232, k270 and ∆k based on methods set out in european commission regulation eec/2568/91. total and individual biophenol content were recorded according to the method described in ital. j. food sci., vol. 31, 2019 454 iooc/t.20 doc. n 29. volatile compounds were measured with the hs-spme-gc-ms technique (fortini et al. 2017). 2.4. statistical analysis data were processed with a one-way anova to assess differences among the three extraction temperatures. where a significant difference (p<0.05) was found, a tukey honest significant difference post-hoc test was carried out. 3. results and discussion 3.1. the effect of paste temperature increase after a malaxation at low temperature for 30 min the evoo samples were analyzed after their extraction by centrifugation of the olive paste, which were malaxed at 20°c for 30 min and then, kept at 20°c for 7 min or heated to 27°c and 35°c in 7 min (tables 1 and 2). in the above evoo samples the oil extraction yields and the eu legal chemical parameters (i.e. able to measure the triglycerides degradation level due the lipase hydrolytic activity and the auto-oxidation and photo-oxidation phenomena) were measured (table 1). table 1. parameters of evoo extracted by centrifugation of the olive paste, which had no malaxation (i.e. at time t = 0) and were malaxed at 20°c for 30 min and then, kept at 20°c for 7 min or heated to 27 and 35°c in 7 min; a and b represent a statistically significant difference (p<0.05) based on the tukey hsd post-hoc test. final temperature after olive paste heating (°c) parameter at time t = 0 20 °c 27 °c 35 °c p oy (%) n.d. 20.7±0.8 b 23.3±0.3 a 23.7±0.9 a 0.01 ey (%)* n.d. 79±4 b 89±2 a 91±6 a 0.01 free fatty acids content (%) n.d. 0.43±0.01 0.47±0.04 0.47±0.06 ns peroxide number (meq/kg) n.d. 11.4±0.7 11±3 11±1 ns k232 n.d. 1.73±0.03 1.67±0.04 1.74±0.03 ns k270 n.d. 0.08±0.02 0.08±0.01 0.09±0.01 ns rk n.d. 0.002±0.001 a 0.003±0.001 ab 0.003±0.001 b 0.03 total phenolic content (mg/kg) 230±31 b 238±23 b 290±10 a 298±37 a 0.04 3,4-dhpea-eda (mg/kg) 21±1 a 23±1 a 29±2 b 46±2 c 0.03 total lox compounds (mg/kg) n.d. 45.36±5.50 46.00±13.45 44.90±6.01 ns e-2-hexenal (mg/kg) n.d. 41.88±4.95 42.20±12.69 41.13±5.15 ns 1-penten-3-ol (mg/kg) n.d. 0.26±0.03 a 0.31±0.01 b 0.33±0.03 b 0.05 e-2-heptenal (µg/kg) n.d. 25±3 a 27±3 a 39±3 b 0.04 *from the oil content of olive paste (%) = 26.2±0.7; n.d. = not determined. the final temperature after the olive paste heating influenced significantly the extraction yields. the oy values increased approximately from 21% at 20°c to 24% at 35°c and the ey values increased approximately from 79% at 20°c to 91% at 35°c; the oy and ei values had no significant differences between 27°c and 35°c final temperatures. these results ital. j. food sci., vol. 31, 2019 455 were consistent with the literature data, which showed a positive relationship between temperature and extraction yield (trapani et al., 2017a). however, in all of these works the temperature was kept constant throughout the process, while our data show that a yield increase can be obtained with a temperature increase that is limited to the centrifugation process. no significant effect of the final temperatures occurred for the free fatty acids content, the peroxide number, the k232 and the k270 uv spectroscopic indexes. although the ∆k spectroscopic index showed a slight increase between 20°c and 35°c, evoo samples were in compliance with the eu legal chemical parameters. the total phenolic content increased significantly with the final temperature (table 1). the evoo samples which were extracted at the 20°c final temperature contained a significant lower content (about 25%) of phenols than those extracted at 27°c and 35°c reached temperatures. the dialdehydic form of decarboxymethyl oleuropein aglycone 3,4 dhpea-eda content (i.e the evoo's most abundant phenolic compound – zanoni, 2014) was also found to significantly increase with the final temperature (table 1); it increased from 23 mg/kg at 20 °c to 46 mg/kg at 35°c. the positive effect of temperature on the phenolic compounds content was already described in the literature. veneziani et al. (2015; 2017) described an increase in both the total phenolic content and the 3,4 dhpea-eda content in relation with the malaxation temperature, due to the combined activities of endogenous enzymes. rodis et al. (2002) found that the partition coefficients (oil/water) of phenolic compounds increased with temperature from 25°c to 45°c; hence, high temperatures were associated with high phenolic compounds content in the oily phase. no significant variation of the total phenolic and 3,4 dhpea-eda contents occurred between evoo samples that were not malaxed (i.e. at time t = 0) and those processed at 20°c. volatile composition of evoo samples was measured to verify the behavior of both the lipoxygenase (lox) pathway compounds, related to positive sensory attributes, and the compounds related to sensory defects (table 1). the experimental data of lox compounds content were similar at all the final temperatures. no significant differences were found for aldehydes and esters and only one alcohol (1-penten-3-ol) was found to increase with the highest temperatures. the olive paste heating after malaxation did not affect the lox pathway, but the c5 and c6 compounds contents in evoo samples seemed only related to the malaxation at 20°c for 30 min; angerosa et al. (2002) reported that malaxation conditions with temperature lower than 25°c for 30-45 min are optimal for the lox pathway. only one volatile compound related to sensory defects (i.e. the used method measured 48 defective compounds; fortini et al., 2017) was significant in the evoo samples (table 1). the (e)-2-heptenal, which is related to the rancid defect (kalua et al., 2006; zhu et al., 2016), occurred in all evoo samples and it increased with the final temperatures after the heating of malaxed olive paste. no significant differences were found between 20°c and 27 °c, but a significant difference was found at 35°c. the content of the (e)-2-heptenal varied approximately from 25 to 40 µg/kg, which are values above the relevant literature odor threshold of 0.005 mg/kg (kotti et al., 2011); values around 40 µg/kg of the (e)-2heptenal content were significantly correlated with olive oil samples with sensory defects (zanoni et al., 2015). ital. j. food sci., vol. 31, 2019 456 3.2. a predicted comparison of evoo yield and quality between the paste temperature increase after a malaxation treatment and the time-temperature malaxation treatment under stationary conditions in this paragraph we compare the effects of a malaxation at 20° c for 30 min, followed by 7 min of olive paste heating to 20, 27 and 35°c (i.e. the tested theses), with the effects of a malaxation carried out under stationary temperature conditions (i.e. at 20, 27 and 35°c) for the same time (i.e. 37 min). for this aim we use the mathematical predictions obtained with the computer program (malaxaction 1.0). the software is the only tool available today in literature (zanoni et al., 2018) for the comparisons of the effects of different olive paste malaxation conditions at laboratory scale. the computer program is based on the kinetic studies of trapani et al. (2017a; 2017b), which were carried out with the reference abencor lab equipment under exposure to air. the computer program is able to predict (i) the oil extraction yield as function of the olive paste oil content, expressed as ei%; (ii) the relative variation of the 3,4 dhpea-eda in the olive oil as the quality index that best represents the effect on the phenolic compounds in the extractable oil. table 2 reports the comparison between our results and the predictive model. to evaluate the ei% during the experimental trials we used the total oil content of the olive paste (i.e. roughly the 26% table 1); to evaluate the change in 3,4 dhpea-eda content in the olive oil samples we used the concentration in non-malaxed olive paste (i.e at time t = 0) of 22 mg/kg. finally we report a semi-quantitative evaluation of the rancid defect obtained using the odor threshold of (e)-2-heptenal in olive oil samples (table 1). the comparison between the ei% values and the relative variation of 3.4 dhpea-eda content showed similar results between the experimental values and the predicted values at 20 °c. it is important to point out that this is the only case among those studied without olive paste heating (i.e. malaxation at 20°c for 37 min). the results predicted by the computer program are consistent with the measured value (table 2), and the malaxaction 1.0 software has been considered suitable for the comparisons. table 2. the predicted comparison of evoo yield and quality between the paste temperature increase after a malaxation treatment and the time-temperature malaxation treatment under stationary conditions. parameter predictive comparison at 20°c predictive comparison at 27°c predictive comparison at 35°c experimental data (final temperature of 20°c) predicted data (malaxation at 20°c for 37 min) experimental data (final temperature of 27°c) predicted data (malaxation at 27°c for 37 min) experimental data (final temperature of 35°c) predicted data (malaxation at 35°c for 37 min) oil extractability index ei (%) 79 80 89 89 91 92 decrease (-) or increase (+) of 3,4 dhpea-eda content in oi (%) 0 0 +30 -61 +110 -4 the values of ei% obtained during our trials were similar those predicted by malaxaction 1.0 (table 2). this result remarks that the paste heating between malaxation and centrifugation allow to obtain the same yield results than the whole process at higher temperature. the model for ei% described by trapani and co-workers (2017a) tends to asymptotically reach the maximum value of 100%; low increases in temperature and short increases in ital. j. food sci., vol. 31, 2019 457 malaxation time have a high impact on ei% when temperature and time values are low. on the other hand, for long times and high temperatures the increase in ei% due to a further increase of such parameters is lower. consistently with the predictive model, a short heating of the olive paste was sufficient to increase the yield between 20°c and 27°c, while no further yield increase was obtained between 27 and 35°c. the model was not able to predict the changes in 3,4-dhpea-eda content (table 2). moreover, the model was built for olive paste under exposure to air, while this test was conducted in a sealed malaxer (i.e. only the oxygen in the malaxer headspace was available). however, the 3,4-dhpea-eda contents can be considered consistent with the general kinetic theory of trapani et al. (2017b). it suggested that the phenolic compound transformation phenomenon during a time-temperature treatment of olive paste is caused by two opposing phenomena during olive paste malaxation: (i) a decreasing phenomenon probably due to enzymatic oxidative damage of the phenolic compounds; (ii) an increasing phenomenon probably due to a physical and enzymatic release of phenolic compounds from the cellular tissues. obviously, the change in the oxygen availability reduces the extent of the decreasing phenomenon. thus, discrepancies between 3,4dhpea-eda measured and predicted values were found. at 27 °c the model predicted a 3,4-dhpea-eda decrease of 61%, while at 35°c of 4%. in the trial data we measured an increase of 30% and 110% of 3,4-dhpea-eda at 27°c and 35°c, respectively. in our trial, the above decreasing phenomenon was inhibited by the reduced oxidative conditions, resulting in a 3,4-dhpea-eda increase. 4. conclusions the current trend in high-quality evoo production is to reduce the malaxation time and temperature. although this enhances quality, it reduces yield. recent works have aimed to improve control of the temperature of the process by introducing a heat exchanger between the crusher and the malaxer. this is designed to improve the control of the process, allowing to maintain quality and increase yield for shorter malaxation times, or improve quality without decreasing yield. a further improvement can be obtained by placing a heat exchanger between the malaxer and the centrifuge and increasing the temperature. this approach obtains a remarkable increase in yield and increases phenols in the olive oil. however, an excessive temperature increase appears to be self-defeating, since at higher temperature off-flavour compounds appeared and no further yield increases were measured. the placement of a heat exchanger between the malaxer and the “decanter” further improves the control of the extraction process and seems to be complementary to the current trend to place a heat exchanger between the crusher and the malaxer. references amirante p, clodoveo m.l., dugo g., leone a. and tamborrino a. 2006. advance technology in virgin olive oil production from traditional and de-stoned pastes: influence of the introduction of a heat exchanger on oil quality. food chem. 98:797-805. angerosa f., mostallino r., basti c. and vito r. 2001. influence of malaxation temperature and time on the quality of virgin olive oils. food chem. 72:19-28. boselli e., di lecce g., strabbioli r., pieralisi g. and frega n.g. 2009. are virgin olive oils obtained below 27 °c better than those produced at higher temperatures? lwt food sci. technol; 42:748-57. ital. j. food sci., vol. 31, 2019 458 cherubini c., migliorini m., cecchi l., trapani s. and zanoni b. 2009. technological ripening indices for olive oil fruits. j. sci. food agric. 89:671. ec 1991 commission regulation eec 2568/1991 and later amendments. official j. eur. communities. 1991, l248(9). espínola f., 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wiley and sons. zanoni b., guerrini s., mari e., migliorini m., cherubini c., trapani s. and vincenzini m. 2015. investigation on microbiology of olive oil extraction process. ital. j. food sci. 27:236-247. zanoni b., breschi c., canuti v., guerrini l., masella p., picchi m. and parenti a. 2018. an original computer program (malaxaction 1.0) to design and control olive paste malaxation under exposure to air. j. food eng. 234:57-62. zhu h., wang s.c. and shoemaker c.f. 2016. volatile constituents in sensory defective virgin olive oils. flavour fragr. j. 31:22-30. paper received september 24, 2019 accepted january 31, 2019 ital. j. food sci., vol. 30, 2018 467 paper effect of ultrasound and chemical compounds on microbial contamination, physicochemical parameters and bioactive compounds of cherry tomatoes j.f.b. de são josé*a, h.s. medeirosb, n.j. de andradeb, a.m. ramosb and m.c.d. vanettic afederal university of espírito santo, vitória, espírito santo, brazil bfood technology departament, federal of viçosa university, viçosa, minas gerais, brazil cmicrobiology department, federal of viçosa university, viçosa, minas gerais, brazil *e-mail address: jackline.jose@ufes.br abstract in this study, different sanitization methods were evaluated to understand their effects on the microbiological and physicochemical characteristics of cherry tomatoes stored at 7°c. microbiological analysis showed that treatment with silver nanoparticles at 6 mg/l in combination with ultrasound treatment demonstrated a significant reduction in the assessed microorganism content of the cherry tomatoes. there were no changes in titratable acidity, ph, soluble solids, instrumental color, firmness, lycopene or beta carotene content after treatment followed by 10 d at 7°c. it was observed that cherry tomatoes treated with ultrasound had lower firmness values. treatment with sodium dichloroisocyanurate, detergent-based surfactants, lactic acid, ultrasound treatment combined with detergent and ultrasound treatment combined with silver nanoparticles all led to increased ascorbic acid content during storage. an application of the silver nanoparticles promoted the best results in terms of microbial count reduction without significantly affecting the quality characteristics of the cherry tomatoes. keywords: food quality, organic acids, sanitizers, silver nanoparticles, vegetables ital. j. food sci., vol. 30, 2018 468 1. introduction a tomato variety with great popularity worldwide is the cherry tomato (lycopersicon esculentum var cerasiforme) (wang et al., 2008; zhao et al., 2010). the cultivation of cherry tomatoes is gaining ground due to the interest in incorporating the plant into modern cuisine (lenucci et al., 2006) as it is tasty and sweet and can be consumed either as a fruit or as an appetizer. the reduced size and great versatility of their applications have made cherry tomatoes very popular in diverse culinary preparations. the use of cherry tomatoes as adornments, appetizers and the main feature of several dishes has led to increased consumption of this vegetable. this vegetable is present in diets because it has many antioxidants, such as carotenoids, ascorbic acid and phenolic compounds that can promote beneficial effects (guerreiro et al., 2016). the microbiological quality of fruits and minimally processed vegetables is directly related to the presence of spoilage microorganisms, which cause changes in sensory characteristics. additionally, the pathogenic micro-organisms in a given population can cause damage to consumer health (rico et al. 2007; letho et al., 2011). fruits and vegetables often possess naturally high amounts of bacteria, yeast and mold (romeo et al., 2010). microorganism contaminants come from various sources such as irrigation water, manure, cultivation, harvesting, postharvest processing and distribution handling (tornuk et al., 2011; guo et al., 2016). washing with sanitizing solutions is considered to reduce the number of spoilage and pathogenic microorganisms, thus contributing to product safety (allende et al., 2008; rahman et al., 2011; são josé & vanetti, 2012). among the sanitizers used in the food industry, especially to wash fresh produce, chlorine and chlorinated compounds are widely used (alvaro et al., 2009). the ease of use, low cost, high antimicrobial activity and complete dissolution in water make chlorinated agents a common choice for disinfectants in the fruit and vegetable industry (allende et al., 2008; sanchez et al. 2015; yamaner et al., 2016). new sanitization treatments have also been evaluated. the choice of sanitizer is important for hygienic and sanitary requirements and to maintain and, where possible, improve the sensory and nutritional characteristics of fruit and vegetables. organic acids are generally recognized as safe (gras) and can inactivate pathogens involved in food contamination (akbas & olmez, 2007; tirawat et al., 2016). lactic acid is an organic acid that may be applied to fresh produce (sagong et al., 2011). ultrasound is a technology recommended for use in the food industry for different applications. among these uses is the removal of particles adhering to surfaces and the inactivation of microorganisms (golmohamadi et al., 2013; bevilacqua et al., 2014; são josé et al., 2014). this inactivation is the result of a process called cavitation, which consists of the formation, growth and collapse of bubbles that generate mechanical energy where chemicals are located. when ultrasound treatment is used in conjunction with chemical agents, the intense pressure gradient allows for penetration of these agents into the cell membrane of microorganisms (piyasena et al., 2003; são josé et al., 2014). the use of detergents / surfactants in sanitizing solutions or washing water reduces the surface tension of the solution and thus improves contact with bacterial cells, causing a greater inactivation / removal of bacteria from vegetable surfaces (keskinen & annous, 2011). the combination of detergents with physical sanitizing agents may enhance their action (sagong et al., 2013). silver has been extensively evaluated as a therapeutic agent and for its use in water disinfection processes. moreover, the interest in the use of silver has increased, especially as a possible replacement for chlorine compounds. other prospective silver applications are related to post-harvest processing of fruits and vegetables (gopal et al., 2010). ital. j. food sci., vol. 30, 2018 469 it is known that fruits and vegetables undergo a series of changes after harvesting due to the environment, nutrient supplies and crop injury from processing. sanitization treatments aim to eliminate spoilage and pathogenic microorganisms to provide consumers with a safe product with a longer shelf life. sanitization should prevent undesired enzymatic reactions as well as microbial growth and multiplication to preserve the nutritional and sensorial aspects of the fruits and vegetables. growing consumer interest in food has led to looking beyond the basic functions of food for energy and nutrient supply and into purchasing products that have properties that promote health and prevent diseases. the quality of these food products is strongly related to their preparation, handling and storage. some studies have shown that these operations have important effects on the content of bioactive compounds (soria & villamiel, 2010; rawson et al., 2011a; rawson et al., 2011b; plaza et al., 2011; zinoviadou et al., 2015). in this context, this study was done to evaluate the effects of different methods of sanitization on the microbiological and physicochemical properties of cherry tomatoes stored at 7°c. 2. materials and methods 2.1. cherry tomato samples cherry tomatoes (lycopersicon esculentum var. cerasiforme) were acquired from local retailers and from a single producer to avoid variation. tomatoes were stored under refrigeration at 7°c for a maximum of 24 h before processing, and damaged or rotten tomatoes were discarded. tomatoes were washed in water to remove dirt adhered to the surface and were then drained for 30 min in a laminar flow hood. 2.2. preparation of sanitizers the effects of the following sanitization methods were evaluated: sodium dichloroisocyanurate (nippon®, indaiatuba, são paulo, brazil) at a concentration of 200 mg/l (sd), 1 % lactic acid (95 %; vetec®, são paulo, brazil) (la), a solution of silver nanoparticles at 6 mg/l (np), and detergent nitrol wv 2640 (nippon®, indaiatuba, são paulo, brazil) (det) with or without ultrasound treatment (us) at a low frequency of 40 khz, 300 w, with a tank size of 6"l x 5.5"w x 4"d (ultrasonic cleaner branson® 1510, st. louis, usa). a 1 % solution of lactic acid (v/v) was prepared. the distilled water used to prepare the solution was previously sterilized and kept under refrigeration. the colloidal dispersions of silver nanoparticles were obtained according to the methodology developed by fernandes (2010). to prepare the sanitizing solutions that were applied in conjunction with ultrasound, we used water at 5°c. previously, it was found that the ultrasonic sanitization time implied a 2 degree increase in water temperature. the next sanitization step consisted of immersing the cherry tomatoes in the sanitizing solution for 5 min at a temperature of 7±1°c. a group of tomatoes exposed to treatment with sterile distilled water and a group of tomatoes exposed to no sanitization treatment were used as controls. for each treatment, 2 kg of cherry tomatoes were sanitized in containers made of polyethylene terephthalate that had previously undergone exposure to ultraviolet light (254 nm) for 30 min in a laminar flow hood. cherry tomatoes were distributed with 250 g in each container and stored at 7°c for 10 d and evaluated at days 1 and 10 for microbiological qualities and on days 1, 2, 4, 6, 8 and 10 for physicochemical and nutritional qualities. ital. j. food sci., vol. 30, 2018 470 2.3. microbiological analysis the procedures used in this step were performed according to the methods of the american public health association (apha) as described in the compendium of methods for the microbiological examination of foods (downes & ito, 2001). after each sanitization treatment, 25 g of cherry tomatoes were homogenized separately in 0.1% peptone water at a dilution of 10-1. homogenization was conducted in a stomacher (seward medical co., london, united kingdom) for 2 min at the normal speed. appropriate dilutions were prepared, and aliquots of these dilutions were transferred to growth media specific for the detection of each microbial group. plating rate experiments were performed in duplicate, and the results were expressed in colony-forming units per gram (cfu/g). to determine the number of aerobic mesophiles, inoculation was performed in standard plate count agar (pca) (difco®), followed by incubation for 48 h at 35±1°c. mold and yeast aliquots were inoculated on potato dextrose agar (pda) (oxoid®) at a ph of 3.5 and incubated at 25±2°c for 5 to 7 d. coliform counts were carried out at 35°c using the petrifilm technique (3m®) according to the recommendations of the association of official analytical chemists (aoac, 2005). plates were incubated at 35°c for 48 h, enumerating e. coli as blue colonies entrapped with gas, and other coliform colonies as those colored red and associated with gas. standard plate counts were used for aerobic psychrotrophics on plate count agar (himedia®), with plates incubated at 7°c±1°c for 7-10 d. lactic acid bacteria counts were performed using the method of plating in depth using 1 ml of the sample and a spilling-out quantity of mrs agar (himedia®) in petri dishes followed by incubation at 30°c for 5 d. 2.4. physicochemical analysis tomato samples were evaluated for their physical and chemical characteristics. these analyses were intended to identify possible changes in the quality of the cherry tomatoes after sanitization treatments compared to non-sanitized samples (controls). the determination of titratable acidity (ta) was performed using titration according to the technique described by aoac (2005). the titration was performed with 0.1 mol/l naoh, and the results were expressed as % citric acid. for ph determination, 10 g of tomato samples were randomly collected and homogenized together with 100 ml of distilled water (ial, 2004). readings were taken using a digimed dm 20 ph meter (são paulo, brazil). total soluble solids (tss) content was determined by refractometry using a portable digital abbe refractometer at 25°c. readings were taken using three drops of pulp juice made with 10 g of tomato. the results were expressed as % (ial, 2004). color change was measured objectively by colorimetry using a minolta colorimeter color reader cr 10 cielab system, calibrated with a direct reflectance reading of l* coordinates (lightness), a* (relative green to red) and b* (relative yellow to blue). for each sample, the average of three measurements in five random cherry tomato areas was used for each repetition of treatments in each evaluation day. the parameters l*, a* and b* were determined using the hue angle (h°) and chroma through the following equations: ºh = tan-1· (b*/a*) chroma = [(a*)2 + (b*)2]1/2 ital. j. food sci., vol. 30, 2018 471 the firmness of the cherry tomatoes was determined using an instron test apparatus (series 3367) with a 3-mm diameter probe, a speed of 5 mm/s and a penetration distance of 19 cm before contact with the sample. five cherry tomatoes were analyzed for each treatment, with two measurements taken per tomato. the results of the firmness analysis were expressed as the maximum force (n). 2.5. extraction and analysis of ascorbic acid ascorbic acid (aa) extraction conditions were optimized by campos et al., (2009). a 5-g sample of the cherry tomatoes and 15 ml of extraction solution (3 % metaphosphoric acid, 8 % acetic acid, 0.3 mol/l sulfuric acid and 1 x 10-6 mol/l edta) were mixed using microtrituration for 5 min, centrifuged at 1789 g for 30 min, and the supernatant was adjusted up to 25 ml with ultrapure water. aa was determined according to campos et al., (2009) using a high-performance liquid chromatograph (shimadzu) equipped with a lichrospher 100 rp-18, 250 x 4 mm, 5 μm chromatographic column. the flow of the mobile phase (1 µmol/l nah2po4, 1 µmol/l edta, ph 3.0) was 1.0 ml/min, and the run time was 5.0 min. elution was detected using a photodiode array detector (shimadzu sod-m10 avp) with the wavelength set to 245 nm. aa identification was done by comparing the retention times obtained for the standard and for samples analyzed under the same conditions. furthermore, the authors compared the absorption spectra of the standard and the peaks of interest in the samples using a diode array detector. the standard used was l-ascorbate (vetec, brazil). quantification of the compound in the samples was taken from the analytical regression curve equation of ascorbic acid (y = 3,870,141.085x + 883,647.051; r2=0.997), and results were expressed in mg of fresh matter. 2.6. extraction and analysis of carotenoids carotenoid extraction was performed according to rodriguez et al. (1976) with modifications. approximately 7 g of tomato were triturated in 60 ml of acetone (divided into three volumes of 20 ml), vacuum filtered on a buchner funnel and transferred to 50 ml of cold petroleum ether. extracts obtained from the samples were then concentrated in a rotary evaporator at a temperature between 35 and 37°c. then, carotenoids were dissolved in 25 ml of petroleum ether and stored in amber glass vials in a freezer (at approximately -5°c) for chromatographic analysis. carotenoids were determined according pinheiro-sant'ana et al. (1998) using a highperformance liquid chromatograph (shimadzu) equipped with a phenomenex c18 rp-18 chromatographic column, 4.6 x 250 mm, 5 μm. the mobile phase was methanol:ethyl acetate:acetonitrile (50:40:10). flow of the mobile phase was 2.0 ml/min, and running time was 10 min. elution was detected using a photodiode array detector (shimadzu sodm10 avp) with the wavelength set to 450 nm. identification of carotenoids was done by comparing retention times obtained for standards and samples analyzed under the same conditions. furthermore, the authors compared the absorption spectra of the standard and the peaks of interest in the samples, using a diode array detector. the standards used were β-carotene (sigma aldrich, usa) and lycopene (sigma aldrich, usa). quantification of the compounds in samples was taken from calibration curves and regression equations for lycopene (y= 6.844.138,9670x – 13.153,9821 r2 = 0.9996) and βcarotene (y= 7.994.514x+ 1.277,372 7; r²= 0.999). the results were expressed in µg/100 g of fresh matter. ital. j. food sci., vol. 30, 2018 472 2.7. statistical analysis a completely randomized design was used, with eight treatment groups and a control group (no sanitizing) with tests run in triplicate. to evaluate the efficiency of sanitization treatments on microbial load at the beginning and end of the storage period, student’s t test was used to compare each treatment. to compare the treatments with each other within each period, the results of the counts (log cfu/g) were analyzed with an analysis of variance (anova), with means compared statistically using a duncan’s test with a 5 % significance threshold. a completely randomized design was used for physicalchemical and nutritional quality with split plots, where treatments were in the plots and time in the sub-plots, with three replicates. the results of the parameters were analyzed using anova, and the means were compared statistically using duncan’s test for qualitative variables (treatment) and regression analysis was performed for quantitative variables (storage time). the significance level used was 5 %. all statistical analyzes were performed using the statistical analysis system (sas institute, north carolina, usa), version 9.1, licensed for use by the federal university of viçosa, minas gerais, brazil. 3. results and discussion 3.1. microbiological analyses after sanitization treatments, the mesophilic aerobic bacteria count was reduced between 0.27 and 2.33 log cfu/g compared to cherry tomatoes that were not sanitized (fig. 1). figure 1. mesophilic aerobic bacteria count and standard deviation after sanitization treatments ( ) and the tenth day ( ) of storage at 7°c. treatments indicated with the same lowercase letter did not differ (p > 0.05) between one another after sanitization treatments. treatments indicated with the same capital letter did not differ (p > 0.05) between one another on the tenth day. ital. j. food sci., vol. 30, 2018 473 on the tenth day of storage at 7°c, counts were 6.83 log cfu/g of aerobic mesophilic bacteria on tomatoes sanitized by ultrasound at 40 khz. treatment with ultrasound did not achieve reduction of 1.0 log cfu/g, a result that was similar to that observed by são josé & vanetti (2012) using ultrasound with a frequency of 45 khz for 10 min. susanarivera, venturini, oria & blanco (2011) observed a reduction of 1.0 log cfu/g of aerobic mesophilic bacteria by applying ultrasound to decontaminate fresh truffles (tuber aetivum). the limitations for the reduction of microorganisms from the vegetable surface can be related to the presence of a hydrophobic cuticle composed of several layers and cutin wax molecules covering the skin of the fruits and vegetables (velázquez et al., 2009). the combination of chemical agents with ultrasound promoted reductions between 1.73 and 2.11 log cfu/g. immediately after sanitization, treatment with detergent promoted a reduction similar to that of sodium dichloroisocyanurate treatment (p>0.05). são josé et al. (2014) and rosário et al. (2017) mentioned that ultrasound has a potential to be used to inactivate bacteria but must be applied in combination with other sanitizing agents. treatments with 1 % lactic acid, silver nanoparticles and both of these sanitizing agents combined with ultrasound demonstrated a superior and significant reduction in aerobic mesophilic bacteria counts when compared to the application of sodium dichloroisocyanurate (p<0.05). however, when lactic acid 1 % was combined with ultrasound treatment, there was no significant difference (p>0,05), indicating that in this combination of treatments, a greater reduction in bacteria was not found. when detergent was combined with ultrasound, we found a significant reduction in bacteria when compared to the reduction promoted by the application of only detergent solution (p<0.05). lactic acid and 1 % silver nanoparticles at 6 mg/l reduced contamination with aerobic mesophilic bacteria by 2.18 and 2.33 log cfu/g, respectively. gopal et al. (2010) investigated the use of silver (as silver nitrate) and electrochemically generated silver in minimally processed lettuce and found that sanitization with silver was more effective than chlorinated solutions for different micro-organisms, such as mold, yeast, enterobactereacea and pseudomonas. at the end of the storage period, the counts of mesophilic aerobic bacteria in the cherry tomatoes treated with lactic acid, detergent, silver nanoparticles and lactic acid + ultrasound and silver nanoparticles + ultrasound were significantly lower than in the sodium dichloroisocyanurate treatment group (p<0.05). fruits and vegetables can also be contaminated by mold and yeast. after harvest, fungal contamination may deteriorate plant material and produce toxic metabolites. some molds and yeasts can also produce mycotoxins as they grow on these products and others, which present pathogenic risks to consumer health (tournas, 2005). untreated cherry tomatoes showed 4.70 and 6.13 log cfu/g of mold and yeast after sanitization and at the end of the storage period, respectively (fig. 2). treatments promoted reductions between 0.37 and 1.58 log cfu/g. at the initial time of storage, the greatest reductions observed in mold and yeast counts were 1.58 and 1.26 log cfu/g, as registered in samples of cherry tomatoes treated with silver nanoparticles and ultrasound combined with 1 % lactic acid, respectively. a smaller reduction was observed and recorded for mold and yeast counts after treatment with ultrasound applied by itself. ital. j. food sci., vol. 30, 2018 474 figure 2. mold and yeast counts and standard deviation after sanitization treatments ( ) and the tenth day ( ) of storage at 7°c. treatments indicated with the same lowercase letter did not differ (p > 0.05) between one another after sanitization treatments. treatments indicated with the same capital letter did not differ (p > 0.05) between one another on the tenth day. of the treatments proposed in this work, only sanitization with silver nanoparticles and ultrasound combined with lactic acid promoted reductions statistically equal to sodium dichloroisocyanurate (p>0.05). this result indicates that these treatments had similar effects in reducing mold and yeast counts. a loss of efficiency in the reduction of the mold and yeast counts was observed when detergent was applied to the silver nanoparticles in combination with the ultrasound. in evaluating the effect of ultrasound on the structure of the nanoparticles (data not shown) it was observed that the action of the ultrasound may have contributed to the removal of the surfactant present in the structure of the silver nanoparticle in order to facilitate the approximation of the silver particles. this aggregation of the particles possibly culminated in an increase in diameter and consequently a lower antimicrobial effect. for coliforms at 35°c, non-sanitized samples showed 5.03 log cfu/g. among the treatments that stood out, 1 % lactic acid, 6 mg/l silver nanoparticles and ultrasound combined with these two sanitizers promoted, respectively, reductions of 2.06, 2.34, 2.86 and 2.59 log cfu/g after the sanitization processes (fig. 3). oliveira et al. (2011) detected coliform bacteria in most samples of minimally processed vegetables, with populations of 3 log mnp/g. the presence of these microorganisms can also contribute to reducing the useful life of products (berbari et al., 2001). brazilian law rdc no. 12 of january 2001 (brazil, 2001) provides a value of 102 mnp/g for testing sanitized vegetable samples. huang and chen (2011) studied different sanitizing treatments in spinach and noted that 1 % lactic acid reduced e. coli o157:h7 to 1.9 log cfu/g. the application of an isolated form of ultrasound was inefficient for inactivating coliform, yielding a reduction of 0.27 log cfu/g. detergent alone promoted a coliform reduction of 0.64 log cfu/g. however, a combination of detergent with ultrasound increased inactivation, resulting in a reduction of 1.53 log cfu/g, and a synergistic effect could be ital. j. food sci., vol. 30, 2018 475 observed. the intense pressure generated by ultrasound can contribute to the penetration of chemical oxidants through the cell membrane, and the cavitation process assists in the breakdown of microorganisms that culminates in increased efficiency of the sanitizer (gogate and kabadi, 2009). it was observed that, at the end of the storage period, the cherry tomatoes treated with lactic acid, ultrasound and ultrasound combined with silver nanoparticles presented lower counts than the tomatoes treated with sodium dichloroisocyanurate. figure 3. coliform count and standard deviation after sanitization treatments ( ) and the tenth day ( ) of storage at 7°c. treatments indicated with the same lowercase letter did not differ (p > 0.05) between one another after sanitization treatments. treatments indicated with the same capital letter did not differ (p > 0.05) between one another on tenth day. treatments promoted a reduction of between 0.41 and 2.64 log cfu/g of lactic acid bacteria (fig. 4). the counts obtained after treatment with 1 % lactic acid, ultrasound and ultrasound combined with silver nanoparticles did not differ statistically compared to the nonsanitized samples (p>0.05). higher inactivation was achieved by treatment with sodium dichloroisocyanurate 200 mg/l, ultrasound combined with 1 % lactic acid and silver nanoparticles, which caused reductions of 2.02, 2.14 and 2.64 log cfu/g, respectively. as these treatments showed statistically similar reductions (p>0.05) in lactic acid bacteria count, this may suggest the possible replacement of sodium dichloroisocyanurate treatment with ultrasound combined with lactic acid or silver nanoparticles. only treatment with silver nanoparticles was efficient in the microbiological control at the end of the storage period. psychotropic count after sanitization was between 3.90 and 6.29 log cfu/g, and at the end of storage, sanitized cherry tomatoes achieved scores of approximately 5 log cfu/g (fig. 5). ital. j. food sci., vol. 30, 2018 476 figure 4. lactic acid bacteria counts and standard deviations after sanitization treatments ( ) and the tenth day ( ) of storage at 7°c. treatments indicated with the same lowercase letter did not differ (p>0.05) between one another after sanitization treatments. treatments indicated with the same capital letter did not differ (p > 0.05) between one another on the tenth day. figure 5. psychrotrophic aerobic counts and standard deviations after sanitization treatments ( ) and the tenth day ( ) of storage at 7°c. treatments indicated with the same lowercase letter did not differ (p > 0.05) between one another after sanitization treatments. treatments indicated with the same capital letter did not differ (p > 0.05) between one another on the tenth day. oliveira et al. (2011) evaluated the contaminant microbiota of minimally processed vegetables and observed large populations of psychrotrophic aerobic bacteria in the samples, suggesting a short shelf life of the products. these counts can be related to the ital. j. food sci., vol. 30, 2018 477 storage condition of the product that favored this group of bacteria. ultrasound, and ultrasound combined with detergent did not lead to significant reductions in bacterial counts (p>0.05). it is worth mentioning that the ultrasound treatment combined with silver nanoparticles promoted a significant reduction of the psychrotrophic aerobic count after the sanitization process and maintained the lower count at the end of the storage period. 3.2. physicalchemical and bioactive compound analysis measurements of ph showed no significant differences (p> 0.05) resulting from sanitization treatments, time or interaction between time and treatments. quality monitoring of this parameter after sanitization and during the storage period was important because sanitizers may influence the ph values of the vegetables, causing changes that may accelerate the deterioration of the product (rico et al., 2007; são josé and vanetti, 2015). values of titratable acidity (ta), total soluble solids (tss), and the ratio of tss/ta had a significant association with storage time. ta values varied significantly with time, which is best represented by linear regression (y= 0,01965x2 + 0,57070, r2 = 0,87). for tss, linear and quadratic models were not significant. for tss/ta, linear regression (y= -0,41861x2 + 12,42302, r2 = 0,99) best represented the data. adekunte et al. (2010) evaluated the effect of ultrasound on tomato juice in different intensities and time and found no change in ph, ta or tss. table 1 shows ph, ta, tss values and the tss/ta ratio for the different treatments. ta values ranged from 0.60 to 0.68 % citric acid. this parameter is involved in the assessment of food preservation, since, in most cases, the decomposition of food alters the concentration of hydrogen ions (ial, 2004) in addition to influencing the sensory characteristics of food. this parameter can vary depending on the degree of maturation and growth conditions (chitarra and chitarra, 2005). cao et al. (2010) treated strawberries with ultrasound at different frequencies and found that ta content decreased with storage time and that fruit treated with ultrasound at 40 khz tended to have higher values of ta compared to the control fruit. average values of tss for cherry tomatoes corroborated the mean value observed by pinho (2011), who recorded 6.1°brix in cherry tomatoes grown in conventional systems. tss is a refractive index that indicates the ratio (%) of dissolved solids in solution. it is the sum of sugars (sucrose and hexose), acids (citrate and malate) and other components (phenols, amino acids, soluble pectins, ascorbic acid and mineral salts) present in minor proportions in tomato fruit pulp (beckles, 2012). according to the abhorticultura (2011), the tomato has between 9 and 12 %, which features the highest concentration of soluble solids, especially sugars. cao et al. (2010) evaluated the effect of ultrasound on tss values and found a decrease in all treatments after strawberry harvest. these authors observed that treatment with 40 khz ultrasound significantly inhibited the decline of tss after six days of storage. in this study, the tss/ta ratio of cherry tomatoes tended to be lower after treatment with ultrasound combined with chemical sanitizers. this relationship is one of the indices most often used to determine the maturation and palatability of fruit and corresponds to fruit sugar and acid content and is thus an appropriate parameter to measure flavor perception by the consumer (suarez et al., 2008; beckles, 2012). javanmardi and kubota (2006) found that the average tss of tomatoes stored at an ambient temperature and at a low temperature ranged from 5.0 to 5.1 %. in this study, treatment with sanitizers, storage time and the interaction between storage time and treatments were not found to significantly affect color parameters (table 1). the color of the food is one of the more attractive attributes for consumers and may vary ital. j. food sci., vol. 30, 2018 478 between species and even between cultivars. the color of a food product is an important freshness-related attribute for consumers when evaluating quality (são josé & vanetti, 2015). gani et al. (2016) observed that ultrasound treatment applied in 10, 20, 30 and 40 min lengths resulted in better retention of color during storage of strawberry samples. muzaffar et al. (2016) observed that ultrasound treatment between 30 and 40 min showed better retention of color of cherries (prunus avium) during the storage period at 4°c. adekunte et al. (2010) observed a decrease in the values of l* and color parameters a* and b* in tomato juice treated with ultrasound under different conditions. they suggested that the color changes observed may have been caused by cavitation, which involves different physical, chemical and biological reactions. in this study, because it involved the sanitization of intact cherry tomatoes, cavitation may have had less of an effect on color. the color of the tomatoes is mainly related to the presence of carotenoid pigments such as lycopene and is influenced by processing (lianfu and zelong, 2008). sagong et al. (2011) evaluated the effects of 1 % lactic acid combined with ultrasound on other vegetables, including fresh lettuce, and observed no significant change in l*, a* or b* after 7 days of storage at 4°c. firmness values did not change significantly (p>0.05) due to the treatment, time or interaction between treatment and time (table 2). the lack of significant changes in the firmness of the cherry tomatoes after the different sanitization treatments during the storage period indicates a preservation of the tomato structure. firmness is a critical attribute of quality in the acceptability of fruit and vegetables by the consumer (cao et al, 2010) and may decrease due to loss of turgor cells due to water loss during storage (akbas and ölmez, 2007). despite the changes in firmness between the different treatments not being statistically significant, it was observed that firmness values were lower after treatment with ultrasound compared to after the application of chemical agents. cao et al. (2010) observed that the application of 40 khz ultrasound markedly inhibited the softening of strawberries, which maintained high levels of firmness during storage through inactivation of polygalacturonase and pectin methyl esterase enzymes. yang et al. (2011) evaluated the effects of ultrasound both as an individual treatment and combined with salicylic acid for 10 min on peaches and observed no change in firmness after 6 days of storage at 20°c. gani et al. (2016) observed that samples treated for 20 and 30 min with ultrasound showed a maximum retention of firmness and the decrease observed at 40 and 60 min could be attributed to the prolonged exposure resulting in cell injury and loss of water. other studies evaluating the influence of chemical sanitizers on the firmness of fruits and vegetables (akbas and ölmez, 2007, sagong et al., 2011; alexandre et al., 2012) have similarly observed maintenance of firmness during storage. fruits and vegetables lose firmness and freshness characteristics when they are kept in refrigerated storage, even for short periods. in this work, maintaining firmness may have been favored by the intact sanitization of cherry tomatoes. this is an important finding for the processing of this product. an ideal sanitization treatment should be simple and easy to apply without causing physical damage and maintain the sensory characteristics of the food (susana-rivera et al., 2011). in addition to the quality features already discussed, the assessment of losses in carotenoid and ascorbic acid contents after sanitization treatments is also important. the content of lycopene and β-carotene in sanitized cherry tomatoes did not change significantly due to sanitization treatments, time, or the interaction of treatment and time (table 3). ital. j. food sci., vol. 30, 2018 479 table 1. mean values* and standard deviations of ph, ta, tss, ratio tss/ta and color parameters of cherry tomatoes under different sanitization treatments stored for 10 d at 7°c. treatment ph ta tss tss/ta l* a* b* hue croma no sanitizer 4.30±0.33 0.64±0.23 6.59±1.57 10.30±1.36 29.19±1.04 19.41±2.66 27.80±1.66 55.17±4.00 33.98±2.07 sd 200 mg l-1 4.27±0.29 0.60±0.22 6.35±1.11 10.58±1.37 29.29±1.01 17.93±2.71 27.40±1.15 56.95±3.94 32.82±1.85 det 4.30 ±0.37 0.63±0.13 6.22±1.33 9.87±1.44 29.78±1.19 16.78±2.80 26.94±3.94 57.95±5.20 31.88±3.76 1 % al 4.29 ±0.35 0.62±0.17 6.05±1.04 9.75±1.95 29.80±1.12 18.10±3.69 27.38±2.02 56.78±5.16 32.95±2.95 np 6 mg l-1 4.31 ±0.34 0.63±0.18 5.83±0.84 9.25±1.64 29.59±1.10 17.71± 3.15 27.11±1.94 57.01±4.75 32.49±2.56 us 40 khz 4.30 ±0.35 0.66±0.18 6.23±1.11 9.43±1.12 28.99±0.76 17.12±2.93 27.07±1.22 57.89±4.10 32.10±2.15 us 40 khz+det 4.33 ± 0.32 0.68 ±0.20 5.77±0.89 8.48±2.98 28.97±1.08 16.50±3.09 25.42±0.72 57.27 ±4.74 30.41±1.95 us 40 khz+1 % al 4.26±0.33 0.60±0.17 6.46±1.24 10.67±2.95 28.95±0.73 17.77±5.73 26.45±1.53 56.82±6.86 32.13±4.25 us 40 khz+np 6 mg l-1 4.31 ±0.35 0.62±0.21 5.66±0.79 9.13±3.11 29.10±0.86 16.90±3.09 25.93±1.12 57.13±4.54 31.05±2.14 *interaction (treatment × storage time) not significant (p > 0.05). effect of the treatments were not significant (p > 0.05). ital. j. food sci., vol. 30, 2018 480 table 2. mean values* and standard deviations of firmness of cherry tomatoes under different sanitization treatments stored for 10 d at 7°c. treatment firmness (n) no sanitizer 2.63±0.43 sd 200 mg l-1 2.32±0.72 det 2.33±0.60 1 % al 2.21±0.58 np 6 mg l-1 2.56±0.75 us 40 khz 2.07±0.75 us 40 khz + det 1.70±0.66 us 40 khz + 1 % al 1.75±0.55 us 40 khz + np 6 mg l-1 1.72±0.63 *interaction (treatment × storage time) not significant (p > 0.05). effect of the treatments were not significant (p > 0.05). table 3. mean* values and standard deviations of lycopene and β-carotene contents of cherry tomatoes subjected to different treatments sanitization stored for 10 d at 7°c. treatment lycopene (µg/100 g fm) β-carotene (µg/100 g fm) no sanitizer 771.82±180.47 646.43±102.72 sd 200 mg l-1 769.24±221.42 704.94±122.04 det 615.26±186.47 634.61±118.59 1 % al 737.31± 216.39 635.95±99.93 np 6 mg l-1 682.81±207.61 643.81±119.75 us 40 khz 797.44±196.56 636.47±120.09 us 40 khz + det 653.77±233.90 651.64±132.59 us 40 khz + 1 % al 730.44±205.15 630.11±182.98 us 40 khz + np 6 mg l-1 831.54±207.54 675.78± 92.56 *interaction (treatment × storage time) not significant (p > 0.05). effect of the treatments were not significant (p > 0.05). fm = fresh matter lycopene degradation can occur due to processing and storage conditions, which cause isomerization and oxidation of this compound. environmental factors such as oxygen, light and temperature have an important role in this process (demiray et al., 2013). free radicals, mechanical energy and hot spots (regions of high temperature and pressure) generated by ultrasonic cavitation can contribute to reducing the contents of nutrients in treated cherry tomatoes. however, this change was not observed, indicating that high retention values were obtained and that the applied sanitizing treatments seemed to have preserved the contents of lycopene and β-carotene. studies with other foods that evaluated other bioactive compounds (alexandre et al.., 2012, tiwari et al., 2010) showed that ultrasound allows for greater retention of anthocyanin content, with the authors suggesting that degradation may occur due to oxidation reactions promoted by the interaction of free radicals formed during sonication. anese et al. (2013) observed that the total concentration of lycopene from tomato pulp was unaffected by processing with ultrasound, and total contents ranged between ital. j. food sci., vol. 30, 2018 481 5.10±0.80 and 6.60±1.10 mg/g of dry mass in untreated and ultrasound-treated tomato samples, respectively. mild heat treatment in tomatoes during food preparation, such as heating, boiling and cooking does not significantly alter carotenoid levels in tomatoes or other vegetables (anese et al., 2013). storage at low temperatures inhibits lycopene formation (javanmardi and kubota, 2006); thus, the maintenance of the values of lycopene, as observed in this study, may also be associated with the storage conditions used here (7°c). it is important to emphasize the maintenance of lycopene and β-carotene content after sanitization using different treatments, because it is known that these carotenoids are directly related to the color displayed by tomatoes. this confirms the results obtained in the analysis of color parameters, which showed no significant changes due to sanitization treatments during storage. although several studies (sass-kiss et al., 2005; kotíková et al., 2011; ilahy et al., 2011) on tomatoes have indicated a higher proportion of lycopene to β-carotene, this study indicated similar concentrations of these two carotenoids. this result may be related to the differences between varieties and/or geographical locations and climates that can promote lycopene production to a greater or lesser extent than the other carotenoids (kotíková et al., 2011). regarding ascorbic acid contents, the interaction between treatment and storage time was significant (p<0.05) (table 4). with regards to this interaction, control treatments, ultrasound and silver nanoparticles showed no significant change over time. the ultrasound treatment combined with 1 % lactic acid exhibited non-significant regression coefficients, which suggests the unsuitability of linear and quadratic regression models to predict the behavior of ascorbic acid contents with respect to time. for the other treatments, regression equations were significant. although vitamin c is the least stable of all vitamins and is easily destroyed during processing and storage, the present study indicated either maintenance or increased ascorbic acid content for some sanitizing treatments. cruz et al. (2008) additionally found that the rate of destruction is increased by the action of metals, particularly copper and iron, enzymes, oxygen availability, prolonged heating in the presence of oxygen and exposure to light. gani et al. (2016) observed a decrease in vitamin c content in strawberries when ultrasound was applied for 10, 20, and 30 min. table 4. regression equation models for ascorbic acid content in cherry tomatoes submitted to different sanitization treatments and stored. treatment regression model r2 p > f no sanitizer ȳ=16.80 sd 200 mg l-1 0.3674x+12.8796 0.42 0.0191 det 0.8653x+12.2803 0.81 0.0024 1 % al 0.5169x+14.2298 0.88 0.0017 np 6 mg l-1 ȳ=15.18 us 40 khz ȳ=15.84 us 40 khz + det 0.4562x+12.6830 0.46 0.0155 us 40 khz + 1 % al y=21.54 us 40 khz + np 6 mg l-1 0.4955x+12.9953 0.75 0.0031 y= mg of ascorbic acid. function of storage time (x), determination coefficients (r2) and probability levels (p). ital. j. food sci., vol. 30, 2018 482 during cavitation, free radicals may be produced, and these compounds may react with food. this interaction may be beneficial or not, depending on the process and the food matrix (soria and villamiel, 2010). rawson et al. (2011a) studied the effect of ultrasound on carrot disks and observed retention of vitamin c content results similar to those observed in the present study, which found that ascorbic acid content was preserved in samples that were not sanitized, treated with ultrasound at 40 khz and treated with silver nanoparticles at 6 mg/l during the storage period. for the other treatments, ascorbic acid contents increased, which may be related to the maturation of the fruit during storage. yahia et al. (2001) stated that ascorbic acid is subjected to oxidation and reduction reactions during tomato ripening. oxidation products are free acid radicals that can be processed again to form ascorbic acid, which allows for the increase of the substance as the fruit ripens. ercan and soysal (2011) observed no significant difference (p> 0.05) in the content of ascorbic acid of tomato extracts after treatment with ultrasound. cao et al. (2010) found higher levels of vitamin c in strawberries treated with ultrasound at 40 and 59 khz compared to controls. the differences observed in research reported earlier might be due to difference in food samples and treatment parameters. 4. conclusions an application of silver nanoparticles promoted the best results in terms of microbial count reduction. in the case of the mesophilic aerobic bacteria, the application of the silver nanoparticles contributed to the control after the sanitization process and until the end of the storage period. for mold and yeast, silver nanoparticle treatment and a combination of ultrasound and lactic acid were also effective. for coliforms, ultrasound associated with nanoparticles promoted the best effect. for control of lactic bacteria and psychrotrophic aerobic counts, silver nanoparticle treatment was more efficient. all of the sanitizing treatments maintained ph values, ta, tss, color parameters, firmness, and lycopene and beta-carotene contents throughout the storage period at 7°c. ascorbic acid content ranged between treatments and during storage and showed an increase for several treatments, including sodium dichloroisocyanurate, detergent-based surfactants, lactic acid, ultrasound combined with detergent and ultrasound combined with silver nanoparticles. these results indicate that some proposed treatments have the potential for use in sanitizing cherry tomatoes and showed greater effects on reducing microbiological contamination without affecting other quality characteristics of the vegetable. it is worth noting that the proposed treatments presented similar or superior results than those promoted by sodium dichloroisocyanurate. this indicates a possibility of replacement. however, when choosing a sanitization method, it is necessary to evaluate not only the efficiency but also the cost benefit of each one. acknowledgements we would like to thank fundação de amparo a pesquisa de minas gerais (fapemig/brazil in process apq-00241-12) and coordenação de aperfeiçoamento de pessoal de nível superior (capes/brazil) for their financial support and granting scholarships, respectively. we are grateful to the nutrition and health department of the federal university of viçosa for allowing the realization of carotenoids and ascorbic acid analyses. references abhorticultura. 2011 tomate cereja sabor e rentabilidade no mesmo produto. disponível em: <http://www.abhorticultura.com.br/news/default.asp?id=4864>. acesso em: 10 de junho de 2012. ital. j. food sci., vol. 30, 2018 483 adekunte a.o. tiwari b.k., cullen p.j., scannell a.g.m and, o’donnell c.p. 2010. effect of sonication on colour. ascorbic acid and yeast inactivation in tomato juice. food chem. 122 (3): 500-507. afari g.k. hung y., king c.h. and hu a. 2016. reduction of escherichia coli o157:h7 and salmonella typhimurium dt 104 on fresh produce using an automated washer with near neutral electrolyzed (neo) water and ultrasound. food control, 63: 246-254. 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ki̇m et al., 2018). commercial kefir is produced using pure starter cultures that contain a mixture of limited number of bacteria and yeasts, which give a similar flavor to the traditional kefir, but do not cause bulge or leakage (o’brien et al., 2016). the most common method in the production of commercial kefir is the use of commercial concentrated lyophilized kefir starter cultures (yaman, 2011). because of the health benefits of kefir, people’s interest onto kefir has been increasing day by day. in the dairy industry, kefir is produced mostly from cow's milk (purnomo and musli̇mi̇n, 2012). however, the researchers used goat milk (purnomo and musli̇mi̇n, 2012; kaczyński̇ et al., 2018), sheep milk (wszolek et al., 2001; cai̇ssokoli̇ńska et al., 2008), camel milk (kavas, 2015), buffalo milk (gul et al., 2015), whey (magalhães et al., 2011), coconut milk (ismai̇el et al., 2011), rice milk (nurli̇yani̇ et al., 2015), soy milk (kesenkaş et al., 2011; ismai̇el et al., 2011), oat milk (di̇nkçi̇ et al., 2015) or peanut milk (bensmi̇ra and ji̇ang, 2012) in addition to cow's milk in the production of kefir. it has been interested in kefir produced from goat milk since the αs1-casein, which has an important responsibility in cow's milk allergy is very low in goat milk. the studies on goat kefir produced with commercial culture mostly focused on physical, chemical and microbiological properties. however, it has been determined that there are not enough studies on the biochemical, organic acid and aroma profile of kefir produced by commercial culture. present study was aimed to determine the changes in chemical, physical, biochemical, microbiological, sensorial properties and also, organic acids and flavor components in kefir samples made from cow and goat milk fermented by kefir starter culture during storage at refrigerated condition. also, the best consumption time of kefir during refrigerated storage was determined in this study. ital. j. food sci., vol. 32, 2020 500 2. materials and methods 2.1. materials cow and goat milk were used in production of the experimental kefir samples. raw cows' milk was purchased from a factory and raw goats' milk from a farmer in bolu. kefir starter culture which is consisted of lactococcus lactis ssp., leuconostoc ssp., streptococcus thermophilus, lactobacillus ssp., kefir yeast, and kefir grain microflora according to the product information were obtained from danisco biolacta (kefir dc1, olsztyn, poland). sterilized bottles made of high-density polyethylene (hdpe) material were used for the preservation of the kefir samples. 2.2. kefir manufacture the milk was transported to the research and development laboratory of department of food engineering, bolu abant i̇zzet baysal university. while the milk was transferred to the pasteurizer, it was filtered through cloth and steel strainer. then, the temperature of the milk was increased to 55°c in the pasteurizer and a portion of the milk was passed through the cream separator to obtain skim milk. the resulting skimmed milk was used to adjust the fat content of the kefir milk to 3.1 %. fat standardized milks were pasteurized at 90°c for 10 min and cooled to 28°c and starter culture was added to both the milks at a level of 0.0065 g l-1. afterwards, inoculated milk was filled into the sterilized hdpe bottles (1 l). all procedures were carried out aseptically as much as possible. after inoculation, kefir milk samples were incubated at 28°c until the ph reached 4.60. after incubation, kefir curd was broken by shaking down 10 times. the stirred kefir samples were kept at a refrigerator (4°c) for 35 days. the chemical, physical, biochemical, microbiological, sensorial properties and also organic acids and flavor components in kefir were determined on 1st, 7th, 14th, 21th, 28th and 35th day of storage period. cow and goat milk kefir were coded as ck and gk, respectively. 2.3. determination of chemical properties analysis of ph, titratable acidity, dry matter, fat and protein contents of kefir samples were done as described by kurt et al. (1993). the ph of milk and kefir samples was directly measured using a calibrated ph meter (wtw 720, germany). 2.4. determination of physical properties apparent viscosity of kefir samples was determined at 15°c, using a sine-wave vibroviscometer sv-10 (a&d company, japan). the syneresis of kefir samples was determined by a procedure modified from sodini, montella, & tong (2005). a sample of about 25 g of kefir (a) was centrifuged for 10 min at 1250g at 4°c. the expelled whey (b) was weighed. the syneresis (%) was calculated as: syneresis (%) = (b/a)x100. a color measurement device (konica minolta cr400, japan) was used to measure color values as cie l*, a* and b* of the milk and kefir samples. ital. j. food sci., vol. 32, 2020 501 2.5. determination of the lipolytic and proteolytic activity the lipolysis as acid degree value (adv) of kefir samples was determined by titrimetric method according to case et al. (1985). the proteolysis values in the milk and kefir samples were determined according to hull (1947). 2.6. enumeration of microorganisms the kefir samples were dispersed and further diluents were made in ringer solution (¼ strength, merck, germany). the lactobacilli counts were determined on mrs medium under microaerophilic condition with anaerocult c (merck, germany) at 30°c for 3 days (irigoyen et al., 2005). the lactococci counts were determined on m 17 medium under microaerophilic condition with anaerocult c at 30°c for 2 days (irigoyen et al., 2005). the leuconostoc counts were determined on mse agar, a selective medium for leuconostoc, under aerobic condition at 25°c for 5 days (garcía fontán et al., 2006). the total mesophilic aerobic bacteria (tmab) counts were determined on pca agar under aerobic condition at 30°c for 2 days (mainville et al., 2001). the acetic acid bacteria counts were determined on apm agar under aerobic condition at 25°c for 5 days (witthuhn et al., 2005). the yeast counts were determined on ygc agar under aerobic condition at 25°c for 5 days (witthuhn et al., 2005). 2.7. determination of organic acids lactic, citric, acetic, pyruvic, orotic and oxalic acid in milk and kefir samples were determined by hplc (perkin elmer flexar, usa) according to güzel-seydi̇m (2000b) with some modifications. four grams of the milk was diluted into 25 ml of 0.013n h2so4 while four grams of kefir samples were diluted into 25 ml of 0.010n h2so4. the mixture was vortexed for 1 min and centrifuged at 7000 g at 4°c for 7 min. the supernatant was passed through filter having a pore diameter of 0.45 µm (millipore) and put into vial. the organic acids were separated in an isocratic system using a c 18 column (perkin elmer, 5µm, 250mm x 4.6mm i.d) at a column oven temperature of 50±1°c. the wavelengths of the uv detection were performed at 210 nm using photodiode array detector. the mobile phase was carried out with 10 mm kh2po4 adjusted to ph 2.5 with phosphoric acid. the flow rate of the mobile phase was adjusted at 0.5 ml min-1. the amount of organic acids was determined on standard curves of individual organic acids. 2.8. determination of volatile flavor components the acetaldehyde, diacetyl, acetoin and ethanol contents of the kefir samples were analyzed at the research center of yeni̇gidam of bolu abant i̇zzet baysal university, according to the method given by güzel-seydi̇m et al. (2000b). the volatile flavor components in the product were determined by gc (shimadzu gc 2010, japan) with flame ionization detector. the kefir samples (5 g) were weighed into 20 ml headspace vials. the prepared vials were heated in a dry block heater at 85°c for 5 min. at the end of the heating period, the air in the headspace of the vial using a gas-tight syringe (supelco) was injected into the capillary column (agilent, db-23, 0.25 id 0.25mm x 60m). the column oven temperature was maintained at 40°c for 1 min and then, increased to 250°c and kept at this temperature for a further 3 minutes. the temperature rise time was 35 minutes. the ital. j. food sci., vol. 32, 2020 502 carrier gas was helium and the flow rate was 0.6 ml min-1. the amount of the volatile components was determined on standard curves of individual volatile components. 2.9. determination of sensory properties sensory evaluation of the samples was determined by modifying the hedonic scale system (drake, 2009) and was performed by 11 trained panelists. the scoring was based on 5 points in three categories: 1) structure, consistence and texture, 2) taste and smell and 3) general appreciation. 2.10. statistical analysis the ck and gk samples were manufactured two times and all the analyses were performed in two parallels. the differences between the characteristics of cow and goat milk kefir samples were determined by t-test and mann whitney u test according to the availability of the dependent variable in each group showing normal distribution. the changes occurred in the kefir samples during storage were analysed by one-way anova based on the tukey hsd test (devore and peck, 1993). statistical analysis of all obtained data was performed with spss 20.0 package program (spss inc., chicago, il) at the significance level 0.05. 3. results and discussion some properties of both cow and goat milk used in kefir production were presented in table 1. as seen from the table, dry matter, fat and protein contents of them were close to each other. the ph, viscosity and lipolysis values of goat milk were higher than that of cow milk. moreover, l* value (lightness) of goat milk was higher than the value of cow milk. goats convert carotene from green fodder completely into vitamin a, and therefore color of goat milk is whiter than cow milk (gürsoy, 2007). the tyrosine value was higher in cow milk. table 1. some properties of standardized milk used in the study (n=2). analyses cow milk (x ̄) goat milk (x ̄) dry matter (%) 11.04 11.09 fat (%) 3.00 3.06 protein (%) 2.96 3.02 ph 6.60 6.64 acidity (la, %) 0.16 0.15 viscosity (mpa.s) 2.032 2.064 color l* 79.378 79.727 color a* -3.212 -3.121 color b* 5.482 5.911 lipolysis (meq koh/100 g fat) 0.359 0.935 proteolysis (mg tyrosine/5 ml milk) 0.171 0.143 x ̄: mean of two repetitions, n: number of repetitions l*: lightness (0= black, 100= white), a*: green (-) or red (+), b*: blue (-) or yellow (+). ital. j. food sci., vol. 32, 2020 503 3.2. chemical changes some chemical changes in the kefir samples made from cow and goat milk during 35-day storage were given in table 2. the kefir samples made from goat milk (gk) had higher general mean dry matter content than the kefir samples made from cow milk (ck) (p<0.05). similar results were observed in cow kefir (kavas, 2015) and goat kefir samples (kaczyński̇ et al., 2018). during storage, dry matter contents in kefir samples did not change (p>0.05). a similar result was obtained by ertekin and güzel-seydim (2010). there was no difference (p>0.05) between the general mean fat values of ck and gk samples. this was thought to be related with the standardization of the fat content of the milk used in the production of both samples. during storage, fat contents in kefir samples did not change (p>0.05). the general mean protein contents of ck and gk samples were close to each other (p >0.05). during the storage, the change in protein values of kefir samples was found to be significant (p<0.05) in gk samples, but not significant (p>0.05) in ck samples. the acidity as lactic acid (%) was higher in ck than gk samples in general (p<0.05). the reason for the slower development of acidity in gk samples compared to the ck sample might be related to the fact that the non-protein nitrogen content of goat milk and its buffering capacity are higher than cow milk (tratnik et al., 2006). in general, the acidity increased during storage and the change was significant (p<0.05) in both samples. it was observed that the ph values of the ck samples were lower than that of the gk samples (p<0.05). the ph values of ck samples were like those determined by ertekin and güzel-seydi̇m (2010) while the ph value of gk samples were similar to those reported by kaczyński et al. (2018). this is related to the high buffering capacity of goat milk (tratnik et al., 2006). the ph value of the samples on the first day of storage was lower than 4.60. this decline was due to the lactic acid bacteria in the sample continued to produce lactic acid from lactose while the internal temperature of the samples was reaching from incubation temperature (28°c) to 4°c. the ph value of ck samples decreased until the 14th day of storage, there was a slight increase on the 21st day of storage, and then decreased again until the 35th day of storage. in the case of gk samples, the ph decreased until the 14th day of storage and then increased slightly up to 35th day of storage. in both kefir samples, the difference between day 1 and day 35 of storage was significant (p<0.05). the ph value of kefir did not change very fast during storage. this is related to yeasts found in kefir (o’brien et al., 2016). some yeast species assimilate lactic acid and cause to rise ph (rattray and o’connell, 2011). 3.3. physical properties the changes in some physical properties of the kefir samples during storage and statistical analysis results of these changes were given in table 3. the general mean viscosity value of ck samples (64.80 mpa.s) was higher than gk samples (9.46 mpa.s) and the difference was significant (p<0.05). the viscosity value of ck samples was like those determined by yildiz-akgül et al. (2018) while the viscosity value of gk samples was similar to those reported by güneşer and karagül-yüceer (2010). similarly, tratnik et al. (2006) and güneşer and karagül-yüceer (2010) reported that the viscosity values of kefir samples made from goat milk were lower than the viscosity values of kefir samples made from cow milk. the reason for this is that the main fraction of goat milk casein is β-casein while the main fraction of cow milk casein is αs1and β-casein (huma et al., 2018). goat milk protein micelles form softer and more brittle gel. as a result, weak texture occurs in ital. j. food sci., vol. 32, 2020 504 goat fermented dairy products (güneşer and karagül-yüceer, 2010). in addition, it has been thought that the difference between the acidity values of the samples affect viscosity of the samples. in general, viscosity values of both ck samples (p<0.05) and gk samples (p>0.05) increased during storage. variability in viscosity during storage might be related to the production of exopolysaccharides and degradation of exopolysaccharides into monomers by microorganisms and enzymes in kefir. the general mean syneresis value of gk samples was higher than that of ck samples (p<0.05). compositional differences of goat milk such as low αs1-casein concentration and smaller diameter fat globule lead to softer structure in fermented dairy products made from goat milk and more syneresis in these products (milani and wendorff, 2011). the changes in syneresis values during storage were found to be nonsignificant (p>0.05) in gk samples and significant (p<0.05) in ck samples. there was an inverse relationship between syneresis and viscosity values of the kefir samples during storage. the general mean l*, a* (negative) and b* values of ck samples were higher than that of gk samples and the difference was significant (p<0.05). l* values of the kefir samples were higher than l* values of milk used in the production of the samples. in general, the l* values of both kefir samples tended to decrease during storage. the changes in l* and b* values of ck and gk samples was found to be significant (p<0.05) during storage. during storage, a* values of ck and gk samples showed fluctuant. however, this change was not significant (p>0.05). gul et al. (2018) reported that milk variety has an effect on a* and b* values while no effect l* value. 3.4. lipolysis and proteolysis the changes in lipolysis and proteolysis of the kefir samples during storage and statistical analysis results of these changes were shown in table 4. the lipolysis values of gk samples were higher than ck samples (p<0.05) and this may be related with some postmilking issues, such as milking time and storage type of goat milk. because, when examining fig. 1, it was seen that the amount of lactic and acetic acids in goat milk was higher than cow milk. this suggested that goat milk had been exhibited high lipolytic activity before reaching to the laboratory. wszolek et al. (2001) reported that milk type used in the production of kefir influences the amount of free fatty acids. the lipolysis values of ck and gk samples increased during storage (p<0.05). cais-sokolińska et al. (2008) reported similar results. the general mean values of proteolysis were found to be 0.597 mg tyrosine 5g-1 kefir for ck samples and 0.397 mg tyrosine 5g-1 kefir for gk samples (p<0.05) and this might be because of the number of microorganisms in kefir, especially the number of lactic acid bacteria. microorganisms can obtain the amino acids needed from proteins and peptides through proteolytic systems (di̇nkçi̇ et al., 2015). proteolysis values of ck and gk samples increased during storage and the increase was significant (p<0.05). di̇nkçi̇ et al. (2015) reported that proteolytic activity increased as long as storage time increased, and storage time affected proteolytic activity. ital. j. food sci., vol. 32, 2020 505 table 2. chemical changes in cow and goat kefir samples during storage. properties kefir storage time (days) (𝒙±sd) (n=2) 1 7 14 21 28 35 general mean drymatter (%) ck 10.78±0.006a* 10.76±0.041a 10.73±0.027a 10.73±0.065a 10.74±0.057a 10.72±0.011a 10.74±0.038b* gk 11.13±0.029a 11.14±0.011a 11.19±0.062a 11.15±0.044a 11.09±0.092a 11.13±0.008a 11.14±0.048a fat (%) ck 3.10±0.071a 3.14±0.159a 3.09±0.059a 3.12±0.057a 2.94±0.035a 3.01±0.124a 3.07±0.100a gk 3.18±0.035a 3.15±0.005a 3.15±0.071a 3.13±0.009a 3.13±0.053a 3.04±0.018a 3.13±0.054a protein (%) ck 3.10±0.013a 3.10±0.020a 3.09±0.000a 3.06±0.047a 3.07±0.008a 3.06±0.002a 3.08±0.026a gk 3.06±0.003c 3.10±0.003a 3.10±0.009a 3.10±0.004a 3.09±0.012ab 3.07±0.002bc 3.09±0.018a acidity (%) ck 0.87±0.006c 0.87±0.006bc 0.90±0.003b 0.94±0.001a 0.94±0.000a 0.95±0.013a 0.91±0.034a gk 0.80±0.003b 0.82±0.003ab 0.84±0.017a 0.84±0.004a 0.84±0.003a 0.82±0.066ab 0.82±0.019b ph ck 4.31±0.011a 4.27±0.020ab 4.26±0.001bc 4.27±0.009ab 4.23±0.007bc 4.21±0.014c 4.26±0.033b gk 4.47±0.002a 4.44±0.009ab 4.38±0.014c 4.40±0.014bc 4.41±0.007bc 4.42±0.012bc 4.42±0.030a ck: cow kefir, gk: goat kefir, 𝑥: mean, n: number of repetitions, sd: standard deviation, a,b: means in each row show statistically difference (p>0.05 or p<0.05) among storage days for each property of the samples. a,b: means in the same column show statistically difference between kefir samples in terms of related property (p<0.05). ital. j. food sci., vol. 32, 2020 506 table 3. physical changes in cow and goat kefir samples during storage. properties kefir storage time (days) (𝒙±sd) (n=2) 1 7 14 21 28 35 general mean viscosity (mpa.s) ck 42.17±2.165c* 52.12±1.539c 48.43±0.798c 64.62±9.117bc 93.23±10.438a 88.24±3.906ab 64.80±20.916a* gk 8.44±0.188a 8.58±0.119a 10.37±0.268a 9.66±0.285a 10.13±0.502a 9.61±1.493a 9.46±0.904b syneresis (%) ck 46.26±0.837a 42.42±0.338cd 45.30±0.414ab 43.83±0.219bc 41.49±0.417d 43.68±0.605bc 43.83±1.723b gk 62.04±0.250a 61.58±0.799a 55.61±1.848a 59.19±0.609a 58.80±0.219a 60.80±4.270a 59.67±2.676a color l* ck 82.810±0.057a 82.723±0.025ab 82.680±0.042ab 82.583±0.081b 82.530±0.050b 82.685±0.057ab 82.668±0.104a gk 81.849±0.014a 81.498±0.122b 81.606±0.049ab 81.562±0.064ab 81.549±0.110ab 81.447±0.035b 81.585±0.145b color a* ck -3.298±0.032a -3.338±0.032a -3.258±0.004a -3.318±0.110a -3.228±0.025a -3.358±0.025a -3.299±0.060b gk -3.103±0.040a 3.047±0.013a -3.189±0.014a -3.189±0.023a -3.152±0.030a -3.174±0.077a -3.142±0.062a color b* ck 6.535±0.000c 6.640±0.028bc 6.743±0.025ab 6.815±0.028a 6.63±0.046bc 6.830±0.035a 6.700±0.112a gk 5.741±0.191ab 5.535±0.052b 5.878±0.032ab 5.960±0.042a 5.891±0.044a 5.804±0.043ab 5.801±0.157b ck: cow kefir, gk: goat kefir, 𝑥: mean, n: number of repetitions, sd: standard deviation, a,b: means in each row show statistically difference (p>0.05 or p<0.05) among storage days for each property of the samples. a,b: means in the same column show statistically difference between kefir samples in terms of related property (p<0.05). l*: lightness (0= black, 100= white), a*: green (-) or red (+), b*: blue (-) or yellow (+) table 4. biochemical changes in cow and goat kefir samples during storage. properties kefir storage time (days) (𝒙sd) (n=2) 1 7 14 21 28 35 general mean lipolysis (meqkoh 100g fat-1) ck 0.45±0.016b* 0.46±0.012b 0.73±0.045ab 1.03±0.018ab 1.16±0.209a 1.23±0.310a 0.84±0.349b* gk 0.99±0.002c 1.12±0.050c 1.33±0.001b 1.63±0.000a 1.43±0.086b 1.42±0.060b 1.32±0.224a proteolysis (mg tyrosine 5g kefir-1) ck 0.440±0.019d 0.489±0.045cd 0.582±0.031bc 0.624±0.009b 0.645±0.013b 0.804±0.006a 0.597±0.124a gk 0.353±0.018b 0.388±0.012ab 0.412±0.014a 0.407±0.009a 0.404±0.009a 0.422±0.006a 0.397±0.025b ck: cow kefir, gk: goat kefir, 𝒙: mean, n: number of repetitions, sd: standard deviation, a,b: means in each row show statistically difference (p>0.05 or p<0.05) among storage days for each property of the samples. a,b: means in the same column show statistically difference between kefir samples in terms of related property (p<0.05). ital. j. food sci., vol. 32, 2020 507 3.5. microbial profile the changes in some microbial properties of the kefir samples during storage and statistical analysis were shown in table 5. as seen from the table, there was no significant (p>0.05) difference in the counts of lactococci between the samples of ck and gk. lactococci counts of the samples were consistent with the results obtained by wszolek et al. (2001). the highest number of lactococci (∼9 log cfu g-1) was determined on the first day of storage in both kefir samples. the change in lactococci count between the 1st and 7th days of storage was significant in ck samples (p<0.05). in gk samples, the change in the lactococci count was statistically significant between the first and last day of storage (p<0.05). the number of lactococci was above 8 log units for both samples during storage. temiz and kezer (2015) reported that the decrease in the number of lactococci was less than about 1.25 log units after 28-day storage. the general mean count of lactobacilli in ck samples was higher (6.74 log cfu g-1) than that of gk samples (6.45 log cfu g-1) and the difference was significant (p<0.05). wszolek et al. (2001) reported the similar results. lactobacilli counts of ck and gk samples decreased during storage (p<0.05). it was determined that 2.7 log-decrease in ck samples and 2.4 log-decrease in gk samples occurred on the 7th day of storage (p<0.05). irigoyen et al. (2005) and grønnevik et al. (2011) reported that there was an approximately 2 log decrease in the number of lactobacilli in kefir samples on day 28 of storage when compared with the first day of storage. the general mean count of leuconostocs of ck samples (6.74 log cfu g-1) was higher than that of gk samples (6.50 log cfu g-1), but not significant (p>0.05). wszolek et al. (2001) reported the similar findings. the leuconostoc counts of the kefir samples decreased during storage and the decrease was significant (p<0.05). a statistically significant decrease was found in the leuconostoc count of ck samples on the 7th day of storage. on the other hand, a statistically significant decrease was found on the 1st, 7th and 14th days of storage of gk samples. at the end of storage, the number of leuconostoc of both kefir samples was over 6 log units. some researchers reported that the number of leuconostoc in the kefir samples decreased below 6 log units at the end of storage (grønnevik et al., 2011; gul et al., 2015). the general mean counts of tmab of each sample were found close to each other (p>0.05). tmab counts of both samples decreased throughout storage time and the decrease was significant (p<0.05) for ck samples but not significant (p>0.05) for gk samples. in ck samples, the decrease was statistically significant between day 1st and day 14th of storage (p<0.05). temiz and kezer (2015) reported that storage time had effect on tmab counts. it was determined that the general mean counts of aab of ck samples was higher than that of gk samples and this was significant (p<0.05). the similar findings were reported by irigoyen et al. (2005). during storage, the number of aab decreased in both samples (p<0.05). approximately 1.2 log reduction (p<0.05) occurred between the 1st and 14th days of storage in gk samples. however, the counts of aab in the gk samples remained almost constant (p>0.05) from the 14th day of storage to the end of storage. leite et al. (2013) reported a 0.6 log unit reduction in the counts of aab during 28-day storage. the general mean counts of yeast of ck and gk samples were 1.95 and 1.10 log cfu g-1, respectively and the difference between them was significant (p<0.05). in general, kefir contains yeasts between 3-6 log cfu g-1 (erteki̇n and güzel-seydi̇m, 2010; di̇nkçi̇ et al., 2015). ital. j. food sci., vol. 32, 2020 508 table 5. microbial changes in cow and goat kefir samples during storage. properties kefir storage time (days) (𝒙sd) (n=2) 1 7 14 21 28 35 general mean lactococci (log cfu g-1) ck 9.23±0.057a* 8.64±0.201b 8.54±0.062b 8.60±0.108b 8.49±0.070b 8.55±0.037b 8.67±0.275a* gk 8.98±0.008a 8.65±0.054ab 8.78±0.038ab 8.68±0.154ab 8.77±0.116ab 8.49±0.043b 8.72±0.168a lactobacilli (log cfu g-1) ck 9.04±0.163a 6.33±0.341b 6.28±0.031b 6.22±0.047b 6.26±0.003b 6.28±0.151b 6.74±1.082a gk 8.62±0.054a 6.20±0.237b 5.96±0.054b 5.96±0.136b 5.96±0.170b 6.01±0.143b 6.45±1.022b leuconostoc (log cfu g-1) ck 8.33±0.231a 6.53±0.112b 6.41±0.082b 6.39±0.011b 6.42±0.023b 6.35±0.364b 6.74±0.759a gk 8.44±0.001a 6.61±0.103b 5.92±0.108c 6.01±0.067c 5.94±0.126c 6.11±0.052c 6.50±0.938a tmab (log cfu g-1) ck 9.24±0.107a 8.66±0.229ab 8.64±0.137b 8.57±0.021b 8.36±0.144b 8.26±0.173b 8.62±0.345a gk 9.35±0.100a 8.55±0.013a 8.71±0.078a 8.20±0.730a 8.64±0.161a 8.20±0.067a 8.61±0.466a aab (log cfu g-1) ck 7.37±0.012a 6.36±0.146b 6.11±0.040bc 6.02±0.010bc 6.03±0.001bc 5.92±0.155c 6.30±0.525a gk 6.98±0.378a 6.12±0.308ab 5.77±0.128b 5.81±0.083b 5.76±0.161b 5.72±0.218b 6.03±0.498b yeast (log cfu g-1) ck gk 2.39±0.017a 1.81±0.096a 2.12±0.232a 1.47±0.172a 1.76±0.086a 0.94±0.060a 1.39±0.144a 0.67±0.180a 1.67±0.048a 0.78±0.042a 2.35±0.657a 0.93±0.690a 1.95±0.443a 1.10±0.477b ck: cow kefir, gk: goat kefir, 𝒙: mean, n: number of repetitions, sd: standard deviation, tmab: total mesophilic aerobic bacteria, aab: acetic acid bacteria, a,b: means in each row show statistically difference (p>0.05 or p<0.05) among storage days for each property of the samples. a,b: means in the same column show statistically difference between kefir samples in terms of related property (p<0.05). ital. j. food sci., vol. 32, 2020 509 however, some researchers reported yeast count between 0.5-3 log cfu g-1 during storage in kefir samples produced from cow and goat milk by using different commercial starter cultures (wszolek et al., 2001; garcίa fontán et al., 2006; kesenkaş et al., 2011). in codex standard for fermented milks (codex stan 243-2003), it is stated that the number of yeasts in kefir should be at least 104 cfu g-1. yeasts produce ethyl alcohol and co2. the high number of yeasts in commercial kefir has caused packaging problems. in addition, due to consumer demand, low yeast counts have been generally preferred in kefir production in some countries. the difference of yeast number in kefir is varied depending on the type of culture used, the inoculation rate of the culture and the number of yeasts of the culture used. the number of yeasts in both ck and gk samples decreased until the 21st day of storage period. then, the yeast count of the samples increased towards the end of the storage. during storage, the number of yeasts in both ck and gk samples was less than 2.5 log. the yeast count of ck samples was higher than that of gk samples during storage. decrease in yeast count of both samples was nonsignificant (p>0.05) during storage di̇nkçi̇ et al. (2015) reported that storage time had no effect on yeast number. 3.6. organic acid content the changes in some organic acid contents of the kefir samples during storage were shown in fig. 1. general average lactic acid content in gk samples was greater than that of ck samples (fig. 1a) and this was significant (p<0.05). türker et al. (2014) found that the lactic acid content of kefir samples from goat milk was higher than that of kefir samples from cow milk. the changes of lactic acid content in ck and gk samples were statistically significant (p<0.05) during storage. it was believed that fluctuations in the amount of lactic acid during storage were related to the amount of lactic acid produced by lactic acid bacteria and the assimilation of lactic acid by some yeast species (rattray and o’connell, 2011). the average acetic acid content of gk samples was approximately 4 times higher than the values of ck samples (fig. 1a) (p<0.05). türker et al. (2014) and gul et al. (2015) reported that milk variety affected the amount of acetic acid in kefir. the acetic acid content of ck and gk samples were similar with values determined by muir et al. (1999) and grønnevik et al. (2011). the acetic acid content of ck samples was highest on day 1st of storage. the difference in the amount of acetic acid between the 1st and 7th days of storage was significant (p<0.05). grønnevik et al. (2011) reported that the change in the amount of acetic acid of kefir samples during storage was not significant. this was thought to be related to the fact that acetic acid is an intermediate product (leite et al., 2013). the cow and goat milk used for kefir production in this study showed citric acid content of 1175 µg g-1 and 433 µg g-1, respectively. while the average amount of citric acid was 108.34 µg g-1 in the gk samples, ck samples contained no citric acid (fig. 1b). citrate is a preferred substrate for the formation of acetoin and diacetyl by some lactic acid bacteria (güzel-seydim et al., 2000a). grønnevik et al. (2011) reported that there was more than 90 % reduction in the amount of citrate in kefir milk during fermentation and it was converted to other volatile components. ismaiel et al., (2011) could not detect citric acid in kefir produced under different fermentation conditions. the amount of citric acid in the gk samples increased up to the 14th day of storage (p<0.05), then suddenly decreased on the 21st day (p<0.05) and reached 35 µg g-1. although there was an increase in the amount of citric acid from the 21st day of storage to the end of storage, this increase was not ital. j. food sci., vol. 32, 2020 510 significant (p>0.05). kesenkaş et al. (2011) reported that the storage time affected the amount of citric acid in kefir. figure 1. lactic and acetic acid (a), citric and pyruvic acid (b), and orotic and oxalic acid (c) content in cow and goat kefir samples during storage (ck: cow kefir, gk: goat kefir, la: lactic acid, aa: acetic acid, ca: citric acid, pa: pyruvic acid, oa: orotic acid, oxa: oxalic acid, a,b,c,d: show statistically difference (p>0.05 or p<0.05) among storage days for each property of the samples. a,b: show statistically difference between kefir samples in terms of related property (p<0.05).) ital. j. food sci., vol. 32, 2020 511 general mean pyruvic acid content of gk samples (22.39 µg g-1) was higher than that of ck samples (15.31 µg g-1) (p>0.05) (fig. 1b). pyruvic acid levels of both ck and gk samples were lower than the milk used in the production. muir et al. (1999) reported higher amounts in traditional and commercial kefir (47 and 60 µg g-1). güzel-seydi̇m et al. (2000b) and beshkova et al. (2003) reported that pyruvic acid was completely consumed during storage. the amount of pyruvic acid significantly (p<0.05) decreased in both kefir samples during storage. reduction in pyruvic acid might be due to the conversion to other organic compounds during storage (güzel-seydi̇m et al., 2000b). orotic acid content was determined as 25 µg g-1 in cow milk used in the production of ck samples and as 5.71-14-74 µg g-1 in ck samples during storage (fig. 1c). the values of ck samples were lower than findings of muir et al. (1999). on the other hand, no orotic acid was detected in both goat milk used in the production of the gk samples and gk samples during storage. in a study, while the amount of orotic acid decreased during fermentation of kefir, it increased during storage (güzel-seydi̇m et al., 2000a,b). during storage time, no significant changes were observed in ck samples (p>0.05). overall mean content of oxalic acid of the gk samples was higher (p<0.05) than that of the ck samples (fig. 1c). ismaiel et al. (2011) could not detect oxalic acid in kefir produced under different fermentation conditions. türker et al. (2014) reported that milk variety affects the amount of oxalic acid in kefir. oxalic acid might be found up to 169.15 mg l-1 in cow kefir and to 119.37 mg l-1 in goat kefir. during storage, fluctuations in the amount of oxalic acid in the kefir samples were determined. the changes in ck samples were not significant (p>0.05) during storage. in gk samples, the increase in the amount of oxalic acid on day 21st of storage was significant (p<0.05). 3.7. aroma content the changes in acetaldehyde, diacetyl, acetoin and ethanol content in the kefir samples during storage were shown in fig. 2. the general mean value of acetaldehyde of ck samples was relatively higher, but insignificant (p>0.05). wszolek et al. (2001) reported similar results. during storage, the amount of acetaldehyde decreased in both samples and the values were between 1.52-2.92 µg g-1 in ck samples and 1.83-2.44 µg g-1 in gk samples. these values were in agreement with values obtained by grønnevik et al. (2011). differences in the amount of acetaldehyde in kefir may vary depending on milk fat ratio, starter culture type, the microbial diversity of the culture, the rate of use of culture, temperature and duration of incubation and storage (ertekin and güzel-seydim, 2010; yildiz-akgül et al., 2018). the changes in acetaldehyde values of kefir samples during storage were found to be significant only in the ck samples (p<0.05). beshkova et al. (2003) reported a similar trend in amount of acetaldehyde during storage. the decrease in the amount of acetaldehyde is related to the conversion of acetaldehyde to ethyl alcohol by the enzyme, which called alcohol dehydrogenase. diacetyl content of the ck samples was higher than that of gk samples (fig. 2a) (p<0.05). diacetyl values obtained in this study were in accordance with the values determined by wszolek et al. (2001) and beshkova et al. (2003). in some studies, diacetyl was not detected during fermentation and storage of kefir (güzel-seydi̇m et al., 2000a,b). during storage, the amount of diacetyl in ck samples increased up to the 14th day of storage and thereafter declined until the end of storage. in the gk samples, it increased until the 21st day of storage and decreased in the following days. the change in diacetyl values of kefir samples during storage was found to be significant (p<0.05) only in the ck samples. it has been reported that the optimum flavour balance for kefir is to be achieved when the ratio ital. j. food sci., vol. 32, 2020 512 of diacetyl to acetaldehyde is 3:1 (grønnevik et al., 2011). the highest ratio of diacetyl to acetaldehyde during storage was determined to be on the 14th day in the ck samples and on the 21st day in the gk samples. however, these rates were below 3. actually, some of researchers found this ratio between 0-2 in their studies (wszolek et al., 2001; grønnevik et al., 2011; yildiz-akgül et al., 2018). acetoin content was determined to be between 13.86-17.26 µg g-1 in the ck samples and 7.81-9.00 µg g-1 in the gk samples during storage (fig. 2b). general mean value of acetoin of ck samples was higher than that of gk samples (p<0.05). the values of the ck samples were similar with the values obtained (16-25 µg g-1) by güzel-seydi̇m et al. (2000b). beshkova et al. (2003) were unable to detect acetoin in kefir samples in their study. acetoin value of the samples tended to decrease during storage. the change in the amount of acetoin was significant (p<0.05) only in ck samples especially on day 21 during storage. grønnevik et al. (2011) reported that the amount of acetoin formed in kefir samples decreased during storage. the decrease in the amount of acetoin may be associated with further degradation, which results in 2.3-butanediol (grønnevik et al., 2011). figure 2. acetaldehyde diacetyl (a), acetoin ethanol (b) contents in cow and goat kefir samples during storage (ck: cow kefir, gk: goat kefir, aa: acetaldehyde, di: diacetyl, ac: acetoin, ea: ethanol, a,b,c,d: show statistically difference (p>0.05 or p<0.05) among storage days for each property of the samples. a,b: show statistically difference between kefir samples in terms of related property (p<0.05).) ital. j. food sci., vol. 32, 2020 513 general mean value of ethanol was higher in the gk samples and this difference was significant (p<0.05) (fig. 2b). the values obtained in this study were consistent with values reported by grønnevik et al. (2011) and gul et al. (2015). yeasts are primarily responsible for alcohol production in kefir, and some heterofermentative lactobacilli, such as lactobacillus kefir, can also produce ethanol (güzel-seydi̇m et al., 2000a; rattray and o’connell, 2011). in some studies, ethanol content in kefir was determined to vary between 0-9700 µg g-1 (güzel-seydi̇m et al., 2000b; wszolek et al., 2001; garcίa fontán et al., 2006; purnomo and muslimin, 2012; temiz and kezer, 2015; yildiz et al., 2018). the amount of ethanol in kefir varies greatly depending on the type of culture used, the inoculation rate of the culture and the microbial diversity of the culture. while a considerable change did not occur in the amount of ethanol in the ck samples until the 28th day of storage period, a sudden increase happened on the last day. the amount of ethanol in the gk samples did not change much during storage. changes in the amount of ethanol in both samples were not significant (p>0.05). the increase in ethanol content of the ck samples on the last day of storage can be attributed to the high number of yeasts on the same day. wszolek et al. (2001) found that storage time did not affect the amount of ethyl alcohol in kefir samples, like our results. 3.8. sensory properties the overall average scores of structure, consistence and texture, taste and smell, and general appreciation of ck samples was higher than the average values of gk samples (fig. 3). while the difference between the structure, consistence and texture, and general appreciation scores of both samples were significant (p<0.05), the difference between taste and smell scores were nonsignificant (p>0.05). in terms of all sensory characteristics, the highest sensory scores were obtained on the 14th day of storage in the ck samples while the lowest sensory scores were on the 35th day of storage. the highest sensory scores were obtained on the 21st day of storage in the gk samples and lowest sensory scores were obtained on the 7th day of storage. in both samples, the change in sensory properties during storage period was significant (p<0.05) except for taste and smell characteristic of gc sample. panelists reported that the gk samples had low consistence, did not give full sensation in the mouth and showed distinct the goat smell. figure 3. sensory changes in cow and goat kefir samples during storage. ital. j. food sci., vol. 32, 2020 514 4. conclusions based on the results of this study, it can be said that kefir samples produced from both cow and goat milk can be stored up to 35 days at refrigerator temperature. all samples had sensory scores above 3 during storage. however, the highest scores by sensory analyses and the highest diacetyl/acetaldehyde ratio in the kefir samples were obtained on the 14th day of storage in ck samples and the 21st day of storage in gc samples. acknowledgements present study was supported by bolu abant i̇zzet baysal university, scientific research projects coordination unit (project no: 2016.09.04.1085). references bensmira m. and jiang b. 2012. effect of some operating variables on the microstructure and 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in scotland and poland using bovine, caprine and ovine milk with different starter cultures. lebensm. wiss. u. technol. 34:251-261. wszolek m., kupiec-teahan b., skov guldager h. and tamime a.y. 2006. production of kefir, koumiss and other related products. in a. y. tamime (ed.), fermented milks (pp. 174-216). ayr, uk: blackwell science. yaman h. 2011. kefir: a fermented milk product and production methods. kocatepe veterinary journal 4(1):43-56. yaygın h. 1994. kefir ve özellikleri. iii. süt ve süt ürünleri sempozyumu, 2-3 haziran 1994, i̇stanbul. yıldız-akgül f., yetişemiyen a., şenel e. and yıldırım z. 2018. microbiollogical, physicochemical, and sensory characteristics of kefir produced by secondary fermentation. mljekarstvo 68(3):201-2013. paper received february 12, 2020 accepted april 30, 2020 ijfs#1444_bozza ital. j. food sci., vol. 31, 2019 661 paper polycyclic aromatic hydrocarbons in selected food items coming from the croatian market t. bogdanovića, s. petričevića, e. listeša and j. pleadin*b acroatian veterinary institute, regional veterinary institute split, poljička cesta 33, 21000 split, croatia bcroatian veterinary institute zagreb, laboratory for analytical chemistry, savska cesta 143, 10000 zagreb, croatia *corresponding author: tel: +38516123626; fax: +38516123670; e-mail address: pleadin@veinst.hr abstract the study aims to evaluate the presence of 15 polycyclic aromatic hydrocarbons (pahs) in fishery products, shellfish, meat products and spices (n = 140). benzo[a]pyrene (bap) was detected in the mean concentration of 0.11 µg/kg in mussels to 4.85 µg/kg in spices. however, none of the samples exceeded the maximal bap and ∑pah4 limit, set out under the european legislation, although high values determined in some food, especially dried herbs and spices, pointed towards a heavy contamination and the necessity for systematic controls. the study showed that processed food samples contained significantly higher (p<0.05) pah levels in comparison to food coming from environmental sources. keywords: croatian market, dried herbs and spices, fresh shellfish, meat products, polycyclic aromatic hydrocarbons, smoked fish ital. j. food sci., vol. 31, 2019 662 1. introduction polycyclic aromatic hydrocarbons (pahs) are represented by roughly 660 different compounds and include highly hydrophobic and diverse organic compounds that have two or more fused aromatic rings (zelinkova and wenzl, 2014; singh et al., 2016). they pose as ubiquitous environmental pollutants that can be found in fresh water and marine sediments, the atmosphere and ice. the major reason for concern as regards human exposure to pahs arises on the grounds of their carcinogenic, mutagenic and teratogenic effects (falcó et al., 2005; reinik et al., 2007; rengarajan et al., 2015). the united states environmental protection agency (us-epa) tagged 16 pahs as priority pollutants based on the frequency of their occurrence and their carcinogenicity (epa, 1994). the 16 pahs include acenaphthene (ace), acenaphthylene, anthracene (anthr), benz[a]anthracene (baa), benzo[b]fluoranthene (bbf), benzo[k]fluoranthene (bkf), benzo[ghi]perylene (b[ghi]p), benzo[a]pyrene (bap), chrysene (chr), dibenz[a,h]anthracene (d[ah]a), fluoranthene (f), fluorene (flr), indeno[1,2,3cd] pyrene (i[cd]p), naphthalene (nap), phenanthrene (phen) and pyrene (pyr). among them, those containing up to four fused benzene rings are known as light pahs, while those containing more than four benzene rings are known as heavy pahs and are more toxic and more stable as compared to their light counterparts. food consumption represents the main route of pah exposure for non-smokers and nonoccupationally exposed adults (alomirah et al., 2011). the routes of pah food contamination include direct contamination from natural and anthropogenic environmental sources present in air, water and soil, as well as contamination with pahs formed throughout thermal food processing (e.g. drying, smoking, heating, baking, frying, roasting, grilling) (efsa, 2008). pahs most commonly found in food are bap, baa, chr, d[a,h]h, pyr, anthr, f and bbf (yebra-pimentel et al., 2015). available profile studies of both unprocessed (seafood, mussels) and processed food have shown the predominance of light over heavy pahs. however, the presence of toxicologically important heavy (high molecular weight) pahs that include chr, baa, bap and bbf, has often been reported in certain whole smoked meat products (smoked pork speck, smoked chicken, smoked pork) and chopped meat products, such as various sausages (reinik et al., 2007; purcaro et al., 2009; kubiak et al., 2015; rozentäle et al., 2015; rozentäle et al., 2018). dominating pahs of toxicological concern present in mussels are chr, bbf and bkf (mercogliano et al., 2016). it has been revealed that spices and herbs, which are important ingredients of many processed food items, are often contaminated with pahs of the similar low molecular profile, with the prevalence of chr similar to that in the above-mentioned foodstuffs (rozentäle et al., 2018). in order to protect public health and to keep food contaminants at toxicologically acceptable levels, food authorities of different countries have established the maximum levels (mls) of pahs for different food categories in which higher pah levels are to be expected. the european union legislation stipulates the limits for bap and the sum of four pahs ∑pah4 (baa, chr, bbf, bap) in more than 10 groups of foodstuffs and is therefore the most comprehensive applicable regulation worldwide (ec regulation no. 835/2011, zelinkova and wenzel, 2016). bivalve molluscs (fresh, chilled or frozen) in which environmental pollution might result in high contamination levels, are allowed mls of 5μg/kg for bap and 30 μg/kg for ∑pah4. the mls of pahs in processed fish and meat products are set at 2 μg/kg for bap and 12 μg/kg for ∑pah4. however, there exists the list of eu countries that are allowed to continue using traditionally smoked fish and smoked meat products containing higher pah levels (5 μg/kg for bap and 30 μg/kg for ∑pah4) (ec regulation no. 1327/2014). despite the application of good smoking practices, lower pah levels in these traditional foodstuffs have not been ital. j. food sci., vol. 31, 2019 663 achieved yet, since these products require smoking practices that significantly change their organoleptic characteristics. a three-year derogation from the obligation to observe and respect lower bap and ∑pah4 mls expired in the autumn of 2017, but in half of the member states the former mls are still in force, pending the adoption of new, reassessment-based provisions. recently, mls of 10 μg/kg and 50 μg/kg were set out for bap and ∑pah4 in dried herbs and spices (ec regulation no. 1933/2015) in response to high pah levels determined in the above in the recent years due to poor drying practices. the main objective of this study was to evaluate the level of contamination of certain food items coming from the croatian market with the ∑pah15 referred to above (except for the non-fluorescent acenaphthylene), and to compare the levels of contamination from two major sources (environmental source vs food processing technique). in order to establish the impact of environmental sources, bivalve molluscs have been investigated, whereas the food processing impact was investigated in fishery products, meat products and spices. in order to establish the occurrence and toxicity of pahs in food under study, low molecular pah (ace, anthr, baa, chr, f, flr, nap, phen, pyr), high molecular pah (bbf, bap, bkf, b[ghi]p, d[a,h]a, i[cd]p), ∑pah4 (baa, chr, bbf, bap) and ∑pah8 (baa, chr, bbf, bap, bkf, b[ghi]p, d[a,h]a, i[cd]p) contents were determined. although the assessment of dietary exposure to pahs does not fall within the scope of this study, for the sake of comparison the pah contents and the respective sums are also expressed as benzo[a]pyrene equivalents (bape), so as to illustrate the toxicity of the investigated pahs, as well as to simplify the interpretation of real-life risk for human health. 2. material and methods 2.1. sampling and sample preparation food samples (n = 140) were obtained from the croatian market during 2017 – 2018 and divided into four groups, as follows: fresh shellfish (n = 42), smoked fishery products (n = 8), meat products (n = 70) and dried herbs & spices (n = 20). sample collection and storage were performed in accordance with the european legislation (ec regulation no. 333/2007, ec regulation no. 836/2011) so as to avoid pah losses (zelinkova and wenzel, 2016). five fresh bivalve species (n = 42), including mussels (m. galloprovincialis, n = 29), oysters (o. edulis, n = 2), variegated scallops (c. varia, n = 4), warty venus shells (v. verruscosa, n = 3) and smooth clam (c. chione, n = 4) were collected from local markets along the croatian coastline. samples containing approximately 4 kg of shellfish were transported to the laboratory in cooled dim containers within 24 hours post sampling. twenty five pieces of variegated scallops, mussels and warty venus shells and fifteen pieces of oysters and smooth clams of similar shell lengths were randomly selected from each sample and put together for the analysis; shells were than discarded, while soft tissues were homogenized (grindomix, gm 200, retsch, haan, germany) and stored at -20°c pending analysis. smoked fishery products (n = 8), including hot-smoked sea bass (n = 3) and sea bream (n = 1) fillets, cold smoked trout (n = 1) and tuna (n = 1) fillet, smoked sardine in sunflower oil (n = 1) and smoked salmon pate (n = 1), were obtained from the local croatian market. in order to ensure a representative sample of fishery products, the whole package content was homogenized (grindomix, gm 200, retsch, haan, germany) and stored at -20°c pending analysis. ital. j. food sci., vol. 31, 2019 664 the meat sample pool (n = 70) consisted of 10 samples of fermented sausages (istrian rožica, dry homemade sausage, kulen, tea sausage and other salami and sausages), 10 samples of semi-dry smoked sausages (bodulska, ham, homemade sausage, kranj sausage, kvarner sausage, grill sausage and peasant sausage), 35 dry-cured meat samples (buđola, dry bacon, dry ham, dry sirloin and other products) and 15 semi-dry-cured meat samples (dry ham, smoked chops, smoked dry porcine shank, smoked rack, smoked ribs, smoked rolled shoulder with skin and other products). the samples were homogenized (grindomix gm 200, retsch, haan, germany) at different speeds for a different length of time depending on the type of meat product, and then stored at -20°c pending analysis. the selected dried herb and spice samples (n = 20) included clove (caryophyllus aromaticus) (n = 1), grounded garlic (allium sativum) (n = 2), grounded ginger (zingiber officinale) (n = 1), laurel (laurus nobilis) (n = 1), grounded red paprika (capsicum spp) (n = 2), grounded smoked red paprika (capsicum spp) (n = 4), grounded black pepper (n = 4), mixed pepper (piper nigrum) (consisting of black, green, white and red pepper, n=1), mixed dried spices (consisting of tomato, rosemary, basil and oregano) (n = 1), parsley (petroselinum crispum) (n=1) and rosemary (rosmarinus officinalis) (n = 2). ungrounded samples were cut or crushed and then sieved through a 1.5-mm sieve. 2.2. standards and reagents all chemicals used (e.g. dichloromethane, acetonitrile) were of a hplc grade. ultrapure water was produced by a millipore, direct-q 3 uv system (millipore, molsheim, france). the glassware was washed with a detergent and water, rinsed with acetone and dichloromethane, and dried at 50°c for an hour before use (european commission, 2007). the certified standard mix solution 16 priority pah, cocktail 3, 16 comp.(10 μg of each/ml in acetonitrile) containing ace, acenaphthylene, (anthr), (baa), (bbf), (bkf), (b[ghi]p), (bap), (chr), (d[ah]a), (f), (flr), i[cd]p, (nap), (phen) and (pyr), was obtained from the chiron, trondheim, norway. standard mix working solutions (concentration ranges 0.15 µg/kg to 8.50 µg/kg) containing pah16 were prepared by virtue of diluting the stock solution with acetonitrile and then stored at + 4°c in darkness. benzo[b]chrysene (bbc) was supplied by interchim (montlucon, france). the reference materials of (frozen) bivalve molluscs (ilc1060, id025), smoked meat (ilc 424, id109) and smoked black pepper (ilc 334, id087), were supplied by the european commission joint research centre european union reference laboratory for polycyclic aromatic hydrocarbons (institute for reference materials and measurements, geel, belgium), while the smoked fish reference material (t0672qc) was purchased from fapas (sand hutton, york, uk). 2.3. extraction and clean-up pahs were determined using the slightly modified method described by wegrzyn and co-authors (2006) pah isolation involves a preparative size-exclusion chromatography allowing for the efficient single-step lipid removal without saponification; within this frame, benzo[b]chrysene is used as an internal quantification standard. a homogenized sample (1 g) was spiked with bbc (50 µg/l, 100 µl) and diluted with dichloromethane to the final 4-ml volume. a sample was homogenized and vortexed for 10 min. the obtained mixture was centrifuged for 10 min at 3 500 rpm and 4°c. the supernatant was decanted and filtered through a 0.22-µm ptfe syringe on-line filter (phenomenex, torrance, usa) and transferred into a glass hplc vial. sample extracts were injected into a hplc agilent 1200 series system (agilent, singapore, singapore) for size-exclusion chromatography (sec) with a fraction collector (gilson, fc203b, middleton, usa). the preparative sec ital. j. food sci., vol. 31, 2019 665 was performed under isocratic conditions (100% dichloromethane) at the flow rate of 1 ml/min and room temperature using 2 size-exclusion columns connected in series (packed with pl gel based on ps/dvb, 300 × 7.8 mm i.d., 5 µm particle size and 50 å) provided by phenomenex (phenomenex, torrance, usa). the injection volume was 400 µl. chromatograms were monitored at 254 nm and fractions were collected within 18 24 min timeframe. aliquots were evaporated to dryness in a rotational vacuum concentrator (rcv2-18hcl; christ, osterode am harz, germany) at the speed of 1,300 rounds per minute; this stage took 40 min and went on at 20°c. the residue was dissolved in 100 µl of acetonitrile, so as to undergo chromatographic analysis (uplc-fld). 2.4. uplc-fld analysis the uplc pah analysis was performed using an ultra pressure liquid chromatograph (agilent 1290 infinity uhplc, agilent, singapore) equipped with a binary gradient pump (g4220a) and an auto-sampler having a thermostated sample compartment (g4226a), a thermostated column compartment (g1316c) and a fluorescence detector (g1321b). the separation of compounds was done in a hypersil green c18 pah analytical column (150 mm x 3.0 mm i.d., 3.0 µm particle size) with a c18 guard hypersil green pah column (10 mm x 3.0 mm i.d., 3.0 µm particle size) supplied by thermo scientific (thermo-scientific, germany), the maintained temperature thereby being 30°c and the injection volume being 15 µl. the mobile phase consisted of a mixture of acetonitrile and acetonitrile/water (1/1) and was operated in the gradient mode at the flow rate of 0.8 ml/min (wegrzyn et al., 2006). the initial composition of 100 % of acetonitrile/water (1/1) increased to 100% of acetonitrile in 30 min. the initial conditions were reached in 5 min. the total run time was 35 min. the excitation and emission wavelength pairs (excitation-ex, emission-em) used with fluorescence detection were as follows: minute 2: ex = 270 nm, em = 340 nm for nap; minute 6.5: ex = 250 nm, em = 310 nm for flr, ace; minute 8.5: ex = 250 nm, em = 380 nm for phen, anthr; minute 11.0: ex = 250 nm, em = 460 nm for f, minute12.1: ex = 270 nm, em = 385 nm for pyr; minute: 15.5 ex = 256 nm, em = 395 nm for baa, chr; minute 19.0: ex = 295 nm, em = 466 nm for bbf; minute 21.5: ex = 250 nm, em = 410 nm for bkf bap, d[ah]a, b[ghi]p; minute 27.4: ex = 295 nm, em = 500 nm for i[cd]p; minute 28.3: ex = 460 nm, em = 250 nm for bbc. the compounds were quantified using internal calibrations curves plotted for each of the 15 pahs at seven concentration levels ranging from 0.15 to 8.5 µg/kg. standard mix working solutions containing pahs in different concentrations and a fixed amount of internal standard (5 µg/kg) were prepared in acetonitrile and injected in duplicates (15 µl per injection) so as to be able to come up with the linear regression lines. each sample of food products under study was analyzed in duplicate; the final pah content was calculated as the mean of two parallel runs and expressed in µg/kg (of wet weight). the pah content calculation also included the toxic equivalency factors (tef) approach, so that pah concentrations are also expressed as benzo[a]pyrene toxic equivalents; to that effect, the converting factors referred to by law et al. (2002) were used. the benzo(a)pyrene equivalent concentration (bape), expressed in µg/kg of food, is calculated as follows: bape = ∑(bape) = ∑(cpahi x tefpahi), where cpahi represents the concentration of the pah congener i in food (µg/kg, while tefpahi represents the toxic equivalency factor of the pah congener i. ital. j. food sci., vol. 31, 2019 666 in order to illustrate the toxic potency of the investigated pahs, the ∑pah15 concentration, ∑pah8 concentration and ∑pah4 concentration are expressed as bape (bape∑pah4, bape∑pah8, bape∑pah15). 2.5. validation of the method and analytical quality assurance in-house validated method for the respective matrices, i.e. dried herbs and spices, fishery products, shellfish and meat products, was applied. the performance assessment criteria included applicability, the limit of detection (lod), the limit of quantification (loq), precision (horratr, horratr), specificity, linearity and recovery. the limit of detection (lod) was calculated from the average of ten pah15-negative samples (consisting of shellfish, smoked fish, smoked meat or spices), earlier analysed for pah presence and used for validation as the blank material; to the above average, the tripled standard deviation was added (lod = mean ± 3sd) (ec regulation no 582/2016). in order to determine the limit of quantification (loq), the mean concentration determined in ten pah-negative samples of each matrix was summed up with the six-fold standard deviation (loq = mean ± 6sd). the precision of the method was assessed using the horwitz equation as the horrat value descriptive of each pah at three concentration levels under repeatable (horratr) and reproducible (horratr) conditions. food samples were spiked at the concentrations of 0.5, 1 and 1.5 of the mls defined for 4 pahs in triplicate. for each pah, the mean recovery of a 36-sample set was calculated and used for the accuracy assessment, evaluated based on the intra-laboratory coefficient of variation. the specificity was checked by analyzing 15 pahs in each of the ten blank samples per tested matrix and verifying the presence of interferences in the region of interest where single pahs were expected to elute. the linearity was checked through the regression coefficients of determination (r2) of the analytical curves using the standard mix working solution containing 15 pahs at seven concentration levels ranging from 0.15 to 8.5 µg/kg. in this study, the presence or absence of matrix effects was identified using calibration curves and a fixed amount of internal standard (5 µg/kg) obtained with matrix-matched calibration standards (all matrices of food groups investigated in the study) and calibration solutions in the solvent. in the first step a three-point calibration curve (1, 2 and 4 µg/kg for meat and fish products; 2.5, 5 and 10 µg/kg for dried herbs and spices and shellfish products) was plotted using linear regression with the calibration standards in the solvent solution. in the next step, another three-point calibration curve of the same concentrations per food group and a fixed amount of internal standard (5 µg/kg) was plotted based on the measurement data of the matrix-matched calibration standards. the slopes of the regression curves representative of the two sets of calibration solutions were evaluated statistically. internal quality control was pursued with each analytical batch using the available reference material (frozen mussels, ilc1060, id025; t0672qc smoked fish, smoked meat, ilc 424, id109 and ilc 334 and id087 smoked black pepper), and was carried out by virtue of spiking the food samples so as to obtain the concentration of 2 µg/kg. within each analytical series, the reference materials and the spiked food samples were analysed in duplicate and checked for recovery. the interpretation of validation and quality assurance results was performed as proposed by the ec regulation no 836/2011. 2.6. statistical analysis data analysis was carried out using the xlstat 2018.3.51141 software package (addinsoft, new york, usa). the kruskal-wallis one-way analysis of variance was ital. j. food sci., vol. 31, 2019 667 performed for each data set so as to detect differences among food groups, considered to be statistically significant if estimated at the level of probability of p = 0.05. 3. results and discussion 3.1. validation of the method and quality assurance results the results concerning linearity, loq, recovery and horratr are presented in table 1 and fig. 1 shows a chromatogram descriptive of the target pahs in a standard (c = 1.06 µg/kg) and a smoked ham sample spiked at the level of 1.96 µg/kg. the lod established for the studied food groups ranged from 0.02 to 1.00 µg/kg, while the loq varied from 0.15 to 1.84 µg/kg. the recoveries obtained within the frame of internal quality control spanned from 50 to 120 %, which is in accordance with the criteria established by the ec regulation no. 836/2011. the mean slopes of the regression curves for the standard and matrix sets of calibration solutions were not significantly different. therefore, matrix-matched standard calibrations were not used. the applied analytical method fulfils all methodological requirements set out by the ec regulation no. 836/2011 and can therefore be considered as suitable for the determination of 15 pahs in food categories under this study. 3.2. shellfish products bivalve molluscs, posing in this study as unprocessed food representatives, are exposed to pahs ubiquitously present in the marine environment due to polluted sediments, spill residues, shipping activities (de-ballasting waters), industrial and urban runoff, and atmospheric pollution (soriano et al., 2006). furthermore, they are widely used in coastal monitoring programmes and pollution assessment studies as filter-feeders having a slow rate of detoxification and the ability to accumulate many toxic contaminants. in this research, shellfish products were divided into two groups: mussels and other shellfish. the mussels were produced on farms, while other shellfish came from natural habitats in the adriatic sea. in cultured mussels, the predominance of phen, flr, f and pyr was established (table 2). other shellfish showed the abundance of phen, ace and f, with the highest phen and ace levels found in warty venus and the f levels in oysters. statistically significant percent-shares of low molecular (78.9 % in mussels and 69.6 % in other shellfish) (fig. 2) as compared to those of high molecular pahs, obtained in this study (fig. 3), were also reported in the studies by bihari et al. (2007), peruggini et al. (2007), serpe et al. (2011), and mercogliano et al. (2016) for shellfish coming from other adriatic and mediterranean areas. the majority of bivalve species collected from the croatian market contained the investigated ∑pah15 in concentrations ranging from 0.71 µg/kg to 14.49 µg/kg in mussels, and from 6.07 µg/kg to 27.45 µg/kg in other shellfish products (table 3). as for toxicologically important pah markers, significantly higher ∑pah4 and ∑pah8 were found in other bivalve species, in particular in oysters, with a predominance of i[cd]p (6.99 µg/kg), bbf (4.02 µg/kg) and chr (3.16 µg/kg). highand mediummolecular pahs in marine ecosystems are mostly of pyrolytic origins (mercogliano et al., 2016), so that their occurrence in other bivalve species under this study may also come from pyrolytic sources (antropogenic pollution coming from the mainland). a statistically significant difference in bap content was found amongst the investigated bivalve ital. j. food sci., vol. 31, 2019 668 molluscs, above all when it comes to the smooth clam (table 3). this study confirmed that farmed shellfish species (mussels) are characterized with lower levels of toxicologically important pahs as compared to native shellfish species, which is in line with the results obtained in the studies by mercogliano et al. (2016) and zelinkova et al. (2015). the majority of bap and ∑pah4 levels found in shellfish harvested along the croatian coast were far below the mls of 5 µg/kg and 30 µg/kg, respectively ec regulation no. 835/2011, as can be seen in table 3, and are in accordance with the findings in mussels harvested along the adriatic, the campanian and the ionian coasts of italy (storelli and marcotrigiano, 2011; serpe et al., 2010). the highest ∑pah15 (27.5 µg/kg and 26.5 µg/kg) and ∑pah8 (14.3 µg/kg) were determined in oysters and warty venus, whereas mussels showed the highest bap content. significant differences in bape∑pah4, bape∑pah8 and bape∑pah15 values were observed, with the highest values established in oysters. literature sources have reported bape∑pah15 values of 1.56 µg/kg ww determined in mediterranean mussels coming from the adriatic sea (perugini et al., 2007) and ranges of 0.1 to 4.5 µg/kg dry wt in shellfish coming from the red sea (el nemr et al., 2016). pah uptake depends on the physiology of the up-taking organisms and cyclic annual variations. in accordance with the eu legislation, monitoring of aqua-cultured shellfish is carried out along the croatian coast, so as to ensure that pah levels are within the consumer safety limits (bogdanović et al., 2014). the study results revealed that sampling techniques developed for pah monitoring in cultivated and wild shellfish found along the croatian coast, minimize any risk for human health coming from the seafood consumption which has generally been tagged as one of the main sources of human exposure to severe pollutants. 3.3. smoked fish the majority of pahs present in smoked food originate from wood smoke. while the amounts of pahs in raw fish are very low due to the fish ability to oxidize and further metabolise pahs absorbed from the environment, cold and hot-smoked fish are generally characterised with higher pah contents that depend on fish properties, methods and parameters of fish smoking, composition of the smoke and the level of exposure of edible fish parts to the smoke released (duedahl-olesen et al., 2010; stolyhwo et al., 2005; zelinkova et al., 2015). furthermore, if a fishery product is canned in oil, the contamination may arise due to vegetable oil. similar to meat products, the skin acts as a barrier against smoke particles, hence preventing any significant pah penetration into fish muscles. the mean pah contents determined in smoked fish samples in this study are reported in table 2. pah profiling of fishery products coming from the croatian market revealed the predominance of four light pahs (ace, phen, flr and anthr) (table 2, fig. 2), which is in accordance with the results reported for commercial smoked fish (varlet et al., 2007; zelinkova et al., 2015; duedahl-olesen et al., 2018). phen, anthr, pyr and f were detected in all samples, the highest concentration of phen thereby being detected in smoked salmon pate (5.76 µg/kg). i[1cd]p (6.63 µg/kg ) was determined in only one smoked sea bass sample. the analysis of the selected fishery products revealed the highest ∑pah15 values in smoked salmon pate (118.05 µg/kg), followed by smoked sea bream (35.29 µg/kg) and smoked trout (32.13 µg/kg), while the lowest value was established in smoked tuna (1.17 µg/kg). the pah amounts found in fish samples positively correlate with the lipid content of the same (singh et al., 2016), which may explain the highest ∑pah15 values obtained in smoked salmon pate within our study frame. as stated above, the amount of pahs transferred by smoke particles into the final fishery product depends on several processing parameters including smoking technology, ital. j. food sci., vol. 31, 2019 669 combustion temperature, smoke composition, type of wood used, and the level of exposure of edible fish parts to the smoke released (stolyhwo et al., 2005, duedahlolesen et al., 2010). among the mutagenic/carcinogenic pahs analysed, the highest bap (0.76 µg/kg), ∑pah4 (1.70 µg/kg) and ∑pah8 (11.56 µg/kg) (table 3) were detected in smoked sea bass fillets, while the lowest bap (< lod µg/kg), ∑pah4 (0.18 µg/kg) and ∑pah8 (0.22 µg/kg) were determined in smoked tuna. the aggregated average ∑pah8 content decreased in the following order: i[cd]p, b[bghi]p, chr, d[ah]a, b[b]f, b[a]p, b[a]a and b[k]f (tables 2 and 3). the investigated fishery products complied to the pah mls of 2 µg/kg for b[a]p and 12 µg/kg for ∑pah4, laid down under the eu legislation. bap-based toxic equivalency factors calculated for ∑pah4, ∑pah8 and pah15 showed a similar decreasing pattern starting with the highest values found in smoked sea bass that declined over smoked sardine in sunflower oil and smoked salmon pate down to the lowest values in smoked trout (table 3). smoked salmon pate, most heavily contaminated with pah15, was characterized with low bape levels, while smoked sea bass fillets, moderately contaminated with pah15, showed the highest bape levels, which can be attributed to the presence of heavy pahs that have higher tef values. a similar bapepahtotal trend has been observed in different food items, for instance in the study by perugini et al. (2007) in fish and shellfish, in the study by santos et al. (2011) in meat products, and in the study by gomes et al. (2013) in traditional meat sausages. 3.4. meat products smoked meat products represent the principal source of pahs that generate during an incomplete wood combustion (alves et al. 2018). as already well known, the amounts and types of pahs present in contaminated smoke meat products depend on a number of factors, such as the fuel used, the smoking technique, the temperature at which the pyrolysis takes place, the air flow through the smoke generator, the distance between the meat sample and the heat source, the smoking chamber design, the smoked meat fat content, the duration of smoking, and the cleanness and maintenance of the equipment used (codex alimentarius commission, 2009). data on pahs in smoked meat products are highly variable, this discrepancy being explained by differences in food smoking procedures and meat product characteristics (whole meat versus chopped meat products). spices used in smoked meat production can also be contaminated with pahs, therefore further increasing the levels of those hazardous contaminants. smoked meat products investigated in this study were divided into four groups, consisting of either whole meat samples in terms of dry-cured and semi-dry-cured meat products, or chopped meat products in terms of dry-fermented and semi-dry sausages. ∑pah4 and ∑pah8 composition pattern was dominated by light, harmless pahs (93.0 % in semi-dry meat products to 98.3 % in dry-fermented sausages). amongst them, the four pahs most abundantly present in dry-cured meat products were (in descending order) phen, flr, ace and anthr. similarly, dry-fermented sausages were also characterized by light pahs supremacy, their representation thereby following virtually the same phen-ace-flr-anthr decreasing pattern described above, while both semi-dry-cured meat products and semi-dry sausages showed a little bit different pah presence pattern dominated by phen, f, ace and flr in decreasing order for the former meat products and by phen, flr, f and pyr in decreasing order of the latter meat products (table 2, fig. 2). ital. j. food sci., vol. 31, 2019 670 table 1. selected performance indicators of the method in use: linearity, limit of quantification (loq), recovery and precision (horratr). pahs a linearity (r2)b dried herbs and spices shellfish smoked fish smoked meat loqc (µg/kg) recovery d horratr e loq (µg/kg) recovery d horratr e loq (µg/kg) recovery d horratr e loq (µg/kg) recovery d horratr e nap 0.998 3.14 71.22 1.88 1.85 76.18 1.66 2.90 77.72 0.99 2.97 119.2 1.08 flr 0.999 3.30 103.5 0.99 3.23 113.9 0.76 2.94 76.88 0.76 3.30 120.0 1.33 ace 0.999 3.27 91.03 1.15 0.63 102.2 1.02 2.61 71.08 0.98 3.00 88.20 1.25 phen 1.000 0.30 69.77 0.79 0.79 117.0 1.32 0.40 82.95 1.44 0.79 98.25 1.06 anthr 1.000 0.43 86.61 1.36 0.50 112.9 0.85 0.69 64.21 0.85 0.50 100.0 0.85 f 0.997 0.50 89.61 2.00 0.40 116.5 1.65 0.59 92.05 1.77 0.30 100.0 0.86 pyr 0.999 0.59 92.06 0.86 0.86 93.87 1.14 0.89 80.34 1.14 0.86 100.0 1.14 b(a)a 0.999 0.26 75.56 1.28 0.07 116.0 1.89 0.23 68.56 1.89 0.07 90.40 1.89 chr 1.000 0.17 94.54 1.66 0.07 117.2 1.18 0.13 76.82 1.18 0.07 88.80 1.18 b(b)f 1.096 0.23 99.99 0.89 0.26 106.4 0.87 0.17 71.86 0.87 0.26 94.71 0.08 b(k)f 1.000 0.23 83.66 1.33 0.20 93.55 1.11 0.17 78.73 1.11 0.20 100.0 1.11 b(a)p 1.000 0.26 76.99 1.65 0.10 94.15 0.71 0.40 75.84 0.71 0.10 82.00 0.71 d[ah]a 0.998 0.63 88.77 1.18 0.66 88.48 0.84 0.69 81.05 0.84 0.76 77.00 0.84 b[ghi]p 0.972 0.56 70.66 1.44 0.56 86.49 1.13 0.53 81.99 1.13 0.50 100.0 1.13 i[cd]p 0.973 0.26 69.05 0.88 0.30 78.32 0.14 0.36 85.24 1.14 0.30 68.75 0.14 apolycyclic aromatic hydrocarbons (pahs): acenaphthene (ace), acenaphthylene, anthracene (anthr), benz[a]anthracene (baa), benzo[b]fluoranthene (bbf), benzo[k]fluoranthene (bkf), benzo[ghi]perylene (b[ghi]p), benzo[a]pyrene (bap), chrysene (chr), dibenz[a,h]anthracene (d[ah]a), fluoranthene (f), fluorene (flr), indeno[1,2,3cd] pyrene i[cd]p, naphthalene (nap), phenanthrene (phen) and pyrene (pyr); bdetermination coefficients (r2) of the analytical seven-point curves constructed for standard solutions (0.25-8.50 µg/kg);climit of quantification; dmean recovery at three concentrations used in the precision assessment (selected food categories were spiked at the concentrations of 0.5, 1 and 1.5 of mls); ehorrat coefficient (ec regulation no. 836/2011) for each pah at three concentrations (selected food categories were spiked at the concentrations of 0.5, 1 and 1.5 of mls value) in reproducibility (r) conditions used for the method precision assessment. ital. j. food sci., vol. 31, 2019 671 table 2. determination of pahs levels in selected food from croatian market. fresh shellfish smoked fish (na = 8) meat products dried herbs and spices (na = 20) mussels (na = 29) other shellfish (na = 13) (na = 13) dry-cured meat products (na = 35) semi-dry cured meat products (na = 15) dry-fermented sausages (na = 10) semi-dry sausasges (na = 10) pahs mean range mean range mean range mean range mean range mean range mean range mean range nap <0.56c <0.56-6.62 <0.56c <0.56 <0.95c <0.88-0.79 1.58b <0.55-27.60 <0.95c <0.55-4.09 2.55a <0.55-11.12 <0.55c <0.55-0.71 1.93b <0.95-27.68 ace 0.24e <0.19-6.85 2.00d <0.19-8.11 22.30b <0.79-99.67 2.75d <0.91-25.97 1.56d <0.91-16.81 11.25c <0.91-45.69 <0.91e <0.91-1.01 76.34a <0.99-807.68 flr 1.21 <0.98-4.01 <1.00 <0.98-1.27 2.79bc <0.89-9.86 4.15b <1.00-51.13 1.04 <1.00-2.33 11.14a <1.00-42.95 1.26c <1.00-4.48 6.56ab <1.00-31.74 phen 1.56e <0.24-4.17 2.16de <0.24-6.29 3.14cde <0.12-5.76 8.88bc <0.91-54.86 3.95cd <0.91-12.93 14.31b <0.91-63.54 3.19d <0.91-9.10 55.44a <0.09-473.05 anthr 0.16f <0.15-3.20 0.28ef <0.15-1.88 0.87de <0.21-2.00 2.10cd <0.24-13.88 0.78e <0.24-3.38 5.38b <0.24-21.95 0.71e <0.24-2.78 13.24a <0.13-74.55 f 0.58cd <0.12-2.21 1.89b <0.12-6.96 0.56d <0.18-1.08 1.54bc <0.15-6.39 2.05b <0.15-10.22 1.80b <0.15-5.39 1.12bc <0.15-3.45 22.87a <0.15-124.63 pyr 0.49d <0.26-1.21 0.84cd 0.30-1.94 0.78d <0.27-1.40 1.13c <0.26-10.88 1.02cd <0.26-4.27 1.94b <0.26-7.07 0.89c <0.26-2.71 26.31a <0.18-163.86 b[a]a 0.10c <0.02-0.62 0.22b <0.02-0.75 0.10c <0.07-0.19 0.15c <0.02-1.41 0.14c <0.02-0.66 0.09c <0.02-0.28 0.07c <0.02-0.21 5.76a <0.08-25.39 chr 0.45b <0.02-0.98 0.53b <0.02-3.16 0.27cd <0.04-0.61 0.30cd <0.02-1.9 0.20cd <0.02-0.65 0.27cd <0.02-0.57 0.18d <0.02-0.38 10.15a <0.05-45.68 b[b]f 0.40bc <0.08-1.27 0.76b <0.08-4.02 0.19de <0.05-0.52 0.26de <0.08-1.78 0.13de <0.08-0.49 0.14de <0.08-0.30 0.08e <0.08-0.25 4.83a <0.07-20.86 b[k]f 0.23b <0.05-0.71 0.40b <0.05-2.14 <0.05c <0.05-0.09 0.15bc <0.06-1.55 0.10c <0.06-0.58 0.09c <0.06-0.17 <0.06c <0.06-0.19 1.55a <0.07-11.02 b[a]p 0.11c <0.12-0.69 0.21b <0.12-0.46 0.16bc <0.12-0.76 0.19b <0.03-1.47 0.11c <0.03-0.40 0.12c <0.03-0.30 0.15bc <0.03-0.37 4.85a 0.15-21.88 d[ah]a 0.11d <0.20-1.39 0.48b <0.20-1.67 0.26c <0.21-1.59 <0.23d <0.23-1.72 <0.23d <0.23-0.52 <0.23d <0.23-0.78 <0.23d <0.23-0.25 8.68a <0.19-39.74 b[ghi]p 0.29c <0.17-1.32 0.69b <0.17-1.81 0.40c <0.16-1.63 0.31c <0.15-1.54 0.37c <0.15-0.70 0.30c <0.15-0.66 0.44c <0.15-1.45 3.70a <0.17-14.29 i[cd]p 0.24c <0.09-2.66 1.14b <0.09-6.99 0.83b <0.11-6.63 0.11c <0.09-1.29 <0.09c <0.09-0.59 <0.09c <0.09-0.53 0.29c <0.09-1.67 3.80a <0.08-22.10 an – number of samples; bpahs polycyclic aromatic hydrocarbons, acenaphthene (ace), acenaphthylene, anthracene (anthr), benz[a]anthracene (baa), benzo[b]fluoranthene (bbf), benzo[k]fluoranthene (bkf), benzo[ghi]perylene (b[ghi]p), benzo[a]pyrene (bap), chrysene (chr), dibenz[a,h]anthracene (d[ah]a), fluoranthene (f), fluorene (flr), indeno[1,2,3cd] pyrene i[cd]p, naphthalene (nap), phenanthrene (phen) and pyrene (pyr); values expressed as < (less than) denote values lower than the detection limit. superscript uppercase letters a, b, c, d denote statistically significant difference (p < 0.05) in the investigated polycyclic aromatic hydrocarbons. ital. j. food sci., vol. 31, 2019 672 table 3. minimum, maximum, median and mean concentrations (µg/kg) of bapa, ∑pah4b, ∑pah8c and pah15d with benzo(a)pyrene toxic equivalents (bapee, µg/kg) detected in different food from croatian market concentration µg/kg bapa ∑pah4b ∑pah8c pah15d ebapepah4 ebapepah8 ebapepah15 mussels (n = 29) minimum <0.03 <0.02 0.18 0.71 <0.03 <0.03 0.03 maximum 0.69 3.56 7.70 14.49 0.63 7.96 8.12 median 0.06 0.95 1.38 5.28 0.12 0.15 0.21 mean 0.11d 1.06bc 1.93cd 6.55c 0.17bc 0.75de 0.82d other shellfish (n=13) minimum 0.09 0.58 1.60 6.07 0.12 0.13 0.20 maximum 0.46 7.94 14.33 27.45 0.83 9.31 9.44 median 0.17 1.34 2.75 9.98 0.32 1.67 1.72 mean 0.21ab 1.72ab 4.42ab 12.08c 0.31a 2.84ab 2.96 ab smoked fish (n=8) minimum <0.12 0.18 0.22 1.17 0.05 0.05 0.14 maximum 0.76 1.70 11.56 118.05 0.80 9.44 9.64 median <0.12 0.59 0.86 18.91 0.11 0.14 0.28 mean 0.16bc 0.72cd 2.23bc 29.64b 0.19b 1.58bc 1.72b dry-cured meat products (n = 35) minimum <0.03 0.07 0.09 4.50 <0.03 <0.03 <0.03 maximum 1.47 6.28 12.13 125.17 1.80 10.54 10.81 median 0.08 0.55 0.92 13.21 0.12 0.19 0.32 mean 0.19cd 0.90de 1.59e 22.04c 0.24bc 0.85e 1.05d semi-dry cured meat products (n=15) minimum <0.03 <0.02 0.31 3.09 <0.03 <0.03 0.18 maximum 0.40 2.15 3.68 30.15 0.48 3.15 3.28 median 0.04 0.36 0.86 9.21 <0.08 <0.08 0.14 mean 0.11d 0.58e 1.18e 12.10c 0.14c 0.44de 0.59cd ital. j. food sci., vol. 31, 2019 673 dry-fermented sausages (n=10) minimum <0.03 0.09 0.31 9.26 <0.03 <0.03 0.18 maximum 0.30 0.97 2.01 171.48 0.33 4.24 4.51 median 0.09 0.67 1.20 28.04 0.13 0.25 0.63 mean 0.12d 0.61cde 1.21de 44.59ab 0.14bc 0.90cd 1.26bc semi-dry sausasges (n=20) minimum <0.03 0.16 0.17 0.28 <0.03 <0.03 <0.03 maximum 0.37 1.10 4.32 23.02 0.39 1.84 2.24 median 0.14 0.35 1.03 6.48 0.15 0.29 0.38 mean 0.15bcd 0.49e 1.29cde 8.76c 0.17bc 0.36de 0.51d dried herbs and spices (n=20) minimum 0.15 1.24 3.63 7.22 0.21 0.31 0.48 maximum 21.88 113.12 207.46 1009.53 27.27 278.36 300.76 median 0.81 4.54 8.38 49.00 1.16 8.02 10.75 mean 4.85a 25.60a 43.34a 246.03a 6.09a 49.93a 53.64a abap: benzo(a)pyrene; b∑pah4: the sum of benzo(a)anthracene baa, chrysene chr, benzo(a)pyrene bap and benzo(b)fluoranthene bbf; c∑pah8: the sum of benzo(a)anthracene baa, chrysene chr, benzo(a)pyrene bap and benzo(b)fluoranthene – bbf, benzo[k]fluoranthene (bkf), benzo[ghi]perylene (b[ghi]p) dibenz[a,h]anthracene (d[ah]a) and indeno[1,2,3cd] pyrene i[cd]p; d∑pah15: the sum of acenaphthene (ace), anthracene (anthr), benz[a]anthracene (baa), benzo[b]fluoranthene (bbf), benzo[k]fluoranthene (bkf), benzo[ghi]perylene (b[ghi]p), benzo[a]pyrene (bap), chrysene (chr), dibenz[a,h]anthracene (d[ah]a), fluoranthene (f), fluorene (flr), indeno[1,2,3cd] pyrene i[cd]p, naphthalene (nap), phenanthrene (phen) and pyrene (pyr) and ebape: the benzo(a)pyrene based toxic equivalency factors expressed to ∑pah4, ∑pah8 and ∑pah15. values expressed as < (less than) denote values lower than the detection limit. different superscript uppercase letters a, b, c, d, e denote statistically significant difference (p < 0.05) in the investigated polycyclic aromatic hydrocarbons and benzo(a)pyrene based toxic equivalency factors of selected food groups (marked in columns). ital. j. food sci., vol. 31, 2019 674 figure 1. chromatogram of the target pahs (acenaphthene (ace), anthracene (anthr), benz[a]anthracene (baa), benzo[b]fluoranthene (bbf), benzo[k]fluoranthene (bkf), benzo[ghi]perylene (b[ghi]p), benzo[a]pyrene (bap), chrysene (chr), dibenz[a,h]anthracene (d[ah]a), fluoranthene (f), fluorene (flr), indeno[1,2,3cd] pyrene i[cd]p, naphthalene (nap), phenanthrene (phen) and pyrene (pyr) in standard sample at the level of 1.06 µg/kg (16 priority pah, cocktail 3, chiron) (a) and smoked ham sample spiked at the level 1.96 µg/kg (b) a similar pah profile was reported in the studies by santos et al. (2011) and roseiro et al. (2012), in which the prevalence of light pahs was attributed to the unique sensorial properties of portuguese traditional meat sausages. alves et al. (2017) also confirmed the similar pattern of low molecular pahs’ domination in fermented sausages of distinctive portuguese and serbian origin. regarding the carcinogenic/mutagenic pahs, bap, ∑pah4 and ∑pah8 levels determined in this study were very low, with the highest values in sirloin (1.47 µg/kg for bap, 6.28 µg/kg for ∑pah4 and 12.13 µg/kg for ∑pah8). ital. j. food sci., vol. 31, 2019 675 significantly higher amounts were reported for bap in smoked meat products produced in latvia (from <0.05 µg/kg to 6.03 µg/kg) (rozentäle et al., 2015) and traditional smoked meat products from the baltic states (from 0.05 µg/kg to 166 µg/kg) (rozentäle et al., 2018), with ∑pah4 in ranges from 0.15-34.65 µg/kg and 0.42-628 µg/kg, respectively. none of the samples investigated in our study exceeded the mls for bap and ∑pah4 stipulated by the pertaining legislation (2 µg/kg and 12 µg/kg, respectively) (ec regulation no. 835/2011). when comparing the investigated meat products based on their pah contents expressed as benzo(a)pyrene toxic equivalencies (bape) (table 3), a slightly higher bape ∑pah15 and bape ∑pah8 were established in dryfermented sausages, while a slightly higher bape∑pah4 was seen in dry-cured meat products. it should also be mentioned that literature sources have revealed significantly higher bape concentrations than those measured in whole meat and chopped meat products analysed in our study; for instance, santos et al. (2011) and gomes et al. (2013) reported that ∑pah16 bape in portuguese traditional meat/blood products range from 2.74 µg/kg to 52.38 µg/kg, while ∑pah8 spans from 0.59 to 55.33 µg/kg and ∑pah4 from 1.43 µg/kg to 20.18 µg/kg. as for the mean total 15 pahs content in the studied meat products, statistically significant differences were observed. the highest average 15 pahs sum of 171.48 µg/kg established in dry-fermented sausages comes as the consequence of the highest low molecular pah content. when it comes to the total pah levels, dry-cured, semi-dry-cured meat products and semi-dry sausages differed significantly from dryfermented sausages (p<0.05). statistically significant differences in toxicologically important bap, ∑pah4 and ∑pah8 (p>0.05) failed to be found within the whole meat products’ groups. therefore, data on croatian meat products examined within the frame of this study argue against the need for exceptions stated under ec regulation no. 1327/2014 under which the mls for bap and ∑pah4 in traditional smoked meat and fish products is set at 5 µg/kg and 30 µg/kg, respectively. however, determination of pahs in processed meat is a permanent process. a true assessment of risk resulting from the constant presence of pahs in food chain requires a versatile and precise analytical method capable of measuring the level of a number of toxic pah compounds, with the possibility of extension to additional compounds in accordance with the recommendation of the scientific committee on food. furthermore, according to the recommendation of the joint fao/who expert committee on food additives (jecfa), benzo(c)fluorene as a compound usually, however inappropriately omitted from pah food analyses, should be included due to its carcinogenic effects and scarce data on its occurrence in food. 3.5. dried herbs and spices dried herbs and spices are defined as vegetable products, or mixtures thereof, which are free from any extraneous matter whatsoever, and are used for flavouring, seasoning and imparting the food aroma; therefore, they are classified as “all natural” (is0, 1995; torre torres et al., 2015). since all spices come from plants, they have generally been recognised as safe (gras). however, even though used in small amounts, spices have also been recognized as a potential source of chemical hazards (rozentäle et al., 2017). very high levels of pahs detected in herbs and spices present in foodstuffs (dg sanco, 2004; efsa, 2008) have recently resulted in new legislation requirements for bap content, which should not exceed 10 µg/kg, and the sum of bap, baa, bbf and bap, which should not exceed 50 µg/kg ec regulation no. 1933/2015). ital. j. food sci., vol. 31, 2019 676 figure 2. contents (µg/kg fresh weight) of low molecular pahs (acenaphthene (ace), anthracene (anthr), benz[a]anthracene (baa), fluoranthene (f), fluorene (flr), naphthalene (nap), phenanthrene (phen) and pyrene (pyr) in selected food from croatian market (food – 1: mussels; 2: other shellfish; 3:smoked fish; 4: semi dry sausages; 5: semi-dry cured meat products; 6: dry-fermented sausages; 7: drycured meat products). figure 3. contents (µg/kg fresh weight) of high molecular pahs (benzo[b]fluoranthene (bbf), benzo[a]pyrene (bap), benzo[k]fluoranthene (bkf), benzo[ghi]perylene (b[ghi]p), dibenz[a,h]anthracene (d[ah]a), indeno[1,2,3cd] pyrene i[cd]p) in selected food from croatian market (food – 1: mussels; 2: other shellfish; 3:smoked fish; 4: semi dry sausages; 5: semi-dry cured meat products; 6: dry-fermented sausages; 7: dry-cured meat products). 1 2 3 4 5 6 7 0 20 40 60 80 100 120 140 160 180 200 µg/kg low molecular pahs in shellfish, smoked fish and meat products mean minimum/maximum 1 2 3 4 5 6 7 0 2 4 6 8 10 12 µg/kg high molecular pahs in shellfish, smoked fish and meat products mean minimum/maximum ital. j. food sci., vol. 31, 2019 677 mean pah concentrations obtained in this study in certain food categories are shown in table 2. the majority of pahs found in randomly sampled spices and herbs circulating on the croatian market were low molecular ace, phen and pyr, present in the total pah15 content in the percent-share of 31.02 %, 22.53%, and 10.69%, respectively. as for the heavy ∑pah4 and ∑pah8, they were detected in all investigated herbs and spices (fig. 4). figure 4. contents (µg/kg fresh weight) of low (acenaphthene (ace), anthracene (anthr), benz[a]anthracene (baa), fluoranthene (f), fluorene (flr), naphthalene (nap), phenanthrene (phen) and pyrene (pyr and high molecular pahs (benzo[b]fluoranthene (bbf), benzo[a]pyrene (bap), benzo[k]fluoranthene (bkf), benzo[ghi]perylene (b[ghi]p), dibenz[a,h]anthracene (d[ah]a), indeno[1,2,3cd] pyrene i[cd]p) in selected dry herbs and spices from croatian market. the highest levels of the above were established in smoked paprika (∑pah4 113.12 µg/kg, ∑pah8 207.46 µg/kg) and the lowest in rosemary (∑pah4 1.24 µg/kg, ∑pah8 3.63 µg/kg) and garlic (∑pah4 2.27 µg/kg, ∑pah8 5.64 µg/kg). bap concentration ranged from 0.15 µg/kg in garlic and 0.21 µg/kg in rosemary to 21.88 µg/kg in smoked paprika (table 3). ∑pah8 most abundantly present in the examined herbs and spices were chr, d[ah]a, b[a]a, b[b]f and b[a]p (in decreasing order) (table 2). the highest chr, d[ah]a and b[a]a values (table 2) were witnessed in smoked paprika, followed by mixed pepper (5.02 µg/kg, 6.09 µg/kg and 2.67 µg/kg, respectively) and rosemary (4.81 µg/kg, 37.92 µg/kg and 0.67 µg/kg, respectively). in the study by rozentäle et al. (2017), the occurrence of four eu-regulated pahs (b[a]p, b[a]a, chr and b[b]f) was checked in 3 groups of herbs and 3 groups of spices. according to the authors, pah concentration found to be the highest in almost all analysed seasonings, with the mean values ranging from 1.73 µg/kg (nutmeg) to 8.68 µg/kg (thyme), was that of chr, its mean values established in red paprika and black pepper thereby being 3.18 µg/kg and 4.63 µg/kg, respectively. similar to our study, the investigated seasonings showed variations in pah levels. contrary to the results of our low pahs 1 low pahs 2 high pahs 1 high pahs 2 0 200 400 600 800 1000 1200 1400 1600 1800 µg/kg low and high molecular pahs in dry herbs and spices mean minimum/maximum ital. j. food sci., vol. 31, 2019 678 study, in the investigation by rozentäle et al. (2017) the highest bap contamination was detected in black pepper at the level of 6.60 µg/kg, while in thyme the ∑pah4 contamination of 37.39 µg/kg was determined. when it comes to bape∑pah4, bape∑pah8 and bape∑pah15 calculated within this study frame, the obtained results exhibited a similar descending pattern with the highest results of 27.27 µg/kg, 278.36 µg/kg and 300.76 µg/kg, respectively, for smoked paprika, to the lowest bape values of 0.21 µg/kg, 0.31 µg/kg and 0.48 µg/kg, respectively, detected in garlic (table 3). traditional smoking and processing methods applied in the production of smoked paprika and smoked cardamom resulted in high pah levels. however, given that the consumption of these spices is low, and to enable these smoked products to remain on the market, they were exempted from the maximal levels set out by the ec (2015). in summary, our results showed statistically significant (p < 0.05) shares of low molecular as compared to high molecular pahs (figure 2). however, the investigated herbs and spices were contaminated with pahs at levels lower than the maximum levels established by the eu. 4. conclusions the results of this study confirmed that certain food items circulating on the croatian market are contaminated with pahs at levels below the established maximal limits set out under the pertaining legislation. a statistically significant difference (p<0.05) in toxicologically important pah markers were found across the investigated food categories. with regard to bap and ∑pah4 contents, only certain spices showed significantly higher levels of the latter. other shellfish, smoked fishery products and spices were characterized with higher ∑pah8 marker values as compared to mussels and meat products. the highest total pah amounts were found in dried herbs and spices, followed by meat sausages and smoked fish. the present study showed that processed foodstuffs are more severely contaminated with pahs in comparison with food contaminated from environmental sources. independent of food category, pah composition pattern was dominated by low molecular pahs. being aware of the fact that the scope of the foodgoverning legislation is limited mostly due to the difficulty to define safe levels for complex pah mixtures, future analyses should be extended to additional pah compounds. a special attention should be paid to benzo[c]fluorene (bcf) as recommended by the joint fao/who expert committee on food additives (jecfa), since data on its occurrence in food are still scarce, but the levels of benzo[c]fluorene-derived adducts are much higher than those of benzo[a]pyrene-derived adducts. also, the margin of exposure (moe) approach would be of interest for our further studies intended to evaluate certain food consumption patterns pursued in croatia. references alomirah h., al-zenki s., al-hooti s., zaghloul s., sawaya w., ahmed n. and kannan k. 2011. concentrations and dietary exposure to polycyclic aromatic hydrocarbons (pahs) from 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martínez-carballo e. and simal-gándara j. 2015. a critical review about the health risk assessment of pahs and their metabolites in foods. crit. rev. food sci. 55:405. zelinkova z. and wenzl t. 2015. the occurrence of 16epa pahs in food a review. polycycl aromat compd. 35:284. paper received november 16, 2018 accepted february 8, 2019 #review_fantozzi_bozza ital. j. food sci., vol. 29, 2017 565 review the italian academic research on beer: past, present and future p. fantozzi department of agricultural, food and environmental sciences, university of perugia, via s. costanzo, perugia, italy *corresponding author. tel.: +39 0755857910; fax: +39 0755857939-5857943 e-mail addresses: paolo.fantozzi@ijfs.eu, paolo.fantozzi@unipg.it abstract in this review, the author wants to share his experience and opinions after 45 years of academic research on beer. the first part of this review is dedicated to the over time situation of the italian beer firms. the main core of the paper deals with the historical aspects of the italian academic research on beer and its relationship with beer industries. the importance of the italian research is also compared with the european situation. the university teaching on this matter is also discussed. finally it is suggested how to further upgrade the quality of the existing university research centres and laboratories. up to 194 references are presented. keywords: beer, malt, hop, humulus lupulus, breweries, research ital. j. food sci., vol. 29, 2017 566 1. the reference framework the scenario of barley, malt and beer production in italy has changed in a very important way in the last 20 years. italian farmers were always involved for producing barley basically for animal feeding (hordeum vulgare) whith high content of starch and protein and in limited quantities as hordeum disticum for selling to the malting industries (agroalimentare sud and saplo) (12) for their utilization in malt production. in this case it was requested a limited presence of nitrogen (<1,6%) and beta-glucans. the italian average year production is ca. 1,2 million of tons (= ca.300.000 ha). this reality did not changed until craft breweries started to enter in the market, often asking for special malts. the malting industries could not easily provide such malts in limited quantities because the size of their producing plants, obviously not satisfying all the different and small requests of craft brewers. while many small craft brewers finally accepted to produce beer with the standard malts available on the italian market, some other new born craft brewers were then obliged to search their malt with particular specific characteristics outside italy, were the elasticity of some malt producers was more present. (3) in force of a further increasing need, few large craft brewers decided to build their own malting plants (e.g.: mastri birrai umbri (pg), cobi (an), la vallescura (pc) (4-6), for their internal utilization and also for specific external customers. in the last 5 years this fact convinced several farmers to modify their crop rotation introducing barley for beer, indirectly answering to the increasing and continuous requests of craft brewers. this particular type of barley, in some particular region, was also considered by the regional agricultural officials as a real and possible alternative utilization to some traditional crops that appears to be not economical anymore. in addition to that, the cultivation of hop (humulus lupulus is also emerging, even if in small dimension. while beer was traditionally considered an industrial product (so then under the control and jurisdiction of the italian ministry of industry), these changes were finally recognized by the italian ministry of agriculture. with its ministerial decree 212/2010 beer was recognized to be an agricultural good, allowing all the products of the processing chain to have also access to italian and eu agricultural advantages (pac, feasr, horizon 2020, etc.). 2. the italian beer industry situation even if the presence of beer industry in italy really started in 1829 (wührer), in 1766 mr. lenz requested h.m. empress of austria authorization to produce theresianer beer in trieste and in 1789 mr. ketter obtained from the savoy state permission of producing beer in at that time italian town of nice (7). many important changes have occurred in the last 20 years, modifying deeply the italian beer firms scenario. at the beginning, the majority of italian beer firms were owned by italian families: 1829. birra wührer (fam. wührer, brescia) 1835. birra venezia (fam. biliotti, venezia) 1837. birra zimmermann (fam. zimmermann, aosta) 1837. birrificio rienzabirra di dobbiaco (fam. reichholf, dobbiaco) ital. j. food sci., vol. 29, 2017 567 1840. birra chiavenna (fam. g.ritter, chiavenna) 1845. birra bosio & caratsch (famm. bosio e caratsch, torino) 1846. birra peroni (fam. peroni, vigevano) 1846. birra menabrea,fam. g. menabrea, biella) 1848. birra perla crova metzger (fam. metzger,torino) 1857. birra forst (fam. wallnöfer e franz tappeiner, (lagundo) 1858. birra bonino (fam. g. bonino, cuneo) 1859. birra moretti (fam. moretti, udine) 1865. birra dreher (fam. prevoltella, trieste) 1877. birrificio angelo poretti (fam. poretti, vedano olona) 1879. birra orobia (fam. von wunster, bergamo) 1893. birra livorno (famm. del moro e di giacomo, chiavenna) 1897. birra pedavena (fam. luciani, canale dìagurdo) 1905. birra busalla (famm. ricchini e poggi, savignone) 1912. birra ichnusa (fam. amsicora capra, cagliari) 1995. birra castello (gruppo e.verardo, s. giorgio di nogaro) 2000. birra theresianer (fam. m. zanetti, nervesa della battaglia) in addition, up to 1959, were existing and operating almost other 80 small craft breweries (8). historically, the first multinational corporation entrance in italy happens in 1959, when heineken acquired 5-10% of dreher. in 1974 dreher was then fully absorbed by heineken. between 1982 and 2002, carlberg progressively acquired the poretti group. today the opposite is true: all the industrial breweries belong to multinational corporations (heineken, anheuser-bush inbev, carlsberg, asahi, sab, etc.), absorbing almost all of the old family firms, while only three of them still remain family owned (castello, forst, theresianer). in the mean time, we observed a massive growth of small-medium craft breweries, almost reaching about a thousand, with a very large variability on their product quality and quantity. their almost exhaustive list could be found in the web sites of assobirra and unionbirrai (9) (10). 3. the italian academic research on beer as far as i can go back in time, beer research was started and carried on by prof. c. cantarelli, university of perugia, in occasion of the 1965 beer monde selection. in this occasion, the institute od food science, university of perugia, was part of the analytical selection of participating beers and started the interest on beer. in 1970, was published the first academic paper on beer (11). according to scopus (12) the papers published in italy by academic scientists on beer from 1971 up to 2017 were 194, divided as follows: from 1971 to 1989 only 2 papers published, 1 on alcohol risk on beverages, including beer, ad 1 on hop botanical characteristics. from 1990 to 1999 : 22 papers, 8 of which in beer technology. ital. j. food sci., vol. 29, 2017 568 from 2000 to 2009: 64 papers, 26 of which in beer technology. from 2010 to 2017. 106 paper on different topic, 93 of them on beer (table 1). for a more complete information, i listed in the addenda a all the found references since 1971) (13). table 1. papers published on beer in the italian universities (years 2010-2017). university # of paper(*) 34 11 11 7 7 5 4 4 3 3 2 1 1 perugia viterbo (tuscia) piacenza (cattolica) ancona (marche) sassari salerno milan udine naples rome florence turin trieste total 93 (*) some papers may be contemporarily listed in different universities (co-authors). all these data do not take into account oral communications or posters in national or international congresses, as suggested by the anvur-vqr (italian evaluation of quality in research) (14) because of their low scientific impact. this 35 years analysis show a progressive increase in papers, but only in the last decade it has been seen an important amount of interests inside the italian universities. furthermore, if we compare papers from the top 20 publishing european countries (only universities), we may observe on table 2 the last 8-years situation. from this table, italy appears to be third in ranking, indicating how the interest of italian academic world increased in the last 8 years on this matter. an important point that needs be further explored, and not still done, should be the comparison between the sum of “citations” received worldwide by the italian papers with the sum of “citations” received by other country papers. in fact, these data will potentially show the true interest (… and indirectly the quality) of the italian scientific research on beer. ital. j. food sci., vol. 29, 2017 569 table 2. distribution of paper on beer published in european universities in the 2010-2017 period. 4. the academic relationship with breweries before 1995, only direct consultancies and assignments existed between single university scientists and breweries. the quantity or quality of them is generally covered by privacy. the first known public italian academic joint research with italian breweries started in 1995, when assobirra, the italian association of malsters and brewers, financed a strategic project on “micronutrients on beer”, having two main consultants (university of perugia and inn rome, the italian institute of nutrition). this project ended in 1998. from 1999 up to 2002, assobirra was the lead partner of the miur-pnr project on “nutritional characteristics of fermented beverages and product innovation”, totally devoted to demonstrate the antioxidant capacity of beer. this project was carried on by the consortium “birraviva” formed by peroni, forst, heineken, poretti, castello and menabrea. several italian research centres and scientists participated as consultants (universities of milan, perugia, rome and inn, rome). considering the outstanding obtained results of the project, assobirra started a deep scientific cooperation with the university of perugia that finally gave birth in 2003 to cerb (centro di eccellenza per la ricerca sulla birra italian brewing research centre) which i directed from the foundation to my retirement in 2015. during this period cerb had the unique possibility to observe all the research (and the related researchers) carried on in italy in the beer sector. moreover, my membership in the country # of papers % germany 280 13,2 united kingdom 202 9,6 italy 194 9,2 spain 191 9,0 france 98 4,6 belgium 183 8,7 czech republic 188 8,9 netherlands 131 6,2 poland 31 1,5 denmark 120 5,7 sweden 115 5,4 portugal 87 4,1 ireland 73 3,4 finland 54 2,6 russian federation 37 1,8 switzerland 42 2,0 romania 23 1,1 austria 21 1,0 bulgaria 34 1,5 greece 10 0,5 total 2116 100,0 ital. j. food sci., vol. 29, 2017 570 ebc brewing science group still provide also the possibility to compare the italian and the european situation on academic research. within respect to the other countries, i may observe that today in italy there is a general lack of public founding for basic and applied public research and it is almost absent a fundamental research partnership with big beer industries. in fact, international beer corporations posses their own large research laboratories and, when there is a need of external assistance, they are used to discuss and get quick answers (no waiting for preliminary studies on the requested matter) with well-established and proactive european academic realities (e.g.: notthingham, ghent, copenaghen, louvain, wheinstephan, berlin) rather than emerging although qualified italian realities (perugia, piacenza, viterbo, ancona, sassari and udine) as a matter of fact, only the following three important projects: a) on mycotoxins (a-mico) (15), b) on cereals (cersuom) both financed by the ministry of agriculture in the years 2008-2011 (16), c) on supply chain gmo traceability, financed by assobirra still outgoing(17), were financed during the last 10 years. meanwhile, and mainly at regional level (directive 2014/23/eu for financing por-fesr 20114_20120 -psrregional developing plans), small projects and consultancies are active between several university departments and small craft breweries on different specific topics. 5. the italian academic teaching on beer “teaching” is an important part of the academic duties. to my knowledge, while “beer” is partially present in the courses in food technology in many italian universities, only two universities have official regular courses on beer: perugia and udine. during my stay at the university of udine, i activated in 1981 the first course in “beer technology” inside the degree of food science and technology at the local department of food science and technology. at the beginning, this course was exceptionally taugh by dr. zangrando and successively by dr. collavo, at that time respectively plant managers of birra moretti and birra pedavena. today the course is under the responsibility of prof. buiatti, university of udine. in perugia the official teaching of beer started on 2007 and the situation was progressively upgraded during the last 15 years. in 2007 cerb started a 5 day “preparatory” internal course (“how to become a brewmaster”) that contributed effectively to the large increase in number, and often in quality, of italian craft breweries. this course reached this year the 20th edition and, in 8 years, received more than 350 students. to enlarge the floor and to raise quality, the department of agricultural, food and environmental sciences of university of perugia created in 2009 a first level master degree on “brewing technology” and in 2014 we enlarged the previous university degree course in agro-food sciences and technologies with an additional 3 years curriculum in “brewing technologies”. during my professor tenure in perugia i considered fundamental, as i did previously in udine, to call for seminars every year specialists from the italian beer companies in order to enrich, with their expertise, the students, allowing them to have a true contact with the industrial external reality, in addition to the internal ital. j. food sci., vol. 29, 2017 571 academic experience. in 2013 together with assobirra and the director of our department in perugia we formally agreed to have, every year, additional distance-learning courses given by specialists from beer companies on applied beer technology. when i retired, i left this important tool to the professors and researchers of my group, hoping they would understand how a continuous scientific exchange between industrial and academic people should be kept alive and not abandoned. 5. conclusions how to allow all “young” italian academic research centres or laboratories to upgrade and to reach the level of others well-established european labs? a brief list of priorities includes: first: increasing their internal scientific force by hiring permanent and multitasking skilled expert scientists, in order to be contemporarily proactive for incoming requests, then: publishing qualified and peer-reviewed scientific researches in high level journals rather than communications or posters in national or international congresses, finally: being principal rather than side-partners in international projects and being ready in solving industrial needs. to accomplish these priorities and to obtain a real upgrade in quality and external credibility, the italian university research centres compulsory need to have availability of money, granted by european or italian public organizations or from the beer industries. consequently, the universities must be professionally dedicated in fund raising. they should not count on money coming from teaching. in fact, while “teaching” is an important mission and an academic duty because is a fundamental preparation of students for their future external position and careers, the real impact on research fund raising is minimal. the net income from student taxes cannot allow its utilization for research project or contracts, being limited in quantity and in availability. the only limited positive point is a utilization of students as a partial support in the research projects when already in progress in the labs. it is obvious that the major responsibility for a scientific growth falls to the involved scientists, because funding comes only from their scientific credibility and from their international well-established reliability. of course, another alternative or parallel route for found rising is to focus on craft breweries, which continuously need a lot of help and advice from university scientists. this choice can allow a safe but slower growth of the italian university research on beer. acknowledgements during my long academic tenure as professor at the university of perugia, many top executives and managers of assobirra, together with skilled technologists of the beer industry (carlsberg, heineken, inbev, peroni, saplo, theresianer), participated as teachers in my courses and as special guests in particular events, believing all in the idea of an italian academic school on beer. hoping i did not miss someone, i want to thank all of them for that. they are: andrea bagnolini, flavio boero, michele cason, roberto cavalli, francesco collavo, leonardo di stefano, leonardo di vincenzo, pietro fontana, alberto frausin, harald fuchs, vittorio gorza, antonio marra, gennaro marturano, lorenzo morena, mauro niada, paolo papetti, rodolfo peroni, piero perron, daniele rossi, ital. j. food sci., vol. 29, 2017 572 raffaele sbuelz, fabio scappaticci, alessandro senia, luigi serino, filippo terzaghi, tullio zangrando, agata zani, giorgio zasio. finally, from the academic side, i want to sincerely thank prof. luigi montanari, university of sassari. he believed and helped me from the beginning, thereby allowing our italian university “beer dream” to become a reality. references 1. http://italmalt.com/ 2. http://saplo.it/ 3. https://www.weyermann.de/ 4. http://www.mastribirraiumbri.com/ 5. http://cobibirragricola.it/ 6. http://www.agrivallescura.it/ 7. zangrando t. 2017, personal communication 8. zasio g. 2017, personal communications 9. http://www.assobirra.it/gli-associati/ 10. http://unionbirrai.it/associati-unionbirrai/ 11. fantozzi p. 1970. determinazione dell’acido citramalico nella birra. birra e malto 6:217. 12. scopus.2017. https://www.scopus.com/search/form.uri?display=basic (key words utilized: “beer”, “malt”, “hop (humulus lupulus)”. 13. addenda a. 2017 italian universities references on beer. 14. miur anvur-vqr. 2017: http://www.anvur.org/index.php?lang=it 15. mipaaf. 2008-2011. project: a-mico. studio pilota sul possibile impiego di anticorpi policlonali fissati a matrice inerte come mezzo innocuo di decontaminazione da presenza occasionale di micotossine nelle fasi preliminari dei processi di preparazione di bevande fermentate. 16. mipaaf. 2012-2015. project: cersuom cereali al servizio dell’uomo: funzionalità integrata dei prodotti e sostenibilità di filiera. 17. assobirra. 2003. ricerca e sperimentazione per la rintracciabilità di materie prime geneticamente modificate (semola di mais) nella filiera produttiva della birra in italia (audit ogm). addenda a. italian universities 194 references on beer from 1980 up to 2017 accorsi c.a., bandini m.m. and forlani l. 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totally incompatible with the meat matrix, resulting in low hardness, yield and incoherent microstructure. fat loss was low, but reduced/eliminated by starch. color was slightly affected by starch, but no major trend was observed. overall, processors should carefully evaluate the functionality of starch they employ. keywords: frankfurters, meat, microstructure, starch, texture ital. j. food sci., vol. 30, 2018 802 1. introduction meat processors use different non-meat ingredients for reasons such as improving water/fat binding, enhancing texture, sliceability, flavor, appearance, and controlling cost (baranowska et al., 2004; brewer, 2012). the functional non-meat ingredients range from proteins (e.g., soy, milk) to carbohydrates (e.g., wheat flour, carrageenan), and spices (barbut, 2015). starches, derived from various sources (e.g., corn, potato, tapioca), represent one of the most diverse groups of non-meat ingredients used to bind water, enhance freeze-thaw stability, and improve sliceability. in general, starch is a naturally occurring substance which is also used as a thickening agent in sauces, yogurt, and emulsion type products (baranowska et al., 2004). different starches are available for industrial application in their natural form or as modified starches; the former type usually have more limited use in meat products. overall, starch functionality may vary due to different botanical origins (e.g., potato, corn, tapioca, rice, pea) and modifications (e.g., enzyme, acid, heat) applied by the food industry (aktaş and gençcelep, 2006). some meat products such as fish surimi have traditionally been produced with starch to increase firmness and water binding especially in such a highly minced, high water added product (verrez-bagnis et al., 1993; fogaça et al., 2013). in other red meat/poultry products, starches are used to enhance gel strength and water holding, to replace fat, and control formulation cost (li and yeh, 2003a, b; brewer, 2012). dexter et al. (1993) reported that starch added to turkey bologna was very effective in decreasing purge while not increasing hardness. they also reported that starch effect depended on the type of starch used, water-to-starch ratio, processing factors, and presence of ingredients such as fat. modified potato starch was reported to improve the texture of low fat bologna (claus and hunt, 1991) and comminuted scaled sausage (pietrasik, 1999). carballo et al. (1995) indicated that adding starch to meat emulsions resulted in more compact and stronger heat induced meat protein network. resconi et al. (2015) reported that using rice starch (0.3-1.5%) helped increase yield and hardness of whole muscle cooked hams. replacing animal fat with vegetable oil is another significant trend seen today, and additives such as starch are important in stabilizing the high moisture added to reduced fat meat products (brewer, 2012; barbut et al., 2016). currently there are some starch suppliers that claim that their products can also enhance fat stabilization in meat products. therefore, the objectives of this study were to compare the effects of potato and corn starches in their native and modified forms (total 8 starches) on cooking losses (moisture and fat), texture, microstructure, and color of emulsified meat products prepared with vegetable oil. 2. materials and methods 2.1. preparation of meat batters lean shoulder blade beef meat was obtained from the university of guelph abattoir. all visible connective tissue and fat were removed from the lean meat. meat was comminuted in a bowl chopper (smk 40; schneidmeister, berlin, germany) at the low speed setting for 1 min to obtain a homogenous mass. the meat (73.6% moisture, 19.6% protein, and 5.9% fat. aoac, 1996) was vacuum-packed and frozen (-18ºc) in polyethylene bags (750g/package) for up to 1 month prior to use. nine different formulas were prepared in 3 independent trials. the starches used include native potato (np) starch (herman laue spice co., uxbridge, on, canada), modified potato starch-1 (mp-1, pencling 530; penford ital. j. food sci., vol. 30, 2018 803 food ingredients co., denver, co, usa), modified potato starch-2 (mp-2, farinex va 15; avebe foods, veendam, netherlands), modified potato starch-3 (mp 3, eliane ve 420; avebe foods, veendam, netherlands), native corn starch (nc, amioca; national starch, westchester, il, usa), modified corn starch-1 (mc-1, em-cap; cargill inc., minneapolis, mn, usa), modified corn starch-2 (mc-2, pencling 570; penford food ingredients co., denver, co, usa), modified corn starch-3 (mc-3, firm-tex; national starch, westchester, il, usa), and a control with no starch (cont). meat was thawed at 5ºc overnight. pure canola oil (no name®; sunfresh limited, toronto, on, canada), was used as the main fat source. the batters were formulated to contain 25% fat/oil (5.9% as beef fat within the lean meat, and the rest added as canola oil), 13.5% protein and 2% starch in all treatments except no starch in the control. meat was initially chopped at the low speed setting for 30 s. later, while chopping at the high speed setting for 30 s, 2.0% salt and 0.25% sodium tripolyphosphate were added. this was followed by a 2 min break (for protein extraction to occur). next, canola oil and the appropriate starch (prepared by dissolving the starch in the 2% water added to the product) were added to the batter while chopping at the high speed setting for 1 min, followed by ice addition and further chopping for 4 min. the temperature of the batter did not exceed 12ºc in any of the treatments. each batter was vacuum-packed (multivac model a300/16; wolfertschwenden, germany) to remove trapped air. for each batter, 35g samples were stuffed into three separate 50 ml polypropylene tubes, which were centrifuged (model 224; fisher scientific, pittsburgh, pa, usa) at the low speed setting for 30 s to remove any remaining small air bubbles. the batters were cooked in a water-bath (w-26; haake, berlin, germany) from 25 to 70ºc within 1.5 h. a thermocouple unit was used to monitor the core temperature of the samples (model 52 kj1, fluke, everett, wa, usa). 2.2. cooking loss test tubes were cooled in ice water for 5 min, and then liquid separated during the cooking cycle was collected and expressed as % cooking loss (liquid expelled (g)/raw batter weight (g) x 100). the next day the volume of the fat (floated to the top overnight) separating out was determined and expressed as fat loss. 2.3. texture profile analysis (tpa) after an overnight storage (5°c), tpa parameters were determined using nine cooked cores (each 16 mm diameter and 10 mm high) per treatment. cores were compressed twice to 75% of their original height by a texture analyzer (stable micro systems, model ta.xt2; texture technologies corp., scarsdale, ny, usa) at a crosshead speed of 1.5 mm/s. the following parameters were determined: hardness, springiness, cohesiveness, chewiness, and gumminess (rosenthal, 2010). 2.4. color the color of fresh cut cross-sections of the cooked meat batters (9 per treatment) was determined (mini scan ms/s; hunter laboratories, reston, va, usa) using the d65 illuminant setting, and 10-degree standard observer. color is expressed according to the commission international de l’eclairage (cie) system and reported as hunter l* (lightness), a* (redness), and b* (yellowness) (wiegand and waloszek, 2003). ital. j. food sci., vol. 30, 2018 804 2.5. microstructure samples (2.0×2.0×0.5 cm) were cut from the centers of cooked meat batters, fixed in 10% neutral buffered formalin for 10 h at room temperature, dehydrated in 70% isopropanol for 2 h, 95% for 1 h, and 100% for 4 h and embedded in paraffin. samples were cut into 4-6 μm sections, stained with hematoxylin-eosin for 4 min, and observed using a light microscope (model bx60f5; olympus optical company, tokyo, japan). black and white pictures were taken (image-pro plus, version 5.1; media cybernetics inc., silver spring, md, usa). 2.6. statistical analysis the experiment was designed as a complete randomized block, with three separate replications. statistical analysis was performed using a software package (sas version 8.02; sas institute, cary, nc, usa). the sas general linear model procedure was used for analysis of variance. tukey’s multiple comparison analysis was performed to separate the means (p < 0.05). 3. results and discussion the addition of all four potato starches resulted in a significant reduction in cooking loss compared to the control (table 1). table 1. effects of native and modified starches on overall cooking loss, fat loss, moisture loss and color parameters (l*=lightness, a*=redness, b*=yellowness) of meat batters prepared with canola oil. treatment* cooking loss fat loss moisture loss color coordinates (#) (%) (%) (%) (lightness) (redness) (yellowness) 1 cont 2.26±0.29b 0.57±0.18a 1.69±0.26b 65.4±0.30b 3.60±0.03c 13.2±0.08b 2 np 0.67±0.09d 0.14±0.05b 0.53±0.10c 63.9±0.16c 3.93±0.03a 13.3±0.05ab 3 mp-1 0.44±0.12d 0.12±0.06b 0.32±0.06c 62.9±0.28d 4.04±0.03a 13.2±0.09b 4 mp-2 0.19±0.07d 0.00±0.00b 0.19±0.07c 63.1±0.22d 3.91±0.04a 13.2±0.07b 5 mp-3 0.29±0.07d 0.00±0.00b 0.29±0.07c 62.4±0.27d 4.00±0.03a 13.2±0.09b 6 nc 1.86±0.20bc 0.19±0.09b 1.67±0.11b 65.0±0.27b 3.69±0.04bc 13.3±0.10ab 7 mc-1 5.89±0.44a 0.00±0.00b 5.89±0.44a 66.5±0.34a 3.35±0.05d 13.5±0.11a 8 mc-2 1.10±0.68cd 0.00±0.00b 1.10±0.68bc 62.4±0.29d 4.04±0.05a 13.2±0.10b 9 mc-3 0.25±0.06d 0.06±0.04b 0.19±0.03c 63.1±0.26d 3.78±0.02b 13.1±0.08b a-dmeans, ± standard error, with no common superscript are significantly different (p < 0.05). *all formulated with 2.0% salt and 0.25% sodium tri-polyphosphate. cont = control; np = native potato starch; mp = modified potato starch; nc = native corn starch, mc = modified corn starch. this can be correlated to starch molecules opening up, during heating, and absorbing moisture; a process known as gelatinization (baranowska et al., 2004). in the case of the added corn starches (treatments 6-9), the native corn starch did not show a significant improvement over the control. examination of the micrographs shows that this treatment did not reach the gelatinization point (i.e., granules shown as compact non-open structures; fig. 1f). ital. j. food sci., vol. 30, 2018 805 figure 1. light micrograph of cooked meat batters (13.5% protein) prepared with canola oil and different starches: (a) control; (b) native potato starch (c) modified potato-1 (mp-1); (d) mp-2; (e) mp-3; (f) native corn; (g) modified corn-1 (mc-1); (h) mc-2; and (i) mc-3. fg-fat globules (i.e., fat removed during sample preparation; prior to paraffin embedding); s-starch; ps-probably starch. bar = 200μm. this is the reason that quite a few of the starches used by the meat industry are pregelatinized, meaning that they are pre-exposed to a certain heat treatment; i.e., to open or partially open their structure and make them capable of absorbing water even at a low temperature (baranowska et al., 2004; resconi et al., 2015). a certain degree of unopened structures is also seen in the mc-2 treatment (fig. 1h) where a number of very dense non-gelatinized starch granules can still be seen after heating the meat batter to 70°c. the mc-2 and mc-3 treatments resulted in a significant reduction in cooking loss compared to the control (2.26 vs. 1.10, a 50% reduction and 0.25, a 90% reduction, respectively). the mc-1 treatment showed the highest cooking loss value (table 1) and revealed extremely small starch granules. however, the high cooking loss seems to be related to the disruption of the entire microstructure (fig. 1g) as seen by the many open channels (discontinuities) and gaps within the matrix. this kind of microstructure has also negatively affected the texture (e.g., hardness, springiness; see below). it should be mentioned that describing the nature of the starches’ modifications (provided by the manufacturers) is very vague. as a result, it is difficult to relate the modifications to specific effects in the meat system. in terms of fat loss, although relatively low in the control (0.5%), all starches helped to lower or eliminate it. this is most likely due to the starch increasing the viscosity of the ital. j. food sci., vol. 30, 2018 806 meat batters rather than actually binding the fat. overall, the fat/oil was held very well within the meat matrix of the control (fig. 1a). the micrograph shows small, stable, and well distributed fat globules, and is in agreement with previously published micrographs (barbut et al., 2016). the addition of starch did not show any interference with the stability of the fat globules and/or any direct interaction with the fat phase. the slight improvement in fat retention seen here is also in agreement with aktas and gençcelep (2006), who noted that some modified potato and corn starches can reduce fat loss from bologna type sausage produced with sheep tail fat. table 2. effects of native and modified starches on texture profile parameters of cooked beef meat batters prepared with canola oil. treatment* hardness springiness cohesiveness chewiness gumminess (#) (n) (cm) (ratio) (n x cm) (n) 1 cont 67.6±1.5c 0.81±0.01a 0.39±0.01a 21.5±0.8a 26.5±0.7ab 2 np 79.4±2.2a 0.75±0.01b 0.35±0.01b 21.3±0.9a 28.3±1.1a 3 mp-1 68.1±1.7bc 0.73±0.02bcd 0.30±0.01d 15.1±0.6cd 20.4±0.5cd 4 mp-2 64.9±1.2cde 0.74±0.01bc 0.29±0.01d 14.1±0.4d 19.0±0.4d 5 mp-3 62.9±1.4def 0.69±0.01d 0.26±0.01e 11.6±0.43e 16.6±0.5e 6 nc 72.2±1.6b 0.76±0.01b 0.34±0.01b 19.3±0.7b 25.2±0.7b 7 mc-1 59.5±1.4f 0.59±0.02e 0.29±0.01d 10.1±0.2e 17.1±0.3e 8 mc-2 66.3±1.1cd 0.76±0.01b 0.31±0.01c 16.1±0.4c 21.0±0.5c 9 mc-3 61.0±1.2ef 0.70±0.01cd 0.27±0.01e 11.7±0.5e 16.5±0.5e a-fmeans, ± standard error, with no common superscript are significantly different (p < 0.05). *all formulated with 2.0% salt and 0.25% sodium tripolyphosphate. cont = control; np = native potato starch; mp = modified potato starch; nc = native corn starch, mc = modified corn starch. texture profile analysis results (table 2) show that using the two native starches (potato and corn) significantly increased hardness values above the ones seen in the control. verrez-bagnis et al. (1993) and li and yeh (2003a) also reported that adding native starch to meat products increased hardness/storage modulus. the other modified starches either did not influence hardness or caused a reduction. sanjeewa et al. (2010) reported that in some of the canadian varieties of chickpea flours (contain 36-41% starch) they added to low-fat bologna, they observed increased tpa hardness values, while in others they did not. in the present study, the lowest hardness value was seen in the mc-1 treatment, which also lost the highest amount of water. as indicated earlier, this is probably due to formation of channels/disruptions within the meat matrix (fig. 1g), and resulted in a weaker physical structure. a similar result was also observed for the springiness value, which was the lowest for this treatment (table 2). overall, the control (no starch) showed the highest springiness value. all starches caused the formation of less elastic cooked meat structures as evidenced by the lower springiness value (table 2). this might be due to some discontinuities imparted by the starch (gelatinized or still granular) within the meat matrix. the same was observed for cohesiveness values. however, it should be mentioned that differences between the starches exist and the two native starches (potato and corn) resulted in higher values than the modified starches. chewiness and gumminess followed the same trend in which the native potato starch was actually not significantly different from the control, but both native starches (potato and corn) resulted in higher values compared to the modified starches. this could be due to more ital. j. food sci., vol. 30, 2018 807 interactions of the modified starches with meat proteins (i.e., because some are pregelatinized and can interact with the proteins before they become heat denatured; barbut, 2015). however, this point needs further investigation. the color of the potato starch added treatments was not as light as the one in the control (lower l* values; table 1). however, the difference of about 2 l* units (scale: 0=black, and 100=pure white) should not be expected to cause a major obstacle in terms of consumer acceptance. in the case of corn starch, the mc-1 ended up lighter than the control, and mc-2 and mc-3 were darker. again these differences are not expected to be a problem in terms of marketing. red color (a*) did not show a major change except for the mc-1 treatment which had the highest cooking loss values (i.e., twice as high as the control; table 1). this resulted in more of the water soluble red pigment (myoglobin) leaching out of the product. yellowness values were basically unchanged by starch addition. 4. conclusions the study demonstrates the positive effect of using modified potato and corn starches in an emulsified meat product. the mp-2 showed the best performance in terms of minimum cooking loss and hardness compared to the control. the study also highlights the fact that attention should be given to starch selection for a specific application. in addition, it should be mentioned that some ingredient suppliers sell blends of 2-3 starches to cover various aspects within the same formula, and the meat processor should be aware of the composition, cost, and added value of each component. acknowledgements the author would like to thank the ontario ministry of agriculture, food and rural affairs for financial support and mr. youssef for technical support. references aktaş n. and gençcelep h. 2006. effect of starch type and its modifications on physicochemical properties of bolognatype sausage produced with sheep tail fat. meat science 74 (2):404. aoac. 1996. “official methods of analysis” 16th ed. association of official analytical chemists, washington, dc. baranowska h.m., rezler r., poliszko s., dolata w., piotrowska e. and piątek m. 2004. starch as a functional addition in meat batters. in: “starch: from starch containing sources to isolation of starch and their applications”. v.p. yuryev, p. tomasik and h. ruck (eds.), p. 115. nova science publishers, new york. barbut s., wood j. and marangoni m. 2016. potential use of organogels to replace animal fat in comminuted meat products. meat science. 122(2): 155. barbut s. 2015. non-meat ingredients. in: “the science of poultry and meat processing”. p. 13-11. www.poultryandmeatprocessing.com pp. 13-11. free download. accessed 10.24.17. published by the university of guelph, on, canada. brewer m.s. 2012. reducing the fat content in ground beef without sacrificing quality: a review. meat science 91(4):385. carballo j., mota n., baretto g. and colmenero f.j. 1995. binding properties and colour of bologna sausage made with varying fat levels, protein levels and cooking temperatures. meat science 41(3):301. claus j.r. and hunt m.c. 1991. low fat, high added-water bologna formulated with texture-modifying ingredients. journal of food science 56(3):643. dexter d.r., sofos j.n. and schmidt g.r. 1993. quality characteristics of turkey bologna formulated with carrageenan, starch, milk and soy protein. journal of muscle foods 4(3):207. ital. j. food sci., vol. 30, 2018 808 fogaça f.h.s., trinca, l.a., bombo, a.j. and silvia sant'ana, l. 2013. optimization of the surimi production from mechanically recovered fish meat (mrfm) using response surface methodology. journal of food quality 36(3):209. li j.y. and yeh a.i. 2003a. effects of starch properties on rheological characteristics of starch/meat complexes. journal of food engineering 57(3):287. li j.y. and yeh a.i. 2003b. gelation properties and morphology of heat‐induced starch/salt‐soluble protein composites. journal of food science 68(2):571. pietrasik z. 1999. effect of content of protein, fat and modified starch on binding textural characteristics, and colour of comminuted scalded sausages. meat science 51(1):17. resconi v.c., keenan d.f., gough s., doran l., allen p., kerry j.p. and hamill r.m. 2015. response surface methodology analysis of rice starch and fructo-oligosaccharides as substitutes for phosphate and dextrose in whole muscle cooked hams. food science and technology 64(2):946. rosenthal a.j. 2010. texture profile analysis–how important are the parameters? journal of texture studies 41(5):672. sanjeewa w.t., wanasundara j.p., pietrasik z. and shand p.j. 2010. characterization of chickpea (cicer arietinum l.) flours and application in low-fat pork bologna as a model system. food research international 43(2):617. verrez-bagnis v., bouchet b. and gallant d.j. 1993. relationship between the starch granule structure and the textural properties of heat-induced surimi gels. food structure 12(3):309. wiegand c. and waloszek g. 2003. “color glossary a-c”. www.sapdesignguild.org/resources/glossary_color/index1.html#norm_cs accessed 01.12.17. paper received november 3, 2017 accepted june 6, 2018 ijfs#1521_bozza ital. j. food sci., vol. 31, 2019 731 paper antioxidant capacity and heat damage of powder products from south american plants with functional properties a. brizzolaria1, a. brandolini*b, p. glorio-pauletc and a. hidalgo*d a department of health sciences, università degli studi di milano, via di rudinì 8, 20142 milan, italy bconsiglio per la ricerca in agricoltura e l’analisi dell’economia agraria unità di ricerca per la zootecnia e l’acquacoltura (crea-za), via forlani 3, 26866 s. angelo lodigiano (lo), italy c universidad nacional agraria la molina, facultad de industrias alimentarias, departamento de ingeniería de alimentos y productos agropecuarios. av. la molina s/n, lima 12, peru d department of food, environmental and nutritional sciences (defens), university of milan, via celoria 2, 20133 milan, italy 1present address: dan europe research division, dan europe foundation, roseto degli abruzzi (te), italy *corresponding author: andrea.brandolini@crea.gov.it; alyssa.hidalgovidal@unimi.it abstract aim of the study was to evaluate color, total polyphenol content (tpc), antioxidant capacity (abts, frap, dpph), reducing sugars and heat damage (furosine, hydroxymethylfurfural, glucosylisomaltol) of 21 commercial powder products obtained from south-american fruits (mesquite, lucuma, camu camu), seeds (amaranth, purple maize), roots and tubers (yacon, maca, mashua, tocosh), bark (cat’s claw) and leaves (graviola). tpc and antioxidant capacity were maximum in camu camu and cat’s claw powders, and minimum in tocosh, amaranth, lucuma and maca; graviola, mashua, purple maize and mesquite also showed good antioxidant properties. yacon, mashua and lucuma powders had high reducing sugars content (40.9, 34.4 and 21.2 g/100 g dm, respectively) and heat damage (hmf 146.6 mg/kg, furosine 2399.8 and 2228.4 mg/100 g protein, respectively). overall, camu camu powders and cat’s claw were the most interesting products, having high levels of total polyphenols and antioxidant capacity together with very low heat damage. keywords: camu camu, cat’s claw, maca, mashua, mesquite, yacon ital. j. food sci., vol. 31, 2019 732 1. introduction a major threat to human wellbeing is the oxidative stress, an “imbalance between oxidants and antioxidants in favour of the oxidants” (sies, 1997), which can lead to cellular damage and facilitate the insurgence of cardiovascular and neurodegenerative diseases, diabetes mellitus, cancer and inflammatory illness (uttara et al., 2009). an effective approach to prevent oxidative stress is to include in the daily diet products rich in antioxidants, which can quench the oxygen free radicals, preventing the oxidation of the cell membrane. plants and plant-derived ingredients have been used as medical remedies from prehistoric ages, and still are a major source of health-promoting elements. in recent years, the interest in the identification and utilization of plants rich in antioxidant compounds to limit the oxidative stress (almeida et al., 2011; krishnaiah et al., 2011) has been steadily growing, because they may behave as preventive medicine. several authors have reviewed the beneficial uses of underexploited and little-known plant species used in food production but also in traditional medicine (e.g. biel et al., 2017; campos et al., 2013; chirinos et al., 2013; contreras-calderón et al., 2011; krishnaiah et al., 2011). peru, thanks to its widely diversified climatic zones, is home to a broad array of endemic plants, which show huge differences in the content and type of nutrients and that are potential sources of valuable bioactive compounds (campos et al., 2018). the antioxidant capacities of plant-derived products vary depending on their content in polyphenols, vitamin c, tocols and carotenoids, (saura-calixto and goñi, 2006), as well as on the different processing conditions. while in some cases the plant products are consumed fresh, most often they undergo some type of transformation and/or drying to improve shelf life, to lower transport costs and to reach far off consumers (cinar, 2018). accordingly, powders from south-american plants with known health-promoting features (supplementary table 1) are manufactured by several industries to find new market niches and to foster the consumption of health-promoting natural products. these innovative powder products, obtained from fruits (mesquite, lucuma and camu camu), seeds (amaranth and purple maize), roots and tubers (yacon, maca, mashua and tocosh), bark (cat’s claw) and leaves (graviola), are currently used for the preparation or enrichment of infusions, juices, shakes/smoothies, yogurts, desserts, as well as ingredients in cosmetic and pharmaceutical recipes. the aim of our study was to evaluate some characteristics of these powder products for their possible utilization as enhancing ingredients in wheat-based oven products. to achieve this goal, 21 commercial powder samples of the above-mentioned species were assessed for color, total polyphenol content, antioxidant capacity, reducing sugars and heat damage. 2. materials and methods 2.1. samples the powders analyzed were acquired in 2016 at an industrial fair dedicated to peruvian export products (expoalimentaria, lima, peru; www.expoalimentariaperu.com) except amaranth, obtained from the peruvian market, and two maca samples, bought from the italian market. several samples (3-5) of each powder product were collected. a detailed list of the products tested is presented in table 1. ital. j. food sci., vol. 31, 2019 733 2.2. physical and chemical analyses 2.2.1 color the color coordinates l* (luminosity), a* (red-green) and b* (yellow-blue) of the samples were scored with a tristimulus colorimeter (chroma meter cr-300, minolta italia s.p.a., italy) using the standard-white reflector plate and illuminant c. four measurements for each sample were performed. table 1. samples analyzed: species, brands, codes, average dry matter and protein contents (g/100 g). product species brand code sample code dry matter protein bark cat’s claw uncaria tomentosa l. a cat’s claw 1 91.8 0.3 cat’s claw bio uncaria tomentosa l. b cat’s claw 2 92.8 2.7 cat’s claw tea uncaria tomentosa l. b cat’s claw tea 92.5 3.0 seeds amaranth flour amaranthus caudatus l. c amaranth fr 90.4 11.5 amaranth flakes amaranthus caudatus l. d amaranth fs 90.4 8.8 purple maize zea mays l. b purple maize 90.3 7.0 roots yacon smallanthus sonchifolius (poepp. &t endl.) h. robinson b yacon 87.8 1.8 maca gluten free lepidium meyenii chacon e maca 1 85.9 10.6 maca bio lepidium meyenii chacon b maca 2 87.3 9.0 maca hp lepidium meyenii chacon b maca 3 90.3 8.5 maca extract lepidium meyenii chacon a maca 4 85.4 9.3 maca lepidium meyenii chacon a maca 5 86.6 7.7 maca root lepidium meyenii chacon f maca 6 92.9 12.0 maca energia lepidium meyenii chacon g maca 7 90.4 7.0 tubers tocosh solanum spp. h tocosh 83.4 2.2 mashua tropaeolum tuberosum ruiz & pav. e mashua 84.7 9.0 leaves graviola bio annona muricata l. b graviola 91.9 10.8 graviola tea annona muricata l. b graviola tea 91.0 11.0 fruits mesquite prosopis spp. b mesquite 89.3 8.7 lucuma pouteria lucuma ruiz & pav. b lucuma 88.9 3.4 camu camu myrciaria dubia (kunth) mcvaugh b camu camu 87.4 5.4 ital. j. food sci., vol. 31, 2019 734 2.2.2 dry matter and protein content dry matter was determined following the gravimetric method, drying 2 g of product at 130 °c for 90 min; protein content was assessed by kjeldahl (n x 6.25). these and all the following analyses were performed in triple. 2.2.3 samples preparation for total polyphenols content and antioxidant capacity analysis all the reagents, of analytical grade, were purchased from sigma-aldrich co. (milan, italy). two different solvents were tested for the extraction of total polyphenols and the evaluation of the antioxidant capacity, i.e. ethanol:h2o (etoh:h2o; 80:20) and methanol:h2o:acetic acid (meoh:h2o:acet; 50:42:8). exactly 0.15 g of powdered product were weighed in 2 ml tubes and subjected to three extractions, adding 1 ml of an etoh:h2o solution each time. in the first extraction, the samples were stirred with a vortex (reax 2000, meindolph heidolph, schwabach, germany) for 1 min and sonicated (f5200b, decon, uk) twice for 20 min; in the second extraction the samples were stirred with a vortex for 1 min, an orbital shaker (multirotator grant-bio, cambridge, uk) for 20 min and sonicated for another 20 min; in the third extraction the samples were stirred with a vortex for 1 min and sonicated for 5 min. after each extraction, the samples were centrifuged with a 4224 centrifuge (alc apparecchi per laboratori chimici srl, milan, italy) for 5 min at 8048 g and all the supernatants were mixed in a single tube. the extractions were performed at 10 °c and away from light as far as possible. following the same procedures, 0.3 g of powdered product underwent three extraction cycles, adding respectively 1.5, 1.5 and 1.0 ml of a meoh:h2o:acet solution. 2.2.3.1 total polyphenol content total polyphenol content (tpc) in samples extracted with etoh:h2o and meoh:h2o:acet was assessed with the folin-ciocalteu method as described by brandolini et al. (2013) using a du-62 beckman spectrophotometer (beckman coulter, nyon, vd, switzerland). the tpc, in mg gallic acid equivalent (gae)/kg dm, was computed from a reference curve obtained from six gallic acid concentrations (range: 0-150 mg/l). 2.2.3.2 assessment of antioxidant capacity using the abts method the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (abts) radical scavenging capacity was analysed as described by yilmaz et al. (2015). a stable stock solution of the abts radical cation was prepared by reacting 10 ml of an aqueous solution of 2-2’azinobis-3-etilenbenzotiazoline 7 mm and 176 µl potassium persulfate 140 mm in the dark at room temperature for 12-16 h. the etoh:h2o or meoh:h2o:acet extracts (150 ml) were reacted with 5 ml of a diluted abts radical solution in ethanol (absorbance: 0.70±0.02 au at 734 nm); the absorbance was measured at 734 nm, after 6 min at 30 °c, with a v650spectrophotometer (jasco, japan), using ethanol as blank. the antioxidant capacity was evaluated as percentage of absorbance decrease (inhibition percentage). a reference curve was built with 11 concentrations (from 0.05 to 0.72 mm) of trolox. the results are expressed as mmol trolox equivalents (te)/kg dm. ital. j. food sci., vol. 31, 2019 735 2.2.3.3 assessment of the reduction power using the frap method the ferric reducing antioxidant power (frap) was determined as described by yilmaz et al. (2015), following the method proposed by benzie and strain (1996). briefly, 200 ml of etoh:h2o or meoh:h2o:acet extracts were mixed with 4.5 ml frap reagent. absorption was measured with a v650 spectrophotometer (jasco, japan) at a wavelength of 593 nm after 60 min incubation at 37 °c; acetate buffer 0.3 m ph 3.6 was used as blank. the frap reagent, prepared daily, consisted of 0.3 m acetate buffer (ph 3.6), 10 mm 2,4,6-tris(2pyridyl)-s-triazine (tptz) in 40 mm hcl and 20 mm fecl3 (10:1:1 v/v/v). frap values were obtained by comparing the results to a calibration curve built with 18 concentrations (0.06 0.90 mm) of trolox. the antioxidant capacity was expressed as mmol te/kg dm. 2.2.3.4 assessment of antioxidant capacity using the dpph method the 2,2-diphenyl-1-picrylhydrazyl (dpph) radical cation scavenging capacity of etoh:h2o and meoh:h2o:acet extracts was evaluated according to brandolini et al. (2013) using a du-62 spectrophotometer (beckman, usa). for each extract five different dilutions were analysed. a dose-response line was computed for each sample and the powder quantity needed to scavenge 50% of the radical (i50) was determined. a reference regression line was computed for the antioxidant trolox, with concentrations between 3 and 50 μm. the antioxidant capacity was expressed as ratio between i50 of trolox and i50 of the sample, i.e. mmol te/kg dm. 2.2.4 sugars content fructose, glucose, maltose and sucrose were assessed by hplc, following hidalgo and brandolini (2011). for peak quantification, sugars calibration curves were constructed using 15 different concentrations (between 0 and 155 mg/l) of fructose, 19 different concentrations (between 0 and 428 mg/l) of glucose, 19 different concentrations (between 0 and 385 mg/l) of maltose, and 15 different concentrations (between 0 and 153 mg/l) of sucrose standards (sigma, st. louis, mo, usa). the calibration curves, after log transformation, were linear (r2 = 1.00; p≤0.001) in the concentration ranges considered. the results are reported as g/100 g dm. 2.2.5 heat damage indices furosine was determined by hplc as described by hidalgo and brandolini (2011). a calibration curve was built using nine different concentrations (between 0.33 and 5.13 mmol/l of furosine dihydrochloride (neomps, polypeptide laboratories, strasbourg, france) in 3 n hcl. the calibration curve was linear (r2 = 1.00; p≤0.001) in the concentration ranges considered. the results are expressed as milligrams of furosine/100 g of protein. hydroxymethlfurfural (hmf) and glucosylisomaltol (gli) were determined following the hplc method of rufián-henares et al. (2008) as described by hidalgo and brandolini (2011). for peak quantification, a calibration curve was constructed using 13 different concentrations (between 0 and 6.25 mg/l) of hmf (safc, st. louis, mo, usa). the calibration curve was linear (r2 = 1.00; p≤0.001) in the concentration range considered. gli quantification was computed considering the response factor of hmf at 280 nm. the results are expressed as mg/kg dm. ital. j. food sci., vol. 31, 2019 736 2.3. statistical analysis the data were processed by one-way analysis of variance (anova) considering the samples as factors. the distribution of the data was checked and, for normalization purposes, l* and b* values were squared, while the other parameters were log10transformed; however, for easier comprehension, in tables and figures the original data are reported. when significant differences were found (p≤0.05), fisher’s lowest significant difference (lsd) was computed at a 95% significance level. to compare the results of the two solvents used for the preparation of the extracts, the t-test was applied (p≤0.05). anova, lsd test and t-test were conducted using the statistical program statgraphics® centurion. mean, standard error and coefficient of variation were computed using the program excel (microsoft® office excel 2007). principal components analysis (pca), performed considering the mean values of the 21 samples and all the parameters, was carried out with the software the unscrambler x 10.2 (camo software as, norway). 3. results and discussion 3.1. powders color supplementary table 2 shows the average values and the results of the lsd test for the color coordinates l*, a*, b* of the twenty-one samples. the results obtained grouping the samples by species are reported in fig. 1. figure 1. colour coordinates (l*, a*, b*) of powdered products from 11 species. different letters indicate significant differences (lsd, p≤0.05) among species. the broad heterogeneity of the samples led to an anova (not presented) showing significant differences for all the parameters. in fact, a preliminary visual control gave the following color characterization: mashua and purple corn were purple; maca, yellowc e a d g g d cd g f b 0 10 20 30 40 50 b* c bc bc ef bc g e d ab f a 0 10 20 30 40 50 a* d c d f g a b c e b e 0 20 40 60 80 100 camu camu lucuma mesquite graviola mashua tocosh maca yacon purple maize amaranth cat's claw l* ital. j. food sci., vol. 31, 2019 737 orange; cat's claw, mesquite and yacon, orange; graviola, green-brown; camu camu, yellow-brown; tocosh, white; amaranth, cream-white; lucuma, ocher. the tocosh powder was the brightest (l*: 86.6), followed by most maca samples (76.2-83.9) and amaranth (78.5-80.3). one maca (maca 4) had an l* of 72.0, lower than the other maca samples. camu camu had a l* like the lyophilized samples (60.45±2.78) and higher than the spouted bed dried samples (36.6-40.8) described by fujita et al. (2013). overall, mashua presented the lowest brightness (28.3), followed by graviola (43.3-41.0). cat’s claw and purple corn scored the highest a* red component values (12.1-13.5 and 10.8, respectively), while tocosh presented the lowest (0.5). the variation among the different maca samples was quite limited, ranging from 1.2 (maca 7) to 5.1 (maca 4). mesquite presented the highest b* yellow component (34.1), followed by cat’s claw (on average 31.6), five maca samples (22.2-26.2) and graviola (on average, 25.6); maca 1 and maca 4 had values different from the other maca (28.3-30.2). purple corn presented the lowest b* value, hence the major blue component (2.8), followed by mashua (5.3) and tocosh (9.7). the differences observed between maca samples may be due either to the different treatments utilized for their preparation (onwude et al., 2017) or to cultivars with different chromatic characteristics. no information or comparisons for the color components are available in literature. 3.2. total polyphenol content supplementary table 3 reports the results of tpc, performed on the etoh:h2o and meoh:h2o:acet extracts, as well as the results of the lsd test comparing the products. the great heterogeneity of the samples led to anovas (not shown) always with significant differences. the average values, obtained by grouping the samples according to the species, are depicted in fig. 2. figure 2. total polyphenol content (tpc) of the ethanol 80% (etoh:h2o) and methanol:h2o:acetic acid (meoh:h2o:acet) extracts of powdered products from 11 species. different letters indicate significant differences (p ≤0.05) among species. a g ef cd bc i g f de h b 0 10 20 30 40 50 60 70 80 90 tpc (mg gae/kg dm) meoh:h2o:acet a e d c bc g e d c f b 0 10 20 30 40 50 60 70 80 90 camu camu lucuma mesquite graviola mashua tocosh maca yacon purple maize amaranth cat's claw tpc (mg gae/kg dm) etoh:h2o ital. j. food sci., vol. 31, 2019 738 etoh:h2o showed a lower tpc extraction capacity than meoh:h2o:acet, but the information provided was similar, as demonstrated by their very high linear coefficient of correlation (r=0.98). tpc was maximum for camu camu (38.3 and 76.4 g gae/kg dm, respectively), followed by cat's claw 2 (24.2 and 53.6 g gae/kg dm, respectively); the lowest tpcs were recorded in tocosh (2.5 and 2.8 g gae/kg dm), amaranth (on average, 3.1 and 2.8 g gae/kg dm), lucuma (2.8 and 7.5 g gae/kg dm) and six maca samples (on average, 3.6 and 6.7 g gae/kg dm). the values generally fell within the range of variation reported in the literature for camu camu (fujita et al., 2013), cat’s claw (berlowski et al., 2013; galvez ranilla et al., 2010), amaranth (repo-carrascovalencia et al., 2010), lucuma (fuentealba et al., 2016), mesquite (cardozo et al., 2010), maca (galvez ranilla et al., 2010; campos et al., 2013), mashua (chirinos et al., 2007; chirinos et al., 2013), yacon (campos et al., 2013), but were lower than those described for graviola frozen pulp (zielinski et al., 2014). for peruvian purple maize the information available on tpc is reported in chlorogenic acid equivalent and is not directly comparable to our results, while for tocosh no similar information was found in literature. the folin-ciocalteu method sometimes overstates total phenolics content because other compounds, including reducing sugars (e.g. glucose and fructose), may interfere with the results; however, in this research the powders with the highest sugars content (yacon, mashua, lucuma and maca), generally have low tpc; conversely, the two highest tpc values were from camu camu and cat’s claw, which showed very low sugars content. 3.3. antioxidant capacity the antioxidant capacity of the samples, assessed by the abts, frap and dpph tests carried out on the etoh:h2o and meoh:h2o:acet extracts are shown in supplementary table 3, along with the results of the lsd test. the great heterogeneity among samples led to anovas (not presented) always indicating significant differences, as previously remarked for color and total polyphenols content. the average antioxidant capacities obtained by grouping the samples according to the type of product are presented in fig. 3. the abts, frap and dpph tests give similar and highly correlated results (r between 0.98 and 1.00 for both etoh:h2o and meoh:h2o:acet extracts). a higher antioxidant capacity was observed in the meoh extracts, the exceptions being amaranth (for all three methods), maca and tocosh (abts), graviola tea and maca 5 (dpph). camu camu, which had the highest tpc concentration (fig. 2) but also an outstanding vitamin c content (fujita et al., 2013), showed the highest antioxidant capacity (fig. 3), followed by cat’s claw, graviola, mashua, purple maize and mesquite. on the other hand, tocosh, amaranth, yacon, maca and lucuma had low antioxidant activities, like that of wheat (yilmaz et al., 2015). comparable results were reported for camu camu (dpph: 153-185 mmol te/g fw; chirinos et al., 2010), cat’s claw (abts: 513 mmol te/kg dm; berlowski et al., 2013), graviola (abts: about 200 mmol te/kg dm; berlowski et al., 2013), mashua (abts: 24.3-247.7 mmol te/g dm; dpph: 23.2-157.1 mmol te/g dm; chirinos et al., 2013), mesquite (abts: 57.0-61.6 μmol te/g dm; cardozo et al., 2010), yacon (abts 23-136 mmol te/g dm; campos et al., 2012), purple maize (dpph: 23.1 mmol te/g dm; cevallos-casals and cisneros-cevallos, 2003), lucuma (abts: 5.6-304.6 mmol te/g dm; dpph: 0.7-132.9 mmol te/g dm; fuentealba et al., 2016) and amaranth (abts: 3.7 mmol te/g dm; dpph: 1.2 mmol te/g dm; chirinos et al., 2013). on the other hand, those of maca were slightly lower than the levels (abts: 67 mmol te/g dm) observed by fuentealba et al. (2016). ital. j. food sci., vol. 31, 2019 739 figure 3. antioxidant capacity (abts, frap and dpph tests,)of the ethanol 80% (etoh:h2o) and methanol:h2o:acetic acid (meoh:h2o:acet) extracts of powdered products from 11 species. different letters indicate significant differences (p ≤0.05) among species. 3.4. sugars content the anova (not presented) showed the existence of significant differences for sugars content among samples. the average values and the results of the lsd test for the different sugars are reported in supplementary table 2. the reducing sugars results obtained grouping the samples by species are presented in fig. 4. a e d c cd h f ef d g b 0 400 800 1200 1600 2000 camu camu lucuma mesquite graviola mashua tocosh maca yacon purple maize amaranth cat's claw abts (mmol te/kg dm) etoh:h2o a e d c bc g e e cd f b 0 400 800 1200 1600 2000 abts (mmol te/kg dm) meoh:h2o:acet a e d c bc g e d c f b 0 400 800 1200 1600 2000 camu camu lucuma mesquite graviola mashua tocosh maca yacon purple maize amaranth cat's claw frap (mmol te/kg dm) a * g ef cd bc i g f de h b 0 400 800 1200 1600 2000 frap (mmol te/kg dm) a e cde abc abc g e cde bcd f ab 0 100 200 300 400 camu camu lucuma mesquite graviola mashua tocosh maca yacon purple maize amaranth cat's claw dpph (mmol te/kg dm) a f de d bc h g e cd h b 0 100 200 300 400 dpph (mmol te/kg dm) ital. j. food sci., vol. 31, 2019 740 figure 4. reducing sugars (fructose, glucose and maltose) and heat damage indices (furosine; hydroxymethylfurfural, hmf; glycosylisomaltol, gli) of powder products from 11 species. different letters indicate significant differences (p ≤0.05) among species. fructose and glucose were detected in all samples, and were particularly abundant in yacon, mashua and lucuma; maltose was detected only in cat’s claw, most maca samples, lucuma and graviola. overall, yacon (40.9 g/100 g dm), mashua (34.4 g/100 g dm) and lucuma (21.2 g/100 g dm) showed the highest content of reducing sugars, which, on the other hand, were almost absent in tocosh (0.11 g/100 g dm) and amaranth (0.30 g/100 g dm). sucrose (a non-reducing sugar, but a possible source of monosaccharides) was present in moderate quantities in mesquite, maca, mashua, lucuma and yacon (42.5, 25.9, 16.4, 8.30, 8.20 g/100 g dm, respectively) and was very scarce in all the other products. the presence of reducing sugars is important, because they are one of the basic reactants involved in the formation of amadori products during the maillard reaction, when exposed to high temperatures (e.g. oven drying, cooking, baking): therefore, higher reducing sugars concentrations forebode higher heat damage during products manufacturing. among the plants tested, yacon is a well-known source of fructo-oligosaccharides (campos et al., 2012) and our results are confirmed by the observations (49.2 g/100 g) of scher et al. (2009). the reducing sugars content found in mashua is de a c c a f bc b de e de 0 500 1000 1500 2000 2500 3000 camu camu lucuma mesquite graviola mashua tocosh maca yacon purple maize amaranth cat's claw furosine (mg/100 g protein) f bcd bcd de bc de bcd a f e cd 0 20 40 60 80 100 120 140 160 hmf (mg/kg dm) 0 2 4 6 8 10 12 gli (mg/kg dm) bc a bc bc a e b a cd d c 0 5 10 15 20 25 30 35 camu… lucuma mesquite graviola mashua tocosh maca yacon purple… amaranth cat's claw fructose (g/100 g) bcd a cd de a f c ab de ef cd 0 5 10 15 20 25 30 35 glucose (g/100 g) ​ ab ​ c ​ ​ a ​ ​ ​ bc 0 5 10 15 20 25 30 35 maltose (g/100 g) ital. j. food sci., vol. 31, 2019 741 analogous to the quantity (6.4-45.3 g/100 g dm, average 28.4 g/100 g dm) reported by guevara-freire et al. (2018), while those of maca are slightly lower than the value (13.10±0.17 g/100 g dm) described by rondán-sanabria and finardi-filho (2009), and that of mesquite is slightly inferior to the data (3.17-3.74 g/100 g dm) reported by cardozo et al. (2010) for different prosopis spp. for fructose and glucose content our lucuma results are within the broad range of variation (1.28-12.71 and 2.48-17.37 g/100 g dm) reported by fuentealba et al. (2016), and the amaranth ones are very similar to those (0.12 and 0.34 g/100 g dm) presented by gamel et al. (2006). 3.5. heat damage the anova (not presented) showed the existence of significant differences for heat damage among the samples. the average values and the results of the lsd test for heat damage indices, i.e. furosine, gli and hmf, are reported in supplementary table 2. the results obtained grouping the samples by species are reported in fig. 4. non-enzymatic browning in dried products may be influenced by water activity, drying temperature, ph and chemical composition of foods (sagar and suresh kumar, 2010). furosine is an index of the first steps of maillard reaction, while gli and hmf are markers of intermediate phases; gli is formed by the heating of maltose and aminoacids (especially glutamine), while hmf is created not only by degradation of amadori compounds but also of sugars. furosine content was very high in mashua and lucuma (>2000 mg/100 g protein), high in yacon and most maca samples and low in camu camu, amaranth, purple maize as well as in two cat’s claw samples. maca 6 and maca 7 had significantly lower furosine content than the other maca samples. hmf was high only in yacon, but was detected, at lower levels, in several other samples, while gli was found only in maca 3, maca 6 and cat’s claw 1. no maillard reactions developed in tocosh (a characteristic food, obtained by natural bacterial fermentation of straw-wrapped potatoes kept in running water for several months) as furosine and gli were lower than the detection limit and hmf was very low, while camu camu, purple maize, amaranths, and cat’s claw tea had limited heat damage (low furosine levels and generally below-detection gli and hmf). furfural, an indicator of more advanced maillard reaction stages mainly produced by pentose degradation or thermal degradation of hmf during caramelization, was absent in all samples, even if the method used for gli and hmf analysis is able to determine its presence. since water activity values for all the samples were very similar, ranging between 0.492 and 0.585, and no correlation between heat damage indices and protein content (table 1) exists, the development of the maillard reaction seems completely attributable to processing conditions and reducing sugar concentration. the lofty heat damage of yacon (981.3 mg/100 g protein of furosine and 146.6 mg/kg dm of hmf) is justified by its high fructose and glucose content and by the strong thermal treatment needed to inactivate its highly thermostable polyphenol oxidase enzyme (neves da silva, 2007) for a luminous color (fig. 1). these conditions lead to the degradation of the amadori compounds and to the formation of intermediate compounds (i.e. hmf). the furosine levels of the other samples correlate well to the concentration of reducing sugars (r = 0.93). thus, mashua, lucuma and most of maca powders with relevant content of reducing sugars have high furosine levels while samples with low reducing sugars content present limited furosine. however, furosine alone is not suitable to completely describe the heat damage of all powder products. the presence of hmf (0.9-11.8 mg/kg dm) in most samples points to ital. j. food sci., vol. 31, 2019 742 an intermediate development of maillard reaction. the samples with detectable gli (maca 3, maca 6 and cat’s claw 1) showed the highest maltose levels but relatively low reducing sugar concentrations (4.0-7.7 g/100 g dm). the simultaneous formation of hmf suggests higher-than-the-average processing temperatures for these samples. a similar hypothesis can be made for some samples with very low reducing sugar concentrations (0.2-2.5 g/100 g dm) and significant hmf content (1.3-6.0 mg/kg dm). to the best of our knowledge, no information on heat damage in this type of powder products is available. with reference to other vegetables, furosine contents of 14-262 mg/100 g protein (rufián-henares et al., 2013) and of 457-1172 mg/100 g protein (bignardi et al., 2016) are reported in sweet pepper and in dried red chili pepper, respectively; similarly, ríos-ríos et al. (2018) describe furosine concentrations of 46.6110.1 mg/100 g protein in black garlic powder, while hmf levels of 1.3-9.5 mg/kg dm are recorded by soria et al. (2009) in carrots dried under different conditions. 3.6. principal components analysis fig. 5 depicts the biplots of scores and loadings obtained by the principal components analysis (pca) performed considering all samples and parameters. pc1 and pc2 describes 46% and 16% of variation (fig. 5a), while pc3 and pc4 10% and 9% (fig. 5b); therefore, the initial four pc explain 81% of total variation. the pca unmistakably separates the different species. pc1, characterized by antioxidant properties, differentiates camu camu and cat’s claw along the left side, while pc2, mainly related to heat damage, positions mashua, yacon and lucuma in the upper side (high furosine, hmf, glucose and fructose contents), and maca 3, maca 6, amaranth and tocosh in the bottom side (high l*, gli, maltose and protein). pc3 further splits purple maize, graviola, amaranth and tocosh, from mesquite and maca samples, while pc4 divides mashua (top of the plot, characterized by high protein and furosine contents) from yacon (bottom, high hmf and l*); maca 3 sits alone in a spot defined by high gli content. ital. j. food sci., vol. 31, 2019 743 figure 5. bi-plot of scores and loads for the first four principal components (pc1 vs. pc2, a; pc3 vs. pc4, b) of the principal component analysis carried out on colour coordinates (l*, a*, b*), protein content, total polyphenol content (tpc) and antioxidant capacity (abts, frap and dpph tests) of the ethanol:h2o (et) and methanol:h2o:acetic acid (me) extracts, reducing sugars, furosine, hydroxymethylfurfural (hmf), and glycosylisomaltol (gli) of 21 powder products. 4. conclusions our results show that, for the traits analysed, camu camu and cat’s claw powders are excellent products, because they have high levels of total polyphenols and antioxidant capacity together with low heat damage. other interesting products are the powders of graviola, purple maize and mesquite, while the high antioxidant properties of mashua are coupled to severe heat damage. for an effective use in the food industry it will be necessary to evaluate the stability of the antioxidant capacity of the powders during the manufacturing process and the digestion of innovative high-nutritional-value foods, as well as to assess the sensorial quality of the end products. supplementary table 1. beneficial properties of the species analyzed, and bibliographic references. species putative properties references cat’s claw immune-modulatory, antioxidant, antiviral, antibacterial and anti-inflammatory steinberg, 1995; heitzman et al., 2005 amaranth balanced aminoacid composition repo-carrasco et al., 2010; bressani et al., 1993 purple maize anti-inflammatory, prevention of obesity, diabetes, hyperglycemia and hypertension tsuda et al., 2003; finkel et al., 2013 yacon low glycemic index, resistance to infections and allergic reactions, prevention of colon cancer delgado et al., 2013 ; de moura et al., 2012 maca sexual dysfunctions, menopausal symptoms, osteoporosis, benign prostatic hyperplasia, lipid and glucose metabolism, skin protection from uv gonzales et al., 2005; gonzales-castañeda and gonzales, 2008; rubio et al., 2011; vecera et al., 2007 ; zhang et al., 2006 ital. j. food sci., vol. 31, 2019 744 tocosh antimicrobial lopez campos, 2017 mashua anticancer noratto et al., 2004 graviola anti-inflammatory and anti-malarial, prevention of some cancers foong and hamid, 2012; mishra et al., 2013; yang et al., 2015 mesquite antidiabetic, anti-inflammatory, anticancer and antimicrobial henciya et al., 2017 lucuma antioxidant, antihyperglycemic fuentealba et al., 2016 camu camu antioxidant, anti-inflammatory, antimicrobial and anti-diabetic akter et al., 2011; fujita et al., 2015 akter, m., oh, s., eun, j., and ahmed, m. 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(2010). flavonoids and other phenolic compounds in andean indigenous grains: quinoa (chenopodium quinoa), kañiwa (chenopodium pallidicaule) and kiwicha (amaranthus caudatus). food chemistry 120:128-133. rubio, j., qiong, w., and liu, x. (2011). aqueous extract of black maca (lepidium meyenii) on memory impairment induced by ovariectomy in mice. evidence-based complementary and alternative medicine, 7. steinberg, p.n. (1995). cat's claw: an herb from the peruvian amazon. sidahora: un proyecto del departamento de publicaciones del pwa coalition, ny, usa. pp. 35. tsuda, t., horio, f., uchida, k., aoki, h., and osawa, t. (2003). dietary cyanidin 3o-beta-d-glucoside-rich purple corn color prevents obesity and ameliorates hyperglycemia in mice. journal of nutrition 133:2125-2130. vecera, r., orolin, j., and skottová, n. (2007). the influence of maca (lepidium meyenii) on antioxidant status, lipid and glucose metabolism in rat. plant foods in human nutrition, 62:59-63. yang, c., gundala, s. r., mukkavilli, r., vangala, s., reid, m. d., and aneja, r. (2015). synergistic interactions among flavonoids and acetogenins in graviola (annona muricata) leaves confer protection against prostate cancer. carcinogenesis, 36:656-665. zhang, y., yu, l., and ao, m. (2006). effect of ethanol extract of lepidium meyenii walp. on osteoporosis in ovariectomized rat. journal of ethnopharmacology, 105:274-279. ital. j. food sci., vol. 31, 2019 745 supplementary table 2. mean±standard error and lds results for colour coordinates (l*, a*, b*), sugars content (fructose, glucose, maltose and sucrose, g/100 g dm) and heat damage indices (furosine, mg/100 g protein; hydroxymethylfurfural, hmf, mg/kg dm; and glycosylisomaltol, gli, mg/kg dm) of 21 commercial powder samples. l* a* b* fructose glucose maltose saccharose furosine hmf gli cat’s claw 1 66.3l±0.4 13.2ab±0.2 31.1c±0.3 0.7l±0.003 1.1il±0.02 2.2b±0.08 0.9n±0.01 ndp 11.8b±0.7 1.2c±0.03 cat’s claw 2 57.7o±0.4 12.1b±0.1 30.3d±0.1 1.0h±0.01 1.2i±0.07 0.1g±0.01 1.0l±0.03 324.5j±20.4 ndl ndd cat’s claw tea 50.2q±0.5 13.5a±0.2 32.8b±0.2 0.8i±0.03 1.1il±0.09 0.1h±0.01 0.9n±0.01 88.6l±0.6 0.9j±0.02 ndd amaranth fr 80.3c±0.3 2.2l±0.0 19.6m±0.1 0.1q±0.002 0.1r±0.002 ndi 1.8h±0.03 34.9n±1.1 3.1h±0.2 ndd amaranth fs 78.5ef±0.2 1.9m±0.0 19.3m±0.2 0.2p±0.01 0.2q±0.005 ndi 1.9h±0.02 29.0o±0.2 ndk ndd purple maize 55.3p±0.3 10.8c±0.1 2.8o±0.1 0.5n±0.01 0.6o±0.01 ndi 1.5i±0.02 79.5l±2.0 ndk ndd yacon 74.9h±0.5 3.9g±0.2 26.8f±0.5 32.7a±2.09 8.2c±0.04 ndi 8.2g±0.13 981.3cd±1.9 146.6a±0.1 ndd maca 1 77.8f±0.2 2.0m±0.1 28.3e±0.1 5.4d±0.01 5.8e±0.2 0.5e±0.01 28.8c±0.35 825.3e±6.8 3.3h±0.1 ndd maca 2 76.2g±0.3 3.8g±0.1 24.6h±0.2 4.2e±0.19 3.6g±0.15 0.5ef±0.03 35.4b±0.43 971.9cd±8.2 5.5de±0.8 ndd maca 3 78.4ef±0.2 4.0g±0.1 26.2fg±0.3 0.2o±0.01 0.7n±0.01 4.2a±0.04 21.2e±0.25 728.5f±17.5 4.4fg±0.2 27.3a±0.3 maca 4 72.0i±0.2 5.1f±0.1 30.2d±0.3 5.3d±0.06 4.6f±0.15 0.4f±0.05 24.4d±0.96 877.7de±20.8 10.4b±0.3 ndd maca 5 83.9b±0.5 1.9m±0.0 22.2l±0.2 5.6d±0.04 7.0d±0.20 ndi 26.4cd±0.47 1008.6c±0.0 4.3fg±0.03 ndd maca 6 79.4d±0.3 2.8i±0.1 23.5i±0.2 5.4d±0.002 0.5p±0.02 1.8c±0.01 27.8c±0.16 378.0i±6.0 3.0h±0.2 8.9b±0.4 maca 7 78.7de±0.1 1.2n±0.0 24.8h±0.1 0.6m±0.005 0.5o±0.004 ndi 17.1f±0.14 167.7k±3.7 5.0ef±0.07 ndd tocosh 86.6a±0.7 0.5o±0.1 9.7n±0.2 0.01r±0.002 0.1s±0.004 ndi 0.1o±0.003 ndp ndk ndd mashua 28.3t±0.4 7.7d±0.1 5.3o±0.0 17.5b±0.27 16.9a±0.25 ndi 16.4f±0.05 2399.8a±107.6 7.5c±0.1 ndd graviola 43.3r±0.3 3.1h±0.0 26.1g±0.1 nds 0.6o±0.03 0.1g±0.001 0.9n±0.03 445.6h±23.4 1.3i±0.02 ndd graviola tea 41.0s±0.3 2.0lm±0.0 25.1h±0.1 1.3g±0.08 1.0l±0.05 0.1h±0.01 1.0lm±0.07 608.2g±6.0 1.3i±0.02 ndd mesquite 63.4n±0.4 8.1d±0.2 34.1a±0.2 1.6f±0.04 0.9m±0.02 ndi 42.5a±0.1 450.5h±0.1 6.0d±0.6 ndd lucuma 72.3i±0.2 8.0d±0.1 22.2l±0.2 10.3c±0.1 9.6b±0.02 1.3d±0.03 8.3g±0.02 2228.4b±334.5 4.1g±0.2 ndd camu camu 64.6m±0.3 6.6e±0.1 28.0e±0.1 1.4g±0.02 1.8h±0.19 ndi 0.9mn±0.04 48.2m±0.7 ndl ndd for each parameter, diverse letters indicate significant differences (lsd, p≤0.05) among samples; nd, not detectable. ital. j. food sci., vol. 31, 2019 746 supplementary table 3. mean and lds results for total polyphenols content (tpc; mg gae/kg dm) and abts, frap and dpph antioxidant capacity (mmol te/kg dm) of 21 powder samples extracted with ethanol 80% (etoh:h2o) and methanol:h2o:acetic acid (meoh:h2o:acet). tpc abts frap dpph etoh:h2o meoh:h2o:acet etoh:h2o meoh:h2o:acet etoh:h2o meoh:h2o:acet etoh:h2o meoh:h2o:acet cat’s claw 1 16.1c±0.5 30.7c±0.0 267.4c±0.0 331.5c±6.1 229.9c±4.2 391.9c±10.0 26.2c±0.1 43.4c±0.04 cat’s claw 2 24.2b±0.4 53.6b±0.8 590.9b±21.0 802.6b±10.6 460.7b±52.8 872.8b±2.3 49.6b±0.2 125.9b±1.1 cat’s claw tea 9.2f±0.1 20.1e±0.6 135.6f±0.2 280.7d±14.1 110.0ef±4.0 255.1e±2.6 9.9e±0.02 32.3e±0.3 amaranth fr 3.5m±0.0 2.9q±0.0 5.7o±0.0 4.9o±0.0 5.1n±0.1 4.6s±0.0 0.2q±0.0 nd amaranth fs 2.6o±0.0 2.6r±0.0 5.5o±0.0 4.6o±0.0 5.2n±0.1 3.3t±0.0 nd nd purple maize 7.8g±0.3 13.7g±0.1 103.1g±0.2 148.8g±0.3 97.3f±0.8 200.1g±3.1 5.8h±0.1 21.1f±0.3 yacon 4.8i±0.1 11.7h±0.1 31.6l±0.3 34.5i±1.7 42.2g±1.2 65.2i±0.7 3.8i±0.01 7.1j±0.1 maca 1 3.5m±0.3 7.8mn±0.0 31.4l±0.4 26.0m±0.7 13.9m±0.3 24.3r±0.7 0.8o±0.002 0.8n±0.001 maca 2 3.4m±0.1 9.2i±0.1 32.8l±0.0 31.7il±0.7 20.5l±0.3 30.1p±0.1 0.9n±0.002 1.5m±0.001 maca 3 3.4m±0.1 5.9o±0.1 31.6l±0.7 26.1m±0.7 23.6i±0.4 35.6n±1,1 1.5k±0.003 1.5m±0.004 maca 4 4.0l±0.2 7.8lm±0.1 48.0i±1.5 33.4il±0.1 29.3h±0.7 49.3l±1.4 1.6j±0.01 1.5m±0.004 maca 5 3.0n±0.1 6.0o±0.2 32.2l±0.5 31.2l±2.0 14.3m±0.2 26.1q±0.1 1.0m±0.002 0.9n±0.001 maca 6 10.4de±0.2 8.1l±0.0 28.7m±0.5 25.8m±0.8 24.6i±0.2 38.4m±0.3 1.1l±0.02 1.6l±0.0 maca 7 4.2l±0.1 3.9p±0.1 20.2n±0.6 13.3n±0.1 18.2l±0.4 32.7o±0.8 0.4p±0.0 0.8n±0.0 tocosh 2.5o±0.0 2.8q±0.0 2.1p±0.0 1.0p±0.0 nd nd nd nd mashua 10.0ef±0.2 21.4d±0.5 129.9f±1.2 264.2de±10.1 152.3d±2.1 410.6c±3.9 8.7g±0.02 36.6d±0.01 graviola 11.2d±0.5 19.7e±0.5 155.3e±3.5 198.1f±1.0 116.3e±10.2 215.3f±3.5 9.0f±0.1 19.9g±0.1 graviola tea 14.8c±0.7 21.0d±0.1 209.9d±1.1 253.8e±1.0 155.2d±5.1 327.6d±1.4 17.6d±0.02 16.6h±0.03 mesquite 6.8h±0.1 15.3f±0.2 51.0h±0.7 95.3h±0.1 47.7g±0.3 115.0h±0.2 3.8i±0.04 13.4i±0.02 lucuma 2.8no±0.0 7.5n±0.0 19.5n±0.4 31.5il±1.5 18.2l±0.2 33.5o±0.1 0.9n±0.001 3.5k±0.03 camu camu 38.3a±0.1 76.4a±0.5 868.6a±16.8 1473.5a±90.3 996.0a±97.8 1786.5a±60.9 75.7a±0.4 184.33a±1.1 for each parameter, diverse letters indicate significant differences (lsd, p≤0.05) among samples; 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zielinski a.a.f., ávila s., ito v., nogueira a., wosiacki g. and haminiuk c.w.i. 2014. the association between chromaticity, phenolics, carotenoids, and in vitro antioxidant activity of frozen fruit pulp in brazil: an application of chemometrics. j. food sci. 79:c510-c516. paper received february 8, 2019 accepted may 9, 2019 ijfs#1572_bozza ital. j. food sci., vol. 32, 2020 181 paper effect of different illumination sources on colour and oxidative stability of seasoned coppa di parma pgi g. minelli*a,b, d.p. lo fiegoa,b, p. macchionic and p. favaa,b adepartment of life sciences, university of modena e reggio emilia, via amendola 2, 42122 reggio emilia, italy binterdepartmental research centre for agri-food biological resources improvement and valorisation (biogest-siteia), university of modena and reggio emilia, via amendola 2, 42122 reggio emilia, italy cdepartment of agricultural and food sciences (distal), university of bologna, viale fanin 44, 40127 bologna, italy *corresponding author: tel.: +39(0)522522085; fax: +39(0)522522027 email: giovanna.minelli@unimore.it abstract the influence of different lighting durations, lamps and modified atmosphere packaging (map) on the colour and oxidative stability of lipids was studied in coppa di parma pgi. the samples were stored (4°c) in darkness or lighted by uv-free lamps. in trials 1 and 2, the samples were lighted 24 and 12 h/day, respectively, and were packaged in air. in trial 3, samples were packaged in ma (70% n2/30% co2) and lighted 12 h/day. in air, illumination reduced oxidative stability, redness, colour saturation and increased the hue angle. in map, the lighting conditions did not affect colour and oxidative stability. during storage the lipid oxidation increased. overall, light negatively affected the studied parameters. keywords: colour, cured salami, lighting conditions, lipid stability, modified atmosphere packaging ital. j. food sci., vol. 32, 2020 182 1. introduction the colour of meat plays a pivotal role in determining the decision of the consumer to buy a particular product. indeed, it is perceived as relating to the freshness, integrity and quality of the food. thus, maintaining an attractive colour during storage is a key of success in the selling of meat. discolouration in retail-fresh meats during display is ascribable to muscle pigment oxidation (oxymyoglobin to metmyoglobin). this is influenced by oxygen concentration, ph and secondary products of lipids peroxidation (papuc et al., 2017). in case of nitrites addition, as in the processing of coppa di parma pgi (protected geographical indication), nitrosylmyoglobin formation occurs; this is an unstable pigment that, in presence of oxygen in cured meat, is oxidized to brownish nitrosylmetmyoglobin nommb (fox, 1966): this evidence was confirmed in scientific literature, in which the discolouration of nitrosylmyoglobin has been linked to the combination of the presence of both oxygen in the headspace surrounding the product and light exposure during the display life (møller and skibsted, 2002; zanardi et al., 2002). indeed, lipid oxidation, which takes place in intramuscular fat and/or membrane phospholipids, besides causing an unpleasant odour and flavour, brings about the loss of desirable colour, thereby reducing display life (buckley et al., 1995; papuc et al., 2017; ruiz et al., 1999) and has deleterious effects on the organoleptic properties of meat and meat products as well as the digestibility of main nutrients (garcía-lomillo et al., 2017; patrakova and gurinovich, 2015). therefore, preventing or delaying both pigment and lipid oxidation enables the duration in which the meat maintains its bright-red colour to be extended. the oxidation of both lipids and pigments in meats are strictly related to similar processes (faustman et al., 1989; papuc et al., 2017) and, in this regard, light may play a critical role. in fact, oxidative reactions can be initiated also by physical factors such as radiation and light (amaral et al., 2018). the effect of light on lipid oxidation has been shown in various foods, such as oils, butter, milk and meat (aurand et al., 1977; chahine and deman, 1971; kiritsakis and dugan, 1985; luby et al., 1986; whang and peng, 1988); uv-light is more effective than visible light in inducing oxidation of lipids and pigments (andersen and skibsted, 1991; bertelsen and boegh-soerensen, 1986; zhu and brewer, 1998). although the amount of radiation below 400 nm is small in fluorescent lamps used in display cabinets, it must be taken into account due to its deleterious effects on the display-life of meat (djenane et al., 2001). meat products on retail shelves are provided in transparent packaging and with residual oxygen inside the packages; in association with the cabinet display light, these can cause discolouration of the packaged meat products (gibis and rieblinger, 2011; mcmillin, 2008). to overcome this problem, in the last few decades the use of modified atmosphere packaging (map) for meat and sliced meat products is increasingly widespread as a tool to extend their shelf-life. however, the effect of lighting on the shelf-life of cured seasoned pork products has not been widely investigated. in particular, the coppa di parma pgi has never been studied under this profile. coppa di parma pgi is obtained from subjects of at least nine months of age weighing approximately 160 kg. after hand-salting, the muscular portion of the neck adhering to the cervical and the first two thoracic vertebrae is placed in a closed-ended beef gut casing and hand-tied with string. after two/three months of curing, coppa is marketable. the product, cylindrical in shape, is 25-40 cm long and weighs at least 1.3 kg. the aim of this work was to study the effects of display under different lighting times and lamps on the colour parameters and oxidative stability of coppa di parma pgi packaged in air as well as in a modified atmosphere. ital. j. food sci., vol. 32, 2020 183 2. materials and methods for the purposes of the study, a total of 18 coppa di parma pgi, obtained from a local retailer, were used. three distinct trials were carried out. for each trial, three replications were performed. in each replication, 2 different coppa were sampled and sliced. in the first two trials, the slices of coppa were air-packaged in trays made of a pet/evoh/pe structure (oxygen permeability < 0.5 cm3 m-2 24h-1 bar-1), lidded with a pet/pe film (oxygen permeability 80 cm3 m-2 24h-1 bar-1). in the third trial, sliced coppa was packaged with the same material (bottom and top) in a modified atmosphere (nominally 70/30 n2/co2), using caveco equipment and working with the technique of vacuum compensation. the initial and final atmosphere composition was determined by a handheld gas analyzer checkpoint (dansensor, denmark). in each replication, 26 trays were filled with 10 slices each of coppa. twenty-four trays were put in refrigerators at 4±1°c. six trays were maintained in the dark, the other 18 were put into another refrigerator, divided into 3 sections separated by horizontal black screens. each section (estimated area of 0.33 m2) was illuminated with a specific lamp, whose characteristics are summarized in table 1. table 1. characteristics of the different lamps used. lamp identifier (code) colour temperature °k colour rendering wattage (w) luminous flux (lm) illuminance* (lx) 640 basic cool white (cw) 4000 62 18 1200 3640 827 lumilux interna warm white (ww) 2700 80 18 1350 4090 76 natura neutral white (nw) 3500 75 18 750 2270 *the illuminance (lx) values have been calculated simply dividing the nominal luminous flux (lm) of each lamp by the exposed area (0.33 m2) of the fridge shelves. the distance between the lamp and the samples was about 40 cm. the samples contained in the remaining 2 trays were immediately assigned to the analyses (described below). the samples were exposed either to continuous lighting (24/24 hours) (trial 1), or to a 12 hours on/12 hours off light cycle (trials 2 and 3). eight trays (two for each lighting condition) were removed from the refrigerators after 24, 48, and 120 hours and then submitted to the analytical determinations. on each coppa, the lipid content was determined according to a.o.a.c. (1995) and the average lipid content was 29.3±5.5. lipid oxidation was measured by the 2-thiobarbituric acid reactive substances (tbars) method (siu and draeper, 1978). each sample was minced, and an aliquot of 2.5 g was homogenised in 12.5ml of distilled water at 9500 rpm for 2 min, using an ultra turrax tissue homogenizer and then vortexed for 1 min at high speed. samples were centrifuged for 20 min at 2000 rpm at 4°c with 12.5 ml of 10% trichloroacetic acid (tca) and the supernatant decanted through a paper filter (whatman 541). four ml of clear filtrate were transferred into 15 ml pyrex screw cap test tubes and 1 ml of 0.06m 2-thiobarbituric acid (tba) was added. a distilled water-tca-tba reagent ital. j. food sci., vol. 32, 2020 184 blank was prepared and treated in the same way as the samples. the samples were heated in a water bath at 80°c for 90 min and then cooled. absorbance was measured at 532 nm in spectrophotometer (jasco model v550, uv/vis, tokyo, japan). results were expressed as mg of malondialdehyde (mda)/kg. the surface colour of coppa slices was determined using a cm-600d spectrophotometer (konica minolta holdings, inc., osaka, japan) with a window diameter of 8 mm and d65 as the source of illumination. before colour measuring, carried out on both lean and fat tissue, the spectrophotometer was calibrated against a white plate supplied by the manufacturer. colour measurements complied with the cie colour convention (cie, 1986), where the three fundamental outputs are l*“lightness”, a*“redness”, b* “yellowness” values. chroma (c*), also referred to as saturation index and colour intensity, was calculated as: [(a*2+b*2)] 0.5 and hue angle (h*) calculated as follows: tan-1 (b*/a*). overall colour change (∆e) was calculated as [(∆l*2 + ∆a*2 + ∆b*2) 0.5] where ∆l*, ∆a* and ∆b* are the difference between time 0 and the values l*, a* and b*, respectively, at 24 (∆e1), 48 (∆e2) and 120 (∆e3) hours. the average values for both fat and lean were the mean of 25 determinations in each fraction. the data were submitted for analysis of variance, with the lamps and exposure times as independent variables (sas, 1996). in addition, interaction effect between exposure time and lamp was evaluated; this was statistically significant for none of the examined parameters (p>0.05) and was thus removed from the model. 3. results and discussion 3.1. trial 1 in this trial, the samples were packaged in air and illuminated 24h/d (table 2). l* values did not vary with lighting conditions, either in the lean fraction or in the fat. however, the values of most of the other colour parameters differed between the samples kept in the dark and those exposed to the lamps. in fact, in the lean component, light exposure led to a significant reduction of both a* (p<0.01) and c* (p<0.05) values and an increase in the h* value (p<0.01), whereas the b* value was unaffected. very similar trends were reported for fresh pork studied by zhu and brewer (1998). cierach and niedźwiedź (2014) reported a decrease of 4-7 units of a* values in semitendinosus muscle of beef after few days of light exposure using a lamp of 3000 k and 3 lightning intensity (500, 1000 and 1500 lux), demonstrating that intensity of 500 lx has less influence on the beef colour changes. in the scientific literature on this topic, light intensity has been chosen as a parameter to be standardized, by adjusting the lamp to product distance (böhner et al., 2014; böhner and rieblinger, 2016; haile et al., 2013; sørheim et al., 2017). in our work a fixed lamp to product distance has been chosen, in order to simulate the actual lighting condition of packaged coppa in the retail display. it is evident that, despite the lighting intensity values, the simultaneous presence of oxygen (20.9%) and the continuous lighting over 120 hours deeply affect the colour of the product, without no appreciable difference among the three lamps used. display time, too, had no effect on the l* value, as reported by other authors (cierach and niedźwiedź, 2014); however, all the other colour characteristics in the lean fraction changed significantly during the 120 hours display period (table 2). the a* value decreased over time (p<0.01), while the b* value increased, significantly (p<0.05) only at 48 hours of storage. ital. j. food sci., vol. 32, 2020 185 table 2. effect of lighting and display time on colour parameters and tbars values (mg mda/kg) in air packaged coppa di parma pgi with 24 hours lighting/day. trial 1 lighting /lamp time of display (h) r-mse (df 66) darkness cw ww nw 24 48 120 lean: l* 44.40 45.90 44.78 45.36 43.65 45.87 45.80 5.02 a* 16.58a 12.06b 12.81b 12.41b 14.69a 13.99a 11.72b 1.69 b* 16.62 17.76 17.05 17.26 16.16b 18.32a 17.03ab 3.24 c* 23.76a 21.65b 21.64b 21.51b 22.15ab 23.38a 20.90b 2.86 h* 44.50b 55.31a 52.04a 53.62a 47.37bb 51.80aba 54.93aab 6.16 fat: l* 63.90 65.17 63.26 62.32 63.58 63.71 63.69 4.41 a* 8.17a 4.52b 5.76b 5.47b 6.23 6.27 5.44 2.06 b* 12.96b 13.94ab 14.30a 14.03ab 13.21b 14.31a 13.89ab 1.87 c* 15.53 14.87 15.63 15.28 14.93 15.85 15.20 2.38 h* 59.32b 73.63aa 69.56ab 70.48aab 66.68b 67.75ab 70.31a 5.74 tbars 0.288bb 1.414aa 0.741abb 0.717abb 0.323b 0.413b 1.635a 0.91 a,b: p<0.05; a, b: p< 0.01. cw: 640 basic cool white lamp; ww: 827 lumilux interna warm white lamp; nw: 76 natura neutral white lamp. the opposite trends of a* and b* values brought about a significant increase of the h* value during storage (p<0.01). the value of c* decreased significantly (p<0.01) between 48 and 120 hours. changes in a*, b*, c* and h* values, and hue angle (h*) indicated that the lean fraction of the samples tended to be less red and more grey as storage time increased, probably due to a progressive loss of nitrosylmyoglobin and the consequent increase of nommb, according with the findings of other authors (böhner et al., 2014; haile et al., 2013). as shown in fig. 1, 24 hours under the basic cool white (cw) led to an a* value lower (p<0.05) than under the other lamps, whereas at 48 and 120 hours a* values were similar regardless the lamps. also with regard to the fat fraction (table 2), the light lowered the a* value (p<0.01), which did not differ among lamps; instead, it increased the values of b* (p<0.05) and h* (p<0.01) without affecting the c* value. indeed, storage time led to an increase of b* and h* values (p<0.05), as observed in the lean fraction. overall, the lipid fraction of the sliced coppa tended to yellow, as a consequence of both light exposure, oxygen in contact with the product and storage time, which promote the fat fraction oxidation. samples under the cw lamp showed tbars values significantly higher than those kept in the dark (p<0.01) or illuminated by the other two lamps (p<0.05). at 24 and 48 hours, tbars values were similar, while they increased significantly at 120 hours (p<0.01). the evolution of tbars values during display, depending on the different lighting conditions, is shown in fig. 2. ital. j. food sci., vol. 32, 2020 186 figure 1. values of cie a* (redness) in coppa di parma pgi packaged in air, displayed under continuous lighting. cw: 640 basic cool white lamp; ww: 827 lumilux interna warm white lamp; nw: 76 natura neutral white lamp. figure 2. tbars values (mg mda/kg) in coppa di parma pgi packaged in air, displayed under continuous lighting. cw: 640 basic cool white lamp; ww: 827 lumilux interna warm white lamp; nw: 76 natura neutral white lamp. ital. j. food sci., vol. 32, 2020 187 lipid oxidation did not develop in the dark. during storage, tbars values increased in all the lighted samples. at 120 hours, the samples under the cw lamp gave values higher (p<0.01) than those under the other two lamps. these results may be explained considering the different emission spectra of the three lamps tested in this work (data from technical data sheet by osram). cool white (cw) has a strong emission in the short wavelengths (blue and green zone of the spectrum – from 430 to 560 nm); instead, warm white (ww) and natura neutral white (nw) lamps are characterized by an emission in the blue zone significantly lower respect to cw; nw also shows a good emission in the red zone (630-700 nm). some authors underlined the importance of the lamps’ emission spectra and the correlated energy. böhner and rieblinger (2016) demonstrated that shorter wavelengths and higher irradiance provoke increased oxygen absorption with concomitant colour changes of bologna sausages. the continuous lighting promotes also lipid oxidation, but low energy emitting lamps will affect to a lesser extent this product degradation, as demonstrated in figure 2, where the tbars values in coppa stored under nw and ww lamps are lower than those determined on the samples stored under cw lamp. 3.2. trial 2 in this trial, the slices of the coppa were also packaged in air, but using a 12 hours on/12 hours off light cycle. as observed in trial 1, the l* value (table 3) did not vary in any tissue, either with the type of illumination or with the storage time. for the other colour parameters studied, the trends observed did not differ markedly from the first trial, though only a* and h* values varied significantly. table 3. effect of lighting and display time on colour parameters and tbars values (mg mda/kg) in air packaged coppa di parma pgi with 12 hours lighting/day. trial 2 lighting /lamp time of display (h) r-mse (df 66) darkness cw ww nw 24 48 120 lean: l* 40.63 42.46 41.61 42.00 40.74 41.35 42.93 3.86 a* 18.23a 14.72b 15.73b 15.04b 17.67aa 15.83abb 14.29b 2.81 b* 14.95 15.86 15.55 15.38 14.99 15.05 16.27 3.80 c* 23.77 21.93 22.38 21.75 23.38 22.10 21.89 4.19 h* 38.56bb 46.60a 43.67aba 44.58a 39.55b 42.23b 48.28a 5.88 fat: l* 61.70 62.34 60.16 59.74 59.77 61.86 61.32 6.73 a* 9.39aa 6.32b 7.45abb 7.98ab 8.97a 7.78ab 6.60b 2.50 b* 12.85 14.07 13.89 14.33 13.20 14.56 13.59 0.83 c* 16.23 15.80 16.13 16.72 16.34 16.88 15.44 2.82 h* 54.13b 66.78a 63.52a 62.31a 57.29bb 62.66aba 65.12a 7.95 tbars 0.340a 1.044b 0.790ab 0.658ab 0.340b 0.519b 1.258a 0.73 a,b: p<0.05; a, b: p< 0.01. cw: 640 basic cool white lamp; ww: 827 lumilux interna warm white lamp; nw: 76 natura neutral white lamp. ital. j. food sci., vol. 32, 2020 188 the statistical data processing demonstrates that illuminated samples showed a lower a* value (p<0.01) and a higher h* value (p<0.05) in the lean fraction; whereas, only h* changed in the fat fraction (p<0.01), and no significant differences were found among the lamps. at increased storage time, a* values decreased, significantly at 120 hours (p<0.01), both in lean and fat components, with a corresponding increase (p<0.01) of the h* values in both tissues. in fig. 3, the a* value evolution on the surface of coppa slices exposed to different light sources or stored in the dark is reported. the colour of samples stored in the dark changes, even if slightly, probably because of high partial pressure of oxygen in the headspace of the packages. if the samples stored under light are considered, it is possible to observe a different influence of the lamps used. samples stored under cw lamp shows a more pronounced decrease of the a* value between 48 and 120 hours of discontinuous lighting in comparison with the results obtained with ww and nw lamps. discontinuous lighting also affects the colour of coppa slices, but highlights the influence of different emitting lamps on the product colour fading. figure 3. values of cie a* (redness) in coppa di parma pgi packaged in air, displayed under 12 h/d lighting. cw: 640 basic cool white lamp; ww: 827 lumilux interna warm white lamp; nw: 76 natura neutral white lamp. tbars values showed the same path observed in trial 1. thus, the samples kept in the dark provided the lowest values, but only the cw lamp caused significantly higher values (p<0.01). moreover, as in trial 1, tbars increased with time, significantly at 120 hours (p <0.01). tbars evolution during storage under different lighting conditions is shown in fig. 4: darkness preserved samples from oxidation, which developed more markedly in the ital. j. food sci., vol. 32, 2020 189 illuminated samples, showing the highest value under the cw lamp. these data are consistent with those obtained for the colour changes. figure 4. tbars values (mg mda/kg) in coppa di parma pgi packaged in air, displayed under 12h/d lighting. cw: 640 basic cool white lamp; ww: 827 lumilux interna warm white lamp; nw: 76 natura neutral white lamp. 3.3. trial 3 in table 4 are shown the data concerning the samples packaged in modified atmosphere (map) exposed to continuous lighting 12h/d. as regards the effect of the type of lighting, the results showed no statistically significant variation (p>0.05) of the values of the parameters taken into account, neither in the lean nor the fat fraction. as concerns the a* value, our results agree with the findings of martínez et al. (2007) on fresh pork sausages and of djenane et al. (2001; 2003) on fresh beef steaks, and partially with the results of haile et al. (2013) on colour stability of cooked ham. the effect of residual oxygen and light exposure on the quality of cured boiled sausages (böhner et al., 2014) and of norwegian salami (sørheim et al., 2017) has been studied. in our work the residual oxygen inside the ma packages was not measured, so we can only hypothesize a complete oxygen depletion during the packaging process, which makes irrelevant the lighting of the product concerning the colour fading. another consideration is that the cured products examined in the cited works differ from coppa, and it is well known that the type of meat and the production process may influence the specific sensitivity against oxygen and light. if storage times are considered, the few significant differences observed in the lean fraction showed an erratic path, difficult to explain. these same considerations also apply to the evolution of a* value in the samples displayed under different lightings; as fig. 5 clearly shows, map maintained the red colour, regardless of light exposure and source; though it must be noted that the a* values at 0 hours were lower in this trial. ital. j. food sci., vol. 32, 2020 190 table 4. effect of lighting and display time on colour parameters and tbars values (mg mda/kg) in coppa di parma pgi packaged in a modified atmosphere with 12 hours lighting/day. trial 3 lighting /lamp time of display (h) r mse (df 66) darkness cw ww nw 24 48 120 lean: l* 41.24 40.92 43.00 41.48 41.48 41.99 41.51 3.36 a* 13.45 13.84 13.03 13.94 13.64ab 14.05a 13.00b 1.60 b* 12.30 12.05 12.50 12.44 12.12 12.48 12.38 1.37 c* 18.43 18.52 18.18 18.81 18.37 ab 18.92a 18.17 b 1.13 h* 42.63 41.46 44.29 42.07 41.87 42.04 43.93 5.66 fat: l* 56.35 57.37 57.09 56.22 56.04 57.19 57.04 5.61 a* 6.63 5.95 6.17 6.93 6.97a 6.78ab 5.51b 2.32 b* 10.39 10.99 11.06 11.01 10.82 10.85 10.92 1.13 c* 12.68 12.87 13.07 13.40 13.23 13.15 12.64 1.92 h* 59.83 64.02 63.64 60.66 59.83b 60.90ab 65.38a 7.82 tbars 0.312 0.306 0.283 0.277 0.254b 0.271b 0.358a 0.07 a,b: p<0.05; a, b: p< 0.01. cw: 640 basic cool white lamp; ww: 827 lumilux interna warm white lamp; nw: 76 natura neutral white lamp. figure 5. values of cie a* (redness) in coppa di parma pgi packaged in a modified atmosphere, displayed under 12 h/d lighting (1c). cw: 640 basic cool white lamp; ww: 827 lumilux interna warm white lamp; nw: 76 natura neutral white lamp. ital. j. food sci., vol. 32, 2020 191 at increasing storage time, a significant decrease at 120 hours (p<0.05) of the a* value and a corresponding significant increase (p<0.05) of the h* value took place in the adipose fraction. the tbars values did not differ between the samples maintained in the dark and those exposed to light. also in this case, as well as the colour evolution, eliminating oxygen in contact with coppa means protect the product against lipid oxidation, even in presence of a luminous source emitting high energy wavelengths, such as cw lamp (refer to fig. 6). our findings are consistent with the results of martínez et al. (2007) on fresh pork sausages, with djenane et al. (2001; 2003) on fresh beef steaks and with gimenez et al. (2004; 2005) on gilt-head sea bream and salmon fillets. as in the two previous trials, after 120 h of storage, tbars values increased significantly (p<0.01), though changes were rather slight. our results conflict with those of møller et al. (2000) who could not detect any difference among tbars values in samples lighted or kept in the dark. the authors stated that this could be due to the low fat content of their hams, much lower than the one found in our samples of coppa, which was well above 29%. figure 6. tbars values (mg mda/kg) in coppa di parma pgi packaged in a modified atmosphere, displayed under 12h/d lighting. cw: 640 basic cool white lamp; ww: 827 lumilux interna warm white lamp; nw: 76 natura neutral white lamp. table 5 shows changes in the value ∆e that expresses, in the cie l*, a*, b*, the quantification of the colour difference. as regards the type of lighting, it can be observed that at 24 hours (∆e1) the cw lamp brought about a colour variation greater than the other lighting conditions, which was significant only when compared with the dark (p<0.01). regarding the ∆e2 values, they were greater than ∆e1, as expected, and were significantly lower in the samples kept in the dark (p<0.05). at 120 hours (∆e3) the colour difference was still greater and although ital. j. food sci., vol. 32, 2020 192 the samples kept into the dark gave the lowest value, no statistically significant variation among lighting conditions was found (p>0.05). table 5. effect of lighting and display time on colour change (∆e) in the lean of coppa di parma pgi. lighting /lamp treatment r-mse (df 66) darkness cw ww nw trial 1 trial 2 trial 3 δe1 5.28b 7.63a 6.21ab 6.38ab 7.73a 6.37ab 5.02b 2.60 δe2 5.68b 8.80a 8.76a 8.15a 9.64a 8.76a 5.14b 3.58 δe3 7.73 9.54 9.10 9.08 11.59a 9.98a 5.01b 3.69 a,b: p<0.05; a, b: p< 0.01. cw: 640 basic cool white lamp; ww: 827 lumilux interna warm white lamp; nw: 76 natura neutral white lamp. ∆e1, ∆e2 and ∆e3 are calculated colour differences between time 0 and 24, 48, 120 hours, respectively with regard to the colour difference among the trials, data showed that the presence of oxygen caused a significant increase of ∆e2 and ∆e3 values (p<0.01), while at 24 hours (∆e1) only permanent lighting gave colour changes higher (p<0.01) than in map stored samples. no visual evaluations have been performed, in order to establish if the colour changes can be detected by consumers, but it was demonstrated that if ∆e>2 a small difference is observed, and when ∆e>5 the changes are well distinguished (fleishman et al., 1998; lindström, 2008). 4. conclusions from our results it may be concluded that the lighting with uv-free lamps negatively affects colour characteristics of coppa di parma pgi sliced and then packaged in air. further, illumination causes a significant increase of lipid oxidation and the basic cool white lamp appears to be somewhat more detrimental. when the product is packaged in a modified atmosphere, colour and lipid stability are not affected by light exposure or source. one hundred and twenty hours of storage led mainly to a loss of redness and to a significant increase of lipid oxidation. acknowledgements the research was carried out with private funds. the 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1988. photosensitized lipid peroxidation in ground pork and turkey. j. food sci. 53:1596-1598. zanardi e., dorigoni v., badiani a. and chizzolini r. 2002. lipid and colour stability of milano-type sausages: effect of packaging conditions. meat sci. 61:7-14. zhu l.g. and brewer m.s. 1998. discoloration of fresh pork as related to muscle and display conditions. j. food sci. 63: 763-767. paper received april 11, 2019 accepted october 13, 2019 paper 208 ital. j. food sci., vol. 27 2015 keywords: betalain, microbial population, quality, red beetroot, sanitation, total phenolic physicochemical, microbial and sensory quality of fresh-cut red beetroots in relation to sanitization method and storage duration dulal chandra*, ae jin choi, yong phil kim and ji gang kim postharvest research team, national institute of horticultural and herbal science, rural development administration, 203 cheoncheon-ro, jangan-gu, suwon 440-706, republic of korea *corresponding author: dchandra@korea.kr; dchandrajp@gmail.com abstract effects of sanitization and storage on fresh-cut beetroots (beta vulgaris l.) were evaluated following sanitation – peeling cutting (spc), peeling – sanitation – cutting (psc) and peeling – cutting – sanitation (pcs) methods with (cl), or without (tw), 100 ppm chlorine solution, then packaged in polyethylene bag and stored at 5°c for up to 14 days. chroma values of fresh-cut beetroots significantly declined whereas whiteness index and titratable acidity values increased, however, texture and total soluble solid contents showed no significant variation. betalain contents decreased gradually and total phenol content showed inconsistence trend. pcs-cl treated samples accounted for higher betalains decline and received lower visual quality scores despite its lower total aerobic bacterial count. minimum microbial population was observed in psc-cl methods along with higher levels of betalain contents. considering pigment retention, microbial and visual qualities, beetroots sanitized with chlorine water following psc method was the best processing way for fresh-cut beetroots and therefore, psc-cl treatment could commercially be used for processing of fresh-cut beetroots. mailto:dchandra@korea.kr mailto:dchandrajp@gmail.com ital. j. food sci., vol. 27 2015 209 introduction beet is a root vegetable of the chenopodiaceae family whose edible part is its tuberous root. its purple-red color is due to the presence of betalain pigments. these pigments are similar to anthocyanins and flavonoids, which were wrongly termed in the old literature as anthocyanins containing nitrogen. betalains are water-soluble, vacuolar pigments and are found only in ten families of the centrospermae group and are divided into two classes: red betacyanin and yellow betaxanthin (fenena, 1995). in addition to their color, betalains possess several desirable biological activities including antioxidant, antiinflammatory, hepatoprotective, and antitumor properties (escribano et al., 1998; winkler et al., 2005). although some studies have indicated their potential as antioxidant pigments, betalains have not been much explored because of their relative scarceness in nature. the bioavailability of betalains was reported to be high in humans, and they remain stable in the gastrointestinal tract without any significant loss in antioxidative properties, which increases their value as health food additives (frank et al., 2005; pavlov et al., 2005). among other compounds with antioxidant properties, phenolics are believed to act as antioxidant, anti-carcinogenic, anti-microbial, anti-mutagenic and anti-inflammatory, as well as in the reduction of cardiovascular diseases (yang et al., 2001; kim et al., 2003; vali et al., 2007). because of these nutritional importances of plant phenolics, there has been an increasing interest in the evaluation of their changes with postharvest treatment (chaudry et al., 1998; lewis et al., 1999). however, literatures are scanty on phenolic content of plant foods, especially of roots vegetables, which are important constituents of diets in many countries. storage and processing can reduce the content of phenolic compounds as some of them are easily oxidized, while others are more stable. processing in the form of simple peeling of fruit and vegetables can remove a major portion of the phenols, as the concentrations of these substances are often higher in the outer than the inner parts (manach et al., 2004). pigment distribution in beetroots, on the other hand, also varied substantially among the outer, middle and center tissues, the former contained much higher pigments than the later tissues (gaertner and goldman, 2005). beet was traditionally used as a vegetable boiled in stews, baked in tarts, roasted as a whole and cut into salads. this vegetable has recently been using as a minimally processed food in many countries. however, the main technological problems of processing fresh-cut beet roots are the significant discoloration and dehydration of the minimally processed material. washing and rinsing operations carried out after slicing have favored the loss of betacyanin and betaxanthin, since these pigments are soluble in water (nilson, 1970). moreover, minimally processed produces are more perishable than their whole counterparts, due to undergoing severe physical stresses especially during peeling and cutting procedures. minimal processing comprises selection, washing, peeling and cutting procedures that are aimed at producing a fresh and convenient product to prepare and consume (burns, 1995). the quality of fresh-cut produce depends on many factors, such as the state of the original plant (variety, plant part and maturation stage), harvesting date and storage, environmental factors and processing techniques (moure et al., 2001). however, rapid quality deterioration and shorter shelf-life are major problems facing the industry for maintaining the quality and safety of fresh-cut produces. several studies have shown that fresh-cut produces are particularly susceptible to microbial growth owing to the removal of plant protective tissues and the release of cellular fluids from cutting (carlin et al., 1989; heard, 2002). the cut slices of beetroot become vulnerable to microbial attack, moisture loss and dehydration because of their large surface area. to ensure the microbial safety, use of proper sanitizing agents along the processing line is an important step in fresh-cut produce processing. chlorine has been used for sanitation purposes in food processing industry for several decades and perhaps the most widely used sanitizer in food industry. it is inexpensive, convenient to use and works against many food borne pathogens. liquid chlorine and hypochlorite generally used in the 50 to 200 mg l-1 concentration range with a contact time of 1 to 2 min. adams et al. (1989) reported that washing lettuce leaves with 100 mg l-1 free chlorine can reduce population of aerobic mesophiles by more than 98% as compared to 92% reduction in tap water without chlorine. the use of beetroots as a fresh-cut produce is relatively new, especially in asian countries. the information on various quality parameters of fresh-cut beetroots processed with different sanitization methods is limited. hence, the objectives of this study were to find out the best processing and sanitation way for maintaining some physicochemical, microbial and sensory qualities, especially pigment and phenolic contents of fresh-cut beetroots during storage at 5°c. materials and methods preparation of sample beetroots (beta vulgaris l. var. udan) samples were harvested from a commercial farm in jeju island, republic of korea, and were transported to our laboratory within two days. samples were thoroughly washed in running tap water and subjected to different ways of sani210 ital. j. food sci., vol. 27 2015 tation treatments such as sanitation – peeling cutting (spc), peeling – sanitation – cutting (psc) and peeling – cutting – sanitation (pcs) with (cl), or without (tw), 100 ppm chlorinated solution. chlorinated solution was prepared with naocl (100 ppm free chlorine, ph 7.0) as a standard industrial disinfection treatment for fresh-cut produces. after peeling out, beetroot samples were cut into small pieces (ca. 4 × 5 × 1 cm) excluding the top and bottom most portions. sanitation treatments lasted for 2 min. to remove the excess surface water, all sanitized samples were spread over a sieve like plastic tray (40 × 50 cm) previously washed and disinfected with the same sanitizing solution and allowed at room temperature. fresh-cut beetroots sample of about 300 g were packaged in 80 µm nylon polyethylene bag (25 × 20 cm) and thermally sealed. four replicates of each bag per treatment and storage duration (0, 3, 7, 10 and 14 days of storage) were prepared and stored in a dark cold room at 5°c. on each evaluation day, outer and inner tissues were separated from the fresh-cut red beetroots slices discarding the middle portion and were stored at -80°c until needed for biochemical analyses. color and texture measurement using a chromameter (minolta cr-400, minolta, osaka, japan), color readings were taken from the middle portion of both sides of sliced red beetroots on each evaluation day. three pieces of sliced beetroot from each pouch were randomly selected and six readings (from both sides of each piece) were taken from each replicate and a total of 24 readings were averaged from each treatment on the measurement day. the chromameter was calibrated using the manufacturer’s standard white plate (y 93.5, x 0.3155, y 0.3320). color changes were quantified in the l*, a*, b* color space. l* refers to the lightness and ranges from black = 0 to white = 100. a negative value of a* indicates green, while a positive number indicates red color. positive and negative b* indicate yellow and blue color, respectively. the color values were further converted into chroma value (ozturk et al., 2009) and ‘whiteness index’ (wi) (bolin and huxsoll, 1991) and were computed by the formulae: chroma = (a*2 + b*2)1/2; wi = 100 – [(100 l*)2 + a*2 + b*2]1/2 the texture of fresh-cut red beetroot was measure in term of force required to make a puncture hole horizontally on the sliced beetroots. the test was conducted through destructive puncture test performance using a texture analyzer (ta plus, lloyd instruments, ametek inc., uk) following the method of hampshire et al. (1987). each sliced beetroots sample was placed horizontally on the stationary platform of the analyzer for the test. tests were carried out on the middle part of each sliced beetroots with a 4 mm diameter stainless steel cylindrical probe. the movement of the probe was adjusted to 5, 2 and 10 mm/s as the pre-test, test and post-test speed, respectively. with a running load cell of 100 n, the probe was attached to a creep meter equipped with the software (nexygen™mt v 4.5, lloyd instruments, ametek inc., uk) for automatic analysis using a computer. the maximum amount of force (n) needed to make a puncture hole on the sliced beetroot was recorded. five pieces of sliced beetroots from each pouch were tested and a total of 20 data from four replicates were averaged from each treatment. all measurements were taken at room temperature (20°±2°c). determination of total soluble solid, ph, and titratable acidity total soluble solid (tss), ph and titratable acidity (ta) were measured according to the aoac (1980) procedures. on each evaluation day, about 5 pieces of sliced beetroot from each bag were cut into small pieces and wrapped with 2 layers of cotton cloth and placed in a juice maker (fru-x80, goojung chromatech inc., korea) attached to an air supplier (evaa– 0300) of the same company. then pressure was created by the air supplier to obtain a homogenized solution of beetroots sample. tss of the resultant cleared juice was measured in terms of °brix using a refractometer (pal–1, atago co. ltd, tokyo, japan). the ph was determined using a ph meter (d-55122, schott instruments gmbh, germany) with a glass electrode. titratable acidity was measured by potentiometric titration with 0.1 n naoh up to ph 8.2 using 10 ml juice and the results were expressed as percentage of citric acid. determination of betalains (betacyanin and betaxanthin) and total phenolic contents the methodology used for the determination of betacyanin and betaxanthin were adopted from von elbe (2001). five gram of previously frozen samples from the outer and inner parts of sliced beetroots were macerated separately in 15 ml distilled water using an ultra-turrax tissue homogenizer (t 25 b, ika works sdn. bhd, malaysia) at a moderate speed for about 1 min. the homogenate was transferred to a volumetric flask filtered through whatman no. 1 filter paper placed on a glass funnel. the filter cake was washed several times with distilled water until the extract became colorless. the extract volume was adjusted with distilled water. the resulting beetroots juice or tissue extract was diluted with 0.05 m phosphate buffer, ph 6.5 such that the absorbance of beetroots juice at ital. j. food sci., vol. 27 2015 211 538 nm was in between 0.4 and 0.5 au. finally, the absorbance of beetroots juice was measured at 476, 538 and 600 nm with an uv-vis recording spectrophotometer (du 650, beckman coulter™, usa) and 0.05 m phosphate buffer, ph 6.5 was used as the blank. values of betacyanin and betaxanthin amounts were obtained through the equation: x = 1.095 × (a – c), y = b – z – x/3.1, z = a – x where a = sample reading at 538 nm; b = sample reading at 476 nm; c = sample reading at 600 nm; x = betacyanin absorption; y = betaxanthin absorption; z = impurities absorption (von elbe, 2001). total phenolic compound was determined based on the method described by singleton and rossi (1965) with few modifications. five gram previously frozen sample from the outer and inner parts of fresh-cut red beetroots slices were separately homogenized with 80% ethanol using an ultra-turrax tissue homogenizer (t 25 b, ika works sdn. bhd, malaysia) at a moderate speed for about 1 min. the homogenate was incubated at 60°c water bath for 30 min and centrifuged at 15,000 × g for 15 min at 20°c and then the supernatant was collected in a volumetric flask. the homogenized tissue was re-extracted with 80% ethanol at the same way and the resulting supernatants were mixed together and carefully made known volume with 80% ethanol. for the determination, folin-ciocalteu reagent was diluted with distilled water to make 1 n phenol reagent. in a test tube, 1 ml supernatant was diluted with 8 ml distilled water and 1 ml of 1 n phenol reagent was added followed by mixing. after 5 min, 1 ml 15% sodium carbonate solution was added, mixed well and allowed the mixture at room temperature (~20°c) for 2 h. the absorbance was read at 725 nm using an uv-vis recording spectrophotometer (du 650, beckman coulter™, usa). the concentration of total phenol was calculated using standard curve of gallic acid and expressed as gallic acid equivalents in mg 100 g-1 fresh weight. monitoring of microbial population microbiological counts were carried out on every sampling day including washing day. fresh-cut beetroots pouches were aseptically opened using a sterilized scissors dipped in 95% ethanol and then ignited in the flame of a bunsen burner. twenty grams beetroots sample was weighed out from each pouch and placed in a stomacher bag (masher-bag p-lts, bacct ®, nbt, japan). the beetroots sample was diluted 1:9 in double distilled autoclaved water and homogenized in a stomacher (seward stomacher 400c, brinkmann, usa) for 1 min at 230 rpm. a 10-fold serial dilution was also made from the homogenate and 1 ml of homogenate solution was inoculated onto total aerobic bacterial (tab) count petrifilmtm (3m microbiology products, st. paul, minn., usa). the plates were then incubated at 35°c for 48 h and the developing colonies (about 25 – 250) were counted with the assistance of a 3m microbial colony counter (same company as of plate) and reported as colony forming units (cfu) per gram of sample. similarly, 1 ml homogenate was plated onto 3m petrifilmtm yeast and mold (ym) count plates (same company) and incubated for 72 h at 25°c. after incubation, yeast and mold colonies were counted manually according to the instruction guide of the company. colonization data were then converted to log cfu per gram of fresh sample. sensory evaluation the sensory analysis of fresh-cut beetroots sample was carried out by an 8-member (aged 24 48) expert panel. the members of the panel were trained to recognize and score off-odor and overall visual quality of fresh-cut beetroots prior to the test. off-odor was evaluated immediately after opening the packages and scored on a 5-point scale in which 0 = none, 1 = slight, 2 = moderate, 3 = strong, and 4 = extremely strong (lopez-galvez et al., 1997); a score of 3 was considered non-acceptable. overall visual quality was evaluated by using 9-point scale (9 = excellent, 7 = good, 5 = fair, 3 = poor and 1 = unusable) (gonzalez-aguilar et al., 1999). a score of 6 was considered as the limit of marketability. statistical analysis the experiment was conducted with four replications per treatment. statistical analyses of the data were carried out using sas software (sas institute, cary, nc, usa). the level of significance was calculated from the f value of anova. mean comparison was achieved by duncan’s multiple range test. prior to the final experiment, two preliminary experiments were conducted with limited replications that resulted similar trend. results and discussion texture, total soluble solids, ph and titratable acidity the effects of different washing methods and storage duration on the textural properties of fresh-cut red beetroots are presented in table 1. the forces were almost similar for all the sanitization methods, except for an insignificant (p>0.05) lower value was recorded in psc-cl treatment on washing day. however, the values were slightly increased to an 212 ital. j. food sci., vol. 27 2015 insignificant level at the end of 14 days storage. pcs-cl treated sample exhibited the highest value among the treatments at the end of storage. maintaining texture of fresh-cut produces is one the main criteria which is affected by morphology, cell turgor, cells wall-middle lamella structure, water content, biochemical components and also by the genetic background of plant species (harker et al., 1997). peeling and cutting of vegetable exposes the interior tissues and drastically increase the rate of evaporation of water. however, we observed less than 1% moisture loss of the original weight at the end of storage (data not shown). apart from water loss, the slight increase in texture value during storage of freshcut beetroots in our study might be due to the lignification of cells that rendered harder tissues and as a result of wound healing at cut surfaces. packaging fresh-cut produces with suitable polyethylene film and selecting appropriate storage temperature have shown to preserve quality. the insignificant increases in texture of fresh-cut beetroots indicate that our packaging film and storage temperature were appropriate. there was no significant difference (p>0.05) found in total soluble solid (tss) content in fresh-cut beetroots regardless of sanitization treatment and storage duration, whereas slight variations were observed in ph and titratable acidity (ta) values among the treatments and table 1 changes in some physicochemical qualities of fresh-cut redbeet root during storage at 5°c after processed with different sanitization methods. parameter/storage day sanitization method spc-tw spc-cl psc-tw psc-cl pcs-tw pcs-cl texture (n) 0 52.58aa 52.22aa 49.92aa 47.61aa 51.13aa 52.49aa 7 52.83aa 53.80aa 51.40aa 49.51aa 52.47aa 53.85aa 14 53.43aa 54.97aa 53.39aa 50.36aa 54.29aa 55.36aa °brix 0 8.50aa 8.67aa 7.83aa 8.70aa 8.33aa 8.23aa 7 8.23aa 8.60aa 8.37aa 8.03aa 8.57aa 8.53aa 14 7.90aa 8.67aa 8.03aa 8.27aa 8.43aa 8.37aa ph 0 6.28aa 6.25aab 6.24bab 6.28aa 6.22aab 6.16cb 7 6.42aab 6.38abc 6.38abc 6.30ac 6.35abc 6.50aa 14 6.26aa 6.28aa 6.21ba 6.30aa 6.21aa 6.36ba ta (% citric acid) 0 0.10ba 0.10ba 0.10ba 0.12aa 0.09ba 0.09ba 7 0.09bab 0.08bb 0.08bb 0.10aab 0.11ba 0.10bab 14 0.17aa 0.15aa 0.15aa 0.16aa 0.18aa 0.14aa spc = sanitation peeling cutting, psc = peeling sanitation cutting, pcs = peeling cutting sanitation, cl = sanitation with 100 ppm chlorinated water, tw = sanitation with tap water. values represent the mean of four replicates. means under the same heading in each column or row with different small or capital letters, respectively are significantly different according to the duncan test (p<0.05). different storage durations (table 1). these reflect that although tss was maintained throughout the storage, ta values increased significantly at the end of the storage that resulted smaller decline in ph values. the lowest tss (7.83°brix) was found in psc-tw treated sample on washing day while the highest value (8.70°brix) was recorded in psc-cl treated sample on the same day. it was reported that tss of fresh-cut beetroots are varied depending on the cut type (kluge et al., 2006), sanitization period (vitti et al., 2011) and storage temperature (vitti et al., 2005). although our tss data were little higher than that of vit ti et al. (2011) and vitti et al. (2005) studies with fresh-cut beetroots, we found both similar amount and changing pattern of tss during storage as reported by kluge et al. (2006). the differences in tss in this study and other studies might be due to the differences in soil condition, cultivar and sowing time (feller and fink, 2004). the ph and ta values ranges from 6.16 to 6.50 and 0.08 to 0.18, respectively among the processing methods and throughout the storage (table 1). in general, ph values slightly increased in the middle of storage and decreased later to their initial values. ta values expressed as percentage of citric acid, on the other hand, remained unchanged until the middle of storage and then increased about 1.5 fold at the end of storage to their initial levels. among the treatments, pcs-cl treated samital. j. food sci., vol. 27 2015 213 ple showed the lowest values of ph and ta on washing day and 14 days of storage, respectively. in agreement with our results, lopez osornio and chaves (1998) found no variation in ph values over a 7-day storage period of raw grated beetroots at 4°c and packaged in trays wrapped with polyvinylchloride film. however, they found steadily increased values of ta throughout the storage. again, pilon et al. (2006) reported that the contents of titratable acidity were not affected by the storage period in minimally processed carrot while the ph values decreased at the beginning of storage and increased thereafter to their initial levels at the end of 3-week storage period. the smaller range of changes in citric acid content during storage of fresh-cut beetroots might be accounted for lower respiratory activity, which is the indicator of retardation of overall metabolic activities. color parameters produce color is one of the most important quality factors that directly affect consumers’ choice. in this study, the effects of different sanitization methods on the color parameters of fresh-cut beetroots were measured in term of chroma value as well as whiteness index and presented in fig. 1. among the treatments, spc-cl and psc-tw showed significantly higher (p<0.05) chroma value on washing day. however, chroma values declined gradually in all treatments when storage progressed except few fluctuations on 7 and 14 days of storage. there was a significant (p<0.05) decline in chroma value in pcs-cl treatment when storage progressed exhibiting the lowest values of chroma throughout the storage among the treatments. this result implies that sanitation after cutting yielded higher loss of pigment in beetroots slices therefig. 1 changes in chroma value (above), whiteness index (below) of fresh-cut red beetroot after processing with different sanitation methods and during storage at 5°c. legends: spc = sanitation–peeling-cutting, psc = peeling–sanitation–cutting, pcs = peeling–cutting–sanitation, cl = sanitation with 100 ppm chlorinated water, tw = sanitation with tap water. 214 ital. j. food sci., vol. 27 2015 by decreased the intensity of color. between the other two sanitation treatments using chlorine water, psc-cl showed no significant decline in chroma value until the end of storage except on 10-day while spc-cl showed gradual decrease until 10-day followed by a slight increase at the end of storage (fig. 1). the decrease in color is a consequence of loss of betalains, the main pigments of red beetroots (von elbe, 2001). therefore, we found significant variation and decrease in betalain contents both on washing day and subsequent storage of fresh-cut beetroots in this study (table 2 and later discussion). in consistence with our result, gradual decrease in color index of fresh-cut beetroots was reported previously (vitti et al., 2005; kluge et al., 2006; vitti et al., 2011). since chroma represents color saturation, which varies from dull (low value) to vivid color (high value), we used chroma value as an indicator of freshness and purity of the color of beetroots slices. however, lopez osornio and chaves (1998) found significant increase in chroma values during storage of grated beetroots. this might be attributed to the differences in processing, subsequent packaging and storage condition of beetroots in the present study with their study. whiteness index (wi) on the other hand, sharply increased on the third day of storage in all samples (fig. 1). both of the sanitized and water washed samples of peeling – cutting sanitation (pcs) method exhibited significantly (p<0.05) higher values of wi from the washing day to 10 days of storage. this result also indicates that higher pigment loss was occurred when beetroots samples were subjected to sanitize after cutting. other two methods showed similar values of wi both on processing day and throughout the storage. at the end of the storage, wi values reached nearly similar level for all samples. although wi was first measured for lignin formation on the surface of fresh-cut carrot slices (bolin and huxsoll, 1991), lopez osornio and chaves (1998) reported that the whitish substances in grated beetroots are a protective lignin biochemically synthesized after peeling. it was also reported that wi is the most sensitive and easily measured indicator of sensory quality of fresh-cut carrot. betalains (betacyanin and betaxanthin) content significantly higher betalain contents were found in the outer tissues than in the inner tissues of fresh-cut beetroots slices (table 2). beetroots samples processed with peeling – cutting sanitation (pcs) method exhibited significantly (p<0.05) lower amount of betalain (betacyanin and betaxanthin) contents on washing day compared to other methods. in general, sanitation – peeling – cutting (spc) method ensured higher amount of betalain contents than other methods whereas the betalain contents ta b le 2 c h a n ge s in b et a la in ( b et a cy a n in a n d b et a x a n th in ) co n te n t in t h e o u te r a n d i n n er t is su es o f fr es h -c u t re d b ee tr o o ts p ro ce ss ed w it h d if fe re n t sa n it iz a ti o n m et h o d s a n d d u rin g st o ra ge a t 5 °c . tr ea tm en t b et al ai n co nt en t/s to ra ge d ay b et ac ya ni n (m g 10 0g -1 f w ) b et ax an th in (m g 10 0g -1 f w ) to ta l b et al ai n (m g 10 0g -1 f w ) 0 3 7 10 14 0 3 7 10 14 0 3 7 10 14 o ut er ti ss ue s p c -t w 74 .6 5a a 65 .4 8a a b 55 .7 9a b c 54 .5 7a b c 49 .4 1a c 60 .5 4a a 54 .2 6a a 51 .6 4a b 47 .3 3a b c 42 .9 5a c 13 5. 19 aa 11 9. 74 aa b 10 7.4 3a b c 10 1. 90 ab c 92 .3 6a c s p c -c l 72 .8 5a ba 62 .0 8a a b 60 .3 7a a b 59 .7 0a a b 51 .9 1a b 56 .3 0a a 54 .5 3a a 51 .1 5a a b 49 .1 2a a b 44 .1 3a b 12 9. 15 ab a 11 6. 61 aa b 11 1. 52 aa b 1 08 .8 2a a b 96 .0 4a b p s c -t w 65 .3 5a ba 55 .8 0a a 53 .3 1a a 52 .4 9a a 49 .0 3a a 52 .5 0a a 51 .2 1a a 47 .3 5a a 41 .6 2a a 42 .4 9a a 11 7.8 5a ba 10 7.0 1a a 10 0. 66 aa 94 .1 1a a 91 .5 2a a p s c -c l 66 .7 2a ba 55 .5 0a a 53 .3 6a a 54 .9 6a a 54 .9 5a a 50 .9 1a a 45 .6 0a a 45 .4 9a a 43 .4 2a a 41 .8 8a a 11 7.6 3a ba 10 1. 10 aa 98 .8 5a a 98 .3 8a a 96 .8 3a a p c s -t w 58 .0 6b a 53 .7 5a a 52 .4 7a a 49 .1 3a a 47 .9 2a a 46 .4 6a a 41 .7 8a a 39 .7 8a a 44 .3 7a a 39 .7 4a a 10 4. 52 ba 95 .5 3a a 92 .2 5a a 93 .5 0a a 87 .6 6a a p c s -c l 57 .6 5b a 51 .5 2a a 48 .4 2a a 45 .0 2a a 42 .8 0a a 47 .8 7a a 43 .8 6a a 45 .1 7a a 40 .3 4a a 39 .2 5a a 10 5. 52 ba 95 .3 8a a 93 .5 9a a 85 .3 6a a 82 .0 5a a in ne r tis su e s p c -t w 37 .4 5a ba 34 .1 8a a b 34 .7 8a a b 30 .6 5a a b 25 .3 8a b 29 .8 2a ba 28 .3 1a a 28 .6 0a a 23 .7 4a a b 18 .7 2a b 67 .2 7a ba 62 .4 9a a 63 .3 8a a 54 .3 9a a b 44 .1 0a b s p c -c l 39 .8 8a a 31 .6 1a a b 32 .4 6a ba b 2 9. 43 ab 29 .2 4a b 32 .3 0a a 23 .9 1a b c 26 .8 8a a b 21 .4 1a b c 18 .0 8a c 72 .1 8a a 55 .5 2a b 59 .3 4a ba b 5 0. 84 ab 47 .3 2a b p s c -t w 30 .8 8b ca 30 .5 3a a 30 .8 9a ba 26 .0 3a a 25 .2 7a a 25 .3 1a ba 22 .8 0a a 24 .0 9a a 19 .3 4a a 18 .9 3a a 56 .1 9b ca 53 .3 3a a 54 .9 8a ba 45 .3 7a a 44 .2 0a a p s c -c l 28 .5 0c a 27 .7 1a a 31 .9 3a ba 26 .7 6a a 28 .1 5a a 25 .9 3a ba 21 .1 9a a b 21 .0 4a a b 20 .7 6a a b 18 .3 5a b 54 .4 3b ca 48 .9 0a a 52 .9 7a ba 47 .5 2a a 46 .5 0a a p c s -t w 28 .0 6c a 26 .8 8 aa 24 .0 3b a 24 .7 2a a 25 .2 9a a 22 .1 9b a 20 .3 3a a 21 .5 2a a 19 .6 8a a 17 .9 6a a 50 .2 5c a 47 .2 1a a 45 .5 5b a 44 .4 0a a 43 .2 5a a p c s -c l 25 .4 8c a 25 .7 9 aa 24 .3 9a ba 26 .1 8a a 23 .3 7a a 22 .1 7b a 22 .9 8a a 21 .7 5a a 19 .7 6a a 18 .4 5a a 47 .6 5c a 48 .7 7a a 46 .1 4a ba 45 .9 4a a 41 .8 2a a s p c = s an ita tio n – pe el in g c ut tin g, p s c = p ee lin g – sa ni ta tio n – cu tti ng , p c s = p ee lin g – cu tti ng – s an ita tio n, c l = s an ita tio n w ith 1 00 p pm c hl or in at ed w at er , t w = s an ita tio n w ith ta p w at er . va lu es re pr es en t t he m ea n of fo ur re pl ic at es . m ea ns u nd er th e sa m e he ad in g in e ac h co lu m n or ro w w ith d iff er en t s m al l o r c ap ita l l et te rs , r es pe ct iv el y ar e si gn ifi ca nt ly d iff er en t a cc or di ng to th e d un ca n te st (p <0 .0 5) . ital. j. food sci., vol. 27 2015 215 of peeling – sanitation – cutting (psc) methods were in between the betalain contents levels of spc and pcs methods. however, betalain contents gradually decreased when storage progressed in all washing/sanitation treatments. the rate of betalain decline during storage was higher in spc sanitization method compared to other two methods thereby significant (p<0.05) declines were observed in all components of both tissues at the end of storage (table 2). the highest (34.4%) decline of total betalain was found in both of spc-tw and spc-cl treated samples of inner tissues while the lowest (12.2%) decline was observed in pcs-cl treated sample of same portion of sliced beetroots. overall, the decline in betacyanin was little higher than betaxanthin in the outer tissues whereas opposite trend was found in the inner tissues. at the end of the storage, total betalain content was almost similar in spc-tw and psc-tw treated samples as well as in spc-cl and psc-cl treated samples of both tissues (table 2). samples of pcs treatments showed the lowest amount of total betalain contents at the end of storage in both tissues. it appears that although the declining rate of betalain was lower in pcs method than other methods, the total amount of betalain content was comparatively lower than other methods at the end of storage probably due to the higher loss of betalain in this method on washing day. in this study, sanitization and washing favor larger pigment losses of beetroots slices owing to their exposure to water or sanitized solution and therefore, we observed significant variation in betalain contents on washing day among the processing/sanitization methods. similar to our results, the variations and decreases in betalains were also found in few studies (vitti et al., 2005; kluge et al., 2006; vitti et al., 2011). in contrast our pigment decline rate, lopez osornio and chaves (1998) found about 40-50% decreases in betalain amount in grated beetroot after 7 days of storage at 0°c, whereas at 4°c the decreases were greater. however, these authors did not measure the pigment contents at different tissues of beetroots, which we did in the present study. betalains accumulate in cell vacuoles of the leaves, flowers and fruits of the plants that synthesize them, mainly in epidermal and/or subepidermal tissues (jackman and smith, 1996). moreover, it was reported that the total phenolic contents and the main betacyanin present in red beetroots distributed mostly towards the outer parts of the root and decreasing in the order peel, crown and flesh (kujala et al., 2000). this localized distribution of betalains was also found in our study. however, due to large number of samples, we did not use the middle part of beetroots tissues and only the outer and inner tissues were used for biochemical measurement. nevertheless, we assume that our fresh-cut beetroots slices were larger in size than that of grated beetroot which might prevented higher decline of pigments compared to that of lopez osornio and chaves (1998) findings. nilson (1973) observed the contents of betacyanin and betaxanthin is cultivar dependent and the levels of these pigments are around 45 to 210 and 20 to 140 mg 100 g-1, respectively. total phenolic contents total phenolic contents of sliced beetroots (fig. 2) followed similar distribution trend at different tissues that we found in betalain contents and is supported by the results of kujala et al. (2000). on the washing day, total phenolic contents also followed similar pattern as we observed for betalains contents among the treatments. the amount of total phenolic contents in the outer and inner tissues of beetroots samples ranged from 102.4 to 116.4 mg gae 100 g-1 fw and 52.1 to 80.0 mg gae 100 g-1 fw, respectively on washing day (fig. 2). higher amount of total phenolic content was observed in spc method followed by psc and pcs methods. however, we did not find any specific trend in total phenolic content of sliced beetroots during storage at low temperature. in the outer tissue, total phenolic contents declined on the third day of storage and then slightly increased the content or keep constant throughout the storage. at the end of the storage, total phenolic contents of this portion slightly increased (p>0.05) than the levels observed on washing day. pcs-tw treated sample showed the lowest amount of total phenolic contents both on washing day and throughout the storage. at the end of storage, spc-tw treated sample showed the highest amount of total phenolic content (118.1 mg gae 100 g-1 fw) followed by psc-cl treated sample (116.8 mg gae 100 g-1 fw). variations in total phenolic contents were also observed in the inner tissues on washing day and the contents either increased or decreased or maintained its level after fluctuating in between the storage. although the highest amount of total phenol (79.9 mg gae 100 g-1 fw) was determined in spc-tw treated inner tissues’ sample on washing day, psc-cl treated sample retained the highest amount (71.3 mg gae 100 g-1 fw) among the treatments at the end of storage (fig. 2). kujala et al. (2000) observed decreased amount of total phenolic content until 63 days and the amount remained almost unchanged until 196 days of storage of raw beetroots. the effects of different storage temperatures on the total phenolic content in plants have been studied and, like our study, both increases and decreases in phenolic contents have been reported. for example, lewis et al. (1999) observed an increase in total phenolic contents of colored potato tubers during storage at 4°c, whereas cordenunsi et al. (2005) found that total phenolic contents of strawberry remained 216 ital. j. food sci., vol. 27 2015 fig. 2 total phenol content of outer (above) and inner (below) tissues of fresh-cut red beetroots after processing with different sanitation methods and during storage at 5°c. legends: same as shown in fig. 1. constant or even slightly decreased during storage at different temperatures. microbial quality figure 3 shows the changes in total aerobic bacteria (tab) count and yeast and mold (ym) count of fresh-cut red beetroots on washing day and during successive storage at low temperature. significant variation (p<0.05) was found in tab among the washing treatments both on washing day and throughout the storage. the range of tab was found 2.6 log cfu g-1 in psccl to 3.4 log cfu g-1 in spc-tw treated samples on washing day. this result indicates that sanitation of beetroots with 100 ppm chlorinated water ensured a significant reduction in tab both in psc and pcs methods. in order to reduce the microbial population, chlorine solution has been used as sanitizer in many fresh-cut vegetables including beetroots (lopez osornio and chaves, 1997; vitti et al., 2011) and sweet potatoes (erturk and picha, 2006). however, studies on microbial safety or monitoring of microbial population in fresh cut beetroots are still limited as compared to other fresh produces. since beetroots contain several bioactive compounds, its use as fresh-cut produce is promising where microbial safety should be addressed and ensured. beetroots washed with tap water either before or after peeling had nearly similar number of tab which indicates the necessity of sanitation of fresh-cut beetroots with appropriate sanitizer. sanitation with chlorinated water beital. j. food sci., vol. 27 2015 217 fore peeling showed almost no effect in reducing tab as compared to after peeling or after slicing sanitation. erturk and picha (2006) reported that chlorination of sweet potatoes before slicing could not ensure acceptable microbiological quality of fresh-cut sweet potatoes. however, in our study we found significant reduction in tab when beetroot samples were sanitized after peeling but before slicing though the numbers of tab were significantly lower in after slicing sanitation throughout the storage (fig. 3). lopez osornio and chaves (1997) found significant reduction in yeast and total aerobic count in grated beetroots after washing treatment with chlorinated water. the number of tab gradually increased until the end of storage with few fluctuations. among the treatments, pcs-cl treated sample showed the lowest numbers of tab throughout the storage followed by psc-cl treated sample (fig. 3). however, we found higher losses of betalain (table 1), lower level of total phenol contents especially in inner tissues (fig. 2) as well as lower visual quality score (later discussion) in pcscl treated samples which limited the potentiality of this treatment. all tap water washed samples regardless of processing methods showed similar pattern of changes in tab. sanitation of beetroot with 100 ppm chlorine water following spc method exhibited similar number of tab until 7 days of storage and the number increased thereafter to reach the similar number with psc-tw treatment. yeast and mold (ym) count, on the other hand, showed different patterns of changes that we obfig. 3 effects of different sanitation methods on total aerobic bacteria (tab) count and yeast and mold (ym) count of freshcut red beetroots during storage at 5°c. legends: same as shown in fig. 1. 218 ital. j. food sci., vol. 27 2015 served in tab (fig. 3). among the treatments, psc-cl treatment ensured the lowest number of ym count on the washing day (1.1 log cfu g-1) and throughout the storage. this result indicates that about 1.1 log cfu g-1 reduction in ym count was occurred in psc-cl treatment on the washing day as compared to psc-tw treatment in which the highest ym count (2.2 log cfu g-1) was observed on that day. unlike tab count, there was no remarkable increase found in ym count when storage progressed. it appeared that survival and growth pattern of aerobic bacteria as well as yeast and mold are different in fresh cut beetroots. the decontamination effect of chlorinated water depends on dipping time, concentration of active chlorine, water temperature and the type of produce used (erturk and picha, 2006; vitti et al., 2011). in agreement with our results, erturk and picha (2006) found no significant reduction in yeast and mold count in fresh-cut sweet potato among control, chlorination of peeled whole roots and chlorination after slicing the roots regardless of water temperatures and concentration of chlorine, except for a significant reduction in chlorination after slicing at 20°c using 200 ppm chlorine solution. however, these authors found significant reduction in initial mesophilic population using both 100 and 200 ppm chlorine water washing of peeled whole sweet potato roots. lopez osornio and chaves (1997), on the other hand, found significant decrease in the initial yeast counts in grated beetroots after washing in 252 ppm active chlorine solution at 8°c. these findings may suggest that higher concentration of active chlorine solution might be effective in reducing yeast and mold count in root vegetables. however, higher concentration of chlorine and longer dipping time caused a substantial decline in pigment contents in beetroots (vitti et al., 2011). our ym count result suggests that higher surface area exposed to washing treatment were more susceptible to ym contamination and thereby unlikely tab count, we found significantly (p<0.05) higher levels of ym count in pcs-cl treatment (fig. 3). among the three sanitation methods used in our study, peeling-sanitation-cutting (psc) was the best method for reducing ym count both on washing day and throughout the storage. sensory quality visual quality of sliced beetroot decreased gradually when storage time elapsed (fig. 4). however, we did not notice off-odor in any sample until the end of storage (data not shown). although all samples retained marketable limit until 7 days of storage, samples processed only with spc and psc methods retained their marketable limit which was set at 6 in a 9-point scale until 10 days of storage. in a previous study, vitti et al. (2005) also reported that minimally processed beetroots could be stored until 10 days at low temperature and the produce quality drastically reduced corresponding with the increases in storage temperatures. due to fig. 4 visual quality scores of fresh-cut red beetroots during storage at 5°c after processing with different sanitation methods. legends: same as shown in fig. 1. ital. j. food sci., vol. 27 2015 219 the earlier development of whitish substances on the surface of beetroots slices, samples processed with pcs method received lower visual quality scores from 7 days of storage. moreover, we found higher losses of betalain and as a consequence higher values of whiteness index in samples treated with this method (table 2 and fig. 1). however, the chlorine treated sample of pcs methods showed the lowest number of tab among the treatments (fig. 3). this result indicates that maintenance of visual quality of fresh-cut beetroots may not depend on microbial load at least until certain limit. in general, consumer can only assess the sensory appearance. hence, there is an increasing demand for the development of improved methods that guarantee a high produce quality and safety until the end of shelf life, especially for fresh-cut produces. at the end of storage, samples of all treatments exhibited the lower values of marketable limit. overall our results indicate that fresh-cut beetroots processed with spc and psc methods could be marketable until 10 days of storage at low temperature whereas samples processed with pcs could have only 7 days of marketable life. conclusions significantly lower values of chroma and higher values of whiteness index were found in samples processed with peeling – cutting –sanitation (pcs) method, especially in pcs-cl treatment. although pcs-cl treatment showed the lowest values of tab throughout the storage, higher betalain decline along with lower level of total phenol and visual quality score limited the storability of beetroots in this treatment only until 7 days. beetroots processed with spc method exhibited the highest amount of betalain and total phenol contents, but showed higher number of microbial population. samples of psc method exhibited intermediate levels of betalain and psc-cl treatment ensured minimum number of microbial population. based on measured parameters, we conclude that psc would be the most suitable method for processing of fresh-cut beetroots and psccl would be the best treatment for ensuring minimum number of microbial population and maintaining betalain and total phenolic contents. therefore, psc-cl treatment could commercially be use for processing and storing of fresh-cut beetroots at low temperature. acknowledgements the first author gratefully acknowledges the financial support from the national institute of horticultural and herbal science (nihhs), rural development administration (rda), republic of korea received through the postdoctoral fellowship program for foreign researcher. 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sanitization time for fresh-cut beet root. rev. iber. tecnología postcosecha 12: 215. von elbe j.h. 2001. betalains. curt. protoc. food analyt. chem. f3.1.1. winkler c., wirleitner b., schroecksnadel k., schennach h. and fuchs d. 2005. in vitro effects of beet root juice on stimulated and unstimulated peripheral blood mononuclear cells. am. j. biochem. biotechnol. 1: 180. yang c.s., landau j.m., huang m.t. and newmark h.l. 2001. inhibition of carcinogenesis by dietary polyphenolic compounds. ann. rev. nutr. 21: 381. p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (1): 73–83 issn 1120-1770 online, doi 10.15586/ijfs.v33i1.1939 73 p u b l i c a t i o n s codon garlic greening: pigments’ biosynthesis and control strategies alberto de iseppi, andrea curioni, matteo marangon, simone vincenzi* and giovanna lomolino department of agronomy, food, natural resources, animals and environment (dafnae), university of padova, viale dell’università, 16-35020 legnaro (padova), italy *corresponding author: simone vincenzi, department of agronomy, food, natural resources, animals and environment (dafnae), university of padova, viale dell’università 16, 35020 padova, italy. email: simone.vincenzi@unipd.it. received: 25 july 2020; accepted: 14 january 2021; published: 13 february 2021 © 2021 codon publications open access review abstract greening is a major problem for garlic’s quality. this phenomenon leads to discoloration of the product and is directly related to the alliinase-catalyzed conversion of isoalliin into 1-propenyl-containing thiosulfates. garlic crushing, refrigeration, and storage in normal atmosphere, as well as in the presence of monocarboxylic acids, are established the main factors that promote its greening. in last decades, the study of biochemical pathway of this phenomenon has allowed to effectively understand the main steps and key enzymes involved, and to identify optimum conditions for chemical and enzymatic reactions leading to discoloration. these findings have, in some cases, determined the development of new tools for the control of garlic greening on large scale. after providing an updated description of the biochemistry of green pigments produced in garlic, this review reports an overview on the strategies for controlling discoloration of garlic at industrial level. keywords: control strategies, discoloration, garlic, greening, isoalliin, s-alk(en)ylcysteine sulfoxides introduction garlic (allium sativum) is a world-spread plant being used for centuries as a food for its health benefits (banerjee et al., 2003). its health benefits have been exploited to treat diseases starting from ancient sumerians, egyptians, greeks, and romans up to the modern times (zang et al., 2013). garlic cultivation was probably started in central asia for more than 10,000 years ago (block, 2010). nowadays also, garlic cultivation is primarily concentrated in the same region, with china, india, and bangladesh accounting for 86% of the world’s production (faostat, 2016). garlic is a fundamental ingredient of many food preparations. indeed, thanks to its antimicrobial properties and distinctive taste, it is used as a food preservative and/or seasoning in many typical recipes, and has become a key component of the mediterranean cuisine (lanzotti, 2006). essentially, owing to its high content of sulfur aroma compounds (speranza and morelli, 2012), garlic is widely used as a taste and flavor enhancer of recipes all over the world. in these preparations, fresh garlic cloves are usually crushed immediately before use, a process that is essential to release the sulfur-containing compounds responsible for its typical flavor (block, 1992). indeed, crushing allows enzyme alliinase, which is originally found in cell vacuole, to get in contact with s-alk(en)ylcysteine sulfoxides, such as isoalliin and alliin, and convert them into 1-propenyl-containing thiosulfinates and allicin, respectively (kubec and velíšek, 2007). this latter compound is in turn the precursor for many flavoring molecules such as diallyl disulfide and diallyl sulfide (yoshimoto et al., 2019). in addition, the isoalliin degradation pathway leads to the synthesis of 1-propenylcontaining thiosulfinates, which are recognized as “color developers,” i.e., compounds responsible for garlic greening (kubec and velíšek, 2007; kubec et al., 2004). this phenomenon of discoloration, generally desirable for many asiatic preparations (i.e., laba garlic; mailto:simone.vincenzi@unipd.it 74 italian journal of food science, 2021; 33 (1) de iseppi a et al. plants is described in figure 2 (imai et al., 2006b; zang et al., 2013). these different steps are discussed in detail in the following sections. biosynthesis of s-alk(en)ylcysteine sulfoxides s-alk(en)ylcysteine sulfoxides are present in all plants belonging to the genus allium, especially alliin (sallylcysteine sulfoxide) and isoalliin (s-1propenylcysteine sulfoxide) (martins et al., 2016). these compounds comprise analk(en)yl group, probably originating from valine catabolism, and a cysteine moiety derived from glutathione. the currently proposed alliin and isoalliin biosynthesis pathway (figure 3 [yoshimoto et al., 2019]), recently reviewed by yoshimoto et al. (2019), includes: (i) the conjugation of glutathione to methacrylic acid or methacrylic-coa (both derived from valine catabolism) to form s-(2-carboxypropyl) glutathione (granroth, 1970; lancaster and shaw, 1989; suzuki et al., 1962); (ii) the formation of γ-glutamyl-s-1 propenylcysteine and γ-glutamyl-s-allylcysteine (precursors of isoalliin and alliin, respectively) after removal of glycyl from s-(2-carboxypropyl) glutathione and its conversion into the s-1-propenyl and/or s-2-propenyl group by oxidative decarboxylation; (iii) the sulfide forma-tion after removal of the γ-glutamyl group catalyzed by γglutamyl transpeptidases enzyme (ggt); and (iv)  the soxygenation of intermediate sulfides into alliin and isoalliin catalyzed by a flavin-containing s-oxygenase. the reactions described at above-mentioned (i) and (ii) are probably catalyzed by still unknown enzymes. conversely, the roles of ggt and flavin-containing s-oxygenase were assessed at molecular level (cho et al., 2012b; yoshimoto et al., 2015), and recently a consensus has been reached on this point (yoshimoto et al., 2019). even though it is known that s-alk(en)ylcysteine sulfoxides are accumulated in cytosol, the subcellular localization of several of the biosynthetic reactions described above has not been completely clarified (yoshimoto et al., 2019). indeed, many of the above-mentioned enzymes and intermediates are probably located in different cell compartments of allium plants. in garlic, the flavincontaining s-oxygenase is located in the cytosol (yoshimoto et al., 2015), and in onion (allium cepa), which presents a similar s-alk(en)ylcysteine sulfoxides biosynthetic pathway and cell structure, γ-glutamyl peptides have been detected also in the cytosol, while glutathione was found in both cytosol and chloroplasts (lancaster and collin, 1981; lancaster and shaw, 1989). conversely, ggt enzyme and methacrylic-coa have never been precisely localized in allium spp., although in plant cells they are usually found in vacuoles and mitochondria, respectively (binder, 2010; grzam et al., 2007). figure 1), is undesirable in western countries, where any variation from the white color negatively affects garlic’s marketability (tao et al., 2016). formation of pigment cannot be inhibited drastically (i.e., by interventions at the genetic level) as it shares the same biosynthetic pathway of bioactive and flavor compounds. therefore, the phenomenon of greening needs to be controlled by preventive practices during garlic storage and/or processing (zang et al., 2013). many papers, reporting on the mechanisms of production of allicin (and related compounds), have primarily focused on their flavoring and health properties. in contrast, fewer and less recent works have dealt with the problem of garlic discoloration by analyzing the main greening controlling strategies employed at industrial level. in this review, an updated biochemical description of the biosynthetic pathway leading to the production of pigments in garlic is reported. subsequently, the available and possible new technologies to control garlic greening are described, and discussed critically. pigments’ formation the starting point of pigments’ biosynthesis is the conversion of isoalliin and other s-alk(en)ylcysteine sulfoxides into 1-propenyl-containing thiosulfinates (kubec and velíšek, 2007; kubec et al., 2004). this biochemical process gained more and more interest starting from the middle of the 20th century when s-alk(en)ylcysteine sulfoxides, including alliin, isoalliin, and methiin, as well as their conversion into allicin by the enzyme alliinase, were first described (stoll and seebeck, 1948, 1949a, 1949b, 1951). a schematic overview on the main biochemical steps leading to the pigment’s biosynthesis in allium sativum figure 1. greening in laba garlic (retrieved from commons. wikimedia.org). http://commons.wikimedia.org http://commons.wikimedia.org italian journal of food science, 2021; 33 (1) 75 strategies for controlling discoloration of garlic at industrial level alliin allicin pyruvic acid green color color developer pigment precursors blue pigments yellow pigments amino acids alliinase isoalliin figure 2. main steps involved in pigments’ biosynthesis pathway in garlic (allium sativum) plants. allium plants synthesize s-alk(en)ylcysteine sulfoxides in a manner to accumulate nitrogen and sulfur as well as a defense mechanism against insects and pathogens (aghajanzadeh et al., 2019; yoshimoto et al., 2019). biosynthesis of thiosulfinates in allium plants, thiosulfinates are produced from the hydrolysis of s-alk(en)ylcysteine sulfoxides, which are catalyzed by the enzyme alliinase, a pyridoxal 5′-phosphate (plp)-dependent c–s lyase (ec 4.4.1.4). since alliinase is located in the vacuole and the s-alk(en)ylcysteine sulfoxides are stored in the cytosol, this reaction takes place only after crushing, cutting, maceration, or other practices leading to cell disruption (lancaster and collin, 1981; li et al., 2015). upon contact, alliinase removes a proton attached to the chiral carbon of the s-alk(en)ylcysteine sulfoxide, cleaving the c–s bond to yield sulfenic acid, pyruvic acid, and ammonia (shimon et al., 2007). sulfenic acids are then converted into thiosulfinates by spontaneous self-condensation (yoshimoto et al., 2019). in onion, this step could be partially hindered by the activity of the enzyme lachrymatory factor synthase (lfs), which converts isoalliin into propanethial s-oxide, the “lachrymatory factor,” which is the compound involved in lachrymation during slicing of onions (imai et al., 2002; silvaroli et al., 2017). in garlic, 1-propenyl-containing thiosulfinates derived from isoalliin are called “color developers.” a minor but significant role is played in the greening of garlic by the alliin-derived thiosulfinate allicin (figure 4 [kubec et al., 2017]). allicin is established as the main thiosulfinate in garlic, where it is the principal responsible for the development of typical flavors and health-benefit properties (borlinghaus et al., 2014; kopec et al., 2013; martins et al., 2016; rahman, 2007). however, for the greening phenomenon, the main role is played by isoalliin-derived 1-propenyl-containing thiosulfinates (“color developers”), as they constitute the basic structure for the formation of pigment precursors (figure 2; kubec et al., 2004, 2017; zang et al., 2013). 76 italian journal of food science, 2021; 33 (1) de iseppi a et al. hooc i ii iii iv hooc hooc hooc hooc hooc hooc hooc cooh cooh cooh cooh cooh hooc cooh hooc o o o o o o o s s s s o s s hs nh2 nh2 nh2 nh2 nh2 nh2 h2n nh2 nh2 n h n h h n h n h n h n methacrylic acid glutathione glycine glutamic acid alliin isoalliin s-allycysteine s-oxygenase ggt s-(2-carboxylpropyl)glutathione γ-glutamyl-s-allylcysteine γ-glutamyl-s-1-propenylcysteine figure 3. biosynthetic pathways for alliin and isoalliin formation. italian journal of food science, 2021; 33 (1) 77 strategies for controlling discoloration of garlic at industrial level hooc hooc hooc cooh hooc hooc hooc hooc cho di-1-propenyl thiosulfinate s o o o o o o ho ho s s s s s s o nh2 nh2 nh2 nh2 n n n n n n alliin allicin thioacrolein acrolein alanine pp-167 pigment precursor pp-221 pigment precursor cc-332 yellow pigment cc-385 blue pigment alliinase alliinase alanine isoalliin color developer figure 4. formation of yellow (cc-332) and blue (cc-385) pigments from alliin and isoalliin. other yellow and blue pigments can be formed, not shown in the figure. 78 italian journal of food science, 2021; 33 (1) de iseppi a et al. of green pigments but rather arises from a mixture of yellow and blue pigments, originating following different pathways (wang et al., 2008b). even if a full consensus has not been reached on the single step, proposed mechanisms suggest that both alliin and isoalliin should be present for the onset of green coloration (imai et al., 2006a; kubec et al., 2017; wang et al., 2008; zang et al., 2013). according to wang and colleagues (2008), yellow pigments result from the interaction between precursors derived from isoalliin and pyruvic acid originated from alliinase activity (figure 2). in the same study, precursors derived from different amino acids (ppaa) give rise to yellow pigments absorbing visible light in the range of 418–460 nm, following at 440 nm this order of intensity: pp-ala > pp-val > pp-gly > pp-ile > pp-leu  > pp-phe. conversely, the blue pigments contributing to green color were attributed to the interaction between different precursors and allicin. even if several precursors developed a blue dye, only pp-gly, pp-ala, and pp-val could determine a significant blue pigmentation in garlic (absorption in the range 590–610 nm). however, owing to their instability, blue dyes could in part be degraded into their yellow analogues (wang et al., 2008; zang et al., 2013), although it is unlikely that this hypothesized phenomenon is sufficient to prevent garlic discoloration. recently, a slightly different mechanism for the synthesis of both yellow and blue pigments was proposed by kubec and colleagues (2017). in their study, two precursors and six color compounds (cc) were identified after a reaction between garlic extract and the enzyme alliinase. the proposed pathway for the formation of these color compounds (cc-332 and cc-385) should not involve pyruvic acid and exclude the possibility of mutual interconversion of blueand yellow-colored species (figure 4). the control of garlic discoloration with the exception of “laba” preparation, in which garlic greening is desired (tao et al., 2016), this phenomenon strongly affects the consumers’ perception, reducing garlic marketability (zang et al., 2013). different strategies have been adopted by farmers, sellers, and retailers to control greening, including management of environmental conditions during storage and usage of additives added directly on garlic cloves (madhu et al., 2019). discoloration control during fresh garlic storage discoloration in garlic is affected by several environmental variables that should be controlled during its storage and processing (zang et al., 2013). among these variables, acting on temperature offers the possibility of formation of pigments thiosulfinates are highly reactive, and thus they easily undergo nonenzymatic reactions yielding numerous sulfur-containing compounds responsible for sensorial attributes and bioactivities of allium plants (yoshimoto et al., 2019). examples are acyclic sulfides, dithiines, and thiolanes that demonstrate bioactive properties similar to those of allicin (block et al., 2018; kubec et al., 2018; martins et al., 2016; štefanova et al., 2019). 1-propenyl-containing thiosulfinates (“color developers”) are converted into n-substituted 3,4-dimethylpyrroles, also called pigment precursors (pps), which can further react with different (thio)carbonyl compounds generating colored compounds that are responsible for the pigmentation of many allium bulbs, including garlic (allium sativum), onion (a. cepa), and leek (a. porrum). among these species, the biosynthetic pathway of these pyrroles is very similar even if it leads to different pigmentations, typically green for garlic and pink/red for onion and leek (comparini et al., 2018; kubec et al., 2015, 2017). on the other hand, the red pigment formed upon crushing of another allium species, giant onion (a. giganteum), is not derived from isoalliin but from a different amino acid precursor, s-(2-pyrrolyl)cysteine sulfoxide (kučerová et al., 2011). in general, almost all abundant free amino acids present in onion, leek, and garlic, including alanine, glutamine, glutamic acid, asparagine, aspartic acid, arginine, and lysine, are largely involved in the production of pigments (cho et al., 2009; kubec and velíšek, 2007; lee et  al., 2013). however, it has been established that different pigments and color intensities are related to different amino acids involved in formation of pyrroles. in 2010, lee and colleagues detected several pink–red pigments, originated from onion juice added with 21 free amino acids, indicating that onion discoloration could be due to these different pigments. lee et al. (2010) also demonstrated that some amino acids could be the precursors of more than one pigment, while others, such as histidine, serine, and cysteine, are not involved in the biosynthesis of pigments. in another study with hydrophobic amino acids, it was hypothesized that the intensity of garlic greening could be related to the dimension of the amino acid r group, namely, the smaller the size of the r  group, the larger the color intensity (wang et al., 2008b). nevertheless, this hypothesis was not confirmed for hydrophilicamino acids as, in a following study, glutamic acid yielded a more intense green color compared to asparagine, which has a bigger r-group (hu et al., 2010). the typical discoloration that occurs in garlic cloves is represented by the development of a green pigmentation (figure 1). this color is not due to the production italian journal of food science, 2021; 33 (1) 79 strategies for controlling discoloration of garlic at industrial level however, it is important to consider that the control of both temperature and atmosphere composition during storage does not appear to be resolutive; indeed, even if the activity of enzymes is inhibited and the production of s-alk(en)ylcysteine sulfoxides is limited, all conditions favorable for developing greening phenomenon could be restored at the end of the storage and/or during transportation and retail. discoloration control during fresh garlic processing in mediterranean countries, garlic is primarily consumed fresh, while in asia and other regions it is generally processed to obtain products such as paste, powder, puree, and macerate cloves (li et al., 2015). owing to crushing, these processes could increase the risk of greening of these products. nevertheless, the addition of additives and/or the adoption of specific techniques, which are normally not available for fresh garlic, during the production process can offer some possibilities to control discoloration. control by acidification, heat treatments, and drying the level of acidity (ph) affects both enzymatic and nonenzymatic reactions leading to pigment formation in garlic. nonenzymatic reactions are promoted in strong acidic conditions (ph 2–3), but they still take place at ph 5–6, which makes these difficult to control by acting on acidity (zang et al., 2013). conversely, enzymatic reactions, in general, and alliinase activity, in particular, are inhibited at ph < 3. since the normal ph of garlic is in the range 4.5–5.5, acidification can represent a relevant tool to control greening of some garlic-based products (bai et al., 2006). the ph drop could be achieved by adding acidifying agents, such as organic acids, during maceration or immediately after cutting or crushing, resulting in the inhibition of alliinase activity. however, it is important to consider that monocarboxylic acids such as acetic acid are great promoters of garlic greening, since they facilitate tonoplast permeability, allowing alliinase to come in contact with its substrate s-alk(en)ylcysteine sulfoxides (zang et al., 2013). as observed previously, once imported into the cell, the dissociated form of acetic acid is able to damage tonoplast and other cellular structures (bai et al., 2006). indeed, for some processed garlic products (such as “laba”), maceration of cloves with vinegar promotes the desired greening in spite of low ph (between 2 and 3) of vinegar (li et al., 2015). this is due to the slow acidification of the inner parts of intact cloves during maceration that allows the acetic acid to permeate tonoplast before alliinase inhibition. therefore, control of greening by organic acid additions should involve only non-monocarboxylic acids such as ascorbic acid, malic acid, and citric acid, which do not affect tonoplast’s integrity, possibly because plants develop specific systems to controlling the activity of ggt enzyme involved in the biosynthesis of greening precursors s-alk(en)ylcysteine sulfoxides. indeed, the inhibition of this enzyme would lead to an early interruption of the pigments’ biosynthesis pathway. to prevent germination, garlic is usually stored at a temperature of around –3°c for up to 9 months (volk et al., 2004). under these conditions the ggt enzyme (also involved in germination) is almost inactive, resulting in low alliin and isoalliin accumulation (lancaster and shaw, 1991). after sale, storage of garlic at refrigeration temperatures (0–12°c) should be avoided, because the enzyme establish its maximum activity at 4°c, thus resulting in a high production rate of s-alk(en)ylcysteine sulfoxides. it was demonstrated that, upon a long-term refrigerated storage, garlic cloves developed, after crushing, much greener dye compared to those stored for a shorter time at the same conditions. on the contrary, it was observed that storing garlic at 35°c drastically reduced the activity of ggt, resulting in the decrease of greening and improvement in product’s shelf-life (li et al., 2008). despite the fact that temperature of 35°c appears to be optimum for alliinase activity (mishra et al., 2001), the concentration of its substrate (s-alk(en)ylcysteine sulfoxides) is reduced due to the thermal inhibition of ggt. in addition, the temperature of 35°c favors the spontaneous conversion of isoalliin (one of the major s-alk(en)ylcysteine sulfoxides) into cycloalliin, which is not involved in the greening phenomenon as it is not a substrate of alliinase. this mechanism could be exploited also in the case of refrigerated storage. indeed, if cold storage conditions (below 0°c) are not available, cloves can be stored at refrigerated conditions and then subjected to warm storage (35–40°c for 2 weeks) to allow the conversion of isoalliin to cycloalliin and produce garlic bulbs without noticeable changes in color (yamazaki et al., 2012). another important factor that could be controlled during storage is the concentration of oxygen and carbon dioxide present in the atmosphere. indeed, the storage in controlled atmosphere, normally applied to prevent spoilage, also aids in controlling garlic greening. encouraging results in terms of final quality, sprout growth, spoilage delay, and discoloration of garlic bulbs have been reported after keeping garlic cloves for up to 6.5 months at 0–1°c in a controlled atmosphere containing 0.5% o2, 5–15% co2, and 60–70% relative humidity, compared to normal atmosphere and modified atmosphere, in which only o2 or co2 concentration was adjusted (0.5% o2/ normal co2 and normal o2/10% co2) (cantwell et al., 2003). the positive effect of low o2/high co2 conditions combined with cold storage was also demonstrated for onion, in which decrease in discoloration was attributed to a slowdown of alliinase activity (uddin and mactavish, 2003). 80 italian journal of food science, 2021; 33 (1) de iseppi a et al. compared with more sophisticated techniques such as freeze-drying and microwave treatment. the last two methods reported effective results in terms of color maintenance by reducing the effect of the maillard reaction, which is promoted at high temperatures/for long durations typical of conventional drying. in particular, microwave drying of garlic appeared to be suitable for industrial applications as it may offer the possibility of reducing treatment time and energy costs (baysal et al., 2003; i̇lter et al., 2018). control by addition of additives as mentioned above, isoalliin is involved in the discoloration process of garlic and other allium plants. in onion, its corresponding sulfenic acid (1-propenesulfenic acid) can be converted into di-1-propenyl thiosulfinate (a pigment precursor) by a nonenzymatic reaction. in allium species, this step could be partially inhibited by the action of the enzyme lachrymatory factor synthase (lfs). indeed, lfs catalyzes the conversion of 1-propenylsulfenic acid into propanthials-oxide (lachrymatory factor). therefore, lfs is able to remove isoalliin from the pathway of formation of pigments, thus inhibiting discoloration (cho et al., 2012a; zang et al., 2013). by considering this discovery and that isoalliin is a key compound for garlic greening (lukes, 1986), some authors hypothesized that the addition of lfs in garlic (where it is not naturally present) could prevent the appearance of green pigmentation. with the aim to suggest the application of lfs as a natural additive, in these studies dried onion was used as a source of lfs instead of adding the purified enzyme. it was reported that 5–15 g of onion powder per kilogram of fresh garlic prevented greening by decreasing the content of isoalliin-derived thiosulfinate (cho et al., 2012a; lee et al., 2012). compared to others, this approach has the advantage of preserving the antioxidant activity of allicin because it allows this compound to remain available in garlic. other compounds have been applied to protect garlic from both greening and browning (zang et al., 2013). addition of cysteine, ascorbic acid, sodium metabisulfite, and trisodium phosphate resulted in a significant drop of green pigmentation in ground garlic, and sodium metabisulfite was the most effective of all these compounds. in addition, it was demonstrated that binary, ternary, and quaternary mixtures of these additives, if containing sodium metabisulfite, were more effective than individual compounds in preventing both greening and browning (kim et al., 1999). control by other technologies the need to control greening without affecting sensory properties of garlic has led to the application of other more advanced technologies. γ-irradiation is a manage these acids stored naturally in vacuole (bai et al., 2006). indeed, when tested on garlic, ascorbic acid, malic acid, and citric acid reported a substantial inhibition of green color development (bai et  al., 2006; kim et  al., 1999). as many other enzymes, ggt and alliinase can be inactivated by high temperature treatments. in fact, in garlic processing the inactivation of both the enzymes can be achieved by blanching. this treatment involves heating at 70–100°c for 1–30 min with hot water or microwaves and is applied to processed vegetables to achieve enzymatic inactivation and reduce microbial contamination. blanching conditions are poorly tolerated by both ggt and alliinase, whose activity rapidly drops after 10 min at 70°c (huang et al., 2019; yin et  al., 2009), whereas complete inactivation was reported after 30 min at 70°c for ggt and after 15 min at 90°c for alliinase (mochizuki et al., 1988; rejano et al., 1997). however, heat treatments also cause a decrease in the contents of allicin and other antioxidant molecules, leading to impoverishment in the bioactive properties of garlic. a recent study calculated the kinetics of ggt and alliinase inactivation along with that of decrease in concentration and antioxidant activity of allicin (huang et al., 2019). following a holistic approach, these authors proposed a blanching treatment of 4 min at 90°c, which seemed to be a good compromise to obtain an effective enzyme inactivation and limited losses of allicin and other valuable compounds. nevertheless, it should be established that blanching is effective only if performed before the accumulation of thiosulfinates formed by the catalytic action of alliinase because the following nonenzymatic reactions leading to production of pigments have established to be strongly promoted by high temperatures (imai et al., 2006b). while the techniques to control greening based on ph modification and heat treatments negatively reduce the accumulation of allicin, drying could represent a compromise between the control of greening and the accumulation of bioactive compounds. it is well known that the drying process can limit many enzymatic and nonenzymatic reactions responsible of food spoilage and degradation by decreasing the activity of water (baysal et al., 2003; fante and noreña, 2015). in the case of garlic, recent studies have investigated the possibility to preserve its original color while maintaining a good content of allicin by drying fresh garlic immediately after cutting. all these studies established that the involvement of allicin (which was formed soon upon cutting) in pigment formation could be dramatically reduced by a drop in activity of water (baysal et al., 2003; duan et al., 2015; fante and noreña, 2015; i̇lter et al., 2018; utama-ang et al., 2018). conventional air drying of garlic was also italian journal of food science, 2021; 33 (1) 81 strategies for controlling discoloration of garlic at industrial level banerjee s.k., mukherjee p.k. and maulik s.k. 2003. garlic as an antioxidant: the good, the bad and the ugly. phytotherapy research 17(2):97. https://doi.org/10.1002/ptr.1281 baysal t., icier f., ersus s. and yildiz h. 2003. effects of microwave and infrared drying on the quality of carrot and garlic. journal of european food research and technology 218(1):68. https://doi. org/10.1007/s00217-003-0791-3 binder s. 2010. branched-chain amino acid metabolism in arabidopsis thaliana. arabidopsis book 8:e0137. https://doi. org/10.1199/tab.0137 block e. 1992. the organosulfur chemistry of the genus allium – implications for the organic chemistry of sulfur. angewandte chemie international edition english. 31(9):1135. https://doi. org/10.1002/anie.199211351 block e. 2010. “garlic and other alliums – the lore and the science”. royal society of chemistry (rsc), cambridge, uk. block e., dethier b., bechand b., cotelesage j.j.h., george g.n., goto k., et al. 2018. ajothiolanes: 3,4-dimethylthiolane natural products from garlic (allium sativum). journal of agricultural and food chemistry 66(39):10193. https://doi.org/10.1021/acs. jafc.8b03638 borlinghaus j., albrecht f., gruhlke m.c.h., nwachukwu i.d. and slusarenko a.j. 2014. allicin: chemistry and biological properties. molecules 19(8):12591. https://doi.org/10.3390/ molecules190812591 cantwell m.i., hong g., kang j. and nie x. 2003. controlled atmospheres retard sprout growth, affect compositional changes, and maintain visual quality attributes of garlic. acta horticulturae 600:791. https://doi.org/10.17660/actahortic.2003.600.122 ceci l.n., curzio o.a. and pomilio a.b. 1992. effects of irradiation and storage on the γ-glutamyl transpeptidase activity of garlic bulbs cv ‘red.’ journal of the science of food and agriculture 59(4):505. https://doi.org/10.1002/jsfa.2740590413 cho j., lee e.j., yoo k.s. and lee s.k. 2012. role of precursors on greening in crushed garlic (allium sativum) bulbs, and its control with freeze-dried onion powder. journal of the science of food and agriculture 92(2):246. https://doi.org/10.1002/jsfa.4568 cho j., lee e.j., yoo k.s., lee s.k. and patil b.s. 2009. identification of candidate amino acids involved in the formation of blue pigments in crushed garlic cloves (allium sativum l.). journal of food science 74(1):c11. https://doi. org/10.1111/j.1750-3841.2008.00986.x cho j., park m., choi d. and lee s.k. 2012. cloning and expression of γ-glutamyl transpeptidase and its relationship to greening in crushed garlic (allium sativum) cloves. journal of the science of food and agriculture 92(2):253. https://doi.org/10.1002/jsfa.4610 comparini d., nguyen h.t. h., ueda k., moritaka k., kihara t. and kawano t. 2018. effect of different light spectra on the pigmentation of stored elephant garlic. journal of the science of food and agriculture 98(7):2598. https://doi.org/10.1002/jsfa.8752 duan x., liu w.c., ren g.y., yang x.t. and liu y.h. 2015. atmospheric freeze drying garlic slices based on freezing point depression. international journal of agricultural and biological engineering 8(4):133. fante l. and noreña c.p.z. 2015. quality of hot air dried and freeze-dried of garlic (allium sativum l.). journal of food widespread technology that is used to process plant foods (for review, see farkas, 2006). first attempts were made to evaluate the impact of application of both γ-irradiation and high hydrostatic pressure on development of green color during storage of garlic (ceci et al., 1992; hong and kim, 2001). thanks to the slowing of certain metabolic processes and inhibition of enzymes such as polyphenol oxidase, both technologies were effective in preventing germination, senescence, and browning (farkas, 2006; hong and kim, 2001; madhu et al., 2019), but no impact on the development of greening and its related enzymes was recorded. in addition, high-pressure treatments probably damaged garlic tissues, thus promoting the contact between alliinase and its substrates salk(en)ylcysteine sulfoxides (tao et al., 2016). more recent studies have evaluated the impact of light of different spectra on developing greening during storage of garlic (comparini et al., 2018; he et al., 2019). a  significant delay in the process of greening was reported after the application of green, blue, and yellow lights, and a combination of them, during storage. even if not resolutive, light-control could represent an additional tool to be integrated in strategies aimed to prevent greening. indeed, the application of light has a negligible impact on garlic integrity, thus making this technique accessible and easily acceptable. conclusions greening represents a major technological problem significantly affecting garlic marketability. in spite of some missing information, great advancements have been made during the last decade in understanding the biosynthetic pathways leading to the formation of pigments in allium plants. some of these findings have improved the available strategies aimed at controlling garlic greening. innovative approaches that would deserve more attention and research are those based on the use of natural additives, as in the case of dried onion addition. however, new strategies to be developed to prevent greening of garlic should in any case also consider the preservation of the compounds known to impact both 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italy cdepartment chemical engineering materials environment, sapienza university of rome, via eudossiana 18, 00184 rome, italy *corresponding author: tel.: +39 089964334 email: gadiletta@unisa.it abstract in a previous paper the effect of chemical pre-treatment on quality attributes of ‘annurca’ apple slabs dried at different temperatures was investigated. herein, we evaluated the effect of the same pre-treatment on the quality attributes of the same dried ‘annurca’ apple samples rehydrated at two temperatures. specifically, slabs were initially pretreated in a dipping solution containing trehalose, sodium chloride, sucrose. then, they were dried by using a convective dryer at 50°, 55°, 60°, and 65°c, and rehydrated at 30° and 70°c by immersion in water. the combination of pre-treatment, drying at 65°c and rehydration temperature of 30°c enabled to obtain the best preservation of rehydration indices (i.e, water absorption capacity), structure and colour properties. on the contrary, the highest antioxidant activity (ec50) in treated samples was found at the lowest drying temperatures (50° and 55°c) among those investigated and rehydration temperature of 30°c. the pca provided different behaviours among untreated and treated dried apples when rehydrated at 30° and 70°c, demonstrating that this pre-treatment combined with drying/rehydration temperatures influenced the quality attributes of rehydrated samples. keywords: ‘annurca’ apple, pre-treatment, rehydration, drying, pca ital. j. food sci., vol. 32, 2020 797 1. introduction the ‘annurca’ apple (malus x domestica borkh. cv annurca rossa del sud) is a temperate and traditional fruit, cultivated in southern italy, in particular in the campania region. ‘annurca’ appleis rich in bioactive components such as polyphenols, flavonoids and anthocyanins which can contribute to fruit quality in terms of nutritional values, flavour and colour (d’abrosca et al., 2017). drying is a common method used for apple’s preservation and for their consumption over long periods of time on the global markets. drying contributes to extend the shelf life (more than one year) (nowackaet al., 2014) and improves food stability by reducing content-microbial growth, water activity, and minimizing chemical deterioration. furthermore, drying process creates new processed products, such as rehydrated apples, and reducing the cost of transportation and storage (proietti et al., 2018; rojas and augusto, 2018; wang et al., 2018; baeghbali et al., 2019; russo et al., 2019;). however, drying can cause some undesirable changes in fruits including browning and oxidation reactions, case hardening and degradation of nutritional compounds. these changes affect the overall quality of dried fruits, as well as the consumer acceptability (wang et al., 2018; önal et al., 2019). moreover, the structure of tissue is partially destroyed, which may affect water permeability, and as a result the rehydration ability and the product texture (krokida et al., 2000; cox et al., 2012). most of the dried foods and particularly apples, are usually rehydrated before their consumption, i.e, in bakery products, instant products (soup), milk products (yogurt, icecream), fruit tea – infusion and liqueurs. rehydration is a complex process, which aims for reconstituting the fresh food’s properties by contacting dried product with water or other liquids, i.e, fruit juice, sucrose or glucose solution. the rehydration process consists of three steps at the same time: dried food absorbs water, swelling occurs in the rehydrated product, and soluble components are lost or are diffused through the solution (lewicki, 1998; moreira et al., 2008; cox et al., 2012). rehydration characteristics of dried fruits are considered as quality parameters. such characteristics are influenced by the samples’ composition (i.e, protein content, volume and density of dried products), the drying conditions (i.e, temperature and type of process), and the pre-treatment. moreover, rehydration temperature is the factor that plays major role on rehydration rate, water uptake and volume changes. on the other hand, during rehydration, immersion of the dried food products in water could cause loss of nutrients (i.e, phenolic content, antioxidant activity) and colour pigments (amin et al., 2006; moreira et al., 2008; tunde-akintunde, 2008). in this context, the determination of the best rehydration conditions can be appropriate for deeper understanding of rehydration process. besides, the improvement of quality attributes such as rehydration indices, volume changes, colour, antioxidant activity seems to be crucial to produce high quality rehydrated new products. the combination of drying temperatures and pre-treatments has been widely implemented in literature leading to improvements in the drying/rehydration process and the quality of final products (i.e, structure, colour, bioactive compounds, sensorial evaluation) and energy savings (lewicki, 1998; vega-gálvez et al., 2008; adiletta et al., 2015; adiletta et al., 2016a; da costa ribeiro et al., 2016; adiletta et al., 2018; dermesonlouoglu et al., 2018; önal et al., 2019). in literature there are many studies focused on the application of different dipping pre-treatment solutions to improve fruits and vegetables’ rehydration process: ethyl oleate alkaline solution for tomatoes ital. j. food sci., vol. 32, 2020 798 (doymaz, 2007); citric acid solution and blanching for apple slices (doymaz, 2010); ethyl oleate and sodium carbonate for cape gooseberries (junqueira et al., 2017). nevertheless, to our knowledge, no work has fully investigated the effect of a pretreatment on the quality attributes of the rehydrated food product. in this sense, an-indepth understanding of rehydration process and how it is affected by pre-treatment conditions is essential for the improvement of process design and rehydrated product quality, as well as, for development of new products. in this work carbohydrate/salt solution is investigated as alternative process protective agents of food products during the drying/rehydration processes. the novel aspect of this work is to clarify the effect of carbohydrate/salt solution on the rehydration phase of dried apples. trehalose is a naturally occurring and known as one the non-reducing sugars. trehalose substitutes water molecules in membrane and thereby preventing the phase transition. trehalose has unique properties on the preservation of the biostructures during the drying process. moreover, trehalose plays an important role in the minimization of quality deteriorations such as loss of nutrients, protect the colour and flavour caused by maillard browning reactions (pati̇st and zoerb, 2005; adi̇letta et al., 2016a; aktas et al., 2017). the mixture of trehalose with other compounds or trehalose alone becomes important because of its numerous advantages in food drying application (i.e, shorter drying time, enhancement of drying rate, protection of flavour and colour, improvement of reconstitution ofdried foods properties, inhibition of protein denaturation and higher nutritional content) (patist and zoerb, 2005; ataréset al., 2008; ohtake and wang, 2011; xin et al., 2013; betoretet al., 2015; önalet al.; 2019). most of works are concentrated on the use of trehalose for osmo-dehydrated or freezedried food materials (dermesonlouoglou et al., 2007; xi̇n et al., 2013). however, detailed information on the effects of trehalose of the rehydrated dried fruits is still lacking, particularly for apple. therefore, in this paper, trehalose was used as a stabilizer in the preparation of a chemical pre-treatment solution. the addition of sodium chloride salt to this solution aims for improving the texture preservation and the rehydration ability (lewicki, 1998; dermesonlouoglou et al., 2007). finally, regarding to rehydration process, several papers deal with the modelling of rehydration process of foodstuffs dried with different methods: hot air drying garcíapasqual et al., 2006, zura-bravo et al., 2013); freeze-drying (lopez qui̇roga et al., 2019; wallach et al., 2011); swell drying and vacuum multi flash drying benseddi̇k et al., 2019). however, limited information is currently available on the effect of rehydration process temperature on the quality attributes of dehydrated products, such as dried edible irish brown seaweed (cox et al., 2012) and dried apple slices (zura-bravo et al., 2013). therefore, the present work aims to study the quality of rehydrated ‘annurca’ apples, both untreated and treated, at two rehydration temperatures (30° and 70°c) (doymaz, 2010). these temperatures resemble the rehydration at approximately room temperature (e.g. milk) and in hot water (e.g. soup, tea, infusion). in this framework, a combined pre-treatment of trehalose, sodium chloride and sucrose solution was here utilised and its effect, and that of drying and rehydration temperatures, was investigated on the rehydration indices, colour, volume changes and antioxidant activity of the rehydrated apples. from an industrial perspective, the development of dried foods is a key to provide for commercialization of innovative rehydrated products and to increase consumers’ demand of healthier, convenience and ready-to-eat-foods. ital. j. food sci., vol. 32, 2020 799 2. materials and methods 2.1. raw material preparation ‘annurca’ apples were obtained from supermarket in campania region, italy, after reddening–ripening treatment (lo scalzo et al., 2001). uniform size, without mechanical damage and freshness were used to select the best samples. several apple fruits were washed with tap water, peeled by using knife and cylinders (30±0.22 mm for diameter and 5±0.01 mm for thickness) were prepared. two different type of samples were analysed in this research: apple slabs without any pretreatment (utr) and apple slabs treated (tr) by dipping in carbohydrate/salt solution (0.8% trehalose, 0.1% nacl and 1.0% sucrose); dipping temperature of 25°c and dipping time of 15 min (önal et al., 2019). 2.2. drying experiments the treated (tr) and untreated (utr) slabs were dried in a convectional drier (zanussi fcv/e6l3) at four different drying temperatures (50°, 55°, 60°, and 65°c), with a constant air velocity of 2.3 m/s. drying experiments were stop when the moisture content of slabs was about 0.04 kg water/kg db. in order to evaluate the drying kinetics, for each type of samples, three slabs were continuously weighted using a sensor (phidgets inc., canada). this weight sensor is a load cell composed of a transducer, which is able to convert mechanical force into electrical signals. the samples’ weight loss was recorded online every 5 min. 2.3. rehydration experiments rehydration experiments of apple slices, previously dried, were performed at the specified rehydration temperatures in distilled water by using water bath to evaluate rehydration capacities (doymaz, 2010; barrera et al., 2016). the rehydration temperatures were selected as 30°c and 70°c on the basis of the literature (doymaz, 2010) for the experimental design of rehydration process. the water to apple ratio was about 100:1 (weight basis). at specific time, samples were taken out from liquid, blotted with tissue paper and measured by using an electronic balance. slabs were weighted every 15 min in the initial phase of rehydration process (up to 120 min) and then every 30 min. the rehydration test was performed in triplicate for each apple sample. the rehydration capacity was calculated as percentage water gain (adiletta et al., 2016a), as follows: weight gain % = (weight of rehydrated samples-weight of dried samples) (weight of dried samples) ×100 (1) in order to have more information about the amount of absorbed water, the amount of removed solutes, and the degree of cellular and structural disruption during rehydration, four quality indices were investigated (lewicki, 1998; barrera et al., 2016), as follows: (1) water absorption capacity, wac; (2) dry matter holding capacity, dhc, (3) rehydration ability, ra; and (4) water holding capacity, whc. the wac index explains the ability of food material to absorb water that replaces the water removed during drying. the dhc index is a measure of food material ability to hold soluble solids after rehydration; it gives information on tissue destruction and on tissue permeability to solutes. the ra index ital. j. food sci., vol. 32, 2020 800 describes the rehydration ability of dried product, and it indicates the total tissue destruction caused by both drying and rehydration conditions (maldonado et al., 2010). the whc index measures the ability of product to maintain its own and added water during the rehydration process. also, whc has an important role in the food structure modifications (zayas, 1997). three indices were calculated by using eq. (2) to eq. (4), which proposed to describe the food’s behaviour after rehydration process (barrera et al., 2016). these indices are water absorption capacity (wac), dry matter holding capacity (dhc), rehydration ability (ra), defined as follows: wac=!! .!! !! !!.!! ! !!!!! (2) dhc= !! .(!!!! !) !! .(!!.!! !) (3) ra = wac⋅dhc (4) where: m is the total mass in g, and xi is the mass fraction of i component in g/g, the subscripts 0, d and r state the fresh sample, the completely dried and rehydrated samples, respectively, while superscript w states water. it was also calculated the water holding capacity (whc) of the rehydrated structure from the soluble solids content of its liquid phase (z) and the amount of liquid (mcf) removed by centrifugation at 4000 rpm for 10 min (eq. 5). whc=!! .!! ! – mcf .(1-.!! !!) !! .!! ! (5) 2.4. surface colour measurement the colour parameters of both untreated and treated fresh and rehydrated apple slabs were measured using a colourimeter (chroma meter ii reflectance cr-300 triple flash mode aperture 10 mm minolta, japan), calibrated previously with a white standard ceramic plate. to analyze the colour change of fresh and rehydrated samples, cie lab colour coordinates (l*, a* and b*) were recorded and the average values were calculated for each sample. the lightness value (l*) indicates the lightness/darkness of the sample, a* index represents green when negative and red when positive, and b* index represents blue when negative and yellow when positive. white index (wi), the whiteness degree of samples, was determined as follows (adiletta et al., 2016b): wi= 100 – (100 − 𝐿 ∗!) + (𝑎 ∗! ) + (𝑏 ∗!) (6) ital. j. food sci., vol. 32, 2020 801 2.5. dpph radical scavenging activity extracts from fresh and rehydrated apple samples were obtained according to d’abrosca et al. (2017) with some modifications. methanol solution (10 ml, 80% v/v) (chromasolv®, for hplc, ≥99.9, sigma-aldrich, usa) was added to fresh (5 g ± 0.01) and rehydrated samples (3 g ± 0.01), after reducing to small pieces. the mixture was homogenized throughout an ultraturrax at 10 rpm and then stirred by vortex for 10 min. supernatant was filtered by using a whatman no:2 filter paper. all extractions were performed in triplicate. the total antioxidant activity of allapple slabs was determined by the dpph radical scavenging method (brand-williams et al., 1995). different extracts volumes were mixed with 3.5 ml of 6×10-5 m of dpph methanol solution in cuvettes. the obtained solution was shaken properly and left for 30 min at room temperature in the dark. the absorbance of solution was measured at 517 nm by using uv-vis spectrophotometer at 517 nm (lambda bio 40; perkinelmer, waltham, ma, usa). methanol was used as the blank, while the control sample was without adding any extract. percentage of inhibition of dpph radical was calculated as follows: % antioxidant activity: (abscontrol-abssample/abscontrol) x 100 (7) where abscontrol is the absorbance of control and abssample that of the sample. the results were showed in terms of the ec50 value, which was identified as the sample concentration (mg/ml) required to inhibit 50% of the dpph radical scavenging activity. ec50 was determined from a graph of antioxidant capacity (%) versus extract concentration (mg/ml). 2.6. diameter and thickness evolution to determine the reconstitution of volume of dried apples, diameter and thickness were measured every 30 min during the rehydration experiments at 30° and 70°c. the average thickness and diameter of utr and tr samples were calculated as the average of the measured values of 5 slabs for each sample through image analysis (nh image/ image j software 1.8.0). both dimensions were measured at different positions of the slices and their average value was considered. in particular, the measurement positions on rehydrated apple slabs were as follows: four positions for diameter (along two perpendicular axes and two diagonal axes) and eight positions for thickness (ponkham et al., 2012). 2.7. statistical analysis all results were repeated three times and they were reported as the mean±standard deviation (s.d). one way analysis (anova) and tukey test were applied for comparing mean values by using spss 24 software statistics program (spss inc., chicago, usa). any statistical difference was considered significant with p<0.05 and it was indicated with different letters. principal component analysis (pca) was used to identify the principal components contributing to most of the variations within the dataset, evaluating the impact of dipping pre-treatment and drying/rehydration temperatures on the quality ital. j. food sci., vol. 32, 2020 802 characteristics of rehydrated apples (utr and tr). all analyses were performed with spss software package, version 24 (spss inc., chicago, usa). 3. results and discussion in our previous paper (önal et al., 2019), the impact of a novel and natural dipping pretreatment containing trehalose, sucrose and sodium chloride, and air drying temperatures (50°, 55°, 60° and 65°c) was evaluated on drying characteristics and quality properties of dried apples. the results demonstrated that, the dipping pre-treatment containing trehalose allowed to decrease drying times, to better retain physico-chemical, nutritional and sensorial attributes (i.e, colour, shrinkage, microstructure, total phenolics compound and antioxidant activity) and obtain high quality of dried apple snacks. this study is a continuation of our work already published (önal et al., 2019), and it will provide information on rehydration characteristics of hot-air dried ‘annurca’ apple, as well as, rehydrated apples’ quality attributes. 3.1. rehydration kinetics and rehydration indices the rehydration is an important process, which is used for understanding the quality of dried food products. the physico-chemical changes and structural modifications that occurred during drying, generate cell collapse and volume reductions, reduce the absorption of water, thereby avoiding the complete rehydration of dried products (lewicki, 1998; krokida et al., 2003; moreira et al., 2008; aral and meşe, 2016). this rehydration process is hence affected by several factors, for instance, physicochemical properties of food, pre-treatment, drying process and conditions. results reported in the following refer to apple samples (utr and tr) dried at four different temperatures (50°, 55°, 60°, and 65°c), and rehydrated at 30° and 70°c. in order to evaluate the damage of drying and the impact of rehydration temperature on the rehydration behaviour, the weight gain (%) during rehydration was shown in fig. 1ab and fig. 2a-b for both utr and tr dried apples at temperatures of 30° and 70°c, respectively. figure 1. rehydration kinetics of untreated (a) and treated (b) dried samples (50°, 55°, 60° and 65°c) at rehydration temperature of 30°c. ital. j. food sci., vol. 32, 2020 803 figure 2. rehydration kinetics of untreated (a) and treated (b) dried samples (50°, 55°, 60° and 65°c) at rehydration temperature of 70°c. it was noticed that at any rehydration temperature all the samples showed the same trend. all rehydration curves showed a clear logarithmic trend, and as expected, the rehydration time decreased with increasing temperature from 30° to 70°c. the total rehydration time, that is the time at which a constant water amount was reached, was found as 270 and 210 min for both treated and untreated dried samples, and for rehydration temperatures of 30° and 70°c, respectively. at the end of the experiments at 30° and 70°c, untreated and treated samples reached almost the same water gain of 420% and 460%, respectively. when rehydration time proceeded, it was observed a reduction in the driving force for water transfer and the system slowly reached equilibrium. the initial rapid water uptake lasted different times: for both samples at 70°c it was about 30 min with respect to 30°c where it was about 100 min. the fast initial water uptake of curves was detected at rehydration times of 30 and 100 min and that the moisture content on the apples’ surface attains the saturation value, almost instantaneously. hot air dried apple samples exhibited an initial high rate of water uptake followed by a slower rehydration stage that lead to equilibrium moisture values in the rehydration curves– approximately after those times. the fast initial water uptake is due to the filling of cavities and capillaries near the surface (önal et al., 2019). then, the diffusion of water in the pores inside the sample is dominant. regarding to the drying conditions, in a previous work (önal et al., 2019), which analyzed the effect of drying temperatures on drying kinetics and quality properties of dried apples, the optimal drying temperature was found 65°c for preserving the principal quality attributes (i.e, colour, shrinkage, sensorial evaluation and rehydration capacity). the tr slices showed higher rehydration capacity compared with the utr ones at both rehydration temperatures. less rehydration capacity of untreated apples was correlated to the collapse of tissue by higher exposure time during drying. the structure of untreated ones was significantly modified by drying. in other words, the carbohydrate/salt solution containing trehalose here proposed is able to protect apple structure during drying. this is in agreement with the findings reported by atarés et al. (2008), doymaz (2010), ital. j. food sci., vol. 32, 2020 804 vásquez-parra et al. (2013), junqueira et al. (2017) and adiletta et al. (2016a,b; 2018) that found higher rehydration capacity during the rehydration experiments, when different pre-treatments were used to preserve the food structure during drying. on the contrary, some studies have mentioned that an increment of rehydration capacity of dried fruits such as for hawthorn (aral and meşe, 2016), apple (doymaz, 2010; zurabravo et al., 2013), red pepper (vegagalvez et al., 2008) and lemon (wang et al., 2018) was observed by increasing the rehydration temperature. this because the higher rehydration temperatures cause the tissue collapse and cell damage, creating larger spaces in dried fruits and in this way enhancing the rehydration ability of the dried materials (wang et al., 2018). in tables 1 and 2, the rehydration quality indices (wac, dhc, ra and whc) of both tr and utr dried apples (50°, 55°, 60° and 65°c) at rehydration temperatures of 30° and 70°c, respectively, were reported. at 30°c, the wac and dhc indices showed significantly (p<0.05) higher values for treated samples dried at 60° and 65°c. this trend indicates that the treated samples were able to absorb more water at low rehydration temperature with regard to the untreated ones. similar findings were reported by barrera et al. (2016), which found higher values of wac and dhc indices in rehydrated apples treated previously with vacuum impregnation with sucrose solution. they stated that these higher indices are correlated to higher rehydration ability of apple samples. furthermore, the highest ability to rehydrate (ra index) and the highest whc values were observed in treated samples dried at 60° and 65°c and rehydrated at 30°c. accordingly, increasing rehydration temperature (70°c) leads to texture damage likely due to the fact that the breaking or the denaturation of polysaccharides of cell wall promotes a remarkable reduction of mechanical resistance in the apples. similar whc results were obtained by zura-bravo et al. (2013), which found that the highest rehydration temperature (60°c) resulted in lower whc of rehydrated apple slices. table 1. rehydration indices of both untreated (utr) and treated (tr) dried samples (50°, 55°, 60° and 65°c) at rehydration temperatures of 30°c. rehydration at 30°c wac dhc ra whc utr 50°c 0.781±0.03a 0.233±0.007a 0.182±0.02a 0.794±0.013a tr 50°c 0.806±0.02ab 0.243±0.004a 0.196±0.009ab 0.822±0.02ab utr 55°c 0.784±0.024a 0.240±0.008a 0.188±0.005ab 0.796±0.06a tr 55°c 0.827±0.03ab 0.247±0.003a 0.204±0.014ab 0.859±0.002abc utr 60°c 0.816±0.014ab 0.241±0.005a 0.197±0.03ab 0.844±0.03abc tr 60°c 0.847±0.02b 0.268±0.003b 0.227±0.02ab 0.902±0.015c utr 65°c 0.831±0.02ab 0.243±0.006a 0.202±0.009ab 0.877±0.02bc tr 65°c 0.853±0.013b 0.274±0.004b 0.234±0.012b 0.911±0.02c data are the average of three replicates±standard deviation. different superscript letters in the same column mean significant differences (p<0.05). barrera et al. (2016) revealed that the vacuum impregnation (vi) with an isotonic sucrose solution significantly improved rehydration process of apple. this explanation was confirmed by higher values of wac, dhc and whc reached by vi sucrose samples. ital. j. food sci., vol. 32, 2020 805 after rehydration experiments at higher temperature 70°c, no significant differences (p>0.05) were found in the following indices: dhc, ra, whc between all untreated and treated samples; except for the treated samples dried at 60°c which showed the highest wac value. the combination of lower rehydration temperature (30°c) and higher drying temperatures (60° and 65°c) with this pre-treatment was proven to be useful to preserve the rehydrated apple structure by reducing cellular damage and promoting the absorption of great amount of water. table 2. rehydration indices of both untreated (utr) and treated (tr) dried samples (50°, 55°, 60° and 65°c) at rehydration temperature of 70°c. rehydration at 70°c wac dhc ra whc utr 50°c 0.767±0.02a 0.222±0.02a 0.177±0.005a 0.807±0.02a tr 50°c 0.811±0.023ab 0.236±0.003a 0.191±0.012a 0.825±0.02a utr 55°c 0.799±0.03ab 0.213±0.003a 0.170±0.02a 0.813±0.14a tr 55°c 0.822±0.02ab 0.237±0.02a 0.195±0.03a 0.823±0.03a utr 60°c 0.801±0.02ab 0.218±0.014a 0.181±0.02a 0.821±0.02a tr 60°c 0.830±0.01b 0.241±0.02a 0.200±0.003a 0.870±0.015a utr 65°c 0.790±0.015ab 0,240±0.02a 0.190±0.004a 0.843±0.009a tr 65°c 0.817±0.01ab 0.239±0.01a 0.195±0.008a 0.879±0.005a data are the average of three replicates±standard deviation. different superscript letters in the same column mean significant differences (p<0.05). 3.2. diameter and thickness evolution diameter and thickness evolution is another important factor that should be analyzed during the rehydration tests of apples. the average diameter and thickness of all dried apples were evaluated during the rehydration. all samples had similar increasing trend of the diameter and thickness during the experiments at 30° and 70°c. in figs. 3a-b and 4a-b the diameter and thickness of utr and tr samples dried at 65°c were compared. in all the conditions investigated, they did not reach the diameter and thickness of fresh samples, which were respectively 30 mm and 5 mm. obviously, the fastest increment of diameter and thickness took place in the initial period of the rehydration process. in the further stage of process, water absorption slowed down since rehydrated samples got close to the state of balance with equilibrium moisture content. there were significant increments of diameter and thickness of both samples (tr and utr) up to 120 min of the rehydration process (for 30° and 70°c). higher increases in diameter and thickness were observed in treated samples than untreated ones at both temperatures: at 30°c the recovered volume (with respect to fresh one) of tr samples was 78% while that of utr samples was 71%. in additon, table 3 showed the diameter and thickness values of untreated (utr) and treated (tr) apple samples dried at 65°c reached at the end of rehydration step at 30° and 70°c. the final diameter values were significantly different from each other. the ‘65 tr 30°c’ sample exhibited higher diameter than the samples ‘65 utr 30°c’, ‘65 utr 70°c’, and ‘65 tr 70°c’. ital. j. food sci., vol. 32, 2020 806 figure 3. diameter (a) and thickness (b) of untreated (utr) and treated (tr) rehydrated samples dried at 65°c during rehydration at 30°c. mean values in treated samples with different lower letters are significantly different (p<0.05) during the rehydration time, and mean values in untreated samples column with different capital letters are significantly different (p<0.05) during the rehydration time. these results indicated that the pre-treatment solution and rehydration temperature had great impact on final size, shape and appearance of apple samples, therefore on the consumer acceptability. similarly, in concern with final thickness, no significant differences were found between the samples ‘65 utr 70°c’ and ‘65 tr 70°c’, while the‘65 tr 30°c’ sample had the highest final thickness values. such behaviour is probably due to use of dissacharides, particularly trehalose which effect on pectic cell components results mainly on protecting functionality of proteins and stabilisng the three-dimensional structure of protein (lewicki, 1998). in this way, the cell membrane is protected and upon rehydration its functionality is restored. the structure changes and loss of nutritional compounds in untreated samples may be associated with the observation of crack internally in the later stages of rehydration at 70°c. moreover, the diameter changes were more significant than thickness ones in all investigated samples because the ital. j. food sci., vol. 32, 2020 807 shrinkage due to the drying occurs preferentially along the diameter than the thickness of the slabs. among the two investigated temperatures, the best temperature was 30°c with regards to the increment of diameter-thickness of samples. figure 4. diameter (a) and thickness (b) of untreated (utr) and treated (tr) rehydrated samples dried at 65°c during rehydration at 70°c. mean values in treated samples with different lower letters are significantly different (p<0.05) during the rehydration time, and mean values in untreated samples column with different capital letters are significantly different (p<0.05) during the rehydration time. the ability to be reconstituted of food products depends especially on the inner structure of the dehydrated samples. the rehydration temperature increament induces a structural damage, increasing that caused during the dehydration process. moreover, increasing temperature results in low mechanical resistance and elasticity in the products. it is seen that, treated apples have homogenous structure which is protected by the pre-treatment and upon rehydration its functionally is restored. this maybe be attributed to the trehalose solution which replaces water in membrane and prevent the phase transition in dried apples. moreover, trehalose is able to form glassy matrices, and direct the sugar ital. j. food sci., vol. 32, 2020 808 interaction with polar groups in phospholipids and proteins, thereby maintaining and stabilizing cellular structure (crowe et al., 1988; lewicki, 1998; betoret et al., 2015). several studied investigated the impact of pre-treatments and of drying/rehydration conditions on the dimension changes of dried fruit and vegetable during their rehydration. winiczenka et al. (2014) found that the temperature of drying influenced the relative increase of the volume of dried apples during rehydration at 20°c. according to bilbao-sáinz et al. (2005), apple cylinders dehydrated in microwave oven with vacuum impregnation showed that the recovered volume (%) of impregnated samples was higher than the recovered volume of non-impregnated ones. a justification to this result is that the major part of the initial gas (air) present in the pores is released by vacuum impregnation and entrance of the isotonic solution of the apple juice in the pores increases the mass of sample. table 3. final diameter and thickness values of untreated (utr) and treated (tr) rehydrated samples dried at 65°c during rehydration at 30° and 70°c. sample diameter (mm) thickness (mm) 65 utr 30°c 27.82±0.05c 4.17±0.04b 65 utr 70°c 26.81±0.05a 3.97±0.04a 65 tr 30°c 28.42±0.05d 4.36±0.04c 65 tr 70°c 27.37±0.05b 4.05±0.04a 3.3. colour evaluation colour parameters are also important as quality indices for rehydrated food products and they should closely resemble the colour characteristics of fresh food material to increase consumer acceptability (cox et al., 2012). the evaluation of rehydration conditions with the aim of minimizing the colour changes during drying/rehydration process is crucial from an economic perspective. the effects of pre-treatment and drying/rehydration temperatures on colour parameters of fresh and rehydrated apple slabs were presented in table 4. lightness (l*) and white index (wi) were reported. according to results, the colour values of rehydrated apple slabs were significantly different (p<0.05) in relation to fresh ones. it was observed a decrease in l* and wi values in all samples, indicating a reduction of lightness respect to fresh ones. this is an unexpected result since it is believed that an increase in water gain would normally lead to a higher luminosity (gowen et al., 2006; moreira et al., 2008). based on these results, it is argued that some modifications in the optical properties of the apples occurred during the rehydration. oxidation processes or other chemical reactions such as maillard reactions probably led to formation of browning agents (gowen et al., 2006; moreira et al., 2008; lemus-mondaca et al., 2009; deng et al., 2017). treated rehydrated samples had higher l* and wi values than untreated rehydrated ones at both rehydration temperatures (30° and 70°c), demonstrating that the pre-treatment preserves the colour stabilization of the final rehydrated apples. furthermore, drying temperatures have a key role on colour attributes of dried products, as well as of rehydrated foodstuffs (link et al., 2017). the higher drying temperature resulted in the best colour preservation in terms of lightness and white index at both rehydration temperatures. on the contrary, moreira et al. (2008) and ital. j. food sci., vol. 32, 2020 809 zura-bravo et al. (2013) for rehydrated chestnut and apples, respectively, showed a reduction of l* values when the rehydration temperature increased. as a consequence, the pre-treatment combined with higher drying temperatures (60° and 65°c) had significant effect on colour of rehydrated samples, while both used rehydration temperatures did not significantly influence the colour changes of rehydrated slabs. table 4. colour parameters for untreated (utr) and treated (tr) fresh and rehydrated samples (drying temperatures, 50°, 55°, 60° and 65°c) at rehydration temperatures 30° and 70°c. samples l* wi fresh apples utr fresh 81.73±0.66f 72.13±1.79g tr fresh 84.79±2.89f 76.91±1.02h rehydrated apples at 30°c utr 50°c 53.26±1.97a 45.30±2.00a tr 50°c 60.45±1.15bc 59.48±1.31cde utr 55°c 53.59±1.29a 46.82±0.59a tr 55°c 65.98±0.77de 58.31±0.90cd utr 60°c 57.64±1.53ab 45.68±0.89a tr 60°c 66.31±0.62de 62.09±0.34ef utr 65°c 59.15±2.79bc 52.55±1.52b tr 65°c 66.81±0.25de 62.88±1.60ef rehydrated apples at 70°c utr 50°c 57.55±2.15ab 56.25±1.93bc tr 50°c 65.51±1.71de 62.57±0.31def utr 55°c 59.35±0.61bc 57.27±0.53c tr 55°c 65.39±0.55de 64.74±1.61f utr 60°c 62.77±1.59cd 59.71±0.51cde tr 60°c 66.12±0.80de 66.01±1.23f utr 65°c 60.31±0.50bc 58.13±1.38cd tr 65°c 68.13±0.18e 65.75±1.83f values are expressed as mean±standard deviation. all measurements are performed in triplicate. values with different letters in a given column are significantly different (p<0.05). 3.4. dpph radical scavenging activity the knowledge of the content and stability of apple antioxidant components after rehydration treatments is essential to assess the nutritional values prior to its consumption (cox et al., 2012). the radical scavenging activity for all analyzed samples was showed in fig. 5 at the two rehydration temperatures (30° and 70°c). as expected, the lowest ec50 values (the highest antioxidant activity) were found as 16.06 and 18.66 mg/ml db in tr and utr fresh samples, respectively. antioxidant activity of all untreated and treated rehydrated samples decreased after both rehydration processes at 30° and 70°c. for treated apples rehydrated at 30°c the dpph activity was higher than those at 70°c. the pre-treatment combined with the lower drying ital. j. food sci., vol. 32, 2020 810 temperatures (50° and 55°c) and the lower rehydration temperature (30°c) can better protect the antioxidant activity of rehydrated apple slabs. the possible explanation of this trend is that during the drying and rehydration processes at higher temperatures modifications of the chemical structure of the main antioxidant compounds in apple (i.e., chlorogenic acid, quercetin, gallic acid, αtocopherol) or interactions between antioxidant compounds and other apple constituents, such as proteins occurred (önal et al., 2019). in addition, trehalose – dried and rehydrated foods showed a higher nutritional value with respect to foods processed by conventional system (colaça and roser, 1994). in this case, lower rehydration temperature (30°c) had positive effect on the radical scavenging activity of rehydrated apples: higher water temperatures promoted significant loss of antioxidant compounds also into the water. similar findings were stated by moldanado et al. (2010), which reported that increases in temperature above 40°c resulted in higher loss of solid compounds. in contrast to these results, cox et al. (2012) evaluated the influence of the rehydration temperatures (20°, 40°, 60°, 80° and 100°c) on the antioxidant activity of seaweed. they reported that the seaweed rehydrated at 80°c showed the highest antioxidant activity increment. higher rehydration temperatures positively effect on the dpph activity of seaweeds. zura-bravo et al., (2013) also found a higher antioxidant activity of rehydrated apple slices (granny smith) at 60°c rather than 20 and 40°c. this increment at higher rehydration temperature is attributed to the accumulation of melanoidins from maillard reaction which have different antioxidant activity values. hence, the higher rehydration temperature promoted the penetration of water, thus resulting in a high antioxidant activity (miranda et al., 2009; vega-gálvez et al., 2009). figure 5. antioxidant activity of both untreated (utr) and treated (tr) fresh and rehydrated apples (drying temperature: 50°, 55°, 60° and 65°c) at 30°c and 70°c. 3.5. effect of pre-treatment, drying/rehydration temperatures by pca the effect of pre-treatment and drying/rehydration temperatures on the qualitative traits of rehydrated apples were evaluated by pca analysis. covariance matrix showed that the eigenvalues accounted for 67.63% of the total variance in the dataset using two principal ital. j. food sci., vol. 32, 2020 811 components (pcs). pc1 explained 38.52% of the variance in the dataset, whereas pc2 explained an additional 29.11% of the variance. all loadings and scores were shown in the same pca plot (fig. 6). wac (r2= 0.844), dhc (r2=0.851), ra (r2= 0.831), whc (r2= 0.590) were positively correlated to pc1; while l* (r2= 0.807), wi (r2=0.876), ec50 (r2=0.585) indicated a positive correlation to pc2. as shown in pca plot, according to the rehydration temperature of 30°c, treated slabs dried at 50° and 55°c are more similar than those dried at 60° and 65°c. furthermore, those dried at 60° and 65°c were more correlated with quality parameters in terms of wac, dhc, ra and whc along pc1, indicating that the drying temperature had significant impact on the ability to reconstitute the water content of apples during rehydration process. at the same rehydration temperature (30°c), untreated apples dried at 50° and 55°c were closer to each other than untreated apples dried at 60° and 65°c. as it is seen from fig. 6, these latter (utr dried at 60° and 65°c) were more correlated with quality parameters of rehydrated apples along pc2, i.e, colour parameters. it is clear that the higher drying temperatures (60° and 65°c) showed the better rehydration results in both utr and tr apples. with regard to the rehydration temperature of 70°c, treated dried samples at 50°, 55° and 60°c were similar. scoring and loading plot enabled to differentiate the behaviour only for treated apples dried 65°c which were more correlated with pc2. untreated samples rehydrated at 70°c shifted from negative values to positive ones along pc2. in conclusion, higher rehydration temperature (70°c) negatively affected quality parameters of rehydrated apples. the samples rehydrated at 30°c showed the better correlations with quality parameters. figure 6. two-dimensional principal component analysis of rehydration properties for untreated (utr) and treated (tr) samples dried at 50°, 55°, 60° and 65°c and rehydrated at 30° and 70°c. (wi = white index; l= lightness; wac = water absorption capacity; dhc = drying matter holding capacity; ra = rehydration ability; whc = water holding capacity; ec50 = antioxidant activity). ital. j. food sci., vol. 32, 2020 812 4. conclusions the investigation of drying/rehydration process conditions and of an alternative dipping pre-treatment on the rehydration curves and qualitative traits of rehydrated ‘annurca’ apple slabs, has shown valuable findings related to keeping quality of apples. rehydration temperatures (30° and 70°c) did not significantly affect the weight gain of dried apples. on the other hand, lower rehydration temperature (30°c) with the pre-treatment had positive effect on the rehydration indices (wac, dhc, ra and whc). higher increases in volume were observed in treated rehydrated samples in comparison with the untreated ones. the pre-treatment was able to preserve the colour properties at both rehydration temperatures, while the lower the drying temperature and the lower rehydration temperature, the higher antioxidant activity for both samples was measured. the results clearly highlighted that the rehydration process and quality of rehydrated apples are influenced by pre-treatment, drying and rehydration temperatures. the combination between dipping pre-treatment with trehalose and optimal drying temperature (65°c) at lower rehydration temperature (30°c) allowed to obtain the best overall reconstitution properties of the rehydrated apples in terms of the rehydration characteristics and structure. thereby, it is recommended to combine the natural pre-treatment and those drying/rehydration process conditions to achieve the high quality attributes of dried/rehydrated apples to meet consumer expectation. these findings, combined with sensorial analysis with trained panel, will contribute to industrial applications and to literature information on the quality attributes of rehydrated apples. thus, the understanding and characterisation of 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botrytis l.). j. food eng. 119:640-647. doi: doi.org/10.1016/j.jfoodeng.2013.06.035 zayas j., 1997. functionality of proteins in food. heidelberg: springer-verlag. zura-bravo l., ah-hen k., vegagálvez a., garcia-segovia p. and lemus-mondaca r. 2013. effect of rehydration temperature on functional properties, antioxidant capacity and structural characteristics of apple (granny smith) slices in relation to mass transfer kinetics. j. food process. eng. 36:559-571. doi: doi.org/10.1111/jfpe.12018 paper received may 17, 2020 accepted september 23, 2020 paper 260 ital. j. food sci., vol. 27 2015 keywords: canned vegetables, extra-virgin olive oil, oxidation, preservation, soybean oil, sunflower oil comparative study of oxidation in canned foods with a combination of vegetables and covering oils e. bravi*, a. mangione, o. marconi and g. perretti department of agricultural, food and environmental science, university of perugia, via s. costanzo n.c.n., 06126 perugia, italy * corresponding author: tel. +39 075 5857923, fax +39 075 5857946, email: e_bravi@yahoo.it abstract the effects of sunflower (sfo), extra-virgin olive (evo), and soybean oils (sbo), in combination with canned aubergins and dried tomatoes were studied during an accelerated shelf-life trial. hydrolytic and oxidative quality parameters was determined and a sensorial test was run. for both canned vegetables, the sbo showed greater resistance to the oxidation at the end of the shelflife trial. the sbo in both vegetables yielded similar results for peroxide formation, whereas a reduced formation of secondary oxidation products was observed in aubergins. the results highlighted a higher oxidation stability of canned vegetables in sbo and evo than those in sfo. the sensorial test underlined differences between the oils, in aubergins and dried tomatoes, after 30 days of accelerated storage (corresponding to the sell-by date). flavour and texture were judged better for vegetables in sbo. ital. j. food sci., vol. 27 2015 261 1. introduction canned foods are products packed and hermetically sealed in metal (tin, aluminum), glass, or polymer containers that are thermally treated to destroy spoiling microorganisms and their enzymes with the aim to prolong the shelf-life. (leistner, 1992). moreover, the process of preparation and preservation could increase the quality of vegetable products because of the presence of added oils as cover liquids (leistner, 1992). in canned vegetables: i) the covering oil promotes anaerobic conditions (i.e., less than 2% oxygen); ii) an adequate blanching treatment reduces numbers of contaminating microorganisms, inactivates enzymes, modifies texture, preserves color, flavor, and nutritional value, and removes trapped air; and iii) pasteurization guarantees commercial sterility (reyes de corcuera et al., 2004; baiano et al., 2005a). in the food industry, virgin and extra-virgin olive oil, olive oil, seed oils, or various oil blends, are used as covering oils to prepare preserved vegetables. the quality of canned vegetables in-oil depends on the complex interactions between traits of the vegetables and those of the covering oils. during storage, many bioactive molecules migrate from the vegetable to the oil and vice versa, in a dynamic equilibrium that depends on the characteristics (e.g., chemical composition, structure, size, and shape) of vegetables and oils as well as on the technology used for preparation and storage (lucchetti et al., 2011). spices and aromatic herbs, generally used as flavor enhancers in preparation of canned vegetables, also contribute to the quality of the canned product. they contain substances with documented antimicrobial, antioxidant and anti-inflammatory activities (gambacorta et al., 2007). the shelf-life of in-oil canned vegetables depends on the quality of the vegetable and the covering oil and its composition (baiano et al., 2005a). during processing and over the time, vegetables and oils undergo modifications because of mechanical and thermal degradation and hydrolytic and oxidative degradation (affecting the quality of oil and preserved food) (de giorgi et al., 2000; baiano et al., 2005b). some studies have examined the oxidative and hydrolytic reactions that occur in the covering oil, and on the effects of the use of different oils as a covering medium on the canned vegetables preservation (leistner, 1992; baiano et al., 2005a; baiano et al., 2005b; baiano et al., 2005c). the aim of the present paper was to compare the effects of the combination of different vegetables and covering oils in canned foods. sunflower oil (sfo), extra-virgin olive (evo) and soybean oil (sbo) in combination with canned aubergins and dried tomatoes were studied. the quality of covering oils was studied to verify the effect of these combinations during storage. hydrolytic and oxidative quality parameters were measured together with sensory analysis. sfo was examined because of its wide use in the production of canned foods, evo was examined because of its sensorial and nutritional properties, and sbo was examined for its nutritional value, widespread use, and low cost. moreover, the effects of aromatic herbs and spices (ingredients included in the recipes for canned vegetables) on oxidative degradation of the three oils were examined as a separate test on pure oils. the herbs and spices in the recipe were: i) garlic, rich in flavonoids and sulfurcontaining compounds (leelarungrayub et al., 2006); ii) chili pepper, containing allicin, carotenoids, ascorbic acid, and phenolic compounds (suhaj, 2006); iii) oregano, containing various flavonoids (kyoji et al. 2006); and iv) mint, rich in polyphenols and flavonoids (kanatt et al., 2007; padmini et al., 2008). 2. materials and methods both commercial in-oil canned aubergins and dried tomatoes were prepared according to traditional recipes with three different vegetable oils (sfo, evo, and sbo). the oil samples were provided by vizzino “orto buono,” minervino di lecce, italy. all canned products were produced on the same day and analysed, at time zero (t0) and 15 days after processing (t1). 2.1. preparation of in-oil canned vegetables the canned vegetables used were aubergins in sfo (ausfo), evo (auevo), or sbo (ausbo), and dried tomatoes in sfo (dtsfo), evo (dtevo), or sbo (dtsbo). traditional recipes included the addition of chili pepper (0.001 g kg-1 of total product), oregano (0.001 g kg-1), mint (0.001 g kg-1), and garlic (0.004 g kg-1) for canned aubergins and mint (0.001 g kg-1) and garlic (0.004 g kg-1) for canned sun-dried tomatoes. a batch of 150 kg of 1 cm slices of peeled aubergins were previously treated with coarse salt (20% w/w), then drained and washed with water and centrifuged. then the slices of aubergins dehydrated were mixed with powdered herbs and spice (chili pepper, garlic, oregano, and mint) and a dose of 280g was put in transparent glass vessels, wrapped with metal caps. vessels were then filled in the three considered oils (vegetables 65%, oil 33%, w/w) and hermetically sealed. a batch of 150 kg of dried tomatoes were blanched in boiling white vinegar for 30 seconds, then drained and dried, mixed with aromatic herbs (garlic and mint). transparent glass vessels were filled with 280g of tomatoes and in the three considered oils (vegetables 65%, oil 33%, w/w), and hermetically sealed. the canned samples were pasteurized at 90°c for 40 minutes, and then quickly cooled to room temperature. 262 ital. j. food sci., vol. 27 2015 2.2. aromatic herbs and spices added to oils a case study (separate test on pure oils with herbs and spices) was conducted to investigate the efficacy as antioxidant, and to verify the protective effects of aromatic herbs and spices present as ingredients in the traditional recipes for considered canned foods. for this purpose sfo, evo, and sbo, used in canned food preparation, were prepared with the different aromatic herbs and spice used in the recipe: chili pepper, garlic, oregano and mint (to investigate the effect of each single herb or spice); a mixture of mint and garlic (to simulate the dried tomatoes recipe); and a mixture of all the aromatic herbs and spice (to simulate the aubergins recipe). the spice and aromatic herb contents were chosen to simulate the same concentrations used in the recipes for canned vegetables in oils. the samples to analyze were: i) sfo, evo, or sbo, with added garlic (0.1 g/100ml of oil); ii) sfo, evo, or sbo with added chili pepper (0.03 g/100ml); iii) sfo, evo, or sbo with added oregano (0.03 g/100ml); iv) sfo, evo, or sbo with added mint (0.03 g/100ml); v) sfo, evo, or sbo with added mint and garlic (0.03 g/100ml and 0.1 g/100ml, respectively); and sfo, evo, or sbo with all aromatic herbs and spice under consideration added. the samples were subjected to the same accelerated shelf-life test (aslt) for canned vegetables and the extent of the hydrolytic and oxidative degradation of oils was determined by assessing peroxide numbers and p-anisidine values. 2.3. accelerated shelf-life test (aslt) to evaluate the shelf-life of the canned vegetables and of the pure oils with added herbs and spices, an accelerated shelf life test (aslt), in which the storing of canned foods for 1 day at 55 °c corresponds to 18 days at room temperature (20°c), was performed (robertson, 1993; kilcast, 2000; man, 2015). accelerated aging was defined according to a common industrial method based on the arrhenius equation, which defines the relationship between product shelf-life and the temperature (robertson, 1999; giménez et al., 2012; marconi et al., 2014;). the glass vessels with the vegetables canned in different covering oils were kept in a laboratory oven (thermo fisher scientific, milan, italy) at 55°c for 10, 20, 30, and 40 days, which corresponds to 6 (t1), 12 (t2), 18 (t3) and 24 (t4) months at room temperature, respectively. the vegetable oils were then subjected to chemical and sensorial analysis. 2.4. chemical analyses the quality of the crude oils and the ongoing hydrolysis and oxidation of the covering oils were monitored by measurements of the acidity, expressed as g of oleic acid per 100 g of oil; peroxide values, expressed as milliequivalents (meq) of active oxygen per kg of oil (e. u, 1348/2013), p-anisidine values (aocs, 1998). the hydrolytic and oxidative parameters were determined by conventional methods of analysis. after separation from the vegetable matrices, the covering oil samples were filtered on anhydrous sodium sulfate and analysed after different storage times. 2.5. sensory evaluation sensorial test was conducted to evaluate the palatability of the canned vegetables preserved in different oils at t0 and t3 (at the sell by date of canned food). nine trained panelists (six women and three men), 30–50 years, evaluated the canned vegetables using the quantitative descriptive analysis technique. the panelists were trained in 10 sessions, using standards and similar food products, to identify and determine descriptors relating to appearance, taste, and texture. the terms and the corresponding definitions (table 1) were available to the panelists during all sessions. the evaluation of the canned vegetables was performed over two days in two sessions. canned vegetables were served at room temperature. crackers were used as a carrier for tasting. different canned vegetables, in the different covering oils, were evaluated for sensory attributes, which included appearance (color), flavor (rancid, salty, and bitter), and texture (hardness and chewiness). a 10-point table 1 definition of physical and flavor descriptors used in the quantitative descriptive analysis. descriptor definition physical color intensity of vegetable color. hardness by steadily compressing the vegetable between the molars, the force required for compression. chewiness the lenght of the time required to masticate the vegetable to a state of swallowing. flavour rancidness unpleasant, stale smell or taste proper of oils and fats. saltiness taste of salt perceptible on the tip of the tongue and on the sides around it. bitterness harsh, disagreeably acrid taste. ital. j. food sci., vol. 27 2015 263 scale (from 0 to 9) was used where color (0 = extremely light to 9 = extremely dark); hardness (0 = extremely soft to 9 = extremely hard); chewiness (0 = none to 9 = extremely gummy); and rancidity, saltiness, and bitterness (0 = none to 9 = extremely strong) were assessed. 2.6. statistical analysis all data were analysed using sigmastat (version 11, jandel scientific, san rafael, ca) software to perform the appropriate statistical tests. comparisons of the different vegetable and oil samples were made using one-way repeated measures analysis of variance, and the results obtained were further analysed using the holm-sidak test. each canned vegetable sample was produced in duplicate and all chemical analyses were performed in triplicate. values were considered significantly different at p < 0.05. 3. results and discussion 3.1. oils the quality parameters (free acidity, peroxide values, p-anisidine values, and fatty acid composition) of the three oils, employed as covering oils in the preparation of canned vegetables, are discussed. the acidity values were lower than 0.3% in all samples (0.11, 0.28, and 0.15 g oleic acid/100g for sfo, evo, and sbo, respectively), which confirms low levels of hydrolytic activity in the oils comparable with cold pressed and virgin oils (codex-stan 210-1999; ec, 1513/2001). the peroxide values were low for all oils (4.22, 13.55, and 2.93 meqo 2 /kg, for sfo, evo, and sbo, respectively), even if comparatively high in evo (a value that was below the legal limits (eec, 2568/91). the p-anisidine values in the three oils were low according to gupta (2005) and list et al. (1974), and the lowest value was observed for sbo (6.39, 6.42, and 1.95 for sfo, evo, and sbo, respectively). 3.2. preserved vegetables the free acidity values of the covering oils for canned aubergins and dried tomatoes during the aslt are reported in fig. 1. free fatty acids (ffa) are formed by chemical or enzymatic hydrolysis of triglycerides and may get promoted by the reaction of oil with moisture (naturally present in vegetables), ffas content is an important measure of alteration for oils. in all considered sample the free acidity increased during the accelerated aging. fig. 1 free acidity of covering oils in canned aubergins (a) and in canned dried tomatoes (b) as a function of storage. n = 6; bars in the figures represent standard deviation values; evo = extra-virgin olive oil. t0: 0 days; t1: 10 days; t2: 20 days; t3: 30 days; t4: 40 days 264 ital. j. food sci., vol. 27 2015 for canned aubergins (fig. 1a), the ffas percentage increased from 0.21 to 0.68% for sfo, from 0.35 to 0.60% for evo, and from 0.30 to 0.46% for sbo. for canned dried tomatoes (fig. 1b) and the three different covering oils used, the free acidity increased during the aslt. the ffas contents increased from 0.27 to 0.67% for sfo, from 0.43 to 0.92% for evo, and from 0.28 to 0.58% for sbo. despite contact with the moist vegetable matrix, the acidity values were satisfactory. after 30 days of aslt (18 months of conventional storage at room temperature), the changes in acidity for both aubergins and tomatoes were less than 1%. for aubergins, the acidity value for evo was under the maximum limit for extravirgin olive oil (eu, 61/2011), and for tomatoes, the value exceeded the maximum legal limit after 30 days of accelerated aging, anyway after the sell-by date of the canned product. fig. 2 shows the peroxide values and the panisidine numbers of the different covering oils for the canned aubergins (fig. 2a) and dried tomatoes (fig. 2b), respectively. the peroxide value indicates the level of rancidity that normally occurs in oils because of progressive unsaturated fatty acid oxidation. for canned aubergins (fig. 2a), at t0, which corresponds to the fresh product, the peroxide values were unaltered or, for sbo, slightly increased with respect to the pure oils used as covering medium. the peroxide values for the product at the beginning of the shelf-life trial was similar for the canned dried tomato covering oils, nearly the same for sfo and evo, and slightly increased for sbo as compared to the peroxide values of the pure oils (fig. 2b). in all preserved vegetables, the peroxide values decreased after the beginning of the shelf-life trial while peroxide compounds were progressively decomposed into secondary oxidation products (aldehydes and ketones and polymers) which corresponds in an increase of p-anisidine value. the p-anisidine value reveals the presence of secondary oxidation products. in canned aubergins, the peroxides were reduced to one-half the initial value at the end of aslt (fig. 2a); in canned dried tomatoes, the decrease in peroxides was more rapid and was about 1 meq o 2 /kg after 40 days of accelerated aging (fig. 2b). as shown in fig. 2, the p-anisidine values of the oils used for covering the preserved vegetafig. 2 peroxide number and p-anisidine value in canned aubergins (a) and in canned dried tomatoes (b), covered with sunflower oil, extra-virgin oil, and soybean oil, as a function of storage. n = 6; bars in the figures represent standard deviation values. t0: 0 days; t1: 10 days; t2: 20 days; t3: 30 days; t4: 40 days ital. j. food sci., vol. 27 2015 265 bles showed a progressive increase during the aslt. for canned aubergins (fig. 2a), p-anisidine values for evo and sbo covering oils were significantly lower (p < 0.05) than that for sfo for the period of storage, and the decomposition of the hydroperoxides proceeded at a greater rate in sfo than in the evo or sbo. as compared with the p-anisidine values found for evo, the p-anisidine values of soybean oil were lower from two to four weeks of accelerated storage. sbo had the lowest oxidative rancidity during the aslt. comparing the results of the two types of canned vegetables, the p-anisidine values of the covering oils for dried tomatoes were higher than those for aubergins during aslt. however, as in canned aubergins, the p-anisidine numbers of tomatoes in sbo were significantly lower than those of the other oils considered at the sell-by date (fig. 2b). since the data underlined the lowest content of hydroperoxides and the simultaneous lowest value of p-anisidine for aubergins and dried tomatoes in sbo and since the sbo showed the lowest value of free acidity, we may conclude that sbo has appreciable characteristics of stability to oxidation as covering oil in canned vegetables. a high stability of sbo under the similar conditions of storage was underlined by warner et al. (1989) in a previous study. dried tomatoes exhibited a higher index of secondary oxidation (p-anisidine value) than aubergins. at the sell-by date (18 months, 30 days of accelerated aging), the p-anisidine value of canned dried tomatoes was 12.66 ± 0.02, 17.38 ± 0.10, and 17.53 ± 0.10 for sbo, evo, and sfo respectively. conversely, in aubergins, the highest p-anisidine value at the sell-by date was 10.80 ± 0.13 in sfo. differences in oxidation of covering oils for aubergins and dried tomatoes could justify consideration of the additive protective effects of antioxidant compounds present in aubergins (such as phenolic compounds, flavonoids, ascorbic acid, and vitamin a) and in dried tomatoes (lycopene, ascorbic acid, phenolic, flavonoids, and vitamin e) (hung and duy, 2012), and the antioxidant effects of aromatic herbs and spices added (chili pepper, garlic, oregano, and mint). aubergin is one of the most active vegetables in its free radical scavenging capacity because of its phenolic constituents (jung et al., 2011; hung and duy, 2012). considering aromatic herbs and spices, the antioxidant effects of the pungent component of chili pepper, capsaicin, has been documented in previous studies (reyes-escogido et al., 2011). henderson and henderson (1992) observed that the oxidation of oleic acid at cooking temperatures was inhibited by the presence of capsaicin. in addition, capsaicin is reported to inhibit lipid peroxidation (salimath et al., 1986; pulla reddy and lokesh, 1992; asai et al., 1999; henderson et al., 1999; okada and okajima, 2001; kogure et al., 2002). garlic has been reported to reduce free radical-induced oxidative damage in animal and human models. extensive studies performed on garlic extracts (allium sativum l.) highlighted the presence of two main classes of antioxidants: flavonoids and sulfur-containing compounds (leelarungrayub et al., 2006). the four main garlic antioxidant compounds are alliin, allyl cysteine, allyl disulfide, and allicin (benkeblia, 2005; el shenawy et al., 2008). oregano (origanum majorana) is one of the aromatic herbs known to possess antioxidant compounds such as rosmarinic acid, caffeic acid, and various flavonoids. oregano extracts have shown a pronounced effect on stabilizing lipids against autoxidation (kyoji et al. 2006). the effectiveness of mint, a common aromatic herb, as a natural antioxidant is widely documented (kanatt et al., 2007; padmini et al., 2008; sazhina et al., 2011). for canned aubergins, the antioxidant capacity arises from the typical antioxidant compounds of the vegetable and of garlic, mint, oregano and chili pepper; for preserved dried tomatoes, the antioxidant capacity arises from the vegetable and garlic and mint. the antioxidant capacity of aubergin is higher than that of dried tomato (wu et al., 2004). the aromatic herbs added to canned dried tomatoes were mint and garlic, and the additive effect of these two herbs may be lower than that of the canned aubergins and the herbs and spices used for it. different results for the two canned foods may have been caused by their different antioxidant properties and the different added aromatic herbs and spices. to confirm the above hypothesis, aromatic herbs and spices were added to the pure oils used as packing oils for the vegetables considered. each oil used for each vegetable had different aromatic herbs and spices in the recipe (chili pepper, garlic, oregano, and mint), a mixture of mint and garlic, or a mixture of all aromatic herbs and spices together, and was analysed for peroxide numbers and p-anisidine values. the results are reported in figs. 3, 4 and 5. the peroxide numbers and p-anisidine values of the three oils considered were influenced by the added aromatic herbs and spices. in particular, during the aslt, the oil samples with added garlic, with added mint and garlic, and with all herbs and spices added presented lower values of the oxidation parameters. in sfo, a higher peroxide number was observed in the sample without spices and herbs, whereas chili pepper, oregano, and mint, showed an antagonistic effect on peroxide formation. for p-anisidine, lower values were observed in oil with added garlic and in oil with all herbs and spices added, whereas the oil with added mint and garlic had higher values; however, these values were lower than those found with other added herbs and spices. 266 ital. j. food sci., vol. 27 2015 fig. 3 peroxide number and p-anisidine value of extra-virgin olive oil (a), sunflower oil (b), and soybean oil (c), without herbs and spices and with different herbs and spices, as a function of storage. n = 6; bars in the figures represent standard deviation values. evo = extra-virgin olive oil. t0: 0 days; t1: 10 days; t2: 20 days; t3: 30 days; t4: 40 days. a similar result was found for evo where a low peroxide number was observed in oil samples with added garlic, and with all herbs and spices added. samples with added mint and garlic had the same results as evo with added oregano. the lower p-anisidine value for evo was observed with added garlic, added mint and garlic, and when all herbs and spices were added. for sbo, each of the aromatic herbs and spices influenced the peroxide number and p-anisidine value. a higher protective effect was found for added garlic and for samples with all herbs and spices added. the oil with mint added had a panisidine value similar to the sample with addital. j. food sci., vol. 27 2015 267 fig. 4 peroxide number and p-anisidine value of sunflower oil, without herbs and spices and with different herbs and spices, as a function of storage. ed mint and garlic, which was lower than that for other herbs and spices. for sbo, the worst p-anisidine values were found in sbo with added oregano (the oregano showed, in the case of sbo, an unusual pro-oxidant effect). the sensorial test underlined differences among the covering oils for each vegetable at t0 and at t3 (fresh product and product at its sellby date). table 2 shows the results for quantitative descriptive analysis for ausfo compared to auevo and with ausbo at t0 and at t3. at t0, auevo was significantly different (p < 0.05) from ausfo and ausbo with respect to its color and bitterness, which were judged to be higher than 268 ital. j. food sci., vol. 27 2015 fig. 5 peroxide number and p-anisidine value of soybean oil, without herbs and spices and with different herbs and spices, as a function of storage. those found for the other oils. these differences could be explained by the characteristics of evo. ausbo was found to have lower rancidity when compared with the other samples. at the sell-by date, the quality of ausbo differed from the other oils in its hardness, chewiness, and bitterness. ausbo was judged slightly harder, less gummy, and less bitter than the other products. table 3 shows the results for dtsfo as compared to dtevo and dtsbo at t0 and at t3. at t0, dtevo was judged to differ from dtsfo and dtsbo with respect to its bitterness, which was slightly higher than that of the other products. dtsbo was found to have lower ital. j. food sci., vol. 27 2015 269 rancidity and saltiness than the other samples. at t3 the dtevo was judged to differ from the other samples in its color and rancidness, and dtsbo was reported to be less gummy and less rancid than the other oil samples. 4. conclusions the results obtained from this study underline the higher resistance to oxidation of vegetables canned in sbo and in evo. a higher concentration of secondary oxidation catabolites was observed in oils covering dried tomatoes than in oils covering aubergins. this might be related to the additive protective effects of antioxidant compounds found in the vegetables and the antioxidant effects of added aromatic herbs and spices (hung and duy, 2012). data obtained from the case study, conduct to investigate the efficacy as antioxidants of aromatic herbs and spice table 2 sensory evaluation scores of ausfo, auevo and ausbo at t0 and t3. sample colora hardnessb chewinessc rancidnessd saltinessd bitternessd ausfo t0 6.70a 6.93a 5.96a 1.33a 4.46a 2.67a auevo t0 7.27b 7.10a 5.6a 1.13b 4.16a 3.13b ausbo t0 6.63a 7.03a 5.30a 0.91c 4.13a 2.47a ausfo t3 6.30a 5.39a 6.65a 1.22a 4.78a 3.96a auevo t3 6.57a 5.48a 6.21b 1.30a 4.48a 3.60a ausbo t3 6.29a 6.43a 5.43a 1.45a 4.71a 2.58b n = 18; t0 = 0 months; t3 = 18 months. pairs (ausfo t0 compared with auevo t0 and with ausbo t0; ausfo t3 compared with auevo t3 and with ausbo t3) with the different letters within the same column are significantly different (p < 0.05). ausfo: aubergins in sunflower oil. auevo: aubergins in extra-virgin olive oil. ausbo: aubergins in soybean oil. a = color: 0 = extremely light to 9 = extremely dark. b = hardness: 0 = extremely soft to 9 = extremely hard. c = chewiness: 0 = none to 9 = extremely gummy d = rancidness, saltiness, and bitterness: 0 = none to 9 = extremely strong present as ingredients in the traditional recipes for considered canned vegetables, confirm the higher antioxidant power found upon addition of all herbs and spices considered in the recipe, of an added mixture of mint and garlic, or of added garlic alone, as compared to the pure oils or the oils with other aromatic herbs and spices added, and the minor effects on secondary oxidation of the oils with added mint and garlic. the sensorial investigation partially confirmed the analytical data at t0 with the finding of lower rancidity and, at the sell-by date, a better consistency and lower bitterness of aubergins in sbo. a lower rancidity at t0 and at t3 a better consistency and a lower rancidity were found for dried tomatoes in sbo. the lowest free acidity value, peroxides number, and p-anisidine value for aubergins and dried tomatoes in sbo, and the results of sensorial investigation underline the stability to oxidation and the validity of sbo as covering oil in canned vegetables production. table 3 sensory evaluation scores of dtsfo, dtevo and dtsbo at t0 and t3. sample colora hardnessb chewinessc rancidnessd saltinessd bitternesse dtsfo t0 6.88a 6.73a 4.73a 1.01a 5.98a 2.66a dtevo t0 6.67a 6.81a 4.83a 1.23a 6.32a 3.12b dtsbo t0 6.65a 6.26a 4.69a 0.86b 7.39b 2.53a dtsfo t3 6.81a 6.63a 4.93a 1.67a 5.57a 3.12a dtevo t3 7.43b 6.43a 5.13a 2.13b 6.37b 3.16a dtsbo t3 7.06a 6.72a 4.13b 1.72c 5.45a 3.06a n = 9; t0 = 0 months; t3 = 18 months. pairs (dtsfo t0 compared with dtevo t0 and with dtsbo t0; dtsfo t3 compared with dtevo t3 and with dtsbo t3) with the different letters within the same column are significantly different (p < 0.05). dtsfo: dried tomatoes in sunflower oil. dtevo: dried tomatoes in extra-virgin olive oil. dtsbo: dried tomatoes in soybean oil. a = color: 0 = extremely light to 9 = extremely dark. b = hardness: 0 = extremely soft to 9 = extremely hard. c = chewiness: 0 = none to 9 = extremely gummy. d = rancidness, saltiness, and bitterness: 0 = none to 9 = extremely strong. 270 ital. j. food sci., vol. 27 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35100, bornova, izmir, turkey *corresponding author: tel.: +90 2323112732 e-mail address: gulfem.unal@ege.edu.tr abstract the effect of inulin, polydextrose and hi-maize resistant starch on probiotic viability under simulated gastrointestinal conditions and sensory characteristics of abt (lactobacillus acidophilus la-5 and bifidobacterium animalis subsp. lactis bb-12, and streptococcus thermophilus) fermented milk was investigated during 28 days. b. animalis presented higher survival rates under gastrointestinal stress than l. acidophilus. although inulin addition enhanced viable counts of l. acidophilus more than those of other prebiotics during the gastric and enteric-1 phases, all samples showed similar l. acidophilus survival after the enteric-2 phase. the supplementation with hi-maize indicated a protective effect on b. animalis tolerance to simulated gastrointestinal conditions on the 14th and 28th days. inulin or hi-maize did not affect the sensory properties of fermented milk whereas the product supplemented with polydextrose had the lowest scores specifically at the end of the storage period. keywords: in vitro gastrointestinal survival, fermented milk, prebiotic, probiotic ital. j. food sci., vol. 30, 2018 569 1. introduction probiotics are live microorganisms, which when administered in adequate amount, confer a health effect on the host (fao/who, 2002). to exert their functional properties, probiotics need to be delivered to the desired sites in an active and viable form. probiotic viability should be at a minimum level during the shelf life, which can range from 106 to 108 cfu/ml, and must survive through the gastrointestinal (gi) tract by tolerating acid, bile, and gi tract enzymes (pepsin, lipase, pancreatin) and then adhere and colonize the intestinal epithelium (casarotti et al., 2015). most studies on probiotics have focused on lactic acid bacteria, especially the genus lactobacillus and bifidobacterium. lactobacillus acidophilus and bifidobacterium animalis subsp. lactis are the frequently used probiotics in the production of fermented milks. using different probiotic combinations (ranadheera et al., 2014), microencapsulation (de araujo etchepare et al., 2016), supplementation with prebiotics (oliveira et al., 2009; nobakhti et al., 2009; casarotti and penna, 2015), and the use of different matrices (casarotti et al., 2015) have been proposed to increase probiotic survival in the gi tract and in the product until the time of consumption. among these options, the addition of prebiotics has been preferred in many studies to increase probiotic viability and their resistance to gi conditions. prebiotics are defined as “non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon” (gibson et al., 2004). different prebiotics (e.g., inulin, hi-maize resistant starch, lactulose, polydextrose, β-glucan, lactilol, and maltodextrin) have been used as supplements in the manufacture of fermented dairy products to improve the growth and activities of selected lactobacillus and bifidobacterium strains (oliveira et al., 2009; nobakhti et al., 2009; heydari et al., 2011) in related studies. inulin, a compound extracted from the chicory root, is a fructan and cannot be digested by α-amylase or other hydrolases in the upper section of the intestinal tract (oliveira et al., 2009; gonzález-herrera et al., 2015). aside from its prebiotic property, inulin also presents some technical characteristics, such as being a fat replacer, sugar replacer, and emulsion and foam stabilizer (gonzález-herrera et al., 2015). polydextrose is a low molecular weight randomly bonded polysaccharide of glucose with an energy contribution of 1 kcal/g (do carmo et al., 2016). this low-calorie content of polydextrose is a result of its poor digestibility in the small intestine and incomplete fermentation in the large intestine (oliveira et al., 2009). however, resistant starch is a small starch fraction that has the ability to resist digestion and can be fermented by the beneficial microbiota in the colon (zaman and sarbini, 2016). all inulin, polydextrose and hi-maize resistant starch have been already reported (gonzález-herrera et al., 2015; zaman and sarbini, 2016; de araujo etchepare et al., 2016) as prebiotics and have been showed to enhance the viability of l. acidophilus and b. animalis in fermented dairy products (oliveira et al., 2009; nobakhti et al., 2009; bedani et al., 2013; padilha et al., 2016). however, there is no knowledge about the effect of these prebiotics on probiotic in vitro gastrointestinal tolerance in abt-cultured (streptococcus thermophilus, lactobacillus acidophilus and bifidobacterium animalis) fermented milk. the aim of this study was to investigate the influence of the addition of the inulin, polydextrose and hi-maize resistant starch on the viability of starter culture bacteria, probiotic survival under in vitro simulated gastrointestinal conditions, and sensory characteristics in abt-fermented milk throughout 28 days of storage at 4°c. ital. j. food sci., vol. 30, 2018 570 2. materials and methods 2.1. cultures and ingredients the abt-10 culture (chr. hansen a/s, hørsholm, denmark), composed of streptococcus thermophilus, lactobacillus acidophilus la-5 and bifidobacterium animalis subsp. lactis bb-12, skim milk powder (smp) (pinar dairy products, izmir, turkey), inulin (fibruline® instant, cosucra, warcoing, belgium), polydextrose (litesse® ip powder, danisco, usa), and resistant starch (hi-maize, ingredion, hamburg, germany), were used in this study. the abt-10 culture (pack size of 200u) was poured into 1 l sterilized reconstituted milk at 40°c and mixed thoroughly, and then 1 l of each milk base was inoculated with 1 ml of the culture. the reconstituted milk was prepared from skim milk powder and has 130g/l of total solids. this procedure gave initial counts after milk inoculation of approximately 7 log cfu g-1 for l. acidophilus la-5 and b. animalis subsp. lactis bb-12 and 8 log cfu g-1 for s. thermophilus. 2.2. production of fermented milk in the production of fermented milk, cow’s milk containing 31 g/l fat and 29.2 g/l protein was supplied from ege university, agricultural faculty (izmir, turkey). after standardizing it with skim milk powder to obtain 110 g/l of nonfat milk solids, the milk was divided into four lots. the control milk was not supplemented with prebiotics, whereas the other three groups were supplemented with 20 g/l inulin, polydextrose and resistant starch. after they were mixed properly, each milk base was heated to 90°c for 10 min by circulating it in a hot water bath and cooled to 42-43°c in an ice bath. at this point, they were inoculated with the abt-10 culture. the mixtures were put into 100-ml plastic containers and incubated at 40°c until a ph of 4.75 was reached. after fermentation, the fermented milk samples were cooled and transferred to a refrigerator at 4°c, then stored at this temperature for 28 days during the analyses. 2.3. determination of ph and microbiological analyses the ph of the fermented milk was determined using a ph meter (model ph 211; hanna instruments, woonsocket, ri). the viability of bacteria in the abt-10 culture was determined according to akalin and ünal (2010). the counts of s. thermophilus were enumerated on m-17 agar (merck, darmstadt, germany) after incubating the plates aerobically at 37°c for 48 h. b. animalis subsp. lactis bb-12 was enumerated using mrs-nnlp (nalidixic acid, neomycin sulfate, lithium chloride, and paramomycin sulfate) agar. the inoculated plates were incubated anaerobically at 37°c for 72 h using an oxygen-free milieu and a co2 atmosphere in anaerobic jars (merck, darmstadt, germany). the counts of l. acidophilus la-5 were enumerated on mrs-sorbitol (merck, darmstadt, germany) agar after incubating the plates anaerobically at 37°c for 48 h in anaerobic jars. 2.4. survival of l. acidophilus and b. animalis subsp. lactis under simulated gastrointestinal conditions the probiotic survival in the fermented milk samples subjected to gastric and enteric simulated conditions was evaluated after 1, 14 and 28 days of refrigerated storage according to the methods described by bedani et al. (2014) and casarotti and penna (2015), but with some modifications. each fermented milk sample was placed into ital. j. food sci., vol. 30, 2018 571 3 sterile flasks in order to perform the phases of simulated gastrointestinal conditions. 10 ml of sample, which was diluted in 0.5% nacl, was used for the method. prior to the gastric stage the sample is brought to ph 2.2-2.6 with 0.5 m hcl. pepsin (from porcine gastric mucosa, sigma-aldrich) and lipase (amano lipase g from penicillium camemberti, aldrich chemical company, st. louis, mo, usa) solutions were added to the sample to reach a concentration of 3 g/l and 0.9 mg/l, respectively. the sample is placed for 2 h at 37°c in a shaking (150 rpm) waterbath (mikrotest, mcs series, ankara, turkey), leading to the simulated gastric phase. after then, the ph was adjusted to 4.3-5.2 with an alkaline solution (150 ml of 1 n naoh and 14 g of po4h2na.2h2o and distilled water up to 1 l), which contained 10 g/l of bile (bovine bile, sigma-aldrich) and 1 g/l of pancreatin (from porcine pancreas, sigma-aldrich) in the final mixture. the sample was incubated again at 37°c in the water bath for 2 h for enteric phase 1. for the last stage, the ph level was adjusted to 7.0-7.3 using the same alkaline solution and the concentrations of bile and pancreatin were adjusted to 10 g/l and 1 g/l, respectively in the final mixture. the sample was then incubated at 37°c for the last 2 h for enteric phase 2. the viable counts of l. acidophilus and b. animalis were determined after each phase. 2.5. sensory evaluation a sensory evaluation of the samples was carried out according to the method modified from turkish yogurt standard (1989) and martín-diana et al. (2003). the panel group consisted of 8 experienced academicians from the department of dairy technology (ege university, izmir, turkey) who were familiar with attributes of fermented milk samples. sensory evaluation consisting of appearance, aroma, taste, texture, and overall acceptability were based on 5-point hedonic scales (1: dislike extremely; 5: like extremely). each sample was scored individually, and the samples were presented to the panelists inside individual plastic containers. fermented milks, coded with 3 digits, were randomly presented to the panel group at each session. panelists evaluated all of the samples after storage for 1, 14, and 28 d at 4°c. 2.6. statistical analysis the experiments, including fermented milk making, were performed in triplicate. six values for each sample were averaged (n = 6). the results were analyzed using a one-way analysis of variance (anova) and the general linear model (glm) procedure of the spss software (version 11.05; spss inc., chicago, il). the treatments were compared among each other in the same storage day, and the fermented milks of the same treatment were compared throughout the time in terms of in vitro simulated gastrointestinal tolerance. the means were compared using the duncan multi-comparison test at the p < 0.05 level. 3. results and discussion 3.1. ph values and microbiological characteristics the ph values of fermented milk samples during refrigerated storage are shown in fig. 1. the values for all fermented milk types ranged from 4.73 to 4.37 during storage. although some fluctuations are observed, all products presented significant ph reduction (p < 0.05) at the end of the storage term when compared to the beginning. ital. j. food sci., vol. 30, 2018 572 figure 1. changes in ph values in fermented milk control (fmc) without addition of prebiotic (◊), fermented milk with addition of 2% inulin (fmi) (□), fermented milk with addition of 2% polydextrose (fmp) (∆), fermented milk with addition of 2% hi-maize (fmh) (x). the ph values of the control fermented milk were found to be lower than those of prebiotic added samples during 28 days, which can be attributed to the buffering capacity of ingredients used in the fortification of the fermented milk samples (helland et al., 2004). similar results were obtained for the control product when compared to prebiotic added samples in other studies (nobakhti et al., 2009; bedani et al., 2013). in contrast to our study, there were no significant differences between the ph values of the control products and prebiotic added products in some studies that could be attributed to the type of product or prebiotic used in these studies (oliveira et al., 2009; heydari et al., 2011; srisuvor et al., 2013). the viability of l. acidophilus, b. animalis subsp. lactis and s. thermophilus during refrigerated storage lasting 28 days is presented in table 1. the population of s. thermophilus remained above 8 log cfu/g throughout the storage period. however, the counts of probiotic bacteria (l. acidophilus and b. animalis) were maintained at the minimum effective dose for beneficial health effects, which has been suggested to be between 106-108 cfu/g, in all treatments during the storage time. in general, the addition of inulin, polydextrose and hi-maize provided a protective effect on the survival of probiotic bacteria by not allowing any decline in viability during the 28 days. viable counts of l. acidophilus have been reported as lower in the hi-maize added fermented milk drink than that of the control sample (nobakhti et al., 2009), which parallels our results. l. acidophilus has also been shown to not be stimulated by inulin in acidophilus-bifidus yoghurt by ozer et al. (2005) and in fermented soy product by bedani et al. (2013). similar results were observed in some other studies (buriti et al., 2010; heydari et al., 2011). however, supplementation with polydextrose enhanced the survival rate of l. acidophilus more than supplementation with inulin throughout the 28 days of our study. allgeyer et al. (2010) obtained parallel results for yoghurt drinks containing both l. acidophilus la-5 and b. animalis bb-12 during 30 days of storage. the viable counts of b. animalis bb-12 significantly decreased throughout the storage in all treatments except for the sample containing hi-maize. the highest viability of bb-12 was ital. j. food sci., vol. 30, 2018 573 detected in the control sample on 1st and 14th days, whereas fermented milk fortified with hi-maize had the highest value at the end of the storage period (p < 0.05). table 1. changes in the viable counts of l. acidophilus, b. animalis, and s.thermophilus during refrigerated storage of fermented milks (log cfu/ml). storage days products 1 14 28 l. acidophilus fmc 7.45±0.03ab 6.91±0.03ac 7.51±0.02aa fmi 7.38±0.02ba 6.94±0.03ab 6.96±0.17cb fmp 7.42±0.02aa 6.82±0.08abb 7.36±0.07ba fmh 6.84±0.05cb 6.77±0.18bb 7.47±0.07aba b. animalis fmc 7.95±0.01aa 7.80±0.03ab 7.63±0.07bc fmi 7.41±0.03db 7.61±0.05ca 7.22±0.07dc fmp 7.85±0.05ba 7.70±0.05bb 7.42±0.01cc fmh 7.50±0.04cc 7.61±0.04cb 7.74±0.09aa s. thermophilus fmc 8.82±0.09aa 8.31±0.07cb 8.43±0.14ab fmi 8.30±0.03bb 8.48±0.07ba 7.99±0.13cc fmp 8.40±0.09bb 8.74±0.07aa 8.15±0.16bcc fmh 8.10±0.11cb 8.09±0.09db 8.32±0.11aba values are means of triplicates. fmc: fermented milk control without addition of prebiotic; fmi: fermented milk with addition of 2% inulin; fmp: fermented milk with addition of 2% polydextrose; fmh: fermented milk with addition of 2% hi-maize a-cmeans ± standard deviations in the same row with different superscript lowercase letters are significantly different (p < 0.05). a-dmeans ± standard deviations in the same column with different superscript uppercase letters are significantly different (p < 0.05). nobakhti et al. (2009) reported that the addition of hi-maize significantly increased the bacteria level of b. animalis bb-12 in the fermented milk drink immediately after fermentation. however, there were no significant differences (p > 0.05) in b. animalis bb-12 counts between aby-type probiotic yoghurt samples supplemented with 1.5% inulin and 1.5% hi-maize during 21 days of storage in another study (heydari et al., 2011). even though some fluctuations were observed in the population of s. thermophilus, in general, the counts significantly reduced at the end of the storage term when compared to the 1st day. similar fluctuations in viable counts of this microorganism were also reported in other studies (akalin and ünal, 2010; bedani et al., 2013; casarotti and penna, 2015). the viable counts were mostly lower in supplemented fermented milk samples compared with those of the control fermented milk during storage in our study; thus, it is obvious that the addition of the prebiotic did not improve the viability of s. thermophilus. in contrast, inulin addition improved the survival of s. thermophilus in fermented soy abt milk during 28 days of storage. this difference can be related to the high ability of this bacterium to metabolize soy oligosaccharides (donkor et al., 2007). ital. j. food sci., vol. 30, 2018 574 3.2. the survival of l. acidophilus and b. animalis subsp. lactis under simulated gastrointestinal conditions the survival of l. acidophilus and b. animalis exposed to in vitro simulated gastrointestinal conditions is shown in figs. 2 and 3, respectively. in general, there was a significant reduction (p < 0.05) in the population of both la-5 and bb-12 during the simulation of the in vitro gi stress, which was also observed in other studies (casarotti and penna, 2015; casarotti et al., 2015). b. animalis bb-12 presented higher survival rates during the in vitro assay than l. acidophilus, especially on the 1st and 14th days, this was also observed in many studies (casarotti and penna, 2015; casarotti et al., 2015). the resistance of both probiotic bacteria to simulated gi conditions significantly decreased during storage time for all treatments (data not shown). this behavior can be related to the sensitivity of bacteria in that cells were more stressed and damaged by the cold storage at the end of the storage period compared to the beginning (wang et al., 2009). vinderola et al. (2011) also reported a significant reduction in probiotic resistance to gastric stress in fermented milk throughout 20 days of refrigerated storage. it has been also reported that the bile resistance of probiotic bacteria can be poor when used in the presence of each other compared with monoculture. this might probably be related to the potential antagonism between each other in bile salt stress (ranadheeta et al., 2014). a competition between bacteria probably occurs so that each bacterium can use essential nutrients for its growth and survival (srisuvor et al., 2013). the counts of l. acidophilus decreased by 2-3 log cycles after 2 h of the gastric phase. this shows that l. acidophilus is highly susceptible to simulated gastric juice containing hcl and pepsin, because the highest reduction in survival was observed during the gastric phase during all storage days. it can be related to the acid tolerance of lactic acid bacteria, which varies by species and strains, as well as exogenous conditions, growth medium, and incubation parameters (madureira et al., 2011). no recovery of viability of this microorganism was detected after the ph level was increased in the enteric phases of the assay. although there were significant differences among fermented milk samples for the gastric phase, the viability of l. acidophilus was generally similar after 6 h of assay on the 1st, 14th, and 28th days. on day 1, the supplementation with polydextrose and hi-maize protected the l. acidophilus cells in the presence of low ph (2.2-2.6); however, fermented milk fortified with hi-maize had the highest counts. there were no significant differences among all treatments when ph was increased to 4.3-5.2 (p > 0.05) at the beginning of the storage. however, fortification with inulin caused an increase in the survival of l. acidophilus during gastric and enteric phase 1 on the 14th and 28th days compared to the fortification with polydextrose and hi-maize. the protective effect of inulin can be attributed to the resistance of inulin to hydrolysis by the gi tract enzymes and to its high degree of polymerization (dp) when compared to short chain fructooligosaccharides. ital. j. food sci., vol. 30, 2018 575 (2a) (2b) (2c) figure 2. survival of l.acidophilus la-5 (log cfu/ml) in fermented milk after 1, 14, and 28 days of storage (a, b, and c, respectively), before (black bar) and during exposure to in vitro simulated gastric conditions, for 2 h (dark gray bar) and enteric conditions, for 4 h (light gray bar) and 6 h (white bar). for the same storage period, a-cdifferent superscript capital letters denote significant differences between formulations for the same sampling period of the in vitro assay (p < 0.05); a-ddifferent superscript lowercase letters denote significant differences between different sampling periods of the in vitro assay for the same formulation (p < 0.05). fmc: fermented milk control, without addition of prebiotic; fmi: fermented milk with addition of 2% inulin; fmp: fermented milk with addition of 2% polydextrose; fmh: fermented milk with addition of 2% hi-maize. aa bb ac bd ba bb ac ac aa abb ac ac ca ab ac ac aa ab ac ad aa ab ac ad aba bb bc ac ba bb bc ac aa ab ac ad ca ab ac ad ba bb bc ac aba bb bc ac ital. j. food sci., vol. 30, 2018 576 (3a) (3b) (3c) figure 3. survival of b. animalis bb-12 (log cfu/ml) in fermented milk after 1, 14, and 28 days of storage (a, b, and c, respectively), before (black bar) and during exposure to in vitro simulated gastric conditions, for 2 h (dark gray bar) and enteric conditions, for 4 h (light gray bar) and 6 h (white bar). for the same storage period, a-cdifferent superscript capital letters denote significant differences between formulations for the same sampling period of the in vitro assay (p < 0.05); a-ddifferent superscript lowercase letters denote significant differences between different sampling periods of the in vitro assay for the same formulation (p < 0.05). fmc: fermented milk control, without addition of prebiotic; fmi: fermented milk with addition of 2% inulin; fmp: fermented milk with addition of 2% polydextrose; fmh: fermented milk with addition of 2% hi-maize. aa ab ac bd da aa ab bc ba ab ac abd ca aa ab ac aa bb bc bcc ca bb bd bc ba cb ac cd cb aa ac ac ba ab ac bd da ab ac cd ca cb bc cd aa bb ac ac ital. j. food sci., vol. 30, 2018 577 inulin with high a dp has low solubility and an increased capacity to form a tridimensional network of microcrystals in the food matrix in which it is added (buriti et al., 2010). this structure containing small aggregates can act as a protective physical cover for bacterial cells against acid and bile (casarotti et al., 2015). on the other hand, hernandez-hernandez et al. (2012) reported that resistance to bile in lactobacillus strains is dependent on carbon source. hydrophobic index of bacteria, which is related to their adhesion capacity to intestinal cells, has been also reported to vary depending on the lactobacillus strain by the same researchers. the addition of a mixture of inulin and fructooligosaccharide in the petit-suisse cheese containing the abt-4 culture resulted in a protective effect for the probiotic survival during 6 h of in vitro simulated assay (padilha et al., 2016). the authors emphasized that this protective effect of prebiotics might be specific for the food matrix. in this study, even though the prebiotics used had significantly different effects on the viability of l. acidophilus during in vitro simulated gi conditions among each other, they generally maintained the viable counts. b. animalis bb-12 was highly resistant to simulated gastric conditions in all fermented milks on the 1st and 14th days, whereas the viability decreased (p < 0.05) 1-2 log cycles after gastric phase at the end of the storage term. although b. animalis showed higher survival rates in the presence of bile and pancreatin than l. acidophilus, significant reductions in the viability of b. animalis were observed during enteric phases of the assay. the higher survivability of b. animalis bb-12 compared to that of l. acidophilus during in vitro simulated gi conditions has also been reported by other authors (bedani et al., 2013; 2014; casarotti and penna, 2015). crittenden et al. (2001) demonstrated that b. animalis bb-12 was both acid and protease tolerant among commercial strains and able to survive well in an in vitro model. the ability of bifidobacterium strains to improve their own tolerance to gastrointestinal environment has been revealed, which is a considerable factor in the performance of strains in the gi tract. bifidobacteria can adapt their enzymatic systems to the different barriers found along gi tract. they have the ability to increase the activity of the membrane-bound f0f1-atpase enzyme, which pumps protons from cytoplasm to the extracellular environment. when the cells are previously exposed to acidic conditions, the f0f1-atpase enzyme is overproduced, and better control of the intracellular ph is observed (sanchez et al., 2013). this can be the reason for the higher tolerance of b. animalis bb-12 to simulated gastrointestinal conditions in this study. as some strains of bifidobacteria may have acid stress throughout the gastric conditions (huang et al., 2014), the intrinsic tolerance of the strains has a decisive influence. the high resistance of b.animalis subsp. lactis to both oxygen and gastrointestinal stress was also verified by other researchers (perrin et al., 2000; andriantsoanirina et al., 2013; ambalam et al., 2014). bile and bile components have been reported to affect the adherence of bifidobacteria in the gastrointestinal tract (kociubinski et al., 2002). the improved tolerance of b. animalis bb-12 in this study can also be attributed to having bile salt hydrolase activity, which is active during its transit through the gastrointestinal tract (picard et al., 2005). however, begley et al. (2005) reported that the tolerance of grampositive probiotic bacteria to bile is a strain-dependent characteristic that should not be generalized in terms of species. supplementation with hi-maize caused a significant increase in the survival of bb-12 during the assay; however, the most effective improvement of hi-maize on bb-12 survival was observed in enteric phase 2 during all storage days. this probably can be caused by the slow degradation of resistant starch in the first part of the gi tract and so, it can reach the distal part of the colon and show a prebiotic effect (zaman and sarbini, 2016). the authors also reported that the amylose to amylopectin ratio is an important property to determine the resistance of a starch and its enzymatic digestibility. the related mechanism has been explained as the interaction of amylose molecules with amylopectin which can ital. j. food sci., vol. 30, 2018 578 influence the accessibility of enzymes to hydrolyze starch molecules. in addition, nugent (2005) reported that hi-maize, which belongs to class 2 of resistant starches, comprises specially structured granules that prevent digestive enzymes from hydrolyzing them. according to this study, supplementation with inulin may increase the viability of l. acidophilus la-5, whereas hi-maize resistant starch can be preferred as a prebiotic ingredient to enhance the viable counts of b. animalis bb-12 during the simulation of gi conditions. therefore, choosing a suitable prebiotic for the manufacture of fermented dairy products can contribute to maintain the viability of probiotic bacteria in the gut. the in vitro analysis used in this study gives information about the survival rate of probiotic bacteria under gastrointestinal conditions but not about their probiotic activity. so, the probiotic activity of these bacteria should also been assessed by appropriate analyses. the species and strain specificity of probiotics to acid and bile tolerance should also not be forgotten (ranadheera et al., 2014). 3.3. sensory evaluation the results of the sensory evaluation of the fermented milk samples are shown in figs. 4a (d1), 4b (d14), and 4c (d28). (4a) (4b) ital. j. food sci., vol. 30, 2018 579 (4c) figure 4. sensory scores of (a) 1-d-old-fermented milks, (b) 14-d-old fermented milks, and (c) 28-d-old fermented milks; ◊ = fermented milk control without addition of prebiotic (fmc), □ = fermented milk with addition of 2% inulin (fmi), ∆ = fermented milk with addition of 2% polydextrose (fmp), x = fermented milk with addition of 2% hi-maize (fmh). in general, the effect of storage time on the sensory characteristics of fermented milk samples was not found to be significant (p > 0.05). there were no significant differences (p > 0.05) among fermented milk samples in terms of taste, flavor, texture, and overall acceptability during the 14 days of storage. the addition of inulin or polydextrose at a ratio of 20 g/l also did not affect the flavor property of low-fat set yoghurts with probiotic-cultured banana purée (srisuvor et al., 2013). the addition of inulin also did not negatively affect the sensory quality of sponge-cake products (zbikowska et al., 2017). similarly, no significant inulin effect could be observed on the “firmness”, measured as the force required to lift a spoonful of yoghurt in another study (guggisberg et al., 2009). fermented milk fortified with polydextrose had the lowest sensory scores among the samples at the end of the storage term (p < 0.05). the lowest appearance values were obtained again in the samples fortified with polydextrose throughout the storage period which can be attributed to the high solubility of polydextrose in water, thus resulting a non-viscous solution. having a neutral taste without reflecting sweetness can also be a reason for indicating low scores (do carmo et al., 2016). an artificially sweetened misti dahi, which is a popular fermented dairy product of eastern india, containing polydextrose was found to have lower flavor and lower overall acceptability scores when compared to the control sample and samples supplemented with other sweeteners (raju and pal, 2011). allgeyer et al. (2010) also observed worse sensory attributes for a symbiotic yoghurt drink containing polydextrose compared with the control (p < 0.05). the addition of inulin or hi-maize did not negatively affect the sensory properties of the fermented milk samples throughout the 28 days. similar results were also obtained in other studies (kip et al., 2006; rinaldoni et al., 2012). 4. conclusions the importance of this study is that it provides knowledge about the efficacy of different prebiotics on the viability of probiotic bacteria in the fermented milk during in vitro simulated gastrointestinal conditions. according to the results obtained in the present ital. j. food sci., vol. 30, 2018 580 study, the supplementation with prebiotics showed a protective effect and did not allow a decline on the viability of l. acidophilus la-5 and b. animalis bb-12 in abt fermented milk, and the probiotics maintained the recommended level for the beneficial health properties, ranging from 6 to 8 log cfu/g during the 28 days of storage. b. animalis bb-12 was more resistant to the simulated gastrointestinal conditions than l. acidophilus la-5 throughout the storage period. although inulin added fermented milk had higher viable counts of l. acidophilus la-5 during the gastric and enteric-i phases, there were no significant differences among products at the end of the in vitro assay, except on the 1st day. however, it was obvious that the use of hi-maize improved b. animalis bb-12 resistance when subjected to simulated gastrointestinal conditions. the addition of inulin or hi-maize to fermented milk had no influence on the sensory characteristics whereas the lowest scores were obtained for the sample supplemented with polydextrose especially on day 28. therefore, the findings of this study suggest that an appropriate prebiotic can be suitable to ensure the viability of the probiotic strains during the manufacturing time and shelf-life of the product, and protect the probiotics during the passage through the gastrointestinal tract so that they can reach the colon. this can be a strategy for providing a broad range of health benefits of probiotic microorganisms to the host and for the development of future functional foods. in addition, further studies are necessary to follow which components of the prebiotics and/or technological processes might influence the probiotic tolerance to simulated gastrointestinal juices in fermented dairy products. the use of higher ratios of the prebiotics can be tested in the future studies and clinical trials should also be needed to support in vitro studies. acknowledgements the authors thank the ege university agricultural faculty, scientific research fund council (project no: 2015-zrf-020) for financial support. references akalın a.s. and ünal g. 2010. the influence of milk supplementation on the microbiological stability and textural characteristics of fermented milk. milchwissenschaft 65:291-294. allgeyer l.c., miller m.j. and lee 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and agricultural biotechnology (ibba), pisa unit, research area of pisa, via moruzzi 1, 56124, pisa, italy bchemistry and industrial chemistry department, university of pisa, via moruzzi 13, 56124, pisa, italy cdepartment of veterinary sciences, viale delle piagge 2, 56124, università di pisa, italy dnational research council (cnr), institute of sciences of food production (ispa), turin unit, largo braccini 2, 10095, grugliasco (to), italy evia carlini s. 73, 56127 pisa, italy *corresponding author: tel. +39 050 2219215/267, fax +39 050 2219260, email: valentina.domenici@unipi.it abstract within the apicultural products, the honey bee-pollen is growing in commercial interest due to its high nutritional properties. for the first time, bee-pollen samples from tuscany (italy) were studied to evaluate botanical origin, phytochemical composition and antioxidant activity. the investigated pollen loads were composed of three botanical families: castanea, rubus and cistus. the highest levels of proteins and lipids were detected in rubus pollen. castanea pollen contained greater polyphenols, flavonoids and anthocyanins content, while the highest flavonols level was detected in cistus pollen. these results were also confirmed by front-face fluorescence spectroscopy, used here, for the first time, as a fast tool to characterize bee-pollens. mailto:valentina.domenici@unipi.it ital. j. food sci., vol. 27 2015 249 1. introduction for centuries the apicultural products have been used in phytotherapy as well as in diet for their health positive implications (kroyer and hegedus, 2001; ferreres et al., 2010; abouda et al., 2011). bee-gathered pollen (bee-pollen) is an apicultural product of great commercial interest owing to its high nutritional value and physiological properties representing an important source of energy and proteins for human nutrition (abouda et al., 2011). worker-bees gather pollen from flowers and, after processing it with some proteic gland-secretion, they package it on their curbicula forming pollen loads before returning to the hive (pinzauti et al., 2002; scarselli et al., 2005). generally, a single pollen load is a one colored little pellet reflecting a homogeneous and monospecific pollen content. bee-pollen loads are stilled from the worker bees at their entrance to the hive by special pollen loads traps. in accordance to the season, to the timing of collection by the beekeepers and to the post collection management is possible to obtain monoor poly-pollen species loads. amounts of pollen loads ready to be commercialized and consumed by human and animals is called bee-pollen. so it is possible to find bee-pollen of one specific flower (monoflora) or belonging to several flower species (polyflora), as well is possible to blend different monofloral in order to create mixtures of bee-pollen with characteristic organoleptic properties and quality attributes. nowadays, bee-pollen represents the richest and most complete natural food supplying high levels of carbohydrates (13-55%), proteins (1040%), particularly free aminoacids, enzymes, cofactors, lipids (1-13%), including fatty acids and sterols, minerals, trace elements and vitamins, especially b group, a, c and e. fresh and dry bee-pollen loads hold a different water content, ranging from 20-30% in the original form and 4-10% if dried, affecting organoleptic and “shelflife time” properties (campos et al., 2008; pascoal et al., 2014). moreover, it is also an excellent source of bioactive compounds, such as phytosterols, carotenoids and polyphenols (especially flavonoids), that exert antioxidant, anti-inflammatory, antimicrobial, anti-allergic and antitumoral effects (morais et al., 2011; pérezpérez et al., 2012; fratini et al., 2014). very recently, several research works were published showing that bee products, such as propolis and pollens, possess a sedative effect and may be effective in protecting humans against depression and similar diseases (yildiz et al. 2014); additionally, preliminary studies show that pollens have a hepatoprotective potential (yildiz et al. 2013). pollens effects on improving immune, cardiovascular and digestive systems as well as their therapeutic effects have been mainly related to the polyphenol content and chemical composition (pascoal et al., 2014). in particular, the phenolic profile of bee-pollen consists of flavonol, glycosides and aglycones, and hydroxycinnamic acids, that can be present in free forms or combined with other pollen components (chantarudee et al., 2012; fanali et al., 2013). however, as well as chemical composition, the phytochemical profile is affected by soil type, beekeeping management, climatic and preservation conditions, and especially by botanical origin (almeida-muradian et al., 2005; arruda et al., 2013; campos et al., 2008). aromatic aminoacids, many polyphenols, some enzyme cofactors, but also some wateror lipid-soluble vitamins and pigment’s derivatives contained in bee-pollen are fluorescent intrinsic compounds (jørgensen et al., 1992; lakowicz, 2006). front-face (ff) fluorescence spectroscopy is a non-destructive, rapid and sensitive technique suitable for complex and opaque samples, otherwise traditional right-angle florescence (karoui et al., 2007; zandomeneghi et al., 2005). in particular, this technique has been proved to be very effective in studying powders, crystalline or amorphous samples, and complex matrix, such as food (zandomeneghi, 1999; zandomeneghi and zandomeneghi, 2009; airado-rodríguez et al., 2011; kulmyrzaev et al. 2005). in this context, ff fluorescence could be a useful instrument to obtain a fingerprint of different bee-pollen types, advantageous to study and compare them. in this study, we examined the botanical origin, the chemical composition (moisture, proteins, carbohydrates, lipids, ash) and the antioxidant profile (the total polyphenols and the flavonoids, flavonols and anthocyanins subclasses) of different color fractions of an organic tuscan (italian) bee-pollen sample. moreover, we proposed for the first time a rapid qualitative evaluation of emission, excitation and synchronous spectra, obtained by front-face spectroscopy, of bulk state and ethanolic extracts of the different pollen types showing the main classes of identified fluorescent molecules. lastly, to evaluate the synergic effect of the bee-pollen bioactive components we analyzed by dpph and orac assay the free radical scavenging activity and the antioxidant capacity of both separate and mixed color fractions. 2. materials and methods 2.1 chemicals and reagents all standards and reagents were of analytical grade. absolute ethanol, methanol, hydrochloric acid, trichloroacetic acid, diethyl ether, sodium carbonate, sodium idrosside, potassium chloride, sodium acetate, folin-ciocalteu rea250 ital. j. food sci., vol. 27 2015 gent, catechin, gallic acid, 6-hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid (trolox), 1,1-diphenyl-2-picrylhydrazyl (dpph) and fluorescein sodium salt were purchased from fluka-sigma-aldrich, inc. (st. louis, mo), as well as the solid standards used in spectroscopic analysis tryptophan, b-carotene, gallic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, vanillic acid, caffeic acid, ferulic acid, pcoumaric acid, quercetin dihydrate and the vitamins c, b3, b2, b6 and b9. hydroxide peroxide, sulfuric acid, boric acid and kjeldahl tablets were purchased from merck (readington, nj). sodium nitrite and aluminum chloride were purchased from carlo erba (milan, it), while 2,2’-azobis (2-amidinopropane) dihydrochloride (aaph) was purchased from polysciences, inc. (warrington, pa). 2.2 plant materials bee-pollen resulted from the blend of pollen loads collected during sunny days by the beekeeper using 10 beehives equipped with bottomfitted pollen traps located in massa macinaia (latitude 43.80638longitude 10.54213) in lucca province (tuscany, italy) between april and july 2013. in total 1 kg of bee-pollen was collected. the blended fresh bee-pollen was stored at -20°c in the dark until further analysis. 2.3 palynological analysis within the bee-pollen sample the pollen loads were divided by colour into three groups (fig. 1). twenty single pollen loads of each colour were analyzed by microscope. each single pollen load prepared by washing the pollen with distilled water and using glycerin jelly for permanent preparations. pollen grains identification was performed by optical microscope with total magnification (400x and 1000x). a reference collection of pisa university and different pollen morphology guides were used for the recognition of the pollen types. 2.4 chemical composition dry matter, proteins, lipids and ash quantification were performed for the pollen-load samples according to aoac guidelines (aoac, 2000) and values were expressed as percentage on fresh matter basis. dry matter content was made through gravimetry until constant weight, using oven at 105°c. protein content was obtained using kjeldahl method, while lipid content was determined by soxhlet extractor using diethyl ether as solvent. ash content was determined by gravimetry until constant weight, using oven at 550°c for one day. moisture content was obtained by subtracting the dry matter from 100, while total carbohydrates content was determined according to the following formula: carbohydrates (g) = 100 [protein (g) + fat (g) + moisture (g) + ash (g)] (ketkar, rathore, lohidasan, rao, paradkar, and mahadik, 2014). 2.5 bee-pollen extraction and phytochemical characterization pollen-loads were separated in three color groups and the mixed pollen sample was made blending the three pollen type in equal part. bee-pollen extracts were obtained after 1 hour incubation at room temperature in 95% ethanol while being shaken gently. then samples were centrifuged 10 minutes at 3500 rpm at 4°c and the supernatants were collected and kept in the dark at 4°c. total polyphenols were determined by the folin-ciocalteu colorimetric method (singleton et al.,1999). briefly, 100 μl of each natural extract were mixed with 500 μl of 0.2 n folin-ciocalteu reagent and incubated in the dark for 5 minutes. then, 400 μl of 0.7 m sodium carbonate (na2co3) were added. the absorbance was measured at 760 nm, after 2 hours incubation at room temperature in the dark. five serial dilutions of gallic acid (0.009, 0,017, 0,043, 0,085, 0.17 mg/ml) were used to obtain the standard calibration curve with the following equation: conc=0.079abs-0.004 (r2=0.996). total polyphenols were expressed as mg of gallic acid equivalents (gae)/g dry weight (dw). the aluminum chloride colorimetric method was used for the total flavonoids determination (kim d.o., chun, kim y.j., moon, and lee, 2003). briefly, 200 μl of extracts were mixed with 800 μl of dh 2 o and 60 μl of 5% nano 2 , followed by incubation of 5 minutes at room temperature. then 60 μl of 10% alcl 3 were added, incubated for 6 min and finally reactions were neutralized with 400 μl of 1m naoh. absorbfig. 1 picture of the original blended bee-pollen sample and castanea sp. (yellow), rubus sp. (green) and cistus sp. (ochre) pollen. ital. j. food sci., vol. 27 2015 251 ance was measured at 430 nm after 30 minutes of incubation and catechin was used to make the calibration standard curve (0.016, 0.032, 0.063, 0.125, 0.25, 0.5 mg/ml). flavonoids concentration was obtained from the following calibration curve: conc=0.464abs-0.005 (r2=0.998) and expressed as mg catechin equivalent (ce)/g dw. flavonols content was measured according to the method described by romani, mancini, tatti and vincieri (1996). briefly, 25 μl of extracts were mixed with 225 μl of 10% etoh, 250 μl of 0.1% hcl in 95% etoh and 1 ml of 2% hcl. after incubation at room temperature for 30 minutes, the absorbance of the reaction mixture was measured at 360 nm and quercetin was used to make the calibration standard curve (0.0006, 0.00125, 0.0025, 0,0050, 0.0075, 0.01, 0.0125, 0.015 mg/ml). flavonols content, derived from the calibration curve equation: conc=0.536abs-0.0001 (r2=0.997), was expressed as mg quercetin equivalent (qe)/g dw, total monomeric anthocyanins were determined according to the ph differential method described by lee, durst, and wrolstad (2005), a spectrophotometric method based on the change in pigmentation ph-dependent of anthocyanins. absorbance was measured at 520 and 700 nm and anthocyanin concentration was expressed as mg cyanidin-3-glucoside equivalents (c3ge)/g dw (cyd-glu, molar extinction coefficient of 26,900 l cm-1 mol-1 and molecular weight of 449.2 g mol-1). 2.6 front-face fluorescence spectroscopy analysis each pollen sample was studied previous in its bulk state by ff fluorescence spectroscopy, then their ethanolic extracts were studied by uvvis absorption and ff fluorescence spectroscopies. the precipitate obtained after the extraction was also studied by ff fluorescence spectroscopy. to recognize the main classes of fluorescence compounds in pollen loads, standard solutions of the reagents reported in paragraph 2.1 were studied by uv-vis absorbance and ff fluorescence spectroscopy. for bulk analysis pollen loads were finely powdered with a pestle in a mortar. a little amount of powder was put between two quartz windows of 1 mm optical path with the help of few water drops to homogenized the sample (about 1:1 mg/ ml sample: water). these quartz windows are held against a support in the spectrofluorometer by a laminar spring. for extraction procedure 2 ml of ethanol were added to 100 mg of pollen loads finely powdered. they were stirred for 30 minutes with a magnetic stir bar and then filtered through filter paper 0.45 mm pore size (sartorious), to obtain a clear solution. to do it, spectrum s-25-10 stirred cell device for ultra-filtration was used. a quartz cell with 2 mm optical path was used to record absorbance spectra and low capacity quartz cells with 5 mm optical path was used to record ff fluorescence spectra. the residual material was kindly removed from the filter paper and it was put between two quartz windows of 1 mm optical path. fluorescence spectra were recorded using a isa fluoromax ii photon counting spectrofluorometer, with xenon arc lamp and a device for front-face measurements with a cell holder designed to set the incident angle of the excitation beam at 31°, eliminating or reducing selfabsorption effects, light-reflected and scattering (zandomeneghi et al., 2005). the excitation and the emission slits were 2 and 5 nm, respectively. the integration constant time was 0.5 s and the wavelength increment was 1 nm. the intensity of the spectra was determined as the ratio between the emisson signal (counts per second, cps) and the intensity of light from the excitation monochromator (ma), measured by means of a photomultiplier and a photodiode, respectively. for each sample emission spectra (280 nm < lambda_ex < 550 nm, with a step of 10 nm), excitation spectra (at the wavelength of the maximum position of fluorescence emission spectra, l em ) and synchronous spectra (20 nm < deltalambda < 120 nm, with a step of 10 nm) were recorded. for bulk analysis emission spectra with l ex =650 nm were recorded. absorbance spectra were recorded with a jasco v-550 spectrophotomer, between 200 nm and 750 nm with scanning speed of 400 nm/s, band width and data pitch of 1 nm and 0.5 nm, respectively. 2.7 antioxidant activity 2.7.1 dpph radical scavenging assay the free-radical scavenging activity of ethanolic bee-pollen extracts was evaluated using the 1,1-diphenyl-2-picrylhydrazyl (dpph) assay (frassinetti et al. 2011). the reduction of dpph radicals was recorded at 517 nm and the radical scavenging activity (rsa) was calculated as percentage of dpph inhibition according to the following equation: % rsa = [(a dpph -a s )/a dpph ] x 100, where a dpph is the absorbance of dpph solution and a s the absorbance of sample. the extract concentration corresponding 50% of dpph inhibition (ec 50 ) was measured by interpolation from the graph of rsa percentage versus beepollen concentration (morais et al., 2011). lower ec 50 values indicate higher antioxidant activities. 2.7.2 oxygen radical absorbance capacity (orac) assay the antioxidant capacity of ethanolic bee-pollen extracts was quantified using the oxygen radical absorbance capacity (orac) assay, modifying some reagent concentration adapted to our requirements (ninfali et al., 2005). the final reaction mixture of our assay contained 0·04 mm fluorescein sodium salt in 0.075 252 ital. j. food sci., vol. 27 2015 m phosphate buffer, ph 7.4, at diluted sample or 5 mm trolox. the control was 0.075 m phosphate buffer, ph 7.4. aaph was used as peroxyl radicals generator and fluorescein as probe. fluorescein fluorescence decay was read at 485 nm excitation and 514 nm emission using a victortm x3 multilabel plate reader (waltham, ma) and trolox was used as antioxidant standard. orac values were expressed as micromoles of trolox equivalents (te)/g dw. 2.8 statistical analysis the statistical analysis was performed using graphpad prism, version 5.00 for windows (graphpad software, san diego, ca). assays were carried out in triplicate and results were expressed as mean values ± standard deviation (sd). differences between bee-pollen samples were analyzed by one-way analysis of variance (anova) followed by tukey’s post test. a p-value lower than 0.05 is considered as statistically significant. interdependence between the antioxidant capacity and the phytochemical profile was evaluated by pearson’s correlation coefficient (r). 3. results 3.1 palynological analysis the blended bee-pollen sample resulted to be a mixture of castanea sp. (yellow), rubus sp. (green) and cistus sp. (ochre). each pollen load owned a homogeneous and monospecific pollen content. the castanea sp. was the most representative (70%), followed by rubus sp. (23%) and cistus sp. (7%). 3.2 chemical composition the chemical composition of castanea, cistus and rubus pollen samples is listed in table 1 and values are expressed as percentage on fresh matter basis. the nutritional content measured is in agreement with literature values (balkanska and ignatova, 2012; carpes, mourão, alencar, and masson, 2009; nogueira, iglesias, feás, and estevinho, 2012). significant variations among samples were showed, with the highest protein (p<0.01) and lipid (p<0.001) content in rubus compared to all other pollens. no difference among groups was found for moisture, dry matter, ash and carbohydrates content (p=ns). 3.3 phytochemicals profile of ethanolic pollen-load extracts pollen-load extracts were screened for total polyphenols, flavonoids, flavonols and monomeric anthocyanins content. phytochemical profile of castanea, cistus and rubus pollen-load samples is listed in table 2 and significant differences were found (p<0.001). in particular, castanea pollen extracts contained the highest levels of polyphenols (24.75±0.78 mg gae/g fw), flavonoids (15.86±0.62 mg ce/g fw) and anthocyanins (77.37±2.55 mg c3ge/l), while the highest levels of flavonols (4.93±0.05 mg qe/g fw) were detected in cistus pollen samples. otherwise, rubus pollen extracts showed the lowest table 1 moisture, dry matter, proteins, lipids, ash and carbohydrates of pollen-load samples. a,b,c different superscript letters indicate statistical differences among the bee-pollen extracts (p<0.001 anova). assays were carried out in triplicate and results were expressed as mean values ± sd. ital. j. food sci., vol. 27 2015 253 content of polyphenols, flavonoids and flavonols. the discrepancies in phytochemical composition observed among the different pollen samples might depend on their botanical origin (arruda et al., 2013; campos et al., 2008; morais et al., 2011). 3.4 fluorescence spectroscopy 3.4.1 bulk analysis pollen is a complex matrix and, as a consequence, the fluorescence spectra are characterized by broad and overlapped bands, caused by the presence of many fluorophores, which limit the quantification and identification of all of them. however, it is possible to get several information as well as qualitative features, which can be used as fingerprint of the investigated pollen. the fluorescence spectra are indeed characterized by three main intervals of excitation wavelength: 280-290 nm, 320-370 nm and 420-480 nm. in fig. 2a the emission spectra obtained with excitation wavelength (l ex ) of 280 nm are reported. two bands can be recognized: the first one, less intense, is centered at 340 nm for rubus and at 360 nm for castanea and cistus pollens. this band is probably due to the aromatic aminoacids, which can be residues of proteins or free. the second one is the dominant band and its shape is different between rubus and castanea/cistus pollens: it is more intense in the range 420-620 nm for rubus and red shifted of about 40 nm for castanea and cistus pollens. this emission band is probably due to hydroxycinnamic acids, compounds belonging to polyphenols family, fluorescent water soluble vitamins such as b6, b9 and b2, and flavoinoid compounds. all these classes of compounds have two bands of absorption (three in the case of b2 vitamin), the first one centered in the 280-290 nm interval. this hypothesis is supported by the emission spectra obtained at highest l ex 320-370 nm, where the above mentioned substances are excited in their second absorption band; these pollens’ bands are indeed less intense but retain the same shape of previous broad band. it is interesting to observe that castanea and cistus’s spectra are again much different from rubus (fig. 2b), probably due to the presence of characteristic fluorophores in the latter pollen type. for all pollens, fluorescence intensity decreases until λ ex =400 nm; thereafter, it slightly increases again, to define a new band centered at 530 nm for cistus and castanea and at 510 nm for rubus (λ ex =450 nm). at this wavelength, b2 vitamin has its third absorption band, moreover, other fluorophores are excited, such as xanthophylls and carotenoids derivatives, although they are very weak emitter (jørgensen et al., 1992). changing the λ ex to 550 nm, only cistus pollen presents a weak fluorescence at about 600 nm, probably caused by the presence of other polar carotenoids derivatives. at higher excitation wavelength, collected to investigate the eventual presence of chlorophyll derivatives (λ ex =650 nm), no relevant fluorescence is recorded. the synchronous spectra obtained with 60 nm offset (fig. 2c) underlines the presence of three different bands of absorption and they are useful to compare the pollen types. the fluorescence profile of rubus pollen is much different than that of castanea/cistus’ one, especially in the second absorption bands (iir and iic respectively). in this spectrum, also the third band differs between rubus and castanea/cistus, centered table 2 total polyphenols, flavonoids, flavonols and anthocyanins concentration of ethanolic pollen-loads extracts. a,b,c different superscript letters indicate statistical differences among the bee-pollen extracts (p<0.001 anova). assays were carried out in triplicate and results were expressed as mean values ± sd. 254 ital. j. food sci., vol. 27 2015 fig. 2 comparison between fluorescence spectra of the different pollen types in their bulk state. (a) emission spectra obtained with l ex =280 nm; the arrow points out the first emission band. the peak at 560 nm is related to lamp; the peaks between wavelength 460-490 nm and at 765 nm are artefacts of lamp. (b) emission spectra obtained with l ex =350 nm. (c) synchronous spectrum obtained with dl ex =60 nm. the roman numerals point out the principal absorption bands, as explained in the text. this spectrum is normalized. at 440 nm (iiir) and 480 nm (iiic), respectively. moreover, the cistus pollen is the only one presenting an absorption centered at 550 nm (iv). all above data are consistent with the emission spectra. vitamins c and b 3 did not show relevant fluorescence so they resulted undetectable. 3.4.2 extracts analysis in order to facilitate the spectral interpretation, an extraction procedure to separate the water soluble fraction from the lipid soluble one was adopted. the solvent used are not strong and the treatment of samples is such to limit the alteration of the matrix, and to use the potentiality of a rapid and direct spectroscopic technique. in the present work ethanolic extracts were studied. the most interesting region of the uv-vis absorption spectra is in the visible range (400-500 nm). here, only cistus extract presents an absorption spectrum with the typical shape of carotenoid or xanthophyll pigments, as shown in fig. 3a. considering fluorescence, the emission spectra at λ ex =280 nm (fig. 3b) are characterized by a broad band, extended to the interval 340-520 nm. castanea and rubus pollen show maximum emission at about 420-430 nm, while the cistus one shows a different profile with an emission band centered at about 400-410 nm and a shoulder at 520 nm. in this region, the fluorescence is probably due to the hydroxycinnamic acid, that at this wavelength begin to absorb. the polyphenols belonging to the hydroxybenzoic family (e.g. gallic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, vanillic acid) are not the mainly fluorophores. in fact, they have their absorption and emission maxima included in the interval 280-297 nm and 325-370 nm, respectively. in fig. 3b emission spectra of two representative compounds of the hydroxybenzoic’s family (gallic and 4-hydroxybenzoic acids) compared with pollen’s spectra are reported. the hypothesis of the presence of hydroxycinnamic acid is supported by the emission spectra recorded with highest λ ex (310-340 nm), characterized by the same shape, but higher intensity. in fact, polyphenols belonging to hydroxycinnamic family (e.g. caffeic acid, ferulic acid, p-cumaric acid) have their absorption and emission maxima included in the interval 310-350 nm and 410-440 nm, respectively. emission spectra of two representative compounds of this group (caffeic and p–coumaric acid) recorded with λ ex =310 nm are reported in fig. 3c and compared with pollen’s spectra. castanea pollen spectrum differs from the two others for the higher intensity. ital. j. food sci., vol. 27 2015 255 considering the excitation spectra recorded with the emission wavelength fixed in the maximum of emission (430 nm for castanea/rubus and 410 nm for cistus) it is clear that their fluorescence is due to different fluorophores. in particular, they are a single fluorophore, with an absorption band centered at 330 nm for rubus, a single fluorophore, with two absorption bands centered at 285 and 330 nm for castanea, and two different fluorophores, centered at 290 and 325 nm excitation wavelength for cistus. this latter hypothesis is confirmed by synchronous spectra obtained with offset 100 nm, in which it is possible to recognize two different contributions with absorption at 295 and 320 nm, respectively. changing the λ ex to 350 nm (fig. 3d), the fluorophores involved are others, thus confirming the complexity of the pollen matrix. castanea and rubus samples present the same profile, while cistus’ spectrum shows a particular shape characterized by three shoulders, most likely due to three different fluorophores, confirmed by excitation spectra recorded with λ em =460nm. these fluorophores are probably other hydroxycinnamic compounds (in the cases of emission at 430 nm and 460 fig. 3 spectra of ethanolic extracts of the different pollen types. (a) uv-vis absorpotion spectra (optical path=1 cm) of pollens compare to a b-carotene solution (3.2 mg/ml). series of emission spectra of pollens compare to: (b) solutions of gallic acid and 4-hydroxybenzoic acid (0.01 mg/ml 90% h 2 o-10% ch 3 oh) (l ex =280 nm); (c) solutions of caffeic acid and p-coumaric acid (0.01 mg/ml 90% h 2 o-10% ch 3 oh) (l ex =310 nm); (d) solution of quercetin (20 mg/ml in ch 3 oh) (l ex =370 nm). all the fluorescence spectra are normalized. caffeic and p-cumaric’ spectra intensity are multiplied by 2.4. nm) and flavonoids compounds (in the case of emission at 500-520 nm). for example, the flavonol quercetin has a weak emission, but the shape of its fluorescence spectrum is compatible with that of cistus pollen, as reported in fig. 3d. the richness in fluorescent compounds of cistus pollen is confirmed by the synchronous spectrum obtained with offset 120 nm, where it is possible to recognize four bands of absorption, centered at 295-320 nm, 350 nm, 370 nm and 400 nm. in the case of the first broad band it is possible to discriminate the contribution of two fluorophores with offset 100 nm, like previously discussed. with higher excitation wavelength, collected to check the presence of carotenoids or xanthophyll with front-face fluorescent of liquid extract, no relevant fluorescence was recorded. it is important to underline that there are several xanthophylls and carotenoids derivatives, and only some of them have weak emission, as previous discussed in section “bulk analysis”. finally is interesting to analyze the fluorescence spectra obtained for solid samples. for example in fig. 4 the comparison between emission spectra (λ ex = 280 nm) of rubus in its bulk 256 ital. j. food sci., vol. 27 2015 state with the precipitate after ethanol extraction (dotted line) is reported. the first band due to water soluble aminoacids and proteins, not extracted in ethanol, is present into both spectra. instead the shape of main broad emission band centered at 500 nm, differs from each other. this can be due to the lack of the classes of compounds extracted with the solvent: hydroxycinnamic acid and flavonol compounds. it is important to remember that fluorescence sensitive changes in relation of the physical state of compounds and their environment (lakowicz, 2006). although the broad emission band, these results are coherent with extracts’ spectra. 3.5 dpph radical-scavenging activity the dpph assay has been widely used to test the free radical scavenging activity of apicultural products, either honey, propolis or bee-pollen. as reported in literature the radical scavenging activity of bee-pollen is very dissimilar among different flower species with a value range of 0-97% that also depends on their chemical composition and solvent extraction (basuny et al.,2013; leblank et al., 2009; silva et al., 2006). we evaluated both percentage of dpph radical scavenging activity (% rsa) and ec50, the extract concentration providing 50% dpph inhibition. as shown in table 3, ethanolic bee-pollen extracts exhibited high free radical scavenging activity with dpph inhibition values ranged from 37.95±0.19% (ec 50 = 641.3±11.4 μg/ml) of rubus extracts and 94.45±0.01% (ec 50 = 215.2±2.7 μg/ml) of castanea extracts. anova with tukey post-test showed significant differences among samples with the highest and lowest activity detected in castanea and rubus extracts. as well as radical quenching capacity, castanea, cistus and mixed bee-pollen fig. 4 emission spectra obtained with l ex =280 nm for solid rubus samples differently processed. the dotted line shows the precipitate after ethanol extraction (spectrum intensity is divided by 1.7). the peak at 560 nm is related to lamp; the peaks between wavelength 460-490 nm and at 765 nm are artefacts of lamp. table 3 orac and dpph assay results of ethanolic pollen-load extracts expressed as μmol te/g fw and % rsa and ec 50 (μg/ml), respectively. a,b,c different superscript letters indicate statistical differences among the bee-pollen extracts (p<0.001 anova). assays were carried out in triplicate and results were expressed as mean values ± sd. ital. j. food sci., vol. 27 2015 257 possessed similar ec 50 values, three times lower than rubus extracts, suggesting a lower radical scavenging activity of rubus bee-pollen as antioxidant. our results also revealed a strong relation between the antiradical activity and the total phenolics, flavonoids and flavonols content, resulting in a significant positive correlation (r=0.9645, r=0.9888, and r=0.9847, respectively). lastly, a moderate correlation was obtained between the anthocyanins and the dpph scavenging activity (r=0.5541). 3.6 oxygen radical absorbance capacity (orac) the antioxidant capacity of ethanolic bee-pollen extracts was also screened using orac assay and expressed as orac units (μmol trolox equivalents/g fw). the orac values were listed in table 3 and ranged from 519.45±15.07 μmol te/g of rubus fraction and 677.70±12.92 μmol te/g of mixed bee-pollen. one-way analysis of variance with tukey’s post test showed a significant increase of orac values in mixed bee-pollen extracts respect to each one separate fractions (p<0.001), suggesting a synergic or additive effect among castanea, cistus and rubus antioxidant compounds. in fact, besides to their specific effects, many antioxidants can interact in synergistic ways, maybe protecting another against oxidative degradation, exhibiting greater antioxidant effects (mărghitaş et al., 2009). the results showed no strong correlation between the orac values and the anthocyanin (r=0.1524) and flavonoid (r=0.4586) compounds; however, a moderate interdependence was obtained between the antioxidant capacity and the polyphenols (r=0.6638) and flavonols (r=0.5572) content. 4. conclusions in this study, an organic bee-pollen sample from tuscany was analysed for the first time investigating the botanical origin, the chemical composition, the phytochemicals profile and the antioxidant activity. different techniques were used. in particular, we propose an original application of ff fluorescence spectroscopy, a promising approach to put in evidence differences and analogies among pollens with different floral origin. this unconventional technique presents the advantage to require no particular sample pre-treatment; moreover, it is economic, fast and easy to use and it could be useful for both further scientific researches and commercial applications. the ff fluorescence results are coherent and uphold the spectrophotometric data obtained in this investigation. specifically, the differences among castanea/cistus and rubus fluorescence profiles, arisen from bulk study, are comparable and in agreement with the significant differences found in lipids and proteins composition. furthermore, the ethanolic extracts fluorescence analysis, as well spectrophotometric results, displays a higher content of flavonols and polyphenols in cistus and castanea, respectively. moreover, the ff fluorescence analysis shows the greater presence of hydroxycinnamic acids than hydroxybenzoic acids, in agreement with literature data (fanali et al., 2013; ketkar et al., 2014). lastly, uvvis ethanolic extracts’ spectra reveal the presence of carotenoid or xanthophyll pigments only in cistus, confirmed also by bulk state’s emission and synchronous spectra λ ex =550 nm and offset 60 nm, respectively). besides nutritional and phytochemical composition, the antioxidant and free radical scavenging activity of tuscan bee-pollen and its monofloral groups was measured. the redox properties of phenolic compounds, especially flavonoid components, play a key role in decomposing peroxides and quenching oxygen, as well as in absorbing and neutralizing free radicals (mărghitaş et al., 2009). moreover, specific bioactive compounds or a combination of them can exert a different antioxidant activity, strongly dependent on structure and polyphenols composition, rather that the phytochemical concentration (mărghitaş et al., 2009). in particular, we showed that mixed bee-pollen exhibit a much better antioxidant activity than the separate fractions with an orac value significantly greater than other samples; whereas, castanea, cistus and mixed bee-pollen showed a comparable dpph radical scavenging activity greater than rubus antiradical capacity. these results are in agreement with literature data that strongly associate the high ability to neutralize reactive oxygen species to the phenolic compounds structure, mainly flavonoids and cinnamic acid derivatives (leja et al., 2007), maybe more representative in cistus and castanea pollen. furthermore, according to mărghitaş et al. (2009) the antioxidant effects of bioactive compounds change differently depending on the antioxidant method used. therefore, we suppose a synergic or additive effect among castanea, cistus and rubus antioxidant compounds or resulting from new antioxidant substances with greater antioxidant activity. in conclusion, the use of spectroscopic techniques applied to bee-pollen samples is a suitable tool to underline differences and analogies in their micronutrient composition. the data obtained with different or complementary techniques are coherent and in agreement with literature concerning the variability in chemical composition and antioxidant activity of pollens from different floral sources (leblanc et al., 2009; pascoal et al., 2014). further investigations should be performed to identify and quantify the main fluorescence 258 ital. j. food sci., vol. 27 2015 compounds present, as well as to investigate on the nature of cistus’ pigments. moreover, future analysis is required to 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2013. hepatoprotective potential of chestnut bee pollen on carbon tetrachloride-induced hepatic damages in rats. evidence-based complementary and alternative medicine. 2013, article id 461478. doi.org/10.1155/2013/461478 yildiz o., karahalil f., can z., sahin h. and kolayli s. 2014. total monoamine oxidase (mao) inhibition by chestnut honey, pollen and propolis. journal of enzyme inhibition and medicinal chemistry. 29, 690-694. ijfs#1291_bozza ital. j. food sci., vol. 31, 2019 531 paper quality of broiler chicken meat during frozen storage a. augustyńska-prejsnar*1, m. ormian1 and r. tobiasz-salach2 1university of rzeszow, faculty of biology and agriculture, department of animal production and poultry products evaluation, 35-601 rzeszów, poland 2university of rzeszow, faculty of biology and agriculture, department of crop production, rzeszow 35-601, poland *corresponding author: augusta@ur.edu.pl abstract the objective of the study was to assess the influence of frozen storage (1mth and 8 mth) and thawing methods on the breast meat quality of broiler chickens. tests revealed significant changes in meat quality in defrosted raw breast muscles as well as in those subjected to thermal treatment. defrosting by using microwave oven proved to be more useful for breast muscles stored for a period of eight months regarding the volume of drip loss, ash content of the raw breast muscles as well as the cooking loss, desirable aroma, and the brittleness of meat subjected to thermal treatment. keywords: breast muscles, broiler chickens, freezing storage, quality, thawing methods ital. j. food sci., vol. 31, 2019 532 1. introduction the poultry meat market is a classic example of a market displaying increased diversification. poultry meat remains a valuable component of human diet with its consumption in europe steadily rising. fast growing broiler chickens are dominant in poultry meat production due to their efficient feed use (augustyńska-prejsnar et al., 2014). the choice of poultry is greatly influenced by its nutritive and sensorial values, as well as its low price, bountiful supply, varied assortment, price relative to that of red meat and other animal products. the high popularity of poultry meat is also determined by its culinary values and the ease of preparation. in addition, the promotions and advertising methods, as well as the increasing awareness of the nutritive value of foods and proper dietary practice enhance the attractiveness of poultry meat as a food. quality issues have attracted greater attention in the much saturated market of meat products. a specific characteristic of meat products is their short shelf life (benli, 2016). majority of the poultry meat for culinary purposes in home markets is offered as frozen meat. freezing is the most commonly applied method in managing its excess production. an accruing benefit of freezing in respect of the transportation of chilled meat is its possibility of prolonged storage as well as its greater handling flexibility in both retail and wholesale trade. freezing of meat effects quality changes that are direct consequences of freezing processes and frozen storage (akhtar et al., 2013). the frozen state does not entirely eliminate the bio-physicochemical processes taking place in meat, but only minimizes them. freezing results in the slowing down or halting of post-slaughtering meat maturity processes, while in-meat water freezing and crystallization processes become intensified (leygonie et al., 2012, gambuteanu et al., 2013). the quality of frozen stored meat depends on on-going changes both during the initial pre-freezing and in later stages of freezing treatment and storage. the quality of frozen products depend on the quality of the primary material, adequate freezing parameters, storage conditions, stability of storage temperature and its duration as well as the applied thawing methods (ali et al., 2016; chen et al., 2017; fernandes et al., 2016). the last stage in the chilling technological process is thawing, aimed at restoring the meat’s properties to conditions similar to that of fresh meat. the procedural parameters like temperature and efficient thawing play significant roles during this stage. the thawing process is acclaimed to be more difficult to control than the freezing process. any improprieties in the thawing process may result in quality deterioration (zhang et al., 2017). thawing in atmospheric air has become a popular method applied in meat thawing. the growing role in industry-led deep freezing of meat products necessitates the application of modern and fast methods of meat thawing in order to shorten the process and have a better control of its parameters. the microwave thawing procedure does fulfil this criteria (chwastowska and kondratowicz, 2005). the aim of the study was to assess the influence of frozen storage duration and thawing methods on the breast muscles quality of broiler chickens. 2. material and methods the research materials were breast muscles obtained directly after slaughtering of minimum 36-day old ross 308 broiler chickens. the chickens were reared on litter and fed on complete standard feed mixtures (starter, grower and finisher), for broiler chickens with full access to water supply. the average weight of slaughtered chickens was 2.30kg. the slaughter and dissection of chickens was carried out under prevailing production specifications at a local slaughterhouse. breast muscles (musculus pectoralis) ital. j. food sci., vol. 31, 2019 533 were manually cut out from the chilled carcasses (ziołecki and doruchowski, 1989). the samples (n=80 and mean weight of 200±50g) were weighed (balance type ed 423s0ce, accuracy 0,001g, company sartorius mechatronics, poland) labelled and vacuum packed in polyethylene bags. the meat was subjected to freezing at -20°c and were stored in deep freeze conditions in freezer compartments (gn 3056 of liebherr company, germany) over a 1 month period for group i and 8 months for group ii. on completing the storage period the samples from group i (n=40) and group ii (n=40) were thawed in atmospheric air and in a microwave oven. the atmospheric air method involved thawing the sample under atmospheric conditions in refrigerated cabinet (fkv 36110, from liebherr company, germany) at 4°c without removing them from the bags until the samples achieved internal temperature of 4°c±1°c. the thawing process with a microwave involved placing the samples in a microwave oven (type 29z013, 800 w, zelmer company, poland) and subsequently subjected electromagnetic wave treatment over a 5 minute period. the samples internal temperature was 0°c±1°c after thawing, but was 5°c ±1°c on its outer surface. the sample’s internal temperature was stabilized at 4°c ±1oc. the laboratory assessment of raw breast muscles from group i (n=20) and group ii (n=20) covered the following: volume of drip loss, ph, water-holding capacity, chemical composition (protein, fat, and mineral salts content) as well as the meat’s colouration and brittleness. the volume of drip loss was determined by placing the sample in a cuvette fitted with a 5 mm thick grid spacer to prevent the leakage from getting in contact with the sample. the amount of leakage was calculated by the difference in weight before freezing and after thawing process using the formula: w!(%) = m! − m! m! ×100% where: wr the amount of drip loss (%), m1 sample weight before freezing (g), m2 sample weight after thawing (g) (marchel et al., 2013). ph measurements were undertaken using a ph-meter fitted with a dagger electrode (hi 99163, hanna instruments, usa) calibrated with duracal buffer solutions: ph 4.01, ph 7.00. (hmilton bonaduz ag, switzerland). the water-holding capacity (whc) was determined based on the volume of free water squeezed from sample (280-300mg) using the whatman no 2 filter papers and 2kg load for 5 minutes (grau and hamm, 1953). whc was calculated on the basis of the mass difference of the sample before squeezing to the mass of the sample after squeezing in relation to the sample weight before squeezing x 100%. the nitrogen content was determined using kjeldah method (foss tecator, höganäs, sweden), and converted to protein using a multiplying factor of 6.25. the fat content was determined using soxhlet method (büchi extraction system b-811 apparatus, flawil, switzerland). after drying the samples (5g±0.001g) at 105°c, they were subjected to extraction using n-hexan as the solvent. the fat amount was determined by weighing after the solvent has been separated. total ash content was measured after the complete mineralization of 5g of the meat sample at 550-6505°c in a muffle carbolite oven type aaf1100, hope valley, uk. the colour of the raw meat’s cross-section was assessed using the chrome meter colorimeter (konica minolta osaka, japan), fitted with a cr 400 head (ø=11mm) set at illumination levels compatible with d65, calibrated with konica minolta calibration plate (observer 20, illuminant d65, y=93.5, x=0.3160, y=0.3324). the readings of measurement results and their conversion in real time was achieved in the cie lab (cie, 1978) colorimetric system, where l* represents brightness, a* relative redness, on red-green axis, b * relative yellowness, on yellow-blue axis. three repetitions were performed for each sample. the brittleness was assessed by measuring the share force (fmax), using the ital. j. food sci., vol. 31, 2019 534 zwick/roell bt1-fr1.oth.d14 resistance machine (zwick cmbh & co. kg. ulm, germany), using a warner-bratzler v-blade knife with a head speed of 100 mm··min-1 and initial force of 0.2 n. meat portions with cross-sectional diameter of 100 mm2 and 50 mm in length were subjected to cutting. after the thawing of samples from both group i (n=20) and group ii (n=20) they were weighed (balance type ed 423s-0ce, accuracy 0,001g, company sartorius mechatronics, poland) with accuracy of 0.1g and then subjected to boiling at a ratio 1:2 (meat/ water) until the attainment of meat internal temperature of 805°c±25°c. assessments of physicochemical properties after cooking were conducted in the same way to those applied for the raw breast muscles. cooking losses were calculated based on the differences in weight prior to and after thermal treatment. the sensory assessment of the quality of thawed breast meat was conducted using the scaling method (baryłko-pikielna and matuszewska 2009). a five-point scale assessment was applied, covering such quality indicators as the meat’s aroma and flavour (especially its desirability and intensity), juiciness, brittleness and general appearance (1 point being the lowest score, and 5 points being the highest). in order to conduct the sensory assessment, the thermally treated samples were cooled to 205°c±25°c, cut into 1.5 cm thick slices, perpendicular to the run of meat fibers and then placed in plastic containers. the samples were randomly assessed after they had been encoded. the sensory assessment process was conducted, in two repetitions, by a 7-member assessment team with proven sensory sensitivity, trained in accordance with iso 8586-2:2008 and iso 8587:2006 standards. the results obtained were statistically analysed using the statistica 13.1 (statsoft, inc. 2018) program, taking account of the arithmetic mean (x), standard deviation (sd), standard mean error of measurement (sem), and the principal effect, namely (c impact of storage duration, r influence of thawing method, c x r impact of storage duration and thawing method), using the two-factor analysis of variance anova. the significance of differences between the mean values within groups was verified using the tukey test. statistically significant differences were assumed at a significance level of p<0.05, while the lack of significance was designated with „ns” (statistically not significant). 3. results and discussion the freezing process leads to the denaturation of myofibril proteins, which results in the deterioration of meat functional properties. as a result of the water transformation phase, there is increased concentration of ions, ionic strength, as well as ph changes (ali et al., 2015). the current studies have indicated significant (p<0.05) influence of frozen storage duration on the ph value (table 1). according to chwastowska-siwiecka (2011) and leygonie et al. (2012) the ph of broiler chicken breast muscles in appropriate conditions decreases along with the prolongation of frozen storage. likewise, results obtained by ali et al. (2015), chen et al. (2017) and wei et al. (2017) have indicated significant declines in ph value of breast muscles over subsequent weeks of frozen storage. santos kumar et al. (2014), however, noted rising ph values of breast muscles of broiler chickens obtained from varied sources, stored frozen. the current studies did not indicate any significant (p>0.05) influence of thawing methods on the ph value. similar results for the thawing methods, but with varying intensity of microwaves, were obtained by kim et al. (2011) for broiler chickens breast muscles, chwastowskasiwiecka et al. (2013) for rabbit meat, while chwastowska and kondartowicz (2005) obtained same for pork meat. drip loss is a watery solution that issues from frozen meat without the application of any external force serving as a significant indicator of the quality of meat subjected to frozen storage (gambuteanu et al., 2013; leygonie et al., 2012). it is acclaimed that the volume of meat leakages while being thawed using various ital. j. food sci., vol. 31, 2019 535 methods may serve as one indicator of the degree of damage to meat muscle tissues during freezing and also as an indirect evaluation of various thawing methods (chwastowska and kondratowicz, 2005). table 1. physicochemical properties of frozen raw breast muscles, including the frozen storage duration and thawing methods (x± s). traits frozen storage duration, months sem impact (group i) (group ii) thawing method c r c x r atmospheric air microwave oven atmospheric air microwave oven ph 6.00±0.02 5.91±0.03 5.84±0.18 5.87±0.17 0.01 * ns ns drip loss (%) 3.34±0.30 2.78±0.1 5.04±0.28 4.74±0.35 0.14 * * * water-holding capacity (%) 15.58±1.37 16.11±1.54 18.34±2.47 17.72±1.23 0.20 * ns ns colour l*lightness a*redness b*yellowness 53.12±2.30 1.76±0.66 6.06±1.07 52.07±2.48 1.98±0.51 6.58±1.39 50.32±1.85 2.21±0.88 7.32±1.28 50.60±2.04 3.30±0.79 8.04±1.62 0.49 0.07 0.18 * * * ns * * n s* ns fmax (n) 13.56±2.02 15.12±1.96 11.86±1.88 13.98±2.44 0.25 * * ns crude protein (%) 23.84±1.13 23.75±1.04 24.04±0.65 23.92±0.58 0.08 ns ns ns fat (%) 1.16±0.16 1.19±0.12 1.18±0.20 1.17±0.16 0.04 ns ns ns ash (%) 1.15±0.10 1.21±0.15 1.13±0.18 1.15±0.12 0.02 * * ns explanations: (x ± s) arithmetic mean±standard deviation, group i 1 month freezing storage (n=20) , group ii 8 months freezing storage (n=20); atmospheric air (n=10), microwave oven (n=10); c impact of frozen storage duration; r impact of freezing method; c x r impact of frozen storage duration and freezing method; *statistically significant differences p<0.05; nsdifferences statistically not significant. the current studies have shown the significant (p<0.05) influence of frozen storage duration, thawing methods, including the interplay of these factors on the volume of drip loss (table 1). the volume of drip loss increases proportionately with frozen storage duration. the study finding corroborates those obtained by ali et al. (2016) with samples thawed in atmospheric air. studies by wei et al. (2017) indicated lower values for this factor, although with a similar increasing trend of the value for drip loss and with an 8month long frozen storage. the current studies have shown that the amount of drip loss of breast muscles using the microwave method was significantly (p<0.05) lower than in case of meat thawed in atmospheric air. the results obtained are similar to those obtained by oliveira et al. (2015) in studies conducted using the same thawing methods on breast meat of broiler chickens and by chwastowską et al. (2013) on rabbit meat as well as by chwastowska and kondratowicz (2005) on pork meat. yu et al. (2005) has posted that the amount of drip loss is influenced by the thawing temperature, with the amount increasing with the growing temperature. the current studies have indicated significant (p<0.05) impact of frozen storage duration on the water-holding capacity of the breast muscles. the results of measurements for the meat water-holding capacity, determined using the forced leakage method point to the existence of dependency between changes in the meat properties and the amount of drip loss. greater rate of water loss during frozen storage stage limited the amount of forced ital. j. food sci., vol. 31, 2019 536 leakage from the meat, which could translate to mean better water absorption during prolonged meat storage, irrespective of the thawing method applied. the findings of the study corroborate those obtained in studies by wei et al. (2017) conducted on the breast muscles of broiler chickens as well as those by chwastowska and kondratowicz (2005) conducted on pork meat. an important criterion of the meat’s technological quality is its colour. the degree of change in colour of frozen meat relies mainly on the availability of atmospheric oxygen and depends on the conditions of frozen storage (akhtar et al., 2013). the results of colour assessment of raw breast muscles after the freezing stage, considering the frozen storage duration as well as the thawing methods are illustrated in table 1. the studies have demonstrated that a 8-month frozen storage duration resulted in a significant (p<0.05) reduction of l* (brightness) parameter, which corroborates the study findings of wei et al. (2017). however, ali et al. (2016) and galobart and moran (2004) demonstrated the impact of shorter frozen storage duration on the increased l*(brightness) and b*(yellowness) parameters. the current study demonstrated significant (p<0.05) impacts of both the duration of frozen storage and thawing methods on the colour saturation in favour of a*(redness) and b*(yellowness). a darker colouration was characteristic of breast muscles stored over a 8-month period and thawed using a microwave method in contrast to breast muscles stored for a month duration and thawed using the atmospheric air method. similar values for the a*(redness) parameter were obtained by kim et al. (2011), using the microwave thawing method on the breast muscles of broiler chickens. a similar trend in colour change during a shorter frozen storage period was posted by chwastowska and kondratowicz (2005) that conducted studies on pork meat thawed using the same methods. colour change in post thermally treated meat depend on the level of myoglobin denaturation as well as the level of temperature applied for the thermal treatment (zhuang and savage, 2010). the colour assessment of post thermally treated meat indicated that frozen storage lead to increased l*(brightness) but a decline b*(yellowness) parameters (table 2). similar results were obtained by galobart and moran (2004). the studies demonstrated a significant (p<0.05) influence of thawing methods on the colour saturation towards redness while retaining the tendency of being similar to raw meat. the current studies have demonstrated the significant (p<0.05) influence of both the frozen storage duration and thawing methods on the share force of raw breast muscles (table 1). it was demonstrated that the brittleness of raw meat measured by its share force increased with the frozen storage duration. the research findings correspond to those of śmiecińska et al. (2015) conducted on turkey breast muscles. in shanks et al. (2002) and farouk et al. (2003) opinions, the process of frozen storage increases the brittleness of meat, especially in case of unprocessed meat. changes in the meat’s brittleness during the frozen storage could be attributable to protein changes in the meat tissue (lee et al., 2008). gambuteanu et al. (2103) argue that the brittleness of poultry meat depend on the thawing methods applied. the current study have demonstrated a significantly (p<0.05) less share force was characteristic of both raw breast muscles, thawed in atmospheric air and subjected to thermal treatment compared to those thawed using microwave method (tables 1 and 2). similar findings were obtained by oliveira et al. (2015), who evaluated the brittleness of breast muscles applying the same thawing methods. the thawing temperature, according to yu et al. (2005), has impacts on the brittleness of broiler chickens breast muscles in which lower share force was characteristic of those samples thawed at lower temperatures. the brittleness of post thermally treated meat depends on the quality of the initial raw material, type of thermal treatment as well as the heating duration and temperature (akhtar et al., 2013). the current study did not identify any significant (p>0.05) impact of frozen storage duration on the brittleness of ital. j. food sci., vol. 31, 2019 537 post thermally treated breast muscles. however, lee et al. (2008) observed increasing toughness in breast muscles stored frozen and boiled. table 2. physicochemical properties of defrosted breast muscles subjected to thermal treatment, including the frozen storage duration and thawing methods (x ± s). traits frozen storage duration, months sem impact (group i) (group ii) thawing method c r c x r atmospheric air microwave oven atmospheric air microwave oven cooking loss (%) 23.47±1.13 25.98±0.44 26.23±1.05 28.21±1.05 0.15 * * * colour l*lightness a*redness b*yellowness 80.79±1.04 1.27±0.15 13.08±0.20 81.64±0.84 1.45±0.13 12.91±0.37 82.71±0.42 1.28±0.09 11.13±0.16 82.68±0.77 1.50±0.07 10.80±0.35 0.56 0.02 0.04 * ns * ns * ns ns ns ns fmax (n) 19.58±2.36 20.01±1.14 18.04±1.84 19.36±1.74 0.27 ns * ns crude protein (%) 30.95±0.71 29.54±0.83 31.02±0.65 30.62±0.51 0.15 ns ns ns fat (%) 1.26±0.16 1.25±0.12 1.24±0.20 1.23±0.16 0.08 ns ns ns ash (%) 1.29±0.10 1.30±0.15 1.23±0.20 1.26±0.18 0.03 * ns ns explanations: (x ± s) arithmetic mean±standard deviation, group i 1 month freezing storage (n=20), group ii 8 months freezing storage (n=20); atmospheric air (n=10), microwave oven (n=10); cimpact of frozen storage duration; rimpact of freezing method; c x r impact of frozen storage duration and freezing method; *statistically significant differences p<0.05; nsdifferences statistically not significant. akhtar et al. (2013) and gambuteanu et al. (2013) postulate that protein metabolism which takes place under specific conditions during the freezing process is not significant and are limited only to minor loss of proteins and amino-acids as a result of drip loss. frozen storage induces protein carbonylation, carboxylation as well as the formation of schiff bases in chicken meat (utrera and estevez, 2013). the current study did not indicate any significant (p<0.05) influence of the duration of frozen storage and freezing methods on the total protein, and fat content in the raw meat (table 1). these findings are corroborated by those from wei et al. (2017) study. kim et al. (2011) did not demonstrate the impact of applied thawing methods on the protein content in raw breast muscles. bustamante-vargas et al. (2016), however, observed that greater protein losses were noted during thawing using microwave than in atmospheric air. protein losses increased proportionately with increasing freezing temperature. deep freezing storage can result in muscle proteins forming into gel, which is associated with a higher solubility of meat protein in frozen meat compared to fresh meat (farouk et al., 2003). the aforementioned dependency was observed in studies by śmiecińska et al. (2015), that indicated increased total and soluble protein content following a 6-week long deep freeze storage of turkey breast muscles. both the deep storage and thawing procedures affect the activity of endogenous proteolytic enzymes responsible for the degradation and relaxation of muscle protein structures of meat tissues (farouk et al., 2003). studies conducted by chan et al. (2011) noted increased content of soluble protein in frozen vacuum-packed turkey meat, stored for 3 weeks and thawed in atmospheric air at 4°c. both chwastowska and kondratowicz (2005) demonstrated the effect of freezing storage and both thawing methods on the total protein and ash content of raw pork meat. ital. j. food sci., vol. 31, 2019 538 higher protein content, but lower ash content was demonstrated in meat stored for a 3month period and thawed using the atmospheric air method. the current study also demonstrated the influence of deep freeze storage on the ash content of raw breast muscles (table 1). the observed decrease in ash content during the 8-month long deep freeze storage in breast muscles was due to increased meat leaks in the process of thawing, thus resulting in greater loss of minerals. the thermal treatment of raw meat leads to changes in its nutrient content (akhtar et al., 2013). the current studies demonstrated similar change tendencies in the nutrients of breast muscles thawed and subjected to thermal treatment as in raw meat (table 2). a significant (p<0.05) influence of the storage duration, thawing methods as well as their interactions on the volume of cooking loss was observed. higher cooking losses were observed following the 8-month long deep freeze storage in meat thawed using the microwave method. similar results for an 8-month long deep freeze storage were obtained by wei et al. (2017) in breast muscles thawed using atmospheric air method. chen et al. (2017) and śmiecińska et al. (2015), on the other hand, observed slight decreases in thermal leakages during the last phase of the freezing process. a similar trend was demonstrated in studies conducted by chwastowska and kondratowicz (2005) on pork meat stored deep frozen over a 3-month period. yu et al. (2005) found that the volume of thermal leakage varied in relation to the thawing temperature applied. the magnitude of the thermal leakage has a huge influence on the quality characteristics of culinary meat and the yield of the finished product (olivier et al., 2015). several changes affecting sensory qualities may take place in meat and its allied products stored frozen. such changes should be explained by way of the physical (recrystallization, denaturation, freeze-thawing burns), chemical (hydrolysis, auto-oxidation) as well as microbiological and enzymatic (hydrolysis, oxidation, dehydration) transformations. the extent of such transformations depends on the temperature and duration of freezing, including the conditions of storage (akhtar et al., 2013; gambuteanu et al., 2013). the sensory properties of breast muscles subjected to thermal treatment following the freezing period, depending on the duration of the deep freeze storage as well as the thawing methods are presented table 3. table 3. sensory properties of breast muscles after thermal treatment, including the frozen storage duration and thawing methods (x ± s). traits frozen storage duration, months sem impact (group i) (group ii) thawing metod c r c x r atmospheric air microwave oven atmospheric air microwave oven odour intensity 3.67±0.46 3.67±0.48 3.58±0.49 3.42±0.49 0.05 ns ns ns flavour intensity 4.48±0.47 4.55±0.43 3.37±0.36 3.53±0.50 0.05 * ns ns odour desirability 3.83±0.37 3.75±0.43 3.67±0.44 3.58±0.49 0.04 ns ns ns flavour desirability 4.05±0.50 4.80±0.37 3.33±0.48 3.40±0.49 0.05 * * ns juiciness 4.42±0.49 4.68±0.49 3.17±0.32 3.20±0.37 0.04 * * ns tenderness 3.88±0.49 3.74±0.47 3.78±0.45 3.68±0.49 0.05 ns ns ns explanations: (x ± s) arithmetic mean±standard deviation, group i 1 month freezing storage (n=20) , group ii 8 months freezing storage (n=20); atmospheric air (n=10), microwave oven (n=10); cimpact of frozen storage duration; rimpact of freezing method; c x r impact of frozen storage duration and freezing method; *statistically significant differences p<0.05; nsdifferences statistically not significant. ital. j. food sci., vol. 31, 2019 539 sensory scale: odour; flavour intensity: 1negatively changed, 2moderately changed, 3typical, weak; 4typical, strong, 5 typical, very strong; odour; flavour desirability: 1not desirable, 2fairly desirable, 3desirable, 4very desirable, 5highly desirable; juiciness: 1very dry, 2dry, 3slightly juicy, 4juicy, 5very juicy; tenderness: 1very hard, 2hard, 3slightly tender, 4tender, 5very tender. the current studies have demonstrated that a significantly (p<0.05) lower intensity of taste desirability and less juiciness was characteristic of meat following a 8-month deep freeze storage. a similar trend in sensory changes was demonstrated in studies conducted by śmiecińska et al. (2015) and santosh kumar et al. (2014), where the prolongation of the deep freeze storage was associated with the deterioration of the taste and juiciness of broiler chickens’ meat. the microwave thawing method has, in the current studies, been shown to be more suitable than the atmospheric air method in respect of taste desirability and juiciness of broiler chickens’ meat. 4. conclusions long deep freeze storage (8 mth) resulted in significantly (p<0.05) higher drip loss, ph decline, darker colouration (less value of l* parameter and higher saturation toward redness and yellowness), reduced ash content and improved brittleness, measured by the shear force in broiler chicken meat. deep freeze storage also resulted in significant (p<0.05) changes in the raw meat following the thermal treatment. long frozen storage contributed to the increased cooking losses, increased saturation of the l*(brightness) parameter and reduction in the b*(yellowness) tone as well as a decrease in ash content and reduced sensory properties compared to the 1-month long frozen storage duration. raw breast muscles that has been thawed using the microwave method was characterized by a significantly (p<0.05) lower drip loss, higher saturation toward redness (a*) and yellowness (b*), higher ash content but with less brittleness compared with breast muscles thawed using atmospheric air. significantly (p<0.05) higher cooking losses, higher saturation toward redness (a*) and higher shear force were characteristics of breast muscles thawed using the microwave method and subjected to thermal treatment compared to meat thawed in atmospheric air. the sensory assessment of breast muscles subjected to thermal treatment following its freezing has demonstrated that microwave is, for reasons of taste desirability and juiciness of meat, a more suitable thawing method (p<0.05) than atmospheric air. acknowledgments the authors would like to thank faculty laboratory for analysis of environment health and materials of agricultural origin for technical support and assistance. references akhtar s., khan m.i. and faiz f. 2013. effect of thawing on frozen meat quality: a comprehensive review. pakistan j. food sci. 23(4):198211. ali s., zhang w., rajput n., khan m., li c.b. and zhou g. 2015. effect of multiple freeze-thaw cycles on the quality of chicken breast meat. food chem. 18: 808-814. ital. j. food sci., vol. 31, 2019 540 ali s., rajput n., li c.b., zhang w. and zhou g. 2016. effect of freeze-thaw cycles on lipid oxidation myowater in broiler chickens. braz. j. poultry sci. 1:35-39. augustyńska-prejsnar a., ormian m. and gajdek g. 2014. market choices for broiler chicken meat in the opinion of students (in polish). journal of agribusiness and rural development 3(33):5-13. benli h. 2016. consumer attitudes toward storing and thawing chicken and effects of the common thawing practices on some quality characteristics of frozen chicken. asianaustral. j. anim. sci. 29(1):100-108. baryłko-pikielna n. and matuszewska i. 2009. sensory testing of food. basics methods application (in polish), publishing: polish society of food technologists. bustamante-vargas c.e., trentini m.s., backes g.t., cansian r.l., mello r.o., kubota e.h., demiate i.m. and prestes r.c. 2016. eletrophoretic profile of exudates of chicken breast submitted to different thawing methods. international food research journal 23(1):55-60. chan j.t.y., omana d.a. and betti m. 2011. effect of ultimate ph and freezing on the biochemical properties of proteins in turkey breast meat. food chem. 127:109117. chen t., zhu y., han m., wang p., wei r., xu x. and zhou g. 2017. classification of chicken muscle with different freezethaw cycles, using impedance and physicochemical properties. j. food eng. 196:94-100. cie 1976. supplement no 2 to: recommendations on uniform color spaces color difference equations. psychometric color terms. commission internationale de eclarage (1971) to 1-1. cie publication no 15 (e-1.3.1), paris france. chwastowska i. and kondratowicz j. 2005. technological properties of a frozen pork meat depending on the storage time period and a thawing method (in polish). food. science. technology. quality. vol. 3, no. 44, supl. 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meat color lightness values. poultry sci. 89(5):1049-1055. ziołecki j. and doruchowski w.1989. evaluation method of the poultry slaughter value (in polish). cobrd poznań. paper received june 4, 2019 accepted february 28, 2019 ijfs#1853_bozza ital. j. food sci., vol. 32, 2020 734 survey wine intolerance in italy: a pilot study f. ceruttia, m.i. crescio*a, a. costantinib, p.l. acutisa, e. vaudanob and s. pelettoa aistituto zooprofilattico sperimentale del piemonte, liguria e valle d'aosta, via bologna 148, 10154, torino, italy bconsiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca viticoltura ed enologia, via pietro micca 35, 14100, asti, italy *corresponding author: mariaines.crescio@izsto.it abstract intolerance to wine has been investigated under different aspects. in our study, we explored intolerance/allergy reactions in italy by means of a survey including 901 questionnaires. prevalence of wine or beer intolerance was 6%, of which 11 reported that was verified by a doctor. odds ratio for wine intolerance with another declared allergy/intolerance is 1.6. in conclusion, we observed a high prevalence of intolerance to wine in italy, highlighting that some sections of the population (young people and women) may be more exposed to these phenomena. keywords: alcoholic drinks, allergy, intolerance, italy, survey, wine ital. j. food sci., vol. 32, 2020 735 1. introduction over the last few years, consumers have become increasingly interested not only in safety of food and beverages, but also in their healthiness. at the same time, the cultural conception of the consumed products has gained importance in relation to shared cultural and social significance and practice in a given environment or geographical area. when dealing with wine, the cultural connotation is well defined: in mediterranean culture, the emotional and cultural aspects of wine have always been strongly connected. moreover, in western culture, wine is often associated with festive and/or convivial moments. the effects of wine consumption have been extensively studied and a moderate alcohol consumption was proven to have positive healthy effects (ronksley et al., 2011); however, as for other food, allergic reactions and intolerances were also reported for wine (niestijl jansen et al., 1994; vally et al., 1999; armentia, 2008; böhn et al., 2013; jaeckels et al., 2015; bansal et al., 2017; wüthrich, 2018). a recent study reported prevalence of 74% for upper airway symptoms and 51% for lower respiratory symptoms after alcohol ingestion in patients with aspirin-exacerbated respiratory disease (cardet et al., 2014). additional risk factors include female sex, history of allergic rhinitis, chronic obstructive pulmonary disease, and asthma (nihlen et al., 2005). the etiology of this reaction is still unclear, because wine is a complex beverage, with several potential allergens. potential allergens include proteins from grape, molds, yeasts, as well as proteins from insects that have contaminated the mash. milk derivatives as caseine and potassium caseinate, and other animal-derived products as isinglass and gelatin are used in the fining process to remove phenolic and tannin compounds from white wine, and egg white (albumin) is used to remove tannin compounds from red wine. non-grape-derived tannins such as those from the bark and galls of trees are also used. these fining agents are added to the wine and the precipitates removed (rolland et al., 2006). allergic reactions have been described also for components like ethanol, acetaldehyde, acetic acid and sulfites. ethanol, acetaldehyde and acetic acid, flavonoids (anthocyanins and chatechines), sulfites, histamine and other biogenic amines are the main causative agents of intolerance reactions (pseudoallergic reactions) to wine (vally et al., 1999; wüthrich, 2018). actually, the presence of histamine in red wine as a source of intolerance has been controversial. several studies observed a clear correlation between the red wine consumption (more rich in histamine than white wine) and the insurgence of the typical symptoms for food intolerance, such as sneezing, flush, headache, diarrhea, skin itch, and shortness of breath (jarisch and wantke, 1996; wantke, hemmer, haglmüller, et al., 1996; wantke, hemmer, haglmuller, et al., 1996). moreover, well trained wine assessors were able to identify elevated histamine concentrations in wine (rohn et al., 2005). nevertheless, other studies did not observe such correlation between wine intolerance and histamine content of wine (kanny et al., 2001). in either cases, sample size was very low, and this may be the reason for this contradiction. similar challenges were conducted to investigate the role of sulfites on the pathogenesis of wine-induced asthma, with no clear correlation, but more likely, this phenomenon appears to be a complex phenomenon, involving several different mechanisms (vally et al., 1999). in asthmatics, alcoholic drinks trigger asthma symptoms in more than one third of these patients (ayres and clark, 1983; vally et al., 2000). with regard to the prevalence of wine intolerance/allergy in general population, two surveys were conducted in germany and denmark (linneberg et al., 2008; wigand et al., 2012). the german study was ital. j. food sci., vol. 32, 2020 736 localized in mainz, the capital of rhineland-palatinate, a wine-producing region of germany, and was conducted by mean of a questionnaire survey (wigand et al., 2012). the intolerance to wine was reported to be 3.2%, but only two persons reported that a wine allergy was verified by a physician. a cross-sectional study by means of postal questionnaire was conducted in copenhagen, denmark, in 2006, involving over 4,000 respondents out of 6,000 invited people (linneberg et al., 2008). the survey investigated the hypersensitivity reactions following consumption of alcoholic beverages divided by symptoms from the nose, lower airways, and the skin, with a prevalence of 7.6%, 3.2%, and 7.2%, respectively, and a cumulative prevalence of 13.9%. this study investigated all types of alcoholic beverages, including beer, red wine, white wine, dessert wine, and spirits. to the best of our knowledge, no data are available about the prevalence of intolerance/allergy symptoms induced by wine in the mediterranean countries, like italy, that have a strong tradition in wine consumption. according to a 2014 fao report, italy is at the first place in the world ranking of wine producing countries, followed by spain and france. aim of the study was to explore whether intolerance/allergy reactions are also present in the general population of a wine-producing country, such as italy, and to estimate the prevalence by means of a questionnaire survey. 2. materials and methods the questionnaire about wine consumption and intolerance developed by wigand an colleagues (wigand et al., 2012) was translated in italian, slightly modified and transferred to google surveys (https://surveys.google.com). questionnaires were submitted either by face-to-face interview or self-submission after receiving the link to the google form. for face-to-face interview, the answers were either recorded on a printed survey and then added later on the google form by our personnel, or directly entered on the online survey from a portable device. subjects were included in the study with a convenience sampling, enrolling for the face-toface interview 170 people attending to two events held in september 2017 in piedmont (cheese 2017: http://cheese.slowfood.it/en/ and european researchers' night 2017: http://nottedeiricercatori.piemontevalledaosta.it). snowball sampling starting from researchers involved in the study was used to enroll additional 731 people, using the online form. as described by wigand and colleagues, the questionnaire, in addition to standard questions on age and sex, included questions on whether allergy-like symptoms had occurred after wine consumption: participants had to choose among a list of symptoms (table 1) experienced after the consumption of red, white and rosé wine. the intensity and the time of occurrence of each symptom was also assessed by the participants (intensity: none, weak, moderate, strong, very strong; time: <15 minutes, 15 minutes to 1 hour, 1 to 2 hours, >2 hours). in addition, participants were asked about intolerance to various known allergens, listed in table 2. for each reported intolerance, participants had to declare if it was medically diagnosed. according to wigand and colleagues (wigand et al., 2012), all reported symptoms, except for headache, were considered as symptoms of intolerance and a score was given (table 1). each subject scoring more than 10 was defined as “intolerant to wine”. ital. j. food sci., vol. 32, 2020 737 table 1. symptoms of wine intolerance, their score and prevalence with ci 95%. symptoms score number of participants prevalence ci 95% circulatory collapse 10 2 0.2 0-0.8 shortness of breath/asthma 8 25 2.9 1.9-4.2 tachycardia 7 50 5.9 4.4-7.7 itching 6 48 5.6 4.2-7.4 flushed skin 5 160 21.6 18.7-24.7 low blood pressure 5 51 6 4.5-7.8 rhinorrea 4 30 3.4 2.3-4.9 burning sensation in lips, palate, neck 4 47 5.5 4.1-7.3 stomach or intestinal cramps 3 99 12.3 10.1-14.8 diarrhea 3 48 5.6 4.2-7.4 vomiting 3 33 3.8 2.6-5.3 skin rash, hives, oedema 23 2.6 1.7-3.9 headache 353 64.4 60.2-68.4 other symptoms 48 5.6 4.2-7.4 once ended the survey, we performed a descriptive analysis of the characteristics of participants, of the frequency and of the preferences in alcohol consumption. then, we calculated the prevalence and the 95% confidence interval (95% ci) of each symptom, and the prevalence of people intolerant to wine. odds ratio (or) and their 95% ci were calculated applying a logistic regression model to verify if there was some gender or age difference among people classified as intolerant to wine. the presence of allergies among participants was described and gender or age differences were assessed by calculating or and their 95% ci. finally, or and their 95% ci were calculated to describe the relationship between intolerance to wine and the presence of allergies. all the statistical analyses were carried out using the software stata 15.1 (statacorp llc, college station tx77845, texas, usa, www.stata.com). 3. results a total of 901 questionnaires were included in the study, and sex was equally distributed with 52% (n=486) of females and 48% (n=429) males. the geographical proportion and the proportion for age among participants do not reflect the proportion in the general population, as reported in fig. 1, with an overrepresentation of people under 55 years of age. as shown in fig. 2, most of participants affirmed to drink wine more than once a month, and only a minority declared to have never drank wine. generally, men drank more than women (or 2.78, 95% ci 2.09-3.70) in our survey, 54 (6%) participants declared to have an intolerance to wine or beer, most of which were females; 11 of them reported that the allergy was verified by a doctor. the 36% of participants reported at least one symptom of intolerance after drinking wine. the prevalence of each symptom consequent to drinking wine, reported by participants is summarized in table 1. ital. j. food sci., vol. 32, 2020 738 figure 1. comparison between the geographical distribution of the italian general population at jan, the 1st 2018 (right), and people enrolled in the study (left). figure 2. alcohol consumption: proportion of alcohol consumption by sex among people enrolled in the study. never: never drunk wine; sometimes: once a month; regularly: more than once a month. ital. j. food sci., vol. 32, 2020 739 having achieved a wine intolerance score of at least 10, a total of 138 subjects were classified as intolerant to wine (18%, 95% ci 15-21%), with an increased risk of intolerance to wine in females (or 1.89, 95%ci 1.29-2.75), without differences in the age classes considered or in the type of wine. the 61% of participants declared to have at least one allergy/intolerance, with a risk of reporting at least one allergy/intolerance increased in females (or 1.96, ci95% 1.49-2.58), without differences in the age classes considered or in the type of wine. the prevalence of each allergy/intolerance self-declared by participants is reported in table 2. table 2. prevalence of each allergy/intolerance self-declared by participants and declared as medically diagnosed. self-declared medically diagnosed allergens prevalence ci 95% prevalence ci 95% alcohol 4 2.8-5.6 1.2 0.6-2.2 banana 2.2 1.3-3.3 1.5 0.8-2.5 beer 3.8 2.6-5.3 1.3 0.7-2.3 carrots 1.6 0.9-2.6 1.3 0.7-2.3 celery 1.2 0.6-2.2 1.1 0.5-2.1 cherries 2.3 1.4-3.5 1.5 0.8-2.5 crustaceans 3.8 2.6-5.3 1.9 1.1-3.1 eggs 2.5 1.6-3.8 1.6 0.9-2.6 fish 1.7 1-2.8 1.3 0.7-2.3 gluten 5.3 3.9-7 1.9 1.1-3.1 grapes 1.6 0.9-2.6 1.2 0.6-2.2 house dust 36.1 32.4-39.9 16.9 14.3-19.7 kiwi 5.1 3.8-6.8 1.8 1-2.9 latex 4.8 3.4-6.4 1.7 1-2.8 lupines 1.5 0.8-2.5 1.1 0.5-2.1 medications 13.6 11.3-16.2 9.2 7.3-11.4 milk and derivates 15.5 13-18.2 4.2 2.9-5.7 mustard 1.8 1-2.9 1.1 0.5-2.1 nickel 13.3 11-15.9 5.3 3.9-7 nuts 6.9 5.3-8.8 3.3 2.2-4.7 peaches 4 2.8-5.6 1.9 1.1-3.1 peanut 3.3 2.2-4.7 1.9 1.1-3.1 pears 2.2 1.3-3.3 1.5 0.8-2.5 peppers 4 2.8-5.6 1.5 0.8-2.5 plums 1.5 0.8-2.5 1.2 0.6-2.2 pollen 39.7 35.9-43.6 21.4 18.5-24.6 seafood 4.9 3.5-6.6 2.3 1.4-3.5 sesame 1.6 0.9-2.6 1 0.5-1.9 soya 1.8 1-2.9 1 0.5-1.9 strawberries 4 2.8-5.6 2.3 1.4-3.5 sulphites 11.9 9.8-14.4 1.6 0.9-2.6 tomatoes 4.9 3.5-6.6 1.8 1-2.9 wine 4.5 3.2-6.1 1.2 0.6-2.2 ital. j. food sci., vol. 32, 2020 740 participants classified as intolerant to wine showed an increased risk to have a medicallydiagnosed allergy/intolerance (table 3). the or of being intolerant to wine when declaring one medically diagnosed allergy/intolerance is 1.6 (ci95% 1.1-2.3). table 3 reports the or of being intolerant to wine for each medically diagnosed allergy/intolerance. table 3. odds ratio and their 95% confidence interval of having a medically diagnosed allergy/intolerance when classified as intolerant to wine. wine intolerants only allergens odds ratio ci95% banana 33 7-307.4 carrots 29.7 6.2-280.3 celery 53.2 7.2-2332.6 cherries 33 7-307.4 crustaceans 14.4 4.6-53 eggs 14.8 4.2-65.4 fish 29.7 6.2-280.3 gluten 10.9 3.6-36.5 grapes 59.5 8.3-2586.2 house dust 2.2 1.3-3.4 kiwi 26.3 7.1-145.3 latex 16.4 4.8-71.5 medications 2.7 1.5-4.6 milk and derivates 5.5 2.6-11.5 nickel 3.7 1.8-7.2 nuts 5.6 2.4-12.8 peaches 8.4 2.8-26.5 peanut 10.9 3.6-36.5 pears 33 7-307.4 peppers 33 7-307.4 plums 59.5 8.3-2586.2 pollen 1.5 0.9-2.4 seafood 11.2 4.1-33.8 strawberries 11.2 4.1-33.8 sulphites 21.9 5.7-123.4 tomatoes 18.1 5.3-77.7 4. conclusions wine is one of the oldest beverages, whose production can be traced back to 5,000 bc and it is nowadays often associated with convivial moments. intolerance to wine has been investigated under different aspects, albeit the unclear etiology. the prevalence of such intolerance was investigated in nordic countries like germany and denmark, but no information about mediterranean countries has been ital. j. food sci., vol. 32, 2020 741 published. our survey aimed to fill this lack and showed that wine intolerance is present also in italy. the prevalence of intolerants to wine (6%) is higher than the prevalence reported in germany (3.2%), but lower than the one reported in denmark (13%). this high percentage may be explained by the inclusion of all the alcoholic drinks in the danish study without differentiation, although the authors reported that red wine was listed as the main cause for hypersensitivity symptoms, as well as spirits (linneberg et al., 2008). both the previous studies reported that symptoms of intolerance were more frequent after drinking red wine rather than white wine, while we found no differences among the type of wine. wüthrich (2018) reported that the most frequent reactions are intolerance reactions to sulfites, which occur particularly after the ingestion of white wine and in asthma patients, and to histamine and other biogenic amines, mainly after ingestion of red wine. particularly in white wine, allergy-like intolerance reactions are caused by sulfite (vally et al., 2000; vally et al., 2001). asthma patients are especially sensitive. in agreement with the previous studies, we also found an increased risk of being classified as intolerant to wine in females than in males. in line with what reported in the literature (nihlen et al., 2005; cardet et al., 2014), our study highlighted a greater risk of being intolerant to wine in people who claim to have allergies or intolerances diagnosed by the doctor. on the other side, participants classified as intolerant to wine showed an increased risk to have a medically-diagnosed allergy/intolerance. specifically, the highest associated risks (or>50) were detected for celery, plums, and (as predictable) grapes. even in this case, however, it must be borne in mind that the analyzed data were reported by participants and were not investigated through the execution of clinical tests, therefore they might be subject to distortions. furthermore, the selection of a sample of convenience could cause a loss of external validity of the study, therefore the quantitative results of this study must be interpreted with caution. the wine also contains molecules (biogenic amines like histamine and putrescine, and sulphites), which can cause phenomena mimicking allergies, making it difficult to distinguish between false allergy phenomena (for example histamine reactions) and real allergies. in fact, allergy/intolerance to sulfites had a significant or (21.9) in those persons classified as intolerant to wine. in conclusion, although with some limitations, our study indicates a high prevalence of the phenomenon of intolerance to wine, highlighting that some sections of the population (young people and women) may be more exposed to these phenomena. however, the relationship between the presence in the wine of molecules that can cause false allergy phenomena must be further investigated. acknowledgments this study was funded by the italian ministry of health [grant number izs plv 16/15 rc]. references armentia a. 2008. adverse reactions to wine: think outside the bottle. curr. opin. allergy clin. immunol. 8:266-269. ayres j.g. and clark t.j.h. 1983. alcoholic drinks and asthma: a survey. br. j. dis. chest. 77:370-375. bansal r.a., tadros s. and bansal a.s. 2017. beer, cider, and wine allergy. case reports immunol. 2017:1-4. ital. j. food sci., vol. 32, 2020 742 böhn l., störsrud s., törnblom h., bengtsson u. and simrén m. 2013. self-reported food-related gastrointestinal symptoms in ibs are common and associated with more severe symptoms and reduced quality of life. am. j. gastroenterol. 108:634-641. cardet j.c., white a.a., barrett n.a., feldweg a.m., wickner p.g., savage j., bhattacharyya n. and laidlaw t.m. 2014. alcohol-induced respiratory symptoms are common in patients with aspirin exacerbated respiratory disease. j. allergy clin. immunol. pract. 2:208-213.e2. jaeckels n., bellinghausen i., fronk p., heydenreich b., saloga j. and decker h. 2015. assessment of sensitization to grape and wine allergens as possible causes of adverse reactions to wine: a pilot study. clin. transl. allergy 5:21. jarisch r. and wantke f. 1996. wine and headache. int. arch. allergy immunol. 110:7-12. kanny g., gerbaux v., olszewski a., frémont s., empereur f., nabet f., cabanis j.c. and moneret-vautrin d.a. 2001. no correlation between wine intolerance and histamine content of wine. j. allergy clin. immunol. 107:375-378. linneberg a., berg n.d., gonzalez-quintela a., vidal c. and elberling j. 2008. prevalence of self-reported hypersensitivity symptoms following intake of alcoholic drinks. clin. exp. allergy 38:145-151. niestijl jansen j.j., kardinaal a.f.m., huijbers g., vlieg-boerstra b.j., martens b.p.m. and ockhuizen t. 1994. prevalence of food allergy and intolerance in the adult dutch population. j. allergy clin. immunol. 93:446-456. nihlen u., greiff l.j., nyberg p., persson c.g.a. and andersson m. 2005. alcohol-induced upper airway symptoms: prevalence and co-morbidity. respir. med. 99:762-769. rohn l., page l., borck h., horr b. and diel f. 2005. can histamine be tasted in wine? inflamm. res. 54:7-9. rolland j.m., apostolou e., deckert k., de leon m.p, douglass j.a., glaspole i.n., bailey m., stockley c.s. and o'hehir r.e. 2006. potential food allergens in wine: double-blind, placebo-controlled trial and basophil activation analysis. nutrition 22:882-8. ronksley p.e., brien s.e., turner b.j., mukamal k.j. and ghali w.a. 2011. association of alcohol consumption with selected cardiovascular disease outcomes: a systematic review and meta-analysis. bmj 342:479. vally h., carr a., el-saleh j. and thompson p. 1999. wine-induced asthma: a placebo-controlled assessment of its pathogenesis. j. allergy clin. immunol. 103:41-46. vally h., de klerk n. and thompson p.j. 2000. alcoholic drinks: important triggers for asthma. j. allergy clin. immunol. 105:462-467. vally h. and thompson p.j. 2001. role of sulfite additives in wine induced asthma: single dose and cumulative dose studies. thorax. 56:763-769. wantke f., hemmer w., haglmuller t., gotz m. and jarisch r. 1996. histamine in wine: bronchoconstriction after a double blind placebo controlled red wine provocation test. j. allergy clin. immunol. 97:238-238. wantke f., hemmer w., haglmüller t., götz, m. and jarisch, r. 1996. histamine in wine. int. arch. allergy immunol. 110:397-400. wigand p., blettner m., saloga j. and decker h. 2012. prevalence of wine intolerance. dtsch. arztebl. int. 109:437-444. wüthrich b. 2018. allergic and intolerance reactions to wine. allergol. sel. 2:1-9. paper received april 8, 2020 accepted september 18, 2020 ijfs#1756_bozza ital. j. food sci., vol. 32, 2020 631 paper krachai dam (kaempferia parviflora) drinks: physicochemical properties, consumer acceptance, purchase intent, and emotional and wellness responses p. hanmontree and a. sae-eaw* department of food technology, faculty of technology, khon kaen university, khon kaen, thailand *corresponding author: sampor@kku.ac.th abstract the rhizomes of krachai dam (kaempferia parviflora) and their main effective methoxyflavones (5,7-dimethoxyflavone and 5,7,4’-trimethoxyflavone) have been reported to have beneficial medicinal effects. additionally, emotions and wellness can play leading roles in product development in addition to liking. this study aims to formulate the krachai dam drink and to assess the effects of health benefit information on consumer acceptance, purchase intent, and emotional and wellness responses. the optimal formulation of krachai dam drink was 165 mg/100 ml dried krachai dam, 16% sugar and 0.32% citric acid, which had a high total phenolic content (tpc), total flavonoid content (tfc), and overall liking score. after consumers received the health benefit information of the product, increases were recorded in consumer acceptance, purchase intent, the highest price that consumers were willing to pay, and positive emotional and wellness responses. keywords: krachai dam drinks, kaempferia parviflora, emotion, wellness, purchase intent ital. j. food sci., vol. 32, 2020 632 1. introduction herbs have been recognized as having health benefits (farzaneh and carvalho, 2015). herbs and their extracts contain different bioactive compounds that can provide therapeutic effects. kaempferia parviflora or krachai dam (kd) is a thai herb that belongs to the zingiberaceae family. its rhizomes have been reputed for their beneficial medicinal effects. pharmacological studies have claimed various benefits of kd and its main effective methoxyflavones, including cellular metabolism regulating activity, anticancer activity, vascular relaxation and cardioprotective activity, sexual enhancing activity, neuroprotective activity, antiallergic, anti-inflammatory, and antioxidative activity, antiosteoarthritis activity, antimicrobial activity, and transdermal permeation activity (chen et al., 2018). normally, kd is used as a food supplement or traditional medicine in the form of capsules and tablets (chen et al., 2018; kang et al., 2016; mekjaruskul and sripanidkulchai, 2019). however, using kd in food products is limited, such as wine (vichitphan et al., 2007), yogurt (kang et al., 2016), and fermented juice (chaiyasut et al., 2018). in thailand, the use of kd in food supplements is approved by the thai fda (thai food and drug administration), which recommends using kd water extracts of no more than 200 mg of kd per day (thai fda, 2017). at higher doses, food products consisting of kd need more research and development to support preventive health care, improve new product options for consumers, and add value to agricultural products. classical sensory evaluations, such as consumer preference tests, seem to be insufficiently accurate at predicting whether a newly developed product will be successful in the market. food-elicited emotions are introduced as a useful tool for new product development. because emotions influence the eating behavior of people, including food choice, motivation to eat, and amount of food intake (jiang et al., 2014), jiang and others (2014) proposed that it is necessary for food industries to understand the emotional denotation of their products and use this connotation in the packaging, branding, and commercials for the marketing of their products. in recent years, several methods for measuring emotions associated with foods have been developed and reported, such as the essense profile® (king et al., 2010), consumer defined check-all-that-apply (cd-cata) (ng et al., 2013), emosemio (spinelli et al., 2014) and the emosensory® wheel (schouteten et al., 2015). in addition, the health and wellness of food products is an interesting issue for consumers. thus, king and others (2015) developed the wellsense profiletm, which is a questionnaire for measuring wellness associated with foods from a consumer perspective (king et al., 2015). accordingly, the emotional and wellness responses of consumers should be considered in food product development. there are many kinds of herbal drinks in thailand. some herbal drinks are very popular, but some are not, even though they have health-promoting abilities. one reason is that there are many factors that influence buying decisions. an important factor is the emotional and wellness responses of consumers. this research aimed to formulate the krachai dam drink and to assess the effects of health benefit information on consumer acceptance, purchase intent, and emotional and wellness responses. ital. j. food sci., vol. 32, 2020 633 2. materials and methods 2.1. materials dried krachai dam rhizomes were purchased from naikrajok farm, khao kho, phetchabun province, thailand. the krachai dam used in this research was cultivated in march 2018 and harvested in january 2019. sugar was mitr phol brand from united farmers & industry co., ltd., thailand. citric acid (food grade) was purchased from union science co., ltd., thailand. 2.2. formulation and optimization of krachai dam drinks optimization of the herbal drink formulation was conducted using a central composite design (ccd), and the data were analyzed by using response surface methodologies (rsms). the three factors, including dried krachai dam (100-200 mg/100 ml), sugar (10-16%), and citric acid (0.2-0.4%), varied in 3 levels as low, middle, and high, which were coded as -1, 0, and 1, respectively (table 1). a central composite design (face-centered design; alpha=1) consisted of a total of 18 runs (table 2). four replicates (runs 15, 16, 17, 18) at the center of the design were used to allow for estimation of the pure error as the sum of the squares. the samples of 18 runs were prepared by using the process as follows. dried krachai dam (kd) was extracted in water at 90°c for 10 min and filtered through two layers of white cloth. sugar and citric acid were added to the kd extract, and the solution was adjusted to a specified volume by adding water and then pasteurized at 85°c for 10 min. a sample of the boiled kd drink was poured into a glass bottle while hot, closed with a bottle cap, and immediately cooled in cold water. the samples were stored at 4°c and subjected to analysis within 48 hours. 2.3. determination of the physicochemical qualities of krachai dam drinks to obtain the physicochemical properties, three replicate analyses were carried out for each sample. the color of the krachai dam drink was measured at room temperature using a colorimeter (miniscan ez, hunter association laboratory inc., usa). cie l*, a* and b* were determined and reported (la peña et al., 2010). the ph of the krachai dam drink was determined using a ph meter (fiveeasy plus, mettler toledo, switzerland) (ayala-zavala et al., 2004). the total acidity of krachai dam drinks was determined as the titratable acidity (ayalazavala et al., 2004). total acidity was determined by diluting each 5 ml aliquot of krachai dam drink with 95 ml of distilled water and then titrating to ph 8.2 using 0.1 mol/l naoh. the total soluble solids content of krachai dam drinks was determined at 20°c using a digital refractometer (0-85°brix, hi96801, hanna instrumants inc., usa) (ayalazavala et al., 2004). the viscosity of approximately 500 ml of each herbal drink was measured using a rotary viscometer (brookfield ametek dv1, usa) at 60 rpm and 5°c (adapted from la peña et al., 2010). ital. j. food sci., vol. 32, 2020 634 table 1. factors and their levels for central composite design (face-centered design; alpha=1). level code level true level krachai dam sugar citric acid krachai dam (mg/100 ml) sugar (%) citric acid (%) low -1 -1 -1 100 10 0.2 middle 0 0 0 150 13 0.3 high 1 1 1 200 16 0.4 table 2. central composite design arrangement (face-centered design; alpha=1). run code level true level krachai dam sugar citric acid krachai dam (mg/100 ml) sugar (%) citric acid (%) 1 -1 -1 -1 100.00 10.00 0.20 2 -1 -1 1 100.00 10.00 0.40 3 -1 1 -1 100.00 16.00 0.20 4 -1 1 1 100.00 16.00 0.40 5 1 -1 -1 200.00 10.00 0.20 6 1 -1 1 200.00 10.00 0.40 7 1 1 -1 200.00 16.00 0.20 8 1 1 1 200.00 16.00 0.40 9 -1 0 0 100.00 13.00 0.30 10 1 0 0 200.00 13.00 0.30 11 0 -1 0 150.00 10.00 0.30 12 0 1 0 150.00 16.00 0.30 13 0 0 -1 150.00 13.00 0.20 14 0 0 1 150.00 13.00 0.40 15 0 0 0 150.00 13.00 0.30 16 0 0 0 150.00 13.00 0.30 17 0 0 0 150.00 13.00 0.30 18 0 0 0 150.00 10.00 0.30 the total flavonoid content of the krachai dam drinks was determined by using a modified colorimetric method, according to kamtekar et al. (2014) with some modifications. quercetin was used as a standard, and a series of standard solutions were prepared in the range of 100-1000 µg/ml. briefly, 1 ml of sample or quercetin standard solution was mixed with 4 ml of distilled water, subsequently mixed with 0.3 ml of 5% sodium nitrite solution, and was allowed to react for 5 min. then, 0.3 ml of 10% aluminum chloride was added and further reacted for 6 min before the addition of 2 ml of 1 m sodium hydroxide. distilled water was added to bring the final volume of the mixture to 10 ml, and the solution was mixed well. the absorbance of the mixture was immediately measured at a wavelength of 415 nm against a prepared blank using a uvvis spectrophotometer (libra s70, biochrom libra, uk). the flavonoid content was determined by a quercetin standard curve and expressed as the mean milligrams of quercetin equivalents per ml of sample±sd in triplicate. ital. j. food sci., vol. 32, 2020 635 the total phenolic content of krachai dam drinks was determined using the folinciocalteu method, according to rahman et al. (2018) with some modifications. gallic acid was used as a standard, and a series of standard solutions were prepared in the range of 0-125 µg/ml. a 1 ml sample of each krachai dam drink was transferred to a test tube and then mixed thoroughly with 2 ml of folin-ciocalteu reagent (10-fold diluted with distilled water). after mixing for 3 min, 10% (w/v) sodium carbonate (8 ml) was added. the mixture was mixed by using a vortex mixer and then allowed to stand for 30 min in the dark at room temperature. the absorbance of the mixture was measured at 760 nm using a uv-vis spectrophotometer. the blank consisted of a solution with only folinciocalteu reagent (without sample). determinations were carried out in triplicate and calculated from a calibration curve obtained from gallic acid. the total phenolic content was expressed as milligrams of gallic acid equivalents (mg gae/ml sample). data are expressed as the mean values±standard deviation (sd). the antioxidant activity of krachai dam drinks was determined using the scavenging activity of the stable radical dpph (1,1-diphenyl-2-picrylhydrazyl) according to the method of iqbal et al. (2015) with some modifications. ascorbic acid was used as a standard, and a series of standard solutions were prepared in the range of 1-8 µg/ml. a series of sample concentrations with different ratios of samples to ethanol, i.e., 10:10, 8:10, 6:10, 4:10, 2:10, and 1:10 were prepared. then, a solution of standard or sample (2 ml) was mixed with 2 ml of 0.1 mm dpph-ethanol solution at room temperature. after incubation for 30 min in the dark, the absorbance of the mixture at 517 nm was determined using a uv-vis spectrophotometer. the control was prepared without sample, while the blank contained dpph solution. the percent dpph radical scavenging activity of the standard solution and each sample were calculated as: dpph radical scavenging activity (%)=[(acontrol asample)/acontrol] × 100 the percent dpph radical scavenging activity was plotted against the sample concentration to determine the amount of sample necessary to inhibit the dpph radical concentration by 50% (ic50). the assay was carried out in triplicate, and the data are expressed as the mean values±standard deviation (sd). 2.4. assessment of the overall liking of krachai dam drinks sensory evaluation for the overall liking of 18 samples of krachai dam drinks was performed by 128 consumer panelists (51.6% male, 48.4% female, mean age=40.3). panelists between the ages of 18 and 65 years were screened for potential food allergies, and they consumed herbal drinks at least once a week. each sample of krachai dam drink (30 ml; table 2) was put in a clear plastic cup, labeled with a three-digit code, and presented to all panelists in a random order. the samples were divided into three sets (6 samples per set) with a 10 min break between each set to prevent sensory fatigue. panelists were asked to rate their overall liking of each sample using a 9-point hedonic scale (9=like extremely, 5=neither like nor dislike, 1=dislike extremely), and they were given drinking water to wash their palates before testing the next sample. 2.5. consumer acceptance testing of the formulated krachai dam drink the optimal formulation of krachai dam drink from the ccd was assessed for consumer acceptance by using a questionnaire that contained 3 sections, as shown in table 3. a 30 ital. j. food sci., vol. 32, 2020 636 ml sample of the optimally formulated krachai dam drink was put in a clear plastic cup and labeled with a three-digit code before being presented to all consumers (n=406; table 8). table 3. details of the questionnaire for assessing consumer acceptance of the optimally formulated krachai dam drink. section topic assessment 1 before consumers received the details of the product and the benefits of krachai dam drinks. emotional and wellness responses consumer acceptance purchase intention 2 after consumers received the product details and health benefit information of krachai dam drinks as following: "the product that you are testing is krachai dam drink, which was produced from natural raw materials, no preservatives, no coloring agents, and no flavoring agents." krachai dam (kaempferia parviflora) or black ginger is a thai herb which belongs to the zingiberaceae family. its rhizomes have been researched for beneficial health effects, such as cellular metabolism regulating activity, anticancer activity, vascular relaxation and cardioprotective activity, sexual enhancing activity, neuroprotective activity, antiallergic, antiinflammatory, and antioxidative activity, antiosteoarthritis activity, antimicrobial activity, and transdermal permeation activity (chen et al., 2018). emotional and wellness responses consumer acceptance purchase intention 3 demographic information of the participants. gender age educational level occupation monthly income emotional and wellness responses were assessed by using the rate-all-that-apply (rata) approach (adapted from jaeger et al., 2018). emotional terms from the essense profile® (king et al., 2010) and wellness terms from the wellsense profiletm (king et al., 2015) were screened for relevance to the krachai dam drink using check-all-that-apply (cata) (n=239; 46% male and 54% female). emotional and wellness terms that were selected for at least 18% of participants were considered to have relevance to krachai dam drinks. the results of the term screening showed that 12 terms were selected, including seven positive emotion terms (active, energetic, good, happy, polite, satisfied, and warm) and five wellness terms (comforted, healthy, invigorated, relaxed, and refreshed). additionally, three negative emotion terms (bored, disgusted, and worried) were added to the questionnaire to include both positive and negative emotions. traditionally, at least 20% frequency of use was recommended for term screening by using the cata questionnaire (king et al., 2010). however, some research has used lower than 20% frequency for screening relevant terms to products (schouteten et al., 2015). in addition, if the study objective was to assess the negative side of food experience, screening criteria in terms of usage frequency of negative emotions may be lower than that of positive emotions (jiang et al., 2014). thus, a total of 15 emotional and wellness terms were listed randomly in the questionnaire. consumers were asked to select the emotional and wellness terms that described how they felt after consuming the product and then rate the intensity of the ital. j. food sci., vol. 32, 2020 637 selected terms on a 5-point intensity scale from 1=slightly to 5=extremely (rata approach). consumer acceptance was assessed by using a yes/no scale. purchase intention was evaluated using a 3-point scale (1=purchase, 2=not sure, and 3=no purchase). 2.6. statistical analysis the physicochemical properties and overall liking data were analyzed by using anova followed by dmrt to determine significant differences between treatments. statistical analyses were performed by using ibm spss statistics 19. the results were considered significant at a level of p<0.05 (95% confidence interval). response surface methodologies were conducted to determine the regression coefficients and statistical models for the experimental data, aiming at an overall optimal region for the response variables. after removing the nonsignificant coefficients from the initial model, three-dimensional surface plots were used to explain the effects of the independent variables (ingredients) on the response variables (product qualities). graphical and numerical optimum formulations and predicted values for the response variables were based on the response optimizer. for the consumer acceptance test, differences in the emotional and wellness responses before and after consumers had received the details and benefits of the krachai dam products were analyzed using a t-test. consumer acceptance and purchase intent were analyzed using a frequency count, and the differences before and after the consumers had received the details and benefits regarding the krachai dam products were analyzed using the mcnemar and mcnemar-bowker tests, respectively. statistical analyses were performed using ibm spss statistics 19. the results were considered significant at a level of p<0.05 (95% confidence interval). 3. results and discussion 3.1. formulation and optimization of the krachai dam drink the physicochemical properties and overall liking of the kd drink products were evaluated. table 4 shows that all physicochemical properties were significantly different between the 18 treatments, including l*, a*, b*, ph, % acidity, total soluble solids, viscosity, total phenolic content (tpc, µg gae/ml), total flavonoid content (tfc, µg qu/ml) and antioxidant activity as dpph scavenging activity (ic50, ml/ml). additionally, the overall liking showed significant differences between the 18 treatments. the relationship between the responses and variables of ccd from response surface methodology are shown in table 5. it should be noted that the adjusted r-square value of the b* value is very low, so this predicted equation is not appropriate to predict the b* value. the adjusted r-square values of % acidity, tss, viscosity, tpc, and tfc are higher than 0.9, which means that their predicted equations provide confidence in the prediction of the values of the responses. then, the optimized formulation was predicted. the results show that the optimized formulation consisted of 165 mg/100 ml dried kd, 16% sugar and 0.32% citric acid (table 6), which had a high predicted value of total phenolic content (tpc=40.10 µg/ml), total flavonoid content (tfc= 77.85 µg/ml), and overall liking (oa=7.1). ital. j. food sci., vol. 32, 2020 638 table 4. physicochemical properties and overall liking of krachai dam drinks from the central composite design treatments. run* l* a* b* tss (°brix) viscosity (cp) ph % acidity tpc (µg gae/ml) tfc (µg qu/ml) ic50 (ml/ml) oa 1 70.69a±0.81 5.63e±0.34 -4.13cde±0.11 10.23gh±0.38 3.10fgh±0.10 2.64a±0.05 0.19l±0.00 24.61i±0.54 34.32m±1.35 1.48bc±0.01 5.42fg±1.46 2 70.39a±1.19 6.25de±0.22 -4.09bcde±0.13 10.73g±0.47 3.13efgh±0.15 2.43fg±0.04 0.43b±0.01 24.06i±0.61 36.63l±1.97 1.43d±0.01 5.23g±1.41 3 69.94ab±0.97 6.17de±0.22 -4.07bcde±0.06 15.30bc±0.95 3.43ab±0.12 2.61ab±0.04 0.20k±0.00 26.90h±0.07 40.60k±1.11 1.56a±0.01 5.90e±1.11 4 68.31cd±0.11 5.75e±0.23 -3.86abc±0.13 14.83c±0.06 3.40abc±0.17 2.39h±0.01 0.44a±0.00 26.15h±1.52 43.81j±2.14 1.52ab±0.05 5.37fg±1.16 5 65.21f±0.44 10.28a±0.77 -4.50f±0.16 9.93hi±0.06 3.07gh±0.15 2.61ab±0.03 0.21j±0.01 37.72d±0.42 76.24e±1.18 1.22i±0.05 5.27fg±1.23 6 64.62fg±0.57 9.70ab±0.69 -4.00abcd±0.29 9.30i±0.70 2.97h±0.21 2.41gh±0.01 0.44a±0.00 43.30c±0.24 99.45a±2.94 1.36ef±0.02 5.13g±1.55 7 66.07ef±0.64 10.49a±0.87 -4.02a-e±0.37 15.97ab±0.06 3.47a±0.12 2.60b±0.02 0.23i±0.00 44.14bc±0.57 82.14d±1.55 1.37e±0.02 6.37d±1.28 8 65.88ef±0.35 10.47a±1.06 -4.03a-e±0.19 15.43bc±0.06 3.33a-e±0.06 2.47def±0.02 0.42c±0.00 47.67a±0.53 91.12fb± 1.02 1.34efg±0.04 6.61bcd±1.38 9 69.54abc±0.65 5.63e±0.28 -3.75ab±0.14 13.93d±0.40 3.30a-f±0.10 2.50cd±0.01 0.31g±0.00 26.86h±0.26 45.09j±0.59 1.50b±0.04 5.64ef±1.60 10 63.49g±0.38 9.15bc±0.67 -3.98abcd±0.17 13.90d±0.10 3.33a-e±0.06 2.48de±0.04 0.36d±0.01 44.91b± 0.53 87.40c±1.94 1.22i±0.01 6.43d±1.71 11 69.48abc±0.30 8.25c±0.74 -4.08bcde±0.17 10.40gh±0.44 3.07gh±0.15 2.54cd±0.01 0.32f±0.00 33.74g±0.10 68.04f±1.02 1.44cd±0.01 6.57cd±1.51 12 68.69bc±0.59 8.82bc±0.38 -4.32def±0.06 16.20a±0.10 3.37abcd±0.06 2.43efg±0.01 0.35e±0.01 34.20fg±0.19 66.24fg±0.44 1.30gh±0.01 7.03a±1.39 13 63.60g±0.66 7.02d±0.34 -3.69a±0.07 13.87d±0.12 3.20c-g±0.10 2.54c±0.02 0.24h±0.00 34.36fg±0.45 54.71i±0.22 1.31fg±0.02 6.94abc±1.46 14 67.25de±0.42 9.65ab±0.69 -4.37±0.07 13.03e±0.31 3.17d-h±0.06 2.43fgh±0.01 0.42c±0.00 37.64d±0.37 56.12i±0.38 1.22i±0.02 6.91abc±1.33 15 66.09ef±2.42 8.64bc±0.48 -4.50±0.45 12.30f±0.20 3.20c-g±0.10 2.45efg±0.02 0.32f±0.00 35.14f±0.14 64.71g±0.22 1.31fg±0.02 7.25a±1.53 16 64.70fg±0.39 8.35c±0.54 -4.13cde±0.06 13.87d±0.64 3.27a-g±0.06 2.48de±0.01 0.32f±0.00 37.55d±0.71 59.83h±0.22 1.26hi±0.04 7.10a±1.73 17 64.82fg±0.44 8.36c±0.56 -4.29def±0.03 13.07e±0.15 3.23b-g±0.15 2.45efg±0.01 0.32f±0.00 36.24e±0.40 63.94g±0.22 1.24i±0.01 6.97ab±1.97 18 64.73fg±0.37 8.35c±0.59 -4.25def±0.17 13.00e±0.26 3.23b-g±0.06 2.46def±0.01 0.32f±0.00 37.01de±0.43 65.60g±0.80 1.25hi±0.01 6.86abc±1.93 *corresponding to formulation runs listed in table 2. values are means±standard deviations (sd). values in the same column with different letters are significantly different (p<0.05). abbreviations: tss, total soluble solid; tpc, total phenolic content; tfc, total flavonoid content; ic50, antioxidant activity as dpph scavenging activity; oa, overall acceptability. ital. j. food sci., vol. 32, 2020 639 table 5. the relationship between the responses and variables of central composite design. response predicted equation* adjusted r2 l* y=65.698-2.360k+0.205k2-0.150s+2.775s2+0.094c-0.885c2 0.70 a* y=8.295+2.066k-0.775k2+1.159s-0.370s2+0.223c+0.170c2 0.84 ph y=2.46+0.03k2-0.009s+0.005s2-0.087c+0.025c2 0.87 %acidity y=0.328-0.009k-0.0002k2+0.005s-0.0002s2+0.108c-0.054c2 0.96 tss (°brix) y=13.409-0.049k+0.158k2+2.714s-0.457s2-0.198c-0.307c2 0.92 viscosity (cp) y=3.236-0.019k+0.075k2+0.166s-0.190s2-0.027c-0.054c2-0.030kc 0.96 tpc (µg/ml) y=36.213-8.917k-0.057k2+1.563s-1.967s2+1.110c+0.061c2+1.303kc 0.97 tfc (µg/ml) y=63.275-23.590k+3.213k2+0.923s+4.110s2 + 3.910c-7.621c2+3.333kc 0.97 ic50(ml/ml) y=1.278-0.099k+0.075k 2+0.015s-0.081s2-0.007c-0.020c2 0.65 overall liking y=7.046+0.224k-1.012k2+0.367s-0.247s2-0.064c-0.122c2+0.244ks 0.94 *y=response, k=dried kd, s=sugar, c=citric acid. abbreviations: tss, total soluble solid; tpc, total phenolic content; tfc, total flavonoid content; ic50, antioxidant activity as dpph scavenging activity. in both adults and children, the who recommends reducing the intake of free sugars to less than 10% of total energy intake (who, 2015), which is equivalent to 50 g for a person of healthy body weight consuming approximately 2000 calories per day. in this research, the sugar level of the optimal formulation that consists of 16 g of sugar per serving (100 ml) is quite high but less than the sugar intake recommendation of the who. moreover, 12 brands of krachai dam drinks from a market survey in thailand consist of 8-20% sugar (data not shown). additionally, the percent sugar in the optimal formulation is in the range of traditional krachai dam drinks. however, we recommend that future studies reduce the sugar or substitute the sugar using no calorie materials such as stevia. table 6. the optimal formulation of the krachai dam drink. ingredient code level true level dried krachai dam 0.4 165 mg/100 ml sugar 1 16% citric acid 0.3 0.32% next, the formulated kd drink products were produced, and the physicochemical properties and overall liking were evaluated. the data were analyzed by using a t-test to compare the differences between the predicted values from the rsm and the actual value of the formulated product. the results (table 7) show that none of the physicochemical properties, except % acidity, were significantly different between the formulated kd drinks and their predicted values. 3.2. consumer acceptance of the formulated krachai dam drink a consumer acceptance test of kd drinks was administered to 406 consumers whose demographic information is shown in table 8 using a central location test in khon kaen ital. j. food sci., vol. 32, 2020 640 province. fifty-four percent of the participants were female and 45% were male, and the participants were mostly over the age of 22 years. their education was mostly at the bachelor’s degree level. most were self-employed. their monthly income was generally in the range of 10,000-20,000 baht. differences in emotional and wellness responses before and after the consumers received the details of the krachai dam product and the health benefit information were analyzed using a t-test, and the results are shown in figure 1. after receiving the product details, all positive emotional and wellness responses were clearly higher than before the consumers had received product details (p<0.05). the participants did not have the “bored” emotion, while “disgusted” and “worried” responses had mean scores of nearly zero and no differences before and after receiving the product details (data not shown). generally, most emotions elicited from foods are positive emotions, even when evaluating not wellliked products, and the intensity of negative emotions is relatively low (spinelli and jaeger, 2019). consumer acceptance and purchase intent were analyzed using % frequency, and the differences before and after the consumers received the details of the product and its benefits were analyzed using the mcnemar test and the mcnemar-bowker test, respectively. the highest price that consumers were willing to pay was reported as the mean value±standard deviation (sd). the differences between the highest price before and after consumers received the product details and health benefit information were compared using a t-test. the results (table 9) show that most participants accepted the product (97.77% before and 100.00% after), and consumer acceptance before and after receiving the product details was not significantly different (p>0.05). after consumers received the product details and health benefit information, the purchase intention (88.92% before and 96.31% after) and the highest price that consumers were willing to pay (17.58 baht/100 ml before and 29.02 baht/100 ml after) were significantly higher (p<0.05). table 7. physicochemical properties and overall liking of formulated kd drinks. physicochemical properties predicted value actual value (mean±sd) t-test (p-value) l* 67.59 66.59±0.67 0.122 a* 9.63 9.18±0.67 0.364 b* -4.25 -4.35±0.10 0.216 ph 2.45 2.43±0.01 0.130 % acidity 0.36 0.35±0.0015 0.006 tss (°brix) 15.68 15.53±0.06 0.251 viscosity (cp) 3.37 3.37±0.15 0.973 tpc (µg gae/ml) 40.10 38.13±0.85 0.057 tfc (µg qu/ml) 77.85 76.12±1.02 0.098 ic50(ml/ml) 1.34 1.32±0.04 0.516 overall liking 7.17 7.07±1.19 0.499 abbreviations: tss, total soluble solid; tpc, total phenolic content; tfc, total flavonoid content; ic50, antioxidant activity as dpph scavenging activity. ital. j. food sci., vol. 32, 2020 641 table 8. demographic information of participants (n=406) in consumer tests of formulated kd drinks. demographic information frequency count percent 1. gender female 220 54.2 male 186 45.8 2. age 18-21 years 23 5.7 22-40 years 134 33.0 41-59 years 128 31.5 ≥60 years 121 29.8 3. education level secondary school 129 31.8 bachelor’s degree 227 55.9 postgraduate 50 12.3 4. occupation student 30 7.4 government employee 71 17.5 self-employed/merchant 118 29.0 employee 60 14.8 private company employee 33 8.1 retiree 65 16.0 unemployed 21 5.2 other 8 2.0 5. monthly income <10,000 baht 109 26.9 10,001-20,000 baht 137 33.7 20,001-30,000 baht 83 20.4 30,001-40,000 baht 50 12.3 40,001-50,000 baht 15 3.7 >50,000 baht 12 3.0 in summary, the product details and health benefit information of the kd drink increased the emotion and wellness responses, consumer acceptance, purchase intention, and the highest price that consumers were willing to pay. this result may be influenced by the relationship between emotions and food choice. emotions can provide an internal stimulus or state that elicits a useful, corrective food choice. on the other hand, food may influence emotions through various factors, such as sensory and hedonic effects, social context, cognitive expectations, psychological distraction, appetite alteration, and brain function (gibson, 2006). food companies must understand the emotional meaning of their food products and use these messages in the forms of packaging, branding, and advertisements to market their products. this also helps with finding target consumers, modifying emotional needs towards different consumers and discovering new markets (jiang et al., 2014). therefore, the sensory-emotional optimization of products has been suggested as an alternative to sensory-hedonic product optimization based on the relation of a sensory property and an emotion (spinelli and jaeger, 2019). in addition, a ital. j. food sci., vol. 32, 2020 642 combination of sensory intensities and emotional responses have been used to predict the consumer acceptance of commercial products such as vegetable juice (samant and seo, 2019). figure 1. positive emotional and wellness responses of the optimal formulated krachai dam drink before and after the consumers received the product details and benefits of kd (t-test, p<0.05). table 9. consumer acceptance, purchase intention, and the highest price that consumers were willing to pay for the krachai dam drink before and after they received the product details and health benefit information (n=406). consumer test before after statistical analysis consumer acceptance accept 97.77% 100.00% mcnemar test, not accept 1.23% 0.00% p-value=0.063 purchase intention purchase 88.92% 96.31% mcnemar not sure 10.34% 3.45% bowker test, no purchase 0.74% 0.24% p-value=0.000 highest price for 100 ml/bottle (baht)* 17.58±11.12 29.02±15.17 t-test, p-value=0.000 values are means±standard deviations (sd). 4. conclusions the optimal formulation of the kd drink was 165 mg/100 ml dried kd, 16% sugar and 0.32% citric acid, which had high total phenolic content (tpc=38.13 µg/ml), total ital. j. food sci., vol. 32, 2020 643 flavonoid content (tfc= 76.12 µg/ml), and overall liking (oa=7.07). most of the consumer panels (97.77%) were satisfied with the formulated product. after the consumers received the product benefit information, consumer acceptance, purchase intent, the highest price that consumers were willing to pay, and their positive emotional and wellness responses increased. acknowledgments the authors gratefully appreciate sakon nakhon rajabhat university for financial 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(kaempferia parviflora) wine. kmitl science and technology journal 7(s2):97-105. welbat j.u., chaisawang p., chaijaroonkhanarak w., prachaney p., pannangrong w., sripanidkulchai b. and wigmore p. 2016. kaempferia parviflora extract ameliorates the cognitive impairments and the reduction of cell proliferation induced by valproic acid treatment in rats. annals of anatomy 206:7-13. who. 2015. guideline: sugars intake for adults and children. geneva: world health organization. yenjai c., prasanphen k., daodee s., wongpanich v. and kittakoop p.p. 2004. bioactive flavonoids from kaempferia parviflora. fitoterapia 75:89-92. youn k., lee j., ho c.-t. and jun m. 2016. discovery of polymethoxyflavones from black ginger (kaempferia parviflora) as potential β-secretase (bace1) inhibitors. journal of functional foods 20:567-574. paper received december 12, 2019 accepted april 21, 2020 ijfs#1826_bozza ital. j. food sci., vol. 32, 2020 712 short communication occurrence of deoxynivalenol in beers commercialised in italy m. grumi, a. kunova, m. isotti, a. barbiroli and m. pasquali* defens, dipartimento di scienze per gli alimenti, la nutrizione e l’ambiente, università degli studi di milano, via celoria 2, 20133 milano, italy *corresponding author: matias.pasquali@unimi.it abstract deoxynivalenol (don) is the most frequently detected mycotoxin in beer. this study represents a comprehensive assessment of don occurrence in beers from the italian market. seventy-two craft and industrial beer samples were tested using the ridascreen don® elisa method. don was found in all samples. the average don contamination was 34.3 μg/l (range: 6.1 111.2 μg/l). the highest contamination level was found in a wheat-based sample. our study determined that wheat-based beers have a higher don contamination than barley-based beers. further studies are needed to verify the role of single ingredients on the risk of don accumulation in beers. keywords: consumption level, elisa, fermented beverage, food safety, mycotoxins, wheat ital. j. food sci., vol. 32, 2020 713 1. introduction deoxynivalenol (don) is one of the most frequently detected mycotoxins in cereals and cereal-based products (pedroso pereira et al., 2019). don belongs to type-b trichothecenes mycotoxins and it is mainly produced by fusarium graminearum and fusarium culmorum (pasquali et al., 2016). type-b trichothecenes involve don, its acetylated derivatives (3-acetyl-don and 15-acetyl-don) nivalenol, and deoxynivalenol-3-glucoside (d3g). d3g might be the result of plant metabolism of don or of some food processing operations (berthiller et al., 2013). although don and its derivatives are not classified as being carcinogenic to humans, an exposure to this type of mycotoxins may be associated with a wide range of adverse health complications (efsa contam panel, 2017). the main effect of deoxynivalenol is the inhibition of protein synthesis. this leads to acute gastro-intestinal symptoms (e.g. emesis and diarrhoea) as well as, in case of long-term exposure, to immune system diseases or disorders (pedroso pereira et al., 2019). considering its potential to cause serious health issues, the european commission established a range of maximum limits for don in cereals and cereal-based foods (ferrigo et al., 2016). in 2006, the european commission proposed the maximum level for don in cereals intended for direct human consumption of 750 μg/kg (efsa contam panel, 2017). the joint fao/who expert committee on food additives (jecfa) has also established a provisional maximum tolerable daily intake (pmtdi) of 1 μg/kg body weight for the sum of don and its derivatives (pedroso pereira et al., 2019). since don is largely detected in cereals and malt, which represent the key ingredients of beer, a don contamination of this type of alcoholic beverages seems unavoidable (rodríguez-carrasco et al., 2015). it is worth specifying that maximum levels for don in beer have not been set so far. monitoring don in beer is important considering that this widely popular fermented beverage may significantly contribute to the intake of mycotoxins and even exceed the safety levels when following a regular diet (papadopoulou-bouraoui et al., 2004; rodríguez-carrasco et al., 2015). therefore, exposure of consumers to don and its derivatives through beer consumption should not be underestimated, especially in case of “heavy drinkers” (pascari et al., 2018). the occurrence of deoxynivalenol in beer has been studied in various surveys all around the world: in belgium (papadopoulou-bouraoui et al., 2004), in poland (kuzdraliński et al., 2013), in austria, hungary, croatia and serbia (varga et al., 2013), in spain (rodríguez-carrasco et al., 2015), in brazil (piacentini et al., 2015), in germany (bauer et al., 2016), in paraguay (arrúa et al., 2019) and in mexico (wallmartínez et al., 2019 b). occurrence of don in beers from the italian market was assessed in two surveys with limited number of samples: by pietri et al. (2003) and by peters et al., (2017). the surveys showed that the small number of italian samples analysed, compared with samples produced in other countries, had the lowest don contamination levels (below than 10 μg/l). in 2018 the italian beer consumption increased by 3.2%. therefore, per capita consumption reached its historical peak of 33.6 l a year (assobirra, 2018). considering both the limited number of surveys focusing on the occurrence of don in beers purchased from the italian market and the significant raise of italian craft breweries the aims of this work were: 1. to assess the level of deoxynivalenol in beer samples sold on the italian market from may 2018 to december 2018 in order to update and enrich the available data; ital. j. food sci., vol. 32, 2020 714 2. to compare the contamination of industrial beers to craft beers, taking into account the findings of peters et al., 2017 who identified higher don incidence in craft beers collected all over europe; 3. to define whether wheat-based beers had higher don contamination compared to barley based beers. 2. materials and methods seventy-two beer samples were purchased from may to december 2018 in pubs, supermarkets and beer shops located in the north of italy. some of them were home brewed from semi-processed products. most samples (53) were produced by italian companies whereas nineteen samples came from different european countries. the foreign beers were produced in germany (8), belgium (5), austria (1), czech republic (1), france (1), netherlands (1), sweden (1) and united kingdom (1). none of the beers exceeded their expiration date. 1.5 ml of each sample were placed in separate test tubes and stored at -80°c for at least 24 hours, in order to reach a complete degassed condition. 50 μl of co2-free samples were subjected to the analysis. the commercial competitive elisa ridascreen don® (r-biopharm ag, germany) was used. the declared detection limit of ridascreen don® is 3.7 ppb for beer samples, with a cross-reactivity to don (100%), 3-acetyldeoxynivalenol (>100%), 15-acetyldeoxynivalenol (approximatively 19%), nivalenol (approximatively 4%), fusarenon-x (<1%) and t-2 toxin (<1%). all reagents required for the analysis – including standards – were contained in the kit. the pbs-tween washing buffer was prepared by dissolving the provided salt in milli-q® ultra-pure water. the test procedure issued by the producer was strictly followed. results were obtained by reading sample or standard absorbances at 450 nm using a synergy (h1) microplate reader (biotek®, us) spectrophotometer. the absorbance was inversely proportional to the don concentration in the samples. absorbance was expressed as a percentage value with respect to the zero standard (100 × (absorbance sample or standard)/(absorbance zero standard)). the values calculated for the standards were entered in a system of coordinates on a semilogarithmic graph against the don concentrations (expressed in μg/l) using the online editor line of best fit generator “plot.ly” (plotly technologies, 2015). this allowed obtaining a calibration curve from which the don concentration, actually contained in all the samples, was defined. all samples were measured at least twice in each analysis. samples resulted off the chart were diluted using ultra-pure water, in ratio 1:2 and 1:5 and then reanalysed. the ph of each sample was also measured using an xs instruments® benchtop phmeter supplied with an automatic temperature compensation and a microelectrode that was fit for the low-volume samples. the statistical analysis was carried out using the statistical software jasp version 0.11.1 (jasp team, 2019). a linear regression was performed to assess relationship among don, ph and alcohol content values. furthermore, analysis of variance (anova) followed by bonferroni post hoc test was performed in order to determine the significance of fixed factors “wheat”, “type of brewing process” and “type of fermentation” on the don concentration. all tests were executed at a significance level of p<0.05. ital. j. food sci., vol. 32, 2020 715 3. results with reference to the whole collection of beer samples, 46 samples were classified as “craft beer” whereas 26 samples were classified as “industrial produced beer”. wheat was one of the ingredients in 21 samples. three of the selected samples were “gluten free” beers. the results of the analysis are summarised in table 1. table 1. samples description and results of the analysis. sample country of origin 1%abv type of fermentation type of brewing process wheat special features ph 2don (𝝁𝒈/𝑳) b1 germany 7.5 bottom industrial 4.66 46.9±7.2 b2 germany 5.3 top industrial 4.36 71.5±15.7 b3 germany 5.4 top industrial 4.40 35.6±3.3 b4 germany 5 bottom industrial 4.48 25.7±8.4 b5 germany 4.9 bottom industrial 4.49 43.3±9.6 b6 germany 5 top industrial yes 4.47 32.1±7.7 b7 germany 5 bottom industrial 4.50 57.2±19.5 b8 italy 4.7 top industrial 4.58 17.2±0.5 b9 italy 6.5 top craft yes 4.51 45.0±2.9 b10 italy 5.5 top craft 4.57 55.9±14.6 b11 italy 4.5 top craft yes 4.31 54.3±13.5 b12 italy 5 top craft yes 3.82 61.4±5.4 b13 italy 6.9 top craft yes 4.50 49.3±12.9 b14 italy 5.2 top craft yes 4.64 25.8±0.8 b15 italy 7.8 top craft 4.96 54.8±7.1 b16 italy 5.6 top craft 4.58 50.5±15.6 b17 italy 3.9 top craft 4.17 18.1±2.7 b18 italy 5 bottom industrial 4.93 18.6±10.2 b19 italy 4.5 bottom craft 4.43 19.9±7.3 b20 italy 5 bottom craft yes 4.64 44.5±17.8 b21 italy 6 top craft 4.64 18.4±2.1 b22 italy 4.5 top craft gluten free 4.50 17.8±8.8 b23 italy 5.6 top craft yes 4.46 20.7±0.6 b24 italy 5.6 top craft 4.36 13.1±2.1 b25 italy 4.7 bottom industrial 4.39 10.8±2.0 b26 italy 4.7 bottom industrial gluten free 4.66 6.1±0.1 b27 italy 5.5 bottom industrial 4.60 20.0±0.8 b28 italy 4.5 bottom industrial 4.59 16.1±1.2 b29 italy 4.7 bottom industrial 4.44 15.7±1.4 b30 italy 5 bottom industrial 4.72 22.7±0.7 b31 italy 5.5 top craft gluten free 4.53 13.1±1.7 b32 italy 7 top craft 4.31 95.8±5.7 b33 italy 5 top craft 4.41 31.9±1.0 b34 italy 0.49 n.d. industrial alcohol-free 4.83 9.5±0.4 ital. j. food sci., vol. 32, 2020 716 b35 italy 5 top craft yes 4.05 65.9±12.2 b36 belgium 4.9 top industrial yes 4.46 60.8±6.8 b37 italy 5 top industrial yes 4.19 76.3±9.6 b38 italy 5.1 bottom industrial 4.22 12.0±0.8 b39 belgium 8 top craft yes 4.40 27.8±4.0 b40 italy 5.2 bottom craft 4.80 10.7±0.5 b41 italy 4.6 bottom craft 4.55 22.3±3.2 b42 belgium 7.5 top industrial yes 4.77 50.7±7.1 b43 germany 12 top industrial yes 4.73 56.3±9.6 b44 italy 8 top industrial 4.62 36.4±7.9 b45 italy 8 bottom craft 4.70 80.3±2.1 b46 italy 7.5 bottom industrial 4.92 19.1±0.8 b47 nether-lands 6.5 top craft yes 4.35 37.6±15.3 b48 united kingdom 4.6 bottom craft 4.55 9.3±0.6 b49 italy 5.4 bottom craft 4.56 54.5±9.8 b50 italy 5 bottom craft 4.73 18.9±1.8 b51 belgium 9.5 top craft 4.36 24.3±8.4 b52 italy 8 top craft 4.80 24.1±14.1 b53 france 5.5 bottom industrial 4.43 12.7±3.1 b54 italy 9 top craft 4.67 10.1±0.2 b55 czech republic 4.4 bottom industrial 4.69 15.0±0.7 b56 belgium 6.5 spontaneous craft yes 3.45 111.2±14.1 b57 italy 6 top craft 4.66 10.3±1.3 b58 sweden 5 bottom craft yes 4.55 23.3±3.2 b59 italy 9 top craft 4.53 45.0±6.2 b60 italy 4.9 top craft 4.56 50.5±15.7 b61 italy 4.9 bottom craft 4.79 48.5±8.2 b62 italy 9.7 top craft 4.80 82.6±13.8 b63 italy 8.7 top craft 4.60 35.2±5.1 b64 italy 4.7 top craft 4.78 7.5±1.2 b65 italy 5.2 top craft yes 4.38 17.2±1.0 b66 italy 4.5 bottom craft 4.57 37.8±1.0 b67 italy 8.5 top craft 4.49 46.4±4.8 b68 italy 4.6 top craft yes 4.31 25.5±2.2 b69 italy 4.6 top craft 4.39 28.9±2.8 b70 italy 6.5 top craft 3.98 35.0±6.9 b71 italy 6.5 top craft 4.77 19.1±4.9 b72 italy 5.5 bottom craft 4.51 20.5±5.6 1percentage alcohol by volume; 2 mean value±standard deviation. ital. j. food sci., vol. 32, 2020 717 don contamination levels obtained by ridascreen® don elisa don was found in all samples with a contamination incidence of 100%. the contamination ranged from 6.1 μg/l to 111.2 μg/l. the average contamination level was 34.3 μg/l, with a median of 25.8 μg/l. only in 43.06% of samples (31) the don contamination was greater than the average contamination level (34.3 μg/l). the highest contamination level (111.2 μg/l) was found in a sample that included wheat among the ingredients (table 1). ph values ranged from 3.45 to 4.96 with an average value of 4.51 and a median of 4.53. the percentage alcohol by volume (%abv) ranged from a minimum value below 0.5% (non-alcoholic beer sample) to a maximum of 12%. the average alcohol content was 5.8% with a median of 5.2%. in order to assess the influence of the two variables ph and %abv on don contamination levels, a linear regression model was computed. both %abv and ph were partially correlated with samples don content (p>0.001) but r2 were negligible (r2 = 0.109, r2 = 0.123). analysis of variance (anova) was performed in order to determine the potential impact of both wheat (as ingredient) and the type of brewing process (industrial or craft beer) on don content. the anova revealed a positive effect of wheat as ingredient on don contamination of the samples (p value = 0.001), with an average don contamination of wheat-based beers of 46.6 μg/l (±23.1) and an average don contamination of beers without wheat of 29.9 μg/l (±20.8). on the contrary, there was no difference between the types of brewing process on the final don content (p value = 0.966). 4. conclusions this study represents the first comprehensive assessment of the don level in beers sold on the italian market. moreover, it identifies wheat-based beers as potentially contributing to higher level of don accumulation in consumers. the screening results showed a weak correlation between the alcohol content (%abv) and the don contamination levels. higher alcohol levels were related to significantly higher don levels in beers by other researchers such as papadopoulou-bouraoui et al., 2004; kostelanska et al., 2009; peters et al., 2017; ksieniewicz-woźniak et al., 2019; wall-martínez et al., 2019 b. the requirement of a higher input of fermentable sugars in malt wort, in order to reach higher alcohol levels, seems to be a possible explanation. indeed, the further supplement of grains may be associated with a higher risk of mycotoxins contamination (kostelanska et al., 2009; peters et al., 2017; pascari et al., 2018; wall-martínez et al., 2019 b). a previous study (wall-martínez et al., 2019 a) did not find any correlation between ph and don contamination values. however, in our study we observed a slightly negative correlation that suggests that higher beer ph is correlated to lower don content. further studies are needed to decipher this phenomenon. given the minor ph diversity it is not possible to postulate that alkaline ph are the cause of decreased don stability as it was found for baking products (young et al., 1984). as it is known that the brewing process can include a ph correction before selling, our observations may not be associated to any specific processing of the beer. according to our surveillance, wheat-based beers represent higher risk for consumers. as stated by a recent report of the u.s. department of agriculture economic research ital. j. food sci., vol. 32, 2020 718 service, wheat may represent 5-10% of the whole malts used by u.s. breweries. the increased use of wheat may be related to the significant growth of craft beer production along with the increased popularity of several wheat beers produced by leading international brewers (jin et al., 2018). the scientific report of the european food safety authority (efsa, 2013) states that wheat has an average don contamination about three times higher (434.4 μg/kg) than barley (176.1 μg/kg). that might explain the greater don contamination values of wheat-based beers when compared to other beer types. similar results were reported by ksieniewicz-woźniak et al. (2019), who found a very high percentage of wheat-based beer samples positive to don. previously published studies (peters et al., 2017; wall-martínez et al., 2019 b) found a higher risk of mycotoxin contamination in craft beers. diversely, our analysis did not reveal any significant differences between industrial and craft beers for what concerns don contamination, which is in accordance with the study conducted by arrúa et al., 2019. efsa estimates that the contribution of don deriving from beer, in adult population, is approximatively 0.5-5.3% (efsa, 2013). the average contamination value obtained in this study (34.3 μg/l) is three times higher than the average value (13.5 μg/l) taken into consideration by efsa, 2013. based on the average don value found in our study, the consumption of a heavy drinker of 70 kg of body weight, consuming 0.5 l of beer per day, will determine a don daily intake of 24.5 %. considering the 2018 beer per capita consumption of 33.6 l (0.092 l of beer per day) of the italian consumers 4.5% of the tdi will be reached. these numbers are substantially higher than those found by pietri et al. (2003). annual climate and weather variability may contribute to modify levels of mycotoxins in field crops (beyer et al., 2014), which will eventually lead to modified levels of don in beer. for this reason, annual monitoring of grains for don contamination would be essential to investigate the variability of malts contamination. for the year 2018, our data suggests that through the consumption of beer, the italian population received a higher percentage of don from beer consumption compared to the average estimation from efsa. hence, our study suggests that the intake of don through beer consumption should be updated for each nation, possibly on a yearly based manner. to verify if this can be simply attributed to a “year effect” or to the changes in the ingredients used for beer production further monitoring is needed. future studies should also focus on the impact of other grains on final don levels in beer. specifically, since maize is used in beer production and it has an average don contamination (1041.9 μg/kg) significantly higher than barley (176.1 μg/kg) (efsa, 2013), an investigation of maize impact on don contamination would be of interest. moreover experimental studies focused on the effects of different technological processes on don accumulation will contribute to have a complete assessment of the factors that determine don accumulation in beers. the article processing charge was partially covered by the university of milan. references arrúa a.a., mendes j.m., arrúa p., ferreira f.p., caballero g., cazal c., kohli m.m., peralta i., ulke g. and fernández ríos d. 2019. occurrence of deoxynivalenol and ochratoxin a in beers and wines commercialized in paraguay. toxins. 11(6):308. doi: doi.org/10.3390/toxins11060308 ital. j. food sci., vol. 32, 2020 719 assobirra. 2018. assobirra annual report. 24-27. www.assobirra.it/wp-content/uploads/2019/05/annualreport_2018_ paginesingole.pdf bauer j.i., gross m., gottschalk c. and usleber e. 2016. investigations on 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audenaert k., balmas v., basler r., boutigny a.l., chrpová j., czembor e., gagkaeva t., gonzález-jaén m.t., hofgaard i.s., köycü n.d., hoffmann l., levic j., marin p., miedaner t., migheli q., moretti a., müller m.e.h., munaut f., parikka p., pallez-barthel m., piec j., scauflaire j., scherm b., stankovic s., thrane u., uhlig s., vanheule a., yli-mattila t. and vogelgsang s. 2016. a european database of fusarium graminearum and f. culmorum trichothecene genotypes. front. microbiol. 7:406. doi: doi.org/10.3389/fmicb.2016.00406 pedroso pereira l.t., putnik p., tadashi iwase c.h. and oliveira rocha l. de. 2019. deoxynivalenol: insights on genetics, analytical methods and occurrence. curr. opin. food sci. 24:1-8. doi: doi.org/10.1016/j.cofs.2019.01.003 peters j., dam r. van, doorn r. van, katerere d., berthiller f., haasnoot w. and nielen m.w.f. 2017. mycotoxin profiling of 1000 beer samples with a special focus on craft beer. plos one. 12(10):1-27. doi: doi.org/10.1371/journal. pone.0185887 piacentini k.c., savi g.d., olivo g. and scussel v.m. 2015. quality and occurrence of deoxynivalenol and fumonisins in craft beer. food control. 50:925-929. doi: doi.org/10.1016/j.foodcont.2014.10.038 pietri a., bertuzzi t., barbieri g. and fortunati p. 2003. analisi di micotossine in birre nazionali ed estere mediante hplc e gc-ms. conference paper: v congresso di chimica degli alimenti, parma (italy) (in italian). ital. j. food sci., vol. 32, 2020 720 plotly technologies inc. 2015. collaborative data science. https://help.plot.ly/make-a-line-of-best-fit/ rodríguez-carrasco y., fattore m., albrizio s., berrada h. and mañes j. 2015. occurrence of fusarium mycotoxins and their dietary intake through beer consumption by the european population. food chem. 178:149-155. doi: doi.org/10. 1016/j.foodchem.2015.01.092 varga e., malachova a., schwartz h., krska r. and berthiller f. 2013. survey of deoxynivalenol and its conjugates deoxynivalenol-3-glucoside and 3-acetyl-deoxynivalenol in 374 beer samples. food addit. contam. part a. 30(1):137146. doi: doi.org/10.1080/19440049.2012.726745 wall-martínez h.a., pascari x., bigordà a., ramos a.j., marín s. and sanchis v. 2019 a. the fate of fusarium mycotoxins (deoxynivalenol and zearalenone) through wort fermenting by saccharomyces yeasts (s. cerevisiae and s. pastorianus). food res. int. 126:108587. doi: doi.org/10.1016/j.foodres.2019.108587 wall-martínez h.a., pascari x., ramos a.j., marín s. and sanchis v. 2019 b. frequency and levels of mycotoxins in beer from the mexican market and exposure estimate for deoxynivalenol mycotoxins. mycotoxin res. 35(2):207-216. doi: doi.org/10.1007/s12550-019-00347-x young j.c., fulcher r.g., hayhoe j.h., scott p.m. and dexter j.e. 1984. effect of milling and baking on deoxynivalenol (vomitoxin) content of eastern canadian wheats. j. agric. food chem. 32(3):659-664. doi: doi.org/10.1021/jf00123a058 paper received january 14, 2020 accepted april 28, 2020 ijfs#1733_bozza ital. j. food sci., vol. 32, 2020 321 paper effect of mixed starter cultures on biogenic amine formation during the ripening of tunisian dry fermented camel meat sausage s. el adab*1, w. ben wadda1, a. tekiki1, o. ben moussa1, m. boulares1, s. sadok2 and m. hassouna1 1research unity “bio-preservation and valorization of agricultural products–ur 13 agr02”, high food industries school of tunisia, 58 avenue alain savary, tunis el khadra 1003, university of carthage, tunisia 2laboratory of blue biotechnology and aquatic bio-products, national institute of marine sciences and technologies, la goulette center, tunis 2060, tunisia *corresponding author: eladabsabrina@gmail.com abstract the effect of mixed starter cultures on biogenic amine production was examined during the ripening process of dry camel meat sausage. changes in ph, moisture content, proteolysis, microbial counts and lipid oxidation were also studied. the combination of three amine-negative bacteria, resulted in a drastic reduction of biogenic amine production. the highest total free amino acid concentration was observed in batches manufactured with mixed starter cultures. the bactericidal properties of l. sakei improved the hygienic quality of sausages by decreasing the number of enterobacteriaceae. inoculation of sausages with a mixture of strains, significantly delayed lipid oxidation and enhanced sensory characteristics. keywords: biogenic amine, fermented sausages, starter cultures, quality, ripening ital. j. food sci., vol. 32, 2020 322 1. introduction the formation of biogenic amines (bas) in fermented sausages could affect the quality of the final products. bas are mainly formed by the decarboxylation of amino acids. biogenic amines could be harmful on health when they are consumed in large quantities; these molecules are responsible for food poisoning (latorre-moratalla et al., 2010; lorenzo et al., 2017). in fact, “tyramine and phenylethylamine have been associated with food histaminic intoxications and severe hypertensive crisis (marine-font et al., 1995)”. moreover, bover-cid et al. (1999) have reported that putrescine and cadaverine could contribute to the formation of heterocyclic carcinogenic nitrosamines. many factors contribute to the formation of bas in dry fermented sausages such as ingredients, technological ripening conditions, acidification, and proteolysis during the ripening of dry fermented sausage. many studies have reported that the production of biogenic amines in fermented meat products was related to the growth of pseudomonas, enterobacteria, enterococci, and lactobacilli (durlu-özkaya et al., 2001; suzzi et al., 2003; latorre-moratalla et al., 2012). strains of lactic acid bacteria (lab) and coagulase negative staphylococci (cns) are usually used in the manufacture of dry fermented sausages. these strains should have the most important technological properties such as the adaptation to meat environment, development of red color, texture, and flavor of fermented meats (talon et al., 2011). moreover, the use of amine-negative strains with bacteriostatic activity can stopped the formation of bas in the initial fermentation stage (bover-cid et al., 2000). in the present study, the influence of mixed starter cultures of l. sakei, s. carnosus and s. xylosus on biogenic amine formation during the ripening of dry fermented camel meat sausage was studied. besides the biogenic amine contents, evolution of microbial population, amino acids content, total lipid and total protein contents, ph, thiobarbituric acid (tba), moisture content, textural and sensory characteristics were determined during the ripening of a dry fermented camel meat sausage. 2. materials and methods 2.1. sausage preparation “the sausage formulation included 3.750 kg of camel meat (75%), 1.250 kg of hump fat (25%), 260 g of salt, 15 g of black pepper, 15 g of paprika, 60 g of glucose and 0.8 g of potassium nitrate. after chopping and mixing the ingredients, the mixture was divided into two batches (2.5 kg for each batch): batch 1, inoculated with a commercial starter culture starter a (20 g/200 kg): l. sakei + s. carnosus + s. xylosus (texel sa-201, danisco, paris, france) and batch 2, control without inoculation. starter a was added to sausages according to manufacturer’s recommendations. the mixture of each batch was stuffed into artificial casings, giving approximately 500 g as the final mass of each sausage and then placed in a fermentation chamber (bcr, cf 1 b, antony, france). the sausages were fermented for 5 days at 24 °c and 80% relative humidity (rh). after 5 days of processing, the temperature was decreased to 14 °c for 23 days and the rh value was 80%. for sampling, three sausages of each batch at 0 day (mix before stuffing) and after 7, 14, 21 and 28 days of ripening were taken for microbiological, physicochemical and textural analysis. all reported values represent the mean of three random measurements of the sausage sample.” ital. j. food sci., vol. 32, 2020 323 2.2. microbiological analysis “sausage samples (10 g) of each batch were homogenized with 90 ml of sterile peptone water (biolife, milan, italy) and decimal dilutions were prepared. mesophilic lab were enumerated on mrs (de man, rogosa and sharpe) agar (biolife) after 48 h of incubation at 30 °c. the number of staphylococci was determined on mannitol salt agar (biolife) after incubation at 37 °c for 48 h. yeasts and molds were enumerated on sabouraud dextrose agar (biokar, beauvais, france) at 28 °c for 4 days. total viable counts were determined on standard plate count agar (biolife) at 30 °c for 48 h. enterobacteriaceae were determined on violet red bile glucose (vrbg) (biokar) at 37 °c for 24 h.” 2.3. ph, moisture, weight loss, total lipid and total protein contents “the ph values were measured in homogenates prepared by blending 10 g of sausage (moulinex dpa141, lyon, france) with 50 ml of distilled water for 2 min. measurements were taken with a ph meter (microprocessor ph meter bt-500, boeco, hamburg, germany). the moisture content was calculated by weight loss experimented by the sample (5 g) maintained in an oven (memmert, ul 60, schwabach, germany) at 105 °c, until constant weight according to the iso recommended method (iso, 1973). weight loss was expressed as the percentage of the initial weight (liaros et al., 2009).” “total lipids were extracted from 5 g of minced sausage according to the method of folch et al. (1957). total nitrogen was determined according to the kjeldahl method and total protein estimated by multiplying the nitrogen content by 6.25.” 2.4. lipid oxidation analysis “lipid oxidation was assessed by measuring thiobarbituric acid reactive substances (tbars) during the ripening period. this analysis was performed according to the method of genot (1996)” as described by el adab et al. (2016). 2.5. free amino acid (faa) content “free amino acids were extracted and analyzed by reverse phase high-performance liquid chromatography (hplc) (agilent l 100 system, province, canada) equipped with a hypersil ods c18 column (250 mm × 4.6 mm dimensions of the column, 5 μm porosity)”, as described by el adab et al. (2016). 2.6. biogenic amines analysis biogenic amines were determined using the method described by boulares et al. (2017). “biogenic amines are first extracted from the test sample by chloridric acid (0.1 m) and then practice derivatization. briefly, 4 ml of hcl (0.1 m) were added to 1 g of sausage. after homogenization, samples were centrifuged at 12.000 rpm for 20 min (sigma, 6-16s, munich, germany) before being filtered. after that, 2 ml of the obtained aqueous fraction were homogenized with 1 ml of sodium bicarbonate and 2 ml of dansyl chloride, and then the mixture was heated during 1 h at 40 °c. after addition of 2 ml of diethyl ether, the organic fraction was collected and evaporated under nitrogen liquid stream. the mixture was then dissolved in 1 ml of acetonitrile. a standard solution of amines was prepared similarly and used as control. finally, 20 µl of each derivatized solution were ital. j. food sci., vol. 32, 2020 324 injected onto hplc column where the components will be retained unequally depending on their size and composition. a knauer eurosphère 100-rp18 reversed-phase column (250 x 4.6 mm, 5 µm, berlin, germany) was used for chromatographic separation. therefore, the detection was performed at 254 nm wavelength using acetonitrile/water as mobile phase at a constant flow rate of 0.8 ml/min for 20 min.” 2.7. texture profile analysis (tpa) “texture profile analysis (tpa) of the samples was performed with a texture analyzer (ta-xt2 stable micro systems, haslemere, uk) equipped with a cylindrical probe of 50 mm in diameter. the sausages were cut in a cylinder 1 cm thick and 3 cm in diameter and compressed twice to 50% of their original thickness. force-time curves were recorded at a crosshead speed of 1 mm/s. texture profile parameters (hardness, cohesiveness, springiness, gumminess and chewiness) were evaluated during the ripening of dry fermented sausages using the method of bourne (1978).” 2.8. color measurement “color measurements were carried out using a cr-300 colorimeter (minolta chroma meter cr-300, tokyo, japan). each sausage was cut and the color of the slices was measured three times for each analytical point l*, a* and b* scale coordinates were obtained: l* (lightness), a* (redness) and b* (yellowness). before each series of measurements, the instrument was calibrated using a white ceramic tile.” 2.9. sensory evaluation “the sensory analysis was performed by a sensory panel of ten assessors who had undergone professional training. a slice of each sample batch (5 mm thick approximately) was served to the assessors. the sensory evaluation was based on a six point hedonic scale to determine red color (10 = red and shiny; 1 = dark and dull), after taste (10 = extremely desirable; 1 = extremely undesirable), fat intensity (10 = high; 1 = low), acidity (10 = strong acidity; 1 = light acidity), hardness (10 = firm; 1 = soft) and overall acceptability (10 = high; 1 = low).” 2.10. statistical analysis “data were statistically analyzed using one-way analysis of variance (anova) procedure of spss 17.0 (spss, inc., chicago, il). duncan’s multiple range test was used to determine any significant difference between mean values and evaluations were based on a significance level of p<0.05.” 3. results and discussion 3.1. microbiological analysis the evolutions of microbial population during the ripening of control and inoculated sausages are shown in fig. 1. the total viable counts (tvc) and lab counts increased (p<0.05) during the ripening period. the numbers of lab and tvc in starter-mediated ital. j. food sci., vol. 32, 2020 325 sausages were significantly higher than those in control ones (p<0.05), which could be explained by the prior inoculation of sausages by lactobacillus sakei, staphylococcus carnosus and staphylococcus xylosus. results showed that the number of lactic acid bacteria in dry fermented sausages increased (p<0.05) during the two first weeks of ripening. a decrease of their number (p<0.05) was observed during the two last weeks of ripening which could be due to the exhaustion of the sugar. these results were similar to those reported by qinxiu et al. (2016) and mejri et al. (2017). enterobacteriaceae counts decreased (p<0.05) during ripening in both control and startermediated sausages. this result was similar to that reported by lu et al. (2015). the number of enterobacteriaceae is lower (p>0.05) in inoculated sausages than those measured on control ones. enterobacteriaceae numbers dropped below 1 logarithmic unit in startermediated batches. this drop is due to bactericidal properties of starters (lorenzo et al., 2007; ciuciu et al., 2014). staphylococci profiles showed no significant differences (p>0.05). at day 0, staphylococci counts in starter-mediated sausages were more than five logarithmic units higher than those of the control samples. a decrease of the number of staphylococci in sausages was observed after seven days of ripening. our results match with those found by zhao et al. (2011) in dry fermented mutton sausages. yeast and molds counts increased (p>0.05) during the first seven days of ripening and then decreased (p>0.05) to reach at day 28 values of 4.15 and 3.77 log cfu/g, respectively, for control and inoculated batches. figure 1. evolution of microbial population during the ripening of control and inoculated sausages: c (control sausage), s (sausage inoculated with mixed starter cultures). 3.2. ph, moisture, weight loss, total lipid and total protein contents the ph of control and starter-mediated sausages decreased (p<0.05) from 6.22 to 5.79 and 5.01 after 14 days of ripening, respectively (fig. 2a). the ph decrease could be attributed to lactic acid production by lab (nie et al., 2014). beyond the 14th day, ph values increased for both control and inoculated sausages. this may be caused by proteolytic ital. j. food sci., vol. 32, 2020 326 processes and mold growth on the sausage surface (essid and hassouna, 2013). the weight of control sausages and those inoculated with mixed starter cultures decreased (p<0.05) during the ripening period (fig. 2b). these results match with those found by jin et al. (2010) and liaros et al. (2009). the moisture content decreased (p<0.05) in all the samples (fig. 2c). however, no significant difference (p>0.05) was found between the different batches during the ripening process. this water loss is due to the elevated temperature of fermentation (24°c) and to the decrease of ph of sausages to their isoelectric ph (hamoen et al., 2013). figure 2. evolution of ph (a), weight loss (b) and moisture (c) during the ripening of control and inoculated sausages: c (control sausage), s (sausage inoculated with mixed starter cultures). changes in total lipid and total protein contents during ripening of dry fermented sausages are summarized in table 1. table 1. evolution of the chemical composition during ripening of the control batch and starter-inoculated sausages. samples ripening time (days) 0 7 14 21 28 chemical composition protein (%) c 15.75±0.21a 19.25±0.44b 20.13±0.88b 21.02±0.88b 27.78±0.22c s 15.97±0.44a 19.47±0.22b 21.22±0.66c 22.97±0.22d 28.87±0.44e fat (%) c 18.1±0.1a 21.50±0.1b 26.65±0.05c 34.7±0.5d 36.1±0.7e s 18.2±0.2a 22.1±1.9b 30.1±1.3c 37.8±0.6d 39.1±0.7d samples: c, control camel meat sausage; s, sausage inoculated with mixed starter cultures. data are means±standard deviation. different letters in the same row indicate significant differences (p<0.05). ital. j. food sci., vol. 32, 2020 327 results showed that protein content and lipid content increased (p<0.05) during ripening of control and inoculated sausages. however, protein content and lipid content showed no significant differences (p>0.05) between control batches and those inoculated with a mixture of strains. dalla santa et al. (2014) reported that there was significant difference between control and inoculated italian sausages in protein content at the end of ripening. however, they did not show any significant difference in lipid content. 3.3. lipid oxidation analysis the tbars values increased (p<0.05) during ripening from 0.25±0.04 to 0.95±0.16 and 0.8±0.1 mg mda/kg of sample, respectively, in control and inoculated sausages (fig. 3). results showed that there was no significant difference (p>0.05) between the different samples. many factors could affect lipid oxidation such as “chemical composition of raw material, processing conditions, light, and access to oxygen (ahn et al., 2002)”. the tbars values are lower (p>0.05) in starter-mediated sausages than those found in control ones. similar results were found by kargozari et al. (2014) and el adab et al. (2016) who reported that s. xylosus and s. carnosus could limit lipid oxidation in dry fermented sausages due to their antioxidant activity. figure 3. changes in thiobarbituric acid (tba) values during the ripening of control and inoculated sausages: c (control sausage), s (sausage inoculated with mixed starter cultures). 3.4. faa content the contents of total faas during ripening are shown in table 2. the total faas increased through ripening (p<0.05); they reached at the day 28 values of 398.82 and 534.06 mg/100 g, respectively, for control and inoculated sausages. the highest total free amino acid content was found in inoculated samples. similar results were found by essid and hassouna (2013) and mejri et al. (2017). many factors could affect the proteolysis in fermented sausages such as product formulation, technological ripening conditions and mixed starter cultures (huges et al., 2002). the main amino acids present in the initial mixture were arginine, threonine, glycine, alanine and glutamine with a concentration higher than 148.02 mg/100 g of dry matter. “the faa content affects sensory properties impacting on fresh taste (glutamic acid and aspartic acid), sweet taste (glycine and alanine), bitter taste (arginine and leucine), sweet and bitter (lysine) and sour or salty (other faa)” (domínguez et al., 2016). ital. j. food sci., vol. 32, 2020 328 table 2. free amino acid content (mg amino acids/100 g of sausage) during ripening of the control batch and starter-inoculated sausages: c (control camel meat sausage), s (sausage inoculated with mixed starter culture). faa ripening time (days) 0 14 28 c c s c s aspartic acid 14.12±0.49e 22.84±1.26d 42.12±2.23b 30.14±0.58c 49.22±1.23a glutamic acid 28.34±0.34e 43.62±1.80d 63.22±1.56b 58.78±2.41c 81.12±1.12a serine + glutamine + histidine 8.62±1.30d 9.02±2.31cd 10.22±1.14c 23.15±0.05b 28.12±2.12a arginine + threonine + glycine 84.32±2.50e 95.56±2.14d 130.02±0.77b 120.45±1.36c 145.02±0.89a alanine 35.36±0.25e 43.14±1.11d 50.41±0.93c 60.98±0.12b 90.45±2.14a lysine 10.65±1.18e 22.34±2.23d 25.17±1.25c 26.23±0.04b 28.78±1.45a tryptophane 18.42±0.32e 23.55±0.22d 29.47±2.24b 37.45±1.86c 51.78±1.23a isoleucine 3.12±0.45e 4.65±1.01d 9.12±0.88b 7.52±1.03c 13.12±0.58a leucine 13.68±2.08d 22.14±0.55c 33.45±0.78b 34.12±2.02b 46.45±1.66a total 216.63±0.79 286.86±0.71 392.2±0.58 398.82±0.85 534.06±0.49 data are means±standard deviation. different letters in the same row indicate significant differences (p<0.05). 3.5. biogenic amines contents total biogenic amine content in starter-mediated sausages was lower than in control ones during ripening (p<0.05) (fig. 4). the total biogenic amine content was 285.67 mg/kg in sausages at the beginning of fermentation, which increased significantly during the first seven days of ripening to reach values of 338.28 and 310.14 mg/kg respectively, in the control sausages and sausages inoculated with l. sakei, s. xylosus and s. carnosus. beyond the 7th day, total ba concentrations decreased significantly (p<0.05) to reach at the end of ripening respectively, values of 256.06 and 116.91 mg/kg. after 28 days of ripening, the total ba content in starter-mediated sausages was 54.3% lower than that in the control samples (p<0.05). the combination of three amine-negative bacteria, resulted in a drastic reduction of biogenic amine production. lee et al. (2016) reported that l. sakei and s. xylosus could degrade bas formed during fermentation through biogenic amine oxidases enzymes. these findings match with those found by hu et al. (2007) and nie et al. (2014). changes in putrescine, cadaverine, spermine, spermidine and histamine concentrations during ripening of camel meat sausages are shown in fig. 5. there was a decrease in all biogenic amines in the analyzed samples during the ripening. spermine and spermidine were the predominant amine compounds in the sausages, followed by putrescine, histamine, and cadaverine. our result is not in agreement with the study of gonzalezfernandez et al. (2003) and lu et al. (2010), who reported that cadaverine and putrescine were the predominant amine compounds respectively, in spanish pork sausage and traditional chinese smoked horsemeat sausage. many factors could contribute to the variability between the different types of products such as the microbiological quality of raw materials, ingredients, diameter of sausage, acidification, proteolysis and technological ripening conditions (bover-cid et al., 2001: bozkurt and erkmen, 2002; latorre-moratalla et al., 2012). ital. j. food sci., vol. 32, 2020 329 figure 4. changes in the total concentration of biogenic amines (mg/kg) in control and inoculated sausages: c (control sausage), s (sausage inoculated with mixed starter cultures) during ripening. spermidine and spermine are natural amines that always appear in fresh meat (herández-jover et al., 1997). bover-cid et al. (2001) showed that these two bas could be a source of nitrogen for some microorganisms, which could explain the decrease of spermidine and spermine concentrations during the ripening of dry fermented sausage. the histamine concentrations were much lower in the starter-mediated sausages than that in the control batches (fig. 5). at the end of ripening, the histamine content in inoculated sausages was 41.1% lower than that of the control ones (p<0.05). many studies showed that histamine toxicity depended on the concentration upon absorption and it could be enhanced by the presence of cadaverine and putrescine (bover-cid et al., 2001; renes et al., 2014). the drastic reduction of biogenic amine production is related to the drop of the ph of inoculated samples, which contribute to the decrease of the number of enterobacteriaceae (p<0.05). “cadaverine can be used as an indicator of food hygiene (chang et al., 2012).” cadaverine contents increased (p<0.05) during the first seven days of ripening from 0 mg/kg to 2.84 and 1.8 mg/kg for the control and starter-mediated sausages, respectively (fig. 5). at the end of ripening, cadaverine accumulation was significantly (p<0.05) inhibited by 63.8% in starter-mediated sausages compared to the spontaneously fermented sausages. results showed that there was a significant difference (p<0.05) between control batches and those inoculated with mixture of starter cultures. komprda et al. (2009) and rabie et al. (2009) reported that the formation of cadaverine in fermented sausages is related to the presence of enterobacteria, which could be used as a chemical indicator of raw material and manufacturing practice hygiene. values obtained for cadaverine indicate application of good hygiene in all phases of production. the putrescine concentrations increased (p<0.05) during the initial 7 days. at the end of ripening, the putrescine concentration in inoculated sausages was 58.8% lower than that of the control (p<0.05) (fig. 5). ital. j. food sci., vol. 32, 2020 330 figure 5. changes in the amounts of putrescine (a), cadaverine (b), histamine (c), spermidine (d), and spermine (e) (mg/kg) in control and inoculated sausages: c (control sausage), s (sausage inoculated with mixed starter cultures) during ripening. our results showed that the combination of three bacterial strains could more effectively inhibit the formation of biogenic amines. many studies reported the important role of l. sakei in the inhibition of the formation of biogenic amines (genϛcelep et al., 2012; latorre-moratalla et al., 2010; baka et al., 2011). “l. sakei is highly adapted to the fermented meat products and the optimal temperature of growth is between 15 and 25˚c, which is the temperature range for sausages manufacture (bover-cid et al., 2001).” moreover, gonzalez-fernandez et al. (2003) reported that l. sakei could reduce the formation of biogenic amine due to its strong acidifying activity. “bover-cid et al. (2001) ital. j. food sci., vol. 32, 2020 331 also reported that this specie is able to inhibit the production of biogenic amines in spanish fermented sausage. however, when l. sakei was combined with s. carnosus or s. xylosus an even more effective reduction of amine accumulation was achieved compared with the effect of each strain used alone (latorre-moratalla et al., 2012).” “masson et al. (1996) showed that s. carnosus and s. xylosus can be used as safe starter cultures. whereas, straub et al. (1995) found that s. carnosus contribute to the production of biogenic amines, but they did not find this for s. xylosus.” 3.6. texture profile analysis (tpa) fig. 6 shows the hardness, gumminess, chewiness, springiness, cohesiveness and resilience of control and inoculated sausages. results showed that there were no significant differences between batches in any of the textural parameters studied. figure 6. changes in textural parameters during the ripening of control and inoculated sausages: c (control sausage), s (sausage inoculated with mixed starter cultures). hardness and springiness increased (p<0.05) during the ripening of control and inoculated sausages. similar results were found by gonzalez-fernandez et al. ital. j. food sci., vol. 32, 2020 332 (2006) and bozkurt and bayram (2006) who reported that the increase of hardness could be explained by the elevated temperature during fermentation (24°c). the increase of springiness indicated that elasticity of camel meat sausage increased during the ripening period. gumminess and chewiness values increased (p<0.05) during the whole process. these results are in agreement with those found by qinxiu et al. (2016). 3.7. color properties the color parameters, lightness (l*), redness (a*) and yellowness (b*) are shown in fig. 7. l* values decreased (p<0.05) during ripening due to weight loss and higher myoglobin content (kadim et al., 2008; olivares et al., 2010). moreover, results showed that l* values were significantly affected (p<0.05) by ripening time and not by the addition of mixed starter cultures (p>0.05). in relation to a* values, an increase (p<0.05) was observed during the first two weeks of ripening of dry fermented sausages followed by a significantly decrease which probably due to partial or total denaturation of nitrosomyoglobin because of the production of lactic acid (perez-alvarez et al., 1999; rubio et al., 2008). the evolution of a* and l* values found in this study was similar to that described by other authors (kayaardi et al., 2003; mejri et al., 2017). the b* values decreased (p<0.05) during ripening of both control and inoculated sausages. this finding was similar to that found by bozkurt and bayram (2006) during the ripening of sucuk. figure 7. changes in lightness (a), redness (b) and yellowness (c) during the ripening of control and inoculated sausages: c (control sausage), s (sausage inoculated with mixed starter cultures). 3.8. sensory evaluation the results of a sensorial evaluation of control and inoculated sausages are shown in fig. 8. in fact, the starter-mediated sausages showed a more pronounced red color when ital. j. food sci., vol. 32, 2020 333 compared to control ones (p<0.05). ravyts et al. (2010) reported that the red color of fermented sausages was often related to the nitrate reductase activity of cns. moreover, inoculated sausages had higher scores of acidity (p<0.05) which could be explained by the lactic acid produced from bacterial carbohydrate metabolism. additionally, sausages inoculated with mixed starter cultures showed a significantly greater overall aromatic intensity than that noted on control samples, which could be due to the lipolytic, acidifying and proteolytic activities of strains of s. xylosus, s. carnosus and l. sakei inoculated in meat products. our results showed that there was no significant difference (p>0.05) in fat intensity. inoculated sausages showed a significantly higher firmer texture (p<0.05) than those found on control samples. similar results were found by fonseca et al. (2013). figure 8. sensory evaluation of control sausages and sausages inoculated with mixed starter cultures. 4. conclusion this study focused on the effect of mixed starter cultures on microbiological, biochemical and sensory characteristics of a dry fermented camel meat sausage. the bactericidal properties of l. sakei improved the hygienic quality of sausages by decreasing the number of enterobacteriaceae. inoculation of dry sausages with a mixture of strains, significantly delayed lipid oxidation and improved sensory characteristics. moreover, the total biogenic amine concentration in starter-mediated sausages was much lower than that in the control samples. these results suggest that l. sakei, s. xylosus and s. carnosus could be used as safe mixed starter cultures in dry sausage production to inhibit biogenic amine formation and enhance sensory quality. acknowledgements “this study was sponsored by the «tunisian ministry of higher education and scientific research», tunisia. the 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y., ma c., song h., li h., wang z. and xiao s. 2011. physico-chemical characteristics and free fatty acid composition of dry fermented mutton sausages as affected by the use of various combinations of starter cultures and spices. meat sci. 88:761-766. paper received november 18, 2019 accepted january 15, 2020 paper 166 ital. j. food sci., vol. 27 2015 keywords: extra virgin olive oil, diglycerides,1,2/1,3-dgs ratio, gc, storage conditions extra virgin olive oil stored in different conditions: focus on diglycerides z. ayyad1, e. valli*2, a. bendini1,2, s. adrover-obrador3, a. femenia4 and t. gallina toschi1,2 1department of agricultural and food sciences, alma mater studiorum university of bologna, cesena (fc), italy 2interdepartmental centre for agri-food industrial research, alma mater studiorum university of bologna, cesena (fc), italy 3institute of agricultural and fishing research and training (irfap) of the government of the balearic islands, c/ d’eusebi estada, 145, 07009, palma, spain 4department of chemistry, university of the balearic islands, ctra. valdemossa, km 7.5, 07122, palma, spain *corresponding author: tel. +390547338121, fax +390547382348, email: enrico.valli4@unibo.it abstract the effects of storage conditions of extra virgin olive oil (evoo) on the isomerization of diglycerides (dgs) have been investigated. aliquots of evoo were stored for 14 months under four different conditions: at 20°c in darkness and in light, at 4-6°c in light and at 20°c in light with argon in the headspace. samples were analysed bimonthly: 12 dgs with c34 and c36 (1,2 and 1,3 isomers) were tentatively identified and quantified by gc-fid. after 14 months, a clear tendency towards a decrease of 1,2-dgs and a significant increase of 1,3-dgs during storage was observed for all samples. 1,2-dgs were always predominant compared to 1,3-dgs and, for both types, c36 dgs were prevalent compared to c34 dgs. overall, evoo stored at 4-6°c in light showed the highest preservation of 1,2-dgs. ital. j. food sci., vol. 27 2015 167 introduction extra virgin olive oil (evoo) is fresh olive (olea europaea l.) juice obtained by mechanical and physical processes (lozano-sanchez et al., 2012), and it is well known as one of the major components of the diet of mediterranean countries. evoos consist of triglycerides as the main components (about 98%) and other minor components including diglycerides, free fatty acids, squalenes, sterols, phospholipids, phenolics and different volatile compounds (boskou, 1996). some of these minor components, in addition to a high content of mono-unsaturated fatty acids, play a major role in keeping evoo more stable against oxidation during storage compared to other vegetable oils (bendini et al., 2009a). elimination of air in the head space, either by fully filling the evoo bottles or by its replacement with inert conditioning gas, has been found to add marked improvement in terms of oxidation quality, stability, shelf life and slow down the oxidation process of evoo (urdaromacho, 2009; giovacchino et al., 2002). newly produced evoo contains a low concentration of diglycerides (dgs) (1-3%), which are formed as intermediate products of the incomplete biosynthesis of triglycerides (spyros et al., 2004) and partial hydrolysis of triglycerides. during storage many changes may occur in dg composition due to isomerisation of 1,2-dgs, the predominant form in fresh evoo, to 1,3-dgs (sacchi et al., 1991). the effects of storage temperature and exposure to light during different periods of time on the quality of evoo have been investigated by different authors (velasco and dobarganes, 2002; mendez and falque, 2007), while other studies have assessed the amount of dgs as an indicative parameter of the freshness of evoo. cat alano et al. (1994) investigated dgs isomerisation occurring in evoo stored in darkness, at room temperature and at 4°c. in particular, the results revealed that the 1,2-dgs remained less than 1.5 % after one year of storage for all samples analysed, while about 10% and 45% of the samples stored at room temperature and at 4°c, respectively, contained less than 0.4% 1,3-dgs. furthermore, pérez-camino et al. (2001) studied the evolution of the two dg isomer classes in oils obtained from olives of different qualities stored at different temperatures, concluding that triacylglycerol hydrolysis and dg isomerisation depended not only on the value of free acidity, but also on the storage temperature. in addition, the 1,3/1,2-dg ratio was a useful parameter for assessing the genuineness of evoos with low free acidity during early storage stages. another interesting study was carried out by spyros et al., (2004), assessing olive oil through investigation of 1,2 and 1,3-dg isomerisation during 18 months of storage at room temperature, at 5°c with light and in darkness. the result of the isomerisation process was mainly dependent on the initial quality parameters of the oil, and in particular the free acidity. another study based on the evaluation of olive oil quality in relation to storage conditions through the analysis of dg isomerisation was carried out by cossignani et al. (2007) on samples produced from different olive cultivars stored at 15°c and at 30°c in darkness for 12 months. the results showed important differences in the percentage of each individual dg and in the ratio among classes; in particular, samples analysed at time zero exhibited the highest percentage of 1,2-dgs and the lowest of 1,3-dgs, whereas samples stored at 30°c showed the highest content of 1,3-dgs suggesting that temperature plays an important role in the isomerisation process. more recently, a study carried out by caponio et al. (2013) investigated the effects of storage of evoo in green glass bottles in light and darkness for 24 months, providing evidence that the degree of isomerisation was affected by the initial hydrolysis level of the oil and by the storage time, although other storage conditions did not show any effect. overall, these results suggest that the content of dgs and the ratio between isomers might be considered as possible markers to establish the freshness state of an evoo alongside with other quality parameters defined by official regulations (eu reg. 61/2011). therefore, the main aim of this study was to investigate the isomerisation processes related to diacylglycerols, and in particular the amounts of 1,2and 1,3-dgs and relative c34 and c36 sub-classes as well as the 1,2/1,3-dg ratio in evoo during storage under different conditions for 14 months. the purpose was to investigate how these compounds were influenced by different variables such as temperature, light and headspace gases. materials and methods samples evoo samples used in this study were produced from olives of the arbequina cultivar (coop. sant bartomeu, soller, spain) using an industrial plant working with a three-phase decanter. once in the laboratory, the evoo was poured into 250 ml transparent glass bottles. the headspace in each bottle was about 2 ml. the bottles were hermetically sealed and divided into four batches. the first batch was stored in darkness inside a thermostatic chamber at 20°c (cond. 1); the second batch was stored at 20°c under diffuse light (600 lux for 12 h/ day 11 w; 595 lm; 6400°k) simulating the conditions of a supermarket shelf (cond. 2); the 168 ital. j. food sci., vol. 27 2015 third batch was stored in a refrigerated chamber at 4-6°c with diffuse light (cond. 3); finally, the fourth batch was stored with argon in the headspace of bottles at 20°c with diffuse light (cond. 4). samples were analysed in triplicate after 2, 4, 6, 8, 10, 12 and 14 months of storage after production. basic chemical analysis free acidity, peroxide value and uv absorption (k 232 , k 270 ) were determined according to the official methods described in eec reg. 2568/91 for all samples at the initial period of storage (2 months) and after the end of storage simulation (14 months). gas chromatographic (gc) determination of diglycerides the silylated samples were prepared according to a previous work (sweeley et al., 1963) and dgs were determined according to a modified version of the method suggested by serani et al., (2001) using a gc carlo erba mfc500 with a rtx-65tg (restek, bellefonte, pa) fused silica capillary column (30 m length x 0.25 mm i.d. x 0.10 μm f.t.) coated with 35 % dimethyl-65 % diphenylpolysiloxane. the oven temperature was programmed from 250 to 320°c at a rate of 2°c min-1 and then increased to 365°c at a rate of 5°c min-1. the final temperature was maintained for 21 min. the injector and fid temperatures were both set at 360°c. helium was used as carrier gas at a pressure of 130 kpa. the split ratio was 1:70. identification of dgs was carried out by comparing peak retention times and gc traces with those of dg standards and chromatograms reported in the literature (serani et al., 2001; bendini et al., 2009b). the results, expressed as mg of each dg per 100 mg of oil, were quantified with respect to dilaurin, added as internal standard (0.5 ml of a solution 2 mg ml-1 of dilaurin dissolved in chloroform, added to 100 mg of oil). statistical analysis the software xlstat 7.5.2 version (addinsoft, usa) was used to elaborate the data by analysis of variance (anova, fisher lsd, p < 0.05). results and discussion the free acidity, peroxide values and extinction coefficients (k 232 and k 270 ), shown in table 1, indicated that at the end of the storage period all samples were within the accepted limits established by eu regulations for the evoo category (eu reg. 61/2011). fig. 1 shows a comparison between the gas chromatography traces of dg fractions of evoo stored for 2 and 14 months in dark at 20°c. twelve different dgs were tentatively identified and quantified as 1,2 and 1,3 isomers with 34 or 36 carbon atoms (c34, c36). only a co-elution was present (peak 11) between 1,3 isomers of the oleic-linoleic and linoleic-linoleic couples. the peaks numbered from 1 to 6 (fig. 1) were relative to c34 dgs whereas from 7 to 11 belonged to c36, and palmitic-oleic (po) and oleic-oleic (oo) were the most abundant dgs for the two classes, respectively. observing the gc traces (fig. 1), it is also possible to note that the 1,2 isomers eluted before the 1,3 ones for both groups with 34 and 36 carbon atoms. fig. 2 illustrates the evolution of 1,2/1,3dg ratios, and tables 2-5 highlight the trends of 1,2-dgs (c34, c36) and 1,3-dgs (c34, c36) for evoos stored under the four different experimental conditions. for the samples kept at 20°c in darkness (cond. 1), a rapid and significant decrease was observed in the 1,2/1,3-dg ratio for the first 8 months; this ratio continued to decrease slowly until the end of storage period (fig. 2). a similar trend was also seen for the 1,2dgs c34 and c36 under the same condition (table 2), and the rapid decrease continued for up to 8 months. at the end of storage period, total 1,2-dg remained about 60 % (data not shown) of table 1 results for free acidity (fa, g of oleic acid per 100 g of oil), peroxide values (pv, meq o 2 kg -1) and extinction coefficient at 232 and 270 nm (k 232 , k 270 ) at time zero and after 14 months of storage under the four different conditions (cond. 1 4)*. * cond. 1, stored at 20°c in dark, cond. 2, stored at 20 °c in light, cond. 3, stored at 4-6°c in light, cond. 4 stored at 20°c in light with argon in the headspace. different letters (a-e) represent significant differences among mean values for a same parameter during the storage time (from 2 to 14 months). different letters (x-z) indicate significant differences among the four storage conditions after 2 and 14 months of storage. 2 months of storage 14 months of storage fa pv k232 k270 fa pv k232 k270 cond. 1 0.15 ± 0.01 b,x 11.63 ± 1.29 a,xy 2.11 ± 0.03 b,x 0.10 ± 0.00 b,z 0.20 ± 0.01 a,x 12.74 ± 0.55 a,y 2.34 ± 0.02 a,x 0.15 ± 0.01 a,y cond. 2 0.15 ± 0.01 b,x 14.00 ± 0.04 a,x 2.00 ± 0.09 b,xy 0.17 ± 0.01 b,x 0.20 ± 0.01 a,x 14.74 ± 1.02 a,xy 2.19 ± 0.07 a,y 0.18± 0.01 a,x cond. 3 0.15 ± 0.01 b,x 10.59 ± 0.01 b,y 1.94 ± 0.12 b, y 0.13 ± 0.00 b,y 0.17 ± 0.01 a,y 15.47 ± 0.80 a,x 2.19 ± 0.06 a,y 0.14 ± 0.00 a,y cond. 4 0.16 ± 0.01 b,x 14.00 ± 0.15 a,z 2.15 ± 0.04 b,x 0.17 ± 0.00 b,x 0.20 ± 0.01 a,x 14.70 ± 0.40 a,xy 2.24 ± 0.03 a,xy 0.18 ± 0.01 a,x ital. j. food sci., vol. 27 2015 169 fig. 1 example of full chromatogram of the evoo sample at 20°c in dark. a) gc tracing of the diglyceride fraction of evoo stored for 2 months at condition 1; b) gc trace of the diglyceride fraction of evoo stored for 14 months at condition 1. 1, 1,2-po; 2, 1,2-poo; 3, 1,2-pl; 4, 1,3-po; 5, 1,3-poo; 6, 1,3-pl; 7, 1,2-oo; 8, 1,2-ol; 9, 1,3-oo; 10, 1,2-ll; 11, 13-ol + 1,3-ll. p = palmitic acid; po = palmitoleic acid; o = oleic acid; l = linoleic acid. fig. 2 trends of 1,2/1,3 dgs during the evoo storage of 14 months at the four different conditions (cond 1-4)*. the concentration of dgs was calculated as mg dilaurin per 100 mg of oil. different letters (a-e) represent significant differences among mean values for a same condition during the storage time (from 2 to 14 months). different letters (x-z) indicate significant differences among the four storage conditions after 14 months. * cond. 1, stored at 20°c in dark, cond. 2, stored at 20°c in light, cond. 3, stored at 4-6°c in light, cond. 4 stored at 20°c in light with argon in the headspace. total dgs with higher amounts of c36 isomers, in particular diolein, which is considered the predominant dg in olive oil (boskou, 1996). a comparable behaviour was observed for samples stored at 20°c in light (cond. 2). accordingly, the ratio of 1,2/1,3-dgs decreased significantly from 5.43 to 1.69 after 10 months (fig.2). moreover, the 1,2-dg c36 isomer (table  3) decreased significantly from 0.79 to 0.57 mg per 100 mg oil at the end of storage period, although this decrease slowed after 10 months. on the other hand, the 1,3-dg c36 isomer showed steady significant increase up to 12 months (table 3) and then remained with slight changes, until the end of storage. however, 1,3dg c34 isomers showed a significant slight change toward increases, after 6 months of storage, reaching about 0.14 mg per 100 mg sample after 14 months of storage (table 2). the results for samples stored at low 170 ital. j. food sci., vol. 27 2015 temperature (4-6°c) (cond. 3) showed that, at the end of the storage period, the 1,2/1,3-dg ratio remained about 2 times higher than the values for evoo samples stored at 20°c (fig. 2). furthermore, the 1,2-dgs isomers c36 and c34 showed a significant decrease from 2 to 14 months (table 4). regarding the samples stored with argon in the headspace (cond. 4), the 1,2/1,3-dgs ratio decreased significantly during the first 8 months of storage, and minor changes were detected up to the end of storage (fig. 2). similarly, 1,2-dgs for both c36 and c34 classes decreased after 14 months of storage compared to the initial value, with a fluctuation trend ( table 5), while 1,3dg c36 isomers showed a significant increase throughout the entire storage period. by comparing the different conditions, after 2 months of storage the highest 1,2/1,3-dg ratio corresponded to the sample stored at low temperature (4-6°c), followed by the sample stored under light at 20°c with argon in the headspace table 3 evolution of 1,2 and 1,3 isomers of c34 and c36 diglycerides during the evoo storage of 14 months under condition 2 (at 20 °c in light). the concentration of dgs was calculated as mg dilaurin per 100 mg of oil. different letters (a-e) represent significant differences among mean values for a same isomer during the storage time (from 2 to 14 months). different letters (x-z) indicate significant differences among the four storage conditions after 14 months of storage. cond. 2 months of oil storage 1,3 c34dgs 1,3 c36dgs 1,2 c34dgs 1,2 c36dgs 2 0.06 ± 0.00 e 0.15 ± 0.01 e 0.35 ± 0.01 ab 0.79 ± 0.15 cd 4 0.09 ± 0.02 d 0.21 ± 0.02 d 0.38 ± 0.05 a 1.06 ± 0.14 a 6 0.12 ± 0.02 cd 0.25 ± 0.02 d 0.37 ± 0.02 a 0.98 ± 0.05 ab 8 0.15 ± 0.01 ab 0.30 ± 0.01 c 0.32 ± 0.01 b 0.85 ± 0.07 bc 10 0.15 ± 0.01 a 0.36 ± 0.01ab 0.22 ± 0.01 c 0.69 ± 0.02 de 12 0.13 ± 0.00 bc 0.39 ± 0.00 a 0.23 ± 0.00 c 0.68 ± 0.00 de 14 0.14 ± 0.01 ab,y 0.32 ± 0.06 bc,y 0.21 ± 0.01 c,z 0.57 ± 0.04 e,z table 2 evolution of 1,2 and 1,3 isomers of c34 and c36 diglycerides during the evoo storage of 14 months under condition 1 (at 20oc in dark). the concentration of dgs was calculated as mg dilaurin per 100 mg of oil. different letters (a-e) represent significant differences among mean values for a same isomer during the storage time (from 2 to 14 months). different letters (x-z) indicate significant differences among the four storage conditions after 14 months of storage. cond. 1 months of oil storage 1,3 c34dgs 1,3 c36dgs 1,2 c34dgs 1,2 c36dgs 2 0.09 ± 0.01 f 0.19 ± 0.03 e 0.48 ± 0.06 a 1.25 ± 0.14 a 4 0.11 ± 0.01 e 0.25 ± 0.02 de 0.47 ± 0.05 a 1.27 ± 0.16 a 6 0.13 ± 0.01 de 0.26 ± 0.01 d 0.38 ± 0.05 b 0.89 ± 0.07 b 8 0.13 ± 0.00 cd 0.33 ± 0.01 c 0.28 ± 0.01 c 0.77 ± 0.02 b 10 0.15 ± 0.00 bc 0.40 ± 0.07 b 0.28 ± 0.01 c 0.75 ± 0.05 b 12 0.16 ± 0.01 b 0.37 ± 0.03 bc 0.27 ± 0.01 c 0.74 ± 0.10 b 14 0.19 ± 0.02 a,x 0.49 ± 0.02 a,x 0.27 ± 0.02 c,yz 0.73 ± 0.05 b,y table 4 evolution of 1,2 and 1,3 isomers of c34 and c36 diglycerides during the evoo storage of 14 months under condition 3 (at 4-6°c in light). the concentration of dgs was calculated as mg dilaurin per 100 mg of oil. different letters (ae) represent significant differences among mean values for a same isomer during the storage time (from 2 to 14 months). different letters (x-z) indicate significant differences among the four storage conditions after 14 months of storage. cond. 3 months of oil storage 1,3 c34dgs 1,3 c36dgs 1,2 c34dgs 1,2 c36dgs 2 0.08 ± 0.01 c 0.14 ± 0.01 d 0.58 ± 0.08 a 1.09 ± 0.18 ab 4 0.08 ± 0.00 c 0.17 ± 0.01 c 0.46 ± 0.02 b 1.25 ± 0.06 a 6 0.12 ± 0.01 ab 0.18 ± 0.01 c 0.41 ± 0.02 bcd 1.01 ± 0.11 b 8 0.12 ± 0.01 ab 0.16 ± 0.01 cd 0.34 ± 0.04 d 0.9 ± 0.12 b 10 0.10 ± 0.03 bc 0.22 ± 0.00 b 0.38 ± 0.01 cd 1.08 ± 0.03 ab 12 0.14 ± 0.02 a 0.25 ± 0.02 b 0.45 ± 0.02 bc 1.03 ± 0.17 b 14 0.13 ± 0.01 a,y 0.28 ± 0.03 a,y 0.39 ± 0.04 cd,x 1.07 ± 0.03 ab,x ital. j. food sci., vol. 27 2015 171 table 5 evolution of 1,2 and 1,3 isomers of c34 and c36 diglycerides during the evoo storage of 14 months under condition 4 (at 20 °c in light with argon in the headspace). the concentration of dgs was calculated as mg dilaurin per 100 mg of oil. different letters (a-e) represent significant differences among mean values for a same isomer during the storage time (from 2 to 14 months). different letters (x-z) indicate significant differences among the four storage conditions after 14 months of storage. cond. 4 months of oil storage 1,3 c34dgs 1,3 c36dgs 1,2 c34dgs 1,2 c36dgs 2 0.07 ± 0.00 c 0.14 ± 0.02 d 0.41 ± 0.08 a 1.07 ± 0.18 ab 4 0.07 ± 0.01 c 0.18 ± 0.01 d 0.32 ± 0.02 bc 0.82 ± 0.13 cd 6 0.14 ± 0.00 b 0.31 ± 0.06 c 0.46 ± 0.05 a 1.09 ± 0.11 a 8 0.15 ± 0.01 b 0.37 ± 0.04 bc 0.29 ± 0.01 c 0.82 ± 0.02 d 10 0.21 ± 0.01 a 0.46 ± 0.04 ab 0.38 ± 0.01 ab 1.06 ± 0.21 abc 12 0.17 ± 0.01 b 0.48 ± 0.06 a 0.25 ± 0.01 c 0.70 ± 0.05 d 14 0.21 ± 0.04 a,x 0.53 ± 0.10a,x 0.31 ± 0.06 bc,y 0.84 ± 0.15 bcd,y (fig. 2). moreover, during the first 4 months, when evoos were stored at 20°c under light without headspace modification (cond. 2), the sample exhibited a lower ratio than the respective sample stored in darkness (cond. 1). the results also highlighted the positive effect of using inert gas in the head space. the total 1,2-dgs remained after 14 months (data not shown) of storage was about 1.5 times higher, in comparison with their presence in evoo stored under the same conditions, but with air in the head space. the findings are in accordance with spyros et al. (2004), suggesting that the length of storage time plays an important role in isomerisation changes of dgs, which is accelerated by temperature. the formation of oxidation products by photo-oxidation was confirmed by the high values of k 270 obtained for samples stored under diffuse light, especially for those stored at 20°c after 14 months of storage (table 1). it should be noted that, at the end of storage period, all the samples remained within evoo category parameters. as expected, free acidity (table 1), which is considered to be the main driving factor affecting dg isomerisation (pérez-camino et al., 2001), showed only a minor increase after 14 months of storage. the results of this study showed that the isomerisation of dgs in evoos depends not only on the length of storage, but also on the temperature of storage. this finding is in agreement with the studies of pérez-camino et al. (2001) and cossignani et al. (2007). moreover, the results showed that after 14 months of storage at 20°c (cond. 1, 2 and 4) there were slight but not significant differences in the 1,2/1,3 ratio among samples stored under diffuse light (cond. 2 and 4) and for those stored in darkness (cond. 1), in spite of the fact that light exposure has an adverse effect on the oxidation of evoo (significantly higher k 270 values were found for samples stored under diffuse light). this result is in agreement with considerations noted by afaneh et al. (2013). conclusion the results of this study confirmed that the isomerisation of dgs in evoo depends not only on the length of storage, but also on the temperature. by comparing the different conditions, it was found that after 10-14 months of storage the 1,2/1,3-dg ratio remained higher for samples stored at low temperature (4-6°c). moreover, the presence of argon gas in the headspace of the sample was not sufficient to protect it from dg isomerisation when the evoo was exposed to light. acknowledgments the authors would like to thank enhancement of the palestinian university system (eplus) phd scholarship grants financed by the italian ministry of foreign affairs-directorate general for cooperation and development (coordinated by the university of pavia) and inia pre-doctoral scholarship grant (simón adrover-obrador). thanks also to the financial support provided by the “ministerio de economía y competitividad” of spain (project agl 2012-34627) and to the balearic government (57/2011). references afaneh i.a., abbadi j., ayyad z., sultan w. and kanan k. 2013. evaluation of selected quality indicators of extra virgin olive oil bottled in different packaging materials upon storage under different lighting conditions. j. food sci. eng. 3: 267. bendini a., cerretani l., salvador m.d., fregapane g. and lercker g. 2009a. stability of the sensory quality of virgin olive oil during storage: an overview. ital. j. food sci. 21: 389. bendini a., valli e., cerretani l., chiavaro e. and lercker g. 2009b. study on the effects of heating of virgin olive oil blended with mildly deodorized olive oil: focus on the hydrolytic and oxidative state. j. agric. food chem. 57: 10055. boskou, d. 1996. olive oil: chemistry and technology. aocs press. champaign, il. catalano m., de leonardis t. and comes s. 1994. diacylglycerols in the evaluation of virgin olive oil quality. grasas aceites. 45: 380. caponio f., paradiso v.m., bilancia m.t., summo c., pas172 ital. j. food sci., vol. 27 2015 qualone a. and gomes t. 2013. diacylglycerol isomers in extra virgin olive oil: effect of different storage conditions. food chem. 140: 772. cossignani l., luneia r.m. and damiani p. 2007. analysis of isomeric diacylglycerolic classes to evaluate the quality of olive oil in relation to storage conditions. eur. food res. technol. 224: 379. eu commission regulation no. 61/2011 amending regulation no. 2568/1991 on the characteristics of olive oil and olive pomace oil and on the relevant methods of analysis. official journal of the european communities, l 23, 1-14. giovacchino di l., mucciarella n., constantini n., ferrante m.l. and surricchio g. 2002. use of nitrogen to improve stability of virgin olive oil during storage. j. amer. oil chem. soc. 79: 339. guil-guerrero j.l. and urda-romacho j. 2009. quality of extra virgin olive oil affected by several packaging variables, grasas aceites. 60: 125. mendez a.i. and falque e. 2007. effect of storage time and container type on the quality of extra virgin olive oil. food control. 18: 521. perez-camino m.c., modera w. and cert a. 2001. effects of olive oil fruit quality and oil storage practices on the diacylglycerols content of virgin olive oils. j. agric. food. chem. 49: 699. sacchi r., paolillo l., giudicianni i. and addeo f. 1991. rapid 1hnmr determination of 1,2 and 1,3 diglycerides in virgin olive oils. ital. j. food sci. 3: 253. serani a., piacenti d. and staiano g. 2001. analytical system for the identification of deodorized oils in virgin olive oils. note 2: kinetics of diacylglycerol isomerization in virgin olive oils. riv. ital. sost. grasse. 78: 525. spyros a., philippidis a.m. and dais b. 2004. kinetics of diglyceride formation and isomerization in virgin olive oils by employing 31p nmr spectroscopy. formulation of a quantitative measure to assess olive oil storage history. j. agric food. chem. 52: 157. sweeley c.c., bentley r., makita m. and wells w.w. 1963. gasliquid chromatography of trimethylsilyl derivatives of sugars and related substances. j. am. oil chem soc. 85: 2497. velasco j. and dobarganes c. 2002. oxidative stability of virgin olive oil. eur. j. lipid sci. technol. 104: 661. paper received february 27, 2014 accepted july 11, 2014 ijfs#198_sevindik_bozza   ital. j. food sci., vol 28, 2016 314 paper characterization of bioactive compounds in rosehip species from east anatolia region of turkey z.t. murathan1, m. zarifikhosroshahi2, e. kafkas2 and e. sevi̇ndi̇k3 1ardahan university, faculty of engineering, food engineering department, ardahan, turkey 2çukurova university, faculty of agriculture, department of horticulture, adana, turkey 3 adnan menderes university, faculty of agriculture, department of agricultural biotechnology, aydın, turkey *corresponding author: ph.d-emre@hotmail.com   abstract the objective of this work was to determine some bioactive compounds for four different rosehip species (rosa l.), growing in the east anatolia region of turkey. it was determined that the average fruit weights of the species varied between 9.8 g (r. dumalis) and 34.5 g (r. canina). the total soluble solids showed statistically significant variations among the rosehip species (14-22 ˚brix). the acidity was inversely proportional to total soluble solids and ranged between 1.00% (r. canina) and 2.67% (r. villosa). the highest total phenolic, lascorbic acid contents and the highest total antioxidant capacity were found in r. canina. the total phenolic, total anthocyanin, total dry matter, and l-ascorbic acid contents and the total antioxidant capacity of the rosehip species ranged as follows 1081-6298 mg gallic acid equivalent/100 g, 2.43-3.72 mg/100 g, 40.1–56.7%, 24.93-754.48 mg/100 g, and 10.0497.95 mmol trolox equivalent/g, respectively. glucose was the most common sugar in rosa species (5.99-12.48 g/100 g), the major organic acid in the rosehip species was citric acid (0.48-1.05 g/100 g). a dendogram based on some pomological and biochemical characteristics of the rosehip species were grouped into 2 main clusters. findings on the biochemical characteristics of the species will provide insights to plant breeders /growers and for further research. keywords: antioxidant, organic acids, phenolic, rosehip, sugars   ital. j. food sci., vol 28, 2016 315 1. introduction rosehip plants are not selective in terms of climate and soil requirements and grow in several areas, including europe, africa, middle and west asia and russia (nilson, 1997; ilisulu, 1992). rosehips grow in almost all regions of turkey and are well-known and consumed fruits in anatolia. they are perennial plants belonging to the genus rosa in the rosaceae family. the genus rosa includes numerous species and varieties, and each country has its own endemic rosehip species. rosa pisiformis and rosa dumalis subsp. antalyensis are endemic species for turkey (ercisli, 2005). out of about 100 rosehip species occurring all around the world, 27 species grow in turkey (turkben, 2003; ercisli and guleryuz, 2005). red fruits are rich in phytochemicals such as phenolic substances, flavonoids, anthocyanin and carotenoids (qian et al., 2004; trappey et al., 2005; cieslik et al., 2004). rosehips contain more and a greater variety of phytochemicals compared to other fruit species (halvorsen et al., 2002; olsson et al., 2004). also, they contain minerals, high-capacity antioxidants, carotenoids, phenolic compounds, tocopherol, bioflavonoids, tannins, pectins, organic acids, amino acids, ascorbic acid, and fatty acids (gao et al., 2000; demir and ozcan, 2001; larsen et al., 2003; chrubasik et al., 2008; jablonska et al., 2009; barros et al., 2010). the fact that rosaceae fruits have important physiological functions may be due to abundant phenolic substances, because it is known that the spectra of biochemical activity of phenolic substances, including their antioxidant activity, antimutagenic and anti-carcinogenic effects, are wide (tapiero et al., 2002; nakamura et al., 2003). these compounds also contribute to the quality and nutritional value of the plant (ercisli, 2007). moreover, it has been reported that rosehip fruits are used to cure illnesses such as influenza, other infections, inflammatory diseases, chronic pain and ulcer and that they have a protective effect on health (guimaraes et al., 2010). despite species variation, rosehips contain about 20to 30-fold more vitamin c compared to oranges. besides, rosehips, which are a valuable source of minerals, are quite rich in phosphorus and potassium (nojavan et al., 2008; szentmihalyi et al., 2002; kovacs et al., 2004). therefore, rosehip fruits are widely used in food and pharmaceutical industries. in turkey, numerous foods such as marmalade, jam, churchkhela, nectar and tea are made from rosehip fruits (ercisli and guleryuz, 2005; yildiz and alpaslan, 2012). besides, rosehip fruits are added to probiotic beverages, fruit yogurts and soup (demir et al., 2014). recently, naturality and bioavailability have been considered among the most important characteristics of food products (ercisli, 2007). in turkey, rosehip fruits grow naturally, without requirement of chemical compounds and fertilizers. in this study, we aimed to determine and compare some important bioactive compounds and biochemical features of four different rosehip species growing naturally in high altitudes of ardahan city located in eastern anatolia in turkey. so far, there is little information about sugar and acidity in rosehips, and no previous scientific studies have been carried out on rosehip species in the region. 2. materials and methods 2.1. plant material mature fruits of r. pimpinellifolia, r. villosa, r. canina, and r. dumalis were collected at the same ripening stage in two locations in ardahan province in september 2014 (table 1). the fruits were immediately transferred to the laboratory in polyethylene bags and stored   ital. j. food sci., vol 28, 2016 316 at –20°c until analysis. rosehip species have been identified based on fruit, flower and leaf of the collected genotypes as described by davis (1972). all analyses except sugar analyses were carried out in triplicate. in total, 75 fruits were used for each species, and each replicate consisted of 25 fruits. table 1: the collection areas of species. species collection areas r. pimpinellifolia l. ardahan, çıldır, gölebakan village, fields, 2010m, september 2014 r. canina l. ardahan, posof baykent village, alaybeyi located, 1950m, september 2014 r. villosa l. ardahan, posof baykent village, alaybeyi located, 1950m, september 2014 r. dumalis l. ardahan, posof gönülçalan village, gönülçalan forest, 2000m, september, 2014 2.2. fruit weight, total soluble solids, total dry matter, ph and titratable acidity ten hips of every species were weighted on a digital scale with a sensitivity of 0.01 g (tx4202l, shimadzu, japan). the seeds of hips of every species were counted (n=10). total soluble solids in ten hips of every species were determined using a digital refractometer (mettler toledo 30p, usa) and expressed in ˚brix at 22°c. the total dry matter in ten hips of every species was measured according to the aoac (1984) reference method. acidity in ten hips of every species was determined titrimetrically according to cemeroglu (1992) and expressed as a percentage of citric acid. 2.3. total anthocyanin, total phenolic content and total antioxidant capacity determination of the total anthocyanin content was done according to giusti and wrolstad (2001) with slight modifications. fresh fruits (5 g) were homogenized in 10 ml of methanol containing 1% hcl for 2 min, then kept overnight, and filtered through whatman no. 2 filter paper. two extracts were prepared, one with potassium chloride buffer, ph 1.0 (1.86 g of kcl in 1 l of distilled water), and the other with sodium acetate buffer, ph 4.5 (54.43 g of ch3co2na.3h2o in 1 l of distilled water). absorbance of the extracts was measured at 510 and 700 nm (sq2800, unico uv visible spectrophotometer, usa) after 15 min of incubation at room temperature. the content of total anthocyanin was calculated from the molar absorption of cyanide 3-glucoside. the total phenolic content was determined by the folin-ciocalteu method (spanos and wrolstad, 1992). a fruit sample (5 g) was homogenized (t18, ika homogeniser, germany) in 25 ml of ethanol and centrifuged (nf 400, nüve, turkey) at 3.500 g for 3 min. the supernatant was collected, purified by filtration through filter paper, and 2 ml of 10% folin-ciocalteu reagent was added to 0.4 ml of the extract, followed by incubation for 2-3 min. then, 1.6 ml of 7.5% na2co3 solution was added to the mix and incubated for 1 hour in the dark. absorbance was measured at 765 nm on a spectrophotometer (sq2800, unico uv visible spectrophotometer, usa) against the blank solution (0.4 ml of water, 2 ml of folin-ciocalteu reagent, and 1.6 ml of na2co3). the total amount of phenolic   ital. j. food sci., vol 28, 2016 317 compounds was calculated as a mg gallic acid equivalent (gae)/100 g by using the gallic acid standard. the ferric reducing antioxidant power (frap) assay was performed according to benzie and strain (1996). samples (1 g) were homogenized in 50 ml of 80% methanol solution in a flask wrapped in aluminum foil. the flasks were incubated in an incubator shaker (ika, germany) at 30°c and 150 g for 24 hours. the samples were centrifuged at 3.200 g for 20 min. the supernatant was collected, and 200 μl of supernatant was mixed by vortexing (ika, germany) with 3 ml of the frap reagent (300 µm acetate buffer, ph 3.6, 10 µm 2,4,6-tripyridyl-s-triazine (tptz) in 40 µm hcl, and 20 µm fecl3, 10:1:1 (v/v/v)). the samples were incubated in a water bath (st30, nüve, turkey) at 37°c for 30 min, and the absorbance was determined at 593 nm. standard curve was prepared using different concentrations of trolox and expressed in mmol trolox equivalent (te)/g frozen sample. 2.4. sugar and organic acid contents determination of sugar contents in rosehips was done according to miron and schaffer (1991) by hplc (hp agilent 1100 series, usa) using a shim-pack hrc nh2 column (300 × 7.8 mm, 5 µm) with a refractive index detector (rid). frozen samples (1 g) were powdered in liquid nitrogen with a mortar and pestle, transferred to an eppendorf tube, and 20 µl of aqueous ethanol (80%, v/v) was added. the mixture was placed in an ultrasonic bath (sonorex digital 10p, switzerland) sonicated for 15 min at 80°c, then filtered, and the procedure was repeated three times. all filtered extracts were combined and evaporated to dryness in a boiling water bath. the residue was dissolved with 2 ml of distilled water and filtered before hplc analysis. the sugar contents in the samples were calculated using calibration curves plotted by using external standards. identification of organic acids and determination of their contents were done by hplc using an hpx 87h (300 × 7.8 mm, 5 µm) column and a uv detector. for carboxylic acid and l-ascorbic acid detection, 1 g of a frozen sample was powdered in liquid nitrogen with a mortar and pestle and mixed with 20 ml of aqueous meta-phosphoric acid (3%) at room temperature for 30 min on a shaker. the acidic extract was filtered, made up to 25 ml with the same solvent, and then used for hplc analysis. external standards were used to identify and calculate organic acid contents from the retention times and calibration curves (bozan et al., 1997). 2.5. statistical analysis all results were analyzed using the spss (version 15) statistical analysis package and the mean ± standard error values obtained from triplicate measurements. data were subjected to analysis of variance (anova) and significant differences between the groups were determined by the multiple comparison procedure according to duncan (1955). differences at p<0.05 were considered significant. the cluster analysis applied to evaluate relationships among species was performed by ward’s method using euclidean distances. 3. results and discussions 3.1. pomological and biochemical characterization the fruit weights of the samples, total soluble solids, total dry matter, ph and acidity values are given in table 2. statistically significant differences (p<0.05) in these parameters between the species were determined (table 2). it was also determined that the average   ital. j. food sci., vol 28, 2016 318 fruit weights of the species varied between 9.8 g (r. dumalis) and 34.5 g (r. canina), and that the average seed numbers of 10 hips varied between 10 (r. villosa) and 23 (r. canina). the total soluble solids showed statistically significant variations among the rosehip species (table 2). the lowest total soluble solids were found in r. villosa (14 ˚brix), while the highest value was found in r. canina (22 ˚brix), followed by r. pimpinellifolia and r.dumalis (20 ˚brix). the total dry matter content of the fruits was between 40.1% (r. villosa) and 56.7% (r. canina) (table 2). demir and ozcan (2001) found that total dry matter amounts in r. canina were in the range between 20.5 and 23.5%. ercisli (2007) reported that the total soluble solids of different rosehip species growing in the erzurum region ranged between 29.4 and 37.3 ˚brix, and that the highest total soluble solids were determined in r. dumalis (37.3 ˚brix), while the lowest content was found in r. villosa. the author also found that the highest total dry matter content was shown by r. dumalis (40.4%) and the lowest total dry matter content was shown by r. villosa (29.4%). the total soluble solids of rosehip species were reported to range between 14 and 40 ˚brix in several studies carried out in different regions of turkey (sen and gunes, 1996; misirli et al., 1999; demir and ozcan, 2001). in our study, the acidity was inversely proportional to total soluble solids and ranged between 1.00% (r. canina) and 2.67% (r. villosa). the lowest ph value was observed in r. villosa (2.86), while the highest ph value was observed in r. canina (3.50). demir and ozcan (2001) demonstrated that the acidity of r. canina hips collected from two different regions was 1.17% in hadim and 1.44% in kastamonu, while the ph values were 5.12 in hadim and 4.34 in kastamonu. different rosehip species, cultivars, climate and geographical conditions are known to affect total soluble solids, acidity and ph values (ercisli, 2007). also, high altitude causes acidity levels to increase in fruits. table 2: some pomological and biochemical properties of rosehip species. different letters (a-d) for same line are statistically significantly differences among sampling dates by duncan’s multiple range test at p<0.05. 3.2. determination of total anthocyanin, total phenolic content and total antioxidant capacity the total anthocyanin, total phenolic content and total antioxidant capacity of the rosehip species are given in table 3. r. pimpinellifolia, known as a ‘black rosehip’ in the region, had species localities fruit shape flesh colour peel colour fruit weight (g) r. pimpinellifolia çıldır round purple black 17.3±0.6b r. villosa posof round orange red 10±0.1c r. canina posof elliptic orange red 34.5±0.9a r. dumalis posof elliptic orange red 9.8±0.2c species average seeds/1 hip total soluble solids (˚brix) total dry matter (%) acidity (%) ph r. pimpinellifolia 11±0.9ab 20±0.9ab 55.3±2.5a 1.30±0.10b 3.00±0.05b r. villosa 10±1.1ab 14±0.8b 40.1±6.7b 2.67±0.09a 2.86±0.04c r. canina 23±0.7a 22±1.4a 56.7±5.5a 1.00±0.02b 3.50±0.05a r. dumalis 18±0.8a 20±1.6ab 55.9±8.2a 1.45±0.02ab 3.06±0.01b   ital. j. food sci., vol 28, 2016 319 the highest anthocyanin content (3.72 mg/100 g), whereas r. dumalis and r.villosa had the lowest values (2.43 and 2.45 mg/100 g, respectively). it was previously reported that the major anthocyanin in r. canina fruits was cyanidin-3-o-glucoside (guimaraes et al., 2013). guerrero et al. (2010) found that the total anthocyanin content in rosehip fruits was 0.38 mg/100 g, and the total phenolic content was 145.7 mg/100 g. anthocyanins give color to fruits and they have therapeutic and antioxidant activity. cyanidin-3-o-glucoside was reported to have the highest oxygen radical scavenging effect (wang et al., 1997). in our study, the lowest total phenolic content was found in r. pimpinellifolia (1081 mg gae/100 g), and the highest content was found in r. canina (6298 mg gae/100 g). various researchers determined that the amounts of total phenolic compounds were between 176–9600 mg gae/100 g in ripe rosehips (ercisli, 2007; su et al., 2007; egea et al., 2010; fattahi et al., 2012; roman et al., 2013). similar to our data, yoo et al. (2008) found the total phenolic content in rosehips to be 815.5 mg gae/100 g, and fattahi et al. (2012) reported it to be 176.48–225.65 mg gae/100 g. demir et al. (2014) detected the highest total phenolic content among rosehip samples collected in gumushane, turkey in r. dumalis subsp. boissieri (5200 mg gae/100 g) and the lowest total phenolic content in r. canina (3100 mg gae/100 g). the total phenolic content results obtained in our study were found to be higher than those reported in the literature. the differences may be due to different extraction methods, the ripening stage of the hips, environmental conditions, the harvest season, altitude or plant genotype. the frap (ferric reducing antioxidant power) method was developed by benzie and strain (1996) and is based on the reduction by antioxidants of fe3+ complexed by tptz (tripyridyl triazine) to fe2+ in a low-ph environment. the results showed that there were statistically significant differences (p<0.05) in the total antioxidant capacities between the rosehip species. r. pimpinellifolia was found to have the lowest antioxidant capacity (10.04 mmol te/g), and r. canina was found to have the highest antioxidant capacity (97.95 mmol te/g). the values found in our study were lower than those found by demir et al. (2014). the authors reported that the total antioxidant capacity of r. dumalis subsp. boissieri was 194.36 mmol te/g and that of r. canina was 103.56 mmol te/g. these differences may be due to factors such as the geographical area, the degree of ripening, climate conditions and experimental conditions. cunja et al. (2015) reported that the highest antioxidant capacity was observed in r. canina fruits harvested in september and that frost damage occurring in the following months decreased antioxidant capacity. in addition, it was shown that antioxidant capacities of r. canina fruits ranged from 63.35 to 127.8 μm te/100 g as determined by the dpph method.   ital. j. food sci., vol 28, 2016 320 table 3: total anthocyanin, total phenolic content, total antioxidant capacity (frap), organic acid and sugar contents of rosehip species. sugars organic acids r. pimpinellifolia r. villosa r. canina r. dumalis total anthocyanin (mg/100g) 3.72±0.06a 2.45±0.03c 2.75±0.07b 2.43±0.09c total phenolic (mg gae/100g) 1081±12.8 d 2944±70.8c 6298±116.7a 4411±16.9b frap (mmol te/g) 10.04±0.47d 37.84±1.55b 97.95±2.12a 26.45±6.98c l ascorbic acid (mg/100g) 24.93±4.0d 119.83±3.3c 754.48±100.2a 254.81±12.5b sucrose (g/100g) 0.38c 0.42b 0.55a 0.41b glucose (g/100g) 5.99d 12.48a 8.05b 6.79c fructose (g/100g) 4.38c 4.90b 5.03a 4.15d sorbitol (g/100g) 4.17c 6.25a 5.15b 3.94d total sugar(g/100g) 14.92d 24.05a 18.78b 15.29c oxalic acid (g/100g) 0.14±0.01c 0.25±0.02b 0.38±0.04a 0.29±0.01b tartaric acid (g/100g) 0.21±0.08c 0.26±0.03c 0.65±0.13a 0.30±0.01b malic acid (g/100g) 0.65±0.04b 0.45±0c 0.48±0.06c 0.73±0.16a citric acid (g/100g) 0.48±0.05c 1.05±0.05a 0.94±0.14b 0.95±0.3b succinic acid (g/100g) 0.092±0.01a 0.007±0c 0.010±0b 0.006±0c fumaric acid (g/100g) 0.015±0b 0.011±0c 0.033±0.01a 0.014±0b different letters (a-d) for same line are statistically significantly differences among sampling dates by duncan’s multiple range test at p<0.05.   ital. j. food sci., vol 28, 2016 321 3.3. l-ascorbic acid, sugar and organic acid contents sugar and organic acid contents are the most important factors determining fruit quality and taste. organic acids increase bioavailability of ascorbic acid by inhibiting ascorbic acid oxidation (padayatty and levine, 2001; kobus et al., 2005). in this study, there were significant differences in l-ascorbic acid, sugar and organic acid contents between the rosehip species, as presented in table 3. the l-ascorbic acid contents of the species were found to range between 24.93 mg/100 g (r. pimpinellifolia) and 754.48 mg/100 g (r. canina). the l-ascorbic acid values obtained in our study were higher than those reported in the literature. roman et al. (2013) revealed that the ascorbic acid contents in ripe rosehips ranged between 112.2 and 360.2 mg/100 g. barros et al. (2010) found the ascorbic acid content in r. canina to be 68.04 mg/100 g. nojavan et al. (2008) determined that the ascorbic acid content increased upon ripening to 417.5 mg/100 g in rosehip species and that the value was 6-fold of that found in oranges. demir et al. (2014) determined that the ascorbic acid content was lowest in r. dumalis (65.75 mg/100 g) and highest in r. gallica (160.30 mg/100 g). in addition, celik et al. (2009) found the ascorbic acid contents in rosehip species in van, turkey to be 604-1.032 mg/100 g. it was also shown that there were significant differences between rosehip species in ascorbic acid content, which could be affected by ecologic factors, the degree of ripening and soil conditions (mabellini et al., 2011; adamczak et al., 2012). rosehip species growing in high altitude regions are rich in ascorbic acid due to higher light exposure and lower oxygen amounts. light exposure increases the amount of carotene and thus protects ascorbic acid in the fruit, while the lack of oxygen reduces oxidative stress and lessens ascorbic acid breakdown (yamankaradeniz, 1983). ascorbic acid contents of rosehips vary depending on climate conditions, fruit types and years (demir and ozcan, 2001). glucose was found to be the most common sugar in rosa species, and the lowest glucose content was found in r. pimpinellifolia (5.99 g/100 g) while the highest content was found in r. villosa (12.48 g/100 g). the amounts of sucrose ranged in the species between 0.38– 0.55 g/100 g (r. pimpinellifolia and r. canina, respectively), the fructose contents ranged between 4.15-5.03 g/100 g (r. dumalis and r. canina, respectively), and the sorbitol contents ranged between 3.94-6.25 g/100 g (r. dumalis and r. villosa, respectively). also, the lowest total sugar amount was found in r. pimpinellifolia (14.92 g/100 g) while the highest amount was found in r. villosa (24.05 g/100 g). other studies reported glucose contents in rosehip fruits to range between 7.45-12.94 g/100 g and fructose contents to range between 7.96-18.44 g/100 g. similar to our results, sucrose contents were reported to range between 0.88-5.61 g/100 g and total sugar contents were reported to range between 12.05-20.46 g/100 g (yoruk et al., 2008; barros et al., 2011; rosu et al., 2011; ozrenk et al., 2012). likewise, demir et al. (2014) revealed glucose amounts in rosa species to range between 9.54 g/100g (r. dumalis) and 17.25 g/100g (r. gallica) and fructose amounts to range between 10.78 g/100g (r. dumalis) and 18.84 g/100g (r. canina). the fructose and sucrose values obtained in our study were found to be lower than those reported in the literature, whereas the total sugar amounts were found to be higher. the differences in organic acid and sugar values between our and other studies might be due to different soil and climate conditions of the region and the differences in experimental analysis. also, differences in the harvest season are thought to affect the results. the major organic acid in the rosa species was citric acid (0.48 to 1.05 g/100 g). in this study, we found that oxalic acid was most abundant in r. canina (0.38 g/100 g) and least abundant in r. pimpinellifolia (0.14 g/100 g). fumaric acid was also most abundant in r. canina (0.033 g/100 g) and least abundant in r. villosa (0.011 g/100g). the tartaric acid values among the species were 0.21-0.65 g/100 g, the malic acid values were between 0.45 and 0.73 g/100 g, and the succinic acid values were between 0.006-0.092 g/100 g. in a   ital. j. food sci., vol 28, 2016 322 previous study, the citric and malic acid amounts in rosa species were 4.76-9.12 g/100 g and 0.45-1.10 g/100 g, respectively (demir et al., 2014). adamczak et al. (2012) found that the citric acid content in r. tomentosa was 4.34 g/100 g. thus, the organic acid values obtained in our study were lower compared to those found in previous studies. a dendogram based on the pomological and biochemical characteristics studied of the rosehip species can be seen in fig. 1. figure 1: cluster analyse of rosehip species according to their pomological and biochemical properties. the species were grouped into 2 main clusters. in the first cluster, r. villosa and r. dumalis were found to be the closest species based on the characteristics analyzed. fruit weights, total anthocyanin contents and succinic acid contents of both species were low. r. pimpinellifolia was found in the same cluster, while r. canina fell in a separate cluster, for its characteristics were different from those of the other species. this study aimed to determine and compare some important bioactive compounds and biochemical features of 4 different rosehip species growing naturally in ardahan (eastern anatolia, turkey) and mostly consumed by the locals. the differences in acidity and sugar contents of the species, compared with previous studies, are thought to be due to different altitudes. moreover, it was found that the l-ascorbic acid, total anthocyanin and total phenolic content values, known to increase with altitude, were high in this study. the total antioxidant capacities of these species were also high. this study is important as a foundation for further research. besides, knowing biochemical characteristics of the species will facilitate the work of plant breeders and growers. it is known that bioactive components of fruits positively affect health. it is suggested that rosehip fruits are good sources of bioactive compounds and phytonutrients. their consumption may prevent some illnesses and protect health. acknowledgements this research was supported by a grant (2012/07) from ardahan university.   ital. j. food sci., vol 28, 2016 323 references aoac. 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turker m., kazankaya a., erez m.e., battal p. and celik f. 2008. fatty acid, sugar and vitamin contents in rose hip species. asian j. chem. 20 (2): 1357. paper received june 22, 2015 accepted october 18, 2015 paper 142 ital. j. food sci., vol. 27 2015 keywords: metabolomics, nmr, wholegrain, rye, wheat, metabolites, biomarkers metabolomics study of cereal grains reveals the discriminative metabolic markers associated with anatomical compartments m. coulomb1, a. gombert1,2 and a.a. moazzami1,3* 1department of chemistry and biotechnology, swedish university of agricultural sciences, p.o. box 7015, se 75007, uppsala, sweden 2department of food science, swedish university of agricultural sciences, p.o. box 7051, se 75007, uppsala, sweden 3nmr-metabolomics core facilities, uppsala biocentrum, swedish university of agricultural sciences, uppsala, sweden *corresponding author: tel. +46 18672048, fax +46 18672995, email: ali.moazzami@slu.se abstract this study used nmr-based metabolomics to compare the metabolic profile of different anatomical compartments of cereal grains i.e. bran and endosperm in order to gain further insights into their possible role in the beneficial health effects of whole grain products (wg). polar watersoluble metabolites in 64 bran and endosperm, samples from rye and wheat were observed using 600 mhz nmr. bran samples had higher contents of 12 metabolites than endosperm samples. a comparative approach revealed higher contents of azelaic acid and sebacic acid in bran than in endosperm. in a pilot study, the consumption of wg rye bread (485 g) caused nmr signals in 24h urine corresponding to azelaic acid. the relatively high abundance, anatomical specificity, pattern of metabolism, urinary excretion in human, antibacterial, and anticancer activities suggest further studying of azelaic acid when exposure to wg or beneficial effects of wg are investigated. mailto:ali.moazzami%40slu.se?subject= ital. j. food sci., vol. 27 2015 143 introduction epidemiological studies have consistently shown that intake of whole grain (wg) can protect against the development of chronic diseases (slavin et al. 2001), e.g. type 2 diabetes (t2d) (de munter et al. 2007, murtaugh et al. 2003), cardiovascular disease (cvd) (flint et al. 2009; jacobs et al. 2007; mellen et al. 2008), and certain cancers (chan et al. 2007, haas et al. 2009; larsson et al. 2005; schatzkin et al. 2008). the american association of cereal chemists provided the following scientific and botanical definition of wg in 1999: “whole grain shall consist of the intact, ground, cracked or flaked caryopsis, whose principal anatomical component-the starchy endosperm, germ and bran-are present in the same relative proportion as they exist in the intact caryopsis” (international 1999). whole grains are a rich source of fiber and bioactive compounds, including tocopherols, b vitamins, minerals, phenolic acids, and phytoestrogens (fardet, 2010). it is generally recognized that the synergistic action of compounds mainly present in the bran and germ fractions of cereals accounts for the protective effects of wg products (fardet, 2010; liu, 2007). recently, the composition and the diversity of bioactive compounds in different anatomical components of cereal grains have been systematically investigated in a large number of different species and varieties within the healthgrain project (nystrom et al. 2008; shewry et al. 2010; ward et al. 2008). however, that project screened the cereal samples for bioactive compounds already documented in cereals using a targeted approach, and made no comparison of the untagged profile of the metabolites in different compartments of cereal grains. metabolomics is an untargeted approach in which the profile of metabolites in a biospecimen is measured using high-throughput analytical methods, e.g. nmr and mass spectrometry (lenz and wilson, 2007; nicholson and wilson 2003). we have used this approach previously to examine the complex physiological/biochemical effects of wg rye products in humans (moazzami et al. 2012; moazzami et al. 2014; moazzami et al. 2011). the aim of the present study was to search for the discriminative metabolites in the two major anatomical compartments in cereal grain, endosperm and bran, using an untargeted nmr-based metabolomics approach and with the emphasis on wheat and rye to gain further insights into their possible role in the beneficial health effects of whole grain products. nmr analysis can potentially provide characteristic structural data, which can be used for elucidation and eventual identification of unknown compounds found to discriminate between the metabolic profiles of bran and endosperm in cereals. materials and methods serial sample collection and extraction a total of 64 cereal samples, comprising 18 wheat endosperm, 24 wheat bran, 8 rye endosperm, and 14 rye bran were obtained from the healthgrain (ward et al. 2008) project or from a local market. the endosperm and bran samples originated from healthgrain projects were from the same grain sample material and therefore were matched (wheat samples n = 18; and rye samples n = 8). the healthgrain project rye varieties (and populations) included potugaise-3, potugaise-6, haute loire, grandrieu, nikita, rekrut, dankowskie-zlote, and lovaszpatonai-1. the details about rye varieties are given in nystrom et al. (2008). the healthgrain project wheat varieties included disponent, herzog, tommi, campari, tremie, san pastore, gloria, spartanka, avalon, claire, malacca, maris huntsman, rialto, riband, obriy, cf99105, chinese-spring, and cadenza. the details about wheat varieties are given by shewry et al. (2010). all rye and wheat varieties were grown in the field at martonvasar, hungary, in 2005. full details of the site including soil type, mineral composition, and weather condition has been given by shewry et al. (2010). all samples were milled, and 0.5 g milled material was extracted in 5 ml milli-q water for 18 h. the samples were then centrifuged (5 min1500 g), and 2 ml supernatant was extracted, mixed with 8 ml ethanol and centrifuged (15 min-1,500 g) in order to precipitate the soluble viscose polymers. a 5 ml portion of the ethanol supernatant was dried using an evacuated centrifuge (savant, svc 100h, savant instrument inc, nj) and dissolved in phosphate buffer (280 µl, 0.25 mol/l, ph 7.0), d2o (40 µl), and sodium-3-(trimethylsilyl)-2,2,3,3-tetradeuteriopropionate solution (tsp, 30 µl, 23.2 mmol/l) (cambridge isotope laboratories, andover, ma). the mixture was then used for 1h nmr analysis. an internal standard was added to the mixture in order to ensure semi-quantitative measurements of metabolites captured by 1h nmr. for 2d nmr analysis the mixture was freeze-dried and dissolved in d2o before analysis. human experiment and the preparation of urine sample for nmr analysis in a pilot study, a male subject (age 35; bmi = 23.4) consumed refined wheat bread 485 g for 6 days (breakfast 2 portions, lunch 1 portion, dinner 1 portion). on day six, 24-hour urine was collected. on day seven, he substituted the 485 g refined wheat bread with 485 g of whole grain rye bread and the urine was collected for 24 hours. during the seven days of experiment, any other cereal products were avoided. the choice of consuming refined wheat bread vs whole grain rye bread was made to 144 ital. j. food sci., vol. 27 2015 replicate the condition of previous human interventions in which refined wheat bread was used as the control diet (moazzami et al. 2011; moazzami et al. 2012; bondia-pons et al. 2013). the refined wheat bread was prepared from commercial refined wheat flour and whole grain rye bread was prepared from commercial whole grain rye flour. the whole trial was repeated twice, in two different times. this study complied with the helsinki declaration, as revised in 1983. the urine samples were kept in -80°c freezers before analysis. the urine samples (500 µl) were mixed with phosphate buffer (250 µl, 0.25 m, ph 7.0) containing 5 mmol/l sodium-3-(trimethylsilyl)-2,2,3,3-tetradeuteriopropionate (tsp) (cambridge isotope laboratories, andover, ma) as an internal standard. resulting solutions were centrifuged to remove particulate matter. the supernatant was then transferred into 5-mm nmr tubes for 1h nmr analysis. for 2d-nmr analysis, 600 µl of the supernatant was freeze-dried and dissolved in 600 µl d 2 o before 2d-nmr analysis. nmr measurements and the identification of signals the 1h nmr analyses (cereal extracts and human urine) were performed on a bruker spectrometer operating at 600 mhz (karlsruhe, germany). 1h nmr spectra were obtained using zgesgp pulse sequence (bruker spectrospin ltd.) at 25°c with 128 scans and 65,536 data points over a spectral width of 17942.58 hz. acquisition time was 1.82 s and relaxation delay was 4.0 s. the nmr signals which were found discriminating between different anatomical compartments were identified primarily using the nmr suite 7.1 library (chenomx inc, edmonton, canada), human metabolome data base and biological magnetic resonance data bank. in the event of multiplicity, the identity was confirmed with 2d nmr. in human experiment, the identity of phytochemical in the urine originating from the cereals in the diet was also confirmed using 2d-nmr. phasesensitive tocsy and cosy with presaturation (2k × 512 experiments) were performed with 32 scans and a spectral width of 7195 hz for both f1 and f2. the mixing time for tocsy was 80 ms. hsqc was performed using 32 scans and a spectral width of 7211 hz and 250002 hz for proton and carbon, respectively. all cereal extracts and urine samples were reconstituted in d2o before 2d nmr analysis. the 1h nmr spectra data (cereal extracts) were processed using bruker topspin 1.3 software and were fourier-transformed after multiplication by a line broadening of 0.3 hz and referenced to tsp at 0.0 ppm. spectral phase and baseline were corrected manually. each spectrum was integrated using amix 3.7.3 (bruker biospin gmbh, rheinstetten) into 0.01 ppm integral regions (buckets) between 0.5-10 ppm, in which area between 4.60-5.18 ppm containing residual water was removed. each spectral region was then normalized to the intensity of internal standard (tsp). statistical analysis principal component analysis (pca) and orthogonal partial least squares-discriminant analysis (opls-da) were performed using simca-p+ 12.0.1 software (umetrics, umeå, sweden) after centering and pareto-scaling of the data as previously described (moazzami et al. 2011). the presence of outliers was investigated using pca-hotelling t2 ellipse (95% ci) and the normality of multivariate data was investigated using the normal probability plot of the pca model. variable influences on projection (vip) values of the opls-da model were used to determine the most important discriminative nmr bucket (signals). nmr buckets (signals) with vip > 1 for which the corresponding jack-knife-based confidence intervals were not close to or including zero were considered discriminative. the significance of opls-da model was tested using cross-validated anova (cvanova), which assesses the reliability of opls models (cv-anova p<0.05 means the oplsda model is reliable) (eriksson et al. 2008). the absolute concentrations of metabolites with corresponding nmr signals that were found to be discriminative in opls-da were calculated from the nmr spectra using the nmr suite 7.1 profiler (chenomx inc, edmonton, canada) and internal standard after correction for overlapping signals. the absolute concentrations of the discriminative metabolites were further investigated using anova in the case of normal distribution, and the mann-whitney test when the distribution was skewed (anderson-darling test, p<0.05). results and discussion pca model was fitted using nmr spectral data (buckets) obtained for the bran and endosperm extracts. three outliers were identified and excluded from the data set based on pca-hotelling t2 ellipse (95% ci). the first and the second component explained 67.4% and 18.1% of spectral variation (r2x) respectively (figure not shown). an opls-da model was fitted including three predictive and six orthogonal components. the first, second, and third predictive components explained 61%, 15.2%, and 1.0% of spectral variation respectively (model parameter: r2y=0.937; q2y=0.876; cross-validated anova p-value = 2.98 × 10-38) (fig. 1). the first component in each model basically separated the bran samples obtained from rye ital. j. food sci., vol. 27 2015 145 fig. 1 opls-da separated different anatomical compartments of cereal grains based on their profile of metabolites measured using nmr. the model parameters were as follow: r2y=0.937; q2y=0.876. crossvalidated anova p-value = 2.98 × 1038. score t[1] (component 1) and score t[2] (component 2) are new variables summarizing the x-variables (the intensity of nmr signals corresponding to metabolites). table 1 discriminative metabolites along the first predictive component of the opls-da model (n = 64)1,2. metabolite nmr signal (ppm)3 vip (confidence interval)4 azelaic acid & sebacic acid5 1.304 ; 1.530 ; 2.179 2.6 (0.67) ; 1.6 (0.41) ; 1.6 (0.40) acetate 1.928 1.3 (0.26) alanine 1.494 1.1 (0.08) betaine 3.269 6.9 (0.59) choline 3.206; 4.085 4.4 (0.46); 2.3 (0.24) citrate 2.545 1.4 (0.13) isoleucine 0.942 1.0 (0.18) leucine 0.964 1.4 (0.24) malate 2.663 1.1 (0.31) maltose 3.280; 3.727; 3.997 1.4 (0.24); 4.4 (0.49) succinate 2.412 1.3 (0.16) unknown signals6 3.778; 4.054; 4.155; 4.020 2.3 (1.07); 2.3 (0.18); 2.2 (0.23); 4.6 (0.32) 1opls-da score scatter plot: the first component separated the bran samples (right) from the endosperm samples (left). all metabolites present in higher concentrations in bran. the model parameters for three predictive component fitted were as follow: r2y=0.937; q2y=0.876. cross-validated anova p-value = 2.98 × 10-38; 2wheat endosperm (n = 18), wheat bran (n = 24), rye endosperm (n = 8), and rye bran (n = 14); 3one nmr signal from the corresponding spectral bucket with the highest vip values was reported when several buckets covered a distinct nmr signal; 4nmr signals with vip > 1 for which the corresponding jack-knife-based confidence intervals were not close to or including zero were considered discriminative; 5concentration equivalent of azelaic acid; 6unknow signals are located in sugar region. and wheat from the endosperm samples (fig. 1, table 1). the second component separated rye samples (both endosperm and bran samples) from wheat samples (fig. 1; table 2). bran samples contained a higher content of 12 metabolites and four unknown signals than endosperm samples (table 1), and their contents contributed to composing the first predictive component of the opls-da model. the content of eight metabolites and five unknown signals changed along the second predictive component of the opls-da model (table 2) separating wheat samples i.e. both anatomical compartments from rye samples. the concentrations of all eight metabolites were found higher in wheat compared with rye. the metabolic signature of wheat and rye samples acquired from the local market did not deviate from those acquired from healthgrain project as all sample tightly accumulated in their corresponding species-compartment cluster (fig. 1). the absolute concentrations of metabolites that were found to differ between different anatomical compartments and species were calculated from nmr spectra and further investigated using anova or the mann-whitney test. a total of 12 metabolites were found to differ between different species and different anatomical compartments in the same species, e.g. bran compared vs endosperm (table 3; fig. 2). 146 ital. j. food sci., vol. 27 2015 table 2 discriminative metabolites along the second predictive component of the opls-da model (n = 64)1,2. metabolite loading3 nmr signal (ppm)4 vip (confidence interval)5 azelaic acid & sebacate6 1.304; 1.545; 2.179 3.3 (0.34); 2.1 (0.25); 1.9 (0.20) betaine 3.269; 3.904 5.2 (0.84); 3.1 (0.39) choline 3.206; 4.085 3.5 (0.42); 1.9 (0.15) citrate 2.545 1.0 (0.18) leucine 0.964 1.1 (0.16) maltose 3.278; 3.727; 5.257 6.1 (0.92); 3.2 (0.49); 1.0 (0.78) succinate 2.414 1.1 (0.23) unknown signals7 + 3.778; 4.054; 4.155; 3.915; 4.043 3.7 (0.75); 1.7 (0.25); 1.6 (0.21); 2.4 (0.46); 2.7 (0.32) 1opls-da score scatter plot: the second component separated the wheat samples (below) from the rye samples (above). the model parameters for three predictive component fitted were as follow: r2y=0.937; q2y=0.876. cross-validated anova p-value = 2.98 × 10-38; 2wheat endosperm (n = 18), wheat bran (n = 24), rye endosperm (n = 8), and rye bran (n = 14); 3loadings: (+): higher concentration in rye samples. (-): higher concentration in wheat samples; 4one nmr signal from the corresponding spectral bucket with the highest vip values was reported when several buckets covered a distinct nmr signal; 5nmr signals with vip > 1 for which the corresponding jack-knife-based confidence intervals were not close to or including zero were considered discriminative; 6concentration equivalent of azelaic acid; 7unknow signals are located in sugar region. table 3 absolute concentrations of metabolites (µmol/g) found to be discriminative along the first and second predictive components1. concentration µmol/g (mean ± sd) metabolite 1 : rye endosperm 2 : rye bran 3 : wheat endosperm 4 : wheat bran azelaic acid & sebacic acid 0.68 ± 0.21a 1.70 ± 0.27a 0.70 ± 0.16a 4.32 ± 1.25b acetate 1.19 ± 0.26a 3.98 ± 4.08b 0.73 ± 0.32c 2.70 ± 0.91d alanine 0.40 ± 0.07a 1.81 ± 0.62b 0.38 ± 0.13a 1.25 ± 0.47 betaine 10.52 ± 2.80a 28.23 ± 6.77b 3.70 ± 2.13c 34.53 ± 9.79d choline 0.78 ± 0.13a 6.70 ± 1.09b 1.12 ± 0.25c 6.91 ± 1.18d citrate 0.55 ± 0.05a 5.25 ± 1.55b 0.62 ± 0.29c 4.93 ± 1.72b isoleucine 0.15 ± 0.03a 0.58 ± 0.21b 0.16 ± 0.03a 0.48 ± 0.13b leucine 0.37 ± 0.10a 1.52 ± 0.48b 0.35 ± 0.08a 1.40 ± 0.35b malate 6.04 ± 0.85a 6.22 ± 3.21b 7.43 ± 2.73a 10.24 ± 5.47a maltose 17.22 ± 0.182a 24.35 ± 8.46b 0.86 ± 1.52c 21.14 ± 7.27b succinate 0.54 ± 0.08a 1.34 ± 0.70b 0.43 ± 0.15a 1.43 ± 0.47a 1anova was performed for betaine, succinate, citrate, alanine, leucine, isoleucine, and maltose. mann-whitney test was performed for malate, acetate, and choline. metabolite means followed by different letters are significantly different (p<0.05). (mean ± sd). fig. 2 (a) a typical 1h nmr spectrum from rye bran polar extract and (b) magnified region 0.5 3.0 ppm. annotated metabolites: leucine (1), isoleucine (2), azelaic acid and sebacic acid (3), alanine (4), acetate (5), malate (6), succinate (7), citrate (8), choline (9), betaine (10) and sugar region (11). ital. j. food sci., vol. 27 2015 147 multivariate statistical analysis (opls-da model) also included signals discriminating between bran and endosperm, which appeared as a multiplet at 1.304 ppm, a multiplet at 1.545 ppm, and a triplet at 2.179 ppm (fig. 2; table 1). using 2d nmr and spiking with authentic standard, these signals were assigned to two saturated, straight-chain dicarboxylic acids, namely azelaic acid (c9h16o4) and sebacic acid (c10h18o4). tocsy nmr indicated that these signals were in the same spin system (fig. 3). cosy nmr also confirmed coupling between (-ch2-) signals at 1.304 ppm and 1.545 ppm, and between (-ch2-) signals at 1.545 ppm and 2.179 ppm. no coupling to a ch3 group was observed on the tocsy and cosy spectra, confirming dicarboxylic structure. the carbon chemical shifts were assigned from coupling to the corresponding hydrogen in hsqc nmr. there was a cross-peak between protons at 1.304 ppm and carbon at 31.484 ppm, between protons at 1.545 ppm and carbon at 28.926 ppm, and between protons at 2.179 ppm and carbon at 40.735 ppm. after applying new processing consisting of lining broadening (-1) and gaussian broadening (0.6), the triplet at 2.179 appeared to be two overlapping triplets from azelaic acid and sebacic acid, the chemical shifts of which deviated from each other by 1.54 hz. the identity of azelaic acid was further confirmed using authentic standard. the molar concentration of total dicarboxylic acids (azelaic + sebacic acid) was then calculated and expressed as equivalent to azelaic acid (table 3). no signal corresponding to azelaic acid was detectable in the 24h urine of a male subject affig. 3 tocsy nmr spectrum of typical rye bran polar extract presenting coupling between a multiple at 1.304 ppm (a), a multiple at 1.545 ppm (b), and a triplet at 2.179 ppm (c), which belong to azelaic acid and sebacic acid, and the assignment of the corresponding -ch2groups on azelaic acid molecule. 148 ital. j. food sci., vol. 27 2015 ter the consumption of refined wheat bread (485 g). however, after the consumption of whole grain rye bread (485 g), nmr signals corresponding to azelaic acid with similar coupling pattern as azelaic acid in the bran extract were detected in 24h urine (fig. 4). the concentrations of azelaic acid and sebacic acid were found to be higher in wheat and rye bran compared with the corresponding endosperm. in addition, wheat bran had higher dicarboxylic acid contents than rye bran (table 3). to our knowledge this is the first study to report comparative differences in these dicarboxylic acids in different anatomical compartments of wheat and rye. in humans, 60 and 17 % of the administered dosage of azelaic acid and sebacic acid, respectively, are excreted in the urine within the first 12 hours (passi et al. 1983). it has been suggested that the dicarboxylic acids are to some extent subjected to β-oxidation, since dicarboxylic acids found in serum and urine possess 2, 4, or 6 carbon atoms shorter than the corresponding administered dicarboxylic acids (passi, nazzaro-porro, picardo, mingrone and fasella 1983). recently bondia-pons et al. using metabolomics approach have shown that azelaic acid beside alkylresorcinols metabolites and enterolactone are the most discriminate metabolites, and are found in higher concentration urine after the intervention with whole grain rye bread compared with refined wheat bread (bondia-pons et al. 2013). the present study showed that the main source of azelaic acid detected in the urine is bran, and that azelaic acid is found in both wheat and rye. the relatively high abundance, anatomical specificity and localization in bran, pattern of metabolism, and previous findings regarding the identification of azelaic acids as discriminative metabolite in the urine after whole grain vs refined grain consumption (bondia-pons et al. 2013) suggest that these dicarboxylic acids can be further investigated as biomarkers of exposure to wg products. further studies are needed to investigate the correlation between azelaic acid concentration and alkylresorcinols concentration, which are validated biomarkers of wg intake (ross 2012) in different biofluids after the intake of whole grain products. little is known about the possible metabolic effects of the dicarboxylic acids in mammals. however, azelaic acid is known for its antibacterial (yu and van fig. 4 tocsy nmr spectrum of 24 hour urine of a 35 year old man after the consumption of 485 g whole grain rye bread. the pattern of coupling between signals at 1.304 ppm (a), at 1.545 ppm (b), and at 2.179 ppm (c) was similar to that observed in rye bran polar extract. ital. j. food sci., vol. 27 2015 149 scott 2004) and anticancer activities (manosroi et al. 2007), which might contribute to the benefits attributed to wg intake. three amino acids i.e. alanine, isoleucine, and leucine, were also present in higher concentrations in bran than in endosperm. these amino acids gave rise to sharp and distinct nmr signals, distinguishing them from the amino acids in proteins, which possess broad nmr signals. in addition, higher contents of succinate, citrate, and malate were observed in bran than in endosperm. these metabolites are associated with the citric acid cycle and central carbon metabolism. the higher levels of amino acids and citric acid may indicate higher metabolic activities in bran compared with endosperm. consistent with previous studies, we observed higher levels of betaine and choline in bran than in endosperm (bruce et al. 2010). in addition, wheat bran had higher levels of betaine than rye bran. circulating betaine is reported to be increased postprandially in animal models (bertram et al. 2009; yde et al. 2012) and in fasting plasma of humans after a 6-8 week intervention with wg rye products (moazzami et al. 2011; moazzami et al. 2012). betaine acts as a methyl donor in the betaine-homocysteine methyl transferase reaction (bhmt-r), which converts homocysteine and betaine to methionine and n,n-dimethylglycine (delgado-reyes and garrow 2005). recently, in two separate human studies, we observed an increase in bhmt-r, as indicated by higher n,n-dimethylglycine levels (moazzami et al. 2011; moazzami et al. 2012) and lower homocysteine levels (moazzami et al. 2011), after a 6-8 week intervention with wg rye products compared with refined wheat products, which highlights the metabolic effects of betaine located in the bran of cereals. in the present study, we used nmr-based metabolomics as an untargeted approach to gain further insights into the metabolic profile of different anatomical compartments of cereal grains. nmr profiling covers metabolites with µmol/g concentration. nmr also proved useful for identification and structural determination of unknown metabolites associated with different anatomical compartments. abbreviations bhmt, betaine homocysteine methyl transferase; bhmtr, betaine-homocysteine methyl transferase reaction; cvd, cardiovascular disease; opls-da, orthogonal partial least squares-discriminant analysis; pca, principal component analysis; t2d, type 2 diabetes; wg, whole grains. acknowledgements we thank the healthgrain project for providing us with wheat and rye samples. we would like to thank the dr. håkansson foundation for supporting the study. we also acknowledge dr. peter agback’s help in interpretation of 2d nmr spectra, and dr. annica andersson for coordinating the present study with healthgrain project. this study was sponsored by dr. håkansson foundation. m.c. and a.a.m. wrote the manuscript. m.c. performed the nmr analysis and metabolomics data analysis. a.g. performed 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boros d., rakszegi m., bedo z. and shewry p.r. 2008. the healthgrain cereal diversity screen: concept, results, and prospects. journal of agricultural and food chemistry 56(21), 9699-9709. yde c.c., westerhuiis j.a., bertram h.c. and bach knudsen k.e. 2012. application of nmr-based metabonomics suggests a relationship between betaine absorption and elevated creatine plasma concentrations in catheterised sows. br. j. nutr. 107(11), 1603-1615. yu r.j. and van scott e.j. 2004. alpha-hydroxyacids and carboxylic acids. journal of cosmetic dermatology 3(2), 76-87. paper received january 23, 2014 accepted june 23, 2014 p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33i1.1964 46 p u b l i c a t i o n s codon chemical characterization of ‘pecorino di farindola’ cheese during ripening serena niro, alessandra fratianni, annacristina d’agostino, ivan notardonato and gianfranco panfili dipartimento di agricoltura, ambiente e alimenti, università degli studi del molise, via f. de sanctis, 86100 campobasso, italy *corresponding author: allesandra fratianni, dipartimento di agricoltura, ambiente e alimenti, università degli studi del molise, via f. de sanctis, 86100 campobasso, italy. email: fratianni@unimol.it submitted: 28 september 2020; accepted 13 january 2021; published 01 february 2021 © 2021 codon publications open access paper abstract this study evaluated the nutritional and sensorial characteristics of pecorino di farindola cheese at different commercial ripening stages. moreover, in order to assess effectively the peculiar features of this product, the evolution of proteolysis and lipolysis, together with that of free amino acids (faas), was studied throughout ripening. a marked proteolysis of pecorino di farindola was found. at the end of ripening, faas with the highest content were glutamic acid, valine, leucine and lysine. long-ripened cheeses had a light spicy feature that distinguishes them from other italian pecorino cheeses. keywords: free amino acids, lipolysis, pecorino di farindola, pig rennet introduction the term ‘pecorino’ refers to a cheese obtained from ewe milk, in most cases of protected origin. in italy, however, several cheeses, even not of protected origin, have typical features and are prepared in limited geographical areas (coda et al., 2006). pecorino cheeses are often produced traditionally in central and south italy and are characterised by different ripening stages (di cagno and gobbetti, 2011; schirone et al., 2011), and the use of different milk, rennet and technology of production (gobbetti, 2016). ‘pecorino di farindola’ is a traditional food product (prodotto agroalimentare tradizionale – pat, published in the official gazette of the italian republic on 20th february 2020, general series n. 42 , ordinary supplement n. 9) and has a limited production in the eastern part of the gran sasso area, abruzzo, italy. this is an original pecorino cheese, since it is made from pig rennet that gives the cheese particular flavours and taste. the milk comes from sheep of pagliarola appenninica breed, which are bred in the wild, with a limited milk production. the cheese can be found at a short ripening time (3 months), demonstrating a soft texture and a yellow crust, or at a long ripening time (over 12 months), having a harder texture, a more intense and spicy flavour and a darker crust (schirone et al., 2011). the cheese-making process is reported by schirone et al. (2011). it starts from raw milk without the addition of natural cultures or selected starters, added with porcine rennet. the microbiota, derived mostly from mesophilic lactobacilli coming from the raw milk and the cheese-making environment, plays an important role during ripening, contributing to the development of typical aromas of this cheese (aquilanti et al., 2007; de angelis et al., 2001; tofalo et  al., 2015). the chemical and microbiological features of 10 pecorino di farindola cheeses coming from different dairies of the consortium, at 90 days of ripening, are reported by schirone et al. (2011). information about the chemical and microbiological characteristics of pecorino di farindola, together with that of proteolytic and lipolytic phenomena during its ripening process is still limited (di giacomo et al., 2013; suzzi et al., 2014; tofalo et  al., 2015). the aim of this work is to have a deeper insight into the chemical and nutritional characterization of the pecorino di farindola cheese and to evaluate its proteolysis and lipolysis at different ripening stages. italian journal of food science, 2021; 33 (1): 46–51 mailto:fratianni@unimol.it 47 italian journal of food science, 2021; 33 (1) s. niro et al. sensorial analysis each sample was evaluated for three times. samples were evaluated by a panel comprising 10 trained components. sensory evaluation was conducted according to the etana method described by bozzetti et al. (2004) modified by chiavari et al. (2006). the evaluated attributes were flavour and aroma: odour intensity, aroma intensity, hardness, solubility, sweet, salty, bitter, spicy and acidic. the definition of the descriptive attributes is reported in niro et al. (2014). samples were served at room temperature. the intensity of each attribute was rated on an increasing scale from 1 to 10 (from absence to maximum). statistical analysis an anova was applied to the data. least significant differences were obtained using the least significant difference test (p < 0.05). results and discussion the chemical composition of different pecorino cheeses (g/100 g fresh weight), at different ripening stages, is reported in table 1. the moisture value ranged from 29.6% in 3r samples to 28.5% in 5r and 23.2% in 12r samples. significant differences (p < 0.05) were found in proteins and fats, depending on different raw milk, which, in 3r and 5r samples, came from grazing sheep, while in 12r samples from sheep fed with forage. different ash values could depend on the variability of salting process. the cheese composition is in accordance with the values reported in the ‘production disciplinary’ and the ranging values are from literature for pecorino di farindola (bellocci et al., 2018). schirone et al. (2011) and tofalo et al. (2015) have reported lower fat values in several pecorino di farindola cheeses. different pecorino cheeses were found to have similar or higher fat contents (coda et al., 2006; di cagno et al., 2007). the proteolysis and lipolysis indices are shown in table 2. the value of sn/tn% increased significantly during ripening, ranging from 32.5% at 3 ripening months (3r) materials and methods sample collection and preparation three batches of pecorino di farindola cheeses were analysed. the three batches were different according to ripening times: three samples were ripened for 3 months (3r), three for 5 months (5r) and three for 12 months (12r). samples came from a dairy in the area of pecorino of farindola, which includes nine towns located within the provinces of pescara and teramo (reported in the ‘production disciplinary of pecorino di farindola’https:// www.pecorinodifarindola.it/disciplinare/). all cheeses were produced following the cheese-making phases stated in the production disciplinary. the 3and 5-month cheeses were produced during spring and summer, respectively, while the 12-month ripened samples in the autumn season. cheeses were grounded and carefully mixed. three bulk samples were prepared by combining the samples of each ripening month and stored at –20°c until analysis. chemical–physical analysis for each ripening stage, each sample was analysed in triplicate. cheese samples were analysed, following the international methods of association of official analytical chemists (aoac, 2000), for fat (method: 933.05), protein (method: 920.123), moisture (method: 948.12) and ash (method: 935.42). proteolysis was assessed by determining the content of water-soluble nitrogen (sn) and non-protein nitrogen (npn), as done in niro et  al. (2014). the amount of sn and npn, expressed as a percentage of total nitrogen (tn) (sn/tn% and npn/tn%) indicates the extent of proteolysis. the nitrogen content was determined by the kjeldahl method (aoac, 2000; method: 920.123). free amino acids (faas) were analysed by a biochrom 30 series amino acid analyzer (biochrom ltd., cambridge science park, uk), with a li-cation-exchange column (20 × 0.46 cm). a mixture of basic, acid and neutral amino acid (aa) of a known concentration (sigma chemical co., st. louis, mo) was used as standards. the faa extraction procedure is reported in niro et al. (2017a). lipolysis was expressed as acid degree value (adv) (deeth and fitz-gerald, 1976). table 1. proximal composition of pecorino di farindola cheeses at different ripening times (g/100-g fresh weight) (mean ± s.d). ripening time moisture proteins fats ash 3 months 29.6 ± 0.04a 25.7 ± 0.29a 39.0 ± 0.09a 4.1 ± 0.03a 5 months 28.5 ± 0.03b 27.7 ± 0.16b 36.3 ± 0.12b 5.4 ± 0.02b 12 months 23.2 ± 0.08c 31.6 ± 0.27c 40.1 ± 0.27c 5.0 ± 0.00c different letters within the same column indicate a significant difference (p < 0.05). https://www.pecorinodifarindola.it/disciplinare/� https://www.pecorinodifarindola.it/disciplinare/� italian journal of food science, 2021; 33 (1) 48 chemical characterisation of ‘pecorino di farindola’ cheese during ripening table 2. proteolysis and lipolysis indices of pecorino di farindola cheeses at different ripening times (mean ± sd). ripening time proteolysis indices lipolysis index sn/tn% npn/tn% adv (meq koh/100-g fat) 3 months 32.5 ± 0.75a 12.1 ± 0.61a 2.9 ± 0.34a 5 months 33.7 ± 1.91a 13.8 ± 1.68b 4.0 ± 0.23b 12 months 37.6 ± 0.12b 19.6 ± 0.24c 7.0 ± 0.05c different letters within the same column indicate a significant difference (p < 0.05). table 3. free amino acid (faa) content of pecorino di farindola cheeses at different ripening times (mg/100-g fresh weight). faa 3 months* 5 months 12 months aspartic acid 10.74a 32.20b 45.26c threonine 3.24a 10.06b 17.49c serine 0.00a 5.68b 11.01c asparagine 10.15a 13.27b 54.20c glutamic acid 15.36a 99.14b 146.21c glutamine 4.56a 4.01a 9.64b glycine 4.34a 6.45a 20.89b alanine 11.35a 14.13b 43.82c valine 30.71a 42.08b 88.03c methionine 7.95a 14.62b 33.68c isoleucine 12.03a 25.50b 60.10 leucine 42.97a 57.66b 115.54c tyrosine 0.00a 2.27b 12.41c phenylalanine 26.75a 37.07b 63.56c γ-aminobutyric acid 21.55a 30.02b 56.98c ornithine 14.33a 15.08a 10.64a lysine 20.34a 38.92b 115.87c histidine 0.00a 9.13b 31.15c total 236.37a 457.28b 936.48c *different letters within the same raw indicate a significant difference (p < 0.05). to 37.6% at 12 ripening months (12r). this value is an indicator of hydrolysis of casein caused by the action of rennet and milk proteases present at the beginning of the ripening process. the sn is very variable for composition, including high-, medium-, low-molecular weight peptides and aas. moreover, a significant part of sn is produced during curd acidification and, consequently, it is partly lost into the water or brine (alichanidis and polychroniadou, 2008). the breaking off of casein and highand medium-molecular mass peptides by microorganisms, endogenous enzymes and rennet into low molecular mass peptides and aas, which are soluble in 12% trichloroacetic acid (tca), is expressed by the npn/ tn% (corradini, 1995). this value increased significantly from 12.1% in 3r samples to 19.6 % in 12r samples. these results demonstrated a marked proteolysis of pecorino di farindola cheese, similar of that of common italian pecorino cheeses (di cagno and gobetti, 2011). the principal proteolytic agents in the curd are coagulant, microbial proteinases and peptidases and indigenous milk proteinase (plasmin) (di cagno and gobetti, 2011). the proteinases and peptidases of rennet are among some of the proteolytic enzymes acting during the cheese-making process and the ripening phase (fox and stepaniak, 1993). tofalo et al. (2015) found faster casein breaking off in pecorino di farindola cheeses, attributed to higher proteolytic activity of the enzymes of pig rennet. on the contrary, a lower proteolytic activity of pig rennet than that of calf rennet was found by di giacomo et al. (2013) in pecorino di farindola cheese samples. a measure of lipolysis is represented by the determination of adv. this value is a measure of the content of free fatty acids (ffa) dissolved in a certain amount of fat by lipases that can be correlated to the sensorial quality of finished products (deeth and fitz-gerald, 1976; mcsweeney and sousa, 2000). the 3-, 5and 12-month ripened cheeses demonstrated an adv value of 2.9, 4.0 and 7.0 meq koh/100-g fat, respectively (table 2). similar values were reported for ewe’s cheeses (georgala et al., 2005), while higher indices were found in mixed cow/ewe caciocavallo cheeses by niro et al. (2014). the evolution of faas of cheeses during ripening is shown in table 3. the concentration of total free amino acids (tfaa) increased significantly (p < 0.05) to 936.48 mg/100 g at 12 months of ripening. at different ripening stages, the average tfaa content is in accordance with literature data for other italian pecorino cheeses (coda et  al., 2006). no proline and arginine were found in all analysed samples. the latter evidence could be due to its consumption by bacteria; in fact, many species of lab are able to convert arginine to citrulline and ornithine (diana et al., 2014; niro et al., 2017a). during ripening, the content of single faas increased, with the exception of ornithine. as also reported by di giacomo et al. (2013), at 12-month ripening, the highest faas found were glutamic acid (glu), valine (val), leucine (leu) and lysine (lys) in pecorino di farindola cheese at 6-month 49 italian journal of food science, 2021; 33 (1) s. niro et al. flavour intensity aroma intensity hardness solubility spicy 5r 3r 12r bittersalty acid sweet 7 6 5 4 3 2 1 0 figure 1. sensorial profile of pecorino di farindola cheeses at different ripening times. 3r: three-month ripening; 5r: fivemonth ripening; 12r: 12-month ripening. 3r 5r 12r flavour intensity* a b c hardness a b c solubility a a b spicy a b c bitter a a a salty a a b acid a a a sweet a b c aroma intensity a b c *different letters within the same row indicate a significant difference (p < 0.05) ripening and in long-ripened pecorino cheese (coda et al., 2006; mangia et al., 2008). glutamic acid is one of the most abundant amino acids in milk protein and, as almost free, in mature cheeses (redruello et al., 2020) and is strictly correlated with the ‘umami’ taste (mcsweeney and sousa, 2000). leu, phenylalanine (phe) and lys e val can be found in other long-ripened cheeses such as caciocavallo cheeses (corsetti et al., 2001; niro et al., 2017b; succi et al., 2016), idiazabal (barcina et al., 1995), picante (freitas et al., 1998), serra da estrela (tavaria et al., 2003), teleme (pappa and sotirakoglou, 2008), goat cheeses (poveda et al., 2016) and italian hard cheeses (niro et al., 2017b). leu, with the other branched chain aa, isoleucine (ile) and val, the aromatic aa, phe and tyrosine (tyr), and metionine (met) are the main precursors of key aroma compounds (yvon and rijnen, 2001). some authors have attributed the bitter flavour in cheese to the concentration of arginine (arg) (barcina et al., 1995; pappa and sotirakoglou, 2008). in all samples, ornithine (orn) and γ-aminobutyric acid (gaba) were found; they do not originate from casein but are the products of microbial metabolism and are established functional aas (diana et al., 2014; tofalo et al., 2019). as for gaba, its anti-hypertensive and anti-diabetic properties and its italian journal of food science, 2021; 33 (1) 50 chemical characterisation of ‘pecorino di farindola’ cheese during ripening be conducted to have a deeper insight into the fatty acid evolution and the influence of lipolysis and proteolysis on the profiles of volatiles during ripening. references alichanidis e. and polychroniadou a. 2008. characteristics of major traditional regional cheese varieties of east-mediterranean countries: a review. dairy sci. technol. 88:495–510. https://doi. org/10.1051/dst:2008023 aquilanti l., silvestri g., zannini a., osimani s., santarelli s. and clementi f. 2007. phenotypic, genotypic and technological characterization of predominant lactic acid bacteria in pecorino cheese from central italy. j. appl. microbiol. 103:948. https:// doi.org/10.1111/j.1365-2672.2007.03513.x association of official analytical chemists (aoac). 2000. official methods of analysis. vol. 2, 17th ed. aoac, washington, dc. isbn-13 978-093558467-7 barcina y., ibanez f.c. and ordonez a.i. 1995. evolution of free amino acids during idiazabal cheese ripening. food contr. 6:161. https://doi.org/10.1016/0956-7135(94)00007-z bellocci m., stramenga a., marchegiani f., cianciavicchia s., d’aloise a., melai v. and migliorati g. 2018. caratteristiche chimiche e valore nutrizionale del pecorino di farindola. in: “il pecorino di farindola. indagine fisica, chimica e microbiologica. riferimenti di territorialità”, p 85-97. izsam-istituto zooprofilattico sperimentale dell’abruzzo e del molise “g. caporale”. teramo, italia. bozzetti v., morara b. and zannoni m. 2004. etana: un modello per definire il profilo organolettico di tutti i formaggi. il latte 11:66. http://dx.doi.org/ 10.3168/jds.2013-7550 chiavari c., nanni m., ferri g., morara b. and qualizza g. 2006. formare assaggiatori per la valutazione sensoriale della mozzarella di bufala. il latte 11:66. coda r., brechany e., de angelis m., de candia s., di cango r. and gobetti m. 2006. comparison of the compositional, microbiological, biochemical, and volatile profile characteristics of nine italian ewes’ milk cheese. j dairy sci. 89:4126. https://doi. org/10.3168/jds.s0022-0302(06)72458-4 corradini c. 1995. chimica e tecnologia del latte. tecniche nuove. isbn 13: 978884810005 corsetti a., m. r. corbo, m. albenzio, r. di cagno, m. gobetti, and p. f. fox. 2001. microbiology and biochemistry of caciocavallo silano cheese. ital. j. food sci. 3:297. issn : 1120-1770 de angelis m., corsetti a., tosti n., rossi j., corbo m.r. and gobbetti m. 2001. characterization of non-starter lactic acid bacteria from italian ewe cheeses based on phenotypic, genotypic, and cell wall protein analysis. appl environ microbiol. 67:2011. https://dx.doi.org/10.1128%2faem.67.5.2011-2020.2001 deeth h.c. and fitz-gerald c.h. 1976. lipolysis in dairy products: a review. aust j dairy technol. 31:53. di cagno r., buchin s., de candia s., de angelis m., fox p.f. and gobetti m. 2007. characterization of italian cheeses ripened under nonconventional conditions. j dairy sci. 90:2689. https:// doi:10.3168/jds.2006-654 ability to reduce stress and anxiety are widely recognised (redruello et al., 2020; tofalo et al., 2019). gaba is synthesised by glutamate decarboxylase through the decarboxylation of l-glutamate; milk origin, milk treatment, proteolytic activity, fat content, texture, ripening time and climate are reported to be the key factors governing its accumulation (redruello et al., 2020). tofalo et al. (2019), in pecorino di farindola cheeses made from pig rennet, reported similar amounts of gaba than those found in this study, higher than the values of different commercial cheeses. regarding sensorial analysis, all samples were characterised by low acid and bitter attributes and a high salty score (figure 1). as reported by suzzi et al. (2014), cheeses made from pig rennet demonstrated the lowest elasticity, bitter taste and fruity and hay flavour intensities, compared with the cheeses made from calf and kid rennet. these data were also confirmed by di giacomo et al. (2013). low-ripened cheeses had a higher solubility, and tasted sweeter, less hard and less spicy than the corresponding long-ripened cheeses (p < 0.05). flavour and aroma intensity increased during ripening, reaching the highest score in pecorino at 12-month ripening (p < 0.05). the 12-month ripened cheeses had a light spicy feature that distinguishes them from other italian pecorino cheeses, probably because of the use of pig rennet instead of the commonly used lamb rennet. as reported by different authors (di giacomo et  al., 2013; kindstedt et al., 2004), cheeses with a more evident lipolysis differ for strong flavours. the effects of faas on taste and flavour are reported by mcsweeney and sousa (2000) and yvon and rijnen (2001). in raw milk cheeses, similar to pecorino di farindola, the native microbiota may have played an important role and contributed to the distinct sensorial characteristics (mcsweeney and sousa, 2000; niro et al., 2014). conclusions different pecorino di farindola cheeses were characterised by certain variability because of different composition of the used raw milk, the low standardization of the cheese making process and different salting and ripening conditions. the use of raw ewe’s milk and pig rennet contributed to the peculiar features of lipolysis and proteolysis and the sensorial attributes of the pecorino of farindola cheese. sensorial analysis confirmed pecorino of farindola as sweet, lightly spicy and never bitter cheese, even when the ripening is extended a distinctive feature that is appreciated by the consumer. data emerging from this work could add new knowledge to the investigations of this cheese, giving an effective characterisation of the final nutritional and sensorial quality of the product. an additional investigation could https://doi.org/10.1051/dst:2008023� https://doi.org/10.1051/dst:2008023� https://doi.org/10.1111/j.1365-2672.2007.03513.x� https://doi.org/10.1111/j.1365-2672.2007.03513.x� https://doi.org/10.1016/0956-7135(94)00007-z� http://dx.doi.org/� https://doi.org/10.3168/jds.s0022-0302(06)72458-4� https://doi.org/10.3168/jds.s0022-0302(06)72458-4� https://dx.doi.org/10.1128%2faem.67.5.2011-2020.2001� https://doi:10.3168/jds.2006-654� https://doi:10.3168/jds.2006-654� 51 italian journal of food science, 2021; 33 (1) s. niro et al. niro s., fratianni a., tremonte p., sorrentino e., tipaldi l., panfili  g., et al. 2014. innovative caciocavallo cheeses made from a mixture of cow milk with ewe or goat milk. j dairy sci. 100:1. https://doi.org/10.3168/jds.2013-7550 pappa e.c. and sotirakoglou k. 2008. changes of free amino acid content of teleme cheese made with different types of milk and culture. food chem. 111:606. https://doi.org/10.1016/j. foodchem.2008.04.027 redruello b., zwengiel a., madero v., del rio b. and alvarez m.a. 2020. identification of technological/metabolic/environmental profiles of cheeses with high gaba contents. food sci technol (lwt) 130:1. https://doi.org/10.1016/j.lwt.2020.109603 schirone m., tofalo r., mazzone g., corsetti a. and suzzi g. 2011. biogenic amine content and microbiological profile of pecorino di farindola cheese. food microbiol. 28:128. https://doi. org/10.1016/j.fm.2010.09.005 succi m., aponte m., tremonte p., niro s., sorrentino e., iorizzo m., et al. 2016. variability in chemical and microbiological profiles of long ripened caciocavallo cheeses. j dairy sci. 99:9521. https:// doi.org/10.3168/jds.2016-11585 suzzi g., sacchetti g., patrignani f., corsetti a., tofalo r., schirone  m., et al. 2014. influence of pig rennet on fatty acid composition, volatile molecule profile, texture and sensory properties of pecorino di farindola cheese. j sci food agr. 95(11):2252. https://doi.org/10.1002/jsfa.6944 tavaria f., franco k.i., carballo f.j. and malcata f.x. 2003. amino acid and soluble nitrogen evolution throughout ripening of serra da estrela cheese. int dairy j. 13:537. https://doi:10.1016/ s0958-6946(03)00060-8 tofalo r., perpetuini g., battistelli n., pepe a., ianni a., martino g., et al. 2019. accumulation γ-aminobutyric acid and biogenic amines in a traditional raw milk ewe’s cheese. foods. 8:401. https://doi:10.3390/foods809040.1 tofalo r., schirone m., fasoli g., perpetuini g., patrignani f., manetta a.c., et al. 2015. influence of pig rennet on proteolysis, organic acids content and microbiota of pecorino di farindola, a traditional italian ewe’s raw milk cheese. food chem. 175:121. https://doi.org/10.1016/j.foodchem.2014.11.088 yvon m. and rijnen l. 2001. cheese flavour formation by amino acid catabolism. int dairy j. 11:185. http://doi.org/10.1016/ s0958-6946(01)00049-8 di cagno r. and gobetti m. 2011. hard italian cheese. in: “encyclopedia of dairy science.” roginski h., fox p.f. and fuquay j.w. 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accepted: 05 october 2020; published: 01 february 2021 © 2021 codon publications open access paper abstract drying kinetics, water-soluble vitamins, total phenolic content (tpc), antioxidant capacity (ac) of the jujube fruits dried at 50, 60, and 70°c, and degradation kinetics of the quality parameters were investigated. the models fitted to drying were determined as page at 50 and 70°c, parabolic at 60°c. increment in the drying temperature increased the drying rate and decreased the drying time. water-soluble vitamins, tpc, and ac were significantly reduced by the drying process. degradation of water-soluble vitamins increased with the drying temperature, although tpc and ac were not significantly affected by temperature. thermal degradations of quality parameters were fitted to first-order kinetic. keywords: antioxidant capacity; degradation kinetics; drying kinetics; total phenol content; water-soluble vitamins introduction jujube fruit (zizyphus jujuba mill.), belonging to the rhamnaceae family, is a drupe, which has a roundelliptic shape, apple-like taste, and is rich in various bioactive compounds and nutrients such as vitamin c, polysaccharides, minerals (especially potassium), and phenolic compounds (chen et al., 2015; gao et al., 2011; wojdylo et al., 2016). the jujube fruit has higher vitamin c content than the fruits that are known as sources of vitamin c such as kiwi, strawberry, and lemon (frenich et al., 2005; wu et al., 2012). in addition, the jujube fruit is a good source for thiamine, riboflavin, niacin, and pyridoxine as b complex vitamins (gao et al., 2013). b complex vitamins have an important role as coenzymes for enzymatic reactions in different biological systems (calderón-ospina and nava-mesa, 2020). moreover, the jujube fruit has been considered as a good source of phenolic compounds compared to common fruits, which are widely known for being a source of phenolic compound such as berries (gao et al., 2011). in traditional chinese medicine, the jujube fruits have been used as a crude drug for analeptic, palliative, and antibechic purposes for thousands of years (li et al., 2007; rostami and gharibzahedi, 2017). the jujube fruits are also used for pharmaceutical benefits such as antioxidant, anticancer, antiinflammatory, antiepileptic, hepatoprotective, and neuroprotective effects (choi et al., 2012; ji et al., 2017). the pharmaceutical benefits of the jujube fruit have been associated with chemical ingredients, which mainly consist of vitamin c, phenolics, polysaccharides, triterpenic acids, and nucleosides (ji et al., 2017). the jujube fruits have been consumed as fresh, dried, tea, alcoholic beverages, pickle, jam, compote, or candy (elmas et al., 2019; wang et al., 2016; wojdylo et al., 2016). although the jujube fruits are generally consumed as fresh, the postharvest shelf-life of the jujube fruit is very short. thus, the commercial value of the jujube fruits is less (wang et al., 2016; zozio et al., 2014). chemical italian journal of food science, 2021; 33 (1): 1–15 mailto:begumotag@gmail.com 2 italian journal of food science, 2021; 33 (1) begüm tepe and raci ekinci ac, content of vitamin c, and tpc. in that study, it was notified that the highest ac and tpc were observed in jujube fruits dried at 70°c, while the highest vitamin c content was obtained at 50°c (anjum et al., 2020). the effects of different drying methods (convective, vacuum microwave, convective pre-drying, and vacuum–microwave combination) on phenolic compounds, ac, and the color of jujube fruits were investigated by wojdylo et al. (2019). convective drying carried out at 50°c has been reported to be the best method in terms of polyphenol content, ac, and color parameters. in this study, it was aimed to: (i) determine the drying characteristics of whole jujube fruits at different air temperatures (50, 60, 70°c); (ii) investigate the effect of drying on vitamin c and b complex content, tpc, and ac of fresh and dried whole jujube fruits; and (iii) determine thermal degradation kinetics of these bioactive compounds during the drying process. materials and methods sample preparation fresh jujube fruits (zizyphus jujuba mill.) were provided from a local producer in denizli, a province in turkey. the fresh jujube fruits were carefully selected in terms of the same ripening stage (fully mature) and the same size. before the analysis, fresh jujube fruits were washed to remove foreign materials. the fresh jujube fruits were stored at 4°c in a refrigerator. determination of the initial moisture content of the samples was carried out in a drying oven at 105°c till any changes in the sample weight. the initial moisture content of whole jujube fruits was 65.26% ± 0.6. drying experiment a cabinet dryer (yücebas¸ makine ltd. inc., izmir, turkey) was used for the drying experiments. for the condition stabilization, the cabinet dryer was turned on approximately 30 min before drying. samples (500  g) were weighted on a drying tray and placed in a cabinet dryer for each drying experiment. drying temperatures were selected as 50, 60, and 70°c, similar to other researches on the drying of jujube fruits (fang et al., 2009a; motevali et al., 2012; wojdylo et al., 2016, 2019). besides, air velocity and relative humidity were 2 m s−1 and 20%, respectively. in practical application, the moisture content of the dried jujube fruits needs to be below 25% on wet basis (wb) (fang et al., 2009a). therefore, the drying experiments were continued until the moisture content of the samples achieved to 21% on wb, similar to the results of the studies by fang et al. (2009a) reactions, microbiological activity, and physical alterations in the preor post-harvest period of many plantbased foods mostly require high water content (tepe and tepe, 2020). therefore, some preservation methods such as drying can be suggested to extend their shelf-life. drying, which is also called dehydration, is one of the oldest methods of food preservation, used since ancient times. the beneficial properties of drying can be ordered as reducing the water activity to microbiological safety zone and transport costs, providing easier process and extending shelf life (elmas et al., 2019). selection of drying methods has great importance for the preservation of quality parameters such as the content of phenolic compounds, vitamins, and antioxidant capacity (ac). sun drying and hot air drying are traditional methods for jujube drying (wang et al., 2016). the main mechanism of hot air drying is mass and heat transfer and phase transition (tepe and tepe, 2020). hot air drying provides some advantages such as being free from the climate effects, reducing the drying cycle, and hygienic conditions in comparison to sun drying (elmas et al., 2019). however, long drying time, loss of nutritional and bioactive value, and changes in sensory properties are disadvantages of hot air drying (elmas et al., 2019; onwude et al., 2017; wang et al., 2019). in addition to the hot air drying method, microwave, vacuum, and freeze drying methods have been regarded as alternative drying methods. condurso et al. (2019) notified that decrement in drying time and better product quality could be provided by microwave drying. in addition, vacuum drying is useful for drying of easily oxidizing foods. besides, freeze drying has also many advantages such as preserving food quality, especially in heat-sensitive foods. these drying methods can be used alone or combined with each other such as microwave–hot air or vacuum–microwave drying. the combined drying methods may be useful for increasing the efficiency (energy efficiency, environment friendly, product quality) of these drying methods (sun et  al., 2019). to select the most appropriate drying method, mathematical modeling is needed along with the determination of product quality, energy efficiency, etc. in this context, thin-layer drying has been used for the determination of the drying kinetics of fruits and vegetables. the most appropriate drying conditions can be selected by using thin-layer drying technology, a kind of mathematical modeling. thus, a drying process can be designed and optimized (onwude et al., 2016). there are some recent researches on the drying of jujube fruits in the literature. elmas et al. (2019) have reported that moisture content, water activity, and the total phenolic content (tpc) decreased as the drying temperature increased in hot air drying. in addition, anjum et al. (2020) have investigated the effects of drying methods (sun and hot air drying) on several physical properties, italian journal of food science, 2021; 33 (1) 3 drying charateristics of jujube fruits (r2), root-mean square error (rmse), and reduced chi-square (χ2). rmse is a statistical parameter, which expresses the deviation between the predicted and the experimental values. the best equation predicting experimental data is determined according to the lower values of χ2 and rmse, and the higher value of r2. the rmse (eq. 3) and chi-square (χ2) (eq. 4) values were calculated as follows: =   = −    ∑ 1 2 2 , , 0 1 rmse ( ) n pre i exp i i mr mr n (3) χ = − = − ∑ 2, ,0( )2 n pre i exp ii mr mr n n (4) mrpre,i: predicted mr; mrexp,i: experimental mr; n: number of observation data; n: constants of thin layer drying models. thin-layer modeling and statistical parameters were calculated using the matlab software (r2015a, version 8.5) non-linear curve fitting toolbox with the trust-region algorithm. determination of effective moisture diffusivity and activation energy in hot-air drying fick’s diffusion equation has been accepted to describe the drying characteristics of biomaterials. crank (1975) has suggested a solution to this equation, which can be used for spherical products. eq. (5) has been recommended for spherical products by assuming constant effective diffusivity and no shrinkage (doymaz, 2006). ∞ π π =  − =      ∑ 2 2 2 2 2 1 6 1 exp eff n n d t mr n r (5) deff: effective moisture diffusivity (m 2 s−1); r: arithmetical average of radius of samples at measured intervals (m). eq. (5) can be simplified for the first term of the series (saravacos and raouzeos, 1986). the new equation is written as given below, eq (6): ( ) π π    = −        2 2 2 6 ln eff d ln mr t r (6) and yi et al. (2012). all of drying experiments were performed in triplicate. drying characteristics of whole jujube fruits to design the best drying conditions, thin-layer drying models are very important. the thin-layer mathematical models selected in the current study are listed in table 1. significant information about drying temperature and time can be provided with these models (demiray et al., 2017). moisture ratio (mr) of whole jujube fruits was calculated using eq. (1): − = − mr t e i e m m m m (1) mi: initial moisture content of the samples (g water g −1 dry matter); mt: moisture content at any point of time (g water g −1 dry matter); me: equilibrium moisture content (g water g −1 dry matter). me can be ignored because of its insignificant value in comparison to mi and mt (fang et al., 2009a). drying rate (dr) was determined using eq. (2): +∆ −= ∆ t t tm mdr t (2) mt+δt: moisture content at time difference; δt: difference of time between two measuring points. the relation between the predicted and the experimental data of whole jujube fruits dried at different drying temperatures is explained with determination coefficient table 1. thin-layer mathematical models. model name model references logaritmic aexp(-kt) + c demiray et al. (2017) lewis exp(-kt) demiray et al. (2017) henderson and pabis aexp(-kt) tepe and tepe (2020) page exp(-ktn) demiray et al. (2017) parabolic a + bt + ct2 bi et al. (2015) wang and sing 1 + at + bt2 tepe and tepe (2020) 4 italian journal of food science, 2021; 33 (1) begüm tepe and raci ekinci the content of water-soluble vitamins was calculated using an equation obtained from a calibration curve consisting of different concentrations of stock solutions (5, 10, 25, 50, 75, and 100 ppm) with a high r2 (0.9999). results were given as mg 100 g−1 in dry weight (dw) for vitamin c and µg 100 g−1 for niacin, pyridoxine, thiamine, and riboflavin. each analysis was performed in triplicate. analyzes of tpc and ac analyses of tpc and ac were performed with methanolic extraction with a slight modification, as suggested by choi et al. (2012). jujube fruit samples (5 g) and 45 ml of 90% methanol were homogenized using a laboratory type blender. the homogenate was centrifuged at 2355 × g for 10 min. after centrifugation, the supernatants were collected and filtrated using a filter paper. tpc analysis was performed according to singleton and rossi (1965) with a slight modification. folin-ciocalteu solution (1500 µl) (10% v/v) was added into 300 µl of the extract, and the mixture was kept in a dark place for 5 min. after adding 1200 µl of aqueous 7.5% na2co3 into the mixture, the mixture was incubated at room temperature in a dark place for 2 h. at the end of the incubation, the absorbance of samples was measured at 760 nm using a spectrophotometer (t80, pg ins. uk.). each analysis was carried out in triplicate, and tpc was expressed as mg gallic acid equivalent (gae) 100 g−1 in dw. the ac analysis was carried out using a method suggested by thaipong et al. (2006) with slight modification. extracts (150 µl) and dpph methanolic solution (2850 µl), whose absorbance is 1.1 at 515 nm, were mixed. after incubation for 60 min at room temperature in a dark place, the absorbance of samples was measured at 515 nm. each sample was analyzed in triplicate, and ac was expressed as mmol trolox equivalent (mmol te) g−1 in dw. color measurement reflectance color value of the whole jujube fruit skin was measured using hunter lab color miniscan xe (45/0-l, usa). the samples were placed on a white background, and the measurement was done by covering a transparent glass. δe was calculated with the equations below (horuz et al., 2017): ( ) ( )∆ = − + − + −2 2 20 0 0e ( )l l a a b b (10) δe: total color differences; l: lightness (0 = black, 100 = white) value at the end of the drying process; the plot gives a straight line with a slope as follows, eq. (7): π = − 2 2slope eff dr (7) arrhenius equation in hot air drying process was used for the calculation of activation energy (fang et al., 2009a): −  =     0 exp a eff e d d rt (8) r: universal gas constant (8.314 j mol−1 k−1 or 1.987 cal mol−1 k−1); t: absolute temperature (k); ea: activation energy (kj mol −1 or kcal mol−1); d0: pre-exponential constant (m 2 s−1). after regulation of the natural logarithm of eq. (8), eq. (9) can be written as given below: = −0 a eff e lnd lnd rt (9) natural logarithm of effective moisture diffusivity versus t−1 gives a straight line with a slope, which represents activation energy. analysis of water-soluble vitamins an extraction method, recommended by dönmez (2015), was used for water-soluble vitamins. in order to determine the water-soluble vitamins, 5 g of each sample was weighted. after homogenization with distilled water (1:9, w:v), the homogenate was centrifuged at 2355 × g for 10 min (nüve nf 800r). the supernatant obtained from centrifugation was filtrated using a 0.45 µm filter to be injected into the high performance liquid chromatography (hplc). a micro syringe was used for injecting 20 µl of the last filtrate into the hplc column. mobile phase consisted of 0.1 m hplc grade kh2po4 at ph 7. an hplc device (shimadzu), column oven at 25°c (shimadzu cto-20a), column ace c18 (7.8 × 300 mm), pump (shimadzu lc-20ad), degasser (shimadzu dgu20a3), photo-diode array (pda) detector (spd-m20a) at 254, 261, 324, 234 nm for ascorbic acid, niacin, pyridoxine, and thiamin, respectively were used for analysis. the mobile phase was isocratic with 0.7 ml min−1 flow rate. for riboflavin analysis, the column is macherey-nagel nh2 (4.6 × 250 mm), column oven temperature is 40°c, and the wave length is 266 nm. the same mobile phase was isocratic with 1 ml min−1 flow rate. italian journal of food science, 2021; 33 (1) 5 drying charateristics of jujube fruits r: universal gas constant (8.314 × 10−3 kj mol−1 k−1 and 1.987 × 10−3 kcal mol−1 k−1); t: absolute temperature (k); ea: activation energy (kcal mol −1 or kj mol−1). quotient indicator (q10) expresses the temperature dependence of reaction rate and is calculated with eq. (16) (kadakal et al., 2017):     − = 2 1 10 2 10 1 ( ) t t k q k (16) half-life time, a time required for half of the concentration, for each temperature is calculated with eq. (17) for the first-order kinetic (kadakal et al., 2017); t1/2 = –ln(0.5)xk –1 = 0.693xk–1 (17) d represents the time that it takes for the compound or quality criterion to lose 90% of its quality and is calculated for first-order kinetics as written below (eq. 18): d = 2.303xk–1 (18) statistical analysis all of the data were statistically analyzed using the spss software (ver. 22 spss inc., chicago, il, usa) and expressed as mean ± standard deviation (sd). analysis of variance (anova) was used to evaluate differences between treatments, with a significance level of p = 0.05. the differences between groups were determined using the duncan test. results and discussion drying characteristics of whole jujube fruits during hot air drying mr and dr of whole jujube fruits during hot air drying are shown in figure 1. as understood from figure 1, drying temperature has statistically affected dr and the drying time of whole jujube fruits. it was clearly observed that dr increased with the increment in drying temperature. accordingly, the drying time was reduced and found to be 48, 30, and 18 h for 50, 60, and 70°c, respectively. likewise, yi et al. (2012) reported that drying times of whole jujube fruits at 45, 55, and 65°c for constant air velocity (2 m s−1) were about 45, 25, and 20 h, respectively. it could be explained with increasing of the heat transfer coefficient by increment in drying temperature. generally, two periods as constant rate and falling rate are the main l0: initial lightness value of the fresh jujube fruits; a: redness (a+ = red, a= green) value at the end of the drying process; a0: initial redness value of the fresh jujube fruits; b: yellowness (b+ = yellow, b= blue) value at the end of the drying process; b0: initial yellowness value of the fresh jujube fruits. calculation of kinetic parameters labuza and riboh (1982) and kadakal et al. (2017) have suggested eq. (11) as the general equation to describe the reaction rate of compounds degrading or forming: [ ]= mdc k c dt (11) for the zero-order kinetic model, eq. (12) can be written as below: c = c – kt (12) when eq. (1) is integrated, if m equals one, eq. (3) is written as follows: lnc = lnc0 – kt (13) ln c: natural logarithm of the residual vitamin c, b complex vitamins, tpc, and ac; ln c0: initial content of vitamin c, b complex vitamins, tpc, and ac; k: rate constant (h−1); t: time. temperature dependence of vitamin c, b complex vitamins, tpc, and ac can be calculated with eq. (14) (kadakal et al., 2017; labuza and riboh, 1982): − = 0 ae rtk k x e (14) when the eq. (14) is regulated, eq. (15) is written as follows:    = − +        0 1aelnk x lnk r t (15) k0: frequency factor (h −1); 6 italian journal of food science, 2021; 33 (1) begüm tepe and raci ekinci 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 10 20 30 40 50 m r time (h) 50°c 60°c 70°c 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 (b) (a) 0.3 0.8 1.3 1.8 2.3 d r (g w at er g –1 m at te r h– 1 ) moisture content (g water g–1 dry matter) 50°c 60°c 70°c figure 1. moisture ratio and drying rate of whole jujube fruits during hot air drying. table 2. models, constants, and statistical parameters of thin-layer drying curves. models temperature model constants χ² rmse r² lewis 50°c k = 0.03638 0.00043784 0.020710 0.9936 60°c k = 0.06996 0.000355573 0.018550 0.9953 70°c k = 0.10140 0.000841207 0.028230 0.9899 page 50°c k = 0.02349 n = 1.135 2.00099e–05 0.004381 0.9997 60°c k = 0.05734 n = 1.074 0.00022413 0.014480 0.9972 70°c k = 0.06731 n = 1.182 5.10587e–05 0.006759 0.9995 henderson and pabis 50°c k = 0.03820 a = 1.041 0.000225899 0.014720 0.9968 60°c k = 0.07249 a = 1.032 0.00024309 0.015080 0.9970 70°c k = 0.10720 a = 1.049 0.000524835 0.021670 0.9944 logaritmic 50°c k = 0.05256 a = 0.9309 c = 0.1427 0.001495566 0.037470 0.9794 60°c k = 0.10390 a = 0.9326 c = 0.1428 0.001457264 0.036280 0.9825 70°c k = 0.14750 a = 0.9333 c = 0.1439 0.002635481 0.047110 0.9733 wang and singh 50°c a = −0.03016 b = 0.0002595 2.90427e–05 0.005278 0.9995 60°c a = −0.05971 b = 0.0010510 4.66771e–05 0.006608 0.9994 70°c a = −0.08348 b = 0.0019720 0.000105507 0.009716 0.9988 parabolic 50°c a = 1.003 b = −0.03039 c = −0.0002634 2.92706e–05 0.005242 0.9996 60°c a = 1.008 b = −0.06080 c = 0.0010800 3.94992e–05 0.005973 0.9995 70°c a = 1.017 b = −0.08723 c = 0.0021410 6.1355e–05 0.007188 0.9994 rmse, root-mean square error. constituents of the drying process of agricultural products such as fruits and vegetables. in the current study, the falling rate period was observed. this statement was found to in sync with other studies, in which the jujube fruits were dried with hot air, by fang et al. (2009a), yi et al. (2012), baomeng et al. (2014), and chen et al. (2015). mr of whole jujube fruits during hot air drying was used to be fitted mathematical models that are listed in table 1. statistical parameters to describe the most suitable model are presented in table 2. demiray et al. (2017) reported that the lower rmse and χ² and the higher r2 are required for goodness of the fit. as seen from table 2, the parabolic model was the best model for predicting the experimental mr of whole jujube fruits for 60°c, while experimental mrs of jujube dried at 50 and 70°c were described with page model with the lowest rmse and χ² and the highest r2 values. effective moisture diffusivity and activation energy of whole jujube fruits during hot air drying deff and ea values of whole jujube fruits are presented in table 3. deff values of whole jujube fruits were calculated in the range of 6.43 × 10−11 and 1.80 × 10−10 m2 s−1. in comparison to the other drying temperatures, the highest value of deff was obtained from the drying process italian journal of food science, 2021; 33 (1) 7 drying charateristics of jujube fruits a, and b values of whole jujube fruits, these parameters were significantly decreased depending on the drying process (p < 0.05). the lowest l, a, and b values were obtained at 50°c. it could be because of the longer time of the drying process at 50°c. δe represents differences between the colors of the samples (horuz et al., 2017). δe values of dried whole jujube fruits depending on drying temperature and time ranged from 16.84 to 20.17. the effect of the drying process on the water-soluble vitamins, tpc, and ac the effect of the drying process on the watersoluble vitamins of whole jujube fruits is given in table 5. watersoluble vitamins were significantly affected by the drying process. vitamin c content of the jujube fruit differs depending on some factors such as geographical conditions and cultivars. in the current study, vitamin c content of fresh whole jujube fruits was determined as 78.90  ± 0.96 mg 100 g−1 dw. vitamin c content of the whole jujube fruit was considerably reduced by the drying process, mainly the drying temperature (p < 0.05). fang et al. (2009b) have similarly reported decrement in vitamin c content of the whole jujube fruit during hot air drying. reduction in vitamin c content of sliced jujube fruit during hot air drying was also notified by chen et al. (2015). vitamin c is a heat-sensitive compound and, thus, might be degraded by a heating process such as drying (chin et al., 2015). in addition, vitamin c oxidation can occur more rapidly at higher temperatures (orikasa et  al., 2014; santos and silva, 2008). the highest loss of vitamin c content of whole jujube fruits was 81.91% at 70°c, while the lowest loss was 55.51% at 50°c. loss of vitamin c content of whole jujube fruits increased with the increment in drying temperature. this result was similar to chen et al. (2015) but contrary to fang et al. (2009b). in other fruits, vitamin c has been reported to be more degraded with the increasing air temperature (chin et al., 2015; kaya et al., 2010; orikasa et al., 2014; vega-galvez et al., 2009). initial content of thiamine (b1), riboflavin (b2), niacin (b3), and pyridoxine (b6) in fresh whole jujube fruits were determined as 27.33 ± 1.52, 41.00 ± 1.00, 883.33  ± performed at 70°c. it is a fact that drying temperature is one of the most important factors affecting the deff value. increment in deff value means more easy evaporation of moisture content of the sample and consequently an increment in dr. in addition, a proportional relationship between deff and dr was reported by demiray et al. (2017). elmas et al. (2019) and fang et al. (2009a) notified deff values of jujube fruits ranging from 1.27 × 10 −9 to 3.55 × 10−9 and 5 × 10−11 to 2 × 10−10 m2 s−1, respectively. when compared to these studies, the result of the current study was less than that reported by elmas et al. (2019) and very similar to the findings notified by fang et al. (2009a). this difference might be because of drying conditions, equipment, and the shape of dried fruits (sliced or whole). arrhenius relation between deff and t−1 is presented in figure 2. ea value of whole jujube fruits was found to be 47.11 kj mol−1 and 11.33 kcal mol−1. various ea values were reported for drying of the jujube fruit. fang et al. (2009a) have reported that the ea value of the jujube fruit was 54.51 kj mol−1. on the contrary, elmas et al. (2019) have found the ea value of sliced jujube fruit to be 28.183 kj mol−1. besides, motevali et al. (2012) have notified that the ea value of the jujube fruit ranged from 34.97 to 74.20 kj mol−1. color properties of whole jujube fruits during hot air drying color properties of fresh and dried whole jujube fruits are shown in table 4. when compared to the initial l, table 3. d eff and e a values of whole jujube fruits. temperature d eff (m2 s–1) e a (kj mol−1) e a (kcal mol−1) 50°c 6.43 × 10−11 60°c 1.11 × 10−10 47.41 11.33 70°c 1.80 × 10−10 y = 0.003e–5.702x r² = 0.9996 0 2e-11 4e-11 6e-11 8e-11 1e-10 1.2e-10 1.4e-10 1.6e-10 1.8e-10 2e-10 2.9 2.95 3 3.05 3.1 3.15 e ff ec tiv e m oi st ur e di ff us iv ity (m 2 s –1 ) t–1x10–3 figure 2. arrhenius type relation between d eff and t−1. table 4. the color properties of whole jujube fruits. l a b δe fresh 21.92 ± 0.01a 15.95 ± 0.12a 9.91 ± 0.12a 0.00 50°c 10.31 ± 0.05b 2.04 ± 0.04b 1.04 ± 0.02b 20.17 60°c 11.09 ± 0.03c 2.23 ± 0.02c 1.34 ± 0.07c 19.47 70°c 12.25 ± 0.04d 4.57 ± 0.02d 2.12 ± 0.08d 16.84 *the different letters in the same column are significantly different (p < 0.05). 8 italian journal of food science, 2021; 33 (1) begüm tepe and raci ekinci drying; however, no there were no significant differences between the drying temperatures (horuz et al., 2017). on the contrary, yaşa (2016) has reported that the tpc of whole jujube fruits was reduced by hot air drying and the loss of tpc increased with an increment in the drying temperature. likewise, elmas et al. (2019) notified more decrement in the tpc of sliced jujube fruits based on an increment in the drying temperature. furthermore, ac of whole jujube fruits dried at 50, 60, and 70°c decreased with the percentage of 61.22, 60.75, and 59.35, respectively. no significant difference was found between the drying temperatures (p > 0.05). long drying times might decrease ac of foods (garau et al., 2007). on the contrary, wojdylo et al. (2016) have notified that ac of three different jujube cultivars decreased with hot air drying, and an increment in the drying temperature increased the reduction of ac in these cultivars. a decrement in ac of hot-air-dried red pepper was reported by vega-galvez et al. (2009). however, no significant differences between drying temperatures were notified in the same study. kinetic parameters of vitamin c to the best of our knowledge, degradation of vitamin c in whole dried jujube fruits was investigated for the first time. thermal degradation of vitamin c in whole dried jujube fruits is shown in figure 3. as seen in figure 3, thermal 15.27, and 80.33 ± 2.08 µg 100 g−1 dw, respectively. yaşa (2016) had reported thiamine (b1), riboflavin (b2), niacin (b3), and pyridoxine (b6) content of jujube cultivated in denizli, a province of turkey, to be 0.018, 0.036, 0.82, and 0.076 mg 100 g−1, respectively. results of the current study were in good agreement with those reported by yaşa (2016). in addition, li et al. (2007) have reported thiamine and riboflavin content of five chinese jujube cultivars in the range of 0.04–0.09 mg 100 g−1 and 0.05– 0.09 mg 100 g−1, respectively. these values were also similar to those reported by gao et al. (2013), pareek (2013), yaşa (2016), and li et al. (2007). drying temperature has a great impact on b complex vitamins. as seen from table  5, b complex vitamins of whole jujube fruits remarkably decreased at the end of the drying process (p < 0.05). the highest losses in thiamin (b1), riboflavin (b2), and niacin (b3) content occurred at 70°c as 39.74, 62.37, and 28.57% (p < 0.05), respectively, whereas the drying process at 50°c resulted in the lowest losses with the percentages of 27.07, 51.88, and 13.77 (p < 0.05), respectively. on the other hand, pyridoxine was the highest affected compound among b complex vitamins. no pyridoxine content of whole jujube fruits was determined at the end of the drying process (p < 0.05). it could be under the limit of detection. it was similarly notified by yaşa (2016) that thiamin (b1), riboflavin (b2), and niacin (b3) content of whole jujube fruits decreased during drying. in addition, no pyridoxine content was reported at the end of the drying process by yaşa (2016). the effect of the drying process on tpc and ac of whole jujube fruits are presented in table 6. as seen in table 6, tpc and ac of fresh whole jujube fruits were found to be 1911.4 ± 47.32 mg gae 100 g−1 dw and 0.214 ± 0.001 mmol te g−1 dw. tpc and ac of whole jujube fruits significantly decreased with hot air drying (p < 0.05). the loss percentages of tpc in whole jujube fruits dried at 50, 60, and 70°c were calculated as 78.10, 76.26, and 74.68, respectively. in the current study, the increment in the drying temperature has no significant effect on the reduction in tpc (p > 0.05). likewise, vega-galvez et al. (2009) noted no significant change in the tpc of red pepper during hot air drying. similarly, tpc of sour cherries was also notified to have decreased during table 5. the effect of the drying process on water-soluble vitamins of whole jujube fruits. vitamin c % loss thiamine (b1) % loss riboflavin (b2) % loss niacin (b3) % loss pyridoxine (b6) % loss fresh 78.90 ± 0.96a 0 27.33 ± 1.52a 0 41.00 ± 1.00a 0 883.33 ± 15.27a 0 80.33 ± 2.08b 0 50°c 35.10 ± 0.36b 55.51 19.93 ± 0.21b 27.07 19.73 ± 0.15b 51.88 761.67 ± 4.04b 13.77 nd* 100 60°c 20.93 ± 0.64c 73.47 18.63 ± 0.15b 31.83 18.13 ± 0.15c 55.78 689.33 ± 4.16c 21.96 nd 100 70°c 14.27 ± 0.55d 81.91 16.47 ± 0.21c 39.74 15.43 ± 0.15d 62.37 631.00 ± 2.00d 28.57 nd 100 *nd, not detected; vitamin c was expressed as mg 100 g−1; dw, b complex vitamins were expressed as µg 100 g−1 dw. the different letters in the same column are significantly different (p < 0.05). table 6. the effect of the drying process on tpc and ac of whole jujube fruits. tpc % loss ac % loss fresh 1911.40 ± 47.32a 0 0.214 ± 0.001a 0 50°c 418.59 ± 12.18b 78.10 0.083 ± 0.001b 61.22 60°c 453.71 ± 4.61b 76.26 0.084 ± 0.001b 60.75 70°c 484.03 ± 6.21b 74.68 0.087 ± 0.001b 59.35 *tpc was expressed as mg gae 100 g−1 dw; ac was expressed as mmol te g−1 dw. the different letters in the same column are significantly different (p < 0.05). tpc, total phenolic content; ac, antioxidant capacity. italian journal of food science, 2021; 33 (1) 9 drying charateristics of jujube fruits activation energy reflects the reaction’s temperature sensitivity. higher ea indicates higher sensitivity to temperature changes. besides, higher ea means higher stability to thermal degradation (bell, 2020; kadakal et  al., 2017). arrhenius equation, which was used for the calculation of the ea of vitamin c thermal degradation, is given in figure 4. the ea of vitamin c thermal degradation in whole dried jujube fruits was calculated as 80.87 kj mol−1. this value was higher than 46.248 and 46.99 kj mol−1 by akdaş and başlar (2015) and demiray et al. (2013), respectively, meaning that vitamin c was more stable to thermal degradation, and its thermal degradation reaction was more sensitive to temperature changes in whole dried jujube fruits. q10 value, a criterion reflecting the effect of raising temperature by 10°c on the rate of reaction, is also used as an indicator of the reaction’s temperature sensitivity. higher q10 values denote greater temperature sensitivity (bell, 2020; kadakal et al., 2017). in the current study, the value of q10 from 50 to 60°c was found to be slightly higher than from 60 to 70°c. this means that the thermal degradation of vitamin c was more affected by an increment of temperature from 50 to 60°c than from 60°c to 70°c. kadakal et al. (2017) and demiray et al. (2013) have similarly reported that the q10 value of vitamin c thermal degradation of vitamin c in whole dried jujube fruits followed the first-order kinetic model. thermal degradation of vitamin c is reported to frequently fit to the first-order reaction model in different dried foods by demiray et al. (2013), kadakal et al. (2017), orikasa et al. (2014), and kurozawa et al. (2014). kinetic parameters of vitamin c are listed in table 7. the rate constant of vitamin c thermal degradation increased depending on an increment in the drying temperature. accordingly, the values of t1/2 and d decreased. likewise, demiray et  al. (2013) and akdaş and başlar (2015) have reported an increment in the degradation rate constant of vitamin c in tomato and mandarin, respectively, as drying temperatures were raised. in addition, the values of t1/2 decreased with an increase in the degradation rate constant. kadakal et al. (2017) have notified an increment in degradation rate constant and decrement in the values of t1/2 and d of vitamin c thermal degradation in rosehip nectar during thermal treatment. also, in another study, vitamin c degradation in hot-airdried kiwi fruits showed an increment with an increase in the drying temperature (orikasa et al., 2014). kurozawa et al. (2014) have indicated a rate constant of vitamin c thermal degradation in papaya with an increment in temperature during the drying process. the result of the current study is in good agreement with other reports. table 7. first-order kinetic parameters of water-soluble vitamins, tpc, and ac of whole dried jujube fruits. compound temperature k (h−1) t 1/2 (h) d (h) r2 e a (kcal mol−1) e a (kj mol−1) q 10 (50–60°c) q 10 (60–70°c) vitamin c 50°c 0.0170 40.76 135.47 0.9865 60°c 0.0445 15.57 51.75 0.9894 19.33 80.87 2.62 2.21 70°c 0.0983 7.05 23.43 0.9890 thiamine 50°c 0.0066 105.00 348.94 0.9798 60°c 0.0125 55.44 184.24 0.9776 16.23 67.90 1.89 2.31 70°c 0.0289 23.98 79.69 0.9869 riboflavin 50°c 0.0162 42.78 142.16 0.9810 60°c 0.0275 25.20 83.75 0.9817 13.23 55.37 1.70 1.96 70°c 0.054 12.83 42.65 0.9918 niacin 50°c 0.0034 203.82 677.35 0.9733 60°c 0.0084 82.50 274.17 0.9856 18.83 78.78 2.47 2.24 70°c 0.0188 36.86 122.50 0.9922 pyridoxine 50°c 0.0505 13.72 45.60 0.9898 60°c 0.0886 7.82 25.99 0.9886 15.63 65.41 1.75 1.72 70°c 0.1524 4.55 15.11 0.9999 tpc 50°c 0.0332 20.87 69.37 0.9815 60°c 0.0503 13.77 45.78 0.9648 9.33 39.02 1.51 1.54 70°c 0.0775 8.94 29.72 0.9796 ac 50°c 0.0194 35.72 118.71 0.9619 60°c 0.0314 22.07 73.34 0.9683 10.68 44.68 1.62 1.63 70°c 0.0512 13.54 44.98 0.9684 tpc, total phenolic content; ac, antioxidant capacity. 10 italian journal of food science, 2021; 33 (1) begüm tepe and raci ekinci 60 (a) (b) (e) (c) (d) 50403020100 2 2.5 3 3.5 4 4.5 50°c 60°c 70°c 50°c 60°c 70°c 50°c 60°c 70°c 50°c 60°c 70°c time (h) ln c 403020100 2 2.5 3 3.5 4 4.5 50°c 60°c 70°c time (h) ln c 6050403020100 2.7 2.9 2.8 3.1 3 3.2 3.3 3.4 time (h) ln c 6050403020100 2.6 2.8 3 3.2 3.6 3.8 3.4 4 time (h) ln c 6050403020100 6.4 6.5 6.45 6.75 6.7 6.65 6.6 6.55 6.8 6.85 time (h) ln c figure 3. first-order plots of (a) vitamin c, (b) thiamine, (c) riboflavin, (d) niacin and (e) pyridoxine during drying of the whole jujube fruits degradation decreased with an increment in the process temperature. kinetic parameters of b complex vitamins to the best of our knowledge, no data on b complex vitamin degradations in jujube fruits during hot air-drying process have been published as yet. thermal degradation kinetics of b complex vitamins in jujube fruits during hot air-drying process was investigated for the first time in this study. thermal degradation of b complex vitamins is given in figure 3. however, no pyridoxine content was determined at the end of the process. therefore, kinetic modeling of pyridoxine thermal degradation was conducted until the last point where pyridoxine was italian journal of food science, 2021; 33 (1) 11 drying charateristics of jujube fruits 6 5 4 3 2 1 0 2.9 3.153.13.0532.95 t–1×10–3 l nk vitamin c thaimine riboflavin pyridoxinenaicin figure 4. arrhenius plots of water-soluble vitamins of whole dried jujube fruits. detected. thermal degradation of b complex vitamins followed the first-order kinetic model. likewise, kadakal et al. (2017) have reported that thermal degradation of thiamine and riboflavin in rosehip nectar fitted to the first-order kinetic model during thermal treatment for 30 min. thermal degradation of niacin in cooked potato for 60 min in the temperature range of 50–120°c was notified to follow the first-order kinetic model by nisha et al. (2009). nisha et al. (2005) and rekha et al. (2004) have reported a first-order kinetic of thermal degradation of riboflavin and thiamin in cooked spinach and red gram splits at a temperature range of 50–120°c for 60 min, respectively. kinetic parameters of b complex vitamins’ thermal degradation are presented in table  7. in the current study, degradation rate constants of b complex vitamins increased with the increment in drying temperature. accordingly, degradation rapidly occurred at higher temperatures. in consequence, the values of t1/2 and d of b complex vitamins decreased. in the current study, degradation rate constants of thiamine, riboflavin, niacin, and pyridoxine ranged from 0.0066 to 0.0289 h−1, 0.0162 to 0.054 h−1, 0.0034 to 0.0188 h−1, and 0.0505 to 0.1524 h−1, respectively. kadakal et al. (2017), nisha et al. (2009), nisha et al. (2005), and rekha et al. (2004) have also reported an increment in degradation rate constant of b complex vitamins in different foods with an increase in process temperatures. figure 4 shows arrhenius equation of b complex vitamins’ thermal degradation. the eas of thiamine, riboflavin, niacin, and pyridoxine were found to be 67.90, 55.37, 78.78, and 65.41 kj mol−1, respectively. sensitivity of a reaction to temperature change can be explained with the ea as stated before. accordingly, niacin was more sensitive to temperature changes but shows the highest stability due to the highest ea value in comparison to other b complex vitamins in jujube fruits. nisha et al. (2005) have found the ea of riboflavin thermal degradation in cooked spinach to be 21.72 kj mol−1. kadakal et  al. (2017) have also reported that the eas of thiamine and riboflavin thermal degradation in rosehip were 36.38 and 37.15  kj  mol−1, respectively. nisha et al. (2009) have notified the ea of niacin in cooked potato cubes to be 16.70 kj mol−1. the eas of thiamine, riboflavin, niacin, and pyridoxine of jujube fruits were found to be higher than those reports. this means that thiamine, riboflavin, niacin, and pyridoxine in jujube fruits were more sensitive to temperature changes; however, they were more stable to thermal degradation when compared to those reports. on the other hand, niacin was the highest affected compound by 10°c temperature increment from 50 to 60°c due to the highest q10 value when compared to q10 values. kinetic parameters of tpc and ac the current study presented the first data on tpc thermal degradation in whole dried jujube fruits. tpc thermal degradation is shown in figure 5. it was found that tpc thermal degradation followed the firstorder kinetic model. akdaş and başlar (2015) have similarly reported a first-order reaction of tpc of mandarin during the oven drying process. sarpong et al. (2018) have notified that tpc thermal degradation in banana samples during convective drying was described using the first-order kinetic model. the result of the current study was in good agreement with reports by akdaş and başlar (2015) and sarpong et al. (2018). kinetic parameters of tpc thermal degradation are listed in table  7. rate constant of tpc thermal degradation showed an increment as drying temperatures increased. it was obvious that this increment caused a decrement in the values of t1/2 and d. rate constant of tpc thermal degradation ranged from 0.0332 to 0.0775 h−1. akdaş and başlar (2015), sarpong et al. (2018), and kadakal and duman (2018) have reported an increment in tpc thermal degradation in different foods with an increase in temperature. tpc thermal degradation was slightly affected by 10°c temperature increment. ea was calculated using the arrhenius equation as given in figure 6. the ea of tpc thermal degradation was calculated as 39.02 kj mol−1. sarpong et al. (2018) have reported the ea of convective dried banana at 60, 70, and 80°c as 14.29 kj mol−1. likewise, the eas of tpc thermal degradation in starking delicious, golden delicious, and granny smith apple cultivars dried at 65, 70, and 75°c were notified as 27.52, 29.84, and 32.48 kj mol−1, respectively (ertekin filiz and seydim, 2018). akdaş and başlar (2015) have reported the ea as 55.037 kj mol −1 for oven-dried mandarin at 55, 65, and 75°c. the result of the current study was higher than those reported by sarpong et al. (2018) and ertekin filiz and seydim (2018); however, it was lower than those reported by akdaş and başlar (2015). on the other hand, thermal degradation of tpc was not affected by the 10°c temperature increment. 12 italian journal of food science, 2021; 33 (1) begüm tepe and raci ekinci other hand, a 10°c temperature increment had no significant effect on ac thermal degradation. figure 6 presents the arrhenius equation of ac thermal degradation that was used for the calculation of the ea. the ea of ac thermal degradation was 44.68 kj mol−1. conclusions in the current study, drying characteristics and some quality parameters of the health-promising fruit, jujube (zizyphus jujuba mill.), in turkey were investigated under different drying conditions. 1. dr of jujube was highly influenced by the drying temperature. the longest drying time was found to be 48 h at 50°c, and the shortest was 18 h at 70°c. 2. the best predicting models of experimental mr were determined as parabolic model for 60°c and page model for 50 and 70°c. 3. effective moisture diffusivity showed an increment with an increase in the drying temperature. the most effective moisture diffusivity was obtained at 70°c. 4. when compared to dried jujube fruits regardless of the drying temperature, water-soluble vitamins, tpc, and ac of fresh jujube fruits were determined to be higher. while the highest loss of water-soluble vitamin occurred at 70°c because of a more rapid enzymatic and nonenzymatic degradation, tpc and ac were not significantly affected by the drying temperature. 5. vitamin c and b complexes, tpc, and ac thermal degradation were fitted to the first-order kinetic model. 6. vitamin c and niacin were very susceptible to temperature change. on the contrary, tpc and ac were the lowest sensitive compounds. ac thermal degradation could be described using the first-order kinetic model (başlar et al., 2014; oancea et al., 2017; sarpong et al., 2018). in contrast to this, orikasa et al. (2014) and ertekin filiz and seydim (2018) have reported that zero-order kinetic model may also be used to describe ac thermal degradation. to the best of our knowledge, ac thermal degradation in whole dried jujube fruits was investigated for the first time. ac thermal degradation is given in figure 5. in the current study, ac thermal degradation followed the first-order kinetic model. oancea et al. (2017), başlar et al. (2014), and sarpong et al. (2018) have reported the first-order kinetic of ac in sour cherry extracts, oven-dried pomegranate, and convective dried banana during the thermal process. kinetic parameters of ac thermal degradation are given in table 7. rate constants of ac thermal degradation increased with an increment in the drying temperature as expected. rate constant of ac thermal degradation was reported to increase with process temperature by oancea et al. (2017), başlar et al. (2014), and sarpong et al. (2018). accordingly, the values of t1/2 and d were reduced. on the (a) (b) 50°c 60°c 70°c 6050403020100 6050403020100 5 –1.4 –1.6 –1.8 –2 –2.2 –2.4 –2.6 –2.8 5.5 6 6.5 7.5 8 7 time (h) 50°c 60°c 70°c time (h) ln c ln c figure 5. first-order kinetics of total phenolic content (a) and antioxidant capacity (b) of whole dried jujube fruits. 4 3.5 2.5 4.5 3 2 1.5 1 0.5 0 2.9 3.153.13.0532.95 t–1×10–3 l n k tpc ac figure 6. arrhenius plots of total phenolic content and antioxidant capacity of whole dried jujube fruits. italian journal of food science, 2021; 33 (1) 13 drying charateristics of jujube fruits chin, s.k., siew, e.s. and soon, w.l., 2015. drying characteristics and quality evaluation of kiwi slices under hot air natural convective drying method. international food research journal 22(6): 2188–2195. choi, s.h., ahn, j.b., kim, h.j., im, n.k., kozukue, n., levin, c.e., et al., 2012. changes in free amino acid, protein, and flavonoid content in jujube (ziziphus jujube) fruit during 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campobasso, italy *corresponding author: tel.: +48 523749739, fax. +48 523228158 e-mail address: winiarska@utp.edu.pl abstract effects of sex and age on fatty acids profile in the meat of carassius spp. were evaluated. carassius auratus fillets displayed a higher content of sfa and mufa than carassius carassius. in contrast, carassius carassius had a higher content of pufa (higher proportion of linoleic, arachidonic, eicosapentaenoic, docosatetraenoic and docosapentaenoic acids) and total n-3 and n-6 pufa, compared with carassius auratus. moreover, γ-linolenic acid was higher in fillets from carassius auratus. what is more, fillets from females had a greater content of eicosapentaenoic acid than males. finally, 4-year-old fish had a lower content of ala, higher mufa content and better nutritional indexes (n-3/n-6, ai) than 3-year-old fish. keywords: age, carassius, fatty acids, sex ital. j. food sci., vol. 31, 2019 140 1. introduction fish meat is considered a high quality-food for human consumption. it is also considered as a functional food that can promote superior health (hosomi et al., 2012). fish can be a great source of high amounts of protein, vitamins and minerals and most of all polyunsaturated fatty acids (pufa) such as omega-3 (n-3) long-chain pufa, including eicosapentaenoic acid (c20:5 n-3, epa) or docosahexaenoic acid (c22:6 n-3, dha), considered as ‘essentials’ (huynh and kitts, 2009). in fact, these long-chain pufa can be synthesized by humans from α-linolenic acid (c18:3 n-3, ala), but due to low conversion efficiency, it is recommended to obtain epa and dha from additional sources, such as seafood. epa and dha are dietary fats with an array of health benefits. indeed, a daily intake of 250–500 mg of epa+dha decreases the risk of mortality from coronary heart disease and sudden cardiac death (efsa 2010). moreover, epa and dha are also the precursors of several metabolites that are potent lipid mediators, considered by many investigators to be beneficial in the prevention or treatment of several diseases (serhan et al., 2008). in contrast, epa in blood is an extremely potent antithrombotic factor. there are various studies, which have examined the effects of exogenous (shirai et al., 2002; luzia et al., 2003; guler et al., 2008; kalyoncu et al., 2009; jabeen and chaudhry, 2011) and endogenous (akpinar et al., 2009; jabeen and chaudhry, 2011) factors on the fatty acids composition of fish meat. it should be noted that fish body composition is determined by different factors, such as sex (akpinar et al., 2009), age (alemu et al., 2013), feed availability, fishing season, location of reservoir, type of tissue and species (kalyoncu et al., 2009, 2010; jabeen and chaudhry, 2011). to our best knowledge, no research has been found on the effects of age and sex on muscle fatty acid composition in two different species of carassius. thus, the aim of this study was to analyze the impact of sex and age on the fatty acids profile in the meat of carassius carassius l. and carsassius auratus gibelio bloch collected in the same exogenous conditions, in the autumn, from the lake gopło. 2. materials and methods 2.1. study area lake gopło is located in the southern part of kuyavian-pomeranian province. the western part of this lake is a strict nature reservoir. the main morphometric indicators of lake gopło are as follows; surface area: 22 km2, maximum depth: 16 m, average depth ca 4.7 m and length of shoreline: 90 km (łuczyńska et al., 2008). based on the limnological classification, it is an eutrophic reservoir, and, based on fishing classification, it is a zander type of the lake. 2.2. biological material collecting the study involved 56 individuals of carassius (28 individuals of carassius carassius l. and 28 carassius auratus gibelio bloch), selected according to the age (n= 28 for fishes > 3-yearold and n= 28 for fishes > 4-year-old) and sex (females: n= 28; males: n= 28). fish were caught in natural conditions (lake gopło), in autumn (november 2015) and body weight and length were determined for each fish (table 1). ital. j. food sci., vol. 31, 2019 141 table 1. body weight and length of carassius carassius l. and carassius auratus gibelio b. according to the age and sex. carassius carassius l. carassius auratus gibelio b. body weight (g) length (cm) body weight (g) length (cm) age >3 years 176.9-206.4 16.3-17.4 176.5-209.0 16.0-17.5 >4 years 267.1-297.5 18.0-19.6 255.3-317.0 19.5-20.8 sex male 176.9-287.8 16.3-19.1 176.5-288.6 16.0-19.8 female 178.6-297.5 16.6-19.6 175.1-317.0 16.4-20.8 the fillet samples for analyses were taken from the large side muscle of the fish body above the lateral line. the samples were vacuum packaged and stored frozen (-20°c) until analyzed for fatty acid composition. 2.3. fatty acids analyses lipid extraction from muscle samples was performed by modification of the bligh and dyer (1959) and folch et al. (1957) methods. briefly, 100 mg of freeze-dried muscle was treated with chloroform/methanol/water (1:2:0.8) and stirred for 4 hours. then, to obtain a better separation between the two phases, chloroform, 2n kcl/0.3 n hcl, and h2o were added consecutively, and the samples were centrifuged for 5 min at 3000 g. the lower lipid-containing phase was separated from the upper phase, and retained for use. subsequently, another extraction with chloroform was repeated. the extracted lipids were esterified and then analyzed by gas chromatography (gc trace 2000 thermoquest ec instruments) equipped with a flame ionization detector (260°c) and a fused silica capillary column (omegawax 320, phenomenex, torrance, ca, usa) 30 m x 0.32 mm x 0.25 μm film thickness. helium was used as the carrier gas at a flow rate of 1.0 ml/min with constant flow compensation. gc inlets were held at a temperature of 240°c, and the detector was maintained at a temperature of 250°c. the oven temperature was programmed from 150°c, and was followed by a ramp-up at a rate of 5°c/min till 240°c with a final hold of 15 min (the total analysis time was 33.00 min). the individual fatty acids peaks were identified by comparison of retention times with those of known mixtures of standard fatty acids (pufa-2, supelco, bellofonte, pa, usa) run under the same operating conditions. results were expressed as percentage of the total fatty acids identified. an example of gas chromatography analysis (gc profile) is presented in fig. 1. to assess the nutritional implications, the n-6 pufa/n-3 pufa and the pufa/sfa ratios were calculated. the group of the fatty acids analyzed included saturated fatty acids (sfa) (c14:0, c16:0, c18:0), monounsaturated fatty acids (mufa) (c16:1 n-7, c18:1 n-9, c18:1 n7, c20:1 n-9,) and polyunsaturated fatty acids (pufa) (c18:2 n-6, c18:3 n-6, c18:3 n-3, c20:3 n-3, c20:4 n-6, c20:5 n-3, c22:4 n-6, c22:5 n-3, c22:6 n-3). in order to evaluate the risk of atherosclerosis and the potential aggregation of blood platelets, respectively, the atherogenic index (ai) and the thrombogenic index (ti) were calculated, according to the formulas suggested by ulbricht and southgate (1991). ai=[12:0+(4×14:0)+16:0]/[n6 pufa+n-3pufa+mufa]; ti=[14:0+16:0+18:0]/[(0.5×mufa)+(0.5×n-6 pufa)+(3×n3pufa)+(n-3 pufa/n-6 pufa)]. ital. j. food sci., vol. 31, 2019 142 figure 1. an example of gas chromatography profile. 2.4. statistical analyses fatty acid composition and nutritional ratio data were analysed by 2×2×2 factorial anova with interactions, where species, sex and age were the main factors. each individual fish was considered as the experimental unit. data analysis was performed using the spss package (spss, 2010). data are presented as means±sem, and a value of p<0.05 was used to indicate statistical significance. 3. results the fillet fatty acids composition and nutritional ratios are presented in table 2. overall, the mufa were the most abundant acids (39.86±0.42%), followed by sfa (31.79±0.39%) and pufa (27.22±0.38%). the factor of greatest relevance affecting sfa content was fish species. c. auratus recorded significantly higher (+3%) sfa concentration compared to c. carassius (p<0.001), and particularly, higher proportions (p<0.001) of myristic (c14:0) and stearic (c18:0) acids, as well as palmitic acid (c16:0; p>0.05). as analyses indicated, sex did not significantly affect the total content of sfa or the content of the single sfa. the age factor affected only the palmitic acid content, which was higher (p<0.05) in fillets from 3year-old fish compared to 4-year-old ones. age did not influence the total content of sfa. in general, the palmitic acid was the most abundant (25.7±0.37%) among sfa, followed by the stearic (4.0±0.10%) and myristic acids (2.1±0.09%). ital. j. food sci., vol. 31, 2019 143 table 2. effects of species, sex and age on fatty acids composition (% of total fatty acids) and nutritional ratios of fish fillets. fatty acids‡ species (sp)† sex (s) age (a) sem significance cc ca male female >3 years >4 years sp s a spxs spxa sxa spxsxa c14:0 1.65 2.56 1.99 2.22 2.14 2.07 0.09 *** ns ns ns * ns ns c16:0 25.14 26.23 25.48 25.90 26.46 24.92 0.37 ns ns * ns ns ns ns c18:0 3.50 4.50 4.13 3.87 3.91 4.08 0.10 *** ns ns ns ** ns ns c16:1 3.66 3.02 3.41 3.27 3.49 3.19 0.05 *** ns ** ns ns ns ns c18:1n-9 28.06 29.49 28.75 28.80 27.22 30.33 0.41 ns ns *** ns ns ns ns c18:1n-7 5.72 7.11 6.36 6.48 6.52 6.32 0.11 *** ns ns ns ns ns ns c20:1n-9 1.31 1.35 1.24 1.41 1.32 1.33 0.05 ns ns ns ns ns ns ns c18:2n-6 10.72 8.23 9.57 9.38 9.84 9.11 0.19 *** ns ns * ns ns ns c18:3n-6 0.26 0.36 0.32 0.30 0.32 0.30 0.02 * ns ns ns ns * ns c18:3n-3 0.18 0.24 0.23 0.19 0.26 0.16 0.02 ns ns * ns ns ** ns c20:3n-3 0.43 0.37 0.43 0.37 0.43 0.37 0.03 ns ns ns ns ns ns ns c20:4n-6 4.95 4.41 4.60 4.76 4.85 4.51 0.10 * ns ns ns ns ns ns c20:5n-3 1.67 0.57 0.95 1.29 1.15 1.09 0.06 *** ** ns ** ns ns ** c22:4n-6 0.32 0.24 0.29 0.28 0.30 0.26 0.01 * ns ns ** ns ns ns c22:5n-3 1.12 0.72 0.97 0.86 0.90 0.94 0.05 *** ns ns * ns ** ns c22:6n-3 10.13 9.52 9.80 9.85 9.62 10.02 0.17 ns ns ns ns ns ns ns partial sums sfa 30.28 33.29 31.59 31.99 32.51 31.07 0.39 *** ns ns ns ns ns ns mufa 38.76 40.97 39.76 39.96 38.56 41.17 0.42 * ns ** ns ns ns ns pufa 29.79 24.66 27.16 27.29 27.68 26.77 0.38 *** ns ns ** ns ns ns total n-6 16.25 13.25 14.78 14.72 15.31 14.19 0.25 *** ns * * ns ns ns total n-3 13.53 11.41 12.38 12.57 12.36 12.58 0.22 *** ns ns ** ns ns ns nutritional ratios n-3/n-6 0.84 0.87 0.85 0.87 0.82 0.90 0.02 ns ns * ns ns ns ** p/s 1.00 0.75 0.88 0.87 0.87 0.88 0.02 *** ns ns ** ns ns ns ai 0.46 0.56 0.50 0.52 0.53 0.49 0.01 *** ns * ns ns ns ns ti 0.44 0.54 0.49 0.50 0.51 0.47 0.01 *** ns ns * ns ns ns †cc = carassius carassius l.; ca = carassius auratus gibelio bloch; ‡sfa = saturated fatty acids; mufa = monounsaturated fatty acids; pufa = polyunsaturated fatty acids; p/s = pufa/sfa ratio; ai = atherogenic index; ti = thrombogenic index. significance: *** p<0.001; ** p<0.01; * p<0.05; ns: not significant. ital. j. food sci., vol. 31, 2019 144 age effect was evident in the oldest c. auratus fish for both myristic and stearic acid contents, which were in lower (p<0.05 and p<0.01) amounts in 4-year-old fish. the total mufa content was higher (p<0.05) in c. auratus fillets compared to those of c. carassius (+2.21%), with higher (p<0.001) content of vaccenic acid (c18:1n-7) and slightly higher (p>0.05) oleic acid (c18:1n-9). in contrast, percentage content of palmitoleic acid (c16:1) was lower in c. auratus fillets compared to those of c. carassius (p<0.001). sex did not significantly affect total mufa and the proportions of the single mufa. as opposed to c. carassius, age has been a relevant factor for the total mufa content. in fact, with the increase of the age of the fish, total mufa content increased (<0.01), as did oleic acid (p<0.001), whereas palmitoleic acid decreased (p<0.01). c. carassius also had higher content of total pufa (+5.13%), in comparison with fillets of c. auratus, and this difference was statistically significant at p<0.001. a higher percentage (p<0.05 and p<0.001) content of linoleic (la, c18:2n-6), arachidonic (aa, c20:4n-6), eicosapentaenoic (epa, c20:5n-3), docosatetraenoic (dta, c22:4n-6) and docosapentaenoic (dpa, c22:5n-3) acids, and content of total n-3 and n-6 fatty acids was found in the fillets of c. carassius, while γ-linolenic acid (gla, c18:3 n-6) was higher (p<0.05) in fillets from c. auratus. a high content of docosahexaenoic acid (dha, c22:6 n3) was also detected in both species, but no significant differences were found. sex and age had a minimal influence on pufa composition. fillets from females had a higher (p<0.01) content of epa than males. comparison among ages showed that fillets of 3-year-old fish had a higher (p<0.05) content of α-linolenic (ala, c18:3 n-3) and total n-6 fatty acids, compared to those of 4-year-old fish. significant interactions were found between species and sex for total pufa (p<0.01), la (p<0.05), epa (p<0.01), dta (p<0.01), dpa (p<0.05), total n-6 (p<0.05) and n-3 (p<0.01), between sex and age for gla (p<0.05), ala (p<0.01) and dpa (p<0.01), as well as among species, sex and age for epa (p<0.01). analyses indicated that the n-3/n-6 ratio (ranging from 0.82 to 0.90) was affected only by the age, being higher (p<0.01) in 4-year-old fish than in 3-year-old. in addition, significant interaction was found among species, sex and age in n-3/n-6 ratio (p<0.01). the pufa/sfa ratio was affected only by species, being higher (p<0.001) in c. carassius than in c. auratus fish. however, a significant interaction (p<0.01) was found among species and sex for pufa/sfa ratio. in the present study, ai and ti were lower (p<0.001) in c. carassius than in c. auratus fish, and ai was lower (p<0.05) in 4-year-old fish than in younger individuals. in addition, a significant (p<0.05) interaction was found among species and sex for ti. 4. discussion the results on the fillet fatty acids composition (31.79% of sfa, 39.86% of mufa and 27.22% of pufa) are in line with the values obtained by kołakowska et al. (2000) in roach (rutilus rutilus l.), caught in the odra and regalica river (poland), with higher amounts of total sfa (34.98%) and mufa (46.83%) than total pufa (18.19%). different trends were reported by polak-juszczak and komar-szymczak (2009), on roach caught from the vistula lagoon (25.10% of sfa, 32.49% of mufa and 42.41% of pufa), and by stanek et al. (2008) on perch (perca fluviatilis l.) from włocławski reservoir (46.02-47.53% of sfa, 23.83-32.24% of mufa and 21.73-28.56% of pufa). regarding the proportion of single sfa, palmitic acid was the most abundant, followed by stearic and myristic acids, respectively. the highest presence of palmitic acid (14.6-16.6%) was found in the meat of carp (cyprinus carpio l.), caught in all four seasons (guler et al., 2008). in the meat of roach originating from the mazurian great lakes region (poland), palmitic acid content was the dominant sfa (25.38%) and the total amount of sfa for this ital. j. food sci., vol. 31, 2019 145 species accounting for 35.38% (łuczyńska et al. 2008). the same results were confirmed by özogul et al. (2007) for the fish called kutum (rutilus frisii) caught in the seyhan dam lake in adana (turkey). it is reported that sfa with 12-16 carbon atoms increase serum concentrations of ldl cholesterol (mensink and katan, 1992). in particular, palmitic acid increases total serum cholesterol, but its effect is lower than that of the myristic acids (daley et al., 2010), which, in the present study, is approximately 2%. stearic acid is considered a “neutral” fatty acid since it does not affect the plasmatic level of ldl or hdl cholesterol in humans (mensink and katan, 1992; williamson et al., 2005). this effect of stearic acid has been attributed to its reduced digestibility and easy desaturation into oleic acid. from a nutritional point of view, the oleic acid, the most common mufa present in considerable quantities in both animal and plant sources, has a relevant importance in the human diet because it acts on lipaemia, reducing both ldl cholesterol and the triglycerides (mozaffarian and clarke, 2009) and providing other health benefits (reviewed in sales-campos et al., 2013). in the present study, oleic acid ranged from 27.2% to 30.3 %, with the highest values in the fillet of 4-year-old fish. regarding the composition of the single pufa, the high amount of dha in comparison with the low level of α-linolenic acid observed in carassius is in agreement with the results obtained by murzina et al. (2016). dha cannot be synthesized de novo in vertebrates, it is biosynthesized from its precursor α-linolenic acid during desaturation and elongation on biochemical pathways (steffens and wirth, 2005; tocher, 2015). it is well known that the freshwater food system contains higher levels of linoleic and α-linolenic acids (tocher, 2003, 2010) leading to evident differences between freshwater and marine fish in terms of fatty acids distribution. in fact, the marine food chain is rich in epa and dha. however, some fish species from freshwater can also be a valuable source of epa and dha (steffens and wirth, 2005; reiter and grimm, 2012), which derive from freshwater plankton (brett et al., 2009) and by endogenous metabolism (tocher, 2003). the dominant pufa found in roach from brda river (poland) was linoleic acid (7.41-10.11%) (stanek et al., 2012). other studies in the meat of roach have found, among pufa, the highest proportion of epa, ranging from 7 to 12% (ahlgren et al., 1994), 10% (grahl-nielsen et al., 2011) and 9% (uysal et al., 2008). however, variable values (ranging from 1.54 to 20.15%) were also found in 9 freshwater fish species from the tigris river (cengiz et al., 2010). a diet rich in n-3 epa is believed to shift the physiological state to one that is less inflammatory than that of a diet containing high amounts of n-6. in addition, marine-derived omega-3 fatty acids are recommended for the treatment and prevention of many chronic diseases, including cardiovascular disease and metabolic syndrome (harris et al., 2010). as analyses indicated, sex and age of carassius had a minimal influence on pufa composition. analyses carried out by stanek et al. (2012), concerning roach caught in fall and spring, indicated that the total pufa content in the fish meat, ranged from 19.96 to 27.42%, and was not affected by sex. concerning the nutritional ratios, the n-3/n-6 ratio (ranging from 0.82 to 0.90) was affected only by age, being higher in 4-year-old fish. the value of n-3/n-6 ratio was found low (0.23) in the fillet of nile tilapia (oreochromis niloticus) (herath et al., 2016) and in common carp (0.47) reared in natural temperatures with water from lake trasimeno (geri et al., 1995). guler et al., (2008) found in fillets of carp caught from lake beysehir, values of n-3/n-6 ratio near 1 in winter, spring and summer and 0.5 in autumn. in contrast, in the meat of carp, caught in ivriz dam lake, n-3/n-6 ratio were found to be 1.08, 1.43, 1.64 and 1.60 in spring, summer, autumn and winter, respectively (kalyoncu et al., 2010). the n-3/n-6 ratio has been suggested to be a useful indicator for comparing relative nutritional values of fish oils (pigott and tucker, 1990). furthermore, an ital. j. food sci., vol. 31, 2019 146 increase in the human dietary n-3/n-6 fatty acids ratio is essential to prevent cardiomyopathy (by reducing plasma lipids) and to reduce cancer risk. until recently, the dietary balance between n-3 and n-6 pufa has been evenly balanced with a n-3/n-6 ratio of approximately 1; the modern western diet is now dominated by a n-3/n-6 ratio of approximately 0.06 (simopoulos, 2002). the ratio of n-3/n-6 pufa in lipids of freshwater fish varies mostly between 0.5 and 3.8, whereas it varies between 4.7 and 14.4 in marine fish (guler et al., 2008). the pufa/sfa is used for the assessment of lipids on the basis of the proportions of the different fatty acid groups. in our study, the pufa/sfa ratio was affected only by species, being higher in c. carassius than in c. auratus fish. the pufa/sfa ratios obtained in the present study are similar to those reported in fillets of nile tilapia (0.83–1.32; herath et al., 2016). however, the pufa/sfa ratio value of the fillets of the present study, ranged from 0.75 to 1.00, is well above the minimum value of 0.45 recommended by the department of health of the uk (hmso, 1994) and confirms that these freshwater fish species are suitable for human consumption. the ai and ti represent the criteria for evaluating the level and interrelation through which some fatty acids may have atherogenic or thrombogenic properties, respectively. in particular, these indexes take into account the different effects that single fatty acid might have on human health, and, in particular, on the probability of increasing the incidence of pathogenic phenomena, such as atheroma and/or thrombus formation (garaffo et al., 2011). in the present study, ai and ti were lower in c. carassius than in c. auratus fish, and ai was lower in 4-year-old fish than in younger. the ai (ranging from 0.46 to 0.56) and ti (ranging from 0.44 to 0.54) values found in the current study can be considered low according with literature reports (ulbricht and southgate, 1991; jankowska et al., 2010; stanek et al., 2012), and not comparable with the values reported in the meat of ruminants (ai: 1.29±0.26, ti: 1.54±0.179, d’alessandro et al., 2012) but is similar to poultry meat (ai: 0.56±0.13, ti: 0.55±0.14, laudadio and tufarelli, 2010; ai: 0.43±0.02, he et al., 2015; ai: 0.42±0.01, tavaniello et al., 2018). 5. conclusions in conclusion, the results of this study indicate that the meat from c. auratus and c. carassius is characterized by a favourable fatty acids profile from the human health point of view. considering that fish were caught in the same season, sex had a negligible effect on fatty acids composition, while species and age had a marked effect on it. furthermore, c. auratus fillets displayed significantly higher contents of sfa and mufa, and a lower content of pufa compared to those of c. carassius. fillets of 4-year-old fish had a lower content of ala, higher mufa 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26, 2018 accepted september 9, 2018 ijfs#1414_bozza ital. j. food sci., vol. 31, 2019 323 paper lipid autoxidation of fish, lard, corn and linseed oils by isothermal calorimetry n. haman, m. bodner, g. ferrentino and m. scampicchio* free university of bolzano, faculty of science and technology, piazza università, 1, 39100 bolzano, italy *corresponding author: tel.: +39 0471017210, fax: +39 0471017009 e-mail address: matteo.scampicchio@unibz.it abstract this work proposes an approach to characterize lipid oxidation of oils based on the measurement of isothermal calorimetry data, including information on the duration of the monomolecular, bimolecular and termination periods. the approach has been exploited with fish and lard oil samples at temperatures from 40 to 80°c and with corn and linseed oils at 80°c. the length of the monomolecular period was the most sensitive to the variation of temperature. accordingly, the monomolecular period was used as index of oxidative stability of oils. thus, the highest oxidative stability was observed for corn oil samples at 80°c (tmono = 2060 min), followed by linseed (tmono = 390 min), fish (tmono = 40 min) and lard oils (tmono = 30 min). the different stability of the samples was discussed with their fatty acids profile and antioxidant activity. the results confirmed that the content in natural antioxidants is the key responsible for the final oxidative stability of the samples. keywords: autoxidation periods, fish oil, edible oils, isothermal calorimetry, oxidative stability ital. j. food sci., vol. 31, 2019 324 1. introduction lipid autoxidation is a radical chain mechanism that involves a reaction between unsaturated fatty acids and oxygen (johnson and decker, 2015; tuorila and cardello, 2002). this reaction is of great importance for the food industry, as its occurrence is often associated with quality defects, like rancidity or nutritional loss. the mechanism of reaction is generally described in three main stages: an “initial pahse”, comprising the monomolecular phase of hydroperoxide formation. this is followed by the “propagation phase”, where radicals are formed with autocatalytic, monomolecular and bimolecular or branching reactions, resulting in the exponential increase of the rate of lipid autoxidation. then, the process ends with a “termination phase”, where the rate of lipid reaction slow down and increase the formation of secondary oxidation products (ghnimi, 2017). between the first and second phase, the abrupt change in the rate of lipid autooxidation is often defined as the induction period (ip). this value is widespread used by food industry as index to characterize the oxidative stability of an oil sample (frankel, 2014). the measurement of the induction period is generally obtained by differential scan calorimetry (dsc) (shahidi and zhong, 2015; litwinienko et al., 1999) or differential thermal analysis (dta) (bacha et al., 2015). however, the ip value obtained with these techniques is often difficult to interpret when the samples have a complex composition. one drawback of dsc is that, at low temperatures, the exothermic peak of oxidation is flat, and the ip value cannot be determined unambiguously. moreover, when dsc is run at high temperatures, the oxidative behavior of foods become dependent on the temperature gradient used and also on the geometry of the samples (fox and stachowiak, 2007; kousksou et al., 2011). furthermore, the analysis of oil samples that contain antioxidants at high temperatures may lead to misleading results when the antioxidants are volatile and can leave the sample before they can exert their protective action (hamama and nawar, 1991). an alternative approach for the analysis of lipid autoxidation is offered by isothermal calorimetry (haman et al., 2017a). this technique has some advantages respect dsc and dta, as it can be used to monitor in real-time the rate of autoxidation of unsaturated fatty acids under mild temperatures. its theoretical background is relatively simple and welldescribed by willson (willson et al., 1995) and hansen (hansen et al., 1990). recent applications of isothermal calorimetry include the study of oxidation of lipid oxidation (dridi et al., 2016), the study of the radical scavenging activity of lipophilic antioxidants (haman et al., 2017b), antioxidant and pro-oxidant activity of spent coffee extracts (haman et al., 2018) and the study of shelf-life of foods (riva et al., 2001). however, in these studies, the calorimetric trace was mainly used to provide the cumulative enthalpy evolution of the sample, or the induction time. in such way, only a small fraction of the information contained in the calorimetric trace is used. for this reason, this work aims to propose a methodological approach that can extend the information normally achievable with calorimetric data, providing information about the duration of the monomolecular, bimolecular and termination phase of the lipid autoxidation process. for this purpose, fish and lard oil samples were analyzed by isothermal calorimetry at different temperatures (from 40 to 80°c), and the results compared with those obtained with linseed and corn oils at 80°c. the relevance and utility of this approach was discussed considering the fatty acid composition and the antioxidant capacity of the oils. ital. j. food sci., vol. 31, 2019 325 2. material and methods 2.1. materials refined corn oil, fish oil, lard oil, cyclohexane (purity ≥ 99%), sodium thiosulphate (purity ≥ 99%) and potassium iodide (purity ≥ 99%) were purchased from sigma-aldrich (milan, italy). refined linseed oil (cold pressed) was from sabo italia s.r.l. (firenze, italy). chloroform (stabilized with ethanol) was purchased from panreac applichem (milan, italy). acetic acid (purity ≥ 99.7%) was purchased from fluka (milan, italy). 2.2. fatty acid profile by near infrared spectroscopy fatty acids were determined by near infrared (nir) spectrometer with a multipurpose analyzer (mpa) (bruker optik gmbh, germany). acquisition software was opus 7.5 (bruker optik gmbh, germany). the background spectrum consisted of empty cell at the same temperature of analysis. opus/quant package was used to quantify the composition of fatty acids (palmitic, stearic, oleic, linoleic and linolenic acid) in fish, lard, linseed and corn oils. the analysis was performed at the same conditions of the calibration kit (calibrations based on 8084 spectra) in the range of 12500-4000 cm−1 (resolution, 16 cm−1; sample, 64 scans) at 40°c. 2.3. isothermal calorimetry a micro-calorimeter (thermal activity monitor, model 421 tam iii, ta instruments), equipped with 24 channels, was used to measure the heat rate. each channel of the instrument is a twin calorimeter where the two units are positioned above each other. in isothermal mode, the oil in the thermostat was maintained at a constant temperature with an absolute accuracy of ±0.0005 °c (manufacturer’s data). the micro-calorimeters were equipped with built-in metal reference specimens having a heat capacity approximately equal to that of a vial. the heat generated or absorbed was continuously measured over time. following the manufacturer’s instructions, each channel was calibrated prior to measurement using a gain calibration procedure with electric impulses. ampoules were first lowered into the thermal equilibration position and left there for 15 min. then, the ampoules were lowered into the measurement position and the heat flow rates recorded for up to 5 and 8 days at 10-second intervals. the auto-oxidation reaction was monitored by calorimetry with close glass ampoules containing 100±3 mg of oil in isothermal conditions in the presence of air in the head space. 2.4. dpph assays for the determination of the antioxidant activity the antioxidant activity of the oils was evaluated using 1,1-diphenyl-2-picrylhydrazil (dpph) as described by brand williams (brand-williams et al., 1995) with small modifications. dpph is a free radical that can react directly with most of the antioxidants and be captured by them (koleva et al., 2002). the reduction of dpph was measured by the decrease in absorbance at a characteristic wavelength (517 nm) and a determined time during the reaction (minimum 60 min). in the radical form (dpph•) presented a violet color at 517 nm. with the addition of a reduced antioxidant (ah), its color changed from violet to yellow and consequently the absorption decreased as a result of the decrease of the dpph concentration. briefly, 10 mg of dpph were dissolved in 250 ml of ethanol and sonicated for 1 min. a defined amount of oil was dissolved in 1 ml ethanol and sonicated ital. j. food sci., vol. 31, 2019 326 (du-06, china) for 5 min. to perform the measurements, 1.9 ml of dpph solution were transferred into the cuvettes and 100 µl of the extracts solutions were added. the samples were kept in dark for 1 h at room temperature (ca. 25 °c) and subsequently the absorbance was recorded at 517 nm with a spectrophotometer (cary 100 series uv-vis spectrophotometer, agilent technologies, italy). the antioxidant capacity of the oils was determined as the ic50 the concentration of antioxidant, which reduces the radical form (dpph•) of 50%. a triplicate assay was performed for each sample. 2.5. determination of peroxide value the peroxide value (pv) of the samples was measured according to the aoac official method 965.33 (horwitz, 2002). the method is based on an iodometric titration, which measures the iodine produced from the potassium iodide by the peroxides present in the oil. the measurements were performed in triplicate for each sample and pv values were expressed as meqo2 per kg of oil. 2.6. statistical analysis calorimetric results were reported as the mean of six independent experiments. all other measurements (nir and dpph) were performed in triplicate (n = 3). all the results are reported as mean, together with the relative standard deviation. to compare the means values, data were subjected to the analysis of variance (anova) using fisher’s least significance difference method with a 95 % confidence level. 3. results and discussion 3.1 oxidative stability based on monomolecular and bimolecular periods figure 1 shows the heat-flow signal of a fish oil sample recorded at 40°c. at any point, the intensity of the heat-flow signal reflects the rate of the autoxidation process that is occurring to the sample, accordingly to the following general relationship (eq. 1) (willson et al., 1995): heat flow = rate x enthalpy (1) at the beginning of the experiment, the signal is negligible (from the beginning to the point (a), fig. 1). this corresponds to a slow autoxidation process. at about 25 h (point (a) of fig. 1), the rate of heat production suddenly increases, at point (b), reaching a maximum heat flow value at point (c). after that, the signal fades down to a minimum steady state, at point (d). the final heat-flow signal has an intensity close to that recorded at the beginning of the experiment. figure 1 also shows the changes in the apparent reaction order as a function of time. the apparent reaction order was determined at any time following an approach previously described by haman (haman et al., 2017a). briefly, for a reaction a à b, where a represents unsaturated fatty acids and b is their oxidation products, the heat-flow signal (dq/dt) is equal to k·h·xn, where k is the rate constant, h is the reaction enthalpy, x is the number of moles of product formed at time t and n is the reaction order. since x can be obtained, at any time, as the ratio between the heat (q) and the enthalpy (h), then this equation becomes (eq. 2): ital. j. food sci., vol. 31, 2019 327 𝑑𝑞 𝑑𝑡 = 𝑘 · 𝐻 · 𝑞 𝐻 ! = 𝑘 · 𝐻!!! · 𝑞! the log-log plot of the heat flow with the heat leads a slope, which corresponds to the reaction order (n). thus, by following the changes of n as a function of time, it is possible to identify three distinct phases of the autoxidation process. for fish oil, the first phase is characterized by a reaction order equals to ~ 0.5. this order corresponds to the monomolecular period, during which the reaction is catalyzed by monomolecular decomposition of the hydroperoxides (looh + lh → lo* + l* + h2o) (labuza et al., 1969; ghimni et al., 2017). the duration of such phase lasts in about 25±0.3 h. interestingly, this phase ends with the onset point of the calorimetric trace (also called induction point). this point is very informative since can be interpreted as the moment when a food become unacceptable for human consumption (labuza and dugan jr, 1971). after this point, the reaction order becomes close to 1. it is during this period that the rate of lipid autoxidation rises exponentially. in this exponential stage, the bimolecular decomposition of hydroperoxides is the dominant mechanism (looh + looh → lo* + loo* + h2o). this process is known to follow the bimolecular period (labuza and dugan jr, 1971). the length of such phase lasts in about 25±0.3 h (from point (a) to point (b) of fig. 1). figure 1. calorimetric trace of fish oil in isothermal conditions at 40°c. legend: (a) end of the monomolecular phase; (b) end of the bimolecular phase; (c) the maximum heat flow and (d) the end of the termination phase; legend: m stands for monomolecular phase; b stands for bimolecular phase and t stands for termination phase. after this point, the reaction order, suddenly, declines towards negative values. negative reaction orders correspond to the so-called “termination phase”, where the rate of the reaction is either inhibited by the high concentration of the reactants (i.e. radicals species) or by the absence of oxygen in the ampoule. in this stage, radical-radical combinations occur, and hydroperoxides start to exponentially decompose to secondary oxidation products, including aldehydes and ketones (ghinmi at al., 2017). in the experimental ital. j. food sci., vol. 31, 2019 328 setup used here, the termination phase occurred when the concentration of oxygen in the ampoule was limiting. this was verified in a control experiment, where, successively to the analysis of a fish oil sample, the ampoule was opened without removing the sample, fresh air was left to enter into the headspace of the ampoule, and the recording of the calorimetric signal re-started. in such way, we observed that a new exothermic peak could be observed. this result confirms that the termination phase was limited by oxygen and that further monomolecular and bimolecular process could occur provided that oxygen is available. accordingly, the termination period of the observed autoxidation process in the ampoule was defined as the period occurring from point (b), where the reaction order starts decreasing, to point (d), where the reaction is likely terminated because of oxygen depletion in the headspace of the ampoule. at times above 80 h, the reaction was completed. the measure of the reaction order become unpredictable because of the heat flow values were nearly constant, with slopes equals nearly to zero. 3.2. oxidative stability of corn and linseed oil the same approach used before with fish oil was next applied to characterize the oxidative stability of different oil samples, including fish, lard, linseed and corn oils. all the sample showed similar initial peroxide values. the resulting heat flows were plotted as a function of time (fig. 2). figure 2. heat flow vs. time curve plot of a) fish oil, b) lard oil c) linseed oil and d) corn oil, placed isothermally at 80°c into closed glass ampoule. table 1 reports the duration of the monomolecular, bimolecular and termination step. by comparing the duration of the monomolecular period at 80°c, the highest oxidative stability was observed for the corn oil sample (2060 min), followed by linseed (390 min), fish (50 min) and lard (30 min) oil samples. ital. j. food sci., vol. 31, 2019 329 table 1. kinetic and thermodynamic parameters of fish, lard, corn and linseed oil. phase duration oil temperature (°c) monomolecular (min) bimolecular (min) termination (min) δh (kj/mol o2) fish 40 2220±20 802±10 1780±35 43.1±0.1 fish 50 600±8 653±11 1102±24 59.3±0.4 fish 60 220±2 450±4 802±8 71.9±0.5 fish 70 90±4 311±2 648±6 84.0±0.6 fish 80 40±3 224±4 470±9 97.8±0.3 lard 40 2490±35 1519±25 5161±71 36.4±0.3 lard 50 550±2 190±3 3120±59 47.8±0.7 lard 60 200±3 121±3 2402±53 66.2±0.9 lard 70 600±3 60±2 1570±31 74.7±0.8 lard 80 30±4 104±18 1143±21 67.7±0.5 linseed 80 390±7 1102±18 1681±31 77.3±0.2 corn 80 2060±25 1121±11 1160±13 46.2±0.1 furthermore, table 1 shows the changes of the monomolecular, bimolecular and termination durations as a function of temperature. for fish and lard oil samples, higher temperatures corresponded with faster reaction rates and, consequently, shorter duration of these periods. moreover, the relationship between the duration of such phases follows the so-called arrhenius function, where the logarithm of the duration of each phase leads a linear relationship when plotted as a function of the inverse of temperature (ln(t) vs 1/t). for fish oil samples, the slopes of such linear relationships were 1.1·104 k, 3.6·103 k and 3.5·103 k, respectively, for the monomolecular, bimolecular and termination phase. similar results were obtained for lard oil samples. the slopes were in this case equal to 1.2·104 k, 3.7·103 k and 4.1·103 k, respectively, for the monomolecular, bimolecular and termination phase of lard oil samples. these values suggest that the most sensitive phase to the changes in temperature is the monomolecular phase. this information is important for food manufacturers, since it provides an evidence that the antioxidants needed to stabilize such oils are those with the highest capacity to retard or stabilize the monomolecular phase. last experiments aimed to understand the reasons of the different oxidative stability observed between the oil samples. the ranking observed cold be explained considering either the quality of fatty acids composition (i.e. presence of monoor poly-unsaturated fatty acids) or the antioxidant content of the samples. table 2 reports the fatty acids profile and the antioxidant content of the oil samples. what is striking is that the ranking of oxidative stability of the oil samples obtained according to the duration of the monomolecular phase cannot be predicted simply by looking at the polyunsaturated fatty acid composition of the samples. instead, the oxidative stability of the samples matches perfectly with the antioxidant capacity determined by the dpph assay on the methanolic extracts of the samples. in practice, the oil samples with the highest oxidative stability (i.e. longer duration of the monomolecular phase, as determined by calorimetry) are also those with the highest antioxidant capacity (i.e. shorter value of the dpph assay). ital. j. food sci., vol. 31, 2019 330 table 2. fatty acid composition, peroxide value and antioxidant capacity of fish, lard, corn and linseed oil. corn oil linseed oil fish oil lard oil fatty acids (%, w/w) palmitic acid (16:0) 14.7±0.6b 6.3±0.5c 15.6±1.4b 26.6±1.6a stearic acid (18:0) 2.9±0.3b 4.2±0.6b 3.5±0.8b 10.7±0.9a oleic acid (18:1) 30.8±1.8b 18.8±1.6c 11.1±0.9d 46.8±2.8a linoleic acid (18:2) 48.4±1.7a 17.9±1.3b 2.5±0.5d 9.3±0.7c linolenic acid (18:3) 1.1±0.2b 50.4±3.1a 1.5±0.3b 1.8±0.2b total sfa 17.6±0.5b 11.1±0.7b 23.1±1.1b 37.3±1.5a total pufa 49.5±2.3b 68.3±3.9a 51.7±2.3b 11.1±0.6c total mufa 30.8±1.4b 19.2±1.8d 22.6±2.1c 49.6±2.4a others 2.1±0.2a 1.4±0.4a 2.6±0.6a 1.9±0.2a peroxide value (meqo2/kg oil) 2.9±1.2 b 1.6±0.5c 4.2±0.3a 5.4±1.7a dpph (ic50) 70.3±1.5 d 83.9±2.7c 251.8±6.4a 98.9±3.4b legend: sfa is saturated fatty acids; pufa is polyunsaturated fatty acids; mufa is monounsaturated fatty acids. values with different letters in the same raw are significantly different (p < 0.05). values are presented as mean±standard deviation (n = 3). 4. conclusions the results of the present study show the potential of isothermal calorimetry to monitor the oxidation processes of edible oils and exemplify a procedure to determine the monomolecular, bimolecular and termination periods according to the changes in the apparent reaction order of the overall process. in addition, we observed that the monomolecular phase was the most sensitive to the changes in temperature. this implied that the duration of the monomolecular phase is very informative to explain the oxidative stability of an oil sample. according to corn, linseed, lard and fish oil samples, their oxidative stability was not correlated with their qualitative composition in fatty acids. instead, their oxidative stability was explained with their antioxidant capacity. on a more general perspective, this work demonstrated the potential of isothermal calorimetry to predict the oxidative stability of oil samples and characterizing the duration of the monomolecular, bimolecular and termination phase of the autoxidation process. acknowledgements we are grateful to the province of bolzano for financial support (landesregierung mittels beschluss nr. 1472, 07.10.2013). references bacha k., ben-amara a., vannier a., alves-fortunato m. and nardin m. 2015. oxidation stability of diesel/biodiesel fuels measured by a petrooxy device and characterization of oxidation products. energy fuels 29:4345-4355. brand-williams w., cuvelier m. and berset c. 1995. use of a free radical method to evaluate antioxidant activity. food sci. technol. 28:25-30. dridi w., toutain j., sommier a., essafi w., gargouri m., leal-calderon f. and cansell m. 2016. 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lipophilic antioxidants and extra-virgin olive oil by isothermal calorimetry. therm. acta. 658:1-6. horwitz w. 2002. peroxide value of oils and fats. off. meth. anal. aoac int. 41:16. johnson d.r. and decker e.a. 2015. the role of oxygen in lipid oxidation reactions: a review. annual rev. food sci. technol. 6:171-190. koleva i.i., van beek t.a., linssen j.p., de groot a., evstatieva l.n. 2002. screening of plant extracts for antioxidant activity: a comparative study on three testing methods. phytochem. anal. 13:8-17. kousksou t., jamil a., el omari k., zeraouli y. and le guer y. 2011. effect of heating rate and sample geometry on the apparent specific heat capacity: dsc applications. therm. acta. 519:59-64. labuza t.p., tsuyuki h. and karel m. 1969. kinetics of linoleate oxidation in model systems. j. am. oil chem. soc. 46:409-416. labuza t.p. and dugan jr, l., 1971. kinetics of lipid oxidation in foods. crit. rev. food sci. nutr. 2:355-405. litwinienko g., daniluk a. and kasprzycka-guttman t. 1999. a differential scanning calorimetry study on the oxidation of c 12-c 18 saturated fatty acids and their esters. j. am. oil chem. soc. 76:655-657. riva m., fessas d. and schiraldi a. 2001. isothermal calorimetry approach to evaluate shelf life of foods. therm. acta. 370:73-81. shahidi f. and zhong y. 2015. measurement of antioxidant activity. j. of functional foods, 18:757-781. tuorila h. and cardello a. 2002. consumer responses to an off-flavor in juice in the presence of specific health claims. food qual. pref. 13:561-569. wadsö l. and galindo f.g. 2009. isothermal calorimetry for biological applications in food science and technology. food contr. 20:956-961. willson r.j., beezer a.e., mitchell j.c. and loh w. 1995. determination of thermodynamic and kinetic parameters from isothermal heat conduction microcalorimetry: applications to long-term-reaction studies. j. phys. chem. 99:7108-7113. paper received october 1, 2018 accepted october 30, 2018 ijfs#1864_bozza ital. j. food sci., vol. 32, 2020 983 paper chemical characterization and bioactive potential of essential oil isolated from rhanterium suaveolens desf. species growing in tunisian arid zone m. hitana*1, h. najaa1, s. fattouch2, t. ghazouani2, c. ben sassi2, c. dupas-farrugia3, n. oulahal3 and m. neffati1 1laboratoire d’écologie pastorale, institut des régions arides, km 22.5, route du djorf, 4119 médenine, tunisia 2laboratory of protein engineering and bioactive molecules (lip-mb), national institute of applied sciences and technology, university of crathage tunis, 1080, tunisia 3université de lyon, université claude bernard lyon 1, biodymia (bioingénierie et dynamique microbienne aux interfaces alimentaires), equipe mixte d'accueil. université lyon 1, isara lyon n°3733 *corresponding author: mhitana@yahoo.com abstract the purpose of this work is to assess the antioxidant and the antibacterial activities of essential oil from flowers of rhanterium suaveolens desf. and to investigate its chemical composition. the gc-ms analysis revealed the identification of a total of thirty-one compounds representing 98.4% of the total oil. spathulenol (18.3%), carvacrol (12.1%), linalool (9.4%), α-terpineol (7.10%), α-terpinolene (6.3%) and pinocarvone (5.6%) were identified as major constituents. the tested oil exhibited weak activities in both dpph and abts radical scavenging assays and ferric reducing power test. however, it showed a good lipid peroxidation activity using the β-carotene/linoleic acid assay with an ic50 value of 26.20±1.01 µg/ml. in addition, the highest antibacterial effect was recorded against staphylococcus aureus ((mic=37.5 μg/ml). these findings show that essential oil of r. suaveolens flowers can be used as a promising source of natural food and drug preservatives. keywords: antioxidant, antibacterial, essential oil, rhanterium suaveolens, chemical composition ital. j. food sci., vol. 32, 2020 984 1. introduction food preservatives are usually used to extend the shelf life of food products and to limit their deterioration caused by oxidation and growth of foodborne pathogens (russell and gould, 2003). as harmful effects caused by the extensive use of chemical preservatives and the increase of microbial resistance to a wide number of antibacterial drugs (shan et al., 2007), the search of new bioactive substances with interesting biological activities is required. in this purpose, several studies have been carried out for the prospection of new products derived from plants and their potential use as ingredients in food and pharmaceutical industries (ben salah et al., 2019). essential oils are natural products known for their multi-propose applications (de martino et al., 2015). they have shown a big interest as agents with several healthypromoting activities such as antibacterial, antioxidant, anti-carcinogenic and antimutagenic properties (gutierrez et al., 2009). therefore, their investigation proves to be a relevant choice in order to limit the use of chemical or synthetic preservatives and minimize their toxic effect (caillet and lacroix, 2007). the use of essential oils can improve food safety and protect our body against bacteria causing food poisoning (ultee et al., 2000). in fact, many studies have demonstrated the potential use of essential oils as natural antimicrobial agents in cheese-making industry (khorshidiana et al., 2018). as an alternative of specific applications, the essential oils can also be prepared in a large number of formulations, which can be used in food preservation. recently, girardi et al. (2018) reported that the application of microencapsulated peumus boldus essential oil was useful to prevent peanut deterioration caused by food spoilage microorganisms. this biological potential is mainly attributed to the presence of several constituents such as oxygenated derivatives and terpenoids (aberrane et al., 2019; bida et al., 2019). tunisian flora is characterized by a wide variety of aromatic and medicinal species producing several bioactive substances with multiple interests (salem et al., 2018). however, only few of these species have been investigated for their antioxidant and antibacterial potential. for example, rhanterium suaveolens desf. from the asteraceae family is an endemic species from north africa growing in algerian sahara (quezel and santa, 1963) and arid zone of tunisia (chaieb and boukhris, 1998). three species of the genus rhanterium; namely, r. epapposum oliver, r. adpressum coss. & durieu and r. suaveolens desf. have been reported in literature. r. suaveolens commonly known as “arfadj” is a forage plant, grazed on by sheep and camel in the desert. it is used by the local population in the production of cheese and in folk medicine as an antidiuretic (hamia et al., 2013). to the best of our knowledge, only few studies have been conducted on the phytochemistry of the r. suaveolens essential oil (rseo) and information on its biological activities, particularly, antioxidant potential, are still scarce in literature. therefore, the main purpose of this study was to investigate the chemical profile of essential oil collected from the flowers of r. suaveolens growing in arid zone of tunisia and to evaluate its antioxidant and antibacterial activities. ital. j. food sci., vol. 32, 2020 985 2. materials and methods 2.1. plant material flowers of r. suaveolens were collected during the flowering period in april 2014 from a single population of this species growing in gorthab from the tataouine region situated in the south east of tunisia. taxonomic identification of the plant material was confirmed by a local botanist at the institute of arid zone research in medenine (tunisia). a voucher specimen (irabs1865) was prepared and deposited in the herbarium of the laboratory of pastoral ecology. the collected plant material was cleaned and then air-dried at room temperature for eight to ten days. .the dried flowers were ground to powders and stored in air-tight glass. 2.2. essential oil extraction air dried flowers (100 g) of r. suaveolens were subjected to hydrodistillation in a clevenger-type apparatus for 3h. the obtained oil was dried over anhydrous sodium sulfate (na2so4) to remove water traces and stored in amber glass vials at 4°c. the oil yield (%) was expressed as volume of essential oil vs. dry weight basis (v/w). 2.3. gas chromatography/mass spectrometry (gc-ms) analysis the gc-ms analysis of the essential oil was carried out using an agilent 6890n network gc system combined with agilent 5975 b inert msd detector (quadrupole) with electron impact ionization (70 ev). ahp-5ms (5% phenyl methyl siloxane) column (30 m×0.25 mm i.d, film thickness 0.25 mm). the analysis was performed using helium (purity ˃ 99.99 vol.%) as a carrier gaz at a flow rate of 1.0 ml.min-1. the column temperature was programmed to rise from 50 to 280°c at a rate of 7 °c/min. injector and detector temperatures were maintained at 220 and 240°c, respectively. essential oil (1 µl) was injected in a split mode ratio of 1:10. scan time and mass range were 2.2 s and 50–550 m/z, respectively. 2.4. identification of the essential oil constituents identification of the r. suaveolens essential oil (rseo) components was based on their linear retention indices (ris) and comparison of their mass spectra with those of the computer library (wiley 275 library and nist98 database/chemstation data system) provided by the instrument software and ms literature data (joulain et al., 2001; adams, 2001). ris were calculated using n-alkane series (c6–c22) analysed under the same gc–ms conditions as for the samples. 2.3. antioxidant assays 2.3.1 scavenging effect on dpph (2,2-diphenyl-l-1-picrylhydrazil) radical the dpph assay was estimated as described by dhaouadi et al. (2014), with slight modifications. different concentrations of the rseo were prepared in pure methanol, then 50 µl of each of them were added to 950 µl of a 40 µmol/l (v/v) dpph methanolic solution in methanol. after vigorous shaking, the resulting mixtures were left in the dark ital. j. food sci., vol. 32, 2020 986 at room temperature for 30 min. the absorbance of the resulting solutions was measured at 517 nm. and the radical scavenging ability of rseo was measured as shown below: 𝐷𝑃𝑃𝐻 𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑒𝑓𝑓𝑒𝑐𝑡 % = 𝐴𝑜 − 𝐴𝑡 𝐴𝑜 ×100 where 𝐴0 is the absorption of the control sample after 30 min and 𝐴t is the sample absorption after 30 min. the antioxidant activity was expressed as ic50 value (mg/ml). 2.3.2 scavenging effect on abts (2,20 azinobis-3-ethylbenzthiazoline-6sulphonic acid) radical cation the abts+ assay was performed according to a slight modified version of the method described by tuberoso et al. (2007). the radical cation was produced by mixing the abts+ solution (7 mmol/l) with potassium persulfate aqueous solution (2.45 mmol/l). the abts+ solution was kept in the dark at room temperature for 12-16 h, then, was diluted with phosphate buffer to the absorbance of 0.7±0.02 at 734 nm. different concentrations of rseo were prepared in methanol. to 50 µl of each test concentration, 950 µl of diluted abts solution were added. the resulting mixtures were allowed to incubate in the dark for 10 min at room temperature. the absorbance of the mixtures was recorded at 734 nm. the antioxidant activity was calculated as follows: 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 % = 1 − a − b c ×100 where, a is the absorbance of the mixture containing the sample, b is the absorbance of the blank reagent and c is the absorbance of the blank sample.the concentration providing 50% of inhibition (ic50) was calculated using a calibration curve in the linear range by plotting the extract concentration. 2.3.3 reducing power assay the reducing power of the rseo was assessed following the method described by singh et al. (2012). one ml of phosphate buffer (0.2 m ‘w/v’, ph 6.6) and 1 ml of potassium ferricyanide [k3fe (cn)6], 1% ‘w/v’ was mixed with 1 ml of different concentrations of rseo (10, 20, 30, 40 and 50 mg/ml). the obtained mixtures were incubated at 50°c for 20 min. then 1 ml of trichloroacetic acid (tca) (10% ‘w/v’) was added. the resulting mixtures were revolved at 3000 rpm for 10 min. the supernatant was recovered and mixed with 1.5 ml of distilled water and 150 µl of fecl3 (0.1% ‘w/v’). the absorbance was measured at 700 nm and the butylated hydroxyanisole (bha) was used as standard. the result was expressed as ic50 (mg/ml). 2.3.4 lipid peroxidation activity the lipid peroxidation activity of rseo was carried out by β-carotene/linoleic method according to dapkevicius et al. (1998), which is based on the inhibition of the products resulting from the oxidation of linoleic acid. a stock solution of β-carotene/linoleic acid was prepared by mixing 200 mg of tween 40, 0.5 mg of β-carotene, 25 µl of linoleic acid and 1 ml of chloroform. after chloroform evaporation, under low pressure at 40°c, 100 ital. j. food sci., vol. 32, 2020 987 ml of oxygenated distilled water were added to the mixture with vigorous shaking. an aliquot of the resulting solution (2.5 ml) was dispersed to test tubes and 0.5 ml of prepared sample with different concentrations (5-40 µg/ml) in methanol and water were added. the obtained emulsion was incubated for 2 h at 50°c. two controls were prepared, one with the standard bha (positive control) and the other without bha or extract (blank). the absorbance of each sample was immediately measured at 490 nm after 30 min, 60 min, 90 min and 120 min. the bleaching rate r of β-carotene was determined according to the following equation: 𝑅 = ln(ab) t were ln=natural log, a=absorbance at time 0, b=absorbance at time t (30 min, 60 min, 90 min and 120 min). antioxidant activity was calculated in terms of inhibition percentage using the following equation: 𝐴𝑛𝑡𝑖𝑜𝑥𝑖𝑑𝑎𝑛𝑡 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (%) = (𝑅𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝑅𝑠𝑎𝑚𝑝𝑙𝑒)×100 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 results were expressed as ic50 value (µg/ml). 2.4. in vitro evaluation of antibacterial activity 2.4.1 tested bacterial strains and growth conditions the antibacterial activity of rseo was tested against a range of bacterial strains collected from american type culture collection (atcc, rockville). gram-positive: staphylococcus aureus (atcc 25923), listeria monocytogenes (atcc 19115) and bacillus cereus (atcc 14579) and gram-negative: eescherichia coli (atcc 35218), salmonella typhimurium (nrlb 4420) and pseudomonas aeruginosa (atcc 27853). all bacterial strains were cultured at 37°c for 24h in mueller-hinon agar (mha). the cultures were started by adjusting the bacterial suspension in broth to 0.5 mc farland turbidity. then the bacterial suspension was diluted using 10 fold serial dilution method in order to obtain an inoculum of 108 colony-forming units (cfu) per plates (dhaouadi et al., 2015). 2.4.2 disk diffusion method the in vitro antibacterial activity of rseo was estimated using the disk diffusion method described by dhaouadi et al. (2015) with slight modifications. 10 µl of rseo were placed onto sterilized paper disc (6 mm ∅), and placed onto the inoculated agar surface. the petri dishes were placed at 4 °c for 1 h and then incubated at 37 °c for 24 h. after incubation, the diameters of the resulting inhibition zones were determined. tests were performed in triplicate. gentamicin (10 µg per disk) was used as positive control and sterile water as negative control. ital. j. food sci., vol. 32, 2020 988 2.4.3 microdilution method the antibacterial activity of rseo was also assessed by the determination of minimum inhibitory and bactericidal concentrations (mic and mbc) using broth microdilution method. the minimal inhibition concentration (mic) was determined as described by gulluce et al. (2007) with slight modifications. rseo sample previously dissolved in 10% dimethylsulfoxide (dmso) was first diluted to the highest concentration (3 mg/ml) to be tested. the 96 well plates were prepared by dispensing into each well 95 µl of the nutrient broth and 5 µl of the inoculum. an aliquot from the stock solution of rseo (100 µl) was added into the first well. then, 100 µl from the serial dilutions were transferred into eleven consecutive wells. the last well containing 195 µl of nutrient broth without rseo and 5 µl of the inoculum on each strip were used as the negative control. after that, the plates were incubated at 37°c for 24 h. all samples were screened two times against each microorganism.the mic is defined as the lowest concentration of the sample that did not allow any visible growth of the tested bacterial strain (ben salah et al., 2019). to the determination of the mbc value an aliquot (25 µl) was spreaded onto mha plates and then incubated for 12–16 h at 37°c. the determination of surviving bacterial strains allowed the estimation of the mbc at 99.9 % of bacterial death (fattouch et al., 2007). 2.5. statistical analysis all experiments were repeated in triplicate and the results were reported as mean values and standard deviation (mean±sd). significance differences between the results were performed by analysis of variance (anova) using tukey’s multiple comparison tests at a level of significance set at p<0.05. data analysis was performed using minitab 18 statistical software (minitab inc., u.s.a.). 3. results and discussion 3.1. essential oil composition the volatile oil extracted from r. suaveolens flowers has yellow color with an agreeable intense smell. its extraction yield was about 0.23%±0.02 (volume/dry weight), which was similar to that reported by ben salah et al. (2019) (0.22%), and was slightly, higher than that obtained from the aerial parts of algerian r. suaveolens (0.14%) (chemsa et al., 2016). as depicted in table 1, thirty-one components have been identified in the rseo which represent 98.4% of the total composition. this oil contains a complex mixture dominated by oxygenated monoterpenes (46%) followed by oxygenated sesquiterpenes (23.6%) and monoterpenes hydrocarbons (17.5%). the major components of the rseo were identified as spathulenol (18.3%), carvacrol (12.1%), linalool (09.4%), α-terpineol (7.10%), αterpinolene (6.3%) and pinocarvone (5.6%). compared to previous studies, our findings differ from those reported by ben salah et al. (2019), with α-pinene (25.84 %), β-pinene (17.57 %), 1-octen-3-ol (16.23 %), camphene (12.28 %), limonene (8.03 %) and β-myrcene (5.13 %) as major compounds. also, the composition of the algerian r. suaveolens essential oil showed a significant difference in the chemical composition (chemsa et al., 2016). ital. j. food sci., vol. 32, 2020 989 table 1. chemical composition of the essential oil from the flowers of r. suaveolens analysed by gc-ms. acompounds are listed in order of their elution from a hp-5ms column. b experimental linear retention index on a hp-5ms capillary column using the homologous series of n-alkanes. c linear retention index from literature. dpeak area of the essential oil components. ecompounds were identified based on their ri on hp5ms capillary column and gc-ms data. values are given as mean± s.d. (n=3). *values with different letters with in the same column indicate significant difference (p<0.05). no. compoundsa riexp b rilit c % area identification methods 1 α-thujene 926 924 0.5±0.02 ri, ms 2 α-pipene 936 939 2.5±0.03 ri, ms 3 camphene 955 956 1.8±0.01 ri, ms 4 β-pinene 981 979 1.9±0.01 ri, ms 5 β-myrcene 990 993 0.5±0.02 ri, ms 6 α-terpinene 1016 1017 2.4±0.04 ri, ms 7 limonene 1030 1029 1.2±0.01 ri, ms 8 γ-terpinene 1060 1059 0.4±0.01 ri, ms 9 α-terpinolene* 1088 1089 6.3±0.10d ri, ms 10 linalool* 1095 1098 9.4±0.08c ri, ms 11 trans-sabinol* 1140 1142 4.1±0.01e ri, ms 12 p-menth-4(8)-ene 1157 1160 1.6±0.02 ri, ms 13 pinocarvone* 1160 1164 5.6±0.21de ri, ms 14 α-terpineol* 1190 1192 7.10±0.02d ri, ms 15 trans-carveol 1220 1217 1.5±0.01 ri, ms 16 carvone 1242 1250 0.5±0.01 ri, ms 17 geraniol 1255 1252 1.5±0.03 ri, ms 18 α-thujenol 1287 1290 2.6±0.01 ri, ms 19 carvacrol* 1298 1299 12.1±0.21b ri, ms 20 trans-caryophyllene 1415 1419 0.6±0.1 ri, ms 21 aromadendrene 1437 1437 3.3±0.1 ri, ms 22 alloaromadendrene 1463 1458 0.7±0.2 ri, ms 23 eremophilene 1511 1512 2.4±0.1 ri, ms 24 ð-cadinene 1522 1523 0.1±0.2 ri, ms 25 α-calacorene 1541 1546 0.4±0.1 ri, ms 26 spathulenol* 1577 1577 18.3±0.1a ri, ms 27 caryophyllene oxide* 1581 1582 4.8±0.2de ri, ms 28 α-cadinol 1676 1652 0.5±0.01 ri, ms 29 myristic acid 1762 1767 0.6±0.1 ri, ms 30 palmitic acid methyl ester 1908 1909 0.3±0.1 ri, ms 31 palmitic acid 1970 1968 2.9±0.02 ri, ms monoterpene hydrocarbons 17.5 oxygenated monoterpenes 46.0 sesquiterpene hydrocarbons 7.50 oxygenated sesquiterpenes 23.60 others (%) 3.80 total identified (%) 98.4 ital. j. food sci., vol. 32, 2020 990 the presence of spathulenol, linalool and carvacrol mentioned in this work as major constituents, had never been, already, reported for the r. suaveolens species. the differences observed between our findings and those previsouly reported by ben salah et al. (2019) and chemsa et al. (2016) can be attributed to the environmental, agronomic, age and geoclimatic factors (season, location, fertility regime, soil type and climate) as well as the experimental extraction conditions (boukhatem et al., 2014; singh et al., 2012). 3.2. antioxidant activity as depicted in figs. 1, 2 and 3, the inhibition of the dpph and abts radicals, the reducing power and the inhibition of lipid peroxidation activities of the rseo, respectively, are dose dependent. figure 1. dpph•− and abts•+ free radical-scavenging properties of the essential oil of the r suaveolens flowers. data were presented as means±sd (n=3). figure 2. reducing power of the essential oil of the r. suaveolens flowers. ital. j. food sci., vol. 32, 2020 991 figure 3. antioxidant activity (%) of essential oil of the r. suaveolens flowers measured by β-carotene–linoleic acid method. values expressed are means±s.d. (n=3) as can be seen in table 2 the ic50 values obtained for both dpph and abts assays (11.48±0.11 mg/ml, 18.58±0.39 mg/ml, respectively) are significantly (p<0.05) higher than those observed for the tested standard bha (0.041±0.002 mg/ml, 0.032±0.004 mg/ml, respectively). in other words, the activity of the selected essential oil is lower than that of the bha standard. our results are in agreement with the findings of the rseo isolated from algeria, which exhibited a weak dpph scavenging activity (chemsa et al., 2016). as depicted in table 2, the ferric reducing power of the rseo (ic50=58.95±1.21 mg/ml) was significantly (p<0.05) lower than that of the bha standard (ic50=0.052±0.001 mg/ml). table 2. antioxidative capacities of the essential oil of the rhanterium suaveolens flowers. ic50 dpph (mg/ml) abts (mg/ml) reducing power (mg/ml) β-carotene/linoleic acid (µg/ml) essential oil 11.48±0.11b 18.58±0.39b 58.95±1.21b 26.15±1.01b bha 0.041±0.002a 0.032±0.004a 0.052±0.001a 5.95±0.82a bha standard was used as a reference. all the values are means±sd (standard deviation) of three parallel measurements. different letters in the same column indicate a significant difference (p<0.05). the inhibition of the lipid peroxidation activity of the tested essential oil was carried out using the β-carotene bleaching test. as shown in table 2, the activity of the rseo was lower (ic50=26.15±1.01 µg/ml) than that of the synthetic standard bha (ic50=5.95±0.82 µg/ml). also, this activity is less important than that previously reported for the essential oil of the algerian r. suaveolens aerial parts with an ic50 of 17.97±5.40 µg/ml (chemsa et al., 2016). compared to the radical scavenging effects and the reducing power, the rseo is more active on the inhibition of the lipid peroxidation. this can be, probably, due to the ital. j. food sci., vol. 32, 2020 992 high specificity of the test to lypophilic molecules (harkat-madouri et al., 2015). it has been reported that the lipid peroxidation activity may be due to the richness of the tested oil on conjugated sesquiterpenoids. indeed, these compounds can scavenge the singlet oxygen and consequently, protect the β-carotene color against bleaching, indirectly (chemsa et al., 2016). the weak antioxidant activity observed for the rseo can be related to its chemical composition as well as the abundance of ineffective compounds such as the monohydroxylated compounds which are unable to chelate ferrous ions (harkatmadouri et al., 2015; aidi wannes et al., 2010; dzami et al., 2013). the low antioxidant potential of the tested oil can, also, be attributed to the degradation of bioactive compounds during their extraction. indeed, during hydrodistillation process, plant material is usually extracted in boiling water for a long period which could cause thermal decomposition of the thermolabile target molecules inducing, therefore, a decrease in the antioxidant capacity of the extract (bagheri et al., 2014). 3.3. antibacterial activity the antibacterial activity was evaluated against six foodborne pathogens (3 gram-positive and 3 gram-negative), using the dilution and disk diffusion methods. as shown in table 3, the rseo was sensitive to all tested bacteria and exhibited a variable antibacterial activity dependent on the tested strains. s. aureus was the most susceptible bacteria with the largest inhibition zone (iz=18.25±0.35 mm) followed by l. monocytogene (iz =17.37±0.53 mm) and b. cereus (16.0±0.0 mm). however, the highest resistance to the rseo was observed for the s. typhimurium with the lowest (p<0.05) inhibition zone (iz=12.25±0.35 mm). results showed that the tested essential oil was slightly more active against gram-positive than gram-negative bacteria. this can be explained by the complexity of their double membrane containing cell envelope, which can limit the diffusion of hydrophobic compounds through its lipopolysaccharide covering. generally, the bacteriostatic and/or bactericide action of the plant extracts is attributed to their ability to disrupt cell membrane structures, disturb their permeability barrier and, consequently, to cause the chemiosmotic control loss (bagamboula et al., 2004). as depicted in table 3, the mic and mbc values of the rseo ranged from 75 to 300 µg/ml for the tested bacterial strains. the highest antibacterial activity was observed against s. aureus with the lowest (p<0.05) mic and mbc values (37.5 µg/ml and 75 µg/ml, respectively). there are a few reports on the antibacterial activity of the r. suaveolens essential oil for comparison. recently, ben salah et al. (2019), developed the antibacterial activity of tunisian rseo against a broad spectrum of bacterial strains. our findings showed discrepancies between their published data. larger inhibition zones values were reported against e. coli and p. aeruginosa (19 mm, 23 mm, respectively). the corresponding mics were found 230µg/ml for e.coli and 46 µg/ml for p. aeruginosa (ben salah et al., 2019). in addition, a moderate antibiofilm potential has been reported against six gram positive bacteria for the essential oil collected from the aerial parts of algerian r. suaveolens (chemsa et al., 2016). the differences between our findings and those prevesiouly reported by other authors, may result from different chemical compositions and percentage content of active constituents in the tested essential oils. factors such as the choice of bacterial strains and their sensitivity, the experimental conditions and the choice of methods used for in vitro antibacterial activity could also be related to the variation in the experimental results (siddique et al., 2017). ital. j. food sci., vol. 32, 2020 993 table 3. antibacterial activity of essential oil of the r. suaveolens flowers using disc diffusion method and determination of mic and mbc values. bacterial strains iz (mm±sd) gentamicine mic (µg/ml) mbc (µg/ml) gram positive bacteria s. aureus (atcc 25923) 18.25±0.35a 34.50±0.71a 37.5 75 l. monocytogenes (atcc19115) 17.37±0.53a 31.00±0.00b 75 150 b. cereus (atcc14579) 16.00±0.00ab 24.00±0.00d 150 300 gram negative bacteria e. coli (atcc35218) 15.62±0.17b 26.00±0.00c 75 150 s. typhimurium (nrlb4420) 12.25±0.35d 21.50±0.71e 150 300 p. aeruginosa (atcc27853) 14.00±0.00c 32.50±0.71b 150 150 iz: the diameter of the inhibition zones (mm), including the well diameter (6 mm), are given as mean±sd (n=3). gentamicine: is used as positive control for bacteria. different letters in the same column indicate a significant difference (p<0.05). the appreciable antibacterial potential of the rseo against some bacterial strains could be related to the presence of a high amount of phytochemicals such as monoterpenes and oxygenated monoterpenes (aggarwal et al., 2002). effectively, many studies have proved the presence of a relationship between the chemical composition of the major components of the essential oils and the antibacterial activity (bel-hadj et al., 2017). the major compounds identified in the rseo such as spathulenol, carvacrol, linalool, αterpineol, α-terpinonene and pinocarvone have not been tested for their antibacterial activity in the present study. however, some reports have approved their antibacterial properties. indeed, a number of researchers have shown that carvacrol and linalool are well-known substances with pronounced antimicrobial activity against several pathogenic bacteria (bozin et al., 2006). likewise, it was found that spathulenol and linalool exhibited moderate to strong activities against several microorganisms (magiatis et al., 2002). in addition, it has been revealed that interactions between the constituents of some essential oils may contribute to different effects such as additive, synergistic, or antagonistic (delaquis et al., 2002). a study conducted on the release of the cellular materials test, showed that α-terpineol/linalool combination treatments have shown a strong effect on the release of cell constituents both from gram-negative and grampositive bacteria (zengin and baysal. 2014). 4. conclusions this paper reports the chemical composition and the in vitro antioxidant and antibacterial properties of the essential oil collected from the r. suaveolens flowers. the gc–ms analysis revealed the identification of 31 constituents. spathulenol, linalool and carvacrol identified as major compounds, were reported for the first time in the essential oil of this species. results obtained from the β-carotene/linoleic acid bleaching assay were found to be stronger than those obtained from dpph, abts and cuprac systems. 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myrtaceae) and its major constituents. lwtfood sci. technol. 48: 237-241. ital. j. food sci., vol. 32, 2020 996 tuberoso c.i.g., kowalczyk a., sarritzu e. and cabras p. 2007. determination of antioxidant compounds and antioxidant activity in commercial oil seeds for food use. food chem. 103:1494-1501. ultee a., slump r.a., steging g. and smid j. 2000. antimicrobial activity of carvacrol toward bacillus cereus on rice. j. food prot. 63:620-624. paper received april 14, 2020 accepted october 8, 2020 46 issn 1120-1770 online, doi 10.15586/ijfs.v33i2.2012 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (2):46–53 sichuan province of china revealed its popularity than crw among young people. japanese sake, a japanese alcoholic beverage, is generally prepared from the polished japonica rice and koji with an alcohol content of 13–17% v/v (sato and kohsaka, 2017). it has a refreshing taste and fruity aroma, and its market is widely distributed in japan, the united states, southeast asia and some european countries (mimura et  al., 2014). the production of jns requires partial grinding of the outer layer of rice to produce “polished rice.” the rate of polished rice refers to the ratio of the mass of polished rice to that of original mass. the lower the rate of polished rice, the lower the content of protein and lipid in rice, thereby making it more suitable for brewing high-quality sake such as daiginjo (okuda, 2019; yamashita, 1997). crw has originated in china and is one of the world’s oldest alcoholic beverages with a history of thousands of years (mcgovern et al., 2004). it is brewed using glutinous rice, wheat qu (fermentation starter) and water. crw has a mellow and rich taste, and contains various nutrients such as amino acids, p u b l i c a t i o n s codon composition and nutritional evaluation of amino acids in mimai qu rice wines kaizheng zhang1*, wenchi wu1,2, wei zou1, zhenhui kang1, qin yan1, and yue qin1 1sichuan university of science & engineering, zigong city, sichuan province, china; 2chengdu institute of product quality inspection co. ltd, chengdu, china *corresponding author: kaizheng zhang, sichuan university of science & engineering, zigong city, sichuan province, china. email: kai7766@126.com received: 25 january 2021; accepted: 22 april 2021; published: 11 may 2021 © 2021 codon publications open access paper abstract an amino acid analyzer was used to detect free amino acids (faa) in mimai qu rice wines (smw and dmw) and control wine samples (chinese rice wine [crw] and japanese sake [jns]). it was found that crw had the highest total amino acid (taa) content (~2814 mg/l), followed by smw (~2509 mg/l) and dmw (~1474 mg/l), while jns had the least (~917 mg/l). amino acid ratio coefficient method (srcaa), linear regression method, cluster analysis (ca) and principal component analysis (pca) were used for evaluating the nutritional value of amino acids in wine samples, giving similar results. smw had the highest nutritional value, followed by crw and dmw and jns. keywords: brewed wine, cluster analysis, linear regression analysis, principal component analysis, ratio coefficient method introduction mimai qu rice wines, prepared from crushed wheat malt (25%) and steamed rice (75%) using the japanese koji process with aspergillus oryzae su-16 and aspergillus oryzae aok139 as inoculum (china application patent no.: cn201810216324), is a novel fermentation starter (http://pss-system.cnipa.gov.cn/sipopublicsearch/portal/ uilogin-forwardlogin.shtml). as for chinese rice wine (crw), the brewing properties of mimai qu are similar to those of traditional wheat qu. however, mimai qu rice wine is made from purified microbes, and the hygiene of the production environment and food safety are much higher than that of wheat qu. mature mimai qu rice wine is white in color (the wheat qu of crw shows yellow color) with a delicate fragrance that is distinct from the complex aroma of wheat qu. mimai qu rice wine is transparent, light yellow in color and has a fresh and elegant mild fruity flavor, but without the so-called “soy sauce flavor” of crw (wang and xu, 2005). its taste and flavor are between those of crw and japanese sake (jns). a small-scale tasting survey conducted in the mailto:kai7766@126.com http://pss-system.cnipa.gov.cn/sipopublicsearch/portal/uilogin-forwardlogin.shtml� http://pss-system.cnipa.gov.cn/sipopublicsearch/portal/uilogin-forwardlogin.shtml� italian journal of food science, 2021; 33 (2) 47 composition and nutritional evaluation of amino acids in mimai qu rice wines water. the alcohol content of this wine was 13–16% v/v, and the total sugar content was 20–30 g/l. the age of the wine was 3–5 years. in all, three crw wine samples, such as guyuelongshan, kuaijishan and tapai, were analyzed in this study. japanese sake: sakes were purchased from japan. the raw materials included water, yamadanishiki rice and rice koji, with a polishing ratio of 50–70%. the alcohol content was 14–16% v/v and the total sugar content was 20–35 g/l. the age of sake was 1–5 years. three sake wine samples were analyzed in this study: company a, b, and c produced in the cities of yamaguchiken, kobe, and kyoto of japan, respectively. mimai qu rice wine (dry type), abbreviated as dmw, was brewed in 2018 with mimai qu rice and water as raw materials at a polishing ratio of 70%. brewing method: the rice was soaked for 45 min after polishing and then steamed, followed by spreading and cooling to 35–40°c. mimai qu, equal to 12% of rice, and commercial mineral water at a volume equal to that of rice were added as raw materials. the initial temperature of the mash was controlled at 20–25°c, and after 6 h, lactic acid equivalent to 0.7% of the mass of mash was added to perform acidification. saccharomyces cerevisiae (pre-activated into yeast mash) equivalent to 0.1% of raw material was then added and mixed evenly. seeding mash was achieved after a day of culture. the feeding was carried out as per the threestep feeding method described in table 1. after the third feeding, fermentation of dmw was commenced officially and completed in 20 days (figure 1). dmw was light yellow and transparent in color, with a fresh and elegant flavor without the so-called “soy sauce flavor” of crw. additionally, it had a light fruity aroma. its alcohol content was 16–18% v/v and the total sugar content was 6–9 g/l. the wine storage time was 1 year. four dmw wine samples were analyzed in this study. mimai qu rice wine (semi-sweet; smw): the raw materials and brewing process were essentially the same as that for dmw, except that in the course of each feeding, 30% of the brewed water was replaced by aged crw (15% v/v; total sugar content: 6 g/l). therefore, smw oligosaccharides, short peptides etc., and therefore is also called “liquid cake” (wang, 1998). currently, 90% of crw consumption is concentrated in eastern china, including zhejiang province, jiangsu province and shanghai, which pose regional restrictions for aging consumer groups (jiao et al., 2017; xu et al., 2013). after the 2010s, because of lack of innovation in the production process of crw, its taste and flavor are not considered attractive by the current young and middle-aged consumers, thus gradually losing these huge main consumer groups (jiao et al., 2017). in 2018, two mimai qu rice wines (dmw and smw) were creatively developed using mimai qu and polished japonica rice varieties as raw materials by combining the processing techniques of sake and crw in our laboratory to attract young people and broaden the consumer groups and consumption areas of crw. amino acids are basic components of protein, providing significant nutrition, and are divided into essential amino acids (eaa) and non-essential amino acids (neaa) (cui et al., 2014; omar et al., 2017). generally, men prefer to drink moderate-alcohol beverages, such as crw, with an average intake of 1000–1500 ml/d (yang, 2021), containing 3–5 g/d of free amino acids (faa). moreover, faa is a non-negligible nutrition source, as it can be easily assimilated and utilized by the human body. contents and composition of amino acids are important factors in the nutritional value of foods (gonzález-castro et al., 1997; okada et al., 2017). therefore, this study intended to detect the content and composition of faa in mimai qu rice wine, and assess its amino acid nutritional value using different analytical methods. this study would provide a theoretical basis for further analyzing the nutritional profiles of this novel rice wine. materials and methods materials chinese rice wine (crw): traditional crws produced in the shaoxing city of china were purchased from walmart supermarket, zigong city, sichuan province. the raw materials included glutinous rice, wheat qu and table 1. three-step feeding method for dmw brewing. ingredients seeding mash first feeding second feeding third feeding total rice (kg) 50 100 200 400 750 mimai qu (kg) 6 20 40 80 146 water (l) 50 160 320 640 1170 lactic acid (l) 7.5 0 0 0 7.5 yeast culture solution (l) 5.5 0 0 0 5.5 dmw: mimai qu rice wine (dry type). 48 italian journal of food science, 2021; 33 (2) zhang k et al. japonica rice steeping/washing steeping/washing pasteurization polishing steaming steaming steamed rice steamed rice wheat malt main mash seed mash water su-16 and aok139 spores miami qu saccharomyces cerevisiae filtration pressing saccharification and fermentation polished rice dmw brown rice figure 1. the brewing process of dmw (mimai qu rice wine, dry type). was with a brewing characteristic of “wine made from wine” and contained more oligosaccharides with a semisweet taste. the alcohol content in smw was 14–16% v/v and the total sugar content was 40–60 g/l with the storage age of 1 year. four smw wine samples were analyzed in this study. methods amino acids determination method free amino acids in the test sample were analyzed by l-8900 amino acid automatic analyzer (hitachi, japan), with a 53-min short program (including the time for washing and rinsing columns). wine sample pretreatment: after filtration, 40 ml of 10% trichloroacetic acid (sigma-aldrich trading co. ltd, shanghai, china) was added to every 10 ml of wine sample to remove proteins, followed by centrifugation in a high-speed centrifuge at 5000 rpm for 10 min. the supernatant solution was dried thrice in an oven at 50°c to remove alcohol, after which 0.02 mol/l of hcl (china national sinopharm chemical reagent co. ltd., shanghai, china) solution was added to a final volume of 10 ml. after centrifugation, 1 ml of supernatant was taken and tested directly on the machine. evaluation methods for amino acid nutrition amino acid ratio coefficient method the who/fao meeting in 1973 pointed out that the amino acid composition of proteins in eggs and human milk is most suitable for absorption by human body and has high nutritional value. therefore, the composition and content of eaa in eggs and human milk were used as templates to evaluate the amino acid ratio (raa; equation 1) and the ratio coefficient (rc; equation 2) in foods to derive the score of ratio coefficient (src; equation 3) of amino acids. this is called the score of ratio coefficient of amino acid (srcaa) method, which is employed to evaluate the amino acid nutritional value of the food (fao/who, 1973). eaa content of the food to be evaluated raa , the corresponding eaa content recommended by who/fao = (1) raa rc , mean value of raa = (2) src = 100 – (cv × 100), (3) where cv is the standard deviation coefficient of rc. cv = rc standard deviation/rc mean. linear regression analysis linear regression is a statistical analysis method that analyzes the strength of correlation between two or more sets of data. the correlation coefficient “r” reflects the degree of correlation in the data. linear regression analysis is able to investigate the degree of correlation between the content of seven types of eaa in wine samples and the recommended pattern of who/fao through the r-value. the larger the r-value, the greater the degree of correlation and higher nutritional value of the wine sample (zhang et al., 2017). italian journal of food science, 2021; 33 (2) 49 composition and nutritional evaluation of amino acids in mimai qu rice wines the mean of three to four samples, which could reduce the analysis and table width. apart for methionine (met) that was not detected in jns and dmw, other 16 amino acids were present in all wine samples. as shown in table 2, among the tested samples, the highest taa (2814.4 mg/l) was found in crw. taa in smw was the second highest at 2509.8 mg/l, while dmw had a taa content of 1474.1 mg/l. the lowest taa was found in jns at 916.5 mg/l (p < 0.05). the most abundant amino acid was proline (pro), followed by alanine (ala) and asparagine (asp). the most abundant amino acid found in jns was arginine (arg). the amino acids that ranked second and third highest were ala and asp, respectively. ala had the highest content in smw, followed by asp and leucine (leu) (pro). the highest amino acid content in dmw was that of pro, with ala and asp ranking second and third highest, respectively (p < 0.05). crw uses traditional wheat qu as a starter with a protein content of about 8–11% (ye et al., 2018; zhang et al., 2019a), which is higher than that of rice. moreover, since rice is not polished, its protein content is higher than that of polished rice. therefore, more protein was decomposed or hydrolyzed into amino acids by microorganisms under acidic conditions during the fermentation process, resulting in a higher taa in crw. rice koji is used as a starter to produce jns, which has a lower protein content than wheat koji. when the polishing ratio reached 50%, the amount of external protein was greatly reduced, resulting in a lower taa content in brewed sake to only one-third of crw. since some wheat was included in both smw and dmw koji, the taa in these wines was higher than that in jns but lower than that in crw. in the brewing process, rice wine partially replaced brewing water, which also added some amino acids, resulting in higher taa in smw when compared with dmw. zhang et al. (2017) measured the amino acid content of hulless barley zajiu using the automatic amino acid analyzer, and found that the taa content in traditional zajiu was 2875 mg/l, which was equivalent to smw. the eaa/taa of smw was 33.84%, about 10% lower than traditional zajiu. this may be due to the raw material for zajiu being hulless barley, while that for the wine samples in this study were mainly rice and a small amount of wheat. difference in brewing process may also contribute to this variation (zhang et al., 2019b). luo et al. (2017) determined the amino acid content of beer sold in lanzhou, china, by capillary electrophoresis. it was found that the total amount of amino acids in beer was about 260–370 mg/l. kabelová et al. (2008) compared the amino acid content found in czech republic beer and foreign brand beer by the hplc method and found that the taa content of czech republic beer was cluster analysis and principal component analysis the score of ratio coefficient of amino acid and linear regression analysis methods were adopted to examine similarities between seven types of eaa in the wine samples and the who/fao-recommended model, which provided a good evaluation parameter. however, the manual calculation workload of the data was large and no investigation was carried out on the proportion of eaa, namely eaa/taa and eaa/neaa. therefore, for a more comprehensive evaluation of the nutritional value of amino acids in brewed wines, eaa/taa and eaa/ neaa were also included within the scope of investigation (the two indicators were 0.4 and 0.6 according to the who/fao-recommended model). cluster analysis (ca) and principal component analysis (pca) were employed for analysis and evaluation of eaa/taa and eaa/neaa. since software is used in evaluation, the amount of manual calculation is greatly reduced. ca (euclidian distance) is a method for data classification analyzing the similarity of observed data and clusters data to optimal groups. observations in each category are different, with some level of similarity. the system clustering method can cluster wine samples and the who/fao-recommended model separately. the wine sample that was grouped with the recommended model first had higher similarity with the recommended model as well as higher nutritional value (biglari et al., 2009). pca is a factor analysis method that reduces the number of factors in a dataset into several major components and ensures that most of the information is retained. using the load map of pca enables one to identify the wine sample that is closer to the who/fao-recommended model. in the coordinate load map, the wine sample that is closer to the who/fao-recommended model point has a greater nutritional value (shin et al., 2010). data analysis all analyses were performed in triplicate. all statistical analyses were accomplished using dps statistical software for windows, version 15.10 (dps 15.10, hangzhou rui feng information technology co. ltd., zhejiang, china) (tang and zhang, 2013). significant differences were analyzed by duncon’s analysis of variance with p < 0.05. results and discussion amino acids and their contents in the tested wine samples table 2 lists 17 faas found in wine samples (tryptophan was degraded at low ph and was not detected). the result indicated that the value for each type of wine was 50 italian journal of food science, 2021; 33 (2) zhang k et al. table 2. individual amino acids in the tested wine samples (mg/l). faa smw (n = 4) dmw (n = 4) jns (n = 3) crw (n = 3) thr 90.2 ± 7.8a 55.4 ± 6.1b 28.8 ± 6.6c 94.2 ± 11.2a val 158.6 ± 13.3a 68.5 ± 7.4c 38.4 ± 9.7d 116.2 ± 16.6b met 20.4 ± 3.6b nd nd 28.4 ± 5.5a ile 105.4 ± 9.2a 45.2 ± 6.9b 24.1 ± 5.2c 91.1 ± 12.6a leu 257.1 ± 19.3a 110.7 ± 9.4b 71.9 ± 11.2c 240.1 ± 28.7a phe 89.4 ± 7.7b 60.7 ± 7.3c 37.6 ± 6.3d 116.3 ± 15.7a lys 128.4 ± 16.1a 72.9 ± 8.2b 31.2 ± 6.6c 124.0 ± 18.2a asp 317.3 ± 19.6a 178.9 ± 20.2b 108.3 ± 14.7c 282.5 ± 32.9a ser 143.7 ± 9.7b 88.1 ± 6.6c 48.6 ± 8.5d 176.4 ± 22.6a glu 102.2 ± 11.5b 53.9 ± 4.1c 29.2 ± 6.3d 167.7 ± 21.4a gly 225.2 ± 16.1a 139.8 ± 17.2b 73.8 ± 11.1c 208.9 ± 27.2a ala 355.0 ± 23.8a 187.6 ± 24.4b 112.2 ± 24.0c 325.6 ± 41.3a cys 19.1 ± 2.4a 9.1 ± 1.1b 3.8 ± 1.1c 4.7 ± 1.6c tyr 155.0 ± 16.2a 124.0 ± 11.5b 83.7 ± 12.5c 150.3 ± 19.6a his 70.9 ± 8.8a 36.4 ± 3.7b 21.7 ± 4.3c 62.5 ± 11.3a arg 14.8 ± 2.2d 58.5 ± 7.4c 124.7 ± 18.0b 231.7 ± 17.2a pro 257.1 ± 22.8b 184.3 ± 16.6c 78.6 ± 11.6d 393.8 ± 27.8a taa 2509.8 ± 19.4b 1474.1 ± 17.7c 916.5 ± 13.7d 2814.4 ± 17.3a eaa 849.5 ± 7.5a 413.5 ± 3.2b 231.9 ± 4.4c 810.3 ± 9.6a neaa 1660.3 ± 12.4b 1060.6 ± 9.7c 684.6 ± 4.5d 2004.1 ± 11.2a eaa/taa (%) 33.8 ± 2.2a 28.0 ± 1.8b 25.3 ± 1.4bc 28.8 ± 2.2b eaa/neaa (%) 51.2 ± 3.3a 39.0 ± 3.4b 33.9 ± 2.4bc 40.4 ± 3.9b faa: free amino acid; smw: mimai qu rice wine (semi-sweet); dmw: mimai qu rice wine (dry type); jns: japanese sake; crw: chinese rice wine; taa: total amino acids; eaa: essential amino acids; neaa: non-essential amino acids; nd: not detected; thr: threonine; val: valine; met: methionine; ile: isoleucine; leu: leucine; phe: phenylalanine; lys: lysine; asp: asparagine; ser: serine; glu: glutamine; gly: glycine; ala: alanine; cys: cysteine; tyr: tyrosine; his: histidine; arg: arginine; pro: proline. higher than that of foreign beer (~450 mg/l vs ~257 mg/l). this result was consistent with the results of redruello et al. (2017). the amino acid content in beer was much lower than that in the wine samples used in this study. this may be due to the brewing process of beer, as the amount of water added was four to seven times of the mass of raw materials, while for the mimai qu wine or jns production in this study, the amount of water did not exceed twice the mass of raw materials. results of srcaa method the nutritional value of amino acids in brewed wine is reflected by the proportion of taa and eaa. a better estimate is the degree of compatibility of eaa and the standard pattern of amino acids most easily absorbed by the human body. who/fao pointed out that when cysteine and tyrosine (tyr) are abundant in diet, they can partially replace methionine and phenylalanine (phe). therefore, phenylalanine and tyrosine, and methionine and cysteine (cys) were consolidated in the srcaa method. based on this, the optimal ratio of eaa in food was proposed as follows: threonine:cystine + methionine:valine:isoleucine:leucine:phenylalanine + tyrosine: lysine:tryptophan = 8:7:10:8:14:12:11:2 (fao/who, 1973). when the ratio of eaa in food is close to this ratio, it is more easily absorbed by the body, with higher nutritional value. as shown in table 3, among the wine samples, smw presented the smallest rc variation coefficient with the highest src (~66) and higher nutritional value, followed by crw (~60) and dmw (~46). the src value of jns (~33) was only half of that for smw, showing the lowest nutritional value (p < 0.05). the first limited amino acids in the wine samples were methionine + cystine. evaluation results based on linear regression method the r-value was used to establish the correlation between contents of seven eaa in the wine samples and the recommended pattern of who/fao. the r-value of smw was the largest (0.938), indicating that its eaa is closest to the who/fao-recommended model, with higher nutritional value. the r-value of crw ranked italian journal of food science, 2021; 33 (2) 51 composition and nutritional evaluation of amino acids in mimai qu rice wines table 3. raa, rc and src of the tested wine samples from srcaa methods. wine sample indicator smw dmw jns crw raa rc raa rc raa rc raa rc thr 0.564 0.819 0.347 0.958 0.180 0.866 0.589 0.914 val 0.793 1.152 0.343 0.947 0.192 0.924 0.581 0.902 lle 0.657 0.957 0.283 0.781 0.151 0.726 0.570 0.884 leu 0.918 1.334 0.395 1.092 0.257 1.236 0.858 1.332 lys 0.584 0.848 0.331 0.916 0.142 0.683 0.564 0.876 met + cys 0.282 0.410* 0.065 0.180* 0.027 0.131* 0.237 0.367* phe + tyr 1.019 1.480 0.770 2.127 0.505 2.434 1.111 1.725 src 66.818a 46.409c 33.807d 60.692b raa: amino acid ratio; rc: ratio coefficient of amino acids; src: the most limiting amino acids of corresponding wine sample; smw: mimai qu rice wine (semi-sweet); dmw: mimai qu rice wine (dry type); jns: japanese sake; crw: chinese rice wine. *mean values within rows without a common superscript lowercase letter indicate significant differences (p < 0.05). second (0.900), while no significant difference was observed between r-values of crw and smw (p < 0.05). the ranked order of the other two wine samples with respect to the r-value was dmw (0.775) and jns (0.752) (p < 0.05). these results were consistent with the srcaa results except for the significant difference between crw and smw in the srcaa method (p < 0.05). cluster analysis and principal component analysis evaluation results cluster analysis and pca are the analytical tools in current statistical analysis to study the hidden connections between samples with unclear data and grouping relationships (granato et al., 2018). it can solve many problems of data analysis involving food science by in-depth mining and data analysis (brown, 2017). the ca is an exploratory data analysis tool that investigates the similarity of data’s potential structure and then divides them into groups (govender and sivakumar, 2019). the pca reduces multiple variables in the sample into several main components through dimensionality reduction, with most of the information being retained for data analysis. based on the results of ca analysis of amino acid data, the wine samples and the who/fao-recommended model were divided into two categories (figure 2). the first category consisted of smw, crw and who/faorecommended model, while the second category included jns and dmw. the first category could be divided into two sub-categories: smw and crw in one group, and who/fao-recommended model in another group. it was concluded that the amino acid nutritional values of smw and crw were similar and were initially grouped into one class with the who/fao-recommended model, indicating that the amino acid composition of the 0 smw crw who/fao jns dmw 5 10 15 20 25 figure 2. cluster analysis results of the tested wine samples based on their amino acid data. who/fao: the who/ fao-recommended amino acid model; jns: japanese sake; crw: chinese rice wine; smw: mimai qu rice wine (semisweet); dmw: mimai qu rice wine (dry type). two wine samples was closer to that of the who/faorecommended model. since the amino acid nutritional values of dmw and jns were similar, they were placed in the same group, and were quite different from the who/ fao-recommended model. based on the amino acid test data and via pca, it was found that pc1 and pc2 explained more than 90% of the total variance. therefore, pc1 and pc2 were selected for further investigation. among the wine samples, smw was the most related sample to the who/ fao-recommended model (figure 3), with a significant coefficient of 0.001, followed by crw with a significant coefficient of 0.002. dmw ranked third (with a significant coefficient of 0.006), and jns ranked last (with a 52 italian journal of food science, 2021; 33 (2) zhang k et al. indicating the highest nutritional value of amino acids in smw, followed by crw, dmw and jns. disclosure statement authors have no potential conflicts of interest. acknowledgments this work was supported by the sci-tech programme of the sichuan province of china (no. 2020jdrc0097); the r&d programme of the neijiang city of sichuan province of china (no. 2020kjfh008) and liquor making biotechnology and application key laboratory of the sichuan province of china (nj2016-02). references biglari, f., alkarkhi, a.f.m. and easa, a.m., 2009. cluster analysis of antioxidant compounds in dates (phoenix dactylifera): effect of long-term cold storage. food chem. 112(4): 998–1001. https:// doi.org/10.1016/j.foodchem.2008.06.063 brown, s., 2017. the chemometrics revolution re-examined. j chemomet. 31(1): 1–23. https://doi.org/10.1002/cem.2864 cui, y., jiang, z., sun, j.y., yu, j., li, m.h. and li, m.j., 2014. enantiomeric purity determination of (l)-amino acids with pre-column derivatization and chiral stationary phase: development and validation of the method. food chem. 158: 401–407. https://doi.org/10.1016/j.foodchem.2014.02.133 fao/who, 1973. energy and 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https://doi.org/10.1016/j.jef.2017.05.005 shin, e.c., craft, b.d., pegg, r.b., phillips, r.d. and eitenmiller,  r.r., 2010. chemometric approach to fatty acid https://doi.org/10.1016/j.foodchem.2009.07.058� https://doi.org/10.1016/j.foodchem.2009.07.058� https://doi.org/10.1111/j.1744-7917.2012.01519.x� https://doi.org/10.6013/jbrewsocjapan1988.92.310� https://doi.org/10.6013/jbrewsocjapan1988.92.310� https://doi.org/10.1016/j.saa.2017.08.055� https://doi.org/10.1111/1750-3841.14768� https://doi.org/10.1111/1750-3841.14768� https://doi.org/10.1002/jib.464� https://doi.org/10.1016/j.foodchem.2019.04.053� https://doi.org/10.1016/j.foodchem.2019.04.053� https://doi.org/10.1016/j.jfca.2008.06.007� https://doi.org/10.1016/j.jfda.2017.03.003� https://doi.org/10.1073/pnas.0407921102� https://doi.org/10.1016/j.jbiosc.2014.04.006� https://doi.org/10.1016/j.jbiosc.2014.04.006� https://doi.org/10.1016/j.je.2016.06.003� https://doi.org/10.1016/j.je.2016.06.003� https://doi.org/10.1080/09168451.2019.1574552� https://doi.org/10.1080/09168451.2019.1574552� https://doi.org/10.1016/j.foodchem.2016.07.060� https://doi.org/10.1016/j.foodchem.2016.08.040� https://doi.org/10.1016/j.foodchem.2016.08.040� https://doi.org/10.1016/j.jef.2017.05.005� ole_link20 ole_link85 ole_link12 ole_link13 ole_link19 ole_link22 ole_link1 ole_link16 ole_link17 ole_link15 ole_link62 ole_link3 ole_link50 ole_link51 ole_link26 ole_link36 ole_link37 ole_link38 ole_link4 ole_link29 ole_link77 ole_link78 ole_link79 ole_link82 ole_link81 ole_link6 ole_link30 ole_link73 ole_link74 ole_link75 ole_link76 ole_link7 bau4 bau5 ole_link9 ole_link11 ole_link10 ole_link35 ole_link14 ole_link18 ole_link52 ole_link53 ole_link54 ole_link55 ole_link21 ole_link2 ole_link32 ole_link28 ole_link27 ole_link57 ole_link58 ole_link59 ole_link60 ole_link56 ijfs#1354_bozza ital. j. food sci., vol. 31, 2019 301 paper utilization of jerusalem artichoke powder in production of low-fat and fat-free fermented sausage c.o. özer department of food engineering, faculty of engineering and architecture, nevsehir hacı bektaş veli university, 50300, nevsehir, turkey *corresponding author: tel.: +90 3842281000-15077; fax: +90 3842281123 e-mail address: cemokanozer@nevsehir.edu.tr abstract the effects of jerusalem artichoke powder (jap) on the quality characteristics and storage stability of fermented sausage were investigated. the replacement of added beef fat with jap were carried out during sausage manufacturing. replacement of beef fat with jap resulted in a significant decrease in tbars and ph values and an increase in moisture and protein content in sausages during fermentation and storage (p<0.05). l* values decreased and a* values increased by adding jap (p<0.05). jap addition decreased hardness values and increased adhesiveness (p<0.05). the use of jap encourages the development of lactic acid bacteria and positively affected their counts during the fermentation (p<0.05). keywords: sausage, jerusalem artichoke powder, functional food, storage stability ital. j. food sci., vol. 31, 2019 302 1. introduction in recent years, functional meat products have attracted the attention of the consumer and their demands are rapidly increasing. researchers and producers have tried to meet these demands with different strategies they have developed such as low fat, cholesterol, sodium chloride and nitrite and improved composition of meat products with incorporated health enhancing specific natural food ingredients (demeyer and astiasarán, 2007). natural additives have been more preferred than synthetic additives in food products because of their low toxicity, safety and cost efficiency (sirocchi et al., 2017; van haute et al., 2016). several natural and functional components mainly from plant origins have been introduced to meet consumer preferences and technological requirements. therefore, improvement of meat products with some natural vegetable source compounds has been studied extensively over the past decades (hygreeva et al., 2014; olmedilla-alonso et al., 2013). vegetable sources with functional properties can be allowing many strategies to be carried out together in meat products and so many effects can be shown together such as reduction of beef fat, cholesterol and energy, enrichment with antioxidants, plant protein and dietary fiber and some technological improvements (grasso et al., 2014; olmedilla-alonso et al., 2013). jerusalem artichoke (helianthus tuberosus l.) powder also may be a versatile alternative functional additive in dry fermented sausage to both reduction of beef fat and a consequent reduction of cholesterol, saturated fatty acid and energy value and increasing nutritional value and textural properties without adversely affecting quality characteristics. jerusalem artichoke contains high amounts of dietary fiber and most of it is inulin. additionally, it contains some antioxidant compounds such as polyacetylenic derivatives, sesquiterpenes and coumarins (furlan et al., 2014). there are many studies about using of inulin and jerusalem artichoke (gedrovica and karklina, 2013) in many food products such as sausages, frankfurters, cookies, pasta and some dairy products and also successful results about technological and nutritional improvements have been reported in the literature (afoakwah et al., 2015, furlan et al., 2014; praznik et al., 2002). however, study about the effects of jerusalem artichoke powder in meat fermentation is limited in the literature. therefore, the present study is aimed to assess the effect of jerusalem artichoke powder as beef fat replacers on the quality characteristics and storage stability of dry fermented sausage, including physicochemical composition, lipid oxidation, color, textural and microbiological properties. 2. material and methods 2.1. ingredients a 24 h post-mortem longissimus thoracis et lumborum cuts and beef fat (beef back fat) was purchased from a local butcher (birlik market, nevsehir, turkey) for each of 3 replications on separate production days. all meat cuts were trimmed from all subcutaneous and intermuscular fats. the lean beef and fat sources were separately ground in a 2 mm and 3 mm plate meat grinder, respectively (arı makine a.ş, i̇stanbul). the freeze-dried culture mix, which contains lactobacillus curvatus, staphylococcus xylosus, staphylococcus carnosus and pediococcus pentosaceus, was used as starter culture mix. jerusalem artichoke (helianthus tuberosus l.) tubers were obtained from a local producer (nevsehir, turkey) during harvest season. jerusalem artichoke was washed, peeled and then dried using freeze dryer at −80°c and 0.01 mbar (operon, opr-fdu-8612, korea). ital. j. food sci., vol. 31, 2019 303 dried samples were ground to a fine powder using laboratory grinder (yucebas makine, izmir, turkey). physicochemical properties, which are moisture, fat, ash, protein, carbohydrate and dietary fiber contents of jerusalem artichoke powder was determined. 2.2. sausage preparation the production and ripening of sausages were carried out as described in ozer and kilic (2014). five batches were prepared from ground beef. the control group incorporated with %72 lean beef and 20% beef fat was prepared without jerusalem artichoke powder. the other experiment groups were given codes as jap25, jap50, jap75 and jap 100 in relation to the ratio of jap powder used replacement of beef fat. jap25, jap50, jap75 and jap100 groups were produced with 15% beef fat and 5% jap, 10% beef fat and 10% jap, 5% beef fat and 15% jap and 0% beef fat and 20% jap, respectively. the other ingredients were added into sausages as follows: 2.5 % nacl, 1.5 % garlic, 1.5 % red pepper, 0.8 % cumin, 0.5 % sucrose, 0.5 % black pepper, 0.5 % allspice and 150 ppm nano2. during the mixing, the starter culture mixture was added at a dose of 4-5 log cfu/g of sausage dough. after the filling up to cases of sausages samples, sausages were ripened at 95-70 % relative humidity (rh) at 25-18 ºc during 7 days; 24 h at 95 % rh at 24 ºc; 24 h at 90 % rh at 22 ºc; 12 h at 85 % rh at 20 ºc; 12 h at 80 % rh at 20 ºc; 48 h at 75 % rh at 18 ºc; and 48 h at 70 % rh at 18 ºc. sausages were vacuum-packaged and then stored at 4 ºc for 30 days. the entire experiment was replicated three times on separate processing days. 2.3. physicochemical composition fat, protein, ash and moisture content of sausages were measured at manufacturing and after fermentation day (aoac 2005). ph of sausages were measured at manufacturing day, after fermentation and during storage period (7, 15, 30d) (aoac 2005). also, moisture, fat, ash, protein and total dietary fiber content of jap according to aoac (2005). additionally, the total carbohydrate content of jap was calculated by difference. furthermore, total phenolic compounds were determined as described by takeuchi and nagashima (2011) and total phenol concentration was quantified against a gallic acid (sigma-aldrich, st. louis mo) standard curve. finally, the water holding capacity of jap was determined and expressed as g water per g of jap (takeuchi and nagashima 2011). 2.4. tbars analysis thiobarbituric acid reactive substances (tbars) values of samples were determined as described by kilic and richards (2003) to evaluate of oxidation stability during fermentation and storage period (7, 15, 30 d) and tbars values were expressed as μmol tbars per kg of meat. 2.5. color measurement color measurement was conducted by a minolta chroma meter cr-200 (minolta, osaka, japan) colorimeter using d65 as a standard daylight illuminant and a standard observer position of 10°. 8-mm-diameter circle and the specular component included (sci) mode was used to measure. the colorimeter was standardized against a white calibration plate (d65, cie l*=97.79, a*=−0.11, b*=2.69). three readings were taken and averaged for each ital. j. food sci., vol. 31, 2019 304 of the three replications. color values were determined at manufacturing day, after fermentation and during storage period (7, 15, 30 d). 2.6. texture profile analysis samples were cut into slices (10±0.5 mm thick), wrapped with plastic film, and then held for equilibration to room temperature (20 ºc) for texture profile analysis (tpa). tpa tests and conditions were carried out as described in kılıç and özer (2017), using a texture analyzer (ta-xt2í, stable micro systems, uk). test conditions were briefly as follows: rectangular probe (5 cm · 4 cm); pre-test speed 2 mm/s, test speed 5 mm/s, post-test speed 2 mm/s, 70 % compression and 50 kg load cell. hardness (n), adhesiveness (ns), springiness, cohesiveness, chewiness index, and resilience value of sausages were determined using 6 sausage slices per treatment. 2.7. microbiological analysis sausage samples (10 g) were homogenized with sterile buffered peptone water (90 ml) in a stomacher at room temperature. decimal dilutions in buffered peptone water were prepared and duplicate 0.1 ml samples of appropriate dilutions were spread. the following groups were investigated: total viable aerobic (tvac) on plate count agar (merck, darmstadt, germany), incubated at 30°c for 48 h; lactic acid bacteria on de man, rogosa and sharpe agar (merck, darmstadt, germany), incubated in anaerobic jar at 37°c for 48 h; yeast and molds on potato dextrose agar (merck, darmstadt, germany), incubated at 25°c for 72 h; coliform on violet red bile agar (merck, darmstadt, germany), incubated at 37°c for 24 h. 2.8. statistical analysis the results were expressed as mean values with standard errors from the three replications. the statistical evaluation of the results was performed using the spss 22.0.0 (spss inc., chicago, usa). data collected for physicochemical properties of sausages were analyzed by one-way analysis of variance (anova). a completely randomized design was used with 5 treatment groups and 3 replications on separate production days. the treatments were one control group and four groups, which were assigned, and the data were analyzed using general linear model (glm) procedure, in which treatment groups and storage time were assigned as fixed effects and replications as a random effect. duncan multiple comparison test was used to compare means values and differences among mean values were considered significant when p<0.05. 3. results and discussion 3.1. microbiological analysis replacement of beef fat by jap showed no significant effect on mold and yeast, total viable and coliform bacteria count during fermentation and storage periods (data is not presented) (p<0.05). the microbiological counts of sausage groups at the end of the fermentation ranged from 8.71 to 9.37 log cfu/g for total viable, from 5.61 to 5.82 log cfu/g for mold and yeast, and from 0.42 to 1.28 log cfu/g for total coliform bacteria, respectively. however, results of lactic acid bacteria count showed that the use of 50% and a higher rate ital. j. food sci., vol. 31, 2019 305 of jap significantly increased lactic acid bacteria count during the fermentation period (p<0.05) (fig. 1). figure 1. lactic acid bacteria count in sucuk during the fermentation and storage periods. a, b; different letters in same day are significantly different (p<0.05). this may be associated with the prebiotic effect of dietary fiber especially inulin and nutritional value of jerusalem artichoke for lactic acid bacteria. it has reported that certain species of lactobacilli can ferment fructo-oligosaccharides and degrade even long-chain inulin-type fructans (choi et al., 2012). additionally, choi et al. (2012) indicated that some l. paracasei strains could convert carbohydrates in jerusalem artichoke to lactic acid without any pretreatment. there is numerous study about the prebiotic effect of added inulin to food products in literature and they indicated that inulin promotes the growth and activities of probiotic microorganisms in some dairy products such as yogurt, ice cream and cheese (cardarelli et al., 2008). 3.2. physicochemical composition analysis physicochemical properties of used jap were determined and results showed that it contained 70.43%±0.68 carbohydrates, 8.18%±0.21 moisture, 9.46%±0.11 protein, 4.19%±0.18 ash and 7.13%±0.14 total dietary fiber. the determined mean proximate compositions for jap are consistent with the literature (afoakwah et al., 2015; praznik et al., 2002). furthermore, jap contained 103.41±21.6 mg gae/kg total phenolic compounds. takeuchi and nagashima (2011) have reported that peels of jerusalem artichoke tuber were the main source of polyphenols and peels contains 10 times more polyphenols than peeled tubers and our result is similar to theirs. jap revealed having the capacity to absorb at least 4.57±0.11 g of water/g at 20°c. the proximate compositions of sausages formulated with different levels of jap are given in table 1. the use of jap had shown significant differences in protein, ash, moisture and fat content of sausage dough. in addition, replacement of beef fat by jap had shown significant differences on all components except ash values after fermentation. jap75 and jap100 had the highest protein content (p<0.05). additionally, as expected, there were significant differences in the fat content of sausage dough (p<0.05) due to variation in the amount of beef fat used in sausage manufacture. similar differences were also determined after fermentation (p<0.05) and control samples no containing jap had the highest fat content after fermentation and storage (p<0.05). the moisture content for sausages after 7 7,2 7,4 7,6 7,8 8 8,2 8,4 8,6 8,8 9 9,2 manufacturing day end of the fermentation end of the storage control jap25 jap50 jap75 jap100 a a a b b a a a b b log cfu/g ital. j. food sci., vol. 31, 2019 306 fermentation was lower in the control group than other treatment groups (p<0.05). these results may be related to protein and carbohydrate content and water holding ability of jap. it was also previously reported that compounds having water holding ability decelerate water loss and the drying rate during the ripening period in dry fermented meat products (choi et al., 2009). additionally, garcia et al. have reported that dietary fibers or additives containing rich carbohydrate and protein may cause a lower degree of water loss during ripening of fermented meat products such as dry sausage (garcia et al., 2002). as a result, the protein content proportionally increased by approximately 13% due to the drying process during the fermentation period, and fat content decreased by approximately 36% due to replacing beef fat with jap in the final product. ph of all sausage samples decreased during fermentation period (p<0.05) and were found to be at the same level for all sausage groups during the storage period (table 2). table 1. chemical composition* of sausage treatment groups. groups manufacturing day end of the fermentation manufacturing day end of the fermentation protein (%) fat (%) control 18.64±0.24 cb 25.91±0.24 ca 21.51±0.10 ab 31.35±0.18 aa jap25 18.98±0.10 cb 26.64±0.39 ca 19.39±0.27 bb 29.88±0.26 ba jap50 19.25±0.13 bb 27.83±0.14 ba 16.11±0.16 cb 25.71±0.13 ca jap75 19.70±0.16 ab 28.77±0.28 aa 12.91±0.20 db 23.12±0.27 da jap100 20.11±0.25 ab 29.41±0.43 aa 10.52±0.19 eb 19.91±0.19 ea groups ash (%) moisture (%) control 1.91±0.04 db 4.06±0.09 ba 56.94±0.28 ea 36.68±0.61eb jap25 2.08±0.04 cb 4.28±0.14 aba 59.54±0.67 da 39.20±0.39db jap50 2.19±0.16 bcb 4.52±0.21 aa 62.45±0.41 ca 41.94±0.27cb jap75 2.40±0.13 bb 4.12±0.19 aba 64.99±0. 87 ba 43.99±0.74bb jap100 2.76±0.08ab 4.50±0.24 aa 66.60±0.48 aa 46.18±0.62ab *all values are the mean ± standard error of three replicates. a, b, c, d, e (↓) different letters within a column are significantly different (p<0.05). a,b (→) different letters within a row are significantly different (p<0.05). table 2. ph values* of sausage during the manufacturing, fermentation and storage periods. groups manufacturing day end of the fermentation storage days (d) 7d 15d 30d control 5.84±0.00 ba 5.11±0.00 ab 5.08±0.00 ab 5.07±0.00 ab 5.06±0.01 ab jap25 5.85±0.00 aba 5.06±0.02 bb 5.04±0.02 bb 5.02±0.03 bb 5.01±0.02 bb jap50 5.86±0.01 aba 5.02±0.01 cb 4.99±0.01 cb 4.98±0.02 bb 4.96±0.01 cb jap75 5.85±0.00 aba 4.94±0.00 db 4.94±0.00 db 4.93±0.00 cb 4.92±0.01 db jap100 5.87±0.00 aa 4.90±0.00 eb 4.88±0.00 eb 4.84±0.00 db 4.84±0.00 eb *all values are the mean ± standard error of three replicates. a, b, c, d, e (↓) different letters within a column are significantly different (p<0.05). a, b, c, d, e (→) different letters within a row are significantly different (p<0.05). ital. j. food sci., vol. 31, 2019 307 replacement of beef fat by jap had shown no significant differences in sausage dough at manufacturing day. however, replacement of beef fat by jap in sausage production affected the ph of sausages during the fermentation period. ph values of sausages decreased depending on the amount of replacement of beef fat by jap in the sausage formulation and significant differences were determined among the treatment groups at the end of fermentation day and storage period (p<0.05). mendoza et al. (2001) have reported a conflicting result with us that the use of inulin as a fat substitute in low fat-dry fermented sausages did not affect the ph during the fermentation period. however, the reason for decreases in ph of sausages in the present study is may be related to protein, carbohydrates and dietary fiber content of jap. these components in jap could be used as a nutrient by lactic acid bacteria and produced more lactic acid. additionally, jap may have created a suitable environment with high moisture content for development of starter culture by decelerating water loss and the drying rate during the fermentation period. despite the effects of replacement of beef fat by jap on the physicochemical properties of sausages produced in the present study were in accordance with the values reported by previous studies and turkish standards for sausage (bozkurt and bayram, 2006; tse, 2002). 3.3. tbars analysis tbars values of sausages were measured throughout fermentation and the storage period shown in table 3. the tbars values of all sausage samples increased during fermentation and storage periods (p<0.05). table 3. tbars values* of sausage (µmol/kg) during the manufacturing, fermentation and storage periods. groups manufacturing day end of the fermentation storage days (d) 7d 15d 30d control 0.74±0.02 be 2.19±0.04 ad 2.82±0.02 ac 3.49±0.02 ab 4.88±0.02 aa jap25 0.78±0.07 abe 1.81±0.01 bd 2.46±0.10 bc 3.12±0.02 bb 4.26±0.10 ba jap50 0.78±0.02 abe 1.59±0.01 cd 2.22±0.02 cc 2.73±0.05 cb 3.82±0.06 ca jap75 0.82±0.03 abe 1.47±0.02 dd 2.01±0.09 dc 2.43±0.06 db 3.47±0.03 da jap100 0.85±0.01 ae 1.35±0.02 ed 1.89±0.02 dc 2.24±0.02 eb 2.92±0.02 ea *all values are the mean ± standard error of three replicates. a, b, c, d, e (↓) different letters within a column are significantly different (p<0.05). a, b, c, d, e (→) different letters within a row are significantly different (p<0.05). there were no differences among tbars values among all treatment groups for sausage dough. however, highest tbars values in control group and lowest tbars values in jap100 group were determined compared with other treatments at the end of fermentation and storage period (p<0.05). according to tbars results, it can be said that the replacement of beef fat by jap in sausage production significantly decreased tbars values (p<0.05). the decrease in tbars levels may be a result of both reducing the beef fat and increasing the jap content. additionally, it is conceivable that bioactive components such as flavonoids and phenolic compounds such as polyacetylenic derivatives, sesquiterpenes and coumarins in jerusalem artichoke exhibiting antioxidative activities and contributing to decrease in tbars values (furlan et al. 2014). ital. j. food sci., vol. 31, 2019 308 3.4. color analysis the color properties of used jap were determined and results showed that l* value is 81.18±0.51, a* value are 2.32±0.11 and b* value is 8.71±0.23. replacement of beef fat by jap influenced cie l* and cie a* values in sausage dough, at the end of fermentation and during storage period (p<0.05) (table 4). however, non-significant differences on cie b* values were determined during fermentation and storage period (p<0.05). table 4. color values* of sausages during the manufacturing fermentation and storage periods. groups manufacturing day end of the fermentation storage days (d) 7d 15d 30d l* v al ue s control 58.02±2.70aa 50.07±2.46 ab 49.35±2.42 ab 49.91±2.45 ab 50.40±2.48 aab jap25 46.89±1.42 ba 42.67±1.30 bcb 42.06±1.28 bcb 42.53±1.30 bcb 42.96±1.30 bcb jap50 47.44±1.44 ba 43.99±0.42 bb 43.36±1.40 bb 43.85±1.42 bb 44.29±1.43 bb jap75 43.50±0.67 ca 40.09±0.66 cb 39.51±0.65 cb 39.96±0.65 cb 40.36±0.66 cb jap100 41.21±1.06 ca 37.08±1.20 db 36.55±2.17 db 36.96±2.19 db 37.33±2.22 db a* v al ue s control 12.33±1.37 ca 11.22±1.24 cb 11.11±1.23 cb 11.38±1.26 cb 11.50±1.28 cab jap25 14.85±0.49 ba 13.51±0.44 bb 13.37±0.44 bb 13.70±0.45 bb 13.84±0.46 bb jap50 15.72±0.15 ba 14.31±0.13 bb 14.16±0.13 bb 14.51±0.13 bab 14.65±0.14 bab jap75 15.91±0.30 ba 14.48±0.28 bb 14.33±0.36 bb 14.68±0.32 bb 14.83±0.01 bab jap100 17.34±0.22 aa 16.78±0.20 aab 15.62±0.20 ab 16.00±0.20 aab 16.16±0.80 aab b* v al ue s control 10.65±0.71 aa 10.86±0.72 aa 10.80±0.72 aa 10.75±0.72 aa 10.86±0.73 aa jap25 12.32±2.76 aa 12.56±2.82 aa 12.50±2.80 aa 12.43±2.79 aa 12.56±2.82 aa jap50 12.95±1.04 aa 13.21±1.06 aa 13.14±1.06 aa 13.08±1.05 aa 13.21±1.06 aa jap75 11.63±1.04 aa 11.86±1.06 aa 11.80±1.05 aa 11.74±1.05 aa 11.86±1.06 aa jap100 10.89±1.49 aa 11.11±1.57 aa 11.05±1.55 aa 11.00±1.53 aa 11.11±1.57 aa *all values are the mean ± standard error of three replicates. a. b. c (↓) different letters within a column are significantly different (p<0.05). a. b. c. d (→) different letters within a row are significantly different (p<0.05). when the replaced of beef fat by jap, cie l* values decreased and cie a* values increased in sausage samples. it was determined that the control group had the highest cie l* and lowest cie a* values at all manufacturing period (p<0.05). additionally, cie l* values for all treatment groups decreased during fermentation period (p<0.05). some researchers have reported that non-meat ingredients may affect the color properties of minced meat products because of the dilution of meat pigments rather than the color of the additives (sarteshnizi et al., 2015; trespalacios and pla, 2007). these results in the present study were similar to some previous studies about the use of some vegetable source ingredient in meat products (alakali et al., 2010; ergezer et al., 2014). 3.5. texture profile analysis table 5 shows the tpa results of the sausages. replacement of beef fat by jap in sausage had shown a significant effect on hardness and adhesiveness. however, there were no significant changes in resilience, cohesiveness, chewiness index and springiness index among all sausage treatment groups. results revealed that replacement of beef fat by jap ital. j. food sci., vol. 31, 2019 309 resulted in an increase in adhesiveness values of sausages and a decreased in the hardness values compared control group (p<0.05). it is reported in many studies that fat reduction in comminuted meat products results in significant changes in textural properties and generally, hardness values of products increased (colmenero, 1996; keeton, 1994; stehle, 2009). however, results of the present study showed that 50%, 75% and 100% replacement of beef fat by jap in sausage resulted in a decrease in hardness values of sausages. jap25 group, which contains 25% jap and 75% beef fat, and the control group had the same level of hardness values. table 5. texture profile analysis* of sausage at the end of the fermentation period. groups hardness (n) adhesiveness (mj) resilience cohesiveness springiness index chewiness index (n) control 61.69±2.39 a 0.43±0.05 c 0.02±0.00 a 0.40±0.07 a 0.60±0.09 c 0.90±0.25 a jap25 60.92±1.07 a 0.64±0.07 b 0.03±0.00 a 0.36±0.02 a 0.75±0.03bc 0.88±0.26 a jap50 52.40±0.88 b 0.76±0.04 a 0.03±0.01 a 0.44±0.02 a 0.81±0.02 b 1.28±0.42 a jap75 35.20±0.63 c 0.80±0.00 a 0.05±0.01 a 0.44±0.07 a 0.88±0.02 b 1.35±0.33 a jap100 27.94±1.13 d 0.83±0.03 a 0.04±0.02 a 0.36±0.07 a 1.07±1.16 a 1.22±0.24 a *all values are the mean ± standard error of three replicates a, b, c (↓) different letters within a column are significantly different (p<0.05) the decrease in hardness values may be a result of increasing the proteins, carbohydrates and dietary fiber that have functional properties such as water binding and retention properties. researchers have reported that fat replacer containing proteins, carbohydrates and dietary fiber may interact with water and fat of meat products and therefore lead to a change in textural properties (ergezer et al., 2014). the effects of using jap on the moisture content of sausage also support this idea. 4. conclusions regarding to results of lipid oxidation analysis, this study proved that improved lipid oxidation stability in fermented sausages during fermentation and storage can be achieved by replacing beef fat with jerusalem artichoke powder. however, some textural differences, which can be undesirable by the consumer, can occur. nevertheless, it can be concluded that meat products manufacturers should consider replacing beef fat with up to 25% jerusalem artichoke powder in low-fat fermented sausage production to enhance positive nutritional effects such as lower beef fat and energy value and rich dietary fiber and improve the shelf life of sausage. acknowledgements author thank to ayşegül gündem, dudu göçmen and dilek kazan for all assistance. additionally, author would like to thank the tubitak (1139b411600477) for the financial support of this study. ital. j. food sci., vol. 31, 2019 310 references afoakwah n.a., dong y., zhao y.s., xiong z.y., owusu j., wang y. and zhang j.y. 2015. characterization of jerusalem artichoke (helianthus tuberosus l.) powder and its application in 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accepted november 15, 2018 24 issn 1120-1770 online, doi 10.15586/ijfs.v33i2.1918 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (2): 24–34 p u b l i c a t i o n s codon the influence of starter cultures on the lactic acid bacteria microbiota of petrovac sausage bojana milićević1*, vladimir tomović2, bojana danilović1 and dragiša savić1 1faculty of technology, university of nis, leskovac, serbia; 2faculty of technology, university of novi sad, novi sad, serbia *corresponding author: bojana milićević, faculty of technology, university of nis, leskovac, serbia, email: bojanamilicevic@rocketmail.com received: 11 june 2020; accepted: 18 february 2021; published: 6 april 2021 © 2021 codon publications open access paper abstract petrovac sausage (petrovská klobása) is a high-quality fermented dry sausage produced traditionally in the municipality of bački petrovac (vojvodina, serbia). the product is characterised by specific and recognised texture, aroma and colour, produced without additives or preservatives. lactic acid bacteria (lab) microbiota plays an important role in production of the sausage. the aim of the paper is to monitor the changes in lab during the production of petrovac sausage. samples of sausages were prepared without and with the addition of starter culture staphylococcus xylosus as well as combined starter culture lactiplantibacillus plantarum and s. xylosus, and produced at two different temperature ranges. a total number of 495 strains were isolated from 33 samples of petrovac sausage during 120 days of production process. characterisation of the isolates was performed by phenotypic tests, while molecular identification of the representative strains was done by 16s ribosomal dna sequencing. the total number of lab was about 8 log (colony forming unit (cfu))/g in all samples, while the number of staphylococci was about 4 log cfu/g. molecular identification confirmed that all isolates belonged to the following species: levilactobacillus brevis, leuconostoc mesenteroides, lactiplantibacillus plantarum and pediococcus pentosaceus. lactobacilli and leuconostoc spp. dominate the total lab strains, while p. pentosaceus was isolated at the lowest frequency. keywords: fermented sausages, lactic acid bacteria, petrovac sausage, 16s rdna sequencing introduction the production of fermented sausages correlate with the diversity of microbiota present in meat batter as well as those added in the form of starter culture (cocconcelli and fontana, 2008; toldra, 2002). starter cultures contribute to the functional properties of fermented products and play a major role in the improvement of organoleptic, technological, nutritional and health characteristics of fermented sausages (laranjo et al., 2017). different types of microorganisms (lactic acid bacteria [lab], staphylococci and micrococci, mould, and yeasts) can be used for autochthonous and commercial starter cultures in the production of fermented sausages (casaburi et al., 2008; kovacevic et al., 2010). combination of lactobacillus spp. and staphylococcus spp. used as starter cultures in the sausages production can contribute to the pleasant aroma of sausages and they possess antimicrobial properties against unwanted microorganisms and pathogen microbiota (hosseini and pilevar, 2017). owing to acidification, lipolysis, and proteolysis, and production and development of volatile aroma compounds, lab microbiota (lactobacillus sakei, lactiplantibacillus pentosus, lactobacillus curvatus, lactiplantibacillus plantarum, lacticaseibacillus paracasei, levilactobacillus brevis etc.) play an essential role of a starter culture in meat fermentation (ammor and mayo, 2007; kumar et al., 2017). production of lactic and acetic acid reduces ph of the meat batter resulting in the formation of characteristic sausage aroma and consistency. the acidification process plays an important role in the inhibition and inactivation of pathogenic microorganisms contributing to the prolonged shelf life and safety of fermented sausages (leistner, 1995; martinovic and veskovic-moracanin., mailto:bojanamilicevic@rocketmail.com italian journal of food science, 2021; 33 (2) 25 the influence of starter cultures on the lactic acid bacteria microbiota of petrovac sausage ‘bačka topola’ (vojvodina, serbia). meat batter was made of minced pork (85%) and solid back fat tissue (15%) with addition of the following ingredients (w/w): red hot pepper (2.5%), salt (1.8%), garlic (0.2%), caraway seeds (0.2%) and sucrose (0.1%). the meat batter was divided into three equal parts: control sausages (h) (without addition of starter), sausages (i) (with the addition of combined starter cultures of s. xylosus and l. plantarum) and sausages (j) (with the addition of starter culture of s. xylosus). the initial number of starter cultures in meat batter was the same for lab and coagulase-negative cocci (cnc) (4.5–5.0 log (cfu)/g). autochthonous starter cultures were previously isolated from traditionally produced petrovac sausage (danilovic, 2012). the mixture was stuffed into artificial collagen casings. smoking, drying and ripening of the sausages were carried out under controlled conditions in the ripening chamber at the temperature range of 14–16°c (tag 1) and ~10°c (tag 2). all experiments were performed in triplicate. samples were collected after 0 (meat batter), 6, 15, 60, 90 and 120 days of production. isolation and enumeration of bacteria for microbiological analysis, 10 g of each sausage sample was aseptically homogenised in 90 ml of sterile saline peptone water (8 g/l nacl + 1 g/l peptone) (urso et al., 2006). the enumeration of microorganisms was performed in triplicate by the successive serial dilution method and represented as the mean value. dilutions were prepared and plated on nutrition agar (na, torlak, belgrade, serbia), de man, rogosa and sharpe (mrs) agar (torlak, belgrade, serbia) and mannitol salt agar (msa) plates for determining the total number of mesophilic bacteria, lab and staphylococci, respectively. after the incubation of plates (48  h, 30°c) and enumeration, randomly selected colonies from mrs agar plates were streaked to new mrs agar plates for purification. phenotypic identification and characterisation of lab isolates basic characterisation of the isolates was performed through gram reaction, cell morphology and catalase test with h2o2 (30% v/v). gram-positive and catalase-negative isolates were subjected to the following physiological tests: co2 production, arginine and esculin hydrolysis, bacterial growth on mrs agar plates at different temperatures (15°c and 45°c) for 72 h, bacterial growth on mrs agar plates supplemented with nacl (4%, 6.5% and 8%) for 72 h, bacterial growth on bile esculin agar, synthesis of exopolysaccharides and the synthesis of bacteriocines. 2006). lab species can produce bacteriocins as antimicrobial products of fermentation. l. sakei, l. curvatus, l. plantarum and l. paracasei, which are often used as starter cultures, contribute to the safety and stability of fermented sausages because of strong antibacterial activity against escherichia coli and listeria monocytogenes (pidcock et al., 2002; veskovic-moracanin, 2010). in addition to the safety of product, some strains of lactobacilli used as starter cultures (l. sakei, l. curvatus and l. plantarum) promote the degradation of peroxide (martinovic and veskovic-moracanin., 2006). gram-positive cocci used as starter cultures (staphylococcus carnosus, staphylococcus xylosus and micrococcus varians) play an important role in the reduction of nitrates and nitrites, decomposition of peroxides, lipolysis stabilisation and development of texture (skocińska et al., 2016). s. carnosus and s. xylosus as starter cultures contribute to the development of desirable colour and aroma in fermented sausages. owing to antioxidant properties, growth on optimal salt concentrations and growth on optimal ph, s. carnosus and s.  simulans are often used as starters in fermented sausages (casaburi et al., 2005). dry-fermented sausages represent the result of physical, chemical, biochemical, microbiological and sensory changes that occur during the ripening of meat batter (hammes et al., 2008). petrovac sausage (petrovská klobása) is a traditional dry-fermented product made in bački petrovac (vojvodina, serbia). as a high-quality fermented product with appropriate texture, aroma and colour, it is produced without additives or preservatives, and protected by protected denomination of origin (pdo) at the national level (ikonic et al., 2015; petrovic et al., 2007). petrovac sausage can be produced without adding starter cultures (danilovic et al., 2018). the traditional production excludes the addition of starter cultures (ikonic et al., 2016; jokanovic et al., 2017, 2010). the aim of this work was to monitor the changes in lab microbiota in the samples of petrovac sausage (petrovská klobása) prepared without and with the addition of starter culture s. xylosus and combined starter cultures l. plantarum and s. xylosus and produced under controlled conditions in two different temperature ranges. for this purpose, isolation, characterisation and identification of lab microbiota were performed. materials and methods fermented sausage technology and sampling procedure fermented sausages were produced according to the traditional recipe in the agro-industrial complex (aic) 26 italian journal of food science, 2021; 33 (2) milićević b et al. by applying ccd camera biometra bdr2/5/6 (bio doc analyze). specific pcr products were analysed by electrophoresis on 1% agarose gel and purified using qiaquick pcr purification kit/250 (qiagen, hilden, germany). purified pcr amplicons were sequenced using macrogen sequencing service in seoul, south korea. the results were compared with the data stored in the national centre for biotechnology information (ncbi) gene databank using blast algorithm (www. ncbi.nlm.nih.gov/blast). results and discussion petrovac sausage is an indigenous fermented sausage produced of minced meat and spices without preservatives with specific and recognisable characteristics. the sausage fermentation process is greatly affected by the changes in the development and composition of lab and staphylococci microbiota. in order to determine the changes in lab microbiota during the production of petrovac sausage with the addition of starter cultures, sausage samples were prepared without starter culture (sausages h), with combined starter culture l. plantarum and s. xylosus (sausages i) and with starter culture s. xylosus (sausages j). production of the sausages was performed under controlled conditions at a temperature range of 14–16°c (tag 1) and ~10°c (tag 2). during the production of petrovac sausage, the change in the number of mesophilic bacteria was almost identical to the change in lab regardless of using starters. in the sausages prepared without starter cultures (sausages h), the number of initial lab and aerobic mesophilic bacteria was about 5 log cfu/g, while the number of staphylococci ranged about 4 log cfu/g. the maximum value of lab and aerobic mesophilic bacteria (8–9 log cfu/g) was reached after 15 days and it remained stable till the end of the production process. the number of staphylococci at the end of the process was lower and was about 3 log cfu/g (figure 1h). similarly, in the sausages prepared with combined starter culture of s. xylosus and l. plantarum (sausages i), the initial number of lab was almost identical to the initial number of aerobic mesophilic bacteria (about 5 log cfu/g). during the production of sausages  i, similar changes in the number of both lab and aerobic mesophilic bacteria were observed as in sausages h. the number of staphylococci in sausages i was in the same range as in sausages prepared without starter cultures (3–4 log cfu/g) (figure 1i). in sausages prepared with the addition of starter culture s. xylosus (sausages j), the number of staphylococci at the end of production was higher in all samples produced at 14–16°c (about 3 log cfu/g) than in the samples produced at ~10°c (about 2 log cfu/g). changes in the number of lab and aerobic mesophilic bacteria were almost identical as in sausages i (figure 1j). arginine hydrolysis was performed in arginine broth (g/l: tryptophan 5, l-arginine 3, glucose 0.5 and k2hpo4  2), while esculin hydrolysis was performed in esculin broth (torlak, belgrade, serbia). after incubation, a few drops of phenyl-red were added to the arginine broth (red colour indicates a positive reaction, and yellow colour a negative one), and a few drops of 2% fecl3 solution to the esculin broth (a positive reaction is the appearance of a black precipitate). for preliminary identification of enterococci, isolates were grown on bile esculin agar (rocheux’s medium, himedia, mubai, india). the appearance of black colonies indicate the presence of enterococci. exopolysaccharide production was detected visually (appearance of mucous colonies) after incubation of isolates on a modified mrs medium supplemented with maltose, sucrose, galactose, fructose, lactose and glucose (merck gmbh, darmstadt, germany) at a temperature of 30°c for 48 h. the bacteriocinogenic activity was performed using the agar well diffusion assay. soft nutrition agar (0.7% w/v), containing indicator strain, was poured into plates with thin layer of mrs agar. after hardening of the medium, small diameter wells (10 mm) were made into plates. into each well, aliquot (50 µl) of the supernatant of overnight culture (16 h) was poured. also, a crystal of pronase e was added close to the edge of the bacteriocin-containing well. the plates were incubated at 30°c for 24 h. appearance of a clear inhibition zone around the well was recorded as a positive signal for production of bacteriocin. for detecting bactericiongenic activity, bacillus subtilis, listeria monocytogenes and e. coli were used as pathogenic microorganisms. production of bacteriocin against any of the analysed strains was stated as positive. molecular identification of lab isolates isolation of the total genomic dna as well as (gtg)5pcr fingerprinting was performed as described previously (nikolic et al., 2008). for 16s ribosomal dna (rdna) sequencing method, pcr amplifications with primers uni 16sf (5’-gag agt ttg atc ctg gc-3) and uni 16sr (5’-agg agg tga tcc agc cg-3’) were performed with a taq dna polymerase kit (fermentas uab, vilnius, lithuania). the amplification of the samples was performed through geneamp® pcr system 2700 (applied biosystems) operated with the following parameters: the initial duration of dna for 7 min at 95ºc, 32 cycles of denaturation of 1 min at 94ºc, polymerisation with a duration of 8 min at 65ºc, and the final extension of incomplete product with a duration of 16 min at 65ºc. plasmide profiles were monitored on 1.5% (w/v) agarose gel with ethidiumbromide at a constant voltage of 60 v (at 4ºc for 20 h) (versalovic et al., 1994). visualisation of pcr products was performed www.ncbi.nlm.nih.gov/blast� www.ncbi.nlm.nih.gov/blast� italian journal of food science, 2021; 33 (2) 27 the influence of starter cultures on the lactic acid bacteria microbiota of petrovac sausage mixed starter culture of s. xylosus and l. plantarum (8.8 cfu/g) (el adabi et al., 2014). also, the maximum level of number of lab during the first 15 days (8–9 log cfu/g) was in accordance with the results obtained for tunisian sausages produced with combined starter culture of s. xylosus and l. plantarum (8.1 log cfu/g) (essid and hassouna, 2013). rapid increase in the total number of aerobic mesophilic bacteria in all samples during the first days of production process was in accordance with the results obtained for the samples of petrovac sausages produced under traditional conditions (5–8.5 log cfu/g) and for the samples produced under controlled conditions (5–7 log cfu/g) (danilovic et al., 2018). the total number of aerobic mesophilic bacteria was in the number of lab in sausages produced without starter culture (h) during the first days of fermentation was in accordance with the results obtained for tunisian dry-fermented sausage produced without starter culture (4.3 log cfu/g). however, the initial number of lab in sausages i and j (about 5 log cfu/g) was lower than the number of lab obtained for tunisian sausages produced with combined starter culture of s.  xylosus and l.  plantarum (7.3 log cfu/g) (essid and hassouna, 2013). the rapid increase in lab during the first days of fermentation is also in accordance with the rapid increase in dry-fermented poultry sausages prepared without starter cultures (8.3 cfu/g), with starter culture of s. xylosus or l. plantarum (8.9 cfu/g) and 9 (h) (i) (j) 8 7 6 5 4 3 2 0 20 b ac te ri al n um be r, lo g c fu /g 9 8 7 6 5 4 3 2b ac te ri al n um be r, lo g c fu /g 9 8 7 6 5 4 3 2 b ac te ri al n um be r, lo g c fu /g 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 days of fermentation 80 100 120 figure 1. the number of aerobic mesophilic bacteria (, red line), lab (, green line) and staphylococci (, blue line) during the production of petrovac sausages prepared without starter cultures (h), with combined starter culture s. xylosus and l. plantarum (i), and with s. xylosus (j) and produced at 14–16°c (full line) and ~10°c (dashed line). vertical error bars represent standard deviation. 28 italian journal of food science, 2021; 33 (2) milićević b et al. accordance with the results obtained for petrovac sausages produced under traditional and controlled conditions (7–8 log cfu/g; danilovic et al., 2018) and for the sausages produced from hot deboned meat (danilovic et al., 2011). results obtained for petrovac sausages indicated that lab microbiota, being the dominant microbiota during the production process, were in accordance with the results of casaburi et al. (2008), casquete et al. (2012) and zdolec et al. (2008). domination of lab microbiota in petrovac sausages was in accordance with the results obtained for alheira-fermented sausage produced in portugal (albano et al., 2009), traditional greek dry-fermented sausages (ambrosiadis et al., 2004; papamanoli et al., 2003), dry-fermented sausages produced with l. sakei (bolumar et al., 2006) and tunisian dry-fermented beef sausage produced with combined starter culture of s. xylosus and l. plantarum (essid and hassouna, 2013). the initial number of staphylococci in sausages h, i and j (about 4 log cfu/g) was lower than the number of the same microbiota in tunisian beef sausage produced without starter culture (5 log cfu/g) and with combined starter culture of s. xylosus and l. plantarum (7 log cfu/g) (essid and hassouna, 2013). the lower number of staphylococci at the end of production process was probably due to reduction in ph caused by lactobacilli (johansson et al., 1994; lizaso et al., 1999). addition of starter culture had no effect on the total number of staphylococci. the higher number of staphylococci in the samples produced at higher temperature range (14–16°c) than the number presented in samples produced at lower temperature (~10°c) was in accordance with the results obtained for italian fermented sausages, where the growth of s. xylosus was better at higher temperatures (fiorentini et al., 2010). other results confirmed that increasing temperature from 10°c to 26°c increased growth of s. xylosus, s. carnosus and s. equorum, with strong synergy between temperature and ph (søndergaard and stahnke, 2002). the number of both aerobic mesophilic bacteria and lab was identical regardless of the addition of starter cultures s. xylosus and l. plantarum. these results were in accordance with the results obtained for tunisian dry-fermented sausages produced with the addition of starter cultures s. xylosus and l sakei (najjari et al., 2020). a total of 495 gram-positive and catalase-negative strains were isolated from 33 samples during the production of petrovac sausage. phenotypic grouping of strains by cell morphological characteristics divided all isolates into five groups (table 1). the identity of the isolate was confirmed by (gtg)5-pcr and 16s rdna sequencing. the 16s ribosomal rna (rrna) gene sequence analysis confirmed that all isolates belonged to l. brevis, l.  mesenteroides, l. plantarum and p. pentosaceus species. dna analyses of the pcr-amplified 16s rrna gene fragments obtained from purified isolates during sausage production provided the fingerprints shown in figure  2. the (gtg)5 fingerprints didn’t show intraspecific biodiversity. gram-positive, catalase-negative and rod-shaped cells were classified as lactobacilli. arginine-negative group of lactobacilli had the ability to grow well at 15ºc and in the presence of 6%, 5% and 8% of nacl. this group didn’t table 1. characterisation of lab isolated during the production of petrovac sausage. group i ii iii iv no. of isolates 188 172 131 4 cell morphology rods rods coccoid cocci co 2 formation – – + – growth at 45°c – – – – 15°c + + + + growth on nacl 4% – – – 6.5% + + + + 8% + + + + hydrolysis of arginine – – – – hydrolysis of esculin – – + + black colonies on bile esculin agar – – – – production of eps from sucrose – + + – production of bacteriocines v v – v identified by 16s rdna gene sequencing l. brevis l. plantarum l. mesenteroides p. pentosaceus ‘+’: positive; ‘−‘: negative; ‘v’: variable, ‘eps’: exopolysaccharides. italian journal of food science, 2021; 33 (2) 29 the influence of starter cultures on the lactic acid bacteria microbiota of petrovac sausage 1 2 3 4 5 6 7 8 9 figure 2. reference pcr profiles of the amplified 16s rrna gene of the isolates: l. brevis (1, 2, 4), l. plantarum (3, 5), p. pentosaceus (6, 7) and l. mesenteroides (8, 9). produce co2 and was not able to grow at 45ºc. on the basis of morphological characteristics, two groups of lactobacilli were observed. (gtg)5-pcr fingerprinting (figure 2) confirmed that two groups belonged to l. brevis and l. plantarum. some l. brevis and l. plantarum strains synthesised bacteriocines, which was in accordance to the data found in literature that these species could be active against l. monocytogenes (tosukhowong et al., 2011). also, nitrite-reduction capability is one of the most important characteristics of l. brevis (paik and lee, 2014). additionally, l. plantarum leads to rapid decrease of ph in fermented sausages and contributes to the organoleptic properties of the fermented product (heinz and hautzinger, 2007). the arginine-negative and esculin-positive isolates that produced co2 from glucose and had the ability of forming slimy colonies on mrs agar plates with sucrose were identified by 16s rdna sequencing as l. mesenteroides. leuconostoc spp. produce lactic acid, acetic acid, dextran, acetaldehyde, diacetyl, ethanol and other metabolites that contributes to the development of aroma and flavour in production of fermented sausages (lee et al., 2006). as heterofermentative strains, leuconostoc spp. produce co2, which is considered as one of the main causes in forming holes in meat products; this property classifies them as undesirable microbiota (ammor and mayo, 2007). leuconostoc spp. may synthesise spectra of bacteriocines (mesentericin y105, produced by l.  mesenteroides spp. mesenteroides; leucocin a-ual 187, produced by l. gelidum; carnosin 44a, produced by l. carnosum; and leuconocin s, produced by l. paramesenteroides) that exhibit strong microbial activity against listeria spp (stiles, 1994). the prevalence of leuconostoc spp. in sausages is in correlation with the results obtained for petrovac sausages produced from hot deboned meat (danilovic et al, 2011) as well as for sausages ripened under the traditional and controlled conditions (danilovic et al., 2018). only four isolates (0.8%) were esculine-positive cocci. they all had the ability to grow at 15ºc as well as on mrs agar plates with addition of nacl (6%, 5% and 8%). some of cocci produced bacteriocines (table 1). esculinepositive cocci, which formed tetrads, were identified by 16s rdna sequencing as p. pentosaceus (figure 2). as a result of low catabolism of amino acids, pediococci don’t play a major role in the formation of organoleptic properties in fermented sausages (leroy et al., 2006). among pediococci, p. acidilactici and p. pentosaceus were often isolated from european sausages (albano et al. 2007; kozachinski et al. 2008). p. acidilactici produced pediocin that inhibits the growth of food-borne pathogens l. monocytogenes and clostridium perfringens in spanish dry-fermented sausages (nieto-lozano et al., 2010). in addition, p. pentosaceus showed strong inhibitory effect against s. aureus (erdogrul et al., 2002). p. pentosaceus and p. acidilactici are commonly used as starters in the united states in producing dry sausages (rantsiou and cocolin, 2006). besides bacteriocines, some strains of pediococci produce eps (semjonovs and zikmanis, 2008). the low frequency of isolation of pediococci is correlated with the results obtained for bosnian sudzuk, alheira sausage and croatian sausage (albano et al., 2009; kozachinski et al., 2008). in petrovac sausages produced from hot deboned meat, pediococci were isolated at the highest percentage after ninth day of production process (danilovic et al., 2011). total isolated lab microbiota constituted l. brevis (37.9%), l. plantarum (34.7%), l. mesenteroides (26.4%) and p. pentosaceus (0.8%). sausages prepared without starter cultures (h1 and h2) were characterised by the prevalence of leuconostoc spp. during the first 15 days of fermentation regardless of temperature. complete replacement of leuconostoc spp. was observed after 15 days and lactobacilli were the dominant microbiota. on the 60th day of production process, l. plantarum rapidly increased up to 80% in sausages h1, while in sausages h2, almost equal distribution of l. brevis and l. plantarum was detected. later stages of production process were characterised by the prevalence of l. plantarum. p. pentosaceus was isolated only from sausages h2 in a 90-dayold sample with a representation of 1.4% (figure  3). on the other hand, in sausages prepared with the addition of combined starter cultures of s. xylosus and l.  plantarum (sausages i1 and i2), the highest percentage of 30 italian journal of food science, 2021; 33 (2) milićević b et al. h1 h2 i1 i2 j1 j2 fr eq ue nc y of is ol at io n 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 6 15 60 90 120 fr eq ue nc y of is ol at io n 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 6 15 60 90 120 fr eq ue nc y of is ol at io n 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 6 15 60 90 120 fr eq ue nc y of is ol at io n 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 6 15 60 90 120 fr eq ue nc y of is ol at io n 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% fr eq ue nc y of is ol at io n 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 6 15 time, days time, days 60 90 120 0 6 15 60 90 120 p.pentosaceus l.mesenteroides l.plantarum l.brevis figure 3. changes in microbial population during the production of petrovac sausages prepared without starter cultures (sausages h), with combined starter cultures of s. xylosus and l. plantarum (sausages i) and with starter culture of s. xylosus (sausages j) produced at 14–16°c (samples with tag 1) and ~10°c (samples with tag 2). italian journal of food science, 2021; 33 (2) 31 the influence of starter cultures on the lactic acid bacteria microbiota of petrovac sausage leuconostoc strains was detected only in the meat batter. leuconostoc spp. decreased immediately after preparation of sausage mixture but remained still up to the 15th day of production in sausages i1 and up to the 60th day of production in sausages i2. after this period, depletion of leuconostoc strain was observed and lactobacilli were the dominant microbiota (figure 3). pediococci were isolated only at the end of production process in sausages i2 with a share of 1%. in sausages prepared with the addition of starter culture of s. xylosus (sausages j1 and j2), the domination of l. brevis was detected at all stages of production process except in the meat batter, where the full presence of l. mesenteroides (100%) was detected. p. pentosaceus was detected on the 60th and 90th day of production in sausages j1 and j2, respectively. this increase in the content of lactobacilli was observed during production, with the presence of 100% lactobacilli in the sample after 120 days of production. during the production of petrovac sausage, the prevalence of l. mesenteroides was observed in the meat batter prepared with and without adding starter cultures. also, l. mesenteroides strains were present during the early stages of fermentation process regardless of temperature. the high frequency of l. mesenteroides at the beginning of production process was in accordance with the results obtained for serbian traditional fermented sausages sremski kulen, lemeski kulen (vasilev et al., 2015) and užička sausage (borovic et al., 2017). on the contrary, these results were not in accordance with the results obtained for italian fermented sausage (comi et al., 2005; urso et al., 2006), where low frequency of leuconostoc spp. was detected at the beginning of the production process. regardless of the production conditions, in all sausages, lactobacilli were the dominant microbiota from 15 days till the end of production process. this is in accordance with the results obtained for užička sausage (borovic et al., 2017). also, the high frequency of lactobacilli was presented in sremski and lemeški kulen (77.1 and 54.3%, respectively). l. brevis was the most dominant lactobacilli species in these sausages (61.5% and 57.9%, respectively) (vasilev et al., 2015). high frequency of l. brevis was in accordance with the results obtained for traditional fermented užička sausage (borovic et al., 2017); p. pentosaceus was isolated in the smallest percentage in the final stages of production, while in sausages ripened under traditional and controlled conditions, pediococci were present only in the meat batter (1.7% of the total microbiota) (danilovic et al., 2018). pediococci were isolated in small percentage from iberian dry-fermented sausages— salcichon and chorizo (benito et al., 2008) and italian fermented sausages—salami (bonomo et al., 2008). on the contrary, pediococci were isolated at high frequency from the fermented sausages produced in the united states, where p. acidilactici and p. pentosaceus are commonly added as starter cultures (anba-mondoloni et al., 2015). conclusion petrovac sausage is an artisanal serbian sausage appreciated for its sensory characteristics. in order to preserve the quality of the industrial production process, there is a need to understand the effect of starter cultures on the level of microbiota and composition. the results indicate that application of starter culture s. xylosus and combined starter culture s. xylosus and l. plantarum didn’t influence the total number of lab during process. on the other hand, temperature range of 14–16°c increased the number of staphylococci, compared with the application of ~10°c temperature. comparison of the effect of different starter cultures with the composition of microbiota resulted in the achievement of similar microbiota composition as for traditional sausages when combined starter culture was used. according to the results, combined starter culture of s. xylosus and l. plantarum could be the most promising solution for the production of petrovac sausage, although further sensory analysis is required to be conducted. acknowledgments this work was performed at the faculty of technology, university of nis, leskovac, republic of serbia, and funded by the ministry of education, science and technological development of the republic of serbia, grant no. 451-03-68/2021-14/200133. references albano h., todorov s.d., van reenen c.a., hogg t., dicks l.m. and teixeira p. 2007. characterization of two bacteriocins produced by pediococcus acidilactici isolated from “alheira,” a fermented sausage traditionally produced in portugal. int j food microbiol. 116(2):239–47. https://doi.org/10.1016/j. ijfoodmicro.2007.01.011 albano h., van reenen c.a., todorov s.d., cruz d., fraga l., hogg t.,et al. 2009. phenotypic and genetic heterogeneity of lactic acid bacteria isolated from ‘’alheira,” a traditional fermented sausage produced in portugal. meat sci. 82:387–398. https://doi. org/10.1016/j.meatsci.2009.02.009 ambrosiadis j., soultos n., abrahim a. and bloukas j.g. 2004. physicochemical, microbiological and sensory attributes for the characterization of greek traditional sausages. meat sci. 66:279–287. https://doi.org/10.1016/s0309-1740(03)00100-1 ammor m.s. and mayo b. 2007. selection criteria for lactic acid bacteria to be used as functional starter cultures in dry sausage production: an update. meat sci. 76:138–146. https://doi. org/10.1016/j.meatsci.2006.10.022 anba-mondoloni j., champomier-vergès m.c., zagorec m., leroy  s., dordet-frisoni e., planchon s. and talon r. “the genetics of microbial starters,” in handbook of fermented meat https://doi.org/10.1016/j.ijfoodmicro.2007.01.011� https://doi.org/10.1016/j.ijfoodmicro.2007.01.011� https://doi.org/10.1016/j.meatsci.2009.02.009� https://doi.org/10.1016/j.meatsci.2009.02.009� https://doi.org/10.1016/s0309-1740(03)00100-1� https://doi.org/10.1016/j.meatsci.2006.10.022� https://doi.org/10.1016/j.meatsci.2006.10.022� 32 italian journal of food science, 2021; 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33 (2) milićević b et al. zdolec n., hadziosmanovic m., kozacinski l., cvrtila z., filipovic i., skrivanko m. and leskovar k. 2008. microbial and physicochemical succession in fermented sausages produced with bacteriocinogenic culture of lactobacillus sakei and semi-purified bacteriocin mesenterocin y. meat sci. 80:480–487. https://doi. org/10.1016/j.meatsci.2008.01.012 versalovic j., schneider m., de brujin f. and lupski j.r. 1994. genomic fingerprinting of bacteria using repetitive sequence based pcr (rep-pcr). methods mol biol. 5:25–40. veskovic-moracanin s. 2010. lactic acid bacteria bacteriocins as natural food protectors: possibilities of application in meat industry. meat technol. 51(1):83–94. https://doi.org/10.1016/j.meatsci.2008.01.012� https://doi.org/10.1016/j.meatsci.2008.01.012� 28 issn 1120-1770 online, doi 10.15586/ijfs.v34i2.2126 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (2): 28–33 p u b l i c a t i o n s codon study on the role of nutrients in food to improve the motion state of athletes hongkai zhou1,2 1pingdingshan university, pingdingshan, henan, china; 2sports college, graduate university of mongolia, ulaanbaatar, mongolia corresponding author: hongkai zhou, pingdingshan university, south section of future road, xincheng district, pingdingshan city, henan province, china. email: k6k262@163.com received: 22 september 2021; accepted: 28 march 2022; published: 20 april 2022 © 2022 codon publications open access paper abstract this study aims to analyze the role of nutrients in food in improving athletic performance and to understand the feasibility of food supplementation in sports training. twenty athletes were randomly divided into two groups, a and b. the athletes in the two groups had the same diet and the same training content and differed only in the supplemented food. group a was supplemented with siraitia grosvenorii water, and group b was supplemented with pure water. after 3 months of training, the body composition, exercise status, and blood indexes of the athletes in the two groups were compared. compared with the athletes in group a before the experiment as well as in group b after the experiment, the athletes in group a showed a significant increase in fat-free weight, improved athletic performance, increased levels of hemoglobin (hb) and red blood cell (rbc), and decreased levels of blood lactic acid (bla) and blood urea nitrogen (bun) (p < 0.05). nutrients in food can effectively improve the body composition and exercise status of athletes and inhibit the decrease of hb and rbc as well as the increase of bla and bun, which have good usability in sports training. keywords: athletes; food; motion state; nutrient introduction in the process of improving sports performance, athletes need to carry out a lot of training and fierce competition; in such a case, the body state of athletes declines (chuckravanen et al., 2019), free radicals accumulate, fatigue appears, and immune function declines, which may cause sports injury and directly affect the training effect and the results of competition. therefore, the question of how to reduce the impact of a rigorous training on athletes and improve motion state has been widely concerned by researchers. in nature, many foods are rich in nutrients, which can be used as nutritional supplements for athletes. guo (2015) studied jujube polysaccharides and carried out the weight-bearing swimming experiment on rats. they found that rats had significantly longer swimming time, larger body weight, and higher glycogen content after taking jujube polysaccharides. liu et al. (2015) studied the polysaccharide components of hericium erinaceus, analyzed its anti-fatigue activity, compared various indexes of mice under different doses of hericium erinaceus polysaccharide, and found that it could reduce the content of blood lactic acid (bla) and malondialdehyde (mda). zhang et al. (2015) simulated the plateau environment and analyzed the anti-fatigue effect of astragalus membranaceus on mice. it was found that the swimming time of mice was prolonged, the lactic acid decreased, and the glycogen increased under the influence of astragalus membranaceus, which indicated that astragalus membranaceus could reduce the fatigue of mice in a plateau environment. ren et al. (2017) studied the effect of soy whey protein supplementation on sports performance after long-term training. it was found that the average fatigue time of rats was longer, mailto:k6k262@163.com italian journal of food science, 2022; 34 (2) 29 study on the role of nutrients sweet or slightly sweet substances have high sweetness and low heat, which can substitute for sucrose (abdelhamid et al., 2020). it also contains flavonoids, but the content is not high. moreover, there is a lot of protein and amino acids in siraitia grosvenorii, including eight kinds of essential amino acids for the human body. the sugar content in siraitia grosvenorii is very high, and the content of fructose is 14%. it contains 24 kinds of inorganic elements. the content of oil or fat is also very high, and the main component is squalene. it also contains rich vitamin c and vitamin e, suggesting high nutritional values. the most important medicinal value of siraitia grosvenorii is clearing away heat and moistening the lung (gong et al., 2019). according to traditional chinese medicine, siraitia grosvenorii is sweet and cool; therefore, it has a good therapeutic effect on lung heat and dry cough. the medicinal value of siraitia grosvenorii also includes: (1) resistance to diabetes (xu et al., 2020). siraitia grosvenorii has high sweetness and low calorie. it can be used as an ideal sugar substitute for diabetes mellitus patients. also, some components in siraitia grosvenorii can reduce blood glucose and blood lipid and improve renal function. the activity of lactate dehydrogenase was higher, and the level of mda was lower after supplementation of soybean whey protein. in this study, the nutrients in siraitia grosvenorii were studied, and a comparative experiment was carried out. the indicators of the athletes, such as body composition and motion status, were analyzed to understand the application prospect of siraitia grosvenorii in sports training. nutrients in siraitia grosvenorii and their functions siraitia grosvenorii is a kind of medicinal and edible material unique to china (tu et al., 2015). it is mainly produced in provinces such as guangxi and guangdong in southern china. the fruits of siraitia grosvenorii are spherical or oblong (figure 1, left). the mature fruits have dark green pericarp, yellowing petioles, and high water content. after being processed and dried (figure 1, middle), the surface is brown or yellowish-green with dark patches; it is light, brittle, and easy to crack, and the seeds are oblate (figure 1, right), light red to reddish-brown, with a sweet taste. siraitia grosvenorii contains many cucurbitane triterpenoids (niu et al., 2017; qiao et al., 2019), which are sweet or slightly sweet substances (shi et al., 2019). the figure 1. siraitia grosvenorii. 30 italian journal of food science, 2022; 34 (2) zhou h et al. blood indicators: including hemoglobin (hb), red blood cell (rbc) count, bla, and blood urea nitrogen (bun). blood samples were sent to hangzhou second people’s hospital for testing. statistical analysis the data were statistically processed by spss17.0 and expressed as x sd± . the comparison between groups was conducted using the t-test. if the value of p was smaller than 0.05, then it suggested that there was a statistical difference. experimental results comparison of body composition before and after the experiment, the changes in body composition of athletes in the two groups are shown in table 3. as shown in table 3, before the experiment, there was no significant difference in body weight and fat-free weight between group a and group b (p > 0.05); after the experiment, the fat-free weight and body weight of athletes in the two groups increased slightly, and the fat-free weight of group a was 59.33 ± 3.25 kg, significantly higher than that before the experiment (p < 0.05) and group b, indicating that drinking siraitia grosvenorii water was effective in improving athletes’ fat-free weight. comparison of motion state before and after the experiment, the comparison of the motion state between the two groups of athletes is shown in table 4. as shown in table 4, before the experiment, there was no significant difference in different motion states between the two groups; after the experiment, the standing long jump of group a was 2.62 ± 0.71 m, which showed a significant improvement compared with that before the experiment and group b; the number of push-ups in group a was 38.71 ± 3.59, and p < 0.05 compared to (2) protect the cardiovascular system. siraitia grosvenorii is rich in unsaturated fatty acids, which can resist fatty liver; inorganic salts, such as selenium and magnesium, can protect myocardial cells and are also effective in expanding blood vessels and preventing thrombosis. (3) improve immunity. siraitia grosvenorii has antibacterial and anti-inflammatory effects (li et al., 2018), which can enhance the cellular and humoral immune functions of the body and coordinate the immune system. experimental methods research subjects twenty athletes were randomly selected from the ministry of public sports of zhejiang shuren university. they were informed of the intention and process of the experiment. all the participants were randomly divided into group a and group b. group a drank water soaked with siraitia grosvenorii, while group b drank pure water. the athletes in both groups were in good health and had no physical injury within half a year. they took no strenuous exercise and did not take any caffeine or tea 24 h before the experiment. they were in a good mental state. there was no significant difference in general data, as shown in table 1. research methods the two groups of athletes were trained for 3 months, including sprinting, long-distance running, standing long jump, etc. they were trained twice a day, 2 h each session. the athletes took three meals in the school canteen every day, without any additional nutrition. the athletes in group a drank the water soaked with siraitia grosvenorii at 8:00, 12:00, 16:00, and 20:00 every day, while the athletes of group b drank pure water. the dosage of siraitia grosvenorii soaking water was 1/4 siraitia grosvenorii and 1 l water, for four times, 250 ml each time. before and after the experiment, blood samples and motion state tests were carried out on the athletes. the indexes are shown in table 2. measurement index body composition: body weight and fat-free weight. movement state: as shown in table 2. table 1. comparison of general information. group a (n = 10) group b (n = 10) age/years 22.78 ± 2.31 23.08 ± 1.56 height/cm 178.64 ± 2.78 177.59 ± 3.08 weight/kg 76.21 ± 5.29 75.94 ± 6.12 table 2. measurement indexes of motion state. number content x1 standing long jump x2 one minute push-ups x3 one minute sit-ups x4 50-m running x5 sit and reach measurement index italian journal of food science, 2022; 34 (2) 31 study on the role of nutrients b decreased, suggesting significant differences compared with that before the experiment, and the hb value of group a was significantly higher than that of group b (p < 0.05); after the experiment, the rbc value of group a significantly increased, and p < 0.05 compared to that before experiment and group b. it was found that drinking siraitia grosvenorii water could increase the rbc count and inhibit the decrease of hb value. the comparison of bla and bun between the two groups is shown in figure 2. as shown in figure 2, before the experiment, the bla values of the two groups were 3.27 ± 0.21 mmol/l and 3.26 ± 0.33 mmol/l, respectively, suggesting no significant difference; after the experiment, the bla of group a increased to 8.16 ± 0.78 mmol/l, while that of group b increased to 11.27 ± 0.56 mmol/l, which were significantly different from those before the experiment, and the bla value of group a was significantly smaller before the experiment and group b; the number of situps in group a was 41.29 ± 5.57, which was significantly different from that before the experiment and group b; the performance of the 50 m running of group a was 5.88 ± 0.41 s (p < 0.05 compared to before the experiment and group b), and the performance of group b also significantly improved after the experiment; finally, there was no significant difference in sit and reach before and after the experiment and between the two groups (p > 0.05). it was found that drinking siraitia grosvenorii water was effective in improving athletes’ performance and motion state. comparison of blood indexes before and after the experiment, the comparison of hb and rbc count between the two groups is shown in table 5. it was seen from table 5 that there was no significant difference in the comparison of hb and rbc between the two groups before the experiment; after the experiment, the hb of group a increased, while that of group table 3. changes in body composition. group a group b before the experiment after the experiment before the experiment after the experiment weight/kg 76.21 ± 5.29 77.33 ± 4.62 75.94 ± 6.12 76.89 ± 5.78 fat-free weight/kg 53.64 ± 0.86 59.33 ± 3.25*# 54.68 ± 1.12 56.48 ± 2.86 *compared with that before the experiment, p < 0.05. #compared with group b, p < 0.05. table 4. comparison of motion state. group a group b before the experiment after the experiment before the experiment after the experiment x1/m 2.43 ± 0.83 2.62 ± 0.71*# 2.51 ± 0.76 2.53 ± 0.48 x2/n 33.68 ± 4.86 38.71 ± 3.59*# 34.12 ± 4.77 35.62 ± 4.63 x3/n 37.12 ± 3.08 41.29 ± 5.57*# 37.25 ± 3.12 39.68 ± 4.51 x4/s 6.32 ± 0.33 5.88 ± 0.41*# 6.41 ± 0.27 6.03 ± 0.39* x5/cm 15.33 ± 0.71 16.21 ± 0.33 16.12 ± 0.78 16.23 ± 0.56 *compared with that before the experiment, p < 0.05. #compared with group b, p < 0.05. table 5. comparison of hb and rbc. group a group b before the experiment after the experiment before the experiment after the experiment hb (g/l) 136.77 ± 1.87 141.34 ± 0.64*# 137.64 ± 0.92 131.66 ± 1.91* rbc (1012/l) 4.63 ± 0.12 5.09 ± 0.26*# 4.57 ± 0.19 4.77 ± 0.21 *compared with that before the experiment, p < 0.05. #compared with group b, p < 0.05. 32 italian journal of food science, 2022; 34 (2) zhou h et al. body and destroy rbc. therefore, after a lot of exercise, the content of hb in the human body decreases. as shown in table 5, the hb of athletes in group b decreased from 137.64 ± 0.92 g/l to 131.66 ± 1.91 g/l, while group a drank siraitia grosvenorii water and had increased hb value rather than decreased hb value. the results showed that the nutrients in siraitia grosvenorii could protect rbc, maintain the function of rbc, inhibit the decline of hb, and improve the human body’s movement state. when the body exercises, lactic acid generated by the skeletal muscles enters the blood and is later eliminated by the process of metabolism. in the case of a large amount of exercise, the bla in the human body increases rapidly, resulting in the accumulation of lactic acid (romadhona et al., 2019), and the metabolic level decreases, which leads to the decline of exercise ability. the results showed that the bla value of group a and group b increased significantly after the experiment, but the bla value of group a was smaller than that of group b (p < 0.05), which showed that nutrients in siraitia grosvenorii, such as flavonoids, protected the cardiovascular system, and enhanced the metabolic capacity of the body, thus reducing the accumulation of lactic acid, maintaining the balance of the internal environment, relieving fatigue, and improving the exercise state of the human body. bun can reflect the body’s protein metabolism. with the progress of exercise, the protein metabolism in the body increases, and the urea content becomes higher, which is not conducive to maintaining physical fitness. the results than that of group b (p < 0.05); before the experiment, the bun value of two groups were 4.31 ± 0.78 mmol/l and 4.29 ± 0.64 mmol/l, respectively; after the experiment, the bla values of the two groups were 4.33 ± 0.64 mmol/l and 4.56 ± 0.74 mmol/l, respectively, which showed a slight increase, but the difference was not obvious. it was found that drinking siraitia grosvenorii water could inhibit the growth of bla. discussion in exercise, the nutrients in the body are consumed rapidly, and the demand for protein and sugar increases. to improve the sport’s ability of the human body, the supplement of nutrients is very important. by supplementing some foods with rich nutrients, the metabolic needs of athletes can be ensured to have good health state. many foods contain a lot of nutrients, which can be widely used to improve the state of human movement. in this study, the nutrients in siraitia grosvenorii were studied. based on the data shown in tables 3 and 4, it was found that after the experiment, the fat-free weight of athletes who drank siraitia grosvenorii water increased, and their standing long jump and push-up performance also improved. it shows that the motion state of athletes in group a significantly improved, and their sports performance was better after the experiment. hb is the carrier of o2 and co2. in the process of long exercise sessions, a lot of free radicals appear in the human figure 2. comparison of bla and bun. *p < 0.05 compared to before experiment. #p < 0.05 compared to group b. italian journal of food science, 2022; 34 (2) 33 study on the role of nutrients li, x., xu, l.y., cui, y.q. and pang, m.x., 2018. anti-bacteria effect of active ingredients of siraitia grosvenorii on the spoilage bacteria isolated from sauced pork head meat. iop conference, 292: 012012. https://doi.org/10.1088/1757-899x/292/1/012012 liu, j., du, c., wang, y. and yu, z., 2015. anti-fatigue activities of polysaccharides extracted from hericium erinaceus. experimental & therapeutic medicine, 9(2): 483–487. https:// doi.org/10.3892/etm.2014.2139 niu, b., ke, c.q., li, b.h., li, y.y., yi, y.j., luo, y.w., shuai, l., yao, s., lin, l.g., li, j. and ye, y., 2017. cucurbitane glucosides from the crude extract of siraitia grosvenorii with moderate effects on pgc-1α promoter activity. journal of natural products, 80: 1428–1435. https://doi.org/10.1021/acs.jnatprod.6b01086 qiao, j., luo, z., gu, z. and zhang, y., 2019. identification of a novel specific cucurbitadienol synthase allele in siraitia grosvenorii correlates with high catalytic efficiency. molecules, 24(3): 627. https://doi.org/10.3390/molecules24030627 ren, g.x., yi, s.q., zhang, h.r. and wang, j., 2017. ingestion of soy– whey blended protein augments sports performance and ameliorates exercise-induced fatigue in a rat exercise model. food & function, 8(2): 670. https://doi.org/10.1039/c6fo01692h romadhona, n.f., sari, g.m. and utomo, d.n. 2019. comparison of sport massage and combination of cold water immersion with sport massage on decrease of blood lactic acid level. journal of physics conference series, 1146: 012012. https://doi. org/10.1088/1742-6596/1146/1/012012 shi, h., liao, j., cui, s. and luo, z., 2019. effects of forchlorfenuron on the morphology, metabolite accumulation, and transcriptional responses of siraitia grosvenorii fruit, molecules, 24(22): 4076. https://doi.org/10.3390/molecules24224076 tu, d.p., mo, c.m., ma, x.j., zhao, h., tang, q., huang, j., pan, l.m. and wei, r.c., 2015. [selection of reference genes of siraitia grosvenorii by real-time pcr]. journal of chinese materia medica, 40(2): 204–209. xu, x., lu, f., yang, z. and yan, x., 2020. comparative study on the antidiabetic efficacy of unripe and ripe fruit extracts of siraitia grosvenorii and the possible mechanism of action. european journal of medicinal plants, 31(9): 10-18. https://doi. org/10.9734/ejmp/2020/v31i930266 zhang, g., zhou, s.m., zheng, s.j., liu, f.y. and gao, y.q., 2015. astragalus on the anti-fatigue effect in hypoxic mice. international journal of clinical & experimental medicine, 8(8): 14030. showed that the bun values of the two groups increased slightly after the experiment, and the value of group a was slightly smaller than that of group b, but the difference was not obvious. it indicated that siraitia grosvenorii water could stabilize the protein level, and inhibit the increase of bun, thus improving the movement state of the human body. conclusion this paper mainly studied nutrients in food with siraitia grosvenorii as an example and its role in improving the athletic performance of athletes. through the experiment and the analysis of the results, it was found that drinking siraitia grosvenorii water could improve the athletes’ fat-free weight, enhance the exercise state, inhibit the decrease of hb, maintain the stability of rbc, reduce the production of bla, restrain the increase of bun, which had a positive role in improving the motion state of athletes. siraitia grosvenorii water can be promoted and applied in practice. references abdel-hamid, m., romeih, e., huang, z., enomoto, t., huang, l. and li, l., 2020. bioactive properties of probiotic set-yogurt supplemented with siraitia grosvenorii fruit extract. food chemistry, 303: 125400.1–125400.7. https://doi.org/10.1016/j. foodchem.2019.125400 chuckravanen, d., bulut, s., kürklü, g.b. and yapali, g., 2019. review of exercise-induced physiological control models to explain the development of fatigue to improve sports performance and future trend. science & sports, 34(3): 131–140. https://doi.org/10.1016/j.scispo.2018.10.017 gong, x., chen, n., ren, k. and jia, j., 2019. the fruits of siraitia grosvenorii: a review of a chinese food-medicine. frontiers in pharmacology, 10: 1400. https://doi.org/10.3389/fphar.2019.01400 guo, x., 2015. effect of jujube date polysaccharide in resisting sports fatigue. advance journal of food science & technology, 9(12): 939–943. https://doi.org/10.19026/ajfst.9.1778 https://doi.org/10.19026/ajfst.9.1778 https://doi.org/10.3892/etm.2014.2139 https://doi.org/10.3892/etm.2014.2139 https://doi.org/10.1021/acs.jnatprod.6b01086 https://doi.org/10.3390/molecules24030627 https://doi.org/10.1039/c6fo01692h https://doi.org/10.1088/1742-6596/1146/1/012012 https://doi.org/10.1088/1742-6596/1146/1/012012 https://doi.org/10.3390/molecules24224076 https://doi.org/10.9734/ejmp/2020/v31i930266 https://doi.org/10.9734/ejmp/2020/v31i930266 https://doi.org/10.1016/j.foodchem.2019.125400 https://doi.org/10.1016/j.foodchem.2019.125400 https://doi.org/10.1016/j.scispo.2018.10.017 https://doi.org/10.3389/fphar.2019.01400 https://doi.org/10.19026/ajfst.9.1778 96 issn 1120-1770 online, doi 10.15586/ijfs.v33i1.1874 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (1): 96–105 p u b l i c a t i o n s codon influence of sugar concentration and sugar type on the polyphenol content and antioxidant activity in spiced syrup preparation mohd nazri zayapor1,2, aminah abdullah3, wan aida wan mustapha2* 1center of food science and technology research, mardi johor baharu, johor, malaysia; 2department of food sciences, faculty of science and technology, universiti kebangsaan malaysia, selangor, malaysia; 3natural medicine research center, universiti islam malaysia, selangor, malaysia *corresponding author: wan aida wan mustapha, department of food sciences, faculty of science and technology, universiti kebangsaan malaysia, 43600 bangi, selangor darul ehsan, malaysia. email: wanaidawm@ukm.edu.my received: 26 april 2020; accepted: 25 january 2021; published: 22 february 2021 © 2021 codon publications open access paper abstract besides their culinary roles, spices in the eastern traditional medical practices serve as a medicinal diet therapy. the polyphenol-rich content of these spices contributes to their antioxidant properties, which convey the therapeutic elements. traditionally, these phytonutrients are orally delivered via herbal decoction, including sugar-syrup-based decoctions. however, the impact of the addition of sugar into polyphenol content and their antioxidant activities are still insufficiently researched. therefore, this study aimed to evaluate the influence of sugar concentration and its types (refined and unrefined) on the polyphenol content and their antioxidant activities. the results of the principal component analysis (pca) did not exhibit any specific trend on either sugar concentration or its type. as indicative evidence, reducing sugar by less than 25% in such products can be considered for lower-calorie beverage development as a means for healthier diet choice. keywords: decoction syrup; low-sugar; polyphenol–sugar interaction; sugar-sweetened introduction spices are traditionally served as culinary condiments and habitually used in ayurveda, persian tebb-e sonnati, traditional chinese medicine (tcm), and unani (graeco-arabic) medicinal preparations. phytochemicals in spices can provide a great deal of medicinal therapy in preventing or treating common ailments. for instance, a mixture of cinnamon, clove, ginger, allspice, nutmeg, and star anise is used in the sugar-sweetened beverage for diet therapy in alleviating metabolic syndrome (block, 2015). their phytotherapeutic benefits are administered mostly in crude forms, either orally as infusions (herbal teas), tinctures (alcoholic extracts), decoctions (boiled extracts), and syrups (extracts of herbs made with syrup or honey), or topically applied as poultices, balms, and essential oils (ahmad et al., 2016; ramalingum and mahomoodally, 2014). among the common liquid forms, sharbat or sérbét, which has the arabic root word shariba or “to drink,” is made from either decoction, infusion of herbal remedies, fruit juice, or flower petal mixtures. there are various types of sharbat preparation, depending on the type of the substrate to be extracted. commonly, dry substrates, like most culinary spices, are boiled until one-third of the water is left and allowed to cool before being filtered. the filtered decoction is usually sugar sweetened with two to three parts and further boiled to obtain a syrupy consistency. mailto:wanaidawm@ukm.edu.my italian journal of food science, 2021; 33 (1) 97 influence of sugar concentration and sugar type cause an alarming glycemic response in patients with metabolic syndrome. nevertheless, the negative health impact of commonly consumed sugary beverages on the risk factors for metabolic syndrome cannot be validated with adequate clinical evidence (angelopoulos et al., 2016; della corte et al., 2018) to confirm that the added sugar products are the chief culprits for the metabolic imbalance. while it is health-wise beneficial to consume added sugars beyond the average level, the reduction in sugar intake accompanied by restricted calorie intake may be more beneficial (rippe and angelopoulos, 2016). therefore, this preliminary study was to evaluate the effect of sugar concentration and the sugar type by examining their influence on polyphenol contents and their antioxidant activities. material and method four culinary spices comprising chinese cinnamon (cinnamomum cassia), dried ginger (zingiber officinale), green cardamom (elettaria cardamomum), sweet fennel (foeniculum vulgare), and six types of sugar, namely, refined white sugar (rws), filtered sugarcane brown sugars (fbs), unfiltered sugarcane brown sugar (nbs), organic molasses (omo), javanese coconut sugar (jcs), and malaccan coconut sugar (mcs) were acquired from jaya grocer store, bangi gateway. spiced syrup preparation dirt and debris were removed from the spices by cleaning under running tap water, and the excess water was drained before they were air-dried. a mixture of spices, comprising 10% (w/v) cinnamon and 5% (w/v) other spices, was added to boiling sugar syrup (110°c) before it was simmered for 240 min at 80°c. the spice mixture was directly used without pounding into smaller sizes based on a traditional technique to avoid a strong flavor. the spiced syrup was prepared (figure 1) according to a traditional recipe, whereby sugars were added to filtered water at ratios of 1:2 kg/l, 1:3 kg/l, 1:4 kg/l, and 1:6 kg/l to produce syrups at the corresponding concentrations (table 1). the range of sugar additions was according to traditional practices in preparing a sugar-based decoction. however, the most common practice is to mix one part of sugar with four parts of water (25%, w/v). the non-spiced 25% sugar syrups (all selected sugar types) were boiled and simmered in the same way as the spiced syrup was prepared and were considered as the positive control (control, ctrl [sugar type]), and the spice decoction without sugar syrup (0% sugar) was considered as the negative control. the spiced syrup was then filtered with a clean muslin cloth before being bottled and addition of sugar to the phytochemical extraction step is relatively similar to the fruit osmotic dehydration technique, whereby sugar syrup is used as an osmotic agent (pereira et al., 2014) to produce an aromatic and flavorful herbal syrup or extract (geng and zhou, 2010). the range of osmotic agent additions were from 2 to 50% of the herbal substrate, but the study did not indicate as to when the sugar should be added. this syrup extraction was traditionally meant to improve the flavor and aroma of the herbal extract but was not intended to enhance the extractability of antioxidant compounds or specifically the polyphenols. previously, it was known that a hot aqueous extract of several spices (non-sugar addition) had a high antioxidant activity that was probably due to the phenolic and flavonoid compounds (kim et al., 2011). later, a study revealed that the addition of sugar before or during processing might play a significant role in enhancing the polyphenol extraction, and simultaneously the antioxidant activities instead of improving only the gastronomic qualities (loncaric et al., 2014). however, studies that evaluate the impact of sugar addition on polyphenols’ content and their antioxidant activities are still limited to ensure this positive enhancement. the latest findings showed that there were only slight polyphenol losses during food processing (szymanowska et al., 2017; zeng et al., 2017) to unapprehensive changes in antioxidant capacities (baroni et al., 2018) upon the introduction of sugar, which was probably due to some degree of protective mechanism. a similar mechanism was observed in chokeberry juice that was added with polysaccharide as the clarifying agent (lachowicz et al., 2018). however, the polyphenolic content became less as soon as the natural sedimentation appeared, resulting in polyphenol binding to polysaccharides. a list of sugar-loaded syrups showed promising therapeutic potentials as novel sources of antioxidant polyphenols. among the examples are the traditional prickly pear (opuntia ficus indica) and the pink tecoma (tabebuia impetiginosa) inner-bark syrups were advocated for their anti-tumor potential (dhaouadi et  al., 2013; pires et al., 2015), and the english plantain (plantago lanceolate) leaf syrup was recommended for combating common cold (mansoor et al., 2017). a polyphenol-rich syrup can potentially be a novel functional ingredient like the bog bilberry (vaccinium uliginosum) syrup (malvidin-3-o-glucoside, petunidin-3-oglucoside, and delphinium-3-o-glucoside), which is the chief flavonoid glucoside that is used to enrich wine for antioxidant capacities (liu et al., 2015). carbohydrates can enhance the bioactivity and the bioavailability of polyphenol compounds through increased fermentation by microbiota in the large intestine, especially the oligosaccharides (zhang et al., 2014). however, the primary concern is monosaccharide and disaccharide, which are readily absorbed and digested in the small intestine and may 98 italian journal of food science, 2021; 33 (1) zayapor mz et al. table 1. sugar ratio to sugar percentage conversion. sugar to water ratio (kg/l) sugar used (g/1000 ml) sugar percentage, % (w/v) 1:2 500.0 50.0 1:3 333.3 33.3 1:4 250.0 25.0 1:6 166.7 16.7 sugar and filtered water mixture boiling simmering at 80°c, 240 minutes filtration bottling addition of cleaned and air-dried mixture of spices figure 1. flow chart of spiced syrup processing stored at room temperature until use. each spiced syrup was prepared in triplicates. the uncertainty due to spice sampling heterogeneity was kept below 13%, as recommended by bodnar et al. (2013). determination of the total phenolic content the folin–ciocalteu (fc) method (singleton et al., 1999) was used to quantify the phenolic content with slight modification on the reactant volumes for 96-well microplate suitability. a hundred microliters of 10% fc reagent were mixed with 20 µl of blank (deionized water)/ standard/filtered syrup (diluted 50-fold with deionized water). after 5 min, 80 μl of 7.5% sodium carbonate was added and incubated at 37⁰c for 120 min. this spectrophotometric measurement was carried out at λ = 765 nm (spectrostar nano, bmg labtech, germany). the quantification of tpc by using the calibration curve of gallic acid was calculated by mars data analysis software version 2.01. the concentration for the calibration curve ranged from 250 to 4000 ppm. the results were expressed as micrograms of gallic acid equivalents (gae)/ml of syrup. antioxidant activity determination multiple spectrometric techniques were employed to evaluate the spice polyphenol antioxidant activities by three different mechanisms: organic radical scavenging, ferric reducing capacity, and peroxyl radical scavenging. it was essential to evaluate the antioxidative compounds by using various techniques due to the unique identity of each polyphenol with different action mechanisms against oxidative substances. with these multiple antioxidant determinations, an almost complete profile of antioxidants can be attained. the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities assay the 2,2-diphenyl-1-picrylhydrazyl (dpph) radical scavenging activities were determined according to kodama et al. (2010). the 0.5 mm dpph was pre-adjusted with methanol (1.00 ± 0.1 absorbance unit) before being mixed with diluted syrup (20-fold with distilled water) at a ratio of 9:1. after 30 min, the absorbance of mixtures was measured at 515 nm, and the dpph radical scavenging activities were calculated as mm trolox equivalent (te)/ml of syrup. ferric reducing antioxidant power assay a ferric reducing antioxidant power (frap) assay of spiced syrup was based on musa et al. (2011), which required the frap reagent to be freshly prepared by sequentially mixing one part of 20 mm fecl3. 6h2o with one part of 10 mm 2,4,6-tri(2-pyridyl)-s-triazine (tptz) and thoroughly mixing with 10 parts of 300 mm acetate buffer, at ph 3.6. a freshly prepared frap reagent at a 1950 μl was mixed with 50 μl diluted syrup before a 30-min incubation at room temperature. the absorbance changes against the blank (distilled water) were measured at 595 nm. the trolox calibration curve (3.125 mm to 100 mm) was used to equivalently estimate the frap reducing capacities, with results expressed as mm trolox equivalents (te)/ml of syrup. oxygen radical absorbance capacity assay for the oxygen radical absorbance capacity (orac) value determinations, 150 µl of 10 nm fluorescein solution was mixed with 25 µl diluted syrup in a 96-well black microplate, as described by payne et al. (2013). after incubation for 30 min at 37⁰c, the fluorescence degradation measurements were executed at the emission and excitation of 520 nm and 485 nm, respectively, by using a microplate reader (fluorstar omega, bmg labtech, germany). automatic injection of 240 mm aaph (25 µl) at the fourth cycle was made before measurement resumption, and the calculated results were expressed as μm trolox equivalents (te)/ml of syrup. italian journal of food science, 2021; 33 (1) 99 influence of sugar concentration and sugar type statistical analysis data collected as means of triplicate readings were subjected to analysis of covariance (ancova), correlation analysis by pearson’s correlation coefficient, and data analysis by principal component analysis (pca) by using xlstat premium 2018.1.4913 (addinsoft inc., new york, ny, usa). the ancova was applied to evaluate whether tpcs were varied with sugar concentrations (quantitative) and types (qualitative), and verify a sensible linear model. the exploratory statistical tool, pca, was applied to observe any typical or divergent features or degree of similarities among a set of variables. results and discussions effect of sugar concentration and sugar type on the tpc of spiced syrups figure 2 shows that all ctrl sugars had a tpc that ranged from 500 to 1000 gae mg/ml, except for rws, which was the least with < 500 gae mg/ml. java coconut sugar, jcs had the highest tpc among the selected sugar types of approximately 1000 gae mg/ml. most brown or unrefined sugars contained a significant amount of polyphenol (p < 0.05), compared to refined sugar (kongkaew et al., 2014; choong et al., 2016). brown sugar from sugarcane and coconut are commonly used in traditional medicine preparation as a sweetener because it is believed to possess therapeutic benefits. however, the 0% sugar decoction had 2-fold to 5-fold higher tpc than each ctrl sugar, and showed that sugar syrup of any type could contribute to a smaller portion of tpc value in the spiced syrup. overall, the higher the sugar concentration, the higher was the polyphenol content, except for omo, which showed an opposite trend. this contradictory effect on omo might be attributed to the omo polyphenol antagonistic effect, reflecting the reduction of content. meanwhile, in the rws syrup, the tpc showed a unique bell-shaped trend, whereby intermediate sugar concentrations of 25 and 33.3% were exhibited among the highest contents (> 2500 gae mg/ml). at higher sugar concentrations, the tpc high and low values were possibly due to the osmotic pressure, which allowed for desorption or prevention of spice polyphenols into the sugar syrup. it was postulated that the tpc decrement in the spiced syrup as the sugar concentration decreased was due to spice polyphenol degradation in the low solute matrix. however, the enhancement of the polyphenolic content in pumpkin by using concentrated flowering quince juice was attributed to the penetration of the polyphenol-rich osmotic solution into the pumpkin, which provided some stability (lech et al., 2018). the inconsistent variance among tpc in a syrup of different sugar concentrations and types might occur due to the interaction of folin– ciocalteu reagents with any reducing or readily oxidizable substances of nonphenolic compounds, causing an aberration of the total phenolic content (essawet et al., 2015; ho et al., 2017; ramachandran and nagarajan, 2014). it was postulated that different profiles of monosaccharide and disaccharide could play some interaction with the spice-polyphenols via a covalent or noncovalent bond, and thus, reflecting some variation in free polyphenol content. the ancova analysis showed that 63% of tpc variability (table 2) was explained by sugar concentrations and types. however, the latter variability and interaction between sugar concentrations and types did not 0.00 c t r l ( 1 0 % s u g a r ) c t r l r w s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l f b s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l n b s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s sugar concentration (%, w/v) 1 6 .7 % r w s c t r l o m o ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l m c s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l j c s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s 500.00 1000.00 1500.00 2000.00 t p c ( g a e µ g /m l ) 2500.00 3000.00 3500.00 4000.00 figure 2. total phenolic content (tpc) of spiced syrups prepared from different type of sugars and concentrations. 100 italian journal of food science, 2021; 33 (1) zayapor mz et al. significantly explain the tpc variability. furthermore, the equation of the tpc linear model is proposed, tpc = 733.60 × type of sugar-c-fbs + 1070.85 × type of sugar-c-jcs + 602.73 × type of sugar-c-mcs + 872.03 × type of sugar-c-nbs + 880.13 (1) since the type of sugar did not carry significant information, the proposed model (equation 1) was rejected. based on type 3 sum of squares, the interaction variable (type of sugar × sugar concentration) was the most influential contributor to the insignificant variation. therefore, rws can still be utilized in the production of the spiced syrup without a considerable loss in the tpc. one can avoid brown sugar in the mass production of the spiced syrup as it was uneconomically feasible. antioxidant capacities (dpph scavenging, frap reducing activities, and orac values) of spiced syrups from the summary of variable analysis (table 2), 44% of the orac value variability was explained by the two explanatory variables (sugar concentration and type). the variability of the orac values in the spiced syrup was similar to tpc variability, which was not significantly explained by the type of sugar and the interaction variables of sugar concentration and sugar type. it may explain that the tpc content may have a corresponding influence on the orac values. all the spiced sugar syrups showed decrement trends in the orac values, excluding three negligible samples that showed increments (figure 3). meanwhile, the dpph scavenging and the frap reducing capacities had 40 and 27% variabilities, respectively, explained. the sugar concentration did not give significant variation information in dpph scavenging capacity (p > 0.05). however, the type of sugar played an influential role in explaining the variation in the capacity based on type 3 sum of squares. it contradicted the frap reducing capacity, whereby the sugar concentration significantly influenced its variability. the dpph scavenging capacity of the spiced syrup showed decreasing trends in the rws (0.6 to 72%) but the opposite trend in the mcs-based spiced syrup (31–53%), as compared to the control zero-sugar spiced decoction (figure 4). the rest of the four-brown sugar-based decoctions exhibited mixed trends in the scavenging capacities. all frap reducing capacities showed reductions as negatively influenced by sugar, excluding two samples from the different types of sugar (figure 5). visualization of the correlations between variables by using pca for distinguishing the spiced syrup-based tpc and antioxidant activities, the pca was employed on the pearson correlation matrix. as a classification/discrimination method, pca permits a simplified data representation over a complex data matrix of the spiced syrup with different sugar concentrations and types of sugar use. the first two principal components (pc), pc1 and pc2, explained a total of 78.4% variance (figure 6). both pcs were almost equally explained, whereby the initial pc, explained up to 41.7% and the latter up to 36.7%. pc1 has a strong positive correlation with the tpc and all antioxidant variables, whereby the orac values have the highest correlation with the tpc (p < 0.514) (table 3). the dpph scavenging and frap reducing capacities have the least correlation with the other two variables and are negatively correlated with each other (p < −0.465). table 2. summary of ancova for all variables. tpc dpph sc frap rc orac v r² 0.627 0.403 0.273 0.439 f 13.098 14.186 2.019 5.681 pr > f < 0.0001 < 0.0001 0.067 0.000 sugar concentration (%) f 0.730 10.740 pr > f 0.401 0.003 sugar type f 1.814 2.726 1.106 pr > f 0.150 0.018 0.395 sugar concentration (%) * sugar type f 2.234 1.226 1.262 pr > f 0.084 0.321 0.320 ancova, analysis of covariance; tpc, total phenolic content; orac, oxygen radical absorbance capacity; dpph, 2, 2-diphenyl-1picrylhydrazyl; frap, ferric reducing antioxidant power. italian journal of food science, 2021; 33 (1) 101 influence of sugar concentration and sugar type o r a c a ct iv ity ( t rx e µ g /m l ) c t r l ( 1 0 % s u g a r ) c t r l r w s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l f b s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l n b s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s sugar concentration (%, w/v) 1 6 .7 % r w s c t r l o m o ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l m c s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l j c s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s 0.00 5000.00 10000.00 15000.00 20000.00 25000.00 30000.00 35000.00 40000.00 45000.00 50000.00 figure 3. orac values of spiced syrups prepared from different type of sugar and concentration. c t r l ( 1 0 % s u g a r ) c t r l r w s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l f b s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l n b s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s sugar concentration (%, w/v) 1 6 .7 % r w s c t r l o m o ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l m c s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l j c s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s 0.00 10 00.00 2500.00 3000.00 4000.00 6000.00 5000.00 d p p h r a d ic a l s ca ve n g in g a ct iv ity (t rx e µ g /m l ) figure 4. dpph radical scavenging activities of spiced syrups prepared from different type of sugar and concentration. c t r l ( 1 0 % s u g a r ) c t r l r w s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l f b s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l n b s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s sugar concentration (%, w/v) 1 6 .7 % r w s c t r l o m o ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l m c s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s c t r l j c s ( 5 0 % ) 5 0 .0 % r w s 3 3 .3 % r w s 2 5 .0 % r w s 2 0 .0 % r w s 1 6 .7 % r w s 0.00 –2.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 f r a p a ct iv ity ( t rx e m m /m l ) figure 5. frap values of spiced syrups prepared from different type of sugar and concentrations. 102 italian journal of food science, 2021; 33 (1) zayapor mz et al. table 3. correlation matrix (pearson [n – 1]). variables tpc dpph sc frap rc orac v tpc 1 0.201 0.255 0.514 dpph sc 0.201 1 −0.465 0.220 frap rc 0.255 −0.465 1 0.156 orac v 0.514 0.220 0.156 1 values in bold are different from 0 with a significance level alpha = 0. ancova, analysis of covariance; tpc, total phenolic content; orac, oxygen radical absorbance capacity; dpph, 2, 2-diphenyl-1picrylhydrazyl; frap, ferric reducing antioxidant power. figure 6. pca biplots of spiced syrups made from different type of sugar and concentrations versus tpc and antioxidant parameters. from the biplot (figure 6), there was no specific classification based on either concentration of sugar or the type of sugar exhibited on both the pcs. however, two clusters can be observed in the positive and negative loadings of pc2 without specific classification in each cluster. the pca analysis results showed that either sugar concentration or sugar type significantly influences the tpc and their antioxidant activities. only hard ingredients, either from bark, seed, or heartwood, are traditionally subjected to simmering. in contrast, the soft or leafy ingredients are infused in boiled water to extract bioactive compounds. the duration of the former may take hours as compared to the latter, which only takes a few minutes. the decoction process started after the sugar syrup was boiled and the spices were added and terminated after 4 h of simmering. the spices were introduced into the boiled sugar syrup to obtain the glassy clear syrup in culinary practice. the point at which the sugar was added did not significantly impact taste (madhavamenon and maliakel, 2015). however, from the study observation, the addition of sugar toward the end of the decoction process only retained the liquid turbidity, which was visually less appealing. previous studies showed that mild thermal exposure, such as in the fortification of sugar syrup with functional fruitbased ingredients, retained its high polyphenol content (tarko et al., 2015). those that were traditionally prepared fruit-based beverages also showed more pronounced antioxidant capacities than the commercially processed juice (marjanović et al., 2015). a traditional calabrian fig syrup made from fresh figs’ (ficus carica) aqueous slurry, prolong boiling until the syrup concentrate has a high polyphenol content (3.92 mg of gae per gram of syrup) with desirable sensorial qualities (puoci et al., 2011). it seemed to be not much affected by the lengthy thermal treatment. therefore, the effect of the thermal treatment on the polyphenol content of the spiced syrup was not carried out. the decrement trend in radical scavenging capacities italian journal of food science, 2021; 33 (1) 103 influence of sugar concentration and sugar type the oxidation of phenolic compounds (le bourvellec and renard, 2012), which probably occurred in the spiced syrups and highly corresponded to the low antioxidant activities. it showed that both types of association have a determinant effect on the quality level of polyphenol-rich beverages. sugars engage in the stability of polyphenols in the form of conjugated sugar or glycoside via a glycosidic bond (excluding the subclass of catechins) to one or more hydroxyl groups (martin, 2009). therefore, the addition of sugar may inhibit the autoxidation of polyphenols and contribute to high antioxidant activities in some spiced syrups. however, the profound antioxidant activities were not solely contributed by inhibition of autoxidation. it may partially be due to the advantage of additive or synergistic effect of different phenolics, flavonoids, reducing sugars, and vitamin c profiles like in the must of dried grapes (peinado et al., 2010), whereby an antagonist effect may be found in the spiced syrup for the reduced antioxidant activities. the glucoside forms (sugar conjugated) are likely to offer more therapeutic benefits than the aglycones (nonsugar conjugated polyphenol), as they are more bioaccessible, bioavailable, and stable under in vivo conditions (fernandes et al., 2017). in addition, the sugar moiety of glycoside can enhance the bioavailability, but dimerization weakens the ability. however, glycosides are usually weaker in vitro antioxidants than aglycones (shashank and pandey, 2013); this explains why some spiced syrups have lower antioxidant activities than the zero-sugar spiced decoction. conclusions various sugar concentrations and different types of sugar used in the extraction of polyphenols to produce the spiced syrup may influence the tpc and antioxidant activities to a particular extent with mixed variabilities, as observed from the results of the ancova and the pca. the weaving trends may uniquely contribute to the different profile components of sugar and water-soluble phytochemicals from the spice mixtures. these biochemical complexities of molecular weight and structure related reaction produced a different level of antioxidant activities. from the compiled results of the ancova and the pca, it can be suggested that a sugar concentration of less than 25% is adequate to prepare the spiced syrup with many antioxidant activities, even while using the rws. moreover, further studies are required to better conclude the sugar–polyphenol interaction as in vivo. acknowledgment the authors are grateful for the research grant from the natural medicine research center, universiti islam malaysia, cyberjaya, and antioxidant lab, faculty might be attributed to the glycosidic bond formation through the condensation reaction between a hydroxyl group of sucrose molecule and the hydroxyl group of phenolic compounds to form glycoside compounds. however, the increments were probably the result of phenolic reduced-form compounds due to the oxidation of phenolic compounds and sucrose molecules (shalaby et al., 2016). in addition, jlassi et al. (2016) suggested that the dose-dependent manners of the polyphenol bioactivities may vary due to the polyphenol functional site reaction with ionized sucrose. besides, the polyphenol profile may influence disaccharide action and give variability in antioxidant activities (pengseng et al., 2011). sugar-sweetened beverage consumption still gives a stir to the national health issues. simultaneously, polyphenol-rich food intakes are linked with a positive impact on consumers’ health. however, sugar addition to polyphenol-rich food products, especially beverages, can change the health benefits. some suggested that the presence of sugars generally increases the bioavailability of polyphenols, but product formulation may influence the sugar impact to a certain degree (ackar et al., 2013). this study observed a specific range of sugar concentration and sugar type that accounts for a certain extent of the tpc. these two variables may introduce different profiles of disaccharides, which in turn react to a certain extent. it was not only the type of disaccharide that was reported to have an essential impact on phenolics but also their chemical isomers since the chemical behavior can be different in complex food matrices (zlatić et al., 2017). for example, maltose and trehalose are isomers of sucrose. the latter isomer has a more hydrophilic site than the former, leading to a higher interaction with hydrophilic polyphenols. in the study spice syrup, hydroxycinnamic acids derived from cinnamon, which was hydrophilic (teixeira et al., 2013), probably had more sugar interaction. in addition to the saccharide structure-related factors, evidence showed that the polyphenol interaction with saccharides was influenced by the polyphenol molecular weight (monomeric or polymeric) (amoako and awika, 2016). it was found that polymeric polyphenol (molecular weight, mw > 1000) polysaccharides via hydrogen bonding and hydrophobic interactions formed nondigestible complexes. this positive 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https://www.academia.edu/36703896/antioxidant_potential_of_selected_traditional_plant-based_beverages_in_bosnia_and_herzegovina https://www.academia.edu/36703896/antioxidant_potential_of_selected_traditional_plant-based_beverages_in_bosnia_and_herzegovina https://www.academia.edu/36703896/antioxidant_potential_of_selected_traditional_plant-based_beverages_in_bosnia_and_herzegovina https://doi.org/10.2147/nds.s6422� https://doi.org/10.1007/s12161-010-9139-3� http://doi.wiley.com/10.1002/fsn3.71� https://doi.org/10.1007/s00217-010-1279-6� https://doi.org/10.1155/2014/157427� https://doi.org/10.3390/molecules201219885� _goback ijfs#1551_bozza ital. j. food sci., vol. 32, 2020 438 paper impact of hydrocolloids on the physico-chemical and sensory properties of gluten-free instant noodles from rice flour and mung bean starch s. sutheeves, p. chai-uea and d. thirathumthavorn* department of food technology, faculty of engineering and industrial technology, silpakorn university, nakhon pathom, thailand *corresponding author: thdoungjai@yahoo.com abstract the physico-chemical properties of gluten-free (gf) instant noodles prepared from rice flour and mung bean starch containing hydrocolloids that were carboxymethyl cellulose (cmc), hydroxypropylmethyl cellulose (hpmc), guar gum (gg) and xanthan gum (xg) had been investigated. the results were found that the sample that contained cmc had the least fat uptake and cooking time. different hydrocolloids had no effect on the gelatinization parameters of the noodle dough. the addition of gg improved the textural properties and cooking yields of the gf instant noodles. the samples that were enriched with hpmc showed a significantly lower cooking loss than the others did. an undesirable characteristic was found in the gf noodles that incorporated xg. microstructural image of sample containing xg revealed a non-continuous matrix. the addition of either gg or hpmc improved the sensory properties of the gf instant noodles. keywords: gluten-free, guar gum, hydrocolloid, instant noodles, sensory evaluation, thermal properties ital. j. food sci., vol. 32, 2020 439 1. introduction instant noodles are a popular food consumed in asian countries due to their convenience, taste, prolonged shelf life, nutrition and low cost. they are an internationally recognized food (gulia et al., 2014). moreover, 100 million servings of instant noodles were consumed worldwide in 2017 (wina, 2018). the main ingredients of instant noodles are wheat flour, water, salt and alkaline salt. however, consuming wheat-containing foods, which contains gluten, may cause an allergenic response, especially in those who have celiac disease. in addition, there is a rising demand for gluten-free (gf) products over the past decade (christoph et al., 2018; houben et al., 2012). one way to develop such a product is by using non-gluten flour, such as rice flour, as a raw material to substitute wheat flour. rice is hypoallergenic, mild and colorless (garcia et al., 2016; wu et al., 2019). it is also a staple food and the most important crop in thailand. rice flour, however, cannot form a cohesive dough structure since rice protein lacks the functionality of wheat gluten (elasticity) (kawamura-konishi et al., 2013; sozer, 2009). therefore, the application of heat moisture treated rice flour (chaichaw et al., 2011), guar gum (gg) (sabbatini et al., 2014), xanthan gum (xg) (sabbatini et al., 2014; yalcin and basman, 2008), locust bean gum (yalcin and basman, 2008) and hydroxypropylmethyl cellulose (hpmc) (sabbatini et al., 2014) are added to obtain a viscoelastic dough when preparing the gf noodles. hydrocolloids provide a broad range of functional properties that make them suitable for each application (anton and artfield, 2008). carboxymethyl cellulose (cmc), gg, locust bean gum and alginates are hydrocolloids that widely used in instant wheat noodle processing (gulia et al., 2014). hydrocolloids mimic the viscoelastic properties of gluten (anton and artfield, 2008; suwannaporn and wiwattanawanich, 2011) and improve cooking quality and textural properties of gluten-free pasta (marti and pagani, 2013; padalino et al., 2013; susanna and prabhasankar, 2013). the improvement of cooking and textural properties of gf noodles that contains gg has been shown (kunyanee et al., 2015; sabbatini et al., 2014). the incorporation of xg and gelatinized rice flour improved the dough forming ability and the cooking and sensory properties of the rice noodles (yalcin and basman, 2008). the hpmc and cmc can form thermo-gelling films (mellema, 2003), which result in a reduction of oil absorption in instant fried wheat noodles (rekas and marciniak-lukasiak, 2015) and deepfried legume snack foods (priya et al., 1996). however, cmc has a small effect on the reduction of fat uptake in instant fried wheat noodles (choy et al., 2012). wheat-rice noodles that contain cmc have an improved textural quality and they possess sensory qualities that are comparable to wheat noodles (suwannaporn and wiwattanawanich, 2011). in addition, cmc and hpmc may have quite different properties i.e. solubility, thermal gelation, thickening capacity that make them adequate as food additives (correa et al., 2010). the ingredients, formulation and processing parameters have an influence on the quality of instant noodles (gulia et al., 2014). this study, therefore, aims to investigate the effect of hydrocolloids including gg, hpmc, cmc and xg on the qualities of gf instant noodles. ital. j. food sci., vol. 32, 2020 440 2. materials and methods 2.1. materials rice flour was purchased from varavoot industry co. ltd. in angthong, thailand. mung bean starch was purchased from a local market in nakhon pathom, thailand (tonson, sitthinan co. ltd). the hydrocolloids used in this study included gg (union chemical 1986 co. ltd., bangkok, thailand), hpmc (methocel k4m, dow chemical co. ltd., samut prakan, thailand), cmc (chemipan corporation co., ltd., bangkok, thailand) and xg (thai food and chemical co. ltd., bangkok, thailand). 2.2. preparation of instant fried noodles the dough was formulated by mixing 55.6% rice flour and 2.8% mung bean starch, 0.5% salt (nacl), 0.5% alkaline salt (sodium carbonate), 5.6% pasteurized liquid whole eggs, 1.7% hydrocolloid and 33.3% water. rice flour, mung bean starch and hydrocolloid were mixed in a mixer (kitchen aid k5ss, usa). the pasteurized liquid whole eggs and water containing dissolved salts and alkaline salt were then added, respectively. the mixer was operated at speed 2 until the resultant dough became crumbly (10 minutes) and then it was manually kneaded to form a dough ball. it was then sheeted using a pasta machine (marcato atlas 150, italy) to obtain a final thickness of 1.0 mm, and it was cut into strips 15 cm in length and 0.18 cm in width. the noodle strands were steamed for 10 minutes, cooled at room temperature and fried in palm oil (morakot industries pcl., thailand) at 150°c for 45 seconds. the fried noodles were cooled at room temperature and the excess oil was drained. finally, the instant fried noodles were stored in resealable plastic bags for analysis. 2.3. chemical analysis the proximate composition (moisture, protein and ash) of the instant noodles was analyzed using the association of official analytical chemists method no. 925.10, 920.87 and 923.03, respectively (aoac, 2000) and each sample was carried out in triplicate. an analysis of fat content was performed using automated soxhlet extraction according to a modified method that is described by rekas and marciniak-lukasiak (2015). the total carbohydrate content was determined by calculation using the difference method (100 (weight in grams [protein + fat + ash] in 100 g of food (dry solid))). 2.4. scanning electron microscopy (sem) the cross-section images of the instant fried noodles prior to cooking in boiling water were observed using a field emission scanning electron microscopy (fe-sem, tescan mira3, kohoutovice, czech republic) at the operating voltage of 5.0 kv. the instant noodle samples were fractured into pieces of an approximately 1 cm in length and defatted using the automated soxhlet extraction. the defatted instant fried noodles were attached to a circular specimen stub with double-sided adhesive tape and coated with gold using a sputter coater. ital. j. food sci., vol. 32, 2020 441 2.5. thermal properties an analysis of the thermal properties of the fresh noodles before they were steamed and fried was conducted using a differential scanning calorimeter (dsc8000, perkin elmer, shelton, ct, usa). an empty stainless steel pan was used as a reference and each noodle dough sample was weighed in a stainless steel pan. distilled water was added into the sample pan to bring the water content to 70%. the sample pan was sealed hermetically and equilibrated for 1 hour at room temperature before the dsc measurement. the sample pan was heated from 25°c to 130°c at a ramp rate of 10°c/min to obtain the characterization of gelatinization. the onset temperature (to), peak temperature (tp1 and tp2), final temperature (tf), and enthalpy of gelatinization (∆h) were determined. 2.6. cooking properties the optimum cooking time, cooking loss and cooking yield (or water absorption) of the instant noodles were determined according to the american association of cereal chemists official methods (aacc, 2000) with slight modification. the noodle samples, which was approximately 10g were broken into pieces with an approximate lengths of 5 cm, they were placed in a beaker that contained 120ml of boiling distilled water and then the timing started. the noodle strands were removed from the cooking water at 10 seconds time intervals and they were squeezed between two pieces of glass plates. the time required to the sample as having an “optimum cooking time” was when the opaque central core of the sample disappeared. samples that were 15g were cooked at optimum cooking time in 180 ml of boiling distilled water. after cooking, the samples were rinsed with 50 ml of distilled water, placed in the water at room temperature for one minute and drained before the weight was recorded. the results were calculated for percentage of “cooking yield.” the cooking loss was the amount of solid loss in the cooking water. the cooking water was added to a pre-weighed beaker and evaporated over a steamed bath, and then it was put into hot air oven at a temperature of 105°c until a constant weight was obtained and reported as a percentage of “cooking loss.” the analysis was performed in triplicate for each sample. 2.7. texture profile analysis the texture measurements of the cooked instant noodles were evaluated using a ta-xt2 texture analyzer (stable micro system, london, england) and the procedure described by choy et al. (2012) with modifications. the samples were cooked for the optimum cooking time as described above and cooled in tap water (~17°c) for 1 minute. then, the cooked noodles were retained at room temperature in a covered plastic container. the noodles were compressed using a cylinder probe (p/50) at 2.00 mm/s speed (pre-test, test and post-test) and 75% strain. the parameters that were obtained from the force-time curve of the texture profiles analysis (tpa) were hardness, adhesiveness, springiness, cohesiveness and chewiness. 2.8. sensory evaluation the instant noodles were served after cooking and evaluated by fifty untrained panelists who like eating instant noodles (48 females and 2 males). the sensory evaluations were ital. j. food sci., vol. 32, 2020 442 performed using a 9-point hedonic scale. the panelists scored each sample and assigned scores on a scale of 1 (extremely disliked) to 9 (extremely liked) for appearance, color, firmness, springiness and overall acceptability. 2.9. statistical analysis a statistical analysis was conducted on the experimental data using a one-way analysis of variance (anova), and a comparison of the means was completed by tukey’s test with a significance level of p<0.05. analysis of variance (anova) was carried out using the spss 10.0 (spss inc., usa). 3. results and discussion 3.1. proximate composition of instant noodles the chemical composition of the gf instant noodles are shown in table 1. moisture, fat, ash and carbohydrate contents of all samples were 5.01-7.80%, 14.09-18.09%, 1.56-1.94% and 73.98-77.83%, respectively. the moisture content of the noodles was reduced from around 40% to 5-8% when they were fried in oil. the fat content of the fried noodles was generally in a range of between 15-20% (gulia et al., 2014). all the samples met the standard requirements as specified by the notification of ministry of industry, thailand. hydrocolloid type had an influence on moisture and fat contents of the final products. the gf instant noodles that contained gg had the lowest moisture content (p<0.05). the lowest fat content was obtained in the sample containing cmc. fat uptake are related to two main mechanisms: condensation and capillary mechanisms; in both, oil penetrates through the pores inside the product (mellema, 2003). the differences in fat content of instant fried noodles come from the microporous structure of the product and from the amount of water absorbed in the evaporation process (marciniak-lukasiak et al., 2019; mellema, 2003). the ability of cmc to reduce oil absorption was linked to its hydrophilic character (ang and miller, 1991) and the final product porosity (pinthus et al., 1995). table 1. proximate composition of the gf instant fried noodles containing different hydrocolloids. samples content (g/100 g of dry basis) moisture proteinns fat ash carbohydrate gg 5.01±0.09c 6.16±0.02 18.09±0.19a 1.56±0.02b 74.19±0.16b hpmc 7.80±0.02a 6.29±0.01 18.09±0.08a 1.67±0.02b 73.96±0.07b cmc 7.72±0.07a 6.15±0.04 14.09±0.81b 1.94±0.03a 77.83±0.80a xg 5.97±0.08b 6.20±0.09 16.74±0.65a 1.86±0.04a 75.21±0.51b guar gum (gg), hydroxypropylmethyl cellulose (hpmc), carboxymethyl cellulose (cmc) and xanthan gum (xg) were used in this study. results are expressed as mean values ± standard deviations. means with same superscripts in a row are not significantly different (p≥0.05) as assessed by tukey’s test. ns = values in the same column are not significantly different (probability value, p≥0.05). ital. j. food sci., vol. 32, 2020 443 in addition, cmc had greater hydrophilicity than hpmc (adedeji a.a. and mo, 2011). the thermal gelation of this hydrocolloid also created an oil-resistant film around the fried product and resulted in an increased in its water holding capacity because it entrapped the food moisture inside and lowered the fat absorption (ang and miller, 1991; sakhale et al., 2011; yazdanseta et al., 2015). 3.2. sem the microstructure in the cross section of the gf instant noodle strands (after frying) is shown in fig. 1. generally, during the frying process, many microporous were created as the water was quickly removed, which left empty spaces in the hole structures of the noodles that were replaced by oil (hou, 2001; ziaiifar et al., 2008). the cross-section structure of the gf instant fried noodles that were incorporated with different hydrocolloids had many pores with various sizes and thicknesses. the structure of the instant fried noodles with cmc (fig. 1c) presented small pore sizes and a fewer number of voids and hollows compared to the others. the sample with xg (fig. 1d) showed a noncontinuous matrix noodle structure, a more open area and a large voids and hollows. (a) (b) (c) (d) figure 1. scanning electron microscopy (sem) of the gf instant fried noodles that contained (a) guar gum (gg), (b) hydroxypropylmethyl cellulose (hpmc), (c) carboxymethyl cellulose (cmc) and (d) xanthan gum (xg). ital. j. food sci., vol. 32, 2020 444 3.3. thermal properties the gelatinization temperature and enthalpy changes of the dough are important for an understanding of these phenomena during the thermal process (sabanis and tzia, 2011). all the gf instant fried noodles did not observe an endothermic peak of gelatinization, which indicates that the starches in those samples were fully gelatinized after frying (data not shown). dsc thermograms of the fresh noodles (before steaming and frying) with different hydrocolloids showed biphasic endotherm referred to as g and m1 endotherms, which is in accordance with the finding of prakaywatchara et al. (2018). the first peak (tp1 or g endothermic peak) is attributed to the swelling of partially degraded starch chains and the second peak (tp2 or m1 endothermic peak) reflects the ‘‘melting’’ of the remaining crystallites (kim et al., 2014; xing et al., 2017). the samples with different hydrocolloids were not significantly changed in all thermal properties (p≥0.05) (table 2). it indicated that the hydrocolloids used in this study had no effect on the thermal properties of the noodle dough. table 2. thermal properties of fresh gf noodles that contained different hydrocolloids. samples to (°c) ns tp1 (°c) ns tp2 (°c) ns tf (°c) ns δh (j/g)ns gg 68.57±0.15 74.10±0.01 82.96±0.04 90.43±0.10 9.31±0.41 hpmc 68.55±0.10 74.39±0.09 82.86±0.29 90.35±0.03 8.08±0.64 cmc 68.66±0.13 74.33±0.01 83.29±0.08 90.49±0.08 8.60±0.05 xg 68.54±0.06 74.10±0.16 82.72±0.23 90.43±0.07 8.26±0.23 guar gum (gg), hydroxypropylmethyl cellulose (hpmc), carboxymethyl cellulose (cmc) and xanthan gum (xg) were used in this study. results are expressed as mean values ± standard deviations. ns = values in the same column are not significantly different (probability value, p≥0.05). 3.4. cooking qualities of the instant noodles the cooking quality characteristics of the gf instant fried noodles, including optimum cooking time, cooking loss and cooking yield, are presented in table 3. the optimum cooking times of all the samples ranged from 2.50 to 3.10 minutes. the samples that contained xg had the longest optimum cooking time. the longer optimum cooking times may be attributed to the limited availability of water to the starch granules, which resulted in the retardation of starch swelling (kaur et al., 2015). on the other hand, the noodles that were incorporated with cmc had the shortest optimum cooking time. this may be due to the carboxyl and hydroxyl groups in the structure of the gum, which allowed them to bind with the readily available water and caused an increment in the swelling index and water absorption (gull et al., 2016). cooking loss was related to the structural strength of the noodles, and a higher value indicated a lower structural strength. because the soluble starch and other soluble components, including non-starch polysaccharides, leached out into the water during cooking (gull et al., 2016). the cooking loss for a good quality noodle should be lower than 12% (ugarcic-hardi et al., 2007). in this study, the cooking loss of the gf instant noodles was between 8.50 and 14.46% (table 3). the sample that contained hpmc was the ital. j. food sci., vol. 32, 2020 445 most effective for reducing the cooking loss because the hydrated hpmc network may have been surrounded in the starch-protein matrix and confined the excessive swelling and diffusion of the amylose content (purnima et al., 2012). finally, the noodles that contained xg had the highest cooking loss, perhaps because the xg interfered with the gel compactness (han et al., 2011) as observed in fig. 1(d). the cooking yield explains the ability of the noodles to absorb water during the cooking process (tan et al., 2016). the highest cooking yield was observed in the samples in which gg was added, followed by cmc, hpmc and xg, respectively. this result may be due to the ability of the gg to absorb water in its interrelated network and interact with starch granules (rodge et al., 2012). table 3. the cooking qualities of the gf instant fried noodles incorporating different hydrocolloids. samples optimum cooking time (min) cooking loss (%) cooking yield (%) gg 3.00 10.84±0.86b 297.26±6.13a hpmc 3.00 8.50±0.79c 270.32±5.43c cmc 2.50 10.34±0.59bc 281.11±5.29b xg 3.10 14.46±1.70a 228.79±7.49d guar gum (gg), hydroxypropylmethyl cellulose (hpmc), carboxymethyl cellulose (cmc) and xanthan gum (xg) were used in this study. results are expressed as mean values ± standard deviations. means with same superscripts in a row are not significantly different (probability value, p≥0.05) as assessed by tukey’s test. 3.5. textural properties of the instant noodles textural characteristics are an important parameter that determines the acceptance of the noodles (wu et al., 2015). hardness, springiness, cohesiveness and chewiness of the instant gf noodles with gg addition presented the highest values (table 4). these results indicated that a strong network could be formed by an interaction between amylose in the molecules of starch and gg, resulting in a three-dimensional structure and an increase in gel hardness (kunyanee et al., 2015). the springiness referred to the elasticity of the noodles and assessed the ability of the noodle to regain its original shape after compression (epstein et al., 2002). the addition of xg presented the lowest amount of springiness in the instant noodles. similar results have been reported by suwannaporn and wiwattanawanich (2011). xg did not enhance the elasticity of wheat-rice noodles (suwannaporn and wiwattanawanich, 2011) and increase the dough resistance during extension (collar et al., 1999). hpmc was effective for reducing the cooking loss of the instant gf noodles, but it showed a relatively high adhesiveness in the cooked samples. similarly, han et al. (2011) also observed the same trend in noodles that contained locust bean gum and they reported that the soluble starch that resided on the noodle surface directly affected their adhesiveness. finally, they found that this hydrocolloid was also effective for the promotion of a gel matrix formation (han et al., 2011). ital. j. food sci., vol. 32, 2020 446 table 4. the textural characteristics of the gf instant noodles containing different hydrocolloids. samples hardness (n) adhesiveness (n.sec) springiness cohesiveness chewiness gg 34.12±2.19a 0.78±0.13b 0.88±0.02a 0.71±0.02a 21.17±1.94a hpmc 30.18±2.63b 1.02±0.16a 0.85±0.05ab 0.64±0.02b 16.52±2.13b cmc 25.86±2.73c 1.07±0.10a 0.84±0.05ab 0.69±0.04a 15.02±2.23bc xg 27.70±2.60c 0.67±0.13c 0.82±0.05b 0.61±0.03b 13.86±2.17c guar gum (gg), hydroxypropylmethyl cellulose (hpmc), carboxymethyl cellulose (cmc) and xanthan gum (xg) were used in this study. results are expressed as mean values ± standard deviations. means with same superscripts in a row are not significantly different (probability value, p≥0.05) as assessed by tukey’s test. 3.6. sensory evaluation the results of the sensory evaluation of the cooked gf instant noodles with mixed hydrocolloids are presented in table 5. the instant noodle that contained gg and hpmc had significantly higher (p<0.05) scores for color, firmness, springiness and overall acceptability. the scores for springiness of the cooked instant noodles were improved with the addition of gg and hpmc. they have medium correlation with hardness (r = 0.621, p<0.05), springiness (r = 0.588, p<0.05) and chewiness (r = 0.631, p<0.05) evaluated by instrument (table 3). the samples that contained gg and hpmc exhibited the highest overall acceptability scores. on the other hand, the samples that contained cmc and xg showed lower overall qualities, namely that they possessed less elasticity and softer texture than the others. table 5. the sensory evaluation of the gf instant noodles containing different hydrocolloids. samples appearancens colorns firmness springiness overall acceptability gg 6.58±1.18 6.92±1.07 6.34±1.38ab 6.18±1.41a 6.58±1.11ab hpmc 6.90±1.22 7.00±1.07 6.58±1.07a 6.18±1.55a 6.70±1.22a cmc 6.64±1.10 6.68±1.08 5.92±1.61b 5.56±1.77ab 6.14±1.37bc xg 6.52±1.30 6.78±1.15 6.08±1.58ab 5.20±1.71b 5.98±1.44c guar gum (gg), hydroxypropylmethyl cellulose (hpmc), carboxymethyl cellulose (cmc) and xanthan gum (xg) were used in this study. results are expressed as mean values ± standard deviations. means with same superscripts in a row are not significantly different (probability value, p≥0.05) as assessed by tukey’s test. ns = values in the same column are not significantly different (probability value, p≥0.05). 4. conclusions the gf instant noodles that consisted of gg and hpmc provided a comparable sensory score. compared with hpmc, the sample with gg had a significantly higher cooking ital. j. food sci., vol. 32, 2020 447 yield, cooking loss, hardness, cohesiveness and chewiness, and it also presented a significantly lower adhesiveness. cmc was effective for the reduction of fat uptake and required the least cooking time. thermograms of the fresh noodles with different hydrocolloids before they were steamed and fried showed no significant difference. acknowledgments this work was financially supported by the silpakorn university research and development institute, thailand. references aacc. 2000. 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bayoumi2013@aswu.edu.eg abstract metabolomic profiling, antioxidant, anticancer and antimicrobial activity of three parts (leaves, male parts and fruits) of the medemia argun palm were evaluated. secondary metabolites content showed a significant difference among the evaluated parts. multivariate data analysis (mvda) classified the parts into three groups based on their metabolomic profiling and the total antioxidant capacity (tac) of their extracts. the highest content of secondary metabolites, particularly in the leaves and fruits, was reflected in the dpph radical scavenging activity and consequently in the ic50 of their extracts. the leaves and fruits extracts showed the lowest ic50, followed by the male parts extract (62.8, 78.9 and 134.4 µg/ ml, respectively). individual polyphenols were also determined by hplc, which revealed the dominance of rutin, spigenin-7-glucoside, vanillic and rosmarinic acids, and kaempferol in the leaves. p-hydroxybenzoic, caffeic, syringic, sinapic, and cinnamic acids and chrysin were the dominant polyphenols in the male parts extract. ferulic acid, luteolin and apigenin were the dominant polyphenols in the fruits extract. medemia argun extracts showed very strong antiproliferative activity against hepatocellular carcinoma (hepg-2) and lung cancer cell lines (a549). of the three parts that showed very strong antiproliferative activity, the male parts extract showed prominent antiproliferative activity against hepg-2, while the leaves extract showed more ital. j. food sci., vol. 32, 2020 929 prominent activity against a549 (ic50 was 0.587 and 1.038 µg/ml, respectively). the leaves and fruits extracts showed antimicrobial activity against bacillus subtilis and staphylococcus aureus (gram-positive bacteria) and pseudomonas aeruginosa (gram-negative bacteria). the male parts showed moderate antibacterial activity only against b. subtilis. no extracts affected the growth of escherichia coli, candida albicans and aspergillus flavus. the biological activity of the m. argun palm will be discussed in the light of secondary metabolites content in the plant. to the best of our knowledge, this is the first study concerning the biological activity of m. argun. keywords: antiproliferative activity, antimicrobial activity, dpph, hplc, medemia argun, polyphenols ital. j. food sci., vol. 32, 2020 930 1. introduction recently, the search for natural compounds as antioxidants from plant materials has become a topic of interest for scientists due to the safe nature and low cost of these sources (lindenschmidt et al., 1986; gurib-fakim, 2006); to avoid the utilisation of synthetic compounds which have negative side effects; and because of the very high costs of the available synthetic compounds (oduje et al., 2016). polyphenols is a group of natural plant compounds that showed very strong antioxidant activity against free radicals, which cause many oxidative processes (langley-evans, 2000; pandey and rizvi, 2009). more than 2000 years ago, the consumption of food rich in antioxidant compounds such as polyphenols, anthocyanins, saponins and carotenoids was recommended in traditional chinese medicine due to their health benefits (langley-evans, 2000; mohamed, 2009). the medemia palm tree is a rare and mysterious genus found in the south of egypt and the north of sudan. it has only one species (m. argun), which resembles hyphaene, particularly in its leaf, flower and inflorescence morphology (ibrahim and baker, 2009). there is a lack of information about the metabolomics and biological activity of m. argun. very few studies have been conducted on medemia argun, and most of these focused on the profiling of essential oils and the proanthocyanidin fraction of the fruits by targeted metabolomics analysis, which was unable to reveal the whole picture of the medemia metabolome. the essential oils from different parts of the fruits (mesocarp and headspace of seeds) were profiled and the results indicated that there was a significant variation in essential oils among the evaluated parts of the fruits (hamed et al., 2012). higher performance liquid chromatography and electrospray ionisation mass spectroscopy revealed that the m. argun nut is a rich source of proanthocyanidin (hamed et al., 2014). in another study, incubation of the proanthocyanidin fraction with blood platelets and plasma reduced the formation of 3-nitrotyrosine and diminished the oxidation of thiol groups, in addition to the reduction of the level of carbonyl groups in proteins caused by treatment with peroxynitrite (morel et al., 2014). masullo et al. (2016) investigated the butanol extract of medemia fruits, revealing the presence of eight compounds. neither a complete picture of medemia metabolome of different parts such as leaves, fruits and male parts, nor the biological activity of these parts is evaluated. the objective of this study is to evaluate different parts such as leaves, male parts and fruits for their metabolomic content using targeted and non-targeted analysis in combination with mvda, and to assess their biological activity against different microorganisms and against human carcinogenic cell lines. 2. materials and methods 2.1. plant materials parts of the m. argun palm (leaves, male parts and fruits) were collected from aswan university desert garden. they were dried, separately ground into powder and stored in closed containers until used. ital. j. food sci., vol. 32, 2020 931 2.1.1 plant extraction 100 mg of dried plant materials was dissolved in 4 ml of methanol-water (80%). the mixture was vortexed for one min and then placed in a 60ºc water bath for 1 h. the mixtures were centrifuged at 800 rpm for 10 min and the supernatants were used for determination of secondary metabolites such as saponins, phenolics, flavonoids, flavonols and tannins. 2.2. plant analysis 2.2.1 determination of carbohydrates carbohydrates were determined where the absorbance was read at 620 nm with a spectrophotometer (thermo spectronic genesys 5) (morris, 1948). the concentrations of carbohydrates in different parts of m. argun were calculated and expressed as mg/ g dw. 2.2.2 determination of anthocyanins plant samples were dissolved in acidified methanol in brown tubes or in well-closed tubes covered with aluminum foil and incubated at +4ºc for 24 h (padmavati et al., 1997). the absorbances of the supernatants after centrifugation were recorded at 530 nm and 657 nm. the anthocyanins content was calculated using the following equation: anthocyanin concentration (μmol/ g) = ([a5300.33 x a657]/31.6) x (volume [ml]/weight [g]). 2.2.3 determination of saponins saponins were determined using vanillin reagent and the absorbance of samples and standard was read at 473 nm. total saponins content was expressed as mg saponins equivalent (mg se/ g extract) (ebrahimzadeh and niknam, 1998). 2.2.4 determination of total phenolics content folin-ciocalteu reagent was used to determine total phenolics content in different parts of m. argun. the absorbance of samples and standard (gallic acid) was read at 700 nm (singleton et al., 1999). total phenolics content was expressed as mg gallic acid equivalent (mg gae/ g extract). 2.2.5 determination of flavonoids aluminium chloride was used to determine flavonoids content in different parts of m. argun. the absorbance of samples and standard (quercetin) was measured at 510 nm (zhishen et al., 1999). the content of flavonoids was expressed as mg quercetin equivalent (mg qe/ g extract). ital. j. food sci., vol. 32, 2020 932 2.2.6 determination of flavonols flavonols content was determined spectrophotometrically using aluminium chloride and sodium acetate. the absorbance of samples and standard (quercetin) was read at 440 nm (kumaran and karunakaran, 2007). the content of flavonols was expressed as mg of quercetin equivalent (mg qe/ 100 g extract). 2.2.7 determination of total tannins tannins content was determined using vanillin reagent. the absorbance of samples and standard (catechol) was read at 550 nm (julkunen-titto, 1985). the amount of total tannins was expressed as mg of catechol equivalent (mg ce/ g extract). 2.2.8 determination of total antioxidant capacity (tac) and 2,2-diphenyl-1-picrylhydrazyl (dpph) free radical scavenging activity assay total antioxidant capacity and dpph radical scavenging activity were estimated based on prieto et al. (1999) and blois (1958), respectively. the experimental details of tac and dpph radical scavenging activity were reported (abdel-farid et al., 2014). dpph radical scavenging activity (%) of different concentrations of the plant crude extracts was calculated from the equation: %dpph = (a0-as) / a0 x 100, where a0 is the absorbance of the control and as is the absorbance of the evaluated sample. then the % inhibitions were plotted against concentrations and ic50 was calculated from the graph. the experiment was performed in triplicate and average absorption was recorded for each concentration. 2.3. profiling of polyphenols in different parts of m. argun using hplc hplc analysis was performed using agilent technologies 1100 series liquid chromatography equipped with an auto sampler and a diode-array detector. the mobile phase was formed from acetonitrile (solvent a) and acetic acid in water (2% v/v) (solvent b). 0.45 µm acrodisc syringe filters (gelman laboratory, mi) were used to filtrate samples before injection. 20 µl injection volume, 0.8 ml/ min flow rate and 60 min run time was used with the following gradient programme: 100% b to 85% b for 30 min, 85% b to 50% b for 20 min, 50% b to 0% b for 5 min and 0% b to 100% b for 5 min. simultaneously, peaks were monitored at 280, 320 and 360 nm and identified by congruent retention times and uv spectra with comparison with the standards (kim et al., 2006; mansour et al., 2018). 2.4. biological activity 2.4.1 antiproliferative activity 2.4.1.1 cancer cell lines, medium and in vitro antiproliferative activity human hepatocellular carcinoma hepg-2 and lung carcinoma cell lines (a549) were obtained from an american culture collection. prmi-1640 medium supplemented with 10% foetal bovine serum (fbs), 2 ml glutamine containing 100 u/ml streptomycin and 100 u/ml penicillin at 37ºc/5% co2 were used for cell culturing. from each m. argun extract, several concentrations were prepared in 1% dmso (0.049, 0.098, 0.195, 0.391, 0.781, 1.56, 3.13, 6.25, 12.5 and 25 µg/ ml). ital. j. food sci., vol. 32, 2020 933 2.4.1.2 determination of inhibition concentration 50% (ic50) for medemia palm extracts using sulforhodamine b (srb) colorimetric assay the antiproliferative activity of m. argun extracts was assessed using srb assay based on vichai and kirtikara (2006). the antiproliferative test procedures, from cell harvesting to measurement of colour intensity using a microplate reader and calculation of ic50 of each extract, were described in vichai and kirtikara, 2006 and el-naggar et al., 2015. 2.4.2 antimicrobial activity antimicrobial activity of the evaluated extracts was determined using disc diffusion assay (bauer, et al., 1966). discs were impregnated with the stock solutions of the evaluated parts of m. argun, where each disc received 500 µg from the medemia extracts. positive control was also prepared where each disc received 200 µg of ampicillin and amphotericin (antibacterial and antifungal agents), respectively. mueller-hinton agar plates were inoculated with gram (+) bacteria (staphylococcus aureus and bacillus subtilis); gram (-) bacteria (escherichia coli and pseudomonas aeruginosa) were incubated at 35-37ºc for 24-48 h. candida albicans was incubated at 30ºc for 24-48 h and filamentous fungi as aspergillus flavus was incubated at 25ºc for 48 h (bauer et al., 1966). the inhibition zone was measured in millimetres, each experiment was repeated three times, and the average of three readings of the diameters of the inhibition zones was calculated. 2.5. statistical analysis spectrophotometer data was subjected to multivariate data analysis (mvda) such as principal component analysis (pca) and hierarchical clustering analysis (hca) using simca-p software (version 12.0). the statistical differences among the content of secondary metabolites in different parts of m. argun were evaluated using analysis of variance (anova) from minitab (version 12.21). the correlation between the determined metabolites in different parts and tac was assessed using pearson's correlation test. data was presented as the average of three readings ± the standard deviation. 3. results 3.1. phytochemical analysis and metabolomic profiling of different parts of m. argun (argun palm) using spectrophotometer and hplc combined with multivariate data analysis the metabolites content of different parts (leaves, male parts and fruits) of m. argun is shown in table 1. the flavonols content differed significantly in the leaves and fruits (p<0.05). carbohydrates showed no significant difference among the evaluated parts (p>0.05). fruits were characterised by a higher content of total saponins, total phenolics, total flavonoids and total condensed tannins. phenolics and flavonoids content showed significant differences among the evaluated parts, while for total saponins and tannins content the only significant difference was observed between the leaves and fruits and the male parts and fruits (table 1). male parts were characterised by higher anthocyanins content, which showed a significant difference among the evaluated parts (p<0.05). ital. j. food sci., vol. 32, 2020 934 table 1. phytochemical analysis, total antioxidant capacity, dpph radical scavenging activity and ic50 of different parts of medemia argun palm. metabolites leaves male parts fruits carbohydrates (mg/g dw) 124.5±0.2a 122.9±0.05a 122.2±0.41a saponins (mg saponins equivalent/g) 15.3±0.7a 16.2±0.7a 18.7±0.04b phenolics (mg/ gallic acid equivalent/ g) 13.3±0.9a 36.04±0.06b 72.3±3.9c flavonoids (mg quercetin equivalent/ g) 13.3±0.9a 27.6±3.6b 43.1±4.4c flavonols (mg quercetin equivalent/ 100 g) 76.12±1.2a 67.9±9.35ab 45.2±7.0b anthocyanin (µmole/ g) 0.34±0.04a 0.41±0.01b 0.26±0.02c tannins (mg catechols equivalent/ g) 2.28±0.45a 2.47±0.09a 5.53±0.5b tac (ascorbic acid equivalent µg/ g) 0.305±0.007a 0.285±0.032a 0.153±0.006c dpph (100 µg/ ml) 85.7±1.34a 37.5±4.9b 72.5±0.42c ic50 (µg/ ml) 62.8±9.9 a 134.4±3.5b 78.9±2.9a different letters in the same row means significant difference at p<0.05. to reduce the dimensionality of the metabolomic data and to evaluate the similarity and dissimilarity between different parts of m. argun from the perspective of their phytochemical composition and metabolomic profiling, the data of the three parts of m. argun was subjected to pca, followed by hca. pca separated the parts into three groups: leaves, fruits and male parts, as shown in the score scatter plot of pc1 vs. pc2 (fig. 1a). the score scatter plot also showed that there is a similarity between the leaves and male parts resulting from the approximation of the two parts in the negative part of pc1 (left hand side of the ellipse) (fig. 1a). the score loading plot (fig. 1b) revealed the metabolites contributed to the separation obtained in the score scatter plot. leaves had higher content of carbohydrates, flavonols, tac and dpph radical scavenging activity. fruits had higher content of saponins, tannins, flavonoids and phenolics. the male parts had higher content of anthocyanins. the score biplot confirmed the results of the pca score scatter and score loading plots (fig. 2a). hca showed the classification of the three parts in three groups with a similarity between the leaves and male parts (fig. 2b). metabolomic profiling of individual polyphenols in different parts of the m. argun palm was evaluated using hplc. 24 standards were injected and their peaks are shown in fig. 3. in the evaluated parts, 18 individual polyphenols were detected and their concentrations were determined (table 2). rutin, apigenin-7-glucoside, kaempferol, and rosmarinic and vanillic acids were the dominant polyphenols in the leaves extract of m. argun. caffeic, p-hydroxybenzoic, sinapic, syringic, and cinnamic acids and chrysin were the dominant polyphenols detected in the male parts extract, where ferulic acid, apigenin and luteolin were the dominant polyphenols in the fruits (table 2). metabolomic profiling of individual polyphenols in different parts of m. argun palm was evaluated using hplc. 24 standards were injected and their peaks are shown in fig. 3. in the evaluated parts, 18 individual polyphenols were detected and their concentrations were determined (table 2). rutin, apigenin-7-glucoside, kaempferol, rosmarinic and vanillic acids were the dominant polyphenols in leaves extract of m. argun. caffeic, phydroxybenzoic, sinapic, syringic, cinnamic acids and chrysin were the dominant polyphenols detected in male parts extract, where ferulic acid, apigenin and luteolin were the dominant polyphenols in fruits (table 2). ital. j. food sci., vol. 32, 2020 935 (a) (b) -4 -2 0 2 4 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 (p c 2 = 25 .6 % ) (pc1 = 56.6%) r2x[1] = 0.566972 r2x[2] = 0.256172 ellipse: hotelling t2 (0.95) ml ml ml mfmfmf mm mm mm simca-p+ 12.0.1 2018-04-08 04:38:44 (utc+5) fruits male parts leaves -0.4 -0.2 -0.0 0.2 0.4 0.6 -0.4 -0.3 -0.2 -0.1 -0.0 0.1 0.2 0.3 0.4 (p c 2 = 25 .6 % ) (pc1 = 56.6%) r2x[1] = 0.566972 r2x[2] = 0.256172 saponin flavonoids phenolics carbohydra portein flavonols anthocyan dpph tac simca-p+ 12.0.1 2018-04-08 04:41:06 (utc+5) figure 1. score scatter plot of pc1 vs. pc2 (a), and the score loading plot of pc1 vs. pc2 of the metabolomic profiling of different parts of medemia argun palm (b). ml = medemia argun leaves, mm = medemia argun male parts and mf = medemia argun fruits. ital. j. food sci., vol. 32, 2020 936 figure 2. score biplot of pc1 vs. pc2 (a), and hierarchical clustering analysis (hca) (b) of the metabolomic profiling of different parts of medemia argun palm. labeling of each group is the same as in fig. 1. ital. j. food sci., vol. 32, 2020 937 figure 3. hplc chromatogram of 24 polyphenols standards at 280 nm (taha et al., 2020). table 2. individual polyphenols (mg/ g) in different parts of m. argun palm. leaves male parts fruits protocatechuic acid 0.167 0.152 nd p-hydroxybenzoic acid 0.158 0.371 0.110 catechins 0.316 0.295 nd chlorogenic acid 0.071 nd nd caffeic acid nd 0.206 nd syringic acid 0.112 0.267 0.081 vanillic acid 0.066 nd 0.05 ferulic acid 0.05 0.010 0.033 sinapic acid nd 0.179 0.169 rutin 12.55 2.352 0.538 p-coumaric acid 0.821 0.722 0.70 apigenin-7-glucoside 2.85 0.288 0.255 rosmarinic acid 0.631 0.127 0.111 cinnamic acid 0.012 0.088 0.017 luteolin nd nd 0.033 apigenin nd 0.079 0.229 kaempferol 0.211 0.131 0.073 chrysin nd 0.044 nd nd = non-detectable. ital. j. food sci., vol. 32, 2020 938 3.2. antioxidant, dpph free radical scavenging activity and ic50 of m. argun palm dpph values differed significantly among the evaluated parts (p<0.05). tac showed a significant difference between the leaves and fruits and the male parts and fruits (table 1). the leaves and fruits had the highest values of dpph radical scavenging activity (85.7 and 72.5%, respectively), whereas the male parts showed the lowest percentage (37.5%). the richness of leaves and fruits in bioactive metabolites was reflected in the dpph radical scavenging activity and also in the ic50 of the extracts under evaluation. the leaves and fruits showed the lowest ic50 compared to the male parts (62.8, 79.9 and 134.4 µg/ ml, respectively) (table 1). the higher the free radical scavenging activity, the lower the ic50 and vice versa. this means that leaves and fruits are more active than male parts as antioxidant agents. pearson's correlation was performed to assess the relation between the estimated metabolites and tac in the three evaluated parts of the m. argun palm. tac positively correlated with the total flavonols and anthocyanins content (p<0.05), and r values: 0.799 and 0.913, respectively. 3.3. antiproliferative activity of different parts of m. argun the results of the ic50 of different parts of the m. argun palm are shown in table 3. the three evaluated parts of m. argun showed very strong antiproliferative activity against hepatocellular carcinoma (hepg-2) and lung cancer cell lines (a549). the male parts of the m. argun palm showed the lowest ic50 against hepg-2 cell lines (0.587 µg/ ml), followed by the fruits and leaves extracts with ic50: 1.247 and 1.476 µg/ ml, respectively. the leaves extract of m. argun was the strongest antiproliferative extract against lung carcinoma cell lines (a549) with the lowest ic50 (1.038 µg/ml), followed by the male parts and fruits extracts (ic50: 2.369 and 3.551 µg/ ml, respectively) (table 3). the dead cells in both cell lines incubated with m. argun were dose dependent as they increased with the concentrations used (fig. 4). although all extracts showed very strong antiproliferative activity against hepatocellular carcinoma and lung cancer cell lines, the effect of the male parts and fruits extracts was more pronounced than that of the leaves extract on hepg-2 cell lines, and the leaves and male parts extracts exerted stronger antiproliferative activity against lung cancer cell lines than the fruits extract (table 3 and fig. 4). hepatocellular carcinoma (hepg-2) revealed more susceptibility to m. argun extracts than lung cancer cell lines (a549) (fig. 4). table 3. ic50 (µg/ ml) of m. argun extracts against hepatocellular carcinoma (hepg-2) and lung cancer cell line (a549). hepg-2 a549 leaves 1.476 1.038 male parts 0.587 2.369 fruits 1.247 3.551 ital. j. food sci., vol. 32, 2020 939 (a) (b) vi ab ili ty ( % ) concentration (µg/ml) 0 10 20 30 40 50 60 70 80 90 100 0 0.049 0.098 0.195 0.391 0.781 1.56 3.13 6.25 12.5 25 leaves male parts fruits vi ab ili ty ( % ) concentration (µg/ml) 0 10 20 30 40 50 60 70 80 90 100 0 0.049 0.098 0.195 0.391 0.781 1.56 3.13 6.25 12.5 25 leaves male parts fruits ic50= 1.476 µg/ml ic50= 0.587 µg/ml ic50= 1.247 µg/ml ic50= 2.369 µg/mlic50= 1.038 µg/ml ic50= 3.551 µg/ml figure 4. the viability percentage (%) of different parts of medemia palm extracts against hepatocellular carcinoma hepg-2 (a) and lung cell line carcinoma a549 (b). each experiment was repeated three times. 3.4. antimicrobial activity of different parts of m. argun the antimicrobial activity of different parts of an m. argun palm was evaluated using paper diffusion disc assay. only the leaves and fruits extracts of m. argun exerted antimicrobial activity against b. subtilis, s. aureus and p. aeruginosa (table 4). the effect of the fruits extract was more prominent than the leaves extract on gram-positive bacteria. the leaves extract showed more effect than the fruits extract against p. aeruginosa (table 4). gram-positive bacteria showed more susceptibility than gram-negative bacteria to the methanol extracts of the m. argun palm (table 4). only the male parts extract showed antibacterial activity against b. subtilis and no extract from the evaluated parts showed antimicrobial activity against c. albicans and a. flavus, meaning that fungal strains had more resistance than bacterial strains against m. argun palm extracts. ital. j. food sci., vol. 32, 2020 940 table 4. antimicrobial activity (in term of clear zones) of different parts of m. argun. zone of inhibition (mm) control leaves male parts fruits gram +ve bacteria bacillus subtilis 28.0±2.0 11.3±2.5* 9.0±0.0* 12.7±0.06* staphylococcus aureus 31.0±6.5 12.3±2.3 * 0.0±0.0* 12.3±2.3* gram -ve bacteria pseudomonas aeruginosa 32.4±1.1 12.7±1.5 * 0.0±0.0* 11.0±2.0* escherichia coli 29.0±1.7 0.0±0.0* 0.0±0.0* 0.0±0.0* fungi aspergillus flavus 14.6±1.5 0.0±0.0* 0.0±0.0* 0.0±0.0* candida albicans 16.3±1.1 0.0±0.0* 0.0±0.0* 0.0±0.0* + control is ampicillin as antibacterial agent and amphotericin b as antifungal agent. each disc was impregnated with 500 µg from each extract; whereas the disc of positive control was impregnated with 200 µg from each control. the data presented is a mean of 3 replicates with the standard deviation. *mean there a significant difference between the diameter of clear zones with the plant extracts and that of the positive control at p<0.05. 4. discussion under normal conditions, plants produce many bioactive water soluble secondary metabolites such as polyphenols, terpenoids, steroids, alkaloids, saponins, glucosinolates, isothiocyanates and tannins (kruse et al., 2000; dixon, 2001). these metabolites are very important not only for a plant itself for its defense against pathogens and herbivores and tolerance for different biotic and abiotic stresses (pandey and rizvi, 2009), but also for humans, as the metabolites are the main source of medicines as antioxidant and free radical scavenging activities for different oxidative stress linked diseases (olajuyigbe and afolayan, 2011). polyphenols are groups of secondary metabolites such as phenolic and cinnamic acids, flavonoids, anthocyanins, stilbene and lignans; they are considered as potentially health beneficial antioxidants. consumption of a polyphenolrich diet provides protection against a series of dangerous diseases such as cancers, diabetes, cardiovascular diseases, neurodegenerative disease and osteoporosis (pandey and rizvi, 2009). to assess the biological activities of plant extracts, the phytochemical and metabolomic profiling of plants is very important in attributing any biological activity to the plants’ metabolites content. to reveal the whole picture of metabolomics of a given plant, many analytical techniques should be used in combination with mvda. m. argun has received very little attention from researchers, and search engine queries for the plant found only five articles that focus only on the targeted phytochemical studies of m. argun (hamed et al., 2012, 2014, morel et al., 2014; masullo et al., 2016; said et al., 2017). the significant variations in the bioactive secondary metabolites content in different parts of m. argun were in line with many previous reports, which showed clearly that each part of a given plant has its own phytoanticipins and metabolomic content (hamed et al., 2012; abdelfarid et al., 2014; taha et al., 2020). the highest percentages of dpph free radical scavenging activity of the leaves and fruits of m. argun can probably be attributed to the richness of these parts in bioactive secondary metabolites such as polyphenols, saponins and tannins, as explored by spectrophotometer ital. j. food sci., vol. 32, 2020 941 and hplc analysis. the positive correlation between tac and dpph free radical scavenging activity and secondary metabolites content, particularly polyphenols, was well documented in many previous studies (basar et al., 2013; abdel-farid et al., 2014; diaconeasa et al., 2015; mansour et al., 2016). the highest content of these previously mentioned secondary metabolites not only affected the dpph radical scavenging activity and tac, they also positively affected the antiproliferative activity of m. argun extracts. the cytotoxic activity of some saudi and egyptian desert plants against hepatocellular, breast and lung cancer cell lines was attributed to the highest content of these secondary metabolites in their extracts (alenad et al., 2013; el-naggar et al., 2015; taha et al., 2020). the individual polyphenols in m. argun extracts such as rutin, catechin, kaempferol, rosmarinic, vanillic, rosmarinic, caffeic, p-hydroxybenzoic, protocatechuic, sinapic, syringic, ferulic, cinnamic acids, apigenin-7glucoside, luteolin, apigenin and chrysin were well known as anticancer agents that worked individually or in combination (synergistic effect) (fuchs and milbradt, 1993; velika and kron, 2012; fonseca et al., 2015; wilkins et al., 2017; chandra and viswanathswamy, 2018; yamagata et al., 2018). the interaction of some of these active secondary metabolites with cancer-associated receptors seems to trigger specific mechanisms and consequently causes the death of cancer cells (wang et al., 2008). the antimicrobial activity of m. argun was also attributed to its active metabolites content, particularly polyphenols. the suppression of proteases and/or inactivation of the microbial adhesions may be the mechanism of polyphenols toxicity against microorganisms (cowan, 1999). the correlation between the content of secondary metabolites such as polyphenols and saponins and the antimicrobial potentiality was reported (cisowska et al., 2011; alves et al., 2013; nayaka et al., 2014; gull et al., 2015). the different responses of gram-negative and -positive bacteria to m. argun extracts may be due to the nature of the cell wall, which varies between gram-positive and negative bacteria. not only the type of bacteria but also the metabolites content of the plant part used and also the extraction method and the extraction solvent used may control the response of bacteria to the extract (gull et al., 2015). m. argun extracts showed no antifungal activity. similar to these results, the proanthocyanidin fraction isolated from the m. argun nut has not affected the growth of cephalosporium gramineum (martyniuk et al., 2017). 5. conclusion m. argun has high bioactive secondary metabolites content, such as polyphenols (with its different classes) and saponins, which were affected positively on potentiality against hepatocellular carcinoma and lung cancer cell lines. the highest content of these metabolites was reflected in the antiproliferative activity of m. argun against carcinogenic cell lines, and it also extended to its tac and free radical scavenging activity. moreover, the high secondary metabolites content, particularly polyphenols in the m. argun palm, was affected positively on potentiality as antibacterial activity. m. argun will be a promising plant in future for industrial and pharmaceutical medicines. pharmacological and pharmaceutical studies are required in which separated individual polyphenols as well as plant extracts should be tested against different carcinogenic cell lines in vitro and in vivo in animals in amelioration experiments. the effect of m. argun extracts on diabetes in mice is also desirable. ital. j. food sci., vol. 32, 2020 942 acknowledgments we would like to thank the 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expression in a549 human lung cancer cells. mol. cell. biochem. 441:9-19. doi: doi.org/10.1007/s11010017-3171-1. zhishen j., mengcheng t. and jianming w. 1999. the determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. food chem. 64:555-559. doi: doi.org/10.1016/s0308-8146(98)00102-2 paper received may 3, 2020 accepted june 16, 2020 survey ital. j. food sci., vol. 27 2015 271 keywords: aspergillus, carry-over, elisa a one-year survey on aflatoxin m1 in raw milk m. schirone1, p. visciano1*, a.m.a. olivastri2, r. tofalo1, g. perpetuini1 and g. suzzi1 1facoltà di bioscienze e tecnologie agro-alimentari e ambientali, università di teramo, via c.r. lerici 1, 64023 mosciano sant’angelo, teramo, italy 2azienda sanitaria unica regionale marche ascoli piceno, italy * corresponding author: tel. +39 0861 266911, fax +39 0861 266940, email: pvisciano@unite.it abstract in the year 2012, 288 raw milk samples were collected from six different dairy cow farms and analyzed for the presence of aflatoxin m 1 (afm 1 ) using the elisa technique. the afm 1 levels ranged from 5 to 25 ng/kg and the highest concentrations were found in autumn, with a significant difference (p<0.05) between february and november. the eu legal limit of 50 ng/kg has never been exceeded. even if the results of the present study show a low risk for afm 1 , its occurrence in dairy products has to be regularly monitored due to their importance as foodstuffs for people and children above all. mailto:pvisciano@unite.it 272 ital. j. food sci., vol. 27 2015 introduction nowadays food industry has the responsibility to develop and implement a hazard analysis and critical control point (haccp) system aiming at identifying and preventing important hazards to food safety. the presence of aflatoxins (afs) in dairy products is one of the most important critical control points to be checked in raw milk supplies. aflatoxins are secondary metabolites mainly produced by three species of aspergillus including aspergillus flavus, aspergillus parasiticus and aspergillus nomius (crappy, 2002). even if eighteen afs have been identified, only four out of them have been found in food and feed, i.e. afb 1 , afb 2 , afg 1 and afg 2 (heshmati and milani, 2010). the toxic effects of these compounds can be both acute, thus causing hepatitis, oedema or hemorrhagic necrosis, and chronic, thus resulting in liver, lung and kidney carcinomas as well as immunosuppression (williams et al., 2004). in particular, afb 1 shows different toxic activities, including teratogenicity, mutagenicity and carcinogenicity (mclean and dutton, 1995). therefore, the international agency for research on cancer (iarc) has included afb 1 in group 1 as a human carcinogen (iarc, 1993). animals eating contaminated feed rapidly adsorb and transfer afb 1 to the liver, where it is metabolized into the 4-hydroxylated derivate afm 1 and excreted through faeces and urine (polonelli et al., 2011). consequently, afm 1 may be secreted in mammalian milk by means of a carry-over process, within 12-24 h after the ingestion of afb 1 (kav et al., 2011). afm 1 exhibits a high level of genotoxic activity due to its possible accumulation and linkage to dna (shundo and sabino, 2006). it can cause dna damage, gene mutation, chromosomal anomalies and cell transformation in in-vitro mammalian cells, insects, lower eukaryotes and bacteria (prandini et al., 2009). for that reason, afm 1 has been included in group 1 (iarc, 2002) and removed from group 2b (i.e. as a possible human carcinogen). sources of afs contamination in animal foodstuffs may vary geographically, with prevalence in areas with favorable environmental and climatic conditions. aspergillus flavus and a. parasiticus colonize plants when still in the field, mainly when damaged; the highest afs production occurs at temperatures between 20° and 30°c. in particular, a. parasiticus prefers a soil environment and can be found more commonly on peanuts, while a. flavus is better adapted to an aerial environment and colonizes cotton and corn (prandini et al., 2009). these molds can also colonize products in post-harvest if not adequately stored. however, the relationship between the amount of afb 1 ingested by animals and the quantity of afm 1 in milk is quite variable, as many factors – such as the individual variability among animals, the presence of udder infections, and the lactation period (at the beginning the carry-over is 3.3-3.5 times greater compared to the advanced lactation) – can affect carry-over (van egmond, 1989). the maximum limit for afm 1 concentration in food varies in the legislation of different countries (godič torkar and vengušt, 2008). the european community has prescribed a limit of 50 ng/ kg in raw milk, heat-treated milk and milk for the manufacture of milk-based products (ec, 2006) while, according to us regulations, the action level of afm 1 in milk should not be higher than 500 ng/kg (ghanem and orfi, 2009). afm 1 is relatively stable in raw and processed milk products and is not affected by pasteurization or cheesemaking processes performed in dairy industry (kav et al., 2011). it has been reported that afm 1 concentration in dairy products can be 3 to 4 times higher than in milk, as it is associated with milk proteins (battacone et al., 2003). the present study aims at detecting the afm 1 levels in bovine raw milk designed to a dairy factory located in the marche region, central italy. the preventive action used by such factory to control this hazard will also be discussed. the samples were analyzed by means of an enzymelinked immunosorbent assay (elisa) that is the most representative method for the fast screening analysis of afs. materials and methods a total of 288 samples of raw milk collected from six different suppliers (named 1 to 6) located in the marche region, central italy, were examined over the year 2012. raw milk samples from each farm were provided to that dairy factory four times in a month and were transported in tanks at 0-4°c. all samples were analyzed in duplicate. such raw milk samples (10 ml) were at first centrifuged at 3,500 g for 10 min at 4°c, then the upper cream layer was completely removed. a sample unit of 100 µl was used for the quantitative analysis of afm 1 using the commercial kit ridascreen (r-biopharm, germany). such kit includes microtiter plates coated with capture antibodies, afm 1 standard solutions used for the construction of the calibration curve, peroxidase-conjugated afm 1 , substrate (urea peroxidase), chromogen (tetramethylbenzidine) and stop reagents 1 n sulfuric acid. the test procedure was performed according to heshmati and milani (2010). the evaluation of afm 1 was obtained dividing the absorbance values of the standards and the samples by the absorbance value of the first standard (zero standard), then multiplying the result by 100 (percentage of maximum absorbance). the adsorption was inversely proportional to the afm 1 concentration in samples. the limit of quantitation according to the kit was 5 ng/kg. ital. j. food sci., vol. 27 2015 273 a statistical analysis was carried out by graphpad instat version 3.0, graphpad software (san diego, california, usa). all the obtained data were assessed for normality by means of kolmogorov-smirnov test. since the values were not normally distributed, non-parametric tests were applied. the differences among the values obtained from the six different suppliers and among the milk samples collected over 12 months were evaluated by kruskal-wallis test (non-parametric anova). when the p value was lower than 0.05, the dunn’s multiple comparisons test was used. results and conclusions the mean afm 1 concentrations in four collections (analyzed in duplicate) of raw milk over a month (for a total of 12 months) from each dairy farm are reported in table 1. the levels ranged from the limit of quantitation (5 ng/kg) to a maximum of 25 ng/kg, with the highest values observed in the months of september, october and november. however, no sample exceeded the maximum levels (50 ng/kg) set for afm 1 in milk by eu legislation (ec, 2006). no significant difference (p>0.05) was also observed among the afm 1 concentrations in samples from the different suppliers, while a significant difference (p<0.05) was noticed only between the values obtained in february and november. according to the haccp plan implemented in the dairy factory of the present study, afm 1 content is regularly monitored four times in a month but, when it results to be higher than 10 ng/kg, the supplier is contacted (as a preventive action) and analyses of raw milk from the matching dairy farm are repeated at the next supply. in this study (table 2), 68.4% of the samples contained afm 1 in the range of 5-10 ng/kg, while 27.1% was in the range of 11-19 ng/kg, exceeding the above mentioned preventive limit (10 ng/kg). moreover, the dairy factory has set an internal system of corrective actions when afm 1 content exceeds 20 ng/kg, defined as action limit. in the present study, the action limit was exceeded only in 4.5% of the samples collected from some dairy farms in different months (i.e. january, september, october, november and december), with values ranging from 20 ng/kg to a maximum of 25 ng/kg. in that case the supply of milk from the dairy farm is suspended until concentrations return to regular values. whereas, if such value exceeds 50 ng/kg (the maximum level by law), the positive sample is analyzed by means of the hplc as confirmatory assay, and milk has then to be intended as “category 2 material” according to the eu regulations on animal by-products (ec, 2009). however, as a preventive measure, the hplc procedure is routinely performed every four months. t a b le 1 c o n ce n tr a ti o n s o f a f m 1 ( n g/ k g) a n d r a n ge ( in p a re n th es es ) fo r ea ch s ix s u p p li er s in t h e ye a r 2 0 1 2 . s up pl ie r ja nu ar y fe br ua ry a m ar ch a pr il m ay ju ne ju ly a ug us t s ep te m be r o ct ob er n ov em be rb d ec em be r 1 n d n d 12 .5 ±2 .9 * (5 .0 -1 5. 0) 10 .0 ±3 .3 (5 .0 -1 5. 0) n d 7.5 ±2 .9 (5 .0 -1 5. 0) n d 9. 10 ±3 .2 (5 .0 -1 5. 0) 7.5 ±2 .9 (5 .0 -1 5. 0) 9. 0± 3. 2 (5 .0 -1 5. 0) 11 .8 ±4 .8 (5 .0 -2 2. 0) 10 .0 ±3 .6 (5 .0 -1 8. 0) 2 8. 8± 4. 3 (5 .0 -2 0. 0) n d 6. 8± 2. 0 (5 .0 -1 2. 0) 9. 3± 2. 9 (5 .0 -1 5. 0) 9. 0± 3. 2 (5 .0 -1 5. 0) n d 10 .0 ±3 .3 (5 .0 -1 5. 0) 13 .4 ±2 .8 (5 .0 -1 7.0 ) 15 .8 ±0 .9 (1 5. 018 .0 ) 15 .2 ±4 .1 (5 .0 -2 5. 0) 9. 3± 2. 9 (5 .0 -1 5. 0) 10 .0 ±3 .3 (5 .0 -1 5. 0) 3 13 .0 ±5 .5 (5 .0 -2 4. 0) n d n d 7.5 ±2 .9 (5 .0 -1 5. 0) 9. 0± 3. 2 (5 .0 -1 5. 0) n d n d n d 17 .3 ±0 .9 (1 5. 018 .0 ) 10 .4 ±4 .3 (5 .0 -1 9. 0) 11 .0 ±4 .1 (5 .0 -1 9. 0) 7.5 ±2 .9 (5 .0 -1 5. 0) 4 6. 8± 2. 0 (5 .0 -1 2. 0) 7.0 ±2 .6 (5 .0 -1 5. 0) n d n d 9. 0± 3. 2 (5 .0 -1 5. 0) 6. 8± 2. 0 (5 .0 -1 2. 0) n d 8. 0± 2. 6 (5 .0 -1 5. 0) 10 .8 ±3 .9 (5 .0 -1 8. 0) 10 .6 ±4 .7 (5 .0 -2 3. 0) 14 .0 ±6 .1 (5 .0 -2 5. 0) 12 .8 ±3 .0 (5 .0 -1 6. 0) 5 n d n d 9. 3± 2. 9 (5 .0 -1 5. 0) n d 7.6 ±3 .4 (5 .0 -1 8. 0) n d 7.5 ±2 .9 (5 .0 -1 5. 0) 9. 2± 3. 3 (5 .0 -1 6. 0) 17 .8 ±1 .2 (1 5. 020 .0 ) 17 .6 ±1 .7 (1 5. 022 .0 ) 15 .8 ±4 .5 (5 .0 -2 3. 0) 10 .0 ±3 .3 (5 .0 -1 5. 0) 6 n d n d 7.5 ±2 .9 (5 .0 -1 5. 0) n d 8. 8± 4. 9 (5 .0 -2 4. 0) n d n d 10 .0 ±4 .1 (5 .0 -1 5. 0) n d n d 7.5 ±2 .9 (5 .0 -1 5. 0) 9. 3± 4. 9 (5 .0 -2 2. 0) *t he se d at a ar e ex pr es se d as m ea n ± st an da rd e rr or ; n d = a fm 1< 5 n g/ kg ; a ,b (p <0 .0 5) 274 ital. j. food sci., vol. 27 2015 in figure 1 the mean content of afm 1 in milk per month was reported, without considering the different suppliers. in the present study, the overall contamination levels of afm 1 in milk samples were lower than those reported by other authors (han et al., 2013; hussain and anwar, 2008; rahimi and ameri, 2012; tajik et al., 2007). these differences could be due to several factors, including different analytical techniques, samples size, season of the year, livestock management, and dairy processing systems. moreover, the afm 1 levels in milk seemed to be significantly influenced by the geographical region. the outcomes of some studies carried out in italy showed an afm 1 concentration range of 2-90 ng/kg (nachtmann et al., 2007) and < 23 ng/kg (galvano et al., 2001). in 2003, the risk of mycotoxins was brought to public attention following the indication of the presence of unusual amounts of afm 1 in milk, in northern italy in particular. at the beginning controls aimed at checking that the levels in milk did not exceed the limit established by law, but special monitoring plans were coordinated for milk and feed towards the end of 2003 due to an alarming amount of positivity in the self-check plan carried out on milk. maybe the positive levels found in feed at the end of 2003 were the consequence of particularly unusual climatic conditions (high temperatures and drought lasting more than four months) that characterized the summer in the year 2003 (decastelli et al., 2007). such approach – i.e. paying particular attention to the correlation in milk-feed monitoring procedures – could be considered particularly valid in order to find contaminated batches starting from controls on milk. in fact, many countries in europe have shown relatively low levels of afm 1 contamination in milk samples as a result of stringent rules on afb 1 in dairy cattle feed (trucksess, 2006). in the present study afm 1 concentrations were not very high and that result could be due to the feeding practices in dairy cow farms. the lower limits adopted by this dairy factory could be particularly effective, above all when the italian ministry of health established an increase in milk analyses in order to detect afm 1 following a series of notifications on afb 1 in maize of european origin by the rapid alert system for food and feed (rasff) since the last maize harvest in autumn 2012 (anonymous, 2012). in order to control afm 1 levels in milk it is necessary to reduce afb 1 contamination of feed for dairy cattle by preventing fungal growth and afb 1 formation in agricultural commodities. that purpose can be achieved through some agricultural practices, such as the choice of hybrids, seeding time and density, suitable ploughing and fertirrigation, and stricter chemical or biological controls. cereals harvested with the lowest possible moisture and conservation moisture close to or less than 14% are necessary to reduce contamination risks. furthermore, kernel mechanical table 2 distribution of afm 1 in raw milk samples. months range of afm 1 concentrations (ng/kg) 5-10 11-19 20-25 number of samples (%) number of samples (%) number of samples (%) january 20 (83.4) 2 (8.3) 2 (8.3) february 23 (95.8) 1 (4.2) march 17 (70.8) 7 (29.2) april 19 (79.2) 5 (20.8) may 16 (66.7) 8 (33.3) june 22 (91.7) 2 (8.3) july 21 (87.5) 3 (12.5) august 15 (62.5) 9 (37.5) september 9 (37.5) 14 (58.3) 1 (4.2) october 9 (37.5) 12 (50.0) 3 (12.5) november 12 (50.0) 8 (33.3) 4 (16.7) december 13 (54.2) 10 (41.6) 1 (4.2) = no sample. fig. 1 mean content of afm 1 in milk per month. ital. j. food sci., vol. 27 2015 275 damage, grain cleaning practices and conservation temperature are also factors which need to be carefully controlled (prandini et al., 2009). a marked seasonal variation in afm 1 levels in milk has been previously reported (kamkar, 2005; rahimi and ameri, 2012; ruangwises and ruangwises, 2010). it has been reported that afs levels in feed are higher in rainy than in dry seasons. moreover, the use of high amounts of contaminated concentrates is more frequent in cold months (kamkar et al., 2011). although no significant differences were observed in afm 1 levels among the different months, except between february and november, the results of the present study shows that the mean concentrations in raw milk samples collected in autumn were higher than in other seasons. such variation may be a result of toxin accumulation when storage occurs in hot and humid conditions. many authors (blanco et al., 1988; lopez et al., 2003; kamkar, 2005) reported on a higher number of yeasts, moulds and consequently on a higher concentration of mycotoxins in ensiled feed, mostly used in autumn or winter. also dashti et al. (2003) observed that the contamination levels in the samples from local companies were higher in winter than in summer. that could be explained by the prolonged storage required for feed, which would provide favorable conditions for fungi to grow; or by the use of contaminated feed for the animals in winter, in addition to other factors such as temperature and relative humidity, agricultural products used as animal feed as well as seasonal effects from the country of origin of feed. two other studies showed similar results – i.e., afm 1 contamination is higher in winter than in summer. the first study was conducted in five regions of iran on ninety-eight samples of raw milk analyzed in order to observe the possible presence of afm 1 . all samples resulted positive for afm 1 with an overall mean level of 53 ng/l. the levels of afm 1 were also higher in winter and spring than in summer and autumn (tajkarimi et al., 2007). the second study was carried out in sarab city, iran, and showed that 76.6% of 111 raw milk samples was positive, with afm 1 levels ranging between 15 and 280 ng/l. the lowest afm 1 levels (24 ng/l) were found in august and the highest (118 ng/l) in december (kamkar, 2005). in conclusion, the occurrence of afm 1 in milk intended for human consumption is a critical control point to be steadily monitored in dairy products. controls of the supply chain from feedstuffs for lactating cows to milk production represents the key to guarantee the safety of the end product, due to the large variation in the content of afb 1 in animal feed and consequently of afm 1 in milk. even if the risk of a high afm 1 content appears limited, it is certainly of great interest to implement a valid system of regular monitoring in order to have always safe raw materials. establishing more restrictive limits, as those chosen by the dairy factory, taken as a case-study in the present paper, could be a good approach to achieve a better food quality for consumers. acknowledgements the authors thank francesca rosati, university of teramo, for her support in editing the english text. references anonymous 2012. rapid alert system for food and feed (rasff). available at: http://ec.europa.eu/food/food/ rapidalert/rasff_portal_database_en.print.htm. battacone g., nudda a., cannas a., cappio borlino a., bomboi g. and pulina g. 2003. excretion of aflatoxin m1 in milk of dairy ewes treated with different doses of aflatoxin b1. j. dairy sci. 86: 2667-2675. blanco j.l., dominguez l., gómez-lucia e., garayzabal j.f., garcia j.a. and suarez g. 1988. presence of aflatoxin m1 in commercial ultra-high temperature treated milk. appl. environ. microbiol. 56: 1622-1623. crappy e.e. 2002. update of survey, regulation and toxic effects of micotoxins in europe. toxicol. lett. 127: 19-28. dashti b., al-hamli s., alomirah h., al-zenki s., abbas a.b. and sawaya w. 2009. levels of aflatoxin m1 in milk, cheese consumed in kuwait and occurrence of total aflatoxin in local 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(ed.), micotoxins in dairy products, elsevier applied science, london and new york, pp. 1155. williams j.h., phillips t.d., jolly p.e., stiles j.k., jolly c.m. and aggarwal d. 2004. human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. am. j. clin. nutr. 80: 1106-1122. paper received february 14, 2014 accepted august 6, 2014 ijfs#1898_bozza ital. j. food sci., vol. 32, 2020 1018 paper effect of harvesting time on hemp (cannabis sativa l.) seed oil lipid composition s. marzocchi* and m.f. caboni department of agricultural and food sciences and technologies, university of bologna, piazza goidanich 60, 47521 cesena, fc, italy *corresponding author: silvia.marzocchi4@unibo.it abstract the most common food using hemp (cannabis sativa l.) is hempseed oil (hso) because it is a rich source of nutrients with nutritional and functional beneficial effects for human body. harvesting time can affect the quality of hso, consequently the aim of this study was to evaluate the composition of lipid fraction, fatty acids, tocopherols and sterols, during ripening. two cultivars, futura 75 and carmagnola, were collected at three ripening stages during august and september 2015 and their lipid composition was determined by analytical techniques. among the fatty acid identified, the linoleic acid was the preponderant, followed by oleic, α-linolenic and palmitic acid. linoleic:α-linolenic acid and polyunsaturated:saturated fatty acid ratios decreased and increased, respectively, in both varieties with ripening. γ-tocopherol was the preponderant tocopherol identified, futura 75 showed the highest content in the middle of maturation while carmagnola at the beginning. β-sitosterol was the predominant sterol identified in both varieties, followed by campesterol, ∆5-avenasterol, stigmasterol and ∆7-stigmasterol. total sterol content increased and decreased with ripening in futura 75 and carmagnola, respectively. the study confirms that ripening stage affects the quality of hempseed oil, important parameter to consider for hemp seed producers. keywords: cannabis sativa l., hempseed oil, fatty acids, tocopherols, sterols ital. j. food sci., vol. 32, 2020 1019 1. introduction cannabis sativa l., belonged to cannabaceae family, is an annual plant known by its long, thin flower and spiky leaves (montserrat-de la paz et al., 2014). cannabis sativa subsp. sativa is characterized by a low content of thc (∆9-tetrahydrocannabinol), it must be lower than 0.2% on dry basis to be cultivated in most european countries (official journal of european union, 2008). industrial hemp with a low thc content has no psychoactive effects (siudem et al., 2019). the most common food using hemp is hempseed oil (hso); it is a rich source of nutrients that provide nutritional and functional support for humans (crescente et al., 2018). hso represents the 25-35% of hemp seed (callaway, 2004; montserrat-de la paz et al., 2014; crimaldi et al., 2017) and it contains more than 80% of polyunsaturated fatty acids (pufas) (petrović et al., 2015; siudem et al., 2019) including essential fatty acids usually not contained in oils used for human diet (petrović et al., 2015); in particular ω-6 linoleic acid (la) and ω-3 α-linolenic acid (ala). in addition, la:ala ratio is 3:1 which agrees with european food safety agency recommendations (efsa, 2009). hso is composed of 1.5-2% unsaponifiable fraction, a source of interesting minor compounds like tocopherols, fat-soluble vitamin d and e (siudem et al., 2019) and phytosterols (montserrat-de la paz et al., 2014). therefore, hso has a lot of beneficial effects: cancer and cardiovascular disease prevention, cholesterol level normalization, blood pressure lowering (devi et al., 2019) and rheumatoid arthritis and dermatitis treatment (oomah et al., 2002; chow, 2008). for all these reasons industries are attracted by hso for drugs, cosmetics, body care products and dietary supplement production (callaway, 2004; kolodziejczyk et al., 2012). to the best of our knowledge, in literature are reported only two studies about the effect of maturation on yield, quality of fiber and oil of industrial hemp. höppner et al. (2007) studied the yield and the quality of fiber and oil of different hemp cultivars in germany at two different harvest times (“intensive flowering” and “initial seed maturity”). the different harvest stages did not have effect on stem diameter, fiber content and yield; on the other hand, seed yield and seed oil content increased with maturation while the γlinolenic acid content decreased. burczyk et al. (2009) studied the effect of sowing density and date of harvest (beginning of panicle forming, full bloom and full seed maturity) on industrial hemp yields. they observed that the maximum yields of biomass, cellulose and fiber can be obtained at 30 kg/ha sowing density at full bloom; while considering hemp for seed or panicles the highest yields were obtained at 10-20 kg/ha of density at full maturity of panicles. the aim of this study was the characterization of lipid fraction from seeds of two different cultivars of cannabis sativa l., futura 75 and carmagnola, harvested at three different harvest stages; in order to investigate the quality of hempseed oil during the maturation, which was never investigated before. to this end, fatty acids, tocopherols and sterols content were determined using fast chromatographic techniques, hplc and gc equipped with different detectors. ital. j. food sci., vol. 32, 2020 1020 2. materials and methods 2.1. fruit harvest and sample preparation cannabis sativa l. seeds from two different varieties, futura 75 and carmagnola, were picked in ancona area in italy approximately 43° 30' 34286" n, 13°15' 33052" e. fruits were harvested at three different harvesting stages during august and september 2015. at first harvest, futura 75 were collected on august 26th (f1) and carmagnola on september 3rd (c1); at the second harvest, futura 75 were collected on september 3rd (f2) and carmagnola on september 15th (c2); and at third harvest, futura 75 were collected on september 15th (f3) and carmagnola on september 23rd (c3). at each sampling time 20 g of seeds were picked from different field’s area; all of them were grounded before analyses. 2.2. oil extraction and moisture content determination the lipid content was obtained by grounding the samples of cannabis sativa l. seeds (5 g) and the oil was extracted with n-hexane in a soxhlet apparatus according to iso method 659:1998. the remaining solvent was removed under vacuum and the oil was taken up with n-hexane/isopropanol (4:1 v/v) solution and stored at −18°c until use. each extraction was carried out two times for each cultivar. moisture content (%) was evaluated on hemp seeds samples in an oven at 105°c until constant weight was receached. for each samples, 3 replicates of 3 g weighted for each extraction were dried (n=6) (aoac, 1995).. 2.3. fatty acid analysis the fatty acid composition was determined as fatty acid methyl esters (fames) by capillary gas chromatography analysis after alkaline treatment (christie, 1982). methyl tridecanoate (c13:0, 2 mg/ml) was used as internal standard and fames were measured on a gc 2010 plus gas chromatograph (shimadzu corporation, kyoto, japan) equipped with a flame ionisation detector (fid) and an aoc-20s auto sampler (shimadzu corporation), at the same conditions reported in marzocchi et al. (2018). peak identification was accomplished by comparing peak retention time with glc-463 standard mixture from nu-check (elysian, mn, usa) and fame 189-19 standard mixtures from sigma-aldrich chemicals (st. louis, mo, usa) and expressed as weight percentage of total fames. fames composition was measured in 3 replicates for each lipid extract (n = 6) and each analysis lasted 7 minutes. 2.4. tocopherols analysis approximately 50 mg of hempseed oil was dissolved in 0.5 ml of n-hexane. after homogenization, the solution was filtered through a 0.2 μm nylon filter and 2.5 μl was injected in a hplc 1200 series (agilent technologies, palo alto, california, usa) equipped with a fluorimeter detector (agilent, palo alto, ca, usa). the excitation wavelength was 290 nm and the emission wavelength was 325 nm. the separation of tocopherols was performed by a hilic poroshell 120 column (100 mm × 3 mm and 2.7μm particle size; agilent technologies, usa), in isocratic conditions, using an n-hexane/ethyl acetate/acetic acid (97.3:1.8:0.9 v/v/v) mobile phase. the flow rate was 0.8 ml/min. tocopherols were identified by co-elution with the respective standards. the calibration ital. j. food sci., vol. 32, 2020 1021 curve used for quantification was constructed with α-tocopherol standard solutions. tocopherols composition was measured in 3 replicates for each lipid extract (n = 6) and expressed in mg/100g of oil; each analysis lasted 8 minutes. 2.5. sterols determination in order to determine the sterols content, 0.5 ml of dihydro-cholesterol (2 mg/ml) was added to 250 mg of oil and saponification was conducted at room temperature (sander et al., 1989). after about 20 h, the organic fraction was washed with 10 ml of diethyl ether and 10 ml of water. the unsaponifiable fraction was further extracted twice with 10 ml of diethyl ether, 10 ml of 0.5 n aqueous koh and 10 ml of distilled water, respectively. the organic solvent was removed under vacuum and the unsaponifiable fraction was used for the sterols analysis. before injection, samples were silylated according to sweeley et al. (1963) and the sterol separation was performed by gc/ms (gcms-qp2010 plus, shimadzu, tokyo, japan) in the same chromatographic conditions reported by cardenia et al. (2012). sterols identification was achieved by comparing peak mass spectra with peaks of standard mixture and by comparing them to the gc–ms data reported by pelillo et al. (2003). an internal standard was used to quantify all the sterols identified in seed samples. sterols composition was measured in 3 replicates for each lipid extract (n = 6) and expressed in mg/100g of oil. 2.6. statistical analysis relative standard deviation was obtained, where appropriate, for all data collected. oneway analysis of variance (anova) was evaluated using statistica 10 software (statsoft, tulsa, ok, usa). the differences between the means of data for the two different cultivars at the three different harvesting stages were compared at the 5% level of significance (p<0.05) using tukey honest significant difference (hsd) test. 3. results and discussion 3.1. oil and moisture content table 1 shows oil content of futura 75 and carmagnola at three different harvest stages expressed on dry basis. futura 75 showed a significantly higher (p<0.05) oil content during the second harvest stage (29.9%) rather than the other two harvestings, 22.9% and 26.4% for the first and the third harvest, respectively. this trend should be related to a non homogeneous maturation, in fact cannabis sativa l. shows a maturation from the bottom to the top of the plant and for this reason is not suitable wait the complete seeds maturation because they tend to fall from the plant. this can be solved operating a constant plant selection to make a more uniform maturation and by adopting appropriate cultivation techniques. in addition, also climatic conditions should be responsible of this trend, in fact cannabis sativa l. shows higher yield in raining condition (madia et al., 1998). on the other hand, carmagnola did not show any significant differences (p<0.05) in oil content during maturation, in fact it was 24.5%, 24.8% and 24.9% at first, second and third harvest, respectively. our results are in line, or slightly lower, with literature, latif et al. (2009), da porto et al., (2012) and kostić et al. (2014) that reported an oil content of 26-32%, 30% and 25-29%, respectively. the lower oil content may be attributed to cultivation ital. j. food sci., vol. 32, 2020 1022 techniques and climatic conditions; in addition, seeds were completely covered by a green skin that certainly influenced the oil content, it took up space which was reflected in the overall oil content. as regard moisture (table 1), an expected reduction in percentage of moisture was recorded in both varieties with the increasing of maturation; it is well known that immature seeds contain a higher water content rather than mature seeds (matthäus et al., 2006). futura 75 starting from a moisture value of 50% at f1, that decreased significantly (p<0.05) at f2 (32%) reaching a final value of 20% at full maturity. moisture content in carmagnola had the same trend, starting from a value of 31% at c1 and decreasing significantly (p<0.05) at c2 (17%) and finally at c3 (10%). moisture value reported in various studies, oomah et al. (2002), tang et al., (2006) and da porto et al. (2012), are much lower than ours; in fact, they reported values of 7.7, 6.7 and 7.8%, respectively. this could be due to the green skin, as previously mentioned, that covered the seeds that certainly had a high impact on moisture content. sacilik et al. (2006) conducted a study on physical properties of hemp seeds (size, density and porosity) as a function of moisture content and found out values more similar to ours (ranging from 8.6 to 20.9%). table 1. oil (%) and moisture content (%) of the two different cultivars of hemp at three harvest stages. futura 75 carmagnola f1 f2 f3 c1 c2 c3 oil content 22.9±0.6 c 29.9±0.6 a 26.4±0.4 b 24.5±0.2 a 24.8±0.2 a 24.9±1.1 a moisture 50.0±1.0 a 32.0±0.4 b 20.7±1.3 c 31.1±2,1 a 17.5±0.2 b 10.0±0.1 c abbreviations: f1, futura 75 first harvest stage; f2, futura 75 second harvest stage; f3, futura 75 third harvest stage; c1, carmagnola first harvest stage; c2, carmagnola second harvest stage; c3, carmagnola third harvest stage. data are reported as mean (n=6)±standard deviation. results of the analysis of variance by tukey’s test are showed: p<0.05, lowercase letters on the same row show significantly different values within each cultivar at three different harvest stages. 3.2. fatty acid profile a total of 17 and 15 fatty acids in futura 75 and in carmagnola, respectively, were identified and quantified by fast gc-fid analysis, within a run time less than 7 minutes. as showed in table 2, the predominant fatty acid in all samples was linoleic acid (c18:2, ω-6), ranging from about 49 to 54%. oleic acid (c18:1 cis9) was the second major fatty acid detected (~15-16%), followed by α-linolenic acid (c18:3, ω-3, ~ 12-15%), palmitic acid (c16:0, ~ 7-9 %), stearic acid (c18:0, ~ 2-3%) and γ-linolenic acid (c18:3, ω-6, ~2-3%). in futura 75, the saturated fatty acids (sfa) were present in significantly (p<0.05) greater amount at the first harvesting time (16.7%), whereas mono-unsaturated fatty acids (mufa) did not showed significant differences among the three different harvest stages (18.7%, 18.3% and 18.1% for f1, f2 and f3, respectively). polyunsaturated fatty acids (pufa), on the other hand, increased significantly (p<0.05) their concentration with hemp maturation, accounting for 64.6%, 69.6% and 69.6% for f1, f2 and f3, respectively. these trends reflect those of the main fatty acid: in fact, pufa was the most abundant class because of linoleic acid concentration. in carmagnola, sfa had a significantly higher (p<0.05) concentration at first harvest stage (12.2%), than the other two (11.8% and 11.6% ital. j. food sci., vol. 32, 2020 1023 in c2 and c3, respectively); while mufa did not show significant differences between first and third harvest stages (18.2% and 18.0%, respectively), with a slightly decrease in the middle stage (16.7%). pufa, on the contrary, showed a significant increase (p<0.05) at the second harvest stage (71.6%), between first and third harvestings (69.7% and 70.5%). comparing our results with literature, sfa content is generally higher than data already reported for hemp native from italy (7%, da porto et al., 2012) and from other countries like croatia (9-11%, petrović et al., 2015), spain (11%, montserrat-de la paz et al., 2014) and turkey (9-10%, kiralan et al., 2010); this because of palmitic and stearic acid content that is higher than reported literature. only in a study conducted by devi et al. (2019) is reported a similar concentration of palmitic acid compared to our results, 10% referred to hemp cultivated in india. mufa content was higher in our study than literature; 12-16% (kiralan et al., 2010), 11% (da porto et al., 2012), 13% (montserrat-de la paz et al., 2014) and 10-14% (petrović et al., 2015). this because of oleic acid, that represent almost the entire mufa content. on the contrary, pufa determined in this study was lower than the already cited literature; in fact, kiralan et al. (2010) showed a 73-78%; da porto et al. (2012) a 80-81%, montserrat-de la paz et al. (2014) a 75% and petrović et al. (2015) a 74-80%. for this reason, the ratio between unsaturated and saturated fatty acids reported in our study, 3-5 in futura 75 and 5-6 in carmagnola is slightly lower than that reported in literature (6,7 showed by montserrat-de la paz et al., 2014). in general, this characteristic high ratio between unsaturated and saturated fatty acids can reduce serum cholesterol and atherosclerosis; and prevent heart diseases (reena et al., 2007). on the other hand, because of its high unsaturation level and susceptibility to oxidation, hemp seeds oil has a short shelf life (kiralan et al., 2010) and it is not suitable for hot uses (da porto et al., 2012). as regard to the ratio between the two essential polyunsaturated fatty acids, linoleic and α-linolenic acid, both varieties, futura 75 and carmagnola, showed the highest value at the first harvest, 4.05 and 3.90, respectively (table 2). this even though linoleic and α-linolenic acids did not have the same trend in the two different varieties. in futura 75 linoleic acid showed a significant (p<0.05) increase from the beginning to the end of maturation (49.2%, 52.3% and 52.5% for f1, f2 and f3); in carmagnola, instead, its concentration increased significantly (p<0.05) from first (53.7%) to second harvest (54.7%) and then decreased again at the third harvest stage (53.9%). α-linolenic acid, instead, in futura 75 increased significantly (p<0.05) between first (12.1%) and second harvest (14.3%), before decreased again at third harvest stage (13.9%); in carmagnola its concentration increased significantly during all the maturation; 13.8%, 14.6% and 14.9% for c1, c2 and c3, respectively. the high quantity of α-linolenic acid improves hemp oil quality for its positive nutritional implications and beneficial effects against coronary disease and cancer (arshad et al., 2011; fretts et al., 2013). in general, our results about these two fatty acids are slightly lower than results in literature. in fact linoleic acid content is reported in a range between 48 and 59% and α-linolenic acid in a range between 16 and 26% (montserrat-de la paz et al., 2014; orsanova et al., 2015; petrović et al., 2015; mikulcová et al., 2017; devi et al., 2019; siudem et al., 2019). 3.3. tocopherols the individual tocopherols identified are showed in table 3; a total of 49.8, 86.9 and 41.2 mg/100g of oil were quantified in f1, f2 and f3, respectively; while a total of 94.8, 87.7 and 77.8 mg/100 g of oil were quantified in c1, c2 and c3, respectively. it is recognized ital. j. food sci., vol. 32, 2020 1024 that tocopherols are the most important natural antioxidants because of their free radical scavenge activity, involving a tocopherol-tocopheryl semiquinone redox system (montserrat-de la paz et al., 2014). tocopherols, as well as phenols compounds, have shown different beneficial effects, on degenerative diseases such as atherosclerosis, cardiovascular disease, alzheimer’s disease and certain type of cancer (fromm et al., 2012). table 2. fatty acids composition (mg/100 mg fames) of the two different cultivars of hemp at three different harvest stages. futura 75 carmagnola f1 f2 f3 c1 c2 c3 c14:0 0.1±0.0 a 0.1±0.0 a 0.1±0.0 a n.d. n.d. n.d. c16:0 9.3±0.8 a 7.7±0.1 b 7.5±0.2 b 7.4±0.1 a 7.3±0.1 c 7.3±0.1 b c16:1 t 0.1±0.0 a 0.1±0.0 a 0.1±0.0 a n.d. n.d. n.d. c16:1 c 0.2±0.0 a n.d. n.d. 0.1±0.0 a 0.1±0.0 a 0.1±0.0 a c17:0 n.d. 0.05±0.0 ab 0.0±0.1 a 0.04±0.3 a 0.06±0.0 a 0.07±0.0 a c18:0 3.4±0.5 a 2.7±0.1 b 2.9±0.1 ab 3.3±0.1 a 3.0±0.0 b 3.0±0.2 c c18:1 t n.d. n.d 0.03±0.0 a 0.2±0.1 a 0.1±0.0 a 0.12±0.0 a c18:1 c9 16.7±0.6 a 16.7±0.3 a 16.3±0.9 a 16.5±0.2 a 15.1±0.1 b 16.4±0.1 a c18:1 c11 1.1±0.1 a 1.0±0.1 a 1.0±0.2 a 0.9±0.1 a 0.9±0.1 a 0.8±0.1 a c18:2, ω-6 49.2±1.4 b 52.3±0.2 a 52.5±0.6 a 53.7±0.3 b 54.7±0.1 a 53.9±0.1 b c18:3, ω-6 2.9±0.1 a 2.9±0.1 a 3.0±0.1 a 2.2±0.1 a 2.2±0.1 a 1.5±0.1 b c18:3, ω-3 12.2±0.4 b 14.3±0.1 a 13.9±0.2 a 13.8±0.1 c 14.6±0.1 b 14.9±0.1 a c20:0 1.3±0.1 a 0.9±0.2 b 1.1±0.1 a 1.0±0.2 a 0.9±0.1 b 0.9±0.1 c c20:1 c 0.7±0.3 a 0.5±0.0 a 0.7±0.1 a 0.4±0.1 b 0.5±0.1 a 0.4±0.0 b c20:2, ω-6 n.d. 0.03±0.0 a 0.04±0.0 a 0.02±0.0 a 0.02±0.0 a 0.05±0.0 a c20:3, ω-6 0.4±0.1 a 0.1±0.0 b 0.2±0.0 ab 0.02±0.0 b 0.1±0.0 ab 0.1±0.0 a c22:0 1.9±0.7 a 0.6±0.1 b 0.7±0.1 b 0.5±0.0 a 0.5±0.0 a 0.4±0.0 b sfa 16.7±1.6 a 12.1±0.2 b 12.3±0.2 b 12.2±0.1 a 11.8±0.1 b 11.6±0.1 c mufa 18.7±0.5 a 18.3±0.2 a 18.1±0.7 a 18.2±0.3 a 16.7±0.1 b 18.0±0.1 a pufa 64.6±2.0 b 69.6±0.2 a 69.6±0.8 a 69.7±0.4 c 71.6±0.1 a 70.5±0.1 b omega-6 52.5±2.4 55.3±3.2 55.7±2.9 55.9±1.8 57.0±1.3 55.6±2.4 la/ala 4.05 3.66 3.76 3.90 3.75 3.61 pufa/sfa 3.87 5.66 5.65 5.70 6.08 6.06 abbreviations: f1, futura 75 first harvest stage; f2, futura 75 second harvest stage; f3, futura 75 third harvest stage; c1, carmagnola first harvest stage; c2, carmagnola second harvest stage; c3, carmagnola third harvest stage. data are reported as mean (n=6)±standard deviation. results of the analysis of variance by tukey’s test are showed: p<0.05, lowercase letters on the same row show significantly different values within each cultivar at three different harvest stages. as expected, considering the literature, in all samples γ-tocopherol was the predominant compound followed by β-tocopherol, α-tocopherol and α-tocotrienol. in futura 75 γtocopherol showed a significative increase (p<0.05) between first and second harvest (40.8 and 82 mg/100 g of oil in f1 and f2, respectively) and then significantly (p<0.05) decrease ital. j. food sci., vol. 32, 2020 1025 at last maturation time (f3, 37.1 mg/100 g of oil). the other tocopherols were present in very low concentration; in fact, β-tocopherol was present only at the first harvesting stage (9.0 mg/100 g of oil) and α-tocotrienol just in the last one (3.3 mg/100 g of oil). αtocopherol was not detected at first maturation stage, but only in the other two, with a significative (p<0.05) decrease between them, 4.9 and 0.8 mg/100 g of oil for f2 and f3, respectively. for this reason, the total tocopherols content had the same trend of γtocopherol, increasing significantly (p<0.05) the total concentration at the second harvest stage (f2) reaching a value about 87 mg/100 g of oil (table 3). this trend is related to oil content trend, in fact it increased at the second maturation stage, increasing the total tocopherols content. also in carmagnola γ-tocopherol was the predominant tocopherol with a significative decrease (p<0.05) during maturation, 90.2, 81.8 and 72.9 mg/100 g of oil for f1, f2 and f3, respectively. β-tocopherol, on the contrary, was not detected during the entire maturation. α-tocopherol was present in every maturity stage with a significant increase (p<0.05) in the middle of maturity; 3.5, 5.9 and 4.9 mg/100 g of oil in c1, c2 and c3, respectively. finally, α-tocotrienol was present just at the beginning of maturity (c1) with a concentration of 1.1 mg/100 g of oil. total tocopherol concentration follows γtocopherol trend, so a constant significative decrease during maturation was recorded; 94.8, 87.7 and 77.8 mg/100 g of oil in c1, c2 and c3, respectively. the total tocopherols content showed a different trend in the two cultivars analyzed and considering they were cultivated in the same field this is related to the cultivars different origin and their gene pool. our results agree with literature where γ-tocopherol represents the 90% of the total tocopherols content (oomah et al., 2002; anwar et al., 2006; latif et al., 2009; montserrat-de la paz et al. 2014). in a study conducted by kriese et al. (2004), where hempseed oil was extracted by supercritical fluids, the general tocopherol content was much lower than ours, so, in addition to botanical characteristics, climatic and cultivation conditions, also the extraction method affects the tocopherols content. table 3. tocopherols content (mg/100 g of oil) of the two different cultivars of hemp at three different harvest stages. futura 75 carmagnola f1 f2 f3 c1 c2 c3 α-tocopherol n.d. 4.9±0.9a 0.8±0.1 b 3.5±1.0 b 5.9±0.1 a 4.9±0.2 b α-tocotrienol n.d n.d 3.3±0.1 a 1.1±0.1 a n.d. n.d. β-tocopherol 9.0±0.3 n.d n.d n.d n.d n.d γ-tocopherol 40.8±1.8b 82.0±4.9 a 37.1±8.2 b 90.2±4.8 a 81.8±3.8 ab 72.9±2.0 b total 49.8±0.1 b 86.9±5.7 a 41.2±1.0 b 94.8±6.7 a 87.7±3.8 ab 77.8±0.9 b abbreviations: f1, futura 75 first harvest stage; f2, futura 75 second harvest stage; f3, futura 75 third harvest stage; c1, carmagnola first harvest stage; c2, carmagnola second harvest stage; c3, carmagnola third harvest stage. data are reported as mean (n=6)±standard deviation. results of the analysis of variance by tukey’s test are showed: p<0.05. lowercase letters on the same row show significantly different values within each cultivar at three different harvest stages. 3.4. sterols analysis of the trimethylsilyl derivatives of phytosterols led to identify nine compounds in both varieties of cannabis sativa l. such campesterol, campestanol, stigmasterol, ital. j. food sci., vol. 32, 2020 1026 clerosterol, β-sitosterol, sitostanol, ∆5avenasterol, ∆5-24-stigmastadienol and ∆7stigmasterol (table 4). the total sterols content showed different trend in the two varieties of cannabis sativa l. considered; in fact, it significantly increased (p <0.05) in futura 75 during maturation (642.8, 679.1 and 913.4 mg/100 g of oil in f1, f2 and f3, respectively) and, on the other hand, significantly decreased in carmagnola (532.7, 518.7 and 456.1 mg/100 g of oil in c1, c2 and c3, respectively). these trends reflect the β-sitosterol trends, the predominant sterol detected. in fact, it represented the 63% of the total content in both varieties, but its concentration significantly increased and decreased in futura 75 and carmagnola, respectively, with maturation (table 4). in futura 75 it had an initial increase about 5% from f1 to f2 and about 36% between f2 and f3; in carmagnola, on the other hand, β-sitosterol decrease about 6% from first to second harvest stage and then, again about 10%, between second and third harvest. in futura 75 campesterol, campestanol, stigmasterol and clerosterol had the same trend reported for β-sitosterol; instead the other sterols detected decreased between the first and the second harvest stage and then increased at full maturity (table 4). in carmagnola, instead, campesterol and clerosterol decreased significantly (p<0.05) during the maturation; campestanol and stigmasterol did not show significant differences during the three harvesting stages. table 4. sterols content (mg/100 mg of oil) of the two different cultivars of hemp at three different harvest stages. futura 75 carmagnola f1 f2 f3 c1 c2 c3 campesterol 74.5±2.5 c 88.6±4.5 b 123.6±17.0 a 66.6±4.6 ab 69.8±1.3 a 61.8±0.4 a campestanol 4.5±0.6 b 6.9±2.9 a 8.2±1.4 a 2.4±0.8 a 3.7±0.7 a 3.3±0.3 a stigmasterol 34.1±1.3 b 33.7±3.5 b 41.8±1.9 a 16.1±2.7 a 16.2±1.5 a 15.7±1.6 a clerosterol 6.1±0.5c 6.3±0.3 b 8.6±0.4 a 4.3±0.7 a 3.9±0.8 a 3.4±0.3 a β-sitosterol 402.1±4.3 c 423.7±6.6 b 575.9±6.1 a 344.1±3.9 a 324.2±4.7 b 293.6±1.4 c sitostanol 30.3±1.7 a 27.5±3.6 a 33.1±5.3 a 14.8±0.7 a 11.3±0.4 b 14.2±0.2 a δ5-avenasterol 50.6±2.3 c 54.6±1.2 b 71.8±19.0 a 47.3±2.1 a 52.8±8.5 a 38.8±0.8 b δ5-24-stigmastadienol 8.7±0.3 a 8.4±0.6 a 9.0±0.8 a 7.2±0.7 a 6.4±0.3 a 4.8±0.1 b δ7-stigmasterol 31.9±0.9 b 29.4±0.3 b 41.5±1.8 a 29.8±1.3 a 30.4±6.1 a 23.1±0.2 b total 642.8±14.4 b 679.1±21.5 b 913.4±35.7 a 532.7±17.5 a 518.7±34.3 a 456.1±0.8 b abbreviations: f1, futura 75 first harvest stage; f2, futura 75 second harvest stage; f3, futura 75 third harvest stage; c1, carmagnola first harvest stage; c2, carmagnola second harvest stage; c3, carmagnola third harvest stage. data are reported as mean (n=6)±standard deviation. results of the analysis of variance by tukey’s test are showed: p<0.05. lowercase letters on the same row show significantly different values within each cultivar at three different harvest stages to the best of our knowledge, only montserrat-de la paz et al. (2014) reported a study of sterols in hemp seed oil. our results were higher than what is reported by these authors for spanish hempseed oil (total content of 279.4 mg/100 g of oil) but β-sitosterol (190.5 mg/100 g of oil) and campesterol (50.6 mg/100 g of oil) were the same predominant sterols. comparing sterols composition of hso with other oils it is possible to see some differences. considering other vegetable oils, olive oil, linseed oil and hazelnut oil contain ital. j. food sci., vol. 32, 2020 1027 an higher amount of campesterol, β-sitosterol and ∆5-avenasterol than hso. campesterol reachs values of 40, 50-95, 785 mg/kg; stigmasterol values of 20, 10-18, 343 mg/kg; βsitosterol values of 750, 1050-1700, 1600 mg/kg and ∆5-avenasterol value of 40-140, 20-80, 369 mg/kg in olive oil, hazelnut oil (azadmard-damirchi et al., 2015) and linseed oil (matthäus et al., 2017), respectively. in addition, linseed oil contains cholesterol, brassicasterol and 5,24-stigmasterol that are not present in hso. on the other hand, hso is richer in sterols that sunflower oil, in fact campesterol, stigmasterol, β-sitosterol and ∆5avenasterol report value of 20, 28, 186 and 20 mg/100g, respectively (yilmaz et al., 2019). corn oil has the same stigmasterol content of hso (about 33 mg/100g); is poorer in βsitosterol than hso (266 mg/100 g) and, on the other hand, is richer in campesterol (191 mg/100 g) (yang et al., 2018). in general, even if sterols are minor constituents of vegetable oils and are present in the unsaponifiable fraction (gusakova et al., 1998), it is well known that they have a lot of beneficial effects on human health. in fact, they can reduce the serum level of cholesterol concentration, atherosclerotic risk (ntanios et al., 2003; patel et al., 2006), low-density lipoprotein cholesterol and they are related to a lower risk of myocardial infarction (klingberg et al., 2013). 4. conclusion this study confirms that ripening stage affects the quality of hempseed oil extracted from cannabis sativa l.; oil content was constant during the maturity only in cultivar carmagnola and moisture content decreased constantly for both cultivars; essential fatty acids (la and ala) increased with ripening while tocopherols decreased. sterols showed a different trend in the two varieties considered, in futura 75 the total concentration increased and in carmagnola decreased. knowledge of the influence of ripening stage on hempseed oil quality has important consequences for industrial output, as harvest could be programmed when oil, tocopherols and sterols are most abundant. futura 75 seemed to have a higher concentration of oil, sterols and la/ala ratio; while carmagnola a higher concentration of tocopherols and pufa/sfa ratio. the comparison between the two varieties and the results obtained are very important for hemp seeds producers since they can choose the best variety to plant from a production and quality point of view aknowledgements the authors are 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74:26-32. yilmaz e. and erden a.k. 2019. purification of degummed crude sunflower oil with selected metal-organic frameworks as adsorbents. grasas y aceites 70(4):e323. doi: doi.org/10.3989/gya.0930182 paper received may 18, 2020 accepted august 5, 2020 156 issn 1120-1770 online, doi 10.15586/ijfs.v33i2.2040 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (2): 156–165 p u b l i c a t i o n s codon determining consumption preferences of consumers considering quality attributes of drinking water: case of iğdır emine aşkan1,*, yavuz topcu2, ayça nur şahin1 1department of agricultural economics, college of agriculture, iğdır university, iğdır, turkey; 2department of agricultural economics, college of agriculture, ataturk university, erzurum, turkey *corresponding author: emine aşkan, department of agricultural economics, college of agriculture, iğdır university, iğdır, turkey, email: emine.askan@igdir.edu.tr received: 22 march 2021; accepted: 15 july 2021; published: 9 august 2021 © 2021 codon publications open access paper abstract the aim of the study was to determine the main factors affecting the consumption preferences of consumers by considering the quality characteristics of drinking water according to the regions where they reside. for this purpose, the data obtained from 400 consumers living in the central districts of iğdır province were used for factor analysis and two-step clustering analysis. research results reported that physiological needs of the consumers residing in region i were based on the physiological and physical quality of the water in their drinking water consumption preference, consumers in region ii relied on the chemical quality of the tap water, and consumers in region iii focused on the cost advantages of tap water depending on the chemical quality of tap water. therefore, supplying differentiated bottled drinking water in hygienic conditions according to physical and physiological quality standards of drinking water for consumers in region i, ensuring the protection and access of local water resources suitable for chemical quality characteristics to consumers in region ii, and maintaining the reliability of local water resources with good chemical quality and low cost of supply and sustainability of their use for consumers in region iii can have positive effects on consumption satisfaction. keywords: drinking water consumption; factor analysis; two-step cluster analysis; water quality introduction water is one of the most important natural resources for all living creatures that have no substitute as a natural resource. it is the second most important indispensable component after oxygen for the existence of human life on earth. oceans and seas make up 97.5% of the world’s water, while 2.5% is fresh water. about 87% of fresh water cannot be used without treatment (yılmaz and peker, 2013). water resources are scarce and the world population which constantly demands these resources is increasing rapidly. in addition, industrialization weakens the sustainability of natural resources and makes it difficult for people to access safe drinking water (tanellari et  al., 2015). organizations such as the united nations world water council (uncww), the world resources institute (iwr), and the world health organization (who) stated that in the 1950s, only a few countries had water problems; in the 1990s, 26 countries with 300 million people suffered from thirst; and by 2050, there will be severe water shortage in 66 countries where 2/3 of the world’s population lives (anonymous, 2015). on the other hand, the recent global climate changes and global warming, as well as the developments in mining and waste recycling technologies, significantly reduce and pollute both the water resources used for agricultural purposes and the water supply resources mailto:emine.askan@igdir.edu.tr italian journal of food science, 2021; 33 (2) 157 drinking water consumption preferences plain. in all wells drilled in a shallow aquifer, the ec and total salt values are above the permissible values. in addition, most of them have ca, mg, so4, cl, and hardness values above the permissible values. for this reason, shallow well waters were not found suitable for drinking, and chemical heavy metal analyzes are carried out by regularly taking water samples from gaziler, acıçay, hamurkesen, yazlık village, gürgöre, and hıdırlı spring waters in the iğdır province. although there are no pollutants in the mentioned water resources, considering the heavy metal contents of the sources other than the hamurkesen spring, they were not found suitable to be used as drinking and utility water. therefore, this research area constitutes a rare micro-area used as drinking water in terms of underground resources in turkey and is immediately accepted as the only sampling region at the national level (anonymous, 2015). due to all these negative reasons, people living in iğdır are trying to make the water usable safely by using personal purifiers in both apartments and houses in order to use the city tap water. however, various spring waters, which have a very high usage area, are far from meeting the needs of consumers, by means of purifiers. for this reason, consumers use well water as drinking water without objective measurements regarding the usability and safety of water as drinking water according to usable quality criteria. in addition to this, initiatives for ready water supply also constitute a very important domestic market in iğdır. in this case, consumers, who have to meet their water consumption needs, exhibit different attitudes and behaviors in terms of their residential areas, taking into account the effects of both water resources and drinking water on health. when the factors affecting the water consumption of consumers are examined, the number of households, the behavior of the consumers, the existence of the garden and swimming pool of the household, and the plant type in the garden are reported as important factors. at the same time, it has been determined that in regions where water scarcity is experienced, besides water price and income, social and demographic characteristics of consumers are effective (domene and saurí, 2007). in addition, it has been analyzed that body weight and temperature and physical activities have an effect on water consumption rates (şen and altunkaynak, 2009). in the study conducted by arslan and mendeş in 2004, it was reported that gender did not affect water consumption and that there was no conscious water consumption in the society (arslan and mendeş, 2004). milton et al. (2006) reported that drinking water is exposed to various pollutants (such as arsenic, lime, and metal-based minerals), making it difficult to access healthy water supply sources, and therefore the amount of drinking water consumption is significantly reduced. available to people. when countries are classified according to water availability, countries with less than 1000 m3 of usable water per capita per year are considered “water poor,” countries with less than 2000 m3 of water are considered “water scarce,” and countries with more than 8000–10,000 m3 are considered “water rich” (dpt, 2007). according to the world average, usable annual amount of water per person is 7.600 m3 (anonymous, 2015). turkey is seen as a country that suffers from water scarcity when approximately 1.519 m3 of usable fresh water per person is taken into account. the total amount of usable water in turkey is around 112 billion m3. the amount of water per person per day for drinking and use in turkey is 216 l (tuik, 2020). the water footprint of an individual, society, or line of business is the total amount of clean water resources used for the production of goods and services consumed by the individual or society, or used by the producer for the production of goods and services. according to the water footprint approach, it is stated that the daily direct and indirect water consumption of a person in turkey is 5416 l (wwf report, 2014). the rapid increase in both the number and population of cities in turkey has made it difficult to meet the water needs of the cities only from available sources and groundwater. for this reason, the water needs of rapidly growing cities are tried to be met by purification from rivers, dams, and lakes, as well as spring and groundwater (özgüler, 1997). it may be possible that the water is supplied from an unhealthy source or the water transferred from a healthy water source is exposed to various microbiological contaminants before reaching the consumer. on the other hand, both natural resource destruction with chemical pesticides and genetic modifications, and improper practices in mining operations as well as factory waste and polluted air emissions cause intensive pollution of the environment and groundwater resources. it is imperative that these contaminated water resources reach usable drinking and utility water standards in terms of chemical, physical, and microbiological water quality (li and wu, 2019; liu et al., 2017; zlatanovic et  al., 2017). the process of distillation and purification of heavily polluted drinking water with various technological approaches and the grading of drinking water with various quality characteristics and its presentation to consumers are of great importance. limit values of usable fresh water are determined according to hygiene, toxic, technical, and aesthetic criteria. in addition, heavy metals, calcium, magnesium, and nitrate values must be reached to levels that will not harm human health (kılıç, 2008). wells were drilled in the shallow aquifer (6–10 m) and in  the deep aquifer during the studies carried out to obtain information about the groundwater in the iğdır 158 italian journal of food science, 2021; 33 (2) aşkan e et al. with the help of following equation (newbold, 1995; şahin, 2019). σ − = − + − p 2 p n (1 p) n (n 1) p(1 p) in equation; n: sample size, n: iğdır city center population (person), r: deviation from the mean (margin of error) (5%), zα/2: z table value (1.96), p: the proportion of those who prefer tap drinking water (50%), σ2p: major population variance. 2 2 2 2 p /2 r 0.05 (0.0255) z 1.96α σ    = = =       it is calculated as, 2 137 613 0.5 0.5 n 384 consumers (137 612 (0.0255) ) (0.5 0.5) × × = = × + × in order to reach the maximum sample volume in the study, p: 0.50 and q: 0.50 were taken. on the other hand, taking into account the number of people in the neighborhoods to be surveyed in each region, how many surveys will be conducted in which neighborhoods with proportional method was determined and given in table 1. during data collection, face-to-face interviews were conducted with 404 consumers by increasing the number of questionnaires by 5% in order to compensate for errors caused by the respondent or the researcher. while counting and cleaning the survey data, the questionnaires containing incorrect and missing data were removed from the data set and the survey data of 400 consumers in total were coded and made ready for analysis in the digital environment. methods used in the preparation of the questionnaires participants of the survey were asked to respond to each statement indicating the significance level of their drinking water consumption preferences. a likert-type scale was used (where 1 refers to the least important attribute, and 5 refers to the most important attributes). the questions related to the drinking water consumption preferences of the consumers consisted of 52 variables belonging to main factors such as physiological, physical, and chemical quality attributes, confidence, and advantages of tap water (table 2). on the other hand, the questions with open and closed-ended scale types for the socioeconomic and demographic factors influencing their drinking water consumption preferences were used (table 3 and table 4). consumers who believe that the drinking water provided by the local and administrative administrations in iğdır is not at a sufficient level in terms of purification from harmful substances, hygiene, quality, and reliability, integrates purification devices into the water lines within the residence, tends to use bottled drinking water, and turns to neighborhood fountains or well waters that they find reliable. among the factors that determine the behavior of consumers towards these alternatives, factors such as physiological effect; chemical, physical, and microbiological quality of water; hygiene; safety of water; access to spring or tap water; physical quality and preservation of tap water; and reliability of drinking water distribution networks are of great importance (dönderici et al., 2010; li and wu, 2019; liu et al., 2017; zlatanovic et al., 2017). consumers residing in different regions of the province of iğdır experience significant problems in accessing network, spring, and ready-made bottled drinking water with alternative usable physical and chemical quality. therefore, in the research, the following question appears: which quality characteristics determine the drinking water consumption of consumers residing in different regions of iğdır province, ready-made water, tap water, tap water connected to the purification device, or spring (fountain) water? in addition to answering the question, this research has been planned in order to help local and administrative administrations and water supply retailers create strategic plans for target audiences. materials and methods material the primary data of the study were obtained from faceto-face surveys conducted with households residing in the city center of iğdır in 2019 (table 1). on the other hand, secondary data obtained from the various domestic and foreign researches and reports, from the public institutions and organizations such as turkey statistics institution (tsi) and fao, and iğdir municipality and governorship records were also used. method sampling method taking into account the 2018 address based population registration system (adnks), it was determined that the central population of iğdır was 137.613 people (turkstat, 2019), which was accepted as the main population. the sample size, which takes into account the probability of the population, whose main population has been determined, to benefit from the local drinking water source, was calculated using the simple random sampling method based on population proportions, italian journal of food science, 2021; 33 (2) 159 drinking water consumption preferences table 1. number of surveys conducted in the neighborhoods of iğdır city center. name of the neighborhood number of surveys 14 kasım neighborhood 28 ali kamerli neighborhood 9 atatürk neighborhood 14 bağlar neighborhood 50 cumhuriyet neighborhood 23 emek neighborhood 36 hakveyis neighborhood 4 karaağaç neighborhood 30 konaklı neighborhood 24 özgür neighborhood 42 pir sultan abdal neighborhood 17 söğütlü neighborhood 27 topcular neighborhood 37 yeni neighborhood 6 bahçelievler neighborhood 5 özvatan neighborhood 6 said nursi neighborhood 7 turgut özal neighborhood 4 yunus emre neighborhood 8 faruktoka neighborhood 4 yeni neighborhood 4 güneşli neighborhood 6 mehmet akif ersoy neighborhood 5 namık kemal neighborhood 4 total number of surveys 400 methods used in the statistics analyses after editing and coding, factor analysis (fa) was first used to determine the main factors related to the product attitudes affecting the consumers’ drinking water consumption preferences and purchase patterns. fa is a data technique that reduces the number of variables used in analysis by creating new factors that combine redundancy in the data (kurtuluş, 2004; spss, 20.0, 2020; tekin, 2007). the first step in fa is to determine the number of relevant factors. this was conducted by fa using the varimax rotation method under an orthogonal rotation method minimizing the number of variables that have high loading on each factor (karagöz, 2017; karpati and szakal, 2009). fa was used initially to identify the underlying aspect explaining a correlation among a set of the drinking water attributes. the purpose of fa was to identify those attributes accounting for a relatively large proportion of the variance in the sample. in the second step of the statistical analyses, the main factors obtained from fa were used for two-step cluster analysis. two-step cluster analysis, which gives the ideal number of clusters between the factors obtained and the consumption groups desired to be formed, is the most effective clustering technique (doğan, 2008; özdamar, 2004; şekerler, 2008; topcu and baran, 2017). in the present study, therefore, the target consumers were separated to three homogenous clusters as regions i, ii, and iii by considering the relationships between the main factors obtained from fa and the location of the neighborhoods according to the distances to city center where the consumers who prefer clean drinking water resided in iğdır province. in the final step of the statistical analysis, descriptive statistics such as compare means, crosstabs, descriptive and frequencies were used to determine demographic and socioeconomic profiles of three consumer clusters segmented by two-step cluster analysis. all analyses were run in sequence with the spss 20.0 statistical software. these techniques were frequently used to analyze the product attributes playing the important roles on the consumers’ consumption preferences in marketing studies (drugova et al., 2020; egbueri et al., 2020; harwood et al., 2020; kusa et al., 2020; topcu, 2006; topcu and baran, 2017). results and discussion the results of fa related to consumers’ drinking water consumption preferences kaiser normalization (kmo), which compares the observation and partial correlation coefficients, including the consumers’ attitudes and behavior towards drinking water consumption preferences, was calculated as 0.723. on the other hand, bartlett’s sphericity test statistics was calculated as 2529.433 (p = 0.000). these two statistics evaluating the sample data showed that the data related to the factors affecting the consumers’ drinking water consumption preferences was at a good level for fa. of the 52 variables, 33 were excluded from the data set, and 19 variables were used in fa. because the 33 variables had not the sufficient loads to explain the common variance, overlap and factor dimensions on the consumers’ drinking water consumption preferences. considering eigen-values greater than 1 and the explanatory variance rates in the factor dimensions created by 19 variables effecting their preferences, 5 main factors were obtained, which explained approximately 62% of the total variance (table 2). physiological quality of water as the first preference factor explained 15.72% of the total variance. it indicated that people should drink at least 2 l water a day 160 italian journal of food science, 2021; 33 (2) aşkan e et al. table 2. the results of fa and fit statistics factor interpretations and variables factor and variable loads* f1 f2 f3 f4 f5 physiological quality of water effect of water on digestion 0.786 0.102 −0.023 0.011 0.011 drinking water aids weight loss and enhances skin 0.786 0.087 −0.035 −0.013 0.049 physiological functions of water in the body 0.782 0.064 −0.021 −0.030 0.079 drinking water removes toxins and cleanses the blood 0.659 0.039 0.124 0.021 −0.022 at least 2 l water should be consumed daily for health. 0.573 0.019 −0.032 0.003 0.153 men and women should daily drink 3 and 2.5 l water, respectively 0.558 0.033 −0.014 0.019 0.005 physical quality of water muddy water after tap water outage 0.068 0.773 −0.002 −0.046 0.034 purified water tastes better and doesn’t contain harmful chemicals. 0.080 0.725 −0.304 −0.191 0.079 packaged waters are clearer and cleaner 0.118 0.707 −0.111 −0.142 0.123 tap water is sometimes yellow and smells bad 0.049 0.700 0.074 0.129 −0.005 confidence in tap water we purify the tap water and drink it more safely −0.039 −0.038 0.875 −0.026 −0.123 purified water is much healthier than bottled water 0.016 −0.090 0.817 −0.011 −0.034 purified tap water tastes more pleasant 0.032 −0.063 0.786 −0.084 0.008 chemical quality of water the amount of calcium in quality water is close to 250 mg 0.012 −0.198 −0.005 0.881 0.100 the amount of magnesium in quality water is close to 75 mg −0.074 −0.234 0.041 0.855 0.156 the ph of treated water is basic 0.083 0.290 −0.201 0.672 0.029 advantage of tap water low cost and availability of tap water 0.094 −0.016 −0.072 0.094 0.876 the fact that the tap water is cold in summer and winter 0.031 0.033 −0.024 −0.011 0.865 transfer difficulty of tap water 0.116 0.201 −0.055 0.193 0.656 factorization statistics eigen-value 2.987 2.370 2.230 2.090 2.051 explained variance (%) 15.719 12.475 11.737 10.999 10.796 cumulative explained variance (%) 15.719 28.194 39.932 50.931 61.727 fit statistics kmo (kaiser–meyer–olkin) statistic 0.723 [chi-square (λ 2df : 171): 2529.43] (p = 0.000) 400 bartlett’s test of sphericity number of samples (n) *bold numbers indicate the largest loading for each variable for a healthy and balanced diet, because water helps the digestive system to work better and cleans the blood by removing harmful chemicals and toxins from the body, is the main substance and vitality source in all physiological functions in the body, makes the skin beautiful, and helps people to lose weight. previous researches, therefore, declared that men and women should drink 3 and 2.5 l water , respectively (cuvelier and bartell, 2021; i̇brahim et al., 2021). explaining the physiological effects of drinking water on human health in all its dimensions, mukate et al. (2019) evaluated the direct and indirect effects of various problems of unsuitable drinking of water in human physiology with many dimensions. on the other hand, water’s physical quality as the second factor explained 12.48% of the total variance. it consisted of variables such as the water provided by the treatment processes not affecting drinking water’s chemical quality, the packaged water from the source, the tap water’s turbid flow, and sometimes being yellowish after being interrupted and not being as pure and clean as it appeared (table 2). the consumers, therefore, gave much italian journal of food science, 2021; 33 (2) 161 drinking water consumption preferences table 3. final cluster center scores and sample numbers in each cluster main factors drinking water consumption preference groups* region i region ii region iii physiological quality of water 0.08*** 0.06*** −0.17*** physical quality of water 0.26*** −0.08*** 0.03*** confidence in tap water −0.11*** 0.07*** −0.05*** chemical quality of water −0.12*** 0.09*** 0.07*** advantage of tap water −0.01*** −0.06*** 0.08*** number of samples in each segment 148 132 120 sample percent in each cluster (%) 37 33 30 *bold numbers indicate the largest final cluster center scores for each factor. ***according to f statistics, the final cluster center scores were found very importance (p<0.01). table 4. demographic and socioeconomic characteristics of the participants in each cluster. demographic and socioeconomic factors drinking water consumption preference groups total region i region ii region iii gender male 57 66 74 197 female 63 66 74 203 occupation white-collar 36 35 44 115 worker 23 24 18 65 self-employment 6 10 14 30 housewife 28 27 26 81 student 15 19 19 53 household (person) mean 4.06 3.93 3.90 3.96 n 120 132 148 400 std. deviation 1590 1514 1446 1512 age (year) mean 37.52 34.27 37.71 36.51 n 120 132 148 400 std. deviation 15.13 13.06 14.06 14.13 education (year) mean 10.58 11.05 11.21 10.97 n 120 132 148 400 std. deviation 4415 4136 4355 4299 income (tl) mean 4295.59 4218.03 4697.01 4418.52 n 120 132 148 400 std. deviation 2226.88 2096.33 2411.85 2260.86 food expenditure (tl) mean 672.92 645.83 659.32 658.95 n 120 132 148 400 std. deviation 265.52 296.61 268.76 276.84 total expenditure (tl) mean 2389.04 2333.63 2417.74 2381.37 n 120 132 148 400 std. deviation 1020.85 1000.31 927.38 978.50 water consumption (liters) mean 7.13 7.39 7.47 734 n 120 132 148 400 std. deviation 2631 2.48 2428 2505 *bold numbers indicate the highest socioeconomic and demographic variable values and frequencies 162 italian journal of food science, 2021; 33 (2) aşkan e et al. effective cost by providing procurement cost advantage on the consumer demands due to the water being at a drinkable temperature and always accessible throughout all year. however, it could be exposed to significant problems through various storage and transport vehicles with various microbiological contaminations. exploring these issues in detail, mcguinness et al. (2020) highlighted that transportation methods with closed systems from safe water sources should be investigated instead of using transportation and storage vehicles to be able to prevent various microbiological contaminants. the results of cluster analysis target consumer masses were segmented to three homogenous groups such as regions i, ii, and iii by considering the relationships among five main factors based on the consumers’ drinking water consumption preferences and their residence neighborhoods. the distributions of their five preference factors in each homogeneous cluster were given in table 3. the ratios of the consumers residing in regions i, ii, and iii of iğdır province were determined as 37% (148 households), 33% (132 households), and 30% (120 households), respectively (figure 1). the consumers residing in region i shaped the purchasing models responding to their physiological needs under drinking water’s physical and physiological quality attributes. the consumers, therefore, aimed to maintain a healthy life with optimal drinking water consumption meeting their physiological needs by considering its physical and physiological qualities. for all these reasons, there was a significant demand for bottled water with higher physical quality and for purified tap water. in order to increase the water consumption satisfaction of the target group, therefore, it should expand the supply of bottled drinking water with high physical and importance to drinking water hygiene. similar to these results, it was been reported that hygiene was an important factor in the consumption of packaged and purified drinking water in india (suganthi, 2014). in addition, it was pointed out that quality attributes such as taste and flavor, smell, clarity, and hygiene revealing the consumers’ drinking water preferences was similar to the results found by tümer et al. (2011) and doria (2006). the third factor explaining 11.74% of the total variance represented confidence in tap water. the factor covered in the variables such as city tap water preserving through treatment, and believing being safer than alternative drinking water supply sources. pointing to the issue of treating the tap water in various ways and supplying it through reliable and healthy distribution systems, liu et al. (2017) and zlatanovic et al. (2017) highlighted that the water source and network distribution systems and their elements were effective on the physiochemical and microbiological quality of water. the chemical quality of water, the fourth preference factor, explained 10.99% of the total variance. good-quality potable water should include 250 mg calcium and 75 mg magnesium per liter; however, not only these ratios but also the ph levels for treated drinking water were lower. similarly, the research conducted by li and wu (2019) emphasized that continuity of standardized essential nutrients and mineral substance amounts in drinking water’s chemical quality was of great importance and had major effects on public health. dönderici et al. (2010) also reported that drinking water’s physical and chemical quality attributes had great importance in order to determine potable water resources deteriorating under the effect of various environmental factors. on the other hand, the fifth preference factor under tap water advantage explained 10.80% of the total variance, and thus the biggest advantage of fountain water was of a drinkable temperature without a real purchase cost versus a remarkable transfer cost. indeed, it does not require an 30% cluster sizes cluster 1 2 3 37% 33% size of smallest cluster 120 (30%) size of largest cluster 148 (37%) ratio of sizes lagest cluster to smallest cluster 148 (37%) figure 1. cluster sizes and distributions. italian journal of food science, 2021; 33 (2) 163 drinking water consumption preferences on the other hand, the results of the descriptive statistics indicated that the average family size of the consumers residing in region i was 4.06 individuals and their average monthly food expenditure was 672.92 tl. they ranked the first, therefore, in terms of these results. besides, the average age and education duration of the participants in region iii reached the highest values with 37.71 and 11.21 years, respectively, average monthly income and total expenditure with 4697.01 and 2417.74 tl, respectively, and monthly water consumption amounted to 7.47  l. those dwelling in region ii were at the lowest levels, however, in terms of all socioeconomic factors (table 4). conclusion the lack of substitution for water as a natural resource makes it an indispensable element for all living creatures throughout human history. in addition to the scarcity of water resources, increasing world population, industrialization, and intensive use of water in agriculture, as well as the negative effects of climate change on water resources make it difficult to access safer drinking water for people. water consumption regimes, therefore, are regulated for crop and animal production in agricultural production, and alternation systems saving water are adopted. on the other hand, various saving and quality-enhancing measures have been implemented to make drinking water possible to people, and significant changes have occurred, therefore, in their attitudes and behaviors towards drinking water. this study was planned to determine the factors affecting the attitude and behaviors of the consumers considering drinking water’s quality characteristics toward its consumption, and to determine the consumption preferences of homogeneous consumer masses in iğdır. in order to achieve this goal, the data obtained from faceto-face surveys with 400 consumers residing in iğdır were used in fa and cluster analysis. the research results indicated that the consumers with middle income, high food expenditure, and lower education levels under the lowest water consumption amount in region i tried to meet their physiological needs in a healthy way from bottled ready water and purified tap water by considering the microbiological and physical quality of drinking water. similarly, it was determined that consumers in region ii including mostly workers with water consumption amount at moderate levels under low income and food expenditure realized their drinking water consumption by considering the chemical quality parameters under the critical confidence interval values. it should be delivered sanitary drinking water to their residences by using the effective treatment systems at the tap water collection and distribution centers, physiological qualities, and focus on the sales of water with these quality attributes by retailers. on the other hand, the consumers dwelling in region ii formed their drinking water consumption preference patterns by considering the reliance on tap water depending on the chemical quality of water. they took an approach that prioritizes the consumption of treated tap water by taking into account the chemical quality parameters of drinking water. if tap water’s chemical quality analyses could be frequently made, and the precautions to maintain its standard quality attributes could be taken by the local governments, the consumers’ water consumption satisfaction could be increased dramatically. similarly, the consumers living in region iii developed their preference models with the basic advantage of fountain water depending on water’s chemical quality. they attributed a bigger importance to water temperature being a physical quality parameter, and tried to supply drinking water from fountains providing spring water with the higher quality features by accounting for its chemical quality parameters. being lower of its supply costs, therefore, and providing alternative selection possibility to their consumers according to chemical quality attributes could create the significant advantages to target consumers. being exposed to various microbiological contaminants of the vehicles used during transportation and storage of fountain waters, however, could cause a vital threat. delivering to the target consumer masses through various closed mains lines of fountain waters with high chemical quality attributes could considerably increase their drinking water consumption satisfaction. the results of the consumer cluster profiles demographic and socioeconomic characteristics of three different homogenous consumer masses such as regions i, ii, and iii segmented by cluster analysis in iğdır province are given in table 4. the results of the frequency statistics showed that considering the gender demographics of the participants, men and women accounted for 49% and 51%, respectively. while the distribution of the women and men in regions ii and iii were equally analyzed, the proportion of the women in region i was determined as 52%. when the consumers’ occupational statutes in the research regions were analyzed, the ratios of the white-collars, housewives, workers, and students were determined as 29%, 20%, 16%, and 3%, 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http://doi.org/10.24925/turjaf.v5i7.822-831.1223� http://www.tuik.gov.tr/prehaberbultenleri.do?id=30668� https://adres.nvi.gov.tr/vatandasislemleri/adressorgu� http://awsassets.wwftr.panda.org/download/su_ayak_izi_raporweb.pdf� http://awsassets.wwftr.panda.org/download/su_ayak_izi_raporweb.pdf� https://dergipark.org.tr/tr/pub/ckuiibfd/issue/32891/365377� https://dergipark.org.tr/tr/pub/ckuiibfd/issue/32891/365377� http://doi.org/10.1016/j.watres.2017.07.019� http://doi.org/10.1016/j.watres.2017.07.019� https://doi.org/10.1016/j.ecolind.2019.01.034� http://doi.org/10.5505/pajes.2016.81542� https://doi.org/10.1016/j.eswa.2009.04.028� https://doi.org/10.1002/2013wr014934� _goback _hlk73278926 _hlk73440834 paper 160 ital. j. food sci., vol. 27 2015 keywords: deep-frying, fats, oils, restaurant, total polar materials assessment of total polar materials in frying fats from czech restaurants j. mlcek1*, h. druzbikova1, p. valasek1, j. sochor2, t. jurikova3, m. borkovcova4, m. baron2 and s. balla3 1department of food chemistry and analysis, faculty of technology, tomas bata university in zlin, namesti t. g. masaryka 275, cz-762 72 zlin, czech republic 2department of viticulture and enology, faculty of horticulturae, mendel university in brno, valticka 337, cz-691 44 lednice, czech republic 3department of natural and informatics sciences, faculty of central european studies, constantine the philosopher university in nitra, drazovska 4, sk-949 74 nitra, slovakia 4department of zoology, fisheries, hydrobiology and apiculture, faculty of agronomy, mendel university in brno, brno, cz-61300 czech republic * corresponding author: tel. +420 576 033 030; fax +420 576 031 444; email: mlcek@ft.utb.cz abstract deep-frying is commonly used as convenient technique for the preparation of foods. the frying oils and fats are absorbed by fried food and become a part of diet. the content of total polar materials was determined in frying oils and fats in 46 restaurants from south moravia and the olomouc regions. twenty-eight samples were found with total polar materials with limit of rejection over 24%. the highest total polar materials values were observed in cooking fat; the lowest one was in vegetable shortening oil. this conclusion corresponds with frying temperatures, which were highest in cooking fat. mailto:mlcek@ft.utb.cz ital. j. food sci., vol. 27 2015 161 introduction fried foods are consumed worldwide with increasing popularity since their unique sensory properties, such as colour, flavour, texture and palatability, are highly appreciated by consumers. oils and fats are used as means of heat transfer from the fryer to the food. the quality of oils and fats during the frying process has a major influence on the quality of the final product (andrikopoulos et al., 2003). although many studies have dealt with the mechanism and products of fat deterioration under laboratory conditions, relatively little attention has been paid to changes that occur in cooking oils and fats during use in restaurants and other establishments. in relation to frying operations of fried foods, most of them are conducted at elevated temperatures (160°-195°c) in presence of air, metal container and moisture, resulting in both thermal and oxidative disintegration of the oil (bansal et al., 2010b). the thermal treatment of the cooking oils results in oxidative and hydrolytic reactions i.e. hydrolysis, cyclisation or polymerisation. these chemical and physical changes take place and lead to the formation of numerous decomposition products (volatile and non-volatile compounds) (fritsch, 1981; friedman, 2000). furthermore, the extent and nature of these decomposition products are affected by the food being fried, the type of the fat used as well as by the choice of the fryer design and the operating conditions (temperature, oxygen exposure, heating time, turnover rate) (al kahtani, 1991). regarding the majority of the non-volatile by-products, they are categorized as the total polar materials (tpm). the tpm constituents include dimeric fatty acids, triglyceride monohydroperoxides, polymerized triglycerides (ptg), cyclic fatty acid monomers and aldehydic triglycerides (márquez-ruiz et al., 1998; gertz, 2000). during the reactions mentioned above, the functional, sensory and nutritional qualities of frying fats are changed and may reach a point where it is no longer possible to prepare high quality fried foods and the frying fat will have to be discarded (vahčič and hruškar, 1999). the discarding point of the oil that is used repeatedly for frying of food is very closely related to the health issues. the compounds formed during deep-frying e.g. enzyme inhibitors, vitamin destroyers, lipid oxidation products, gastrointestinal irritants and/or potential mutagens are harmful to human health and can therefore become a chemical and physical hazard (soriano et al., 2002). there are many studies examining how the quality of frying oils and fats, which are consumed in a diet, influences the health of live animals, especially the rats. many surveys dealing with the effect of these oils on the growth, liver size, cholesterol or phospholipids in rats showed the relevant negative changes of mentioned parameters by rats fed with oils containing the higher percentage of tpm. however, no epidemiological or public health investigations have directly proved the effect of abused frying oils on the healthy persons (billek, 2000; bansal et al., 2010b). therefore, it is important to observe the quality of frying oils and fats in view of the fact that they are absorbed by frying food and so become a part of our diet. the uptake of absorbed oil in food ranges in percentage from 4% to 14% of the total weight, depending on the food and the type of frying medium (andrikopoulos et al., 2003). nevertheless, it is necessary to test the oil quality and establish the cut-off point at which the oil should be discarded in order to protect public health. since there are health and safety issues related to the reuse of frying oils, several countries have established relevant laws, regulations or recommendations regarding the further use or the discarding of such oils. these countries have set limits for parameters such as frying temperature, acid value, smoke point, polar compounds and polymers (bansal et al., 2010b). in general, the percentage (%) of tpm in the cooking oil has been shown to be almost identical to the one present in the oil absorbed by the food. thus, by measuring tpm % in frying oil, the direct content of tpm in the fried food could be reflected. moreover, most european countries have established the limits for the rejection and replacement of cooking oil in restaurants as the content of tpm% where its maximum values range from 24 to 27% (caldwell et al., 2001). the limit 24% for tpm was recommended as the most appropriate for rejection while the limit of 20% tpm (gertz, 2000) has been recommended for the replenishment of the oil or fat. the concentration of tpm is so rapidly becoming the most widely accepted parameter for the determination of used frying oil quality (dobarganes et al., 2000; gertz, 2000). as to the present study, the tpm% of six different types of cooking oils and fats from 46 restaurants in south moravia and the olomouc regions are reported indicating the quality status of these oils and fats used under current catering practice. materials and methods samples the quality of frying oils and fats was examined during one month in 46 restaurants and fast-food outlets of various types from south moravia and the olomouc regions, in the czech republic. the measurements were performed in daily frying operations of these restaurants and were analysed repeatedly for the relevance of results. in total were evaluated 46 samples of 162 ital. j. food sci., vol. 27 2015 oils and fats that were divided according to raw materials. the types of oils and fats used for frying are summarized in table 1. the most of restaurants used only one type of frying fat during measurements. methods the amount of tpm was determined by using testo 270 (testo inc., germany). this instrument has to provide the content of tpm in percentage with accuracy +/2% tpm. the samples were analysed by inserting the sensor into oil heated to frying temperature and reading the temperature and the tpm content in percentage from the display after about 30 s. the sensor was calibrated with the calibration oil supplied by the manufacturer before analysing the frying oils. the equipment was cleaned with warm water and neutral detergent and dried well between the measurements. each test was performed in three times. the limit value for the replacement of frying oils and fats was established on 24% for tpm according to german regulations (bansal et al., 2010b). table 1 oils and fats used for deep-frying in 46 selected restaurants and fast-food outlets in south moravia and olomouc region, czech republic. producer (trademark) type of oil or fat n a canola oil 2 b canola oil 2 c canola oil 6 d canola oil 2 e canola oil 4 f canola oil 2 g canola oil 2 h canola oil with palmolein 4 i canola oil with palmolein 2 j palm oil 2 k palm oil 2 l palm oil 4 m sunflower oil 2 n sunflower oil 2 o vegetable cooking fat 2 p vegetable shortening oil 2 q vegetable shortening oil 4 total 46 table 2 the content of tpm in examined frying oils and fats in percentage. canola oil canola oil with palmolein palm oil sunflower oil vegetable cooking fat vegetable shortening oil n 138 50 66 34 18 28 mean 15.3a 18.1b 17.9b 14.9a 19.6c 12.3d sd ±6.08 ±7.47 ±8.41 ±7.22 ±8.47 ±6.06 minimum 4.5 5.5 6.3 5.0 8.2 5.0 median 16.0 20.4 15.0 11.8 22.5 10.7 maximum 26.6 30.8 31.8 27.1 32.5 24.0 n number of the measurements from the 1st day of frying time to day of used oil replacement. statistical analysis the data obtained were statistically analysed by the analysis of variance (anova) and tukey’s multiple range test for comparison of means. other functions were calculated using the unistat, v. 5.1 statistical package and office excel® microsoft 2010. results and discussion the mean values, the medians and the range of the parameter studied (total polar materials) for oils and fats used for frying from 46 restaurants are shown in table 2. the mean values for tpm were determined below 20% in all types of oils and fats and therefore did not reach the limit for rejection. the minimum of tpm observed was about 5% in canola oil and vegetable shortening oil at the 1st day of frying, while the maximum value increased to 33% in vegetable cooking fat after 9 days of frying. the analogous wide range was also found by andrikopoulos et al. (2003), who mentioned in their papers that the minimum of tpm is about 3% and maximum reaches the level of 40%. total polar materials reflect the total level of breakdown products from the frying process. the amount and character of these products are affected by some frying parameters such as fat and food composition, frying conditions (temperature, oxygen exposure, heating time, turnover rate) and the design and material of frying equipment (al-kahtani, 1991; vahčič and hruškar, 1999). the reported cases of oils and fats that exceed the tpm limit for rejection (24%) are presented in table 3. approximately 60% of all samples (12 samples of canola oil, 6 samples of canola oil with palm olein, 4 samples of palm oil, 4 samples of sunflower oil and 2 samples of vegetable cooking fat) were found over the tpm limit for rejection. in case of canola oil with palm oil, sunflower oil and vegetable cooking fat was found out, that all the average values for tpm were above the limit for rejection. the values above 24% tpm were measured already after 6 days of frying by vegetable cooking fat, 7 days by canola oil with palm olein, palm oil and 9 days by sunflower oil. ital. j. food sci., vol. 27 2015 163 on the other hand, the average content of total polar materials in the samples of vegetable shortening oil was observed below this limit. in the study related to frying oils and fats from 63 restaurants in athens, greece were determined only (17%) of all samples over the tpm limit for rejection. the most samples above this limit were observed in vegetable cooking fat (40%) and then in sunflower oil (30%) and palm oil (18%) (andrikopoulos et al., 2003). approximately (41%) of frying oils used in the restaurants in zagreb, croatia reached the oil discard level. however, there was no information about the type of frying oils and fats (vahčič and hruškar, 1999). the extent of oxidative degradation in frying oils and fats can be reliably determined using the content of total polar materials. the results of this study showed that the content of tpm increased with frying time (fig. 1). the initial values of the tpm were below 10%. at the end of frying time (the 9th of frying) the content of tpm reached above 24%, which is oil discard level set in many european countries. these values were observed after 5 days of frying at the earliest. after 6 up to 9 days of deep frying, the final tpm levels were: “19,8”% in vegetable shortening oil, “23,1”% in canola oil, “25,8”% in sunflower oil, “28,8”% in canola oil with palm olein, “31,8”% in palm oil and “32,5”% in vegetable cooking fat (fig. 1). the maximum content of tpm in frying oil was accepted as 24%. from this point of view the percentage (%) of tpm in determined oils would be ranged in ascending sequence: vegetable shortening oil > canola oil > sunflower oil > canola oil with palm olein > palm oil > vegetable cooking fat. in the other study the highest amount of tpm in frying oils was established at 27% and was found out that sunflower oil reached lower value for %tpm than palm oil (xu et al., 1999). there are many factors that impact the amount of total polar content in frying oils and fats. for example the fatty acid composition of oil has marked effects on its frying performance as well as on its physical and chemical behaviour (brinkmann, 2000). the formation of polar compounds during repeated frying operations has been shown to increase with the degree of oil unsaturation, both during repeated frying and during the heating of oils (takeoka et al., 1997; romero et al., 1998). the next significant parameter that influences the formation of polar compounds in heated oils is the ratio of the surface oil area to, oil volume in the fryer. the specific surface also plays an important role in behaviour of oils during frying, as the overall deterioration is an oxidation process rather than an interaction with frying foods according to bracoo et al., (1981). the differences in temperatures do not cause significant changes in frying oils (jorge et al., 1996). the table 3 the summary of examined oil and fat samples that exceed tpm limit for rejection. type of oil or fat n tpm < 24% tpm > 24% canola oil 20 8 12 canola oil with palmolein 6 6 palm oil 8 4 4 sunflower oil 4 4 vegetable cooking fat 2 2 vegetable shortening oil 6 6 total 46 18 28 fig. 1 the content of total polar materials during frying in examined types of oils and fats 164 ital. j. food sci., vol. 27 2015 oil alteration depended on the frying procedure mainly as a result of different surface area to volume ratios and specific areas because panfrying caused more marked changes than deepfrying on all the parameters studied including the content of total polar materials (andrikopoulos et al., 2002). the next important factor is the temperature which should be in the range of 160 180°c for frying operations (soriano et al., 2002). in this research the highest temperatures were observed for vegetable cooking fat (173.6°c) while the lowest temperatures were examined for using vegetable shortening oil (124.1°c) (table 4). similar conclusion was found also by aladedunye and przybylski (2009), who mentioned in their paper that the extent of oxidative deterioration, as measured by the tpm formation, was faster during frying at 215°c compared to 185°c. soriano et al., (2002) recommended the continuous heating as the intermittent heating is much deleterious due to an increased rate of oil breakdown. their team was concerned with the daily oil turnover too which should be ranged between 15 to 25 weight per cent in food service kitchens. this suggestion was determined with regard to much longer turnover periods influenced by fluctuations in the demand for fried foods. in our study only 40% of restaurants replenished the frying oils and fats. the replenishment of frying medium was made between the 4th and the 7th day of using. the average volume of replenished oil was 1.5litres. our finding about catering practice in examined restaurants showed, too, that oils and fats were replaced after 9 days of using while the content of tpm reached the oil discard level already on the sixth day (fig. 1). conclusion among 46 restaurants, the samples of oils and fats over the rejection limit comprise a relatively high part of samples examined (60% regarding tpm). there was observed higher content of total polar materials with increasing frying temperature. the catering practice in czech restaurants as such has some lacks, like the turnover rate or the late replacement of used oils and fats, too. from all the findings of the presented study it appears, that it is necessary to survey the conditions of the usage of frying oils and fats more in detail. in addition, more frequent controls and the application of strict regulations by food authorities are important as well. acknowledgements this study was funded by internal grant agency of tomas bata university in zlín, project no. iga/ft/2015/010. 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soc., 76: 1092-1099. http://www.scopus.com/authid/detail.url?origin=resultslist&authorid=6505854305&zone= http://www.scopus.com/record/display.url?eid=2-s2.0-0033471021&origin=resultslist&sort=plf-f&src=s&st1=vahcic&sid=377a67213c38a2175e0a3d9412cdd114.wlw7nkkc52nnqnxjqaqrla%3a1630&sot=b&sdt=b&sl=19&s=author-name%28vahcic%29&relpos=39&relpos=39&citecnt=2&searchterm=author-name%28vahcic%29 http://www.scopus.com/record/display.url?eid=2-s2.0-0033471021&origin=resultslist&sort=plf-f&src=s&st1=vahcic&sid=377a67213c38a2175e0a3d9412cdd114.wlw7nkkc52nnqnxjqaqrla%3a1630&sot=b&sdt=b&sl=19&s=author-name%28vahcic%29&relpos=39&relpos=39&citecnt=2&searchterm=author-name%28vahcic%29 http://www.scopus.com/source/sourceinfo.url?sourceid=15632&origin=resultslist http://www.scopus.com/source/sourceinfo.url?sourceid=15632&origin=resultslist 39 issn 1120-1770 online, doi 10.15586/ijfs.v33i1.1933 p u b l i c a t i o n s codon p u b l i c a t i o n s codon the antimicrobial activity of two phenolic acids against foodborne escherichia coli and listeria monocytogenes and their effectiveness in a meat system oluwatosin ademola ijabadeniyia*, austin govendera, omotola folake olagunjua, ajibola bamikole oyedejib* adepartment of biotechnology and food technology, durban university of technology, durban, south africa; bdepartment of biotechnology and food technology, faculty of science, university of johannesburg, gauteng, south africa *corresponding authors: oluwatosin ademola ijabadeniyi, department of biotechnology and food technology, durban university of technology, s9 level 1, steve biko campus, durban 4000, south africa. email: oluwatosini@dut. ac.za; ajibola bamikole oyedeji, department of biotechnology and food technology, faculty of science, university of johannesburg doornfontein campus, p.o. box: 17011, gauteng 2028, south africa. email: jibanky2@gmail.com received: 21 july 2020; accepted after revision: 14 december 2020; published: 1 february 2021. © 2021 codon publications open access paper abstract ready-to-eat meats are susceptible to pathogenic contamination during their production, distribution, and sale. this study evaluated the antimicrobial effects of two phenolic acids (caffeic and ferulic acids) against foodborne pathogens in cold-cut meat at low-temperature conditions. the individual and combined antibacterial activities of caffeic and ferulic acids against escherichia coli o157:h7 atcc 43888 and listeria monocytogenes atcc 7644 were determined by diffusion disk assay in broth media and cold-cut meat. broth media and meat samples already inoculated with e. coli and l. monocytogenes were treated with caffeic acid, ferulic acid, and their combination at the concentrations of 150 ppm and 200 ppm and stored at 4°c. microbial growths were monitored at 0, 24, 48, and 72 h. caffeic acid at 200 ppm exhibited a zone of inhibition of 12.33 mm on e. coli, and ferulic acid revealed a zone of inhibition of 11.00 mm on l. monocytogenes. the combination of caffeic-ferulic acid at a concentration of 200  ppm was most effective against e. coli, demonstrating a synergistic effect over 72 h at 4°c in both broth media and meat. for meat samples, the combination of caffeic acid and ferulic acid exhibited a log reduction of 3.63 cfu/g at 150 ppm and 2.51 cfu/g at 200 ppm against e. coli o157:h7 at the end of cold storage. caffeic acid alone exhibited an overall log reduction of 2.48 cfu/g at 150 ppm and 2.75 cfu/g at 200 ppm against l. monocytogenes. these results indicate the ability of caffeic and ferulic acids, individually and in combination, to reduce pathogenic contamination and improve safety of cold-cut meats. keywords: antibacterial activity; caffeic acid; ferulic acid; cold-cut meat; e. coli; l. monocytogenes introduction cold-cut meat products such as ham are one of the most commonly consumed ready-to-eat products. they are often in sliced forms and used as fillings for sandwiches and similar foods. they are manufactured from raw pork and/or beef and characterized by physical and biochemical changes that occur during curing (marušić et al., 2014). in most modern cold-cut meats, nitrites (either sodium nitrite or potassium nitrite) are employed during curing to prevent bacterial growth and survival and enhance safety and storage stability. however, owing to the toxicity of nitrites, some country regulations specify maximum allowable contents in the final product (campion et al., 2017). this indulges public health concern because, under certain conditions, nitrites in meat can react with degradation products of amino acids, forming nitrosamines, which are known carcinogens italian journal of food science, 2021; 33 (1): 39–45 mailto:oluwatosini@dut.ac.za mailto:oluwatosini@dut.ac.za mailto:jibanky2@gmail.com italian journal of food science, 2021; 33 (1) 40 the antimicrobial activity of two phenolic acids against foodborne e. coli and l. monocytogenes produced by plants and can be classified into three different groups based on their chemical structures. these classes include non-flavonoids, flavonoids, and tannins in their simple or complex forms (pernin et al., 2018). in plant-based foods, phenolic compounds are responsible for the color of red fruits, juices, and wines, substrates involved in enzymatic browning and are also considered to contribute to the health benefits associated with dietary consumption of fruits and vegetables (cheynier, 2012). in particular, caffeic and ferulic acids possess aromatic rings with a hydroxyl group (-oh) on the fifth position of their rings while their difference is in the presence of methoxy group (-h3co) for ferulic acid and hydroxyl group (-oh) at the sixth position of aromatic rings (sanchez-maldonado et al., 2011). they are well distributed in plants, and they possess antioxidant and prooxidant properties (nystrom et al., 2005) substantiated by their functions in the reduction of auto-oxidation by acting as radical scavengers, inhibitors of lipid peroxidation, and low-density lipoproteins (maurya & devasagayam, 2010). their antioxidant and prooxidant abilities in foods have been adduced to factors such as concentration, chemical structure, and the nature of substrates or foods (maurya & devasagayam, 2010). previous studies have validated the antimicrobial properties of phenolic compounds against bacteria, including pathogenic species. these findings justified that their use could be an alternative means of inhibiting or inactivating the growth of l. monocytogenes and e. coli (vaquero et al., 2007). when combined, phenolic acids could reflect greater antimicrobial efficacy against foodborne pathogens, compared to individual phenolic acids. therefore, this study was conducted to investigate the individual and combined antimicrobial effects of caffeic and ferulic acids on the survival of l. monocytogenes atcc 7644 and e. coli o157:h7 atcc 43888 in cold-cut meat products under low-temperature storage conditions. materials and methods preparation of inoculum the bacterial strains l. monocytogenes atcc 7644 and e. coli o157:h7 atcc were obtained from the microbial culture collection of the department of biotechnology and food technology, durban university of technology. stock cultures of l. monocytogenes were grown aerobically for 24 h at 30°c in brain heart infusion (bhi) broth. for e. coli, stock cultures were grown at 37°c for 24 h in tryptic soy broth (tsb). a loopful of the stock culture of each microorganism was transferred to 10 ml of bhi and tsb, as applicable, and incubated at 37°c for (shpazier et al., 2018). furthermore, foodborne pathogenic bacteria may persist and replicate in cold-cut meats due to their low effective doses, antimicrobial resistance, and capability of adapting to stress conditions without observable sensorial changes in the product (rybarczyk et al., 2017). listeria monocytogenes (l. monocytogenes) is a grampositive bacterium that thrives in water, soil, and foods (chen et al., 2017). it is the causative organism for listeriosis and has the potential to contaminate different forms of foods, including low-moisture foods, at different stages within the food chain. it has been implicated in many foodborne disease outbreaks, and some of the most common vehicles involved in these outbreaks include readyto-eat meals, unpasteurized milk, dairy products, meat, fruits, and vegetables (pernin et al., 2019). ingestion of foods contaminated with l. monocytogenes could result in different symptoms from mild gastroenteritis to severe central nervous system infections. high-risk individuals prone to l. monocytogenes infection include pregnant women and immunocompromised people (hunjak et al., 2019). escherichia coli (e. coli) is also a gram-negative, rod-shaped, ubiquitous bacterium, which also lives in the gut of humans and suppresses the growth of harmful bacteria (saxena et al., 2015). one of its pathogenic strains, e. coli o157:h7, is listed by the united states centers for disease control (cdc) as a pathogen that has attracted increasing attention due to the number of victims who had to be hospitalized following its ingestion (de souza et al., 2018). the cdc estimated that in the us, there were 10,000 illnesses, thousands of hospitalizations, and hundreds of deaths attributed to the e.  coli o157:h7 yearly. most e. coli o157:h7 outbreaks are associated with fecal-oral transmission due to poor hygiene and food handling practices. sources of human infection include poorly cooked meat, apple juice, water, and milk products such as yogurt and unpasteurized milk. these bacteria have been adjudged the most threatening foodborne pathogens because of their severe debilitating effects on humans upon ingestion (buchanan et al., 2017). although various measures have been implemented in the past to control and reduce the prevalence of l. monocytogenes and e. coli o157:h7, reports of foodborne disease outbreaks as a result of their infection subsists. chemical preservatives are capable of inhibiting or inactivating bacterial growth in foods. however, the use of natural preservatives is currently encouraged due to increasing bacterial resistance to chemical preservatives, food safety regulations, and increased awareness of the adverse effects of chemicals (pernin et al., 2019). phenolic compounds have attracted much interest due to their antioxidant potential and antimicrobial properties. phenolic compounds are secondary metabolites 41 italian journal of food science, 2021; 33 (1) oluwatosin ademola ijabadeniyi et al. in each broth treatment was determined at 12, 24, 48, and 72 h by pour-plating six serial dilutions on appropriate growth agar. plates were incubated at 37°c for 24  h before enumeration. each experiment was repeated at least thrice. meat study cold-cut meat samples were bought from a trusted retail outlet and sliced into 5 g portions. for sterilization, the samples were dipped into 95% ethanol solution and left to dry in a laminar flow chamber. afterward, 0.1 ml of each bacterial suspension was spot-inoculated onto the meat samples and spread out onto the surface of the meat using sterile spreader. inoculated meat samples were then dried for 1 h at room temperature. thereafter, samples were dipped in phenolic acid solutions containing a concentration of 150 ppm and 200 ppm of each phenolic acid and their combination (1:1) for 60 s. broth inoculum containing a bacterial suspension of 1x108 cfu/ml for each strain was used as the control. the samples were stored at 4°c for a 72-hour period in sterilized stomacher bags containing phosphate buffer solution (pbs). microbial survival was also evaluated at 12, 24, 48, and 72 h. plates were incubated at 37°c for 24 h prior to enumeration. each experiment was repeated at least three times. statistical analysis all experiments were performed in triplicate. experimental data were analyzed by anova (analysis of variance) (p<0.05). the mean values of the experimental growth data were compared using duncan’s multiple range test. results and discussion antibacterial activity of phenolic acids using disk diffusion assay the effects of caffeic acid, ferulic acid, and their combination on the growth of l. monocytogenes and e. coli o157:h7 are shown in table 1. they demonstrated various degrees of inhibition against the two pathogenic strains. ferulic acid was found to be most effective against the e. coli o157:h7 strain at 150 ppm concentration, with a zone of inhibition of 10 mm, compared to caffeic acid and their combination at the same concentration. there was no difference (p<0.05) in the inhibitory actions of caffeic acid and the combination of caffeic and ferulic acids at this same concentration (150 ppm). with an increase in the ferulic acid concentration to 200 ppm, the zone of 24 h. turbidity of cultures was adjusted to match that of a 0.5 mcfarland standard to achieve an approximate 108 cfu/ml in each case. preparation of phenolic acid extracts caffeic acid and ferulic acid (>98% purity) were purchased from sigma-aldrich (johannesburg, south africa). to elucidate the antimicrobial properties, these phenolic compounds were dissolved in 99.8% ethanol (merck, johannesburg, south africa). however, for broth and meat experiments, the phenolic acids were dissolved in water at 1% w/v (zhang et al., 2016). the solutions were filtered through 0.22-μm membrane filters in each case and stored at 4°c in sterilized glass containers until needed. antibacterial activity determination for agar disk diffusion assay, 0.1 ml of each bacterial culture suspension was transferred into petri dishes containing mueller hinton agar (merck, johannesburg, south africa) and were uniformly spread onto the surface with the help of a sterile plate spreader. fifty microliters of phenolic acids solutions, at different concentrations (150 and 200 ppm), were pipetted on 6 mm sterile filter paper disks (whatman no. 1) and air-dried in a laminar flow chamber. distilled water was used as the negative control, while ciprofloxacin (merck, johannesburg, south africa) was used as the positive control for its effectiveness against both gram-positive and gram-negative bacteria. these dried disks were then transferred into inoculated plates and incubated at 30°c for 24 h for l.monocytogenes and 37°c for e. coli. antibacterial activity for each phenolic acid and their combination was determined by measuring the inhibition zone around the disk following a 24-hour incubation period. each experiment was replicated at least three times. broth study in each case, 1 ml bacterial suspension containing 1x108 cfu/ml e. coli and l. monocytogenes were transferred into either bhi broth for l. monocytogenes or tsb broth for e. coli o157:h7. thereafter, 50 µl of individual solutions of caffeic and ferulic acid of 150 ppm and 200 ppm concentration as well as a combination (1:1) of caffeic and ferulic acid solutions (150 ppm:75 ppm of each and 200 ppm:100 ppm of each) were added to inoculated broths. an inoculum containing a bacterial suspension of 1x108 cfu/ml of each bacterium that was not treated by the phenolic acids served as the control samples. the samples were then stored at 4°c for 72 h. microbial survival italian journal of food science, 2021; 33 (1) 42 the antimicrobial activity of two phenolic acids against foodborne e. coli and l. monocytogenes 7.87x108 after 12 h to 8.21x108 at the end of the incubation period. at a concentration of 150 ppm, ferulic acid was the most effective antimicrobial agent against e. coli o157:h7, showing a reduction of 2.98 log cfu/ml, while caffeic acid was the most effective against l. monocytogenes with a log reduction of 2.17 log cfu/ml following the incubation period. the combination of caffeic acid and ferulic acid in a 1:1 ratio demonstrated a synergistic effect whereby the viability of microorganisms diminished by 2.42 and 2.14 log cycles, respectively. at a concentration of 200 ppm, the order of effectiveness remained the same for both gram-positive and gram-negative strains; however, a higher inhibition rate was observed (table 2) – a reduction of 3.49 log cfu/ml against e. coli o157:h7 by ferulic acid and 2.35 log cfu/ml against l. monocytogenes by caffeic acid. the synergistic effect of the combination of caffeic acid and ferulic acid enhanced the reduction of viability of microorganisms by 2.42 and 2.25 log cfu/ml (at 150 ppm) as well as 3.14 and 2.62 log cfu/ml for e. coli and l. monocytogenes, respectively. in a previous study, the combination of caffeic acid and gallic acid, at 100 ppm and 200 ppm solution produced 1 log cfu/ml and 2 log cfu/ml reduction of l. monocytogenes, respectively, in meat (rodriguez-vaquero et al., 2011). in broth media, phenolic acids were less effective against l. monocytogenes. a possible explanation could be that the microorganism can proliferate under refrigeration conditions, allowing it to be more adaptable to the stress imposed by phenolic acids. inhibitory effect of phenolic acids against e. coli o157:h7 and l. monocytogenes in cold-cut meats table 3 illustrates the growth response of e. coli o157:h7 and l. monocytogenes in cold-cut meats treated with phenolic acids and stored at 4°c for over 72 h. the number of viable cells in the control samples increased from 7.49x108 to 7.75x108 cfu/ml for e. coli o157:h7 and inhibition was found to increase further. for l. monocytogenes, caffeic acid was the most effective antibacterial agent at the two concentrations tested, giving zones of inhibition of 7.66 and 11 mm at 150 and 200 ppm, respectively. the structure-function relationships between the antimicrobials and the respective microorganisms could justify the contrasting activities of the phenolic acids against gram-positive and gram-negative bacteria (sánchez-maldonado et al., 2011). distilled water did not inhibit the growth of organisms as no inhibition zones was detected during screening. ciprofloxacin, used as the positive control, demonstrated greater inhibitory potential for l. monocytogenes, possibly due to differences in the cell wall structures of gram-positive and gram-negative bacteria. the combination of caffeic acid and ferulic acid exhibited a joint effect at both concentrations, suggesting an affinity relationship between the two antimicrobials. however, they tend to possess a greater antimicrobial activity individually than in combination. inhibitory effect of phenolic acids against e. coli o157:h7 and l. monocytogenes in broth the growth response of e. coli o157:h7 and l. monocytogenes in broth media treated with phenolic acids at 150 or 200 ppm and stored at 4°c for a 72 h period is presented in table 2. the high levels of l. monocytogenes and e. coli inocula in broth and meat samples in this study are necessary to clearly understand the efficiency of the different concentrations of phenolic acids and the extent of their activity in highly contaminated food materials. inoculum levels excess of 107–109 have been used by previous authors (de souza et al., 2018; kwon et al., 2019; rodriguez-vaquero et al., 2007). in the control medium, the number of viable cells of e. coli o157:h7 increased from 7.70x108 to 8.01x108 cfu/ml at the end of the incubation period (72 h). for l. monocytogenes, the number of viable cells in the control medium increased from table 1. antibacterial activity of phenolic compounds against e. coli o157:h7 and l. monocytogenes determined by disk diffusion assay. treatments concentration zone of inhibition (mm) organism e. coli o157:h7 (atcc 43888) l. monocytogenes (atcc 7644) caffeic acid 150 ppm 7.00 ± 1.73b 7.66 ± 2.51b 200 ppm 9.33 ± 1.15a 11.00 ± 3.00a ferulic acid 150 ppm 10.00 ± 2.00b 6.00 ± 1.72b 200 ppm 12.33 ± 2.51a 8.33 ± 2.52a combination (1:1) 150 ppm 7.00 ± 1.73b 6.67 ± 1.15b 200 ppm 8.67 ± 1.16a 7.66 ± 2.51a control positive 26.67 ± 2.88a 36.67 ± 2.89a negative nd nd superscripts indicate significant differences (p < 0.05) across the columns only for each phenolic acid and concentration. 43 italian journal of food science, 2021; 33 (1) oluwatosin ademola ijabadeniyi et al. table 2 the effect of phenolic compounds (at 150 ppm and 200 ppm) against e. coli o157:h7 and l. monocytogenes at 4°c for over 72 h in a broth medium. phenolic acids (150 ppm) population (log cfu/ml) over storage period (h) of 12 24 48 72 e. coli o157:h7 (atcc 43888) control 7.70 ± 0.04a 7.80 ± 0.04a 7.92 ± 0.06a 8.01 ± 0.04a caffeic acid 6.08 ± 0.07b 5.73 ± 0.05b 5.71 ± 0.05b 5.63 ± 0.06b ferulic acid 5.89 ± 0.03c 5.57 ± 0.03b 5.55 ± 0.05b 5.03 ± 0.05b combination (1:1) 6.01 ± 0.03b 5.70 ± 0.13b 5.61 ± 0.03b 5.59 ± 0.07b l. monocytogenes (atcc 7644)         control 7.87 ± 0.03a 8.08 ± 0.05a 8.17 ± 0.04a 8.21 ± 0.03a caffeic acid 6.24 ± 0.02b 6.13 ± 0.02b 6.08 ± 0.03b 6.04 ± 0.05b ferulic acid 6.36 ± 0.03b 6.18 ± 0.02b 6.14 ± 0.03b 6.11 ± 0.03b combination (1:1) 6.33 ± 0.03b 6.18 ± 0.03b 6.12 ± 0.02b 6.07 ± 0.04b phenolic acids (200 ppm) e. coli o157:h7 (atcc 43888) control 7.70 ± 0.04a 7.80 ± 0.04a 7.92 ± 0.06a 8.01 ± 0.04a caffeic acid 6.30 ± 0.02b 6.27 ± 0.02b 5.34 ± 0.01b 5.29 ± 0.02b ferulic acid 6.29 ± 0.03b 6.21 ± 0.02b 4.85 ± 0.04c 4.52 ± 0.06c combination (1:1) 6.33 ± 0.05b 6.26 ± 0.03b 4.97 ± 0.04c 4.87 ± 0.04c l. monocytogenes (atcc 7644) control 7.87 ± 0.03a 8.08 ± 0.05a 8.17 ± 0.04a 8.21 ± 0.03a caffeic acid 6.19 ± 0.02b 6.01 ± 0.03b 5.92 ± 0.06b 5.86 ± 0.08b ferulic acid 6.24 ± 0.03b 6.09 ± 0.03b 6.03 ± 0.05b 6.01 ± 0.02b combination (1:1) 6.22 ± 0.02b 6.05 ± 0.03b 6.01 ± 0.04b 5.96 ± 0.03b superscripts indicate significant differences (p<0.05) across the columns only for each phenolic acid used. 7.51x108 to 7.96x108 cfu/ml for l. monocytogenes. at a concentration of 150 ppm, the combination of caffeic acid and ferulic acid was found to be most effective against e.  coli o157:h7 in comparison to caffeic acid, and ferulic acid was applied individually, with a log reduction of 3.38 cfu/ml at 72 h. as observed in broth media, caffeic acid revealed the greatest effects against l. monocytogenes in cold-cut meats with a log reduction of 2.44 cfu/ ml at the maximum incubation period. at a concentration of 200  ppm, a similar pattern of effectiveness was documented for both gram-positive and gram-negative strains, with a higher inhibition rate (table 3). the survival rate was lower for both strains, indicating that the synergistic effect of the combination of caffeic acid and ferulic acid was more effective in the meat compared to the broth. this could be attributed to the presence of macromolecules (proteins, lipids) and micromolecules (vitamins) in the food matrix (meat), which provided enhanced affinity for phenolic compounds. arima et al. (2002) reported that the combinations of quercetin and quercitrin, quercetin and morin, and quercetin and rutin portrayed synergistic effects resulting in improved efficacy than individual flavonoids against bacillus cereus and salmonella enteritidis. the inhibitory effect of phenolic compound mixtures was greater at 4°c incubation temperature than at 20°c for meat (rodriguez-vaquero et al., 2011) and fish (rodríguez-vaquero et al., 2013), reducing the viability of l. monocytogenes at two concentrations (100 and 200 mg/l). this is because their mode of action depends on their migration into bacterial membranes, which reduce fluidity at lower temperatures (ultee et al., 2000). beuchat et al. (1994) substantiated the improved antibacterial effect of phenolic compounds at low storage conditions. the shelf lives of foods could be further preserved by hurdle technology, such as in the case of combining refrigeration temperatures between 0°c and 4°c with modified atmosphere packaging to preserve foods (leistner & gorris, 1995). conclusions caffeic acid, ferulic acid, and their combination have potentials for the effective reduction or inhibition of foodborne pathogens; thus, providing a good alternative to chemical additives. zones of inhibition of e. coli and l. monocytogenes were widened with an increase in the italian journal of food science, 2021; 33 (1) 44 the antimicrobial activity of two phenolic acids against foodborne e. coli and l. monocytogenes table 3 the effect of phenolic compounds (at 150 ppm) against e. coli o157:h7 and l. monocytogenes at 4°c for over 72 h in meat. phenolic acids (150 ppm) population (log cfu/g) over storage period (h) of 12 24 48 72 e. coli o157:h7 (atcc 43888) control 7.49 ± 0.06a 7.70 ± 0.05a 7.76 ± 0.06a 7.75 ± 0.11a caffeic acid 5.67 ± 0.04b 5.51 ± 0.04b 5.22 ± 0.05b 4.98 ± 0.05b ferulic acid 5.49 ± 0.02c 5.31 ± 0.04c 5.06 ± 0.08b 4.51 ± 0.11b combination (1:1) 5.42 ± 0.03c 5.21 ± 0.04c 4.91 ± 0.02b 4.37 ± 0.04c l. monocytogenes (atcc 7644) control 7.51 ± 0.05a 7.71 ± 0.05a 7.87 ± 0.04a 7.96 ± 0.02a caffeic acid 6.29 ± 0.02c 6.09 ± 0.03b 5.76 ± 0.03b 5.52 ± 0.04b ferulic acid 6.52 ± 0.02b 6.39 ± 0.02b 6.12 ± 0.03b 5.97 ± 0.06b combination (1:1) 6.41 ± 0.03c 6.27 ± 0.08b 5.99 ± 0.03b 5.68 ± 0.04b phenolic acids (200 ppm) e. coli o157:h7 (atcc 43888) control 7.49 ± 0.06a 7.70 ± 0.05a 7.76 ± 0.06a 7.75 ± 0.11a caffeic acid 5.34 ± 0.08b 5.21 ± 0.05b 4.94 ± 0.07b 4.67 ± 0.08b ferulic acid 5.24 ± 0.05b 5.10 ± 0.02b 4.76 ± 0.08b 4.23 ± 0.04c combination (1:1) 5.19 ± 0.02b 5.07 ± 0.06b 4.66 ± 0.08b 4.09 ± 0.02c l. monocytogenes (atcc 7644) control 7.51 ± 0.05a 7.71 ± 0.05a 7.87 ± 0.04a 7.96 ± 0.02a caffeic acid 6.02 ± 0.04b 5.63 ± 0.02b 5.52 ± 0.05b 5.25 ± 0.02c ferulic acid 6.19 ± 0.02b 5.77 ± 0.04b 5.74 ± 0.04b 5.63 ± 0.04b combination (1:1) 6.12 ± 0.03b 5.77 ± 0.04b 5.68 ± 0.03b 5.49 ± 0.02c superscripts indicate significant differences (p<0.05) across the columns only for each phenolic acid used. concentration of phenolic acids, with ferulic acid having the greatest inhibition effect on e. coli at 200 ppm. for the two concentrations tested, phenolic acids were more efficacious against e. coli in broth and meat as the storage hours increased, compared to l. monocytogenes. however, the greater effects were obtained at 200 ppm for the two phenolic acids and their combination for both microorganisms. these results show the effectiveness of phenolic acids tested, at the stipulated concentrations, against the pathogenic organisms investigated and, as such, may allow the formulation of new antimicrobial products for potential use as food preservatives. further studies involving sensory evaluation may be necessary to determine consumer acceptability of caffeic and ferulic acid-treated meats. acknowledgments this work is based on research supported in part by the national research foundation of south africa, sa (nrf)/russia (rfbr) joint science and technology research collaboration (grant number: 118910). references arima, h., ashida, h. and danno, g.i., 2002. rutin-enhanced antibacterial activities of flavonoids against bacillus cereus and salmonella enteritidis. bioscience, biotechnology, and biochemistry 66(5):1009–1014. https://doi.org/10.1271/bbb.66.1009 beuchat, l.r., brackett, r.e. and doyle, m.p., 1994. lethality of carrot juice to listeria monocytogenes as affected by ph, sodium chloride and temperature.  journal of food protection 57(6): 470–474. https://doi.org/10.4315/0362-028x-57.6.470 buchanan, r.l., gorris, l.g., hayman, m.m., jackson, t.c., and whiting, r.c., 2017. a review of listeria monocytogenes: an update on outbreaks, virulence, dose-response, ecology, and risk 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https://doi.org/10.1016/j.foodcont.2005.08.010� https://doi.org/10.1098/rstb.2015.0024� https://doi.org/10.1098/rstb.2015.0024� https://doi.org/10.1016/j.vetmic.2016.05.010� https://doi.org/10.1111/j.1365-2672.2011.05141.x� https://doi.org/10.1111/j.1365-2672.2011.05141.x� https://doi.org/10.1016/j.diagmicrobio.2015.03.015� https://doi.org/10.1016/j.diagmicrobio.2015.03.015� https://doi.org/10.1021/acs.jafc.8b04549� https://doi.org/10.1021/acs.jafc.8b04549� https://doi.org/10.4315/0362-028x-63.5.620� https://doi.org/10.4315/0362-028x-63.5.620� https://doi.org/10.1016/j.fshw.2015.11.003� https://doi.org/10.1016/j.fshw.2015.11.003� https://doi.org/10.1016/j.fshw.2017.06.002� https://doi.org/10.1016/j.fshw.2017.06.002� https://doi.org/10.1007/s11101-012-9242-8� https://doi.org/10.1007/s11101-012-9242-8� https://doi.org/10.1016/j.ijfoodmicro.2017.11.024� https://doi.org/10.1016/j.ijfoodmicro.2017.11.024� https://doi.org/10.1016/j.ijid.2018.11.221� https://doi.org/10.1016/j.fm.2019.03.012� https://doi.org/10.1016/j.fm.2019.03.012� https://doi.org/10.1016/s0924-2244(00)88941-4� https://doi.org/10.1016/j.meatsci.2013.12.003� https://doi.org/10.1016/j.fct.2010.09.006� https://doi.org/10.1021/jf048051t� https://doi.org/10.1002/jsfa.9082� https://doi.org/10.1002/jsfa.9082� https://doi.org/10.1016/j.fm.2018.12.010� https://doi.org/10.1016/j.fm.2018.12.010� _hlk55821032 _hlk55821512 _hlk55818494 _hlk55819205 _hlk55819256 _hlk53738592 _hlk54082044 _hlk55478207 _hlk54356156 _hlk55478097 ijfs#208_gamli_bozza   ital. j. food sci., vol 28, 2016 178 review the health effects of oleuropein, one of the major phenolic compounds of olives, olea europaea l. ö.f. gamli korkut ata university food engineering department osmaniye, turkey *corresponding author. tel.: +90 328 827100/3652 e-mail address: farukg69@gmail.com abstract olives (olea europaea l.) and olive oils have a significant place in the daily diet in the mediterranean region and contain significant amounts of polyphenolic compounds, of which oleuropein, hydroxytyrosol and tyrosol are dominant. harvest time, geographical region and climate all affect the phenolic content. experimental, clinical and epidemiological studies show the beneficial effects of oleuropein, such as antioxidant, antimicrobial, anticancer anti-inflammatory, antineuropathic and other properties. in particular, olive leaves, roots, virgin olive oil and olive mill waste (vegetation and wastewater) are potential sources of oleuropein. this review focuses on the pharmacological effects of oleuropein and extraction procedures for oleuropein. keywords: olive, phenolic compounds, oleuropein, pharmacological effects, olea europaea, extract   ital. j. food sci., vol 28, 2016 179 1. introduction olives are fruits and are a significant part of the daily diet, playing a vital role in the agricultural sector especially in the mediterranean countries and having high economic value in this region. in addition to their popular usage as table olives, they have become extremely valuable because they have important nutrient properties and have shown positive benefits for human health. the majority of olives produced globally are used in oil production (approximately 85-90%), and the rest is used in the production of table olives. although turkey comes behind spain, italy and greece in oil flesh and table olive production (fao, 2011), it is reported that olive producing regions in this country are in order of aegean (55.11%), marmara (27.72%), mediterranean (14.94%) and black sea region (2.22%) and that 75-80% of total oil production of olive oil is made in the aegean region (arslan and ozcan, 2011). it is reported that as of 2010, turkey has 6 million of olive trees and 2 million of table olive trees, and 73% of total olive areas and olive production is made of oil types and 27% comprises table olives. olive oil production was approximately 1,040,000 tons and table olive production was 375,000 tons according to statistical data in 2011 (dogaka, 2011). olive trees (olea europaea l.) are included in the oleaceae family. the most important characteristic of olive fruits is their high oil content, chemical composition and a unique bitterness in comparison to other fruits with hard seeds (othman et al., 2008; mafra and barros, 2006). the typical bitterness of olive fruits results from their rich content of phenolic compounds, especially of oleuropein, in olive cultivars (fig. 1). these phenolic compounds and oleuropein content of olive cultivars increase the interest in their antioxidant properties and their positive effects on health (arslan and ozcan, 2011; barbaro et al., 2014;cardeno et al., 2013). it has been reported that, in addition to their mono-unsaturated fatty acid content, lower-concentration compounds are the reason for their positive effects on nourishment. the amount of phenolic compounds ranges between 1-2% in fresh fruits, and that these are mainly secondary metabolites in olives (othman et al., 2008). phenolic acids, phenolic alcohols, flavonoids and secoiridoids are also important phenolic compounds in olive cultivars. iridoids and secoiridoids are compounds that are bound to glycosides and are formed from the secondary metabolism of terpenes as precursors of various indole alkaloids. it is reported that the secoiridoides in oleaceae are usually derived from the oleoside type of glycosides, which are characterized by an exocyclic 8,9-olefinic functionality. oleuropein is an ester that forms from 3-4dihydroxyphenyl ethanol and hydroxytyrosol and has the oleosidic skeleton. it is reported that demethyloleuropein, oleuropein aglycone, elenolic acid and verbascoside are essential compounds of hydroxycinnamic acid derivative in olive cultivars (romani et al., 1999; servilli et al., 2004; romero et al., 2002; esti et al., 1998; amiot et al., 1986).   ital. j. food sci., vol 28, 2016 180 figure 1: chemical structures of some of the phenolic compunds that are found in the oleacea family (yildiz and uylaser, 2011). factors such as variety, geographical location, maturing progress, harvest time and seasonal conditions all affect phenolic substance amounts in olives. it was stated that the total precipitation content during the period until harvest time affected phenolic substances (romero et al., 2003; esti et al., 1998), and there were decreases in phenolic concentrations of olive oils from fruits of trees cultivated in irrigated areas or under high rainfall conditions when compared to the oils from non-irrigated or low rainfall regions (tovar et al., 2002; yousfi et al., 2006; botia et al., 2001). contrary to this situation, it was indicated that phenolic contents were higher in olives in the alanya region, which receives a relatively high rainfall (arslan and ozcan, 2011). the phenolic contents of olives that were cultivated in higher altitude locations were greater than those from lower altitude regions (mousa et al., 1996), and that high altitude did not affect the content of phenolic substances of sariulak olives that were grown in different regions. it was stated that harvest time affected the phenolic substances in olives; and phenolic contents changed based on olive harvests made at different times during september-december months. it was indicated that at the second harvest period around the october-november months, rutin, verbascoides, taxifolin and tyrosol contents increased, and in addition, phenolic contents decreased toward the last periods of the harvest time. it was reported that among the phenolic substances present in sariulak olives, apigenin, cinnamic acid and p-coumaric and 4-hydroxybiphenyl carboxylic acid contents decreased, whereas ferulic and vanillic acid contents increased as the harvest time progressed (arslan and ozcan, 2011). 2. oleuropein and its formation olive fruits contain a high level of mono-unsaturated oils and there are significant amounts of phenolic substances. phenolic compounds are good antioxidants and contribute to the prevention of illnesses due to their important biological activity characteristics (othman et al., 2008). the antioxidant capacities of phenolic substances   ital. j. food sci., vol 28, 2016 181 originate from their reducing characteristics, which enable them to react with mono oxygen (rice-evans et al., 1997). it is stated that there are approximately 40 phenolic compound when olive plants are considered as a whole as a tree, branch, root, and olive fruit. among the phenolic compounds the concentrations of, especially, oleuropein, hydroxytyrosol, lutolein, rutin, trans cinnamic acid and tyrosol are higher compared to other phenolic substances (yorulmaz et al., 2013; arslan, 2012; arslan and ozcan. 2011). oleuropein, dimethyl-oleuropein, ligstride and oleoside compounds indicate dominance of phenolic oleosides in olive fruits and, in addition to this, verbascoside is the main derivative of hydroxycinnamic acid in olives. oleuropein is the most important phenolic compound in olive cultivars and its concentrations can reach up to 140 mg g-1 (db) levels in some olive species; and the content ranged between 60-90 mg g-1 (db) of dry matter in young olive cultivars (amiot et al., 1986; le tutour and guedon, 1992). oleuropeins, classified within the secoiridoids, exist in varying concentrations in olive plants, mostly in tree stems, branches, leaves and olive fruits. iridoid and secoiridoids that are bound to glycosides are compounds formed in the secondary metabolism of terpenes which are the precursors of various indole alkaloids. it is reported that oleuropein (fig. 2) is an ester of 2-ethanol (3,4-dihydroxyphenyl) and oleuropein aglycone have an oleoside structure known as secoiridoide glucoside (soler-rivas et al., 2000). figure 2: chemical structures of oleuropein and oleuropein aglycone (yıldız and uylaser, 2011). oleuropein production in olives (fig. 3) occurs by branching in the mevalonic acid cycle in the secondary metabolism of terpenes during the formation of oleosides (damtoft et al., 1992). secoiridoids are derived from these compounds and the carbon skeleton is derived from mevalonic acid. geraniol, 10-hydroxygeraniol and iridoidal are regarded as the precursors of loganin. deoxyloganic acid, 7-epiloganic acid and loganic acids are the precursors of oleuropein, which are incorporated in to the ligstrosides (damtoft et al., 1993).   ital. j. food sci., vol 28, 2016 182 figure 3: formation of oleuropein in the olea family (yildiz and uylaser, 2011). in studies conducted on the contents of oleuropein which is responsible for the bitter taste in olive cultivars, its content ranged between 2.1-134 mg g-1(db) in olive leaves, 11-18.9 mg g-1(db) in olive branches, 1.9-6 mg g-1 (db) in olive roots, 0.3-21.7 mg g-1 (db) in olive fruits and 0-11.2 mg g-1 (db) in extra virgin olive oil (ansari et al., 2011; savournin et al., 2001; tayoub et al., 2012; malik and bradford, 2006; altinyay and altun, 2006; yorulmaz et al., 2013). in non-virgin oils, oleuropein contents range between 0-0.4 mg g1 after oil extraction and oleuropein is also found in pomace and in the liquid portion (vegetation and waste water) except for oil obtained as a result of olive oil pressing that is found in ratios between 6.5-8.7 mg g-1 (goldsmith et al., 2014; allouche et al., 2004; yildiz and uylaser, 2011). oleuropein formation in olive fruits takes place at the order of growth stage, green maturation stage and black maturation stage. during the growth stage, oleuropein accumulates in olive fruits and amounts of chlorophyll and oleuropein decrease at the green maturation stage. at the black maturation stage in olives, a decrease in oleuropein contents takes place and anthocyanin pigments are formed (amiot et al., 1989). it is proposed that oleuropein content is high at the beginning of the fruit growth in young olive fruits (yorulmaz et al., 2013). although there is a significant amount of oleuropein in olives that are harvested when the olive fruits are green, the oleuropein content decreases rapidly with the black maturation phase (bianco et al., 1993). although phenolic compounds in table olives are initially higher, in the production of table olives the oleuropein concentration decreases during maturation and disappears at full ripeness. the oleuropein concentration also decreases sharply after salt treatment and disappears after lye treatment. it is also reported that the phenolic contents of table olives showed different phenolic profiles such as hydroxytyrosol concentration of olive drupes, which decline rapidly during the black maturation phase, which are explained by the fact of climate, soil type and growing region (othman et al., 2008).   ital. j. food sci., vol 28, 2016 183 3. oleuropein and its pharmacological effects the significant effects of phenolic compounds that are present in plants have increased interest for the use of these substances in the preparation and processing of foodstuffs. olive fruits and oil that are produced in the mediterranean region have a key position in the daily diet and they are good resources in terms of oleuropein, which is the most prominent phenolic substance (arslan and ozcan, 2011). oleuropein, which is found in varying quantities in olive fruits, branches, leaves and roots of olive trees, has many positive effects on human health according to clinical, epidemiological and experimental investigations conducted on the health effects, such as antioxidant (visioli et al., 2002; gonzales-hidalgo et al., 2012), anti-atherogenic, anti-inflammatory (visioli et al., 1998), anti-cancer, antimicrobial and antiviral effects (tripoli et al., 2005; fredrickson and group, 2000). effects of oleuropein on lipid oxidation are shown in the decrease in by-products (malondialdehyde and 4-hydroxynonenal) that react with 2-thiobarbituric acid (tba) and produced by oxidation. it is stated that antioxidant potential of oleuropeins is related to the ability of radical stability development by forming a hydrogen bond within the molecule between the free hydrogen of hydroxyl groups and phenoxy radicals and the higher content of oleic acid that are present in virgin oils compared to other edible oils and the presence of high level of antioxidant compounds in olives and olive oils (hydroxytyrosol and oleuropein) have less sensitivity to oxidation in comparison to n-6 unsaturated fatty acids. (barbaro et al., 2014). oleuropein has protective effects in the decrease of plasmatic levels of cholesterol in the total, free and ester forms in rabbits for improving the resistance of low density lipoproteins to oxidation (coni et al., 2000) and also oleuropeins have the ability to clear nitric oxide (no). in addition, this causes an increase in inducible nitric oxide synthesis in cells (de la puerta et al., 2001). hypochlorous acid, which is formed as a result of oxidation by neutrophil myeloperoxidases at the site of inflammation and can cause damage to proteins including enzymes (visioli et al., 2002). there are many studies that have considered the effects of oleuropein on health. the consumption of virgin olive oil, which is rich in nutrients, have an observed antitumor effect and decrease the proliferation of cancer cells, especially of the colon, breast and skin (cardeno et al., 2013; psaltopoulou et al., 2011). it is reported that an application of a 400 µm dose of oleuropein and hydroxytyrosol cause a significant decrease in the cell proliferation of colon cancer (ht-29) 24-hour after treatment (cardeno et al., 2013). it is stated that the oleuropein aglycone compound is the most effective phenolic compound for decreasing the viability of cancer cells in breast, colon and kidney cancer types (menendez et al., 2007;hamdi and castellon, 2005). oleuropein that has been extracted from olive leaves has marked antitumor effects on prostate, breast and hepatoma cancer cells, depending on the amount of dose, 24-72 hours after application (kockar et al., 2010). the effects of oleuropeins on the various cancer types and cells are shown in table 1.   ital. j. food sci., vol 28, 2016 184 table 1: oleuropein-induced anti-tumor effects in different cancer cells. cell line cancer type references mcf-7;mda;t-47d breast adenocarcinoma and ductal carcinoma (hamdi and castellon, 2005) 786-o renal cell adenocarcinoma (hamdi and castellon, 2005) rpmi 7951 melanoma (hamdi and castellon, 2005) ht-29,caco-2,lovo colorectal adenocinoma (cardeno et al., 2013;corona et al., 2007) pc-3,mcf-7,hep3b prostate,breast,hepatoma (kockar et al., 2010) lncap and du145 prostate cancer (acquaviva et al., 2012) a549 lung carcinoma (mao et al., 2012) the antitumor effect of oleuropein is related to its anti-angiogenic function. there are many different varieties of tumors that are connected with other cells and these are formed by complex interactions in a permeable microstructure. the development of a tumor mass is formed in the ‘nutrition’ environment, and in growing initially forms a deoxygenated environment. the formation of new blood vessels occurs by stimulating multiplication and activities of endothelial cells (barbarro et al., 2014). skin that is exposed to solar uv radiation is damaged and also skin thickness and elasticity are reduced, which makes it more vulnerable to skin cancers. it is reported that oleuropein and olive leaf extracts from were administered orally twice for 30 weeks in hairless mice. both the extract and oleuropein (0.3 and 1 g kg-1) significantly inhibit the increases in skin thickness, reductions in skin elasticity, skin carcinogenesis and tumor growth (kimura and sumiyoshi, 2009). antimicrobial agents are used to control microbial growth and many synthetic antioxidant substances are used for this purpose. there is an increase in the use of natural substances instead of synthetic substances due to their side effects on human health. oleuropein has been considered as a natural antioxidant substance to reduce the growth rate of microorganisms. it has been reported that the phenolic compound oleuropein in virgin olive oil has antimicrobial activity towards l. plantarum, b. cereus and s. enteritidis among both gram positive and gram negative bacteria (cicerale et al., 2012; aziz et al., 1998). the toxic effect of oleuropein is higher for gram (+) bacteria than for gram (-) bacteria, especially s. aureus and e. coli (furneri et al., 2002). oleuropein also has antimycoplasmal properties and consequently has positive effects against mycoplasma bacteria strains, which are resistant to antibiotic applications (furneri et al., 2002). although the antimicrobial activity mechanism of the oleuropein component has not been explained completely, it has been suggested that the microbial activity mechanism of oleuropein originates from the presence of an ortho-diphenolic (catechol) system in the environment and that it changed the ability of a glycoside group, enabling it to enter through the cell membrane and reach the target region (saija and uccella, 2001). it was expressed that oleuropein that had been extracted from olive leaves also has antimicrobial characteristics towards campylobacter jejuni, helicobacter pylori and staphylococcus aureus, which are resistant to methicillin, and extracts of olive leaves played a crucial role in the arrangement of the composition of stomach flora by reducing h. pylori and c. jejuni microorganisms (sudjana et al., 2009).   ital. j. food sci., vol 28, 2016 185 4. different analytical approaches for oleuropein extraction from olive leaves oleuropein is the most significant component among phenolic compounds present in olive cultivars. it is present in a significant amount in the development phases of olive fruits, but its content starts to diminish along with maturation and decreases to near zero after the olive fruits turn from green to black. there is a demand for extracting and purifying of oleuropein existing in the olive cultivars, especially from olive leaves, because of their pharmacological characteristics and favorable effects on health. there are many methods for extracting such phenolic substances from olive leaves, such as extraction by using organic solvents at high temperature and pressure, liquid-liquid extraction (japonlujan and luque de castro, 2006), super-critical liquid extraction (sahin et al., 2011), polar substances extraction (malik and bradford. 2008), solid phase extraction, acidified de-ionized water extraction (stamatopoulos et al., 2012), steam blanching, hot water and ultrasound extraction (ansari et al., 2010). oleuropein is more readily extracted from olive leaves due to their higher content of oleuropein compared to the fruits. prior to the extraction of oleuropein, olive leaves are dried at temperatures not exceeding 60°c and then the dried leaves are crushed to granules with their dimensions ranging between 0.9-2 mm. following this, the phenolic substances and oleuropein are extracted. the stability and content of oleuropein differs, depending on the extraction method used. it is reported that a higher amount of oleuropein is extracted by using 80% methanol compared to extraction by hexane, ethanol, hot water or a mixture of methanol/hexane (sahin et al., 2011; malik and bradford, 2008). the oleuropein is quite stable for 30 days at room conditions and also stable in aqueous extracts for 7 days when stored at room temperatures but is degraded after 17 days (malik and bradford, 2008). drying of olive leaves at 25°c has no effects on the oleuropein and verbascosides contents at room conditions but drying leaves at greater than 60°c causes a decrease in phenolic substances (malik and bradford. 2008). the extraction of oleuropein from leaves by using acidified de-ionized water (ph: 3, 60°c) has a marked effect on the color of the leaf extract (green) and oleruopein content; and also this method has the advantage of the non-toxic characteristics of acidified de-ionized water (ansari et al., 2011). among the treatments of steam blanching, hot water and uvc irradiation, the steam extraction method provides an increase in oleuropein yield from 25 to 35 times compared to non-steam blanching samples whereas antioxidant activity increases from 4 to 13 times. the particle size of leaves (1-3 mm) used affects the extraction of phenolic contents and oleuropein and the ethanolic extraction of hot water blanched leaf fractions produces higher amounts of oleuropein compare to the untreated samples (stamatopoulos et al., 2012). ethanol, methanol and water are considered as cosolvents in 20% (v/v) for the extraction of oleuropein from olive leaves, whereas the maximum oleuropein yield is obtained by supercritical fluid (co2) extraction modified with 20% methanol (sahin et al., 2011). 5. results and conclusions olives have a crucial place in the daily diet in the mediterranean region. when they are examined in terms of ingredients, in addition to mono-unsaturated fatty acids, there are 15-40 phenolic substances in olive fruits, leaves, branches and roots; and oleuropein is found to be the most prominent substance among the phenolic compounds. the amount of rainfall, geographical region, harvest time and altitudes affect the phenolic contents in olive cultivars and there is a close relationship between phenolic compounds and olive   ital. j. food sci., vol 28, 2016 186 maturation. it is crucial that when prompted to maximum extract of oleuropein, color changes of olive fruits should be monitored in terms of phenolic contents and oleuropein. oleuropein has many pharmacological effects such as antioxidant, antimicrobial, antiinflamatory, antitumor and neuroprotective effects on neural disorders emerging with aging (table 2). table 2: biological activities of oleuropein that found in olive fruits,leaf and virgin olive oils. activity effects antioxidant inhibition of oxidation of ldl,improvement of radical stability anti-tumor antiproliferative,anti-migration,angiogenesis and apoptosis effect antimicrobial bacterial cell membrane damage neuroprotective tau fibrilization inhibition,oxidative stress reduction anti-inflammatory lipoxygenase and cytokine inhibition it has been observed that oleuropein extracted from olive cultivars can be used in the prevention of proliferation of different cancer cells such as breast, skin, lung, colon cancers and also neuronal disorders occurring due to aging. it is possible to obtain oleuropein naturally and due to its pharmacological effects it is likely to be used as an antioxidant and antimicrobial agent in the food preparation or preservation and health sectors. among the available extraction methods of oleuropein from olive cultivars, especially from leaves, polar substances are being used according to color, antioxidant activity and stability of oleuropein at room conditions. some pre-treatments of the extraction procedure can be applied to increase extraction yield from olive leaves such as use of supercritical fluid, steam blanching and grinding dried olive leaves to the size of 0.9-3 mm. acknowledgements the author would like to convey his thanks to prof. i̇brahim hayoglu at the faculty of agriculture, university of harran, şanliurfa, turkey for his critical comments and suggestions. references acquaviva r., di giacomo c., sorrenti v., galvano f., santangelo r., cardile v., gangia s., d’orazio n., abraham n.g. and vanella l. 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(2006). changes in quality and phenolic compounds of virgin olive oils during objectively described fruit maturation, eur. food res. tech. 223: 117-124. paper received july 10, 2015 accepted september 26, 2015 ijfs#1438_bozza ital. j. food sci., vol. 31, 2019 437 paper effects of probiotic fermentation of selected milk and whey protein preparations on bioactive and technological properties k. skrzypczak1*, e. fornal2, a. waśko3 and w. gustaw1 1department of plant food technology and gastronomy, department of fruits, vegetables and mushrooms technology, faculty of food science and biotechnology, university of life sciences in lublin, skromna 8, 20-704 lublin, poland 2department of pathophysiology, medical university of lublin, jaczewskiego 8b, 20-090 lublin, poland 3department of biotechnology, microbiology and human nutrition, faculty of food science and biotechnology, university of life sciences in lublin, skromna 8, 20-704 lublin, poland *corresponding author: katarzyna.skrzypczak@up.lublin.pl abstract the objective of this study was to compare the effect of probiotic fermentation (conducted by l. acidophilus la-5) of milk containing an addition of selected whey or milk protein preparations (whey protein concentrates, whole milk, whey protein isolate, casein glycomacropeptide or α-lactalbumin) on the technological and functional properties of obtained milk beverages. determination of the antioxidative activities and identification sequences of biologically active peptides generated in selected protein preparations during probiotic fermentation also constituted the aim of the research. the results obtained indicate that the addition (1%) of α-lactalbumin into regenerated skimmed milk in the highest degree reduced the syneresis level in fermented products. also, αlactalbumin hydrolysates exhibited the strongest antioxidative properties (57.57±6.6%), while casein glycomacropeptide hydrolysates allowed us to obtain the highest amount of various biopeptides. keywords: biopeptides, lactic acid fermentation, probiotics ital. j. food sci., vol. 31, 2019 438 1. introduction the area of functional food that becomes a part of an everyday diet (nagpal et al., 2012) and the significance of many dairy products, especially fermented ones, is dynamically developing. nowadays, it is observed that the consumers’ awareness of the role of diet and beneficial health effects of some food compounds is on the increase, a fact also reflected in higher consumption of fermented milk products. simultaneously, the requirements of and demand for various the health-promoting features of those kinds of products have been growing too. therefore, to meet those expectations, scientists and the dairy industry are aiming at improving the technological and functional properties of dairy products as well as creating new ones. proteins are the most valuable constituent of whey protein isolates (wpi) and whey protein concentrates (wpc) that supply good nutritional quality and enhance the functionality of many product formulations (gustaw and mleko, 2007; jeewanthi et al., 2015). it is suggested that selected whey protein constituents (e.g.α-lactalbumin, lactoferrin, serum albumin lactoperoxidase, β-lactoglobulin, bovine) and peptides deriving from them exhibit, among others, an anticarcinogenic activity, may stimulate the immune system and influence metabolic processes (gobbetti et al., 2002). therefore, they constitute an important source of bioactive ingredients that might find an application, e.g. in the preparation of some of the functional food and therapeutic formulations (kabašinskienė et al., 2015). furthermore, apart from nutritional aspects, whey (as well as products deriving from it) is also a functional component that might influence some products’ properties, including colour, flavour, and texture (liutkevičius et al., 2016). thus, incorporation of selected milk and whey protein preparation might not only enhance the technological properties of a product but might also positively influence the functional properties. this is largely associated with the fact that milk and whey proteins are precursors of biologically active peptide sequences (biopeptides), which exhibit a wide range of desired beneficial health effects affecting the regulation of the human body’s system functions (mohanty et al., 2016; lucarini, 2017). however, biopeptides enclosed within a native structure of milk and whey proteins are inactive, whereas enzymatic hydrolysis with digestive enzymes as well as fermentation process conducted by a starter culture with an efficient proteolytic enzyme system contributes to the release of various sequences of active peptides (korhonen and pihlanto, 2003; michaelidou, 2008). moreover, bacteria involved in the fermentation processes not only contribute to the formation of bioactive substances that give food product a functional characteristic, but determined strains meeting certain requirements may also constitute a functional food component. the microorganisms claimed as probiotics constitute another important functional food constituent that might be introduced to milk products. an activity of probiotics contributes to the modification of the microbiota composition in the host’s intestines, which influences some of the physiological process leading to favourable health consequences. the health-promoting effects caused by probiotics concern their desired influence on maintaining proper intestine micrflora (normalization of the bacteria composition, e.g. after antybioticoteraphy), hypocholesterolemic or anticarcinogenesis effects, inhibition growth of some pathogens, stimulating the immunity system and also alleviation of some of the allergy, including food allergies and lactose intolerance (sarkar et al., 2016). the objective of this study was to compare the effect of probiotic fermentation (conducted by l. acidophilus la-5) of milk containing the addition of chosen whey and milk protein preparations on technological and functional properties of obtained milk beverages. the study was also focused on evaluation of the antioxidative activities and determination of ital. j. food sci., vol. 31, 2019 439 the biologically active peptide sequences generated during probiotic fermentation of selected protein preparations. 2. material and methods 2.1. microorganism and growth conditions the probiotics strain lactobacillus acidophilus la-5 (chr. hansen, poland) was stored at -80°c in manrogosasharpe broth (btl, łódź, poland) containing 15% (v/v) glycerol stock. for further analysis, the strain culture was activated and routinely cultured (2%v/v of inoculum) in mrs broth and incubated at 37°c for 18h under aerobic conditions. 2.2. fermentation of milk with the addition of protein preparations the samples of 13% regenerated skim milk (osm krasnystaw, poland) were enriched by the 1% (w/v) addition of one of the following protein preparations: whole milk powder (osm krasnystaw, poland) wmp; whey protein concentrates (polsero, poland) wpc 30 and wpc40; whey protein isolate (milei gmbh, allgau, germany) wpi; casein glycomacropeptide (arla food, denmark) cgmp and α-lactalbumin (arla food, denmark) α-la. samples of milk without any protein addition (rsm) were used as a control variant. all obtained sample variants were pasteurized (80°c/30min), then cooled down to ambient temperature and inoculated by 1% (v/v) of lactobacillus acidophilus la-5 cell suspension, which was previously prepared as follows: an overnight culture of analyzed probiotic strain (grown in mrs broth) was used to inoculate fresh medium (sterile mrs broth) to obtain an optical density at 600 nm (od600) of 0.05 (helios gamma; thermo fisher scientific), subsequently the bacteria strain was cultivated at 37 °c until an od600 of 0.7 (exponential phase). afterwards, bacteria cells were harvested by centrifugation at 8000g for 10 min at 4 °c (mpw 350-r; mpw). the pellet was washed twice with sterile phosphate-buffered saline (pbs, ph 7.0) and resuspended (also in pbs) to obtain cell suspension of od600 equal to 0.3 that was used for inoculation. then, inoculated samples of all milk variants were transferred in equal volumes (40ml) into sterile packages and sealed. after the process of fermentation (42°c/12h, thermostatic method), all samples were cooled down up to 4°c and maintained in this temperature for another 12h before further analysis. 2.3. texture profile analysis to compare the textural properties of the fermented products received, the texture profile analysis (tpa) was performed, using a ta-xt2i (stable micro systems, godalming, uk), according to the procedure described by gustaw et al. (2016). the parameters hardness, fracturability springiness, cohesiveness, gumminess and chewiness were included in the analyses. 2.4. spontaneous syneresis assay the acidic milk gels (the fermented final products) obtained were analyzed also in terms of their ability for water binding. therefore, the level of syneresis in received products was measured according to the method described by amatayakul et al. (2006). ital. j. food sci., vol. 31, 2019 440 2.5. hydrolysis of milk and whey protein preparations aqueous solutions (1% w/v) of skim milk powder (smp) and all above-mentioned protein preparations were pasteurized in a water bath (80 ˚c/30 min). after cooling down up to 35 °c, all variants of solutions were inoculated (1% v/v) by previously prepared inoculum (uninoculated samples were control variants) and incubated at 37 °c for 24 h. subsequently, all samples were heated at 100 °c for 5 min in water bath to inhibit the hydrolysis process and inactivation of bacterial enzymes. then, all samples were filtered (syringe filter ø = 0.45 μm) and subjected to further analysis. 2.6. antioxidant activity an antioxidant activity in obtained filtrates (of fermented and nonfermented 1% (w/v) aqueous solutions of skim milk powder [smp] and all analyzed protein preparations) were determined as an ability for free radical scavenging, using alcoholic solution of dpph (1,1-diphenyl-2-picrylhydrazyl, sigma-aldrich, poland) in accordance with the method described by namdari and nejati (2016). briefly, the analyzed samples were diluted with phosphate buffer (0.1 m) in ratio 1:4. afterwards, to 1 ml of each of the diluted samples 2.5 ml of 0.1 mm dpph (in 60% methanol) was added and vigorously mixed. after 30 min of sample incubation (in darkness at ambient temperature), the absorbance was measured at λ=517 nm. the determination of the value of the analyzed activity for tested samples was carried out in a five-fold repetition (n=5). antioxidant activity (radical-scavenging activity) was expressed as % inhibition of dpph-absorbance that was calculated using the equation: inhibition [%] = [(ac-as)/ac] x100 where, as absorbance (λ=517 nm) of the test sample, and ac the absorbance (λ=517 nm) of the control sample consisting of 1 ml phosphate buffer mixed with 2.5 ml of dpph (the methanolic solution of free radicals). 2.7. liquid chromatography-high-resolution mass spectrometry (lc-hrms) and peptide sequencing to analyze the biopeptides content, those protein preparations characterized by the highest antioxidative activities and the ones that allowed obtaining the fermented products with the most desirable textural properties were selected. the analysis was conducted through the previously described procedure (skrzypczak et al., 2017) using agilent mass hunter acquisition b.05.01 software to acquire data. the data analysis and peptide mapping were performed using the agilent mass hunter qualitative analysis b.07 with integrated bioconfirm add-on software. 2.8. determination of the biological activities of peptide sequences to determine the profiles of potential biological activities, the obtained peptide sequences were subjected to further analysis that was performed according to the procedure included in the databases biopep-uwmp (minkiewicz et al., 2008; dziuba et al., 2009; www.uwm.edu.pl/biochemia/index.php/pl/biopep) and biopepdb (bis.zju.edu.cn/ biopepdbr). ital. j. food sci., vol. 31, 2019 441 2.9. statistical analysis statistical analysis was performed using the statistica 8.0 (statsoft, poland) program. to evaluate the differences between means values, the data were subjected to analysis of variance (anova) using tukey’s test with a level of significance set at p<0.05. the similarities of the textural properties analyzed between obtained fermented final products were determined on the basis of the results of bottom-up hierarchical cluster analysis using average linkage clustering as a linkage criterion (upgma). to avoid the effect of the differences in measurement units between the parameters on the values of euclidean distances, the data were standardized prior to the analysis. 3. results and discussion 3.1. effect of addition of selected protein preparation on the textural properties and syneresis level of fermented products the fermented milk products obtained with the addition of analyzed protein preparations exhibited some variety in terms of texture properties as well as level of spontaneous syneresis (table 1). analyzing hardness parameter, there were no statistically significant differences (p<0.05) between control variant and products containing the addition of wpc30, wmp or α-la additions. the highest values of measured parameter were exhibited by samples with addition of cgmp, but simultaneously these fermented products were characterized by the most intensified syneresis effect (table 1). this might be caused by the formation of a strong bond between generated products of hydrolyzed cgmp that influenced the moulding a strong structural network increasing the strength (hardness) of the gels and enhancing water disposal from the structure of curds. regarding the values of fracturability as well as cohesiveness (table 1), there were no differences (p<0.05) between analyzed products. however, comparative analysis of all textural parameters indicated that, in terms of similar of texture profiles, the obtained variants of fermented products might be divided into three clusters consisting of pairs of fermented products’ variants containing one of the tested protein preparations’ addition (wmp with α-la, control variant and wpc30, wpi and cgmp) (fig. 1). moreover, the results of cluster analysis indicated that in terms of values of analyzed texture profile parameters (springiness hardness, fracturability, cohesiveness, gumminess and chewiness), fermented beverages containing a wpi or cgmp additive were distinguished from all tested products and constituted a separate and farthest cluster that represents textural properties (fig. 1). the addition of whey protein concentrate (wpc 30 and wpc40) or cgmp to milk increased the intensity of syneresis in fermented products, while acidic milky gels containing the α-la or wmp addition were characterized by the highest water-binding capacity exhibiting the lowest syneresis effect (table 1). the obtained results are in accordance with jovanović et al. (2005), who suggested that α-la influence the increase in the hydrophilic properties of the coaggregates at ph 4.5. moreover, the study of matumoto-pintro et al. (2011) indicates that a higher proportion of α -la in the yoghurt ingredients or partial hydrolysis of whey protein polymers inhibit the rate of sedimentation in the fermented product. ital. j. food sci., vol. 31, 2019 442 figure 1. the result of upgma analyses. diagram expresses the similarity of texture characteristics between fermented products regarding all parameters measured in texture profile analysis; the similarity between particular groups of products was expressed as distance [%]; rsm is the control sample (fermented regenerated skim milk [13 %] without addition of any protein preparation). it was claimed that the gelation properties of whey proteins are dependent on hydrolysis conditions and the degree of conducted enzymatic process (jeewanthi et al., 2015). the process of gel formation is an effect accompanying the fermentation conducted by starter cultures. therefore, the modification conditions of the hydrolysis process enable the creation of gels with various rheological properties (creusot and gruppen 2007; pouliot et al., 2009; jeewanthi et al., 2015). moreover, the results obtained might suggest that preferences of the probiotic strain to composition of fermenting medium (rsm samples containing selected protein preparations) and the specificity of bacterial enzymes toward selected milk and whey proteins might also influence the degree of hydrolysis and various characteristics of received gels (fermented products). it was implied that some of the generated peptide sequences might also initiate the aggregation process of whey protein hydrolyzates that influence the textural and rheological characteristic of fermented milk products. in wpi hydrolysates obtained using bacterial enzymes, creusot and gruppen (2007) identified such peptides and determined their sequences: β-lg [f1-45], β-lg ab[f(90-108)]-s-s-α-la [f(50-113)], α-la[f(12-49)]-s-s-α-la [f(50-113)], β-lg ab[f(90-108)]-s-s-α-lg ab[f(90-108)], β-lg a[f(90-157)] and β-lg ab[f(135-157/158)]. it was reported that in the production of yoghurt, the addition of wpi to milk enhances the formation of disulfide bonds during fermentation, which influences the increase in the mechanical resistance of received acidic gel (matumoto-pintro et al., 2011). however, in the presented study the results indicate that the 1% addition of wpi (compared with the control sample) has no significant effect on the improvement of textural property in terms of gel hardness. study results presented by dąbrowska et al. (2017) revealed that the addition of whey protein hydrolysates (instead of other protein preparations like smp or wpc80) to milk in yoghurt production might contribute to the increase in counts of starter culture bacteria at the initial stage of fermentation. moreover, the addition of some whey protein ital. j. food sci., vol. 31, 2019 443 hydrolysates into milk improves the viability of probiotic bacteria in the final products (zhao et al., 2006; dąbrowska et al., 2017). also, the prebiotic-like properties of some whey proteins (including wpc) have been confirmed (gustaw et al., 2016). this positive effect of selected milk and whey protein preparations, as well as their hydrolysates on the growth of the desired strains of probiotic bacteria, may strengthen the potential of the health-promoting properties of various fermented products. 3.2. comparing the antioxidant activity of milk and whey protein preparations’ hydrolyzates the fermentation process conducted using the probiotic strain (l. acidophilus la-5) influenced the increase in the ability of analyzed protein preparation solutions to free radical scavenging (fig. 2). unfermented samples of smp, wmp, wpc 30 and wpc40 exhibited a similar level of dpph radical scavenging activity (differences, not statistically significant, p> 0.05). moreover, it was noted that the hydrolysis process carried by tested bacteria improved the antioxidative properties of all analyzed protein preparations (fig. 2). figure 2. antioxidant activity of analyzed protein preparations in dpph assay. differences between mean values (x; n=5) of antioxidant activity in obtained filtrates of tested samples (fermented and unfermented aqueous solutions (1% w/v) of skim milk powder [smp] as control variant of samples and all analyzed protein preparations) denoted by different letters are statistically significant (p<0.05); error bars express the standard deviation (± sd). the samples of α-la and cgmp fermented by probiotic strain exhibited the strongest hydrogen-donating capacity among analyzed solutions of protein preparations (57.57±6.6% and 54.87±1.3%, respectively). differences in the value of antioxidative activity recorded for both types of samples were not statistically significant (p> 0.05), while the lowest values were noted for samples of smp before (24.90±0.81%) as well as after hydrolysis (29.53±3.0%). these results are in accordance with rahmawati and suntornsuk (2016), who suggested that increased antioxidant activities in yoghurt (compared to raw milk material) might be associated with the released peptides (antioxidative) by bacterial proteolitic enzymes from protein molecules during the fermentation process. the peptides generated through protein hydrolysis as well as some products of the bacteria metabolism exhibit properties of electron donors and react with free radicals (dpph) to achieve more stable molecule (kullisaar et al., 2002). 0 20 40 60 80 smp wmp α-la wpc40 wpc30 cgmp wpi in hb it io n [% ] type of analyzed protein preparation fermented unfermented ital. j. food sci., vol. 31, 2019 444 3.3. biological activities of peptide sequences identified in hydrolyzes of selected protein preparations the analysis of identified biopeptides indicates that diversity of amino acid sequences as well as the quantity of the peptides in tested hydrolyzates depended on the protein matrix, namely the type of protein preparation (table 2). the results of liquid chromatography-high-resolution mass spectrometry (lc-hrms)-and peptide sequencing revealed that most of the identified biopeptides sequences in analyzed hydrolyzates of protein preparations possess potential antihypertensive activities (table 2) while in only one hydrolyzate (wpi) the presence of the sequence (releelnvpgeiveslssseesitr) with potential of mineral-binding was confirmed. furthermore, some of the biopeptide sequences possess more than one biologically active function, for instance, ttmplw detected in hydrolyzate of cgmp or ayps presented in hydrolyzed α-la (table 2). the analysis of peptide sequences revealed the presence of different biopeptides with antioxidant properties (ikh, ipnpigse, nen, ayps, llr) in all analyzed hydrolyzate samples (table 2). however, fermented cgmp was characterized by the largest number of different peptides with antioxidative properties. moreover, within 21 of various biopeptides identified in this hydrolyzate samples, the potential of their biologically active properties also involved the following activities: antihypertensive, antithrombotic, antibacterial, immunoand cytomodulatory peptide as well as the function of opioid and ace inhibitor. furthermore, the antioxidant peptide ipnpigse [αs1 -cn, f(182-189)], previously identified by gútiez et al. (2013) as a major peptide fragment in supernatants of e. faecalis strains grown in bovine skim milk has also been detected in cgmp hydrolized by the analyzed probiotic l. acidophilus strain. these results are of particular importance in the creation of functional food products. it is claimed that peptide abilities to scavenge free radicals is connected with the presence of hydrophobic amino acid residues like: met (m), ile (i), val (v), leu (l), phe (f), trp (w), ala (a), tyr (y) and pro (p) (penaramos et al., 2004; cheison et al., 2007; ren et al., 2008). an example of such peptide sequence is ayps that was identified in hydrolyzate of α-la. it was also proved that the presence of amino acids with aromatic residues enhances the ability for radical scavenging (rajapakse et al., 2005). the findings of the investigations indicate that the probiotic strain l. acidophilus la-5 is also capable of degrading whey and milk proteins and generating peptide sequences exhibited (among others) the ace-inhibitory activities (table 2). obtained results are in accordance with gútiez et al. (2013), who noticed that proline residue is present in most amino acid sequences of ace-inhibitory peptides. it was also suggested that this residue is favourable for peptide binding to the active site of ace. moreover, gútiez et al. (2013) identified the peptide lhlplp that is a competitive inhibitor of ace exhibiting resistance toward gastrointestinal enzymes (quirós et al., 2009). interestingly, another sequence lpypyy (identified as angiotensin-i-converting enzyme inhibitory peptide) identified in analyzed cgmp hydrolyzates was detected through esi-ms/ms in samples of yak milk casein hydrolysates exhibiting a high level of ace inhibition (83.16±1.37 %) (jiang et al., 2007). moreover, hernández-ledesma et al. (2007) described the β-lg-derived dipeptide, wy [f(19−20)] as a sequence with ace-inhibitory bioactivity and radicalscavenging capacity. the same biopeptide was detected in the samples of wpi hydrolyzates obtained using l. acidophilus la-5 (table 2). ital. j. food sci., vol. 31, 2019 445 table 1. texture profiles and spontaneous syneresis levels exhibited by obtained fermented milk products. added protein preparation texture parameter syneresis [%] hardness [n] fracturability [n] springiness cohesiveness gumminess chewiness control* 0.153±2.59ab 4.59±0.84a 0.82±0.05c 0.45±0.10a 5.32±0.21ab 4.70±0.44bc 19.79±1.86ab cgmp 0.192±0.35a 4.23±1.08a 0.92±0.00ab 0.44±0.09a 7.69±0.45ab 7.32±0.07a 31.79±3.17a wpi 0.128±0.30b 4.25±0.51a 0.99±0.01a 0.64±0.07a 8.26±0.48a 7.59±0.50a 11.27±1.29bc wmp 0.133±1.02ab 5.66±1.04a 0.94±0.03ab 0.55±0.05a 6.43±0.44ab 6.76±0.69ab 7.33±0.08c α-la 0.136±2.37ab 4.11±0.52a 0.94±0.01ab 0.51±0.02a 6.95±1.00ab 6.52±0.96ab 6.95±0.66c wpc30 0.147±2.39ab 3.94±0.40a 0.89±0.01b 0.50±0.07a 5.03±0.86ab 5.91±0.82abc 26.07±3.8a wpc40 0.096±1.00b 5.14±0.49a 0.92±0.01abc 0.49±0.06a 3.97±0.77b 3.73±0.37c 27.63±6.03a *control samples consisting of 13% regenerated skim milk without any addition of protein preparation. differences between mean values (n = 8) ± standard deviation in the same column with the same letter designation are not statistically significant (p <0.05). table 2. the sequences of biopeptides identified in analyzed hydrolysates obtained using lactobacillus acidophilus la-5. source of peptides (analyzed hydrolysate) identified peptide sequence mass (da) id of the bioactive peptide in the data base activity/function reported in the data base cgmp lpypyy 814.30 biopep00859/biopepdbb antihypertensive cgmp sppein 655.30 biopep01254/biopepdb antihypertensive cgmp wpi erf 450.22 biopep00189/biopepdb antihypertensive cgmp vrsp 457.26 biopep01460/biopepdb antihypertensive α-la sry 424.21 biopep01260/biopepdb antihypertensive cgmp α-la rpkhpikhqglpqevlnen 2233.22 biopep01215/biopepdb antihypertensive cgmp leql 501.28 biopep00755/biopepdb antihypertensive wpi wy 367.15 biopep01540/biopepdb antihypertensive wpi gkekv 559.33 biopep00360/biopepdb antihypertensive wpi vapfpevfgke 1218.63 biopep01336/biopepdb antihypertensive wpi fvapfpev 904.47 biopep00301/biopepdb antihypertensive cgmp rpk 399.26 biopep01206/biopepdb antihypertensive wpi vlnenl 700.37 biopep01413/biopepdb antihypertensive wpi vapfpevfgke 1218.63 biopep01336/biopepdb antihypertensive wpi lqpevmgvsk 1086.57 biopep00867/biopepdb antihypertensive wpi lvypfpgpi 1001.56 biopep00927/biopepdb antihypertensive ital. j. food sci., vol. 31, 2019 446 cgmp hkempfpkypvepf 1744.86 biopep00457/biopepdb antihypertensive cgmp wpi sqskvlpvpq 1081.61 biopep01258/biopepdb antihypertensive wpi lqsw 532.26 biopep00875/biopepdb antihypertensive cgmp tqslvyp 806.42 biopep01306/biopepdb antihypertensive wpi tedelqdkihp 1323.62 biopep01279/biopepdb antihypertensive cgmp wpi maippkk 783.46 biopep00946/biopepdb 3294/biopep-uwmc antihypertensive, antithrombotic cgmp ipnpigse 825.42 biopep00561/biopepdb antioxidative wpi ikh 396.25 biopep00532/biopepdb antioxidative cgmp nen 375.32 9363/biopep-uwm antioxidative α-la ayps 436.20 8472/biopep-uwm 8380/biopep-uwm antioxidative, ace inhibitor cgmp wpi llr 400.28 8484/biopep-uwm biopep00827/biopepdb antioxidative, antihypertensive cgmp lktvyqhqkamkpwiqpktkvipyvryl 3455.96 8337/biopep-uwm antibacterial wpi releelnvpgeiveslssseesitr 2801.39 biopep04772/biopepdb mineral-binding cgmp α-la yqepvlgpvrgpfpiiv 1880.06 biopep04801/biopepdb biopep04091/biopepdb biopep01621/biopepdb immunoand cyto-modulatory peptides antimicrobial antihypertensive cgmp nlhlplp 802.47 2669/biopep-uwm, biopep01010 /biopepdb ace inhibitor antihypertensive cgmp wpi vtstav 576.30 7481/biopep-uwm biopep01475/biopepdb ace inhibitor, antihypertensive α-la llyqepvlgpvrgpfpiiv 2106.22 8174/biopep-uwm immunomodulating cgmp ttmplw 747.30 3530/biopep-uwm, 3127/biopep-uwm, 8172/biopep-uwm, biopep0479 6/ biopepdb, biopep01313/biopepdb ace inhibitor, opioid, immunomodulating immunoand cytomodulatory peptide, antihypertensive cgmp α-la maippkknqdkteiptintiasgeptstptteavestvatl edspeviesppeintvqvtstav 6703.37 biopep03480/biopepdb antibacterial α-la yyqqkp 825.40 8383/biopep-uwm ace inhibitor cgmp wpi vqvtstav 803.40 biopep01445/biopepdb, 8264/biopep-uwm antihypertensive, antibacterial bdata base: bis.zju.edu.cn/biopepdbr/ cdata base: www.uwm.edu.pl/biochemia/index.php/pl/biopep ital. j. food sci., vol. 31, 2019 447 hydrolysis of cgmp conducted with the application of the probiotic l. acidophilus strain allowed to receive the sequence ttmplw exhibited multi-directional biological activities (opioid, ace inhibitor, antihypertensive, immunoand cytomodulatory peptide) (table 2). the results of analysis of ace-inhibitory and antihypertensive activity in spontaneously hypertensive rats of biopeptides (generated through tripsine hydrolysis of milk proteins) claimed that this sequence was effective in decreasing the level of systolic blood pressure (karaki et al., 1990). the concentration of peptide (ttmplw) needed to inhibit 50% ace activity (ic50) achieved the value of 16 μm, whereas for sequences lqsw [β-cn, f(155−158)], yqepvlgpvrgpfpiiv [β-cn, f(208−224)] and maippkk [κcn f(106-112)], the ic50 values were 500, 101 and 4785 μm, respectively (karaki et al., 1990; maeno et al., 1996; miguel et al., 2007; hernández-ledesma et al., 2011). all the sequences mentioned above were also identified in whey and milk protein preparations hydrolyzed by l. acidophilus la-5 (lqsw in samples of hydrolyzed wpi; maippkk in cgmp and wpi hydrolyzates; yqepvlgpvrgpfpiiv in cgmp and α-la hydrolyzates). the presented results of the research have practical relevance because health-promoting properties provided in food by biopeptides may find an application in personalized nutrition as well as individual dietary practices (korhonen and pihlanto, 2006). biologically active peptides are important constituents of food and allow the design of novel foods, including functional food products, supplements, nutraceuticals or even pharmaceuticals (meisel, 2005; liutkevičius et al., 2016; lucarini et al., 2017). 4. conclusions and future work the obtained study findings demonstrate that analyzed protein preparations are an important source of dietary antioxidants. furthermore, due to their influence on fermented product, texture properties may find a wider application in the formulation of new dairy products, especially functional food. the use of 1% α-la additive into regenerated skimmed milk is conducive to the reduction of the level of syneresis, with 1% addition of cgmp leaded to obtain the strong, hard gels in fermented milk products, but with a weaker capacity to water-binding. in addition, the hydrolysis process of protein preparations carried out by the probiotic strain improved their antioxidative properties. furthermore, using the l. acidophilus la-5 to hydrolysis of cgmp and α-la seems to be an effective method of obtaining products with high antioxidant properties as well as some various biopeptide sequences. among all analyzed hydrolysates, the sequences with potential antihypertensive activity were the most numerous biopeptides, whereby fermented (by the probiotic l. acidophilus strain) cgmp allowed acquiring the largest amount of different biologically active peptides. however, despite the promising research results, undoubtedly, further investigations are necessary to verify in vivo the biological activity of biopeptide sequences identified in presented study. also, further analyses might yield new biologically active substances or contribute to a more efficient use of l. acidophilus la-5 in the production of functional foods, nutraceuticals or pharmaceuticals. ital. j. food sci., vol. 31, 2019 448 acknowledgements the research was funded by the national science centre, poland (research grant no. 2014/ 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κ-casein as a source of short-chain bioactive peptides generated by lactobacillus helveticus. journal of food science and technology 54(11):3679-3688. doi: doi.org/10.1007/s13197-017-2830-2. zhao q.z., wang j.s., zhao m.m., jiang y.m. and chun c. 2006. effect of casein hydrolysates on yogurt fermentation and texture properties during storage. food technology and biotechnology 44(3):429-434. paper received november 12, 2018 accepted february 20, 2019 ijfs#1858_bozza ital. j. food sci., vol. 32, 2020 945 paper properties of strawberries puree stored in the freezer v. obradović, m. ergović ravančić*, h. marčetić and s. škrabal agricultural department, study of food technology, polytechnic in požega, vukovarska 17, 34 000 požega, croatia *corresponding author: mergovic@vup.hr abstract four different strawberry varieties were used for puree production and stored at -18 ºc for 12 months. every three months samples were tested for rheological parameters, polyphenols content and antioxidant activity. mathematical models for rheological behaviour of the samples were determined together with consistency coefficient (k) and flow behaviour index (n). fluidity of the samples increased over time, but pseudoplastic behaviour remained through tested period. the biggest decrease of polyphenol content was observed between 9 and 12 months of storage, while antioxidant activity decreased the most during first three months by dpph and abts method. keywords: antioxidant activity, polyphenols, rheology, strawberries ital. j. food sci., vol. 32, 2020 946 1. introduction current guidelines for fruit and vegetable consumption recommend five portions per day (hartmann et al., 2008). due to seasonal character of many fruits, as well as hectic lifestyle, these recommendations are not easily achievable especially when targeting fresh fruit and vegetables. thus, replacement of one or several of portions by fruit juices, concentrates or purees is suggested (hartmann et al., 2008). consumers have increased interest to high-value food products, especially fruit, vegetables and other functional foods (bisharat et al., 2013). therefore, food manufacturers are facing the challenge to create new products which, beyond basic nutrients, also provide certain health-promoting properties (obradović et al., 2015). strawberries are one of the most consumed berries worldwide. in european market average strawberries intake is 2.16 kg/year per person including raw and processed fruit (gasperotti et al. 2015). these popular fruits are favoured for their attractive taste, and are considered as rich source of micronutrients and phytochemical compounds such as water soluble vitamin c and polyphenols (phenolic acids, anthocyanins, flavonols, tannins and other) (klopotek et al., 2005; oszmiański et al., 2009; bodelón et al., 2013; żebrowska et al., 2019). diet rich in fruit and vegetables is beneficial for human body. it lowers the risk of many diseases: diabetes, atherosclerosis, cardiovascular disease, inflammatory-related illnesses, and cancer. this is attributed to vitamins, dietary fiber and polyphenols (nowicka et al., 2019). strawberries have very strong antioxidant activity. they have 1.3 times activity of oranges, 2 times that of red grapes, 5 times that of apples and bananas and 13 times that of honeydew melon (oszmiański et al., 2009). for all mentioned reasons, strawberries are considered as a functional food although exact mechanism involved is still generally unclear (gasperotti et al., 2015). unfortunately, strawberries are very perishable due to high water content and soft structure, and consequently have extremely short postharvest shelf-life (holzwarth et al., 2012; peinado et al., 2012). therefore, there is a huge demand for strawberries puree for use as a base product for preparation of juices and soft drinks, for addition to ice-creams and yoghurts (bodelón et al., 2013), or it can be sold directly to consumers in canned or frozen forms (diamante et al., 2016). purees are usually preserved by freezing or by heat. freezing results in cell destruction allowing reactions between genuine enzyme activities and their corresponding substrates. thawing is especially critical since polyphenoloxidases (ppo) are responsible for polyphenols destruction (holzwarth et al., 2012). in order to create puree of satisfying quality and nutritional value it is necessary to determine optimal storage time. therefore, the objective of this study was to explore influence of freezing on strawberry purees. rheological properties, polyphenol content and antioxidant activity were evaluated. changes in the rheological properties of fruit purees that have undergone freezing or freeze–thaw treatments are of practical significance for their acceptance and consumption (diamante et al., 2016). reduction in antioxidant activity during processing and storage may reduce the health beneficial effects of food products (oszmiański et al., 2009) and that was the reason for test puree samples for above mentioned parameters. some works have reported the rheological characterization of different fruits like mango and papaya (el-mansy et al., 2005), blueberry (antonio et al., 2007, nindo et al., 2007), raspberry, strawberry, prune, peach (maceiras et al., 2007, ergović ravančić et al., 2012), nectarine and blackberry (ergović et al., 2009; ergović et al., 2010), but to the best of our knowledge they haven’t followed rheological parameters together with nutritional characteristics over a period of time in order to determine how storage time in the freezer affects mentioned characteristics. ital. j. food sci., vol. 32, 2020 947 2. materials and methods 2.1. sample preparation sample s-1 was prepared from wild strawberries harvested in woods near požega town, slavonia region, croatia. strawberries for sample s-2 (albion variety) and s-3 (clery variety) were purchased from the local farmers and for sample s-4 (joly variety) in the local supermarket. fruits were cleaned and blended in kitchen blender at room temperature for 3 minutes, divided in small portions (100 ml), sealed in polyvinyl chloride freezer bags (at atmospheric pressure and temperature, vacuum or modified atmosphere haven’t been used) and kept in chamber freezer at -18 ºc, until the analysis. every three months three bags were thawed at room temperature as parallels for the analysis. 2.2. sugar and acidity determination total and reducing sugars were determined according to the luff-schoorl method (gafta, 2018). total acidity was determined by potentiometric titration. 2.3. extract preparation 1 g of strawberry puree was extracted with 20 ml of acidified methanol (methanol/2% hcl, 95:5) at room temperature for 60 min with constant shaking in temperaturecontrolled shaker (kottermann labortechnik) at 200 rpm and centrifuged (tehtnica, centric 322a). glasses were covered with aluminium folium to prevent evaporation of solvent. 2.4. total phenol content polyphenols were determined according the folin-ciocalteu method (obradović et al. 2015, with modifications). an aliquot of the extract (200 µl) was mixed with 2 ml water and 100 µl folin-ciocalteu reagent (kemika, croatia). the mixture was allowed to equilibrate for 5 min, and then 300 µl of sodium carbonate solution (20%) was added. after incubation at room temperature in dark for 30 min, the absorbance of the mixture was read at 725 nm (camspec m501, uk). acidified methanol was used as a blank. total polyphenols were determined with 3 replications. gallic acid (carlo erba reagents, italy) was used as a standard (calibration curve y = 1.1979x 0.0188, r2 = 0.9984), and results were expressed in mg of gallic acid equivalents per 100 g of sample. 2.5. antioxidant activity determination (abts) abts·+ radical was obtained by mixing 7.4 mm abts (fluka, switzerland) solution and 2.6 mm solution of ammonium persulfate in 1:1 ratio. solution was left in dark through the night in order to develop stable radical, and then radical solution was diluted with ethanol in 2:70 ratio to obtain absorbance approximately 1.100 (aabts). an aliquot of extract (0.2 ml), was mixed with 3.2 ml of diluted abts·+ radical. after incubation at room temperature in dark for 95 min, the absorbance of the mixture was read at 734 nm (aextr), and ∆a was calculated as aabts aextr. trolox (sigma aldrich, usa) was used as a standard. decrease in absorbance caused by trolox was done in the same way as for the samples, and standard curve ∆a/trolox concentration was created (y = 489.13x 17.903, r2 = 0.9952). ital. j. food sci., vol. 32, 2020 948 determination of antioxidant activity was done in 3 replications. results were expressed in µmol of the trolox equivalents per gram (obradović et al., 2015). 2.6. antioxidant activity determination (dpph) an aliquot of extract (50 µl) was mixed with 2 ml dpph radical solution (0,1mm in ethanol). the absorbance of the mixture was read at 517 nm during period of 30 min, results were expressed as the mean of 3 replications. pure ethanol was used as a blank. % inhibition = [(a0 at)/a0] x 100 (1) a0 absorbance of dpph radical solution, at – absorbance after 30 minutes. 2.7. rheological properties determination the rheological properties were measured before storage in the freezer and after 3, 6, 9 and 12 months of storage by rotation rheometer, model vt 550 362-0001 haake with concentric cylinders (rheowin pro 2.91 software). diameter of inner rotating cylinder was 36 mm, inner diameter of outer stationary cap was 40 mm, gap between cylinders was 4 mm. the measurements were carried out in triplicate at 40°c, at shear rates 0 – 60 1/s. temperature of 40°c has been selected as the closest to the temperature of “ready to eat” food containing puree. puree is not expected to be eaten alone, it is usually used as a filling for some cakes like strudel, or as a dressing for pancakes and this is temperature of “ready to eat” product, close to the body temperature. for each shear rate computer recorded shear stress which was provided by the strawberry puree during rotation of the measuring cylinder of the rheometer. flow curves are presented as the mean value of recorded results. rheological parameters determined with experimental flow curves were fitted to ostwald-de waele (power law) model using a software (excel 2016, usa). τ = k · dn (2) τ shear stress (pa), k consistency coefficient (pasn), d shear rate (1/s), n flow behavior indeks. samples were taken from the freezer and after thawing and reaching room temperature, rheological parameters were determined. relation between shear rate and shear stress were presented graphically and determination coefficient (r2) was calculated for each curve. 2.8. data analysis chemical composition data were analysed by statistica 12 software, using post hoc lsd at 95% level. ital. j. food sci., vol. 32, 2020 949 3. results and discussion initial composition of strawberries puree samples is presented in table 1. parameters were tested as an indicator of ripeness to initially assess the starting material. s-1 sample, wild strawberries, had the highest sugar content and the lowest acidity. sample s-2 is following with slightly lower content of sugars, but with much higher acidity, and the samples s-3 and s-4 were similar in sugar content, but sample s-3 had the highest acidity among all samples. these parameters were not expected to be significantly changed during storage, so they were not measured every three months. table 1. composition of purees before storage. s-1 s-2 s-3 s-4 total sugars (%) 15.40±0.22 13.42±0.16 8.22±0.26 8.64±0.04 reducing sugars (%) 10.56±0,06 8.96±0.12 7.64±0.08 8.16±0.06 total acidity (mmol/100g) 7.96±0.04 17.52±0.02 24.40±0.06 13.60±0.10 there are diverse phenolic compounds in strawberries, not only coloured anthocyanins, but also colourless phenols like ellagic acid, ellagitannins, p-coumaric acid and quercetins (hartmann et al., 2008). gasperotti et al. (2015) identified and quantified 56 individual compounds in strawberries, with concentrations ranging from 1 µg/100 g to 40 mg/100 g. they also highlighted that this is not a complete list of polyphenols present in strawberries. total phenol content in puree samples before and after 3, 6, 9 and 12 months of storage is presented in table 2. table 2. total phenol content in samples during 12 months of storage a, b. sample total phenols (mggae/100 g) before storage after 3 months after 6 months after 9 months after 12 months s-1 422.59e±4.72 415.10d±2.82 404.60c±5.06 400.89b±1.55 368.98a±0.44 s-2 196.92d±6.96 192.66c±0.16 189.96b±0.98 188.12b±0.46 170.49a±1.47 s-3 164.68d±0.99 152.15c±0.83 151.62c±3.39 147.49b±1.41 135.21a±0.47 s-4 185.70d±3.12 165.69c±0.89 163.35b±3.94 163.91b±4.53 151.47a±1.19 aresults are expressed as mean of three repetitions ± standard deviation. bmeans followed by the same letter in the lines are not statistically different at 5% probability. as presented by yildiz et al (2014) and diamanti et al (2014), initial polyphenols content in wild strawberries puree (sample s-1) was more than double compared to cultivated strawberries purees (samples s-2 till s-4). polyphenols content is in direct relation to the ripeness stage (sugar content and acidity presented in table 1). obtained results are similar to the values presented by galoburda et al. (2014) and klopotek et al. (2005). it is already documented that strawberry phenolics such as pelargonidin, ellagic acid, p-coumaric acid, quercetin and kampferol derivatives are very unstable and undergo destruction during fruits transformation in frozen products especially in the thawing process by native and microbiological enzymes and by nonenzymatic oxidation ital. j. food sci., vol. 32, 2020 950 (aaby et al., 2007; oszmiański et al., 2009), but we couldn’t find recommendations for storage time in freezer in order to preserve reasonable high level of polyphenols, and if initial value influences degree of degradation. as it can be seen in table 3, samples s-1 and s-2 had relatively low level of polyphenols destruction during first three months in the freezer (1.77 and 2.17%, respectively). level of degradation continued over next months of storage, so both samples had percentage of degradation approximately 5% after 9 months of storage, which can be considered as good result. between 9 and 12 months of storage a large decrease in polyphenol content can be seen and percentage of degradation during that period was higher than in previous 9 months. on the other hand, samples s-3 and s-4 had relatively high degradation during period of first three months (7.61 and 10.78%, respectively), which haven’t changed significantly until the 9 months, so at the end of that period degradation was 10.44 and 11.74%. still, the highest degradation was between 9 and 12 months. therefore, it can be concluded that storage time of strawberry puree shouldn’t be longer than 9 months in order to preserve phenolic compounds. in the end, percentage of degradation after 12 months of storage was higher in samples with lower initial values of polyphenols. it can be explained by the protective role of polyphenols (higher level of polyphenols-higher level of protection). similar effect can be found in wines where red wines are less susceptible to degradation and require less chemical protection than white wines. hartmann et al. (2008) concluded that every processing step during production of juices and purees reduces the content of polyphenols, but they are better retained in purees than in juices. they also recommended a short enzymatic treatment of the mash with maceration enzymes in order to achieve maximal yield of polyphenols and antioxidant capacity. it is very important to obtain short enzymatic treatment because longer mash standing actually increases the loss. at the same time enzymatically treated puree was less viscous and smoother. while the nonenzymatically treated puree registered a water phase separation in less than 3 weeks of storage, which wasn’t the case in this research. holzwarth et al. (2012) reported that freezing technique did not have significant influence on polyphenols as well as on colour and ascorbic acid. thawing method was, on the other hand, very important factor affecting mentioned parameters. thawing at 20ºc and microwave thawing were favourable methods (compared to 4 ºc and 37ºc thawing). as previously explained, in this research samples were thawed at room temperature. table 3. percentage of polyphenols degradation during storage compared to the initial value. sample degradation (%) after 3 months after 6 months after 9 months after 12 months s-1 1.77 4.26 5.13 12.68 s-2 2.17 3.54 4.47 13.42 s-3 7.61 7.93 10.44 17.90 s-4 10.78 12.04 11.74 18.43 abts and dpph methods have gained popularity for the study of antioxidant activity due to their speed and simplicity, and both of them are based on free-radical scavenging activity. antioxidant activity by abts method is presented in table 4 and by dpph method in table 5. values are in accordance with the results presented by klopotek et ital. j. food sci., vol. 32, 2020 951 al. (2005) and nowicka et al. (2019). as expected, wild strawberries puree (s-1) had much higher antioxidant activity than other samples. samples with higher content of polyphenols also had higher level of antioxidant activity by both methods. contrary to polyphenols degradation, antioxidant activity by abts method in sample s-1 had the biggest decrease in first six months of storage. sample s-3 (which had the lowest antioxidant activity at the beginning) lost almost 50% of the initial value by the end of a storage. results obtained by dpph method also show the biggest decrease in antioxidant activity during first three months. during the rest of the storage period further decrease was slower compared to first three months. although polyphenols are the most important and the most popular antioxidants, there are also other molecules with antioxidant properties like products of maillard reactions (obradović et al., 2015), so direct correlation between polyphenols content and antioxidant activity is not always the case and it depends on method used. baiano et al. (2009) showed low correlation between amount of polyphenols in wines and antioxidant activity. they also concluded that beside previously mentioned antioxidants, antioxidant activity depends not only on the phenolic concentration, but also on the specific chemical structure of each phenolic compound. table 4. antioxidant activity of samples during 12 months of storage (abts method) a, b. sample antioxidant activity (abts) (µmol te/g) before storage after 3 months after 6 months after 9 months after 12 months s-1 30.20d±0.59 27.65c±0.53 24.31a±0.21 25.70b±0.45 25.71b±0.32 s-2 10.79e±0.37 9.96d±0.56 9.16c±0.33 7.98b±0.20 7.16a±0.18 s-3 7.62e±0.08 6.22d±0.04 5.46c±0.15 4.52b±0.39 3.94a±0.29 s-4 8.63d±0.04 5.52c±0.14 5.59c±0.13 5.10b±0.48 4.70a±0.41 aresults are expressed as mean of three repetitions ± standard deviation bmeans followed by the same letter in the lines are not statistically different at 5% probability. table 5. inhibition of dpph radical after 30 minutes a, b. sample inhibition (%) before storage after 3 months after 6 months after 9 months after 12 months s-1 80.02d±1.82 71.80c±2.04 70.54b±1.94 68.38a±2.20 68.13a±1.56 s-2 49.39c±0.96 39.63b±1.14 39.93b±1.55 39.49b±1.24 38.95a±0.93 s-3 43.94d±1.06 36.14c±0.58 36.60c±1.86 32.75b±0,64 31.44a±0.88 s-4 49.74d±1.58 40.35c±0.98 38.20b±0.60 38.27b±1,46 37.21a±1.38 aresults are expressed as mean of three repetitions ± standard deviation. bmeans followed by the same letter in the lines are not statistically different at 5% probability. beside chemical and nutritional properties, knowledge of the rheological properties of food products is important for process design, control of the process, and consumer acceptability of a product. rheological properties provide information on how to control flow properties of the product so that the desired product can be prepared. rheological ital. j. food sci., vol. 32, 2020 952 properties are explained by rheological parameters: flow behavior index (n) and consistency coefficient (k) (lovrić, 2003; osorio et al., 2008). fruit purees are suspensions of solid matter in fluid media and have been categorized as time-independent non-newtonian fluids showing a pseudoplastic behavior (rudra et al., 2007; sorour et al., 2016). rheological properties of strawberry puree samples are presented in fig. 1. shape of the curve in fig. 1. shows that strawberry puree is pseudoplastic system. pseudoplastic nonnewtonian flow behavior occurs when shear stress is increasing at a diminishing rate, while increasing shear stress decrease when fluid is subjected to higher shear rates. it leads to a convex profile curve in which tangential slope is decreasing with increasing shear rate (kreith, 1999; figura and teixeira 2007). this behavior is caused by decreasing molecular interactions within the molecular structure of the fluid during flow. pseudoplastic behavior of all samples remains regardless of storage time. it can be seen that freezing caused decrease of shear stress values in all samples compared to the starting sample. deviations presented in stress-strain graphs are result of a samples’ inhomogeneity, but still, measured values fit well to the ostwald de waele model for pseudoplastic systems (table 6, r2 ranging from 0,911 till 0,994). the same trends were obtained by several authors. alvarez et al. (2006) studied the rheological behavior of strawberry jam, maceiras et al. (2007), bukurov et al. (2012) and yalçinöz and erçelebi (2016) researched the rheological properties of strawberry puree. all mentioned authors fitted results of rheological measurements to the same model. figure 1. rheological properties of strawberry purees during storage in the freezer. ital. j. food sci., vol. 32, 2020 953 shear stress values were the highest in sample s-4 before and during storage with maximum shear stress 921 pa and the lowest in sample s-3 with maximum shear stress 379 pa at the shear rate 60 s-1. table 6. rheological parameters of strawberries puree samples during storage. sample s-1 s-2 s-3 s-4 before storage n 0.573 0.572 0.753 0.758 k, pa·sn 91.285 79.818 19.289 41.910 r2 0.953 0.911 0.994 0.991 after 3 months of storage n 0.575 0.545 0.837 0.769 k, pa·sn 56.559 58.425 8.322 32.848 r2 0.985 0.930 0.994 0.979 after 6 months of storage n 0.588 0.646 0.878 0.778 k, pa·sn 45.301 36.274 5.785 32.674 r2 0.941 0.953 0.988 0.983 after 9 months of storage n 0.701 0.675 0.873 0.873 k, pa·sn 26.044 24.786 5.957 17.495 r2 0.987 0.962 0.961 0.971 after 12 months of storage n 0.895 0.831 0.879 0.867 k, pa·sn 11.622 12.956 6.067 14.375 r2 0.990 0.9863 0.998 0.961 as shown in table 6, flow behavior index values are within 0 and 1 (0,545-0,878), characteristic for pseudoplastic systems. pseudoplastic fluid behavior is explained by cracking of the molecule structure when exposed to hydrodynamic forces and increasing the alignment of the constituent molecules. it can be seen that from the start samples s-1 and s-2 had lower values for flow behavior index than samples s-3 and s-4. this difference is obvious till 9 months of a storage, while after that flow behavior index is practically the same for all samples. strawberry purees before storage in the freezer had the lowest values of flow behavior index and the highest values of consistency coefficient. consistency coefficient is constantly decreasing during time in all samples. before storage, samples s-1 and s-2 had higher k values than samples s-3 and s-4 (lower fluidity). decrease in k value overtime indicates that fluidity of the sample increased during storage time. moreover, weak physical bonds like electrostatic and hydrophobic forces might have been destroyed easily during shearing (isanga and zhang, 2009). destruction of cellular structure during freezing and thawing caused increase of flow behavior index value and decrease of consistency coefficient value. although it is obvious that rheological behavior is changed during storage with increased fluidity, pseudoplastic behavior remained. depending on the final purpose of puree, improvement of fluidity can be achieved by addition of hydrocolloids (ergović et al., 2010). ital. j. food sci., vol. 32, 2020 954 4. conclusions based on the results of this paper it can be concluded that strawberry puree shouldn’t be stored in the freezer longer than 9 months to avoid excessive polyphenol degradation because after 9 months of storage degradation of 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91.1% less than 44 years old; 81.2% high-educated). most of the respondents (90.8%) had high concerns related to food waste. bakery products are the most wasted foods. monthly economic value of food waste is 5-25 euro. raising montenegrins’ awareness about environmental, ethical and economic implications of household food wastage is crucial to address this issue. keywords: behavior change, food labelling, food waste cost, household food waste, montenegro, online survey ital. j. food sci., vol. 31, 2019 275 1. introduction food is lost or wasted throughout the supply chain, from initial agricultural production down to final household consumption (fao, 2011; czarniawska and löfgren, 2013; fao, 2017; hlpe, 2014). the causes of food losses and waste are mainly connected to financial, managerial and technical limitations in harvesting techniques, storage and cooling facilities, infrastructure, packaging and marketing systems (fao, 2011; hlpe, 2014; oecd and fao, 2015). food wastage represents a missed opportunity not only to improve global food security (hlpe, 2014; oecd and fao, 2015; kader, 2005), but also to mitigate environmental impacts and resources use from food chains (hlpe, 2014; oecd and fao, 2015; parfitt et al., 2010; jereme et al., 2013; fao, 2013; chapagain and james, 2013; hodges et al., 2010; smil, 2004; grizetti et al., 2013; kummu et al., 2012). montenegro is a service-based economy; in 2016, the tertiary sector accounted for 71.9% of total gross domestic product (gdp), while the primary production – agriculture, forestry and fishing – accounted for 9.0% (ec, 2018). the green economy concept has a prominent place in the revised national strategy for sustainable development of montenegro for the period 2014–2020; however, montenegro does not have a strategic document that would explicitly state the country’s commitment to green economy (radovic-markovic et al., 2015; ministry of tourism and environmental protectionmontenegro, 2007). several laws are dealing with waste policies, and montenegro has made notable efforts to harmonize its legislation with the acquis of the european union (eu). the 2011 law on waste management (government of the republic of montenegro, 2011, 2016) requires the waste producer to make all efforts to prevent and reduce waste generation. the law sets up ambitious targets in several areas of waste management and for biodegradable waste by 2020 – the aim is to reduce biodegradables by 50%. other laws such as the law on environment, a key legal act on the management and protection of the environment aim to align with obligations resulting from montenegro’s international commitments and relevant eu’s directives dealing with waste policy issues. nevertheless, these laws cover waste in general and are not specifically addressing food waste. government strategies and actions – e.g. waste management plan 2014–2020, national policy on waste management from 2004 and the 2005 strategic master plan for solid waste management of montenegro – do not recognize food waste as an important subject (radovic-markovic et al., 2015). although several nongovernmental organizations (ngos) deal with environmental protection and waste, they have rarely initiatives related to food waste (e.g. in 2014, ecological movement ozon promoted action “stop food waste” during the european week for waste reduction), with the exception of the ngo “food bank” dealing with decreasing food waste and fight against hunger. in several occasions (e.g. primary milk producers in 2013 and watermelon producers in 2011, who demonstratively destroyed their products due to poor government management policies), lack of effective market management resulted in significant food losses. in general, there is a lack of data about food waste and losses in montenegro, as responsible authorities and relevant strategies do not deal with these issues and data on industrial and municipal solid waste do not seem to realistically reflect food waste generation. thus, attention should be focused on waste generated by households and at consumer level, as these may be more reactive and likely to yield results faster (grethe et al., 2011). in mediumand high-income countries, such as montenegro, food is to a significant extent wasted at the consumption stage, meaning that it is discarded even if it is still suitable for human consumption (hlpe, 2014; lundqvist, 2010). there is a growing body of literature dealing with household food waste in different countries and regions (e.g. ital. j. food sci., vol. 31, 2019 276 bygrave et al., 2017; evans, 2011; graham-rowe et al., 2014; jereme et al., 2013; lebersorger and schneider, 2011; mondéjar-jiménez et al., 2016; neff et al., 2015; principato, 2018; principato et al., 2015; quested et al., 2013; secondi et al. 2015; stenmarck et al., 2016; williams et al., 2012; wrap, 2011), but the balkan region in general and montenegro in particular are largely underserved. therefore, in order to address this literature gap, this exploratory study aims to provide a general overview about household food waste in montenegro. 2. material and methods the paper is based on the results of a voluntary self-administered online survey that was adapted to montenegrin context and designed through elaboration of questionnaires previously used for similar research purposes e.g. office of environment and heritage in the state of new south wales (nsw), australia (nsw-epa, 2012), and the university of bologna (last minute market, 2014). the questionnaire on food waste (fw) was available in montenegrin online through survio website (www.survio.com) from january until april 2015 (87 days in total) and the participation was entirely on a voluntary basis. potential adult respondents were contacted using direct emails and social networks (facebook, twitter). the online questionnaire included 25 one-option and multiple-choice questions dealing with: (i) food purchase behaviour and household food expenditure estimation; (ii) knowledge of food labelling information; (iii) attitudes towards fw; (iv) extent of household fw; (v) economic value of household fw; and (vi) willingness and information needs to reduce fw. the concept of fw was briefly presented in the introductory part of the online questionnaire to inform the respondents more about the topic and the research purpose (the following statement was included in the questionnaire: “for the purpose of the present survey, food waste is considered food that was purchased by the household for human consumption but was thrown away i.e. was not consumed”), as well as about approximate time needed to complete the survey (10-15 minutes). the response rate (aapor, 2017) was used to provide essential information about the quality of the survey. received unfinished questionnaires, contradictory or bad quality data, were excluded from further data processing (table 1). answering questionnaire on fw required time so that might be the reason why many of those who received the questionnaire did not answer (41.9%). table 1. online survey visits and response rate. detail no % total visits 825 100 just seen by respondents but not answered 346 41.9 total questionnaires answered (completed and unfinished) 479 58.1 total questionnaires completed by respondents 371 45.0 total unfinished questionnaires 108 13.1 responses included in final analysis 371* 45.0* source: authors’ elaboration based on survey results. * the number of complete questionnaires divided by the number of total visits (those eligible in the sample). ital. j. food sci., vol. 31, 2019 277 the questionnaire was disseminated all over montenegro but most of those who completed the questionnaire (69.5%) were from the capital of montenegro podgorica. quantitative data collected through the questionnaire survey were analyzed using descriptive statistics (e.g. means, max, min, percentages), in order to get a general picture of frequencies of variables, using microsoft excel spreadsheets. besides descriptive analysis, chi-square test was used to analyze the association between different variables in spss 16. the null hypothesis was that there is no relation between tested variables (gender, age, level of education, occupation, frequency of food purchasing, estimated amount of food waste, use of shopping list, knowledge about labelling, habits of respondents in terms of food preparation and use) as well as relation between the amount of uneaten food and frequency of throwing away food by households. the major constraint faced during the research was the shortage and/or difficult access to secondary data on fw in montenegro. a further limitation of the study is the nonprobabilistic sampling design used for data collection as respondents were recruited on a voluntary basis. this also implies the non-representativeness of the recruited sample for the adult population in montenegro. moreover, while household food waste surveys are methodologically simple, they are mainly useful to provide qualitative information, because quantification of food wastage (cf. weight of food purchased and discarded, so not consumed) is prone to error as consumers often tend to underestimate their waste (and food waste) when self-reporting (e.g. beretta et al., 2013; neff et al., 2015; simunek et al., 2015; ventour, 2008). moreover, it should be highlighted that the questionnaire was prepared in english then translated into montenegrin and this may have affected the understanding of respondents of issues regarding food wastage and, consequently, their answers. 3. results and discussion 3.1. main characteristics of respondents most of the interviewees were females (70.1%), quite young (77.4% less than 44 years old) and had high education level (more than 80% were holders of bachelor, master or phd degrees). taking into consideration obtained results, it is clear that mainly younger respondents use social media; the main tool utilized in the online questionnaire survey dissemination. around 70% of respondents declared they have full-time or part-time paid work, 16.7% are unemployed persons (including housewives), 12.1% are students, while 1.1% is retired. more than a third of the respondents are married with children (37.2%). a significant share of the respondents still lives with parents (36.9%). as for household composition, they mainly consist of four or more members (47.7%). 3.2. food purchase behavior and household food expenditure estimation this part refers to respondents’ food behavior and an estimation of their food expenditures in order to understand their attitudes towards food. it was found that about 77.4% of the respondents purchase their food from supermarkets and hypermarkets, 12.1% purchase from mini/small markets and 9.2% from small markets. the majority is buying food once a day (36.9%) and once a week (20.5%), while those who buy twice a week and every second day represent 15.4% and 17%, respectively. food wastage could be attributed to poor planning when purchasing food as only 32.3% of the respondents use list prior to purchasing food. about 70.1% of the respondents spend monthly over 150 eur for food. ital. j. food sci., vol. 31, 2019 278 meanwhile, more than a half of the respondents (53.4%) are attracted to special food offers, which normally take place at superand hypermarkets. 3.3. knowledge of food labelling information and attitudes towards food waste the results show respondents’ knowledge about food labels, which might eventually affect food wastage among consumers and the respondents’ attitudes towards food waste and food habits. it was indicated that 86.5% of the respondents understand and have knowledge about “use by” label as food must be eaten or thrown away by this date. this result could be attributed to the high educational level of the respondents. whereas only 11.1% regarded the “best before” label as food is still safe to eat after this date. it was evident that 90.8% of the respondents do worry about food waste and they try to avoid it, while 6.5% are aware about food waste problems but have no intention to change their current habits. moreover, 82.3% of the respondents indicated that they dispose of “very little”, or “reasonable amount” of uneaten food (fig. 1). figure 1. quantity of uneaten food thrown away by montenegrin households. regarding food waste management, 49.9% of the respondents give the remained food to animals and 44.7% said they dispose of it in the garbage. meanwhile, when they were asked about how often they throw away leftovers or food, 56.1% of the respondents declared that they waste food at least once a week (fig. 2). tracking consumers’ food habits could explain their attitudes towards food waste and its quantity. in that regard, the survey results showed that 60.9% of the respondents cook a main meal from raw ingredients from 3 to 6 times/week. furthermore, 73% of the respondents eat a meal left over from a previous day (less than twice/week), and 56.9% eat out of home less than two times a week. 4% 29% 15% 38% 14% much more than it should be reasonable amount almost nothing very little more than it should be ital. j. food sci., vol. 31, 2019 279 figure 2. frequency of throwing away food. 3.4. quantity and value of food wasted and extent of household food waste the analysis regarding the main reasons that contribute to throwing food showed that 45.6% of the respondents throw food because of ‘expiration date’ (without any differentiation between best-before and use-by dates); 46.6% of the respondents throw food leftovers; and 43.7% states that the main reason of throwing food is its long storage in the refrigerator. in addition, this part of the results deals with the amount, extent and value of food waste. in table 2 are presented the results of purchased food, which is thrown in households. the most wasted food group is bakery products while pulses and oilseeds, roots and tubers as well as fish and seafood are the least wasted food products by montenegrin households. table 2. respondents’ estimation for food groups wastage (in percentage). items less than 2% 3 to 5% 6 to 10% 11 to 20% over 20% cereals and bakery products (bread, rice, pasta, etc.) 146 (39.4%) 96 (25.9%) 50 (13.5%) 33 (8.9%) 46 (12.4%) vegetables 227 (61.2%) 81 (21.8%) 41 (11.1%) 14 (3.8%) 8 (2.2%) milk and dairy products 231 (62.3%) 80 (21.6%) 36 (9.7%) 16 (4.3%) 8 (2.2%) fruits 240 (64.7%) 75 (20.2%) 31 (8.4%) 14 (3.8%) 11 (3.0%) meat and meat products 236 (63.6%) 76 (20.5%) 39 (10.5%) 11 (3.0%) 9 (2.4%) roots and tubers (potatoes, etc.) 258 (69.5%) 57 (15.4%) 36 (9.7%) 16 (4.3%) 4 (1.1%) pulses and oil seeds (e.g. peas, chickpeas, olives, sunflowers) 286 (77.1%) 48 (12.9%) 28 (7.5%) 4 (1.1%) 5 (1.3%) fish and seafood 311 (83.8%) 39 (10.5%) 12 (3.2%) 6 (1.6%) 3 (0.8%) source: authors' survey. 28% 56% 9% 7% one to two times a week less than once a week never more than twice a week ital. j. food sci., vol. 31, 2019 280 as for the extent of food waste, 48.8% of the respondents do not throw away food that is still consumable, 23.5% throw less than 250 gr per week, and 18.1% throw between 250 and 500 gr per week. regarding the economic value of wasted food, it was revealed that for about 52.8% of the respondents the value of wasted food is between 5 and 25 eur per month. 3.5. willingness to behavioral change to reduce household food wastage this part deals with the notion of consumers’ willingness to change their behavior regarding food waste. thus, the first step was to explore the respondents’ perception of food waste reasons. it was evident from the results that most of the respondents are familiar with such reasons; for instance, 43.7% mentioned food is left in the fridge for too long time, followed by 45.6% of them that said ‘food expired’, 30.2% indicated food does not look eatable/good, and 46.6% referred to leftovers. while many respondents mentioned “food is left in the fridge for too long time” or “food has expired”, it is important to consider the true reasons or root causes that led to this result and, consequently, to food wastage. these reasons are mainly related to inappropriate meals planning and inadequate food preseveration; which surprisingly were less mentioned by the respondents. eventually, since the respondents have clear vision about food waste causes or reasons then they could be willing to reduce such waste. though, willingness is affected by information availability and other factors; so, 39.4% of the respondents mentioned that they will reduce food waste if the packaging was more suitable, followed by if they were better informed about the negative impacts of food waste on the environment (39.6%) or on the economy (21.3%). finally, as for the information needed to reduce food waste, 28.8% of the respondents said that they need recipes with leftovers, 48% need tips on how to conserve food properly, 33.7% need information about organizations and initiatives that deal with food waste prevention and reduction, and, finally, 36.7% need information on how to assess the freshness of products. 3.6 relations between tested variables the independence of variables was analysed by using chi–square test. in particular, relations between the following variables were tested: gender, age, level of education, occupation, frequency of food purchasing, estimated amount of food waste, use of shopping list, knowledge about labelling, habits of respondents in terms of food preparation and use. all the tested relations were not statistically significant. according to the conducted online survey, the following results were obtained when it comes to the quantity of uneaten food and frequency of throwing away food by households in montenegro (table 3). regarding the frequency of throwing away food, regardless of the quantity of food, most respondents throw food less than once a week or one to two times a week. also, regardless of the frequency of throwing away food, most respondents answered that they throw very little quantity of food. furthermore, the relation between two key questions was statistically tested by pearson’s chi-square test: (1) in general, how much of uneaten food your household usually throws away? (2) how often your household throws away leftovers and food that you consider not usable? ital. j. food sci., vol. 31, 2019 281 the value of pearson’s chi-square test of independence was 45.525, which is statistically significant at p<0.01 (table 4). therefore, there is a significant relation between the quantity of uneaten food and the frequency of throwing away food (table 4). in other words, with increasing quantity of uneaten food, also the frequency of throwing away food increases. therefore, it is crucial to pay particular attention to meals planning in order to reduce the quality of uneaten food and leftovers. moreover, another strategy consists in providing households with more information (especially recipes) about the use of leftovers. table 3. cross tabulation – the relation between quantity of uneaten food and frequency of throwing away food. how often your household throws away leftovers and food that you consider not usable? (choose one answer) never less than once a week one to two times a week more than twice a week total in general, how much of uneaten food your household usually throws away? (choose one answer) very little 28 125 38 5 196 reasonable amount 4 59 35 11 109 more than it should be 2 24 31 9 66 total 34 208 104 25 371 source: authors’ survey. table 4. chi-square test of independence. value df asymp. sig. (2-sided) pearson chi-square 44.525 6 .000** likelihood ratio 45.744 6 .000** no of valid cases 371 asymp. sig.: asymptotic significance. ** statistically significant at p< 0.01. 3.7. food wastage in montenegro: urgent action is needed results show that most of the respondents have high concerns related to food waste. according to the respondents, food waste is prevalent in montenegro and the most wasted foods are bakery products. more than half of montenegrin respondents declared that the economic value of food waste generated each month is 5–25 eur. meanwhile, almost half of the interviewees declared that they throw food that is still edible/consumable. food waste is a serious issue that undermines food security and food system sustainability in the mediterranean region (berjan et al., 2018; capone et al., 2016; el bilali, 2018). the results of the present survey are in line with those obtained in similar studies on household food waste in the mediterranean. these include surveys carried out in countries such as algeria (ali arous et al., 2017), egypt (elmenofi et al., 2015; abdelradi et al., 2018), lebanon (charbel et al., 2016), morocco (abouabdillah et al., 2015), tunisia (sassi et al., 2016) and turkey (yildirim et al., 2016). in fact, all these ital. j. food sci., vol. 31, 2019 282 studies made the case for addressing urgently household food wastage given its negative environmental (fao, 2013; chapagain and james, 2013; quested et al., 2013; wrap 2011), economic (hlpe, 2014; principato, 2018; rutten, 2013) as well as ethical (stuart, 2009) implications. despite that, food waste is still not covered by overarching waste strategic documents in montenegro. in fact, the national strategy for sustainable development of montenegro by 2030 defines the strategic goals and measures for introducing green economy by, among others, improving waste management towards circular economy. using the instruments of circular economy is possible to connect the activities and initiatives of producers, retailers, consumers and recyclers. those strategic goals and measures are related to management of all kinds of waste, but there are no specific strategic goals and measures that address food waste management within circular economy. the new document entitled “a comprehensive assessment of the current waste management situation in south east europe and future perspectives for the sector including options for regional cooperation in recycling of electric and electronic waste” (eunomia research & consluting ltd, 2017) deals with national waste assessment and roadmap for improving of waste management in montenegro. furthermore, according to the ministry of sustainable development and tourism of montenegro (msdt), there are no existing initiatives on food waste reduction. the only novelty, and this per se is a complex task, is waste separation (on wet and dry fraction), as well as composting possibility. this task is covered with waste management strategy by 2030, developed by msdt. in addition to this should be mentioned that the administration of capital city, podgorica, announced recently the start of a pilot project related to primary waste selection in households, in cooperation with ngos and international organizations in montenegro. the initiative should promote waste selection in households through informing citizens about existing recycling yards in podgorica (total 6) as well as about possibility of selecting waste at these locations. in addition, big investments are expected in construction of sanitary waste baths, facilities for purification of water, production of energy from landfill biogas. local self-government authorities in charge of waste management are poorly staffed and trained, and are in need of stronger capacities. very often, consumer health and food safety are at the center of regulators’ attention and responsibilities for managing waste are broadly separated between government bodies leading to lack of coordination in implementation of policy on food waste reduction (box 1). focusing attention on the reduction of food waste generated by households is likely to yield results faster. therefore, communication campaigns should target consumers with the objective to raise public awareness on the issue of food waste in order to change the behaviour of consumers towards food wastage. some potential causes of food waste result from business practices and private standards sometimes set at much higher levels than those set by the government. for instance, the “best before” date displayed on food products is not set by law but rather the result of industry practice that seeks to adapt to business liability constraints (nrdc, 2013). likewise, marketing and sale strategies influence negatively the waste behaviour of individuals, especially youths (mondéjar-jiménez et al., 2016), so that retailers can play an important role in preventing food waste generation. therefore, the private sector is engaged to reduce food waste throughout the food supply chain through various initiatives such as innovation (e.g. technologies, packages, production processes), corporate initiatives and consumer education via social media and other platforms (biac, 2013; bygrave et al., 2017; di terlizzi et al., 2016). some supermarkets in montenegro (e.g. voli, aroma/conto, idea) have introduced the practice of promotional discount due to expiry date or sale of two products for the price of one. these initiatives are directed into improving of products sale as well as reduction of food wastage. ital. j. food sci., vol. 31, 2019 283 besides the state institutions responsible for environmental protection as well as for waste management, also ngos should have a more active role in food waste reduction initiatives. there are three leading ngos in montenegro i.e. green home (www.greenhome.co.me), ozon (www.ozon.org.me), zero waste montenegro (www.zerowastemontenegro.me/me) that have undertaken activities related to waste management in the form of implementing projects and organizing roundtables. ozon currently implements “establishing of system for waste management in budva municipality” project whose activities encompass waste collection, selective disposal and recycling of waste. the activities of zero waste management include raising awareness on the concept of circular economy, promoting zero waste practices, lobbying against waste incineration, providing technical expertise to recycling facilities to increase their recycling rate, establishing a zero waste community, supporting establishment of first zero waste municipality. in previous years, these ngos organized roundtables on waste management within the projects funded by the european union, but never exclusively on food waste management. ngos – in cooperation with public institutions and the private sector – can play an important role in initiatives such as educational campaigns directed to consumers and industry and food recovery as well as research and knowledge dissemination activities. national campaigns, such as consumer education campaigns on reading “use by” or “best before” date labels, can help change consumer behaviour (nrdc, 2013) thus contributing to the prevention and/or reduction of household food wastage. such campaigns should focus on youths, who proved to be the population segment most inclined to waste food (mondéjar-jiménez et al., 2016; principato et al., 2015), and focus on concrete practices such as waste sorting, which was found to be positively associated with food waste reduction (secondi et al., 2015). however, actions against food wastage, especially educational campaigns, should also target social marketers, retailers and policy makers (principato et al., 2015). one possible way for rising the awareness of the new generations regarding the issues of waste in general and food waste in particular is the reform of the education system in montenegro, with the aim of introducing more environment-related disciplines into educational programs at schools and universities (north country ngo, 2016). box 1. institutions dealing with environmental protection and waste management in montenegro. the main governmental authority responsible for policymaking on environment and sustainable development is the ministry of sustainable development and tourism within which operates the environmental protection agency (epa) that is responsible for implementation of environmental legislation. while the framework legislation related to food waste is under the responsibility of the ministry, waste management is the responsibility of local governments and municipalities (ministry of tourism and environmental protection-montenegro, 2007). in general, lack of investment and poor capacities of local self-government authorities and public enterprises responsible for waste management have been commonly recognized as restricting factors for implementation of the waste management policy. the ministry of agriculture and rural development (mard) has responsibilities for food safety and supervises the authorities responsible for policy implementation, in particular, the phytosanitary administration, responsible for food safety. “project – consulting” ltd (procon) was founded by the government in 2008 to provide expert support in implementation of projects on environmental protection and communal services, adopted by the government and/or local self-government authorities and supported by international financial institutions. in early 2014, the centre for sustainable development was established as a programme, jointly implemented by the montenegrin government and united nations development programme (undp) (procon, 2008). ital. j. food sci., vol. 31, 2019 284 4. conclusions food losses can take place along the whole food chain from production, handling and storage, processing and packing, distribution and marketing, to final consumption. the purpose of this study was to assess food losses at consumer level (i.e. food waste) in montenegro. results show that household food wastage is high in montenegro and that the most wasted foods are cereals and bakery products. similarly, economic value of food waste is rather high (generally 5–25 eur monthly). despite the evident negative environmental and economic impacts of food waste, it is still not covered by waste management strategic documents in montenegro. the paper highlights that focusing attention on waste generated at consumer level is likely to yield good results in food waste prevention and reduction strategies. therefore, awareness raising initiatives should target consumers with the objective to change their attitude and behaviour towards food wastage. however, strategies for food wastage reduction can be effective only if there is a better coordination among all actors of the food chain i.e. public institutions, civil society (cf. ngos) and the private sector (e.g. retailers). meanwhile, responsibilities of local selfgovernment authorities should be optimized in order to ensure exchange of information. in fact, data are rarely reported on a regular basis through national statistical databases in montenegro. in order to establish a reliable food waste dataset in the country, an important first step is to develop a regular inventory for food waste estimation. for that, a more stringent enforcement of separate collection of waste in all montenegrin municipalities is crucial. involving broader range of stakeholders in food waste reduction will result in moving from the concept of ‘managing food waste’ to improving overall sustainability of the food chain in montenegro. references aapor [american association for public opinion research] 2017. response rates an overview. www.aapor.org/education-resources/for-researchers/poll-survey-faq/response-rates-an-overview.aspx abdelradi f. 2018. food waste behaviour at the household level: a conceptual framework. waste manag. 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journal of food science, 2021; 33 (sp1): 163–169 issn 1120-1770 online, doi 10.15586/ijfs.v33isp1.2093 163 replacement of meat by mycoproteins in cooked sausages: effects on oxidative stability, texture, and color narges shahbazpour1, kianoush khosravi-darani2*, anousheh sharifan1, hedayat hosseini2 1department of food science and technology, science and research branch, islamic azad university, tehran, iran; 2department of food science and technology, faculty of nutrition science and food technology, national nutrition and food technology research institute, shahid beheshti university of medical sciences, tehran, iran *corresponding author: kianoush khosravi-darani, department of food science and technology, faculty of nutrition science and food technology, national nutrition and food technology research institute, shahid beheshti university of medical sciences, tehran, iran. email: k.khosravi@sbmu.ac.ir, kiankh@yahoo.com received: 23 june 2021; accepted: 16 november 2021; published: 12 december 2021 © 2021 codon publications open access paper abstract processed meat is one of the most consumed products worldwide. naturally, production of proteins with animal origins includes limitations such as costs, energy, time, and environmental problems. thus, replacement of meats by alternative biomaterials such as mycoproteins can be promising. mycoproteins with hyphal morphologies, including branches and lengths, have close structures to meat and can be a potential alternative for meat products. therefore, the major objectives of this study included complete replacement of sausage meats by mycoproteins and comparing characteristics of the novel formula with those of meat. in general, physicochemical, microbial, nutritional, and mechanical characteristics of the formulas were assessed. results showed that the mycoprotein substitution improved the nutritional and health effects due to the higher valuable protein and lower lipid contents. besides, it had a high content of essential amino acid and unsaturated fatty acid, compared to meat sausage. absence of yeasts, molds, salmonella spp., eshrichia (e.) coli, and staphiloccocus (s.) aureus verified the effectiveness of the heat treatment and also the effectiveness of the hygienic procedures in both samples. with regard to phycicochemical properties, more contents of moisture and lipids in sausages containing mycoprotein were linked to further water binding capacity (wbc) (p < 0.05) and oil binding capacity (obc) in them, compared to beef samples. besides, the mycoprotein sample had lower (p < 0.05) values of carbohydrates, ash, and ph, compared to the beef sample. in contrast, beef sausages had better textural characteristics, such as hardness, cohesiveness, gumminess, and springiness indexes, compared to mycoprotein sausages. higher water and obc values of the mycoproteins led to the filling of the protein interstitial spaces as well as decreasing of the textural attributes. thus, it resulted in the use of less oil and water in mycoprotein formulations. in conclusion, mycoproteins can be addressed as appropriate replacements for meats in sausages. keywords: meat alternatives; mycoproteins; nutritional values; sausages; textural properties introduction in recent decades, the world population has significantly increased from 2.6 to 8 billion individuals (gabriel et al., 2014). if the global population grows at the recent rate, it may reach 9 billion individuals by 2042, which could pose serious problems in providing food to all (upadhyaya et  al., 2016). approximately, 1 billion people globally will not be able to properly access food sources with sufficient energy and proteins (godfray et al., 2010). this can result in serious medical problems such as defective immune system and stunted growth. in contrast, high 164 italian journal of food science, 2021; 33 (sp1) shahbazpour n et al. blood cholesterol. toxin analysis and allergy assays have shown no general concerns (hashempour‐baltork et al., 2020a). however, a little information is available on various formulations for the replacement of food meats by mycoproteins. hence, the major objective of the current study was to compare physicochemical, microbial, nutritional, and mechanical characteristics of sausages containing mycoproteins with those containing meat. materials and methods preparation of sausages sausage samples (40% red meat) were prepared in three replications in a famous meat production factory, tehran, iran. frozen beef samples were defreezed at 4°c for 16 h before use. then, beef samples were minced twice using laboratory mincer (model mk-g1800, panasonic, japan) equipped with 6–10 mm steel plates. mycoprotein masses were provided by ghazabon paya, iran. two sausages of mycoproteins and meats were prepared for 4 kg batters (table 1) based on the guidelines from meat producers. formulations of both samples were mostly similar, with differences in meat and mycoprotein contents. to prepare sausages, minced beef/mycoprotein was transferred into a bowl chopper (robot coupe model r-10, france) and mixed slowly with the dry ingredients, except spices. ice was continuously added to the mixture in the chopping process to control the temperature. then, oil and spices were added to the mixture, respectively. the total time of mixing was 10 min while the final temperature of the batters was set below 12°c. then, the batters were stuffed into impermeable cellulose casings using a hydraulic piston-type stuffer. then, the sausages were cooked at 76°c for 60 min using a steam chamber and then cooled down to a final temperature of 10°c using ice-water bath and stored at 4°c overnight (kamani et al., 2019). in general, each sausage type was prepared in two batches. totally, two sausages from each batch were chosen for further analysis. consumption of meat products can pose serious health problems as well (qian et al., 2020). research and development of meat replacements majorly focuses on the production of products that imitate the physical characteristics of meat such as appearance, taste, and texture, as well as providing its nutritional values. muscle products, such as chicken and steaks, minced products, such as burgers and nuggets, and emulsion products, such as frankfurter and mortadella sausages, are the major meat replacements (kyriakopoulou et al., 2021). however, excessive meat consumption can significantly affect the global climate change (hashempour-baltork et al., 2020b). thus, the quest for novel substitutes for animal proteins requiring less financial resources, energy, and time consumption can be promising. one of the best meat substitutes includes mycoproteins with relatively similar textures to meat (upadhyaya et al., 2016). the common source of mycoproteins is usually fusarium (f.) venenatum, a filamentous fungus generally recognized as safe (gras) (hashempour-baltork et al., 2020b). generally, mycoproteins contain 10 g of carbohydrates, 13 g of fats, 25 g of fibers, and 45 g of proteins, as well as various vitamins, carotenes, minerals, and essential amino acids (eaa) in 100 g of dry matter (finnigan et al., 2019). recent studies on human volunteers have demonstrated that biological values of proteins from mycoproteins are similar to biological values of proteins from milks. commonly, sensory characteristics, such as texture, taste, and overall appearance of the final products, are critical for their overall acceptance. fusarium biomass is virtually odorless and tasteless and is appropriate to imitate the consistency and taste of regular meats. comparing mycoproteins with textured soya and poultry muscle tissues, recent studies using microscopy techniques have shown that large cable-like fibers (especially in vegetable protein textures) result in rubbery textures, which are unfavorable when chewing. technically, further fibrous and rubbery eating qualities occur in poultry and mycoprotein products due to their tight packaging laminations, compared to those that occur in products of vegetable protein origins (hashempour-baltork et al., 2020b). ideally, mycoproteins can be good replacements for meat since their dry weights include nearly 50% of proteins, similar to grilled steaks. however, the fungi include lower fat quantities (∼13%) than those steaks do. furthermore, this is a vegetable fat with cholesterol alternative (ergosterin) and good fiber content (∼25%), which are increasingly accepted by the health-conscious people (finnigan et al., 2017). based on the literature, mycoproteins can mitigate the problem of food unavailability worldwide. moreover, even routine production of mycoproteins would need only lower water resources and occupy less  land (hashempour-baltork et al., 2020b). the use of mycoproteins can limit foodborne diseases and lower table 1. sausage ingredients. ingredient content (% w/w) meat/mycoprotein 40 sunflower oil 10 ice 20 mixed spices 3.5 soy protein isolate 5 gluten 10 flour 10 salts 1.5 italian journal of food science, 2021; 33 (sp1) 165 physicochemical assessment of sausages containing mycoproteins one-hundred microliters of this hydrolysate were carefully injected into the hplc instrument (waters, usa) at 40°c. the hplc instrument was equipped with zorbax eclipse-aaa column (4.6 mm × 150 mm, 5 μm) (agilent technologies, usa) as well as a fluorescence detector. furthermore, sodium dihydrogen phosphate (nah2po4) solution (40 mmol l−1) was used in the instrument as mobile phase a and acetonitrile:methanol:water solution (45:45:10 v/v/v) as mobile phase b. fatty acids generally, the folch method (chloroform:methanol 2:1 v/v) was used for the sample lipid extraction (folch et  al., 1957). fatty acid methyl esters (fame) were methylated based on the european official methods of analysis (godfray et al., 2010; hashempour-baltork et al., 2018). the fame were analyzed using gas chromatography (gc) (agilent 6890, agilent technologies, usa) equipped with capillary column (30 m per 0.25 mm id, 0.25-μm film thickness) and flame ionization detector (fld) (thermo tr-5, thermofisher scientific, usa). the instrument used helium (he) as the carrier gas with 0.2 ml min−1 flow rate based on a method described by hashempour-baltork et al. (2017). fatty acid (fa) was identified by comparing the sample and the reference methyl ester chromatograms (sigmaaldrich, usa). mechanical characteristics texture profile analysis (tpa) was performed using stable micro systems texture analyzer model ta.xt plus (stable micro systems, uk). the analyzer was equipped with a 50-kg load cell. the sample was cut into pieces of 25 mm and axially fixed on the platform. twocycle compression assay was carried out with up to 50% of the strain compression of the original height using steel probes. return speed, distance, and contact force were 2 mm s−1, 50 mm, and 20 g, respectively. the various attributes of the food, including cohesiveness, hardness (n), adhesiveness (n.s), springiness (%), gumminess, chewiness, and springiness, were assessed (kamani et al., 2019). statistical analysis descriptive data of the study were recorded as mean ± sd (standard deviation) using spss software v.17 (ibm analytics, usa). duncan’s test was used for the comparison of means and different letters represent significant statistical differences (p < 0.05). for each sample of each test, three replicates were used. physicochemical characteristics proximate ph, moisture, protein, lipids, carbohydrate, ash, peroxide value, water holding capacity (whc), oil binding capacity (obc), and water binding capacity (wbc) of the two sample types were assessed using official methods (aocs, 2017). microbial characteristics the count of microorganisms, escherichia (e.) coli, salmonella spp., staphylococcus (s.) aureus, bacillus (b.) cereus, clostridium (c.) perfringens, yeasts, and molds were enumerated based on the iso protocols (iso, 2013). nutritional characteristics vitamins briefly, 10 g of the ground samples were weighed using a 50-ml glass beaker. then, 20 ml of fresh 5% (w/v) metaphosphoric acid solution were added to the vessel and mixed well. the mixture was then homogenized by stirring at room temperature (rt) for 2 min. the homogenate was centrifuged at 3000 rpm for 5 min; then, the upper solution was filtered using albet no. 1305 filter papers and re-filtered using 0.45-µm millipore filters for liquid chromatography (lc) analysis (valls et al., 2001). then, stock solutions of 100 µg ml−1 vitamin b5 (pantothenic acid), vitamin b9 (folic acid), vitamin b2 (riboflavin), and vitamin b7 (biotin) (sigma-aldrich, st. louis, mo, usa) were prepared and stored in 4  °c until use. the vitamin content of the sample was assessed using the standard curve, and high-performance liquid chromatography (hplc) (waters, usa) was used for the assessment of vitamin b according to sasaki et al. (2020). the method included the use of capcell pak c18 sg120 hplc column (250 nm × 4.6 mm with 5-μm particle sizes) (osaka soda, japan), gradient elution of phosphate buffer-acetonitrile (ph 3), ion-pairing reagent (mobile phase) with 1.0 ml min−1 flow rate and uv detection (210 nm). the vitamin b compound was separated within 60 min. the value of 0.01 µg g−1 was set as the detection limit. amino acids briefly, 10 g of dried sausage sample were hydrolyzed using 1 n hydrochloric acid (50 ml) based on an original protocol by czauderna et al. (2003). amino acid (aa) standards were purchased as a cell-free aa mixture from sigma-aldrich, st. louis, missouri, usa. the sample was then centrifuged (10,000 g) to collect the hydrolysate. 166 italian journal of food science, 2021; 33 (sp1) shahbazpour n et al. of yeasts, molds, salmonella spp., and e. coli verified the effectiveness of the heat treatment in both samples. due to the absence of s. aureus, the hygienic procedures seemed to be effectively preventive. the number of b. cereus and c. perfringens were similarly reported to be less than 10 cfu g−1 in both samples. researchers demonstrated that the presence of nacl and phosphate might inhibit the bacterial growth (kim et al., 2021). moreover, a similar report has been published on increased load of pseudomonas spp. in fish sausages enriched with mycoproteins during refrigerated storage (bahmani and movanes, 2021). nutritional assessments vitamins levels of vitamins b2, b5, b7, and b9 were assessed in both samples (table 3). contents of vitamin b9 showed no significant difference (p < 0.05) between the samples. for other vitamins, mycoprotein sausages significantly achieved higher scores (p < 0.05). according to hashempour-baltork et al. (2020), who comprehensively compared the vitamin b content in meats and mycoproteins, contents of riboflavin, niacin, pyridoxine, and pantothenic acid was 9, 14, 5, and 10 µg g−1 in mycproteins and 0.018, 0.5, 0.052, and 0.35 µg g−1 in meats. these differences were seen in the formulated products as well. results and discussion physicochemical assessments proximate analyses of sausages formulated with mycoproteins and beef are shown in table 2. results of moisture contents demonstrated that moisture in sausages formulated with mycoproteins significantly was higher (p < 0.05) than moisture in sausages formulated with beef. protein and lipid contents seemed to increase with the substitution of mycoproteins in the formulation of sausages. higher contents of moisture and lipids were linked to further wbc (p < 0.05) and obc in mycoprotein samples, compared to beef samples. based on the results, mycoprotein formulation included lower values of carbohydrates, ash, and ph, verified by the previous studies (hashempour-baltork et al., 2020). the low ph in mycoprotein samples was associated with low ph of mycoproteins (4.7) in comparison to ph of meat (5.6). in another report, use of mycoproteins significantly increased nutritional values of fish sausages (e.g., ash, carbohydrate, fat, and protein) (p < 0.05) (bahmani and movanes, 2021). in this study, the ph of fish sausages enriched with mycoproteins increased during storage (bahmani and movanes, 2021). no significant difference (p < 0.05) was generally reported between the two formulations of sausages in whc and peroxide values (p > 0.05). characterization of these two sausage samples indicated that lesser oil and water should be used in the formulation due to the higher wbc and obc values in mycoproteins than beef. in addition to higher obcs, higher proteins and lower carbohydrates could be used in mycoprotein samples as appropriate meals for obese people. these findings verified previous findings, which addressed mycoproteins as healthy nutritious proteins (finnigan et al., 2019). microbial assessments microbiological analysis was carried out on day 1 after cooking to understand heat behaviors of mycoproteins and compare microbial patterns of the samples. absence table 2. proximate analysis and ph of beef sausage. treatment moisture (%) protein (%) lipid (%) carbohydrate (%) ash (%) ph peroxide (meq/kg) whc** (%) wbc** (ml/g) obc** (ml/g) mycoproteinsausage 59 ± 1.9a* 12.5 ± 1.0a 13.9 ± 1.2a 9.8 ± 1.1b 2 ± 0.5b 5.7 ± 0.01b 1.2 ± 0.2a 54 ± 2.5a 0.98 ± 0.09a 0.37 ± 0.08a beef sausage 47.5 ± 1.5b 11.6 ± 1.1b 8.9 ± 1.3b 10.9 ± 1.2a 3 ± 0.4a 6.5 ± 0.01a 1.3 ± 0.2a 55 ± 3.3a 0.79 ± 0.1b 0.3 ± 0.08b *mean ± sd, different letters represent significant differences (p < 0.05). **whc: water holding capacity, wbc: water binding capacity, obc: oil binding capacity. table 3. the level of vitamin b group in mycoprotein/beef sausages (µg/g). treatment vit b2 vit b5 vit b7 vit b9 mycoprotein sausage 3.31 ± 0.5a* 0.02 ± 0.05a 8.81 ± 1.45a 0.48 ± 0.05a beef sausage 1.51 ± 0.5b 0.01 ± 0.04b 2.57 ± 1.5b 0.51 ± 0.03a *mean ± sd, different letters represent significant differences (p < 0.05). italian journal of food science, 2021; 33 (sp1) 167 physicochemical assessment of sausages containing mycoproteins fatty acids the fa composition of the sausage samples are presented in table 5. the total sfas in mycoprotein and beef sausages were 29.7 and 60.7% (w/w), respectively. in fact, unsaturated fatty acid (ufa) levels in mycoprotein sausages (65.44) were significantly (p < 0.05) higher than ufa levels in beef sausages (35.54% w/w). these contents as well as previous contents highly verified the results, especially for the ratio of ufa to saturated fatty acids (sfa) of 2:1 (reihani and khosravi-darani, 2018). in 2009, hosseini et al. (2009) reported 3.2–3.5:1 ratio of ufa to sfa. higher consumption of usfas provides health benefits to patients with cardiovascular diseases (cvd). naturally, the ratio of polyunsaturated fatty acid (pufa) to sfa in beef is typically 0.1. however, the ratio decreases with an increase in meat fats (vahmani et  al., 2015). naturally, the ratio reaches 1.44 in mycoproteins. chicken fat naturally includes 30% of sfas, 45% of monounsaturated fatty acids (mufa), and 21% of pufas (hashempour‐baltork et al., 2020; usda, 2008). these values are close to those of mycoproteins (table 5). mechanical assessments table 6 represents mechanical properties of the cooked samples. hardness of mycoprotein sausages was significantly lower than that of meat sausages (p < 0.05). similar decreases were reported for cohesiveness when meat was totally replaced by mycoproteins. it was reported that beef sausages needed a greater force of chewing, compared to that of nonmeat samples. this was possibly due to the occurrence of stronger networks in myofibril proteins, increasing the product resistance to compression. mycoprotein sausages showed lower values for gumminess and springiness (p < 0.05). lower springiness values were reported by youssef and barbut (2011), when soy protein extracts were used as meat alternatives in emulsified meat batters. kamani et al. (2019) recorded lower levels of food hardness, cohesiveness, gumminess, and springiness by replacement of meats by proteins of amino acids based on the nutritional and physiological roles, aas can be differentiated as eaas, including valine, tryptophan, threonine, phenylalanine, methionine, lysine, isoleucine, leucine, histidine (essential for infants), arginine (semi-essential), and nonessential amino acids (neaa), including tyrosine, serine, proline, glycine, glutamine, glutamic acid, cysteine, asparagine, aspartic acid, and alanine (damodaran and parkin, 2017). the aa profiles showed that mycoprotein sausages included almost all eaas, compared to beef sausages (table 4). these findings were similar to those of other studies, reporting the presence of eaas in single-cell proteins (scp) of f.  venenatum (hashempour‐baltork et al., 2020). in fact, three food categories totally provide 80.9% of daily protein needs of humans (górskawarsewicz et al., 2018). the significance of protein nutrition includes eaa content, biological value, digestibility, net protein use, and protein efficiency ratio. hashempour-baltork et al. (2020) compared the quality of the mycoproteins with that of meat proteins and demonstrated that nutritional indices of these two sources were almost similar. monteyne et al. (2020) reported that mycoproteins were good food sources enriched with eaas. table 4. the amino acid profile in mycoprotein/beef sausages (µg/g). amino acid content in mycoproteinsausage (%g per 100 g protein) content in beef sausage (mg per 100 g protein) l-alanine 4.84 ± 0.99b* 14.90 ± 0.59a* l-arginine 6.74 ± 0.07a 6.0 ± 0.18a aspartic acid 5.25 ± 0.03b 15.20 ± 0.10a l-cystine 11.21 ± 0.15b 96 ± 0.05a l-glutamic 12.92 ± 0.52b 29.02 ± 0.12a glycine 4.15 ± 0.23b 9.05 ± 0.35a l-histidine 3.20 ± 0.75a 1.10 ± 0.85b l-isolucine 5.90 ± 0.63a 3.00 ± 0.53b l-leucine 7.80 ± 0.35a 5.01 ± 0.33b l-lysine 7.50 ± 0.61a 3.0 ± 0.66b l-methionine 1.90 ± 0.80a 1.2 ± 0.11b l-serine 5.35 ± 0.23a 1.04 ± 0.17b l-theronine 13.75 ± 0.77a 11.95 ± 0.95b l-tyrosine 4.35 ± 0.97a 4.950 ± 0.907a l-valine 5.90 ± 0.70a 4.10 ± 0.07b phenyl alanin 9.78 ± 0.87a 3.15 ± 0.50b proline 5.30 ± 0.77b 17.1 ± 0.60a tryptophan 6.7 ± 0.60a 1.5 ± 0.70b *mean ± sd, different letters represent significant differences (p < 0.05). table 5. fatty acid profile of mycoprotein/beef sausage. fatty acid content in mycoprotein sausage (% w/w) content in beef sausage (% w/w) palmitic (c16:0) 18.8 ± 0.20b 32.4 ± 0.29a* stearic (c18:0) 10.90 ± 0.16a 28.3 ± 0.20b oleic (c18:1) 24.95 ± 0.11a 9.13 ± 0.365b linoleic (c18:2) 25.35 ± 0.15a 21.31 ± 0.353b α-linolenic (c18:3) 15.14 ± 0.18a 5.10 ± 0.748b *mean ± sd, different letters represent significant differences (p < 0.05). 168 italian journal of food science, 2021; 33 (sp1) shahbazpour n et al. the production of meat alternatives seems necessary due to the preference of consumers for vegetarian diets, and the increasing nutritional awareness of the populace. like other functional foods that were unknown in the past, after numerous studies and production of various products, today there is a unique response to these products. in the past 2 years, the covid-19 pandemic has drawn the public attention to food security and meat supply worldwide with further global demands for meat alternatives with plant origins. acknowledgment this study was supported financially by the national nutrition and food technology research institute, school of nutrition sciences and food technology, shahid beheshti university of medical sciences, tehran, iran. conflict of interest the authors report no conflict of interest. references aocs, 2017. official methods and recommended practices of the aocs, reapproved 2017. available at: https://www.aocs.org/ attain-lab-services/methods/methods/search-results?subject=b. bahmani, z. and movanes, f., 2021. enrichment of carp sausage with fusarium venatum mycoprotein. utilization and cultivation of aquatics 10(11–25). czauderna, m., kowalczyk, j., niedzwiedzka, k. and wasowska, i., 2003. a highly efficient method for determination of some amino acids and glutathione by liquid chromatography. journal of animal and feed sciences 12(1): 199–214. https://doi. org/10.22358/jafs/67697/2003 damodaran, s. and parkin, k.l., 2017. amino acids, peptides, and proteins. in: fennema’s food chemistry. crc press, pp. 235–356. edited by srinivasan damodaran, kirk l. parkin. boca raton. finnigan, t., needham, l. and abbott, c., 2017. mycoprotein: a healthy new protein with a low environmental impact. in: sustainable protein sources. elsevier, pp. 305–325. sudarshan r. nadathur givaudan flavors, cincinnati, oh, united states; janitha p.d. wanasundara agriculture and agri-food canada, saskatoon sk, canada; laurie scanlin colorado state university, fort collins, co, united states plant origin in chicken sausages. researchers concluded that proteins of nonmeat origin could include further fat and water contents, which might fill the protein interstitial spaces and decrease the product springiness (kamani et al., 2019; youssef and barbut, 2011). this is also addressed for mycoprotein replacement regarding wbc and obc (table 2). textural analysis demonstrated association of sample meats with lower values of adhesiveness. it could be interpreted that a decrease in meat quantity might lead to a significant decrease in the consistency of the cooked emulsions. conclusion this study was carried out to investigate the appropriateness of mycoproteins as complete substitutes for meat in beef sausages. the results showed that mycoprotein substitution improved nutritional and health effects due to the high-value proteins with eaas and less lipid content (mostly ufas). also, it has high contents of eaa and ufa, compared to meat sausages. absence of yeasts, molds, salmonella spp., eshrichia (e.) coli, and staphiloccocus (s.) aureus verified the effectiveness of heat treatment and also hygienic procedures in samples. phycicochemical evaluations show higher contents of moisture and lipids in sausages containing mycoprotein due to wbc and obc, compared to beef samples. besides, mycoprotein samples had lower values (p < 0.05) of carbohydrates, ash, and ph, compared to beef samples. however, mycoprotein sausages achieved lower scores of hardness, cohesiveness, gumminess, and springiness in mechanical assessments, compared to beef sausages. mycoproteins could hold excessive water and fat caused by higher wbc and obc, filling the protein interstitial spaces and decreasing the springiness. this suggested less use of oil and water in mycoprotein formulations. this study has provided valuable information for increasing public awareness on the characteristics of mycoprotein products, including nutritional, textural, and formulation characteristics. this is the first study that substituted meat with mycoproteins in sausages. however, further studies are necessary to optimize mycoprotein sausages using texture improvement ingredients to enhance their gel-forming and textural characteristics. these are currently the major problems in the manufacturing of meat-free sausages. table 6. the hardness, adhesiveness, springiness, cohesiveness, chewiness and gumminess of mycoprotein/beef sausages. treatments hardness (n) adhesiveness (n.s) springiness (%) cohesiveness gumminess chewiness mycoprotein-sausage 23 ± 1.1b* 1.5 ± 0.19a 0.49 ± 0.21b 0.19 ± 0.02b 20.1 ± 1.2b 1.0 ± 0.01b beef-sausage 37.12 ± 1.5a 0.5 ± 0.10b 0.75 ± 0.2a 0.24 ± 0.01a 25.4 ± 1.5a 2.1 ± 0.2a *mean ± sd, different letters represent significant differences (p < 0.05). italian journal of food science, 2021; 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the case of poland. nutrients 10(12): 1977. https://doi.org/10.3390/nu10121977 hashempour‐baltork, f., hosseini, s.m., assarehzadegan, m.a., khosravi‐darani, k. and hosseini, h., 2020a. safety assays and nutritional values of mycoprotein produced by fusarium venenatum ir372c from date waste as substrate. journal of the science of food and agriculture 100(12): 4433–4441. https:// doi.org/10.1002/jsfa.10483 hashempour-baltork, f., khosravi-darani, k., hosseini, h., farshi,  p. and reihani, s.f.s., 2020b. mycoproteins as safe meat substitutes. journal of cleaner production 253: 119958. https:// doi.org/10.1016/j.jclepro.2020.119958 hashempour-baltork, f., torbati, m., azadmard-damirchi, s. and savage, g.p., 2017. quality properties of sesame and olive oils incorporated with flaxseed oil. advanced pharmaceutical bulletin 7(1): 97. https://doi.org/10.15171/apb.2017.012 hashempour-baltork, f., torbati, m., azadmard-damirchi, s. and savage, g.p., 2018. chemical, rheological and nutritional characteristics of sesame and olive oils blended with linseed oil. advanced pharmaceutical bulletin 8(1): 107. https://doi. org/10.15171/apb.2018.013 hosseini, s., khosravi-darani, k., mohammadifar, m. and nikoopour, h., 2009. production of mycoprotein by fusarium venenatum growth on modified vogel medium. asian journal of chemistry 21(5): 4017. iso, 2013. making food safer according to iso methods, culture media and associated products for pathogen detection and enumeration. available at: http://www.oxoid.com/pdf/iso-foodsafety-brochure.pdf. kamani, m.h., meera, m.s., bhaskar, n. and modi, v.k., 2019. partial and total replacement of meat by plant-based proteins in chicken sausage: evaluation of mechanical, physico chemical and sensory characteristics. journal of food science p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33isp1.2075 127 p u b l i c a t i o n s codon characterization of functional fish ham produced from silver carp (hypophthalmichthys molitrix) surimi enriched with natural antioxidant and vegetable fiber ebrahim mahdavi1, peiman ariaii2,* 1department of food science & technology, nour branch, islamic azad university, nour, iran; 2department of food science & technology, ayatolla amoli branch, islamic azad university, amol, iran *corresponding author: peiman ariaii, department of food science & technology, ayatolla amoli branch, islamic azad university, amol, iran, email: p.aryaye@yahoo.com received: 23 may 2021; accepted: 27 june 2021; published: 23 august 2021 © 2021 codon publications open access paper abstract in this study, the effect of grape pomace (ge), orange peel extract (oe), and nisin (n) with modified atmosphere packaging (map: 70% co2 + 30% n2) was investigated on the quality and shelf life of ham produced from low fat silver carp surimi (containing inulin fiber [fi] and salatrim [s]) kept in the refrigerator with t1: control (containing nitrite), t2: control + map, t3: ge 0.5%+ fi 5% +oe 0.5% +n 0.5%, t4: s 5%+ oe 0.5%+ ge 0.5% +n 0.5% + map, t5: fi 2.5%+ s 2.5%+ ge 0.5%+ oe 0.5%+ n 0.5%+ map. the texture characteristics of ham at the beginning of storage, cooking loss, chemical indices (peroxide value, ph, color index), and microbial (total count bacteria, psychrotrophic bacteria, mold and yeast, clostridium botulinum) during 42 days storage in the refrigerator (4 ± 1°c) were evaluated. the results of the tests were analyzed according to duncan’s by spss software with 95% confidence. the results showed that inulin and salatrim fibers have a positive effect on the texture and cooking loss of ham and inulin had a better effect, so the value of cooking loss in treatment 3 was 1.62% and control treatment was 2.09%. using three natural preservatives along with map could slow down the oxidative spoilage, microbial and color index changes in ham. these combinations also inhibited the growth of c. botulinum. in most tests examined, treatment 3 showed no significant difference with treatment 1 (ham containing nitrite) (p > 0.05), so this treatment shows that natural additives (nitrite replacement) improve the quality properties of low-fat ham. keywords: fat replacement; fish ham; inulin fiber; nisin; plant extracts introduction seafood is one of the most important sources of protein and have a considerable role in the health of consumers. surimi is one of the minced fish products, which has been a traditional method of fish preservation. in general, surimi refers to minced and washed fish, which can be used for a variety of food products such as fish sausage, fish ham, fish cake, fish burger, and other products (jin et al., 2017; ozpolat and patir, 2015; shabanpour et al., 2007; zhang et al., 2020). one of the meat products is ham which is very common in different parts of the world. these products have low oxidative stability and are sensitive to fat rancidity during storage. therefore, some additives are used to control microbial load and oxidative changes in meat products. one of the main ways of processing meat products is the use of nitrate and nitrite. these compounds increase the shelf life and also prevent the spoilage of products during storage (riazi et al., 2016; nogueira et al., 2019). salt and nitrite are also used to increase the shelf life and taste of the meat. due to the chemical structure of nitrite in sausage and ham, its carcinogenic potential and also in response to consumer demand for natural products, reducing nitrite utilization italian journal of food science, 2021; 33 (sp1): 127–136 mailto:p.aryaye@yahoo.com 128 italian journal of food science, 2021; 33 (sp1) ariaii p sensory satisfaction. in general, an increase in fat and oil increases the frying ability and reduces the fragility of the tissue. however, its reduction results in a firm and gummy texture with low moisture content (akalin and  erisir, 2008). on the other hand, a high intake of fats and oils can increase blood triglycerides, leading to cardiovascular disease and heart failure, so their consumption should be reduced. inulin is a non-digestible carbohydrate-containing natural fructooligosaccharides and 1–2 glucopyranose residues. it has dietary fiber properties and due to its specific health and technological properties, there is a great interest in its use. the characteristic of inulin as a mimetic lipid is related to its ability to bind to water molecules and form a gellike network. inulin also makes the mouth feel greasy. this property has been used in the production of fatfree yogurt, chocolate, imitated cheeses, ice cream, and low-fat fermented sausages, and the results indicate no change in the organoleptic properties of the products (ognean et al., 2006; shi et al., 2020). another fat replacement is salatrim. salatrim (derived from small and large molecules of triacylglycerides) is a generic name for a family of triglycerides that contains a mixture of at least one short-chain fatty acid (mainly c4:0, cs:0, c2:0) and at least one long-chain fatty acid (mostly stearic acid cus: 0) that locates randomly on glycerol. this triglyceride has the physical properties of fat, but only contains 5 calories per gram instead of 9 calories per gram of natural fat (surendra babu et al., 2018) menegas et al. (2013) reported that fermented chicken sausages formulated with standard amounts of corn oil, reduced amounts of oil, and reduced amounts of oil containing inulin as a partial oil substitute remained stable and had no substantial loss of physical, chemical, microbiological, or sensory attributes during storage at 4°c for 45 days. in this study, we evaluated the effect of natural antioxidants as a nitrite substitute and the use of fat substitutes to reduce oil in ham formulations produced from silver carp surimi as well as map for the increased shelf life of the ham. material and methods raw materials at first, 30 kg of silver carp were caught from the farms (1 hectare) and transported to the laboratory with ice boxes (0°c), followed by washing and peeling. after this step, the underlying meat was removed without contact with the viscera and then it was ground first through a 10 mm plate and then through a 5 mm plate in a meat grinder (mkg1300p, panasonic, japan). or replacing all or part of it in meat products with natural compounds have been considered (shu et al., 2020). here are some of the compounds that have antioxidant and antimicrobial activity as nitrite substitutes (araújo et al., 2019). citrus (citrus reticulata, blanco) is one of the most important fruits in the world. it contains minerals, phenolics, pectin, and dietary fiber, and is rich in vitamins b, a, and c; as a result, they have nutritional and medicinal properties. nearly a hundred industries use citrus to produce their products (drosou et al., 2015). one notable point in the citrus industry is the added value of these products through the production of by-products such as citrus peel. these are rich in flavones, polymethoxylates, and phytochemicals, which are rare in other plants, resulting in recent years, special attention has been paid to the use of citrus peel. grape pomace (ge) is one of the many wastes (5000 tonnes per year) produced by juice factories, a rich source of several valuable compounds such as citric acid, tartrate, dietary fiber, and phenolic compounds. anthocyanin (malvidin and penonidine), flavonol (quercetin and meristine), and phenolic acids are the major phenolic compounds and flavan-3-l, catechin, and epi-catechin, and gallic acid are the predominant phenolic compounds in ge (batpho et al., 2017). bacteriocin–nisin (n) is from group a antibiotics with 34 amino acids that have a ring structure. in the nisin structure, there is a part called the hinge area which is capable of disassembling the ring systems and is characterized by its flexibility. nisin has no inhibitory effect on gram-negative bacteria, yeasts, and fungi (siroli et al., 2016). this bacteriocin was first synthesized by lactococcus lactis in england in 1928 by rogers and whitier and was reported by the food and agriculture and world health organization (fao/who) in 1969, for its low toxicity to humans, as a gars1 and food preservative. since 1987, it has been used as a permitted additive in food and dairy products (hematian sourki et al., 2012). packaging is another way to increase food shelf life. modified atmospheric packaging (map) is used for the shelf life of fresh (non-frozen) food. the use of vacuum, map, and high co2 packaging is easily feasible for processed meats, but high levels of co2 will have negative effects on product quality, especially texture changes and increased blood loss (ashraf et al., 2011; ghosh and dash 2020). fat is one of the important constituents that affect the sensory properties of food products, including flavor, color, texture, oral sensation, and overall 1generally recognized as safe. italian journal of food science, 2021; 33 (sp1) 129 characterization of functional fish ham produced from silver carp surimi after these, with inulin fiber, salatrim concentrations of 5% were chosen as optimal for the following study. fat decreased by 5%. also, extracts of orange peel, grape pomace, and nisin in the concentration of 0.5% (concentration approved by sensory evaluators unchanged in the taste of fish ham) were added combined as a substitute for part of nitrite and then, same steps were taken as that of control treatment and finally the treatments were packed into three multilayer flexible pouches (3 and 4 layers) under modified atmosphere (map: 70% co2 + 30% n2). the treatments were kept at refrigerator temperature (4 ± 1°c) for 42 days. on the 0, 7, 14, 28, 35, and 42 days of storage, three hams from each section were randomly selected and tested to determine qualitative parameters (physicochemical and microbiological). all experiments were performed with three replications. in total, five treatments were studied: t1: control treatment (with nitrite) t2: control treatment + map t3: ge 0.5%+ fi 5% +oe 0.5% +n 0.5% t4: s 5%+ oe 0.5%+ ge 0.5% +n 0.5% + map t5: fi 2.5%+ s 2.5%+ ge 0.5%+ oe 0.5%+ n 0.5%+ map cooking loss test the cooking loss test was performed according to the method given by hayes et al. (2009) with slight modification. thirty grams of samples were stuffed into screw-top test tubes and were heated in a steam bath at 70°c for 30 min. the cooked samples were quickly immersed in cool water for 10 min. cooking loss was determined by weighing individual samples before and after cooking, and the difference was expressed as a percentage of the original weight. texture analysis to measure the texture of the ham, the cubic pieces were cut into 1 × 1 × 1 cm3 dimensions and subjected to compression test by a texture analyzer with a flat probe profile of 40 × 40 mm and a load of 10 kg. the force required to compress the samples to 70% of their initial height was measured at a constant rate of 200 mm/min (vural, 2003). chemical analyses peroxide value peroxide values of different treatments were determined according to the bagheri et al. method (2016). results were expressed in meq oxygen kg-1 lipids. preparation of treatments to prepare the surimi, minced meat was washed with drinking water at a temperature below 10°c for 10 min in three stages. rinsing was performed in 0.2% brine in the third step. the later step caused better dehydration and reduced solubility of sarcoplasmic proteins in the salt solution. surimi obtained during experiments was stored at refrigerator temperature (shabanpour et al., 2007). for samples preparation (control treatment), the raw materials were weighed and blended to obtain a uniform paste according to the formulations (table 1). for this purpose, the meat was placed with a third of the ice in the cutter (talsa, e-46950, eu) and was mixed with the high-speed cutter, followed by nitrate, protein residues with ice, carbohydrates, fillers, vitamin c, and finally, spices were added into the meat. after mixing, the components of the cutter and paste mixture were filled into the wrapper, the ham was incubated in the baking chamber at 85°c for 45 min (bourne, 2002; hayes et al., 2009). inulin and salatrim fibers were added to the primary ham formulation as a fat substitute. in addition, a ranking test previously performed comparing ham samples with inulin fiber and salatrim at different concentrations showed significantly lower acceptability of the samples incorporating 6% or 7% of them when compared to the rest (5% or lower) (data not shown). table 1. formulation and components of ham (control treatment). row components % 1 fish meat (surimi) 55 2 oil 10 3 egg yolk 1 4 starch 1 5 gluten 1 6 skim milk 3 7 wheat 3 8 spice 1 9 salt 1.2 10 garlic 1 11 sugar 0.5 12 phosphate 0.4 13 vitamin c 0.05 14 nitrite 0.012 15 ice+ water 20 16 citrate 1 17 casein 0.5 18 sodium glutamate 0.288 19 glucono delta lactone 0.05 130 italian journal of food science, 2021; 33 (sp1) ariaii p ph value the ph values of different treatments were measured with a digital ph meter calibrated to ph 4 and 7 standards (valipour kootenaie et al., 2017). color test the color of the sausage was measured using the hunterlab color flex colorimeter. the color test results include three hunter indices a*, b*, and l*; where, l* is a light symbol which is black (0) and white (100), a* is a green-to-red symbol, where -a is green and + a is red, and b* is blue to yellow symbol, where + b is yellow and -b is blue. the experiment was performed in triplicates (choi et al., 2009). microbial analyses ten grams of each sample was mixed and homogenized with 90 ml sterile sodium chloride solution and the required dilutions were prepared. one milliliter of each dilution was used for culture by the pour plate method. total count and psychrotrophic bacteria were counted on plate count agar at 37°c for 2 days and 7°c for 10 days, respectively. the results were reported as log cfu/g (javadian et al., 2017). mold and yeast test dilution of the sample was first prepared in peptone water broth, then transferred to a plate containing drbc2 medium. plates were aerobically incubated at 25°c for 5 days (isiri, 2008). inoculation and enumeration of clostridium botulinum to ham samples approximately 108 cfu/ml of clostridium botulinum was added to the ham samples. the samples were then massaged to ensure complete mixing of the bacterium with the hams and placed in especially filled coatings in the baking chamber. at least 10 hams were considered for each treatment. all treatments were packed in zippered nylon bags and stored at refrigerator temperature (4 ± 1°c) during the experiment. for bacterial count at each sampling time, 1 g of sample was mixed with 9 ml of physiological serum and suspended for half an hour. depending on the sample, the dilutions range from 102 to 104. one milliliter of diluted sample was poured into the petri dish and then 10–15 ml of sc agar medium at 44 to 47°c was added to the petri dish and thoroughly mixed. after solidification of the medium, about 10 ml of the same medium was poured into the petri dish. plates were then placed in an anaerobic 2dichloran rose bengal agar. jar and incubated at 37°c for 22 h. at the end of incubation, all plates containing less than 150 colonies were selected and the black colonies on each plate indicating the probability of c. botulinum were counted (isiri, 1994). statistical analysis all experiments were performed in a completely randomized design with three replications and the result was reported as mean ± standard deviation. statistical analysis of treatments was performed using spss 16.0 software by anova. significant differences were determined by the duncan test at the level of 0.05 and figures were drawn using microsoft excel software. results and discussion texture analysis according to the results (figure 1a), the replacement of oil with inulin fiber and salatrim increased the firmness b 1 0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5 5.1 1 2 3 4 5 6 7 8 9 10 (a) (b) 2 3 treatment f rir m ne ss ( kg ) 4 5 1 2 3 treatment e la st ic ity ( cm ) 4 5 b b b b a a a a a figure 1. texture analysis of different treatments of sausage [firmness (a); elasticity (b)] (t1: control, t2: control + map, t3: ge 0.5%+ fi 5% +oe 0.5% +n 0.5%, t4: s 5%+ oe 0.5%+ ge 0.5% +n 0.5% + map, t5: fi 2.5%+ s 2.5%+ ge 0.5%+ oe 0.5%+ n 0.5%+ map). values with different superscripts are significantly different at p < 0.05. italian journal of food science, 2021; 33 (sp1) 131 characterization of functional fish ham produced from silver carp surimi moisture during the frying process and are reduced cooking loss. this is due to their ability to form hydrogen bonds with water molecules, that is, to entrap water molecules, which prevents moisture outflow during the frying process (farajzadeh et al., 2013; marchetti et al., 2013). amina et al. (2014) also reported that the addition of apple fiber reduced the cooking time of nugget meat. peroxide value lipid oxidation in meat is one of the causes of damage to meat tissue during storage. oxidation of lipids in meat has a complex mechanism. during this process, in addition to the adverse effects on taste and color, protein solubility is also reduced and eventually nutritional value is declined (valipour kootenaie et al., 2017). according to the results of the present study (figure 3a), the peroxide value increased in all treatments with increasing time (p <0.05), the increase of peroxide value in meat products was also reported by other researchers (javadian et al., 2017; valipour kootenaie et al, 2017). according to the results, the lowest peroxide values were observed in the nitrite treatments with the map during storage (p < 0.05). this is due to the high amount of carbon dioxide used in map, which prevents the growth of aerobic and anaerobic bacteria. the highest level of carbon dioxide in the map has been reported up to 50% (silbande et al., 2018). the change in peroxide value in the inulin treatment was also slower than salatrim treatment. the lower peroxide value in the inulin treatment was due to the lower fat content which reduced oxidative spoilage. also, using three preservatives together effectively controlled the increasing trend of peroxide value. the lowest values were observed in all days after t2, in t1 and t3. most of the time, the latter treatments did not have significant differences (p < 0.05). the antioxidant properties of plant extracts depend on phenolic compounds. polyphenols are capable of trapping free radicals, especially proxy radicals, which are one of the key intermediate chain reactors, thereby terminating the cycle of oxidative spoilage reactions (valipour kootenaie et al., 2017). also, bacteriocin–nisin can decrease peroxide value by decreasing the population of lipase-producing bacteria (such as pseudomonas species) (dehbandi et al., 2014). in total, these three preservatives together with map can have similar effect with nitrite on ham. the permitted level of peroxide value in the aquatic product for human consumption is 5 meq oxygen kg-1 lipids (yanar, 2007). according to the results at the end of the storage period, peroxide value was lower than the acceptable range in all samples except t4. values and the effect of inulin was significantly higher than salatrim. the lowest values were observed in control treatments (t1 and t2) and the highest values were observed in t3 (5% inulin fiber) (p < 0.05). the final texture of food products is affected by the composition of the food. the interactions between proteins, starch, and other constituents are important for the final quality of the product. on this basis, compounds used probably enhance the ability of the meat proteins to bind and, as a result, tighten the ham texture. it also appears that lower oil content in t3 affects texture firmness than other treatments (amina et al., 2014). by definition, elasticity refers to the rate of return of the sample to its original state after removing the deformation force. elasticity is directly related to the degree of rigidity of the treatment (amina et al., 2014). according to the results (figure 1b), the highest values were observed in control treatments (t1 and t2) and the lowest values were observed in t3. the results of the present study were consistent with the results of menegas et al. (2013) by applying inulin fiber in chicken sausage fermented with corn oil to increase texture firmness and reduce sausage elasticity. cooking loss the results of cooking loss (figure 2) in the present study showed that with increasing time, the values of cooking loss increased in all treatments. replacing the oil with inulin fiber and salatrim reduced the cooking loss. the effect of inulin on the reduction of cooking loss was significantly higher than salatrim. the highest values were observed in control treatments (t1 and t2) on all days of storage and the lowest values were observed in t3 (5% inulin fiber) (p < 0.05). in general, the fibers retain 1 0 0 0.5 1 1.5 2 2.5 7 14 21 storage time (day) c oo ki ng lo ss ( % ) 28 35 42 2 3 4 5 figure 2. cooking loss of different treatment during storage (t1: control, t2: control + map, t3: ge 0.5%+ fi 5% +oe 0.5% +n 0.5%, t4: s 5%+ oe 0.5%+ ge 0.5% +n 0.5% + map, t5: fi 2.5%+ s 2.5%+ ge 0.5%+ oe 0.5%+ n 0.5%+ map). 132 italian journal of food science, 2021; 33 (sp1) ariaii p ph values even during frozen storage that the microorganisms are inactive, the previously produced enzymes and the endogenous enzymes in the meat are active. the results of ph values in ham (figure 3b), in the present study, showed that with increasing time, ph values increased in all treatments. the increase in ph during storage can also be attributed to the production of volatile nitrogenous bases (such as ammonia and trimethylamine) resulting from the activity of meat-spoilage bacteria (valipour kootenaie et al., 2017). the use of map slowed the ph increasing trend. since high levels of carbon dioxide reduce bacterial growth, therefore, the ph increasing trend is slower than the control treatment. in general, the lowest ph values were observed in nitrite + map treatment (t2) (p < 0.05). in general, the lowest values were observed in all days after t2 in t1 and t3 (p < 0.05). the latter treatments were not significantly different (p > 0.05). the equivalence of ph values in the sample containing extract compared to the nitrite treatments can be related to the antibacterial activity of the extract. as the microbial flora decreases, the number of secondary metabolites produced decreases and the ph increasing trend is slowed (vilela et al., 2016). the use of nisin also influences spoilage bacteria due to its antibacterial properties and reduces the capacity of the bacteria to oxidative deamination non-protein nitrogen compounds (such as ammonia and trimethylamine), thereby reduces the ph-increasing process (dehbandi et al., 2014). color test the color index l indicates the brightness symbol (black to white). so the more l the meat is lighter. the color index a is the indicator of the color change from green to red. the color index b represents the color change from blue to yellow. results of color index showed that replacement of nitrite with preservatives reduced color index l and a and increased color index b. sodium nitrate and nitrite are used to create a bright red (pink) and to prevent the darkening of the meat products, as well as antimicrobial preservatives and to create a special flavor. so it seems natural to replace nitrite with other compounds and change its color. according to the results of using modified atmosphere, the color index l decreases (figure 4a). overall, the highest values were observed in control treatments. the other three treatments had no significant difference indicating a decrease in the oxidation process in ham. the brightness values of the ham samples are correlated with the peroxide values. as the number of peroxides increases, the brightness decreases and the samples darken. it can be stated that the use of three preservatives prevents the oxidation of pigments and act as a chemical preservative such as nitrite. the color index a (figure 4b) also decreased in all treatments. generally, one of the causes of oxidation in meat products is the presence of compounds such as myoglobin and hemoglobin which in the presence of metals such as iron, act as peroxidants. this is one of the factors affecting the oxidation of oxymyoglobin (light red) to metmyoglobin (brown color) during the storage of meat products. therefore, as a result of redox oxidation, color index a is reduced (jin et al., 2007). the color index b (figure 4c) decreased and increased during the storage period, which was consistent with the results of giatrakou et al. (2010). they reported that yellow index changes in meat products did not have a specific pattern and can change during storage by product type, formulation, and form of packaging. total and psychrotrophic bacteria the results of total count bacteria (tvc) (figure 5a) and psychrotrophic bacteria (ptc) (figure 5b) were somewhat consistent (p < 0.05) and were increased during storage time (p < 0.05). the map decreased the growth of tvc and ptc so that the lowest values were observed in 1 0 0 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 1 2 3 4 5 6 (a) (b) 7 14 21 storage time (day) p v ( m eq /k g) 28 35 42 0 7 14 21 storage time (day) ph 28 35 42 2 3 4 5 1 2 3 4 5 figure 3. peroxide value (pv) (a) and ph (b) of different treatment during storage period (t1: control, t2: control  + map, t3: ge 0.5%+ fi 5% +oe 0.5% +n 0.5%, t4: s 5%+ oe 0.5%+ ge 0.5% +n 0.5% + map, t5: fi 2.5%+ s 2.5%+ ge 0.5%+ oe 0.5%+ n 0.5%+ map). italian journal of food science, 2021; 33 (sp1) 133 characterization of functional fish ham produced from silver carp surimi nitrite + modified atmosphere treatment (t2). gases used in modified atmospheres such as carbon dioxide have antimicrobial activity and their mechanism is to dissolve in the water of the food and produce carbonic acid, which enters the cell membrane of the microorganism and after ionization, it disrupts the intracellular electrical balance and ultimately causes bacterial death (ghosh and dash 2020). the use of three preservatives together more effectively slowed down the growth of tvc. the lowest values were observed in all days of t2 and four other treatments had no significant difference (p > 0.05). similar results were observed with ptc bacteria indicating a positive effect of three preservatives as a substitute for nitrite in sausages. the lower tvc and ptc in the extract containing treatments can be due to phenolic compounds such as cineol. phenolic compounds in plant extracts destroy the microorganisms and cause liposaccharides to exit and increase the permeability of the cytoplasmic membrane to atp3. atp release leads to the termination of cell 3adenosine triphosphate. 1 (a) (b) (c) 2 3 4 5 1 2 3 4 5 1 2 3 4 5 0 7 14 21 storage time (day) 28 35 42 0 7 14 21 storage time (day) 28 35 42 0 7 14 21 storage time (day) 28 35 42 35 5 4 5 6 7 8 9 10 5.5 6 6.5 7 7.5 8 37 39 41 43 45 47 c ol or in de x (l ) c ol or in de x (a ) c ol or in de x (b ) figure 4. color index l, b, and a (a, b, and c, respectively) of different treatment during storage period (t1: control, t2: control + map, t3: ge 0.5%+ fi 5% +oe 0.5% +n 0.5%, t4: s 5%+ oe 0.5%+ ge 0.5% +n 0.5% + map, t5: fi 2.5%+ s 2.5%+ ge 0.5%+ oe 0.5%+ n 0.5%+ map). 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 0 7 14 21 storage time (day) 28 35 42 0 7 14 21 storage time (day) 28 35 42 0 7 14 21 storage time (day) 28 35 42 0 1 2 3 4 5 6 7 8 (a) (b) (c) t v c ( lo g c f u /g ) 0 1 2 3 4 5 6 7 8 9 c lo st rid iu m b ot ul in um ( lo g c f u /g ) 0 1 2 3 4 5 6 7 p t c ( lo g c f u /g ) figure 5. total viable count (tvc) (a), psychrotrophic bacteria count (ptc) (b), and clostridium botulinum (c) of different treatment during storage period (t1: control, t2: control  + map, t3: ge 0.5%+ fi 5% +oe 0.5% +n 0.5%, t4: s  5%+ oe 0.5%+ ge 0.5% +n 0.5% + map, t5: fi 2.5%+ s 2.5%+ ge 0.5%+ oe 0.5%+ n 0.5%+ map). 134 italian journal of food science, 2021; 33 (sp1) ariaii p texture and cooking loss of ham, which had a higher effect of inulin fiber. also, the use of three natural preservatives combined with map in most treatments had almost similar chemical and microbial properties compared to conventional ham treatment (containing nitrite). in all tests, among the treatments containing natural preservatives, the best results were obtained in t3 (5% oil + 5% inulin fiber + 0.5% ge extract + 0.5% orange peel extract (oe) + 0.5% n + map), so the results can produce a functional low-fat, nitrite-free, fiber-containing product and natural antioxidants. references akalin, a.s. and erisir, d., 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y.s., choi, j.h., han, d.j., kim, h.y., lee, m.a., kim, h.y., et al. 2009. characteristics of low-fat meat emulsion systems with pork fat replaced by vegetable oils and rice bran fiber. meat science 82: 266–271. https://doi.org/10.1016/j.meatsci.2009.01.019 dehbandi, a., motallebi, a.a., razavilar, v. and pourgholam,  r., 2014. effect of nicin z on some of spoilage chemical and bacterial energy storage and cell death (burt, 2004). bacteriocin– nisin also acts by creating gaps in the plasma membrane due to the wide inhibitory spectrum shown on bacteria in vegetative cells, so that cytoplasmic components seep out through the gaps, and after atp hydrolysis, cell death occurs. the anionic lipid present in the membrane is an active site for nisin binding (li et al., 2016). in the present study, no mold and yeast were observed. clostridium botulinum clostridia are bacteria that occur in the environment and on the flora of the intestines of humans and animals (wagner et al., 2006). these bacteria have different resistance to adverse environmental factors. however, it has been shown that sulphite-reducing clostridia can persist against high salt levels of ham and can tolerate subsequent processes such as pasteurization and high water activity (aw) reactivates them and risks to consumer health (wagner et al., 2006). therefore, the inactivation of these bacteria using natural preservatives is important. results of c. botulinum (figure 5c) showed that with increasing time, c. botulinum decreased in all treatments. according to the results of the map, the process of growth of c. botulinum was slowed so that on day 14 the lowest value of the bacterium was observed in control + map treatment (t2). the use of the preservative effectively inhibited the bacterium as no treatment was observed on day 21 due to the antimicrobial activity of the extracts. phenolic compounds in plant extracts destroy microorganisms and result in the release of liposaccharides and increased permeability of the cytoplasmic membrane to atp. atp withdrawal results in the depletion of cell energy storage and cell death (burt, 2004). clostridium botulinum is a gram-positive bacterium, and the fact that nisin is effective against gram-positive bacteria and not effective against gram-negative bacteria has been well established. nisin is specifically active against the vegetative cells and heat-resistant spores of bacillus, clostridium, and listeria monocytogenes. the mechanism of nisin antimicrobial activity against the vegetative walls of cells is binding to the cytoplasmic membrane. nisin is a cationic antimicrobial that binds to electrons with a negative charge through electrostatic bonding. after binding, nisin enters the membrane and creates a temporary small cavity. this process causes the rapid release of ions, amino acids, and cellular atp (vongsawasdi et al., 2012). conclusions the results of the present study showed that the addition of inulin fiber and salatrim had a 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https://doi.org/10.1007/s00217-003-0727-y� https://doi.org/10.1007/s00217-003-0727-y� https://doi.org/10.1007/s00217-003-0727-y� https://doi.org/10.1111/j.1745-4573.2007.00094.x� https://doi.org/10.1111/jfs.12295� https://doi.org/10.1016/j.fpsl.2016.04.002� _goback 74 issn 1120-1770 online, doi 10.15586/ijfs.v34i2.2204 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (2): 74–79 p u b l i c a t i o n s codon policaptil gel retard® reduces body weight and improves insulin sensitivity in obese subjects giorgia centorame1, maria pompea antonia baldassarre1*, giulia di dalmazi1, francesca gambacorta2, fabrizio febo2, agostino consoli1,2, gloria formoso1,2 1department of medicine and aging sciences, center for advanced studies and technology, university g. d’annunzio of chieti-pescara, chieti, italy; 2endocrinology and metabolic disease clinic of pescara, pescara, italy *corresponding author: maria pompea antonia baldassarre, md-phd, department of medicine and aging sciences, center for advanced studies and technology, university g. d’annunzio of chieti-pescara, via luigi polacchi, 11 66100 chieti (ch), italy. email: marbaldassarre@gmail.com received: 24 march 2022; accepted: 18 may 2022; published: 14 june 2022 © 2022 codon publications open access paper abstract policaptil gel retard® (pgr), a natural fiber-based molecule, has been shown to prevent weight gain and ameliorate insulin-resistance indices in obese children and adolescents. the aim of this study was to compare the effects of 12 weeks of low calories and low glycemic index (lc-lgi) diet associated or not with the intake of pgr on anthropometric, bioimpedance, and metabolic parameters. data from 20 obese adult subjects (10 per group) were analyzed. an lc-lgi diet with or without pgr intake reduced weight, bmi, and waist circumference. pgr intake elicited a reduction in fasting plasma insulin and insulin resistance index together with an improvement in insulin sensitivity. keywords: caloric intake; dietary fiber; glycemic index; mediterranean diet; obesity introduction the world health organization (who) declared obesity as the largest global chronic health disease in adults (frühbeck et  al., 2013). obesity is a metabolic disease (icd-10 code), with epidemic proportions, becoming one of the leading causes of cardiovascular disease, disability, and death worldwide (blüher, 2019). obesity is the result of individual behaviors and environmental factors leading to excessive caloric intake and inadequate physical activity. it is characterized by a pro-inflammatory milieu leading to hyperinsulinemia, hyperglycemia, and hyperlipidemia, which can foster insulin resistance and metabolic abnormalities (ceriello, 2003; finer, 2015; who, 2015). appropriate goals of weight management involve achieving a realistic weight loss (at least 5% of baseline body weight) to promote a reduction in health risks and should include, besides weight loss, weight maintenance and prevention of weight regain (frühbeck et al., 2013). to date, conventional treatment for obesity is based on nutritional therapy, low-calories and low-glycemic index (lc-lgi) diets, combined with regular physical activity. nevertheless, results of several clinical studies indicate that is not often feasible to achieve and maintain weight loss (dwyer et al., 2000). an elevated consumption of fibers slows down the absorption of carbohydrates, thus reducing the extent and the velocity of post-prandial blood glucose increase (weickert and pfeiffer, 2008). for this reason, integrating an lc-lgi diet with the intake of natural fiber-based molecules, such as the polysaccharide complex pgr, might improve the success rate of dietary intervention. italian journal of food science, 2022; 34 (2) 75 policaptil gel retard and obesity data relative to body weight, bmi, waist circumference, bioimpedance data, fasting plasma glucose (fpg), plasma insulinemia levels and insulin resistance index (homeostatic model assessment for insulin resistance, homa-ir), and sensitivity index (quantitative insulin sensitivity check index, quicki) before and after 12 weeks of treatment were collected. the study was conducted in compliance with the declaration of helsinki and european guidelines on good clinical practice. ethical approval (ethical code pe 08) was obtained from the chieti and pescara provinces ethics committee. bioimpedance analysis and insulin sensitivity bioimpedance analysis was performed using a bc-420 ma class iii body composition analyzer (class iii: compliant with the european directive on medical devices) and the european nawi standard relating to nonautomatic weighing instruments, tanita, tokyo, japan. body composition was evaluated by the instrument through a frequency of 50 khz. the margin of error for the measure ments performed corresponded to ±2%, which corresponds to a variation of approximately ±0.5% of the fat mass measurement in a standard figure (esparza-ros et al., 2019). insulin resistance (homa-ir) and sensitivity (quicki) indexes were calculated according to the following formulas as previously reported: • homa-ir: fpg (expressed in mg/100 ml) × fasting insulin (expressed in μu/ml)/405 (matthews et  al., 1985); • quicki: 1/(log (fasting insulin μu/ml) + log (fpg mg/dl.) (katz et al., 2000). dietary intervention a personalized diet plan prescription was elaborated taking into consideration the subject’s ideal body weight, lifestyle, eating habits, food preferences, and working shifts. the energy intake was fixed by reducing by 25% the estimated caloric intake, which was calculated on the basis of food history and basal metabolism assessed by bioimpedance analysis. the total amount of energy intake never fell below 1200 kcal per day. the diet composition was formulated in compliance with the indications provided by the italian recommended dietary allowances (rads) (sinu, 2019): pgr is a patented complex of macromolecules produced by concentrating specific polysaccharide fractions obtained from: cellulose, opuntia ficus indica, amorphophallus konjac, althaea officinalis, linum usitatissimum, tilia platyphyllos, and cichorium intybus. pgr, with or without association with metformin, has been shown to prevent weight gain, and to ameliorate insulin-resistance indices, in obese children and adolescents (stagi et  al., 2016, 2017). recently, it has been observed that a single intake of pgr is associated with a significant reduction in appetite, ghrelin, and triglycerides in the postprandial period in obese children (fornari et al., 2020). guarino and coll. published results of a randomized controlled clinical trial showing that pgr supplementation and metformin have comparable effects in terms of glycemic control in obese adult subjects affected by metabolic syndrome (ms) or type 2 diabetes (t2d). moreover pgr supplementation was associated with a greater serum lipid-lowering capacity and tolerability as compared to metformin (guarino et al., 2021). the aim of this study was to compare the effects of an lc-lgi diet plus pgr assumption versus an lc-lgi diet alone on anthropometric, bioimpedance, and metabolic parameters in obese adults. materials and methods study design and subjects this was a retrospective pilot single center study conducted at the endocrine and metabolic disease unit, pescara town hospital, italy. data of obese adults (body mass index, bmi ≥ 30 kg/m2; age ≥18 years) treated for at least 12 weeks with an lc-lgi diet with or without pgr in the period between 01/01/2016 and 31/12/2020 were retrospectively collected. we excluded from analysis subjects with t2d, thyroid dysfunction, treated with medications associated with weight gain or weight loss, affected by genetic syndromes associated with obesity or by autoimmune, chronic, or systemic diseases. patient’s data were anonymously extracted from an electronic medical record system (mystar connect/smart digital clinic, meteda srl, san benedetto del tronto, italy) and divided into two groups according to whether or not pgr was part of patients’ treatment (lc-lgi diet plus pgr group/lc-lgi diet alone group). 76 italian journal of food science, 2022; 34 (2) centorame g et al. results baseline characteristics the primary demographic, clinical, and biochemical characteristics of the two study groups are shown in table 1. all baseline characteristics were similar in both groups. effects of 12-week intervention of an lc-lgi diet versus an lc-lgi diet plus pgr anthropometric measurements after 12 weeks of intervention, there was a significant reduction in body weight, bmi, and waist circumference both in patients following an lc-lgi alone diet and in those on an lc-lgi diet plus pgr, as shown in figure 1 and table 2. the magnitude of the intervention effects on these parameters was not different between the two groups (table 2). body composition variables the effects of an lc-lgi diet and an lc-lgi diet plus pgr on body composition measurements are shown in • carbohydrates 50–53 % kcal/day (<10% simple sugars); • lipids 25–30 % kcal/day (<10% saturated fatty acids); • protein 15–20 % kcal/day (about 0.9 g/kg/day); • fibers at least 30 g/day. the alimentary plan consisted of three main meals (breakfast, lunch, dinner) and one to three snacks (variable according to subject’s habits) to avoid prolonged fasting between main meals (>5 h). the low glycemic index was guaranteed by the presence of a balance among macronutrients and fibers. moreover, dieticians strongly encouraged consumption of low glycemic index foods. as per clinical practice, all subjects were encouraged to accumulate at least 30 min or more of moderate-intensity physical activity per day and underwent a follow-up visit every month to monitor weight changes, compliance with physical activity, diet and supplement prescribed for the entire duration of the treatment. pgr supplementation pgr® is a patented complex of macromolecules produced by aboca spa company (sansepolcro, arezzo, italy). this complex contains specific polysaccharide fractions obtained from: cellulose, opuntia ficus indica, glucomannan (amorpho phallus konjac), althaea officinalis, linum usitatissimum, tilia platyphyllos, and cichorium intybus. the pgr group patients consumed three pgr tablets with a large glass of water before their two main meals for a period of at least 12 weeks. statistical analysis variables distribution normality was checked using the shapiro–wilk test. normally distributed data are shown as mean values ± standard deviation (sd), while data with nonnormal distribution are presented as median values and interquartile ranges. since the distributions of most of the quantitative variables were significantly different from the normal distribution (shapiro-wilk test), nonparametric tests were used. the wilcoxon signed rank test was used to compare baseline and follow-up parameters within the study group. the mann whitney u test was used to compare differences between independent groups. differences with p < 0.05 were considered statistically significant. statistical analysis was performed using the statistical software package stata (version 16.1, statacorp, 4905 lakeway drive, college station, tx, usa). table 1. baseline demographic, clinical, and biochemical characteristics of the two study groups. parameters lc-lgi diet (n = 10) lc-lgi diet plus pgr (n = 10) p value age (years) 54.5 ± 26 59.5 ± 11 ns gender (m/f) 2/10 4/6 ns height (cm) 160 ± 14 165 ± 5 ns weight (kg) 90.8 ± 12.5 97.1 ± 20.1 ns bmi (kg/m2) 35.6 ± 8.9 36.7 ± 6.6 ns wc (cm) 115.5 ± 29 111 ± 8.5 ns fm (kg) 37.8 ± 10.4 45.3 ± 11.3 ns ffm (kg) 50.9 ± 22.7 53.7 ± 10.1 ns mm (kg) 48.3 ± 21.6 51 ± 9.9 ns tbw (l) 36 ± 1.5 38.5 ± 7.7 ns fpg (mg/dl) 95.5 ± 13 102.5 ± 10 ns fasting insulin (μu/ml) 18.5 ± 8 14.7 ± 7.8 ns homa-ir 4.5 ± 2.8 3.7.4 ± 1.9 ns quicki 0.31 ± 0.03 0.31 ± 0.02 ns pgr (weeks) – 13 ± 1 – data shown as medians ± iqr. abbreviations: iqr, interquartile range; lc-lgi, low-calorie and low-glycemic index; pgr, policaptil gel retard; ns, not significant; bmi, body mass index; wc, waist circumference; fm, fat mass; ffm, fat free mass; mm, muscle mass; tbw, total body water; fpg, fasting plasma glucose; homa-ir, homeostatic model assessment for insulin resistance; quicki, quantitative insulin-sensitivity check index. italian journal of food science, 2022; 34 (2) 77 policaptil gel retard and obesity lgi group. the difference between the two study groups with respect to quicki was statistically significant (p = 0.029), as shown in table 2. discussion this retrospective pilot study shows that while an lc-lgi diet both with or without pgr intake reduce, as expected, weight, bmi, and waist circumference, as compared to the diet only intervention, 12 weeks of pgr intake induce a significant improvement in insulin circulating levels, in insulin resistance calculated by homa-ir index, as well as in insulin sensitivity calculated according to quicki. these effects of pgr may be related to a reduction in the post meal glycemic and insulinemic peaks as suggested by stagi and collaborators who demonstrated an amelioration of homa-ir in obese children and adolescents after 1 year of pgr intake (stagi et al., 2016, 2017). moreover, greco and colleagues recently observed an improvement table 2. fat mass (fm) and muscle mass (mm) slightly decreased after 12 weeks in the lc-lgi group. however, the difference between the two study groups with respect to fm, fat free mass (ffm), and mm loss was not statistically significant. there was no change in total body water (tbw) in the study subjects (table 2). metabolic profile fpg did not change after 12 weeks of intervention in both study groups. compared to an lc-lgi diet, the lc-lgi diet plus pgr elicited a greater decrease in fasting insulin (−1.5 ± 1 vs −5.8 ± 4.3, p = 0.025, table 2) and homa-ir index (−0.2 ± 0.9 vs −1.5 ± 1, p = 0.043, table 2), with a percent change from baseline of −36 % and –37 %, respectively (figure 1d and 1e). quicki was significantly ameliorated in an lc-lgi plus pgr group (0.31 ± 0.02 vs 0.33 ± 0.02, p < 0.001, table 2) with an increase of 7.1 % (figure1f ) but not in figure 1. median change from baseline (t0) in (a) body weight (bw), (b) body mass index (bmi), and (c) waist circumference (wc) to 12 weeks (t1) in obese subjects treated with an lc-lgi diet or an lc-lgi diet plus pgr. change in (d) fasting insulin, (e) homa-ir, and (f) quicki after 12-week intervention of an lc-lgi diet versus an lc-lgi diet plus pgr. 78 italian journal of food science, 2022; 34 (2) centorame g et al. for all these reasons, further studies with suitable study design on larger samples with a longer follow-up period are needed to confirm our preliminary results. conclusions pgr associated with a low calorie and low glycemic index diet may be useful to reduce body weight and improve insulin sensitivity in adult subjects affected by obesity. references blüher, m., 2019. obesity: global epidemiology and pathogenesis. nature reviews endocrinology 15(5): 288–298. https://doi. org/10.1038/s41574-019-0176-8 ceriello, a., 2003. new insights on oxidative stress and diabetic complications may lead to a “causal” antioxidant therapy. diabetes care 54(1), 1–7. https://doi.org/10.2337/diacare.26.5.1589 dwyer, j.t., melanson, k.j., sriprachy-anunt, u., cross, p. and wilson, m., 2000. dietary treatment of obesity. south dartmouth (ma). endotext. mdtext.com, inc. esparza-ros, f., vaquero-cristóbal, r. and marfell-jones, m., 2019. international standards for anthropometric assessment (2019). p. 115. available at: https://www.researchgate.net/publication/ 236891109_international_standards_for_anthropometric_ assessment of parameters defining metabolic syndrome in a mouse model fed with high-fat diet treated with pgr (greco et al., 2020). similar results were obtained by guarino and colleagues in adults in whom pgr supplementation induced a better effect on serum lipid and tolerability as compared to metformin (guarino et al., 2021). it is worth noting that in our study, similar observation has been made in a clinical setting, without the “trial effect,” thus confirming the potential effectiveness of pgr as a valid clinical tool in obesity management. insulin resistance and hyperglycemia are known risk factors of cardiovascular disease (laakso and kuusisto, 2014). therefore, a treatment that improves insulin action leading to a considerable amelioration of metabolic profile in obese subjects could represent an efficacious strategy in the prevention of cardiovascular disease. the small sample size is the main limitation of our study. furthermore, even if our short observation period suggests an effect of pgr in improving the carbohydrate metabolism, we cannot exclude a concomitant effect given by the weight loss obtained through an lc-lgi diet. a randomized placebo-controlled study would be useful to better single out the effects of this macromolecular complex. table 2. comparison of differences between baseline and 12 weeks in anthropometric, body composition, and metabolic parameters between the lc-lgi diet and lc-lgi plus pgr groups. parameters lc-lgi diet lc-lgi plus pgr lc-lgi vs lc-lgi plus pgr baseline (t0) follow-up (t1) change (∆t0-t1) baseline (t0) follow-up (t1) change (∆t0-t1) p value weight (kg) 90.8 ± 12.5 88.3 ± 9.5 −2.8 ± 1.9* 97.1 ± 20.1 92.8 ± 22.6 −3.8 ± 5.1** 0.393 bmi (kg/m2) 35.6 ± 8.9 33.8 ± 9.5 −1.1 ± 0.7* 36.7 ± 6.6 36.1 ± 8.1 −1.4 ± 1.6** 0.413 wc (cm) 115.5 ± 29 111 ± 28 −3.5 ± 2* 111 ± 8.5 105.5 ± 9 −4.5 ± 3.5** 0.518 fm (kg) 37.8 ± 10.4 36.3 ± 9.4 −1.5 ± 1* 45.3 ± 11.3 45.4 ± 12.5 −1.6 ± 3.8 0.837 ffm (kg) 50.9 ± 22.7 50.7 ± 22.4 −1 ± 0.3 53.7 ± 10.1 49.5 ± 10.8 −1.5 ± 2.8 0.517 mm (kg) 48.3 ± 21.6 47.8 ± 21.5 −0.6 ± 0.1* 51 ± 9.9 49.3 ± 10.6 −1 ± 2.35 0.731 tbw (l) 36 ± 1.5 35.5 ± 2.5 −0.5 ± 0.3 38.5 ± 7.7 37.5 ± 9.3 −0.25 ± 1.9 0.865 fpg (mg/dl) 95.5 ± 13 91 ± 22 2.5 ± 10 102.5 ± 10 95.5 ± 11.5 −5 ± 12.5 0.402 insulinemia (μu/ml) 18.5 ± 8 19 ± 11 −1.5 ± 1 14.7 ± 7.8 9.3 ± 3.3 −5.8 ± 4.3** 0.025 homa-ir 4.5 ± 2.8 4.5 ± 3.6 −0.2 ± 0.9 3.7.4 ± 1.9 2.3 ± 0.8 −1.5 ± 1** 0.043 quicki 0.31 ± 0.03 0.31 ± 0.04 0.00 ± 0.01 0.31 ± 0.02 0.33 ± 0.02 0.02 ± 0.01** 0.029 data are shown as medians ± iqr. wilcoxon signed rank test was used for intragroup comparisons (baseline vs follow-up). for intergroup analyses (lc-lgi diet vs lc-lgi plus pgr) the mann whitney u test for independent groups was applied. *p < 0.05; **p < 0.001. abbreviations: iqr, interquartile range; lc-lgi, low-calorie and low-glycemic index; pgr, policaptil gel retard; bmi, body mass index; wc, waist circumference; fm, fat mass; ffm, fat free mass; mm, muscle mass; tbw, total body water; fpg, fasting plasma glucose; homa-ir, homeostatic model assessment for insulin resistance; quicki, quantitative insulin-sensitivity check index. italian journal of food science, 2022; 34 (2) 79 policaptil gel retard and obesity laakso, m. and kuusisto, j., 2014. insulin resistance and hyperglycaemia in cardiovascular disease development. nature reviews endocrinology 10(5): 293–302. https://doi.org/10.1038/ nrendo.2014.29 matthews, d.r., hosker, rudenski, naylor, treacher, and turner, 1985. homeostasis model assessment: insulin resistance and β-cell function from fasting plasma glucose and insulin concentrations in man. diabetologia 28(7): 412–419. https://doi. org/10.1007/bf00280883sinu, 2019. livelli di assunzione di riferimento di nutrienti ed energia per la popolazione italiana (larn). available at: https://sinu.it/larn/ stagi, s., lapi, e., seminara, s., pelosi, p., del greco, p., capirchio, l., strano, m., giglio, s., chiarelli, f. and de martino, m., 2016. policaptil gel retard® significantly lowered body mass index and hyperinsulinemia, and reduced the risk of diabetes mellitus type 2 (dm2) in children and adolescents with obesity, family history and dm2. pediatriya 56(2): 38–45. https://doi.org/ 10.1186/s13052-015-0109-7 stagi, s., ricci, f., bianconi, m., sammarco, m.a., municchi, g., toni, s., lenzi, l., verrotti, a. and de martino, m., 2017. retrospective evaluation of metformin and/or metformin plus a new polysaccharide complex in treating severe hyperinsulinism and insulin resistance in obese children and adolescents with metabolic syndrome. nutrients 9(5): 1–17. https://doi.org/10.3390/nu9050524 weickert, m.o. and pfeiffer, a.f.h., 2008. metabolic effects of dietary fiber consumption and prevention of diabetes. journal of nutrition 138(3) 439–442. https://doi.org/10.1093/jn/138.3.439 who, 2015. obesity: preventing and managing the global epidemic. who. finer, n., 2015. medical consequences of obesity. medicine (united kingdom) 43(2): 88–93. https://doi.org/10.1016/j.mpmed.2014. 11.003 fornari, e., morandi, a., piona, c., tommasi, m., corradi, m. and maffeis, c., (2020). policaptil gel retard intake reduces postprandial triglycerides, ghrelin and appetite in obese children: a clinical trial. nutrients 12(1): 214. https://doi.org/10.3390/ nu12010214 frühbeck, g., toplak, h., woodward, e., yumuk, v., maislos, m. and oppert, j.m., 2013. obesity: the gateway to ill health – an easo position statement on a rising public health, clinical and scientific challenge in europe. obesity facts 6(2): 117–120. https://doi.org/10.1159/000350627 greco, c.m., garetto, s., montellier, e., liu, y., chen, s., baldi, p., sassone-corsi, p. and lucci, j., 2020. a non-pharmacological therapeutic approach in the gut triggers distal metabolic rewiring capable of ameliorating diet-induced dysfunctions encompassed by metabolic syndrome. scientific reports 10(1): 12915. https://doi.org/10.1038/s41598-020-69469-y guarino, g., della corte, t., strollo, f. and gentile, s., 2021. policaptil gel retard in adult subjects with the metabolic syndrome: efficacy, safety, and tolerability compared to metformin. diabetes & metabolic syndrome 15(3): 901–907. https://doi. org/10.1016/j.dsx.2021.03.032 katz, a., nambi, s.s., mather, k., baron, a.d., follmann, d.a., sullivan, g. and quon, m.j., 2000. quantitative insulin sensitivity check index: a simple, accurate method for assessing insulin sensitivity in humans. the journal of clinical endo crinology & metabolism 85(7): 2402–2410. https://doi.org/ 10.1210/jcem.85.7.6661 140 issn 1120-1770 online, doi 10.15586/ijfs.v34i1.2171 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (1): 140–148 p u b l i c a t i o n s codon is coffee powder extract a possible functional ingredient useful in food and nutraceutical industries? ancuta nartea1, paolo lucci2, monica rosa loizzo3*, rosa tundis3, mariarosaria leporini3, luigia gervasi3, benedetta fanesi1, oscar núñez4, natale giuseppe frega1, dennis fiorini5, deborah pacetti1 1dipartimento di scienze agrarie, alimentari e ambientali, università politecnica delle marche, ancona, italy; 2department of agri-food, animal and environmental sciences, university of udine, udine, italy; 3department of pharmacy, health and nutritional sciences, university of calabria, rende, italy; 4department of chemical engineering and analytical chemistry, university of barcelona, barcelona, spain; 5chemistry division, school of science and technology, university of camerino, camerino, italy *corresponding author: monica rosa loizzo, department of pharmacy, health and nutritional sciences, university of calabria, 87036 rende (cs), italy. email: monica_rosa.loizzo@unical.it received: 12 january 2022; accepted: 21 february 2022; published: 8 march 2022 © 2022 codon publications open access paper abstract the present study aimed to assess the phytochemical content and in vitro bioactivity of ethanolic extracts of arabica (a) and/or robusta (r) coffee powder having different geographical origins. for this purpose, total phenols (tpc) and flavonoids (tfc) content as well as aand b-tocopherol were quantified. the antioxidant activity was assessed by using a multi-target approach in which the radical scavenging potential, the protection from lipid peroxidation, and the involvement of the iron-reducing mechanism were applied. the carbohydrate hydrolyzing enzymes’ (a-amylase and a-glucosidase) inhibitory activities were also assessed. arabica coffee sample (c2-a) showed the highest tpc, tfc, and a-tocopherol content with values of 63.1 mg chlorogenic acid equivalents (cae)/g dry powder, 16.2 mg of quercetin (qe) equivalents/g dry powder, and 5.6 mg/100 g dry powder, respectively. relative antioxidant capacity index (raci), used to statistically integrate results from 2,2′azino-bis(3 ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts), 2,2-diphenyl-1-picrylhydrazyl (dpph), ferric reducing ability power (frap), and protection of lipid peroxidation assays, evidenced that sample c4-r derived from robusta from guatemala showed the highest antioxidant potential with a value of –0.61. arabica from puerto rico was the most active against a-amylase, whereas the blend arabica/robusta sample (c5-a60r40) showed the highest inhibitory activity against a-glucosidase with ic50 values of 120.2 and 134.6 mg/ml, respectively. the  results show how the qualitative-quantitative composition of the extracts is strongly associated not only with the variety but also with the geographical origin of the samples. keywords: antioxidant activity; coffee; ethanolic extract; hypoglycemic effect; phenols; tocopherols introduction coffee is consumed worldwide with a total production of 16,868 million of 60-kg bags in 2019/2020. the two commercially produced coffee species are coffea arabica linn. (known as arabica) and coffea canephora pierre ex froehner (known as arabica). more than 70 countries produce coffee, but most of the global output comes from the top five producers: brazil, vietnam, colombia, indonesia, and ethiopia. arabica coffee is mainly cultivated in colombia, whereas robusta is mainly cultivated in vietnam and ethiopia. brazil cultivates mailto:monica_rosa.loizzo@unical.it italian journal of food science, 2022; 34 (1) 141 is coffee powder extract useful in food and nutraceutical industries? ingredients in formulations of nutraceutical/functional foods rich in antioxidant compounds. anyway, the standardization of raw material for their preparation is quite a challenge. in fact, it is known that the overall composition of bioactive substances of roasted coffee, as well as their functional activity, is heavily influenced by agronomical and technological factors (kròl et al., 2020). the genetic background (species and variety), geographical origin, and roasting conditions of the coffee beans play a pivot role in defining the final bioactivity of the derived coffee products. within this frame, the present study aimed to assess the phytochemical content, in terms of total phenols, total flavonoids, aand b-tochopherol, and in vitro antioxidant activity evaluated by abts, dpph, frap, and b-carotene bleaching test of ethanol extracts obtained from roasted coffea arabica and c. robusta beans. these samples are characterized by different geographical origins (brazil, colombia, vietnam, india, ethiopia, guatemala, puerto rico, and costa rica) and commercial coffee blend (arabica/robusta) powders. furthermore, the hypoglycemic effect of the ethanol extracts was assessed by testing the inhibition of carbohydrate-hydrolyzing enzymes against a-amylase and a-glucosidase. materials and methods reagents and standard all chemicals and reagents used in this study were purchased from sigma-aldrich chemical co. ltd (milan, italy) and vwr international (milan, italy) and, unless specified otherwise, were of analytical grade or higher. coffee samples and preparation of ethanolic extract the roasted coffee powder from coffea arabica, c. canephora var. robusta, and roasted coffee blends were supplied by a local industrial coffee roaster (caffè del faro, robin s.r.l., montegranaro, italy) able to confirm their botanical and geographical origin, as well as the general type of postharvest processing (dry/wet process). both arabica and robusta coffees had different geographic origins. all samples were roasted under the same conditions (175°c, 15 min). the compositional features of coffee samples investigated are reported in table 1. seven grams of coffee powder were added to 30 ml of anhydrous ethanol and the mixture was magnetically stirred in the dark, at 25°c for 12 h. then, the top phase was filtered and dried in a rotary evaporator. for each coffee sample, the ethanol extraction procedure was repeated in triplicate. copious amounts of both species (international coffee organization, 2019). roasted coffee contains many bioactive compounds that have beneficial effects on human health. most investigations have so far focused on the potential therapeutic effect of caffeine (farah and de paula lima, 2019). cafestol and kahweol, present in the coffee unsaponifiable matter, have positive effects against cancer and diabetes. other secondary metabolites of coffee beans, such as tocopherols and polar phenolic compounds, have well-known antioxidant abilities. among them, chlorogenic, ferulic, cinnamic, and caffeic acids are the most frequently investigated (dórea and da costa, 2005). furthermore, the presence of tyrosol in spent espresso coffee grounds in rather high concentration, from 0.83 to 11.85 mg/100 g of dry spent powder, was recently reported (balzano et al., 2020). according to different studies, tyrosol demonstrates a wide spectrum of biological effects on physiological processes. based on the most recent epidemiological and research data, consumption of coffee is associated with a lower risk of developing type ii diabetes in healthy individuals, probably requiring a series of mechanisms of action involving the antioxidant activity and interventions on glucose homeostasis (osama et al., 2021). diabetes mellitus (dm) will reach pandemic proportions in the next 20 years. the international diabetes federation estimated that in 2025, 371 million people will be affected by dm (idf, 2019). chronic hyperglycemia, insulin resistance in target tissues, and a relative deficiency of insulin secretion from pancreatic b-cells are the major features of type 2 dm (t2dm). the strict linkage between ros and metabolic disorder was recently demonstrated (carrier, 2017). nutritional stress, caused by excess of carbohydrate and/or high-fat diets, promotes oxidative stress and results in decreased antioxidant status. oxidative stress, in fact, may contribute to the long-term deterioration of pancreatic islet b-cell by affecting mitochondrial atp production, which is necessary for insulin secretion. the consequent mitochondrial dysfunction influenced insulin sensitivity within muscle, liver, and adipose tissue. to reduce sugar intestinal absorption, treatment of t2dm patients with carbohydratehydrolyzing enzymes (a-amylase and a-glucosidase) inhibitors has been reported (loizzo et  al., 2017). regarding chronic diseases, including hyperglycemia, the market of nutraceutical ingredients, as phytochemical and plant extracts, is projected to reach a compound annual growth rate of 7.5%. due to the rising global demand for nutraceuticals made up of natural ingredients (global nutraceutical statistics, 2019), the formulation of new natural extracts and the study of their bioactivities and their content of bioactive molecules become a fundamental task. from this point of view and considering the safety and acceptability for the human use of ethanol compared to other solvents, the coffee powder ethanol extracts could represent suitable 142 italian journal of food science, 2022; 34 (1) nartea a et al. in vitro antioxidant assays abts and dpph radicals scavenging tests were performed following the procedures reported by loizzo et al. (2019), to investigate the radical scavenging potential of coffee powder samples. for the abts test, a solution of abts radical cation was prepared by mixing 7 mm abts solution with 2.45 mm potassium persulfate and stored at room temperature. after 12 h, the solution was diluted with ethanol to an absorbance of 0.70 at 734 nm using a uv-vis jenway 6003 spectrophotometer. dilution of extracts in ethanol was added to 2 ml of diluted abts+ solution in order to test the following concentrations from 400 to 1 μg/ml. after 6 min, the absorbance was read at 734 nm by using an uv-vis jenway 6003 spectrophotometer (carlo erba, milan, italy). in the dpph test, an aliquot of 1.5 ml of 0.25 mm dpph radical (dpph·) in ethanol was mixed with 12 μl of coffee extract to test concentrations ranging from 1000 to 1 μg/ml. the mixture was shaken and allowed to reach a steady state at 25°c for 30 min. after that, the absorbance was read at 517 nm by using the uv-vis jenway 6003 spectrophotometer (carlo erba, milan, italy). ascorbic acid was used as positive control in both tests, and ic50 values are reported in table 4. protective effect on lipid peroxidation was assessed by using the previously described β-carotene bleaching test (loizzo et al., 2019). one milliliter of β-carotene (0.2 mg/ ml in chloroform) was mixed with linoleic acid (20 μl) and 100% tween 20 (200 μl). after evaporation of the solvent and dilution with water, the emulsion (288 μl) was added to a 96-well microplate containing 12 μl of coffee extract in ethanol (concentrations ranging from 100 to 2.5 μg/ml). the plate was shaken and placed in a water bath at 45°c for 30 and 60 min of incubation. the absorbance was measured at 470 nm by using uv-vis jenway 6003 spectrophotometer (carlo erba, milan, italy). propyl gallate was used as positive control, and ic50 is reported in table 4. the frap test was performed following the procedure previously described by loizzo et al. (2019). the frap value represents the ratio between the slope of the linear plot for reducing fe3+-tptz reagent by different coffee powder ethanol extracts, compared to the slope of the plot obtained for feso4. butylated hydroxytoluene (bht) was used as positive control. for the preparation of the frap reagent, a mixture of 2.5 ml of 10 mm tripyridyltriazine (tptz) solution, 40 mm hcl, 2.5 ml of 20 mm fecl3, and 25 ml of 0.3 m acetate buffer (ph 3.6) was prepared. sample at a concentration of 2.5 mg/ml in ethanol (100 μl) was mixed with 2.0 ml of frap reagent bioactive phytochemicals content in coffee powder ethanol extracts total phenol (tpc) and flavonoid (tfc) contents were quantified using the spectrophotometric methods already published elsewhere (loizzo et al., 2019). coffee extract at a concentration of 1.5 mg/ml (0.1 ml) was mixed with a solution of folin–ciocalteu reagent (0.5 ml) and water (1 ml). after 1 min of incubation, 1.5 ml of 20% sodium carbonate was added, and the mixture was incubated at room temperature. the absorbance was measured at 765 nm using a uv-vis jenway 6003 spectrophotometer (carlo erba, milan, italy). the total phenol content was expressed as milligrams of chlorogenic acid equivalents (cae)/g dry powder. in the total flavonoid content (tfc) determination, coffee extract was mixed with aluminum chloride solution (2%) in a 1:1 ratio and incubated at room temperature for 15 min. the absorbance was measured using a uv-vis jenway 6003 spectrophotometer (carlo erba, milan, italy) at 510 nm. the tfc was expressed as milligrams of quercetin equivalents (qe)/g dry powder. tocopherols’ profile was determined following the procedure reported by giardinieri et al. (2019). coffee ethanolic extract was dissolved in acetonitrile and analyzed by means of ultra-high performance liquid chromatography fluorescence (uhplc-fld) using ascentis® express c18 (75 × 4.6 mm, 2.7 μm, from supelco, milan, italy) as the analytical column. the mobile phase was acetonitrile/ methanol (90:10, v/v), at a flow rate of 0.45 ml/min. the injection volume was 1 µl. fld was set with an excitation wavelength of 290 nm and an emission wavelength of 330 nm. calibration curves (25–250 µg/ml) were prepared for quantitative analysis with r2 higher than 0.996 for both tocopherols. table 1. composition and geographical origin of coffee blend powders used for preparing ethanol extracts. sample code coffee blend composition c1-a arabica 100% (puerto rico) c2-a arabica 100% (50% brazil, 20% colombia, 20% guatemala, 10% ethiopia) c3-a arabica 100% (colombia) c4-r robusta 100% (guatemala) c5-a 60 r 40 60% arabica (20% brazil, 20% colombia, 10% guatemala, 10% costa rica)/40% robusta (15% vietnam, 25% india) c6-a 10 r 90 10% arabica (brazil)/90% robusta (20% india, 70% vietnam) c7-a 75 r 25 decaffeinated coffee blend arabica 75% (unknown)/robusta 25% (unknown) italian journal of food science, 2022; 34 (1) 143 is coffee powder extract useful in food and nutraceutical industries? windows (graph pad software, san diego, ca, usa). differences within and between groups were evaluated by one-way analysis of variance test (anova) followed by a multicomparison dunnett’s test compared with the positive control at a significance level of p < 0.001. pearson’s correlation coefficient (r) and linear regression, assessment of repeatability, calculation of average and relative standard deviation were performed using microsoft excel 2010 software. the relative antioxidant capacity index (raci) was used as a statistical approach to compare the antioxidant activity of coffee powder ethanol extracts obtained by different applied tests (loizzo et al., 2019). results and discussion ethanol extraction yield ethanol was chosen as the extraction solvent because it complies with the legislation on extraction solvents to be used in the production of foodstuffs and food ingredients (directive 2009/32/ec, 2009), and also considering the effectiveness toward the extraction of bioactive substances, the toxicity, and environmental impact (socaci et al., 2018). the investigated coffee powders provided an ethanol extract yield ranging from 43.5 to 111.0 g/kg dry powder. generally, arabica coffee powders gave higher extraction yields than robusta samples, with the blends containing higher percentage of robusta (40 and 60%) giving lower yields. results are similar to those obtained from spent ground coffee, reported by balzano et al. (2020), where samples of spent ground coffee extracted in the same experimental conditions gave a range of 58 to 112 g/kg of dry powder. bioactive phytochemicals in coffee ethanolic powder table 2 shows the average values of tpc and tfc in coffee powder ethanolic extracts. tpc content ranged from 11.0 to 63.1 mg cae/g dry powder, whereas tfc varied from 4.5 to 16.2 mg qe/g dry powder. generally, samples obtained from the arabica variety had higher tpc and tfc than robusta or arabica/robusta mixtures. these results are in agreement with those previously reported by other researchers. in particular, bobková et al. (2020) investigated the effect of roasting process on the tpc of arabica and robusta coffee beans from different geographical origins. tpc values ranged from 49.19 to 74.05 mg cae/g dry powder. water coffee extracts showed the highest levels of tpc in green and light roasted coffees where values ranged from 49.19 to 74.05 g gae/kg for vietnam queen and ethiopia sidamo, respectively, and from 38.34 to 59.79 g gae/kg for india monsooned malabar and ethiopia sidamo, respectively. interestingly, and 900 ml of water; the absorbance was measured at 595 nm by using the uv-vis jenway 6003 spectrophotometer (carlo erba, milan, italy) after 30 min of incubation at room temperature. carbohydrate hydrolysis enzymes inhibition the hypoglycemic potential of coffee powder extracts was assessed by using the a-amylase and a-glucosidase inhibitory tests (loizzo et al., 2019). the enzyme solution was prepared by adding 0.0253 g of enzyme in 100 ml of cold water, and the starch solution was prepared by stirring (at 65°c for 15 min) 0.125 g of potato starch in 25 ml of sodium phosphate buffer (20 mm) and sodium chloride (6.7 mm). samples were dissolved in ethanol at concentrations ranging from 1000 to 25 μg/ml, added to starch solution, and left to react with the enzyme at 25°c for 5 min. the absorbance was read at 540 nm by using the uv-vis jenway 6003 spectrophotometer (carlo erba, milan, italy). in the a-glucosidase inhibitory test, a maltose solution was prepared by dissolving 12 g of maltose in 300  ml of 50 mm sodium acetate buffer; α-glucosidase (ec 3.2.1.20) solution was prepared by adding 1 mg of enzyme (10 units/mg) in 10 ml of ice-cold distilled water; and o-dianisidine (dian) solution was prepared by dissolving 1 tablet in 25 ml of distilled water [15]. the peroxidase/glucose oxidase (pgo) system-color reagent solution was obtained by dissolving one capsule in 100 ml of ice-cold distilled water. a mixture of 5 μl of the sample (at concentrations ranging from 1000 to 25 μg/ ml), 250 μl maltose solution, and 5 μl enzyme was left to incubate at 37°c for 30 min. then, 50 μl of perchloric acid was added, and the mixture was centrifuged. the supernatant was collected and mixed with 5 μl of dian and 300 μl of pgo and left to incubate at 37°c for 30 min. the absorbance was read at 500 nm by using the uv-vis jenway 6003 spectrophotometer (carlo erba, milan, italy). acarbose was the positive control in both tests, and ic50 is reported in table 5. in the α-glucosidase inhibitory activity test, acarbose was used as a positive control in both tests. statistical analysis data were expressed as means ± standard deviation (s.d.) (n = 3). differences of polar phenolic substances and tocopherol content among samples were calculated using one-way analysis of variance (anova) with tukey’s post hoc procedure (p < 0.05). the inhibitory concentration of 50% (ic50) was calculated by nonlinear regression with the use of prism graph pad prism version 4.0 for 144 italian journal of food science, 2022; 34 (1) nartea a et al. decaffeinated sample c7-a75r25 and in the sample c1-a from puerto rico, respectively). the values referred to the ethanol extract range between 36 and 362 mg/100 g (table  3). β-tocopherol is predominant, and the ratio β-tocopherol/α-tocopherol varies from 1.6 to 4.0. the overall results are in agreement with several studies (alves et al., 2009; gonzález et al., 2001; górnaś et al., 2014). górnaś et al. (2014) found an average total tocopherol content in roasted robusta of 11.54  mg/100  g, and 28.8 mg/100 g in roasted arabica, and an average ratio b-/a-tocopherol of 1.2 in roasted robusta and 3.1 in roasted arabica. alves et al. (2009) reported an average total value of aand β-tocopherol of 9.7 mg/100 g in roasted arabica and 3.1 mg/100 g in roasted robusta, with a ratio β-/a-tocopherol of 3.0 and 1.0 for arabica and robusta, respectively. gonzález et al. (2001), found a total value of aand b-tocopherol ranging from 1.9 to 4.6 mg/100 g in roasted robusta and from 11.5 to 19.3 mg/100 g in roasted arabica, with a ratio β-/atocopherol ranging from 2.1 to 6.1 and from 3.5 and 6.0 in roasted arabica and robusta, respectively. the variability found in the investigated samples did not allow to highlight any statistical relationships between the tocopherol content and the arabica/robusta composition of the powder. a similar situation was observed also by górnaś et al. (2014) and alves et al. (2009). coffee powder ethanolic extract bioactivity antioxidant effects oxidative stress in humans arises from an imbalance between radical oxygen species (ros) and endogenous defense enzymes such as superoxide dismutase, catalase, glutathione peroxidase, etc. besides these defenses, consumption of dietary antioxidants is fundamental to prevent the development of several diseases. by using different in vitro tests, we have checked the ability of the roasting process led to a reduction in tpc values from the green to dark roasting stage. this evidence confirmed that the coffee-growing region has probably an important influence on the development of this class of phytochemicals in coffee. the impact of the roasting process was the object of investigation by sulaiman et al. (2011) that evidenced how tpc decreased linearly over the roasting temperature from 63.51 mg cae/g coffee beans (roasted at 200°c) to 42.56 mg cae/g coffee beans (roasted at 240°c). similarly, kròl et al. (2020) demonstrated that coffees roasted in light and medium roasting conditions are richer in tpc in comparison to dark roast coffee. furthermore, organic coffee beans showed higher tpc and tfc content than conventional coffee beans (8.95 vs 8.28 mg/g and 1.35 vs 0.94 mg/g, respectively). the same observation was done by acidri et al. (2020) that found a decline in tpc from 146.8 to 87 mg gae/g dw in indonesian arabica coffee after the roasting process. the great variability of tpc found in literature confirmed that the content of these phytochemicals may be related to the varieties, the cultivation method, as well as to the coffee origin. tocopherol content tocopherols are very important molecules effectively inhibiting lipid oxidation in foods and the biological system. in coffee beans, the tocopherol content is approximately 3–10 mg/100 g (górnaś et al., 2014). to investigate the relation between the overall antioxidant activity of the coffee extracts and their main chemical contributors, tocopherol composition and content were determined by means of rp-uplc-fld. as a result, aand βwere the tocopherols largely predominant in the ethanol coffee extract samples investigated, with a certain variability of the total content, ranging, in the dry powder form, between 3 and 27 mg/100 g (in the table 2. total phenols content (tpc) and flavonoids content (tfc) in coffee powder ethanol extracts. sample tpc° tfc^ c1-a 51.5 ± 0.9b 15.6 ± 0.5b c2-a 63.1 ± 1.1a 16.2 ± 0.7a c3-a 25.9 ± 0.4e 13.3 ± 0.4c c4-r 11.0 ± 0.3g 4.5 ± 0.0g c5-a 60 r 40 35.5 ± 0.5d 11.0 ± 0.2d c6-a 10 r 90 19.9 ± 0.4f 10.0 ± 0.2e c7-a 75 r 25 38.6 ± 0.6c 9.5 ± 0.1f data are expressed as mean ± standard deviation (n = 3). °mg chlorogenic acid equivalents (cae)/g dry powder; ^mg of quercetin (qe) equivalents/g dry powder; and values in the same column with different superscript letters are significantly different (p < 0.05). table 3. content of αand β-tocopherols in coffee ethanol extracts. sample α – tocopherol§ β – tocopherol§ c1-a 5.4 ± 0.5b 21.3 ± 0.2a c2-a 5.6 ± 0.5a 10.6 ± 0.2c c3-a 4.1 ± 0.0c 13.9 ± 0.5b c4-r 4.1 ± 1.1c 10.5 ± 0.5c c5-a 60 r 40 2.3 ± 0.0d 4.9 ± 0.1b c6-a 10 r 90 5.4 ± 1.1b 10.8 ± 0.4c c7-a 75 r 25 1.0 ± 0.0e 1.6 ± 0.0d data are expressed as mean ± standard deviation (n = 3). §mg/100 g dry powder. values in the same column with different superscript letters are significantly different (p < 0.05). italian journal of food science, 2022; 34 (1) 145 is coffee powder extract useful in food and nutraceutical industries? promising activity was also observed with decaffeinated coffee powder extract (c7-a75r25), followed by the blend c5-a60r40. raci was calculated to evaluate and create a ranking clustering of the antioxidant capacity of different samples. based on this parameter, the following antioxidant ranking could be evidenced: c4-r>c5-a60r40>c7a75r25>c6a10r90>c2-a>c1-a>c3-a (figure 1). a positive pearson’s correlation coefficient was found between tfc and dpph (r = 0.70) and b-tocopherol and both dpph and abts tests (r = 0.87 and 0.76, respectively). a significant positive correlation coefficient was also observed for tfc and b-carotene bleaching test after 30 and 60 min of incubation (r = 0.61 and 0.79, respectively). based on raci statistical approach c4-r, richest in phenols and b-tocopherol resulted the most active as antioxidant. tocopherols are able not only to react toward free radicals and hydroperoxides but also with many other possible side reactions which are affected by tocopherol concentrations, type of substrate, and by other chemical species acting as pro-oxidants and synergists in the system. carbohydrate hydrolysis enzymes’ inhibitory effect by coffee powder extracts the inhibition of carbohydrate hydrolyzing enzymes, a-amylase and a-glucosidase, given by coffee powder extract was concentration-dependent (table 5). generally, a-amylase enzyme was the most sensitive to the action of the extracts (see selectivity index, si). coffee powder ethanolic extract to act as an antiradical or antioxidant agent. the approach with multiple tests is recommended for measuring antioxidant properties of food matrix to better reflect their potential protective effects. the antiradical activity characterizes the ability of phytocomplex to react with free radicals, while the antioxidant activity represents the ability to inhibit the oxidation process, which usually occurs through different reactions (tirzitis and bartosz, 2010). generally, a concentration-effect relationship was found in all the tests except in the frap assay (table 4). the extract obtained from the robusta sample (c4-r) showed the highest radical scavenging potential with ic50 values of 1.1 and 9.2 mg/ml for abts and dpph assay, respectively. a promising radical scavenging activity was also observed in the decaffeinated sample c7-a75r25 (ic50 values of 8.6 and 16.4 mg/ml for abts and dpph assay, respectively). both samples are also able to react as reductants in the frap assay (frap values of 56.8 and 56.9 mm fe (ii)/g at 2.5 mg/ml). these values are quite lower than that reported for the positive control bht (63.2 mm fe (ii)/g at 2.5 mg/ml). the antioxidant activity of coffee samples was also tested, using the b-carotene bleaching method. this assay evaluated the ability of the phytocomplex to protect against lipid peroxidation. since no high temperatures are required, the antioxidant activity of thermosensitive phytochemicals may be determined and quantitatively evaluated. the sample c4-r showed a promising protection against lipid peroxidation with ic50 values of 5.3 and 5.9 mg/ml at 30 and 60 min of incubation, respectively. a table 4. in vitro antioxidant activity of coffee powder ethanol extracts from arabica and robusta varieties and their blend. sample dpph test ic 50 (μg/ml) abts test ic 50 (μg/ml) frap# test μm fe (ii)/g β-carotene bleaching test ic 50 (μg/ml) 30 min 60 min c1-a 809.5 ± 2.9**** 411.3 ± 3.8**** 16.9 ± 1.3**** 34.6 ± 0.7**** 38.9 ± 1.4**** c2-a 212.6 ± 2.4**** 101.2 ± 3.7**** 6.8 ± 0.7**** 20.5 ± 0.8**** 94.1 ± 1.9**** c3-a 460.4 ± 3.5**** 445.5 ± 3.1**** 13.5 ± 0.7**** 52.1 ± 0.7**** 59.6 ± 1.7**** c4-r 9.2 ± 0.8** 1.1 ± 0.4ns 56.8 ± 2.7** 5.3 ± 0.6* 5.9 ± 0.9* c5-a 60 r 40 138.5 ± 2.6**** 83.6 ± 1.2**** 2.4 ± 0.6**** 11.0 ± 0.2*** 18.3 ± 1.2**** c6-a 10 r 90 178.9 ± 3.5**** 52.8 ± 1.3**** 15.0 ± 0.6**** 50.9 ± 1.6**** 43.9 ± 1.6**** c7-a 75 r 25 16.4 ± 1.1*** 8.6 ± 0.9*** 57.9 ± 1.8** 10.9 ± 1.4*** 16.8 ± 1.7**** positive controls ascorbic acid 5.0 ± 0.8 1.7 ± 0.06 – – – bht – – 63.2 ± 4.3 – – propyl gallate – – – 1.0 ± 0.04 0.09 ± 0.004 data are given as media ± s.d. (n = 3); #at 2.5 mg/ml; dpph radical scavenging activity assay; antioxidant capacity determined by radical cation (abts+). b-carotene bleaching test. ferric ion reducing antioxidant power (frap); ascorbic acid. bht and propyl gallate were used as positive controls in antioxidant tests. differences within and between groups were evaluated by one-way anova followed by a multicomparison dunnett’s test (a = 0.01): ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.1 compared to the positive controls. 146 italian journal of food science, 2022; 34 (1) nartea a et al. 0.8 0.6 0.4 0.2 r a c i v al ue 0 c1 -a c2 -a c3 -a c4 -r c5 -a 60 r4 0 c6 -a 10 r9 0 c7 -a 10 r9 0 –0.2 –0.4 –0.6 –0.8 figure 1. relative antioxidant capacity index (raci) of coffee powder ethanol extracts from arabica and robusta varieties. table 5. carbohydrates hydrolyzing enzymes inhibition by coffee powder ethanol extracts from arabica and robusta varieties and their blend. sample α-amylase ic 50 (μg/ml) α-glucosidase ic 50 (μg/ml) si α-amylase/ α-glucosidase c1-a 120.2 ± 3.1**** 412.5 ± 6.1**** 0.3 c2-a 880.3 ± 7.4**** 362.2 ± 6.3**** 2.4 c3-a 132.6 ± 5.2**** 445.3 ± 8.1**** 0.3 c4-r 122.4 ± 5.0**** 320.4 ± 7.2**** 0.4 c5-a 60 r 40 800.3 ± 8.3**** 134.6 ± 2.8**** 5.9 c6-a 10 r 90 970.5 ± 8.9**** 244.7 ± 3.5**** 4.0 c7-a 75 r 25 130.9 ± 2.8**** 472.4 ± 8.6**** 0.3 positive control acarbose 52.4 ± 3.9 35.3 ± 3.7 1.5 data are expressed as mean ± s.d. (n = 3). one-way anova ****p < 0.0001 followed by a multicomparison dunnett’s test: ****p < 0.0001 compared with acarbose. c1-a extract showed the best activity with ic50 value of 120.2  mg/ml followed by guatemalan robusta extract (c4-r) and decaffeinated sample (c7-a75r25) with ic50 values of 122.4 and 130.9 mg/ml, respectively. except sample c5-a60r40 that showed ic50value of 134.6 mg/ml against a-glucosidase, all other samples are less active (ic50 values in the range 320.4–472.4 mg/ml). sample c1-a showed the highest tpc content. among phytochemicals able to interfere with carbohydrate hydrolyzing enzymes, phenols represent the main studied compounds (loizzo et al., 2017). several studies pointed out that cgas, that are reported as the most abundant phytochemicals in coffee extract (jeon et al., 2019; jeszkaskowron et al., 2016), inhibited both a-amylase and a-glucosidase with ic50 values of 25 and 26.07 m, respectively (oboh et al., 2015). cgas also stimulate glucose uptake in skeletal muscle and suppression of hepatic glucose production by ampk activation (ong et al., 2013). in addition, it has been found that cgas could modulate glucose in both genetically and healthy metabolic related disorders including dm (naveed et al., 2018). differently nyambe-silavwe and williamson (2018) demonstrated that both cgas are only weak inhibitors of human salivary α-amylase despite several publications claiming otherwise. in fact, more recently, herawati et al. (2019) demonstrated that robusta coffee beans extract obtained after roasting, grinding, and brewing process was able to inhibit α-glucosidase activity up to 69% and exerted anti-glycation activity. conclusion the present work investigated the bioactive phytochemicals content, in vitro antioxidant activity, and hypoglycemic potential of ethanol extracts deriving from arabica and robusta coffee powders, as well as their mixture, from different geographical origins. a great variability in terms of total phenol, flavonoid, and tocopherol content was observed, and this evidence strongly influenced the bioactivity although it is not possible to identify any relationship with the coffee variety and blend composition. our findings strongly emphasize that coffee ethanol extracts should be used as a value-added ingredient for formulations of nutraceutical or functional products useful for the prevention of disease associated with oxidative stress and hyperglycemic condition. declarations 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different levels. international food research journal 26: 1305–1313. international coffee organization, 2019. growing for prosperity. economic viability as the catalyst for a sustainable coffee sector, coffee development report 2019. available at: https://www. internationalcoffeecouncil.org/media/coffeedevelopment report.pdf coffee market report 2021. accessed 11 may 2021. conflicts of interest the authors have no conflicts of interest to disclose with the present submission. availability of data and material the data sets supporting the results of this article are available from the corresponding author (m.r.l.) upon reasonable request. code availability not applicable. author contributions d.p. conceptualized and supervised the study; m.r.l. prepared the original draft; a.n., m.l., l.g., and b.f. did the formal analysis; r.t., p.l., and d.f. performed data curation; o.n., p.l., and d.f. edited the manuscript; n.g.f and d.p. were concerned with funding acquisition. all authors have 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https://doi.org/10.18388/abp.2010_2386� https://doi.org/10.18388/abp.2010_2386� https://doi.org/10.1016/j.jare.2019.01.002� https://doi.org/10.1007/s00217-016-2643-y� https://doi.org/10.1007/s00217-019-03388-9� https://doi.org/10.1002/9783527805921.ch6� https://doi.org/10.3390/antiox8010023� https://doi.org/10.1016/j.biopha.2017.10.064� https://doi.org/10.1016/j.biopha.2017.10.064� p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33i2.1921 35 p u b l i c a t i o n s codon quality and safety evaluation of new tomato cultivars mattia rapa*, salvatore ciano, laura gobbi, roberto ruggieri and giulina vinci department of management, sapienza university of rome, via del castro laurenziano 9, 00161, rome, italy *corresponding author: mattia rapa, department of management, sapienza university of rome, via del castro laurenziano 9, 00161, rome, italy. email: mattia.rapa@uniroma1.it received: 17 may 2020; accepted: 12 march 2021; published: 7 april 2021 © 2021 codon publications open access paper abstract this paper is aimed to provide a quality and safety assessment of new cherry tomato cultivars (solanum lycopersicum var. cerasiforme): bamano, dulcemiel, and sugarland. eight biogenic amines, total phenolics, total carotenoids, lycopene, and antioxidant activity (2,2-diphenyl-1-picrylhydrazyl [dpph] and 2,2-azino-bis (3ethylbenzo thiazoline-6-sulfonic acid) diammonium salt [abts] assays) were determined. comparison with control cultivars demonstrated lower ph values, and total contents of biogenic amines and antioxidant compounds while having higher soluble solid concentration. moreover, multivariate statistical analyses (principal component analysis and cluster analysis) were applied to the results. different results allowed for a successful differentiation of new cultivars. therefore, the chosen compounds resulted in suitable markers for quality and safety assessment of tomatoes. keywords: antioxidants, biogenic amines, carotenoids, food safety and quality, phenolics, tomato introduction tomato (solanum lycopersicum) is an annual plant whose berries are used widely, either processed or raw, for food and beverage. tomato plants are native to south america, and their cultivation in the mediterranean countries dates back to the 17th century (peralta and spooner, 2014). italy is the first tomato-producing country in europe and one of the top 10 producers in the world (food and agriculture organization corporate statistical database [faostat], 2019). italy also tops the list for global export of processed tomatoes ahead of china (istituto servizi mercato agricolo alimentare, 2017). in italy, tomatoes are cultivated especially in the central and southern regions, where small-size tomato varieties are much appreciated (masetti et al., 2014; carillo et al., 2019). tomato is of great value in the global vegetable consumption, and recently, there has been an increase in the spread of new small-size tomato cultivars. small-size tomatoes are preferred for fresh consumption than regular-size tomatoes, and consumers choose the same for their organoleptic proprieties (liu et al., 2019). tomato is one of the most studied crops, and their genetic improvement is constant. the breeding programs led to the offspring of new varieties that can meet the industry and/or consumer preferences. moreover, through new cultivars, disease resistance is achieved. recently, new hybrid cultivars of tomatoes named bamano, dulcemiel, and sugarland have been introduced in central italy. these cultivars are trying to expand the fresh agronomic market with products characterized by unique organoleptic and nutritional properties. usually, seed companies evaluate prime properties (e.g., size, color, sugars, etc.) under different stress and environmental conditions, followed by researchers’ early characterization (ingallina et al., 2020c). however, in order to valorize the final product, quality and safety assessment is highly recommended. quality assessment is necessary to determine molecular markers, typical of italian journal of food science, 2021; 33 (2): 35–45 mailto:mattia.rapa@uniroma1.it 36 italian journal of food science, 2021; 33 (2) rapa m et al. hydrophilic antioxidant fraction was tested by in vitro antioxidant activity through scavenging of dpphand abts-free radicals and total phenolic content by the folin–ciocâlteu method. total contents of carotenoids and lycopene were analyzed in lipophilic fraction by uv-vis methods. moreover, the profile of eight bas was evaluated in tomato samples by high-performance liquid chromatography with fluorescence detection (hplc-fd) after dansyl chloride derivatization. the bas studied were spermine (spm), spermidine (spd), putrescine (put), and cadaverine (cad) for polyamines, whereas β-phenylethylamine (β-pea), his, ser, and tyr were studied for monoamines. the above-mentioned analyses were also conducted on samples from two traditional cultivars of tomatoes used for fresh market and canning industry. finally, a multivariate statistical analysis (principal component analysis [pca] and cluster analysis [ca]) was conducted on the bioactive compound profiles of tomatoes to highlight natural differentiation of samples coming from the new cultivars. materials and methods materials methanol (ch3oh), n-hexane (c6h14), water (hplc grade), acetonitrile (hplc grade), folin–ciocâlteu reagent (h3[p(w3o10)4]/h3[p(mo3o10)4]), abts, dpph, potassium persulfate, sodium bicarbonate (nahco3), gallic acid (c7h6o5), perchloric acid (hclo4), sodium hydroxide (naoh), sodium carbonate (na2co3), and ammonium hydroxide (nh4oh) were purchased from sigma aldrich chemical co. the eight bas—his, ser, spm, spd, put, β-pea, cad, and tyr—were supplied by supelco (bellefonte, pa, usa) as well as the derivatizing agent, dansyl chloride, and the internal standard, 1,7-diaminoheptane (is). sampling tomato samples were supplied by eight different farmers with similar pedo-climatic conditions, located in the south of lazio region (italy), which were harvested in 2016. tomato seeds (bamano and dulcemiel) were supplied by syngenta, basel, switzerland and rijk zwaan, de lier, the netherlands (sugarland). two samplings per cultivar were prepared for each farm. a total of 48 samples were collected (bamano, n =16; dulcemiel, n = 16; and sugarland, n = 16). moreover, other 11 samples were collected from selected cultivars for fresh market and canning industry. after acquisition, samples were a sample, that can establish the sample’s origin or the good state of storage (giuggioli et al., 2016). antioxidant compounds are usually used to evaluate food quality (armenta and de la guardia, 2016). phenolic compounds, secondary metabolites of many plants, are ubiquitous in the vegetable domain and they are one of the most extensively studied groups of natural compounds. their dietary intake is highly recommended, and they have anti-microbial and anti-carcinogenic effects (coyagocruz et al., 2018). these compounds have already been detected in good quantity in commercial tomatoes, especially in small-size varieties (selli et al., 2014). tomatoes have a significant antioxidant activity afforded by phenolic compounds and antioxidants such as carotenoids, lycopene, and vitamins (szabo et al., 2018). total phenolic content, 2,2-diphenyl-1-picrylhydrazyl (dpph), and 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts) assays are established as quick and robust tools for characterizing antioxidants in the fractions of hydrophilic tomatoes. they are used widely in an explorative and preliminary assessment of vegetables and fruits (fanasca et al., 2006; preti et al., 2017). besides, to characterize the antioxidants present in the lipophilic fraction, such as carotenoids (including lycopene), ultraviolet–visible spectroscopy (uv-vis) methods are used generally (ingallina et al., 2020a). other metabolites present in small amounts in food are often used as markers of food safety. among these compounds, biogenic amines (bas) are widely used as food safety markers because of their presence in food and their effect on the human body. bas are the result of the decarboxylation of amino acids, but their presence in food can also be related to spoilage. in addition, bas can induce several negative physiological reactions, and the investigation of their levels in food is important for consumers’ health and the formulation of diets (kalač, 2014). some bas, such as histamine (his) and tyramine (tyr), pose potential risks to human health, that is, ‘scombroid food poisoning’ and ‘cheese crisis’ (al bulushi et al., 2009). however, not all bas are dangerous for human health. for example, serotonin (ser) plays an essential role to regulate mood, sleep, body temperature, sexuality, and appetite in the central nervous system (hano et al., 2017). therefore, its presence in food could be an exciting feature. notwithstanding that presence of bas is regulated in some foods and drinks, some authors have suggested bas to be food quality markers (silla santos, 1996). in this work, a quality and safety assessment of three new tomato cultivars is proposed. at first, soluble solid concentration (ssc) and ph were determined to evaluate physicochemical characteristics of the tomato cultivars. thereafter, an evaluation of antioxidants present in hydrophilic and lipophilic fractions was carried out. the italian journal of food science, 2021; 33 (2) 37 quality and safety evaluation of new tomato cultivars table 1. physical characteristics of new tomato cultivars. cultivar color size fruit weight picture bamano bright orange elongated shape (2.5 ± 0.5 × 4.5 ± 0.5 cm) 11 ± 1 g dulcemiel green with honey shades round shape (2.5 ± 0.5 × 3.5 ± 0.5 cm) 15 ± 1 g sugarland deep shiny red round shape (1.5 ± 0.5 × 2.5 ± 0.5 cm) 12 ± 1 g homogenized by an ultra-turrax system and stored at –18°c. the physical characteristics of each cultivar are reported in table 1. physicochemical parameters the ssc was determined with a portable refractometer (rs pro; milan, italy) at 20°c and expressed as °brix. the ph was measured with a ph-meter (hach company; loveland, co, usa).  determination of biogenic amines extraction biogenic amines were extracted according to a previously optimized tomato products method (chiacchierini et al., 2006). about 8 g of sample, previously added with 0.1 ml of is (100 mg/l), was extracted with 10 ml of 0.6 m hclo4, homogenized for 3 min, and centrifuged at 2,700 g for 10 min. the supernatant was filtered through a 0.20-µm membrane millipore filter and collected in a flask. the residue was added with 10 ml of 0.6 m hclo4, mixed, and again centrifuged for 10 min. then the second extract was filtered and added to the first one. the final volume was adjusted to 25 ml with 0.6 m hclo4. derivatization an aliquot of 1 ml of the final extract was derivatized according to procedures reported by ingallina et al. (2020b). about 200 µl of 2m naoh, 300 µl of saturated nahco3 solution, and 2 ml of dansyl chloride solution (10 mg/ml in acetone) were added in a tube. after shaking, the samples were left in dark for 60 min at 45°c. about 100 µl of 25% nh4oh was added to stop the derivatizing reaction. the final volume was adjusted to 5 ml by adding acetonitrile. the dansylated extract was filtered using 0.22-µm filter (polypro acrodisc, pall gelman laboratory, usa) and injected into the hplc system (ingallina et al., 2020b). chromatographic setup chromatographic separation was achieved by a system consisting of a lc-10 atvp binary hplc pump, a supelcosil lc-18 column (supelco, 5-μm particle size, 150 × 2.1-mm i.d.) equipped with a supelguard lc-18 guard column (supelco inc., bellefonte, pa, usa), and an rf-10axl fluorescence detector (shimadzu, kyoto, japan). the injector was fitted with a 20-μl loop. the chromatographic data were collected and processed using class-vp software (shimadzu). the analysis was conducted as described in previous work. fluorescence detection was set at 320 nm for excitation and 523 nm for emission. identification of the bas was based on their retention time and adding of standards. the quantification was performed using the internal standard calibration method by linear regression analysis (r2 > 0.995). extraction of hydrophilic antioxidant compounds sample extractions for antioxidant activity and total phenolic content were prepared from 2 g of tomatoes in 20 ml of methanol. samples were homogenized in an ultraturrax for 3 min and centrifuged at 2,400 g for 5 min (fratoddi et al., 2018). determination of total phenolic content (folin–ciocâlteu) total phenolic content (tpc) was determined using the folin–ciocâlteu method (fratoddi et al., 2018), modified for tomatoes as follows: 1 ml of methanolic extract was 38 italian journal of food science, 2021; 33 (2) rapa m et al. where abs is the absorbance reading, mw is the molecular weight, 2.7 refers to the volume (in ml) of the hexane phase, w is the sample weight, and e is the molar extinction coefficient of lycopene in hexane (185.3 mm/cm). results were expressed as mg/kg of lycopene (fresh weight, fw) (antolinos et al., 2020). statistical analysis all the experiments were conducted in triplicate and expressed as mean ± standard deviation. t-test, correlations, and chemometric data analyses (pca and ca) were performed with jmp software (ver. 15.2, sas institute, cary, nc, usa). results and discussion physicochemical properties the presence of phytochemicals in tomatoes, such as carotenoids and phenolic compounds, mineral salts, and organic and fatty acids content is closely related to their health-promoting properties. therefore, these are used in quality and safety assessment. these compounds are biosynthesized and accumulated in fruits, and their content is influenced by environmental factors, cultural practices, and genetic aspects, such as different cultivars (antolinos et al., 2020). in this respect, ph and ssc were evaluated in the examined samples, and the results are reported in figure 1. the new cultivar samples had significantly lower ph values (p < 0.01) compared to control cultivars, even if the difference was 8–10%. these results suggest a possible use of specific consumers satisfaction related to their organoleptic and sensorial features. moreover, the highest ssc was found for sugarland cultivar, followed by bamano and dulcemiel. the resulting ssc values of new cultivars were statistically different from that of the control, indicating greater soluble solid compounds. biogenic amines the evaluation of bas in fresh vegetables has been recently explored in literature, tomatoes included (sánchez-pérez et al., 2018). according to sánchezpérez et al. (2018), the bas found in tomatoes were his (n.d.–22 mg/kg fw); tyr (n.d.–6.38 mg/kg fw); put (5.3–35.5 mg/kg fw), and cad (n.d.–2.33 mg/kg fw). in this study, contents of eight bas were determined in three new cultivars of cherry tomatoes; their profiles are shown in figure 2. the chromatograms exhibited different trends for the three cultivars. an appreciable peak resolution was achieved (palomino-vasco et al., 2019; ramos et al., added to 0.25 ml of folin–ciocâlteu reagent and 0.5 ml of na2co3 water solution (7.5% w/v) in a 10-ml volumetric flask. the final volume was reached with purified water. spectrophotometric analysis was performed at λ = 750 nm after 45 min of incubation in dark at room temperature. tpc was expressed as milligrams of gallic acid equivalent (gae) per kg. the final results were obtained through a calibration curve ranging from 15 to 500 mg/l (r2 = 0.9925). determination of antioxidant activity the dpph and abts assays were based on the same mode of action, and they are common in vitro antioxidant tests (tonolo et al., 2019). the disappearance of radical was determined by measuring absorbance at 515 nm (dpph) and 734 nm (abts) as described previously (preti et al. 2017); the absorbance was measured in 1-cm path length cuvettes, using a uv-vis spectrophotometer (jenway, stone, uk). results were expressed as inhibition rate and were calculated based on equation 1: − = ×0 f 0 a a i% 100, a (1) where a0 is the radical cation’s initial absorbance, and af is the absorbance after the addition of sample extract. lipophilic antioxidant extraction briefly, 7 ml of ethanol:hexane mixture (4:3 v:v) was added to 0.1-g homogenized sample in a glass tube (protected from light). the lycopene extraction was conducted by agitating the mixture for 1 h (darkness) at 200 rpm. thereafter, 1 ml of distilled water was added to the mixture and stirred by inversion. the hexane fraction was then collected in an amber vial. total carotenoids the total carotenoid content was determined at 449 nm (ingallina et al., 2020a). the results were compared with a standard solution of β-carotene in n-hexane, and the quantification of total carotenoids was achieved by the linear regression (r2 = 0.9962) and expressed as milligram of β-carotene (mg bce). lycopene determination the lycopene determination was performed by measuring the hexane phase absorbance at 472 nm in a spectrophotometer. the lycopene content was calculated with the lambert–beer law as described in equation 2: abs mw 2.7lycopene (mg / kg) , w e × × = × (2) italian journal of food science, 2021; 33 (2) 39 quality and safety evaluation of new tomato cultivars 8 7 6 5 4 3 c c c c,d d b ph ssc (°brix) bamano dulcemiel sugarland control fresh control canning b b a a figure 1. determination of physicochemical properties of different tomato cultivars: ph and soluble solid concentration (ssc) (°brix). samples not connected by the same letter are significantly different. 2020). the quantification of bas in new cultivars and control tomatoes is summarized in table 2. table 2 also describes the t-test results (α = 0.95) of each variable for the five categories of the sample analyzed. among new cultivars, the highest total ba contents in tomatoes was determined in sugarland (275.2 ± 11.10 mg/ kg), followed by dulcemiel (201.01 ± 1.71 mg/kg) and bamano (137.36 ± 1.98 mg/kg). these contents were comparable with the control canning cultivar, in spite of the fact that the fresh control had a higher total ba values. the amount of his, put, and cad of the new cultivars (<loq: 0.57 mg/kg, 0.16–5.75 mg/kg, and 1.15–2.41 mg/ kg, respectively) was in agreement with results from literature, while tyr was below the limit of quantification for all the samples. compared with the control cultivars, the his values were comparable with that of the canning cultivar and were lower than that of the fresh cultivar 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 5 6 7 8 9 10 11 12 minutes cad b-pea his ser tyr spd spm is put dulcemiel bamano sugarland 13 14 15 16 17 18 v ol ts figure 2. chromatographic profiles of biogenic amines determined in tomato samples: sugarland (red trace), bamano (green trace), and dulcemiel (dark yellow trace). β-pea: β-phenylethylamine; put: putrescine; cad: cadaverine; his: histamine; is: internal standard; ser: serotonin; tyr: tyramine; spd: spermidine; and spm: spermine. 40 italian journal of food science, 2021; 33 (2) rapa m et al. table 2. quantitative results of biogenic amines in tomato samples (mg/kg). samples not connected by the same letter are significantly different. bamano dulcemiel sugarland control fresh control canning β-pea 1.16b ± 0.05 0.17c ± 0.001 0.17c ± 0.01 1.47a ± 0.12 1.13b ± 0.10 put 0.16c ± 0.01 0.56c ± 0.03 5.75b ± 0.14 11.17a ± 4.17 4.98b ± 0.21 cad 2.41a ± 0.09 1.15b ± 0.01 1.22b ± 0.02 0.94c ± 0.15 0.70d ±0.03 his <loqc 0.57a,b± 0.02 0.33b,c ± 0.01 1.01a ± 0.41 0.10b,c ± 0.06 ser 132.47d ± 2.05 197.27c ± 1.71 266.87b ± 11.16 379.51a ± 4.06 146.81c,d ± 8.67 tyr <loqc <loqc <loqc 1.29a ± 0.09 0.67b ± 0.08 spd 0.29b ± 0.01 0.37b ± 0.02 0.33b ± 0.01 8.32a ±0.40 8.32a ± 0.85 spm 0.87b ± 0.04 0.91b ± 0.06 0.53c ± 0.02 0.80b,c ± 0.03 1.16a ± 0.47 total bas 137.36d ± 1.98 201.01c ± 1.71 275.2b ± 11.10 404.53a ± 9.45 164.67c,d ± 10.53 β-pea: β-phenylethylamine; put: putrescine; cad: cadaverine; his: histamine; ser: serotonin; tyr: tyramine; spd: spermidine; spm: spermine; total bas: total biogenic amines; loq: limit of quantification. (1.01 ± 0.41 mg/kg). besides, in the control cultivar, tyr was also observed (0.67 – 1.29 mg/kg). it is essential to underline bas’ shallow levels such as his and tyr, frequently reported as dangerous in the human diet (linares et al., 2016). although, the his and tyr contents were not dangerous, a lower concentration in new cultivars allowed products with lesser contamination at the processing stage. polyamines, such as spd and spm, play a role in increasing shelf life of tomatoes. the gene expression related to spd and spm would reduce the post-harvest senescence and decay (handa and mattoo, 2010; nambeesan et al., 2010). thereafter, the low amount of spd (0.29–0.37 mg/kg) and spm (0.53–0.91 mg/kg) found in all new cultivars could be a desirable feature. they could be related to a natural over expression of some metabolic pathways in these cherry tomato varieties, contributing to the elongation of shelf life. also, the lowest concentrations in new cultivars, compared to the control, suggest these tomato cultivars’ eligibility in the supply chain. finally, an interesting remark should be made about the ser content. the ser content was the major contributor to the total contents of bas established in the samples, starting from 132.47 ± 2.05 mg/kg (96%) for bamano to 197.27 ± 1.71 mg/kg (98%) for dulcemiel and 266.87 ± 11.16 mg/kg (97%) for sugarland. these results were in agreement with already published results (riga et al., 2016). a similar trend in ser content was also found in control cultivars (90–93%), although in slightly lower proportions. however, the excellent ser content in tomato fruits is related to its several physiological functions in plants (e.g., growth regulator, protection against pathogens, etc.). in plants, ser is produced from tryptophan, and demonstrates some positive effects on the human body. daily assumption of ser-rich vegetable varieties has demonstrated, inter alia, useful anti-obesity and anxiety control effects (islam et al., 2016). moreover, it has been proved that the ser content tends to decrease in processed tomato products. therefore, tomato cultivars relatively rich in ser could be of interest for the tomato industry (hano et al., 2017). antioxidants evaluation nowadays, several features, such as being rich in nutrients or having physiological benefits, are searched in foods. among these, antioxidant compounds are the most interesting nutrients for human health, and are considerably present in fruits and vegetables (dudonné et al., 2009). moreover, these compounds are widely used to evaluate food quality. the hydrophilic and lipophilic fractions were examined to evaluate antioxidants in new tomato cultivars. tpc assay was chosen to quantify phenolics’ content in hydrophilic fraction, essential components of antioxidant compounds in tomatoes (fanasca et al., 2006). the antioxidant activity was also tested by two different in vitro anti-radical assays—abts and dpph (campestrini et al., 2019). moreover, these two radicals are sensitive to different types of antioxidants. consequently, their combined use consented to an effective evaluation of antioxidant activity. the results and significant differences are shown in table 3. for tpc, sugarland had the highest results with 303.15 ± 21.62 mg gae/kg, followed by dulcemiel and bamano (256.39 ± 6.63 and 242.18 ± 6.6 mg gae/kg, respectively). tpc results were in accordance with previously reported tomato results, especially for cherry tomatoes (raffo et al., 2002; riga et al., 2016). italian journal of food science, 2021; 33 (2) 41 quality and safety evaluation of new tomato cultivars marker of the tomato variety was investigated (bajoub et al., 2016). for this purpose, pca and ca were used to explore data matrices in order to highlight a natural grouping among samples (marengo et al., 2017). autoscaling pretreatment was conducted in the data matrix composed of experimental results (nur azira et al., 2014). the pca results are reported in figure 3: sugarland is represented by circles, bamano by squares, dulcemiel by crosses, control fresh by stars, and control canning by triangles. in pca, the first two principal components (pc1 and pc2) accounted for 82.9% of the total variability (liu et al., 2013). in the scores plot, all cultivars were clearly separated (šamec et al., 2016). new cultivars are located in the left part of the diagram, sugarland and dulcemiel in the upper part, while bamano in the lower one. control cultivars were located on the right, fresh cultivar samples on the top, and the canning ones on the lower part. as highlighted by the scores plot, pc1 differentiated new cultivars (sugarland, bamano, and dulcemiel) from the control. this pc was highly influenced by physicochemical properties (ph and ssc), carotenoids (including lycopene), phenolic compounds, and spd and tyr for bas. separation among each cultivar was enabled by pc2, whereby bas (ser, bai, his, and β-pea) and dpph anti-radical assays were the major contributors. to characterize new cultivars, pca was recalculated by excluding control cultivars. the scores and loadings’ plot of this analysis are given in figure 4. the loadings’ plot pointed out for dulcemiel samples was positively correlated with dpph, spd, and his variables. it is clear in tables 2 and 3 that dulcemiel cultivar had the highest content in these compounds. sugarland demonstrated a positive correlation with ser, bai, put, tpc, ssc, total carotenoids, and lycopene. moreover, sugarland had a high negative correlation with spm. samples of the bamano cultivar were positively correlated with cad, β-pea, ph, and abts content. therefore, the trend of tpc results agreed with dpph radical scavenging assay for antioxidant activity, proving a high radical inhibition by the three cultivars. the lowest result was achieved by bamano cultivar (88.61, i%). a similar result was reported by lu et al. (2020), who established that tpc values could be positively correlated with the dpph values (lu et al., 2020). however, bamano variety had demonstrated the highest abts scavenging activity results (60.34, i%), followed by sugarland (32.50, i%) and dulcemiel variety (26.63, i%). in bamano samples, these results could be explained by a more significant presence of other chemical compounds with antioxidant activity not included in the phenolic compounds, such as vitamin c or anthocyanins (pataro et al., 2015; marengo et al., 2017). total carotenoids and lycopene contents were evaluated in the lipophilic fraction of antioxidants by uv-vis methods. among new cultivars, sugarland had the highest content of carotenoids (54.12 ± 1.36 mg bce/kg) and lycopene (48.88 ± 2.95 mg/kg), followed by bamano (40.12 ± 2.69 mg bce/kg, 29.25 ± 7.48 mg/kg) and dulcemiel (33.12 ± 0.99 mg bce/kg, 12.12 ± 1.25 mg/kg). these values of compounds in new tomato cultivars were compared with the literature data (d’evoli et al., 2013). it is also appropriate to highlight that lycopene is the major carotenoid in cherry tomatoes, representing 40–90% of the total carotenoid contents in new cultivars. the values obtained were significantly lower than that of control, except for dpph assay results for sugarland and dulcemiel cultivars. multivariate analysis different profiles of bioactive compounds found in tomatoes had suggested the hypothesis that some of the compounds detected for quality and safety assessment could also be typical of a cultivar (uarrota et al., 2014). therefore, their presence as a potential authenticity table 3. quantitative results of evaluation of antioxidants in tomato samples. samples not connected by the same letter are significantly different. bamano dulcemiel sugarland control fresh control canning tpc (mg gae/kg) 242.18d± 6.60 256.39d± 6.63 303.15c ± 21.62 369.98b ± 12.37 458.97a ± 3.11 dpph (i%) 88.61b± 3.42 93.38a± 3.47 91.25a ± 1.36 93.46a ± 1.34 92.04a ± 0.74 abts (i%) 60.34b± 1.92 26.63d ± 1.54 32.50c± 2.49 96.82a ± 0.29 98.64a ± 0.38 tcc (mg bce/kg) 40.12c ± 2.69 33.12d ± 0.99 54.12b ± 1.36 142.00a ± 5.05 143.17a ± 4.99 lycopene (mg/kg) 29.25c ± 7.48 12.12d ± 1.25 48.88b ± 2.95 127.80a ± 1.79 128.50 a ± 1.38 tpc: total phenolic content; tcc: total carotenoids content; dpph: 2,2-diphenyl-1-picrylhydrazyl; abts: diammonium salt; gae: gallic acid equivalent. 42 italian journal of food science, 2021; 33 (2) rapa m et al. 8 (a) (b) 6 4 2 0 –2 –4 –6 –8 –10 –5 0 5 10 –1.0 –1.0 spm ph bpea abts °brix put tpc baiserdpph his cad tyr –0.5 –0.5 0.0 0.0 1.00.5 1.0 0.5 p c 2 (2 7, 2% ) p c 2 (2 7, 2% ) pc1 (55,7%) pc1 (55,7%) figure 3. (a) principal components analysis (pca) scores and (b) loading plots of tomato samples: sugarland (circles), bamano (squares), dulcemiel (crosses), control fresh (stars), control canning (triangles). 8 (a) (b) 6 4 2 0 –2 –4 –6 –8 –10 –5 0 5 10 –1.0 –1.0 spm ph bpea abts totalc carotenoids lycopene °brix put tpc bai ser dpph his spd cad tyr –0.5 –0.5 0.0 0.0 1.00.5 1.0 0.5 p c 2 (2 9, 9% ) p c 2 (2 9, 9% ) pc1 (56,7%) pc1 (56,7%) figure 4. (a) principal components analysis (pca) scores and (b) loading plots of tomato samples: sugarland (circles), bamano (squares), and dulcemiel (crosses). results of the pc explorative analysis were in accordance with the experimental ones (guerreiro et al., 2013). the good results of pca analysis were also confirmed by ca, reported in figure 5. this analysis pointed out general similarities or differences in the profile of bioactive compounds of the investigated cultivars. the first level of dendrogram demonstrates two clusters: the first one comprises control cultivars, and the second one by three new cultivars (bamano, dulcemiel, and sugarland). at the lower level of dendrogram, the first cluster is divided in two parts by separating control cultivars as the samples used for fresh market and that for canning industry. the second cluster (three new cultivars) was also divided into two parts: the first part consisting of bamano samples, and the other one comprising dulcemiel and sugarland samples. therefore, these two cultivars exhibited similar contents to the compounds examined herein. ca and pca results demonstrated differentiation among new cultivars and the control. italian journal of food science, 2021; 33 (2) 43 quality and safety evaluation of new tomato cultivars conflict of interest the authors declare that they have no conflict of interest in the subject matter or materials discussed in this manuscript. references al bulushi i., poole s., deeth h.c. and dykes g.a. 2009. biogenic amines in fish: roles in intoxication, spoilage, and nitrosamine formation-a review. crit. rev. food. sci. nutr. 49(4):369. https:// doi.org/10.1080/10408390802067514 antolinos v., sánchez-martínez m.j., maestre-valero j.f., lópezgómez a. and martínez-hernández g.b. 2020. effects of irrigation with desalinated seawater and hydroponic system on tomato quality. water. 12(2):518. https://doi.org/10.3390/ w12020518 armenta s. and de la guardia m. 2016. analytical approaches for the evaluation of food protected designation of origin. in: espiñeira, m. and santaclara, f.j. 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because of production factors (e.g., climate, soil, etc.). bas and antioxidant fractions were investigated in bamano, dulcemiel, and sugarland varieties, besides evaluation of physicochemical characteristics. the results were also compared with two control cultivars usually involved in the fresh market and canning industry. the new cultivars had meager amount of his (<loq 0.57 ± 0.02 mg/kg) and tyr (<loq) as well as an interesting amount of ser (132.47 ± 2.05 mg/kg–266.87 ± 11.16 mg/kg). therefore, quality features were assessed in addition to the absence of spoilage indicators. the antioxidant evaluation was conducted by tpc, anti-radical assays, and total carotenoids and lycopene contents. comparison with control cultivars demonstrated different physicochemical properties, lower content in total bas, and lower antioxidant compounds. finally, a chemometric evaluation of bioactive compounds was conducted by pca and ca. different profiles of analyzed compounds enabled a successful 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(z. tenuior), ferulago angulata (f. angulata), and bunium persicum (b. persicum)) all in three levels (5, 7, and 10% w/w) and tertiary butyl hydroquinone (100, 200, and 300 ppm) to evaluate the oxidation stability of iranian animal oil (iao). z. tenuior, f. angulata, and b. persicum (5, 7, and 10% w/w) and tertiary butyl hydroquinone (100, 200, and 300 ppm) were added to iao. the physicochemical properties and color analysis, and sensory characteristics (odor, taste, rancidity, and overall acceptability) of the treatments were investigated on days 0, 7, 14, 21, and 28. the results showed that increasing the amounts of z. tenuior, f. angulata, and b. persicum was associated with reducing acid, peroxide, and thiobarbituric acid values. it also lowered brightness and yellowness while the oxidative stability of the iao significantly was increasing. it was concluded that the incorporation of b. persicum had the highest effectiveness regarding the proposed criteria with the least effect on sensory properties. keywords: bioactive compounds; herbal antioxidant; oil stability; natural preservative; color analysis introduction oxidation in food, especially in fats and oils, is a destructive process with adverse effects on their nutritional values and chemical composition (henry and heppell, 2002). it can also have undesirable effects on the color, taste, and texture of foods, while degrading the essential vitamins and fatty acids or creating toxic compounds (stoilova et al., 2007; wang et al., 2008). antioxidants are natural or synthetic substances added to food products to prevent or delay the damage caused by oxygen (sharififar italian journal of food science, 2021; 33 (sp1): 69–77 mailto:fatemeh_fazeli2002@yahoo.com mailto:mohammadi@sbmu.ac.ir mailto:mousavi@unicamp.br 70 italian journal of food science, 2021; 33 (2) azizi m et al. materials and methods extraction of essential oil fresh iao, free of any antioxidants, was purchased from a local market. the dried leaves of z. tenuior, f. angulata, and b. persicum were obtained from the iranian institute of medicinal plants, karaj, iran. the essential oils were extracted from these leaves using a clevenger-type apparatus (biogenic, brasilia, brazil), and based on the steam distillation method of gharibzahedi et al. (2015). the brief protocol is as follows: one-hundred grams of the leaves were placed in the distillation flask, and the oil was extracted from the vegetal substrate at 5-, 15-, 30-, 60-, and 100-min intervals. in order to completely isolate the water from the essential oil, 0.5 g of sodium sulfate was added to the separation column each time. the extracted essential oils were stored at 4 ± 1°c until experiment time. tbhq and other chemicals were purchased from merck co. (darmstadt, germany). preparation of the treatments different concentrations of the essential oils and tbhq were added to 100 g of iao based on the values indicated in table 1 and mixed using rh basic 2 magnetic stirrer (staufen, staufen germany). staufenthe iao samples were evaluated on days 0, 7, 14, 21, and 28 of storage. all tests were performed in three replications. reactions. its phenolic group stabilizes free fatty radicals by using hydrogen and thus preventing the oxidation of fats (wang et al., 2008). however, some concerns have been raised regarding the mutagenicity and the role of synthetic antioxidants in the disruption of liver enzymes’ activity as well as the development of diseases such as cancer and cardiovascular disease (iqbal and bhanger, 2007; tavakoli et al., 2018). as a result, replacement of these compounds with natural antioxidants of plant origin has been considered. antioxidant properties of plant extracts mainly depend on their phenolic compound content (ahmed et al., 2019; burt, 2004). in this regard, herbs and their derivatives (essential oils and extracts) with potent antioxidant properties are widely used to prevent the oxidative degradation of food (hashemi et al., 2017; tiwari et al., 2009). “kermanshahi roghan,” known as the “roghan-eheyvani,” is an iranian animal oil (iao) and a yogurt by-product, like ghee and yayik butter commonly used in india and turkey, respectively (mostafaie et al., 2018). it is a traditional anhydrous milk fat product with a golden yellow color because of carotenoids. as an expensive dairy product, iao is one of the most favorable oils consumed in many regions of iran (salarabadi et al., 2015). produced from cow, sheep, or goat’s milk, iao is exported to many countries worldwide (chalabi et al., 2018). the fatty acid composition of iao (99% fat and < 0.2% moisture) is different from that of butter, as the lipids are extracted directly from milk. therefore, it has lower long-chain fatty acids and cholesterol (najafi et al., 2011). z. tenuior, ferulago angulata (chavill), and bunium persicum (black caraway) belong to lamiaceae, apiaceae, and chattrian, respectively. z. tenuior (bakhtiar et al., 2021) and f. angulata (ghasemi pirbalouti et al., 2016) are widely distributed in iran. also, b. persicum grows in areas with a mediterranean climate such as central and western asia (e.g., iran) (hassanzadazar et al., 2018). due to their phenolic and flavonoid compounds, these plants have antioxidant and antibacterial activities, and gastrointestinal transit accelerating properties (amiri and joharchi, 2016; dakah et al., 2019; ehsani et al., 2016; hazrati et al., 2019; mahboubi, 2019). considering the increasing awareness among consumers regarding the harmfulness of synthetic additives in food, as well as studies on the use of herbal extracts to increase the shelf life of vegetative oils, the present study is designed to compare the oxidation of iao treated with z. tenuior, f. angulata, b. persicum, with that of synthetic antioxidant tbhq. table 1. iranian animal oil treatments with incorporation of different essential oils and tbhqa. treatments z. tenuior (%) f. angulata (%) b. persicum (%) tbhq (ppm) t1 5 – – – t2 7 – – – t3 10 – – – t4 – 5 – – t5 – 7 – – t6 – 10 – – t7 – – 5 – t8 – – 7 – t9 – – 10 – t10b – – – 100 t11b – – – 200 t12b – – – 300 atbhq: tertiary butyl hydroquinone, artificial antioxidant. bt10–t12 treatments were used as control. italian journal of food science, 2021; 33 (2) 71 animal oil stability with medicinal herb oil iao analysis iao with a peroxide value of less than 5 meq o2/kg was purchased from the pegah golpayegan dairy company (golpayegan, iran). it was kept at 4°c during the whole study, as recommended by the producer. acid value the av was defined as the amount (mg) of potassium hydroxide (koh) required to neutralize the free fatty acids in 1 g of iao samples dissolved in a mixture of ethanol. the titration method was used to determine the av according to iso 660 (iso, 2009) using the following equations: × × = × 56.1 n v acid value (ml koh/g iao) 100 m (1) n: koh normality; v: consumed koh volume (ml); m: iao weight (g); 56.1 = koh molecular weight (g/mol). peroxide value iao was dissolved in a solution of isooctane and glacial acetic acid and then mixed with potassium iodide. free iodine via peroxides was measured (iso 3960, iso, 2017) using iodometry in the presence of starch and sodium thiosulfate solution. pv was determined through sodium thiosulfate titration and was calculated using the following equation: ×= ×2 v n peroxide value (meq o /kg iao) 1000 m (2) v: consumed thiocyanate volume; n: thiosulfate normality; m: iao weight. thiobarbituric acid value thiobarbituric acid (tba) value shows the amount of malondialdehyde (mda) present in each 100 g of oil. mda, commonly used as an oxidation marker, is one of the most abundant aldehydes generated during secondary lipid peroxidation in foods (reitznerová et al., 2017). this method allows direct measurement of the tba levels in fats and oils without the mda’s need for prior isolation. mda reacts with tba and forms a color complex with absorption maxima at a wavelength of 530 nm (aocs, 2017). 50 (a b)tba (mg mda/kg iao) m × − = (3) a: absorption of iao sample; b: absorption of tba as the blank sample; m: iao weight oxidative stability the ability of the oil against oxidation is known as os and is expressed as h. the test was performed based on the rancimat technique using a metrohm 743 rancimat (fanazma gostar, alborz, iran) at 110°c and 20 l/h airflows (iso 6886, iso, 2016). oxidation resistance ends when a rapid increase is seen in the specific conductance due to the decomposition of the carboxylic acids resulting from lipid oxidation and their absorption in deionized water. color analysis the color evaluation was performed using hunter lab colorimeter (hunter lab d25-dp9000, germany) with black and white calibration plates. l* (lightness) from black (0) to 100 (white), a* (green-red), and b* (blueyellow) was calculated from −120 to 120 (guo et al., 2016). sensory evaluation the sensory characteristics of the treatments such as odor, taste, rancidity, and overall acceptability of coded iao samples during 28 days of storage were evaluated based on a 5-point hedonic scale from 1 (dislike very much) to 5 (like very much) (mohammadi et al., 2011). statistical analysis a randomized complete block design was applied. the data were analyzed by analysis of variance (anova) using sas 9 (sas institute inc., cary, nc, usa) followed by duncan’s multiple range test (koushki et al., 2011). values were reported as mean ± standard deviation (sd) of six repetitions for each treatment. p values < 0.05 were considered statistically significant for all comparisons (amiri-rigi et al., 2011). results and discussion av, pv, tba values and os analysis of different iao treatments av value depends on the type and the amount of the applied essential oils. in other words, the difference in the av is due to the difference in the antioxidant content of the active compounds as well as their ability to trap water. according to figure 1a, there was a significant difference between the av values of the controls (t10–t12) and that of the treatments containing essential oils (p < 0.05). a decreasing trend was observed in av values with increasing concentrations of essential oils. treatments containing b. persicum and z. tenuior were reported to have significantly lower av values than those containing f. angulata. this is mainly because the high polyphenolic, flavonoid, and antioxidant content of treatments containing b. persicum deactivates free radicals derived from the environmental oxidation process (hassanzadazar et  al., 2018). av indicates the progress of the oxidation process, and therefore its value decreases with the increasing flavonoid content of the oil. this is mainly because of the increased concentration of applied essential oils. 72 italian journal of food science, 2021; 33 (2) azizi m et al. 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 0 7 14 21 28 a v (m l k o h /g ia o ) time (d) t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 t11 t12 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 0.11 0.12 0 7 14 21 28 tb a v al ue (m g m d a /k g ia o ) time (d) t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 t11 t12 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 0 7 14 21 28 p v (m eq o 2 /k g ia o ) time (d) t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 t11 t12 6 7 8 9 10 11 12 0 7 14 21 28 o s (h ) time (d) t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 t11 t12 (a) (b) (c) (d) figure 1. acid value (av) (a), peroxide value (pv) (b), thiobarbituric acid (tba) value (c), and oxidative stability (os) (d) of iranian animal oil treated with herbal essential oils during storage time. treatments (t1–t12) are described in table 1. there was an overall increase in the av of iao treatments with increased storage time that negatively affects the oil quality. this can be secondary to the increase of iao’s free fatty acid content during this time, either because of the hydrolysis of triglycerides or their formation as an end product of the oxidation process (guo et al., 2016). moreover, the increased exposure of oil to oxygen, light, and acidic compounds as the oxidation process progresses could also be responsible for the significant increase noted in the av values. for instance, the increase noted in av of iao treatments containing b. persicum and z. tenuior was lower than that of controls containing tbhq. treatments containing 10% b.  persicum and z. tenuior had the lowest av enhancement (28 days) (p < 0.05). on the other hand, the av of treatments containing f.  angulata increased significantly during storage but was negatively correlated with the amount of f. angulata (p < 0.05). compared with b. persicum and z. tenuior, f. angulata is high in aromatic compounds but low in polyphenolic ones. in line with our findings, najafi et al. (2011) showed similar results while studying the antioxidant effect of olive leaf and its extract on soybean oil stability (najafi et al., 2011). unsaturated fatty acids such as oleic and linoleic acids with double bonds in their chemical structure are highly susceptible to oxidation-accelerating factors such as heat, light, oxygen, and lipoxygenase activity (pradhananga and manandhar, 2018). hydroperoxides are the primary products of the oxidation reactions and are thus used to evaluate the oxidation reactions’ progression (alizadeh et al., 2016). iao is very susceptible to oxidative corruption. according to figure 1b, the use of essential oils changed the pv of the treated iao significantly compared with the tbhq-containing controls (p < 0.05). the treatments containing b. persicum had the lowest pv. this can be due to the presence of active compounds such as limonene and trans-carveol and limonene derivatives (mohammadi et al., 2021). pv of all treatments increased with longer storage time, depending on the type and the amount of applied essential oil. among the applied essential oils, f. angulata had the least effect on pv values. in other words, there was no significant difference between the pv of the f. angulata–treated iao and the control containing 100 ppm tbhq. the least amount of change in pv values was reported in iao containing 10% b. persicum after 28 days of storage (p < 0.05). darughe et al. (2012) also found that the antioxidant compounds italian journal of food science, 2021; 33 (2) 73 animal oil stability with medicinal herb oil iao’s color indices treated with herbal essential oils as part of the quality control study. in this study, a* value did not change significantly due to the absence of red pigment and lycopene compounds in z. tenuior, f. angulata, and b. persicum. l* and b* values of treated iao samples during the 28 days of storage are shown in figure 2 (a,b). figure 2a illustrates that iao samples treated with z. tenuior and f. angulata had lower l* values than the control and b. persicum–treated ones. in addition, treatment with higher concentrations of f. angulata and z. tenuior resulted in a lower l* value than those containing 5% essential oils. therefore, it could be concluded that the l* value is significantly but inversely correlated with the concentration of the essential oils (p < 0.05). this could be due to the significant but negative effects of the chlorophyll content and pigment content of herbal essential oils on the light reflection, and thus the l* value. compared with b. persicum–treated iao samples, the control iao samples were not reported to have significantly different l* values. the l* values of treatments decreased significantly with increasing storage time (p < 0.05). this reduction, which is believed to be secondary to oxidative and lipolytic corrosion, was not significant in iao samples treated with in b. persicum significantly inhibited any increase in pv values of the cake during storage (darughe et al., 2012). pv breaks down after reaching a certain level and the by-product compounds are formed (pradhananga and manandhar, 2018). for this reason, tba value, alongside pv, was also investigated. according to figure 1c, there was a significant difference between the tba values of different iao treatments based on the type and the amount of applied essential oil (p < 0.05). tba followed an increasing trend during the 28 days of storage. the treatment containing 5% f. angulata had the highest tba value, whereas treatments with b. persicum had the lowest increase in tba value during storage (p < 0.05). this can be due to the increase in the latter’s phenolic and limonene compounds that inhibited oxidation more effectively (mohammadi et al., 2021). the high aromatic compound content of f. angulata, on the other hand, was not as effective in inhibiting the oxidation process. the increase noted in the tba values of the iao treatments indicates the formation of products such as aldehydes and ketones that have an adverse effect on the organoleptic properties of the oil (serfert et al., 2010). in corroboration with our results, hassanzadazar et al. (2018) also concluded that b. persicum could be used as a natural alternative to synthetic antioxidants based on the results of pv and tba values of their investigation (hassanzadazar et al., 2018). figure 1d illustrates a significant difference between the treatment’s oxidation resistance time, and the controls (p < 0.05). treatments containing 10% of each essential oil had the highest os compared with those with 5% and 7% of the corresponding essential oils. the os rate in treatments containing b. persicum, regardless of their concentration, was higher than that of iao samples treated with f. angulata and z. tenuior. the lowest os was reported in the iao samples treated with f. angulata. also, it was observed that the treatments with b. persicum had an os equivalent to tbhq. while increasing the storage time was associated with a significant decrease in the os values, increasing the concentration of essential oils had the opposite effect (p < 0.05). the former could be secondary to the partial inactivation of phenolic compounds as well as an increase in the levels of secondary hydroperoxide compounds due to storage-related oxidation (hashemi et al., 2016). our results were also supported by the research on rosemary extract’s antioxidant effects on soybean oil (casarotti and jorge, 2014). color analysis of iao treatments color is one of the apparent qualitative properties of the food products with a substantial impact on their marketability. this points out the importance of evaluating 17 18 19 20 21 22 23 0 7 14 21 28 l* time (d) t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 t11 t12 42 43 44 45 46 47 48 49 50 51 52 0 7 14 21 28 b* time (d) t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 t11 t12 (a) (b) figure 2. l* value (a) and b* value (b) of iranian animal oil treated with herbal essential oils during storage time. treat ments (t1–t12) are described in table 1. 74 italian journal of food science, 2021; 33 (2) azizi m et al. essential oils at concentrations higher than 5%. these findings are in line with that of guo et al. (2016), who reported that the rosemary ethanol extract had a significant effect on the l* value of treated palm oil samples (guo et al., 2016). they also reported that an increase in rosemary ethanol extract could decrease the l* value of palm oil treatments. similarly, hozhabri et al. (2014) investigated the effect of fish oil and z. tenuior on the quality and oxidation rate of the mahabadi goat meat. they showed that the addition of z. tenuior could significantly reduce l* value (hozhabri et al., 2014). figure 2b shows significant differences observed between the b* values of various treatments (p < 0.05). the highest b* value was observed in the treatment containing 5% f. angulata essential oil, whereas the lowest values were found in those with 10% of b. persicum, f. angulata, and z. tenuior (p < 0.05). an increasing trend in b* values was reported in the treated iao samples with increased storage time; the increase, however, was lower in the iao samples containing 10% of essential oils and 300 ppm tbhq. similar results were reported in previous research into the antioxidant effects of rosemary extract on soybean oil (casarotti and jorge, 2014). sensory evaluation of iao treatments according to figure 3a, a significant decrease in taste scores of treatments increased the essential oil concentration. one of the reasons for this may be the dominance of the plant’s taste over iao’s special taste. guo et al. (2016) also found that rosemary ethanolic extract in high amounts in palm oil caused a vegetable taste in palm oil. with increasing storage time, there was a decreasing trend in the taste scores of the treatments. treatments containing higher than 5% essential oils had lower taste scores on day 0, but they kept their scores steadily during storage time. treatments with f. angulata and then z. tenuior showed the lowest scores, but the treatments containing b. persicum had the same score as the control treatment with 300 ppm tbhq. even treatments with 200 and 100 ppm tbhq had lower taste scores than those with b. persicum during storage until the end of day 28 (p < 0.05). according to figure 3b, increasing essential oil concentration led to a significant decrease in the odor scores in iao treatments. treatments with b. persicum had the least effect on the odor characteristic because the aromatic compounds in z. tenuior and f. angulata are 0 1 2 3 4 5 t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 t11 t12 day 0 day 7 day 14 day 21 day 28 0 1 2 3 4 5 t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 t11 t12 day 0 day 7 day 14 day 21 day 28 0 1 2 3 4 5 t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 t11 t12 day 0 day 7 day 14 day 21 day 28 0 1 2 3 4 5 t1 t2 t3 t4 t5 t6 t7 t8 t9 t10 t11 t12 day 0 day7 day 14 day 21 day 28 (a) (b) (c) (d) figure 3. taste scores (a), odor scores (b), rancidity scores (c), and overall acceptability (d) of iranian animal oil treated with herbal essential oils during storage time. treatments (t1–t12) are described in table 1. italian journal of food science, 2021; 33 (2) 75 animal oil stability with medicinal herb oil a significant decrease in av, pv, tba value, l*, and b* of the treated iao. the addition of herbal antioxidants, regardless of their concentrations, also improved the os of the treated iao; their effects though started to decrease after 28 days of storage. moreover, by increasing the essential oils’ content, taste and odor of iao decreased, while rancidity and overall acceptability were increased. the bioactive effects of the applied essential oils was found to be in the order b. persicum > z. tenuior > f. angulata. the addition of 10% b. persicum to the iao was found to be an applicable and safe replacement for tbhq. our results are beneficial for developing strategies for producing edible oils and lipid-rich foods containing natural antioxidants with appropriate oxidative stability and pleasing sensory characterizations. in general, more study should be done to evaluate the antimicrobial effects of edible oils and emulsions enriched with the studied essential oils. acknowledgments the authors would like to thank the national nutrition and food technology research institute (iran, tehran) for providing the equipment required for this study. conflict of interest the authors of this manuscript wish to declare no conflict of interest associated with the submission and its content. references ahmed, a.f., attia, f.a.k, liu, z., li, c., wei, j. and kang, w., 2019. antioxidant activity and total phenolic content of essential oils and extracts of sweet basil (ocimum basilicum l.) plants. food science and human wellness 8(3): 299–305. https://doi. org/10.1016/j.fshw.2019.07.004 alizadeh, l., nayebzadeh, k. and mohammadi, a., 2016. a comparative study on the in vitro antioxidant activity of tocopherol and extracts from rosemary and ferulago angulate on oil oxidation during deep frying of potato slices. journal of food science and technology 53(1): 611–620. https://doi.org/10.1007/ s13197-015-2062-2 aocs, 2017. “american oil chemists’ society” 2-thiobarbituric acid value. direct method. official method cd 19-90. the american oil chemists’ society ; 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rui, chen gang chongqing academy of chinese materia medica, chongqing, china; chongqing engineering research center for fine variety breeding techniques of chinese materia medica, chongqing, china; chongqing key laboratory of chinese medicine resources, chongqing, china; chongqing sub-center of national resources, center for chinese materia medica, china academy of chinese medical sciences, chongqing, china *corresponding author: li long yun, chongqing engineering research center for fine variety breeding techniques of chinese materia medica, chongqing, china. email: lilongyun8688@163.com received: 5 november 2020; accepted: 18 january 2021; published: 15 february 2021 © 2021 codon publications open access paper abstract this study aimed to optimize the stir-baked technology for flos sophorae immaturus tea (fsit) and evaluate the quality of fsit. the optimum stir-baked conditions were found to be as follows: amount, 3.9 kg; rotation speed, 400 r/min; and time required to reach the temperature of 120°c, 5 min and maintained for 3.9 min after adding 15 ml of 1% stevioside. the machine-made fsit soup was clear and golden in color, with charred taste, no bitterness, no peculiar smell, and improved sensory quality under the above-mentioned conditions. heavy metal contents and microorganisms did not exceed the national standards. keywords: flos sophorae immaturus, quadratic regression rotation-orthogonal design, quality, stir-baked introduction flos sophorae immaturus (fsi), which originated from the flower buds of sophora japonica l., is a medicinal food homology crude drug planted throughout china. china, which has rich resources, is the origin of flos sophorae immaturus. it is bitter, slightly cold, nontoxic, and distributed in the large intestine and liver channel. it is effective in cooling blood and stopping bleeding, clearing the liver, and reducing fire. flos sophorae immaturus contains large amounts of flavonoids and glycosides, such as rutin, narcissoside, quercetin, isorhamnetin, kaempferol, genistein, total polysaccharides, saponin, tannin, sterol, and vitamin a (liu et al., 2016; xie et al., 2014; krishna, et al., 2012), and has good curative effect on myocardial circulation. moreover, the drug clears body heat, detoxifies, lowers blood fat and blood pressure levels, softens the blood vessels, reduces inflammation, tones the kidney, prevents arteriosclerosis, and has beauty-holding and antiaging properties (chua, 2013; he et al., 2016). flos sophorae immaturus has an extremely high nutritional value and contains 19 kinds of amino acids, including essential amino acids required for the human body. the protein content in flos sophorae immaturus is as high as 19.03%, which is 2.2fold that of common health food silver almond (wang and wang, 2009). with emphasis on healthcare, the research and development of functional foods containing flos sophorae immaturus has become extensive, especially the development of flos sophorae immaturus tea (fsit), which has become a focus of research by the flos sophorae immaturus processing industry. the stir-baked method for flos sophorae immaturus was first recorded in the song dynasty, and the chinese pharmacopoeia (edition 2015) has also recorded mailto:lilongyun8@163.com italian journal of food science, 2021; 33 (1) 85 stir-baked technology and quality evaluation of fsi tea drug control. as a standard solution, the lead (pb) and cadmium (cd) standard solutions were purchased from american sigma company. acetonitrile, methanol, and acetic acid of chromatographic grade were purchased from tedia company (usa). all other solvents and chemical reagents were of analytical grade or effective. instruments and equipment instruments and equipment included are as follows: agilent 1263 hplc (agilent company, usa), uv-2600 ultraviolet spectrophotometer (shimadzu corporation, japan), milli-q integral 5 pure water meter (millipore company, usa), bsa 124-s electronic scales (sartorius company, germany), kq-250 db numerical control ultrasonic cleaning instrument (kunshan ultrasonic instrument co. ltd., china), xmtb digital display electric thermostatic water bath (shanghai yuejin medical instrument co. ltd., china), g2x-9240 mbe electricity heat drum wind drying oven (shanghai boxun industrial co. ltd., china), zdhw temperature regulating electric heating sleeve (beijing zhongxing weiye instrument co. ltd., china), and cy-900 drum-type medicine stir-fry machine (kanghua pharmaceutical machinery co. ltd., china). fsit preparation high-quality flos sophorae immaturus with full granules and large grain size was selected, cleaned, placed in a self-made sterilizing device (patent application number: 201920071851.3), piled up, steamed at a thickness range of 4–6 cm, and cooked for 15–20 min after boiling. then it was taken out and dried for use. according to the stirbaked temperature, rotation speed, and time designed for the experiment, an appropriate amount of flos sophorae immaturus was placed in a drum stir-baked machine for frying, and an appropriate amount of 1% stevia glycoside was added to it for flavor mixing. at the end of frying, after cooling for 1 h with a blower, the sample was placed for two times in a double-layer vibrating screening machine through 90-mesh screen. finally, the sample was placed in the color selection machine for removing burnt paste and black flos sophorae immaturus, and the finished product of fsit was obtained. single factor design of stir-baked process for fsit stir-baked amount the fixed stir-baked temperature was 120°c, and the motor synchronous speed was 400 r/min. at 120°c, the selected stir-baked amounts were 2, 3, 4, 5, and 6  kg. we analyzed water extract, total polysaccharides, total flavonoids, and rutin, narcisin, quercetin, and stir-baked flos sophorae immaturus. in chinese medicine, stir-baked flos sophorae immaturus can reduce the side effects of bitter cold, and is used in patients with spleen deficiency. the stir-baked substitute tea prepared from flos sophorae immaturus has evident curative effects on diabetes, hypertension, vascular sclerosis, constipation, various hemorrhoids, pharyngeal dryness, sore throat, red eyes and heat, and heart irritability (li et al., 2017). at present, domestic research on fsit is primarily focused on handmade or simple mechanism and the development of new compound fsit (jiang and chen, 2008), although scientific reports on the key technologies of the stir-baked process are relatively few. the evaluation method of fsit is primarily limited to sensory evaluation, and the fsit production cannot be evaluated through modern methods of measurement. in the present study, the shape, soup color, aroma, and taste of fsit are used as sensory indicators. water extract content, total polysaccharides, total flavonoids, and rutin, narcisin, quercetin, and isorhamnetin contents are used as chemical indicators. the weight distribution was determined through projection pursuit clustering (ppc) with objective assignment, and comprehensive vector distance (ci), used as an evaluation index, was obtained by analyzing the sensory and chemical indices through the technique for order preference by similarity to ideal solution (topsis) method. on the basis of single factor, the quadratic regression rotation-orthogonal combination design was applied for the optimization of the process conditions for stir-baked fsit, development of an fsit variant with burnt flavor, and without bitterness and peculiar smell, and evaluation of sensory, physical, and chemical indices of production. this design can provide reference for the development and utilization of flos sophorae immaturus and quality control of fsit. materials and methods materials and reagent the flos sophorae immaturus sample was collected in 2018 at the flos sophorae immaturus base of maojiawan, changping township, wanzhou district, chongqing city. the sample was identified as a dry flower bud of s. japonica l. by a researcher (longyun li) of chongqing academy of chinese materia medica. rutin reference substance (batch number: must−16070511, purity 99%), quercetin reference substance (batch number: must−16072203, purity 98%), and isorhamnetin reference substance (batch number: must−16092001; purity of 98%) were purchased from chengdu mansite biotechnology co. ltd. narcissoside reference substance (batch number: taqt−lrla; purity of 93.1%) was purchased from national institutes for food and 86 italian journal of food science, 2021; 33 (1) shang f.h. et al. criteria mentioned in table 2, and the average of the results was taken as the final sensory evaluation. determination of physical and chemical indicators for fsit determination of moisture: refer to the direct drying method in gb 5009.3-2016, ‘determination of moisture in foods’. determination of ash: refer to the first method in gb 5009.4-2016, ‘determination of ash in foods’. determination of aflatoxin b1 content: refer to the second method in gb/t 5009.22-2016, ‘determination of aflatoxin b and g in foods’. determination of lead, arsenic and cadmium content: refer to the inductively coupled plasma-mass spectrometric method in gb/t 35876, inspection of grain and oils— determination of sodium, magnesium, kalium, calcium, chromium, manganese, iron, copper, zinc, arsenic, selenium, cadmium, and lead in cereals and derived products’. determination of water extract, total polysaccharides and total flavonoids: the sample processing and detection methods were tested according to the relevant methods furnished in the 2015 edition of the pharmacopoeia of the people’s republic of china (national pharmacopoeia commission, 2015). determination of rutin, narcisin, quercetin, and isorhamnetin: refer to the method introduced in a previous report (tan et al., 2018). determination of microbial indicators for fsit the total number of bacterial colonies and coliforms of salmonella and staphylococcus aureus of s. japonica were determined with gb 4789-2016. shigella assay was performed with gb 4789-2012. isorhamnetin contents, and performed sensory evaluation for fsit for determining preferred stir-baked amount. stir-baked temperature the fixed stir-baked amount was 4 kg, the motor synchronous speed was 400 r/min, the selected stir-baked temperatures were 80, 100, 120, and 140°c. the sample was stir baked for 4 min after the target temperature was reached. water extract, total polysaccharides, total flavonoids, and rutin, narcisin, quercetin, and isorhamnetin contents were analyzed, and sensory evaluation was performed for fsit for determining the preferred stir-baked temperature. stir-baked rotation speed the fixed stir-baked amount was 4 kg, and the stir-baked temperature was 120°c. the stir-baked time needed to reach the target temperature was 4 min, and the selected motor synchronous rotation speeds were 200, 300, 400, 500, and 600 r/min. we analyzed the water extract, total polysaccharides, total flavonoids, and rutin, narcisin, quercetin, and isorhamnetin contents, and performed sensory evaluation for fsit to determine the preferred stir-baked rotation speed. stir-baked time the fixed stir-baked amount was 4 kg, the stir-baked temperature was 120°c, and the motor synchronous rotated speed was 400 r/min. after stir-baking for 5 min and reaching 120°c, the stir-baked timings were 1, 2, 3, 4, 5, and 6 min for the test. we analyzed the water extract, total polysaccharides, total flavonoids, and rutin, narcisin, quercetin, and isorhamnetin contents, and performed sensory evaluation for fsit to determine the preferred stir-baked time. secondary orthogonal rotating combination design of stir-baked process for fsit according to the results of the single factor test, four factors for stir-baking that is, amount, temperature, rotation speed, and time, were designed, thereby applying the quadratic regression rotation-orthogonal combination design with four factors and five levels to optimize the fsit stirbaked process (ye and liu, 2017), see table 1. deployment of flavor for fsit according to the best process parameters of the test, different volumes of 1% stevioside were sprayed with regard to time, and stir-baking was continued for a certain period. ten professional tasters were invited to score different samples according to the sensory evaluation table 1. coding list of levels of various factors. levels code factors amount (kg) x 1 temperature (°c) x 2 time (min) x 3 rotate speed (r/min) x 4 +2 6 140 6 600 +1 5 130 5 500 0 4 120 4 400 -1 3 110 3 300 -2 2 100 2 200 italian journal of food science, 2021; 33 (1) 87 stir-baked technology and quality evaluation of fsi tea application of technique for order preference by similarity to ideal solution (topsis) method topsis, as proposed by hwang and yoon in 1981 (hwang and yoon, 1981), is a sorting method based on the proximity of finite evaluation objectives to ideal objectives. topsis is an effective method that is used commonly in multi-objective decision-making analysis, whose basic idea is to explore optimal and worst solutions (represented by the optimal vector di + and worst vector di –, respectively) in the finite solutions on the basis of normalized original matrix. then the distance of each evaluation objective to the optimal and worst solutions is calculated. thus, the relative proximity between the evaluation objective and optimal solution (represented by the ci) as the basis of optimal or worst solution evaluation was obtained (wang et al., 2019; tang, 2010). the specific method is as follows: n evaluation objectives and m evaluation indices are set, and the original data could be written as matrix x = (x ij)n×m. high(the larger the better) and low-quality indices (the smaller the better), that is, ijij n 2 iji 1 x z x − = ∑ and ijij n 2 iji 1 x z , (1 / x ) = = ∑ respectively, were normalized. the normalized matrix was z = (zij)n×m, with the optimal and worst vectors comprising maximum and minimum values in each column labeled as z+ = (zmax1, zmax2 … zmax) and z = (zmin1, zmin2 … zmin), respectively. the distances between the evaluation objective i and the optimal and worst solutions were 2m i max j ijj 1 d (z z )+ = = −∑ and m 2 i max j ijj 1 d (z z ) ,+ = = −∑ respectively. the relative proximity between the evaluation objective i and optimal solution is as follows: i i i ic d / (d d ). − + −= + sensory evaluation of fsit according to gb/t 23776-2018, ‘methodology for sensory evaluation of tea’, the method of password evaluation was adopted for sensory evaluation and score. the review procedure is as follows: sampling → comment on the shape → weighing 3 g → brewing 200 ml of boiling water → turn over the soup → look at the soup color → sniffing aroma → taste. the product quality consisted of four factors, namely, shape, aroma, soup color, and taste, and each factor was evaluated at three levels, that is, poor, medium, and good. the scores were presented in percentage; the scores for shape, aroma, soup color, and taste were 25%, 30%, 10%, and 35%, respectively. ten professional tasters were invited to evaluate taste according to the scoring criteria, and the average was taken as the final sensory score. the sensory score criteria are demonstrated in table 2. weight determination by the projection pursuit clustering method pursuit clustering is a new statistical method for processing and analyzing high-dimensional data. the basic idea is to project high-dimensional data onto low-dimensional subspace and determine the optimal projection direction reflecting data structure or characteristics in solving the comprehensive evaluation of high-dimensional problems (tang, 2010). according to the size of the projection direction, the weight coefficient of each evaluation index can be determined. in this study, the weight of the quality index of fsit was calculated by the ppc method in dps software. table 2. sensory scoring standards for fsit. factor grade index shape (25%) good (18–25 points) with charred taste, even and coking yellow in color, full grain, texture solid, crisp middle (9–17 points) light fragrance, uneven color, brownish yellow, individual grain broken poor (0–8 points) light yellow or brownish brown, with obvious burnt smell and severe grain breakage soup color (10%) good (8–10 points) clear and golden, no turbidity, no scattered particles middle (4–7 points) brownish yellow, no turbidity, no scattered particles poor (0–3 points) light green or tan, turbid, with scattered particles aroma (30%) good (21–30 points) strong taste with charred smell middle (11–20 points) light fragrance, no charred smell poor (0–10 points) with charred smell taste (35%) good (26–35 points) no astringency middle (13–25 points) with astringency poor (0–12 points) with paste taste and strong astringency 88 italian journal of food science, 2021; 33 (1) shang f.h. et al. index weight was introduced into the topsis of dps; the weight coefficient of the chemical indices was calculated using ppc; and the quality indices of the stir-baked fsit were evaluated comprehensively by combining with topsis. the larger the value of ci, the better the effect of experimental method. results single-factor test on stir-baked process of fsit the factors influencing the stir-baked process of fsit primarily included stir-baked amount, temperature, time, and speed. the assignment of water extract, total polysaccharides, total flavonoids, and rutin, narcissoside, quercetin, and isorhamnetin contents, as well as the sensory score in the preparation of fsit, were evaluated by the objective ppc method and analyzed through the topsis method using dps software. the results are demonstrated in table 3. water extract, total polysaccharides, total flavonoids, and rutin, narcissoside, quercetin, and isorhamnetin contents, as well as the sensory scores, were used as evaluation indicators. according to the ranking of ci, the fsit sample with the best quality was obtained after the stir-baked amount was 4 kg, stir-baked temperature was 120°c, stir-baked rotation speed was 400 r/min, and stir-baked time was 4 min at a temperature of 120°c or above. therefore, the following tests selected the stir-baked amount of 4  kg, the stir-baked temperature of 120°c, the stir-baked speed of 400 r/min, and the stir-baked time of 4 min at zero level. optimization of stir-baked process of fsit by quadratic regression rotation-orthogonal combination design according to single-factor experiments, the quadratic regression rotation-orthogonal combination design with four factors and five levels was used to optimize the stirbaked process of fsit. a total of 36 combinations at five levels for each factor were present, and the experimental design and results are demonstrated in table 4. according to the test results (table 4), the statistical analysis software dps 17.10 was used to obtain the quadratic regression model of ci for four experimental factors, such as stir-baked amount, temperature, time, and rotation speed, as follows: y = −19.80272 + 0.64512x1 + 0.25508x2 + 0.98855x3 + 0.00966x4 − 0.11938x 2 1 − 0.00106x 2 2 − 0.1095 2x 2 3 − 0.00001x24 + 0.00189x1x2 − 0.00309x1x3 + 0.00021x1x4 − 0.00071x2x3 − 0.00001x2x4 − 0.00004x3x4 anova was performed with f1 = mean square loss/ mean square error and f2 = mean square regression/ mean square residual. as demonstrated in table 5, flf = f1 = 0.3916 < f0.1(10,11) = 2.25 and the p-value was higher than 0.05 (0.9245), thereby suggesting that the lack of fit was not significant, that is, the regression equation fitted all the test points well, and no other unknown factors affected the results. fregression = f2 = 11.2178 > f0.01 (14,21) = 3.03 and p = 0 < 0. 01, thereby indicating that the regression equation was extremely significant, that is, the quadratic regression model was suitable. among them, the influences of stir-baked temperature (x2) and all quadratic terms on ci were extremely significant at a = 0.01, stir-baked time (x3) and stir-baked speed (x4) were significant at a = 0.05, and the stir-baked volume (x1) was significant at a = 0.1. however, all interaction terms were not significant. therefore, at the significant level of a = 0.1, the regression equation was simplified by eliminating the nonsignificant terms as follows: y = −19.80272 + 0.64512x1 + 0.25508x2 + 0.98855x3 + 0.00966x4 − 0.11937x 2 1 − 0.00106x 2 2 − 0.1095 2x 2 3 − 0.00001x24 the p-value influenced by the main effect of each factor can reflect the importance of each factor to the test index. the smaller the p-value, the greater the influence of the factor on test result, that is, the greater will be the importance (sun et al., 2016). as demonstrated in table 5, the primary and secondary influences of the four factors on ci followed the following order: stir-baked temperature (x2) > stir-baked time (x3) > stir-baked rotation speed (x4) > stir-baked amount (x1). the stir-baked temperature had a significant influence on ci, followed by the stirbaked time, and the effect of stir-baked rotation speed was relatively small, but all reached a significant level (p < 0.05), and the stir-baked amount had no significant effect on experimental results (p > 0.05). the relationship between various factors and ci is demonstrated in figure 1. as demonstrated in figure 1, the relationship between stir-baked amount (x1) and ci was nearly linear, thereby indicating that the stir-baked amount had minimal influence on ci. the three other factors (i.e., x2, x3, and x4) also had the same trend with increase in temperature, time, and rotation speed. ci established an upward trend, among which the changed trend of temperature was the most evident one, thereby indicating that temperature had the most significant influence on ci. the frequency analysis method was used to find the good stir-baked conditions, and the frequency analysis results are demonstrated in table 6. as demonstrated in table 6, in the 95% confidence interval (ci), the average of ci was more than 0.50. the optimized stir-baked scheme is as follows: stir-baked amount = 3.9 kg, stir-baked temperature = 121°c, stir-baked time after reaching the target temperature = 3.9 italian journal of food science, 2021; 33 (1) 89 stir-baked technology and quality evaluation of fsi tea ta bl e 3. r es ul ts o f si ng le -f ac to r te st . fa ct or p ar am et er c on te nt (% ) s en so ry -s co re c i r an k w at er ex tr ac t to ta l po ly sa cc ha ri de s to ta l fla vo no id s r ut in n ar ci ss os id e q ue rc et in is or ha m ne tin a m ou nt (k g) 2 32 .3 0 1. 18 31 .4 1 25 .9 9 0. 79 0. 49 0. 34 56 0. 04 5 3 31 .6 3 1. 89 32 .7 0 26 .7 5 0. 88 0. 69 0. 35 70 0. 74 2 4 35 .3 9 1. 89 32 .8 9 27 .5 7 0. 93 0. 69 0. 35 83 0. 99 1 5 32 .4 7 1. 74 32 .9 7 27 .5 4 0. 91 0. 49 0. 34 79 0. 57 3 6 30 .8 5 1. 88 32 .5 3 26 .4 4 0. 81 0. 50 0. 33 72 0. 51 4 t em pe ra tu re (℃ ) 80 30 .9 8 1. 66 30 .2 4 26 .1 0 0. 86 0. 64 0. 33 49 0. 35 4 10 0 31 .6 3 1. 89 32 .7 0 26 .7 5 0. 88 0. 69 0. 35 70 0. 79 3 12 0 39 .9 2 2. 03 33 .1 2 27 .2 8 0. 88 0. 69 0. 36 75 0. 90 1 14 0 35 .0 8 2. 14 34 .4 8 26 .6 7 0. 85 0. 69 0. 34 65 0. 79 2 16 0 33 .7 2 1. 89 30 .9 2 25 .5 8 0. 81 0. 63 0. 33 32 0. 15 5 r ot at e sp ee d (r /m in ) 20 0 29 .3 1 1. 68 26 .0 8 27 .4 2 0. 81 0. 49 0. 28 60 0. 04 5 30 0 31 .6 3 1. 89 32 .7 0 26 .7 5 0. 88 0. 69 0. 35 70 0. 67 4 40 0 38 .2 1 1. 78 32 .4 4 26 .1 7 0. 87 0. 62 0. 38 78 0. 91 1 50 0 38 .7 0 1. 50 35 .0 6 26 .1 6 0. 88 0. 61 0. 35 78 0. 73 2 60 0 38 .1 5 1. 24 34 .4 4 25 .4 7 0. 85 0. 59 0. 34 79 0. 68 3 ti m e (m in ) 1 31 .7 3 2. 12 32 .0 0 26 .0 1 0. 88 0. 58 0. 30 37 0. 30 6 2 33 .9 9 1. 63 32 .1 8 26 .5 1 0. 89 0. 59 0. 32 50 0. 31 5 3 31 .6 3 1. 89 32 .7 0 26 .7 5 0. 88 0. 69 0. 35 70 0. 72 3 4 39 .6 2 1. 90 34 .4 8 27 .4 0 0. 92 0. 63 0. 38 78 0. 84 1 5 34 .3 7 1. 94 34 .3 2 25 .6 2 0. 87 0. 60 0. 35 73 0. 75 2 6 33 .8 4 1. 66 32 .7 4 26 .2 4 0. 79 0. 47 0. 33 69 0. 55 4 n ot e: d i+ is th e op tim al v ec to r d is ta nc e; d i– is th e di st an ce o f th e w or st v ec to r; c i is th e re la tiv e pr ox im ity o f op tim al v al ue . 90 italian journal of food science, 2021; 33 (1) shang f.h. et al. ta bl e 4. e xp er im en ta l d es ig n an d re su lts o f qu ad ri c re gr es si on o rt ho go na l r ot at io n co m bi na tio n w ith fo ur fa ct or s an d fiv e le ve ls . te st n um be r x 1 x 2 x 3 x 4 c on te nt d et er m in at io n (% ) s en so ry s co re c i w at er ex tr ac t to ta l po ly sa cc ha ri de s to ta l fla vo no id s r ut in n ar ci ss os id e q ue rc et in is or ha m ne tin 1 1 1 1 1 33 .8 0 1. 67 38 .6 8 26 .8 8 1. 03 0. 56 0. 33 82 0. 28 2 1 1 1 –1 34 .5 5 1. 34 38 .4 3 31 .1 6 1. 00 0. 53 0. 29 81 0. 20 3 1 1 –1 1 47 .0 2 1. 26 34 .8 1 29 .8 3 0. 98 0. 54 0. 32 79 0. 27 4 1 1 –1 –1 38 .2 0 1. 58 33 .3 1 28 .4 0. 96 0. 55 0. 33 75 0. 23 5 1 –1 1 1 37 .0 0 1. 31 33 .7 5 27 .6 1 0. 94 0. 47 0. 30 72 0. 16 6 1 –1 1 –1 35 .7 4 1. 45 33 .2 1 26 .7 9 0. 97 0. 47 0. 32 80 0. 19 7 1 –1 –1 1 35 .5 8 1. 73 30 .6 7 27 .3 4 0. 95 0. 5 0. 31 78 0. 24 8 1 –1 –1 –1 35 .8 5 1. 25 34 .4 2 28 .0 8 0. 96 0. 44 0. 28 77 0. 15 9 –1 1 1 1 39 .1 6 1. 42 37 .1 5 27 .3 8 0. 96 0. 43 0. 28 81 0. 21 10 –1 1 1 –1 34 .9 9 1. 81 38 .1 8 30 .5 3 1. 07 0. 42 0. 27 69 0. 30 11 –1 1 –1 1 35 .5 3 1. 43 34 .7 1 30 .7 2 1. 10 0. 44 0. 28 80 0. 23 12 –1 1 –1 –1 36 .8 3 1. 87 40 .8 6 30 .8 8 1. 10 0. 47 0. 29 81 0. 35 13 –1 –1 1 1 34 .5 2 1. 8 38 .2 4 31 .0 1 1. 08 0. 45 0. 28 76 0. 30 14 –1 –1 1 –1 33 .2 5 1. 74 37 .4 30 .5 6 1. 10 0. 48 0. 30 75 0. 30 15 –1 –1 –1 1 28 .1 0 1. 69 38 .0 4 29 .8 2 1. 12 0. 47 0. 30 70 0. 28 16 –1 –1 –1 –1 30 .3 4 1. 58 38 .4 1 31 .7 7 1. 10 0. 45 0. 29 79 0. 26 17 –2 0 0 0 28 .9 6 1. 31 39 .1 3 31 .5 4 1. 07 0. 46 0. 29 81 0. 21 18 2 0 0 0 31 .5 1 1. 97 41 .8 8 31 .8 5 1. 09 0. 43 0. 27 78 0. 35 19 0 –2 0 0 34 .2 7 1. 99 40 .4 4 30 .3 3 1. 08 0. 44 0. 28 70 0. 35 20 0 2 0 0 34 .4 0 1. 81 41 .0 1 31 .9 7 1. 09 0. 42 0. 27 71 0. 31 21 0 0 –2 0 31 .7 0 1. 67 41 .8 8 32 .0 9 1. 15 0. 40 0. 26 78 0. 31 22 0 0 2 0 34 .3 0 1. 86 40 .4 7 28 .4 9 1. 06 0. 53 0. 31 75 0. 33 23 0 0 0 –2 37 .2 2 1. 9 37 .3 3 27 .1 2 0. 83 0. 68 0. 35 73 0. 30 24 0 0 0 2 38 .8 6 1. 84 34 .8 8 26 .4 4 0. 83 0. 73 0. 37 78 0. 31 25 0 0 0 0 38 .6 2 1. 91 39 .9 4 29 .1 8 1. 28 4 0. 73 0. 37 83 0. 49 26 0 0 0 0 37 .3 8 1. 96 38 .1 7 30 .5 2 1. 18 0. 64 0. 40 88 0. 44 (c on tin ue s) italian journal of food science, 2021; 33 (1) 91 stir-baked technology and quality evaluation of fsi tea ta bl e 4. c on tin ue d te st n um be r x 1 x 2 x 3 x 4 c on te nt d et er m in at io n (% ) s en so ry s co re c i w at er ex tr ac t to ta l po ly sa cc ha ri de s to ta l fla vo no id s r ut in n ar ci ss os id e q ue rc et in is or ha m ne tin 27 0 0 0 0 39 .9 3 1. 83 44 .6 2 30 .7 9 1. 94 0. 58 0. 37 85 0. 73 28 0 0 0 0 39 .5 6 1. 91 42 .9 35 .6 7 1. 97 0. 58 0. 37 89 0. 74 29 0 0 0 0 38 .8 0 1. 97 39 .2 1 30 .4 4 1. 97 0. 58 0. 37 80 0. 73 30 0 0 0 0 37 .2 0 1. 92 5 43 .3 8 29 .3 4 1. 97 0. 59 0. 37 85 0. 73 31 0 0 0 0 38 .5 8 2. 15 44 .8 4 31 .3 1. 96 0. 62 0. 39 81 0. 77 32 0 0 0 0 38 .6 8 1. 89 44 .1 9 33 .1 7 1. 77 0. 94 0. 49 82 0. 79 33 0 0 0 0 37 .6 2 1. 96 41 .9 8 34 .2 2 1. 74 0. 99 0. 52 83 0. 78 34 0 0 0 0 38 .3 3 1. 96 39 .9 7 30 .0 8 1. 74 0. 93 0. 47 81 0. 77 35 0 0 0 0 39 .1 3 2. 06 43 .6 9 31 .0 1 1. 85 0. 74 0. 44 80 0. 80 36 0 0 0 0 37 .8 5 2. 16 42 .0 4 30 .0 3 1. 78 0. 93 0. 49 80 0. 81 92 italian journal of food science, 2021; 33 (1) shang f.h. et al. table 5. anova table of regression model. sources of variation sum of squares degrees of freedom average square standard regression coefficient f-values p-values x 1 9.9885 1 9.9885 2.3281 1.7559 0.0937 x 2 156.1570 1 156.1570 9.2051 5.4658 0 x 3 23.4535 1 23.4535 3.5674 2.6906 0.0137 x 4 22.4100 1 22.4100 3.4871 2.6301 0.0156 x 1 2 22.3462 1 22.3462 –3.4822 6.6377 0 x 2 2 156.1020 1 156.1020 –9.2035 5.9017 0 x 3 2 18.8064 1 18.8064 –3.1945 6.0894 0 x 4 2 19.8862 1 19.8862 –3.2849 6.2617 0 x 1 x 2 1.3810 1 1.3810 0.8657 0.7441 0.4650 x 1 x 3 0.0075 1 0.0075 –0.0637 0.1214 0.9045 x 1 x 4 0.3599 1 0.3599 0.4419 0.8424 0.4091 x 2 x 3 0.1944 1 0.1944 –0.3248 0.2791 0.7829 x 2 x 4 0.4848 1 0.4850 –0.5129 0.4408 0.6638 x 3 x 4 0.0133 1 0.0133 –0.0851 0.1622 0.8727 regression 14 0.1161 f 2 = 11.2178 0 surplus 21 0.0103 loss of quasi 10 0.0057 f 1 = 0.3916 0.9245 error 11 0.0146 sum 35 x1 stir-baked amount x2 stir-baked temperature x3 stir-baked time x4 stir-baked rotation speed –2 –22.5 –22 –21.5 –21 –20.5 –20c i –19.5 –19 –18.5 –18 –1.5 –0.5 level of factor 0.5 1.5–1 0 1 2 figure 1. influence of various factors on comprehensive vector distance (ci). min, and stir-baked rotation speed = 393 r/min. however, considering the practical operability of the equipment and production, the optimization scheme was appropriately modified. the modification scheme is as follows: stir-baked amount = 3.9 kg, stir-baked temperature w= 120°c, stirbaked time after reaching the target temperature = 3.9 min, and stir-baked rotation speed = 400 r/min. study on taste blending of fsit in section 2.2.4, we obtained the best stir-baked process parameters, that is, the stir-baked amount was 3.9 kg, the stir-baked rotation speed was 400 r/min, and the stirbaked time after reaching 120°c was 5 min; 1% stevia glycoside of different volumes was added, and the stir-baked italian journal of food science, 2021; 33 (1) 93 stir-baked technology and quality evaluation of fsi tea table 6. value frequency distribution of stir-baked amount (x 1 ), temperature (x 2 ), time (x 3 ), and rotation speed (x 4 ). level x 1 (kg) x 2 (°c) x 3 (min) x 4 (r/min) number frequency number frequency number frequency number frequency –2 0 0 0 0 0 0 0 0 –1 3 0.2 3 0.2 2 0.13 3 0.2 0 10 0.67 8 0.53 12 0.8 10 0.67 1 2 0.13 4 0.27 1 0.067 2 0.13 2 0 0 0 0 0 0 0 0 95% confidence interval (ci) –0.3569~0.2236 –0.2774~0.4107 –0.2905~0.1571 –0.3569~0.2236 stir-baked conditions 3.63~4.22 117.22~124.11 3.71~4.16 364.31~422.36 average 3.9 121 3.9 393 was conducted for 3.9 min. according to the results of scoring, the product obtained by adding 15 ml of 1% stevia glycoside tasted good. therefore, the result of the taste blending scheme of fsit is as follows: 15 ml of 1% stevia glycoside was added per 3.9 kg of flos sophorae immaturus. study on quality of fsit the sensory evaluation of fsit was conducted according to the requirements of sensory indices of the national industry standard of substitute tea. we observed the appearance of the obtained tea: the color was burnt yellow, the grain was full, the texture was firm and crisp, and the smell had a strong coke flavor. under natural light, the soup of fsit was clear and golden in color; had no turbidity; had no scattered particles; had no astringency; and had charred taste through watching, smelling, and tasting. the physicochemical and microbial indicators of fsit were determined according to the requirements of the chinese pharmacopoeia and the national industry standard on physical and chemical indices of substitute tea and microbiological indicators on food microbial national standards. the results are demonstrated in table 7; all the indicators complied with the relevant provisions of the industry standards, and the contents of all kinds of microorganisms did not exceed the values specified in the national standards. discussion in this experiment, the sensory and chemical indices of the multicomponent content were used to: evaluate the stir-baked process of the new mechanism of fsit comprehensively; optimize the best process; avoid the limitation and inaccuracy caused by the evaluation of single index; make the stir-baked process of fsit reasonable; table 7. physical and chemical indices of fsit. project index measured value total flavonoids (%) >20 36.87 ± 0.38 rutin (%) >15 29.49 ± 0.72 moisture (%) ≤13 2.43 ± 0.25 total ash (%) ≤12 5.23 ± 0.25 aflatoxin b1 (µg·kg -1) ≤5 3.20 ± 0.10 lead (mg·kg-1) ≤5 0.8 ± 0.10 total arsenic (mg·kg-1) ≤0.5 0.13 ± 0.06 cadmium (mg·kg-1) ≤0.5 0.26 ± 0.06 total number of colonies (cfu·g-1) ≤30,000 17,000 coliform (mpn·g–1) ≤30 15 staphylococcus aureus not detected not detected salmonella not detected not detected shigella not detected not detected and perfect the quality evaluation system. for the selection of the content evaluation indices, water extract was the general term of soluble substances that could be dissolved in hot water in the tea soup and a comprehensive index that represented the overall level of flavor components in the tea soup (liu et al., 2014). flavonoids could not only enhance the capillary and has anti-inflammatory, antispasmodic, antiulcer, hypolipidemic, and other pharmacological effects but could also cool the blood to stop bleeding, clear the liver, and purge fire and heat. flavonoids are one of the substances with evident biological activity and physiological functioning in fsit (liu et  al., 2018). although the total content of polysaccharides in flos sophorae immaturus is not high but has anti-inflammatory and antiviral effects, maintains vascular resistance, is rouge, and inhibits aldose reductase. the total content of polysaccharides is also one of the basic substances involved in life activities (zhou and xia 2011). the single-factor experiment selects ppc weight analysis combined with topsis as a comprehensive evaluation 94 italian journal of food science, 2021; 33 (1) shang f.h. et al. conclusions the combined ppc-topsis method with the quadratic orthogonal rotation combination design method was used to investigate the four factors, namely, stir-baked amount, temperature, time, and rotation speed, of the machine-made fsit. the contents of seven components and sensory scores were taken as inspection indices to study the key technology of stir-baked fsit, and the optimum stir-baked conditions are as follows: stir-baked amount: 3.9 kg, stir-baked rotation speed: 400 r/min, and time needed to reach the temperature of 120°c: 5 min and maintained for 3.9 min after adding 15 ml of 1% stevioside. the technology had high stability and simple operation, and the results established that the machine-made fsit soup was clear and golden in color, with charred taste, no bitterness, no peculiar smell, and improved sensory quality under the above-mentioned conditions. the sensory, that is, physical and chemical indicators, and microbial indicators agreed with relevant national standards, and the technical standards for the production of stir-baked fsit could be formulated accordingly. this research provides additional insight into the types of substitute teas and technical support for enterprises to develop the stir-baked technology of fsit for industrial application, which has practical guiding significance. acknowledgments this work was supported by the national industry technical system of tradition chinese medicine (grant no. cars-21), the forestry key technology r&d project of chongqing (grant no. 2016-14), the industry technical system of tradition chinese medicine of chongqing (grant no. 2017-[5]), the natural science foundation project of chongqing (grant no. cstc2018jcyjax0669, cstc2020jcyj-msxmx0828), and the fundamental research funds of chongqing (grant no. cstc2018jxjl-jbky120002). the authors also thank liu y for valuable discussions and helpful suggestions. references chua, l.s., 2013. a review on plant-based rutin extraction methods and its pharmacological activities. journal of ethnopharmacology 150: 805–817. https://doi.org/10.1016/j. jep.2013.10.036 he, x.r., bai, y.j. and zhao z.f., 2016. local and traditional uses, phytochemistry, and pharmacology of sophora japonica l.: a review. journal of ethnopharmacology 187: 160–182. https:// doi.org/10.1016/j.jep.2016.04.014 hwang, c. l. and yoon, k. p., 1981. multiple attribute decision making: methods and applications. new york: springer-verlag. method for initial screening and stir-baked conditions. given the difference in the contribution of sensory and chemical indices in evaluating the quality of fsit in the stir-baked process, the direct use of subjective valuation method to determine the weight coefficient is biased. the ppc method in dps software is a relatively objective weight assignment method that avoids artificial interference factors of expert grading and eliminates grading steps. therefore, single-factor experiment is more scientific, reasonable, and advantageous than the subjective weight assignment method. the topsis method could provide effective solutions for the optimization of different evaluation indices and comprehensive evaluation of target groups (ning et al., 2018) and simplify the statistical analysis of multi-index variable data. the topsis method could be used as an auxiliary analysis method to compare different cooking conditions of flos sophorae immaturus in improving work efficiency and accuracy. orthogonal and uniform 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http://dx.doi.org/10.1590/s0102-695x2012005000043 http://dx.doi.org/10.19664/j.cnki.1002-2392.2017.03.035 http://dx.doi.org/10.19664/j.cnki.1002-2392.2017.03.035 https://doi.org/10.3390/molecules21030296� https://doi.org/10.7501/j.issn.0253-2670.2018.19.027 http://dx.doi.org/10.7506/spkx1002-6630-201402031 http://dx.doi.org/10.3969/j.issn.1002-2481.2018.12.04 ole_link11 ole_link13 ole_link27 ole_link16 ole_link17 ole_link14 ole_link12 ole_link18 ole_link21 ole_link22 ole_link23 ole_link24 ole_link25 ole_link26 ole_link29 ole_link28 ijfs#1470_bozza ital. j. food sci., vol. 31, 2019 669 paper chemical-nutritional composition, microbiological analysis and volatile compound content of fossa cheese ripened in different pits f. siano1, g. fasulo2, l. giaramita3, a. sorrentino1, f. boscaino1, m. sprovieri3, m. di stasio1, r. coccioni4 and m.g. volpe*1 1istituto di scienze dell’alimentazione, consiglio nazionale delle ricerche (cnr), via roma 64, 83100 avellino, italy 2besana spa, via ferrovia 210, 80040 san gennaro vesuviano (na), italy 3istituto per l'ambiente marino costiero uos capo granitola, consiglio nazionale delle ricerche (cnr), via del mare 3, 91021 campobello di mazara (tp), italy 4dipartimento di scienze pure ed applicate, università degli studi di urbino “carlo bo”, campus scientifico, località crocicchia, 61029 urbino, italy *corresponding author: tel.: +39 0825299513; fax: +39 0825781585 e-mail address: mgvolpe@isa.cnr.it abstract fossa cheese samples were ripened for 90 days in two different pits and analysed to evaluate the influence of the environment on the chemical and nutritional characteristics. the significant changes were recorded only for certain parameters, particularly the contents of fatty acids and volatile molecules. in the fatty acid profiles, the sum of monounsaturated fatty acids showed a significant decrease in the mature cheeses due to a strong decrease in oleic acid. even the sum of polyunsaturated fatty acids and the ratio between the sums of saturated fatty acids and polyunsaturated fatty acids decreased after ripening in both pits. hp-spme-gc/ms analysis allowed the identification of 77 volatile compounds that increased in the cheese samples during ripening. the results of this study indicated that there are substantial differences between the chemical and chemical/physical parameters, and certain fatty acids of the just curdled cheese samples and the cheese ripened in the two pits showed different geologicalgeochemical parameters. keywords: fossa cheese, microclimate conditions, cheese composition, microbiological analysis, volatile compounds ital. j. food sci., vol. 31, 2019 670 1. introduction fossa cheese, literally pit cheese, is a typical italian product of the montefeltro area, specifically from talamello, sogliano al rubicone and other towns located in a small geographical area (the emilia romagna and marche regions of central italy) (gobbetti et al., 1999; barbier et al. 2012). the cheese is variously produced from sheep, bovine or a mix of sheep and bovine milk; it is a hard cheese, produced in limited quantities (approximately 200 tons/year), and it has great economic importance in its market niche. the first phase of maturation takes place at dairy farms for a period of approximately 60 days at 6-14°c and a relative humidity of 7592 %, after which it is aged for a period of 90-100 days in pits dug in tuffaceous rock sanitized by fire and smoke. wooden boards are laid on the bottom of the pit forming a floor, and the pit walls are lined with a 15-20-cm-thick layer of straw before the cheeses are placed inside. the pits are then filled with the cheeses and hermetically sealed from august to november (avellini et al., 1999). the denomination of protected origin "formaggio di fossa di sogliano al rubicone and talamello" is reserved for cheese that meets the requirements of this specification. when put on the market, the “fossa cheese from sogliano al rubicone and talamello” dop has the following characteristics: the colour of the outer part of the finished product varies from ivory white to amber yellow. at the end of ripening, the cheese exhibits irregular forms, with typical bumps and depressions. the cheese surface is primarily wet and greasy and, in some cases, may be covered by butterfat and mould that is easily scraped off. the presence of small cracks and possible yellow ochre stains, more or less intense, on the surface, fits all the characteristics of the product. a skin is absent or barely visible. the internal consistency is easily friable, with a white or slightly yellow amber colour. the smell is typical and lingering, sometimes intense, with a rich aroma reminiscent of woodland undergrowth with hints of mould and truffles. the aging process gives the product its unique, highly appreciated flavour, which is different from that of cheeses not aged in pits. the geological characteristics of the pits play a key role in the process of cheese ripening and affect the quality. the present study aimed to test the influence of the geological-geochemical nature of two different pits on the chemical, microbiological, nutritional and olfactory properties of a product in the italian culinary tradition, very peculiar in its organoleptic characteristics, which are intimately related to the process of ripening (gobbetti et al., 1999). the two geographical areas in which cheese ripening took place were those of talamello (province of rimini, emilia-romagna region) and cartoceto (pesaro-urbino marche region). the first area is characterized by soils composed of bipolar cross-laminated sandstonetype mediumto coarse-grained “fishbone” material, belonging to the formation of “arenarie di monte perticara” (pliocene). the second area is characterized by soils composed of very thick arenaceous layers intercalated with thin pelitic layers belonging to “formazione a colombacci” (miocene superiore). ital. j. food sci., vol. 31, 2019 671 2. material and methods 2.1. cheese samples the fossa cheese was produced in a single cheesemaking process (valmetauro fattorie marchigiane amandola, fm, italy) at the same dairy farm and ripened for 60 days. after this period, two italian companies located in emilia romagna (talamello) and marche (cartoceto) provided the cheese ripening environment as imposed by the consortium of fossa cheese regulations (gobbetti et al., 1999); the samples previously ripened at the dairy farm were equally distributed in the companies’ respective pits for 90 days. the analyses were performed in triplicate on each different batch consisting of three distinct samples. in this paper, the following samples were analysed: sheep milk, cheese after curdling, cheese ripened at the dairy farm for sixty days and after ripening in two different pits for ninety days. all samples were homogenized with a laboratory mixer (msm87160 maxomixx, bosch gmbh, germany) before being subjected to the chemical analyses. all analyses were performed in triplicate. 2.2. microclimate measurements microclimate measurements were carried out using relative humidity and temperature sensors (wm33 and 52, michell instruments) located at the surface and the bottom of each pit. a digital signal converter and a pc transformed the sensor signal to be compatible with a specific software program developed to acquire and store data. to analyse the microclimate dynamics inside the pits regularly, the downloaded data were controlled weekly using a umts/gprs modem and the remote control software “teamviewer”. 2.3. chemical-physical analysis 2.3.1 dry matter method the free water content in the samples was determined by drying an aliquot (5 g) of the sample to a constant weight in an oven at 105°c. the weight loss corresponded to the loss of moisture. the result was expressed as a percentage (aoac, 1990). 2.3.2 ash methods the sample (approximately 2 g) was dried in an oven at 100°c and thereafter calcinated in a muffle at 525°c; the weight obtained after calcination was the ash content (aoac, 1990). 2.3.3 ph determination the ph determination was performed by potentiometric analysis. the ph of the milk was measured without dilution; regarding the cheese samples, 100 ml of distilled water previously brought to a boil was added to 10 g of cheese, and the mixture was vortexed on a magnetic plate for 15 min. the mixture was centrifuged for 5 min and left to decant to separate the supernatant. finally, the ph of the supernatant was measured (aoac, 1990). ital. j. food sci., vol. 31, 2019 672 2.4. determination of the total protein content the total nitrogen content of the samples was determined with the kjeldahl method (aoac, 1990). one gram of homogenized sample was digested inside an appropriate reaction tube with 10 ml of sulfuric acid (96 %), 5 ml of hydrogen peroxide (30 %) and a catalyst based on copper sulphate pentahydrate and heated at a high temperature (250°c) to destroy all the organic material. afterwards, 50 ml of distilled water and 50 ml of sodium hydroxide (30 %) were added. adding an excess of sodium hydroxide solution, the ammonium ions were released in the form of ammonia, distilled and added to a boric acid solution. the ammonia content was determined with a volumetric acid solution or by back titration with a sodium hydroxide solution of a known concentration. the percentage of total protein was calculated using a conversion factor of 6.38. 2.5. determination of the sodium chloride content (the mohr method) approximately 2 g of the dried (in an oven at 105°c until constant weight) sample (cheese and milk) was added to 40 ml of bi-distilled water for 2 h under stirring at room temperature, followed by centrifugation for 10 min at 2683 g, and finally filtered. the ph of the solution was adjusted with 0.1 n sodium hydroxide up to a value of 8.0. twenty millilitres of distilled water containing 5 drops of a 5 % k2cro4 indicator was added to the mixture, which was then titrated with 0.1 n silver nitrate until the colour changed (from white to brick red). twenty millilitres of sample with a few drops of indicator was titrated with silver nitrate until the colour changed from yellow to red brick (johnson and olson, 1985). when the silver chloride was completely precipitated, the excess of titrant formed a silver chromate precipitate, which indicated the end point. 2.6. extraction of the total lipids from milk after vortexing for 90 seconds, 300 ml of a solution composed of a dichloromethane:ethanol mix in a 2:1 ratio (v/v) was added to 30 g of sample, and the mixture was centrifuged for 10 min at 2683 g. the supernatant was removed, and the extraction was repeated twice (stefanov et al., 2010). the lower organic phase was recovered and filtered into a round bottom flask, and the dichloromethane was removed using a rotary evaporator at 35°c (model hei-vap value; heidolph, schwabach, germany). the residue was placed in a drier and regularly weighed until a constant value was reached. 2.7. extraction of the total lipids from cheese forty millilitres of hydrochloric acid (25 %) and 40 ml of ethanol (95 %) were added at approximately 12 g of sample. the mixture was stirred for 30' in a water bath at 50°c. after cooling, 100 ml of a solution of n-heptane:diethyl ether (1:2, v/v) was added, and the mixture was maintained under stirring for 15 min at room temperature. then, the mixture was allowed to decant, and the supernatant (organic phase) was recovered. the extraction procedure was carried out three times, and the supernatant was gathered. the solvent was removed by a rotary evaporator (model hei-vap value; ital. j. food sci., vol. 31, 2019 673 heidolph, schwabach, germany) (romano et al., 2011). the residue was placed in a drier and regularly weighed until a constant value was reached. 2.8. fatty acid methyl ester analysis fatty acid methyl esters (fames) were prepared according to aoac 996.06 (2011). briefly, to 200 mg of a lipid extract, 2 ml of a 1.25 m hcl/ch3oh solution was added and the mixture was heated for 60 min at 90°c. then, the methyl esters were extracted with 1 ml of n-hexane and 1 µl of the methyl esters was injected in a trace gc ultra gas chromatograph (thermo scientific, waltham, ma, usa) equipped with a flame ionization detector (fid) and sp-2560 capillary column (100 m × 0.25 mm × 0.20 μm, supelco, bellefonte, pa, usa). helium was used as the carrier gas with a constant flow rate of 1.5 ml min-1. the samples were introduced with a split-splitless injection system in split mode (ratio 1:100) using an as 3000 autosampler (thermo scientific, waltham, ma, usa). the operating conditions that were followed corresponded to those observed by siano et al. (2016). the ramp started at a temperature of 140°c, which was stabilized for 5 min; the temperature was increased at a rate of 4°c per minute up to a temperature of 240°c for 15 min. the run lasted 45 min. the temperature of the injector and detector was 260°c. to perform the qualitative and quantitative analysis, the retention times of the fatty acids detected in the cheese samples were compared with those of a mixture of fatty acid methyl esters (fame mix-37, supelco, bellefonte, pa, usa). 2.9. microbiological analysis a microbial analysis of the milk used in the fossa cheesemaking process was not performed because, according to the production regulations, the milk had been pasteurized at 72°c for 15 min, cooled, inoculated with the selected starter and added to the rennet calf powder. under sterile conditions, 10 g of each cheese sample was placed in sterile stomacher bags, 90 ml of a sterile peptone-saline solution (bacteriological peptone 0.1 %; nacl 0.85 %) was added, and the mixture was homogenized in a stomacher apparatus (lab-blender 400, pbi international, italy). the homogenates were serially diluted 10-fold. to count the bacterial population, the following media, and temperature and time conditions of incubation were used. one millilitre of each dilution was inoculated on mrs agar and m17 agar plates (oxoid, thermo fisher, italy), incubated under anaerobic conditions (anaerogen, oxoid, thermo fisher, italy) at 28°c for 72 h lactobacillus and lactococcus spp. in plate count agar (pca) (oxoid, thermo fisher, italy) and incubated at 28°c for 72 h, to enumerate the total microbial mesophilic bacteria (tmc). the sulphite-reducing clostridia (srcs) on sps agar plates incubated under anaerobic conditions at 28°c for 5 days were counted; the total and faecal coliforms were evaluated on violet red bile glucose agar and violet red bile lactose agar (oxoid, thermo fisher, italy) after incubation at 36 and 44°c, respectively, for 48 h. in addition, 100 µl of the diluted solution was streaked onto mannitol salt agar plates (oxoid, thermo fisher, italy) and incubated at 28°c for 3-5 days to count micrococcaceae and on ypd plates (yeast extract 1 %; bacteriological peptone 2 %; dextrose 2 %; and agar 2 %), incubated at 28°c for 3-5 days, for the detection of yeasts and moulds. to evaluate the bacterial load of the fossa cheese, the plates that contained between 15 and ital. j. food sci., vol. 31, 2019 674 300 colonies were counted. the values obtained were expressed as the colony forming units of a gram of sample (cfu/g). the microbial counts were carried out in triplicate. 2.10. determination of the profile of volatile molecules the extraction of volatile compounds was carried out using the headspace solid-phase microextraction technique (hs-spme) combined with gas chromatography paired with mass spectrometry (hs-spme-gc/ms). five grams of sample (pre-equilibrated to 45°c for 10 min) was weighed in vials of 20 ml containing 5 μl of 3-octanol (internal standard, 100 mg/l standard solution) and the volatile compounds (vocs) were extracted from the samples by a fibre dvb/car/pdms; the compounds were held for 45 min and block heated to 45°c in the headspace of the sample. the analysis of the volatile compounds was performed using an agilent 7890a/5975c gc/ms with a gerstel mps2 autosampler, using an innowax capillary column (30 m × 0.25 mm × 0.50 μm) and the following temperature programme: 40°c for 2 min, 5°c/min to 230°c for 10 min. the injector, quadrupole, source and transfer line temperatures were 240, 150, 230 and 200°c, respectively. the electron ionization mass spectra in full-scan mode were recorded at an electron energy level of 70 ev in the range of 20-400 amu (2 s/scan). the volatile molecules were identified by comparing the recorded values to the mass spectra present in the wiley 07/nist 98 libraries and the retention index present in the database or in the literature. afterwards, to calculate the ri value of the compounds, the n-alkanes (c5-c25) were also analysed under the same conditions using gc-ms (van den dool and kratz, 1963). the results were expressed as the relative peak area (rap) with respect to the internal standard. 2.11. statistical analysis the experimental data (the moisture content, ash, ph, lipid content and sodium chloride concentration) were statistically analysed using statistica software version 10 (statsoft, tulsa, usa). one-way repeated measures analysis of variance (rm anova) was used to estimate the significant differences during the manufacture and ripening of the cheeses. to isolate the group or groups that differed from the others, multiple comparisons versus control group (the holm-sidak method) were used. anova followed by kruskal-wallis one-way analysis of variance on ranks was used to estimate the differences in the fatty acid content (p < 0.05). the averages and the standard deviations were calculated with microsoft office excel 2016. 3. results and discussion 3.1. microclimate measurements microclimate measurements were performed during the cheese ripening in both pits (78 days, in the talamello pit and in cartoceto pit). the results are shown in figs. 1 and 2. different microclimate characteristics were found between the pits. in talamello, the pit temperature remained constant over time at both depths (18°c at the surface and 20-21°c at the bottom), while in the cartoceto pit, the temperature increased over time (increasing from 12°c to 23°c at the surface and from 17°c to 27°c at the bottom). on the other hand, the relative humidity was variable at all depths and in both pits. in the talamello pit, the ital. j. food sci., vol. 31, 2019 675 surface values were in the range of 88 to 99 % and the bottom values varied between 85 and 90 %, whereas in the cartoceto pit, the relative humidity ranged between 90 and 99 % at the surface and between 80 and 88 % at the bottom. figure 1. trend of the temperature and humidity inside the talamello pit. figure 2. trend of the temperature and humidity inside the cartoceto pit. 3.2. chemical-nutritional composition the average values (± sd) of the ph, the moisture, lipid, and protein content, and the ash and salt concentration of the fossa cheese samples are shown in table 1. rm anova showed significant differences between treatments (f = 18.642 with 3 degrees of freedom, p = 0.004). multiple comparisons versus control group (the holm-sidak method) showed significant differences in the comparison of “cheese ripened at the dairy farm for sixty days” versus “cheese from the talamello site” (p = 0.004) and “cheese ripened at the dairy farm for sixty days” versus “cheese from the cartoceto site” (p = 0.027); in contrast, there was no statistically significant difference in the comparison between “cheese from the talamello site” versus “cheese from the cartoceto site” (p > 0.05). ital. j. food sci., vol. 31, 2019 676 the ph of the milk was 6.51, while the ph of the cheese curds decreased to 5.43, a value similar to that of the talamello-ripened cheese and slightly different from that of the cartoceto-ripened cheese (5.19). the protein, lipid, ash and nacl content of the cheese after ripening in the two pits increased significantly with respect to the cheese curd values, but no significant differences were recorded between the ripened cheese in the two pits and the cheese ripened at the dairy farm for sixty days. table 1. the gross composition of the fossa cheese during production and ripening. milk cheese after curdling cheese ripened at the dairy farm for sixty days cheese from the talamello site cheese from the cartoceto site statistical significanc e ph 6.51±0.02 5.43±0.04 5.42±0.01 5.42±0.27 5.19±0.15 ns moisture (%) 83.02±4.98 42.33±1.52 a 35.46±1.28 b 34.33±6.71 b 32.59±3.81 c ** lipid1 (%) 33.92±5.71 49.26±2.75 a 52.13±4.19 b 51.25±2.40 b 51.10±2.20 b * protein1 (%) 32.27±0.59 32.35±5.77 a 42.10±3.95 b 40.61±6.58 c 40.32±3.50 c * ash1 (%) 2.35±1.88 5.10±0.78 a 5.77±1.08 b 8.14±0.61 c 8.58±0.67 c * nacl1 (%) 0.00±0.00 2.25±0.00 a 4.91±0.01 b 6.85±0.56 c 6.67±0.50 c * a, b, c, d: the different letters in the same row indicate the statistically significant differences (p<0.05). ns = not significant, *p<0.05; **p<0.01. 1the concentrations are expressed in terms of the dry matter. the fatty acid contents in the cheese samples during the production and ripening, together with the results of the variance analysis, are shown in table 2. significant differences between the free fatty acid contents of the fossa cheese samples during the production time and the ripening phase (f = 81.093 with 20 degrees of freedom, p ≤ 0.001) were observed. for all types of cheese, the major saturated fatty acids (sfa) were myristic (c14:0), palmitic (c16:0) and stearic (c18:0) acid; all the cheese showed highly significant different values (p < 0.001) during the production time. moreover, cheese ripening in the talamello pit resulted in high values of sfa compared to cheese from the cartoceto pit. three monounsaturated fatty acids (mufa) were identified: myristoleic (c14:1), palmitoleic (c16:1), and oleic (c18:1 ɷ-9,cis) acid. the most important differences observed in the content of mufa were the strong decreases during ripening, up to values of approximately 15 %. among the polyunsaturated fatty acids (pufa), only linoleic (c18:2 ɷ-6,cis) and linolenic (c18:3 ɷ-3) acid were identified, but only the c18:2 ɷ-6,cis content was significantly different during ripening (p < 0.001). in particular, the fatty acid profile of the cheese after ninety days of ripening in the pits showed a percentage increase of the butyric, caproic, caprylic and capric acid content compared to the cheese after sixty days of ripening at the dairy farm, with respect to just the cheese curds, confirming the fact that the short-chain fatty acids are produced by lipolysis phenomena; in parallel, a decrease in the percentage of long chain fatty acids (oleic, linoleic and linolenic acids) in the cheese after sixty days at the dairy farm was observed compared to the cheese after ninety days of ripening in the pits, with respect to ital. j. food sci., vol. 31, 2019 677 just the cheese curds. the values of the long chain fatty acid content were almost constant except for oleic acid and palmitic acid, as suggested by various authors (olmedo and coll-hellin, 1976; najera et al., 1993), highlighting the higher values in linoleic and linolenic acids. the sum of saturated fatty acids showed no substantial differences between the milk and mature cheese, even if there was a decrement measured in the cheese curds. instead, the sum of mufa showed a significant decrease from 21.39 % in the milk to approximately 16.70 % in the ripened cheeses. according to alewijn et al. (2005), this decrease is due to the conversion of fatty acids into aldehydes, ketones, and lactones as a result of β-oxidation followed by decarboxylation. the most significant decrease was observed for the oleic and stearic acids, the main components of the total fatty acids in the analysed samples. even the sum of pufa and the ratio between the sums of pufa and sfa decreased after ripening in the pits at both sites. table 2. concentration of fatty acids in the milk and fossa cheese expressed in %. milk cheese after curdling cheese ripened at the dairy farm for sixty days talamello cheese cartoceto cheese statistical significanc e butyric c4:0 1.80±0.12 0.68±0.08 1.33±0.10 2.10±0.14 1.76±0.10 ** caproic c6:0 1.92±0.15 1.04±0.12 1.61±0.09 2.17±0.11 1.77±0.12 ** caprylic c8:0 1.96±0.09 1.42±0.10 1.92±0.12 2.51±0.13 2.10±0.10 ** capric c10:0 6.06±0.25 5.13±0.26 6.24±0.26 8.10±0.22 6.86±0.24 ** lauric c12:0 3.49±0.16 3.13±0.12 3.56±0.15 4.39±0.20 3.89±0.18 ** myristic c14:0 10.89±0.24 10.19±0.28 10.96±0.28 11.28±0.22 11.33±0.24 ** myristoleic c14:1 0.16±0.06 0.14±0.02 0.55±0.05 0.20±0.08 0.22±0.09 * pentadecanoic c15:0 1.25±0.18 1.28±0.14 1.22±0.10 1.23±0.11 1.38±0.14 ** palmitic c16:0 26.38±0.16 25.74±0.08 26.42±0.36 25.49±0.32 25.10±0.48 ** palmitoleic c16:1 1.06±0.10 1.39±0.05 0.99±0.08 0.76±0.10 0.79±0.12 * heptadecanoic c17:0 0.78±0.08 0.87±0.04 0.83±0.08 0.73±0.04 0.83±0.10 * stearic c18:0 11.83±0.66 11.56±0.78 11.56±0.65 9.98±0.88 10.29±0.87 ** oleic c18:1 ɷ-9,cis 20.17±0.95 21.89±0.86 21.03±0.88 15.43±0.98 15.92±0.77 ** linoleic c18:2 ɷ-6,cis 2.24±0.22 2.44±0.42 2.37±0.36 1.89±0.42 1.80±0.37 ** arachidic c20:0 0.34±0.04 0.40±0.08 0.39±0.09 0.28±0.02 0.33±0.07 * linolenic c18:3 ɷ-3 1.47±0.21 1.54±0.23 1.44±0.26 0.89±0.23 1.15±0.18 ns σ-sfa 66.70±1.14 61.44±1.37 66.04±1.28 68.26±1.77 65.63±1.98 σ-mufa 21.39±1.51 23.42±1.22 22.57±1.29 16.39±1.61 16.93±1.01 σ-pufa 3.71±0.15 3.98±0.38 3.81±0.32 2.77±0.41 2.95±0.28 σ-pufa/σ -sfa 0.06 0.06 0.06 0.04 0.04 oleic c18:1 ɷ-9,cis linoleic c18:2 ɷ-6,cis 9.00 8.97 8.87 8.18 8.83 note: *, ** indicate the significant differences during ripening, with p<0.05 and p<0.001; ns = not significant. ital. j. food sci., vol. 31, 2019 678 3.4. microbiological analysis the data on the microbial population of the different fossa cheese samples are shown in fig. 3. figure 3. bacterial dynamics of the principal microbial groups in the fossa cheese during the manufacturing and ripening process in the pits located in different geographical areas. data are the means±sd of the three cheese samples. in summary, in the cheese ripened for 24 h and sixty days, the total microbial count (tmc) was between 109 and 108 cfu/g. however, the mesophilic lactic microflora count was approximately 109 and 107 cfu/g; in this case, the higher load of lactic acid bacteria (lab) and, consequently, the higher total bacterial count were related to the addition of the starter during cheesemaking, and the content of sulphite-reducing clostridia (srcs) was approximately 105 cfu/g. instead, the contents of the yeast and moulds grown during ripening were approximately (on the order of) 104 and 105 cfu/g, respectively. a very low load of micrococcaceae was detected in the fossa cheese, as well as coliforms and escherichia coli. moreover, in the cheeses that were ripened for 3 months in underground pits (fossa) located at two different sites (talamello and cartoceto), the microbial population showed certain changes in the order of magnitude relative to the total mesophilic microbial count, which dropped from 109 to 107 cfu/g in the cheese of the talamello pit, while the count decreased from 109 to 108 cfu/g in the cartoceto cheese; the same trend was recorded for the lactic microflora (lactobacilli and lactococci). the high relative humidity, fairly high temperature and size of the pit environment may influence the oxygen pressure during ripening and may affect the metabolic pathways of cheese microflora that characterize the finished product. through enzymatic processes, the ital. j. food sci., vol. 31, 2019 679 elimination of fat and residual moisture occurs, and at the same time the drying of the product is limited (pozzetto, 2000). conversely, the content of sulphite-reducing clostridia increased up to one order of magnitude, and the same trend was shown by yeast and mould contents, probably because of the cheese ripening process and the pit habitat benefitting their growth compared to the other microbial components. finally, enterobacteriaceae and escherichia coli were not detected in the 10 g cheese samples. these results suggest that the content of fossa cheese microflora during ripening decreased because the chemical-physical parameters changed. in fact, during maturation, the cheeses undergo a considerable decrease in weight (approximately 20 %) and take on irregular shapes. the surface of the shapes is wet and greasy, and in some cases, the surface is covered by mould; a skin is absent (gobbetti et al., 1999). the microflora involved in the fossa cheese-making process were composed of starter cultures and native microflora that played important roles during the manufacturing of the cheese at the dairy farm and during its ripening in the pit. in particular, the starter microbiota carried out a rapid acidification by the production of lactic acid but also produced enzymes that are important for flavour development during ripening (leroy and de vuyst, 2004). furthermore, non-starter lactic acid bacteria (nslabs), which are complex mixtures of bacteria, yeasts and moulds, play an important role, together with environmental factors, in achieving the specific characteristics of cheese varieties (fox and wallace, 1997; beresford et al., 2001). additionally, filamentous fungi may reach the cheeses in the environment of the natural caves during ripening (lopez-diaz et al., 1996; budak et al., 2016). the content of nonstarter lactic acid bacteria usually increases from a low number in fresh curds to eventually dominate the microflora in the mature cheese because the bacteria tolerate the hostile environment during the cheese ripening well. furthermore, the heterogeneity of the nslab strains together with a pool of enzymatic activities, such as proteolytic and lipolytic activities, may determine a higher complexity in cheese flavour (fox et al., 1998; mcsweeney and sousa, 2000; de angelis et al., 2001). 3.5. volatile compounds the hp-spme-gc/ms analysis of the cheese samples allowed the identification of 77 compounds belonging to seven groups of volatile compounds. in this work, we quantified 5 aldehydes, 16 ketones, 15 esters, 16 alcohols, 12 acids, 7 terpenes, 3 lactones and 3 scompounds. the relative amounts of the individual compounds were expressed in terms of their relative peak area (rap) (table 3). the total quantities of the volatile compounds typically increase in almost all cheeses during ripening while the profile changes (massouras et al., 2006). additionally, the addition of spice plants during cheese manufacturing enhances the volatile compounds both qualitatively and quantitatively (cakir et al., 2016). aliphatic aldehydes (hexanal, heptanal and nonanal) are transitory compounds and do not accumulate in cheese because they are rapidly transformed to alcohols or to the corresponding acids (hayaloglu et al., 2007). branched chain aldehydes are normally found in cheese, 3-methylbutanal is formed by the strecker degradation of leu amino acid (urbach, 1995) and has been found to be a potent odour compound in different cheese varieties (curioni and bosset, 2002; hayaloglu et al., 2007). benzaldehyde, mainly derived from the metabolism of phenylalanine in cheese, which has the aromatic note of bitter almond, is commonly found in cheese and is formed by the oxidative reactions of cinnamic acid or phenylacetaldehyde (molimard and spinnler, 1996). ital. j. food sci., vol. 31, 2019 680 table 3. relative peak area (area of the compound/is area)×100±the standard deviation for the volatile compounds of the fossa cheese (n = 3). cheese after curdling cheese ripened at the dairy farm for sixty days cheese from the talamello site cheese from the cartoceto site odour description* ri aldehydes 1165 hexanal 13.7±0.5b 38.7±0.2a nd nd herbaceous 1264 heptanal 20.3±0.9b 73.8±3.8a nd nd sour milk 1466 nonanal 26.0±0.03a 10.5±0.07b nd nd floral, citrus 1009 butanal, 3-methyl nd nd 2.7±0.2b 4.2±0.1a mild, oil 1589 benzaldehyde nd nd 44.9±4.9a 7.9±0.5b sweet ketones 872 2-propanone 208.3±6.7b 240.9±2.3a 101.7±7.2d 189.0±5.2c apple, pear 993 2-butanone 20.8±0.1a 44.5±0.5c 33.4±0.5b 41.3±2.0c chemical, fruity 1066 2-pentanone 39.3±0.9d 965.5±31.3a 263.3±4.6c 501.7±35.2b sweet, floral 1069 2,3-butanedione 100.1±7.2a 82.2±1.5b nd nd fruity, buttery 1165 2-hexanone nd nd 12.2±0.4a 7.6±0.2b fruity, fungal 1261 2-heptanone 68.5±0.7d 645.8±27.1c 1254.7±19.1a 1034.9±29.5b blue cheese 1277 2-heptanone, 3-methyl nd nd 2.0±0.2a 2.3±0.2a 1303 5-hepten-2-one nd nd 13.1±0.4a 2.9±0.1b 1359 2-octanone nd nd 22.3±1.7b 28.1±0.9a dairy, waxy 1360 acetoin 342.8±18.6a 159.1±7.1b 4.7±0.2c 7.3±0.03d cream, dairy 1410 6-methyl-5-hepten-2-one nd 2.7±0.04 a 1.1±0.1b 2.5±0.1a citrus 1461 2-nonanone 31.3±1.6d 438.4±16.6c 1139.6±81.4a 1294.4±80.4a fruity, floral 1497 5-nonen-2-one nd nd 2.1±0.2b 3.8±0.2a fruity 1512 8-nonen-2-one nd nd 67.6±3.6a 94.9±2.6b fruity, baked 1556 2-decanone nd nd 7.2±0.2 a 7.3±0.2a orange, fatty 1663 2-undecanone nd 17.9±0.1c 56.8±1.1a 43.5±0.8b waxy, fruity esters 975 ethyl acetate 63.5±3.5a 45.3±0.2b nd 3.2±0.2c fruity 1047 propanoic acid, ethyl ester 13.7±0.2 a 7.3±0.08b nd nd fruity 1137 butanoic acid, 2-methyl, methyl ester 2.5±0.1 a 3.7±0.1b nd nd fruity 1124 butanoic acid, ethyl ester 19.3±0.4 d 23.7±0.8c 63.7±0.8a 45.1±0.6b fruity, cheesy 1135 butanoic acid, 2-methyl, ethyl ester 6.7±0.5 a 6.5±0.4a nd nd 1156 acetic acid, butyl ester 12.7±0.6a 12.3±0.3a nd nd fruity 1253 butanoic acid, 2-propenyl ester nd nd 0.9±0.1 a 1.1±0.03a fruity, green 1286 butanoic acid, 1-methyl, butyl ester nd nd 1.0±0.1 a 0.8±0.01a fruity 1310 hexanoic acid, ethyl ester 8.2±0.03 d 20.7±0.1c 28.0±1.8a 25.1±0.6b sweet, pineapple 1390 butanoic acid, 3-methyl, butyl ester 2.8±0.02 b 3.4±0.1a nd nd apple, fruity 1505 octanoic acid, ethyl ester nd 7.2±0.06 c 24.1±0.8a 9.7±0.4b sweet, fruity 1701 decanoic acid, ethyl ester nd nd 33.8±1.8 a 13.6±0.3 b waxy, fruity 1423 formic acid, hexyl ester 4.0±0.3a 4.1±0.1a nd nd ethereal, sweet 1348 acetic acid, hexyl ester 11.6±0.5a 16.3±0.1b nd nd fruity, green 1942 butanoic acid, propyl ester nd nd 10.2±0.5 b 19.5±0.9a sweet, fruity ital. j. food sci., vol. 31, 2019 681 alcohols 1023 ethanol 440.9±13.3a 343.8±1.7b 169.8±17.0c 204.9±13.0d alcoholic 1226 1-butanol 8.7±0.1a 7.5±0.1b 1.3±0.1d 3.5±0.2c banana, fusel 1230 isobutanol nd nd 3.4±0.3b 9.4±0.1a fusel 1208 2-pentanol nd nd 44.2±2.7b 88.1±4.0a mild, green 1284 3-methyl-1-butanol 12.0±0.1b 42.3±0.2a 2.07±0.1c 1.9±0.1d fusel, fermented 1297 1-hexanol nd nd 1.5±0.1a 1.6±0.1a green, fruit 1324 1-pentanol 25.6±0.9a 21.3±0.5b 2.6±0.2c 0.9±0.02d fusel, fermented 1391 2-heptanol nd nd 71.1±3.4a 58.7±2.6b fresh, lemon 1392 3-methyl-2-buten-1-ol 15.9±1.3a 9.4±0.1b nd nd 1518 1-octen-3-ol 7.7±0.03b 11.2±0.1a nd nd mushroom, earthy 1556 2-ethyl-hexanol 6.8±0.2a 5.1±0.01b nd nd sweet, fatty 1638 2,3-butandiol 18.4±0.9b 68.5±0.4a nd nd fruity, creamy 1640 2-octanol nd nd 1.5±0.1b 5.6±0.3a fresh, woody 1647 2-nonanol nd nd 51.4±3.9a 41.9±1.2b waxy, green 1721 2-furanmethanol nd nd 1.6±0.01b 4.1±0.2a burnt, sweet 1016 2-propanol nd nd 7.3±0.8b 18.2±0.4a must, woody acids 1524 acetic acid 141.4±4.9c 249.3±16.5a 95.9±3.2d 154.0±5.8b pungent, sour 1610 propanoic acid nd 2.7±0.1c 4.8±0.3b 16.1±0.3a acidic, dairy 1634 propanoic acid, 2-methyl nd nd nd 5.5±0.2a sour, cheese 1695 butanoic acid 132.2±8.9d 1022.9±58.5c 1386.9±78.5b 1902.1±69.6a sharp, cheese 1735 butanoic acid, 3-methyl nd nd 7.8±0.3a 6.3±0.2b cheesy, dairy 1736 pentanoic acid nd 11.6±0.3c 20.4±0.6a 19.6±0.3b sweet, rancid 1909 hexanoic acid 120.8±3.8d 584.3±35.3c 1337.4±72.9b 2028.3±100.1a sickening, sour 2012 heptanoic acid 5.9±0.03c 6.3±0.05c 12.3±0.6b 15.1±0.4a rancid, cheese 2117 octanoic acid 24.3±0.6d 44.3±0.6c 346.1±8.5b 444.3±14.9a fatty, waxy 2220 nonanoic acid nd nd 6.1±0.2a 5.7±0.3a waxy, dirty 2326 decanoic acid nd 7.5±0.06c 115.7±6.8a 74.6±1.5b fatty 2384 9-decenoic acid nd nd 3.1±0.03a 2.7±0.03a waxy, green terpenes 1119 dihydromyrcene 22.7±1.5a 21.4±0.1a 2.1±0.1c 2.4±0.1b citronellol, herbal 1178 p-menth-4(8)-ene 43.8±1.4a 45.4±0.7a nd 2.7±0.2b 1238 α-phellandrene 11.4±0.3a 6.4±0.1b 1.5±0.1c 1.4±0.03c citrus, lime 1272 limonene 152.6±12.7b 360.6±7.6a 6.3±0.2c 2.3±0.1d pine, peppery 1107 α -pinene 20.4±0.1a 20.5±0.1a 1.2±0.1c 1.9±0.1b woody, pine 1319 γ -terpinene 3.7±0.02a 4.7±0.1b nd nd citrus, lime 1195 sabinene 2.2±0.1b 3.4±0.1a nd 3.7±0.04c woody, citrus lactones 1766 γ-caprolactone nd 5.4±0.5c 7.0±0.3b 13.0±0.5a herbal, coconut 2246 δ-decalactone nd nd 2.6±0.3a 4.2±0.2a coconut, sweet 1974 γ-octalactone nd nd nd 2.4±0.1a sweet, coconut s-compounds 761 dimethyl sulphide 35.9±0.1b 44.1±0.2a nd nd vegetable, dairy 1308 methyl propyl disulphide 7.4±0.03b 12.8±0.5a nd nd onion, radish 1958 dimethyl sulfone 10.3±0.3a 6.6±0.1b 0.03±0.00c 2.2±0.1c sulphurous, burnt mean data for the three batches of fossa cheese, cheeses analysed in triplicate. a, b, c, d: the different letters in the same row indicate the statistically significant differences (p< 0.05). nd = not detected; ri= retention index; * www.thegoodscentscompany.com/ ketones are formed by the enzymatic oxidation of fatty acids to keto-acids and their consequent decarboxylation to methyl ketones (mcsweeney and sousa, 2000). moreover, the content of longer chain methyl ketones increases during cheese ripening. the ketones have distinctive odours and low perception thresholds. according to ital. j. food sci., vol. 31, 2019 682 giocchini et al. (2010), in fossa cheese, the major ketones are 2-heptanone and 2nonanone, which contribute to the aroma with blue cheese notes. different esters were detected in the volatile fraction of the fossa cheese, namely, 7-ethyl ester, 1-propyl ester, 3-butyl ester, 2-hexyl ester, 1-propenyl ester and 1-methyl ester. esterification reactions may occur between the shortto medium-chain fatty acids and the alcohols. nevertheless, the esters might also be synthesized directly from triglycerides and alcohols via an alcoholysis reaction. in particular, the proliferation of esters after ripening is due to the presence of ethanol and the abundance of short-chain ffa. these compounds are probably the result of microbial metabolism of the fatty acids. they play an important role in the formation of the fruity feature and characterize the flavour of certain italian cheeses (panseri et al., 2008). they also contribute to the balance of the flavour by minimizing the sharpness imparted by the free fatty acids. we observed a variability in the level of esters during ripening. the more representative esters in the fossa cheese samples that increased were butanoic acid ethyl ester, hexanoic acid ethyl ester, octanoic acid ethyl ester, decanoic acid ethyl ester and butanoic acid propyl ester. ethyl esters, due to their high content, probably contributed to the overall flavour of fossa cheese because they have low detection thresholds (delgado et al., 2010). the alcohol class showed higher variability during ripening, and the different patterns could be associated with the different metabolic pathways involved in the formation of alcohols in cheese, namely, lactose metabolism, methyl ketone reduction, amino acid metabolism and degradation of linoleic and linolenic acids (delgado et al., 2010). twelve acids were identified in the samples, and they had a positive contribution to the typical flavour in a majority of the cheeses (panseriet al., 2008). during the ripening of cheese, carboxylic acids could originate from three main biochemical pathways: (i) lipolysis (hydrolysis of the triglycerides into free fatty acids), (ii) proteolysis (cracking of the caseins into peptides and amino acids) and (iii) lactose fermentation (curioni and bosset, 2002). based on this information, we have found acids with a microbial origin (acetic acid and propanoic acid), acids with an origin of lipolysis (butanoic, pentanoic, hexanoic, heptanoic, octanoic, nonanoic and decanoic acid), and acids with an origin in amino acids (propanoic acid, 2-methyland 3-methylbutanoic acid). finally, the trend of the acids increased during the ripening process of the fossa cheese. hexanoic and butanoic acids were the most abundant acids identified. due to their low aroma thresholds, they are considered important contributors to the flavour profile in a wide variety of cheeses (moio and addeo, 1998; delgado et al., 2010; delgado et al., 2011). the branched-chain fatty acids (propanoic acid, 2-methyland 3-methylbutanoic acid) are characteristic odour-active compounds that have an impact on goat and sheep milk cheeses. terpenes are important volatile compounds with origins in plants that constitute the forage mixture of pastures (delgado et al., 2011). three lactones, namely, γ-caprolactone, δ-decalactone, and γ-octalactone, were detected in the cheese ripened for 90 days, but γ-octalactone was only detected in the cheese from the cartoceto pit. the lactones have fruity sweet, creamy, fermented notes, and they could contribute pleasant odour notes to the aroma of fossa cheese (delgado et al., 2010). moreover, three s-compounds were identified (dimethyl sulphide, methyl propyl disulphide and dimethyl sulfone), and these compounds decreased during ripening. finally, the cheese from the cartoceto pit contained more volatile compounds than the cheese from the talamello pit, in particular for the alcohol, acid and lactone classes. the results of this study showed that there were substantial differences between the chemical and chemical/physical parameters, and many fatty acids of the just curdled ital. j. food sci., vol. 31, 2019 683 cheese samples and the cheese ripened for 90 days in the two pits were characterized by different geological-geochemical parameters. slight differences in the nutritional parameters between the cheeses ripened in the different pits could be identified in certain components of the fatty acids and in the content of certain groups of volatile molecules. in particular, the cheese ripened in the talamello pit showed high values of sfa compared to the cheese from the cartoceto pit, while the sum of pufa in the cartoceto cheese was higher. the cheese from the cartoceto pit had more volatile compounds than the cheese from the talamello pit, in particular for the alcohol, acid and lactone class. in conclusion, although the cheeses ripened in the two different pits had been prepared with the same milk, the pedo-climatic environment in the pits significantly influenced only certain nutritional parameters. acknowledgements the authors thank prof. francesca rosati (sworn translator) for her support in checking the manuscript for english form. references alewijn m., sliwinski e.l. and wouters j.t.m. 2005. production of fat-derived (flavour) compounds during the ripening of gouda cheese. int. dairy j. 15:733-740. 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(aoac). 2001. association of official analytical chemists official method 996.06. in: official methods of analysis, 17th ed., revised; aoac: gaithersburg, md, usa. avellini p., clementi f., trabalza marinucci m., cenci goga b., rea s. and branciari r. 1999. pit cheese: compositional, microbiological and sensory characteristics. ital. j. food sci. 11:317-333. barbier e., schiavano g.f., de santi m., vallorani l., casadei l., guescini m., gioacchini a.m, rinaldi l., stocchi v. and brandi g. 2012. bacterial diversity of traditional fossa (pit) cheese and its ripening environment. int. dairy j. 23:62-67. beresford t.p., fitzsimons n.a., brennan n.l. and cogan t.m. 2001. recent advances in cheese microbiology. int. dairy j. 11:259-274. budak s.o., figge m.j., houbraken j. and de vries r. p. 2016. the diversity and evolution of microbiota in traditional turkish divle cave cheese during ripening. int. dairy j. 58:50-53. cakir y., cakmakci s. and hayaloglu a.a. 2016. the effect of addition of 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g., boscaino f. 2016. volpe m. g. physico-chemical properties and fatty acid composition of pomegranate, cherry and pumpkin seed oils. j. sci. food agric. 96:1730-1735. stefanov i. and vlaeminck b. 2010. fievez v. a novel procedure for routine milk fat extraction based on dichloromethane. j. food compost. anal. 23:852-855. urbach g. 1995. contribution of lactic acid bacteria to flavour compound formation in in dairy products. int. dairy j. 5:877-903. van den dool h. and kratz i. 1963. generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. j. chromatogr. 11:463-471. paper received december 18, 2018 accepted march 14, 2019 p u b l i c a t i o n s codon italian journal of food science, 2023; 35 (1): 41–48 issn 1120-1770 online, doi 10.15586/ijfs.v35i1.2219 41 qualitative characteristics of four sicilian monofloral honeys from apis mellifera ssp. sicula paola bambina1, francesca malvano2, claudio cinquanta2, donatella albanese2, andrea cirrito1, francesca mazza1, onofrio corona1* 1department of agricultural, food and forest sciences university of palermo, viale delle scienze, 90128 palermo, italy; 2department of industrial engineering, university of salerno, via giovanni paolo ii, 84084 fisciano, italy *corresponding author: onofrio corona, department of agricultural, food and forest sciences university of palermo, viale delle scienze, palermo, italy. email: onofrio.corona@unipa.it received: 29 april 2022; accepted: 24 november 2022; published: 2 february 2023 © 2023 codon publications open access paper abstract four monofloral honeys, obtained from the sicilian black bee by foraging on thistle, sulla, chestnut and eucalyptus, were studied. results showed that the phenolic composition of chestnut honey was the highest (316 mg gallic acid equivalent gae/kg), while that of sulla honey was the lowest (122 mg gae/kg). data confirmed a correlation between the total phenol content and colour intensity in chestnut honey, which was the darkest of the four samples. sulla honey showed the highest antioxidant activity, while eucalyptus honey had the highest mineral content (k, ca, mg, and na). thistle honey showed the most intense floral and fruity aromas, as well as an intense yellow colour. principal component analysis showed the potential to discriminate different honeys in three different quadrants. keywords: colour; honey; sensory analysis; sicilian black bee introduction the composition of honey varies due to differences in botanical, geographical and entomological origins, and is also influenced by the seasonal, production and storage conditions (wang et al., 2022). the european union (eu, 2014) has imposed strict labelling rules on honey, requiring that the botanical and geographical origins of honey be correctly labelled before sale (thrasyvoulou et  al., 2018). based on their botanical origins, honey products can be classified as monofloral or multifloral. the commercial value of monofloral honey is much higher than that of multifloral honey due to its unique aroma and taste, which are in greater demand by consumers (marcazzan et al., 2014). to confirm the typicality of honey, it is necessary to identify its botanical origin, generally through melissopalynological analysis. honey can also be classified according to its geographical origin, considering that, in addition to the influence of the flower species, the physico-chemical and sensory characteristics of honey may vary according to the subspecies of bees (silva et al., 2016). the sicilian black bee (apis mellifera ssp. sicula) is an african subspecies of a. mellifera that has adapted to the warm lands of the mediterranean, including sicily (italy) (mannina et  al., 2015). it differs from the more common a. mellifera ssp. ligustica in the colour and size of its wings, as well as in the fact that it is more resistant to high temperatures, allowing it to tolerate temperatures above 40°c, whereas other subspecies of bees do not produce honey under such extreme conditions (attanzio et  al., 2016). a. mellifera ssp. sicula also possesses considerable immunological resistance, enabling it not to succumb to varroasis nor virosis (franck et  al., 2000). a. mellifera ssp. sicula has a pronounced pollination capacity that ensures the continuity of many plant species, including some that are in danger of extinction. this subspecies risked extinction in the 1970s, when beekeepers imported the ligustica subspecies into sicily. following this massive introduction, a group of entomologists and beekeepers recovered the 42 italian journal of food science, 2023; 35 (1) bambina p et al. meter. the ash content was determined according to official methods (aoac, 1999): about 5 g of honey was placed in a combustion pot preheated in the dark with a gas flame to prevent the honey from foaming. then, the sample was incinerated (burned) at a high temperature (550°c) in a burning muffle furnace for 5 h. after cooling to room temperature, the ash obtained was weighed. the total polyphenol content was determined spectrophotometrically using a folin– ciocalteu method as reported by singleton et al. (1999), with some modification. the results were expressed as gallic acid equivalents (gae) per 100/g honey. colour measurements were performed using a konica minolta chroma meter cr-c2500 (konica minolta sensing singapore pte ltd, singapore). results were recorded as a*, b*, and l* values, where a* is an index of redness (+) or green (−), b* an index of yellow (+) or blue (−), and l* indicates brightness on a scale of 0–100 (adiletta et  al., 2020). the overall colour difference (δe), chroma (c) and hue angle (h°) were also calculated, where chroma indicates the dullness or vividness and hue angle indicates how an object’s colour is perceived by the human eye (red, orange, green or blue). the samples were placed in an optical glass cell for measurement. glucose and fructose were determined by the hplc system using an agilent 1100 chromatograph with a refractive index  detector (agilent, santa clara, usa) equipped with a eurokat, 300 × 8 mm, 10 μm column (knauer, berlin, germany). the mobile phase was a water solution with a flow rate of 1 ml/min and a column temperature of 80°c. the results were expressed as “mg glucose/g honey.” the antioxidant activity of honey was evaluated using the ddph radical scavenging activity (larrauri et  al., 1998) and expressed as μmol trolox equivalents (te)/g honey. all measurements were repeated three times. all results represent the average of three measurements per sample. mineral content the mineral content of each honey sample was determined according to the procedure of chudzinska et al. (2011), with some modifications. two grams of each sample were dispersed in 5 ml hno3 (65 %) and 1 ml h2o and then digested in a microwave digestion system (mars 6, cem, matthews, nc, usa) by increasing the temperature up to 210°c. at the end of the procedure, after appropriate dilutions with bi-distilled h2o, samples were analysed by inductively coupled plasma spectroscopy (icap 6200 duo, thermo scientific, waltham, ma, usa), and ca, fe, k, mg, na, cu, mn and zn content were determined. each sample was analysed in triplicate, and the reported results are the average of the three measurements. genetically pure sicilian subspecies by transferring some old hives to the island of ustica (pa), where the selected bees were bred without risk of contamination. a reintegration plan was then launched in western sicily by the slow food presidium founded in 2008. the plan included fertilization stations for pure reproduction of a. mellifera ssp. sicula. purity was periodically checked through genetic screening (attanzio et al., 2016). the physico-chemical and sensorial properties of honey produced by a. mellifera ssp. sicula has thus been of scientific interest. the aim of this study was to evaluate, by means of sensory and physico-chemical analysis, the differences between four monofloral honeys obtained by a. mellifera ssp. sicula. materials and methods honey samples honey samples of thistle (silybum marianum l.), sulla (hedysarum coronarium l.), chestnut (castanea sativa mill.) and eucalyptus (eucalyptus  globulus labill.) were collected from sicily from the 2019 production by nettare di sicilia, caltavuturo (pa). nettare di sicilia is a company situated inside the madonie park and part of the slow food presidium. the various monofloral honeys were produced by moving the bees to the most suitable environments in sicily with the specific botanical species. precisely, the honeys analysed were chestnut honey, produced in the nebrodi nature park in the province of messina at an altitude of over 1000 m above sea level; sulla honey, produced in a hilly area in the madonie park at around 700 m above sea level; eucalyptus honey, produced in the province of agrigento in a fairly arid area with small woods; and thistle honey, produced in the province of palermo at sea level where this plant grows wild. all samples were classified by melissopalynological analysis (soares et  al., 2017), whereby the pollen grains of the different botanical species were distinguishable by microscopic observation. three different samples of honey from different hives were analysed for each of the four botanical species, all processed in the same way. honey samples were kept away from sunlight at room temperature before analysis. physico-chemical parameters the moisture content of each honey sample was determined from its refractive index using a digital refractometer (nr 101 spain) thermostated at 20°c and regularly calibrated with distilled water (bogdanov, 2009). the ph was assessed by the crison glp 21 ph italian journal of food science, 2023; 35 (1) 43 qualitative characteristics of sicilian monofloral honeys was controlled during processing (table 1). moisture in each analysed honey sample was around 14–15%, which is the optimal level, ensuring stability and spoilage resistance against yeast fermentation (bacandritsos et  al., 2006) while prolonging shelf life and limiting granulation (singh and kuar bath, 1997). the four monovarietal honey samples were all acidic, with ph values ranging between 2.96 (sulla honey) and 5.21 (chestnut honey). all of these values fall within the standard limit (ph 3.40– 6.10) (codex alimentarius, 2001), ensuring the freshness of the honey samples (table 1). the low ph of honey is related to the fermentation of the sugars, which results in two important characteristics of honey: flavour and stability against microbial spoilage (bogdanov, 2009). the ash content of honey is often used to determine its botanical origin (floral, blend or honeydew). ash concentrations in the honey samples ranged from 0.10 (sulla honey) to 0.79% (chestnut honey), which are all values within the limits allowed for floral honeys (0.60%) except for chestnut honey, in which ashes were present in a higher percentage, showing that it belongs to the dark honeys (oddo et  al., 1995). the high ash content could explain the high ph value of the chestnut honey samples, being that ash depends on the constituents of the flora type, geographical area, physiology of the plants and soil type on which the plants from which the bees collect nectar grow. the total phenol content (figure 1) was highest in chestnut honey (316.3 mg gae/kg), followed by eucalyptus (193.5 mg gae/kg), thistle and sulla honey; these results are in agreement with those reported by preti and tarola (2022). the phenolic content of monofloral honeys of a. mellifera ssp. sicula varied according to the botanical origin of the plants from which the nectar was collected (al-mamary et  al., 2002; amiot et  al., 1989). in sicilian environments characterised by a warm climate and a high level of exposure to sunlight, the plants may contain many more total phenols than the same plant varieties grown in colder environments (spayd et  al., 2002). phenolic compounds are responsible for the colour and taste characteristics of honey and for multiple biological sensory analysis the different honeys were judged by a trained panel of nine tasters (7 males and 2 females, aged between 24 and 48 years), consisting of technical experts. twenty grams of honey was weighed into 200 cc transparent glasses, sealed with foil and kept at 20°c for 2 h before tasting. samples were presented to each taster in random order. a descriptive sensory profile test based on quantitative descriptive analysis was used for the evaluation. based on the frequency of citation (>60%), 17 descriptors were identified: three visual (yellow intensity, amber intensity and crystallisation), nine olfactory (ripe fruit, herbaceous, floral, caramel, liquorice, beeswax, hay, medicinal and off-flavour), three gustatory (sweet, sour and bitter), one taste persistence and one overall liking. each of the descriptors was measured on a structured intensity scale of 1–9, with 1 denoting absence and 9 denoting maximum perception. because all types of honey were suitable for trade, the council of ethics exempted the authors to ask for a formal ethical approval. the panelists did, however, give verbal informed consent prior to participation. statistical analysis analysis of variance (anova) and tukey’s honest significant difference test at a 5% level were used to compare analytical differences between samples. principal component analysis (pca) was performed to reduce the multidimensionality of the dataset, generating new principal components that accounted for most of the total variation. all statistical analyses were done using the spss software package, version 20.0 (spss inc., chicago, il, usa). results and discussion physico-chemical traits the moisture content did not differ significantly (p  <  0.05) among the honey samples, as this parameter table 1. physico-chemical traits of different monofloroal honeys. parameters thistle sulla chestnut eucalypt water % 85.5 ± 0.501a 85.1 ± 0.47a 84.90 ± 0.50a 85.5 ± 0.48a ash (g/100 g) 0.32 ± 0.02b 0.10 ± 0.01a 0.79 ± 0.06c 0.10 ± 0.01a ph 3.26 ± 0.04b 2.96 ± 0.04 a 5.21 ± 0.04c 3.30 ± 0.04b sucrose (mg/g) 62.16 ± 4.51b 61.79 ± 5.63b 53.60 ± 6.87a 57.14 ± 7.85a glucose (mg/g) 287.31 ± 16.22b 287.03 ± 17.51b 232.10 ± 19.58a 312.36 ± 28.11c fructose (mg/g) 293.87 ± 22.76a 392.52 ± 34.26b 356.81 ± 41.85b 391.23 ± 49.27b dpph (μmol te/100g) 16.57 ± 0.05a 17.19 ± 0.01b 16.27 ± 0.04a 17.01 ± 0.03a,b mean ± sd (n = 3) (different letters in the same row indicate significant differences for p ≤ 0.05, anova, tukey’s test). 44 italian journal of food science, 2023; 35 (1) bambina p et al. but lowest for the sulla sample. the colour saturation (c) was highest for chestnut honey and was lowest for the sulla sample. for the parameter indicating colour hue, no significant variations were found in the three samples of thistle, sulla and eucalyptus, while chestnut had a slightly lower value than these three. sucrose was highest in thistle and sulla honey, and lowest in the chestnut sample. glucose was present in concentrations of 232–312 mg/g of honey in chestnut and eucalyptus, while fructose was present in concentrations of 293–393  mg/g of honey in thistle and sulla. the antioxidant activity of honey measured by dpph protocol showed values between 16.27 and 17.19 μmol te/100g, with sulla honey showing the highest activity. properties, such as antioxidant, antibacterial and radical scavenging activities. results reported by karabagias et al. (2014) and preti and tarola (2022) confirmed the correlation between the total phenol content and colour intensity, with darker honeys having a higher phenolic content and antioxidant capacity. in this regard, it should be noted that the average value of the parameter a* (index of red) was about 9.3 for chestnut honey (rodríguez-flores et al., 2019), compared to values below 1.0 for the other three samples (figure 2). the value of b* (yellow index) was also highest for chestnut honey, while sulla honey showed the lowest value. the l* (lightness) value was highest for chestnut honey, b a d c a a c b b a c bb a c b c c a b 0 5 10 15 20 25 30 35 40 thistle sulla chestnut eucalyptus mono�oral honeys l* a* b* c hue figure 2. colour traits of different monofloral honeys. different letters indicate significant differences for p ≤ 0.05, anova, tukey’s test. figure 1. total phenol content (mg gallic acid equivalent/kg) in different monofloral honeys. different letters indicate significant differences for p ≤ 0.05, anova, tukey’s test. 0 50 100 150 200 250 300 350 400 t o ta l p h e n o l c o n te n t (m g a c id g a lli c /k g ) thistle ab a b c sulla chestnut eucalyptus mono�oral honeys italian journal of food science, 2023; 35 (1) 45 qualitative characteristics of sicilian monofloral honeys thistle samples showed no significant (p < 0.05) differences in the content of all analysed macroand microelements except for k, which was higher in sulla honey. all the other elements, such as fe, cu, mn and zn, were present in traces in all samples. sensory analysis honey from different floral sources may have distinct aromas and flavours due to differences in volatile composition, which in turn may depend on the geographical origins (manyi-loh et  al., 2011). sensory analysis of the sulla honey showed a high yellow intensity, with an advanced state of crystallisation (figure 3). for the sense of smell, this honey registered the highest floral value, while ripe fruitiness, beeswax and hay were less marked, owing to the presence of norisoprenoids mineral content chemical evaluation of the most common minerals present in honey samples was performed. according to the literature (alves et al., 2013; bontempo et al., 2017; lobos et al., 2022), for all types of honey, the most abundant elements were, in decreasing order of concentration, k, ca, mg and na (table 2). eucalyptus honey contained the highest amount of potassium (884.55 mg/kg), which was significantly different (p  <  0.05) from thistle (527.93 mg/kg), chestnut (518.08 mg/kg) and sulla (422.38 mg/kg) samples. ca and mg were the most abundant macroelements in eucalyptus and chestnut honey, with respective values of 113.90 mg/kg and 14.46 mg/kg for eucalyptus and 120.38  mg/kg and 31.28 mg/kg for chestnut. moreover, eucalyptus honey was also very rich in na. sulla and table 2. mineral content of different monofloroal honeys. sample ca (mg/kg) fe (mg/kg) k (mg/kg) mg (mg/kg) na (mg/kg) cu (mg/kg) mn (mg/kg) zn (mg/kg) sulla 80.36 ± 4.56a 0.28 ± 0.02a 422.38 ± 23.24a 8.47 ± 1.02a 5.50 ± 0.95a 0.10 ± 0.02a 0.50 ± 0.10a 0.50 ± 0.08a eucalyptus 113.90 ± 8.49b 1.47 ± 0.09c 884.55 ± 52.57c 14.46 ± 1.12b 84.10 ± 10.66b 0.20 ± 0.07b 1.00 ± 0.09b 0.80 ± 0.13b thistle 90.19 ± 7.34a 0.27 ± 0.04a 527.93 ± 34.36b 9.50 ± 1.04a 6.30 ± 1.29a 0.10 ± 0.04a 0.40 ± 0.03a 0.60 ± 0.09a chestnut 120.38 ± 10.12c 0.83 ± 0.11b 518.08 ± 42.31b 31.28 ± 3.12c 7.47 ± 0.84a 0.30 ± 0.06b 2.00 ± 0.24c 0.90 ± 0.16b mean ± sd (n = 3) (different letters in the same row indicate significant differences for p ≤ 0.05, anova, tukey’s test). figure 3. sensory analysis of different monofloral honeys. different letters indicate significant differences for p ≤ 0.05, anova, tukey’s test. amber intensity (a-a-c-b) crystallization (b-b-a-b) ripe fruit (a-a-b-a) herbal (b-b-a-ab) floral (c-c-a-b) caramel (a-a-b-b) licorice (a-a-b-b) beeswax (a-b-a-a)hay (a-b-a-b) medicinal (a-a-c-b) off-flavor sweet (c-c-a-b) acid (ab-bc-c-a) bitter (ab-a-c-b) gustatory persistence (ab-a-b-ab) overall satisfaction (b-ab-a-ab) yellow intensity (b-b-a-ab) thistle sulla chestnut eucalyptus 8 7 6 5 4 3 2 1 0 46 italian journal of food science, 2023; 35 (1) bambina p et al. the thistle sample was characterised by the following sensory parameters: yellow intensity, crystallization, and ripe fruit odour. conclusion the analysed sicilian honeys, produced using the same techniques and obtained from black bees, showed different sensory and quality profiles. these differences reflect the botanical species and environmental characteristics in which the bees developed. the phenolic composition of honey, which is important from taste and health points of view, was the highest in chestnut honey and lowest in sulla honey. ca and mg were the most abundant macro nutrients in chestnut and eucalyptus honey, with the latter also containing the highest amount of potassium. the sensory analysis, which considered 17 descriptors (visual, olfactory, taste and persistence), showed that the overall liking was higher for thistle honey, followed by sulla, eucalyptus and finally chestnut. references adiletta, g., di matteo, m., albanese, d., farina v., cinquanta, l., corona, o., magri, a. and petriccione, m., 2020. changes in physico-chemical traits and enzymes oxidative system during cold storage of “formosa” papaya fresh cut fruits grown in the mediterranean area (sicily). italian journal of food science. 32: 845–857. 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35 (1) bambina p et al. soares, s., amaral, j.s., oliveira, m.b.p.p. and mafra, i.a., 2017. a comprehensive review on the main honey authentication issues: production and origin. comprehensive reviews in food science and food safety. 15: 1072−1100. https://doi.org/ 10.1111/1541-4337.12278 spayd, s.e., tarara, j.m., mee, d.l. and ferguson, j.c., 2002. separation of sunlight and temperature effects on the composition of vitis vinifera cv. merlot berries. the american journal of enology and viticulture. 3: 171–182. thrasyvoulou, a., tananaki, c., georgios, g., karazaphiris, e., dimou, m., liolios, v., kanellis, d. and gounari, s., 2018. legislation of honey criteria and standards. journal of apiculture research. 1: 88–96. https://doi.org/10.1080/00218839.2017.1411181 wang, x., yaxi hu, c., jinhui zhou, j., chen, l. and lu, x,. 2022. systematic review of the characteristic markers in honey of various botanical, geographic, and entomological origins. acs food science and technology. 2: 206–220. https://doi.org/10.1021/ acsfoodscitech.1c00422 24 issn 1120-1770 online, doi 10.15586/ijfs.v33isp1.1990 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (sp1): 24–33 p u b l i c a t i o n s codon an assessment of cuminum cyminum (boiss) essential oil, nacl, bile salts and their combinations in probiotic yogurt noushin mohajeri1, peyman mahasti shotorbani2*, afshin akhondzadeh basti3, zaleh khoshkhoo4, ali khanjari5 1student of food science and technology, department of food science and engineering, tehran north branch, islamic azad university; 2department of food quality control and hygiene, science and research branch, islamic azad university, tehran, iran; 3department of food hygiene, faculty of veterinary medicine, university of tehran, tehran, iran; 4department of food science and technology, tehran north branch, islamic azad university, tehran, iran; 5department of food hygiene, faculty of veterinary medicine, university of tehran, tehran, iran *corresponding author: peyman mahasti shotorbani, department of food quality control and hygiene, science and research branch, islamic azad university, tehran, iran. email: p-mahasti@srbiau.ac.ir received: 27 december 2020; accepted: 5 february 2021; published: 11 february 2021 © 2021 codon publications open access paper abstract this article is prepared to investigate the impacts of cuminum cyminum essential oil (ceo), nacl, bile salts, and their combinations on the viability of lactobacillus casei in probiotic yogurt. the water distillation method was used to extract the ceo, and gc/ms was used to determine its constituents. then, the ceo’s antibacterial activity, together with nacl and bile salts, was investigated via the microdilution technique by determining the minimum inhibitory concentration (mic) against l. casei. further, the stress effects of 50% mic on ceo, nacl, and bile salts were examined by comparing the stress treatments with the control in terms of the l. casei population, ph, acidity, and syneresis percentage in probiotic yogurt during storage in the refrigerator for 28 days. according to the results, the l. casei population and ph decreased in all the treatments during the storage time, such that the intensity of the decrease in the control and ceo treatments was lesser than in other stress treatments (p<0.05). the acidity and percentage of syneresis during the storage time increased for all the treatments, with the increase being less in control and ceo than in the other stress treatments (p<0.05). the control and ceo treatments scored the highest in the sensory evaluation (p<0.05). applying stresses below the mic resulted in the survival of l. casei in the recommended amount (105–106 cfu ml–1) in the probiotic yogurt until the end of 28 days. keywords: cuminum cyminum, probiotic, yogurt, lactobacillus casei, stress, essential oil introduction today, particular attention is being given to functional foods, which have nutritional value as well as positive effects on human health. people are excited about eating products containing probiotics. however, products containing probiotics are amongst the ones in which there have been health allegations. these allegations are even advertised in the media during the last few years (zendeboodi et al., 2020). various studies on probiotics for humans’ advantages have focused on the innovative formulation, and some even provided valuable information on probiotics linked to health and well-being. (roobab et al., 2020). probiotics are microorganisms that improve the gut microbial balance. these often include the lactobacillus and bifidobacterium species, mailto:p-mahasti@srbiau.ac.ir italian journal of food science, 2021; 33 (sp1) 25 an assessment of cuminum cyminum (boiss) essential oil, nacl, bile salts and their combinations in probiotic yogurt probiotic bacteria in yogurt by applying different procedures to increase the resistance of bacterial cells against stresses. hence, the usage of herbal essential oils and probiotic strains in dairy products is a new strategy to overcome pathogenic bacteria and stimulate probiotics. this study aimed to evaluate the effects of stresses less than the mic of  cuminum cyminum  boiss. essential oil (ceo), nacl (nc), and bile salts (bs) or a combination of them on the viability of probiotic l. casei and later we monitored through the storage time, physicochemical and sensory properties of probiotic yogurt. materials and methods materials cow’s milk (3% fat) was supplied from pegah, tehran plant, and starter culture of yogurt (streptococcus thermophilus and lactobacillus bulgaricus sub spp. delbrukii) was purchased from hanson company, denmark. extraction and analysis of the essential oil the cuminum cyminum was collected from the kerman province of iran, and the iranian institute approved its scientific name of botanical garden research. the plant›s essential oil was extracted by the water vapor distillation method, and then it was analyzed by colorimeter attached to a mass spectrometer (model hp-6890, usa). hp-5ms capillary column with 30 m length, 0.25 mm inner diameter, and 0.32 μm inner layer thicknesses was used. the regulated programming for identification and quantification was set up as follows: the temperature was elevated from 60 to 265°c, with a flow rate of 2.5°c per min, and then the column was maintained at 265°c for 30 min. the injection room temperature was 250°c, and the flow rate of helium as a carrier gas was 1 mm/min. finally, the flame ionization detector (fid) identified the essential oil components with an electrical capacity of 70 ev and an ionization source temperature of 250°c. preparation of inoculums lactobacillus casei  (atcc39392) was supplied from the microbial collection of pasteur institute of iran. it remained in laboratory samples in a glycerol stock at –70°c and transferred in brain heart infusion (bhi) broth (merck, germany) at 37°c without shaking. working cultures were prepared from stock cultures in two successive transfers (1% inoculum) in bhi broth at 37°c for 18 h. l. casei cells were inoculated from working cultures to bhi broth. after 18 h incubation at 37ºc, optical density (od) (absorbance) of 0.1 at 600 nm, as they have a historically prolonged and reliable value. also, identified as generally recognized as safe (gras), all are the predominant inhabitants in the human intestines. (al-okbi and mohamed, 2012; lucatto et al, 2020). lactobacillus casei is a gram-positive, mesophilic, microaerophilic, catalase-negative, and spore-free bacterium and a facultative hetero-formative bacterium with high acid production capacity (fontana et al., 2018). l. casei is a probiotic strain linked with antihypertensive antioxidant, antihypocholesterolemic and anticarcinogenic, characteristics (balthazar et al., 2018; garcia et al., 2019). fermented milk, such as yogurts, has the potential to act as a medium for producing value-adding materials because of its favorable sensory attributes, nutritional properties, and high-grade harmony, and ample content of essential nutrients; besides, yogurt consumption enhances gut macrobiotic activity, mitigates immune responses, and increases gastrointestinal functionality by adjusting lactose intolerance. technologists and manufacturers have reviewed distant fortifications by combining probiotics and nutraceutical compounds (alizadeh khaledabad et al., 2020; lucatto et al., 2020). today, probiotic yogurt is the most popular and widely consumed probiotic product in the world. the survival of probiotic bacteria in yogurt and similar products is an important challenge during storage in probiotic products. the minimum acceptable concentration of probiotic strains for beneficial and therapeutic effects should be at least 106–107 cfu g -1 or ml in the final product (azizkhani and parsaeimehr, 2018). the main problem in production was maintaining the survival rate of probiotic strains during storage of product with due attention to high acidity, oxygen stress, and nutrient deficiencies. the main reasons for reducing the viability of probiotic strains in the stomach were low ph and bile salts in the intestines (el-shafei et al., 2010). many studies have been carried out on the viability of probiotics under the stomach’s acidic conditions, bile salts of the small intestine (sahadeva et al., 2011), and survival rate of probiotics were studied in the cold storage of foods (mortazavian et  al., 2007). various methods such as microencapsulation, the addition of prebiotics and essential oils (capela et al., 2006), and different procedures were used to increase the survival rate of probiotic strains during storage of functional products applied stresses were less than the minimum inhibitory concentration (mic). they produced resistance-inducing genes (maragkoudakis et al., 2006). some essential oils improve probiotics’ viability, and others may also decrease the viability of probiotics (calsamiglia et al., 2007). in many cases, the viability of probiotic bacteria is not sufficient, and it is necessary to evaluate the viability of 26 italian journal of food science, 2021; 33 (sp1) mohajeri n et al. 37°c for 48 h. furthermore, all experiments have been done twice. physicochemical properties of yogurt samples the experiments for physicochemical properties were ph, acidity%, and syneresis%. ph and titratable acidity concluded based on the method described by yangilar and yildiz (2018). moreover, the percentage of syneresis was averaged according to the method defined by wacherrodarte et al. (1993). five milliliters of the yogurt sample was centrifuged at 2.208 g for 20 min at 4°c, and the volume of isolated whey was calculated after 1 min. lastly, we displayed the syneresis rate (%) as the separated whey volume per 100 g of yogurt (wacher-rodarte et al., 1993). sensory evaluation sensorial tests were executed based on a 5-point hedonic scale. the lowest score was intensely disliked, and the highest score was 5 as remarkably like samples (shahdadi et al., 2015). sensory properties were measured as follows: flavor, texture, and overall acceptability. sensory evaluations were carried out during 28 days of storage. ten trained panelists performed judgments. analytical study  all experiments were performed in completely randomized design as triplicates and the result was reported as mean ± sd. the comparisons of data mean were performed by tukey test. two-way anova was used for determination of significance or non-significance of data (p<0.05). results the chemical combination of cuminum cyminum boiss. essential oil accordingly, 15 compounds were identified that constitute 100% of the essential oils in total. the most abundant essential oil component was propanal, 2-methyl-3phenyl. the concentration of them was 24.2%. next, gammaterpinene, phenylethanediol, and 2-beta-pinene had the highest level of essential oil components, with 18.94%, 18.88%, and 12.59%, respectively (table 1). mic results the mic values of the ceo, nc, and bs against l. casei were 1%, 4%, and 0.3% (v/v), respectively. using a spectronic 20 spectrophotometer (varian, usa) was applied for determining the population of l. casei. cell concentration of l. casei was 2.2×1010 cfu ml-1 for inoculation. the enumeration of l. casei  was performed according to the serial dilution method and cultivating on (bhi) agar (merck, germany) after incubation for 24 h at 37ºc. determination of minimum inhibitory concentration (mic) of cuminum cyminum boiss. essential oil, nacl, and bile salt a 96-well plate with a volume of 300 μl was used in this experiment. sequential concentrations of essential oil of c. cyminum boiss (0, 500, 1000, 2000, 3000, 4000, 5000, 7500, and 10. 000 mg l-1), bile salt (0%, 0.05%, 0.02%, 0.03%, 0.06%, 0.07%, 0.08%, 0.1%, 0.2% and 0.3%) and nacl (0%, 1%, 2%, 3%, 4% and 5%) were used in de man, rogosa and sharpe agar (mrs) broth (merck, germany). media contained 5% dmso and were transferred to 96-well plates. then, 250 μl of different concentrations of essential oil, nacl, and bile salts along with 20 μl of l. casei suspension (5×106 cfu ml-1) were added to all well. the contents of each well were mixed with a shaker for 2 min. the microplates were closed by parafilm and then incubated for 24 h at 37°c in an anaerobic jar (merck, germany). at the end of incubation time, turbidity or non-turbidity was evaluated in the wells. adaptation and challenging conditions cultures were at the logarithmic phase. bacterial cells were separated by centrifugation (hettich, germany) and resuspended in fresh bhi broth (non-adapted control culture). the inoculation content of l. casei was 1 × 1010 cfu ml-1. adaptation time was conducted in the same medium at 37°c (i) for 120 min with 1 ml included in 100 ml of 5% dmso (ceo), (ii) 10 g per 100 ml in mueller hinton broth medium (merck, germany) (nc), (iii) 0.05 g per 100 ml in mueller hinton broth medium (bs), (iv) 1 ml in 100 ml containing 5% dmso plus 10 g per 100 ml in mueller hinton broth medium (ceonc), (v) 1 ml in 100 ml containing 5% dmso plus 0.05 g per 100 ml in mueller hinton broth medium (ceobs), (vi) 10 g per 100 ml plus 0.05 g per 100 ml in mueller hinton broth medium (ncbs) and (vii) 1 ml in 100 ml containing 5% dmso, 10 g per 100 ml plus 0.05 g per 100 ml in mueller hinton broth medium (ceoncbs). after centrifugation, adapted and non-adapted cells were inoculated to yogurt (108 per ml). enumeration of l. casei was conducted by serial dilution method (most probable number) at 0, 7, 14, 21, and 28 days of storage to evaluate survival rate. all the plates were incubated at italian journal of food science, 2021; 33 (sp1) 27 an assessment of cuminum cyminum (boiss) essential oil, nacl, bile salts and their combinations in probiotic yogurt table 1. gc/ms results of cuminum cyminum (boiss) essential oil no. name area (%) 1 α-pinene 1.12 2 sabinene 0.74 3 2-beta-pinene 12.59 4 β-myrcene 0.93 5 l-phellandrene 1.10 6 cymene 6.91 7 limonene 1.73 8 gamma-terpinene 18.94 9 (e)-4-(cyclohex-1’-enyl)but-2-en-1-ol 1.88 10 propanal, 2-methyl-3-phenyl24.22 11 2-caren-10-al 8.80 12 phenylethanediol 18.89 13 gamma-cadinene 0.57 14 trans-beta-farnesene 0.72 15 carotol 0.86 survival of lactobacillus casei the results showed that the interaction effects of treatment and storage time on  the l. casei population (figure 1) were significant (p<0.05). the viability of l. casei  decreased in all treatments except for control and sample under ceo stress at 7 days of storage time (p<0.05). the viability of  l. casei  of all samples significantly decreased during 28 days of storage (p<0.05). following 28 days of storage time, the highest survival rate of l. casei  was detected for ceo (6.05±0.03 log cfu  ml-1) and control (6.01±0.02 log cfu ml-1) treatments, which was significantly different from others (p<0.05). no significant difference was observed within nc and bs treatments (p>0.05). nevertheless, the lowest survival rate of l. casei was observed in the stress condition treated with ceoncbs at all days of storage time (p<0.05). ph the data for ph (figure 2) showed that the effect of time and type of samples were significant (p<0.05). the ph of all the yogurt samples decreased significantly during the storage period (p<0.05), and higher ph was attributed to samples under stress (except for the ceo) (p<0.05). at the end of 28 days of storage, the highest and lowest ph values for ceoncbs and the control treatments were 4.23±0.06 and 3.91±0.06, respectively (p<0.05). the comparison among all stress treatments showed the highest and lowest ph of samples were attributed to ceo and ceoncbs during storage time, respectively (p<0.05). no significant difference was observed between nc and bs treatments on the same day of storage (except on day 14) (p>0.05). figure 1. effect of cuminum cyminum essential oil (ceo), nacl (nc), bile salts (bs), and their combinations on the survival of l. casei atcc-39392 (log cfu ml-1). deviation bars designate the standard error of the method (n = 3). a a a a a ab a a a a b b b b b b bc bc bc b c c c bc c c c c c bc c c d b d d d d d e 4.5 5 5.5 6 6.5 7 7.5 8 0 7 14 21 28 lo g 10 c ou nt ( c f u /m l) storage time(days) control ceo bs nc ceobs ceonc bsnc ceoncbs 28 italian journal of food science, 2021; 33 (sp1) mohajeri n et al. a a a a a b a a b b b b b c c c c c c c c c d c d c bc cd d d b c e d e c d f e de 3.5 3.7 3.9 4.1 4.3 4.5 4.7 0 7 14 21 28 p h time (days) control ceo bs nc ceobs ceonc bsnc ceoncbs figure 2. effect of cuminum cyminum essential oil (ceo), nacl (nc), bile salts (bs), and their combinations on changes in ph of probiotic yogurt. deviation bars designate the standard error of the method (n = 3). d e e e f d e d d e c d c c d b c c c cd b c b b c b b b b c bc b 1.37 ab b a a a a a 0.7 0.9 1.1 1.3 1.5 1.7 0 7 14 21 28 ta ( % ) storage time (days) control ceo bs nc ceobs ceonc bsnc ceoncbs figure 3. effect of cuminum cyminum essential oil (ceo), nacl (nc), bile salts (bs), and their combinations on acidity changes of probiotic yogurt. deviation bars designate the standard error of the method (n = 3). titratable acidity (ta%) the titratable acidity (figure 3) results showed that interaction between group and time was significant (p<0.05). the acidity of all samples significantly increased during storage time (p<0.05). the acidity of the control sample was less than those of stress treatments (p<0.05). treatments ceo and ceoncbs had the highest and lowest acidity, respectively (p<0.05). after 28 days of the storage time, the lowest and highest acidity values were related to treatments under the stress of ceoncbs (1.19±0.008) and control (1.50±0.006), respectively. no significant difference was observed between nc and bc treatment during 28 days of storage (p>0.05). syneresis results showed that exchange within-group and time was notable (p<0.05). as shown in figure 4, the amount of syneresis significantly rose in all samples during storage time (p<0.05). the increase in intensity for control and ceo treatments was less than that for stress treatments (p<0.05). among the stressful treatments, the treatments under stress with ceo and ceoncbs had the lowest and highest percentage of syneresis, respectively, during 28 days of storage (p<0.05). no significant difference was observed between treatments with nc and bc on the same day of storage time (p>0.05). after 28 days of storage time, the lowest and the italian journal of food science, 2021; 33 (sp1) 29 an assessment of cuminum cyminum (boiss) essential oil, nacl, bile salts and their combinations in probiotic yogurt effect of concentrations less than mic was also seen on pathogenic bacteria. hence, the usage of concentrations of essential oils, nacl, and bile salts less than mic may also eliminate pathogens without harm to probiotics (calsamiglia et al., 2007). the viability of probiotic strains during production, food storage, and passage through the gastrointestinal tract is a major challenge in fermented dairy products. researchers reported that enumeration of probiotic strains for beneficial and therapeutic effects should be at least 106 to 107 cfu g-1 or ml in products (azizkhani and parsaeimehr, 2018). treatments with stresses of nc, bs, ceonc, ceobs, and ceoncbs maintained viability until 21 days of storage. however, control with ceo sample-maintained viability up to 28 days of storage time. the survival rate of l. casei in control and stress treatments with the ceo increased at 7 days of storage. a rise in the survival rate of  l.  casei  was observed to reduce ph at 7 days of storage. the increased survival rate of  lactobacillus acidophilus  la5,  lactobacillus fermentum, and  bifidobacterium  bb-12 in yogurt at 7 days of storage was reported  by azizkhani and parsaeimehr  . the bacterial population decline was attributed to the accumulation of organic acid during growth and fermentation. the main reasons for reducing ph are converting lactose to lactic acid, type of starter culture, duration of storage, and fermentation temperature (singh et al., 2011). several studies reported a decline in probiotic strains’ survival rate during storage time (azizkhani and parsaeimehr, 2018; yangilar and yildiz, 2018). indeed, decreased ph of products during storage was due to activation of the beta-galactosidase enzyme at 0–5°c, as well as post-acidification. ph values decreased to 4.2. the viability of probiotic bacteria is affected by increased highest percentages of syneresis were observed for control (8.30±0.09) and stress with ceoncbs (9.25±0.13) treatments, respectively. sensory properties table 2 showed the results of sensory characteristics (flavor, texture, and general acceptance) during 28 days of storage time. the interaction between group and time on sensory characteristics was significant (p<0.05). the sensory scores of examples decreased during storage time. a more severe drop in sensory score was observed for stress treatments except for the ceo (p<0.05). flavor scores for all treatments showed there were no significant differences among all samples on the first day of storage (p>0.05). the most leading and lowest scores were attributed to control (4±0) and ceoncbs (3.11±0.33) at 28 days of storage time, respectively (p<0.05). the texture feature results showed no significant difference within samples on the first day of storage (p>0.05). the highest and lowest scores were attributed to control (4.33±0.50) and ceoncbs (3.22±0.44) at 28 days of storage time, respectively (p < 0.05). overall acceptance of samples indicated no significant difference among all samples on the first day of storage (p > 0.05). the highest and lowest scores were attributed to control (4±0) and ceoncbs (3.220 ± 0.44) at 28 days of storage time, respectively (p < 0.05).  discussion and conclusion the effect of mic of the essential oils and salts in return to the survival of l. casei was quite high; the inhibitory f f e e e e e d d d d e cd c c d d c c c b c c d b c c bc c b bc b b b b a a a a a 6 6.6 7.2 7.8 8.4 9 9.6 0 7 14 21 28 s y ( g/ 10 0g ) storage time (days) control ceo bs nc ceobs ceonc bsnc ceoncbs figure 4. effect of cuminum cyminum essential oil (ceo), nacl (nc), bile salts (bs), and their combinations on syneresis changes in probiotic yogurt. deviation bars designate the standard error of the method (n = 3). 30 italian journal of food science, 2021; 33 (sp1) mohajeri n et al. table 2. sensory properties of probiotic yogurt samples with cuminum cyminum essential oil (ceo), nacl (nc), bile salts (bs), and their combinations. sensory properties yogurt samples storage time (days) 0 7 14 21 28 flavor control 5 ± 0a 5 ± 0a 5 ± 0a 4.67 ± 0.50a 4 ± 0a ceo 5 ± 0a 5 ± 0a 5 ± 0a 4.75 ± 0.44a 4 ± 0a bs 5 ± 0a 4 ± 0b 4 ± 0b 4 ± 0b 3.33 ± 0.50b nc 5 ± 0a 4 ± 0b 4±0b 3.78 ± 0.44b 3.33 ± 0.50b ceobs 5 ± 0a 4 ± 0b 4 ± 0b 3.78 ± 0.44b 3.33 ± 0.50b ceonc 5 ± 0a 4.33 ± 0.50b 3.78 ± 0.44b 3.78 ± 0.44b 3.33 ± 0.50b ncbs 4.67 ± 0.50a 4 ± 0b 3.78 ± 0.44b 3.22 ± 0.44c 3.11 ± 0.33b ceonsbs 4.75 ± 0.44a 4.33 ± 0.50b 4.33 ± 0.50b 3.22 ± 0.44c 3.11 ± 0.33b texture control 5 ± 0a 5 ± 0a 5 ± 0a 4.67 ± 0.23a 4.33 ± 0.50a ceo 5 ± 0a 5 ± 0a 4.56 ± 0.53b 4.33 ± 0.50bc 4.33 ± 0.50a bs 5 ± 0a 4 ± 0b 4.33 ± 0.50bc 4.33 ± 0.50bc 3.44 ± 0.53b nc 5 ± 0a 4.33 ± 0.50b 4.33 ± 0.50bc 4.33 ± 0.50bc 3.22 ± 0.44b ceobs 4.56 ± 0.53a 4 ± 0b 4 ± 0c 4 ± 0c 3.44 ± 0.55b ceonc 5 ± 0a 4 ± 0b 4 ± 0c 4 ± 0c 3.44 ± 0.44b ncbs 4.67 ± 0.50a 4.33 ± 0.50b 4 ± 0c 3.22 ± 0.44d 3.44 ± 0.44b ceonsbs 4.67 ± 0.50a 4 ± 0.70b 3.78 ± 0.44b 3.22 ± 0.44d 3.22 ± 0.44b overall acceptability control 5 ± 0a 5 ± 0a 4.89 ± 0.33a 4.67 ± 0.50a 4 ± 0a ceo 5 ± 0a 5 ± 0a 4.56 ± 0.53a 4.33 ± 0.50ab 3.78 ± 0.44ab bs 5 ± 0a 4 ± 0b 4 ± 0b 4 ± 0.70b 3.22 ± 0.44b nc 5 ± 0a 4 ± 0.70b 4 ± 0b 4 ± 0b 3.22 ± 0.44b ceobs 4.75 ± 0.44a 4 ± 0b 4 ± 0b 4 ± 0b 3.22 ± 0.44b ceonc 5 ± 0a 4 ± 0b 4 ± 0b 4 ± 0b 3.22 ± 0.44b ncbs 4.67 ± 0.50a 4.33 ± 0.50b 4 ± 0b 3.56 ± 0.53c 3.22 ± 0.44b ceonsbs 4.67 ± 0.50a 4 ± 0.70b 4.33 ± 0.50b 3.22 ± 0.44c 3.22 ± 0.44b each observation is a mean ± sd of three replications. in each column and row, means with the same letters had no significant difference at p > 0.05. hydrogen ions compared to lactate ions (kailasapathy, 2006). compared to treatments with stress, treatment with ceo had higher population viability of l. casei than others during storage time (p < 0.05). the susceptibility of microorganisms to essential oils depends on the details of essential oil and the type of microorganisms. the antimicrobial activity of essential oils is complicated due to their volatility, insolubility in water, and complex chemical structure (calsamiglia et al., 2007). the antibacterial effects of cuminum cyminum boiss. essential oil on different microorganisms depends on the concentration and composition of nutrients, storage temperature, and nature of the organism’s metabolites (mahmoudi, 2013). the survival rate of lactobacillus acidophilus in bioyogurt containing different concentrations of the essential oils, mentha piperita and ziziphoraclinopodioides, was significantly reduced 7 days of storage time at 4°c. one of the main criteria for selecting probiotic bacteria is resistance to nacl and bile salts (sarabi jamab and niazmand, 2009). according to the obtained results, treatments under stress with 0.15% bs were to be effective in the food at end of the 21st day. however, the survival rate of l. casei in bs stress treatment was significantly decreased from the 21st to the 28th day of storage time. it was less than the acceptable limit (p < 0.05). probiotic bacteria have different mechanisms of protection against stress, one of which is the bile hydrolysis system. the resistance of some strains to bile salts is associated with the bile salt hydrolysis activity. therefore, the hydrolysis of the bile salts will reduce their toxicity and side effects (sahadeva et al., 2011). according to taranto et al. (2006), lactobacillus delbrucium subsp. bulgaricus treated with different concentrations of thiorodoxylate (one of the bile salts) showed different levels of activity of the hydrolysis system (taranto et al., 2006). the researchers reported that in some bacterial cells, the bile hydrolysis system’s activity was significantly stronger than others, which resulted in cells showing greater resistance to higher concentrations and longer exposure times to these bile salts (taranto et al., 2006). when probiotic bacteria are exposed to bile salts, cellular homeostasis disorders occur. destruction of lipid membranes and cell italian journal of food science, 2021; 33 (sp1) 31 an assessment of cuminum cyminum (boiss) essential oil, nacl, bile salts and their combinations in probiotic yogurt of the yogurt during storage (kailasapathy, 2006). at the same time, akgun (2018) found that the syneresis rate in probiotic yogurt samples increased during storage at 4°c. the consistency of yogurt increased by stabilizers, an increase in the amount of milk casein concentration, and a reduction of acidification rate (everett and mcleod, 2005). samples with stress under essential oil had a lower syneresis percentage than others. the main reason for lower syneresis was acidification of yogurt containing herbal essential oils, which might cause high-strength gels, low permeability, a fine protein mesh, and higher water uptake. consequently, the syneresis of probiotic yogurt was decreased (ozer et al., 2007). in the overall acceptability of food products by consumers, sensory characteristics represent a vital role. researches affirm that flavor is the first criterion for food acceptance, followed by health considerations as the second rule (alizadeh khaledabad et al., 2020). the reduction of flavor scores in all samples during storage might be related to increasing acidity and reduced starter bacteria activity, which induced flavor components (yangilar and yildiz, 2018). the percentage of syneresis causes a change in the firmness of the sample. the firmness of yogurt decreased with increasing of syneresis. indeed, the percentage of syneresis was inversely correlated to yogurt’s firmness (ayar and gurlin, 2014). treatments that contained more probiotic counts had better flavor and texture than treatments with less probiotic counts, so the overall acceptance scores of yogurts with more probiotic counts were higher than the yogurt with less probiotic counts (under stress). proteolytic strains of lactobacillus could produce taste through carbohydrate metabolism, proteolysis, and low lipolysis processes. enzymes of lactobacillus hydrolyzed casein and produced large and medium bioactive peptides. proteolytic enzymes may subsequently degrade these peptides from starter bacteria, non-acidic lactic acid probiotic bacteria to small peptides and free amino acids, which are the major contributors to taste in dairy products (everett and mcleod, 2005; grom et al, 2020). all in all, the highest and lowest sensory scores were attributed to control and ceoncbs at 28 days of storage time, respectively. according to silva et al. (2018), salt is also expressed as a factor affecting the formation and development of aroma compounds and impacts the dairy product’s sensory properties. won-young et al. (2020) described that olive leaf extract to yogurt reduces the yogurt’s sensory score. according to the results, the ph and population of l. casei decreased for all the treatments during the storage time, while their acidity and percentage of syneresis increased. the enumeration of l. casei was in the range of the recommended amount (106_107 cfu ml-1) for probiotic yogurt with stresses less than mic during 28 membrane proteins leads to bacteria’s death (sahadeva et al., 2011). the survival rate of l. casei with considering salt stress was within acceptable limit until the end of 21 days of storage, and these findings are following other research that studied the viability of probiotic strain more than 2% of concentration (fortin et al., 2011). other researchers reported that probiotic strains’ viability decreased in samples with high concentrations of salt (4%) (hekmat et al., 2009). the suitable ph for commercial yogurt is 4.5. ph enhanced shelf life of the yogurt maintained mild taste and optimum appearance. undesirable ph (less than 4), which had been resulted by lactobacillus bulgaricus, produced large amounts of lactic acid, acetaldehyde, and byproducts from proteolytic activity (mahmoudi et al., 2014). the main reason for the lower ph of ceo, bs, and nc salts compared to the control sample was due to the presence of some phenolic compounds in the c.  cyminum boiss essential oil, which may have inhibitory effects on the growth of l. casei and changed ph values (mahmoudi, 2013). salt also affected ph and acidity values by influencing the growth of microorganisms, which may cause lactic acid production during storage time (fortin et al., 2013). some studies reported that probiotic yogurt’s ph decreased during storage time (mahmoudi, 2013 ; azizkhani and pasaeimehr, 2018). the acidification degree of probiotic yogurt is a crucial process control model that directly impacts the gel intensity and the commercially available fermentation period (alizadeh khaledabad et al., 2020). the production of organic acids by lactic acid bacteria was the main reason for increased acidity during storage time (mahmoudi et al., 2014). salt also influenced the acidity of samples because it impacted the growth of microorganisms, which influenced lactic acid production during storage time (fortin et al., 2013). the consumption of lactose by lactic acid bacteria led to lactic acid production, and then the acidity of samples increased. the production of lactic acid in yogurt is the main factor in producing a unique flavor. due to casein instability, conversion of the colloidal calcium phosphate complex to soluble calcium phosphate, calcium excretion, and casein coagulation take place at ph 4_4.6 (ramasubramanian et al., 2008). significant factors such as heterogeneity, high acidity, storage, breakage of protein strand, and structural rearrangement that will induce yogurt’s whey to leakage are recognized as substantial defects (yildiz and ozcan, 2020). the main reason for increased syneresis in probiotic yogurt during storage might be the activation of microorganisms of starter culture and their effects on long-chain biopolymers, which could be an important factor in reducing the softness and enhancing the syneresis 32 italian journal of food science, 2021; 33 (sp1) mohajeri n et al. yogurt . international dairy journal. 15(11): 1175–1183. . https://doi.org/10.1016/j.idairyj.2004.12.004 fontana, a., zacconi, c. and morelli, l., 2018. genetic signatures of dairy lactobacilluscasei group. frontiers in microbiology. 9: 2611. https://doi.org/10.3389/fmicb.2018.02611 fortin, m.h., champagne, c.p., st-gelais, d., britten, m., fustier, p. and lacroix, m., 2011. effect of time of inoculation, starter addition, oxygen level and salting on the viability of probiotic cultures during cheddar cheese production. international dairy journal. 21(2): 75–82. https://doi.org/10.1016/j.idairyj.2010.09.007 garcia, c., bautista, l., rendueles, m., and diaz, 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https://doi. org/10.1016/j.idairyj.2005.02.009 mortazavian, a.m., ehsani, m.r., mousavi, s.m., rezaei, k., sohrabvandi, s. and reinheimer, j.a., 2007. effect of refrigerated storage temperature on the viability of probiotic micro organisms in yogurt. international journal of dairy technology. 60(2): 123–127. https://doi.org/10.1111/j.1471 0307.2007.00306.x ozer, b., kirmaci, h.a., oztekin, s., hayaloglu, a. and atamer, m., 2007. incorporation of microbial transglutaminase into non-fat days of storage. therefore, it is possible to produce novel functional products such as probiotic yogurt containing herbal essential oil. the usage of combined c. cyminum boiss, nacl, and bile salts in cool conditions and mic up to 50% during 21 days of storage is recommended for probiotic yogurt containing l. casei. acknowledgments this work was supported by the department of food science and technology, islamic azad university, science and research branch, tehran, iran. references akgun, a., yazici, f., 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https://doi.org/10.1111/j.1745-4549.2011.00528.x� https://doi.org/10.1111/j.1745-4549.2011.00528.x� https://doi.org/10.1016/j.resmic.2006.04.002� https://doi.org/10.1017/s0022029900027564� https://doi.org/10.1080/19476337.2019.1640797� https://doi.org/10.1080/19476337.2019.1640797� https://doi.org/10.1111/jfpp.13332� https://doi.org/10.1111/jfpp.13332� https://doi.org/10.1111/1471-0307.12566� https://doi.org/10.1111/1471-0307.12566� https://doi.org/10.1016/j.cofs.2020.03.009� https://doi.org/10.1016/j.cofs.2020.03.009� https://doi.org/10.1016/j.idairyj.2006.02.007� https://doi.org/10.3168/jds.2008-1354� https://doi.org/10.3168/jds.2008-1354� https://doi.org/10.1016/j.cofs.2020.01.003� https://doi.org/10.3168/jds.2018-14819� _heading=h.gjdgxs _heading=h.30j0zll _heading=h.3znysh7 bookmark=id.2et92p0 bookmark=id.tyjcwt _heading=h.3dy6vkm _goback _heading=h.1t3h5sf _heading=h.4d34og8 _heading=h.2s8eyo1 _heading=h.17dp8vu _heading=h.3rdcrjn _heading=h.26in1rg _heading=h.lnxbz9 _heading=h.35nkun2 _heading=h.1ksv4uv _heading=h.44sinio _heading=h.2jxsxqh _heading=h.z337ya _heading=h.3j2qqm3 _heading=h.1y810tw _heading=h.4i7ojhp _heading=h.2xcytpi _heading=h.1ci93xb _heading=h.3whwml4 _heading=h.2bn6wsx _heading=h.qsh70q _heading=h.3as4poj _heading=h.1pxezwc _heading=h.49x2ik5 _heading=h.2p2csry _heading=h.147n2zr _heading=h.3o7alnk paper ital. j. food sci., vol. 27 2015 173 keywords: sweet cherry, quality, shelf life, edible coating effects of alginate edible coating on quality and antioxidant properties in sweet cherry during postharvest storage v. chiabrando* and g. giacalone department of agriculture, forest and food science, university of turin, largo braccini 2, 10095 grugliasco (to), italy *corresponding author: tel. +39 011 6708938, fax 0039 011 6708658, email: valentina.chiabrando@unito.it abstract two sweet cherry (prunus avium l.) cultivars (“big lory” and “grace star”), were treated with 1%, 3% and 5% sodium alginate as an edible coating before storage. analytical determinations were made after 7, 14 and 21 days of storage at 4°c. cherries were analyzed for the following quality parameters: firmness, weight loss, titratable acidity, soluble solid content, external color, anthocyanin content, phenolic content and total antioxidant capacity. alginate treatment at 3% delayed changes in most of the ripening parameters, weight and acidity losses, softening and color changes. however, the soluble solids content was not affected by the alginate fruit coating. in terms of the antioxidant properties, no significant results were obtained with the use of the alginate coating. the results of this study suggest that alginate treatments at 1% and 3% could be used as natural postharvest treatments in cherry cultivars with the aim of delaying the postharvest ripening process and maintaining fruit quality. mailto:valentina.chiabrando%40unito.it?subject= 174 ital. j. food sci., vol. 27 2015 introduction sweet cherry is one of the most appreciated fruits by the consumer due to its precocity and quality. among the factors determining consumer acceptability, soluble solids, skin color and acidity are the most important (díaz-mula et al., 2012). color is an indicator of quality and ripening of fresh sweet cherry and depends on the accumulation and profile of anthocyanins (crisosto et al., 2003). the antioxidant properties of sweet cherry are associated with the ascorbic acid and polyphenolic content (chaovanalikit and wrolstad, 2004; serrano et al., 2005). sweet cherries deteriorate rapidly after harvest and, in some cases, do not reach consumers with the optimal organoleptic quality. both the fruit and the stem consist largely of air and water, and the water is lost very quickly. the main causes of sweet cherry deterioration are weight loss, loss of acidity, softening, color changes, surface pitting and stem browning, along with changes in the soluble solids content (bernalte et al., 2003). adequate postharvest technologies combined with cold storage are fundamental. several preand postharvest technologies have been used to control cherry decay. in this sense, the use of an edible coating could be a new technological alternative to the use of modified atmosphere packaging to maintain fruit quality during storage, especially in minimally processed cherries (commercialized without the steam) (campos et al., 2011). edible coatings are traditionally used to improve fruits appearance and conservation. coatings on products create a semipermeable barrier to external elements that can reduce moisture loss, solute migration, respiration and oxidative reactions and retard the natural physiological ripening process (vargas et al., 2008). maintenance of fruit quality has been achieved by the utilization of different coatings such as chitosan in peach (li and yu, 2001) and nectarines (chiabrando and giacalone, 2013), pectin coating in melon (ferrari et al., 2013), alginate in apple (olivas et al., 2007; rojas-grau et al., 2007; chiabrando and giacalone, 2012), and hydroxypropylmethylcellulose and whey protein in plum (navarro-tarazaga et al., 2008; reinoso et al., 2008). in particular, alginate is a hydrophilic biopolymer that has a coating function because of its unique colloidal properties, which have allowed its use as a thickening agent, in forming suspensions and gels, and for stabilizing emulsions (acevedo et al., 2012). sodium alginate has been effective in maintaining postharvest quality in tomato (zapata et al., 2008) and peach (maftoonazad et al., 2008). in sweet cherry, some effects on fruit quality have been obtained with edible coatings based on chitosan (romanazzi et al., 2003), sodium alginate (diaz-mula et al., 2012), semperfreshtm (yaman and bayindirli, 2001) and with the use of aloe vera gel (ravanfar et al., 2012). thus, the aim of this study was to analyze the effect of sodium alginate applied as an edible coating at three concentrations (1%, 3% and 5% w/v) on the quality properties and antioxidant activity in grace star and big lory sweet cherry cultivars during storage at 4°c. materials and methods plant material sweet cherry (prunus avium l. cv “grace star” and “big lory”) fruits were harvested from a commercial plot. fruits were picked at commercial maturity stage, with a score of 4 on the color chart from the centre technique interprofessionel de fruits et légumes (ctifl, paris). fruits were transported immediately to the laboratory, and only whole and unwounded fruits were selected for the experiment. the following treatments were used: 0% (control), 1%, 3% and 5% (w/v) alginate coating. sodium alginate (sigma-aldrich co., steinhein, germany) was prepared according to previous reports (diaz-mula et al. 2012; chiabrando and giacalone, 2013), dissolved in hot water (45°c) with continuous shaking until the solution became clear. after cooling to 20°c, glycerol at 20% v/v was added as a plasticizer. fruits were dipped twice in the fresh coating solution for 1 min to assure the uniformity of the coating of the whole surface. after, fruits were dried for 30 min under an air-flow heater at 25°c. control fruits were dipped in distilled water. after drying, 30 cherries were placed in polypropylene (pp) punnets, weighed, and stored in a controlled chamber at 4°c and relative humidity of 90-95%. six punnets for each treatment were prepared, and after 7, 14 and 21 days of cold storage, two punnets per treatment were taken at random and used for the analysis. quality properties measurements and weight loss quality measurements were determined at day 7, 14 and 21 of storage. total acidity (meq/l), ph and the percentage of soluble solids (°brix) were measured according to official methods (aoac, 1995). the total soluble solids content (tss) was determined using the juice from five cherries at 20°c. three replicates were used for each treatment. titratable acidity (ta) was determined by titration with 0.1 n naoh up to ph 8.1, using 10 ml of diluted juice in distilled h 2 o. three replicates were used for each treatment. for fruit firmness measurements, a hand-held shore durometer (t.r. turoni, italy) was used and 30 fruits (replicates) per treatment were anital. j. food sci., vol. 27 2015 175 alyzed (kappel et al., 1996). weight loss was determined in each punnet by the percentage weight loss with respect to day 0. color measurements the color of coated cherries was measured at day 7, 14 and 21 of storage, individually for each fruit (30 for each treatment). the surface color was analyzed with a tri-stimulus cr-400 chroma meter (konica minolta sensing) with d75 illumination and observation angle of 10° calibrated with a standard white plate (y = 94.00, x = 0.3158, y = 0.3322, l* = 97.79, a* = -0.43, b* = + 2.25). two readings of l* (lightness), b* (yellow chromaticity), and a* (green chromaticity) coordinates were recorded for each cherry. numerical values of a* and b* parameters were employed to calculate the hue angle (h° = tan-1 (b*/a*)2) and chroma (c = (a*2 + b*2)0.5). the reported values are the mean ± sd of 60 determinations. anthocyanin content, phenolic content and total antioxidant capacity the anthocyanin content, phenolic content and total antioxidant capacity were measured at day 0 and after 21 days of storage. for determination of the anthocyanin content, phenolic content and total antioxidant capacity, extracts were prepared by weighing 10 g of fresh cherries into a centrifuge tube, adding methanol (25 ml) and homogenizing the sample for 1 min. extractions were performed under reduced light conditions. tubes were centrifuged (3000 rpm for 15 min) and the clear supernatant fluid collected and stored at -26°c. for identification and quantification, extraction was performed as three replicates. the anthocyanin content was quantified according to the ph differential method of cheng and breen (1991). anthocyanins were estimated by their difference in absorbance at 515 and at 700 nm in buffer at ph 1.0 and at ph 4.5, where a = (a515 a700) ph1.0 (a515 a700) ph4.5 . results are expressed as mg of cyanidin-3glucoside (c3g) per 100 g of fresh cherries. total phenolics were determined with folinciocalteu reagent following the method of slinkard and singleton (1977), using gallic acid as the standard. absorption was measured at 765 nm. results are expressed as mg gallic acid equivalents (gae) per 100 g of fresh cherries. the antioxidant activity was determined using a ferric reducing antioxidant power (frap) assay, following the methods of pellegrini et al. (2003) with some modifications. the antioxidant capacity of the diluted cherry extract was determined by its ability to reduce ferric iron to ferrous iron in a solution of tptz prepared in sodium acetate at ph 3.6. results are expressed as mmol fe2+/kg of fresh cherries. statistical analysis the basic experimental design consisted of four coating treatments, each having three replicates. for each parameter evaluated, two punnets containing 30 fruits each were considered a replicate and all determinations were performed in triplicate. data were analyzed by analysis of variance using statistical procedures in statistica ver. 6.0 (statsoft inc., tulsa, ok, usa). the sources of variance were the coating treatments. tukey’s test hsp (honestly significant differences) was used to determine significant differences among treatment means. mean values were considered significantly different at p ≤ 0.05. the mean values were calculated and reported as the mean ± sd (n = 3). results and discussion quality properties measurements and weight loss texture is a major factor defining the quality of fruit and strongly influences acceptability by consumers. the firmness values of cherries decreased, demonstrating texture softening during storage for both coated cultivars and control fruits, as shown in table 1. in cv big lory, the alginate coatings had a beneficial effect on fruit firmness. retention of firmness can be explained by retarded degradation of the components responsible for the structural rigidity the fruit, primarily insoluble pectin and proto-pectin. the fruit firmness of cv big lory at harvest was 67.24 n; after 14 days of storage, this value increased and then decreased at the end of storage at 4°c, reaching a final value of 41.41 n for the control and 57.08 n, 53 n and 48.72 n for the 1%, 3% and 5% coatings, respectively (table 1). by the end of the storage period, all the coating treatments gave rise to fruit with greater flesh firmness than the untreated fruit (p < 0.05) (table 1). in addition, significant differences were noted between the alginate coating treatments: higher values for flesh firmness were found for fruit coated with 1% and 3% alginate. the beneficial effect of the alginate concentration on firmness has also been reported for strawberry (hernández-muñoza et al., 2008), peach, japanese pear, kiwifruit (du et al., 1997) and citrus (chien et al., 2007). the coating of fruits can be expected to modify the internal gas composition of fruits, especially reducing the oxygen concentration and elevating the carbon dioxide concentration, which might explain the delayed textural changes in the coated fruits. in cv grace star, fruits tended to be less firm than cv big lory. at harvest, fruit firmness was 44.47 n; after 7 days of storage, this value increased and then decreased during storage at 4°c, reaching a final value of 32.87 n for the 176 ital. j. food sci., vol. 27 2015 table 1 changes in the quality parameters of coated cherries (cv big lory and grace star) stored at 4°c for 21 days. different letters in the same column indicate significant differences (p ≤ 0.05). column without letters have no significant differences. values at storage days cv big lory quality parameter treatments 0 7 14 21 firmness (n) control 67.24 65.80 80.60 b 41.41 b sodium alginate (1%) 67.24 65.06 85.26 a 57.08 a sodium alginate (3%) 67.24 64.80 84.31 a 53.00 a sodium alginate (5%) 67.24 66.01 84.05 a 48.72 ab total soluble solids content (°brix) control 16.43 15.67 a 14.85 b 14.37 b sodium alginate (1%) 16.43 16.97 a 14.37 b 16.17 a sodium alginate (3%) 16.43 13.83 b 14.47 b 16.00 a sodium alginate (5%) 16.43 15.47 a 16.93 a 14.23 b titratable acidity (meq/l) control 69.27 46.81 b 46.2 b 41.77 a sodium alginate (1%) 69.27 51.49 a 41.41 b 40.46 a sodium alginate (3%) 69.27 42.31 c 42.23 b 43.92 a   sodium alginate (5%) 69.27 47.79 b 48.25 a 31.56 b cv grace star quality parameter treatments 0 7 14 21 firmness (n) control 44.47 77.06 b 54.96 a 32.87 b sodium alginate (1%) 44.47 78.83 b 56.18 a 37.4 a sodium alginate (3%) 44.47 69.47 c 49.97 b 29.97 b sodium alginate (5%) 44.47 82.01 a 53.76 a 36.15 a total soluble solids content (°brix) control 17.87 18.16 18.3 17.17 sodium alginate (1%) 17.87 18.27 17.03 16.13 sodium alginate (3%) 17.87 18.3 18.5 17.97 sodium alginate (5%) 17.87 17.43 18.27 17.7 titratable acidity (meq/l) control 127.6 113.59 113.24 b 105.01 b sodium alginate (1%) 127.6 112.48 113.95 b 101.53 b sodium alginate (3%) 127.6 112.6 123.36 a 114.82 a   sodium alginate (5%) 127.6 110.91 113.03 b 116.43 a control and 37.4 n, 29.97 n and 36.15 n for the 1%, 3% and 5% coatings, respectively (table 1). fruit softening was delayed in the 1% and 5% alginate-treated cherries, while the control and 3% alginate-treated cherries exhibited a significantly higher reduction in firmness (table 1). for this parameter, the alginate concentration of 1%, according to the results obtained in cv big lory, was more effective than 3% and 5% alginate in reducing softening, especially at the last sampling date. the effects of edible coatings on decreasing softening have been also found in sweet cherry coated with semperfreshtm (yaman and bayindirli, 2002) and with aloe vera gel (martínez-romero et al., 2006). according to the results obtained for cv big lory, a 1% alginate edible coating significantly slowed down the softening process compared to the other coated treatments. in cv big lory, the tss at harvest was 16.43° brix, which decreased slightly during storage. at the end of storage, the 1% and 3% alginate coatings had a significant effect on tss (table 1), with significantly higher values compared with the control and 5% alginate coating. in this case, the 1% and 3% alginate coatings delayed the degenerative processes of the treated fruits. grace star showed higher tss values compared to the cv big lory at harvest and during storage with similar values in all the samples. no significant differences in tss values were found in relation to specific treatments, in accordance with the results of yaman and bayoindirli (2002). a decrease in total acidity is typical during postharvest storage of fleshy fruit and has been attributed to the use of organic acids as substrates for respiratory metabolism (valero and serrano, 2010). in cv big lory, ta values decreased from 69.27 meq/l at harvest to 41.77 meq/l in control fruits after 21 days at 4°c, and 40.46 meq/l, 43.92 meq/l and 31.56 meq/l in cherries coated with 1%, 3% and 5% alginate, respectively (table 1). only samples coated with 5% alginate showed significantly lower ta values. in this case, the use of the coating did not limit the degradation of organic acids. in cv grace star, the storage period led to a similar drop in the acidity of all samples, including the control. however, at the end of storage, acidity losses were significantly higher in the 1% algiital. j. food sci., vol. 27 2015 177 nate coated cherries and control, since after 21 days of storage was ≈ 20% and ≈ 18% respectively (table 1) respect to ≈ 9% in the 3% and 5% coated fruits. a decline in acidity demonstrates advanced maturation, thus the coating on the fruits contributed to delaying fruit maturation/ripening. in cv big lory, weight loss increased during storage, reaching values of 7.35% in control fruits after 21 days of cold storage and 8.15%, 7.4%, 8.25% in fruits coated with alginate at 1%, 3% and 5%, respectively, without significant differences between treatments. in cv grace star, after 21 days of storage, weight loss ranged from ≈10% in the control and 1% alginate coated cherries, to ≈ 12% in the 3% and 5% alginate coated cherries. greater losses were found in grace star compared to big lory, probably due to the larger size of the fruits of this cultivar. in both cultivars, no significant reduction of weight loss was detected in cherries treated with the coatings (data not shown). the use of an edible coating did not lead to a general reduction in weight loss, as expected. color measurements it is accepted that the most important quality parameters determining sweet cherry visual quality and acceptability by consumers are a bright red color and firmness (crisosto et al., 2003). hue angle is an indicator of ripeness and it is expressed as tan-1 (b*/a*)2. at harvest, big lory cherries had a red bright color with a hue angle of 24.44. during storage, an increase in hue angle was observed in all samples, in particular the coated cherries (table 2) with final values of 27.43, 27.56 and 27.65 in cherries coated with alginate at 1%, 3% and 5%, respectively, similar to that found in other sweet cherry cultivars (serrano et al., 2009). at the end of storage, the hue angle was significantly higher for all the coated fruits than in the controls (table 2). a decrease in hue angle could indicate the senescence process of sweet cherry, which is considered detrimental. in big lory, the coating treatments maintained the typical bright red color of recently harvested fruits with high table 2 changes in color parameters of coated cherries (cv big lory and grace star) stored at 4°c for 21 days. different letters in the same column indicate significant differences (p ≤ 0.05). column without letters have no significant differences. values at storage days cv big lory color parameter treatments 0 7 14 21 chroma control 33.88 27.91 b 31.66 b 26.26 b sodium alginate (1%) 33.88 33.76 a 34.56 a 31.56 a sodium alginate (3%) 33.88 33.89 a 30.96 b 33.34 a sodium alginate (5%) 33.88 33.78 a 28.72 b 30.46 a hue angle control 24.44 23.39 b 26.32 24.56 b sodium alginate (1%) 24.44 26.03 a 26.64 27.43 a sodium alginate (3%) 24.44 25.09 a 25.47 27.56 a sodium alginate (5%) 24.44 25.53 a 24.85 27.65 a lightness control 33.01 30.23 a 28.33 a 26.23 b sodium alginate (1%) 33.01 30.66 a 29.64 a 26.57 b sodium alginate (3%) 33.01 32.09 a 28.32 a 27.2 a   sodium alginate (5%) 33.01 27.73 b 27.87 b 26.74 b cv grace star color parameter treatments 0 7 14 21 chroma control 22.48 16.01 b 19.79 b 16.66 b sodium alginate (1%) 22.48 19.98 a 21.11a 18.57 a sodium alginate (3%) 22.48 15.62 b 18.46 b 16.87 b   sodium alginate (5%) 22.48 21.50 a 19.20 b 17.92 a hue angle control 21.92 22.2 26.38 a 27.08 sodium alginate (1%) 21.92 22.28 25.81 a 25.72 sodium alginate (3%) 21.92 22.05 26.77 a 26.55   sodium alginate (5%) 21.92 22.1 23.46 b 26.64 lightness control 23.81 24.77 18.39 b 21.16 sodium alginate (1%) 23.81 25.56 21.68 a 22.66 sodium alginate (3%) 23.81 24.9 20.17 b 22.33   sodium alginate (5%) 23.81 24.11 22.18 a 21.88 178 ital. j. food sci., vol. 27 2015 hue angle values during postharvest storage and even after 21 days of cold storage. in this case, the use of an alginate coating contributed to maintaining the original color of the cherries. the same trend was also observed in cv grace star, where hue angle values increased during storage, but without significant differences between treatments, in agreement with the results of yaman and bayoindirli (2002) with the semperfreshtm coating, but in disagreement with the results of cv big lory, where the use of the alginate coating contributed to maintaining the original color of the cherries. a decrease in l (lightness) is an indicator of fruit darkening. during storage, big lory darkened slightly as evidenced by decreasing values of l for control and all treated cherries (table 2). by the end of the storage period, l decreased by around 21% for control fruit, by around 19% for fruit coated with 1% and 5% alginate and by 17% for fruit coated with 3% alginate. this result confirms that the alginate coating exert significant effects in maintaining the original color of big lory cherries. in grace star, the lightness values of the cherries increased after 7 days of storage and then showed a decreasing trend until the end of cold storage. after 21 days of storage at 4°c, there were no significant (p≤ 0.05) differences in lightness values between the treated samples and the control (table 2). we may therefore conclude that the use of an alginate coating on grace star sweet cherries did not significantly alter, but rather improved the skin color or its evolution during storage at 4°c. the changes in the chroma values (c) of big lory cherries during storage are presented in table 2. fruit developed less vivid coloration, as evidenced by lower values of c in cherry samples during storage. the reduction in c values was significantly greater for uncoated fruit, and significant differences were found between control and coated cherries (p≤ 0.05). regarding the coated fruits, significant differences were found among samples treated with different concentrations of alginate, since 3% coated cherries showed higher c values. chroma was reduced by around 30% for control and 10% for coated cherries. in cv grace star, the main color changes were observed in the c values, which diminished during cold storage at 4°c, in particular in the control and 3% coated fruits. the values at 21 days were 25% and 24% lower, respectively, than those found at day 0 (table 2). anthocyanin content, phenolic content and total antioxidant capacity anthocyanins are responsible for the red color in sweet cherry (gardiner et al., 1993) and are beneficial to human health. in cv big lory, the anthocyanin content at harvest was 28.5 mg (c3g) and decreased significantly during storage (table 3), in agreement with bernalde et al. (2003). after 21 days of storage, significantly lower levels were found in 5% alginate coated fruits with mean values of 18.12 mg (c3g). in cv grace star, during post-harvest storage, anthocyanin significantly increased. thus, the cherries became darker during storage as ripening progressed. anthocyanin values showed that 5% alginate treatment delayed the ripening process, with significantly lower anthocyanin accumulation during storage compared with the other treatments (table 3). cherries coated with 1% and 3% alginate showed the greatest anthocyatable 3 values of anthocyanin contents, phenolic contents and total antioxidant capacity of coated cherries (cv big lory and grace star) at harvest and at the end after 21 days of storage. different letters in the same column indicate significant differences (p ≤ 0.05). storage (days) anthocyanins polyphenols antioxidant activity (mg cyanidin-3glucoside (c3g) (mg gallic acid equivalents (gae) (mmol fe2+/kg) per 100 g) per 100 g) cv big lory treatments harvest 0 28.5 57.49 13.64 control 21 15.11 b 42.17 a 12.62 a sodium alginate (1%) 21 15.14 b 41.41 a 12.00 a sodium alginate (3%) 21 14.32 b 35.68 b 10.92 b sodium alginate (5%) 21 18.12 a 39.88 ab 12.91 a cv grace star treatments harvest 0 53.85 194 15.33 control 21 64.65 b 156.56 a 15.17 a sodium alginate (1%) 21 85.96 a 140.44 a 15.76 a sodium alginate (3%) 21 76.15 a 128.22 b 15.07 a sodium alginate (5%) 21 58.15 c 93.83 c 15.12 a ital. j. food sci., vol. 27 2015 179 nin accumulation after cold storage. post-harvest increases in anthocyanin have been previously reported for cherries and for other small red fruits like raspberries, plums and strawberries (wang and stretch, 2001; serrano et al., 2009; díaz-mula et al., 2012). anthocyanin accumulation during storage is attributed to normal sweet cherry ripening. wong et al. (1992) suggested that the edible coating film forms a gas barrier, probably due to the dense structure of the film, so a possible modification of the internal atmosphere in coated samples due to film application could explain this behavior; this seemed to delay anthocyanin synthesis and/or degradation. polyphenols are important non-color compounds present in sweet cherry at harvest and during storage. these compounds not only contributed to the flavor but may also influence fruit color (mazza and brouillard, 1990). the total polyphenol content decreased in both cultivars after 21 days of storage at 4°c and total polyphenol contents were significantly different between treatments (table 3). in big lory, more pronounced changes were observed for the 3% and 5% coated samples, resulting in 37% and 30% losses, respectively (table 3). in grace star, there was a 19-51% loss, depending on the treatment (table 3). in the control and 1% alginate coated fruit, the decrease in the polyphenol content was significantly lower. the polyphenol content in the 5% coated fruit decreased during storage, and the value at 21 days of storage was 51% lower than that found at 0 days. no changes in antioxidant capacity were observed during cold storage (table 3). in particular, in big lory samples, the total antioxidant capacity at harvest was 13.69 mmol fe2+/kg and at the end of storage period this was 12, 10.92 and 12.91 mmol fe2+/kg in cherries coated with alginate at 1%, 3% and 5%, respectively. similarly, in grace star, the antioxidant activity remained stable during storage without differences between treatments (table 3). conclusions alginate treatments can be used as a natural postharvest treatment in sweet cherry cultivars with the aim of delaying the postharvest ripening process and maintaining fruit quality. alginate treatment at 1% and 3% was effective in delaying weight and acidity losses, softening and color changes in the cultivars big lory and grace star. in terms of the 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ripening process and to maintain tomato (solanum lycopersicon mill) quality. j. sci. food agric. 88: 1287. paper received february 7, 2014 accepted july 27, 2014 24 issn 1120-1770 online, doi 10.15586/ijfs.v34i1.2109 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (1): 24–32 effect of extraction process and storage time on the quality attributes of pomegranate juice of two local pomegranate varieties amal n. alkuraieef, amani aljahani* department of physical sport science, college of education, princess nourah bint abdulrahman university, riyadh, saudi arabia *corresponding author: amani al-juhani, department of physical sport science, college of education, princess nourah bint abdulrahman university, p.o. box 84428, riyadh 11671, saudi arabia. email: ahaljahani@pnu.edu.sa received: 8 november 2021; accepted: 30 december 2021; published: 28 january 2022 © 2022 codon publications open access paper abstract this study investigates the impact of two extraction processes (squeezing the whole fruit and centrifuging the seeds) of pomegranate juice and storage on sweet and sour pomegranate quality attributes. the ph, acidity, and levels of organic acids, sugars and anthocyanin differed in both varieties and changed during the storage period. fructose and glucose were the primary sugars, and citric acid was the dominant organic acid in the juice of both cultivars. a high level of established anthocyanin content was 15.40, 18.53, 18.03, 16.92, 16,68 and 15.47 mg/l when the storage period was 0, 5, 15, 32, 48 and 72 h, respectively, in the juice of sweet fruits obtained by squeezing the whole fruit. the juice prepared from the sweet fruits by squeezing method outscored, in all sensory quality attributes, the juice prepared by centrifuging process. keywords: anthocyanin; centrifugation of seeds; organic acids; sensory attributes; squeezing whole fruit introduction pomegranate (punica granatum) fruit is known as ‘miracle fruit’ because of its vast food and pharmaceutical applications. the seeds are wrapped with juicy edible pulp and are consumed as food, pressed for juice production, and used as functional foods (coronado-reyes et al., 2021; hegazi et al., 2021). pomegranate fruit, peel, seeds and juice have high medicinal applications for treating and preventing various diseases such as inflammation, diabetes, diarrhea, obesity, dysentery, dental plaque and malaria (ismail et al., 2012). in addition, parts of pomegranate fruit are also used as food additives, functional food materials and active ingredients in nutraceutical products (akhtar et al., 2015). pomegranate juice is a primary commercial product of pomegranate fruit with high consumer preferences because of its comprehensive nutritional and phytochemical components. it contains substantial amount of dietary polyphenols, tannins, anthocyanins and flavonoids with high potential antioxidant activities (fahmy et al., 2020). topalović et al. (2021) identified among 97 phenolic compounds, 23 anthocyanins and their derivatives, 33 ellagitannins and derivatives of ellagic acid, 12 flavonols, 4 flavonol glycosides, 1 flavone, 17 hydroxybenzoic acids and 7 hydroxycinnamic acids and their derivatives. cyanidin-3,5-diglucoside and cyanidin-3-glucoside are reported as primary anthocyanins in pomegranates (kostka et al., 2020). in addition, it was found that juice is a rich source of flavan-3-ols (2,650–9,820 mg/l), ellagitannins (2,010–6,420 mg/l) and hydroxybenzoic acids (720–3,390 mg/l) (topalović et al., 2021). therefore, pomegranate juice is favored as a healthy juice with great applications for treating and preventing obesity, diabetes, blood pressure and inflammation (hegazi et al., 2021). factors such as cultivars, maturity stage, harvest season, climatic and agronomical conditions, post-harvest processing and juice extraction processes greatly affect the nutritional and phytochemical composition of pomegranate juice (hegazi et al., 2021; mphahlele et al., 2014). italian journal of food science, 2022; 34 (1) 25 quality attributes of two-varieties of pomegranate juice washed thoroughly with tap water and rinsed thrice in distilled water before extraction of juice by the two processes. extraction of pomegranate juice the two processes used for the extraction of pomegranate juice from both sweet and sour fruits were as follows: 1. peels of the fruits (10 fruits of each type) were removed manually, and the seeds were separated. the juice was extracted from the seeds using an electric centrifuge (philips electric hr2738/01 citrus press juicer, france). 2. the fruits (10 fruits of each type) were cut with knives into two halves and squeezed using juice maker (philips viva collection juicer-hr1863, france) to obtain the juice. the extraction processes were repeated for six times, and the resulting juice was transferred into sterilized bottles and stored in the dark at 4°c for 72 h. the samples were collected at intervals of 0, 5, 15, 32, 48 and 72 h for analyzing physicochemical properties. determination of acidity and ph the acidity was measured by titrating 10-ml juice against 0.1-n naoh, and the results were expressed as percentage of citric acid (association of official analytical chemists [aoac], 2000). the ph was measured using digital ph meter. determination of organic acids organic acids (tartaric, citric, malic and oxalic acids) of pomegranate juice samples were analyzed using the highperformance liquid chromatography (hplc) method as described in the aoac (2000) standard methods with some modifications. briefly, 10-g juice sample was centrifuged at 6,000 relative centrifugal force (rcf) for 20 min, and the supernatant was collected and filtered using 0.45-μm filters (millipore). then, 20-μl supernatant was injected into reverse phase (rp) column (250 × 4.6 mm) and processed using 50-mm potassium phosphate buffer and 70% methanol at a flow rate of 1 ml/min. the peaks were detected at 210 nm, identified and quantified by comparing their retention time with the authentic standards of tartaric, citric, malic and oxalic acids. determination of sugars the sugar contents of pomegranate juice were analyzed using the shimadzu hplc system (lc-10advp, shimadzu, optimization of these conditions is highly important to produce pomegranate juice with good quality attributes. juice extraction is one of the critical steps that affect its functional properties, and this step is influenced by fruit genotype and other factors (hegazi et al., 2021; mena et al., 2014). generally, optimal time for fruit harvest and preparation process is the factor that plays an important role in the taste acceptability of consumers. in addition, the composition of organic acids and sugars in fruit juice plays a key role in flavor and sensory characteristics, such as ph, total acidity and sweetness (ikegaya et al., 2019). to date, several extraction processes have been used such as pressing the whole fruit or fruit halves by hand presser or squeezer, peeling off the fruit and extraction of juice using pressing and centrifugation, crushing the seeds and arils using the juice blender, and quartered cut fruit and pressing using rack and cloth (hegazi et al., 2021). of these processes, the hand pressing of fruit halves gave the highest yield of phenolic contents, anthocyanins and antioxidant activity (mphahlele et al., 2016). crushing the whole fruit or fruit halves resulted in a bitter taste because of the extraction of more tannins in the juice (miguel et al., 2004). in addition to the extraction process, fruit type (sweet, sweet–sour and sour) affected pomegranate juice’s nutritional and sensory quality attributes (hegazi et al., 2021; mphahlele et al., 2014). in saudi arabia, great interest has been found in recent years in producing and consuming pomegranate juice. consequently, cultivation of pomegranate has increased greatly, and several genotypes are cultivated throughout the country. sweet (taifi) and sour (bidah) genotypes are the primary pomegranate cultivars produced in western and southern saudi arabia. today, studies on production of juice from these cultivars are scarce. therefore, the present study was conducted to investigate the effect of juice extraction process (squeezing or centrifugation) and storage period (up to 3 days) on the quality attributes of pomegranate juice of taifi and bidah cultivars. material and methods materials the samples of sweet and sour pomegranate fruits were obtained from two different locations in saudi arabia. sweet pomegranate fruits were obtained from a farm of taif city located in western saudi arabia whereas sour fruits were obtained from bidah village of southern saudi arabia. the samples were transferred to laboratory on the same day of harvesting under controlled conditions. after sorting of samples by removing damaged fruits and selecting the same size and maturity stage fruits, they were 26 italian journal of food science, 2022; 34 (1) alkuraieef an and al-juhani a sensory analysis the sensory analysis of pomegranate juice samples of sweet and sour fruits extracted by above-mentioned two processes was conducted using 20-point scaling method (0–4: unacceptable, 5–8: acceptable, 9–12: good, 13–16: very good and 17–20: excellent; chen et al., 1991). a panel comprising 30 trained staff of the college of home economics, princess nourahbint abdulrahman university, saudi arabia, evaluated the color, smell, taste, texture and overall acceptance of pomegranate juice samples. the data were collected and subjected to statistical analysis. statistical analysis the data of three experiments were collected and analyzed using one-way analysis of variance (anova). the mean was calculated using student’s t-test, and p < 0.05 was considered as statistically significant (roscoe, 1975). results and discussion effect of storage and extraction process on the ph and acidity of pomegranate juice the ph and acidity values of pomegranate juice of sweet and sour varieties as affected by extraction processes and storage period are shown in table 1. kyoto, japan) equipped with a spherisorb 5 nh2 column (30 × 0.65 cm) and a 1530 refractive index detector (rid) (shimadzu). before analysis, 1-ml juice sample was centrifuged at 13,000 rpm for 20 min. the supernatant was collected and filtered using 0.45-μm millipore filters. then, 10-μl sample was injected into the column having a temperature of 35°c and separated using 75% acetonitrile as mobile phase at a flow rate of 1 ml/min. the sugars were identified by comparing their retention time with authentic standards run under the same conditions, and the concentration was calculated using the standard curves of sugars. determination of anthocyanin contents the anthocyanin contents of pomegranate juice samples were determined using the hplc system described in the aoac (2000) standard method. briefly, 1-ml juice sample was centrifuged (3,000 rcf, 20 min), and the supernatant was filtered using 0.45-μm millipore filters. the filtrate (20 μl) was injected into a 100-rp 10 lichrocart® column and separated using a linear gradient of 5% formic acid (a) and methanol from 15% to 35% (b) for 15 min, followed isocratic application to a total run time of 20 min. the flow rate was 1 ml/min, and the anthocyanin peaks were detected at 510 nm. the anthocyanin content of the samples was detected by comparing their retention time with that of authentic standard anthocyanins quantified from the standard curves generated using 0-, 0.01-, 0.02-, 0.04and 0.08-mg/l of authentic standard. table 1. effect of storage and juice production method on the ph and acidity of local sweet (taif) and sour (bidah) pomegranate juice. parameters storage (hours) 0 5 15 32 48 72 ph squeezing sweet pomegranate fruit 4.26 ± 0.03a,a 4.20 ± 0.02a,a 4.26 ± 0.02a,a 4.20 ± 0.05a,a 4.12 ± 0.08a,b 3.87 ± 0.04a,c centrifuging sweet pomegranate seeds 3.90 ± 0.01b,a 4.01 ± 0.09b,a 3.96 ± 0.09b,a 3.87 ± 0.10b,a 3.05 ± 0.15b,c 3.63 ± 0.20b,b squeezing sour pomegranate fruit 3.80 ± 0.03c,a 3.84 ± 0.03c,a 3.86 ± 0.09b,a 3.88 ± 0.08b,a 3.60 ± 0.15b,b 3.68 ± 0.11c,b centrifuging sour pomegranate seeds 3.60 ± 0.06d,a 3.66 ± 0.05d,a 3.60 ± 0.05c,a 3.67 ± 0.029c,a 3.44 ± 0.05b,b 3.50 ± 0.07c,b total acidity (mg/100 ml) squeezing sweet pomegranate fruit 288.1 ± 5.98c,b 289.8 ± 7.78c,b 302.1 ± 5.97c,a 296.0 ± 3.50c,a,b 294.2 ± 0.00d,a,b 294.2 ± 0.00d,a,b centrifuging sweet pomegranate seeds 394.1 ± 29.21b,a 381.8 ± 36.41b,a 404.6 ± 26.42b,a 385.3 ± 0.00b,a 385.3 ± 0.00c,a 378.3 ± 9.89c,a squeezing sour pomegranate fruit 394.1 ± 29.21b,b,c 381.8 ± 36.41b,b,c 404.6 ± 26.42b,a,b,c 448.3 ± 55.29a,a,b 453.6 ± 8.80b,a 444.3 ± 4.04b,a,b centrifuging sour pomegranate seeds 449.2 ± 16.26a,a 453.6 ± 23.99a,a 452.7 ± 24.81a,a 458.8 ± 4.041a,a 462.3 ± 5.7a,a 462.3 ± 13.78a,a means ± sd of 10 samples followed by different superscript letters are significantly different at p < 0.05. the small letters indicate differences in the treatments (columns), while the capital letter indicate differences in the storage time (rows). italian journal of food science, 2022; 34 (1) 27 quality attributes of two-varieties of pomegranate juice found in pomegranate juice (p < 0.05). similarly, citric acid was the primary organic acid found in the juice of various pomegranate varieties, and its content was affected by these varieties as reported by other studies (aarabi et al., 2008; türkyılmaz, 2013). moreover, citric acid was found higher in sour cultivars than sweet ones (ghaderighahfarokhi et al., 2016). the citric acid content in sour fruit juice remained unchanged for 72 h of storage, while it was reduced to minimum values at 15 h of storage in sweet fruit juice but increased again with the progress of storage period (p < 0.05). changes in citric acid during 60 days of storage differed due to variation in the fruit type, chemical changes and extraction processes used, as reported previously for various pomegranate cultivars (aarabi et al., 2008). tartaric acid and malic acid are the second major organic acids found in pomegranate juice of different varieties, as reported by previous studies (aarabi et al., 2008). content of tartaric acid was affected more by the extraction process than the fruit type. the highest values were observed in the juice prepared by squeezing of sweet and sour fruits (p < 0.05). the increased tartaric acid in the juice obtained by squeezing the whole fruits could be due to the fact that some amount of acid is found in the peels of the fruit and peeling off could lead to releasing of this amount. the storage period did not affect the tartaric acid content found in both types of pomegranate juices. the fruit type and the extraction process also affected the content of malic acid in pomegranate juice, and a pronounced effect was observed in sour fruit juice. the highest level of malic acid was found in the juice prepared by squeezing the whole sour fruits, while the least value was found in the juice prepared from the centrifugation of sour pomegranate fruit seeds (p < 0.05). variation in the content of malic acid was reported in the juice prepared from different pomegranate cultivars, suggesting the influence of cultivar on malic acid (aarabi et al., 2008; gundogdu and yilmaz, 2012; türkyılmaz, 2013). malic acid levels were gradually reduced with the storage time of the juice prepared by centrifuging the seeds of sour fruits. decrease in malic acid during storage was likely due to its metabolism by indigenous microflora present in the juice. decrease in the content of organic acids after pressing step was observed in pomegranate juice (akyıldız et al., 2020). the level of oxalic acid also varied between fruit types and extraction processes of the juice, with the highest value found in the juice prepared by squeezing whole sweet pomegranate fruits, and the lowest value found in the juice prepared by centrifugation of sour fruit seeds (p < 0.05). similarly, different levels of oxalic acid were reported in pomegranate juice obtained from different cultivars and by various extraction processes (aarabi et al., 2008; gundogdu and yilmaz, 2012; türkyılmaz, comparing the pomegranate types demonstrated that sweet pomegranate juice had a higher ph than that of sour juice. similarly, a previous report indicated that sweet pomegranate varieties have higher ph values than sour varieties (fadavi et al., 2005). the ph of juice was significantly affected by the extraction process and pomegranate variety, and high ph was determined in the juice prepared by squeezed method of sweet fruit seeds, followed by that of centrifuged sweet fruit seeds, whereas the least ph was found in the juice prepared from the centrifuged sour fruit seeds (p < 0.05). the storage period of up to 32 h did not affect the ph of juice; however, as the storage period increased to 48 h and 72 h, the ph of both types of pomegranate juice was decreased. the change in ph during extended storage period of pomegranate fruit juice was likely due to the formation of acids because of enzymatic and microbial activities during storage. the acidity was also higher (p < 0.05) in juice prepared from sour pomegranate fruits than that of sweet fruits, which agreed with the previous report of the juice prepared from 10 pomegranate varieties (fadavi et al., 2005). the highest acidity and the lowest ph of pomegranate juice of sour fruit types could be due to high acid content of sour varieties (fadavi et al., 2005). in this study, the extraction process greatly influenced the acidity of juice, with the highest values being found in the juice prepared from centrifuged sour fruit seeds, followed by that of squeezed sour fruit seeds, whereas the least acidity was observed in juice prepared from squeezed sweet fruits. again, the storage did not affect the acidity of the juice extracted by both processes of pomegranate fruits. it has been reported that the acidity of pomegranate juice obtained by centrifuging the seeds decreased after 32 h of storage whereas decrease in acidity of the juice obtained by squeezing method was less noticeable (miguel et al., 2004). difference between the results of these studies could be due to variation in the genetic background of used pomegranate fruits. effect of storage and extraction process on the content of organic acids of pomegranate juice the content of organic acids in the pomegranate juice prepared from two types of fruits and extraction processes during 72 h of storage at 4°c is presented in table 2. both fruit types (sweet and sour) and extraction processes (squeezing the fruit or centrifuging the seeds) affected the content of organic acids in different manners (p < 0.05) whereas the effect of storage period on content of organic acids was limited. citric acid is the major organic acid in pomegranate juice. its level was higher in the juice prepared from sour fruits than that prepared from sweet variety regardless of the extraction method, suggesting that the fruit type influenced the content of citric acid 28 italian journal of food science, 2022; 34 (1) alkuraieef an and al-juhani a the highest total organic acids content was found in the juice prepared by squeezing the whole sour fruits whereas the lowest organic acids content was found in the juice obtained by centrifuging the seeds of sweet fruits. 2013). during cold storage, the oxalic acid content fluctuated except in the case of juice prepared from the whole sweet pomegranate fruits, which demonstrated no change during storage. table 2. effect of storage and juice production method on the organic acids of local sweet (taif) and sour (bidah) pomegranate juice. parameters storage (hours) 0 5 15 32 48 72 citric acid (mg/100 ml) squeezing sweet pomegranate fruit 357.88 ± 65.76b,a,b 354.14 ± 11.15b,b 261.73 ± 9.98b,c 367.57 ± 4.66b,a,b 374.17 ± 9.67b,a 379.32 ± 7.12b,a centrifuging sweet pomegranate seeds 344.63 ± 6.40b,b 435.10 ± 45.87b,a 292.87 ± 57.08b,c 369.49 ± 15.17b,b 373.21 ± 20.83b,b 374.31 ± 25.82b,b squeezing sour pomegranate fruit 471.13 ± 51.66a,a 476.84 ± 2.27a,a 474.53 ± 3.43a,a 481.34 ± 4.92a,a 487.07 ± 1.363a,a 484.98 ± 6.04a,a centrifuging sour pomegranate seeds 469.36 ± 1.81a,a 472.48 ± 1.45a,a 477.14 ± 9.39a,a 479.58 ± 7.73a,a 479.48 ± 3.62a,a 476.98 ± 1.40a,a tartaric acid (mg/100 ml) squeezing sweet pomegranate fruit 159.30 ± 6.69a,a 185.98 ± 6.86a,a 156.99 ± 7.38a,a 155.11 ± 8.04a,a 155.05 ± 7.13a,a 154.15 ± 7.42a,a centrifuging sweet pomegranate seeds 135.64 ± 6.12b,a 134.76 ± 5.67b,a 133.34 ± 6.09b,a 131.93 ± 6.2b,a 131.31 ± 6.02b,a 129.62 ± 6.46b,a squeezing sour pomegranate fruit 157.30 ± 0.38a,a 158.79 ± 13.61a,a 158.81 ± 13.58a,a 152.28 ± 4.47a,a 147.24 ± 0.147a,b 155.32 ± 10.03a,a centrifuging sour pomegranate seeds 133.91 ± .0.17b,a 135.16 ± 0.12b,b 134.87 ± 0.45b,b 125.05 ± 0.28b,c 124.66 ± 0.26b,c 123.10 ± .0.91b,d malic acid (mg/100 ml) squeezing sweet pomegranate fruit 116.38 ± 2.16b,a 116.59 ± 5.29a,a 108.66 ± 11.56b,a 109.34 ± 8.69a,a 104.81 ± 7.87a,b,a 104.75 ± 7.05b,a centrifuging sweet pomegranate seeds 104.86 ± 10.54b,a 102.35 ± 11.39b,a 100.22 ± 10.95b,a 97.11 ± 11.30b,a 97.42 ± 9.95b,a 95.28 ± 11.58c,a squeezing sour pomegranate fruit 123.33 ± 0.57a,a 122.66 ± 1.54a,a 116.77 ± 0.71a,a 112.61 ± 0.56a,b 112.77 ± 7.24a,b 123.44 ± 9.12a,a centrifuging sour pomegranate seeds 92.77 ± 0.68c,a 87.87 ± 0.46c,b 88.12 ± 0.58c,b 85.67 ± 0.64c,c 85.15 ± 0.29c,c 85.44 ± 0.81d,c oxalic acid (mg/100 ml) squeezing sweet pomegranate fruit 20.62 ± 2.61a,a 20.36 ± 2.75a,a 18.61 ± 2.78a,a 21.91 ± 2.26a,a 19.97 ± 2.25a,a 20.02 ± 2.59a,a centrifuging sweet pomegranate seeds 11.19 ± 1.41b,a,b 10.61 ± 1.21b,a,b 9.83 ± 0.80b,b 12.06 ± 1.25b,a 11.19 ± 1.08b,a,b 11.47 ± 0.91b,a,b squeezing sour pomegranate fruit 11.73 ± 0.42b,a,b 11.30 ± 0.20b,a,b 10.90 ± 0.86b,b,c 10.64 ± 0.43b,c 10.41 ± 0.59b,c 12.57 ± 1.35b,a centrifuging sour pomegranate seeds 4.33 ± 1.54c,c 5.72 ± 1.61c,a,b 6.45 ± 0.69c,a,b 6.37 ± 0.61c,a,b 5.80 ± 0.45c,b,c 8.15 ± 0.54a,a total organic acids (mg/100 ml) squeezing sweet pomegranate fruit 654.18 ± 4.78c,a 650.07 ± 10.81c,a 454.98 ± 15.58c,b 653.92 ± 4.11c,a 654.00 ± 11.77c,a 658.24 ± 6.6c,a centrifuging sweet pomegranate seeds 596.32 ± 59.53da 592.81 ± 54.79da 536.25 ± 65.42 c a 610.59 ± 24.58da 613.13 ± 29.28da 610.68 ± 35.03da squeezing sour pomegranate fruit 763.49 ± 2.29aa 769.60 ± 11.60aa 761.00 ± 16.49aa 756.87 ± 3.74ba 757.48 ± 7.16aa 776.30 ± 12.47aa centrifuging sour pomegranate seeds 700.37 ± 2.48bb 701.23 ± 0.69bb 706.58 ± 9.04bb 969.68 ± 7.62aa 695.09 ± 3.33bb 693.67 ± 2.34bb means ± sd of 10 samples followed by different superscript letters are significantly different at p < 0.05. the small letters indicate differences in the treatments (columns), while the capital letters indicate differences in the storage time (rows). italian journal of food science, 2022; 34 (1) 29 quality attributes of two-varieties of pomegranate juice juice. however, as the storage period extended to 72 h, chemical complexing reactions and microbial metabolism might occur, which reduced the extractability of fructose and thereby its concentration. the content of glucose was highest in the juice obtained by centrifugation of seeds of sweet fruits (p < 0.05). storage greatly affected the glucose content of the juice of both types of fruits and extraction processes. glucose content generally increased to the maximum at 15 h (juice obtained by centrifugation process of sweet fruit seeds) and 48 h (juice of whole sweet and sour fruits and the seeds of sour fruits). the fruit type and the extraction process did not affect the total sugar level on 0 day of storage. however, as the storage time progressed, significant changes were observed in juice depending on the fruit types and extraction processes, especially at 72 h of storage, suggesting the combined effects of fruit type, extraction process and extraction time on the total sugar contents of pomegranate juice. previous reports have also indicated that sugar content in pomegranate juice decreased at 5 h of storage and then increased as the storage time progressed to the maximum at 48 h, and reduced again by the end of storage period (aarabi et al., 2008). this was attributed to decrease in carbohydrates because of the de novo synthesis of anthocyanin (aarabi et al., 2008). this might be the same reason for changes in the content of sugars during storage of pomegranate juice in the present study. effect of storage and extraction process on the anthocyanin content of pomegranate juice the anthocyanin content of pomegranate juice of two fruit types (sweet and sour) and two extraction processes (squeezing whole fruit and centrifuging of seeds) as affected by the storage time of the juice is shown in table 4. the level of anthocyanin in pomegranate juice was affected by fruit type, extraction process and storage time (p < 0.05). the highest anthocyanin level was observed in the juice prepared from whole sweet fruits, followed by that from whole sour fruits. the least level of anthocyanin was found in the juice prepared by centrifuging of seeds of both fruit types. this could be attributed to the fact that anthocyanin is found in pomegranate fruit peels, and removing the peels reduced its content in the juice. compared the fruit types, the highest anthocyanin content was observed in the juice prepared from whole sweet fruits than that from the whole sour ones. during storage, the anthocyanin content of the juice prepared from sweet fruits increased to the maximum at 5 h (whole fruit juice) and 15 h (seed juice). although the anthocyanin content decreased with increase in storage time, it decreased concomitantly in the case of sour fruits with increase of storage time (p < 0.05). the increase in anthocyanin content similarly, a higher level of organic acids was reported in the juice obtained from sour pomegranate fruits than that obtained from sweet fruits (ghaderi-ghahfarokhi et al., 2016). among the fruit types, the highest values of organic acids were found in the juice prepared from whole fruit, indicating that removal of peels reduced total organic acids. the content of total organic acids in pomegranate juice was not influenced by storage, except in the case of the juice prepared from whole sweet fruits, which demonstrated decrease at 15 h and then increased to the same level, and the juice prepared from sour fruit seeds, which increased to the maximum at 32 h and thereafter decreased to the initial level with progression in storage time (p < 0.05). the changes, i.e., increasing and decreasing trends, in the level of organic acids during storage were likely due to occurrence of different chemical and enzymatic reactions. content of organic acids fluctuated during storage, indicating decrease in the first 15 h of storage followed by an increment with progress in storage time (aarabi et al., 2008). variations in the levels of organic acids in pomegranate juice of different varieties and prepared by various processes have been reported in numerous studies (aarabi et al., 2008; gundogdu and yilmaz, 2012; türkyılmaz, 2013). the differences in the level of organic acids of pomegranate juice among these studies were likely due to variations in the genetic makeup, growing region and conditions, season and maturity stage, cultural and post-harvest practices, processes of preparing juice, and analysis of organic acids. effect of storage and extraction process on the content of sugars in pomegranate juice the content of sugars in pomegranate juice prepared from sweet and sour fruits using two extraction processes as affected by storage time is shown in table 3. fructose and glucose were the primary sugars found in pomegranate juice, and content of both was affected by fruit type and extraction and processing processes. previous studies have also indicated fructose and glucose as dominant carbohydrates in pomegranate juice (aarabi et al., 2008; fadavi et al., 2005; hasnaoui et al., 2011). on 0 day of storage, the highest (p < 0.05) level of fructose was found in the juice prepared from seeds of sweet pomegranate fruit by centrifugation extraction, whereas the least value was observed in the juice prepared by squeezing whole sweet fruit, suggesting the influence of extraction method on the fructose content of juice. the fructose content in the juice was also affected (p < 0.05) by the storage time in a fluctuated manner, reaching the highest level at 15 h (juice from the seeds of sweet fruits), 32 h (juice from whole sour fruits) and 48 h (juice from whole sweet fruits and seeds of sour fruits). the increase in the content of fructose during storage could be due enzymatic hydrolysis of disaccharides and polysaccharides present in the 30 italian journal of food science, 2022; 34 (1) alkuraieef an and al-juhani a table 3. effect of storage and juice production method on the sugar contents of local sweet (taif) and sour (bidah) pomegranate juice. parameters storage (hours) 0 5 15 32 48 72 fructose (g/100 ml) squeezing sweet pomegranate fruit 7.22 ± 0.34c,c 7.56 ± 0.58a,b 7.67 ± 0.32b,b 6.57 ± 1.44d,d 8.82 ± 2.55a,a 7.65 ± 0.34a,b,b centrifuging sweet pomegranate seeds 7.86 ± 0.12a,a,b 7.95 ± 0.08a,a,b 8.10 ± 0.24a,a 7.73 ± .010c,b,c 7.50 ± 0.39a,c 7.73 ± 0.19a,b,c squeezing sour pomegranate fruit 7.71 ± 0.01b,a 7.82 ± 0.04a,a 7.94 ± 0.04a,b,a 8.05 ± 0.03a,a 7.88 ± 0.12a,a 6.98 ± 0.81b,b centrifuging sour pomegranate seeds 7.69 ± 0.00b,b 7.68 ± 0.02a,b 7.81 ± 0.02a,b,a,b 7.84 ± 0.03b,a,b 7.89 ± 0.04a,a 7.81 ± 0.08a,a,b glucose (g/100 ml) squeezing sweet pomegranate fruit 6.93 ± 0.21b,a,b 7.12 ± 0.45a,b,a,b 7.37 ± 0.41b,a,b 6.25 ± 1.19d,b 8.66 ± 2.45a,a 7.36 ± 0..20a,a,b centrifuging sweet pomegranate seeds 7.45 ± 0.17a,b 7.56 ± 0.31a,b 7.98 ± 0.03a,a 7.55 ± 0.09a,b 7.47 ± 0.29a,b 7.62 ± 0.25a,b squeezing sour pomegranate fruit 7.07 ± 0.01b,c 7.16 ± 0.03a,b,b 7.32 ± 0.01b,a 7.35 ± 0.03b,a 7.38 ± 0.05a,a 6.67 ± 0.73b,d centrifuging sour pomegranate seeds 6.89 ± 0.01b,d 6.99 ± 0.03b,c 7.13 ± 0.03c,b 7.16 ± 0.02c,b 7.26 ± 0.03a,a 7.24 ± 0.05a,b,a total sugars (brix) squeezing sweet pomegranate fruit 16.0 ± 0.00a,c 16.8 ± 0.00b,a 16.5 ± 0.00 b,b 16.0 ± 0.00c,c 15.0 ± 0.00b,d 8.5 ± 0.00 c,e centrifuging sweet pomegranate seeds 16.0 ± 0.00a,b 17.3 ± 0.50a,a 17.0 ± 0.00a,a,b 17.3 ± 0.29a,a 16.5 ± 0.58a,a,b 17.3 ± 0.50a,a squeezing sour pomegranate fruit 16.0 ± 0.00a,b 16.0 ± 0.50b,a,b 16.0 ± 0.00b,b 16.5 ± 0.00b,a 16.0 ± 0.41a,b 9.3 ± 1.50 c,c centrifuging sour pomegranate seeds 16.0 ± 0.00a,a 16.0 ± 0.00b,a 16.0 ± 0.00b,a 16.6 ± 0.25b,a 15.8 ± 0.29b,b 13.1 ± 0.85b,c means ± sd of 10 samples followed by different superscript letters are significantly different at p < 0.05. the small letters indicate differences in the treatments (columns), while the capital letters indicate differences in the storage time (rows). at the beginning of storage (5 h and 15 h) could be due to the de novo synthesis of anthocyanin from carbohydrates (aarabi et al., 2008) whereas decrease in its concentration with increase in storage time could be likely due to chemical reactions and microbial metabolism. previous studies have also demonstrated the varying levels of anthocyanin in pomegranate juice of different origins such as tunisian (hasnaoui et al., 2011), iranian (alighourchi et al., 2008) and turkey (türkyılmaz, 2013) varieties. these studies established the influence of genotypes, environmental conditions, maturity stage, post-harvest processing and juice extraction processes on the levels of anthocyanin found in pomegranate juice. in addition, similar changing trend during storage of pomegranate juice extracted by two processes (squeezing and centrifugation) was also reported by aarabi et al. (2008). sensory attributes of pomegranate juice the sensory attributes of pomegranate juice prepared from two local varieties (sweet and sour) using two table 4. effect of storage and juice production method on the anthocyanin contents of local sweet (taif) and sour (bidah) pomegranate juice. parameters storage (hours) 0 5 15 32 48 72 alkali treatment squeezing sweet pomegranate fruit 15.40 ± 0.49a,c 18.53 ± 0.43a,a 18.03 ± 0.20a,a 16.92 ± 0.44a,b 16.68 ± 0.035a,b 15.47 ± 6.86a,c centrifuging sweet pomegranate seeds 11.07 ± 1.65c,c 14.32 ± 2.81b,a 14.92 ± 0.07b,a 14.12 ± 0.53b,a,b 13.55 ± 0.68b,b 12.15 ± 0.58a,c squeezing sour pomegranate fruit 14.74 ± 0.77b,a 12.79 ± 0.09c,b 12.47 ± 0.08c,b 11.99 ± 0.11c,c 11.70 ± 0.12c,d 11.11 ± 0.36a,e centrifuging sour pomegranate seeds 9.78 ± 0.82c,a 9.48 ± 1.00d,a 9.15 ± 1.09d,a 8.64 ± 1.14d,a,b 8.32 ± 1.13d,a,b 7.19 ± 0.87bb acid treatment squeezing sweet pomegranate fruit 0.56 ± 0.02a,b 0.76 ± 0.11a,a 0.75 ± 0.14a,a 0.71 ± 0.00a,a 0.70 ± 0.01a,a 0.68 ± 0.01a,a centrifuging sweet pomegranate seeds 0.34 ± 0.06c,b 0.50 ± 0.00b,a 0.54 ± 0.25b,a 0.53 ± 0.00b,a 0.54 ± 0.03b,a 0.51 ± 0.05b,a squeezing sour pomegranate fruit 0.47 ± 0.00b,a 0.46 ± 0.12b,a 0.45 ± 0.00b,a 0.43 ± 0.00c,a 0.43 ± 0.00c,a 0.42 ± 0.01c,a centrifuging sour pomegranate seeds 0.30 ± 0.00c,a 0.30 ± 0.05c,a 0.29 ± 0.02 c,a 0.28 ± 0.01d,a,b 0.27 ± 0.01d,b 0.26 ± 0.01d,b means ± sd of 10 samples followed by different superscript letters are significantly different at p < 0.05. the small letters indicate differences in the treatments (columns), while the capital letters indicate differences in the storage time (rows). italian journal of food science, 2022; 34 (1) 31 quality attributes of two-varieties of pomegranate juice peels. storage duration affected the quality attributes in a fluctuated manner. overall, squeezing unpeeled pomegranate fruit is the most economical and easy process to produce acceptable and stable juice, especially from sweet pomegranate fruits. the future studies must specifically address the effect of sterilization and long storage conditions on the quality attributes of pomegranate juice. acknowledgments this research was funded by princess nourah bint abdulrahman university researchers supporting project number pnursp2022r249, princess nourah bint abdulrahman university, riyadh, saudi arabia. references aarabi a., barzegar m., and azizi m.h. 2008. effect of cultivar and cold storage of pomegranate (punica granatum l.) juices on organic acid composition. asean food j. 15(15): 45–55. akhtar s., ismail t., fraternale d., and sestili p. 2015. pomegranate peel and peel extracts: chemistry and food features.  food chem.  174: 417–425. https://doi.org/10.1016/j. foodchem.2014.11.035 akyıldız a., karaca e., ağçam e., dündar b., and çınkır n.i. 2020. changes in quality attributes during production steps and frozen storage of pomegranate juice concentrate.  j food compos anal. 92: 103548. https://doi.org/10.1016/j.jfca.2020.103548 alighourchi h., barzegar m., and abbasi s. 2008. anthocyanins characterization of 15 iranian pomegranate (punica granatum l.) varieties and their variation after cold storage and pasteurization.  eur food res technol.  227: 881–887. https://doi. org/10.1007/s00217-007-0799-1 association of official analytical chemists (aoac). 2000. official methods of analysis. 17th ed. aoac, gaithersburg, md. borochov-neori h., judeinstein s., tripler e., harari m., greenberg  a., shomer i., and holland d. 2009. seasonal and cultivar variations in antioxidant and sensory quality of pomegranate (punica granatum l.) fruit.  j food compos anal.  22: 189–195. https://doi.org/10.1016/j.jfca.2008.10.011 extraction processes are shown in table 5. generally, the juice prepared from sweet fruits outscores the one prepared from sour fruits in all sensory attributes. this is due to the above-mentioned results, which indicated the superiority of sweet pomegranate juice over sour variety considering the levels of chemical components, namely sugars, organic acids and anthocyanins. similarly, it has been reported that the juice prepared from sweet pomegranate fruits received higher preferences than that prepared from sweet–sour and sour fruits (mayuonikirshinbaum et al., 2013). the scores of overall acceptability and all sensory attributes of all juice types were higher than 13, suggesting that the panelists ranked all juice types as very good and excellent, with the highest preference to the juice prepared from whole sweet fruits and the least preference for the juice prepared from the seeds of peeled off fruits. previous studies have demonstrated that the sensory attributes of pomegranate juice depended on the factors such as genotype, maturity, season, agronomical practices, climatic conditions and processing methods (borochov-neori et al., 2009; mayuoni-kirshinbaum et al., 2013; vázquez-araújo et al., 2014). overall, the produced juices could have high consumer acceptability regardless of the fruit type and the extraction process. conclusion this study investigated the production of pomegranate juice from local varieties using two different extraction processes (squeezing whole fruit, and centrifuging fruit seeds after removal of fruit peels). the results established that the quality attributes of the produced juice were affected by genotype, extraction method and storage period at different magnitudes. the juice prepared from the sweet variety had higher ph and sensory acceptability, and lower acidity and anthocyanin level than the juice prepared from sour fruits. concerning the processing methods, the juice prepared by squeezing the whole fruit outscore, in most of quality attributes, the juice prepared by centrifuging the seeds after removal of fruit table 5. sensory attributes of local sweet (taif) and sour (bidah) pomegranate juice produced by two different methods and from two different types of local verities. treatments sensory attributes stability color flavor texture smell overall acceptance squeezing sweet pomegranate fruit 17.7 ± 2.78a 17.5 ± 2.92a 17.76 ± 2.82a 17.00 ± 3.83a 17.66 ± 3.06a 17.96 ± 2.39a centrifuging sweet pomegranate seeds 15.93 ± 3.03 ab 15.76 ± 3.6 ab 16.70 ± 3.14 ab 14.93 ± 4.20 ab 16.86 ± 3.54 a 16.26 ± 2.80 a squeezing sour pomegranate fruit 15.16 ± 3.75 b 14.36 ± 4.24 b 15.20 ± 4.12 ab 13.70 ± 5.03ab 16.63 ± 2.97 a 15.26 ± 2.91 a centrifuging sour pomegranate seeds 13.50 ± 4.99 ab 14.53 ± 5.42ab 14.13 ± 5.09 b 13.16± 5.65 b 16.36 ± 3.66 a 14.20 ± 4.72 a means ± sd of 10 samples followed by different superscript letters are significantly different at p < 0.05. 32 italian journal of food science, 2022; 34 (1) alkuraieef an and al-juhani a scavenging activity and influence on cellular oxidative stress. foods. 9: 1617. https://doi.org/10.3390/foods9111617 mayuoni-kirshenbaum l., bar-ya’akov i., hatib k., holland d., and porat, r. 2013. genetic diversity and sensory preference in 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bioactive compounds in pomegranate (punica granatum l.)—a review.  sci hortic.  178: 114–123. https://doi.org/10.1016/j. scienta.2014.08.010 roscoe j.t. 1975. fundamental research statistics for the behavioral sciences. holt, rinehart and winston, new york, ny. topalović a., knežević m., gačnik s., and mikulic-petkovsek m. 2020. detailed chemical composition of juice from autochthonous pomegranate genotypes (punica granatum l.) grown in different locations in montenegro. food chem. 330: 127261. https://doi.org/10.1016/j.foodchem.2020.127261 topalović a., knežević m., ivanović l., gačnik s., and mikulicpetkovsek m. 2021. phytochemical screening of wild pomegranate (punica granatum l.) juices from the market. j. food compost. anal. 100: 103933. https://doi.org/10.1016/j.jfca.2021.103933 türkyılmaz m. 2013. anthocyanin and organic acid profiles of pomegranate (punica granatum l.) juices from registered varieties in turkey.  int j food sci technol. 48: 2086–2095. https:// doi.org/10.1111/ijfs.12190 vázquez-araújo l., nuncio-jáuregui p.n., cherdchu p., hernández  f., chambers iv, e, and carbonell-barrachina á.a. 2014. physicochemical and descriptive sensory characterization of spanish pomegranates: aptitudes for processing and fresh consumption. int j food sci technol. 49: 1663–1672. https://doi. org/10.1111/ijfs.12472 chen j., serafin f.l., pandya r.n., and daun h. 1991. effects of extrusion conditions on sensory properties of corn meal extrudates. j food sci. 56: 84–89. https://doi.org/10.1111/j.1365–2621.1991. tb07981.x coronado-reyes j.a., cortés-penagos c.d.j., and gonzálezhernández j.c. 2021. chemical composition and great applications to the fruit of the pomegranate (punica granatum): a review. food sci technol. 2021: 1–8. https://doi.org/10.1590/fst.29420 fadavi a., barzegar m., azizi m.h., and bayat m. 2005. note. physicochemical composition of ten pomegranate cultivars (punica granatum l.) grown in iran.  food sci technol int.  11: 113–119. https://doi.org/10.1177/1082013205052765 fahmy h., hegazi n., el-shamy s., and farag m.a. 2020. pomegranate juice as a functional food: a comprehensive review of its polyphenols, therapeutic merits, and recent patents. food funct. 11: 5768–5781. https://doi.org/10.1039/d0fo01251c ghaderi-ghahfarokhi m., barzegar m., and nabil m. 2016. geographical discrimination of iranian pomegranate cultivars based on organic acids composition and multivariate analysis. j agr sci tech. 18: 1221–1232. http://hdl.handle. net/123456789/3788 gundogdu m. and yilmaz h. 2012. organic acid, phenolic profile and antioxidant capacities of pomegranate (punica granatum l.) cultivars and selected genotypes. sci hortic. 143 38–42. https:// doi.org/10.1016/j.scienta.2012.05.029 hasnaoui n., jbir r., mars m., trifi m., kamal-eldin a., melgarejo p., and hernandez f. 2011. organic acids, sugars, and anthocyanins contents in juices of tunisian pomegranate fruits.  int j food prop.  14: 741–757. https://doi. org/10.1080/10942910903383438 hegazi n.m., el-shamy s., fahmy h., and farag m.a. 2021. pomegranate juice as a super-food: a comprehensive review of its extraction, analysis, and quality assessment approaches.  j food compos. anal.  97: 103773. https://doi.org/doi: 10.1016/j. jfca.2020.103773 ikegaya a., toyoizumi t., ohba s., nakajima t., kawata t., ito s., and arai e. 2019. effects of distribution of sugars and organic acids on the taste of strawberries. food sci. nutr. 7: 2419–2426. https://doi.org/10.1002/fsn3.1109 ismail t., sestili p., and akhtar s. 2012. pomegranate peel and fruit extracts: a review of potential anti-inflammatory and anti-infective effects.  j ethnopharmacol.  143: 397–405. https://doi. org/10.1016/j.jep.2012.07.004 kostka t., ostberg-potthoff j.j., briviba k., matsugo s., winterhalter p., and esatbeyoglu t. 2020. pomegranate (punica granatum l.) extract and its anthocyanin and copigment fractions-free radical paper ital. j. food sci., vol. 27 2015 151 keywords: sea bass, sea bream, quality parameters, season, farming location influence of season and farming location on the quality parameters of sea bass (dicentrarchus labrax) and sea bream (sparus aurata) marinko petrovića, greta krešićb, snježana zrnčićc, dražen oraićc, natalija džafićd and jelka pleadine* afood control center, faculty of food technology and biotechnology, university of zagreb, jagićeva 31, 10000 zagreb, croatia bfaculty of tourism and hospitality management, department of food and nutrition, university of rijeka, primorska 42, 51410 opatija, croatia ccroatian veterinary institute, laboratory for fish pathology, savska cesta 143, 10000 zagreb, croatia dcroatian veterinary institute, veterinary institute rijeka, laboratory for food and feed microbiology, podmurvice 29, 51000 rijeka, croatia ecroatian veterinary institute, laboratory for analytical chemistry, savska cesta 143, 10000 zagreb, croatia *corresponding author: tel. +38516123626, fax +38516123670, email: pleadin@veinst.hr abstract the study determined chemical composition of sea brass (dicentrarchus labrax) and sea bream (sparus aurata) farmed in adriatic sea, together with variation caused by seasonal variations and farming location. samples were collected from four different fish farms at three times: june 2012, october 2012 and january 2013. the presented results clearly show seasonal variations of moisture and fat content in the edible part of the fish, while the farming location was proven not to have any significant impact (p>0.05). fatty acid composition was significantly influenced both by the season and the farming location (p<0.05). the resulting n-3/n-6 ratios were lower than those reported in other studies, which can be attributed to differences in diet the fish were fed on. seasonal variations and farming location did not affect fish mineral composition, but mutual differences between the two species were significant. mailto:pleadin%40veinst.hr?subject= 152 ital. j. food sci., vol. 27 2015 introduction european sea bass (dicentrarchus labrax) and sea bream (sparus aurata) are some of the most important finfish species farmed in the mediterranean region (grigorakis et al., 2002; alasalvar et al., 2002; kyrana and lougovois, 2002; fuentes et al., 2010). fish as human seafood is widely consumed because of its high nutritional value, i.e. high protein content, low saturated fatty acid content and high n-3 fatty acid content. studies have reported that the consumption of only one fatty fish meal per day can result in an approximate n-3 fatty acid intake of 900 mg/day (epa and dha), sufficient to have a protective effect on cardiovascular system (kris-etherton et al., 2002). furthermore, fish is a rich source of minerals (e.g. sodium, calcium, iron, magnesium, copper, zinc and selenium) and vitamins (e.g. vitamin a, e and d) (fao/who, 2002; capelli et al., 2008; custódio et al., 2011). organoleptic properties and nutritional value are two sets of characteristics that, together with freshness, are accountable for fish quality. both of them strongly depend on the chemical composition of fish and are affected by many factors including intrinsic fish characteristics (such as species, age, sex, etc.), environmental factors (seasonal changes in temperature, salinity, etc.) and feeding regimen (composition of the feed in use, feeding ratio, etc.) (pirini et al., 2000; cordier et al., 2002; grigorakis et al., 2004; grigorakis, 2007). the quality of farmed fish strongly depends on factors involved into the production processes (periago et al., 2005). studies have also shown that, like most mediterranean fish, farmed fish spawns between december and march, correspondent to the peak sex steroid levels in their plasma. this period is featured by ovarian growth and a substantial reduction in food intake (cordier et al., 2002; cerdà et al., 1995). feeding on commercial nutritive products directly influences growth rates and meat quality, especially its lipid content and composition that can both be modified by diet (izquierdo et al., 2005). at the same time, an intense production of cultured fish has raised concerns over the quality of such fish in comparison to the wild one (alasalvar et al., 2002). an advantage of farmed fish over a wild-caught is that the first is produced and harvested under controlled conditions, so that hazards associated with fish consumption get to be reduced. a number of previous studies dealing with physico-chemical and organoleptic properties of wild and cultivated sea bream and sea bass have drawn attention to differences in fish quality, especially to the differences in lipid content and saturated and unsaturated fatty acid composition (grigorakis et al., 2002; alasalvar et al., 2002; fuentes et al., 2010; custódio et al., 2011; periago et al., 2005; saglik et al., 2003; orban et al., 2003). however, no data on the quality of these two species cultivated in the area of the eastern adriatic coast under our study, nor data on possible differences in fish meat composition between the two, arising from farming locations or seasonal variations, have been published insofar. therefore, the aim of this study was to evaluate the influence of season and geographical location on quality parameters of farmed sea bass and sea bream by virtue of analysing their chemical parameters, mineral and fatty acid composition. materials and methods growth conditions and fish sampling three individual samples of market size fish containing ten specimens of sea bass with mean body weight 342.77±86.90 g (92.92-473.18 g) and sea bream with mean body weight 294.70± 49.46 g (184.85-351.35 g) were collected in june 2012, october 2012 and january 2013 from four different commercial marine fish farms. two of these farms (1 & 2) are situated in the northern part of the adriatic coast (istria), while the remaining two (3 & 4) are situated in the mid-eastern coast (dalmatia). despite of their common location on the eastern adriatic coast, each of these sites has its particularities. site 1 is situated in a very long bay protected from the influence of an open sea, unlike site 2, which is situated at the entrance of the bay and is exposed to currents. at both sites, the temperature of the sea water fluctuates from 6°c in the winter to 2°c in the summer due to water shallowness. the remaining two sites are situated in the midcoast, on the islands exposed to currents and waves, so that the sea water temperature fluctuates less strikingly. its lowest temperature registered in the winter is about 12°c, while its highest temperature registered in the summer is about 25°c. fry is referred to on-growing originated from different commercial hatcheries and was cultivated in floating net cages of different size and shape for 22 to 28 months, the cultivation density thereby ranging from 5 to 12 kg per cubic meter. the fry was fed on a commercially available fish feed, with a daily ratio of 4.0-1.5 % of body weight for juveniles and 1.5-0.6% of body weight for premarket size. daily ratio was dependent on the sea temperature, photoperiod and oxygen saturation according to manufacturer’s recommendations. all samples of sea bass and sea bream were fed on a same commercial feed manufactured by international feed mills. according to the manufacturer’s declaration, the feed on which the sea bass was fed contained 40% of proteins, 24% of fat, 3% of fibres, 6.3% of ash and 0.9% of phosphorus from fish meal, as well as soybean meal, corn gluten, wheat and ital. j. food sci., vol. 27 2015 153 wheat by-products. the composition of the feed on which the sea bream was fed was very similar, with slight differences in the share of proteins (43%) and fat (16%). the fish was killed by virtue of immersing into ice/water slurry and transported to the laboratory on ice. before eviscerating and filleting the edible part of the fish for analysis, body weight and body length were measured to the closest g (184.85 to 473.18 g) and cm (23.9 to 31.7 cm). analysis of compositional parameters ten fillets per individual sample were homogenized separately using grindomix gm200 (retch, germany), so as to obtain a homogeneous sample for the determination of chemical parameters. for the sake of analysis, the entire meat portion was employed as edible. the sea bass and sea bream samples were analysed using standard analytical methods: iso 1442:1997 (moisture), iso 936:1998 (ash), iso 937:1978 (crude protein) and iso 3496:1994 (hydroxyproline/collagen). sodium and calcium content were determined by using an inhouse-validated titration methods described by trajković et al. (1983). for the determination of sodium, 2 g of the sample were homogenized with sand and 3 ml of water, transferred into a 100 ml-volumetric flask, stirred and placed into a water bath at 100 °c for 15 min. after cooling, the mixture was made up to volume with water and filtered. an aliquot (25 ml) of the filtrate was transferred into an erlenmeyer flask containing a few drops of k 2 cro 4 (62 g/100 ml of water) as indicator and titrated with 0.1 m-agno 3 until a persistent reddish colour was obtained. sodium content was calculated based on the volume of titration reagent used and its concentration. for the determination of calcium, the sample was heated in a furnace at 550 ºc up to white ashes were obtained. after cooling, ashes were transferred into a 250 ml-glass, into which 40 ml of hcl (30%), 60 ml of water and few drops of hno 3 (65%) were added. the solution was boiled for 30 min, cooled, and transferred into a 250 ml-volumetric flask and filled with water up to the mark. an aliquot of the filtered solution (100 ml) was transferred into a 250 ml-glass into which 1 ml of citric acid (300 g/l) and 5 ml of ammonium chloride solution (50 g/l) were added and made up to 100 ml with water. the solution was shortly boiled; then, 10 drops of bromocresol green solution and 30 ml of hot ammonium oxalate solution (4,5 g/100 ml of water) were added. the solution was neutralized by the addition of ammonia (25%) with constant steering up to ph 4.4-4.6, when the colour of the solution turned light blue. the solution was then left in a dark place for 30 min and filtered. after addition of 80 ml of h 2 so 4 (20%), the solution was heated to 80 ºc till all precipitates were dissolved and then titrated with kmno 4 (0.1 n) till the pink colour of the solution remained stable for 1 min. calcium content was calculated based on the expenditure of titration reagent and its concentration. for the determination of phosphorus, iso 13730:1996 spectrophotometric method was employed. all chemicals used for the analyses were of an analytical grade. lipid analysis total lipid contents were determined gravimetrically after extraction in a soxhlet apparatus according to the aoac method 948.22:2000. homogenized samples (5 g) had been extracted with petrol ether for six hours. the solvent was evaporated to dryness in a heated oven at 105°c, following which total lipid content was calculated. for fatty acid analyses, extracted triacylglycerols were converted into corresponding fatty acid methyl esters (fame) by trans-esterification with methanolic solution of potassium hydroxide. approximately 60 mg of the sample was weighed into a test tube equipped with a glass stopper, and dissolved in 4 ml of isooctane. after that, 200 μl of potassium hydroxide solution in methanol (2 mol/l) was added; the reaction was carried out at the room temperature, enhanced by vigorous shaking (2 x 30 s). the solution was neutralized by adding 1 g of sodium hydrogen sulphate monohydrate and transferred into a 2 ml vial. gc analyses were performed on cp-3800 (varian, palo alto, usa) using split/splitless injector and flame-ionisation detector. capillary column db 23ms 60 m x 0.25 mm, film thickness 0.25 μm, was used, the temperature thereby being first set at 60°c, then risen up to 210°c at the rate of 4 °c/min, and then rested at 210°c for 15 minutes. helium was used as a carrier gas at a flow rate of 1 ml/min. the temperature of the split/splitless injector was 250 °c; the same applies to the flame-ionisation detector, while the split ratio equalled to 1:20. the samples were injected manually (1.0 μl). the detector flow rates were as follows: hydrogen 30 ml/min, air 300 ml/ min, and detector makeup gas was helium with flow rate of 27 ml/min. a detailed description of the employed method and its suitability for the given purpose was published elsewhere (petrović et al., 2010). statistical analysis the statistical significance of difference between samples was tested by analysis of variance (anova) using statistica ver. 10.0 software (statsoft inc. 1984-2011, usa). a pvalue of 0.05 was considered statistical significance (p=0.05). 154 ital. j. food sci., vol. 27 2015 results and discussion mean values and standard deviations of chemical parameters determined in farmed sea bass and sea bream sampled at different adriatic coast sites and at three different times, are shown in table 1 and table 2, respectively. a significantly higher total fat content was observed for sea bream in comparison to sea bass, with a significant rise in total fat content in both species sampled in october in comparison to those sampled in june and january. results of our study also showed the same proportion of fat and moisture in both species, with fat content in range from 3.2 to 12.3% in sea bass and 4.2 to 15.0% in sea bream, dependent on, and highly varying according to, the farming season. our results respective to the seasonal variations of seam beam’s fat content are in agreement with the results of cardinal et al. (2011). previous data have shown the approximate composition of sea bass to be 70.71% of moisture, 20.35% of protein, 6.10% of total fat and 1.66% of ash (erkan and özden, 2007) or, according to another source, 76.72% of moisture, 19.43% of protein, 4.81 % of total fat and 1.23% of ash (kyrana and lougovois, 2002); the aforementioned data are similar to the results of this study obtained for the sea bass sampled in june and january. in 2001, huidobro et al. reported the chemical composition of the spanish sea bream to be 71.83% of moisture, 22.31% of protein, 5.28% of total fat and 1.27% of ash, whereas alasalvar et al. (2002) gave the approximate composition of 74.74% of moisture, 18.80% of protein, 6.53% of fat and 1.53% of ash. these results are also comparable to the values obtained in this study for the sea bream sampled in june. as fish is included in the category of ectothermic poikilotherms, the content of fat in a certain period of the year can be explained by physiological process of fat stock saving or spending. high values of fat obtained in october for all sampling sites and in both species, indicate the preparation of fish bodies for the winter period that comes after a long period of intense feeding. reduction of fat content in the winter mirrors the spending of fat consequent to the diet minimized due to the low sea temperature and slow fish metabolism. in early summer, when a change in the environmental conditions occurs, primarily in terms of temperature rises, fish metabolism is accelerated, and the energy taken from the food is used for fish growth, which, as seen in both this and earlier studies (james, 1995), resulted in a lower fat content. the analysis of variance (anova) revealed statistically significant differences (p<0.05) in moisture, fat and crude protein content of the sea bass, and statistically significant differ ences in moisture and fat content in the sea bream dependent on the sampling season (tatable 1 mean chemical composition (±sd) of the sea bass. june 2012 october 2012 january 2013 parameter farm 1 farm 2 farm 3 farm 1 farm 2 farm 3 farm 1 farm 2 farm 3 moisture (%) 73.2±2.4 74.0±1.8 75.5±2.3 67.5±1.6 72.0±2.6 68.8±2.28 70.9±2.87 72.4±2.01 72.6±1.85 ash (%) 1.16±0.13 1.24±0.15 1.42±0.07 1.32±0.12 1.40±0.09 1.27±0.10 1.62±0.11 1.44±0.06 1.40±0.09 fat (%) 6.6±0.3 3.7±0.4 3.2±0.2 12.3±1.6 8.0±1.1 11.9±0.9 6.0±0.6 5.5±0.4 5.1±0.2 crude protein (%) 20.64±0.82 21.50±0.71 22.52±1.21 19.30±1.18 19.09±0.98 18.89±1.02 21.47±1.22 20.51±0.95 20.82±1.03 hydroxyproline (%) 0.059±0.007 0.064±0.005 0.059±0.010 0.061±0.008 0.059±0.005 0.060±0.004 0.061±0.007 0.065±0.006 0.064±0.008 collagen (%) 0.47±0.06 0.51±0.04 0.47±0.08 0.49±0.06 0.47±0.04 0.48±0.03 0.49±0.06 0.52±0.05 0.51±0.06 ca (mg/kg) 701±12.1 685±11.4 731±9.2 754±12.3 813±17.1 662±18.4 723±20.5 802±21.8 725±24.3 p (mg/kg) 3665±33.6 3913±28.5 3843±17.5 3751±30.1 3656±26.5 3721±25.3 3616±41.3 3856±34.5 3589±29.4 na (mg/kg) 626±7.5 621±9.3 568±7.3 515±6.6 711±10.4 625±8.6 638±7.1 558±6.9 738±8.9 table 2 mean chemical composition (±sd) of sea bream. june 2012 october 2012 january 2013 parameter farm 1 farm 2 farm 4 farm 1 farm 2 farm 4 farm 1 farm 2 farm 4 moisture (%) 72.7±2.1 71.1±1.7 74.5±1.3 66.2±1.8 64.3±2.0 66.5±1.8 67.2±1.7 68.3±2.1 66.3±2.4 ash (%) 1.33±0.14 1.34±0.09 1.33±0.17 1.32±0.08 1.61±0.13 1.36±0.07 1.71±0.09 1.32±0.11 1.41±0.06 fat (%) 6.9±0.15 7.4±0.22 4.2±0.17 13.5±0.26 15.0±0.34 14.2±0.28 10.9±0.17 11.3±0.29 11.6±0.22 crude protein (%) 21.06±1.09 20.32±1.15 21.73±1.08 19.42±1.15 19.65±1.66 18.76±1.09 20.39±1.23 19.14±1.31 20.76±0.92 hydroxyproline (%) 0.069±0.008 0.070±0.012 0.063±0.008 0.071±0.006 0.073±0.010 0.070±0.011 0.061±0.009 0.062±0.007 0.065±0.006 collagen (%) 0.55±0.06 0.56±0.10 0.50±0.06 0.57±0.05 0.58±0.08 0.56±0.09 0.49±0.07 0.50±0.06 0.52±0.05 ca (mg/kg) 281±5.6 255±8.7 246±9.1 268±7.4 303±5.6 241±3.8 269±4.5 307±5.2 261±3.9 p (mg/kg) 3551±19.6 3456±25.8 3503±22.7 3321±18.4 3410±25.7 3545±28.8 3412±16.3 3478±23.4 3388±17.6 na (mg/kg) 312±4.7 365±3.4 311±6.2 268±2.5 338±3.8 341±4.5 283±5.1 298±3.9 319±4.8 ital. j. food sci., vol. 27 2015 155 table 3 statistical analyses (anova) of chemical parameters witnessed on various farming locations and during different farming seasons. parameter of analysis sea bass (p values) sea bream (p values) season location season location moisture 0.026* 0.193 0.008* 0.623 ash 0.243 0.998 0.615 0.833 fat 0.012* 0.207 0.002* 0.460 crude protein 0.031* 0.814 0.090 0.485 hydroxyproline 0.250 0.466 0.053 0.647 collagen 0.250 0.534 0.059 0.609 ca 0.583 0.431 0.558 0.154 p 0.472 0.442 0.512 0.785 na 0.867 0.793 0.430 0.170 *significantly different (p<0.05). ble 3). farming location had no significant effect (p>0.05) on chemical parameters and mineral composition of either of the two. del coco et al. (2009) studied the difference between the nutritional value of sea bream produced within three different farming systems and the wild fish. their results showed a significant difference in protein, lipid and cholesterol content between the fish grown within different farming systems. among fatty acids only oleic acid varied significantly. fatty acid composition, expressed as mean values and standard deviations obtained for sea bass and sea bream samples is shown in table 4 and table 5. the most represented fatty acid in both analysed species was oleic acid (c18:1 n-9, oa), followed by linoleic acid (c18:2 n-6, la) and palmitic acid (c16:0, pa). strobel et al. (2012) have recently reviewed studies that table 4 fatty acid composition (%) of the sea bass1. june october january fatty acid farm 1 farm 2 farm 3 farm 1 farm 2 farm 3 farm 1 farm 2 farm 3 c14:0 3.53±0.24 2.56±0.16 3.71±0.21 2.92±0.18 3.34±0.09 2.85±0.10 4.05±0.36 2.51±0.15 3.84±0.16 c15:0 0.35±0.01 0.30±0.02 0.47±0.17 0.30±0.11 0.43±0.01 0.30±0.01 0.37±0.03 0.25±0.01 0.35±0.05 c16:0 16.97±0.12 15.49±0.35 16.61±0.39 15.11±0.38 18.23±0.35 15.60±0.68 16.37±0.27 15.74±0.42 16.74±0.44 c16:1n-7 4.45±0.06 3.77±0.14 5.28±0.29 3.64±0.19 3.63±0.06 3.77±0.14 4.30±0.15 3.31±0.41 4.97±0.26 c17:0 0.55±0.02 0.52±0.11 0.65±0.09 0.53±0.05 0.67±0.02 0.55±0.02 0.55±0.04 0.43±0.03 0.37±0.02 c17:1n-7 0.12±0.03 0.17±0.01 0.32±0.09 0.18±0.01 0.14±0.01 0.17±0.01 0.18±0.03 0.16±0.01 0.17±0.02 c18:0 3.55±0.27 3.86±0.22 3.87±0.11 3.64±0.11 5.07±0.22 3.82±0.25 2.94±0.04 4.12±0.20 3.96±0.18 c18:1n-9 25.53±0.37 26.70±0.19 26.68±0.23 30.31±0.47 31.84±0.54 31.16±0.21 24.38±0.33 29.50±0.58 21.86±0.36 c18:1n-7 2.59±0.14 2.36±0.06 2.66±0.23 2.54±0.08 2.71±0.09 2.61±0.10 3.07±0.22 1.92±0.32 2.76±0.12 c18:2n-6 18.16±0.37 25.81±0.34 20.92±0.29 22.30±1.19 16.01±0.34 21.66±0.91 16.49±0.57 23.83±0.69 21.49±0.28 c18:3n-6 0.08±0.01 0.10±0.02 0.28±0.01 0.19±0.07 0.21±0.04 0.18±0.01 0.22±0.03 0.22±0.01 0.20±0.02 c18:3n-3 2.69±0.11 3.22±0.14 3.21±0.23 5.61±0.19 2.71±0.04 4.42±0.68 3.04±0.11 4.49±0.71 2.67±0.18 c18:4n-3 0.90±0.03 0.57±0.05 0.76±0.02 0.56±0.02 0.46±0.01 0.51±0.07 1.23±0.02 0.53±0.03 0.88±0.23 c20:0 0.31±0.02 0.28±0.02 0.31±0.02 0.31±0.03 0.49±0.02 0.30±0.02 0.16±0.02 0.28±0.02 0.16±0.05 c20:1n-9 3.75±0.28 1.93±0.19 1.48±0.14 2.17±0.35 2.24±0.38 2.09±0.11 5.27±0.32 2.54±0.40 1.75±0.08 c20:2n-6 1.13±0.27 0.95±0.03 0.87±0.08 0.73±0.17 0.25±0.01 0.76±0.03 0.68±0.02 0.76±0.08 0.82±0.20 c20:3n-6 nd nd 0.08±0.03 0.08±0.04 0.06±0.00 0.08±0.01 0.05±0.03 0.08±0.02 0.18±0.01 c20:4n-6 0.33±0.02 0.33±0.01 0.33±0.02 0.30±0.03 0.58±0.02 0.24±0.01 0.35±0.07 0.28±0.06 0.44±0.02 c20:3n-3 nd nd 0.09±0.02 0.15±0.06 0.10±0.01 0.11±0.04 0.16±0.02 0.12±0.02 0.19±0.02 c20:4n-3 0.46±0.02 0.42±0.02 0.43±0.13 0.30±0.01 0.44±0.04 0.29±0.01 0.48±0.02 0.29±0.02 0.37±0.02 c22:0 nd 0.09±0.02 nd 0.15±0.01 0.29±0.12 0.16±0.02 0.07±0.01 0.16±0.01 0.32±0.03 c20:5n-3 3.46±0.90 3.64±0.54 2.88±0.14 2.61±0.35 2.18±0.15 2.72±0.15 3.78±0.44 2.55±0.27 6.65±0.29 c22:1n-11 3.24±0.15 1.04±0.09 0.86±0.39 1.03±0.05 1.57±0.23 0.93±0.14 3.53±0.32 1.16±0.42 0.60±0.06 c22:1n-9 0.38±0.02 0.20±0.01 0.39±0.02 0.26±0.03 0.46±0.01 0.24±0.03 0.45±0.02 0.26±0.03 0.29±0.10 c22:5n-3 0.92±0.14 0.80±0.22 1.81±0.16 0.71±0.16 0.71±0.05 0.71±0.02 1.00±0.32 0.70±0.21 1.70±0.14 c24:1n-9 0.41±0.03 0.24±0.02 0.40±0.08 0.20±0.12 0.71±0.02 0.21±0.03 0.23±0.05 0.17±0.01 0.13±0.03 c22:6n-3 6.14±0.21 4.65±0.34 4.66±0.22 3.21±0.55 4.49±0.72 3.58±0.53 6.58±0.23 3.62±0.42 6.15±0.20 sfa 25.26±0.19 23.09±0.64 25.61±0.88 22.96±0.29 28.52±0.64 23.56±0.97 24.52±0.32 23.49±0.48 25.70±0.97 mufa 40.46±0.38 36.41±0.26 38.07±0.24 40.35±0.72 43.28±0.47 41.18±0.24 41.41±0.29 39.03±0.47 32.58±0.34 pufa 34.28±0.57 40.50±0.74 36.32±0.75 36.69±0.91 28.20±0.68 35.25±1.21 34.06±0.41 37.48±0.58 41.72±0.62 total n-3 14.58±0.40 13.30±0.11 13.85±0.70 13.14±0.62 11.09±0.29 12.34±1.02 16.27±0.37 12.30±0.20 18.60±0.54 total n-6 19.70±0.37 27.20±0.48 22.47±0.35 23.55±0.35 17.11±0.48 22.91±0.76 17.79±0.25 25.18±0.21 23.13±0.18 total n-3/total n-6 0.74±0.01 0.49±0.01 0.62±0.03 0.56±0.03 0.65±0.04 0.54±0.04 0.91±0.02 0.49±0.01 0.80±0.04 hh 2.87±0.01 3.70±0.09 3.02±0.13 3.46±0.03 2.71±0.09 3.60±0.14 2.82±0.01 3.63±0.02 3.01±0.04 1each value represents the mean value ± standard deviation of three samples of the extracted fat per group, analysed twice (n=6). nd – not detected, detection limit 0.05% abbreviations: sfa, saturated fatty acids, mufa, monounsaturated fatty acids, pufa, polyunsaturated fatty acids, hh, hypocholesterolaemic/hypercholesterolaemic ratio = (c18:1 n-9+ c18:2 n-6+c20:4 n-6 + c18:3 n-3+c20:5n-3+ c22:5 n-3+ c22:6n-3)/(c14:0+c16:0) 156 ital. j. food sci., vol. 27 2015 confirmed an increase in fatty acids with 18c such as oa, la and ala in farmed fish, gained through the use of vegetable oils in their feed. the concentration of longchain polyunsaturated acids – eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) in total fatty acids was in range of 2.18-6.65% for epa and 3.21-6.58% for dha in sea bass samples, and in the range of 0.79-1.48% for epa and 0.94-3-19% for dha in sea bream samples. these results are due to the feeding strategy, since it is well known that fatty acid composition in fish meat reflects dietary fatty acid profile. over the last decades, fish nutrition research has devoted continued effort to the development of sustainable feeds that can provide long-chain n-3 fatty acid levels adequate for human nutrition (izquierdo et al., 2005; kris-etherton et al., 2003). it has been suggested that “n-3/n-6 ratio” reptable 5 fatty acid composition (%) of the sea bream1. june october january fatty acid farm 1 farm 2 farm 4 farm 1 farm 2 farm 4 farm 1 farm 2 farm 4 c14:0 3.67±0.13 4.29±0.16 3.09±0.10 5.41±0.30 2.75±0.11 2.95±0.30 2.71±0.34 3.32±0.44 2.77±0.45 c15:0 0.30±0.02 0.43±0.03 0.38±0.04 0.38±0.05 0.24±0.03 0.27±0.06 0.24±0.02 0.26±0.01 0.23±0.01 c16:0 15.87±0.31 17.99±0.26 15.37±0.44 20.77±0.11 13.77±0.24 14.55±0.51 14.09±0.42 15.25±0.24 15.00±0.42 c16:1n-7 4.61±0.16 5.35±0.27 4.59±0.33 6.19±0.16 4.41±0.17 4.43±0.22 3.65±0.24 4.85±0.23 3.99±0.16 c17:0 0.40±0.02 0.58±0.02 0.68±0.04 0.49±0.04 0.44±0.02 0.46±0.02 0.42±0.14 0.57±0.04 0.41±0.03 c17:1n-7 0.06±0.01 0.20±0.02 0.29±0.04 0.11±0.01 0.21±0.02 0.20±0.03 0.21±0.03 0.32±0.02 0.20±0.02 c18:0 3.92±0.27 4.55±0.29 3.46±0.21 3.94±0.17 3.28±0.19 3.40±0.12 3.67±0.13 4.07±0.54 3.29±0.27 c18:1n-9 25.74±0.37 27.11±0.34 26.51±0.27 30.68±0.30 28.02±0.34 30.02±0.91 27.79±0.32 25.27±0.46 28.44±0.73 c18:1n-7 2.50±0.04 2.67±0.15 2.42±0.12 3.15±0.12 2.32±0.05 2.46±0.17 2.01±0.20 2.97±0.57 2.44±0.53 c18:2n-6 22.96±0.38 22.61±0.28 24.93±0.27 10.92±0.18 24.20±0.32 22.46±0.44 27.32±0.46 25.19±0.62 25.80±0.19 c18:3n-6 0.22±0.01 0.05±0.01 0.26±0.01 0.09±0.01 0.21±0.02 0.23±0.08 0.23±0.01 0.21±0.05 0.30±0.02 c18:3n-3 2.90±0.15 2.09±0.15 3.41±0.17 1.43±0.15 4.51±0.25 5.21±0.54 5.37±0.10 3.01±0.07 5.34±0.07 c18:4n-3 0.76±0.10 0.46±0.01 0.60±0.04 0.45±0.02 0.57±0.03 0.59±0.02 0.52±0.11 0.63±0.11 0.51±0.03 c20:0 0.31±0.01 0.41±0.02 0.28±0.03 0.31±0.02 0.38±0.04 0.35±0.02 0.27±0.03 0.23±0.02 0.21±0.01 c20:1n-9 2.86±0.28 1.85±0.24 1.97±0.10 5.00±0.20 1.74±0.14 1.72±0.36 1.54±0.23 1.67±0.31 1.59±0.38 c20:2n-6 0.67±0.07 0.61±0.02 0.80±0.05 0.14±0.03 0.76±0.21 0.64±0.22 0.66±0.02 0.59±0.12 0.65±0.05 c20:3n-6 0.26±0.07 0.07±0.02 0.08±0.03 0.11±0.01 0.24±0.10 0.19±0.08 0.22±0.02 0.22±0.02 0.21±0.01 c20:4n-6 0.28±0.02 0.32±0.11 0.38±0.03 0.14±0.02 0.25±0.11 0.23±0.04 0.22±0.03 0.33±0.01 0.21±0.01 c20:3n-3 0.20±0.02 0.40±0.12 0.06±0.01 0.11±0.03 0.31±0.17 0.26±0.02 0.28±0.05 0.16±0.01 0.27±0.02 c20:4n-3 0.52±0.06 0.48±0.15 0.42±0.11 0.32±0.02 0.35±0.03 0.33±0.01 0.47±0.02 0.49±0.02 0.47±0.02 c22:0 nd 0.11±0.02 0.07±0.01 0.15±0.01 0.19±0.05 0.16±0.04 0.20±0.02 0.16±0.02 0.16±0.00 c20:5n-3 2.04±0.26 1.48±0.15 3.46±0.38 0.79±0.26 2.45±0.02 2.15±0.40 1.96±0.22 2.96±0.43 1.97±0.09 c22:1n-11 2.81±0.15 1.40±0.12 1.00±0.03 5.64±0.19 1.04±0.31 1.04±0.08 0.70±0.13 0.76±0.14 0.72±0.04 c22:1n-9 0.55±0.03 0.43±0.02 0.19±0.02 0.81±0.32 0.41±0.09 0.39±0.02 0.32±0.02 0.29±0.04 0.31±0.01 c22:5n-3 1.49±0.27 1.07±0.16 0.82±0.11 0.60±0.07 2.01±0.26 1.45±0.43 1.36±0.31 2.04±0.43 1.34±0.41 c24:1n-9 0.54±0.04 0.49±0.11 0.14±0.04 0.65±0.13 0.36±0.11 0.37±0.04 0.32±0.02 0.28±0.02 0.14±0.03 c22:6n-3 3.58±0.28 2.48±0.23 4.34±0.40 0.94±0.18 4.56±0.23 3.47±0.47 3.19±0.33 3.89±0.32 3.04±0.32 sfa 24.52±0.21 28.42±0.28 23.34±0.34 31.46±0.15 21.06±0.48 22.14±0.52 21.60±0.88 23.86±0.29 22.08±0.64 mufa 39.61±0.29 39.51±0.47 37.11±0.24 52.33±0.29 38.50±0.47 40.64±1.04 36.52±0.34 36.40±0.38 37.76±0.46 pufa 35.87±0.41 32.07±0.31 39.56±1.21 16.21±0.34 40.44±0.24 37.22±0.56 41.87±0.75 39.73±0.57 40.16±0.58 total n-3 11.49±0.37 8.46±0.20 13.11±0.65 4.64±0.31 14.77±0.29 13.46±0.69 13.13±0.70 13.18±0.40 12.94±0.49 total n-6 24.39±0.25 23.61±0.21 26.45±0.48 11.57±0.19 25.67±0.21 23.76±0.87 28.74±0.25 26.55±0.37 27.22±0.48 totak n-3/total n-6 0.47±0.01 0.36±0.01 0.50±0.04 0.40±0.01 0.58±0.01 0.57±0.02 0.46±0.03 0.50±0.01 0.48±0.01 hh 3.08±0.03 2.63±0.08 3.54±0.09 1.84±0.03 4.00±0.08 3.77±0.08 4.04±0.02 3.42±0.02 3.78±0.02 1each value represents the mean value ± standard deviation of three samples of the extracted fat per group, analysed twice (n=6). nd – not detected, detection limit 0.05% abbreviations: sfa, saturated fatty acids, mufa, monounsaturated fatty acids, pufa, polyunsaturated fatty acids, hh, hypocholesterolaemic/hypercholesterolaemic ratio = (c18:1 n-9+ c18:2 n-6+c20:4 n-6 + c18:3 n-3+c20:5n-3+ c22:5 n-3+ c22:6n-3)/(c14:0+c16:0) resents a reliable index for inter-species comparisons of relative nutritional values (piggot and tucker, 1990). according to sargent (1997), the optimum n-3/n-6 pufa ratio should be 1:5 (0.2). generally fish have this ratio more favourable as also evident from the results shown in tables 4 and 5. however, our results showed lower n-3/n-6 ratios in comparison to comparative studies of farmed and wild sea bass and sea bream (alasalvar et al., 2002; fuentes et al., 2010; periago et al., 2005; saglik et al., 2003). orban et al. (2003) studied lipid quality of wild fish and fish farmed in north adriatic, and also obtained n-3/n-6 ratio higher than ours. similar or even lower n-3/n-6 ratios were presented by cardinal et al. (2011), who studied seasonal variations of physico-chemical and sensory characteristics of sea bream coming from the market. these differences in n-3/n-6 ital. j. food sci., vol. 27 2015 157 muscle protein content may be less important than the fat one, but proteins, especially those interacting with water, contribute to organoleptic quality of the fish meat (zayas, 1997). from the nutritional point of view, fish proteins are important since, according to the recent fao data, fish accounts for 15.7% of the global population’s animal protein intake and 6.1% of the entire protein intake (fao, 2012). in this study, crude protein content varied in range from 18.89% to 22.52% for sea bass, and 18.76% to 21.73% for sea bream, in accordance with the proximate values obtained in earlier studies, summarized in the review of grigorakis (2007). depending on the species, collagen and hydroxyproline fish meat contents vary in range from 0.28% to 0.79% and 30 to 98 mg/100 g, respectively (morrisey and fox, 1981). values ratios are caused by the differences in formulation of diets used in fish farms. it is obvious that farms that were compared to the wild fish have better adapted their fish meals. however, the recent research conducted by sofi and co-workers (2013) confirmed that the intake of fish with similar epa+dha content but different n-3/n-6 ratio has different effects on lipid, inflammatory and haemoreological parameters of healthy subjects. as it is formally recommended to humans to take 0.3 to 0.5 g of n-3 fatty acids (epa + dha) per day (pratoomyotet et al., 2010), consumers´ weekly needs should be satisfied with the consumption of approximately 600 g of sea bass or sea bream. consumers with coronary heart disease should be encouraged to increase their daily consumption to even 200 g of sea bass or sea bream. due to the known effects of specific fatty acids on cholesterol metabolism, one of the indicators of nutritional quality may also be the ratio between hypocholesterolaemic and hypercholesterolaemic fatty acids (hh) (santos-silva et al., 2002; testi et al., 2006). generally, sea bream had higher hh index due to the lowest share of saturated fatty acids. the highest hh value, i.e. the most desirable one (4.04), was found in the sea bream sampled in january. the influence of season and location on fatty acid composition of both fish species is presented in table 6. statistical analysis showed the influence of these parameters on the sea bass to be stronger than on the sea bream. as for the sea bass, significant difference (p<0.05) in the content of almost all n-3 fatty acids was shown, resulting in differences in both total n-3 fatty acid content and n-3/n-6 ratio. although diet is the main factor that affects n-3 and n-6 pufa content in fish, location, species, season and environmental conditions may also play a role (hossain, 2011). cordier et al. (2002) also reported differences in n-3/n-6 ratio, especially the difference in epa over aa ratio. that is in accordance with our results, although within the frame of our study arachidonic acid (p=0.012) was influenced by the season, and not by the farming location. epa over aa ratio is considered to be an important parameter, since dietary intake of n-3 pufas helps replacing, at least to a point, n-6 fatty acids in cell membranes, most importantly in platelet, erythrocyte and neutrophil cell membranes (simopoulos, 2002). cardinal et al. (2011) also reported significant season-dependent differences in fatty acid content, but this was attributed to the rearing conditions, feed formulation included, which were actually not controlled within their study. significant variations in some fatty acids present in fish meat, especially variations in linoleic acid (c18:2 n-6) (p=0.013), possibly arise as a consequence of different plant contents present in the diets selected as common feed by various farms. table 6 statistical analyses of fatty acid profile (anova) of the sea bass and sea bream. sea bass (p values) sea bream (p values) fatty acid season location season location c14:0 0.715 0.184 0.521 0.315 c15:0 0.556 0.392 0.216 0.947 c16:0 0.258 0.463 0.672 0.473 c16:1n-7 0.841 0.476 0.344 0.278 c17:0 0.244 0.719 0.408 0.859 c17:1n-7 0.909 0.597 0.665 0.523 c18:0 0.225 0.196 0.538 0.514 c18:1n-9 0.069 0.497 0.132 0.778 c18:1n-7 0.565 0.238 0.795 0.171 c18:2n-6 0.210 0.013* 0.200 0.310 c18:3n-6 0.183 0.220 0.610 0.683 c18:3n-3 0.714 0.528 0.262 0.151 c18:4n-3 0.043* 0.106 0.764 0.596 c20:0 0.089 0.419 0.083 0.477 c20:1n-9 0.052 0.001* 0.380 0.216 c20:2n-6 0.406 0.859 0.538 0.333 c20:3n-6 0.417 0.095 0.650 0.932 c20:4n-6 0.012* 0.153 0.242 0.913 c20:3n-3 0.004* 0.015* 0.722 0.133 c20:4n-3 0.053 0.407 0.001 0.563 c22:0 0.243 0.433 0.027 0.255 c20:5n-3 0.120 0.328 0.762 0.717 c22:1n-11 0.153 0.002* 0.329 0.200 c22:1n-9 0.983 0.090 0.257 0.209 c22:5n-3 0.009* 0.009* 0.681 0.868 c24:1n-9 0.117 0.112 0.311 0.313 c22:6n-3 0.041* 0.310 0.892 0.800 sfa 0.301 0.624 0.627 0.437 mufa 0.153 0.915 0.246 0.431 pufa 0.066 0.135 0.420 0.419 total n-3 0.033* 0.193 0.736 0.549 total n-6 0.326 0.014* 0.415 0.560 total n-3/total n-6 0.038* 0.092 0.442 0.439 *significantly different (p<0.05). 158 ital. j. food sci., vol. 27 2015 obtained in this study for both fish types are within this range. other studies reported lower collagen values in farmed fish in comparison to the wild one, which was presumed to be related to the swimming behaviour (sato et al., 1986), as well as to other factors such as a higher number of muscle fibres which predestines for greater collagen content (sikorski et al., 1984). the main functions of fish minerals include skeleton structuring, maintenance of colloidal system and regulation of acid-base equilibrium; in addition, these minerals also represent the important hormone, enzyme and enzyme activator constituents (belitz and grosch, 2001). literature data have demonstrated that the origin of fish and their feeding pattern did not have any effect on mineral composition, except for that on calcium content (fuentes et al., 2010). calcium (ca) and phosphorus (p) are necessary for maintaining optimal bones development, being a higher intake of both minerals required during childhood and growing ages so as to prevent rickets and osteomalacia (erkan and özden, 2007). the content of these minerals determined in sea bass of this study, ranged from 662 to 813 mg/kg and from 3589 to 3913 mg/kg, respectively, while that in sea bream from 241 to 281 mg/kg and from 3321 to 3551 mg/kg, respectively. as reported earlier by erkan and özden (2007), mean contents of these minerals in sea bass were also significantly higher (p<0.05) than those determined in sea bream. furthermore, obtained results for ca content in sea bream are in a good agreement with results published by orban et al. (2003) whose results ranged from 220 to 230 mg/kg. as for ca, sodium (na) content found in sea bass was significantly higher than in sea bream. results obtained for sea bass ranged from 515 to 738 mg/kg, while for sea bream ranged from 268 to 365 mg/kg. literature have reported sea bream na contents ranging from 280 to 370 mg/kg and sea bass na contents of 773±1.8 mg/kg (orban et al., 2003). other sources reported na content about 289±1.6 mg/kg in sea bream (erkan and özden, 2007), which is quite similar to the data obtained within this study. conclusion results of our study clearly show seasonal variations of moisture and fat content in both fish species. as for sea bass, differences in fatty acid composition were shown, while in sea bream these differences were not observed. the n-3/n-6 ratios were lower than those previously reported for farmed and wild fish. fish nutritional value is related to n-3 fatty acids, and is heavily dependent on the production process. since fish is promoted as a good source of n-3 fatty acids, efforts should be made to properly tailor lipid quality, basically relying on dietary manipulation so as to fit fat deposition and fatty acid profile. seasonal and location changes did not affect mineral composition, but mutual difference between the two species under study was significant. references alasalvar c., taylor k.d.a., zubcov e., shahidi f. and alexis m. 2002. differentiation of cultured and wild sea bass (dicentrarchuslabrax): total lipid content, fatty acid and trace mineral composition. food chem. 79: 145-150. belitz h.d. and grosch w. 2001. lehrbuch der lebensmittelchemie, berlin heidelberg new york, springer verlag. capelli r., das k., pellegrini r.d., drava g., lepoint g., miglio c., minganti v. and poggi r. 2008. distribution of trace elements in organs of six species of cetaceans from the lingurian sea (mediterranean), and the relationship with stable carbon and nitrogen rations. sci. total environ. 390: 569-578. cardinal m., comet j., donnay-moreno c., gouygou j.p., berge j.p., rocha e., soares s., escorcio c., borges p. and valente l.m.p. 2011. seasonal variation of physical, chemical and sensory characteristic of sea bream (sparusaurata) reared under intensive conditions in southern europe. food contr. 22: 574-585. cerda` j., zanuy s., carrillo m., ramos j. and serrano r. 1995. shortand longterm dietary effects on female sea bass (dicentrarchus labrax): seasonal changes in plasma profiles of lipids and sex steroids in relation to reproduction. comp. biochem. physiol. c 111:83–91. cordier m., brichon g., weber j.-m. and zwingelstein g. 2002. changes in the fatty acid composition of phospholipids in tissues of farmed sea bass (dicentrarchuslabrax) during an annual cycle. roles of environmental temperature and salinity. comp. biochem. physiol. b133: 281-288. custódio p.j., pessanha s., pereira c., carvalho m.l. and nunes m.l. 2011. comparative study of elemental content in farmed and wild life sea bass and gilthead bream from four different sites by faas and edxrf. food chem. 124: 367-372. del coco l., papadia p., de pascali s.a., bressani g., storelli c., zonno v. and fenizzi f.p. 2009. comparison among different gilthead sea bream (sparusaurata) farming systems: activity of intestinal and hepatic enzymes and 13c-nmr analysis of lipids. nutrition 1: 291-301. erkan n. and özden ö. 2007. proximate composition and mineral contents in aqua cultured sea bass (dicentrarchuslabrax), sea bream (sparusaurata) analyzed by icp-ms. food chem. 102: 721-725. fao. 2012. the state of world fisheries and aquaculture, fisheries and aquaculture department, rome. http:// www.fao.org/docrep/013/i1820e/i1820e.pdf. fao/who. 2002. “human vitamin and mineral requirements” report of a joint food and agriculture organisation of the united nations/world health organisation expert consultation, bangkok, thailand. http://www.fao. org/documents fuentes a., fernández-segovia i., serra j.a. and barat j.m. 2010. comparison of wild and cultured sea bass (dicentrarchuslabrax) quality. food chem. 119: 1514-1518. grigorakis k. 2007. compositional and organoleptic quality of farmed and wild gilthead sea bream (sparusaurata) and sea bass (dicentrarchuslabrax) and factors affecting it: a review. aquaculture 272: 55-75. grigorakis k., alexis m., gialamas i. and nikolopoulou d. 2004. sensory, microbiological, and chemical spoilage of cultured common sea bass (dicentrarchuslabrax) stored in ice: a seasonal differentiation. eur. food res. technol.219: 584-587. http://www.ncbi.nlm.nih.gov/pubmed?term=minganti%20v%5bauthor%5d&cauthor=true&cauthor_uid=18035400 http://www.ncbi.nlm.nih.gov/pubmed?term=poggi%20r%5bauthor%5d&cauthor=true&cauthor_uid=18035400 ital. j. food sci., vol. 27 2015 159 paper received january 27, 2014 accepted june 23, 2014 grigorakis k., alexis m.n., anthony taylor k.d. and hole m. 2002. comparison of wild and cultured gilthead sea bream (sparusaurata); 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(http://www.lipidworld.com/ content/11/1/144) testi s., bonaldo a., gatta p.p. and badiani a. 2006. nutritional traits of dorsal and ventral fillets from three farmed fish species. food chem. 98: 104-111. trajković j., mirić m., baras j. and šiler s. 1983. analysis of food. faculty of technology and metallurgy, belgrade, serbia. wall r., ross r.p., fitzgerald g.f. and stanton c. 2010. fatty acids from fish: the anti-inflammatory potential of long-chain omega 3fatty acids. nutr. rev. 68: 289-289. zayas j.f. 1997. functionality of proteins in food, springer, verlag berlin heidelberg. http://www.lipidworld.com/content/11/1/144 http://www.lipidworld.com/content/11/1/144 ijfs#1794_bozza ital. j. food sci., vol. 32, 2020 562 paper evaluation of the chemical and nutritional properties of tunisian almond cultivars h. gouta*a, e. ksiab, i. laaribia, f. molinoc, g. estopañanc, t. juanc, o. kodadd, p. martínez-gómeze and p.j. martínez-garcíae aolive tree institute, po. box.1087, 3000 sfax, tunisia bfaculty of sciences tunis, campus universitaire 2092 el manar, tunis, tunisia cinstituto agroalimentario de aragón ia2, cita de aragón, zaragoza, spain decole nationale d’agriculture de meknes, meknes, morocco edepartment of plant breeding, cebas-csic, po box 164, e-30100 murcia, spain *corresponding author: zallaouz@yahoo.fr abstract the aim of this research was to evaluate for the first time protein, oil content, fatty acid profile and sugar composition for the main commercial almond cultivars in tunisia in comparison to foreigners. thus, fruits from twelve locals and five introduced cultivars from france, italy and spain were analyzed over two years. in fact, total oil content varied from 52.28% (‘blanco’) to 60.95% (‘lsen asfour’) in the first year and from 47.75% (‘zahaaf’) to 56.15% (‘mahsouna’) in the second. however, the highest oleic acid content was noted in ‘francoli’ (76.2%) for both years. it was followed by ‘sahnoun’ (75.11%) firstly and ‘abiodh’ (73.02%) secondly. likewise, the highest linoleic acid content was observed in ‘porto’ for both studied years (22.87% and 23.67%). the highest palmitic acid content was detected in ‘porto’ (7.02%) and in ‘tuono’ for the consecutive years. sugars profile was quite distinctive among cultivars. the cultivar ‘porto’ presented the highest total sugars (5.8 g/100g dw) and sucrose contents (4.96 g/100g dw). nevertheless, protein content doesn’t show extreme values. for both years, the local cultivar ‘zahaaf’ presented the highest protein content (27 g/100g dw) while introduced french cultivar ‘fournat de breznaud’ presented the lowest protein content (17 g/100g dw). all the analyzed components were different significantly according to cultivar and year effects. results evidenced that the local tunisian cultivars are highly rich in oil and fatty acids particularly oleic and linoleic acids, confirm the almond kernel as a high nutritional dietetic source and underline the high adaptability of some introduction. keywords: fatty acid composition, local cultivars, oil quality, prunus dulcis l., sugar content ital. j. food sci., vol. 32, 2020 563 1. introduction the cultivated almond [prunus dulcis (miller) webb] is a tree species whose domestication and spread has closely paralleled the rise of eurasian civilizations. this tree-crop species is mainly planted for its edible seeds (kernels). today, almonds are cultivated in more than 50 countries (http://faostat.fao.org), with approximately 95% produced in california, australia and the mediterranean basin. in tunisia, almond cultivation is present around the country mainly under rainfed conditions (gouta et al., 2019). moreover, the almond kernel represents the main nutrient source for many rural populations in the central and southern parts of the country. the high nutritive value of the almond kernel comes mainly from its high lipid content. in fact, it contains 52% of lipids, 20% of proteins and 20% of carbohydrates including 5% of water and 3% of soluble sugars (kader, 1996). almond quality was formerly related to the kernel flavor in addition to its physical parameters such as kernel size, percentage of double kernel and kernel rate without any attention to its nutraceutical composition (romojaro et al., 1988; nanos et al., 2002). at this moment, however, the nutritive value of almond kernel related to lipid, sugar, protein and mineral richness are being evaluated as main component of the almond kernel quality. different studies have reported that almonds consumption can significantly lower total and low-density-lipoprotein (ldl) cholesterol in plasma, reduce risk for heart disease and prevent several forms of cancer and inflammation (jenkins et al., 2008). the beneficial health effect of almond was attributed to its high content of mono and poly-unsaturated fatty acids (ros and mataix, 2006). moreover, the high (oleic acid/linoleic acid) ratio is used in determining the kernel quality due to its preventive effect on lipid oxidation and oil stability (kodad et al., 2010). in addition, negative cholesterol effects can be treated by an equilibrate lipid diet based on nut consumption including almonds (musa-velasco et al., 2016). furthermore, almond oil contains antioxidants and fat-soluble bioactive compounds that make it oil with interesting nutritional and cosmetic properties (roncero et al., 2016). in this context several studies have been published about total oil and fatty acid profile of some almond cultivars (özcan et al., 2010; yildirim et al., 2016; čolić et al., 2017; socias i company et al., 2018). in addition, sugars composition in the almond kernel has been reported in many studies (kazantzis et al., 2003; balta et al., 2009). however, as far as we know, very few researches were carried out to characterize the nutraceutical values together with these chemical compositions (socias i company et al., 2010; kodad, 2017). this information is null regarding the rich almond germplasm from tunisia considered as an almond diversification center. the objective of this study was to determine for the first time the chemical and nutritional composition (including total oil, protein contents, fatty acid and sugar composition) of most important tunisian almond cultivars. moreover, the interaction of genotype x environment would be deeply discussed. findings of the present work will be important for selecting cultivars with more stable macronutrients composition from year to year and consequently less subject to climate changes. 2. materials and methods 2.1. plant material plant material assayed included twelve tunisian almond cultivars (‘dillou’, ‘khoukhi’, ‘blanco’, ‘abiodh’, ‘lsen asfour’, ‘achaak’, ‘zahaaf’, ‘fekhfekh’, ‘ksontini’, ‘sahnoun’, ital. j. food sci., vol. 32, 2020 564 ‘porto’, and ‘mahsouna’) and five almond cultivars originating from italy (‘mazetto’ and ‘supernova’), spain (‘francoli’), france (‘lauranne’ and ‘fournat de breznaud’) assayed as reference. the local cultivars used in this work (fig. 1) are early flowering, autoincompatible and their pomological and agronomical characteristics were previously well described (gouta et al., 2011; gouta et al., 2019). all studied almonds were collected from the national collection in sidi bouzid in central-western part of tunisia during two consecutive years 2009 and 2010. figure 1. pomological characteristics among the native tunisian almond cultivars. 2.2. oil and fatty acid determination kernels were preliminary blanched for 3 min in boiling water eliminating seed coat. the kernels were dried at 25°c until constant weight and then ground. oil was extracted using about 5g of ground almond in a soxtec avanti 2055 fat extractor (foss tecator, höganäs, sweden) for 2 h using 70 ml of petroleum ether as solvent and keeping temperature at 135°c. to remove any residual ether, the extract was subject successively to vacuum evaporation for 15 min in a vacuum desiccator. ten microliters of butylated hydroxyl toluene methanol solution (bht) as an antioxidant agent was added to each oil sample which was kept in an amber vial at -20°c until analysis. the percentage of the different fatty acids in oil samples was determined by capillarity gas chromatography of the fatty acid methyl esters (fames). methyl esters of the corresponding fatty acids were obtained by trans-esterification with koh of each almond oil sample according to the official method une-en (isao 5509, 2000). they were separated using a flame ionizing detector ital. j. food sci., vol. 32, 2020 565 (fid) gas chromatograph hp-6890 equipped with hp-innowax column (30 m × 0.25 mm i.d.) and 0.25 µm film thinness (agilent technologies, waldron, germany). the fames identification was realized by comparison with relative chromatographic retention times of standard methyl esters mixture (sigma-aldrich, madrid, spain). 2.3. sugar determination free sugar profiles were determined by a high performance liquid chromatography (hplc, agilent 1100, germany) during the two consecutive years 2009 and 2010. in first step kernels samples were dried in an oven at 25°c until weight stabilized, ground in a mortar and then defatted using a soxhlet and ether petroleum as a solvent. once defatted, a sample of 0.7 to 1.3 g of the remaining powder was moved to a falcon tube and mixed with of 9 ml milliq water. for protein denaturation, 0.5 ml of carrez i (potassium ferrocyanide 15% w/v) and 0.5 ml of carrez ii (zinc acetate 30% w/v) solutions were added and kept under agitation in an agitator (reax, madrid, spain) for 10 min. the resulting suspension was centrifuged at 8000 rpm for 20 min. the supernatant was recuperated and passed throw a nylon filter 0.45 µm before injection in a hplc apparatus. a volume of 20 µl of the filtrate was injected in an interchange cationic column (pb) cho682 (transgenomic, madrid, spain). sugar detection was performed according to the detection time of reference samples (sigma, madrid, spain) of raffinose, sucrose, glucose and fructose. 2.4. total protein determination protein fraction was obtained by the following formula: protein percentage = total nitrogen percentage x kc with kc presenting a conversion factor equal to 6.25 for almond. the total nitrogen content was obtained by the dumas method (dumas, 1826). almond kernels for each genotype were defatted as already mentioned (using soxhlet and ether petroleum solvent) and then analyzed by a leco fp-528 protein/nitrogen analyzer (leco cooperation, saint joseph, mi, usa). a sample of 0.2 g of the resulting powder was incinerated at 850°c and the gases generated were passed through hot copper to remove oxygen. nitrogen molecules with helium were measured in a cell differential thermo-conductivity. then, data were read and interpreted with cpu-car-02 software. results were expressed as percentage of nitrogen by kernel powder weight. 2.5. statistical analysis three replicates of 20 kernels from each genotype were evaluated. the significance of cultivar, year and cultivar × year interaction effects for all studied components were tested on the 17 cultivars by anova using spss 20.0. differences between means were evaluated by using duncan multiple range test. correlations between traits were calculated from raw data of the two years using pearson correlation coefficient. trait mean values were used to perform a principal component analysis (pca). ital. j. food sci., vol. 32, 2020 566 3. results 3.1. effect of the year and its interaction with the cultivar the analysis of variance showed significant effect of cultivar and year for the fatty acids and sugars compositions and oil and protein contents in the seventeen almond cultivars assayed during two consecutive years. in addition, the interaction cultivar × year exhibited considerable variation for all analyzed parameters (table 1). besides the significant effect of the cultivar, a clear and significant environmental effect was noted in the oil content for all studied cultivars due to the specific climatic conditions of years tested. some almond cultivars have shown high year to year stability in their fatty acids content compared to other cultivars. these results indicate that the year effect on the fatty acids composition in almond mainly depends on genotype. stable values for some fatty acids were observed in cultivars such as ‘dillou’, ‘sahnoun’, ‘mazetto’ and ‘mahsouna’ for arachidic acid; ‘dillou’, ‘khoukhi’, ‘lsen asfour’, ‘sahnoun’, ‘super nova’, ‘lauranne’ and ‘mahsouna’ for linolenic acid; ‘porto’, ‘abiodh’ and ‘mahsouna’ for palmitic acid; ‘lsen asfour’ and ‘mahsouna’ for palmitoleic acid; ‘lauranne’ and ‘mahsouna’ for stearic acid (table 2). the year effect was significant for different sugar amounts except raffinose percentage (table 1). moreover, studied cultivars show stable and similar year to year sugar percentage excepting the glucose percentage, confirming that the year to year stability depends on the specific characteristics of the genotype. 3.2. oil content the mean value of oil content over the 2 years varied from 47.75% for ‘zahaaf’ to 60.95% for ‘lsen asfour’ (table 2). in 2009, the mean value of total lipid was 56.23%, ranged for the local cultivars from 52.28% for blanco to 60.95% for ‘lsen asfour’ and for the foreign cultivars from 53.36% for ‘francoli’ to 55.93 % for ‘breznaud’. in 2010, the mean value of total oil was 51.39%, ranged for the local cultivars from 47.75% for ‘zahaaf’ to 56.15% for ‘mahsouna’ and for the foreign cultivars total lipid content ranged from 48.37% for ‘francoli’ to 54.45% for ‘lauranne’. the values of total lipid content were found to be low for the european cultivars compared to the values registered in the tunisian local cultivars. in fact, it was found in the range of 47-56% for ‘francoli’ (spain), ‘super nova’ and ‘mazetto’ (italy) and ‘laurane’ and ‘fournat de breznaud’ (france). 3.3. protein content the mean value of the protein content was for almost studied cultivars higher in 2010 than in 2009, contrarily to the oil content which was higher in 2009 (table 2). for the year 2009, the lowest contents were showed by the local cultivars ‘lsen asfour’ (14.49%) and ‘mahsouna’ (17.34%) and the french cultivar ‘fournat de breznaud’ (17.84%) while the highest values ranged between 23 and 21.2% for ‘francoli’, ‘zahaaf’, ‘ksontini’, ‘super nova’ and ‘mazetto’, respectively. in 2010 these same cultivars showed the highest protein content with a greater range of variation (27.15-23.35%). likewise ‘mahsouna’, ‘fournat de breznaud’ and ‘lsen asfour’ showed the lowest protein content (17.14-18.11%) the second year of study. however, the cultivars ‘dillou’, ‘mahsouna’ and ‘fournat de breznaud’ showed stable mean value of the protein content over the two year. ital. j. food sci., vol. 32, 2020 567 table 1. analysis of variance of fatty acid (palmitic, palmitoleic, stearic, oleic and linoleic) content, total lipid content, sugar composition (raffinose, sucrose, glucose, fructose), total sugar content and protein content in the 17 assayed almond cultivars. source of variation mean squares df1 palmitic palmitoleic stearic oleic linoleic total lipid raffinose sucrose glucose fructose total sugar protein genotype (g) 16 0.636 0.027 1.722 32.18 28.40 25.25 0.541 2.908 0.028 0.021 4.33 37.55 year (y) 1 1.547 0.048 1.498 189.36 172.58 596.627 0.065 15.514 0.036 0.003 19.651 50.28 g × y 16 0.141 0.006 0.238 8.055 5.56 11.365 0.054 1.144 0.005 0.003 1.511 8.723 error 68 0.000 0.000 0.013 0.587 0.788 0.328 0.012 0.042 0.000 0.000 0.065 0.159 mean squares in bold case present a level of significance of p<0.001. 1df: degree of freedom. table 2. fatty acid (palmitic, palmitoleic, stearic, oleic, linoleic, arachidic, α-linolenic), protein content and total lipid for each almond cultivar assayed during two consecutive years (2009 and 2010). miristic palmitic palmitoleic margaric margaroleic stearic oleic 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 tunisian almond cultivars dillou 0,04a 0,05b 6,19a 6,46b 0,52a 0,56b 0,04a 0,05b 0,09a 0,10b 1,36a 1,53b 72,10a 69,49b khoukhi 0,05a 0,06b 6,32a 7,23b 0,51a 0,48b 0,04a 0,06b 0,09a 0,10b 1,31a 1,70b 73,59a 66,70b blanco 0,04a 0,04b 6,14a 6,29b 0,41a 0,43b 0,04a 0,05b 0,09a 0,09b 1,40a 1,35b 70,65a 70,42b abiodh 0,03a 0,06b 6,88a 6,85a 0,59a 0,54b 0,05a 0,02b 0,09a 0,05b 1,75a 1,62b 73,56a 73,02b lsen asfour 0,03a 0,04b 6,56a 6,85b 0,54a 0,52a 0,04a 0,05b 0,07a 0,09b 2,84a 1,82b 71,22a 67,62b achaak 0,02a 0,05b 6,62a 7,25b 0,53a 0,40b 0,05a 0,03b 0,08a 0,06b 2,69a 2,40b 73,06a 67,95b zahaaf 0,04a 0,11b 6,61a 6,44b 0,55a 0,43b 0,04a 0,02b 0,09a 0,05b 1,46a 1,72b 75,06a 71,91b fekhfekh 0,01a 0,05b 5,86a 6,12b 0,35a 0,31b 0,05a 0,04b 0,06a 0,06a 2,82a 2,53b 72,95a 69,65b ksontini 0,03a 0,05b 7,01a 6,91b 0,34a 0,39b 0,06a 0,02b 0,08a 0,07b 3,75a 2,66b 67,50a 67,90b sahnoun 0,03a 0,03a 6,28a 6,42b 0,53a 0,46b 0,05a 0,05b 0,09a 0,10b 2,00a 1,92b 75,11a 71,05b porto 0,03a 0,03b 7,02a 7,04a 0,52a 0,43b 0,05a 0,06b 0,08a 0,10b 2,38a 2,13b 66,70a 66,28b mahsouna 0,02a 0,02a 6,77a 6,85a 0,50a 0,43a 0,04a 0,05b 0,07a 0,09b 2,58a 2,43a 73,14a 70,38a ital. j. food sci., vol. 32, 2020 568 international reference almond cultivars mazetto 0,02a 0,03b 6,77a 7,53b 0,47a 0,30b 0,05a 0,07b 0,08a 0,09b 2,63a 2,52b 72,56a 65,29b francoli 0,02a 0,04b 6,25a 6,40b 0,50a 0,49b 0,05a 0,06b 0,08a 0,10b 3,05a 2,50b 76,21a 76,15b supernova 0,02a 0,02b 6,61a 7,24b 0,46a 0,48b 0,05a 0,06b 0,08a 0,10b 2,72a 2,17b 73,15a 69,89b lauranne 0,02a 0,02b 6,63a 6,79b 0,62a 0,55b 0,05a 0,05b 0,10a 0,10a 1,79a 1,79a 73,77a 70,67b f. breznaud 0,03a 0,03b 6,76a 6,84b 0,52a 0,51b 0,05a 0,05b 0,08a 0,09b 2,32a 1,94b 69,94a 69,56b min 0,01 0,02 5,86 6,12 0,34 0,30 0,04 0,02 0,06 0,05 1,31 1,35 66,70 65,29 max 0,05 0,11 7,02 7,53 0,62 0,56 0,06 0,07 0,10 0,10 3,75 2,66 76,21 76,15 mean 0,03 0,04 6,55 6,79 0,50 0,45 0,05 0,05 0,08 0,08 2,29 2,04 72,37 69,64 sd 0,01 0,02 0,33 0,39 0,07 0,08 0,01 0,01 0,01 0,02 0,70 0,40 2,53 2,65 cv 31,64 48,45 4,97 5,76 14,65 16,82 11,36 31,47 12,09 20,70 30,58 19,81 3,50 3,80 table 2. continues. linoleic arachidic α-linolenic gadoleic protein total lipid o/l ratio usfa 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 tunisian almond cultivars dillou 19,40a 21,37b 0,05a 0,03a 0,02a 0,02a 0,06a 0,07b 19,75a 19,49a 55,22a 52,81b 3,72a 3,25b 91,50a 90,86b khoukhi 17,86a 23,27a 0,05a 0,06b 0,01a 0,01b 0,06a 0,06a 20,48a 21,65b 55,80a 50,60b 4,12a 2,87b 91,45a 89,96b blanco 21,01a 20,84b 0,06a 0,06b 0,02a 0,02b 0,06a 0,07b 19,67a 18,39b 52,28a 53,83b 3,36a 3,38b 91,65a 91,26b abiodh 16,85a 17,54b 0,07a 0,07b 0,01a 0,07b 0,06a 0,03b 19,94a 18,48b 58,66a 48,61b 4,36a 4,16b 90,41a 90,55b lsen asfour 18,40a 22,27b 0,09a 0,06b 0,02a 0,02a 0,07a 0,07a 14,49a 18,12b 60,95a 52,51b 3,90a 3,04b 89,62a 89,89a achaak 16,60a 21,51b 0,09a 0,09b 0,02a 0,06b 0,07a 0,03b 17,91a 18,37b 58,26a 55,53b 4,40a 3,16b 89,66a 89,46b zahaaf 15,91a 19,03b 0,06a 0,07b 0,02a 0,08b 0,06a 0,00b 22,89a 24,83b 54,01a 47,75b 4,712a 3,78b 90,97a 90,94a fekhfekh 17,60a 20,53b 0,10a 0,09b 0,02a 0,07b 0,07a 0,00b 17,74a 22,84b 57,76a 52,57b 4,14a 3,39b 90,55a 90,17b ksontini 20,89a 21,68b 0,12a 0,10b 0,02a 0,06a 0,08a 0,04b 21,95a 24,28b 54,33a 50,77b 3,23a 3,13b 88,38a 89,57b sahnoun 15,67a 19,34b 0,08a 0,08a 0,02a 0,02a 0,06a 0,07a 18,84a 20,17b 56,69a 49,96b 4,80a 3,67b 90,78a 90,39b porto 22,87a 23,67a 0,08a 0,08b 0,02a 0,00b 0,07a 0,07a 19,92a 22,71b 56,99a 49,44b 2,92a 2,80b 89,57a 89,95b mahsouna 16,54a 18,93a 0,10a 0,10a 0,02a 0,02a 0,08a 0,07a 17,35a 17,15a 60,37a 56,15b 4,70a 3,72a 89,67a 89,31a ital. j. food sci., vol. 32, 2020 569 international reference almond cultivars mazetto 17,06a 23,67b 0,12a 0,12a 0,02a 0,03b 0,07a 0,06a 21,27a 27,15b 54,30a 48,82b 4,25a 2,76b 89,62a 88,96b francoli 13,45a 13,92b 0,11a 0,10b 0,02a 0,03b 0,08a 0,07b 23,02a 23,35b 53,36a 48,37b 5,66a 5,47b 89,66a 90,07b supernova 16,58a 19,69b 0,11a 0,11b 0,03a 0,03a 0,08a 0,08a 21,63a 26,33b 55,48a 50,60b 4,41a 3,55b 89,73a 89,58b lauranne 16,79a 19,75b 0,08a 0,08b 0,03a 0,01a 0,06a 0,08b 20,55a 18,02b 55,52a 54,45b 4,39a 3,58b 90,55a 90,42b f. breznaud 20,08a 20,77b 0,08a 0,07b 0,02a 0,00b 0,06a 0,07a 17,84a 17,79a 55,93a 50,91b 3,43a 3,35b 90,02a 90,33b min 13,45 13,92 0,05 0,03 0,01 0,00 0,06 0,00 14,49 17,15 52,28 47,75 2,92 2,76 88,38 88,96 max 22,87 23,67 0,12 0,12 0,03 0,08 0,08 0,08 23,02 27,15 60,95 56,15 5,67 5,47 91,65 91,26 mean 17,86 20,46 0,08 0,08 0,02 0,03 0,07 0,06 19,72 21,12 56,23 51,39 4,15 3,47 90,22 90,10 sd 2,34 2,42 0,02 0,02 0,00 0,02 0,01 0,03 2,20 3,25 2,39 2,55 0,67 0,64 0,87 0,61 cv 13,11 11,81 28,85 27,81 22,02 79,53 12,56 46,97 11,18 15,39 4,24 4,97 16,24 18,33 0,96 0,68 mean values of each parameter in each genotype in different years followed by a different lower-case letter are significantly different at p=0.01 by the duncan test. ital. j. food sci., vol. 32, 2020 570 3.4. fatty acid composition the fatty acid profile of almond oil consisting of mystiric (c14:0), palmitic (c16:0), palmitoleic (c16:1), margaric (c17:0), margaroleic (c17:1 n-8), stearic (c18:0), oleic (c18:1 n-9), linoleic (c18:2 n-6), α-linolenic (c18:3 n-3), arachidic (c20:0), and gadoleic (c20:1 n11) (table 2). fatty acid composition of studied almond kernel oil has shown three predominant fatty acids regardless of cultivar or year. the oleic acid is the main monounsaturated fatty acid, followed by linoleic acid the main polyunsaturated fatty acid and the palmitic acid the main saturated fatty acid. the ranges of variation of these three fatty acids were 65-76%, 13-23% and 5.8-7.5%, respectively. the contents of stearic and palmitoleic acids were <4%, and ranged between 1.3-3.7% and 0.3-0.6%, respectively. in both years, the oleic, linoleic and palmitic acids varied among cultivars. in 2009, the highest values of oleic acid content were determined in ‘francoli’ (76.21%), followed by cultivars ‘sahnoun’ (75.11%) and ‘zahaaf’ (75.06%). however, the lowest values were found in ‘porto’ (66.70%) and ‘breznaud’ (69.94%). for linoleic acid, ‘porto’ represented the highest value (22.87%) and ‘francoli’, ‘sahnoun’ and ‘zahaaf’ showed the lowest value (13.45-15.91%). for palmitic acid, ‘porto’ and ‘ksontini’ demonstrated the highest palmitic content (7%) while ‘fekhfekh’ showed the lowest value (5.86%). in 2010, the cultivar ‘francoli’ showed the highest value of oleic acid content (76.15%), followed by ‘abiodh’, ‘zahaaf’ and ‘sahnoun’ while ‘mazetto’ recorded the lowest value (65.29%). for linoleic acid, the highest value was obtained for ‘porto’ (23.67%) whereas the lowest value was obtained for ‘francoli’ (13.92%). ‘mazetto’ represented the highest palmitic acid content (7.53%) and ‘fekhfekh’ represented the lowest palmitic content (6.12%). thus the varieties ‘sahnoun’, ‘zahaaf’ and ‘francoli’, are superior in marketing quality with high oleic acid content and low linleic and palmitic contents. the oleic/linoleic (o/l) ratio showed a large variability among cultivars because of the high variability in oleic and linoleic acids contents. this ratio was generally higher in 2009 than in 2010 (table 2). the cultivars ‘francoli’ showed the higher (oleic/linoleic) ratio (5.65.4) during the two consecutive years followed by the cultivars ‘sahnoun’, ‘zahaaf’ and ‘mahsouna’ (table 2). owing to their highest (o/l) ratio, these cultivars represented the greatest stability of almond kernels and oil. however, ‘porto’ cultivar showed the lowest (oleic/linoleic) ratio (2.8-2.9) followed by the varieties ‘ksontini’ and ‘mazetto’ in 2009 and 2010, respectively. for the local cultivars it was noted that oleic and linoleic acids together accounted from 88.38 to 91.65% of the total extracted almond oil. 3.5. sugar composition total sugar content varied from 2.3 to 6.5 g 100 g-1 of dry weight (dw), with an average content of 3.98 g 100 g-1dw (table 3). sucrose, raffinose, glucose and fructose contents were analyzed separately. sucrose was the sugar present at the highest concentration in all studied cultivars (1.9 to 5.8 g 100 g-1dw) followed by raffinose (0.04 to 1.36 g 100 g-1dw), glucose (0.019 to 0.43 g 100 g-1dw) and fructose (0.007 to 0.322 g 100 g-1dw). the year effect was significant on the total sugar content (table 1). the mean value of total sugar was higher in 2009 than in 2010 for all studied cultivars excepting ‘blanco’ (table 3). ital. j. food sci., vol. 32, 2020 571 table 3. sugar composition (raffinose, sucrose, glucose, fructose), total sugar content and percentage of each type of sugars for each almond cultivar in two consecutive years (2009 and 2010). raffinose sucrose glucose fructose total sugar %raffinose %sucrose %glucose %fructose 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 tunisian almond cultivars dillou 1,049a 0,958b 3,592a 3,558a 0,061a 0,062a 0,010a 0,010a 4,712a 4,589b 0,223a 0,209a 0,762a 0,775a 0,013a 0,014a 0,002a 0,002b khoukhi 0,720a 0,724a 3,447a 3,456a 0,120a 0,114a 0,010a 0,010b 4,297a 4,304a 0,168a 0,168a 0,802a 0,803a 0,028a 0,026a 0,002a 0,002a blanco 1,046a 1,365a 3,131a 4,139b 0,067a 0,053a 0,010a 0,010b 4,254a 5,567a 0,246a 0,245a 0,736a 0,743a 0,016a 0,010b 0,002a 0,002b abiodh 0,612a 0,301b 5,807a 2,741b 0,076a 0,069a 0,010a 0,040a 6,505a 3,151b 0,094a 0,095a 0,893a 0,870a 0,012a 0,022b 0,002a 0,013a lsen asfour 0,151a 0,369b 3,737a 3,265b 0,050a 0,041a 0,010a 0,010b 3,949a 3,684b 0,038a 0,100b 0,946a 0,886b 0,013a 0,011a 0,003a 0,003b achaak 0,516a 0,390b 3,256a 2,722b 0,058a 0,031b 0,007a 0,010b 3,837a 3,154b 0,134a 0,124b 0,849a 0,863b 0,015a 0,010b 0,002a 0,003b zahaaf 0,318a 0,145b 2,846a 2,202a 0,034a 0,028a 0,010a 0,010b 3,207a 2,385a 0,099a 0,061b 0,887a 0,923b 0,010a 0,012a 0,003a 0,004a fekhfekh 0,369a 0,182b 3,977a 2,760b 0,092a 0,026b 0,010a 0,010b 4,449a 2,978b 0,083a 0,061b 0,894a 0,927b 0,021a 0,009b 0,002a 0,003b ksontini 0,134a 0,472b 3,569a 2,576b 0,042a 0,046a 0,010a 0,010b 3,755a 3,104b 0,036a 0,152b 0,950a 0,830b 0,011a 0,015b 0,003a 0,003b sahnoun 0,279a 0,227a 4,657a 2,790b 0,063a 0,023b 0,010a 0,040b 5,009a 3,080b 0,056a 0,074a 0,930a 0,906b 0,013a 0,007b 0,002a 0,013b porto 0,714a 0,789a 5,074a 4,845a 0,060a 0,063a 0,010a 0,050a 5,858a 5,747b 0,122a 0,137b 0,866a 0,843b 0,010a 0,011a 0,002a 0,009b mahsouna 0,450a 0,318a 2,411a 1,976b 0,065a 0,033b 0,010a 0,010b 2,936a 2,338b 0,153a 0,136a 0,821a 0,845a 0,022a 0,014b 0,003a 0,004b international reference almond cultivars mazetto 0,413a 0,225b 3,286a 2,428b 0,027a 0,019a 0,026a 0,024b 3,752a 2,695b 0,110a 0,083b 0,876a 0,901b 0,007a 0,007a 0,007a 0,009b francoli 0,416a 0,214b 3,990a 2,536b 0,141a 0,032b 0,116a 0,036b 4,663a 2,818b 0,089a 0,076b 0,856a 0,900b 0,030a 0,011b 0,025a 0,013b super nova 0,326a 0,152b 3,238a 2,611b 0,056a 0,052a 0,038a 0,038a 3,658a 2,854b 0,089a 0,053b 0,885a 0,915b 0,015a 0,018a 0,010a 0,013b lauranne 0,234a 0,042b 3,903a 3,128b 0,434a 0,212b 0,322a 0,174b 4,893a 3,556b 0,048a 0,012b 0,798a 0,880b 0,089a 0,060b 0,066a 0,049b f. breznaud 0,230a 0,244a 5,083a 4,025b 0,161a 0,077b 0,104a 0,067a 5,578a 4,413b 0,041a 0,055b 0,911a 0,912a 0,029a 0,017b 0,019a 0,015a min 0,134 0,042 2,411 1,976 0,027 0,019 0,007 0,010 2,936 2,338 0,036 0,012 0,736 0,743 0,007 0,007 0,002 0,002 max 1,049 1,365 5,807 4,845 0,434 0,212 0,322 0,174 6,505 5,747 0,246 0,245 0,950 0,927 0,089 0,060 0,066 0,049 mean 0,469 0,419 3,824 3,045 0,095 0,058 0,042 0,033 4,430 3,554 0,108 0,108 0,863 0,866 0,021 0,016 0,009 0,009 mean values of each parameter in each genotype in different years followed by a different lower-case letter are significantly different at p=0.01 by the duncan test. ital. j. food sci., vol. 32, 2020 572 taking into account both years of study, the tunisian variety ‘porto’ and french variety ‘fournat de breznaud’ represented the higher sugar (5.8 and 4.9 g 100 g-1dw) and sucrose content (4.9 and 4.5 g 100 g-1dw), while the varieties ‘zahaaf’ and ‘mahsouna’ showed the lowest contents. ‘blanco’, ‘dillou’, ‘porto’ and ‘khoukhi’ have the highest raffinose levels, in decreasing order, for both years. concerning the fructose and glucose percentages, the french varieties ‘lauranne’ and ‘fournat de breznaud’ demonstrated the highest mean values for the two years of study (table 3). however, the italian variety ‘mazetto’ represented the lowest glucose content (0.02 g 100g-1dw). the two local varieties ‘achaak’ and ‘porto’ are the two most appreciated almond kernel by consumers. ‘porto’ seems to be sweeter than ‘achaak’ and showed two times more total sugar and four times more fructose percentage. 3.6. correlation among nutraceutical properties the correlation among oil content and fatty acids of the studied almond kernel cultivars is reported in table 4. high significant negative correlation was found between linoleic and oleic acid contents (r= -0.969). significant positive correlations were also found between stearic and arachidic acid contents (r= 0.848) and in margaric versus margaroleic and gadoleic (r= 0.686 and r= 0.705, respectively). a significant negative correlation was found between gadoleic versus myristic and linolenic (r= -0.704 and r= -0.810, respectively). for the sugar composition, total sugar content was positively and highly correlated with sucrose (r= 0.974) and raffinose (r= 0.539). also, a significant and high correlation was found between glucose and fructose contents (r= 0.933). moreover, significant and negative correlations were observed between total sugar content and arachidic acid (r= -0.537) and linolenic acid (r= -0.547). similarly, a significant and negative correlation was also found between raffinose and stearic acid (r= -0.527) and arachidic acid (r= -0.589). significant positive correlation was found between glucose and palmitoleic acid (r= 0.511). these relationships between different biochemical traits of almond suggest that the selection for one of these fatty acids or sugars could negatively or positively modify the amount of the other. finally, a significant negative correlation was found between the oil and protein contents (r= -0.647). 3.7. chemical diversity analysis a principal component analysis (pca) was performed on biochemical data (fatty acid, total oil and protein contents and sugar composition) for screening and describing the similarities among the 17 studied almond cultivars (fig. 2). the pca yielded six significant components with eigenvalues ≥ 1 and accounting for 91% of the total variance in the dataset (table 5). the first two pcs (pc1 and pc2) accounted for 48.24% of the total of variance. pc-1 and pc-2 represented 27.41% and 20.83% of the variance, respectively. eigen analysis of the correlation matrix revealed that pc-1 was mainly contributed by total sugar, sucrose and raffinose contents. pc-2 was correlated to arachidic, gadoleic, margaric and stearic acids. the third and fourth pc accounted for 16.83% and 11.35%, respectively. pc-3 was represented by oleic, linoleic, fructose and glucose contents while pc-4 was highly correlated to oil and protein contents. ital. j. food sci., vol. 32, 2020 573 table 4. correlations between fatty acid and oil content composition, protein content, total lipid, sugar composition and total sugar content. palmitic palmitoleic stearic oleic linoleic arachidic α-linolenic protein total lipid raffinose sucrose glucose fructose total sugar palmitic 1 palmitoleic -0,047 1 stearic 0,165 -0,400 1 oleic -0,607 0,458 -0,047 1 linoleic 0,474 -0,405 -0,175 -0,968 1 arachidic 0,237 -0,436 0,848 0,026 -0,232 1 α-linolenic 0,006 -0,322 0,055 -0,010 0,001 0,143 1 protein 0,252 -0,373 0,094 -0,119 0,080 0,285 0,297 1 total lipid -0,192 0,294 0,223 0,260 -0,294 0,055 -0,322 -0,647 1 raffinose -0,217 0,060 -0,458 -0,117 0,250 -0,521 -0,264 -0,128 0,065 1 sucrose -0,080 0,346 -0,066 0,005 0,032 -0,238 -0,485 -0,271 0,362 0,305 1 glucose -0,067 0,455 -0,164 0,205 -0,170 -0,131 -0,166 -0,136 0,175 -0,103 0,287 1 fructose 0,010 0,374 -0,045 0,178 -0,181 0,069 -0,109 -0,002 0,014 -0,269 0,166 0,896 1 total sugar -0,136 0,363 -0,206 -0,005 0,078 -0,361 -0,503 -0,277 0,336 0,530 0,961 0,333 0,181 1 correlations shown in bold case are significant at p<0.05. ital. j. food sci., vol. 32, 2020 574 figure 2. score plot showed the principal component analysis (pca) based on nutraceutical data (fatty acid, total oil and protein contents and sugar composition) describing the similarities among the 17 studied almond cultivars. dillou khoukhi blanco abiodh lsen asfour achaak zahaaf fekhfekh ksantini sahnoun porto mahsouna mazetto francoli supernova lauranne breznaud -3 -2 -1 0 1 2 3 4 5 -4 -3 -2 -1 0 1 2 3 4 f2 ( 20 ,8 3 % ) f1 (27,42 %) dillou khoukhi blanco abiodh lsen asfour achaak zahaaf fekhfekh ksantini sahnoun porto mahsouna mazetto francoli supernova lauranne breznaud -3 -2 -1 0 1 2 3 -4 -3 -2 -1 0 1 2 3 4 5 f4 ( 11 ,3 6 % ) f3 (16,83 %) ital. j. food sci., vol. 32, 2020 575 table 5. eigenvectors of the four principal components axes from pca analysis of the 17 almond cultivars for fatty acid and oil content composition, protein content, total lipid, sugar composition) and total sugar content. eigenvalues and their contribution to total variation are listed at the bottom of columns. variable f1 f2 f3 f4 palmitic -0,010 -0,400 -0,310 0,042 palmitoleic -0,588 0,161 0,581 0,114 stearic 0,570 -0,663 -0,230 0,121 oleic 0,228 0,160 0,860 -0,158 linoleic -0,375 0,062 -0,823 0,115 arachidic 0,581 -0,777 -0,056 -0,050 α-linolenic 0,856 0,312 0,154 0,063 protein 0,419 -0,133 -0,056 -0,842 total lipid -0,084 -0,133 -0,027 0,935 raffinose -0,485 0,440 -0,435 -0,156 sucrose -0,682 0,152 -0,203 -0,028 glucose -0,504 -0,183 0,586 0,114 fructose -0,391 -0,367 0,643 0,021 total sugar -0,798 0,240 -0,228 -0,067 eigenvalue 4,935 3,749 3,030 2,045 variance (%) 27,415 20,827 16,831 11,359 cumulative (%) 27,415 48,243 65,073 76,433 ital. j. food sci., vol. 32, 2020 576 based on the pca results (fig. 2), same studied almond cultivars could be described by similarities in chemical characteristics considering oil and sugar composition while others had different chemical profile. pc-1 allowed the separation of ‘porto’, ‘fournat de breznaud’, ‘blanco’, ‘dillou’, ‘khoukhi’ and ‘lauranne’ which are rich in total sugar, sucrose and raffinose. the cultivars ‘sahnoun’, ‘zahaaf’, ‘francoli’ and ‘lauranne’, separated along the positive direction of pc-3, were characterized by high oleic, fructose and glucose contents and low linoleic content. ‘mahsouna’, ‘achaak’, ‘lsen asfour’, ‘fekhfekh’ and ‘lauranne’ were situated in the positive side of pc-4 owing their high oil content opposing to ‘mazetto’, ‘supernova’, ‘zahaaf’, ‘francoli’ and ‘ksontini’ on the negative direction with the highest protein content. this data suggests that almond kernels of ‘lauranne’ cultivar offer unique nutritional potential, with high oil content, oleic acid and oleic to linoleic acids ratio and with superior total sugar content, especially fructose content. moreover, the cultivars ‘lsen asfour’, ‘achaak’ and ‘mahsouna’ were associated together and represented some similarities in their composition. 4. discussion the results showed that the main cultivated almonds in tunisia are a potentially rich source of protein, unsaturated fatty acids and sugars. however, their contents on nutritional compound was affected by both genotype and harvest year. the year-to-year variation in fruit quality parameters may be explained by the differences in annual temperatures and precipitation over the two years of study (data not shown). the hard climatic conditions prevailing (dry and hot season) during 2010 were believed to be a contributing factor to the reported variation in sugar and oil content. significant genotypic and environmental effects were noted in the oil content for studied cultivars in the present study. the discrepancies in the possible year effect on oil content could be the result of the specific climatic conditions of the years tested (socias i company et al., 2008). the variation between years indicated that climatic conditions had an effect on almond fruit development and thus severe deficiencies influenced lipid content (zhu et al., 2015). therefore, the oil content trait appears to be under polygenic control (font i forcada et al., 2011), with a clear environmental effect (abdallah et al., 1998; sathe et al., 2008; kodad et al., 2010). moreover, the effect of harvest year on almond kernel oil content has been widely reported in the literature to be significant (barbera et al., 1994; abdallah et al., 1998; sathe et al., 2008). yildirim et al. (2016) reported that the total oil content changed significantly by year in fifteen commercial almond cultivars with the exception of cultivar ‘sonora’. however, no significant year effect was found by kodad et al. (2011) in extensive two-year studies, although the interaction of genotype × year was significant. the magnitude of the effect of the external factors such as the climatic condition of the year probably depends on the genetic background of each cultivar, explaining the significant effect of the interaction genotype × year (kodad et al., 2011). the variability range in total oil content in the present study was similar to the range of variability reported in previous studies. sathe et al. (2008) have reported that oil content for eight almond californian cultivars varied from 49.10% to 66.38%. askin et al. (2007) reported that kernel oil content of 26 almond genotypes from eastern anatolia (turkey) varied from 25.19% to 60.77%. čolić et al. (2017) reported that the range in total oil content for twenty almond spontaneous selections varied between 36.3 and 62.8%. oil content of local almond genotype from argentine varied from 48% to 57.5% (maestri et ital. j. food sci., vol. 32, 2020 577 al., 2015). kodak et al. (2008) found that total lipid contents ranged from 54 to 64.5% for european cultivars. they reported also that total lipid contents ranged from 35 to 53% for australian cultivars and from 35 to 61% for californian cultivars. similarly, yada et al. (2011) reported the variation range of kernel lipid contents of the most important commercial and local almond cultivars growing in usa-california (35-66%), greece (5661%), italy (42-57%), portugal (48-59%), spain (40-67%), turkey (25-61%), afghanistan (4363%), egypt (55-59%), india (44-56%) and iran (55-62%). the heritability described for oil content is high (0.57) indicating an additive gene action, being a trait less influenced by environmental effects (font i forcada et al., 2011). consequently, selection for this trait will be more effective because it is less influenced by the environment (kodad et al., 2013). the local tunisian cultivars with high and stable oil content could be incorporated into the almond breeding program in order to increase the oil content. in addition, the lipid portion, followed by the protein fraction, is the main component of the almond kernel, and is a major determinant of kernel flavor particularly following roasting (socias et al., 2008). however, kernels with a relatively low percentage of oil such as ‘blanco’; ‘francoli’, ‘ksantini’ and ‘ zahaaf’ are required to produce almond milk, a dietetic product; because it’s caloric level must be similar to that of cow’s milk. low lipid contents (‘lsen asfour’, ‘fourna de breznaud’) are also suitable for production of almond flour because of their correlation with high protein content (longhi, 1952). for protein content, stability from year to year was observed for the cultivars ‘dillou’ ‘mahsouna’ and ‘fournat de breznaud’. drogoudi et al. (2012), studying protein and mineral nutrient contents in kernels of 72 sweet almond cultivars and accessions grown in france, greece and italy, reported that the higher temperatures may have favored growth and nutrient utilization, resulting in greater nutrient contents in warmer year. protein content in the seventeen studied almond cultivars ranged from 14 to 27%, which presented an interested range of variability compared with previous studies. in fact, protein contents ranged from 18.5 to 24.0 g 100g-1 of almond among all samples for the top ten almond-producing varieties in california and presently account for about 80% of the total commercial almond acreage (yada et al., 2013). kodad et al. (2013) reported that the protein content ranging between 14.1 and 35.1% for 41 native almond genotypes grown in different geographical regions in morocco. özcan et al. (2011) noted that crude protein content of five turkish almonds varied from 12.7% to 16.3%. askin et al. (2007) reported a wider range of protein content variability (16-31%) in 26 native genotypes from turkey. all these results indicate the high range of variability of protein content depending on the genotype and the environmental conditions of the growing region (kodad et al., 2006). font i forcada et al. (2011) reported that the heritability estimate of protein content in almond is very low (h2= 12.1%), confirming the strong effect of environmental conditions on its expression. almond oil has been reported to be very rich in monounsaturated fatty acids (mufas), especially in oleic and linoleic acids, whereas saturated fatty acids, especially palmitic, palmitoleic and stearic, are very low (yada et al., 2011). in commercial almond cultivars grown in various regions of the world, oleic and linoleic acids together accounts for about 90% of the total lipids, whereas, other fatty acids, including saturated fatty acids accounts for less than 10% (yada et al., 2011). this was consistent for the cultivars originate from the north of tunisia that are ‘dillou’, ‘khoukhi’, ‘blanco’ and ‘abiodh’. but overall the fatty acid composition, in the present paper, was in agreement with previous studies on almond grown around the world (sathe et al., 2008; maestri et al., 2015; zhu et al., 2015; čolić et al., 2017). ital. j. food sci., vol. 32, 2020 578 the variety ‘mahsouna’ appears to present the most stable oil composition. moreover, it presented stable value for oleic and linoleic acid contents. however, the oil composition of the varieties ‘blanco’, ‘achaak’, and ‘francoli’ was more affected by the climatic conditions of the year studied. this confirmed that the year-on-year stability of each fatty acid depended on the specific characteristics of the genotype (abdallah et al., 1998; sathe et al., 2008; kodad et al., 2008, 2010; yada et al., 2011). kodad et al. (2010) reported stable values for some fatty acids in some genotypes such as ‘marcona’, ‘del cid’, and ‘castilla’ for palmitic acid; ‘marcona’ and ‘khoukhi’ for palmitoleic acid; desmayo largueta and ‘del cid’ for stearic acid; ‘brézenaud’ and ‘vivot’ for oleic acid; and ‘desmayo largueta’, ‘khoukhi’, ‘marcona’, ‘retsou’, and ‘vivot’ for linoleic acid. abdallah et al. (1998) reported that the year effect was significant for all fatty acids except palmitoleic acid in twenty one californian cultivars growing at four different sites. kodad et al. (2010), after studying seventeen almond cultivars, reported that the year effect was significant for all fatty acids, except palmitic acid. similarly, kodad et al. (2011) noted that the year effect was not significant for palmitic and stearic acids. furthermore, kodad et al. (2010) reported that the genotype× year interaction was significant for all fatty acids except oleic acid, showing that the magnitude of the values changed each year. yildirim et al. (2016) reported also that the effect of the cultivar, year and the interaction cultivar×year were significant for all fatty acids except heptadecanoic acid in fifteen commercial turkish almond cultivars. finally, sathe et al. (2008) reported that the year effect was significant for all fatty acids in californian cultivars growing at different sites, but stated that the year-to-year variability in fatty acid composition depended on the specific climatic conditions in that year. comparing linoleic acid levels in spanish, mediterranean, californian and australian almonds, zhu et al. (2015) noticed that the regions producing almonds with lower linoleic acid were not irrigated, whereas californian and australian regions routinely apply irrigation to their orchards. nanos et al. (2002), based on oil composition data, noted that irrigation resulted in almonds with superior oil quality as the oil had higher oleic acid content and oleic/linoleic acid ratio than almonds from non-irrigated trees. consequently, irrigation can affect almond kernel oil composition. for the others fatty acids, nanos et al. (2002) reported that irrigation decreased the amounts of palmitic and palmitoleic acids, but did not affect the amount of stearic acid in ‘ferragnès’ and ‘texas’. the high content of unsaturated fatty acids, mainly of oleic acid, increases the phytonutrient value of the almond because this type of fatty acids does not contribute to the formation of cholesterol (kodad et al., 2011). moreover, high levels of oleic acid and low levels of linoleic acid have been associated with prolonged shelf-life of almonds and are often advocated (zhu et al., 2015). thus the varieties ‘sahnoun’, ‘zahaaf’ and ‘francoli’, are superior in marketing quality with high oleic acid content and low linoleic and palmitic contents. furthermore, the higher oleic/linoleic (o/l) ratio was reported on ‘francoli’ followed by ‘sahnoun’, ‘zahaaf’ and ‘mahsouna’ cultivars. this ratio is considered a significant quality criterion of the oil kernel due to its preventive effect on lipid oxidation especially where almonds will be stored for long periods (kodad et al., 2010). in fact, a high o/l ratio is considered as an important factor providing stability in oils as well as a higher nutritional value and healthiest almond lipids (kodad et al., 2013; yildirim et al., 2016). for this, all oils of ‘abiodh’, ‘francoli’, ‘mahsouna’, ‘sahnoun’ and ‘zahaaf’ can be considered of highly perfromant (oleic/linoleic ratio > 4.2). the two cultivars ‘achaak’ and ‘francoli’ were proved to be highly affected by the climatic conditions for sugars composition while ‘dillou’ and ‘khoukhi’ presented the most stable ital. j. food sci., vol. 32, 2020 579 sugar composition regarding harvest year. sugar composition of almond kernel has vital value for good flavor and taste (nanos et al., 2002). it depends on the cultivar as well as the maturity stage but some sugar composition changes during maturation are cultivarspecific (nanos et al., 2002; kazantzis et al., 2003). in fact, kazantzis et al. (2003) indicated that early harvested ‘ferragnes’ almonds had higher raffinose content than late harvested almonds (due to sucrose accumulation with maturation and the preferential production of sucrose from raffinose and the other sugars) while the opposite held true for ‘texas’ almonds. however, the effect of the year was reported to be non-significant on the expression of the sucrose content (yada et al., 2013). sánchez-bel et al. (2008) reported that sucrose and glucose contents in kernels of ‘guara’ grown under drip-irrigated orchards were higher than those from non-irrigated orchards. the effect of year was reported to be significant on the total sugar content of the kernel of ‘ferragnes’ and ‘mazeratto’ varieties (barbera et al., 1994). soluble sugars, while present in relatively low amounts, are sufficient to make kernels sweet-tasting (schirra, 1997). free sugars are important nutritional components that affect the kernel flavor of almond (balta et al., 2009). ‘porto’ and ‘fournat de breznaud’ represented the higher sugar and sucrose content. data regarding sucrose contents of this study were similar to those by kazankaya et al. (2008) and balta et al. (2009). the prevalence of sucrose as the main sugar in almond is in agreement with previous works (fourie and basson, 1990; kader et al., 1996; nanos et al., 2002; kazankaya et al., 2008; barreira et al., 2010). they found, also, that sucrose was the main sugar constituent in almond followed by raffinose, glucose and fructose. fourie and basson (1990) obtained individual sugar contents of five almond cultivars ranging between 3.10 to 4.68 g 100 g-1dw, 0.02 to 0.07 g 100 g-1dw and 0.05 to 0.13 g 100 g-1dw for sucrose, glucose and fructose, respectively. yada et al. (2011) reported that the range variation of almond sugar contents (percentage of total weights) of commercially and locally almond cultivars growing in california is from 2.1 to 7%, in greece from 2.6 to 4%, in india from 3.6 to 12%, in italy from 2.1 to 5.5%, in portugal from 2.5 to 7.1%, in spain from 1.8 to 7.6%, and in turkey from 2.5 to 13%. regarding relationships among the different nutraceutical almond parameters some interesting correlations were demonstrated in this work. oleic and linoleic acids presented a conversely relationship. in the literature it has been reported that the proportion of oleic acid among total fatty acids is highly and negatively correlated with the linoleic acid levels with similar correlation coefficient (r= -0.9) (abdallah et al., 1998; askin et al., 2007; sathe et al., 2008; kodad et al., 2011; zhu et al., 2015). this high correlation between the two predominant fatty acids of almond kernels would allow accurate future predictions of total fatty acid composition by analyzing only linoleic acid level (abdallah et al., 1998). this higher correlation, could be considered as an index in any almond breeding program to improve almond quality (wang et al., 2019). in addition, the proportion of oleic acid was negatively correlated with palmitic level (r= -0.607). similar results were reported in other almond cultivars (askin et al., 2007; sathe et al., 2008; kodad et al., 2011). moreover, the correlation between stearic and arachidic was also approved by other authors (sathe et al., 2008). correlation coefficients, greater than 0.71 or smaller than -0.71, have been suggested to be biologically meaningful showing that this correlation is not influenced by climatic and environmental conditions and is genotype-dependent (kodad et al., 2011). no correlations were observed between the oil content and the percentages of the different fatty acids, even with the major fatty acid, which was also consistent with previous studies (sathe et al., 2008; kodad et al., 2011). ital. j. food sci., vol. 32, 2020 580 the negative correlation between protein and oil contents observed was previously reported by kodad et al. (2013). on the other hand, balta et al. (2009) reported a positive correlation between maltose, glucose and fructose in sweet almond while this relationship was negative in bitter almond. accordingly, these correlation findings indicate that inter-relationship among sugar contents vary according to kernel taste. 5. conclusions this work represents one of the most complete chemical and nutritional studies in almond characterizing the main almond cultivars grown in tunisia. results evidenced that oil, sugar and protein contents in almond depend of a polygenic background with a clear environment effect. local tunisian cultivars are highly rich in oil and fatty acids particularly oleic and linoleic acids with percentages between 88.4 and 91.6% of the extracted oil. in addition, the local cultivar ‘mahsouna’ identified after a prospecting effort in the region of sfax (south tunisia) presented the most stable characteristics over the years regarding oil composition and protein content. the cultivar ‘porto’ from the north of the country was performing in terms of sucrose and total sugar contents. this information would be essential to increase our knowledge on the local tunisian almond diversity and their biochemical performance regarding traits to select adequate parents for future breeding programs. these results also support the importance of the characterization and preservation of genetic diversity being the granary for selecting in the coming future cultivars with high quality in a context of global warming offering valuable information for breeders about limits and capacities of the tunisian almond cultivars to be used for breeding programs. acknowledgements projects a/027075/09 and a/031665/10 from the spanish agency for international cooperation (aeci), resgenolamab from the institution for agricultural research and higher education of tunisia and nut4drought from arimnet-2 european program. references abdallah a., ahumada m.h. and gradziel t.m. 1998. oil content and fatty acid composition of almond 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yildirim a.n., akinci-yildirim f., şan b. and sesli y. 2016. total oil content and fatty acid profile of some almond (amygdalus communis l.) cultivars. polish j. food nut. sci. 66:173-178. zhu y., taylor c., sommer k., wilkinson k. and wirthensohn m. 2015. influence of deficit irrigation strategies on fatty acid and tocopherol concentration of almond (prunus dulcis). food chem. 173:821-826. paper received december 9, 2019 accepted march 13, 2020 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (1): 13–23 issn 1120-1770 online, doi 10.15586/ijfs.v34i1.2080 13 p u b l i c a t i o n s codon characteristics of non-gluten noodles from modified cocoyam (xanthosoma sagittifolium) and porang (amorphophallus oncophyllus) dedin finatsiyatull rosida*, ulya sarofa, delbra aliffauziah department of food technology, universitas pembangunan nasional veteran surabaya, east java, indonesia *corresponding author: dedin finatsiyatull rosida, department of food technology, universitas pembangunan nasional veteran surabaya, east java, indonesia. email: dedin.tp@upnjatim.ac.id received: 30 may 2021; accepted: 8 december 2021; published: 26 january 2022 © 2022 codon publications open access paper abstract foods product were developed after the discovery of gluten-free flour. this study aimed to determine the proper formulation of dry noodles from different proportions of modified cocoyam flour and porang flour. in addition, the use of egg was evaluated in terms of physicochemical and organoleptic properties. the first factor was the proportion of modified cocoyam flour and porang flour (90%:10%, 85%:15%, and 80%:20%). the second factor was the addition of eggs (5%, 10%, and 15%). the result showed that the best formulation according to chemical, physical, and organoleptic parameters was made with modified cocoyam flour and porang flour, 85%:15%, with 10% eggs. this non-gluten-based formulation of noodles showed both high dietary fibers content derived from glucomannan (60.14%) and high protein content (12.75%). keywords: cocoyams tubers, eggs, modified flour, noodles, porang, soybean introduction noodles are long thin pieces of food made from flour, water, and eggs. noodles usually cooked in soup or boiling water. moreover, noodles are composed of wheat flour with a lot of gluten. wheat flour should not be consumed by people who have certain digestive disorders. hence, in this research, non-gluten flour was used. nongluten flour is obtained from tubers such as cocoyam (xanthosoma sagittifolium). however, some limitations during production of noodles using non-gluten flour occurred. therefore, other ingredients must be added to enhance the texture of noodles. porang (amorphophallus oncophyllus) flour is added to increase the texture elasticity of noodles. cocoyam is a commodity with an appreciable nutritional profile, high productivity, and better storability than other indigenous roots and tubers. in addition, cocoyam has the potential for sustainable food security. cormels, flours, and starches could be explored in the snack and complimentary food industries by utilizing processing properties, ease of crop production, storability, and nutritional value (boakye et al., 2018). the utilization of cocoyam flour as a substitute for wheat flour in production of noodles has physical and sensory limitations such as dull color, too much stickiness when added with water, very low solubility and swelling strength, and soft texture. therefore, modification of flour is required to enhance the physical and sensory properties of noodles. the physical and sensory properties of noodles can be improved in several ways. one of them is by fermenting the flour using lactic acid bacteria (lab). fast-growing microbes produce pectin lytic and cellulolytic enzymes. these enzymes can destroy the tuber cell wall allowing the liberation of starch granules (subagio, 2008). these conditions change the characteristics of flour, including increased viscosity, gelation ability, rehydration, and dissolving ability. mailto:dedin.tp@upnjatim.ac.id 14 italian journal of food science, 2021; 34 (1) rosida df et al. hydration. the egg white protein forms a layer that is strong enough to cause a better binding of water to noodles and increase suppleness (diniyah et al., 2017). the addition of eggs at low concentrations does not meet the indonesian national standard for dry noodles. therefore, they must be supplemented with other ingredients with high protein content. soybean is a food with a high nutritional content. it is the best source of protein among nuts. the addition of soybeans to the noodles production is expected to meet the protein quality standards of non-gluten dry noodles (widaningrum and soekarto, 2005), and produce a better texture and noodles taste. for this reason, a fixed proportion of soybean flour (15%) is added to the dry noodles composition. this study aimed to determine the exact proportion between the use of modified cocoyam flour, porang flour, and egg on the noodles quality. methods raw material properties cocoyam tuber, porang flour, and soda ash were used as raw materials to produce dry noodles. cocoyam tubers and porang flour were obtained from sleman, indonesia. lactobacillus plantarum fncc 0027 strain (agricultural product technology laboratory, gadjah mada university, indonesia) was used for the fermentation method. hydrochloric acid (sigma aldrich), sodium hydroxide (merck), fehling a, fehling b, sulfuric acid, de man, rogosa and sharpe (mrs) broth (oxoid ltd.), bromocresol green, boric acid (ho3bo3), distilled water, 95% alcohol, isopropyl alcohol, and aluminum sulfate were used as analysis materials. modified cocoyam and porang flour cocoyam flour was made based on the method of rosida et al., 2020. cocoyam tubers were peeled, washed, and sliced for 0.5-cm thickness. sliced tubers were soaked in 7.5% nacl solution for 1 h to remove calcium oxalate in the tuber tissue, which can cause itching (rozali et al., 2021). sliced tubers were washed with tap water until clean and drained. these were dried using a cabinet dryer at 60°c for 12 h. dried tubers were ground using a grinding machine. finely knotted tubers were sifted using 80-mesh sieves. a suspension of 2500-g cocoyam flour and 7500-ml distilled water was fermented using 7% lactobacillus plantarum fncc 0027 starter for 96 h at room temperature (25 ± 2°c). the fermented products were washed with distilled water at a neutral ph. fermented flour the tuber flour modified by fermentation method resulted in lower water content, brighter flour color, and higher amylose content compared to the ordinary cocoyam flour. the increase of amylose content in cocoyam flour impacts production of dry noodles. starch with high amylose content can absorb water due to amylase, which can form hydrogen bonds that are greater than amylopectin (hidayat et al., 2007). additionally, amylose plays a role in increased firmness compared to amylopectin. therefore, the higher amylose content reduces the stickiness of noodles (supriyadi, 2012). noodles from 100% modified cocoyam flour have less elastic properties of dough. thus, the need for additional ingredients plays an essential role in the chewy texture of noodles. a binder can be added to obtain the rubbery texture of noodles. moreover, binder can trap water to form an elastic and springy texture (saha and bhattacharya, 2010). in this case, porang flour and eggs were used as a binder. tubers of porang (amorphophallus oncophyllus), or often called “iles-iles” in indonesia, from the araceae family, grow in a warm subtropical climate such as in east asia, south china, japan, and indonesia (ambarwati et  al., 2000). porang flour contains high content of glucomannan (15–64% on a dry basis) (faridah et al., 2012) therefore, porang flour can absorb water, form a gel (gelling agent), and increases the elasticity of noodles (wang et al., 2012). the addition of porang flour improves the texture, elasticity, and flexibility of noodles. porang flour increases the thickness of noodles, thus can be used as a substitute for thickening chemicals in noodles. therefore, porang flour can be a gelling agent (retnaningsih and hartayani, 2005). formation of gel occurs when dispersed in water, as hydrocolloids and water molecules are trapped in the complex structures through hydrogen cross-linking. this condition leads to hydration process (citra et al., 2012). glucomannan is a soluble dietary fiber with high water but low calorie contents (yang et al., 2006). nugraheni and puspitaningrum (2013) examined the positive effect of porang tuber (amorphophallus konjac k. koch) on the liver. the results demonstrated that administering porang tuber flour to rats at a dosage of 2000 mg/kg alters the levels of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase as well as the liver histopathology images of wistar male rats induced by paracetamol. the eggs added in production of noodles create more resilient dough so that the noodles will not break easily. moreover, the noodles turbidity during boiling will be prevented. the function of egg yolk is to accelerate water italian journal of food science, 2021; 34 (1) 15 properties of non-gluten noodles from modified cocoyam and porang cooking loss test cooking loss was determined by boiling approximately 5  g of noodles in 150-ml water for 3  min and draining. then noodles were dried at 100°c until their weight was constant. other 5-g noodles were weighed for water content to calculate dry weight of the sample. the percentage of cooking loss was calculated based on the difference between dry weights of the sample before and after boiling divided by dry weight of the sample before boiling. elasticity elasticity was measured using a ruler. the cooked sample was placed on a ruler and measured (the initial length, p1); then it was pulled off until it broke and measured again (the final length, p2). elasticity was calculated by the following formula: (p1 − p2/p1) × 100%. glucomannan levels using gravimetric method the sample (25 g) and aluminum sulfate salt (2.5 g) were dissolved in 75-ml water 1:10 (w/v) with continuous stirring for 35 min. the precipitated samples were separated using a 2000-rpm centrifuge for 30 min. supernatant was added with isopropyl alcohol in a ratio of 1:1 (v/v) with stirring until lumps were formed (rhim and wang, 2013; yan et al., 2012). the lumps were filtered using a filter paper and dried at 60°c for 24 h and weighed. glucomannan content was the percentage of the dry weight to the initial weight of the sample. organoleptic test food quality can be measured by three properties: chemical, physical, and sensory. consumer acceptance is primarily determined by quality factors, especially organoleptic (agustia et al., 2019; akonor et al., 2017). organoleptic properties are determined using the human senses: sight, smell, and taste (chauhan et al., 2018). the organoleptic properties of the dry noodle products tested in this study were color, taste, texture, and aroma. according to friedman’s test performed for the color, taste, and texture of non-gluten dry noodle products, there were significant differences (x2count  ≥  x2table). panelists (n = 25) gave an assessment of color, taste, and aroma using the following evaluating scale: 5 = liked extremely, 4 = liked moderately, 3 = liked slightly, 2 = disliked moderately, and 1 = disliked extremely. data analysis data from all test parameters (except for ash content) were obtained and processed for variance analysis. variance analysis was used to determine the interaction between each composition. further tests were performed was dried at 60°c for 12 h. the modified cocoyam flour was analyzed for moisture, ash, protein (association of official agricultural chemists [aoac], 2004), starch (sudarmadji et al., 1997), and amylose (international rice research institute [irri], 1978). cocoyam tubers and porang flour were obtained from sleman, indonesia. similarly, porang flour was evaluated in terms of moisture, ash, protein (aoac, 2004), starch (sudarmadji et al.,1997), amylose (irri, 1978), and glucomannan (widjanarko and megawati, 2015). dry noodle production dry noodles were produced with the composition of modified cocoyam flour and porang flour (90%:10%, 85%:15%, or 80%:20%) and egg addition (5%, 10%, or 15%). steps for dry noodles production are as follows: modified cocoyam flour and porang flour (90%:10%,  85%:15%, or 80%:20%), soy flour (15%), soda ash (1%), and salt (2%) were mixed while adding water and egg (5%, 10%, or 15%) until it became an homogeneous dough. the dough was put into a noodle extruder machine. noodles were steamed at 100°c for 5 min and dried using a cabinet dryer at 60°c for 7 h. experimental design the experimental design was a completely randomized design with a factorial pattern that comprises two factors. factor i comprised the three levels of the proportion of modified cocoyam flour and porang flour (90%:10%, 85%:15%, or 80%:20%), and factor ii comprised the three levels of egg addition (5%, 10%, or 15%). dry noodles were analyzed for proximate composition such as moisture, ash content, protein content (aoac, 2004), starch content (sudarmadji et al., 1997), rehydration (ramlah, 1997), cooking loss (subarna and muhandri, 2013), elasticity (ramlah, 1997), glucomannan level, and organoleptic properties (wulandari et al., 2008). dry noodles properties physical properties rehydration the measurement of rehydration was conducted using the weighing method. rehydration is the ability of noodles to absorb water after gelatinization. measurements were made by (a) weighing 5  g of raw noodles, and (b) boiling for 4  min or until the noodles were completely gelatinized, and then weighing again. rehydration was the percentage difference between (a) raw noodles and (b) gelatinized noodles. 16 italian journal of food science, 2021; 34 (1) rosida df et al. the absorption of water by proteins is related to the sidechain polar groups such as carbonyl, hydroxyl, amino, carboxyl, and sulfhydryl groups. this condition causes hydrophilic proteins to form hydrogen bonds with water (kilara, 1994). these proteins bind water molecules by the formation of hydrogen bonds (sari, 2017). glucomannan absorbs maximum water compared to other food fibers (charoenrein et al., 2011). a previous study (chan, 2011) confirmed this property, reporting that porang flour contains glucomannan and absorbs up to 200 times of water. if more porang flour is added, the holding capacity of water becomes greater, resulting in higher water content of dry noodles. regarding the quality of dry noodles, indonesia national standard (sni) stated that the maximum water content is 10%. in the present study, dry noodles in each category satisfied this standard (sni no. 01-2974-1996). ash levels of dry noodles were found in the range 1.68– 1.84%. the smaller modified cocoyam flour resulted in the higher porang flour proportion and ash content. the ash content of porang flour was 3.33%. it was higher than the modified cocoyam flour raw material, which was 1.08%. the higher the ash content, the higher the minerals contained in a food ingredient. it is because the ash content from mineral elements and chemical composition does not evaporate during the drying process. the addition of eggs further increases the ash content in noodles. it is due to the mineral content of eggs (juanda and cahyono, 2000). a total of 1% of ash content is found in eggs (muchtadi et al., 2010). egg yolk contains several minerals, especially phosphorus (p), manganese (mn), iron (fe), iodine (i), copper (cu), calcium (ca) and a small portion of zinc (zn), which are more than those found in egg white. maximum mineral component found in egg yolk is p, which binds phospholipids, especially lecithin. more than 60% of p in egg yolk is found in lecithin. on the other hand, egg white contains minerals such as chlorine (cl), magnesium (mg), potassium (k), sodium (na), and sulfur (s) in higher proportion than found in egg yolk (andriani et al., 2015). using duncan’s multiple range test (5%) if there were significant differences. results and discussion raw material properties table 1 shows the characteristics of modified cocoyam flour (rosida et al., 2020) and porang flour compared to the previous research (arifin, 2001; aryanti and abidin, 2015; kumoro et al., 2018; dan widjanarko, 2015). the differences between the raw materials in this study and previous studies were harvesting period, tuberose varieties, and flour production method. according to table 1, porang flour based on the data of previous studies contained 12.32% of water, 3.9% of ash (aryanti and abidin, 2015), 24.6% of starch, 18.2% of amylose (kilara, 1994), 3.42% of protein (kumoro et al., 1994), and 63.49% of glucomannan (widjanarko and megawati 2015). dry noodles’ proximate properties water and ash contents of dry noodles the water content of dry noodles ranged from 6.05% to 7.49%. the lowest water content was 6.05 % in dry noodles. this result was obtained with the modified cocoyam flour 90%, porang flour 10%, and 5% egg yolk. in contrast, the formulation of modified cocoyam flour of 90%, porang flour of 15%, and the addition of 15% egg yolk produced the highest water content (7.49%). table 2 shows the relationship of the proportions of modified cocoyam flour, porang flour as well as the addition of eggs to the water content of noodles. in addition, as shown in table 2, the water content of noodles increases with the lower composition of modified cocoyam flour, the higher composition of porang flour, and more addition of egg yolk. this condition is caused by the presence of glucomannan, which is a water-soluble fiber. similarly, more addition of eggs increases the water level of noodles. eggs contain hydrophilic proteins. table 1. raw material properties according to previous studies. component modified cocoyam flour modified cocoyam flour data in the literature porang flour water content (%) 8.92 ± 0.023 8.28 11.10 ± 0.030 ash (%) 1.076 ± 0.021 1.05 3.33 ± 0.171 starch (%) 63.31 ± 0.247 76.74 24.82 ± 0.182 amylose (%) 22.77 ± 0.164 26.28 19.74 ± 0.158 protein (%) 3.09 ± 0.008 − 3.47 ± 0.005 glucomannan (%) − − 60.14 ± 0.192 italian journal of food science, 2021; 34 (1) 17 properties of non-gluten noodles from modified cocoyam and porang table 2. water content, protein, starch, and amylose level of each composition of modified cocoyam flour and porang flour with addition of egg. formula composition modified cocoyam flour and porang flour (%) egg (%) water content (%) protein (%) starch (%) amylose (%) 5 6.05a ± 0.10 12.23a ± 0.01 65.32e ± 0.84 23.50f ± 0.05 90:10 10 6.35a,b ± 0.22 12.93b± 0.02 64.45d ± 0.76 23.14e ± 0.11 15 7.14d ± 0.12 13.65c ± 0.01 64.03d ± 0.24 22.79c,d ± 0.12 5 6.57b,c ± 0.19 12.55c ± 0.03 64.13d ± 0.07 23.14e ± 0.04 85:15 10 6.78c ± 0.19 13.22d ± 0.02 63.16b,c ± 0.10 22.67c ± 0.08 15 6.71b,c ± 0.34 14.19e ± 0.08 62.59b ± 0.16 22.14b ± 0.18 5 6.77c ± 0.24 12.87f ± 0.07 63.86c,d ± 0.63 22.91d ± 0.04 80:20 10 7.16d ± 0.13 13.53g ± 0.02 62.76b ± 0.10 22.26b ± 0.14 15 7.49d ± 0.20 14.49h ± 0.01 60.98a ± 0.27 21.45a ± 0.16 products from animals contain high ash content because it contains several minerals such as ca, fe, and p. the heating process also affects the amount of ash content in the product produced. the drying process causes the decomposition of water molecular bonding components. moreover, drying process increases sugar, fat, and minerals, thereby increasing ash content. the ash content should be only 3% according to the quality standard of dry noodles. in the present study, dry noodles in each category satisfied this standard (sni no. 01-2974-1996). protein content the protein content of dry noodles was 12.23–14.49%. the proportions of modified cocoyam flour of 90% and porang flour of 10% with an egg addition of 5% produced the lowest protein content (12.23%) whereas the proportions of modified flour of 80% and porang flour of 20% with an egg addition of 15% produced the highest protein content (14.49%). table 2 shows the relationship of the proportions of modified cocoyam flour, porang flour as well as the addition of egg to the protein content of noodles. moreover, the smaller proportion of modified cocoyam flour, the higher proportion of porang flour, and the addition of eggs increased the protein content of dry noodles (table 2). it was due to the higher protein content in porang flour than in modified cocoyam flour, thereby increasing the protein content of dry noodles. the protein content of raw material in porang flour was higher (3.47%) than the protein content of modified cocoyam flour (3.09%). similarly, the addition of more eggs increased protein level in dry noodles. according to muchtadi et al. (2010), eggs have 12.7% of protein. the egg added in the manufacture of dry noodles improves the quality of noodle protein and creates a more resilient dough (astawan, 2008). moreover, addition of soy flour to dry noodles increases protein content because soybean is a good source of protein. a total of 41.7% of protein is found in soybean. according to the quality standard of dry noodles, the standard protein content must be at a minimum of 8%. in this study, the dry noodles in each category satisfied this standard (sni number 01-2974-1996). starch content the starch content of dry noodles ranged from 60.98% to 65.32%. the proportions of modified cocoyam flour of 80% and porang flour of 20% with an egg addition of 15% produced minimum starch content (60.98%) whereas the proportions of modified cocoyam flour of 90% and porang flour of 10% with an egg addition of 5% produced maximum starch content (65.32%). table 2 shows the relationship of the proportions of modified cocoyam flour, porang flour as well as the addition of eggs to the starch content of dry noodles. furthermore, the smaller proportion of modified cocoyam flour, the higher proportion of porang flour, and the addition of eggs decreased starch level in noodles (table 2). this was because the starch level in porang flour was less than that in modified cocoyam flour. the starch level of dry noodles decreased by increasing the proportion of porang flour. similarly, more eggs decreased starch levels in dry noodle products because eggs did not contain starch. the starch content in modified cocoyam flour was 63.31%. it was higher than in porang flour (24.82%); hence, the lower proportion of modified cocoyam flour in noodle production reduced its starch content. 18 italian journal of food science, 2021; 34 (1) rosida df et al. 90% and porang flour of 10% with the egg addition of 5% produced the lowest rehydration (114.56%) whereas the proportions of modified cocoyam flour of 80% and porang flour of 20% with the egg addition of 15% produced the highest rehydration (120.46%). the relationship of the proportions of modified cocoyam flour, porang flour as well as the addition of eggs to the rehydration of dry noodles is presented in table 3. the less proportion of modified cocoyam flour, the more proportion of porang flour, and the addition of eggs increased the rehydration of noodles (table 3). these conditions show that porang flour contains glucomannan, which has high water absorption properties. similarly, the addition of more eggs increased rehydration. it is indicated by lecithin in egg yolks that are hydrophilic. moreover, the addition of eggs causes higher water absorption. lambrecht et al. (2014) reported that lecithin in egg yolks have polar and nonpolar groups. the polar group contains phosphate ester, which is hydrophilic and tends to dissolve in water. the nonpolar group contains fatty acid ester, which is lipophilic and tends to dissolve in fat or oil. it is supported by a previous study (winarno, 2004) that reported that the use of eggs for non-gluten noodles accelerate hydration time. the presence of polar and nonpolar groups of egg yolk determines the water absorption rate and noodles’ elasticity. a previous study (chan, 2011) revealed that porang flour has a high content of glucomannan. it absorbs up to 200 times of water and inhibits syneresis. water-soluble polysaccharides increase the water absorption capacity of the product. therefore, increased proportion of porang flour increases the rehydration of noodles (faridah and widjanarko, 2014). glucomannan comprises monomer α,β-1,4-mannose and α-glucose. glucomannan in ilesiles tubers strengthens the gel, improves texture, and increases thickness (sande et al., 2009). the rehydration of dry noodles with cocoyam flour and the addition of mung bean flour ranged from 138.26% to 145.01% (kartini and widya, 2018). difference in the rehydration of dry noodles is caused by different starch content, protein content, and processing activities. cooking loss the cooking loss value ranged from 8.22% to 9.58%. the proportions of modified cocoyam flour of 90% and porang flour of 10% with the egg addition of 15% produced the lowest cooking loss value (8.22%) whereas the proportions of modified cocoyam flour of 90% and porang flour of 10% with the egg addition of 5% produced the highest cooking loss value (9.58%). the relationship amylose content amylose levels of dry noodles ranged from 21.45% to 23.50%. the proportions of modified flour of 80% and porang flour of 20% with an egg addition of 15% produced the lowest amylose content (21.45%) whereas the proportions of modified cocoyam flour of 90% and porang flour of 10% with an egg addition of 5% produced the highest amylose content (23.50%). table 2 shows the relationship of the proportions of modified cocoyam flour, porang flour as well as the addition of eggs to the amylose content of noodles. these results indicated that the smaller proportion of modified cocoyam flour, the higher proportion of porang flour, and the addition of eggs resulted in the lower level of amylose in noodles. this was because the amylose content in porang flour was less than that in the modified cocoyam flour. therefore, the amylose content of dry noodles decreased by increasing the proportion of porang flour. similarly, the addition of more eggs decreased the amylose levels of dry noodles. the amylose content of modified cocoyam flour was 22.77%. it was higher than that of porang flour (19.74%). the high content of amylose in modified flakes was due to the activity of enzymes produced during fermentation. therefore, the breakdown of amylopectin branch chain in α-1,6 glycosidic bonds caused the formation of new amylose. the lab has amylolytic properties that produce amylase and pullulanase enzymes. these enzymes hydrolyze starch. the release of amylopectin by pullulanase enzyme produces a straight-chain glucose polymer, which is amylose with a smaller degree of polymerization (asha et al., 2013). this pullulanase enzyme can be used to degrade glycosidic α-1,6 branch bonds to amylopectin and produce high amylose (chen et al., 2010).microbes that produce pullulanase enzyme can break down high amounts of amylopectin into simple sugars, resulting in higher amylose concentrations of flour (akbar and yunianta, 2014). low amylose content in porang flour and high protein content in eggs cause the gel structure to form weakly. additionally, these conditions cause higher dissolved solids, stickiness, and inelasticity in dry noodles. amylose plays an important role in the gelatinization process and the character of starch paste (rahim, 2007). physical properties rehydration the rehydration of noodles ranged from 114.56% to 120.46%. the proportions of modified cocoyam flour of italian journal of food science, 2021; 34 (1) 19 properties of non-gluten noodles from modified cocoyam and porang elasticity the elasticity of noodles ranged from 9.30% to 14.66%. the proportions of modified cocoyam flour of 90% and porang flour 10% with the egg addition of 5% produced the lowest elasticity value (9.30%) whereas the proportions of modified cocoyam flour of 80% and porang flour of 20% with the egg addition of 15% produced the highest elasticity value (14.66%). table 3 presents the relationship of the proportions of modified cocoyam flour, porang flour as well as the addition of eggs to the noodles elasticity. our results showed that the less proportion of modified cocoyam flour, the more proportion of porang flour, and the addition of eggs increased noodles’ elasticity (table 3). porang flour contains glucomannan compounds, which form an elastic gel to hold water strongly. similarly, the addition of eggs increased noodles’ elasticity. egg white forms a strong layer or strong adhesion and enhances the texture of dry noodles. moreover, the less proportion of modified cocoyam flour the more proportion of porang flour result in the higher elasticity of noodles. this is because porang flour contains glucomannan compounds, which form an elastic gel and hold water. the formation of hydrocolloid gel occurs due to the formation of a mesh or a three-dimensional gel matrix network by a primary molecule. the primary molecule extends to the entire volume of gel formed by trapping an amount of water, thus making noodles’ texture more elastic (winarno, 2004). the gel in porang flour contains 99.90% water and has special properties such as solidity and especially elasticity. the elasticity of noodles prepared from kimpul flour, tapioca, and tempeh flour ranged from 20.00% to 43.33% (kustanti et al., 2013). the difference in the elasticity of the proportions of modified cocoyam flour, porang flour as well as the addition of eggs to the cooking loss of noodles is presented in table 3. our results demonstrated that the less proportion of modified cocoyam flour, the more proportion of porang flour, and the addition of eggs increased the value of cooking loss. it was due to less amount of amylose in porang flour. however, addition of more egg decreased the cooking loss value. it was due to the presence of protein in eggs that made dough more compact. porang flour has lower amylose content (19.74%) than modified cocoyam flour (22.77%). setyani et al. (2017) stated that the loss of solids because of cooking loss in noodles is influenced by the presence amylose content in the raw materials used. the higher the amylose content, the stronger the gel structure formed. therefore, the smaller total loss of solids happens in noodle strands during the cooking process. owing to the addition of eggs, the higher value of cooking loss decreased in non-gluten dry noodles. it was due to egg protein enhancing the noodle mixture compact and reducing water turbidity during cooking. reza et al. (2008) stated that the higher the level of egg concentration added, the less the cooking loss in dried noodles, because eggs make the dough compact. if the noodle mixture is more compact, less solid is lost during cooking. eggs use in gluten-free products reduces cooking loss (jayadi, 2019). egg protein (albumin) produces a thin and strong layer on the surface of noodles. the coating is effective in reducing water turbidity during cooking. the higher cooking loss resulted in more undesirable dry noodle products. a higher cooking loss causes water turbidity during cooking and the noodles leave a feeling of stickiness in the mouth (uba’idillah, 2015). table 3. rehydration, cooking loss, and elasticity of each composition of modified cocoyam flour and porang flour with addition of egg. formula composition modified cocoyam flour and porang flour (%) egg (%) rehydration (%) cooking loss (%) elasticity (%) 5 114.56a ± 0.11 9.35a ± 0.04 9.30a ± 0.11 90:10 10 115.29b ± 0.18 8.72b ± 0.02 10.47b ± 0.12 15 115.86c ± 0.07 8.22b ± 0.02 11.50c ± 0.35 5 116.38d ± 0.15 9.47c ± 0.11 10.43b ± 0.14 85:15 10 117.64e ± 0.14 8.75c ± 0.01 11.67c ± 0.08 15 117.91f ± 0.08 8.39c ± 0.02 12.70d ± 0.30 5 118.35g ± 0.13 9.58d ± 0.03 13.74e ± 0.18 80:20 10 119.44h ± 0.20 8.76e ± 0.01 13.85e ± 0.09 15 120.46i ± 0.07 8.43f ± 0.01 14.66f ± 0.25 20 italian journal of food science, 2021; 34 (1) rosida df et al. of porang flour, the noodles smell fishier (sumarwoto, 2005). moreover, the panelists’ preference for the taste of noodles tended to be high in the proportion of modified cocoyam flour and porang flour (90%:10%) with the egg addition of 5%, which was 153.5% (table 4). the taste of dry noodles tended to decrease with the addition of more eggs (biyumma et al., 2017). the use of eggs caused a savory taste to noodles due to the presence of lecithin in egg yolk, but the panelists did not like the excessive use of eggs because it made noodles too rancid (a fishy smell). as shown in table 4, the level of texture preference for dry noodles was obtained from the proportion of modified cocoyam flour and porang flour (85%:15%). moreover, the egg addition of 5% was the most preferred composition to meet the preference of consumers. the higher proportion of porang flour and eggs decreased the preference level of panelists for the texture of dry noodles. starch has a high amylose content and hydrogen bond strength because of the large number of straight chains in granules. therefore, it requires more energy for gelatinization and making the noodles chewier (smith, 1982). since porang flour has a low amylose content (19.7%), its addition to dry noodles reduces the texture value. additionally, the more the proportion of porang flour and eggs used, the higher the softness of noodles that have been rehydrated. the level of softness of dry noodles is determined by the presence of glucomannan hydrocolloids in porang flour. it absorbs up to 200 times water of its weight. an increase in porang flour increases the noodle water content and produces softer noodles (citra et al., 2012). egg yolk causes more water absorption. additionally, egg white has crystallization control power because of albumin. it prevents water evaporation and makes the dough softer value of dry noodles was due to the content of amylose, amylopectin, and binder in different flours. additionally, different processing activities cause differences in elasticity. organoleptic test table 4 shows the mean organoleptic value of dry noodle products. our results showed that the proportion of modified cocoyam flour and porang flour (90%:10%) with the egg addition of 10% produced dry noodles with the highest level of color preference (brown). additionally, the proportion of modified cocoyam flour and porang flour (90%:10%) with the egg addition of 15% produced dry noodles with the lowest level of color preference (blackish brown). the color of dry noodles was brown to blackish brown, obtained from the modified cocoyam flour and porang flour. the color of cocoyam flour was white whereas the porang flour was brown. an increase in the proportion of porang flour decreased the level of panelist acceptance of color in dry noodles. the color change in dry noodles occurred because of the addition of porang flour; hence, if more proportion of porang flour was added, then the color of the dry noodles produced increasingly turned brown. porang flour color tends to be brown, and if applied to the product, a darker color is produced (sumarwoto, 2007). hence, the addition of more porang flour produced less liked colors. table 4 shows that the panelists liked the aroma of noodles prepared with the modified cocoyam flour and porang flour formulation (90%:10%) with the egg addition of 5%. owing to the 15% addition of egg, the aroma of noodles produced with the modified cocoyam flour and porang flour formulation (80%:20%) was similar to that of porang. porang flour is light brown with a distinctive odor like that of a fish; hence, with a higher proportion table 4. organoleptic values. formula composition score proportion of modified cocoyam flour and porang flour (%) egg addition (%) color aroma taste texture 5 3.28 3.08 3.12 3.16 90:10 10 3.28 3.00 2.96 3.04 15 3.04 2.64 2.72 2.68 5 3.20 3.04 3.04 3.12 85:15 10 3.20 2.96 3.00 3.04 15 2.76 2.60 2.52 2.72 5 3.00 3.00 2.88 2.88 80:20 10 2.96 2.88 2.68 2.76 15 2.48 2.48 2.56 2.52 italian journal of food science, 2021; 34 (1) 21 properties of non-gluten noodles from modified cocoyam and porang aryanti n. and abidin k.y. 2015. glucomannan extraction from local porang (amorphophallus oncophyllus and amorphophallus muerelli blume). metana. 11(1):21–30. asha r., niyonzima f.n., and sunil s.m. 2013. purification and properties of vpullulanase from bacillushalodurans. int res j biol sci. 2:35–43. association of official agricultural chemists (aoac). 2004. official method of analysis, 12th ed. aoac, washington, dc. astawan m. 2008. instant noodle 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https://doi.org/10.1016/j.phpro.2012.05.026� _hlk87422185 _hlk81020218 _hlk71168835 _hlk80971783 _hlk30452801 _hlk30452841 ijfs#1867_bozza ital. j. food sci., vol. 32, 2020 721 review characterization of lipid substances of rose hip seeds as a potential source of functional components: a review c. mannozzi, r. foligni*, a. scalise and m. mozzon department of agricultural, food and environmental sciences, università politecnica delle marche, via brecce bianche 10, 60131 ancona, italy *corresponding author: r.foligni@staff.univpm.it abstract functional foods receive the greatest attention for nutritional needs of specific consumers. the rose hip fruit, besides carotenoids and polyphenols, are also good sources of lipid substances (fatty acids, sterols and tocopherols), which can be used as functional foods instead of being discarded as waste. the aim of this review is to present an overview of the lipid characterization of rosehip seeds as affected also by the oil extraction procedure. the rosehip seeds oil is proven to be rich in polyunsaturated fatty acid (pufa), sterols and tocopherols, which provide specific biological activities (anti-inflammatory, anti-obesity, antioxidant, anti-diabetic activity). in particular, the oil content of rose hip seeds ranges from 5 to 18 % and is composed of unsaturated fatty acids such as linoleic acid (36-55 %) which is the most abundant one, linolenic (17-27 %) and oleic acid (15-22 %) respectively. as for the sterols, its content ranges around 5 g/kg constituting predominantly βsitosterol, whereas, the tocopherols amount to around 1 g/kg with γ-tocopherol being the most abundant. keywords: rosehip oil, oil extraction, fatty acids, unsaponifiable matter components ital. j. food sci., vol. 32, 2020 722 1. introduction nowadays, the optimization of food production in sustainable way is a requirement with the rise in world population and the reduction of natural resources. as such, the use of abundant natural food sources could be a solution, since food stuffs provide not only nutrition but also exercise a great potential for health benefits (patel, 2015). moreover, the global interest in functional foods increased owing to their promising response to numerous diseases. in accordance, numerous works have reported the potential health properties of plant based products that are rich sources of nutrients and phytochemicals as evidenced through their biological activities (tylewicz et al., 2019; nadpal et al., 2016; mozzon et al., 2015; guimarães et al., 2013; deliorman orhan et al., 2007; rein et al., 2004). in recent times, food wastes are classified as valuable constituents with the emerging technologies ensuring the extraction of target compounds through the food chain (galanakis, 2012).among fruit and vegetable products, rose hips have gained considerable attention due to their highest content of bioactive compounds (ilyasoğlul, 2014). rose hips are included in the rosaceae family, with the rose species being large shrubs or small trees that grow in various regions of the world. they are perennial woody plants, with more than 200 species and 18000 cultivars in the world, geographically distributed mainly in europe, asia and north america (patel, 2017). the rose hips constitute the aggregate fruit of the rose bush plants and are formed of a stretched, pulpy shell called “hypanthium” which encloses the real fruits known as “achenes” (winther et al., 2016) (fig. 1). figure 1. anatomical scheme of rose hip. the achenes represent the thin membranes close to the seeds of rose hip, which are 30-40 % of the overall weight of fruit. the fleshy part of the rose hips is usually utilized in production of different kind of food products (juice, jam, bakery products, candies etc.), while the seeds are discarded as waste. the nutritional characterization has proven that rose hips are rich in nutrients and are good source for dietary supplements, for direct use as functional food product or as food ingredients and additives such as natural colorants for industrial production process (patel, 2017; rosu et al., 2011). rose hip seeds furthermore, have been evidenced to exhibit a series of biological activities such as gastrointestinal protection, diabetic (insulin mimicking), anti-aging, enhancing ital. j. food sci., vol. 32, 2020 723 immunity properties. these can be attributed to their rich chemical composition which include not only phenolic components, mainly flavonoids and proanthocynidins,(fascella et al., 2019; koczka et al., 2018), but also other nutrients such as fatty acid, terpenes, tocopherol, carotenoids, proteins, sugars and minerals (bhave et al., 2017; demir et al., 2014; nadpal et al., 2016). the expression of the studied biological activities and the functional compounds are strictly related to the harvest period, genotypes and species (barros et al., 2011; çelik et al., 2010; güneş et al., 2017). in addition, parts of plant such as fruits, seeds, nuts and sprouts could represent a rich sources of oil with valuable components, which usually have discarded as waste (patel, 2017). in particular, the rose hip seeds acquire oil content from 5 to 18 % which includes varying amounts of unsaturated fatty acids such as linoleic acid (36-55 %), followed by linolenic and oleic acid respectively (17-27 % and 15-22 %). due to their higher favourable polyunsaturated fatty acids and sterols, rose hip seeds oil represents a high value added compound that can be extracted from vegetable wastes and reused in food processing (salgin et al., 2016). the oil constituents were found to exhibit anti-inflammatory, antibacterial, antioxidant activity and potentiality in the cosmetic products production (nadpal et al., 2016; rein et al., 2004). in consideration, the seed oil extraction methods are extremely important as well as their efficiency in order to preserve the bioactive compounds such as the unsaturated fatty acids, and also to obtain an economical oil yield. generally, cold pressing is used in the production of seeds oil. however, many other technologies have been investigated such as soxhlet, organic solvent extraction, ultrasound, microwave and supercritical co2 extraction with the latter exhibiting a higher solubility of the oils and short extraction times, thus minimizing the degradation of bioactive compounds due to thermal and oxygen exposure (dąbrowska et al., 2019a; salgin et al., 2016). the objective of this review is to summarize the state of the research, to underline the attention in the lipophilic components extracted from rose hip seeds oil for further use in the food process and pharmaceutical industries in order to enhance the health benefits. 1.1. proximate and energy profile of rose hip seed oil the different nutritional profiles and assessed energy values of rose hip seeds are shown in table 1. the predominant constituent in the seed was the carbohydrate while the protein represented the lowest content. high carbohydrate content and low protein content avoids protein emulsification, which may limit the oil release. the difference among the nutritional profiles of rosehip seed is mainly due to the different species and the growing conditions (temperature, rainfall and harvest stage) (ilyasoğlul, 2014; du et al., 2017; concha et al., 2006; özcan, 2002). 2. oil extraction methods standard procedures for the oil extraction of plant tissues are cold pressing and solvent extraction. the cold pressing method ensures the production of safe oil since neither heat nor chemical substances are used. on the contrary, this procedure results in low oil yield and hence economically inconvenient in case of rose hip seeds oil (20 % of oil). whereas, ital. j. food sci., vol. 32, 2020 724 solvent extraction requires large amounts of solvent, long extraction times and high temperatures, which may provoke the destruction of sensitive bioactive compounds. table 1. nutritional composition and energy value of the rosehip seeds. references ilyasoǧlu (2014) du et al. (2017) concha et al. (2006) özcan (2002) samples origin turkey (rosa canina) china (rosa acicularis) chile (rosa affinis rubiginosa) turkey (rosa canina) parameters moisture g/100g, dry weight 10.3 (g/100 g of fresh weight) 9.0 6.0 5.7-6.6 carbohydrate g/100g, fresh weight 89.1 40.4 crude fiber 56.0 crude fiber 47.1-65.1 crude fiber fat g/100g, dry weight 6.3 6.7 9.0 13.4-17.8 ash g/100g, dry weight 1.6 3.9 2.0 1.2-2.1 protein g/100g, dry weight 3.0 3.8 3.0 9.6-11.5 energy value kcal/100g, dry weight 425.0 3797.0-5086.0 cal/g studies show that the extraction efficiency of rosa affinis rubiginosa can be increased by enzymatic pre-treatment (up to 30 %) (concha et al., 2006). hydrolytic enzymes are useful tools to selectively depolymerize and break down the cell walls. among the innovative technologies ultrasound, microwave, subcritical and supercritical fluid extraction are used for the recovery of rose hip seeds oil fatty acids (jahongir et al, 2019; szentmihályi et al., 2002). microwave extraction at 40°c for 30 min, supercritical co2 extraction (35°c, 250 bar for 80 min.) and subcritical co2 + c3h8 (28°c, 100 bar for 35 min.) enhanced the oil extractability compared to soxhlet and ultrasound methods. efficiency of supercritical fluid extraction (sfe) and the bioactive components extractability are ascribed to many factors such as temperature, pressure and flow rate (salgin et al., 2016; del valle et al., 2004). in contrast to the solvent extraction, sfe works at low temperature and short process times, thus reducing the thermal damages and degradation of oxygen sensitive compounds. carbon dioxide (co2) is commonly used as solvent for sfe which is a non-toxic, inert, non-flammable, odourless and cheaper compound. fatty acids in the oil are soluble in supercritical co2 at 40°c and up to 280 bar and subsequently increased solubilities can be reached with co-solvent addition (salgin et al., 2016; szentmihályi et al., 2002). 3. lipophilic components of the rosehip seeds oil the oil extracted from rose hip seeds presents high level of bioactive compounds and for this reason it could be used for the enrichment of food products. in particular, the lipophilic part of oil is rich in polyunsaturated fatty acid (pufa), sterols and tocopherols. ital. j. food sci., vol. 32, 2020 725 3.1. fatty acid oil composition the fatty acid composition of the different seed oils extracted from rose hips is affected to the different rose species, environmental factors, agronomic practices, cultivation area and oil extraction methods. table 2 provides an overview of the fatty acid composition of rosehip seed oil subjected to different oil extraction methods. the traditional method for oil extraction from plants is a cold pressing procedure (a). this extraction method employs neither the use of chemical solvents nor heat treatment, thus allows the production of natural and safe oils without compromising their quality. however, the cold pressing method produces low oil yield therefore economically inconvenient for raw materials such as rosehip seeds, which contain a low percentage of oil. in fact, concha et al. (2006) obtained 30-40 % of oil from rosa affinis rubiginosa by cold pressing procedure, however, the yield was increased to 72 % by the enzymatic pre-treatment (h) which allows the cell wall degradation enhancing the extractability of oil. nevertheless, several authors reported similar results for what concerns the fatty acid composition of oil by using cold pressing with or without enzymatic pre-treatment. indeed, the fatty acid composition of oils originating from r. canina and r. rubiginosa grown in poland and chile were characterized by high content of linoleic content (42.20-51.70 %) followed by α-linolenic (21.50-34.00 %) and oleic (12.3618.42 %) rather than palmitic (3.33-4.97 %), stearic (0.11-3.00 %) and arachidonic (0.6-0.7 %) acids (concha et al., 2006; dąbrowska et al., 2019; grajzer et al., 2015; prescha et al., 2014). soxhlet method (b) by using different solvents for extraction such as hexane (ҫelik et al., 2010; dąbrowska et al., 2019; fromm et al., 2012; salgin et al., 2016; szentmihályi et al., 2002), petroleum (kazaz et al, 2009), methanol, chloroform (yilmaz et al., 2011) and diethyl ether (özcan, 2002) had been widely used for rose seeds. the oil yield ranged from 2.75-12.90 % and varied strongly depending on the solvent type, variety and origin of the raw material. ҫelik et al. (2010) reported seed oil composition of five different rose hip species growing in turkey (r. dumalis var. boissieri, r. pulverulenta, r. canina l., r. iberica and r. heckeliana subsp. vanheurckiana). among the studied species, soxhlet extraction yield was highest for r. heckeliana subsp. vanheurckiana (7.95 %) and the lowest for r. canina l. (4.97 %). although in all reported rose species the main unsatured fatty acids were linoleic, α-linolenic and oleic acids, r. heckeliana subsp. vanheurckiana contained high quantitie of linoleic acid (51.06 %), while r. iberica oil was caractherized by high α-linolenic (23.83 %) and oleic acid (23.03 %) contents, respectively. in contrast to these values, other studies reported lower linoleic and α-linolenic acids that ranged from 35.94-36.7 and 14.3-21.15 %, respectively (fromm et al., 2012; szentmihályi et al., 2002) these discrepancies in studiescan be attributed to the different climatic conditions, geological location and other agronomic factorswhich might have influenced the biosynthesis of fatty acids. dąbrowska et al., (2019) reported fatty acid oil profile, obtained from rosa canina grown in bulgaria, by soxhlet with hexane as extraction solvent, and the oil was found to be rich in palmitic (17.80 %) and linoleic (52.60 %) acids and poor in α-linolenic (2.10 %) and oleic acids (1.60 %). when petroleum was used as solvent, theoil content was respectively 7.15 and 2.75 % for rosa canina and rosa damascena. despite the higher oil yields detected, only 48.84 % linoleic and 22.14 % oleic acids have been found in oil of rosa canina, whereas, 54.18 % linoleic and 23.91 % oleic acids have been found in oil of rosa damascena, respectively (kazaz et al., 2009). the specific fatty acid profile is also strongly dependent on the species of plant but ital. j. food sci., vol. 32, 2020 726 even on the growing conditions. in fact, özcan, 2002 found different percentage of fatty acids for rose seeds coming from different regions of turkey. several studies proposed supercritical fluid extraction (sfe), as a solid-liquid process for extraction of seed oil as an alternative to the traditional methods. contrary to the traditional solvent extraction, sfe works with low temperature and short process times, thus, allowing for reduced thermal and oxygen degradation of sensitive compounds. moreover, sfe is widely recognized as a valuable technology due to the extraction efficiency for the use of the fluid (mostly carbon dioxide) which has a high density, a high diffusivity and low viscosity thus leading to rapid solute mass transfer (dassoff and li, 2019). salgin et al. (2016) investigated the influence of particle size (125-1000 µm), pressure (2040 mpa), volumetric flow rate of fluid solvent (0.75-3.5 ml min-1) and temperature (4060°c) on the extraction yield and oil composition of rosa canina by sfe. the oil yields obtained with seed particles lower than 500 µm were about 50 % higher compared to the bigger particle sizes (>1000 µm). this is probably due to the difficulty in extraction of smaller particles (around 30 µm) closer to the bigger fractions, which may limit the mass transfer inside the pores, causing less enhanced release of oil. extraction rate of oils increased with decreasing particle sizes (18 % at 250 bar, 50°c and 3 ml min-1) (machmudah et al., 2007). separation process carried out with co2 (e) provoked the highest extraction yield (16.5 % oil) by the application of 30 mpa, 40°c, 0.75 ml min-1 and 355-500 m in extraction time of 150 min. however in the case where 5 % vol. ethanol (g) was used as solvent, the same amount of oil was extracted in about 90 min. concerning these oils, no significant differences were found in the fatty acid profiles for the different sfe extraction processes, and were composed mainly by linoleic (48.3-49.0 %), α-linolenic (19.9-21.2 %) and oleic (19.5-20.7 %) acids (salgin et al., 2016). szentmihályi et al. (2002) emphasized the oil yield extractability of rosa canina hip seeds by sfe with co2+c3h8 (f) (28°c, 100 bar and 35 min.) and sfe with co2 (e) (35°c, 250 bar and 80 min.), respectively. the yields were determined to be around 6.68 and 5.72 % for (f) and (e) respectively, and were higher compared to that of soxhlet, microwave and ultrasound water bath extraction (4.85, 5.26 and 3.25 %). thus, determined an enhanced fatty acids percentage of linoleic acid from 8.18 to 18.81 % rather than oleic and α-linolenic acids, which reported similar amount compared to the other extraction method. the effect of temperature, pressure and co2 flow rate on fatty acids content of rosehip seeds was investigated (machmudah et al., 2007). the linoleic acid (47.02-49.14 %) and α-linolenic (33.02-40.21 %) significantly increased with increasing temperatures (40, 60 and 80°c). above 300 bar the linoleic acid content tends to be steady; this is probably due to the increased co2 density at higher pressure causing the enhancement of acid dissolution, while α-linolenic acid significantly increased with increasing pressure applied. however, palmitic and stearic acids were not affected by any of the considered parameters for sfe, rather they were not quantified at 150 bar. rosa woodsii provided a rose seed oil, extracted with folch method (i), rich in linoleic (37.10 %), α-linolenic (30.75 %) and oleic (19.70 %) acids (anwar et al., 2008). ital. j. food sci., vol. 32, 2020 727 table 2. fatty acid composition of rose hip seed oils under different oil extraction methods. references grajzer et al. (2015) presha et al. (2014) szentmihályi et al. (2002) ҫelik et al. (2010) salgin et al. (2016) du et al. (2017) yilmaz (2011) concha et al. (2006) origin of raw materials poland poland hungary turkey turkey china turkey chile oil extraction system a a b, c, d, e, f b b, e, g c b-h a-h n. of species 2 4 5 5 6 4 9 fatty acid (%) c16:0 4.2-4.8 3.8 3.60-7.87 4.25-5.15 2.3-3.8 4 1.18-3.39 3.33-4.97 c16:1 0.22-0.89 0.50-1.88 c18:0 2.1-3.0 1.8 2.45-3.27 1.80-2.87 1.9-2.5 2.9 0.84-2.58 traces 0.11-1.75 c18:1 14.7-16.3 14.6 l6.25-22.11 20.35-23.03 19.5-20.5 34.2 0.48-29.96 12.36-14.82 c18:2 44.4-51.7 44.1 35.94-54.75 41.l4-51.06 47.0-49.2 56.5 3.21-36.33 42.20-47.87 c18:3 21.5-31.8 34.0 20.29-26.48 19.66-23.83 19.9-22 1.7 0.22-0.31 26.41-31.09 c20:0 nd 0.7 0.6 0.94-1.29 0.8-1 0.65-0.79 c20:2 nd 0,4 others 1.7-2.4 0.4 oil content (%) 3.25-6.68 4.97-7.95 16.5 σ sfa 7.1-8.0 6.5 4.63-5.08 σ mufa 15.2-16.4 15.2 12.36-14.82 σ pufa 73.3-76.3 78.4 73.29-76.21 a: cold pressing procedure; b: soxhlet; c: ultrasound; d: microwave; e: sfe with co2; f: sfe with co2+c3h8; g: fse with 5%vol. ethanol; h: enzymatic pretreatment; i: folch procedure. ital. j. food sci., vol. 32, 2020 728 table 2. continues. references kazaz et al. (2009) machmudah et al. (2007) dabrowska et al. (2019) özcan (2002) fromm et al. (2012) anwar et al. (2008) ilyasoǧlu (2014) origin of raw materials turkey france bulgary germany hungary poland turkey turkey germany canada turkey oil extraction system b e a, b, e b b-h i e n. of species 2 9 1 1 1 3 1 fatty acid (%) c16:0 5.26-5.30 0-4.68 2.72-17.80 1.71-3.17 3.1 3.70 3.34 c16:1 0.04-2.60 0.24-1.01 0.6 0.57 c18:0 2.02-3.13 0-2.88 2.05-8.80 1.69-2.47 2.2 1.59 1.69 c18:1 22.14-23.91 13.17-52.60 14.71-18.42 18.8 19.70 19.50 c18:2 48.84-54.18 47.02-50.25 2.10-55.70 48.64-54.41 36.7 37.10 54.05 c18:3 15.09-20.65 33.02-40.21 1.60-31.80 16.42-18.41 14.3 30.75 19.37 c20:0 0.23-3.50 1.87-2.61 1.3 0.80 1.00 c20:2 0.10 others oil content (%) 2.75-7.15 18 3.1-12.90 13.37-17.82 10 6.29 σ sfa 7.1 8.20 σ mufa 20.1 21.73 σ pufa 51.0 68.10 a: cold pressing procedure; b: soxhlet; c: ultrasound; d: microwave; e: sfe with co2; f: sfe with co2+c3h8; g: fse with 5%vol. ethanol; h: enzymatic pretreatment; i: folch procedure. ital. j. food sci., vol. 32, 2020 729 3.2. sterols profile the recent interest for enriched functional foods with plant sterols is due to their demonstrated reducing effects of cholesterol level well as anti-inflammatory and anticarcinogenic properties (alvarez-sala et al., 2018). in particular, among the several plant sterols, approved by european commission, β-sitosterol, campesterol and stigmasterol are allowed to be used in a higher proportions than the sterol content commonly added as ingredients in functional foods (barriuso et al., 2016). the rosehip seed oil was characterized by higher sterol contents than economically available vegetable oils such as soybean and sunflower (< 5 g/kg) (amarowicz and pegg, 2019). characterization of phytosterols profile of rosehip seed oil is reported in table 3. grajzer et al. (2015) observed that the total content of sterols was high in both rosehip oils (obtained from two different manufacturers), which are respectively 5.891 and 6.485 g/kg compared to camellia (2.312 g/kg) and walnut (1.791 g/kg) oils. no difference in the β-sitosterol and ∆ 5 –avenasterol content of rosa canina oil was found between cold pressing and folch procedure (grajzer et al., 2015; ilyasoǧlu, 2014). however, ∆ 7 stigmasterol and clerosterol were quantified by the dgf official method (ilyasoǧlu, 2014)and not confirmed by gc-ms method, whereas up to 0.6 g/kg of cycloartenol was found (grajzer et al., 2015). beside that zlatanov (1999) in rosa canina oil from bulgaria β-sitosterol followed by ∆ 5 –avenasterol (81.5 and 4.6 g/kg respectively), which were the main sterols, and were found twenty times more compared to the one obtained by ilyasoǧlu (2014) and grajzer et al. (2015). while zlatanov (1999) reported values of phytosterol, which are not comparable with the other data available from similar studies. table 3. phytosterol composition of rose hip seed oils. references zlatanov (1999) ilyasoǧlu (2014) grajzer et al. (2015) turan et al. (2018) phytosterols (g/kg) brassicasterol 5.4 nd 1 campesterol 1.8 0.233 0.192-0.205 43 cholesterol 0.5 4 clerosterol 0.014 stigmasterol 3.5 0.189 0.077-0.060 β-sitosterol 81.5 5.44 4.753-5.297 780 δ5 -avenasterol 4.6 0.316 0.242-0.379 39 δ7-stigmasterol nd 0.414 nd 43 δ7.25-stigmasterol 1.8 δ7-avenasterol 0.9 0.019 0.037-0.056 15 cycloartenol 0.589-0.649 ital. j. food sci., vol. 32, 2020 730 3.3. tocopherol content tocopherols are important compounds of the unsaponifiable fraction, which are present as liposoluble phenols in vegetable oils. different isomer forms could be found (α, β, γ, and δ) depending on the number and position of methyl groups in the phenolic ring (hernandez, 2015). they exhibit antioxidant properties and that ensure the oxidative stability of oils. table 4 provides a summary of different tocopherols composition of rosehip seed oils. the cold-pressed oil of rosehip obtained from two different manufacturers were characterized respectively by 1.0 and 1.1 g/kg of total tocopherols content, which were higher compared to camellia and walnut oils (0.7 and 0.4 g/kg) (grajzer et al., 2015). these results were in agreement with the one obtained by fromm et al. (2012) for rosa canina oil extracted with soxhlet procedure. however, the tocopherols amount was not comparable with the results obtained by zlatanov (1999). besides, a high level of tocopherols is associated with high oil rich in pufa rose woodsii oil obtained with folch procedure was constituted by a high amount of αtocopherol (0.4 g/kg) followed by δ-tocopherol and γ-tocopherol (0.09 and 0.002 g/kg) (anwar et al., 2008). instead, in experiments with rosa canina oils, the most abundant isomer form was γ-tocopherol ranging from 0.6 to 0.9 g/kg (grajzer et al., 2015; fromm et al., 2012). in all rosehip oils, β-tocopherol was not detected (anwar et al., 2008; grajzer et al., 2015; fromm et al., 2012). table 4. total tochopherols of rosehip seed oils. 4. conclusion the improvement of food products is directed towards ensuring nutritional and functional benefits. therefore, an adequate description of lipid food components of rosehip seed oil was provided in order to develop their possible re-use as bioactive component in the functional food production. considering the possibility of their combination, which may permit an improved solubility, stability or bioactivity than the single one. in particular, the oil content of rose hip seeds ranges from 5 to 18 % and it basically includes different amount of unsaturated fatty acids as linoleic, linolenic and oleic acid. however, for what refereces zlatanov (1999) anwar et al. (2008) grajzer et al. (2015) fromm et al. (2012) tocopherols (g/kg) α-tochopherols 19.0 0.4±34.9 0.1-0.2 0.2±5.1 β-tochopherols nd nd nd ɣ-tochopherols 71.0 0.002±84.0 0.6-0.8 0.9±55.6 δ-tochopherols 1.8 0.09±10.4 0.2-0.3 0.03±3.7 total tochopherols 91.8 0.002±50.2 1.0-1.1 1.0±55.9 ital. j. food sci., vol. 32, 2020 731 concern the sterols content around 5 g/kg with the most abundant β-sitosterol and tocopherols amount with γ-tocopherol were observed. references alvarez-sala a., blanco-morales v., cilla a., garcia-llatas g., sánchez-siles l.m., barberá r. and lagarda m.j. 2018. safe intake of a plant sterol-enriched beverage with milk fat globule membrane: bioaccessibility of sterol oxides during storage. journal of food composition and analysis. 68:111-117. doi: doi.org/10.1016/j.jfca.2017.03.011 amarowicz r. and pegg r.b. 2019. natural antioxidants of plant origin. in: advances in food and nutrition research. academic press inc. 90:1-81 doi: doi.org/10.1016/bs.afnr.2019.02.011 anwar f., przybylski r., rudzinska m., gruczynska 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essential oils and methanolic extract l. rezig1, m. saada2, n. trabelsi3, s. tammar2, h. limam2, i. bettaieb rebey2, a. smaoui3, g. sghaier2, g. del re4, r. ksouri2 and k. msaada*2 1high institute of food industries, 58 alain savary street, el khadra city, tunis, 1003, tunisia 2laboratory of aromatic and medicinal plants, biotechnology center in borj cedria technopole, bp. 901 hammam-lif 2050, tunisia 3laboratory of olive biotechnology, biotechnology center of borj-cedria, p.o. box 901, 2050 hammam-lif, tunisia 4università dell'aquila, dipartimento di ingegneria industriale e dell'informazione e di economia, piazzale ernesto pontieri, monteluco di roio, 67100 l'aquila, italy *corresponding author: tel.: +21622205878; fax: +21679412638 e-mail address: msaada.kamel@gmail.com abstract the essential oil variability in aerial parts of aloysia triphylla, collected from four different tunisian regions was assessed. in addition, total polyphenols, flavonoids, and condensed tannins as well as antioxidant, antibacterial, and antifungal activities of methanolic extract and essential oils were determined. chromatographic analysis of aloysia triphylla essential oils showed the predominance of monoterpene aldehydes represented mainly by neral and geranial. rp-hplc analysis of aloysia triphylla methanolic extract revealed the predominance of p-coumaric acid among the 15 phenolic acids identified. antiradical activity was region-dependent and the methanolic extract of kairouan sample had the strongest activity. concerning the reducing power, the methanolic extract of kairouan, siliana, kairouan, and gabes samples were less active than the positive control. siliana sample showed the least ability to prevent the bleaching of β-carotene, whereas kairouan sample exhibited the strongest activity. obtained results of antimicrobial and antifungal activities showed that aloysia triphylla essential oil was endowed with important antibacterial and antifungal properties. overall, based on its methanolic extract and essential oil features, aloysia triphylla may be considered as a valuable source of new multipurpose products for industrial, cosmetic, and pharmaceutical utilization. keywords: aloysia triphylla l., regions, phenolic, essential oils, antioxidant activity, antimicrobial activity ital. j. food sci., vol. 31, 2019 557 1. introduction the lemon verbena, aloysia triphylla (l’herit), britt’s = lippia citriodora (lam.), is a perennial shrub belonging to the verbenaceae family (sharma et al., 2016). it grows naturally in south america and was introduced into north africa (tunisia) and in southern europe in the late seventeenth century (carnat et al., 1999; bensabah et al., 2014). leaves of aloysia triphylla are used for culinary purposes as food seasoning and beverage flavoring (dominguez-avila et al., 2016). furthermore, due to their aromatic, antispasmodic and digestive properties, leaves of aloysia triphylla are commonly used as infusion or herbal tea (bensabah et al., 2014). traditional applications of aloysia triphylla include its use as herbal remedy for colds, fever, spasms asthma, flatulence, colic, diarrhea, indigestion, insomnia, anxiety, analgesic and sedative (valentao et al., 1999). these therapeutic benefits were attributed to phenolic compounds (mainly flavonoids, phenolic acids, and phenylpropanoids) (pascual et al., 2001; quirantes-piné et al., 2009). antioxidants acting as radical scavengers are able to protect the human body as well as processed foods from oxidative damage. presently, much attention has been focused on the antioxidant effect of plant natural compounds due to their wide application in food. medicinal plants, being a promising source of phenolics, flavonoids, anthocyanins and carotenoids, are usually used to add flavor and improve the shelf life of dishes and processed food products. regarding these beneficial effects, low cost and properties of plant phenolics, the interest is to increase research on natural antioxidants, in order to improve on their use in the food industry and as preventive medicine (el babili et al., 2013). antioxidant properties of extracts of aloysia triphylla leaves, as well as chemical composition of their essential oil obtained from different localities were investigated by several authors (pascual et al., 2001; catalan and lampasona, 2002; crabas et al., 2003; kim and lee, 2004; santos gomez et al., 2005; choupani et al., 2014). the essential oil has been shown to exhibit antimicrobial and anti-candida activities (duarte et al., 2005; teixeira et al., 2007). according to ali et al. (2011), antimicrobial activity of aloysia triphylla essential oil may be attributed to the presence of high concentration of long-chain alcohols and aldehydes especially citral, citronellol, menthol, and βcaryophyllene. otherwise, antifungal activity is related to eugenol, camphene, and βcaryophyllene contents (magwa et al., 2006). there is hardly any available datum concerning aloysia triphylla cultivated in tunisia. in our present study, we have studied essential oil and methanolic extracts composition of aloysia trihpylla cultivated in tunisia and gathered from four distinct regions (kairouan, korba, siliana, and gabes). moreover, we have evaluated their antioxidant, antimicrobial, and antifungal activities. this report also stressed on the underlying variability of aloysia triphylla essential oil and methanolic extracts and their biological activities as affected by the collection site. 2. material and methods 2.1. plant material the aerial parts of four accessions of tunisian aloysia triphylla were gathered from four different regions in tunisia, namely kairouan (center), korba (northest), siliana (northwest), and gabes (southeast) (table 1). the aerial part samples were airdried and conserved in a desiccator at room temperature (~25°c) in darkness for further extraction. ital. j. food sci., vol. 31, 2019 558 table 1. geographical coordinates and bioclimatic classification of the collecting zone of aerial parts of aloysia triphylla. longitude latitude elevation (m) bioclimatic zone kairouan 10°05’30,93’’e 35°40’33,29’’n 62 arid superior korba 10°51’43,49’’e 36°34’50,18’’n 12 semi-arid superior siliana 9°21’52,32’’e 36°05’19,39’’n 425 semi-arid superior gabes 10°05’51,08’’e 33°53’17,08’’n 7 arid inferior 2.2. essential oil extraction dried aerial parts (100 g) were subjected to hydro distillation in a clevenger type apparatus for 3h according to the european pharmacopoeia (2017). 2.3. essential oil analysis analysis of volatile compounds by gc was carried out on a hewlett-packard 6890 gas chromatograph (palo alto, ca, usa) equipped with a flame ionization detector (fid) and an electronic pressure control (epc) injector. a polar polyethylene glycol (peg) hp innowax capillary column (30 m × 0.25 mm, 0.25 mm film thickness; hewlett-packard, ca, 127 usa) was used. the flow of the carrier gas (n2) was 1.6 ml/min. the split ratio was 60:1. the analysis was performed using the following temperature program: oven temperature kept isothermally at 35°c for 10 min, increased from 35°c to 205°c at the rate of 3°c/min and kept isothermally at 205°c for 10 min. injector and detector temperatures were held at 250°c and 300°c, respectively. the individual peaks were identified by comparing their relative retention indices to n-alkanes (c6-c22) with those of literature (adams, 2004) and/or with those authentic compounds available in our laboratory. volatile aroma compounds analysis by gc/ms was performed on a gas chromatograph hp 5890 (ii) interfaced with a hp 5972 mass spectrometer (palo alto, ca, usa) with electron impact ionization (70 ev is the ionization energy). a hp-5 ms capillary column (30 m × 0.25 mm, coated with 5% phenyl methyl silicone, 95% dimethylpolysiloxane, 0.25 mm film thickness; hewlett-packard, ca, usa) was used. the column temperature was programmed to rise from 50°c to 240°c at a rate of 5°c/min. the carrier gas was helium with a flow rate of 1.2 ml/min; split ratio was 60:1. scan time and mass range were 1 s and 40-300 m/z, respectively. identification of aroma compounds was made by matching their recorded mass spectra with those stored in the wiley/nbs mass spectral library of the gc/ms data system and other published mass spectra. 2.4. polyphenols extraction the air-dried aerial parts of aloysia triphylla were finely ground with a blade-carbide gringing (ika-werk type: a: 10). triplicate sub-samples of 1 g of each ground organ were separately extracted by stirring with 10 ml of pure methanol for 30 min. the extracts were then kept for 24 h at 4°c, filtered through a whatman no. 4 filter paper, evaporated under vacuum to dryness and stored at 4°c until analyzed (mau et al. 2004). 2.5. total phenolic content total phenolic contents were assayed using the folin-ciocalteu reagent, following the singleton’s method, which was slightly modified by dewanto et al. (2002). an aliquot ital. j. food sci., vol. 31, 2019 559 (0.125 ml) of a suitable diluted methanolic organ extract was added to 0.5 ml of deionized water and 0.125 ml of the folin-ciocalteu reagent. the mixture was shaken and allowed to stand for 6 min, before adding 1.25 ml of 7% na2co3 solution. the solution was then adjusted with deionized water to a final volume of 3 ml and mixed thoroughly. after incubation for 90 min at 23°c, the absorbance versus prepared blank was read at 760 nm. total phenolic contents of aerial parts (three replicates per treatment) were expressed as mg gallic acid equivalents per gram (mg gae/g of dw) through the calibration curve with gallic acid. the calibration curve range was 50-400 mg/ml (r2 = 0.99). all experiments were performed in triplicates. 2.6. total condensed tannins content the total tannin content was measured using the modified vanillin assay described by sun et al. (1998). a total of 3ml of 4% methanol vanillin solution and 1.5ml of concentrated h2so4 were added to 50 𝜇l of suitably diluted sample. the mixture was kept for 15 min, and the absorbance was measured at 500 nm against the blank (methanol). the amount of total condensed tannins was expressed as milligrams of (+)-catechin equivalent per gram of dry weight (mg of ce/g of dw) through the calibration curve with catechin. triplicate measurements were taken for all samples. 2.7. total flavonoid content total flavonoid content was measured according to dewanto et al. (2002). the 250 μl appropriately diluted methanolic aerial extract was mixed with 75 μl nano2 (5%). after 6 min, 150 μl of 10% alcl3 and 500 μl of naoh (1 m) were added to the mixture. finally, the mixture was adjusted to 2.5 ml with distilled water. the absorbance versus prepared blank was read at 510 nm. total flavonoid contents of aerial parts (three replicates per treatment) were expressed as mg catechin equivalents per gram (mg ce/g of dw) through the calibration curve with catechin. the calibration curve range was 50-500 mg/ml. 2.8. isolation and identification of phenolic compounds dried samples from aerial parts of aloysia triphylla were treated and used to hydrolize phenolic compounds following the method of proestos et al. (2006). thereafter, the analysis was carried out using an agilent technologies 1100 series liquid chromatograph (rp-hplc) coupled with an ultraviolet (uv)-vis multi wavelength detector. the separation was carried out on a 250 × 4.6-mm, and 4 µm hypersil ods c18 reversed phase column at ambient temperature. the mobile phase consisted of acetonitrile (solvent a) and water with 0.2% sulphuric acid (solvent b). the flow rate was kept at 0.5 ml/min. the gradient program was as follows: 15% a/85% b 0-12 min, 40% a/60% b 12-14 min, 60% a/40% b 14-18 min, 80% a/20% b 18-20 min, 90% a/10% b 20-24 min, and 100% a 24-28 min. phenolic compounds were identified based on their retention times and spectral characteristics of their peaks against those of the standards, as well as by spiking the sample with standards. analyses were performed in triplicate. 2.9. 2,2-diphenyl-1-picrylhydrazyl (dpph) scavenging assay the scavenging capacity of the obtained extracts was measured by bleaching of the purple-colored solution of the dpph radical according to the method of hanato et al. (1988). a total of 1 ml of different concentrations of extracts prepared in methanol was ital. j. food sci., vol. 31, 2019 560 added to 0.5 ml of a 0.2 mmol/l dpph methanolic solution. the mixture was shaken vigorously and kept at room temperature for 30 min. the absorbance of the resulting solution was then measured at 517 nm after 30 min. the antiradical activity was expressed as ic50 (μg/ml), which is the concentration required to cause 50% dpph inhibition. the ability to scavenge the dpph radical was calculated using the following equation: dpph scavenging effect % = [(ao – a1)/ao] x 100 where ao is the absorbance of the control at 30 min and a1 is the absorbance of the sample at 30 min. bht was used as a positive control. 2.10. reducing power assay the method of oyaizu (1986) was used to assess the reducing power of different extracts. a total of 1 ml of different concentrations of extracts in methanol was mixed with 2.5 ml of a 0.2 m sodium phosphate buffer (ph 6.6) and 2.5 ml of 1% potassium ferricyanide [k3fe(cn)6] and incubated in a water bath at 50°c for 20 min. then, 2.5 ml of 10% trichloroacetic acid was added to the mixture that was centrifuged at 650g for 10 min. the supernatant (2.5 ml) was then mixed with 2.5 ml of distilled water and 0.5 ml of 0.1% ferric chloride solution. the intensity of the blue-green color was measured at 700 nm. results were expressed as ec50 (mg/ml), which was the extract concentration at which the absorbance was 0.5 for the reducing power and was calculated from the graph of absorbance at 700 nm against the extract concentration. ascorbic acid was used as a positive control. 2.11. β-carotene bleaching test the β-carotene bleaching method is based on the loss of the yellow color of β-carotene due to its reaction with radicals formed by linoleic acid oxidation in an emulsion. the rate of βcarotene bleaching can be slowed down in the presence of antioxidants (kulisic et al., 2004). a modification of the method described by koleva et al. (2002) was employed. βcarotene (2 mg) was dissolved in 20 ml of chloroform and to 4 ml of this solution, linoleic acid (40 mg) and tween 40 (400 mg) were added. chloroform was evaporated under vacuum at 40°c and 100 ml of oxygenated ultra-pure water was added, thereafter, the emulsion was vigorously shaken. reference compound (bht), sample extracts were prepared in methanol. the emulsion (3 ml) was added to a tube containing 0.2 ml of different concentrations of extract (1, 10, 100 and 200 µg/ml). the absorbance was immediately measured at 470 nm and the test emulsion was incubated in a water bath at 50°c for 120 min, when the absorbance was measured again. bht was used as the positive control. for the negative control, the extract was substituted by an equal volume of methanol. the antioxidant activity (%) of the aloysia triphylla aerial parts extracts was evaluated in terms of bleaching of the β-carotene using the following formula: % inhibition = !"!!" !!!!" where at and ct are the absorbance values measured for the test sample and control, respectively, after incubation for 120 min. c0 is the absorbance values for the control measured at zero time during the incubation. the results are expressed as ic50 values (µg/ml), which is the concentration required to cause a 50% β-carotene bleaching inhibition. tests were carried out in triplicate. ital. j. food sci., vol. 31, 2019 561 2.12. screening of antibacterial and antifungal activities antibacterial activity was analyzed by the disc diffusion method against four human pathogenic bacteria including escherichia coli (atcc 35218), pseudomonas aeruginosa (atcc 4141), enterococcus faecalis (atcc 2212), and bacillus subtilis (cip 5265) according to the method of rios and recio (2005). the same agar-disc diffusion method was used for screening the antifungal activity of aloysia triphylla aerial parts extracts. four yeast strains (candida albicans (atcc 2091), candida kefyr, candida parapsilosis, and candida glabrata (atcc 90030)) were first grown on sabouraud chloramphenicol agar plate at 30°c for 18-24 h. several colonies of similar morphology of the clinical yeast were transferred into api suspension medium and adjusted to 2 mcfarland turbidity standards with a densimat. the inocula of the respective yeast were streaked on to sabouraud chloramphenicol agar plates at 30°c using a sterile swab and then dried. a sterilized 6 mm paper disc was loaded with 20 𝜇l (10 mg/ml) of aerial parts extract. the treated petri dishes were placed at 4°c for 1-2h and then incubated at 37°c for 18-24h. the inhibition of fungal growth was also evaluated by measuring the diameter of the transparent inhibition zone around each disc. the susceptibility of the standard was determined using a disc paper containing nystatin. 2.13. data analysis all analyses were performed in triplicate and the results expressed as mean values±standard deviations (sd). the data were subjected to statistical analysis using statistical program package statistica (statsoft, 1998). the one-way analysis of variance (anova) followed by duncan multiple range test was employed and the differences between individual mean values were significant at 𝑃 <0.05. 3. results and discussion 3.1. essential oil yields essential oil yields were determined by hydrodistillation of 100 g of aloysia triphylla dry aerial parts gathered from different localities. from these results, the optimal yield was observed in the sample of korba (0.56%) followed by the samples of kairouan, siliana and gabes. statistical examination revealed significant differences in the essential oil yield of aloysia triphylla originated from korba, and those from siliana, kairouan, and gabes. these values are closer to those reported by di leo lira et al. (2013) on essential oil yields of aloysia triphylla from argentina. when compared with other studies reported by moein et al. (2014) and el-hawary et al. (2011) on aloysia citriodora palau leaves and on lippia citriodora kunth fresh leaves, essential oil yields are higher than those of tunisia region, bearing essential oil contents of 1.3% and 0.9%, respectively. according to taghi ebadi et al. (2015), el-hawari et al. (2015), and bensabah et al. (2014), variation in the essential oil yield of aloysia triphylla is attributed to cultivation climates, edaphic variables, water quality irrigation, ripening stages, and drying methods. 3.2. variability in chemical composition of essential oil essential oils analyzed are divided into seven classes based on their chemical functional groups (table 2). ital. j. food sci., vol. 31, 2019 562 table 2. essential oils composition (peak area % w/w±sd) and analysis of variance analysis results (p-values) of essential oil aloysia triphylla aerial parts. compound* rri collection region p kairouan korba siliana gabes α-pinene 1031 0.42 c±0.03 0.55 a±0.04 0.35 d±0.02 0.44 b±0.03 0.002** sabinene 1132 0.22 a±0.01 0.13 d±0.01 0.20 b±0.01 0.19 c±0.02 0.268ns myrcene 1176 0.38 d±0.04 0.41 c±0.03 0.45 b±0.05 0.52 a±0.06 0.001** limonene 1202 7.27 c±0.54 6.34 d±0.43 7.52 b±0.74 7.80 a±0.81 0.000*** (z)-β-ocimene 1245 1.59 d±0.13 2.00 c±0.21 2.20 b±0.19 2.41 a±0.31 0.309ns γ-terpinene 1265 0.21 c±0.05 0.22 b±0.03 0.23 a±0.08 0.19 d±0.02 0.150ns cis -limonene oxide 1450 0.42 b±0.04 0.44 a± 0.03 0.39 c±0.03 0.35 d±0.03 0.501ns transsabinene hydrate 1474 0.26 a±0.02 0.15 d±0.01 0.19 c±0.02 0.24 b±0.01 0.918ns cissabinene hydrate 1556 0.23 a±0.02 0.22 b±0.02 0.20 c±0.01 0.22 b±0.02 0.034* linalol 1552 0.50 d±0.04 0.55 c±0.05 0.58 b±0.06 0.60 a±0.05 0.444ns terpinene-4-ol 1611 0.12 c±0.01 0.13 b±0.01 0.11 d±0.01 0.15 a±0.01 0.483ns α-cadinol 1620 0.49 d±0.05 0.54 c±0.05 0.58 a±0.06 0.57 b±0.07 0.009** trans-chrysanthenol 1684 0.89 a±0.07 0.77 b±0.06 0.69 b±0.07 0.68 bc±0.06 0.042* α-terpineol 1705 0.74 c±0.06 0.70 d±0.05 0.78 a±0.07 0.75 b±0.08 0.480ns cis-chrysanthenol 1762 0.58 a±0.06 0.51 b±0.05 0.40 c±0.03 0.38 d±0.03 0.004** nerol 1798 0.25 a±0.02 0.24 b±0.02 0.20 d±0.01 0.23 c±0.02 0.774ns geraniol 1856 5.57 d±0.54 6.02 b±0.59 5.87 c±0.61 6.42 a±0.54 0.024* geranyl acetate 1765 1.79 a±0.14 1.80 a±0.12 1.72 b±0.15 1.70 c±0.17 0.296ns chrysanthenone 1507 0.55 d±0.05 0.66 a±0.06 0.65 b±0.06 0.61 c±0.06 0.192ns neral 1240 17.22 b±1.80 14.81 d±1.25 15.60 c±1.44 18.73 a±1.75 0.000*** geranial 1742 25.15 c±2.15 26.85 b±2.17 27.41 a±2.45 24.85 d±2.51 0.000*** 1-8-cineole 1213 1.62 d±0.14 1.70 c±0.15 1.77 b±0.18 1.78 a±0.16 0.051ns α-cubebene 1350 0.29 b±0.03 0.33 a±0.02 0.25 c±0.03 0.28 bc±0.01 0.075ns β-cubebene 1466 0.11 c±0.01 0.10 cd±0.01 0.12 b±0.01 0.15 a±0.01 0.223ns δ-elemene 1479 0.45 d±0.03 0.48 c±0.05 0.50 b±0.06 0.55 a±0.06 0.318ns α-copaene 1497 0.16 c±0.01 0.18 b±0.01 0.19 ab±0.02 0.20 a±0.02 0.069ns β-bourbonene 1535 0.86 b±0.07 0.88 a±0.08 0.85 c±0.07 0.85 c±0.08 0.110ns cis-α-bergamotene 1560 0.46 a±0.05 0.38 b±0.03 0.35 c±0.03 0.34 c±0.02 0.003** β-copaene 1580 0.30 b±0.03 0.32 a±0.03 0.28 c±0.02 0.25 d±0.02 0.000*** α-cedrene 1583 0.52 b±0.05 0.50 d±0.04 0.53 a±0.03 0.51 c±0.06 0.910ns β-gurjunene 1597 0.36 a±0.03 0.26 b±0.02 0.21 c±0.02 0.19 d±0.01 0.036* α-caryophyllene 1610 1.64 d±0.15 2.21 c±0.20 2.23 b±0.21 2.24 a±0.25 0.244ns β-caryophyllene 1612 1.06 a±0.12 1.00 b±0.11 0.98 cd±0.07 0.99 c±0.08 0.000*** allo-aromadendrene 1630 0.48 a±0.04 0.44 b±0.03 0.41 c±0.05 0.48 a±0.05 0.000*** aromadendrene 1661 1.03 d±0.11 1.22 b±0.10 1.12 c±0.12 1.25 a±0.10 0.459ns α-amorphene 1679 1.23 d±0.12 1.40 b±0.11 1.33 c±0.10 1.52 a±0.14 0.279ns β-acoradiene 1688 0.41 b±0.03 0.40 c±0.05 0.44 a±0.04 0.38 d±0.03 0.255ns α-zingiberene 1720 0.94 a±0.08 0.85 b±0.07 0.84 c±0.08 0.83 d±0.09 0.000*** germacrene-d 1725 0.44 d±0.05 0.45 c±0.03 0.47 b±0.05 0.49 a±0.05 0.439ns bicyclogermacrene 1755 4.54 a±0.42 4.21 c±0.41 4.25 b±0.39 4.12 d±0.40 0.000*** ar-curcumene 1760 5.24 a±0.45 4.31 d±0.32 5.22 b±0.61 5.13 c±0.44 0.861ns α-cadinene 1773 0.42 c±0.04 0.40 d±0.03 0.47 b±0.05 0.48 a±0.04 0.344ns δ-cadinene 1776 0.63 d±0.05 0.68 c±0.06 0.72 b±0.07 0.77 a±0.08 0.621ns ital. j. food sci., vol. 31, 2019 563 caryophyllene oxide 2008 0.91 b±0.08 0.85 d±0.06 0.92 a±0.07 0.90 c±0.08 0.485ns (e)-nerolidol 2050 1.41 d±0.12 1.54 b±0.14 1.56 a±0.16 1.47 c±0.13 0.995ns spathulenol 2150 1.06 c±0.10 1.20 a±0.11 1.08 b±0.09 1.09 b±0.09 0.030* t-cadinol 2185 0.53 d±0.04 0.62 a±0.05 0.55 c±0.05 0.59 b±0.06 0.023* isospathulenol 2230 2.60 c±0.24 2.65 b±0.25 2.64 bc±0.26 2.66 a±0.29 0.995ns chemical classes monoterpene hydrocarbons 10.77 c±1.34 10.46 d±2.01 11.73 b±1.32 12.36 a±2.45 0.000*** monoterpene alcohols 9.14 d±0.76 9.46 b±0.95 9.21 c±0.83 9.78 a±0.89 0.000*** monoterpene esters 1.79 a±0.14 1.80 a±0.12 1.72 b±0.15 1.70 c±0.17 0.296ns monoterpene ketones 0.55 a±0.05 0.66 a±0.06 0.65 a±0.06 0.61 a±0.06 0.192ns monoterpene aldehydes 42.37 d±4.37 41.66 c±3.98 43.01 b±4.38 43.58 a±5.12 0.000*** monoterpene ethers 1.62 d±0.14 1.70 c±0.15 1.77 b±0.18 1.78 a±0.10 0.051ns sesquiterpenes 28.08 d±2.56 27.86 a±3.11 28.51 c±2.73 28.71 b±3.54 0.000*** total identified 94.29 c±7.52 93.60 d±8.64 96.60 b±8.72 98.52 a±7.24 0.000*** rri: relative retention index; ∗compounds in order of elution on hp-innowax; values of volatile essential oil percentages are the average of three determinations (n = 3). these values with different letters (a-d) are significantly different at p <0.05. ns: not significant. ∗∗p < 0.01. ∗∗∗p < 0.001. p: probability. a total of 48 compounds were identified representing 94.29%, 93.6%, 96.6%, and 98.52% of total volatiles in the samples of kairouan, korba, siliana, and gabes, respectively. these different identified compounds vary significantly (p < 0.05) from one region to another and are highly (p <0.001) affected by the regional factor (table 2). the major contribution was attributed to the monoterpene aldehydes fraction which represents 42.3%, 41.66%, 43.01%, and 43.58% of all compounds detected in kairouan, korba, siliana, and gabes samples, respectively. indeed, this latter fraction is dominated by geranial (24.85% in gabes and 27.41% in siliana) and neral (14.81% in korba and 18.73% in gabes). these results are in agreement with previous studies reported by moein et al. (2014) and taghi ebadi et al. (2015). our studies have reported that limonene was the third major compound in the essential oil of aloysia triphylla samples with a content ranging between 6.34% and 7.8% for korba and gabes samples, respectively. this result is similar to that reported by moein et al. (2014), on essential oil of aloysia citriodora palau cultivated in gardens where neral, geranial, and limonene rates reached 13.46%, 16.7%, and 12.41%, respectively. some authors reported citral to be present at a higher percentage than limonene in l. citriodora (kim and lee, 2004), while others reported the opposite (özek et al., 1996). santos-gomes et al. (2005) reported that the percentage of citral exceeded that of limonene. the ar-curcumene is the main component of sesquiterpene fraction with a rate ranging between 4.31% and 5.24% for the samples of korba and kairouan, respectively. from a statistical point of view, this compound seemed not to be affected by any regional factor. geraniol represented the main constituent in the monoterpene alcohols fraction with a percentage of 5.57%, 6.02%, 5.87%, and 6.42% in the sample of kairouan, korba, siliana, and gabes, respectively. with regard to the 1-8 cineole recognized for its antimicrobial and antifungal properties, this component showed lower amounts in all localities studied varying between 1.62% and 1.78% for kairouan and gabes samples, respectively. taghi ebadi et al. (2015) reported higher levels of 1-8 cineole in lippia citriodora kunth essential oil than those reported in our study. the 1-8 cineole content amounted to 4.5% and 7.3% in freeze dried and oven dried leaves, respectively. it must be pointed out that a variety of geographical and ecological factors can lead to qualitative and quantitative differences in the essential oil produced. at the ital. j. food sci., vol. 31, 2019 564 same time, essential oil composition can be affected by a number of other factors such as the development stage of the plant, its physiology, the age of leaves, and the growing conditions (santos gomes et al., 2005) as well as, the conditions and isolation method (crabas et al., 2003; kim and lee, 2004; santos gomes et al., 2005). 3.3. total polyphenols, flavonoids, and condensed tannins the aerial parts of aloysia triphylla were gathered from different localities, ranging from the center (kairouan), north (korba and siliana), and south (gabes) in tunisia characterized by diverse geographic and climatic conditions as mentioned in table 1. depending on its geographical origin, total polyphenols, flavonoids, and condensed tannins contents of aloysia triphylla extracts are illustrated in fig. 1. figure 1. total polyphenols (tpp), total flavonoids (tf), and total condensed tannin (tct) contents of different regions of aloysia triphylla aerial parts. gae: gallic acid equivalents; ce: catechin equivalents. the letters (a-d) indicate significant differences (p < 0.05). the results showed that the plant is a valuable source of phenolics with content ranging from 26.58 mg gae/g for kairouan to 14.25 mg gae/g for gabes, respectively. these results are lower than those cited by cheurfa and allem (2016) on hydro-alcoholic and aqueous extracts of algerian aloysia triphylla leaves. moreover, zhang and wang (2001) reported a total phenolic content of 1.55 mg gae/ g of fresh weight on aloysia triphylla herb extracted with phosphate buffer. statistical analysis revealed that the total polyphenol contents varied significantly (p < 0.05) between the four studied localities. likewise, the importance of the solvent type used in the extraction has been mentioned by choupani et al. (2014) and bettaieb rebey et al. (2012). according to vasco et al. (2008), differences in total phenol content may also be attributed to varieties, ripening stage, and post-harvest conditions (msaada et al., 2007). besides, the highest total flavonoid content was observed in kairouan (19.26±0.74 mg ec/ g) followed by korba (16.72±0.59 mg ec/g), siliana (15.3±1.46 mg ec/g), and gabes 10.59±1.1 mg ec/g), respectively. these results are much higher than those reported by vinha et al. (2012) on the aqueous extract of aloysia triphylla, showing a total flavonoid content of 43.38 mg c/100 g). it is well known that an important function of flavonoids and phenolic acids is 0 5 10 15 20 25 30 35 tpp mg gae/g dw tf mg ce/g dw tct mg ce/g dw korba kairouan gabes siliana a b c d a b c d a a b a ital. j. food sci., vol. 31, 2019 565 their action in plant defense mechanisms (dixon and paiva, 1995). indeed, flavonoids have several biological activities such as the inhibition of plasma platelet aggregation and cyclooxygenase activity, the suppression of histamine release, potent nitric oxide radical scavenging activity and exhibiting antibacterial, antiviral, anti-inflammatory and antiallergenic effects (cook and samman, 1996). among the different localities studied, no significant differences (p > 0.05) were found in the total condensed tannins contents of aloysia triphylla methanolic extracts. kairouan showed the highest total condensed tannins (1.10±0.17 mg ce/g of dw), followed in a descending order by korba (1.04±0.11 mg ce/g of dw), siliana (1.01±0.10 g ce/g of dw), and gabes (0.89±0.09 g ce/g of dw). interestingly, no condensed tannins were observed in aqueous extracts (infusion and decoction) of aloysia citriodora aerial parts (portmann et al., 2012). this finding was attributed to these authors in the absence of proanthocyanidins. meanwhile, el babili et al. (2013) reported a condensed tannins content of 1.97 g ce/kg dry in aqueous extract of verbena (verbena officinalis l.) belonging to the same botanical family (verbenacea). 3.4. individual phenolic compounds the phenolic compounds in methanol extracts of the aerial parts of aloysia triphylla were identified by a rp-hplc system. this system is a high resolution chromatographic technique widely used for simultaneous separation and quantification of phenolic substances. the results related to phenolic compounds are summarized in table 3. it is fair to say that methanol extracts of aloysia triphylla are rich in phenolics. in total, 15 compounds were identified. statistical analysis revealed that the identified compounds were significantly affected (p < 0.001) by the regional factor. the p-coumaric acid was the predominant phenolic compound. the highest content of p-coumaric acid was observed in kairouan (54.90%), followed by korba, siliana, and gabes. the second major compound was catechol with a content ranging from 6.00% to 12.23% for kairouan and korba, respectively. likewise, rp-hplc analysis was used for the identification and quantification of phenolic compounds in leaves of aloysia triphylla after extraction with a mixture of 62.5% aqueous methanol. authors reported the presence of only four compounds: caffeic acid (0.84%), ferulic acid (0.82%), hydroxytyrosol (0.4%), and apigenin (0.24%). the presence of caffeic and ferulic acids in methanol extracts of aloysia triphylla is worth noting with concentrations ranging between 0.28% and 2.39%, and 0.36% and 3.28% for korba and gabes, and gabes and siliana, respectively. furthermore, apigenin and hydroxytyrosol were not identified in our present study. the o-hydroxybenzoic acid, hydroxycaffeic acid, and 3-nitro-phthalic acid were also identified by gc-ms after sialylation according to proestos et al. (2006). three phenolic compounds; two phenolic acids, dihydrocaffeic acid and 4-hydroxycinnamic acid and a flavonoid glycoside, luteolin7-o-glucoside, were isolated and identified from the ethyl acetate fraction of the fresh aerial parts of lippia citriodora kunth cultivated in egypt (el hawary et al., 2012). luteolin 7-o glucoside was also identified in methanol extracts of the aerial parts of aloysia triphylla, which originated from korba (2.46%), siliana (0.16%), gabes (2.10%), and kairouan (0.35%). this flavonoid glycoside is one of the main flavonoid constituents in many herbs and is known to possess low oxygen radical absorbance capacity (orac) values (zhang and wang, 2001). 3.5. antioxidant activities of methanol extracts the results for antioxidant activities from the different accessions are displayed in table 4. anova analysis (table 4) showed that the methanol extracts are highly influenced by the ital. j. food sci., vol. 31, 2019 566 regional effect (antiradical activity was region-dependent). methanolic extract of kairouan sample shows the highest antioxidant activity (ic50 = 5.78±0.08 µg/ml), which was stronger than that of the positive control: bht (11.5±1.23 µg/ml). the reduced activity was observed in the sample of gabes (30.67 µg/ml). cheurfa and allem (2016) have reported antiradical of aqueous extract of algerian aloysia triphylla leaves activity with an ic50 of 27.4 mg/ml. this value was significantly higher (p < 0.05) than that of hydroalcoholic extract witnessing an ic50 of 23.52 mg/ml. on the other hand, the positive control bht exhibited a significantly lower ic50 value (p < 0.05) when compared to the two studied extracts (6.96 mg/ml). these antiradical activities are lower than those reported in our present study. other studies conducted on culinary decoction of verbena (verbena officinalis l.), belonging to the same botanical family of aloysia triphylla, showed an ic50 of 15.76 mg/ml (el babili et al., 2013). it is worth mentioning now that there is a linear correlation between total polyphenols, flavonoids, and condensed tannins, and the scavenging activity against dpph for the methanolic extracts of the aerial parts of aloysia triphylla. in fact, the methanolic extract of kairouan sample, rich in polyphenols, flavonoids, and condensed tannins, was a more effective scavenger of dpph radicals than the poor ones observed in korba, siliana, and gabes samples. consistency can be seen in our results and those obtained by cheurfa and allem (2016) who analyzed the total polyphenols, flavonoids, and scavenging activity of dpph in aqueous and hydro-alcoholic extracts of aloysia triphylla leaves. meanwhile, el-babili et al. (2013) failed to show any positive correlation between phenol contents and anti-oxidant activities according to the abts/dpph assays on culinary decoction of verbena officinalis l. besides, table 4 showed that the fe3+ reducing power of aloysia triphylla methanolic extracts differs greatly depending on accession provenance. gabes sample showed the higher reducing capacity (ec50 = 482.00 µg/ml) followed by siliana (ec50 = 371.00 µg/ml), korba (ec50 = 322.66 µg/ml), and kairouan (ec50 = 209.33 µg/ml). furthermore, in comparison with the positive control: ascorbic acid (ec50 = 37.33 µg/ml), kairouan, korba, siliana, and gabes samples methanolic extracts exhibited 6, 9, 10, and 13 fold lower activities, respectively. these results indicate that the different methanolic extracts are able to act as electron donor and, therefore, react with free radicals, converting them to a more stable products and, thereby, terminating radical chain reactions. on the other hand, statistical analysis revealed a higher region effect (p < 0.001) on reducing power (table 4). the antioxidant activity of aloysia triphylla methanolic extract was also evaluated by the βcarotene-linoleate bleaching method (table 4). this method was based on the loss of the yellow color of β-carotene due to its reaction with radicals formed after linoleic acid oxidation in emulsion. the rate of β-carotene bleaching can be slowed down in the presence of antioxidants (kulisic et al., 2004). siliana sample showed the lowest ability to prevent the bleaching of β-carotene (ic50 = 4066.67 µg/ml), whereas kairouan sample exhibited the strongest activity (ic50 = 500 µg/ml). on the other hand, all the methanolic extracts had lower antioxidant activities than bht with ic50 of 75.00 µg/ml (table 4). in addition, the β-carotene-linoleate bleaching values were highly (p < 0.001) affected by the accession provenance. it is worth mentioning that differences in antioxidant activities according to the dpph/βcarotene bleaching inhibition/reducing power might reflect differences in the ability of anti-oxidant compounds to act against the different radicals present or formed during each specific reaction. 3.6. antibacterial activity the test results of the antibacterial effect are summarized in table 5. the results showed that the diameter of the inhibition zone (iz) is highly affected by the region’s factor for ital. j. food sci., vol. 31, 2019 567 escherichia coli (atcc 35218) and bacillus subtilis (cip 5265) strains (p < 0.001). on the other hand, essential oils of aloysia triphylla did not exhibit an antibacterial activity against pseudomonas aeruginosa (atcc 4141) and enterococcus faecalis (atcc 2212) strains. the highest antibacterial activity was observed against bacillus subtilis (cip 5265) witnessing an inhibition zone equal to 85±7.67 mm for the aloysia triphylla essential oil of kairouan, followed by that of siliana (iz = 32±3 mm), korba (iz = 26±4.08 mm), and gabes (iz = 25.33±9.62 mm). the lower antibacterial activity of aloysia triphylla essential oils against escherichia coli, when compared to that against bacillus subtilis, was attributed to the cellwall of the gram-negative bacteria covered by an outer membrane (lipopolysaccharide, phospholipid and some proteins) (chao et al., 2000). this latter prevents uptake of oils or protect peptidoglycan layer from oils. hence, lipopolysaccharide (lps) membrane of gram-negative bacteria presents a permeability barrier to hydrophobic substances that can enter and inhibit the gram-positive bacteria. in gram-positive bacteria, the peptidoglycan layer is on the outside and more in contact with the oils. our results are in compliance with those reported by ali et al. (2011) who reported a very strong antibacterial activity of aloysia triphylla essential oil against bacillus subtilis (caicc) (iz ≥ 16 mm) and a negative one against escherichia coli (atcc 25922) (iz = 0 mm). the interesting antibacterial activity against bacillus subtilis was attributed to the presence of a high concentration of long-chain alcohols especially geranial and neral, particularly active against gram-positive bacteria (delaquis et al., 2002). in addition, ali et al. (2011) reported that the antibacterial nature of aloysia triphylla essential oil was apparently related to the presence of β-caryophyllene, showing in vitro activity against escherichia coli, pseudomonas aeruginosa, and staphylococcus aureus (sacchetti et al. 2004). 3.7. antifungal activity aloysia triphylla essential oils had a significant antifungal activity against the four strains of candida species studied as shown in table 5. the highest antifungal activity was recorded for the aloysia triphylla essential oil of kairouan, showing inhibition diameter zones of 85±8.27 mm, 85±7.89 mm, 85±6.59 mm, and 85±8.57 mm against candida albicans (atcc 2091), candida kefyr, candida parapsilosis, and candida glabrata (atcc 90030), respectively. these antifungal activities were higher than that observed for nystatin (iz = 25±2.68 mm) and used as a reference substance. higher antifungal activities were also observed for the aloysia triphylla essential oil of siliana with inhibition diameter zones of 85±8.04 mm for candida albicans (atcc 2091), 85±8.45 mm for candida parapsilosis, and 85±8.81 mm for candida glabrata (atcc 90030), respectively. moreover, aloysia triphylla essential oil of gabes, showed an antifungal activity higher than that of korba against candida albicans (iz = 85±2.48 mm vs 46.33±2.61 mm) and lower than those observed for the three other strains of candida studied. our results are higher than those observed by ali et al. (2011) on antifungal activity of aloysia triphylla essential oil against candida albicans caicc 51, witnessing an inhibition diameter zone between 10 and 15 mm. antifungal activity presented by aloysia tripylla essential oils could be associated with the presence of citral. in fact, literature points to the fact that citral acts as a fungicidal agent because it is capable of forming a charge transfer complex with an electron donor of fungal cells, resulting in fungal death (kurita et al., 1981). ital. j. food sci., vol. 31, 2019 568 table 3. phenolic composition (peak area %, w/w) and analysis of variance results (p-value) of methanol extracts of aloysia tripyllla aerial parts. compound collection region p kairouan korba siliana gabes resorcinol 1.19 d±0.07 1.96 a±0.07 1.72 b±0.02 1.25 c±0.01 0.000*** catechol 6.00 d±0.39 12.23 a±0.34 11.14 b±0.08 8.23 c±0.11 0.000*** epigallocatechin 1.30 b±0.06 0.09 cd±0.03 0.10 c±0.01 1.34 a±0.02 0.000*** caffeic acid 1.50 b±0.10 0.28 d±0.07 1.11 c±0.09 2.39 a±0.03 0.000*** syringic acid 1.92 b±0.28 2.10 a±0.13 1.49 c±0.13 0.96 d±0.36 0.000*** p-coumaric acid 54.90 c±1.33 38.91 a±0.53 38.81 a±2.50 38.23 b±3.34 0.000*** sinapic acid 3.97 a±0.36 1.82 c±0.18 0.86 d±0.10 3.73 b±0.32 0.000*** ferulic acid 0.38 c±0.03 1.24 b±0.04 3.28 a±0.26 0.36 d±0.03 0.000*** luteolin 7-oglucoside 0.35 c±0.02 2.46 a±0.10 0.16 d±0.03 2.10 b±0.07 0.000*** coumarin 1.45 c±0.06 0.22 d±0.02 1.63 b±0.05 1.92 a±0.21 0.000*** rutin 0.52 b±0.03 0.25 c±0.01 1.03 a±0.10 1.04 a±0.36 0.000*** rosmarinic acid 1.20 b±0.09 0.22 d±0.01 1.33 a±0.03 0.49 c±0.16 0.000*** resveratrol 0.20 d±0.01 0.61 b±0.02 0.44 c±0.11 0.76 a±0.06 0.000*** ellagic acid 0.28 d±0.13 1.26 a±0.11 0.31 c±0.02 0.70 b±0.06 0.000*** quercetin 0.26 d±0.06 0.45 b±0.05 1.39 a±0.03 0.38 c±0.40 0.000*** the values of the levels and percentages of phenolic compounds represent the average of three replicates (𝑛 = 3). letters (a-d) indicate significant differences at 𝑃 < 0.05. *** significant effect at 𝑃 < 0.001. 𝑃: probability. ital. j. food sci., vol. 31, 2019 569 table 4. dpph scavenging activity (ic50 µg/ml), reducing power (ec50 µg/ml), and β-carotene bleaching (ic50 µg/ml) and analysis of variance results (p-value) of methanol extracts of aloysia tripyllla aerial parts. collection region bht ascorbic acid kairouan korba siliana gabes p value dpph scavenging activity (ic50 µg/ml) 5.78 d±0.08 6.07 c±0.13 13.23 b±0.28 30.67 a±1.31 0.000*** 11.5±1.23 reducing power (ec50 µg/ml) 209.33 d±1.30 322.66 c±2.84 371.00 b±1.13 482.00 a±2.26 0.000*** 37.33±3.41 β-carotene bleaching (ic50 µg/ml) 500.00 c±11.32 3966.67 a±65.33 4066.67 a±65.33 1566.67 b±130.66 0.000*** 75±5.12 values are means of triplicates±sd. values in the same row with different superscripts (a–d) are significantly different at p < 0.05. *** p <0.001. table 5. antibacterial and antifungal (iz mm) activities and analysis of variance results (p-value) of essential oils of aloysia tripyllla aerial parts. collection region p tetracycline 10 µg/ml nystatin 10 µg/ml kairouan korba siliana gabes bacteria escherichia coli (atcc 35218) 12 a±1.96 11 c±1.13 8.67 d±0.65 11.33 b±0.65 0.000*** 23±2.55 pseudomonas aeruginosa (atcc 4141) na na na na 22±2.71 enterococcus faecalis (atcc 2212) na na na na 24±2.64 bacillus subtilis (cip 5265) 85 a±7.67 26 c±4.08 31.67 b±3.27 25.33 d±9.62 0.000*** 25±2.11 fungi candida albicans (atcc 2091) 85 a±8.27 46.33 a±2.61 85 a±8.04 85 b±2.48 0.000*** 25±2.68 candida kefyr 85 a±7.89 21.67 c±4.71 28.33 b±1.73 19.33 d±1.31 0.000*** 24±2.18 candida parapsilosis 85 a±6.59 28.67 b±9.15 85 a±8.45 25 c±2.56 0.000*** 25±3.05 candida glabrata (atcc 90030) 85 a±8.57 18 b±4.08 85 a±8.81 14 c±1.96 0.000*** 23±2.33 results are the mean of three replications. the diameter of disc was 6 mm. values with different superscripts (a-d) are significantly different at 𝑃 < 0.05. na: not active. ns: not significant. 𝑃: probability. ** 𝑃 < 0.01. *** 𝑃 < 0.001. ital. j. food sci., vol. 31, 2019 570 4. conclusions this study has revealed that aloysia triphylla methanolic extracts and essential oils features are markedly influenced by the regional factor. korba sample have high potential for selecting variety rich in essential oil, while kairouan sample was suitable for being a valuable source of antioxidants, including condensed tannins, flavonoids, phenolic compounds, and polyphenols. the high content of these bioactive compounds both highlights their nutritional and medicinal values and provides a high protection against oxidation phenomenon. essential oil of aloysia triphylla, which originated from kairouan stood out for presenting the best selective antibacterial and antifungal performances. additional researches on the bioactivities of aloysia triphylla might pave the way for its broad application in industrial, pharmaceutic, and cosmetic fields. references adams r.p. 2004. identification of essential oil components by gas chromatography/mass spectrometry. allured, il: carol stream. ali h.f.m., el-beltagi h.s. and nasr n.f. 2011. evaluation of antioxidant and antimicrobial activity of aloysia triphylla. elec. j. envir. agric. food chem. 10:2689-2699. bensabah f, lamiri a, and naja j. 2014. effect of purified wastewater from the city of settat (morocco) on the quality of lippia citriodora essential oil and infusion. j. saudi soc. agric. sci. 14:101-108. bettaieb rebey, i, bourgou s, ben slimen debez i, jabri karoui i, hamrouni sellami i, msaada k, limam f, and marzouk b. 2012. effects of extraction solvents and provenances on phenolic contents and antioxidant activities of cumin (cuminum cyminum l.) seeds. food bioprocess technol. 5:2827–2836. carnat a., carnat a.p., fraise d. and lamaison, j.l. 1999. the aromatic and polyphenolic composition of lemon verbena tea. fitoterapia 7:44-49. catalan c.a.n., de and lampasona m.e.p. 2002. the chemistry of the genus lippia (verbenaceae). in: kintzios, s.e. 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of aqueous extracts of medicinal plants from portugal. eur. j. med. plants 2:335-347. zhang w. and wang s.y. 2001. antioxidant activity and phenolic compounds in selected herbs. j. agric. food chem. 49:5165-5170. paper received october 10, 2018 accepted february 1, 2019 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (3): 13–24 issn 1120-1770 online, doi 10.15586/ijfs.v34i3.2180 13 p u b l i c a t i o n s codon effect of pulsed electric field treatment on cell-membrane permeabilization of potato tissue and the quality of french fries seung-hyun lee1, hafiz muhammad shahbaz2, se-ho jeong1, seok-young hong1, dong-un lee1* 1department of food science and biotechnology, chung-ang university, anseong, republic of korea; 2department of food science and human nutrition, university of veterinary and animal sciences, lahore, pakistan *corresponding author: dong-un lee, department of food science and biotechnology, chung-ang university, anseong 17546, republic of korea. email: dong-un.lee@cau.ac.kr received: 4 february 2022; accepted: 2 june 2022: published: 3 august 2022 © 2022 codon publications open access paper abstract the effect of pulsed electric field (pef) treatment on the cell membrane permeabilization of potato tissue and the quality of french fries was investigated. pulses with an electric field strength of 0.5, 1.5, and 2.5 kv/cm and a width of 20 μs were applied to the potato. pef treatment permeabilized the membrane of potato cells. the magnitude of cell-membrane permeabilization was estimated by ion leaching and biological impedance tests and verified by microscopic observation. as the pef field strength increased, the accumulation of neutral red dye decreased due to increased cell rupture. the index z-values (relative cell membrane breakdown values) for 0.5-, 1.5-, and 2.5-kv/ cm pef-treated samples were 0.01, 0.28, and 0.52, respectively. pef treatment at 2.5 kv/cm reduced the cutting force of potatoes by 33%; it also increased the degree of the crispness of french fries by 64% and decreased crude fat content by 28%. the total reducing sugar content was decreased by pef treatment, which could be attributed to increased lightness and yellowness after frying. therefore, pef treatment improved the quality of french fries by increasing crispness, improving color, and reducing crude fat content. keywords: cell membrane permeabilization; fat content; french fries quality; potato; pulsed electric field introduction potato is one of the most available foods worldwide and can contribute to the daily supply of carbohydrates, minerals, vitamins, and proteins. french fries are among the most popular potato products because of their texture and characteristic taste. frying is a complicated process that involves the heat and mass transfer between fried foods and the surrounding oil. the frying process results in developing a thin outer layer and crust, which are essential in heat and mass transfer during frying and for the sensory characteristics (moreira et al., 2009). dehydration toward the center of the potato causes pore creation and shrinking, resulting in the production of this thin layer (kalogianni and smith, 2013). during crust development, complex chemical and physical events have been reported such as starch gelatinization, cell shape and size changes, and tissue disruption (pedreschi and aguilera, 2002). in the french-fry industry, blanching is a widely used thermal process. it is usually performed in water at 70–90°c for 3–10 min to inactivate the polyphenol oxidase enzyme and soften the tissue (ignat et al., 2015). starch is partially gelatinized during blanching, and cell walls are weakened (moyano et al., 2007). however, blanching is time-consuming and requires a large amount of water. in addition, chlorogenic acid and iron in potatoes may form a colorless compound, ferri chlorogenic acid, resulting in an undesirable darkening of the blanched tissue (wang-pruski and nowak, 2004). sulfate is added to the blanching solution to prevent this mailto:dong-un.lee@cau.ac.kr 14 italian journal of food science, 2022; 34 (3) lee s-h et al. potato samples fresh potatoes (superior) were obtained from a local marketplace in anseong, south korea, and before use, were stored at 4°c. potatoes were sliced into quarters, and the two cut pieces were placed in the batch chamber for pef treatment. pef treatment pef treatment was done using a pulse generator of 5-kw (hvp-5; dil, quakenbrueck, germany) with batching equipment and continuous treatment chambers. the pulse generator creates bipolar and rectangular pulses. the batch chamber had parallel stainless-steel electrodes (10 × 5 cm) separated by 8 cm. potato samples were submerged in the pef treatment chamber containing 300 ml tap water. pef was performed using an out voltage of 1–70%, frequency electrical, 500 pulses, 20 μs pulse width, and electrical field strengths of 0.5 to 2.5 kv/cm. the strength of the electric field in the treatment chamber was estimated by the following equation (1) (zhang et al., 2021): ( ) ( ) kv electrical field strength e, cm output voltage kv distance between the electrode cm   =    (1) the electric field strengths ([e] 0.5–2.5 kv/cm) were used to attain irreversible cell disruption—an e of > 1 kv/cm leads to permanent pore development in the membranes of plant cells. pef treatment was conducted at room temperature. untreated samples were immersed in tap water for 1 min. the pef treatment conditions are listed in table 1. pef-treated potato pieces were sliced into cuboids (1 × 1 × 4 cm) using a commercial cutter with an adjustable frame (jg-04, chromecater, south africa). blanching blanching was performed at 90°c for 3 min by immersing 50 g of raw potato slices in 100 ml of tap water. for 20 min, the blanched potato was immersed in 300 ml of process. however, the sulfate may leave a chemical residue in the product, which can cause an allergic reaction in sensitive humans. therefore, improving the quality of french fries while minimizing chemical additives and reducing the excessive usage of energy and water are important considerations. the food industry has focused on nonthermal processing technologies for reducing energy consumption and shortening the treatment duration (shahbaz et al., 2018). pef is a nonthermal processing technology that involves electrical stimulation using pulsations of high voltage and short intervals (1–100 μs) to food or food ingredients (hill et al., 2022; vorobiev and lebovka, 2009). the processing parameters for pef include the pulse width (μm), pulse number (n), pulse frequency (hz), and electric field strength (kv/cm). membrane conductivity increases immediately after pef treatment, and membrane permeability increases with increasing conductivity (angersbach et al., 2000; pereira et al., 2009). pef damages only the cell membrane (vorobiev and lebovka, 2009), having lipid components as the site of electric interaction. the degree of cell-membrane permeability is determined by the cell membrane’s properties and the electrical pulse (kandušer et al., 2008). pef treatment can induce reversible or irreversible membrane permeability, depending on the conditions. in general, reversible breakdown occurs when pores stay small in proportion to the membrane area and can lead to variations in metabolic responses by increasing the development of secondary metabolites, causing sublethal stress to cells. pef-induced irreversible permeability can significantly improve the transfer of mass in various processes, such as drying (parniakov et al., 2016), extraction, and concentration (gagneten et al., 2019). pef treatment induces softening of plant tissues such as in apples, carrots, and potatoes (boussetta et al., 2013). pef treatment has also been used to improve mass-delivery processes, e.g., drying, extraction, and cooking of various fruits and vegetables (amami et al., 2008; donsì et al., 2010). however, little data are available on the comprehensive results of the pef processing effects on fresh potatoes such as membrane permeabilization, textural properties, and pef-frying effects of french fries, including texture, color, fat contents, and reducing sugar contents. in this study, we examined the pef processing impact on the physical properties of raw potatoes and determined whether it affects the final quality of french fries. materials and methods reagents all chemicals used were purchased from sigma-aldrich, south korea. table 1. pef treatment conditions. size (g) field strength (kv/cm) out voltage (%) pulse width (µs) frequency (hz) pulse number 40 ± 2 0.5 14 20 50 500 1.5 42 2.0 70 italian journal of food science, 2022; 34 (3) 15 pef treatment of potatoes for french fries ion leaching measurement cell membrane permeabilization of potato tissue was estimated indirectly by measuring ion leaching. potato samples were cut into cuboids (1 × 1 × 3 cm) and submerged in 250 ml of deionized water. a conductivity meter (cm-21p, toa-dkk, japan) was used to measure water conductivity at room temperature for 6 h. texture properties texture profile analysis a texture analyzer (ta-xt, stable micro systems ltd., surrey, uk) and a cylindrical probe (20 mm diameter, p/20) performed texture profile analysis. samples were cut into cubes (1 × 1 × 1 cm) in two cycles, and compressed to 15% of their initial height. the test was performed at a pretest speed of 5 mm/s, posttest speed of 5 mm/s, and during test speed of 1 mm/s. the obtained data were analyzed using texture expert software (stable micro systems, uk) and expressed as chewiness, hardness, cohesiveness, springiness, and resilience values. cutting force cutting force was evaluated using a texture analyzer (tahdi/500, godalming, uk) and a warner-bratzler flat blade (caine et al., 2003). samples were sliced into cuboids (1 × 1 × 4 cm), and the maximum shear force (n) was measured. the test was performed at a pretest speed of 5 mm/s, a posttest speed of 5 mm/s, and a test speed of 10 mm/s. frying conditions a commercial deep-fat fryer (dk-201, delki, china) was used for frying. potato cubes were cut into cuboids (1 × 1 × 4 cm) and fried in soybean oil at 180°c for 5 min (potato– oil ratio 1:30, w/w). after frying, potato cubes were drained for 3 min in a frying sieve to remove excess oil. moisture content of french fries the oven-drying method was used to measure the moisture content of fries at 105°c temperature for 18 h. the moisture content of the french fries was measured at 1-min intervals from 1 to 5 min during frying. the crispness of french fries a texture analyzer (tahdi/500, tahd, godalming, uk) equipped with a warner-bratzler flat blade was used for measuring crispness. the crosshead speed was 10 mm/s. color measurement of french fries a colorimeter (ultra scan pro, hunter lab) was used to determine the color differentiation of the french fries. a standard whiteboard was used to calibrate the colorimeter, and values for the l* (lightness), a* (+, redness/−, tap water at room temperature. untreated samples were immersed in tap water for 20 min. microscopic observation of potato tissue sample thickness was determined to be 300 μm using a hand microtome (fb1262, finn science). neutral red solution (0.5% aqueous neutral red) diluted to 0.04% with 0.2 m mannitol−0.01 m 4-(2-hydroxyethyl)-1 piperazineethanesulfonic acid (hepes) buffer at ph 7.8 was used for visualization (caine et al., 2003). tissue was immersed in a 0.04% diluted dye solution for 2 h and rinsed for 1 min in 0.2 m mannitol–0.01 m hepes buffer (ph 7.8). neutral red staining tissue was examined by using an optical microscope (cx22ledrfs1, olympus, japan). the stained cells were observed using an electron microscope at magnifications of 40× and 100×. estimation of cellular damage based on potato tissue conductivity measurements electrical conductivity (s/m) was measured to estimate the pef-induced membrane permeabilization of potatoes. samples of potatoes were cut into cylinders (diameter of 1 cm, thickness of 1 cm) using a cork borer and measured with an lcr meter (lcr-8000g, gw instek, taiwan) at 1 khz to 1.9 mhz frequencies. the electrical conductivity of potato samples was estimated by the following equation (2) (angersbach et al., 2000): s s l ( ) a z( j ) σ w w = (2) where l is the length of the sample, a is the area perpendicular to the electrical field, and z(jw)s is the system impedance. tissue rupture was estimated from the electrical conductivity index z-value calculated by the following equation (3) (vorobiev and lebovka, 2009): i d i z σ σ σ σ − = − (3) where σ (s/m) is the measured electrical conductivity and subscripts i and d indicate intact and damaged tissue (freeze-thaw or heat treatment), respectively. the value is the electrical conductivity of untreated potato tissue, and value is obtained by thawing after freezing for 24 h in the refrigerator and measuring the electrical conductivity. the z-value of the intact tissue is 0 and that of damaged tissue is 1. 16 italian journal of food science, 2022; 34 (3) lee s-h et al. preference evaluation of french fries preference was evaluated by examining the color, texture, and overall acceptability of french fries on a 9-point hedonic scale by 20 panelists. the panelists comprised students and graduate students at the department of food science and biotechnology, chung-ang university who were 20–30 years of age and trained in the inspection method. the sample was placed in a white dish to evaluate color, followed by assessing texture and overall preference. color, texture, and overall acceptability were rated from very disliked (1 point), neither good nor bad (5 points), to very good (9 points). statistical analysis data were analyzed using analysis of variance (anova) in ibm spss statistics 19. results were expressed as means ± standard deviation. differences in means were assessed by duncan’s multi-range test and considered significant at p < 0.05. results and discussion microscopic observation of potato tissue neutral red (nr) staining visualized cell membrane permeabilization of potato tissue. in the control (i) and blanched (ii) samples, nr remained in the cytoplasm because the cell membrane was intact (figure 1). the greenness), and b* (+, yellowness/−, blueness) were determined. the color differentiation between untreated and pef-treated samples was shown as δe, calculated by the following equation (4): 2 2 2e l (( ) a) ( b)∆ = ∆ + ∆ + ∆ (4) total reducing sugar content of potato tissue five grams of raw potato sample was homogenized in 50 ml of distilled water for 1 min and centrifuged at 4°c for 20 min at 8000 rpm. the supernatant was filtered through a membrane filter (0.45 μm; millipore) and diluted twofold with distilled water. samples were inserted into a test tube containing 1 ml of dinitroalicylic acid (dns), and heated for 10 min. after cooling, 3 ml of distilled water was added, and 200 µl of every sample was transferred to a 96-well plate. at 550 nm, the absorbance was measured using a spectrophotometer. total crude fat content of french fries the folch method with several modifications was used to extract crude fat from milk powder to allow fat to be removed without being affected by moisture. chloroform-methanol (300 ml; 2:1, v/v) was added to 15 g of homogenized sample and shaken. next, 60 ml of distilled water was added, shaken, and impurities in the lower layer were removed using filter paper (whatman no. 4). the filtered solvent was concentrated using a decompression condenser and the crude fat content was calculated by weighing. figure 1. microscopic observation of potato tissue. (a) control, (b) blanched, and pef-treated at (c) 0.5 kv/cm, (d) 1.5 kv/cm, and (e) 2.5 kv/cm. (a) (d) (b) (e) (c) italian journal of food science, 2022; 34 (3) 17 pef treatment of potatoes for french fries samples were 0, 0.01, 0.01, 0.28, and 0.52, respectively. the low z-values of the control blanched and 0.5-kv/ cm-treated potato samples indicate intact tissues. however, 1.5and 2.5-kv/cm pef treatment increased z-values, indicating tissue destruction. similarly, the z index values of red beet were 0.6 and 0.9, following pef at 375 and 1000 v/cm, respectively (loginova et al., 2011). ion leaching from potato the breakdown of the potato cell membrane was assessed by measuring the elution of ionic materials (figure 4). the conductivity values of the control, blanched, and 0.5-, 1.5-, and 2.5-kv/cm pef-treated samples after 6 h of immersion were 0.006, 0.01, 0.02, 0.06, and 0.07 s/m, respectively. untreated potato showed very slowly increasing conductivity, indicating little leaching of ionic materials and intact cell membranes. by contrast, the pef-treated samples showed rapidly increasing solution conductivity, indicating that disrupted cell membranes accelerated the release of ionic materials. this finding is consistent with a prior report on pef-treated red pepper (won et al., 2015). therefore, membrane permeabilization of potato cells was increased by pef treatment. effect of pef treatment on potato textural properties the maximum n values of the control, blanched, and 0.5-, 1.5-, and 2.5-kv/cm pef-treated samples were 43.14, 37.88, 36.30, 32.45, and 28.75, respectively (figure  5). samples subjected to pef treatment at 0.5 kv/cm also showed nr-stained cells, implying an intact membrane (figure 1c). pef treatment at 1.5 to 2.5 kv/cm exhibited unstained areas (figure 1d, e). this is consistent with a previous report on onions treated with pef (ersus and barrett, 2010). therefore, pef treatment may induce membrane breakdown and be affected by membrane permeability. electrical conductivity of potato figure 2 shows potato samples’ frequency-dependent electrical conductivity spectrum from 1 khz to 1.9 mhz. increased membrane permeability reduces the electrical resistance, altering the impedance of plant cells. therefore, determining the impedance of plant samples enables the assessment of the magnitude of pef-induced damage (oey et al., 2016). the conductivity-frequency spectrum of the potato samples treated at 0.5 kv/cm was similar to those of the untreated control and blanched pieces. conductivity is degraded by the electrical resistance of an intact cell membrane. however, samples subjected to pef at 1.5 and 2.5 kv/cm had high conductivity, indicating membrane damage. figure 3 shows the z-values obtained by measuring electrical conductivity (s/m). the z-value reflects the degree of disintegration of tissue (lebovka et al., 2002). the index z-value of freeze-thawed tissue is 1.0, and that of untreated tissue is 0. the z-values of the control, blanched, and 0.5-, 1.5-, and 2.5-kv/cm pef-treated 1.0 0.8 0.6 0.4 0.2 0.0 0.0 0.5 1.0 1.5 frequency (log hz) e le ct ric c on du ct iv ity ( s /m ) 2.0 2.5 3.0 control blanching 0.5 kv/cm 1.5 kv/cm 2.5 kv/cm 3.5 figure 2. electrical conductivity spectra of potato samples. bars are standard errors of the mean. 18 italian journal of food science, 2022; 34 (3) lee s-h et al. con 0.0 0.1 0.2 0.3 0.4 0.5 blanching field strength (kv/cm) c on du ct iv ity d is in te gr at io n in de x z 0.5kv/cm 1.5kv/cm 2.5kv/cm figure 3. cell membrane disintegration index z values of potato samples. the conductivities of 1-khz pef-treated, intact, and freeze-thawed samples were used. control blanching 0.5 kv/cm 1.5 kv/cm 2.5 kv/cm 0.00 0 1 2 3 time c on du ct iv ity ( s /m ) 4 5 6 7 0.02 0.04 0.06 0.08 figure 4. effect of pef treatment on ion release from potato. bars are standard errors of the mean. the  pef-treated samples had significantly decreased maximum n values (p < 0.05); the 2.5-kv/cm peftreated sample was reduced by 33%. the hardness values [n] of the control, blanched, and 0.5-, 1.5-, and 2.5-kv/cm pef-treated samples were 21.05, 21.62, 18.96, 17.09, and 14.06, respectively (table  2). the hardness value decreased as the pef-treated field strength increased; the 2.5-kv/cm pef-treated sample decreased by 33%. similarly, the chewiness value of 2.5kv/cm pef-treated potato significantly decreased from 12.96 ± 2.94 to 7.86 ± 1.40 (p < 0.05). however, pef treatment did not significantly affect the springiness, cohesiveness, and resilience values. the tissues became weaker, and the elastic modulus decreased with increasing pef treatment time (lebovka et al., 2004). indeed, the hardness and chewiness of potato samples reportedly decreased with increasing pef field strength (icier et al., 2010). therefore, pef treatment of potatoes may change their textural properties and result in membrane breakdown. effect of pef treatment on the moisture content of french fries figure 6 shows moisture content according to frying time (1–5 min). the moisture content of pef-treated samples rapidly decreased compared to the control but without a significant difference. the moisture content of the control sample at 4 min was similar to that of the 2.5-kv/ cm pef-treated sample at 2.3 min. in pef-treated pieces, diffusion of liquid from the core to the surface might be greater during frying because of increased cell membrane permeability. the blanched samples exhibited slow water diffusion from the core to the surface due to structural changes. blanching promoted starch gelatinization and maintained the integrity of the native pectin network by deactivating pectolytic enzymes (pedreschi and moyano, 2005). therefore, pef increased the rate of water diffusion from the core to the surface during frying. effect of pef treatment on the crispness of french fries the crispness of potato chips is the most critical indicator of their freshness, which is also reflected by their italian journal of food science, 2022; 34 (3) 19 pef treatment of potatoes for french fries control a b b c d blanching 0.5 kv/cm 1.5 kv/cm 2.5 kv/cm 20 30 40 50 10 0 m ax fo rc e (n ) figure 5. maximum cutting force [n] of potato. bars are standard errors of the mean; means with different letters are significantly different (p < 0.05). table 2. texture profile analysis of potato samples. hardness springiness cohesiveness chewiness resilience control 21.05 ± 1.39a 0.82 ± 0.16a 0.75 ± 0.04a 12.96 ± 2.94ab 0.96 ± 0.10b blanched 21.62 ± 1.76a 0.85 ± 0.11a 0.74 ± 0.02a 13.57 ± 1.70a 0.99 ± 0.05b 0.5 kv/cm 18.96 ± 1.80b 0.74 ± 0.11a 0.73 ± 0.02a 10.45 ± 2.73bc 1.03 ± 0.06b 1.5 kv/cm 17.08 ± 1.76b 0.75 ± 0.12a 0.72 ± 0.01a 9.19 ± 1.44c 1.04 ± 0.03b 2.5 kv/cm 14.06 ± 0.94c 0.75 ± 0.11a 0.75 ± 0.02a 7.86 ± 1.40c 1.13 ± 0.08a *values are means ± standard deviation. *means with different superscript letters within the same column indicate significant difference (p < 0.05). hardness (salvador et al., 2009). the crispness of french fries is influenced by their desirable quality characteristics (kita et al., 2007). the crispness values of french fries are shown in figure 7. the crispness value increased with increasing pef field strength. it was the highest for 2.5-kv/cm pef and significantly lower for the untreated control and blanched samples. although the moisture content of samples was varied but without a statistically significant difference (figure 6), it cannot be the only reason for the difference in crispness. during frying, moisture evaporates due to the difference in partial vapor pressure between the product and the frying oil. the diffusion of liquid from the core to the surface in peftreated potato cubes was enhanced by cell membrane permeabilization. pef treatment increases the water vapor pressure, thickens the surface vapor layer, and increases the crispness (janositz et al., 2011). effect of pef treatment on the appearance and color values of french fries an image of fried potato cubes is shown in figure 8. pef treatment led to uniform and brightly colored french fries. the untreated control and blanched samples showed uneven dark colors with brown edges. the color of fried potato is affected by its sugar content. most consumers expect french fries to be golden-brown in color, and higher l* and b* values are preferable for french fries. table 3 shows the l* (lightness), a* (greento-red), and b* (yellow-to-blue) color values of the french fries. pef-treated french fries showed higher lightness and b* values than the untreated control and blanched samples. the french fries treated with 2.5-kv/cm pef showed significantly increased brightness (51.49 ± 1.21 20 italian journal of food science, 2022; 34 (3) lee s-h et al. control blanching 0.5 kv/cm 1.5 kv/cm 2.5 kv/cm 50 60 70 80 90 m oi st ur e (% ) 0 min 1 min 2 min 3 min 4 min 5 min time figure 6. the moisture content of french fries according to frying time. bars are standard errors of the mean. control blanching 0.5 kv/cm 1.5 kv/cm 2.5 kv/cm 0 2 4 6 8 10 12 14 16 m ax fo rc e (n ) ab a ab b c figure 7. the crispness of french fries. bars are standard errors of the mean; means with different letters are significantly different (p < 0.05). (a) (b) (c) (d) (e) figure 8. appearance of french fries after frying at 180°c for 5 min. (a) control, (b) blanched, and (c) 0.5-kv/cm, (d) 1.5-kv/cm, and (e) 2.5-kv/cm pef-treated samples. italian journal of food science, 2022; 34 (3) 21 pef treatment of potatoes for french fries table 3. color values of french fries after frying at 180°c for 5 min. l* a* b* δe control 51.49 ± 1.21d 11.46 ± 0.81c 15.80 ± 1.82c – blanching 57.29 ± 1.57b 14.17 ± 0.87a 23.71 ± 2.86b 9.64 0.5 kv/cm 55.54 ± 1.28c 13.29 ± 0.60ab 23.04 ± 1.73b 7.97 1.5 kv/cm 57.38 ± 0.90b 12.88 ± 0.57b 23.66 ± 1.60b 9.44 2.5 kv/cm 65.81 ± 1.05a 11.01 ± 0.93c 28.51 ± 1.59a 18.80 *values are means ± standard deviation. *means with different superscript letters in the same column are significantly different (p < 0.05). 0 2 4 6 8 10 12 14 16 to ta l r ed uc in g su ga r (m g/ g) control blanching 0.5 kv/cm 1.5 kv/cm 2.5 kv/cm a b c d e figure 9. the total reducing sugar content of potato tissue before frying. bars are standard errors of the mean; means with different letters are significantly different (p < 0.05). to 65.81 ± 1.05; p < 0.05) and yellowness (15.80 ± 1.82 to 28.51 ± 1.59; p < 0.05). therefore, pef treatment affected the color of french fries, as indicated by the δe values. effect of pef treatment on the total reducing sugar content of potato tissue pef treatment of potato cubes significantly decreased their reducing sugar content by disrupting cell membranes (figure 9). treatment of potato cubes with 2.5kv/cm pef decreased the total reducing sugar content from 14.11 ± 0.08 to 5.06 ± 0.08 (p < 0.05). in a previous study, pef treatment at 1.5 kv/cm and 20 pulses stimulated glucose and fructose release (janositz et al., 2011). therefore, pef treatment could be an adjunct to heat treatment for removing reducing sugars linked to the maillard reaction and acrylamide formation. acrylamide formation during french fries production is linked with maillard reactions from reducing sugars and asparagine, as acrylamide precursors, and depends on the frying temperature (yang et al., 2016). it was reported that during the processing, when the sugar concentration was relatively high, acrylamide formation was proportional to the sugar content. in contrast, when the sugar level was low, acrylamide formation was proportional to the asparagine content (halford et al., 2012). efforts are being made by researchers for the reduction of acrylamide content in potato chips. ostermeier et al. (2021) reported that combining pretreatment of pef with ultrasound assisted frying caused a reduction of 66% in acrylamide content of potato chips. in another study, a reduction of 30% of acrylamide content was reported by pef treatment (genovese et al., 2019). pretreatment with yeast followed by pef was found useful in reducing the acrylamide content in potato chips (schouten et al., 2020). a combined treatment of pef and blanching reduced the acrylamide content of french fries (zhang et al., 2021). effect of pef treatment on the total crude fat content of french fries the total crude fat contents of the untreated control, blanched, and 0.5-, 1.5-, and 2.5-kv/cm pef-treated samples were 15.67, 16.00, 15.00, 12.67, and 11.33, 22 italian journal of food science, 2022; 34 (3) lee s-h et al. the color of french fries is closely linked to their freshness (troncoso and pedreschi, 2009). also, the desirable characteristics of potato chips are influenced by their color and crispness, as is the overall taste (salvador et al., 2009). therefore, pef treatment at 2.5 kv/cm increased the overall preference scores of french fries. conclusions pef treatment increased cell membrane permeability in potato tissue proportionately with field strength. the neutral red dye did not accumulate in the cytoplasm after pef treatment, and cell membrane permeability increased with increasing field strength. pef treatment also reduced the maximum cutting force and hardness of potato tissue. the increased cell membrane permeability improved the quality of french fries, which were of uniform and bright color, because of the decreased sugar content resulting from pef treatment. during frying, water rapidly diffused from the core to the surface, thickening the surface vapor layer and reducing oil uptake, and increasing the crispness of french fries. in conclusion, pef treatment could prevent the need for blanching and chemical treatment and reduce the processing time and water and energy consumption. conflicts of interest the authors declared no potential conflict of interest with respect to the research, authorship, and/or publication of this article. funding this research was supported by the chung-ang university graduate research scholarship in 2020. data availability data will be available on request. references amami, e., khezami, l., vorobiev, e. and kechaou, n., 2008. effect of pulsed electric field and osmotic dehydration pretreatment on the convective drying of carrot tissue. drying technology 26: 231–238. https://doi.org/10.1080/07373930701537294 angersbach, a., heinz, v. and knorr, d., 2000. effects of pulsed electric fields on cell membranes in real food systems. innovative food science and emerging technologies 1: 135–149. https:// doi.org/10.1016/s1466-8564(00)00010-2 respectively (table 4). the total crude fat content of peftreated samples decreased significantly with increasing field strength (p < 0.05). this is in agreement with a previous report (ignat et al., 2015) that control and blanched samples had similar oil contents, which were significantly higher (p < 0.05) than those of pef-treated pieces. during frying, water vapor prevents oil penetration (van loon et al., 2007), and pef treatment reduces oil uptake by enhancing water diffusion from the core to the surface of potato strips, creating a thicker water vapor layer and suppressing oil uptake. the decreased oil content of peftreated french fries may also result from their smoother surface, which promotes the draining of oil after frying (thanatuksorn et al., 2005). therefore, pef treatment reduced the crude fat content of french fries. effect of pef treatment on the preference scores of french fries the maximum color, crispness, and overall acceptability scores of french fries were achieved by 2.5-kv/cm pef treatment (table 5). the control samples showed significantly lower scores for color, crispness, and overall acceptability. the scores of the blanched and 0.5-kv/cm pef-treated samples were similar to those of the control. there is reportedly no difference in the color of french fries according to storage temperature and duration. table 4. the total crude fat content of french fries. sample total crude fat content (%) control 15.67 ± 2.31a blanching 16.00 ± 1.00a 0.5 kv/cm 15.00 ± 1.00a 1.5 kv/cm 12.67 ± 0.58b 2.5 kv/cm 11.33 ± 0.58b values are means ± standard deviation. *means with different letters are significantly different (p < 0.05). table 5. preference scores of french fries. sample color crispness overall acceptability control 4.42 ± 2.17c 2.95 ± 1.31d 3.47 ± 1.58c blanching 4.68 ± 1.86bc 3.74 ± 1.24c 4.37 ± 1.38bc 0.5 kv/cm 5.53 ± 1.95abc 4.63 ± 1.34b 5.16 ± 1.80b 1.5 kv/cm 6.00 ± 2.00ab 7.21 ± 0.98a 7.26 ± 0.93a 2.5 kv/cm 6.42 ± 2.19a 7.89 ± 1.20a 7.58 ± 1.46a *values are means ± standard deviation. *means with different letters in the same column are significantly different (p < 0.05). https://doi.org/10.1080/07373930701537294� 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https://doi.org/10.1016/b978-0-12-811447-6.00007-2� https://doi.org/10.1016/j.ifset.2020.102397� https://doi.org/10.1016/j.ifset.2020.102397� https://doi.org/10.1002/jsfa.2300� https://doi.org/10.1002/jsfa.2300� https://doi.org/10.1016/j.lwt.2009.01.008� https://doi.org/10.1016/j.lwt.2009.01.008� https://doi.org/10.1007/s00217-006-0463-1� https://doi.org/10.1007/s00217-006-0463-1� _hlk60588858 _hlk60588923 _goback _hlk89602890 _hlk23862102 _hlk98151240 _hlk98151205 _hlk98151211 _hlk98151054 _hlk98151121 _hlk98151698 _hlk107563485 paper 236 ital. j. food sci., vol. 27 2015 keywords: moulds, sensory defects, virgin olive oil, volatile compounds, yeasts investigation on microbiology of olive oil extraction process s. guerrini, e. mari, m. migliorini1, c. cherubini1, s. trapani, b. zanoni*, m. vincenzini department of agricultural, food and forestry management systems gesaaf food science and technology and microbiology section, university of florence, via donizetti 6, 50144 florence, italy 1promofirenze laboratorio chimico merceologico, special agency of the florence chamber of commerce, via orcagna 70, 50121 florence, italy *corresponding author: bruno.zanoni@unifi.it abstract several batches of approx. 200 kg olives from frantoio and moraiolo cultivars were processed in an oil mill at two dates of harvesting. samples were collected in several steps of extraction process for sensory, chemical and microbial analyses. all extracted olive oil from the second olive harvesting date was affected by sensory defects and hence classified as being “non-extra virgin”. a distinction between extra virgin olive oil and nonextra virgin olive oil obtained from both harvesting dates was explained by the volatile compounds content of olive oil samples and by yeast and mould counts collected at different processing steps. mailto:bruno.zanoni@unifi.it ital. j. food sci., vol. 27 2015 237 introduction the absence of sensory defects is necessary for olive oil to be marketed as ‘‘extra virgin’’ in the eu. extra virgin olive oil (evoo) is characterized by pleasant sensory notes. they are mainly originated by aldehydes, esters, alcohols and ketones, which are responsible for oil sensory attributes such as “green” and “fruity” (aparicio and morales, 1998; morales et al., 2005, bendini et al., 2012). however, several phenomena can alter the initial pleasant flavour, giving rise to unpleasant sensory notes. the current olive oil regulations (eu reg. 1348/2013) classify the most frequent sensory defects into four groups as follows: “fusty”, “musty”, “winey–vinegary”, and “rancid”. storage of olive fruits in piles before being processed is a cause of sensory alterations in evoo. olive transpiration during storage is known to increase pile temperature, enabling microbial cells to grow and to affect the chemical composition of olives (morales et al., 2005). both biogenesis of volatile compounds and transformation phenomena of phenolic compounds can be significantly influenced by microbial contamination of olives. effects of olive microbiota on oil characteristics are considered even greater than time-temperature conditions of malaxation (vichi et al., 2011). oil quality may be affected by microorganisms, according to their metabolic activities. during olive crushing, microorganisms might migrate into oil through both solid particles of olive fruit and micro-drops of vegetation water (ciafardini and zullo, 2002). some microorganisms do not survive a long time, but others may persist and become a typical microbiota of olive oil. for example, yeasts may remain metabolically active during olive oil storage and thus modify olive oil characteristics (zullo et al., 2010). enzymatic activities of yeasts and moulds isolated from either olives or evoo have been reported to include β-glucosidase, β-glucanase, polyphenoloxidases, peroxidase and, in some cases, lipase and cellulase activities (ciafardini and zullo, 2002; ciafardini et al., 2006; zullo and ciafardini, 2008; romo-sanchez et al., 2010). enzymes such as β-glucosidase are known to improve oil quality by increasing phenolic compound extractability, while others such as lipase, polyphenoloxidases and peroxidase are known to cause detrimental effects (palomares et al., 2003; romo-sanchez et al., 2010; vichi et al., 2011; migliorini et al., 2012). penicillium and fusarium spp. isolates have been shown to produce amounts of exogenous lipoxygenase (fakas et al., 2010) that, together with endogenous lipoxygenase, is the key enzyme of lox pathway (angerosa et al., 2004). extraction process control should include monitoring activities on microbial contamination in olives and evoo, as associated with sensory and chemical analyses. the study of the microbial populations occurring at different steps of evoo extraction process, as well as their role in affecting oil characteristics, appears to be increasingly useful. the aim of this work was to investigate both microbial ecology throughout olive oil processing and a possible relationship between evoo volatile compound content and microbial contamination. materials and methods experimental design during 2011 crop season, several batches of approx. 200 kg olives from frantoio and moraiolo cultivars were processed in an oil mill (azienda agricola buonamici, fiesole, florence, italy). plant for oil extraction (tem, florence, italy) consisted of a cleaning and water washing system, an olive grinding cutter crusher (mod. fr350), a controlled-temperature vertical axis malaxation equipment (500 kg capacity) (mod. v500), a “decanter” (two-step mod. d1500) with 1500 kg/h maximum capacity and a cardboard filter press (15 μm cut-off). plastic residue or “alperujo” from decanter was subjected to separation by centrifugation of stone fragments to obtain destoned pomace (fig. 1). olives were processed within 12 h from harvest at two dates (hd): november 16, 2011 (hd1) and november 23, 2011 (hd2). oil extraction trials were carried out in quadruple. olives were crushed at 2,500 rpm (crusher holes 6.5 mm in diameter); malaxation was carried out at half capacity under vacuum (residual pressure of 20 kpa) at 22 ± 1°c for a mean time of 15 min to work under low oxidative stress impact conditions; decanter worked with a screw conveyor rotating at a slower speed than that of the bowl. samples were collected in several steps of extraction process for sensory, chemical and microbial analyses, as shown in fig. 1. chemical analyses olives a homogeneous olive sample was crushed with a laboratory crusher, and resulting olive paste was used for chemical analyses. the water content (g kg−1 of dry matter) was measured on olive paste by gravimetric method (cherubini et al., 2009). the total sugar content was determined by the uni 22608 method, modified as described in a previous study (cherubini et al., 2009). results for sugar content obtained from the equipment (compact titrator, crison, modena, italy) were expressed as g kg−1 of dry matter. 238 ital. j. food sci., vol. 27 2015 fig. 1 overview of extraction process and analyses carried out. the total oil content was determined with hexane in an automatic randall extractor (mod. 148, velp scientifica, milan, italy), following the analytical technique described in a previous study (cherubini et al., 2009). results were expressed as g kg−1 of dry matter. the total phenolic compounds content was determined by weighing 4 g crushed olives and adding 80 ml methanol:water (60:40) solution; two series of stirring for 30 min and centrifugation at 4,000 rpm for 15 min were performed, and the supernatant was collected. the phenolic extract was adjusted to volume of 200 ml by methanol:water (60:40) and placed in the freezer for 2 h. after thawing, the phenolic extract was filtered. one ml filtered extract, 5 ml folin ciocalteu reagent, and 20 ml sodium carbonate were placed into a 100 ml flask and adjusted to volume with distilled water. sixty minutes were waited for colour development; after one hour, uv reading (uv/vis, varian model cary 1e, the netherlands) was performed at 765 nm wavelength. the total phenolic compounds content was expressed as mg kg−1 of dry matter on a calibration curve. olive oil acidity (% oleic acid), peroxide value (meq o 2 kg-1) and spectroscopic indices were measured according to eu official method (ec reg. 1989/2003). extraction, identification and determination of phenolic compounds were performed in agreement with ioc official method (ioc, 2009) by an hplc equipment consisting of a hewlett packard 1200 diode-array detector system and a hewlett packard model 1200 autosampler (agilent technologies, santa clara, california, usa). secoiridoids, lignans, flavonoids and phenolic acids were quantified in mg tyrosol kg oil -1. the total phenolic compounds content (mg tyrosol kg oil -1) was determined using the sum of the peak areas of phenols recorded at 280 nm. the tocopherol content was determined according to iso 9936:2006 (iso, 2006) using a hewlett packard mod. 1050 liquid chromatograph with quaternary pump and fluorescence detector, provided with hewlett packard mod.1100 autosampler (agilent technologies, santa clara, california, usa). quantitative analysis was carried out using the external standard method. results were expressed as mg of total tocopherols per kg of oil. the volatile compound content was determined according to the literature (vichi et al., 2003), using hs-spme-gc-ms technique (solid phase microextraction of the head space, couital. j. food sci., vol. 27 2015 239 pled with a gas chromatograph with a mass spectrometer as a detector). analysis was performed using the trace cg instrument combined with a trace dsq thermo finnigan instrument (fisher scientific sas, illkirch, france). quantitative analysis was performed using 4-methyl-2-pentanol as an internal standard. results were expressed as mg of volatile compound per kg of oil. sensory analyses sensory evaluation of olive oil was performed by a panel test according to the eu official method (eu reg. 1348/2013). samples were analyzed by a panel of professional tasters (8 tasters and a panel leader) of cciaa (chamber of commerce, industry, handcraft and agriculture) of florence. the panel has been recognized by mipaaf (ministry of agricultural policies, food and forestry) since 2002. intensity of both sensory defects and “fruity”, “bitter” and “pungent” attributes was assessed and expressed as the median of tasters score on a scale range from 0 to 10. microbiological analysis paste and oil samples from each batch were sterilely withdrawn and then transported to the laboratory under refrigerated conditions (4°c). ten g of olive paste or 10 ml of unfiltered oil were transferred into 90 ml of sterile saline and homogenized for 10 min with a stomacher lab blender 400 (seward ltd, worthing, west sussex, uk) and a magnetic stirrer, respectively. after decimal dilutions, 100 µl suspension was plated on specific growth media for cell enumeration in triplicate using the spread plating technique. yeasts were counted on mypg agar (zullo and ciafardini, 2008) integrated with ampicillin and sodium propionate in order to inhibit growth of bacteria and moulds, respectively (romo-sanchez, 2010). the plates were incubated at 30°c for 48-72 h. moulds were counted on mypg agar without inhibitors (kawai et al., 1994) after incubation at 30°c for 24-48 h. finally, total mesophilic microorganisms were counted on plate count agar (oxoid ltd, basingstoke, hampshire, uk) after incubation at 30°c for 48 h. filtered oil samples (100 ml) were microfiltered through nitrocellulose filters with a porosity of 0.45 µm (minisart nml-sartorius, göttingen, germany), which was able to retain yeasts and moulds. then the nitrocellulose filters containing the microorganisms were washed with 10 ml saline and placed onto the specific media described above. data processing chemical, sensory and microbiological determinations were processed according to one-way anova followed by tukey’s test (significance level: p = 0.05). principal component analysis (pca) was used to classify samples by statistica 7.0 software package (stasoft gmbh, hamburg, germany). correlation studies between microbial cell density and the volatile compounds content of oil samples were carried out by calculating both pearson and spearman rank correlation coefficients (significance level: α = 0.05). results characteristics of olive and olive oil samples chemical characteristics of processed olives are given in table 1. they show a slight increase in olive ripening level between the two dates of harvesting. as reported in the literature (ryan et al., 2002; servili et al., 2004; cherubini et al., 2009), a significant decrease in phenolic compounds content occurred, and a decrease in sugar content, even if significant only for frantoio cultivar, was also observed. no significant variations were measured in both water and olive oil contents during the harvesting interval. sensory and chemical characteristics of extracted olive oil are given in table 2, while their volatile compounds content is reported in table 3. samples are encoded in relation to olive cultivar, harvesting date and batch. table 2 shows that all olive oil samples extracted from olives of the first harvesting date were extra virgin. they had much lower values table 1 olive characteristics on two harvesting dates (hd1 and hd2). sd: standard deviation; different letters in the same row indicate significant differences (p < 0.05) for the same cultivar; dm: dry matter. frantoio cultivar moraiolo cultivar hd1 hd2 hd1 hd2 mean value sd mean value sd mean value sd mean value sd phenolic compounds (mg/kg dm) 33000 a 2285 26000 b 1811 37000 a 2579 30000 b 2097 sugar content (g/kg dm) 75 a 5 54 b 4 77 a 5 69 a 5 water content (g/kg) 391 b 20 437 a 22 411 a 21 430 a 22 oil content (g/kg dm) 440 a 31 460 a 32 500 a 35 450 a 31 240 ital. j. food sci., vol. 27 2015 than eu legal chemical limits, no sensory defects and a value of “fruity” attribute with a medium intensity of perception, as reported in eu reg. 1348/2013. conversely, all olive oil samples extracted from olives of the second harvesting date were not extra virgin, as they had significant sensory defects. despite this, they were in compliance with all legally established (eu reg. 1348/2013) chemical characteristics and “fruity” attribute. as a result of malaxation operating conditions at low oxidative stress impact, olive oil resulted in high phenolic compounds content and a phenolic profile characterized by slightly degraded phenolic compounds (servili et al., 2004; gomezrico et al., 2009). the total phenolic compound content was approx. 670 mg/kg; the dialdehydic form of decarboxymethyl oleuropein aglycone (3,4-dhpea-eda) was the most abundant phenolic compound, and its content was approx. 150 mg/kg; low (approx. 0.7 mg/kg) hydroxytirosol content (3,4-dhpea) was found. no significant differences were observed between samples at the two harvesting dates; the medium intensity of “bitter” and “pungent” attribute perception can be explained by phenolic compounds values (eu reg. 1348/2013). volatile compounds content of olive oil samples were subdivided into chemical classes, as reported in table 3. compounds that have been shown (kalua et al., 2007; di giacinto et al., 2010; aparicio et al., 2012) to be significantly related to oil defects are reported. underlined volatile compounds are intermediate of lox pathway and they are considered (di giacinto et al., 2010; kotti et al., 2011; aparicio et al., 2012) to be responsible for olive oil “fruity” positive attribute. a sum of underlined compound contents is reported in table 2 as “fruity volatile compounds”; “fruity” attribute, measured by panel test, can be explained by these values. ta b le 2 c h em ic a l a n d s en so ry c h a ra ct er is ti cs o f ex tr a ct ed o li ve o il . fr an to io c ul tiv ar m or ai ol o cu lti va r h d 1 h d 2 h d 1 h d 2 ba tc h co de (1 ) f1 a f1 b f1 c m ea n va lu e f2 a f2 b f2 c f2 d m ea n va lu e m 1a m 1b m 1c m 1d m ea n va lu e m 2a m 2b m 2c m 2d m ea n va lu e ± sd ± sd ± sd ± sd eu le ga l c ha ra ct er is tic s fr ee a cid ity ( % o lei c a cid ) 0. 20 0. 20 0. 22 0. 21 a ± 0. 01 0. 19 0. 19 0. 23 0. 24 0 .2 1a ±0 .0 3 0. 20 0. 22 0. 22 0. 22 0. 22 b ± 0. 01 0. 25 0. 25 0. 27 0. 27 0. 26 a ± 0. 01 pe ro xid e va lue (m eq o 2/k g oil ) 3. 6 3. 6 3. 5 3. 8b ±0 .1 6. 2 4. 9 5. 3 4. 6 5. 2a ±0 .7 3. 9 4. 0 4. 3 4. 6 4. 2b ±0 .3 5. 3 4. 5 5. 2 4. 9 5. 0a ±0 .4 k 2 32 1.7 6 1.7 1 1.7 4 1.7 4a ±0 .0 3 1.7 7 1.8 0 1.8 7 1.8 5 1.8 2a ±0 .0 5 1.8 0 1.7 4 1.8 2 1.8 0 1.7 9a ±0 .0 3 1.7 9 1.8 6 1.7 9 1.8 3 1.8 2a ±0 .0 3 k 2 70 0. 15 0. 15 0. 16 0. 15 b ± 0. 01 0. 19 0. 19 0. 23 0. 24 0 .2 1a ±0 .0 3 0. 17 0. 16 0. 17 0. 17 0. 17 a ± 0. 01 0. 15 0. 17 0. 17 0. 17 0. 17 a ± 0. 01 ∆k 0. 00 0. 00 0. 00 0. 00 a ± 0. 00 0. 00 0. 00 0. 00 0. 00 0 .0 0a ±0 .0 0 0. 00 0. 00 0. 00 0. 00 0. 00 a ± 0. 00 0. 00 0. 00 0. 00 0. 00 0. 00 a ± 0. 00 p.a .: f ru ity * ( 010 ) 3. 5 3. 4 4. 1 3. 7a ±0 .4 n. d. 2. 9 3. 3 4. 0 3. 4a ±0 .6 3. 8 3. 9 4. 4 3. 7 4. 0a ±0 .3 2. 9 3. 2 3. 4 4. 1 3. 4a ±0 .5 p.a .: b itte r* (0 -1 0) 2. 6 3. 6 3. 6 3. 3a ±0 .6 n. d 3. 6 3. 3 5. 1 4. 0a ±1 .0 2. 6 3. 6 4. 6 3. 7 3. 6a ±0 .8 3. 8 3. 5 3. 5 3. 5 3. 6a ±0 .1 p.a .: p un ge nt * ( 010 ) 3. 7 4. 5 4. 9 4. 4a ±0 .6 4. 1 4. 1 4. 2 5. 0 4. 4a ±0 .4 4. 0 4. 2 4. 7 4. 6 4. 4b ±0 .3 4. 9 5. 8 5. 6 5. 6 5. 5b ±0 .4 n. a. : r an cid * ( 010 ) 0. 0 0. 0 0. 0 0. 0b ±0 .0 0. 0 1.9 1.6 1.6 1.3 a ± 0. 9 0. 0 0. 0 0. 0 0. 0 0. 0b ±0 1.2 1.4 1.4 1.1 1.3 a ± 0. 2 n. a. : f us ty* (0 -1 0) 0. 0 0. 0 0. 0 0. 0b ±0 .0 2. 1 1.6 1.3 1.0 1.5 a ± 0. 5 0. 0 0. 0 0. 0 0. 0 0. 0b ±0 1.1 1.2 0. 8 1.0 1.0 a ± 0. 2 n. a. : w ine y-v ine ga ry * ( 010 ) 0. 0 0. 0 0. 0 0. 0b ±0 .0 1.7 1.7 0. 6 1.0 1.3 a ± 0. 5 0. 0 0. 0 0. 0 0. 0 0. 0b ±0 1.2 1.4 1.4 1.1 1.3 a ± 0. 2 bi oc om po un ds (m g/ kg ) to ta l p he no l c on te nt 72 0 64 0 72 0 69 0a ±4 6 56 0 61 0 70 0 69 0 64 0a ±6 8 69 0 67 0 73 0 72 0 70 0a ±3 0 57 0 70 0 61 0 70 0 64 0a ±6 5 ol eu ro pe in 39 32 32 34 a ± 4 30 40 50 50 40 a ± 11 40 50 70 60 55 a ± 11 40 50 60 60 50 a ± 10 3, 4 dh pe aed a (2 ) 23 0 17 0 17 0 19 0a ±3 2 13 0 13 0 15 0 14 0 14 0a ±1 0 15 3 14 2 15 2 15 7 15 1a ±6 10 0 14 0 10 0 11 0 11 0b ±1 9 php ea -e da (3) 92 84 84 83 a ± 4 11 0 90 60 50 80 a ± 27 67 59 46 47 55 a ± 10 40 38 34 26 34 b ± 6 3, 4 dh pe aea (4) 31 29 26 29 a ± 2 41 38 58 51 47 a ± 9 26 36 33 33 32 b ± 4 48 48 40 49 46 a ± 4 ph pe aea (5) 8. 4 8. 1 8. 0 8. 1a ±0 .2 8. 0 8. 0 19 .0 18 .0 13 a ± 6 8. 6 8. 2 7.7 7.4 8. 0a ±0 .5 17 15 11 12 14 b ± 2 3, 4 dh pe a (6 ) 0. 5 0. 5 0. 7 0. 6a ±0 .1 0. 4 0. 6 0. 8 0. 7 0. 6a ±0 .2 0. 8 0. 6 0. 7 0. 6 0. 7a ±0 .1 0. 6 0. 7 1.2 0. 9 0. 9a ±0 .3 to co ph er ols 29 0 25 0 24 0 26 0a ±2 5 29 2 29 2 28 7 28 8 29 0b ±3 24 9 25 6 25 2 25 7 25 4a ±4 28 0 28 0 25 0 25 0 27 0a ±1 7 fr uit y v ola tile co m po un ds (7) (m g/ kg ) 13 .4 14 .0 13 .4 13 .6 a ± 0. 4 9. 8 10 .2 10 .9 10 .5 10 .3 b ± 0. 4 13 .6 13 .4 13 .1 14 .2 13 .6 a ± 0. 5 11 .7 11 .5 13 .0 10 .9 11 .8 b ± 0. 9 h d : h ar ve st in g da te ; s d : s ta nd ar d de vi at io n; a, b d iff er en t l et te rs in th e sa m e ro w in di ca te s ig ni fic an t d iff er en ce s (p < 0 .0 5) fo r t he s am e cu lti va r; n. d. : n ot d et er m in ed ; * m ed ia n of th e ta st er s sc or e; p .a . a nd n .a .: po si tiv e an d n eg at iv e a ttr ib ut es . (1 ) f : f ra nt oi o; m : m or ai ol o; 1 : f irs t h ar ve st in g da te ; 2 : s ec on d ha rv es tin g da te ; a , b , c , d : o liv e ba tc he s. (2 ) d ia ld eh yd ic fo rm o f d ec ar bo xy m et hy l o le ur op ei n ag ly co ne ; ( 3) d ia ld eh yd ic fo rm o f d ec ar bo xy m et hy l l ig st ro si de a gl yc on e; (4 ) o le ur op ei n ag ly co ne ; ( 5) l ig st ro si de a gl yc on e; (6 ) h yd ro xy ty ro so l; (7 ) s um o f t he u nd er lin ed v ol at ile c om po un d co nt en ts in t ab le 3 . ital. j. food sci., vol. 27 2015 241 t a b le 3 v o la ti le c o m p o u n d s co n te n t o f ex tr a ct ed o li ve o il . h d : h a rv es ti n g d a te ; n .d . n o t d et er m in ed . a. c las s o f e ste rs , a cid s a nd h yd ro ca rb on s     m et hy l a ce ta te et hy l a ce ta te bu ty l a ce ta te ci s3he xe ny l tr an s2he xe ny l bu ty ric a ci d pe nt an oi c ac id he xa no ic a ci d o ct an oi c ac id h ep ta n o ct an ac et at e ac et at e   hd ba tc h (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) co de fr an to io 1 f1 a 0. 03 6 0. 01 9 0. 00 2 0. 14 4 0. 00 3 nd 0. 01 0 0. 27 4 0. 07 0 0. 00 7 0. 04 8 f1 b 0. 02 2 0. 01 7 0. 00 2 0. 13 1 0. 00 2 nd 0. 01 3 0. 33 3 0. 08 9 0. 00 6 0. 04 3 f1 c 0. 01 5 0. 02 0 0. 00 2 0. 13 4 0. 00 1 nd 0. 01 1 0. 25 5 0. 04 7 0. 00 4 0. 03 8 2 f2 a 0. 01 5 0. 04 2 0. 00 2 0. 07 0 nd 0. 01 1 0. 00 7 0. 21 0 0. 12 1 0. 00 5 0. 05 3 f2 b 0. 00 7 0. 03 5 0. 00 2 0. 40 9 nd 0. 01 0 0. 00 8 0. 24 3 0. 13 5 0. 00 2 0. 03 9 f2 c 0. 00 8 0. 02 7 0. 00 1 0. 61 4 nd 0. 01 2 0. 01 3 0. 23 7 0. 17 0 0. 00 3 0. 03 6 f2 d 0. 00 6 0. 02 3 0. 00 1 0. 61 9 nd 0. 01 0 0. 01 0 0. 23 5 0. 16 6 0. 00 3 0. 03 6 m or ai ol o 1 m 1a 0. 00 5 0. 01 8 0. 00 1 0. 74 7 0. 00 3 nd 0. 00 4 0. 23 6 0. 09 5 0. 00 3 0. 03 2 m 1b 0. 00 5 0. 01 6 0. 00 2 1.0 70 0. 02 7 nd 0. 00 7 0. 25 9 0. 08 3 0. 00 5 0. 03 1 m 1c 0. 00 6 0. 01 9 0. 00 3 1.1 73 0. 00 4 nd 0. 00 8 0. 27 7 0. 06 5 0. 00 3 0. 03 3 m 1d 0. 00 5 0. 01 5 0. 00 1 0. 48 0 0. 00 6 nd 0. 00 4 0. 21 4 0. 06 5 0. 00 5 0. 02 8 2 m 2a 0. 00 6 0. 02 1 0. 00 1 1.0 32 nd 0. 01 3 0. 01 2 0. 27 7 0. 18 5 0. 00 3 0. 03 5 m 2b 0. 00 5 0. 02 0 0. 00 2 0. 85 8 nd 0. 01 3 0. 01 1 0. 26 1 0. 16 0 0. 00 4 0. 04 0 m 2c 0. 00 8 0. 02 2 0. 00 1 0. 92 7 nd 0. 01 2 0. 00 8 0. 20 1 0. 11 9 0. 00 2 0. 03 6     m 2d 0. 00 6 0. 02 3 0. 00 1 0. 70 1 nd 0. 01 4 0. 01 5 0. 26 2 0. 17 1 0. 00 3 0. 03 7 b. c las s o f a ld eh yd es va le ra ld he yd e he xa na l tr an s2 ci s3 he pt an al tr an s2 oc ta na l tr an s2 2. 4 tr an s2 be nz al de hy de tr an s2 tr an s2 pe nt en al he xe na l he xe na l he pt en al he xa di en al oc ta na l no ne na l de ce na l hd ba tc h (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) co de fr an to io 1 f1 a 0. 12 8 0. 58 6 0. 03 1 1.4 21 0. 02 5 10 .0 90 0. 13 1 0. 03 8 0. 29 2 0. 01 5 0. 03 1 0. 19 0 0. 22 9 f1 b 0. 10 6 0. 64 4 0. 03 5 1.8 22 0. 02 6 10 .2 17 0. 15 9 0. 03 3 0. 31 3 0. 01 0 0. 03 1 0. 19 6 0. 23 2 f1 c 0. 08 7 0. 59 6 0. 03 0 1.5 69 0. 02 5 9. 96 3 0. 13 4 0. 02 4 0. 27 0 0. 01 0 0. 03 0 0. 18 5 0. 23 7 2 f2 a 0. 09 4 0. 48 1 0. 02 3 1.0 85 0. 01 7 7.3 31 0. 06 3 0. 04 2 0. 19 8 0. 01 0 0. 03 0 nd 0. 13 2 f2 b 0. 07 9 0. 50 4 0. 02 8 1.0 84 0. 01 7 6. 93 8 0. 06 6 0. 03 4 0. 18 9 0. 01 5 0. 03 2 nd 0. 12 5 f2 c 0. 07 0 0. 54 7 0. 03 5 1.1 57 0. 02 3 6. 78 5 0. 09 0 0. 03 1 0. 17 7 0. 01 4 0. 03 3 nd 0. 07 8 f2 d 0. 07 1 0. 53 5 0. 03 5 1.0 50 0. 02 2 6. 54 5 0. 10 3 0. 03 0 0. 17 2 0. 01 6 0. 03 4 nd 0. 09 2 m or ai ol o 1 m 1a 0. 09 2 0. 55 6 0. 03 6 1.7 42 0. 02 8 8. 75 0 0. 14 8 0. 01 3 0. 27 1 0. 01 9 0. 02 8 0. 18 7 0. 20 3 m 1b 0. 07 9 0. 44 3 0. 03 6 2. 01 6 0. 02 2 7.6 84 0. 13 7 0. 00 9 0. 26 2 0. 01 6 0. 02 9 0. 19 7 0. 18 m 1c 0. 10 0 0. 48 9 0. 04 2 1.9 37 0. 02 9 7.3 71 0. 15 2 0. 02 0 0. 24 6 0. 01 4 0. 03 1 0. 19 4 0. 20 4 m 1d 0. 08 7 0. 56 3 0. 03 2 2. 04 5 0. 02 5 9. 43 5 0. 11 7 0. 02 3 0. 27 6 0. 01 9 0. 02 8 0. 20 2 0. 18 0 2 m 2a 0. 05 3 0. 53 8 0. 03 9 1.3 20 0. 02 7 6. 59 5 0. 11 7 0. 02 4 0. 20 6 0. 01 2 0. 03 6 nd 0. 08 1 m 2b 0. 04 4 0. 47 4 0. 04 3 1.8 03 0. 02 2 6. 17 2 0. 09 3 0. 02 1 0. 22 9 0. 00 9 0. 03 5 nd 0. 05 8 m 2c 0. 04 2 0. 41 6 0. 04 5 4. 05 1 0. 01 5 5. 12 1 0. 07 2 0. 01 0 0. 34 8 0. 02 2 0. 03 4 nd 0. 05 9    m 2d 0. 07 0 0. 58 5 0. 03 8 1.1 23 0. 02 5 6. 60 7 0. 12 6 0. 03 6 0. 19 2 0. 01 8 0. 03 9 nd 0. 09 9 242 ital. j. food sci., vol. 27 2015 a multidimensional map of all samples related to volatile compounds was obtained by pca. the relevant sample loading and score plots are reported in fig. 2. the model explained 60% of data variability along the first (factor 1) and second (factor 2) principal components. a comparison between the score plot and the loading plot showed that olive oil samples extracted from olives of the second harvesting date were all positioned on the left side of the plot. they were characterized by high values of benzaldehyde, 2-butanone, butyric acid, 2-heptanol, octanoic acid, 1-octen-3-ol, 1-octen-3-one and 2-octanone. all these compounds are related to olive oil defects: these compounds have been associated with “musty”, “winey–vinegary” and “fusty” defects by some literature data (kalua et al., 2007; aparicio et al., 2012), whereas they have been associated with “rancid” defect by di giacinto et al. (2010). microbial ecology of oil extraction process cell concentrations of dominant microbial populations at different steps of oil extraction process from frantoio and moraiolo cultivar olives are shown in tables 4 and 5, respectively. yeasts and/or moulds were always the dominant populations, independently of the sampling point. cell density of bacteria only accounted for 1% of the total microbial counts on pca plates. the cell concentrations in olive paste after crushing (p) and in extracted olive oil (d) ranged between values below 10 and above 104 cfu/g or ml. these values were higher than that obtained from filtered olive oil (o), which, in most cases, was < 102 cfu/100 ml. microbial counts of each olive batch were often affected by high standard deviation values, as it typically occurs in manufacturing processes of raw materials (such as olives) at industrial scale. a rough general pattern for microbial evolution during olive processing could nonetheless be drawn. mould counts in olive paste after crushing (pm) were always significantly higher than those in extracted olive oil (dm), while yeast counts showed a different behaviour. in most olive batches (from both frantoio and moraiolo cultivars) of the first harvesting date, yeast counts decreased by about one order of magnitude from olive paste after crushing (py) to extracted olive oil (dy), as expected on the basis of olive oil yield. at the second harvesting date, yeast counts remained almost unchanged from olive paste (py) to olive oil (dy), or even increased in extracted olive oil (dy), suggesting a progressive yeast colonization of the malaxation equipment and/or “decanter”. indeed, at the second harvesting date, olive paste (py) harboured almost the same yeast concentration as that at the first harvesting date, with values ranging be-c . c las s o f a lco ho ls, ke to ne s a nd p he no ls 1pe nt en -3 -o l 2he pt an ol pe nt an ol 1oc te n3ol tr an s3 ci s3 ci s2 2bu ta no ne 2 -o ct an on e 1oc te n 1pe nt en 6m et hy l gu ai ac ol ph en ol et hy l 4et hy l he xe no l he xe no l pe nt en ol 3on e 3on e 5he pt en -2 -o ne gu ai ac ol ph en ol hd ba tc h (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) (m g/ kg ) co de fr an to io 1 f1 a 0. 44 6 nd 0. 00 5 0. 00 2 0. 00 4 0. 24 2 0. 33 7 0. 43 3 0. 00 3 0. 00 1 0. 43 3 0. 00 4 0. 00 5 0. 26 1 0. 13 1 nd f1 b 0. 45 4 nd 0. 00 5 0. 00 2 0. 00 4 0. 26 0 0. 35 8 0. 47 7 0. 00 4 0. 00 1 0. 47 7 0. 00 5 0. 00 5 0. 27 5 0. 12 4 nd f1 c 0. 47 5 nd 0. 00 4 0. 00 2 0. 00 3 0. 20 0 0. 35 3 0. 48 4 0. 00 4 0. 00 1 0. 48 4 0. 00 4 0. 00 8 0. 25 3 0. 15 8 0. 08 0 2 f2 a 0. 40 3 0. 22 5 0. 00 4 0. 00 5 0. 00 4 0. 08 9 0. 28 8 0. 36 0 0. 01 4 0. 00 3 0. 36 0 nd 0. 00 2 0. 19 8 nd nd f2 b 0. 49 1 0. 29 4 0. 00 4 0. 00 7 0. 00 5 0. 25 9 0. 31 0 0. 48 1 0. 01 2 0. 00 2 0. 48 1 nd 0. 00 4 0. 20 0 nd nd f2 c 0. 59 7 0. 35 1 0. 00 5 0. 00 8 0. 00 6 0. 43 1 0. 38 2 0. 70 8 0. 01 0 0. 00 1 0. 70 8 nd 0. 00 5 0. 19 3 nd nd f2 d 0. 58 2 0. 34 6 0. 00 5 0. 01 0 0. 00 7 0. 45 4 0. 37 6 0. 71 2 0. 03 5 0. 00 4 0. 71 2 nd 0. 00 6 0. 20 3 nd nd m or ai ol o 1 m 1a 0. 55 4 nd 0. 00 6 0. 00 2 0. 00 6 0. 52 1 0. 42 8 0. 58 6 0. 00 8 0. 00 2 0. 58 6 0. 00 4 0. 00 4 0. 24 5 nd nd m 1b 0. 56 9 nd 0. 00 6 0. 00 2 0. 00 9 0. 69 5 0. 43 5 0. 59 0 0. 00 4 0. 00 2 0. 59 0 0. 00 2 0. 00 4 0. 25 1 0. 11 7 nd m 1c 0. 58 4 nd 0. 00 5 0. 00 2 0. 00 8 0. 70 9 0. 44 4 0. 60 6 0. 00 6 0. 00 2 0. 60 6 0. 00 4 0. 00 3 0. 35 2 0. 11 4 0. 06 7 m 1d 0. 57 1 nd 0. 00 6 0. 00 2 0. 00 6 0. 34 6 0. 44 7 0. 53 3 0. 00 5 0. 00 1 0. 53 3 0. 00 3 0. 00 4 0. 23 8 nd nd 2 m 2a 0. 65 8 0. 37 5 0. 00 5 0. 00 9 0. 00 8 0. 71 4 0. 42 0 0. 71 5 0. 00 9 0. 00 2 0. 71 5 nd 0. 00 6 0. 20 8 nd nd m 2b 0. 57 5 0. 36 9 0. 00 5 0. 00 7 0. 00 5 0. 74 5 0. 41 2 0. 71 1 0. 00 3 0. 00 2 0. 71 1 nd 0. 00 4 0. 21 0 nd nd m 2c 0. 60 3 0. 36 1 0. 00 4 0. 00 6 0. 00 6 0. 85 3 0. 40 2 0. 72 0 0. 00 3 0. 00 2 0. 72 0 nd 0. 00 2 0. 19 6 nd nd     m 2d 0. 63 6 0. 38 1 0. 00 6 0. 01 2 0. 00 7 0. 50 9 0. 39 4 0. 75 3 0. 01 1 0. 00 4 0. 75 3 nd 0. 00 6 0. 20 5 nd nd ital. j. food sci., vol. 27 2015 243 fig. 2 principal component analysis carried out on volatile compounds content of olive oil samples. a: similarity map determined by principal component (factor) 1 and 2; b: projection of the variables on the factor plane. samples are coded as reported in table 2. tween 102 and above 103 cfu/g. on the contrary, the extracted olive oil of the second harvesting date (dy) harboured yeast concentrations, in most cases, of about one or two orders of magnitude higher than the extracted olive oil of the first harvesting date. correlation studies demonstrated that mould counts in olive paste after crushing (pm) and in extracted olive oil (dm) were positively related to each other, suggesting that mould contamination of unfiltered oil could be affected by the hygienic level of olives (table 6). on the contrary, yeast cell densities in olive paste (py) and in olive oil (dy) were statistically unrelated, sug244 ital. j. food sci., vol. 27 2015 table 4 microbial cell counts at different steps of oil extraction process on two harvesting dates (hd) for frantoio cultivar. p = olive paste after crushing; d = olive oil after extraction by “decanter”; o = olive oil after filtration; tmc = total microbial count; different letters indicate significant differences between different extractive steps of the same olive batch (p < 0.05); when no letter is reported, no significant difference was found. yeasts moulds tmc hd batch sampling code point mean sd mean sd mean sd 1 f1a p (cfu/g) 1.60 x 103a 1.40 x 102 1.00 x 102 0 1.40 x 103a 1.40 x 102 d (cfu/ml) 4.50 x 101b 7.07 <10 4.00 x 101b 2.82 o (cfu/100ml) <1 <1 <1 f1b p (cfu/g) 8.50 x 102a 2.12 x 102 <10 1.60 x 103a 5.66 x 102 d (cfu/ml) 1.00 x 102b 2.80 x 101 <10 4.00 x 101b 0 o (cfu/100ml) <1 <1 <1 f1c p (cfu/g) 1.10 x 103a 1.41 x 102 8.00 x 102 0 2.00 x 103 1.41 x 103 d (cfu/ml) 3.25 x 102b 3.54 x 101 <10 5.00 x 102 2.83 x 102 o (cfu/100ml) <1 <1 <1 2 f2a p (cfu/g) 1.00 x 102a 1.40 x 101 4.20 x 104a 2.82 x 103 4.80 x 104a 2.83 x 103 d (cfu/ml) 3.00 x 102b 2.80 x 101 4.00 x 101b 2.82 3.00 x 102b 2.88 x 101 o (cfu/100ml) 5.00 x 101c 1.41 <1 5.00 x 101b 2.82 f2b p (cfu/g) 2.70 x 103 1.84 x 103 2.85 x 104 2.32 x 104 8.75 x 103a 3.18 x 103 d (cfu/ml) 2.92 x 103 1.99 x 103 3.33 x 101 3.27 x 101 1.00 x 102b 0 o (cfu/100ml) 5.50 x 101 1.41 <1 6.50 x 101b 1.41 f2c p (cfu/g) 2.30 x 103 9.90 x 102 2.50 x 104 2.25 x 104 1.10 x 103a 1.41 x 102 d (cfu/ml) 3.26 x 103 1.60 x 103 9.67 x 101 8.96 x 101 1.81 x 103a 7.66 x 102 o (cfu/100ml) 5.50 x 101 2.82 <1 1.00 x 101b 2.82 f2d p (cfu/g) 4.00 x 102a 2.83 x 101 3.45 x 104 3.32 x 104 7.00 x 103a 1.41 x 102 d (cfu/ml) 1.38 x 104b 6.36 x 102 1.20 x 102 2.83 x 101 1.35 x 104b 9.90 x 102 o (cfu/100ml) 1.50 x 101a 1.40 5.00 0 4.00 x 101c 2.82 table 5 microbial cell counts at different steps of olive oil extraction process on two harvesting dates (hd) for cultivar moraiolo. p = olive paste after crushing; d = olive oil after extraction by “decanter”; o = olive oil after filtration; tmc = total microbial count; different letters indicate significant differences between different extraction steps of the same olive batch (p < 0.05); when no letter is reported, no significant difference was found. yeasts moulds tmc hd batch sampling code point mean sd mean sd mean sd 1 m1a p (cfu/g) 1.10 x 103a 1.41 x 102 4.00 x 102a 0 1.45 x 103 6.36 x 102 d (cfu/ml) 4.50 x 101b 7.07 <10 4.00 x 101 1 o (cfu/100ml) <1 4.00 x 101b 1.41 1.00 x 101 1 m1b p (cfu/g) 3.75 x 103a 3.54 x 102 5.50 x 102 5.36 x 102 5.35 x 103 2.33 x 103 d (cfu/ml) 5.00 x 101b 1.40 <10 <10 o (cfu/100ml) <1 2.00 x 101 1.00 2.00 x 101 1.40 m1c p (cfu/g) 1.10 x 103a 1.41 x 102 4.00 x 102a 0 2.35 x 103a 9.19 x 102 d (cfu/ml) 6.90 x 103b 2.82 x 102 <10 1.50 x 104b 3.54 x 103 o (cfu/100ml) <1 1.00 x 101b 2.82 1.00 x 101a 1.40 m1d p (cfu/g) 1.10 x 103 1.41 x 102 4.00 x 102a 1.41 x 101 1.45 x 103 7.78 x 102 d (cfu/ml) 3.20 x 102 3.11 x 102 <10 3.50 x 101 2.12 x 101 o (cfu/100ml) <1 2.00 x 101b 0 2.00 x 101 1.40 2 m2a p (cfu/g) 1.70 x 103 2.83 x 102 2.60 x 103a 1.98 x 103 2.70 x 103a 4.24 x 102 d (cfu/ml) 1.04 x 103 7.45 x 102 6.00 x 101a 5.66 x 101 9.73 x 102ab 8.60 x 102 o (cfu/100ml) <1 5.50 x 101b 2.82 5.50 x 101a 1.41 m2b p (cfu/g) 2.45 x 103a 7.78 x 102 6.00 x 102 5.66 x 102 2.35 x 103a 9.19 x 102 d (cfu/ml) 3.27 x 102b 1.42 x 102 3.50 x 101 2.12 x 101 3.80 x 102b 2.31 x 102 o (cfu/100ml) 1.00 x 101c 0 1.60 x 102 2.82 x 101 7.50 x 101b 3.53 m2c p (cfu/g) 7.45 x 103 2.19 x 103 1.00 x 103 2.82 x 102 4.15 x 103a 2.12 x 102 d (cfu/ml) 9.72 x 103 5.04 x 103 4.00 x 101 3.66 x 101 1.16 x 103b 1.07 x 103 o (cfu/100ml) 8.00 x 101 14.00 <1 1.65 x 102b 3.00 m2d p (cfu/g) 1.65 x 103 1.61 x 103 6.75 x 103 1.77 x 103 5.80 x 103 3.11 x 103 d (cfu/ml) 3.08 x 103 3.02 x 103 6.00 x 101 5.66 x 101 2.53 x 103 2.04 x 103 o (cfu/100ml) 1.10 x 103 2.00 <1 5.50 x 101 1.41 ital. j. food sci., vol. 27 2015 245 gesting that yeast growth could be encouraged by malaxation and/or “decanting” steps. finally, no correlation was found between yeast and mould concentrations in both olive paste (py and pm, respectively) and filtered oil (oy and om, respectively). according to pca of all microbiological data (fig. 3), processed olive batches clustered into two different groups, independently of the olive cultivar: the samples of the first harvesting date, harboring the lowest microbial cell densities, clustered in group a), while all batches of the second harvesting date resulted to be included in group b). it is worth noting that both the pca resulting from all microbiological data (fig. 3) and the pca resulting from volatile compounds (fig. 2) are in full agreement, as olive batches from both statistical analyses are clustered in the same way. finally, some statistically significant correlations were found between microbial cell densities at the different steps of oil processing and some volatile compounds of olive oil. the significant correlations between yeast (y) and mould (m) counts, in both extracted (d) and filtered olive oil (o), and volatile compounds content of the final olive oil samples are reported in table 7. in particular, correlation coefficients (i.e. pearson and spearman) agreed on indicating significant positive correlations between yeast and mould counts in olive oil, both before and after filtration, and some volatile compounds; among the latter, the highest significance was related to ethyl acetate, 2-butanone, butyric acid, pentanol, 2-heptanol, octanoic acid and 1-octen-3-ol contents. since most of these compounds were identical to those correlated to olive oil batches with sensory defects, as described in the previous paragraph, yeast and mould contamination may have been responsible for those sensory defects. which specific sensory defects were associated with the above-mentioned compounds could not be explained, as in the literature “rancid”, “fusty”, “winey–vinegary” and “musty” defects have been associated with both yeasts and moulds. as an example, a recent study demonstrated the capability of some oil born strains of candida fig. 3 principal component analysis of the various olive batches tested by considering as variables the microbial cell concentrations during various extraction process steps. samples are coded by combination of letters which identify both samples at processing steps (p = olive paste after crushing; d = olive oil after centrifugation by “decanter”; o = olive oil after filtration) and microorganisms (tmc = total microbial count; y = yeasts; m = moulds). a: similarity map determined by principal component (factor) 1 and 2; b: projection of the variables on the factor plane. table 6 correlation coefficients calculated between microbial contaminations (y = yeasts; m = moulds) of olive paste after crushing (p) and microbial contaminations of extracted (d) and filtered olive oil (o). statistically significant correlations (p<0.05) are underlined. dy dm oy om   spearman pearson spearman pearson spearman pearson spearman pearson r r r r r r r r pm 0.8304 0.7347 -0.1575 -0.2485 py 0.08641 0.05563 0.2841 0.1241 246 ital. j. food sci., vol. 27 2015 table 7 correlation coefficients calculated between yeast (y) and mould (m) counts of extracted and filtered olive oil (d) and volatile compounds of the final olive oil samples (o). statistically significant correlations (p<0.05) are underlined. dy dm oy om   spearman pearson spearman pearson spearman pearson spearman pearson r r r r r r r r esters, acids and hydrocarbons methyl acetate -0.006737 -0.3253 -0.08564 -0.4042 0.01485 -0.2926 -0.8317 -0.5387 ethyl acetate 0.4464 0.2619 0.6978 0.5603 0.7348 0.6665 -0.4953 -0.4413 butyl acetate -0.3046 -0.27 -0.5824 -0.5091 -0.3481 -0.3366 -0.1901 -0.1524 cis-3-hexenil acetate 0.3194 0.325 0.1589 0.2075 -0.0114 0.01915 0.652 0.6335 trans-2-hexenil acetate -0.5621 -0.4382 -0.862 -0.4494 -0.7775 -0.3811 0.243 0.2902 butyric acid 0.5532 0.5918 0.8694 0.9727 0.7434 0.8125 0.03821 -0.0028 pentanoic acid -0.0685 -0.1613 -0.1662 -0.1045 -0.3671 -0.331 0.5344 0.5813 hexanoic acid -0.1944 -0.3239 -0.337 -0.3943 -0.4902 -0.5172 -0.01623 octanoic acid 0.4624 0.4945 0.8818 0.9251 0.6282 0.6242 0.1469 0.1633 heptan -0.6824 -0.5102 -0.5535 -0.4601 -0.5756 -0.4022 -0.0887 -0.218 octan -0.1092 -0.2505 0.0174 -0.04 0.205 0.0699 -0.6035 -0.5001 aldehydes valeraldheyde -0.5583 -0.4607 -0.708 -0.7013 -0.5681 -0.5144 -0.291 -0.388 hexanal -0.3341 -0.3271 -0.3477 -0.3659 -0.4185 -0.4153 -0.3585 -0.315 trans-2-pentenal 0.3599 0.3423 0.2134 0.1599 0.06842 0.02669 0.5254 0.464 cis-3-hexenal -0.2487 0.1017 -0.59 -0.1577 -0.4412 0.03461 0.3088 0.07831 heptanal -0.3023 -0.3369 -0.4689 -0.5529 -0.7091 -0.7582 0.2273 0.2932 trans-2-hexenal -0.6784 -0.6681 -0.8182 -0.8242 -0.6991 -0.7192 -0.2628 -0.1959 octanal -0.405 -0.4349 -0.6645 -0.7347 -0.7142 -0.7745 0.1745 0.2041 trans-2-heptenal -0.0552 -0.01303 0.1559 0.1205 0.2061 0.1596 -0.6411 -0.5767 2.4 hexadienal -0.4503 -0.3867 -0.7002 -0.6627 -0.4882 -0.42 -0.0957 -0.0823 trans-2-octanal -0.0244 0.1057 -0.0918 -0.1199 0.08817 0.08237 -0.0718 -0.1306 benzaldehyde -0.5041 0.3715 0.5785 0.4958 0.4283 0.3689 -0.1509 0.1584 trans-2-nonenal -0.6304 -0.6281 -0.8605 -0.9855 -0.7762 -0.8358 0.07103 0.04037 trans-2-decenal -0.5946 -0.615 -0.8119 -0.895 -0.6682 -0.7092 -0.2806 -0.2243 alcohols, ketones and phenols 1-penten-3-ol 0.6681 0.4847 0.5822 0.4303 0.3147 0.1095 0.5055 0.5417 2-heptanol 0.6178 0.6498 0.8857 0.9774 0.7328 0.7846 0.06237 0.0213 pentanol 0.4111 0.4182 0.8213 0.8118 0.7221 0.7422 -0.1372 -0.2154 cis-3-hexenol 0.3252 0.2959 0.2486 0.216 0.07623 0.05016 0.6277 0.5872 trans-3-hexenol 0.3032 0.2647 0.2875 0.1636 0.0247 0.5309 0.4904 1-octen-3-ol 0.6212 0.6381 0.9304 0.9199 0.7286 0.7176 -0.0491 -0.0339 cis-2-pentenol 0.0486 0.08362 -0.1142 -0.16 -0.2467 -0.2997 0.685 0.618 2-butanone 0.5204 0.5477 0.782 0.7111 0.5529 0.5283 0.00517 -0.1297 1-penten-3-one 0.6461 0.5539 0.6247 0.5717 0.412 0.3304 0.3666 0.4029 2-octanone 0.264 0.4949 0.5565 0.5658 0.4097 0.3878 -0.06146 -0.0623 1-octen-3-one 0.2545 0.3958 0.5142 0.5573 0.5459 0.5804 0.1314 0.0085 6-methyl-5-hepten-2-one -0.5882 -0.5785 -0.8678 -0.9186 -0.7828 -0.779 -0.1013 -0.1062 guaiacol -0.0316 -0.1852 0.08201 -0.1711 -0.2404 -0.3518 -0.1757 -0.1935 phenol -0.4724 -0.258 -0.8245 -0.8028 -0.8142 -0.7034 0.1395 0.0637 ethyl-guaiacol -0.3957 -0.4097 -0.6943 -0.7573 -0.6262 -0.6423 -0.3215 -0.2771 4-ethyl-phenol -0.0820 -0.1033 -0.4472 -0.4672 -0.3997 -0.3962 -0.1999 -0.2132 spp. to induce defects such as “musty” and/or “rancid” in oil (zullo et al., 2013). conclusions this study was carried out on several olive oil samples extracted by olive batches from frantoio and moraiolo cultivars, harvested on two different dates. all extracted olive oil samples from the second olive harvesting date were classified as “non extra virgin”, as they were affected by sensory defects. by combining chemical, sensory, and microbiological data, it can be assumed that the olive oil samples with sensory defects were significantly correlated with specific volatile compounds (i.e., 2-butanone, butyric acid, 2-heptanol, octanoic acid, 1-octen-3-ol). the same volatile compounds were correlated to both yeast and mould counts. it could not be evidenced whether a specific sensory defect might result from specific volatile compounds, which in turn can be produced by specific yeasts and moulds. different processing steps were also identified, which resulted to be the most critical steps to cause the measured sensory defects: (i) the ital. j. food sci., vol. 27 2015 247 mould contamination of olives; (ii) the two central steps of olive oil processing (i.e. malaxation and extraction by “decanter”), which were likely to have enabled some yeast species to grow. a study on identification of yeast isolates and determination of their enzymatic properties is being carried out to further investigate the incidence of yeast populations during olive oil extraction process. acknowledgements research was supported by “bando misura 124 programma di sviluppo rurale (psr) 2007-2013” of tuscany region, which was published in the gazzetta ufficiale of tuscany region (burt) no. 7 of 16/02/11. references angerosa f., servili m., selvaggini r., taticchi a., esposto s. and monteodoro g. 2004. volatile compounds in virgin olive oil: occurrence and their relationship with the quality. j. chromatogr. a. 1054:17. aparicio m., morales m.t. and garcìa-gonzàles d.l. 2012. towards new analyses of aroma and volatiles to understand sensory perception of olive oil. eur. j. lipid sci technol. 114:1114. aparicio r. and morales m.t. 1998. characterization of olive ripeness by green aroma compounds of virgin olive oil. j agric food chem. 46: 1116. bendini a., valli e., barbieri s. and gallina toschi t. 2012. sensory analysis of virgin olive oil. ch. 6. in “olive oil constituents, quality, health properties and bioconversions”. d. boskou (ed.), p. 109. intech open access publisher. cherubini c., migliorini m., mugelli m., viti p., berti a., cini e. and zanoni b. 2009. towards a technological ripening index for olive oil fruits. j. sci food agric. 89:671. ciafardini g. and zullo b.a. 2002. microbiological activity in stored olive oil. int. j food microbiol. 75:111. ciafardini g., zullo b.a., cioccia g. and iride a. 2006. lipolytic activity of williopsis californica and saccharomices cerevisiae in extra virgin olive oil. int. j food microbiol. 107: 27. commission regulation (ec) no 1989/2003. ojeu. l295:57. commission implementing regulation (eu) no 1348/2013. ojeu. l338:31. di giacinto l., di loreto g., di natale c., guianni g., guasti s., migliorini m., pellegrino m., perri e. and santonico m. 2010. caratterizzazione analitica degli attributi sensoriali degli oli 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yeast biodiversity from oleic ecosystems: study of their biotechnological properties. food microbiol. 75:487. ryan d., antolovich m., prenzler p., robards k. and lavee s. 2002. biotranformations of phenolic compounds in olea europea l.. hort. sci. 92:147. servili m., selvaggini r., esposto s., taticchi a., montedoro g.f. and morozzi g. 2004. health and sensory properties of virgin olive oil hydrophilic phenols: agronomic and technological aspects of production that affect their occurence in the oil. . j. chromatogr. a. 1054:113. vichi s., castellote a.i., pizzale l., conte l.s., buxaderas s. and lòpez-tamames e. 2003. analysis of virgin olive oil volatile compounds by headspace solid-phase microextraction coupled to gas chromatography with mass spectrometric and flame ionization detection. j. j. chromatogr. a. 983:19. vichi s., romero a., tous j. and caixach j. 2011. the activity on healthy olive microbiota during virgin olive oil extraction influences oil chemical composition. j. agric. food chem. 59:4705. zullo b.a. and ciafardini g. 2008. lypolitic yeast distribution in commercial extra virgin olive oil. food microbiol. 25:970. zullo b.a., cioccia g. and ciafardini g. 2010. distribution of dimorphic yeast species in commercial extra virgin olive oil. food microbiol. 27:1035. zullo b.a., cioccia g. and ciafardini g. 2013. effects of some oil borne yeasts on the sensory characteristics of italian virgin olive oil during storage. food microbiol. 36:70. paper received july 30, 2014 accepted november 12, 2014 http://www.sciencedirect.com/science/article/pii/s0740002010001802 http://www.sciencedirect.com/science/article/pii/s0740002010001802 http://www.sciencedirect.com/science/article/pii/s0740002010001802 p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33i2.2005 117 a number of human diseases, including autoimmune diseases, diabetes, tumours, alzheimer’s disease and stroke (boston et al., 2004; fritsche, 2006; wall et al., 2010). α-linolenic acid (ala, 18:3 n-3) is a precursor of n-3 pufa group; epa, docosapentaenoic (dpa, 22:5 n-3) and dha fatty acids are synthesized from ala through consecutive elongation and desaturation. in general, animal fats are lacking in n-3 pufa, while contain n-6 pufa abundantly. hence, pork products provide too much of n-6 while lacking in n-3 pufa (wood et al., 2008; kouba and mourot, 2011). owing to a high content of saturated fatty acids (sfa) and an p u b l i c a t i o n s codon effects of high linolenic acid diet supplemented with synthetic or natural antioxidant mix on live performance, carcass traits, meat quality and fatty acid composition of longissimus thoracis et lumborum muscle of medium-heavy pigs anna maria belmonte1, paolo macchioni2, giovanna minelli1,3,*, corina scutaru1, luisa antonella volpelli1,3 and domenico pietro lo fiego1,3 1department of life sciences, university of modena and reggio emilia, via amendola 2, 42122 reggio emilia, italy; 2department of agricultural and food sciences (distal), university of bologna, viale fanin 44, 40127 bologna, italy; 3interdepartmental research centre for agri-food biological resources improvement and valorisation (biogestsiteia), university of modena and reggio emilia, via amendola 2, 42122 reggio emilia, italy *corresponding author: giovanna minelli, department of life sciences, university of modena and reggio emilia, via amendola 2, 42122 reggio emilia, italy. email: gminelli@unimore.it received: 19 january 2021; accepted: 3 may 2021; published: 30 june 2021 © 2021 codon publications open access paper abstract we studied the effect of a high linolenic acid diet supplementation with synthetic (vitamin e + selenium) or vegetal mix rich in natural antioxidants (grape skin + oregano) on live performances, carcass and meat quality, fatty acid composition and oxidative stability of intramuscular lipids of longissimus thoracis et lumborum muscle in medium-heavy pigs. neither carcass traits nor chemical proximate composition of meat was affected by dietary treatments. linseed dietary inclusion reduced the n-6:n-3 polyunsaturated fatty acids ratio and increased longchain n-3 precursor, fundamental for human health. our results offer new opportunities to use products more acceptable by consumers and are more eco-friendly. keywords: extruded linseed, fatty acids profile, grape skin extract, oregano extract, oxidative stability of pork, vitamin e introduction polyunsaturated fatty acids (pufa), including n-6 and n-3 groups, are essential for physiological functioning and health of humans and domestic animals (delgadolista et al., 2012). western diets are deficient in n-3 pufa and contain excessive amounts of n-6 pufa, resulting in a high n-6:n-3 ratio ranging from 10:1 to 20:1, while an optimal recommended ratio is 1:1–4:1. high values of ratio can be the cause of various diseases (simopoulos, 2002, 2010; leslie et al., 2015). many studies have shown that n-3, mainly eicosapentaenoic (epa, 20:5 n-3) and docosahexaenoic (dha, 22:6 n-3) fatty acids (fa), have an anti-inflammatory effect and play a beneficial role in italian journal of food science, 2021; 33 (2): 117–128 mailto:gminelli@unimore.it 118 italian journal of food science, 2021; 33 (2) belmonte am et al. the aim of this research was to study the effects of inclusion of linseed in the growing–finishing diet of medium-heavy pigs and of the supplementation with supranutritional levels of a synthetic antioxidant complex (vitamin e and selenium) or vegetal mix rich in natural antioxidants (grape skin and oregano) on live performance, carcass and meat quality, and on fatty acid composition and oxidative stability of intramuscular lipids of longissimus thoracis et lumborum (ltl) muscle. materials and methods ethics approval all the experimental procedures performed in this study were in accordance with the italian legislation and did not require special animal care authorizations, that is the decision of the animal welfare committee of consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria (crea; 14 september 2016), according to the italian legislative decree of 4 march 2014 n. 26 art. 2, point f. animals, diets and growth performances forty-eight italian large white pigs, balanced for gender and weight, housed in 16 pens (9 m2 concrete floor pens) with three animals in each pen, were evenly assigned to four different dietary treatments (12 pigs per diet; four replicates). the diets were similar for energy and protein levels with the same lysine/digestible energy ratio. the composition of the diets is shown in table 1. the control group (c) received barley/soybean diet. in the three treatment (linseed) groups (experimental groups), 5% of barley was substituted for 5% of extruded linseed, either unsupplemented (l) or supplemented with a synthetic antioxidant complex (lsa) containing 200 ppm of α-tocopheryl acetate and 0.21 ppm of selenium (supported on calcium carbonate), or added with 5 g/kg of vegetal mix rich in natural antioxidants (lna), providing 3 g/ kg of grape skin extract and 2 g/kg of oregano extract, adhering to the amounts suggested by the manufacturer for food supplementation. water was always available. the trial lasted for 104 days starting from an average live body weight (lbw) of 79.9 ± 5.8 kg (6 months of age), till slaughter at 150.5 ± 9.9 kg lbw. from starting weight and up to 113 ± 10.6 kg lbw, the subjects were fed at 7.5% of metabolic weight, calculated as lbw0.75; thereafter, till slaughtering, the pigs were fed at 8.5% of lbw0.75. the natural extracts from grape skin are normally used as supplement, nutraceutical, or for food colouring; the total amount of polyphenols in the antioxidant mix of lna group was 14.3 g/l expressed as gallic acid unfavourable n-6:n-3 pufa ratio (liu and kim, 2018), pork consumption has been associated with an increased risk of chronic diseases (egeberg et al., 2013; klurfeld, 2015). however, genetic factors (cameron et al., 2000; piedrafita et al., 2001; wood et al., 2008), sex, age, live weight at slaughter (lebret and mourot, 1998; lo fiego et al., 2005b; minelli et al., 2019) and feeding strategies (lo fiego et al., 2005a) can influence deposition of lipids in pig tissues and their fatty acid composition. it is widely accepted that nutrition is the main factor influencing deposition of lipid and fatty acid in monogastric animals. dietary fa can significantly modify the fatty acid profile of adipose tissues in pigs. in fact, many studies have confirmed the enrichment of pig tissues with n-3 pufa by incorporating linseed in pig diets (kouba et al., 2003; corino et al., 2008; minelli et al., 2020) or its derivatives rich in ala (guillevic et al., 2009; musella et al., 2009; corino et al., 2014). however, increasing pufa and lowering sfa contents raises the susceptibility of meat to lipid oxidation, leading to undesirable effects such as deterioration of its sensorial quality and nutritional value (rivas-cañedo et al., 2013; chamorro et al., 2015). to counteract this effect, dietary addition of antioxidants, such as vitamin e and selenium, is widely used in swine feeding. supranutritional levels of vitamin e may improve oxidative and colour stability of meat during storage (de la fuente et al., 2009; kasapidou et al., 2012; muíño et al., 2014). moreover, the combination of vitamin e and selenium can build a complex antioxidant system capable of protecting against free radicals and lipid oxidation products (surai and fisinin, 2015). however, now consumers feel more reasonable the use of natural antioxidant products in animal feeding (gladine et al., 2007; brenes et al., 2016). among these, oregano extract has shown a considerable antioxidant effect (botsoglou et al., 2003) as well as antimicrobial and anti-inflammatory activity (cheng et al., 2017). oregano antioxidant action is attributed to the presence of different phenolic active compounds such as thymol and carvacrol (tuttolomondo et al., 2013). another very rich natural source of antioxidants is represented by various by-products of winery, such as grape skin and seeds, that are rich in polyphenolic compounds characterized by a high antioxidant activity (selani et al., 2011). furthermore, the wine industry produces a large amount of residues in a short period of the year whose disposal is expensive and involves problems related to pollution (bustamante et al., 2008). the inclusion of these by-products as an alternative to synthetic antioxidants could be an interesting nutritional and environment-friendly strategy. italian journal of food science, 2021; 33 (2) 119 effects of high linolenic acid diet supplemented with synthetic or natural antioxidant mix table 1. ingredients (%), proximate composition (% on dry matter basis) and fatty acid composition (% of total fatty acids) of dietary treatments. c l lsa lna 1st 2nd 1st 2nd 1st 2nd 1st 2nd ingredients extruded linseed % 0.00 0.00 5.00 5.00 5.00 5.00 5.00 5.00 barley meal % 85.50 91.00 80.50 86.60 80.30 86.40 80.50 86.60 soybean meal % 11.00 5.50 11.00 5.00 11.00 5.00 11.00 5.00 l-lysine % 0.31 0.29 0.30 0.29 0.30 0.29 0.30 0.29 dl-methionine % 0.06 0.04 0.06 0.03 0.06 0.03 0.06 0.03 l-threonine % 0.05 0.04 0.05 0.03 0.05 0.03 0.05 0.03 calcium carbonate % 1.18 1.13 1.19 1.15 0.89 0.85 1.19 1.15 dicalcium phosphate % 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 salt (nacl) % 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 vitamin/mineral pre-mix1 % 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 vit e + se pre-mix2 % — — — — 0.50 0.50 — — vegetal ext. (grape skin + oregano)3 % — — — — — — 0.3+0.2 0.3+0.2 proximate composition dry matter (dm) % 88.30 89.50 88.60 89.80 88.70 89.90 88.80 90.00 (on dm basis) digestible energy mj/kg 13.35 13.26 13.63 13.54 13.60 13.52 13.63 13.54 crude protein % 16.87 12.55 17.89 13.20 17.98 13.58 17.93 13.03 crude fat % 2.00 1.73 4.30 3.86 4.21 4.00 4.41 3.98 crude fibre % 4.76 4.50 4.91 5.08 5.01 4.65 5.26 4.97 ashes % 5.87 4.50 5.86 5.89 6.26 5.98 6.15 5.40 fatty acid (fa) composition % (of total fas) c14:0 % 0.47 0.39 0.25 0.21 0.25 0.22 0.26 0.22 c16:0 % 29.01 24.25 18.13 15.20 17.78 15.59 18.80 15.31 c16:1 % 0.49 0.34 0.17 0.15 0.17 0.17 0.02 0.15 c18:0 % 2.03 1.51 4.00 3.18 3.88 3.34 4.16 3.23 c18:1n-9 % 14.92 13.50 20.60 18.12 20.24 18.45 21.29 18.26 c18:2n-6 % 47.55 53.67 33.50 34.69 33.91 34.09 32.52 34.47 c18:3n-3 % 4.77 5.70 22.83 28.02 23.25 27.73 22.38 27.95 c20:1 % 0.74 0.64 0.53 0.41 0.52 0.42 0.57 0.41 c: control group; l: experimental group with 5% of extruded linseed; lsa: experimental group with 5% of extruded linseed, 200 ppm vitamin e + 0.21 ppm selenium; lna: experimental group with 5% of extruded linseed and vegetal extract 5 g/kg of feed (3.00 g of grape skin extract + 2.00 g of oregano extract). 1st = feed administered from an average weight of 79.9 to 113.4 kg (growing period); 2nd = feed administered from an average weight of 113.4 kg to the slaughter (finishing period). 1vitamin/mineral: providing the following nutrients (per kg diet as-fed): vitamin a, 15.000 iu; vitamin d3, 2.000 iu; vitamin e (α-tocopheryl acetate), 50 mg; vitamin k, 2.5 mg; vitamin b1, 2 mg; vitamin b2, 5 mg; vitamin b5, 15 mg; vitamin b6, 4 mg; vitamin b12, 0.036 mg; niacin, 25 mg; folic acid, 1 mg; biotin, 0.15 mg; choline, 346 mg; cu, 15 mg; fe, 150 mg; mn, 25 mg; co, 0.4 mg; i, 1.5 mg; zn, 100 mg; and se, 0.1 mg. 2vitamin e + selenium: providing the following nutrients (per kg diet as fed): vitamin e (α-tocopheryl acetate), 200 mg, and se, 0.21 mg, supported on calcium carbonate. 3vegetal extract was added directly to the water of the diet and not mixed with the complete feed. equivalent (gae). a complete characterisation of phenolic compounds of vegetal extracts used is reported in a previous paper (martini et al., 2020). grape skin extract was supplied by enocianina fornaciari s.n.c. (reggio emilia, italy) and oregano extract was supplied by phenbiox s.r.l. (bologna, italy). all diets were distributed in wet form (water:feed ratio of 3:1) and the vegetal extract mix was diluted in the water of the diet. during the trial, the average daily feed intake (adfi) per pen was monitored, and the feed conversion rate (fcr) per pen was calculated. 120 italian journal of food science, 2021; 33 (2) belmonte am et al. longitudinal orientation of muscle fibres. the measurements were averaged, and the peak force was expressed as kilogram. the working conditions of the zwick z50 kn testing machine (model bt1-fb050tn, zwick roell, kennesaw, ga, usa) were as follows: 1-kn load cell equipped with a v-shaped blade with a triangular hole of 60°; and a constant speed of 250 mm/min. lipid oxidation analysis the lipid oxidation of fresh and cooked ltl samples was evaluated in duplicate according to siu and draper (1978, slightly modified). minced sample, 2.5 g, was blended and homogenised with 12.5 ml of distilled water for 2 min at 9,500 rpm using an ultra-turrax tissue homogenizer (ika, germany). before centrifugation, at 2,000 rpm at 4°c for 20 min, 12.5 ml of 10% trichloroacetic acid (tca) solution (sigma-aldrich, milan, italy) was added. the supernatant was collected after decantation through a paper filter (whatman no. 541), and 4 ml of clear filtrate was transferred into 15-ml pyrex tubes; 1-ml 0.06 m 2-thiobarbituric acid (tba, sigma-aldrich, milan, italy) was added and the samples were kept for 90 min in a water bath at 80°c; the samples were cooled before reading. at the same time, the blank was run (2-ml distilled water + 2-ml tca solution + 1-ml tba). absorbance at 532 nm was measured against blank sample using a jasco spectrophotometer (model v550, uv/ vis, tokyo, japan). using 1,1,3,3 tetraethoxypropane (tep, sigma-aldrich, milan, italy) as a standard, thiobarbituric acid reactive substances (tbars) was expressed as milligram of malondialdehyde (mda) per kilogram of muscle. chemical composition of fresh meat and feed the analyses of moisture, ether extract and crude protein were performed on ltl muscle according to the association of official analytical chemists methods (aoac, 1995). the results were expressed as percentage of wet matter. analyses for the determination of proximate composition of feeds (dry matter, ash, crude protein, crude fat and crude fibre) were carried out according to the aoac methods (aoac, 1995) and the results were expressed on dry matter basis. energy values of feeds were calculated according to sauvant et al. (2004). fatty acid profile of fresh meat total lipids from ltl muscle were extracted according to the folch et al. (1957) method. according to slaughtering and sampling the pigs were weighed individually after an overnight fasting, and slaughtered in a commercial abattoir by exsanguination after electrical stunning, in agreement with the council regulation (ec) no. 1099/2009 on the protection of animals at the time of killing. each carcass, after slaughtering, was graded in agreement with europ grid carcass grading, using fat-o-meater device (mipaaf, 2018). the hot carcasses were weighed, the ph1 (45 min post-mortem) value at the last rib level was measured and the hot carcass yield was calculated as hot carcass weight (kg)/slaughter lbw (kg)×100. subsequently, each carcass was dissected into commercial cuts, which were weighed and cold-stored at 0–4°c for about 24 h. at 24 h post-mortem, the refrigerated ltl muscle from each half left carcass was transported to laboratory and sliced in four subsamples: the first to be used for ph2 (24 h post-mortem), colour and drip loss analysis; the second for evaluation of oxidative stability, moisture, intramuscular lipid and protein content of raw meat; the third one for cooking loss and shear force analysis and oxidative stability of cooked meat; and the last one was vacuum-packed (elegen, reggio emilia, italy) and stored at –20°c until lipid extraction for fatty acid analysis. instrumental analysis at 24 h post-mortem, ph values and colour parameters were measured directly on the fresh muscle. the ph value was recorded using a portable crison ph-meter equipped with a xerolite electrode (crison instruments, alella, spain). meat colour was measured by minolta cm-600d spectrophotometer (konica minolta holdings inc., osaka, japan) using illuminant d65 and an 8-mm diameter aperture. after calibration with a white calibration plate, five different points on each sample were analysed and the measurements were averaged. the results were expressed as the cie lab three coordinates: l* – ‘lightness’, a* – ‘redness’, and b* – ‘yellowness’. further, chroma (c*), the expression of saturation index and colour intensity, was calculated as +2 22 a * b * , and the hue angle (h*) was calculated as arctan (b*/a*). drip loss (%) was evaluated on ltl muscles (starting at 24 h post-mortem) according to honikel (1998, slightly modified). to evaluate cooking loss, a sample (approx.100  g) of ltl muscle was vacuum-packed and cooked in a water bath till the core temperature reached 70°c. after cooling, the samples were weighed, and the cooking loss was calculated as the percentage of initial sample weight. the warner–bratzler shear force (wbsf) was determined on cooked samples according to honikel (1998). briefly, from each ltl-cooked muscle, six cylindrical cores (ø, 1.50 cm) were cut parallel to the italian journal of food science, 2021; 33 (2) 121 effects of high linolenic acid diet supplemented with synthetic or natural antioxidant mix statistical analysis the statistical analysis was performed using the mixed model procedure of sas (sas institute inc., cary, nc, usa). the statistical model included dietary treatments, gender and their interactions as fixed effects and pen as a random effect. the interactions were not statistically significant; therefore, they were removed from the statistical model. live performance data as average daily gain (adg), adfi, fcr, and slaughter weight were covariate for starting lbw. moreover, for adfi and fcr, the pen was considered as an experimental unit. carcass weight and dressing percentage were covariates for slaughter weight, while the fatty acid composition was a covariate for intramuscular fat content (ifc). when a significant (p < 0.05) treatment effect was observed, tukey’s multiple comparison test was performed to compare mean values. results and discussion performance and carcass characteristics the experimental diets did not influence adg and slaughter lbw (p > 0.05) (table 2). these results confirm the results reported by corino et al. (2008) in pigs fed with a diet containing 5% of extruded linseed and slaughtered at 100 or 160 kg, and kouba et al. (2003) in pigs fed for 20, 60 or 100 days with a diet containing 6% of whole crushed linseed. moreover, juàrez et al. (2010), feeding pigs with 5%, 10% or 15% of extruded flaxseed, reported no statistical differences on adg and final lbw but found a slight improvement of feed efficiency with an increased dietary level of flax. in our study, the control group showed the highest and the lna group the lowest fcr values (p < 0.05). the ficarra et al. (2010), 25 mg of lipid extract was methylated with methanolic potassium hydroxide (koh) solution 2n (koh supplied by carlo erba, milan, italy, and methanol supplied by itw reagents, barcelona, spain) and an aliquot of tridecanoic acid (c13:0) (larodan fine chemicals ab, malmö, sweden) was added as internal standard. for determining the fa profile, tracetm gas chromatograph (gc) ultra (thermo electron corporation, rodano, milano, italy) equipped with flame ionization detector, a pvt injector and tr-fame column (30-m long, 0.25-mm i.d., 0.2-µm film thickness), supplied by thermo scientific (rodano, milano, italy), was used. methylated sample, 1 µl, was injected into gc with a split flow rate of 10 ml/min, operating at a constant flow of 1 ml/min of helium as a carrier gas. detector and injector had the same operating temperature of 240°c. after 2  min, the program temperature was increased at a rate of 4°c per minute from 140°c to 250°c and maintained for 5  min. the chrom-card software (version 2.3.3, thermo electron corporation rodano, milano, italy) was used to record, identify and integrate the peaks of fatty acid methyl esters (fames). to identify the retention period of fames, a solution of standard fa mixed with the known quantity of standard was used (supelcor 37 component fame mix, pufa standard n.2, animal source, supelco, bellafonte, pa, usa, and single fames standard, larodan, fine chemicals ab, malmö, sweden). the amount of each fame was expressed as fame relative percentage with respect to the total amount of fames. moreover, atherogenic index, ai = [c12:0 + (4 × c14:0) + c16:0] / [n-6 pufa + n-3 pufa + monounsaturated fa (mufa)] (ulbricht and southgate, 1991), and thrombogenic index, ti = [c14:0 + c16:0 + c18:0] / [(0.5 × mufa) + (0.5 × n-6 pufa) + (n-3 pufa / n-6 pufa)] (ulbricht and southgate, 1991), were calculated. table 2. effect of dietary treatment and gender on live performance (data covariate for initial lbw). dietary treatments gender c l lsa lna sem gilts barrows sem initial lbw (kg) 77.4 82.3 80.1 79.7 2.59 79.8 79.9 1.64 adg (kg) 0.67 0.68 0.68 0.70 0.035 0.70 0.68 0.025 adfi (kg/day) 2.50 a 2.46ab 2.46a,b 2.42 b 0.019 — — — fcr (kg/kg-–1) 3.81a 3.51b 3.59b 3.47b 0.066 — — — slaughter lbw (kg) 148.9 150.9 150.6 151.7 2.52 151.5 149.6 1.86 c: control group; l: experimental group with 5% of extruded linseed; lsa: experimental group with 5% of extruded linseed, 200 ppm vitamin e + 0.21 ppm selenium; lna: experimental group with 5% of extruded linseed and vegetal extract 5 g/kg of feed (3.00 g of grape skin extract + 2.00 g of oregano extract). sem: standard error of mean values; adg: average daily gain; adfi: average daily feed intake; fcr: feed conversion rate (adfi/adg); lbw: live body weight. feed conversion ratio: pens were considered as experimental units. a,bdifferent letters in the same line indicate statistically different mean values (p < 0.05). 122 italian journal of food science, 2021; 33 (2) belmonte am et al. gender had no effect (p > 0.05) on any of the parameters evaluated. since feed intake was recorded per pen, and both sexes were housed in each pen, the gender-wise statistical analysis of adg and fcr was not possible. inclusion of dietary linseed (table 3) did not affect (p > 0.05) carcass characteristics, as already observed in previous studies (kouba et al., 2003; karolyi et al., 2012). although differences were not statistically significant, the backfat thickness was slightly higher in the control group, and the lean meat percentage was lesser than in the linseed-fed groups (33.5 mm vs. avg. 30.9 mm, and 50.2% vs. avg. 51.4%). the same trend was recorded by duan et al. (2014), who reported a significant decrease in adipose tissues and an increase in lean tissue masses with a decrease in dietary n-6:n-3 ratio from 10:1 to 1:1. the effect of gender was significant only on the percentage of perirenal fat, higher in barrows (1.95% vs.1.59%; p < 0.05). adfi was higher in the control group with statistical differences (p < 0.05) only when compared with the lna group (2.50 vs. 2.42 kg*day–1). we could assume that the high content of polyphenols was the main reason for these results. fiesel et al. (2014) stated that the presence of plant products in growing pigs’ diet improves the gain:feed ratio, since polyphenols not only cause alteration in the microbial composition of the gut but also exert anti-inflammatory action. in our study, dietary n-6:n-3 pufa ratios were 9.8:1 in the control group and averaged as 1.35:1 in linseed groups (data not reported in the table). the groups with lower n-6:n-3 pufa ratios showed slight improvement in feed efficiency (p < 0.05), and this result agrees with duan et al. (2014) and li et al. (2015). the authors stated that n-6:n-3 ratios of 1:1 and 1:5 compared with 10:1 improved feed efficiency of finishing pigs. these authors assumed that improvement in feed efficiency could be due to the anti-inflammatory effect of n-3, which spares energy and nutrients for tissue deposition. table 3. effect of dietary treatment and gender on carcass traits (carcass weight and dressing percentage were covariate for slaughter weight, while all carcass traits were covariate for hot carcass weight). dietary treatments gender c l lsa lna sem gilts barrows sem hot carcass weight (kg) 124.82 129.26 127.03 127.75 2.493 128.11 126.32 1.765 hot carcass yield (%) 85.02 84.59 84.28 84.23 0.503 84.36 84.70 0.359 backfat thickness (mm) 33.53 31.09 30.72 30.93 1.615 31.45 31.68 1.109 lean meat content (%) 50.23 51.33 51.31 51.41 0.815 51.08 51.06 0.278 lean cuts (%)1 • thigh 26.89 26.73 27.06 27.17 0.314 27.09 26.84 0.222 • loin 18.61 17.62 18.02 17.62 0.369 18.00 17.94 0.259 • neck 7.90 8.17 8.00 7.79 0.249 8.06 7.87 0.169 • shoulder 14.48 14.52 14.15 14.34 0.182 14.35 14.40 0.128 • total lean cuts 67.90 67.03 67.23 66.92 0.480 67.55 66.99 0.340 adipose cuts (%)1 • backfat 4.50 5.06 5.22 5.53 0.285 5.17 4.98 0.200 • belly 13.38 13.47 13.49 12.98 0.389 13.37 13.29 0.275 • jowl 6.35 6.49 6.78 6.69 0.240 6.49 6.67 0.165 perirenal fat 1.73 1.74 1.81 1.81 0.115 1.59b 1.95a 0.080 total adipose cuts 25.94 26.77 27.31 26.98 0.513 26.54 26.96 0.363 others (head, feet, tail) (%)1 6.13 6.20 5.46 6.10 0.580 5.91 6.05 0.410 c: control group; l: experimental group with 5% of extruded linseed; lsa: experimental group with 5% of extruded linseed, 200 ppm vitamin e + 0.21 ppm selenium; lna: experimental group with 5% of extruded linseed and vegetal extract 5 g/kg of feed (3.00 g of grape skin extract + 2.00 g of oregano extract). sem: standard error of mean values. 1percentage of hot carcass weight. a,bdifferent letters in the same line indicate statistically different mean values (p < 0.05). italian journal of food science, 2021; 33 (2) 123 effects of high linolenic acid diet supplemented with synthetic or natural antioxidant mix table 4. effects of dietary treatment and gender on chemical and physical characteristics of raw and cooked longissimus thoracis et lumborum muscle. dietary treatments gender c l lsa lna sem gilts barrows sem ph 1 6.49 6.37 6.37 6.37 0.096 6.34 6.47 0.062 ph 2 5.60 5.55 5.58 5.54 0.020 5.55 5.58 0.014 l* 52.78 52.69 53.34 53.20 0.755 53.66 52.34 0.533 a* 3.16a 2.71a,b 1.88b 2.07a,b 0.379 2.02b 2.89a 0.268 b* 12.40a 11.94a,,b 11.59b 11.92a,b 0.277 11.85 12.08 0.196 chroma (c*) 12.88a 12.35a,b 11.79b 12.16a,b 0.312 12.08 12.51 0.221 hue angle (h*) 76.04b 77.80a,b 81.01a 80.51a,b 1.672 80.73a 76.95b 1.18 drip loss (%) 2.45 3.37 2.89 2.63 0.464 3.05 2.62 0.329 cooking loss (%) 21.88 23.23 21.15 20.95 0.985 21.34 22.27 0.698 mda, raw meat (mg/kg) 0.104 0.115 0.083 0.164 0.044 0.094 0.138 0.116 mda, cooked meat (mg/kg) 0.401 0.501 0.401 0.380 0.0505 0.393 0.448 0.0358 moisture (%) 68.98 68.24 67.98 68.75 0.655 68.65 68.33 0.461 ether extract (%) 1.62 1.58 1.39 1.75 0.158 1.40b 1.78a 0.109 protein (%) 23.40 23.62 23.81 22.88 0.344 23.42 23.44 0.243 wbsf (kg) 6.58 6.40 6.33 5.93 0.366 6.28 6.34 0.259 c: control group; l: experimental group with 5% of extruded linseed; lsa: experimental group with 5% of extruded linseed, 200 ppm vitamin e + 0.21 ppm selenium; lna: experimental group with 5% of extruded linseed and vegetal extract 5 g/kg of feed (3.00 g of grape skin extract + 2.00 g of oregano extract). sem: standard error of mean values; mda: malondialdehyde; wbsf: warner bratzler shear force. a,bdifferent letters in the same line indicate statistically different mean values (p < 0.05). raw and cooked meat characteristics the effects of dietary treatment and gender on chemical and physical characteristics of ltl muscle are shown in table 4. in agreement with corino et al. (2014) and minelli et al. (2020), the ph values of ltl muscle were not influenced by dietary treatments. the colour parameters a*, b*, c*, and h* were the only parameters affected by dietary treatments. the control group showed higher a* (3.16 vs. 1.88), b* (12.40 vs. 11.59) and c* (12.88 vs. 11.79) values but lower h* (76.04 vs. 81.01) values with respect to the lsa group (p < 0.05). the control group yielded redder and yellower meat than the lsa group, whilst the other two diets with linseed showed intermediate and not statistically different values. probably, vitamin e might have affected variation of colour parameters. our data conflicted with the results of hasty et al. (2002), who reported a tendentially (p > 0.05) linear increase of b* value with increasing levels of dietary α-tocopheryl acetate, while asghar et al. (1991) observed that a dietary supplementation of vitamin e at a level of 200 iu/kg led to an increase in the redness of pork without affecting yellowness. in general, we found that all linseed groups have tendentially lower a*, b* and c* values and higher h* value, and this indicated that extruded linseed could produce tendentially decoloured meat. the moisture, ether extract, drip loss, mda and protein content of raw meat, and shear force and mda content in cooked meat were not affected by dietary treatments. oxidative stability during meat storage and cooking is an important factor for both shelf-life and consumer safety. in this research, the mda content increased by about four times with the cooking process, but no difference ascribable to diet or sex was found in raw or cooked meat, although unsupplemented extruded linseed showed the highest value (p > 0.05). these results confirmed the previous findings in pigs fed with similar diets (minelli et al., 2020). yet by imposing more challenging experimental conditions, other authors obtained quite different results. among these authors are botsoglou et al. (2012), who evaluated pork chops containing lipids with far higher proportions of n-6 and n-3 pufa than our ltl samples. they observed that lipid oxidation in both raw and cooked chops was significantly alleviated in the samples obtained from the subjects that had received a dietary supplementation of α-tocopheryl acetate, 200 mg/kg feed. 124 italian journal of food science, 2021; 33 (2) belmonte am et al. group and statistically different (p < 0.05) compared with the l group. the thrombogenic index (ti) decreased (p < 0.05) with linseed feeding but only in the l group compared with the control group. in general, main variations in fa composition ascribable to linseed feeding are as follows: an increase of n-3 pufa, mainly ala and its derivates epa and dpa, originated from elongase and desaturase reactions; decrease of n-6 pufa, mainly 22:4 n-6, probably accounted for by the higher affinity of ∆6 desaturase for n-3 substrates (lee et al., 2016) and decrease of n-6:n-3 ratio below 4, the maximum threshold indicated by simopoulos (2002) to avoid adverse health consequences. our results agree with previous works (riley et al., 2000; minelli et al., 2020). the fa composition of intramuscular fat did not differ significantly between lsa and lna groups. regarding the n-6:n-3 ratio, it did not vary in linseed-fed groups, although antioxidant supplementation led to a numerical increase of this ratio, with the highest value found in the lsa group. this could be due to different effects of diets enriched with n-3 pufa and natural or synthetic antioxidants in modulating the expression of pig skeletal muscle genes involved in de novo synthesis of fa and metabolism of lipids. vitali et al. (2019) reported a more evident stimulation of the expression of genes involved in controlling muscle metabolism when n-3 dietary pufa are administered in association with polyphenols-enriched diet. pigs’ gender affected the total mufa content, higher in barrows than in gilts (47.11 vs. 45.29; p < 0.05), specifically 18:1 n-7 and 18:1 n-9. moreover, barrows had the lowest amount of pufa (14.34 vs. 16.52, p < 0.05) and total n-6 content (11.80 vs. 13.57; p < 0.05), confirming previous findings (alonso et al., 2009; lo fiego et al., 2010; okrouhlá et al., 2013). no difference was observed for total n-3, n-6:n-3 pufa ratio, and atherogenic and thrombogenic indices. conclusion our study confirms that 5% of dietary-extruded linseed in pig diet is a suitable means to increase n-3 pufa content and reduce n-6:n-3 pufa ratio in ltl muscle, an important aim from the point of view of human nutrition, without affecting live performances and carcass traits. the technological characteristics of carcass and meat were not impaired by the ameliorated pufa ratio. the main qualitative characteristics and chemical composition of the muscle were neither affected by linseed feeding nor by inclusion of synthetic or natural further, martini et al. (2020), evaluating a subsample of this research, reported in meat grilled at 140°c for 5 min a dramatic increase of about eight times in mda with respect to raw meat. the sharpest increase was found in the l group that showed statistically different results from all other groups, while no difference was found in c, lsa and lna groups. thus, rearing and processing conditions play a pivotal role in the oxidative stability of meat enriched with n-3 pufa. however, in our experimental conditions, the natural antioxidants were as effective as the synthetic ones in preventing formation of advanced lipid oxidation end products. all groups showed an mda content lower than 1.0 mg/ kg of meat, which is considered the maximum acceptable threshold for rancidity (rahman et al., 2015). regarding the effect of gender on colour parameters, the redness value was higher in barrows than in gilts (2.89 vs. 2.02; p < 0.05) and h* was lower in barrows (76.95 vs. 80.73; p < 0.05). our results are in agreement with previous studies (alonso et al., 2009; daza et al., 2018). these authors reported that a* value was directly related to myoglobin content that was higher in barrows than in gilts, and consequently their meat resulted redder globally. ltl muscle in barrows had higher content of ether extract (1.78% vs. 1.40%; p < 0.05). this matched with other findings (alonso et al., 2009; lo fiego et al., 2010; daza et al., 2018) and was largely expected, given that castration promoted the intramuscular fattening of meat (barton-gade, 1987). intramuscular fatty acid composition dietary treatments did not significantly affect the proportion of total sfa and mufa (p > 0.05) (table 5). among sfa, the percentage of 12:0 and 16:0 tended to be lower in the control group but statistically different (p < 0.05) from the lsa group (–0.01 and –0.95 percentage points, respectively). among mufa, 17:1 tended to be higher in the control group but statistically different from the l group (+0.06; p < 0.05). irrespective of the type of antioxidant used, inclusion of extruded linseed in the diet increased (p < 0.05) the proportion of all n-3 pufa except dha, in agreement with guillevic et al. (2009) and minelli et al. (2020). further, dha was lower (p < 0.05) in the lsa group than in the l group (0.07% vs. 0.10%, respectively). moreover, linseed dietary inclusion reduced (p < 0.05) the n-6:n-3 pufa ratio and the proportion of all n-6 pufa, except 18:2 n-6 and 20:2 n-6 (p > 0.05). the total content of pufa was lowest in the lsa italian journal of food science, 2021; 33 (2) 125 effects of high linolenic acid diet supplemented with synthetic or natural antioxidant mix table 5. effect of dietary treatments and gender on fatty acid profile (% of total fatty acid) of longissimus thoracis et lumborum muscle. dietary treatments gender c l lsa lna sem gilts barrows sem c10:0 (capric) 0.12 0.11 0.12 0.11 0.005 0.11b 0.12a 0.004 c12:0 (lauric) 0.07b 0.08a,b 0.08a 0.08a,b 0.003 0.07b 0.08a 0.002 c14:0 (myristic) 1.19 1.23 1.27 1.28 0.034 1.20b 1.29a 0.025 c16:0 (palmitic) 23.24b 23.41a,b 24.19a 24.00a,b 0.319 23.49 23.93 0.233 c17:0 (heptadecanoic) 0.23 0.21 0.25 0.23 0.018 0.23 0.23 0.012 c18:0 (stearic) 12.66 12.69 12.94 13.11 0.354 12.95 12.75 0.258 c20:0 (eicosanoic) 0.14 0.15 0.14 0.14 0.006 0.14 0.15 0.004 c16:1 (palmitoleic) 3.18 3.07 3.10 2.96 0.124 2.94 3.22 0.090 c17:1 (heptadecenoic) 0.30a 0.24b 0.26a,b 0.28a,b 0.014 0.27 0.26 0.010 c18:1n-7 (vaccenic) 4.15 4.05 4.05 3.91 0.101 3.92b 4.17a 0.074 c18:1n-9 (oleic) 38.88 37.27 39.03 37.48 0.651 37.54b 38.78a 0.475 c20:1 (eicosenoic) 0.62 0.64 0.67 0.66 0.033 0.62 0.68 0.023 c18:2n-6 (linoleic) 9.45 9.70 8.38 9.37 0.514 9.80a 8.65b 0.374 c18:3n-3 (α-linolenic) 0.49b 1.85a 1.62a 1.84a 0.086 1.53 1.37 0.062 c18:3n-6 (γ-linolenic) 0.22a 0.19a,b 0.16b 0.19a,b 0.014 0.20 0.18 0.010 c20:2n-6 (eicosadienoic) 0.23 0.24 0.21 0.24 0.010 0.24a 0.22b 0.007 c20:3n-3 (eicosatrienoic) 0.08b 0.22a 0.20a 0.22a 0.010 0.19 0.17 0.007 c20:4n-6 (arachidonic) 3.54a 2.86a,b 2.03c 2.37b,c 0.252 2.96 2.44 0.184 c20:5n-3 (eicosapentaenoic) 0.14c 0.58a 0.40b 0.48a,b 0.046 0.44 0.36 0.033 c22:4n-6 (docosatetraenoic) 0.53a 0.31b 0.24b 0.28b 0.029 0.37 0.31 0.021 c22:5n-3 (docosapentaenoic) 0.45b 0.80a 0.59b,c 0.69a,c 0.067 0.69 0.57 0.049 c22:6n-3 (docosahexaenoic) 0.09ab 0.10a 0.07b 0.08a,b 0.009 0.10a 0.07b 0.006 total saturated 37.65 37.88 38.99 38.95 0.648 38.19 38.55 0.472 total monounsaturated 47.13 45.27 47.11 45.29 0.778 45.29b 47.11a 0.567 total polyunsaturated 15.22ab 16.85a 13.90b 15.76a,b 0.965 16.52a 14.34b 0.703 total n-6 13.97a 13.30c 11.02b 12.45a,b,c 0.789 13.57a 11.80b 0.575 total n-3 1.25c 3.55a 2.88b 3.31a,b 0.198 2.95 2.54 0.145 n-6:n-3 ratio 11.39a 3.73b 3.95b 3.79b 0.142 5.74 5.70 0.103 atherogenic index ai 0.45 0.46 0.48 0.48 0.012 0.46 0.48 0.009 thrombogenic index ti 1.05a 0.91b 1.00a,b 0.95a,b 0.035 0.96 0.10 0.026 c: control group; l: experimental group with 5% of extruded linseed; lsa: experimental group with 5% of extruded linseed, 200 ppm vitamin e + 0.21 ppm selenium; lna: experimental group with 5% of extruded linseed and vegetal extract 5 g/kg of feed (3.00 g of grape skin extract + 2.00 g of oregano extract). sem: standard error of mean values. atherogenic index, ai = [c12:0 + (4 × c14:0) + c16:0] / [n-6 pufa + n-3 pufa + mufa] (ulbricht and southgate, 1991). thrombogenic index, ti = [c14:0 + c16:0 + c18:0] / [(0.5 × mufa) + (0.5 × n-6 pufa) + (n-3 pufa / n-6 pufa)] (ulbricht and southgate, 1991). a,bdifferent letters in the same line indicate statistically different mean values (p < 0.05). antioxidants. absence of further improvement with the addition of natural antioxidants may be due to the low quantity used in this study, based on the quantities used in human diets. further research should investigate the effects of higher levels of natural antioxidants, added to n-3 pufa-enriched diets, on pork quality under different storage conditions and cooking methods. acknowledgements the authors thank dr. giacinto della casa (crea-research centre for animal production and aquaculture – modena 126 italian journal of food science, 2021; 33 (2) belmonte am et al. chamorro s., viveros a., rebolé a., 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https://doi.org/10.1016/0140-6736(91)91846-m� https://doi.org/10.1016/0140-6736(91)91846-m� https://doi.org/10.1371/journal.pone.0212449� https://doi.org/10.1111/j.1753-4887.2010.00287.x� https://doi.org/10.1111/j.1753-4887.2010.00287.x� https://doi.org/10.1016/j.meatsci.2007.07.019 https://doi.org/10.17221/6826-cjas� https://doi.org/10.1016/s0309-1740(00)00078-4� https://doi.org/10.5851/kosfa.2015.35.6.772� https://doi.org/10.5851/kosfa.2015.35.6.772� https://doi.org/10.1017/s1357729800055454� https://doi.org/10.1017/s1357729800055454� https://doi.org/10.1016/j.meatsci.2012.08.017� https://doi.org/10.1016/j.meatsci.2012.08.017� https://doi.org/10.3920/978-90-8686-668-7� https://doi.org/10.1016/j.meatsci.2011.01.017� _goback p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33i2.1969 1 p u b l i c a t i o n s codon quality perception and willingness to pay: the case of red wine with health-beneficial effects jelena ruso1, jovan filipović1, milica maričić1*,vesna spasojević-brkić2 1university of belgrade, faculty of organizational sciences, belgrade, serbia; 2university of belgrade, faculty of mechanical engineering, belgrade, serbia *corresponding author: milica maričić, department of operational research and statistics, university of belgrade, faculty of organizational sciences, jove ilića 154, belgrade, serbia, email: milica.maricic@fon.bg.ac.rs received: 15 october 2020; accepted: 14 february 2021; published: 1 april 2021 © 2021 codon publications open access paper abstract this paper attempts to identify consumers’ preferences toward red wine quality cues and willingness to pay (wtp) for wine with health-beneficial effects, thus clarifying the complex process of purchasing habits and patterns. the data were analyzed using structural equation modeling. the findings from a case study conducted in serbia show that consumer health-effect consciousness and household income are significant predictors of their wtp. on the other hand, health-conscious consumers are more inclined to intrinsic quality cues, while those who are willing to pay a higher price for a bottle of red wine are prone to extrinsic wine quality cues. keywords: consumer quality perception, red wine, structural equation modeling, willingness to pay, wine preferences introduction the choice-making of wine is a difficult and complicated process since there is a diverse and vast range of wines available in the market (agnoli et al., 2016). accordingly, numerous papers aimed to segment and cluster the wine market (caracciolo and furno, 2020; hu and ruimei, 2019), and thus indicate the importance of diverse wine quality attributes in the overall consumer preference. quality perception is seen as a mediator between product characteristics and consumer preferences (steenkamp, 1989; tomic et al., 2017). consumer judgment about an entity’s overall superiority (product, service, and process) is based on the cues of excellence (lee and hwang, 2016; snoj et al. 2004). one of the biggest challenges for businesses and wine industry is to meet consumer requirements and the need to understand their decision-making when purchasing wine. a extensive body of literature sheds light on the quality cues of wine (rodrigues and parr, 2018; sáenz-navajas et al., 2013; verdú jover et al. 2004). for example, verdú jover et al. (2004) observed consumer preferences through 15  red wine quality cues and divided them into extrinsic and intrinsic elements. intrinsic cues are defined as inherent characteristics of wine, including taste, color, acidity, level of alcohol, etc. (hu and baldin, 2018; valentin et al., 2016). on the other hand, extrinsic attributes are often associated with noninherent characteristics such as brand, price, and year of production, country of origin, grape variety, label, tradition, awards, and recommendation (boncinelli et al., 2019; lu et al., 2017; sáenz-navajas et al., 2013, 2016; williamson et al. 2016). verdú jover et al. (2004) determined seven quality dimensions that contain the majority of aspects used in the 15 initial cues: origin, image, presentation, age, harvest, sensitivity, and acuteness of bouquet. next, jaeger et al. (2009) recognized 13 intrinsic and extrinsic purchasing decision-making wine cues such as award, brand, origin, grape variety, taste, recommendation, information presented on the label, alcohol level, etc. for instance, heatherly et al. (2019) and valentin et al. (2016) emphasized color as a strong intrinsic predictor of consumer preference. however, different research studies have indicated that extrinsic cues are becoming the main determinants of wine quality (balestrini and gamble, 2006; reynolds et al., 2018). italian journal of food science, 2021; 33 (2): 1–12 mailto:milica.maricic@fon.bg.ac.rs 2 italian journal of food science, 2021; 33 (2) ruso j et al. (bisson et al., 2002; higgins and llanos, 2015; samoggia, 2016). therefore, high values of health-related substances in wine tend to lead to increased consumption of the particular wine type (fiore et al., 2019). finally, samoggia (2016) observed that consumers, who doubted the favorable effect of wine on one’s health, failed to perceive the positive relation between higher price and the health properties of wine. scientific literature has frequently examined the above-mentioned topics. to the best of our knowledge, however, few authors have jointly observed relationship between consumer consciousness of healthbeneficial effects of wine, quality cues preferences, and wtp. hence, the paper attempts to create and verify a conceptual model which explores consumers’ perception regarding the following constructs: intrinsic wine quality perception (iwqp), extrinsic wine quality perception (ewqp), health effect consciousness (hec), and wtp, while also considering monthly household income (mhi). the model tested on wine consumers in serbia aimed to improve the conceptual models on the topic devised so far and fill in the literature gaps related to the habits and patterns of consumption of red wine. serbia is one of the major producers and consumers of grapes and wines in southeast europe (radovanović et al., 2019). according to statistical report on world vitiviniculture (oiv, 2019), serbia ranks 30th in the world in terms of wine consumption with 1.1 million hectolitres. also, in 2018, serbia imported $31.6 million worth of wine (workman, 2019), that is, 0.1% of the world’s total worth. the growth of wine market in serbia thus presents a challenge for marketing and quality management professionals. proper understanding of the consumer quality perception would therefore help retailers, producers, and supplying organizations to successfully design their marketing strategies for evolving markets in order to have a significant competitive advantage. the next section of this paper provides a detailed overview of the proposed conceptual model and the related hypotheses. the results and discussion are given in the third section, while the concluding remarks are outlined in section four. methodology the proposed conceptual model in our research, we attempted to broaden some of the current conceptual models for exploring the factors that impact the consumer behavior of red wine. the proposed conceptual model consists of the following four besides quality cues related to hedonistic and social beliefs, consumer beliefs about the health benefits of wine are gradually becoming an important factor of purchase (samoggia, 2016). nowadays, health-conscious customers are looking for food and beverages that are not only nutritional but also have extraordinarily health benefits (rathi, 2018). the health effects of wine were pointed at as far back as in ancient greece and rome (fiore et al., 2019). recent research has highlighted that red wine is considered a healthy drink (chang et al., 2016; vecchio et al., 2017), because it contains ingredients that support cardiovascular, neurodegenerative, and aging health, and reduce the risk of cancer, diabetes, parkinson’s, and alzheimer’s disease (fiore et al., 2019; krstonošić et al., 2019; kuršvietienė et al., 2016; liberale et al., 2019; soares et al., 2015). nevertheless, customer opinions differ. chinese consumers tend to drink more red wine for its potential health benefits, whereas australian consumers are less likely to rate wine as a healthy product (yoo et al., 2013). the same belief about the positive health effects of wine was noticed among young adults in portugal (patrícia silva et al., 2014). however, thach and olsen (2006) revealed that a minority of youth in the united states highlighted the health-beneficial effects as the main reason for wine-consuming. conversely, agnoli et al. (2016) conclude that novice consumers fail to completely recognize the beneficial effects of wine on health (mueller and szolnoki, 2012). regarding wine purchasing reasons, bazzani et al. (2019) acknowledge that health-oriented customers prefer extrinsic cues referring to quality assurance information (marked on the label) such as hand-picked grapes, sustainable product certifications, and unfiltered wine. in the research conducted by cavaliere et al. (2016), however, customers are keener on reading nutrition information on the bottle label. furthermore, bazzani et al. (2019) and martin-moreno et al. (2013) also found out that health-conscious consumers have an aversion toward a higher percentage of alcohol content. most of the previously mentioned studies highlight red wine benefits, show that most people are aware of the curative effects of this wine, and indicate that health-oriented consumers pay more attention to information provided on label. the positive consumer perception of wine quality and willingness to pay (wtp) can be associated with health-enhancing wines (samoggia, 2016), organic wines (jorge et al., 2020), sustainable wines (sellers-rubio and nicolau-gonzalbez, 2016), or old world producer wines (giacomarra et al., 2020). researchers often point to consumers’ attitudes toward tasty, healthy, and eco-friendly food that influence their shopping behavior (jorge et al., 2020). for example, consumers in spain are willing to pay for resveratrol-enriched wine (barreiro-hurlé et al., 2008), while it was also found that health-oriented consumers are willing to pay more for health-enhancing wine italian journal of food science, 2021; 33 (2) 3 riboflavin removal by commercial bentonites and charcoals in white and red wines wtp and consumer consciousness (bisson et al., 2002; higgins and llanos, 2015; samoggia, 2016). accordingly, following is our next hypothesis: h5. consumer consciousness of red wine health effect influences consumer willingness to pay. high willingness to pay for a bottle of wine may also relate to a higher household income (hofmann et al., 2018; sogari et al., 2016). research indicates that the respondents with a higher household income are ready to pay more for a bottle of wine and turn to premium wine (camillo, 2012; onofri et al., 2015). consequently, following is the final hypothesis: h6. monthly household income influences consumer willingness to pay. the proposed conceptual model is graphically presented in figure 1. for more details on the questions used to measure each construct, see appendix 1. data analysis to test the validity of the proposed conceptual model, we opted for the structural equation modeling (sem). the sem analysis is a multivariate statistical analysis based on the principles of factor analysis and regression or path analysis (hox and bechger, 1998; kline, 2005). on one side, sem reduces the dimensionality of the observed phenomenon, while on the other it provides insights on the relationship between the newly formed latent variables or constructs. taking into account the benefits of the sem analysis and the clear theoretical concept it is based on, this analysis has become a vastly applied approach for representing dependency in multivariate data (kline, 2005). so far, the sem analysis has been conducted in the research field of wine consumption. for example, vilela et al. (2018) used sem to explore how the sensory profile of the respondent impacted his/her observation of the wine aroma, mouth feel, and flavor for three different types of portuguese wine. pestar bizjak et al. (2018) observed how respondents from two slovenian wine regions perceived the value of wine through emotional-social dimension, quality-price, and terroir and the impact of regiocentrism. further, bianchi (2015) proposed a conceptual model of consumer brand loyalty based on wine knowledge, wine experience, wine brand satisfaction, wine brand trust, and wine brand loyalty, and tested this in chile. the survey inspiration for the items of a questionnaire came from bruwer et al. (2002); cholette and castaldi (2005); constructs: hec, ewqp, iwqp, and wtp, and also takes into account mhi. hec construct points to the degree of consumer awareness expressed through the attitudinal questions on health issues and the impact of red wine on human health (appendix 1). another construct, ewqp, indicates the level of importance of extrinsic quality in consumer’s wine purchasing decision, including tradition, price, awards, recommendation, grape variety, brand, label, year of production, and country of origin. by investigating purchase intention, huang et al. (2018) stated that wine lovers prefer to use extrinsic characteristics. so, the first hypothesis we test in our study is as follows: h1. consumer consciousness of red wine health effect influences extrinsic wine quality perception. the third construct, iwqp, points to the degree of importance of intrinsic quality in consumer wine purchasing decision such as flavor, health ingredients, wine color, additives, percentage of alcohol, and acidity. according to bazzani et al. (2019), cavaliere et al. (2016), and martin-moreno et al. (2013), consumers who are conscious of the positive effects of wine on their health are more inclined to pay attention to intrinsic cues. therefore, the next hypothesis we propose is as follows: h2. consumer consciousness of red wine health effect influences intrinsic wine quality perception. the fourth construct, wtp, is determined by questions linked to the amount of money buyers are willing to spend on a bottle of red wine. some of the researchers observed the effects of extrinsic cues on the willingness to pay (lee et al., 2018; nowak et al., 2006; veale and quester, 2009). for instance, nowak et al. (2006) indicate that consumers are willing to pay more for a famous wine brand. hence, following is our third hypothesis: h3. consumer willingness to pay influences extrinsic wine quality perception. generally, some studies have found that consumers are more willing to pay for intrinsic quality cues such as nutritional contents or freshness (balineau, 2018). the same conclusion is observed in a study conducted by barreiro-hurlé et al. (2008) and gabrielyan et al. (2014). our next hypothesis is as follows: h4. consumer willingness to pay influences intrinsic wine quality perception. even though some studies failed to find a significant relationship between health attitudes and willingness to buy, most authors discovered a positive relationship between 4 italian journal of food science, 2021; 33 (2) ruso j et al. results and discussion sample characteristics a total of 605 responses were collected after closing the questionnaire. in order to obtain a sample of true wine consumers, we asked respondents whether they declare themselves as red wine consumers in the first place. in reply, 496 respondents (81.9%) declared themselves as consumers of red wine, while the remaining 109 (18.1%) responded in negative. the non-consumers of red wine were not eligible for our research, so their answers were removed from further analysis. the sample consisted of 312 female respondents, who formed 62.9% of the sample, and 184 were male respondents (37.1%). a slight disproportion in the respondents’ gender was observed, but the same was also found in the work of bruwer (2004). most of the respondents (64.7%) are in the age group 18–32 years, followed by the age group 33–45 years (24.8%). the remaining respondents were aged more than 46 years. this indicates that our sample comprised younger population, namely genxers and millennials. it is expected that genxers would become the largest consumers of wine in 2021, while millennials would take over in 2026 (mcmillan, 2018). accordingly, it johnson et al. (2017); keller (2009); lim et al. (2013); sjostrom et al. (2016); werdelmann (2014); and yoo et al. (2013). the questionnaire was distributed online between september 2019 and january 2020. some of the questions were adopted for the purpose of the study, while some scales were used in the original form. all the questions regarding respondents’ opinions on red wine cues were measured on a 5-point likert scale ranging from 1 (strongly disagree) to 5 (strongly agree). for more details on the questions used to measure each construct, and the obtained mean values and standard deviations (sd), see appendix 1. the questionnaire was distributed on facebook groups related to wine consumption and on linkedin. the questionnaire comprised the following five groups of questions: demographic information, habits of red wine consumption, health characteristics, price, and quality perception. in the first section of the questionnaire, the respondents were asked some basic demographic information regarding gender, age, residence, highest completed degree, and household income. the rest of the questions were related to the frequency, quantity, occasions and place of consumption, and purchase of red wine, its characteristics, price, health effects, and perceptions of respondents about red wine consumption. afterwards, statistical analysis using spss 25 and amos 22 was performed. iwqp1 iwqp6 iwqp ewqp ewqp1 ... ewqp9 hec wtp mhi h6 h3 h4 h1 h2 h5 wtp1 wtp3 hec1 hec10 figure 1. conceptual model. italian journal of food science, 2021; 33 (2) 5 riboflavin removal by commercial bentonites and charcoals in white and red wines is reasonable to focus on all these segments. similar age distribution of respondents within a sample was found in wine consumption research conducted by bruwer (2004) and bruwer et al. (2012). respondents mainly came from serbia (73.2%), followed by consumers from other countries of the region (montenegro, bosnia and herzegovina, and croatia). taking a look at the highest educational degree, 51.0% of the respondents had a bachelor’s degree, 22.4% had a master’s degree, 15.7% completed high school, while 10.9% had phd. we observed that the sample consisted of highly educated individuals, with 60.0% of surveyed millennials having at least a bachelor of science (bsc). this indicated that in this regard the sample was unbalanced. we believe that this occurred due to the sampling method used. although this might be a limitation of the study, we believe that it would not distort the conclusions as we were observing health enhanced wine which was more expensive and regarded as premium wine, which respondents with lower educational attainment and income did not often consume. most respondents earned a monthly income between €1000 and €2000 (36.1%), 35.1% earned income below €1000, while the rest had a monthly income of above €2000. it was concluded that we covered a segment of the population which was young, educated, and had more than serbia’s average monthly income. sample characteristics are provided in table 1. to provide additional insights on the consumers’ attitudes toward the four explored constructs of the proposed model, we provide mean value and sd of each item of the constructs in appendix 1. furthermore, we aimed to obtain insight on the respondents’ attitudes and habits regarding consumption of red wine. most of the respondents (32.7%) were consuming red wine for 1–5 years, followed by those who had been consuming for 6–10 years (28.8%). as for frequency of consumption of red wine, most of the respondents consumed it once every 2 months (38.3%), followed by those who consumed it on a monthly basis (19.4%) or once in a fortnight (16.3%). as for purchasing habits, most respondents (32.3%) consumed one to three bottles per year, followed by those who consumed over 10 bottles (27.4%). on the other hand, 39.3% of the respondents buy one to three bottles a year as a gift. half of the respondents (50.8%) believed that the reasonable price of a red wine bottle is between €5 and €10, while 27.6% were ready to pay between €10 and €40. this suggests that a large percentage of our respondents had multiple years of moderate consumption and purchasing habits and were ready to pay for a good bottle of wine. sem model the primary step in sem analysis is to observe the internal consistency of the proposed latent variables. the table 1. characteristics of the respondents who participated in the survey. variable frequency proportion gender female 312 62.9% male 184 37.1% age group 18–32 321 64.7% 33–45 123 24.8% 46–64 48 9.7% 65–75 4 0.8% educational attainment high school 92 15.7% bsc 253 51.0% msc 111 24.2% phd 54 10.9% monthly household income less than €1000 174 35.1% €1000–2000 179 36.1% €2000–3000 51 10.2% €3000–4000 34 6.9% €4000–5000 17 3.4% over €5000 41 8.3% most commonly used metric for internal consistency and scale reliability is cronbach’s alpha (cronbach, 1951). the cronbach’s alpha provides a metric level up to which all the measured variables in a latent construct measure the same concept, and it takes values from 0 to 1. the closer the cronbach’s alpha is to 1, the higher the internal consistency (peterson, 1994). as reported by relevant literature, the acceptable levels of cronbach’s alpha are in the range of 0.70–0.95 (tavakol and dennick, 2011). besides cronbach’s alpha, average variance extracted (ave) and construct reliability are used (fornell and larcker, 1981). the closer these indices are to 1, the better the internal consistency; thus, this shows that the scale is more reliable. the threshold for the acceptable level for ave is above 0.5, while for composite reliability, it is above 0.7 (fornell and larcker, 1981; wong, 2013). the calculated cronbach’s alpha per latent variable and the number of items per scale are given in table 2. as presented, the internal consistency ranges from 0.751 (wtp) to 0.984 (hec). the composite reliability for all constructs is at the threshold of 0.7 or above. however, when it comes to ave, ewqp’s ave is less than the threshold (0.447). having in mind the obtained metrics of reliability and validity, we can conclude that the data are suitable for sem analysis. additionally, we explored the normality of variables in the model, having in mind that the 6 italian journal of food science, 2021; 33 (2) ruso j et al. concerning the construct iwqp, it initially had two predictors, hec and wtp. however, wtp proved to be statistically insignificant, which would mean that hypothesis h4 was not confirmed. the obtained standardized coefficient of the impact of hec on iwqp was 0.776, with c.r. = 12.888, indicating that hec significantly impacted iwqp and the impact was positive and medium in strength, taking into account that the standardized coefficient could take a value of 0–1. this finding confirmed the assumption h2. the r2 of this construct was 0.601, meaning that one predictor explained 60.1% of the variability of iwqp, thus creating a solid model. these results showed that more health-conscious consumers valued more the intrinsic characteristic of wine quality (focusing on flavor, healthy ingredients, color, percentage of alcohol, additives, and acidity). the results were in accordance with the research done by bazzani et al. (2019) and martin-moreno et al. (2013). the obtained result might be due to the fact that health-oriented consumers were more likely to undertake actions and behaviors that could contribute to their health improvement and thus pay more attention to the intrinsic quality of wine cues (cavaliere et al., 2014). similar to iwqp, ewqp also initially had two predictors, hec and wtp. in this case, hec proved to be statistically insignificant, thus hypothesis h1 was not proved. non-normality might significantly impact the result of analysis. according to muthén and kaplan (1985), ‘if most variables have univariate skewness and kurtosis in the range –1.0 to +1.0, not much distortion is to be expected’. in our sample, out of 28 variables in the model, six proved to have issues with skewness and kurtosis in the range from –2 to 3. taking into account that just several variables expressed skewness and kurtosis out of the suggested range, we continued with the analysis (hallow, 1985). the initial model had relatively poor fit to the data (chisquare = 2643.028, df = 372, p < 0.000, root mean square error of approximation (rmsea) = 0.111, comparative fit index (cfi) = 0.862, tucker-lewis index (tli) = 0.849). to evaluate the significance of the paths and indicators, we used critical ratios (c.r.). the value of c.r. above 1.96 or below –1.96 points out a two-sided significance at the 5% level (hox and bechger, 1998). paths between hec and ewqp and between iwqp and wtp had a c.r. below the defined threshold, so they were removed from the model. additionally, we used modification indices to fine-tune our model. the final model had relatively good fit to the data (chi-square = 1721.177, df = 328, p < 0.000, rmsea = 0.092, cfi = 0.915, tli = 0.895). the detailed model assessment is given in table 3. table 2. obtained cronbach’s alpha, average variance extracted (ave), and composite reliability per construct and the number of items per construct. hec iwqp ewqp wtp no. of items 10 6 9 3 cronbach’s alpha 0.984 0.862 0.886 0.751 ave 0.501 0.470 0.447 0.484 composite reliability 0.904 0.841 0.876 0.697 note: hec health effect consciousness; iwqp intrinsic wine quality perception; ewqp extrinsic wine quality perception; wtp willingness to pay table 3. assessment of the model: construct, predictors, obtained unstandardized and standardized coefficients, c.r., r2, and the decision on the related hypothesis. construct predictors unstd. coeff. std. coeff. c.r. r2 hypothesis iwqp hec 0.203 0.776 12.880 0.601 h2—approved wtp not significant h4—rejected ewqp hec not significant 0.613 h1—rejected wtp 0.542 0.783 16.449 h3—approved wtp hec 0.710 0.975 33.908 0.998 h5—approved mhi 0.124 0.221 13.202 h6—approved note: c.r. – critical ratio, r2 – r square, hec health effect consciousness; iwqp intrinsic wine quality perception; ewqp extrinsic wine quality perception; wtp willingness to pay; mhi monthly household income italian journal of food science, 2021; 33 (2) 7 riboflavin removal by commercial bentonites and charcoals in white and red wines other research on wine consumption (bisson et al., 2002; higgins and llanos, 2015; samoggia, 2016), the findings indicated that health-conscious consumers were willing to pay more for a bottle of red wine as well as for health-enhancing wine. in corroboration of the previous claims made by hofmann et al. (2018), hu and ruimei (2019), onofri et al. (2015), and sogari et al. (2016), results included the observation that respondents with higher mhi seemed to have higher wtp. in other words, people who earned more were willing to pay more for a bottle of red wine. hence, we can say that hypotheses h5 and h6 were proved. the graphical interpretation of the results and the final model are given in figure 2. conclusion the way a user views the quality of a product is closely related to his/her needs and requirements. these needs include not only the physical benefit of the product but also the one that relates to the image a user creates of himself/herself for the use of a specific brand and all other aspects of the benefits of the delivered product that are considered valuable. furthermore, a user’s perception the obtained standardized coefficient of the impact of wtp on ewqp was 0.783 with c.r. = 16.449, indicating that wtp significantly impacted ewqp statistically and that the impact was positive and medium in strength. the r2 of this construct was 0.613, meaning that one predictor explained 61.3% of the variability of ewqp, thus creating a solid model. the results suggested that consumers who were more willing to pay a higher price for a bottle of red wine and its health effect were more inclined to extrinsic wine cues such as price, year of production, country of origin, brand image, grape variety, label, tradition, recommendation, and award. correspondingly, hypothesis h3 hypothesis, about a positive impact of wtp on ewqp, was also confirmed. a similar relation was observed by lee et al. (2018) and veale and quester (2009). the construct wtp had two predictors, hec and mhi, and both proved to be statistically significant. in the final model, hec and mhi had a statistically significant positive impact with respective standardized coefficients of 0.975 and 0.221. mhi was left in the model, although its impact was weak as it was statistically significant and the goal was to obtain a good measurement model (allen et al., 2019; milenković et al., 2019). the r2 of this construct was 99.8%, indicating that the two predictors explained a high percentage of variability of wtp. as in iwqp6iwqp1 iwqp ewqp ewqp1 h3 (0.783*) h2 (0.776**) h1 (0.975**) h6 (0.221**) ewqp9 hec wtp mhi wtp1 wtp3 hec1 hec10 ... figure 2. final conceptual model. **p < 0.01. 8 italian journal of food science, 2021; 33 (2) ruso j et al. comparative analysis, thus significantly improving the quality of the result. also, another possible direction of the study could be the extension of the conceptual model by including new constructs such as wine knowledge, wine experience, or purchase intention (bianchi, 2015). the paper provided important information for policy makers to identify barriers, develop rules, policies, and initiatives, as well as labeling schemes to engage consumers in sustainable consumption of wines with health benefits. our findings could benefit companies, wineries, and managers who plan to enter the growing wine market in serbia and the balkans. we believe that the proposed conceptual model for exploring the relationship between quality perceptions and willingness to pay would initiate further research on both factors that affected consumer decision-making process when purchasing red wine and the future improvements of the conceptual models regarding this topic. acknowledgment this paper presented research funded by the innovation fund from the budget of the republic of serbia from the division of the ministry of education, science and technological development, through the serbia competitiveness and jobs project (loan agreement with the world bank), grant no. 50138. conflict of interest there are no conflicts of interest. the authors of this research strictly followed all ethical guidelines provided by the university of belgrade. references agnoli, l., capitello, r. and begalli, d. 2016. behind intention and behaviour: factors influencing wine consumption in a novice market. brit food j. 118:660–678. https://doi.org/10.1108/ bfj-05-2015-0181 allen, j., eboli, l., forciniti, c., mazzulla, g. and ortúzar, j. de d. 2019. the role of critical incidents and involvement in transit satisfaction and loyalty. transp policy. 75:57–69. https://doi. org/10.1016/j.tranpol.2019.01.005 balestrini, p. and gamble, p. 2006. country-of-origin effects on chinese wine consumers. brit food j. 108:396–412. https://doi. org/10.1108/00070700610661367 balineau, g. 2018. engel curves for fair trade consumption and development perspectives for producers: evidence from data on real fairtrade purchases. j dev stud. 55:894-916. https://doi.org/ 10.1080/00220388.2018.1499894 barreiro-hurlé, j., colombo, s. and cantos-villar, e. 2008. is there a market for functional wines? 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(2010) health effects consciousness item mean sd source 4. red wine can slow down the aging process and extend human life 3.33 2.024 yoo et al. (2013) 5. red wine can reduce the risk of certain diseases (cardiovascular and metabolic diseases and cancer) 3.52 2.047 6. red wine can cure certain diseases 2.61 2.130 7. i think, red wine is a healthy alcoholic beverage 3.45 2.221 8. red wine has better health properties than other alcoholic beverage 3.67 2.227 9. it is important to limit the amount of alcohol you consume 4.35 2.258 10. i understand how much alcohol is considered healthy 3.66 2.335 11. i know what moderate drinking is 4.22 2.430 12. red wine has more health-enhancing properties 3.56 2.453 13. i would drink more red wine if i thought it was healthy for me 3.24 2.574 intrinsic wine quality perception item mean sd source 14. flavor 4.43 1.104 bruwer et al. (2002); werdelmann (2014); yoo et al. (2013) 15. health ingredients 3.51 1.239 16. color of wine 3.47 1.391 17. alcohol (%) 3.15 1.461 18. additives 3.08 1.569 19. acidity 3.43 1.540 extrinsic wine quality perception item mean sd source 20. price 3.80 1.116 bruwer et al. (2002); werdelmann (2014); yoo et al. (2013) 21. year of production 3.05 1.268 22. country of origin 3.17 1.342 23. brand image 2.93 1.328 24. grape variety 3.28 1.414 25. label 2.91 1.159 26. tradition 3.21 1.568 27. recommendation 3.95 1.500 28. awards 3.08 1.627 _hlk59434284 _hlk59433553 _hlk59450899 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (4): 107–117 issn 1120-1770 online, doi 10.15586/ijfs.v33i4.2123 107 p u b l i c a t i o n s codon goldenberry (physalis peruviana l.) seed oil: press extraction, optimization, characterization, and oxidative stability pedro p. ugarte-espinoza1, victor delgado-soriano1, lorenzo estivi2, alyssa hidalgo2*, gloria pascual-chagman1 1departamento de tecnología de alimentos y productos agropecuarios, facultad de industrias alimentarias, av. la universidad s/n. la molina, universidad nacional agraria la molina (unalm), lima, perú; 2department of food, environmental and nutritional sciences (defens), università degli studi di milano, milan, italy *corresponding author: alyssa hidalgo, department of food, environmental and nutritional sciences (defens), università degli studi di milano, italy. email: alyssa.hidalgovidal@unimi.it received: 18 october 2021; accepted: 10 december 2021; published 26 december 2021 © 2021 codon publications open access paper abstract in order to optimize the screw-press extraction conditions of oil from goldenberry (physalis peruviana l.) seeds obtained from nectar processing waste, a face centered design was applied. the oil was extracted at different temperatures (60, 80, and 100°c) and seed moisture contents (8, 10, and 12%). oil recovery (or) increased and residual oil in the cake decreased significantly as moisture content and temperature were reduced; oil moisture and volatile matter as well as acid value, k232, k268, and p-anisidine, respectively, decreased proportionally with the moisture extraction. thus, the highest or (86.4%) and the best quality were obtained at 8% moisture content and 60°c pressing temperature. under these conditions, the extracted oil presented high linoleic acid (76.0%), iodine value (140.0 mg i2/g), and refractive index (1.4769). the oil stability index, measured by rancimat, varied from 3.65 h (120°c) to 14.87 h (100°c); the predicted shelf life at 25°c was 120.4 days and the activation energy was 85.6 kj/mol. the results highlighted that screw-pressing of goldenberry seeds provides quality oil without employing polluting and hazardous solvents. keywords: cape gooseberry; expeller; oil recovery; oxidation kinetics; rancimat; shelf life introduction physalis peruviana l., commonly known as goldenberry or cape gooseberry, is a perennial herb native of the andean highlands belonging to the solanaceae family. p. peruviana has become one of the most promising tropical fruits and has received growing interest from all over the world due to its potential as an intensive crop with a high content of bioactive compounds (etzbach et al., 2018; ramadan, 2020). the fruit when fresh is used as decoration in meals, salads, and desserts; when processed is used in sauces, jam, syrup, and yogurt (chasquibol silva and yácono llanos, 2015; ramadan and mörsel, 2003); and when dehydrated is used in baked foods, cocktails, snacks, and cereal breakfast (vásquez-parra et al., 2013). the food industry produces juices and nectars from goldenberry pulp, discarding seeds and peels (ca. 27% fruit fresh weight, ramadan, 2020) as by-products. the amount of seeds compared to the fresh fruit weight is rather variable: ramadan and mörsel (2003) reported that seeds constituted 17% of the fruit’s weight, whereas popova et al. (2020) found this to be 7.3 and 11.5% in two different genotypes. the seeds are a potential raw material for oil production because of their high oil content (aslanov et al., 1995; chasquibol silva and yácono llanos, 2015; popova et al., 2020) and high nutritional value. in addition, mailto:alyssa.hidalgovidal@unimi.it 108 italian journal of food science, 2021; 33 (4) ugarte-espinoza pp et al. k268), and p-anisidine value (p-av). the experiments were carried out following a face centered design (fcd), considering temperature (−1 = 60°c, +1 = 100°c) and moisture (−1 = 8%, +1 = 12%) as independent variables. the fcd was performed considering three central points, with a total of 11 runs according to table 1. to avoid systematic errors, the experiments were performed randomly. oil extraction the seeds were hydrated with distilled water until moisture levels of 8, 10, and 12%, according to the methodology indicated by singh and bargale (2000). the hydrated seeds were packed in hermetic containers and stored at room temperature for approximately 48 h to reach equilibrium. the containers were shaken at regular intervals to distribute the moisture evenly throughout the seeds. the amount of water necessary for hydration was determined by applying the following formula (mridula et  al., 2019): 1 0w s 1 (h h ) m m 100 h − = − , where mw is the mass of water to be added (g), ms is the mass of seeds to be hydrated (g), h0 and h1 are, respectively, the initial and the final moisture content (wb) of the seeds. the seeds were pressed at 60, 80, and 100°c, using a komet screw press (ca 59 g, ibg monforts, germany), at a screw speed of 15 rpm and a nozzle diameter of 4 mm. before introducing the seeds into the feed hopper, the press was operated for 15 min with heating through the electric resistance ring fixed around the press head to raise the temperature of the cylinder to the selected temperature. after each run, all press devices were cleaned and dried. the oils obtained were centrifuged (rotofix 32a, hettich, germany) at 2,701 g for 30 min and subsequently stored in amber bottles at 4°c until analysis. the cakes obtained were stored at 4°c in hermetic low-density polyethylene bags until analysis. analyses all the following determinations were performed in triplicate. seed characterization the moisture, crude fat, ash, crude fiber, and crude protein (n × 6.25) of the seeds were determined following the aoac methods, 935.29, 945.16, 950.49, 962.09, and 950.48 (aoac international, 2016), respectively. the total carbohydrate content was determined by difference, i.e. by subtracting all the above mentioned compounds from the total. they are an important source of linoleic acid (omega 6) and vitamins a, e, and k (chasquibol silva and yácono llanos, 2015; ramadan and mörsel, 2003), as well as of phenolic compounds. furthermore, no adverse effects or toxicity are reported (nocetti et al., 2020). in previous studies, oil from goldenberry pomace (seeds, peels, and pulp remnants) was obtained by aqueous enzymatic extraction and solvent extraction (mokhtar et al., 2018; ramadan et al., 2008; ramadan and moersel, 2009). however, these methods have certain disadvantages related to performance, economy, and safety. for instance, the aqueous enzymatic extraction has low yields and high costs of enzymes (mwaurah et al., 2020). on the other hand, solvent extraction requires expensive facilities and equipment, and has the risk of fire and explosion associated with the flammable nature of solvents (deli et al., 2011). they are also harmful to both human health and environment as pollutants (mwaurah et al., 2020). hence, mechanical extraction using a screw press is a cheaper, safer, and simpler alternative. although goldenberry seed oil (gso) has already been extracted using this method (chasquibol silva and yácono llanos, 2015), the effect of extraction conditions on oil yield and quality was not investigated. likewise, no characterization of gso oxidation kinetic or shelf-life prediction by rancimat is available in literature. therefore, the objective of this research was to optimize the screw-press extraction process of oil from goldenberry seeds, as well as to characterize and evaluate the oxidative stability by rancimat of the oil obtained at the best extraction conditions. materials and methods raw material the goldenberry pomace (peel, seed, and pulp remains) obtained during the nectar production process at the agroindustrial development institute-indda (limaperu) was used. after removing the peels and pulp residual by washing with water, the seeds were dried at 50°c for 17 h, sieved, and stored at 4°c in hermetic lowdensity polyethylene bags until the extraction trials. optimization trials for oil screw-press extraction response surface methodology was used to evaluate the effect of different extraction conditions on the response variables, namely, oil recovery (or), residual oil (ro), oil moisture and volatile matter, acid value (av), peroxide value (pv), specific extinction at 232 and 268 nm (k232 and italian journal of food science, 2021; 33 (4) 109 goldenberry (physalis peruviana l.) seed oil by gas chromatography as described by rodríguez et al. (2021). evaluation of oil oxidative stability (osi) rancimat test the osi of each oil and of the blends were evaluated by the method aocs cd 12b-92 (aocs, 1998) using a 743 rancimat equipment (metrohm schweiz ag, zofigen, switzerland). the assays were carried out using 3.0 ± 0.1 g of oil sample with an air flow of 20 l/h at 100, 110, and 120°c. oil shelf life the prediction of shelf life was determined by extrapolation of the linear correlation of the logarithm of osi vs temperature (t) for a temperature of 25°c, as described by heidarpour and farhoosh (2018): log osi = at + b, where a and b represent the slope and intercept, respectively. oxidation kinetics the reaction rate constant (k) was calculated as the reciprocal of osi (k = 1/osi), as indicated by aktar and adal (2019). the temperature coefficient (q10), which indicates the increase in reaction rate due to a 10°c rise in temperature, was calculated according to symoniuk et al. (2017): q10 = (k2/k1) 10/(t2–t1). the relationship between k and temperature was defined by the arrhenius equation: ln k = ln a – ea/rt, where a is the frequency factor (h−1), ea is the activation energy (kj/mol), r is the universal gas oil recovery (or) and residual oil (ro) the oil recovery (or) of the oil extracted was calculated using the formula indicated by mridula et al. (2019): ( )   = −    oil content on cake (g) or % 1 x 100 oil content in seeds (g) the oil content in the seeds before pressing extraction and in the cake was assessed following method 945.16 (aoac international, 2016). the residual oil (ro) was determined from the oil content in the cake after pressing. physicochemical analyses of the oils moisture and volatile matter, acid value (av), peroxide value (pv), refractive index at 20°c, p-anisidine value (p-av), iodine value (iv), saponification value (sv), and unsaponificable matter were determined following the methods ca 2d-25, ca 5a-40, cd 8-53, cc 7-25, cd 18–90, cd 1d-92, cd 3-25, and ca-40 (aocs, 1998), respectively. specific extinction at 232 and 268 nm (k232 and k268) was determined following the method, iso 3656 (iso, 2011). the fatty acids’ (fa) composition was determined as fatty acid methyl esters (fame) by gas chromatography after transesterification of the oils with 2 n koh in methanol, according to iupac standard method 2.302 (iupac, 1987). the fatty acid profile was determined table 1. experimental design and average results for oil recovery (%), residual oil (% dm), moisture and volatile matter (%), acid value (mg koh/g), p-anisidine value, extinction coefficients k 232 , k 268 of goldenberry seed oil obtained at different press extraction conditions (temperature, °c; moisture, g/100 g). standard order independent variables response variables temperature moisture oil recovery residual oil moisture and volatile matter acid value p-anisidine k 232 k 268 1 60 (−1) 8 (−1) 86.43a 6.62h 0.072d 0.237c 0.72d 1.33e 0.17e 2 100 (1) 8 (−1) 65.46d 15.66e 0.075d 0.234c 0.73d 1.36de 0.19de 3 60 (−1) 12 (1) 56.15f 18.57d 0.097a 0.360ab 1.00b 1.49a 0.25a 4 100 (1) 12 (1) 34.98i 25.26a 0.097a 0.382a 1.04a 1.50a 0.26a 5 60 (−1) 10 (0) 78.27b 10.01g 0.087b 0.350ab 0.83c 1.40bc 0.21c 6 100 (1) 10 (0) 48.95g 21.01c 0.087b 0.373a 0.84c 1.42b 0.22b 7 80 (0) 8 (−1) 73.03c 13.13f 0.075d 0.236c 0.73d 1.34e 0.19de 8 80 (0) 12 (1) 43.51h 22.45b 0.097a 0.361ab 0.99b 1.49a 0.26a 9 80 (0) 10 (0) 57.35e 18.46d 0.086bc 0.338b 0.84c 1.41b 0.21bc 10 80 (0) 10 (0) 56.42f 18.82d 0.088b 0.369ab 0.84c 1.38cd 0.20cd 11 80 (0) 10 (0) 56.83ef 18.79d 0.084c 0.371ab 0.84c 1.40bc 0.20cd different letters in the same column indicate significant differences (p ≤ 0.05) among trials following the lsd test. 110 italian journal of food science, 2021; 33 (4) ugarte-espinoza pp et al. g/100 g dm ash, 32.48 ± 0.40 g/100 g dm crude fiber, and 16.98 ± 0.04 g/100 g dm total carbohydrates. similar protein, ash, and fiber levels were reported in the seed cake by chasquibol silva and yácono llanos (2015). optimization trials for oil screw-press extraction table 1 reports the results of the oil extraction trials, while figure 1 shows the response surface plots. the anova (table 2) highlighted significant effects of temperature and moisture on oil recovery and residual oil in the cake; the moisture presented the highest influence. while oil recovery showed a linear behavior, the residual oil showed a quadratic effect of temperature. even if the lack-of-fit for both variables was significant, the adjusted-r2 and predicted-r2 were very high. during the extraction, the oil quality parameters were mainly influenced by the seed moisture and by its quadratic term for acid value and p-anisidine. all these models showed nonsignificant lack of fit and high r2. the or increased and the residual oil decreased significantly as the moisture content of the seeds and the pressing temperature decreased from 12 to 8% and from 100 to 60°c, respectively (figure 1). similar trends were reported by mridula et al. (2015) and singh et al. (2002) during the screw-pressing oil extraction from, respectively, linseed (moisture 6–10% wb; 50–90°c) and crambe seeds (moisture 3.6–9.2% dm; 120°c). similarly, silvia et al. (2012) observed an increase of or from nigella seeds with a pressing temperature decrease from 100 to 50°c. according to martínez et al. (2017) and constant (8.314 j/mol k), and t is the absolute temperature (k). statistical analysis the analyses of variance (anova) of data and construction of response surface plots were performed using design expert software v.12 (stat-ease inc., minneapolis, usa). before the anova, normal data distribution was verified. the data were also processed by one-way anova; when significant differences at p ≤ 0.05 were found, fisher’s least significant difference (lsd) at p ≤ 0.05 was determined. these statistical analyses were performed with the statgraphics® centurion xvi program (statpoint technologies, usa). the data are presented as mean ± standard deviation (sd) of three replicates, computed using the software excel (microsoft® office excel 2016). results and discussion seed composition the proximate chemical composition of the goldenberry seeds was 33.63 ± 0.03 g/100 g dry matter for crude fat, similar to the level (32.7 g/100 g, i.e., 18.09 g/100 g extracted oil + 14.63 g/100 g in the cake) observed by chasquibol silva and yácono llanos (2015), but much higher than the value (18 g/100 g) reported by aslanov et al. (1995) in seeds from azerbaijan. the seeds also contained 14.46 ± 0.05 g/100 g dm crude protein, 2.45 ± 0.01 90 (a) 80 70 60 50 40 30 12 11 o il re co ve ry ( % ) 10 b: moisture a: temperature 9 8 60 70 80 90 100 0.40 0.35 0.30 0.25 0.20 (d) 12 11 a ci d va lu e (m g k o h /g ) 10 b: moisture a: temperature 9 8 60 70 80 90 100 0.7 0.8 0.9 1.0 1.1 (e) 12 11 pan is id in e va lu e 10 b: moisture a: temperature 9 8 60 70 80 90 100 1.30 1.35 1.40 1.45 1.50 1.55 (f) 12 11 k 23 2 10 b: moisture a: temperature 9 8 60 70 80 90 100 0.16 0.18 0.20 0.22 0.24 0.26 (g) 12 11 k 26 8 10 b: moisture a: temperature 9 8 60 70 80 90 100 30 (b) 25 15 20 10 5 12 11 r es id ua l o il (% ) 10 b: moisture a: temperature 9 8 60 70 80 90 100 (c) 0.070 0.075 0.080 0.085 0.090 0.095 0.100 12 11m oi st ur e an d vo la til e m at te r (% ) 10 b: moisture a: temperature 9 8 60 70 80 90 100 figure 1. response surface plots for oil recovery (a), residual oil in the cake (b), and physicochemical quality (moisture and volatile matter, c; acid value, d; p-anisidine value, e; k 232 , f; k 268 , g) of goldenberry seed oil as a function of moisture and temperature of press extraction. italian journal of food science, 2021; 33 (4) 111 goldenberry (physalis peruviana l.) seed oil oxidation of linolenic acid as well as from dehydration of hydroxy-linoleate or -linolenate (frankel, 2012). the k232 and k268 ranged from 1.33 to 1.50 and 0.17 to 0.26, respectively (table 1). these values are in contrast with those of ramadan et al. (2008), who reported k232 = 0.57 and k268 = 1.02–1.12 in goldenberry pomace oil processed by enzyme-aided aqueous and solvent extractions. however, the oil obtained by ramadan et al. (2008) appeared to contain secondary oxidation products. in fact, as reported by spatari et al. (2017) for several edible oils (soybean, olive, corn, linseed, sunflower, and peanut), uv absorbance spectra are always higher around 230 nm than around 270 nm, and the primary oxidation mainly affects the absorbance in the former region. the p-avs were in the range of 0.72 and 1.04, which is below the maximum limit (10.0) indicated by matthäus (2010) for refined oils. the p-anisidine test detects high molecular weight carbonyl compounds (frankel, 2012), thus a low value indicates that the oil is not in an advanced stage of oxidation, thereby supporting the fact that the applied treatments did not induce oil oxidation. the values of all the physicochemical parameters decreased with the reduction of seeds’ moisture from 12 to 8% except for av, whose values remained almost constant between 12 and 10% and decreased from 10 to 8% following a quadratic relation. in general, this parameter was not influenced by the pressing temperature (table 2). lipase, lipoxygenase, and phospholipase are activated at high moisture (>10%; gupta, 2002): this may explain why a predominant moisture effect on oil degradation indicators was observed. besides, lipase exhibits hydrolytic activity only if sufficient water is available both as savoire  et  al.  (2012), both high moisture content and high pressing temperature may result in poor or due to excessive softening of the tissues, which makes seeds to stick and reduces friction. the moisture and volatile matter were, respectively, in the range of 0.072 and 0.097%, below the maximum limit (0.2%) established by the codex alimentarius (1999a) for vegetable oils. the avs were in the range of 0.234 and 0.382 mg koh/g, below the maximum limit established by the codex alimentarius (1999a) for cold-pressed oils (4 mg koh/g) and refined oils (0.6 mg koh/g). we obtained quite low av when compared to mokhtar et al. (2018; 2.36 mg koh/g), indicating that the applied treatments did not greatly increase the hydrolysis of fatty acids. in fact, unlike mokhtar et al. (2018), in the present study, the seeds were separated from the pomace before grinding, probably reducing the contact between the oil and the endogenous hydrolytic enzymes. the hydroperoxides were below the detection limit (0.5 meq o2/kg; frankel, 2012), suggesting that the treatments did not accelerate the oxidative process, likely due to the presence of natural antioxidants that may have delayed it. in fact, gso is extremely rich in total tocopherols (2400–5100 mg/kg), with β>γ-δ>α (popova et al., 2020, 2021; ramadan et al., 2008). specific extinctions in uv (k232 and k268) are highly sensitive indices that measure the extent of oil oxidation. conjugated dienes are detected at 232 (or 234) nm and derive from primary oxidation of linoleic acid, following the same trend of peroxides. conjugated trienes are detected at 268 (or 270) nm and derive from primary table 2. analysis of variance (mean square and significance) for oil recovery, residual oil in the cake, and physicochemical quality of goldenberry seed oil. source df oil recovery residual oil moisture and volatile matter k 232 k 268 df acid value p-anisidine a-temperature 1 850.9*** 119.2*** 1.4 × 10−6 0.0004 0.00023 1 0.00029 0.00059* b-moisture 1 1358.1*** 158.9*** 0.00076*** 0.035*** 0.00737*** 1 0.026*** 0.12*** ab 1.37 1 0.00015 0.00023 a² 11.95* 1 0.00004 0.00006 b² 0.03 1 0.009*** 0.00205** residual 8 10.8 1.27 2.3 × 10−6 0.00018 0.00005 5 0.00016 0.00009 lack of fit 6 14.4* 2.10* 1.6 × 10−6 0.00018 0.00007 3 0.00003 0.00015 pure error 2 0.22 0.04 4.1 × 10−6 0.00019 0.00001 2 0.00035 2.7 × 10−6 r2 0.96 0.98 0.98 0.96 0.95 0.98 1.00 adj-r2 0.95 0.96 0.97 0.95 0.93 0.96 1.00 pred-r2 0.93 0.81 0.96 0.93 0.92 0.94 0.96 c.v. % 5.50 5.58 1.75 0.96 3.46 3.85 1.11 df, degrees of freedom; adj-r2, r2 adjusted by df; pred-r2, r2 in prediction; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. 112 italian journal of food science, 2021; 33 (4) ugarte-espinoza pp et al. et  al. (2018) reported an iv of 109.5 g i2/100 g, despite their fatty acid composition being very similar to ours. we determined a saponification value of 188.1 mg koh/g, similar to those observed by mokhtar et al. (2018) in goldenberry pomace oil and by anwar et al. (2002) in safflower oil, i.e., 186.2 and 189.0 mg koh/g, respectively. the gso’s sv was lower than the sv of coconut, babassu, and palm kernel oils, where medium chain fatty acids (mainly lauric and myristic; codex alimentarius, 1999b) predominate. this indicates that long-chain fatty acids (c18) were more abundant in gso, as sv decreases with fatty acid chain length. the unsaponificable matter is the sum of minor but valuable components such as tocopherols, carotenoids, squalene, and phytosterols, which not only impart oxidative stability to the oils but also enhance their nutritional value (raziq et al., 2012). the value we found, 1.67 g/100 g, is the average among edible oils, being very close to soybean, sunflower (both regular than high oleic), safflower, coconut, and cottonseed oil; conversely, corn oil has superior values, near to 3 g/100 g (codex alimentarius, 1999b; gunstone, 2004). higher values were reported by ramadan et al. (2008) and popova et al. (2021): 2.13–2.25 and 3.02 g/100 g, respectively. according to popova et al. (2021), the peel is the part richest in unsaponificable matter (61.33 g/100 g of peel oil), despite containing twofold less tocopherols and far less sterols than seeds. in our opinion, this is explained by the wax covering the fruit, which distorts the result. instead, our experiments were conducted separating the seeds from the rest of the pomace. the l*, a*, and b* coordinates were 30.21, −2.13, and 10.03, respectively. this indicated that the oil was slightly dark with the presence of yellow and, in lower degree, green compounds that likely correspond to pigments such as carotenoids and chlorophylls, respectively. the gso coordinates in the cielab system were lower than those reported for other oils, such as chia (ixtaina et al., 2011), pistachio (ling et al., 2016), and linseed (varas condori et al., 2020). fatty acid composition linoleic acid (c18:2 ω-6) was the most abundant fatty acid, followed by oleic (c18:1), palmitic (c16:0), stearic (c18:0), and vaccenic (c18:1 ω7) acids (table 4). the remaining unsaturated fatty acids (palmitoleic and α-linolenic), as well as the long-chain saturated fatty acids (arachidic, behenic, and lignoceric) were found in low concentrations (<0.5%). similar results were reported by mokhtar et al. (2018), ramadan and moersel (2009), ramadan and mörsel (2003), and chasquibol silva and yácono llanos reactant and to form a water–oil interface (brockman, 2013). to find out the experimental conditions that maximize oil recovery and minimize other responses, a multi-response optimization by rsm was performed by using the desirability function. the solution we found had a desirability equal to 0.98 and corresponded to the treatment performed with 8% moisture at 60°c. the maximum oil recovery (86.43%) was far higher than the yield obtained by ramadan and moersel (2009; 42.1%) with an enzymatic-aided aqueous extraction; this might be due to nonfatty matter retaining oil in the pomace. the or observed in the present research was similar to those reported for screw-pressed flaxseed (82.9%) and sesame (74.2%) seeds (martínez et al., 2017; mridula et al., 2015). therefore, screw-pressing of goldenberry seeds separated from pomace guarantee higher yields and better quality of oil. characterization of the oil extracted at the optimized conditions physicochemical characteristics table 3 shows the physicochemical characteristics of  the gso extracted at 60°c with 8% of final moisture. the  iodine value, 140.5 g i2/100 g, is consistent with the theoretical value calculated as the average, weighted on the fatty acid composition, of the values provided by gunstone (2004; 85.6, 173.2, and 260.3 for methyl oleate, linoleate, and linolenate, respectively). this places the goldenberry oil in a straddling position between semi siccative and siccative oils. the degree of unsaturation was also reflected by ri (1.4769 at 20°c). the iv and ri values were higher than those (116.3 g i2/100 g and 1.4481, respectively) reported by aslanov et al. (1995), likely due to a lower degree of unsaturation of the oil they analyzed. in fact, both iv and ri increase as the number of double bonds increases (raziq et al., 2012). mokhtar table 3. physicochemical characteristics of goldenberry seed oil corresponding to the treatment with the highest oil recovery and the best quality (8% of final moisture, 60°c). parameter iodine value (g i 2 /100 g) 140.50 ± 0.24 refractive index (20°c) 1.4769 ± 0.0001 saponification value (mg koh/g) 188.10 ± 0.20 unsaponifiable matter (g/100 g) 1.67 ± 0.02 color coordinates: l* 30.21 ± 0.08 a* −2.13 ± 0.01 b* 10.03 ± 0.11 italian journal of food science, 2021; 33 (4) 113 goldenberry (physalis peruviana l.) seed oil (2015), although they found lower contents of α-linolenic and vaccenic acids. aslanov et al. (1995) reported higher values of palmitic, oleic, and α-linolenic acids, lower values of linoleic acid, but similar percentage of stearic acid. our results were also quite similar to embaby et al. (2022), who found slightly higher amounts of stearic, behenic, lignoceric, and α-linolenic acids offset by lower contents of linoleate and palmitate. differences in oil composition may be explained by different origin, environment, growing conditions, and oil extraction process (varas condori et al., 2020). in fact, for two genotypes of goldenberry, popova et al. (2020) reported extremely discordant values for fatty acids: palmitic 20.6–20.9%, stearic 13.0–17.5%, oleic 5.4–29.4%, linoleic 5.3–11.3, and αlinolenic 5.4– 9.2%. comparing other oil species, gso appears very similar to regular safflower oil (anwar et al., 2002; codex alimentarius, 1999b; gunstone, 2004). linoleic acid derivatives serve as structural components of the plasma membrane and as precursors of some metabolic regulatory compounds. in addition, studies suggest that linoleic acid consumption is inversely correlated with the risk of cardiovascular diseases (marangoni et al., 2020); thus, introducing gso in diet could help maintain an adequate health. shelf-life prediction the osi at 100°c was 14.87 h (table 5), higher than the values reported for other crude oils such as camelina (5.21 h; ratusz et al., 2016), linseed (4.07 h; varas condori et al., 2020), chia (3.03 h; villanueva et al., 2017), and sacha inchi (1.59 h; rodríguez et al., 2015) but lower than crude pumpkin oil (18.2 h; vujasinovic et al., 2010). these differences could be due to the different fatty acids’ profile, since osi decreases as the degree of unsaturation increases (shadyro et al., 2017). the relationship between osi and temperature was linearized through logarithm transformation. the line of regression showed a high coefficient of determination (r2 = 0.9994), thus 99.94% of osi variation was explained by the model. the predicted shelf life at 25°c was 120 days (approximately 4 months), very close to the 118.9–123.2 days of two chia:sesame oil blends studied by rodríguez et al. (2020), higher than the 91 days reported for crude linseed oil by varas condori et al. (2020), but lower than the 386 and 211 days of, respectively, pistachio (dini et  al., 2016) and avocado (aktar and adal, 2019) crude oils. in comparison with commercial oils, whose shelf life generally is between 12 and 15 months under normal storage conditions, gso predicted shelf life was low (kochhar and henry, 2009). taking into account that the gso started with very low levels of deterioration (table 1), the short time obtained can be attributed to the high concentration of linoleic acid, which oxidizes 10– 40 times faster than the oleic acid (symoniuk et al., 2016). oxidation kinetics the k constant increased as a function of the temperature (table 5) since the oxidation rate was accelerated by the temperature increase. the magnitude of the effect of temperature on k was demonstrated by q10, which had a value of 2.02. similar values were reported in refined soybean oils (1.99–2.08; farhoosh, 2007) and crude linseed oils (1.99–2.05; symoniuk et al., 2017), while lower values were reported in crude canola oils (1.84–1.86; symoniuk et al., 2016). a high q10 implies that small changes in temperature induce a greater increase in the reaction rate, so that high q10 values indicate lower oxidative stability (farhoosh et al., 2008; symoniuk et al., 2016). the gso oxidation kinetics obeyed the arrhenius equation (table 5) in the temperature range from 100 to 120°c (r2 = 0.9988). the activation energy gives an indication of the minimum amount of energy needed to initiate the oxidation reaction. the ea of gso was 85.56 kj/mol, slightly higher than the levels reported for camelina (70.39–79.08 kj/mol; ratusz et al., 2016), linseed (74.03–77.76 kj/mol; symoniuk et al., 2016), canola (75.73–77.64 kj/mol; symoniuk et al., 2017), and chia (82.0 kj/mol; rodríguez et al., 2020) crude oils but below the ea of sesame (96.2 kj/mol; rodríguez et al., 2020), hazelnut (94.75 kj/mol; gülmez and şahin, 2019), and avocado (99.6 kj/mol; aktar and adal, 2019) crude oils. these differences may be due to several factors, such as the degree of unsaturation and the presence of different table 4. fatty acid profile (% of total fame) of goldenberry seed oil corresponding to the treatment with the highest oil recovery and best quality (8% of final moisture, 60°c). fatty acid palmitic acid (c16:0) 6.43 ± 0.01 palmitoleic acid (c16:1 ω-7) 0.40 ± 0.02 stearic acid (c18:0) 3.23 ± 0.01 oleic acid (c18:1 ω-9) 10.97 ± 0.06 vaccenic acid (c18:1 ω-7) 1.67 ± 0.01 linoleic acid (c18:2 ω-6) 75.99 ± 0.02 α-linolenic acid (c18:3 ω-3) 0.23 ± 0.00 arachidic acid (c20:0) 0.40 ± 0.01 behenic acid (c22:0) 0.16 ± 0.01 lignoceric acid (c24:0) 0.16 ± 0.00 saturated fatty acids 10.37 ± 0.01 unsaturated fatty acids 89.25 ± 0.11 monounsaturated 13.03 ± 0.04 polyunsaturated 76.22 ± 0.02 114 italian journal of food science, 2021; 33 (4) ugarte-espinoza pp et al. endogenous antioxidants or prooxidants (symoniuk et al., 2017). conclusions the pressing temperature and the moisture content of the seeds exerted a significant, but negative, effect on the or. on the other hand, the seeds’ moisture content affected the physicochemical quality of the oil to a greater extent than the pressing temperature. all the extraction conditions allowed to obtain oils with good physicochemical quality, but the best quality and the highest or were achieved at 60°c with 8% of moisture content. under these extraction conditions, the oil exhibited a yellow tone with low luminosity, presented high iodine value and refractive index, and low saponification value. in addition, gso consisted mainly of unsaturated fatty acids, with a high percentage of linoleic acid (ω-6), which makes it an important source of this essential fatty acid. the oil presented a low oxidative stability that resulted in a shelf life of 120 days at 25°c; the ea was 85.56 kj/mol. finally, the results highlighted that screw-pressing of goldenberry seeds provides quality oil without employing polluting and hazardous solvents. authors’ contribution pedro p. ugarte-espinoza designed the study, collected and analyzed the data, and drafted the manuscript; victor delgado-soriano supervised the work, collected and analyzed the data, and revised the manuscript; lorenzo estivi and alyssa hidalgo statistically elaborated the data, and drafted and revised the manuscript; gloria pascual-chagman planned and designed the study, supervised the work, provided resources, and revised the manuscript. conflict of interest the authors declare that they have no conflicts of interest concerning this article. there was no financial support, except those mentioned in the acknowledgments. references aktar, t. and adal, e., 2019. determining the arrhenius kinetics of avocado oil: oxidative stability under rancimat test conditions. foods 8: 236. https://doi.org/10.3390/foods8070236 anwar, f., bhanger, m.i., nasir, m.k.a. and ismail, s., 2002. analytical characterization of salicornia bigelovii seed oil cultivated in pakistan. journal of agricultural and food chemistry 50: 4210–4214. https://doi.org/10.1021/jf0114132ta bl e 5. o xi da tiv e st ab ili ty in de x (o s i) at d if fe re nt te m pe ra tu re s an d ox id at io n ki ne tic p ar am et er s of g ol de nb er ry s ee d oi l c or re sp on di ng to th e tr ea tm en t w ith h ig he r oi l r ec ov er y an d be tt er q ua lit y (8 % of fi na l m oi st ur e, 6 0° c ). s he lf li fe a t 2 5° c (o s i 25 ) w as e xt ra po la te d. te m pe ra tu re (° c ) li ne o f re gr es si on s lo pe in te rc ep t r 2 s he lf li fe o s i 25 (d ) q 10 e a (k j/ m ol ) 10 0 11 0 12 0 o s i ( h) 14 .8 7 ± 0. 08 7. 52 ± 0 .1 1 3. 65 ± 0 .0 6 lo g o s i = a t + b −0 .0 30 5 ± 0. 00 03 4. 22 3 ± 0. 03 2 0. 99 94 12 0. 44 k ×1 03 (h −1 ) 67 .2 7 ± 0. 35 13 2. 9 ± 1. 9 27 3. 8 ± 4. 8 ln k = ln a – e a/ r t −1 02 91 ± 1 45 24 .8 7 ± 0. 38 0. 99 86 2. 02 85 .5 6 https://doi.org/10.3390/foods8070236� https://doi.org/10.1021/jf0114132� italian journal of food science, 2021; 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aceite de chía (salvia hispanica l.) por rancimat. scientia agropecuaria 8: 19–27. https://doi.org/10.17268/sci.agropecu. 2017.01.02 https://doi.org/10.1007/s11746-010-1630-x� https://doi.org/10.1007/s11746-010-1630-x� https://doi.org/10.17268/sci.agropecu.2017.01.02� https://doi.org/10.17268/sci.agropecu.2017.01.02� _hlk66139061 _hlk66139450 _hlk63112566 _hlk70278146 _hlk43653533 _hlk64128899 _hlk51519656 _hlk21777727 _hlk26214373 _hlk25582034 bau005 _goback p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33i4.2104 33 p u b l i c a t i o n s codon analysis of aroma compounds of nine autochthonous and non-autochthonous loquat cultivars grown in sicily diego planeta, vittorio farina, paola bambina, ilenia tinebra, roberta passafiume, luciano cinquanta, onofrio corona* department of agricultural, food, and forest sciences (saaf), università degli studi di palermo, viale delle scienze, palermo, italy *corresponding author: onofrio corona, department of agricultural, food and forest sciences (saaf), università degli studi di palermo, viale delle scienze, palermo, 90128 italy. email: onofrio.corona@unipa.it received: 23 july 2021; accepted: 15 october 2021; published: 11 november 2021 © 2021 codon publications open access paper abstract loquat cultivation in sicily is mainly based on nonnative cultivars and local ecotypes characterized by high nutraceutical value and appreciable physicochemical characteristics. increased interest in commercial loquat production has increased the intention to provide premium quality loquat cultivars that include volatile substances capable of conditioning the sensorial properties and, therefore, the acceptability of fruits by consumers. this study determined the content of volatile compounds in nonnative and local loquat fruits grown in sicily. analyses were performed on five international cultivars and four local cultivars. keywords: international and local cultivars, loquat, volatile compounds introduction in recent years, there has been a growing demand for fruit products from tropical and subtropical countries. among the attractive characteristics that fuel this demand from consumers is the increased interest in products with a high nutraceutical value, importantly their characteristic taste and flavour (gentile et al., 2019). it is well known that the taste and quality of food are determined by aromatic compounds, which in turn, influence consumer preferences and attract the attention of farmers who require more information and analytical tools to enable them to select the most suitable cultivars to grow (baldwin, 2004; schwab et al., 2008). the aroma is a critical quality parameter that differentiates one fruit from the other and it is associated with many volatile compounds (lo bianco et al., 2010; ye et al., 2017; yuan et al., 2018) belonging to different chemical groups, such as esters, alcohols, terpenes, ethers, aldehydes, etc. one of the fruits belonging to the subtropical species, which is well adapted to the temperate zones of the mediterranean and whose production is concentrated in spain and italy, is the loquat (eriobotrya japonica lindl). it is an evergreen subtropical species (family rosaceae subfamily mathat loideae) that originates from southern china. today, 90% of the cultivation is concentrated in the region of sicily, particularly in the province of palermo (farina et al., 2016), with an extended harvest period (from april to june) based on several cultivars and local ecotypes (farina et al., 2011; gentile et al., 2016). loquat cultivation in sicily is based especially on nonnative cultivars and local ecotypes characterized by a high nutraceutical value (gentile et al., 2019) and appreciable physico-chemical characteristics. today, the interest in loquat commercial production has risen, and it is geared towards loquat cultivars of premium quality (badenes et al., 2013). the most important characteristic for the market is fruit size. the value of the crop (goulas et al., 2014) is in line with the italian journal of food science, 2021; 33 (4): 33–42 mailto:onofrio.corona@unipa.it 34 italian journal of food science, 2021; 33 (4) planeta d et al. bundessortenamt and chemische industrie (bbch) scale). the analyses were carried out on four local cultivars (brt20, claudia, sanfilippara, and nespolone di trabia) and five international cultivars (algerie, bueno, el buenet, golden nugget, and peluche). a sample of 30 fruits per cv was submitted for laboratory analyses. commercial classification primarily the loquat fruits were divided into international and local cultivars and later were classified based on the flesh color (yellow and white) and diameter (ggg, gg, g, and m), according to testa et al. (2020). volatile compounds analysis three replicates of the pulp (about 200 g) of 10 fruits were separated from the peel and seeds with the addition of 100 mg of sodium metabisulfite. the pulps were crushed with a laboratory blender by a high-speed ultraturrax t25 (ika labortechnik, staufen, germany) and centrifuged twice at 4500gand 4°c for 15 minutes and later the solid residue was washed with 70 ml of ethanol:water solvent (12:88). the final extract (250 ml) was then clarified with 0.1 g of the pectolytic enzyme without secondary glycosidase activity (rapidase x-press, dsm, the netherlands) at room temperature for 2 hours. 1-heptanol was added as internal standard (0.2 ml of 40 mg/l in 10% ethanol) to the samples and was loaded onto a 5-g c18 reversed-phase solid-phase extraction (spe) cartridge (isolute, spe columns, uppsala, sweden), previously activated with 20 ml of methanol,50 ml of deionized water using a flow-rate of ca. 3 ml/min, and then rinsed with 100 ml of deionized water to eliminate sugars, acids, and other low molecular weight polar compounds. the free aromatic fraction was then eluted with 25 ml of dichloromethane. the eluate was dried over anhydrous sodium sulfate (na2so4) and was concentrated to about 0.2 ml under a stream of nitrogen. this extract, containing free volatile compounds, was immediately analyzed by gas chromatography/mass spectrometry (gc/ms). then, the glycoconjugates aromas were finally eluted from the cartridge with 20 ml of methanol, and the eluate was concentrated to dryness using a vacuum rotary evaporator set at 30 °c (buchi r-210, switzerland). these dried glycosides extract were dissolved in 5 ml of citrate-phosphate buffer (0.2 m, ph 5) and subjected to enzymatic hydrolysis with 50 mg of an ar-2000 commercial preparation with glycosidase side activities (dsm oenology, the netherlands) at 40 °c for 24 hours. later, 0.2 ml of 1-heptanol was added as internal standard, and the volatiles generated by the enzymatic hydrolysis of glycosylated precursors were then extracted following the spe method previously described. the dichloromethane commercial classification (testa et al., 2020). as a result, fruits are divided into four classes based on their diameter: ggg for fruits over 53 mm; gg for fruits between 46 and 52 mm; g for fruits between 32 and 45 mm; and m for fruits between 31 and 28 mm. quality is a complex of chemical and physical parameters and aromatic composition. volatile flavor compounds are likely to play a key role in determining the perception and acceptability of products by consumers (pott et al., 2020). fruits produce a range of volatile compounds that make up their characteristic aromas and contribute to their flavor. the differences in volatile compounds may be because of their ripening phase during the harvest time (agozzino et al., 2007) and differences in the studied cultivars (farina et al., 2020). many studies on the volatile component of many fruits can be found in literature, while only a few studies have been conducted to date for the mediterranean loquat. in this regard, shaw and wilson (1982) identified the following volatile compounds in loquat fruits; 2-phenylethanol, 3-hydroxy-2-butanone, phenylacetaldehyde, isomeric hexen1-ols, ethyl acetate, methyl cinnamate, and β-ionone. hexanal and (e)-2-hexenal and benzaldehyde have also been identified by fröhlich and schreier (1990). chen et al. (2011) reported that β-ionone, decanoic acid, propanoic acid, bicycle nonane, and heptadecane are the major volatile compounds in the zaozhong 6 cultivar. these studies highlighted that volatile compounds such as 2e-hexenol, 3z-hexenol, and hexanol contribute to green notes; methyl cinnamate and eugenol contribute to the spicy note; ethyl and methyl 2-methylbutanoates are responsible for fresh fruity notes; a sweet watery aroma was also detected by traces amount of phenylacetaldehyde, vanillin, and βionone (hideki et al. 1998). finally, takahashi et al. (2000) reported that the tanaka cultivar presents phenylacetaldehyde as the most aromatic compound, while small traces of hexanal, (e)-2-hexanal, hexanoic acid, and β-ionone have also been found. nevertheless, many other volatile compounds are present in traces which can be detected not only by analytical instruments but also by human olfaction (goff et al., 2006). the aim of this study was to determine for the first time the content of volatile compounds in non-native and local loquat fruits grown in sicily. material and methods plant material two hundred and seventy loquat fruits (eriobotrya japonica lindl.) were harvested in an experimental orchard located in santa maria di gesù (palermo, italy, 38°04’n, 13°22’e, 150 m a.s.l.) between april and june at commercial ripening, using fruit peel color as a ripening index (807-809 degree of biologische bundesantalt, italian journal of food science, 2021; 33 (4) 35 analysis of aroma compounds of nine autochthonous and non-autochthonous loquat cultivars grown in sicily volatile compounds analysis gc-ms analysis of the concentrated flesh extract of spe was performed to evaluate the aromatic compounds of loquat fruit flesh, and 35 free volatile compounds (table 2) and 17 glycosylated compounds (table 3) were detected. of which, 14 were acids, 10 alcohols, two aldehydes, one benzenoid, and eight esters. among which four were terpenes, four c13-norisoprenoids, and nine benzenoids. the latter were released after enzymatic hydrolysis from aromatic precursors linked to sugars. during maturation, the volatile compounds of the two cultivars, algerie and golden nugget, have been studied, and a strong similarity was identified in terms of aroma, flavor, and parameters related to physiological-qualitative traits (pino et al., 2002; besada et al., 2013, 2017). the heritability of loquat aromas was assessed by jiang et al. (2014) by examining the composition of the volatile compounds of xiantgtian and jiefangzhong cultivars and two-hybrid progenies (xiangzhong no.11 and zhongxiang no. 25). they concluded that the level of volatile compounds in the fruit of the progeny was like the values known in their parents. previous studies showed that the maturity stage determines the qualitative and quantitative volatile substances of many fruit species (mattheis et  al. 1992; perez et al. 1992), for loquat, very little is known about their association with other ripening or quality characteristics that vary between cultivars (jiang et al., 2014). free volatile compounds among the free volatile compounds, minimal presence of volatile compounds that can characterize the aroma of loquat fruit was observed. it is a predominance of compounds belonging to the class of acids and alcohols, followed by esters, aldehydes, and finally a benzenoid extract obtained was dried using anhydrous na2so4, concentrated to 0.2 ml, and kept at −20 °c until further analysis. gc/ms analysis was performed with an agilent 6890 series gc system and agilent 5973 network mass selective detector (agilent technologies, palo alto, ca) equipped with a db-wax column (30 m, 0.250 mm i.d., film thickness 0.25 μm; agilent technologies). the gc-ms conditions used were as reported by corona et al. (2019). the detection was carried out by electron impact mass spectrometry in total ion current (tic) mode using ionization energy of 70 ev. the mass acquisition range was m/z 30–330. volatile organic compounds were identified by comparing their mass spectra and gc retention times with those of the pure commercial standard compounds and those within the nist/ epa/nih mass spectral library database (version 2.0d, build 2005). the concentration (µg/kg pulp) of volatile compounds was determined as 1heptanol equivalents. all solvents and reagents were purchased from wwr international (milan, italy). statistical analysis the data were presented as mean ± standard deviation and analysed using the tukey test at p ≤ 0.05. all statistical analysis was conducted using xlstat software version 9.0 (addinsoft, paris, france). results and discussion commercial classification according to commercial classification, our data showed that local ecotypes sizes were larger than international ecotypes (amorós et al., 2003; testa et al., 2020). however, only peluche had the highest value as a wellknown large fruit cultivar (barone et al., 2010). varieties with a larger size are more appreciated by consumers because of their small portion size and high sugar/acid ratio. (agusti et al. 2000; testaet al. 2020). more sugar in local fruits versus international cultivars shows an increased degree of acidity (gentile et al., 2016). in this study, all the international cultivars had yellow flesh, whereas, among the local cultivars, only nespolone di trabia showed more similar behavior with that of the international cultivars (yellow flesh). on the other hand, brt 20, claudia, and sanfilippara, showed white flesh with highest commercial classification (table 1). table 1. commercial classification based on origin, size, and color of analyzed loquat fruits: ggg> 53mm, gg 46-52mm, g 32-45 mm, and m 31-28 mm. origin cultivars commercial classification color classification local brt 20 ggg white flesh claudia ggg white flesh sanfilippara gg white flesh nespolone di trabia gg yellow flesh international algerie gg yellow flesh bueno g yellow flesh el buenet g yellow flesh golden nugget gg yellow flesh peluche ggg yellow flesh 36 italian journal of food science, 2021; 33 (4) planeta d et al. ta bl e 2. fr ee v ol at ile c om po un ds r el ea se d by e nz ym at ic h yd ro ly si s of g ly co sy la te d pr ec ur so rs (μ g/ kg p ul p) . c om po un ds lo ca l c ul tiv ar s* in te rn at io na l c ul tiv ar s* b r t 20 c la ud ia s an fil ip pa ra n es po lo ne d i t ra bi a a lg er ie b ue no e l b ue ne t g ol de n n ug ge t p el uc he a ci ds                   b ut yr ic a ci d 4. 0 ± 0. 0 6. 2 ± 0. 1 0. 6 ± 0. 0 0. 7 ± 0. 1 0. 4 ± 0. 1 3. 6 ± 0. 2  6. 4 ± 0. 4 n. d. 0. 5 ± 0. 1 pe nt an oi c ac id 10 .3 ± 1 .6 7. 2 ± 0. 1 3. 5 ± 0. 1 9. 4 ± 0. 3 8. 1 ± 0. 7 5. 5 ± 0. 5  15 .7 ± 1 .8 8. 9 ± 0. 7 8. 5 ± 0. 7 h ex an oi c ac id 10 60 .9 ± 4 4. 7b 11 90 .9 ± 3 1. 0b 55 4. 4 ± 34 .3 a 51 1. 1 ± 29 .4 a 47 5. 2 ± 57 .6 ab 64 5. 8 ± 49 .3 ab 35 8. 4 ± 7. 3a 59 8. 5 ± 50 .4 ab 50 6. 1 ± 34 .9 ab h ep ta no ic a ci d 2. 2 ± 0. 6b 2. 2 ± 0. 2b 2. 2 ± 0. 1b 0. 8 ± 0. 0a 0. 1 ± 0. 0a 3. 4 ± 0. 2b 3. 8 ± 0. 2b 0. 8 ± 0. 0a 0. 4 ± 0. 0 a o ct an oi d ac id 13 .6 ± 1 .3 b 11 .9 ± 1 .1 b 9. 9 ± 0. 1a 11 .9 ± 0 .2 b 13 .8 ± 2 .0 b 10 .5 ± 1 .6 ab 12 .1 ± 0 .7 ab 4. 2 ± 0. 2a 10 .1 ± 0 .7 ab n on an oi c ac id 18 .4 ± 1 .7 b 17 .4 ± 1 .9 17 .4 ± 0 .7 16 .4 ± 1 .2 13 .2 ± 1 .5 20 .4 ± 1 .5 18 .9 ± 1 .8 20 .8 ± 1 .3   19 .0 ± 1 .5   d ec an oi c ac id 16 .8 ± 1 .0 b 8. 0 ± 0. 1a 8. 2 ± 0. 5a 17 .4 ± 0 .2 b 33 .5 ± 1 .3 b 12 .0 ± 0 .7 a 8. 0 ± 0. 3a 20 .1 ± 0 .8 ab 11 .4 ± 1 .1 a d od ec an oi c ac id 3. 9 ± 0. 1a 2. 4 ± 0. 3a 4. 2 ± 0. 1a 9. 9 ± 0. 1b 13 .3 ± 0 .8 ab 8. 4 ± 0. 6a b 39 .1 ± 3 .6 b 12 .8 ± 0 .2 ab 4. 4 ± 0. 1a te tra de ca no ic a ci d 7. 5 ± 0. 1 1. 9 ± 0. 2 3. 7 ± 0. 2 3. 2 ± 0. 0 7. 4 ± 0. 4  14 .1 ± 1 .4   15 .6 ± 1 .2   6. 7 ± 0. 7  1. 8 ± 0. 2  pe nt ad ec an oi c ac id 7. 6 ± 0. 3a 19 .8 ± 1 .2 b 15 .9 ± 0 .2 b 45 .3 ± 1 .0 c 4. 0 ± 0. 0a 10 .8 ± 0 .8 ab c 5. 3 ± 0. 2a b 25 .9 ± 0 .1 d 1. 3 ± 0. 1a h ex ad ec an oi c ac id 24 0. 6 ± 17 .4 a 21 9. 4 ± 21 .1 a 44 4. 9 ± 95 .5 b 34 1. 7 ± 21 .3 b 63 6. 4 ± 16 .0 b 66 7. 6 ± 26 .8 b 61 7. 8 ± 13 .7 b 52 5. 0 ± 10 .5 ab 10 5. 0 ± 4. 2a h ep ta de ca no ic a ci d 4. 3 ± 0. 2 n. d. 0. 3 ± 0. 0 2. 1 ± 0. 1 5. 4 ± 0. 1  3. 4 ± 0. 1 3. 8 ± 0. 3  1. 5 ± 0. 1  n. d. o ct ad ec an oi d ac id 12 4. 5 ± 11 .6 12 0. 3 ± 10 .2 13 3. 4 ± 21 .7 11 6. 1 ± 4. 8  14 3. 4 ± 4. 2b 17 1. 7 ± 12 .0 b  13 7. 0 ± 10 .3 b  11 8. 8 ± 13 .7 b  73 .9 ± 2 .6 a  b en zo ic a ci d 10 53 .3 ± 5 3. 0b 11 85 .2 ± 1 30 .4 b 54 7. 8 ± 33 .5 a 50 4. 5 ± 25 .7 a 46 4. 2 ± 55 .2 ab 64 0. 5 ± 61 .0 ab 35 3. 6 ± 7. 9a 59 4. 4 ± 40 .2 ab 50 2. 8 ± 35 .2 ab to ta l 25 67 .8 ± 1 33 .6 b 27 92 .8 ± 1 97 .9 b 17 46 .3 ± 1 87 .0 a 15 90 .6 ± 8 4. 4a 18 18 .4 ± 1 38 .9 ab 22 17 .4 ± 1 57 .7 ab 15 95 .3 ± 4 9. 7a b 19 38 .4 ± 1 18 .9 ab 12 45 .2 ± 8 1. 4a a lc oh ol s                   1pe nt an ol o 2. 4 ± 0. 0b 2. 9 ± 0. 1b 1. 6 ± 0. 1a 2. 7 ± 0. 0b 1. 7 ± 0. 1a b 1. 7 ± 0. 1a b 5. 6 ± 0. 2b 2. 7 ± 0. 0a b 4. 3 ± 0. 2a b 3h ep ta no l 0. 6 ± 0. 0 n. d. 0. 4 ± 0. 0 n. d. 0. 3 ± 0. 0a 0. 7 ± 0. 0a 2. 8 ± 0. 1b n. d. a 0. 2 ± 0. 0a 2h ex an ol 1. 3 ± 0. 0  n. d. 0. 4 ± 0. 0 1. 6 ± 0. 0 1. 3 ± 0. 1a 54 .7 ± 4 .3 b 6. 3 ± 0. 2a b 1. 3 ± 0. 1a 2. 0 ± 0. 1a 1h ex an ol 1. 5 ± 0. 0a 1. 4 ± 0. 0a 4. 7 ± 0. 1b 5. 5 ± 0. 0b 12 .4 ± 0 .3 b 2. 0 ± 0. 0a 3. 9 ± 0. 1a 3. 5 ± 0. 1a 3. 3 ± 0. 2a 3e th yl -3 -h ep ta no l 0. 3 ± 0. 0 n. d.   n. d. n. d. n. d. 0. 2 ± 0. 0  0. 2 ± 0. 0  n. d. 0. 3 ± 0. 1 c is -3 -h ex en -1 -o l 2. 0 ± 0. 0a 12 .5 ± 0 .1 bc 6. 7 ± 0. 1b 22 .2 ± 1 .2 c 20 .0 ± 1 .4 ab 5. 9 ± 0. 3a b 2. 8 ± 0. 1a 5. 7 ± 0. 2a b 4. 8 ± 0. 0a b 2b ut ox ye th an ol 5. 4 ± 0. 0b 3. 4 ± 0. 1a 3. 5 ± 0. 0a 2. 4 ± 0. 0a 5. 0 ± 0. 1a b 3. 9 ± 0. 1a b 2. 0 ± 0. 0a 2. 3 ± 0. 1a b 3. 3 ± 0. 3a b tr an s3he xe n1ol 2. 7 ± 0. 0a 6. 5 ± 0. 1b 4. 0 ± 0. 0a 9. 4 ± 2. 6b 26 .5 ± 0 .3 c 11 .2 ± 0 .2 ab 8. 1 ± 0. 1a b 7. 5 ± 0. 2a b 14 .6 ± 0 .2 b 2e th yl h ex an ol 5. 3 ± 0. 0 2. 4 ± 0. 0 3. 9 ± 0. 1 2. 3 ± 0. 0  5. 6 ± 0. 2  4. 7 ± 0. 2  2. 5 ± 0. 1  3. 3 ± 0. 1  2. 8 ± 0. 1  italian journal of food science, 2021; 33 (4) 37 analysis of aroma compounds of nine autochthonous and non-autochthonous loquat cultivars grown in sicily to ta l 21 .6 ± 0 .0 a 29 .1 ± 0 .4 a 25 .4 ± 0 .4 a 46 .2 ± 3 .9 b 72 .5 ± 2 .5 c 85 .1 ± 5 .1 bc 34 .2 ± 0 .9 ab c 26 .5 ± 0 .8 ab c 35 .6 ± 1 .2 ab c a ld eh yd es                   c is -2 -h ex en al 6. 2 ± 0. 1a 8. 7 ± 0. 1a 9. 2 ± 0. 2 a 20 .5 ± 0 .2 b 92 .1 ± 8 .9 b 7. 9 ± 0. 6a n. d. a 5. 0 ± 0. 1a 30 .3 ± 1 .7 ab n on an al 1. 6 ± 0. 0 ab 3. 5 ± 0. 1b 2. 4 ± 0. 1a b 1. 0 ± 0. 0a b 0. 6 ± 0. 0a 1. 8 ± 0. 1a b 0. 4 ± 0. 0a 1. 4 ± 0. 2a b 1. 3 ± 0. 2a b to ta l 7. 8 ± 0. 1a 12 .3 ± 0 .2 ab 11 .6 ± 0 .3 ab 21 .5 ± 0 .2 b 92 .7 ± 8 .9 b 9. 7 ± 0. 7a b 0. 4 ± 0. 0a 6. 4 ± 0. 3a 31 .6 ± 1 .9 ab b en ze no id s                   va ni lli n 11 .7 ± 0 .2 b 8. 7 ± 0. 1a 8. 0 ± 0. 1a 8. 2 ± 0. 0a 11 .1 ± 1 .1 ab 27 .9 ± 2 .4 b n. d. a 11 .8 ± 0 .2 ab 10 .1 ± 0 .1 ab e st er s                   e th yl h ex an oa te 1. 9 ± 0. 1a n. d. a 5. 0 ± 0. 1a b 0. 6 ± 0. 0a 3. 3 ± 0. 5a 3. 8 ± 0. 4a 9. 6 ± 0. 3b 1. 0 ± 0. 0 a 3. 2 ± 0. 2a e th yl o ct an oa te 3. 4 ± 0. 0a 1. 4 ± 0. 0a 9. 4 ± 0. 3a 3. 7 ± 0. 0a 11 .3 ± 1 .8 a 6. 7 ± 0. 3a 25 .2 ± 0 .3 b 3. 1 ± 0. 2a 7. 4 ± 0. 2a e th yl -3 -h yd ro xy bu ty ra te 23 0. 8 ± 8. 7d 10 .0 ± 0 .1 a 18 1. 1 ± 9. 6c d 7. 1 ± 0. 1a 83 .3 ± 4 .5 b 20 3. 9 ± 23 .1 d 12 0. 8 ± 10 .8 c 5. 2 ± 0. 3a 4. 6 ± 0. 0a e th yl d ec an oa te 0. 2 ± 0. 0a 0. 2 ± 0. 0a 1. 3 ± 0. 0a b 1. 2 ± 0. 0a b 2. 8 ± 0. 2b 0. 5 ± 0. 0a 2. 4 ± 0. 1b 0. 6 ± 0. 0a 0. 5 ± 0. 1a d ie th yl s uc ci na te 0. 8 ± 0. 0a n. d. a 1. 3 ± 0. 1a b 0. 1 ± 0. 0a 3. 3 ± 0. 1b 0. 7 ± 0. 0a 0. 3 ± 0. 0a 0. 2 ± 0. 0a 2. 5 ± 0. 1b b ut an oi c a ci d2m et hy l 13 7. 4 ± 10 .9 a 11 8. 7 ± 9. 1a 8. 7 ± 0. 1a 65 .7 ± 1 .8 a 62 3. 9 ± 7. 1c 41 5. 1 ± 36 .2 b 30 8. 0 ± 28 .6 b 7. 3 ± 0. 2a 30 1. 3 ± 3. 8b h ex ad ec an oi c ac id m et hy l e st er 4. 4 ± 0. 4a 25 .3 ± 0 .2 b 2. 7 ± 0. 2a 7. 4 ± 0. 2a 4. 3 ± 0. 3a 4. 6 ± 0. 3a 2. 9 ± 0. 1a 1. 6 ± 0. 1a 6. 4 ± 0. 2b h ex ad ec an oi c ac id et hy l e st er 1. 6 ± 0. 0  2. 6 ± 0. 0 1. 4 ± 0. 1 2. 4 ± 0. 2 7. 0 ± 0. 3 1. 5 ± 0. 1  5. 4 ± 0. 2  2. 4 ± 0. 2  2. 8 ± 0. 1  to ta l 38 0. 5 ± 20 .1 cd 15 8. 2 21 0. 9 ± 10 .5 ab c 88 .1 ± 2 .3 a 73 9. 3 ± 14 .8 c 63 6. 7 ± 60 .4 c 47 4. 7 ± 40 .4 b 21 .5 ± 1 .0 a 32 8. 8 ± 4. 7b *m ea n ± st an da rd d ev ia tio n (n = 3 ). d iff er en t s up er sc rip te d le tte rs in di ca te s ig ni fic an t d iff er en ce s fo r p ≤ 0 .0 5 (a na ly si s of v ar ia nc e or t uk ey te st ). n. d. , n ot d et er m in ab le . 38 italian journal of food science, 2021; 33 (4) planeta d et al. ta bl e 3. g ly co sy la te d vo la til e co m po un ds r el ea se d by e nz ym at ic h yd ro ly si s of g ly co sy la te d pr ec ur so rs (μ g/ kg p ul p) . lo ca l c ul tiv ar s* in te rn at io na l c ul tiv ar s*  c om po un ds b r t 20 c la ud ia s an fil ip pa ra n es po lo ne d i tr ab ia a lg er ie b ue no e l b ue ne t g ol de n nu gg et p el uc he te rp en es                   tr an s8di id ro lin al oo l 6. 1 ± 0. 0b 8. 0 ± 0. 1b 4. 0 ± 0. 0a 3. 3 ± 0. 0a 10 .7 ± 0 .2 c 6. 9 ± 0. 0b 3. 4 ± 0. 1a b 6. 1 ± 0. 2b 1. 4 ± 0. 0a tr an s8hy dr ox yl in al oo l 5. 5 ± 0. 1a 15 .1 ± 0 .3 b 7. 0 ± 0. 2a 15 .5 ± 0 .3 b 12 .8 ± 0 .1 bc 9. 5 ± 0. 2b 7. 1 ± 0. 2b 16 .0 ± 1 .4 c 1. 0 ± 0. 0a c is -8 -h yd ro xy lin al oo l 1. 5 ± 0. 0a 4. 1 ± 0. 1 ab 8. 5 ± 0. 2b 7. 6 ± 0. 3b 3. 2 ± 0. 0b 4. 3 ± 0. 2b c 5. 7 ± 0. 1c 3. 7 ± 0. 2b 1. 1 ± 0. 0a g er an ia l 3. 4 ± 0. 0a b 2. 6 ± 0. 2a 6. 9 ± 0. 2b 4. 2 ± 0. 2a b 6. 0 ± 0. 0b c 3. 8 ± 0. 1a 4. 8 ± 0. 1b 7. 6 ± 0. 2c 5. 6 ± 0. 2b to ta l 16 .5 ± 0 .1 a 29 .8 ± 0 .7 b 26 .4 ± 0 .6 b 30 .6 ± 0 .8 b 32 .7 ± 0 .3 c 24 .5 ± 0 .5 b 21 .1 ± 0 .5 b 33 .4 ± 2 .0 c 9. 2 ± 0. 2a c 13 -n or is op re no id s                   3ox oaio no l 59 .2 ± 4 .8 a 62 .4 ± 1 .2 a 94 .4 ± 4 .9 ab 20 7. 5 ± 23 .0 b 46 1. 0 ± 41 .5 d 28 6. 6 ± 19 .8 c 21 7. 9 ± 19 .3 b 37 3. 4 ± 34 .0 c 11 2. 3 ± 9. 8a 34di hy dr o3ox oaio no l 19 .8 ± 1 .1 a 41 .9 ± 1 .1 ab 32 .7 ± 2 .9 ab 74 .1 ± 1 4. 0b 92 .1 ± 7 .2 c 55 .6 ± 4 .2 b 27 .7 ± 2 .0 a 72 .9 ± 1 7. 1b c 24 .8 ± 1 .1 a 3o h -b -io no ne 2. 0 ± 0. 0a 28 .5 ± 1 .3 ab 34 .1 ± 1 .6 b 64 .7 ± 3 .7 c 23 .4 ± 2 .9 c 13 .0 ± 1 .8 bc 7. 8 ± 1. 0a b 22 .2 ± 1 .6 c 2. 6 ± 0. 1a vo m ifo lio l 13 5. 0 ± 10 .4 a 14 9. 2 ± 0. 8a 17 8. 0 ± 12 .2 a 28 3. 4 ± 23 .8 b 58 1. 6 ± 26 .7 c 79 .9 ± 2 .2 a 10 1. 2 ± 9. 1a 28 3. 8 ± 10 .9 b 12 0. 0 ± 11 .5 a to ta l 21 5. 9 ± 16 .3 a 28 1. 9 ± 4. 4a b 33 9. 2 ± 21 .6 b 62 9. 6 ± 64 .5 c 11 58 .1 ± 7 8. 3c 43 5. 1 ± 28 .0 ab 35 4. 5 ± 31 .4 a 75 2. 3 ± 63 .6 bc 25 9. 7 ± 22 .5 a b en ze no id s                   e ug en ol 32 .3 ± 1 .6 a 21 .7 ± 0 .1 b 15 .9 ± 0 .2 a 27 .9 ± 0 .3 b 55 .0 ± 3 .6 b 59 .2 ± 4 .1 b 24 .7 ± 1 .9 a 12 1. 3 ± 9. 5c 16 .1 ± 0 .2 a 4vi ny lg ua ia co l 54 .4 ± 4 .1 c 35 .2 ± 0 .3 b 30 .1 ± 0 .2 ª 32 .5 ± 0 .4 a 29 .0 ± 2 .4 a 25 .1 ± 2 .1 a 21 .4 ± 0 .7 a 78 .0 ± 8 .4 b 16 .9 ± 0 .9 a is oe ug en ol 2. 6 ± 0. 1a 7. 3 ± 0. 1b 7. 9 ± 0. 1b 5. 2 ± 0. 1a 23 .0 ± 0 .1 c 22 .0 ± 1 .0 c 24 .5 ± 1 .5 d 10 .8 ± 0 .7 ab 7. 0 ± 0. 1a m et hy lv an ill at e 30 .5 ± 0 .2 a 19 2. 5 ± 11 .9 b 24 .7 ± 0 .2 ª 45 .1 ± 0 .4 a 19 4. 9 ± 10 .7 c 32 .0 ± 2 .9 a 19 .8 ± 1 .7 a 13 4. 8 ± 0. 4b 11 .8 ± 0 .2 a b en zy la lc oh ol 5. 9 ± 0. 2a 7. 9 ± 0. 2a 6. 8 ± 0. 1a 12 .2 ± 0 .2 b 11 .3 ± 1 .1 c 3. 4 ± 0. 2a b 4. 8 ± 0. 2b 12 .2 ± 0 .1 c 2. 5 ± 0. 1a 2ph en yl et ha no l 22 5. 4 ± 13 .6 b 44 3. 8 ± 24 .3 c 17 7. 4 ± 13 .7 a 23 4. 1 ± 21 .1 b 28 5. 1 ± 18 .7 c 92 .4 ± 2 .1 b 84 .7 ± 2 .0 b 24 1. 7 ± 13 .0 c 77 .2 ± 6 .2 a va ni lli n 18 .8 ± 0 .2 c 13 .8 ± 0 .7 b 3. 9 ± 0. 2a 13 .4 ± 1 .8 b 12 .4 ± 0 .8 ab 10 .2 ± 1 .0 ab 9. 1 ± 0. 7a b 25 .4 ± 2 .3 c 2. 8 ± 0. 1a m et ho xy eu ge no l 5. 7 ± 0. 2b   5. 3 ± 0. 1b   3. 9 ± 0. 1a 2. 2 ± 0. 2a 2. 3 ± 0. 0b 3. 4 ± 0. 1c 5. 6 ± 0. 3d 0. 4 ± 0. 0a   3. 8 ± 0. 1c s yr in ga ld eh yd e 56 .3 ± 0 .4 ab 4. 6 ± 0. 1a 45 .9 ± 2 .2 ab 36 .8 ± 3 .2 ab 68 .7 ± 3 .4 b 16 .7 ± 0 .9 ab 26 .3 ± 1 .9 ab 10 .2 ± 0 .7 ab 7. 0 ± 1. 0a to ta l 43 1. 9 ±2 0. 6b 73 2. 3 ± 37 .8 c 31 6. 5 ± 17 .0 a 40 9. 4 ± 27 .7 b 68 1. 7 ± 40 .8 c 26 4. 3 ± 14 .4 ab 22 1. 0 ± 10 .9 ab 63 4. 8 ± 35 .1 c 14 5. 1 ± 8. 9a *m ea n ± st an da rd d ev ia tio n (n =3 ). d iff er en t s up er sc rip te d le tte rs in di ca te s ig ni fic an t d iff er en ce s fo r p ≤ 0 .0 5 (a na ly si s of v ar ia nc e or t uk ey te st ). n. d. , n ot d et er m in ab le . italian journal of food science, 2021; 33 (4) 39 analysis of aroma compounds of nine autochthonous and non-autochthonous loquat cultivars grown in sicily table 4. odor descriptor and odor threshold volatile compounds. compuonds odor descriptor odour threshold (ppb) reference acids butyric acid rancid, cheese 173 fariña et al. (2015) pentanoic acid sweet 70 pino and mesa (2006) hexanoic acid fatty, cheese 420 fariña et al. (2015) heptanoic acid waxy, cheese, fruity – www.thegoodscentscompany.com/ octanoic acid fatty, cheese 500 fariña et al. (2015) nonanoic acid green, fatty 3000 pino and mesa (2006) decanoic acid rancid, fatty 1000 fariña et al. (2015) dodecanoic acid fatty, waxy – www.thegoodscentscompany.com/ tetradecanoic acid waxy, oily, fatty – www.thegoodscentscompany.com/ pentadecanoic acid waxy – www.pherobase.com hexadecanoic acid oily – www.pherobase.com heptadecanoic acid oily – www.pherobase.com octadecanoid acid oily – www.pherobase.com benzoic acid balsamic – alcohols 1-pentanol green, grassy, powerful 4,000 pino and mesa (2006) 3-heptanol herbal – www.thegoodscentscompany.com/ 2-hexanol chemical, winey 500 pino and mesa (2006) 1-hexanol fatty, green, resin, flower, sweet 500 bonneau et al. (2016) 3-ethyl-3-heptanol – cis-3-hexen-1-ol green, moss, fresh 110 bonneau et al. (2016) 2-butoxyethanol – trans-3-hexen-1-ol green, grass, fruity 70 bonneau et al. (2016) 2-ethyl-hexanol oily, rose, sweet – li et al. (2011) aldehydes trans-2-hexenal green, banana-like 17 pino and mesa (2006) nonanal fatty, citrus, green, floral, sweet, soapy 1 bonneau et al. (2016) benzenoid vanillin vanilla-like, sweet 25 bonneau et al. (2001) esters ethyl hexanoate apple peel, fruity 1 pino and mesa (2006) ethyl octanoate fruity, fat 194 pino and mesa (2006) ethyl-3-hydroxy butyrate fruity, grape 1000 moyano et al. (2002) ethyl decanoate sweet, oily, nutlike, grape 6300 pino and mesa (2006) diethyl succinate overripe melon, lavender 100,000 fariña et al. (2015) 2-methylbutanoic acid cheese 250 fariña et al. (2015) methyl hexadecanoic waxy – www.thegoodscentscompany.com/ ethyl hexadecanoic waxy – www.thegoodscentscompany.com/ (vanillin only, according to hideki et al. 1998). the identified acids range from c4 to c18, the esters from c4 to c10 and c16; they contribute to the taste sensations perceptible in the mouth during tasting and eating of the flesh, giving taste, fat sensation, and different aromatic sensations, ranging from fruity to floral (table 4). the greater acids concentration are: hexanoic (1060.9±44.7 in brt20, local cultivar), benzoic (1185.2  mg/kg flesh fruit in claudia, local cultivar), hexadecanoic (667.6 mg/ kg flesh fruit in bueno, international cultivar), and octadecanoic (171.7 mg/kg flesh fruit also in bueno cultivar). in terms of bad tastes, the highest values of butyric and decanoic acid, commonly detectable in cheese and fat www.thegoodscentscompany.com/� www.thegoodscentscompany.com/� www.thegoodscentscompany.com/� www.pherobase.com� www.pherobase.com� www.pherobase.com� www.pherobase.com� www.thegoodscentscompany.com/� www.thegoodscentscompany.com/� www.thegoodscentscompany.com/� 40 italian journal of food science, 2021; 33 (4) planeta d et al. demonstrating that loquat does not have a high supply of sugar-related flavors, which can be released and perceived after swallowing the flesh (flavor). among the identified glycosylates, there is a clear predominance of compounds belonging to the class of benzenoids and c13-norisoprenoids followed by terpenes, the latter is present at low concentrations and with significant differences between cultivars. among the benzenoids, the presence of eugenol, 4-vinylguaiacol, vanillin, syringaldehyde, methylvanillate, and 2-phenylethanol was shown according to the study outcome of chen et al. (2011) and shaw and wilson (1982). among the c13-norisoprenoids and terpenes presence of vomifoliol and 3-oxo-α-ionol and their dehydroxylated forms is recorded. important compounds that contribute to aromatic sensations range from fruity to floral (table 5). the international cultivars tend to have a higher concentration of these compounds, especially algerie and golden nugget. among the local cultivars, claudia has the highest values. higher values for total benzenoids are observed in claudia (local cultivar), golden nugget, and algerie (international cultivars); higher c13-norisoprenoids in algerie and golden nugget; the latter also had higher total terpenes. as in all cultivars, the synthesis of terpene compounds is shifted towards dehydroxylated forms (trans-8-hydroxylinalool and cis-8-hydroxylinalool), and geraniol is present only among monohydroxylates. notes was observed only in bueno and el buenet (both international cultivars). among the esters, higher amounts of ethyl-3-oh-butyrate and 2-methyl-butanoic acid were observed. the very limited presence of c6 alcohols, deriving from the enzymatic activities of lipoxygenase, highlights how loquat is poorly endowed with these enzymes, which lead to the formation of herbaceous aromas that are not always pleasant, or that the sample preparation was done correctly. the different cultivars examined show significant differences in ester and alcohol content: international algerie and bueno cultivars tend to have the highest values. the total contents of acids, aldehydes and benzenoids show a similar profile in most cultivars. significant differences were recorded for total acid content in claudia (local cultivar) which represent the highest values (2792 mg/kg flesh fruit) and in peluche (international cultivar), which represent the lowest values (1245 mg/kg flesh fruit); in total aldehydes, algerie has significantly higher values and brt 20, el buenet and golden nugget are lower; finally, benzenoids are present in higher amounts in bueno. glycosylated volatile compounds a limited number of glycosylates, released by enzymatic hydrolysis, have been recorded in the studied cultivars, table 5. odor descriptor and odor threshold volatile compounds. compounds odor descriptor odor threshold (ppb) reference terpenes linalool floral, lavender 6 pino and mesa (2006) trans-8-hydroxy-linalool sweet, floral, creamy – www.thegoodscentscompany.com/ cis-8-hydroxy-linalool sweet, floral, creamy – www.thegoodscentscompany.com/ geranial citrus-like, flowery, fruity 32 pino and mesa (2006) c13-norisoprenoids 3-oxo-a-ionol spicy, woody, violet – www.pherobase.com 3,4-dihydro-3-oxo-actinidol 1 – www.pherobase.com 3-oh-b-ionone flower, violet – www.thegoodscentscompany.com/ vomifoliol fruity – www.pherobase.com benzenoids eugenol clove, spicy, balsamic 6 pino and mesa (2006) 4-vinylguaiacol clove, curry 3 pino and mesa (2006) isoeugenol flower 6 escudero et al. (2007) methyl vanillate caramel, butterscotch, vanilla 990 escudero et al. (2007) benzylalcohol sweet, flower – www.pherobase.com 2-phenylethanol hawthorne, honey, sweet 1100 pino and mesa (2006) methoxyeugenol sweet, flower – www.pherobase.com syringaldehyde sweet, cocoa, chocolate 50,000 escudero et al. (2007) www.thegoodscentscompany.com/� www.thegoodscentscompany.com/� www.pherobase.com� www.pherobase.com� www.thegoodscentscompany.com/� www.pherobase.com� www.pherobase.com� www.pherobase.com� italian journal of food science, 2021; 33 (4) 41 analysis of aroma compounds of nine autochthonous and non-autochthonous loquat cultivars grown in sicily conclusion among local cultivars, only cv claudia had higher values of glycosylated compounds highlighting the floral and acidic notes that are appreciated in the market. regarding the international cultivars, all yellow fleshed cultivars had a higher number of free aromatic compounds that showed cheese and fat notes (butyric and decanoic acid) and other odors and flavors less appreciated by consumers. they have fewer acids and a greater number of glycosylated compounds that showed characteristic floral and woody notes. this article tends to highlight the importance of local and international cultivars in mediterranean environments that are grown with the best market characteristics. references agozzino p., avellone g., filizzola f., farina v. and bianco r.l. 2007. changes in quality parameters and volatile aroma compounds in ‘fairtime’ peach during fruit development and ripening. ital. j. food sci. 19(1): 3. available from: https://www.researchgate. net/publication/236969721 agusti m., juan m., almela v. and gariglio n. 2000. loquat fruit size is increased through the thinning effect of naphthalene acetic acid. plant growth regul. 31: 161–171. https://doi. org/10.1023/a:1006376219543 amorós a., zapata p., pretel m.t., botella, m.a. and serrano m. 2003. physico-chemical and physiological changes during fruit development and ripening of five loquat (eriobotrya japonica lindl.) cultivars. food sci. technol. 9(1): 43–51. https://doi.org/ 10.1177%2f1082013203009001007 badenes m.l., janick j., lin s., zhang z., liang g.l. and wang w. 2013. breeding loquat. in j. janick (ed.). plant breeding reviews, ch. 5, vol. 37. wiley-blackwell, hoboken, nj. baldwin e.a. 2004. flavor. in k.c. gross, c.y. wang and m.e. saltveit (eds.), the commercial storage of fruits, vegetables, and florist and nursery stocks. agricultural research service, washington, dc. barone f., farina, v. and lo bianco r. 2010. growth, yield and fruit quality of peluche loquat under windbreak nets. in iii international symposium on loquat acta. hortic. 887: 155–159. https://doi.org/10.17660/actahortic.2011.887.25 besada c., salvador a., sdiri s., gil r. and granell a. 2013. a combination of physiological and chemometrics analyses reveals the main associations between quality and ripening traits and volatiles in two loquat cultivars. metabolomics. 9(2): 324–336. https://doi.org/10.1007/s11306-012-0447-z besada c., sanchez g., gil r., granell a. and salvador a. 2017. volatile metabolite profiling reveals the changes in the volatile compounds of new spontaneously generated loquat cultivars. int. food res. 100 (1): 234–243. https://doi.org/10.1016/j. foodres.2017.06.068 bonneau a., boulanger r., lebrun m., maraval i. and gunata  z. 2016. aroma compounds in fresh and dried mango fruit (mangifera indica l. cv. kent): impact of drying on volatile composition. int. j. food. sci. technol. 51: 789–800. https://doi. org/10.1111/ijfs.13038 chen f.x., lui x.h., chen l.s. and zheng s.x. 2011. the determination of volatile constituents of loquat fruit and leaf by gas chromatography-mass spectrometry coupled with solid-phase microextraction. acta. hortic. 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corona2, a. magri3 and m. petriccione3 1department of industrial engineering, university of salerno, via giovanni paolo ii, 84084 fisciano, sa, italy 2department of agricultural, food and forest sciences university of palermo, viale delle scienze, 90128 palermo, pa, italy 3council for agricultural research and economics. research centre for olive, citrus and tree fruit, via torrino 3, 81100 caserta, italy *corresponding author: luciano.cinquanta@unipa.it abstract in this study, the effects of cold storage (5±0.5°c and relative humidity of 90±1%) on the quality of fresh papaya slices packed in a passive atmosphere with a semi-permeable film were evaluated. physico-chemical traits such as total soluble solids, reducing sugar, ph increased during storage as well as the polyphenols, carotenoid content and antioxidant activity that reaching the highest values at end of trials. changes in colorimetric parameters resulted in a significant decrease after 4 days of hue angle values, which then remained constant. the cutting process enhanced the antioxidant enzymes activity such as superoxide dismutase, catalase and ascorbate peroxidase. the analysis of the main components showed physical-chemical, qualitative, and enzymatic changes in papaya samples during cold storage, showing a shift from negative to positive values along the pc1 and indicating a qualitative decay of sliced papaya. keywords: papaya, minimally processing, enzymes, color, antioxidant, packaging ital. j. food sci., vol. 32, 2020 846 1. introduction papaya (carica papaya l.) is the fifth most widely produced tropical fruit worldwide after mango, banana, pineapple, and avocado, with 13.0 million tons per year (liu et al., 2019). papaya is a perennial herbaceous plant recently spread in spain and italy (farina et al., 2020a), where, in recent years, papaya has adapted to the mediterranean climate under sheltered structures (farina et al., 2020b) with good quality results (farina et al., 2020a), like other new crops (niro et al., 2017). its cultivation is supported by a constant demand for freshly cut papaya, especially in europe, mainly to young consumers and "baby boomers", who eat it as a snack (james et al., 2010). papaya is a type of climacteric fruit whose maturation after harvest is accompanied by tissue softening and microbial growth (gonzalez-aguilar et al., 2009). the proximity to european markets makes it possible to harvest the climacteric fruits close to their ripening stage, allowing for excellent organoleptic attributes (gentile et al., 2019) that reduce the number of kilometers of food and greenhouse gas emissions. in this context, the search for methods that use simple operations and equipment to improve the shelf life of minimally processed papaya is of interest to farmers and consumers (jayathunge et al., 2014). several studies have demonstrated an increase in the reactive oxygen species (ros) level after the cutting process in fresh fruit such as carrot (jacobo-velázquez et al., 2011), zizania latifolia (liu et al., 2012), and pitaya (li et al., 2017). ros have harmful effects in various cellular compartments at high levels, but these compounds may also act as signaling molecules in ripening and senescence processes in response to cutting stress (jacobo-velázquez et al., 2011). however, the balance between ros generation and scavenging is closely modulate by antioxidant defense system characterized by enzymatic and non-enzymatic components in fresh-cut fruit (hodges et al., 2008). different enzymes control the intracellular metabolism of ros, such as superoxide dismutase (sod), ascorbate peroxidase (apx), glutathione peroxidase (gpx) and catalase (cat), which modulate the ros level. the cutting process induces an increase of ros above threshold levels, with damages to cell membranes due to lipid peroxidation with a high lipoxygenase activity and malondialdehyde content (karakurt and huber, 2003; souza et al., 2015; wu et al., 2019). furthermore, in fresh cut fruit have been observed a high enzymatic browning due to loss of cellular compartmentalization that allows to the polyphenol oxidases (ppos) come in contact with phenolic compounds causing fruit color changes (wu et al., 2019). the aim of this research was to study the effect of cold storage (5°c and rh of 90±1%) and passive atmosphere packaging on the shelf-life of minimally processed papaya fruit grown in mediterranean climate under greenhouse, by monitoring the variations in physico-chemical, microbiological and nutraceutical traits. in addition, in order to assess the physiological stress induced by the cutting and slicing process, the enzymatic oxidative system and the markers of oxidative damage have also been studied. 2. materials and methods 2.1. fruit samples and experimental design ‘formosa’ papaya fruits were picked in a commercial orchard in palermo, italy (33s 0333746 m.e, 4217131.00 m.n). the fruits were harvested at stage 3 (one or more orangecolored stripes in skin; pulp almost completely orange in color, except near skin, still hard but contains less latex), using the skin color of the fruit as a maturity index (basulto et ital. j. food sci., vol. 32, 2020 847 al., 2009). the subsample of 25 fruits (5 per tree) was selected. fruits were stored before the evaluation under a controlled temperature (18°c, 90% rh) and analyzed when papayas reached the stage 4 (basulto et al., 2009) after 3 days. 9 subsamples of the fruit were submitted to physical-chemical determination whereas 6 subsamples of the fruit were washed with cold tap water and peeled with the help of a stainless-steel knife; the seeds were removed, and the fruits were cut into small pieces approximately 2.5 cm thick. about 120 g of the cut samples were placed inside sanitized plastic trays (142 x 95 x 50 mm) and wrapped with a film cryovac sealappeal psf. the characteristics of the film are as follows: thickness 25 μm; transmission rate of co2 800 cm3m−2day-1 at 23°c and 0% rh; transmission rate of o2 72 cm3m−2day-1 at 4°c and 0% rh; moisture transmission vapor 47 gm−2day-1 at 38°c and 100% rh. three replicates were prepared for each sampling time (9 packages) that were stored for 12 days at 5°c and rh of 90±1%. samples were analyzed every four days and for each biological replicate were realized two technical replicates. the chosen time of storage adopted in this study emerged from preliminary assays performed to determine the probable shelf life resulted in early mold formation on the 12th day at 5ºc (data is not shown). 2.2. determination of physico-chemical traits 2.2.1 skin color (raw fruit) for the skin cover color index (cc) evaluation (at harvest and consumption points), we used the fruit analysis system (fas) procedure in agreement with farina et al. (2011). 2.2.2 flesh color chromaticity values l* (lightness), a* (green to red), and b* (blue to yellow) of flesh color fruit was determined with a colorimeter (minolta c2500. konica, ramsey, ny), chroma (chr) and hue angle (hue) were also calculated (farina et al., (2020c). 2.3. physico-chemicals analyses a digital scale (gibertini, italia) was used for determining the fresh weight (fw) and the size code was determined (cbi, 2018). flesh firmness (ff) was measured using a digital penetrometer tr5325 (turoni, forlì, italy) with a cylindrical needle (8 mm diameter) and values expressed in newtons (n). flesh juice was used to detect the total soluble solids content (tss) with an optical digital refractometer (atago, japan), titratable acidity (ta) using a compact titrator (crisom, spain) and expressed in g citric acid/100 g fresh fruit and ph with a digital ph-meter (model 2001, crison, barcelona, spain). reducing sugars were evaluated through a volumetric fehling assay as described previously by adiletta et al. (2018). 2.4. carotenoids, polyphenols content and antioxidant activity spectrophotometric detection of carotenoids content, xanthophylls plus carotenes (car), was determined in agreement with petriccione et al. (2015) and results were expressed as µg 100 g−1 fw applying wellburn equations (wellburn, 1994). the total phenolic compounds (tp) were assessed as reported by magri et al. (2020). the free radical ital. j. food sci., vol. 32, 2020 848 scavenging activity was gauged by 1,1-diphenyl-2-picryl-hydrazil (dpph) according to cinquanta et al. (2013). 2.5. evaluation of antioxidant enzymatic system total soluble proteins were obtained from fresh papaya (1:3 w/v) blended in an extraction buffer prepared in agreement with magri et al. (2020) and determined by the bradford assay. catalase activity (ec 1.11.1.6) (cat) was estimated in according to the method described by adiletta et al. (2019) using 100 µl of crude enzyme extract. the activity was expressed in µmol of h2o2 g−1 fw. superoxide dismutase activity (ec 1.15.1.1) (sod) was evaluated with the method of nitroblue tetrazolium (nbt) reduction inhibition in agreement with adiletta et al. (2018a) using 50 µl of crude enzyme extract. sod activity was expressed as u mg-1 fw, considering that one sod unit corresponds to the amount of enzyme that, in the assay conditions, inhibits 50% the nbt reduction. guaiacol peroxidase activity (ec 1.11.1.7) (gpx) was determined according to petriccione et al. (2015) and expressed as nmol g−1 fw. ascorbate peroxidase activity (ec 1.11.1.7) was assessed as reported by adiletta et al. (2018b). the activity was expressed as µmol g−1 fw. hydrogen peroxide content was estimated with the method reported by goffi et al. (2020) and expressed as nmol g-1 fw. 2.6. oxidative damage markers and enzymatic browning polyphenoloxidase activity (ec.1.10.3.1) (ppo) was established with the method described by adiletta et al. (2018b) and ppo assay was carried out with 10 µl of crude enzyme extract. ppo activity was expressed as µmol g−1 fw. malondialdehyde content (mda) was determined as described by adiletta et al. (2018b) and expressed as nmol g-1 fw. lipoxygenase activity (ec 1.13.11.12) (lox) was determined in according to adiletta et al. (2018b) and expressed in nmol m-1g-1 fw, as the specific rate of molar change of hydroperoxides. 2.7. microbiological analysis total aerobic bacteria (tab) and yeast and mold count (ym) were conducted using a method designated by yousuf and srivastava (2015). results were reported as log colony forming units per gram. 2.8. statistical analysis analysis of variance (anova) and duncan’s test at a 5% level were used to compare the differences between samples analysed during storage. principal components analysis (pca) was realized to reduce the multidimensionality of dataset generating new principal components that account for most of the total variation. all statistical analyses were realized by spss software package, version 20.0 (spss inc., chicago, il, usa). ital. j. food sci., vol. 32, 2020 849 3. results and discussion 3.1. fresh fruits the examined fruits were classified in h size (1101 1500 g) in according to cbi size codes a–j used for marketing channels (codex stan, 2001). consumers tend to prefer smaller papayas, particularly in northwestern europe; because they suit individual consumption better and consider the fresh-cut papaya more convenient than the whole fruit (which should be peeled, deseeded and sliced before consumption) and they find the large size of some cultivars off-putting (rivera-lópez et al., 2005). fruit with <55% yellow skin was the best to slicing and deseeding while those with <25% yellow skin showed no soft and edible flesh. in this study, papaya fruit were harvested at mature green stage 3 (basulto et al., 2009) and was characterized by some orange-colored stripes in skin; pulp almost completely orange in color, except near the skin, but still hard for consumption. in this stage, the color of the skin changes from dark green to light green and one or more yellow streak begins to develop from the base upwards. the cover color analysis revealed 33.3% of skin cover color at picking; afterwards fruits were let to ripe at room temperature reaching 59.6 % of yellow skin color after 4 days. our results agree with yahia (2011), who suggested to select whole fruit with 55-80% of yellow skin which ensures > 50% of edible flesh recovery for production of fresh-cut papaya because fully ripe fruit were easily bruised and difficult to handle yahia (2011). nonetheless, after these storage conditions the fruit reached good physico-chemical characteristics for freshcut processing (table. 1). l*, chroma and hue values were comparable with those reported by gayosso-garcía et al. (2011) in the flesh of raw papaya fruit and by farina et al. (2020b) in fresh-cut coated fruit; similar tss, ta (zuhair et al., 2013) and ph values were observed by zuhair et al. (2016). finally, flesh firmness values were in agreement with other studies carried out on papaya fruit at the same ripening stage grown in tropical (farina et al., 2020a) and mediterranean area (farina et al., 2020b). 3.2. physico-chemical traits in fresh-cut fruits formosa papaya has fruit with a large size and orange flesh, for these features are generally valued as minimally processed fruits. in papaya fruit, flesh colour changes can be attributed to different regulation of carotenoid biosynthesis, a secondary metabolic pathway that yield metabolites also destined to other important fruit qualitative features (yahia, 2011; shen et al., 2019). furthermore, the increase in metabolic activities also causes flesh colour changes during fresh-cut processing (rivera-lópez et al., 2005). the intensity and uniformity colour of flesh fruit affect its quality and consumer’s choice and preference (rivera-lópez et al., 2005). samples showed a significant decrease in l*after 4 days of cold storage, followed by a constant trend up to 12 days (fig. 1a). furthermore, the results showed a significant increase in redness a* (from 46.3 to 52.3) and decrease in yellowness b* (from 56.2 to 48.6) after 12 days of storage (figs. 1b and c). waghmare and annapure (2013) observed a similar trend in passive atmosphere packaging, and jayathunge et al. (2014), using micro-perforated polyvinyl chloride containers. as a result, changes in colorimetric parameters resulted in a significant decrease after 4 days of hue angle values, which then remained constant (fig. 1d), while no significant differences were registered in chroma values during storage conditions (data not shown). in other word, red colour increased its intensity in all samples during the storage period; this was due to the degradation of chlorophylls or unmasking of preformed pigments during fruit ital. j. food sci., vol. 32, 2020 850 development. the colour changes ranged from yellow to reddish orange and were associated with ripening progress, as well as at the onset of browning. tss in fresh-cut papaya tended to increase during cold storage with significant differences during storage time (table 1). the highest tss (8.51%) value in papaya samples was found on day 12. waghmare and annapure (2013) reported a similar trend in tss, explaining it by the solubilisation and synthesis of carbohydrates in fresh-cut papaya packed in polypropylene film with and without modified atmosphere and stored for 25 days at 5°c. moreover, the low temperature helped to maintain a low level of respiration rates stopping the decrease in tss content (rivera-lópez et al., 2005). reducing sugar slightly increased in fresh-cut papaya during storage showing a high positive correlation (r2=0.969 p < 0.01) with tss (table 2). the ph values increased significantly during storage, ranging from 5.51 to 5.64 after 12 days of cold storage. figure 1. evaluation of colorimetric traits, lightness l*(a), redness index a* (b), yellowness index b* (c) and hue angle h° (d) in cut ‘formosa’ papaya fruit packaged in passive atmosphere stored at 5°c for 12 days. means ± standard deviations followed by the same letter do not differ significantly at p = 0.05 (duncan test). 0 days 4 days 8 days 12 days l* 0 10 20 30 40 50 60 70 b a a a a 0 days 4 days 8 days 12 days a* 0 10 20 30 40 50 60 70 a ab b b b 0 days 4 days 8 days 12 days b* 0 10 20 30 40 50 60 70 b a a a c 0 days 4 days 8 days 12 days h ue a ng le ( h ∞) 0 10 20 30 40 50 60 70 b a a a d ital. j. food sci., vol. 32, 2020 851 table 1. pomological traits of the formosa papaya cultivar after three days of ripening at room temperature (18°c rh 90%). values represented as mean ±sd (n=9). fruit weight (fw), solid soluble content (tss), titratable acidity (ta), firmness (f), juiciness (j), skin cover color (cc), lightness(l*), chroma (chr), hue angle (hue). table 2. changes in total soluble solid (tss), reducing sugar (rs) and ph in cut ‘formosa’ papaya fruit packaged in passive atmosphere stored at 5°c for 12 days. storage time tss rs ph time 0 8.03±0.02 a 2.23±0.11 a 5.51±0.01 a 4 days 8.02±0.02 a 2.29±0,22 b 5.56±0.01 ab 8 days 8.09±0.01 b 2.42±0.13 c 5.59±0.01 bc 12 days 8.51±0.01 c 2.79±0.41 d 5.64±0.01 c means followed by the same letter do not differ significantly at p = 0.05 (duncan test). 3.2.1 microbial growth in cut fruit fresh-cut fruit is more prone to the rapid growth of spoilage microorganisms as well as the pathogens of public health significance. peeling process eases cross-contamination and the transfer of microflora from peel to the flesh fruit that represents an optimal substrate for microbial growth. cold storage of fresh-cut papaya leads to an increase in tab and ym values. tab increased in all stored samples ranging from 1.8 (0 day) to 5.6 log cfu/g (12 days) (data not shown). these values were lower than the critical limit for total microbial loads of vegetables (8.0 log cfu/g) (jacxsens et al., 2002). ym values increased from 1.3 to 5.2 log cfu/g, overcoming the critical limits of 5 log cfu/g for yeasts (jacxsens et al., 2003) after 12 days of storage. increased trend in microbial counts of tab and ym throughout the storage of fresh-cut papaya were also reported by gonzalez-aguilar et al. (2009) and waghmare and annapure (2013), correlated to the packaging systems, storage temperatures and different cut types of fresh-cut fruits. 3.2.2 free radical scavenging power components in fresh cut fruits papaya is a tropical fruit with a high concentration of bioactive compounds such as polyphenols, vitamins and carotenoids, whose interactions contribute to the overall antioxidant activity of this fruit. carotenoids are a group of fat-soluble molecules responsible to yellow-red color of fruits and vegetables. ripening, cold storage and postharvest treatments can influence carotenoids content in fresh-cut fruit (supapvanich et al., 2020). the samples tested showed an increase in the carotenoids content during storage time, ranging from 570±12 (0 days) to 1600±92 µg 100 g fw-1 (12 days) (fig. 2a). our results were in agreement with fajar falah et al. (2015) who evaluated fresh-cut ‘bangkok’ papaya at different storage temperatures suggesting that this climacteric fruit continues to ripe during storage. in addition, it can also be assumed that carotenoids are more extractable due to changes in cell structure during storage time. tp fw (g) tss (brix°) ta (g/l) f (n) j (ml/100 g) cc (%) l* chr hue 1380±20 8.03±0.9 0.8±0.21 33.42±3.8 46.21±2.6 59.6% 62.85±7.9 60.96±7 1.02±0.3 ital. j. food sci., vol. 32, 2020 852 content significantly increased during 12 days of cold storage in fresh-cut ‘formosa’ papaya ranged from 19.1±1.8 (0 day) to 54.3±4 (12 days) mg gae 100 g-1 fw (fig. 2b). wounding stress caused by cutting process might contribute to an increase of secondary metabolites such as phenolic compounds. several studies have evaluated tp content in different papaya fruit highlighting that several factors such as fruit ripening, agronomic practices and post-harvest storage conditions affect the content of these bioactive compounds (ali et al., 2014; zuhair et al., 2013). throughout the 12 days of cold storage, the dpph radical-scavenging activity significantly increased (p<0.05) in stored samples (fig. 2c). bioactive compounds can act as antioxidants and our results indicated that high antioxidant activity in fresh-cut papaya fruit was related to the increase of polyphenols (r2 = 0.840; p<0.01) content due to ripening process occurring during storage. figure 2. carotenoids content (a; µg 100 g-1 fw), polyphenols content (b; mg gae 100 g-1 fw), antioxidant activity (dpph assay) (c; µmol te g-1 fw), malondialdehyde content (d; nmol g-1 fw) in cut ‘formosa’ papaya fruit packaged in passive atmosphere stored at 5°c for 12 days. means followed by the same letter do not differ significantly at p = 0.05 (duncan test). 0 days 4 days 8 days 12 days µm ol /g f w 0 1 2 3 4 5 a b c d c 0 days 4 days 8 days 12 days nm ol / g f w 0 20 40 60 80 100 a b b bd ital. j. food sci., vol. 32, 2020 853 3.2.3 antioxidant system in cut fruits the stress by cutting process induces a physiological disorder with an alteration in cellular homeostasis increasing the ros production in damaged cells. in fresh-cut fruit, the imbalance between production and accumulation of ros could be due to enhancing the respiration rate and activation of amineand nadph-oxidases (mittler, 2002). h2o2 level increased rapidly during the first 4 days from cutting process and this trend continued up to 12 days of storage. h2o2 acts as a second messenger for the induction of several defense genes in crops in response to wounding (table 3). our results highlighted marked changes in enzymatic oxidative system due to the exposure to wounding stress in fresh-cut fruit. the superoxide dismutase (sod) activity of fresh-cut papaya showed a slow increase through storage time, with minimal significant changes from 15.4±2 u mg-1 fw (0 day) to 20.2±4 u mg-1 fw (12 days) (table 3). during the first 8 days of storage, the ascorbate peroxidase (apx) activity had no significant changes until 8 days, with an average value of 0.87±0.12 µmol g-1 fw, while its activity significant increased at the end of storage (12 days) (table 2). a significant increase was registered in catalase (cat) activity that during storage time up to 14.9±2 µmol g-1 fw (table 3). our results suggest that an increase of activities of antioxidant enzymes such as sod, cat, and apx, could improve the ability of the fresh-cut fruit to dismutate superoxide radicals and to eliminate hydrogen peroxide. in papaya, these enzymes prevent oxidative damage and reduce the susceptibility to chilling injury at low-temperature storage (hanif et al., 2020). in cut fruit, antioxidant enzymes can modulate ros levels and cat and apx activities increased when ros reached toxic levels. at low and moderate levels, ros can act as signaling molecules and in cut fruit such as pitaya and strawberry mediating woundinginduced phenolic accumulation (li et al., 2017; jacobo-velazquez et al., 2015). 3.2.4 oxidative damage in cut fruits the cut fruit showed a high perishability due to the peeling and cutting processes that caused cell disruption and membrane damage with decompartmentation of cellular structures, cellular functions and quality loss (pal et al., 2004; jacobo-velazquez et al., 2015). in cut tissues, several enzymes come into physical contact with their substrates afterwards the cellular damages due to the cutting process (karakurt and huber, 2003). browning is the result of enzymatic oxidation of phenolic compounds in lightly processed fruit (jacobo-velazquez et al., 2015). polyphenoloxidase (ppo) and gpx are the main intracellular oxidative enzymes involved in enzymatic oxidation in stored fresh-cut fruit. ppo and gpx activities increased significantly during storage in the freshcut papaya up to 2.5and 3.1-fold, respectively at the end of the experiment (table 3). this suggests that the browning of fresh-cut papaya is primarily due to phenolic compounds oxidation caused by ppo and gpx activities. in the cut papaya, fruit lipoxygenase (lox) activity significantly increased throughout cold storage with values ranging from 12.4±1 nmol g-1fw (0 days) to 36.2±3 nmol g-1fw (12 days) (table 3). instead, mda content increased rapidly (47 %) during the first 4 days and then was stable around 88.9±5 nmol g-1 fw up to the end of storage (fig. 2d). as suggested by karakurt and huber (2003), lox activity is involved in peroxidative lipid metabolism with a possible relationship with tissue softening in fresh-cut and whole ‘sunrise solo’ papaya during cold storage. several studies have demonstrated that loxs are ripening-related enzymes in papaya fruit (farina et al., 2011). our results confirm that in fresh-cut papaya the membrane lipid peroxidation occurred during cold storage. mda content is useful to ital. j. food sci., vol. 32, 2020 854 evaluate the cell oxidative damage during storage and the effectiveness of post-harvest treatments in several fresh-cut fruit samples (gonzalez-aguilar et al., 2009; souza et al., 2015; jacobo-velázquez et al., 2011). table 3. evaluation of superoxide dismutase (sod; u mg-1 fw), catalase (cat; µmol g-1 fw), ascorbate peroxidase (apx; µmol g-1 fw), guaiacol peroxidase (gpx; nmol g-1 fw), polyphenoloxidase (ppo; µmol g-1 fw), lipoxygenase (lox; nmol g-1 fw) activity and h2o2 content (nmol g-1 fw) in cut ‘formosa’ papaya fruit packaged in passive atmosphere stored at 5°c for 12 days. storage time sod cat apx gpx ppo lox h2o2 time 0 15.4±2a 6.1±0.9a 0.8±0.1a 39.9±3a 0.4±0.05a 13±1.2a 0.02±0.01a 4 days 16.8±3ab 8.9±1b 0.9±0.1a 82.7±2b 0.4±0.1b 20±2.1b 0.06±0.01b 8 days 19.6±3ab 12.9±1c 0.9±0.1a 97.6±2c 0.6±0.1c 31±2.9c 0.11±0.03c 12 days 20.2±4c 14.9±1d 1.3±0.2b 124.8±7d 1.1±0.12d 39±3.8d 0.11±0.02c means followed by the same letter do not differ significantly at p = 0.05 (duncan test). 3.3. pca in cut fruit a dimensional map with loadings and scores plot obtained by pca analysis is shown in fig. 3. figure 3 principal component analysis of the physico-chemical, nutraceutical, and enzymatic traits in cut ‘formosa’ papaya fruit packaged in passive atmosphere stored at 5°c for 12 days. (tss: total soluble solid; ph; l*: lightness; a*: redness; b*: yellowness; hue: hue angle; chr: chroma; tp: total polyphenol content; car: carotenoid content; dpph: antioxidant activity; sod: superoxide dismutase, cat: catalase, apx: ascorbate peroxidase; ppo: polyphenoloxidase; gpx: guaiacol peroxidase; lox: lipoxygenase; mda: malondialdehyde content; h2o2: hydrogen peroxide content. ital. j. food sci., vol. 32, 2020 855 the first two principal components (pcs) explained 60.20% and 23.67% of the total variance, respectively. tss, ph, a*, cat, gpx, sod, apx, ppo, lox, h2o2 content, tp and car were positively correlated to pc1. pc2 showed a positive correlation with mda and dpph and a negative correlation with l*, b* and hue. a shift along the first two pcs of score values highlighted physico-chemical, qualitative and enzymatic changes in fresh-cut papaya samples during cold storage. at the beginning of cold storage, fresh-cut samples were situated in iii quadrant after four days a shift from negative to positive ones along pc2 was registered. as cold storage progressed, samples displayed a shift from negative values to positive ones along pc1 showing qualitative decay of the fresh-cut papaya. the quality of fresh-cut papaya fruit, which includes physico-chemical, microbiological and nutritional traits, was preserved for 8 days of cold storage, while oxidative damages and qualitative decay were observed as the storage period progressed (12 days). 4. conclusions the semi-permeable packaging and cold storage (5±0.5°c) have extended the post-harvest period up to 12 days of minimally processed 'formosa' papaya, preserving its microbiological and qualitative decay. physico-chemical and nutritional traits changed as storage progressed due to the physiological processes associated with the ripening stage in papaya fruit. cold storage leads to an increase in total aerobic bacteria, with lower values to the critical limit for total microbial loads of vegetables (8.0 log 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yahia e.m. 2011. postharvest biology and technology of tropical and subtropical fruits: fundamental issues. woodhead publishing, cambridge. yousuf b. and srivastava a.k. 2015. psyllium (plantago) gum as an effective edible coating to improve quality and shelf life of fresh-cut papaya (carica papaya). ijsrit 9(7):702-707. zuhair r.a., aminah a., sahilah a.m. and eqbal d. 2013. antioxidant activity and physicochemical properties changes of papaya (carica papaya l. cv. hongkong) during different ripening stage. int. food res. j. 20(4):1653. zuhair r.a., aminah a., sahilah a.m. and khalid h.m. 2016 evaluation of fruit leather made from two cultivars of papaya ital. j. food sci. 28:73-82. paper received may 14, 2020 accepted october 7, 2020 50 issn 1120-1770 online, doi 10.15586/ijfs.v34i2.1954 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (2): 50–66 p u b l i c a t i o n s codon exploring the potential of bottle gourd (lagenaria siceraria) flour as a fat mimetic in biscuits with improved physicochemical and nutritional characteristics and anti-diabetic properties syed muhammad ghufran saeed1, syed arsalan ali1*, rashida ali1,2,3, syed asad sayeed2 and lubna mobin1 1department of food science and technology, university of karachi, karachi, pakistan; 2department of food science and technology, jinnah women university, karachi, pakistan; 3english biscuits manufacturer (pvt.) limited, korangi industrial area, karachi, pakistan *corresponding author: syed arsalan ali, department of food science and technology, university of karachi, karachi, pakistan. email: 786syedarsalanali@gmail.com received: 20 october 2020; accepted: 12 may 2021; published: 28 april 2022 © 2022 codon publications open access paper abstract bottle gourd (lagenaria siceraria) is a naturally rich source of several phytochemicals that play a vital role in the prevention of diseases. in the present study, the effect of bottle gourd flour (bgf) as a fat mimetic in biscuits at different concentrations (10, 15, 20, 25, and 50% w/w) was explored. rheological properties, the microstructure of dough, bioactive compounds, anti-diabetic potential, storage stability, and the nutritional, physical, and sensory properties of samples were investigated. the addition of bgf in wheat flour (wf) showed increased water absorption (58.70–72.43%) and reduced dough stability time (11.71–5.01 min). trough (1165.11–1096.10 torque) and set back viscosities (471.21–448.09 torque) decreased significantly (p ≤ 0.05) with the increased level of bgf in wf. furthermore, gluten content was also reduced. the baking process decreased the ic50 values, although total phenol content and total flavonoid content decreased. shelf life study of 2 months revealed no significant (p ≤ 0.05) difference in bioactive compounds, and alpha-amylase inhibition activity of fat-replaced biscuits. moreover, the anti-hyperglycemic activity of bgf-wf biscuits in human subjects also showed positive results. the percentage increase in both peroxide value (0.2–2.87 milliequivalent o2/kg of fat) and free fatty acid (0.1–0.9%) during storage was significantly (p ≤ 0.05) lower than control biscuits (0.9–5.53 mill-equivalent o2/kg of fat and 0.2–1.1%), which correlates to the enhanced activity of bioactive compounds in bgf-wf biscuits, which in turn relates to decreasing the rate of oxidation. the crude fiber and protein contents of bgf-wf biscuits were improved from 0.21 to 17.30% and 11.20 to 18.70%, respectively. sensory and textural performance exhibited that the biscuits were acceptable after fat replacement of up to 15% bgf. based on this study, bgf may be suggested as an excellent natural fat replacer to be used in nutraceuticals and functional foods. keywords: anti-diabetic potential; antioxidant; bottle gourd flour; dough rheology; physical properties; sensory profile introduction obesity is one of the biggest challenges of the present world. people are becoming highly conscious of their body weight due to the increased risk of obesity-linked diseases like diabetes mellitus, hypertension, cardiovascular disease, and cancer. possible ways to control obesity are reducing the intake of fat, sugar, salt, limiting kilocalories in the daily diet, and increasing the consumption of high fiber vegetables and fruits. that’s why ingredients mailto:786syedarsalanali@gmail.com italian journal of food science, 2022; 34 (2) 51 bottle gourd flour as a fat mimetic in biscuits materials and methods raw materials commercial wheat flour (wf) made from wheat triticum aestivum sp. vulgare (hexaploid) was received from graib sons private limited, karachi. icing sugar, bg, whole fresh egg, and salt were purchased from the local market of karachi. semi-solid fat (partially unsaturated) was purchased from paracha mills ghee textile unit, karachi. glucose, sodium bicarbonate, and soy lecithin were obtained from sulop chemicals located in karachi. all chemicals used for the study were of analytical reagent grade procured from dae-jung chemicals, south korea and sigma-aldrich, germany. preparation of bgf the bgs were thoroughly washed in distilled water, peeled, and then deseeded. bg was manually cut into thin slices of thickness 0.002 cm and dried in a hot air oven (dso-300 d, digisystem laboratory instruments, taiwan) at 60°c for 8 h. dried bg flakes were milled by a laboratory miller (3100, perten instruments) and then passed through a sieve of 60 µm. the fine bottle gourd flour (bgf) was stored in airtight glass bottles at 4°c. varying concentrations of bgf [10, 15, 20, 25, and 50% (w/w)] were added to wheat flour as a fat mimetic to have sample flours of varying wheat to bgf ratios, represented as bgf-wf. proximate composition proximate profiles of bgf-wf blends were analyzed according to the method of ali et al. (2018). moisture was determined by moisture analyzer (brabender 51–55, cw brabender, duisbury, nj, usa). protein and ash contents were estimated by brabender kernelyzer (brains instruments, germany). fat content was estimated by aacc method 30-25 (aacc, 1999). carbohydrate content was calculated by subtracting the contents of protein, fat, ash, and moisture from 100. wet gluten (wg), dry gluten (dg), gluten index (gi), and total gluten contents (tgc) were estimated by glutomatic (2200, perten instruments). each sample was tested in triplicates for proximate analysis. dough rheological properties the effect of the incorporation of bgf in wheat flour in various concentrations on rheological properties was studied by using farinograph (mixer bowl 300g, brabender ohg, duisburg, germany) by the aacc that could replace fats and sugars are being extensively explored for applications in food industries. lagenaria siceraria is the most popular fruit vegetable throughout the world. it belongs to the family cucurbitaceae and is commonly called as bottle gourd (bg) or lauki. among the different vegetables grown in india and pakistan, bg is more commonly consumed because it is inexpensive and well-known for its medicinal properties (attar and ghane, 2019). several studies have revealed that bg served as an antidiabetic and anticancer agent. besides, bg is also used to treat various neurological disorders like alzheimer’s disease. nutritional evaluation reveals that it is a rich source of various nutrients such as vitamin b, c, pectin, fibers, beta-carotene, amino acids, proteins, and glycosides (attar and ghane, 2019). its pulp and seeds have been used to manufacture different food products; high fiber and protein-rich biscuits were prepared by incorporating bg seed powder in wheat flour (patel and pradhan, 2015). in another study, bg pulp was used for the preparation of bg sweet meat and was evaluated for sensorial and textural properties (saini and sharma, 2018). on the basis of its diverse functional properties, bottle gourd flour (bgf) can be considered as a great source of fat mimetic. for instance, the presence of the nonpolar compounds confers lipid soluble flavor-carrying capacity while polar groups facilitate water-binding, which together help to generate creaminess and lubricity in foods similar to that found in full-fat products (saeed et al., 2020a). numerous studies have been conducted based on the utilization of dietary fiber or inulin as a fat replacer in biscuits and analyzed for its chemical, physical, and sensory properties. saeed et al. (2021) studied the effect of fat replacement by date pit flour on the rheological and other quality parameters of the biscuits. furthermore, saeed et al. (2020c) studied the utilization of lotus root flour as a fat replacer in biscuits, while roman et al. (2015) proved that extruded wheat flour paste with the addition of emulsifier could be an active fat replacer in cakes formulation. although some studies have demonstrated favorable health effects of bakery products that contain fat replacer or fat mimetic components, no information is available on the use of bgf as a fat mimetic. therefore, this study was proposed to evaluate the functional properties of bgf and the effect of its incorporation at different levels (10–50%) on the physicochemical properties, rheological properties, microstructure of dough, nutritional profile, physical parameters (dimensional, color, and textural), and sensorial attributes of biscuits. furthermore, the dough and biscuits were evaluated for their antioxidant activity, bioactive compounds, anti diabetic properties, and storage stability, so that new type of functional biscuits with optimum sensory and nutritional benefits could be introduced at the industrial level. 52 italian journal of food science, 2022; 34 (2) saeed smg et al. electron microscopic (sem) studies were carried out on an applied voltage of 15 kv at 500´ magnification. biscuit preparation formulations of biscuits samples are presented in table 1. control and bgf-wf biscuits were prepared by incorporating different levels of fat. for the preparation of biscuit samples, fat was replaced from the recipe by bgf at concentrations of 10, 15, 20, 25, and 50%. the standard biscuits recipe was followed, which consists of flour 100 g (vmf and wheat flour mixed according to different ratios of fat replacement), sugar 40 g, fat 40 g, salt 0.5 g, egg 13.4 g, glucose 0.5 g, sodium bicarbonate1 g, soya lecithin 0.25 g, and water 20±5 ml. biscuit samples were prepared, as reported by ali et al. (2018). briefly describing, the dough was sheeted at a fixed thickness of 7.2 mm and was cut into circular shapes using a biscuit cutter having a fixed diameter of 36.3 mm. biscuits were baked at 180°c for 15 min in an oven. about 25 biscuits were baked per batch, and each batch was prepared in triplicates. antioxidant activity and phytochemicals antioxidant activity of bgf-wf blends, bgf-wf biscuits, and control biscuits was determined by free radical scavenging activity and ferric reducing power. extracts of bgf-wf blends, control, and bgf-wf biscuit samples were prepared by the method of saeed et al. (2021). the blends of bgf-wf and biscuit powder (control and bgf-wf) were added to methanol at a concentration of 60 mg/ml, 125 mg/ml, 185 mg/ml and 250 mg/ml, and the mixtures were analyzed. 2,2-diphenyl-1-picrylhydrazyl-dpph radical scavenging activity the technique mentioned by saeed et al. (2020c) with slight modification was utilized for determining the method 54-21(aacc, 2000). the parameters measured include water absorption (wa), dough development time (ddt), the degree of softening (dos), dough stability time (dst), and farinograph quality number (fqn). pasting properties of flour the pasting properties were studied by microviscoamylo-graph (brabender, duisburg, germany) according to the aacc method 22-10 (aacc, 2000). the parameters measured for each sample included the average values for peak viscosity (pv), final viscosity (fv), and breakdown and setback viscosities. functional properties water absorption capacity (wac) and oil absorption capacity (oac) of flour samples were determined by the method of saeed et al. (2020a). briefly, the wac was determined by taking 1 g of the flour sample in a 20 ml centrifuge, and 10 ml of distilled water was added to it; the mixture was vortexed for 2 min. the tube was centrifuged at 2200×g for about 20 min and the supernatant was decanted while the residual pellets were weighed and analyzed for wac. the same procedure was used to determine oac using 10 ml soya bean oil. micromorphology of biscuits dough the microstructure of biscuit dough was evaluated using scanning electron microscope (joel, analysis system, model # jsm-6380, japan), as described by ali et al. (2018). briefly, the samples were first frozen at −20°c, then transferred into a freeze dryer (laboratory freeze dryer vaco 2, germany), which operated at −50°c and 0.1m pa. freeze-dried dough samples were cut transversally into slices with a sharp blade without damaging the structure. samples were mounted on the sample holder and sputter-coated with gold (2 min, 2 mbar). scanning table 1. composition of biscuits prepared from bottle gourd flour (bgf). s.no. ingredients (g) control bgf (10%) bgf (15%) bgf (20%) bgf (25%) bgf (50%) 1 flour 100 100 100 100 100 100 2 fat 40 36 34 32 30 20 3 icing sugar 40 40 40 40 40 40 4 egg 13.4 13.4 13.4 13.4 13.4 13.4 5 soya lecithin 0.25 0.25 0.25 0.25 0.25 0.25 6 baking powder 1 1 1 1 1 1 7 salt 0.5 0.5 0.5 0.5 0.5 0.5 8 glucose 0.5 0.5 0.5 0.5 0.5 0.5 9 water 20±5 20±5 20±5 20±5 20±5 20±5 10 bgf – 4 6 8 10 20 italian journal of food science, 2022; 34 (2) 53 bottle gourd flour as a fat mimetic in biscuits where abs control is the abs of all reagents except the test sample, and abs sample is the absorbance of the test sample. all the experiments were carried out in triplicates. anti-hyperglycemic of bgf biscuits in-vivo the anti-hyperglycemic activity of biscuits was determined in human subjects using the oral glucose tolerance test (ogtt) according to the method of fombang and saa (2016). incremental area under the blood glucose response curve (iuac) was calculated according to the method recommended by the food and agricultural organization (1998). glycemic index value was calculated as follows: iuac for the test food glycemic index (gi) 100 iuac for the standard food = × (3) evaluation of changes in biscuits during storage bgf biscuits and control biscuits were stored at room temperature (27 ± 2) in an air tight container at relative humidity of 67%. after every 15 days, shelf life study was performed for 2 months. and, biscuits samples were analyzed for free fatty acid value (ffa), peroxide value (pv), antioxidant activities, total phenolic content (tpc), total flavonoid content (tfc), and water activity (aw) determination of free fatty acid (ffa) and peroxide value (pv) the ffa content of biscuit samples were analyzed by aoac method cc 5a-40 (aoac, 2001). the ffa content was calculated as the percentage of oleic acid according to the following equation: ml naoh naoh normality 28.2 ffa as % oleic acid weight of sample (g) × × = (4) pv of biscuit samples was analyzed by the aoac method cd 8-53, (aoac, 2001) evaluating the effect of storage changes on antioxidants, alpha amylase inhibition, and phytochemicals the impact of storage conditions on the antioxidant activity was determined by frap and dpph tests, while alpha amylase inhibition, tpc, and tfc were determined as described earlier in the above sections (2.9 and 2.10). water activity to evaluate the possibility of microbial growth water activity (aw) of biscuits samples was determined in two duplicates of each formulation, using a decagon antioxidant activity of extracts of bgf-wf blends, control, and bgf biscuits. 2, 2-diphenyl-1-picrylhydrazyl (dpph) solution was prepared by dissolving 33.9 mg of dpph in 100 ml of methanol. one milliliter of each of the prepared samples (having concentrations of 60 mg/ ml, 125 mg/ml, 185 mg/ml, and 250 mg/ml) was mixed with 1 ml of dpph solution in a test tube and was placed in the dark for about 30 min. absorbance (abs) values were estimated at the wavelength of 517 nm using a spectrophotometer (perkin elmer, lambda 25, and uv-vis spectrophotometer). the percentage scavenging activity was determined as follows. abs of control abs of sample scavenging activity % 100 abs of control − = × (1) ferric/ferricyanide (fe3+) reducing antioxidant power ferric/ferricyanide reducing antioxidant power (frap) values of the samples were analyzed according to the method of gawlik-dziki et al. (2014) with slight modification adapted by saeed et al. (2021). perl’s prussian color was measured at an absorbance of 700 nm, where the increase in abs is an indication of increased antioxidant activity. total phenolic content total phenolic content (tpc) of samples were determined by using folin–ciocalteu reagent, as described by salar and purewal (2017). the abs of the extracts was recorded at 765 nm against a blank, and the results were expressed as milligram gallic acid equivalent/100 g (mg gae/100g) of the extract on dry weight (dw) basis obtained from the standard calibration curve. total flavonoid content tfc of bgf-wf and biscuit samples were determined by the method of gbenga et al. (2018). the abs of the extracts was measured at 510 nm using catechin as standard, and results were expressed as milligram catechin equivalent (mg cae)/100 g of the extract on dry dw basis. in vitro alpha-amylase inhibition in bgf biscuits alpha-amylase inhibition test by elshibani et al. (2020) based on the starch–iodine technique was used to determine the in-vitro enzyme inhibition in bgf biscuits. abs was estimated at a wavelength of 630 nm .the alpha amylase inhibitory activity was determined as follows: inhibition of alpha amylase (%) abs sample – abs control 100 abs sample − = × (2) 54 italian journal of food science, 2022; 34 (2) saeed smg et al. factor system: carbohydrate (4 kcal/g), lipid (9 kcal/g), and protein (4 kcal/g). sensory evaluation sensory examination was carried out in the baking laboratory following the method of ali et al. (2018). biscuit samples were evaluated by 40 trained panelists, male and female (age 24–45), comprising mainly of students and staff members of the department of food science and  technology, university of karachi (karachi, pakistan). the panelists were trained by utilizing the sensory profiles method (lawless and heymann, 2010) with the commercial biscuits and prototypes prepared in the baking laboratory. by means of this training, a specific terminology for the sensory characteristics and ranges for each attribute was agreed upon. the trained sensory panel passed the basic taste test, the odor test, and the color vision test, and their evaluation capacity were routinely verified by way of individual control cards. panelists used 9 points hedonic scale (1 = extremely dislike to 9 = extremely like) for analyzing the desirability of biscuit samples for taste, color, appearance, texture, and overall acceptability statistical analysis all the analyses were performed in triplicate and the average value was calculated. the results were expressed as mean ± standard deviation. the data were analyzed by analysis of variance (anova) using spss (version 17.0. inc, chicago, usa) statistical program. duncan’s multiple range test (dmrt) was applied to identify any significant differences among the treatments at p ≤ 0.05. furthermore, dmrt involves the computation of numerical boundaries that allow for the classification of the difference between any two treatment means as significant or nonsignificant. this requires calculation of a series of values each corresponding to a specific set of pair comparisons. results and discussion proximate composition table 2 illustrates proximate compositions of bgf-wf blends. the moisture content of the refined wheat flour (14.1%) was slightly higher compared to the bgf-wf blends which ranged from 11.9 to 12.9%, and the decrease in moisture was due to less moisture content of raw bgf (8.01%) (table 12). the increased ash content (0.93 to 2.02%) was due to the presence of a high amount of minerals in bgf than in the wheat flour. similar results were reported by saeed et al. (2020b) when black gram flour was incorporated in wheat flour as fat replacer. since aqua lab meter (pullman, wa, usa) at room temperature (25 ± 2°c), calibrated with a saturated potassium acetate solution (aw = 0.22) (saeed et al., 2020a). sufficient amount of sample was taken in the sample holder, and precaution was taken so that the sample does not touch the sensor. evaluation of biscuits quality color analysis color was measured according to the method of saeed et al. (2020b) by using nh3 colorimeter (china). color values l*, a*, and b* were recorded, each value being the average of four measurements at different points of the biscuits. l* value represents the lightness variable from 100 for perfect white to zero for black, while as a* and b* values are the chromaticity values that indicate (+) redness/(-) greenness and (+) yellowness/(-) blueness, respectively. textural analysis biscuits sample were studied for the effect of fat mimetic on its breaking strength (hardness) and fracturability using texture analyzer (utm, zwick/roell, germany) as per the method described by kuchtová et al. (2018) utilizing three points bend rig technique (load cell: 5 kg, pre-test speed: 1.0 mm/s, test speed: 5.0 mm/s, post-test speed: 10.0 mm/s, distance: 10 mm, trigger force: 50 g). dimensional analysis the diameter or the width of biscuits was measured with the help of a venire caliper (twice by rotating the biscuit at 90°c). the thickness of biscuits was measured by stacking three biscuits on top of one another and the total height was divided by three to get the average value. the spread ratio of biscuits was calculated from the fraction of diameter and thickness (kohajdová et al., 2014). nutritional analysis nutritional analysis of samples included the analysis of protein, fat, carbohydrate, ash, moisture, crude fiber, and kilocalories. protein and ash contents were determined by the kjeldahl apparatus (thermo fisher scientific) and muffle furnace (thermo fisher scientific), respectively, according to aacc methods 08-01 and 46-10, respectively (aacc, 2000). fat content and crude fiber were determined by the soxhlet apparatus (thermo fisher scientific) and fiber digester (marconi, ma-444, brazil), respectively, by using aacc methods 30-25 and 32-10, respectively (aacc, 2000). moisture content was determined by moisture analyzer (brabender 51–55, cw brabender, duisbury, nj, usa). the total carbohydrate was determined by difference: carbohydrate = 100 − (% moisture + % protein + % fat + % ash + % crude fiber). calories were measured by applying the atwater general italian journal of food science, 2022; 34 (2) 55 bottle gourd flour as a fat mimetic in biscuits of bgf decreased with the increase in their concentration in wheat flour. similar findings were reported by klunklin and savage (2018). generally protein contains both hydrophobic and hydrophilic groups which are responsible for oil and water absorption (saeed et al., 2021), which is also proved by our results as the amount of bgf increased in wheat flour, degree of nonpolarity, and hydrophobicity decreased so did the oac. hence, bgf contains more water interactive proteins. dough rheological properties table 4 presents the dough mixing properties of bgf-wf blends. a continuous increase in wa (63.6 to 72.4%) and stability time was observed with increased amount of bg. increased wa was probably due to the presence of dietary fiber in bgf. similar findings of wa and stability were reported by ahmadet al. (2015) when different ratios of green tea powder were incorporated in wheat flour. ddt and dos also increased with the inclusion of bgf in wheat flour. the increase in ddt is due to the rise in wa of the dough. in general, high wa means excellent protein in bgf has remarkable water holding property, the percentage moisture, wac (%), and farinograph wa increased correspondingly (table 3–4); the same results were observed by saeed et al. (2020b). the wheat flour contained less protein content (9.8%) than the bgf as increasing the amount of bgf in wheat flour significantly increased (p ≤ 0.05) the protein content (13.8 to 18.5%). gluten content (dry and wet) and gluten index also decreased as the level of incorporation of bgf increased in wheat flour, which might be due to dilution of gluten network which was also confirmed by micrograph scanning images (figure 1). all these findings were in agreement with the study conducted by saeed et al. (2020b). functional properties water absorption and oil absorption capacities wac and oac are the critical functional properties of the food ingredients because they determine the texture, mouthfeel, and yield of the product (ram and singh, 2004). table 2 represents the wacs of bgf-wf blends. the wac ranged from 146 to 162%. the highest wac was observed for bgf 50% (162%) followed by bgf 25% (155.43%) and lowest for bgf 10% (146%). the study revealed that more the amount of bgf incorporated, the more was the resultant wac. in previous research, purple rice flour incorporated in wheat flour also showed similar behavior (klunklin and savage, 2018). hence, result suggested that the addition of bgf to wheat flour affected the amount of water absorption, as bgf competes with wheat flour for water absorption, which may be due to the difference in chemical composition and structure of the bgf. probably, carbohydrate structure and the polarity of amino acids are responsible for the wac (saeed et al., 2021). the oac ranged from 100.2 to 137.3% in bgf-wf blends. oac of the raw bgf was determined as 100%, whereas wheat flour showed maximum oac, i.e., 153.03%. the data reported in table 2 proved that oac table 2. effect of bottle gourd flour (bgf) addition on proximate and chemical properties of wheat flour. samples moisture content % ash % protein % gluten dry gluten wet gluten gluten index wheat flour 14.10 ± 0.13e 0.43 ± 0.01a 9.81 ± 0.10a 9.20 ± 0.2d 25.83 ± 0.26e 95.00 ± 1.2d bgf 10% 11.91 ± 0.11a 0.93 ± 0.03b 13.82 ± 0.15b 9.00 ± 0.18d 24.00 ± 0.22d 92.00 ± 1.42f bgf 15% 12.10 ± 0.12b 0.98 ± 0.05b 14.10 ± 0.18bc 8.80 ± 0.16c 24.20 ± 0.25d 88.00 ± 1.21e bgf 20% 12.21 ± 0.10b 1.05 ± 0.08c 14.42 ± 0.19c 8.40 ± 0.15bc 23.60 ± 0.21c 86.00 ± 1.08c bgf 25% 12.50 ± 0.13bc 1.09 ± 0.09c 14.91 ± 0.19d 8.30 ± 0.13b 18.70 ± 0.19b 84.00 ± 1.06b bgf 50% 12.90 ± 0.12d 2.02 ± 0.09d 18.52 ± 0.21e 7.70 ± 0.11a 11.30 ± 0.14a 78.00 ± 1.03a values expressed are the mean ± standard deviation (n = 3). means in the column with different superscripts are significantly (p ≤ 0.05) different. table 3. functional properties of wheat flour and different ratios of bottle gourd flour (bgf) incorporated in wheat flour. samples wac% oac% wheat flour 145.61 ± 2.08a 153.03 ± 2.05g bgf 500.16 ± 3.52g 100.21 ± 1.1a bgf 10% 150.26 ± 2.11b 152.36 ± 2.25f bgf 15% 157.42 ± 2.15c 150.22 ± 2.1e bgf 20% 164.26 ± 2.05d 148.36 ± 1.05d bgf 25% 170.26 ± 2.11e 146.81 ± 1.17c bgf 50% 199.32 ± 2.57f 137.34 ± 1.11b values expressed are the mean ± standard deviation (n = 3). means in the column with different superscripts are significantly (p ≤ 0.05) different. wac, water absorption capacity; oac, oil absorption capacity. 56 italian journal of food science, 2022; 34 (2) saeed smg et al. highest peak viscosity was observed for bgf 10% and lowest for bgf 50% because of increment in nonstarch content (i.e., protein) upon addition of higher amount of bgf in wheat flour. the less breakdown viscosity was estimated for wheat flour which was related to the limited swelling of the starch granules. however, breakdown viscosity significantly (p ≤ 0.05) increased when the level of bgf increased in wheat flour. the final viscosity decreased with an increased amount of bgf which resulted in less potential for the formation of viscous paste (kuchtová et al., 2018). in addition, trough and set back viscosities decreased significantly (p ≤ 0.05) as the concentration of bgf increased in wheat flour. the decrease in trough and set back viscosities reflected the less retrogradation tendency and hence contributed to less staling rate of the biscuits (saeed et al., 2020b). the microstructure of biscuits dough the micrograph of the control biscuits dough showed (figure 1a) a thin sheet representing protein matrix along with small and large starch granules embedded in it. sem image of dough with bgf 10% (figure 1b) was very similar to control biscuit dough; however, with the gradual increase in the concentration of bgf in dough baking performance. on the other hand, wheat flour showed lower ddt, lower dos, and higher dst (except bgf10%) than the bgf-wf blends. inconsistency in the mixing properties of flour samples was attributed to an increase in fiber and protein content with the increased level of bgf (bae et al., 2014). another reason could be the increased fqn, which gave a hardening effect to flour and strength to the dough and ultimately caused delay in ddt, ds, and dos (ali et al., 2018). generally molecules of fat gives strength and elasticity to the dough matrix (ali et al., 2018). however, similar functions were performed by the protein moles of bgf in the absence of fat molecules. pasting properties of flour samples the results of the pasting properties of wheat flour and bgf-wf blends are shown in table 5. an increase in starch gelatinization was found with an increased amount of bgf in the wheat flour. it was due to the quick rupture of starch granules leading to lower pasting temperatures and higher paste consistency (kuchtová et al., 2018). similar findings were reported when grape skin was added in the wheat flour (kuchtová et al., 2018). the table 4. farinograph properties of wheat flour and different ratios of bottle gourd flour (bgf) incorporated. samples wa (%) ddt (min) dst (min) dos (icc) fqn wheat flour 58.70 ± 0.2a 1.67 ± 0.01a 8.58 ± 0.18d 35.12 ± 0.90a 88.21 ± 1.12e bgf 10% 63.61 ± 0.20b 2.21 ± 0.04b 11.71 ± 0.18e 78.11 ± 1.11b 136.31 ± 1.52f bgf 15% 64.52 ± 0.33c 5.20 ± 0.08c 5.10 ± 0.15a 129.10 ± 0.81c 65.01 ± 1.10b bgf 20% 64.90 ± 0.51c 5.71 ± 0.03d 5.01 ± 0.11a 143.09 ± 1.72d 62.72 ± 1.11a bgf 25% 65.83 ± 0.58d 5.82 ± 0.01e 6.01 ± 0.13b 153.32 ± 4.50e 72.01 ± 1.12c bgf 50% 72.43 ± 0.81e 5.80 ± 0.01e 8.22 ± 0.16c 168.11 ± 3.21f 86.41 ± 1.14d means with different letters in superscript within a column differ significantly, calculated by the duncan method (p ≤ 0.05). each value was expressed as mean ± sd (n = 3). wa; water absorption, ddt; dough development time, dst; dough stability time, fqn; farinograph quality number. table 5. microvisco-amylo-graph properties of wheat flour and different ratios of bottle gourd flour (bgf) incorporated.means with different letters in superscript within a column differ significantly and are calculated by duncan method (p ≤ 0.05), each value is expressed as mean ± sd (n = 3). samples gelatinization (torque) peak viscosity (torque) trough (torque) final viscosity (torque) breakdown (torque) setback viscosity (torque) pasting temperature (°c) wheat flour 24.01 ± 0.11a 1037.04 ± 10a 717.08 ± 4.11a 1542.32 ± 18.11f 378.18 ± 4.11a 479.21 ± 6.11e 57.91 ± 0.23a bgf 10% 29.21 ± 0.32d 1253.10 ± 14d 1165.11 ± 11.01b 1367.11 ± 16.21e 456.14 ± 8.30f 471.21 ± 7.42f 76.01 ± 0.3d bgf 15% 30.01 ± 0.40e 1222.01 ± 15e 1116.15 ± 12.13d 1299.12 ± 16.13d 466.13 ± 7.50e 493.41 ± 5.32d 81.13 ± 0.34e bgf 20% 32.12 ± 0.60f 1221.13 ± 18f 1112.18 ± 13.10e 1240.21 ± 13.01c 485.13 ± 7.20d 481.71 ± 5.10b 81.13 ± 0.15e bgf 25% 33.31 ± 0.21b 1217.30 ± 12c 1099.11 ± 14.10f 1120.91 ± 12.61b 551.15 ± 6.42c 457.14 ± 3.23c 72.04 ± 0.17c bgf 50% 36.10 ± 0.36c 1215.11 ± 12b 1096.10 ± 10.00c 1131.12 ± 11.21a 613.19±5.11b 448.09±2.21a 68.03 ± 0.21b italian journal of food science, 2022; 34 (2) 57 bottle gourd flour as a fat mimetic in biscuits (a) (b) (c) (d) (e) (f) figure 1. scanning electron micrograph of biscuits dough (magnification, 500×): (a) control, (b) 10% bottle gourd flour, (c) 15% bottle gourd flour, (d) 20% bottle gourd flour, (e) 25% bottle gourd flour, and (f) 50% bottle gourd flour. (figure 1c), the bgf proteins were concentrated, and more and more large and small wheat starch granules were seen trapped in protein fibrils of bgf (figure  1d and e). similar observations were also depicted by dachana et al. (2010) in their study on moringa leave powder which was used for the manufacturing of biscuits dough. higher amount of fat substitution (figure  1f) by higher level of fiber in bgf resulted in disrupted gluten matrix. similarly, a reduction in gluten matrix was observed by indrani et al. (2010) upon addition of 20% multi-grains. the literature revealed that even though microstructural properties of fat mimetics are quite different from fat when they are used to replace fat in food products, the end products show comparable qualities to the standard that is in agreement with our observations (patel et al., 2020). free radical scavenging activity the ic50 value of flour blends and biscuits samples are given in tables 6 and 7, respectively. as predicted, the control biscuits demonstrated the lowest antioxidant activity and the ic50 value of bgf-wf biscuits reduced with the increment in the quantity of the bgf in biscuit samples. the increase in antioxidant activity of bgf-wf biscuits was probably due to the presence of bioactive compounds with hydrogen-donating ability such as vitamins, flavonoids, and phenols (wojtunik-kulesza et al., 2020). results showed that storage time had no impact on antioxidant activity till the second week; however, it became obvious after the fourth week. caleja et al. (2017) reported similar findings when fennel and chamomile extracts were used in biscuit samples; however, storage time did not influence antioxidant activity until the eighth week of storage. the development of antioxidative melanoidin pigments during baking further improved the free radical scavenging capacity (sharma and gujral, 2014). similar observations were reported by mabogo et  al. (2021) when varying concentrations of unripe banana flour was incorporated in wheat-based biscuit samples. ferric/ferricyanide (fe3+) reducing antioxidant power the results of reducing the power of bgf-wf blends and biscuit samples are shown in tables 6 and 7, respectively. frap value of biscuit samples increased with the incorporation of bgf. bgf-wf biscuits showed maximum reducing capacity, on the other hand, control biscuit samples demonstrated the highest ic50 value, hence the lowest reducing ability, which further became undetectable 58 italian journal of food science, 2022; 34 (2) saeed smg et al. table 6. antioxidant activity (dpph and ferric-reducing antioxidant power) and bioactive compounds (total phenol content (tpc) and total flavonoid content (tfc) of different levels of bottle gourd flour (bgf)) incorporated in wheat flour. antioxidant activity bioactive compounds samples dpph (ic 50 ) frap (ic 50 ) tpc (mg gae/100 g dw) tfc (mg ce/100 g dw) wheat flour 488.12 ± 3.15f 327.21 ± 4.52f 16.73 ± 0.11a 31.16 ± 1.01a bgf 10% 301.40 ± 1.29e 127.35 ± 0.51e 334.64 ± 1.18b 174.82 ± 2.81b bgf 15% 245.62 ± 1.21d 121.34 ± 0.21d 437.81 ± 2.31c 385.30 ± 2.14c bgf 20% 221.04 ± 1.01c 89.32 ± 0.45c 506.69 ± 3.42d 456.83 ± 4.45d bgf 25% 200.60 ± 0.92b 78.81 ± 0.13b 543.37 ± 3.55e 484.67 ± 4.61e bgf 50% 187.21 ± 0.43a 57.62 ± 0.10a 667.16 ± 3.81f 583.65 ± 4.72f means with different letters in the column differ significantly. they were calculated by the duncan method (p ≤ 0.05). each value was expressed as mean ± standard deviation (n = 3), nd, not detected. where, gae, gallic acid equivalent. ce, catechin equivalent. dw, dry weight of the sample. nd, not detected. table 7. dpph radical scavenging activity, ferric/ferricyanide (fe3+) reducing antioxidant power (frap), and alpha amylase inhibition about storage period at different levels of bottle gourd flour (bgf) incorporated in biscuit samples. samples initial second week fourth week sixth week eighth week dpph-scavenging activityic 50 (mg/ml) control 500.02 ± 3.15p nd nd nd nd bgf 10% 151.63 ± 1.29j 152.12 ± 1.37jk 166.44 ± 1.55l 183.82 ± 1.82o 185.02 ± 1.87o bgf 15% 139.91 ± 1.21i 140.03 ± 1.23i 154.24 ±1.28k 178.87 ± 1.33m 180.51 ± 1.41m bgf 20% 93.95 ± 1.01c 93.98 ± 1.09c 131.69 ±1.17gh 167.87 ± 1.36l 168.39 ± 1.21l bgf 25% 88.14 ± 0.92b 88.94 ± 1.11b 122.75 ±1.16f 133.89 ± 1.25gh 134.89 ± 1.28hg bgf 50% 68.36 ± 0.43a 69.00 ± 0.81a 96.56 ± 0.94d 105.57 ± 1.11e 108.55 ± 1.12e frapic 50 (mg/ml) control 350.14 ± 4.52o nd nd nd nd bgf 10% 52.44 ± 0.4i 53.14 ± 0.42hij 56.60 ± 0.44l 59.74 ± 0.48m 60.82 ± 0.52m bgf 15% 50.88 ± 0.3h 52.88 ± 0.33h 55.24 ± 0.36k 57.45 ± 0.37l 58.71 ± 0.44l bgf 20% 42.53 ± 0.15e 43.24 ± 0.17e 54.06 ± 0.25j 55.40 ± 0.28k 57.61 ± 0.31kl bgf 25% 33.87 ± 0.13d 35.30 ± 0.1d 45.08 ± 0.14f 49.80 ± 0.17g 50.13 ± 0.24g bgf 50% 12.69 ± 0.12a 13.11 ± 0.11a 22.51 ± 0.12b 28.89 ± 0.14c 29.14 ± 0.15c alpha amylase inhibition -ic 50 (mg/ml) control nd nd nd nd nd bgf 10% 105.78 ± 4.04i 109.88 ± 4.21i 121.56 ± 4.32l 128.59 ± 4.41m 132.02 ± 4.60n bgf 15% 96.50 ± 2.12g 100.67 ± 3.11gh 119.51 ± 3.20kl 124.38 ± 3.28m 128.51 ± 3.35m bgf 20% 62.42 ± 1.46c 68.06 ± 1.52c 92.22 ± 1.73f 112.80 ± 3.00j 120.62 ± 3.23l bgf 25% 50.04b ± 1.57a 54.94b ± 1.40a 89.15 ± 1.56e 90.79 ± 2.12f 100.50 ± 3.17h bgf 50% 47.94 ± 1.11a 50.41 ± 1.70a 80.81 ± 2.04d 89.62 ± 2.09e 93.02 ± 3.15fg means with different letters in the row differ significantly. they are calculated by duncan method (p ≤ 0.05). each value expressed as mean ± sd (n = 3). nd, not detected. control represents biscuits without fat mimetic. during storage. results of storage stability also showed a similar pattern as that of dpph for bgf-wf biscuits, there was no significant (p ≤ 0.05) difference between the ic50 value of initial and second week of storage, although reducing power decreased from the fourth week of storage and further decreased to sixth week. however, there was no significant (p ≤ 0.05) difference in ic50 values between sixth to eighth weeks of storage. similar findings were reported by caleja et al. (2017) when fennel and chamomile extracts were used in biscuit samples. italian journal of food science, 2022; 34 (2) 59 bottle gourd flour as a fat mimetic in biscuits like the dpph test, baking further improves the frap as lower ic50 values were witnessed in biscuits (table 7) when compared to flour samples (table 6). an increase in reducing power upon baking has been reported by mabogo et al. (2021) for biscuits prepared by incorporation of unripe banana flour in wheat flour. total phenolic content table 8 represents the tpc of bgf-wf biscuit samples during storage. biscuits prepared with bgf 50% showed the highest tpc throughout the storage period of 8 weeks followed by bgf 25% and bgf 20%. it was observed that there is no significant (p ≤ 0.05) difference between the first day and second week of storage. although tpc reduced from the fourth week of storage, all bgf-wf biscuit samples have higher tpc as compared to control biscuit samples which exhibited the least tpc (18.56 mg gae/100g dw). despite greater decrease in tpc of bgf-wf biscuits during baking was found compared to bgf-wf blends (table 6), the tpc value in bgf-wf biscuits was still higher than that of control biscuits. jan et al. (2015) also found a decrease in tpc of buckwheat flour when heated to a temperature of 180°c. the decrease in tpc was may be due to alteration in the chemical structure of the phenolic compounds (pintado et al., 2021). in previous research, various natural sources of phenolic compounds were added to bakery products to increase the nutraceutical value (pattnaik et al., 2021). total flavonoid content from table 8, it can be observed that tfc in biscuit samples increased with the level of addition of bgf. biscuits prepared with bgf 50% showed the highest tfc throughout the storage period of 8 weeks, followed by bgf 25% and bgf 20%. natural flavonoidcontaining ingredients were also added in bakery products to increase the bioactive compounds, as reported by many researchers; for instance, a study reported that biscuits prepared with water chestnut flour demonstrated enhanced tfc (shafi et al., 2016). inhibition of alpha-amylase activity in-vitro the methanolic extract of bgf-wf biscuits samples showed significant (p ≤ 0.05) inhibitory activity against alpha-amylase. from table 7, it can be observed that ic50 values decreased with increasing ratio of bgf. the follow-up of storage changes in biscuits samples revealed that there was no significant (p ≤ 0.05) change in enzyme inhibition up to 2 weeks of storage; however, ic50 of all bgf biscuits gradually increased after the second week of storage, and the inhibition activity was almost undetectable in control biscuits. petrus et al. (2012) reported that antioxidant compounds such as catechin, phenolics, flavonoids, alkaloids, and triterpenoids possess antidiabetic activity. epicatechin, a flavonoid is known to possess insulin-like properties, while epigallocatechin gallate is table 8. total phenol content (tpc) and total flavonoid content (tfc) concerning storage period at different levels of bottle gourd flour incorporated in biscuit samples. samples initial second week fourth week sixth week eighth week tpc-(mg gae/100 g dw) control 27.430a ± 0.12 nd nd nd nd bgf 10% 326.66f ± 2.18 323.33f ± 1.31 255.5c ± 1.14 230.00b ± 1.12 225.50b ± 1.1 bgf 15% 383.33h ± 2.21 380.66h ± 1.43 319.33e ± 1.25 280.00d ± 1.21 278.00d ± 1.08 bgf 20% 413.83j ± 3.12 410.88j ± 1.97 391.66i ± 1.45 371.66g ± 1.32 368.50g ± 1.11 bgf 25% 525.00p ± 3.15 520.00p ± 2.48 469.33n ± 2.35 421.66k ± 3.00 418.50k ± 2.41 bgf 50% 638.33q ± 3.21 634.00q ± 3.11 486.00o ± 3.07 460.00m ± 3.17 454.16l ± 3.04 tfc-(mg ce/100 g dw) control 18.56a ± 1.01 nd nd nd nd bgf 10% 104.32e ± 2.21 100.84e ± 1.44 90.24c ± 1.25 76.16b ± 1.23 74.24b ± 1.22 bgf 15% 121.60f ± 2.74 119.84f ± 2.46 104.32e ± 2.42 88.96d ± 1.14 87.04d ± 1.12 bgf 20% 380.51i ± 3.45 376.66i ± 3.21 338.83h ± 3.19 330.5g ± 3.14 326.66g ± 3.11 bgf 25% 451.66l ± 4.51 447.16l ± 4.43 397.18k ± 3.32 371.66j ± 3.32 366.00j ± 3.25 bgf 50% 558.33n ± 4.82 552.00n ± 4.89 460.00m ± 4.61 397.16k ± 3.41 394.00k ± 3.33 means followed by different letters in the raw differs significantly (p ≤ 0.05). calculations were made using the duncan method. each value is expressed as mean ± sd (n = 3), where gae, gallic acid equivalent. ce, catechin equivalent. dw, dry weight of the sample. nd, not detected. *control represents biscuits without fat replacement. 60 italian journal of food science, 2022; 34 (2) saeed smg et al. considered to be an effective hypoglycemic agent (sun et al., 2020). data reported in table 8confirmed that bgf-wf biscuits have a significant (p ≤ 0.05) amount of bioactive phenolics and flavonoids. being inhibitors of alpha-amylase activity, these antioxidant compounds in the bgf biscuits could induce antidiabetic potential in the biscuits and hence are helpful in controlling diabetes. anti-hyperglycemic of bgf biscuits in-vivo table 9 shows the mean blood glucose responses after the consumption of glucose and the test foods, that is, biscuits prepared with bgf 15% (selected due to good sensorial profile) and the control biscuits in healthy human subjects. the iauc for glucose was found to be 143 mmol/l, and for bgf 15% and control were 31.05 mmol/l and 59.25 mmol/l, respectively. gi of control and bgf biscuits were 41.18 and 21.58, respectively. although both control and bgf biscuits contained an equal quantity of sugar, biscuits with bgf 15% significantly (p ≤ 0.05) resulted in a lower glycemic response at the 120th minute of intake. apart from phytochemicals, bg is a blend of soluble and insoluble fiber; however, impact on a glycemic level could be credited more for the presence of soluble fiber (luan and hong, 2016). the mechanism of action may be achieved through a reduction in both fasting blood glucose and insulin concentrations. this occurs because of water-soluble gel-forming fibers. these dietary fibers form a viscous solution in the small intestine, which reduces the contact and mixing of macronutrients with digestive enzymes, and this delays the absorption of glucose, which consequently reduces the postprandial plasma glucose and insulin levels (mcrae, 2018). another reason for decrease in blood glucose profile was credited to phenolics present in bgf-wf biscuits samples as these phenolic compounds are bioactive compounds with antioxidant potential, and hypoglycemic, hypolipidemic, and anti-tumor properties (fombang and saa, 2016). a similar trend of blood glucose modulation was observed in a study in which moringa tea was ingested by healthy human subjects for determination of glycemic response (fombang and saa, 2016). in previous research, various solvent extracts of bg have also been shown to exhibit antihyperglycemic activity in animal subjects, because of the presence of secondary metabolites; for instance, phenolic and flavonoids (luan and hong, 2016). peroxide value of biscuit samples figure 2a shows that pv was less than three meq o2/kg of fat for all the bgf biscuit samples during storage which is considered safe value according to poilish standard, pn-a-86908:1966. less pvs of the bgf-wf biscuit samples were because of the two main reasons: first, the presence of antioxidants that contributed by controlling oxidation and hence peroxide formation. second, fat replacement, the greater the amount of fat replaced by bgf, lesser the amount of fat that remained for the process of oxidation. in a previous study, addition of chokeberry polyphenols extract to butter cookies resulted in a higher pv than the recommended value after ninth week of storage (bialek et al., 2016) which is contradictory to our findings. however, pv range after 6 weeks of preparing the biscuits with the addition of grape pomace extract was comparable to our findings (zaky et al., 2020). there was a gradual increase of pv in all the bgf-wf biscuits samples during storage; on the other hand, control biscuits had the highest pv of 5.53 at the eight week of storage. free fatty acid in biscuit samples data reported in tables 6, 7, and 8 demonstrated that bgf-wf blends and bgf-wf biscuits possessed significant (p ≤ 0.05) antioxidant activity and hence were helpful for the inhibition of oxidation of fats in biscuits. during storage, increased ffa value was observed in all the biscuit samples. the increase was remarkably greater table 9. blood glucose responses (mmol/l), incremental area under the curve (iuac), and glycemic index (gi) in normal healthy volunteers following glucose, tested, and control biscuits. samples time intervals (min) iauc gi 0 30 60 90 120 glucose 5.90 ± 0.13b 7.83 ± 0.24c 8.43 ± 0.25c 5.74 ± 0.12b 5.23 ± 0.1b 143.00 – bgf 15% 5.30 ± 0.11a 5.45 ± 0.13a 5.80 ± 0.16a 5.63 ± 0.14a 5.19 ± 0.1a 31.05 21.58 control* 5.96 ± 0.17b 6.75 ± 0.22b 6.83 ± 0.25b 6.25 ± 0.18c 5.91 ± 0.14c 59.25 41.18 means with different letters in the column differ significantly. they are calculated by the duncan method (p ≤ 0.05). each value expressed as mean ± sd (n = 15 healthy female volunteers). bgf, bottle gourd flour. iauc, incremental area under the curve. gi, glycemic index. *control represents biscuits without fat mimetic. italian journal of food science, 2022; 34 (2) 61 bottle gourd flour as a fat mimetic in biscuits biscuits. the water requirement for the growth of microorganisms is expressed in terms of moisture available or water activity ( macedo et al., 2020). in this study, even at 10% bgf, the water activity was found to be 0.58 at the eighth week of storage which assured the safety of product with respect to microbial profile (morais et al., 2018). rodríguez et al. (2013) also reported a similar trend of decreased water activity when fat was replaced by inulin in biscuit samples. lower water activity of bgf biscuits might be due to higher water-binding capacity of bgf, as less water evaporated during baking, and less free water remained in the biscuits, thus contributing to longer shelf life (rodríguez et al., 2013). no significant (p ≤ 0.05) change was observed in water activity of control and bgf biscuits from first day till the second week of storage. although water activity increased after the fourth week of storage and this remained constant up to 8 weeks. morais et al. (2018) also reported a similar trend of water activity in biscuit samples. the increased water activity can be attributed to the increased moisture of biscuits with respect to storage, but it remained limited under the value of 0.6 for 2 months to inhibit microbial growth in control (1.1%) as compared to bgf-wf biscuit samples. from figure 2b, it was clear that initially the ffa was not detected in bgf-wf biscuit samples; however, a gradual increase (up to 0.9 %) was observed during storage indicating the capability of antioxidants present in bgf in reducing the formation of ffa. therefore, the increment of ffa level in bgf biscuits was relatively slow. similar observations were reported when a flavonoid-rich extract from green tea was incorporated in biscuit samples (navaratne and senaratne, 2014). another study in which pomegranate peel was added in different ratios in biscuit samples was also in agreement with our findings (ismail et al., 2014). hence, it can be concluded that bgf was capable of controlling the formation of ffa in biscuit samples when used as a fat mimetic. water activity to evaluate the possibility of microbial growth table 10 shows that an increase in the storage period increases the water activity of control and bgf-wf 6 (a) (b) 5 4 3 2 1 0 0 2 4 period of storage (weeks) p er ox id e va lu e m ili eq ui va le nt o 2 / kg o f f at 6 10% bgf 15% bgf 20% bgf 25% bgf 50% bgf control 8 10 0.2 0.4 0.6 0.8 1 1.2 0 0 2 4 storage period (weeks) f re e f at ty a ci d (% ) 6 10% bgf 15% bgf 20% bgf 25% bgf 50% bgf control 8 10 figure 2. (a) peroxide value levels in control and bottle gourd flour incorporated biscuits stored for 2 months. (b) free fatty acid (%) levels in control and bottle gourd flour incorporated biscuits stored for 2 months. 62 italian journal of food science, 2022; 34 (2) saeed smg et al. table 10. water activity concerning storage period at different levels of bottle gourd flour (bgf) incorporated in biscuit samples. samples initial second week fourth week sixth week eighth week control* 0.62 ± 0.02h 0.61 ± 0.02h 0.70 ± 0.02i 0.71 ± 0.02i 0.71 ± 0.02i bgf 10% 0.55 ± 0.08e 0.55 ± 0.01e 0.57 ± 0.04ef 0.57 ± 0.02ef 0.58 ± 0.01f bgf15% 0.52 ± 0.01d 0.52 ± 0.03d 0.56 ± 0.02ef 0.56 ± 0.01ef 0.56 ± 0.01ef bgf 20% 0.51 ± 0.02d 0.52 ± 0.02d 0.55 ± 0.05ef 0.56 ± 0.02ef 0.57 ± 0.01ef bgf 25% 0.47 ± 0.05c 0.47 ± 0.08c 0.50 ± 0.03d 0.51 ± 0.02d 0.51 ± 0.01d bgf 50% 0.35 ± 0.01a 0.34 ± 0.01a 0.38 ± 0.07b 0.38 ±0.07b 0.38 ± 0.01b means with different letters in the row differ significantly (p ≤ 0.05). they are calculated by the duncan method. *control represents biscuits without fat mimetic. (morais et al., 2018) and ensure product stability. on the other hand, water activity of control biscuits increased from 0.6 to 0.71 during 2 months of storage. physical properties of biscuit samples table 11 summarized the effects of the bgf as a fat mimetic on the physical properties of biscuits such as diameter, thickness, spread ratio, hardness, and fracturability. a standard decrease was observed in the diameter of fat mimetic biscuit samples, although biscuits prepared with bgf 10% and bgf 15% showed a maximum increase in diameter when compared with the control sample. in addition to this, biscuits containing bgf 10%, bgf 15%, and bgf 20% showed less thickness than control. bgf significantly (p ≤ 0.05) increased the spread ratio at the level of 10 and 15%, which was more than the control. similarly, kuchtová et al. (2018) and mildner et al. (2013) reported an increase in the spread ratio of biscuit samples produced by incorporation of grape skin and white grape pomace, respectively. an increased amount of dietary fiber in bgf biscuits could be the reason for increased thickness, reduced diameter, and decreased spread ratio as a higher amount of fat was replaced with bgf. it has already been reported that the addition of dietary fiber from various sources and substitutes has a negative effect on the diameter, thickness, and spread ratio of biscuits/ cookies (saka et al., 2020). another reason for the variation in these physical parameters could be the dilution of gluten network in biscuit dough (kohajdová et al., 2014). hardness and fracturability are textural properties that attract significant attention in the evaluation of baked goods; these parameters should be as low as possible (saka et al., 2020). the increased amount of bgf from 10 to 15% resulted in considerable less hardness and fracturability; however, hardness increased when fat was replaced beyond 20%. this is possibly related to the high wac of bgf, as it has been reported by saeed et al. (2021) that components that enhance the water absorption of dough results in the development of the complex gluten network and ultimately hardens the texture of the biscuit samples. color characteristics of biscuits color plays an essential role in determining the quality of products in food processing industries and food engineering research (palamthodi et al., 2019). results of color measurements of biscuits made with different levels of bgf are given in table 12. it was found that the lightness l* of the biscuits exhibited a decreasing trend with an increasing level of bgf. the reducing values of table 11. effect of bottle gourd flour (bgf) on physical properties (dimension and texture) of biscuits. sample diameter (mm) thickness (mm) spread ratio (mm) breaking force (n) fracturability (mm) control* 42.67 ± 0.56d 9.07 ± 0.52d 4.71 ± 0.10d 22.90 ± 0.39d 2.10 ± 0.12b bgf 10% 42.74 ± 0.40e 8.06 ± 0.2a 5.30 ± 0.14f 17.42 ± 0.2a 0.87 ± 0.11a bgf 15% 42.84 ± 0.88f 8.26 ± 0.20b 5.18 ± 0.12e 20.87 ± 0.23b 0.90 ± 0.12a bgf 20% 41.74 ± 0.51c 8.68 ± 0.21c 4.67 ± 0.13c 24.77 ± 0.30c 2.45 ± 0.14c bgf 25% 41.59 ± 0.42b 9.32 ± 0.24e 4.37 ± 0.10b 27.42 ± 0.41e 2.74 ± 0.17d bgf 50% 40.78 ± 0.33a 10.68 ± 1.02f 3.81 ± 0.10a 31.51 ± 0.44f 2.81 ± 0.19e values expressed are the mean ± standard deviation (n = 3). means in the column with different superscripts are significantly (p ≤ 0.05) different. *control represents biscuits without fat mimetic. italian journal of food science, 2022; 34 (2) 63 bottle gourd flour as a fat mimetic in biscuits table 12. effect of bottle gourd flour (bgf) on the color of biscuit samples. samples l* a* b* control* 73.12 ± 0.82f 6.13 ± 0.01a 43.12 ± 0.21f bgf 10% 70.41 ± 0.67e 6.91 ± 0.03b 38.11 ± 0.15e bgf 15% 64.33 ± 0.34d 7.30 ± 0.02c 33.14 ± 0.13d bgf 20% 58.29 ± 0.13c 7.81 ± 0.01d 26.31 ± 0.10c bgf 25% 52.14 ± 0.11b 8.11 ± 0.02e 18.35 ± 0.10b bgf 50% 46.21 ± 0.10a 10.32 ± 0.05f 8.21 ± 0.06a values expressed are the mean ± standard deviation (n = 4). means in the column with different superscripts are significantly (p ≤ 0.05) different. *control represents biscuits without fat mimetic. table 13. nutritional analysis of bottle gourd flour (bgf), control, and fat mimetic biscuits. samples moisture content % ash % protein % fat % dietary fiber % carbohydrate % kcal/100 g control* 5.02 ± 0.12a 0.53 ± 0.01a 11.20 ± 0.10a 24.02 ± 0.20g 0.21 ± 0.26a 64.04 ± 0.80g 517 bgf 8.01 ± 0.30d 4.23 ± 0.10f 20.21 ± 0.50f 0.43 ± 0.01a 26.30 ± 0.42f 48.83 ± 0.30b 280 bgf 10% 4.68 ± 0.14c 2.73 ± 0.03b 13.70 ± 0.12b 20.21 ± 0.14f 13.80 ± 0.20b 49.56 ± 0.42c 435 bgf 15% 4.68 ± 0.10c 2.77 ± 0.01b 13.90 ± 0.11bc 19.11 ± 0.10e 13.60 ± 0.11b 50.62 ± 0.57d 430 bgf 20% 4.65 ± 0.11b 3.82 ± 0.02c 13.60 ± 0.15b 16.32 ± 0.21d 14.00 ± 0.21c 52.26 ± 0.64f 410 bgf 25% 4.64 ± 0.12b 3.86 ± 0.06cd 14.40 ± 0.20d 15.01 ± 0.10c 14.60 ± 0.13d 52.13 ± 0.60e 401 bgf 50% 4.65 ± 0.13b 3.87 ± 0.09ed 18.70 ± 0.22e 12.54 ± 0.11b 17.30 ± 0.17e 47.59 ± 0.54a 378 values expressed are the mean ± standard deviation (n = 3). means in the column with different superscripts are significantly (p ≤ 0.05) different. *control represents biscuits without fat mimetic. l* indicated that the biscuits were darker at higher levels of bgf compared to the control sample. the effect might be due to the presence of natural pigments or the maillard reaction products that were formed from amino acids and reducing sugars during baking (mildner et al., 2013). furthermore, it was shown that the higher levels of bgf in biscuits increased redness (higher a* value) and decreased yellowness (lower b* value). this may be because bg contains a good amount of anthocyanin, a red-blue natural pigment (palamthodi et al., 2019). kuchtová et al. (2018) and sharma and gujral (2014) reported a decrease in the color values of l*, an increase in redness a*, and decreased yellowness b* when grape seeds and buckwheat flour were incorporated in biscuits, respectively, and it is in agreement with our findings. nutritional analysis of biscuits the nutrient composition of bgf, control, and fat mimetic biscuits are summarized in table 13. bgf was found to be an excellent source of crude fiber (26.30%), protein (20.21%), and minerals (ash content 4.23%). all the fat mimetic biscuits showed higher protein, ash, and crude fiber contents than the control. as expected, fat content reduced with the level of addition of bgf than the control sample. as the concentration of bgf increased, the moisture content of biscuit samples reduced. according to camelo-méndez et al. (2018), moisture reduction was due to the decrease in the gluten network with the increase in the amount of bgf. the crude fiber, protein, and ash content increased with the increased amount of bgf in biscuit samples. the control sample showed a low value of crude fiber (6.0%) while the biscuits containing bgf 50% and bgf 25% possessed the highest crude fiber. the carbohydrate content and energy (kcal) values were the highest in control (59.04% and 255.92 kcal) and lowest in biscuits with bgf 50% (43.25% and 219.68 kcal). therefore, bgf biscuit samples showed less energy value and the reason was fat substitution. sensory characteristics of biscuits figure 3 presents the sensory scores of the biscuit samples (50% replacement of fat in biscuits excluded from the data reported because of the undesirable sensory profile). biscuits with bgf10% demonstrated similar results as that of control sample in terms of taste, texture, color, and overall acceptability. on the other hand, at the level of bgf15%, score for color was higher than the control biscuits, although appearance, taste, texture, and overall acceptability were not significantly (p ≤ 0.05) different from control biscuits. as the level of bgf increased beyond 15%, taste which is one of the essential attributes, declined due to unpleasant mouthfeel, bitterness, and the texture also became harder. the bitterness was may be due to the interaction between a high amount of phenolic compounds in bgf and saliva present in the mouth (kuchtová et al., 2018) while the hard texture could be due to the low amount of fat in the biscuit recipe as fat 64 italian journal of food science, 2022; 34 (2) saeed smg et al. taste 10 8 6 4 2 0 control bgp 10% bgp 15% bgp 20% bgp 25% overall acceptability texture appearance color figure 3. effect of fat replacement by bottle gourd flour (bgf) on the sensorial quality of biscuits. control represents biscuits without fat mimetic. helps to lubricate and soften the structure of food and contributes desirable textural properties (hasmadi et al., 2014). moreover, in the study conducted by serin and sayar (2017), maltodextrin and polydextrose were used as a fat replacer in biscuits which also resulted in increased hardness with the level of fat replacement. conclusion the bakery industry is in constant innovation, and biscuits are products that are consumed worldwide by different classes of consumers. therefore, the production of this type of product having nutraceutical effects may be attractive for consumers who are concerned about the choice of healthy foods. the result showed that the addition of bgf as a fat mimetic in the proportion of 10 to 15% produced desirable biscuits, and the functional properties of the wheat flour were not affected. bgf was observed to be an excellent source of phenolic and flavonoids, which makes it a valuable source of antioxidants. incorporation of bgf raised the bioactive compounds in biscuits’ enhanced antioxidant activity, and inhibited alpha-amylase. biscuits with 15% bgf showed excellent physical properties and sensorial attributes. therefore, administration of this formulation of biscuits in healthy volunteers for the determination of anti-hyperglycemic activity revealed a lower blood glucose response curve and glycemic index as compared to the control biscuits. moreover, further studies are needed to improve the functional behavior and quality of biscuits. acknowledgment the help of ms. uroosa ejaz in proof reading of this manuscript is acknowledged. the authors are grateful to the university of karachi for partial funding through a dean research grant, and to english biscuit manufacturers management, particularly dr. zeelaf munir and madam saadia naveed, for their encouragement and support. ethics declarations conflict of interest there is no conflict of interest. compliance with ethics requirements this article contains in-vivo studies with human participants (anti-hyperglycemic activity and sensory 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_enref_45 _enref_46 _enref_47 _enref_59 _goback _enref_56 _enref_57 _enref_66 ijfs#213_koral_bozza   ital. j. food sci., vol 28, 2016 230 paper quality changes of spotless shad during storage at different conditions s. koral1*, s. köse2, f. yavuz2 and y.e. şen2 1 recep tayyip erdogan university, faculty of fisheries, 53100 rize, turkey 2 karadeniz technical university, faculty of marine sciences, 61530 çamburnu, trabzon, turkey *corresponding author. tel.: +90 4642233385 (1434); fax: +90 4642234118 e-mail address: serkan.koral@erdogan.edu.tr abstract this study investigates the effect of using ice in combination with refrigeration on the sensory, physico-chemical and microbiological attributes of spotless shad during storage. spotless shad kept in ice under refrigerated conditions had better sensory, physicochemical and microbiological quality as compared with control groups. the shelf life of samples kept at ambient temperature without ice was 2 days. using ice and refrigeration only extended the shelf-life for 3 days and 4 days, respectively, while ice application with refrigeration further increased the shelf-life by 10 days. physico-chemical and microbiological results usually supported sensory values. histamine values were below eu (european union) and fda permitted levels for the shelf-life of fish. keywords: spotless shad, biogenic amines, quality changes, sensory values, shelf life   ital. j. food sci., vol 28, 2016 231 1. introduction spotless shad, alosa immaculata, bennett, 1835 is the largest representative species of the clupeidae family, it has a pelagic life and is classified under diadromous fish species (fao, 2014). this shad species is the most abundant in eastern europe both in inland and marine waters (navodaru et al., 2003). it is a commercially important species in turkey (tui̇k, 2013) and some other european countries (fao, 2014). the fishing period of shad was recorded to take place between march and october for the black sea. various catching methods and materials are used for shad (ergüden et al., 2007) and the total catch value was 1,541 tons for the year 2013 in turkey (tui̇k, 2013). some other european countries also reported a total catch of about 1,029 tons (fao, 2014). however, this species is commonly caught by local fishermen and therefore, most of their catch is likely not to be recorded. fish spoilage relates to both seafood quality and safety that further affects the market value of the product as well as its nutritional value. spoilage is mainly caused by bacterial activity, other than sensory quality loss (such as its appearance, flavour, odour and texture), some compounds, such as biogenic amines (bas), can be formed by bacterial decarboxylation of the precursor amino acids, thus, leading to food safety issues. most reports have focused on histamine, which is reported to cause scombrotoxin poisoning, closely linked to the consumption of fish kept in abused time/temperature conditions and species belonging to different families, including clupeidae, due to high content of free histidine in their muscle (lehane and olley, 2000; eu directive, 2005). putrescine and cadaverine are also known to enhance the toxicity effect of histamine. other than food safety issues, several bas are known to be associated with fish decomposition and the formation of cancerous nitrosamines (lehane and olley, 2000; pons-sánchezcascado et al., 2006). although, previous studies demonstrated the advantage of using chilled storage conditions for various fish species and the different shelf lives were observed (varlik and heperkan, 1990; veciana-nogués et al., 1990; carache et al., 2002; köse and erdem, 2004; chotimarkorn, 2014; koral and köse, 2012), and limited studies are available for the quality changes of spotless shad stored at chilled temperatures. so far, duyar et al. (2012) investigated the shelf life of this species at refrigerated temperature and obtained 6 days of shelf life. in our previous study, we studied physico-chemical and sensory quality of shad, obtained in a market in the months of july and august and kept in refrigerated storage in open air for 3 days. we gathered that the quality of shad varies depending on the initial quality of fresh fish (köse et al., 2000). however, storage quality and development of biogenic amines under various chilled temperature conditions of this species are still unknown. previous literature, including our past studies, showed that keeping fish in ice during refrigerated storage improves the storage quality, while extending the shelf life. similarly, this type of application retarded bas development (koral and köse, 2012). shad is known to contain high fat, up to 26%, which affects its storage quality more than less fatty fish (boran and karaçam, 2011). moreover, this species is highly susceptible to the formation of histamine and other biogenic amines, which result in seafood health risk for the consumers. therefore, we aimed to investigate the effect of different ice application under refrigerated temperature of this species on its shelf life and biogenic amine development.   ital. j. food sci., vol 28, 2016 232 2. materials and methods 2.1. materials and experimental design fresh, spotless shad samples used in this study were caught in the south-eastern black sea in the month of january 2013. the samples that were previously kept in ice and placed in eps (expanded polystyrene) boxes for 3hours after the catch, were collected directly from a boat at the port of trabzon, turkey. the fishes were then kept in eps boxes containing ice and were transferred to the laboratory within 1hour. the fishes which weighed 30kg (125 fishes) in total were immediately washed with tap water and then, the fishes were randomly divided into 4 groups. each group was kept in plastic containers. ice application on fish was carried out layer by layer. the containers were covered with plastic wrap (cling film). the sample groups were; (i) control: fish stored at ambient temperature without ice (c): the container temperature: 17.0°c ± 3.0°c, fish average temperature: 16.0°c ± 4.0°c. the average length and weight of the fishes weighing a total of 5.40 kg (24 fishes) are 29.59 cm ± 1.98 cm, 225.59 g ±37.38 g, respectively. (ii) fish stored in ice, ratio is at 1:1 (w: w), and at ambient temperature (iat) 17.0°c ± 3.0°c: fish temperature fluctuated between 4.0°c and 14.0°c with daily ice replacement (fish temperature dropped to 4°c in 2 hours after ice addition). the average length and weight of the fishes weighing a total of 7.2 kg (30 fishes) are 30.19 cm ± 2.30 cm, 238.98 ± 53.17 g, respectively. (iii) refrigerated (in a refrigerator: arçelik, 8810nf, turkey) fishes without ice (rt): storage in cold air at 4.8°c ± 0.3°c. fish temperature 3.8°c ± 0.4°c. the average length and weight of the fishes weighing a total of 7.2 kg (30 fishes) are 29.88 ± 2.15 cm, 241.2 ± 59.94 g, respectively. (iv) iced storage in refrigerator (irt) at 4°c ±1°c: fish and ice ratio, (1:1 w:w). fish temperature, 0.5°c ± 0.3°c. the average length and weight of the fishes weighing 10kg (40 fishes) are 30.58 cm ± 1.76 cm, 246.25g ± 40.8 g, respectively. ice was replaced daily. sampling was carried out daily by randomly choosing spotless shad, until the products were spoilt based on sensory results. the ice was the flake iced type obtained freshly from an ice maker (hoshizaki, fm-80ee, amsterdam, holland). the internal temperatures of the fishes in each group were measured on a daily basis using a digital thermometer (taylor, 9842n waterproof digital thermometer, usa) by injecting the upper dorsal part of the fish. 3. methods 3.1 sensorial evaluation sensory analysis was carried out using eight trained panellists who judged the freshness of the samples using a 10-point scale (archer, 2010). the panellists were comprised of 6 males and 2 females (5 academicians and 3 administrative staff). the subjects were qualified after passing the screening tests stated by botta (1995). the panellists are 20 in number and they were chosen from previously trained people who are regular shad consumers based on the criteria given by botta (1995). table 1 shows the sensory score sheets used to evaluate spotless shad samples. this structured category scale is based on the traditional freshness, quality grading system for   ital. j. food sci., vol 28, 2016 233 the whole iced and refrigerated spotless shad. according to scale, 4 is the limit for acceptable/unacceptable, <4: unacceptable. the results were presented as the means of data obtained from 8 panellists. table 1: sensory evaluation criteria of shad samples. score appearance odour texture eyes gills skin flesh appearance of belly walls 10 slightly convex, clear, black pupil. dark red, purple, clear slime bright colours, iridescent, few loose scales. glossy, rosy hue, fresh bright blood on fillet. no belly burst oily, marine, fresh blood, sulphide, weak odour. firm, stiff, smooth 9 flat, slightly convex, clear black pupil. dark red, pink, slightly faded. bright, slight iridescence, few loose scales. slight translucency, rosy hue, bright blood on fillet. no belly burst. oily, marine, fresh fruit, sulphide. loss of stiffness, still firm, smooth. 8 flat, slightly convex, slightly cloudy dark red, pink, slight brown, slight reddening on gill covers loss of brightness, some loose scales. slight translucency, slight discoloration of belly flaps. no belly burst or very slight belly burst oily, musty, leathery, slight sulphide. loss of stiffness, slight softening, smooth. 7 red, pink, slight bleaching, slight reddening on gill covers. dull, slight bronzing, some loose scales. slightly opaque, slightly brown, slight discoloration of belly flaps. slight belly burst. oily, musty, sulphide, slightly sour. limp, slightly soft, slightly gritty. 6 flat, slightly cloudy red, pink, brown, slight bleaching, reddening on gill covers. dull, slight bronzing, some dirty scales. opaque, dull, brown, reddening on belly flaps. musty, stale fruit, stale grass, malty, sour. 5 flat, slightly sunken, slightly cloudy, grey pupil. pink, brown slime, bleached, reddening on gill covers. dull, bronzing, dirty scales. definite belly burst. stale fruit, stale grass, sour, malty, slightly rancid. limp, soft, gritty. 4 flat, slightly sunken, cloudy, bloodshot, discoloured. opaque, brown, discoloured belly flaps and tail. sweaty, sour sink, stale meat,h2s 3 flat, slightly sunken, wrinkled, discoloured, blood shot. pink, bleached, brown slime, bronzing on gill covers. dull, bleached, brown slime. opaque, general discoloration. sweaty, cheesy, sour sink, byre-like, rotten fruit.   ital. j. food sci., vol 28, 2016 234 triplicate samples (3 randomly selected fish) were taken per day at other intervals from each group to evaluate their quality criteria at the sensory laboratory section. fresh spotless shad was also included in the evaluation, starting from the 2nd day, to support the judgement of the assessors as a reference sample (although, the panellists were blind to its code). different samples were simultaneously presented in plates coded. they were expected to give the highest score for the blinded reference sample (freshly caught spotless shad). after the sensory analysis, the fish were further used for physico-chemical analysis. 3.2. physico-chemical analysis total volatile basic nitrogen (tvb-n) was determined using the method of lucke and geidel as described by inal (1992). thiobarbituric acid value (tba) was estimated according to smith et al. (1992). the method of boland and paige (1971) was used for analyzing trimethylamine nitrogen (tma-n). the ph measurements were taken with a digital ph meter (jenco 6230n, ca, usa) by placing the electrode into the homogenized samples (köse et al., 2012). biogenic amines were analyzed using a high performance liquid chromatography (hplc) method according to köse et al. (2012) and koral and köse (2012). hplc equipment was shimadzu prominence lc-20 at series (japan) with auto sampler (sıl20ac, shimadzu, japan), a diode array detector (spd-m20a, shimadzu, japan) and intertsil column (gl sciences, ods-3, 5µm, 4.6×250mm). all physico-chemical analysis was carried out in triplicate sampling, and results were represented as means ±sd. 3.3. bacterial analysis total aerobic viable bacteria (tvb) were counted using plate count agar according to köse and erdem (2004). mesophilic and psychrophilic microorganisms were counted after incubation at 4°c for 8 days for psychrophilic microorganisms and at 37°c for 48 hours for mesophilic microorganisms. histamine-forming bacteria (hfb) were determined in accordance with the method used by köse (1993). total mesophilic and psychrophilic hfb were determined using the same condition applied for tvb with the exception of using niven’s medium, instead of plate count agar. triplicate samples were used for each type of analysis, while each analysis was performed in duplicate. counts of bacteria were expressed as log cfu/g. the results were presented as means of all counts ±sd. 3.4. statistical analysis all statistical analysis was performed using jmp software (version 8.02, 2009, sas institute. inc., cary, nc, usa) (sokal and rohlf, 1987). analysis of variance (anova) was used to compare the results within the groups as well as during storage period. when significant differences were found, comparisons among data were carried out using turkey test. a significant level of 95% (p<0.05) was used throughout the analysis. 4. results and discussion sensory analysis is important in any food quality evaluation program because the ultimate criterion for judgment is the human response (xu et al., 2014). in this study, sensory evaluation was based on appearance, texture and odour and the results are presented in table 2. sensory scores decreased significantly in both control and experimented samples   ital. j. food sci., vol 28, 2016 235 with increasing storage (p<0.05). significant changes occurred between the control group and others, starting from the 1st day of storage (p<0.05) for the relating sensory attributes. table 2: sensory values of shad groups stored under various conditions. day of storage sample type appearance odour texture over all mean 0 fresh 9.66±0.21 9.80±0.10 9.62±0.36 9.69±0.09 1 c 8.28±0.19aa 7.28±0.15 a a 8.30±0.16 a a 7.95±0.58 a a iat 9.84±0.09ba 9.28±0.29 b a 9.30±0.23 b a 9.47±0.32 b a rt 9.68±0.23ba 9.40±0.37 b a 9.76±0.11 b a 9.61±0.19 b a irt 9.78±0.13ba 9.80±0.10 b a 9.94±0.09 b a 9.84±0.13 b a 2 c 6.04±0.11ab 6.12±0.16 a b 7.22±0.22 a b 6.46±0.66 a b iat 8.18±0.13bb 8.16±0.11 b b 9.10±0.22 b a 8.48±0.54 b b rt 9.12±0.13cb 9.12±0.23 c a 9.50±0.10 c a 9.24±0.45 c b irt 9.48±0.22ca 9.64±0.45 c a 9.74±0.09 c a 9.65±0.10 c a 3 c 4.08±0.08ac 3.22±0.13 a c 4.14±0.11 a c 3.81±0.51 a c iat 7.18±0.08bc 7.16±0.17 b c 8.16±0.18 b b 7.50±0.57 b c rt 9.06±0.15cc 8.70±0.16 c c 8.88±0.25 c b 8.71±0.45 c c irt 8.74±0.15cb 8.62±0.09 c b 9.10±0.11 c b 8.77±0.16 c b 4 c 3.04±0.05ad 2.02±0.11 a d 3.04±0.11 a d 2.70±0.59 a d iat 6.12±0.13bd 6.10±0.10 b d 7.22±0.13 b c 6.48±0.64 b d rt 8.18±0.11cd 8.24±0.19 c d 8.56±0.19 c b 8.32±0.49 c d irt 8.44±0.29cb 8.40±0.14 c b 8.70±0.07 c c 8.50±0.08 c b 5 c 1.06±0.09ae 0.54±0.11 a e 0.88±0.15 a e 0.83±0.26 a e iat 5.04±0.15be 5.26±0.11 b e 6.30±0.21 b d 5.53±0.67 b e rt 7.14±0.11ce 7.28±0.13 c e 7.26±0.11 c c 7.23±0.08 c e irt 8.26±0.15db 8.12±0.08 c c 8.38±0.19 c d 8.12±0.17 d c 6 iat 3.34±0.42af 4.36±0.21 a f 4.12±0.13 a e 3.94±0.53 a f rt 6.08±0.15bf 6.40±0.19 b f 6.16±0.15 b d 6.21±0.17 b f irt 8.04±0.32cb 7.92±0.29 c c 7.80±0.08 c e 7.82±0.12 c d 7 iat 2.98±0.08ag 3.22±0.16 a g 3.16±0.09 a f 3.12±0.12 a g rt 5.14±0.22bg 5.00±0.19 b g 4.90±0.07 b e 5.02±0.11 b g irt 7.16±0.17cd 7.14±0.19 c d 7.04±0.09 c f 7.11±0.06 c e 8 iat 1.12±0.15ah 1.00±0.07 a h 1.02±0.11 a g 1.05±0.06 a h rt 3.02±0.23bh 3.02±0.25 b h 3.20±0.33 b f 3.45±0.13 b h irt 6.78±0.33ce 7.04±0.15 c d 6.84±0.23 c g 6.89±0.14 c f 9 rt 0.76±0.18ai 1.06±0.23 a i 0.72±0.40 a h 0.85±0.19 a i irt 6.34±0.15bf 6.50±0.19 b e 6.58±0.16 b h 6.47±0.13 b g 10 irt 5.98±0.31f 6.20±0.31ef 6.18±0.16i 6.12±0.16h 11 irt 5.44±0.29fg 5.92±0.29f 5.56±0.10i̇ 5.81±0.10i 12 irt 4.90±0.23g 4.96±0.27g 5.04±0.11j 4.97±0.07i̇ 13 irt 3.94±0.21h 3.92±0.08h 3.78±0.27k 3.88±0.09j ‘4’ is the limit for acceptability/unacceptability of the samples. different superscript small letters (a,b,c) represents statistical differences among groups (p<0.05). different superscript capital letters (a,b,c,d,..) represents statistical differences amongst different days within the same group during storage (p<0.05).c: control, iat: fish stored in ice at ambient temperature; rt: refrigerated fish without ice; irt: fish stored in ice at refrigerator temperature.   ital. j. food sci., vol 28, 2016 236 the changes were also found to be significant between samples stored in ice at ambient temperature and the samples stored at refrigerated temperatures with and without ice, starting from the 2nd day of storage (p<0.05). the sensory scores of both groups kept at refrigerated temperatures were similar up to the 4th day, thereafter, the variations occurred. according to the overall sensory data, the control group sample got spoilt on the 3rd day, whereas using ice extended its shelf life for 3 more days. refrigerated temperature without ice also supported sensory values having a shelf life of 7 days, although, a dryness on the surface of the fish occurred. the samples kept in ice at refrigerated temperature received the highest sensory scores compared to other samples with a shelf life of 12 days. this may be attributed to the stable as well as cold temperature of the fish under this condition. in our previous study with fresh atlantic bonito, we demonstrated that filleting can improve the sensory quality of fish for about 6 days if kept in ice and under refrigerated temperature (koral and köse, 2012). we also demonstrated that sensory quality of shad can vary at refrigerated temperature depending on the initial quality of fish (köse et al., 2000). duyar et al. (2012) investigated the quality changes of shad (alosa tanaica) at refrigerated storage packed in stretch film without ice. according to sensory values, they obtained one day higher shelf life than present study for the shad samples kept at refrigerated temperature without ice. the difference can be explained in the protecting effect of using stretch film, since we obtained dryness on the surface of fish at open air in the refrigerator. xu et al. (2014) determined the effect of electrolyzed oxidizing water and chitosan on the microbiological, physico-chemical, and sensory attributes of american shad (alosa sapidissima) during refrigerated storage (4±1°c) kept in air-proof polyethylene pouches. they showed that the shelf life of fish can further be improved by 9-10 days during refrigerated storage with this treatment. the ph can be used as a spoilage indicator of fish and fish products (xu et al., 2014). the ph of fresh fish after death is usually reported as close to neutral and varies between 6.0 and 7.1 during post-mortem storage depending on the fish species, diet, season, type of muscle, and other factors (huss, 1988; xu et al., 2014). the ph of fresh shad was recorded as 6.8 and it has increased significantly throughout storage period for all groups (p<0.05) (table 3). the ph values of control group were significantly higher than the experimental groups and reached 7.18 at the time of spoilage. similar result was obtained on the 5th day for the group stored in ice at ambient temperature, while higher values were recorded for fish samples stored at refrigerated temperatures on the day when the fish was organoleptically unfit for consumption. although, increase in ph values correlates well with the spoilage during storage, the values do not support sensory values. although, different authors obtained lower ph values for fresh shad species, they likewise obtained similar trend for ph values during spoilage (köse et al., 2000; duyar et al., 2012; xu et al., 2014). tvb-n has been used as a quality indicator of fish products (huss 1988; connell 1990). it includes ammonia; primary, secondary, and tertiary amines; and products of protein breakdown. an increase in tvb-n is attributed to an increase in the activity of endogenous enzymes and bacterial growth (xu et al., 2014). the european union sets varying tvb-n limits as 25-35 mg/100 g for unprocessed fishery products that is regarded as unfit for human consumption, where organoleptic assessment has raised doubts as to their freshness (eu directive 2005 and 2008). the changes in the tvb-n values during storage are shown in table 4. the values increased significantly with the increasing storage time for all groups (p<0.05). this increase may be attributed to the production of ammonia. significant differences were also observed between control group and the experimental groups beginning from the 1st day of storage (p<0.05). tvb-n value of the control group was close to the suggested limit on the 3rd day of storage, when the fish   ital. j. food sci., vol 28, 2016 237 became organoleptically unfit for consumption. the levels reached 82.6 mg/100g on the 4th day. however, tvb-n values did not support sensory results obtained for the experimental groups and the levels reached unacceptable limit on the 6th and 8th day of storage for samples stored in refrigerator and ice at ambient temperature, respectively. tvb-n values of the samples stored in ice at refrigerated temperature were found within the acceptable quality throughout the storage. this result confirms our earlier findings with fresh bonito on the beneficiary effect of using ice during refrigerated storage (koral and köse, 2012). previous studies also demonstrated that filleting and addition of electrolyzed, oxidized water and chitosan can retard the development of tvb-n in fish (köse and koral, 2012; xu et al., 2014). however, higher tvb-n values were found by köse et al. (2000) with shad species kept at refrigerated temperature and packed in stretch film without ice. the higher values can be explained by the sampling season, being carried out in summer in previous study, while sampling was done in winter in the current study, since the water temperature can affect the initial microbial load and variations in bacterial species, leading to differences in tvb-n formation. duyar et al. (2012) also obtained higher tvb-n values for a. tanaica, which was kept in refrigerated temperature without ice application. however, they did not state the sampling time and the initial conditions of fish before storage. therefore, we assumed that the initial conditions of fish may be the reason for differences in the results. trimethylamine is a pungent, volatile amine often associated with the typical "fishy" odour of spoiling seafood. its presence in spoiling fish is due to the bacterial reduction of trimethylamine oxide, which is naturally present in the living tissue of many marine fish species. although, tma is believed to be generated by the action of spoilage bacteria, its correlation with bacterial numbers is often not very good (huss 1995). a suggested, acceptable level is reported as 12 mg/100 g (goulas and kontominos 2005). initial tma-n values was 0.28 mg/100g and it significantly increased throughout the storage period for all experimental groups (table 4). significant variations were observed between control and other groups, beginning from the 1st day of storage, and also within the groups (p<0.05). the lowest tma-n values were found in the group stored in ice at refrigerated temperature, while the highest levels were received by the control group. the values were within the acceptable levels for this parameter throughout the storage period. therefore, tma values did not support the sensory results for this species. the results were in agreement with our previous findings for fresh bonito stored under refrigerated conditions (koral and köse, 2012). tba is a by-product of lipid oxidation. therefore, it is a common method for assessing lipid oxidation in fish products (xu et al., 2014). the tba value for a good quality chilled fish is reported to be between 5 and 8 mg malonaldehyde/kg, while the levels of 8 mg malonaldehyde/kg fish flesh are generally regarded as the limit of acceptability for most fish species (schormüller, 1969). the values of tba increased significantly depending on time, and significant variations were also observed between the control group and the other groups (p<0.05), beginning from the 1st day of storage (table 4). significant differences were observed within the experimental groups beginning from the 2nd day of storage. the control group reached an unacceptable limit on the 4th day, while fish stored at refrigerated temperature without ice got spoilt on the 5th day. the sample kept in ice and stored at ambient temperature had a slightly favourable condition and tba value reached an unacceptable level on the 7th day. fish kept in ice and in refrigerated conditions had the lowest values that were within the acceptable quality throughout the storage period. these results suggest that storage of fish in ice should also be applied in refrigerated/cold storage conditions to avoid lipid oxidation. cold storage without ice application is not suitable to retard oxidation. tba values were only in support of control group in terms of its overall acceptability of sensory values. lower values were recorded from previous   ital. j. food sci., vol 28, 2016 238 studies for bonito and indian shad during refrigerated storage (köse et al., 2000; köse and koral, 2012; massa et al., 2012; xu et al., 2014). the differences in tba values might be attributed to the fat content in different fish species. the results of duyar et al. (2012) supported our findings for tba results. the results of the total mesophilic and psychrophilic viable bacteria and hfb are shown in table 5. initial counts of mesophilic and psychrophilic total viable bacteria, and the total mesophilic and psychrophilic hfb of fresh fish were observed as 1.95, 2.18, 2.15 and 2.45 log cfu/g, respectively. significant differences were found amongst the groups along with the increasing levels throughout the storage with some exceptions (p<0.05). storage in ice and under refrigerated conditions significantly decreased the total mesophilic viable bacteria and hfb load of the samples in the first day of storage, then significant increase occurred in the counts during further storage (p<0.05). the lowest mesophilic counts were obtained for samples containing ice at refrigerated temperatures within a variation of 1.303.18 log cfu/g. table 3: the changes in ph values of spotless shad groups stored under various conditions. day of storage c iat rt irt fresh 6.80±0.08 6.80±0.08 6.80±0.08 6.80±0.08 1 7.02±0.02aa 6.88±0.03 b a 6.87±0.04 b a 6.86±0.05 b a 2 7.14±0.03ab 6.90±0.06 b a 6.97±0.02 b b 6.88±0.03 b a 3 7.18±0.09ac 6.94±0.04 b a 7.07±0.04 c c 6.90±0.03 b a 4 7.37±0.02ad 7.02±0.03 b b 7.17±0.02 c d 6.94±0.04 d a 5 7.64±0.02ae 7.08±0.06 b b 7.21±0.03 c e 6.98±0.04 d a 6 * 7.12±0.03abc 7.26±0.03 b e 6.99±0.04 c a 7 * 7.18±0.04ac 7.38±0.02 b f 7.00±0.02 c a 8 * 7.24±0.08ac 7.50±0.09 b g 7.05±0.01 c b 9 * * 7.40±0.07ad 7.09±0.02 b c 10 * * * 7.18±0.03d 11 * * * 7.23±0.04d 12 * * * 7.26±0.05d 13 * * * 7.34±0.06e *: not analyzed, different superscript small letters (a,b,c) represents statistical differences among groups (p<0.05). different superscript capital letters (a,b,c,d,…) represents statistical differences amongst different days within the same group during storage (p<0.05).c: control, iat: fish stored in ice at ambient temperature; rt: refrigerated fish without ice; irt: fish stored in ice at refrigerator temperature.   ital. j. food sci., vol 28, 2016 239 table 4: the changes in the levels of chemical quality parameters of spotless shad groups stored at various conditions. day of storage sample type chemical criteria tvb-n (mg/100 g) tma (mg/100 g) tba (mg mda/ kg) 0 fresh 9.11±0.30 0.28±0.13 0.46±0.28 1 c 14.71±0.28aa 1.36±0.06 a a 0.83±0.10 a a iat 11.21±0.28ba 1.08±0.18 b a 0.57±0.05 b a rt 12.61±0.15ba 0.53±0.04 c a 0.63±0.08 b a irt 10.51±0.16ca 0.39±0.08 d a 0.47±0.09 b a 2 c 21.01±0.16ab 2.58±0.10 a b 1.68±0.06 a b iat 14.85±0.34bb 1.78±0.05 b b 1.09±0.08 b b rt 14.48±0.26bb 1.01±0.08 c b 1.47±0.09 c b irt 11.91±0.15ca 0.65±0.13 d b 0.63±0.02 d b 3 c 28.72±0.18ac 2.94±0.08 a c 4.62±0.23 a c iat 14.71±0.22bb 2.08±0.16 b c 2.06±0.17 b c rt 17.02±0.20cc 1.33±0.19 c c 3.55±0.05 c c irt 13.31±0.12bb 0.77±0.08 d b 0.90±0.14 d c 4 c 82.65±0.21ad 3.86±0.18 a d 9.98±0.16 a d iat 17.51±0.10bc 2.98±0.06 b d 4.86±0.12 b d rt 21.18±0.20cd 2.42±0.03 c d 7.13±0.07 c d irt 14.01±0.12db 0.86±0.14 d c 1.18±0.10 d d 5 c 119.07±0.16ae 5.89±0.21 a e 14.31±0.08 a e iat 18.21±0.23bd 3.86±0.07 b e 6.14±0.18 b e rt 24.86±0.15ce 3.25±0.18 c e 11.15±0.07 c e irt 16.11±0.10dc 1.01±0.09 d d 1.38±0.16 d e 6 iat 23.81±0.14ae 5.14±0.22 a f 7.67±0.03 a f rt 29.40±0.20bf 4.67±0.12 b f 13.24±0.05 b f irt 17.51±0.26cd 1.25±0.14 c e 2.02±0.16 c f 7 iat 26.62±0.28af 7.68±0.18 a g 10.09±0.18 a g rt 33.26±0.20bg 5.49±0.09 b g 15.35±0.19 b g irt 20.31±0.17ce 1.40±0.03 c f 2.46±0.16 c g 8 iat 32.92±0.15ag 8.12±0.07 a h 11.72±0.17 a h rt 42.82±0.20bh 6.99±0.08 b h 18.36±0.25 b h irt 21.71±0.22ce 1.57±0.07 c g 3.06±0.14 c h 9 rt 37.82±0.22ah 9.28±0.16 a i 13.19±0.28 a i irt 22.41±0.12bf 1.71±0.10 b h 3.86±0.18 b i 10 irt 24.51±0.30g 1.86±0.08h 4.98±0.20i 11 irt 25.21±0.28g 1.81±0.14h 5.55±0.55i̇ 12 irt 25.91±0.16g 2.03±0.12i 6.14±0.14j 13 irt 26.62±0.34g 2.42±0.10i̇ 6.82±0.18k different superscript small letters (a,b,c) represents statistical differences among groups (p<0.05). different superscript capital letters (a,b,c,d,…) represents statistical differences amongst different days within the same group during storage (p<0.05).c: control, iat: fish stored in ice at ambient temperature; rt: refrigerated fish without ice; irt: fish stored in ice at refrigerator temperature. tvb-n: total volatile basic nitrogen, tba: thiobarbituric acid value, tma-n: trimethylamine nitrogen.   ital. j. food sci., vol 28, 2016 240 the recommended limit for total viable bacteria counts (tvc) for fresh fish consumption is reported to be between 6-7 log cfu/g (icmsf 1992; chotimarkorn, 2014). the results of this study suggest that although, mesophiles reached the unacceptable level on the 3rd day of storage for the control group, psychrophilic bacteria counts were still within an acceptable level for further storage, possibly due to unfavourable conditions. better quality was obtained from the samples kept in ice at ambient conditions in comparison with samples stored without ice at refrigerated conditions, in terms of bacteria growth, indicating the washing effect of ice during melting at ambient conditions. tvc exceeded the limit for psychrophilic bacteria of the samples stored under refrigerated conditions without ice on the 7th day, while samples with ice at ambient conditions were still acceptable up to the 9th day. however, the best quality was found for shad which was kept in ice and under refrigerated conditions, the tvc was still within the acceptable level throughout the storage period. sensory results supported tvc for control group and the bacteria load are still under the suggested limit for other groups within their acceptable sensory quality. with the exception of tvb-n, the results of mesophilic and psychrophilic viable counts did not support chemical quality parameters. uddin et al. (2001) observed a higher bacteria load from the muscle of indian shad (tenualosa ilisha) stored in ice, and placed in insulated polystyrene and wooden boxes. they also reported higher counts for fish stored in wooden boxes than fish stored in the initial. the total bacterial count in the muscle of the fish stored in insulated boxes was 3.5 x 104 cfu/g and it increased to 1.8 x 109cfu/g after 18 days, when the fish became organoleptically unfit for consumption. they also reported that the bacterial flora of the fishes stored in wooden boxes showed comparatively higher initial bacteria counts and increased rapidly during storage. no study exists on the microbial changes of fresh, spotless shad, either at ambient or chilled conditions. therefore, this study presents new data on the possible microbial activities during storage of this species under different conditions. initial levels of hfb in fish is important since previously formed histamine decarboxylases, are likely to continue to decarboxylase histidine to histamine, even when histamine decarboxylase positive bacteria are no longer viable (köse, 2010). in this study, initial hfb counts were 2.15 log cfu/g for mesophiles and 2.45 log cfu/g for psychrophilic bacteria. the counts also increased significantly throughout the storage period with the exception of mesophiles, which significantly dropped on the 1st day for samples stored in ice at ambient temperatures and without ice at refrigerated temperatures (p<0.05). significant variations were observed between control group and other groups as well as within the experimental groups (p<0.05). the highest hfb counts was recorded for control group, while the lowest were observed in samples stored in ice at refrigerated temperatures. the results of bas contents are shown in table 6. biogenic amines contents varied significantly depending on storage life and the groups, with some exceptions (p<0.05). histamine is the only bas that is regulated for fish and fisheries products. the european regulation (eu) permits up to 100 mg/kg for fresh and processed fish, and 200 mg/kg for fishery products which have undergone enzyme maturation treatment in brine (eu directive, 2005), while fda allows for less than 50 mg/kg (fda, 2011). shad is listed among the regulated fish species due to its high histidine content (fda, 2011). histamine contents in samples representing control and in all experimental groups, were under the detection limit at the beginning of the storage trial and the 1st day of storage, and significantly increased throughout storage (p<0.05). detectable levels started on the 2nd day for control group as 7.41 mg/kg and the values reached an unacceptable level set by the fda on the 5th day. detectable values of histamine were obtained on the 3rd day for the samples stored in ice at ambient temperature and without ice at refrigerated temperatures as 4.93 mg/kg and 1.50 mg/kg, respectively.   ital. j. food sci., vol 28, 2016 241 table 5: microbial changes of the shad groups stored under various conditions. day of storage sample type total viable bacteria (log cfu/g) total histamine forming bacteria (log cfu/g) mesophiles psychrophilic mesophiles psychrophilic 0 fresh 1.95±0.07 2.18±0.10 2.15±0.23 2.45±0.13 1 c 3.12±0.14aa 2.20±0.09 a a 3.36±0.12 a a 2.74±0.04 a a iat 2.95±0.10aa 2.76±0.11 b a 1.95±0.15 b a 2.57±0.07 b a rt 1.60±0.08ba 2.43±0.12 a a <1.47 2.72±0.16 a a irt <1.47 2.72±0.07ba 2.36±0.17 d a 2.30±0.15 b a 2 c 5.22±0.13ab 2.24±0.10 a a 4.41±0.23 a b 2.98±0.10 a b iat 3.27±0.15bb 3.04±0.12 b b 2.81±0.09 b a 2.85±0.13 a b rt 1.90±0.12cb 3.44±0.07 c b 1.60±0.12 c b 4.31±0.15 b b irt 1.60±0.05db 2.61±0.20 d b 1.78±0.08 c b 2.18±0.02 c a 3 c 6.34±0.25ac 2.26±0.04 a a 5.18±0.13 a c 3.10±0.09 a b iat 3.45±0.07bc 3.00±0.28 b b 3.73±0.11 b b 3.30±0.21 a c rt 2.30±0.10cc 3.85±0.17 c c 2.23±0.14 c c 4.28±0.01 b b irt 1.85±0.03dc 2.68±0.12 d b 1.95±0.10 d b 2.89±0.12 c b 4 c 7.26±0.29ad 2.43±0.12 a b 5.58±0.27 a d 3.32±0.14 a c iat 3.95±0.10bd 3.30±0.21 b c 3.40±0.07 b c 3.45±0.13 a c rt 4.19±0.13cd 5.21±0.12 c d 3.57±0.09 b d 5.36±0.09 b c irt 2.20±0.09dd 2.80±0.06 d a 2.48±0.23 c a 2.93±0.12 c b 5 c 9.43±0.21ae 2.38±0.04 a b 6.23±0.17 a e 3.40±0.18 a c iat 4.12±0.17bd 3.54±0.13 b d 3.60±0.05 b b 3.69±0.12 a d rt 4.24±0.22bd 5.48±0.19 c e 3.83±0.10 b e 5.60±0.13 b d irt 3.02±0.18ce 2.93±0.04 d c 2.57±0.20 c a 3.19±0.06 c b 6 iat 4.42±0.05ae 3.70±0.09 a e 3.85±0.14 a b 4.26±0.30 a e rt 5.04±0.17be 6.30±0.32 b f 3.60±0.13 a d 5.90±0.15 b e irt 2.91±0.02ce 3.04±0.09 c c 2.86±0.17 b c 3.30±0.05 b c 7 iat 4.82±0.13af 4.19±0.15 a f 3.93±0.15 a b 4.39±0.17 a e rt 5.28±0.07bf 7.39±0.06 b g 3.78±0.12 a e 6.69±0.01 b f irt 2.48±0.11cd 2.77±0.07 c a 2.48±0.16 b a 3.37±0.15 c c 8 iat 5.60±0.06ag 4.24±0.11 a f 4.20±0.29 a d 5.48±0.04 a f rt 5.85±0.09bg 7.85±0.13 b h 4.40±0.08 a f 6.48±0.09 b g irt 2.70±0.12cd 2.82±0.08 c a 2.00±0.17 b b 3.54±0.08 c d 9 iat 6.94±0.30ah 4.46±0.10 a g 4.18±0.05 a d 6.18±0.12 a g irt 2.95±0.11be 3.11±0.10 b c 2.30±0.12 b a 3.27±0.06 b c 10 irt 2.90±0.08e 2.98±0.11c 3.11±0.09d 3.59±0.08d 11 irt 3.18±0.07f 2.89±0.06a 3.30±0.07e 3.79±0.10e 12 irt 3.10±0.10f 3.04±0.03c 3.18±0.05d 3.28±0.09c 13 irt 3.00±0.11e 3.29±0.14c 3.23±0.16e 3.48±0.07d different superscript small letters (a,b,c) represents statistical differences among groups (p<0.05). different superscript capital letters (a,b,c,d,…) represents statistical differences amongst different days within the same group during storage (p<0.05).c: control, iat: fish stored in ice at ambient temperature; rt: refrigerated fish without ice; irt: fish stored in ice at refrigerator temperature.   ital. j. food sci., vol 28, 2016 242 according to fda, the values were unacceptable on the 8th and 9th day, for samples stored at refrigerated temperature without ice and ambient temperature in ice, respectively. the lowest histamine values were recorded for the samples stored in ice at refrigerated temperature. the detectable levels were obtained for this group on the 7th day, and the values were under 10 mg/kg throughout the storage period. therefore, this condition is found to be the most suitable method used to store fresh shad in order to avoid histamine health risk. histamine values were well correlated with sensory and chemical quality parameters in terms of food safety. al-bulushi at al. (2009) reported that mesophilic bacterial count of log 6–7 cfu/g has been associated with 50 mg/kg histamine. the suggested level reached by the control group on the 3rd day. on the other hand, the results of mesophilic hfb count on the 5th day were correlated with the amount of histamine obtained on the relating day for control group. the results of mesophilic bacteria counts obtained for samples stored in ice at ambient temperature supported the statement of albulishi at al. (2009), while the counts of psychrophilic total viable bacteria and hfb were in agreement with this statement, for the group which stored in refrigerator without ice, indicating a favourable temperature for such bacteria. since both bacteria counts and histamine values were well below the permitted levels for the group stored in ice and at refrigerated temperature, the results gotten from this group also supports the above statement. although, significant variations were obtained for tryptamine levels of some groups on certain days, the values were found insignificant amongst the groups. tryptamine values were observed to be below 7 mg/kg throughout the storage. the values of phenylethylamine decreased at the beginning of storage, thereafter, fluctuations in the levels occurred. putrescine value of fresh fish was 0.73 mg/kg at the beginning of trial, then, the levels increased sharply up to 72.5 mg/kg for control group. although, the putrescine levels of other groups also increased significantly, the rate was significantly slower for these groups, with the lowest values being at 2.49 mg/kg, representing the samples kept in ice at refrigerated temperature at the end of the storage period. higher values were observed for cadaverine with the highest levels corresponding to control group. the values reached up to 166.69 mg/kg for control group on the 5th day, while the levels were 56 mg/kg and 120.02 mg/kg on the 8th and 9th day, for groups stored in ice at ambient temperature and without ice at refrigerated temperature, respectively. fig 1. shows the samples stored in ice at refrigerated temperature which also contained the lowest cadaverine value and the result was 25.18 mg/kg at the end of storage. tyramine is a naturally occurring monoamine compound derived from the amino acid, tyrosine. fresh fish contains little or no tyramine, but a large amount can be found in spoiled or fermented fish (fao, 2013). tyramine values also increased significantly during storage (p<0.05) with the highest values representing the control group at 54.63 mg/kg on the 4th day. the values of other groups were significantly lower in comparison to control group (p<0.05). the lowest values were attributed to the samples stored at refrigerated temperatures. spermidine values of fresh fish was 12.32 mg/kg, and the levels decreased significantly in all groups throughout storage (p<0.05). significant variations were also observed amongst the groups (p<0.05). the changes in the spermine values are usually insignificant up to the 6th day of storage with some exceptions, thereafter, fluctuations in the values were observed. the highest spermine level was determined to be 4.70mg/kg on the 7th day for the sample stored in ice at ambient temperature. biogenic amines in foods are reported to be indicators of freshness or spoilage in fish. moreover, some migraines are known to bas, particularly, tyramine and phenylethylamine. although, tyramine might also act as a histamine potentiator (taylor and lieber, 1979), the contribution of this biogenic amine to histamine poisoning is not clear. however, around 100-800 mg/kg of tyramine and 30 mg/kg of   ital. j. food sci., vol 28, 2016 243 phenylethylamine have been reported to be toxic doses in foods (koral and köse 2012). our study showed that the levels of tyramine and phenylethylamine did not reach toxic levels within the storage period of this research fig. 1: chromatogram belongs to shad samples (irt group, 13th days).   ital. j. food sci., vol 28, 2016 244 table 6: the values of biogenic amines for the shad samples stored under various conditions. day of storage results of biogenic amines (mg/kg) sample groups tryptamine phenylethylamine putrescine cadeverine histamine tyramine spermidine spermine 0 fresh 5.60±0.21 18.21±0.26 0.73±0.13 2.80±0.08 <0.86* 2.12±0.19 12.32±0.11 2.49±0.23 1 c 5.04±0.62aa 17.11±0.44 a a 1.32±0.16 a a 4.28±0.37 a a <0.86* 1.83±0.21 a a 9.34±0.20 a a 2.37±0.18 a a iat 6.45±0.50ba 15.82±0.54 b a 1.37±0.14 a a 4.82±0.23 b a <0.86* 2.10±0.15 a a 11.70±0.42 b a 2.67±0.10 a a rt 5.98±0.31ba 16.07±0.35 b a 0.95±0.12 b a 5.35±0.29 c a <0.86* 1.95±0.21 a a 10.52±0.34 a a 3.34±0.19 b a irt 6.02±0.03ba 17.62±0.05 a a 0.78±0.04 c a 3.40±0.14 d a <0.86* 2.01±0.01 a a 12.28±0.10 c a 3.65±0.02 b a 2 c 5.13±0.23aa 8,74±0,21 a b 6,83±0,22 a b 31,44±0,80 a b 7.41±0.15a 6,97±0,13 a b 5,29±0,10 a b 2,73±0,18 a a iat 5.43±0.18ab 16,83±0,39 b b 2,94±0,24 b b 15,60±0,71 b b <0.86* 4,74±0,22 b b 9,75±0,36 b b 2,67±0,31 a a rt 5.85±0.37aa 17,75±0,49 b b 1,66±0,11 c b 9,25±0,37 c b <0.86* 2,82±0,20 c b 9,74±0,35 b a 3,08±0,11 b a irt 5.35±0.49ab 18,86±0,42 c b 2,38±0,11 d b 6,57±0,30 d b <0.86* 1,96±0,11 d a 11,06±0,52 c a 3,32±0,40 b a 3 c 5.15±0.21aa 8,87±0,27 a b 12,76±0,65 a c 42,25±0,93 a c 15.88±0.60 a b 14,31±0,83 a c 4,11±0,28 a c 2,36±0,11 a a iat 4.93±0.32ac 12,59±1,00 b c 8,89±0,44 b c 35,70±0,71 b c 4.93±0.13 b a 6,67±0,47 b c 6,18±0,28 b c 1,81±0,11 b b rt 5.73±0.38ba 18,06±0,77 c b 1,70±0,11 c b 7,59±0,52 c c 1.55±0.11 c a 3,27±0,16 c c 9,30±0,44 c a 2,85±0,21 c a irt 5.71±0.42bb 17,25±0,64 c a 0,92±0,10 d a 4,82±0,28 d c <0.86* 2,02±0,15 d a 8,13±0,25 d b 3,34±0,30 d a 4 c 5.48±0.25aa 8,10±0,28 a b 55.91±1,32 a d 126.32±1,38 a d 35.86±0.64 a c 54,63±0,89 a d 4,12±0,30 a c 2,83±0,25 a a iat 4.91±0.13bc 12,20±0,40 b c 3,94±0,11 b d 25,83±0,86 b d 8.26±0.60 b b 6,57±0,33 b c 6,35±0,37 b c 3,10±0,14 a c rt 5.17±0.23aa 16,90±0,28 c a 2,70±0,12 c c 10,41±0,61 c b 1.88±0.14 c a 3,16±0,28 c c 7,53±0,32 c b 2,92±0,16 a a irt 5.18±0.29ab 17,54±0,61 c a 1,69±0,18 d c 8,13±0,24 d d <0.86* 2,76±0,14 c b 8,25±0,42 d b 2,37±0,12 b b 5 c 5.38±0.42aa 9,85±0,35 a c 72,57±0,98 a e 166,69±1,81 a e 48.82±1.68 a d 46,31±0,98 a e 3,39±0,25 a d 2,27±0,15 a a iat 4.92±0.47ac 16,04±0,80 b b 4,36±0,29 b e 27,80±0,71 b e 16.88±0.25 b c 6,37±0,36 b c 7,65±0,40 b d 2,44±0,30 a a rt 5.25±0.34aa 16,76±0,39 b a 3,36±0,21 c d 12,70±0,57 c d 2.59±0.18 c b 3,16±0,25 c c 8,50±0,72 b b 2,64±0,31 a a irt 5.10±0.20ab 15,31±0,63 b c 3,52±0,47 c d 19,35±0,94 d e <0.86* 3,67±0,43 c c 6,87±0,25 b c 2,62±0,33 a b different superscript small letters (a,b,c) represents statistical differences among groups (p<0.05). different superscript capital letters (a,b,c,d,..) represents statistical differences amongst different days within the same group during storage (p<0.05).c: control, iat: fish stored in ice at ambient temperature; rt: refrigerated fish without ice; irt: fish stored in ice at refrigerator temperature*: lod(limit of detection for histamine). (lod values for histamine:0.86; for putrescine:0.56; for tryptamine: 1.80; phenylethylamine: 1.50; cadeverine: 0.66; tyramine: 0.87; spermidine: 0.65; spermine: 0.71 mg/kg.).   ital. j. food sci., vol 28, 2016 245 limited studies exist for the biogenic amine contents of spotless shad. in our previous studies on salted shad products, obtained from turkey and greece, we determined histamine values to be below 20 mg/kg, although, higher amounts of tyramine and cadaverine which is about 85mg/kg and 100 mg/kg, respectively, were observed for products stored at ambient temperatures, while low values correspond to the samples kept at chilled conditions (köse et al., 2011; koral et al., 2013). 5. conclusions good sensory quality was observed in fresh shad with the addition of ice during refrigerated storage. ice application and refrigerated storage increased the shelf-life for 10 days. the worst sensory results, represented control group which was without ice and ambient temperature storage shelf life less than three days. storing fresh shad in ice also helped to improve its physico-chemical quality and decrease biogenic amine development. therefore, keeping fresh shad in ice under refrigerated storage is advised, in order to delay histamine formation and retard quality loss prior to processing or fresh market. the results suggest that using ice can improve the shelf-life of shad, stored at ambient and refrigerated temperatures as relating to food quality and safety. references al-bulushı i., poole s., deeth h.c. and dykes g.a. 2009. biogenic amines in fish: roles in intoxication, spoilage, and nitrosamine formation-a review. crit. rev. food sci. nutr. 49(4): 369-377. archer m. 2010. sensory assessment score sheets for fish and shellfish torry & qim. research & development department of seafish. http://www.seafish.org/media/publications/sensory_assessment_scoresheets_14_5_10.pdf boland f.e. and paige d.d. 1971. collaborative study of a method for the determination of trimethylamine nitrogen in fish. j. aoac. 54: 725-727. boran g. and karaçam h. 2011. seasonal changes in proximate composition of some fish species from the black sea. turkish journal of fisheries and aquatic sciences. 11 (1): 01-05. botta j.r. 1995. evaluation of seafood freshness quality. new york: vch publishers inc. careche m., garcía r. and borderías j. 2002. anchovy shelf-life as affected by different chilling methods during distribution. j. food protect. 65(2): 353-362. chotımarkorn c. 2014. quality changes of anchovy (stolephorus heterolobus) under refrigerated storage of different practical industrial methods in thailand. j. food sci. technol. 51(2): 285-93. connel j.j. 1990. methods of assessing and selecting for quality. in control of fish quality. 3rd ed. fishing news books, oxford. p. 122-150. duyar h.a., gargacı a. and altınelataman c. 2012. determination of chemical composition and shelf life of shad (alosa tanaica grimm, 1901) in refrigeration conditions. journal of fisheriessciences.com. 6 (1): 1-8. ergüden d., turan c. and çevik c. 2007. the growth features of pontic shad alosa pontica (eichwald, 1838) in the sea of marmara. turkey. j biologic. sci. 7(4) : 685-688. eu directive. 2005. official journal of the european union. commission regulation (ec) no 2074/2005 of 5 december 2005. l 338/36. eu directive. 2008. commission regulation (ec) no 1022/2008 of 17 october 2008 amending regulation (ec). no 2074/2005 as regards the total volatile basic nitrogen (tvb-n) limits, 277, 18-20. fao, 2014. fao yearbook, fishery and aquaculture statistics. rome/italy, fao. 76 pp. fda. (2011). fish and fisheries products hazards and controls guide. chap.13 (4th ed.).   ital. j. food sci., vol 28, 2016 246 goulas a.e. and kontominos m.g. 2005. effect of salting and smoking method on the keeping quality of chub mackerel (scomber japonicus): biochemical and sensory attributes. food chemistry, 93: 511-520. http://www.fda.gov/downloads/food/guidancecomplianceregulatoryinformation/guidancedocuments/seafood/ ucm251970.pdf accessed 2014 march 21. huss h.h. 1988. fresh fish, quality and quality changes. fao fisheries series, no. 29. fao. 132p. rome, italy. huss h.h. 1995. quality and quality changes in fresh fish. fao fisheries series, technical paper, no. 348. rome, italy. icmsf. 1992. microorganisms in food. in: sampling for microbiological analysis. icmsf (ed). university of toronto press, toronto, canada. inal t. 1992. besin hijyeni. hayvansal gıdalarda kalite kontrol. (2nd ed.) i̇stanbul, turkey: final ofset. koral s. and köse s. 2012. the effect of filleting and ice application on the quality and safety of atlantic bonito (sarda sarda) at refrigerated storage. int. j. food sci. technol. 47 (1): 210-220. koral s., tufan b., scavnicar a., kocar d., pompe m. and köse s. 2013. investigation of the contents of biogenic amines and some food safety parameters of various commercially salted fish products. food control 32: 597-606. köse s. 1993. investigation into toxins and pathogens implicated in fish meal production. phd thesis, uk: loughborough university of technology. köse s. and erdem m.e. 2004. an investigation of quality changes in anchovy (engraulis encrasicolus, l. 1758) stored at different temperatures. turkish j. vet. animal sci. 28 (3): 575-582. köse s., 2010. evaluation of seafood safety health hazards for traditional fish products: preventive measures and monitoring issues, turkish journal of fisheries and aquatic sciences, 10: 139-160. köse s., ay s., and uzuncan y. 2000. trabzon’da satılan bazı balık türlerinin buzdolabı koşullarında depolanmalarında meydana gelen kimyasal ve duyusal kalite değişimleri üzerine bir araştırma. ege su ürünleri dergisi. 17 (3-4): 35-48. köse s., kaklıkkaya n., koral. s., tufan b., buruk k.c. and aydın f. 2011. commercial test kits and the determination of histamine in traditional (ethnic) fish products evaluation against an eu accepted hplc method. food chemistry, 125: 1490-1497. köse s., karaçam h., kutlu s. and boran m. 2001. investigating the shelf-life of the anchovy dish called 'hamsikusu' in frozen storage at -18 +/-1 degrees c. turkish j. vet. animal sci. 25 (5): 651-656. köse s., koral s., tufan b., pompe m., scavniçar a. and koçar d. 2012. biogenic amine contents of commercially processed traditional fish products originating from european countries and turkey. eur. food res. technol. 235 (4): 669-683. lehane l. and olley j. 2000. histamine (scombroid) fish poisoning. a review in a risk-assessment framework. national office of animal and plant health canberra 1999. revised 2000. agricultural, fisheries and forestry of australia. massa a.e., manca e. and yeannes m.i. 2012. development of quality index method for anchovy (engraulis anchoita) stored in ice: assessment of its shelf-life by chemical and sensory methods. food science and technology international. 18: 339-351. navodaru, i., and waldman, j.r. 2003. shads of eastern europe from the black sea: review of species and fisheries. in: biodiversity, status, and conservation of the world’s shads, limburg, k.e. and waldman, j.r (eds). international conference on status and conservation of shads world wide. vol. 35, american fisheries society symposium, pp. 6976. pons-sánchez-cascado s., veciana-nogués m.t., bover-cid s., mariné-font a. and vidal-carou m.c. 2006a. use of volatile and non-volatile amines to evaluate the freshness of anchovies stored in ice. j. sci. food agri. 86, 699-705. schormüller j. 1969. handbuch der lebensmittelchemie (band iii/2). triesrische lebensmittel eier, fleisch, fisch, buttermich, berlin/heidelberg, springer verlag, germany/new york. ny. 1584 p. smith, g., hole m. and hanson s.w. 1992. assessment of lipid oxidation in indonesian salted-dried marine catfish (arius thalassinus). j. sci. agri. 51, 193-205. sokal r.r. and rohlf f.j. 1987. introduction to biostatistics. (2nd ed.). new york, usa: w.h. freeman and company.   ital. j. food sci., vol 28, 2016 247 taylor s.l. and lieber e.r. 1979. in vivo inhibition of rat intestinal histamine-metaboliz ing enzymes. food cosmetic toxicol., 17(3): 237-240. tui̇k, 2013. fishery statistics book 2013, turkish statistical institute (tui̇k), ankara/turkey, 61 pp. uddin m., moin uddin a., tanaka m. and kamal m.d. 2001. bacteriological changes of indian shad (tenualosa ilisha) during ice storage. journal of food science and technology, india. 38 (6): 639-642. varlık c. and heperkan d. 1990. preservation of anchovies in ice. j. aquatic products. 4(1): 53-58. vecıana-nogués m.t., vıdal-carou m.c. and marıné-font a. 1990. histamine and tyramine during storage and spoilage of anchovies (engraulis encrasicholus): relationships with other fish spoilage indicators. j. food sci. 55 (4): 1192-1195. xu g., xue t., shihan t., huabin y., huawei s. and ruobo g. 2014. combined effect of electrolyzed oxidizing water and chitosan on the microbiological, physicochemical, and sensory attributes of american shad (alosa sapidissima) during refrigerated storage. food control 46: 397-402. paper received july 10, 2015 accepted october 18, 2015 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (4): 25–32 issn 1120-1770 online, doi 10.15586/ijfs.v34i4.2259 25 pea protein isolates: emulsification properties as affected by preliminary pretreatments g. d’alessio1, f. flamminii2, m. faieta1, p. pittia1, c.d. di mattia1* 1department of bioscience and technology for agriculture, food and environment, university of teramo, teramo, italy; 2department of innovative technologies in medicine and dentistry, university “g. d’annunzio” of chieti-pescara, chieti, italy *corresponding author: c.d. di mattia, department of bioscience and technology for agriculture, food and environment, university of teramo, via renato balzarini 1, 64100 teramo, italy. email: cdimattia@unite.it received: 13 july 2022; accepted: 16 november 2022; published: 2 december 2022 © 2022 codon publications open access paper abstract the surface and emulsifying properties of a commercial pea protein isolate in oil-in-water model emulsions and the role of insoluble residues in emulsion stability were investigated. droplet size distribution, flocculation index, microstructure, and protein coverage of the emulsions were evaluated. the insoluble fraction positively contributed to the pea proteins’ emulsifying properties, allowing the formation of emulsions with higher dispersion degree, especially at low isolate concentration, with an enhancement of the physical stability. keywords: emulsion stability; pea protein; plant proteins; oil-in-water emulsions; technological properties introduction the growing consumer concern on issues, such as climate change, environmental sustainability, health, and well-being, has led to the development and spread of new eating styles, such as vegetarians, vegans, and flexitarians, which are considered more environment-friendly and sustainable. in parallel, the food industry started to invest in the use of alternative food ingredients and the development of innovative meat-analogue formulations based on alternative protein sources such as plants, insects, and algal proteins. the importance of proteins in food technology is due not only to their nutritional value but also to the technological properties that they exert in the systems in which they are either naturally present or intentionally added for formulation/processing purposes. the majority of foods are complex colloidal systems in which the emulsifying properties are of particular interest. the latter ones are determined by the amphiphilic nature of proteins, which allows them to arrange and organize, based on the specific type of emulsion, at either the oil–water or the water–oil interface leading to a reduction in surface tension, promoting the dispersion of immiscible phases and improving the physical stability of the system. thanks to these properties, proteins are excellent emulsifying agents to be used both in the formulation of common foods, such as seasoning sauces (e.g. mayonnaise or vinaigrettes), ice creams, and snacks, and in the design and development of “new generation” products, such as meat-analogues, in which they are both structuring and emulsifying agents (de angelis et al., 2020; zhu et al., 2021). among vegetable proteins, pea proteins have become particularly popular in recent years; they are extracted from the seed of pisum sativum l., one of the most widely cultivated legumes in the world, due to their excellent tolerance to low temperatures during germination and growth, utilized for both human and animal nutrition (lu et al., 2020). pea seed is characterized by starch and fiber content of 40–50% and 10–20% (d. m.), respectively. the protein fraction (about 20–30%) consists of 55–65% of globulins, 18–25% of albumins, 4–5% prolamins, and 3–4% of glutelin (karaca et al., 2011; lu et al., 2020; tulbek et al., 2016). globulins are the principal reserve 26 italian journal of food science, 2022; 34 (4) d’alessio g et al. physical stability of the corresponding emulsions were evaluated. materials and methods materials the ppi was kindly donated by victa food srl (mogliano veneto, italy) on behalf of cosucra (warcoing, belgium). ppi was obtained through a process consisting of different phases including alkaline extraction, decantation, pasteurization, purification, and spray-drying, with 0.8% of carbohydrates, 2.4% of fibers, and 86% of proteins, as reported in the technical data sheet. sunflower oil was obtained from a local supermarket (oleificio zucchi, cremona, italy). ultrapure water was used to prepare the aqueous protein dispersions; all other reagents were of analytical grade. methods preparation of the ppi dispersions the ppi dispersions were prepared in two different ways, depending on their use in emulsion: for the aqueous non-centrifuged (nc) protein suspension, the ppi was dispersed in distilled water, in a concentration range of 1.0–4.0% (w/w), and left to stir overnight and used as it is; the centrifuged (c) ppi suspension was prepared in the same concentration range as the nc and then submitted to a centrifugation step at 5000 rpm for 20 min. the protein concentration of the dispersions was checked by the bradford assay. surface and interfacial tension surface tension was measured with a tensiometer attension sigma 700/701 (biolin scientific oy, espoo, finland) equipped with a wilhelmy plate t107 (width: 19.44 mm; thickness: 0.1 mm; height: 65 mm). measurements were carried out at 25°c on centrifuged ppi dispersions at different concentrations (0.001% – 0.005% – 0.01% – 0.05% – 0.1% – 0.5% – 1.0% w/v) for 10  min, after 1 min of equilibration time. interfacial tension was measured by the du noüy platinum ring (d:  120.39 mm), between sunflower oil and the centrifuged protein dispersions at the same concentrations, using the same process conditions. emulsion preparation oil-in-water model emulsions were prepared with sunflower oil (20% w/w) and the aqueous dispersions of proteins and include three fractions: legumin, composed of six subunits; vicilin, consisting of poorly glycosylated trimer; and, lastly, convicilin, also consisting of three subunits and which has a great homology with vicilin in the protein core (barać et al., 2015; lu et al., 2020). due to their high lysine content and high nutritional value, pea proteins are considered an excellent candidate to produce meat substitutes and plant-based products (tömösközi et  al., 2001.). generally, globulins have poor solubility, and various studies have highlighted how different environmental conditions (ph, ionic strength, and protein concentration) can have a deep impact on their technological properties (burger and zhang, 2019; kimura et al., 2008; liang and tang, 2013). furthermore, the quantity and the ratio between globulins and albumins and/or legumin and vicilin can vary according to cultivar, species, and production methods, and this can determine substantial differences in the physical-chemical properties and therefore also in the corresponding technological functionality (burger and zhang, 2019). the growing demand for plant-based products has resulted in an increased request for plant protein–based ingredients (e.g. flours, isolates) with desired functionalities for exploitation in food formulations. currently, the production of protein isolates is carried out by the use of rather diverse extraction technologies leading to finished products with different compositions in terms of proteins and insoluble fraction content, including fibers, starch, and large protein aggregates. consequently, uneven technological and functional properties can be expected, making difficult their standardization, with the consequence of variable qualitative characteristics of the final products in which they are intended to be used (boye et al., 2010; karaca et al., 2011; tanger et al., 2020). insoluble particulate materials from plant sources, however, have been recently shown to play an interesting role in colloidal systems through the ability to physically stabilize oil-in-water emulsions, acting through two concomitant mechanisms, that is, particle stabilization of the oil/water interface and increase of the viscosity of the continuous phase (schröder et al., 2021). despite this, limited studies have been carried out to unravel the role of the insoluble fractions of pea protein isolates (ppis) in the formation and stabilization of dispersed emulsions, which, thus, represents the aim of the present work. to this scope, a preliminary centrifugation step was carried out on the isolate suspensions to eliminate insoluble particulate materials; both centrifuged and not centrifuged protein dispersions were then used as emulsifying agents to formulate oil-in-water model emulsions. surface and interfacial properties of the aqueous protein dispersions as well as droplet size distribution, flocculation index, microstructure, protein coverage, and italian journal of food science, 2022; 34 (4) 27 pea protein isolates emulsifying properties microstructural analysis microstructural observation of emulsions made of 2.0% w/w centrifuged and nc pea protein dispersions was carried out under an optical microscope (olympus bx53, tokyo, japan) at 100× magnification, and images were acquired by a digital camera (qimaging fast 1394, surrey, bc, canada) connected to the microscope. viscosity of emulsions the viscosity of e-nc and e-c was determined by using a rheometer (mcr 302, anton paar, graz austria) equipped with a concentric cylinder geometry. flow curves were measured at 20°c at increasing shear rates from 3 to 250 s−1. statistical analysis all experiments were carried out in triplicate; results are reported as mean and standard deviation. a one-way analysis of variance (anova) and tukey’s test were used to establish the significance of differences among the mean values at the 0.05 significance level; data analysis and modeling were carried out by originpro 2016 software (originlab corporation, northampton, ma, usa). results and discussion interfacial and emulsifying properties of ppi the knowledge of the surface and interfacial properties of a protein is important to understand and foresee its behavior in multiphasic systems, such as foams or emulsions; indeed, the amphiphilic nature of proteins allows them to adsorb at the water–oil interface and thus to lower the surface and interfacial tension, hence facilitating the stabilization of dispersed systems (burger and zhang, 2019; mcclements, 2016). however, besides proteins, pea isolates can include a consistent amount of particulate material of different nature, like fibers and starch particles, which may affect by different mechanisms the stabilization of complex colloidal systems. therefore, in this study, centrifugation was applied as a pretreatment to obtain ppi dispersions without the insoluble residue in order to evaluate the contribution of both the pea proteins and insoluble fractions of the isolate on the surface properties and the stabilization of model oil-in-water emulsions. the surface (st) and interfacial (it) tension of ppi suspensions at their native ph (6.5–6.8) after centrifugation is reported in figure 1a and b. the analysis of the pea protein suspensions before centrifugation did not allow to obtain reliable results (data not shown). indeed, as ppi (e-nc) and centrifuged ppi (e-c) section 2.2.1. emulsions were prepared in two steps: the aqueous and oil phases were preliminarily pre-homogenized with a rotor-stator device (yellowline di 25 basic, ika werke gmbh & co, germany) at 13,500 rpm for 1 min and then emulsified using a high-pressure homogenizer (panda plus 2000, gea niro soavi, parma, italy) at 150 bars, for 10 cycles. particle size and flocculation index emulsifying capacity was evaluated by measuring particle size and distribution of oil-in-water emulsions using a laser diffraction particle size analyzer (mastersizer 3000; malvern, worcestershire, uk). for the analysis, a refractive index of 1.474 was chosen for sunflower oil, 0.01 as the absorption index, and water as the dispersing phase, with a refracting index of 1.330. droplet size was expressed as d[4; 3], that is, the de brouckere mean diameter. the stability of the two different emulsions was also evaluated by monitoring the droplet size after 7 days of storage at different temperatures (4°–10°–22°c). the flocculation index of the e-nc and e-c emulsions was obtained according to the formula reported by peng et al. (2016): fi % in water in % sds ( ) ( ) ( ) ; ; � � � � � � � � � � � d d 4 3 4 3 1 1 100 interfacial protein concentration (γ) and percentage of adsorbed proteins (ap%) interfacial protein concentration (γ) and percentage of adsorbed proteins (ap%) at each concentration of the two model emulsion series were determined according to the method described by peng et al. (2016). briefly, an aliquot of 1.5 ml of the emulsion was centrifuged at 10,000 g for 30 min, to separate the cream and the aqueous fractions: the latter was carefully recovered with a syringe and filtered in a 0.45 µm filter. then, the corresponding protein concentration was determined by using the bradford assay. the interfacial protein concentration was computed by using the following equation: mg m d in sds c cini ser 2 3 2 1 6 � � � � � � � � �( )( )( ) ( ) ; � � where cini is the initial protein concentration of the emulsions, cser is the protein concentration determined in the aqueous phase after centrifugation, and φ is the volume fraction of the oil phase. then, the percentage of adsorbed protein was calculated as follows: ap % c c c ini ser ini ( ) ( ) � � �100 28 italian journal of food science, 2022; 34 (4) d’alessio g et al. similarly, a decrease in the it values at the oil–water interface was observed when the isolate concentration was increased. at neutral ph, which is close to the value of the isolate solutions under investigation, pea proteins open their structure and acquire a negative charge, which allows them to adsorb more easily at the interface and lower the value of interfacial tension. values found in the present study are similar to those obtained by amine et al. (2014) at protein concentrations of 0.5% (w/v) and 1.0% (w/v) (amine et al., 2014). the emulsifying capacity of the ppis was then evaluated. contrary to the surface and interfacial activity, both the ppi dispersions (nc and centrifuged) allowed to obtain fine and stable oil-in-water emulsions and, thus, in figure 2, the particle size distributions of the systems produced with the nc (e-nc, figure 2a) and centrifuged ppis (e-c, figure 2b) are shown. reported in preliminary investigations (d’alessio et al., 2022), this commercial protein isolate showed a very low solubility in water (ca. 25%), thus making it difficult to obtain a stable signal in the detection of the surface and interfacial tension values due to precipitation phenomena. on the contrary, in the aqueous dispersions obtained from the ppi after centrifugation, as expected, a concentration-dependent behavior was observed for both the parameters, with a reduction of the surface attractive forces with the increase of protein content. at the lowest concentration tested, the st had a value very close to that of water, and, by increasing the protein isolate concentration, a progressive decrease of the surface tension due to the adsorption of the pea proteins at the air/water interface was observed. moreover, for protein concentrations higher than 0.5% (w/v), a plateau value was observed as a consequence of the surface saturation by the progressive adsorption of pea proteins. non-centrifuged 12 10 8 6 4 2 0 1.0% 1.5% 2.0% 2.5% 3.0% 3.5% 4.0% 0.01 0.1 v o lu m e d e n s it y ( % ) 1 10 �m 100 1000 centrifuged 12 10 8 6 4 2 0 1.0% 1.5% 2.0% 2.5% 3.0% 3.5% 4.0% 0.01 0.1 v o lu m e d e n s it y ( % ) 1 10 �m 100 1000 figure 2. distribution of emulsions formulated at different concentrations (range between 1.0 and 4.0% w/w) of non centrifuged and centrifuged pea protein solutions. (a) (b) 75 70 65 60 55 50 45 40 0 0.001 0.005 0.01 0.05 0.1 0.5 [isolate %] s t ( m n /m ) 1 figure 1. surface (a) and interfacial tension (b) of centrifuged pea protein isolate dispersion as a function of protein concentration. (a) (b) 30 25 20 15 10 5 0 0 0.001 0.005 0.01 0.05 0.1 0.5 [isolate %] it ( m n /m ) 1 italian journal of food science, 2022; 34 (4) 29 pea protein isolates emulsifying properties the occurrence of flocculation phenomena was also evaluated and the flocculation index (%) was calculated from the droplets’ size value of emulsions just after preparation, obtained using different dispersing media. at the lowest isolate concentration (1.0 and 1.5% w/w), e-c showed limited flocculation with values of 10.54 ± 2.27% and 10.63 ± 2.73%, respectively, while very low indices (∼3.5 ± 0.5%) were found when higher isolate amounts were used, as a consequence of protein concentration increase, and in agreement with the literature (peng et al., 2016). the e-nc emulsions did not show flocculation phenomena, regardless of the concentrations of pea proteins isolate used, with values lower than 1.0%. the physical stability of the emulsions was investigated by evaluating the droplet size after 7-days of storage, under different storage temperatures, and results are reported in figure 4 where data are compared with those of the the e-nc systems were characterized by monomodal droplet distributions, with a maximum peak moving toward higher diameters (10 µm) when the lowest concentration was used (1.0% w/w) and with a progressive shift toward smaller particle diameters, corresponding to more finely dispersed emulsions, as the concentration of protein increased. on the contrary, at the lowest protein concentrations (1.0 and 1.5% w/w), the emulsified systems stabilized with centrifuged pea proteins (e-c) showed polydisperse population (figure 2b), becoming monomodal only with the increase in protein concentration, with a concentration-dependent behavior. the different behavior of the two types of emulsion systems may be due to the effect of the insoluble fraction present in the nc protein suspension, which could therefore play a stabilizing role at the interface and improve its emulsifying capacity also at the lowest concentrations tested. particulate plant materials made of slightly hydrophobic particles were indeed proven to exert emulsifying properties (schröder et al., 2021). based on the technical data sheet provided by the manufacturer, in addition to proteins, the commercial protein isolate also contains fibers and starch (1.4 g/100 g of product and 0.7 g/100 g of product, respectively). it can be supposed that starch particles could either be localized at the interface, behaving like colloidal surfactants as in pickering emulsions (sun et al., 2022), or could interact with pea proteins, by improving their surface properties and helping in the oil droplets’ formation and stabilization. on the other hand, the size of oil droplets (reported as d[4;3]) of both e-nc and e-c systems prepared at the lowest isolate amount (1.0% w/w) showed no significant differences (p > 0.05). the increase in protein concentration (>1.5%) led to small yet significant differences in the d[4;3] value (p < 0.05), confirming the contribution of the insoluble fraction in the stabilization of the emulsified system (figure 3). non-centrifuged [isolate %] 1.0% 1.5% 2.0% 2.5% 3.0% 3.5% 4.0% t0 4°c 10°c 22°c d [4 ,3 ] ( � m ) 12 10 8 6 4 2 0 centrifuged [isolate %] 1.0% 1.5% 2.0% 2.5% 3.0% 3.5% 4.0% t0 4°c 10°c 22°c d [4 ,3 ] ( � m ) 12 10 8 6 4 2 0 figure 4. stability of emulsions formulated with non-centrifuged (a) and centrifuged (b) pea protein isolate solutions, after 7 days of storage at different temperatures (4°–10°–22°c). (a) (b) figure 3. droplet sizes (d [4,3] ) of emulsions prepared with non-centrifuged and centrifuged pea protein solution. centrifuged [isolate %] 1.0% 1.5% b b b b b b a a a a a a a a 2.0% 2.5% 3.0% 3.5% 4.0% d [4 ,3 ] ( � m ) 3.0 2.5 2.0 1.5 1.0 0.5 0.0 non-centrifuged 30 italian journal of food science, 2022; 34 (4) d’alessio g et al. indicates that more proteins could adsorb at the oil– water interface per unit of interfacial surface (peng et al., 2016; shao and tang, 2014). besides the interface composition, the different physical stabilities of the e-nc and e-c systems upon storage may be related to other physical properties of the systems and in particular to the viscosity. flux curves of both emulsified systems prepared with the same protein concentration (2.0% w/w) were carried out and data are reported in figure 5. indeed, shear stress data were higher for the systems stabilized by the nc suspensions that could exert a positive effect on emulsions’ stability, thanks to the overall reduced mobility of the systems. moreover, it was interesting to observe that the presence of the insoluble fraction determined a shift in the flow behavior from newtonian (e-c) to shear-thickening (e-nc). corresponding samples just after preparation. the droplet size of e-nc (figure 4a) remained quite constant over time; only when samples were stored at 22°c, an increase in the d[4;3] at the lowest protein content was observed, while for the other tested temperatures and concentrations, particle size remained almost unchanged during storage. on the contrary, a clear increase in droplet size was seen in e-c (figure 4b), especially at the lowest protein concentrations (<2.0% w/w), while it was progressively reduced at increasing protein concentrations. considering the droplet size of the systems just after preparation, these results may be related to the action of the insoluble fraction at the droplet interface, which probably reduced particle–particle interactions and therefore destabilization phenomena of the system during the time. indeed, as reported in the literature, particle-stabilized emulsions are more stable over time and against droplet coalescence (skelhon et al., 2012; sun et al., 2022). to deepen this aspect, the protein adsorption (γ) at the oil droplet interface was evaluated and the results are reported in table 1. as hypothesized, the adsorption behavior is extremely different between the two series of emulsion systems: the e-nc showed very low values of protein adsorption suggesting that the oil–water interface is stabilized also by other components (starch) and that proteins contributed only in part, with a trend not dependent on the isolate concentration. this behavior is also supported by the results obtained for the adsorbed proteins (ap%) at the interface; indeed, e-nc shows very different values at the different protein concentrations analyzed and without a concentration-dependent trend. conversely, in e-c emulsions, the interfacial protein concentration (γ) increased with the increase in the concentration of the protein isolate used, even though for concentrations equal or higher than 2.0% similar values were found, as if an adsorption plateau had been reached at the interface. the progressive increase of this parameter parallel to the increase of the protein concentration table 1. interfacial protein concentration and percentage of adsorbed proteins of the two different emulsions formulated with noncentrifuged and centrifuged pea protein solutions at different concentrations. [pea protein isolate] % γ (mg/m2) ap % non-centrifuged centrifuged non-centrifuged centrifuged 1.0% (w/w) 0.01 ± 0.03 aa 0.70 ± 0.03b a 0.39 ± 2.49 aa 90.85 ± 1.11 b a 1.5% (w/w) 0.97 ± 0.05 ab 0.81 ± 0.04 aa 54.57 ± 3.52 ab 82.50 ± 1.57 bbc 2.0% (w/w) 0.49 ± 0.35 aab 2.50 ± 0.37 bb 20.30 ± 13.84 ac 91.29 ± 1.65 bac 2.5% (w/w) 0.58 ± 0.33 aab 3.82 ± 0.38 bbc 19.55 ± 10.37 acd 91.40 ± 0.30 bc 3.0% (w/w) 1.10 ± 0.21 abc 3.46 ± 0.56 bcd 40.06 ± 4.02 abc 88.28 ± 1.71 bac 3.5% (w/w) 0.11 ± 0.04 aab 4.34 ± 0.71bcd 3.78 ± 1.48 aa 89.60 ± 2.42 bac 4.0% (w/w) 0.25 ± 0.07 aab 3.25 ± 0.16 bbd 7.17 ± 1.94 aa 89.14 ± 0.74 bac means with different lowercase letters in the same row are significantly different (p < 0.05) for the two parameters considered. different capital letters in the same columns indicate significant differences (p < 0.05) among different isolate concentrations. figure 5. flow curves of e-nc and e-c emulsions prepared with non-centrifuged and centrifuged pea protein isolate (2% w/w). centrifuged shear rate (s–1) s h e a r s tr e s s ( p a ) 0 50 100 150 200 250 2.0 1.5 1.0 0.5 0.0 non-centrifuged italian journal of food science, 2022; 34 (4) 31 pea protein isolates emulsifying properties references amine, c., dreher, j., helgason, t. and tadros, t., 2014. investigation of emulsifying properties and emulsion stability of plant and milk proteins using interfacial tension and interfacial elasticity. food hydrocolloids 39: 180–186. https://doi. org/10.1016/j.foodhyd.2014.01.001 barać, m.b., pešić, m.b., stanojević, s.p., kostić, a.z. and čabrilo, s.b., 2015. techno-functional properties of pea (pisum sativum) protein isolates – a review. acta periodica technologica 46: 1–18. https://doi.org/10.2298/apt1546001b boye, j.i., aksay, s., roufik, s., ribéreau, s., mondor, m., farnworth, e. and rajamohamed, s.h., 2010. comparison of the functional properties of pea, chickpea and lentil protein concentrates processed using ultrafiltration and isoelectric precipitation techniques. food research international 43(2): 537–546. https://doi.org/10.1016/j.foodres.2009.07.021 burger, t.g. and zhang, y., 2019. recent progress in the utilization of pea protein as an emulsifier for food applications. trends in food science and technology 86: 25–33. https://doi. org/10.1016/j.tifs.2019.02.007 d’alessio, g., flamminii, f., faieta, m., pittia, p. and carla daniela,  d.m., 2022. proteine di pisello: tecnologie di produzione simili, ma funzionalità tecnologiche differenti. industrie alimentari 61(635): 7–24. . de angelis, d., kaleda, a., pasqualone, a., vaikma, h., tamm, m., tammik, m.l., squeo, g. and summo, c., 2020. physicochemical and sensorial evaluation of meat analogues produced from dry-fractionated pea and oat proteins. foods 9(12): 1754. https://doi.org/10.3390/foods9121754 karaca, a.c., low, n. and nickerson, m., 2011. emulsifying properties of chickpea, faba bean, lentil and pea proteins produced by isoelectric precipitation and salt extraction. food research international 44(9): 2742–2750. https://doi.org/10.1016/j.foodres.2011.06.012 kimura, a., takako, f., meili, z., shiori, m., maruyama, n. and utsumi, s., 2008. comparison of physicochemical properties of 7s and 11s globulins from pea, fava bean, cowpea, and french bean with those of soybean-french bean 7s globulin exhibits microstructure of the oil-in-water emulsions microimages of the two series of oil-in-water emulsions were acquired to evaluate any eventual differences in the colloidal system microstructure due to the effect of the insoluble fraction present in the protein isolate. at a microscopic level, the e-nc (figure 6a) shows a homogeneous distribution with smaller and more spherical oil droplets than e-c (figure 6b). this result is consistent with the d[4;3] values and the droplet size distribution (see figures 2 and 3). furthermore, in e-nc, a more compact network compared to e-c is highlighted, justifying the higher viscosity determined in the corresponding emulsions. a similar behavior was observed in other studies reported in the literature, in which the use of insoluble fractions of various nature as emulsion stabilizers led to the formation of a thick and solid layer surrounding the oil droplets and to the creation of a network between them, allowing the stabilization of the system, also through the increase in viscosity and the decrease in droplet sizes (pirozzi et al., 2021; ren et al., 2019). conclusions in this study, the technological properties of a commercial ppi were evaluated in model oil-in-water emulsions with a special focus on the role of the insoluble residues on systems properties’ and stabilization. the insoluble fraction improved the emulsifying properties of pea proteins and enhanced the physical stability of the emulsions, by contributing to the stabilization of the oil-water interface and by increasing the viscosity of the emulsions. with regard to the first aspect, further investigations are needed to better understand the adsorption behavior of the insoluble fractions on the oil-water interface and the eventual role of high-pressure homogenization on their emulsifying properties. figure 6. optical microscope images (100× magnification) of emulsions prepared with 2.0% (w/w) of non-centrifuged (a) and centrifuged (b) pea protein dispersion. 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the abts and the dpph assays. phytochemical composition of the 27 fig ecotypes was found to be very diverse, as the total polyphenols varied from 51.50 (‘bouholi’) to 100.23 (‘nasri’) mg gallic acid equivalent/100 g fresh weight. total flavonoids also varied from 0.33 (‘bayoudhi1’) to 17.59 (‘soltaniahmar’) mg quercetin equivalent/100 g fresh weight, and total anthocyanins extended from 1.61 (‘besbessi’) to 11.67 (‘zidi2’) mg/100 g fresh weight. additionally, dpph % inhibition ranged from 11.37 (‘besbessi’) to 64.73 % (‘bouharrag’) and abts from 38.50 (‘sawoudi5’) to 676.13 (‘nemri’). the ecotypes ‘zergui’ and ‘nasri’ had the highest contents of glucose (5.68 and 4.83 g/ 100 g fw, respectively) and fructose (5.43 and 4.69 g/ 100 g fw, respectively). the results also showed that fig fruits are a good and valuable source of natural antioxidants that can be used in food and medical sectors. keywords: anthocyanins, antioxidant activity, ecotypes, ficus carica, flavonoids, fruits, polyphenols ital. j. food sci., vol. 32, 2020 756 1. introduction fig (ficus carica l.), which belongs to the moraceae family, is considered to be one of the oldest cultivated fruit species and an important crop worldwide for both fresh and dry consumption (duenas et al., 2008; bachir bey and louaileche, 2015). the world production of figs is about one million tons, and it is mostly concentrated in the mediterranean area (veberic et al., 2008). tunisia produces about 29 000 tons, which represents 3 % of total world production (faostat, 2015). in tunisia, figs have been grown traditionally for several centuries (aljane et al., 2018). local fig ecotypes are numerous and well adapted to the local agro-ecological conditions (aljane and ferchichi, 2010). their denominations relate to the fruit color, the period of fruit maturation or to their geographic origin (aljane, 2016). exchange of plant material was frequent between regions of which synonymy and homonymy may be encountered (chatti et al., 2004; mars, 2003; aljane and ferchichi, 2010). since several decades, the cultivated areas decreased due to the extinction of many ecotypes, the intensive urbanization as well as the biotic and abiotic stresses (mars et al., 1998; mars, 2003) despite the installation of many new plantations (mars et al., 2008). whether fresh or dried, figs constitute an important part of the human diet; they are especially rich in fiber, minerals, proteins, sugars, organic acids and antioxidant compounds (ercisli et al., 2012). fig fruit is an important source of minerals, vitamins and polyphenols (duenas et al., 2008; aljane and ferchichi, 2009; adiletta et al., 2019). in addition, solomon et al. (2006) recorded high polyphenols contents, especially flavonoids and anthocyanins, the highest being their antioxidant activity. the contents of total polyphenols, anthocyanins as well as total antioxidant activity and other properties such as skin color are strongly influenced by the ecotype (solomon et al., 2006; veberic et al., 2008; caliskan and polat, 2011; ercisli et al., 2012). similarly, several reports have highlighted the influence of fruit variety, harvest season and growing technology in the fields of phenolic contents (treutter, 2010; vallejo et al., 2012). moreover, antioxidant activity and phenolic compounds varied considerably depending on the part of the fruit. indeed, several authors have reported the great contribution of fruit skin (compared to pulp) to these compounds especially in darker varieties (veberic et al., 2008; duenas et al., 2008). the aim of the present work was to study the phytochemical characteristics and sugar composition of 27 fig ecotypes grown in tunisia. 2. materials and methods 2.1. fruit fig material ripe fig fruits from 27 tunisian fig ecotypes (different fig-growing traditional geographic regions) were harvested in 2015 from the experimental field for germplasm collection of the institute of arid regions (ira) of medenine, tunisia (table 1). the experimental orchard of 10 years old, included 3 replicates of 5 x 5 m cultivated understandard cultural practices. within 2 h after harvest, whole fruits were stored at 20°c for further analysis. triplicate of 10 frozen fruits samples from each ecotype were homogenized in a blender and used for phytochemical and nutritional analysis. ital. j. food sci., vol. 32, 2020 757 table 1. ecotype’s name, types, localities of origin of the studied 27 tunisian fig fruits. ecotype’s name types localities of origin (governorate) bither1 san pedro ghadhabna (mahdia) jebali1 smyrna islands of kerkenah (sfax) mahdoui smyrna islands of kerkenah (sfax) bayoudhi1 common beni kheddache (médenine) bayoudhi2 common toujen (gabès) besbessi san pedro masjedaissa (sousse) bither2 san pedro islands of kerkenah (sfax) jemâaoui smyrna beni kheddache (médenine) rogabi smyrna beni kheddache (médenine) gaa zir smyrna gafsa (gafsa) temri smyrna islands of kerkenah (sfax) zergui smyrna djébba (béja) baghali2 smyrna ghadhabna (mahdia) baghali3 smyrna islands of kerkenah (sfax) chetoui akhal common ghadhabna (mahdia) croussi smyrna beni kheddache (médenine) kahli2 smyrna islands of kerkenah (sfax) nemri smyrna djébba (béja) soltani ahmer smyrna djébba (béja) wedlani smyrna beni kheddache (médenine) bouharrag smyrna djébba (béja) bouholi san pedro djébba (béja) kahli1 smyrna ghadhabna (mahdia) nasri smyrna toujen (gabès) sawoudi3 smyrna bir amir (tataouine) sawoudi5 smyrna gafsa (gafsa) zidi2 smyrna djébba (béja) 2.2. determination of phenolics composition of fig fruits 2.2.1 methanolic extraction a total of 1 g of fruit samples was homogenized in 25 ml of extraction solution and 80% methanol. it was stirred for 2 h in the dark at room temperature. the obtained mixture was centrifuged two sequential times for 15 min at 3500 rpm, and supernatant was filtered and taken for further analysis. 2.2.2 total polyphenols (tp) total polyphenols (tp) contents of fig fruits were determined spectrophotometrically using the folin-ciocalteu method as previously described by slingard and singleton (1977) with some modifications. the absorbance of each sample was measured at 760 nm using a spectrophotometer (shimadzu 1600-uv, japan). ital. j. food sci., vol. 32, 2020 758 quantifications were calculated using a calibration curve daily prepared with known concentrations of gallic acid standards, and results are expressed as mg gallic acid equivalents (gae) on fresh weight (fw) basis (mg gae/100 g fw). 2.2.3 total anthocyanins (ta) total anthocyanins (ta) contents were quantified in accordance with the ph differential method using two buffer systems as previously described by cheng and breen (1991). in brief, methanolic extract were diluted with two buffer solutions of ph 1 and 4.5. anthocyanins were estimated using absorbance measurement at 530 and 657 nm in buffers at ph 1.0 and 4.5, respectively; where absorbance (a) was measured using this formula: a = [(a530 – a657) ph 1.0 (a530 – a657) ph 4.5] with a molar extinction coefficient of cyanidin-3-glucosid of 29.600. total anthocyanin quantities were expressed as mg of cyanidin-3-glucoside equivalents (cge) per g fresh weight of fig fruit (mg cge/100 g fw). 2.2.4 total flavonoïds (tf) total flavonoïds were determined using a colorimetric method previously described by karadeniz et al. (2005). methanolic extract (1 ml) was added to 5 ml of distilled water and mixed. then, 5% sodium nitrite solution (0.3 ml) was added, followed by 10% aluminium chloride solution (0.3 ml), mixed and incubated at room temperature for 5 min. after incubation, 2 ml of 1m sodium hydroxide were added to the mixture and thenthe volume of reaction mixture was made up to 10 ml with distilled water. the mixture was thoroughly vortexed and the absorbance was determined at 510 nm. flavonoid contents were calculated using a standard calibration curve, prepared from quercetinand expressed as quercetin equivalent in mg per g fresh weight of fruit (mg quercetin/100 g fw). 2.3. determination of antioxidant properties of fig fruits the dpph (1,1 diphenyl 2 pycrilhydrazil (dpph) radical-scavenging activity of the extract was measured as described by rebai et al. (2012) and bachir bey et al. (2013). an aliquot (200 µl) of the extract was added to 1 ml of a methanolic dpph solution (500 µm). the decolorizing process was measured at 517 nm after 30 min of reaction. the scavenging activity percentage of dpph (%) of the fig extract was calculated using this formula: a = (a blank – a sample)/ (a blank) * 100. for the standard teac (trolox equivalent antioxidant capacity) assay, abts (2, 2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) was dissolved in methanolic solution (14 mm) and prepared with 10 ml ammonium persulfate (nh2 2s2o8) (4.9 mm) as described by ozgen et al. (2009). the mixture was diluted in methanol to an absorbance of 1.00±0.01 at 734 nm for long stability (ozgen et al., 2009). for the spectrophotometric assay, 30 µl of fig fruit extract and 2.97 ml of abts+ solution were mixed and incubated for 1 h in darkness. the absorbance was determined at 734 nm using a spectrophotometer (specord 210 plus-analytik jena, japan). the teac was expressed as mg equivalent vitamin c (acid ascorbic) per 100 g fresh weight of fig fruit (mg evc/100 g fw). ital. j. food sci., vol. 32, 2020 759 2.4. determination of reducing sugars of fig fruits reducing sugars (glucose and fructose) were determined according to the method described by melgarejo et al. (2003) and gundogdu et al. (2011). briefly, 10 g fruit was centrifuged at 12000 rpm for 2 min at 4°c, thereafter, the supernatant was filtered and transferred into a vial and used for analysis. analysis of glucose and fructose was performed by hplc (knauer type) with eurospher 100 nh2 column and refractive index detector (ri detectors k-2301) using 80% acetonitrile as a mobile phase. the calculation of concentrations was based on standards solutions of glucose (2%) and fructose (2%). the results were expressed in g/100 g fw and all the samples were analysed in triplicate. 2.5. statistical analysis all analyses were performed with r software (r core team, 2019). dpph inhibition %data were arcsine transformed to meet assumptions of analysis of variance (anova) for homogeneity of variance and normality and are reported in tables as untransformed values. data were analyzed using one-way analysis of variance (anova) considering them as factor ecotypes or ecotype groups, followed by post-hoc tukey multiple comparison to determine if differences (p < 0.05) between fig ecotypes were significant. additionally, pearson’s correlation coefficients were also performed based on phytochemical compositions and antioxidant activity of the 27 fig ecotypes. 3. results and discussion 3.1. fruit skin color the 27 tunisian local fig ecotypes revealed great morphological variability in their external fruit color (fig. 1) and consequently were classified into 6 groups which are: (green yellowish, green, red greenish, brown purplish, purple greenish and purple blackish). among the studied fruit fig ecotypes, fifteen had variably intense purple skin (eight had purple-greenish and seven purple blackish). additionally, seven ecotypes showed skin color ranging from green to yellow. the remaining ecotypes: ‘jemâaoui’ and ‘rogabi’ presented red greenish and ‘gaa zir’, ‘temri’ and ‘zergui’ were brown purplish (table 2). color is one of the most important indicators of maturity and quality of fruits, which is influenced by the concentration and distribution of various anthocyanins (gao and mazza, 1995). 3.2. fruit phenolic compound contents the level of phenolic compounds of the 27 tunisian fig ecotypes are given in table 2, while the mean values obtained for each skin color group are shown in fig. 2. the oneway anova analysis followed by post-hoc tukey multiple comparison test of total polyphenols, total anthocyanins and total flavonoids showed highly significant differences (p < 0.001) among the 27 fig ecotypes. when we applied anova analysis to the six skin color groups, the total anthocyanins showed highly significant differences (p<0.001), ital. j. food sci., vol. 32, 2020 760 whereas the total flavonoids were only significant (p<0.05). unlike these compounds, the total polyphenols revealed no significant differences among the six groups. figure 1. morphological variability in external fruit color of the 27 studied fig ecotypes (a: green yellowish, b: green, c: red greenish, d: brown purplish, e: purple greenish and f: purple blackish). ital. j. food sci., vol. 32, 2020 761 3.2.1 total polyphenols the total polyphenols (tp) have been reported to be the main phytochemical responsible for the antioxidant activity of figs. the tp contents of fig ecotypes varied from 51.50 (‘bouholi’) to 100.22 (‘nasri’) mg gae/ 100 g fw. the highest tp levels were observed, in descending order, in the following ecotypes (‘nasri’, ‘bayoudhi2’, ‘zidi2’, baghali3’, ‘rogabi’, sawoudi5’) (table 2). the results of the total polyphenols contents are higher than those obtained in previous studies conducted by aljane and sdiri (2014). nevertheless, thesecontents are inferior to those found by vallejo et al. (2012) and capanoglu (2014), who reported concentrations of 331.93 and 169.4 mg gae/ 100 g fw in indian and turkish figs, respectively, but are comparable to the results of piga et al. (2008). on the contrary solomon et al. (2006), caliskan and polat (2011) and debib et al. (2014) showed that the dark fig fruits contain higher total polyphenols than the light ones. we did not obtain significant differences in total polyphenols based on fruit skin color groups (fig. 2). this discrepancy might be explained by the fact that total polyphenols contents are greatly influenced by various parameters such as weather conditions, ripening stage, degree of fruit maturation, and postharvest storage conditions (vallejo et al., 2012; bachir bey and louaileche, 2015). 3.2.2. total anthocyanins the total anthocyanins (ta) are natural pigments belonging to the flavonoid family and are responsible for the red, blue and purple color of many fruits. the total anthocyanins amounts of the studied fig ecotypes varied from 2.57 (‘baghali3’) to 11.67 (‘zidi2’) mg cge/100 g fw (table 2). ‘zidi2’ ecotypes had the highest contents (11.67) followed by ‘sawoudi3’ (9.7) and then ‘bouholi’ (8.17). it is apparent that purple blackish ecotypes contain more anthocyanins, with average value of 7.11 mg cge/ 100 g fw. the other fruit ecotypes varied within 3.17 in green-yellowish fruit skin color group to 5.08 mg cge/100 g fw in red greenish (fig. 2). these levels are similar to those obtained in our previous study on tunisian fig varieties, where we found ta to be between 0.55 and 9.16 mg cge/100 g fw (aljane and sdiri, 2014). solomon et al. (2006) reported that the dark fig ‘mission’ variety has eight times higher total anthocyanins (10.9 mg cge/100 g fw) than the red-brown turkey one (1.3 mg cge/ 100 g fw), while these compounds were not detected in ‘brunswick’ and ‘kadota’ ecotypes, which have light fruit skin color. the ta content of the majority purple-blackish ecotypes is higher than that found by ouchemoukh et al. (2012) in black figs (5.9 mg cge/ 100 g fw). in addition, the total anthocyanins content of our samples was lower than that of other studies on commercial fig ecotypes (del caro and piga, 2007; piga et al., 2008; duenas et al., 2008; ercisli et al., 2012). the results showed that total anthocyanins (ta) contents were strongly influenced by fruit skin color. indeed, the purple blackish fig ecotypes (‘zidi2’, ‘sawoudi3’ and ‘bouholi’) had the highest contents and might be used as good sources of anthocyanins. such result is in good agreement with those advanced by solomon et al. (2006), who reported a large contribution of fig fruit skin to the total anthocyanins accumulation. ital. j. food sci., vol. 32, 2020 762 table 2. total polyphenols, total anthocyanins and total flavonoids of 27 tunisian fig ecotypes. ecotype’s name total polyphenols mg gae/ 100 g fw total anthocyanins mg cge/ 100 g fw total flavonoids mg qe/ 100 g fw fruit skin color group bither1 60.50±0.74 ef 3.75±0.19 ade 5.68±0.30 ef green yellowish jebali1 76.47±0.10 lm 3.43±0.40 ad 11.50±0.90 i mahdoui 63.21±0.31 g 3.73±0.12 ade 15.26±0.9 j bayoudhi1 76.62±0.05 lm 3.00±0.1 ab 0.33±0.11a green bayoudhi2 88.45±0.47 p 5.61±0.10 ghi 5.68±0.30 ef besbessi 56.29±0.76 bc 3.33±0.27 ac 12.16±0.30 i bither2 65.53±1.45 h 6.80±0.82 ij 8.59±0.3 h jemâaoui 76.15±0.24 lm 6.20±0.1 hj 2.77±0.68 bc red greenish rogabi 79.03±0.15 no 3.96±0.24 bcdf 3.76±0.19 cd gaa zir 71.75±0.07 ij 4.54±0.38 cdfg 5.42±0.11 def brown purplish temri 60.61±0.03 ef 3.67±0.47 ade 5.68±0.3 ef zergui 54.60±1.36 b 5.57±0.08 ghi 16.57±0.14 jk baghali2 69.93±0.10 i 3.75±0.08 ade 6.14±0.9 fg purple greenish baghali3 79.42±0.61 no 2.57±0.04 a 4.36±0.36 ce chetoui akhal 73.35±0.59 jk 7.03±0.72 jk 12.29±0.19 i croussi 74.57±0.52 kl 4.28±0.24 cdf 5.68±0.41 ef kahli2 62.33±0.09 fg 4.21±0.08 bcdf de 12.75±0.30 i nemri 59.34±0.56 de 3.78±0.34 ade 5.76±0.82 eg soltani ahmer 61.59±0.66 eg 4.67±0.12 dfg 17.59±0.14 k wedlani 63.56±0.3 gh 3.55±0.10 c 1.78±0.19 ab bouharrag 58.20±1.58 cd 5.12±0.24 fh 7.47±0.24 gh purple blackish bouholi 51.50±1.49 a 8.17±0.90 i 1.78±0.90 ab kahli1 61.29±0.62 eg 6.21±0.06 hj 11.70±0.07i nasri 100.22±0.38 q 3.95±0.94 bcdf 1.85±0.94 ab sawoudi3 75.44±0.41 km 9.70±0.47 l 8.99±0.48 h sawoudi5 77.37±0.44 mn 4.90±0.25 efg 11.70±0.26 i zidi2 81.25±0.99 o 11.67±0.15 m 5.62±0.16 ef total mean 69.58±11.14 4.08±2.11 7.73±4.72 f value 731.6 85.1 222.1 p value *** *** *** '***' 0.001 '.values in the same column with different lowercase letters are significantly different at p<0.05 according to post-hoc tukey multiple comparison, gae: gallic acid equivalent, cge: cyanidin-3-glucoside equivalent, qe: quercetin equivalent, fw: fresh weight. 3.2.3 total flavonoids the purple-greenish ecotype ‘soltani ahmar’ had the highest contents (17.59 mg qe/100 g fw) followed by ‘zergui’ from the brown purplish group (16.57 mg qe/100 g fw) and ‘mahdoui’ from the green-yellowish with an amount of 15.26 mg qe/100 g fw. whereas, the lowest contents were observed in the following ecotypes (‘bayoudhi1’, ‘bouholi’, ‘wedlani’, ‘nasri’, ‘jemâaoui’ and ‘rogabi’) (table 2). ital. j. food sci., vol. 32, 2020 763 gy: green yellowish, g: green, rg: red greenish, bp: brown purplish, pg: purple greenish, pb: purple blackish figure 2. total phenolic content (a): total polyphenols, total anthocyanins, total flavonoids, antioxidant capacity (b): dpph: 1.1 diphenyl 2 pycril hydrazil, abts: acid 2.2-azino-bis-3 ethylbenzothiazoline-6sulfonique and sugar compositions (c): glucose and fructose of 6 fig fruit skin color groups. different letters indicate significant differences by post-hoc tukey multiple comparison at p< 0.05. ital. j. food sci., vol. 32, 2020 764 the obtained values of total flavonoids are lower than those found by bachir bey and louaileche (2015) who have advanced contents of 87.24 and 126.55 mg/100 g fw for algerian light and dark varieties, respectively. the green yellowish group, which is light figs, has the highest total flavonoid contents, followed by green, brown purplish, purple greenish and purple blackish groups (fig. 2). such result is quite different from those reported by solomon et al. (2006) and vallejo et al. (2012) who found that the total flavonoids contents of dark-purple fig varieties were greater than those of light ones. 3.3. antioxidant activities the antioxidant activities of the 27 tunisian fig ecotypes are summarized in table 3. the one -way anova analysis of abts and ddph followed by post-hoc tukey multiple comparison test indicated highly significant differences among the 27 fig ecotypes and also between the six groups. 3.3.1 dpph radical-scavenging activity data of the scavenging activity against ddph indicated that the best antiradical effect was achieved by the ‘bouharrag’ ecotypes (64.73%), whereas, ‘besbessi’ had the least activity (14.59%) (table 3). the results clearly revealed a stronger dpph scavenging activity in purple blackish ecotypes compared to green ones, with average values of 50.25% and 26.95%, respectively (fig. 2). these results are in accordance with those obtained by bachir bey and louaileche (2015), who reported a ddph radical scavenging activity varying from 28.33% to 45.25% in ‘taghanimt’ and ‘bouankik’ varieties, respectively. the study of ddph scavenging activity of algerian fig varieties clearly showed that dark varieties have stronger ddph scavenging activities than the light one, with mean values of 41.63 and 31.3%, respectively (bachir bey and louaileche, 2015). 3.3.2 abts radical cation scavenging activity the results of the scavenging activity of abts radical ranged from ‘mahdoui’ (263.7 evc mg/100 g fw) to ‘nemri’ (676.13 evc mg/100 g fw). it is apparent that antioxidant activity (abts) was lower in green yellowish and purple-blackish groups, whereas, the purple greenish showed the highest value (fig. 2). the current results are comparable to the data obtained by solomon et al. (2006), who indicated that dark fig varieties had high abts antioxidant capacities. 3.4. reducing sugars compositions the analyses of variance for glucose (gluc) and fructose (fruc) revealed significant differences among the 27 studied ecotypes and within the fruit skin color groups. the ecotypes ‘zergui’ and ‘nasri’ had the highest contents of glucose (5.68 and 4.83 g/100 g fw, respectively) and fructose (5.43 and 4.69 g/100 g fw) values. nevertheless, gluc and fruc were very low for the ‘mahdoui’ ecotype (1.12 and 0.86 g/100 g fw, respectively) (table 3). these results were lower than those obtained by melgarejo et al. (2003), as the glucose contents of ‘tio antonio’ and ‘calar’ variety were 15.89 and 13.41 g/100 g fw, respectively. similarly, caliskan and polat (2012) reported that gluc and fruc contents obtained in ‘sarilop’ variety were 10.7 and 7.8 mg 100/ g fw, ital. j. food sci., vol. 32, 2020 765 respectively. the sugar composition of figs, especially fructose, can influence perceived fruit sweetness (setser, 1993). table 3. effects of genotype on antioxidant activity (dpph and abts) and sugar compositions for 27 tunisian fig ecotypes. ecotype name dpph inhibition % abts mg evc/ 100 g fw gluc g/ 100 gfw fruc g/ 100 gfw fruit skin color group bither1 40.74±0.65 l 412.96±6.60 def 1.79±0.29 ab 1.96±0.21 acd green yellowish jebali1 28.99±0.99 f 480.26±26.04 hi 3.30±0.90 bde 3.16±0.42 cef mahdoui 28.63±0.54 f 263.70±9.31 a 1.12±0.88 a 0.86±0.12 a bayoudhi1 30.38±0.53 g 496.76±5.68 ij 4.66±0.10 ef 3.52±0.09 eg green bayoudhi2 26.55±0.50 e 376.40±5.55 cd 2.52±0.30 ad 2.47±0.10 bce besbessi 14.59±0.52 b 407.96±3.61 de 3.20±0.31 bde 2.89±0.28 bcef bither2 30.37±0.54 g 601.13±1.02 k 2.30±0.29 ad 2.18±0.81 ae jemâaoui 38.49±0.50 j 448.60±10.28 fgh 3.37±0.67bde 2.45±0.11bce red greenish rogabi 38.49±0.50 j 493.76±6.26 ij 3.21±0.23 bde 2.41±0.26 bce gaa zir 45.49±0.50 m 384.06±5.47 cd 3.32±0.11 bde 3.34±0.31 cef brown purplish temri 27.42±0.51 e 441.56±7.76 eg 3.32±0.30 bef 2.32±0.45 bce zergui 46.61±0.53 n 409.36±10.96 de 5.68±0.16 f 5.43±0.10 h baghali2 29.47±0.50 fg 378.73±1.55 cd 2.00±0.90 abc 1.96±0.10 acd purple greenish baghali 3 26.48±0.50 e 658.96±10.15 l 2.57±0.90 ad 2.39±0.08 bce chetoui akhal 15.46±0.50 b 465.16±4.19 gi 2.56±0.30 ad 1.98±0.80 ade croussi 35.54±0.50 i 575.86±3.58 k 2.52±0.40 ad 2.32±0.30 acd kahli2 45.30±0.60 m 275.60±39.57 a 2.26±0.30 ad 2.15±0.10 ae nemri 41.05±1.07 l 676.13±13.85 l 4.33±0.12 ef 3.96±0.35 fg soltani ahmer 48.07±1.00 o 490.43±10.50 ij 1.73±0.18 ab 1.56±0.11 ab wedlani 31.52±0.50 h 519.70±1.47 j 3.63±0.20 cde 2.90±0.12 bcef bouharrag 39.63±0.65 k 347.16±4.07 bc 3.58±1.05 cde 3.29±0.95 deg purple blackish bouholi 64.73±0.55 s 264.70±7.59 a 3.19±0.22 bde 3.05±0.95 cef kahli1 19.62±0.54 d 407.80±5.63 de 2.25±0.90 ad 2.19±0.10 ae nasri 56.55±0.51 q 465.16±4.19 gi 4.83±0.12 ef 4.69±0.95 ghe sawoudi3 52.42±0.52 p 462.83±7.00 gi 3.12±0.30 ade 2.52±0.45 bcef sawoudi5 62.45±0.51 r 383.50±15.05 cd 2.24±0.90 ad 1.82±0.25 ac zidi2 56.37±0.54 q 322.20±1.92 b 3.84±0.30 de 3.22±0.17 cef total mean 36.62±13.42 441.13±105.74 3.00±1.15 2.63±1.04 f value 1454 763.1 10.47 13.47 p value *** *** *** *** 0 '***' 0.001. values in the column with different lower-case letters are significantly different at p< 0.05 according to post-hoc tukey multiple comparison. dpph: 1.1 diphényl 2 pycrilhydrazil. abts: acide 2.2azino-bis-3-ethylbenzothiazoline-6-sulfonique, evc: equivalent vitamin c, gluc: glucose, fruc: fructose, fw: fresh weight. ital. j. food sci., vol. 32, 2020 766 it is more likely that the gluc and fruc contents depended on fruit skin color (fig. 2). similarly, caliskan and polat (2012) observed that fig genotypes with green or brown fruit skin color had higher gluc and fruc than the genotypes with black skin fruit. abidi et al. (2011) and caliskan and polat (2011) have also mentioned that several parameters like: climate variables, cultural practices and harvest time could introduce variability among sugar compositions of fig fruits. 3.5. correlations between phytochemical and antioxidant activities parameters obtained results revealed the existence of a significant positive correlation between gluc and fruc (r = 0.889). similar results between fructose and sucrose contents in fig fruits havealso been reported by caliskan and polat (2011; 2012). in addition, we detected slightly positive correlations between gluc and dpph (r = 0.374) and between ta and ddph antioxidant activity (r = 0.292). the later correlation was not significant as reported by bachir bey and louaileche (2015) and solomon et al. (2006), who recorded a high correlation (r=0.91).it is also worthy to mention a slightly negative correlation between tp and tf, with value of r=-0.370 (table 4). table 4. pearson’s linear correlation coefficients between total polyphenols (tp), total anthocyanins (ta), total flavonoids (tf), antioxidant capacity (dpph and abts) and sugar composition (gluc and fruc) in fig fruits (n =30). tf -0.370 ta 0.053 0.037 dpph 0.144 -0.038 0.292 abts 0.187 -0.225 -0.294 -0.213 gluc 0.185 -0.171 0.012 0.374 0.104 fruc 0.082 -0.236 -0.118 0.284 0.154 0.889* parameters tp tf ta dpph abts gluc dpph: 1.1 diphényl 2 pycrilhydrazil, abts: acide 2.2-azino-bis-3-ethylbenzothiazoline-6-sulfonique; *, p<0.05. 4. conclusions since all fig trees were grown under the same environmental and edaphic conditions and subjected to uniform cultural practices (irrigation, fertilization, pruning), the observed differences in the phytochemical composition, antioxidant activity and sugar contents on fig fruits are largely dependent on the biochemical characteristic of each ecotype and to a lesser extent on the ripening stage and postharvest storage conditions. our results revealed a considerable variation in the phytochemical, antioxidant activity and sugar compositions were observed in the 27 tunisian fig ecotypes. the ecotypes with purpleblackish skin ‘bouholi’, ‘sawoudi3’ and ‘zidi2’ had the highest contents of ta. skin color had a highly significant effect on total anthocyanins and was the major tissue that contributed to anthocyanin compositions in figs fruits. among all studied ecotypes, ‘nasri’ showed the highest amount of tp. in addition, ‘bouholi’ ecotype presented the highest antioxidant activity of ddph and ‘nemri’, ‘baghali3’ and ‘bither2’ ecotypes ital. j. food sci., vol. 32, 2020 767 showed the highest abts radical scavenging activity. regarding the sugar contents, the ecotypes with higher values of gluc and fruc were ‘zergui’ and ‘nasri’, respectively. due to high contents of bioactive substances and antioxidant activities, figs (particularly dark varieties) are an interesting alternative for antioxidant additives that could be used in pharmaceutical and food industry. acknowledgements authors wish to acknowledge the efforts of their colleagues of fig germplasm collection of the arid land institute of médenine established in “el gordhab”, tataouine. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. references abidi w., jiménez s., moreno m.a. and gogorcena y. 2011. evaluation of antioxidant compounds and total sugar content in a nectarine (prunuspersica l. batsch) progeny. int. j. mol. sci. 12:6919-6935. adiletta g., zampella l., coletta c. and petriccione m. 2019. chitosancoating to preserve thequalitativetraits and improveantioxidantsystem in freshfigs (ficus carica l.). agric. 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farming and breeding—visions and constraints int. j. mol. sci. 11:807-857. vallejo f., marin j.g. and tomas-barberan f.a. 2012. phenolic compound content of fresh and dried figs (ficuscarica l.). food chem. 130:485-492. veberic r., colaric m. and stampar f. 2008. phenolic acids andflavonoids of fig fruit (ficuscarica l.) in the northern mediterranean region. food chem. 106 (1):153-157. paper received january 10, 2020 accepted may 18, 2020 p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33i2.2049 129 p u b l i c a t i o n s codon changes in physicochemical characteristics, polyphenolics, and amino acids of wax apple cider vinegar during prolonged storage somwang lekjing and karthikeyan venkatachalam* faculty of innovative agriculture and fishery establishment project, prince of songkla university (surat thani campus), makham tia, muang, surat thani 84000, thailand *corresponding author: karthikeyan venkatachalam, faculty of innovative agriculture and fishery establishment project, prince of songkla university (surat thani campus), makham tia, muang, surat thani 84000, thailand. email: karthikeyan.v@psu.ac.th; drkarthikeyan.v@outlook.com received: 8 april 2021; accepted: 29 june 2021; published: 8 july 2021 © 2021 codon publications open access paper abstract several quality attributes of wax apple cider vinegar (wacv) were determined every 30 days for six months at ambient temperature. acetic acid fermentation significantly increased the acetic acid content in wacv. the lightness and yellowness gradually decreased, whereas redness increased during storage. density, viscosity, and ph of wacv continuously retarded, and total acidity, volatile acidity, electrical conductivity, and total nitrogen content increased during storage. phenolic and flavonoid contents and antioxidant potentials of wacv were affected by storage. various amino acids and volatile compounds were observed in wacv during storage. throughout the storage period, the microbial growth in wacv was considerably low. keywords: wax apple cider vinegar, functional properties, storage, quality introduction fruit processing plays a significant role in controlling the quality loss of numerous fruits and vegetables and generates various unique products with added economic value. wax apple is a fruit susceptible to damage from handling, physiological, and physicochemical changes, which adversely influence its quality and value (techakanon and sirimuangmoon, 2020; venkatachalam et al., 2018). they are one of the unique fruits in peninsular thailand. their special characteristics include apple-like crispness, watery sweetness, low pungency, and have a bit of roselike aroma. they are available in different colors, from green to dark red. usually, a vibrant red color indicates readiness for consumption. wax apple fruit has a different names depending on the region; common names include rose apple, water apple, wax jambus, lianwu, chomphu, and jamalac. furthermore, wax apple contains lots of phytochemicals that can possess potent antioxidant activities with health benefits. they are consumed mainly as whole fruit or in fresh-cut form. to date, no products or alternatives are developed nor under development on wax apples. this study initiated developing a wax apple cider (wac). cider is a fermented alcoholic (1–12% alcohol by volume (abv)) beverage produced by a specific cider yeast. ciders are popular in western countries, and their consumption is also increasing in southeast asia (wood and anderson, 2006). wac possesses low acidity and has improved minerals, increased essential amino acids, phytochemicals, and antioxidants (techakanon and venkatachalam, 2021). typically, alcoholic beverages are highly resistant against spoilage and most pathogenic microorganisms. the cider is slightly unstable because of its rich sugar and low alcohol content, depending on the fruits and preprocessing conditions. several traditional and modern techniques have already been used in various ciders to improve their italian journal of food science, 2021; 33 (2): 129–141 mailto:karthikeyan.v@psu.ac.th mailto:drkarthikeyan.v@outlook.com 130 italian journal of food science, 2021; 33 (2) lekjing s and venkatachalam k wac into wacv and to extensively study the stability of various chemical components, including phenolics, amino acids, volatile compounds, and antioxidant properties in wacv under prolonged storage. material and methods materials and reagents fully matured wax apples (syzygium agueum alston cv. taaptimjan) were purchased from a commercial orchard in surat thani province, in southern thailand. the cider yeast strain (saccharomyces bayanus) was purchased from fermentis (france). acetobacter estunensis for vinegar production was obtained from thailand bioresources research center, thailand. folin-ciocalteu reagent, 2,4,6-tri (2-pyridyl)-s-triazine (tptz), 2,2-diphenyl-2-picrylhydrazyl (dpph), 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate (abts), ethylene diamine tetraacetic acid (edta), trichloroacetic acid (tca), l-ascorbic acid, chlorogenic acid, gallic acid, caffeic acid, ferulic acid, vanillic acid, catechin, quercetin, cyanidin-3-o-glucoside, cyanidin-3-o-rutinoside, and amino acid standard solution were purchased from sigma aldrich (st. louis, mo, usa). ethanol, methanol, and acetic acid were purchased of analytical grade from j. t. baker (phillipsburg, nj, usa). potato dextrose agar and plate count agar were purchased from himedia laboratory (mumbai, india). wax apple juice extraction matured wax apples of uniform size, color, total soluble solids (10-14 °brix), and free from any apparent damage or disease were selected for cider production. the procured fruits were taken to the laboratory within eight hours. fruits were cleaned of dust with tap water and then washed again with distilled water. later, the fruits were thoroughly juiced using a food processor in a cold environment, roughly strained and fermented to produce cider. cider and cider vinegar preparation wax apple juice (6 l) was fermented to cider in a fermenter by previously followed method (venkatachalam et al., 2018). filtered cider samples proceeded to vinegar production. the cider (4.7% alcohol) sample (10 l) was transferred to a sterile fermenter and inoculated with bacterial culture, specifically acetobacter estunensis, in a ratio of 10:1. the fermenter was placed in the dark at ambient temperature and was continuously aerated by an electric aeration pump. the acidification was continued quality and characteristics. an alternative approach is to proceed to acetic acid fermentation or conversion of cider into cider vinegar (lea, 1989). among the various processed foods, cider vinegar could be a new product suitable for small or large-scale production and a valuable extension to thailand’s food industries. cider vinegar is a newly developed product with several health benefits and has extensive culinary applications. typically, vinegar is the result of failed cider fermentation or poor cider storage conditions and are produced by aerobic fermentation with the acetic acid bacteria, wherein ethanol gets converted into acetic acid. the microorganism is a crucial factor for controlling the fermentation and the product’s quality and functional properties (mas et al., 2014). wax apple cider vinegar (wacv) is obtained by the double fermentation of alcohol and acetic acid. initially, apple juice is subjected to alcoholic fermentation to produce the required acetic acid (dabija and hatnean, 2014). later, acetic acid fermentation takes place in two steps. in the first step, ethanol is oxidized to acetaldehyde by alcohol dehydrogenase. then in the second step, acetaldehyde is oxidized to acetic acid by aldehyde dehydrogenase (raspor and goranovic, 2008). the reaction is exothermic, thus increasing the temperature in the medium. the acetic acid can be further oxidized to carbon dioxide when the ethanol concentration is limited in the tricarboxylic cycle, an unwanted process in vinegar production (johnston and gaas, 2006). cider vinegar is an amazingly versatile cooking ingredient. it adds a tangy taste to many drinks and deepens the flavors of various foods. it is important to note that vinegar is a significant ingredient in most condiments. mayonnaise and tomato ketchup account for more than 10% of the vinegar production in america. cider vinegar benefits include many external that include soothing sunburns and insect bites, shiny hair, and dandruff treatment. cider vinegar has functional therapeutic properties, such as antioxidative, antibacterial activity, promoting recovery from exhaustion, and regulating blood pressure and blood glucose (johnston and gaas, 2006; verzelloni et al., 2007). with increasing interest in the potential health effects of cider vinegar worldwide, there have been many reports confirming the antioxidative activity of various kinds of cider vinegars (budak et al., 2014). phenolic compounds play an essential role in vinegar’s antioxidative activity (shimoji et al., 2002; verzelloni et al., 2007; zhao et al., 2018). several fruits, particularly apple, perry, pear, pome, and orange, are used to produce cider and vinegar products (techakanon and sirimuangmoon, 2020). however, no prior research is attempted to produce cider vinegar from wax apples. also, no study about the stability of such products during prolonged storage is available. therefore, the present study aimed to explore the possibilities of converting the italian journal of food science, 2021; 33 (2) 131 changes in physicochemical characteristics, polyphenolics, and amino acids of wax apple cider vinegar during prolonged storage for 30 days. every 24 hours, samples were collected and tested for acetic acid content and fermentation efficiency (figure 1). after fermentation, cider vinegar was filtered to get rid of the slime. then, the filtered samples were racked in brown bottles for 6 months to study their stability under ambient storage conditions. every month, samples were measured for various quality criteria. quality analysis physicochemical analysis the color coordinates lightness (l*), redness (a*), and yellowness (b*) were measured for wac using a hunterlab colorimeter. electrical conductivity was measured using an electrical conductivity meter, and the results were expressed in ms/cm2. the vinegar sample’s density was measured using a hydrometer, and the results were expressed in g/cm3. the sample’s viscosity was analyzed using a brookfield viscometer, and the results were expressed in cp. the ph of the cider vinegar was measured using a ph meter. the fermentation efficiency of cider vinegar during fermentation was measured following the method of kaur et al. (2011). the results are expressed as percentages. the total nitrogen content in cider vinegar was determined by the kjeldahl method following aoac (2000). the results are expressed in g/l. the ethanolic and acetic acid contents in cider before acetic acid fermentation and during the fermentation were analyzed using the chromatographic method proposed by dias et al. (2016). the results are expressed as percentages. the cider vinegar’s total acidity and volatile acidity were measured using the method of kaur et al. (2011). the results are expressed as percentages. phytochemicals, phenolic compounds, and antioxidant activities phytochemical analysis for total phenolic content (alberti et al., 2014), 100 µl of wacv, 8.4 ml of distilled water, and 500 µl of folin-ciocalteu reagent were added to a test tube. later, 1  ml of sodium carbonate (20%) was added and mixed well. then, the reaction mixture was incubated at room temperature for 30 minutes and was measured using a spectrophotometer at 720 nm. the absorbance was compared with the calibration curve made using gallic acid (10–100 µg/ ml; the coefficient of determination (r2)  = 0.9970; p < 0.0001). the results were expressed as µg gallic acid equivalent (gae)/ml. for total flavonoid content (alberti et al., 2014), 250 µl of wacv was placed in the test tube, and 2.72 ml of 30% ethanol and 120 µl of 0.5 mol/l sodium nitrite were added, mixed, and incubated for 5 minutes. later, 120 µl of 0.3 mol/l aluminum chloride was added to the mixture, followed by 800 µl of 1 mol/l sodium hydroxide, and mixed well. then the reaction mixture was measured at 510 nm using a spectrophotometer. the absorbance was compared with the calibration curve made using catechin (10–100 µg/ ml; r2 = 0.9960; p < 0.0001). the results were expressed as µg catechin equivalent (ce)/ml. for ascorbic acid (asa; bavisetty and venkatachalam, 2021), 1 ml of wacv and 24 ml of 0.4% oxalic acid were mixed in a flask. after that, the reaction mixture was titrated against the 0.04% aqueous sodium dichlorophenolindophenol solution to obtain the first pink shade (endpoint). then, the ascorbic acid in wacv was calculated based on the method of kabasakalis et al. (2000). the results were expressed as µg asa/ ml. phenolic compound analysis a sample of wacv (2 ml) was mixed with 4 ml of 100% methanol and vortexed at high speed for 1 minute, incubated at 60°c for 15 minutes, and was centrifuged at 8160 g for 20 minutes at an ambient temperature. later, the supernatant was collected and filtered through a 0.22 µm nylon syringe filter (waters, milford, ma, usa). high-performance liquid chromatography (hplc) was performed with the collected filtrate to analyze the phenolics (alberti et al., 2014). the phenolic compounds mainly gallic acid, chlorogenic acid, caffeic acid, ferulic acid, vanillic acid, cyanidin-3-o-glucoside, and cyanidin 3-o-rutinoside; and the flavonols such as catechin and quercetin were identified and quantified in the wacv samples. the standard calibration samples for the listed phenolic compounds in this study were prepared at seven concentrations (10–70 µg/ml; r2 ≥ 0.998; p < 0.0001). the retention time and ultraviolet spectra of the standards were used to identify and quantify the phenolic compounds in the samples. the results were expressed in μg/ml. antioxidant activities for dpph radical scavenging assay (brand-williams et al., 1995), 100 µl of wacv and 3.9 ml of 60 µmol/l dpph were mixed well in a test tube, the reaction mixture was incubated for 30 min in the dark at an ambient temperature and was measured at 515 nm using a spectrophotometer. the results were expressed as a percentage of dpph radical scavenging ability. for abts radical cation scavenging assay, 100 µl of wacv and 100 µl abts reagent (as described in lee et al., 2015) in a 96 well microplate incubated for 6 minutes at room temperature. later, the sample was measured at 734 nm using a microplate reader. the results were expressed as a percentage of abts radical scavenging ability. for ferric reducing antioxidant potential assay (frap), 100 µl of wacv was mixed with 3 ml of frap reagent 132 italian journal of food science, 2021; 33 (2) lekjing s and venkatachalam k transfer line was set to 290°c, splitless. about 1.5 ml sample was injected into gc via an automatic injector with a column flow rate of 1 ml/min. leco pegasus 4d mass spectrophotometer was used to record the electronic ionization source at −70 ev. the solvent delay was set to 9 minutes, acquisition rate to 10 spectra/second, the ion source temperature to 250°c, the mass range was 50–1000 amu, detector voltage 1800 v, and the scan time was 1.5 seconds. the data was recorded in total ion chromatogram mode. the agilent chemstation software was adopted to identify the volatile compound name, chemical formula, and % peak area of unknown compounds and were compared with the known data from the gc-ms library (nist mass spectral library). microbial analysis wacv was examined each week for microbiological counts. plate count agar (pca) determined the total counts for aerobic microbes. triplicate serial dilution in peptone water of each sample bag was prepared, 1 ml from each dilution were aseptically inoculated on a petri dish with 20 ml of sterilized agar and mixed thoroughly. upon agar solidification, the petri dishes were inverted and incubated for 48 h at 30°c. colonies were counted and are reported as log10 cfu/ml (american public health association, 1978). yeast and mold counts were determined using acidified (ph 3.5) potato dextrose agar. dilutions and samples were prepared as described in the pca method. the sterilized agar’s ph was adjusted to 3.5, with sterilized 10% (w/v) tartaric acid and poured on petri dishes, mixed thoroughly, inverted, and incubated for two days at 30°c. later, colonies were counted and reported as log10 cfu/ ml (beever and bollard, 1970). escherichia coli (e. coli) was determined using petrifilm e. coli count plate. onto the center of the bottom film, 1 ml of sample was added by lifting the top film and was slowly rolled down onto the sample to prevent air bubble entrapment. the plate was left undisturbed for one minute to permit solidification of the gel and then incubated at 37°c for two days. after incubation, the colonies were counted and reported as log10 cfu/ml (aoac, 2002). statistical analysis all the experiments were carried out in triplicates. the data were presented as the mean and standard error of the mean (sem), one-way analysis of variance studied the differences during the storage. duncan’s multiple range test identified the significant differences between the means at p < 0.05. spss v6 for windows (spss, chicago, il, usa) was used to run all the statistical analyses. (as described in alberti et al., 2014). the reaction mixture was incubated for 20 minutes for a blue-colored complex development and was measured at 593 nm using a spectrophotometer. the absorbance of sample was compared with the calibration curve made for ferrous ion (fe2+;10– 100 mmol/ml; r2 = 0.0997; p < 0.0001). the results were recorded in mmol fe2+ equivalent (fe2+e) /ml. for hydroxyl radical scavenging assay (halliwell et al., 1987), 1 ml of wacv was added to a test tube containing 1 ml of ferrous ammonium sulfate (0.13%– 0.26%) (edta), 0.5 ml of 0.018% edta, 1 ml of 0.85% dimethyl sulfoxide, and 0.22% ascorbic acid, mixed well, and incubated in a water bath at 90°c for 10 min. later, 1 ml of ice-cold and 3 ml of nash reagent wax were added to the reaction mixture and incubated at room temperature for 15 minutes for yellow color development. finally, the reaction mixture was measured at 412 nm using a spectrophotometer, and the outcomes were expressed as a percentage of hydroxyl radical scavenging ability. amino acid profile about 3 ml sample of wacv was mixed well with 2 ml of 0.25 mmol/l norleucine and centrifuged at 13,000 g for 20 minutes at 4°c. the supernatant was collected and filtered through a nylon syringe filter (0.2 µm, waters) and was used to identify the amino acids by the chromatographic method proposed by alberti et al. (2016). the amino acids were derivatized using a waters accq tag™ reagent kit (flask 1: 200 mmol/l borate buffer, ph 8.8; flask 2a: 6-aminoquinolyl-n-hydroxysuccinimidyl carbamate; and flask 2b: acetonitrile). then the identification and quantification were accomplished by hplc using the accq tag reagent kit methodology with a picotag column (4 mm, 3.9 × 150 mm). the identified amino acids in wacv were quantified and were reported in mg/100 ml. analysis of volatile compounds the volatile compounds in the wacv were extracted and analyzed by gas chromatography-mass spectroscopy (gc-ms; agilent gc: 6890, with a 7683b autosampler, agilent technologies, palo alto, ca) using the static headspace method proposed by pietrowski et al. (2012) with some modifications. the capillary column (phenomenex column with 30 m in length with 25 mm internal diameter and 0.25 µm thick zbwax film) was attached directly to the agilent gc. the analysis conditions were programmed with an initial temperature of 40°c for 5 minutes and then increased to 10°c/min to 300°c with an isothermal state of 10 minutes. the sample injector port temperature was set to 250°c; the italian journal of food science, 2021; 33 (2) 133 changes in physicochemical characteristics, polyphenolics, and amino acids of wax apple cider vinegar during prolonged storage results and discussion acetic acid production and fermentation efficiency wac was studied for acetic acid production to understand the acetic acid bacteria’s efficiency and the cider medium’s suitability for acetic acid production for up to 29 days (figure 1). the fermentation was ceased at 29  days, as no significant changes in acetic acid levels were noted after 20 days. during the continuous acetic acid fermentation of wac, a gradual increase in acetic acid content was noted. in the initial stages, a gradual increase in the production was recorded on day 11 of the fermentation, an increase in the pace of acetic acid production was noted until day 21. later it was found to be almost constant till day 29. concerning acetic acid production, the ethanol content in wac gradually decreased during the formation of acetic acid in the wac. a similar finding was reported by štornik et al. (2016) in a study of apple cider vinegar production. kocher et al. (2006) reported that the rapid conversion of ethanol into acetic acid by acetic acid bacteria is facilitated by aeration, stirring, and heating. at the end of the fermentation and before bottling for storage and/or maturation for six months, wacv had about 2.3% acetic acid. on the other hand, the ethanol content decreased from 4.8% to 2.1% as it was used up by the bacteria to produce acetic acid during the fermentation. the fermentation efficiency (fe) tended to increase with fermentation time (figure 1). though a continuous boost in fe was observed during the fermentation, the level of acetic acid was stable after 21 days because of the nature of the acetic acid-producing bacteria. joshi et al. (2016) reported that some acetic acid-producing bacterial strains could over-oxidize acetic acid into carbon-dioxide and water, which could halt the acetic acid production in the wac during the fermentation. moruno et al. (1993) reported three main factors that could adversely affect the production of acetic acid during fermentation: (1) the genetics of the yeast strain, (2) the presence of polyphenols, especially catechin and anthocyanins, and (3) the presence of unsaturated fatty acids. physicochemical properties wacv was bright yellowish. during the vinegarmaking process, the usual pinkish-yellow wac wholly turned yellow (figure 2a). furthermore, the color changes were consistent during the prolonged storage of wacv (the yellowness increased during storage). on the other hand, the lightness representing luminosity and redness, with negative values representing the darkness, tended to decrease throughout the storage period without many fluctuations in the results. the vinegar samples’ color changes could be caused by maturation, with the degradation of anthocyanin residues in wacv and potential effects from acidification. mas et al. (2014) reported that polyphenolic compounds are responsible for the color and astringency of vinegar. tarazona-diaz and aguayo (2013) observed the reduced redness in the fruit juice during acidification, which agreed with the present study as the ph of wacv increased during storage (figure 2c). the density decreased steadily, along with viscosity 5 4 3 2 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 0 20 40 60 80 100 120 fe (%) acetic acid (%) ethanol (%) a ce tic a ci d pr od uc tio n (% ) fe rm en ta tio n e ff ic ie nc y (f e % ) 0 figure 1. acetic acid production and fermentation efficiency of wax apple cider. 134 italian journal of food science, 2021; 33 (2) lekjing s and venkatachalam k –14 (a) (b) (c) (d) –16 –18 –22 –24 –26 –28 –30 0 1 c ol or (a *) c ol or (l * an d b* ) ph 2 3 storage period (months) storage period (months) 4 5 6 0 1 2 3 storage period (months) 4 5 6 0 10 redness (a*) lightness (l*) electrical conductivity ph yellowness (b*) 20 30 40 50 60 3.0 3.1 3.2 3.3 3.4 3.5 –20 4.4 4.2 4.0 3.6 3.4 e le ct ri ca l c on du ct iv ity (m s /c m 2 ) 3.8 1.016 1.014 1.012 1.010 1.008 1.006 1.004 1.002 1.000 0 1 d en si ty (g /c m 3 ) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 a ci di ty (% ) v is co si ty (c p ) 2 3 4 5 6 storage period (months) 0 1 2 3 4 5 6 6.08 6.10 6.12 6.14 6.16 6.18 6.20 6.22 6.24 6.26 6.28 density viscosity total nitrogen total acidity volatile acidity to ta l n itr og en (g /l ) 5.0 5.2 5.4 5.6 5.8 6.0 figure 2. physiochemical qualities of wax apple cider vinegar during the storage period. throughout the storage of wacv (figure 2b). the vinegar density is linked to acetic acid density directly in case it has been distilled. the present study shows that density decreased with storage time, indicating that organic and inorganic substances in wacv could lose their binding potential and sediment at the bottom of the bottle, consequently affecting density and viscosity. the electrical conductivity of wacv (figure 2c) shows a linear pattern with storage time. during the study period, it increased from 3.58 to 4.11 ms/cm2. at the end of the study, the conductivity of the samples was slightly lowered. the ph of wacv steadily changed to acidic during prolonged storage under ambient temperature (figure 2c). the ph was acidic from the beginning (3.44) and gradually decreased to 3.22 at the end of storage. a decrease in ph is the direct influence of organic acid’s presence in the samples. in this study, a continuous increase of acid content (total acidity and volatile acidity) in wacv during the storage was observed (figure 2d), which agreed with the study of vithlani and patel (2010). although there is a significant change in the ph and organic acid level in the wacv, it still stayed in the cider vinegar range recommended by the codex (ho et al., 2017). the total acidity was slightly higher in wacv than in volatile acidity. the increased acidity and decreased ph in the samples indicate that prolonged storage tended to increase the acidification of wacv. typically, the volatile acidity represents the odorous fatty acids in vinegar, mainly acetic acid. chidi et al. (2018) and jackson (2008) reported that the acetic acid level in vinegar increase during aging. furthermore, the total nitrogen content in the wacv showed a steady increase during storage (figure 2d). phenolic contents and antioxidant activities phytochemicals of wacv are shown in figure 3(a). among the various phytochemicals in the wacv, the total phenolic content (tpc) was the most abundant, followed by total flavonoids (tfc) and asa. prolonged storage significantly affected the phytochemicals in the samples. tpc and tfc contents steadily increased in wacv during prolonged storage, whereas asa steadily decreased. various phenolic acids and flavonoids in wacv were quantified, and the results are presented in figure 3(b). phenolic acids such as gallic italian journal of food science, 2021; 33 (2) 135 changes in physicochemical characteristics, polyphenolics, and amino acids of wax apple cider vinegar during prolonged storage 0 0 10 20 30 40 50 tpc (µg gae/ml) gallic acid chlorogenic acid caffeic acid ferulic acid dpph abts hydroxyl radical frap vanillic acid catechin quercetin cyanidin-3-o-glucoside cyanidin-3-o-rutinoside tfc (µg ce/ml) asa (µg/ml) 1 2 3 storage period (months) p hy to ch em ic al s 0 5 10 15 20 p he no lic c om po un ds (m g/ m l) 0 20 40 60 80 100 120 0 5 10 15 20 25 30 r ad ic al s ca ve ng in g (% ) fr a p (m m ol f e2 + e /1 00 m l) 4 5 6 0 1 2 3 storage period (months) 4 5 6 0 1 2 3 storage period (months) 4 5 6 (a) (b) (c) figure 3. phytochemicals, phenolic compounds, and antioxidant activities of wax apple cider vinegar during the storage period. acid, chlorogenic acid, caffeic acid, ferulic acid, and vanillic acid were observed in wacv. among the various phenolics, caffeic acid, ferulic acid, and gallic acid were the highest in wacv (p < 0.05). chlorogenic acid tended to decrease throughout the storage period. vanillic acid gradually increased in wacv until four months and was constant later. however, flavonoids such as catechin and quercetin concentrations highest among the various phenolic compounds. during storage, catechin and quercetin steadily increased in waca, whereas the lesser components cyanidin-3-o-glucoside and cyanidin-3-o-rutinosidecontinuously decreased. 136 italian journal of food science, 2021; 33 (2) lekjing s and venkatachalam k hornedo-ortega et al. (2017) found that anthocyanin content in the vinegar was more susceptible than in the cider. anthocyanin (91%) continuously decreased in the acetic acid fermentation versus alcoholic fermentation (19%). figure 3(c) presents the radical scavenging activity of wacv during the prolonged storage period. among the various scavenging activities, dpph activity in wacv was higher at the beginning of the storage. for the first four months of storage, minimal changes in the scavenging activity of wacv was noted. when the storage reached between 5 and 6 months, the dpph activity significantly decreased. instead, hydroxyl and abts radical scavenging activities gradually increased throughout the storage. wacv showed a higher potential of scavenging hydroxyl radicals than abts radicals. davies et al. (2017) have found that the asa content was the primary contributor of the dpph activity in vinegar. a reduction of asa in the vinegar during storage was probably the cause of chemical changes by the bio-oxidative process. furthermore, the abts activity in the vinegar could be influenced by the synergic effect of phenolics, ascorbic acids, and some amino acids (campodonico et al., 1998). overall, all types of radical scavenging activities tested in wacv were maintained for at least 40% of the initial level during the storage. however, the frap of wacv was minimal and steadily increased throughout the storage (p < 0.05). frap activity is strongly correlated with the polyphenolics present in a food product, especially in beverages, which agreed with the finding of schlesier et al. (2002). amino acid profile table 1 shows the amino acid content in wacv during the prolonged storage period. about 18 amino acids were observed in wacv during storage. the storage period significantly affected the levels of amino acids in wacv. glutamic acid, hydroxyproline, histidine, lysine, methionine, proline, serine, threonine, tyrosine, valine, and glutamine levels increased in wacv during the storage. however, alanine, arginine, aspartic acid, glycine, isoleucine, leucine, and phenylalanine levels decreased. alanine, histidine, isoleucine, leucine, and valine were found in very minimal contents in wacv. acetic acid bacteria could metabolize the amino acids from cider vinegar, particularly alanine, arginine, glycine, and leucine (valero et al., 2005; wang et al., 2015). the storage period could limit the suitable conditions for the bacteria to survive and reproduce. hence, it catalyzed the available resources from the vinegar to sustain. arginine, lysine, methionine, proline, threonine, and glutamine were the primary amino acids found in the wacv. the other listed amino acids in table 1 were found at moderate levels. in our previous study (venkatachalam et al., 2018), we observed 23 amino acids in the wac. the present study shows that acetic acid fermentation significantly influenced the amino acid contents. cysteine, hydroxylysine, tryptophan, asparagine, and glutamine, were not detected in the wacv. furthermore, ardö (2006) reported that the aminotransferase enzyme in vinegar could catabolize amino acids (aspartic acid, isoleucine, leucine, and phenylalanine) for flavor formation (buttery, malty, fruity, sweaty, floral, chemical, and fecal). volatile profile the volatile profile of wacv during prolonged storage is presented in table 2. normally, volatile compounds of fermented beverages are by-products of the catabolic processing of amino acids by microorganisms. the volatile profile of wacv was significantly influenced by acetic acid fermentation and by prolonged storage. about 31 volatile flavor compounds were observed in wacv. the present study showed that cider vinegar exhibited complex flavors, including strawberry, apple, raspberry, vinegar, acetic acid, fruity, wine, plastic, sweet, floral, acidic, feet, gruyere cheese, fusty, ripened cheese, rancid, rose, sour, spicy, potato, burnt, floral, honey, wax, caramel, and clover, that agreed with the research of charles et al., 2000. most volatile compounds in wacv gradually decreased with storage time. among the different volatile compounds, acid, and fatty acid-related volatile compounds were predominant, followed by esters, ketones, alkanes, phenol, and benzothiazole. ethyl acetate and acetoin were the major volatile compounds found in the wacv, which agreed on the studies of ubeda et al. (2011) and valero et al. (2005) reporting that acetoin is the predominant volatile compound present in cider vinegar and tends to decrease as the vinegar ages. the other volatiles were minimal. volatiles that increased during storage included boranemethyl sulfide, ethyl acetate, 2, 3-butanedione, isoamyl alcohol, 3-acetoxy-2-butanone, dimethyl sulfone, butanoic acid, 2-propenoic acid, benzyl alcohol, 2-ethyl hexanoic acid, benzothiazole, phenol, 2-ethyl heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid. however, 2,4,5-trimethyl-1,3-dioxolane, acetoin, 2-hydroxyethyl propanoate, 1-phenylpropane-1,2-dione, propanoic acid, 3-methyl butanoic acid, β-phenethyl acetate, hexanoic acid, 2-isopropyl-2,3-dimethylbutanoic acid, 2-ethyl-2,5-dimethylhexanoic acid, (+)-curdione and 4-ethyl-phenol decreased in wacv during the storage. furthermore, during the storage study, some volatile compounds such as (3,4-diphenylisothiazol5yl)-phenylmethanone and 2-methylpropylmethyl ether italian journal of food science, 2021; 33 (2) 137 changes in physicochemical characteristics, polyphenolics, and amino acids of wax apple cider vinegar during prolonged storage ta bl e 1. a m in o ac id c on te nt s in w ax a pp le c id er v in eg ar d ur in g pr ol on ge d st or ag e. a m in o ac id (m g/ 10 0 m l) s to ra ge p er io d (m on th s) 0 1 2 3 4 5 6 a la ni ne 6. 78 ± 0 .5 6a 6. 48 ± 0 .1 1a 4. 56 ± 0 .7 4b 4. 01 ± 0 .0 5b c 3. 25 ± 0 .1 2c 3. 01 ± 0 .1 9c 1. 47 ± 0 .2 8d a rg in in e 77 9. 05 ± 5 .3 0a 72 5. 14 ± 3 .5 1b 70 1. 29 ± 4 .1 1c 68 9. 21 ± 0 .5 6d 67 8. 17 ± 3 .1 1e 66 2. 41 ± 1 .2 7f 65 4. 39 ± 0 .9 9g a sp ar tic a ci d 44 .8 9 ± 0. 80 a 43 .5 6 ± 0. 73 ab 42 .1 5 ± 0. 27 b 40 .1 9 ± 0. 69 c 39 .8 7 ± 1. 27 c 36 .5 4 ± 0. 59 d 34 .4 4 ± 1. 75 e g lu ta m ic a ci d 71 .8 9 ± 0. 43 f 77 .8 9 ± 0. 21 e 81 .4 5 ± 0. 67 d 83 .5 6 ± 0. 59 cd 84 .5 9 ± 1. 51 c 87 .1 5 ± 1. 21 b 90 .1 6 ± 0. 78 a g ly ci ne 37 .8 9 ± 2. 64 a 36 .5 1 ± 1. 41 ab 35 .4 4 ± 2. 41 b 35 .0 1 ± 1. 17 b 34 .4 5 ± 0. 89 b 31 .8 7 ± 1. 66 c 30 .9 9 ± 0. 92 d h is tid in e 8. 94 ± 1 .1 1b 9. 01 ± 0 .4 8b 9. 22 ± 0 .9 5b 10 .4 1 ± 0. 27 ab 10 .7 8 ± 0. 67 ab 11 .0 1 ± 1. 66 a 11 .5 0 ± 1. 10 a h yd ro xy p ro lin e 12 .4 1 ± 0. 59 d 13 .5 6 ± 0. 44 c 14 .7 8 ± 0. 62 bc 12 .4 6 ± 0. 79 d 13 .8 9 ± 0. 75 c 15 .6 6 ± 0. 30 b 16 .7 4 ± 0. 89 a is ol eu ci ne 2. 77 ± 0 .5 0a 2. 01 ± 0 .2 4b 1. 45 ± 0 .1 9c 1. 25 ± 0 .1 2c d 1. 05 ± 0 .1 4d 0. 89 ± 0 .0 6e 0. 77 ± 0 .0 9e le uc in e 8. 88 ± 0 .8 0a 8. 01 ± 0 .5 3a b 7. 55 ± 0 .8 5b 7. 17 ± 0 .8 2c 6. 89 ± 1 .2 0c 6. 04 ± 0 .2 4c d 5. 89 ± 0 .6 4d ly si ne 21 5. 89 ± 1 8. 00 g 23 5. 11 ± 1 3. 00 f 24 1. 56 ± 4 .0 0e 25 6. 78 ± 1 9. 00 d 28 8. 71 ± 7 .8 0c 30 1. 41 ± 3 .7 4b 31 7. 84 ± 4 .5 6a m et hi on in e 25 61 .8 2 ± 88 .0 0g 25 71 .8 8 ± 61 .0 0f 25 86 .1 9 ± 90 .0 0e 26 01 .8 2 ± 56 .0 0d 26 57 .4 4 ± 32 .0 0c 27 15 .5 3 ± 17 .0 0b 27 23 .5 1 ± 48 .0 0a p he ny la la ni ne 29 .1 8 ± 1. 00 a 28 .5 6 ± 0. 40 a 27 .1 5 ± 2. 00 b 24 .5 6 ± 0. 86 c 22 .1 4 ± 0. 44 d 20 .1 5 ± 0. 69 e 18 .9 5 ± 0. 17 f p ro lin e 28 8. 79 ± 1 2. 00 g 29 5. 78 ± 1 5. 00 f 31 2. 41 ± 3 .4 0e 33 3. 45 ± 7 .0 0d 34 5. 87 ± 3 .0 0c 35 1. 51 ± 8 .5 0b 35 9. 87 ± 7 .1 2a s er in e 55 .4 8 ± 8. 00 g 57 .8 9 ± 7. 00 f 58 .9 1 ± 3. 60 e 62 .1 5 ± 0. 78 d 64 .5 2 ± 1. 00 c 67 .1 4 ± 0. 19 b 68 .1 1 ± 0. 91 a th re on in e 18 9. 78 ±9 .0 0g 21 7. 11 ± 8 .4 0f 23 4. 56 ± 3 8. 00 e 24 7. 81 ± 1 4. 00 d 25 1. 41 ± 7 .8 9c 25 5. 87 ± 6 .4 1b 25 7. 49 ± 9 .1 9a ty ro si ne 10 .4 1 ± 0. 50 d 12 .1 4 ± 0. 90 c 13 .2 1 ± 0. 90 bc 13 .5 6 ± 0. 20 b 14 .0 1 ± 0. 73 ab 14 .4 5 ± 0. 14 ab 14 .9 8 ± 0. 56 a va lin e 1. 67 ± 0 .0 9e 2. 07 ± 0 .0 6d 2. 45 ± 0 .5 0c d 2. 78 ± 0 .1 7c 3. 05 ± 0 .0 5b 3. 56 ± 0 .8 7a 3. 78 ± 0 .5 1a g lu ta m in e 11 14 .1 1 ± 94 .7 8g 11 27 .5 6 ± 27 .3 9f 11 56 .1 5 ± 61 .1 8e 11 74 .2 1 ± 24 .1 2d 11 91 .1 4 ± 17 .1 0c 12 01 .4 5 ± 2. 77 b 12 24 .5 1 ± 17 .3 1a #d at e re pr es en te d as m ea n ± st an da rd d ev ia tio n fro m tr ip lic at es . t he d iff er en t a lp ha be t i n th e co lu m n in di ca te s a si gn ifi ca nt d iff er en ce . 138 italian journal of food science, 2021; 33 (2) lekjing s and venkatachalam k ta bl e 2. th e vo la til e pr ofi le o f w ax a pp le c id er v in eg ar d ur in g pr ol on ge d st or ag e. vo la til e c om po un d r t p ea k ar ea % s to ra ge p er io d (m on th s) 0 1 2 3 4 5 6 b or an em et hy l s ul fid e 3. 06 29 0. 47 ± 0 .0 1e 1. 16 ± 0 .0 8d 1. 31 ± 0 .0 5c 1. 51 ± 0 .0 1b c 1. 74 ± 0 .0 4b 1. 78 ± 0 .0 1b 2. 04 ± 0 .0 5a e th yl a ce ta te 3. 76 55 3. 38 ± 0 .0 1d 3. 69 ± 0 .0 5c 3. 84 ± 0 .0 7b c 4. 00 ± 0 .0 3b 4. 19 ± 0 .0 0a 4. 19 ± 0 .0 0a 4. 21 ± 0 .0 0a 2, 4, 5tr im et hy l-1 ,3 -d io xo la ne 4. 60 20 0. 69 ± 0 .0 1a 0. 68 ± 0 .0 7a 0. 61 ± 0 .0 1a 0. 58 ± 0 .0 4a b 0. 54 ± 0 .0 1a b 0. 54 ± 0 .0 3a b 0. 51 ± 0 .0 7b 2, 3b ut an ed io ne 5. 15 06 1. 61 ± 0 .0 2d 1. 66 ± 0 .0 4d 2. 27 + 0 .0 6c 2. 51 ± 0 .0 4b 2. 57 ± 0 .0 1b 2. 55 ± 0 .0 3b 2. 79 ± 0 .0 7a is oa m yl al co ho l 6. 71 46 0. 61 ± 0 .0 1c 0. 68 ± 0 .0 8c 0. 74 ± 0 .0 1b 0. 77 ± 0 .0 1b 0. 73 ± 0 .0 1b 0. 77 ± 0 .0 6b 0. 98 ± 0 .1 1a a ce to in 15 .2 34 8. 86 ± 0 .0 7a 8. 56 ± 0 .1 7a b 8. 33 ± 0 .2 2b 7. 94 ± 0 .1 0c 7. 94 ± 0 .5 5c 8. 01 ± 0 .1 1c 7. 99 ± 0 .0 7c 2hy dr ox ye th yl p ro pa no at e 17 .5 72 0. 76 ± 0 .0 7a 0. 64 ± 0 .0 4b 0. 55 ± 0 .1 0c 0. 52 ± 0 .0 8c 0. 47 ± 0 .0 3c d 0. 43 ± 0 .0 2c d 0. 41 ± 0 .0 4d 3ac et ox y2b ut an on e 18 .8 70 0. 25 ± 0 .0 5b 0. 26 ± 0 .0 5b 0. 30 ± 0 .0 1a 0. 31 ± 0 .0 4a 0. 31 ± 0 .0 1a 0. 31 ± 0 .0 9a 0. 32 ± 0 .0 1a 1ph en yl pr op an e1, 2di on e 19 .6 66 0. 28 ± 0 .0 1a 0. 28 ± 0 .0 3a 0. 20 ± 0 .0 0b 0. 17 ± 0 .0 1c 0. 17 ± 0 .0 0c 0. 13 ± 0 .0 1d 0. 11 ± 0 .0 1d p ro pa no ic a ci d 21 .0 21 0. 30 ± 0 .0 3b 0. 37 ± 0 .0 1a 0. 33 ± 0 .0 2a b 0. 33 ± 0 .0 6a b 0. 32 ± 0 .0 7a b 0. 19 ± 0 .0 0c 0. 17 ± 0 .0 2c (3 ,4 -d ip he ny lis ot hi az ol -5 -y l)ph en yl m et ha no ne 21 .9 49 0. 09 ± 0 .0 0b 0. 15 ± 0 .0 1a 0. 16 ± 0 .0 2a n d n d n d n d 2m et hy l p ro pa no ic a ci d 22 .0 96 1. 17 ± 0 .0 8a 1. 18 ± 0 .0 0a 1. 15 ± 0 .0 0a 1. 10 ± 0 .0 0b 1. 05 ± 0 .0 0b 1. 07 ± 0 .0 0b 1. 05 ± 0 .0 0b 2m et hy lp ro py lm et hy l e th er 22 .4 81 0. 09 ± 0 .0 0n s 0. 08 ± 0 .0 0n s 0. 08 ± 0 .0 0n s n d n d n d n d d im et hy l s ul fo ne 22 .5 96 0. 12 ± 0 .0 0d 0. 23 ± 0 .0 1c 0. 24 ± 0 .0 4c 0. 32 ± 0 .0 2b 0. 35 ± 0 .0 4a b 0. 37 ± 0 .0 2a 0. 37 ± 0 .0 3a b ut an oi c ac id 23 .2 65 0. 20 ± 0 .0 3 n s 0. 20 ± 0 .0 1n s 0. 20 ± 0 .0 4n s 0. 21 ± 0 .0 1n s 0. 21 ± 0 .0 1n s 0. 22 ± 0 .0 1n s 0. 22 ± 0 .0 0n s 2p ro pe no ic a ci d 23 .3 89 0. 21 ± 0 .0 0d 0. 33 ± 0 .0 2c 0. 43 ± 0 .0 8b 0. 47 ± 0 .0 2b 0. 48 ± 0 .0 4b 0. 55 ± 0 .0 8a 0. 57 ± 0 .0 2a 3m et hy l b ut an oi c ac id 23 .7 63 4. 43 ± 0 .0 7a 4. 36 ± 0 .0 8a b 4. 22 ± 0 .0 5b 4. 12 ± 0 .0 9b 3. 97 ± 0 .1 0c 4. 03 ± 0 .0 8c 4. 01 ± 0 .1 2c βp he ne th yl a ce ta te 24 .3 69 0. 40 ± 0 .0 1a 0. 38 ± 0 .0 3a 0. 36 ± 0 .0 4a b 0. 35 ± 0 .0 7a b 0. 33 ± 0 .0 2a b 0. 27 ± 0 .0 6b 0. 25 ± 0 .0 1b h ex an oi c ac id 25 .5 25 1. 34 ± 0 .0 1a 1. 34 ± 0 .0 0a 1. 33 ± 0 .0 0a 1. 30 ± 0 .0 0a b 1. 30 ± 0 .0 0a b 1. 26 ± 0 .0 0b 1. 24 ± 0 .0 0b b en zy l a lc oh ol 26 .1 41 0. 07 ± 0 .0 0 n s 0. 07 ± 0 .0 0n s 0. 08 ± 0 .0 0n s 0. 08 ± 0 .0 0n s 0. 09 ± 0 .0 0n s 0. 09 ± 0 .0 0n s 0. 10 ± 0 .0 0n s 2et hy l h ex an oi c ac id 26 .9 17 0. 09 ± 0 .0 0b 0. 09 ± 0 .0 0b 0. 14 ± 0 .0 0a b 0. 14 ± 0 .0 0a b 0. 14 ± 0 .0 0a b 0. 14 ± 0 .0 0a b 0. 19 ± 0 .0 0a b en zo th ia zo le 27 .0 77 0. 32 ± 0 .0 1b 0. 36 ± 0 .0 4a b 0. 36 ± 0 .0 7a b 0. 38 ± 0 .0 1a b 0. 40 ± 0 .0 4a b 0. 43 ± 0 .0 1a 0. 45 ± 0 .0 3a p he no l 27 .4 22 0. 34 ± 0 .0 3b 0. 38 ± 0 .0 1a b 0. 40 ± 0 .0 2a 0. 40 ± 0 .0 1a 0. 40 ± 0 .0 1a 0. 41 ± 0 .0 1a 0. 43 ± 0 .0 1a 2et hy l h ep ta no ic a ci d 27 .5 90 0. 13 ± 0 .0 1b 0. 14 ± 0 .0 0b 0. 14 ± 0 .0 1b 0. 15 ± 0 .0 0b 0. 18 ± 0 .0 0a 0. 18 ± 0 .0 0a 0. 18 ± 0 .0 1a 2is op ro py l-2 ,3 -d im et hy lb ut an oi c ac id 27 .6 26 1 0. 19 ± 0 .0 2a 0. 19 ± 0 .0 0a 0. 18 ± 0 .0 2a 0. 16 ± 0 .0 0a 0. 14 ± 0 .0 0b 0. 14 ± 0 .0 0b 0. 14 ± 0 .0 0b 2et hy l-2 ,5 -d im et hy lh ex an oi c ac id 27 .6 83 0. 18 ± 0 .0 0a 0. 17 ± 0 .0 0a 0. 16 ± 0 .0 0a 0. 14 ± 0 .0 0a b 0. 12 ± 0 .0 0a b 0. 13 ± 0 .0 0a b 0. 10 ± 0 .0 0b o ct an oi c ac id 27 .8 05 0. 13 ± 0 .0 1d 0. 77 ± 0 .0 0c 0. 97 ± 0 .0 1b 1. 04 ± 0 .0 0a b 1. 08 ± 0 .0 6a b 1. 14 ± 0 .0 9a 1. 18 ± 0 .0 2a (+ )c ur di on e 27 .9 02 0. 37 ± 0 .0 4a 0. 37 ± 0 .0 1a 0. 37 ± 0 .0 4a 0. 36 ± 0 .0 2a 0. 34 ± 0 .0 4a b 0. 34 ± 0 .0 6a b 0. 27 ± 0 .0 7b n on an oi c ac id 28 .1 86 0. 11 ± 0 .0 0c 0. 13 ± 0 .0 2b 0. 13 ± 0 .0 0b 0. 13 ± 0 .0 1b 0. 13 ± 0 .0 0b 0. 14 ± 0 .0 0a b 0. 16 ± 0 .0 1a 4et hy lp he no l 29 .2 27 5 0. 22 ± 0 .0 1a 0. 22 ± 0 .0 0a 0. 21 ± 0 .0 4a 0. 20 ± 0 .0 0b 0. 20 ± 0 .0 1b 0. 20 ± 0 .0 0b 0. 16 ± 0 .0 1c d ec an oi c ac id 30 .4 92 6 0. 35 ± 0 .0 5d 0. 54 ± 0 .0 4c 0. 58 ± 0 .0 4c 0. 58 ± 0 .0 1c 0. 76 ± 0 .0 3a 0. 75 ± 0 .0 4a 0. 70 ± 0 .0 1b # d at a pr es en te d as m ea n ± st an da rd d ev ia tio n fro m th re e re pl ic at io ns . t he d iff er en t s up er sc rip ts in a c ol um n in di ca te s ig ni fic an t d iff er en ce s (p < 0 .0 5) . n d , n ot d et ec te d; r t, m ea ns re te nt io n tim e; n s , n on si gn ifi ca nt d iff er en ce . italian journal of food science, 2021; 33 (2) 139 changes in physicochemical characteristics, polyphenolics, and amino acids of wax apple cider vinegar during prolonged storage were nondetectable in wacv after two months of storage. volatile compounds in the vinegar could be influenced by the environmental conditions, light intensity, constituents formed during vinegar production, and aging (valero et al., 2005; chen et al., 2020). ribéreaugayon et al. (2006) reported that oxygen plays a key role in the formation of volatile compounds in vinegar. kang et al. (2020) observed that extended storage of fruit cider vinegar significantly decreased the alcoholic, bitterness, and sweetn flavors and significantly increased sourness and astringency flavors. microbial growth microbial growth in wacv was minimal to absent during the prolonged storage (table 3). total plate count showed active microbial growth in the samples at the beginning of the storage period, which could be because of the active stage of the acetic acid-producing bacteria. however, during the prolonged storage, the bacterial growth in wacv decreased because of acidity and the anaerobic conditions. furthermore, pathogenic bacteria, particularly e. coli, were absent in the wacv throughout the storage. similarly, yeast and mold also did not survive in the wacv. the results showed that wacv potently inhibited microbial growth. yagnik et al. (2018) reported that cider vinegar could possess multiple antimicrobial properties against various microbial species, especially e.  coli, staphylococcus aureus, and candida albicans, controlling the microbial growth and suppressing mononuclear cytokine and phagocytic responses. gomezgarcia et al. (2019) observed various organic acids and their antifungal effects. their study reported that acetic acid inhibited (45.21%) fungal growth compared with other organic acids. yang et al. (2016) observed that phenolics and flavonoids from vinegar exhibited higher antimicrobial activity, especially against pathogens. table 3. microbial growth in wax apple cider vinegar during prolonged storage. storage time (months) total plate count (log cfu/ml) * yeast and mold (log cfu/ml) e. coli (log cfu/ml) 0 2.4 ± 0.3a nd nd 1 2.3 ± 0.3a nd nd 2 1.8 ± 0.0b nd nd 3 <10c nd nd 4 <10c nd nd 5 <10c nd nd 6 nd nd nd nd, not detected. #data presented as mean ± standard deviation from three replications. the different superscripts in a column indicate significant differences. conclusion this study was the first to understand the physiochemical characteristics, flavor, and functional properties of wax apple cider vinegar during prolonged storage. overall, the study found that the storage period or aging of wacv significantly influenced its qualities. organic acids, polyphenolics, and flavonoids in wacv increased significantly, and consequently, the antioxidant and antimicrobial activities were higher in the wacv. the flavor profile of wacv was significantly influenced by continuous changes in the amino acid contents during storage. author contributions lekjing s conducted the experiment, performed a literature review, and complied the first draft. venkatachalam  k developed the experimental design, conducted the experiments, reviewed, revised the manuscript, and performed the final revision. all authors agreed to publish this manuscript. acknowledgments the authors acknowledge the food innovation and product development (fipd) laboratory for providing laboratory space and equipment support. furthermore, the authors thank associate professor dr. seppo karrila for proofreading the draft manuscript. conflicts of interest no potential conflict of interest was reported by the authors. funding the study received research grants from the research and development office (rdo), prince of songkla university, hatyai campus, and surat thani campus (project grant no. sit590720s). references alberti, a., santos, t.p.m., zielinski, a.a.f., santos, c.m.e., braga, c.m. and demiate, i.m. 2016. impact on chemical profile in apple juice and cider made from unripe, ripe and senescent dessert varieties. lwt-food. sci. technol. 65:436–443. https:// doi.org/10.1016/j.lwt.2015.08.045 alberti, a., zielinski, a.a.f., zardo, d.m., demiate, i.m., nogueira,  a. and mafra, l.i. 2014. optimisation of the https://doi.org/10.1016/j.lwt.2015.08.045� https://doi.org/10.1016/j.lwt.2015.08.045� 140 italian journal of food science, 2021; 33 (2) lekjing s and venkatachalam k gomez-garcia, m., sol, c., de nova, p.j.g., puyalto, m., mesas, l., puente, h., et al. 2019. antimicrobial activity of a selection of organic acids, their salts and essential oils against swine enteropathogenic bacteria. porc. health. manag. 5(32):1–8. https://doi. org/10.1186/s40813-019-0139-4 halliwell, b., gutteridge, jmc and aruoma, o.i. 1987. the deoxyribose method: a simple “test-tube” assay for determination of rate constants for reactions of hydroxyl radicals. anal. biochem. 165:215–219. https://doi.org/10.1016/0003-2697(87)90222-3 hornedo-ortega, r., alvarez-fernandez, m.a., cerezo, a.b., garcia-garcia, i., troncoso, a.m. and garcia-parrilla,  m.c. 2017. influence of fermentation process on the anthocyanin composition of wine and vinegar elaborated from strawberry. j. food. sci. 82(2):364–372. https://doi.org/10.1111/1750-3841. 13624 ho, w.c., lazim, m.a., fazry, s., zaki, h.h.k.u. and lim, s. 2017. varieties, production, composition and health benefits of vinegars: a review. food. chem. 221:1621–1630. https://doi. org/10.1016/j.foodchem.2016.10.128 jackson, r.s. 2008. biochemistry of alcoholic fermentation. in: wine science, principles and applications. academic press, san diego, usa, pp. 358–363. johnston, c.s. and gaas, c.a. 2006. vinegar: medicinal uses and antiglycemic effect. med. gen. med. 8(2):61. joshi, v.k., yadav, j., sharma, r., joshi, d. and gupta, r.k. 2016. effect of nutrients and growth stimulators on acetic acid fermentation using natural consortia. int. j. food. ferment. technol. 6: 81–95. https://doi.org/10.5958/2277-9396.2016.00030.1 kabasakalis, v., siopidou, d. and moshatou, e. 2000. ascorbic acid content of commercial fruit juices and its rate of loss upon storage. food. chem. 70(3):325–328. https://doi.org/10.1016/ s0308-8146(00)00093-5 kang, m., ha, j. and lee, y. 2020. physiochemical properties, antioxidant activities and sensory characteristics of commercial grape vinegars during long term storage. food. sci. technol. 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https://doi.org/10.3390/molecules23112949� https://doi.org/10.1021/jf302290k� https://doi.org/10.1021/jf302290k� https://doi.org/10.1080/07388550802046749� https://doi.org/10.1002/0470010398� https://doi.org/10.1002/0470010398� https://doi.org/10.1080/10715760290006411� https://doi.org/10.1080/10715760290006411� https://doi.org/10.1021/jf020458f� https://doi.org/10.1021/jf020458f� https://doi.org/10.17113/ftb.54.01.16.4082� https://doi.org/10.1002/jsfa.6332� https://doi.org/10.3390/beverages6030043� https://doi.org/10.3390/beverages6030043� https://doi.org/10.12982/cmujns.2021.034� _goback _hlk69118852 _hlk511075901 142 issn 1120-1770 online, doi 10.15586/ijfs.v33i2.2055 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (2): 142–155 p u b l i c a t i o n s codon comparison of some functional properties and protein profiles of different protein sources with egg components mine köstekli büyükcan, sibel karakaya* department of food engineering, faculty of engineering, ege university, bornova 35100, i̇zmir, turkey *corresponding author: sibel karakaya, department of food engineering, faculty of engineering, ege university, bornova, 35100 i̇zmir, turkey. e-mail: sibel.karakaya@ege.edu.tr received: 17 april 2021; accepted: 7 july 2021; published: 31 july 2021 © 2021 codon publications open access paper abstract emulsifying and foaming properties of plant and animal-sourced proteins; wheat protein hydrolysates (wp1, wp2, and wp3), potato protein isolates (pp1, pp2), pea proteins isolates (pep1, pep2), whey protein concentrate (wpc), and buttermilk powder (bmp) were compared with the egg white powder (ewp) and egg yolk powder (eyp). foaming capacity, stability, emulsion activity, stability, heat stability, morphology, and electrophoretic protein profiles were determined. the proteins representing competitive emulsifying functions were pep1, wpc, and bmp. heat treatment for 30 min at 80°c remarkably reduced the emulsion activity (ea) of eyp. our findings demonstrated that patatin-rich potato protein (pp1), an allergen-free and vegan option, has great potential to replace the foaming function of the egg white. the relationship between the protein profiles of the samples and their functional properties was further discussed in detail. keywords: dairy protein; egg replacement; emulsifying; foaming; plant protein; protein profile introduction proteins have critical functional properties for the food industry, such as emulsification, thickening, gelation, foaming, and water binding. although dietary proteins are available in both plant and animal sources, in recent years, there has been increasing attention toward utilizing novel plant proteins to support human nutrition at a low cost (ogunwolu et al., 2009; shah et al., 2019). in addition, maintaining sustainability by balancing food production against increasing food demand and nutritional requirements is one of the most important topics, especially in developing countries (butt and batool, 2010). for this purpose, several studies have focused on replacements of the ingredients in formulated foods. the egg is one of the major constituents in the food industry, especially in the bakery. it brings about various techno-functional properties such as foaming, emulsifying, binding, leavening, tenderization, volume, texture, stabilization, coagulation, flavor, color, and nutritional value to the food products (abdul hussain and al-oulabi, 2009; geera et al., 2011). however, consumers’ needs and expectations have led the food industry to focus on egg alternatives. those needs and expectations include ethical values, allergen-free/-reduced food products, affordable prices, extended shelf life, no refrigeration requirement, and lesser microbiological concerns (kohrs et al., 2010). furthermore, fluctuating egg prices, consumer concerns about clean labeling, animal welfare, environment, and sustainability reveal a great need to investigate an alternative ingredient to replace egg used in food formulations (abdul hussain and al-oulabi, 2009; jarpa-parra, 2018; lin et al., 2017). physicochemical interactions between protein and other food system components such as oil, water, air, and other proteins contribute to the functional properties of the proteins. those functions are related to several intrinsic and extrinsic factors, namely, shape, size, amino acid content mailto:sibel.karakaya@ege.edu.tr italian journal of food science, 2021; 33 (2) 143 comparison of some functional properties and protein profiles therefore, as performed in this present study, examining different samples regarding their techno-functional properties by comparing egg white powder (ewp) and egg yolk powder (eyp) with a standard method in the same conditions becomes crucial. in this study, the investigation of plant and animal sourced proteins’ main functions was aimed at. besides, another aim was to compare them with the egg and to present the relationship among the function and protein profiles individually. materials and methods materials dr oetker gıda sanayi tic. aş., turkey, kindly provided the commercial samples (wp1, wp2, wp3, pp1, pp2, pep1, pep2, whey protein concentrate [wpc], buttermilk powder [bmp], ewp, and eyp) and whole egg powder (wep). the sunflower oil and the fresh eggs were supplied from a local market. all chemical reagents were analytical grade and supplied from merck kgaa. distilled water was used in all analyses, except for the sds-page analysis in which ultrapure water was used. technical data provided by individual producers on a wet basis (wb) are given in table 1. determination of protein and moisture content according to the instrument’s instruction, moisture content was determined with a halogen moisture analyzer (mettler toledo, he73) in triplicates. protein content was and sequence, distribution of net charges, hydrophobicity and hydrophilicity ratio, structure, solubility; interaction with other substances represent the former while ph, temperature, moisture, additives, enzymes, salt concentration, ionic strength, and extraction method is included in the latter (butt and batool, 2010; ivanova et  al., 2014; shah et al., 2019). moreover, protein concentration, oil phase volume, and processing conditions have a crucial effect on functional properties (barać et al., 2015). the replacement of an ingredient can be achieved by identifying the target ingredient’s functions in food matrices. some egg replacers composed of whey protein isolates, soy compounds, wheat gluten, and several thickening agents are commercially available. they used to claim a 100% egg replacement. however, due to the unique structure of the egg and its function, such claims remain unrealistic (geera et al., 2011). there is an increasing demand for value-added food products with low-cost protein sources (chandi and sogi, 2006). attention to sustainable and cost-optimized egg alternatives for industrial application is increasing. current literature provides valuable data for the functions of the various proteins. however, applied methods and conditions (concentration, ph, equipment, oil phase, oil type) are diverse in the current literature. therefore, the determination of different protein sources’ functional properties should contribute to challenging egg replacement studies. unfortunately, different methodologies lead to limitations in comparing the results for industrial purposes. the opposite results for the same protein sample can be explained by the sample purity, isolation methods, parameters used for analyses, genotypes of the protein source, and environmental conditions (barać et al., 2015). table 1. technical data of the samples. sample maximum % general description protein moisture wp1 79 4.5 enzymatic partly hydrolyzed wheat protein wp2 79 4.5 enzymatic partly hydrolyzed wheat protein wp3 85 7 wet extracted and enzymatically hydrolyzed wheat protein pp1 90.5 9 potato protein isolate pp2 93.2 9 potato protein isolate pep1 79 7 pea protein isolate from yellow pea (pisum sativum) pep2 81.7 5 pea protein isolate wpc 79 5 whey protein concentrate bmp 36 5 sweet buttermilk powder, evaporated and spray dried eyp 30 5 egg yolk powder, pasteurized and spray dried ewp 82 8 egg albumen powder, pasteurized and spray dried from partially de-lysozymed liquid egg white bmp: buttermilk powder, ewp: egg white powder, eyp: egg yolk powder, wpc: whey protein concentrate. 144 italian journal of food science, 2021; 33 (2) büyükcan mk and karakaya s determination of emulsifying properties the emulsion activity was determined as described by ivanova et al. (2014) with slight modifications. protein solutions (2g/100 ml) were prepared in distilled water and were homogenized (heidolph, diax 900, 900w) for 30 seconds (sec) at 8000 rpm. sunflower oil (100 ml) was colored with sudan blue ii (0.15%) and added to the protein solution. homogenization for 30 sec was applied. immediately after homogenization, the emulsion was centrifuged (hettich zentrifugen rotofix 32 a) at 1200  rpm for 5 min. emulsion activity (%) was determined using the below formula. height of emulsified layer (cm) ea (%) 100 height of total liquid (cm) = × (3) the method proposed by shen and tang (2012) with slight modifications was used to determine es (%). in brief, oil in water emulsion (40:60 v/v) was used. protein solution (3%) was prepared in distilled water, and 120 g protein solution was combined with 80 g sunflower oil colored with sudan blue ii. the mixture was homogenized (heidolph, diax 900, 900w) for 2 min at 11,600 rpm. then, the emulsion was transferred into the tubes (50 ml) without taking up the foam phase. the volume of the emulsion was recorded after 1 h and 24 h. emulsion stability (%) was calculated using the below formula. 0 1;24 0 es (%) (emulsion volume t liquid volume separated t ) 100 emulsion volume t − = × (4) where emulsion volume t0: the volume of the emulsion at the beginning (50 ml) liquid volume separated t1: the volume of the emulsion after 1 h; t24: the volume of the emulsion after 24 h. emulsion heat stability (ehs) was determined by the method proposed by ivanova et al. (2014). samples were prepared as described in the ea method (2.5.1). before centrifugation, emulsions were heated in a water bath at 80°c, for 30 min. then, the tubes were cooled to room temperature under tap water. then the tube’s content was centrifuged at 1200 rpm for 5 min. the emulsion heat stability (%) also represented ea determined after heat treatment and was calculated as follows: ehs 80 c (%) height of remaining emulsified layer (cm) 100 height of total liquid cm) ° ( = × (5) determined by dumas combustion method with fp 528 nitrogen/protein determinator (leco). samples were weighed individually to tin foil cups and analyzed in parallel. conversion factors were 6.38 for bmp, 6.15 for wpc, 5.7 for wp1, wp2, wp3, and 6.25 for the rest of the samples. determination of protein profiles the samples’ protein profiles were determined with sdspage electrophoresis (laemmli, 1970) with bio-rad miniprotean electrophoresis system and mini protean® tgxtm 12% separation gel. samples were first diluted with ultrapure water to a final protein concentration of 3 mg/ml and then with a sample buffer (1:2 v/v). diluted samples were incubated at 95°c for 5 min at 200 rpm in an eppendorf thermomixer c. the gel was run at 50 v and was stained. the gel was washed with the destaining solution after staining to remove excess dye. the protein bands’ molecular weight (mw) was calculated with imagej (1.52a, national institutes of health, usa). determination of foaming properties the foaming capacity was determined with the method described by ivanova et al. (2014) with slight modifications. in brief, 100 ml protein solution (3%, w/v) was prepared in distilled water by stirring continuously with a magnetic stirrer (2mag, magnetic emotion) and poured in a 250 ml measuring cylinder. for foam formation, a foaming apparatus (matestar, mat-101w) was located at the center of the measuring cylinder above a constant height and operated for 3 min. the volume after foaming (v1) was directly recorded at once. foaming capacity (%) was calculated using the below formula. 1 0 0 v v fc (%) 100 v − = × (1) the foam stability was defined as the foam’s ratio that remained after a certain period to the initial foam volume at room temperature. total volume after 1 hour (h) (v2:1) and after 24 h (v2:24) at room temperature was recorded. foam stability (%) was calculated using the below formula: 2 0 1 0 v v fs (%) 100 v v − = × − (2) where v0 = total volume before whipping, v1 = total volume after whipping, v2:1 = total volume after 1 h, v2:1 = total volume after 24 h, italian journal of food science, 2021; 33 (2) 145 comparison of some functional properties and protein profiles protein profiles protein profiles of the samples were represented in figures 1 and 2. mw of protein bands obtained for wpc (lane 2) were compared with the existing literature (jiménez et al., 2012) and referred to as lactoferrin (lf, 80  kda), bovine serum albumin (bsa, 66 kda), βlactoglobulin (β-lg, 18 kda), and α-lactalbumin (α-la, 14 kda). in terms of functional properties, major wpc proteins, namely, β-lg and α-la, were described as the most basic fractions to use in food applications (svanborg et  al., 2015). β-lactoglobulin and α-la are both small globular proteins, and β-lg contains two disulfide bridges and a free thiol group while α-la contains four disulfide bridges (soltani et al., 2017). the sulfur content and surface hydrophobicity of main whey proteins are essential for their functional properties (abd el-salam et al., 2009). the bands with high mw obtained for bmp (figure 1, lane 3) represented the milk fat globule membrane proteins (mfgmp), namely, xanthine oxidase (xdh, 145 kda) and cd36 (74 kda). the β-lg band was observed in the protein profile of bmp. the main protein fraction of bmp was casein (24–34 kda). the protein profile of bmp in this present research was in accordance with the existing literature (que phan et al., 2014; sodini et al., 2006). pea protein isolates from two different producers exhibited identical protein profiles (lanes  4–5). the bands having mw between 93 kda–19 kda seen in pep1 and pep2 were mainly composed of legumin and vicilin fractions. these fractions were lipoxygenase (lox, 93 kda), major vicilin (v) fractions (76  kda and 50 kda), legumin acidic subunit (lα, 41  kda), and legumin basic subunit (lβ, 25 kda). the bands having mws below 35 kda could be v fractions and disassociated minor v fractions. similar protein profiles for pea protein were reported in the literature (shah et  al., 2019; shand et  al., 2007). sds-page analysis of microscopic observations of the emulsions the microstructure was imaged with a microscope (olympus ch20bimf200, olympus optical co., ltd., japan) according to kawano et al. (2015). the emulsion preparation procedure was the same as applied in ea determination. the emulsion droplet (6 µl) was placed on a concave glass slide immediately after emulsion preparation. the droplet was diluted with sunflower oil (60 µl) colored with sudan blue ii dye to improve the image quality. the concave glass provided the integrity of the emulsion’s natural structure. the software image j (1.52a, national institutes of health, usa) was used to perform the emulsion microstructure evaluation and diameter measurement after proper scaling settings. the emulsion droplet diameter was determined by the average of 50 different droplets from each image as triplicates. statistical analyses all analyses were done in triplicates and parallels except protein content and sds-page. protein analyses were carried out in duplicate. experimental data were analyzed using anova (spss for windows version 24.0, spss inc., chicago, il, usa), and comparisons among the means were performed using tukey’s test. differences between the means were considered significant at p < 0.05. the relationship between the protein sources and the functional properties was analyzed using the cluster analysis and principal component analysis (pca) (trial version of xlstat 2020, addinsoft). result and discussion moisture and protein content the protein sources used in this study were hydrolyzed proteins (wp1, wp2, wp3), protein isolates (pp1, pp2, pep1, pep2), protein concentrate (wpc), and spray-dried powders (bmp, eyp, ewp). the highest protein content was found in pp2, 91.70% (p < 0.05), which was followed by pp1 (89.52%) (table 2). eyp exhibited the lowest protein content (32.47%). in general, protein and moisture content agreed with the technical product specification values. the moisture content of wp1, wp2, and pep2 was slightly higher than the values reported in the technical product specification. the moisture absorption during storage can be the reason for this. total protein content, protein solubility, and moisture content of the proteins have critical importance on the functional properties by affecting the amount of protein adsorbed at the interface. table 2. protein and moisture contents of samples. sample protein (%) moisture (%) pp1 89.52 ± 0.43b 7.37 ± 0.30c pp2 91.70 ± 0.13a 8.17 ± 0.22b wp1 76.09 ± 0.12f 7.28 ± 0.50c wp2 76.79 ± 0.06e,f 6.96 ± 0.17c wp3 77.38 ± 0.25e 5.49 ± 0.07d pep1 78.69 ± 0.04d 8.04 ± 0.23b pep2 80.39 ± 0.18c 8.76 ± 0.24a wpc 81.38 ± 0.19c 5.74 ± 0.01d bmp 36.27 ± 0.00g 3.93 ± 0.21e values were given as mean ± standard deviation (n = 6). a–h different letters in the same column represent statistical significance (p < 0.05) bmp: buttermilk powder, wpc: whey protein concentrate. 146 italian journal of food science, 2021; 33 (2) büyükcan mk and karakaya s 1 2 3 4 5 6 7 8 9 10 mw (kda) 250 150 100 75 50 37 25 15 20 10 a-la la lb b-lg casein cd36 xdh bsa lf lox v v v v pi patt patt agg figure 1. sds–page profile of proteins from different sources. lane 1. standard mw (kda); lane 2. wpc; lane 3. bmp; lane 4. pep1; lane 5. pep2; lane 6. pp1; lane 7. pp2; lane 8. wp1; lane 9. wp2; lane 10. wp3. lf: lactoferrin, bsa: bovine serum albumin, β-lg: β-lactoglobulin, α-la: α-lactalbumin, xdh: xanthine oxidase, cd36, lox: lipoxygenase, v: vicilin, lα: legumin acidic subunit, lβ: legumin basic subunit; patt: patatin; patt agg: patatin aggregates, pi: protease inhibitors, wpc: whey protein concentrate, bmp: buttermilk powder. 1 2 3 4 5 6mw (kda) 250 150 100 75 50 37 25 15 20 10 apo ldl apo ldl otf apo ldl & ly ly ly ova g-livetin & g-livetin & apo ldl b-livetin apo ldl apo hdl apo hdl apo hdl apo ldl apo ldl phosvitin figure 2. sds–page gel of egg components. lane 1. mw standard in kda; lane 2. wep (whole egg powder); lane 3. few (fresh egg white); lane 4. ewp (egg white powder); lane 5. eyp (egg yolk powder); lane 6. fey (fresh egg yolk). otf: ovotransferrin, ova: ovalbumin, ly: lysozyme, apo ldl: low-density lipoprotein apoprotein, apo hdl: high-density lipoprotein apoprotein. pp1 (figure 1, lane 6) showed that the predominant protein fraction was patatin (patt) (39–43 kda). the bands having mws above 75 kda could be patatin aggregates (patt agg), which were easily unfolded even at low temperatures (approximately 60°c) as reported in the literature (schmidt et al., 2018). formation of monomer, dimer, and trimetric aggregates of patatin with mw of 43, 82, and 108 kda after heat treatment was reported (pots et al., 1999). these aggregates could have occurred (1)  due to spray drying used during the production and (2) due to the heating step (95°c, 5 min) during sdspage analysis. the other potato protein (pp2) contained the protein fractions with mw lower than 25 kda (figure  1, lane  7). the bands having mws of 4.3 italian journal of food science, 2021; 33 (2) 147 comparison of some functional properties and protein profiles kda-20.6 kda were identified as protease inhibitors (pi). the weak intensity bands obtained for the sds-page profiles of the wheat proteins (figure 1, lane 8–9–10) and the intensification of the bands below 50 kda for wp1 and wp3, below 25 kda for wp2 were consistent with the structure of the wheat proteins showing that they were hydrolyzed enzymatically (table 1). in a previous study, wang et al. (2006) fractionated proteolytic hydrolysis of wheat gluten and found similar bands in gel electrophoresis. these bands could be occurred due to the enzymatic hydrolysis of the wheat proteins as reported for hydrolyzed wheat, rice (yang et al., 2018), and rapeseed proteins (tessier et al., 2005). figure 2 represents the protein profiles of egg components. wep (lane 2), as a widely used ingredient in the food industry, was included to compare its protein profile with ewp and eyp fractions separately. the protein bands observed for wep were low-density lipoprotein  apoprotein (apo-ldl, 200 kda), low-density lipoprotein apoprotein (apo-ldl, 116 kda), ovotransferrin (otf, 75 kda), ovalbumin (ova, 35–40 kda), high-density lipoprotein apoprotein (apo-hdl, 30 kda), and lysozyme (ly, 14 kda). ovotransferrin, ova, and ly are the egg white proteins, while the others belong to egg yolk (netting et al., 2015). similar protein profiles of fresh and powder forms of ew (figure 2) indicated that spray drying did not influence its protein profile. egg white protein profiles were in accordance with the literature (opazo–navarrete et al., 2018). however, fresh egg yolk (fey) and eyp exhibited different protein profiles. the protein profile of eyp did not include the two protein bands, while the sds-page profile of fey contained 59 kda, phosvitin, and 47 kda, apo hdl (figure 2, lane 5). the missing bands in the spray-dried eyp protein profile were from the ey granule fraction. the granule fraction accounts for only 19%–25% of the ey based on dry matter (denmat et al., 2000). as expected, wep did not contain some of the eyp’s fractions (62 kda, 59 kda, 47 kda, 36 kda, and 31 kda) (lane 2). however, plasma proteins of egg yolk were visible in the protein profile of wep. foaming properties the foam was defined as a two-phase system that consists of air cells separated by a thin continuous liquid layer named a lamellar phase. foam systems are relatively unstable. there is a need for stabilizer surfactant molecules to orient at the air/water interface (makri et  al., 2005). during foam formation, protein molecules tend to unfold their hydrophobic side chain in the air and remaining hydrophilic chains in the water phase. the hydrophilic chain portion of protein holds the water in the aqueous phase. thus, the foam system is prevented from water drainage, coalesces of air bubbles, table 3. foaming properties of the samples. protein source samples fc (%) fs (%) wheat wp1 108.47 ± 1.92c 85.10 ± 2.04b wp2 128.84 ± 3.08b 16.61 ± 2.95e wp3 106.35 ± 2.46c 26.88 ± 0.61d pea pep1 64.29 ± 2.61e 85.16 ± 0.60b pep2 81.75 ± 4.68d 81.50 ± 2.64b potato pp1 129.36 ± 3.58b 96.32 ± 0.10a pp2 126.98 ± 2.46b 7.5 ± 0.15f milk wpc 89.68 ± 1.94d 25.68 ± 2.34d bmp 39.68 ± 2.46f 0.00 ± 0.00g egg eyp 19.05 ± 4.26g 67.22 ± 6.72c ewp 174.76 ± 17.39a 91.67 ± 4.44a values were given as mean ± standard deviation (n = 6). (a–g) different letters in the same column represent significant differences (p < 0.05). bmp: buttermilk powder, ewp: egg white powder, eyp: egg yolk powder, wpc: whey protein concentrate. and obviously, destabilization (rybak, 2013). table 3 represents the foaming properties of the samples. as expected, ewp showed the highest fc (174.76%) due to its ovalbumin content, which is a well-known foaming agent (p < 0.05). it was followed by pp2, pp1, wp2, wp1, wp3, wpc, pep2, pep1, bmp, and eyp (table 3). potato and wheat proteins exhibited the highest fc among the plant proteins. however, pea proteins displayed weak fc. the foaming properties are closely related to the protein’s quickly unfolding structure and its adsorption rate in the interface (alleoni, 2006). furthermore, as applied in wheat protein hydrolysates, hydrolytic treatments to native proteins result in reduced mw, facilitated diffusion, and adsorption at the interface (wouters et al., 2016). the mentioned effects of hydrolytic treatment on the protein profile can be linked to the improved fc of wheat protein hydrolysates (wp1, wp2, and wp3) due to the low mw proteins they contain, as shown in the sds-page profile (figure 1). wheat gluten hydrolysates provided lower mw subunits with higher solubility, and better fc was reported elsewhere (wang et al., 2006). however, different mechanisms can affect fs, such as the relationship between the decrease in mw and lower fs, and hydrophobic interactions and higher fs (wouters et al., 2016). the foaming stability of wheat protein hydrolysates, wp2 and wp3, were lower when compared to the proteins with high fs, such as pea proteins, pp1, and ewp. the loss in protein structure by extensive hydrolysis can lead to impaired film formation (wouters et al., 2016), which is attributed to the obtained lower fs values for wheat protein hydrolysates. 148 italian journal of food science, 2021; 33 (2) büyükcan mk and karakaya s between air and liquid phases, which leads to an increase in viscosity and elasticity of the liquid phase (pęksa et al., 2008). as we detected in this study, the foam volume obtained with protein isolates tends to decrease as a function of time (butt and batool, 2010). the lower fc values can be due to inadequate electrostatic repulsions, lesser solubility, and excessive protein-protein interactions. emulsion properties the stability of an emulsion is related to its resistance to creaming. the emulsion stability is crucial, especially during the shelflife of emulsified food products such as mayonnaise, ready-to-bake cake batter, and sauces. table  4 represents the ea, es, and ehs (%) of samples. there was no difference between the ea of eyp and pep1 (p > 0.05). also, the es of eyp and pep1 was similar. these results showed that pep1 could be a replacer for the ea of eyp effectively at the same concentration and conditions. in contrast to our findings, barac et al. (2010) reported low emulsion activity index for commercial pea protein isolate at ph 7.0 and ph 8.0 compared to in-house isolated proteins. the researchers followed the spectrophotometric method for emulsion functions with lower protein concentration (1 g/kg) and different protein solution: oil ratio (1:3) than our study. the procedure, sample concentration, equipment used, and homogenization duration can be the reason for diverse results. among the protein sources, the wheat protein (wp1) displayed the lowest ea (p < 0.05). animal protein sources, wpc and bmp, showed relatively high ea, and their es values after 1 h were higher when compared to wheat protein wp2, pea protein pep2, and potato protein pp1. the emulsion stability of all proteins decreased after 24 h. the composition and processing conditions of emulsions affect the emulsion stability (hu et al., 2017). eyp formed the most stable emulsions eyp (68.08%, es), and wpc (58.58%), pep1 (57.75%), and bmp (57.50%) followed it, respectively. among the protein sources, es of wp1 was the lowest (p < 0.05). however, the es of wheat proteins at the end of 24 h was not statistically different from each other (p > 0.05). therefore, wheat proteins cannot be an alternative for mimicking ea and es of egg proteins. various heat treatments are applied during food production. therefore, the effect of heat treatment on emulsifying properties was also in our interest. researchers reported the impairing effect of heat treatment on proteins’ emulsifying characteristics due to irreversible protein denaturation (dissanayake & vasiljevic, 2009). however, the partial unfolding of protein may increase proteins’ surface hydrophobicity, leading to enhanced potential protein adsorption at the interface with improved emulsifying properties (dissanayake & although pea proteins (pep1 and pep2) have similar protein profiles, the fc of pep2 was significantly higher than that of pep1 (p < 0.05). however, the fs of pea proteins was not different (p > 0.05). the varying agricultural lines of field pea proteins can explain the significantly different fc results (shevkani et al., 2015). barac et al. (2010) reported different foaming capacity and low foam stability independently from ph for six different pea genotypes. the previous study reported that pea protein isolate demonstrated better fc than a broad bean and lupine protein isolates (makri et al., 2005). in the present study, pea proteins displayed lower fc than other plantbased proteins (table 3). the foaming stability is as crucial as fc in a food system. potato protein isolates displayed high and similar fc, but their fs was different (p < 0.05). protein profiles were the main difference between potato protein isolates (figure  1). higher fs of pp1 could be related to the high mw protein fraction, patt. a similar relationship between the high stability of foams and the globular proteins with high mw was reported elsewhere (pęksa et al., 2008). the criteria for better foaming were reported as (1) rapid adsorption at the air/water interface during bubbling, (2) ability for a conformational change, rearrangement at the interface, and (3) ability to form a cohesive viscoelastic film via intermolecular interactions (makri et al., 2005). furthermore, a higher charge on the proteins has a weakening effect on the hydrophobic reaction, which leads to increased solubility and flexibility. proteins with higher solubility and flexibility can move quickly to the air-water interface (shevkani et al., 2015) and support improved foaming. therefore, better foaming abilities obtained for pp1 may be related to those attributes mentioned above. the foaming capacity of wpc (89.68%) was significantly higher than the fc of bmp (39.68%) (p < 0.05). however, both milk proteins did not form stable foams. a previous study reported comparable fc of both spray-dried wpc obtained from acid and sweet whey with the fc of ew (slack et al., 1986). the well-described factors that affect foaming properties are the existence of lipids, ash, protease peptone, degree of denaturation (el-shibiny et al., 2007), denaturation of β-lg, the origin of α-la (sweet or acid whey), and drying methods of wpc (spray drying or freeze-drying) (slack et al., 1986). in our study, the fat content of bmp could be the reason for unstable foam formed by bmp. a study that reported similar results linked lower fc of bmp to the competition between the fat and protein in buttermilk at the interface (sodini et al., 2006). particularly, foam-forming proteins can create a firm film around the air bubble by lowering the interfacial tension italian journal of food science, 2021; 33 (2) 149 comparison of some functional properties and protein profiles table 4. emulsifying properties of the protein sources. protein source proteins ea (%) es (%) ehs (%) 80°c–30 min 1 h 24 h wheat wp1 11.60 ± 0.99d 79.33 ± 2.80c,d 50.50 ± 1.22e 3.93 ± 0.03g wp2 40.87 ± 1.77c 68.50 ± 2.00e 57.00 ± 3.52b,c,d,e 7.45 ± 0.65f wp3 44.08 ± 0.75b 78.73 ± 2.98c,d 51.70 ± 3.39c.d,e 7.19 ± 0.73f pea pep1 47.15 ± 0.80a 92.67 ± 1.63a 57.75 ± 2.04b,c 44.08 ± 0.74a,b pep2 43.48 ± 0.59b 74.33 ± 2.34d,e 50.83 ± 5.95d,e 40.71 ± 1.19c potato pp1 44.17 ± 0.55b 73.50 ± 4.85d,e 55.33 ± 6.41b,c,d,e 45.30 ± 1.35a pp2 41.24 ± 0.62c 78.67 ± 1.97c,d 56.33 ± 1.63b,c,d,e 35.26 ± 1.21d milk wpc 44.03 ± 1.42b 84.17 ± 4.92b,c 58.58 ± 1.24b 43.05 ± 1.76b bmp 43.57 ± 1.15b 82.67 ± 4.32c 54.70 ± 2.19b,c,d 42.27 ± 1.15b,c egg eyp 49.11 ± 1.18a 90.00 ± 1.41a,b 68.08 ± 3.26a 14.03 ± 0.90e values were given as mean ± standard deviation (n = 6). (a–f) different letters in the same column represent significant differences (p < 0.05). bmp: buttermilk powder, eyp: egg yolk powder, wpc: whey protein concentrate. vasiljevic, 2009; li et al., 2011; peng et al., 2016). our findings indicate that the most heat-resistant emulsions were obtained from pp1 and pep1 (table 4). besides, emulsions prepared with wpc and bmp resisted the heat treatment. the decrease in ea (%) after heat treatment was 2.23% and 2.98% for wpc and bmp, respectively. moreover, whey protein is the frequently used component in food formulations to stabilize foams or emulsions via forming films and producing stable gels (konrad et al., 2005). the improved emulsion stability of heat-treated whey protein was linked to the generation of stable nanoparticles and/or aggregates, which can stabilize the emulsion by increasing the continuous phase’s viscosity and participating in layer formation on the oil droplets (dybowska, 2011). surface hydrophobicity influences the emulsion properties of protein and peptides, and β-lg exhibited higher surface hydrophobicity than α-la, indicating that β-lg is the main contributor to whey protein’s functional properties (schröder et al., 2017). however, a previous study showed that the emulsion stabilities of whey protein (spray dried) and its major fraction β-lg’s (freeze dried) were different. for example, spray drying can lead to partial denaturation in wpc proteins and the formation of strong intramolecular bonds. it was found that wpc mimics composed of freeze-dried proteins (57% β-lg, %37 α-la, %6 bsa) exhibited similar emulsion properties as freeze-dried β-lg (tcholakova et al., 2006). therefore, it is evident that the protein fraction and the production method influence the functional properties. corredig and dalgleish (1998) reported that among the protein fractions, the adsorbed casein ratio was 50% at the interface of the oil in water emulsion prepared with bmp. in addition, caseins and whey proteins covered the fat globule surface area, but caseins reduced the surface tension faster than whey proteins (cano-ruiz and richter, 1997). therefore, the good emulsifying activity obtained from this study can be linked to the casein fraction of bmp. similar to ea and es values, the decrease in heat resistance of wheat proteins’ emulsions were 66%, 81.77%, and 83.69% for wp1, wp2, and wp3, respectively (table 4). wang et al. (2006) reported a decrease in solubility with the increase in temperature above 50°c for the wheat protein hydrolysate fractions. the heat treatment applied in this study (80°c, 30 min) can explain the reason for lower ehs values for wheat protein hydrolysates. the heat resistance of the emulsion prepared with eyp was low as the results were obtained from wheat protein emulsions. the low ehs of the emulsion of eyp may be due to the heat-induced denaturation of apo-ldl and lipovitellins (above 65°c), egg yolk proteins, and the tendency of denatured eyp to form gel network as reported in the literature (campell et al., 2005). besides, heat treatment might cause the disruption of the adsorbed hydrolyzed wheat protein and eyp at the interface. the plasma proteins of the egg yolk are the major contributors to the emulsion formation, especially a lipoprotein apo-ldl (17 kda) (denmat et al., 2000). in our study, the protein bands mentioned above were available for the ey protein profile (figure 2). in contrast, a slight increase was detected for ehs of the emulsion of pp1 attributable to the improved interfacial properties due to enhanced protein-oil reaction by heat-induced changes and exposure to hydrophobic groups (chao and aluko, 2018). similar to our results, the positive effect of heat treatment (80°c, 30 min) on the patatin fraction’s emulsion resistance was linked to the formation of aggregates or gels and enhancement of the interfacial layer resistance (ralet and guéguen, 2000). 150 italian journal of food science, 2021; 33 (2) büyükcan mk and karakaya s protein isolate at varying concentrations (10–15 mg/ml) at ph 5.0 and 7.0 (aluko et al., 2009). our results also indicated that pep1 is a good plant-based candidate for replacing the emulsifying properties of eyp (p > 0.05). furthermore, the ea of pep1 was not influenced by the heat treatment (table 4), made it an alternative to replace eyp. emulsion morphology figure 3 indicates that samples’ emulsion morphology exhibited better emulsifying properties (eyp, pep1, wpc). in general, eyp formed independent and larger emulsion droplets with an average diameter of 170.04 µm, while the emulsion droplets formed by wpc and pep1 were closely distributed, intense, intact, and smaller in size. table 5 lists the average droplet diameters of the emulsions. the average diameters of the droplet size of the emulsions of wpc and pep1 were 56.11 and 44.15 µm, respectively. these values were approximately 3 to 4 times lower than the droplet size of the eyp emulsion (p  < 0.05). homogenously distributed, relatively small-sized emulsion droplets represent the nonflocculated situation positively affecting the es. the resistance although protein profiles of pep1 and pep2 were identical, their foaming and emulsifying activities were different. the production process applied different surface hydrophobicity, solubility, and ability to adhere to the air–water and water–oil interfaces and their agricultural varieties (dissanayake & vasiljevic, 2009; shevkani et al., 2015; li et al., 2011; peng et al., 2016) can explain the difference. among the legume proteins, the pure vicilin fraction exhibited better functional properties than the pure legumin fraction due to different protein structures of 7 s and 11 s proteins (barać et al., 2015). therefore, the pea genotypes with a higher vicilin/legumin ratio can be more suitable for their functional properties. in contrast, barac et al. (2010) reported that the pea genotype with the lowest vicilin/legumin ratio showed the highest emulsion stability. the authors explained this with higher adsorption of legumin at the interface due to its higher surface hydrophobicity than vicilin. therefore, legumin fraction may be responsible for the higher ea values obtained for pep1. in addition, the compact structure of the legumin fraction can resist thermal-unfolding, thus forming heat-stable emulsions as obtained for pep1 and pep2 (ladjal-ettoumi et al., 2016). a previous study reported that emulsifying capability of the pea protein isolate was significantly higher than the soy 250 µm 250 µm 250 µm 250 µm 250 µm 250 µm 250 µm 250 µm 250 µm (a) (b) (c) figure 3. digital photographs of emulsions under light microscope (10×) (a) eyp (b) pep1 (c) wpc. italian journal of food science, 2021; 33 (2) 151 comparison of some functional properties and protein profiles table 5. average emulsion droplet diameter (µm). morphology evaluation eyp (a) pep1 (b) wpc (c) 1. 153.36 ± 39.88b,a 43.91 ± 14.89a,c 65.62 ± 19.92a,b 2. 184.18 ± 55.98a, a 45.56 ± 16.46a,b 48.47 ± 14.83b,b 3. 184.59 ± 51.38a,a 42.99 ± 16.31a,b 54.25 ± 21.46b,b average 174.04 ± 51.35a 44.15 ± 15.83c 56.12 ± 20.14b values were given as mean ± standard deviation (n = 150). 1, 2, 3 represent the number of repeats. (a–c) different letters in the same column represent significant differences (p < 0.05). (a–c) different letters in the same line represent significant differences (p < 0.05). eyp: egg yolk powder, wpc: whey protein concentrate. to creaming is related to factors such as smaller droplet size, the lower difference between the densities of continuous and dispersed phases, and the higher viscosity of the continuous phase (hu et al., 2017). the emulsion droplet size distribution of eyp was in a wide range (figure 4). however, the droplet size distribution of the emulsions of pep1 and wpc was apparently in a narrow range. in close agreement with the size distribution results, the emulsions of wpc and pep1 were relatively resistant to creaming than the emulsion of eyp. although the emulsion droplet average diameter of eyp was large and their distribution was in a wide range, the high emulsion stability of eyp (table 4) can be explained by the different conditions (sample concentration, duration of homogenization, energy input, and water:oil ratio) applied during emulsion formation in es analysis and the morphology evaluation. the continuous phase’s viscosity supports es by limiting the emulsion droplets’ mobility, leading to the prevention of creaming or coalescence (motta-romero et al., 2017). besides, forming smaller oil droplets in a continuous aqueous phase, which has sufficiently high viscosity to prevent oil droplets from coalescence, is crucial in increasing es (rybak, 2013). functionality per dry weight we evaluated the results as functionality (%) per gram of dry weight (dw) of the sample rather than protein amount because of the possible contribution of nonprotein components (phospholipids), namely, bmp, wpc, and eyp to the functional properties (figure 5). results showed that pp1, pep1, pep2, wpc, and bmp could replace egg fractions’ various functions. depending on the importance of the required function for a specific food product (mayonnaise, cake batter, sauces), the proteins mentioned above may be utilized alone or in combination. although bmp has relatively lower protein content, functionality (%) per dw was better in all aspects, except for the fs. bmp provided 87% of ea formed by eyp alone (figure  5). therefore, further isolation and purification of bmp fractions and phospholipids can have great potential for 60 40 20 f re q u e n cy e y p s a m p le s p e p 1 w p c 0 60 40 20 0 60 40 20 0 0.00 100.00 200.00 emulsion droplet diameter (µm) 300.00 400.00 figure 4. the distribution of the emulsion droplet size for eyp, pep1, and wpc. techno-functionality in the food industry. for instance, the good emulsifying capacity of mfgmp found in bmp was reported previously (abdul hussain and al-oulabi, 2009). our results based on the functionality (%)/g dw showed that bmp and wpc could replace ey’s emulsifying function. pea proteins (pep1 and pep2) supplied good functionalities for all functions when evaluated based on dw. however, pep1 had more significant potential in emulsion formation and stability than pep2. therefore, pep1 may be an option for egg replacement studies. regarding potato proteins’ functionality, the fc determined for pp1 was higher than pp2, most probably due to the patt and patt agg fractions. moreover, lower fs obtained for the pi fraction (pp2) brought the patt fraction (pp1) to the fore to be used as an allergen-free alternative to the foaming properties of ewp. similar to our findings, higher foam overrun was determined for patt fraction at ph 3.0 and ph 5.0 than pi (schmidt et al., 2018). however, similar fs for patt and pi fractions were reported in the same study. contrary results should be explained with the diversity of the methods 152 italian journal of food science, 2021; 33 (2) büyükcan mk and karakaya s also, following the functional properties (%), there was a close correlation between pep1 and eyp for es. also, the ea of bmp and wpc were closer. in the case of foaming properties, pp1 displayed the closest similarity with ewp. conclusion this study examined the different proteins from animal and plant sources for their foaming and emulsifying properties separately and their protein profiles. all samples’ protein profiles agreed with the existing literature, indicating the commercial samples’ original characteristics. although spray drying caused ey’s protein profile changes, it did not influence ew’s protein profile. two different branded pea protein isolates demonstrated different functional properties, although their protein profiles were identical. among the samples, ewp showed the highest fc and fs as expected. our results showed that plant proteins used in this study revealed higher foaming properties than animal proteins. the foaming capacity of potato protein isolates arose from the protein fractions patt and pi they contain, and the stability of foam arises from the patt fraction. our results demonstrated the great potential of pp1 providing the foaming function of ewp due to its higher fc and fs. therefore, pp1 should be considered a good candidate and as a nonallergenic and vegan option for foaming properties to replace ewp. the emulsifying properties of samples revealed the pep1 as the best eyp alternative (p < 0.05). however, wpc and bmp also displayed suitable emulsion activities. the stabilities of all emulsions decreased within time at room temperature. however, eyp exhibited the highest and samples’ purity. all wheat proteins exhibited high foaming capacities, but the ehs values were the lowest. therefore, wp1 may be an alternative for the food formulations requiring high volume and stable foam instead of ew. similar to our findings, wang et al. (2006) reported higher fc for enzymatically hydrolyzed wheat proteins due to increased surface activity by the polypeptides produced. these results showed that combining different protein sources might be a good strategy to replace eggs’ functions in different food matrices. cluster and principal component analysis cluster analysis and pca show the differences among samples and the relationship between variables in functional properties (ea, es, fc, and fs). factor analysis showed that four principal components explained the total variation. there were very strong correlations between es and f1 component (−0.899) and fc and f1 component (0.931). in fs, a strong relationship was found for the f2 component (0.780). a moderate relationship between ea and f2 component was also detected (−0.576). these two components explained 74.17% of the total variance. therefore, f1 and f2 were used for pca, and a biplot diagram was obtained. figure 6 shows the cluster and biplot diagrams of pca analysis. three main clusters explained the variability and similarity among the samples’ functional properties. one of them included eyp and pep1, and the other was composed of wpc and bmp in line with the results we found for emulsion functions. wheat protein, wp1, formed the second cluster alone. the third cluster involved ewp, pep2, pp1, pp2, wp2, and wp3. besides, pp1 with better foaming functions formed a segment with ewp in the third cluster. f1 is linked to es and fc, while f2 is linked to ea and fs. 60 70 40 20 30 50 10 0 pp1 pep1 pep2 wpc bmp eyp ewppp2 wp1 wp2 ea (%) es (%) ehs (%) fc (%) fs (%) wp3 functionlity per g sample (dw) figure 5. functionality per gram sample (dw). italian journal of food science, 2021; 33 (2) 153 comparison of some functional properties and protein profiles e y p p ep 1 w p c b m p w p 1 w p 2 w p 3 p p 2 e w p p ep 2 p p 1 0 2 4 6 8 10 12 14 16 18 d is si m ila rit y dendrogram (b)(a) ea es fc fs ewp eyp wp1 wp2 wp3 pep1 pep2 pp1 pp2 wpc bmp –2 –1.5 –1 –0.5 0 0.5 1 1.5 2 2.5 3 –3 –2 –1 0 1 2 3 f 2 (2 6, 63 % ) f1 (47,54 %) biplot (axes f1 and f2: 74,17 %) active variables active observations figure 6. cluster diagram (a) and pca biplot diagram (b). es after 24 h. the average emulsion droplet size of pep1 was the smallest among the samples, and wpc and eyp followed it (p < 0.05). the egg is the key ingredient responsible for foaming and emulsifying properties in various food applications such as cake batter preparation. providing similar functional and sensory properties with alternative plant and animal-based proteins should bring about a new perspective to product development studies. furthermore, obtained successful results should be confirmed by sensory evaluation. this research can be further supported in complex food matrices where interactions between sugar, flour, proteins, and fats occur. in conclusion, pp1 has a high potential to replace ewp’s foaming function, whereas pep1, wpc, and bmp should be considered as alternatives to eyp’s emulsifying function. in addition, pp1 and pep1 should be highlighted as allergen-free, vegan alternatives for the functions mentioned above. furthermore, samples evaluated in this study should be used alone or in combination to provide functional properties required for certain food products. acknowledgements karakaya and köstekli thank dr. oetker gıda sanayi tic. a.ş. for providing materials. this article includes data from some parts of the ph.d. thesis conducted by mine köstekli büyükcan. funding this research did not receive any specific grant from funding agencies in public, commercial, or not-for-profit sectors. conflict of interest the authors declare that they have no conflict of interest. büyükcan holds the research and development manager position at dr oetker gıda sanayi tic. a.ş., i̇zmir, turkey. availability of data and material (data transparency): available authors’ contributions mine köstekli büyükcan and sibel karakaya were involved with 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_hlk77675650 _hlk68556052 _goback _hlk48683844 _hlk68556337 _hlk27320816 _hlk12400894 _hlk75692871 _hlk75694596 _hlk77674720 _hlk77667524 _hlk77667552 _hlk69133888 _hlk69133476 _hlk69134985 ijfs#1877_bozza ital. j. food sci., vol. 32, 2020 912 paper influence of different pretreatments and chaptalization types on the physiological characteristics and antioxidant activity of apricot (prunus armeniaca l.) wine k.-t. choi1, s.-b. lee1, j.-s. choi1 and h.-d. park*1,2 1school of food science and biotechnology, kyungpook national university, 80 daehakro, daegu 41566, south korea 2institute of fermentation biotechnology, kyungpook national university, 80 daehakro, daegu 41566, south korea *corresponding author: hpark@knu.ac.kr abstract the effects of pretreatment (pectinase and caco3) and chaptalization (sugar and puree concentrate) on the quality of apricot wine were investigated. pectinase-treated apricot wines had increased amounts of total phenolics, flavonoid compounds, as well as antioxidant activities. the apricot wine chaptalized with puree concentrate and treated with pectinase (pcp) showed the highest total acidity and some organic acid contents, which resulted in the strongest sourness. in contrast, the apricot wine treated with pectinase and caco3 (scpc and pcpc) showed the lowest total acidity and least sourness. antioxidant activities of pcp and pcpc wines were higher than other wines, and other pectinase-treated wines were also higher than the control wine. volatile higher alcohols and terpenes increased in all the pectinase-treated wines, whereas volatile ester compounds were decreased. sensory evaluation showed that scpc, pcp, and pcpc wines obtained significantly high flavor scores, and scpc and pcpc wines obtained the highest overall preference scores. keywords antioxidant, apricot wine, aroma profile, fruit wine, pretreatment ital. j. food sci., vol. 32, 2020 913 1. introduction apricot (prunus armeniaca l.) is a stone fruit mainly grown in china, the mediterranean european countries, turkey, and the usa (soliman, 2013). consumption of apricot has shown human health benefits because of its antioxidant, anti-inflammatory, and immunestimulating properties, which might be attributed to the presence of various phytochemicals, such as carotenoids, polyphenols, vitamins, and fiber (dragovicuzelac et al., 2007; hegedűs et al., 2010; madrau et al., 2009). due to the various advantages of apricot, the development of apricot wine has good potential for commercialization. despite the excellent functionality, the strong sourness of apricot, associated with its notably high acidity, has still not been acceptable, which prevents the development of apricot wine. pretreatment of high-acid wines by deacidification offers a suitable resolution to this issue, and it is commonly carried out by physicochemical methods, such as carbonic amelioration, blending, chemical neutralization, and precipitation, and by biological methods, such as malolactic fermentation (loira et al., 2018; volschenk et al., 2006). among these methods, chemical neutralization by the addition of salts (caco3) to deacidify fruit wines is usually preferred because it reduces the risk of increasing the ph levels and, additionally, prevents microbial problems (cosme et al., 2018; mattick et al., 1980). pectinases are enzymes that are generally added to maximize juice yield and act by degrading the pectins that interfere with extraction and clarification of most fruit juices (sharma et al., 2017). in addition, treatment of fruit juice with pectinase has been reported to increase the amounts of phenolics and anthocyanins, facilitate filtration, and contribute to the release of the molecules responsible for aroma and color, two of the major components that characterize a wine (pardo et al., 1999; pinelo et al., 2006; watson et al., 1999). some fruits with low sugar content must be chaptalized to obtain sufficient sugar content for making wine (jarvis, 1996; miyawaki et al., 2016). several researchers have used various technologies, such as freeze-concentration and nanofiltration, to decrease the levels of available water in fruits deficient in sugar content, thereby concentrating the sugar content (banvolgyi et al., 2006; clary et al., 2006; miyawaki et al., 2016). puree concentrate can also be a suitable alternative instead of chaptalization because of its concentrated sugar content and using the apricot puree concentrate could reduce labors and enhance productivity by skipping the process of washing the fruit and removing the seed for the industrial mass production of apricot wine. this study aimed to improve the quality of apricot wine. apricot wines were prepared following different types of pretreatments, including pectinase and caco3, and chaptalization, by the addition of sugar and puree concentrate, and their physicochemical parameters, volatile aromatic profiles, antioxidant activities, and sensory characteristics were investigated. 2. materials and methods 2.1. chemicals and reagents 2,2-diphenyl-1-picrylhydrazyl (dpph), 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (abts), trolox, folin–ciocalteu reagent, methanol (hplc grade), potassium ital. j. food sci., vol. 32, 2020 914 metabisulfite (k2s2o5), organic acids, and all other standards were obtained from sigma– aldrich (st louis, mo, usa). white table sugar (cj co., seoul, south korea), used to adjust the sugar content of the must, was bought from the local market. apricot puree concentrate (30.7°bx, ph 4.1, and acidity 1.31%) was procured from aftun gida ltd. (yenisehir, mersin, turkey). rapidase® x-press l (pectinase+hemicellulase, 180,000 avjp/g) was purchased from dsm food specialties (delft, netherlands). the fermentation agent saccharomyces cerevisiae var. bayanus ec-1118 yeast was purchased from lallemand inc. (montreal, canada). caco3 was acquired from daejung co. (siheung, south korea). 2.2. apricot fruit samples fully-ripened apricot fruit (p. armeniaca l.) were bought from local farms in yeongcheon (gyeongsangbuk-do, south korea) during the 2017 harvest season. ”harcot” apricot fruit was selected for uniformity of size, color, and absence of decay or rot. fruit was stored at –18℃ until further use. 2.3. apricot fruit must preparation and pretreatment conditions apricot fruit was washed with tap water, the seeds removed manually. the deseeded fruit was blended using a household juicer (nj-9300a, nuc juicer, daegu, south korea) and then combined immediately with 0.02% (w/v) k2s2o5 to prevent bacterial contamination and oxidation. to determine the most suitable amount of enzyme and caco3 (for deacidification), a part of the apricot fruit pulp was divided into four portions of 300 ml each. the first portion was used as the control while the three remaining portions were treated with pectinase (rapidase® x-press l) at 0.05%, 0.1% and 0.2% (v/w), respectively. for the deacidification process, caco3 was added at 0.1%, 0.2%, and 0.3% (w/w), respectively. pectinase treatment or deacidification occurred for 2 h under constant agitation using a shaking incubator (30℃, 200 rpm). the pulp samples were centrifuged at 3,578 × g for 10 min, and the obtained juices were analyzed and compared for ph level, total acidity, total soluble solids, and reducing sugars. 2.4. apricot wine-making apricot fruit pulp was divided into five wine-making trial batches (5 kg), from which wines were prepared in triplicate and, subsequently, treated before fermentation. the chaptalization and pretreatment conditions are listed in table 1. in the first batch, namely, the control batch (sc), the apricot pulps were chaptalized with white sugar to obtain 22°bx. in the second batch (scp), the apricot pulps were chaptalized to 22°bx with white sugar and then treated with 0.1% (v/w) pectinase. in the third batch (scpc), the apricot pulps were chaptalized to 22°bx with white sugar, treated with 0.1% (v/w) pectinase, and then deacidified with 0.3% caco3. in the fourth batch (pcp), the apricot pulps were chaptalized to 22°bx with apricot puree concentrate and then treated with 0.1% (v/w) pectinase. in the fifth batch (pcpc), the apricot pulps were chaptalized to 22°bx with apricot puree concentrate, treated with 0.1% (v/w) pectinase, and then deacidified with 0.3% (w/w) caco3. each treatment process lasted for 2 h under constant agitation (30℃, 200 rpm), 200 mg/l of k2s2o5 was added to prevent bacterial contamination, and then the batches were centrifuged at 3,578 × g for 10 min. the apricot wine was fermented with 1–2 × 106 cfu ml−1 s. cerevisiae var. bayanus ec-1118 that was rehydrated by sterile distilled ital. j. food sci., vol. 32, 2020 915 water at 40℃ for 30 minutes, according to the manufacturer’s instruction. each sample was fermented without shaking at 20℃ for 7 days until complete fermentation. the final wine samples were filter-sterilized, poured into wine bottles with 50 mg/l of k2s2o5, and stored at 4℃ for further analysis and sensory assessment. table 1. list of ingredients used in apricot wine-making. ingredients (g) chaptalization and pretreatment conditions sc scp scpc pcp pcpc apricot pulp 4,472.5 4,472.5 4,472.5 2,430 2,430 sugar 527.5 527.5 527.5 apricot puree concentrate 2,570 2,570 pectinase 1 1 1 1 caco3 15 15 sc sugar chaptalization, scp sugar chaptalization treated with 0.1% pectinase, scpc sugar chaptalization treated with 0.1% pectinase and 0.3% caco3, pcp puree concentrate chaptalization treated with 0.1% pectinase, pcpc puree concentrate chaptalization treated with 0.1% pectinase and 0.3% caco3 2.5. physicochemical parameters the physicochemical analysis was undertaken on the supernatant obtained from centrifugation of the wine samples at 3,578 × g for 10 min. the ph was measured using a ph meter (mp225k, mettler-toledo ch, seoul, south korea). soluble solids (°bx) were determined using a refractometer (ra250, atago, tokyo, japan). a vinometer was used to evaluate the alcohol content at 15°c. titratable acidity was assayed using naoh solution (0.1 n) until neutralization of the organic acids to ph 8.2-8.3, and the results were expressed as a percentage of citric acid/100 g. 2.6. total phenolic compounds the total phenolic compounds in the apricot wine samples were estimated, as detailed by ough and amerine (1988), with some modifications. wine samples (2 ml) were mixed with 2 ml of 1:1 (v/v) folin–ciocalteu reagent and incubated at room temperature for 3 min. afterward, each tube was added with 2 ml of 10% na2co3, vortexed, and allowed to stand at room temperature for 1 h. the absorbance was measured at 700 nm. the results were expressed as gallic acid equivalents in mg/ml of apricot wine. 2.7. total flavonoid content the total flavonoid contents of the apricot wines were determined, as described by zhishen et al. (1999) with minor modifications. the wine samples were examined spectrophotometrically at 510 nm against a blank solution containing all reagents and 200 μl of distilled water instead of wine samples using a spectrophotometer (uv-1601, shimadzu co.). first, 430 μl of 50% ethanol, 70 μl of wine sample, and 50 μl of 5% nano2 were combined in a test tube. after 30 min of incubation, samples were combined with 50 μl of 10% al(no₃)₃·9h2o. six minutes later, 500 μl of naoh (1 n) was added, ital. j. food sci., vol. 32, 2020 916 and the solutions vortexed. the results were expressed as rutin equivalents in mg/ml of apricot wine. 2.8. dpph radical scavenging activity dpph radical scavenging activity was measured according to the method previously described by oszmiański et al. (2011). here, 100 μm of dpph was dissolved in pure ethanol (96%). the radical stock solution was prepared just before experimentation. then, 1 ml of dpph was added to 1 ml of apricot wine sample and 3 ml of 96% ethanol. the mixture was thoroughly shaken and placed at room temperature in the dark for 10 min. the decrease in absorbance of the resulting solution was observed at 517 nm at 10 min. the results were corrected for dilution and expressed in μm of trolox/ml of apricot wine. absorbance was measured using a spectrophotometer (uv-1601, shimadzu co.). 2.9. abts radical scavenging activity abts radical scavenging activity was measured based on the method previously reported by oszmiański et al. (2011). abts was dissolved in water to make a 7 μm concentration. abts radical cation (abts+) was produced by reacting the abts stock solution with 2.45 of μm potassium persulfate (final concentration) and kept in the dark at room temperature for 12–16 h before use. the radical was stable in this form for more than 2 days when stored in the dark at room temperature. the samples containing abts+ solution were diluted with redistilled water to an absorbance of 0.700±0.02 at 734 nm and equilibrated at 30℃. after adding 3.0 ml of diluted abts+ solution (a734 nm = 0.700±0.02) to 30 μl of apricot wine sample, the absorbance was read at exactly 6 min after initial mixing. the results were corrected for dilution and expressed in μm trolox/1 ml of apricot wine. absorbance was measured using a spectrophotometer (uv-1601, shimadzu co.). 2.10. frap assay ferric ion reducing antioxidant power was measured according to the method previously described by oszmiański et al. (2011). the assay was based on the reducing power of a compound (antioxidant). a potential antioxidant will reduce ferric ions (fe3+) to ferrous ions (fe2+), with the latter forming a blue complex (fe2+/tptz) that increases absorbance at 593 nm. moreover, frap reagent was prepared by mixing with an acetate buffer (300 μm, ph 3.6), a solution of 10 μm of tptz in 40 μm of hcl and 20 μm of fecl3 at a ratio of 10:1:1 (v/v/v). the reagent (300 μl) and apricot wine sample solutions (10 μl) were added to each well and thoroughly mixed. the absorbance was measured at 593 nm after 10 min. a standard curve was plotted using different trolox concentrations. all solutions were prepared on the same day of experimentation. the results were corrected for dilution and expressed in μm of trolox/1 ml of apricot wine. absorbance was measured using a spectrophotometer (uv-1601, shimadzu co.). 2.11. free sugar and organic acid analyses the free sugar and organic acid contents in the wine samples were identified and quantified using a prominence hplc instrument (shimadzu co.) with a refractive index detector (rid-10a, shimadzu co.), as described by kim et al. (2018). the wine samples were centrifuged at 3,578 × g for 10 min, and the resultant supernatants were filtered ital. j. food sci., vol. 32, 2020 917 through a millex-hv 0.45-μm membrane filter (millipore co., bedford, ma, usa) to obtain analytical samples. free sugar content was determined using a sugar-pak i column (6.5 mm × 300 mm, 10 μm; waters, milford, ma, usa). the mobile phase was ca–edta buffer (50 mg/l) at a flow rate of 0.5 ml/min at 90°c. organic acids were quantified using a shodex rspak kc-811 column (8.0 mm × 300 mm, 6 μm; showa denko kk, kawasaki, japan), and a mobile phase of 0.1% h3po4 at a flow rate of 1 ml/min at 65°c. standard curves were plotted using different concentrations of each compound. the results were expressed as each compound’s equivalents in g/l of apricot wine. 2.12. analysis of volatile compounds volatile compounds were analyzed as described by lee et al. (2016) with minor modifications, using a 7890a gc–ms system (agilent, santa clara, ca, usa). volatile compounds were separated using a db-wax column (60 m × 0.25 mm i.d., 0.25 μm film thickness, agilent, santa clara, ca, usa) and detected using an agilent 5975c tad inert xl msd. helium was used as the carrier gas at a constant flow rate of 1 ml/min. the temperature of the gc oven was initially held at 40°c for 2 min, increased at a rate of 2°c/min until 220°c, and then increased at 20°c/min to 240°c, and maintained at 240°c for 5 min. volatile compounds were collected using a headspace (hs) solid-phase microextraction (spme) fiber (10 mm length, 50/30 μm dvb/car/pdms; supelco, bellefonte, pa, usa) with magnetic stirring. five milliliters of each sample was placed in a hs vial (20 ml, 23 × 75 mm, ptfe/silicone septum, magnetic cap, agilent, santa clara, ca, usa) and then 1.25 g nacl was added to increase the efficiency of salting-out of volatile aromatic compounds in the hs. prior to extraction, the sample was shaken in a water bath at 35°c for 20 min to achieve equilibrium. afterward, the spme fiber was inserted into the vial and incubated at 35°c for 40 min. the chemical standards for volatile ester compounds were customized by chem service inc. (west chester, pa, usa). other volatile compound standards were purchased from sigma-aldrich (st louis, mo, usa). volatile compounds were identified by comparing their retention times and mass spectra against the wiley 9 spectral library (john wiley and sons, hoboken, nj, usa) using nist 0.8 (version 5.0; nist, gaithersburg, md, usa). for the quantitative analysis of each compound in the wine, a calibration curve was established by plotting the peak area against the concentration of the chemical standards. some chemicals that were commercially unavailable were quantified using standard curves of volatile compounds that had similar molecular properties. the results were expressed as each volatile alcohol compound’s equivalents in mg/l of apricot wine and each volatile ester and terpene compound’s equivalents in μg/l of apricot wine, respectively. 2.13. sensory evaluation a seven-point hedonic scale was used for sensory evaluation. each apricot wine was placed in a sample bottle and left undisturbed at room temperature for 1 h, with the bottle lid still closed before being subjected to sensory evaluation. after opening the lid, each wine was poured into wine glasses to evaluate color, sweetness, sourness, and overall preference. clarity and turbidity levels were considered as part of the parameters for color evaluation. the well-trained panel was composed of 20 students (13 males and 7 females aged 20–29 years old) from the school of food science and biotechnology, kyungpook national university, korea. each panelist evaluated the apricot wines with at least a 3-min ital. j. food sci., vol. 32, 2020 918 interval between samples, and water was provided to cleanse their palate. sensory scores ranged from 1 (very poor) to 7 (excellent). 2.14. statistical analysis all experiments were conducted at least three times or more. statistical significance was determined by the student’s t-test for independent means using microsoft excel (microsoft, redmond, wa, usa). one-way analysis of variance and duncan's multiple range test were used to determine significant differences between means. statistical significance was set at p<0.05. 3. results and discussion 3.1. effect of different pretreatment conditions on the physicochemical parameters of apricot juice the effects of different pretreatments on the physicochemical parameters of apricot juice are listed in table 2. the yield of apricot juices subjected to pectinase treatment were higher by 5.66%, 10.02%, and 10.38% in apricot pulp containing 0.05%, 0.1%, and 0.2% pectinase, respectively, compared with the control juice, but the yields of juices treated with 0.1% and 0.2% pectinase enzyme were not considerably different. in addition, juices treated with pectinase enzyme had a statistically lower ph and higher total acid contents relative to the control juice. the reducing sugar contents of apricot juices also increased with increasing pectinase enzyme concentrations, but no significant differences were found between pectinase-treated juices. apricot juices treated with 0.05%, 0.1%, and 0.2% pectinase had reducing sugar contents of 15.65%, 15.83%, and 15.90%, respectively. the ph and total acid contents of apricot juices by deacidification significantly increased and decreased, respectively, with increasing caco3 concentration compared with those of nontreated apricot juice. table 2. effects of pectinase enzyme and caco3 concentrations on the physicochemical properties of apricot juices. treatment pectinase enzyme non-treated 0.05% 0.1% 0.2% juice yield (%) 67.20±0.12d 72.86±0.03c 77.22±0.05b 77.58±0.05a ph 3.16±0.02a 3.11±0.01b 3.10±0.04b 3.10±0.07b total acidity (%) 2.56±0.02b 2.62±0.03a 2.63±0.04a 2.64±0.3a soluble solids (°bx) 16.2±0.05b 16.4±0.08a 16.4±0.05a 16.4±0.09a reducing sugars (%) 14.5±0.04b 15.65±0.10a 15.83±0.08a 15.90±0.12a treatment deacidification (caco3) non-treated 0.1% 0.2% 0.3% ph 3.14±0.06d 3.22±0.01c 3.33±0.02b 3.42±0.01a total acidity (%) 2.56±0.10a 2.48±0.03a 2.30±0.05b 2.17±0.04c all data are expressed as mean±standard deviation (n = 3). different letters in the same row indicate significant differences at p<0.05. ital. j. food sci., vol. 32, 2020 919 fruits other than grape, such as apricot, have high acidity, which needs to be controlled before, during, or after fermentation, for producing a suitable final wine (velić et al., 2018). in this study, each 0.1% pectinase treatment and 0.3% caco3 treatment improved the juice yield and appropriate physicochemical changes in apricot juice, so we further investigated the appropriate combination of these pretreatment conditions for apricot wine. 3.2. effects of different chaptalization types and the combination of pretreatments on the fermentation and physicochemical properties of apricot wine the influences of the various chaptalization techniques and the combination of pretreatments on the changes in fermentation characteristics during alcohol fermentation and physicochemical properties of fully fermented apricot wine are provided in fig. 1 and table 3. the soluble solid and alcohol contents of all the apricot wines similarly decreased and increased, respectively, for the first 3 days of fermentation. after then, all the pectinase-treated apricot wines showed higher soluble solid and alcohol contents, compared with the control wine because of increased juice yield and reducing sugar caused by 0.1% pectinase treatment. the ph and total acidity of all the apricot wines decreased and increased, respectively, for first or second days of fermentation, then steadily increased and slightly decreased, respectively, until complete fermentation. the ph and total acidity of apricot wines treated with caco3 (scp and pcp wines) were significantly lower and higher, respectively, than those of other apricot wines from beginning to end of the fermentation process. the total phenolic and total flavonoid contents of all the apricot wines were significantly superior to those of the control wine because pectinase released phenols and polyphenols from the plant cell wall (chang et al., 1995). in addition, pcp and pcpc wines that were chaptalized with puree concentrate presented higher total acidity, as well as total phenolic and flavonoid contents, compared with those of scp and scpc wines that were chaptalized with sugar, because all of these compounds were concentrated in the added apricot puree concentrate. although the total phenolic and total flavonoid contents of pectinase-treated apricot wines were relatively higher than those of other groups, the lower ph and higher total acid content of pcp wine may be negatively associated with the sensory properties. on the contrary, pcpc wine contained similar contents of functional compounds but better palatability compared to pcp wine because of deacidification. figure 1. changes in the soluble solid, alcohol, ph, and total acidity of apricot wines during fermentation. sc sugar chaptalization, scp sugar chaptalization treated with 0.1% pectinase, scpc sugar chaptalization treated with 0.1% pectinase and 0.3% caco3, pcp puree concentrate chaptalization treated with 0.1% pectinase, pcpc puree concentrate chaptalization treated with 0.1% pectinase and 0.3% caco3 ital. j. food sci., vol. 32, 2020 920 table 3. effects of different chaptalization types and pretreatment conditions on the physicochemical parameters of apricot wines. parameter wine sc scp scpc pcp pcpc soluble solids (°bx) 9.80±0.20b 10.75±0.10a 10.75±0.10a 10.80±0.20a 10.70±0.10a alcohol (%) 10.9±0.10b 11.74±0.20a 11.72±0.10a 11.70±0.10a 11.64±0.10a ph 3.53±0.10ab 3.42±0.09b 3.65±0.05a 3.36±0.03c 3.58±0.05a total acidity (%) 1.78±0.04c 2.02±0.01b 1.35±0.01e 2.20±0.03a 1.43±0.04d total phenolic compounds (mg/ml) 11.41±0.37c 16.95±3.11b 17.15±2.03b 21.87±0.96a 21.43±1.21a total flavonoids (mg/ml) 0.39±0.00b 0.41±0.01a 0.42±0.01a 0.43±0.01a 0.43±0.01a all data are expressed as mean±standard deviation (n = 3). different letters in the same row indicate significant differences at p<0.05. sc sugar chaptalization, scp sugar chaptalization treated with 0.1% pectinase, scpc sugar chaptalization treated with 0.1% pectinase and 0.3% caco3, pcp puree concentrate chaptalization treated with 0.1% pectinase, pcpc puree concentrate chaptalization treated with 0.1% pectinase and 0.3% caco3 3.3. free sugar and organic acid contents of apricot wines the impacts of different chaptalization types and the combination of pretreatments on the free sugar and organic acid contents in apricot wines are evident in table 4. after alcoholic fermentation, sucrose, glucose, galactose, and fructose were identified in the apricot wines. fructose was the most abundant reducing sugar (0.599±0.014–4.662±0.019 g/l) in all the apricot wines. marked differences in the organic acids were observed between each apricot wine. citric acid and quinic acid of scp and scpc wines were significantly decreased and increased compared with sc wine, respectively, whereas tartaric acid and malic acid of scpc wine were the lowest among all the apricot wines. citric acid and quinic acid contents of pcp and pcpc wines were significantly higher than other wines because various components of apricot were concentrated during puree concentrate preparation, whereas tartaric acid of pcpc wine was significantly lower than pcp wine due to deacidification. succinic acid levels were comparable among all the apricot wines, and acetic acid of pectinase-treated apricot wines was slightly increased compared with control apricot wine. according to amerine et al. (1965), the decreasing order of sourness intensity of organic acids is malic acid, tartaric acid, citric acid, and lactic acid. caco3 treatment was reported to reduce wine acidity by inducing the precipitation of tartrate and malate (mattick et al., 1980). thus, the combination of pectinase and caco3 treatments increased the yield of apricot juice and reduced the acidity in apricot wine. 3.4. antioxidant activity of apricot wines the various antioxidant activities, such as dpph radical scavenging activity, abts radical scavenging activity, and frap of apricot wines are shown in fig. 2. all of the antioxidant activities were highest in pcp and pcpc wines, followed by scp and scpc wines, and then sc wine, which might be attributed to the release of pigment compounds, such as flavonoids, by pectinase (all the pectinase-treated apricot wines) and the concentration of those compounds in the added puree concentrate (pcp and pcpc wines). ital. j. food sci., vol. 32, 2020 921 table 4. composition of free sugar and organic acid contents (g/l) of apricot wines depending on different chaptalization types and pretreatment conditions. parameter wine sc scp scpc pcp pcpc free sugars sucrose 0.08±0.01a nd nd nd nd glucose 0.16±0.06c 0.28±0.03b 0.25±0.01b 0.70±0.02a 0.67±0.03a galactose 0.23±0.04c 0.86±0.01b 0.82±0.04b 1.58±0.02a 1.50±0.06a fructose 0.60±0.01c 2.66±0.01b 2.64±0.01b 4.56±0.02a 4.66±0.02a organic acid citric acid 11.58±0.35b 9.89±0.25c 9.61±0.34c 14.40±0.51a 14.25±0.48a tartaric acid 2.83±0.08b 2.84±0.11b 0.42±0.04d 3.11±0.12a 0.73±0.06c malic acid 5.61±0.12a 4.39±0.12b 3.20±0.09c 4.29±0.09b 2.96±0.10d quinic acid 7.37±0.16c 11.48±0.34b 11.34±0.33b 34.17±1.03a 32.87±1.17a succinic acid 0.50±0.02a 0.54±0.04a 0.52±0.04a 0.45±0.02b 0.44±0.02b acetic acid 0.18±0.01c 0.31±0.04b 0.29±0.01b 0.42±0.02a 0.40±0.02a all data are expressed as mean±standard deviation (n = 3). different letters in the same row indicate significant differences at p<0.05. sc sugar chaptalization, scp sugar chaptalization treated with 0.1% pectinase, scpc sugar chaptalization treated with 0.1% pectinase and 0.3% caco3, pcp puree concentrate chaptalization treated with 0.1% pectinase, pcpc puree concentrate chaptalization treated with 0.1% pectinase and 0.3% caco3, nd not detected figure 2. effects of different chaptalization types and pretreatment conditions on the dpph radical scavenging activity (a), abts radical scavenging activity (b), and ferric ion reducing power (c) antioxidant activities of apricot wines. different letters indicate significant differences at p<0.05. l-aa l-ascorbic acid, te trolox equivalents, sc sugar chaptalization, scp sugar chaptalization treated with 0.1% pectinase, scpc sugar chaptalization treated with 0.1% pectinase and 0.3% caco3, pcp puree concentrate chaptalization treated with 0.1% pectinase, pcpc puree concentrate chaptalization treated with 0.1% pectinase and 0.3% caco3 ital. j. food sci., vol. 32, 2020 922 apricot contains numerous phenolic compounds, including catechin, epicatechin, pcoumaric acid, caffeic acid, and ferulic acid, that contribute to the antioxidant activity and nutritional benefits (campbell and padilla-zakour, 2013; sochor et al., 2010). arnous et al., (2002) mentioned that total polyphenol and total flavonol compounds could significantly contribute to the overall antioxidant activity of wine. as such, in the present study, the high antioxidant activities displayed by the apricot wines depended on the increased total phenolic and flavonoid compounds released by pectinase pretreatment and concentrated by puree concentrate chaptalization. 3.5. volatile aromatic compounds of apricot wines the volatile aromatic compounds of apricot wines are given in table 5. the volatile higher alcohol compounds were more abundant in pectinase-treated apricot wines than control apricot wine. in pcp and pcpc wines, most of the volatile higher alcohols, except for 1propanol, were detected at levels lower than in scp and scpc wines, respectively. moreover, scpc wine showed the highest amount of 1-propanol, isobutanol, isoamyl alcohol, 1-hexanol, 3-ethoxypropanol, 1-decanol, and benzyl alcohol, among all the apricot wines. a higher amount of 2,3-butanediol, which is an unattractive compound in wine because of its buttery aroma (bartowsky and henschke, 2004), was detected in greater quantities in sc and scp wines than in the other apricot wines examined. total volatile ester compounds were the highest in sc wine, as those of pectinase-treated apricot wines were evaporated during pectinase treatment at 30℃ for 2 h. furthermore, pcp and pcpc wines presented significantly lower total volatile ester compounds than those of the other wines, which is considered to be due to the loss of their corresponding precursors during heat treatment of the puree concentrate production process. sc wine contained the highest amounts of isoamyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl-9decanoate, as well as ethyl decanoate. these compounds primarily influenced the changes in the amount of total volatile ester compounds. volatile terpenes were higher in all the pectinase-treated apricot wines than control apricot wine. in particular, linalool and αterpineol of pcp and pcpc wines were significantly higher than those of the other wines. the group of higher alcohols is well known as one of the dominant chemical constituents in wine, in which they play a major role as ester precursors (lambrechts and pretorius, 2000). esters are well recognized as the most abundant aromatic compounds in wine (rojas et al., 2001) and are produced by yeasts during alcoholic fermentation, whereas terpenes are only present in small amounts in some fruits, such as grape (especially in aromatic cultivars), apricot, and peach. however, terpenes can mostly affect the floral properties of wines with low odor thresholds (100-400 ppb) (maicas and mateo, 2005). in the present study, significantly decreased contents of volatile ester compounds were detected in the pretreated apricot wines compared with non-treated apricot wine, but the levels of volatile higher alcohols and terpenes were greater, which might have assisted in improving the sensory properties of apricot wine. ital. j. food sci., vol. 32, 2020 923 table 5. the concentration of volatile aromatic compounds in apricot wines depending on different chaptalization types and pretreatment conditions. compound odor description threshold (mg/l) amount of volatile aromatic compound sc scp scpc pcp pcpc 1-propanol alcohol, ripe fruity[1] 306[1] 85.48±6.55b 97.17±9.45b 169.76±14.11a 103.27±10.11b 178.63±13.28a isobutanol alcohol, solvent, green, bitter[1] 75 [1] 159.27±12.09b 176.03±16.23ab 199.71±20.56a 141.00±12.24b 162.13±15.06b isoamyl alcohol solvent, sweet, nail polish[2] 60[2] 2605.68±233.17a 2863.76±256.18a 3024.25±306.50a 2729.15±250.06a 2896.57±269.77a 1-hexanol herbaceous, grass, woody[1] 1.1[1] 20.10±1.94c 30.68±3.31b 37.41±3.42a 23.85±2.65c 32.64±3.04ab 3-ethoxypropanol fruity[1] 0.1[1] 10.04±1.11a 10.27±0.98a 10.73±0.94a 6.82±0.61b 6.77±0.64b 1-octanol jasmine, lemon[1] 0.8[1] 13.62±1.26b 89.27±7.24a 10.10±0.88b 8.83±0.77b 5.04±0.62c 2,3-butanediol floral, fruity, herbal, buttery[2,3] 150 [2] 14.70±1.32a 14.24±1.52a 11.21±1.05b 9.52±0.89b 8.16±0.72b 1-decanol floral, fruity, bitter, winey[2] 0.4[2] 5.13±0.44b 6.21±0.56a 6.51±0.52a 4.44±0.41b 4.50±0.39b benzyl alcohol roasted, sweet, fruity[1] 200[1] 20.07±2.12c 52.63±5.10a 60.63±6.12a 35.17±3.41b 41.17±4.41b phenylethyl alcohol rose, honey[1] 14[1] 201.16±19.43a 242.63±22.73a 245.14±26.18a 249.67±24.07a 243.81±23.58a ∑alcohols 3135.25±279.43a 3582.89±323.30a 3775.46±380.28a 3311.73±305.22a 3579.42±331.51a methyl acetate nd 13.93±1.30b 15.67±1.51b 23.25±2.16a 25.17±2.32a ethyl acetate pineapple, fruity, balsamic[2] 12[2] 729.35±74.28a 668.56±64.86a 760.26±71.34a 726.78±70.86a 810.59±78.50a ethyl propionate fruity[4] 1.8[4] 18.65±1.56a 17.24±1.55a 19.18±1.68a 15.43±1.62a 16.22±1.55a ethyl isobutyrate sweet, rubber[4] 0.015[4] 11.51±1.05a 9.51±0.92ab 11.11±1.06a 7.77±0.74b 8.95±0.78b propyl acetate sweet, fruity[4] 4.7[4] 24.83±2.62a 18.04±1.77b 21.16±2.04ab 19.21±1.78b 22.04±2.04ab isobutyl acetate fruity, apple, banana[4] 1.6[4] 42.19±3.84a 30.40±3.13b 34.23±2.99b 23.79±2.24c 25.64±2.82bc ethyl butanoate banana, pineapple, strawberry[1] 0.4 [1] 43.24±4.13a 28.37±2.47b 29.59±3.41b 22.59±2.01b 23.10±1.98b butyl acetate fruity[5] 4.88±0.43a 3.96±0.35ab 4.37±0.56a 3.35±0.36b 3.84±0.33ab isoamyl acetate banana[1] 0.16[1] 2472.35±242.56a 1403.56±142.53b 1541.19±136.04b 871.05±82.60c 972.42±88.09c ethyl pentanoate yeast, fruity[4] 0.094[4] 4.24±0.36a 2.89±0.27b 3.62±0.35a 4.19±0.40a 4.63±0.51a ethyl hexanoate banana, green apple[1] 0.08[1] 749.92±72.65a 515.85±49.06b 535.92±55.50b 388.25±36.12c 442.16±43.69bc hexyl acetate apple, cherry, pear, floral[1] 1.5[1] 60.09±7.32a 47.19±4.53b 58.32±5.36a 18.63±1.92c 22.85±2.12c ethyl heptanoate fruit[4] 0.22[4] 9.26±0.87a 6.41±0.67b 5.27±0.61bc 4.04±0.38c 4.36±0.41c ital. j. food sci., vol. 32, 2020 924 methyl octanoate orange[4] 34.89±3.57a 40.63±3.88a 43.31±4.10a 35.28±3.11a 41.49±3.89a ethyl octanoate fruity, sweet, banana, pear[1,2] 0.24-0.58 [1,2] 2552.69±226.39a 1521.94±126.93b 1668.65±171.03b 890.86±82.62d 1114.51±103.43c geranyl acetate floral, rose[6] 96.95±9.32b 120.62±11.05a 102.99±10.23ab 67.65±6.59c 62.78±6.32c ethyl nonanoate 44.32±4.34a 32.19±3.36b 33.67±3.42b 36.19±3.54b 35.20±3.17b methyl decanoate wine[4] 1.2[4] 14.46±1.28a 12.63±1.01a 13.90±1.21a 9.91±0.79b 10.33±0.92b ethyl decanoate fatty acids, fruity, soap[1,2] 0.2[1,2] 1840.95±156.98a 878.86±90.09b 957.66±92.06b 464.04±42.22c 504.38±48.56c ethyl benzoate heavy, floral, fruity[4] 5.75[4] 315.49±33.65b 577.75±46.60a 589.05±54.98a 606.73±61.17a 631.40±56.77a ethyl 9-decenoate fruity[4] 0.1[4] 231.95±24.25a 32.81±3.14b 27.38±2.67b 7.50±0.73c 5.53±0.50c methyl salicylate pepper, mint[4] 11.38±1.14b 15.12±1.87a 16.64±1.52a 12.13±1.10b 12.44±1.39b ethyl phenylacetate fruity, sweet[4] 2.03±0.15b 1.88±0.23b 2.08±0.19b 3.26±0.33a 3.41±0.31a 2-phenylethyl acetate fruity, rose[1] 1.8[1] 41.38±3.36a 34.34±3.18a 35.77±3.48a 24.39±2.31b 26.72±2.43b ethyl dodecanoate oily, fatty, fruity[1] 1.5[1] 175.20±18.21b 178.56±15.56b 223.61±21.13a 120.02±10.65d 154.12±12.98c ∑esters 9532.22±894.31a 6213.25±580.31b 6754.61±648.47b 4406.30±418.35c 4984.28±465.81c linalool flowery, muscat[1] 0.025[1] 731.91±71.03c 1007.80±96.32b 897.27±90.43bc 1424.51±153.07a 1369.53±128.25a α-terpineol lilac, floral, sweet[1] 0.25[1] 135.46±12.63c 184.23±16.70b 159.49±14.17bc 302.61±28.65a 288.09±26.72a citronellol rose[1] 0.1[1] 18.48±1.72c 28.88±2.64b 27.68±2.60b 58.05±5.57a 60.98±5.78a geraniol citric, geranium[1] 0.02[1] 40.89±4.65b 53.12±5.35a 49.27±4.55ab 56.91±5.43a 51.64±5.33a ∑terpenes 926.75±90.03c 1274.03±121.01b 1133.72±111.75bc 1842.09±192.72a 1770.24±166.08a all data are expressed as mean±sd (n = 3). different letters in the same row indicate statistically significant differences at p<0.05. sc sugar chaptalization, scp sugar chaptalization treated with 0.1% pectinase, scpc sugar chaptalization treated with 0.1% pectinase and 0.3% caco3, pcp puree concentrate chaptalization treated with 0.1% pectinase, pcpc puree concentrate chaptalization treated with 0.1% pectinase and 0.3% caco3, nd not detected [1] cai et al., 2014; [2] butkhup et al., 2011; [3] bartowsky and henschke, 2004; [4] zhang et al., 2015; [5] nattaporn and pranee, 2011; [6] nishimura, 1995 ital. j. food sci., vol. 32, 2020 925 3.6. sensory evaluation of apricot wines the sensory evaluation results of apricot wines are shown in fig. 3. all the pectinasetreated apricot wines obtained higher color scores compared with control apricot wine, due to clarification by pectinase enzyme. the flavor scores of scpc wine, containing the highest amount of total volatile higher alcohols, and pcp and pcpc wines, which recorded the greatest abundance of total volatile terpenes, were significantly higher relative to the other apricot wines. the sweetness scores of pectinase-treated apricot wines were slightly higher than control apricot wine because of some remaining free sugars. the sourness of pcp wine was the strongest, whereas that of scpc wine was the weakest because these wines contained, respectively, the highest and lowest presence of tartaric acid and malic acid, which are the two strongest organic acids. pcpc wine also obtained low sourness score because of its low tartaric acid and malic acid levels. in the overall preference, scpc and pcpc wines, having the most reduced sourness, obtained the highest scores among all the apricot wines. scp and pcp wines also obtained higher scores when compared with sc wine. figure 3. sensory evaluation of apricot wines depending on different chaptalization types and pretreatment conditions. sc sugar chaptalization, scp sugar chaptalization treated with 0.1% pectinase, scpc sugar chaptalization treated with 0.1% pectinase and 0.3% caco3, pcp puree concentrate chaptalization treated with 0.1% pectinase, pcpc puree concentrate chaptalization treated with 0.1% pectinase and 0.3% caco3 in this study, we investigated the effects of puree concentrate chaptalization and various pretreatments on the quality of apricot wine. the results demonstrated that apricot wines chaptalized with puree concentrate have shown not only higher antioxidant activity and total volatile terpene compounds than sugar-chaptalized apricot wines but also higher acidity that negatively affects the sensory properties of wine. pectinase and caco3 pretreatments can clarify the appearance apricot wines and reduce the acidity of apricot wines, indicating that combining puree concentrate chaptalization and various pretreatments may result to improved apricot wine quality. ital. j. food sci., vol. 32, 2020 926 acknowledgments this work was supported by the national research 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breisach, germany zhang s., petersen m.a., liu j. and toldam-andersen t.b. 2015. influence of pre-fermentation treatments on wine volatile and sensory profile of the new disease tolerant cultivar solaris. molecules 20:21609-21625. zhishen j., mengcheng t. and jianming w. 1999. the determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. food chem. 64:555-559. paper received april 20, 2020 accepted september 9, 2020 16 issn 1120-1770 online, doi 10.15586/ijfs.v33i1.1949 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (1): 16–28 p u b l i c a t i o n s codon drying characteristics and degradation kinetics in some parameters of goji berry (lycium barbarum l.) fruit during hot air drying heysem suat batu1,* and çetin kadakal2 1department of food engineering, institute of science, pamukkale university, denizli, turkey; 2department of food engineering, faculty of engineering, pamukkale university, denizli, turkey *corresponding author: h.s. batu, department of food engineering, institute of science, pamukkale university, denizli, turkey. tel: +90 5413181989. email: h.s.batu@gmail.com received: 24 august 2020; accepted: 07 october 2020; published: 01 february 2021 © 2021 codon publications open access paper abstract drying kinetics, color properties, water-soluble vitamins, antioxidant capacity, total phenolic content, and thermal degradation kinetics of bioactive compounds in goji berries were investigated. drying experiments were conducted at 50°c, 60°c, and 70°c. page model was determined as the best model to predict experimental moisture ratio for all temperatures. increment in drying temperature increased effective moisture diffusivity and drying rate values. vitamins c and b6, antioxidant activity and total phenolic content were significantly reduced by drying. thermal degradation of vitamins c and b6, antioxidant capacity and total phenolic content were found to fit the first-order kinetic model. keywords: antioxidant capacity, degradation kinetics, drying kinetics, goji berry, total phenolic content, water-soluble vitamins introduction goji berry (lycium barbarum l.), which belongs to solanaceous family, is grown in china, tibet, and some regions of asia. goji berry is primarily found in east asia and grown particularly in japan, south korea, and south china (gao et al., 2017). lycium barbarum l. is a deciduous woody shrub, mostly thorny, that grows 1–4 m in height (griffiths and huxley, 1992). fruits are two-chambered, mostly orange-red in color, juicy, and fleshy (chen et al., 2018). ripe goji berries are 3–10 mm in diameter, 6–20-mm long, and have oblong or ellipsoid shape (gao et al., 2017). goji berry contains a high amount of anthocyanin (cui et al., 2011), carotenoids (lycopene, zeaxanthin dipalmitate, beta-carotene, zeaxanthin, and lutein), vitamins (tocopherol, glucopyranosyl ascorbic acid, and ascorbic acid), betaine, fatty acids, and peptidoglycans (islam et al., 2017). fatty acids commonly found in goji berry are linoleic, oleic, and palmitic acids (cossignani et al., 2018). the fruit also includes organic acids such as malic, citric, shikimic, and fumaric acids (mikulic-petkovsek et al., 2012), monosaccharides, which are mannose, rhamnose, galactose, xylose, arabinose, and glucose, 18 amino acids, and galacturonic acid (amagase and farnsworth, 2011). owing to bioactive substances and healthy functions, goji berries are popular in the western world (bertoldi et al., 2019). in traditional chinese herbal medicine, goji berry has been used as a supplement for more than 2000 years (burke et al., 2005). it is used to protect the liver, kidneys, and eyes, strengthen the eyesight (shan et al., 2011), and reduce serum lipids and blood glucose levels. also, goji berry has other health-promoting effects such as anti radiation, immune-enhancing, anti-fatigue, and anti aging effects, stimulating hematopoiesis, and treating male infertility (luo et al., 2004; tian et al., 2013). mailto:h.s.batu@gmail.com italian journal of food science, 2021; 33 (1) 17 characteristics and degradation kinetics of goji berry on hot air drying method drying process of samples goji berry samples were dried in a tray drying cabinet (yücebaş makine tic. ltd. şti., i̇zmir, turkey). dryer comprised an electronic proportional controller (euc442 model, enda, turkey), an electric heater, and a centrifugal fan to provide airflow. the dryer’s internal size was 70 cm × 55 cm × 100 cm, the range of workable temperature was 40–120°c, and the range of workable relative humidity was 20%–95%. three different drying air temperatures were used in the experiments: 50°c, 60°c, and 70°c. the cabinet was heated for 1 h before the start of drying process to reach a constant temperature; and 200 g of samples were uniformly placed on the drying tray. the drying process was performed up to the targeted dry matter content at a relative humidity of 20% and air velocity of 2 m/s. the drying experiments were performed in triplicate and weighed at certain time intervals with a 0.001 g precision digital scale. drying characteristics of goji berry fruit empirical models are more useful because theoretical drying models are complicated and the former models offer a direct relationship between drying time and moisture content (moradi et al., 2020). thin-layer drying models have great significance in designing the best drying conditions. moisture ratio (mr) must be calculated by mi, mt, and me values to choose the best model. − = − t e i e m m mr m m (eq. 1) where mi: initial moisture content of samples (g water/g dry matter); mt: moisture content at any time (g water/g dry matter); me: equilibrium moisture content (g water/g dry matter). however, if the equilibrium moisture content (me) is very low than mt and mi, it can be neglected and equation 2 is used (yousefi et al., 2013): = t i m mr m (eq. 2) drying rate (dr) was determined using equation 3: +∆ −= ∆ t t tm mdr t (eq. 3) drying is a protection technique that is widely applied to fresh products. dehydration prolongs stability of fruits and vegetables by decreasing water content and minimizing physicochemical changes and microbial growth (tepe and tepe, 2020). besides, the drying process protects valuable foods under effective conditions, prolongs shelf life, and reduces storage, transportation, and packaging costs because of decrement in the volume and weight of food products (önal et al., 2019). one of the most important steps in the food processing industries is the dehydration process. sun drying is a preferred method because it is economical and does not require investment, but it is a disadvantageous method due to microbial reliability and loss in quality such as color and aroma (göztok and içier, 2017). convective drying is one of the most used drying methods to protect agricultural products compared to other drying methods because it is simple and low costing. on the contrary, this method causes changes in sensory properties and nutritional values (orikasa et al., 2014). the convective drying can be used as an alternative method instead of conventional (sun) drying. it has more advantages than conventional drying in terms of preventing microbial contamination, component protection, and involving lower drying time (lewicki, 2006). adiletta et al. (2015) and fratianni et al., (2018) studied goji berry drying, but no data are available in literature about drying characteristics, degradation kinetics of water-soluble vitamins, antioxidant capacity (ac), and total phenolic content (tpc) of dried goji berry fruit. absence of data on drying characteristics and degradation of some bioactive compounds can be regarded as a gap in the drying process of goji berry fruit. in this context, determining drying characteristics and degradation of some bioactive compounds can be useful for the designing of drying process. thus, the aims of this study are to: determine the drying characteristics of goji berry fruit at 50°c, 60°c, and 70°c, examine the influence of drying process on contents of vitamins c and b6, antioxidant capacity and total phenolic content of goji berries, and calculate the degradation kinetics of these bioactive compounds. materials and methods materials and sample preparation goji berry fruits, of nq1 variety, were obtained from redlife in the çivril district of denizli province of turkey. geographical location of denizli is between 28°30’– 29°30’ east meridians and 37°12’–38°12’ north parallels and is located in the aegean region of turkey. the fruits were carefully collected in july 2019 from 10 randomly selected plants. fresh fruits were washed to remove any foreign material and kept at –18°c before analysis. 18 italian journal of food science, 2021; 33 (1) h.s. batu and ç. kadakal equation 8 shows a straight line with a slope provided in the plot: = − 2 2 effslope dr π (eq. 8) the arrhenius equation of hot air-drying process was used for calculation of activation energy (fang et al., 2009): −  =     0 exp a eff e d d rt (eq. 9) where r: universal gas constant [8.314 j/mol (k) or 1.987 cal/mol (k)]; t: absolute temperature (k); ea: activation energy (kj/mol or kcal/mol); d0: pre-exponential constant (m 2/s). after regulation of natural logarithm in equation 8, equation 9 can be written as follows: = −0ln ln a eff e d d rt (eq. 10) natural logarithm of effective moisture diffusivity versus 1/t gives a straight line with a slope that represents activation energy. analysis of water-soluble vitamins an extraction method proposed by donmez (2015) was used for analysis of water-soluble vitamins. to determine water-soluble vitamins, a sample of 5 g of goji berries was taken, and after homogenization with distilled water (1:9, w:v), the homogenate was centrifuged at 4500 rpm for 10  min (core nf 800r). the supernatant obtained from centrifugation was filtered using a 0.45-μm filter to be injected into a high-pressure liquid chromatography (hplc) column. using a micro syringe, 20 μl of filtrate was injected into the hplc column. mobile phase consisted of 0.1 m hplc grade kh2po4 at ph 7. the hplc device (shimadzu), in which analysis of water-soluble vitamins was performed, consisted of column oven (shimadzu cto-20a, japan), pump (shimadzu lc-20ad, japan), degasser (shimadzu dgu20a3, japan), photodiode array (pda) detector, and hplc software in a computer. the column used in the analysis was ace c18 column (7.8 × 300 mm), column temperature was 25°c, and the flow rate of mobile phase was 0.8 ml/min (isocratic). wavelengths used in analysis were 254 nm, 261 nm, 324 nm, and 234 nm for ascorbic acid, niacin, pyridoxine, and thiamine, respectively. analysis was performed in triplicate. where mt+δt: moisture content at time difference; δt: time difference between two measuring points. the relation between predicted and experimental data of goji berry fruits dried at different drying temperatures is explained with root mean square error (rmse), reduced chi-square (χ2), and determination coefficient (r2). rmse is a statistical parameter that expresses deviation between experimental and predicted values. the best equation predicting experimental data is determined accordingly with lower rmse and χ2 and higher r2 values. the chi-square (equation 4) and rmse (equation 5) values were calculated as follows: ( )= −= − ∑ 2 , exp,2 0 n pre i ii mr mr n n χ (eq. 4) ( )=   = −   ∑ 1/2 2 , exp,0 1 n pre i ii rmse mr mr n (eq. 5) where mrpre,i: predicted mr; mrexp,i: experimental mr; n: number of observation data; n: constants of thin layer drying models. matlab software was used to calculate thin-layer modeling and for statistical analyses. determination of effective moisture diffusivity and activation energy in hot air drying fick’s diffusion equation described the drying characteristics of biomaterials. crank (1975) suggested a solution to this equation to be used for spherical products. equation 6 is recommended for spherical products, provided that there is no shrinkage and constant effective diffusivity (doymaz, 2006): ∞ =  − =      ∑ 2 2 2 2 21 6 1 exp eff n n d mr n r π π (eq. 6) where deff: effective moisture diffusivity (m 2/s)’ r: arithmetical average of radius of samples at measured intervals (m). equation 6 can be reduced (saravacos and raouzeos, 1986) and a new equation is provided below: 2 2 2 6 ln( ) ln eff d mr t r π π    = −        (eq. 7) italian journal of food science, 2021; 33 (1) 19 characteristics and degradation kinetics of goji berry on hot air drying for the zero-order kinetic model, equation can be written as follows: c = c0 – kt (eq. 13) if equation 13 is integrated and m = 1, then equation 14 is written as follows: lnc = lnc0 – kt (eq. 14) where ln c: natural logarithm of the residual vitamins c and b complex, tpc, and ac; ln c0: initial content of vitamins c and b complex, tpc, and ac] k: rate constant (1/h); t: time. temperature dependence of vitamins c and b complex, tpc, and ac can be calculated using equation 15 (labuza and riboh, 1982): − = 0 ae rtk k xe (eq. 15) when equation 15 is regulated, equation 16 is written as follows:    = − +        0 1 ln lna e k x k r τ (eq. 16) where k0: frequency factor (1/h); r: universal gas constant [8.314 × 10-3 kj/mol (k) and 1.987 × 10-3 kcal/mol (k)]; t: absolute temperature (k); ea: activation energy (kcal/mol or kj/mol). quotient indicator (q10) expresses temperature dependence of reaction rate and is calculated using equation 17 (labuza and schimidl, 1985):     −   =     2 1 10 2 10 1 t tk q k (eq. 17) half-life time, time required for half of concentration, for each temperature is calculated using equation 18 for first-order kinetics (labuza, 1984): = − =1/2 1 1 ln(0.5) 0.693t x x k k (eq. 18) time taken by the compound, or quality criterion, to lose 90% of its quality is expressed as d and is calculated for first-order kinetics as follows (equation 19): 1 2.303d x k = (eq. 19) a calibration curve of different concentrations of stock solutions (5, 10, 25, 50, 75, and 100 ppm) with high r2 (0.9999) was obtained. the content of water-soluble vitamins was calculated by the equation obtained from the calibration curve. analysis of antioxidant capacity and total phenolic content the ac and tpc analysis was carried out using the methanolic extraction method proposed by otağ (2015). a sample of 5 g of goji berries was mixed with 45 ml of 90% methanol and homogenized using a laboratory blender. the homogenate was centrifuged at a speed of 4500 rpm for 10 min. after centrifugation, supernatants were collected and filtered using a filter paper. the tpc analysis was performed according to singleton and rossi (1965) with modifications. in this analysis, 1500 µl folin–ciocalteu solution (10% v/v) was added into 300 µl of extract and the mixture was kept in a dark place for 3 min. then 1200 µl aqueous 7.5% na2co3 was added into the mixture. the final mixture was incubated for 2 h at room temperature in a dark place. after incubation, the absorbance measurement of the samples was carried out at a wavelength of 760 nm using spectrophotometer (t80, pg ins., uk.). analysis was carried out in triplicate, and tpc was expressed as mg/100 g gallic acid equivalent (gae) dry weight (dw). the ac analysis was carried out using the method suggested by thaipong et al. (2006) with modifications. here, 150 μl of extracts and 2850 μl of dpph methanolic solution (absorbance value: 1.1 at a wavelength of 515  nm) were mixed. absorbance of samples was measured at a wavelength of 515 nm using spectrometer after 60 min incubation in a dark place at room temperature. each sample was analyzed in triplicate and ac was expressed as mmol trolox equivalent (te)/g dw. color measurement reflectance color value of goji berry skin was measured by using hunter lab color miniscan xe (45/0-l, usa). the samples were placed on a white background and the measurement was performed by covering with a transparent glass. the highest color difference (δe) was calculated using equation 11 (horuz et al., 2017): ∆ = − + − + −2 2 20 0 0( ) ( ) ( )e l l a a b b (eq. 11) calculation of kinetic parameters the following equation (equation 12) was used as a general equation to describe the reaction rate of the compounds that are degraded or formed by labuza (1984): = [ ]m dc k c dt (eq. 12) 20 italian journal of food science, 2021; 33 (1) h.s. batu and ç. kadakal model predicting experimental moisture ratio of goji berry fruits for all drying temperatures (50°c, 60°c, and 70°c), with the lowest rmse and χ² and the highest r2 values. effective moisture diffusivity and activation energy of goji berry fruits during hot air drying the effective moisture diffusivity (deff) and activation energy (ea) values of goji berry fruits are presented in table 2, and the deff values were calculated in the range of 2.98 × 10-8–1.04 × 10-8 m2/s. effective moisture diffusivity is a useful indicator of dehydration effectiveness (chen et al., 2016). when compared with other drying temperatures, the highest deff value was determined in the drying process conducted at 70°c. increase in the deff value means the moisture content in goji berry samples is evaporated more easily. as understood from equation 9 mentioned above, it is a known fact that the drying temperature is an important factor affecting the deff value. no mention of deff value during the drying of goji berry fruits with hot air was found in literature. senadeera et al. (2014) found deff values in the range of 1.32 × 10 -6– 1.34 × 10-6 m2/h because of the drying process executed on different types of grapes at 50°c, 0.5 m/s air velocity, and 20% moisture content. chen et al. (2016) carried out hot air drying of wine grapes, grown in canada, between 25°c and 80°c and determined the deff value at 25°c and 80°c as 0.05 × 10-10 m2/s and 0.49 × 10-10 m2/s, respectively, at mr = 0.2. they observed that the deff values increased 10 times with increase in temperature from 25°c to 80°c. dong et al. (2013) studied the drying process of grapes at 30°c, 35°c, 40°c, and 45°c and found that the deff value was higher at the highest temperature. in other words, the deff value increases with increase in drying temperature, and the data examined in literature support our study. statistical analysis spss 22.0 software (ibm corporation, armonk, ny) was used for statistical analysis and expressed as mean ± standard deviation (sd). analysis of variance (anova) was used to evaluate differences between treatments with the significance level p = 0.05. differences between groups were determined using the duncan test. results and discussion drying characteristics of goji berry fruits during hot air drying the drying rate and moisture ratio values of goji berries during hot air drying are presented in figure 1. drying time and drying rate of goji berry fruits were significantly affected by drying temperature, and it is clearly seen that drying rate increases with the increment in drying temperature. drying time decreased depending on the increment in temperature, so drying time was found to be 24 h at 50°c, 19 h at 60°c, and 9 h at 70°c. adiletta et al. (2015) determined drying time as 21 h at 60°c for hot air-drying treatment of goji berries. fratianni et al. (2018) dried goji berry fruits in a convective dryer and the drying process was finished in 45 h at 50°c, 21 h at 60°c, and 12 h at 70°c, and the velocity of air was 2.1 m/s. it could be that the increment in drying rate with increase in temperature might be due to the increase in heat transfer coefficient. the results of this study were similar to the results of the studies examined in literature. mathematical models used in modeling the drying process, constants, and the statistical data of mathematical models are listed in table 1. demiray et al. (2017) reported that the lower rmse and χ² and the higher r2 values are required for determining the best model. as seen in table 1, the page (1949) model is the best figure 1. moisture ratio and drying rate of goji berries during hot air drying. 50°c 0,2 0,2 0,02 0,015 0,01 0,005 0 1,2 moisture content (g water / g dry matter) (g w at e r /g d r y m at te r .h ) 2,2 3,2 4,2 0,4 0,6 m r d r 0,8 1 0 0 5 10 time (h) 15 20 25 60°c 70°c 50°c 60°c 70°c italian journal of food science, 2021; 33 (1) 21 characteristics and degradation kinetics of goji berry on hot air drying table 1. thin-layer mathematical models, model constants, and statistical parameters of thin-layer drying curves. model names and references model temperature model constants χ² rmse r² lewis / lewis (1921) exp(-kt) 50°c k = 0.1186 0.001344007 0.03592 0.9793 60°c k = 0.1737 0.001277711 0.03484 0.9819 70°c k = 0.3450 0.000251669 0.01505 0.9977 page / page (1949) exp(-ktn) 50°c k = 0.1812 n = 0.8161 0.000264861 0.01561 0.9962 60°c k = 0.2508 n = 0.8107 0.000165378 0.01220 0.9979 70°c k = 0.3574 n = 0.9716 0.000290322 0.01524 0.9979 henderson and pabis / henderson and pabis (1961) aexp(-kt) 50°c k = 0.1075 a = 0.9150 0.000576501 0.02303 0.9918 60°c k = 0.1607 a = 0.9316 0.000866761 0.02793 0.9890 70°c k = 0.3438 a = 0.9964 0.000316013 0.01590 0.9977 logaritmic / doymaz (2011) aexp(-kt) + c 50°c k = 0.1341 a = 0.8742 c = 0.07044 0.000666050 0.02421 0.9910 60°c k = 0.2094 a = 0.8951 c = 0.07613 0.000296302 0.01587 0.9964 70°c k = 0.4267 a = 0.9425 c = 0.07490 0.000811922 0.02384 0.9948 wang and singh / wang and singh (1978) 1 + at + bt2 50°c a = -0.09269 b = 0.002374 0.003999604 0.06066 0.9433 60°c a = -0.13020 b = 0.004536 0.004329174 0.06242 0.9450 70°c a = -0.26180 b = 0.018240 0.001641672 0.03624 0.9880 parabolic / bi et al. (2015) a + bt + ct2 50°c a = 0.8701 b = -0.07149 c = 0.001653 0.001539746 0.03681 0.9800 60°c a = 0.8800 b = -0.10560 c = 0.003485 0.002166198 0.04291 0.9754 70°c a = 0.9556 b = -0.24320 c = 0.016610 0.001492261 0.03232 0.9917 rmse, root mean square error. the arrhenius-type relation between deff and 1/t is presented in figure 2. the ea values of goji berry fruits were found to be 48.37 kj/mol and 11.56 kcal/mol. in literature, no activation energy data for hot air drying of goji berry fruits were found. when compared with similar berry fruits dried with hot air, vega-galvez et al. (2009) found ea = 48.34 kj/mol in blueberries. in another study done on hot air drying, abdulla (2012) found ea = 51.31 kj/mol for golden fruits. lópez et al. (2010) and shi et al. (2008) reported the ea values of blueberry as 57.85 kj/mol and 61.2 kj/mol, respectively. although the values found in some studies are similar to the values found in our study, others were higher. differences between the results of the current study and other studies, in which other fruits were used, may be due to different factors such as different fruit structures, temperature, airflow rate, and relative humidity. effect of drying process on water-soluble vitamins, total phenolic content, and antioxidant capacity effects of drying on water-soluble vitamins of goji berries are provided in table 3. carr and frei (1999) indicated that vitamin c easily scavenges nitrogen species and reactive oxygen and thereby may prevent oxidative damage to nontrivial biological macromolecules such as proteins, lipids, and dna. it is extremely important to maintain vitamin c during the drying process or to carry out this process with minimal loss; however, vitamin c is significantly affected by the drying process. in this study, value of vitamin c in fresh goji berries was found to be 112.75  ± 2.23 mg/100 g dw. donno et al. (2015) found the concentration of vitamin c in goji berries to be 42 mg/100  g fw (fresh weight). the united states department of agriculture (usda) has found the amount of vitamin c in dried goji fruit to be 48.4 mg/100 g (koçyiğit and sanlier, 2017). when compared with literature, it could be said that goji berries grown in turkey are rich in vitamin c. there are statistical losses in values of vitamin c at all drying temperatures (p  < 0.05). vitamin c values at 50°c, 60°c and 70°c were determined as 39.45 ± 2.21, 26.48 ± 1.16 and 21.87 ± 0.971 mg/100 g dw, respectively. since vitamin c has low stability against heat treatments, it is established as table 2. effective moisture diffusivity and activation energy of goji berry fruit. temperature d eff (m2/s) e a (kj/mol) e a (kcal/mol) 50°c 1.04 × 10-8 60°c 1.31 × 10-8 48.37 11.56 70°c 2.98 × 10-8 22 italian journal of food science, 2021; 33 (1) h.s. batu and ç. kadakal a quality index in foods during the processing (discala and crapiste, 2008). besides kinetic parameters, vitamin c may be a significant quality parameter in goji berries’ drying process. lópez et al. (2010) reported that there were significant loss in vitamin c values of blueberries at all drying temperatures, and the highest loss was 92% at 80°c. in a detailed review on ascorbic acid, santos and silva (2008) stated that a significant loss in ascorbic acid was seen due to hot air drying of fruits and vegetables. they even stated that no vitamin c was left in some drying processes applied on tomatoes over 100°c. other studies (araya-farias et al., 2011; kadakal et al., 2017) have demonstrated that the hot air-drying process significantly reduces the amount of vitamin c. in our study, the amount of vitamin b complex was analyzed in fresh goji berry fruits and the kinetic data were obtained during the drying process. the amount of pyridoxine (b6) in fresh goji berries was determined as 2.19 ± 0.046 mg/100 g dw but thiamine, riboflavin, and niacin were not detected. right after hot air-drying process at different temperatures, amount of pyridoxine was determined as 0.937 ± 0.055, 0.681 ± 0.061, and 0.49 ± 0.034 mg/100 g dw at 50°c, 60°c, and 70°c, respectively. the highest loss appears to be in the drying process at 70°c. ryley and kajda (1994) stated that loss in the values of water-soluble vitamins was observed with the effect of heat treatment in various foods. in consequence of drying at different temperatures, an important decrease in the amount of pyridoxine was observed in goji berries. decrease in the amount of water-soluble vitamin b6 increases with the increment in drying temperature. effects of hot air drying on total phenolic content and antioxidant capacity of goji fruits are presented in table 3. the tpc and ac values of fresh goji berries were found as 1838.43 ± 37.47 mg/100 g dw and 0.077 ± 0.002 mmol te/g dw, respectively. islam et al. (2017) determined the tpc value of red goji berry fruits as 217–448 mg gae/100 g. ban et al. (2015) determined the tpc value of fresh goji berries in the range of 449.92–450.48 mg gae/kg fw. zhang et al. (2016) determined the tpc values of goji berry fruits in the range of 5840–7340 mg gae/100 g fw. pedro et al. (2018) investigated tpc by extraction of goji berry fruits in different concentrations of methanol and found it in the range of 1052.53–1736.36 mg gae/100 g. the tpc of goji berry fruits because of drying processes at 50°c, 60°c, and 70°c was determined to be 491.00, 450.17, and 404.45 mg gae/100 g dw, respectively. islam et al. (2017) and zhang et al. (2016) determined the ac value of red goji berry fruits as 16.07–17.47 mg µmol te/g and 77.41–85.46 µmte/g fw, respectively. pedro et al. (2018) found the ac values of goji berry in the range of 0.94–1.51 mmol te/100 g. mikulic-petkovsek et al. (2014) stated that a significant difference in the content of fruits is seen when grown at different locations. the compositional difference seen in the same varieties of fruits and vegetables is influenced by numerous factors such as environmental conditions of the region where the product is grown, especially soil quality, cultivation technique and cultural measures, maturity level, transportation and storage, and so on (gökkür and çelik, 2016). the reason why our results are different from those found in literature may be due to the reasons explained above. table 3. effect of drying process on vitamins c and b 6 , total phenolic content, and antioxidant capacity of goji berries. vitamin c* loss percentage (%) pyridoxine (b 6 )* loss percentage (%) tpc** loss percentage (%) ac** loss percentage (%) fresh 112.75 ± 2.23a 0 2.19 ± 0.046a 0 1838.43 ± 37.47a 0 0.077 ± 0.002a 0 50°c 39.45 ± 2.21b 65.03 0.937 ± 0.055b 56.56 491.00 ± 7.96b 73.29 0.017 ± 0.001b 77.92 60°c 26.48 ± 1.16c 76.84 0.681 ± 0.061c 69.04 450.17 ± 8.26b 75.51 0.014 ± 0.001bc 81.82 70°c 21.87 ± 0.971d 80.5 0.492 ± 0.034c 77.48 404.45 ± 6.89c 78 0.011 ± 0.001c 85.71 *vitamins c and b 6 was expressed as mg/100 g dw. **tpc was expressed as mg gae/100 g dw, ac was expressed mmol te/g dw. ***different letters in the same column are significantly different values (p < 0.05). tpc, total phenolic content; gae, gallic acid equivalent; te, trolox equivalent; dw, dry weight. figure 2. the arrhenius-type relation between effective moisture diffusivity and 1/t. 2,9 0 5e-09 1e-08 1,5e-08 2e-08 2,5e-08 3e-08 3,5e-08 e ffe ct iv e m oi st ur e di ffu si vi ty (m 2 / s) 2,95 3 3,05 3,1 3,15 1/t×10–3 y = 0,6246e-5,818x r2 = 0,8977 italian journal of food science, 2021; 33 (1) 23 characteristics and degradation kinetics of goji berry on hot air drying color properties of goji berry fruit during hot air drying color properties of fresh and dried goji berries were presented in table 4. when compared with initial l*, a*, and b* values of goji fruits, values were significantly decreased due to drying process (p < 0.05) and the lowest l*, a*, and b* values were obtained at 70°c. δe indicates differences between colors of samples (horuz et al., 2017). the δe value of dried goji fruits depends on drying conditions and ranges from 10.87 to 13.91. the highest δe was obtained from the goji berry fruits dried at 70°c. kinetic parameters of vitamins c and b6 to the best of our knowledge, vitamin c degradation in the hot air-drying process of goji berries was investigated for the first time in this study. thermal degradation of vitamin c in goji berries is shown in figure 3; its content in fully dried goji berries is found to fit the first-order kinetic model. it is stated by gamboa-santos et al. (2014), hiwilepo-van hal et al. (2012), kadakal et al. (2017), and wang et al. (2017) that the thermal degradation of vitamin c fits the first-order kinetic model in different dried foods. air-drying may have a negative effect on the physical properties of products and cause degradation of aromatic compounds and nutrients (araya-farias et al., 2011). in other words, losses are observed in the compounds found in all foods, especially vitamin c, by heat treatment. dağhan et al. (2018) studied the hot air drying of isot at different temperatures and found that there was significant loss in vitamin c. they found the highest loss at 75°c and stated that vitamin c is highly sensitive to changes in temperature. marfil et al. (2008) performed tomato hot air drying at different temperatures and reported that the loss of vitamin c in tomatoes increased with increase in drying temperature. kinetic parameters of vitamin c in goji berries are presented in table 5. vitamin c degradation rate constants of goji fruits at 50°c, 60°c, and 70°c were found to be 0.047, 0.075, and 0.182 1/h, respectively. it is clearly observed that the rate constant increased but table 4. color properties of goji berry fruits. l* a* b* ∆e fresh 25.97 ± 0.12a 25.16 ± 0.13a 17.30 ± 0.05a 50°c 23.11 ± 0.09b 16.45 ± 0.07b 11.03 ± 0.11b 10.87 60°c 22.79 ± 0.05c 14.41 ± 0.09c 10.67 ± 0.07c 13.01 70°c 21.99 ± 0.06d 14.67 ± 0.08d 9.62 ± 0.05d 13.91 *different letters in the same column are significantly different values (p < 0.05). 50°c 0 2,5 3 3,5 in c in c in c 4 4,5 0,5 -1 -0,5 0 8 7,5 7 6,5 6 5,5 5 in c -2 -2,5 -3 -5 -3,5 -4 -4,5 5 5 10 15 20 (a) (b) (c) (d) 25 30 time (h) 60°c 70°c 50°c 0 5 10 15 20 25 30 time (h) 60°c 70°c 50°c 0 5 10 15 20 25 30 time (h) 60°c 70°c 50°c 0 5 10 15 20 25 30 time (h) 60°c 70°c figure 3. first-order kinetics of (a) vitamin c, (b) pyridoxine, (c) total phenolic content (tpc), and (d) antioxidant capacity (ac) of goji berries. 24 italian journal of food science, 2021; 33 (1) h.s. batu and ç. kadakal t1/2 and d values of vitamin c decreased due to the increment in temperature. similarly, demiray et al. (2013) stated that the k value increased with the increment in drying temperature. they also stated that the t1/2 value decreased with the increase of drying temperature in drying of tomatoes. kadakal et al. (2017) stated that the degradation rate constant of vitamin c was increased due to the thermal increase in rosehip nectar while the t1/2 and d values were decreased. our results are compatible with literature. also, the q10 value from 60°c to 70°c was found to be higher than from 50°c to 60°c. with this data obtained in our study, it is understood that the thermal degradation of vitamin c is more sensitive to the increment of temperature from 60°c to 70°c. the q10 value of vitamin c thermal degradation increased with the decrement in drying temperature (demiray et al., 2013; kadakal et al., 2017). kadakal et al. (2017) stated that high activation energy of reaction indicates that the reaction sensitivity of temperature is very high. the arrhenius equation of vitamin c thermal degradation is given in figure 4. to the best of our knowledge, the degradation of vitamin b6 in the hot air-drying process in goji berries has been investigated for the first time. thermal degradation of vitamin b6 is shown in figure 3, and the kinetic parameters of vitamin b6 thermal degradation are presented in table 5. the thermal degradation of vitamin b6 content in fully dried goji berries is found to fit the first-order kinetic model. vitamin b6 degradation rate constants of goji berries at 50°c, 60°c, and 70°c have been found to be 0.034, 0.064, and 0.164 1/h, respectively. rate constant increased, but the t1/2 and d values of vitamin b6 decreased due to the increment in temperature. kadakal et al. (2017) stated that the degradation rate constant in vitamin b complex increased due to thermal increase in rosehip nectar. also, the q10 value from 60°c to 70°c was found to be higher than that from 50°c to 60°c. when q10 values vitamins c and b6 were compared, it was understood that vitamin b6 is more sensitive to increase in temperature. the ea value of vitamin b6 was found to be 71.94 kj/mol. when the ea values of vitamins c and b6 were compared, the ea value of vitamin b6 was higher than that of vitamin c, which means that vitamin b6 is more stable than vitamin c. at the same time, vitamin b6 is more sensitivity to changes in temperature than vitamin c. kinetic parameters of total phenolic content and antioxidant capacity there are no data about the kinetic parameters of tpc in dried goji berries. the tpc thermal degradation is shown in figure 3, and the kinetic parameters of tpc thermal degradation are listed in table 5. the tpc thermal degradation rate constant increased and values of t1/2 and d decreased with the increment in drying temperature. thermal degradation of tpc content in fully dried goji berries was found to fit the first-order kinetic model. the rate constant of tpc thermal degradation in goji fruits ranged from 0.057 to 0.179 1/h. tpc thermal degradation increases depending on the increment of temperature (kadakal and duman, 2018; sarpong et al., 2018). lópez et al. (2010) reported that the tpc value decreased with increase in the temperature of drying air. the activation table 5. first-order kinetic parameters of vitamins c and b 6 , total phenolic content, and antioxidant capacity of dried goji berries. compound temperature k t 1/2 d r2 e a e a q 10 q 10 (1/h) (h) (h) (kcal/mol) (kj/mol) (50–60°c) (60–70°c) vitamin c 50°c 0.047 14.62 48.59 0.984 14.75 61.72 1.58 2.44 60°c 0.075 9.28 30.83 0.993 70°c 0.182 3.81 12.66 0.989 pyridoxine (vitamin b 6 ) 50°c 0.034 20.2 67.14 0.986 17.19 71.94 1.85 2.59 60°c 0.064 10.91 36.27 0.982 70°c 0.164 4.22 14.03 0.989 tpc 50°c 0.057 12.07 40.12 0.990 12.43 52.01 1.39 2.25 60°c 0.080 8.72 28.97 0.986 70°c 0.179 3.88 12.90 0.984 ac 50°c 0.060 11.49 38.19 0.989 13.12 54.90 1.53 2.17 60°c 0.092 7.53 25.03 0.976 70°c 0.199 3.48 11.55 0.954 tpc, total phenolic content; ac, antioxidant capacity. italian journal of food science, 2021; 33 (1) 25 characteristics and degradation kinetics of goji berry on hot air drying conclusions in this study, for the first time, drying characteristics and thermal degradation of some ingredients in goji berry (lycium barbarum l.) grown in turkey were investigated under different drying conditions. page model was determined to be the best model to predict experimental moisture ratio at all drying temperatures (50°c, 60°c, and 70°c). drying temperature affects the drying speed and drying time. drying time ranged from 9 to 24 h at 50–70°c. with increase in drying temperature, effective moisture diffusivity increased and the highest effective moisture diffusivity was determined at 70°c. the drying process showed losses in vitamins c and b6, tpc, and ac, and the highest loss was observed at 70°c. the highest percentage loss was found in ac. the thermal degradation of vitamins c and b6, tpc, and ac is found to fit the first-order kinetic model, and the drying rate values of all these in goji berries increased by drying temperature increment. vitamins c and b6 were very susceptible to temperature increment, but tpc and ac were the lowest sensitive compounds in dried goji berries. the highest color difference (δe) was obtained in the goji berries dried at 70°c. the shortest drying time was observed in the goji berries dried at 70°c, and the drying process at 50°c provided the highest retention of bioactive compounds in goji berries. according to the data obtained and evaluated, energy was calculated using the arrhenius equation presented in figure 4 and found to be 52.01 kj/mol. the q10 value from 60°c to 70°c was found to be higher than that from 50°c to 60°c. thus, the thermal degradation of tpc is more sensitive to the increment of temperature from 60°c to 70°c. to the best of our knowledge, ac thermal degradation in dried goji fruits was studied for the first time in the current study. the ac thermal degradation is shown in figure 3, and the kinetic parameters of ac thermal degradation are given in table 5. in the current study, thermal degradation of ac in fully dried goji berries was found to fit the first-order kinetic model. oancea et al. (2017) used the first-order kinetic model on ac thermal degradation in sour cherry extract. owing to temperature increment, the rate constant increased but t1/2 and d values of ac decreased. oancea et al. (2017) and sarpong et  al. (2018) reported that the rate constant of ac increased with increase in temperature in sour cherry extract and banana slices, respectively. the arrhenius equation of ac thermal degradation is presented in figure 4 and ea was found to be 54.90 kj/mol. the q10 values from 50°c to 60°c and from 60°c to 70°c were found as 1.53 and 2.17, respectively. in this context, the increment in q10 value from 60°c to 70°c indicates that the thermal degradation of ac is more sensitive than that in the range of 50–60°c. figure 4. arrhenius plots of dried goji berries: (a) vitamin c, (b) pyridoxine, (c) total phenolic content (tpc), and (d) antioxidant capacity (ac). 3,5 3 2,5 2 1,5 1 0,5 2,9 2,95 3 3,05 3,1 3,15 0 1/t×10–3 -in k y = 7,4233x 19,856 r2 = 0,9597 3,5 4 3 2,5 2 1,5 1 0,5 2,9 2,95 3 3,05 3,1 3,15 0 1/t×10–3 -in k y = 8,6533x 23,356 r2 = 0,9806 3,5 3 2,5 2 1,5 1 0,5 2,9 2,95 3 3,05 3,1 3,15 0 1/t×10–3 -in k y = 6,2561x 16,427 r2 = 0,9347 3,5 3 2,5 2 1,5 1 0,5 2,9 2,95 3 3,05 3,1 3,15 0 1/t×10–3 -in k y = 6,6036x 17,574 r2 = 0,9661 (a) (b) (c) (d) b 26 italian journal of food science, 2021; 33 (1) h.s. batu and ç. kadakal the optimal drying temperature for goji berries is 50°c in hot air drying. as additional studies, research should be conducted on obtaining dried goji berries with different and combined drying method, which could be a more efficient drying process with less component loss. also, the content differences in goji berries grown at different locations should be investigated. acknowledgments this study was supported by pamukkale university with grant number 2018febe026. references abdulla, g., 2012. effect of hot air temperature on drying kinetics of golden berry. zagazig journal of agricultural research 39(4): 665–673. adiletta, g., alam, s.r., cinquanta, l., russo, p., albanese, d. and di matteo, m., 2015. effect of abrasive pretreatment on hot dried goji berry. chemical engineering transactions 44: 127– 132. https://doi.org/10.3303/cet1544022 amagase, h. and farnsworth, n.r., 2011. a review of botanical characteristics, phytochemistry, clinical relevance in efficacy and safety of lycium barbarum fruit (goji). food research international 44(7): 1702–1717. https://doi.org/10.1016/j. foodres.2011.03.027 araya-farias, m., makhlouf, j. and ratti, c., 2011. drying of seabuckthorn (hippophae rhamnoides l.) berry: impact of dehydration methods on kinetics and quality. drying technology 29(3): 351– 359. https://doi.org/10.1080/07373937.2010.497590 ban, z., wei, w., yang, x., feng, j., guan, j. and li, l., 2015. combination of heat treatment and chitosan coating to improve postharvest quality of wolfberry (lycium barbarum  l.). international journal of food science & technology 50(4): 1019–1025. https://doi.org/10.1111/ijfs.12734 bertoldi, d., cossignani, l., blasi, f., perini, m., barbero, a., pianezze, s. and montesano, d., 2019. characterization and geographical traceability of italian goji berries. food chemistry 275: 585–593. https://doi.org/10.1016/j.foodchem.2018.09.098 bi, j., yang, a., liu, x., wu, x., chen, q., wang, q., jian, l., wang, x., 2015. effects of pretreatments on explosion puffing drying kinetics of apple chips. lwt—food science and technology 60(2): 1136–1142. https://doi.org/10.1016/j.lwt.2014.10.006 burke, d.s., smidt, c.r. and vuong, l.t., 2005. momordica cochinchinensis, rosa roxburghii, wolfberry, and sea buckthorn— highly nutritional fruits supported by tradition and science. current topics in nutraceutical research 3(4): 259–266. carr, a. and frei, b., 1999. does vitamin c act as a pro-oxidant under physiological conditions? the faseb journal 13(9): 1007–1024. https://doi.org/10.1096/fasebj.13.9.1007 chen, j., chao, c.t. and wei, x. 2018. gojiberry breeding: current status and future prospects. in: soneji, j.r. and nageswararao,  m. 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tommaso beccari1* 1department of pharmaceutical sciences, university of perugia, perugia, italy; 2department of chemistry, biology and biotechnologies, university of perugia, perugia, italy; 3department of veterinary medicine, university of milan, lodi, italy *corresponding author: tommaso beccari, department of pharmaceutical sciences, university of perugia, via fabretti 48, 06123 perugia, italy. email: tommaso.beccari@unipg.it received: 13 june 2022; accepted: 30 june 2022; published: 2 september 2022 © 2022 codon publications open access paper abstract goji berries are the most cultivated fruit crop in asian countries as they contain many nutrients and healthpromoting bioactive compounds. these health-promoting properties have recently stimulated the interest of food and nutraceutical industries in europe, so this crop has spread within italy, which has become the largest european producer. several works on the chemical composition and biological activities of chinese goji berries are available. in this review, the chemical and the nutritional profile of goji berries from licium barbarum spp. cultivated in italy are reported. keywords: goji berries; licium barbarum spp; bioactive compounds; antioxidant properties; anticancer activities introduction goji berry or wolfberry is a bright orange-red berry produced by perennial plants of the genus lycium, belonging to the solanacee family (aronson, 2006). of late, goji berries (gbs) used in traditional chinese medicine are becoming very popular also in the occidental world because of their properties, such as antioxidant (ma et al., 2019), anti-aging (gao et al., 2017), anti-cancer (ceccarini et al., 2016a; georgiev et al., 2019), neuroprotective (xing et al., 2016), antidiabetic (zhao et al., 2020), immunomodulatory (yang et al., 2013), anti-inflammatory (chen et al., 2020), and others activities, such as hormonal profile and reproductive performance in livestock animals (andoni et al., 2021; brecchia et al., 2021; agradi et al., 2022). among lycium (l.) species (spp), l. barbarum spp is definitely the most commercially widespread species given its high nutritional and medicinal value. the l. barbarum plant is a perennial plant that mainly grows in the ningxia hui region in north-central china, the xinjiang region in western china, and also in tibet and in mongolia (potterat, 2010). this plant is highly tolerant to adverse environmental conditions and grows in salinity regions and at different altitudes ranging from 700 to 2700 m (kruczek et al., 2020). in china, l. barbarum fresh leaves are much used in food dishes such as soups or used as herbal tea (crawford, 2012) while the fruits are squeezed for their juice or used fresh or dried (donno et al., 2015). the dried fruits and leaves have also been used for medicinal purposes, as a traditional chinese medicine, for thousands of years (zhu et al., 2013). according to the traditional chinese pharmacopeia (tpc), lycium spp can be used to treat various diseases including blurry vision, glaucoma, diabetes, kidney failure, cancer, cough, asthma, metabolism/ energy expenditure, and aging (amagase et al., 2009). many studies have shown that goji fruits have antioxidant, hypoglycemic, anticancer, and immunomodulatory effects so that supplementation of a regular diet with these berries can help to prevent many age-related italian journal of food science, 2022; 34 (3): 59–65 10.15586/ijfs mailto:tommaso.beccari@unipg.it 60 italian journal of food science, 2022; 34 (3) ceccarini mr et al. diseases (gao et al., 2017; ma et al., 2019; ceccarini et al., 2016b; menchetti et al., 2020). these health-promoting properties have also recently stimulated the interest of food and nutraceutical industries in europe (bae et al., 2017). this crop has spread within italy, which has become the largest european producer (knowles, 2016). several papers on the chemical composition and biological activities of chinese gbs are available (wang et al., 2021; lu et al., 2021), less of italian gbs (lopatriello et al., 2017). this review aims to analyze the chemical and the nutritional profile of gbs from l. barbarum spp. cultivated in italy and its application as a functional food. nutritional composition of italian gbs the nutritional composition of fresh gbs cultivated in italy was investigated by niro and montesano (niro et al., 2017; montesano et al., 2018). both research groups analyzed gbs samples from southern italy. the nutritional composition of italian gbs is reported in table 1. the results are very similar and the small differences may depend on some variables such as different methods of preparation and analysis of the samples. the nutritional values make gbs an excellent source of macronutrients, especially carbohydrates. they are present as highly branched water-soluble l. barbarum polysaccharides and represent 5–8% of the total dry matter of the berries. several studies indicate that the high biological activity components in gbs are polysaccharides (amagase and farnsworth, 2011). the mixture of l. barbarum polysaccharides exerts a retinal ganglion cell protection and reduces the oxidative stress in retinal/ reperfusion injury (li et al., 2011; mi et al., 2012) and in high-fat diet fed mice (ming et al., 2009) in which a decrease in total cholesterol, ldl-cholesterol, and triglycerides, and an increase in hdl-cholesterol have been observed (luo et al., 2004). thanks to the lipid-lowering and hypoglycaemic effects, the gbs exert a protective effect on the cardiovascular system (ma et al., 2019). moreover, yang et al. demonstrated the immunomodulatory properties of l. barbarum polysaccharides that increased the phagocytosis and nitric oxide production of raw 264.7 macrophages (yang et al., 2015). gbs have a low protein content (2–2.5 g/100g) and therefore they could not be considered a good source for protein. however, it has been shown that these fruits contained 17 of the 20 protein amino acids, including all eight essential amino acids (lu et al., 2021). carotenoid composition of italian gbs the second highly significant group of biologically active molecules present in gbs are carotenoids, the coloured components of goji fruits. the total carotenoid content of different l. barbarum fruits ranges from 0.03% to 0.5% of dried fruits with zeaxanthin dipalmitate representing more than 75% of the total carotenoids (cenariu et al., 2021). table 2 shows the total carotenoid and zeaxanthin dipalmitate content, expressed in milligram per 100 g of italian dried fruits according to bertoldi and niro (bertoldi et al., 2019; niro et al., 2018). the study of bertoldi et al. showed that the average total carotenoid content of asian gbs (197.8 mg/100g) was just over half of the italian ones (355 mg/100g). these data are of particular importance as it means that italian gbs have a biological and nutritional value higher than those of asian origin. instead, the data obtained by niro showed levels of carotenoid and zeaxanthin palmitate similar to those of asian origin. the discrepancy between the data may depend on the different methods used for their determination or to ecological factors and cultural practices (arena and curvetto, 2008). goji antioxidative power has carotenoid content (kulczynski and gramza-michalowska, 2016; chang and so, 2008). many studies have demonstrated a correlation between antioxidant activity of l. barbarum fruit and its antitumor (potterat et al., 2010; ceccarini et al., 2016a; georgiev et al., 2019), anti-inflammatory (amagase and farnsworth, 2011; chen et al., 2020), and immunomodulatory (yang et al., 2013) activities. cenariu et al. have indicated the cytoprotective activity of a zeaxanthin-rich extract from l. barbarum berries on tumor-derived a375 skin cell line through mitogenactivated protein kinase (mapk) influence (cenariu et al., 2021). table 1. average nutritional composition (g/100g) of italian fresh goji berries (mean± standard deviation). moisture fats proteins carbohydrate fiber ash montesano et al., 2018 78±1.5 0.2±0.01 2.5±0.01 16.5±1.8 2.0±0.5 0.8±0.02 niro et al., 2017 77.4±1.8 1.1±0,05 2.5±0.02 15.3±1.3 2.9±0.8 0.8±0.03 table 2. average total carotenoid and zeaxanthin dipalmitate content (mg/100g) in italian dried goji berries (mean± standard deviation). total carotenoids zeaxanthin dipalmitate bertoldi et al., 2019 355±49 245±49 niro et al., 2018 184±4 159±2 italian journal of food science, 2022; 34 (3) 61 licium barbarum cultivated in italy: chemical characterization and nutritional evaluation mineral composition of italian gbs gbs are also extremely rich in many minerals which are essential components for a balanced diet. indeed, adequate mineral intake is essential for many human vital functions such as oxygen transport, muscle contraction, enzyme activation, blood acid–base balance, bone structure, nerve impulse conduction, heart contraction, antioxidant system activity, and immune system activation (williams, 2005). sodium and potassium, for example, are macro elements essential for membrane depolarization and body water balance, calcium for mineral bone structure and muscle and heart contraction, magnesium is a cofactor of more than 300 enzymes and is required for energy production and nucleic acid synthesis (sá et al., 2019). the average values of principal macro elements in dried italian gbs are shown in table 3. it was observed that potassium (k) is the main macro element of the italian and asian gbs. anyway, the k concentration determined by bertoldi in italian gbs was about twice the average values found in asian gbs (endes et al., 2015). high dietary k intake (>3.5g/day) is associated with a decrease in blood pressure as documented in several clinical studies (binia et al., 2015). sodium (na) concentrations were very similar in all italian samples but about a third of that was found in the asian gbs (llorentmartinez et al., 2013). many studies suggest a direct relationship between na intake and blood pressure values (mente et al., 2014). indeed, excessive na intake (defined as >5 g na per day (who, 2012)) has been shown to produce a significant increase in blood pressure and has been linked with the onset of hypertension and its cardiovascular complications (weinberger, 1996; strazzullo et al., 2009). also, phosphorus (p), magnesium (mg), and calcium (ca) contents were rather different in the two studies. the p value of the bertoldi et al. study was particularly high even when compared to data obtained for asian gbs (270 mg/100g). these discrepancies can be due to mineral variation in the soil and water source mineral quality and differences in growing conditions. referring to recommended dietary allowances (rda) and adequate intake (ai), a daily portion of about 30 g of dried gbs provides on average 6–9% of rda for mg and p, 6–9% of ai for k and na, and 1–2% of rda for ca (bertoldi et al., 2019). in addition, gbs can be a good source of microelements. the most abundant micro elements in gbs are reported in table 4. iron (fe) is a very important mineral for living organisms since it participates in a wide variety of metabolic processes such as oxygen transport, electron transport, and dna synthesis (lieu et al., 2001). zinc (zn), copper (cu), and manganese (mn) are essential elements that principally act as cofactors of different enzymes, which participate in all the major metabolic pathways (agget and harries, 1979; cox, 1999; eirkson and aschner, 2003). a similar amount of fe was found by the two research groups and these values were about half of that found in the asian gbs (bertoldi et al., 2019). zn, cu, and mn contents from bertoldi et al. samples were about two times higher than asian berries, whereas the zn, cu, and mn values from niro et al. were similar to those of the asian gbs (bertoldi et al., 2019). referring to rda, a daily portion of about 30 g of dried gbs provides on average 20% of rda for cu, 15% of rda for fe, and 4% of rda for zn, whereas referring to ai, the same portion provides on average 10% of ai of mn (bertoldi et al., 2019). toxic trace elements present in italian gbs gbs may also contain some toxic elements that can have a negative impact on human health (bordean et al., 2011). bertoldi et al. also investigated italian gbs for their toxic metal concentrations by comparing them with asian gbs. the average values of the main toxic trace elements measured in italian gbs are shown in table 5. regarding arsenic (as), a daily portion (30 g) of italian gbs provides around 12 µg which is one-third less than asian goji’s as content (18 µg/30g). daily intake of a portion of 30 g of italian gbs provides 5.8 µg of cadmium (cd) which is about twice the asian goji’s average cd content. thirty grams of italian gbs also provides 0.17 µg of mercurius (hg), a value slightly higher than that found in asian gbs (0.11 µg/30g). finally, a daily portion table 3. average values (mg/100g) of the main macro elements in dried italian gbs (mean± standard deviation). k na ca p mg bertoldi et al., 2019 2079±403 264±7 72±17 317±60 134±27 niro et al., 2018 882±239 209±72 101±23 174±32 46±9 k, potassium; na, sodium; ca, calcium; p, phosphorus; mg, magnesium. table 4. average values (mg/100g) of the main trace minerals in dried italian goji berries (mean± standard deviation). fe zn cu mn bertoldi et al., 2019 3.9±1.8 3.5±1.3 1.3±0.8 1.1±0.2 niro et al., 2018 3.5±1.5 1.5±0.6 0.8±0.2 0.5±0.2 fe, ferrum; zn, zincum; cu, cuprum, mn, manganesum. table 5. average values (µg/100g) of the main toxic trace elements in italian gbs (mean± standard deviation). as cd hg pb bertoldi et al., 2019 40.7±0.5 19.3± 2.7 5.1±0.5 84.3± 5.5 as, arsenicum; cd, cadmium; hg, mercurius, pb, plumbum. 3.5g/day 62 italian journal of food science, 2022; 34 (3) ceccarini mr et al. of italian gbs provides 25.3 µg of plumbum (pb) that represent a significant amount of the daily dietary intake (0.02–3 µg/kg bw/day), but it is one sixth of that found in asian berries (bertoldi et al., 2019). thus, concerning toxic elements, italian gbs are better than asian gbs. however, caution should be used in taking large amounts of these fruits because excessive intake can overload the body with toxic mineral (gogoasa et al., 2014). fatty acid and sterol composition of italian gbs cossignani et al. studied fatty acid and sterol composition of gbs samples from italy, china, and mongolia in order to distinguish goji samples of different production areas (cossignani et al., 2018). table 6 shows average lipid percentage and the average percentage values of saturated (sfa), monounsaturated (mufa), and polyunsaturated (pufa) fatty acids from the previously mentioned three origins. as can be seen, the lipid percentage of italian gbs samples was higher than that of asian samples, in accordance with literature data (endes et al., 2015; rosa et al., 2017). in addition, italian gbs samples compared to asian ones are richer in saturated (18.9% vs 14 and 16.4 % of chinese and mongolian samples) and monounsaturated (31.7% vs 20.8 and 21.2 % of chinese and mongolian samples) fatty acids but poorer in polyunsaturated fatty acids (48.4% vs 64.2 of chinese and mongolian samples). anyway, the pufa was the most abundant fraction in all samples and the linoleic acid (c18:2 n-6) was the most abundant pufa acid for both italian and asian samples, followed by α-linolenic acid (c18:3 n-3), most abundant in italian samples with respect to asian samples (cossignani et al., 2018). the low percentage of lipids combined with the richness in pufa makes gbs a new dietary source of essential fatty acids, especially linoleic acid. cossignani and co-workers also studied the sterol fraction of gbs from italy, china, and mongolia. to this end, alkaline hydrolysis was carried out on goji lipid fraction to obtain data about percentage and quantitative composition (mg/100g) of the main phytosterols identified in italian and asian samples (table 7). it can be noted that δ5-sterols were the major components compared to δ7-sterols and that β-sitosterol was the most abundant sterol in italian and chinese samples while δ5-avenasterol was the most abundant sterol in mongolian samples. β-sitosterol, the predominant component in italian samples (53.7%), is known to lower blood cholesterol levels in humans and also for its strong free radical scavenging activity (lin et al., 2014). in addition to these properties, phytosterols possess anti-inflammatory, anti-cancer, and anti-atherogenicity activities (berger et al., 2004). the nutritional quality of the lipid and sterol fractions of the italian gbs was confirmed through the determination table 7. average values (% and mg/100g) of sterol composition in goji samples of different origin (mean± standard deviation). italy (n=9) italy (n=9) china (n=6) china (n=6) mongolia (n=4) mongolia (n=4) % mg/100g % mg/100g % mg/100g sterols cholesterol 7.4±2.9 3.3±1.8 5.2±0.5 2.9±0.2 4.5±0.4 6.0±0.7 ergosterol 12.7±3.8 5.4±2.1 13.8±0.2 7.8±0.8 12.8±0.8 16.4±1.2 stigmasterol 4.4±2.3 2.1±1.8 11.6±1.3 6.4±0.4 6.0±0.9 8.7±1.1 δ-5,23-stigmastadienol 3.6±2.8 1.2±1.0 12.6±0.3 6.9±0.3 20.1±1.5 25.8±2.2 β-sitosterol 53.7±6.5 22.8±5.4 39.4±2.5 21.4±1.0 25.9±2.0 31.7±3.1 sitostanol 4.8±3.0 2.3±1.9 1.0±0.1 0.6±0.2 0.6±0.2 1.1±0.2 δ5-avenasterol 9.7±3.6 3.9±1.3 14.5±1.5 7.8±07 26.5±2.3 35.5±3.5 δ7-stigmasterol 0.6±0.5 0.2±0.2 0.8±0.2 0.5±0.2 1.2±0.3 1.9±0.5 δ7-avenasterol 2.8±1.8 1.5±1.3 1.2±0.3 0.8±0.1 2.0±0.5 2.7±0.8 table 6. average values (% mol) of fatty acid composition of goji samples of different origin (mean± standard deviation). italy (n=9) china (n=6) mongolia (n=4) lipid % 5.1±2.1 2.0±0.7 3.3±1.2 fatty acid c16:0 14.6±4.6 9.5±2.4 11.7±0.6 c16:1 0.6±0.4 0.4±0.1 0.2±0.1 c18:0 2.9±0.8 3.2±0.2 3.3±0.0 c18:1 31.7±19.7 21.2±0.3 20.8±0.1 c18:2 (n-6) 40.5±14.8 56.5±2.9 57.2±0.2 c18:3 (n-6) 0.6±0.4 2.6±0.1 1.3±0.3 c18:3 (n-3) 7.3±6.0 5.1±0.6 3.8±0.2 c20:0 0.5±0.3 0.5±0.0 0.7±0.1 c22:0 0.4±0.3 0.5±0.0 0.5±0.2 c24:0 0.5±0.2 0.3±0.0 0.2±0.0 italian journal of food science, 2022; 34 (3) 63 licium barbarum cultivated in italy: chemical characterization and nutritional evaluation of the atherogenicity (ai), thrombogenicity (ti) indexes, and hypocholesterolemic (hi) index since the low values of these indexes (0.1% ai, 0.2% ti, and 10% hi, respectively) showed a good amount of anti-atherogenic lipids in the examined samples (ulbricht and southgate, 1991; fernandez et al., 2007). total tocopherols and ascorbic acid amounts in italian gbs niro’s research group also determined the average total tocopherols (vit e) and ascorbic acid (vit c) amounts in fresh and dried italian gbs samples as reported in table 8. taking into account the rda for vit e, which is 12 mg/ day (regulation eu1169/2011) and rda for vit c which is 80 mg/day (regulation eu1169/2011), a portion of dried gbs (30 g) provides about 20% of the rda for vit e and 16% of the rda for vit c. donno et al. (2015) reported an amount slightly higher (42 mg/100 g) of vit c in italian dried gbs from northern italy. health benefits of italian gbs antioxidant properties together with anticancer activity of italian gbs were investigated. this berry has been described as a “super-fruit” thanks to its concentrated levels of beneficial substances and anti-oxidant powers. the antioxidant properties of italian l. barbarum fruits (cultivated in umbria) have been evaluated by determining the total phenolic content (tpc) and the oxygen radical absorbance capacity index (orac) (ceccarini et al., 2016b). the tpc value, expressed as milligram of gallic acid equivalents (mg gae) per 100 g of dry weight (dw) resulted in 1278.247 ± 29.60 mg gae/100g dw. these results showed that italian gbs have a tpc higher than the asian one (712.01 ±29.12ggae/100g), whereas the orac value, expressed as micromoles of trolox equivalent per 100 g dw (µmol te/100g dw), was slightly lower than the asian one (22507.03±1402.02 µmol te/100 g dw vs 26502±3807 µmol te/100g dw). these data showed that gbs cultivated in italy have high antioxidant properties. moreover, italian fruits (cultivated in umbria) have also been tested for their apoptotic and antiproliferative effects on human hepatocellular carcinoma (hepg2) cell line (ceccarini et al., 2016b). to this end, a panel of 96 genes involved in oxidative stress, proliferation, and apoptosis was investigated in hepg2 cells using a quantitative real-time pcr-array analysis. downregulation of genes involved in tumor migration and invasion together with upregulation of tumor suppressor genes suggests that umbrian gbs play an anticancer role in vitro and could play a role against hepatocellular carcinoma (ceccarini et al., 2016b). conclusions despite the limited literature data concerning the chemical and nutritional characterization of gbs cultivated in italy, it is possible to conclude that these fruits are an important source of bioactive compounds. in 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https://doi.org/10.1155/2019/2437397 https://doi.org/10.1155/2019/2437397 https://doi.org/10.1016/j.meatsci.2019.108018 https://doi.org/10.1056/nejmoa1311989 https://doi.org/10.1056/nejmoa1311989 https://doi.org/10.1371/journal.pone.0045469 https://doi.org/10.1371/journal.pone.0045469 https://doi.org/10.1016/j.foodchem.2008.03.064 https://doi.org/10.1016/j.foodchem.2008.03.064 http://researchgate.net/publication/321007006 http://researchgate.net/publication/321007006 p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33i2.1945 13 p u b l i c a t i o n s codon riboflavin removal by commercial bentonites and charcoals in white and red wines veronica vendramin1,2, simona primerano1,3, giampiero leserri2, giulio paniccia2, simone vincenzi1,3* 1centre for research in viticulture and enology (cirve), university of padova, viale xxviii aprile 14, 31015 conegliano (tv), italy; 2department of land, environment, agriculture and forestry (leaf), university of padova, viale dell’università 16, 35020 legnaro (pd), italy; 3department of agronomy, food, natural resources, animals and environment (dafnae), university of padova, viale dell’università 16, 35020 legnaro (pd), italy *corresponding author: centre for research in viticulture and enology (cirve), university of padova, viale xxviii aprile 14, 31015 conegliano (tv), italy. email: simone.vincenzi@unipd.it received: 19 august 2020; accepted: 26 february 2021; published: 1 april 2021 © 2021 codon publications open access paper abstract riboflavin (rf) represents one of the primary molecules undergoing photodegradation in wine, and its excited form acts as an intermediate in light-induced oxidation reactions responsible for the light-struck fault. a recent study has revealed bentonites (bens) and charcoals (chas) as the most promising fining agents for removal of rf in model wine. this work explored their potential on both white and red wines, where polyphenols could interfere in the fining agent–rf interaction. a total of 11 bens and 11 chas were compared. bens exhibited a limited capacity, while decoloring carbons confirmed a great attitude for removal of rf in white wine, even at low dosages. nevertheless, efficiency of chas shows a sensible reduction in red wine. keywords: bentonite, charcoal, red wine, riboflavin removal, white wine introduction shelf life of wine assumes a complicated meaning because of high diversity in both wine styles and typical characters searched by consumers. the quality of final product depends on both winemaking practices (fermentation and post-fermentation fining treatments) and storage in bottles. maturation of wine should be strictly controlled to avoid undesirable chemical reactions, with particular attention to temperature and light conditions during transportation to the final seller and its often long storage. the post-fermentation is particularly critical for red wine because it commonly requires more time for maturation. exposure to light could have two independent effects on wine: first, it could activate oxidative reactions, and second, it could cause heatinduced damages on direct exposure to sunlight (jackson, 2011). in order to limit light-induced oxidation, the best protection is represented by storage in dark conditions and use of ultraviolet (uv)-masking bottles. it is known that green glass bottles can filter a larger spectrum of uv-visible (vis) wavelengths than uncolored bottles, thus reducing the rate of photodegradation reactions (grant-preece et al., 2017). however, this technological requirement is in contrast with the consumer preference for uncolored bottles, which is rapidly growing along with market for rosè wines. riboflavin (rf) assumes a particular relevance in wine’s shelf life because of its implication in intermolecular photoreduction. in fact, rf is one of the principal responsible for the development of light-struck taste, a wine fault characterized by ‘cooked-cabbage’ aroma. rf is thermostable at the temperature of winemaking process, but it is highly photosensitive and can easily undergo photochemical degradation (sheraz et al., 2014). in aqueous solutions, rf is implicated in photosensitization reaction that acts following two mechanisms. the type 1 pathway results in the formation of two charged free radicals, rf and a italian journal of food science, 2021; 33 (2): 13–23 mailto:simone.vincenzi@unipd.it 14 italian journal of food science, 2021; 33 (2) vendramin v et al. materials and methods chemicals and reagents methanol, trifluoroacetic acid (tfa), tartaric acid, acetic acid, and sodium acetate were purchased from sigmaaldrich (milano, italy). enocyanin powder (enocianina gse12 uc) was provided by ever s.r.l. (pramaggiore, italy). water of hplc grade was obtained from milli-q system (millipore filter, bedford, ma, usa). bentonites and charcoals eleven bens, two calcic (cal), one sodic–calcic (sodcal), and eight sodic (sod), and 11 chas, four deodorizing (deo) and seven dec, were provided by different commercial suppliers. wine selection the wines were selected from different wine samples for their medium–high rf content. one white wine (glera base wine, harvest 2018, produced by school of oenology cerletti, conegliano (tv), italy), with a content of 123.9 µg/l of rf, was chosen for ben trials. this wine showed 9.6% alcohol and 6.5 g/l of titratable acidity. one white wine (glera base wine) and another red wine (wildbacher), both produced by collalto winery (susegana (tv), italy), with 104.0 µg/l and 138.6 µg/l of rf, respectively, were chosen for cha treatments. glera wine (harvest 2018) showed 11.6% alcohol and 6.2 g/l of titratable acidity, while wildbacher (harvest 2018) showed 12.5% alcohol and 5.1 g/l of titratable acidity. bentonite’s protein adsorption trial deproteinization capability of bens was evaluated according to modified oeno 441-2011 resolution (the international organization of vine and wine [oiv], 2011). trial solution was prepared using bovine serum albumin protein (bsa) 500 mg/l instead of ovalbumin 5 g/l, and the protein was dissolved into model wine (5 g/l tartaric acid, 12% v/v ethanol, ph = 3), whereas in the oiv method, the ethanol was absent. eight ben dosages (namely 10, 20, 30, 40, 50, 60, 70, 80 g/hl) were tested in order to determinate ben adsorption curves. samples were shaken and maintained in dark at 25°c for 30 min before performing protein quantification using a pierce bca protein assay kit (fischer scientific italia, rodano (mi), italy). after an incubation of 30 min at 37°c, samples were read using microplate reader (euroclone) and data were elaborated by software manta and quantified over a calibration curve of bsa between 12.5 mg/l and 1000 mg/l. target molecule, through hydrogenor electron-transfer reactions between the excited triplet state of rf (3rf1) and the substrate. when this substrate is methionine (met), it leads to the formation of methional. while rf radical is involved in a recycling reaction, methional is unstable, and thus readily decomposes to methanethiol (mesh) and acrolein. in addition, two molecules of methional can combine into dimethyl disulfide (dmds). the type 2 process uses the energy transferred from 3rf1 to oxygen to form singlet oxygen, which can then react with multiple biological substrates. in light-struck reactions, it oxidizes methionine sulfur, generating methionine sulfoxide (silva et al., 2019). mesh and dmds are the main compounds responsible for the ‘cooked cabbage’ aroma of light-struck reaction; they have a perception threshold of 2–10 µg/l and 20–45 µg/l, respectively (fracassetti et al., 2019). in a recent study, fracassetti and colleagues (2019) explored the relationship between met and rf and verified that met degradation could be avoided if rf concentration remains below 50 µg/l. in a different study, the same authors compared different fining agents with the aim to determine the clarification practice that could be the most efficient one for removal or lowering of rf (fracassetti et al., 2017). this study compared several fining agents, namely polyvinylpolypyrrolidone (pvpp), bentonite (ben), zeolite, silica, kaolin, albumin, and charcoal (cha), using different concentrations in model wine, and identified ben and cha as the most promising agents. however, the efficacy of fining agents seems strictly dependent on the media composition, as demonstrated by comparison of ben, cha, and zeolite performance in both model wine and a real chardonnay wine (fracassetti et al., 2017). in this case, the cha removal efficacy was lower in real wine at all the tested dosages. from the applicative point of view, it would be interesting to understand whether different categories of bens and chas could be used successfully in real wines. besides white wines, which are commonly preferred for rf studies because of being more subjected to light-struck fault, red wines represent an interesting case of study because, as reported by lagunes et al. (2017), their extracts could act as photosensitizer if exposed to light, generating 1o2, which in turn is able to oxidize other compounds. moreover, polyphenols make more complex the removal of rf because of the high affinity of phenols to chas (lisanti et al., 2017). a fine balance between the quenching and the photosensitizing nature of red wine polyphenols is of particular interest for monitoring the removal of rf in this kind of wine. in the present study, different commercial bens and chas, provided by different suppliers, were compared in two wines to explore deeply their ability to remove rf. in particular, it was verified whether this ability is correlated to other fining properties, such as protein removal and decolorizing (dec) capacity. italian journal of food science, 2021; 33 (2) 15 riboflavin removal by commercial bentonites and charcoals in white and red wines wine and sum of the 420, 520, and 620 nm absorbance values for the red wine. quantification of riboflavin in high-performance liquid chromatography (hplc) a nexera hplc system (shimadzu) equipped with rf 20-a xs fluorescence detector was used. filtered samples (20 µl) were separated in a kinetex c18 (5 μm, 100  å, 150 × 4.6 mm phenomenex). eluting solvents were as follows: (a) milli-q water and 0.1% of tfa v/v and (b)  gradient-grade methanol and 0.1% of tfa v/v. the gradient program was 0–2 min, 30% b; 2–10 min, 30–60% b; 10–11  min, 60–100% b; 11–14 min, 100% b; 14–15 min, 100–30% b; and 15–18 min, 30% b. the flow rate was set to 0.6 ml/min and the column temperature was kept at 37°c. the rf was detected by fluorescence using 452 and 516 nm as excitation and emission wavelengths, respectively. rf was quantified using the external standard method. data were acquired and processed with labsolutions version 5.93. statistical analyses r software (r version 3.0.1) was used for statistical analysis. differences were evaluated by one-way anova, welch’s anova, and kruskal–wallis h test depending on data distribution. post hoc analyses tukey hsd test and games–howell test were used for anova and welch’s anova, respectively, while dunn test with holm correction was chosen as kruskal–wallis post hoc test. correlations were tested using pearson’s correlation test. statistical significance was attributed with p < 0.05 or a confidence interval of 0.95. results and discussion bentonite’s deproteinization capacity bentonites are mainly used to remove proteins and avoid unpleasant haze in white wines. selected bens have been characterized for determining their performance in protein removal using modified oeno 11/2003 (oiv, 2011) protocol. for this purpose bsa was considered more suitable than egg albumin because of its isoelectric point and absence of glycosylation, which make bsa more similar to wine proteins (sarmento et al., 2000). in addition, 12% ethanol was added to the test solution, as ben is generally used in wine and it has been demonstrated that ethanol can influence the swelling of ben, modifying its protein removal ability (achaerandio et al., 2001). moreover, the quantity of protein used was 500 mg/l, more close to the real protein content in unstable white charcoal’s decolorization power in enocyanin solution decolorizing power (dp) of a commercial cha was evaluated applying the method reported in oiv 7/2007 (oiv, 2007) with little modifications. the enocyanin solution was prepared adding 4.5 g/l of enocyanin, 7 g/l of tartaric acid, 4 g/l of acetic acid, and 7 g/l of sodium acetate. the solution was stirred to allow complete dissolution and centrifuged for 10 min at 14,000 g. supernatant was recovered and its absorbance was read in quartz cuvettes (2-mm path length) at three different wavelengths, namely 420, 520, 620 nm, using spectrophotometer ultrospec 2100pro (amersham bioscience europe gmbh, cologno monzese (mi), italy). the color intensity (ci) was calculated as the sum of the three absorbance values standardized to a length path of 10 mm. each cha was added to 100 ml of enocyanin solution at a final concentration of 1 g/l. samples were stirred for 30 min; after 10 min, they were collected into eppendorf tubes and centrifuged for 10 min at 14,000 g to remove chas. supernatant ci was measured as described for enocyanin solution. finally, dp was expressed as percentage using the following equation: −ci1 ci2 dp = 100 × , ci1 (1) where ci1 is the enocyanin solution color intensity and ci2 is its color intensity after cha treatment. the specific dp of each cha was calculated by three independent replications. bentonites and charcoals riboflavin removal in wine four ben dosages were chosen for removal of rf in white wine (glera wine with 123.9 µg/l of rf), namely 20, 40, 60, and 80 g/hl; the experiment was performed twice in 50-ml falcon tubes. tubes were vigorously shaken to allow dispersion of ben and maintained at 25°c for 10  min before centrifugation at 14,000 g for 10  min. removal assay was repeated in two independent tests. for chas, five dosages were selected for both red and white wines (wildbacher and glera, with 138.6 µg/l and 104 µg/l of rf, respectively), namely 10, 5, 2, 1, and 0.5 g/ hl. the test was repeated for three times. samples were shaken and kept in dark for 24 h. at the end of time contact, samples were mixed and centrifuged for 10 min at 14,000 g in order to assure removal of cha. the supernatant obtained was used for both rf quantification and color intensity determination. for the latter analysis, control ci (cc) and samples ci (cs) were used in eq. (1) in place of ci1 and ci2, respectively. color intensity was calculated as 420-nm absorbance value for the white 16 italian journal of food science, 2021; 33 (2) vendramin v et al. table 1. summary of selected bentonites. for each bentonite, de-proteinization curve slope and related r2 values are reported. sample id category slope r2 bent1 sod 7.97 0.99 bent2 sod 7.17 0.99 bent3 cal 4.11 1.00 bent4 cal 2.44 0.94 bent5 sod-cal 2.61 0.94 bent6 sod 6.75 0.96 bent7 sod 5.34 0.97 bent8 sod 5.30 0.97 bent9 sod 7.00 0.99 bent10 sod 4.99 1.00 bent11 sod 7.14 0.99 wines (marangon et al., 2011) than the 5-g/l of ovalbumin used in the original oiv method. removal curve slopes of 11 bens belonging to cal, sod, and sod-cal categories were used to compare efficiency of bens, the highest slope value corresponded to the best performing ben. data revealed a positive linear relationship between ben dosage and quantity of protein removed in this range of concentration. only in two cases, namely bent4 and bent5, r2 was lower than 0.95 (table 1) because of the lower absorbing capacity of these bens. statistical analyses revealed a significant difference between categories (f(2,8) = 10.5, p < 0.05) and post hoc test-grouped sod-cal with cal being the reason of their low deproteinization capacity (figure 1). this trend is in accordance with jönsson et al.’s (2009) findings, which demonstrated that sod bentonites are characterized by about 10 times major swelling capability than cal ones. the nature of cations arranged between cal a a b p ro te in r em ov al c ur ve s lo pe 0 2 4 6 8 sod sod-cal figure 1. slope index of protein removal curves. leastsquare mean values and standard errors are presented. different capital letters identify significantly different groups (p < 0.05) according to tukey test. montmorillonite lamellae strongly affects physical properties of ben, resulting in a great difference in protein-binding ability. sodium guarantees a major distance between ben layers enhancing protein entrance and a higher availability of binding surfaces. bentonite’s effect on removal of riboflavin attitude of ben to removal of rf was recently studied in comparison to other fining agents (fracassetti et al., 2017). ben was identified as one of the most useful fining agents. in fact, in the study conducted by fracassetti et al. (2017), all the tested bens (six, all from the same supplier) were able to remove about 60% of original rf in an rf-enriched model wine (350 µg/l of rf). for the screening presented in this work, bens were chosen with the aim to explore a major variability of commercial products, and therefore 11 bens furnished from seven different suppliers were selected. in glera wine, bens showed a limited removal of rf even at the highest dosage (on average, 28% of reduction at 80 g/hl) in accordance with the data reported by fracassetti and colleagues (2017) when testing a calcic ben at 100 g/hl in chardonnay wine. interestingly, the treatment with sodcal ben (bent5) at 80 g/hl led to a sensible reduction, corresponding to about 60%, of rf (fig. 2). considering that white wines present a mean rf content of 115 µg/l (cataldi et al., 2002), this is the only ben treatment that assured to decrease rf content below 50 µg/l, which is considered the threshold for light-struck taste risk (fracassetti et al., 2019). nevertheless, several cases of white wine in which concentration of rf overcame 151 µg/l have been reported (ournac, 1968; pilcher, 1996), and it should be taken into account that high dosages of ben could lead to severe side effects, that is, wine aroma depletion (lambri et al., 2012; lira et al., 2015). statistical analysis evidenced a significant difference among bens only at 60 g/hl, and bent5 confirmed to be the most efficient ben. in two cases, bent5 and bent6, a statistically significant effect of dosage was registered (figure 2). as also reported by fracassetti and colleagues (2017), no evidence of different responses was observed between cal and sod. the statistical analysis revealed no significant correlation (r = -0.33, p = 0.328) between the percentage of removed rf at the highest ben dosage and the protein removal curve slope, reinforcing the idea that protein and rf are linked to ben surfaces through different mechanisms. bens are known to link molecules by means of three different mechanisms: the first involves dipole bindings, the second is based on hydrogen bonding through the water bridge mechanism, and the third is based on van der waals forces (luckham and rossi, 1999). probably, the chemical structure of rf, characterized by low polarity, induces the italian journal of food science, 2021; 33 (2) 17 riboflavin removal by commercial bentonites and charcoals in white and red wines bent1 r f re m ov al (% ) 0 20 1,2 1,2 1,2 1,2 2 2 2 2 2 2 b b b b b; a a 1 ab40 60 80 bent2 bent3 bent4 bent5 bent6 bent7 bent8 bent9 bent10 bent11 80 g/hl 60 g/hl 40 g/hl 20 g/hl figure 2. riboflavin removal by bentonites. mean values and standard deviation of two replicates are presented. statistical differences between doses of bentonite are expressed by capital letters, while differences between bentonites at the same dosage are expressed by numbers (tukey test, p < 0.05). establishment of low polar bonds belonging to two latter types (kasimova et al., 2019). charcoal’s decolorizing capacity active chas are commonly used in oenology to reduce organoleptic fault because of phenolic off-odors (lisanti et al., 2017) as well as fining agents to correct color intensity of white wines obtained by the vinification of red grapes. their application depends on physical properties of cha, in particular on the pore size, which strongly affects cha permeability through molecular size exclusion. in fact, decolorizing chas are characterized by 20–500 å macropores, while deodorizing chas show a dominance of cha1 e no cy an in r em ov al (% ) 0 20 40 60 80 100 c a a a e cde cde cde cd b de cha2 cha3 cha4 cha5 cha6 cha7 cha8 cha9 cha10 cha11 figure 3. percentage values of enocyanin removal. mean values and standard deviations are presented. each test was repeated for three times. different capital letters identify significantly different groups (p < 0.05) according to post hoc tukey test. line corresponds to oiv threshold. small pores (yahya et al., 2015). resolution oeno 7/2007 (oiv, 2007) categorizes chas into the two groups based on the percentage of enocyanin removed from a model wine (decolorizing power). in particular, chas are assigned to dec group if they are able to remove more than 40% of initial enocyanin, while they are recognized as deo if the removed enocyanin is less than 40%. the decolorizing power of 11 selected chas was analyzed. for all chas, decolorizing power test confirmed the category assignment declared in the technical datasheets. four chas belonged to deo, namely cha3, cha4, cha8, and cha11, while six chas were clearly assigned to dec (cha1, cha5, cha6, cha7, cha9, and cha10; figure 3). the ambiguous case of cha2 18 italian journal of food science, 2021; 33 (2) vendramin v et al. and 31.25 ± 9.01 µg/l, respectively). on the other hand, deo permitted to achieve the threshold only after treatments with the highest doses and only with two chas (cha4 and cha8 with a residual rf of 39.61 ± 2.80 µg/l and 34.95 ± 2.49 µg/l, respectively; figs 4a and 4b). the percentage of rf reduction in samples treated with dec varied between 85% and 94%, with the only exception of cha2, which is the worst color removal dec. among decs, cha5 evidenced the best performance, reducing the rf value from 104 µg/l to 5.71 µg/l. it could be observed that chas demonstrated interesting variation in their efficiency depending on the dosage. cha5 showed a reduction of only 10%, passing from 10 g/hl to 5 g/hl, other two chas, namely cha1 and cha10, showed a reduction of about 15%, while cha6, cha7, and cha9 showed a marked reduction (higher than 20%). cha2 lost 18% of its removal ability passing from 10 g/hl to 5 g/hl. this resulted in a greater difference registered among dec at 5 g/hl, which showed an rf removal varying between 63.2% and 88.6%, corresponding to 41–15 µg/l of residual rf (figure 4a). deo could be divided into two subcategories. cha4 and cha8 achieved a maximum rf reduction of about 65%. differently, cha3 and cha11 evidenced a very low rf removal, without exceeding 25% even at 10 g/hl, leaving a residual vitamin concentration of 82.78 ± 5.63 µg/l and 79.15 ± 0.21 µg/l, respectively (figure 4b) in the investigated white wine. differences in rf adsorption could be attributed to specific surface availability between the two categories as well as within deo. it is well known that the main difference between dec and deo lies in pore size (yahya et al., 2015). even if mesopores and micropores seem to be the main contributors in cha’s removal power, by extending the surface area, macropores could represent an indispensable way for large molecules to achieve internal surfaces. recently, it has been demonstrated that reduction in large-size pores affects cha affinity toward methylene green, a cationic dye showing high similarity to rf molecule structure (tran et al., 2017). most likely, pore configuration could also affect rf permeability and consequently its removal, confirming the previous finding of having close relationship between pore size and rf permeability (kisler et al., 2001). color intensity of white wine was evaluated after treating with 10 g/hl of cha in order to quantify color depletion (cd) at 420 nm. color depletion significantly differed between cha categories (f(1,31) = 40.42, p < 0.05; data not shown); on average, deo removed 5%, while dec removed 8% of color. pearson’s correlation analysis between percentage of rf removal and color depletion at 10 g/hl revealed a positively significant correlation (r = 0.77, p < 0.05), which indicated that rf and flavan-3-ols/flavonoids have similar interaction with cha. concerning this, gogoi and colleagues (2010) suggested that the catechin sequestration by activated carbon depended on external that reduced 40% of enocyanin content was ascribed to dec in accordance with the statistical analysis, which grouped cha2 with cha6, cha7, cha9, and cha10. statistical analysis evidenced significant differences between samples (f(10, 22) = 108.8, p < 0.01) and allowed to differentiate decolorizing power ability even inside the two categories. for example, different from the other deo, cha8 revealed a singularly higher decolorizing power, which confirmed the specific supplier declaration of presence of high mesopores. among dec, cha5 achieved the highest percentage of subtraction (almost 55%; figure 3). charcoal’s riboflavin removal in white wine in a recent study, cha was recognized as the best fining agent for removal of rf (fracassetti et al., 2017). nevertheless, cha should be carefully used in order to avoid undesirable effects on wine, such as depletion of color and flavor. therefore, in this work five dosages of low-range cha (between 0.5 g/hl and 10 g/hl) have been used for comparison. in a preliminary test performed with different contact periods, chas showed the best vitamin removal property after 24 h of contact (figure si-1); therefore, the cha samples are compared after 24 h. in white wine, the results evidenced that dec have a better performance than deo at all tested doses, achieving about 90% of rf removal (figure si-2). as reported for other absorption kinetics, rf showed a positive but nonlinear trend of reduction with respect to cha concentration increment (ribéreau-gayon et al., 2006). differences between decolorizing and deodorizing chas were statistically significant at all doses, and drop in doses corresponded to a progressive decrease in differences between average of categories (figure si-2). as indicated by fracassetti et al. (2017), in real wine even the highest cha concentration didn’t allow the complete removal of rf. in fact, fracassetti et al. (2017) reported that after 24 h of contact a large-pore cha was able to remove 100% rf at 5 and 10 g/hl in a model wine, while it reached only 58% and 71% of rf removal in chardonnay wine. when compared with fracassetti et al. (2017), the decolorizing chas studied in the present work showed higher rf removal capacity in real wine. this phenomenon probably depends on differences in wine compositions because of grape varieties and winemaking processes. recently, it has been demonstrated that decreasing the final rf concentration below 50 μg/l drastically reduced the risk of light-struck development (fracassetti et al., 2019), and therefore this concentration was chosen as a threshold for the evaluation of efficacy of chas. dec assured sufficient removal of rf at 10 g/hl and 5 g/hl (residual rf concentration of 13.58 ± 5.24 µg/l italian journal of food science, 2021; 33 (2) 19 riboflavin removal by commercial bentonites and charcoals in white and red wines cha3 b ab ab aba a a a a a abc abc ab b b b b b b b 10 g/hl 5 g/hl 2 g/hl 1 g/hl 0.5 g/hl 10 g/hl 5 g/hl 2 g/hl 1 g/hl 0.5 g/hl b bc b bc b b c c c cdc d d cd d b b a a a a a a a a b b b bab ab b b ab ab 0 20 40 60 80 100 0 20 40 60 80 100 cha1 cha2 cha5 cha6 cha7 cha9 cha11 cha4 cha8 cha11 r f re m ov al (% ) r f re m ov al (% ) figure 4. removal of riboflavin in white wine. (a) wine treated with decolorizing chas. different capital letters identify statistically significant differences within carbon according to post hoc games–howell test (ci = 0.95, dosage 10 g/hl) and tukey test (p  > 0.05, other doses). (b) wine treated with deodorizing chas. different capital letters identify statistically significant differences within carbon according to post hoc dunn test (ci = 0.95, dose: 10 g/hl and 0.5 g/hl) and tukey test (p > 0.05, other doses). mean values and standard deviations of three replications are expressed. physicochemical parameters, for example, ph and competing compounds present in the solution that could interfere in the solute–sorbent association. this linkage was supposed to occur as an equilibrium between the hydrogen bonding (less important in the water environment because of high number of water hydrogen bonds) and the π-electron interaction between phenol ring and carbon backbone, which directly determines the bond strength. charcoal’s rf removal in red wine quantity of rf in wine directly depends on grape variety and winemaking process (cataldi et al., 2002). on average, red wines revealed higher vitamin content with respect to both white and rosè wines. although the lightstruck phenomenon is not relevant in red wines, it has been demonstrated that rf-mediated oxidation could play a role in degradation of anthocyanins (kim et al., 2010). moreover, chas are more often used in fining of red wine (lisanti et al., 2008). therefore, cha’s ability to remove rf in red wine is of particular interest. as before, rf quantification was performed after 24 h of treatment, samples were kept in dark condition, and five dosages were tested. as before, dec resulted in a higher rf removal capacity than deo at all selected concentrations (figure si-3). the best performance was shown by dec at 10 g/hl with a significant difference from deo (f(1,32) = 53.82, p < 0.05). in general, chas evidenced a reduced ability of rf removal in red wine in comparison to white wine; in fact, dec registered less than 25% of rf removal at the highest concentration. this effect could depend on other wine components which interfere in the rf–cha interaction. these compounds likely belong to phenolics, which have been studied for their high affinity to cha (da̧browski et al., 2005). deodorizing chas revealed no rf removal capacity in red wine; on the contrary, they seem to display a slight rf protection, as the final rf content in the treated samples was slightly higher than in the control (figure si-3). it is widely reported that spontaneous degradation of rf occurs during the first few hours of opening of bottle (mattivi et al., 2000) and that this phenomenon is accelerated in organic solvents (sheraz et al., 2014). even by keeping the samples in the dark, a slight rf degradation occurred during the treatment; however, the presence of deo seemed to prevent this degradation, as the rf content was greater at higher cha concentrations. it can be assumed that the same rf degradation occurred in all the samples; although in dec-treated samples the rf removal masked this effect, in deo-treated samples the removal power was too low. 20 italian journal of food science, 2021; 33 (2) vendramin v et al. the π electrons of the graphitic structure, and, if ions are present, then electrostatic attraction and repulsion (da̧browski et al., 2005). conclusion this work explored the potential of commercial bens and active chas in lowering of rf in real wines. bens showed a reduced ability to sequester rf which appeared independent from their shied cation nature, and thus from their swelling properties. among 11 different commercial bens, only a sodic-calcic ben (bent5) revealed an interesting higher ability in rf removal. this work represents the first study in which a sodic-calcic ben has been tested for this purpose. the future studies would define whether sod-cal matrices could play a role as rf removal agents. on the other side, activated chas confirmed their high attitude to remove rf, which for the first time was tested in both white and red wines. a great difference between deodorizing and decolorizing cha was registered in both wines, which probably depends on cha porosity. additionally, in comparison to white wine, cha rf removal was dramatically reduced in red wine. on average, decolorizing chas revealed 87% of rf removal in glera wine to 22% in wildbacher wine. this phenomenon has been attributed to complex interactions between cha and wine phenols. phenolic acids, even in deo-treated samples, differences between categories were statistically significant at all dosages. the analyses of individual chas belonging to dec group revealed strong differences in their behaviors. in particular, cha10 was identified as the best one for rf removal. in fact, it is the only one that reduced the rf value by 43% at 10 g/hl (corresponding to a final concentration of 77.96 ± 9.11 µg/l in treated wine; figure 5). considering the initial rf concentration of 138 µg/l, it means that about 60 µg/l was removed, similar to the depletion recorded in white wine at 5 g/hl. two chas, namely cha1 and cha5, exhibited limited ability, reaching a little more than 25% of removal (figure 5). in all other cases, the rf sequestering was very low. color depletion at 10 g/hl significantly differed between cha categories (f(1,31) = 85.19, p < 0.05); deo removed on average 2%, while dec removed 9% of color intensity. the correlation between percentage of removed rf and color depletion at 10 g/hl highlighted a positively significant correlation (r = 0.88, p < 0.05). this suggests that the interaction between rf and polyphenols with cha is characterized by a similar binding mechanism. in a dynamic equilibrium, this mechanism depends on the ionic strength (and ph) of the solvent that probably involved electron donor–acceptor interactions between the aromatic phenolic ring and the surface oxygens, dispersion effect between the aromatic phenolic ring and cha1 bc ab a b de bce c b b ab ab ab ab a a a c bc cd bc –15 5 25 45 65 cha2 cha5 cha6 cha7 cha9 cha10 10 g/hl 5 g/hl 2 g/hl 1 g/hl 0.5 g/hl r f re m ov al (% ) figure 5. riboflavin removal of decolorizing chas in red wine. different capital letters identify statistically significant differences within dec according to post hoc tukey test (p > 0.05) at the dosages of 10 g/hl, 5 g/hl, and 1 g/hl. mean values and standard deviations of three replications are expressed. italian journal of food science, 2021; 33 (2) 21 riboflavin removal by commercial bentonites and charcoals in white and red wines solutions. j mol struc. 1185: 107–111. https://doi.org/10.1016/j. molstruc.2019.02.084 kim, m., yoon, s.h., jung, m. and choe, e. 2010. stability of meoru (vitis coignetiea) anthocyanins under photochemically produced singlet oxygen by riboflavin. new biotechnol. 27(4): 435– 439. https://doi.org/10.1016/j.nbt.2010.01.003 kisler, j.m., antje, d., stevens, g.w. and connor, a.j. 2001. separation of biological molecules using mesoporous molecular sieves. microporous mesoporous mater. 44–45: 769–774. https://doi.org/10.1016/s1387-1811(01)00259-1. lagunes, i., vázquez-ortega, f. and trigos, á. 2017. singlet oxygen detection using red wine extracts as photosensitizers. j food sci. 82(9): 2051–2055. https://doi.org/10.1111/1750-3841.13815 lambri, m., dordoni, r., silva, a. and de faveri, d.m. 2012. comparing the impact of bentonite addition for both must clarification and wine fining on the chemical profile of wine from chambave muscat grapes. int j food sci. technol. 47(1): 1–12. https://doi.org/10.1111/j.1365-2621.2011.02800.x lira, e., rodríguez-bencomo, j.j., salazar, f.n., orriols, i., fornos, d. and lópez, f. 2015. impact of bentonite additions during vinification on protein stability and volatile compounds of albariño wines. j. agric food chem. 63(11): 3004–3011. https://doi. org/10.1021/acs.jafc.5b00993 lisanti, m.t., gambuti, a., genovese, a., piombino, p. and moio, l. 2017. treatment by fining agents of red wine affected by phenolic off-odour. eur. food res. technol. 243(3): 501–510. https:// doi.org/10.1007/s00217-016-2763-4 lisanti, m.t., piombino, p., gambuti, a., genovese, a., 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(% ) figure si-1. trend of removal of riboflavin with cha5 and cha3 in white wine. mean values and standard deviations are expressed. each test was repeated for three times. black: decolorizing cha (cha5), and gray: deodorizing cha (cha3). different capital letters identify statistically significant differences within category according to tukey test (p < 0.05). 10 g/hl a a b c d c c e ab bc r f re m ov al (% ) 0 20 40 60 80 100 5 g/hl 2 g/hl 1 g/hl 0.5 g/hl figure si-2. comparison of removal of riboflavin with chas in white wine. mean values and standard deviations are expressed. each test was repeated for three times. gray: decolorizing cha, and light gray: deodorizing cha. different capital letters identify statistical significant differences within categories according to post hoc dunn’s test (ci = 0.95). ci: color intensity. 10 g/hl –15 5 25 45 65 a a a b ab ab b b bab 5 g/hl 2 g/hl 1 g/hl 0.5 g/hl r f re m ov al (% ) figure si-3. comparison of removal of riboflavin with cha in red wine. mean values and standard deviations are expressed. each test was repeated for three times. gray: decolorizing cha, and light gray: deodorizing cha. different capital letters identify statistically significant differences within category according to dunn’s test (ci = 0.95) and tukey test (p < 0.05) respectively. ci: color intensity. _hlk64534864 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (4): 1–10 issn 1120-1770 online, doi 10.15586/ijfs.v34i4.2267 1 p u b l i c a t i o n s codon assessment of the bioaccessibility of phenolic compounds and antioxidant activity in raw and pickled white cabbage and gherkins müzeyyen berkel kaşıkçı*, neriman bağdatlıoğlu food engineering department, faculty of engineering, manisa celal bayar university, manisa, turkey *corresponding author: müzeyyen berkel kaşıkçı, food engineering department, faculty of engineering, manisa celal bayar university, manisa, turkey. email: muzeyyen.berkel@cbu.edu.tr received: 22 august 2022; accepted: 19 september 2022; published: 6 october 2022 © 2022 codon publications open access paper abstract white cabbage and gherkin are vegetables that are widely consumed as pickles as well as raw vegetables. in this research, we explored the effect of pickling on the bioaccessibility of phenolics and flavonoids and changes in antioxidant activity after in vitro digestion. in general, the pickling process enhances the bioaccessibility of phenolics and flavonoids in white cabbage and gherkin. the bioaccessibility of total phenolics (tp) in cabbages, pickled cabbages, gherkins, and pickled gherkins is determined as 125.2%, 185.1%, 369.2%, and 462%, respectively. in contrast, after in vitro digestion of raw and pickled vegetables, total antioxidant activity is reduced. so it can be concluded that both raw and pickled gherkins are good sources of bioaccessible phenolics and flavonoids. the consumption of these vegetables and their pickles is suggested to promote the reduction of diseases plagued by free radicals. keywords: antioxidant; bioaccessibility; cabbage; gherkin; phenolics; pickle introduction white cabbage and gherkin are consumed substantially throughout the world. white cabbage and gherkin are the most popular pickles and are consumed in raw form. in the united states, 245.85 million americans consumed pickles in 2020. it is expected to increase to 251.03 million in 2023 (statistica research department, 2022). the size of the global market for packed pickles, which was 7.9 billion in 2018, is expected to increase at a compound annual growth rate (cagr) of 3.5% until 2025 (grand view research, 2019). global consumption of cabbages and other brassicas, both raw and pickled, was 70.7 million tonnes in 2015 (indexbox, 2016a). in 2015, 78.5 million tons of gherkins and cucumbers, both raw and pickled, were consumed globally (indexbox, 2016b). in 2015, import and export volumes of pickled gherkins and cucumbers were 557.000 and 554.300 tonnes, respectively (indexbox, 2016c). oxidative stress causes the formation of free radicals and other reactive oxygen species, which are involved in many diseases, particularly chronic degenerative diseases such as cancer, diabetes, cardiovascular disease, and obesity. as a result, reactive compounds must be quenched by antioxidants. phenolic compounds are secondary metabolites of plants and have antioxidant properties. phenolic compounds have several groups such as flavonoids, lignans, and phenolic acids. flavonoids is comprised of quercetin, kaempferol, lutein, and naringenin. some of phenolic acids are gallic acid, chlorogenic acid, and syringic acid (tapia-hernández et al., 2018; deltoro-sánchez et al., 2021; maribel perez-perez et al., 2018). gherkins and white cabbages have several antioxidant compounds, which can be categorized into phenolic compounds and non-phenolic compounds. as shown in figure 1, the main antioxidant compounds in gherkin are flavonoids, tannins, triterpenes, alkoloids, and saponins (mohamed et al., 2022; omokhua-uyi et al., 2020; mailto:muzeyyen.berkel@cbu.edu.tr 2 italian journal of food science, 2022; 34 (4) kaşikçi mb and bağdatlioğlu n therefore, the objective of this research was to assess the effect of the pickling process on the bioaccessibility of phenolics and antioxidant activity in cabbage and gherkin. for achieving this objective, the total phenolic content (tpc), total flavonoid content (tfc), and antioxidant activity (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt [abts], 2,2-diphenyl-1-picrylhydrazyl [dpph], and ferric reducing antioxidant power [frap]) were determined before and after in vitro digestion. materials and methods chemicals phenolphtalein, sodium carbonate, catechin, 2,4,6-tris(2-pyridyl)-s-triazine (tptz), iron(ii) sulphate, 2,2-diphenyl-1-picrylhydrazyl (dpph), 6-hydroxy2,5,7,8-tetramethylchromane-2carboxylic acid (trolox), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts), potassium persulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, haemoglobin, tris (hydroxymethyl) aminomethane (trizma base), p-toluene sulfonyl-larginine methyl ester (tame), pepsin, pancreatin, bile salt, methanol (≥%99.9), hyperoside and 4-hydroxybenzoic acid (4-hba) standards were purchased from sigma dimitry et al., 2022; murthy et al., 2022). the main antioxidant compounds in white cabbage are phenolic acids, flavonoids, lignans, and glucosinolates (kuljarachanan et al., 2021; demir et al., 2023; tao et al., 2019; zhou et al., 2022; cvetković et al., 2019). these vegetables are healthy due to their phenolic content and antioxidant properties (sayın et al., 2015; song et al., 2010; bahorun et al., 2004). phenolic compounds have antiviral, anti-allergic, antiplatelet, anti-inflammatory, antitumor, and antioxidant properties (yao et al., 2004). to exploit these benefits for humans, phenolic compounds should be bioaccessible and bioavailable. bioaccessibility is the fraction of a compound that can reach the intestine by passing through the intestinal barrier. although the bioaccessibility of phenolics in different cabbage species has been studied previously (kaulmann et al., 2016; tomas et al., 2021), no studies about the bioaccessibility of phenolics and changes in antioxidant activity during simulated gastrointestinal digestion for white cabbages, pickled white cabbages, gherkins, and pickled gherkins have been reported in the literature. our hypothesis is that the fermentation process loosens other molecules in the food matrix and allows the bound phenolics to be released from the cell wall, as depicted in figure 2. on the other hand, fermentation can degrade vulnerable phenolics, and the bioaccessible fraction can be decreased. antioxidants compounds in gherkin flavonoids tannins triterpenes alkoloids saponins antioxidants compounds in white cabbage phenolic acids flavonoids glucosinolates lignans figure 1. antioxidant compounds in gherkin and white cabbage. fermentation sugars cellulose hemiellulose pectin lignin phenolic compounds protein structure bound phenolic free phenolic figure 2. conversion of bound phenolics to free phenolics by fermentation (maribel perez-perez et al., 2018). italian journal of food science, 2022; 34 (4) 3 assessment of the bioaccessibility of phenolic compounds and antioxidant activity aldrich (usa). sodium hydroxide, potassium chromate, silver nitrate, sodium chloride, de man rogosa and sharpe (mrs) agar, formic acid, folin-cioceltau, gallic acid, sodium nitrite, aluminium chloride, sodium hydroxide, sodium acetate trihydrate, glacial acetic acid, hydrochloric acid, iron(iii) chloride, and calcium chloride dihydrate were obtained from merck. potassium chloride and ammonium carbonate were acquired from riedel-de haen (germany). sodium bicarbonate, magnesium chloride dihydrate, and acetonitrile (≥99.9%) were purchased from carlo elba (france). pefabloc was obtained from acros (usa). sm-199 lactobacillus plantarum culture was purchased from chris hansen (germany). cabbages and gherkins cabbage and gherkin samples were purchased from a local market in manisa, turkey. small and firm cabbages and gherkins ranging in length from 3 to 6 cm were selected. pickling procedure cabbages and gherkins were placed into 660 ml jars with brine composed of 6% salt and 0.05% vinegar. cabbage and gherkin brine were adjusted to 105 and 106  cfu/ ml lactic acid bacteria (lab), respectively, by using chris hansen sm-199 lactobacillus plantarum culture, respectively. the weights of the vegetables and composition of the brine in the jar is shown in table 1. titratable acidity, ph, salt analyses, and total lab counts were performed in brines and pickles. extraction of phenolic compounds the extraction procedure reported by (bovy et al., 2002) was used with slight modifications. samples were ground in a waring blender, and 4 g of each sample was then homogenized in 10 ml of 75% methanol and incubated for 15 min in an ultrasonic water bath. afterward, samples were subjected to a 2000 g, 10 min, 4°c centrifugation procedure. the extraction procedure was repeated three times, with the second and third extractions that included the addition 5 ml of 75% methanol. the supernatants were transferred to a plastic container, resulting in a final volume of 20 ml by adding 75% methanol. for further analysis, extracts were stored at −86 °c. in vitro gastrointestinal digestion the in vitro gastrointestinal digestion of samples was performed following the method described by (minekus et al., 2014), which involves oral, gastric, and intestinal phases. the only difference is that no salivary α-amylase was used in our protocol. pepsin and pancreatin activity analyses were performed according to (minekus et al., 2014). note that 5 g of each sample were grounded in a waring blender to mimic chewing process and mixed with 3.5 ml simulated saliva fluid (ssf), which was composed of 15.1 mmol/l kcl, 3.7 mmol/l kh2po4, 13.6 mmol/l nahco3, 0.15 mmol/l mgcl2(h2o)6, and 0.06 mmol/l (nh4)2co3. the mixture was then mixed with 25 µl of 0.3 m cacl2(h2o)2. afterward, the ph was adjusted to 7, and distilled water was added to make a total volume of 10 ml. in order to complete oral digestion, the mixture was shaken for 2 min at 100 rpm in a 37°c shaking incubator. following oral digestion, gastric digestion procedures were carried out immediately. to the oral bolus, 7.5 ml of simulated gastric fluid (sgf), containing 6.9 mmol/l kcl, 0.9 mmol/l kh2po4, 25 mmol/l nahco3, 47.2 mmol/l nacl, 0.1 mmol/l mgcl2(h2o)6, and 0.5 mmol/l (nh4)2co3 was added. then, pepsin enzyme of 2000 u/ ml activity and 5 µl of 0.3 m cacl2(h2o)2 were added, and the ph was adjusted to 3 with 6 m hcl. then 20 ml of total volume was obtained by adding distilled water. samples are kept in a shaking incubator at 100 rpm, 37˚c for 2 h to mimic gastric conditions. intestinal digestion was performed immediately after gastric digestion. 11 ml simulated intestinal fluid (sif), which was composed of 6.8 mmol/l kcl, 0.8 mmol/l kh2po4, 85 mmol/l nahco3, and 38.4 mmol/l nacl, and 0.33 mmol/l mgcl2(h2o)6 was added to the gastric bolus. then, pancreatin enzyme (100 u/ml tripsin activity), 10 mm bile extract, and 40 µl of 0.3 m cacl2(h2o)2 were added to gastric bolus and ph was adjusted to 7 with 1 m naoh. then, distilled water was added to make a total volume of 40 ml. finally, samples were incubated for 2 h in a shaking incubator at 100 rpm and 37˚c to simulate intestinal conditions. table 1. formulation of pickles in a jar. cabbage pickle gherkin pickle weight of the vegetables (g) 245 ± 2 310 ± 2 volume of brine (ml) 295 250 volume of the culture solution (ml) 10 10 total volume of brine (ml) 305 260 volume of the jar (ml) 660 660 4 italian journal of food science, 2022; 34 (4) kaşikçi mb and bağdatlioğlu n this is how the percentage inhibition of abts was determined: control sample control a a inhibition of abts (% 10 a ) ) ( 0 − = × where acontrol = absorbance of the control solution and asample = absorbance of the sample solution. a graph showing the inhibition of the abts (%)-amount of sample (mg) was created for each sample. the inbition of the abts (%)-amount of trolox (µg) plot was also created. the abts antioxidant activity was estimated by dividing the slope of the sample graph by the slope of the trolox graph. the results were expressed as mg trolox equivalents/100 g of sample. with some adjustments, the dpph method was assayed in accordance with (brand-williams et al., 1995; singh et  al., 2002). a final volume of 200 µl was created by mixing different extract volumes (1, 10, 25, and 50 µl) with 75% methanol. then, 3.8 ml dpph solution was added. after 30 min of incubation, the absorbance of the mixtures was measured at a wavelength of 515 nm. the identical procedure was carried out for the control using 200 µl of pure deionized water. the same procedure was also carried out by using 200 µl trolox standards. the percent inhibition of dpph was calculated as follows: control sample control a a inhibition of dpph (% 10 a ) ) ( 0 − = × where acontrol = absorbance of control, asample = absorbance of sample a graph of inhibition of dpph (%) versus amount of sample (mg) was drawn for each sample. the inhibition of dpph (%) versus the amount of trolox (µg) was also plotted. the dpph antioxidant activity was calculated by dividing the slope of the sample graph by the slope of the trolox graph. the results were expressed as mg trolox equivalents/100 g of sample. the ferric reducing antioxidant power (frap) method was used according to (liu et al., 2008; wang et al., 2012). statistical analysis the statistical software spss 16.0 was used to conduct the statistical analysis. the results are expressed as the mean ± standard deviation. the paired sample t-test was used to analyze the differences in the mean values between two paired comparisons. differences between the means were considered significant at p < 0.05. correlation coefficients (r2) between different methods were calculated. after all digestion procedures were completed, the enzyme reactions were halted by adding 150 mm pefabloc enzyme inhibitor to the digested samples. the digested samples were subjected to 4000 rpm, 10 min, 4°c centrifugation procedure. the supernatant that remains on top was transferred to sample tubes and stored at −86˚c until further analysis. total phenolic content (tpc) tpcs were assessed using the folin-cioceltau technique with some modifications as described by (miceli et al., 2009). then, 100 ml of appropriately diluted sample extract was combined with 200 ml of 0.2 n folincioceltau reagent. then, 1 ml of 6% naco3 solution and 2 ml of pure deionized water were added and vortexed. the mixture was incubated at room temperature for 2 h before the absorbance at a wavelength of 765 nm was measured with a multiskan go uv/vis microplate spectrophotometer (thermo fisher scientific, usa). the results are given as milligrams of gallic acid equivalents (gae) per 100 g of fresh weight (fw). total flavonoid content (tfc) tfcs were determined using the method provided by (dewanto et al., 2002) with some changes. a total of 250 µl of appropriately diluted sample extract was combined with 1.25 ml of pure deionized water. after adding 75 µl of sodium nitrite and letting it sit for 6 min, 150 µl of aluminium chloride was then added. after 5 min, a total mixture amount of 2.5 ml was obtained by adding 0.5 ml naoh and pure deionized water. prior to the measurement of the absorbance at 510 nm with a multiskan go uv/vis microplate spectrophotometer (thermo fisher scientific, usa), the mixture was vortexed. the results are presented as milligrams of catechin equivalents (ces) per 100 g of fw. total antioxidant activity (taa) three different methods, abts, dpph, and frap, were used to measure the total taa. the abts method was applied (miller et al., 1997) with various adjustments. different extract volumes (1–20 µl) were combined with pure deionized water to create a final volume of 20 µl. following the addition of 180 µl of abts, the absorbance at 734 nm was measured after 30 s. the identical procedure was carried out for the control using 20 µl of pure deionized water. using 20 µl trolox standards, the same procedure was also carried out. italian journal of food science, 2022; 34 (4) 5 assessment of the bioaccessibility of phenolic compounds and antioxidant activity gae/100 g, respectively. the bioaccessibility of tpc is higher for pickled cabbage (185.1%) than cabbage (125.2%) (p < 0.05). the increase in tpc values after in  vitro digestion is probably related to the release of bound phenolic compounds in the matrix of the cabbage. in the literature, no study has investigated the bioaccessibility values for total phenolics (tp) for white cabbage. in a study of red cabbage (vanhoutte, 2014), alkaline hydrolysis was applied to red cabbage, and it was found that the alkaline extract contained a total phenolic content of more than 3 times that of the methanolic extract of red cabbage. this indicates that the majority of phenolics in red cabbage consist of nonextractable phenolics. alkali hydolysis can break ether and ester bonds (vanhoutte, 2014; acosta-estrada et al., 2014; rashmi et al., 2020). similarly, sodium hydroxide (naoh) is used in the intestinal digestion part of our study, as in alkaline hydrolysis. the tpc of white cabbage is increased after in vitro digestion in our study, indicating that some of the phenolics in white cabbage are composed of nonextractable phenolics and that the alkali application leads to the release of phenolics via the breaking of ether and ester bonds. the higher bioaccessibility of tp in pickled cabbages compared to cabbages can be explained by the protective effect of fermentation. although the protective mechanism for fermentation has not yet been fully explained, it is thought that lactic acid produced by fermentation can alleviate the negative effect of ph on phenolic compounds by providing hydrogen ions and creating a buffer environment (zhao et al., 2016). fermentation decreases the tpc for gherkins from 40.3  ± 0.2 to 29.4 ± 1.4 mg gae/100 g (p < 0.05); this value is similar with (kiczorowski et al., 2022), but is lower than that found in another literature (ciniviz et al., 2020). after the in vitro digestion process, tpc values for gherkins and pickled gherkins are increased to 149.1 ± 7.3 and 136.2 ± 11.2 mg gae/100 g, respectively. the bioaccessibility values for gherkin and pickled gherkin are determined to be 369.2% and 462.0%, respectively (p < 0.05). higher bioaccessibility values in pickled gherkins are probably related to the protective effect of fermentation (zhao et al., 2016; leonard et al., 2021; ed nignpense et al., 2022). results and discussion physical and chemical properties of raw vegetables and their pickles the results of humidity (%), ph, acidity (%), and salt (%) analyses are shown in table 2. the humidity (%) of the cabbage, gherkin, pickled cabbage, and pickled gherkin is determined as 92.44% ± 0.93, 95.12% ± 1.25, 92.8 ± 0.92, and 94.1 ± 0.49, respectively. the ph values for cabbage and gherkin are 6.74 and 6.55, respectively, and the pickling process reduces the ph values to 3.55 and 3.73 for pickled cabbage and pickled gherkin, respectively. the salt content (%) for pickled cabbage and pickled gherkin is balanced at 2.85% and 2.55%, respectively. the total lab for pickled cabbage and pickled gherkin is 5.60 ×106 cfu/g and 2.67 ×107 cfu/g, respectively. total phenolic content (tpc) the tpcs of cabbages before and after in vitro digestion and the bioaccessibility values are shown in table 3. the tpc of cabbages and pickled cabbages is 104.3 ± 6.5 and 107.3 ± 1.5 mg gae/100 g, respectively. fermentation with lactobacillus plantarum does not change the tpc of the cabbage (p > 0.05). in a previous study (sayın et al., 2015), the fermentation process was found to increase tpc initially and then decrease the tpc of white cabbage. in another spontaneous fermentation study, fermentation decreased the tpc of white cabbage (parada et al., 2022). sauerkraut production was found to increase tpc and some individual phenolics (ciska et al., 2005a; tlais et al., 2022). in these studies, except a study (tlais et al., 2022), bacterial culture was not used in brine processing, but lactobacillus plantarum was used in our study. different fermentation conditions, such as temperature, time, microorganisms, acidity and ph, are probably responsible for the variation in the tpc after the brining process (sayın et al., 2015; ciska et al., 2005b). after in vitro digestion, the tpc of cabbage and pickled cabbage is increased to 130.2 ± 2.9 and 198.6 ± 8.5  mg table 2. physical and chemical properties of the raw vegetables. humidity (%) ph acidity (%) salt (%) cabbage 92.44 ± 0.93 6.74 ± 0.20 0.14 ± 0.00 0.31 ± 0.03 gherkin 95.12 ± 1.25 6.55 ± 0.17 0.14 ± 0.00 0.19 ± 0.04 pickled cabbage 92.80 ± 0.92 3.55 ± 0.02 0.73 ± 0.02 2.85 ± 0.09 pickled gherkin 94.10 ± 0.49 3.73 ± 0.09 0.57 ± 0.02 2.55 ± 0.03 6 italian journal of food science, 2022; 34 (4) kaşikçi mb and bağdatlioğlu n total flavonoid content (tfc) the tfc of cabbages and gherkins before and after in vitro digestion is shown in tables 3 and 4. the tf values for cabbages (1.85 ± 0.31 mg ce/100 g) and pickled cabbages (1.89 ± 0.36 mg ce/100 g) are similar (p > 0.05). in vitro digestion increases the tfc of raw and pickled cabbage. the tf bioaccessibility of raw and pickled cabbages is 124.3% and 161.2%, respectively (p > 0.05). similar to the bioaccessibility of tpc, the bioaccessibility of tfc is also higher in pickled cabbages, probably due to the protective effect of fermentation (zhao et al., 2016). the tfc of gherkin and pickled gherkin is 0.34 and 0.23 mg ce/100 g, respectively (p > 0.05). after in vitro digestion, the tfc for gherkin and pickled gherkin is increased to 1.19 and 0.78 mg ce/100 g, respectively. the tf bioaccessibility for raw (347.7%) and pickled gherkins (348.2%) is similar. after in vitro digestion, the tf values for both cabbages and gherkins are increased, which show a similar trend to that observed for the tp values. abts, dpph, and frap antioxidant activity the taa of gherkins and cabbages was determined by measuring their free radical scavenging activities using the abts, dpph, and frap methods. the values and after in vitro digestion, it was shown that the tpc value in both the cabbage and cucumber samples is increased. there is no study in the literature that investigates the tpc after in vitro digestion of white cabbage and gherkin samples, but there are many studies that show an increase in the tpc after the in vitro digestion process in various foods. the release of phenolic compounds from the food matrix due to a change in ph values, digestive fluids and enzymes during digestion is thought to be the underlying cause for this increase in tp value after digestion. the activities of enzymes can lead to hydrolysis of phenolics bound to other food components. enzymes can disintegrate high molecular weight compounds such as proteins and carbohydrates, thus enabling the release of phenolics attached to these macromolecules (bouayed et al., 2012; thomas-valdés et al., 2018; celep et al., 2017; ti et al., 2015; tomas et al., 2018; da silva fernandes et al., 2017; ed nignpense et al., 2022). the folin-cioceltau method is a simple and useful method for determining tp. nonphenolic compounds such as ascorbic acid, sugars, aromatic amines, organic acids, and proteins are also likely to react with folin reagent, leading to an overestimation of the amount of phenolic compounds. despite the fact that many different compounds reduce the folin reagent, these interfering compounds remain in the food matrix prior to digestion. as a result, although the folin method is not selective enough, it can be used to demonstrate an increase in the concentration of folin reagent reducing compounds when compared to the control sample (fernandes et al., 2017; prior, 2005). table 3. total phenolic content (tpc) and total flavonoid content (tfc) of cabbages. total phenolic content (tpc) (mg gae/100 g) total flavonoid content (tfc) (mg ce/100 g) before in vitro digestion after in vitro digestion ba (%) before in vitro digestion after in vitro digestion ba (%) cabbage 104.3 ± 6.5a, b 130.2 ± 2.9b, a 125.2 ± 9.4b 1.85 ± 0.31a, b 2.30 ± 0.42a, a 124.3 ± 12.4b pickled cabbage 107.3 ± 1.5a, b 198.6 ± 8.5a, a 185.1 ± 10.0a 1.89 ± 0.36a, b 3.07 ± 0.78a, a 161.2 ± 17.0a values are the mean ± standard deviation. a-b different letters indicate that the difference between the values in the same column is significant at the p < 0.05 level. a-b different letters indicate that the difference between the values in the same row is significant at the p < 0.05 level. table 4. total phenolic content (tpc) and total flavonoid content (tfc) of gherkins. total phenolic content (tpc) (mg gae/100 g) total flavonoid content (tfc) (mg ce/100 g) before in vitro digestion after in vitro digestion ba (%) before in vitro digestion after in vitro digestion ba (%) gherkin 40.4 ± 0.2a, b 149.1 ± 7.3a, a 369.2 ± 17.2b 0.34 ±0.12a,b 1.19±0.45a,a 347.7±5.6a pickled gherkin 29.4 ± 1.4b, b 136.2± 11.2a, a 462.0 ± 17.1a 0.23 ± 0.04a,b 0.78±0.12a,a 348.2±3.2a values are the mean ± standard deviation. a-b different letters indicate that the difference between the values in the same column is significant at the p < 0.05 level. a-b different letters indicate that the difference between the values in the same row is significant at the p < 0.05 level. italian journal of food science, 2022; 34 (4) 7 assessment of the bioaccessibility of phenolic compounds and antioxidant activity table 6. abts, dpph, and frap antioxidant activity values for gherkins. abts (mg te/100 g) dpph (mg te/100 g) frap (mmol fe/100 g) before in vitro digestion after in vitro digestion df (%) before in vitro digestion after in vitro digestion df (%) before in vitro digestion after in vitro digestion df (%) gherkin 16.1 ± 0.8a,a 12.2 ± 0.7a,b (−) 24.2a 8.06 ± 0.10a,a 6.06 ± 0.34a,b (−) 24.8a 0.35 ± 0.02a,a 0.40 ± 0.0b,a (+) 14.7b pickled gherkin 15.2 ± 1.0a,a 10.5 ± 1.6a,b (−) 30.9a 7.03 ± 0.2b,a 5.23 ± 0,22b,b (−) 25.6a 0.40 ± 0.04a,b 0.55 ± 0.0a,a (+) 36.8a values are the mean ± standard deviation. a-b different letters indicate that the difference between the values in the same column is significant at the p < 0.05 level. a-b different letters indicate that the difference between the values in the same row is significant at the p < 0.05 level. df: difference. table 5. abts, dpph, and frap antioxidant activity values for cabbage. abts (mg te/100 g) dpph (mg te/100 g) frap (mmol fe/100 g) before in vitro digestion after in vitro digestion df (%) before in vitro digestion after in vitro digestion df (%) before in vitro digestion after in vitro digestion df (%) cabbage 25.1 ± 0.9a,a 13.8 ± 2.4a,b (−) 45.1 9.7 ± 0.7a,a 9.8 ± 1.1a,a (+) 1.4 0.30 ± 0.02a,a 0.29 ± 0.04a,a (−) 1.42 pickled cabbage 24.3 ± 0.9a,a 13.8 ± 2.8a,b (−) 43.6 9.3 ± 0.5a,a 9.1 ± 1,2a,a (−) 2.8 0.32 ± 0.01a,a 0.34 ± 0.02a,a (−) 6.32 values are the mean ± standard deviation. a-b different letters indicate that the difference between the values in the same column is significant at the p < 0.05 level. a-b different letters indicate that the difference between the values in the same row is significant at the p < 0.05 level. df: difference. changes after in vitro digestion are shown in tables 5 and 6. raw cabbages (25.11 mg te/100 g) and pickled cabbages (24.33 mg te/100 g) show comparable abts antioxidant activity values (p > 0.05). (sayın et al., 2015) found that fermentation first increases and then decreases abts antioxidant activity. abts antioxidant activity was found to remain unchanged by brining in another study, similar to our study (girgin et al., 2015). in vitro digestion decreases the abts antioxidant activity values for raw and pickled cabbage by 45.1% and 43.6%, respectively. the dpph antioxidant activity of raw and pickled cabbages is 9.68 and 9.33 mg te/100 g (p > 0.05), respectively, and no change is observed after the brining process, similar to results reported in the literature (girgin et al., 2015). differently, in a previous research, the dpph antioxidant activity of white cabbage increased on day 1 and then lowered through fermentation, probably due to the passage of antioxidant compounds to the brine (parada et al., 2022). after in vitro digestion, dpph antioxidant activity is found to remain unaffected by in vitro digestion of raw and pickled cabbage. the frap antioxidant activity values obtained for the cabbages were similar. the frap antioxidant activities of raw cabbage, pickled cabbage, digested raw cabbage, and digested pickled cabbage are determined to be 0.30, 0.32, 0.29, and 0.34 mmol fe/100 g, respectively. the abts antioxidant activity values for gherkin and pickled gherkin are determined as 16.11 and 15.19 mg te/100, respectively (p > 0.05). after in vitro digestion, the abts antioxidant activity of gherkin and pickled gherkin is reduced by 24.2% and 30.9%, respectively (p > 0.05). while the dpph antioxidant activity value in gherkin is 8.06 mg/te, this value is decreased to 7.03 mg te/100 g for pickled gherkin. it is observed that dpph antioxidant activity values are decreased after in vitro digestion of gherkin and pickled gherkin compared to before digestion. after the digestion of gherkins and pickled gherkins, the dpph antioxidant values are decreased by 24.8% and 25.6%, respectively. the frap antioxidant activity values for gherkin and pickled gherkin are determined as 0.35 and 0.40 mmol fe/100 g, respectively (p > 0.05). the frap antioxidant activity value before digestion in gherkin, 0.35 mmol fe/100 g, becomes 0.40 after in vitro digestion (p > 0.05). the frap antioxidant value of pickled gherkins (0.40 mmol fe/100  g) is increased to 0.55 mmol fe/100 g (p < 0.05). correlations between tp, tf, abts, dpph, and frap the correlation coefficients between tp, tf, abts, dpph, and frap values were determined by only using the values obtained for the cabbage samples and are 8 italian journal of food science, 2022; 34 (4) kaşikçi mb and bağdatlioğlu n that for pickled vegetables; fermentation probably leads to looser bonds between phenolic compounds and other molecules in the food matrix, and these compounds are easily released after in vitro digestion. fermentation also has a protective effect on phenolics. both raw and pickled white cabbages and gherkins contain highly bioaccessible phenolics. acknowledgments this research was funded by the scientific and technological research council of turkey-tubitak (project number: 117o754). additionally, the first author received a scholarship from tubitak. disclosure statement the authors declare that they have no known competing interests to report. references acosta-estrada b.a., gutiérrez-uribe j.a., serna-saldívar s.o. bound phenolics in foods, a review. food chem. 2014;152:46– 55. https://doi.org/10.1016/j.foodchem.2013.11.093 bahorun t., luximon-ramma a., crozier a., aruoma o.i. total phenol, flavonoid, proanthocyanidin and vitamin c levels and antioxidant activities of mauritian vegetables. j. sci. food agric. 2004;84:1553–1561. https://doi.org/10.1002/jsfa.1820 bouayed j., deußer h., hoffmann l., bohn t. bioaccessible and dialysable polyphenols in selected apple varieties following in vitro digestion vs. their native patterns. food chem. 2012;131:1466– 1472. https://doi.org/10.1016/j.foodchem.2011.10.030 bovy a., vos r. kemper m., schijlen e., pertejo m.a., muir s., 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https://doi.org/10.1016/j.foodchem.2013.11.093 cvetković b.r., pezo l.l., mišan a., mastilović j., kevrešan ž., ilić n., et al. the effects of osmotic dehydration of white cabbage shown in table 7. the strongest correlation is found to occur between the tp and tf in cabbage samples. tp and tf give the highest positive correlation coefficients for frap antioxidant activity among the antioxidant activity tests. it is also observed that there is no strong positive correlation between antioxidant activity values in cabbage samples. in the literature, while a very strong correlation has been found between ascorbic acid and abts, the tp-abts correlation is insignificant (güleç et  al., 2013). in another study conducted using brassica vegetables, including cabbage, the tp-frap and tf-frap correlation coefficients were determined to be 0.357 and −0.0737, respectively (jaiswal et al., 2011). the correlation coefficients between tp, tf, abts, dpph, and frap values were calculated using values obtained for the gherkin sample and are shown in table  8. the strongest positive correlations were found to occur between abts-dpph and tp-tf. according to the data obtained, the phenolic compounds in gherkins have little effect on antioxidant activity. however, frap antioxidant activity is found to have the highest positive correlation with tp and tf among antioxidant activity analyses. conclusion white cabbages, gherkins, and their pickles are healthy foods due to their phenolic and flavonoid contents and antioxidant activity. fermentation does not affect tpc, tfc, or antioxidant activity in white cabbage. for gherkin, tfc and antioxidant activity are also not affected by fermentation, but the tpc is decreased. the bioaccessibility of the tpc and tfc for raw vegetables is lower than table 7. correlation coefficients between tp, tf, abts, dpph, and frap in cabbage. tf abts dpph frap tp 0.768 −0.788 −0.279 0.577 tf – −0.538 0.048 0.663 abts – – 0.289 −0.193 dpph – – – −0.211 table 8. 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ying-chun lin1* 1department of food science and technology, and 2department of pharmacy (with master’s program), chia nan university of pharmacy and science, tainan city, rende dist., taiwan (roc) *corresponding author: ying-chun lin, department of food science and technology, chia nan university of pharmacy and science, no. 60, sec. 1, erren rd., tainan city, rende dist. 71710, taiwan (roc). email: ifelin@mail.cnu. edu.tw received: 28 september 2021; accepted: 8 february 2022; published: 25 february 2022 © 2022 codon publications open access paper abstract cocoa tree (theobroma cacao l.) is a recently planted crop in taiwan, a country located in east asia. taiwanese cocoa beans are appreciated globally because of their distinctive flavor and aroma. the effects of the water extracts of unfermented taiwan cocoa beans (wufcb) and fermented and roasted taiwan cocoa beans (wfrcb) on the anti-oxidation, vascular protection, and variation in phytochemical components were investigated. variations in the catechins components of wufcb and wfrcb were examined by high performance liquid chromatography. the values of catechin compounds in wufcb (epicatechin [ec]: 52.32 ± 0.56 mg/g, and catechin (c): 15.14 ± 0.26 mg/g) were approximately two times higher than those found in wfrcb (ec: 26.22 ± 0.48 mg/g, and c: 4.56 ± 0.10 mg/g), indicating that the fermentation and roasting steps caused decline in catechins compounds of wfrcb. in the range of 50–300 µg ml-1, both wufcb and wfrcb depict noncytotoxicity in endothelial cells; they protect cells from h2o2-induced cytotoxicity as established by mtt assay. meanwhile, nitric oxide (no) levels in endothelial cells were elevated by wufcb. in addition, wufcb displayed radical scavenging in the acellular model and inhibited increase in reactive oxygen species (ros) noted in endothelial cell induced by h2o2. overall, the significant vascular protection of wufcb is associated with increased no formation. the decreased ros generation against oxidative damage was attributed to abundant catechin compounds. this study establishes taiwan cocoa polyphenol as an effective and complementary tool for preventing endothelial dysfunction and cardiovascular disease. keywords: endothelial cells, fermentation and roasting, oxidative damage, taiwan cocoa polyphenol, vascular protection introduction the impression of humans toward chocolate products has gradually shifted from desserts to healthcare products, emphasizing the amount of bioactive substances found in cocoa beans. polyphenols in cocoa beans not only bring out unique astringency and bitterness of chocolate flavor but also possess numerous benefits to human health, for instance, eyesight protection (puell and de pascualteres, 2021); prevention of parkinson’s and alzheimer’s diseases, improved recognition ability, prevention of obesity, increasing the amounts of adiponectin and glucose transporter, decreasing the production of lipid, and insulin resistance (magrone et al., 2017). moreover, buijsse et al. (2010) discovered that polyphenols relax the smooth muscles, having the potential to reduce blood pressure. heiss et al. (2010) demonstrated that polyphenols prevent the aggregation of platelets and moderate inflammation (monagas et al., 2009). all these mentioned benefits are related to a critical neurotransmitter in the mailto:ifelin@mail.cnu.edu.tw mailto:ifelin@mail.cnu.edu.tw italian journal of food science, 2022; 34 (1) 115 phytochemical component, and antioxidant and vasculo-protective activities of taiwan cocoa polyphenols material and methods materials 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt), epicatechin, catechin, theobromine, caffeine, and dimethyl sulfoxide (dmso) were purchased from sigma-aldrich chemical co. (st louis, mo, usa). mouse vascular endothelial cells, svec4-10 (bcrc no. 60220) were purchased from the bio-resource collection and research center (bcrc, food industry research and development institute, hsinchu, taiwan). all chemicals of analytical grade were used in the research. preparation of cocoa beans cocoa pods were purchased from a pingtung farm in taiwan. the husk and damaged parts of the cocoa pods were removed. then 60-kg cocoa beans were fermented for 7 days and sun-dried until the moisture level was 6%. following fermentation and sun-drying, the cocoa beans were randomly sampled for roasting. the beans were roasted for 25 min at a temperature of 130°c. the final roasted cocoa beans were known as fermented and roasted cocoa beans. on the other hand, another 20 kg of harvested cocoa beans were in boiling water for 15 min at 90°c and at once cooled in ice water. then, the boiled cocoa beans were washed and sun-dried until the moisture level dropped to 6%. these bean samples were known as unfermented cocoa beans. extraction a total of 100 g of cocoa beans were peeled manually for each processing method and the nibs were ground into powder in a grinder with liquid nitrogen. the cocoa powder was defatted for 20 min and extracted with boiling water in 1:10 (w/v) ratio. after filtering through advantec no, 2. filter paper, the residue was reextracted using the same steps. the supernatant was collected and concentrated through a rotary evaporator, freeze-dried, and stored under -20°c. two different cocoa extracts were obtained, viz. water extracts of fermented and roasted cocoa beans (wfrcb) and water extracts of unfermented cocoa beans (wufcb). high-performance liquid chromatography (hplc) analysis marker components in cocoa extract the analysis was performed with hplc according to the method enumerated by zhang et al. (2016). hplc cardiovascular system, that is, nitric oxide (no). nitric oxide serves as a second messenger for signaling the cardiovascular smooth muscles. small molecular weight and lipophilicity of no allow it to rapidly pass through cell membranes and reach the smooth muscle cells. sufficient no bioavailability is associated with normal vasodilation and normal blood pressure. the factors influencing no levels are as follows: (1) most no is synthesized from l-arginine in a reaction catalyzed by the enzyme of endothelial no synthase (enos). the no level depends on enos expression and activity. (2) no reacts with superoxide anion (o2-) to form peroxynitrite (onoo-), which can oxidize cell components, eliminate no levels and suppress no bioavailability. (3) the major sources of reactive oxygen species (ros) (including h2o2, oh. and o2-.) are related to mitochondrial electron transport chain and nicotinamide adenine dinucleotide phosphate (nadph) oxidase (nox) pathway. the elimination of ros production is associated with decreasing cellular oxidative stress, generation of onooand increased no levels (fraga et al., 2011 ; oteiza et al., 2021). as indicated, cocoa polyphenol is involved in these phenomena. according to the statistics provided by the international cocoa organization (icco), global cocoa bean production in 2018–2019 season exceeded 4.78 million tons, and the production of 2019–2020 season exceeded 4.69 million tons (international cocoa organization, 2020; olga et al., 2020). in taiwan, the planting area of cocoa has increased in recent years, particularly in neipu and wanluan counties of pingtung, southern taiwan, where cocoa plantation is done in about 350 hectares. nowadays, the distribution of cocoa tree planting area has even reached to the middle of taiwan, indicating cocoa trees have become a recently developed crop in taiwan. this has led the government and academic institutions to conduct cocoa-relevant research, for instance, the effect of choice of fermentation of starter cocoa cultures, roasting temperature of cocoa beans leading changes in flavanols and procyanidins and sensory properties of cocoa end products. all these investigations aim to enrich the competitiveness and value of taiwan’s cocoa products. however, few studies are conducted involving the cytoprotection of taiwan cocoa beans and content levels of catechins and methylxanthine compounds in fermented and unfermented cocoa beans, both of which are expected to have higher phytochemical contents. the present study was designed to prepare two types of cocoa beans under different processing methods (i.e., fermented and unfermented) to explore the effect of their extracts concerning phytochemical level, antioxidant activity, diminishing oxidative stress and vascular protection of endothelial cells. the study also evaluated function of taiwan cocoa beans to effectively improve endothelial function. 116 italian journal of food science, 2022; 34 (1) chu h-l et al. was determined. the samples were added to a methanolic solution (1 ml) of dpph radicals (final concentration of dpph was 0.2 mm). the mixture was shaken vigorously for 30 min at room temperature and then placed in dark. absorbance of the resulting solution was measured at 517 nm. trolox equivalent (te) was used as a reference standard. trolox equivalent antioxidant capacity (teac) assay the 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (abts+) cation-free radical inhibitory activity was measured as described previously (mellinas, et al., 2020). the abts+ was generated by reacting 1-mm abts+ with 0.5-mm hydrogen peroxide and 10 units/ml horseradish peroxidase in dark at 30°c for 2 h. after addition of 1-ml abts+ to cocoa extract, absorbance was measured after 10 min at 734 nm. a lower level of absorbance indicated a stronger inhibitory activity of samples. trolox equivalent was again used to obtain calibration curve. svec4-10 cell line culture svec cells were obtained from bcrc, taiwan. the cells were cultured in dulbecco’s modified eagle medium (dmem) containing 10% heat-inactivated fetal bovine serum and 1.5 g l-1 l-glutamine at 37°c in a humidified 5% co2/95% air-controlled incubator. cell viability assay cell viability was performed by mtt assay. mtt is a tetrazolium salt and was converted into insoluble formazan by mitochondrial succinate dehydrogenase of living cells. briefly, cells were seeded in 96-well plates cultured for 24 h. next, the cells were treated with sample for 1 h, h2o2 was added to the medium and incubated for 24  h. then 5 mg ml-1 mtt stock solution was added in each well and incubated for 2 h. subsequently, the medium was removed and the plates were incubated for 30 min to solubilize colored formazan dye by the addition of dmso. then the absorbance of each well was measured with a thermo model 355 microplate reader at 550 nm. intercellular reactive oxygen species in order to determine the generation of ros in cells, 2’,7’-dichlorofluorescein-diacetate (dcfh-da) was used. it penetrates cell membranes and is hydrolyzed by intracellular esterase to form dichlorodihydro-fluorescein (dcfh). dcfh reacts with ros generated by intracellular stress to produce highly fluorescent dcf, which chromaster (hitachi ltd., tokyo, japan) comprised 7725i injector, 5110 pump and 5430 diode array detector (dad). sample (1 mg ml-1) was filtered through a hydrophilic pvdf 0.22-µm membrane and injected with a 20-µl sample into an rp-18 column (5µm particle, 4.6 × 250 mm, mightysil [kanto chemical, tokyo, japan]). the elution buffer consisted of acetonitrile (solvent a) and 1% (v/v) phosphoric acid in water (solvent b) at a flow rate at 0.8 ml/min. the solvent gradient was as follows: 0–8 min, 5–15% b, 8–30 min, 15–25% b, 30–40 min, 25–30% b followed by equilibration of the column prior to new injection. all compounds were detected at 280 nm. based on the profile of the concentration (x, µg ml-1) versus peak area (y), the linear regression equation and the correlation coefficients (r2) were as follows: theobromine: y = 7,810.1x -14,896 (r2 = 0.997); epigallocatechin (egc): y = 2,700x -21.0 (r2 = 0.999); caffeine: y = 31,668x +9,043.1 (r2 = 0.999); catechin (c): y = 8,278.9x –1,790.9 (r2 = 0.999); epicatechin (ec): y = 9,274.6x +13,850 (r2 = 0.994); epigallocatechin gallate (egcg): y = 9,929.2 x -474.1 (r2 = 0.999); and epicatechin gallate (ecg): y = 10,790x -445.4 (r2 = 0.999). the hplc analysis for each sample was performed in triplicate. total polyphenolic content (tpc) total polyphenol content was analyzed as gallic acid equivalent (gae). different concentrations of cocoa extracts were added in a 10-ml volumetric flask, to which 2-ml sodium carbonate (20% w/v) was added. after 5 min, 0.1-ml folin–ciocalteu reagent (50% v/v) was added and the volume was made up to 10 ml with h2o. after 1-h incubation at 30°c, the absorbance was measured at 750 nm and compared to a gallic acid calibration curve. proanthocyanidins content (pac) the working solution consisted of 0.25-ml cocoa extract, 1.5-ml n-buoh/hcl (95:5, v/v) and 50-µl 2% solution of nh4fe(so4)2 12h2o in 2-m hcl. the reaction mixture was capped, shaken and incubated for 1 h at 95°c. the solution was cooled at room temperature and the absorbance was measured at 550 nm using an enzyme-linked-immunosorbent assay (elisa) reader (molecular devicesvmax, ma, usa). the concentration of proanthocyanidin was measured from a standard curve of cyanidin chloride with the same protocol, and was indicated as mg cyanidin chloride equivalent (cye/g) of cocoa extract. dpph radical scavenging activity the effect of cocoa extracts on reducing the 2,2-diphenyl-1-picrylhydrazyl (dpph) radical scavenging activity italian journal of food science, 2022; 34 (1) 117 phytochemical component, and antioxidant and vasculo-protective activities of taiwan cocoa polyphenols 0.11 mg/g wfrcb), caffeine ( 5.38 ± 0.07 mg/g wufcb and 8.90 ± 0.16 mg/g wfrcb), catechin (15.14 ± 0.26 mg/g wufcb and 4.56 ± 0.10 mg/g wfrcb), epicatechin (52.32 ± 0.56 mg/g wufcb and 26.22 ± 0.48 mg/ g wfrcb), epigallocatechin gallate (egcg, 10.78 ± 0.12 mg/g wufcb and 5.00 ± 0.50 mg/g wfrcb) and epicatechin gallate (ecg, 0.40 ± 0.15 mg/g wufcb and 0.48 ± 0.05 mg/g wfrcb), by comparison between retention time (rt), uv-vis spectrum, the characteristic maximum wavelength (λmax), and literature data from commercial standard and samples. variation in methylxanthine and catechin levels between wufcb and wfrcb was investigated as shown in table 1. the data indicated that wufcb was rich in catechin compounds, including catechin, epicatechin, egcg, and egc. the values of catechin compounds present in wufcb were approximately two times higher than those found in wfrcb, except for ecg. alternatively, there was a lower content of methylxanthines (theobromine and caffeine) in wufcb involved in the pretreatment; it was boiled in hot water at 90°c for 15 min for inactivating polyphenoloxidase and peroxidase. in the latter situation, a lot of methylxanthines (theobromine and caffeine) were found soluble in hot water (febrianto and zhu, 2020) that retained a lower concentration of methylxanthines present in wufcb. theobromine, caffeine, catechin, epicatechin and procyanidins are related to the astringency and bitter taste of cocoa products. the bitter taste of cocoa is largely influenced by the content of theobromine and caffeine, and to a lesser degree by polyphenolic compounds. additionally, theobromine and caffeine have a physiological stimulatory activity on the central nervous system (cns) with beneficial health effects on cognition, satiety function, and mood (letricia et al., 2021). as described in table 1, the main polyphenol present in both wufcb and wfrcb was epicatechin. current research has found that epicatechin can serve as a free radical scavenger, metal chelator, enos activator and arginase inhibitor (fraga et al., 2011), modulating oxidative stress and contributing to the prevention of cardiovascular diseases. therefore, epicathchin was used as a reference control in the following assay. effect of cocoa extracts on antioxidant activity polyphenol and proanthocyanidins naturally occur in cocoa beans, playing important roles in different biofunctional activities and have positive health effects in humans. the tpc values in wufcb and wfrcb are shown in figure 1a, displaying significant difference (p < 0.05). at 400 µg ml-1 of wufcb and wfrcb, tpc was quantified in 16.93 ± 1.71 mg gae and 13.84 ± 1.48 mg gae, respectively. it was suggested that fermentation and emits fluorescence when excited at 485 nm. the svec410 cells were cotreated with cocoa extract and 0.2-mm h2o2. subsequently, dcfh-da (50 µm) was added to the medium and incubated for 240 min. the treatment medium was removed, and the cells were washed twice with phosphate buffer solution (pbs). the ros produced from intracellular stress was detected using a bio-tek flx800 microplate fluorescence reader (winoosky, vt, usa) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. intercellular nitric oxide nitrite levels in cell-cultured media, reflecting the intracellular no synthase activity, were determined by the griess reaction. briefly, the cells were cultured for 18 h with cocoa extracts. then the growth medium was added with the same volume of griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% n-(1-naphthyl)ethylenediamine dihydrochloride in water) at room temperature. then the absorbance of mixture at 550 nm was detected. statistical analysis each experiment was performed at least for three times and the average was calculated. data are expressed as mean ± sd, and anova was conducted using the spss software (version 12.0, spss inc., chicago, il, usa). when a significant f-ratio (mean square between treatments / mean square within treatments) was obtained (p < 0.05), a post hoc analysis was conducted between groups using duncan’s multiple range tests. a significant difference between treatments was considered for p < 0.05. results and discussion principle phytochemicals present in wufcb and wfrcb using hplc phytochemicals are plant-derived small molecules possessing multifunctional effects and significantly inhibit oxidative stress. it is worth evaluating the variation in the phytochemical components of taiwan cocoa beans with different processing procedures. the analysis and separation of the phytochemical components present in wufcb and wfrcb were performed by hplc-dad using an rp-18 analytical column. the qualitative analysis of the phytochemicals utilized linear regression equation of commercial standard compound as described in ‘methods’ section. seven principle compounds were identified and quantified, including theobromine (49.57 ± 1.06 mg/g wufcb and 71.22 ± 1.24 mg/g wfrcb), epigallocatechin (2.22 ± 0.31 mg/g wufcb and 1.06 ± 118 italian journal of food science, 2022; 34 (1) chu h-l et al. table 1. hplc analysis of principle polyphenols present in wufcb and wfrcb. compound retention time (min) λ max (nm) content (mg/g extract) wufcb wfrcb theobromine 10.1 272 49.57 ± 1.06 71.22 ± 1.24 egc 13.6 271 2.22 ± 0.31 1.06 ± 0.11 caffeine 14.9 272 5.38 ± 0.07 8.90 ± 0.16 catechin 15.3 278 15.14 ± 0.26 4.56 ± 0.10 epicatechin 18.1 278 52.32 ± 0.56 26.22 ± 0.48 egcg 18.9 275 10.78 ± 0.12 5.00 ± 0.50 ecg 26.7 276 0.40 ± 0.15 0.48 ± 0.05 the data were indicated with mean ± sd. egc: epigallocatechin; egcg: epigallocatechin gallate; ecg: epicatechin gallate; wufcb: water extracts of unfermented cocoa beans; wfrcb: water extracts of fermented and roasted cocoa beans. roasting of the processing stage significantly decreased tpc, that is, wfrcb is 18.3% lower in tpc after these processing steps. proanthocyanidins have depicted potential beneficial effects on human health, in addition to a pleasant sensory ingredient found in cocoa products (pedan et al., 2018). the effect of wufcb and wfrcb on pac display significant differences, as shown in figure 1b (p < 0.05). pac was influenced by fermentation and roasting steps; it decreased from 3.43 ± 0.02 mg cye/g wufcb to 2.97 ± 0.09 mg cye/g wfrcb. proanthocyanidins play a potential role in the prevention of cardiovascular disease and this includes the monomer, catechin and epicatechin, and oligomer units. the obtained data from figures 1a and b confirm the observation that operational process decreased the levels of pac and tpc. the results were consistent with previous studies, and the following reasons explain the higher levels of pac and tpc in wufcb than in wfrcb: (1) wufcb (unfermented cocoa beans) were raw cocoa beans and had not undergone the process of fermentation and roasting. polyphenolic compounds diffused from cellular compartments and oxidized to produce insoluble high molecular weight tannins during the fermentation period. these oxidation reactions were catalyzed by polyphenol oxidase (ppo), which could be the remaining 5–10% of enzymatic activity between the first and the second day of fermentation (febrianto and zhu, 2020); and (2) the pretreated wufcb were heated at 90°c for 15 min, which almost completely inhibited ppo activity. lower ppo activity retained more tpc and pac. further, lower pac and tpc contents in wfrcb could be because these compounds were susceptible to oxidative degradation during fermentation and roasting (quiroz-reyes and fogliano, 2018). figure 1. determination of (a) total polyphenol content (tpc) and (b) proanthocyanidins content (pac) in water extracts of unfermented cocoa beans (wufcb) and water extracts of fermented and roasted cocoa beans (wfrcb). different superscript letters indicate significant differences (p < 0.05). (a) (b) italian journal of food science, 2022; 34 (1) 119 phytochemical component, and antioxidant and vasculo-protective activities of taiwan cocoa polyphenols in causing oxidative damage and pathological events, including inflammation, aging and cardiovascular diseases. these observations demonstrated that antiradical activities were potentially affected by tpc and pac found in cocoa and the low-processing cocoa extract (i.e., wufcb), rich in tpc and pac and higher antiradical activity, was a promising candidate for becoming a potential health-promoting food. in order to examine whether the antiradical activity is related to tpc and pac, a correlation coefficient test (r2) was performed. previous studies have reported a strong correlation between polyphenolic compounds with the antiradical activity of extracts (carmen l.d.t.s. et al., 2021). based on the concentration of tpc (x, mg gae) or pac (x, mg cye) versus dpph (y, mg te) and abts scavenging activities (y, mg te), the linear regression equation and the correlation coefficient (r2) are as follows: tpc-dpph /wufcb: y = 1.1383x + 5.007 (r2  = 0.992); tpc-abts/wufcb: y = 2.5858x 5.6934 (r2 = 0.999); pac-dpph/wufcb: y = 12.108x + 3.889 (r2 = 0.990); pac-abts/wufcb: y = 31.462x 10.461 (r2 = 0.998); tpc-dpph/wfrcb: y = 1.6322x + 4.1053 (r2  = 0.987); tpc-abts/wfrcb: y = 1.9505x 0.0044 (r2  = 0.965); pac-dpph/wfrcb: y = 16.93x + 3.4375 (r2 = 0.996); pac-abts/wfrcb: y = 15.483x + 0.6961 (r2 = 0.972). the high correlation values observed for antiradical activity are related to both tpc and pac. the r2 varied from 0.965 to 0.999. effect of cocoa extracts on cell viability in endothelial cells endothelial cells are susceptible to oxidative damage, resulting in disturbed cardiovascular homeostasis. dpph and abts methods, associated with free radical scavenging activities, were selected to assay antioxidant activity. the effect of wufcb and wfrcb on dpph and abts radical scavenging activities are shown in table 2. both wufcb and wfrcb exhibit a concentration-dependent effect for these two methods in terms of free radical scavenging assay and display a significant difference (p < 0.05). the values of antiradical activity by dpph were similar to that of abts. although similar results were obtained in both dpph and abts assays, properties of each of the extracted compounds differed. in dpph assay, it easily interacted with hydrophobic compounds; however, it interacted with both hydrophilic and hydrophobic molecules in abts assay (carmen l.d.t.s. et al., 2021). the dpph radical scavenging activities for wufcb and wfrcb at concentrations of 150 µg ml-1 were equal to 9.70 ± 0.82 mg te and 8.86 ± 0.62 mg te, respectively. the abts radical scavenging activities were 11.81 ± 0.26 mg te and 9.26 ± 0.39 mg te, respectively. wufcb exhibited more antiradical activity than wfrcb. the obtained results demonstrated that more of processing steps decreased both free radical scavenging activities. the data obtained in figure 1 and table 2 show that less processing steps implies higher retention of tpc and pac, and dpph and abts radical scavenging activities. the results also established that the antioxidant capacity is directly related to tpc and pac, which, of course, are affected by the process of fermentation, drying and roasting. the main polyphenolic compounds present in cocoa beans are epicatechin and pac, which are composed of aromatic rings and functional groups such as hydroxyl group. these structures allow tpc and pac to chelate metal ions, resulting in the formation of inactive complexes, or the activity to stabilize and reduce hydroxyl, peroxyl or superoxide radicals (noelia et al., 2021). free radicals play a crucial role table 2. the effect of wufcb (water extracts of unfermented cocoa beans), and wfrcb (water extracts of fermented and roasted cocoa beans) on dpph and abts scavenging activity. sample concentration (μg ml–1) dpph scavenging activity (mg te) abts scavenging activity (mg te) epicatechin 50 4.37 ± 0.39e 3.59 ± 0.57e 100 5.43 ± 0.49de 6.82 ± 0.34d 150 6.87 ± 0.90bcd 10.60 ± 0.31ab wufcb 50 7.50 ± 0.85bc 4.48 ± 0.71e 100 8.74 ± 0.94ab 8.25 ± 0.43c 150 9.70 ± 0.82a 11.81 ± 0.26a wfrcb 50 6.68 ± 0.52cd 3.48 ± 0.65e 100 8.09 ± 0.66abc 6.52 ± 0.53d 150 8.86 ± 0.62ab 9.26 ± 0.39bc the data were indicated with mean ± sd. the different letters indicated significant differences (p < 0.05). 120 italian journal of food science, 2022; 34 (1) chu h-l et al. cytoprotection of taiwan cocoa polyphenol was ascribed to the amelioration of oxidative stress, the effects of the wufcb and wfrcb were examined on the intracellular ros formation of svec4-10 damaged by h2o2. figure 3 displays the effect of wufcb, wfrcb and epicatechin on ros formation in endothelial cells. the intensity of florescence dcf was determined in endothelial cells pretreated with samples for 1 h and then exposed to 0.2-mm h2o2. if the endothelial cells were damaged by h2o2 for 30 min, intracellular ros increased to 207.8% compared with the corresponding control value (100%). in addition, pretreatment of endothelial cells with 300 µg ml-1 of wufcb and wfrcb, and 5 µg ml-1 of epicatechin, together with h2o2, decreased intracellular ros to 82.31%, 86.41% and 106.58%, respectively (figure 3). there was a statistically significant decrease in ros formation, indicating that wufcb, wfrcb and epicatechin significantly ameliorated intracellular accumulation of ros. moreover, the identification of catechins compounds by hplc-dad, such as epicatechin, against oxidative damage induced by h2o2 may help researchers to better understand protective association of wufcb with oxidative stress induced by h2o2. this demonstrated that taiwan cocoa polyphenol could be expected to protect endothelial cells against ros formation by h2o2-induced oxidative damage. effect of cocoa extracts on no production in endothelial cell endothelial nitric oxide synthase produces no, a freely diffusible gas, acting as a second messenger for signaling vascular smooth muscle relaxation, causing vasodilation and lowering of blood pressure. however, a fall in no steady-state level results in endothelial dysfunction, failure of smooth muscle relaxation, and consequent hypertension (engler and engler, 2004; fraga, et al., 2011). in this regard, we determined whether wufcb, wfrcb and epicathchin modulated no generation in endothelial cells. no is a short-lived free radical that rapidly reacts with molecular oxygen to yield nitrogen dioxide, dinitrogen trioxide and nitrite. nitrite is the only stable product. nitrite levels in the culture medium were determined as an index of no synthesis by using the griess reagent (chu et al., 2017). incubation with samples for 18 h demonstrated that epicatechin had no significant effect on no generation (figure 4). alternatively, treatment of endothelial cells with 300-µg ml-1 wufcb resulted in 116.38% no generation compared with the untreated cells (100%). in other words, wufcb has the potency to increase no generation in endothelial cells. this result is in agreement with previous reports (engler and engler, 2004) that demonstrated that polyphenol-rich cocoa could activate enos for synthesis no, inhibit arginase and nadph oxidase, leading to lower levels of o2 -.; it for  this reason, the endothelial cell line svec4-10 was used in this study. mtt assay is widely used to measure cell growth. the cytotoxicity of cocoa extract and epicatechin was determined by an mtt assay. figure 2a shows the response of endothelial cells incubated with samples for 24 h. the cell viabilities of two cocoa extracts of 50–300 µg ml-1 and epicatechin at 5–20 µg ml-1 were from 91.46% to 127.31% compared to untreated control cells. in other words, wufcb, wfrcb and epicatechin demonstrated non-cytotoxicity in endothelial cells at all studied concentrations. effect of cocoa extracts on cytoprotection by h 2 o 2 -induced oxidative stress in endothelial cells free radicals, including o2 -.,oh., and h2o2, are toxic to vascular endothelial cells. h2o2 has no charge and can diffuse through the cell membrane. therefore, in this research h2o2 was selected as an oxidant reagent to induce oxidative stress in endothelial cells. in order to evaluate whether taiwan cocoa polyphenol was involved in protecting endothelial cells from h2o2-induced oxidative stress, direct cytotoxicity effect of h2o2 on endothelial cells was examined by mtt assay. this included 50, 100 and 300 µg ml-1 of cocoa extracts and 5, 10 and 20 µg ml-1 of epicatechin. endothelial cells were damaged by h2o2 for 1 h, and the samples were added and incubated for 24 h. the viability of untreated or treated cells by 0.2-mm h2o2 in the absence of samples was 223.85% and 100%, indicating that h2o2 demonstrated significant cytotoxicity on endothelial cells (figure 2b). treatment of 300 µg ml-1 of wufcb and wfrcb and 20 µg ml-1 of epicatechin with 0.2-mm h2o2 resulted in increased survival rates of endothelial cells, viz. 121.52%, 115.75% and 143.97%, respectively, compared with untreated cells (100%). the results in figure 2b indicated the cytoprotective effects of wufcb, wfrcb, and epicatechin from h2o2-induced cytotoxicity and depicted a dose-dependent increase in cell growth. in this study, epicatechin, a major component of taiwan cocoa beans, acted as an effective free radical scavenger in acellular and cellular models, and modulated oxidative stress. effect of cocoa extracts on ros generation in endothelial cells many studies have revealed that the development of endothelial dysfunction and cardiovascular disease displayed altered redox status compared with normal cells, including increased ros generation. additionally, accumulating studies reported that natural phytochemicals, such as egcg, egc, ecg, and proanthocyanin, demonstrate a preventive effect, and are involved in ameliorating intracellular accumulation of ros. to verify whether the italian journal of food science, 2022; 34 (1) 121 phytochemical component, and antioxidant and vasculo-protective activities of taiwan cocoa polyphenols could also raise the level of no (giovanni et al., 2014). moreover, tetramers and higher polymers of procyanidin were related to the activation of enos. according to the analysis of wufcb using hplc-dad (data not shown), the content of tetramer and higher degree of polymerization (dp) procyanidins were abundant in wufcb. conclusion this study demonstrated the basis for the exploitation of polyphenolic compounds extracted from taiwan cocoa beans. the results provided profiles of polyphenol compounds of wfrcb compared to wufcb. wufcb is rich in catechins compounds; additionally, wufcb exerts a marked vascular protective activity in endothelial cells induced by h2o2 that could be attributed to the higher levels of polyphenols, such as epicatechin, catechin, egc, egcg and other compounds, present in wufcb. these exert their protective action against oxidative stress through scavenging ros, and modulate no generation. therefore, wufcb has a great potential for preventing endothelial dysfunction and related complications. declaration of interest the authors declared no conflicts of interest. (a) (b) figure 2. cell viability relative to (a) untreated control and (b) damage of endothelial cells by 0.2-mm h 2 o 2 following different concentrations of water extracts of unfermented cocoa beans (wufcb), water extracts of fermented and roasted cocoa beans (wfrcb) and epicatechin, estimated with mtt assay. the data were indicated as mean ± sd. different markers indicate significant differences (p < 0.05). 122 italian journal of food science, 2022; 34 (1) chu h-l et al. figure 3. effect of water extracts of unfermented cocoa beans (wufcb), water extracts of fermented and roasted cocoa beans (wfrcb) and epicatechin on intercellular ros formation in endothelial cells induced by 0.2-mm h 2 o 2 . ros formation is relative to untreated control of endothelial cells and those treated with different concentrations. the data were indicated as mean ± sd. different markers indicate significant differences (p < 0.05). figure 4. effect of water extracts of unfermented cocoa beans (wufcb), water extracts of fermented and roasted cocoa beans (wfrcb) and epicatechin on intercellular no production in endothelial cell. no production is relative to untreated control of endothelial cells and those treated with different concentrations. the data were indicated as mean ± sd. different markers indicate significant differences (p < 0.05). acknowledgments this work was funded by the ministry of science and technology of the republic of china [most 108-2221-e-041-001]. contribution of authors ying-chun lin designed the study and heuy-ling chu supervised data collection. hong-xuan fu and en-kuang chou analyzed the data. heuy-ling chu and ying-chun lin interpreted the data and reviewed the manuscript for publication. all authors approved the final manuscript. references buijsse b., weikert c., drogan d., and bergmann m. 2010. chocolate consumption in relation to blood pressure and risk of cardiovascular disease in german adults. eur heart j. 31(13):1616–1623. 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ole_link117 ole_link118 ole_link106 ole_link107 ole_link63 ole_link64 ole_link89 ole_link90 ole_link28 ole_link76 ole_link77 ole_link29 ole_link32 ole_link5 ole_link12 ole_link30 ole_link31 ole_link11 ole_link14 ole_link24 ole_link33 ole_link37 ole_link38 ole_link47 ole_link49 ole_link50 ole_link69 ole_link40 ole_link41 ole_link3 ole_link8 ole_link16 ole_link21 ole_link25 ole_link26 ole_link27 ole_link70 ole_link71 ole_link94 ole_link99 ole_link85 ole_link93 ole_link22 ole_link23 ole_link13 ole_link15 ole_link54 ole_link56 ole_link51 ole_link52 ole_link43 ole_link46 ole_link35 ole_link42 ole_link57 ole_link59 ole_link60 ole_link61 ole_link58 ole_link4 ole_link7 ole_link80 ole_link86 ole_link53 ole_link62 ole_link65 ole_link55 ole_link66 ole_link95 ole_link96 ole_link101 ole_link102 ole_link123 ole_link124 ole_link72 ole_link73 ole_link78 ole_link79 bau005 bau010 bau015 bau020 bau025 ole_link44 ole_link48 80 issn 1120-1770 online, doi 10.15586/ijfs.v35i2.2324 p u b l i c a t i o n s codon italian journal of food science, 2023; 35 (2): 80–87 p u b l i c a t i o n s codon effectiveness of bdellovibrio bacteriovorus to contain escherichia coli on milk and temperature impact on predation dynamics silvia pieralisi1#, cristina canonico1#, stefania di lullo1*, gabriele angelico1*, gianluigi cardinali2, elena rocchegiani1, diego maiolatesi1, donatella ottaviani1 1laboratorio controllo alimenti, istituto zooprofilattico sperimentale dell’umbria e delle marche “togo rosati”, ancona, italy; 2facoltà di scienze farmaceutiche, università degli studi di perugia, perugia, italy #these authors contributed equally to this work. *corresponding authors: stefania di lullo, laboratorio controllo alimenti, istituto zooprofilattico sperimentale dell’umbria e delle marche “togo rosati”, via cupa di posatora 3, 60126 ancona, italy. email: s.dilullo@izsum.it; gabriele angelico, laboratorio controllo alimenti, istituto zooprofilattico sperimentale dell’umbria e delle marche “togo rosati”, via cupa di posatora 3, 60126 ancona, italy. email: g.angelico@izsum.it received: 17 january 2023; accepted: 17 march 2023; published: 23 may 2023 © 2023 codon publications open access paper abstract we tested the predation of b. bacteriovorus against escherichia coli in milk samples in three different experiments. in experiment 1, the growth and predatory activity of b. bacteriovorus against e. coli in milk stored at 4°c were evaluated. in experiment 2, the predatory activity of b. bacteriovorus against e. coli in the milk matrix was compared to the optimal one in the medium of choice. in experiment 3, the influence of the native milk microbial community on the predation of b. bacteriovorus against e. coli experimentally added or indigenous grown at 4°c was tested. the predator increased at 4°c by about 1 log in the first 48 hours and caused e. coli decrease by about 2 log after 24 hours. the predator at 30°c reduced e. coli faster (3 log after 6 hours) than at 4°c (2 log after 24 hours). b. bacteriovorus at 30°c preyed on e. coli more in the nutrient broth than in the milk, with the most significant difference by about 4 log after 48 hours. in raw milk contaminated only by the predator, it increased by about 1 log after 48 hours at 4°c, suggesting that it preyed on indigenous microorganisms. b. bacteriovorus could find application in raw milk used as food or raw material during storage at 4°c to reduce the microbial load of spoilage and shigatoxin-producing e. coli (stec) strains of e. coli and therefore increase its shelf-life and healthiness. keywords: bdellovibrio and like organisms; bdellovibrio bacteriovorus; e. coli; milk introduction in italy and other industrialized countries, the consumption of raw milk has been increasing in recent years due to the growing interest of consumers in untreated and locally produced foods (tremonte et al., 2014). indeed, the consumption of raw milk may have a protective association with the development of allergies and it is an important source of vitamin b2 (macdonald et al., 2011). however, the presence of pathogenic microorganisms in raw milk is reported everywhere (boor et al., 2017; macdonald et al., 2011). the legislation dictates to store raw milk at 4°c for 72 hours and to consume it only after boiling to ensure its safety (eu, 2004; italian ministry of health, 2009, 2013). however, the responsibility to sanitize raw milk lies with the consumer, who may underestimate or ignore the microbiological risks associated with it (tremonte et  al., 2014). moreover, when milk is used as a raw material to produce pasteurized, uht (ultra high temperature) milk, and other milk-based foods, mailto:s.dilullo@izsum.it mailto:g.angelico@izsum.it italian journal of food science, 2023; 35 (2) 81 milk and temperature impact on predation dynamics bacteriovorax and bdellovibrio for bio-based food sector intervention strategies (usda, 2018). however, these lines of study have not yet produced published data, to the best of our knowledge. to date, the only data available are those concerning the application of b. bacteriovorus on some sterilized foods and food processing surfaces on a laboratory scale (fratamico and cooke, 1995; lu and cai, 2010; ottaviani et al., 2019; youdkes et al., 2020). the predatory ability of b. bacteriovorus in complex natural habitats, with mixed microbial flora, such as raw milk, is yet to be discovered. this study is the first application of b. bacteriovorus as a predator on milk to obtain laboratory-scale information on its predation potential on a food matrix never tested before. in particular, it was investigated how the refrigeration, “milk matrix,” and the indigenous microbial community could influence the predation of b. bacteriovorus against its preferred prey, that is, e. coli. materials and methods collection and field strains used e. coli atcc 15144 was used as prey. b. bacteriovorus 109 j atcc 15143 was used as a predator. e. coli enrichment and the attack phase of the predator were prepared according to previously standardized methods (ottaviani et al., 2019). for the challenge experiments, we used the predator/prey ratio of 107 pfu/105 cfu per ml (pfu=plaque-forming unit) to activate the best predation (ottaviani et al., 2019). milk sampling each sample was 1460 ml of raw milk from marche’s breed cows from an automatic raw milk machine inside a dairy farm located in the ancona province (the marches region, central italy). samples were stored in a cool box at 4°c and tested within 2 hours from the sampling. four batches were made from each sample, one of 540 ml and three of 300 ml. before the experiment, on other 20 ml of each sample ph (ph meter 300 hanna instruments) was measured, and it ranged between 6.68 and 6.88. raw milk from the same machine had been analyzed for indigenous e. coli, according to the standard method (iso 16649-2:2001). in the previous 6 months, e. coli counts never exceeded 4 log cfu per ml. experiment 1 a 540 ml volume of the first batch was poured into a sterile container and heated until foam appeared, then the heating was stopped. immediately after boiling, the its refrigeration for up to 72  hours is the most efficient method to increase its shelf life and eliminate spoilage by mesophilic bacteria (boor et al., 2017). however, refrigeration of raw milk does not limit the development of psychrotrophic bacteria like pseudomonas (boor et al., 2017). these microorganisms produce thermostable extracellular enzymes that can affect the quality of milk and dairy products after heat treatments when exceeding 106 cfu (colony-forming unit) per ml (boor et al., 2017). to increase the safety and microbiological quality of raw milk, biological control methods based on bacterium-bacterium predation could be tested. bdellovibrio and like organisms (balos) are gram-negative, aerobic bacteria that are predatory toward other gram-negative bacteria (stolp and starr, 1963; williams et al., 2005). balos have been investigated over the past 50 years with promising results in medicine, agriculture, veterinary, and for the treatment of pathogenic and drug-resistant bacteria (dwidar et al., 2012; kadouri et al., 2013). compared to bacteriophages, balos show more non-specific predation as they can attack gram-negative bacteria of distinct genera (dwidar et al., 2012; fratamico and whiting, 1995). in particular, the favorite prey of bdellovibrio bacteriovorus is escherichia coli, including commensal and pathogenic as shigatoxin-producing e. coli strains (stec) (fratamico and whiting, 1995; ottaviani et al., 2019). however, it is also able to attack other pathogenic and spoilage bacteria such as salmonella, shigella, yersinia, serratia, proteus, pseudomonas (dashiff et al., 2011). b. bacteriovorus does not pose a risk to humans as it does not grow in eukaryotic cells (bratanis et al., 2020; dwidar et al., 2012). b.  bacteriovorus does not carry harmful or antibiotic resistance genes and does not prey on gram positive bacteria or fungi playing the role of starter cultures for some food products (dwidar et al., 2012; shemesh and jurkevitch, 2004). finally, b. bacteriovorus can prey on bacteria even if organized in biofilm or viable but non-cultivable (vbnc) (dashiff et al., 2011; dwidar et al., 2012; kadouri and o’toole, 2005; markelova, 2010). previous research has shown that ph, incubation temperature, predator/prey ratio, and the physical and chemical characteristics of the medium can influence predation (fratamico and whiting, 1995; sockett, 2009). most of the published studies have used pure cultures in liquid medium to elucidate the prey-predator interaction. in those experimental contexts, b. bacteriovorus showed preferential predation on e. coli, but the basis for this selection is not known. the interest of the scientific community and legislative bodies in these microorganisms has been increasing in recent years. recently, for the first time, experts from the european food safety agency (efsa) have proposed the use of predatory bacteria such as b. bacteriovorus as one of the measures to be taken in food production environments to contain antimicrobial resistance (efsa, 2021). moreover, the united states department of agriculture (usda) is beginning to test 82 italian journal of food science, 2023; 35 (2) pieralisi s et al. table 1. predator/prey challenge experiments experiment number and aim experimental design storage t°c timescale predator/prey counts—hours 1 evaluate growth and predatory activity of b. bacteriovorus against e. coli in milk stored over time up to 7 days. milk test: boiled milk contaminated with the predator/prey ratio 107 pfu/105 cfu milk control: boiled milk contaminated with 105 cfu prey 4 0, 6, 24, 48, 72, 96, 120, 144, 168 2 (a, b) evaluate predatory activity of b. bacteriovorus against e. coli in milk matrix: (a) during the shelf-life of raw milk; (b) compared to dnb, the medium of choice. a b 30 0, 6, 24, 48, 72 milk test: boiled milk contaminated with the predator/prey ratio 107 pfu/105 cfu milk control: boiled milk contaminated with 105 cfu prey dnb test: dnb contaminated with the predator/prey ratio 107 pfu/105 cfu dnb control: dnb contaminated with 105 cfu prey 3 (a, b) evaluate influence of the native milk microbial community on the predation of b. bacteriovorus against e. coli during the shelf-life of raw milk: (a) by adding e. coli experimentally under the best prey/predator ratio; (b) at natural concentrations of e. coli. a b 4 0, 6, 24, 48, 72 milk test: raw milk contaminated with the predator/prey ratio 107 pfu/105 cfu milk control: raw milk contaminated with 105 cfu prey milk test: raw milk contaminated with 107 pfu predator milk control: raw milk dnb, diluted nutrient broth. graphical scheme of performed experiments: 1, 2 (a, b), 3 (a, b). milk was dispensed into 54 tubes, each with 10 ml of milk. then, 27 tubes were assigned to the control and the same number to the test. to obtain in milk a predator/ prey ratio of 107 pfu/105 cfu per ml, all tubes were homogeneously contaminated by inoculating 0.5  ml of prey suspension at a concentration of about 5 × 106 cfu per ml. the test tubes were then contaminated with 0.5 ml of about 5 × 108 pfu per ml predator. the same amount of peptone salt solution was added to the control. all tubes were stored at 4°c. predator and prey counts were performed at 0, 6, 24, 48, 72, 96, 120, 144, 168 hours from the treatment (table 1). the predator was also tested in the milk control to exclude a cross contamination with  the predator experimentally added to the milk test. for the prey enumeration, 9 ml were diluted 1:10 in buffered peptone water and from the stock suspension, decimal dilutions in peptone salt solution were made (iso 6887-1:2017). then 1 ml of each dilution was inoculated on tryptone bile x-gluc agar (tbx) (biolife, milan, italy) in duplicate, and the plates were incubated at 44°c for 24 hours (iso 16649-2:2001). the data were reported as cfu per ml. b. bacteriovorus enumeration was performed with the plaque assay combining 0.1 ml of prey enrichment and 1 ml of undiluted or diluted milk, and plates were incubated at 30°c for 24  hours up to 5 days (ottaviani et al., 2019). the data were reported as pfu per ml. experiment 2 (a, b) the second batch was heated as in experiment 1. immediately after boiling, the milk was dispensed into 30 tubes, each with 10 ml. fifteen tubes were assigned to the control and the same number to the test. all tubes were homogeneously contaminated as described for experiment 1 and stored at 30°c. the predator and prey counts were performed at 0, 6, 24, 48, 72 hours from the treatment as in experiment 1 (experiment 2a). in parallel, an analogous test and control were prepared, represented by the medium of choice for predator (2b) that was diluted nutrient broth (dnb) consisting nutrient broth (biolife, milan, italy) at a concentration of 0.08 g per liter of distilled water (experiment 2b) (table 1). experiment 3 (a, b) an experiment analogous to experiment 2a was performed on the third batch at 4°c (experiment 3a). italian journal of food science, 2023; 35 (2) 83 milk and temperature impact on predation dynamics predation ability toward e. coli was evaluated. then, we tested boiled milk contaminated with the predator/prey ratio to get the best predation at 4°c. predator and prey counts were performed at 0, 6, 24, 48, 72, 96, 120, 144, 168 hours from the treatment in test and control. the results are summarized in figure 1. the optimal growth temperature of b. bacteriovorus is between 28 and 30°c, making it a mesophile (stolp and starr, 1963; williams et al., 2005). however, in this experiment, the predator multiplied from 24 to 72 hours and then held the experimentally added charge until the end of the experiment at 4°c. lower e. coli levels of the test with respect to the control were observed for all time points, with the greatest significant difference of about 2 log after 24 hours (t = 12.3747; p < 0.0001). previous studies have shown that b. bacteriovorus preyed on e. coli in the liquid medium at the temperature between 12 and 37°c, with the higher activity in the first 7 hours (fratamico and cooke, 1996; fratamico and whiting, 1995). in disagreement with that evidence, in this experiment, b. bacteriovorus preyed on e. coli at 4°c. the e. coli level also progressively decreased in the control, possibly due to refrigeration. however, b. bacteriovorus increased the prey reduction in the test compared to the control throughout the analysis time. experiment 2 (a, b) the aim of this experiment was to evaluate the effect of the milk matrix on the predation of b. bacteriovorus against e. coli for a time corresponding to the shelf-life of raw milk. then we tested at 30°c both the boiled milk (2a) and dnb (2b) contaminated with predator/prey from the microbiological analyses of raw milk carried out in the previous 6 months indigenous e. coli had never reached 5 log, which is the optimal level for predation at maximum efficiency (ottaviani et al., 2019). for this reason, the milk sample was contaminated with 5 log e. coli atcc 15144. in experiment 3b, the growth of b. bacteriovorus and its predation toward natural level of indigenous e. coli were evaluated in raw milk, under normal storage conditions, for a time corresponding to its shelflife. then on the fourth batch, an experiment on raw milk contaminated only with the predator at a concentration of 107 pfu per ml was performed at the same conditions as experiment 3a (table 1). statistical analysis each experiment was repeated in three separate trials, and each trial was carried out in triplicate (n = 9). results of microbiological analyses were reported as mean values (log-transformed) ± standard deviation. mean of plate counts were analyzed for differences in response to predator treatments using the student’s t-test (t). statistical calculations were based on confidence levels (p) equal to or higher than 95%. results and discussion experiment 1 in this experiment, the influence of raw milk storage temperature on the growth of b. bacteriovorus and its bx test 10 9 8 7 6 5 4 3 2 1 0 6 24 48 72 time (h) lo g 1 0c f u /p f u m l–1 96 120 144 168 0 bx control e. coli test e. coli control figure 1. experiment 1. growth of b. bacteriovorus (bx) and e. coli in test (with bx) and control (without bx) boiled milk at 4°c. ♦ e. coli control; ● e. coli test; ▲ bx control; ■ bx test. results of microbiological analyses (n = 9) were reported as mean values (log transformed) ± standard deviation. 84 italian journal of food science, 2023; 35 (2) pieralisi s et al. of b. bacteriovorus against e. coli under the best predation conditions in terms of prey/predator ratio, at the refrigeration temperature for a time corresponding to its shelf-life. then, we tested at 4°c up to 72 hours, the raw milk contaminated with the predator/prey ratio to obtain the best predation. in experiment 3b, the growth of b. bacteriovorus and its predation toward natural level of indigenous e. coli were evaluated in raw milk, under normal storage conditions, for a time corresponding to its shelf-life. predator and prey counts were performed at 0, 6, 24, 48, 72 hours from the treatment in test and control. the results are summarized in figure 3. the predator trends in boiled and raw milk (experiments 1 and 3a) were similar. lower e. coli levels were observed in the test compared to the control for all time points, with the greatest significant difference of about 2 log after 24 hours (t = 5.9747; p = 0.0001). b. bacteriovorus demonstrated a greater (3 log) and faster (after 6 hours) capacity to contain prey in the milk test at 30°c than at 4°c, that is, 2 log after 24 hours. this evidence is realistic as 30°c is the optimum temperature for predation. similar prey concentrations were present in the boiled and raw milk tests (experiments 1 and 3a), at all the time points. this evidence suggests that when e. coli is at an optimal concentration for b. bacteriovorus predation, the indigenous microorganisms of milk potentially competing with e. coli as prey, do not affect the predator’s performance toward its favorite prey. raw milk represents a relevant source of infection by stecs (caprioli et al., 2015; jang et al., 2017). we tested b. bacteriovorus with a non-pathogenic strain of e. coli, however, previous work had shown that the growth rates of pathogenic and non-pathogenic e. coli are similar (cassin et al., 1998). ratios to activate the best predation. for both milk and dnb, predator and prey counts were performed at 0, 6, 24, 48, 72 hours from the treatment in test and control. the results are summarized in figure 2. by comparing predator concentrations at different time points in milk and dnb tests, higher b. bacteriovorus levels for alltime points in dnb than in milk were observed, with the greatest significant difference of about 2 log after 6 hours (t = 3.9497; p = 0.0027). by comparing prey concentrations at different time points in the milk test and control, lower e. coli levels were observed in the test than in the control from 6 to 48 hours, with the greatest significant difference by about 3 log after 6 hours (t = 10.6956; p < 0.0001). by comparing prey concentrations at different time points in milk and dnb tests, lower levels for all-time points in dnb than in milk were observed, with the greatest significant difference of about 4 log at 48 hours (t = 4.0068; p = 0.0025). b. bacteriovorus preyed on e. coli more in dnb than in milk. the higher viscosity of milk than in dnb could make the former less suitable for predation than the latter by hindering b. bacteriovorus to swim and attack the prey. this hypothesis is reinforced by lower predator counts in milk than dnb for the entire analysis period. comparing the growth trends of e.  coli in milk and dnb controls, a lower growth trend was observed in milk than in dnb. this evidence suggests that the milk matrix also slowed prey growth. experiment 3 (a, b) the aim of experiment 3a was to evaluate the influence of the native milk microbial community on the predation bx test 10 (a) (b) 9 8 7 6 5 4 3 2 1 0 6 24 48 72 time (h) lo g 1 0c f u /p f u m l–1 0 bx control e. coli test e. coli control bx test 10 9 8 7 6 5 4 3 2 1 0 6 24 48 72 time (h) lo g 1 0c f u /p f u m l–1 0 bx control e. coli test e. coli control figure 2. experiment 2. growth of b. bacteriovorus (bx) and e. coli in test (with bx) and control (without bx) boiled milk (a) and dnb (b) at 30°c. ♦ e. coli control; ● e. coli test; ▲ bx control; ■ bx test. results of microbiological analyses (n = 9) were reported as mean values (log transformed) ± standard deviation. dnb, diluted nutrient broth. italian journal of food science, 2023; 35 (2) 85 milk and temperature impact on predation dynamics refrigeration and the “matrix of the milk” have limited its performance. at the refrigeration temperature, when e. coli was at an optimal concentration for the predation, the presence of other potential preys in the milk did not affect the predatory efficiency of b. bacteriovorus toward its preferred prey. on the other hand, when e. coli was at a low concentration, the predator grew without predating it, probably by preying on other bacteria present in milk at a high concentration, such as psychrotrophs. our next goal will be to test the predatory activity of b. bacteriovorus against native strains of e. coli and pseudomonas isolated from raw milk and tested both in monoculture and in mixtures. this will help us understand the dynamics of b. bacteriovorus predation and toward which prey the predator shows the greatest affinity. moreover, we are going to test b. bacteriovorus predation against native e. coli in raw milk samples heavily contaminated by this microorganism, extending the study to sheep and goat milk. finally, it will be assessed whether b. bacteriovorus and its metabolites produced during growth have any effect on the organoleptic characteristics, ph, and nutrients of raw milk during its shelf life. it is known that b. bacteriovorus reduces but does not eliminate prey because a balance is established in the growth medium that allows the survival of both the prey, although quantitatively reduced, and the predator (bratanis et al., 2020). in light of this, even if b. bacteriovorus cannot be used to eliminate prey microorganisms, it can still reduce their microbial load and thus improve the microbiological quality of raw milk during its storage at 4°c. it would also be interesting to test this predator in integrated biological-chemical-physical approaches to moreover, in our previous study, b. bacteriovorus showed lytic ability toward stecs and multidrug-resistant e. coli strains of different origins (ottaviani et al., 2019). so it is realistic to think that b. bacteriovorus could similarly contain stecs if they are in raw milk. in experiment 3b, the predator trend was like that of experiment 3a where the prey was experimentally added. in the milk control, predator counts were always not detectable. no significant difference in prey concentration between the test and control was observed. however, in the milk test, b. bacteriovorus grew between 24 and 48 hours, suggesting that it preyed on indigenous microorganisms other than e. coli. previously, it has been reported that b. bacteriovorus showed lytic ability toward psychrotrophic spoilage bacteria like pseudomonas that are commonly found in raw milk (dashiff et al., 2011; fratamico and whiting, 1995; tremonte et al., 2014; vianna et al., 2012). psychrotrophic bacteria and pseudomonas in the raw milk increase during refrigeration, reaching overtime values higher than 6 log cfu per ml (tremonte et al., 2019; vianna et al., 2012). in light of this, in experiment 3b, b. bacteriovorus might have preyed on psychrotrophic microorganisms. this aspect would confirm b. bacteriovorus as a potential candidate to contain gram-negative pathogenic and spoilage bacteria in raw milk, while preserving those gram-positive bacteria, such as streptococci and lactobacilli, which represent part of the natural flora of milk or used as starters in dairy production. this is a preliminary laboratory-scale study to understand whether b. bacteriovorus has the potential as a predator in milk. our results showed that b. bacteriovorus at 4°c survived in milk for the entire analysis time, about 1 week, and preyed for up to 48 hours, although bx test 10 (a) (b) 9 8 7 6 5 4 3 2 1 0 6 24 48 72 time (h) lo g 1 0c f u /p f u m l–1 0 bx control e. coli test e. coli control bx test 10 9 8 7 6 5 4 3 2 1 0 6 24 48 72 time (h) lo g 1 0c f u /p f u m l–1 0 bx control e. coli test e. coli control figure 3. experiment 3. growth of b. bacteriovorus (bx) and e. coli experimentally added in test (with bx) and control (without bx) raw milk at 4°c (a); growth of b. bacteriovorus (bx) and indigenous e. coli in test (with bx) and control (without bx) raw milk at 4°c (b) ♦ e. coli control; ● e. coli test; ▲ bx control; ■ bx test. results of microbiological analyses (n = 9) were reported as mean values (log transformed) ± standard deviation. 86 italian journal of food science, 2023; 35 (2) pieralisi s et al. contain experiments involving human participants and/ or animals. references bonfiglio, g., neroni, b., radocchia, g., marazzato, m., pantanella,  f. and schippa, s., 2020a. insight into the possible use of the predator bdellovibrio bacteriovorus as a probiotic. nutrients 12: 2252. https://doi.org/10.3390/nu12082252 bonfiglio, g., neroni, b., radocchia, g., pompilio, a., mura, f., trancassini, m., et al. 2020b. growth control of adherent invasive escherichia coli (aiec) by the predator bacteria bdellovibrio bacteriovorus: a new therapeutic approach for crohn’s disease patients. microorganisms 8(1): 17. https://doi. org/10.3390/microorganisms8010017 boor, k.j., 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dwidar et al., 2012; iebba et al., 2013). as bdellovibrios are natural residents of the intestinal microbial ecosystem, their functionality and stability should not be affected by the chemical–physical characteristics of the intestinal habitat. probiotics are defined as live microorganisms that confer a health benefit to the host (efsa, 2018). assessment of safety, functionality, and stability are the first points to consider for a microorganism in terms of its use as a probiotic (efsa, 2018). many in vitro and in vivo studies have shown that bdellovibrios meet all these requirements (bonfiglio et al., 2020a, 2020b). as far as our knowledge is concerned, this is the first report on the application of b. bacteriovorus as a predator in milk: we believe our results are promising and that b. bacteriovorus merits further investigation. this approach to decontamination of raw milk based on bdellovibrio is certainly economically sustainable, non-aggressive as it uses microorganisms which are natural components of the human intestinal flora and that also have potential as probiotics. conclusions due to its biological properties and predation mode, b. bacteriovorus could find application in raw milk used as food or raw material during storage at 4°c to reduce the microbial load of e. coli including both spoilage and stec strains and therefore increase its shelf-life, quality, and healthiness. furthermore, if we confirm that b. bacteriovorus preys on the psychrotrophic microorganisms of raw milk and, consequently, also reduces the production of thermostable enzymes, this could also be effective in increasing the shelf life of uht, pasteurized milk, and other milk-derived foods. acknowledgements authors thank general director of izsum for providing necessary facilities for the experiments. this work was supported by italian ministry of health (grant rc 2020-011). declarations conflicts of interest/competing interests the authors declare that they have no financial/non-financial conflict of interest. the manuscript does not https://doi.org/10.3390/nu12082252� https://doi.org/10.3390/microorganisms8010017� https://doi.org/10.3390/microorganisms8010017� https://doi.org/10.3168/jds.2017-12969� https://doi.org/10.3168/jds.2017-12969� https://doi.org/10.3389/fmicb.2020.00662� https://doi.org/10.1128/microbiolspec.ehec-0014-2013� https://doi.org/10.1128/microbiolspec.ehec-0014-2013� https://doi.org/10.1016/s0168-1605(98)00028-2� https://doi.org/10.1016/s0168-1605(98)00028-2� https://doi.org/10.1111/j.1365-2672.2010.04900.x� https://doi.org/10.1111/j.1365-2672.2010.04900.x� https://doi.org/10.5483/bmbrep.2012.45.2.71� https://doi.org/10.5483/bmbrep.2012.45.2.71� https://doi.org/10.2903/j.efsa.2021.6651� https://doi.org/10.2903/j.efsa.2018.5206� italian journal of food science, 2023; 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u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33i4.2090 1 p u b l i c a t i o n s codon colloidal properties and stability of olive oil-in water emulsions stabilized by starch particles umer farooq, carla di mattia*, marco faieta, federica flamminii, paola pittia faculty of bioscience and technology for agriculture, food and environment, university of teramo, teramo, italy *corresponding author: carla daniela di mattia, faculty of bioscience and technology for agriculture, food and environment, university of teramo, via renato balzarini 1, 64100 teramo, italy. email: cdimattia@unite.it received: 17 june 2021; accepted: 21 august 2021; published: 8 october 2021 © 2021 codon publications open access paper abstract in this study, olive oil-in-water emulsions (30% oil, v/v) were prepared by using high-pressure homogenization and different concentrations of modified corn starch particles (6–10% w/v). after a preliminary physical characterization, the modified starch particles were used to produce olive oil-in water (o/w) emulsions whose droplet size and distribution, flow behavior, microstructure, and physical stability were evaluated. the stabilization by pickering phenomena was observed, as well as the formation of a starch network able to entrap oil particles. increasing the starch concentration enhanced the emulsion physical stability by improving the oil particles’ stabilization within the starch network. keywords: corn starch; high pressure homogenization; olive oil; o/w emulsions; pickering emulsions introduction oil-in-water (o/w) emulsions are amongst the most widespread multiphasic systems and, because of their role in providing a proper structure, desirable appearance, and mouthfeel properties to lipid-containing matrices, they are of crucial importance in a wide range of emulsified products from the food, pharmaceutical, and cosmetic industry (yang et al., 2017). in recent years, studies on pickering stabilization of the oil/water interface by solid particles which get absorbed on the interfacial layer and act as a physical barrier have been progressively increasing (tavernier et al., 2016; xiao et al., 2016). indeed, due to the high adhesion energy at the liquid–liquid interface, particle adsorption is considered to be much stronger in comparison to low molecular weight surfactants (mcclements and gumus, 2016; ravera et al., 2020; wu and ma, 2016). solid particles can thus reside at the interface of droplets, thereby providing pickering emulsions with high resistance against coalescence and instability phenomena like ostwald ripening. in preliminary works, inorganic particles such as clay, silica, and latex were used as stabilizers in pickering emulsions (abend et al., 1998; aveyard et al., 2003; binks and lumsdon, 2000); however, due to rapid biodegradability, poor bioavailability, and above all, the increasing attention of consumers toward more natural ingredients, their use in the food industry was largely limited, highlighting the need of using natural biopolymer particles as stabilizers in pickering emulsions (berton-carabin and schroën, 2015; dickinson, 2012). recently, starch has obtained an increased interest as a potential stabilizer biopolymer in pickering emulsions for both food and pharmaceutical applications thanks to its low cost, high natural availability, nontoxicity, ease in production, and high bioavailability (ge et al., 2017; lee and chang, 2019; mcclements and gumus, 2016; tavernier et al., 2016). however, native starch particles are generally italian journal of food science, 2021; 33 (4): 1–10 mailto:cdimattia@unite.it 2 italian journal of food science, 2021; 33 (4) farooq u et al. stabilized by starch-zein nanoparticle complexes were studied as starting material to produce biocomposite edible films (farajpour et al., 2020; qian et al., 2020). the aim of this work was, thus, to study the colloidal properties, flow behavior, and physical stability of olive oil-in-water emulsions obtained by means of hph and stabilized with corn starch granules modified by esterification with citric acid. in a preliminary step, the physical properties, microstructure, and size of corn starch granules after esterification were initially evaluated. moreover, to evaluate any likely effect induced by high dynamic pressure, the properties of the modified starch subjected to hph at conditions similar to those applied during the emulsion preparation were also studied. finally, the o/w emulsions were characterized for droplet size and distribution, flow properties, and physical stability over a storage time of 30 days. material and methods experimental plan the investigation was carried out according to the following steps: step i: starch particles’ modification by esterification with citric acid. step ii: emulsifying activity of modified starch toward olive oil. step iii: characterization of emulsions’ colloidal properties and physical stability. materials native corn starch was provided by roquette italia s.p.a. (cassano spinola, italy). commercial extra virgin olive oil was purchased from a local market. anhydrous citric acid (99.5% purity) was purchased from sigma-aldrich (steinheim, germany). all other chemicals were of analytical grade. methods starch particles’ modification by esterification native corn starch (ns) was esterified with citric acid according to the method of kim et al. (2017) with some modifications. briefly, citric acid (30% on starch dry basis) was dissolved in 50 ml distilled water and subsequently the ph of the solution was adjusted to 3.5 using 10m naoh. an aliquot of 50 g of ns was added in the citric acid solution with continuous mixing and the mixture was then left at room temperature for 16 h. the mixture not hydrophobic and present a low capacity to absorb at oil/water interfaces. among the methods available to increase starch hydrophobicity, esterification is one of the most commonly used (khan and ahmad, 2013). indeed, esterification improves the performance of starches by making them more amphiphilic and hence more suitable for the stabilization of emulsions. esterification is a commonly used process in which an acid or its derivative is used to react with the hydroxyl groups of starch, replacing them with ester groups which are more hydrophobic (fang et al., 2002). as a result, a more hydrophobic starch with reduced retrogradation and improved emulsifying properties is produced (ghanbarzadeh et al., 2011; xie and liu, 2004; zhou et al., 2016). for esterification purposes, among the various organic acids used, citric acid, a trifunctional carboxylic acid, is most widely used due to its low cost, high effectiveness, and environmental sustainability (jiangping et al., 2019). high pressure homogenization (hph) is a technology applied for obtaining finely dispersed o/w emulsions characterized by higher physical stability and resistance. in general, a pre-homogenized, coarse, pre-emulsified mix of the oil and water phases is passed through a small valve under dynamic pressure to produce the final fine dispersion (friberg et al., 2003). it is well known that hph induces starch gelatinization; however, this high pressure-induced gelatinization is significantly different form the heat-induced gelatinization as starch granules retain their structure and only little amount of amylose oozes out (rubens and heremans, 2000; stute et al., 1996). moreover, high-pressure-treated starch granules comprise two different zones: the inner zone, which is completely destroyed due to high pressure and forms a gel-like network, and an outer zone, which largely remains undisturbed (błaszczak et al., 2005). so far the use of hph is largely focused on stabilizing the traditional emulsions using the chemical surfactants and biomolecules while the use of hph in stabilizing the pickering emulsions is rarely investigated. to the best of authors knowledge so far no study is published which investigate the use of hph in stabilizing the pickering emulsions with starch particles, using olive oil as dispersed phase. while hph is largely used to produce emulsions with conventional emulsifiers, its use in the formation of pickering emulsions has been scarcely investigated and, to the best of authors’ knowledge, no studies are available in the literature on its application to olive oil o/w emulsions stabilized with starch particles. currently, very few works are available on starch-stabilized olive oil emulsions as well (farajpour et al., 2020; qian et al., 2020); in the work by quian and coworkers, the use of corn starch nanocrystals with increasing levels of acetylation was explored to produce, by means of a rotor-stator device, pickering olive oil emulsions characterized by different levels of viscoelasticity. in another work, pickering olive oil emulsions italian journal of food science, 2021; 33 (4) 3 colloidal properties and stability of olive oil emulsions experimental data were modeled using the power law equation (eq. 2) σ = kγn (2) where σ = shear stress (pa), k = consistency index, γ = shear rate (1/s), and n = flow behavior (dimensionless). physical stability of o/w emulsions the physical stability of emulsions was measured using the creaming index (ci) during a storage period of 30 days at room temperature. emulsions were photographed at 1-week intervals and the images were processed using imagej software. the cream volume was measured and the creaming index was obtained as a ratio between the total volume (vt) and cream volume (vc) using the following equation c t creaming inde v x 100 v = × (3) optical microscopy the microscopic observation of the pickering emulsions was carried out using an olympus-bx53 light microscope at 10× and 40× magnification. the photographs were taken with a 12-bit digital camera (qiqam fast 1394, surrey, canada) connected with the microscope. the starch was stained with lugol’s solution; samples were placed on the microscope slide and covered with a glass cover before observation. scanning electron microscopy (sem) a scanning electron microscope (model leica cambridge, uk) at 5kv was used to evaluate the microstructural properties of esterified starch. about 100 mg of sample with double-sided adhesive tape was mounted on the sem stub, and to avoid the charging effect due to electron beam it was sputter coated with gold. statistical analysis all experiments were performed at least in triplicates. the analysis of variance (anova) was performed using spss version 24 (spss, chicago, usa). ducan’s test was used for reporting the significance difference results at 95% confidence level (p < 0.05). results and discussion effect of citric acid treatment and hph on corn starch particles the emulsifying ability of starch granules as pickering particles toward o/w emulsions is highly dependent on was than dried in a hot air oven at 60°c for 3–4 h till the 5–10% (w/w) moisture content was achieved followed by grinding and drying at 140°c for 4 h. the dried starch was then ground using a pestle and mortar and washed with distilled water thrice to remove the leftover citric acid. finally, the starch was dried at 45°c for 24 h and finely powdered by a ball-miller (fritsch, idar-oberstein, germany) and passed through a 90-micron sieve. preparation of o/w pickering emulsions the 30% (v/v) o/w emulsions were prepared by mixing the modified starch (ms) suspension at different concentrations (6.0, 8.0, 8.5, 9.0, 9.5, and 10.0%) (w/v) and olive oil with a rotor-stator device (di 25 basic, ika, stufen, germany) at 20,000 rpm for 2 min; the pre-emulsions were then submitted to hph using a panda plus 2000 homogenizer (gea, parma, italy) at 55 bar with a circulation time of 5 min. degree of substitution of esterified starch the method of jeon et al. (1999) with some modifications was used to determine the degree of substitution (ds) of the modified starch. both the native and modified starches (1 g) were slowly dissolved in the 10 ml solution of dmso at 70°c for 10 min and left to cool down at ambient temperature. then, after the addition of a few drops of phenolphthalein indicator, the solution was titrated against a standardized 0.05m naoh solution till a light pink color was attained as the end point. the ds values were calculated using the following equation (zhang et al., 2017): [ ] 0.162(a m / w) 1 0.210 (a m w d / s ) × − × = (1) where, a = volume of naoh used, m = molarity of naoh, and w = weight of the starch sample used. particle size measurement of starch granules and o/w emulsions the particle size distribution of starch granules and oil droplets in the emulsions was measured by laser diffraction (mastersizer 3000, malvern instruments ltd. malvern, uk). for starch, a refractive index of 1.52 was used (loisel et al., 1998). for emulsions, refractive indices of 1.33 and 1.59 were used for water and olive oil, respectively. droplet size measurements are reported as particle size distribution curves and surface mean diameter (d3,2). flow behavior of o/w emulsions the flow curves of pickering emulsions were determined by using a controlled stress–strain rheometer (mcr 302, anton paar, graz austria) equipped with a concentric cylinder configuration. flow curves were measured at 20°c at increasing shear rates from 3 to 300 s−1. the 4 italian journal of food science, 2021; 33 (4) farooq u et al. starch (12.54 ± 0.04 µm and 20.84 ± 0.12 µm, respectively), likely due to the structural modification of corn starch induced by the citric acid pretreatment. the hydrophobic properties of starch particles as a consequence of both esterification and esterification in combination with the hph process were evaluated by measuring the degree of substitution (ds). during the esterification process, hydrophobic groups are introduced to the amylose residues, causing an increase of the ds, resulting in enhanced surface active properties (zhou et al., 2016). the ds of ms was 0.22 ± 0.01, significantly higher than that of ns whose ds value was equal to 0.01 ± 0.00, confirming the efficacy of the esterification process applied. this result is in agreement with literature data where significantly higher ds values were reported after esterification of maca starch with citric acid (lee and chang, 2019). hph did not affect the hydrophobic properties of the starch particles of both native and modified starches as the ds of both hph-ns and hph-ms was not significantly different than the not homogenized sample (data not shown). the changes induced in the morphology and microstructure of the ns and ms are highlighted in the sem images their composition, structure, and granule size (timgren et al., 2013). in order to evaluate any likely effect of hph on starch particles’ morphology and functionality, corn starch was preliminarily modified by using citric acid and then subjected to the homogenization process conditions used for emulsion preparation. it is known that hph can significantly affect starch properties and induce gelatinization (wang et al., 2008) ; however, in this work, pressure of homogenization was kept low (55 bar) to limit state transition of the starch particles dispersed in the aqueous phase. in figure 1, the microstructural properties of native (ns) and citric-acid-modified corn starch (ms) subjected to hph (hph-ns and hph-ms) are shown, while in figure 2 their particle size is reported as d3,2. regardless of the treatments applied, all the starch samples were characterized by a poly-dispersed population. however, hph significantly (p < 0.05) reduced the particle size of both the native starch (figure 1b) and modified corn starch (figure 1d), as also confirmed by the results of the volume/surface (d3,2) and particle size of the samples (figure 2). the size reduction was more evident on the citric-acid-modified corn starch whose median value was significantly lower (p < 0.05) compared to native corn (a) (b) (c) (d) figure 1. micrographs of starch particles taken by light microscope: ns (a), hph-ns (b), ms (c), hph-ms (d). hph: high pressure homogenization; ns: native starch; ms: modified starch. italian journal of food science, 2021; 33 (4) 5 colloidal properties and stability of olive oil emulsions 0 5 10 ns d b c a hph-ns ms hph-ns 15 d 3, 2 (µ m ) 20 25 figure 2. particle size (d 3,2 ) of the differently treated corn starch particles. different letters on each bar indicate significant differences among the mean values (p < 0.05). ns: native starch; ms: modified starch; hph: high pressure homogenization. modified starches at increasing concentrations were used to prepare o/w emulsions made of olive oil as dispersed phase (fixed concentration 30%, v/v), homogenized by means of hph. regardless of the concentration tested, no emulsifying capacity was observed when ns was used as the emulsions readily destabilized; hence, ns was not further taken into consideration. on the contrary, fine olive oil o/w emulsions were obtained when ms was used as emulsifier: figure 4 thus shows the size class and particle size distribution of the olive oil o/w emulsions prepared with ms in the concentration range of 6.0–10.0% (w/v). such a result can be related to the improvement of the emulsifying capacity due to the introduction of ester groups during modification (lee and chang, 2019). indeed, some authors evidenced that esterification increases the amphiphilic properties of starch granules and this leads to a decrease of the surface tension between the oil–water interface with respect to that observed in the corresponding native, unmodified ones (królikowska et al., 2017), favoring the formation and stabilization of the fine oil droplets formed during the homogenization process. with regard to the dispersion degree, of all the ms concentrations tested, the particle size distribution was characterized by a high degree of polidispersity (figure 4a) with at least three oil droplet populations with a relative maximum average at particle sizes of around 2, 5, and 50 mm, respectively. at increasing starch concentrations, a progressive higher particle size was observed along with a relative higher amount of larger particles, with no variation in the size range distribution. this behavior was also reflected in the results of the surface mean droplet size d3,2 of the emulsified samples (figure 4b) with the smaller droplet diameter observed in the emulsions made with lower starch concentration (5.94 ± 0.73 µm). then, by increasing the starch concentration, the droplet particle size increased up to a plateau value of about 2 µm eht = 500w wd = 8.3 mm mag = 5.00 kx signal a = inlens 2 µm eht = 500w wd = 8.3 mm mag = 5.00 kx signal a = inlens figure 3. sem micrographs of ns (a) and ms (b) particles (ns: native starch; ms: modified starch). reported in figure 3. ns granules (figure 3a) present a smooth outer surface while after the citric-acid-modification slight groves and corrosion appeared on the surface of the ms granules (figure 3b). however, overall, the esterification did not significantly alter the main structure of the starch granules or their size. these results are similar to those reported by other authors reporting that chemical modification of native starch only affects the outer surface of the granules while no changes in the morphology and structure of the starch granules occur (mbougueng et al., 2012; zhang et al., 2017). the surface modification of the starch granules induced by the esterification has been related to the weathering effect of acid hydrolysis (alimi and workneh, 2018). emulsifying capacity of modified starch toward olive oil in order to study the emulsifying capacity of starch particles toward olive oil, suspensions of both native and 6 italian journal of food science, 2021; 33 (4) farooq u et al. magnifications are reported in figure 5, as an example of systems’ microstructure. the micrographs highlight that the emulsions present a complex structure where small oil droplets are dispersed in an aqueous phase that contains a large amount of starch (black spots in the images) that are in some cases interacting (figure 5a), likely due to the effect of the high dynamic pressure during emulsification. moreover, it could be clearly evidenced that oil droplets are stabilized by adsorbed starch particles (figure 5b). as previously observed in the literature, the overall emulsified dispersed state of the emulsions can be due to both the pickering mechanism by starch particles adsorbed on the o/w interface as well as the stabilization and immobilization of oil droplets within the starch, gel-like granules network formed during the hph process (dickinson, 2020). indeed, it could be affirmed that during the high-pressure homogenization, the pickering emulsions were stabilized by several mechanisms occurring simultaneously, which are the adsorption of starch particles on the oil/water interface, the release of amylose from the starch granules which formed complexes with the oil droplets, and the formation of the gel-like network of starch granules which immobilized the oil droplets. indeed, these stabilization phenomena have been previously reported in pickering emulsions prepared with corn starch under hph (meng et al., 2014; villamonte et al., 2016). flow behavior of pickering olive oil emulsions flow behavior of pickering emulsions at different ms concentrations is reported in figure 6. all dispersed systems presented a non-newtonian shear-thinning behavior as, at increasing ms concentration, the shear stress increased. the shear-thinning behavior can be explained by a weak droplet association where the network underwent breakage as the shear force was increased, the rate of starch network deformation was higher than the reformation rate, resulting in lower viscosity because of reduced intermolecular resistance, behavior which has been previously observed for starch-stabilized o/w emulsions (ye et al., 2017). the experimental data were fitted by using a power law model (σ = kγn) where σ is the shear stress (pa), k the consistency index, γ the shear rate (1/s), and n = flow behavior (dimensionless). the extent of shear-thinning behavior in a non-newtonian fluid is calculated using the flow behavior (n). if the value of n is <1, it is shear-thinning fluid and >1 indicates a shear-thickening fluid (rezaei et al., 2017). in table 1, the flow behavior (n) and consistency index (k) values of the differently made emulsions are summarized. all samples presented an n value <1 confirming the shear-thinning behavior; it significantly (p > 0.05) increased from 6.0 to 8.0% starch 10 µm. this result is in disagreement with data reported in the literature, as higher starch concentration allows more particles to be able to absorb at the oil/water interface and stabilize the interfacial layer and thus decrease the particle size (li et al., 2013; marefati et al., 2017). this disagreement could be due to several causes, such as a difference in the starch particles’ properties and size; the emulsified systems, like the oil to water volume ratio; and the homogenization conditions used with respect to the current study. however, it is worth observing that emulsions presented a surface mean droplet size lower or equal to 10 µm, which, according to some authors, indicates good emulsification capacity and emulsion stability toward physical destabilization phenomena such as creaming and flocculation, when compared to emulsion with larger droplets (saari et al., 2016). so, it can be generally affirmed that in the range of concentrations tested and under the process conditions applied, the modified starch particles exerted good emulsifying properties, determining the formation of fine o/w olive oil dispersed systems. images of emulsions made with 9.5% ms (w/v) evaluated by light microscopy captured at two different 0.01 0 0 2 4 6 8 10 12 3 6 9 12 (a) (b) 0.1 6.0% a bc bc cd cd d 8.0% 8.50% 9.50% 10.0%9.0% 1 10 size class (µm) starch concentration 6.0% 8.0% 8.5% 9.0% 9.5% 10.0% vo lu m e de ns ity (% ) d 3, 2 (µ m ) 100 1000 10000 figure 4. droplet size distribution (a), and surface mean droplet size (b) of o/w pickering emulsions prepared with different concentrations of modified corn starch. different letters in the same columns indicate significant differences (p < 0.05) (n = 3). italian journal of food science, 2021; 33 (4) 7 colloidal properties and stability of olive oil emulsions (a) (b) figure 5. optical microscope images of pickering emulsions prepared with 9.5% ms at different magnifications (10× in a, 40× in b). ms granules appear to be black in color (ms: modified starch). 0 0 50 100 150 shear rate 1/s s he ar s tr es s p a 200 250 6.0% 8.0% 8.5% 9.0% 9.5% 10.0% power law model 300 350 0.5 1 1.5 2 2.5 3 3.5 figure 6. flow behavior of the o/w pickering emulsions prepared with different concentrations of modified corn starch (6.0– 10.0% w/w). table 1. consistency index and flow behavior of the 30% o/w pickering emulsions stabilized by high pressure homogenization and ms particles. ms 6.0% ms 8.0% ms 8.5% ms 9.0% ms 9.5% ms 10.0% k 0.027a ± 0.01 0.017a ± 0.01 0.022a ± 0.00 0.034a ± 0.01 0.033a ± 0.01 0.036a ± 0.01 n 0.740a ± 0.03 0.856b ± 0.06 0.83ab ± 0.02 0.783ab ± 0.04 0.784ab ± 0.02 0.783ab ± 0.03 k = consistency index, n = flow behavior, *r2 value for all the measurements was greater than 0.977. different letters in the same rows indicate significant difference (p < 0.05) (n = 3). ms: modified starch. concentration while no significant increase was observed afterward. the lowest consistency index (k) was found in emulsions stabilized with 6.0% of ms while an increase was observed at all the remaining concentrations, even though with no significant differences among them (p < 0.05). from these data, it could be concluded that consistency index largely remained unaffected by increasing starch concentration while the flow behavior was only significantly affected up to a certain level (6–8% of ms) and afterward it also remained unaffected by increasing starch concentrations. creaming stability of o/w pickering emulsions stability of the pickering emulsions was investigated by the evaluation of the creaming index (ci) as a parameter that could indicate the extent of droplet aggregation in an emulsion, which in turn affects its physical stability. the higher the creaming index, the more the droplets clump together (sun-waterhouse et al., 2013). figure 7 shows the ci of o/w pickering emulsions over a storage period of 1 month at room temperature. it is possible to note that the higher the starch concentration, the lower 8 italian journal of food science, 2021; 33 (4) farooq u et al. dispersed systems were obtained. the microscopic images demonstrated the emulsions’ stabilization by the occurrence of a mixed mechanism based on pickering phenomena by solid starch particles absorbed on the o/w interface and the formation of a starch network able to entrap oil particles. the flow properties of the pickering emulsions showed a shear-thinning non-newtonian behavior; increasing the starch concentration enhanced the emulsion physical stability as evaluated by the creaming index by improving the oil particles entrapment within the starch network. further work is needed to explore the emulsifying properties and exploitability of starch particles in more complex food systems formulated with olive oil as the lipid phase. references abend, s., bonnke, n., gutschner, u. and lagaly, g., 1998. stabilization of emulsions by heterocoagulation of clay minerals and layered double hydroxides. colloid and polymer science 276(8): 730–737. https://doi.org/10.1007/s003960050303 alimi, b.a. and workneh, t.s., 2018. structural and physicochemical properties of 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https://doi.org/doi.org/10.1016/j.foodchem.2017.02.062� https://doi.org/doi.org/10.1016/j.foodchem.2017.02.062� https://doi.org/10.1016/j.foodchem.2017.02.061� https://doi.org/10.1016/j.foodchem.2017.02.061� https://doi.org/10.1016/j.ijbiomac.2016.06.082� https://doi.org/10.1002/1097-0282(200012)54:7%3c524::aid-bip50%3e3.0.co;2-y� https://doi.org/10.1002/1097-0282(200012)54:7%3c524::aid-bip50%3e3.0.co;2-y� https://doi.org/10.1094/cchem-05-15-0107-r� https://doi.org/10.1002/star.19960481104� https://doi.org/10.1002/star.19960481104� https://doi.org/10.1016/j.foodres.2011.05.030� https://doi.org/10.1016/j.foodres.2011.05.030� https://doi.org/10.1016/j.tifs.2016.01.023� https://doi.org/10.1016/j.tifs.2016.01.023� https://doi.org/10.1002/fsn3.17� https://doi.org/10.1016/j.foodhyd.2015.07.031� https://doi.org/10.1016/j.foodhyd.2015.07.031� _hlk60068089 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (4): 33–43 issn 1120-1770 online, doi 10.15586/ijfs.v34i4.2250 33 p u b l i c a t i o n s codon some technical properties of dried terminalia chebula (kara halile) for use in harvest and post-harvest processing elçin yeşiloğlu cevher department of agricultural machinery and technologies engineering, faculty of agriculture, ondokuz mayıs university, samsun, turkey corresponding author: elçin yeşiloğlu cevher, department of agricultural machinery and technologies engineering, faculty of agriculture, ondokuz mayıs university, samsun, turkey. email: elciny@omu.edu.tr received: 23 june 2022; accepted: 17 november 2022; published: 7 december 2022 © 2022 codon publications open access paper abstract  depending on humidity, some technical properties of t. chebula (black halile) dried fruit were investigated. it was observed that various properties, such as dimension, geometric mean diameter, and arithmetic mean diameter, increased linearly with increasing moisture content. with the increase in moisture content, sphericity increased from 57.2% to 67.7%, surface area increased from 487.65 mm2 to 805.03 mm2, porosity increased from 0.49 to 0.59, and the angle of repose increased from 22.77° to 27.86°. however, moisture content, true density, and bulk density decreased from 1.85% to 3.27%, 1469.54 kg/m3 to 1740.22 kg/m3, and 735.64 kg/m3 to 705.99 kg/m3, respectively. when the moisture content increased from 1.85% to 3.27%, the static and dynamic friction coefficient increased from 0.231 to 0.495 and 0.311 to 0.637, respectively. the lowest static and dynamic friction force values were obtained for stainless steel and the highest for rubber surface. when moisture content increased from 1.85% to 3.27%, tensile strength decreased from 446.46 n to 257.59 n. rupture energy and deformation increased with an increase in the moisture content of the fruit. when the moisture content increased from 1.85% to 3.27%, the rupture energy and deformation increased from 0.09 j to 0.27 j and 0.83 mm to 1.76 mm, respectively. keywords: deformation; friction coefficients; rupture energy; rupture force; terminalia chebula introduction terminalia chebula (t. chebula) belongs to the combretaceae family. the fruit is a yellowish-green oval drupe, 3–6-cm long and 1.3–1.5-cm wide, containing an oval seed. t. chebula can grow in various soils, including clay and shady soils. the trees can be grown at altitudes of about 2000 m above sea level and in regions where the annual precipitation is 100–150 cm and the temperature is 0–17°c. although t. chebula is a native of asia, it is also found in nepal, sri lanka, myanmar, bangladesh, egypt, iran, turkey, pakistan, yunnan, tibet, and guangdong, guangxi province of china. in india, it grows in the deciduous forests of himachal pradesh, tamil nadu, kerala, karnataka, uttar pradesh, andhra pradesh, and west bengal (gupta, 2012). famous as kara halile in turkey, worldwide t. chebula is known as myrobalan, mapki, harad, harada, karkchettu, kadukkaya, king of medicine, halile, harde, harar, and sa mao tchet. t. chebula is rich in organic acids, flavonoid substances, ascorbic acid, protein, amino acids, and minerals (murathan et al., 2020). it is called the “king of medicines” in tibet and ranks first as an ayurvedic medicine for its extraordinary wound-healing power and wide range of medicinal properties. t. chebula has antibacterial, antifungal, antiviral, antidiabetic, antimutagenic, antioxidant, antiulcer, and wound-healing properties. it also prevents heart damage, and is used to treat kidney disease. it is a mild, safe, and mailto:elciny%40omu.edu.tr?subject= 34 italian journal of food science, 2022; 34 (4) cevher e y not determined by their moisture content. the study to assess the engineering properties was carried out by considering the moisture content and the mechanical properties of the product obtained under the compression load which have not been studied before. this was obtained from the mechanical properties of rupture force, rupture energy, and deformation. mechanical effects can damage harvest or post-harvest crops. damage to the outer layers of the product causes faster deterioration. these factors negatively affect the storability and shelf life of products. for this reason, it is essential to know the mechanical properties of agricultural products (yıldız and cevher, 2022). the study was carried out with three different moisture contents, and the relationship between engineering properties was explained through mathematical equations. physical properties of t. chebula dried fruit such as length, width, thickness, geometric-arithmetic mean diameter, sphericity, surface area, bulk density, true density, porosity, and inclination angle were investigated as engineering properties. at the same time, mechanical properties such as fracture force, fracture energy, deformation, and static and dynamic friction coefficients were determined. thus, engineering properties for improving the quality of t. chebula dried fruit have been demonstrated. material and methods material t. chebula dried fruit used in the study was obtained from the local market in samsun, turkey. before starting the experiments, all foreign material in the samples, such as dirt, stones, dust, and cracked dried fruit were cleaned manually. then, the initial and conditioned moisture content values were determined by keeping the dried fruit in a standard hot air oven at 105°c for 24 h. in the study, the samples were conditioned by adding a calculated amount of water according to the following equation to reach different humidity levels (balasubramanian, 2001; pathak et al., 2019; yurtlu et al., 2010): f i f wi(m m ) q , 100 m − = − (1) where q: mass of water to be added (kg), wi: initial mass (kg), mi: initial moisture content of the sample in percent (% wet basis [wb]), and mf: final moisture content of the sample in percent (% wb). in order to ensure a homogeneous moisture distribution, the samples were placed in polyethylene bags and kept effective laxative in traditional medicine. t. chebula and its photo components have therapeutic effects without toxicity. it is an active ingredient in the well-known herbal preparation triphala, which is used to treat liver enlargement, stomach ailments, and eye inflictions (gupta, 2012). triphala’s main component is a seed coat powder of dried t. chebula fruit. therefore, removing the seed from the seed coat must be carried out carefully. it is necessary to design suitable equipment to perform this separation process (pathak et al., 2020). it is essential to know the engineering properties of the material for designing and developing equipment and for processing, transportation, classification, separation, and storage of agricultural products. this includes engineering features such as shape, size, mass, bulk density, true density, porosity, coefficient of static-dynamic friction against various surfaces, and rupture characteristics of the product. the engineering information obtained could be used to determine the efficiency and operation of crop machinery (gharibzahedi et al., 2010; sangamithra et al., 2016). the moisture content of the product is one of the critical parameters in the designing of suitable equipment. moisture-related properties of agricultural products are essential for process design, determination of product quality, processing, and packaging (rao et al., 2005). the physical properties of agricultural products are used in the designing of processing units, such as cleaning, separation, storage, transport, and drying systems. sphericity is used to understand heat and mass transfer. the stagnation angle is an indicator of the flowing ability of a food product. porosity is the property used to characterize and process food products (bajpai et al., 2019). many researchers have conducted research to determine agricultural products’ physical and mechanical properties depending on their moisture content. for example, bajpai et al. (2019), gharibzahedi et al. (2010), pathak et al. (2020), and putri et al. (2015) conducted studies to determine the physical and mechanical properties of jamun seeds, black cumin, myrobalan fruits, and rice, respectively, depending on their moisture content. studies have been conducted using seeds and nuts to determine engineering properties of jatropha curcas l. seeds (herak et  al., 2013), raw cashews (balasubramanian, 2001), mahogany seeds and kernels (aviara et al., 2014), almond seeds (atteh et al., 2021), charoli nut (shelare et al., 2021), bambara groundnut seed (aremu et al., 2022), arugula seed (mirzabe et al., 2021), tamaring seed (mohite et al., 2019), and date nut (ola et al., 2020). investigation into physical and mechanical properties of agricultural products has an essential role in the designing aspects of harvesting and post-harvest machinery as well as sorting, packaging, and transport equipment (bajpai et al., 2019). the literature review assessed the physical properties of t chebula and discovered that engineering features are italian journal of food science, 2022; 34 (4) 35 technical properties of dried terminalia chebula for harvest and post-harvest processing in a refrigerator at 5°c for 1 week. humidity control was conducted before starting the experiments. the study was conducted with kara halile dried fruit with a moisture content of 1.85%, 2.59%, and 3.27% wb (figure 1). measurement of physical properties randomly selected 30 samples of t. chebula dried fruit were used. dimensions of dried fruit were measured with a digital caliper with an accuracy of 0.01 mm (mitutoyo, absolute digimatic, japan) (figure 2). mass measurements were conducted with a kern electronic precision balance with a sensitivity of 0.01 g and a maximum measurement capacity of 2500 g. the arithmetic mean diameter (da), geometric mean diameter (dg), and sphericity (ϕ) values were calculated with the following equations (bulan et al., 2020; mohsenin, 1970, 1980) (figure 3): a l w t d , 3 + + = (2) dg = (lwt) 1/3, (3) figure 1. dried termınalıa chebula fruit. figure 2. size measurement of t. chebula dried fruit. length (l) thickness (t) w id th ( w ) figure 3. dimensions of t. chebula dried fruit. 1/3(lwt) , l φ = (4) where l is the length (mm), w is the width (mm), t is the thickness (mm), and ϕ is the sphericity. following equation was used to calculate the surface area (mohsenin, 1980): s = pd2g, (5) where s: surface area (mm2) and dg : geometric mean diameter (mm). the bulk density (ρb) was determined by filling t. chebula dried fruit to a height of 150 mm into a 500-ml cylindrical carton and weighing it. the true density (ρt) was obtained by displacement method using the container with known mass and volume of the samples. while applying this method, toluene (c7h8), which is less absorbed by the samples, was used instead of water (yurtlu and yeşiloglu, 2011). the porosity (e, %) was determined using bulk density and true density values (mohsenin, 1980; selvi et al., 2020). t b t 100, ρ ρ e ρ − = × (6) where ρb: bulk density (kg m –3), and ρt: true density (kg m–3) (mohsenin, 1980): a 200 mm in diameter and 150 mm in height conical cylinder with two open sides was used to determine the angle of repose (q). the conical cylinder filled with t. chebula dried fruit was lifted slowly, and its height and diameter consisting of fruit samples were measured. the angle of repose (q, degree) is determined by the following equation (pathak et al., 2019): 1 2h q tan , d −  =     (7) where h: height of cone (mm) and d: diameter of the cone (mm). 36 italian journal of food science, 2022; 34 (4) cevher e y for t. chebula dried fruit, this was determined by connecting a wooden box to the load cell of the universal tester. the wooden box (60 × 120 × 100 mm) had an opening at the bottom and was connected to the load cell (lloyd biologicals tester; figure 4) with a pulley mechanism. the test was carried out by moving the box filled with dried fruit horizontally at a speed of 100 mm/min so that the contents were in contact with the friction surface. the opening at the bottom of the box allowed the fruit to touch the friction surface. a 10-mm gap was left between the opening and the surface of the fruit-filled box. horizontal pull (friction force) was recorded with the software of the lloyd device. the friction test was conducted at a sliding speed of 100 mm/min. stainless steel, court fabric galvanized sheet and rubber surfaces were used in the test carried out with 10 replications (yurtlu and yeşiloğlu, 2011). measurement of mechanical properties a universal biological material test device (lloyd instrument lrx plus; lloyd instruments ltd, bognor regis, united kingdom) was used to determine rupture force, rupture energy, and deformation values of t. chebula dried fruit (selvi et al., 2020; yurtlu and yeşiloğlu 2011). the experiments were carried out with a load cell having a capacity of 1000 newton (n) by applying load to the cross-section axis of dried fruit at a compression speed of 10 mm/min. the coordinate system describing the compression position of dried fruit is given in figure 5. data obtained from the compression test experiments were processed using the nexygen plus software (figure 6). statistical analysis of the data was performed with the ibm spss statistics 21 software. t. chebula dried fruit were tested at three different moisture contents (1.85, figure 4. wooden box used in friction tests. applied force thickness of t. chebula figure 5. axis of t. chebula dried fruit under compression load. 2.89, and 3.27% wb) for all traits. each test was performed in triplicate and mean ± standard deviation values were obtained and examined for variance using one-way anova and duncan’s test. in addition, linear regression analysis was performed to obtain regression equation and coefficient of determination (r2) for all parameters. results and discussion dimensions of dried fruit dimensions of t. chebula dried fruit are given in table 1. with increase in moisture content, the three axial dimensions of the fruit also increased. this could be due to expansion in fruit size with moisture filled in the cell spaces of dried fruit. as the moisture content increased from 1% to 3.27%, the length, width, and thickness of dried fruit increased from 21.34 mm to 25.70 mm, 10.57 italian journal of food science, 2022; 34 (4) 37 technical properties of dried terminalia chebula for harvest and post-harvest processing figure 6. lloyd instrument universal testing machine. table 1. means and standard deviation of the axial dimensions of dried t. chebula fruit. moisture content (%) wb axial dimension (mm) average diameter (mm) length (l) width (w) thickness (t) arithmetic mean (d a ) p-value 1.85 ± 2 21.34 ± 2.98a 10.57 ± 0.96a 8.55 ± 0.86a 13.49 ± 0.41a 0.000 2.59 ± 2 24.31 ± 0.53a,b 13.38 ± 0.26b 10.29 ± 0.54b 15.90 ± 0.53b 0.000 3.27 ± 2 25.70 ± 0.90a,b 14.16 ± 0.96c 11.27 ± 0.75c 17.05 ± 0.62c 0.000 note: different letters in superscript indicate the import differences. mm to 14.16 mm, and 8.55 mm to 11.27 mm, respectively. in addition, the average diameter of the fruit increased with the moisture content. with increase in the moisture content from 1.85% to 3.27%, the arithmetic and geometric mean diameters increased from 13.49 mm to 17.05 mm and 12.41–15.99 mm, respectively. t. chebula dried fruit was statistically significantly related to humidity. therefore, depending on the moisture content, the geometric mean diameter (dg) of t. chebula dried fruit is presented graphically in figure 7. the given equation in figure 7 establishes a relationship between the geometric mean diameter and the moisture content. the size and diameter values of t. chebula dried fruit were significantly affected by the moisture content (p ≤ 0.01). sphericity globality, which primarily refers to a social condition, potentially the end-point of globalization, and reveals the degree of global definition of the product, is an important parameter used in bringing products together. the sphericity of t. chebula dried fruit was significantly affected by the moisture content and increased from 57.2% to 18 16 14 12 10 1.5 1.85 2.2 moisture content (% wb) dg = 2.531mc + 7.904 r2 = 0.97 g eo m et ric m ea n di am et er ( m m ) 2.55 2.9 3.25 figure 7. effect of moisture content on the geometric mean diameter of t. chebula dried fruit. 67.7% (p ≤ 0.01) (figure 8). this indicates that relatively proportional changes occur in the size of the dried fruit because of sphericity. the linear equation for sphericity can be formulated as given in figure 8. trends similar to the effect of moisture content on the sphericity of t. chebula dried fruit were reported by vashishth et al. (2020) for horse gram, gharibaahedi et  al. (2010) for black cumin seed, su et al. (2021) for maize kernel, and yurtlu et al. (2010) for laurel seed. surface area in the study, the moisture content of t. chebula dried fruit increased from 1.85% to 3.27% and the surface area increased from 487.65 mm2 to 805.03 mm2. figure 9 shows the linear relationship between moisture content and surface area and the regression equation of relationship between the moisture content and the surface area of the dried fruit of t. chebula. the surface area was significantly (p ≤ 0.01) affected by the moisture content. density in the study, the bulk density of t. chebula dried fruit decreased from 735.64 kg/ m3 to 705.99 kg/m3. this 38 italian journal of food science, 2022; 34 (4) cevher e y 68 62 56 50 1.5 1.85 2.2 moisture content (% wb) ф = 0.074mc + 0.429 r2 = 0.92 s ph er ic ity ( % ) 2.55 2.9 3.25 figure 8. effect of moisture content on the sphericity of t. chebula dried fruit. 300 500 700 900 1.5 1.85 2.2 moisture content (% wb) s = 224.3mc + 84.52 r2 = 0.98 s ur fa ce a re a (m m 2 ) 2.55 2.9 3.25 figure 9. effect of moisture content on the surface area of t. chebula dried fruit. showed that the product’s mass increased due to lower moisture absorption than the volumetric expansion. the relationship between moisture content and bulk density and the regression equation is shown in figure 10. the bulk density of t. chebula dried fruit was significantly affected by the moisture content (p ≤ 0.01). a negative correlation was observed between the moisture content and bulk density of t. chebula dried fruit. similar results were observed in the studies conducted by bhushan and raigar (2020) for rice bean, malik and saini (2016) for sunflower seed, and selvi et al. (2006) for linseed. true density of any product is a physical property that can be used in aerodynamic handling and separation processes. in case of t. chebula dried fruit, this feature refers to its true mass, excluding all its cavities and pores (bhushan and raigar, 2020). the true density of t. chebula dried fruit increased from 1469.54 kg/m3 to 1740.22 kg/m3 and was significantly (p ≤ 0.01) affected by its moisture content. the relationship between moisture content and true density, and regression equation, is given in figure 11. the overall increase in the true density of t. chebula dried fruit could be due to the higher increase in weight than fruit volume. the true density results of t. chebula dried fruit agreed 680 700 720 740 760 1.5 1.85 2.2 moisture content (% wb) ρb = –20.76mc + 776.11 r2 = 0.94 b ul k de ns ity ( kg /m m 3 ) 2.55 2.9 3.25 figure 10. effect of moisture content on the bulk density of t. chebula dried fruit. 1400 1500 1600 1700 1800 1.5 1.85 2.2 moisture content (% wb) ρt = 190.632mc + 1117.02 r2 = 0.99 tr ue d en si ty ( kg /m m 3 ) 2.55 2.9 3.25 figure 11. effect of moisture content on the true density of t. chebula dried fruit. with the results of the studies conducted by singh and meghwal (2019) for ajwain seed and bhushan and raigar (2020) for rice beans. porosity porosity is an essential feature for applying airflow to agricultural grain products, and their packaging and cooling processes. with increase in the moisture content of t. chebula dried fruit from 1.85% to 3.27%, the porosity increased from 0.49 to 0.5. the porosity value of t. chebula dried fruit was significantly (p ≤ 0.00) affected by moisture content. the relation between moisture content and porosity along with the regression equation is given in figure 12. the results obtained for the porosity of t. chebula dried fruit, depending on the moisture content, were consistent with the results obtained by kumar et al. (2016) for chironji nut (buchanania lanzan). angle of repose the angle of repose is the maximum angle at which a granular agricultural product can stand without italian journal of food science, 2022; 34 (4) 39 technical properties of dried terminalia chebula for harvest and post-harvest processing figure  12. effect of moisture content on the porosity of t. chebula dried fruit. 0.4 0.5 0.6 0.7 1.5 1.85 2.2 moisture content (% wb) ε = 0.070mc + 0.359 r2 = 0.99 p or os ity 2.55 2.9 3.25 20 22 24 26 28 30 1.5 1.85 2.2 moisture content (% wb) q = 3.72mc + 15.906 r2 = 0.98 a ng le o f r ep os e (° ) 2.55 2.9 3.25 figure 13. effect of moisture content on angle of repose of t. chebula dried fruit. scattering as a heap. this angle value can be used to design equipment and warehouse structure where mass flow of product is confronted. the angle of repose of t. chebula dried fruit was significantly (p ≤ 0.01) affected by its moisture content. the effect of moisture content on the angle of repose of t. chebula dried fruit and the regression equation are given in figure 13. an increase in the natural agglomeration angle of t. chebula dried fruit was observed. the increased moisture of t. chebula dried fruit decreased its flow ability, which increased fruit’s stickiness. the natural agglomeration angle of t. chebula dried fruit increased from 22.77° to 27.86°. the result obtained agreed with the result of the study conducted by bajpai et al. (2019) for jamun seed (syzgium cuminii). static coefficient and dynamic of friction it is necessary to know the friction coefficient of an agricultural product, mainly when it is transported by a conveyor. the friction coefficient of an agricultural product varies with the used surface. the following four surfaces were used in the study to compute static and dynamic friction coefficient values: the most commonly used stainless steel, court fabric, galvanized sheet, and rubber. the static and dynamic friction coefficient results of t. chebula dried fruit are summarized in table 2. it was observed that static friction coefficients were higher than dynamic friction coefficients for all moisture contents. in addition, static and dynamic friction coefficient values for the tire’s surface were much higher than for other surfaces. this could be explained by the fact that the tire’s surface was rougher than other test surfaces used. a lower adhesion force between smoother stainless-steel surface and t. chebula dried fruit resulted in lower coefficients of static and dynamic friction (shafaei and kamgar, 2017; visvanathan et al., 1996). the coefficient of friction depends on roughness of the material used and frictional forces, which causes an increase in energy consumption. similar results were obtained for the coefficient of friction in other studies (kaliniewicz et al., 2013; shafaei and kamgar, 2017; visvanathan et al., 1996). according to the results presented in table 2, the highest value of dynamic friction coefficient was obtained for rubber surface (0.448), followed by court fabric (0.387), galvanized sheet (0.338), and stainless steel (0.252). similar results were obtained for static friction coefficient. the increase in moisture content of t. chebula dried fruit caused an increase in both dynamic and static friction coefficients. this could be attributed to the increase in adhesion forces between the fruit and the surfaces used, because a rise in humidity increased the stickiness of dried fruit (esgici et al., 2018; ghodki and goswami., 2016). the lowest value of the dynamic friction coefficient was obtained for stainless steel surface (0.361), and the highest value was for rubber surface (0.632). the same results were observed for static friction coefficient. table 2 also includes the results of duncan’s multiple range tests to identify significant differences between average moisture content and the examined surfaces. significant differences were observed between static and dynamic friction coefficients and moisture content. oneway anova demonstrated that variations in moisture content, surfaces used, and interaction between moisture content and surface were significant for both static and dynamic friction coefficients (p ≤ 0.01). both static and dynamic friction coefficients for t. chebula dried fruit increased linearly with moisture content and varied with used structural surfaces. this trend was consistent with the findings of the previous studies (aviara et al., 2014, 2015; shafaei et al., 2016). at higher moisture content, increased roughness was observed in t. chebula dried fruit, resulting in its 40 italian journal of food science, 2022; 34 (4) cevher e y table 2. average static and dynamic friction coefficient values for different moisture content and surfaces used for t. chebula dried fruit. moisture content surface dynamic coefficient of friction static coefficient of friction 1.85 ± 2 stainless steel 0.174 ± 0.011 0.249 ± 0.012 court fabric 0.259 ± 0.005 0.359 ± 0.009 galvanized sheet 0.218 ± 0.012 0.261 ± 0.017 rubber 0.274 ± 0.021 0.374 ± 0.037 2.59 ± 2 stainless steel 0.220 ± 0.021 0.360 ± 0.044 court fabric 0.359 ± 0.021 0.471 ± 0.049 galvanized sheet 0.344 ± 0.024 0.434 ± 0.035 rubber 0.446 ± 0.035 0.664 ± 0.013 3.27 ± 2 stainless steel 0.362 ± 0.028 0.472 ± 0.028 court fabric 0.543 ± 0.017 0.650 ± 0.021 galvanized sheet 0.451 ± 0.023 0.568 ± 0.014 rubber 0.623 ± 0.016 0.858 ± 0.021 mean values stainless steel 0.252a ± 0.084 0.361a ± 0.098 court fabric 0.387c ± 0.120 0.481c ± 0.127 galvanized sheet 0.338b ± 0.099 0.433b ± 0.134 rubber 0.448d ± 0.147 0.632d ± 0.204 1.85 ± 2 0.231a ± 0.041 0.311a ± 0.061 2.59 ± 2 0.342b ± 0.086 0.482b ± 0.119 3.27 ± 2 0.495c ± 0.101 0.637c ± 0.146 p-values moisture content 0.000 0.000 surface 0.000 0.000 moisture content × surface 0.000 0.000 decreased sliding properties and increased coefficient of static friction. in addition, an increase in static friction coefficient was observed due to increased stickiness and adhesion forces between t. chebula dried fruit and material surfaces used at higher moisture content. mechanical properties t. chebula dried fruit with moisture contents of 1.85%, 2.59%, and 3.27% was tested in the cross-section direction. the mechanical properties of t. chebula dried fruit, such as rupture force, rupture energy, and deformation values, were determined. it was observed that moisture content had a statistically significant (p ≤ 0.01) effect on the mechanical properties of the fruit. rupture force the study determined that the force required for rupturing t. chebula dried fruit decreased with increasing moisture content. this was due to increased flexibility of the dry fruit because of increased moisture content; increase in elasticity decreased the rupture force of the fruit. it was concluded that rupture force was minimum when moisture content was maximum. this established that more loading force was required when the moisture content of t. chebula dried fruit was low and vice versa. in t. chebula dried fruit, for a moisture content of 1.85%, the minimum rupture force determined was 405.24 n and the maximum was 487.20 n. for a moisture content of 2.59%, the minimum rupture force was 296.79 n and the maximum was 397.72 n. finally, for a moisture content of 3.27%, the minimum rupture force determined was 214.29 n and the maximum was 309.33 n. one-way anova demonstrated a significant relation between moisture content and rupture force of t. chebula dried fruit (p ≤ 0.01); this along with regression equation is given in figure 14. the results obtained in the present study were compatible with the previous studies (amoah et al., 2017; pathak et al., 2020; putri et al., 2015). italian journal of food science, 2022; 34 (4) 41 technical properties of dried terminalia chebula for harvest and post-harvest processing rupture energy depending on the size of the fruit or seed, increase in rupture energy was observed in t. chebula dried fruit (pathak et al., 2020). increase in size of the fruit or seed increased its rupture energy. at a moisture content of 1.85%, the maximum rupture energy determined was 0.11 j and the minimum was 0.07 j. at a moisture content of 2.59%, the maximum rupture energy determined was 0.18 j and the minimum was 0.13 j. finally, at a moisture content of 3.27%, the maximum rupture energy was 0.31 j and the minimum was 0.22 j. the relationship between moisture content and rupture energy was significant (p ≤ 0.01), which along with the regression equation is given in figure 15. the results obtained for rupture energy in the present study were compatible with the previous studies (olaniyan and oje, 2002; shashikumar et al., 2018; swain and gupta, 2013). deformation deformation in t. chebula dried fruit also increased with the moisture content. at a moisture content of 1.85%, the maximum deformation determined was 0.94 mm whereas the minimum was 0.73 mm. at a moisture content of 2.59%, the maximum deformation determined was 1.64 mm whereas the minimum was minimum 1.22 mm. finally, at a moisture content of 3.27%, the maximum deformation determined was 1.61 mm whereas the minimum was 0.65 j. according to one-way anova, the effect of moisture content on the deformation of t. chebula dried fruit was significant (p ≤ 0.01). the relationship between moisture content and deformation, along with the regression equation, is given in figure 16. it was observed that deformation and rupture energy increased whereas rupture force decreased with increased moisture content of t. chebula dried fruit. similar results were obtained for faba bean (altuntaş and yıldız, 2007), beechwood seed (nyorere and uguru, 2018), walnut (sharifian and derafshi, 2008), and lima bean (aghkhani 150 250 350 450 550 1.5 1.85 2.2 moisture content (% wb) f = –132.97mc + 692.99 r2 = 0.91 r up tu re fo rc e (n ) 2.55 2.9 3.25 figure  14. effect of moisture content on the rupture force of t. chebula dried fruit. et al., 2012). further, if the moisture content of t. chebula dried fruit was low, it was less susceptible to breakage during harvest and post-harvest processing. the effect of moisture content on the crushing resistance of t. chebula dried fruit was also investigated in the present study. the results depicted that the crushing resistance of the dried fruit was a function of fruit’s moisture content, because breaking force of the seed decreased whereas its breaking energy and deformation increased with increase in the moisture content of the fruit. therefore, the crushing resistance of the fruit was found to be useful for designing and development of processing machinery. conclusion the study obtained the following results, including the physical and mechanical properties of t. chebula dried fruit at a moisture content of 1.85%, 2.59%, and 3.27% wb. the moisture content significantly affected dried fruit’s physical and mechanical properties (p ≤ 0.01). 0 0.1 0.2 0.3 0.4 1.5 1.85 2.2 moisture content (% wb) e = 0.122mc – 0.141 r2 = 0.98 r up tu re e ne rg y (j ) 2.55 2.9 3.25 figure 15. effect of moisture content on the rupture energy of t. chebula dried fruit. 0.5 1.0 1.5 2.0 2.5 1.5 1.85 2.2 moisture content (% wb) d = 0.652mc – 0.355 r2 = 0.97 d ef or m at io n (m m ) 2.55 2.9 3.25 figure 16. effect of moisture content on the deformation of t. chebula dried fruit. 42 italian journal of food science, 2022; 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https://doi.org/10.1007/s13197-019-04183-w� https://doi.org/10.1006/jaer.1996.0003� https://doi.org/10.24925/turjaf.v10i8.1565-1570.5332� https://doi.org/10.1501/tarimbil_0000001185� https://doi.org/10.9734/ejnfs/2020/v12i530230� https://doi.org/10.9734/ejnfs/2020/v12i530230� https://doi.org/10.1006/bioe.2002.0049� https://doi.org/10.1007/s13197-019-04162-1� https://doi.org/10.1007/s13197-019-04162-1� https://doi.org/10.1007/s40030-020-00476-y� https://doi.org/10.1007/s40030-020-00476-y� https://doi.org/10.18517/ijaseit.5.5.561� https://doi.org/10.1016/j.biosystemseng.2006.08.008� https://doi.org/10.1016/j.biosystemseng.2006.08.008� https://doi.org/10.1016/j.jare.2017.04.003� https://doi.org/10.1016/j.jare.2017.04.003� ijfs#230_gambacorta_bozza   ital. j. food sci., vol 28, 2016 258 paper volatiles and acceptability of liqueurs from kumquat and grapefruit c. summo, a. trani, m. faccia, f. caponio and g. gambacorta* department of soil, plant and food science, university of bari, via amendola 165/a, 70126 bari, italy *corresponding author. tel.: +39 0805442942; fax: +39 080 5442850 e-mail address: giuseppe.gambacorta@uniba.it abstract the aim of this work was to produce liqueurs from "minor" citrus fruits, such as kumquat and grapefruit, characterize their volatile fraction and evaluate their acceptability by a consumer test. a limoncello sample (lp) was produced under the same conditions and used for comparison. all the new liqueurs were found to be richer in limonene and poorer in oxygenated compounds than the lp. the volatile fraction was mostly represented (85%) by limonene in grapefruit liqueur. liqueur from kumquat peel (kp) was the richest in volatile compounds, whereas the one from kumquat whole fruit (kwf) was the poorest. this latter also had the particular feature to be the richest in sesquiterpene alcohols. octanal and decanal, and two acetals deriving from these aldehydes (1,1-diethoxyoctane and 1,1-diethoxydecane) were most prevalent in kp and lp. the consumer test showed that all liqueurs were judged to be acceptable. nevertheless, limoncello remained the most preferred, while the kwf liqueur obtained the best flavour score in the group of minor citrus fruits. keywords: consumer acceptance, grapefruit, kumquat, liqueur, volatile compounds   ital. j. food sci., vol 28, 2016 259 1. introduction citrus fruits are either consumed as fresh or processed to obtain juices, jams and, in small amounts, to produce liqueurs such as the so-called rosolio. rosolios are liqueurs obtained by alcoholic maceration of flowers or fruits with the final addition of water and sugar. the most famous italian rosolio made from citrus fruits, is limoncello. it is highly requested on the international market and it is manufactured by the alcoholic maceration of lemon (citrus limon l.) peel. the composition of the volatile fraction plays a fundamental role in the development of citrus liqueurs aroma. in particular, the ratios carbonyls-tooxygenated compounds, alcohols-to-oxygenated compounds, and esters-to-oxygenated compounds are indices of flavouring quality (poiana et al., 2006). several investigations have been carried out on the volatile compounds of limoncello in order to find the molecular markers to establish quality and genuineness. versari et al. (2003) reported that the addition of essential oils to limoncello causes an increase of oxygenated compounds and a loss of hydrocarbons. they also reported that compounds such as ethyl acetate, acetaldehyde, and 2-methyl-1-propanol should be related to the occurrence of microbiological activity in the sugar syrup. crupi et al. (2007) found differences in terms of type, amount, and variation range of volatile compounds in 12 commercial limoncello samples and in 2 types of limoncello produced in a laboratory. poiana et al. (2006) reported variations in the volatile profiles of the alcoholic extract of lemon fruit to be a function of the geographic area and season. besides limoncello, there are only a few liqueurs made from citrus fruits, such as rosolio from tangerine and orange, mainly produced for local markets. to our knowledge, no effort has been made for producing liqueurs from "minor" citrus fruits, such as kumquat (fortunella margarita l.) and grapefruit (citrus paradisi l.). kumquat is a vigorous and prolific small bushy tree that produces oval or round fruits with a smooth, bright orange rind (barreca et al., 2011). unlike other citrus fruits, kumquat fruit is eaten without discarding the peel, and this has nutritional relevance since this part is particularly rich in flavonoids (gattuso et al., 2007; tripoli et al., 2007). many citrus flavonoids exhibit antioxidant activity, inhibit angiogenesis, and slow down cancer cell migration and proliferation (barreca et al., 2009; benavente-garcía and castillo, 2008). there are only a few studies on the volatile constituents of kumquat. bernhard and scrubis (1961) found limonene to be the most abundant compound in kumquat oil extracted by steam distillation. aldehydes, ketones, free alcohols, terpene esters, α-pinene, and myrcene were also reported in this study. koyasako and bernhard (1983) identified 71 volatiles in oil obtained by simultaneous distillation and extraction; umano et al. (1994) reported 84 volatiles in steam-distillation extracts; choi (1995) identified 82 volatiles in oil extracted by cold pressing. more recently, peng et al. (2013) identified a total of 43 compounds in the volatile fractions of kumquat essential oils extracted by different methods. the principal constituents of the oils were similar, and differences were only found for minor compounds such as linalool, terpinen-4-ol and αterpineol. grapefruit is a citrus fruit that contributes to human health mainly thanks to its high contents of ascorbic acid and fiber (peiró et al., 2006). unfortunately, the presence of some flavonoids, such as naringin, is responsible for the bitter taste that limits acceptance by consumers. nevertheless, fresh or processed grapefruit may be conveniently mixed with other foods to formulate desirable and wholesome products. the present study aimed to assess the possibility of using kumquat and grapefruit for the production of innovative types of rosolio. the experimental liqueurs were produced on a laboratory-scale and were subjected to volatile profile characterization by headspace-solid   ital. j. food sci., vol 28, 2016 260 phase microextraction (hs-spme) and evaluation of consumer acceptance in comparison with limoncello. 2. materials and methods 2.1. liqueur making fresh fruit peels were utilized for the preparation of kumquat (fortunella margarita) (kp), grapefruit (marsh seedless) (gp), and lemon (femminello comune) (lp) liqueurs. for this purpose, about 500 g of each fruit was accurately peeled, and the peels, consisting of the flavedo part, were put into a jar containing 500 ml of ethanol (95% v/v) and left to steep for 2 weeks. after this period, the peels were taken out of the alcohol and syrup made with 500 ml of water plus 400 g of sugar was added to the ethanol extract. the liqueurs obtained were let to rest for 2 months in the dark at room temperature for maturation. during preparation, it was observed that peeling the kumquat was very difficult, due to the small size of the fruit (about 15 g). in order to assess the possibility of avoiding such step, a liqueur from the maceration of the whole fruit (kwf) was also prepared. in this latter preparation, 500 g of the fruit was directly put into 500 ml of ethanol (95% v/v) and left to steep for 2 weeks; then the preparation followed the same steps as described for the other liqueurs. 2.2. volatile fraction extraction and gc/ms analysis headspace-solid phase microextraction (hs-spme) was chosen as the extraction technique for the present study, since it had been successfully applied to determine the volatile composition of kumquat essential oils (peng et al., 2013) and lemon liquor (crupi et al., 2007). volatile compounds were extracted using a preconditioned 2-cm-long 50/30 mm divinylbenzene/carboxen/polydimethylsiloxane fiber (supelco, bellefonte, pa., u.s.a.). two ml of each liqueur were put in a 12-ml crimped vial, with 0.4 g nacl added, and conditioned for 10 min at 37°c and stirred with a magnetic bar. then the fiber was exposed in the headspace of the vial for 20 min. desorption of analytes from the spme fiber took place in a split/splitless injector set at 250°c with a split ratio of 1:25 using a 3 min desorption time. separation of volatile compounds was performed using an agilent 6890 gas chromatograph (gc) coupled with an agilent 5975 mass spectrometer (ms) (agilent, wilmington, del., u.s.a.) using a hp5-ms column (30 m × 0.25 mm × 0.25 mm). the chromatographic conditions were: (i) oven, 40°c (2 min) to 190°c at 5°c min-1, to 230°c at 15°c min-1, held 2 min; (ii) detector, source temperature 240°c; transfer line temperature 240°c; (iii) carrier gas, helium at constant flow of 1.0 ml min-1. the impact energy was 70 ev. data were acquired using full-scan mode in the range of 20-250 m/z at an acquisition rate of 5 hz. volatile compounds were tentatively identified by comparing the experimental spectra with those reported in the nist library and with those obtained by pure external standard injection when available. each sample was analyzed in triplicate and results were reported as a mean of area counts x 106. the repeatability of the spmegc/ms method was lower than 10% in terms of relative standard deviation (rsd). 2.3. acceptance and preference testing the consumer test was carried out in a conference room where temporary partitions were erected to create isolated booths able to separate testers during analysis, in compliance with the standard no. 8589 of the international organization for standardization (iso   ital. j. food sci., vol 28, 2016 261 1988). testing was performed at room temperature (20 °c) with appropriate and adequate artificial lighting, simulating daylight. a total of 75 consumers (age 19–47, mean 23.1; 45 males and 30 females) were recruited to participate in the consumer test. a 3-digit random code was assigned to the liqueurs, which were served at room temperature in 80-ml white polyethylene glasses. for each sample, about 10 ml was served. mineral water was at each participant's disposal to cleanse mouth during testing. the evaluation form used had 3 sections: the first required information about the sex and age of the panelists; the second section required the evaluation of color, odor, and flavor using a 6-point hedonic scale (1 = extremely dislike; 6 = like extremely); the last section asked the consumers to rank the samples according to overall appreciation. the use of flavor, instead of taste, as a sensorial descriptor was chosen because our purpose was to assess the blend of taste and smell sensations evoked in the mouth. 2.4. statistical analysis the results of color, odor, and flavor assessment were subjected to a one-way analysis of variance (anova). moreover, differences in the preference rank sums between all possible pairs of products were considered. should any of these (absolute) differences exceed a critical value, the preferences for that pair of products would differ from one another at the stated statistical significance level (n = 75, p ≤ 0.05, critical value = 40.6) (basker, 1988). 3. results and discussions 3.1. volatile fraction figures 1 and 2 show the total ion current profile of volatile compounds of liqueurs obtained from kumquat peel (fig. 1a), kumquat whole fruit (figure 1b), grapefruit peel (fig. 2a), and lemon peel (fig. 2b). clearly, the 4 liqueurs were different under a qualitative point of view.   ital. j. food sci., vol 28, 2016 262 figure 1: spme-gc/ms profiles of kumquat peel liqueur (a) and kumquat whole fruit liqueur (b). the peak numbers refer to the compounds in table 1.   ital. j. food sci., vol 28, 2016 263 figure 2: spme-gc/ms profiles of grapefruit peel liqueur (a) and lemon peel liqueur (b). the peak numbers refer to the compounds in table 1. table 1 summarizes the mean values for the volatile compounds expressed as both absolute and relative percentage area. the total area and the sums of the areas of monoterpenes (mts), sesquiterpenes (sts), and oxygenated compounds (ocs) are also reported in table 1.   ital. j. food sci., vol 28, 2016 264 table 1: volatile composition of liqueurs. no. compound kp kwf gp lp bid aarea % area % area % area % 1 α-thujene 0.20 0.01 0.50 0.07 ms 2 α-pinene 7.63 0.47 0.25 0.18 3.88 0.40 3.95 0.54 ms, es 3 camphene 0.15 0.02 ms, es 4 β-sabinene 12.67 0.78 0.66 0.07 2.16 0.29 ms, es 5 β-pinene 1.73 0.11 0.29 0.03 39.85 5.43 ms 6 β-myrcene 36.77 2.26 1.43 1.05 20.92 2.14 4.33 0.59 ms, es 7 α-phellandrene 0.50 0.03 0.28 0.03 0.10 0.01 ms 8 octanal 14.34 0.88 0.08 0.06 0.33 0.03 3.30 0.45 ms, es 9 α-terpinene 0.89 0.05 0.07 0.01 1.72 0.23 ms, es 10 p-cymene 0.08 0.06 0.22 0.02 16.43 2.24 ms, es 11 limonene 1239.37 76.07 104.96 76.96 833.53 85.29 231.73 31.56 ms, es 12 β-cis-ocimene 0.88 0.05 1.46 0.20 ms 13 β-trans-ocimene 9.66 0.59 1.02 0.14 ms 14 γ-terpinene 2.62 0.16 0.13 0.10 0.42 0.04 64.83 8.83 ms 15 trans-sabinene hydrate 0.25 0.02 0.30 0.04 ms 16 1-octanol 2.40 0.15 0.05 0.04 0.30 0.04 ms, es 17 α-terpinolene 0.76 0.05 0.06 0.04 0.15 0.02 4.07 0.55 ms 18 linalool 1.55 0.10 0.79 0.58 0.53 0.05 2.56 0.35 ms, es 19 nonanal 4.02 0.25 0.37 0.27 0.30 0.03 11.73 1.60 ms, es 20 nonanol 0.21 0.01 0.07 0.05 0.13 0.01 ms, es 21 γ-terpinolene 2.06 0.13 0.22 0.02 1.00 0.14 ms 22 camphor 0.39 0.02 0.08 0.01 ms 23 β-citronellal 1.00 0.06 1.85 0.25 ms, es 24 cis-sabinene hydrate 2.60 0.16 6.00 0.82 ms 25 1-decanol, 2-hexyl0.39 0.02 0.08 0.01 ms 26 (-)-4-terpineol 0.93 0.06 0.36 0.26 4.14 0.56 ms 27 α-terpineol 0.55 0.03 0.15 0.11 0.24 0.02 2.43 0.33 ms, es 28 decanal 53.75 3.30 0.59 0.43 1.44 0.15 8.25 1.12 ms, es 29 acetic acid, octyl ester 13.76 0.84 2.04 1.50 6.16 0.63 7.50 1.02 ms 30 nerol 0.50 0.37 0.10 0.01 1.99 0.27 ms, es 31 β-citronellol 0.24 0.01 0.07 0.01 0.89 0.12 ms, es   ital. j. food sci., vol 28, 2016 265 32 z-citral (neral) 0.24 0.01 27.04 3.68 ms 33 trans-geraniol 0.60 0.44 1.78 0.24 ms, es 34 e-citral (geranial) 0.43 0.03 0.06 0.04 39.13 5.33 ms 35 1,1-diethoxyoctane 39.04 2.40 0.23 0.17 0.14 0.01 5.19 0.71 ms 36 anethol 0.21 0.01 1.26 0.92 0.31 0.03 0.36 0.05 ms, es 37 undecanal 1.57 0.10 0.05 0.04 0.14 0.01 3.27 0.45 ms 38 nonyl acetate 0.96 0.06 0.09 0.07 0.47 0.05 0.79 0.11 ms 39 methyl geranoate 0.12 0.01 0.45 0.06 ms 40 δ-elemene 1.37 0.14 ms 41 trans-carvyl acetate 0.18 0.01 ms 42 copaene 1.48 0.09 0.24 0.02 ms 43 α-terpinenyl acetate 5.03 0.31 0.10 0.01 ms 44 citronellyl acetate 2.76 0.17 0.53 0.39 1.76 0.18 8.45 1.15 ms 45 neryl acetate 1.47 0.09 0.57 0.42 1.61 0.16 87.54 11.92 ms, es 46 isoterpinolene 14.77 0.91 0.21 0.02 0.89 0.12 ms 47 geranyl acetate 16.74 1.03 8.40 6.16 24.00 2.46 89.29 12.16 ms, es 48 β-cubebene 10.60 0.65 0.48 0.05 ms 49 β-elemene 0.63 0.04 0.84 0.09 ms 50 citronellal 1.00 0.06 1.86 0.25 ms, es 51 acetic acid, decyl ester 10.53 0.65 0.17 0.12 0.83 0.08 1.70 0.23 ms 52 limonen-10-yl acetate 0.61 0.04 0.52 0.38 1.63 0.17 ms 53 trans-caryophyllene 37.14 2.28 0.18 0.13 0.75 0.08 5.82 0.79 ms 54 α-santalol 0.32 0.23 0.77 0.08 ms 55 α-bergamotene 5.45 0.74 ms 56 cis-caryophyllene 0.56 0.03 0.20 0.15 0.31 0.03 0.29 0.04 ms 57 neryl propionate 2.98 0.18 0.21 0.02 1.64 0.22 ms 58 α-humulene 6.12 0.38 0.08 0.06 0.27 0.03 0.76 0.10 ms 59 β-santalene 0.70 0.07 0.37 0.05 ms 60 geranyl propionate 0.30 0.02 0.19 0.14 0.74 0.08 1.27 0.17 ms, es 61 1,1-diethoxydecane 14.40 0.88 1.79 0.24 ms 62 germacrene d 10.20 0.63 0.73 0.54 47.56 4.87 0.10 0.01 ms 63 trans-β-farnesene 0.61 0.04 0.20 0.15 0.26 0.03 0.45 0.06 ms 64 β-selinene 0.43 0.03 0.30 0.22 0.37 0.04 ms   ital. j. food sci., vol 28, 2016 266 65 valencene 0.91 0.06 0.71 0.07 1.35 0.18 ms, es 66 bicyclogermacrene 4.93 0.30 0.53 0.39 10.03 1.03 3.06 0.42 ms 67 cis-α-bisabolene 2.00 0.12 0.24 0.18 1.08 0.11 1.18 0.16 ms 68 β-bisabolene 2.10 0.13 1.40 0.14 16.20 2.21 ms 69 γ-cadinene 0.24 0.01 0.23 0.17 0.77 0.08 ms 70 δ-cadinene 21.33 1.31 2.45 1.80 1.90 0.19 0.24 0.03 ms 71 longifolene 0.81 0.05 0.16 0.12 ms 72 α-chamigrene 0.84 0.09 ms 73 γ-bisabolene 0.07 0.05 0.27 0.03 0.23 0.03 ms 74 germacrene b 1.45 0.09 0.19 0.14 2.47 0.25 ms 75 nerolidol 0.34 0.02 0.14 0.01 ms 76 palustrol 0.24 0.01 0.10 0.07 0.25 0.03 ms 77 caryophyllene oxide 0.36 0.02 0.15 0.11 ms 78 dodecanoic acid, ethyl ester 0.49 0.03 0.47 0.34 0.50 0.05 0.24 0.03 ms 79 veridiflorol 0.60 0.44 0.19 0.03 ms 80 globulol 0.29 0.21 0.21 0.02 ms 81 fonenol 0.81 0.59 0.15 0.02 ms 82 t-cadinol 0.09 0.01 0.22 0.16 ms 83 aromadendrene 0.11 0.01 0.87 0.64 0.17 0.02 ms 84 cedrenol 0.04 0.01 0.52 0.38 0.27 0.03 0.44 0.06 ms 85 hinesol 0.46 0.03 0.26 0.19 ms 86 torreyol 0.21 0.15 ms 87 α-bisabolol 0.20 0.01 1.43 1.05 0.34 0.03 0.49 0.07 ms total 1629.33 136.39 977.26 734.25 monoterpenes 1333.36 81.83 106.91 78.39 860.85 88.09 380.49 51.82 sesquiterpenes 101.65 6.24 6.43 4.71 72.79 7.45 35.50 4.83 oxygenated compounds 194.32 11.93 23.05 16.90 43.62 4.46 318.26 43.34 kp: kumquat peel; kwf: kumquat whole fruit; gp: grapefruit peel; lp: lemon peel. id: identification. acompounds quantified as total area counts x 106 (mean of 3 repetitions). bms: identification based on the nist ms library; es: identification based on authentic external standards analysed by mass spectrometry. the kp liqueur was the richest in volatile compounds (73 molecules identified), followed by the lp (65), the gp (61), and the kwf (54). the kp also had the highest total integrated peaks area, followed by the gp, the lp, and the kwf. even though the study was not quantitative, under our experimental conditions the area of the kp appears to be about 12fold larger than that of the kwf, and this could be a consequence of the peeling operation due to: i) the breakage of the cells containing the essential oils that favored the better   ital. j. food sci., vol 28, 2016 267 extraction of the volatile compounds during maceration; ii) the higher contact area between the peel and alcohol since only peels had been used in the maceration. in comparison to the lp, the kp total area was about 2 times higher, the kwf was about one fifth lower, and the gp was about 30% higher. among the volatile compounds, mts group was the most abundant in all the samples, constituting 78-80% of the total, with the exception of the lp, where these compounds were found to be about one-half of the total. in fact, the lp was characterized by a remarkable presence of ocs, which represent about the second half of the total volatile compounds. as concerns single volatiles, limonene was the predominant compound identified, even though the peak area strongly varied among liqueurs (from about 105×106 in kwf to about 1239×106 in kp). in terms of relative abundance, this monoterpene represented about 85% of the total area in the gp and about 76% in kumquat samples (both in the kp and the kwf), which are much higher than in the lp (about 32%). the abundance of limonene was expected, since it is the principal component of the volatile fraction of various citrus fruits (versari et al., 2003; crupi et al., 2007; dugo et al., 2010; asikin et al., 2012), including kumquat, in which it represents more than 90% of volatile compounds of the peel essential oil (umano et al., 1994; choi, 2005). the low concentration of limonene detected in the lp liqueur, compared to the kp and the gp, could be explained by the different content in the corresponding essential oils. as reported by caccioni et al. (1998), the lemon essential oils were characterized by a limonene concentration of 60–71% of the total volatile compounds, while the limonene concentrations in the essential oil of other citrus, such as grapefruit, orange, and bitter orange, were always higher than 90%. this monoterpene is associated with odor descriptors such as lemon-like, lemon, and orange, but presents a high odor threshold (choi, 2005; pohjanheimo and sandell, 2009). as regards the other mts, the most representative ones, with peak area > 15×106, were ß-myrcene, isoterpinolene, ß-sabinene, and ß-trans-ocimene in kp, and α-terpinene, ß-pinene, geranial, neral, and p-cymene in the lp. it is well known that neral and geranial (terpenoid isomers known as citral) are responsible for the strong lemon aroma; they were not detected or detected at only very low level in the kp, the kwf, and the gp samples. apart from limonene, ß-myrcene was the monoterpene found in appreciable amounts in the gp and the kwf samples (20.92×106 and 1.43×106, respectively). as far as sts are concerned, the kp contained more compounds with peak area > 10×106, such as trans-caryophyllene, δ-cadinene, β-cubebene, and germacrene d, whereas germacrene d and β-bisabolene were found in the gp and in the lp, respectively. this suggests that the kp liqueur is characterized by greater aroma complexity compared with the other samples investigated. the kwf had the particular feature to be the richest in sts alcohols (α-bisabolol, fonenol, veridiflorol, cedrenol, and globulol). these compounds are the primary constituents of the essential oil, conferring a weak sweet floral aroma, and are used commercially in various fragrances. among the ocs, geranyl acetate and neryl acetate were detected at the highest level in the lp (peak area value about 90×106). they were also found in the other liqueurs under examination, but at lower levels. these esters were associated with fresh and citrusy notes (thi minh tu et al., 2002), and are used in flavor and perfumery products to impart floral and fruity aromas. the kp liqueur also contained high levels of octanal and decanal (peak area values about 14.34×106 and 54×106, respectively). this finding does not agree with the results of previous studies carried out on the volatile fraction of kumquat essential oil, in which octanal and decanal were not detected or detected only at trace level (umano et al., 1994; peng et al., 2013). these two aldehydes are commonly detected both in citrus sphaerocarpa peel oil (thi minh tu et al., 2002) and in orange essential oil (högnadóttir and rousseff, 2003). at gc/olfactometry analysis, octanal was associated to sweet, citrusy, lemon and green descriptors, whereas decanal was perceived as sour, metallic, lemon and fatty (thi minh tu et al., 2002; högnadóttir and   ital. j. food sci., vol 28, 2016 268 rousseff, 2003). in our study, the presence of 2 acetals corresponding to the 2 aldehydes, 1,1-diethoxyoctane, and 1,1-diethoxydecane, was ascertained. 1,1diethoxyoctane has an odor of fatty, oily, green citrus with woody, spicy and fruity nuances (mosciano, 1994a), whereas 1,1-diethoxydecane presents an odor defined as waxy, green, aldehydic and orange with cognac and coconut nuances (mosciano, 1994b). the 2 acetals were most prevalent in the kp and the lp liqueurs, and their presence was due to the high level of the aldehydes. in fact, acetals originate from the reaction between alcohols and aldehydes, giving rise to an unstable semiacetal, which evolves to a stable derivative after reacting with a second alcohol molecule (heydanek and min, 1976). plutowska et al. (2010) found acetals as minor compounds in alcoholic beverages and spirits, with a possible role in enhancing the bouquet of the product. 3.2. acceptance and preference testing figure 3 reports the results of the sensory analysis. no significant difference among samples was perceived regarding color, which ranged between 3.4 and 3.9 (for the gp and the lp, respectively). the lp was the most appreciated sample as to flavor. this result could be due to the higher concentration of volatile compounds with a high olfactory impact (in particular ocs) and the greater familiarity of consumers with this traditional italian liqueur. regarding the liqueurs obtained from kumquat, the odor scores were 2.89 for the kp and 3.20 for the kwf, while the flavor scores were 3.01 and 3.32 highlighting a certain appreciation by consumers. this could be linked to the high amounts of sesquiterpene alcohols. the grapefruit liqueur was more significantly preferred for its odor in comparison to the two kumquat liqueurs. figure 3: mean score values and statistic analyses of the consumer acceptance of the liqueurs. kp, kumquat peel; kwf, kumquat whole fruit; gp, grapefruit peel; lp, lemon peel. values having different subscript letters are significantly different (p<0.05).   ital. j. food sci., vol 28, 2016 269 table 2 shows the ranking of preferences expressed by the panelists (third section of the evaluation form). in the same table the result of the statistical analysis obtained comparing the differences among the rank sums of the single liqueur with the critical value, as proposed by basker (1988), is reported. the rank sums were obtained by adding the preference ranking scores (from 1 to 4 starting from the most appreciated liqueur) expressed by each panelist. as expected, the lp proved to be the most appreciated liqueur, whereas the kwf and the kp were more appreciated than the gp. on the whole, the taste panel data highlighted the possibility of using kumquat for the production of rosolio, even though adjustments in the preparation process (fruit to ethyl alcohol ratio; duration of the maceration step) should be carried out to improve the odor and flavor perception. the use of the whole fruit instead of the peel determined an increase in the preference by the taste panel members; therefore, the peeling step could be avoided. table 2: rank sums of preferences and results of the statistical analysis (basker, 1988) of the liqueurs. kp kwf gp lp rank sums 206 183 230 122 kp bc 206 0 23 24 84 kwf b 183 23 0 47 61 gp c 230 24 47 0 108 lp a 122 84 61 108 0 abc: different letter indicates significant differences (n = 75, p ≤ 0.05, critical value = 40.6). bold font indicates values higher than critical value. 4. conclusions the main result of this study was the assessment of the feasibility of using minor citrus fruits for producing innovative liqueurs. with respect to limoncello, the kumquat and grapefruit liqueurs had greater concentrations of limonene and lower concentration of oxygenated compounds, the latter having high relative flavor activity. these differences justify the preference given to the liqueur obtained from lemon peel, even though all the innovative liqueurs were judged to be acceptable. among them, the kumquat liqueur obtained from whole fruit seems to be the most promising, probably because it had the highest content of sesquiterpene alcohols. references asikin y., taira i., inafuku s., sumi h., sawamura m., takara k. and wada k. 2012. volatile aroma components and antioxidant activities of the flavedo peel extract of unripe shiikuwasha (citrus depressa hayata). j. food sci. 77: c469. barreca d., laganà g., tellone e., ficarra s., leuzzi u. and galtieri a. 2009. influences of flavonoids on erythrocyte membrane and metabolic implication through anionic exchange modulation. j. membrane biol. 230: 163. barreca d., bellocco e., caristi c., leuzzi u. and gattuso g. 2011. kumquat (fortunella japonica swingle) juice: flavonoid distribution and antioxidant properties. food res. int. 44: 2190. basker d. 1988. critical values of differences among rank sums for multiple comparisons. food technol. 42: 79.   ital. j. food sci., vol 28, 2016 270 benavente-garcía o. and castillo j. 2008. update on uses and properties of citrus flavonoids: new findings in anticancer, cardiovascular, and anti-inflammatory activity. j. agr. food chem. 56: 6185. bernhard r.a. and scrubis b. 1961. the isolation and examination of the essential oil of the kumquat. j. chromatog. a 5: 137. caccioni d.r.l., guizzardi m., biondi d.m., renda a. and ruberto g. 1998. relationship between volatile components of citrus fruit essential oils and antimicrobial action on penicillium digitatum and penicillium italicum. int. j. food microbiol. 43: 73. choi h s. 2005. characteristic odor components of kumquat (fortunella japonica swingle) peel oil. j. agr. food chem. 53: 1642. crupi m.l., costa r., dugo p., dugo g. and mondello l. 2007. a comprehensive study on the chemical composition and aromatic characteristics of lemon liquor. food chem. 105: 771. dugo p., ragonese c., russo m., sciarrone d., santi l., cotroneo a. and mondello l. 2010. sicilian lemon oil: composition of volatile and oxygen heterocyclic fractions and enantiomeric distribution of volatile components. j. sep. science 33: 3374. gattuso g., barreca d., caristi c., gargiulli c. and leuzzi u. 2007. distribution of flavonoids and furocoumarins in juices from cultivars of citrus bergamia risso. j. agr. food chem. 55: 9921. heydanek m.g. and min d.b.s. 1976. carbonyl-propylene glycol interactions in flavor system. j. food sci. 41: 145. högnadóttir á. and rouseff r.l. 2003. identification of aroma active compounds in orange essence oil using gas chromatography–olfactometry and gas chromatography–mass spectrometry. j. chromatog. a 998: 201. iso. 1988. sensory analysis. methodology. ranking (iso 8587). geneva (switzerland): international organization for standardization. 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analysis of some italian lemon liquors (limoncello). j. agr. food chem. 51: 4978. paper received july 31, 2015 accepted september 25, 2015 ijfs#1923_bozza ital. j. food sci., vol. 32, 2020 815 paper physicochemical, rheological, and sensory evaluation of voluminous breads enriched by purslane (portulaca oleracea l.) m. delvarianzadeha, l. nouria, a. mohammadi nafchi*b,a, and h. ebrahimic adepartment of food science and technology, damghan branch, islamic azad university, damghan, iran bfood technology division, school of industrial technology, universiti sains malaysia, 11800 usm, penang, malaysia crandomized controlled trial research center, shahroud university of medical sciences, shahroud, iran *corresponding author: amohammadi@usm.my abstract portulaca oleracea (purslane) can be used as a vegetable and herb for medical and food products. the aim of this study was to investigate the psychochemical, rheological, and sensory properties of voluminous breads enriched by different amounts of purslane powder (0, 5, 10, and 15%) were compared to a control group. the results showed that, with an increase in the concentration of purslane in samples, water absorption capacity, stability under mixer, and softening level increased. adding 15% of purslane powder decreased farinograph quality number significantly. addition of purslane powder also improved resistance to extension and decreased extendibility, energy, and viscosity of the dough significantly. in terms of sensory properties, the sample with 15% purslane powder obtained the minimum score and other samples had acceptable conditions in terms of different sensory properties like taste, texture, color, odor, and general acceptability. in summary incorporation of purslane in voluminous bread is feasible and the optimum percentage of the purslane powder is 10% for the best acceptance in sensory evaluation. keywords: bread, portulaca oleracea, purslane, sensory evaluation, fortification, physichochemical properties ital. j. food sci., vol. 32, 2020 816 1. introduction wheat products like flour and bread are good carries for adding nutrients needed by the consumers (el khoury et al., 2018). in addition, bread is the staple food of about one half of the world population and a reliable source in terms of nutrition and inexpensive diet (graham, welch, and bouis, 2001). to improve nutritional support for low income families, it is essential to pay more attention to improving quality and diversity of available breads, minimize the wastes, and produce breads with strong sensory properties (guyot, 2012). along with being a main source of energy, bread also supplies the dietary fibers, some minerals (iron (fe), calcium (ca), and vitamin b group and vitamin e (found in wheatgrass) (kaur, 2011). improvement of texture, volume, crust, and quality of bread is one of the advantages of using fat in bread formulation (el-sohaimy et al., 2019). another role that is filled is adding tastes and energy content of the product. as shown by the literature, fat tastes in breads are more desirable for the consumer than other tastes (heenan et al., 2008). portulaca oleracea (purslane) is a grassy annual plant in the family oleracea with succulent stems, yellow or white small flowers, black seeds, and medicinal properties. the wild plant is a watery weed that prefers warm and arid condition and grows in a wide range of soils and climates. according to the traditional medicine, the plant has a cold and moist humor, styptic, and diuretic and decreases bile sack movement and bile flow in return (asadi gharneh and reza hassandokht, 2008; naeem and khan, 2013). purslane is a well-known plant in traditional medicine with protective effect on the liver that is used for several therapeutic purposes. it preserves the liver against the damages caused by free radical invasion and lipid peroxidation in the endoplasmic grid of cells (zarei et al., 2015) (hadi et al., 2018). in addition, purslane is rich of antioxidant compounds and a good source for flavonoid, and carotenoid (m. alam et al., 2014). in addition, there has been no report of toxic effects of this plant. in addition to the said effects, purslane demonstrated anti-pain and anti-inflammatory effects. it is the richest plant source of w-3 (m. alam et al., 2014; uddin et al., 2014). total fat content of the leaves range from 1.5 to 2.5 mg/g of fresh mass out of which around 60% and 40% is αlinolenic acid (c18:3ω3) (liu et al., 2000). nowadays, researchers are looking for the ways to enrich food products, meet nutritional needs, and improve health in the consumers. so far, different additives have been added to bread formulation such as purslane seed flour (fathnejhad kazemi et al., 2012), flaxseed flour (mervat et al., 2015), fenugreek flour (nasehi et al., 2018), and garlic flour (balestra et al., 2011). in addition, the importance of using medicinal plants in pharmaceutical and economic fields is quite clear (bhat et al., 2013; daneshzadeh et al., 2020; guariguata et al., 2014; whiting et al., 2011). taking into account the high rate of linolenic fat acid in some of oil seeds like purslane seed, the high consumption rate of bread, and low consumption rate of essential fatty acids per capita, the present study is an attempt to survey the possibility of using purslane to enrich voluminous breads to improve physicochemical, rheological, and sensory qualities of the breads. ital. j. food sci., vol. 32, 2020 817 2. materials and methods 2.1. purslane powder preparation shahroud strand of purslane plant was collected from a vineyard in shahroud, semnan province, iran. the plats were washed with distilled water to remove dust, cleaned, and dried at room temperature (35±2°c) for several days till complete dryness and then powdered using a laboratory grinder (pars khazar, rasht, iran). the powder was sieved using a sieve with mesh size of 0.5 at most. the obtained powder was kept in a capped container in fridge (0-4°c). 2.2. voluminous bread samples preparation as the control group, voluminous bread formulation is listed in table 1. to prepare the samples and according different levels of purslane powder to replace wheat flour (0 (control), 5, 10, and 15% w/w) was mixed with wheat flour, yeast suspension (2%) and other ingredients using an electrical mixer and incubated at 30°c for 30 minutes (pars khazar, rasht, iran). water content was determined using farinograph and the dough was mixed for 10min. to mix the dough, spiral mixer was used, and the dough was cut into 150 gr pieces. the baking process took 25-30 min at 180°c in an electrical convection oven (sm-705e model, sinmag, jakarta, indonesia). afterwards, the breads were cooled down and packed in polyethylene packages for further examination (shittu et al., 2009). table 1. voluminous bread formulation. ingredients amounts flour 1 kg sugar 10 g yeast 10 g vegetable oil 10 g salt 5 g water enough 2.3. physicochemical and rheological analysis 2.3.1 analyzing wheat flour, purslane powder, and voluminous breads moisture, ash, total protein, fat, raw fiber, falling number, and moist gluten were determined according to aacc method (2000) under code numbers 44-15a, 08-01, 46-13, 30-10, 32-10, 81-56, 54-11, and 12-38, respectively (aacc, 2000). 2.3.2 dough rheological analysis to examine amylograph properties of dough samples, an amylograph device (brabender, germany) was used based on the standard instruction. rheological properties of different samples were determined using farinograph and amylograph based on aacc method ital. j. food sci., vol. 32, 2020 818 (2000) no. 54-21 and 54-30. then different parameters like water adsorption, dough development time (ddt), stability of dough, and mixture resistance index were plotted on farinograph diagrams and parameters like gelatinization and viscosity were extracted based on amylograph diagram (aacc, 2000). 2.3.3 physicochemical analysis of voluminous bread the bread samples were analyzed using aacc (2000). the moisture, raw protein content, fat content, ash content, and gluten content were determined through 44-15, 46-13, 30-25, 01-08, and 38-11 methods respectively (aacc, 2000). 2.4. sensory evaluation the quality of breads, fresh and cooled down in ambient temperature, was examined by 20 trained examiners (10 men and 10 women) were analyzed. sensory specifications included tastes, texture, color, odor, and general acceptability. the samples were coded randomly and provided to the examiners in separate containers. they scored the samples based on a five-point scoring system (5= very good, 4 = good, 3= moderate, 2= bad, and 1= very bad) (ghanbari and farmani, 2013; yaseen et al., 2010). 2.5. statistical analysis all experiments were done with three replicates using anova in spss 22 (chicago, il, usa). to compare mean score (p<0.05), duncan’s multiple range test was used. the independent variable was different levels of purslane powder and dependent variables were all the experiments on the treatments. figures were developed in ms excel. 3. results and discussion 3.1. physicochemical properties of wheat flour and purslane powder physicochemical properties of wheat flour and purslane are listed in table 2. as listed, moisture of wheat flour is higher than that of purslane powder; while purslane powder has higher fat, protein, total ash, and fiber content compared to wheat. purslane powder did not have gluten and alpha amylase activity. 3.2. dough rheological analysis 3.2.1 farinograph test on the dough results of water absorption (wa) in farinograph test (table 3) showed that by adding purslane powder into the wheat flour, wa capacity decreases notably so that the control sample contained 57.96% water and the sample containing purslane powder (15%) only contained 55.1% water. still, the decrease in wa rate in the samples with 5 and 10% of purslane powder was not significant (p>0.05). since, purslane powder contains hydrophobe chemical compounds like fatty acids, the decrease in wa capacity by adding purslane powder is expectable. ital. j. food sci., vol. 32, 2020 819 as listed in table 3, by increasing the level of purslane powder in dough sample, stability time in mixture decreases. dough stability (ds) time in the control sample was 4.98min and for the samples with 5, 10, and 15% purslane powder, this time was 4.63, 4.26, and 3.65 min respectively. the decrease in stability time by replacing wheat flower by purslane powder is rooted in dilution or degradation of gluten grid. physical break of gluten grid can be a reason for the less stable dough as gluten proteins are responsible for viscoelastic grid and stability of dough and purslane powder does not contain gluten. (macritchie, 2010). table 2. physico-chemical properties of wheat flour and purslane powder. parameters wheat flour purslane powder moisture (%) 13.83±0.59a 4.72±0.45b protein (%) 8.89±0.33b 16.39±0.24a fat (%) 1.49±0.14b 4.79±0.06a ash (%) 0.72±0.11b 18.12±0.08a crude fiber (%) 0.13±0.03b 4.78±0.05a wet gluten (%) 24.16±0.27 felling number (seconds) 353 *different letters in the same row indicate significant differences (p<0.05). the degree of loosening analysis showed that by adding high levels of purslane powder to the dough samples, looseness of the sample increased notably after 12 min (table 3). this increase in looseness can be explained by the dilution of gluten proteins that weakens the dough. purslane powder also contains unsolved fibers that weaken gluten functions. dough weakening levels for the control and samples with 5, 10, and 15% purslane powder were 89.68, 91.29, 95.13, and 100.85 bu respectively. farinograph quality number (fqu) for different samples is listed in table 3. clearly, adding purslane powder to the dough sample (15%) caused a significant decrease in fqu (p<0.05) as adding the powder decreases stability of the dough. in general, there was no significant difference between fqu of the control and purslane samples 5% and 10% (p>0.05). xu et al. (2014) showed that adding linseed flour to wheat flour increased wa, dough expansion time, and dough resistance (xu et al., 2014). garden (1993) consistently found that mixing linseed and wheat flour decreased stability of dough significantly (gardenrobinson, 1993). koca and anil (2007) reported that the reason for the difference in the mixture of dough containing linseed was dilution of gluten protein by fiber and the reaction between fiber materials and gluten, which also affects the mixing process (koca and anil, 2007). farinograph results by koca et al. (2007) showed that by increasing linseed content, wa, dough expansion, and mixture resistant index increased; while ds decreased by adding different levels of linseed (koca and anil, 2007). these findings are consistent with the present study. ital. j. food sci., vol. 32, 2020 820 table 3. farinograph characteristics of dough samples. samples moisture absorption (%) dough stability time (min) degree of loosening (after 12 minutes fermentation) (b.u.) farinograph qualitative number control 57.96±0.42a 4.98±0.32a 89.68±2.42c 58.16±2.37a 5% of purslane powder 56.39±0.59 b 4.63±0.19ab 91.29±2.01bc 57.89±1.14a 10% of purslane powder 55.61±0.32 bc 4.26±0.29b 95.13±1.94 b 53.91±2.02a 15% of purslane powder 55.11±0.48 c 3.65±0.24c 100.85±2.06a 47.96±1.68b p-value 0.000 0.000 0.000 0.013 *different letters in the same column indicate significant differences (p<0.05). 3.2.2 extensograph test on dough rheology tests with large deformation range, including one-side extension test using an extensograph device yielded information about viscoelastic behavior of dough and dilatancy of gluten grid (gilbert, 2002). since, extensograph results have direct relationship with gluten protein properties, changes in dough resistance to extension and extendibility of dough can be attributed to the interactions between fiber structure and gluten protein. the results of the effects of different concentration of purslane powder on extension strength of the dough (fig. 1a) showed that with a longer rest time of 45-135 min, extension strength of the samples increased significantly both in the control and experiment groups (p<0.05). the dough rest was in extensograph container and during resting and due to changes in glutens, the ingredients were revived and a uniform gluten grid was reestablished due to the changes in gluten (xu et al., 2014). therefore, extension strength after the rest time is improved. at different rest times (45, 90, and 135min), adding purslane powder increases tensile strength of dough due to high fiber content of the powder. in general, the highest level of tensile strength happened in the samples with 15% purslane content and fermentation time of 135min (247.75 bu) and the lowest level was with the control sample with fermentation time of 45min (169.77 bu). extendibility levels of different dough samples are demonstrated in fig. 1b. clearly, in the samples with different concentrations of purslane, extendibility is notably less than the control samples (p<0.05). this can be due to the larger size of purslane powder particles compared to wheat flour that causes early rapture of gluten under extension. the second cause might be dilution of dough protein so that changes the ratio protein to starch (macritchie, 2010), the increase in tensile strength and decrease in its extendibility with different concentration of purslane powder is justifiable. in short, with different fermentation times, the highest extendibility was observed with the control sample and the lowest level was observed with the control sample with fermentation time of 45 min (14.73 cm). ital. j. food sci., vol. 32, 2020 821 figure 1. comparison of mean values of (a) dough elasticity (cm), (b) tensile strength of dough (bu), (c) energy (cm2) of different dough samples during different fermentation times. different letters on bars represent significant differences among means. the area under diagram and dough energy indicates the energy or mechanical work needed for extending the dough until rapture. this is a reliable index of dough strength. for academic purposes, the curve height and the area under it are considered as strength index and the higher this index, the higher the strength of dough (gilbert, 2002). the mean area under the diagram or tough energy of different dough samples containing different concentration of purslane powder with fermentation periods 45, 90, an d135min in extensograph is illustrated in fig. 1c. the highest and lowest levels of dough energy with different fermentation time were obtained by the control and purslane powder (15%) respectively. there was no significant difference between the energy level of the samples with 5 and 10% of purslane powder regardless of fermentation time. still, the increase in fermentation concentration time from 10 to 15% had a significant effect on the dough energy (considerable decrease). the sample with different levels of purslane powder ital. j. food sci., vol. 32, 2020 822 demonstrated a gradual increase in energy level of dough with an increase in fermentation time in the extensograph. mariotti et al. (2006) reported rheological and baking performance specifications of bread with different levels of avena sativa flour and showed that adding avena sativa decreased wa and strength of breads (mariotti et al., 2006). stepniewska et al. (2019) examined the quality of breads with rye flour and found that lower protein content, lower unsolved total pentosan content, higher solved pentosan content in water, flour granola, and solved content in water (pentosan in particular) had a significant effect on the hardiness of bread samples (stępniewska et al., 2019). marie and ivan (2017), consistent with our findings, reported that replacing linseed fiber had a significant effect on the energy of extensogram curve (marie and ivan, 2017). 3.2.3 amylograph analysis table 4 lists the results about the effect of different levels of purslane powder on gelatinization of wheat flour-based dough. clearly, despite the trivial increase in gelatinization temperature (gt) caused by the increase in the volume of purslane powder in the samples, there is no significant difference between the control and experiment samples in terms of gt (p>0.05). table 4. amylograph results of dough samples. variables control 5% 10% 15% gelatinization temperature (°c) 58.37±0.08 a 58.39±0.09a 58.46±0.05a 58.51±0.08a viscosity (bu) 1941.2±4.7a 1929±5.4 b 1917.5±2.9c 1906.2±4.1 d *different letters in the same column indicate significant differences (p<0.05). viscosity of the control and experiment samples is listed in table 4. clearly, the control sample has the highest viscosity (1941.2 bu) and adding purslane powder created a significant decrease in viscosity of the samples (p<0.05). that is, viscosity levels in the samples with 5, 10, and 15% of purslane powder were 1929.0, 1917.5, and 1906.2 bu respectively. the reason for this decrease in viscosity after adding purslane powder to the sample can be the decrease in gluten protein content. therefore, with an increase in purslane concentration, the continuous grid of gluten is broken and viscosity declines (salim-ur-rehman, 2006; yousif et al., 2012). salim-ur-rehman et al. (2006) showed that increasing the content of sorghum flour up to 30%, lowered the viscosity of dough (salim-ur-rehman, 2006). indrani et al. (2015) reported that adding ground black gram to dough sample increased viscosity of the dough samples notably. their results are consistent with the results here (indrani et al., 2015). mlakar et al. (2009) showed that replacing amaranthus flour up to 10% did not have any effect on gt of wheat flour dough, while adding 20% amaranthus flour increased starch gt (mlakar et al., 2009). moreover, a significant decrease in viscosity of the dough due to adding amaranthus flour was reported. ital. j. food sci., vol. 32, 2020 823 3.3. bread samples analysis 3.3.1 bread moisture content fig. 2a illustrates results of moisture assessment of the samples with different levels of purslane. clearly, the lowest moisture level is with the control sample (33.53%) and the moisture increases significantly with the increase of purslane content to 5 and 10% (p<0.05). still, the increase in purslane powder level from 10 to 15% decreases moisture content. the increase in moisture content with the lower levels of purslane can be explained by the high fiber content in purslane, which preserves moisture in the samples. still, with further increase in purslane content, gluten grid is degraded and its capacity to store water decreases. the powder contains hydrophobic chemical compounds like fatty acids that decrease water content. demine et al. (2013) showed that adding quinoa flour to flour formulation creates a significant decrease in moisture content of bread samples (demin et al., 2013). still, the increase in quinoa flour did not have a significant effect on bread samples moisture. gohar et al. (2016) stated that replacing a part of wheat flour with quinoa flour decreased moisture content significantly (gewehr et al., 2016). as to the increase in moisture content after adding amaranthus flour to the formulations used by baking industries, inglett et al. (2015) studied cookies containing amaranthus flour. they showed that adding amaranthus flour increased moisture capacity in baking process comparing with other samples (inglett et al., 2015). teutonic and knorr (1985) showed that amaranthus seeds have 3.54% lignin and this increases the capacity to store water (teutonico and knorr, 1985). in addition, elgeti et al. (2014) used quinoa flour and replaced it with rice and corn flour up to 40100% to obtain gluten free bread. the results showed that along with improving the volume of bread, quinoa flour created a softer inner texture, distributed air cell more evenly, had a positive effect on moisture content of the samples and delayed going stale (elgeti et al., 2014). they argued that the increase in moisture content of the samples was due to adding fiber-rich flour (e.g. lignin) to the samples. 3.3.2 breads protein content fig. 2b illustrates protein content of the samples. clearly, the minimum protein content appears in the control sample (10.05%) and since purslane powder contains more protein than wheat flour, adding purslane powder to the formulation created a significant change on protein content (p<0.05) so that samples with 5%, 10%, and 15% purslane powder contained 11.82%, 12.48%, and 12.91% protein content respectively. hossein and salem (2016) studied enrichment of gluten-free snacks using different levels of purslane and showed that adding purslane increased protein content of the products significantly (hussien and salem, 2016). asma and gindy (2017) showed that adding purslane powder to bread samples increased protein content significantly (asma and gindy, 2017). almasoud and eman (2014) found that adding purslane significantly increased protein content of crackers (almasoud and eman, 2014). these findings are consistent with our findings. ital. j. food sci., vol. 32, 2020 824 figure 2. comparison of (a) protein, (b) moisture, (c) ash, (d) fat and (e) fiber content (%) of different bread samples. different letters on bars represent significant differences among means. ital. j. food sci., vol. 32, 2020 825 3.3.3 breads fat content fat content results are demonstrated in fig. 2c; clearly, the control sample has the lowest fat content (0.65%) and adding purslane powder makes a significant change in fat content (p<0.05). that is, samples with 5%, 10%, and 15% purslane powder contain 1.01, 1.25, and 1.53% fat respectively. taking into account the fatty nature of purslane, the increase in fat content is expectable. desta and molla (2020) suggest that the highest oil content was observed in seed (desta, 2020) in the present study, all parts of the plant have been used. hossein and salem (2016) argued that increasing purslane powder in gluten-free stack formulations increased fat content significantly (hussien and salem, 2016). asma and gindy (2017) studied the increase in fat content of breads through increasing purslane level in the formulation (asma and gindy, 2017). 3.3.4 total ash content of breads fig. 2d illustrates total mean ash content of the control and experiment samples. clearly, the lowest ash content is observed with the control sample (2.85%) and since purslane powder contains higher levels of mineral elements, it yields more ash than wheat flour (alam, juraimi, yusop, hamid, and hakim, 2014). by increasing the share of purslane in the formulation, a significant increase in ash content takes place (p<0.05). the samples with 5%, 10%, and 15% of purslane powder yielded total ash volumes of 3.28%, 3.49%, and 3.68% respectively. a study by iglesias-puig et al. (2015) on the breads produced with quinoa total flour showed that with an increase in quinoa flour, ash content increases, which is consistent with our findings (iglesias-puig et al., 2015). a study by hossein and salem (2016) reported similar results so that an increase on purslane powder content in gluten-free snacks increased ash content of the products significantly (hussien and salem, 2016). asma and gindy (2017) showed that an increase of purslane powder in bread formulation significantly increased ash content of the samples, which is consistent with our results (asma and gindy, 2017). as the ash increases, the amount of minerals in the raw material increases (uddin, 2012). 3.3.5 bread fiber content raw fiber content in the control and experiment samples is illustrated in fig. 2e. clearly, the higher fiber content of purslane powder compared to wheat flour increases the fiber content significantly (p<0.05). with an increase in purslane content in the sample, the fiber content increases significantly. the control sample have 0.69% fiber and the samples with 5%, 10%, and 15% purslane powder have 1.02, 1.32, and 1.59% fiber content respectively. hossein and salem (2016) showed that an increase in purslane powder content in gluten-free snack increased fiber content significantly (hussien and salem, 2016). a study by asma and gindy (2017) showed that using purslane powder in the sample increased fiber content (asma and gindy, 2017). almasoud and eman (2014) consistently reported that adding purslane formulation to cracker increased fiber content notably (almasoud and eman, 2014). ital. j. food sci., vol. 32, 2020 826 3.3.6 sensory evaluation of bread mean scores of taste, texture, color, odor, and general acceptability of the samples are illustrated in fig. 3. figure 3. comparison of (a) texture, (b) taste, (c) odor, (d) color and (e) overall acceptance score of different bread samples. different letters on bars represent significant differences among means. ital. j. food sci., vol. 32, 2020 827 the control sample obtained the total score of taste, color, and general acceptability. in addition, the control and 5% samples obtained total score of texture and odor as well. still, there was no significant difference between the control and 5% samples in terms of taste and general acceptability (p>0.05). adding purslane powder to the bread formulation significantly lowered taste and general acceptability scores (p<0.05) so that the sample with 15% purslane content had the lowest score of color. except for the 15% sample, the rest of the treatments were acceptable in terms of sensory indices. by increasing purslane content from 5% to 15%, texture, taste, and general acceptability scores declined (p<0.05) so that the 15% sample obtained the lowest scores of texture, odor, and general acceptability (fathnejhad kazemi et al., 2012). still, all the treatments were acceptable in terms of texture, odor, and general acceptability. high purslane content in the formulation decreased volume and moisture content of the breads and had a negative effect on the texture. the decrease in color score by adding purslane powder content can be explained by the dark color of purslane powder. hossein and salem (2016) showed that adding 5% of purslane powder had a significant effect on sensory acceptability of glutenfree snacks (hussien and salem, 2016). however, adding 10 and 15% of purslane powder resulted in a decrease in sensory score of products. however, all the enriched samples were acceptable in terms of sensory specifications. mervat et al. (2015) maintained that adding 10% of linseed flour with total fat content did not have a significant effect on sensory acceptability (mervat et al., 2015). ganorkar and jain (2014) noted that dry crust, a decrease in tenderness, and feeling roughness in the mouth were the reasons for a decrease in general acceptability score after adding linseed to cookies formulation (ganorkar and jain, 2014). these results show that increasing the additive content increases tenderness of the product due to the higher content of fatty acids content; however, color, odor, and general acceptability decrease, which is consistent with our results. the reason for the noticeable decrease in the sensory score of the samples containing higher percentages of portulaca oleracea was due to the black color of the portulaca oleracea and its effect on the color of the bread samples. but in the melilli et al. (2020) study, the sensory score of 5% obtained the highest sensory score (melilli et al., 2020). it seems that this discrepancy is due to the difference of different varieties in different parts of the world and it is predicted that if the yellow varieties of portulaca are used, such a decrease will not be observed in fortified breads with a higher percentage of 10% portulaca oleracea 4. conclusion hysicochemical, rheological, and sensory properties of voluminous wheat flour breads containing different levels of purslane powder were examined. an increase in purslane content in dough samples decreased ds against mixture and increased looseness level. adding 15% of purslane powder decreased fqu significantly. in addition, despite the increase in extension strength and a significant decrease in dough extendibility, energy, and viscosity of the dough sample, the increase in gt was not notable. moreover, increasing the content of purslane powder increased protein, fat, total ash, moisture, and fiber content of the samples compared to the control samples. in terms of sensory specifications, the samples with 15% purslane powder content obtained the lowest score and the rest of the samples obtained acceptable scores in terms of taste, texture, color, taste, and general acceptability. using purslane in voluminous bread is feasible and the optimum formulation should contain 10% of purslane powder. ital. j. food sci., vol. 32, 2020 828 references aacc, c. 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2embrapa rice and beans, santo antônio de goiás, brazil; 3summerland research and development centre, agriculture and agri-food canada, summerland, bc, canada †retired. *corresponding author: priscila zaczuk bassinello, embrapa rice and beans, po box 179, santo antônio de goiás, goiás, 75375-000, brazil. email: priscila.bassinello@embrapa.br received: 8 march 2021; accepted: 27 august 2021; published: 11 november 2021 © 2021 codon publications open access review abstract the culinary quality of carioca beans is related to their market value and consumer acceptability. the depreciation of the cooking/technological quality of the product occurs mainly because of the integument browning and the longer cooking time of the grains, which are influenced by the storage time and conditions. the loss of culinary quality reduces the market value of carioca beans because consumers reject darkened grains that are attributed to a longer cooking time. as a result, cooking time (resistance to cooking), the color of the integument, and the texture of the cooked beans are determinant factors in the acceptance of carioca bean cultivars. the browning of the grain integument and the cooking time mainly depends on the environmental conditions, storage time, the tegument of each genotype, and the chemical and physical properties of the cotyledons. therefore, this review aims to survey the scientific literature on the extrinsic and intrinsic factors that affect the culinary quality of carioca beans. keywords: browning; cooking time; polyphenolic compounds; quality loss; storage introduction beans are cultivated during the whole agricultural year, in distinct ecosystems, principally in developing countries, which are responsible for 86.7% of world consumption. in 2019, five countries (myanmar, india, brazil, china, and the united republic of tanzania) accounted for 59% of the world’s dry bean production (~30.2 million tons; food and agriculture organization of the united nations, 2019). beans are rich in essential nutrients like (i) proteins with high lysine content (essential amino acid); (ii) high complex carbohydrate content as oligosaccharides, and dietary fibers with their recognized hypocholesterolemic and hypoglycemic effects; and (iii) complex vitamins and minerals (calcium [ca], iron [fe], copper [cu], zinc [zn], phosphorus [p], potassium [k], and magnesium [mg]); bioactive antioxidant compounds including saponins, polyphenols, and anthocyanins (ganesan and xu, 2017; lovato et al., 2017; oliveira et al., 2017; celmeli et al., 2018; yang et al., 2018; jeepipalli et al., 2020; liu et al., 2020; ). therefore, regular consumption of this pulse benefits human health. the us department of health and human services (us-dhhs, 2015) recommends eating about three cups of beans like pinto, kidney, or black beans/per week because of their health benefits. brazil is the largest global producer of common beans, mainly for domestic consumption, with an average per capita bean consumption of 16 kg in 2013 (rawal and italian journal of food science, 2021; 33 (4): 43–56 mailto:priscila.bassinello@embrapa.br 44 italian journal of food science, 2021; 33 (4) bento jac et al. grains, thereby reducing consumer’s acceptance, digestibility, and protein absorption (oliveira et al., 2011). briefly, 52% of surveyed consumers preferred commercial brands of carioca beans for their technological (lighter color grains [53 and 6.2; l* and a* values, respectively], medium-sized beans [28 g], elliptical shape, and semi-full flatness), cooking (fast cooking time ~15 min), and nutritional (high protein and minerals [k, p, ca, zn and cu] contents) quality traits (ribeiro et al., 2019). resistance to cooking/cooking time the time for beans to reach the desired degree of softness, determined as cooking time, is a critical attribute for consumers. cooking time can also be evaluated by the resistance to cooking generally determined using the mattson cooker (figure 1; proctor and watts, 1987; bento et al., 2020a). freshly harvested grains have less resistance to cooking. however, when stored under ambient conditions, carioca beans increase their resistance to cooking or take longer to cook (alvares et al., 2020; bento et al., 2020a, 2020b; de farias et al., 2020; alvares et al., 2020; bento et al., 2021a). navarro, 2019). the national market in brazil predominantly consists of two classes of beans: carioca (70%) and black bean (20%; souza et al., 2020). carioca beans are seasonally produced beans in brazil and many other countries. so, to maintain bean supply throughout the year and prevent scarcity between harvests, the storage of this variety is crucial. however, improper storage can cause undesirable changes in the carioca beans leading to consumer rejections (carbonell et al., 2010; scariot et al., 2017; alvares et al., 2020). these changes occur in the integument of some carioca bean cultivars because some genotypes darken very quickly, and the bean also hardens itself rapidly. these processes reduce the culinary quality of the carioca beans economically, depreciating the product (bento et al., 2020a, 2020b; de farias et al., 2020; bento et al., 2021a). the loss of culinary quality reduces the market value of carioca beans because consumers reject darkened grains that are considered resistant to cooking. consequently, determinant factors in the acceptance of carioca bean cultivars are cooking time (resistance to cooking), the color of the integument, and the texture of the cooked beans. therefore, this review aims to survey the scientific literature on factors that affect the culinary quality of carioca beans during storage. culinary quality culinary quality of beans refers to their sensory attributes, technological properties, such as water absorption before and after cooking, cooking time or resistance to cooking, percentage of soluble solids in the broth, integument color, and the broth. crop production, postharvest drying, and storage conditions are responsible for bean quality when it reaches its destination, either at the consumer’s table or back in the field to be used as seed. generally, beans subjected to improper handling or even storage conditions will influence the culinary quality, resulting in consumer rejection of the product. breeding new carioca bean cultivars emphasize high technological and cooking quality prioritizing preferential selection for lighter grains, 250–300 g 1000 seed weight, elliptical shape, semi-full degree of grain flatness, and cooking time less than 25 minutes (kaur et al., 2009; wani et al., 2013, 2017; yadav et al., 2018; ribeiro et al., 2019; ). bean coats are rich in water-insoluble fibers and polyphenols. their cotyledons have higher soluble fibers, oligosaccharides, and resistant starch (singh, 2017). hardening of the husk during bean storage occurs mainly when the air humidity is high, hindering hydration during preparation for consumption. the increased air humidity accompanied by high temperatures increases the incidence of a hardshell and hard-to-cook (htc) (a) (b) figure 1. mattson apparatus for bean resistance classification to cooking based on the scale defined by proctor and watts (1987). the time (t 13 ) is recorded until the drop of the 13th rod and is converted into a rank (rmc) of resistance to cook, as described by bento et al. (2020a). beans in the mattson apparatus (a). the cooking progress of beans in mattson apparatus (b). italian journal of food science, 2021; 33 (4) 45 factors affecting the cooking quality of stored carioca beans hardness (alvares et al., 2020; bento et al., 2020a; alvares et al., 2021). the htc defect manifests itself differently depending on the cultivar, planting, and storage conditions. these defects reduce the culinary quality of beans, causing depreciation and often a rejection of the product (njoroge et al., 2015, 2016). the htc phenomenon has not yet been completely elucidated, despite several studies trying to understand it. some theories have been postulated to explain the htc phenomenon: (i) polymerization of phenolic compounds or lignification, (ii) production of insoluble pectates or phytase-phytate-pectin, (iii) changes in protein and starch, and (iv) a multiple mechanism theory (nasarabbas et al., 2008; siqueira et al., 2016a; jombo et al., 2018; siqueira et al., 2018). in the lignification theory of cotyledon tissues, the development of grain hardening is related to the polymerization of phenolic compounds, mainly from phenolic-rich seed coats. the polymerization reaction is mediated by oxido-reducing enzymes and by cross-links formation between the phenolic compounds and the cotyledon cell wall proteins (nasarabbas et al., 2008). however, other studies by siqueira et al. (2014, 2016a, 2018) showed the inadequacy of the lignification mechanism. demonstrating that the htc in carioca beans during storage cannot be attributed to changes in the total phenol content or the oxidoreductase activities. according to the most widely accepted “phytase-phytate-pectin” theory (jombo et al., 2018; yang et al., 2018), water-soluble pectin allows water absorption by legume seeds. both phytates and carboxyl groups of soluble pectin can complex with ca or mg ions, but phytates preferably bind to divalent cations. if phytates complex with ca or mg ions, legume seeds are easy to cook. however, phytates can be hydrolyzed by phytase during storage, reducing their chelating potential. then the ca or mg ions complexes with the carboxyl groups of the soluble pectin to form insoluble ca and mg pectates which are not readily dissolved when heated, thus restricting cell separation and inhibiting water absorption resulting in htc defect (jombo et al., 2018; yang et al., 2018; bento et al., 2020a). finally, the theory that associates changes in starch during storage (rupollo et al., 2011) was discredited. since the noninvolvement of starch or protein in the hardening of carioca bean genotypes was known. in addition, the authors obtained results confirming that the hardening of carioca beans is related to structural changes in the cell wall and middle lamella. genotypes susceptible to the htc phenomenon display intense structural changes (shiga, 2004; siqueira et al., 2018). integument color consumers have different requirements. but generally attribute the dark color in carioca beans to prolonged beans are soaked in water (1–12 h [overnight]) before cooking to reduce the cooking time. the beans are hydrated, swollen, and smooth during immersion, which reduces cooking time (yadav et al., 2018). immersion also promotes the uniform expansion of the seed coat and cotyledon. the extent of hydration and swelling of the grains during immersion depends on the hydration capacity of the grains and varies among different bean cultivars and is related to cooking quality (singh et al., 2004; kaur et al., 2009). therefore, the bean water absorption capacity is a pivotal factor for the technical quality of the grains and is causally related to their resistance to cooking, as the cooking time decreases because of its occurrence before cooking (kaur et al., 2009). hence factors like the type of grain (i.e., size or shape), moisture level, genetics, storage time, and storage conditions influence the bean water absorption rate (delfino and canniatti-brazaca, 2010). the hydration kinetics of carioca beans is complex and mostly related to the seed coat. they are associated with bean composition (fat and k contents, protein to lipid ratio that correlates with the lag phase time) and structure (specific surface and seed coat impermeability to water; miano et al., 2018). alkaline conditions (ph 6–12) increase hydration kinetics (rate and water absorption) of the carioca bean (cv. iac eté) and reduce the lag phase (83%), affecting the mass transfer behavior in both the seed coat and cotyledons, indicating variations in proteins and polysaccharides (oladele et al., 2018). hydration with ferrous sulfate (feso4; 0.271% w/v) solution accelerates carioca bean softening and cooking, particularly with ultrasound (91 w/l; 25 khz) at 25 °c (miano and augusto, 2018). this process also fortifies the beans with iron (60 vs. 34 mg fe/100 g wet basis [510 min, with and without ultrasound]). moreover, the iron content of the seed coat, cotyledons, and whole carioca beans (cv. iac imperador) increased 68, 5, and 16 folds, respectively when soaked in feso4solution for 510 minutes compared to conventional hydrate (distilled water) for the same time. one of the defects (hardshell) that is observed in grains stored under conditions of inadequate temperature and humidity is alleviated bean water absorption, even after long periods of maceration. the hardshell phenomenon can be a consequence of grain storage in high humidity and high temperatures, which also causes the htc phenomenon (yadav et al., 2018). that refers to grains requiring a longer cooking time to soften or fail to soften even when subjected to boiling water for extended periods. storage under ambient conditions of temperature and humidity for 6 months induces htc phenomenon in common beans and consequently increases the cooking time. for most carioca bean cultivars, only 3 months of storage is sufficient to observe enhanced 46 italian journal of food science, 2021; 33 (4) bento jac et al. stored under high relative humidity, a factor known to promote browning (rani et al., 2013). the rate of grain darkening is linked to the genetics of the strains, storage conditions, and storage time (coelho et al., 2009, 2013; rani et al., 2013; spitti et al., 2019; de farias et al., 2020 ). considering that small farmers do not always have the technology or resources to invest in controlled storage of beans (under low temperature and humidity), commercial damage is inevitable, leading to the accumulation of low-value grains in the producers’ warehouses, filling stations, and supermarkets. thereby limiting the production and commercialization of good-quality beans. however, some bean genotypes that darken regardless of storage conditions do not necessarily harden or lose nutritional and functional value, and vice versa (alvares et al., 2020; bento et al., 2020b; de farias et al., 2020). the browning of stored beans has been extensively studied, although its association with culinary quality is limited in routine breeding programs (cichy et al., 2019; rodrigues et al., 2019; miklas et al., 2020), which can lead to the development of apparently suitable materials in terms of appearance (color). these results in genotypes that are resistant or tolerant to factors that accelerate grain browning or darkening, but not necessarily resistance to the hardening process, masking its culinary quality (alvares et al., 2020). therefore, bean breeding programs must consider nutritional properties, culinary properties, and color when selecting genotypes. texture textural parameters are also used to assess the culinary quality of beans, as grain hardness or resistance cooking, and therefore low culinary quality (ribeiro et al., 2008; coelho et al., 2009; cichy et al., 2019; rodrigues et al., 2019; alvares et al., 2020; bento et al., 2020a; de farias et al., 2020; ). according to bolsinha de sp (brazilian platform with bean market price information), the market negotiates the price of carioca beans, considering the color of the grain. the color is evaluated visually and subjectively based on an unofficial ascending color grade scale that ranges between 5 and 10, and in practice, the product with a grade below 8.5 is devalued (bento et al., 2020b; bolsinha, 2020). in short, carioca beans with dark coloring are depreciated, as they are considered of low culinary quality. studies generally show that the luminosity (l*) of the grains and the hue angle (h°, color angle) decrease during storage and are influenced by storage time. on the other hand, the value of chroma (c*), a variable that measures the opacity of the grains and the total color difference (δe), increase during storage (siqueira et al., 2014, 2016b; bento et al., 2020a; coelho et al., 2020; de farias et al., 2020;). these changes in the instrumental color parameters indicate the darkening of the carioca bean; the grains leave the cream color with brown streaks and become dark brown (figure 2). changes in color parameters during storage are often associated with grain genotype, indicating that cultivars have a genetic predisposition to faster or slower browning (darkening) when stored. da silva et al. (2008) verified that storage time accentuated the differences between quick and moderate browning carioca beans. moreover, genotype influenced the browning/darkening rate of five carioca bean cultivars stored for 5 months under environmental conditions (siqueira et al., 2014). in short, genotypes are sensitive to the browning process, exhibiting difference in intensity among genotypes even when (a) (b) (c) (d) (e) (f) (g) (h) figure 2. freshly harvested carioca beans from slow darkening cultivar brsmg madrepérola (a), taa dama (b), darkening cultivar brs notable (c), and iac imperador (d). the corresponding grains after 6 months storage (27.5 ± 1.6 °c / 56.9 ± 10.0% relative humidity-rh) (e–h). italian journal of food science, 2021; 33 (4) 47 factors affecting the cooking quality of stored carioca beans beans, respectively). the hotplate (45 or 60 minutes) and autoclave (110 °c/15 minutes) cooking promote grain softening and discriminate fresh and aged beans. hence are, therefore, suitable procedures to prepare carioca beans for instrumental texture analyses (marles et al., 2008). hardness has also been used to evaluate the effects of irradiation on widely consumer accepted commercial carioca beans. irradiation (5 kgy) almost doubled the rupture force or hardness of cooked (autoclave 121 °c/ 15 minutes) carioca beans compared with nonirradiated grains. this increase in grain hardness is presumably associated with starch retrogradation after cooking (mendes et al., 2011). industrial canning (rotating autoclave 120 °c, 2 hours/35 minutes cooking) showed no significant difference in the texture or maximum compression (90% of initial height using 50 kg texture meter analyzer) force (0.60–0.75 n/grain) of carioca beans from three cultivars (schoeninger et al., 2020). bean hardness increases in raw and cooked grains when stored (siqueira et al., 2014, 2016a). this increase in hardness of aged bean is attributed to the loss of cell membrane integrity, which increases soluble solids loss in htc grains during the immersion and cooking processes and may explain the changes in water permeability (siqueira et al., 2013, 2014, 2016a, 2018). these results demonstrate that carioca beans hardening is related to structural changes in the cell wall and middle lamella during storage. field management/environmental influence the management of bean cultivation and grain harvest is the first control point for good culinary quality beans acquisition. adverse environmental conditions, inadequate harvest time, incomplete drying, and severe threshing showed grain breakage (or the appearance of cracks in the seed coat), which favors rotting, the development of fungi, and bruchid attacks (mutungi et al., 2020). grains grown in tropical conditions (for example, high temperature and high humidity) may have long cooking times, and cotyledons have higher resistance to softening during cooking (cichy et al., 2019; kigel, 1999). cichy et al. (2019) recommended future research to differentiate the effect of the growth and storage environment on the culinary quality of beans. in short, the growth conditions affect pests and microflora development cycles and regulate progression rates of the biochemical reactions of bean grains, thereby influencing grain quality (mutungi et al., 2020). however, some genotypes exhibit stable cooking times in different production environments (cichy et al., 2019). the best harvest period for most grains is close to their physiological maturity, wherein the grains to compression strength is one of the primary sensory properties of foods. it refers entirely to the feeling of hardness that the food presents during chewing and can be objectively measured by mechanical means in fundamental units of mass or strength. the texture parameters for beans refer to the evaluation of the hardness of raw or cooked grains, generally using the texturometer with a return-to-start analysis method that determines the strength required to compress/puncture the bean (revilla and vivar-quintana, 2008; siqueira et al., 2013, 2014, 2016a; bento et al., 2020a). other studies evaluate grain texture through texture profile analysis (tpa) that provides information on hardness, cohesiveness, elasticity, gumminess, and resilience of bean samples (koriyama and kasai, 2019; wani et al., 2013, 2017; yadav et al., 2018). the best evaluation of carioca bean texture is the method that measures the force necessary to drill the grain with a 2 mm probe (p2) according to revilla and vivar-quintana (2008) and siqueira et al. (2013). the small area of this probe penetrates the integument. they can differentiate similar samples, even when these grains have soft cotyledons but hard integument. the hardness of raw and cooked common beans varies (10–30 and 0.2–0.8 kgf, respectively). the texture of the cooked grains depends on the degree of cooking, and consequently if the grain is difficult to cook or exhibits htc defect that will present greater hardness. physical and chemical changes such as protein denaturation, carbohydrate solubilization, and starch gelatinization occur when the beans are cooked. starch can exhibit different gelatinization patterns. furthermore, depending on the cultivar, these changes can occur with greater or lesser intensity, favoring the reduction of grain hardness (wani et al., 2017; yadav et al., 2018). moreover, carbonell et al. (2010) associated the increase in the hardness of some cultivars with genetic characteristics of the grain or the susceptibility in the interaction of genetic and environmental factors, which can accelerate with inadequate storage. the method of bean preparation to evaluate the texture of the cooked grain interferes with the results obtained. the difference in hardness of fresh and aged carioca beans after distilled water (1:2 w/v) soaking (18 hours, 25  °c) has been evaluated by five cooking methods: mattson bean cooker, hot air oven, hotplate, boiling water bath, and autoclave (siqueira et al., 2013). generally, cooking time and temperature affect bean hardness. mattson bean cooker and hot air oven undercook the beans with hardness above 4 newtons (n). increasing cooking time from 30 to 60 minutes on a hotplate reduces bean hardness whereas, mild autoclave condition (105 °c/10 minutes) differentiates fresh and aged bean hardness (3 n vs. 3.4 n) and severe environment (115 °c/20 minutes) produce softer beans (0.8 n vs. 1n for fresh and aged 48 italian journal of food science, 2021; 33 (4) bento jac et al. physiological quality, such as 1000 seed weight, germination, and vigor (scariot et al., 2017). there are no current reports on drying methods that aim to reduce the loss of the culinary quality of beans. additionally, few studies report the effects of the drying process on the organoleptic, nutritional, and culinary characteristics of the grains. however, increasing the grain drying temperature to 60 °c increased the cooking time after 225 days of storage than the lower drying temperatures (elias et al., 2016). storage the last step before the commercialization of dry grains is storage. during this period, monitoring of some factors is pivotal to maintain bean quality and quality. while stored, grains undergo chemical and physical changes that alter their characteristics( loss of cooking quality and weight reduction). these are linked to the consumption of dry matter because of breathing and attack by pests. the absence of effective grain handling and storage techniques significantly decreases the quantity and quality of grains during the postharvest phases (affognon et al., 2015; mutungi et al., 2020). hardening and reduction of the permeability of the tegument occur when grains are stored in inadequate conditions of temperature and relative humidity, increasing their resistance to cooking. these quality losses can be considered a hardshell when the tegument loses its water absorption ability and htc when beans have increased resistance to cooking (oliveira et al., 2011). temperature and relative humidity also influence grain quality during storage. the high temperature, combined with high relative humidity (rh) of the air during storage, favors an increase in respiratory rate, culminating in accelerated deterioration rate, as well as increased fungi and insect contamination. therefore, low temperature and relative humidity are necessary to maintain grain quality during storage (rani et al., 2013; coelho et al., 2020; de farias et al., 2020). grains stored in cold rooms (8 ºc and 45% rh, 360 days) show no increase in cooking time. on the other hand, grains stored under normal environmental conditions increased their cooking time with longer storage time (morais et al., 2010). moreover, de almeida et al. (2017) reported shorter cooking times in beans stored for 108 days at 15 ºc and 45% rh than those stored at 27 ºc and 75% rh. some countries, such as australia, brazil, africa, and argentina, have adopted a system of hermetic storage for pulses using polyethylene (pet) silo bags. the silo bag is constructed with high-density polyethylene in three layers: two black internal layers and one white external layer present maximum dry matter accumulation and quality. at this stage the grains have high (30–45%) water contents depending on the type and cultivar (faroni et al., 2006; scariot et al., 2017). thus, the grains are left in the field until they absorb water appropriate for threshing or mechanized harvesting. however, field drying can compromise grain quality since the grains are susceptible to the environmental climate, such as temperature and relative humidity variations that accelerate the respiratory rate of grains, increasing the consumption of reserves and attacks by insects and fungi. according to scariot et al. (2017), beans harvested with 16.6% water content had lower physiological quality and 1000 seed weight than those with higher water content (25.2% and 35.2%). similarly, faroni et al. (2006) confirmed that beans harvested with 11.7% water content had lower technological classification over those with higher water content (18.7%), demonstrating the negative effects of delayed harvest. postharvest management drying the reduction of water content limits the availability of water for physical-chemical and biological processes of the grains, and the development of fungi, bacteria, and insects is mainly responsible for degradation and loss during storage (mutungi et al., 2020). therefore, drying is extremely important before enhanced safe storage. the first stage of bean drying occurs initially at the plant itself in the field, before harvesting, and is called predrying. this is not sufficient to maintain the grain for prolong storage. drying in the sun or at high temperatures (10 °c above ambient air) under tarpaulins or land, mainly by small farmers, is carried out. here the grain layer must not exceed 5 cm, and periodical mixing of grains is necessary to standardize drying. this method may not reduce the water content of the grains to ideal levels or delay this process, causing losses of culinary quality in the product and are contaminated by fungi (rani et al., 2013; scariot et al., 2017). the removal of water performed inappropriately by drying can negatively influence the physical, physiological, chemical, and cooking characteristics of beans. the main factors that affect the quality of the grains are the drying air temperature and the initial water content of the grains. the use of high temperatures for drying the grains, mainly combined with high water contents, can cause damage such as cracks and fissures, which can become a gateway for fungal attack during storage, in addition to reducing quality after drying (faroni et al., 2006; rani et al., 2013; scariot et al., 2017). drying beans at temperatures above 40 ºc reduces their physical and italian journal of food science, 2021; 33 (4) 49 factors affecting the cooking quality of stored carioca beans darkening gene genetic variation occurs in seed coat darkening of many bean classes including carioca beans (rodrigues et al., 2019). the variation can be monogenic or oligogenic, which means their inheritance is controlled by one or a few genes (silva et al., 2018). in carioca beans, the darkening phenotype is controlled by a single recessive gene “sd,” the slow darkening is represented by the “sd” recessive allele (silva et al., 2008). however, oligogenic control has also been proposed (elsadr et al., 2011; silva et al., 2014). a model with two genes interacting under epistasis was suggested by elsadr et al. (2011), where the “j” gene modulates darkening or interacts with a second sd gene, responsible for regulating the darkening rate. similarly, spitti et al. (2019) described that this characteristic is oligogenic or even polygenic from significant results obtained by the genotype and environment interaction. islam et al. (2020) demonstrated yet another allele in the p gene (psd) responsible for the sd trait in common beans, “p” is a transcription factor that restores the seed coat color. the sequence comparison of this gene in several beans differing in the seed coat postharvest darkening provided insights into the molecular mechanism that governs this characteristic and the development of new specific gene markers for potential use in bean breeding programs. selecting lines with slow seed coat darkening is possible despite conflicting results regarding the control of beans darkening (silva et al., 2014). however, selection based on the evaluation of only one environment can generate sufficient gains for this characteristic, even in the presence of genotype and environment interaction, which would explain the inconsistencies between the patterns of genetic control in each location (alvares et al., 2016). the elucidation of the genetic control of grain darkening is of fundamental importance to establish breeding programs for developing cultivars with slow seed coat darkening during storage (silva et al., 2014). microsatellites are notable in genetic diversity studies. polymerase chain reaction (pcr)-based markers are developed for a many plant species, including commercial crops. a panel of 24 microsatellites has been built for the common bean specifically for studying the genetic diversity available to the scientific community and has been routinely used for this type of analysis in brazil (métais et al., 2002; blair et al., 2003; morais et al., 2016). thousands of single nucleotide polymorphism markers are currently available for the common bean. this number increased immensely because of the species genome sequence publication (phaseolus vulgaris v1.0; http://www.phytozome. net; schmutz et al., 2014; morais et al., 2016). even if the choice of breeding lines and the development of cultivars made of titanium dioxide. the level of oxygen within the bag drastically falls, whereas carbon dioxide increases once the product is wrapped and the bag is sealed (hell et al., 2014; freitas et al., 2016). thus, this technology is based on creating storage environments harmful to pests by one of the following methods: bio-generated modified atmosphere and hermetic vacuum or gas hermetic fumigation (freitas et al., 2011; hell et al., 2014; mutungi et al., 2015; freitas et al., 2016). an advantage of the hermetic silo bags is the nonchemical alternative to postharvest quality control in terms of moisture content, specific mass, germination, and electrical conductivity, for up to 120 days (mutungi et al., 2015; magalhaes and de sousa, 2020). common beans (red beans) storage in silo bags with 17.8% rh presented higher cooking time than those stored in pet bottles or a glass recipient (closed with organza fabric; freitas et al., 2011). information is limited on the hermetic storage using pet silo bags, silo bags with temperature control, or even pet bottles influence in cooking culinary quality of carioca beans. type of cultivar/genotype the darkening of common beans is an undesirable characteristic for consumers, generally associated with old beans and consequently extended cooking time. however, the factors that influence darkening include environmental and genetic factors. the latter do not show any darkening pattern among different grain types and do not strongly affect the environment (alvares et al., 2019; spitti et al., 2019). seed coat postharvest darkening depends on beans genotype (islam et al., 2020). postharvest darkening (phd) of seed coat gradually changes the seed coat color of some dry bean market classes during storage. genotypic and environmental factors influence the rate and extent of phd, and darkening occurs rapidly in environments subjected to high temperatures, humidity, and light exposure. there are at least three phd phenotypes: (i) non-darkening (nd), (ii) slow darkening (sd), and (iii) regular darkening (rd). sd and nd genotypes have already been identified in common beans (elsadr et al., 2011). according to spitti et al. (2019), darkening is closely linked to the growing environment regardless of the cultivated genotype, or the genotype and environment interaction affect the bean seed coat darkening, requiring the characteristic evaluation in various climates of genetic control and strains selection (silva et al., 2014). seed coat darkening and cooking resistance increased in different carioca bean genotypes over 6 months of storage under varied environmental conditions (siqueira et al., 2014). http://www.phytozome.net� http://www.phytozome.net� 50 italian journal of food science, 2021; 33 (4) bento jac et al. airway control. another study by siqueira  et al. (2016b) also stressed the importance of analyzing the separate cotyledon integument because of these enzyme variations activities. nevertheless, according to bento et al. (2020a), the presence and activity of sod indicate that oxidative stress occurs during storage. the hardening of beans during storage is also related to low humidity and hydration defects. high temperatures and low relative humidity in the presence of light and oxygen are the main factors that hinder water absorption, in which shows grain hardening consequently, contributing to the htc phenomenon because of changes in phenolic content related to lignification and loss of phytates (junk-knievel et al., 2008). the lignification process relates the hardening with the polymerization of the phenolic compounds from the integument, mediated by polyphenol oxidases, and the formation of cross-links between phenolic compounds and cell wall proteins of the cotyledon. peroxidase is involved in the polymerization reaction of phenolic compounds. an increase in its activity may be associated with the cell wall lignification process (alves et al., 2021). the hardening process can also be explained by the sod activity in oxidative stress (bento et al., 2020a). polyphenolic compounds polyphenolic compounds are associated with the plant defense system, ensuring resistance to pest attacks at considerable levels but, they can accelerate the grain darkening process (spitti et al., 2019). according to chen et al. (2015), bean darkening is rapid and more common in cultivars with high phenolic content in the tegument. other studies have also associated bean darkening to the content of phenolic compounds, where darkening is more prevalent in cultivars with high phenolic content in the tegument, and the degree of darkening is proportional to the loss of phenolics (martín-cabrejas et al., 1997; beninger et al., 2005; luthria and pastor-corrales, 2006; nasar-abbas et al., 2009). in a recent study, the content of phenolic compounds decreased for fast darkening grains but did not change or increased in slow darkening carioca beans during storage (bento et al., 2021a). furthermore, kaempferol was suggested as the marker to differentiate fast and slow darkening cultivars since it decreased in quick darkening cultivars during storage. the bean darkening has also been associated with the presence of proanthocyanidins (condensed tannins) in the seed coat (junk-knievel et al., 2007), which accumulate at higher levels in regular darkening than in the slow darkening genotypes, just like most flavonoids (duwadi et al., 2018). proanthocyanidins are oligomeric flavonoids composed mainly of catechin and epicatechin with the slow darkening trait are relatively simple, given its high heritability and simple genetics, the genetic complexity of many interesting characteristics makes this selection difficult in breeding programs. however, the slow darkening trait is expressed maternally and inherited recessively (silva et al., 2018; alvares et al., 2019). oxidative enzymes related to beans aging during storage, cell damage occurs with advanced grain aging, and the physical barrier that separates enzymes and substrates is lost, enabling the oxidation of phenolic compounds by oxidoreductases. complex processes and enzymatic reactions are activated in postharvest beans and intensify during storage, initiating the aging and darkening phenomenon (siqueira et al., 2016b). postharvest darkening of the bean seed coat, both enzymatic and nonenzymatic, has already been attributed to the presence of phenolic compounds (spitti et al., 2019) because of their involvement in oxidative steps and subsequent changes in the flavonoid skeleton, as well as by the formation of quinones or similar enzyme-mediated reactions (marles et al., 2008; siqueira et al., 2016b; bento et al., 2021a). polyphenol oxidase is associated with the enzymatic activity of peroxidase. it produces a dark compound that causes grain integument darkening called melanin (siqueira et al., 2016b). rd is strongly associated with increased polyphenol oxidase activity in some strains of beans (marles et al., 2008). likewise, alves et al. (2021) evaluating, a fast darkening and hardening cultivar that demonstrated high peroxidase and polyphenol oxidase activity in the process. higher polyphenol oxidase activity is noted in bean cultivars with lighter tegument than in dark tegument cultivars during the complete aging process. this activity confirms that oxidative processes of phenolic compounds linked to polyphenol oxidase activity contribute to color changes in the bean grain coat (siqueira et al., 2016b). polyphenol oxidase and peroxidase activities are attenuated during controlled bean storage at cooling temperature, thereby inhibiting/suppressing grain darkening (demito et al., 2019). therefore, the highest color change occurs at higher storage temperatures because of high enzymatic activity, mainly polyphenol oxidase, which degrades polyphenols and reduces the bioactive value of these grains. oxidation by environmental oxygen can trigger the darkening and hardening process of carioca beans. previous research by bento et al. (2020a) found the predominant superoxide dismutase (sod) activity in the grain integument. but its byproduct, hydrogen peroxide, was only noted in the integument. the high sod activity suggests that the seed tissue (like cotyledons) maintains strict italian journal of food science, 2021; 33 (4) 51 factors affecting the cooking quality of stored carioca beans and obesity, and contributes to overall health and wellness (tyler et al., 2017; de lima et al., 2019; mullins and arjmandi, 2021). conclusion the culinary quality of beans is influenced by intrinsic and extrinsic factors and involves sensory attributes and technological properties that directly reflect consumer choice. the cooking time, color, and texture are the determining properties in the acceptance of the grain, which can be affected during storage mainly by temperature and humidity. the type of cultivar directly influences the darkening and hardening of the grain. the literature presents this control as oligogenic or monogenic. moreover, different cultivars have a varied genetic makeup for darkening. specific phenolic compounds present in the tegument play an important role as a substrate for oxidation reactions, and the type of pigments determine the final color of the grain, that is, the post-harvest darkening rate. the future perspectives to enhance bean quality may be related to the use of different techniques for enhanced preservation of the grains appearance and composition, such as the system of hermetic storage using polyethylene silo bags. additionally, the breeding programs can recommend new bean cultivars in the market, which are resistant to long-term storage without losing their culinary quality. a study of factors influencing bean properties acceptable by the final consumer is critical to encourage breeding programs and increase the demand for this high nutritional food. the use of bean flour as a food ingredient is an alternative for the use of aged beans with low culinary quality and improves food industry diversity. in addition, they are the excellent raw material for protein extraction in plant-based products applications, a market that has been growing in recent years. additionally, aged beans or broken grains have potential for use in gastronomy (as flour) because of their technological, nutritional, and functional properties, as well as their versatile application. it is also an option to enrich school meals and the diet of low-income populations, in addition to specific demands (gluten-free, low glycemic index, vegan, etc.). references affognon h., mutungi c., sanginga p. and borgemeister c., 2015. unpacking postharvest losses in sub-saharan africa: a meta-analysis. world dev. 66: 49–68. https://doi.org/10.1016/j. worlddev.2014.08.002 alvares r., pereira h., melo l., miklas p. and melo p., 2020. induction of seed coat darkening in common beans (phaseolus vulgaris l.) and the association with cooking time after storage. aus. j. crop sci. 14: 21–27. https://doi.org/10.21475/ajcs.20.14.01.p1500 units. the synthesis of proanthocyanidin shares the flavonoids pathway with anthocyanins to leukocyanidin/ cyanidin (duwadi et al., 2018). the presence of catechin and kaempferol was identified by beninger et al. (2005), and the significant increase in aging suggesting that the formation of these compounds occurred during the oxidative process. therefore, these compounds can interfere with the culinary quality of beans once the dimer procyanidin b-type contributes to the seed coat darkening process because of the oxidation of proanthocyanidin in reactive quinones (brown colored compound; ranilla et al., 2007; bento et al., 2021a). alternative uses of beans with low culinary quality the aged beans with low culinary quality and consequently low commercial value can be used as ingredients in food formulation. in the same way, bean byproducts (i.e., broken beans) may be used for food development since they present similar nutritional value compared with whole grains. both can be transformed into flours that constitute a new way of using materials with low added value that can contribute to the sustainability of the bean production chain. the bean flour usage is aligned with the current trends and consumption habits, based on sensory quality, practicality, diversity, and healthiness. bean flours can also be used in the gluten-free and vegan products development, one of the most successful markets in the food industry. thus, various studies report an effective way to apply aged dry beans flour as a base ingredient in many foods, such as tempeh (bento et al., 2020c), baked snacks (tortillas), and instant pasta (bento et al., 2021b), vegan tempeh burger (bento et al., 2021c), mix for cakes (gomes et al., 2015; bassinello et al., 2020). in these studies, the aged beans were heat treated (e.g., extrusion, cooking in an autoclave, or the traditional pressure-cooking method) to mitigate some unpleasant flavors in the bean flours (pasqualone et al., 2020). other studies have also used bean as ingredient for the development of spaghetti and ravioli (gallegos-infante et al., 2010; ringuette et al., 2018), extruded snacks (bassinello et al., 2015), light red kidney bean porridge (nyombaire et al., 2011), snack bars (ramírez-jiménez et al., 2018), cookies (pérez-ramírez et al., 2018), bread and chips (hooper et al., 2019), and extruded snacks made with maize and bean (7:3; félix-medina et al., 2021). in addition, there is significant interest presently in dry and wet fractionation of pulses into starch, protein, and fiber concentrates for use in both food and nonfood products (tyler et al., 2017). additionally, the consumption of bean products is associated with health benefits such as the reduced risk for cardiovascular disease and cancer, the management of type 2 diabetes, metabolic syndrome, https://doi.org/10.1016/j.worlddev.2014.08.002� https://doi.org/10.1016/j.worlddev.2014.08.002� https://doi.org/10.21475/ajcs.20.14.01.p1500� 52 italian journal of food science, 2021; 33 (4) bento jac et al. profile in grains of carioca beans during storage. lwt. 139: 110599. https://doi.org/10.1016/j.lwt.2020.110599 blair m.w., pedraza f., buendia h.f., gaitán-solís e., beebe 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chiara la torre1, vincenzo sicari2, angelo maria giuffré2, alessandro neri2, marco bonesi1, mariarosaria leporini1, alessia fazio1, tiziana falco1, monica r. loizzo1,* 1department of pharmacy, health and nutritional sciences, university of calabria, rende cs, italy; 2department of agraria, university “mediterranea” of reggio calabria, salita melissari, feo di vito, reggio calabria (rc), italy *corresponding author: monica r. loizzo, department of pharmacy, health and nutritional sciences, university of calabria, 87036 arcavacata di rende (cs), italy. tel.: +39-0984-493071. email: monica_rosa.loizzo@unical.it received: 24 july 2020 / accepted: 11 september 2020 / published: 06 february 2021 © 2021 codon publications open access paper abstract this study aimed to evaluate the influence of capsicum baccatum l. aji angelo and bishop crown cultivars to the quality parameters of flavoured olive oils (foos) obtained by the addition of both fresh and dried pepper powders (1%) to dolce di rossano and roggianella monovarietal extra virgin olive oils (evoos). first, pepper extracts were investigated for their total phenolic, flavonoid, carotenoid content as well as phenolic acids, fatty acid profile, and vitamin c and e content. in order to evaluate the impact of both fresh and dried peppers on the oxidative stability of foos, the rancimat test was applied. 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic) acid (abts), 2,2-diphenyl-1-picrylhydrazyl (dpph), β-carotene bleaching (b-cb) and ferric reducing antioxidant power (frap) assays were used to investigate the antioxidant potential. bishop crown dried extracts showed the highest phenolic, carotenoid and vitamin content, whereas aji angelo had the highest amount of capsaicinoids. among evoos, roggianella evoo showed the highest antioxidant activity as well as the highest induction time (39.6 h). remarkably, foo obtained by the addition of bishop crown dried pepper extract to roggianella evoo showed a higher induction time (44.9 h) with respect to the corresponding evoo. keywords: antioxidant activity; capsicum; chemical profile; monovarietal extra virgin olive oil; oxidative stability introduction according to the definition of the european union commission (ec, 2003; eec, 1991), an extra virgin olive oil (evoo) must be extracted “only from olives with superior quality, cannot undergo any treatment other than washing the fruits, and decanting, centrifuging and filtering the extracted olive oil. it excludes oils obtained from seeds by chemical or mechanical methods or the use of solvent extraction or re-esterification methods, and those mixed with oils from other sources.” thus, the addition of herbs, spices or other fruits to an evoo generates a product that cannot be called “extra virgin olive oil,” but can be defined as flavoured olive oil (foo) (baiano et al., 2010). these foos can be characterised by improved nutritional values, enriched sensory characteristics and increased shelf-life. recently, several foos have been introduced into the market (issaoui et  al., 2016). capsicum genus comprises 30 species, however, only five (c. baccatum, c. annuum, c. frutescens, c. chinense and c. pubescens) are those mainly cultivated (tripodi and kumar, 2019). capsicum phytochemicals include phenolics, carotenoids, capsaicinoids and other metabolites (wahyuni et al., 2013). several of these compounds are well-known as antioxidants accounting for the traditional use of peppers as food preserving agents from italian journal of food science, 2021; 33 (1): 61–72 mailto:monica_rosa.loizzo@unical.it 62 italian journal of food science, 2021; 33 (1) plastina p et al. materials, drying process and enrichment procedure dolce di rossano and roggianella monovarietal evoos were supplied in november 2019 by frantoio meringolo, corigliano calabro (cosenza, italy). all samples have accomplished the uni10939, 2001 certification. evoos were stored in green glass bottles without headspace before analysis. fruits of c. baccatum aji angelo and bishop crown cultivars were obtained from miceli s.r.l. farm (scalea, cosenza, italy) that provides its authentication. fruits were picked up at the maturity stage, defined by a visual colour change and size measurement. before analyses, fruits were examined for integrity and absence of insect contamination, devoid of peduncles and seeds and cut into small pieces. to obtain dried peppers, fruits were sun-dried at 35°c for 2 weeks. both fresh and dried peppers were grounded and the powders (50 mg) were added to 5 g of evoos and stirred to obtain foos that were left for 30 days in the infusion. after that, foos were filtered to remove the powder and analysed. samples were stored at −20°c until analysis. evoos and foos quality parameters extra virgin olive oil quality parameters (free acidity, peroxide index, uv light absorption sterol and fatty acid profiles) were analysed according to the ec regulation methods (eu, 2016). to compare oxidative stability of foos and evoos, rancimat equipment (metrohm, basel, switzerland) at 98°c and with airflow of 10–12 l/h was used, according to a known protocol (firestone, 1993). evoo phenolic extract and high-performance liquid chromatography (hplc) analysis the evoo phenolic extract was obtained by using a previously described procedure (montedoro et al., 1992). the obtained extract was dissolved in 1 ml of methanol/water (1:1, v/v) and after filtration injected into a high-performance liquid chromatography (hplc) instrument, knauer instrument (asi – advanced scientific instruments, berlin, germany), coupled with uv-vis detector of waters company (model waters 486 tunable), as previously described (sicari et al., 2010). the mobile phase was constituted by water/acetic acid (98:2, v/v) (a) and methanol/acetonitrile (1:1, v/v) (b), with a flow rate of 1 ml/min. data from three independent experiments were acquired with clarity software (chromatography station for windows) and expressed as mean ± sd. total phenolic, flavonoid, carotenoid and chlorophylls contents total phenolic content (tpc) was determined as previously described by gao et al. (2000). the absorbance was ancient times. in the last decades, our research group investigated the chemical composition and bioactivity of many food plants including different capsicum species collected in calabria (southern italy) (fazio et al., 2018; loizzo et al., 2015, 2017; menichini et al., 2009; tundis et al., 2013). this study aimed to evaluate the effect of capsicum baccatum aji angelo and bishop crown cultivars on the quality parameters of foos obtained by the addition of pepper powder to dolce di rossano and roggianella monovarietal evoos. for this purpose: (i) total phenolic, flavonoid and carotenoid contents, as well as vitamins c and e were assessed in both evoos, pepper extracts and foos; (ii) fatty acids, sterols, phenolics and capsaicinoids were also quantified; (iii) the protective effect of pepper on foo oxidative stability; and (iv) the antioxidant potential was investigated by different in vitro methods. materials and methods chemicals and reagents all reagents were purchased from sigma-aldrich s.p.a. (milano, italy), whereas analytical-grade solvents were obtained from vwr international s.r.l. (milan, italy). the following standards were used: fame mixes-analytical standards (crm47885); cholesterol (pubchem cid: 5997); 24-methylene-cholesterol (pubchem cid: 92113); campesterol (pubchem cid: 173183); campestanol (pubchem cid: 119394); stigmasterol (pubchem cid: 5280794); δ7-campesterol (pubchem cid: 5283646); clerosterol (pubchem cid: 5283638); clerosterol (pubchem cid: 5283638); β-sitosterol (pubchem cid: 222284); sitostanol (pubchem cid: 6743); δ5-avenasterol (pubchem cid: 5281326); δ5,24-stigmastadienol (pubchem cid: 286499); δ7-stigmastenol (pubchem cid: 3080632); δ7-avenasterol (pubchem cid: 12795736); erythrodiol (pubchem cid: 101761); uvaol (pubchem cid: 92802); 4-hydroxybenzoic acid (pubchem cid: 135); p-coumaric acid (pubchem cid: 637542); o-coumaric acid (pubchem cid: 637540); ferulic acid (pubchem cid: 445858); hydroxytyrosol (pubchem cid: 82755); tyrosol (pubchem cid: 10393); (+) pinoresinol (pubchem cid: 73399); 3,4-dhpea-eda (pubchem cid: 18684078); p-hpea-eda (pubchem cid: 11652416); hydroxytyrosol acetate (pubchem cid: 155240); apigenin (pubchem cid: 5280443); luteolin (pubchem cid: 5280445); chlorogenic acid (pubchem cid: 1794427); quercetin (pubchem cid: 5280343); beta-carotene (pubchem cid: 5280489); vitamin c (pubchem cid: 54670067); vitamin e (pubchem cid: 14985); capsaicin (pubchem cid: 1548943); dihydrocapsaicin (pubchem cid: 107982). italian journal of food science, 2021; 33 (1) 63 addition of capsicum baccatum to calabrian monovarietal eoos leads to foos with enhanced oxidative stability evaluation of protection of lipid peroxidation in the β-carotene bleaching (b-cb) test, a mixture of linoleic acid (la), β-carotene and tween 20 was prepared and the resulting emulsion was mixed with samples (loizzo et al., 2019). the absorbance was read after 30 min of incubation at 470 nm. propyl gallate was used as a positive control. ferric reducing activity power (frap) assay both pepper extract and evoo phenolic fraction (at a concentration of 2.5 mg/ml) were tested. also, the ability of samples to protect the iron from redox reaction is evaluated (loizzo et al., 2019). butylated hydroxytoluene (bht) was used as control. calculation of relative antioxidant capacity index (raci) the relative antioxidant capacity index (raci) was used to establish antioxidant rank of samples (sun and tanumihardjo, 2007). statistical analysis data were obtained from three different experiments (n = 3) and expressed as means ± standard deviation (sd). prism graphpad prism version 4.0 for windows (graphpad software, san diego, ca, usa) was used to calculate the concentration that yielded a 50% inhibition (ic50) value as a result of the concentration-response curve. pearson’s correlation coefficient (r) was calculated using microsoft excel 2010 software. anova followed by dunnett’s test (α = 0.05) was applied to evaluate the differences between data and positive control result in biological assays, however, tukey’s test was used to determine any significant difference among all treatments at different levels *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. principal component analysis (pca) is the most common form of factor analysis and is categorised as a multivariate statistical technique. it is used to analyse interrelationships among a large number of variables. for the evaluation of the results of the chemical (tfc, tpc, tcc, dpph, abts, β-carotene-bleaching, frap and induction time), analyses of roggianella and dolce di rossano evoos and foos, pca was applied. results and discussion dolce di rossano and roggianella evoo quality parameters based on the results reported in table 1 and according to ec regulation, both dolce di rossano and roggianella evoos could be classified as “extra virgin” (ec, 2003; eec,  1991). read at 765 nm. for the total flavonoid content (tfc), the method reported by yoo et al. (2008) was applied. absorbance was read at 510 nm. total carotenoid content (tcc) and chlorophyll content were determined following a previously reported procedure (mínguez-mosquera et al., 1900). according to this method, the index k670 assesses the total chlorophylls and their derivatives, whereas the index k470 assesses tcc. capsicum baccatum extraction procedure both c. baccatum bishop crown and aji angelo cultivars (250 g) were subjected to maceration in ethanol (500 ml) for three times. extracts were stored until analysis at −20°c in amber bottles. total phenolic, flavonoid and carotenoid contents total phenolic content, tfc and tcc were determined as described for evoo samples in the previous section. tpc was expressed as mg chlorogenic acid (ca) equivalents/100 g dry weight (dw). tfc was expressed as mg quercetin equivalents (qe)/100 g dw. tcc was expressed as mg β-carotene (βc) equivalents/100 g dw (menichini et al., 2009). capsaicin and dihydrocapsaicin content capsaicin and dihydrocapsaicin contents were determined according to a previously reported method (menichini et al., 2009), by using a gc17a gas chromatograph (gc) (shimadzu, milan, italy) equipped with a flame ionization detector (fid). analyses were performed in isothermal conditions at 210°c. the capsaicinoids content was done and data are expressed as mean ± sd in µg/g dw. vitamin c and e content the content of vitamin c in pepper samples was determined according to a previously reported method (klein and perry, 1982). for determination of vitamin e content gas chromatography-mass spectrometry (gc-ms) analyses were performed (loizzo et al., 2015). vitamin content was expressed as mg/100 g dw. antioxidant activity of evoos and pepper extracts radical scavenging activity by abts and dpph tests both 2,2-diphenyl-1-picrylhydrazyl (dpph) and 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic) acid (abts) tests were applied to examine the radical scavenging activity of capsicum extracts and evoo phenolic fraction using the procedure previously described by leporini et al. (2018). in both cases, ascorbic acid was used as a positive control. 64 italian journal of food science, 2021; 33 (1) plastina p et al. table 1. chemical and qualitative parameters of monovarietal dolce di rossano and roggianella evoos produced in calabria (italy) during 2018–2019 season. quality parameter evoo significance dolce di rossano roggianella free acidity (%) 0.79 ± 0.02a 0.73 ± 0.01b ** peroxide value (meqo 2 /kg) 15 ± 1a 13 ± 1b ** k232 1.9 ± 0.9a 1.8 ± 0.8b ** k270 0.15 ± 0.02a 0.15 ± 0.02a n.s. δk 0.001 ± 0.00a 0.001 ± 0.00a n.s. tpc (ppm) 73 ± 2b 537 ± 6a ** tfc (ppm) 18 ± 1a 15 ± 1b ** tcc (ppm) 3 ± 2a 2 ± 3b n.s. chlorophyll (ppm) 4 ± 2a 4 ± 3a n.s. tpc: total phenolic content; tfc: total flavonoid content; tcc: total carotenoid content. ** significance at p < 0.01, n.s. not significant. results followed by different letters in the same row are significantly different by tukey’s multiple range test. our values agreed with those reported by lavelli and co-workers for evoo pendolino, leccino, moraiolo and taggiasca cultivars (lavelli and bondesan, 2005). a significant difference (p < 0.01) was observed between the phytochemicals content (tpc, tfc and tcc) of two investigated oils. in particular, roggianella evoo showed a tpc value that was seven-time higher than that found for dolce di rossano (table 1). the tpc value is in line with those found by sicari et al. (2017) for roggianella evoo from reggio calabria whereas lower values were recorded for calabrian ottobratica and carolea evoo, respectively (piscopo et al., 2016). a statistically significant difference was also observed in tfc with values of 18 and 15 ppm for dolce di rossano and roggianella evoos, respectively. the pigment content of evoos is an important quality parameter since consumers directly evaluate it based on their colour (gargouri et al., 2013). moreover, their content is strictly related to evoo stability. as reported in table 1, tcc and chlorophyll content were not statistically different for dolce di rossano and roggianella evoos. this tcc content is similar to that reported by borrello and domenici (2019) for tuscan evoos from frantoio, leccino, moraiolo and pendolino cultivars. previously, tuberoso et al. (2016) found chlorophylls values in the range from 6.5 to 10.5 ppm, and from 20.9 to 47.6 ppm with respect to tcc for sardinian evoos semidana and tonda di cagliari, respectively. evoo chemical profile as expected, oleic acid (oa, ω-9) was the main monounsaturated fatty acid (mufa) whereas palmitic acid (pa) was the major saturated fatty acid (sfa) in both evoos (table 2). a statistical difference was observed in oa/ la (ω-6) ratio (p < 0.01), as roggianella evoo showed the highest oa/la ratio, an indicator of the evoo stability (alvarruiz et al., 2003). our data are in line with data found for roggianella evoo from reggio calabria province (sicari et al., 2010). more recently, several italian monovarietal evoos including leccino, frantoio, dolce agogia and moraiolo were investigated by blasi et al. (2019). the percentages of oa and pa were quite similar to those found in our samples. values from 71.84 to 73.20% and from 16.09 to 19.30% were recorded for oa and pa, respectively, in sari hasebi and halhali green evoos from turkey (yorulmaz and konuskan, 2017). in general, the sterolic composition of the two investigated evoos was significantly different (p < 0.01). as expected, β-sitosterol represented the most abundant phytosterol (table 3). two-time higher content of δ5-avenasterol was found in roggianella evoo with respect to dolce di rossano. a statistically significant difference in the campesterol/stigmasterol index was observed between investigated evoos. both evoos showed values of campesterol lower than 4%, the maximum limit established by european regulations and by the ioc (eu, 2011; ioc, 2009). our data were in agreement with those reported for turkish sari hasebi and halhali green evoos, that were found in the range from 80.72 to 87.81% of β-sitosterol, respectively, followed by δ5-avenasterol (3.34–5.29% for halhali and sari hasebi, respectively) (yorulmaz and konuskan, 2017). the total sterol content was 1698 and 1971 mg/kg for dolce di rossano and roggianella, respectively, i.e. much higher than 1000 mg/kg indicated by the european regulations and by the ioc as a minimum sterol content for an evoo (eu, 2011; ioc, 2009). italian journal of food science, 2021; 33 (1) 65 addition of capsicum baccatum to calabrian monovarietal eoos leads to foos with enhanced oxidative stability table 2. fatty acid composition of monovarietal dolce di rossano and roggianella evoos produced in calabria (italy) during 2018–2019 season. fatty acid evoo significance dolce di rossano roggianella myristic acid (c14:0) 0.01 ± 0.00a 0.01 ± 0.00a n.s. palmitic acid (pa, c16:0) 14 ± 2a 13 ± 2b ** palmitoleic acid (c16:1) 1.3 ± 0.1a 0.9 ± 0.1b ** margaric acid (c17:0) 0.02 ± 0.00a 0.02 ± 0.00a n.s. heptadecenoic acid (c17:1) 0.04 ± 0.00a 0.04 ± 0.00a n.s. stearic acid (sa, c18:0) 1.4 ± 0.2b 1.5 ± 0.1a ** oleic acid (oa, c18:1) 72 ± 4b 79 ± 6a ** linoleic acid (la, c18:2) 8.4 ± 0.9a 6.6 ± 0.8b ** α-linolenic acid (ala, c18:3) 0.40 ± 0.02a 0.40 ± 0.02a n.s. arachidic acid (c20:0) 0.90 ± 0.03a 0.80 ± 0.03b * gadoleic acid (c20:1) 0.20 ± 0.01b 0.30 ± 0.01a ** behenic acid (c22:0) 0.10 ± 0.01a 0.10 ± 0.01a n.s. oa/la 8.6b 12.0a ** ∑sfa 16.43a 15.43b ** ∑mufa 73.54b 80.60a ** ∑pufa 8.8a 7.0b ** mufa/pufa 8.36b 11.51a ** sfa: saturated fatty acid; pufa: polyunsaturated fatty acids; mufa: monounsaturated fatty acids. **significance at p < 0.01, * significance at p < 0.05, n.s. not significant. results followed by different letters in the same row are significantly different by tukey’s multiple range test. table 3. sterol composition of dolce di rossano and roggianella evoos produced in calabria (italy) during 2018–2019 season sterol evoo significance dolce di rossano roggianella cholesterol (%) 0.12 ± 0.01a 0.07 ± 0.02b ** 24-methylene-cholesterol (%) 0.07 ± 0.01b 0.29 ± 0.01a ** campesterol (%) 2.23 ± 0.03b 2.67 ± 0.05a ** campestanol (%) 0.16 ± 0.01a 0.11 ± 0.01b ** stigmasterol (%) 0.80 ± 0.02b 1.81 ± 0.04a ** δ7-campesterol (%) 0.08 ± 0.01a 0.08 ± 0.01a n.s. clerosterol (%) 0.87 ± 0.01b 0.98 ± 0.02a ** β-sitosterol (%) 88.21 ± 0.08a 81.71 ± 0.06b ** sitostanol (%) 0.98 ± 0.03a 0.63 ± 0.02b ** δ5-avenasterol (%) 5.01 ± 0.05b 10.3 ± 0.8a ** δ5,24-stigmastadienol (%) 0.62 ± 0.04a 0.51 ± 0.01b ** δ7-stigmastenol (%) 0.37 ± 0.01a 0.39 ± 0.05a n.s. δ7-avenasterol (%) 0.51 ± 0.01a 0.43 ± 0.01b ** apparent β-sitosterol (%) 95.68a 94.16b ** campesterol/stigmasterol 2.79a 1.48b ** β-sitosterol/δ5-avenasterol total sterols (mg/kg) 17.61a 1698b 7.89b 1971a ** erythrodiol (%) 1.52 ± 0.02a 0.92 ± 0.02b ** uvaol (%) 0.79 ± 0.01a 0.16 ± 0.01b ** all the sterols as well as erythrodiol and uvaol are expressed as percentage of the total sterol content. ** significance at p < 0.01, * significance at p < 0.05, n.s. not significant. results followed by different letters in the same row are significantly different by tukey’s multiple range test. 66 italian journal of food science, 2021; 33 (1) plastina p et al. table 4. phenolic composition of monovarietal dolce di rossano and roggianella evoos produced in calabria (italy) during 2018–2019 season. compound amount in evoo (ppm) significance dolce di rossano roggianella 4-hydroxybenzoic acid 0.21 ± 0.01b 0.6 ± 0.9a ** p-coumaric acid 0.8 ± 0.2a 0.20 ± 0.02b ** o-coumaric acid 0.15 ± 0.01b 0.24 ± 0.02a ** ferulic acid 0.27 ± 0.02a 0.14 ± 0.01b ** hydroxytyrosol 3.12 ± 0.03a 2.9 ± 0.8b ** tyrosol 4.04 ± 0.04b 6.2 ± 0.5a ** (+) pinoresinol 8 ± 2b 22 ± 2a ** (+) 1-acetoxypinoresinol 14 ± 4b 52 ± 7a ** 3,4-dhpea-eda 17 ± 2b 48 ± 5a ** p-hpea-eda 5.1 ± 0.9b 18 ± 1a ** hydroxytyrosol acetate 9 ± 1b 21 ± 2a ** 3,4-dhpea-ea 3.6 ± 0.3b 8.26 ± 0.09a ** p-hpea-ea 0.87 ± 0.08b 1.15 ± 0.01a ** apigenin 0.00 ± 0.00b 0.79 ± 0.01a ** luteolin 0.48 ± 0.02b 1.12 ± 0.08a ** ∑ identified phenolics 66.64b 178.52a ** 3,4-dhpea-eda: oleacein, p-hpea-eda: oleocanthal, 3,4-dhpea-ea: oleuropein aglycone, p-hpea-ea: ligstroside aglycone. ** significance at p < 0.01. results followed by different letters in the same row are significantly different by tukey’s multiple range test. the phenolic composition of both dolce di rossano and roggianella evoos is reported in table 4. in general, the total amount of identified phenolic compounds in the two evoos was significantly different (p < 0.01). as expected, secoiridoids and lignans were the most abundant compounds. roggianella evoo resulted to be richer in phenolic compounds than dolce di rossano. 1-acetoxypinoresinol followed by 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde (3,4-dhpea-eda) were the two most abundant compounds in both evoos. the amounts of these compounds in roggianella were 3.7and 2.8-times higher than those in dolce di rossano. roggianella evoo was also characterised by a significant content of pinoresinol and hydroxytyrosol acetate. dolce di rossano evoo variety showed a slightly higher tyrosol content than roggianella (table 4). roggianella evoo showed a twotimes higher content of luteolin than dolce di rossano variety. among phenolic acids, 4-hydroxybenzoic acid was the main abundant acid in roggianella oil whereas p-coumaric acid was the most abundant in dolce di rossano evoo. our data were in line with those previously reported for evoos from roggianella cultivars (giuffrè and louadj, 2013). mean values of 48.54, 38.02 and 16.24 µg/g have been found for 3,4-dhpea-eda, 1-acetoxypinoresinol and hydroxytyrosol acetate, respectively. values in the range 21.11–76.80 µg/g were found for 3,4-dhpea-eda in grignano and leccino evoos (baiano et al.,  2009). the lower content of phenolic compounds has been found in sardinian evoos where 3,4-dhpea-eda ranged from 1.0 to 11.6 µg/g in tonda di cagliari and semidana, respectively (tuberoso et al., 2016). it is interesting to emphasise that 1-acetoxypinoresinol, one of the main phenolics identified in our calabria evoo, was not revealed in tonda di cagliari and found in a very low quantity in tonda di villacidro, semidana and bosana oils. however, bosana contained a high amount of tyrosol (22.5 ppm). a perusal analysis of literature evidenced a great variability in evoo phenolic profile due to several factors including geographical origin, genetic factors and environmental conditions. this is particularly true for hydroxytyrosol that was found in a high amount in colozzese evoo (16.1 ppm), whereas its amount ranged from 0.2 to 8.8 ppm in spina and oliva grossa samples (negro et al., 2019). with respect to apigenin and luteolin, a higher amount of both phenols were found by giuffrè et al. (2010) in roggianella evoo from reggio calabria during the season 2006. phytochemical content in c. baccatum cultivars fresh and dried c. baccatum bishop crown and aji angelo fruits were subjected to extraction by maceration. the highest extraction yields were obtained with dried fruits (table 5). italian journal of food science, 2021; 33 (1) 67 addition of capsicum baccatum to calabrian monovarietal eoos leads to foos with enhanced oxidative stability table 5. investigated capsicum baccatum cultivars. capsicum baccatum colour length (cm) width (cm) extraction yield (%) bishop crown fresh red 5–7 6–8 6.06 dried 29.92 aji angelo fresh red 5–7 6–8 6.06 dried 27.90 table 6. total phenolic, flavonoid, carotenoid, capsaicin and dihydrocapsaicin, vitamin c and vitamin e content in capsicum baccatum cultivars. content bishop crown aji angelo significance fresh dried fresh dried tpc1 6.80 ± 0.02c 67 ± 4a 7.4 ± 0.4c 63 ± 2b ** tfc2 1.40 ± 0.03c 10.90 ± 0.02b 1.10 ± 0.01c 11.9 ± 0.2a ** tcc3 2036 ± 8d 4785 ± 9a 2763 ± 7c 3388 ± 8b ** vitamin c4 3.90 ± 0.22b 5.2 ± 0.3a 3.40 ± 0.09c 4.0 ± 0.1b ** vitamin e4 5.1 ± 0.5b 5.3 ± 0.2b 5.2 ± 0.4b 5.5 ± 0.9a ** capsaicin5 44 ± 3c 125 ± 7b 19 ± 5d 175 ± 9a ** dihydrocapsaicin5 24 ± 4c 58 ± 6b 15 ± 1d 156 ± 7a ** 1 tpc: total phenolic content, expressed as mg of chlorogenic acid equivalents/100 g dried weight (dw), 2 tfc: total flavonoid content, expressed as mg of quercetin equivalents/100 g dw, 3 tcc: total carotenoid content, expressed as mg of β-carotene equivalents/100 g dw. 4 expressed as mg/g dw, 5 expressed as µg/g dw. results followed by different letters in the same row are significantly different (p < 0.01, **) by tukey’s multiple range test. dried peppers contained the highest amounts of all analysed phytochemicals. in particular, bishop crown showed the highest tpc and tcc values whereas aji angelo had the highest tfc (table 6). the same trend was also observed in the extracts obtained from fresh samples. as reported in table 6, dried pepper extracts were rich in both capsaicin and dihydrocapsaicin capsaicinoids with values ranging from 58 to 125 µg/g dw for bishop crown and aji angelo dried pepper extract, respectively. bishop crown dried pepper showed the highest vitamin c content while vitamin e was particularly abundant in aji angelo dried pepper extract. antioxidant activity despite it has been previously reported that antioxidant ability determined by dpph and abts in vitro assays can significantly differ due to different mechanisms of inactivation of the radicals (antolovich et al., 2002; plastina et al., 2018), we observed similar trends for the investigated samples. promising radical scavenging activity was found for roggianella evoo phenolic extracts in both applied assays (table 7). these values are better than that reported for different calabrian monovarietal evoos by sicari (2017) for sinopolese and roggianella and leporini and co-workers (2018) for frantoio evoo (average value of 117.2 and 131.9 µg/ml for abts and dpph, respectively). previously, negro and co-workers found ic50 values in the range 91–160 g/oil for dpph radical scavenging activity for oliva grossa and spina, respectively (negro et al., 2019). the radical scavenging potential of evoos phenolic fraction was positively correlated to tfc and tcc with r values of 1.0. among investigated peppers, bishop crown resulted to be the most active with ic50 values of 148 and 167 µg/ml for dpph and abts tests, respectively. no significant differences between bishop crown and aji angelo dried pepper extracts were evidenced in the protection from lipid peroxidation. promising ferric reducing ability was observed for both dolce di rossano and roggianella evoo, respectively. previously, loizzo et al. (2017) investigated the antioxidant activity of c. annuum and c.  chinense fresh and processed peppers and found the highest dpph radical scavenging potential with effix fresh pepper sample (ic50 value of 3.9 µg/ml). a high radical scavenging potential against abts has been previously observed for dried c. annuum cv pellegrino and idealino samples with ic50 values of 45.2 and 45.7 μg/ml, respectively (loizzo et al., 2017). great variability in antioxidant potential was observed with oleoresin obtained from different varieties of fresh peppers including 68 italian journal of food science, 2021; 33 (1) plastina p et al. table 7. antioxidant activities of dolce di rossano and roggianella evoos and c. baccatum extracts. sample abts1 dpph1 β-carotene bleaching1 frap (µm fe(ii)/g) raci values evoo phenolic extracts dolce di rossano 48 ± 3**** 54 ± 2**** 205 ± 9**** 79 ± 3 71 roggianella 36 ± 1**** 30 ± 1**** 127 ± 5**** 57 ± 3* –71 c. baccatum extracts bishop crown fresh 170 ± 2**** 162 ± 3**** 33 ± 3**** 15 ± 1**** 0.04 dried 167 ± 3**** 148 ± 3**** 21 ± 1*** 14 ± 2**** –0.47 aji angelo fresh 453 ± 4**** 186 ± 2**** 77 ± 3**** 9.1 ± 0.4**** 0.86 dried 256 ± 4**** 157 ± 3**** 20 ± 1*** 9.2 ± 0.8**** –0.43 positive controls ascorbic acid 1.71 ± 0.03 5.0 ± 0.8 propyl gallate 1.00 ± 0.01 bht 63 ± 4 1 ic 50 value (µg/ml). data are expressed as means ± s.d. (n= 3). dpph radical scavenging activity assay, antioxidant capacity determined by radical cation (abts+), β-carotene bleaching test, ferric reducing antioxidant power (frap). ascorbic acid, bht and propyl gallate were used as positive control in antioxidant tests. differences within and between groups were evaluated by one-way anova followed by a multicomparison dunnett’s test (α= 0.05): ****p < 0.0001, ***p < 0.001, compared with the positive controls. c.  annuum and c. chinense. the raci approach was applied to estimate the rank of antioxidant activity for evoos and pepper samples. roggianella evoo resulted as the most active one whereas bishop crown dried pepper extract exhibited the highest antioxidant potential among investigated pepper extracts (table 7). protective effect of pepper extracts against oil oxidation rancimat apparatus was used to investigate and compare the stability of evoos and the corresponding foos (figure 1). in this method, the oxidation is induced by the passage of constant airflow through the oils that is kept under constant temperature. the volatile products of the reaction are collected in deionised water and are measured through the electric conductivity. during the development of the reaction, due to an increase of the conductivity, a curve is drawn from which the induction period is inferred. stability was expressed as oxidation induction time (expressed in hours). roggianella evoo showed a greater induction time than dolce di rossano sample (39.6 versus 19.2 h, respectively). this is probably due to the higher amounts of tpc and identified phenolic compounds found in roggianella evoo. in line with our data, the following trend for resistance to oxidation has been previously observed for calabrian evoos: roggianella > ottobratica > sinopolese (sicari, 2017) and carolea > ottobratica > sinopolese > grossa di gerace (piscopo et al., 2016). remarkably, all foos enriched with pepper extracts had a prolonged induction time with respect to the corresponding evoo. among them, foos obtained by the addition of bishop crown dried extract to roggianella evoo showed the highest induction time of 44.9 h. this possibly depends on the high amount of bioactive phytochemicals, with the only exception of vitamin e and dihydrocapsaicin, found in bishop crown dried extract. moreover, by considering the protection factor quotient (pf), bishop crown fresh pepper-foo had a pf of 5.7 with respect to dolce di rossano evoo followed by foo obtained by the addition of the dried extract from the same pepper in roggianella evoo (pf = 5.3). lower values were observed for foos enriched with aji angelo extracts with pf values ranging from 1.05 to 3.03. it has been previously reported that foos obtained by the addition of c. frutescens extract did not show any protective effect against the oxidation process (gouveia et al., 2006). this was probably due to the low content of capsaicinoids. by contrary, a protective effect towards oxidation was observed when dried c. annuum pepper was added to evoo by infusion (10–20% w/w) up to 30 days (caporaso et al., 2013). the effect of the addition of spices or their extract/essential oils on oil oxidative stability gained attention by researchers working in the food area. the infusion of dried garlic, oregano and rosemary and hot pepper to dauno monovarietal evoo led to foos in which the formation of primary oxidation products was reduced without any modification on acidity parameter and secondary oxidation compounds (gambacorta et al., 2007). more recently, ayadi italian journal of food science, 2021; 33 (1) 69 addition of capsicum baccatum to calabrian monovarietal eoos leads to foos with enhanced oxidative stability figure 1. (a) induction time and (b) protection factor quotient of dolce di rossano (dr) and roggianella (r) evoos and corresponding foos enriched with c. baccatum cv bishop crown (bc) and aji angelo (aa) fresh (f) and dried pepper (d) extracts. drbcf drbcd draaf draad dr rbcf rbcd raaf raad r 0 10 20 induction time (h) 30 40 50 (a) (b) raad raaf rbcd rbcf draad draaf drbcd drbcf 0 1 2 3 protection factor quotient (h) 4 5 6 7 et al. (2009) investigated the quality parameters of foos obtained by the infusion of 5% (w/w) of mediterranean spices to chemlali evoo. although the addition of spices has led to a slight increase in free acidity, an improvement of their thermal resistance and stability with this hierarchy: rosemary > thyme > lemon > basil ≥ evoo control was also observed. this could be caused by the transfer of antioxidant compounds from spices to oil during the infusion. more recently, ammar et al. (2017) evaluated the effect on quality parameters including oxidative stability of the addition of opuntia ficus indica flower extracts to “chemlali” evoo that results in foo1 and foo2 (5 and 15% (w/w), respectively). foo1 was richer in phenolics by 3.4% than foo2. in disagreement with our data, this addition did not improve oxidative stability. also, the induction time obtained by the rancimat method showed values of 2.73 and 2.42 h for foo1 and foo2, respectively, in comparison to the control evoo (2.83 h). the authors speculated that this may be due to a diffusion of undesirable compounds during the flower maceration of olive oil. these compounds can take part in reactions that negatively interfere with oxidative stability (ammar et al., 2017). the unfavourable effect of evoo aromatisation on oxidative stability was also observed by sena-moreno and co-workers who proposed a new evoo aromatisation method. in this study, liquid–liquid extractions of saffron aqueous extract in “arbequina” evoo was done. the sample obtained from the first liquid– liquid extraction was so1, whereas so2, so3 and so4 were those obtained from the second, third and fourth 70 italian journal of food science, 2021; 33 (1) plastina p et al. (fresh and dried), samples and the bioactive attributes associated with foos. the pca model showed that foos were characterised by a higher induction time than the corresponding evoos. conclusions flavoured olive oils gained attention by consumers not only as a new dressing for meat, fish or salad but also for the role of spice phytochemicals on human health. in this context, the protective effect of c. baccatum peppers cultivars on the oxidative stability of foos obtained by the addition of pepper to two calabrian evoos was evaluated. roggianella evoo, characterised by a high phytochemicals content showed a longer induction time. the addition of c. baccatum bishop crown to roggianella evoo led to a foo with an increased induction time with respect to the corresponding evoo. promising results were also obtained with foo resulted by the addition of bishop crown fresh pepper to dolce di rossano. therefore, the addition of c. baccatum bishop crown peppers was able to enhance the oxidative stability of oils, regardless of the quality of the starting evoo. funding this research was funded by por calabria fesrfse 2014–2020, research project “glasoil—glassa extraction, respectively. so1 showed the highest safranal concentration. it is worthy of mention that in the first seven months, foos were characterised by a reduction of quality parameters including oxidative stability. after that foos parameters were still comparable to those of evoo (sena-moreno et al., 2018). the impact of enrichment with phenolic compounds from the olive cake and dried thyme on the quality parameters phenolic composition, oxidative stability and antioxidant activity of “arbequina” evoo was investigated by rubió et al. (2012). flavonoids from thyme were characterised by a higher transference ratio (average 89.7%) with respect to secoiridoids from an olive cake (average ratio of 35.3). in each case, all resulted foos were more resistant to oxidation with values of induction time of ~8 and ~20 h for control and enriched oil, respectively. principal component analysis the projections of the observations on the first two principal component axes are shown in figure 2. the accessions are distributed on the factor plane. these two coordinates represent 87.77% of the total variance (pc1 explained 56.17% of total variation, while pc2 explained 31.59% of total variation). the first component (pc1) was positively correlated with tpc, tfc, frap, β-carotene bleaching, raci and itdr. the second component (pc2) was positively correlated with tpc, itdr and itr. figure 2 shows the space of oils and hot pepper samples 1.0 0.5 0.0 -0.5 -1.0 -1.0 -0.5 0.0 0.5 raci dr fraptfc tpc itdr r itr bcd bcf aaf dpph tcc abts aad pc1 – 56,17 p c 2 – 31 ,6 0 1.0 figure 2. principal component analysis plot based on bioactivity attributes of extra virgin olive oils (evoos) and corresponding flavoured olive oils (foos) enriched with c. baccatum pepper extracts. dr: dolce from rossano; bcf: bishop crown fresh; bcd: bishop crown dried; aaf: aji angelo fresh; aad: aji angelo dried; r: roggianella. italian journal of food science, 2021; 33 (1) 71 addition of capsicum baccatum to calabrian monovarietal eoos leads to foos with enhanced oxidative stability fazio, a., iacopetta, d., la torre, c., ceramella, j., muià, n., catalano, a., carocci, a. and sinicropi, m.s., 2018. finding solutions for agricultural wastes: antioxidant and antitumor properties of pomegranate akko peel extracts and βglucan recovery. food & function 9: 6618−6631. https://doi.org/10.1039/c8fo01394b firestone, d., 1993. official methods and recommended practices of the american oil chemists’ society. 4th ed., american oil chemists’ society, champaign, 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science, 2023; 35 (1): 57–71 issn 1120-1770 online, doi 10.15586/ijfs.v35i1.2286 57 p u b l i c a t i o n s codon effect of region of cultivation, tree age, and harvest time on the quality of lebanese virgin olive oil antonio el chami1, paola conte1, georges hassoun2, antonio piga1* 1department of agricultural sciences, university of sassari, sassari, italy; 2environmental and natural resources department, lebanese university, dekwaneh, lebanon *corresponding author: antonio piga, dipartimento di agraria, università degli studi di sassari, viale italia 39/a, 07100 sassari, italy. email: pigaa@uniss.it received: 1 october 2022; accepted: 3 february 2023; published: 26 february 2023 © 2023 codon publications open access paper abstract in lebanon, olive oil plays an essential role at both economic and social levels. however, factors influencing its quality are rarely addressed. this preliminary study is the first analysis to show a comparison between ancient and adult virgin olive oil trees in lebanon. analysis of pedo-climatic conditions and physicochemical parameters of virgin oil samples taken from ancient and adult trees from three lebanese regions (bechmizine, kfaraaka, and kawkaba) and at two different harvest periods showed that these parameters differed significantly among cultivation regions, while the tree age and harvest time had a lower effect. we observed that the oil obtained from adult trees of kawkaba region during the first harvest period (green to reddish stage of fruit maturity) had the best quality, compared to all other samples. oil produced from these trees showed the highest polyphenol content, a relatively higher composition of tocopherols, oleic acid, monounsaturated fatty acids, and trans fatty acids c18: 1–c18: 2, with relatively low acidity and peroxide values. it was concluded that the quality of virgin olive oil was associated with its chemical composition, and was the result of a complex interaction between several environmental factors associated with the area of cultivation, tree age, and harvest time. keywords: adult trees, ancient trees, harvest date, lebanon, region of cultivation, virgin olive oil introduction olive oil is an integral part of cultural and culinary heritage of mediterranean countries, as it is the oldest crop in their history and their main edible oil, while other countries, such as the united states, chile, and new zealand, have made it a valuable commodity (aparicio and harwood, 2013). olive oil is mainly appreciated for its characteristic taste and therapeutic, dietary, and nutritional values (sotiroudis et al., 2003). composition of olive oil is closely correlated with its production. the quality and quantity of olive oil is influenced by different interrelated factors, leading to a dynamic multivariate structure (inglese et al., 2011). although the genotype is a key factor (ripa et al., 2008), olive trees are subjected to environmental factors (geographic locations, soil type, and soil water content), agronomic practices (crop load, irrigation, and pruning), and climatic conditions (temperature, rainfall, and humidity), all playing crucial roles in determining oil content and composition. in addition, age of tree (chtourou bouchaala et al., 2014, 2017; rouas et al., 2016), and especially oil extraction method (bendini et al., 2012; garcía and jesús, 2012) and storage conditions (ouedrhiri et al., 2017), plays an important role in determining the quantity of some olive oil components. lebanon is historically known for olive cultivation that dates back to the phoenician period. olive trees are on average 150 years old, and centennial trees are distributed in many areas from the north to the south under mailto:pigaa@uniss.it 58 italian journal of food science, 2023; 35 (1) el chami a et al. (21-0-0) in spring (mid-february to first week of march). a total of 5 kg of olives from each tree age in each region were picked at two harvesting periods: the first was at veraison (ripening) stage (ripening index 2 to 3), as it was proved to obtain the best quality of olive oil (bellincontro et al., 2012; chtourou bouchaala et al., 2014; gutiérrez et  al., 1999; inglese et al., 2011), and the second was on harvest date chosen by olive grower of each orchard (table 1). only healthy fruits without any kind of infection or physical damage were harvested manually, placed in a plastic box, and stored in a portable igloo isulated box for transportation to laboratory for processing. the weather data for bechmizzine “b” and kfaraaka “l” regions were obtained online from the website (weatherlink.com, private weather stations) and for kawkaba region from the lebanese agricultural research institute (lari) through their weather station located in hasbaya. soil from each of the selected orchards was analyzed. using a sheathed auger, soil sample was taken randomly at five points under trees from a depth of 30–40 cm. all soil samples were oven-dried at 40ºc for 48 h, ground, and sieved through a 2-mm aperture to remove pebbles– roots–leaves etc. the analyses were performed according to the official methods indicated by the d.m. no. 79 of 11/05/1992 and d.m. no. 185 of 13/09/1999 and subsequent amendments approved by italy’s ministry for agricultural and forestry policies (1999). oil extraction for oil extraction, 5 kg of healthy olives were chosen from each tree age group alone. the oil was extracted within 24 h of olive harvesting using a small-size abencor mill (mc2; ingenieria y sistemas, seville, spain), simulating commercial oil extraction systems. the olives were first washed with water and then crushed in a hammer mill (abencor mm-100, ingenieria y sistemas, seville, spain) equipped with a 4-mm sieve. the obtained paste was then processed using a malaxer (abencor tb-100, ingenieria y sistemas, seville, spain) (30 min at 25ºc) and centrifuged (abencor, cf-100, ingenieria y sistemas, seville, spain) at 3500 rpm for 2 min. the oil was poured different pedo-climatic conditions (chalak et al., 2011). olive fruits are harvested for oil production from adult and ancient olive trees distributed all over the country in mid-october in coastal areas, and from midoctober to mid-november, at higher altitudes, after the first rains (chehade et al., 2012). several agricultural practices applied by olive farmers, such as late harvesting period, managing the harvest by beating tree shoots using wood sticks, collecting of damaged olive fruits from the ground and mixing them with healthy fruits, transporting of olive fruits into plastic bags, and the long time spent prior to processing, decreased the quality of olive oil produced in lebanon (consultation and research institute and acted lebanon, 2018). although few studies have investigated the quality of olive oil produced from lebanese olive cultivars (el  riachy, 2019) as well as the effect of geographical origin (agro-climatic conditions) and harvest time (ripening stage) (dib et al., 2020; el riachy, 2018; merchak, et al., 2017), no study has classified and characterized the influence of tree age on the quality of olive oil. hence, in the present study, effects of age of olive tree (ancient and adult), harvest period (two harvest periods were chosen), and region of cultivation were investigated to find the best growth area and conditions for obtaining high virgin olive oil quality in lebanon. materials and methods plant material the study was conducted during the growing season 2018–2019. two groups of tree age from “baladi” cultivar were chosen: ancient “an” (trees aged more than 150 years old) and adult “ad” (trees aged between 80 and 100 years) from three different lebanese regions, namely bechmizzine “b,” kfaraaka “l” (north of lebanon), and kawkaba “k” (south of lebanon). the choice of these regions was based on their characterization by different microclimates and the presence of two different tree ages in each region. all trees were rain-fed and fertilized twice a year: 20 kg of poultry manure in autumn (mainly after the harvest season) and 5 kg/tree of ammonium sulfate table 1. olive harvest dates for two harvest periods. sample name crop season date of 1st harvest ripening index (ri) date of 2nd harvest ripening index (ri) ban 2018–2019 26 sep 2.5 24 oct 4 bad 2018–2019 26 sep 2.4 24 oct 3.8 lan 2018–2019 02 oct 2.2 29 oct 3.5 lad 2018–2019 02 oct 2.3 29 oct 3.6 kan 2018–2019 25 sep 2.8 13 nov 4.5 kad 2018–2019 25 sep 2.3 17 oct 3.5 italian journal of food science, 2023; 35 (1) 59 chemical composition and environmental factors affecting quality of lebanese virgin olive oil for 3 s. subsequently, the mixed solution was agitated for 10 min using a magnetic stirrer. after agitation, the solution was centrifuged for 10 min at 4ºc and 6000 rpm. after that, the residue obtained was removed and centrifuged for 5 min at 4ºc and 9000 rpm. the obtained hydroalcoholic phase was filtered and collected using a 0.45-mm syringe filter. the extract was subjected to spectrophotometric determination of total polyphenols following singleton and rossi (1965) using the above-mentioned spectrophotometer. results were expressed as milligram of gallic acid per kilogram of oil. antioxidant activity a changed free radical 2,2-diphenyl, 1-picrylhydrazyl (dpph) approach, defined by atoui et al. (2005), was used to assess the antioxidant activity of olive oil. a dpph solution 0.25 g l-1 (0.0634 μmol ml−1) was prepared. the antioxidant activity was determined on the polyphenol extract with the dpph stable radical. under continuous stirring, 50 ml of extract was added to 2.8 ml of ddph solution. decrease in absorbance at 515  nm was recorded every 60 s until the plateau was reached (30 min) against a blank of methanol using the above mentioned spectrophotometer. results were expressed as percentage decrease in absorbance per milligram of oil when 0.17 μmol of dpph was available for reaction. fatty acids the content of fatty acids for olive oil samples was analyzed according to gerber method for milk and the gerber-van gulik method (international organization for standardization [iso], 1975). in brief, 1 g of olive oil was added to 0.4-ml ammonia (nh3) 25%, 2-ml ethyl alcohol (c2h6o) 95%, and 4-ml hexane (c6h14). samples were vortexed for 1 min and centrifuged at 3000 rpm, and the upper layer was collected. another extraction was executed, this time using ethyl alcohol 95% (1 ml) and hexane (5 ml); samples were also vortexed for 1 min and centrifuged at 3000 rpm and the upper layer was collected. a final extraction was done by adding 5 ml of hexane and the samples were vortexed for 1 min and centrifuged at 3000 rpm before obtaining the upper layer. the fatty acid methyl esters (fame) were prepared in conjunction with the fil-idf standard protocol (cantellops et al., 1999) with base-catalyzed transesterification. statistical analysis a principal component analysis (pca) was performed on all physicochemical parameters of oil to investigate the factor–among cultivation region, harvest time, and tree age—that mostly affected the oil parameters analyzed. therefore, to do a more in-depth evaluation of the into amber 225-ml glass bottles without head space and stored at 4ºc in dark until analysis. determination of oil content the oil content of the olive paste was determined by gravimetric analysis using a solvent automatic extractor (series 158 series; velp scientifica, usmate velate mb, italy). olive paste, 2 g, was stored at 4ºc until extraction, and dried for 24 h at 105ºc to determine oil content in dry matter. each sample was placed in previously weighed flasks. petroleum ether was used as a solvent. the total length of the program was 2 h and 30 min. it consisted of the following five cycles: immersion for 1 h, removing for 10 min, washing for 50 min, recovery for 30 min, and cooling for 10 min. once the process was complete, the flasks were removed, placed in an oven at 105ºc for 1 h, and then placed in desiccators for 12 h before being weighed. oil content was expressed as percentage of dry matter. every sample was subjected to three repetitions. analytical determinations routine parameters, such as free acidity (% oleic acid) and peroxide value (pv), were determined according to the methods described by international olive council (ioc, 2017a, 2017b). other analysis was carried out in duplicate to determine the following: chloropylls chlorophylls were measured according to the spectrophotometric method reported by pokorny et al. (2007) using a spectrophotometer (model. 8453; hewlettpackard, palo alto, ca, usa). the chlorophyll content (c) is expressed in terms of milligram of pheophytin a per kilogram of oil. tocopherols tocopherols were determined and quantified by the method proposed by conte et al. (2019) using an hplc agilent 1100 series (agilent technologies, palo alto, ca, usa) equipped with a fluorescence detector (fld; agilent technologies, palo alto, ca, usa) set at 290 nm for excitation and at 330 nm for emission, and a gemini c18 column (100-mm length × 4.6-mm internal diameter; 3-mm particle size; phenomenex, torrence, ca, usa). the mobile phase was methanol–water (98:2 v/v) and the flow rate was 2 ml/min (gimeno et al., 2002). total polyphenols total polyphenols were extracted according to the method proposed by pirisi et al. (2000). in particular, 4 ml of hexane and 4 ml of methanol–water (ratio 70:30 v/v) were added to 10 g of olive oil and mixed on vortex 60 italian journal of food science, 2023; 35 (1) el chami a et al. recorded was 108.2 mm for amioun weather station (figure 2). in kawkaba, temperatures above 30°c were registered for the following 5 months successively: may 30.1°c, june 30.9°c, july 32.6°c, august 32.4°c, and september 32.2°c. temperature below 0°c on any day was not recorded in this region. rainfall took place for 9 months per year, with the rainiest months being january (379 mm) and february (142 mm). the total amount of precipitation recorded at hasbaya weather station was 777 mm (figure 3). differences in temperature were observed, with the highest temperature recorded in bechmizine, followed by kfaraaka and kawkaba. in addition, different amount of rainfall was also recorded in these three regions, with kawkaba having the highest rainfall and kfaraaka experiencing the lowest. these environmental changes had an impact on the physiological behavior of olive trees and consequently on the quality of olive oil (angerosa et  al., 1996; aparicio et al., 1994; ponti et al., 2014). in our study, no clear relationship was observed between climatic conditions and the analyzed olive oil parameters. this could be because our study studied climatic conditions for only 1 year, which was not a sufficient period to confirm a relationship between climatic changes and the quality properties of the analyzed olive oil. therefore, the future work should study climatic conditions for a longer period to confirm the effect of environmental conditions of lebanon on the quality of olive oil. results of pedological characterization of the three studied lebanese regions results of physicochemical soil analysis for the three studied lebanese regions are presented in table 3. kad soil had the highest ph (h2o) value, followed by kan, lad, lan, and soil samples of bechmizzine “b.” the ph (kcl) was measured only for bechmizzine “b” soil samples after obtaining a ph (h2o) value lower than 6.5. data, one-way anova analysis was conducted for each region to evaluate whether the other two factors (tree age and harvest time) could have influenced the chemical parameters at least within the individual region. the statistical analysis was performed using graphpad prism (version 9). fisher’s least significant differences (lsd) test was used to evaluate differences when analyzing factors for different regions of culture. results and discussion results of climatic conditions for the three studied lebanese regions climatic data for the three studied regions are represented in table 2. bechmizzine region “b” has the lowest altitude above the sea level (265 m), with an annual average rainfall of 643 mm/year and an annual average temperature of 21ºc. kfaraaka “l” region, which is also located at 334 m above the sea level, had an annual average rainfall and average temperature of 108 mm/year and 19.5ºc, respectively. kawkaba “k” region has the highest altitude above the sea level (672 m), with an annual average rainfall of 777 mm/year and an annual average temperature of 18ºc. temperatures above 30°c, registered in bechmizzine region for 7 months of the year, are as follows: march 30.4°c, may 34.6°c, june 33.4°c, july 35.7°c, august 34.2°c, september 34.1°c, and october 32.2°c. no temperature below 0°c was recorded in this region. rainfall took place for 10 months per year, with the rainiest months being december (308.32 mm) and february (146.30 mm). at daher el ain station, the total amount of precipitation recorded was 643.09 mm (figure 1). in kfaraaka, temperature above 30°c was recorded only for the following 2 months: july 30.2°c and august 30.4°c. temperature below 0°c on any day was not recorded in this region. rainfall took place for 12 months per year, with the rainiest months being january (16 mm) and december (40 mm). the total amount of precipitation table 2. regions, locations, annual rainfall, temperatures, and altitude of the studied lebanese regions. region location year rainfall (mm/year) temperature (ºc)*† altitude (m) bechmizzine “b” north 2018 643 21 (12.2–29.5) 265 kfaraaka “l” north 2018 108 19.5 (15.5–24.3) 334 kawkaba “k” south 2018 777 18 (12.8–25.3) 672 kawkaba “k” south 2019 424 18.8 (12.1–24.7) 672 *average values of minimum and maximum temperatures of the year. †minimum and maximum temperatures are reported in parentheses. italian journal of food science, 2023; 35 (1) 61 chemical composition and environmental factors affecting quality of lebanese virgin olive oil weather data for bechmizzine 2018 mm rainfall t max 40350 ja nu ar y fe br ua ry ma rch ap ril ma y ju ne ju ly au gu st se pte mb er oc tob er no ve mb er de ce mb er 300 250 200 150 100 50 0 35 30 25 20 15 10 5 0 t min figure 1. minimum and maximum temperature (°c) and rainfall (mm) recorded at daher el ain station near bechmizzine orchards in 2018. ja nu ar y fe br ua ry ma rch ap ril ma y ju ne ju ly au gu st se pte mb er oc tob er no ve mb er de ce mb er weather data for kfaraaka 2018 rainfall t max t min ˚cmm 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0 45.0 40.0 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0 figure 2. minimum and maximum temperature (°c) and rainfall (mm) recorded at amioun station near kfaraaka orchards in 2018. 62 italian journal of food science, 2023; 35 (1) el chami a et al. weather data for kawbaba 2018 mm rainfall t max 350 400 ja nu ar y fe br ua ry ma rch ap ril ma y ju ne ju ly au gu st se pte mb er oc tob er no ve mb er de ce mb er 300 250 200 150 100 50 0 35 30 25 20 15 10 5 0 t min ˚c figure 3. minimum and maximum temperature (°c) and rainfall (mm) recorded at hasbaya station near kawkaba orchards in 2018. table 3. results of chemical analysis of soil for the three studied lebanese regions. chemical analysis of soil chemical parameters of soil unit bechmizzine “b” kfaraaka “l” kawkaba “k” ban bad lan lad kan kad ph (h 2 o) 5.5 5.5 7.4 7.5 7.8 8.1 ph (kcl) 4.8 4.8 ec (ds/m) 0.794 0.794 1.421 0.308 0.287 0.189 total limestone (g/kg) nda nda 252 31 71 504 active limestone (g/kg) nda nda 153 nda nda 210 total nitrogen (g/kg) 1.2 1.2 3.5 1.9 5.9 1.1 carbon 11 11 27 17 43 9 organic matter 19 19 47 29 74 16 c/n 9 9 8 9 7 8 assimilable phosphate (p 2 o 5 ) 46 46 128 18 174 9 exchangeable potassium (k 2 o) 328 328 1455 436 2083 1455 nda: not detectable analytically; ec: electrical conductivity in decisiemens per meter (ds/m); ban: bechmizzine ancient trees; bad: bechmizzine adult trees; lan: kfaraaka ancient trees; lad: kfaraaka adult trees; kan: kawkaba ancient trees; kad: kawkaba adult trees. the highest electrical conductivity was discovered for lan soil, followed by lad, kan, kad, and soil samples of bechmizzine “b.” as for total limestone, it was not detectable analytically in the soil samples of bechmizzine “b” region, while kad soil had the highest value followed by lan, kan, and lad. regarding active limestone, it was not detectable analytically neither in soils of region bechmizzine “b,” nor in lad and kan, while kad had italian journal of food science, 2023; 35 (1) 63 chemical composition and environmental factors affecting quality of lebanese virgin olive oil higher value of active limestone than lan. with respect to total nitrogen, the highest value was detected in kan, followed by lan, lad, bechmizzine “b” soil samples, and finally in kad soil. carbon content was maximum in kan, followed by lan, lad, bechmizzine “b,” and kad soil samples. as for organic matter, kan soil had the maximum value, followed by lan, lad, bechmizzine “b,” and kad soil samples. the carbon:nitrogen (c:n) ratio was the same for bechmizzine “b” and lad soil samples but lower for both lan and kad and the lowest for kan soil sample. the assimilable phosphate (p2o5) had the highest value in kan soil, followed by lan, bechmizzine “b,” and kad. as for exchangeable potassium (k2o), the maximum value was detected in kan, followed by kad, lan, lad, and bechmizzine “b” soil samples. in bechmizzine, soil typology (clay soil) was the same for both ancient and adult trees, since they were planted together in the same olive orchard (table 3). regarding kfaraaka, similar soil type was also registered for adult and ancient trees (loamy clay soil). on the other hand, in kawkaba, soil types were different: the adult trees were grown in a silty clay soil, while the ancient trees were planted in loamy clay soil. in the literature, few studies have established that soil type influences the quality of olive oil, but the relation between soil components and olive oil physicochemical parameters is scarcely debated (çetinkaya and kulak, 2016; rached et al., 2017a, 2017b; rouas et al., 2016). pca results for physicochemical parameters of oil table 4 shows that the first two components account for the majority (76.11%) of the total variance, and therefore these two components can be used in further analyses. scores of first principal component (pc1) and second principal component (pc2) were plotted and color-coded according to the cultivation region, the harvest time, and the tree age (figure 4). in figure 4, a clear clustering at pc1 based on the region of cultivation is observed, where the data from “b” region are clustered on the left-hand side of the x-axis (pc1), the data of region “l” are clustered toward the right-hand side of the same axis, and the data of region “k” are less regrouped but distributed below the x-axis on the right and left-hand sides. these observations were supported by the weight of each variable of oil physicochemical and fatty acid parameters of extra virgin olive oil (evoo) on the two major principal components (table 5). bold values shown in table 5 explain the strong positive or negative loadings of pc1 and pc2 on olive oil parameters. the results showed that, except acidity, all variables such as total polyphenol, antioxidant activity, palmitic acid, palmitoleic acid, linoleic acid, region of cultivation, table 4. physicochemical oil data showing the percentage values of individual variance and cumulative variance for each principal component. principal component pc1 pc2 pc3 pc4 pc5 pc6 pc7 pc8 pc9 pc10 pc11 individual variance (%) 54.37 21.74 12.63 4.12 3.19 2.05 0.82 0.58 0.35 0.13 0.03 cumulative variance (%) 54.37 76.11 88.74 92.86 96.04 98.09 98.91 99.49 99.83 99.97 100 pc scores pc1: 54.37% ad ad an an an an ad ad ad ad an an ad region b l k harvest second first p c 2: 2 1. 74 % 4 2 0 –2 –4 –6 –6 –4 –2 0 2 4 6 figure 4. pca score plot of first principal component (pc1) and second principal component (pc2) illustrating the distribution of all oil physicochemical parameters color-coded according to the region, sized according to the harvest time, and labeled with “an” for ancient trees and “ad” for adult trees. 64 italian journal of food science, 2023; 35 (1) el chami a et al. tree age, and harvest time were negatively associated with pc1, and heptadecanoic acid, heptadecenoic acid, arachidic acid, tocopherols, and the region of cultivation had the maximum impact on pc1 (table 5). on the other hand, the antioxidant activity, palmitic acid, palmitoleic acid, and lignoceric acid had the maximum influence on pc2 (table 5). the pca indicated that the observed diversity was primarily due to the “cultivation region” factor, with the “age” and the “harvest” factors playing a minor role. influence of the studied lebanese regions on the physicochemical parameters of evoos one-way anova analysis showed that region kfaraaka “l” had the maximum oil yield (% of dm) among the three tested regions (table 6). this may be attributed to the higher weight of fresh olive fruits in this region (trentacoste et al., 2010), which reported a positive relation between oil yield and fresh fruit weight. altitude could have probably affected oil yield, as indicated by other authors (lombardo et al., 2008; mousa et al., 1996), who found significantly higher fruit weight and lower oil yield at lower altitudes than at higher altitudes. these findings could explain the results of oil yield obtained in the present study for olive oil obtained in bechmizzine “b,” which has the lowest altitude between the three tested regions. the higher free acidity value in oils from kfaraaka “l” and kawkaba “k,” compared to bechmizzine “b,” was most likely due to the lipase enzyme activity; either by an active lipase existing in olive seeds (peres et al., 2017) or by fruit microbiota (lactic and enteric bacteria, fungi, and pseudomonas), which had the main influence in the extent of hydrolytic process (vichi et al., 2011). these significant differences in free acidity detected among the three tested regions (table 6) could result in differences in the quality of olive oil and its stability during storage (ayton et al., 2012). it is well known that acidity is an important parameter for assessing the quality of olive oil (ioc, 2019). owing to the presence of lipase enzymes in the oil, hydrolysis continues even after extraction and may lead to an increase in oil acidity during storage, thus reducing its quality (ayton et al., 2012). the higher peroxide value obtained in oils from bechmizzine “b,” compared to the other two regions (table 6), could be related to lower content of polyphenols and tocopherols in oils from this region, as indicated in the dedicated section of the paper. it has been shown that polyphenols and tocopherols are natural antioxidants which act as chain-breaking components (shahidi and de camargo, 2016), leading to a removal of free radicals and suppressing peroxidation (roginsky, 2003). the highest chlorophyll content obtained in oils from bechmizzine “b,” compared to the other two regions (table 6), was probably due to the very low—or almost non-detectable—limestone content in this region (table  3). this finding was in line with the results of rouas et al. (2016), who found that chlorophyll content decreased with an increased percentage of limestone in the soil. another factor that could have affected the chlorophyll content was the altitude, as reported previously by other authors (dabbou et al., 2010), who found higher chlorophyll content at lower-altitude cultivation area as in bechmizzine “b.” total polyphenol content was higher in oils from kawkaba “k,” which has the highest altitude of 672 m above the sea level (table 6). these results were in accordance with those reported previously by dabbou et al. (2010), issaoui et al. (2010), ripa et al. (2008), and rouas et al. (2016), who observed a significant higher polyphenols content in higher-altitude cultivated regions. these authors ascribed this difference to the tree cultivar and to the effect of pedo-climatic conditions that differed between regions. table 5. loading weights of each variable on principal components. variable pc1 pc2 acidity 0.75 –0.44 peroxide value –0.68 0.21 chlorophyll content –0.59 –0.01 total polyphenol 0.65 –0.53 tocopherols –0.88 –0.07 antioxidant activity 0.62 –0.70 myristic acid (c14: 0) –0.33 –0.56 palmitic acid (c16: 0) 0.65 0.75 palmitoleic acid (c16: 1) 0.50 0.84 heptadecanoic acid (c17: 0) –0.96 –0.19 heptadecenoic acid (c17: 1) –0.98 0.02 stearic acid (c18: 0) –0.79 –0.59 oleic acid (c18: 1 cis9) –0.66 –0.15 linoleic acid (c18: 2) 0.56 –0.43 arachidic acid (c20: 0) –0.93 –0.26 eicosenoic acid (c20: 1) –0.88 –0.39 linolenic acid (c18: 3) –0.67 0.47 behenic acid (c22: 0) –0.86 –0.20 lignoceric acid (c24: 0) –0.68 0.69 area 0.78 –0.43 age 0.19 –0.04 harvest 0.18 –0.11 italian journal of food science, 2023; 35 (1) 65 chemical composition and environmental factors affecting quality of lebanese virgin olive oil table 6. influence of the studied lebanese regions on evoo’s analytical indices during 2018–2019 crop season. oil parameters regions b l k oil content (% of dm) 35 ± 2c 48 ± 6a 46 ± 6b fruit fresh weight (g) 1.50 ± 0.06c 2.74 ± 0.07a 2.58 ± 0.04b free acidity (% oleic acid) 0.22 ± 0.04c 0.34 ± 0.02b 0.43 ± 0.17a peroxide value (meqo 2 kg-1) 6.50 ± 0.59a 5.81 ± 0.44b 5.66 ± 0.64b chlorophyll content (mg pheophytin a kg-1) 47 ± 11a 27 ± 15b 32 ± 22b total polyphenols (mg gallic acid kg-1) 274 ± 98b 293 ± 110b 550 ± 233a tocopherols (mg kg-1) 325 ± 59a 191 ± 45b 192 ± 47b antioxidant activity (%/mg) 0.67 ± 0.35b 0.73 ± 0.42b 2.16 ± 0.99a mean values ± standard deviation: within rows, values with the same superscript letters do not differ significantly from each other for each oil parameter according to lsd test (p < 0.05). b: bechmizzine region; l: kfaraaka region; k: kawkaba region; evoo: extra virgin olive oil. the higher antioxidant activity observed in region “k,” compared to regions “b” and “l” (table 6), is explained by high polyphenols content discovered in this region. a two-tailed pearson’s correlation test was performed, and high correlation was found between antioxidant activity and total polyphenols content in regions “b” (r = 0.9704, p < 0.001), “l” (r = 0.9974, p < 0.001), and “k” (r = 0.8409, p = 0.0089). the tocopherol content (table 6) was in accordance with those obtained by other authors in oils obtained in low altitude regions (aguilera et al., 2005; jukić špika et al., 2016; mousa et al., 1996). in fact, it was shown that formation of tocopherols was augmented with increase in temperature during fruit maturation (kalogeropoulos and tsimidou, 2014; mailer et al., 2010). the discussion on fatty acids was focused only on major fatty acids (table 7), as minor fatty acids had a less important role in the determination of olive oil quality (douzane, 2012). oleic acid (c18: 1) and linoleic acid (c18: 2) showed an opposite trend in the three regions. the higher content of oleic acid and the lowest of linoleic acid observed in the oil samples from the hottest region “b” were not in agreement with the results of other studies, which discovered lower oleic acid (c18: 1) and higher linoleic acid (c18: 2) levels in oils obtained from areas of hot temperature (kalogeropoulos and tsimidou, 2014; mailer et al., 2010; ripa et al., 2008), and from lower altitude regions (issaoui et al., 2010; piravi-vanak et al., 2012; mansour et al., 2015). these results could be related to the mean daily thermal amplitude as reported by garcía-inza et al. (2018), who found a significant but nonlinear relationship between oleic acid and mean daily thermal amplitude. in this study, the mean daily thermal amplitude was not calculated due to the absence of daily temperature in meteorological data. therefore, the future daily thermal amplitude data need to be collected to confirm the results of the literature. palmitic acid (c16: 0) and σsfa are known to be affected by temperature and altitude of the geographical place; both components increase with increase in temperature (gargouri et al., 2013; ripa et al., 2008). in this study, palmitic acid showed a contradictory behavior with regions’ temperature. there is no clear explanation for the content of palmitic acid, but it could be related to the dilution effect by oleic acid. no significant results were observed for σmufa and σpufa, and mufas–pufa and c18:1–c18:2 ratios and need to be further investigated. influence of tree age and two different harvest periods on the physicochemical parameters and fatty acid composition of evoos in the regions of cultivation analysis of physicochemical parameters and fatty acid composition of olive oil showed a pronounced clustering according to the region of cultivation. moreover, within each region, significant differences were observed in oils from trees of different ages as well as at different harvest periods (tables 8–10). as reported by other authors (bengana et al., 2013; fregapane et al., 2017), lower amounts of polyphenols and tocopherols are detected with the advancement of ripening stage. the results of the present study confirmed this statement by revealing low polyphenol and tocopherol content in olive oil produced from all trees during the second harvest time when the maturity index was high irrespective of the tree age and the region of cultivation. on the other hand, free acidity and peroxide values did not change between ancient and adult trees between the two harvest periods in bechmizzine and kfaraaka. these values were significantly different in kawkaba, where free acidity increased during the second harvest in olive oil produced by both ancient and adult trees, while 66 italian journal of food science, 2023; 35 (1) el chami a et al. table 7. influence of the studied lebanese regions on the composition of fatty acids of evoos during 2018–2019 crop season. fatty acids samples b l k myristic acid (c14: 0) 0.027 ± 0.008b 0.020 ± 0.005a,b 0.031 ± 0.01a palmitic acid (c16: 0) 11 ± 0c 14 ± 0a 11 ± 0b palmitoleic acid (c16: 1) 0.37 ± 0.02c 0.64 ± 0.09a 0.38 ± 0.07b heptadecanoic acid (c17: 0) 0.31 ± 0.02a 0.18 ± 0.01c 0.20 ± 0.04b heptadecenoic acid (c17: 1) 0.34 ± 0.02a 0.23 ± 0.008b 0.22 ± 0.04c stearic acid (c18: 0) 4.84 ± 0.09a 3.63 ± 0.30c 4.26 ± 0.43b oleic acid (c18: 1) 72 ± 1a 69 ± 1c 70 ± 1b linoleic acid (c18: 2) 8.95 ± 1.18c 9.93 ± 0.94b 11 ± 1a arachidic acid (c20: 0) 0.69 ± 0.007a 0.57 ± 0.01c 0.59 ± 0.05b eicosenoic acid (c20: 1) 0.39 ± 0.01a 0.31 ± 0.01c 0.34 ± 0.03b linolenic acid (c18: 3) 0.70 ± 0.04a 0.64 ± 0.07b 0.53 ± 0.07c behenic acid (c22: 0) 0.19 ± 0.009a 0.16 ± 0.003c 0.16 ± 0.02b lignoceric acid (c24: 0) 0.09 ± 0.002a 0.09 ± 0.006b 0.07 ± 0.005c σsfa 17 ± 0b 18 ± 0a 17 ± 1b σmufa 73 ± 1a 70 ± 1a 71 ± 1a σpufa 9.65 ± 1.22a 11 ± 1a 11 ± 2a mufas–pufas 7.56 ± 1.08a 6.04 ± 1.37a 6.09 ± 1.06a c18: 1–c18: 2 8.02 ± 1.07a 6.98 ± 1.35a 6.29 ± 1.02a mean values ± standard deviation: within rows, values with the same superscript letter do not differ significantly from each other according to lsd test (p < 0.05). b: bechmizzine region; l: kfaraaka region; k: kawkaba region. σsfa: sum of saturated fatty acids; σmufa: sum of monounsaturated fatty acids; σpufa: sum of polyunsaturated fatty acids; mufas–pufas: monounsaturated–polyunsaturated fatty acids ratio; c18: 1–c18: 2: oleic acid–linoleic acid ratio. table 8. influence of tree age and harvest time factors on evoo’s physicochemical parameters in region “b” during 2018–2019 crop season. sample name free acidity (%) peroxide value (meqo 2 kg–1) chlorophyll content (mg pheophytin a kg–1) total polyphenols (mg gallic acid kg–1) tocopherols (mg kg–1) antioxidant activity (%/mg) oil yield (dry matter) (%) fruit fresh weight (g) ban1 0.22 ± 0.001a 6.26 ± 0.18b 55 ± 1b 332 ± 0b 282 ± 21c 0.86 ± 0.01b 38 ± 2 1.54 ± 0.08 bad1 0.22 ± 0.001a 6.40 ± 0.07b 59 ± 1a 212 ± 5c 299 ± 8b,c 0.35 ± 0.01c 34 ± 2 1.45 ± 0.05 ban2 0.22 ± 0.0002a 6.42 ± 0.23b 30 ± 0d 345 ± 7a 392 ± 4a 1.01 ± 0.004a 34 ± 0 1.49 ± 0.04 bad2 0.22 ± 0.01a 7.19 ± 0.30a 45 ± 0c 140 ± 0d 347 ± 28a,b 0.27 ± 0.0003d 35 ± 2 1.50 ± 0.08 mean values ± standard deviation: within columns, values with the same superscript letter do not differ significantly from each other for each oil parameter according to lsd test (p < 0.05). ban1: bechmizzine ancient trees, first harvest; bad1: bechmizzine adult trees, first harvest; ban2: bechmizzine ancient trees, second harvest; bad2: bechmizzine adult trees, second harvest. peroxide values also significantly changed, but there was no clear relation with the tree age (table 10). results concerning fatty acids were statistically significant for the three regions; however, no clear relation was observed with respect to the tree age (tables 11–13). oleic acid, mufa, mufas–pufas, and c18: 1–c18: 2 were found to be always higher in the oil produced during the first harvest, regardless of the tree age. linoleic acid and pufas always showed an inverse relation to oleic acid. palmitic acid did not show a clear pattern neither with the tree age nor with the harvest time (tables 11–13). conclusion summarily, it is important to mention that all virgin olive oil samples analyzed in this study were within the limit of evoo according to the standards of ioc (peres et al., italian journal of food science, 2023; 35 (1) 67 chemical composition and environmental factors affecting quality of lebanese virgin olive oil table 9. influence of tree age and harvest time factors on evoo’s physicochemical parameters in region “l” during 2018–2019 crop season. sample name free acidity (% oleic acid) peroxide value (meqo 2 kg–1) chlorophyll content (mg pheophytin a kg–1) total polyphenols (mg gallic acid kg–1) tocopherols (mg kg–1) antioxidant activity (%/mg) oil yield (dry matter) (%) fruit fresh weight (g) lan1 0.34 ± 0.00a 5.55 ± 0.17a 8 ± 0d 265 ± 11b 227 ± 35a 0.58 ± 0.0c 43 ± 2c 2.55 ± 0.08c lad1 0.36 ± 0.04a 5.47 ± 0.30a 47 ± 1a 393 ± 8a 222 ± 9a 1.08 ± 0.00b 50 ± 2b 2.81 ± 0.01b lan2 0.33 ± 0.01a 6.02 ± 0.14a 24 ± 0c 138 ± 5c 128 ± 11b 0.15 ± 0.00d 42 ± 1c 2.50 ± 0.02c lad2 0.33 ± 0.00a 6.21 ± 0.68a 31 ± 0b 377 ± 1a 186 ± 23a,b 1.10 ± 0.01a 56 ± 3a 3.10 ± 0.02a mean values ± standard deviation: within columns, values with the same superscript letters do not differ significantly from each other for each oil parameter according to lsd test (p < 0.05). lan1: kfaraaka ancient trees, first harvest; lad1: kfaraaka adult trees, first harvest; lan2: kfaraaka ancient trees, second harvest; lad2: kfaraaka adult trees, second harvest. table 10. influence of tree age and harvest time factors on evoo’s physicochemical parameters in region “k” during 2018–2019 crop season. sample name free acidity (%) peroxide value (meqo 2 kg–1) chlorophyll content (mg pheophytin a kg–1) total polyphenols (mg gallic acid kg–1) tocopherols (mg kg–1) antioxidant activity (%/mg) oil yield (dry matter) (%) fruit fresh weight (g) kan1 0.22 ± 0.00d 6.41 ± 0.10a 58 ± 0a 227 ± 4c 259 ± 19a 0.55 ± 0.001c 38 ± 2d 1.77 ± 0.21d kad1 0.34 ± 0.00c 5.19 ± 0.03c 41 ± 0b 765 ± 13a 190 ± 4b 2.64 ± 0.02b 49 ± 1b 3.13 ± 0.09b kan2 0.62 ± 0.00a 4.98 ± 0.004c 4 ± 0d 475 ± 2b 179 ± 13b 2.73 ± 0.01a 42 ± 1c 2.14 ± 0.21c kad2 0.56 ± 0.00b 6.06 ± 0.21b 24 ± 0c 734 ± 1a 139 ± 12c 2.70 ± 0.02a 53 ± 2a 3.30 ± 0.30a mean values ± standard deviation: within columns, values with the same superscript letters do not differ significantly from each other for each oil parameter according to lsd test (p < 0.05). kan1: kawkaba ancient trees, first harvest; kad1: kawkaba adult trees, first harvest; kan2: kawkaba ancient trees, second harvest; kad2: kawkaba adult trees, second harvest. table 11. influence of tree age and harvest time factors on fatty acids composition of evoos in region “b” during 2018–2019 crop season. fatty acids samples ban1 bad1 ban2 bad2 myristic acid (c14: 0) 0.023 ± 0.008a 0.027 ± 0.008a 0.034 ± 0.01a 0.35 ± 0.005a palmitic acid (c16: 0) 11 ± 0b 11 ± 0a 11 ± 0a 11 ± 0b palmitoleic acid (c16: 1) 0.37 ± 0.002b 0.35 ± 0.001c 0.40 ± 0.0002a 0.35 ± 0.002c heptadecanoic acid (c17: 0) 0.32 ± 0.001b 0.31 ± 0.001b 0.28 ± 0.003c 0.33 ± 0.0008a heptadecenoic acid (c17: 1) 0.35 ± 0.001a 0.34 ± 0.003b 0.31 ± 0.0001c 0.35 ± 0.005a stearic acid (c18: 0) 4.86 ± 0.03b 4.76 ± 0.004c 4.78 ± 0.002c 4.96 ± 0.02a oleic acid (c18: 1) 73 ± 0b 73 ± 0a 70 ± 0c 70 ± 0c linoleic acid (c18: 2) 8.02 ± 0.005c 7.69 ± 0.01b 10 ± 0b 10 ± 0a arachidic acid (c20: 0) 0.68 ± 0.002b 0.68 ± 0.002b 0.69 ± 0.001b 0.70 ± 0.005a eicosenoic acid (c20: 1) 0.40 ± 0.006a 0.37 ± 0.001c 0.40 ± 0.002a,b 0.39 ± 0.0001b linolenic acid (c18: 3) 0.72 ± 0.02a 0.63 ± 0.005b 0.71 ± 0.0004a 0.71 ± 0.006a behenic acid (c22: 0) 0.18 ± 0.001b 0.19 ± 0.002b 0.18 ± 0.003b 0.20 ± 0.0004a lignoceric acid (c24: 0) 0.09 ± 0.001a 0.09 ± 0.001a 0.09 ± 0.001a 0.009 ± 0.002a (continues) 68 italian journal of food science, 2023; 35 (1) el chami a et al. table 12. influence of tree age and harvest time factors on fatty acids composition of evoos in region “l” during 2018–2019 crop season. fatty acids samples lan1 lad1 lan2 lad2 myristic acid (c14: 0) 0.02 ± 0.0002a 0.01 ± 0.001a 0.02 ± 0.01a 0.01 ± 0.0002a palmitic acid (c16: 0) 15 ± 0.02b 13.39 ± 0.01d 14.91 ± 0.05a 14.21 ± 0.02c palmitoleic acid (c16: 1) 0.73 ± 0.002a 0.50 ± 0.003d 0.71 ± 0.001b 0.59 ± 0.0003c heptadecanoic acid (c17: 0) 0.17 ± 0.001b 0.19 ± 0.001a 0.16 ± 0.001c 0.19 ± 0.0009a heptadecenoic acid (c17: 1) 0.24 ± 0.006a 0.22 ± 0.001c 0.23 ± 0.004b,c 0.23 ± 0.0009a,b stearic acid (c18: 0) 3.41 ± 0.02c 4.02 ± 0.008a 3.32 ± 0.01d 3.75 ± 0.006b oleic acid (c18: 1 cis9) 70 ± 0b 71 ± 0a 68 ± 0d 67 ± 0c linoleic acid (c18: 2) 9.19 ± 0.04c 8.93 ± 0.009d 11 ± 0a 11 ± 0b arachidic acid (c20: 0) 0.56 ± 0.009b 0.59 ± 0.004a 0.56 ± 0.003b 0.57 ± 0.002b eicosenoic acid (c20: 1) 0.31 ± 0.0006a 0.32 ± 0.003b 0.30 ± 0.007b 0.29 ± 0.002b linolenic acid (c18: 3) 0.70 ± 0.01a 0.55 ± 0.006c 0.70 ± 0.01a 0.59 ± 0.001b behenic acid (c22: 0) 0.16 ± 0.002a 0.16 ± 0.002a 0.16 ± 0.001a 0.15 ± 0.0008a lignoceric acid (c24: 0) 0.09 ± 0.001a 0.09 ± 0.008a 0.09 ± 0.002a 0.08 ± 0.002a σsfa 19 ± 0b 18 ± 0c 19 ± 0a 19 ± 0b σmufa 71 ± 0b 72 ± 0a 69 ± 0d 70 ± 0c σpufa 9.90 ± 0.06c 9.49 ± 0.01d 11 ± 0a 11 ± 0b mufas–pufas 7.17 ± 0.05b 7.59 ± 0.01a 5.90 ± 0.01d 6.24 ± 0.0004c c18: 1–c18: 2 7.58 ± 0.04b 7.94 ± 0.01a 6.17 ± 0.01d 6.48 ± 0.001c mean values ± standard deviation: within rows, values with the same superscript letters do not differ significantly from each other according to lsd test (p < 0.05). lan1: kfaraaka ancient trees, first harvest; lad1: kfaraaka adult trees, first harvest; lan2: kfaraaka ancient trees, second harvest; lad2: kfaraaka adult trees, second harvest. σsfa: sum of saturated fatty acids; σmufa: sum of monounsaturated fatty acids; σpufa: sum of polyunsaturated fatty acids; mufas–pufas: monounsaturated–polyunsaturated fatty acids ratio; c18: 1–c18: 2: oleic acid–linoleic acid ratio. table 11. continued. fatty acids samples ban1 bad1 ban2 bad2 σsfa 17 ± 0a 17 ± 0a 17 ± 0a 17 ± 0a σmufa 74 ± 0b 74 ± 0a 71 ± 0c 71 ± 0c σpufa 8.74 ± 0.02c 8.33 ± 0.01d 11 ± 0b 11 ± 0a mufas–pufas 8.46 ± 0.01b 8.91 ± 0.01a 6.69 ± 0.001c 6.64 ± 0.03d c18: 1–c18: 2 9.08 ± 0.005b 9.52 ± 0.01a 7.06 ± 0.002c 7.00 ± 0.02d mean values ± standard deviation: within rows, values with the same superscript letters do not differ significantly from each other for each fatty acid according to lsd test (p < 0.05). ban1: bechmizzine ancient trees, first harvest; bad1: bechmizzine adult trees, first harvest; ban2: bechmizzine ancient trees, second harvest; bad2: bechmizzine adult trees, second harvest. σsfa: sum of saturated fatty acids; σmufa: sum of monounsaturated fatty acids; σpufa: sum of polyunsaturated fatty acids; mufas–pufas: monounsaturated–polyunsaturated fatty acids ratio; c18: 1–c18: 2: oleic acid–linoleic acid ratio. 2017). the present study is the first one conducted in lebanon to show differences between physicochemical and fatty acid composition of virgin oils obtained from ancient and adult trees in the three selected regions during two harvest periods. based on the analysis of virgin olive oil characteristics and in combination with meteorological and soil analysis data, we conclude that the quality of virgin olive oil is associated with its chemical composition. this further is the result of complex interactions between several environmental factors associated with the area of cultivation, tree age, and harvest time, with the maximum influence of the cultivation region, and less influence of both harvest time and tree age. italian journal of food science, 2023; 35 (1) 69 chemical composition and environmental factors affecting quality of lebanese virgin olive oil table 13. influence of tree age and harvest time factors on fatty acids composition of evoos in region “k” during 2018–2019 crop season. fatty acids samples kan1 kad1 kan2 kad2 myristic acid (c14: 0) 0.04 ± 0.01a 0.02 ± 0.005a 0.03 ± 0.01a 0.02 ± 0.004a palmitic acid (c16: 0) 11 ± 0.04c 12.54 ± 0.01b 10.90 ± 0.04d 12.99 ± 0.006a palmitoleic acid (c16: 1) 0.31 ± 0.002c 0.42 ± 0.004b 0.31 ± 0.006c 0.45 ± 0.008a heptadecanoic acid (c17: 0) 0.24 ± 0.004a 0.16 ± 0.0001c 0.23 ± 0.003b 0.15 ± 0.002d heptadecenoic acid (c17: 1) 0.27 ± 0.003a 0.19 ± 0.003c 0.24 ± 0.002b 0.18 ± 0.003d stearic acid (c18: 0) 4.55 ± 0.02b 3.90 ± 0.01c 4.76 ± 0.01a 3.81 ± 0.01d oleic acid (c18: 1) 72 ± 0a 71 ± 0b 68 ± 0d 69 ± 0c linoleic acid (c18: 2) 9.87 ± 0.01c 9.81 ± 0.001d 13 ± 0a 11 ± 0b arachidic acid (c20: 0) 0.63 ± 0.001b 0.54 ± 0.002c 0.64 ± 0.002a 0.54 ± 0.006c eicosenoic acid (c20: 1) 0.36 ± 0.002a 0.31 ± 0.002b 0.36 ± 0.001a 0.31 ± 0.002b linolenic acid (c18: 3) 0.58 ± 0.001b 0.45 ± 0.001d 0.60 ± 0.001a 0.46 ± 0.004c behenic acid (c22: 0) 0.18 ± 0.002a 0.14 ± 0.0001b 0.18 ± 0.002a 0.14 ± 0.003b lignoceric acid (c24: 0) 0.08 ± 0.0001a 0.07 ± 0.002b 0.07 ± 0.002b 0.07 ± 0.0001ns σsfa 17 ± 0c 17 ± 0b 18 ± 0c 18 ± 0a σmufa 72 ± 0a 72 ± 0b 69 ± 0d 70 ± 0c σpufa 11 ± 0c 10 ± 0d 14 ± 0a 12 ± 0b mufas–pufas 6.95 ± 0.0003b 7.05 ± 0.0002a 5.01 ± 0.002d 5.77 ± 0.01c c18: 1–c18: 2 7.27 ± 0.001a 7.28 ± 0.001a 5.17 ± 0.002c 5.92 ± 0.02b mean values ± standard deviation: within rows, values with the same superscript letters do not differ significantly from each other according to lsd test (p < 0.05). kan1: kawkaba adult trees, first harvest; kan2: kawkaba ancient trees, second harvest; kad2: kawkaba adult trees, second harvest. σsfa: sum of saturated fatty acids; σmufa: sum of monounsaturated fatty acids; σpufa: sum of polyunsaturated fatty acids; mufas–pufas: monounsaturated–polyunsaturated fatty acids ratio; c18: 1–c18: 2: oleic acid–linoleic acid ratio. considering the analysis of physicochemical parameters and fatty acids discussed above, we observed that oil obtained from the adult trees of kawkaba region during the first harvest period (greenish to reddish stage of fruit maturity; kad1 sample) had the best quality of olive oil, compared to all other samples. oil produced from these trees showed the highest polyphenol content, a relatively high composition of tocopherols, oleic acid, mufas, and c18: 1–c18: 2, with relatively low acidity and peroxide values. all these parameters combined together reflect a high quality of the olive oil produced from these trees. conflict of interest there was no conflict of interest to declare. author contributions conceptualization, a.p (antonio piga), p.c. a.e., g.h.; methodology, p.c. (paola conte), a.p., a.e.; validation, p a.p (antonio piga), p.c. a.e., g.h.; formal analysis, a.e. (antonio el chami), p.c.; investigation, a.e. (antoino el chami), p.c.; data curation, a.e. (antonio el chami), p.c., ap.; writing—original draft preparation, a.e. (antonio el chami); writing—review and editing, a.e. (antonio el chami), p.c., a.p.; funding acquisition, a.p. 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33 (2): 54–62 the previous year (we are social, 2020). because of the increase in sm users, companies have started to show their presence on sm platforms (smps) to reach users. companies that can promote their products and services with minimal marketing expense have started to continue their marketing activities on sm to increase their brand awareness. the use of sm increases the rate of interaction with existing and potential consumers. sm marketing (smm) is the new media marketing channel that uses smps to interact with customers (yao et al., 2019). smm makes a significant contribution to companies in terms of customer relationship management by allowing rapid consumer feedback. companies prefer smm for fast target audience reach for unique products and for contacting potential customers. today, sm is also a product research tool for conscious and interested consumers in product research. smm has become a preferred marketing channel in many sectors and , has increased marketing food sector products on sm. the global epidemic i.e., the coronavirus (covid-19); has also increased the tendency of companies to use digital marketing channels for marketing food p u b l i c a t i o n s codon comparison of social media platforms in terms of marketing performances of food companies seren celimli and hakan adanacioglu* department of agricultural economics, faculty of agriculture, ege university, bornova, 35100 izmir, turkey *corresponding author: hakan adanacioglu, department of agricultural economics, faculty of agriculture, ege university, bornova, 35100 izmir, turkey. e-mail: hakan.adanacioglu@ege.edu.tr received: 12 march 2021; accepted: 19 april 2021; published: 14 may 2021 © 2021 codon publications open access research article abstract the objective of this study was to evaluate to what extent social media platforms are effective on the marketing performances of food companies. facebook was the most effective platform in terms of some performance criteria such as time-saving, easy access to customers, customer feedback, brand awareness, marketing costs, order taking frequency, and sales amount. the most effective platforms after facebook in terms of marketing performance are instagram and twitter, respectively. marketing costs and product sales are factors that affect the attitude of food companies towards social media platforms. keywords: food, marketing, social media, social media marketing introduction the research of new marketing methods and the rapid development of technology have improved the marketing techniques. more access to people with the web has started to move marketing to the digital environment. the convenience of web marketing for product promotion, services, and to reach potential customers has made companies adopt this method. companies that promote their marketing activities digitally started to offer product and service promotions at a low cost. internet marketing is not limited to space and time, makes it more attractive. technological changes and constantly changing consumer demands have created new avenues in marketing. companies use those sites for marketing which has increased usage. for example use of social media (sm) and the awareness of its ease of use attracted companies to these platforms. according to the report published by the sm analysis company ‘we are social’ for 2020, 4.5/ 7.7 billion world population use the internet, out of which 3.8 billion are active sm users. the active sm users in the world increased by 9.2% compared with mailto:hakan.adanacioglu@ege.edu.tr italian journal of food science, 2021; 33 (2) 55 comparison of social media platforms in terms of marketing performances of food companies generate the secondary data of this study. a survey was conducted by selecting food brands that actively use smps in each subsector. the authors planned to interview 100 companies (20 per sub-sector). additional surveys were also conducted to eliminate the negativity that may arise from incomplete and erroneous surveys. a  total of 101 questionnaires were taken into consideration for data analysis after obtaining the feedback and accuracy levels of the survey. valid questionnaires received from each subsectors included 19 for conf, 21 each for mdp and oop and, 20 each for dfp and ct. during the selection of food companies in each sector, their fb followers were also considered. companies with at least 1000 followers were included in the context of the research. for general evaluation of each subsector, companies with different number of followers were selected (between 1000 and 100,000). an online survey was conducted to obtain data from companies using google form and was shared with the respective company officials for survey completion. statistical analysis the data obtained from the surveyed food companies were presented using the five-point likert scale. the kruskal-wallis (kw) test was used to test whether the data means differ in terms of the food subsectors examined. the reason for using this test is that the data do not show normal distribution. kalaycı (2006) defines the kw test as a nonparametric alternative to a one-way analysis of variance between groups. results and discussion general information about the examined food companies table 1 gives the general information about the examined food companies. the legal structure of food companies showed that most of the companies (56.40%) operated as limited companies. the rate of food companies operating as sole proprietorships and joint-stock companies was 38.60% and 5%, respectively. the activity period of food companies showed that the majority (88.0%) have been operating for 10 years or more, 9.90% of the companies for 4–9 years, and 2% for 1–3 years. grouping based on the number of employees revealed that more than half of the companies (51.50%) have a workforce of 10–49 people, 41.60% of the companies, however, had 50–249 people. the distribution of domestic and foreign sales of food companies showed that the share of companies with products. however, advertising many nonfood products in the digital environment is easier versus food products owing to seasonal variations, fluctuations in production amount, durability of products, and cultural differences. it is not difficult to predict that the impact of covid-19 and similar shocks have increased the high consumers’ demand in the digital environment. however, without considering the short-term effects of these shocks, it is pivotal to study the effects of sm, one of the most important digital marketing channels, on the marketing performance of companies in the food sector. this study studied the effect of smps on the marketing performance of food companies. this evaluation differs from previous studies in some ways. previous studies investigating different aspects of this topic have been analyzed within the scope of one or more smps where facebook is predominant (ainin et al., 2015; aspasia and ourania, 2015; say, 2015; nyarkoa and altıntaş, 2015; saad and badran, 2016; francisco, 2016; yurttadur and sari, 2017; de vries et al., 2018; pantano et al., 2019; bernal jurado et al., 2019). in this study, the marketing performance of food companies was analyzed concerning seven smps, including facebook (fb), twitter (tw), instagram (ig), youtube (yt), google plus (g+), linkedin (li), and blogs. in previous studies, the effect of smm on companies was generally examined within the scope of sales increase (ainin et al., 2015; nyarkoa and altıntaş, 2015; say, 2015; canovi and pucciarelli, 2019) and marketing costs (ainin et al., 2015; yurttadur and sari, 2017; barišić and vujnović, 2018; yao et al., 2019). this study more comprehensively analyzed the effect of smm on companies with performance criteria such as time-saving, easy access to customers, customer feedback, brand awareness, marketing costs, order taking frequency, and sales amount. another different aspect of this study from previous studies is that research was conducted on companies operating in different subsectors of the food industry. in previous studies, it is seen that the food sector has been examined in general terms or together with nonfood sectors. this study examined a total of five different subfood sectors. materials and methods data acquisition the primary data of this study were obtained from survey interviews with different food subsector companies that actively use smps. five subsectors of food that use sm intensely in the food industry in turkey were selected that included confectionery (conf), milk and dairy products (mdp), olive and olive products (oop), dry food and pulses (dfp), and coffee and tea (ct). information obtained from smps, statistics published by sm analysis companies, and previous research on the subject helped 56 italian journal of food science, 2021; 33 (2) celimli s and adanacioglu h table 1. general characteristics of food companies surveyed. variables frequency percentage (%) legal structure joint stock company 5 5.00 limited liability company 57 56.40 sole proprietorship 39 38.60 operating period (years) 1–3 2 2.00 4–9 10 9.90 ≥10 89 88.10 number of employees 1–9 6 5.90 10–49 52 51.50 50–249 42 41.60 ≥250 1 1.00 share of domestic sales as a percentage of total sales (%) 50–75 8 7.92 76–99 40 39.60 100 53 52.48 average domestic sales rate (%) 92.76 share of food products in total sales (%) <50 3 2.97 50–75 6 5.94 76–99 67 66.34 100 25 24.75 average share of food sales (%) 87.38 the turnover of 66.34% of the companies varies between 76–99%, and 24.75% of the examined companies got their entire turnover from the food products sale. evaluation of smps in terms of marketing performance following the opinions of the examined companies, a comparison of smps for marketing performance was conducted. some performance criteria such as time saving, easy access to customers, customer feedback, brand awareness, marketing costs, order taking frequency, and sales amount were used to compare smps. companies need to carry out, follow, and interpret marketing activities in a shorter time. it was observed that companies had started preferring sm applications to perform these activities faster as they wanted to reach more customers in less time. effectiveness of smps in terms of timesaving when the data were analyzed to get an idea about the time-saving platform for companies marketing activities, the fb platform led the list, followed by ig and tw, respectively. in general, companies in different subsector groups find fb, ig, and tw effective for saving time in marketing, and no statistically significant difference was found between the groups. yt and g+ were evaluated as moderately effective, but li and blogs are less effective. however, a statistically significant difference of opinion among subsector groups in terms of time savings was noted (table 2). companies in the ct and conf sectors found these four platforms less effective in terms of saving time. effectiveness of smps for easy customer access when the opinions of companies regarding the effectiveness of smps for easy access to customers were examined, domestic sales was high. the portion is 92.76% on average, and 52.48% of the companies make all their sales domestically. in general, the share of food products in the total turnover of companies was high. the average share of food products in the total turnover of the companies interviewed is 87.38%. the share of food products in table 2. evaluation of social media platforms in terms of time-saving. smps conf mdp oop dfp ct total kw test p value x̄ sd x̄ sd x̄ sd x̄ sd x̄ sd x̄ sd fb 4.26 1.05 4.57 0.60 4.43 0.60 4.25 0.55 4.45 0.69 4.40 0.71 0.483 tw 3.37 1.46 4.24 0.62 3.76 1.22 4.05 0.39 3.70 1.42 3.83 1.12 0.465 ig 3.37 1.57 4.19 0.68 4.00 0.84 3.70 0.86 4.05 1.05 3.87 1.06 0.298 yt 2.16 1.07 3.71 0.90 3.24 1.04 3.60 0.88 3.00 1.12 3.16 1.13 0.000* g+ 2.68 1.29 3.62 0.92 3.43 0.98 3.45 1.00 2.80 1.15 3.21 1.12 0.023** li 2.21 1.08 3.24 1.09 3.14 1.01 3.35 1.09 2.45 1.32 2.89 1.19 0.005* blogs 2.11 1.10 3.33 1.06 3.24 1.00 3.35 1.09 2.70 1.26 2.96 1.18 0.003* x̄ : mean score of the likert scale by the level of effectiveness of each smp (1: not at all effective to 5: highly effective); sd: standard deviation. *statistical significance at 1%. **statistical significance at 5%. italian journal of food science, 2021; 33 (2) 57 comparison of social media platforms in terms of marketing performances of food companies send messages to the relevant company. unlike fb and ig, the sharing of users via text messages is limited on the tw, and the option to send direct messages to the company is often not available. a statistically significant difference was observed between the group evaluations for tw, yt, and blogs platforms. compared with the other groups, companies operating in the confec industry did not find tw, yt, and blogs platforms very effective for customer feedback. effectiveness of smps for brand awareness when the impact of smps for increasing brand awareness was analyzed, the fb platform led with an average score of 4.58. large number of users attribute to its popularity. the other two platforms that were effective in increasing brand awareness were ig (4.22) and tw (4.07), respectively. other smps scored 3.64 (yt ), 3.43 (g+ ), 3.23 ( blogs), and 3.21 (li ) concerning their effect on increasing brand awareness (table 5). the evaluations made according to the 5-point likert scale used in the survey study indicated that the fb with a mean score of 4.52 was determined to be quite effective . platforms found to be moderately effective in terms of easy access to customers were ig, tw, and yt, respectively (table 3). in general, it was understood that the fb platform was more convenient for food companies to reach their customers. the number of fb users in turkey is high, causing many customers to focus on this platform. li (1.87) stands out as the least effective platform concerning easy access to customers as it is primarily business-oriented. effectiveness of smps concerning customer feedback when the opinions of customer feedback on sm were examined, the fb platform was effective in customer feedback with an average score of 4.61 followed by the ig (3.62), and tw was moderately effective (3.29). yt, g+, li, and blogs platforms were found to be minimally effective concerning customer feedback (table 4). the fb, was quite impressive for customer feedback, as it allows users to comment and share, directly message the companies, and provides more notifications from the customers. the ig also offers users the opportunity to comment and table 3. the effectiveness of social media platforms in terms of easy access to customers. smps conf mdp oop dfp ct total kw test p value x̄ sd x̄ sd x̄ sd x̄ sd x̄ sd x̄ sd fb 4.47 1.12 4.62 0.59 4.57 0.60 4.75 0.55 4.20 0.83 4.52 0.77 0.146 tw 3.32 1.16 3.38 1.16 3.29 1.31 3.65 1.04 3.10 1.17 3.35 1.16 0.657 ig 3.63 1.12 3.81 0.98 3.90 1.00 3.90 1.17 4.00 0.97 3.85 1.03 0.829 yt 2.32 1.11 2.52 1.03 2.62 1.02 2.40 1.10 2.75 1.21 2.52 1.08 0.586 g+ 2.32 1.00 2.57 0.98 2.48 0.98 2.20 0.77 2.45 1.23 2.41 0.99 0.841 li 1.84 1.07 2.05 0.92 2.14 0.91 1.70 0.57 1.60 0.88 1.87 0.89 0.170 blogs 1.89 0.94 2.62 1.02 2.52 0.87 2.00 0.65 2.45 1.05 2.31 0.95 0.022* x̄ : mean score of the likert scale by the level of effectiveness of each smp (1: not at all effective to 5: highly effective); sd: standard deviation. *statistical significance at 5%. table 4. the effectiveness of social media platforms in terms of customer feedback. smps conf mdp oop dfp ct total kw test p value x̄ sd x̄ sd x̄ sd x̄ sd x̄ sd x̄ sd fb 4.63 0.76 4.67 0.48 4.71 0.46 4.70 0.47 4.35 0.75 4.61 0.60 0.385 tw 2.47 1.39 3.57 1.08 3.52 1.33 3.75 0.97 3.05 1.43 3.29 1.31 0.027** ig 2.95 1.58 3.90 1.00 3.71 1.27 3.75 1.07 3.75 1.12 3.62 1.24 0.301 yt 1.63 1.01 2.76 1.09 2.33 1.15 2.50 1.00 2.80 1.24 2.42 1.16 0.003* g+ 2.05 1.13 2.48 1.03 2.57 1.21 2.15 0.93 2.45 1.23 2.35 1.11 0.459 li 1.58 1.07 2.00 0.95 2.05 1.02 1.90 0.85 2.30 1.22 1.97 1.03 0.130 blogs 1.53 1.02 2.48 0.98 2.43 1.16 2.30 0.92 2.45 1.23 2.25 1.11 0.004* x̄ : mean score of the likert scale by the level of effectiveness of each smp (1: not at all effective to 5: highly effective); sd: standard deviation. *statistical significance at 1%. **statistical significance at 5%. 58 italian journal of food science, 2021; 33 (2) celimli s and adanacioglu h companies had a positive opinion about the effect of smps on brand awareness. a statistically significant difference was found between groups for other smps other than fb, ig, and tw. the companies in the confec and mdp sectors were effective in this difference. companies in the confec industry found smfs other than fb, ig, and tw had minimal effect of increasing the awareness of their brands. on the other hand, the opinions of the companies in the mdp sector on this issue are more positive versus other subsector groups. effectiveness of smps in terms of marketing cost reduction when the effect of smps on reducing marketing costs was analyzed, fb once again led the race with an average of 4.34, followed by ig (4.14) and tw (4.12). table 6 shows the effectiveness of smps on reducing marketing costs. the effect of these platforms in reducing marketing costs was noted as 3, which is above the neutral value according to the 5-point likert scale average. the kw test was conducted to determine whether there is a difference between the evaluations of the company table 5. the effect of social media platforms on brand awareness. smps conf mdp oop dfp ct total kw test p value x̄ sd x̄ sd x̄ sd x̄ sd x̄ sd x̄ sd fb 4.53 0.96 4.62 0.59 4.57 0.60 4.55 0.60 4.65 0.59 4.58 0.67 0.969 tw 3.68 1.00 4.33 0.66 3.81 1.03 4.40 0.60 4.10 1.12 4.07 0.93 0.070 ig 4.00 1.11 4.48 0.68 4.14 0.79 4.00 1.17 4.45 0.69 4.22 0.91 0.353 yt 2.89 1.24 4.05 0.67 3.57 1.08 3.55 1.05 4.10 1.02 3.64 1.09 0.004* g+ 2.68 1.25 3.86 0.79 3.48 1.03 3.50 1.05 3.55 1.19 3.43 1.12 0.020** li 2.42 1.30 3.81 0.81 3.48 0.87 3.25 1.21 3.00 1.56 3.21 1.24 0.014** blogs 2.26 1.28 3.86 0.79 3.29 0.96 3.25 1.21 3.40 1.47 3.23 1.25 0.003* x̄ : mean score of the likert scale by the level of effectiveness of each smp (1: not at all effective to 5: highly effective); sd: standard deviation. *statistical significance at 1%. **statistical significance at 5%. groups regarding the effect of smps in reducing marketing costs. a statistically significant difference was found for the blogs platform. compared with other food subsectors, companies in the confec industry found the blogs platform’s decreased effect on reducing marketing costs. according to the results mentioned above, the examined food companies think that smps are generally effective in reducing marketing costs. this result is consistent with the findings obtained in previous studies (ainin et  al., 2015; yurttadur and sari, 2017; barišić and vujnović, 2018; yao et al., 2019). in particular, the fb platform was evaluated as highly effective in reducing marketing costs, which can be attributed to its free content sharing and easy tracking of user comments. effectiveness of smps concerning increasing product sales when the effect of increasing product sales was examined, fb again led with an average of 4.11, followed by moderate product sales by ig (3.26) and tw (3.15). table 6. the effect of social media platforms on reducing marketing costs. smps conf mdp oop dfp ct total kw test p value x̄ sd x̄ sd x̄ sd x̄ sd x̄ sd x̄ sd fb 4.21 1.23 4.43 0.60 4.29 0.72 4.35 0.59 4.40 0.82 4.34 0.80 0.926 tw 4.00 1.25 4.24 0.62 4.00 1.00 4.30 0.57 4.05 1.19 4.12 0.95 0.951 ig 3.89 1.33 4.24 0.62 4.24 0.70 4.05 0.76 4.25 0.97 4.14 0.89 0.823 yt 3.37 1.34 3.95 0.67 3.90 0.94 3.85 0.81 4.00 0.92 3.82 0.96 0.478 g+ 3.32 1.34 3.81 0.75 3.90 0.94 3.75 0.79 3.95 0.94 3.75 0.97 0.385 li 2.68 1.38 3.71 0.96 3.67 0.91 3.60 0.88 3.50 1.43 3.45 1.17 0.091 blogs 2.63 1.38 3.71 0.96 3.76 0.89 3.65 0.93 3.75 1.16 3.51 1.14 0.031* x̄ : mean score of the likert scale by the level of effectiveness of each smp (1: not at all effective to 5: highly effective); sd: standard deviation. *statistical significance at 5%. italian journal of food science, 2021; 33 (2) 59 comparison of social media platforms in terms of marketing performances of food companies table 7 reports the impact of yt, g+, blogs, and li platforms in enhancing sales. fb was the most effective platform in the sales increase because of marketing their products via sm, which attributes to the consumers’ emphasis on visuality in marketing food products through sm. ainin et al. (2015) and say (2015) also revealed that fb effectively increased product sales. ainin et al. (2015) showed that the use of fb had a positive effect on the sales volume of smes in malaysia. say (2015) determined that the companies in the convenience food sector in turkey increase their online sales with campaigns supported by fb. ig was placed second concerning the effect of increasing product sales because of its visual density like fb. attitude of food companies towards smm fifteen statements were presented to companies during the survey study to measure the attitude of companies. likert scale responses of companies for these statements were tested with reliability analysis. in the analysis, cronbach’s alpha value, the general reliability coefficient, was determined as 0.873. since this value was between 0.80 ≤ α < 1.00, the scale was found to be reliable. the responses of food companies to some statements through smm are shown in table 8. the statements to which the companies mostly agree were: providing brand awareness, the convenience of offering products and services to target regions, presenting campaigns and activities at the appropriate time, increasing the competitive power, strengthening the status of the company, reducing marketing expenses, increasing loyal customers, providing tips about the market, and increasing sales. the statement that the interviewed companies least agreed was about the price. companies hardly agreed with the view that smm provides a higher price than traditional marketing. besides, companies believe that marketing food products on sm are more difficult than other product categories. in general, there is no statistically significant difference between companies in different food subsectors in terms of their level of agreement with some statements related to smm. there is a statistical difference of opinion among companies for statements that “social media enables customers to make better decisions” and “social media is preferred over other marketing channels.” however, the degree to which companies agree with both statements is high. table 9 shows the correlation analysis results of the relationship between the general attitude of companies towards smm. these results revealed that the attitude of companies towards sm is not in a statistically significant relationship with the size of the companies, operating period of the companies, and the smm experience of the companies. the correlation analysis was used to determine the attitude change toward sm according to the company size. however, there was no significant relationship found between company size and attitude toward sm. this aspect was not examined in previous studies. however, some studies investigated the relationship between company size and sm use. aspasia and ourania’s (2014) study on the greek food sector found a positive relationship between company size and the adoption of sm tools. according to the authors, this is because large firms allocate more staff and budget to sm. braojos-gomez et al. (2015) and pantano et al. (2019) state that small companies with low financial resources must improve their sm skills to gain a competitive advantage in sm. tarsakoo and charoensukmongkol (2019) argue that both small and large companies use sm to add value to their business activities. but many difficulties that limit the capabilities of small companies in terms of effective smm. on the other hand, the increasing effect of sm on reducing marketing costs positively increases the attitude of table 7. the effect of social media platforms in increasing product sales. smps conf mdp oop dfp ct total kw test p value x̄ sd x̄ sd x̄ sd x̄ sd x̄ sd x̄ sd fb 4.37 0.83 4.43 0.87 3.90 1.09 4.20 0.95 3.65 1.35 4.11 1.06 0.206 tw 3.00 1.20 3.57 0.87 3.00 1.38 3.40 1.10 2.75 1.59 3.15 1.26 0.336 ig 3.21 1.36 3.71 0.96 3.05 1.47 3.30 1.34 3.00 1.62 3.26 1.36 0.615 yt 1.63 0.68 2.33 1.11 2.14 1.35 1.90 0.91 1.90 1.21 1.99 1.09 0.309 g+ 2.11 1.05 2.14 0.91 2.05 1.28 1.85 0.81 1.75 0.97 1.98 1.01 0.561 li 1.68 0.67 1.95 0.86 1.76 1.00 1.70 0.73 1.45 0.69 1.71 0.80 0.348 blogs 1.63 0.68 2.10 0.94 1.95 1.12 1.75 0.72 1.50 0.69 1.79 0.86 0.249 x̄ : mean score of the likert scale by the level of effect of each smp (1: not at all effective to 5: highly effective); sd: standard deviation. 60 italian journal of food science, 2021; 33 (2) celimli s and adanacioglu h table 8. level of agreement of food companies with some statements on social media marketing. statements conf mdp oop dfp ct total kw test p value x̄ sd x̄ sd x̄ sd x̄ sd x̄ sd x̄ sd sm increases brand awareness 4.16 0.90 4.52 0.51 4.43 0.68 4.30 0.66 4.40 0.60 4.37 0.67 0.645 it is easier to reach the target audience with sm 3.89 0.99 4.43 0.60 4.43 0.60 4.20 0.62 4.30 0.86 4.26 0.76 0.236 sm is effective for campaigns 3.87 1.13 4.14 0.48 4.38 0.59 4.55 0.60 4.30 0.66 4.25 0.74 0.100 sm gives a competitive edge 3.81 0.93 4.27 0.76 4.36 0.72 4.31 0.62 4.38 0.57 4.23 0.74 0.161 sm strengthens the status of the company 3.84 0.83 4.43 0.51 4.43 0.60 4.15 0.49 4.10 0.79 4.20 0.68 0.059 sm reduces marketing expenses 3.74 0.99 4.38 0.50 4.19 0.75 4.15 0.67 4.40 0.60 4.18 0.74 0.120 sm increases loyal customers 3.69 1.06 4.19 0.60 4.38 0.67 4.05 0.51 4.20 0.77 4.11 0.76 0.163 sm provides marketrelated tips 4.06 0.91 4.14 0.57 4.19 0.60 3.90 0.55 4.20 0.62 4.10 0.66 0.400 sm increases sales 3.95 0.78 4.00 0.77 3.81 1.08 3.75 0.85 4.05 1.05 3.91 0.91 0.704 sm enables customers to make better decisions 3.57 0.76 3.95 0.59 4.05 0.67 3.64 0.74 4.20 0.77 3.89 0.73 0.023** selling on sm is easy 3.84 0.90 3.73 0.62 3.98 0.71 3.68 0.56 4.19 0.70 3.88 0.71 0.078 i prefer sm to other marketing channels 3.28 1.14 3.71 0.64 3.86 0.57 3.38 0.49 4.05 0.69 3.66 0.77 0.008* sm offers special products to customers 3.03 1.00 3.11 0.86 3.05 0.90 3.12 0.21 3.18 1.31 3.10 0.91 0.673 sm is a good option for marketing food products. 2.90 1.07 2.57 1.00 2.52 0.95 2.52 0.73 2.58 1.40 2.62 1.04 0.365 sellers on sm get higher prices 2.01 1.23 1.84 1.13 1.51 0.71 1.73 0.82 2.20 1.34 1.85 1.08 0.526 x̄ : mean score of likert scale by the level of agreement with statements (1: not at all effective to5: highly effective); sd: standard deviation. *statistical significance at 1%. **statistical significance at 5%. table 9. correlation analysis results between the attitude of companies towards social media.   attitude of companies towards sm size of the companies operating period of the companies smm experience of the companies effect of sm on reducing marketing costs effect of sm on increasing product sales attitude of companies towards sm pearson correlation 1.000 0.114 −0.182 −0.030 0.216** 0.317* sig. (2-tailed) – 0.255 0.069 0.763 0.030 0.001 *correlation is significant at the 1% level (2-tailed). **correlation is significant at the 5% level (2-tailed). companies toward smm (r = 0.216; p = 0.030). analysis findings also revealed a statistically significant and positive relationship (r = 0.317, p = 0.001) between the increasing effect of sm on product sales and attitude toward sm. although the relationship between them is not strong, according to the size of the correlation coefficients, the effect of sm, both to reduce marketing costs and increase product sales positively affects the attitudes of companies toward sm. however, the highest degree of relation with the attitude of companies towards sm is the increasing effect of sm on product sales. italian journal of food science, 2021; 33 (2) 61 comparison of social media platforms in terms of marketing performances of food companies conclusion this study examined the effectiveness of smps on the marketing performance of food companies. according to the outcomes, fb is the most effective platform for performance criteria such as time-saving, easy access to customers, customer feedback, brand awareness, marketing costs, order taking frequency, and sales amount. the most effective platforms after fb in terms of marketing performance are ig and tw, respectively. li, blogs, and g+ are the platforms with the least performance. the marketing performance of food companies varies according to smps. the use of all sm platforms for marketing purposes will waste the time of companies. hence, a company should first determine which smp their current and target customers use more. in the next stage, these companies should conduct their marketing activities over the smp chosen. a food company that is engaged in marketing activities on a platform where there are no current and target customers will not reach the sm usage purpose. since the content offered by the food company cannot reach current and target customers, smm will not have an impact on the product sales of the company. besides, companies need to follow the sm activities of their competitor companies while continuing their marketing activities on sm. food companies to examine the content on sm provided by competitors that produce similar products and their feedback. enhancing the company’s knowledge on the use of sm and smm will aid in increasing the marketing effectiveness of food companies on sm. in general, the food companies make intensive marketing initiatives on sm. however, they do show their competence in using smps. it has been observed that some companies have incorrect/ no or nonsuitable information entered in their sm accounts. food companies should start operating on sm after doing a good research on using the functional features of smps as every smp has options specific to the platforms it offers. since marketing strategies will change according to platforms, preliminary research is required on this subject. in addition, visually intensive shares for food products should be presented to the consumer. hence, food companies need to pay attention to the quality and remarkable features of the content offered on sm. rapid and positive feedback of food companies on sm will be a supportive effort to achieve the smm goal. acknowledgments the authors thank the food companies for their valuable contributions in study data collection. conflict of interest no potential conflict of interest was reported by the authors. references ainin s.,  parveen f.,  moghavvemi s.,  jaafar n.i. and mohd shuib, n.l. 2015. factors influencing the use of social media by smes and its performance outcomes. ind. manag.  data  syst. 115(3): 570–588. https://doi.org/10.1108/imds-07-2014-0205 aspasia v. and ourania n. 2014. social media adoption and managers’ perceptions. int. j.  strategic innovative  mark. 1: 61–73. https://doi.org/10.15556/ijsim.01.02.001 aspasia v. and ourania n. 2015. greek food manufacturing firms’ social media 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132 issn 1120-1770 online, doi 10.15586/ijfs.v34i1.2173 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (1): 132–139 p u b l i c a t i o n s codon two cases of black human breast milk not related to minocycline. a sphingolipidomic approach laura cerquiglini1, stefania troiani1, chiara gizzi1, michele dei cas2, rita paroni2, paola signorelli2, antonella mencacci3, maurizio radicioni1, stefano pasquino4, maria cristina de lio5, lina cossignani6, giuseppa verducci6, carmela conte6, tommaso beccari6,samuela cataldi,6 elisabetta albi6* 1struttura complessa di neonatologia e terapia intensiva neonatale, azienda ospedaliera di perugia – santa maria della misericordia, perugia, italy; 2department of health sciences, università degli studi di milano, milan, italy; 3medical microbiology, department of medicine and surgery, university of perugia, perugia, italy; 4department of cardiac surgery, azienda ospedaliera di perugia – santa maria della misericordia, perugia, italy; 5igiene e organizzazione dei servizi ospedalieri, azienda ospedaliera di perugia – santa maria della misericordia, perugia, italy; 6department of pharmaceutical sciences, university of perugia, perugia, italy *corresponding author: elisabetta albi, department of pharmaceutical sciences, university of perugia, perugia 06126, italy. email: elisabetta.albi@unipg.it received: 19 january 2022; accepted: 13 february 2022; published: 4 march 2022 © 2022 codon publications open access opinion paper abstract different colors of human breast milk (hbm) are reported in literature, and black milk is produced only during minocycline therapy. we herein report two cases of black/dark gray color hbm without minocycline involvement. we analyzed both milk samples and compared the data with two control hbm samples taken from two mothers who had the same dietary behaviors and took the same supplements (iron) as done by the mothers under study. results indicated that the black color was not due to iron intake, disease or infection. with sudan iii stain, specific for lipids, dark precipitates were evident. antioxidant power was much higher in studied milk samples than in control samples. as antioxidants at high levels become pro-oxidants, our data suggested possible lipid oxidation. sphingolipid profile of black milk samples demonstrated accumulation of sphingomyelin and ceramide, which could be a sign of impaired lipid metabolism. it was concluded that iron supplementation was not responsible for hbm pigmentation, but altered biochemical mechanisms in the mammary gland could be implicated. in our experience, dark color hbm did not represent an absolute indication for discontinuation of breastfeeding. keywords: black milk, breast milk, ceramide, fatty acids, human milk, sphingomyelin introduction human breast milk (hbm) is universally recognized as the preferred source of neonatal nutrition. it is an extremely complex biological fluid. some components vary with the maternal diet, while others are independent of this and depend on metabolic activity of the mammary gland (dei cas et al., 2020). thus, breast milk is species-specific and reflects the requirement of infants (tripaldi et al., 2021). in fact, hbm protects infants from diseases as they wait for their immune systems to mature (hanson and korotkova, 2002), from dysfunctional lipid metabolism and gut dysbiosis (norris et al., 2019). moreover, hbm has good antioxidant properties (codini  et al., 2020) and it is essential for the cognitive maturation (skinner and narchi, 2021). throughout lactation, hbm undergoes a gradual biochemical modification in lipid and protein concentration so that the milk is commonly classified into colostrum, mailto:elisabetta.albi@unipg.it italian journal of food science, 2022; 34 (1) 133 study of black human breast milk not related to minocycline materials and methods population and sample collection procedure blud in perugia, italy (banca del latte umano donato, struttura complessa di neonatologia e terapia intensiva neonatale—azienda ospedaliera santa maria della misericordia—perugia, italy) provided human milk samples. two black milk samples under investigation and two crt white milk samples collected from healthy iron-supplemented mothers were examined for the study. all procedures were performed according to the indications of bioethics committee, and the women signed informed consent. mothers were invited to answer questions about: (1) age; (2) cigarette use; (3) use of medical products containing nicotine; (4) alcohol use; (5) use of restrictive or specific diets; and (6) medical therapy. immediately after harvesting, milk sample was kept for microbiological analysis and the remaining amount was submitted for holder pasteurization (hop) by heating it to 62.5°c (145°f) for 30 min, and then cooling it back to 4–10°c; it is a globally used method to ensure distribution of microbiologically safe milk to infants (codini et  al., 2020). after pasteurization, microbiological analysis was performed again and the milk samples were stored at –20°c in a freezer before analysis. materials anhydrous sodium sulfate, chloroform, hexane, methanol and potassium hydroxide were purchased from carlo erba reagents (milan, italy). sudan iii, codex s-4131, was obtained from sigma chemical co. (st. louis, mo, usa). supelco™ 37-component fatty acid methyl esters (fame) mix, containing methyl esters of 37 fatty acids, was supplied by supelco (bellefonte, pa, usa). lipids standards were purchased from avanti polar lipids (alabaster, al, usa). chemicals, all analytical grade, were purchased from sigma-aldrich (st. louis, mo, usa). all aqueous solutions were prepared using purified water of milli-q grade (burlington, ma, usa). microbiological analysis milk samples were collected aseptically by using breast pump and an aliquot was taken for gram staining and culture. quantitative culture was performed onto chocolate, blood supplemented with colistin and nalidixic acid, mcconkey, mannitol salt and sabouraud agar plates (all media were acquired from becton dickinson, east rutherford, nj, usa). plates were incubated in co2 or air-incubators and were read for development of colonies after being kept overnight and 48-h incubation. transitional milk and mature milk (jenness, 1979). colostrum, the first milk produced after parturition, is rich in proteins and growth factors and has high concentrations of secretory immunoglobulin, thus indicating its immunological function (castellote et al., 2011). transitional milk is produced around the third–fifth day after childbirth until 20 days–1 month of child birth. after this, mature milk is produced and it lasts for the duration of breastfeeding. transitional milk has high contents of fat and carbohydrates and is low in minerals and proteins. mature milk is truly abundant in fats and carbohydrates and has an optimal protein and minerals intake in relation to the increased nutritional needs of the newborn (riordan and wambach, 2014). the color of hbm changes with lactation. if sometimes, the colostrum appears clear, thin and watery, at other times it is often yellow or orange, thanks to the presence of high levels of beta-carotene (patton et al., 1990). transitional milk  typically changes from yellow to white, and mature milk is first clear or bluish and afterwards white or yellow in color (riordan and wambach, 2014). milk color may vary physiologically depending on diet, nutritional supplements and medications. hbm can appear pink, red and orange with fruit drinks or green with green color beverages, green vegetables, vitamins or mineral supplements  in diet (yazgan et al., 2012) or intake of blue-green algae (naor et al., 2019). however, the color of hbm may change for different reasons, as pink color is described in  colonization of serratia marcescens (ayuzo del valle and treviño salinas, 2014; jones et al., 2014; quinn et al., 2018). moreover, green color is reported after administration of propofol (birkholz et al., 2009; rainone et al., 2018). however, black hbm is reported only in relation to minocycline therapy (basler, et al., 1985; hunt et al., 1996; lawrence, 1985). although different observations have been reported on the colors of hbm, possible modifications in lipid components have not been studied yet. since the sphingomyelin (sm) present in breast milk is important for the maturation of child’s nervous system (jiang et al., 2021), we particularly focused on sphingolipidomic study. we report two cases of black hbm produced during early lactation by two mothers not under antibiotic therapy but both taking iron for anemia. iron intake and breastfeeding were discontinued. milk color in both cases turned white within a few days of iron discontinuation and mothers restarted breastfeeding without any incident. as a control (crt), two clear/white milk samples from two mothers on iron supplementation were examined. we investigated and reported microbiological, cytological and biochemical aspects of both samples. 134 italian journal of food science, 2022; 34 (1) cerquiglini l et al. gas chromatographic analysis of fatty acids milk lipid fraction was extracted using a chloroform– methanol mixture (2:1,  v/v), following the procedure reported previously (codini et al., 2020). the fame of total lipids was prepared by transmethylation with methanolic koh and analyzed using high-resolution gas chromatography. a dani 1000dpc gas-chromatograph (norwalk, ct, usa), equipped with a split–splitless injector and a flame ionization detector, was used. the fame separation was performed with a cp-select cb for famefused silica capillary column (50 m × 0.25 mm i.d., 0.25μm f.t.; varian, superchrom, milan, italy). the injector and detector temperatures were 250°c. the oven temperature was 60°c, held for 5 min, then raised to 225°c at a rate of 3°c/min; the final temperature was held for 10 min. the chromatograms were acquired and processed using the clarity integration software (dataapex ltd., prague, czech republic). a standard solution containing 37 fame was used to identify individual fatty acids. high resolution gas chromatography analysis was conducted in triplicate. liquid chromatography with tandem mass spectrometry sphingolipid (sph) extraction and liquid chromatography coupled with tandem  mass spectrometry (lc–ms/ms) analyses were performed as described by dei cas et al. (2020). sphingolipids were extracted from three independent 25-µl aliquots of hbm using monophasic solvent extraction (chloroform:methanol:water—30:60:10, v/v/v). these were analyzed with liquid chromatography (dionex 3000 ultimate, thermofisher scientific, waltham, ma, usa) coupled with tandem mass spectrometer (ab sciex 3200 qtrap, sciex, vaughn, on, canada). the separation was achieved by a reversed-phase analytical column (acquity beh c8 100 × 2.1  mm × 1.7  μm; waters, milford, ma, usa) through a linear gradient between eluent a (0.2% formic acid, 2-mm ammonium formate in water solution) and eluent b (0.2% formic acid, 1-mm ammonium formate in methanol). quantitative analysis was performed interpolating each peak area of analyte/ area internal standards with a calibration curve for each sphingolipid. statistical analysis statistical analysis of lipidomic study was performed with graphpad prism 7.0 (graphpad software inc, la jolla, ca, usa). statistical differences of fatty acids and sphingolipidomic study were investigated by unpaired t-test. graphs were represented as mean ± sd, and statistical significance was set as p < 0.05. microbial load in milk samples was measured as colony forming units/ml. colonies were identified using the bruker maldi biotyper instrument (bruker daltonik gmbh, bremen, germany), and antimicrobial susceptibility testing was performed with the bd phoenix automatic system (becton dickinson). sudan iii staining saturated sudan iii was prepared by diluting 0.5-g powder in 100-ml 99% isopropanol. the solution was allowed to set for 2 days before using the supernatant. working sudan iii stain was obtained by diluting 6 ml of saturated sudan iii dye in 4 ml of distilled water, and letting it stand for 10 min and filtering the solution before use. each sample, 50 µl, was dispersed on polarized slides and allowed to air-dry for 24 h. the samples were stained with working sudan iii, left at room temperature for 10 min, washed with distilled water, and allowed to air-dry. the stained slides were examined with euromex-holland fe 2915 microscope (bd arnhem, the netherlands) equipped with a dc5000 cmex-5.0 pixel digital usb camera system and analyzed at 40× magnification. antioxidant assay by oxygen radical absorbance capacity (orac) the antioxidant capacity of human milk samples was determined using the orac method as reported by ceccarini et al. (2016). we used orac method because it is a robust and reliable method. in fact, the orac assay, with other common measures of antioxidant capacity, including ferric ion-reducing antioxidant power and trolox equivalence antioxidant capacity assays, is regarded as a preferable method because of its biological relevance (codini et al., 2015). a duplicate extraction was performed for each sample and used to evaluate lipophilic oracfl (l-oracfl) and hydrophilic oracfl (h-oracfl) values. evaluations of lipophilic and hydrophilic oracfl values in the samples were performed separately, and the total antioxidant capacity was calculated by adding l-oracfl and h-oracfl values. oracfl assays were conducted on a fluostar optima microplate fluorescence reader (bmg labtech, offenburg, germany) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. 2,20-azobis (2-methylpropionamide) dihydrochloride was used as a peroxyl radical generator; trolox was used as a reference antioxidant standard; and fluorescein was used as a fluorescent probe. the data were expressed as micromoles of trolox equivalents (te) per gram of sample (μmol te/g). italian journal of food science, 2022; 34 (1) 135 study of black human breast milk not related to minocycline yeasts. organisms identified in the milk of control 1 mother were staphylococcus epidermidis (20.000 colony-forming units [cfu]/ml), acinetobacter junii (>100.000 cfu/ml), acinetobacter ursingii (3.000 cfu/ ml) and staphylococcus aureus (<3.000 cfu/ml). organisms identified in the milk of control 2 mother were staphylococcus epidermidis (10.000 cfu/ml) and staphylococcus haemolyticus (1.000 cfu/ml). organisms identified in the milk sample of mother a were staphylococcus lugdunenesis (20.000 cfu/ml) and staphylococcus aureus (5.000 cfu/ml). organisms identified in the milk sample of mother b were staphylococcus epidermidis (10.000 cfu/ml), staphylococcus lugdunensis (800 cfu/ml) and pseudomonas fluorescens (800 cfu/ml). after pasteurization, all microbiological tests were negative in all milk samples and the black color of milk samples a and b remained unchanged. therefore, we decided to investigate the lipid component of black milk samples and compared them with the light milk samples. first, we performed milk staining with sudan iii. figure 1 shows the presence of fat globules of different sizes in the two crt samples with physiological orange color. in the two samples of black milk, black precipitations appeared in the central or peripheral part of fat globules. the analysis of antioxidant capacity demonstrated that samples a and b had very high antioxidant capacity (figure 2). orac value in crt samples was results mothers two women, a: 26-year-old, and b: 43-year-old , were examined for the study. the women were enrolled because of the dark color of their colostrum. it emerged from the analysis of their questionnaires that both mothers neither smoked nor used alcohol and followed a mediterranean diet without restrictions. mother a was under iron supplementation due to physiological anemic condition during pregnancy. mother b took iron for anemia as well and methyldopa for hypertension arising during pregnancy. moreover, she supplemented the diet with folic acid and docosahexaenoic acid (dha). in order to address possible bias because of the suspected role of iron, we also included in the study two healthy mothers, aged 26 and 43 years, taking iron but producing light color milk, as crt samples. the selected mothers were neither affected by any chronic pathology nor had received any recent transfusion; they were only anemic and followed a mediterranean diet. characteristics of human breast black milk samples microbiology was aspecific. milk samples of control as well as a and b mothers were positive to culture test for bacteria but negative to culture test for figure 1. image of milk samples stained with sudan iii. the arrows indicate black precipitates in fatty granules. magnification 20×. (a) ctr1 10µm ctr2 (b) 136 italian journal of food science, 2022; 34 (1) cerquiglini l et al. 632.97 + 19.24 μmolte/ml. in black milk sample a, the value was 1826.09 + 46.95 μmolte/ml and in black milk sample b, it was 122.49 + 37.34 μmolte/ml. lipid composition of human breast black milk samples first, we measured fatty acids (fa) in hbm. results of black milk samples a and b were compared with those of white milk crt samples. we analyzed the general similarity and difference of total saturated fatty acids (sfa) and unsaturated fatty acids (ufa). black milk sample a contained more ufa but black milk sample b had less sfa than ctr milk samples (figure 3a). interestingly, the sfa:ufa ratio was similar in black milk samples a and b and it was lower than that of crt milk samples (figure 3b). if each species of fa was examined, it became evident that in both black milk samples a and b, c16:1n-9, c18:3n-3, c20:1, c20:4, c22:5 and c22:6 were higher 2000 1500 1000 µm ol t e /m l 500 0 ctr 1–2 a b figure 2. antioxidant potential of human breast milk. crt: control milk sample; a and b: black milk samples. data are expressed as mean ± sd calculated as reported in ‘statistical analysis’. significance of a and b versus ctr 1–2; *p < 0.05. 1800 (a) (b) 1600 1400 1200 1000 m g/ 10 0m l 600 800 400 0 2 s at /u ns at r at io 1 0 200 ctr 1–2 sat unsat saturated and unsaturated fatty acid a b ctr 1–2 saturated/unsaturated fatty acid a b figure 3. composition of fatty acids in human breast milk. (a) total saturated and total unsaturated fatty acids. (b) ratio of saturated and unsaturated fatty acids. crt: control milk samples; a and b: black milk samples. data are expressed as mean ± sd calculated as reported in ‘statistical analysis’. significance of a and b versus ctr 1–2; *p < 0.05. than in crt milk samples (figure 4). moreover, milk sample a was particularly rich in c12:0 and c18:1n-9 content whereas milk sample b was deficient in c14:0 and c16:0 contents (figure 4). we investigated possible changes in the composition of sphingolipids in black milk samples in comparison to ctr milk samples. the results highlight a higher level of sphingomyelin content in both black milk samples a and b (figure 5a) but a higher level of ceramide (cer) content in black milk sample a (figure 5b). in figure 6,  each sphingolipid species  was reported. of note, all sphingomyelin species were increased in milk samples a and b except 24:0 sm compared to crt milk samples. 14:0 cer, 16:0 cer, 18:0 cer, 18:1 cer and 20:0 cer were increased in milk sample a but only 10:0 cer was enhanced in milk sample b. dihydroceramide (dhcer) species was present only in milk sample a. discussion previous studies have demonstrated that color of hbm may vary physiologically, in relation to diet, nutritional supplements, infections and medications, from pink to orange, red and green (ayuzo del valle and  treviño salinas, 2014; jones et al., 2014; naor et al., 2019; quinn et al., 2018; yazgan et al., 2012). the black color of hbm is reported only during minocycline treatment (basler and lynch, 1985; hunt et al., 1996; lawrence, 1985). however, here we report for the first time the case of two mothers who neither suffered from any pathology nor had minocycline therapy but had a black/deep dark color hbm. the only clinical aspect they had in common was iron supplementation. mothers who had dark color milk as well as control mothers who had white color normal milk were using iron for anemia, suggesting that mineral intake was not responsible for milk’s color, and italian journal of food science, 2022; 34 (1) 137 study of black human breast milk not related to minocycline 1200 1000 600 800 400m g/ 10 0 m l 200 0 ctr 1–2 fatty acids c 4: 0 c 6: 0 c 8: 0 c 10 :0 c 12 :0 c 14 :0 c 14 :1 c 15 :0 c 16 :0 c 16 :1 n9 c 18 :1 n9 c 18 :2 c 20 :0 c 18 :3 n3 c 20 :1 c 20 :4 c 20 :5 c 22 :5 c 22 :6 c 16 :1 n7 c 17 :0 c 17 :1 c 18 :0 a b figure 4. fatty acid species in human breast milk. crt: control milk samples; a and b: black milk samples. data are expressed as mean ± sd calculated as reported in ‘statistical analysis’. significance of a and b versus ctr 1–2; *p < 0.05. 140 160 180 200 (a) (b) 120 100 µm 20 40 60 80 0 ctr 1–2 sphingomyelin a b 120 100 µm 20 40 60 80 0 ctr 1–2 ceramide a b figure 5. total sphingomyelins and ceramides in human breast milk. crt: control milk samples; a and b: black milk samples. data are expressed as mean ± sd calculated as reported in ‘statistical analysis’. significance of a and b versus ctr 1–2; *p < 0.05. 100 80 60 µm 20 40 0 ctr 1–2 a b ce r 1 4 ce r 1 6 ce r 1 8 ce r 1 8:1 ce r 2 0 ce r 2 2 ce r 2 4 ce r 2 4:1 dh ce r 1 6 dh ce r 1 8 dh ce r 1 8:1 dh ce r 2 4 dh ce r 2 4:1 sm 16 sm 18 sm 18 :1 sm 24 sm 24 :1 figure 6. species of sphingomyelins, ceramides and dihydroceramides in human breast milk. crt: control milk samples; a and b: black milk samples. data are expressed as mean ± sd calculated as reported in ‘statistical analysis’. significance of a and b versus ctr 1–2; *p < 0.05. 138 italian journal of food science, 2022; 34 (1) cerquiglini l et al. conclusions to the best of our knowledge, we reported the first two cases of black/dark gray color breast milk of women not related to minocycline therapy but having iron supplementation in common. iron intake does not seem responsible for black milk pigmentation. the dark color of milk could be due to an alteration in antioxidant properties that become pro-oxidant. this induces a biochemical modification in lipid components in terms of fatty acids and sphingolipids. however, it was not possible to determine whether this was due to a change in the metabolic activity of the mammary gland. in summary, our data highlights that it is possible to have black/dark color milk from a healthy mother having a normal diet, and this finding does not represent an absolute indication for discontinuation of breastfeeding. references ayuzo del valle c. and treviño salinas e. 2014. pink breast milk:  serratia marcescens  colonization. ajp rep.  4(2): e101– e104. https://doi.org/10.1055/s-0034-1387934 birkholz t., eckardt g., renner s., irouschek a. and schmidt  j. 2009. green breast milk after propofol administration. anesthesiology. 111(5): 1168–1169. https://doi.org/10.1097/ aln.0b013e3181bbc4b1 basler r.s. and lynch p.j. 1985. black galactorrhea as a consequence of minocycline and phenothiazine therapy. case rep arch dermatol. 121(3): 417–478. https://doi.org/10.1001/ archderm.1985.01660030139039 castellote c., casillas r., ramirez-santana c., perez-cano f.j., castell m., moretones m.g., et al. 2011. premature delivery influences the immunological composition of colostrum and transitional and mature human milk. j nutr. 141(6): 1181–1187. https://doi.org/10.3945/jn.110.133652 ceccarini m.r., codini m., cataldi s., vannini s., lazzarini a., floridi a., et al. 2016. acid sphingomyelinase as target of lycium chinense: promising new action for cell health. lipids health dis. 15(1): 183. https://doi.org/10.1186/s12944-016-0351-z codini m., cataldi s., ambesi-impiombato f.s., lazzarini a., floridi a., lazzarini r., et al. 2015. gentamicin arrests cancer cell growth: the intriguing involvement of nuclear sphingomyelin metabolism.  e int j mol sci.  16: 2307–2319. https://doi. org/10.3390/ijms16022307 codini m., tringaniello c., cossignani l., boccuto a., mirarchi a., cerquiglini l., et al. 2020. relationship between fatty acids composition/antioxidant potential of breast milk and maternal diet: comparison with infant formulas. molecules. 25: e2910. https:// doi.org/10.3390/molecules25122910 dei cas m., paroni r., signorelli p., mirarchi a., cerquiglini l., troiani s., et al. 2020. human breast milk as source of sphingo lipids for newborns: comparison with infant formulas and commercial cow’s milk. j transl med. 18: 481–494. https://doi. org/10.1186/s12967-020-02641-0 it could just be an incidental finding of dark color hbm reported by both mothers a and b. as a matter of fact, these are the only two cases of black color hbm among a large number of pregnant/brestfeeding mothers taking this mineral globally. on discontinuation of iron intake and breastfeeding, milk in both cases again became white after a few days and both mothers restarted breastfeeding without any incident. in our experience, dark color of breast milk is not an absolute indication for discontinuation of breastfeeding. microbiology was aspecific. staining with sudan iii, specific for lipids, revealed the presence of dark formations inside fat granules, which were absent in crt milk samples. it was difficult to exactly articulate these formations, but the possibility that they were altered lipids cannot be excluded. we assumed that these could be partially oxidized lipids that precipitated as lipofuscins, which stained with sudan iii (marani and lazarov, 2017). the analysis of antioxidant properties revealed that dark milk samples had a much higher antioxidant power compared to crt milk samples. for analysis, we have chosen orac method was chosen because it is a robust and reliable method. in fact, the orac assay, other common measures of antioxidant capacity include ferric ion reducing antioxidant power and trolox equivalence antioxidant capacity assays and therefore is considered to be a preferable method because of its biologic relevance (codini et al., 2015). a high antioxidant power is generally considered positive for the quality of a food. however, if a food has very high antioxidant properties, it becomes pro-oxidant (mendes-da-silva et al., 2014). in addition, it has been demonstrated that in a large number of women milk has medium level of antioxidant properties (codini et al., 2020), similar to that we observed in two crt milk samples. in addition, crt milk samples had a higher sfa content than ufa, as demonstrated by codini et al. (2020). this is a characteristic of hbm that differentiates it from formula milk, which is particularly enriched in polyunsaturated fatty acids (codini et al., 2020). we assumed that a higher value of ufa in black milk samples predisposed them to easier oxidation. we demonstrated that hbm of mothers who followed a mediterranean diet had richer sphingomyelin contents than found in infant formulas (dei cas et al., 2021). in the present case also, we discovered similar sphingomyelin content in crt milk samples as reported by dei cas et al. 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https://doi.org/10.1111/j.1365-2133.1996.tb06332.x� https://doi.org/10.1093/advances/nmab117� https://doi.org/10.1093/advances/nmab117� https://doi.org/10.1111/1552-6909.12492� https://doi.org/10.1111/1552-6909.12492� https://doi.org/10.1001/archderm.121.3.417� https://doi.org/10.1016/b978-0-12-809324-5.02594-3� https://doi.org/10.1016/b978-0-12-809324-5.02594-3� https://doi.org/10.1016/j.neuropharm.2014.06.027� https://doi.org/10.1016/j.neuropharm.2014.06.027� _hlk90295184 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (3): 105–123 issn 1120-1770 online, doi 10.15586/ijfs.v34i3.2238 105 p u b l i c a t i o n s codon effect of extrusion processing conditions on the techno-functional, antioxidant, textural properties and storage stability of wholegrain-based breakfast cereal incorporated with indian horse chestnut flour farhana mehraj allai1,2, z.r.a.a. azad1*, b.n. dar2*, khalid gul3* 1department of post-harvest engineering and technology, faculty of agricultural science, aligarh muslim university, uttar pradesh, india; 2department of food technology, islamic university of science and technology, awantipora, india; 3department of food process engineering, national institute of technology, rourkela, india *corresponding authors: z.r.a.a. azad, department of post-harvest engineering and technology, faculty of agricultural science, aligarh muslim university, uttar pradesh, india. email: zrazad@gmail.com; b.n. dar, department of food technology, islamic university of science and technology, awantipora, india. email: darnabi@gmail.com; and khalid gul, department of food process engineering, national institute of technology, rourkela, india. email: gulmk@nitrkl.ac.in received: 3 june 2022; accepted: 15 july 2022; published: 27 october 2022 © 2022 codon publications open access paper abstract the central composite rotatable design was used to plan the experiments using feed moisture (fm) content, barrel temperature (bt), screw speed (ss), and concentration of indian horse chestnut flour (ihcf) as process variables varied between 10–18%, 70–150°c, 290–380 rpm and 1.75–4.75%, respectively. surface mechanical energy, water holding capacity, swelling volume, piece density, longitudinal expansion, water activity and colour attributes were used as response variables. the whole grain flours added with 2.5% ihcf and extruded at 12% fm, 130°c bt and 380 rpm ss produced optimum quality breakfast cereals with 0.713 desirability. extrudates packed in aluminum pouches were found to be suitable packaging materials and were shelf-stable for 6 months with better quality retention. keywords: extrusion cooking, fibre, horse chestnut, packaging, shelf-life, whole grains introduction over the decades, people across the world have struggled to ensure a healthy and delicious diet. for this purpose, producers had to adopt different techniques aimed at making food products more appealing and tastier with increased nutritional value and organoleptic attributes (nkhata et al., 2018). the traditional cooking method improves the bioavailability and bioaccessibility of nutrients but at the same time causes significant loss of macro as well as micronutrients (kamau et al., 2020). nutrient utilization can be increased by processing methods such as extrusion cooking. extrusion cooking is the most popular food processing technique in which the ingredients are subjected to amalgamation of temperature, internal pressure, moisture and shear forces, leading to the modification and disruption of molecular structure and texture resulting in the development of an ample range of food products such as breakfast cereals, pasta, snacks, baby foods and texturised meat substitutes (espinosa-ramirez et al., 2021). it is also used to overcome the limitations associated with the processing of traditional cereal-based food products by enhancing its functional, physicochemical and shelf stability (jabeen et al., 2021). the extrusion process improves the food quality by reducing the deterioration of essential nutrients by thermal treatment while increasing the palatability, digestibility by starch gelatinization, modifying the molecular structure of the protein, and inhibiting unacceptable compounds including anti nutritional factors and enzymes (pasqualone et al., 2020). mailto:zrazad@gmail.com mailto:darnabi@gmail.com mailto:gulmk@nitrkl.ac.in 106 italian journal of food science, 2022; 34 (3) allai fm et al. ihcf, and so far, no research has been conducted on the use of wwf, wbf, wcf and ihcf for the development of breakfast cereal. therefore, it is necessary to characterise the wholegrain flours and ihcf to analyse their possible utilisation in food industry and also to optimise the processing conditions, that is, feed moisture content, barrel temperature and screw speed and ihcf for the development of breakfast cereals. in this research, the physicochemical and functional properties, colour, texture and storage stability of the developed extrudates were studied. materials and methods wholegrain flours whole grain wheat (sw-2) and whole white corn (dt-2) were purchased from sher-e-kashmir university of agricultural sciences and technology (skuast), shalimar, jammu and kashmir, (j&k), and whole grain barley (pl-807) was obtained from kargil, india. the whole grains were cleaned to eliminate the impurities. after removing the impurities, the whole grains were milled to obtain flour. the whole grain flours were then packed and stored at −21°c for further use. indian horse chestnut seeds (aesculus indica) were manually harvested from local areas of shalimar, j&k. preparation of indian horse chestnut flour the preparation of ihcf is done according to the method described in our previous research (allai et al., 2022a). conception of experimental design response surface methodology (rsm) consists of different experimental designs based on statistical calculations and mathematical models on raw experimental data, to describe the empirical association between two or more independent variables, including design factors (xi) and design response (yi) that are required to be optimised through prediction approach (moussaoui et al., 2021). statistical software design-expert-12 (stat-ease inc. minneapolis, mn, usa) was applied to determine the influence of process parameters on response variables by using the second-order polynomial model as shown in the below equation. the adjusted and predicted r-square was in a reasonable agreement for all the models as presented in table 1. = = = = = + + +∑ ∑ ∑∑ 4 4 4 4 2 i 0 i i ii i i ij i j i 1 i 1 i 1 i 1 y b b x b x b x x ready-to-eat products such as breakfast cereals are most commonly consumed in the morning as the first meal of the day (oliveira et al., 2017b). traditionally, breakfast cereals are prepared from refined cereal flours due to their high availability, ease of processing, low cost, and their bland taste. technically, refined flours are nutritionally poor in microand macronutrients and contain only the digestible carbohydrate, that is, starch in comparison to wholegrain flours (oliveira et al., 2017a). in food industry, cereals are predominantly used as the base ingredient for the development of extruded products, but the trend is now changing and shifting towards the healthy ingredients due to the increased incidences of chronic diseases (naseer et al., 2021). as such, addition of different whole grains such as whole wheat flour (wwf), whole corn flour (wcf) and whole barley flour (wbf) in breakfast cereals has a promising scope for enhancing the potential health benefits and market acceptability of cereal-based products. wholegrain flours are rich sources of antioxidant activity, bioactive compounds, vitamins, minerals, and dietary fibres such as cellulose, hemi-cellulose, lignin, β-glucan and resistant starch (allai et al., 2021). several researchers have prepared extruded products from non-conventional seeds and evaluated their antioxidant and phytochemical properties (beigh et al., 2019; jabeen et al., 2021). indian horse chestnut is an underutilised nut that has not been used previously with wholegrain flours and can, therefore, be explored for the development of healthy, sugar-free breakfast cereal. indian horse chestnut (aesculus indica) provides an excellent opportunity for the pharmaceutical and food industries for having unique medicinal properties that include anti-obesity, anti-fever, antiviral, anti inflammatory and anti-oedemic (ahmad and gani, 2021). in india, horse chestnut remains unrecognised and is mainly consumed by cattles and wild animals and considered as waste (mishra et al., 2018). the seeds are poisonous and bitter in taste, if consumed without processing (raw), because of the presence of anti-nutrients such as tannins and saponins. so, the seeds must be kept under running water in order to eliminate the bitterness and grind to fine flour. indian horse chestnut flour (ihcf) is simply added with other ingredients, partially replacing the wheat flour, to make wide range of products such as halwa (porridge), chapatti (flat bread), pasta and noodles (rafiq et al., 2021). horse chestnut could be utilised as a nutritional supplement due to their high levels of dietary fibre, starch, minerals, vitamins, polyphenols, flavonoids and other bioactive compounds such as triterpenoids, epicatechin and kaempferol (idris et al., 2020). due to its nutritional quality, ihcf may be considered as a potential non-conventional alternative to conventional flours. very limited research has been done regarding italian journal of food science, 2022; 34 (3) 107 wholegrain-based breakfast cereals table 1. anova for the fit of data to response surface models. dependent variables models r2 adjusted r2 predicted r2 adequate precision cv (%) p lack of fit sme sme = 129.88 + 1.99x 1 – 4.54x 2 – 9.17x 3 + 5.69 x 4 + 2.07x 1 2 + 1.18x 4 2 0.97 0.95 0.90 23.97 1.91 <0.05 ns pd (g/ml) pd = 6.34 + 0.0996x 1 + 0.803x 2 – 0.1388x 3 – 0.407x 4 – 0.210.6x 2 x 4 – 0.0594x 3 x 4 – 0.5430 x 1 2 – 0.186x 2 2 0.99 0.99 0.97 50.10 1.59 <0.05 ns lei lei = 0.286 – 0.011x 1 – 0.010x 2 + 0.0133x 3 + 0.0285x 4 0.79 0.76 0.72 18.67 5.58 <0.05 ns a w a w = 0.3033 + 0.042x 2 – 0.01x 3 0.93 0.87 0.79 16.27 4.77 <0.05 ns whc (g/g) whc = 5.57 + 0.182x 1 + 0.662x 2 + 0.464x 3 – 0.275x 4 – 0.266x 2 * x 3 + 0.11x 1 2 + 0.252x 2 2 + 0.412x 3 2 – 0.134x 4 2 0.96 0.93 0.83 22.87 4.00 <0.05 ns sv (g/ml) sv = 4.81 + 0.093x 1 + 0.264x 2 + 0.205x 3 – 0.154x 4 – 0.121x 2 x 3 – 0.045x 2 2 + 0.471x 3 2 – 0.215x 4 2 0.98 0.97 0.93 49.20 1.99 <0.05 ns l* l* = 66.94 – 0.623x 2 – 0.717x 3 + 1.40x 3 x 4 + 0.833x 1 2 + 0.905x 4 2 0.85 0.71 0.56 9.54 1.68 <0.05 ns a* a* = 4.26 – 0.124x 1 + 0.572x 2 + 0.227x 3 + 0.052x 4 + 0.045x 2 x 3 + 0.077x 1 2 + 0.166x 2 2 + 0.234x 3 2 + 0.036x 4 2 0.99 0.98 0.95 38.40 0.44 <0.05 ns b* b* = 22.64 + 1.15x 2 + 1.08x 3 0.77 0.74 0.65 15.83 3.75 <0.05 ns a*, redness; a w , water activity; b*, yellowness; cv, coefficient of variance; l*, luminosity; lei, longitudinal expansion index; ns, non-significant; pd, piece density; sme, surface mechanical energy; sv, swelling volume; whc, water holding capacity. where yi is the design response, and xi is the independent factors, that is, x1, x2, x3 and x4 represent feed composition (ihcf), feed moisture content, barrel temperature and screw speed, respectively, and the coefficients of regression for constant, linear, quadratic and interaction regression terms were indicated by b0, bi, bii and bij, respectively. in this study, the percentage of wbf and wwf was kept constant (10%) throughout the experiment. in the control, wcf accounted for the remaining 80% of the feed formulation. in the feed mixture, ihcf was used to replace wcf. the central composite rotatable design was used to investigate the effect of the four independent variables. x1, x2, x3 and x4 at each five levels are shown in table 2. the experiment runs along with actual and coded levels for the independent variables are presented in table 3. extrusion cooking the breakfast cereals were prepared in a corotating twin-screw extruder (basic technology pvt. ltd., kolkata, india) with a die width of 3.0 mm and a length to diameter ratio 8:1. the temperatures of the first, second and third zones were maintained at 40°c, 60°c and 80°c, respectively, throughout the experiment, while the fourth zone varied according to the design of experiment. the extrusion process was carried out using different feed table 2. coded levels and experimental ranges of the independent variables generated using central composite rotatable design. numerical variable symbol coded variable levels −2 −1 0 1 2 indian horse chestnut flour (%) x 1 1.75 2.5 3.25 4 4.75 feed moisture content (%) x 2 10 12 14 16 18 temperature (°c) x 3 70 90 110 130 150 screw speed (rpm) x 4 290 320 350 380 410 108 italian journal of food science, 2022; 34 (3) allai fm et al. table 3. effect of independent variables on techno-functional characteristics of extrudates. runs independent variables physical properties functional colour ihcf (%) feed moisture (%) barrel temperature (ºc) screw speed (rpm) sme (wh/kg) pd (g/ml) lei (%) a w whc (g/g) sv (ml/g) l* a* b* 1 2.5(−1) 12(−1) 90(−1) 320(−1) 142.2 4.86 0.28 0.27 5.04 4.48 71 4.1 20.17 2 4(+1) 12(−1) 90(−1) 320(−1) 145.56 5.24 0.22 0.29 5.56 4.92 71.64 3.8 19.64 3 2.5(−1) 16(+1) 90(−1) 320(−1) 130.5 7.09 0.25 0.36 7 5.23 69.75 5.3 23.13 4 4(+1) 16(+1) 90(−1) 320(−1) 137.4 7.23 0.23 0.37 7.11 5.46 70.33 4.7 23 5 2.5(−1) 12(−1) 130(+1) 320(−1) 120.7 4.72 0.32 0.24 6.41 5.06 64.71 4.47 22.67 6 4(+1) 12(−1) 130(+1) 320(−1) 124.3 5.01 0.3 0.25 6.78 5.35 66.92 4.22 22 7 2.5(−1) 16(+1) 130(+1) 320(−1) 110.6 6.83 0.28 0.34 7.34 5.37 67.41 5.74 25.19 8 4(+1) 16(+1) 130(+1) 320(−1) 114.5 7 0.25 0.36 7.55 5.55 66 5.53 24.31 9 2.5(−1) 12(−1) 90(−1) 380(+1) 149.8 4.57 0.33 0.27 4.22 4.3 68.74 4.22 20.69 10 4(+1) 12(−1) 90(−1) 380(+1) 152.6 4.83 0.32 0.3 5.04 4.36 69.32 4.13 18.02 11 2.5(−1) 16(+1) 90(−1) 380(+1) 142.4 5.95 0.3 0.36 6.34 5.08 66.23 5.37 23 12 4(+1) 16(+1) 90(−1) 380(+1) 145.3 6.11 0.28 0.35 6.88 5.17 67 5.05 23.15 13 2.5(−1) 12(−1) 130(+1) 380(+1) 133.4 4.24 0.35 0.24 6 4.96 69.7 4.53 23 14 4(+1) 12(−1) 130(+1) 380(+1) 139.5 4.44 0.33 0.26 6.12 5.11 70 4.44 22.78 15 2.5(−1) 16(+1) 130(+1) 380(+1) 121.5 5.41 0.33 0.34 6.66 5.21 68.36 5.78 25 16 4(+1) 16(+1) 130(+1) 380(+1) 127.4 5.56 0.31 0.36 6.97 5.29 68 5.51 24.82 17 1.75(−2) 14(0) 110(0) 350(0) 134.2 4.03 0.3 0.31 5.46 4.79 68.92 4.73 23.03 18 4.75(+2) 14(0) 110(0) 350(0) 140.3 4.35 0.26 0.31 6.15 5.15 70.65 4.3 22.4 19 3.25 (0) 10 (−2) 110(0) 350(0) 135.6 4.11 0.29 0.24 5.07 4 66.14 3.7 21.65 20 3.25(0) 18(+2) 110(0) 350(0) 120.3 7.12 0.27 0.39 7.68 5.26 63.13 6.04 24.12 21 3.25(0) 14(0) 70(−2) 350(0) 146.55 6.54 0.28 0.33 5.89 6.19 67.42 4.66 21.88 22 3.25(0) 14(0) 150(+2) 350(0) 113.4 6.21 0.31 0.3 8.14 7.2 65.26 5.62 25.33 23 3.25(0) 14(0) 110(0) 290 (−2) 121.06 6.94 0.23 0.31 5.34 4.39 70.15 4.33 23.21 24 3.25(0) 14(0) 110(0) 410 (+2) 146.3 5.49 0.32 0.33 4.31 3.51 70 4.37 20.01 25 3.25(0) 14(0) 110(0) 350(0) 132.5 6.33 0.29 0.32 5.57 4.88 69 4.3 23.43 26 3.25(0) 14(0) 110(0) 350(0) 130.2 6.4 0.26 0.29 5.61 4.8 67.22 4.28 23 27 3.25(0) 14(0) 110(0) 350(0) 127.3 6.32 0.25 0.33 5.5 4.71 65.22 4.32 22.65 28 3.25(0) 14(0) 110(0) 350(0) 125.2 6.41 0.27 0.31 5.46 4.79 68.42 4.25 22.13 29 3.25(0) 14(0) 110(0) 350(0) 130.5 6.3 0.28 0.29 5.87 4.85 66.7 4.2 22.87 30 3.25(0) 14(0) 110(0) 350(0) 133.6 6.29 0.29 0.28 5.41 4.81 65.1 4.22 23 a*, redness; a w , water activity; b*, yellowness; ihcf, indian horse chestnut flour; coded values are in parenthesis; l*, luminosity; lei, longitudinal expansion index; pd, piece density; sme, surface mechanical energy; sv, swelling volume; whc, water holding capacity. compositions and process parameters as given in table 3. the amount of water to be added before the extrusion was calculated in order to adjust the feed moisture content. a homogeneous flour mixture was obtained by mixing properly for 20 min, and water was added to the feed. the prepared feed mixtures were allowed to equilibrate for 12 h prior to extrusion to stabilise the moisture content. the prepared samples were then collected and cooled to ambient temperature, dried and packed in low-density polyethylene (ldpe) bags for further analysis. surface mechanical energy surface mechanical energy (sme) is the amount of energy given to extrudates for the conversion of starch. italian journal of food science, 2022; 34 (3) 109 wholegrain-based breakfast cereals colour analysis the cielab space parameters of extrudates were analysed with a hunter lab colorimeter (cr 300, konica minolta, japan). l*, a* and b* values represent lightness or darkness, redness and blueness or yellowness, respectively. each value is an average of five different independent measurements. functional parameters swelling volume (sv) and water holding capacity (whc) sv and whc of extrudates were determined by following the method by espinosa-ramirez et al. (2021). whc measures the amount of retained water in the extrudates without undergoing any stress, and sv was defined as the ratio of total volume of swollen extrudates to the weight of solids. optimal point and validation optimization of independent variables was done through the highest desirability function in order to validate the developed models, that is, by comparing the predicted and actual values. the optimal conditions considered for numerical optimisation was minimum piece density and maximum longitudinal expansion (le), sme, swelling volume (sv) and whc. the developed extrudates were evaluated for all the selected parameters, and percentage prediction error was measured as follows (scheuer et al., 2016). actual value predicted value pr ediction error (%) 100 pr edicted value − = × texture analysis the texture of extrudates was investigated using a ta-hd plus texture analyser (stable micro system, godalming, surrey, uk) equipped with five blade kramer shear cell (oliveira et al., 2017b) fitted with a 50 kg load cell. the compression test was done by arranging the sample on a single-layer bed, and the test was carried out at a pre-test speed of 1.0 mm/s, test speed of 2 mm/s and post-test speed of 10.00 mm/s. the trigger type used was button type, and the samples were compressed to 50% of the original height. the software recorded the number of peaks produced from the force deformation curve, resulting in the wall fracture of the sample, which represented the crispiness (np) (dogan and kokini, 2007). hardness was measured as the maximum force (n) required for rupturing the sample. also, average crushing force (cf) and crispiness work (cw) were measured using the below equations (igual et al., 2020): n np d = sme was calculated using the following formula (oliveira et al., 2017a), the motor torque was recorded for each treatment displayed in the monitor panel. sme (wh/kg) screws peed (rpm) motor power (kw) torque (%) kg maximumscrewspeed (rpm) mass flow rate 100 h = × ×  × ×    where motor power = 4000 w, and maximum screw speed = 682 rpm. techno-functional parameters physical properties piece density (pd) piece density of breakfast cereals was measured according to the protocol followed by seker (2005). a 250 ml graduated cylinder was filled with 4 g of the sample. the cylinder was filled by adding mustard seeds. the extrudates were then removed, and the volume of leftover mustard seeds was measured and noted. piece density was calculated as follows: mass of extrudates 100 volume of extrudate pd s (g/ml) = × longitudinal expansion index (lei) the lei of the extruded products was determined by the method explained in by alvarezalvarez-martinez et al. (1988). it is the ratio of the velocity of expanded extrudates to the velocity in the die orifice, which is indicated as follows: d d e e 1 m1 ler (%) ser 1 m ρ ρ    −=    −   where, ρd is the density of melt behind the die (ρd= 1400 kg/m3), and ρe is the extrudate density. me is the moisture content of extrudates, and the moisture content of the melt (md) was measured by drying 2–3 g of the samples in an oven at 105°c until constant weight was achieved. water activity (a w ) water activity is a dimensionless number representing the ratio of vapour pressure of the sample to that of pure water at a given temperature. aw of flour and extrudates was measured using a water activity meter (novasina ag ch-8853, lachen). 1.0 g of flour was kept in the sample cup of water activity meter. the lid was closed and the sample was allowed to equilibrate, read as aw. 110 italian journal of food science, 2022; 34 (3) allai fm et al. et  al., 2020). bd was calculated by using mass/volume relationship as given below: g weight of sample (g) bulk density mll volume of sample after tapping (ml)   =    swelling capacity (g/ml) swelling capacity was determined by following the method of okaka and potter (1977) with some modifications. a 100 ml graduated cylinder was taken and filled with sample to the 10 ml mark. distilled water was added to adjust the total volume to 50 ml. the top of the measuring cylinder was covered tightly, and the solution was mixed by inverting the cylinder. after 120 s, the suspension was again inverted and allowed to rest for 30 min. the final volume occupied by the sample was noted after 30 min. shelf-life studies the optimised product was packed in aluminum-laminated (al) pouches and ldpe, and stored at 25°c for 6 months. the stored samples were analysed for water activity, moisture, hardness, crispiness, free fatty acids (ffas), peroxide value (pv), total plate count and overall acceptability. moisture (%) moisture content was determined by the oven method, as per the protocol described by aoac (2005). free fatty acid (%) ffa was estimated by the standard procedure of aoac (1973), with some slight modifications. 5 g of sample was placed in a 250 ml flask, and to this 50 ml of benzene was added. the mixture was kept undisturbed for 30 min for extraction of ffa. then, 5 ml of the extract was taken to which 10 ml of alcohol, 5 ml of benzene and two drops of phenolphthalein indicator were added. it was then titrated against 0.02 n koh till the light pink colour disappeared. ffa was expressed as the percentage of oleic acid, and was calculated using the following formula, ( )ffa % of oleic acid 282 0.02 n koh ml of alkali used dilution factor 100 1000 weight of sample taken = × × × × × peroxide value (%) 30 ml of acetic acid chloroform solution and 0.5 ml of potassium iodide was added to 5 g of sample, and the mixture was allowed to stand for 60 s with occasional shaking. 30 ml of distilled water was added to the mixture. this was then titrated against 0.1 n sodium thiosulphate solution with constant swirling till the yellow colour disappeared. 0.5 ml of starch solution as indicator where, n = number of peaks d = distance travelled by the probe s cw n = where, s = area under deformation curve (upto 50% deformation) s cf d = total phenolic content the procedure by allai et al. (2022b) was used to calculate the total phenolic content in the extrudates. the extraction process was carried out using methanol as solvent.1 g of extrudate was homogenised in 30 ml of methanol. the mixture was placed into an ultrasonic water bath for 15 min. then the homogenate was left undisturbed for 12 h. the resulting mixture was centrifuged for 10 min at 3500 rpm. 0.5 ml of the extract was taken after centrifugation and blended with 2.5 ml of folin–ciocalteu reagent and incubated at room temperature for 5 min. the mixture was then mixed with 2 ml of 7.5% na2co3 and kept aside for 1 h at 25°c. after 1-s reaction time, the absorbance at 750 nm was measured using a spectrophotometer. gallic acid was used to make a calibration curve, and the total phenolic content was expressed as mg gae/100 g of dry sample. antioxidant activity the antioxidant activity of the samples was estimated by the dpph radical scavenging assay according to the method by zhang et al. (2018) with a slight modification. methanol was used to prepare 0.1 mm of dpph solution. extraction of different concentration was made, followed by the addition of 5 ml of dpph solution and was mixed properly. the mixture was left undisturbed for about 45 min in dark at 25°c, and absorbance of the sample at 517 nm was measured. dpph radical scavenging activity was calculated using the following equation: control sample sample a a 100 % inhibition a − × = where, acontrol and asample are the absorbance values of the control and the sample, respectively. techno-functional properties of flours bulk density (bd) bd was measured by filling pre-weighed sample in a 100 ml graduated cylinder. the base of cylinder was gently tapped till a constant volume was achieved (adeloye italian journal of food science, 2022; 34 (3) 111 wholegrain-based breakfast cereals where, a and b represent the average score given to the product and the maximum score obtained for the product, respectively. a product that obtains an ai score of 70% is considered a good product (gusmao et al., 2019). statistical analysis spss (version 20) statistical software was used to analyse the data acquired throughout the studies. the results were expressed as mean ± standard deviation. duncan’s multiple range test and analysis of variance (anova) were used to find a significant difference at (p < 0.05), among the means of samples. results and discussion a fit of models anova was used to select the appropriate models for different dependent responses. statistics of fit summary suggested quadratic models for sme, pd, whc, sv, l*, and a*; and linear models for lei, aw and b*. the regression models were significant (p < 0.05) with a high coefficient of determination (r2 = 0.99–0.77) for all the responses. these dependent variables were significantly influenced by feed composition, feed moisture content, barrel temperature and screw speed. the predicted and adjusted r2 were observed to be in reasonable agreement, and the regression models showed less than 1.91–7.84% of coefficient of variation (cv), suggesting high precision and reproducibility of obtained results. surface mechanical energy sme is the key parameter that determines the molecular degradation or breakdown of ingredients received during the extrusion process (lee et al., 2022). higher sme indicates a rapid conversion of starch, resulting in an increased puffing of extruded products (jabeen et al., 2021). the values of sme varied between 110 and 152 wh/kg for wholegrain breakfast cereals enriched with ihcf (table 3). the fitted regression analysis exhibited quadratic coefficients of feed composition, feed moisture content, barrel temperature and screw speed in extrudates (table 1). the regression analysis showed that barrel temperature had the most dominant effect on sme (table 1) among all parameters. table 1 shows anova results for the fit of data to response surface models. it was noticed that the feed moisture content (x2) and barrel temperature (x3) were inversely proportional to sme. with an increment in x2 and x3, a reduction in sme was recorded, whereas was added and titrated until the blue colour disappeared. blank was also determined. pv was expressed as meq/kg and was calculated using the following formula (tatledgis et al., 1960): titre n 100 pv weight of the sample (g) × × = where, titre = sample reading–blank reading n = normality of sodium thiosulphate solution microbiological analysis total plate count the total plate count of samples was carried out after every 1 month of storage interval, up to 6 months. samples were evaluated for total fungi and bacteria by using the standard serial dilution method. 10 g of grounded sample was dissolved in 90 ml of water and stirred for 5 min. after stirring, the aliquots were serially diluted; 1 ml in 9 ml of sterile saline was prepared in test tubes, and 0.1 ml of dilution was transferred aseptically on sterile plates using nutrient agar as media. the plates were rotated gently for uniform spread of the inoculum before the media solidified. the plates were then incubated at 37°c for 3–7 days. the number of colonies developed on each plate of different dilutions was counted using the digital colony counter and is calculated as: number of colonies dilution factor cfu/g volume of sample used (ml) × = sensory analysis twenty-five consumers who take breakfast cereal daily (15 females and 10 males) were selected randomly. samples were served to the consumers in a three-digit coded manner and were carried out on a nine-point hedonic scale (1 meant ‘dislike very much,’ and 9 denoted ‘like very much’). consumers were guided to rinse their mouths after the consumption of every different treatment to determine different attributes (texture, taste and overall quality) of the extrudates. overall acceptability of breakfast cereals was evaluated as the average score of sensory attributes determined. the purchase intent of each sample was questioned using a five-point scale (5 = certainly would buy, 3 = might or might not buy, 1 = definitely would not buy) to complement the acceptance results. the acceptability index (ai) was calculated using the following formula a ai (%) 100 b = × 112 italian journal of food science, 2022; 34 (3) allai fm et al. x1, x2, x3, x4, x2x4 x3x4, x1 2, and x2 2 were significant at p < 0.01, whereas the model terms x1x2, and x2x3 were non-significant. as evidenced in table 3, the feed composition and feed moisture content showed a positive linear effect, whereas the barrel temperature and the screw speed exhibited negative linear effect on the pd of extrudates. rsm (figure 1) illustrates the effect of independent parameters on pd. . the increase in pd of extrudates with the increased feed composition might be due to the presence of fibre in ihcf that enhances the extrudate density. the fibre tends to breakdown the air cells, which reduces extensibility and expansion, resulting in higher density of extrudates with less porous structure (dos santos et al., 2019). this suggests its suitability and applicability for the development of food products. high feed moisture content also decreases frictional force between screw and mixture leading to reduced expansion and enhanced density (bisharat et al., 2013). the extrusion process does not evaporate whole moisture content at the exit die point. so, the retention of some of the water content makes the product denser with decreased puffing (asare et al., 2004). an increase in barrel temperature and screw speed causes a reduction in the density of the extruded products. this might be because of the higher barrel temperature that enhances the degree of starch gelatinization and also the generated superheated vapours that produce a more expanded structure with lighter weight products (samray et al., 2019). additionally, an increased shear rate disintegrates the structure of macromolecules of proteins and starches that subsequently weaken the structure, leading to a reduced density with increased screw speed (bisharat et al., 2013). figure 1 showed the higher feed composition (x1) and screw speed (x4) showed increased sme values. an increase in feed composition along with an increasing sme was mainly due to the increased starch content of ihcf (meuser et al., 1990). furthermore, the presence of fibre in wholegrain flours increases sme as high fibre content has more water binding affinity and could dilute the concentration of starch in the mixture (singha et al., 2018). feng and lee (2014) reported that an increase in feed moisture content causes a reduction in the viscosity of feed ingredients. viscosity is temperature dependent, and reduction of barrel temperature can reduce the gelatinization of starch, resulting in enhanced viscosity (karkle et al., 2012). thus, increasing barrel temperature causes a reduction in viscosity leading to a decrease in torque and sme values (kesre and masatcioglu, 2022). kaur et al. (2015) described that with an increment in screw speed, sme progressively enhanced due to the high shear force and less residence time that induces viscosity, starch conversion and high sme. the cv assessed the relative dispersion of the experimental points, that is, 1.91% for developed breakfast cereals (table 1). r2, a coefficient of determination, indicated that the correlation among the selected processing conditions for extrudates was good, which showed a value of 0.90. piece density (pd) the pd for developed extrudates was observed to be in the range of 4.03–7.23 g/ml (table 3). the fitted model for pd is shown in table 1. in table 1, the model terms 8 7 6 5 4 380 p d ( g/ m l) 8 7 6 5 4 p d ( g/ m l) 370 360 350 340 d: screw speed (rpm)c: moisture (%) 330 320 380 370 360 350 340 d: screw speed (rpm) 330 320 12 13 14 15 16 c: temperature (˚c)90 100 110 120 130 figure 1. response surface plots of piece density as a function of whole wheat flour, corn flour, barley flour and indian horse chestnut flour. italian journal of food science, 2022; 34 (3) 113 wholegrain-based breakfast cereals and starch gelatinization, which may enhance the expansion of extrudates (yadav et al., 2021). as a result of the increase in speed, less residence time for material was observed, leading to less degradation of the material and higher expansion of extruded products (kaur et al., 2015). water activity (a w ) the aw of the extrudates prepared from blend of wholegrain flours and ihcf varied from 0.24 to 0.39 (table 3). the higher values of aw accelerated the rate of reaction in the food products (shah et al., 2017) that determined the shelf life of foods. fit statistics summary suggested a quadratic model for aw among all parameters. the feed moisture content had a significantly dominant effect (p < 0.01) on water activity. the impact of the feed moisture content on aw is presented by the 3-d surface plots in figures 2a,b. figure 2a showed a straight line with respect to temperature (110°c) along the axis of feed moisture content (14%). a similar observation is shown in figure 2b, where the variation of aw with respect to the feed moisture content (14%) also presents a straight line along the axis of screw speed (350 rpm). generally, water activity above 0.7 promotes microbial load (zhou et al., 2021). thus, from our study, all the extruded samples prepared under different treatment conditions fell in the safe category of shelf-stable products. colour analysis the colour values of extruded products are displayed in table 3. the regression analysis for l*, a* and b* values of extrudates are presented in table 1. the regression negative interaction effect of x2x4 and x3x4. similar results for the interaction terms have been reported by jabeen et al. (2021) in corn-water chestnut extrudates and by pansawat et al. (2008) in fish rice–based snacks. longitudinal expansion index lei values of extrudates varied from 0.22–0.35 (table 3). the regression analysis revealed that the feed composition and feed moisture content had a significantly (p < 0.05) negative linear effect (table 1). this might be due to the enhanced water content in the melt that would soften the molecular structure of amylopectin, reducing its elasticity and thus decreasing the longitudinal expansion (alvarez-martinez et al., 1988). it can be well described by the fact that low moisture content promotes drag forces that enhance the die pressure, leading to more expansion of the developed products (kaur et al., 2022). higher feed moisture content induces a lubrication effect, decreasing the internal barrel temperature as well as the shear rate of the extruder. as a result, a reduction in cooking of the ingredient and expansion can take place (da silva et al., 2014). the addition of ihcf caused a negative effect on the product expansion rate as the presence of fibre in the feed composition binds with some of the water content in the matrix and acts as an interference factor, thus decreasing its availability for longitudinal expansion (witczak et al., 2021). the lei values increased significantly as the barrel temperature enhanced (table 3). a similar trend was also observed for the screw speed response. the highest lei value (3.5) was observed at the highest barrel temperature (130°c), screw speed (380 rpm) and at the lowest feed moisture content (12%) and feed composition (2.5%) (table 3). the increased temperature inside the barrel causes superheating, which implies a higher degree of protein cooking 0.4 (a) (b) 0.38 0.36 0.34 0.32 0.3 0.28 0.26 0.24 130 120 110 100 90 12 13 14 15 16 c: temperature (˚c) b: moisture (%) a w 0.4 0.38 0.36 0.34 0.32 0.3 0.28 0.26 0.24 a w 380 370 360 350 340 d: screw speed (rpm) 330 320 12 13 14 15 16 b: moisture (%) figure 2. response surface plots of water activity as a function of whole wheat flour, corn flour, barley flour and indian horse chestnut flour. 114 italian journal of food science, 2022; 34 (3) allai fm et al. structure (wang et al., 2019a). the increased moisture content and temperature might increase the gelatinization of starch, where the granules of starch are disintegrated and more water remains bound to it, resulting in enhanced whc (wang et al., 2019b). a high shear rate causes disruption of molecular structure, which leads to increase in solubility and decrease in whc of extruded snacks (ek et al., 2021). the regression model elucidates positive linear as well as quadratic terms for x1, x2, and x3, while x4 shows a negative effect on sv. figure 3 shows that the variation of sv with respect to feed moisture content is curvilinear along the axis of barrel temperature. there was a positive correlation between sv and whc of extrudates (r = 0.98). the higher sv in extrudates has been ascribed to the changes in the fibre and starch fractions. this could lead to an increased change in the fibre integrity, resulting in a higher sv for the prepared extrudates (espinosaramirez et al., 2021). the negative interactive effect (p < 0.05) of moisture content and barrel temperature (x2x3) suggested that the moisture content (x2) had a dominant effect over barrel temperature (x3). optimization and model validation the highest desirability (0.713) was obtained on the basis of optimal solutions, suggesting that an ihcf substitution of 2.5%, a feed moisture content of 12%, a barrel temperature of 130°c, and a screw speed of 380 rpm were the optimum criteria to develop good quality sugar-free breakfast cereals with higher sme, lei, whc, sv, and low water activity and pd values (figure 4). table 4 depicts the optimum conditions applied for the optimisation of independent variables. the values for predicted responses were found to be similar to the actual values with less than 4.5% variation. equation for luminosity (l*) (table 1) showed significantly negative(p < 0.01) linear and quadratic effects of feed moisture content (x2) and barrel temperature (x3). it was observed that the barrel temperature and the feed moisture content had the most predominant effect that influenced all the three colour coordinates. the l*, a* and b*values of extrudates developed by different processing conditions were in the range of 63.13–71.64, 3.7–6.04 and 18.02–25.33, respectively. generally, extrusion cooking, that is, increasing feed moisture content and barrel temperature caused reduction in the luminosity (l*) and increments in a* and b* values (table 3). the presence of higher water content leads to the development of extrudates with dense air cells, packed together, which enhances the absorption of light and decreases the luminosity (nakhon et al., 2018). another parameter that affects the colour values is the increased barrel temperature that may cause loss of pigments, that is, non-enzymatic browning reactions such as caramelization and maillard reaction, which can occur during the extrusion process and change the colour of the ingredients (zhang et al., 2020), leading to a decline in the l* value and a subsequent increase in the a* and b* values (jabeen et al., 2022). during the extrusion, the degradation of pigment could also be used to measure the process intensity with regard to chemical and nutritional changes (devrajan et al., 2018). it is also an essential quality parameter as it indicates the degree of cooking as well as the level of chemical reactions that occur during extrusion process. water holding capacity and swelling volume the extrusion enhances the water holding and binding capacity of the extrudates (espinosa-ramirez et al., 2021). whc of extrudates varied between 4.22 and 8.14 g/g (table 3). the impact of independent variables on whc was measured using anova. the fitted model showed a highly significant linear, quadratic as well as interaction effect on whc at p ≤ 0.01, as shown in table 1. the positive effect of the feed composition content, moisture content and the barrel temperature indicates that whc increases with an increase in ihcf, moisture content and temperature, while the negative coefficient for the screw speed showed that an increase in shear rate causes a decrease in whc. the presence of fibre and starch in feed composition also led to a higher whc. fibre-rich flours have a higher whc and can be utilised as functional ingredients to modify the texture and viscosity and also avoid syneresis in food products (repo-carrasco-valencia et al., 2009). the proteins present in wholegrain flours and ihcf may be improved during extrusion treatment, by dissociation and unfolding of molecules that enhance the exposure of hydrophilic sites due to the variation in macromolecular 8 7 6 5 4 3 16 15 14 13 12 s v ( m l/ g dr y sa m pl e) b: moisture (%) a: ihcf (%)2.5 2.8 3.1 3.4 3.7 4 figure 3. response surface plots of swelling volume as a function of whole wheat flour, corn flour, barley flour and indian horse chestnut flour. italian journal of food science, 2022; 34 (3) 115 wholegrain-based breakfast cereals desirability 380 370 360 350 340 330 320 2.5 2.8 3.1 a: ihcf (%) d : s cr ew s pe ed ( r p m ) 3.4 3.7 4 desirability 0.713 0.65 0.6 0.7 0.55 figure 4. desirability plot. table 4. numerical optimisation. variable goal range importance values variation level (%) lower limit upper limit actual predicted ihcf is in range 2.5 4 3 feed moisture is in range 12 16 3 barrel temperature is in range 90 130 3 screw speed is in range 320 380 3 responses sme maximise 110.6 152.6 3 133.0 134.14 0.84 pd minimise 4.03 7.23 3 4.17 4.24 1.65 lei maximise 0.22 0.35 3 0.33 0.34 2.9 a w minimise 0.24 0.39 3 0.22 0.23 4.3 whc maximise 4.22 8.14 3 5.68 5.77 1.55 sv is in range 3.51 7.2 3 4.8 5.0 4 l* is in range 63.13 71.64 3 67.45 69.06 2.33 a* is in range 3.7 6.04 3 4.33 4.5 3.7 b* is in range 18.02 25.33 3 21.92 22.03 0.5 a*, redness; a w , water activity; b*, yellowness; l*, luminosity; lei, longitudinal expansion index; pd, piece density; sme, surface mechanical energy; sv, swelling volume; whc, water holding capacity. techno-functional properties of wholegrain flours, ihcf and optimised extrudates the functional properties of wholegrain flours and ihcf are depicted in table 5. the functional attributes define how a food material interacts with other food ingredients. it also determines its suitability for end use. thus, flour with good functional characteristics can be easily substituted for other foods and will yield good quality with acceptable end products. the bd of flour was similar in all flours, with a bd of 0.774 ± 0.52 in wwf, 0.722 ± 0.44 in wcf, 0.44 ± 0.26 in wbf and 0.6 ± 0.58 in ihcf. these results are similar to those reported by tangariya and srivastava (2022) for wwf, adedeji and tadawus (2019) for corn flour, hamdani et al. (2014) for wbf and shafi et al. (2016) for horse chestnut flour. the higher bd helps to enhance the weight of flour-supplemented foods without affecting the volume and the flours also favour their suitability in processing of different food products, while low bd helps in the preparation of complementary foods (awuchi et al., 2019). wwf had slightly higher swelling power (0.85 ± 0.45 g/ml) than wcf (0.79 ± 0.43 g/ml), wbf (0.7 ± 0.33 g/ml) and ihcf (0.66 ± 0.27 g/ ml) (adegunwa et al., 2014; chaudhary et al., 2018). wwf was characterised by a higher hydration capacity and hydration index (6.72 g/g and 3.36 g/g, respectively) with relative to wcf (5.19 g/g and 2.5 g/g), wbf (4.6 g/g and 2.3 g/g) and ihcf (2.33 g/g and 1.16 g/g). these findings are consistent with the literature (adegunwa et al., 2014; boucheham et al., 2019; rafiq et al., 2021). the differences in functional properties might be due to the variation in the particle size of the flour, their variety and the milling process (das et al., 2019). table 5 depicts the functional properties of optimised product (extrudates). a significantly higher (p < 0.05) bd (4.95), hydration capacity (6.03), hydration index (3.015) and swelling power (0.91) were observed in the developed extrudates than native wholegrain flours and ihcf. the increased capacity of extruded flours to hold water 116 italian journal of food science, 2022; 34 (3) allai fm et al. table 5. functional, colour and textural attributes of individual flour, their blends and optimised extrudates. parameters wwf wbf wcf ihcf blend optimised extrudate techno-functional characteristics bulk density (g/ml) 0.77 ± 0.52b 0.44 ± 0.58f 0.722 ± 0.26c 0.6 ± 0.58e 0.69 ± 0.37d 4.95 ± 0.32a swelling power (g/ml) 0.85 ± 0.45c 0.7 ± 0.33e 0.79 ± 0.43d 0.66 ± 0.27f 0.85 ± 0.31b 0.91 ± 0.52a hydration capacity (g/g) 6.72 ± 0.25a 4.6 ± 0.55e 5.19 ± 0.18d 2.33 ± 0.10f 5.24 ± 0.05c 6.03 ± 0.44b hydration index 3.36 ± 0.03a 2.3 ± 0.05e 2.5 ± 0.06d 1.16 ± 0.11f 2.62 ± 0.06c 3.01± 0.13b colour attributes l* 79.14 ± 0.22c 72.35 ± 0.47e 83.83 ± 0.1b 90.27 ± 0.06a 77.34 ± 0.35d 69.7 ± 0.32f a* 3.09 ± 0.04d 4.29 ± 0.07c 1.09 ± 0.1e 3.09 ± 0.06d 4.33 ± 0.17b 4.53 ± 0.23a b* 15.65 ± 0.20c 16.04 ± 0.17b 12.70 ± 0.3f 12.97 ± 0.06e 13.21 ± 0.65d 23 ± 0.17a textural properties hardness (n) 199.29 ± 0.34 crispiness 56 ± 0.27 crispiness work 1.86 ± 0.47 average crushing force 5.1 ± 0.31 total phenolic and antioxidant content total phenolic content (mg gae/g) 29.38 ± 0.33b 20.02 ± 0.46c 39.78 ± 0.21a 10.66 ± 0.17e 17.24 ± 0.02d 5.87 ± 0.14f antioxidant activity (%) 25.72 ± 2.11f 68.21 ± 1.77b 70.31 ± 2.52a 63.55 ± 0.32c 42.77 ± 2.53d 18.33 ± 0.42e wwf, whole wheat flour; wbf, whole barley flour; wcf, whole corn flour; ihcf, indian horse chestnut flour; wwf:wbf:ihcf:wcf 10:10:2.5:77.5, blended flour. values are mean ± sd. values with different superscripts within same column differ significantly (p < 0.05). content upon rehydration as compared to raw flours might be due to the disintegration of the starch granules or due to the molecular disruption as a result of the shear stress and the thermal extrusion process (martínez et al., 2014). the phenolic compounds act as antioxidants and perform an essential role in stabilising the free radicals. the total phenolic content and the antioxidant activity (dpph radical scavenging) of raw flours, their blends and optimised extrudates are presented in table 5. wcf had the highest total phenolic content and antioxidant activity among the flours, with a total phenolic and antioxidant contents of 39.78 mg gae/g and 70.31, respectively (lopez-martinez et al., 2009; oboh et al., 2010). the total phenolic content of wwf, wbf, ihcf and their blends was 29.38, 20.02, 10.66 and 17.24 mg gae/g, respectively (abozed et al., 2014; baba et al., 2016; shafi et al., 2016; zengin et al., 2017). the antioxidant activities of wwf, wbf, ihcf and their blend were 25.72 (%), 68.21 (%), 63.55 (%) and 42.77 (%), respectively (abozed et al., 2014; horvat et al., 2020). after extrusion, the total phenolic and antioxidant content of optimised extrudates were reduced as compared to raw flours (table 5). the reduction in the total phenolic content and antioxidant activity after extrusion might be attributed to the changes in the molecular structure of bioactive compounds, leading to the reduction in extraction efficiency and chemical reactivity due to the development of polymerised products (pandey et al., 2021). the colour characteristics of wwf, corn flour, wbf, ihcf and optimised extrudates are presented in table 5. generally, extrusion reduces the luminosity (l*) of flour with an increase in a* and b* attributes. the results of this work are in accordance with the previous literature reports and can be mostly ascribed to the degradation of pigments and maillard reaction during extrusion processes (brahma et al., 2016; jafari et al., 2017). for example, carotenoids present in whole grains are heat unstable pigments that are lost into colourless compounds during extrusion (kadian et al., 2013). thus, after extrusion, the colour developed may influence the extrudate acceptability of these flours. textural analysis textural parameters of extruded products depend largely on the ingredient composition and their blend. it is an essential physical property of extrudates. the texture parameters for optimised extrudates are shown in table 5. the hardness, crispiness, crispiness work (cw) and average crushing force (cf) values of optimised ihcf blended with wholegrain flours produced at minimum feed moisture content (12%) and highest barrel italian journal of food science, 2022; 34 (3) 117 wholegrain-based breakfast cereals food safety and standards authority of india (fssai) for dehydrated snacks. the increase in ffa of extrudates packed in al pouches was non-significant for 120 days. the samples packed in ldpe had a significant effect that might be due to the property of al pouches that acts a barrier for the transfer of light, which is mainly responsible for the rancidity of product. furthermore, the lipid hydrolysis leads to the disintegration of longchain ffa into single fatty acids (syed et al., 2019) and also higher permeability to oxygen and water vapour resulting in higher values of ffa in ldpe as compared to al pouches. also, mold growth was absent during the entire period of storage for all the samples either packed in ldpe or al pouches. the reason for low total plate count could be attributed to the low moisture level. the total plate count was too low to count (less than 25 colonies/plate) up to 180 days of storage period. jabeen et  al. (2021) also reported similar results in total plate count of corn-based extrudates enriched with water chestnut during storage period. peroxide value indicates the amount of rancidity because of the oxidation process during storage. it can be observed from figure 5d that the pv of the breakfast cereals increased significantly (p < 0.05) from 0.28 to 0.59 meq/100 g in extrudates packed in ldpe and 0.47  meq/100 g for those packed in al pouches after 180 days of storage. the increase in pv is due to the poor oxygen and moisture barrier property of ldpe in comparison to that of al pouches (raleng et al., 2019). the pv in this study was found to be under the permissible range (<10 meq/kg) given by fao. the hardness (figure 5e) of the breakfast cereals increased from 199.29 to 217.56 n for the extruded product packed in ldpe and 199.29 to 208.56n for the extrudates packed in al pouches. the packaging materials significantly affected (p < 0.05) the hardness of the extrudates during the storage period of 180 days. the hardness values increased due to the increment in water content of the extruded products, thereby modifying the balance in bonds in starch granules. however, there was a non-significant change during the initial months of storage. a similar trend was reported by badding-smithey et  al. (1995) for beef-based extrudates enriched with carrot pomace powder. the crispiness of the extrudates packed in ldpe and al pouches reduced from 56 to 51.32 and 56 to 55.32, respectively (figure 5f). the crispiness of dehydrated foods like snacks, breakfast cereals, chips and crackers is desirable, but the excess absorption of moisture in samples causes sogginess and finally the rejection of extrudates (dar et al., 2014). during storage, absorption of moisture could be due to the packaging material and storage conditions (badding-smithey et al., 1995). moisture gain reduces the storage shelf life and stability of the product. temperature (130°c) and screw speed (380 rpm) were found to be 199.29 n, 56, 1.86 and 5.1, respectively. crispiness work (cw) can be inferred as the work that is needed to break one pore or group of pores, and it is also the sensory attribute of fracturability. average crushing force (cf) is defined as the force required to compress a solid substance and depends mostly on the sensory perception such as hardness during chewing (igual et al., 2020). lower moisture content (12%) had a positive effect on the hardness and crispiness of the extrudates as low water content increases the sme and viscosity, producing soft extrudates with higher crispiness. kesre et al. (2022) reported that higher screw speed and barrel temperature exhibited a inverse relationship with hardness and direct relationship with expansion and crispiness. the expanded extrudates are more puffed with thinner cell walls that results in the easy crushing of extrudates under compression (yao et al., 2006). furthermore, the availability of low residual moisture content during extrusion cooking and the increment in the degree of gelatinization and superheating of water might favour more expansion with softer texture and also improves the crispiness of extruded products (guha and ali, 2006; wang et al., 2005). the results of our work are in agreement with the published literature, which explains that the crispiness of extrudates has a positive and strong correlation with expansion. storage studies the shelf-life stability of breakfast cereals indicates that the storage days and the packaging materials had a considerable influence on the ffa, pv, moisture content, water activity, hardness, crispiness and overall acceptability. the moisture content of the samples packed in ldpe and al pouches was initially 3.04% that enhanced to 5.12 and 3.7%, respectively, during 180 days of storage (figure 5a). the temperature, relative humidity (storage environment) and hygroscopic nature of extruded products, as well as the nature of packaging material, increase the moisture content during storage (sharma et al., 2004). the extrudates packed in ldpe showed increased moisture content compared to al pouches due to its lower barrier characteristic. jan et al. (2017) also reported the quick gain of moisture content in gluten-free extrudates packed in al and ldpe packages. water activity of extrudates was observed to be increased from 0.3 to 0.55 and 0.42 when packed in ldpe and al pouches, respectively (figure 5b) (hussain et al., 2017). the ffa change was noticed in the extrudates with storage period in both the packaging materials. the change in ldpe was higher (0.04–0.32%) as compared to al pouches (0.04–0.21%) (figure 5c). these values were well within the acceptable range of 0.5% as reported by 118 italian journal of food science, 2022; 34 (3) allai fm et al. 3 3.5 4 4.5 5 5.5 (a) 0 2 4 6 m oi st ur e (% w b) storage (months) ldpe al 0.3 0.35 0.4 0.45 0.5 0.55 (b) (c) (d) 0 2 4 6 8 w at er a ct iv ity storage (months) ldpe al 0.04 0.09 0.14 0.19 0.24 0.29 0.34 0 2 4 6 fr ee f at ty a ci d (% ) storage (months) ldpe al 0.25 0.35 0.45 0.55 0 2 4 6 p er ox id e va lu e (m eq /1 00 g) storage (months) ldpe al 199 204 209 214 219 (e) (f) (g) 0 2 4 6 h ar dn es s (n ) storage (months) ldpe 49 50 51 52 53 54 55 56 57 0 1 2 3 4 5 6 c ri sp in es s storage (months) ldpe al 7 72 3.5 8.2 78.58 4 0 20 40 60 80 overall acceptability acceptability index (%) intention to purchase averages of acceptance of extrudates packed in ldpe and al pouches ldpe al figure 5. effect of storage period and packaging material on (a) moisture content, (b) water activity, (c) free fatty acids, (d) peroxide value, (e) hardness, (f) crispiness and (g) over all acceptability of optimised extrudates packed in ldpe and al pouches. italian journal of food science, 2022; 34 (3) 119 wholegrain-based breakfast cereals technology, skuast–kashmir and post-harvest engineering and technology, amu, india for providing the necessary facilities to conduct the study. references abozed, s.s., el-kalyoubi, m., abdelrashid, a. and salama, m.f., 2014. total phenolic contents and antioxidant activities of various solvent extracts from whole wheat and bran.  annals of 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resveratrol with anti-diabetic, anti-obesity and antioxidant properties.  food chemistry. 352: 129323. https://doi.org/10.1016/j.foodchem.2021.129323 allai, f.m., azad, z.r.a.a., dar, b.n., gul, k. and jabeen, a., 2022a. breakfast cereals from whole grain and indian horse chestnut flours obtained through extrusion: physical, mechanical and functional characteristics. applied food research. 2(2): 100137. https://doi.org/10.1016/j.afres.2022.100137 allai, f.m., azad, z.r.a.a., gul, k. and dar, b.n., 2021. wholegrains: a review on the amino acid profile, mineral content, physicochemical, bioactive composition and health benefits. international journal of food science & technology. 57(4): 1849–1865. https://doi.org/10.1111/ijfs.15071 allai, f.m., dar, b.n., gul, k., adnan, m., ashraf, s.a., hassan, m.i., pasupuleti, v.r. and azad, z.r.a.a., 2022b. development of protein rich pregelatinized whole-grain cereal bar enriched with non-traditional ingredient: nutritional, phytochemical, textural and sensory characterization.  frontiers in nutrition. 9: 870819. https://doi.org/10.3389/fnut.2022.870819 alvarez-martinez, l., harper, j.m. and kondury, k.p., 1988. a general model for expansion. journal of food science. 53(2): 609– 615. https://doi.org/10.1111/j.1365-2621.1988.tb07768.x aoac, 1973. official and tentative methods, 3rd ed. american oil chemical society chicago, il, usa. ca 54-40, ed 8-53. aoac, 2005. official methods of analysis, 18th ed. association of official analytical chemists, 10th ed., gaithersburg, md, washington, dc, usa. asare, e.k., sefa-dedeh, s., sakyi-dawson, e. and afoakwa, e.o., 2004. application of response surface methodology the mean values for sensory attributes are presented in figure 5g. the optimised samples packed in ldpe and al pouches showed good scores in colour, texture, taste and overall acceptability. the overall acceptability for the samples packed in ldpe and al pouches ranged from 7.0 to 8.2, representing ‘like slightly’ to ‘like very much’ in terms of the hedonic scale. the test for an index of purchase indicates the probable buying of a product. samples packed in ldpe and al pouches showed non-significant variations, and the panelist suggested that the products are recommended to buy (4 scores). the acceptability index (ai) was evaluated based on the mean scores given by the judges, where the samples packed in al pouches reported the highest ai (78.58%), while samples packed in ldpe reported ai above 70% (figure 5g). a product having at least 70% of approval is considered to be acceptable (dutcosky, 2011). conclusion extrusion cooking is a versatile technology used to produce ingredients with improved functional and textural properties from whole grains and non-conventional flour. this study exhibited that rsm was used to optimise the process conditions and feed composition for the development of fibre-rich wholegrain-based extrudates using ihcf. furthermore, the incorporation of ihcf into wholegrain breakfast cereals could broaden consumer acceptability for a better phytochemical profile and hydration rate. results obtained from this study revealed that independent variables such as feed composition, moisture content, barrel temperature and screw speed had a considerable effect on all the dependent responses. the optimal conditions for the preparation of ihcf incorporated wholegrain-based breakfast cereals were 2.5% ihcf, 12% feed moisture content, 130°c temperature and screw speed of 380 rpm. the hardness and crispiness of the optimised extrudates were found to be 199.29 (n) and 56, respectively, which reported that extrudates enriched with ihcf at 2.5% level improves textural attributes. the antioxidant and total phenolic contents of optimised breakfast cereal were 30.36 (%) and 5.03 (mg gae/g), respectively, which indicated that breakfast cereal enriched with wwf (10%), wbf (10%), wcf (77.5%) and ihcf (2.5%) level improve the phytochemical profile for the consumer’s daily diet. the 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2020. effects of low moisture extrusion on the structural and physicochemical properties of adlay (coixlacryma-jobi https://doi.org/10.1016/j.procbio.2020.05.028� https://doi.org/10.1016/j.ijbiomac.2018.02.084� https://doi.org/10.1111/ijfs.15003� https://doi.org/10.1094/cc-83-0692� https://doi.org/10.1080/10942912.2016.1238391� _goback bbib6 paper ital. j. food sci., vol. 27 2015 221 keywords: conjoint analysis, consumer acceptance, consumer segmentation, nanotechnology, purchase intention, wine consumer perceptions of nanotechnology applications in italian wine n. casolania, g.m. greehy b, a. fantinia, e. chiodoa* and m.b. mccarthyb a facoltà di bioscienze e tecnologie agro-alimentari e ambientali, università degli studi di teramo, via c.r. lerici 1, 64023 mosciano sant’angelo (te), italia b department of food business and development, university college cork, western road, cork, ireland *corresponding author: tel. +39 0861 266898, fax +39 0861 266915, email: echiodo@unite.it abstract this paper examines italian consumer acceptance of nanotechnology applications in wine production, surveying wine consumers from the abruzzo region. conjoint and post-hoc segmentation analysis establishes how consumers value different wine product attributes and place them within the context of applications of nanotechnology. consumers appear relatively unfamiliar with nanotechnology applications, both generally and specifically to food. although, an overall rejection of the concept of “nano wine” is evident, low acceptance scores disguise a somewhat more open attitude to specific applications of the technology. in particular, consumers appear more receptive to applications that enhance certain wine attributes. practical implications are discussed. mailto:echiodo%40unite.it?subject= 222 ital. j. food sci., vol. 27 2015 1. introduction 1.1. nanotechnology applications in food and wine nanotechnology is the science that studies the manipulation of matter at atomic and molecular scales; a nanometre refers to one-billionth of a metre. nanotechnology is perceived to offer many potential benefits (mura et al., 2014), such as producing healthier foods without compromising taste (weiss et al., 2006). applications in food packaging and food contact material include microfilms that incorporate nanomaterials to improve packaging properties, e.g. flexibility and moisture stability, and “smart packaging” that incorporates nano-sensors that detect pathogens and contaminants in food (sorrentino et al., 2007; chaudhry et al., 2008). oberdörster et al. (2005) argue that the properties of materials at the nanoscale can differ considerably from conventional materials. therefore, nanotechnology-based foods have generated significant debate, particularly about potential associated risks (chaudhry et al., 2008; siegrist et al., 2008). specifically, concerns have been expressed regarding potential negative impacts of certain nanoparticles on the health of humans, animals and the environment (kuzma and verhage, 2006). furthermore, the fao/who (2009) argues that when the size of particles decreases, this increases the surface-to-volume ratio and therefore, creates new properties, potentially resulting in altered toxicity profiles. to date, a limited number of “nanofoods” appear to have been made available on the market (siegrist et al., 2008). that said, it is difficult to truly establish the extent of the application of nanotechnology in food and beverage production at present across international markets, as there is currently no legal requirement to declare the use of such ingredients on product labels. nevertheless, there is some indication of nanotechnology being applied within the food domain (momin et al., 2013; durán and marcato, 2013). focusing on the wine sector, nanotechnology could potentially be applied at the following stages of production: grape-growing, wine making and packaging. specifically, nano-compounds could improve grape growth when added to pesticides and fertilizers to increase soil fertility and crop production (allianz ag and oecd, 2005). furthermore, nanoparticle-based pesticides could be more easily absorbed by plants than conventional pesticides, or could equally be programmed to be released more gradually over time, thereby optimising their usage (ibid). hypothetically, yet plausibly, nanotechnology could be applied during wine making to alter the characteristics of the wine including its taste, flavour or other product characteristics, including the calorie or alcohol content of the wine (allianz ag and oecd, 2005; weiss et al., 2006; durán and marcato, 2013). other possible applications of nanotechnology during wine production include the use of nanotechnology-based devices and materials for nano-filtration and water treatment (momin et al., 2013). nanotechnology-based devices could also potentially improve surveillance systems and the tracking of products as they move through the supply chain (weiss et al., 2006), thereby enhancing authenticity measures. finally, pertaining to wine bottling, nanotechnology could be used to produce bottle caps that more effectively regulate gas exchange with the outside environment (durán and marcato, 2013). 1.2. consumer acceptance of nanofoods it is important to understand public perceptions of nanofoods (siegrist et al., 2008). however, these may be difficult to measure at present, as opinions may not yet have formed, given low levels of public awareness of nanotechnology (fell et al., 2009; siegrist, 2010). gaskell et al. (2010) found that approximately ½ of eu27 citizens (46%) and just over 1/ 3 of italian citizens (37%) were aware of nanotechnology. gaskell and colleagues also found that a significant minority (40%) of eu-27 citizens is likely to be unsure about their feeling towards applications of nanotechnology and that awareness generally resulted in more positive views regarding its safety. however, as more information becomes available through mass media, public attitudes will become more solidified (dudo et al., 2010). although several studies have found the impact of awareness on attitudes towards novel food technologies to be mixed (fell et al., 2009; sacchetti et al., 2009); kahan et al. (2007) found a positive relationship between awareness of nanotechnology in general and the belief that associated benefits outweigh potential risks. attitudes towards and, in turn, willingness to buy nanofoods may be influenced by general values, for example risk sensitivity and attitudes towards nature, the environment, science and technology (ronteltap et al., 2007; fell et al., 2009; stampfli et al., 2010). for example, numerous studies suggest that the dichotomy between nature and technology is important in determining receptivity (rozin, 2005; siegrist et al., 2008). in addition to naturalness, other product characteristics, including taste and price may impact consumer acceptance (fell et al., 2009). willingness to buy nanofoods is also strongly influenced by risk and benefit perceptions (stampfli et al., 2010). personal belief in the ability to control exposure to the technology may also influence acceptance (siegrist et al., 2008). http://en.wikipedia.org/wiki/atom http://en.wikipedia.org/wiki/molecular http://en.wikipedia.org/wiki/molecular ital. j. food sci., vol. 27 2015 223 consumers use intrinsic and extrinsic cues to form opinions regarding objective and subjective product quality (veale et al., 2006). grunert (2005), among others, notes that subjective hedonic characteristics, e.g. taste and pleasure, are important determinants of purchase and consumption decisions. this is particularly evident in the case of wine (olsen et al., 2007). wine purchase decisions are based on a complex array of factors including region of origin, grape variety and price (atkin et al., 2006; lockshin et al., 2006), in addition to other aspects including health and authenticity characteristics (chiodo et al., 2011; barreiro-hurlé et al., 2008). that said, given the hedonistic nature of wine, certain health characteristics may not have the same prevalence for wine as they do for other food products. furthermore, a greater focus by consumers on environmental aspects of wine production and distribution systems is emerging (remaud et al., 2008). elsewhere, cardello et al. (2007) and von schomberg and davies (2010) describe how the public may have concerns about novel food technologies, including nanofoods. these concerns, if not addressed, can lead to consumers rejecting these technologies and searching the supermarket shelves for products claiming to be “nano-free” (kuzma and verhage, 2006). some of the applications of nanotechnology in wine production outlined may be negatively perceived by consumers, due to perceptions of unnaturalness and tampering with winemaking traditions. potential concerns may also emerge in terms of the unknown health and environmental consequences of applying nanotechnology in wine production, as indicated in various studies (e.g. kuzma and verhage, 2006; chaudhry et al., 2008). nonetheless, potential associated benefits may be positively perceived. these includes benefits to: 1) consumers, for example improving the wine’s health characteristics (weiss et al., 2006) by, for instance, reducing its calorie or alcohol content; 2) industry, for example improving production processes, such as the bottling process (durán and marcato, 2013); and, 3) the environment, for example decreasing the use of pesticides during grape cultivation (allianz ag and oecd, 2005). in turn, this may lead to nanotechnology application to wine being acceptable to consumers and adopted by industry. following these considerations, the aim of this study was to understand the impact of the application of nanotechnology in wine production on consumers’ wine purchase intention. possible consumer reactions towards nanotechnology application to wine and varying determinants of consumer acceptance were explored, as well as the homogeneity of consumers’ responses. 2. materials and methods 2.1. overview the study involved wine consumers from the abruzzo region of italy completing a faceto-face administered questionnaire. an overall profile of respondents and also profiles using an a-priori segmentation variable (frequency of wine consumption) is presented. following this, consumers’ preferences are analysed using a conjoint analysis (ca) approach. the influence of production methods (conventionally produced versus produced using nanotechnology) and product attributes (e.g. associated with health and naturalness) on product preference are examined. within this study, conventional methods refer to production practices currently in place which comply with present pdo production regulations. “produced using nanotechnology” refers to the use of nanotechnology in any one or more phases of the production chain, e.g. during the cultivation of grapes or packaging of wine. conjoint and post-hoc segmentation analysis establishes how respondents value different wine attributes and place them within the context of the application of nanotechnology. both the a-priori and post hoc segments are profiled based on importance placed on different wine attributes, perceptions of different applications of nanotechnology to wine and demographic variables. the wine used within the experiment was “montepulciano d’abruzzo doc”, the predominant pdo wine in the abruzzo region and one of the largest wine denominations in italy. data collection was completed in october-december 2011. in total, 221 wine consumers completed the survey. no incentive was offered to respondents to complete the questionnaire. similarly to verdü jover et al. (2004), sample stratification was based on previous studies carried out which included a similar number of study items. the sample of wine consumers is representative of the regional population in terms of age and gender, based on demographic data provided by the italian national institute of statistics and referred to the same period (istat, 2014), as follows: 6% of women and 5.7% of men aged 18-24 years; 10% of women and 10.3% of men aged 25-34 years; 12.2% of women and 12.2% of men aged 35-44 years; 11.8% of women and 11.4% of men aged 45-54 years; and, 10.3% of women and 9.8% of men aged 55-64 years. 2.2. questionnaire respondents were screened to ensure: 1) they did not work in the agro-food sector; 2) purchased or consumed wine at least once a month on average; 3) were between the ages of 18 and 64; and, 4) were either an italian citizen or had 224 ital. j. food sci., vol. 27 2015 been living in italy for at least five years. the questionnaire, presented in italian, posed questions regarding frequency of wine consumption and habits; attitudes towards wine production and wine purchasing/consumption habits; factors that influence choice of wines; and, awareness of nanotechnology and its applications in food and beverage production. low levels of public awareness of nanotechnology, as previously outlined, presented a clear challenge in terms of deciding whether to present prior information about the technology to respondents. consequently, in designing this experiment, we looked to those who have examined consumers’ appraisals of novel food technologies in the past and best practice in terms of an appropriate ca approach (e.g. siegrist et al., 2009; schnettler et al., 2012). an underlying principle of conjoint analysis research is that it should be as realistic, reasonable and understandable as is feasibly possible (cox et al., 2008). thus, similar to siegrist et al. (2009), our study was conducted in terms of a “virtual market”, i.e. what consumers would do if they were informed (via a label) that a product is produced using nanotechnology and had some prior awareness of the concept of nanotechnology. therefore, following the aforementioned general questions, in the context of ensuring a minimal level of awareness of nanotechnology among respondents in advance of completing the ca experiment, a brief (neutral) definition of nanotechnology and its potential food applications (appendix 1) was presented. the definition provided is similar in content and structure to that which was included in siegrist et al.’s (2009) study. following the provision of this definition, the 10 wine labels (based on the conjoint analysis profiles generated see section 2.3) were presented for scoring. attitudes towards the use of nanotechnology in wine production were then measured. specifically, questions were posed regarding attitudes towards the use of nanotechnology in wine production in general and attitudes towards different applications in wine production for a variety of purposes. finally, demographic information was gathered. all statements and associated scales are summarised in table 1. 2.3. conjoint analysis conjoint analysis (ca), a market research approach used to support product and service design, has been widely applied to consider the impact of different product attributes on food and beverage purchase decisions (makokha et al., 2006; szolnoki et al., 2010). ca assumes that consumers are able to evaluate a range of products/services along key dimensions, called factors (attributes) and involves constructing a series of different product profiles (concepts) that represent a possible product or service. in the case of this research, the ca experiment involved different combinations of information about wine that may (or may not) be modified using nanotechnology, i.e. different profiles. the aim of this approach is to estimate the importance of each factor (product attribute) presented to consumers. for categorical product attributes, the utility function consists of part-worth estimates for each level of the attribute. market simulation models use this information to predict how each respondent would choose among alternative products. therefore, ca enables an understanding of how people make choices between products or services across different combinations of levels and attributes. the ca method has several advantages, including the possibility to measure consumer preferences for each attribute level using more realistic decision models (schaupp, 2005). using ca, the researcher can answer questions such as what product attributes are important/ unimportant to the consumer. ca has previously been applied to explore consumer perceptions of the application of specific novel food technologies (e.g. ares and gambaro, 2007; bech-larsen and grunert, 2003; cardello et al., 2007; cox et al., 2008; hailu et al., 2009; schnettler et al., 2012; annunziata and vecchio, 2013), including nanotechnoloappendix 1: definition of nanotechnology presented to respondents in advance of conjoint analysis experiment (english version) “new and advanced technologies with applications in food are constantly being developed. nanotechnology is one such technology, which deals with nanoparticles (particles that are 100 nanometres or less in dimension). a nanometre is one-billionth of a metre. a sheet of paper is about 100,000 nanometres thick. some nanoparticles are naturally occurring, for instance, it is nano-size particles that make milk appear white. materials can possess new properties at this nanoscale and this technology makes interesting innovations possible in food. nanotechnology, potentially, has widespread applications in food, including uses in food products, processing and packaging. it can be used to make food products with additional benefits such as better availability of vitamins or longer shelf-life without altering the taste, appearance or texture of food. however, possible consequences or risks of using nanotechnology for humans and the environment are largely unknown. on the one hand, additional benefits may enhance our health and improve products. on the other hand, the use of nanotechnology in food stuffs may be associated with potential risks”. ital. j. food sci., vol. 27 2015 225 gy (siegrist et al., 2009), and associated product attributes. furthermore, various ca studies have explored preferences for different wine attributes (e.g. gil and sánchez, 1997; atkin et al., 2006; martínez-carrasco et al., 2006) including, for instance, price, origin and grape variety/vintage. bearing in mind the attributes examined across these ca studies, within this work, a full profile conjoint analysis was applied in order to determine consumers’ preference (purchase intention) for the following wine attributes: price, method of production and benefits. the conjoint experiment was generated using spss 19. product profiles were presented as wine labels with different information included on each label (appendix 2 includes an example of one of the labels). the text included in each wine label was presented in italian. table 1. type of questions question or statements posed/ attributes listed scales source frequency of consumption. how often do you consume wine on average? 4-point frequency scales (1 is “everyday” and 4 is “at least once a month”) developed by researchers. wine consumption habits. i always drink the same variety of wine. i always drink wine produced in my region. i always drink wine from the same territory. 7p o i n t l i k e r t scales ( 1 i s “ d i s a g r e e strongly” and 7 is “agree strongly”) developed by researchers and adapted from seghieri et al. (2007). attitudes towards wine production and wine purchasing/ consumption habits. wine is an important part of italians’ culture. i am proud of italian tradition in wine production. i spend a lot of time deciding which bottle of wine to purchase. 7p o i n t l i k e r t scales ( 1 i s “ d i s a g r e e strongly” and 7 is “agree strongly”) developed by researchers. attr ibutes influencing wine choice. how important are each of the following when selecting wine? region of production; brand; type of cork; price; age of the wine; grape variety; packaging; territory of origin; alcohol content; sulphite content; and, calorie content. 7-point importance scales (1 is “extremely important” and 7 is “extremely unimportant”) developed by researchers and adapted from and gil & sánchez (1997) and atkins & johnson (2010). awareness of nanotechnology. have you ever heard of nanotechnology? have you ever heard of nanotechnology being used in food and beverage production? yes/no developed by researchers. nanotechnology acceptance. i do not want nanotechnology to be applied in wine production. i consider the use of nanotechnology in wine production to be acceptable. i would be happy to consume wine produced using nanotechnology. 7p o i n t l i k e r t scales ( 1 i s “ d i s a g r e e strongly” and 7 is “agree strongly”) developed by researchers. acceptance of nanotechnology applications in wine production. how acceptable do you consider it to use nanotechnology to: produce lower calorie wine. produce lower alcohol content wine. modify the colour of wine. modify the structure and properties of the cork. enhance the taste of wine. reduce the amount of pesticides used when growing the grape. produce less expensive wine. enhance authenticity. 7-point acceptance scales (1 is “extremely unacceptable” and 7 is “extremely acceptable”) developed by researchers. overview of questionnaire statements and associated scales. http://www.sciencedirect.com/science/article/pii/s0950329305001461 226 ital. j. food sci., vol. 27 2015 similar to o’ connor et al. (2005), sorenson and bogue (2006) and siegrist et al. (2008), a ten-point purchase intention rating scale was used to measure purchase preference. assigning a score from 1 to 10, based on willingness to purchase the product, emulated a real-life wine purchase situation. a rating, rather than ranking, scale was considered most suitable as the former “avoid[s] validity and reliability problems as a consequence of the large number of concepts presented to respondents for evaluation” (sorenson and bogue, 2006: 705) the wine attributes that varied across the profiles are outlined in table 2. in order to make the conjoint labels presented were as realistic as is feasibly possible (cox et al., 2008; siegrist et al., 2009), the labels included additional standardised information. this approach is not novel, as several other ca studies (e.g. laboissière et al., 2007) have included additional attributes in their experiments, which were not then included in the ca plan. each of the labels contained the following standardised information: • name of the producer: “azienda agricola la collina” • designation of origin: “montepulciano d’abruzzo doc” • product description: “this red wine is ideal to serve with roast meat. serve at 18-20°c” the product attributes (e.g. price) that varied were the specific focus of consideration. in terms of the variable attributes, the selected price levels (€5.99 and €11.99) are reflective of two different price segments; premium and super-premium wines, as recommended by heijbroek (2003). furthermore, they are representative of the price points for several brands of montepulciano d’abruzzo wine currently offered in italian supermarkets. where the wine was not produced using nanotechnology, i.e. was produced using conventional methods, the method of production was not stated on the label. in many conjoint studies applied to food labelling (e.g. silayoi and speece, 2007; cox et al., 2008), the level “absence of information” or “no claim” is included for certain attributes. this results in various degrees of information being included on the different product labels (i.e. for some of the product profiles). this lack of information for certain attributes is reflective of real life purchase situations. in comparison, when produced usappendix 2: example of a wine label used in the conjoint experiment (english and italian versions of profile 8). table 2. attribute level 1 level 2 level 3 level 4 price €5.99 €11.99 method of production conventionally produced produced using (method of production nanotechnology not stated on label) (stated on label) benefits lower sulphite levels lower calorie content lower alcohol content no claim on label (sulphite information (9% instead of 12.5%) excluded from label) attributes (and levels) that varied across wine profiles. ital. j. food sci., vol. 27 2015 227 ing nanotechnology, it was explicitly stated on the wine label. within this conjoint experiment, if the wine had a sulphite level lower than 10 mg/l (the limit established from the regulation (eu) no 1169/2011 for omitting the indication of the presence of sulphites from the label), sulphite information was not included on the label. thus, in keep with our research goals, this attribute level best resembles market place situations. therefore, how the “benefit” attribute levels were presented is based on what is practical, relevant and realistic within the marketplace (gil and sánchez, 1997). furthermore, the approach used for the “benefit” attribute levels is similar to that of other published ca studies in the context of the inclusion of a “no claim” or “no information” level (e.g. deliza et al., 2003; krystallis and ness, 2005). the rating task was carried out applying the full-profile conjoint analysis method using spss 19.0. this software calculated the utility values for each level of each factor. ca is useful in evaluating purchase intentions (sánchez and gill, 1998). an “average importance” value was also calculated for each factor that reflects the relative range of utility values for the levels within each factor (cardello et al., 2007). when adopting the full-profile method, the number of possible profiles can increase rapidly due to the various combinations of factors and levels. the design must be balanced with a sufficient rotation of the factors and number of profiles in order to maintain the overall significance of the experiment. therefore, a fractional factorial design (orthogonal array) was used which presented a suitable fraction of all possible combinations of the factor levels. table 3 summarises the 10 profiles generated in spss 19; two holdouts were included to ensure the validity of the test. in the results section, an overall profile of respondents is presented as well as profiling using an a-priori segmentation variable (frequency of wine consumption). following this, perspectives on nanotechnology are considered. conjoint and post-hoc segmentation analysis establishes how respondents value different wine product attributes. the influence of production methods (conventionally produced versus produced using nanotechnology) and other product attributes (e.g. associated with health and naturalness) on product preference are examined. 3. results 3.1 consumers’ behaviours and attitudes to wine fifteen percent, 33% and 24% of respondents indicated that they had a daily, weekly or fortnightly wine consumption habit respectively. the remaining 28% were relatively infrequent consumers, with consumption levels at around once monthly. respondents reported that they do not always drink the same varieties of wine (x̄ = 4.69; s.d. = 1.63), drink wine from their region (x̄ = 4.01; s.d. = 1.91) or drink wine from the same territory (x̄ = 4.07; s.d. = 1.94). generally, participants indicated that they spend table 3. profile price method of production benefits 1 € 5.99 conventionally produced lower sulphite content design (method of production not stated on label) (sulphite information excluded from label) 2 € 11.99 produced using nanotechnology lower sulphite content (sulphite information excluded from label) design 3 € 5.99 conventionally produced (method of production not stated on label) lower calorie content design 4 € 5.99 produced using nanotechnology no claim on label design 5 € 5.99 produced using nanotechnology lower alcohol content design 6 € 11.99 conventionally produced (method of production not stated on label) no claim on label design 7 € 11.99 conventionally produced (method of production not stated on label) lower alcohol content design 8 € 11.99 produced using nanotechnology lower calorie content design 9 € 5.99 produced using nanotechnology lower sulphite content (sulphite information excluded from label) holdout 10 € 11.99 produced using nanotechnology lower alcohol content holdout list of profiles used in conjoint analysis experiment (fractional factorial design). 228 ital. j. food sci., vol. 27 2015 some time selecting which wine to purchase (x̄ = 4.55; s.d. = 1.50). the general sentiment of the sample to italian wine was very positive, which was reflected in their view that wine forms an important part of italian culture (x̄ = 5.84; s.d. = 1.52) and in their expression of pride in italian wine tradition (x̄ = 5.97; s.d. = 1.28). when selecting wine, price, region of production and grape variety were among the most important selection attributes (fig. 1). a paired sample t-test highlighted that as an information cue, price was significantly more important than all other cues (p < 0.001). using frequency of consumption as an a-priori segmentation variable, we observed significant differences in wine behaviour patterns. one-way anova analysis (p ≤ 0.002) highlights that everyday consumers were more likely to drink wine from a variety of territories when compared to the fortnightly and monthly consumers. while the patterns of the daily and weekly consumers were similar, the weekly consumers (p ≤ 0.001) were also more likely to spend time engaging in the selection of wine than their fortnightly or monthly counterparts. in addition to this analysis, evidence of differences in the importance of quality attributes in the selection of wine was also apparent. levels of importance for territory of origin (p = 0.03), cork (p < 0.01), price (p < 0.01), age (p < 0.01), variety (p = 0.018) and packaging (p < 0.01) varied across the segments (anova analysis with post hoc bonferroni). the everyday consumers placed more importance on territory of origin (p ≤ 0.031) and variety (p < 0.02) than the fortnightly consumers; while the weekly consumers placed more importance on the age (p < 0.01) and less importance on the price (p ≤ 0.03) than the fortnightly and monthly consumers. interestingly, irrespective of their consumption level, all held similar sentiments towards italian wine. to assess the relative importance of each attribute for each segment, a paired sample t-test was applied. this indicated that it was only in the case of fortnightly and monthly consumers that price was significantly more important than other key information cues. in the case of both everyday and weekly consumers, no significant differences were identified in terms of price, region, grape variety and territory of origin. this suggests that frequent consumers of wine rely equally on a greater variety of information cues in their selection of wine. the segments differed significantly (χ² = 9.46; p = 0.024) based on gender, with males being more likely to be daily drinkers, accounting for 71% of the everyday category. in comparison, 62% of the monthly category was female. there were no significant differences with regard to age and frequency of consumption. fig. 1 mean importance of attributes influencing wine purchase decisions by frequency of consumption. note: scale from 1 to 7, where 1 is extremely important, 7 is extremely unimportant and 4 is neither important nor unimportant. letters above the bars reflect significant differences between frequencies of consumption groups at the 95% confidence level. ital. j. food sci., vol. 27 2015 229 to provide a rich account of consumer acceptance of adopting nanotechnology in wine production, the next section explores respondents’ awareness of and perspectives on nanotechnology. it examines acceptance of nanotechnology applications in wine, both generally and specifically, at the segment level based on frequency of consumption. 3.2. awareness and attitude towards nanotechnology the majority of the sample was unaware of nanotechnology applications in general (58%). this lack of awareness increased considerably for food applications (84%). to get an initial indication of attitudes towards nanotechnology, following the provision of information on this technology, respondents were asked about their level of acceptance of the use of nanotechnology in wine production using three statements (table 1). unidimensionality of this measure was assured on the basis of principal axis factor analysis, with 84% of variation explained by a single factor and factor loadings ranging from 0.51 to 0.86. reliability of the measure was also good (α = 0.936). an overall acceptance score was calculated based on a mean score for the three statements. widespread acceptance of nanotechnology in wine production is unlikely (x̄ = 3.06; s.d. = 1.75) and was not significantly different across consumption levels. to further understand levels of acceptance, an examination of potential applications of nanotechnology that offer specific benefits was undertaken. although the applications presented are hypothetical at present, they may become a reality in the future. this suggested that certain applications are more acceptable than others, as summarised in fig. 2. enhancing the authenticity of wine, relates to improving the traceability and safety of the wine and ensuring the preservation of product characteristics linked to its origins. this enhancement was considered the most acceptable application, followed by reducing the use of pesticides and enhancing sensory characteristics. paired sample t-tests highlight that the application of nanotechnology to enhance the authenticity of wine was significantly more acceptable (p < 0.001) than its applications for other purposes. however, this disguised differences across consumption levels. while the monthly consumers displayed the same pattern as the overall sample, the everyday and weekly consumers considered applications to reduce the fig. 2 acceptance of applying nanotechnology to obtain the following benefits by frequency of consumption. note: scale from 1 to 7, where 1 is extremely unacceptable, 7 is extremely acceptable and 4 is neither acceptable or unacceptable. letters above the bars reflect significant differences between frequencies of consumption groups at the 95% confidence level. 230 ital. j. food sci., vol. 27 2015 usage of pesticides equally as acceptable as applications to enhance authenticity. enhancing taste was the third most acceptable application and was significantly less acceptable than authenticity improvements. that said, in the case of the fortnightly group, taste along with modifying cork and reducing calories were judged as equally as acceptable as authenticity improvements. furthermore, taste benefits were significantly less acceptable than benefits such as price and reduced alcohol content for everyday consumers. anova analysis confirmed that the everyday and weekly consumers were significantly less accepting (p ≤ 0.01) of taste benefits when compared with the fortnightly consumers. finally, colour modification was the least acceptable application across all consumption levels. anova analysis highlights that the everyday consumers were significantly more accepting of low alcohol benefits when compared to weekly (p = 0.008) or fortnightly (p = 0.019) consumers. furthermore, they were significantly less accepting of modifications to the cork and colour in comparison to the fortnightly (p = 0.018) and weekly (p = 0.013) consumers respectively. the weekly consumers were significantly more accepting of modifying colour than the monthly (p = 0.031) and were significantly less accepting of reducing calories than the fortnightly (p = 0.008) consumers. no other significant differences in acceptance were noted across the segments. the following section further explores wine preferences, presenting the findings of the conjoint experiment which involved wine products based on combination of attributes, one of which was “produced using nanotechnology”. this conjoint analysis therefore provides additional insights into varying levels of acceptance of applications of nanotechnology in wine production across the sample. post-hoc segmentation analysis enables further understanding of how different consumer value different wine attributes and place them within the context of the application of nanotechnology. 3.3. conjoint and post-hoc segmentation analysis the conjoint analysis suggests that, across the sample, price was the most important factor influencing wine preference (47.8%) with a preference for lower priced wine (utility = 1.08) being evident (table 4). method of production (35%) was the second most important attribute. in this case, conventionally produced wine (utility =0.79) was preferred over wine produced using nanotechnology (utility = -0.79). benefits (17.2%) were the least important factor influencing preference. benefits with positive utility values were lower sulphite levels (0.4) and lower calorie content (0.21). in fact, the negative utility of applying nanotechnology (-0.79) may be traded-off against, for example, the positive utility of a lower price (1.08) coupled with either lower sulphite levels (0.4) or lower calorie content (0.21). in terms of the other benefits offered, a negative utility for lower alcohol content (-0.23) indicates that consumers disliked this suggested benefit. table 4. % of sample total sample price sensitive traditionalist indifferent (n = 221) (n = 131) (n = 46) (n = 44) 100% 59.3% 20.8% 19.9% intercept 4.77 4.96 4.40 4.62 price €5.99 1.08 1.61 0.49 0.13 €11.99 -1.08 -1.61 -0.49 -0.13 relative importance (%) 47.80 50.78 20.40 18.44 method of production conventionally produced 0.79 0.90 1.07 0.19 produced using nanotechnology -0.79 -0.90 -1.07 -0.19 relative importance (%) 34.99 28.37 43.95 27.46 benefits lower sulphite levels 0.40 0.12 1.14 0.46 lower calorie content 0.21 0.66 -0.59 -0.29 lower alcohol content -0.23 -0.11 -0.52 -0.27 no claim on label -0.38 -0.67 -0.03 0.11 relative importance (%) 17.20 20.85 35.65 54.10 r of pearson 0.99 0.99 0.96 0.90 utility values of levels in the conjoint experiment and cluster segment. ital. j. food sci., vol. 27 2015 231 the “ideal” profile (i.e. the profile respondents were most willing to purchase) was profile 1, with the following characteristics: €5.99, conventionally produced (method of production not stated on label) and lower sulphite content (sulphite information excluded from label). the least preferred profile is hypothetical and was not presented in the profiles that respondents scored. this hypothetical profile did not include any proposed benefits, was priced at €11.99 and produced using nanotechnology. to identify different consumer segments based on product attribute utility scores derived from the conjoint experiment, “k-means cluster analysis” was employed across two to five clusters. each of these was evaluated and three clusters were identified as best representing the data. of these three segments, the first and largest segment (59.3% of respondents), labelled“price sensitive”; price (50.8%) was the most important product attribute, followed by method of production (28.4% of importance) and subjective benefits (20.8% of importance). low priced (1.61), conventionally produced (0.9), lower calorie (0.66) and lower sulphite (0.12) wine offered the greatest positive utilities. the second segment (20.8% of respondents), labelled“traditionalist”, placed most importance on method of production (43.9%), followed by subjective benefits (35.6%) and price (20.4%). they displayed a strong negative utility for nanotechnology produced wine (-1.07) relative to conventionally produced wine. this negative utility may not be traded-off by the positive utility of a lower price (0.49). the only benefit offering a positive utility was lower sulphite levels (1.14). finally, the third segment (19.9% of respondents), labelled “indifferent”, considered benefits to be the most important attribute (54.1%), followed by method of production (27.5%) and then price, which they considered to be the least important attribute (18.4%). however, not all proposed benefits offered utility; the benefit of interest for this segment was low sulphite levels (0.46), with no other benefits offering utility. compared to the other segments, the “price sensitive” included significantly (χ² = 11.395; p = 0.003) more females (60%) than males (40%) and were among the least frequent consumers of wine, with 73% of them consuming wine, at most, once fortnightly (table 5) compared to 19% and 23% for the “traditionalist” and “indifferent” segments respectively (χ² = 108.092; p < 0.001). furthermore, the consumers belonging to the first segment were more inclined to always purchase the same variety of wine (p ≤ 0.001), from the same territory (p ≤ 0.034) when compared to the consumers of the other two segments. anova analysis with post hoc bonferroni suggests that, in comparison to the two other segments, the price sensitive placed greater importance on price and region and less importance on age and packaging when selecting wine (p ≤ 0.022). they were also the most open to applitable 5. price construct level total sensitive traditionalist indifferent χ2 (%) (%) (%) (%) (p-value) male 50 40 61 66 gender female 50 60 39 34 11.40 (<0.001) total 100 100 100 100 18-24 12 8 0 34 25-34 20 22 0 36 age 35-44 25 33 4 21 108.1 45-54 23 24 35 9 (< 0.001) 55-64 20 13 61 0 total 100 100 100 100 everyday 15 11 28 16 frequency weekly 33 16 52 61 62.92 (<0.001) of consumption fortnightly 24 32 9 14 monthly 28 41 11 9 total 100 100 100 100 segment characteristics. 232 ital. j. food sci., vol. 27 2015 cations of nanotechnology that reduced calorie content and the least open to those to modify colour (p ≤ 0.026) (table 6). they placed greater importance on alcohol and calorie content of wine and were more receptive to applications that reduce alcohol content and enhance taste than the traditionalist (p ≤ 0.032). however, the price sensitive had the lowest overall acceptance score (x̄ = 2.83; s.d. = 1.87), which was significantly lower (p < 0.01) than that of the indifferent segment (x̄ = 3.75; s.d. = 1.73) the traditionalist segment was older, in fact 90% were 45 years or over; this compares to 9% and 37% for the indifferent and price sensitive segments respectively. they also represented the most frequent consumers of wine, with almost 30% consuming wine everyday and almost 80% of the segment consuming wine at least weekly. the traditionalists were the most different to the price sensitive in their perspectives on wine and were less interested in changes to the current characteristics of wine, as indicated by their lower receptivity to many of the suggested benefits associated with the application of nanotechnology. however, no significant difference in overall acceptance (x̄ = 3.08; s.d. = 1.31) was evident between the traditionalist and two other segments. the indifferent segment included predominately younger respondents; 70% were 36 or younger. furthermore, males (66%) were disproportionately represented within the segment. they were also quite frequent consumers of wine, with 77% consuming wine at least weekly. the findings illustrate that utility scores offer an effective means of dividing the market and establishing different perspectives on wine attributes across the post hoc segments. each segment displayed a negative utility for applying nanotechnology. however, the extent of such negative attitudes (utilities) and the relative importance placed on applying nanotechnology in comparison to the other attributes (i.e. price and benefits) varied across the segments. 4. discussion and conclusions in this paper, we sought to understand consumer acceptance of nanotechnology within a product category that is strongly embedded in italian culture. the analysis of consumers indicates that tradition continues to be important in choice decisions in the wine category; however, price plays a more important role in wine choice. these factors combined with region of production and grape variety are key choice attributes. based on the findings, italians are relatively unfamiliar with applications of nanotechnology, both generally and specifically to food. as suggested by others (e.g. fell et al., 2009), we observed a cautious response to the concept of nanotechnology. indeed, within the sample, there was an overall rejection of the concept of “nano wine”. however, low acceptance scores disguised a somewhat more open attitude to specific applications of this technology. it is clear that for many, acceptance is considered on a case by case basis, and the bundle of benefits offered by a product is central to evaluations of the associated technology. acceptance of the technology increases when the specific application satisfies an unfulfilled need. thus, while the concept of the technology results in a reluctant response, this changes when more concrete product examples of personal relevance are considered. within this study, consumers were most receptive to applications that result in improved authenticity and reduced use of pesticides. the findings therefore concur with the views of bruhn (2007) and siegrist (2008) that if an objective of a communication is to successfully market and sell novel food technology products, including nanotechnology-based foods and beverages, attention should be given to communicating explicit, tangible benefits of relevance to consumers. the conjoint analysis results suggest that, across the sample, price was the most signifitable 6. please indicate how acceptable you sample price sensitive traditionalist indifferent consider it to use nanotechnology to: mean mean mean mean (st. dev.) (st. dev.) (st. dev.) (st. dev.) enhancing the authenticity wine. 5.55 (1.58) 5.65 (1.81) 5.24 (1.32) 5.57 (0.97) reducing the amount of pesticides used when growing the grape. 4.96 (1.87) 4.81 (2.09) 4.98 (1.39) 5.41 (1.53) enhancing the taste of wine. 4.62 (1.72) 4.89 (1.76) 3.91 (1.66) 4.57 (1.47) modifying the structure and properties of the cork. 4.57 (1.62) 4.63 (1.85) 4.37 (1.10) 4.64 (1.33) reducing the calorie content of wine. 4.26 (1.75) 4.57 (1.89) 3.35 (1.21) 4.30 (1.47) reducing the alcohol content of wine 4.22 (1.84) 4.50 (2.05) 3.70 (1.41) 3.95 (1.38) producing less expensive wine. 4.10 (1.70) 4.08 (1.72) 3.85 (1.75) 4.43 (1.56) modifying the colour of wine. 2.62 (1.42) 2.29 (1.35) 2.91 (1.26) 3.32 (1.49) acceptance of applications of nanotechnology by segment. ital. j. food sci., vol. 27 2015 233 cant factor influencing wine preference followed by method of production; with consumers displaying a preference for conventionally produced rather than “nano” wine. given the significance of price, it is not surprising that some consumers may be willing to purchase “nano wine” if it is priced lower than its conventional counterpart and additional benefits are offered. this work implies that segmentation is a useful platform for exploring consumer acceptance of nanotechnology application in wine production. for example, while the price sensitive, traditionalist and indifferent segments all displayed negative utility for nanotechnology, the extent of such negative attitudes (utilities) could be traded-off against a lower price and the enhancement of other product characteristics which were valued by particular segments (e.g. lower sulphite levels). however, the extent of “trading-off” between these attributes clearly depended on the segment in question. in addition, the a-priori and post hoc segmentation analysis demonstrates that variation exists in how groups of individuals evaluate and consume wine. in particular, significant variation was evident in wine behaviour patterns and the importance placed on different wine attributes (i.e. region of origin, cork, price, age, variety and packaging) and was also apparent in consumers’ evaluations of the different nanotechnology applications. heterogeneity in behaviour across consumer segments, in addition to variation in terms of the importance placed on wine product attributes have been highlighted in several other studies. empirical evidence supports the finding of this work that frequent consumers of wine rely on a greater variety of information cues in their wine selection. specifically, atkin and johnson (2010) found that core consumers (i.e. those who drink wine at least once a week) draw more heavily on place-of-origin cues than infrequent consumers. elsewhere, perrouty et al. (2006) found that perceived expert consumers make use of a greater number of attributes, particularly region, brand, variety and price when evaluating wine products compared to perceived non-expert (novice) consumers. the former also evaluate relationships between attributes more deeply than novices. within this study, although authenticity improvements were considered the most acceptable application of nanotechnology to wine overall, the segments were not homogenous in their assessments of the other applications presented. for example, the more frequent wine consumers considered applications to reduce the use of pesticides to be as acceptable as those that enhance authenticity. furthermore, the conjoint and post-hoc segmentation analysis illustrate how, although the price sensitive segment had the lowest overall acceptance score, they were more responsive than the indifferent and traditionalist segments to applications that reduce calorie content. this finding, once again, demonstrates that acceptance is lower at the conceptual/abstract level than the product attributes level, thereby illustrating the merits of segmenting the population. both segmentation approaches can guide approaches to targeting different consumer groups. in particular, insights from the utility based segmentation may be useful in designing and developing a “nano wine” that is targeted at the most suitable market segments. based on the findings, a traditionalist segment would be an inappropriate target market for “nano wine”, given the high importance this cautious group places on conventional production methods. conversely, considering optimum commercialisation and marketing strategies for “nano wine”, producers and distributors may be interested in offering a competitively priced “nano wine” that has reduced sulphite levels to an indifferent segment that frequently consume wine and could therefore be a profitable target market. furthermore, another strategy might be to offer a competitively priced “nano wine” with reduced calorie content to price sensitive consumers. the emerging positive reactions towards applications that enhance wine authenticity align with the connotations of wine being a “natural” product, strongly associated with heritage, origin and region, as romano and natilli (2009) have previously argued. this “natural” perception of wine is particularly evident in the case of traditional wine producing and consuming countries including italy, where pdo and pgi wines are prevalent. building on this research, marketers should recognise the influence of perceived “naturalness” on wine preferences and develop communication strategies around emphasising how nanotechnology can, in fact, enhance “natural” qualities of wine, e.g. improve authenticity and lower sulphite levels, rather than tamper with its “natural” properties. to sum, although the application of nanotechnology is not generally positively perceived in wine production, low measures of overall acceptance may conceal greater acceptance of specific applications which enhance valued wine attributes. finally, we recognise the potential limitations of this study. specifically, while this work is in keeping with the approach of siegrist et al. 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(2007), distillation method can be called as molecular distillation if the distance between evaporator and condenser reaches to mean free path of a vapor molecule. lei et al. (2005) described the mean free path, < λ >, with the following equation: where d (m) is the diameter of molecule, n a is avogadro constant (6.023x1023 mol-1), p (pa) is pressure, r is universal gas constant and t (k) is temperature. lutišan and cvengroš (1995) defined molecular distillation as “the safest fig. 1 sample phase diagram of any pure substance. method to separate and purify thermally unstable compounds”. shi et al. (2007) pointed out that risk of thermal decomposition could be reduced with low temperature; as well oxidation could be prevented with air removal by vacuum. in addition, de moraes et al. (2006) drew one’s attention to advantages of molecular distillation (e.g. avoiding toxicity, protect environment) that other chemical agent-based techniques do not have. lutišan and cvengroš (1995) defined the main features of molecular distillation as; short time of exposure to heat, low evaporating temperature and a characteristic mass transfer. according to martinello et al. (2007), “small distance between evaporator and condenser” can also be defined as a feature of molecular distillation. ital. j. food sci., vol. 27 2015 279 high vacuum and molecular distillation equipment there are typically two types of evaporators used in high vacuum distillation, i.e. thin film evaporators (tfe) and short-path evaporators (spe). these equipments have similar designs with few differences. in both evaporators, feed is agitated with a rotor-wiper system and high vacuum is produced by vacuum pumps. in tfe, operating pressure can be reduced to 1-100 mbar (uic gmbh, 2014) and there is no other unit between vacuum and condenser (pilodist, 2014). fig. 2 shows an illustration of a tfe. in spe, condenser is placed in the centre of evaporator unit, so distance between boiling and condensation surface is extremely reduced and pressure drop is minimized. the operating pressure can be reduced up to 0.001 mbar. distillation performed by a short-path evaporator is also called as “molecular distillation” (busssms-canzler gmbh, 2014a; buss-sms-canzler gmbh, 2014b; pilodist, 2014; technoforce, 2014). fig. 3 shows an illustration of a spe. there are many parameters that can affect distillation yield and molecular evaporation rate. molecular evaporation rate, k i , can be calculated by langmuir-knudsen equation (rossi et al., 2011): where ts is evaporation temperature, r is universal gas constant, m i is molecular weight of evaporating component and pvi is vapor pressure of component. xu et al. (2002) describes the most important parameters of molecular distillation as evaporator temperature, flow rate, vacuum and wiper speed. flow rate has an important effect on the contact time of the molecules with hot surface during evaporation. higher flow rates reduce the residence times of molecules being vaporized. wiper speed affects film thickness and viscosity. feed becomes highly turbulent with intensive agitation, which leads to high heat transfer coefficients (buss-sms-canzler gmbh, 2014c). molecular distillation in food processing: some examples of recent studies molecular distillation has many application areas in food industry. some of these applications can be summarized as but not limited to: concentration of ω-3 fatty acids, distillation of monoglycerides from diand triglycerides, concentration of tocopherols and tocotrienols (busssms-canzler gmbh, 2014d), fractionation of squalene (sun et al., 1997), recovery of carotenoids (batistella and wolf-maciel, 1998). as distillation is a separation process, studies about molecular distillation generally focus on either removal of undesired compounds or concentration of valuable compounds. removal of undesired compounds in a study about removal of cholesterol from butter and lard by using molecular distillation (lanzani et al., 1994), researchers reported that cholesterol content of lard was reduced from 988 ppm to 105 ppm in the residue after 2 hours of distillation under 10-4 torr pressure and 250oc evaporator temperature. molecular distillation can also be used for physical deacidification. martins et al. (2006) separated free fatty acids (ffa) from vegetable oil deodorizer distillate. they achieved to reduce fig. 2 illustration of a tfe unit. fig. 3 illustration of a spd unit. 280 ital. j. food sci., vol. 27 2015 ffa content to 6.4% from initial ffa content of raw material with 57.8% at 160oc evaporator temperature, under 10-6 bar pressure and 10.4 g min-1 feed flow rate. they also noted that concentration of tocopherol in residue stream was found 18.3%, while initial tocopherol concentration was 8.97%. ffa elimination was 96.16% and tocopherol recovery was found 81.23%. wang et al. (2010) aimed to separate ffas and diacylglycerols (dag) from enzimatically hydrolyzed soybean oil. they achieved to increase the removal of ffas from 88.8% to 99.44% by increasing evaporator temperature from 125oc to 160oc, under 0.5-1.0 pa process pressure, 200 ml h-1 feed rate and 300 rpm wiper speed. olli et al. (2013) studied removal of organic pollutants in fish oils. their spd system, which has an evaporator temperature of approx. 220oc and operating pressure below 0.03 mbar, achieved to remove total amount of chlorinated pesticides (some of them are ddt and hch) from 215.07 ng g-1 to 21.95 ng g-1, corresponding to 89% reduction. according to meyer et al. (2011), total pesticide traces in rapeseed deodorizer distillate were dropped below 0.05 mg kg-1 from an initial content of 0.968 mg kg-1 by achieving more than 94.8% reduction. spd evaporator temperature was set to 110oc, feed flow rate was 200 ml h-1 and pressures were between 0.006 and 0.01 mbar. researchers stated that it would be a mistake to affirm that all types of pesticides were removed by using spd according to this reduction data, because many different types of pesticides might be present before distillation and analysis of effects on specific compounds has to be performed. concentration and/or fractionation of compounds batistella and wolf-maciel (1998) studied the recovery of carotenoids from palm oil by using a molecular distillator and after a set of distillation trials, they achieved to increase carotene concentration to 19500 ppm from an initial feed concentration of 600 ppm under 9x10-5 torr pressure and 170oc evaporator temperature. sun et al. (1997) fractionated squalene from alkali-refined amaranth seed oil and their highest recovery of squalene was 67.8% with spd conditions of 100 mtorr pressure and 180oc distillation temperature. campos et al. (2003) fractionated milk fat by spd and recorded distillate yields (w/w) as a function of temperature. they observed that distillate yield was 0.3% at 125oc process temperature; however a 42.7% recovery was observed when process temperature was increased to 250oc, which meant a significant and positive effect of temperature on process efficiency. spd was performed on lemongrass essential oil by tovar et al. (2011) and researchers reported that they were able to increase citral concentration in distillate stream from 17.658 mg ml-1 to 33.576 mg ml-1 when evaporator temperature was increased from 60oc to 120oc with a feed flow rate of 1.5 ml min-1 and pressure of 5 pa. mono and diglyceride (mdg) concentration and production are also possible with molecular distillation. fregolente et al. (2010) produced partial glycerides from soybean oil by using molecular distillation. concentration of monoglyceride (mg) in distillate stream increased with elevated evaporator temperature. at 250oc with 10 ml min-1 feed flow rate, mg concentration was increased from initial feed value of 12.75% to 80.00% in distillate stream under 24 pa operating pressure. they also pointed that lower flow rate increased recovery of mg, because molecules contacted with hot evaporator surface for a longer period of time. recovery for any component is defined with following equation: zhang et al. (2013) studied effects of evaporation temperature, feeding rate, feeding temperature and wiper speed on concentration of ω-3 fatty acids by molecular distillation and optimized these parameters with response surface methodology (rsm). researchers reported the optimum conditions as 110.4oc evaporator temperature, 78.7 ml h-1 feeding rate, 350 rpm wiper speed, 10 pa operating pressure and 80oc feed temperature. conclusions separation techniques such as extraction, evaporation, distillation etc. are accepted as unit operations in food industry. vacuum distillation is frequently used both in chemical and food industries; however simple vacuum distillation might not be capable of separation of heat-sensitive materials from food products. in that case, molecular distillation (short-path distillation) should be used for separation of these materials. molecular distillation has been used more in pharmaceutical, chemical and petrochemical applications, but nowadays importance of molecular distillation has increasingly been understood in food industry. separation, concentration and purification of commercially valuable food constituents can be easily performed by molecular distillation; furthermore, healthier food products can be produced by removal of some health damaging compounds such as excess cholesterol, organic pollutants. authors expect an increasing trend in usage of molecular distillation in food industry when taking all these applications into consideration. acknowledgements this review article has not been funded by any organization. ital. j. food sci., vol. 27 2015 281 references batistella c.b. and wolf-maciel m.r.1998. recovery of carotenoids from palm oil by molecular distillation. computers & chemical engineering 22(supplement 1): s53s60. buss-sms-canzler gmbh. molecular distillation. available at: http://www.sms-vt.com/en/technologies/shortpath-evaporator/molecular-distillation.html (accessed 20 may 2014a). buss-sms-canzler gmbh. short path evaporator. 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liu y.f. 2013. concentration of omega-3 polyunsaturated fatty acids from oil of schizochytrium limacinum by molecular distillation: optimization of technological conditions. industrial & engineering chemistry research 52(10): 3918-3925. paper received july 7, 2014 accepted september 23, 2014 84 issn 1120-1770 online, doi 10.15586/ijfs.v33i4.2095 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (4): 84–97 p u b l i c a t i o n s codon effect of addition of tunisian zizyphus lotus l. fruits on nutritional and sensory qualities of cookies youkabed zarroug1*, jazia sriti2, dorra sfayhi3, bechir slimi3, wafa alloucha, kamel zayani4, khaoula hammami2, maryam sowalhia1 and mohamed kharrat1 1université de carthage, laboratoire des grandes cultures (lr16inrat02), institut national de recherche agronomique de tunisie (inrat), ariana, tunisie; 2laboratoire des substances bioactives, centre de biotechnologie, technopole borjcedria, hammam-lif, tunisie; 3laboratoire des nanomatériaux et systèmes pour les energies renouvelables (lanser), centre de recherches et des technologies de l’energie technopole borj cedria, hammam-lif, tunisie; 4centre national des recherches en sciences des matériaux (cnrsm), technopôle de borj-cédria, soliman, tunisie *corresponding author: youkabed zarroug,, université de carthage, laboratoire des grandes cultures (lr16inrat02), institut national de recherche agronomique de tunisie (inrat), ariana, tunisie. email: zarrougyoukabed@yahoo.fr received: 26 june 2021; accepted: 19 october 2021; published: 7 december 2021 © 2021 codon publications open access paper abstract zizyphus lotus, which belongs to rhamnaceae family, has been widely used to formulate many healthy food products. the aim of this work was to formulate new functional cookies enriched with different amounts of zizyphus lotus powder (zlp; 15%, 30%, 45% and 100%). the chemical properties of zlp were also determined. the formulated cookies were evaluated for their physicochemical, textural and sensory characteristics. results revealed that zlp contained various bioactive components, fatty acids, and antioxidants. zlp-added cookies demonstrated higher phytochemical and antioxidant activities than control cookies prepared without zlp. the activity of zlp cookies was enhanced with increase of zlp level. hardness and fracturability (brittleness) of cookies increased with increasing amount of zlp. results of fourier-transform infrared spectroscopy and thermogravimetric analysis also revealed the presence of many bioactive compounds in formulated cookies. all cookie samples were generally accepted, but the panelists indicated a higher preference for cookies containing 15% zlp. keywords: antioxidants; cookies; phytochemical compounds; sensory characteristics; texture; zizyphus lotus. introduction bakery products such as cookies are widely consumed around the world. this is mainly due to their higher energy, low cost, ready-to-eat nature, availability in different tastes and extended shelf life (ajila et al., 2008). in addition, cookies may represent an excellent model product for enhancement of nutritional value and fortification. the main ingredients used for production of cookies are wheat flour, fat and sugar. to obtain homogeneous dough, water is also added. nowadays, the enrichment of these cookies with vitamins, minerals, polyphenols and fibers is considered as a good alternative to produce high nutritional value foods to improve human health. to achieve this goal, many fruits and vegetable powders are used for the formulation of cookies such as papaya pulp flour, choke-berry extract, grape marc extract, sour cherry pomace extract and japanese quince (antoniewska et al., 2019; molnar et al., 2015; pasqualone et al., 2014; šaponjac et al., 2016; varastegani et al., 2015). in addition, many fruits being used are gaining attention because of attractive flavor as well as diverse antioxidant, anticarcinogenic and antimutagenic substances included in them (antoniewska et al., 2019). besides, the consumption of these products has health benefits against several chronic diseases, including cardiovascular disease and certain types of cancers (ludwig et al., 2018). genus zizyphus belonging to rhamnaceae family is widespread in tropical and subtropical regions: asia, africa, north america, south america, oceania and europe, with the center of diversity in asia mailto:zarrougyoukabed@yahoo.fr italian journal of food science, 2021; 33 (4) 85 effect of addition of tunisian zizyphus lotus l. fruits on nutritional and sensory qualities of cookies the water activity (aw) was determined using aqua lab (meter, aqualab series 3te, usa) at 25°c. each assay was carried out in triplicate. techno-functional properties of zlp water absorption capacity (wac) was determined according to the method described by anderson et al. (1970). in a pre-weighed centrifuge tube, 1-g sample was mixed with 10-ml distilled water. the tube was stirred for 2 min and then centrifuged at 3,000 rpm for 15 min. the supernatant was decanted into a tared evaporating dish for determining its solid content and the sediment was weighed. the wac was calculated as weight of sediment (g) per weight of sample (g) on a dry basis. oil absorption capacity (oac) was determined according to the method described by kaur and singh. (2005). an amount of 0.5 g of sample was mixed with 6 ml of corn oil in preweighted centrifuge tubes, stirred for 1 min, left for 30 min and centrifuged at 3,000 g for 25 min. the separated oil volume was recorded and the tubes were inverted for 25 min to drain out the oil prior to reweighting. the oac was expressed as gram of oil bound per gram of sample on a dry basis. swelling index (si) was determined by the procedure described by rosell et al. (2009). an amount of 1 g of sample was transferred into a graduated cylinder, it was gently leveled and the volume was noted. then 10 ml of distilled water was added to the sample; the cylinder was swirled and left to stand for 60 min while the swelling (change in volume) was recorded every 15 min. the swelling index of samples was determined as a multiple of original volume. fatty acid extraction and analysis zizyphus lotus powder (20  g) was extracted in a soxhlet apparatus with 250 ml of ether of petroleum at 40ºc. between each step, the extract was filtered with a whatman filter paper (n°4), concentrated under rotary vacuum evaporator (rotovapor-el, labortechnik ag, büchi, switzerland) at 40 c and conserved at 4°c for analysis fatty acid composition of zlp lipid fraction was determined as fatty acid methyl esters using gas chromatography (agilent 19091s-433) equipped with a flame ionization detector and a polar phenylmethyl-siloxane capillary column (60 m × 25 mm × 0.25 µm film thickness) as described by zarroug et al. (2015). formulation of cookies enriched with zlp cookies were formulated according to the method described of tyagi et al. (2020) with minor modification. (richardson et al., 2004). besides, previous studies have described the secondary metabolites and pharmacology of genus zizyphus. traditionally, zizyphus plant has been used in medicines to treat different diseases such as diarrhea, cholera, bronchitis, diabetes, hypertension, inflammation and intestinal spasms (el maaiden, 2020; le-floc’h, 1983). in tunisia, the commonly known species of zizyphus shrub is zizyphus lotus, which is named as ‘sedra’, and its edible fruit is called ‘nbeg’ (mkadmini et al., 2015). various studies have described the nutritional and beneficial effects of zizyphus lotus fruits (yamada et al., 1985). this small and round fruits are a rich source of sugar (ghedira, 2013), minerals (calcium, magnesium, iron, sodium, potassium and phosphorus), carbohydrates, fatty acids and proteins (abdeddaim et al., 2014). authors have also highlighted the presence of large amounts of vitamins a, c and d (chouaibi et al., 2011; ghedira, 2013). therefore, owing to the presence of high healthy functional components, such as polyphenols, fibers, bioactive compounds, including micronutrients and phytochemicals (da silva et al., 2007), zizyphus lotus fruits are widely consumed in many countries and used to formulate many food products such as pastes, purees, syrups and confections. moreover, zizyphus fruits are usually used as food additive in many bakery products to improve their technological quality and nutritional value. in this context, the aim of the present work was to characterize zizyphus lotus fruit powder and to evaluate its effect on physicochemical properties and sensory evaluation of cookies. materials and methods preparation of zizyphus lotus powder zizyphus lotus fruits were collected in july 2020 from goubollat region of beja in the northwest of tunisia. the edible part of the fruits were manually isolated from seeds, sun-dried for 120 h, and milled. the obtained samples were then sieved through a 75-µm sieve to get fine powder of zizyphus lotus fruits (zlp) and finally stored in plastic containers at 4°c until further use. chemical composition moisture, protein, fat and ash contents of zlp and cookie samples were determined according to the method described by association of official analytical chemists (aoac, 2005). total carbohydrate content was calculated by difference (100 – sum of protein, fat, ash and moisture). the results were expressed as g per 100 g of dry weight. energy was calculated as follows: energy (kcal/100 g) = 4 × (g protein + g carbohydrate) + 9 × (g fat). 86 italian journal of food science, 2021; 33 (4) zarroug y et al. fourier-transform infrared spectroscopy (ftir) functional groups present in cookie samples were characterized by the perkin elmer ftir instrument equipped with the software perkin elmer spectrum version 10.4.3. the samples were grinded with potassium bromide pellets and measured at a wavelength range of 400–4,000 cm˗1. scanning electron microscopy (sem) microstructure of baked cookies was evaluated using sem (jeol/jsm-5400) as described by adebiyi et al. (2016). cookies were milled, sieved and then observed using two magnification levels of ×400 and ×1,500. phytochemical analysis and antioxidant activities of zlp and formulated cookies extract preparation zizyphus lotus powder was extracted by maceration using the following solvents: acetone (60%), ethanol (60%), methanol (60%) and water. this extraction was done according to the method described by mau et al. (2001). triplicate samples of 2.5 g of dry matter were extracted by mixing with 25 ml of solvent. the mixture was stirred for 30 min and kept in darkness for 24 h at 4°c. finally, this mixture was filtered with a whatman filter paper (n°4) and concentrated under rotary vacuum evaporator at 40°c. the dry residues were stored at 4°c for further analysis. each extraction was done in triplicate. for cookie samples, extracts were prepared using the method described by blanco canalis et al. (2020). cookies were milled and defatted in 30-ml hexane at 70°c for 20 min. cookie sample, 100 mg, was extracted with 1 ml of acetone:water (70:30 v/v) mixture, and agitation in vortex was applied for 5 min at room temperature. then the extracts were centrifuged for 10 min at 800× g and supernatants were collected. the supernatants were filtered and stored for further analysis. determination of total polyphenols (tpc), total flavonoids (tfc) and condensed tannins content (ctc) content of tpc was determined using the folin– ciocalteu spectrophotometric method (uv–vis) as described by dewanto et al. (2002). tpc was expressed as milligram of gallic acid equivalent per gram of dry matter (mg eag/g dw) through the calibration curve with gallic acid. for determining tfc, 250 µl of methanolic extract was combined with 75-µl nano2 (5%) (dewanto et al., 2002). tfc levels were expressed in the ingredients used for preparing cookies are as follows: wheat flour, zlp, 25-g powdered sugar, 26-g margarine, 0.6-g baking powder, 12-ml water, 0.7-g salt and 18-g whole eggs. control cookie (cc) samples were prepared with wheat flour (100%) and other ingredients whereas the enriched cookie samples were formulated using combinations of wheat flour and zlp. the zlp was incorporated into cookies at the following five levels: 0% (cc), 15% (c15), 30% (c30), 45% (c45) and 100% (c100) by replacing the equivalent amount of wheat flour from the formulation. for preparation of cookies, the process consisted of mixing wheat flour (or zlp), margarine and sugar using a mixer (kenwood, poland) for 7 min. then whole eggs and required amount of water were added to this mixture with the rest of ingredients to obtain homogeneous dough. prepared dough was rolled out to 2-mm thick circular shapes and biscuit dough was cut with a circular cutter to obtain pieces of 5-cm diameter. finally, cookies were baked in oven (convection, type de dietrich) at 175°c for 15 min. the baked cookies were cooled for 30 min at ambient temperature and stored in air-tight bags for further analysis. physical, color and textural analysis of cookies physical parameters, including weight (g), thickness (t), diameter (d) and spread ratio (d/t) were determined for cookie samples according to the procedure described by hussai et al. (2006). color measurement of the surface of the studied cookie samples was determined by a chromameter (konika minolta, sensing inc, japan) using the hunter l*, a* and b*. the l* values measure black (0) to white (100), a* values measure redness (+100) and greenness (-100), and b* values measure yellowness (+100) and blueness (-100). the hardness of formulated baked cookies was studied by three-point bending tests at room temperature. a texture analyzer taxt2i (perten, tvt 6700, uk) was used with 1,000-n load cell and a sharp blade cutting probe (hdp/bsk blade set with knife). the distance between support bars was 4 cm and the probe travelling speed was set at 1 mm/s. texture analysis were carried out in triplicate for each cookie formulation. thermogravimetric analysis (tga) thermogravimetric analysis of cookie samples was carried out in tga-4000 perkin elmer (curie grant) and analyzed by the pyris manager software. the analysis was carried out by standard protocol. weight loss (weight%) was observed with respect to temperature, and graphs were obtained for each sample with the pyris software. italian journal of food science, 2021; 33 (4) 87 effect of addition of tunisian zizyphus lotus l. fruits on nutritional and sensory qualities of cookies extremely. average of the scores was calculated and rounded off to the nearest whole number. sensory analysis of cookie samples was carried out in triplicate for each sample. statistical analysis each analytical determination was performed at least in triplicate. values of different parameters were expressed as mean ± standard deviation (x – ± sd). statistical analyses were performed with the statistica software. the duncan’s test was used to evaluate the significance of differences between mean values at p < 0.05. results and discussion zlp characterization chemical composition and techno-functional properties of zlp results of chemical composition and techno-functional properties of zlp are presented in table 1. the zlp had a moisture content of 9.55%, which was lower than those reported for zl fruits from algeria (saadoudi et al., 2017), but was within the recommended moisture contents (<14%) for safe storage, minimal microbial growth and chemical deterioration, leading to longer shelf life. protein, fat and ash contents of zlp were 2.64%, 5.37% and 3.01%, respectively. fat and ash contents in tunisian zlp were higher than those found by choi et  al. (2016) for korean jujubes. however, the protein content in zlp was higher than those found by ghalem et al. (2014) (2.10%) and saadoudi et al. (2017) (1.43%) in zizyphus lotus from algeria. the carbohydrates content in tunisian zlp, about 79.43%, was lower than those found by li et al. (2007) in five chinese zizyphus lotus cultivars (ranged from 80.86% to 85.63%). the observed difference in chemical composition of zlp depended on the location and the used species. in comparison to other fruit powders, the protein and ash contents in zlp were lower than those found in the tinospora cordifolia stem powder (proteins: 5.21% and ash: 6.26%) by tyagi et al. (2020). referring to the techno-functional properties of zlp, the values of wac, si and oac in zlp were about 2.81 g/g, 5.5 mg/g and 2.96 g/g, respectively. the obtained wac (about 1.32 g/g) and oac (about 1.20 g/g) values were higher than those detected in pearl millet flour (adebiyi et al., 2016). the wac of zlp was lower than those found in some cereals, such as in rice bran (around 5.21 g/g) (sangnark and noomhorm, 2004) and fruit coproducts such as passion fruit (13.5 g/g) and pineapple milligram of quercetin equivalent per gram of dry matter (mg ec/g dw). the protocol followed in the extraction of ctc was that recommended by sun et al. (1998). ctc was expressed in milligram of catechin equivalent per gram of extract (mg ce/g dw). antiradical activity methanol extract, 1,000 μl, was added to 500 μl of 2,2-diphenyl-1-picrylhydrazyl (dpph, 0.2 mm; hatano et  al., 1988). after vigorous stirring, the mixture was kept at room temperature for 30 min in the dark and the absorbance was measured at 517 nm. the antiradical activity was calculated as percentage of inhibition (pi) of dpph using the following formula: ( ) control extract control do do percentage of inhibition pi , do − = where docontrol is the absorbance of control at 30 min and doextract is the absorbance of extract. the antiradical activity was finally expressed as ic50 (µg ml –1). a lower ic50 value corresponds to a higher antioxidant activity of the extracted sample (patro et al., 2005). all samples were analyzed in three replications. reducing power this method consists of mixing 1,000 μl of fraction at different concentrations with 1,250 μl of phosphate buffer (0.2 mol/l, ph 6.6) and 1,250 μl of k3fe (cn)6 (1%) (ferreira et al., 2007). the solution was then kept in water bath for 20 min at 50°c. to stop reaction, 1,250 μl of trichloroacetic acid (tca) (10%) was added to the solution followed by centrifugation at 650 g for 10 min at 25°c. finally, at the intake of 1,250 μl of supernatant, 1,250 μl of h2o and 250 μl of fecl3 (0.1%) were added to the solution and the absorbance was measured at 700 nm. sensory analysis of cookies sensory analysis of cookies was conducted by 15 semitrained panelists (research students and laboratory staff ) from the field crops laboratory, inrat, tunisia. they were asked to evaluate cookie samples for flavor, color, texture, after taste and overall acceptability. after cooling, the cookies were coded and served to panelists in plastic containers with mineral water to cleanse the palate between each tasted sample. the scores were based on the following criteria: 9 = liked extremely; 8 = liked very much; 7 = liked moderately; 6 = liked slightly; 5 = neither liked nor disliked; 4 = disliked slightly; 3 = disliked moderately; 2 = disliked very much; 1 = disliked 88 italian journal of food science, 2021; 33 (4) zarroug y et al. monounsaturated fatty acids (mainly oleic acid) may contribute to the lower rate of coronary heart disease (chd) and a nutritional perspective (ryan et al., 2007). other representative fatty acids were palmitic (13.12%), stearic (4.72%) and gadoleic (2.64%) acids. in addition, arachidic, behenic, tetracosanoic and 10-octadecenoic acid methyl ester acids were minor fatty acids with contents varying from 0.60% to 1%. these results are similar to those reported by chouaibi et al. (2011) for zl seed oil. phytochemical composition and antioxidant activities of zlp total polyphenols, tfc and ctc of different extracts are summarized in figure 1. the highest contents of tpc, tfc and ctc were obtained in the acetone (60%) extract with values of 3.12 mg gae/g dw, 0.49 mg ce/g dw and 1.09 mg ce/g dw, respectively. the lowest contents were recorded in the methanol fraction. according to wojdyło et al. (2016), zl fruit is considered as a rich source of bioactive components, including polyphenols, triterpenic acids, polysaccharides, nucleosides and nucleobases. in general, phenolic compounds in zl  fruit  contribute to the antioxidant potential of its powder extracts. authors have reported correlation between phenolic compounds and antioxidant activity (elfalleh et al., 2011). antioxidant activity of different extracts of zlp was examined by its radical scavenging activity using the stable radical dpph and by reducing powers (table 2). methanol (60%) and acetone (60%) fractions exhibited the uppermost antiradical capacities with ic50 reaching 70 µg/ml and 75  µg/ml, respectively. the obtained results are lower than those found in moroccan zl (ic50  = 477.6 µg/ml) using methanol solvent (70%) (bakhtaoui et al., 2014). previous study conducted on zl fruit from another region of tunisia revealed ic50 = 289 µg/ml (mkadmini et al., 2015) and 310 µg/ml (ghazghazi et al., 2014). the zizyphus mauritiana fruits from nigeria exhibited ic50 = 338.45 µg/ml (okala et al., 2014), which was higher than the value obtained in the present study. the consequences of solvent on the antioxidant ability were also analyzed by determining reducing powers (figure 1). the highest antioxidant potential was observed in acetone fractions (330 µg/ml). the obtained result was similar to that found in southern tunisian zl fruits (289 µg/ ml) (mkadmini et  al., 2015). the observed variability of phytochemical contents and antioxidant activities of zizyphus genus could be explained by various factors, such as the geographical provenance, grown conditions, types of species and the nature of solvent used for extraction (ksouri et al., 2008). cookies characterization nutritional composition of formulated cookies nutritional composition of enriched cookies with zlp is presented in table 2. results established that the addition table 1. chemical composition and techno-functional properties of zlp. parameters zlp moisture (%) 9.55 ± 0.07 fat (%) 5.37 ± 0.42 protein (%) 2.64 ± 0.07 ash (%) 3.01 ± 0.02 carbohydrates (%) 79.43 ± 1.6 energy (kcal/100 g) 388.65 ± 2.92 si (mg/g) 5.5 ± 0.02 wac (g/g) 2.81 ± 0.07 oac (g/g) 2.96 ± 0.22 fatty acids (% total fatty acid) palmitic acid (c16:0) 13.12 ± 0.41 stearic acid (c18:0) 4.72 ± 0.22 arachidic acid (c20:0) 1.00 ± 0.01 behenic acid (c22:0) 0.92 ± 0.06 tetracosanoic acid (c24:0) 0.60 ± 0.02 oleic acid (c18:1) 55.73 ± 0.85 10-octadecenoic acid methyl ester (c19:1) 0.96 ± 0.05 gadoleic acid (c20:1) 2.64 ± 0.01 linoleic acid (c18:2) 20.31 ± 0.24 ⅀ saturated fatty acids (%) 20.36 ⅀ monousaturated fatty acids (%) 59.33 ⅀ polyunsaturated fatty acids (%) 20.31 ratio: unsaturated:saturated fatty acids 3.91 values are expressed as mean ± sd of three determinations. si: swelling index; wac: water absorption capacity; oac: oil absorption capacity. (14.6 g/g) (martínez et al., 2012). as noted, the hydration properties of zlp were related to the chemical structure of polysaccharides, proteins and the fruit source. however, oac is the capability of vegetable apolar chain protein to physically bind lipids by capillary attraction. this interaction is essential in food applications, because oil is known as retainer of flavor and enhances mouthfeel of food-formulated products. in addition, a better si improved functionality of flour, which would ultimately yield a good product (adebiyi et al., 2016). composition of fatty acids in zlp, saturated fatty acids represented 20.36% of total fatty acids, while monounsaturated fatty acids accounted for 79.64%. nine fatty acids were identified, where oleic acid was the major one accounting for 55.73%, followed by linoleic acid with 20.31% of total fatty acids (table 1). since zl oil was rich in both oleic and linoleic acids, it might be considered healthier for human consumption. it has long been recognized that plant oils containing relatively low concentrations of omega-6 and higher levels of italian journal of food science, 2021; 33 (4) 89 effect of addition of tunisian zizyphus lotus l. fruits on nutritional and sensory qualities of cookies 3.5(a) (b) ascorbic acid reducing power dpph bht water methanol (60%) ethanol (60%) acetone (60%) tpc tfc ctc3 2.5 2 1.5 1 0.5 acetone (60%) c on te nt s ethanol (60%) methanol (60%) water 0 200 400 600 800 0 figure 1. (a) tpc, tfc and ctc of different solvent extracts, and (b) antioxidant activities of zlp. tpc: total polyphenols content (mg gae/g ms); tfc: total flavonoids content (mg ce/g ms); ctc: condensed tannins content (mg ce/g ms); dpph (ic 50 , µg/ml); reducing power (ec 50 , µg/ml); gae: gallic acid equivalents; ce: catechin equivalents. table 2. nutritional analysis of formulated cookies. cookies moisture (%) fat (%) protein (%) ash (%) carbohydrates (cho %) water activity (aw) energy (kcal/100 g) cc 5.14 ± 1.20e 17.10 ± 0.01a 7.10 ± 0.03e 0.84 ± 0.02a 69.82 ± 1.22a 0.50 ± 0.01 461.58 ± 4.57a c15 6.55 ± 0.32d 18.90 ± 0.01b 6.60 ± 0.02d 0.92 ± 0.03b 67.03 ± 0.34b 0.53 ± 0.02 464.62 ± 2.13a c30 7.29 ± 0.37c 19.50 ± 0.02c 6.35 ± 0.02c 1.10 ± 0.03c 65.76 ± 0.41c 0.55 ± 0.13 463.94 ± 1.33a c45 7.62 ± 2.48b 19.70 ± 0.01d 6.06 ± 0.02b 1.03 ± 0.01d 65.59 ± 2.48c 0.57 ± 0.01 463.90 ± 9.29a c100 7.64 ± 0.53a 19.90 ± 0.05e 4.94 ± 0.01a 1.50 ± 0.01e 66.02 ± 0.51d 0.59 ± 0.01 462.94 ± 1.21a mean values with different superscript alphabets in the same column differ significantly. cc: control cookies; c15, c30, c45, c100 are cookie samples containing wheat flour:zlp in the ratio of 85:15, 70:30, 55:45 and 0:100, respectively. of zlp significantly increased (p < 0.05) the moisture, ash, fat and carbohydrates contents in cookies when compared to control cookies whereas protein values were decreased significantly. in all formulated cookies, carbohydrates were the major component, followed by fat, protein, water and ash. results demonstrated statistically significant differences (p < 0.05) between formulated cookies, especially for moisture, fat, protein and ash contents. only for carbohydrates content, a nonsignificant difference was observed. similar results revealed increase in ash and moisture contents in cookies enriched with spinach (5–15%) (galla et al., 2017) and tinospora cordifolia stem (2–12%) powders (tyagi et al., 2020). in the same context, bhat et al. (2020) reported consistent results in cookies containing tomato powder and crude lycopene. the energy values of enriched cookies ranged from 461.58 kcal in cc sample to 464.62 kcal in c15 sample. these results were in consistency with previous studies carried by kaur et al. (2017) and saadoudi et al. (2017) on cookies enriched with zlp from algeria and raw flaxseed flour. about water activity, similar to that observed for moisture content, addition of zlp in cookies provoked a significant increase in this parameter. the highest water activity value was observed in c100 sample (0.59), but it was still below than that allowed for growth of microorganisms (aw > 0.6) (chieh, 2006). these findings were higher than those reported by milićević et al. (2020), who revealed water activity values ranging from 0.297 to 0.411 in cookies prepared with wheat bran gels. physical, textural and color characteristics of formulated cookies according to pareyt and delcour (2008), quality of good cookies is related to large piece diameter, tender but snapping final product, and uniform surface-cracking pattern. the physical, textural and color characteristics of formulated cookies are depicted in table 3. the weight of cookies enriched with zlp varied from 12.5 g in c15 sample to 13.27 g in c100 sample. after addition of zlp, the weight of cookies increased significantly (p < 0.05) in 90 italian journal of food science, 2021; 33 (4) zarroug y et al. comparison to cc sample. this increase in the weight of cookies was related to the presence of dietary fibers in zlp, which enables to bind with water molecules and prevents loss of moisture during baking process. these results are in the trend of those observed by saadoudi et al. (2017) on biscuits made with algerian zlp. in all formulated cookies, the diameter ranged from 56.70 mm to 57.60 mm. maximum diameter was observed in cc sample, while minimum was in c15 cookie sample. these variations could be due to the dilution of gluten present in wheat flour. results also demonstrated increase in the thickness of cookies from 5.03 mm (cc sample) to 6.15 mm (c100 sample). the obtained results of cookies enriched with zlp are in accordance with the studies reported by kaur et al. (2017) and saadoudi et al. (2017) on some fortified cookies. a decrease in the spread ratio of cookies was observed with the inclusion of zlp; this reduction could be due to the water absorption capacity of fibers present in zlp. similar results were obtained by das chagas et al. (2020), ismail et al. (2014) and toledo et al. (2017) when evaluating the effect of addition of by-products of pomegranate peels, pineapple, apple, melon, and camu-camu in cookies. in order to evaluate the acceptability of prepared cookies by consumers, it is very important to determine their color parameters. the photographic images (figure 2) of formulated cookies revealed that zlp was the main ingredient that developed dark color, and c100 sample was the much darker cookies, looking like biscuits made with chocolate. the statistical analysis revealed a significant difference (p < 0.05) between all cookie samples regarding l* and b* values. in addition, as observed in table 4, cc sample had the highest l* and b* values as compared to the cookies enriched with zlp. moreover, addition of zlp decreased l* and b* values but increased a* values of formulated cookies. these effects could be explained by the dark color of cookies because of added zlp, which is related to its richness in natural pigments such as polyphenols (ajila et al., 2008; takeungwongtrakul and benjakul. 2017). according to masmoudi et al. (2021), the dark color of cookies could be also due to the more pronounced maillard and caramelization reactions during the baking process, when wheat flour was substituted by zlp containing higher content of sugar. these findings are in agreement with those of biscuits supplemented with doum dietary fiber (aboshora et al., 2019) and bamboo shoot powder (ajila et al., 2008). regarding texture analysis, results revealed that the hardness and fracturability (brittleness) of cookies were increased significantly (p < 0.05) with the addition of zlp. in all formulated cookies, c100 sample had the highest hardness and fracturability values (74.33 n and 6.8 mm) compared to cc sample (22.33 n and 0.58 mm). similar results were also observed in previous findings in ta bl e 3. p hy si ca l, te xt ur al a nd c ol or a na ly si s of fo rm ul at ed c oo ki es c oo ki es d ia m et er (d ) ( m m ) th ic kn es s (t ) ( m m ) w ei gh t ( g) s pr ea d ra tio (d /t ) l* a* b* h ar dn es s (n ) fr ac tu ra bi lit y (m m ) c c 57 .6 0 ± 0. 24 a 5. 03 ± 0 .0 2a 13 .1 3 ± 0. 21 b 2 .6 0 ± 0. 24 a 66 .7 2 ± 0. 56 c 4. 17 ± 0 .1 2a 47 .8 3 ± 0. 17 e 22 .3 3 ± 1. 53 a 0. 58 ± 0 .0 3a c i5 56 .7 0 ± 0. 92 a 5. 09 ± 0 .0 9a 12 .5 0 ± 0. 95 ab 1. 70 ± 0 .9 2a 46 .6 2 ± 0. 02 d 10 .0 6 ± 0. 07 b 36 .9 7 ± 0. 01 4d 40 .0 0 ± 4. 58 b 0. 99 ± 0 .0 6a b c 30 57 .1 0 ± 0. 43 a 5. 50 ± 0 .3 2b 12 .7 0 ± 0. 36 ab 2. 10 ± 0 .4 3a 39 .7 2 ± 0. 25 c 11 .5 8 ± 0. 08 c 34 .5 6 ± 0. 17 c 51 .3 3 ± 1. 53 c 1. 41 ± 0 .0 5b c 45 57 .2 3 ± 0. 66 a 5. 55 ± 0 .3 3b 12 .8 0 ± 0. 21 b 2. 23 ± 0 .6 6a 36 .8 8 ± 0. 82 b 11 .4 3 ± 0. 29 c 32 .8 6 ± 0. 14 b 65 .0 0 ± 2. 65 d 2. 45 ± 0 .0 6c c 10 0 57 .3 2 ± 0 46 a 6. 15 ± 0 .1 0c 13 .2 7 ± 0. 38 b 2. 32 ± 0 .4 6a 32 .5 3 ± 0. 31 a 12 .3 3 ± 0. 18 b 29 .6 4 ± 0. 05 a 74 .3 3 ± 2. 52 c 6. 80 ± 0 .6 6d m ea n va lu es w ith d iff er en t s up er sc rip ts o n th e sa m e co lu m n di ffe r s ig ni fic an tly (d un ca n’ s ls d te st , p < 0 .0 5) . w he re c c : c on tro l c oo ki es , c 15 , c 30 , c 45 , c 10 0 ar e co ok ie s sa m pl es c on ta in in g w he at fl ou r: zl p (8 5: 15 , 7 0: 30 , 5 5: 45 , 0 :1 00 ). italian journal of food science, 2021; 33 (4) 91 effect of addition of tunisian zizyphus lotus l. fruits on nutritional and sensory qualities of cookies compounds such as protocatechuic acid, gallic acid, chlorogenic acid, caffeic acid and ascorbic acid present in zlp (koley et al., 2016). in all formulated cookies, c100 sample contained the highest contents of tpc (0.11 mg gae/g), tfc (0.05 mg qe/g) and ctc (1.07 mg qe/g). our results were generally in agreement with the findings of other studies regarding the substitution of wheat flour in cookies by some fruit and vegetable powders such as apple, pineapple and melon coproducts (toledo et al., 2017), watermelon rind powder (naknaen et al., 2016) and camu-camu coproduct (das chagas et al., 2020). antioxidant potential of cookies table 4 indicates that the addition of zlp in cookies significantly (p < 0.05) increased the free radical scavenging activity and reducing power as compared to cc sample. this implies that the addition of higher level of zlp improved the antioxidant activity of cookie products. in all formulated cookies, the highest free radical scavenging activity and reducing power were observed in c100 sample. this observation was in accordance with previous studies carried out with incorporation of which cookies were enriched with bamboo shoot, spinach, tinospora cordifolia stem and tomato powder (ajila et al., 2008; bhat et al., 2020; galla et al., 2017; tyagi et al., 2020). this increase in texture parameters are explained by the presence of zlp, with high content of polysaccharides, which may lead to the dilution of gluten and extensive gluten structure. in addition, substituting zlp for wheat flour in cookies formulation decreased its gluten content responsible for the viscoelastic character of dough. das chagas et al. (2020) affirmed in their study on cookies enriched with camu-camu coproduct powder that reduction of glutenin and gliadin proteins could have a remarkable effect on the formation of viscoelastic dough, which may possibly lead to increased hardness. tpc, tfc and ctc of cookies table 4 reveals the tpc, tfc and ctc values of defatted methanolic extract of formulated cookie samples. results revealed that the addition of zlp increased significantly (p < 0.05) tpc, tfc and ctc in enriched cookie samples when compared to cc sample, and this increase was effectively due to the high content of phenolic cc c15 c30 c45 c100 figure 2. photographic images of cookie samples. cc: control cookies, c15, c30, c45, c100 are cookie samples containing wheat flour:zlp in the ratio of 85:15, 70:30, 55:45 and 0:100, respectively. table 4. phytochemical analysis and antioxidant activities of formulated cookies. cookies phytochemical parameters antioxidant activities tpca tfcb ctcb dpphc reducing powerd cc <0.01 0.01 ± 0.01a 0.57 ± 0.14e 454 ± 0.54e 950 ± 3.42e c15 0.01 ± 0.02a 0.02 ± 0.01b 0.80 ± 0.15d 355 ± 0.21a 820 ± 2.45d c30 0.02 ± 0.01b 0.03 ± 0.02c 0.93 ± 0.1b 280 ± 0.15b 410 ± 1.34c c45 0.05 ± 0.01c 0.04 ± 0.01d 1 ± 0.05a 165 ± 0.1c 380 ± 3.5b c100 0.11 ± 0.04d 0.05 ± 0.01e 1.07 ± 0.01c 95 ± 0.24d 320 ± 2.45a synthetic standards bht (ic 50 , mg/ml) – – – 46.6 ± 0.08 ascorbic acid (ec 50 , mg/ml) – – – – 68 ± 0.06 a(mg gae/g), b(mg qe/g), c(ic 50 , µg/ml), d(ec 50 , µg/ml). bht: butylated hydroxytoluene; tpc: total polyphenols content ; tfc: total flavonoids content ; ctc: condensed tannins content; cc: control cookies; c15, c30, c45, and c100: cookie samples containing the wheat flour:zlp in the ratio of 85:15, 70:30, 55:45, 0:100, respectively.bht: butylated hydroxytoluene. an values with different superscripts in the same column differ significantly (duncan’s lsd test, p < 0.05). 92 italian journal of food science, 2021; 33 (4) zarroug y et al. zlp. besides, decrease in the intensity of peaks in cookie samples reflects their change in crystallinity. thermogravimetric analysis in general, when materials are heated, they lose weight because of simple processes, such as drying, or because of chemical reactions that release gasses (blanco canalis et al., 2018). the observed weight loss, which was related to reduction of water by evaporation can provide valuable information about the volatile and heat labile compounds present in cookie samples and their thermal stability. the tga and differential tga (dr-tga) curves of cookie samples are revealed in figure 4(a) and figure 4(b). the tga of all studied cookie samples demonstrated four typical stages of weight loss. figure 4(b) indicates that no significant differences in shape and intensity were observed between c45 and c100 samples. comparison between the cookies enriched with zlp and control samples indicated that the maximum rate of weight loss was in c45 sample (92.69%) followed by c100 (91.72%). the first stage of weight loss ranged from 190°c to 211°c, and was attributed to the loss of free and bound water with increasing temperature. it is suggested that inclusion of zlp in cookies reduced the amount of water available for protein hydration, allowing faster water evaporation. in addition, the observed increase in weight loss suggested the formation of a weak gluten network in cookies dough. according to blanco canalis et al. (2018), water in cookies dough is shared between different components (such as starch, gluten and sucrose) responsible for trapping water until it is released because of heating. the second, third and fourth stages ranged from 229.54°c to 254.38°c, 311.85°c to 373.63°c and 426.29°c to 470.3°c, respectively. the second stage represented freeze-dried japanese quince fruits (antoniewska et al., 2019) and tomato powder in cookies (bhat et al., 2020). several authors also demonstrated that the dpph radical scavenging activity and reducing power increased in cookies with baking temperature, which could be due to the formation of melanoidins produced in the maillard reaction (bhat et al., 2020). in addition, high concentration of bioactive compounds in zlp lead to an increase in the antioxidant potential of formulated cookies. this could be of interest for human health and could provide an extended shelf life of cookies for food industry. ftir analysis the ftir spectra of cookie samples in the range of 400–4,000 cm−1 are revealed in figure 3. results revealed that cookie samples demonstrated common peaks with small variations in intensity. in all cookie samples, major peaks were observed at 3,500 cm−1 and 2,800 cm−1, which indicated the stretch vibrations of o–h groups. in addition, the observed peak at 2,943 cm−1 resulted from the stretching vibration of c–h bond. however, the peaks found between 1,645 cm−1 and 1,742 cm−1 were attributed to the c–o stretch vibration of α,β-unsaturated compound. similar results were reported by tyagi et al. (2020) on cookies enriched with tinospora cordifolia stem powder. the observed peaks at 1,485.71 cm−1 were essentially assigned to water molecules absorbed in the amorphous region. the established peaks at a range of 1,142–1,000 cm-1 could be attributed to the c–o bond and aliphatic c–n stretching; these peaks were with higher intensities in cookie samples enriched with zlp compared to cc sample. these peaks are related to the presence of phenolic compounds, β-glycoside and glucoside (tyagi et al., 2020), which were essentially present in 44.3 40 35 30 25 20 15 10 5 3.7 4000,0 3600 3200 2800 2400 2000 wavenumber (cm–1) tr an sm itt an ce ( % ) 1800 1600 1400 1200 1000 800 600 400,0 cc c15 c30 c45 c100 figure 3. ftir spectra of cookie samples; cc: control cookies; c15, c30, c45 and c100 are cookie samples containing wheat flour:zlp in the ratio of 85:15, 70:30, 55:45 and 0:100, respectively. italian journal of food science, 2021; 33 (4) 93 effect of addition of tunisian zizyphus lotus l. fruits on nutritional and sensory qualities of cookies 100 cc c15 c30 c45 c100 cc c15 c30 c45 c100 80 60 40 20 0 100 200 300 temperature (°c) t g a ( % ) –0,025 –0,020 –0,015 –0,010 –0,005 –0,000 d rt g a ( % ) 400 500 600 0 100 200 300 temperature (°c) 400 500 600 figure 4. (a) thermogravimetric, and (b) differential thermogravimetric curves of cookie samples; cc: control cookies; c15, c30, c45 and c100 are cookie samples containing wheat flour:zlp in the ratio of 85:15, 70:30, 55:45, 0:100, respectively. the depolymerization and degradation of organic matter (starch, gluten and polysaccharides). however, the third stage of weight loss represented the oxidation of organic matter that lead to the formation of ash residue. in the last range of temperature (close to 500°c), the total weight loss measured for cookie samples was not noticeably different. microstructure of cookie samples scanning electron microscopy was used to study the microstructure of baked cookie samples. the microstructure observation indicated that the protein network matrix was well developed and the starch granules were more intact in the case of control cookies (figure  5). however, a change in the structure of cookies was observed with increase in zlp content. this change was strongly observed in the case of c100 sample. alteration in the microstructure of cookies could be due to the richness of zlp in polysaccharides, which have good water retention capacity; this could also be due to the gelatinization of starch and denaturation of protein matrix. these results are in agreement with those found by adebiyi et al. (2016), who reported a similar microstructure of cookies enriched with millet flours. sensory evaluation of cookies the flavor, color, texture, after-taste and overall acceptability scores recorded for the cookies enriched with zl powder are depicted in table 4. results of sensory evaluation revealed that color, flavor and after-taste parameters increased with increase of zlp in cookies, reaching the values of 7.87, 8.89 and 3.20, respectively, noted essentially in c100 sample. the sensory evaluation of cookies’ color surface paralleled the colorimetric measurement (table 3), with zlp-enriched cookies being darker than the control. in addition, increase in zlp concentration demonstrated a decrease in texture parameter in comparison to control cookies; these results were in agreement with the instrumental data of hardness and fracturability cc c15 c30 c45 c100 figure 5. sem of cookies samples. magnification ×400 and ×500; cc: control cookies. c15, c30, c45, c100 are cookies samples containing wheat flour: zlp (85:15, 70:30, 44:45, 0:100). (table 3). similar effect was reported by krystyjan et al. (2015), saadoudi et al. (2017) and šaponjac et al. (2016) with cookies enriched with bee pollen, jujube, and sour cherry pomace extract, respectively. in all enriched cookies, the c15 sample had a sensory score near to the cc 94 italian journal of food science, 2021; 33 (4) zarroug y et al. sample. for the overall acceptability of results, cc and c15 samples were most acceptable, while c100 sample was not preferred by panelists. masmoudi et al. (2021) established that biscuits supplemented with jujube flour had as acceptable quality as the control for all its doses (5%, 10% and 15%). reduction in acceptability of cookies at higher level of substitution with zlp was due to more plant undertones contributed by zlp. similar effect was reported for cookies supplemented with flaxseed flour (kaur et al., 2017) and japanese quince fruit (antoniewska et al., 2019). conclusion the results of the present study reveal that zlp contains several bioactive components, fatty acids, phenolics and antioxidants that are beneficial for human health. results demonstrate that this powder has a great nutritional potential to be used in food industry and to substitute the conventional wheat flour in the formulation of new functional cookies. addition of zlp improves the phytochemical composition and the antioxidant potential of formulated cookies as revealed by the increase in dpph and reducing power values in cookies. the ftir and tga results revealed the presence of many bioactive compounds in zlp and formulated cookies. besides improving the nutritional value, addition of zlp affects the appearance, physicochemical, textural and sensorial properties of formulated cookies. based on the sensory analysis, it is concluded that the acceptability of formulated cookies is directly dependent on the amount of added zlp, and cookies enriched with 15% zlp (c15) seems to have gathered the overall preference of panelists. acknowledgments this work was supported by the ministry of higher education and scientific research, tunisia and the table 5. sensory acceptability of both control and formulated cookies. cookies color flavor after-taste texture overall acceptability cc 4.27 ± 1.12a 6.54 ± 0.71c 8.47 ± 1.30a 6.60 ± 1.60b 7.20 ± 2.04a c15 5.20 ± 1.00b 7.33 ± 0.63c 7.93 ± 0.96a 5.66 ± 1.13a,b 6.47 ± 1.30b c30 5.60 ± 0.64b 7.41 ± 1.03c 7.33 ± 1.05a 5.06 ± 1.03c 5.00 ± 1.31a,b c45 6.67 ± 0.63c 8.27 ± 0.52 d 5.36 ± 1.39b 4.87 ± 1.44a 4.68 ± 1.25a c100 7.87 ± 0.63c 8.89 ± 0.41d 3.20 ± 2.01b 4.07 ± 1.74a 4.20 ± 2.61a values are expressed as mean ± standard deviation of three determinations. mean values with different superscripts in the same column differ significantly (p < 0.05). cc: control cookies; c15, c30, c45 and c100 are cookie samples containing wheat flour:zlp in the ratio of 85:15, 70:30, 55:45, 0:100, respectively. ministry of agriculture, water resources and maritime fisheries, tunisia. the authors wish to thank the technical staff, particularly hanen nasri and nesrine haj yahya, for their valuable contribution in performing various analyses. we are also grateful to tesnim zarroug for her valuable efforts to check proofs of this paper. declaration of interest the authors declare that they have no known competing financial interests or personal relationships that could have influenced the research reported in this paper. references abdeddaim m., lombarkia o., bacha a., fahloul d., abdeddaim d., farhat r., 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antioxidant capacity of spanish jujube (zizyphus jujube mill.) fruits. food chem. 201:307–314. https://doi.org/10.1016/j. foodchem.2016.01.090 yamada h., nagai t., cyong j.c., otsuka y., tomoda  m. and shimizu n. 1985. relationship between chemical structure https://doi.org/10.1016/0008-6215(85)85011-4� https://doi.org/10.1016/0008-6215(85)85011-4� https://doi.org/10.1016/j.foodchem.2016.01.090� https://doi.org/10.1016/j.foodchem.2016.01.090� p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (3): 91–98 issn 1120-1770 online, doi 10.15586/ijfs.v34i3.2225 91 p u b l i c a t i o n s codon determination of aspartame and alitame in liquid dairy products and milk-containing beverages in the chinese market jiasi yu1, wei hu2, dan wu1, yun ding1, xin wang1, and ping liu1* 1department of physical and chemical inspection, school of public health, cheeloo college of medicine, shandong university, jinan, china; 2shinan district health bureau, qingdao, china *corresponding author: ping liu, department of physical and chemical inspection, school of public health, cheeloo college of medicine, shandong university, jinan, 250012 shandong, china. email: liupingp@sdu.edu.cn received: 4 may 2022; accepted: 29 august 2022; published: 6 october 2022 © 2022 codon publications open access paper abstract aspartame and alitame are a type of food additives commonly used in recent years. however, it is difficult to conclude that long-term use of synthetic sweeteners is completely harmless. this study aimed to establish a simple high performance liquid chromatography (hplc) method to detect these two sweeteners, and to measure the concentrations of sweeteners in liquid dairy products and milk-containing beverages in the chinese market. in this experiment, aspartame and alitame had a good linearity, and the average recovery values were also good with the relative standard deviations (rsds) of 0.4–2.2%. the limits of detections (lods) were 0.52 and 0.48 µg g–1 for aspartame and alitame, respectively. according to consumer daily purchase habits, 100 samples were purchased from supermarkets and milk tea shops in jinan, china. the results showed that the amount of sweeteners added in all samples did not exceed the national standards, but there was a problem that the food label content was incomplete. we hope that the relevant departments would strengthen supervision and management of food labelling, and protect the legitimate rights and interests of consumers. keywords: alitame; aspartame; hplc; liquid dairy products; milk-containing beverages introduction with the development of the food industry, more and more food additives are used in food processing to improve the colour, taste, smell, nutritional value, and shelf-life of food. food additives are synthetic or natural substances added to food, which can not only improve food quality, colour, aroma and taste but also meet the needs of anti-corrosion, preservation, and processing technology. there are more than 2000 kinds of food additives permitted by the state (gb 2760-2014), which are divided into 22 types according to their functions, mainly including sweeteners, preservatives, acidity regulators, antioxidants, colorants, bleaching agents, etc. among them, sweeteners are used in all kinds of food to increase the flavour of food. according to different sources, sweeteners can be divided into two categories, one is natural sweeteners such as licorice and stevioside, and the other is synthetic sweeteners. in the past few decades, the global prevalence of obesity has increased rapidly. the survey of lifestyle shows that excessive consumption of sugars such as sugary drinks may lead to obesity, diabetes, and cancer (malik et al., 2010). the world health organization recommends that the intake of free sugars should not exceed 10% of the total energy intake (who, 2015). in order to reduce the intake of sugar, some countries have begun to levy sugar tax and have successfully reduced sugar consumption and obesity rate (nakhimovsky et al., 2016). industry is also responding to this policy by reducing sugar in food processing. sugar is an indispensable ingredient in food processing, because it can improve the structure, colour, mailto:liupingp@sdu.edu.cn 92 italian journal of food science, 2022; 34 (3) yu j et al. sweeteners (stevioside and mogrosides). these sweeteners are considered safe to eat. in the european union, australia, and new zealand, sugar alcohols such as maltitol, lactose alcohol, xylitol, and erythritol are also considered safe to eat. fao, who, and jecfa points out that the use of food additives within the permitted scope will not cause damage to the human body (praveena et al., 2019). china’s standard for the use of food additives also refers to the international standards, which clearly stipulates the amount of sweeteners used in all kinds of food. nevertheless, the safety of synthetic sweeteners with high sweetness is still controversial. emerging epidemiological evidence shows that food additives may have non-benign biological effects and have a negative impact on human intestinal flora, leading to the destruction of intestinal mucus layer (rinninella et al., 2019). eating excessively processed and packaged foods is associated with increased all-cause mortality and increased global incidence of obesity, metabolic syndrome, and inflammatory bowel disease (ibd) (kim et al., 2015; marionletellier et al., 2019; pinget et al., 2019; rico-campà et  al., 2019). for example, with the increase in the sales of processed food, the incidence of ibd in china has increased significantly (ng et al., 2013). recent studies have shown that the metabolic and inflammatory effects of food additives may be induced by changes in intestinal flora. artificial sweeteners in food can change the number of intestinal bacteria, increase the pathogenicity of intestinal bacteria, and interfere with the normal environment of human and animal intestinal mucosa (bian et al., 2017; swidsinski et al., 2009; zuo et al., 2018). studies suggest artificial sweeteners such as saccharin, sucralose and aspartame can induce glucose intolerance in rats. artificial sweeteners have metabolic effects and may lead to type 2 diabetes, obesity, and other diseases (suez et al., 2014). consumption of synthetic sweeteners during pregnancy and lactation has adverse effects on the metabolism of infants, which may lead to metabolic disorders in later life (olivier-van stichelen et al., 2019). plows et al. find that the intake of synthetic sweeteners during pregnancy can lead to glucose intolerance, hyperglycaemia, shortened pregnancy time, male fetal growth restriction, and female fetal hypoglycaemia (plows et al., 2020). in addition, studies have shown that consumption of aspartame during pregnancy can increase the obesity rate and make offspring more prone to anxiety behavior (palatnik et al., 2020). long-term consumption of synthetic sweeteners is closely related to memory loss, alzheimer’s disease, and depression (burke and small, 2015; gao et al., 2018), in addition to carcinogenicity and genotoxicity (mao and song, 2018; purohit and mishra, 2018; zhao and wang, 2018). and taste of food. hence, reducing the sugar content in food may have a negative impact on the taste of food. to ensure sweet taste while reducing sugar, synthetic sweeteners are usually used instead of sugar. the human body does not digest and absorb sweeteners (such as sugar alcohols). the content of sweetener is high and provides little energy, so its dosage is very small. in order to prevent obesity, cardiovascular disease, diabetes, and other chronic diseases, consumers tend to choose foods containing lower calorie sweeteners. as a result, “sugar free” or “no added sugar” products with synthetic sweeteners instead of sugar have become more and more popular. at present, the artificial synthetic sweeteners approved for use in china mainly include aspartame, alitame, saccharin sodium, acesulfame, sodium cyclamate, sucralose, etc. aspartame, the chemical name of l-aspartyl-l phenylalanine methyl ester, also known as proteoglycan, sweetener, aspartame essence, aspartame mother, aspartame, etc., belongs to dipeptide derivatives. at room temperature, it is white crystalline powder. the taste is very similar to sucrose, and the sweetness is 100–200 times of sucrose. in china, aspartame can be used as sweetener and flavour enhancer in dairy products, pastries, seasonings, drinks, jellies, puffed foods, etc. after people consume aspartame, it completely decomposes into aspartic acid, phenylalanine, and methanol in the stomach and intestines, and then is absorbed in the blood. patients with phenylketonuria lack phenylalanine hydroxylase, and their excessive intake of aspartame causes accumulation of phenylpyruvate in the body and damages the nervous system. thus, foods containing aspartame should be marked as follows: aspartame (containing phenylalanine). alitame, the chemical name of l-α-aspartyl-n-(2,2,4,4tetramethyl-3-sulfotrimethyl)-d-alanamide, also known as aspartame, is a dipeptide derivative, which is generally a white crystalline powder without odour and hygroscopicity. the sweetness of alitame is 2000 times higher than that of sucrose, and its properties are relatively stable, especially for heat and acid. alitame is easily soluble in water or hydroxyl containing solvents, but it is difficult to dissolve in lipophilic organic solvents. alitame is often used with other sweeteners and has a good sweetening synergistic effect. china approved the use of alitame in 1994. it can be added to dairy products, frozen beverages, preserves, beverages, gum-based candies, jellies, and other foods as a sweetener (wang and shen, 2019). at present, with the increasing demand for “sugar free” or “low sugar” products, synthetic sweeteners can be an ideal and calorie-free sugar substitute in the food industry. the fda has approved six synthetic sweeteners (edvantame, aspartame, acesulfame, neotame, saccharin, and sucralose) and two natural non-nutritive italian journal of food science, 2022; 34 (3) 93 determination of aspartame and alitame studies have shown that the effect of aspartame may be related to methanol or its metabolites. as a neurotoxin, methanol is harmful to human health. one litre of beverage containing aspartame can produce about 56 mg of methanol, and a can of beverage containing aspartame can produce about 22.4 mg of methanol. the environmental protection bureau recommends that the daily intake of methanol should not exceed 7.8 mg (liu et al., 2012). moreover, long-term consumption of aspartame can cause migraine (sathyapalan et al., 2015; zaeem et al., 2016). some scholars have found that aspartame has become a new type of water pollutant, which can affect the water environment through biochemical reactions (lin et al., 2017; seo et al., 2016). according to national regulations (gb 28050-2011), additives in food must be clearly marked on the label, but there are still phenomena of incomplete and non-standard content of food labels. it is necessary to strengthen the supervision of the use of synthetic sweeteners. at present, the detection methods of aspartame and alitame mainly include ultra-high performance liquid chromatography (uplc) (jin, 2020; lu et al., 2021; yu et al., 2018), ion chromatography (xie et al., 2011; zhu et al., 2005), high performance liquid chromatography (hplc) (chen and li, 2006), and high performance liquid chromatography tandem mass spectrometry (hplc-ms) (tang et al., 2019; wang et al., 2019; yang and chen, 2009), etc. however, most of the above methods require expensive equipment, which is not available in conventional laboratories. in this study, a convenient and reliable hplc method for the determination of aspartame and alitame was developed and validated. furthermore, this study measured the concentrations of aspartame and alitame in liquid dairy products and milk-containing beverages collected from jinan supermarkets and milk tea shops. the results could provide a basis for improving market supervision and regulation in china. materials and methods reagents and chemicals standards: aspartame (purity asis for improving market supervision and regulation solarbio (beijing, china). acetonitrile (hplc grade) was purchased from damao chemical reagent factory (tianjin, china), ethanol (superior purity) from sinopharm chemical reagent co., ltd. (shanghai, china), filter paper (diameter 15 cm) from hangzhou special paper co., ltd. (zhejiang, china), organic filter membrane (0.45 μm) from beijing high purity technology co., ltd. (beijing, china), and the experimental water was milli-q double distilled water (bedford, ma, usa). sample collection and treatment all the samples were selected according to the daily purchasing habits of consumers: 100 samples of different types, including 40 liquid dairy products and 60 milk-containing beverages, were collected randomly from supermarkets and milk tea shops in lixia district of jinan, china, in august 2019. an aliquot of 5 g (accurate to 0.0001 g) of thoroughly and adequately mixed sample was put into a 50 ml centrifuge tube (shimadzu analytical balance aux120, kyoto, japan). ten milliliters ethanol was added to each sample and then sealed. the centrifuge tube was turned upside down five times without oscillating for milk-containing beverages, or was vortexed for 10 s for liquid dairy products. the resulting mixture was left at room temperature for 1 min, and then centrifuged at 4000 rpm for 5 min (eppendorf centrifuge 5430r, hamburg, germany). the supernatant was transferred to a 25 ml volumetric flask. the residue was dissolved with 8.0 ml ethanol/ water (2/1, v/v) and centrifuged at 4000 rpm for 5 min. subsequently, the supernatant was transferred into the same 25 ml volumetric flask and made up to volume with ethanol/water (2/1, v/v). finally, the solution was filtered through a 0.45 μm organic filter membrane, and 20 µl aliquot solution was injected into the hplc system. standard solutions stock standard solutions of aspartame and alitame were prepared at 205.8 and 200 μg ml–1 by dissolving 2.1 and 2.0 mg of powder in 10 ml of water, respectively. all the stock solutions were stored at 4°c for utilization. a mixed standard solution was produced by transferring 2.5 and 1.7 ml of aspartame and alitame stock solutions into a 10 ml volumetric flask and making up to volume with water. thereafter, a series of mixed standard solutions were obtained by further dilution. the concentrations of aspartame in the mixed standard solutions were 0.51, 1.03, 10.29, 34.99, and 51.45 μg ml–1, respectively; the concentrations of alitame were 0.50, 1.00, 10.00, 34.00, and 50.00 μg ml–1, respectively. the mixed standard solutions were stable for 1 month and were stored at 4°c. hplc conditions a lachrom elite hplc system (hitachi, tokyo, japan) was used for the determination of aspartame and alitame and was quantified by diode array detection using an l-2455 diode array detector (hitachi, tokyo, japan) with the wavelength of 200 nm. the sample was separated on a c18 column (4.6 × 150 mm, 5 μm) (elite, dalian, china) 94 italian journal of food science, 2022; 34 (3) yu j et al. 208 nm. in this experiment, the detection wavelength was carried out at 200 nm based on the ultraviolet full wavelength scanning spectrum. mobile phase the composition of the mobile phase plays an important role in elution and chromatographic separation. at the beginning of this project, we planned to perform the hplc analysis of aspartame and alitame using two types of mobile phases, methanol/water (40/60, v/v) and acetonitrile/water (20/80, v/v). it was found that the target peaks overlapped, which may be due to the interference of other impurities in the sample, when the mobile phase methanol/water (40/60, v/v) was used for sample detection. other mobile phases were tested by changing the ratio of acetonitrile and water, but these mobile phases did not deliver a notable improvement to the chromatography. therefore, acetonitrile/water (20/80, v/v) was used as the mobile phase in terms of peak shape, resolution, and overall analysis time. flow rate the fast flow rate can increase column pressure, while slow flow rate can cause tailing deformation of chromatographic peaks. the flow rate of 0.8, 1, and 1.2 ml min–1 was employed for this experiment, and other experimental conditions remained unchanged. when the flow rate was 1.2 ml min–1, the resolution of the chromatographic peak was poor. when the flow rate was 0.8 ml min–1, the retention time (rt) of the chromatographic peak was longer. considering comprehensively, a flow rate of 1  ml  min–1 was suitable for sample analysis, and the analysis time was 10 min (figure 1). at 30°c, with a mobile phase of 20% acetonitrile and 80% water at a flowrate of 1 ml min–1, and an injection volume of 20 µl. method validation the hplc method was validated using the following parameters: linearity, recovery, precision, lod, and limits of quantification (loq). linearity was evaluated from the five concentration levels of mixed standard solution. recovery was assessed by spiking the sample with two sweeteners at three fortified concentrations of 0.285, 0.355, 0.425 µg ml–1 in three replicates, and calculated as the ratio of a measured concentration for spiked sample divided by spiked concentration. the precision tests were conducted using a sample added 71 µg of alitame standard to determine for six times in the same day. the precision was based on the relative standard deviation (rsd%). the lod and loq were defined by signal/ noise ratios of 3:1 and 10:1, respectively. both lod and loq were verified experimentally after injecting blank samples. results and discussion optimization of chromatographic conditions detection wavelength the maximum absorption wavelength of aspartame was 195.2 nm, and that of alitame was 198.7 nm. according to the literature, analytes were mostly detected at 200 or au 0.15 0.10 0.05 0.00 0 1 2 3 4 5 6 7 8 9 10 min aspartame alitame figure 1. chromatogram of aspartame and alitame standard solutions. italian journal of food science, 2022; 34 (3) 95 determination of aspartame and alitame especially for aspartame. in our study, under the lod of this method, no alitame was detected in any sample and it was not identified on the food label. notably, a total of 21 products were marked with aspartame on the food label, while there were far more than these in actual testing. the samples that detected aspartame accounted for nearly half of the total. among them, aspartame was detected in 11 samples of 40 liquid dairy products (27.5%), and the concentrations ranged from 2.41 to 103.67 μg g–1; aspartame was detected in 24 samples of 42 milk-containing beverages (57.1%), and the concentrations ranged from 1.81 to 61.49 μg g–1; aspartame was detected in eight samples of 18 milk drinks on sale (44.4%), and the concentrations ranged from 2.14 to 21.11 μg g–1. table 4 summarized determination results of aspartame and alitame in liquid dairy products and milk-containing beverages from the chinese market. the national standard stipulates a limit of 600 μg g–1 of aspartame in modified milk, protein drinks, and flavored drinks (gb 2760-2014). according to the regression equation, the measured value of aspartame in the sample was calculated, and compared with the national standard, none of them exceeded the standard. through the detection of liquid dairy products and milk-containing beverages samples, there was no phenomenon of excessive use of aspartame and alitame. although alitame was rarely used in the products in this study, most of the products were added with aspartame. as a new type of sweetener in recent years, there is no guarantee that long-term consumption of synthetic sweeteners is safe. therefore, the type of sweetener added should be identified on the food label, especially aspartame (containing phenylalanine). it is recommended that the market management department implement label method validation after verification, the standard curve was plotted using the peak area as the ordinate and the concentrations as the abscissa. the linearity was good using the standard solutions at five concentration levels (table 1). according to the experimental results, the average recovery values of aspartame and alitame were 93.0–107.3% and 97.2– 98.2%, respectively. the repeatability expressed as rsd (%) was 1.1% for aspartame and 2.5% for alitame. the data corresponding to the recoveries and precisions were detailed in tables 2 and 3, and met the needs of analysis with great reliability and repeatability. the lods were 0.52, 0.48 μg g–1 for aspartame and alitame, respectively, and the loqs for same analytes were 1.72 and 1.58 μg g–1, respectively. lu y et al. established an analytical method for the detection of nine kinds of sweeteners and preservatives in baked products by uplc, and the lods of aspartame and alitame in this method were 3.0 mg kg–1, which were higher than our method (lu et al., 2021). in the study of uplc detection of sweeteners in beverages, the lods of aspartame and alitame were 0.75 mg  kg–1 (jin, 2020). for the liquid chromatography coupled with mass spectrometry techniques, the lods of the two sweeteners were sufficient for trace analysis, which were 1.0 and 0.2 ug l–1, respectively (wang et al., 2019). given the requirements of the equipment and the performance of the method, our method has the advantage of being simple, rapid, and low cost. analysis of aspartame and alitame in liquid dairy products and milk-containing beverages among the 100 samples in this survey, except for milk drinks on sale, the remaining 82 beverages all have label instructions. milk tea is immensely popular among young people for its smooth and silky taste, but so far, many businesses lack the label of the ingredient list, table 1. linear equation and linear range of aspartame and alitame. analyte linear equation r linearity (μg ml–1) aspartame y = 11485x + 2613 0.9993 0.51–51.45 alitame y = 9200x − 4606 0.9996 0.50–50.00 table 2. recoveries and rsd values of aspartame and alitame in samples. aspartame alitame spiked (µg ml–1) average recovery % rsd % n=3 spiked (µg ml–1) average recovery % rsd % n=3 0.285 93.0 2.7 0.285 98.2 1.6 0.355 100.1 2.0 0.355 97.8 0.4 0.425 107.3 1.9 0.425 97.2 1.2 table 3. precision test results. analyte measured value (µg g–1) average (µg g–1) rsd % 1 2 3 4 5 6 aspartame 595.53 591.05 587.69 581.18 586.44 578.53 586.73 1.1 alitame 262.58 274.41 267.68 256.39 259.00 262.60 263.78 2.5 96 italian journal of food science, 2022; 34 (3) yu j et al. table 4. concentrations of aspartame and alitame in liquid dairy products and milk-containing beverages. group n(a) food labels sweeteners labels containing sweeteners detected sample concentrations of sweeteners in detected samples (μg g–1) mean p50(c) p90(c) p95(c) max liquid dairy products 40 40 alitame 0 8 –(b) 11 – – – – – aspartame 31.66 8.23 72.10 87.88 103.67 milk-containing beverages (pre-package) 42 42 alitame 0 13 – 24 – – – – – aspartame 24.32 16.27 60.64 61.23 61.49 milk-containing beverages (made-on-site) 18 0 alitame 0 0 – 8 – – – – – aspartame 5.77 3.00 11.33 16.22 21.11 (a) number of samples (b) – indicates did not detect the acesulfame in these samples (c) 50th, 90th, and 95th percentiles of the distribution management on milk drinks, and indicate the types of sweeteners used, which is conducive to protecting the legitimate rights and interests of consumers. conclusions in this paper, a rapid and reliable hplc method for the simultaneous determination of aspartame and alitame in liquid dairy products and milk-containing beverages from the chinese market was developed and validated, which was applied for the analysis of 100 products. since the use of artificial sweeteners has increased, continued monitoring and strengthening the regulation of the sweeteners used in food is essential. in addition, the analysis data showed that 14 samples with food labels were not labelled with aspartame. the mislabelling leads to uncertainty over both the contents and their concentrations, and increases the likelihood of excessive intake of artificial sweeteners. thus, the results of this survey involve public health issues, and require the attention of the chinese government to strengthen market supervision and regulation. however, there are still some uncertainties that should be considered. for example, the number of food types involved in this study is not big enough. 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ole_link211 ole_link212 ole_link217 ole_link218 ole_link219 ole_link242 ole_link243 ole_link244 ole_link213 ole_link214 ole_link277 ole_link278 ole_link274 ole_link275 ole_link272 ole_link273 ole_link23 ole_link24 ole_link113 ole_link138 ole_link281 ole_link282 ole_link128 ole_link129 ole_link283 ole_link284 ole_link134 ole_link135 ole_link136 ole_link137 ole_link130 ole_link131 ole_link132 ole_link133 ole_link21 ole_link22 ole_link268 ole_link269 ole_link78 ole_link79 ole_link69 ole_link154 ole_link165 ole_link224 ole_link225 ole_link264 ole_link66 ole_link172 ole_link203 ole_link207 ole_link208 ole_link82 ole_link83 ole_link39 ole_link40 ole_link76 ole_link77 ole_link51 ole_link52 ole_link250 ole_link251 ole_link61 ole_link63 ole_link262 ole_link263 ole_link271 ole_link276 ole_link279 ole_link280 ole_link226 ole_link227 ole_link285 ole_link286 ole_link305 ole_link306 ole_link301 ole_link302 ole_link72 ole_link73 ole_link25 ole_link26 ole_link372 ole_link373 ole_link374 ole_link375 _goback ole_link376 ole_link377 ole_link378 ole_link379 ole_link380 ole_link382 ole_link384 ole_link385 ole_link386 ole_link27 ole_link28 ole_link387 ole_link388 ole_link74 ole_link75 ole_link389 ole_link390 ole_link391 p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33isp1.2045 55 p u b l i c a t i o n s codon natural protective agents and their applications as bio-preservatives in the food industry: an overview of current and future applications saber amiri1, zahra motalebi moghanjougi1, mahmoud rezazadeh bari1*, amin mousavi khaneghah2* 1department of food science and technology, faculty of agriculture and natural resources, urmia university, urmia, iran; 2department of food science and nutrition, faculty of food engineering, state university of campinas, brazil *corresponding authors: mahmoud rezazadeh bari, department of food science and technology, faculty of agriculture and natural resources, urmia university, urmia, iran. email: m.rezazadehbari@urmia.ac.ir; amin mousavi khaneghah, department of food science and nutrition, faculty of food engineering, state university of campinas, brazil. email: mousavi@unicamp.br received: 5 april 2021; accepted: 30 april 2021; published: 18 may 2021 © 2021 codon publications open access paper abstract today, the usage of natural additives in the food matrix has increased. natural antimicrobial compounds include peptides, enzymes, bacteriocins, bacteriophages, plant extracts, essential oils, and fermented compounds that can be used as alternatives to chemical antimicrobials. plant extracts and essential oils contain terpenes, flavonoids, aldehydes, and phenolic compounds that cause antimicrobial and antioxidant activity. the synergistic activity of compounds synthesized from lactic acid bacteria (lab) prevents the growth of bacteria and fungi. in addition to removing mycotoxins, lab compounds have antioxidant and anticancer potentials and increase food safety and nutritional value. one of these antimicrobial molecules is bacteriocin, which is made by various microorganisms. nisin is one of these bioactive peptides that are used widely in food bio-preservation. antimicrobial peptides can be used alone or along with other compounds to enhance food security. this article reviews natural preservatives and their applications in food products. keywords: bioactive compounds; antioxidants; protective culture; antimicrobial peptides; bacteriocin; essential oils italian journal of food science, 2021; 33 (sp1): 55–68 introduction microorganisms and lipid oxidation are important problems in food safety (aziz and karboune, 2018). bacteria, yeasts, and molds are microorganisms that spoil food products (figure 1) (gonelimali et al., 2018). many strains of aspergillus and penicillium produce mycotoxins that cause disease (varsha and nampoothiri, 2016). in general, the food industry relies on chemicals for food preservation. food additives include the following: preservatives (antimicrobials, antioxidants, and anti-browning agents), nutritional additives, coloring agents (azo compounds, xanthan, chinophthalon derivatives, indigo, and triarylmethane), flavors (sweeteners and flavor enhancers), and textural ingredients (stabilizers and emulsifiers) (carocho et al., 2018). usually, chemical preservatives are applied to prevent lipid oxidation and microorganisms growing in the food industry. the use of nitrite and sulfur dioxide (chemical preservatives) can have side effects on foods and human health. owing to the extensive use of these substances, bacteria have become resistant, and consumers are looking for natural products with nonchemical preservatives, so there is a need to find natural preservatives (amiri et al., 2021a; mahmud and khan, 2018; pisoschi et al., 2018; rai et al., 2016). with regard to these needs, bio preservation, super chilling, and active packaging were mailto:m.rezazadehbari@urmia.ac.ir mailto:mousavi@unicamp.br 56 italian journal of food science, 2021; 33 (sp1) amiri s et al. or put into packaging because the latter is beneficial (aziz and karboune, 2018; mahmud and khan, 2018; bai-ngew et  al., 2021; sohrabpour et al., 2021). all herbal essential oils (eos) and extracts have an inhibitory effect against viruses, bacteria, fungi, oxidative corruption, and insects (xing et al., 2012). various herbal and spice antimicrobial agents are used to eliminate foodborne microorganisms such as listeria monocytogenes, escherichia coli, staphylococcus aureus, bacillus cereus, and salmonella, and to enhance the quality of different foods. the most common sources of eos of clove, cinnamon, oregano, citrus, garlic, coriander, parsley, lemongrass, sage, rosemary, and vanillin are antimicrobial and antioxidant agents that are used in bioactive packaging of foods (gonelimali et al., 2018; mahmud and khan, 2018). bio-preservation is applying bacteria or their bioactive metabolites to inhibit foodborne pathogens (amiri et al., 2021b; azizi et al., 2021; gonelimali et al., 2018; tumbarski et al., 2020). the most crucial advantage of antimicrobial peptides is that they do not alter food quality and are harmless (rai et al., 2016). lactic acid bacteria (lab) is used as a protective culture in dairy foods due to the production of lactic acid, hydrogen peroxide, fatty acids, acetaldehyde, and bacs that inhibit the growth of microorganisms (amiri et al., 2020a, 2020b, 2020c; considered (aziz and karboune, 2018; tumbarski et al., 2020). most natural antimicrobial compounds include extracts (grape seed, tea, strawberry, cranberry, and blueberry), spices (garlic, nutmeg, cinnamon, clove, black pepper, and ginger) herbs (basil, oregano, rosemary, marjoram, and sage), enzymes (lysozyme, lactoferrin), bacteriocins (bacs) (nisin and natamycin), peptides, organic acids (propionic, citric acid, and sorbic), vitamins (α-tocopherol and ascorbic acid), and natural polymers (chitosan) (table 1). they are added directly to foods table 1. natural antimicrobials and antioxidants in the food industry. natural preservatives natural antimicrobial natural antioxidant plant based essential oils cinnamon, oregano, clove, garlic, rosemary, parsley, coriander, lemongrass, rosemary, allspice, litsea cubeba, sage, purple, and bronze muscadine seeds pigments anthocyanins, anthocyanins spices black, white, cayenne peppers, mustard, chillies, paprika, coriander, clove, cumin, dill fennel, nutmeg, mace, cinnamon, garlic, ginger spices cinnamon, cloves, nutmeg, ginger, black pepper, garlic, onion, cumin, turmeric herbs thyme, basil, bay leaf, marjoram, shallot, onion, garlic, oregano, rosemary, sage herbs rosemary, oregano, marjoram, sage, basil, thyme extracts grape seed, tea, strawberry, cranberry, blueberry extracts tea, grape seed, cranberry, blueberry, strawberry, pomegranate, citrus fruits peptides lipid transfer protein 2, snakin1, kalata b1, thionin, defensins vitamins α-tocopherol, ascorbic acid animal based enzymes lysozyme, lactoperoxidase, peptides pleurocidin, protamine, magainins, sarco toxin, hymenoptaecin, attacin, diptericin, coleoptericin, lactoferricin, kappacin, k-casecidin, s1s2a-b caseins natural polymers chitosan, poly-l-lysine microbial based enzymes glucose oxidase bacteriophages listextmp100, ecoshieldtm, salmofreshtm organic acids propionic, citric acid, sorbic, lactic acid bacteriocins nisin, natamycin, enterocin, pediocin, reuterin, food spoilage microorganism bacteria yeasts molds lipid oxidation free radicals figure 1. the cause of food spoilage. italian journal of food science, 2021; 33 (sp1) 57 applications of food bio-preservatives rai et al., 2016; tumbarski et al., 2020). bacs are ribosomally synthesized proteins or peptides from certain bacteria. bacs are efficient against antibiotic-resistant pathogens such as s. aureus and enterococcus faecalis without showing toxicity (ahmad et al., 2017; amiri et al., 2021c; mahmud and khan, 2018). lab produces a diversity of antifungal compounds and may be used as a bioprotective without changing the organoleptic characteristics. lab has been found naturally in various foods for centuries and has been used in fermented foods without side effects. it is known as gras (generally recognized as safe) (amiri et al., 2019a; favaro et al., 2015; maleki et al., 2020; rezazadeh-bari et al., 2019; varsha and nampoothiri, 2016). the food and drug administration of usa (fda) accepted lab as a safe agent for fermented foods (upendra et al., 2016). it is heat resistant and has a tremendous antimicrobial effect against gram-positive (g+) and gram-negative (g−) pathogens after pasteurization and also does not alter the taste, aroma, or texture and physical, chemical, or biological properties of foods (figure 2) (rai et al., 2016). natural antimicrobial agents plant-based antimicrobial agents essential oils essential oils (eos) are natural and volatile compounds determined by odor and taste (mohajeri et al., 2021; ghamari et al., 2021; regnault-roger et al., 2012). eos are include terpenes, alcohols, acids, esters, epoxides, aldehydes, ketones, amines, and sulfides. they are synthesized in the cytoplasm and plastids of plant cells through malonic acid, mevalonic acid, and methyl-d-erythritol4-phosphate pathway. terpenes are hydrocarbons and consist of various isoprene units. at the same time, terpenoids are made from the biochemical alteration of terpenes by enzymes that add oxygen molecules and transfer methyl. terpenoids have high antimicrobial activity (pisoschi et al., 2018). the constituents in eos are the following groups: terpenes and terpenoids that are more effective to g+ bacteria (mahmud and khan, 2018). g− bacteria’s resistance is due to the presence of an outer membrane of lipopolysaccharide around the cell wall (tongnuanchan and benjakul, 2014). however, there are reports that eos of cinnamon, oregano, clove, garlic, rosemary, parsley, coriander, lemongrass, sage, purple, and bronze muscadine seeds are effective on both types of bacteria, many of which are carvacrol, thymol, eugenol, and citral (aziz and karboune, 2018; irkin and esmer, 2015). eos of cinnamon, oregano, clove, garlic, coriander, parsley, lemongrass, rosemary, and sage have good effects. eos with great amounts of eugenol (allspice, clove, and cinnamon), trans-cinnamaldehyde (cinnamon), and citral (litsea cubeba, lemon myrtle, and lime) have strong effects (dussault et al., 2014). the oregano eo has irreversible damage (in 1 min) against e. coli o157: h7 cells (pisoschi et al., 2018). the role of oregano, thyme, and savory as an antimicrobial agent is because of phenolic compounds caracole, thymol ρ-cumene, and у-terpinene (aziz and karboune, 2018; dussault et al., 2014). the existence of a hydroxyl group in the structure of phenolic compounds and its position is the reason for the antimicrobial effect. the antimicrobial effect of sage is caused by terpene thujone. rosemary eo has an inhibitory effect against g− bacteria (e. coli, klebsiella pneumonia) and g+ bacteria (bacillus subtilis, s. aureus) (aziz and karboune, 2018; tongnuanchan and benjakul, 2014). this is because of terpene groups such as borneol, camphor, 1, 8-cineole, a-pinene, camphone, natural antimicrobial essential oils and plant extracts peptides bacteriocins eliminate free radicals attack on cell membrane disruption of enzymatic structures electrostatic interaction with cell membrane oxidation of unsaturated fatty acids and hydroperoxide formation fracturing the chain of the oxidation reaction attach to cell wall ingredients prevent protein or nucleic acid synthesis disrupt cell division destroy potential energy decrease in oxygen concentration stimulation of antioxidant enzymes quenching •o2 chelating metal ions natural antioxidant figure 2. mechanisms of action of natural protective agents. 58 italian journal of food science, 2021; 33 (sp1) amiri s et al. hydrocarbons (cinnamon, cloves, garlic, mustard, and onions) (embuscado, 2015; mahmud and khan, 2018). cinnamon has antibacterial activity against both types of bacteria, such as b. cereus, l. monocytogenes, s. aureus, e. coli, and salmonella anatum (shan et al., 2007). plant extracts grape seed and green tea extracts grape seeds contain high amounts of phenolic compounds, including catechin, epicatechin, epicatechin3o-gallate, dimeric, and trimeric tetrameric procyanidins. green tea leaves have epicatechin, epicatechin gallate, epigallocatechin, teaflavin gallate, teaflavin monogallate a, and b teaflavin digallate. extracts of grape and green tea postpone growing of microorganisms in raw beef (banon et al., 2007). tea phenols have antimicrobial properties against both types of bacteria. the antimicrobial effect of arrowroot tea is related to catechins. green tea has antibacterial activity against pathogens (kim and fung, 2004). it has been reported that grape seed extract decreased e. coli o157: h7 and s. typhimurium, and postponed the growth of l. monocytogenes and aeromonas hydrophila in cooked meat (aziz and karboune, 2018). antibacterial compounds of coffee are caffeic acid, chlorogenic acid, and protocatechuic acid. the growth of e. coli o157: h7 is inhibited by trimethylated alkaloid caffeine (ibrahim et al., 2006). cranberry extracts phenolic compounds, such as low molecular weight phenolic acids, condensed tannins, proanthocyanidins, and flavonoids (anthocyanins, especially proanthocyanidins), which are composed of tetramers of type a and pentamer type a, are the main factors of antimicrobial activity in cranberry (khaneghah et al., 2018). extract of cranberry fruit at 15% concentration inhibited a. hydrophila, b. cereus, enterobacter aerogenes, e. coli, k. pneumonia, proteus vulgaris, p. aeruginosa, s. typhimurium, s.  aureus, and yersinia enterocolitica (sagdic et al., 2006). mechanisms of action an accurate way has not yet been elucidated. nevertheless, plant substances can be affected by attacks on the cell membrane’s phospholipid bilayers, disruption of enzyme activity, endangering the genetic material of bacteria, and oxidation of unsaturated fatty acids (tajkarimi et al., 2010). the inhibitory activity of aromatic and phenolic compounds is due to affecting the cytoplasmic membrane’s structure and function. the outer membranes of e. coli and s. typhimurium was dissipated after exposure to carvacrol and thymol. the antimicrobial effect of nonphenolic isothiocyanates is due to the denaturized of extracellular enzymes. terpenes can disrupt and affect the bacterial cell wall’s fat composition verbenonone, and bornyl acetate. the antimicrobial activity of garlic and onion is due to allicin and mustard caused by allyl and isothiocyanates. allyl isothiocyanate is effective against fungi and g− bacteria (aziz and karboune, 2018). ginger, oregano, rosemary, sage, thyme, and peppermint are significant sources of eos and include alcohols, aldehydes, phenylpropanoids, terpenes, and ketones that are antioxidants. in addition to antioxidant activities, eos can provide antibacterial activity (s. aureus, e. faecalis, e. coli, clostridium perfringens, and clostridium sporogenes) in meat foodstuffs (mahmud and khan, 2018). marei et al. (2012) studied 12 monoterpenes’ antifungal activity against rhizoctonia solani, fusarium oxysporum, and penicillium, and digitatum and aspergillus niger thymol were the good antifungal compounds comparable to fungicide and carbendazim. allyl isothiocyanate, cinnamon oleoresin, cinnamon chinese cassia, and oregano chinese cinnamon eos have antimicrobial activity against e. coli o157:h7, s. aureus, b. cereus, l. monocytogenes, and salmonella enterica serovar typhimurium. the coriander seed oil has antimicrobial efficacy against g+ and g− bacteria, some yeasts, dermatophytes, and molds (silva and domingues, 2017). low ph, temperature, and oxygen content can complete eos’ performance (aziz and karboune, 2018; dussault et al., 2014). these compounds are gras and can be used as food additives (mahmud and khan, 2018; bai-ngew et al., 2021). mechanisms of action plant material affects microorganisms through various mechanisms, such as attacking the cell membrane, disrupting enzymatic structures, compromising bacterial genetic material, and fatty acid hydroperoxide formation by oxygenation of unsaturated fatty acids (tajkarimi et al., 2010). the function of phenolic compounds of eos likely depends on alteration of bacterial cell permeability, damage to the cytoplasmic membrane, disruption of the cellular production system of cellular energy (atp), proton motility, and cell death that occurs by damaging the cytoplasmic membrane (mahmud and khan, 2018). herbs and spices herbs are obtained from the same plant’s leaves, but spices are obtained from different parts of the plant. herbs and spices are divided into the following categories: hot spices (peppers), mild flavor spices (paprika and coriander), aromatic spices (clove, cumin, dill fennel, nutmeg, mace, and cinnamon), and aromatic herbs and vegetables (thyme, basil, marjoram, shallot, onion, and garlic). phytochemicals are bioactive compounds that contain flavonoids and other phenolic ingredients, carotenoids, plant sterols, glucosinolates, and other sulfur compounds. extracts and herbs act against g+ bacteria. also, the shelf life can increase using bioactive ingredients such as phenol, alcohol, aldehyde, ketone, esters, and italian journal of food science, 2021; 33 (sp1) 59 applications of food bio-preservatives thiocyanate should be added. its use in juice storage and food coating is suggested (marcio carocho et al., 2018). considerations of using the lactoperoxidase system in packaging films depend on the cost and the antimicrobial activity of the lactoperoxidase system (thiocyanate and h2o2). if h2o2 levels are exceeded in food products, it may raise toxicological concerns (aziz and karboune, 2018). antimicrobial peptides eukaryotic peptides animal peptides. mammalian antimicrobial peptides are secreted into mucosal, epithelial, and paneth cells. mammalian leukocytes are a rich origin of cationic antimicrobial peptides that prevent bacterial infection. several of them can rapidly destroy the lipid layer of the cell membranes of microorganisms. in addition to inhibition of both types of bacteria, they also have antifungal and antiviral activity. these are considered a suitable option for antibiotic resistance (aires et al., 2009; tiwari et al., 2009). pleurocidin and protamine isolated from fish have antimicrobial activity on g− bacteria such as vibrio parahemolyticus, l. monocytogenes, e. coli o157:h7, saccharomyces cerevisiae, and penicillium expansum. nevertheless, magnesium and calcium prevent their efficacy (burrowes et al., 2004). magainins from amphibians have shown extensive activity against both types of bacteria and are generally used to preserve meat and cheese (rai et al., 2016). insect’s peptides. antimicrobial peptides such as sarco toxin iia, hymenoptaecin, attacin, diptericin, and coleoptericin act against micrococcus luteus, aerococcus viridians, bacillus megaterium, b. subtilis, bacillus thuringiensis, and s. aureus secreted by insects (wang et al., 2016). plant peptides. these peptides are cysteine-rich (molecular weight 2–9 kda), such as lipid transfer protein 2, snakin1, kalata b1, thionin, and potato defensins. they have antimicrobial effects against l. monocytogens, s. typhimurium, and e. coli o157:h7. in particular, defensins have antimicrobial potential against microorganisms (rai et al., 2016; tiwari et al., 2009). milk peptides. lactoferricin (an 80 kda iron-binding glycoprotein), kappacin, and k-casecidin are antimicrobial peptides of milk proteins. lactoferrin (as a gras) is an iron-binding glycoprotein with antimicrobial effect on both types of bacteria, such as salmonella and e. coli, and fungi. in the past, it was used in baby formulas and to treat beef carcasses, but currently, lactoferrin approved in the usa for use on beef (aziz and karboune, 2018; baines and seal, 2012; rai et al., 2016; tiwari et al., 2009). casocidin is a byproduct of the casein hydrolysis and has an antibacterial effect against staphylococcus spp., and ultimately cause bacterium death (aziz and karboune, 2018). carvacrol can split g− bacteria’s outer membrane diffusing lipopolysaccharides and enhancing the permeability to atp. its activity against g+ bacteria is because of its interplay through the bacterial membrane, which changes h+ and k+ cations (tongnuanchan and benjakul, 2014). animal-based antimicrobial agents enzymes lysozyme it is an enzyme and natural antimicrobial agent that hydrolyzes the-beta 1, 4 glycosidic bonds between n-acetylmuramic acid and n-acetyl glucosamine existing in peptidoglycan. g+ bacteria are sensitive to lysozyme since 90% of these bacteria’s cell wall is composed of peptidoglycans. therefore, it is a natural antimicrobial agent (irkin and esmer, 2015). g− bacteria are resistant to lysozyme because of the lipopolysaccharide layer, which prevents lysozyme from approaching the peptidoglycan layer. however, this enzyme can also affect g− bacteria if membrane destabilizing factors, for example, detergents and chelating agents, are present. lysozyme has a high resistance to a wide range of temperatures and ph, which enables its usage in edible active films (bayarri et  al., 2014). inovapure is a commercial lysozyme studied in model studies, either alone or with other compounds, against l. monocytogenes, clostridium botulinum, campylobacter jejuni, pseudomonas spp., salmonella enteritidis, clostridium thermosaccharolyticum, bacillus stearothermophilus, and clostridium tyrobutyricum. lysozyme derived from egg white has been allowed by health canada to be used in hard cheeses to inhibit the gas blowing of c. tyrobutyricum (aziz and karboune, 2018; baines and seal, 2012). lysozyme has also been assayed in eggs, milk, beef, and part in edible films and coatings, along with other antimicrobial materials (bayarri et al., 2014). lacto-peroxidase system one of the natural antimicrobial systems is the lactoperoxidase system obtained from milk, saliva, and tears. it consists of lactoperoxidase, thiocyanate, and hydrogen peroxide. lacto-peroxidase accelerates the oxidation of thiocyanate ions using h2o2. afterward, hypothyroidism and acid hypothyroidism show inhibitory effects on microorganisms through the oxidation of sulfhydryl groups to enzymatic systems and proteins. it shows antimicrobial activity against bacterial and fungal species (campos et al., 2011). the general application of lactoperoxidase is to preserve raw milk (especially if the refrigerator is not available). because thiocyanate in milk is not enough to stimulate the lactoperoxidase system’s activity, 60 italian journal of food science, 2021; 33 (sp1) amiri s et al. laxity. h2o2 can be removed from foods using catalase, which changes it to water and oxygen. microbial glucose oxidase is allowed to remove oxygen and preserve taste and aroma in bottled beverages and is used as an additive in bakery products, flour, yolk, and white egg. d-gluconic acid, a catalytic product of glucose oxidase, is secure, and the who does not have a regulation for it (aziz and karboune, 2018). bacteriophages bacteriophages are viruses that attack bacteria. lithium bacteriophages disrupt the metabolism of bacteria that results in bacterial death. bacteriophages have been shown to be innocuous to mammalian cells, so they can be used for biological control of pathogens in foods (carvalho et al., 2017). bacteriophages are suitable for use in carcasses, vegetables, and fruits to increase various food products’ shelf life. in 2006, listextmp100 and lmp102 bacteriophages were accepted by the fda to control l. monocytogenes contamination, and in 2010, approved the use of listextmp100 against l. monocytogenes in meat, fish and poultry, vegetable, and some dairy products (chibeu et al., 2013). in 2014, canadian health accepted the use of ecoshieldtm in meat to control e.  coli o157: h7 and salmofreshtm to control salmonella growth in all foods (aziz and karboune, 2018). bacteriophages have been investigated as antimicrobial agents in food packaging by immobilization on cellulose membrane or by encapsulating phages in alginate beads (lone et al., 2016). also, chitosan film contains liposome-encapsulated beef meat storage (cui et al., 2017). fermented ingredients fermentation ingredients can be prepared by propionic acid bacteria and lab from raw materials such as milk, sugar, or plant materials. the preservative role of lab is because of producing organic acids (acetic, lactic, propionic, and formic acids) diacetyl, acetylene, hydrogen peroxide, fatty acids, antifungal agents (phenyl-lactate, hydroxyphenyllactate, propionate, cyclic dipeptides, and 3-hydroxy fatty acids), and the bacs (reuterin, reutericyclin, nisin, pediocin, lacticin, and enterocin). labs are used as antimicrobials and sugars, sweeteners, polymers, disinfectants, aromatic compounds, and various enzymes (favaro et al., 2015). lab and bio-preservation the biologically active ingredients in kimchi, such as benzyl isothiocyanate, indole, thiocyanate, and cystosterol, are antibiotics, anticancer, and immune stimulation. lab was used to prevent l. monocytogenes and s. enteritidis in poultry and inhibition of s. typhimurium, e. coli, and l. monocytogenes in lettuce and apples. enterococcus and pediococcus were used to vacuum-package cold-smoked salmon (varsha and nampoothiri, 2016). microgardtm sarcina spp., b. subtilis, diplococcus pneumoniae, and streptococcus pyogenes. casein a and casein b inhibit the growth of enterobacter sakazakii. the peptides containing an s2-casein, an s1-casein, and k-casein have good inhibitory effect on e. coli and b. subtilis (elbarbary et al., 2012). mechanisms of action antimicrobial peptides act through an electrostatic interaction with the membrane and have a permeability function. they can enter the membrane and eventually disrupt it (rai et al., 2016). several mechanisms are responsible for antibacterial activity of lactoferrin: prevention of growing by isolating iron from pathogens, the potential of cations in the surface of lactoferrin for a direct reaction with lipid a, and modifying the permeability of the outer membrane and causing the release of lipopolysaccharide (jenssen and hancock, 2009). chitosan chitosan is natural cationic linear polysaccharides made up of (1, 4) linked 2-amino-deoxy-β-d-glucan. sources of chitosan are shrimp shells, fungi, and green algae (dutta et al., 2009; irkin and esmer, 2015). it is effective against most microorganisms (both types of bacteria, yeasts, and molds). due to the positive charge on c2 of glucosamine monomer at ph below 6, chitosan is soluble than chitin and has higher antimicrobial effect (irkin and esmer, 2015). the positively charged amino group of chitosan can interact with the negatively charged cell membrane, causing leakage of intracellular compounds of microbes. chitosan can inhibit toxin synthesize and bacterial growth by chelating the trace metals. it can activate some protection systems in the host tissue and prevent several enzymes’ activity. also, it can penetrate the nucleus of microorganisms and interfere in the translation process. the inhibitory mechanism of chitosan is diverse in g+ and g− bacteria; antimicrobial effect on e. coli increases with decreasing molecular weight of chitosan, while it has the opposite effect on s. aureus (aziz and karboune, 2018; pisoschi et al., 2018). microbial-based antimicrobial agents glucose oxidase glucose oxidase is synthesized by a. niger and penicillium spp. glucose oxidase is an oxidoreductase that catalyzes the oxidation of d-glucose to h2o2 and d-glucono-δ lactone. d-glucono-δlactone reacts with water to form d-gluconic acid. the antimicrobial role of glucose oxidase is because of the cytotoxicity of h2o2, lowering of ph by the formation of d-gluconic acid. hydrogen peroxide levels may also exceed the acceptable levels by the fda and cause toxic problems. long-dated disposal of foods to h2o2 can enhance lipid oxidation and lead to italian journal of food science, 2021; 33 (sp1) 61 applications of food bio-preservatives salvaricin a, sublancin 168). type b are spherical peptides such as meracidin and cinnamycin belong to globular peptides, and type c are a few details bacs such as lactin, 3147 and cytolysin, that made by more than one part and biological activities are needed. class ii bacs are divided into (iia, iib, and iic) subgroups: type iia is related to pediocin (pediocin pa-1, carnobacteriocin b2, listerocin 743a, and ubericin a), type iib is related to multifactorial bacs (lactococcin g, thermophilin 13, lactacin f, and lactocin 705), and type iic are sundry such as sakacins q, t, and x, and aureocin a53. class iii contains lysins (class iiia) and nonlytic bacs (class iiib). class iv contains circulating inhibitory peptides such as enterocin as-48 (favaro et al., 2015; milicevic et al., 2021). nisin nisin is produced by fermenting milk modified by the strains of l. lactis. nisin is a canadian food and drug and health license and is accepted as a preservative in more than 48 countries. nisin is efficient on most g+ bacteria such as l. monocytogenes and s. aureus, and ethylenediaminetetraacetic acid (edta) can also inhibit g− bacteria (aziz and karboune, 2018; campos et al., 2011; pisoschi et al., 2018). enterocin enterocin as-48 is one of the cyclic bacs that exhibit significant stability in ph and heat. the effectiveness of as-48 for the control of staphylococci in culture has been represented. also, chemical preservatives (sodium nitrite, sodium lactate, and sodium chloride), clusters, and temperate heat are in synergy with as-48 on b.  cereus, s. aureus, salmonella choleraesuis, and e. coli o157:h7 (favaro et al., 2015). enterocin as-48 has the ability to eliminate the parasitic protozoan trypanosoma brucei, and enterocin v effectively decreases c. albicans (martínez-garcía et al., 2018). pediocin pediocins are produced by p. acidilactici and p. pentosaceus. they are thermal resistant and can work in a wide range of ph. pediocins have an antimicrobial effect against l. monocytogenes, e. faecalis, s. aureus, c. perfringens, and oenococcus oeni. pediocin can be used in dairy products or as a film in crushed ham and vegetables (díez et al., 2012; juneja et al., 2012). natamycin natamycin is a polyene macrolide that is synthesized by streptomyces species with an antifungal activity that has almost no effect on bacteria, protozoa, and viruses. natamycin is associated with ergosterol, so it does not develop resistance in fungi. it has been used as a free, encapsulated, or film-forming agent to control yeast growth in cheese, salami sausages, and beverages (pisoschi et al., 2018). is a commercial milk supply that is fermented by dairy microorganisms. microgardtm could be effective against g− bacteria (yersinia, salmonella, and pseudomonas) and some fungi but ineffective against the g+ bacteria (l. monocytogenes, s. aureus, and b. cereus). fda has approved propionibacterium metabolites as gras. the microgardtm products are used in dairy desserts, cheese, yogurt, pasta, sauces, and others (aziz and karboune, 2018; favaro et al., 2015). lab produces antifungal compounds, for example, hydroxyl fatty acids, organic acids, low molecular weight bioactive ingredients, and proteinaceous components. some species of enterococci, lactobacilli, pediococci, and lactococci are also known as antifungal agents (favaro et al., 2015; gholam-zhiyan et al., 2021). the antifungal effect of lactococcus lactis on aspergillus, penicillium, mucor, and rhizopus has been reported (oranusi et al., 2013). also, lactobacillus acidophilus and bifidobacterium bifidum were efficient in preventing a. niger from growing on the surface of brined white cheese (moghanjougi et al., 2020). use of l. plantarum on botrytis cinerea, glomerella cingulate, phytophthora drechsleri, p. citrinum, and f. oxysporum and application of lactobacillus and weisella cultures on a. niger, candida albicans, aspergillus tubingensis, and p. crustosum have been reported (varsha and nampoothiri, 2016). feedtech silage f3000 is a commercial compound of antifungal bacteria (l. plantarum milab 393, p. acidilactici, e. faecium, and l. lactis) that improves growth beneficial bacteria and prevents yeast fungi and clostridia. durafresh (kerry) is also a commercial preservative derived from the fermentation of labs that inhibits g− bacteria, yeast, and mold. alta 2341 is another fermented product. therefore, the use of lab in food has existed for a long time, and recent studies indicate their potential as an alternative to chemical preservatives (varsha and nampoothiri, 2016). bacteriocins bacs are ribosomal antimicrobial proteins that usually have antimicrobial activity against similar genetic strains. the beneficial properties of bacs are stability to high temperature and low ph and sensibility to proteolytic enzymes. their application can decrease the intensity of heat treatment and the use of chemical preservatives. also, bacs and other methods as a hurdle can be helpful (favaro et al., 2015). bacs are used in food products because they are harmless, do not change foods’ nutritional characteristics, are efficient in low concentration, and active in refrigerator storage, and, therefore, an ideal bio-preservation (mahmud and khan, 2018; milicevic et al., 2021). bacs isolated from g+ bacteria are classified into the following groups: class i, class ii, class iii, and class iv. class i are divided into (a, b, and c) subgroups: type a corresponds to linear peptides and is further subdivided into two subgroups including ai subtype contains nisin like (nisin a, nisin u, streptin), and subtype aii contains sa-ff22 like (sa-ff22, lacticin 481, 62 italian journal of food science, 2021; 33 (sp1) amiri s et al. hydroxyquinamic acids, flavonoids including anthocyanins, tannins, lignins, acetylbases, and coumarins) due to high antioxidant ability and health properties can be used as bioactive substances in foods (savaş et al., 2020; carocho et al., 2018). plant-based antioxidant agents vitamins (ascorbic acid and α-tocopherol), spices (cinnamon, cloves, nutmeg, ginger, black pepper, and garlic), herbs (rosemary, oregano, marjoram, sage, and basil), and plant extracts (tea, grape seed, cranberry, blueberry, and strawberry) contain antioxidant ingredients (ahmad et al., 2015; amiri et al., 2019b). the antioxidant role of plant extracts is because they contain phenolic acids (gallic, protocatechuic, caffeic, and rosmarinic acids), phenolic diterpenes (carnosol, carnosic acid, rosmanol, and rosmadial), flavonoids (quercetin, catechin, naringenin, and kaempferol), and volatile oils (eugenol, carvacrol, thymol, and menthol). plant pigments such as anthocyanins and anthocyanins also have antioxidant roles. catechins, epicatechins, phenolic acids, proanthocyanidins, and resveratol confer antioxidant effect in tea and grape seed extract (aziz and karboune, 2018; gonelimali et al., 2018). ferric acid, a hydroxyaminic acid, is used as an antioxidant and other preservatives in edible films and gels (kumar and pruthi, 2014). catechin is a 3-ol flavon that has antioxidant activity. ascorbic acid can be used as an oxygen scavengers to stabilize lipids and oils in foods, regenerating phenolic oxidants and oxidized tocopherols. carotenoids also have antioxidant effects but are sensitive to light oxidation and, therefore, less used. lycopene is the primary carotenoid in tomatoes. beta-carotene is used as a singlet oxygen quencher in cooked foods, eggs, and dairy products. as a maker of vitamin e, tocopherols are powerful antioxidants that are used individually or in combination with ascorbic acid (carocho et al., 2018). herb extracts rosemary the antioxidant role of rosemary is found in phenolic diterpens (carnosic, carnosol, rosmanol, rosmadial, 1, 2-methoxycarnosic acid, and epiand iso-rosmanol) and phenolic acids such as rosmarinic and caffeic acid (brewer, 2011). carnosic acid, a hydroxybenzoic acid derivative found in rosemary extract, has the highest antioxidant effect and is used in oils, sauces, bakeries, meat, and fish (birtić et al., 2015). rosemary extract e 392 is available on the european food additives list (eu regulation 1129/2011). rosemary extracts for food preservation alone or with other antioxidants, such as nisin, polyphenols, bha, and bht, are examples of the application of natural antioxidants (carocho et al., 2018). reuterin reuterin (b-hydroxypropionaldehyde) is a bac produced by l. reuteri that affects both types of bacteria (l. monocytogenes, s. aureus, s. typhimurium, and e. coli). it is soluble in water and stable to temperature and proteolytic and lipolytic enzymes. it is characterized by resistance in a wide range of ph. reuterin is used in combination with lactobacillus in glycerol as starter cultures in cheese or as pure extract at the level of sausages and salmon or inside cheese (gyawali and ibrahim, 2014; montiel et al., 2014). mechanisms of action bacs affect the basic functions of living cells such as transcription, translation, replication, and cell wall biosynthesis, but most of the time, destroy the potential energy of sensitive cells by creating channels or pores in the membrane (milicevic et al., 2021). bacs perform their function on bacteria (both types of bacteria) by disrupting cell division and preventing protein or nucleic acid synthesis, using various mechanisms. bacs can attach to cell wall ingredients using specific or nonspecific receptors, such as the lipid-binding site or the molecular surface, leading to the formation of pores or direct cell lysis and ultimately to death through the loss of the proton motor bacterial system. nisin is a more effective combination with edta attacking the cytoplasmic membrane of g− bacteria due to the chelating effect of edta on cell wall components. by inhibiting the cell wall, mercasidine eliminates g+ bacteria. colicin eliminates g− bacteria’s membrane pore-forming mechanism (ahmad et al., 2017; mahmud and khan, 2018). poly-l-lysine poly-l-lysine is a lysine homopolymer as a gras that is used as a staple in foods. large quantities produce a bitter flavor. it has high antimicrobial power, so it is used in small quantities. it also acts better with other antimicrobial agents. it is used in rice, noodles, soups, salads, cakes, custard, and for external protection of fish and sushi (baines and seal, 2012). natural antioxidant agents oxidation of lipids is the most important cause for the lower quality of foods (prakash et al., 2012). antioxidants inhibit oxidation by preventing the making of free radicals or by stopping their release—large amounts of synthetic antioxidants (bha or bht) may be carcinogenic in some animals. therefore, natural antioxidants are a good option (ahmad et al., 2015). foods that use antioxidants include meat, dairy, oil, fried or extruded foods, and sauces (baines and seal, 2012). all polyphenol derivatives (phenolic-hydroxybenzoic acids or italian journal of food science, 2021; 33 (sp1) 63 applications of food bio-preservatives zingiberene, and pentadecanoic acid. ginger extract exhibits approximately equal antioxidant activity to bha and bht. the main constituents of cumin are cuminal, υ-terpinene, and pinocarveol. the effect of cumin eo on reducing fe3+ ions is better than fresh and dried ginger. turmeric is mainly composed of curcumin, dimethoxycurcumin, bis-dimethoxycurcumin, and 2,5-xylenol, which is associated with vitamin e and bht in the removal of free radical. αand β-turmerone, curlone, and α-terpineol are the most important turmeric oil compounds with antioxidant activity. black pepper, nutmeg, and cloves also have antioxidant activities (brewer, 2011; gonelimali et al., 2018). tea and fruit extracts tea and grape seed extracts green tea extract has the highest total phenolic content, 94% of which contains flavonoids. oolong tea has about 18% total phenols and 4.4% flavonoids. in black tea, teaflavins and thearubigins are the predominant ones. the high antioxidant role of tea is due to flavonoids, tannins, and vitamins. grape seed extract contains catechin and epicatechin. the total phenolic amounts depend on the grape varieties, climatic conditions, degree of maturity, extraction, and solvents (brewer, 2011). pomegranate extracts the pomegranate peel contains a good amount of tannins, anthocyanins, and flavonoids. pomegranate juice has three times more antioxidant effects than green tea and red wine (ahmad et al., 2015). other fruit extracts cranberries have one of the highest levels of total phenol and antioxidant properties among fruits, and citrus fruits have antioxidant efficacy (ahmad et al., 2015). citrus fruits contain flavonoids, especially glycosylated flavanones, and polymethoxy flavones. citrus juice has been reported at about 5–10% to decrease extra nitrite content and degree of lipid oxidation in tending sausages (aziz and karboune, 2018). mechanism of action some of the factors that contribute to the oxidation of lipids are oxygen and metal ions, moisture, heat, and light. eos have different mechanisms, including inhibition of chain initiation and inhibition of hydrogen accumulation, free radical scavengers, termination of peroxides, singlet oxygen formation quenchering, and binding of metal ions (mahmud and khan, 2018). most antioxidants of spices and plants react with free radicals when autoxidation begins, while others form complexes with metal ions. eighty-five percent eos are phenolic compounds such as carvacrol, eugenol, and thymol, which are effective oregano and sage oregano extract has large amounts of phenolic acids (especially rosmarinic acid), phenolic carboxylic acids, apigenin, dihydroquercetin, and glycoside antioxidant agents that help eliminate superoxide anion radicals (brewer, 2011). oregano eo especially reduces lipid and protein oxidation and improves the color of chicken meat (al-hijazeen et al., 2016). marjoram marjoram eo contains high amounts of rosmarinic acid, carnosol, terpinen-4-ol, cis-sabinene hydrate, р-cumene, and υ-terpinene (brewer, 2011). basil and thyme the main constituents of basil aromas include 3,7-dimethyl-1,6 octadien 3-ol, 1 methoxy 4(2-propenyl) benzene, methyl cinnamate, 4 allyl 2 methoxyphenol, and 1,8 cineole. eugenol, thymol, carvacrol, and 4 allyl phenol are antioxidant agents comparable to bht and α-tocopherol (aziz and karboune, 2018). spice extracts cinnamon cinnamon contains the highest amount of polyphenolic compound with the highest antioxidant and anti radical activity. eugenol and cinnamaldehyde are the most important elements known in cinnamon leaf oil and oleoresin in cinnamon bark. vanillic, caffeic, gallic, photochatechuic, p-hydroxybenzoic, p-coumaric, ferulic acids, and phydroxybenzaldehyde are also antioxidant components of cinnamon (brewer, 2011; bai-ngew et al., 2021). garlic and onions garlic and myrrh contain the major antioxidants of flavonoids (flavones and quercetins) and the sulfuric components (allyl-cysteine, diallyl sulfide, and allyl trisulfide) (brewer, 2011). in addition, it has antioxidant roles in  vitro and in vivo due to its antimicrobial efficacy. allicin is one of the major constituents of thiosulfinates in garlic that has a specific garlic odor. when the garlic crumbles, allicin is converted to alliin by alliinase. onion extracts have higher free radical removal than garlic due to their higher total phenolic content (aziz and karboune, 2018). fresh or oil extract and dried powder of garlic can prevent lipid oxidation and increase sausage persistence (sallam et al., 2004). other spices fresh and dried ginger contains a high concentration of volatile oils of camphene, p-cineole, α-terpineol, 64 italian journal of food science, 2021; 33 (sp1) amiri s et al. wiener sausages. bukvički et al. (2014) studied the efficacy of satureja horvatii (containing eos of pcymene, thymol, and thymol methyl ether) on pork meat. in addition to inhibiting l. monocytogenes, these compounds also improved meat color and taste during storage. fish products g− bacteria spoil fish and fish products. clostridium spp. and l. monocytogens are responsible for spoilage in vacuumpacked fish. application of thyme eo and odor leaves increased the shelf life of fish. the combination of map and thyme eo increased the retention time of mediterranean fish fillets. oregano oil has a strong effect against photobacterium phosphoreum in codfish fillets than salmon. eos of aloysia sellowii were effective on both types of bacteria and two yeasts in brine shrimp. coating with eos is a good way to increase the product quality of fish. also, application of chitosan coated with cinnamon eos prolongs salmon fillet and increases texture, odor, and color. nisin can be used to store fish and other seafood. the combination of nisin with microgard is the best way to preserve fish and prevent aerobic microorganisms’ growth (mahmud and khan, 2018; rai et al., 2016). vegetables and fruits some of the methods used to maintain fruit quality and fresh vegetables are immersion, coating, and spraying. alginate coating with eos (lemongrass, cinnamon eos, citral, and cinnamaldehyde) reduced e.  coli o157:h7 in fuji apple slices and increased their shelf life. antimicrobial activity of propionic, maleic, acetic, lactic, and citric acid was also demonstrated in red apple and lettuce against e. coli o157:h7, s. typhimurium, and l. monocytogenes. garlic oil encapsulates in β-cyclodextrin has influenced the microbial and sensory property of fresh-cut tomatoes. fruit juices are susceptible to yeasts such as pichia anomala, s. cerevisiae, and schizosaccharomyces pombe. standard processes such as heat treatment, aseptic packaging, or the use of weak acids prevent yeast. eos are acidic to prevent yeast growth. lemon eo added to apple juice prolongs the time of “open” storage at room temperature and gives a fresh and pleasant taste (mahmud and khan, 2018). lu et  al. (2014) used a washing solution containing thymol that reduced salmonella and total aerobic bacteria in grape tomato without altering its quality. enterococci could be applied as starter cultures or cocultures to inhibit microbial contamination. bac-producing lab are screened in olives, sourdough, miso, sauerkrauts, refrigerated pickles, and mungbean sprouts. nisin is used to prevent the growth of c. thermosacchrolyticum and as primary or fragile chain antioxidants and free radical scavengers (hyldgaard et al., 2012). antioxidants are important components that inhibit autoxidation by preventing the making of free radicals and/or scavenge free radicals by the following mechanisms: eliminating those that begin to peroxidize, chelating metal ions, quenching ⋅o2, which prevent the making of peroxides, fracturing the chain of the oxidation reaction, decreasing oxygen concentration, or stimulating antioxidant enzymes. antioxidants that are able to disrupt free radical chain reactions are most effective. in general, they have aromatic phenolic rings with hydroxyl groups and can donate h● to free radicals that convert themselves to radicals. phenolic acids trap free radicals, and flavonoids can quench free radicals and chelate metals (brewer, 2011). food applications natural antimicrobial compounds can be used as preservatives in foods. there are two major issues for the application of plant compounds in foods, one being the odor caused by the high concentrations of these substances and the other being the cost. the safety of natural antimicrobials for use in foods is paramount, so toxicity tests must be done before application. meat products natural antibacterial components, such as spice and plant extracts, eos, organic acids, salts, and bacs, are used to modify the shelf life of meat. to improve sausages’ retention, lemon and thymol are used with modified atmospheric packaging (map). a combination of bay eo and oxygen-free map showed that it controls l. monocytogenes and e. coli growing and increases chicken meat’s retention time. one study showed that separate extracts of clove, rosemary, cassia bark, and liquorice extracts alone have potent antimicrobial efficacy. the mixture of rosemary and liquorice extracts showed a good repressor effect on l. monocytogenes, e. coli, pseudomonas fluorescens, and lactobacillus sake in freshly packed pork slices with atmospheric packaging and vacuum ham slices (mahmud and khan, 2018). nisin, pediocin pa-1, enterocin as-48, enterocins a and b, sakacin, and leucocin a can be used for bio-preservation of meat products and increase their shelf life. pediocin is a good bio-preservative in meat products. the antimicrobial efficacy of the mixture of lysozyme, nisin, and edta against l.  monocytogenes was observed in packed ostrich slices. immersing meat products in thyme and oregano oil (0.1 and 0.3% concentrations) improves shelf life (gonelimali et al., 2018; rai et al., 2016). massani et al. (2014) showed that active polymers including lactobacillus curvatus crl705 bacs reduced l. monocytogenes levels in italian journal of food science, 2021; 33 (sp1) 65 applications of food bio-preservatives nitrite in the meat which converts to carcinogenic nitrosamines. natural compounds such as eos of cinnamon, cloves, lemongrass, and their active ingredients are gras. the antimicrobial and antioxidant effects of plant extracts and eos owe to have phenolic compounds such as terpenes and flavonoids. however, despite the tremendous potential, flavor problems and toxicity can limit their use. to alleviate these problems and improve effectiveness, encapsulation of eos and active compounds and coatings containing these compounds are the best options. due to the potency of enzymes to produce antimicrobial compounds and their capability to separate some bacteria’s outer membrane, they can be known as natural antimicrobials compounds. however, their use in foods needs to be further studied. bacteriophages are used as an auxiliary process to control some pathogens but do not increase products’ shelf life. bacs are antimicrobial or peptide proteins that are ribosomally encoded and widely used in food production. bacs can be used as bio-preservatives because they are safe. the use of antimicrobial peptides in the form of nanoparticles can be very effective. for example, antimicrobial peptide coatings with metal nanoparticles will be beneficial in removing food contamination. the application of films and coatings, including natural agents, is expanding due to their biodegradability and potency to enhance food safety, quality, and shelf life. this study has shown that eos, bacs, enzymes, organic acids, and coatings can change chemical additives, but more research is needed. references ahmad, s., gokulakrishnan, p., giriprasad, r. and yatoo, m., 2015. fruit-based natural antioxidants in meat and meat products: a review. critical reviews in food science and nutrition 55(11): 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sinop, turkey brize recep tayyip erdogan university, fisheries faculty, depertmant of the fish processing technology, fener mah., 53100 rize, turkey csivas directorate of provincial food agriculture and livestock *corresponding author: eminerdem68@hotmail.com abstract in this study the sensory and chemical parameters of marinated shad (alosa immaculata, bennett, 1838) were determined. fish were marinated with different package methods (in brine, oil and vacuum packed) and stored at 4±1°c. during the storage period, diffusion of proximate composition, acetic acid and sodium chloride into the fish fillets were determined. at the end of 7 months storage period, tvb-n were 8.05, 16.81 and 17.56 (mg/100 g), tma were 2.28, 2.53 and 2.73 (mg/100 g), tba were 7.08, 7.13 and 6.05 (mg malonaldehyde/kg) and ph were 4.42, 4.72 and 4.77 for brine, oil and vacuum packed samples, respectively. throughout the storage period, effect of different package methods on tvb-n, tma, tba, ph, aw, acetic acid, sodium chloride and sensory issues were significant (p<0.05). mailto:eminerdem68@hotmail.com ital. j. food sci., vol. 27 2015 331 introduction fish is really healthy food and is known as the only food having a life-sustaining balanced ratio of protein, fat, carbohydrates, vitamins and minerals, which are essential to maintain good health. in addition, because of having low fat content, cholesterol and calories, fish meat is preferable for consumers (koral, 2006). shad is a close relative to sardines has an average length of 30-33 cm. shad are a black sea fish, but it can be found in the marmara, aegean and the mediterranean seas. shad live close to the coast as herd by shoal, they enter in the river to spawn at reproduction time in the spring. today, shad is also heavily fished in the united states and around. according to turkish statistical institute-tsi (2013), 1699 tons shad were caught in turkey at 2012. turkish consumers generally prefer as fresh, salted or smoked shad. marinating is traditionally a fish preservation method. meat tenderization and flavoring are consequences of the marinating process. sodium chloride, polyphosphates, sugars spices and sauces are considered important ingredients of marinades and they improve meat tenderness and flavor (sindelar et al., 2003; suderman, 1993). the purpose of bringing rigidity to the marinated product, enriching the meat flavor and preserving the meat longer (chen, 1982; resurrección, 2003). the immersion of meat in the marinating solution is done not only in turkey. the physical characteristics of fish change in acid and salt in several days. muscle tissue softens; skin and bones can be removed easily. marinate generally contains 4-5% vinegar and 7-10% salt. acetic acid leads to breakdown of structural proteins and enables the tearing of muscle membrane (meyer, 1965; erdem et al., 2005). muscle tissue, which has been softened during initial days by the joint effect of acid and salt, lost 15-20% of raw material weight at the end of the process (alparslan et al., 2013). the aim of the present study is to determine the sensory and chemical changes of marinated and brine, oil and vacuum packed shad fillets. therefore, the use of shad as an alternative to the other fish species used in marinade production will be investigated. materials and methods a total of 186 pontic shad (alosa immaculata) samples with an average weight of 191.53±33.63 g and average length of 28.71±1.58 cm were purchased from a local fish market in trabzon, turkey. they were stored in a thermally insulated container, brought to the laboratory and stored at –30°c with the purpose of eliminating the risk of parasites until the marinating process. marination process before marinating process, fishes were cleaned at the head, and the integral organs, after that fillet and washed with clean water. approximately 36 kg fillets were immersed into the following different marinating solutions for 48 h (1:2 fish:solution ratio) in a refrigerator. after two days of storage in refrigerator, the fillets were dipped in 4.5% acetic acid, 0.2% citric acid and 10% salt marination solutions, the marinated fishes were divided into tree groups (brine, sunflower oil and vacuum packaging). 70 grams of marinated shad fillets were packed into plastic box of 250 ml capacity with 150 ml brine, 150 ml sunflower oil, vacuum packaged and than were stored at 4±1°c for 7 months. proximate, chemical quality and sensory analyses were performed in triplicate on days 1, 30, 90, 150, and 210th. chemical analysis samples were homogenized and subjected to moisture and ash analyses using aoac (1990) methods. crude protein content was calculated by converting the nitrogen content according to kjeldahl’s method (aoac 1990), and lipid content was determined according to the method of the bligh and dyer (1959). thiobarbituric acid (tba) amounts were determined using the method of tarladgis et al., (1960), expressed as mg malonaldehyde/kg sample using a conversion factor of 7.8. the ph was determined from homogenates of minced samples in distilled water in a ratio of 1:10 (w/v) by using a digital ph meter (hanna, germany) (curran et al., 1980). total volatile basic nitrogen (tvb-n) was determined on steam distillation using the kjeldahl distillation apparatus and until red color was titrated, for clarification (antonocopoulus 1973). the method of aoac (1990) was used for trimethylamine nitrogen (tma-n) analysis. the water activity determination aqualab 3 te (0.100 to 1.000 ± 0.003), were measured by u.s. brand equipment. salt content was measured using the method proposed by karl (1994). 20 g fish were homogenized for 5 minutes with 100 ml distilled water, and than 150 ml distilled water was added and it was filtered. to this solution was added 2.5 ml of 10% k 2 cro 4. until red color was titrated with agno 3 0.1 n and according to the following formula amount of salt (%) was calculated: nacl (%) = a x 0.00585 x 100 x 500 / amount of sample (g) x 50 a: agno 3 consumption (ml) for the sensory evaluation of the marinated products five panelists were used. sensory analysis to assess appearance, odor, flavor and texture criteria were used, and analyises results, 332 ital. j. food sci., vol. 27 2015 which were scored on a scale of 1 to 9. in the scoring system points from 9 to 7 indicates “very good”, from 4.1 to 6.9 indicates “good”, 4 indicates “expendability” (4 is the rejection line), and from 1 to 3.9 indicates unacceptability (varlik et al., 1993). statistical analysis the statistical analysis was performed using minitab release 13.20 (minitab inc., state college, pa, usa). differences were analyzed by one-way analysis of variance and tukey’s test. in all statistical tests, p<0.05 was considered as statistically different (sumbuloglu and sumbuloglu, 2000). results and discussion the proximate composition of the shad fillet in each stage of the process is shown in table 1. these results are wet fish sample and marinated shad. protein, lipid, dry matter and ash of fresh shad fillets were 17.41%, 18.98%, 39.15% and 2.17%, respectively. at the end of the storage, all of these values have increased. maximum protein value was brining sample (18.83%) while maximum values at the lipid and dry matter were 20.30% and 47.11% at oil marinated and brining, respectively. guner et al. (1998), and boran and karacam (2011) reported 22.42% and 19.80% for protein and 15.91% and 9.34% lipid in fresh shad fillets, respectively. in another experiment, at the end of the brining stage the protein and lipid content were 18.32 g.100 g –1 and 3.20 g.100 g –1 of fish fillets (yeannes and casales 2008). the results of proximate analysis of fresh shad fillets were in agreement with our study. tvb-n of raw material was 14.01( mg/100 g) (table 2). at the end of 7 months storage period for marinated shad that were packaged differently in brine, oil and vacuum packed, tvb-n values were 8.05, 16.81 and 17.56 (mg/100 g), respectively. statistically difference was found between the brine group with the other groups on tvb-n value (p<0.05). özden and erkan (2006) reported that tvb-n in fresh fish and marinated trout were 7.35 mg/100 g and 6.78 mg/100 g, respectively. tvb-n values increased to 12.08 mg/100 g and 11.98 mg/ 100 g at the end of storage in vacuum and oil packed samples, respectively. similar results obtained in marinated fish packaged in vacuum and stored in refrigerator at a different time (aksu et al., 1997, meti̇n et al., 2000 and arik et al., 2001). tba was 0.99 malonaldehyde/kg at the beginning of the this study (table 2). this value increased to 7.08 in the brine, 7.13 in the oil and 6.05 mg malonaldehyde/kg in the vacuum packaged group on the last day (210th day) of storage. statistically difference was found between the vacuum group with the other groups on tba value (p<0.05). tba value is an important indicator and is excessively used to determine the level of lipid oxidation in fish (sallam 2007; cadun et al., 2008; turhan et al., 2009). varlik et al. (1993) reported that a consumable limit was between 7-8 mg malonaldehyde/ kg tba in sea fish. özden and erkan (2006) reported 0.45 mg malonaldehyde/kg tba values for fresh rainbow trout and 2.8 mg malonaldehyde/ kg tba values for marinated fish after 90 days, while 9.5 mg malonaldehyde/kg for vacuum and 10.26 mg malonaldehyde/kg for oil packaged marinated trout fillets were determined. water activity (a w ) of the raw material changed from 0.98 to 0.99, indicating that, it is the most suitable period for microbial growth (table 2). the water activities (a w ) at the marination were 0.93-0.94 between each group. the findings regarding to the a w value are in compliance with borgstrom’s (1968) with yeannes and casales (2008). in this study, ph level was 4.42, 4.72 and 4.77 in the brine, oil and vacuum packaged samples at 210 days, respectively and there were no significant differences between groups (p>0.05). the ph of fresh raw fish was initially approximately 6.30 and then changed during the maturation process to 4.29 after 90 days (özden and erkan 2006). in another study, ph values in anchovy marinated with 2% and 4% acetic acid increased was changed from 4.25 to 4.53 (aksu et al., 1997). the sodium chloride content of the fillet remained stable during the marinating stage while the salt level becomes richer in the brintable 1 the proximate composition in percentage of fresh and different marinating process of shad (alosa immaculata,) fillets during storage. storage days protein (%) lipid (%) dry matter (%) ash (%) fresh fish 17.41±0.51a 18.98±0.66a 39.15±0.15 a 2.17±0.16a brine 18.83±0.28b 20.14±0.11c 47.11±0.16 b 10.28±0.04c oil marinated 18.06±0.33b 20.30±0.19c 47.05±0.70 b 8.68±0.11c vacuum marinated 18.16±0.15b 20.21±0.33c 47.05±0.02 b 8.12±0.07b data are expressed as means±standard deviation. ital. j. food sci., vol. 27 2015 333 table 2 effects of brine, oil and vacuum packets on chemical changes of marinated shad (alosa immaculata) fillets during refrigerated storage. storage days after marinated 30 90 150 210 tvb-n mg/100 g brine 14.01±0.00a 8.40±0.0.23ba 5.80±0.00dec 5.95±0.0.49cdc 8.05±0.49bc oil 14.01±0.00a 8.55±0.89aa 12.01±0.00bb 13.61±0.00cb 16.81±0.00ab vacuum 14.01±0.00a 8.86±0.50 aa 14.16±0.49bca 15.66±0.66cda 17.56±0.00ea tma-n mg/100g brine 0.63±0.01ab 0.70±0.01ab 0.73±0.01bc 1.06±0.04cc 2.28±0.04ea oil 0.63±0.01ab 0.69±0.01ab 0.88±0.01abc 1.94±0.04ca 2.53±0.03da vacuum 0.63±0.01ab 0.93±0.03ca 1.22±0.03da 1.48±0.02eb 2.73±0.04fb tba mg malonaldehyde /kg brine 0.99±0.01a 1.82±0.06bca 3.21±0.36da 5.36±0.18ea 7.08±0.06fa oil 0.99±0.01a 1.92±0.06bca 3.66±0.15da 5.56±0.18ea 7.13±0.06fa vacuum 0.99±0.01a 1.28±0.04aa 2.32±0.12cbb 3.75±0.21db 6.05±0.35eb a w brine 0.99±0.001a 0.94±0.001cb 0.93±0.001db 0.93±0.001eb 0.93±0.001cb oil 0.98±0.001a 0.95±0.001dea 0.94±0.001ea 0.94±0.001fa 0.94±0.001fa vacuum 0.99±0.001a 0.95±0.001ba 0.94±0.001da 0.94±0.001cda 0.94±0.001bb ph brine 6.42±0.02a 4.25±0.01cdb 4.31±0.03bcda 4.33±0.04bcdb 4.42±0.03bb oil 6.42±0.02a 4.37±0.01fga 4.53±0.04dea 4.65±0.01bca 4.72±0.01ba vacuum 6.42±0.02a 4.39±0.01fga 4.49±0.01dea 4.59±0.01ca 4.77±0.01ba acidity brine 0.15±0.00a 1.28±0.00ca 1.58±0.11da 1.65±0.00db 1.66±0.01da oil 0.15±0.00a 1.20±0.11ca 1.38±0.11 cdb 1.53±0.11cda 1.65±0.00da vacuum 0.15±0.00a 1.35±0.00cb 1.48±0.11 dc 1.53±0.11da 1.65±0.00ea sodium chloride brine 0.27±0.12a 6.29±0.29ca 6.79±0.16ca 6.00±0.37ca 6.32±0.00ca oil 0.27±0.12a 4.77±0.21bb 4.92±0.50bb 4.77±0.04bb 4.39±0.08bb vacuum 0.27±0.12a 4.75±0.42bb 4.47±0.04bb 4.65±0.37bb 4.51±0.04bb data are expressed as means±standard deviation. a,b,c: differences between groups expressed with different letters in the same column are important (p<0,05). a,b,c: differences between groups expressed with different letters in the same line are important (p<0,05). ing stage and the salt concentration in the marinating solution becomes lower. according to kolakowski and bednarczyk (2002), the acetic acid in the marinating solution caused the decrease in the water content of fillet. sodium chloride in fresh fish muscle was 0.27%, while at the end of the study, it was reached to 6.32%, 4.39% and 4.51% for brine, oil and vacuum package groups, respectively. according to duerr and dyer (1952), fennema (1977) and honibel (1989), myosin denaturation, as measured by salt solubility, occurs at a definite salt concentration, about 8 to 10% sodium chloride in the tissue. meyer (1965) stated that the amount of the acid in high quality marine products should be between 2-3% in fish tissue at the end of the ripening period. in this study the acid amount changed between 1.20-1.66 during the trial. the sensory scores of marinated shad fillets indicated a good quality of the storage period (table 3). there were significant differences between brine, oil and vacuum packaged shad marinades (p<0.05) for sensory value. these results are in agreement with the findings of özden and baygar (2003) for marinated chub mackerel, horse mackerel, sardine and anchovy packaged in jars with vegetable oil and vacuum packed in polyethylene bags, then stored at 4±1°c. in addition to these studies, yeannes and casales (2008) reported that there were not quality changes on sensory analysis of marinated anchovy (engraulis anchoita) throughout storage time. many studies showed that good quality marinated fish is between 3 and 6 months (erkan et al., 2000; varlik et al., 2000; özden and baygar 2003; kilinc and çakli 2004; gökoglu et al., 2004, erdem et al., 2005; kaba et al., 2013). conclusions in this study, the effects of brine, oil and vacuum packing on chemical and sensory changes in marinated shad stored at 4°c were investigated. a quality assessment was performed by monitoring sensory quality, total volatile basic nitrogen and thiobarbituric acid, ph, aw, and salinity count. the results of this study indicate that the shelf life of brine and oil packed marinated shad fillets had a shelf life of 210 days. accord334 ital. j. food sci., vol. 27 2015 table 3 effects of different package methods on sensory property of marinated shad (alosa immaculata) fillets during storage time. storage days after marinated 30 90 150 210 appearance brine 9.55±0.38aa 9.00±0.38 aa 7.05±0.69 ba 6.40±0.98 ca 5.00±0.32 da oil 9.57±0.41 aa 8.50±0.55 ab 7.13±0.52 ba 6.04±0.39 ca 4.53±0.52 db vacuum 9.65±0.35 aa 930±0.61 ac 7.47±0.41 ab 5.81±0.32 da 4.20±0.32 db odor brine 9.25±0.25aa 8.58±0.49 aba 7.42±1.02 bca 5.92±0.92 ca 4.28±0.35 da oil 9.52±0.61 aa 8.60±0.41 aba 7.08±0.92 ba 6.25±0.76 ca 4.57±0.75 db vacuum 9.61±0.41 aa 8.70±0.32 aba 7.83±0.41 cda 5.97±0.26 da 3.91±0.29 ac flavor brine 9.63±0.05aa 8.75±0.35 aba 7.25±0.15 ca 5.90±0.10 da 4.13±0.18 ea oil 9.63±0.05aa 8.50±0.20 bb 7.25±0.20 ca 6.00±0.15 da 4.50±0.06 eb vacuum 9.63±0.05aa 9.25±0.09 abc 7.75±0.18 ba 4.75±0.15 cb 3.50±0.00ec texture brine 9.85±0.39aa 8.58±0.41 ba 7.08±0.44 ca 6.00±0.33 da 4.20±0.13 ea oil 9.88±0.37aa 8.79±0.20 ba 7.67±0.21 cab 5.58±0.27 da 4.40±0.15 ea vacuum 9.93±0.23aa 9.71±0.26 ab 7.93±0.24 bb 5.33±0.15 cb 4.00±0.21db a,b,c differences between groups expressed with different letters in the same column are important (p<0,05). a,b,c differences between groups expressed with different letters in the same line are important (p<0,05). ing to this study, fishbones of the shad which has a large number of them melted as a result of the marination process. acknowledgments this study was supported by the scientific research coordination unit of recep tayyip erdogan university, rize, turkey (project number: rübap 2008.103.03). references aksu h., erkan n., çolak h., varlık c., gökoğlu n. and ugur m. 1997. farklı asit-tuz konsantrasyonlarıyla hamsi marinatı üretimi esnasında olusan bazı degisiklikler ve raf ömrünün belirlenmesi. university of 100.yıl.. j. vet. fac. 8 (1-2): 86. alparslan y., baygar t., hasanhocaoğlu h. and metin c. 2013. effects of scale and skin on chemical and sensory quality of marinated sea bass 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(ed.). fish as food. processing: part 1. new york: academic press, v. 3, p. 165. özden ö. and erkan n. 2006. effect of different packing methods on the shelf life of marinated rainbow trout, archiv für lebensmittelhygiene, 57: 69. özden ö. and baygar t. 2003. the effect of different packaging methods on some quality criteria of marinated fish. turk. j.vet. anim. sci. 27: 899. resurrección a.v.a. 2003. sensory aspects of consumer choices for meat and meat products. meat sci. 66: 11. sallam kh.i., ahmed a.m., elgazzar m.m. and eldaly e.a. 2007. chemical quality and sensory attributes of marinated pacific saury (cololabis saira) during vacuum-packaged storage at 4 °c. food chem., 102: 1061. sindelar j.j., prochaska f., britt j., smith g.l. and osburn w.n. 2003. strategies to eliminate a typical flavors and aromas in sow loins. ii consumer acceptance of loins marinated with sodium tripolyphosphate and sodium bicarbonate. meat sci., 65:1223. suderman d.r. 1993. selecting flavoring and seasoning for batter and breading systems. cereal foods world, 38: 689. sümbüloglu k. and sümbüloglu v. 2000. biostatistics, hatiboglu press:53, 9th edition, ankara, p. 269. tarladgis b., watts b.m. and yonathan m. 1960. distillation method for determination of malonaldehyde in rancidity food. j. am. oil chem. soc. 37: 44. t.s.i. (turkish statistical institute) 2013. tuik, ankara. yeannes m.i. and casales m.r. 2008. modifications in the chemical compounds and sensorial attributes of engraulis anchoita fillet during marinating process, ciênc. tecnol. aliment., campinas, 28(4): 798. varlık c., uğur m., gökoğlu n. and gün h. 1993. principle and methods of quality control in sea products. food technology association press 17, 174 p., ankara. varlık c., erkan n., metin s., baygar t. and özden ö. 2000. determination of the shelf-life of marinated fish balls. turk. j. vet. anim. sci., 24(6): 593. paper received june 26, 2014 accepted october 10, 2014 http://wos15.isiknowledge.com/?sid=d65jgf9ahbp3@5ain7k&func=abstract&doc=1/33 http://wos15.isiknowledge.com/?sid=d65jgf9ahbp3@5ain7k&func=abstract&doc=1/33 http://wos15.isiknowledge.com/?sid=d65jgf9ahbp3@5ain7k&func=abstract&doc=1/33 http://wos15.isiknowledge.com/?sid=d65jgf9ahbp3@5ain7k&func=abstract&doc=1/34 44 issn 1120-1770 online, doi 10.15586/ijfs.v35i3.2381 p u b l i c a t i o n s codon italian journal of food science, 2023; 35 (3): 44–54 anti-dyslipidemic and anti-hypercholesteremic effects of concoction juice: a randomized, double-blind, placebo controlled trial wajiha saeed1, shahid mahmood1, ghulam mueen-ud-din1, muhammad yousaf quddoos2* 1institute of food science and nutrition, university of sargodha, sargodha-pakistan; 2punjab food authority khushab-pakistan *corresponding author: muhammad yousaf quddoos, punjab food authority khushab-pakistan. email: yousafquddoos@gmail.com received: 6 june 2023; accepted: 27 june 2023; published: 1 august 2023 © 2023 codon publications open access paper abstract globally, dyslipidemia is an alarming condition, which may cause death. however, its country-wise ratio varies. in pakistan, it is about 16–20% in both men and women. dyslipidemia can be defined as the up level quantity of lipids in blood than the average value. the treatment of dyslipidemia can be done by dietary interventions with indigenous sources. the (concoction) juice has the ability to reduce the low-density lipoprotein (ldl), total cholesterol (tc), triglycerides (tgs). and raise the high-density lipoprotein (hdl) level. for efficacy studies, dyslipidemic females (n = 1010) were approached in different clinics and hospitals of sargodha. they were approached with biomarkers, that is, lipidemic profile, tc, hdl, urea, creatinine, and tgs. a total of 85 females were selected and they were divided into three groups (t0, l1 and m1). t0 is an experimental (control) group, t1 group has dyslipidemic patients treated with lab-made juice, and m1 has dyslipidemic patients treated with market products. after 120 days of treatment, the data were analyzed statistically to validate the results of the study. the anthropometric and dietary intake was assessed by dyslipidemic volunteers. a significant level of dyslipidemic biomarker was found in l1. the down level was found in tc (216-184 mg/dl), tgs (215.54-138.46 mg/dl), and ldl (145.64-134.34 mg/dl), and raised level was found in hdl (44.15-54.43 mg/dl). other parameters that showed a downward trend were urea (12.73-11.15 mg/dl), uric acid (6.12-5.38 mg/dl), creatinine (1.02-1.0 mg/dl), alt (55-34 u/l), ast (47-27 u/l), alp (91.63-83.76 iu/l), bilirubin (0.65 to 0.57 mg/dl), rbcs, wbcs, and hb. based on these results, it is concluded that lab-made concoction l1 showed best results overall and is also appropriate and cost effective and further research has to be done on a large scale. keywords: anthropometry; dyslipidemia; garlic; ginger; honey; juice; total cholesterol; triglycerides introduction health can be defined as a state of psychological and social well-being and not just the absence of disease (who, 2012). many effects that are interrelated at different points are collectively called the nutritional status of a person (park, 2009). plants are the natural sources for the treatment of diseases (weidner et al., 2012). nowadays extracts that are prepared from biomaterials are used as functional food and nutraceuticals (müller and kersten, 2003). liver produces cholesterol and performs good functions in the body. high cholesterol levels cause health complications like arteriosclerosis (plaque produced in arteries that causes myocardial infarction is termed as arteriosclerosis) (kurian et al., 2013). garlic has a sulfur-containing compound known as allicin ((r, s)-diallyldisulfid-s-oxide), which is prepared when alliinase enzymes act on alliin. this compound has lots of functions that are advantageous for the health of italian journal of food science, 2023; 35 (3) 45 anti-dyslipidemic and anti-hypercholesteremic effects of concoction juice citric acid (2-hydroxy-1,2,3-propanetricarboxylic acid) that is weak tricarboxylic acid. citric acid is used for many purposes like providing sour taste to beverages and food, and acidity to the food. among all food acids, citric acid is present in lime and lemon (villa-ruano et al., 2019). influence of apple cider vinegar has been investigated from hundreds of years. hippocrates (father of modern medicine) prescribed a combination of apple cider vinegar and honey for the treatment of a number of diseases (mindell and mindell, 2002). polyphenolic components of apple cider vinegar have important beneficial effects on health (shahidi et  al., 2008; verzelloni et  al., 2007). antioxidant flavonoid components of apple cider vinegar lower the side-effects of a high cholesterol diet (setorki et al., 2010). the main constituent of apple cider vinegar is acetic acid, which is consumed at a concentration of 3 to 9%. it is used as seasoning and also as a medicine (fushimi et al., 2006). the study of naseem et  al. (2016) revealed that dietary herbal extract has cardioprotective and anti-atherogenic effects devoid of any known side-effects in experimental animal models. in the present study, cardioprotective effects of some of these herbs (apple cider vinegar, honey, garlic, ginger, and lemon) were investigated in experimental animal models of hyperlipidemia. the plasma levels of total cholesterol (tc), triglycerides (tg), high-density lipoprotein-c (hdl-c), low-density lipoprotein-c (ldl-c), and body weight in rabbits fed on normal chow diet (group i) remain stable throughout the experimental period. with hypercholesterolemic diet, body weight increases (p > 0.05), and there was a significant rise in tc, tg, vldl, and aip (p 0.05). plasma glucose and alt levels were increased and plasma gsh decreased in hyperlipidemic animals as compared to the control group. objectives • to make a comparative study between lab-made and market concoctions. • to explore the effects of selected juice treatments in dyslipidemic female adults. materials and methods procurement of materials ingredients (ginger, apple cider vinegar, garlic, and lemon) were purchased from the bazar of sargodha and honey was sourced from the forest. the raw materials were stored humans. allicin possesses recirculatory, hypolipidemic, and anti-platelet actions (leuter et al., 1996). atherosclerosis is one of the foremost origins of primary disability and death. the top recognized independent risk influence implicated in the pathology of atherosclerosis is hypercholesterolemia (libby, 2005). coronary events and atherosclerosis pose a risk to the cardiovascular system and have a robust connection with raised serum lipids (gotto, 2011: siri-tarino et al., 2010). the prevalence and disease incidence report released in india in 2013 showed that >224 billion individuals in india have increased levels of cholesterol (lachhiramka and patil, 2016). the rhizome/stem of ginger, in addition to containing such proximate constituents like ash, protein fiber, moisture, and carbohydrate, has a volatile oil in its stem that contributes to its pleasant aroma. in addition, ginger contains ascorbic acid, β-carotene, curcumin, gingerol, linalool, paradol, γ-terpinene, as well as terpinen-4-ol (shahidi and hossain, 2018). the swollen rhizome/ stem of ginger has been associated with antimicrobial, anti-inflammatory, and anti-carcinogenic properties (samaniego et al., 2011). it was shown by in vitro experiments that lipid peroxidation was decreased and levels of antioxidant enzymes among serum glutathione were increased by the administration of ginger. hence, it has been proved that ginger has an equivalent antioxidant consequence to that of vitamin c (ahmad et  al., 2009). this study showed that the combined extract of zingiber officinalle (ginger) and allium sativum (garlic) retain anti-inflammatory, immune-modulatory, antioxidant, and anti-lipidemic effects and, therefore, can be useful in the management of inflammation-related diseases and useful as an immune booster and in the treatment of dyslipidemia. the combined extract restored the hematological indices and improved the serum lipid profile (adegbola et al., 2021). according to the islamic point of view, honey is a, healthy food. the holy quran intensely demonstrates the latent beneficial value of honey: “and thy lord taught the bee to build its cells in hills, on trees, and in (men’s) habitations; then to eat of all the produce (of the earth), and find with skill the spacious paths of its lord: there issues from within their bodies a drink of varying colors, wherein is healing for men: verily in this is a sign for those who give thought.” the muslim prophet mohammad (s. a. w. w.) suggested honey for the management of diarrhea (molan, 1999). almost 1000 years ago, avicenna, the great iranian scientist and physician, suggested honey as the best medication for tuberculosis (asadi-pooya et al., 2003). lemon contains citric acid, vitamin c (ascorbic acid), flavonoids, and minerals (benavente et  al., 1997). in citrus fruits, there is a naturally occurring compound known as 46 italian journal of food science, 2023; 35 (3) saeed w et al. in glass bottle) which showed best physic-chemical, sensory, microbial and stability study technique eighty-five adults, both men and women, were approached. for the study, different hospitals and institutes were selected. two-stage sampling techniques, that is, convenience sampling and purposive sampling, were adopted for the selection of volunteers. the blood samples of selected volunteers were collected; blood was coded and placed in specified jars for tests in labs muhammad (2000). the clinical controlled trial, randomized research method, was performed by taking samples of blood of all the volunteers in two steps. in the first step, the selected dyslipidemic volunteers were shifted for further research and in the second step, these tests were again performed in dyslipidemic volunteers after 120 days macmahon and trichopoulos, 1996; kumar et al., 2008. the study was performed for 120 days study with test (before and after). the data of dietary intakes assessment and biomarker (lipid profile, cbc, lft and rft, urea, creatinine, cholesterol, ldl, hdl, tg) of selected volunteers were recorded for their nutritional status assessment (figure 1). at suitable temperature at the nutrition lab, institute of food science and nutrition, university of sargodha. preparation of concoction at the university lab, juice was extracted and mixed with honey and apple cider vinegar in equal amounts and then boiled at 60 ± 5°c. after cooling, it is packed in different packing materials and stored. at the time of using, one table spoon of juice was mixed in lukewarm water and provided to volunteers three times a day naseem et  al., 2016. treatment plan is mentioned in table 1. up to 8 month selected treatment of concoction juice (l1: concoction stored at refrigerated temperature (0–4°c) table 1. treatment plan and composition. treatments ginger (%) garlic (%) lemon (%) *acv (%) honey (%) t 0 0 0 5 5 0 l 1 20 10 15 15 40 m 1 – – – – – *acv, apple cider vinegar; t 0 , control group; l 1 , lab-made product; m 1 , market product. weight body muscle body bone meat veget fruits cereals tgs tcjunk fat milk creatin uric hdl ldl ast bmi alt alp rb bilirubi wbcs h urea figure 1. graphical. tc, (total cholesterol); tg, (triglycerides); ldl, (low-density lipoprotein); hdl, (high-density lipoprotein) italian journal of food science, 2023; 35 (3) 47 anti-dyslipidemic and anti-hypercholesteremic effects of concoction juice on days (3.67 ± 0.17 to 2.67 ± 0.13). also, the same trend is found with regard to junk food and snacks (4.05 ± 0.18 to 3.05 ± 0.13). cholesterol levels might be increased with fried or junk food due to the existence of trans fat in them. regular ingesting of such food ultimately results in many diseases like dyslipidemia. anthropometric assessments with respect to days, only significant values were found in weight (66.55 ± 2.37 to 64.48 ± 2.28) and body mass index (bmi) (27.45 ± 0.94 to 26.56 ± 0.9), which were reduced after 120 days. it means that the combined juice may affect weight reduction but no changes were found in the mean values (days) of body muscle mass (bmm) and body water (bw) and body bones mass (bm) (table 3). biomarker of blood samples (dyslipidemia) tc level (mg/dl) the combined juice showed positive outcomes with regard to tc (table 4). in t0 (219 ± 1.18 to 225.43 ± 2.75), the cholesterol levels were increased. cholesterol levels decreased significantly with treatment l1 (216 ± 1.15 to 184 ± 1.69) over the course of 120 days. treatment m1 also decreased tc (217.44 ± 1.77 to 193.25 ± 1.4) with a dose of 1 tablespoon three times a day. statistical analyses with the help of blood samples and sensory evaluation, statistical analyses were performed. significant values were tested by parametric and nonparametric tests. p ≤ 0.05–0.01 is of statistical significance. tables, graphs, and charts were used to present the data. data were checked by descriptive statistics and analyzed by using minitab 16 statistical software to validate the outcomes of the study (steel et al., 1997). results the test parameters of results are mentioned in the graph: anthropometric (blue color), food intake dairy (green color), and blood sample (reddish and red color) parameters are mentioned in this figure, while their complete details are mentioned in the results section. dietary intakes assessment after getting a health assessment of dyslipidemic volunteers, we found nonsignificant values found in days of dietary intake (table 2). per usda recommendation, cholesterol can be decreased by eating fruits and vegetables. with respect to mean values, dyslipidemic volunteers consumed more of meat and meat products. significantly lessened values of fat and oil were consumed table 2. dietary intake of dyslipidemic volunteers before and after. ps cereals fruits vegetables meat and meat products milk and milk products fats and oil junk and snacks 0 days 120 days 0 days 120 days 0 days 120 days 0 days 120 days 0 days 120 days 0 days 120 days 0 days 120 days dyslipidemic 6.43 ± 0.23 6.58 ± 0.23 0.89 ± 0.1 1 ± 0.1 1.73 ± 0.1 1.76 ± 0.07 4.25 ± 0.2 4.2 ± 0.16 4.27 ± 0.2 4.28 ± 0.16 3.67 ± 0.17 2.67 ± 0.13b 4.05 ± 0.18a 3.05 ± 0.13b ps, physiological status. table 3. anthropometric measurements of volunteers before and after. ps age weight (kg) body fat (%) body water (%) bone mass (%) muscle mass (%) bmi 0 days 120 days 0 days 120 days 0 days 120 days 0 days 120 days 0 days 120 days 0 days 120 days dyslipidemic 30–50 ± 7 66.55 ± 2.37 64.48 ± 2.28 37.07 ± 0.57 37.07 ± 0.57 36.76 ± 0.63 36.8 ± 0.63 7.08 ± 0.23 7.08 ± 0.23 25.86 ± 0.26 25.86 ± 0.26 27.45 ± 0.94 26.56 ± 0.9 ps, physiological status. 48 italian journal of food science, 2023; 35 (3) saeed w et al. in l1 (145.64 ± 0.49 to 134.34 ± 0.48). this indicates that treatment l1 reduces the ldl level than the other treatments. garlic facilitates high production of hdl and reduced level of ldl (hongbao and kuan-jiunn, 2006). hdl (mg/dl) the levels of hdl were decreased in the respondents following treatment t0 (45.74 ± 0.53 to 34.59 ± 0.88), while the hdl levels moved upward significantly in treatment m1 and was move more upwards significantly in treatment l1 (44.15 ± 0.43 to 54.43 ± 0.67) (table 7). urea (mg/dl) means squares for effect of juice (table 8) on urea have shown highly changed outcomes. in the control group (t0), means for efficacy study indicated that urea value increased significantly (12.33 ± 0.27 to 14.27 ± 0.43) while tgs (mg/dl) the good ingredients combination of concoction juice showed significant results in treatments and days of tgs (table 4). tg levels of respondents were increased in treatment t0 (206.21 ± 3.7 to 241.64 ± 3.14) while they were significantly decreased in treatment m1 (200.25 ± 3.52 to 161.13 ± 4.67) and significantly decreased in treatment l1 (215.51 ± 3.43 to 138.463 ± 5.31). niacin plays a role in preventing the deposition of tgs in adipose tissue by controlling or holding the diacylglycerol acyltransferase enzyme in the liver. in the production of cholesterol, this enzyme is active in the 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase (hmg-coa reductase) pathway (alagwu et  al., 2011; mushtaq et al., 2011). according to yaghoobi and salomé (2008), daily consumption of honey (70 g) in lukewarm water will reduce the tgs and other hyperlipidemic parameters.yaghoobi ldl (mg/dl) means squares for the effect of juice (table 6) on ldl have shown highly significant results. in the control group (t0), means for efficacy study indicated that ldl value increased significantly (154.15 ± 1.48 to 167.21 ± 1.72) while in other treatments, namely, l1 and m1, values significantly decreased. high values decreased table 4. comparison test of tc (mg/dl) of volunteers before and after. treatment day 0 day 120 mean t 0 219 ± 1.18 225.43 ± 2.75 222 ±1.965 l 1 216 ± 1.15 184 ± 1.69 200 ±1.42 m 1 217.44 ± 1.77 193.25 ± 1.4 205.345 ± 1.585 mean 217.48 ± 1.36 200.89 ± 1.94 – tc, total cholesterol; t 0 , control group; l 1 , lab-made product; m 1 , market product. table 6. comparison test of ldl (mg/dl) before and after. treatment day 0 day 120 means t 0 154.15 ± 1.48 167.21 ± 1.72 160.68 ± 1.6 l 1 145.64 ± 0.49 134.34 ± 0.48 139.99 ± 0.485 m 1 141.21 ± 1.16 138.31 ± 1.25 155.105 ± 1.205 mean 147 ± 1.043 146.62 ± 1.15 – ldl, low-density lipoprotein; t 0 , control group; l 1 , lab-made product; m 1 , market product. table 7. comparison test of hdl (mg/dl) before and after. treatment day 0 day 120 means t 0 45.74 ± 0.53 34.59 ± 0.88 40.165 ± 0.71 l 1 44.15 ± 0.43 54.43 ± 0.67 49.29 ± 0.55 m 1 42.44 ± 0.57 46.74 ± 0.43 44.59 ± 0.5 mean 44.11 ± 0.51 45.25 ± 0.66 – hdl, high-density lipoprotein; t 0 , control group; l 1 , lab-made product; m 1 , market product. table 8. comparison test of urea (mg/dl) before and after. treatment day 0 day 120 means t 0 12.33 ± 0.27 14.27 ± 0.43 13.3 ± 0.35 l 1 12.73 ± 0.43 11.15 ± 0.12 11.94 ± 00.275 m 1 12.31 ± 0.14 12.41 ± 0.38 12.36 ± 0.26 mean 12.45 ± 0.28 12.61 ± 0.31 – t 0 , control group; l1, lab made product; m 1 , market product. table 5. comparison test of tgs (mg/dl) before and after. treatment day 0 day 120 means t 0 206.21 ± 3.7 241.64 ± 3.14 223.925 ± 3.42 l 1 215.51 ± 3.43 138.463 ± 5.31 176.98 ± 4.37 m 1 200.25 ± 3.52 161.13 ± 4.67 180.69 ± 4.095 mean 207.32 ± 3.55 180.411 ± 4.37 – tg, triglycerides; t 0 , control group; l 1 , lab-made product; m 1 , market product. italian journal of food science, 2023; 35 (3) 49 anti-dyslipidemic and anti-hypercholesteremic effects of concoction juice ast (sgot) (u/l) the levels of ast were increased in the respondents following treatment t0 (50 ± 3.45 to 63 ± 3.14) while the ast levels were reduced significantly in treatment m1 (51 ± 3.18 to 36 ± 3.22) and were reduced highly significantly in treatment l1 (47 ± 2.45 to 27 ± 2.75) (table 12). alp (iu/l) alp levels were improved in t0, while the alp (iu/l) levels were reduced significantly in m1 and were reduced highly significantly in treatment l1 (table 13).. bilirubin (mg/dl) bilirubin scores were increased slightly in treatment t0 (0.59 ± 0.02 to 0.63 ± 0.04), while they were decreased slightly in treatment m1 (0.61 ± 0.02 to 0.59 ± 0.02) and treatment l1 (0.65 ± 0.02 to 0.57 ± 0.01) (table 14.). in other treatments, namely, l1 and m1, values significantly decreased. high values decreased in l1 (12.73 ± 0.43 to 11.15 ± 0.12). this indicates that l1 has a greater effect on reducing the urea level than other treatments. uric acid (mg/dl) significant results were found in t0, l1, and m1. uric acid levels were increased a little in t0 respondents (6.53 ± 0.03 to 7.09 ± 0.02), no changes were found in m1, and it was decreased a little in treatment l1 (table 9). ginger positively lowers the uric acid quantity (shokr and mohamed, 2019). creatinine (mg/dl) the levels of creatinine were slightly increased in treatment t0 (1.03 ± 0.01 to 1.09 ± 0.03) while the levels of creatinine showed a downward trend in treatments m1 and l1 with 0.01 and 0.02 difference, respectively (table 10). alt (sgpt) (u/l) means squares for effect of juice (table 11) on alt have shown highly changed outcomes. in the control group (t0), means for efficacy study indicated that alt value increased significantly (60 ± 2.45 to 75 ± 2.74) while in other treatments, namely, l1 and m1, values significantly decreased. high values decreased in l1 (55 ± 3.15 to 34 ± 3.45). this means that l1 has a greater effect on reducing the alt level than m1 (53 ± 4.38 to 39 ± 4.22). table 12. comparison test of ast (sgot) before and after. treatment day 0 day 120 means t 0 50 ± 3.45 63 ± 3.14 56.5 ± 3.29 l 1 47 ± 2.45 27 ± 2.75 37 ± 2.6 m 1 51 ± 3.18 36 ± 3.22 43.5 ± 3.2 mean 49.33 ± 3.02 49.33 ± 3.03 – t 0, control group; l 1 , lab-made product; m 1 , market product. table 13. comparison test of alp (iu/l) before and after. treatment day 0 day 120 means t 0 98.74 ± 4.18 114.68 ± 3.71 106.71 ± 3.94 l 1 91.63 ± 6.32 83.76 ± 4.36 87 ± 5.34 m 1 100.43 ± 4.72 85.94 ± 4.2 93.18 ± 4.46 mean 96.93 ± 5.07 94.79 ± 4.09 – t 0 , control group; l 1 , lab-made product; m 1 , market product. table 9. comparison test of uric acid before and after. treatment day 0 day 120 means t 0 6.53 ± 0.03 7.09 ± 0.02 6.81 ± 0.025 l 1 6.12 ± 0.07 5.38 ± 0.04 5.75 ± 0.05 m 1 6.05 ± 0.07 6.02 ± 0.04 6.035 ± 0.055 mean 6.23 ± 0.056 6.16 ± 0.03 – t 0 , control group; l 1 , lab-made product; m 1 , market product. table 10. comparison test of creatinine (mg/dl) before and after. treatment day 0 day 120 means t 0 1.03 ± 0.01 1.09 ± 0.03 1.06 ± 0.02 l 1 1.02 ± 0.01 1 ± 0.01 1.025 ± 0.01 m 1 1.02 ± 0.01 1.03 ± 0.01 1.025 ± 0.01 mean 1.023 ± 0.01 1.04 ± 0.02 – t 0 , control group; l 1 , lab-made product; m 1 , market product. table 11. comparison test of alt (sgpt) before and after. treatment day 0 day 120 means t 0 60 ± 2.45 75 ± 2.74 67.5 ± 2.59 l 1 55 ± 3.15 34 ± 3.45 44.5 ± 3.3 m 1 53 ± 4.38 39 ± 4.22 46 ± 4.3 mean 56 ± 3.32 49.33 ± 3.47 – t 0 , control group; l 1 , lab-made product; m 1 , market product. 50 italian journal of food science, 2023; 35 (3) saeed w et al. they showed good results in the conversion of hyperlipidemic to hypolipidemic (sobenin et  al., 2010). it is revealed by alizadeh-navaei (2008) that he used ginger capsules (3 g per day) in humans and found that ginger powder decreases endogenous production of cholesterol by increasing the ldl receptors for removing the ldl-c from plasma. the level of pancreatic amylase and lipase increases and lipid hydrolysis decreases by the action of ginger (alizadeh-navaei et al., 2008). our tgs study was also supported by shishehbor et  al. (2008) who explained that hyperlipidemia is also reduced by apple cider vinegar due to the presence of acetic acid, which plays a role in inhibiting hepatic lipogenesis by raising the beta oxidation of fatty acids in rats. sterol regulatory elements binding protein-1 (srebp-1) gene is blocked by acetic acid in diets that reduce the level and function of atp citrate lyase (atp-cl) and mrna, respectively, which try to decrease the distribution of acetyl coa (fushimi et al., 2006). the level of pancreatic lipase moves upward with the bioactive component gingerol of ginger (platel and srinivasan, 2000; ramakrishna et  al., 2003). gingerol plays an important role in increasing the working activity of hepatic cholesterol-7-hydroxylase, which converts the cholesterol into bile acid and finally eliminates it from the body (srinivasan and sambaiah, 1991). djousse et  al. (2004) showed the outcomes of their research: 6–7% ldl was reduced in those volunteers by taking fruits and vegetables as paralleled to those who were not taking enough fruits. this may be because of dietary fibers in fruits that help in the absorption and removal of cholesterol from the blood (ballesteros et al., 2001; sprecher and pearce, 2002). few phytochemicals present in the juice of citrus limon like flavonoids, polyphenols, and terpenes assist in the reduction of serum bilirubin (al-snafi, 2016; muhammad et al., 2013). this outcome is also confirmed by shefalee (bhavsar et al., 2007). it also comprises ascorbic acid (vit c), which acts as an antioxidant that helps in lowering the level of bilirubin (tounsi et al., 2011). rbcs (mcells/mcl) non-significant results were obtained among treatments (table 15). rbc levels were reduced slightly in treatment t0, while they increased slightly in treatment m1 and were better slightly in treatment l1 (table 15). wbcs (cells/mcl) the wbc levels were reduced slightly in treatment t0, while the wbc levels improved slightly in treatment m1 and were better slightly in treatment l1. hb (g/dl) hb levels were reduced slightly in treatment t0, while the hb levels improved in treatment m1 and were better in treatment l1 (table 15). the comparison test of tc, tgs, ldl, and hdl. three treatments, namely, t0, l1, and m1 are mentioned for each disease with their values before and after. discussions sobenin and colleagues have reported that they gave garlic powder (150 mg per day) for a period of 1 year to hyperlipidemic patients and found significant results in the case of ldl, tc, and tgs. sulfur-containing amino acids like ajoene, s-allylcysteine, s-methylcysteine, allicin, sulfoxides, and diallyl disulfide are present in garlic. table 14. comparison test of bilirubin before and after. treatment day 0 day 120 means t 0 0.59 ± 0.02 0.63 ± 0.04 0.61 ± 0.03 l 1 0.65 ± 0.02 0.57 ± 0.01 0.61 ± 0.015 m 1 0.61 ± 0.02 0.59 ± 0.02 0.6 ± 0.02 mean 0.61 ± 0.02 0.59 ± 0.02 – t 0 , control group; l 1 , lab-made product; m 1 , market product. table 15. comparison test of rbcs (mcells/mcl), wbcs (cells/mcl) and hb (g/dl) before and after. treatments rbcs wbcs hb 0 days 120 days 0 days 120 days 0 days 120 days t 0 5.55 ± 0.18 4.8 ± 0.14 6435.25 ± 217 5420.25 ± 225 36.76 ± 0.63 36.8 ± 0.63 l 1 4.87 ± 0.1 5.42 ± 0.14 5963.25 ± 105 6234.5 ± 97.1 11.54 ± 0.13 11.48 ± 0.12 m 1 4.9 ± 0.15 5.12 ± 0.13 5829.08 ± 113 6071.7 ± 78.4 11.77 ± 0.09 12.22 ± 0.08 t 0 , control group; l 1 , lab-made product; m 1 , market product. italian journal of food science, 2023; 35 (3) 51 anti-dyslipidemic and anti-hypercholesteremic effects of concoction juice which in turn converts blood cholesterol into bile acid, resulting in a decrease in blood cholesterol levels. regular intake of vitamin c (1 ml/kg/day) reduces cholesterol and ldl blood tgs and promotes concentration of hdl in blood (yasmin et  al., 2010). el-shenawy and hassan (2008) stated that consumption of garlic prevents the increase in serum urea while producing a prominent reduction in the level of serum urea. gluconeogenesis decreases with consumption of ginger through proteolysis, which ultimately reduces the level of serum urea and creatinine (abd elwahab and ali, 2015). al-jowari (2014) demonstrated that garlic has a tendency of augmenting the level of hb, platelet count, wbcs, and rbcs. it might be possible due to the end product of garlic metabolism in the body, which accelerates the production and secretion of more erythropoietin in the kidney which are powerful stimulators of bone marrow. besides this, garlic possesses few components that are considered active oxygen scavengers that compete with hb in rbcs for o2 causing hypoxia, which ultimately increases the production of rbcs and hb (iranloye, 2002). increase in wbcs is due to the immune stimulant activities of garlic. immune stimulants are fixed precisely to the receptors on the surface of cells of lymphocytes and phagocytes that activate the cell to produce some enzymes that can kill pathogens (fazlolahzadeh et  al., 2011). results are summarized in figure 2. novelty nowadays, most dyslipidemic patients are treated with medicines, which may have some side-effects and high cost. but, this product is prepared with a combination of natural food and herbs (concoction) and has very good shelf life, sensory quality, and is very cost-effective. the recovery time period from disease is short. conclusion dyslipidemia is a very serious condition with a high prevalence rate in both men and women in pakistan. sedentary lifestyle coupled with regular intake of high calories, that is, high fat and carbohydrates, is a key factor. the combined juice contains garlic, ginger, honey, lemon extract, and apple cider. all of these have active ingredients to reduce the level of tc, tgs, ldl, and increase the level of hdl to prevent cardiovascular diseases. they also improve other biomarkers like decreasing urea, uric acid, creatinine, bilirubin, alt, ast, and alp. the combined juice (l1) provides better results as compared to the market product (m1) as it is cost effective, pure, and more reliant. the treatment of dyslipidemia with natural herbs or food sharma et  al. (2010) demonstrated the outcome of garlic extract against the poisonousness of lead nitrate in male mice because it might augment the level of alp. the results showed a substantial reduction in the level of alp in those mice that were treated with ethanolic garlic extract. similarly, the same effect of garlic was tested by d’argenio et al. (2010) as it takes the alp level to a standard value. the same pattern was found by dkhil et  al. (2011). d’argenio et  al. (2012) recently reported a nonsignificant decrease in the amount of alp with the use of garlic. with ginger intake, the level of alp decreases dramatically (shokr and mohamed, 2019). soleimani et  al. (2020) found that after intake of garlic powder, the hepatic steatosis improved significantly and there was a reduction of serum alt (57.8 ± 13.9 to 47.2 ± 16.1) and ast (48.3 ± 11.6 to 42.2 ± 11.2) concentration. alliin, allinase, and other minerals are present in garlic. alliin has properties of organosulfur compounds, which convert allicin to allinase enzyme, which has good anti-inflammatory and anti-oxidant properties. our study of alt supported by rahimlou et  al. (2016) showed that serum alt level was reduced by the ginger group (36.59 to 30.5 u/l) significantly as compared to the placebo group (34.53 to 30.82 u/l). eidi et al. (2006) investigated the effect of garlic in diabetic rats at the alt stage, which was suggestively decreased due to the use of garlic extract. d’argenio et  al. (2010) also evaluated liver functioning in rats. he gave garlic treatment, which showed a significant decrease in alt level in rats. this study confirmed the capability of garlic extract to alleviate the damage of liver and reinforce the therapeutic potential of the diallyl disulfide. dkhil et  al. (2011) highlighted the positive effect of garlic against liver dysfunction because it reduces the elevated alt level to its normal level. our result similar to that of faran et al. (2019) observed significant difference in ast values when given garlic and ginger juice. the group e and d showed significant decreased values except group b in ast jabbari et al. (2005) noticed in their study that the creatinine level was reduced due to the defensive action of garlic against nephrotoxicity. in the research of el-shenawy and hassan (2008), serum creatinine level becomes normal due to the effect of garlic. consumption of ginger regulates gluconeogenesis through proteolysis, which ultimately decreases the level of serum urea and creatinine (abd elwahab and ali, 2015). the hmg-coa reductase has the role in cholesterol formation can be decreased by garlic (kim and kim, 2011). the high quantity of vitamin c present in lemon (citrus aurantifolia l.) stimulates a-hydroxylase enzyme  7, 52 italian journal of food science, 2023; 35 (3) saeed w et al. references abd elwahab, a.h. and ali, f.i., 2015. mitigation of alloxane-induced renal damage by zingiber officinale (ginger) root in rats: an impact on oxidative stress, inflammatory cytokines and tissue damage. al-azhar assiut medical journal 13(1): 158. adegbola, p.i., fadahunsi, o.s., ajilore, b.s., akintola, a.o., & olorunnisola, o.s., 2021. combined ginger and garlic extract improves serum lipid profile, oxidative stress markers and reduced il-6 in diet induced obese rats. obesity medicine 23: 100336. https://doi.org/10.1016/j.obmed.2021.100336 ahmad, a., khan, r.a. and mesaik, m.a., 2009. anti-inflammatory effect of natural honey on bovine thrombin‐induced oxidative burst in phagocytes. phytotherapy research: an international journal devoted to pharmacological and toxicological evaluation of natural product derivatives 23(6): 801–808. https://doi.org/10.1002/ptr.2648 alizadeh-navaei, r., roozbeh, f., saravi, m., pouramir, m., jalali, f. and moghadamnia, a.a., 2008. investigation of the effect of ginger on the lipid levels. a double blind controlled clinical trial. saudi medical journal 29: 1280–1284. al-jowari, s.a.k., 2014. effect of garlic powder (allium sativum) on blood constituents in male rabbits. al-nahrain journal of science 17(3): 132–137. https://doi.org/10.22401/jnus.17.3.18 al-snafi, a.e., 2016. nutritional value and pharmacological importance of citrus species grown in iraq. iosr journal of pharmacy 6(8): 76–108. https://doi.org/10.9790/3013-0680176108 al-waili, n.s. and haq a., 2004. effect of honey on antibody production against thymus-dependent and thymus-independent antigens in primary and secondary immune responses. journal of medicinal food 7(4): 491–494. https://doi.org/10.1089/ jmf.2004.7.491 asadi-pooya, a.a., pnjehshahin, m.r. and beheshti, s., 2003. the antimycobacterial effect of honey: an in vitro study. rivista di biologia 96(3): 491–495. is more important than by medicine. further studies have to be done to address the issue of dyslipidemia at an initial stage in order to avoid the high risk of hospitalization. future recommendations during the research, we found this product offers high efficiency and high recovery rate with low cost. however, it was performed as a small pilot project. it should be expanded to a large scale so that more patients will be benefitted. in this way, we can reduce the number of dyslipidemic and hypercholesteremic patients and also create more job opportunities. ethics approval not applicable. availability of data and materials collective data are available for publishing but cannot be provided by individual authors. conflict of interest the authors declare no conflict of interest and give their consent for publishing the material. funding this research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors. figure 2. the effect of juice on tc, tgs, ldl, and hdl. tc, total cholesterol; tg, triglycerides; ldl, low-density lipoprotein; hdl, high-density lipoprotein. italian journal of food science, 2023; 35 (3) 53 anti-dyslipidemic and anti-hypercholesteremic effects of concoction juice ldl? 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activities and nano-formulations muhammad ishfaq1,2, wanying hu2, zhihua hu1, yurong guan1, and ruihong zhang1* 1college of computer science, huanggang normal university, huanggang, china; 2college of medicine, northeast agricultural university, harbin, china *corresponding author: ruihong zhang, college of computer science, huanggang normal university, huanggang 438000, china. email: jsjrh@hgnu.edu.cn received: 4 april 2022; accepted: 22 june 2022; published: 29 july 2022 © 2022 codon publications open access review article abstract ginger is a rhizome of the family zingiberaceae and is one of the most commonly used spices in food and beverages worldwide. the pharmacological activities of ginger, including antioxidant, anti-inflammatory, anticancer, and protective effects against pain and gastrointestinal tract disorders, are primarily attributed to its phenolic compounds. however, knowledge about the mechanisms of toxicity, absorption, molecular targets, and dose– response relationship of ginger in human clinical studies is still elusive. the aim of this review is to give an overview of the current literature in the context of bioactive compounds and biological activities of ginger. furthermore, recent findings regarding the absorption, tissue distribution, and nano-formulations of ginger bioactive compounds are discussed. the current in vitro and in vivo studies identified and validated ginger extracts and bioactive compounds, including gingerols, zingiberene, shogaols, and zingerone. despite the data available regarding the pharmacological uses of ginger together with a deep mechanistic approach about the pharmacokinetic, pharmacodynamic and dose-response studies in humans is yet to be provided. studies on the absorption, bioavailability, adverse reactions, and safe doses of the bioactive compounds of ginger will additionally improve its therapeutic applications. nonetheless, the use of nano-formulations of bioactive compounds of ginger will be a more effective strategy in drug delivery. these novel evidences may bring ginger to the forefront of nutraceuticals for the treatment and/or prevention of various human health disorders. keywords: absorption; bioactive compounds; biological activities; ginger; nano-formulations introduction ginger (scientific name: zingiber officinale roscoe) is a perennial and yellow flowering plant, belonging to the zingiberaceae family. ginger plant has tuberous roots, named as rhizomes, which have pungent taste and aromatic odor. ginger is primarily used for culinary purposes as a flavoring agent mostly in dried, fresh, powdered, and preserved food and/or pickles (peng et al., 2017). more importantly, ginger has received much attention from researchers because of its therapeutic potential to modulate the activities of several enzymes. ginger is also used in traditional medicines such as unani system, ayurvedic and chinese medicines since ancient times to treat different ailments, including sprains and muscular aches, rheumatoid arthritis, nausea, sore throat, indigestion and constipation, fever, infectious diseases, helminthiasis and cancer (ali et al., 2008). nowadays, ginger plant is grown in tropical regions such as africa, asia, europe and america, and has gained an increasing interest because of its medicinal properties (srinivasan, 2017) and research, including identification and isolation of its bioactive constituents (as shown in figure 1) and its pharmacological, phytochemical and toxicological properties. mailto:jsjrh@hgnu.edu.cn 2 italian journal of food science, 2022; 34 (3) ishfaq m et al. following reasons: (1) suppressing of lipid peroxidation; (2) inhibition of enzymes of arachidonate metabolism: 5-lipoxygenase and 2-cyclooxygenase enzymes; (3) free radical scavenging; (4) stimulating the activities of endogenous antioxidant enzymes; (5) enhancing antioxidant molecules in tissues; (6) inhibition of the activity of inducible nitric oxide synthase and (7) inhibition of low-density lipoprotein (ldl) oxidation (srinivasan 2014). similarly, ginger has been reported to possess antioxidant activities, and has demonstrated a crucial role in scavenging superoxide anion and hydroxyl radicals (ji et al., 2017; mao et al., 2019). it has been analyzed that the trend varies with the content of phenolics found in ginger. for example, dried ginger has higher phenolic contents and indicates stronger antioxidant activities compared to stir-fried ginger and its other forms (dried ginger > stir-fried ginger > carbonized ginger > fresh ginger). on heating, the antioxidant activity decreases due to the conversion of gingerols into shogaols (li et al., 2016). the type of solvents used for extraction could have an effect on the phenolic contents, and subsequently affect the antioxidant activity of ginger (yeh et al., 2014). for instance, an aqueous extract and an ethyl acetate extract had higher antioxidant potential than diethyl ether, nbutanol and ethanol extracts (nile and park, 2015). the underlying mechanism of ginger’s antioxidant potential were investigated, and it was found that ginger extract reduced the generation of ros and lipid peroxidation and stimulated the expression of several antioxidant enzymes (hosseinzadeh et al., 2017). in addition, ginger extract activated the nuclear factor erythroid 2-related factor 2 (nrf2) signaling pathway and its downstream genes, which, in turn, provide defense against oxidative stress and free radicals (peng et al., 2015). the bioactive compound of ginger, 6-shogaol, up-regulated nrf2 target genes and enhanced intracellular glutathione/glutathione bioactive compounds in ginger chemical analysis of ginger revealed that if contains more than 400 compounds. the major constituents of ginger are lipids (3–8%), carbohydrates (50–70%), phenolic compounds and terpenes (grzanna et al., 2005). the nutraceutical properties of ginger are attributed to its bioactive constituents, primarily, phenolic compounds and terpenes. some of these compounds belonging to classes such as shogaols, gingerols, paradols, zingerone and zigiberenes have been reported to possess potential to modulate biological activities. shogaol (18–25%) and gingerols (23–25%) are found in higher quantities compared to other compounds. besides these, water, raw fiber, protein, ash, phytosterols, minerals and some vitamins are also present (ali et al., 2008). gingerols account for the taste of ginger. however, shogaols are formed at high temperature, for instance, during cooking, and provide spicy–sweet aroma. these active compounds have many biological activities through the modulation of enzyme activities in living systems (shukla and singh, 2007; srinivasan, 2017). therefore, in this review, the pharmacological activities (as shown in figure 2) associated with the bioactive compounds, their absorption and distribution, with a special focus on enhancing its bioavailability through nano-formulations are discussed. biological activities of ginger antioxidant activities studies have reported that reactive oxygen species (ros) play a crucial role in the growth of different chronic diseases (poprac et al., 2017). the antioxidant activity of spices could be due to one or more of the 1-dehydro-10-gingerdione 1-dehydro-10-gingerdione 6-paradol 6-gingerol 6-shogaol gingerenone-a gingerdione zingerone ginger figure 1. ginger rhizome and chemical structure of some of its bioactive compounds. italian journal of food science, 2022; 34 (3) 3 nutritional implications of bioactive compounds of ginger anti-inflammatory activities antioxidant activities cardiovascular activities gastroprotective activities anti-cancer activities anti-obesity activities anti-diabetic activities ginger figure 2. schematic diagram showing multiple biological activities exhibited by ginger. disulfide (gsh/gssg), metallothionein 1 (mt1), heme oxygenase-1 (ho-1), aldo-keto reductase family 1 member b10 (akr1b10), γ-glutamyl transferase-like activity 4 (ggtla4), glutamate-cysteine ligase catalytic subunit (gclc), glutamate-cysteine ligase modifier subunit (gclm) and ferritin light chain (ftl) (chen et al., 2014). 6-shogaol, 6-dehydroshogaol and 1-dehydro-6-gingerdione inhibited the synthesis of nitric oxide (no) in macrophages (li et al., 2011). a previous study demonstrated that gingerol reduced the peroxidation of phospholipid liposomes in the presence of ascorbate and ferric ions (fe3+) (aeschbach et al., 1994). another study reported the antioxidant effects of ginger in cerebellum, cerebral cortex, hypothalamus and hippocampus in diabetic mice (shanmugam et al., 2011). it is worthy to mention here that there are several factors, such as individual differences, health conditions, other dietary factors, the lifestyles of people, solubility, route, dosage, and solubility, that could affect the bioavailability and bioaccessibility of antioxidants, which are probably associated with the efficacy of antioxidants in the real world. digestive stimulant and gastroprotective activities ginger exerts potential protective effects on the gastrointestinal membrane and lowers the chances of mucosal injury (prakash and srinivasan, 2010a). it has been reported that dietary ginger altered the permeability and fluidity of the intestinal membrane, resulting in increased absorptive surface of the small intestine with an increase in microvilli length (prakash and srinivasan, 2010b). it was demonstrated that dietary ginger (0.05%) fed for 8 weeks significantly altered the ultrastructural morphology in wistar mice. besides, ginger enhanced the activity of enzymes such as leucine aminopeptidase, g-glutamyl transpeptidase and glycyl-glycine dipeptidase, accompanied by a decrease in the ratio of cholesterol and phospholipid in the small intestinal mucosa. ginger also increased the absorption of zinc, iron, β-carotene and calcium in the intestines (prakash and srinivasan, 2013). in another study, higher intestinal absorption of micronutrients has been noted in ginger-fed animals. this may be presumably associated with alteration in permeation characteristics, including increased absorptive surface and antioxidant status in the intestines (veda and srinivasan, 2011). despite providing increased surface area for absorption, ginger acts as a sialagogue, stimulates the production of saliva and thus enhances the swallowing process (platel and srinivasan, 2004). ginger stimulated bile acid secretion from the liver (bhat et al., 1985), leading to the absorption and digestion of dietary fat. dietary ginger significantly increased the activity of several digestive enzymes, for example, amylase, proteases (chymotrypsin, trypsin and carboxy peptidase) and pancreas’ lipase (platel and srinivasan, 2000). enzymes such as disaccharides are also stimulated by ginger, and food transit time is reduced due to facilitated digestion and increased intestinal motility (platel and srinivasan, 1996). among spices, ginger ranks at the top level based on its pharmacological evidence (platel and srinivasan, 2004), and is used for alleviating the gastrointestinal tract (git) disorders in traditional medicines (afzal et al., 2001). moreover, ginger’s free phenolic and hydrolyzed phenolic fractions inhibited growth of helicobacter pylori and gastric cell proton potassium atpase activity. it has been suggested that these two steps are associated with the inhibition of ulcers (siddaraju and dharmesh, 2007). anti-inflammatory activities currently, gastric ulcer and adverse effects have been observed due to extensive use of nonsteroidal anti inflammatory drugs. in general, the anti-inflammatory mechanism of ginger and its active compounds has been probably associated with the suppression of protein kinase b (akt) and nuclear factor kappa b (nf-κb) 4 italian journal of food science, 2022; 34 (3) ishfaq m et al. 6-gingerol and 6-shogaol enhanced the catabolism of cellular fatty acid through activation of peroxisome proliferator-activated receptor δ (pparδ)-dependent gene expression (misawa et al., 2015). in high-fat diet mice, both orlistat and ginger decreased lipid profile and body weight. however, ginger proved effective than orlistat by reducing the level of high-density lipoprotein cholesterol (hdl-c) (mahmoud and elnour, 2013). in addition, in a double blind, randomized and placebo-controlled study, daily intake of 2 g of ginger powder decreased body mass index (bmi) in obese women (ebrahimzadeh attari et al., 2016). a study reported that ginger oleoresin interfered with cholesterol absorption (gujral et al., 1978). it was demonstrated that fecal cholesterol increased and liver and serum cholesterol levels decreased compared to control group in mice fed with 1% cholesterol diet along with 0.5% ginger oleoresin for 20 days (gujral et al., 1978). in hypercholesterolemic mice, aqueous ginger infusion significantly reduced serum total cholesterol, triglycerides and ldl-cholesterol (elrokh el et al., 2010). however, dietary ginger (0.04% dry powder) did not reduce cholesterol in hypercholesterolemic mice (sambaiah and srinivasan, 1991). it is important to observe that dietary ginger stimulated the absorption and digestion of dietary fat by enhancing the activity of pancreatic lipase and secretion of bile salts (prakash and srinivasan, 2012). it has been observed that gingerol significantly reduced glucose and insulin resistance, body weight gain, expression of inflammatory markers and activity of enzymes of cholesterol biosynthesis, which results in the prevention of high-fat diet (hfd)-induced hyperlipidemia through modulating the expression of enzymes associated with cholesterol homeostasis (brahma naidu et al., 2016). thus, ginger helps in management of weight through suppression of body cholesterol and reducing accumulation of lipids. anti-diabetic activity diabetes mellitus caused by insulin deficiency is a severe metabolic disorder that results in hyperglycemia. numerous studies have evaluated the antidiabetic potential of ginger and its bioactive compounds. in an in vitro study, 6-gingerol and 6-shogaol prevented diabetic complications by inhibiting advanced glycation end products (ages) by trapping methylglyoxal (mgo) (zhu et al., 2015). in addition, 6-gingerol decreased the level of plasma glucose and insulin in mice through nrf2 activation (sampath et al., 2017). in c2c12 myotubes and 3t3-l1 adipocytes, 6-shogaol and 6-paradol promoted glucose utilization via ampk phosphorylation. besides, 6-paradol decreased the level of glucose in the blood of mice fed with high-fat diet (wei et al., 2017). another study reported that 6-gingerol pathway, up-regulation of anti-inflammatory cytokines and inhibition of pro-inflammatory cytokines. a previous study demonstrated that ginger suppressed pro inflammatory cytokines such as tumor necrosis factor-α (tnf-α), interlukin (il)-1 and il-8 (pan et al., 2008). another study established that ginger oil (33 mg/kg, orally) for 26 days significantly alleviated joint swelling in mice during chronic adjuvant arthritis (sharma et al., 1994). ginger extract administered at a dose of 100 mg/kg body weight suppressed the elevated expression tnf-α in mice (habib et al., 2008). besides, ginger inhibited both cyclooxygenase and 5-lipoygenase in vitro and in vivo (kiuchi et al., 1992; mustafa et al., 1993), and reduced the expression of inflammatory genes (ho et al., 2013; tripathi et al., 2008) through the suppression of nf-κb gene expression (kim et al., 2004; oyagbemi et al., 2010; takada et al., 2005). in addition, 6-shogaol suppressed claudin-1 and claudin-2 through pi3k/akt and nf-κb pathway and inhibited tnf-α-induced intestinal barrier dysfunction in human cell models (luettig et al., 2016). it has been noted that 6-dehydroshogaol proved to be more effective than 6-gingerol and 6-shogaol in reducing prostaglandin e2 (pge2) and nitric oxide (no) in mouse macrophage raw 264.7 cell line (zhang et al., 2013). moreover, 6-gingerol decreased the expression of inflammatory markers such as no, tnf-α and myeloperoxidase in the ovaries, uterus and brain of mice treated with chlorpyrifos (abolaji et al., 2017). capsules containing ginger powder (500 mg) alleviated the increased levels of plasma il-1β, il-6 and tnf-α after running exercise in 28 male endurance runners (zehsaz et al., 2014). the anti-inflammatory effects of ginger and its bioactive constituents, such as zingerone, 6-, 8-, 10gingerol and 6-shogaol proved effective against lipopolysaccharide (lps)-induced inflammation in vivo and in vitro (hsiang et al., 2015). these studies revealed that ginger and its bioactive compounds act as a broad spectrum anti-inflammatory agents through suppression of inflammatory cells infiltrates, nf-κb, il-1β and other proinflammatory cytokines. anti-obesity activity previous research has reported that obesity is a risk factor for cardiovascular diseases, hypertension and diabetes (misawa et al., 2015). recently, ginger has gained an increasing interest in the prevention and management of obesity (ebrahimzadeh attari et al., 2018). in 3t3-l1 preadipocyte cells, gingerenone a attenuated diet-induced obesity through modulation of fatty acid metabolism via up-regulation of 5’ amp-activated protein kinase (ampk) in vivo, and exhibited an effective inhibitory effect on lipid accumulation and adipogenesis compared to 6-shogaol and gingerols (ebrahimzadeh attari et al., 2018). in cultured skeletal muscle myotubes, italian journal of food science, 2022; 34 (3) 5 nutritional implications of bioactive compounds of ginger hdl (oh et al., 2017). the hypercholesterolemic properties of ginger attribute to its cardioprotective function. in wistar mice, ginger extract prevented isoproterenol induced myocardial infarction (amran et al., 2015). pretreatment with ginger extract at a dose of 400 mg/ kg for 4 weeks significantly reduced cardiac markers, including enzyme activities of creatine kinase-mb isoenzyme, infarction–troponin, alanine transaminase, aspartate transaminase and lactate dehydrogenase. in addition, significant increase was found in cardiac antioxidant enzymes and improvement in cell membrane integrity in ginger-pretreated mice (srinivasan, 2017). aqueous ginger extract at a low dose (50 mg/kg) administered orally or intraperitoneally did not cause significant reduction in platelet thromboxane-b2 (tbx2) level. however, significant changes were observed in serum prostaglandin-e2 (pge2) levels when ginger extract was given orally. these results provided an insight regarding the anti-thrombotic potential of ginger (thomson et al., 2002). in anesthetized mice, crude extract of ginger (0.3–3 mg/kg) induced a dose-dependent decrease in arterial blood pressure. crude extract depicted a cardiodepressant activity on the force and rate of spontaneous contractions in guinea pig paired atria. in addition, crude extract of ginger at a dose 10 times higher than required against k-induced contraction resulted in the relaxation of phenylephrine-induced vascular contraction in rabbits. these results demonstrated that ginger lowered blood pressure through the blockage of voltage dependent calcium channels (ghayur and gilani, 2005). another study reported that aqueous ginger extract mediated blood pressure lowering effect by inhibition of both ca2+ channels and muscarinic receptors (ghayur et  al., 2005). it has been observed that cardiovascular protective effects of ginger are attributed to its potential to alleviate hypertension and dyslipidemia. anti-cancer activity recently, increasing number of studies have focused on the anticancer effects of ginger and its bioactive compounds in different cancer cell lines, including cervical, breast, leukemia, colorectal, lung, liver, nasopharyngeal, prostate, retinoblastoma and ovarian cell lines. in the breast cancer cell line mda-mb-231, 86.7-μg/ml and 57.5-μg/ml methanolic solution of ginger given for 24 h and 48 h, respectively, exhibited cytotoxic effect in a time-dependent manner (ansari et al., 2016). in mcf-7 cell line, z-6-oxo-6-shogaol and z-6-oxo-8-shogaol treatment with half-maximum inhibition concentration (ic50) values of 6.27-μm and 47.22-μm for 48 h demonstrated significant cytotoxic action (li et al., 2018). in addition, 6-shogaol inhibited growth of both breast and alleviated glucose tolerance through activation of glucagon-like peptide-1 (glp-1) in type 2 diabetic mice and facilitated glucose-mediated secretion of insulin. it has been found that 6-gingerol enhanced cell membrane presentation of glucose transporter type 4 (glut4) and activated glycogen synthase 1, resulting in enhancing storage of glycogen in skeletal muscles (samad et al., 2017). moreover, consumption of ginger could decrease the levels of glycated hemoglobin a (hba1c), plasma glucose, insulin, total cholesterol (tc) and triglyceride (tg) in patients with type 2 diabetes mellitus (arablou et al., 2014). in mice, ginger extract promoted insulin sensitivity during metabolic syndrome, which might be associated with improvement in energy metabolism by the active component, 6-gingerol (li et al., 2014). apart from direct protective effects on diabetes mellitus, ginger demonstrated protective effects against diabetes mellitus induced secondary complications of the kidney, eye, liver and neural system (li et al., 2012). studies have established that ginger and its bioactive compounds possess potential therapeutic properties that could protect from diabetes mellitus. the main mechanism of action could be one of the following: (1) ginger maintains blood glucose homeostasis by promoting glucose uptake in insulin responsive peripheral tissues, (2) ginger enhances insulin sensitivity and release and/or (3) it improves lipid metabolism and inhibits enzymes in carbohydrate metabolism. these mechanisms provide evidence for the future studies to evaluate these steps in the clinical investigations of ginger and its bioactive compounds for treating diabetes mellitus. cardiovascular and blood protective activities ginger has the potential to treat cardiovascular diseases and has preventive effects on the blood. it is well documented that high blood cholesterol is primarily associated with cardiovascular diseases. studies reported that cardiovascular diseases cause 17.9 million premature deaths annually (du et al., 2016). hypertension and dyslipidemia are well known risk factors for stroke, coronary heart disease and cardiovascular diseases (khosravani et al., 2016). several studies have investigated the cardiovascular protective properties of ginger and indicated that ginger treatment primarily decreased blood pressure and level of blood lipids (natalia et al., 2017). in high-fat diet-fed mice, ginger extract increased the level of serum hdl-c and reduced body weight, thus protecting against coronary heart disease. besides, the levels of lecithin-cholesterol acyltransferase and apolipoprotein a-1 mrna were increased in the liver, associated with the formation of 6 italian journal of food science, 2022; 34 (3) ishfaq m et al. hippocampi in mice by the activation of cyclic adenosine monophosphate (camp) response element-binding protein (creb) and nerve growth factor (ngf) (lim et  al., 2014). moreover, ginger alleviated narcotics-induced neurotoxicity and memory deficits in the hippocampus through modulation of cholinergic activity and memory retrieval ability (gomar et al., 2014). a previous study demonstrated that ginger extract effectively alleviated epilepsy-like symptoms. for example, pentylenetetrazole (ptz)-induced incidence of seizures and its duration was reduced by ginger therapy (hosseini and mirazi, 2014). overall, ginger and its compounds alleviated epileptic symptoms, including involuntary movement and recurrent seizures; these findings provided evidence for the application of ginger against epileptic patients. ginger also demonstrated antiallergic, nephroprotective and hepatoprotective effects. a study conducted by rodrigues et al. (2014) demonstrated that gingerol alleviated gentamicin-induced kidney damage by reducing stress and lipid peroxidation and increased superoxide dismutase (sod) activity and levels of glutathione (gsh) in a dose-dependent manner. similarly, ginger extract attenuated biochemical and histological alterations through anti-inflammatory and antioxidant activities in a radiation-induced kidney model of mice (saberi et al., 2017). the above-mentioned discussion established that ginger and its bioactive compounds played a crucial role in treating various diseases and, therefore, must be evaluated additionally for their nutraceutical properties. absorption and tissue distribution of ginger compounds information about the pharmacokinetics and bioavailability profile of an active substance is very important for understanding its nutraceutical properties. a previous study investigated the distribution profile of 6-gingerol in the blood and other tissues of mice. it was observed that this component was absorbed quickly if given orally at a dose of 240 mg/kg (ginger extract having 53% 6-gingerol). the maximum concentration found in the plasma was 4.24 mg/ml after 10 min that declined in a biexponential pattern with time. 6-gingerol is distributed to the heart, brain, spleen, lung, kidney, liver, small intestine and stomach. the highest concentration was found in the git, and the elimination half-life was 1.77 h (jiang et al., 2008). a clinical trial studied the pharmacokinetics of the following ginger constituents: 6-shogaol, 6-gingerol, 8-gingerol and 10-gingerol. in human volunteers, ginger was given orally at a dose of 100–2.0 g. the ginger constituents were absorbed and detected as sulfate and glucuronide conjugates in blood samples after 15 min to colon cancer cells through up-regulation of peroxisome proliferator-activated receptor gamma (pparγ) (tan et al., 2013), and 10-gingerol inhibited orthotopic tumor growth by inducing apoptosis through caspase-3 in breast cancer and suppressed metastasis to lung, brain and bone cancers (martin et al., 2017). researchers demonstrated that ginger and its bioactive compounds were found effective against prostate cancer cell lines such as lncap, du145, c4-2, pc-3 and c4-2b. 10-gingerol with ic50 value of 59.7-μm was more effective than 6-shogaol with ic50 value of 100.0 μm against pc-3 cells (peng et al., 2012). in lncap human prostate cancer cell lines, 6-gingerol induced apoptosis via caspase-3 in timeand dose dependent manner and degraded poly(adp-ribose) polymerase (parp) (kim et al., 2011). in h-1299 lung cancer cells, 6-gingerol demonstrated cytotoxic effect with ic50 value of 136.73-μm for 24 h (lv et al., 2012), and initiated autophagy by inhibiting the akt/mtor pathway (hung et al., 2009). in liver cancer, ginger extract suppressed nf-κb and tnf-α in mice (habib et al., 2008). additionally, z-6-oxo-8-shogaol, z-6-oxo-6-shogaol and e-4-isoshogaol revealed cytotoxic effect against hepg2 cell lines (van breemen et al., 2011). in hl-60 and k562 leukemic cell lines, 6-shogaol (with ic50 values of 24.2 μm and 7.9 μm) proved more effective than 10-gingerol (mahomoodally et al., 2021). in another study, 6-shogaol induced apoptosis and transformed cellular leukemia through eif2α dephosphorylation (liu et al., 2013). it has been noted that 6-gingerol induced apoptosis in colorectal cancer cells by up-regulating g1 and nag-1 cell cycle arrest through down-regulation of cyclin d1 (lee et al., 2008). another study confirmed that p53/p21 contributed to cell cycle arrest by g2/m check point induced by 6-shogaol (radhakrishnan et al., 2014). besides, 6-shogaol induced apoptosis primarily through mitochondrial pathway and bcl-2 family proteins (qi et al., 2015). however, additional studies are required to elucidate the complete molecular mechanisms associated with the anticancer effects of ginger. other beneficial effects in addition to the above-mentioned bioactivities, ginger and its bioactive compounds have several other multiple health benefits. recently, a study has reported that ginger demonstrated several positive effects on neuroinflammatory disorders and memory function (huh et al., 2018). 10-gingerol inhibited neuroinflammation through the suppression of nf-κb, no, il-1β, tnf-α and il-6 in lps-stimulated bv2 microglia culture model (ho et al., 2013). ginger extract alleviated scopolamine-induced memory deficits in mice. in addition, ginger extract promoted the formation of synapses in c6 glioma cells and italian journal of food science, 2022; 34 (3) 7 nutritional implications of bioactive compounds of ginger (behroozeh et al., 2018). similarly, 6-shogaol-made liposomes in a 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol-sodium (dmpg-na) carrier exhibited anticancer action both in vitro and in vivo. it was observed that 6-shogaol was released slower from liposomes compared to ginger oleoresin (ahmad et al., 2015). in a recent in vitro study, drug delivery system was designed with ginger nanoparticles and chitosan for controlled release of 5-amino salicylic acid (5-asa) to treat inflammatory bowel disease. it was concluded that design system for the controlled release of 5-asa was appropriate and favorable at gastrointestinal ph and beneficial in treating inflammatory bowel disease (markam and bajpai, 2020). however, additional studies are required to investigate and develop ginger as an effective candidate of nanomedicines for treating different cancers and neurodegenerative diseases. conclusion and future trends this review demonstrated the biological activities of ginger and its bioactive constituents in a number of diseases, with particular interest for having digestive stimulation and antioxidant potential, and anti-inflammatory, antiobesity, antidiabetic, cardiovascular and blood protective, and anticancer activities. the underlying mechanisms of ginger and its bioactive compounds were discussed from the context of modulating various cellular enzymes, including cytochrome p450, cyclooxygenases, lipoxygenases, nrf2, nf-κb and several other pathways considering apoptotic and cell cycle check points. in addition, available information about the absorption and tissue distribution of ginger and its bioactive compounds was highlighted. the use of ginger and its bioactive compounds in nanomedicine, especially nano-formulations, was viewed in the light of available literature. the future studies should provide a complete understanding of the interactions of ginger and its bioactive compounds. however, lack of knowledge makes it difficult to use ginger in human clinical cases. although nanotechnology provides effective delivery system for ginger and its constituents, more pharmacokinetic and pharmacodynamics trials could provide in-depth information regarding the bioavailability and molecular pathways associated with these nano-formulations. omic studies such as proteomics, transcriptomics and metabolomics are recommended to dig out the possible adverse reactions and molecular targets of ginger and its compounds. there is still a challenge for researchers to evaluate ginger and its constituents as nutraceuticals because of the complex systems that involve multiple signaling pathways, choice of route administration, dose optimization, intensity, and treatment duration. notably, the efficacy of ginger and its active constituents against 72 h. importantly, no free 6-shogaol, 6-, 8-, or 10gingerol was detected and the calculated half-lives of these compounds was less than 2 h (zick et al., 2008). another clinical trial reported the pharmacokinetics of 6-shogaol, 6-, 8-, and 10-gingerols in colon tissues and plasma. free forms of 6-shogaol and 10-gingerol were observed in plasma after oral intake, with a maximum level reaching in 1 h. however, no free forms of 6-gingerol and 8-gingerol were detected in plasma. the half-lives of these ginger constituents in plasma ranged from 1 to 3  h. traces of 10-gingerol sulfate and glucuronide conjugate were detected in colon tissue, while traces of free forms of 6-shogaol and 10-gingerol, as well as glucuronide metabolites of 6-shogaol and 6-, 8-, and 10-gingerol, were detected with a more sensitive technique 1 h after oral intake (yu et al., 2011). in experimental mice, free 6-shogaol, 8-, and 10-gingerols were discovered in the plasma after single oral dose of ginger oleoresin (300 mg/ kg) whereas 6-gingerol administered orally at a rate of 50  mg/kg was excreted in bile as glucuronide conjugate over 60 h (wang et al., 2009). in addition, 6-shogaol (78.5%) was excreted in bile over 48 h after oral intake (asami et al., 2010). nano-formulations recently, owing to certain beneficial effects in treating diseases, nano-formulations in nanomedicine have received increasing interest from researchers (kumar et al., 2017). nanomedicine has certain benefits in drug delivery system, for the drug is delivered at correct sites, thereby making drug absorption more successful (aneja et al., 2014). significant research is in progress in the area to study its pharmacokinetics, mechanisms and delivery along with exploring of new nanocomposites, such as hybrid polymer-metal composites or grapheme, for drug delivery (rahman et al., 2015). rahman et al. (2017) reviewed the therapeutic applications and benefits of nano-formulations in the context of liposomal-based drug delivery. the study demonstrated that pegylated nanoliposomal formulation of gingerol allowed a slower release of drug and enhanced its cytotoxic effect in breast cancer mcf-7 cells compared to standard gingerol and liposomal gingerol. pegylation has the advantage of controlled drug release and ensures longer blood circulation to reach the target site by protecting liposomes against lipase enzymes and immune system (khalili et al., 2013). another research established that pegylated nanoliposomal gingerol proved more effective than the standard drug. the release of gingerol from nanoparticles enclosed in pegylated nanoliposomal formulation (76% gingerol) was fast and instant at the start and then became slower 8 italian journal of food science, 2022; 34 (3) ishfaq m et al. drug metabol drug interact. 18(3–4):159–190. https://doi. org/10.1515/dmdi.2001.18.3-4.159 ahmad 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https:// doi.org/10.2174/1574891x10666150415120506 ansari j.a., ahmad m.k., khan a.r., fatima n., khan h.j., rastogi n., mishra d.p. and mahdi a.a. 2016. anticancer and antioxidant activity of zingiber officinale roscoe rhizome. indian j exp biol. 54(11):767–773. https://doi.org/10.9734/bjpr/2016/26198 arablou t., aryaeian n., valizadeh m., sharifi f., hosseini a.f. and djalali m. 2014. the effect of ginger consumption on glycemic status, lipid profile and some inflammatory markers in patients with type 2 diabetes mellitus. int j food sci nutr. 65(4):515–520. https://doi.org/10.3109/09637486.2014.880671 asami a., shimada t., mizuhara y., asano t., takeda s., aburada t., miyamoto k. and aburada m. 2010. pharmacokinetics of [6]-shogaol, a pungent ingredient of zingiber officinale roscoe (part i). j nat med. 64(3):281–287. https://doi.org/10.1007/ s11418-010-0404-y behroozeh a., mazloumi tabrizi m., kazemi s.m., choupani e., kabiri n., ilbeigi d., heidari nasab a., akbarzadeh khiyavi  a. and seif kurdi a. 2018. evaluation the anti-cancer effect of pegylated nano-niosomal gingerol, on breast cancer cell lines (t47d), in vitro. asian pac j cancer prev. 19(3):645–648. bhat b.g., sambaiah k. and chandrasekhara n. 1985. the effect of feeding fenugreek and ginger on bile composition in the albino rat. nutr rep int. 32(5):1145–1151. brahma naidu p., uddandrao v.v., ravindar naik r., suresh p., meriga b., begum m.s., pandiyan r. and saravanan g. 2016. ameliorative potential of gingerol: promising modulation of inflammatory factors and lipid marker enzymes expressions in hfd induced obesity in rats. mol cell endocrinol 419:139–147. https://doi.org/10.1016/j.mce.2015.10.007 chen h., fu j., chen h., hu y., soroka d.n., prigge j.r., schmidt e.e., yan f., major m.b., chen x. and sang s. 2014. ginger compound [6]-shogaol and its cysteine-conjugated metabolite (m2) activate nrf2 in colon epithelial cells in vitro and in vivo. chem res toxicol. 27(9):1575–1585. https://doi.org/10.1021/tx500211x du h., li l., bennett d., guo y., key t.j., bian z., sherliker p., gao  h., chen y., yang l., chen j., wang s., du r., su h., diseases requires more well-designed human clinical trials. gas chromatography and high-performance liquid chromatography fingerprinting could provide valuable insights into the nutraceutical uses, and more bioactive compounds of ginger could be identified and isolated. nevertheless, we established in this review the potential uses of ginger as a safe herbal medicine and food ingredient for managing various diseases; still this spice deserves to be considered for additional investigations to enhance its bioavailability and controlled release via advance technologies, including nano-formulations. declaration of competing interest none. acknowledgments the work was supported in 2021 by the natural science foundation of hubei province of china (project name: research on brain tumor diagnosis based on capsule neural network), and 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https://doi.org/10.3390/ijms18010168� _hlk37838113 _hlk99541523 ole_link14 ole_link16 ole_link15 ole_link17 _hlk38970032 _hlk95556063 paper ital. j. food sci., vol. 27 2015 351 keywords: cooking, egg, florfenicol, florfenicol amine, residue, storage the effect of cooking and storage on florfenicol and florfenicol amine residues in eggs ayhan filazi1*, ufuk tansel sireli2, begum yurdakok dikmen1, farah gonul aydin1 and asli gul kucukosmanoglu1 1department of pharmacology and toxicology, faculty of veterinary medicine, ankara university, 06110 diskapi, ankara, turkey 2department of food hygiene and technology, faculty of veterinary medicine, ankara university, 06110 diskapi, ankara, turkey *corresponding author: filazi@veterinary.ankara.edu.tr abstract the aim of this study was to evaluate the effects of storage conditions (room temperature, refrigerator) and cooking methods (frying, boiling) on florfenicol (ff) and florfenicol amine (ffa) residue levels in eggs. without any significant difference between storage conditions at 20˚c and +4˚c, residue levels decreased within days, but were still present on day 28. frying and boiling for 1 and 5 min yielded similar results to the storage conditions just described; there was a significant decrease in residue levels, but still not enough for decomposing. these findings indicate that ff and ffa residues are heat-labile. mailto:filazi%40veterinary.ankara.edu.tr?subject= 352 ital. j. food sci., vol. 27 2015 introduction when veterinary drugs are administered to farm animals, either therapeutically or to promote growth, residues remain in their meat, milk or eggs if proper precautions are not followed (botsoglou and fletouris, 2001). antibiotics play an important role among such drugs. in addition to their positive effects, they can also cause health problems, including drug hypersensitivity (paige et al., 1999; donoghue, 2003). antibiotics not only threaten food safety, but also cause the development of some resistant bacterial strains from among sensitive bacteria even when used at moderate doses for long periods of time (paige et al., 1999; filazi et al., 2005). testing for drug residues are ordinarily performed on raw products. almost no edible animal products or byproducts are consumed raw, but require some type of processing or cooking, such as frying, boiling, or roasting, before consumption. these processes can cause denaturation of proteins, elevation of temperature, loss of water and fat, and ph variations that can eventually result in alteration to the concentration, chemical nature, chemical reactions, and solubility of drug residues in a particular food item. many drugs are chemically unstable to varying degrees, and therefore may undergo degradation during storage, cooking or processing in consumable foods (botsoglou and fletouris, 2001). in general, the temperatures achieved during cooking are assumed to degrade antibiotic residues in food; however, ordinary cooking procedures are unreliable for degrading or inactivating several commonly used veterinary drugs. earlier studies have indicated that sulfamethazine (rose et al., 1995; papapanagiotou et al., 2005) chloramphenicol (botsoglou and fletouris, 2001), streptomycin (inglis and katz, 1978; o’brien et al., 1980), neomycin (katz and levin, 1978), gentamicin (sireli et al., 2006), fluoroquinolones (baydan et al., 2000a, b; baydan et al., 2002), penicillin g (nouws and ziv, 1976; boison et al., 1992), nitrofurantoin (cooper and kennedy, 2007), oxacillin, clindamycin, novobiocin, trimethoprim, vancomycin, and azlocillin are heat-stable (traub and leonhard, 1995), whereas oxytetracycline (kitts et al., 1992) and amphenicols (franje et al., 2010) heat-labile. on the other hand, several β-lactams including ampicillin and amoxicillin are partially heat-labile (traub and leonhard, 1995). antibiotics of the same class were reported to vary in heat stability according to the type of matrix and heating treatment involved (kitts et al., 1992; rose et al., 1996; franje et al., 2010). as such, the effect of different matrices on the stability of every veterinary drug should be investigated. although most edible animal products are consumed after cooking or some type of processing, for the licensing of veterinary drugs research concerning the effects of storage and cooking of the drugs on different matrices are lacking. most data on drug residues in edible animal products and government regulation concern raw products. it is therefore essential to determine the effect of processing on all veterinary drugs when assessing human exposure to drug residues in animal food products (ibrahim and moats, 1994; moats, 1988; moats 1999; botsoglou and fletouris, 2001). florfenicol (ff) is a wide-spectrum, synthetic antibacterial that is structurally related to d(−) threo-chloramphenicol; however, ff differs from chloramphenicol in that ff contains a p-methyl sulfonyl group instead of a p-nitro group and it contains a fluorine atom instead of a hydroxyl group in the terminal primary alcohol group (emea, 1999). ff has not been approved for use in laying hens; however it is used in cattle, swine, poultry, and fish (emea, 2000). ff is metabolized into florfenicol amine (ffa), florfenicol oxamic acid, florfenicol alcohol, and mono-chloroflorfenicol in animals. ffa is the longest-lived metabolite in the bovine liver; therefore, ffa can be used as a marker for the calculation of withdrawal time (anadon et al., 2008; xie et al., 2011). in light of the apparent advantages over chloramfenicol and its availability as an additive, the potential for off label use of ff is high. due to its broad spectrum antibacterial activity, ready availability and low cost, it remains a possibility that ff residues will continue to be found in such animal food products as eggs. for example, xie et al. (2011) analyzed 50 egg samples obtained from a local supermarket in china and reported 19 ppb of ff and 36 ppb of ffa in only 1 egg. filazi et al. (2014) reported that the concentration of ff and ffa in eggs were 0.1%, 0.08% respectively regardless of the route of administration. data on the heat stability of ff is essential for food safety; however, the literature contains few data regarding its heat-stability during cooking. under environmental conditions ff is stable at 25 °c, yet photodegradation occurs at varying rates in water under various lighting conditions (ge et al, 2009). ff was shown to rapidly degrade to ffa in the deep sediment of marine environments via biodegradation (hektoen et al, 1995). a few studies on the residue of ff and ffa in eggs have been published (xie et al. 2011; filazi et al., 2014); but the data are insufficient. franje et al. (2010) reported that amphenicols exhibit differential behavior in terms of heatinduced degradation in solutions and protein matrices. although the level of amphenicol degradation in soybean sauce and meat was high, heating may generate product with antimicrobial activity; therefore, heating amphenicol residues in food cannot always be considered safe. ff is the most commonly used veterinary antimicrobial agent in turkey, particularly so due to its illegally use in laying hens. nonetheless, ital. j. food sci., vol. 27 2015 353 few studies have examined ff residue levels in eggs. an earlier study reported that ff and ffa were detected in the eggs of hens administered with ff (filazi et al., 2014) chicken eggs are widely used in the preparation of many types of food, including many baked goods. some of the most common preparation methods include fried in oil, hard-boiled, softboiled and omelets. data regarding ff and its main metabolites in cooked and stored eggs are lacking. as such, the aim of the present study was to determine the effects of different storage conditions (room temperature and refrigeration) on ff residue levels in eggs stored up to 28 days and to determine the effect the different cooking methods (frying and boiling) on ff and ffa residue levels. materials and methods animals the study protocol was approved by the ankara university ethics committee (2007-15-45). the study included 50 isa brown laying hens aged 48 weeks and weighing 1.9-2.4 kg. the hens were housed individually in fiber cages (30x35x45 cm), in a ventilated room maintained at 20°c under 14 h day light condition. the hens received standard commercial layer mash (120 g/d) and water ad libitum. the hens were fed for 1 week and their eggs were collected for preliminary analysis to determine if they were analyte-free. trials a veterinary drug containing 300 mg of ff in 1 ml was used (mediflor 30% oral solution, medicavet company, turkey) for the clinical trials. ff was administered at a dose of 20 mg/kg/ day via gavage for 3 days to the 50 laying hens, and then their eggs were collected daily thereafter. the effect of storage procedures on the residues was determined on the first day using 44 eggs. the effects of cooking procedures were determined on the second day using 32 eggs. in all, 20 of the eggs collected on the first day were kept at 4°c in a refrigerator, and 20 were kept at 15-20°c (room temperature). in addition, 4 eggs were analyzed on day 4, 7, 14, 21, 28 of storage to determine ff and ffa residue levels. lastly, 4 uncooked eggs collected on day 1 were analyzed as a control group; of the eggs collected on day 2, 8 uncooked, 8 fried in oil, 8 undercooked (1 min in boiling water) and 8 overcooked (5 min in boiling water) were then analyzed. sample preparation and analysis ff and ffa were extracted from homogenized eggs via phosphate buffer (ph:7) and ethyl acetate. following purification, the samples underwent high-performance liquid chromatography (hplc) using a photodiode array detector (pda) and c18 column; the method was validated according to ich guidelines, as described elsewhere (filazi et al., 2014). according this method, limits of detection and of quantitation values were 1.94 and 6.45 ppb for ff, respectively, and 0.48 and 1.58 ppb for ffa, respectively. relative standard deviation values of intra-day and inter-day variation below 11% also confirmed the usefulness of the method for analysing ff and ffa in eggs. statistical analysis variance analysis was performed with all data and a multiple range test was used to determine the differences between groups. all analyses were performed using spss v. 17.0 for windows. results and conclusion the effects of different storage temperatures and durations on ff and ffa residue levels in table 1 mean±sd* concentration (in ppb) of florfenicol and florfenicol amine residues in eggs stored at room temperature (15-20°c) and in a refrigerator (+4°c). days florfenicol florfenicol amine (n=4) room temperature refrigerator room temperature refrigerator (15-20°c) (+4°c) (15-20°c) (+4°c) 0 290.65±11.02a 290.65±11.02a 91.79±6.77a 91.79±6.77a 4 151.24±10.69b 167.43±8.18b 58.26±5.98b 58.61±5.85b 7 79.65±9.43cx 105.10±4.25cy 28.95±5.03c 35.40±2.33c 14 68.23±8.74dx 87.84±5.01dy 20.52±3.92d 24.37±1.20d 21 29.43±4.91ex 61.82±2.11ey 10.42±1.54e 8.54±1.04e 28 18.57±3.48f 22.14±0.03f 6.74±0.79f 7.06±1.21f *sd: standard deviation. abcdef: differences between values with different letters in the same columns are significant (p<0.05). xy: differences between values with different letters in the same rows are significant (p<0.05). 354 ital. j. food sci., vol. 27 2015 eggs are shown in table 1. both ff and ffa amine residue levels in eggs were observed on day 28, though their levels had decreased significantly (p<0.05). hektoen et al. (1995) reported that ff rapidly depurated in the sediment of marine environments and that its metabolite (ffa) was isolated from the sediment. this finding suggests that ff is degraded to ffa in the sediment via metabolization or leaching; however, the present study ff residues in eggs following storage for 28 day at room temperature and in a refrigerator were observed. ff residue levels in eggs were higher than ffa residue levels in the present study, which indicates that the in vitro degradation of ff might occur at a very low level or that it differs from its biological degradation. further research would be required to understand the effect of storing on the ff and ffa residues in the eggs. franje et al. (2010) studied the heat stability of amphenicols in chicken meat and reported that 5-min heating of amphenicols in water in a microwave oven generated a comparable percentage of degradation as did boiling in a water bath for 30 min 1 h; ff produced thiamphenicol (tap) as a product of its breakdown, but not ffa. it was reported that although a higher level of degradation of amphenicols was observed in soybean sauce, heating treatment might still generate product with antimicrobial activity (ff to tap) and as such, heating amphenicol residues in food cannot always be safe. ff was reported to be hydrolytically stable and to have a hydrolysis half-life > 1 year at 25°c in natural waters (hayes et al., 2003; pouliquen et al., 2007; ge et al., 2009). ge et al. (2009) performed photodegradation experiments on tap and ff in aqueous solutions under irradiation from different light sources. they reported that under uv-vis irradiation (λ>200 nm) photodegradation in seawater was fastest, followed by pure water and freshwater, whereas under solar or simulated sunlight (λ > 290 nm), photodegradation occurred only in freshwater. under uv-vis irradiation, cl(dominant sea water constituent) was observed to promote singlet oxygen formation and accelerated the photodegradation of phenicols, whereas phenicols did not photolyze under simulated solar irradiation, irrespective of the presence of cl-. in contrast, hayes et al. (2003) reported that ff was stable under a range of simulated field conditions, including various pipe materials and conditions of hard and soft and chlorinated or non-chlorinated water at low or high ph; therefore not only clbut also some other minerals might effect the stability of ff. the effects of different cooking procedures on ff and ffa residues in eggs observed in the present study are shown in table 2. even though, none of the cooking methods completely destroyed ff or ffa residues in eggs, there was a significant decrease in the level of detectable ff and ffa residues (p<0.05). concentrations of both analytes were reduced by 78%97% via frying and boiling. these findings suggest that ff and ffa heat labile in eggs, which indicates that both do not bind to proteins in eggs with high affinity. franje et al. (2010) reported that amphenicol degradation was apparent following as little as 30 min of heating and that it was correlated with the length of heating, implying that as cooking time increased the degree of residual drug present in samples decreased; as such, it could be assumed that there was a strong correlation between the decrease in ff and ffa concentrations in observed eggs during different cooking methods and the duration of cooking (p<0.05, table 2). shakila et al. (2006) studied the stability of chloramphenicol (chp) residues in white shrimp (penaeus indicus) subjected to cooking (100 °c) for 10, 20 and 30 min as well as retorting (121°c) for 10 and 15 min, based on a microbial assay method using photobacterium leiognathi as the test organism. they reported that the loss of chp increased as temperature and duration of heating increased, where the drug could be completely destroyed. on the other hand botsoglu and fleutoris (2010) reported that chp was quite stable under heating conditions when added to water or milk; after 2 h of boiling, it was decreased by <8%. these findings indicate that the heat stability of amphenicols is matrix dependent, where results from different matrices could not be attributed to eggs when interpreting. even though, ff is not approved for use in laying hens, its off label use for severe indications can result in antibiotic residues in eggs that both farmers and consumers should be informed about. as such, drug withdrawal periods should be extended prior to poultry slaughter table 2 mean±sd*, quantity of florfenicol and florfenicol amine residues (in ppb) after different cooking methods. residues (n=8) raw fried undercooked overcooked (1 minute) (5 minutes) florfenicol 265.45±13.67a 56.51±9.68b 35.67±4.57c 5.68±1.17d florfenicol amine 110.31±12.73a 19.77±4.71b 10.20±1.72c 4.57±0.92d *sd: standard deviation. abcd: differences between values with different letters in the same row are significant (p<0.05). ital. j. food sci., vol. 27 2015 355 or egg distribution to avoid antimicrobial resistance. thermal treatments may reduce the concentration of veterinary drug residues in foods and thereby might reduce the pharmacological and/or toxic effects of these compounds. (hsieh et al., 2011). in the current study, ff and ffa were observed to be heat labile in chicken eggs, the level of which depended on cooking method and duration. the findings show that ff and ffa residue levels in eggs from treated laying hens were not completely eliminated via cooking or of up to 28 d; however, cooking did significantly decreased the level of the drug in eggs. acknowledgements authors sincerely thank ankara university scientific research committee for supporting this study by ankara university scientific research projects funding (project no: 10b3338003). this study was presented as a poster presentation in the 12th international congress of the european association for veterinary pharmacology and toxicology (eavpt 2012), 8-12 july2012, noordwijkerhout, the netherlands where the abstract only (not the full text) as a special issue appears in journal of veterinary pharmacology and therapeutics (35(3): 78) which is modified for the current publication. the authors 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http://www.ema.europa.eu/docs/en_gb/document_library/maximum_residue_limits_-_report/2009/11/wc500014280.pdf 356 ital. j. food sci., vol. 27 2015 freshwater and seawater under abiotic conditions. aquaculture, 262: 23-28. rose m.d., bygrave j., farrington w.h.h. and shearer g. 1996. the effect of cooking on veterinary drug residues in food. 4. oxytetracycline. food addit. contam. 13: 275-286. rose m.d., farrington w.h.h. and shearer g. 1995. the effect of cooking on veterinary drug residues in food. iii: sulphamethazine (sulphadimidine). food addit. contam. 12: 739-750. shakila r.j., vyla s.a.p., kumar r.s., jeyasekaran g. and jasmine g.i. 2006. stability of chloramphenicol residues in shrimp subjected to heat processing treatments. food microbiol. 23: 47-51 sireli u.t., filazi a. and cadirci o. 2006. effect of cooking and storage times on gentamicin residues in eggs. ital. j. food. sci. 18: 441-446. traub w.h. and leonhard b. 1995. heat stability of the antimicrobial activity of sixty-two antibacterial agents. j. antimicrob. chemother. 35: 149-154. xie k., jia l., yao y., xu d., chen s., xie x., pei y., bao w., dai g., wang j.and liu z. 2011. simultaneous determination of tiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection. j. chromatogr. b. analyt technol. biomed life sci. 879: 2351-2354. paper received may 7, 2014 accepted november 3, 2014 http://jac.oxfordjournals.org/search?author1=walter+h.+traub&sortspec=date&submit=submit http://jac.oxfordjournals.org/search?author1=birgit+leonhard&sortspec=date&submit=submit ijfs#252_czaplicki_bozza   ital. j. food sci., vol 28, 2016 412 paper sea-buckthorn oil in vegetable oils stabilisation s. czaplicki*, m. tańska and i. konopka chair of plant food chemistry and processing, faculty of food sciences, university of warmia and mazury in olsztyn, pl. cieszyński 1, 10-726 olsztyn, poland *corresponding author. tel./fax: +48 895233466 e-mail address: selek@go2.pl abstract the paper proposes the development of blends of vegetable oils with a high content of easily oxidizable unsaturated fatty acids with sea-buckthorn fruit oil as a natural method of their preservation. the predominant lipophilic compounds in sea-buckthorn oil included β-carotene, α-tocopherol, and β-sitosterol. strong correlations were found between oxidative stability of the blends and αand β-tocopherols, lutein and β-carotene concentration (r ranging from 0.96 to 0.99). the observed effect may result from both the particularly huge increase in the carotenoid content (from 64to 171-fold) in the obtained blends, and the synergistic interaction between tocopherol mixtures and carotenoids. keywords: oils stabilisation, sea-buckthorn fruit oil, natural antioxidants   ital. j. food sci., vol 28, 2016 413 1. introduction consumption of vegetable oils provides the body with energy and ingredients with structural, regulatory and protective functions (ayala et al., 2014). lipid components form, inter alia, the structure of cell membranes; moreover, they are involved in the formation and functioning of nerve cells, regulation of intrasystemic metabolism, and maintaining the oxidation/reduction balance in the cells (murray et al., 1995). the composition of fatty acids and the profile of low-molecular lipophilic compounds depend on the botanical source of origin, and the method of oil production. most oils contain predominantly unsaturated fatty acids which allow them to maintain the liquid state at room temperature. however, the presence of unsaturated fatty acids promotes the initiation of oxidation processes, which are particularly rapid for polyunsaturated acids. with an increase in the number of unsaturated bonds in the cell, the number of carbon atoms separating them also increases. the carbon-hydrogen bond near such a carbon atom is characterized by lower dissociation energy, which leads to easy formation of free radicals. according to cosgrove et al. (1987) the oxidation of oleic acid is 50 times slower than that of linoleic acid, and 100 times slower than that of linolenic acid. the slightly lower rate of the oxidation of oleic acid as compared to linoleic acid (10-40 times lower rate) was specified in the study by mcclement and decker (2008). oxidation of an oil begins at the moment of the extraction thereof from a plant matrix. due to the destruction of natural cell structures, they become susceptible to the action of enzymes, oxygen, light, and other free radical generators (szukalska, 2003). the basic and primary indicator of the oxidation of an oil is an increase in the value of peroxide value (picuric-jovanovic et al., 1999; broadbent and pike, 2003). for coldpressed oils, the value of peroxide value as permitted by codex alimentarius (2005) is 15 meqo2/kg. the value of peroxide value, however, does not allow clear determination of freshness of an oil. only the performance of additional analyzes on the secondary products of oil oxidation e.g. by the tba test, determination of the anisidine value, or the measurement of absorbance of conjugated dienes and trienes allows a more precise determination of the degree of oxidation (jerzewska, 1991). despite the presence of oxidizable unsaturated fatty acids, vegetable oils contain numerous stabilizing compounds exhibiting antioxidant action (czaplicki et al., 2011; ogrodowska et al., 2014; roszkowska et al., 2015). they get to the oil from the plant matrix, or are added at the packaging stage. the compounds of particular significance are terpenoid compounds such as tocols, sterols, carotenoids, and squalene. most often, however, the content of natural antioxidants is not sufficiently high to fully protect the oil against oxidation. a study by czaplicki et al. (2011) on nine popular bio-oils found that the content of unsaponifiable fraction ranged from only 0.48% (poppyseed oil) to 7.12% (amaranth oil). it therefore seems that vegetable oils valued for their unique compositions of fatty acids, such as linseed, borage, and evening primrose oils, should be enriched with either natural or synthetic antioxidants. literature describes attempts to increase the oxidative stability of oils through the addition of compounds such as e.g.: tocopherols, tocotrienols, sesamol, butylated hydroxyl toluene (bht), butylated hydroxyanisole (bha), propyl gallate (pg), tertiarybutylhydroquinone (tbhq) and ascorbyl palmitate (ap) (hamdo et al., 2014; hwang and winkler-moser, 2013; önal-ulusoy and ergin, 2002). similarly, a positive effect on the oxidative stability of oils was observed in tests using grape seed extract, green tea extract, microalgae scenedesmus almeriensis extracts, and extracts of herb such as rosemary (rosmarinus officinalis), oregano (origanum vulgare), marcela (achyrocline satureioides), and carqueja (baccharis trimera) (poiana, 2012; chen et al., 2013; vieitez et al., 2013; limón et al., 2015). however, the introduction of pure substances or lyophilized extracts into an oil results in the need for standardization   ital. j. food sci., vol 28, 2016 414 of their concentrations in the oil, which may be limited by the solubility of the compound or extract being added. it was demonstrated, inter alia, that in corn oil at a temperature of 25°c, only 3% sterols can be dissolved (vaikousi et al., 2007). the practice of introducing into oils substances which do not occur in them naturally gives rise to controversy associated with the loss of the “natural” characteristic of a product. on the other hand, a natural manner of increasing the content of antioxidants in oils may be the development of their blends with oils being particularly rich in natural antioxidants. an oil which is characterized by an exceptionally high content of phytosterols, tocopherols, and carotenoids, is the oil extracted from sea-buckthorn fruits. this study attempted to determine the effects of the addition of sea-buckthorn oil as a stabilizer of linseed, borage, and evening primrose oils which are characterized by different compositions of fatty acids and contents of endogenous antioxidants. the following were assessed: the content of natural terpenoid antioxidants in the obtained blends, the composition of fatty acids, and their oxidative stability. 2. materials and methods 2.1. chemicals chromatography-grade solvents: methanol, methyl tert-butyl ether (mtbe), iso-propanol, hexane, pyridine, n,o-bis (trimethylsilyl) trifluoroacetamide (bstfa) with 1% trimethylchlorosilane (tmcs) were purchased from sigma-aldrich (st. louis, mo, united states, supplier poznań, poland). analytical-grade reagents: methanol, dichloromethane, chloroform, sulphuric acid, potassium hydroxide, sodium sulphate, powdered zinc (poch, gliwice, poland) were used. analytical standards: 5α-cholestane (97%), β-apo-8'carotenal (>96%) and fatty acids mixture were purchased from sigma-aldrich and tocopherols mixture (95%) from merck (darmstadt, germany). deionized water was obtained from hlp 5s deionizer (hydrolab, gdańsk, poland). 2.2. materials the linseed oil, borage seed oil, evening primrose seed oil and sea-buckthorn fruits oil were used in this study. oils were obtained by pressing the raw material on a ibg monforts & reiners, komet ca59g (germany) laboratory expeller. the oils were purified by centrifugation at 8000 x g on a eppendorf centrifuge (type 5810r). the oils blends were prepared by 15% of sea buckthorn oil addition to analysed linseed, borage seed and evening primrose seed oils. in oils and prepared mixtures fatty acids compositions and bioactive compounds (carotenoids, sterols and tocopherols) concentrations were analysed as well as oxidative stability was also examined. 2.3. determination of fatty acid composition ten micrograms of sample was dissolved in 1.5 ml of chloroform-methanol-sulphuric acid (100:100:1, v/v/v), transferred into 2 ml-pharmaceutical vials and sealed hermetically over a gas burner (zadernowski and sosulski, 1978). the fatty acids methylation was carried out by heating the vials at 70°c for 2 hours. after cooling, the vials were opened and the powdered zinc was added to decompose remained sulphuric acid. obtained methyl esters were dried in a stream of nitrogen, purified in a hexane extraction and analysed by gas chromatography with a gc-ms qp2010 plus (shimadzu,   ital. j. food sci., vol 28, 2016 415 japan) system. separation was performed on a bpx70 (25 m x 0.22 mm x 0.25 µm) capillary column (sge analytical science, victoria, australia) with helium as the carrier gas at a flow rate of 0.9 ml/min. the column temperature was programmed as follows: a subsequent increase from 150°c to 180°c at the rate of 10°c/min, to 185°c at the rate of 1.5°c/min, to 250°c at the rate of 30°c/min, and then 10 min hold. the interface temperature of gc-ms was set at 240°c. the temperature of the ion source was 240°c and the electron energy 70 ev. the total ion current (tic) mode was used in 50-500 m/z range. according to the shares of individual fatty acids oils oxidation index (u) was calculated using the formula given by cosgrove et al. (1987): u = (0.02 · (c16:1+c18:1) + 1 · c18:2 + 2 · c18:3)/100. 2.4. determination of carotenoids carotenoids in oils were analysed with a reversed phase high performance liquid chromatography (rp-hplc) technique. the sample of oils was diluted in hexane contained β-apo-8'-carotenal as an internal standard and saponified with 6 ml of 40% methanolic koh solution in a shaker at room temperature in the dark for 16 h. next, 30 ml of hexane to the sample was added and then the tube was filled up to 50 ml with 10% na2so4. the lower phase was separated, triple-rinsed with 10 ml of hexane and collected with the upper organic phase. the organic solvent was evaporated at 40°c under a nitrogen stream and dissolved in 2 ml of a methanol: dichloromethane (45:55 v/v) solution. the chromatographic analysis of carotenoids was conducted according to modified emenhiser et al. (1995) method. briefly, the analysis was carried out using a 1200 series liquid chromatograph manufactured by agilent technologies (palo alto, ca, usa), equipped with a diode array detector (dad) from the same manufacturer. separation was performed at 30°c on a ymc-c30 250 x 4.6 mm, 5 µm column and ymcc30 10 x 4.6 mm, 3 µm precolumn (ymc-europe gmbh, germany). a methanolmethyl tert-butyl ether (mtbe) gradient was programmed as it is presented in table 1. the absorbance was measured at the wavelength of 450 nm. carotenoids were identified, based on retention times of available standards (sigma-aldrich, usa), and by comparing the uv–visible absorption spectra. table 1: hplc gradient conditions established for analysis of carotenoids. time [min] methanol [%] mtbe [%] flow rate [ml/min] 0-5 95 5 1 25 72 28 1.25 33 5 95 1.25 40 95 5 1 60 95 5 1 2.5. determination of phytosterols the content of sterols in oils was determined by gas chromatography coupled with mass spectrometry (gc-ms qp2010 plus, shimadzu, japan) according to the method described   ital. j. food sci., vol 28, 2016 416 by vlahakis and hazebroek (2000). the sample was saponified by adding a 0.5 ml 2m naoh methanolic solution at ambient temperature for 2 hours. unsaponifiables were extracted with diethyl ether which was evaporated under nitrogen conditions. the dry residues were re-dissolved in 1.5 ml of n-hexane and a 0.2 ml of 5α-cholestane internal standard solution was added (0.4 mg/ml). after evaporation, the residues were redissolved in 100 µl of pyridine and 100 µl bstfa (n,o-bis (trimethylsilyl) trifluoroacetamide) with 1% tmcs (trimethylchlorosilane) and left in 60°c for 60 minutes to complete derivatization. one ml of hexane was then added to the sample and 1 µl of the obtained mixture was analysed. a zb-5msi capillary column was used for the separations of sterols with helium as a carrier gas at a flow rate of 0.9 ml/min. the injector temperature was set at 230ºc and the column temperature was programmed as follows: 70°c for 2 min, a subsequent increase to 230°c at the rate of 15°c/min, to 310°c at the rate of 3°c/min, and then 10 min hold. the interface temperature of gc-ms was set at 240°c. the temperature of the ion source was 220°c and the electron energy 70 ev. the total ion current (tic) mode for quantification (100-600 m/z range) was used. the quantifications using the internal standard method were done. 2.6. determination of tocopherols the tocopherols analysis was carried out by high performance liquid chromatography (hplc), according to the method described by czaplicki et al. (2011). briefly, 0.1 g of oil (± 0.001 g) was diluted in hexane in a 10 ml measuring flask. after subsequent centrifugation (10 min. at 25000 x g) in a 5417r-type eppendorf centrifuge (eppendorf ag, hamburg, germany), the sample was transferred to a chromatographic vial and 20 µl was injected into the chromatographic system. the analysis was performed using a 1200 series liquid chromatograph manufactured by agilent technologies (palo alto, ca, usa), equipped with a fluorescence detector from the same manufacturer. the separation was done on a merck lichrospher si 60 column, 250 mm x 4 mm, 5 µm. a 0.7% isopropanol solution in hexane at a 1 ml/min flow rate was used as the mobile phase. the fluorescence detector was set at 296 nm for excitation and 330 nm for emission. peaks were identified on the basis of retention times determined for α-, β-, γand δ-tocopherol standards (merck, darmstadt, germany) separately, and their content was calculated using external calibration curves. 2.7. determination of induction time induction time of oils was tested on a rancimat apparatus 743 (metrohm, herisau, switzerland). the analysis was performed according to method described by farhoosh (2007). briefly, 2.5 g of oil in a reaction vessel was weighed and after capping the vessel was placed in a thermostated electric heating block at temperature 110°c. an air flow rate of 20 l/h was given. determination of the induction time was based on the conductometric detection of volatile oxidation products. the time that elapsed until these oxidation products appeared was saved as the induction time. 2.8. determination of initial state of oils rancidity the acid (av), peroxide (pv), and p-anisidine (p-av) values were determined in accordance with procedures of cen iso 660:2009, cen iso 3960:2010, and cen iso 6885:2008, respectively.   ital. j. food sci., vol 28, 2016 417 2.9. statistical analysis the obtained results of all analysis (performed in triplicate) were statistically analysed using statistica 12.0 pl software (statsoft inc., kraków, poland). in order to indicate the significance of differences between oil samples, unvaried analysis of variance (anova) with a duncan test at p≤0.05 significance level was used. the intra-sample quality variation of fresh oils and their blends with the sea buckthorn oil was assayed using principle component analysis (pca) at p ≤ 0.05 significance level. 3. results and discussions the initial rancidity of three used in this study highly unsaturated oils (linseed, borage seed, and evening primrose seed) was relatively low, with an av value from 1.37 to 3.15 mg koh/g, and a pv value from 0.93 to 3.76 meq o2/kg (table 2). according to requirements of the codex alimentarius commission these results met the standard for cold-pressed and virgin oils, determined as 4 mg koh/g, and 15 meq o2/kg of oil, respectively. values of a p-av of these oils, which reflect the content of secondary products of lipid oxidation, varied from 2.00 to 7.85 (table 2), and were similar for example to results for raspberry seed oil (oomah et al., 2000). determined av, pv, an pav values showed that used oils were of food-grade quality typical for other cold pressed and virgin oils. table 2: rancidity indices of linseed, evening primrose, and borage oils before enrichment with seabuckthorn oil. acid value [mg koh/g oil] peroxide value [meq o2/kg oil] anisidine value [-] sd sd sd linseed 1.37 0.02 0.93 0.05 2.00 0.26 evening primrose 1.57 0.01 3.76 0.11 7.85 0.61 borage 3.15 0.22 1.45 0.10 4.61 0.52 the fatty acid compositions of the studied linseed, borage seed, and evening primrose seed oils, and of their blends with sea-buckthorn oil, are presented in table 3. it was found that linseed, borage seed, and evening primrose seed oils were characterized by a high percentage of polyunsaturated fatty acids (pufa). the oils being richest in these acids included linseed oil (73%) and evening primrose oil (82%), and the total share of acids containing at least one unsaturated bond in these oils amounted to nearly 90%. in turn, borage oil was characterized by the highest share of γ-linolenic acid (7%) of all the studied oils, and unsaturated fatty acids were also represented by oleic (26%) and linoleic acid (33%). the oils used in the experiment are valued for their composition of fatty acids; however, both the great number of unsaturated bonds and the degree of their unsaturation have an adverse effect on the oxidative stability of an oil. the susceptibility of the oils under study to oxidation is expressed as an oxidizability index calculated according to the formula proposed by cosgrove et al. (1987). borage oil turned out to be the oil being least susceptible to oxidative changes; the oxidizability index for this oil amounted to 0.61. in turn, linseed oil, which contained almost 60% of α-linolenic acid, exhibited the highest value of the oxidizability index (1.33). as compared with e.g.   ital. j. food sci., vol 28, 2016 418 rapeseed oil (roszkowska et al., 2015), the oxidizability indices of the oils under study had values higher by 65, 143, and 259% for, respectively, borage oil, evening primrose oil, and linseed oil. table 3: fatty acids composition (%), the main bioactive compounds content in oils [mg/100 g of oil], and their oxidation index [-] and induction time [h]. linseed oil borage seed oil evening primrose seed oil sea buckthorn fruit oil compound sd sd sd sd palmitic (c16:0) 7.33 a 1.13 19.60b 1.25 7.98a 0.11 36.31c 0.01 palmitoleic (c16:1) nda nda nda 40.97b 0.04 stearic (c18:0) 3.28a 0.75 7.46b 0.44 2.28c 0.05 0.55d 0.01 oleic (c18:1) 15.88a 1.8 26.40b 1.44 7.12c 1.34 8.77c 0.11 linoleic (c18:2) 14.49 a 0.32 32.64b 1.41 75.47c 1.88 12.12d 0.13 α-linolenic (c18:3) 59.02 a 3.36 ndb ndb 1.28c 0.04 γ-linolenic (c18:3) nd a 13.91b 1.73 7.17c 0.39 nda σ pufa 73.51a 3.68 46.55b 3.14 82.64c 2.27 13.40d 0.17 oxidizability index 1.33a 0.07 0.61b 0.05 0.90c 0.03 0.16d 0.00 lutein 0.22a 0.01 0.09a 0.01 0.07a 0.01 3.24b 0.49 all-trans β-carotene 0.22a 0.05 0.07a 0.05 0.14a 0.04 118.36b 9.58 other carotenoids 0.04a 0.01 0.01a 0.01 0.09a 0.08 74.82b 6.25 total carotenoids 0.48a 0.06 0.18a 0.07 0.30a 0.12 206.04b 15.63 α-tocopherol 6.21a 0.30 ndb 26.42c 0.17 144.14d 4.10 β-tocopherol nda nda nda 3.98b 0.23 γ-tocopherol 37.02a 1.01 11.95b 0.21 40.41c 0.86 4.63d 0.32 δ-tocopherol nda 100.25b 0.50 nda 0.75a 0.00 total tocopherols 43.23a 1.32 112.20b 0.71 66.83c 0.69 153.50d 4.10 campesterol 40.44a 0.63 38.30a 3.04 57.35b 3.08 7.87c 0.45 δ5-avenasterol 13.37a 0.53 43.42b 0.77 62.92c 5.95 20.46d 1.54 β-sitosterol 110.21a 0.29 37.96b 2.05 595.94c 6.46 536.30d 1.25 δ7-stigmastenol nda nda 5.95b 0.28 nda δ7-stigmasterol 14.75a 0.35 ndb ndb ndb cycloartenol 162.38a 0.53 51.02b 4.74 ndc 28.50a 0.63 other sterols 45.44a 1.64 37.13a 1.22 8.58b 0.19 262.81c 16.61 total sterols 386.58a 0.83 207.81b 7.49 730.74c 4.06 855.94d 8.95 induction time 2.44a 0.23 3.91b 0.25 3.97b 0.20 >48c nd – not detected values within a row with different letters are significantly different (p ≤ 0.05).   ital. j. food sci., vol 28, 2016 419 sea-buckthorn oil, used as a stabilizer, owed its resistance to oxidation to, inter alia, the relatively low share of polyunsaturated fatty acids. the oxidizability index calculated for this oil only amounted to 0.16, and the induction time in the rancimat test was longer than 48 h (in a temperature of 110°c), which demonstrates its extraordinary resistance to oxidative changes. however, this resistance is owed not only to the characteristics of fatty acids but also to the abundance of natural antioxidants. the average content of carotenoids in sea-buckthorn oil amounted to 206 mg/100 g, and the predominant one was β-carotene (65%). in addition to β-carotene, sea-buckthorn oil also contained, inter alia, α-carotene, lutein, zeaxanthin, and β-cryptoxanthin. sea-buckthorn oil also contained tocopherols at an amount of 144 mg/100 g, with the predominant α homologue (94%), and phytosterols at an amount of 856 mg/100 g, with the predominant β-sitosterol (63%). linseed, evening primrose, and borage oils were characterized by significantly lower contents of these antioxidants. the carotenoid content did not exceed the value of 0.48 mg/100 g and, in the extreme case, was ca. 1100 times lower than that in seabuckthorn oil. sea-buckthorn oil was also significantly richer in tocopherols, as it contained, respectively, 3.6-, 1.4-, and 2.3-times more of these compounds than, in turn, linseed, borage seed, and evening primrose seed oils. in the group of these compounds, γtocopherol (linseed and evening primrose oils), and δ-tocopherol (borage seed oil) were predominant. as regards phytosterols, the total content thereof being similar to that of sea-buckthorn oil was found in evening primrose seed oil, while linseed oil and borage seed oil contained 2.21and 4.12-times less of those compounds, respectively. the predominant phytosterols in the enriched oils included cycloartenol (linseed oil and borage seed oil) and β-sitosterol (evening primrose seed oil). the addition of sea-buckthorn oil resulted in an over 64-, 171-, and 103-fold increase in the carotenoid content of linseed oil, borage seed oil, and evening primrose seed oil, respectively, with a particularly apparent increase in the share of all-trans β-carotene (table 4). the enrichment with sea-buckthorn oil resulted in an increase in the content of this compound to a level ranging from approx. 3 mg/100 g in an evening primrose seed oil blend to almost 5 mg/100 g in a linseed oil blend. the enrichment with sea-buckthorn oil also contributed to an increase in tocopherol content. this change was biggest for linseed oil (45%), and the content of α-tocopherol in this oil increased over three-fold. borage seed oil, which was the only one containing no α-tocopherol, was enriched with this component to an amount of almost 29 mg/100 g. at the same time, the addition of seabuckthorn oil caused a significant increase in the content of β-sitosterol in linseed oil (58%) and borage seed oil (197%). evening primrose seed oil was the only oil in which no significant change to the concentration of this sterol was noted, with the total increase in the share of sterols by 2.57%. at the same time, the enriched oils were characterized by significantly lower oxidizability index (a decrease by 12-30%), and the induction time being increased by approx. 21–32% (table 4). the noted relative increase in the stability of oil was statistically significant, and reached the highest value for linseed oil. however, the actual induction time of this oil only increased to a value of 3.21 h (from the initial value of 2.44 h), which may be explained by the particularly high content of pufa. this phenomenon may be explained not only by the change to fatty acid concentration but also by the more than 3-fold increase in the concentration of α-tocopherol, and over 60-fold increase in the concentration of carotenoids.   ital. j. food sci., vol 28, 2016 420 table 4: fatty acids composition (%), the main bioactive compounds content [mg/100 g of oil] in oils blends with sea buckthorn oil and their oxidation index [-] and induction time [h]. linseed oil borage seed oil evening primrose seed oil compound sd sd sd palmitic (c16:0) 10.87 a 0.83 27.15b 1.97 13.69c 1.99 palmitoleic (c16:1) 5.68a 0.01 7.10b 0.53 7.17b 0.69 stearic (c18:0) 2.96 a 0.37 6.74b 0.47 1.87c 0.19 oleic (c18:1) 15.19 a 0.17 25.22b 0.75 8.19c 0.29 linoleic (c18:2) 14.39 a 0.21 24.94b 1.11 63.32c 2.09 α-linolenic (c18:3) 50.91a 1.18 ndb ndb γ-linolenic (c18:3) nda 8.87b 0.05 5.77c 5.77c σ pufa 65. 30a 1.39 33.81b 1.16 69.09a 3.15 oxidizability index 1.17a 0.03 0.43b 0.01 0.75c 0.04 lutein 0.67a 0.04 0.56a 0.10 0.55a 0.13 all-trans β-carotene 17.94a 0.20 17.81a 0.77 17.87a 0.47 other carotenoids 11.26a 0.16 11.23a 0.80 11.30a 0.46 total carotenoids 31.31a 0.29 31.06a 1.9 31.16a 0.46 α-tocopherol 26.90a 2.18 21.62b 1.26 44.08c 0.60 β-tocopherol 0.60a 0.43 0.60a 0.44 0.60a 0.06 γ-tocopherol 32.16a 1.05 10.85b 0.54 35.04c 0.19 δ-tocopherol 0.11a 0.00 85.33b 1.64 0.11a 0.00 total tocopherols 59.77a 1.65 118.40b 2.87 79.83c 0.85 campesterol 35.55a 1.81 33.74a 2.12 49.93b 3.94 δ5-avenasterol 14.43a 3.25 39.98b 2.15 56.55c 3.52 β-sitosterol 174.12a 6.16 112.71b 8.99 586.99c 29.61 δ7-stigmastenol nda nda 5.06b 0.44 δ7-stigmasterol 12.54a 0.66 ndb ndb cycloartenol 142.30a 6.05 47.64b 3.51 4.28c 0.39 other sterols 78.05a 7.55 70.98a 5.30 46.71b 2.45 total sterols 456.98a 11.25 305.03b 10.15 749.52c 14.28 induction time 3.21a 0.23 4.80b 0.25 4.81b 0.18 nd – not detected values within a row with different letters are significantly different (p ≤ 0.05). a pca analysis confirmed strong correlation between the induction time for the oil and the shares of palmitoleic acid (0.98) and palmitic acid (0.77) being typical of sea-buckthorn oil (shafi et al., 2008) (fig. 1a), and between the induction time and the content of αand β-tocopherol as well as lutein and β-carotene – correlations within a range of 0.96–0.99 (fig. 2a). stability of the oils was affected, to a much smaller extent, by the content of βsitosterol, with the correlation coefficient being only equal to 0.41.   ital. j. food sci., vol 28, 2016 421 linoleic acid pufa -1.0 -0.5 0.0 0.5 1.0 factor 1 : 47.14% -1.0 -0.5 0.0 0.5 1.0 f ac to r 2 : 3 1. 40 % palmitic acid palmitoleic acid stearic acid oleic acid α-linolenic acid γ-linolenic acid oxidation index induction time a) l lsb b bsb ep epsb sb -4 -3 -2 -1 0 1 2 3 4 5 6 7 factor 1: 47.14% -3 -2 -1 0 1 2 3 4 5 fa ct or 2 : 3 1. 40 % b) figure 1: a) pca loading plot of tested variables; b) score plot of the two first principal components after pca analysis of fatty acid composition, oxidation index and induction time of fresh oils and their blends with sea buckthorn fruit oil. b – borage seed oil, bsb – borage seed oil enriched with sea buckthorn fruit oil, ep – evening primrose seed oil, epsb evening primrose seed oil enriched with sea buckthorn fruit oil, l – linseed oil, lsb linseed oil enriched with sea buckthorn fruit oil, sb sea buckthorn fruit oil.   ital. j. food sci., vol 28, 2016 422 oxidation index γ-tocopherol campesterol δ5-avenasterol ß-sitosterol δ7-stigmastenol δ7-stigmasterol total sterols -1.0 -0.5 0.0 0.5 1.0 factor 1 : 53.84% -1.0 -0.5 0.0 0.5 1.0 f ac to r 2 : 2 6. 11 % induction time lutein β-carotene total carotenoids α-tocopherol β-tocopherol δ-tocopherol total tocopherols cycloartenol a)   l lsb b bsb ep epsb sb -6 -4 -2 0 2 4 6 8 10 factor 1: 53.84% -5 -4 -3 -2 -1 0 1 2 3 4 fa ct or 2 : 2 6. 11 % b) figure 2: a) pca loading plot of tested variables; b) score plot of the two first principal components after pca analysis of bioactive compounds content, oxidation index and induction time of fresh oils and their blends with sea buckthorn fruit oil. b – borage seed oil, bsb – borage seed oil enriched with sea buckthorn fruit oil, ep – evening primrose seed oil, epsb evening primrose seed oil enriched with sea buckthorn fruit oil, l – linseed oil, lsb linseed oil enriched with sea buckthorn fruit oil, sb sea buckthorn fruit oil. the relationship between the induction time for an oil and its potential oxidizability is rarely noted, since an oil is a complex mixture of compounds. strong correlations could   ital. j. food sci., vol 28, 2016 423 possibly be noted for a pure phase of proper lipids. however, bhatnagar et al., (2009) demonstrated that the addition of coconut oil to refined sunflower oil and rice bran oil had a positive effect on the stability of blends due to a change to the proportions of fatty acids. correlations between the induction time (stability of an oil) and the antioxidants content were found significantly more often (mateos et al., 2005). hovewer, the in the case of mixture of antioxidants the resultant activity depends not only on their content and composition, but also on their synergistic or antagonistic activity, as well as lipophilic or hydrophilic properties (kmiecik et al., 2011). a relationship between the stability of an oil and the content of β-carotene, being similar to that noted in this study, was found earlier by goulson and warthesen (1999) in relation to high oleic rapeseed oil. they demonstrated that β-carotene significantly inhibited the oxidation of this oil in the dark at a concentration of approx. 5 mg/100 g, while with the simultaneous exposure, the antioxidant effect was already observed at a concentration of approx. 2.75 mg/100 g. however, the impact of particular antioxidant components is not only dependent on the concentrations at which they occur. synergistic interactions which were demonstrated, inter alia, between α-tocopherol and β-carotene, are also important (schroeder et al., 2006). tocopherols are considered to be the main lipid antioxidants. choe and min (2006), referring to numerous studies, report that the tocopherols’ capacity to quench free radicals depends on their structure and the concentration in the oil. according to the cited authors, the highest activity as regards quenching free radicals is exhibited by δtocopherol, and the value for this activity decreases for γ-, βand α-tocopherol, respectively. the addition of α-tocopherol at an amount of 10 mg/100 g of oil may, in certain cases, even accelerate the oxidation (choe and min, 2006). in turn, with concentrations of tocopherols exceeding 4·10-3 m, the activity of particular homologues does not differ significantly (jung et al., 1991). sterols were the predominant group of compounds in the unsaponifiable fraction of all oils under study. the noted low coefficient of correlation between their concentrations and the induction time indicates a weaker resultant effect of the action of these compounds on the oxidation mechanism. earlier studies indicate that these compounds may exhibit both proand antioxidative action. a weak pro-oxidative action was demonstrated for, inter alia, β-sitosterol (lampi et al., 1999), while ergosterol, lanosterol, stigmasterol and cholesterol exhibit no antioxidative properties in relation to thermally oxidized safflower oil (sims et al., 1972). on the other hand, antioxidative activity is exhibited by sterols containing an ethylidene group within their structure (lampi et al., 1999). this group is found in, inter alia, ∆5-avenasterol which, in the oils tested as part of this study, occurred at an amount ranging from approx. 13 mg/100 g (linseed oil) to 63 mg/100 g (evening primrose oil). the noted increase in the content of ∆5-avenasterol by approx. 8% in enriched linseed oil could have affected the increase in the stability of this oil in relation to a non-enriched oil. the mechanism of antioxidative action of sterols having an ethylidene group involves their capacity to easily split off a proton, and form stable tertiary radicals. the resulting compounds are so durable that they do not initiate autooxidation (małecka, 1995). our recent study showed that the thermal degradation of rapeseed oil sterols was faster than tocopherols and carotenoids (roszkowska et al., 2015). analysis of plot score (fig. 1b) confirmed a close similarity of linseed oil and evening primrose seed oil, and blends of these oils with sea-buckthorn oil. then, a separate group comprised both borage seed oils (enriched and natural). both groups of oils were clearly distinguished from sea-buckthorn oil in terms of the composition of fatty acids. on the other hand, other chemical components analysis (fig. 2b) showed a close similarity of linseed oil and borage seed oil, and their blends with sea-buckthorn oil. in this case, a separate group comprised natural and enriched evening primrose seed oils.   ital. j. food sci., vol 28, 2016 424 4. conclusions results of the study suggest that the development of blends of oils containing valuable and unique polyunsaturated fatty acids with sea-buckthorn oil being rich in antioxidant compounds increases the oxidative stability of these oils. in addition, the enrichment with sea-buckthorn oil contributes to both significant increase in the content of carotenoids (mainly β-carotene), α-tocopherol and β-sitosterol, and relative decrease in the share of polyunsaturated acids. enriched oils gain new sensory qualities (the color derived from carotenoids) and health-promoting properties (the inhibition of radical formation, and the presence of numerous antioxidants); moreover, they have an extended shelf life. acknowledgements the authors gratefully acknowledge the financial support from the national science centre, poland (project 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variation in the composition and oxidative stability of commercial rapeseed oils during their shelf life. eur. j. lipid sci. tech. 117:673. schroeder m.t., becker e.m. and skibsted l.h. 2006. molecular mechanism of antioxidant synergism of tocotrienols and carotenoids in palm oil. j. agric. food chem. 54:3445. shafi n., kanwal f., siddique m., ghauri e.g. and akram m. 2008. chemical variability of fatty acid composition of seabuckthorn berries oil from different locations by gc-fid. pak. j. sci. ind. res. 51:250. sims r.j., fioriti j.a. and kanuk m.j. 1972. sterol additives as polymerization inhibitors for frying oils. j. am. oil chem. soc. 49:298. szukalska e. 2003. the chosen problems of fats oxidation. tłuszcze jadalne. 38:42 (in polish). vaikousi h., lazaridou a., biliaderis c.g. and zawistowski j. 2007. phase transitions, solubility, and crystallization kinetics of phytosterols and phytosterol−oil blends. j. agric. food chem. 55:1790. vieitez i., mailhe i., braun m. and jachmanian i. 2013. stabilizing edible oils with supercritical extracts from herbs. inform. 24:494. vlahakis c. and hazebroek j. 2000. phytosterol accumulation in canola, sunflower, and soybean oils: effects of genetics, planting location, and temperature. j. am. oil chem. soc. 77:49. zadernowski r. and sosulski f. 1978. composition of total lipids in rapeseed. j. am. oil chem. soc. 55:870. paper received september 4, 2015 accepted january 5, 2016 p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33i3.2077 33 p u b l i c a t i o n s codon quality of veiled olive oil: role of turbidity components carlotta breschi, lorenzo guerrini*, ferdinando corti, luca calamai, paola domizio, alessandro parenti, and bruno zanoni department of agriculture food, environment and forestry (dagri), università degli studi di firenze, 50121 florence, italy *corresponding author: lorenzo guerrini, department of agriculture food, environment and forestry (dagri), università degli studi di firenze, 50121 florence, italy. email: lorenzo.guerrini@unifi.it received: 28 may 2021; accepted: 2 september 2021; published: 18 september 2021 © 2021 codon publications open access paper abstract this study investigated the effects of water and content of solid particles, taken together as well as separately, on stability of veiled olive oil. the following oil samples were obtained through four different separation treatments: veiled, filtered, ‘solid-only’, and ‘water-only’. changes in chemical, microbial, and sensory characteristics were evaluated during storage (240 days). a significant effect of hydrolysis was shown in veiled and ‘water only’ oils; in ‘solid-only’ oils, a slow increase of phenols was observed. a notable microbial activity, with resulting formation of volatile metabolites and sensory defects, was observed in veiled samples. filtered oils underwent less significant changes. keywords: biophenols, hydrolysis, oil-borne microorganism, olive oil quality, volatile compounds introduction preservation of quality during storage is an important subject for extra-virgin olive oil (evoo) producers (international olive council [ioc], 2018). good preservation practices are essential to maintain quality of evoos up to shelf-life. moreover, sensory profile and contents of phenolic compounds change during storage, leading to a decrease in hedonic and health characteristics. filtration is one of the most used stabilization processes for evoos (guerrini et al., 2015). interest in unfiltered oils has increased during last few years (bimbo et al., 2020). cloudy aspect of veiled extra-virgin olive oils (vevoo) is due to the presence of micro-droplets of water and fragments of olive pulp and stone suspended/dispersed in the oil phase (lercker et al., 1994; koidis et al., 2008). furthermore, different combinations of water and insoluble solids can lead to different ‘turbidities’ in vevoos (breschi et al., 2019). the same degree of turbidity of a vevoo could be characterized by different water contents, insoluble solid contents, water activity, and/or microbial contamination. therefore, vevoo turbidity is not a dichotomous variable, but it is a continuous variable of different proportions of water, insoluble solids, microbial contamination, and water activity (breschi et al., 2019). the difference between vevoo and filtered extra virgin olive oil (fevoo) during storage is still a controversial and widely studied topic for the quality of olive oil (cayuela-sànchez and caballero-guerrero, 2019). some authors have proclaimed that suspended particles play a stabilizing function during storage because most phenolic compounds present in olive oil, having hydrophilic nature, are located in water droplets and insoluble solids (lonzano-sànchez et al., 2010). therefore, the presence of suspended particles acts as an antioxidant, providing greater oxidative stability (lercker et  al., 1994; ambrosone et al., 2002; koidis and boskou, 2006; migliorini et al., 2009). moreover, the suspended italian journal of food science, 2021; 33 (3): 33–46 mailto:lorenzo.guerrini@unifi.it 34 italian journal of food science, 2021; 33 (3) breschi c et al. to plan separation techniques during crop seasons and storage of olive oil. materials and methods olive oil samples evoo samples were extracted in october–november 2017 in an industrial continuous plant (tem, florence, italy) in azienda agricola la ranocchiaia (florence, italy). the plant was equipped with the following: olive cleaner, blade cutter crusher, sealed vertical malaxer (300 kg), and two-phase horizontal centrifuge (i.e., decanter). the malaxation was carried out at 18°c for 20 min. six different 300-kg batches of blend of olive cultivars, harvested in tuscany, were processed on three different days in 2017: olive oils #1 and #2 were processed on october 31, 2017; olive oils #3 and #4 were processed on november 7, 2017; and olive oils #5 and #6 were processed on november 28, 2017. six 20-kg batches of oil from each batch of blended olive cultivars were collected at the end of ‘decanter’, immediately transferred to the laboratory, and subjected to the following four different water and solid particle separation treatments: (1) first ¼ of oil batches (5 kg of oil) were untreated, forming vo samples for this study (i.e., samples vo#1–vo#6); (2) second ¼ of oil batches (5 kg of oil) were filtered using a portable filter press (colombo inox 12, rover pompe, padua, italy) equipped with five filter sheets (rover 8, 3-μm cut-off, rover pompe, padua, italy), forming fo samples for this study (i.e., samples fo#1–fo#6); (3) third ¼ of oil samples (5 kg of oil) were freeze-dried (modulyo, edwards, milan, italy), forming the ‘solid particle-only’ (so) samples for this study, that is, freshly extracted olive oil containing solid particles only without water (i.e., samples so#1–so#6); and (4) last ¼ of oil samples (5 kg of oil) were filtered with glass wool using a filter aid to separate solid particles, forming ‘water-only’ (wo) samples for this study, that is, freshly extracted olive oil containing water only without solid particles (i.e., samples wo#1–wo#6). all oil samples obtained (4 treatments × 6 different oil batches = 24 oil samples) were bottled in 0.25-l clear glass bottles with a headspace of about 8% of bottle’s volume, and immediately analyzed to measure turbidity characterization parameters (i.e., degree of turbidity, water content, water activity, solid particles content, and microbial cell count) as described in breschi et al. (2019). chemical characteristics (ffa, peroxide value [pv], ultraviolet [uv] spectroscopic indices [k232, k270, and ∆k], and content of phenolic and volatile compounds) and sensory attributes were also measured. particles act as buffer against increase in free fatty acid (ffa) and hydrolytic degradation (frega et al., 1999). on the other hand, in literature, improvement in shelf life because of elimination of sediment by filtration was evidenced. in vevoo, solid particles and water micro-droplets trap microorganism, mainly yeasts, and constitute a perfect environment for microbial survival (guerrini et al., 2015; ciafardini and zullo, 2002a; ciafardini and zullo, 2002b; zullo and ciafardini, 2020a; zullo and ciafardini, 2020b). in veiled oils (vos), microbial metabolism promoted by a water activity of more than 0.6 (breschi et al., 2019; bubola et al., 2017) was responsible for fast behavior of sensory defects, such as ‘fusty’ and ‘muddy-humidity’, and oil debittering phenomena (zullo and ciafardini, 2020b ; zullo et al., 2013; zanoni, 2014; cayuela et al., 2015; guerrini et al., 2020a; zullo et al., 2020). moreover, the yeast present in vevoo was responsible for oxidation of phenolic compound and hydrolysis of triacylglycerol (zullo et al., 2013; romo-sánchez et al., 2010; el haouhay et al., 2018; ciafardini and zullo, 2018). water content also affects the hydrolytic activity of olive oil; hydrolysis is faster at the interface between the two phases of oil and water (xenakis et al., 2010). this effect has been demonstrated with a higher increase of hydroxytyrosol and tyrosol in veiled olive oils than in filtered oils (fo) (brenes et al., 2001; fregapane et al., 2006; fortini et al., 2016; guerrini et al., 2020b). given the conflicting results about the role of turbidity on the stability of vevoos, in this work, an original research was carried out on the different role of water and insoluble solid particles content during storage of evoo by testing a wide spectrum of olive oil ‘turbidities’. the present work is a part of wide study on the turbidity and stabilization of olive oil. the first contribution (breschi et al., 2019) allowed defining a set of analyses useful to study turbidities of olive oil based on its physical– chemical and microbiological characterization. then a specific research (guerrini et al., 2020b) was carried out on the role of water and microorganisms, the two factors that mostly compromise the stability of vevoos. then the dynamics of development of ‘fusty’ sensory defect and the hydrolysis of phenolic compounds were studied (guerrini et al., 2020a), since these phenomena were always present in the analyzed vevoos, with the aim of establishing an adequate filtration schedule. finally, the present work aimed (i) to study the contribution of dispersed water droplets or solid particles, which, to different extent, contribute to turbidity in vevoos and affect the qualitative characteristics of olive oil during a simulated medium storage period, and (ii) how important the qualification of olive oil turbidity could be italian journal of food science, 2021; 33 (3) 35 quality of veiled olive oil: role of turbidity components and autosampler. the analytical conditions were as follows: hplc column: lichrocart® 250-4.6 purospher® star rp-18e, 5 µm (250 × 4.6-mm id; merck kgaa) equipped with a lichrocart® 4-4 purospher® star rp-18e, 5-µm pre-column (4 × 4 mm). contents of phenolic compounds in oil samples were studied as total content, content of polyphenols from different family groups (sum of oleuropein and its derivates, sum of ligstroside and its derivates, phenolic acids, flavonoids, and lignans), and content of single representative compounds in evoo (hydroxytyrosol and tyrosol). moreover, r-index, which relates the content of the more hydrolysed phenols (hydroxytyrosol and tyrosol) to the less hydrolysed ones (oleuropein and its derivates and ligstroside and its derivates) was calculated as follows (fiorini et al., 2018): hydroxytyrosol tyrosol r-index= oleuropein and its derivates ligstroside and its derivates + + the content of volatile organic compounds in olive oil was determined using the combination of headspace solid phase microextraction (hs-spme) and gas chromatography–mass spectrometry (gc–ms) technique as described in literature (fortini et al., 2017). analyses were carried out by weighing 4.3 g of sample and 0.1 g of internal standard mixture (istd mix) in 20-ml screwcap vials fitted with a ptfe/silicone septum. after 5 min of equilibrium at 60°c, the spme fiber (50/30 µm dvb/ car/pdms by supelco, darmstadt, germany) was visible in the vial headspace for 20 min while being subjected to orbital shaking (500 rpm). then the fiber immediately desorbed for 2 min in a gas chromatograph injection port operating in split less mode at 260°c. the identification of volatile compounds was performed by gas chromatography coupled with quadrupole mass spectrometry using a gc-ms scientific trace system (thermo fisher, waltham, ma, usa) equipped with a 30 m × 0.25 mm id and 0.25-µm df zb-ffap capillary column (phenomenex, torrance, ca, usa). the mass detector was operated in scan mode within a mass range of 30–330 thomson (th) at 1,500 th/s, with an ionization energy (ie) of 70 ev. compounds were identified and quantified (mg/kg) by comparing their mass spectra and retention period with those of istd mix. these consisted of the following 11 compounds: 3,4-dimethyl phenol, 4-methyl2pentanol, hexanoic acid-d11, 1-butanol-d10, ethyl acetate-d8, toluene-d8, ethyl hexanoate-d11, acetic acid-2,2,2-d3, 6-chloro-2-hexanone, 3-octanone, trimethylacetaldehyde. the panel test was carried out according to the official ioc method (ioc/t.20/doc.15/rev.10; international olive council [ioc], 2018b). three women and five men, aged 29–58 years, comprised the panel. all panelists were for storage test, all olive oil samples (4 treatments × 6 different oil batches × 4 storage periods  = 96 oil samples) were bottled in 0.25-l clear glass bottles with a headspace of about 8% of bottle’s volume. these were stored at room temperature (20°c) in a chamber (1.3 × 1.0 × 0.8 m) with internal walls covered with reflective material and a light intensity of 1,900 lux (master tl-d 90 graphica lamp, 35 w/390, philips, amsterdam, the netherlands) for 12 h per day. after 45, 120, 180, and 240 days of storage, the olive oil samples were analyzed to measure ffa, pv, k232, k270, ∆k, and phenolic and volatile compounds content and sensory parameters. analyses turbidity characterization parameters and microbial cell count the degree of turbidity was measured in nephelometric turbidity unit (ntu) using a hach model 2100 turbidimeter (hach, loveland, co, usa). water content, calculated as percent of water content weight/100-g olive oil sample (% w/w), was analyzed with a karl fischer kit for visual water determination without titrator (37858 hydranal – moisture test kit, honeywell fluka, bucharest, romania). water activity (aw) was measured using a rotronic hygroskop dt hygrometer (michell italia srl, milan, italy). the solid particles content, calculated as the difference in weight and quantified as percentage of solid particles weight/100-g olive oil sample (% w/w), was measured using the method described in literature (zullo and ciafardini, 2018), and calculated by weighing the difference and quantified as % w/w. microorganisms were enumerated according to the method reported in literature (zullo et al., 2010): an aliquot of each sample (i.e., ≈20 ml) was taken from each bottle under sterile conditions and filtered through a 0.45-μm sterile nitrocellulose membrane. then the filtered content was transferred into a 50-ml sterile falcon tube containing 20-ml sterile physiological solution (0.85% nacl) and homogenized using ultraturrax (mod. t25 homogenizer, ika milan, italy). of each homogenized sample, 200-μl serial dilution was placed on ypd agar medium. colonies were counted after 48–72 h of incubation at 28°c. chemical and sensory parameters the ffa (% oleic acid), pv (meq o2 kg −1), and uv spectroscopic indices (k232, k270, and δk) were measured according to the official eu method (reg. 2016/2095). extraction, identification, and determination of phenolic compounds was performed in agreement with the official ioc method (ioc/t.20/doc.29/rev.1; international olive council [ioc], 2017) using an hplc apparatus comprising agilent 1200 series system (agilent technologies, santa clara, ca, usa). the system was composed of a quaternary pump equipped with a diode-array detector 36 italian journal of food science, 2021; 33 (3) breschi c et al. w/w for wo samples; and 0.02–0.04% w/w for so samples), water activity value (0.45–0.75 for wo samples; and 0.30–0.40 for so samples), and insoluble solids content (0.00% w/w for wo samples; and 0.15–0.40% w/w for so samples). the microbial cell counts for wo and so olive oil samples were 0.5–3.0 log cfu g-1 and 0.0–.7 log cfu g-1, respectively. chemical parameters and microbial cell count all olive oil samples resulted from the values of chemical parameters, ffa, pv, k232, k270, and ∆k, in the ‘extra-virgin’ category during whole storage (table 1). however, the spectroscopic indices (k232, k270, and ∆k) significantly increased during storage for all treatments (p ≤ 0.01). vo samples had statistically higher ffa and ∆k values than fo, so, and wo samples. however, the highest value of k270 was determined in so olive oil samples. microbial cell count was statistically significant for treatment. vo samples had a microbial cell count higher than fo samples; wo olive oil samples had a microbial cell count between vo and fo samples. so olive oil samples had a microbial cell count between wo and fo samples (i.e., no significant difference than both wo and fo). no statistically significant variation occurred during storage time. however, interactions between time and treatment were statistically significant. in wo and so olive oil samples, the microbial cell count decreased during storage, in fo samples it did not change, and in vo samples, the microbial contamination increased up to 120 days, then decreased (figure 1). content of phenolic compounds the content of phenolic compounds of oil samples was studied as total content, content of different family groups of polyphenols, and content of single representative compounds in evoo, as described in literature (breschi et al., 2019; el riachy et al., 2011) (table 2). the total phenolic content was statistically significant (p ≤ 0.001) for treatment. the content of total phenolic compounds was statistically higher in so samples than in vo and wo samples, which had a higher content of total phenolic compounds than in fo samples (table 2). the statistically significant higher content of total phenolic compounds in so samples was also determined by the sum of oleuropein and its derivates and the sum of ligstroside and its derivates (table 2). instead, the content of hydroxytyrosol, tyrosol, and phenolic acids was statistically higher (p ≤ 0.001) in vo samples than in wo and so samples, which had higher content of hydroxytyrosol, tyrosol, and phenolic acids than in fo samples (table 2). trained following the official ioc procedure (ioc/t.20/ doc.14/rev.5; international olive council [ioc], 2018a). the panelists worked for the taste commission of the ministerodelle politiche agricole alimentari, forestali e del turismo (mipaaaft—italian ministry of agri-food and forestry policy and tourism). for the safety of panelists, wo samples, filtered on glass wool, were not tasted but only smelt out. data processing a linear model that included two tested variables (treatment and storage period) and their interactions were used to fit the experimental data. data were analyzed with matlab r2017b software (mathworks, natick, ma, usa). a two-way mixed effect anova was performed to assess significant differences (p ≤ 0.05). treatment was considered a fixed effect variable, while storage period was taken as a random effect variable. six olive oil samples for each treatment were used as replicated for storage study. this choice was done to understand both the behavior of unfiltered oils related to filtered oils, regardless of individual oil turbidity characteristics, and the separated role of water and solid particles during storage of unfiltered olive oils. results turbidity characterization immediately after production, the six vevoo samples (vo#1–vo#6) used in this study were characterized for different ‘turbidities’ (breschi et al., 2019). the turbidity grade ranged between 800 and 1,700 ntu, with water content between 0.15 and 0.40% w/w, water activity between 0.60 and 0.85, and insoluble solids content between 0.10 and 0.45% w/w. microbial cell count was between 2.5 and 4.5 log cfu g-1. after treatments, turbidity characteristics of olive oil samples changed radically. fevoo samples (fo#1– fo#6) were characterized by a degree of turbidity grade (10–20 ntu), water (0.04–0.05% w/w), and insoluble solids content (0.00% w/w), water activity (0.30–0.45), and microbial cell count (0.00 log cfu g-1), which were statistically (p > 0.05) lower than vo samples. the wo olive oil (wo#1–wo#6) and so olive oil (so#1–so#6) samples were characterized by turbidity characteristics, which were between vevoo and fevoo samples. the degree of turbidity grade for wo olive oil samples was between 40 and 90 ntu and that for so olive oil samples between 150 and 240 ntu. these turbidity grades were characterized by different water content (0.10–0.11% italian journal of food science, 2021; 33 (3) 37 quality of veiled olive oil: role of turbidity components ta bl e 1 m ea n va lu es o f ch em ic al p ar am et er s of a ll ol iv e oi l s am pl es fo r ea ch s ep ar at io n tr ea tm en t. d if fe re nt s up er sc ri be d le tt er s (a ,b ,c ) i n th e sa m e ro w in di ca te s ig ni fic an t d if fe re nc es (p < 0 .0 5) fo r di ff er en t t re at m en ts . d if fe re nt s up er sc ri be d le tt er s (x ,y ,z ) i n th e sa m e co lu m n in di ca te s ig ni fic an t d if fe re nc es (p < 0 .0 5) fo r di ff er en t s to ra ge p er io ds . f ol lo w in g ar e re po rt ed in th e la st fi ve c ol um ns : st an da rd e rr or ; p -v al ue fo r th e st or ag e tim e (p -v al ue t) ; p -v al ue fo r th e tr ea tm en t ( pva lu e t) ; p -v al ue fo r tim e– tr ea tm en t i nt er ac tio ns (p -v al ue t* t) ,, an d lim it va lu e fo r ‘e xt ra -v ir gi n’ c at eg or y (r e g . 20 16 /2 09 5) . ti m e (d ay s) fo #1 –f o #6 vo #1 –v o #6 s o #1 –s o #6 w o #1 –w o #6 s t. e rr . pva lu e t pva lu e t pva lu e t* t r 2 a d jr 2 li m it va lu e fo r ‘e xt ra -v ir gi n’ ca te go ry a ci di ty (% o le ic a ci d) 0 0. 19 a, x 0. 22 b, x 0. 16 a, x 0. 17 a, x 0. 01 n. s. ** * n. s. 0. 64 90 0. 60 96 45 0. 18 a, x 0. 24 b, x 0. 18 a, x 0. 20 a, x 12 0 0. 17 a, x 0. 25 b, x 0. 20 a, x 0. 18 a, x ≤0 .8 18 0 0. 17 a, x 0. 24 b, x 0. 21 a, x 0. 19 a, x 24 0 0. 18 a, x 0. 25 b, x 0. 16 a, x 0. 20 a, x pe ro xi de v al ue (m eq o 2/k g) 0 5. 4a ,x 6. 3a ,x 5. 9a ,x 5. 8a ,x 0. 2 n. s. n. s. n. s. 0. 12 02 0. 02 16 ≤2 0 45 7. 6a ,x 6. 4a ,x 7. 5a ,x 7. 2a ,x 12 0 5. 9a ,x 5. 9a ,x 6. 2a ,x 7. 2a ,x 18 0 7. 5a ,x 5. 8a ,x 5. 4a ,x 6. 9a ,x 24 0 9. 2a ,x 7. 5a ,x 7. 2a ,x 6. 3a ,x k 23 2 0 1. 69 a, x 1. 68 a, x 1. 77 a, x 1. 70 a, x 0. 01 ** n. s. n. s. 0. 55 42 0. 50 42 ≤2 .5 0 45 1. 76 a, xy 1. 74 a, x 1. 80 a, x 1. 79 a, y 12 0 1. 79 a, y 1. 78 a, y 1. 84 a, xy 1. 80 a, y 18 0 1. 81 a, y 1. 78 a, y 1. 82 a, x 1. 81 a, y 24 0 1. 84 a, y 1. 79 a, y 1. 87 a, y 1. 87 a, y k 27 0 0 0. 13 a, x 0. 15 a, x 0. 19 b, x 0. 15 a, x 0. 01 ** ** * n. s. 0. 53 40 0. 48 18 ≤0 .2 2 45 0. 15 a, xy 0. 16 a, x 0. 18 b, x 0. 16 a, x 12 0 0. 18 a, y 0. 17 a, xy 0. 21 b, x 0. 17 a, xy 18 0 0. 17 a, y 0. 17 a, xy 0. 20 b, x 0. 17 a, xy 24 0 0. 18 a, y 0. 18 a, y 0. 20 b, x 0. 18 a, y δk 0 –0 .0 05 a, x –0 .0 04 a, x –0 .0 04 a, x –0 .0 05 a, x 0. 00 0 ** * ** n. s. 0. 52 15 0. 46 78 ≤0 .0 1 45 –0 .0 05 a, x –0 .0 02 b, y –0 .0 03 ab ,x y –0 .0 03 ab ,y 12 0 –0 .0 02 a, y 0. 00 0b ,z –0 .0 02 a, y –0 .0 01 ab ,z 18 0 –0 .0 02 a, y 0. 00 0b ,z –0 .0 01 ab ,y –0 .0 01 ab ,z 24 0 –0 .0 02 a, y 0. 00 0b ,z –0 .0 02 a, y –0 .0 01 ab ,z n. s. , * , * *, a nd * ** in di ca te s ig ni fic an t d iff er en ce s by tw ow ay a n o va a t p > 0 .0 5, p ≤ 0 .0 5, p ≤ 0 .0 1, a nd p ≤ 0 .0 01 . n um be r o f re pl ic at es = 6 . v o : v irg in o il; w o : o liv e oi l c on ta in in g w at er o nl y; s o : o liv e oi l co nt ai ni ng s ol id p ar tic le s; f o : fi lte re d oi l. 38 italian journal of food science, 2021; 33 (3) breschi c et al. 5.36 4.95 3.25 4.22 3.61 1.10 0.48 0.00 0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 15 30 45 60 m ic ro bi al c el l c ou nt (l og c fu g –1 ) 75 90 105 time (days) 120 135 150 165 180 195 210 225 240 1.14 0.75 0.00 0.23 0.37 0.00 0.42 0.92 0.00 0.26 0.00 figure 1 mean contents and standard error of microbial cell count in samples of virgin oil (vo; red circle), olive oil containing water only (wo; blue diamond), olive oil containing solid particles (so; purple triangle), and filtered oil (fo; green square) during storage. the r2 and adj-r2 values of microbial cell count were 0.8522 and 0.8356, respectively. significant interactions between storage period and treatment (p ≤ 0.001) were determined for hydroxytyrosol and tyrosol contents, which statistically increased faster in vo samples than in wo > so > fo samples during storage (table 2). immediately after production (time = 0), the content of both hydroxytyrosol and tyrosol was lower than 10 mg kg–1 and 5 mg kg–1, respectively, in all samples. during the 240 days of storage, the contents increased statistically in all samples except fo samples. vo samples had content of hydroxytyrosol and tyrosol statistically (p ≤ 0.001) higher than in fo samples. content of both hydroxytyrosol and tyrosol in wo and so samples was statistically different (p ≤ 0.001) and was between the content determined in vo and fo samples. the contents of hydroxytyrosol, tyrosol, oleuropein, and ligstroside and their derivates were used to calculate r-index (r-index = [hydroxytyrosol + tyrosol]/[oleuropein and its derivates + ligstroside and its derivates]), a useful marker of the hydrolysis of secoiridoids (fiorini et al., 2018). during storage r-index increased significantly (p ≤ 0.001) in all treatments, demonstrating degradation of phenols (figure 2). the difference between treatments was statistically significant (p ≤ 0.001); except at the beginning of storage, the r-index of vo samples was always higher than that of fo and so samples. wo samples had intermediate value of r-index. moreover, time– treatment interactions were also statistically significant: in vo samples, the r-index gain was faster than in wo samples, which was faster than so and fo samples. the ratio of oxidized form of phenolic compounds to not oxidized form (ox:not ox) during storage period (figure 3) was determined to observe the effect of oxidation on phenolic compounds. immediately after production, fo samples showed the ox:not ox ratio value statistically (p ≤ 0.05) lower than in vo, wo, and so samples. after 240 days of storage, increase in oxidized forms of phenolic compounds made a statistically significant difference in treatment: the ox:not ox ratio value was higher in fo and so samples than in wo and vo samples. content of volatile compounds the content of volatile compounds in olive oil samples was studied as described in literature (guerrini et al., 2020a): pleasant lipoxygenase pathway (lox pathway) volatile compounds with five (c5) and six (c6) carbon atoms; unpleasant volatile compounds related to ‘fusty’/‘mouldy’/‘vinegary’ defects; and unpleasant volatile compounds related to ‘rancid’ defect. some statistically significant differences (p ≤ 0.05) were identified in c5 and c6 branches of lox pathway volatile compounds. a statistically significant main effect of filtration was detected in 1-hexanol, e-2-hexenol, z-3hexenol, 1-penten-3-one, and e-2-penten-1-ol volatile compounds (figure 4). the content of all these volatile compounds was higher in vo samples than in fo, wo, and so samples. the same statistically significant difference was also determined in 3-methyl-butanal, 2-octanol, and 2-nonanone unpleasant volatile compounds related to ‘fusty’ defect (figure 5). moreover, a statistically significant italian journal of food science, 2021; 33 (3) 39 quality of veiled olive oil: role of turbidity components ta bl e 2 m ea n va lu es o f to ta l c on te nt , c on te nt o f gr ou ps , a nd c on te nt o f si ng le r ep re se nt at iv e ph en ol ic c om po un ds o f al l o il sa m pl es fo r ea ch s ep ar at io n tr ea tm en t. d if fe re nt s up er sc ri be d le tt er s (a ,b ,c ) in th e sa m e ro w in di ca te s ig ni fic an t d if fe re nc es (p < 0 .0 5) fo r di ff er en t t re at m en ts . d if fe re nt s up er sc ri be d le tt er s (x ,y ,z ) i n th e sa m e co lu m n in di ca te s ig ni fic an t d if fe re nc es (p < 0 .0 5) fo r di ff er en t s to ra ge pe ri od s. in th e la st fo ur c ol um ns a re r ep or te d st an da rd e rr or ; p -v al ue fo r th e st or ag e tim e (p -v al ue t) ; p -v al ue fo r th e tr ea tm en t ( pva lu e t) ; a nd p -v al ue fo r tim e– tr ea tm en t i nt er ac tio ns (p -v al ue t* t) . ti m e (d ay s) fo #1 –f o #6 vo #1 –v o #6 s o #1 –s o #6 w o #1 –w o #6 s t. e rr or pva lu e t pva lu e t pva lu e t* t r 2 a d jr 2 h yd ro xy ty ro so l ( m g/ kg ) 0 2. 7a ,x 5. 0b ,x 6. 5b ,x 4. 4a b, x 1. 5 ** * ** * ** * 0. 79 85 0. 77 59 45 3. 1a ,x 14 .3 b, y 8. 1a b, x 8. 4a b, xy 12 0 4. 7a ,x 20 .0 b, yz 9. 4a b, x 11 .7 ab ,y 18 0 4. 7a ,x 20 .0 b, yz 9. 1a b, x 13 .5 ab ,y 24 0 5. 9a ,x 27 .9 b, z 15 .4 ab ,y 17 .5 ab ,y ty ro so l ( m g/ kg ) 0 2. 4a ,x 2. 9a ,x 3. 1a ,x 3. 1a ,x 0. 6 ** * ** * ** * 0. 75 04 0. 72 24 45 2. 8a ,x 5. 4a ,x 3. 5a ,x 3. 6a ,x 12 0 3. 0a ,x 7. 9b ,x y 4. 2a b, xy 4. 6a b, x 18 0 2. 9a ,x 10 .2 b, y 3. 8a b, xy 4. 1a ,b x 24 0 4. 1a ,x 11 .8 b, y 5. 4a b, y 7. 1a b, y s um o f ol eu ro pe in a nd it s de riv at es (m g/ kg ) 0 29 0. 9a ,x 36 9. 5b ,x 43 7. 9c ,x 38 4. 8b c, x 13 .9 n. s. ** * n. s. 0. 66 46 0. 62 70 45 24 8. 5a ,x 30 7. 8b ,x 42 7. 5c ,x 34 6. 1b c, x 12 0 30 7. 3a ,x 30 8. 4a ,x 43 8. 8b ,x 27 8. 3a ,x 18 0 29 8. 5a ,x 28 2. 6a ,x 42 5. 6b ,x 32 6. 8a ,x 24 0 32 5. 9b ,x 28 6. 6a ,x 44 4. 9c ,x 34 3. 5b ,x s um o f lig st ro si de a nd it s de riv at es (m g/ kg ) 0 15 2. 4a ,x 18 1. 4a ,x 14 9. 7a ,x 16 3. 7a ,x 6. 7 n. s. ** * n. s. 0. 38 22 0. 31 29 45 10 1. 4a ,x 19 8. 6b ,x 18 6. 4b ,x 17 3. 5b ,x 12 0 13 2. 6a ,x 17 8. 3a b, x 20 6. 4b ,x 14 9. 4a b, x 18 0 13 8. 1a ,x 17 6. 3a b, x 22 9. 9b ,x 16 1. 3a b, x 24 0 15 6. 2a ,x 17 8. 7a b, x 21 4. 8b ,x 17 8. 5a b, x to ta l c on te nt s (m g/ kg ) 0 54 8. 4a ,x 70 1. 2c ,x 72 4. 6c ,x 67 1. 5b ,x 20 .3 n. s. ** * n. s. 0. 61 79 0. 57 51 45 44 5. 5a ,x 65 5. 1b ,x 73 2. 7c ,x 64 4. 6b ,x 12 0 54 3. 6a ,x 64 6. 6b ,x 76 9. 6c ,x 55 6. 2a ,x 18 0 54 3. 3a ,x 63 8. 5b ,x 77 8. 5c ,x 60 9. 2b ,x 24 0 59 7. 6a ,x 63 7. 7b ,x 79 8. 0c ,x 67 6. 2b ,x n. s. , * , * *, a nd * ** in di ca te s ig ni fic an t d iff er en ce s by tw ow ay a n o va a t p > 0 .0 5, p ≤ 0 .0 5, p ≤ 0 .0 1, a nd p ≤ 0 .0 01 . n um be r o f re pl ic at es = 6 . v o : v irg in o il; w o : o liv e oi l c on ta in in g w at er o nl y; s o : o liv e oi l co nt ai ni ng s ol id p ar tic le s; f o : fi lte re d oi l. 40 italian journal of food science, 2021; 33 (3) breschi c et al. 0.00 0 60 120 time (days) r -in de x 180 240 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 figure 2. mean value, standard error of r-index in samples of virgin oil (vo; red circle and line), olive oil containing water only (wo; blue diamond and line), olive oil containing solid particles (so; purple triangle and line), and filtered oil (fo; green square and line) during storage. the r2 and adj-r2 values of r-index were 0.8343 and 0.8157, respectively. 0.25 0 60 120 time (days) o x /n ot ox 180 240 0.30 0.35 0.40 0.45 0.50 0.55 figure 3. mean value and standard error of phenolic oxidized–not oxidized form ratio (ox:not ox) in virgin oil vo (red circle), olive oil containing water only wo (blue diamond), olive oil containing solid particles so (purple triangle), and filtered oil fo (green square) samples during storage. the r2 and adj-r2 of ox/not ox were 0.1957 and 0.1055, respectively. effect of treatment was determined in some single and some c5 and c6 lox pathway volatile compounds, with lower content in so samples than in fo, wo, and vo samples because of stripping caused by freeze-drying. no statistically significant differences during storage period and no significant interactions between filtration and storage period were determined in all the evaluated volatile compounds of lox pathway and those related to ‘fusty’ defect. the main effect of treatment and storage period and their interactions were not statistically significant for the unpleasant volatile compounds related to ‘rancid’ defect. sensory evaluation the sensory attributes were evaluated and a significant (p ≤ 0.05) effect of treatment and storage period was determined. the positive ‘fruity’ attribute decreased during storage period in all samples. the vo and so samples were significantly less fruity than fo and wo samples after 120 days of production (table s1). the negative ‘fusty’ and ‘winey’ defects, both related to microbial activity, and ‘rancid’ defect, related to oxidation, showed significant (p ≤ 0.001) increase during storage, and were of higher values in vo samples than in fo, so, and wo samples after 45 days (table s1). furthermore, interactions between filtration and storage period were statistically significant for ‘fusty’, ‘winey’ and ‘rancid’ defects. indeed, these defects increased faster in vo samples than in fo, wo, and so samples (figure 6). the bitterness and pungency attributes significantly (p ≤ 0.001) decreased in intensity during storage (table s1). the vo samples were significantly (p ≤ 0.001) less bitter and pungent than so and fo samples after 45 days. wo samples were not tasted due to filtration with glass wool. italian journal of food science, 2021; 33 (3) 41 quality of veiled olive oil: role of turbidity components 00.0 0.2 0.4 0.6 0.8 1.0 1.2 60 120 time (days) 1-hexanol e-2-hexanol c on ce nt ra tio n (m gk g– 1 ) c on ce nt ra tio n (m gk g– 1 ) 180 240 0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 60 120 time (days) 180 240 00.0 0.2 0.6 0.4 0.8 1.0 1.4 1.2 1.6 60 120 time (days) z-3-hexanol 1-penten-3-one e-2-penten-1-ol c on ce nt ra tio n (m gk g– 1 ) c on ce nt ra tio n (m gk g– 1 ) 180 240 00.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 60 120 time (days) c on ce nt ra tio n (m gk g– 1 ) 180 240 0 0.0 0.2 0.1 0.3 0.4 0.5 0.6 60 120 time (days) 180 240 figure 4. mean contents and standard error of lipoxygenase (lox) pathway volatile compounds in virgin oil vo (red circle), olive oil containing water only wo (blue diamond), olive oil containing solid particles so (purple triangle), and filtered oil fo (green square) samples during storage. only compounds statistically significant different (p ≤ 0.05) for time and/or treatment are reported. the r2 and adj-r2 values for lox pathway volatile compound are as follows: 1-hexanol, r2 = 0.5003, adj-r2 = 0.4442; e-2-hexenol, r2 = 0.6473, adj-r2 = 0.6077; z-3-hexenol, r2 = 0.7068, adj-r2 = 0.6740; 1-peten-3-one, r2 = 0.5996, adj-r2 = 0.5547; and e-2-penten-1-ol, r2 = 0.7460, adj-r2 = 0.7175. discussion the experimental data highlighted that water and solid particles had some specific roles to play in the quality evolution of evoo during storage. the obtained results demonstrated that two degradation phenomena, hydrolysis and microbial activity, were faster in vo samples than in fo, wo, and so samples. the presence of water micro-droplets dispersed in oil matrix increased the water/oil exchange surface, and the hydrolysis reaction occurred to a significant extent (xenakis et al., 2010). the enzymatic hydrolysis of triglycerides produced, not esterified fatty acids, that increased the ffa value more in vo samples than in fo, so, and wo samples. furthermore, the formation of phenolic compounds with low molecular weight, such as hydroxytyrosol and tyrosol (due to chemical hydrolysis of phenolic compounds (zanoni, 2014; cinquanta et al., 1997)), was higher in vo samples than in fo, so, and wo samples. r-index confirmed the above trend in vo samples and established that wo 42 italian journal of food science, 2021; 33 (3) breschi c et al. 0 0.00 0.05 0.10 0.15 0.20 0.25 0.35 0.30 60 120 time (days) 3-methyl-butanal c on ce nt ra tio n (m gk g– 1 ) 180 240 0 0.00 0.05 0.10 0.15 0.20 0.25 0.35 0.40 0.45 0.50 0.30 60 120 time (days) 2-octanol c on ce nt ra tio n (m gk g– 1 ) 0 0.00 0.05 0.10 0.15 0.20 0.25 0.35 0.40 0.30 60 120 time (days) 2-nonanone c on ce nt ra tio n (m gk g– 1 ) 180 240 180 240 figure 5. mean contents and standard error of volatile compounds related to ‘fusty’ defect in virgin oil vo (red circle), olive oil containing water only wo (blue diamond), olive oil containing solid particles so (purple triangle), and filtered oil fo (green square) samples during storage. only compounds statistically significant different (p ≤ 0.05) for time and/or treatment are reported. the r2 and adj-r2 values for ‘fusty’ defect volatile compounds are as follows: 3-methyl-butanal, r2 = 0.4201, adj-r2 = 0.3551; 2-octanol, r2 = 0.7852, adj-r2 = 0.7611; and 2-nonanone, r2 = 0.5197, adj-r2 = 0.4659. samples, with intermediate water content, had intermediate hydrolytic activity (figure 2). the cause and effect relationship between the presence of micro-droplets of water in vo samples and the chemical hydrolytic phenomena of phenolic compounds were in accordance with the experimental data given in literature (guerrini et al., 2020b). the ‘fusty’ and ‘winey’ sensory defects and their related volatile compounds were strictly connected to the microbial activity. the microorganism cell count in vo samples was higher than in fo, so, and wo samples during storage; the microbial survival was due to the favorable environment of vo samples, starting with water activity of >0.6 (derossi et al., 2011), resulting in unpleasant volatile microbial metabolites, such as 3-methyl-butanal, 2-octanol, 2-nonanone (figure 5). the microbial activity was also helped by the content of solid particles. our results highlight that water has to be combined with solid particles for microbial growth. wo and so samples were not good for microbial survival, and only vo samples had favorable conditions for microbial growth (figure 1). the content of solid particles could be involved in promoting the transfer of phenols transfer from solid particles to oil. the so samples were able to show the above effect, thanks to both absence of water and slow hydrolytic phenomena of phenolic compounds. the significant higher contents of both total phenolic compounds and sum of oleuropein and its derivatives in so samples (table 2) could be explained by the mass transfer of phenolic compounds from solid particles to oil. solid particles consist of olive pulp and core fragments that are rich in high molecular weight phenolic compounds (jerman klen et al., 2015; cecchi et al., 2018; morales et al., 2005). however, the freeze-drying conditions led to initial oxidation, as shown in ox:not ox ratio values (figure 3), and stripping of volatile compounds which affected quality parameters, such as k270 (table 1), and development of ‘rancid’ defect (figure 6). derived from the experimental results, following are the other functions of water and solid particles in the quality evolution of evoo during storage, although they had some uncertain aspects. the water content seemed to promote the lox enzymatic pathway, which is responsible for ‘fruity’ positive italian journal of food science, 2021; 33 (3) 43 quality of veiled olive oil: role of turbidity components 0.00 0.05 0.10 0.15 0.20 0.25 0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 time (days) fusty fu st y va lu e 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 time (days) rancid r an ci d va lu e 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 time (days) winey w in ey v al ue figure 6. mean contents and standard error of the ‘fusty’, ‘winey’, and ‘rancid’ defect scores in virgin oil vo (red circle), olive oil containing water only wo (blue diamond), olive oil containing solid particles so (purple triangle), and filtered oil fo (green square) samples during storage. the r2 and adj-r2 for each sensory attribute are reported below: ‘fusty’, r2 = 0.4908, adj-r2 = 0.4337; ‘winey’, r2 = 0.6216, adj-r2 = 0.5792; ‘rancid’, r2 = 0.5960, adj-r2 = 0.5507. sensory attributes. the content of c5 and c6 volatile compounds of lox pathway was higher in vo samples than in fo and wo samples (figure 4); however, vo samples had a significant low level of ‘fruity’ sensory attributes than determined in fo and wo samples. we suppose that significant appearance of ‘fusty’ defect led panelists to measure decrease in the ‘fruity’ score of vo samples (guerrini et al., 2020a). the water content also seemed to protect evoo against negative oxidative phenomena during storage. the ox:not ox ratio of phenolic compounds (figure 3) was higher in fo and so samples than in wo and vo samples because of the stabilizing effect of water on oxidative degradation, as demonstrated in literature (lercker et al., 1994; ambrosone et al., 2002; koidis and boskou, 2006; frega et al., 1999). however, the protective effect of water was not shown for chemical parameters, k232, k270, and ∆k, which did not increase significantly during storage as a function of treatments. the effect of treatments was not statistically significant for unpleasant volatile compounds, commonly related to ‘rancid’ sensory defect. instead, the ‘rancid’ sensory defect behavior during storage demonstrated an opposite trend to the above oxidation phenomena: the ‘rancid’ scores were higher in vo samples than in fo, so, and wo samples. the significant appearance of ‘fusty’ defect led panelists to measure an increase in the ‘rancid’ score of vo samples, since these two defects are characterized by some common volatile compounds (morales et al., 2005). conclusions in this study, an original approach was carried out to understand the significance of vo in terms of preservation of evoo quality during storage. a clear effect of water content on hydrolytic phenomena and microbial activity was evidenced. effect of content of solid particles to promote microbial activity was also demonstrated, potentially resulting in the loss of evoo quality. the results of the present study asserted that the recommended technique to avoid significant degradation during storage was to quickly filter freshly produced olive oil. however, an immediate filtration is not always possible as veiled olive oil is the product sought for bottling by producers. therefore, a qualification of oil turbidity, 44 italian journal of food science, 2021; 33 (3) breschi c et al. characterization of veiled extra virgin olive oil turbidity for degradation risk assessment.  eur. j. lipid sci. tech. 121(11), 1900195, 1–6. https://doi.org/10.1002/ejlt.201900195 bubola, k.b., lukić, m., mofardin, i., butumović, a., and koprivnjak, o. 2017. filtered vs. naturally sedimented and decanted virgin olive oil during storage: effect on quality and composition. lwt. 84, 370–377. https://doi.org/10.1016/j.lwt.2017.05.069 cayuela, j.a., gómez-coca, r.b., moreda, w., and pérez-camino, m.d.c. 2015. sensory defects of virgin olive oil from a microbiological perspective.  trends food sci. tech. 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profiles of olive oil produced in traditional mills in morocco. j. mat. env. sci. 2508, 854–863. https://doi.org/10.26872/jmes.2018.9.3.94 el riachy, m., priego-capote, f., león, l., rallo, l., and luque de castro, m.d. 2011. hydrophilic antioxidants of virgin olive oil. part 1: hydrophilic phenols: a key factor for virgin olive oil quality.  eur. j. lipid sci. tech. 113(6), 678–691. https://doi. org/10.1002/ejlt.201000400 european union commission. 2003. commission implementing regulation (ec) no. 2016/2095 of 26 september 2016 amending regulation no. 2568/91 on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis. off. j. eur. union. l 295, 57–77. based on separate measurement of water and insoluble solids contents, is suggested during different processing steps of olive oil chain, such as vo storage in mills, vo supply and storage in oil blenders, and transportation and distribution of veiled evoo. it follows that, for olive oil producers, the qualification of veiled olive oil in potentially different combinations of water and solid contents (i.e., high–high, high–low, low–high, or low–low) could be useful to plan and control both water/solid separation techniques and storage of oil. author contributions lorenzo guerrini, alessandro parenti, and bruno zanoni did conceptualization; carlotta breschi and lorenzo guerrini curated the data. formal analysis was done by carlotta breschi, ferdinando corti, and luca calamai. funding acquisition was done by alessandro parenti and bruno zanoni. methodology was done by luca calamai and paola domizio; and software handling was done by carlotta breschi and lorenzo guerrini. supervision was carried out by alessandro parenti and bruno zanoni. original draft was written by carlotta breschi and lorenzo guerrini; and the final writing—review and editing—was done by lorenzo guerrini, alessandro parenti, and bruno zanoni. funding this study was supported by the ager 2 project, grant no. 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https://doi.org/10.1002/ejlt.201700309� https://doi.org/10.1002/ejlt.201700309� https://doi.org/10.3390/colloids4010011� https://doi.org/10.3390/microorganisms8030402� https://doi.org/10.3390/microorganisms8050663� https://doi.org/10.3390/microorganisms8050663� https://doi.org/10.1016/j.foodres.2017.11.005� https://doi.org/10.1016/j.talanta.2016.12.082� https://doi.org/10.1002/ejlt.201500229� https://doi.org/10.1007/s11746-999-0239-4� https://doi.org/10.1002/ejlt.200501175� https://doi.org/10.1002/ejlt.200501175� https://doi.org/10.3390/foods9081067� https://doi.org/10.3390/foods9081067� https://doi.org/10.3390/molecules25020420� https://doi.org/10.14674/1120-1770/ijfs.v190� https://doi.org/10.14674/1120-1770/ijfs.v190� https://doi.org/10.1021/jf506353z� https://doi.org/10.1021/jf506353z� https://doi.org/10.1002/ejlt.200500319� 46 italian journal of food science, 2021; 33 (3) breschi c et al. ta bl e s 1 m ea n va lu es o f se ns or y at tr ib ut es a nd d ef ec ts o f al l o il sa m pl es fo r ea ch s ep ar at io n tr ea tm en t. s up er sc ri be d di ff er en t l et te rs (a ,b ,c ) i n th e sa m e ro w in di ca te s ig ni fic an t d if fe re nc es (p < 0 .0 5) fo r di ff er en t t re at m en ts . s up er sc ri be d di ff er en t l et te rs (x ,y ,z ) i n th e sa m e co lu m n in di ca te s ig ni fic an t d if fe re nc es (p < 0 .0 5) fo r di ff er en t s to ra ge p er io ds . f ol lo w in g ar e re po rt ed in th e la st fo ur c ol um ns : st an da rd e rr or ; p -v al ue fo r th e st or ag e tim e (p -v al ue t) ; p -v al ue fo r th e tr ea tm en t ( pva lu e t) ; a nd p -v al ue fo r tim e– tr ea tm en t i nt er ac tio n (p -v al ue t* t) . ti m e (d ay s) fo #1 –f o #6 vo #1 –v o #6 s o #1 –s o #6 w o #1 –w o #6 s t. e rr . pva lu e t pva lu e t pva lu e t* t r 2 a d jr 2 fu st y 0 0. 00 a, x 0. 00 a, x 0. 00 a, x 0. 00 a, x 0. 16 ** * ** * * 0. 49 08 0. 43 37 45 0. 28 a, x 0. 68 b, xy 0. 00 a, x 0. 05 a, x 12 0 0. 75 a, xy 1. 96 b, yz 0. 38 a, x 0. 59 a, x 18 0 1. 08 a, y 1. 79 b, y 1. 73 b, y 0. 53 a, x 24 0 0. 60 a, xy 2. 23 c, z 1. 39 b, y 0. 61 a, x m ud dy /h um id ity 0 0. 00 a, x 0. 00 a, x 0. 00 a, x 0. 00 a, x 0. 06 ** * n. s. n. s. 0. 20 23 0. 11 29 45 0. 00 a, x 0. 00 a, x 0. 00 a, x 0. 00 a, x 12 0 0. 63 a, y 0. 78 a, y 0. 50 a, y 0. 66 a, y 18 0 0. 00 a, x 0. 30 b, xy 0. 08 a, x 0. 00 a, x 24 0 0. 00 a, x 0. 39 b, xy 0. 10 a, x 0. 00 a, x w in ey 0 0. 00 a, x 0. 00 a, x 0. 00 a, x 0. 00 a, x 0. 09 ** * ** * ** 0. 62 16 0. 57 92 45 0. 00 a, x 0. 55 b, xy 0. 08 a, x 0. 00 a, x 12 0 0. 00 a, x 1. 03 b, y 0. 12 a, xy 0. 08 a, x 18 0 0. 17 a, y 1. 08 b, y 0. 19 a, y 0. 14 a, x 24 0 0. 15 a, y 1. 32 b, y 0. 14 a, xy 0. 12 a, x r ac id 0 0. 00 a, x 0. 00 a, x 0. 00 a, x 0. 00 a, x 0. 26 ** * ** * n. s. 0. 59 60 0. 55 07 45 0. 65 ab ,x 1. 33 b, y 0. 83 ab ,x 0. 00 a, x 12 0 1. 70 a, y 3. 18 c, z 2. 36 b, y 0. 43 a, xy 18 0 1. 57 a, xy 3. 14 c, z 2. 38 b, y 0. 82 a, y 24 0 1. 65 a, xy 3. 25 c, z 2. 42 b, y 0. 95 a, y fr ui ty 0 3. 40 a, y 3. 37 a, y 3. 12 a, y 3. 57 a, z 0. 19 ** * ** * n. s. 0. 53 39 0. 48 16 45 2. 98 a, xy 2. 33 a, xy 2. 63 a, xy 3. 03 a, xy 12 0 2. 31 b, x 1. 03 a, x 1. 83 a, x 1. 97 b, x 18 0 2. 48 b, x 1. 18 a, x 1. 16 a, x 2. 65 b, xy 24 0 2. 51 b, x 1. 05 a, x 1. 01 a, x 2. 11 b, x b itt er 0 3. 42 a, z 3. 30 a, z 2. 80 a, y n. d. 0. 27 0. 74 10 0. 71 19 45 2. 85 a, y 2. 03 a, y 2. 72 a, y n. d. 12 0 1. 93 b, x 0. 47 a, x 2. 27 b, xy n. d. 18 0 2. 99 b, y 1. 68 a, y 1. 95 b, x n. d. 24 0 2. 58 b, xy 1. 12 a, xy 1. 90 b, x n. d. p un ge nt 0 4. 96 a, z 4. 53 a, z 4. 90 a, z n. d. 0. 39 0. 83 27 0. 81 39 45 3. 53 b, y 2. 73 a, y 3. 93 b, y n. d. 12 0 1. 78 b, x 0. 63 a, x 2. 63 b, x n. d. 18 0 3. 40 b, y 1. 21 a, x 3. 08 b, x n. d. 24 0 2. 75 b, xy 1. 05 a, x 2. 97 b, x n. d. n. s. , * , * *, a nd * ** in di ca te s ig ni fic an t d iff er en ce s by tw ow ay a n o va a t p > 0 .0 5, p < 0 .0 5, p < 0 .0 1, a nd p < 0 .0 01 . n .d . = n ot d et ec te d. n um be r o f re pl ic at es = 6 . v o : v irg in o il; w o : o liv e oi l c on ta in in g w at er on ly ; s o : o liv e oi l c on ta in in g so lid p ar tic le s; f o : fi lte re d oi l. ole_link1 ole_link2 _goback p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (2): 43–49 issn 1120-1770 online, doi 10.15586/ijfs.v34i2.2106 43 p u b l i c a t i o n s codon the role of whey protein in myogenic differentiation rui xu1*, yuejuan xiao2, ying zhang2, xiyan zhao1 1college of food science and technology, hebei normal university of science and technology, changli, qinhuangdao, china; 2college of marine resources and environment, hebei normal university of science and technology, changli, qinhuangdao, china *corresponding author: rui xu, college of food science and technology, hebei normal university of science and technology, changli, qinhuangdao, 066600, china. email: robust100@163.com received: 25 july 2021; accepted: 28 march 2022; published: 26 april 2022 © 2022 codon publications open access paper abstract whey protein has been shown to prevent exercise-induced protein degradation and muscle damage. we hypothesized that whey protein would regulate muscle cell differentiation. adding various concentrations of whey protein to c2c12 myoblasts induced cell differentiation and myod (myogenic differentiation protein) expression as well as the phosphorylation of akt (protein kinase b). whey protein-induced differentiation-specific markers increased the enzymatic activities of creatine kinase and citrate synthase and the expression of muscle-specific microrna. whey protein elevated akt phosphorylation on thr308 and ser473, which was inhibited by ly294002 (a non selective phosphoinositide 3-kinases inhibitor), suggesting that whey protein acts via pi3k (phosphatidylinositol 3-kinase). blocking of the pi3k/akt pathway with specific inhibitors revealed its requirement in mediating the promotive effects of whey protein on c2c12 cell differentiation. these effects of whey protein on myoblast differentiation suggest its positive influence in preventing muscle atrophy. keywords: c2c12, myoblast differentiation, whey protein introduction whey protein, including essential branched-chain amino acid, is one of the latest dietary supplements for promoting/aiding the increase in strength and improving the lean body mass. the branched-chain amino acid has been shown to increase muscle hypertrophy by inhibiting muscle degradation (aoyama et al., 2019). a change in lean muscle mass is often attributed to a changed balance between protein synthesis and degradation rates in tissues. however, the direct effect of whey protein on myogenic mechanisms has never been studied. here we examined the effects of whey protein on the differentiation of c2c12 myoblasts. skeletal muscle formation or myogenesis is a complex and highly regulated process that involves the proliferation of myoblasts, exit of these myoblasts from the cell cycle, and their differentiation into muscle fibers (hernández-hernández et al., 2017). the sequential expression of myogenic regulatory factors (mrfs), a group of basic helix-loop-helix (bhlh) transcription factors that include myod, myf-5 (myogenic factor 5), myogenin, and mrf4 is required (seale and rudnicki, 2000). myod and myf-5 are the primary mrfs required for the formation, proliferation, and survival of myoblasts whereas myogenin and mrf-4 act later to control myoblast differentiation and induce the expression of important muscle-specific genes, such as myosin heavy chain and creatine kinase. micrornas (mirnas) are a class of highly conserved, non-coding rnas molecules that play key roles in posttranscriptional gene regulation. a small number mailto:robust100@163.com 44 italian journal of food science, 2022; 34 (2) xu r et al. enzyme assays the activities of two marker enzymes of differentiation, creatine kinase (ck) and citrate synthetase (cs), were assayed in cellular extracts using a spectrophotometric based kit from nanjing jiancheng bioengineering institute (nanjing, china) and shanghai genmed pharmaceutical co., ltd (shanghai, china), respectively. detection of mature mirnas to detect mirna in all rna samples, the all-in-one mirna q-pcr detection kit and pcr primer sets for mir-1, -133a, and -206 were used according to the manufacturer’s directions (genecopoeia, guangzhou, china). further, the expression of all samples was normalized to rnu6 (u6 small nuclear rna) levels to account for possible differences in the amount of initial rna. quantitative real-time pcr isolation of all rna and rt-pcr was done as described by dogra et al. (2006), but with a minor modification. briefly, an rnaiso plus kit (takara, dalian, china) was used to extract rna from cells. all rna samples were reverse-transcribed using gene-specific primer and fermentas’s reverse transcription kit according to the manufacturers’ instructions. the sequences of primers used were as follows: myod, 5΄-tgcggtgcacccaggcccag-3΄ (forward) and 5΄-ccgcctcactgtagtaggcg-3΄ (reverse); myf5, 5΄-caagagtagcagccttcgga-3΄ (forward) and 5΄-ggagcttttatctgcagcac-3΄(reverse); myogenin, 5΄-gctatgagcggactgagctc-3΄ (forward) and 5΄-ggagtgcagattgtgggcgt-3΄ (reverse); gapdh, 5΄-ccttcattgacctcaactac-3΄ (forward) and 5΄-agccccacggccatcacgcc-3΄ (reverse). quantitative real-time pcr was conducted using the same primers as described above and following a method as detailed by dogra et al. (2006). data normalization was accomplished using the endogenous control (gapdh) and the normalized values were subjected to a 2–δδct threshold cycle formula to calculate the fold change between the control and experiment groups. western blot analysis cells were extracted with (radio immunoprecipitation assay) (ripa) buffer and lysates were cleared by centrifugation. equal amounts of protein were loaded and resolved by 12% (w/v) sds-page, then transferred to nitrocellulose membranes (whatman). the membranes were blocked with 5% nonfat dry milk in trisbuffered saline-0.1% tween (tbst) and probed overnight with primary antibodies in tbst supplemented with 5% bovine serum albumin. after reaction in tbst with horseradish peroxidase-conjugated secondary antibodies (boisynthesis, beijing, china), bands were visualized using enhanced hrp-dab reagents (tiangen, beijing). of muscle-specific mirnas have been shown to have a crucial role in myoblast proliferation and differentiation (wang et al., 2018). numerous mirna expression profiling studies have consistently shown that mirna-1 (mir-1), mir-133a, and mir-206 have to be muscle-specific (motaei et al., 2019). analysis of the presumptive mir-1 promoter has shown that mir-1 expression is regulated by srf (serum response factor), myod, mef2 (myocyte enhancer factor-2), and dorsaland twist-binding sites, all factors known to be important in conferring muscle specific gene expression (zhang et al., 2021). mir-1 promotes the differentiation and exit of cardiac and skeletal progenitors from the cell cycle in mammals. the phosphoinositide 3΄-kinase (pi3k) plays a crucial role in effecting alterations in a broad range of cellular functions in response to extracellular signals. akt phosphorylates a variety of substrates involved in the regulation of key cellular functions including cell growth and survival and protein translation. the pi3k/akt pathway has been reported to play a major role in inducing cell differentiation and hypertrophy (rommel et al., 2001). given the importance of the muscle-specific mirnas and pi3k/akt signaling in muscle development, we must have the interest to determine what role they may have in skeletal muscle plasticity. to gain insight into the roles of whey protein in muscular hypertrophy, we analyzed the profiles of mir-1, mir-133a, and mir-206 expression as well as pi3k/akt pathway in c2c12 cells. materials and methods materials c2c12 myoblast was purchased from china center for type culture collection. dulbecco’s modified eagle’s medium (dmem) and fetal bovine serum (fbs) were purchased from gibco (grand island, usa). whey protein concentration contained 80.05% protein, which was measured using the kjeldahl method. ly294002 is ly 294002 hydrochloride, purchased from beyotime institute of biotechnology (jiangsu, china). cell culture the cells were grown in dmem containing 10% fbs. once cells had reached 80% confluency, the medium was changed to dmem supplemented with 2% horse serum. c2c12 myoblasts were differentiated into myotubes by incubation in the differentiation medium (2% horse serum in dmem). myotubes were maintained in the differentiation medium, and the medium was changed every 48 h. the inhibitors ly294002 (20 μm) was added before whey protein, which was used at 0.1 mg/ml or 0.4 mg/ml for c2c12 myoblasts. italian journal of food science, 2022; 34 (2) 45 the role of whey protein myogenic differentiation by influencing the mrf transcript levels. mirna expression there was no difference in the level of expression for mirna-206, which was 2.6-fold higher in the low-dose group relative to the control group. as shown in figure 2, expression of mirna-1 and mirna-206 increased by ~2.5fold in response to whey protein, whereas mirna-133a expression increased by ~1.7-fold. in contrast to the other mirna, mirna-206 and mir-133a expression did not change significantly after the low-dose treatment. the observed change in mirna expression suggested there would be a similar change in the expression of the mature muscle-specific mirnas, mir-1, mir-133a, and mir-206. pi3k/akt signaling pathways given our results, we sought to determine whether whey protein induces signaling pathways such as that of pi3k/akt. in serum-starved c2c12 cells, akt phosphorylation on amino acid ser473 was upregulated after incubation with whey protein in an elevated manner. akt phosphorylation was induced in c2c12 cells in a dose-dependent manner (figure 3). whey protein induced the phosphorylation of akt phosphorylation sites, ser473 and thr308, both are required for full kinase activity. this phosphorylation was completely abolished in the presence of the specific pi3k inhibitor ly294002. ly294002 treatment completely abolished the whey-induced akt phosphorylation, implying the requirement of pi3k activation for this response. increases in protein synthesis require activation of the translational machinery and in addition may consist of indirect effects after stimulation of gene transcription. to test whether akt phosphorylation is required for whey protein’s stimulatory effect on myoblast protein synthesis, c2c12 myoblasts were treated with 0.1 and 0.4 mg/ml whey protein for 24 h with or without ly294002, a stable pi3k inhibitor, which was added 30 min before the whey protein addition. a western blot analysis for mhc (myosin heavy chain) revealed that ly294002 addition prevented the whey-induced mhc levels in c2c12 cells (figure 3). in the absence of ly294002, treatment with 0.4 mg/ml whey protein increased baseline protein synthesis compared to untreated controls for serine and threonine phosphorylation, respectively. these results indicate a strong impact of pi3k on baseline protein synthesis. the whey-induced increase in protein synthesis was reduced by ly294002 above the corresponding control conditions, indicating that pi3k contributes to whey protein-induced protein synthesis. the whey-induced pi3k-dependent akt phosphorylation is required for whey’s promotive effect on the after blocking, the membranes were incubated with the following primary antibodies: polyclonal anti-akt (cell signaling), anti-phospho-akt (thr308), anti-phospho-akt (ser473), and monoclonal anti-myosin heavy chain (santa cruz), and polyclonal anti-β-actin antibody (boisynthesis). statistical analysis results are expressed as mean ± se. a value of p < 0.05 was considered statistically significant unless otherwise specified and statistical analyses were performed using origin 8.0. results assay of marker enzyme the addition of various concentrations of whey protein to serum-starved c2c12 myoblasts for 24 h enhanced the enzymatic activity of the muscle differentiation-specific markers creatine kinase and citrate synthase in a dose-responsive manner, with the highest levels being observed at 0.4 mg/ml whey protein. the enzyme activities of cs and ck are shown in table 1. there was no significant change in cs activities for the concentration of 0.1 mg/ml. comparisons of the activities of markers of differentiated skeletal muscle cs and ck revealed increases from 2.61 to 5.87 u/mg·prot and from 41.7 to 68.4 μmol/mg·min, respectively. myogenic gene expression since the activation of mrfs is a prerequisite for myogenesis, we investigated the effect of whey protein on the expression of myod, myogenin, and myf-5. c2c12 myoblasts were incubated in dm alone or with 0.1 and 0.4 mg/ml whey protein. the expression of myod, myogenin, and myf-5 mrna was measured using qrt-pcr. as shown in figure 1, whey protein increased the expression of myod, myf5, and myogenin in c2c12 myoblasts. the mrna levels of these genes were significantly higher in the experimental group as compared with the control group. these data suggest that whey protein regulates table 1. enzymatic activity of two marker enzymes of differentiation. activity control 0.1 mg/ml whey 0.4 mg/ml whey ck(u/mg·prot) 2.61 ± 0.12 3.94 ± 0.25* 5.87 ± 0.39+ cs(μmol/mg·min) 41.7 ± 3.09 53.2 ± 3.22 68.4 ± 4.41* data are expressed as means ± se. * statistically significant difference from control (p < 0.05). + significantly different from control (p < 0.01). 46 italian journal of food science, 2022; 34 (2) xu r et al. and cs, the well-known and accepted markers of differentiation, cultures treated with a differentiation medium showed a higher activity, evidencing a higher degree of differentiation. as expected, a dose-dependent promotion in both ck and cs activity was seen in the presence of whey protein in c2c12 cultures. many literatures (griffen et al., 2022; thalacker-mercer et al., 2020) have shown that whey protein can promote muscle hypertrophy because it contains branched-chain amino acids. in the pre-experiment, we found that when whey protein concentration was 0.1 mg/ml, it showed an effect compared with the control group, so we chose these two doses (0.1 and 0.4 mg/ml). skeletal muscle differentiation can be regulated by at least three possible mechanisms, which include alteration in differentiation of myoblasts. taken together, these data suggest that whey protein has a beneficial effect on adult skeletal muscle, both during normal growth and under stress conditions. discussion our study demonstrates that whey protein promotes general protein accretion, muscle-specific gene expression, and myoblast fusion during myogenic differentiation. moreover, our data suggest that stimulation of these processes by whey protein relies on positive regulation of akt because increased akt phosphorylation and a subsequent increase in mhc protein were observed in response to whey protein. in the enzymatic assay of ck figure 1. whey protein increases markers of myogenic differentiation. c2c12 myoblasts were incubated in the absence or presence of whey protein at various concentrations for 24 h, and mrna expression levels of myogenin, myod, and myf5 were analyzed by quantitative real-time pcr using gapdh as an internal control. *statistically significant difference from control (p < 0.05). myod myf5 group myogenin control 0.1 mg/ml whey 0.4 mg/ml whey m r n a r e la tiv e e xp re ss io n 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 figure 2. whey protein increases the expression of muscle-specific mirna. c2c12 myoblasts were incubated in the absence or presence of whey protein at various concentrations for 24 h, and mirna expression levels were analyzed by fluorescence quantitative real-time pcr using u6 as an internal control. *statistically significant difference from control (p < 0.05). m ir n a r e la tiv e e xp re ss io n mirna-1 mirna-206 control 0.1 mg/ml whey 0.4 mg/ml whey mirna-133a group 0 0.5 1 1.5 2 2.5 3 italian journal of food science, 2022; 34 (2) 47 the role of whey protein mir-1, mir-133a, and mir-206 are transcribed during myogenesis and seem to be regulated by myod and myogenin (rosenberg et al., 2006). mir-206 is transcribed independently of mir-1 and mir-133a, which are transcribed as a common pri-mirna precursor from the mir-1/mir-133a locus, followed by alternative splicing to generate different primary transcripts (taylor and hughes, 2017). this is consistent with the present finding that mir-1 and mir-133a expression showed similar patterns in their induction and increase during differentiation. these observations suggested that mir-206 might be induced by myod whereas mir-1 and mir-133a might be induced by myogenin, as the onset and temporal sequence of mir-206 and mir-1/mir-133a expression seem to coincide with those of myod and myogenin expression during myogenesis. after their induction by myod and myogenin, however, other factors must be involved in the expression of these mirnas at later stages. the challenge for future studies will be to identify relevant target genes of each muscle-specific mirna and how they contribute to the regulation of skeletal muscle growth and phenotype. further studies are needed to address these complex regulatory roles of mirnas, and the identification of bona fide, biologically relevant targets for mirnas will be an important goal for mirna researchers in the future. whey protein has been observed to influence muscle strength via various mechanisms, such as decreasing protein degradation and attenuating the proteosome– ubiquitin degradation pathway on the one hand, and increasing protein synthesis on the other. taken together, our findings suggest that, at least in culture, whey protein can drive c2c12 myoblasts into the cell cycle, thus acting as a mitogen in these cells. the differential response of c2c12 cells to whey protein suggests this mitogen’s species-specific effect. many investigators have reported the stimulatory effect of essential amino acids on mrna translation (sans et al., 2021), which generally relies on the enhanced activity of the ribosomal protein s6 kinase (s6k1) resulting from signaling through the pi3k/akt/ mtor pathway. whey protein increased the protein levels of mhc in a dose-dependent manner. the signaling pathways through which whey protein exerts its effects are not fully understood. in this study, we demonstrate for the first time the promotive effect of whey protein on akt phosphorylation and the requirement of this pathway to mediate its effects, as demonstrated by employing specific inhibitors. by blocking the pi3k/akt pathway with specific inhibitors, we demonstrate that this pathway is required in mediating the promotive effect of whey protein on muscle cell differentiation. the crucial role of akt in protein degradation, mrna stabilization, and gene transcription. although evidence exists to support the first two mechanisms (lee et al., 2019; vaisid and kosower, 2013), modulation in gene expression appears to be most important during differentiation. this occurs via the activation of various phosphatases and kinases, which in turn alter the activity of downstream regulatory factors such as mef2, myf-5, myogenin, myod, serum response factor, and nuclear factor of activated t cells. since the presence of whey protein increased the transcripts of myod, myf-5, and myogenin (figure 1) and also promoted the protein synthesis of mhc in myoblasts (figure 3), our data suggest that the increased myogenesis is the result of increased transcription of mrfs. the current investigation was undertaken as an initial survey of muscle-specific mirna expression in response to whey-induced differentiation to determine if these novel trans-factors have a potential role in adult skeletal muscle hypertrophy. the results of this study comprehensively provide the first evidence of mirnas functions during whey-induced muscle hypertrophy and the foundation for future studies directed at elucidating the role of specific mirnas during muscle growth. the possibility that mir-206 may have a role in the regulation of skeletal muscle hypertrophy is supported by a recent study demonstrating that mir-206 contributes to the hypertrophic phenotype of texel sheep (qin et al., 2017). quantitative trait loci mapping revealed texel sheep have a single nucleotide polymorphism (snp) within the 3΄-utr of the myostatin gene that results in the formation of a functional mir-1/mir-206 target site. figure 3. the pi3k/akt pathway is induced by whey protein. akt phosphorylation of serum-starved c2c12 myoblasts in response to various concentrations of whey protein. akt phosphorylation was analyzed by western blot with the antiphospho-akt antibody followed by densitometric analysis normalized to levels of β-actin. the western blot analysis for mhc in c2c12 cells treated for 24 h with different concentrations of whey protein without or with ly294002 (25 μm). ly294002 whey (mg/ml) akt p-thr p-ser actin mhc 0 0.1 0.4 + 0.1 + 0.4 48 italian journal of food science, 2022; 34 (2) xu r et al. in healthy active older men: a randomised, double-blind, placebo-controlled trial. experimental gerontology 158: 111651–111657. https://doi.org/10.1016/j.exger.2021.111651 hernández-hernández, j.m., garcía-gonzález, e.g., brun, c.e. and rudnicki, m.a., 2017. the myogenic regulatory factors, determinants of muscle development, cell identity and regeneration. seminars in cell & developmental biology 72: 10–18. https:// doi.org/10.1016/j.semcdb.2017.11.010 kwak, s.s., kang, k.h., kim, s., lee, s., lee, j.-h., kim, j.w., byun, b., meadows, g.g. and joe, c.o.et al, 2016. amino acid-dependent nprl2 interaction with raptor determines mtor complex 1 activation. cellular signalling 28(2): 32–41. https://doi.org/10.1016/j.cellsig.2015.11.008 lee, s.-j., bae, j.h., lee, h., lee, h., park, j., kang, j.-s. and bae, g.-u., 2019. ginsenoside rg3 upregulates myotube formation and mitochondrial function, thereby protecting myotube atrophy induced by tumor necrosis factor-alpha. journal of ethnopharmacology 242: 112054–112059. https://doi.org/10.1016/ j.jep.2019.112054 motaei, j., yaghmaie, m., ahmadvand, m., pashaiefar, h. and kerachian, m.a., 2019. micrornas as potential diagnostic, prognostic, and predictive biomarkers for acute graft-versushost disease. biology of blood and marrow transplantation 25(12): e375–e386. https://doi.org/10.1016/j.bbmt.2019.08.004 qin, y., peng, y., zhao, w., pan, j., ksiezak-reding, h., cardozo, c., wu, y., pajevic, p.d., bonewald, l.f., bauman, w.a. and qin, w. et al, 2017. myostatin inhibits osteoblastic differentiation by suppressing osteocyte-derived exosomal microrna-218: a novel mechanism in muscle-bone communication. journal of biological chemistry 292(26): 11021–11033. https://doi. org/10.1074/jbc.m116.770941 rommel, c., bodine, s.c., clarke, b.a., rossman, r., nunez, l., stitt, t.n., yancopoulos, g.d. and glass, d.j., 2001. mediation of igf-1-induced skeletal myotube hypertrophy by pi(3)k/akt/ mtor and pi(3)k/akt/gsk3 pathways. nature cell biology 3: 1009–1013. https://doi.org/10.1038/ncb1101-1009 rosenberg, m.i., georges, s.a., asawachaicharn, a., analau, e. and tapscott, s.j., 2006. myod inhibits fstl1 and utrn expression by inducing transcription of mir-206. journal of cell biology 175: 77–85. https://doi.org/10.1083/jcb.200603039 sans, m.d., crozier, s.j., vogel, n.l., d’alecy, l.g. and williams, j.a., 2021. dietary protein and amino acid deficiency inhibit pancreatic digestive enzyme mrna translation by multiple mechanisms. cellular and molecular gastroenterology and hepatology 11(1): 99–115. https://doi.org/10.1016/j.jcmgh.2020.07.008 seale, p. and, rudnicki, m.a., 2000. a new look at the origin, function, and “stem-cell” status of muscle satellite cells. developmental biology 218: 115–124. https://doi.org/10.1006/ dbio.1999.9565 taylor, m.v. and hughes, s.m., 2017. mef2 and the skeletal muscle differentiation program. seminars in cell & developmental biology 72: 33–44. https://doi.org/10.1016/j.semcdb.2017.11.020 thalacker-mercer, a., riddle, e. and barre, l., 2020. chapter two – protein and amino acids for skeletal muscle health in aging. advances in food and nutrition research 91: 29–64. https://doi. org/10.1016/bs.afnr.2019.08.002 myogenic differentiation and hypertrophy induced by whey protein has been well demonstrated in this study. recent studies have reported that a class iii pi3k mediates the amino acid activation of the mammalian target of rapamycin (mtor) and its downstream molecules, which is distinct from the effect of insulin (kwak et al., 2016). our findings indicate that whey protein fully activates akt by inducing phosphorylation on both thr308 and ser473, while the latter phosphorylation is inhibited by ly294002, a specific pi3k inhibitor. however, we cannot rule out the possibility that whey protein activates the mtor/p70s6k pathway in a manner similar to other amino acids. therefore, we believe that the promotive effects, shown here, of whey protein on muscle cell differentiation, together with its previously demonstrated anti-catabolic effects, justify its supplement as a therapeutic strategy to prevent muscle loss in myopathies as well as in aging and trauma. however, together with mrna content, it could be expressed by showing the differences in cell morphology and quantifying the level of index fusion of the number of nuclei by another measurement method. moreover, the pi3k/akt axis such as p38 mapk could also be discussed and we would have a great interest in it in the future. acknowledgments this project was sponsored by the research project of hebei education department, china (zc2021230). conflict of interest there are no conflicts of interest. references aoyama, s., hirooka, r., shimoda, t. and shibata, s., 2019. effect of different sources of dietary protein on muscle hypertrophy in functionally overloaded mice. biochemistry and biophysics reports. 20: 100686–100693. dogra, c., changotra, h., mohan, s. and kumar, a., 2006. tumor necrosis factor-like weak inducer of apoptosis inhibits skeletal myogenesis through sustained activation of nuclear factor kappab and degradation of myod protein. journal of biological chemistry 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https://doi.org/10.1016/bs.afnr.2019.08.002 https://doi.org/10.1074/jbc.m511131200 https://doi.org/10.1074/jbc.m511131200 italian journal of food science, 2022; 34 (2) 49 the role of whey protein muscle development. gene 668: 107–113. https://doi. org/10.1016/j.gene.2018.05.039 zhang, h., wang, y., tang, x., dou, s., sun, y., zhang, q. and lu, j., 2021. combinatorial regulation of gene expression by uorfs and micrornas in drosophila. science bulletin 66(3): 225–228. https://doi.org/10.1016/j.scib.2020.10.012 vaisid, t. and kosower, n.s., 2013. calpastatin is upregulated in non-immune neuronal cells via toll-like receptor 2 (tlr2) pathways by lipid-containing agonists. biochimica et biophysica acta (bba) – molecular cell research 1833(10): 2369–2377. https://doi.org/10.1016/j.bbamcr.2013.06.006 wang, j., yang, l.z., zhang, j.s., gong, j.x., wang, y.h., zhang, c.l., chen, h. and fang, x.t., 2018. effects of micrornas on skeletal https://doi.org/10.1016/j.gene.2018.05.039 https://doi.org/10.1016/j.gene.2018.05.039 https://doi.org/10.1016/j.scib.2020.10.012 https://doi.org/10.1016/j.bbamcr.2013.06.006 p u b l i c a t i o n s codon italian journal of food science, 2023; 35 (3): 55–74 issn 1120-1770 online, doi 10.15586/ijfs.v35i3.2339 55 p u b l i c a t i o n s codon grape-based traditional foods produced in turkey simge aktop1*, pınar şanlıbaba1, yalçın güçer2 1department of food engineering, engineering faculty, ankara university, 50th year settlement, gölbaşı, ankara, turkey; 2department of cookery, beypazarı vocational school, ankara university, beypazarı, ankara, turkey *corresponding author: simge aktop, department of food engineering, engineering faculty, ankara university, 50th year settlement, 06830, gölbaşı, ankara, turkey. email: saktop@ankara.edu.tr received: 22 february 2023; accepted: 12 july 2023; published: 2 august 2023 © 2023 codon publications open access review article abstract turkey is considered the gene centre of the vine, and it has remained one of the most important trading centres of viticulture and grape-based foods for a long time. the grapes grown in anatolia and the foods obtained by processing these grapes contribute greatly to the economy of the nation. in addition to economic outcomes, grape-based traditional foods provide anti-carcinogenic, anti-inflammatory, antioxidant and cardiovascular disease– preventing benefits when consumed. a few examples of these grape-based traditional foods are molasses, wine, turkish rakı, hardaliye and pestil. in this review, the production processes of certain grape-based turkish traditional foods are summarized. keywords: grapes; process; traditional foods; turkey introduction traditional foods of a society or region are representative of its lifestyle, culture, history, climate and geography, and are, therefore, considered one of the most permanent elements of cultural heritage. the traditional foods passed down from generation to generation present several health benefits due to their richness in nutrition and flavour. therefore, present-day consumers worldwide are opting for traditional foods, resulting in a sharp increase in the demand for these products (ademoğlu and özkaya, 2021; bulut-solak, 2021; tamer et al., 2019). the history of grapes dates back to 5000 bc, rendering it one of the oldest fruits cultivated on earth. the asia minor region, which includes anatolia and the caucasus, is considered the homeland of grapes. consequently, viticulture is an ancient and deep-rooted culture in the anatolian lands, and as well as in turkey (batu, 2006; yakar, 2018). over 10,000 varieties of grapes exist, among which nearly 1200 are cultivated in anatolian lands, while only 50–60 are cultivated commercially (kop, 2021). grapes are a rich source of vitamins, minerals, carbohydrates and proteins, as well as phenolic compounds (coskun, 2017; dolgun et al., 2020). the phenolic compounds present in grapes, including catechin, gallic acid, myricetin, quercetin, kaempferol and coumaric acid, exert several positive effects on human health due to their richness in anthocyanins and other flavonoids (amoutzopoulos et al., 2013). a few of these positive effects of grapes on health are antioxidant, cardiovascular disease-preventing, anti-carcinogenic, anti-inflammatory and anti-allergic effects (coskun, 2017). the majority of the grapes produced in turkey are consumed fresh and sun-dried, although a small proportion is also processed into various grape-based traditional foods. the perishable nature of grapes, the shortness of the grape harvest season and the necessity of cold storage requirements have effectively led to the popularity of grape-based traditional foods. the processing of grapes into traditional foods not only extends their shelf life but also adds several sensory and healthenhancing properties to the foods (nakilcioğlu-taş et al., 2018; mailto:saktop@ankara.edu.tr 56 italian journal of food science, 2023; 35 (3) aktop s et al. hardaliye hardaliye is a traditional non-alcoholic, grape-based, fermented beverage that is produced widely in the thrace region of turkey (bayram et al., 2015; bulut-solak, 2021). it is produced to consume the huge amounts of grapes harvested in the limited period of the summer season and allow for their consumption in other seasons. kırklareli province is the region where hardaliye is geographically indicated (aşkın, 2018; bayındır and önçel, 2019). hardaliye is a salt-free and low-fat beverage that is preferred by various consumer groups. it has a bitter taste and a refreshing characteristic. it is reported that a cup of hardaliye provides 170 kcal of energy to one who consumes it. hardaliye may also be consumed by children as it lacks alcohol (aktaç et al., 2015; amoutzopoulos et al., 2013; coskun, 2017). moreover, because hardaliye does not contain milk and dairy products, it may also be consumed by those with milk intolerance and vegans. the raw material used for producing hardaliye is the black grapes, which have a high nutritional content, and consequently, it is also reported to be rich in monomeric anthocyanins and phenolic compounds (aşkın, 2018; coskun, 2017). hardaliye is obtained by malolactic fermentation of grape juice, mainly performed by lactic acid bacteria (lab). therefore, hardaliye serves as a non-dairy probiotic beverage (bayram et al., 2015). lactobacillus casei spp. pseudoplantarum and l. paracasei spp. are frequently isolated from hardaliye (altay et al., 2013; bulut-solak, 2021; otles and ozyurt, 2019). it is reported that the number of lab during fermentation ranges from 1 × 102 tamer et al., 2019). a few examples of these grape-based food products are must, hardaliye, wine, molasses, tarhana and pickles (bayram et al., 2015; cangi and yağcı, 2012). the production of these traditional foods may or may not include the fermentation process. the production regions for certain grape-based traditional foods in turkey are depicted in figure 1. in this review, the production processes of certain grape-based turkish traditional foods, such as hardaliye, wine, molasses, must, raisins, walnut sausage (köme), köfter, pestil, grape tarhana, unripe grape pickle, verjuice, bulama, brined vine leaf and turkish rakı, are summarized. fermented grape-based traditional foods fermentation is one of the most common methods used for food preservation since ancient times. several people around the world include fermented foods in their daily diet. microorganisms play an important role in food fermentation. fermentation adds certain peculiar properties to the foods, and the aroma, acidity and textural properties of the food product also usually change after fermentation. the final product often has a better taste, with increased health benefits, has a greater shelf-life and the development of pathogens is also inhibited in most cases (altay et al., 2013; coskun, 2017; piergiovanni, 2012). the most important grape-based turkish traditional foods produced using fermentation are hardaliye, wine, unripe grape pickle, brined vine leaf and turkish rakı. hardaliye, must, wine brined vine leaf, pestil, bulama walnut sausage, molasses, unriped grape pickle : hardaliye must, wine, pestil, walnut sausage, molasses : grape tarhana, brined vine leaf pestil, must, raisins, wine, molasses, walnut sausage, brined vine leaf, walnut sausage, molasses, must, wine, pestil wine, must, pestil, walnut sausage, molasses, raisins, turkish raki raisins, wine, pestil, walnut sausage, molasses, verjuice, must must, wine, pestil, walnut sausage, molasses, turkish raki kirklareli marmara region aegean region black sea region tokat central anatolia region nevşehir : köfter gaziantep southeastern anatolia region eastern anatolia region mediterranean region : bulama n s w e nw ne sw se figure 1. production sites for certain grape-based traditional foods in turkey (modified from anonymous, 2021). italian journal of food science, 2023; 35 (3) 57 grape-based traditional foods in it serves as a chemo preventive agent against cancer (coşkun, 2017; gucer et al., 2009; zhang, 2010). the allyl isothiocyanate is also reported to reduce the number of microorganisms, and its presence is actually responsible for the inhibition of alcoholic fermentation in hardaliye. in addition, the compounds released from sour cherry leaves were reported to play a role in preventing coronary heart diseases and have an effect on the digestive system (amoutzopoulos et al., 2013; bayram et al., 2015; tamer et al., 2019). the production of hardaliye includes several steps. the grapes are collected, washed, separated from their bunch and then crushed. meanwhile, a mixture of black mustard seeds and benzoate is prepared. the crushed grapes are then placed in an oak barrel and fermented (bayındır and önçel, 2019; bayram et al., 2015). a layer of the prepared mixture is then added to the barrel. if desired, crushed sour cherry leaves may also be added to this mixture to enhance the aroma. at this stage, most of the components in the fermented grape juice have passed into hardaliye. after the fermentation begins, the hardaliye in the barrel is collected through the tap and added back to the barrel from the top. this cycle is continued several times on the first day (bayram et al., 2015). the entire process of day 1 is continued for 5–10 days to ensure the complete fermentation of the product (yılmaz, 2022). finally, the fermented hardaliye is filtered, bottled and stored under appropriate cold conditions. the flow chart for hardaliye production is presented in figure 2. wine turkey is one of the most important nations in the world in terms of its vineyard regions and the amount of grapes produced. in terms of vineyard regions, turkey ranks alongside nations such as italy, spain and france. however, despite the huge amount of grapes produced in turkey, only 2.5–3.0% of the grapes are used for wine production (şenuyar et al., 2014). to 4 × 104 cfu/ml. l. acetolerans, l. brevis, l. sanfranciscensis and l. pontis have also been isolated from hardaliye (arici and coskun, 2001). grape varieties, such as papazkarası, cardinal, cabernet, hamburg muscat, cinsault, shiraz, merlot and alphonse lavallée are generally used as raw materials in the production of hardaliye (bayındır and önçel, 2019; bayram et al., 2015; bulut-solak, 2021). in hardaliye production, black mustard seeds (brassica nigra (l.) koch) are usually mixed with the grape marc as a whole or as crushed grains at various concentrations, even though heat-treated black mustard seeds may also be used (tamer et al., 2019). substances such as sour cherry leaf (prunus cerasus l.) and benzoic acid may be used as the preservative and aroma source during hardaliye production (bulut-solak, 2021; coskun, 2017; otles and ozyurt, 2019). hardaliye is usually produced and stored in stainless steel tanks, oak barrels or large glass or plastic barrels (bayındır and önçel, 2019). its production is generally conducted using traditional small-scale processing methods, due to which only the traditional methods for the production of hardaliye are generally reported in the literature (aydoğdu et al., 2014; gucer et al., 2009; tamer et al., 2019). hardaliye has antioxidant properties owing to its high content of phenolic compounds and ascorbic acid. it, therefore, has protective effects against cardiovascular diseases. in addition, its high antioxidant effect prevents the formation of cancer cells by preventing oxidative stress and causes a decrease in the plasma lipid peroxidation parameters (aşkın, 2018; bulut-solak, 2021; coskun, 2017). however, due to its short shelf-life, it is necessary that we pay attention to its storage conditions. the health benefits of hardaliye are also associated with the essential oil content of mustard seeds. mustard oil has antibacterial properties that are effective in the control of diseases, such as bronchitis, cold and circulatory disorders (bulut-solak, 2021). sinigrin in mustard seeds exerts an anti-carcinogenic effect, while the allyl isothiocyanate raw material (grape) washing and sorting pressing storage (in cold environment) bottling fermentation obtaining grape juice add: mixture of black mustard seeds and benzoate figure 2. the flow chart for hardaliye production (bayındır and önçel, 2019; bayram et al., 2015). 58 italian journal of food science, 2023; 35 (3) aktop s et al. moreover, it has been associated with the health of the cardiovascular system. the first epidemiological observation was that the french population suffers a relatively low incidence of coronary heart disease (chd), in spite of a relatively high dietary intake of saturated fatty acids and high intake of dietary cholesterol. this situation was first described by the irish physician samuel black in 1819, and then named the “french paradox.” several researches have been conducted and several hypotheses have been made in order to explain it (ellison, 2011; ferrie`res 2004; ghahremani and salami, 2021; lippi et al., 2010). the only clear message is that moderate alcohol drinking has a protective effect against chd. moreover, alcohol intake raises high density lipoprotein (hdl) cholesterol concentrations, and also approximately 50% of the risk reduction contributed to alcohol consumption is explained by changes in hdl cholesterol (lippi et al., 2010). in the past, wine was produced using only traditional methods. however, several modern methods for wine production are available currently. one of the most important steps in wine production is the inoculation of selected yeasts into the grape must. inoculation with pure culture ensures microbiological control during the fermentation and standardization of the product. s.  cerevisiae is generally used for inoculation (gülgör and korukluoğlu, 2015; kaya, 2017). s. cerevisiae is the dominant microflora in the environment, which prevents the development of other wild yeasts in the environment, and in addition contributes to the completion of fermentation. inoculation with yeast cultures other than a pure culture of s. cerevisiae into the must is not recommended (jouhten et al., 2016). as this might induce the risk of formation of compounds such as acetaldehyde, ethyl acetate, acetic acid and acetone in huge amounts due to uncontrolled fermentation which would then negatively affect the sensory properties of the wine produced (gülgör and korukluoğlu, 2015). the components responsible for the colour, taste and aroma of the wine are present in the grape skin, from where these components are transferred to the wine during the production processes. therefore, small and thick-skinned grape varieties are generally preferred for wine production. the main grape varieties used in wine production which are cultivated in turkey include kalecik karası, öküzgözü, semillion, sultaniye, narince, emir, viognier, malbec, ada karası, mourvedre, marsanne, bornova misketi, boğazkere, grenache and gamay (kaya, 2017; kayikcioglu and arikan, 2020). the different steps followed in wine production are illustrated in figure 3. unripe grape pickle another grape-based traditional food produced in turkey is the unripe grape pickle. unripe grape pickle is prepared using bunches or separated grape grains, grape the data obtained from archaeological excavations have revealed that wine is one of the oldest beverages in history. while the exact date of the first wine production remains unknown, it is thought to date back to 7000 years (kocaadam and acar-tek, 2016; yılmaz and akay, 2020). wine production is believed to have spread to different civilizations, such as the hittites and lydians from asia minor, which is, at present, considered the homeland of the vine. the thousands of years-old goblets, jugs, inscriptions, pictures and coins (acropolis ancient coins) extracted from anatolian lands have confirmed anatolia as an important centre of viticulture and wine trade (kayikcioglu and arikan, 2020). wine is a traditional fermented drink obtained by converting the glucose present in grape juice into ethyl alcohol using microorganisms (çam and yıldırım, 2018; kayikcioglu and arikan, 2020). at the initial stage of fermentation, the activity of the microorganisms that are intolerant to alcohol is usually inhibited (gülgör and korukluoğlu, 2015). yeasts, however, are generally able to tolerate a small amount of alcohol and, therefore, play an effective role in the initiation of natural grape fermentation. therefore, 3–4 days after the onset of alcoholic fermentation, yeasts such as saccharomyces cerevisiae become dominant in the fermentation medium. these yeasts play a role in the continuation and completion of the fermentation (ciani et al., 2006; sun et al., 2015). the chemical composition of wine may vary according to the ecological and physiological properties of the raw material used for its production, which then exerts an impact on the sensory properties and the quality of the wine. in addition, the type and the amount of aroma of wine are affected by various factors. yeast variety, fruit condition, culturing type, environmental factors, must acidity and sulfur dioxide (so2) amount are a few of these factors (gülgör and korukluoğlu, 2015). wine contains components that affect human health positively, such as polyphenols, resveratrol and quercetin. wine is also a good source of vitamins a, b and c, in addition to being rich in important minerals such as magnesium, calcium, iron, potassium, phosphorus, zinc, sulfur, copper, manganese, iodine, sodium, cobalt and chlorine that are involved in body metabolism (barbaros and kabaran, 2014). wine has several health benefits owing to the beneficial properties of its components. the components in wine are reported to provide anti-carcinogenic, antioxidant and anti-inflammatory effects, thereby playing a role in the regulation of cholesterol levels. regular and moderate consumption of wine is reported to have positive effects on health during diseases such as cancer, hypertension and type-ii diabetes (barbaros and kabaran, 2014). italian journal of food science, 2023; 35 (3) 59 grape-based traditional foods raw material (grape) seperating grapes from stems yeast grape skin maceration and fermentation pressing raw wine (1st press and 2nd press) malolactic fermentation must fermentation yeast separation from sediment maturation filtration bottling aging sales r ed w in e w hi te w in e figure 3. the flow chart for the production of red and white wine (anlı, 2022). juice and the roots of armoracia rusticana (horseradish). this product differs from other pickles in that no salt is used in its production. traditionally, the production of the unripe grape pickle is conducted at homes, and that too has now been limited to a few towns in the thrace region of turkey. the commercial production of the unripe grape pickle is also conducted locally at the scale of small businesses (adınır, 2011; becerikli, 2015). pickles have been produced traditionally since ancient times to increase the durability of foods and ensure the long-term preservation of food items. various vegetables and fruits are used for the production of pickles. the preliminary preparation steps for processing fruits and vegetables into pickles include washing, selecting and shredding the fruits and vegetables, after which the fruits and vegetables are left to ferment in a brine containing salt at certain concentrations. the juices of fruits and vegetables may also be used as raw materials in the above process (adınır 2011; tokatlı et al., 2012). under normal conditions, the fermentation period lasts for 2–3 weeks. lab are particularly effective during the fermentation stage. among the various lab species, l. plantarum, l. brevis, pediococcus cerevisiae, enterococcus faecalis and leuconostoc mesenteroides are the ones frequently involved in pickle production. among these, l. plantarum generally constitutes the dominant microflora in the fermented product, and this species is resistant to low ph values (adınır, 2011; karagöz and güllü, 2017). the lactic acid fermentation that occurs in pickles has certain other advantages besides increasing the durability of fruits and vegetables, such as improvement in the sensory and textural properties of fruits and vegetables (tokatlı et al., 2012). moreover, the lactic acid formed as a result of fermentation lowers the ph of the environment, thereby limiting the development of pathogens in the product. in addition, with pickle formation, the minerals and vitamins in fruits and vegetables are preserved and their digestibility is increased (adınır, 2011). 60 italian journal of food science, 2023; 35 (3) aktop s et al. vine leaves contain phenolic compounds, amino acids, organic acids, sugars, minerals, and vitamins (gülcü, 2010; gülcü and torçuk, 2016). in the production of brined vine leaves, the organic substances in the vine leaves are altered biochemically by the action of microorganisms. in the fermentation stage, lab are particularly active. the lab develop in the brine and produce lactic acid, which increases the acidity of the product and leads to a decreased ph value, thereby limiting the growth of unwanted microorganisms in the product (gülcü and torçuk, 2016; gülcü et al., 2009). this increases the durability of the vine leaves, extends their shelf life, and also contributes to the development of their popular organoleptic properties. the digestibility of the product also increases with fermentation (gülcü et al., 2009). in addition, during the production of brined vine leaves, easily accessible substances such as water and salt are required, while the requirement for machinery and equipment is minimal. commercially, brined vine leaves are packaged using vacuum packaging systems and then sent for sale to consumers in every region of turkey (gülcü and torçuk, 2016). turkish rakı turkish rakı is a traditional drink produced through the distillation of suma in different regions of turkey followed by aromatization using anise (anli et al., 2007). suma is a liquid produced by fermenting the must prepared from dried or fresh grapes (özden, 2015). traditionally, turkish rakı is consumed after mixing it with cold water. it may also be consumed along with other beverages, such as ayran, turnip juice and soda (süren and kızıleli, 2021). while it remains unclear when rakı production first began in anatolia, the earliest mention of rakı is in the work of fuzuli from the 16th century. therefore, it is believed that rakı from the anatolian lands has a history of at least 500 years, and throughout history, rakı-like beverages have been produced from grapes using the distillation method (kesmez and aydın, 2014). the anisebased drinks such as rakı are particularly produced and consumed in mediterranean nations. a few examples of these drinks from different regions of the world are ouzo from greece, pastis from france, sambuca from italy, zebib from egypt, and anesone from spain (süren and kızıleli, 2021). however, such anise-based drinks differ from each other in terms of the production process used and the traditional purposes of usage. therefore, in 2009, the turkish patent institute obtained the geographical indication registration to protect turkish rakı and distinguish it from other anise-based distilled beverages. the registered production area of turkish rakı is the “republic of turkey” (bergama, 2017). brined vine leaf turkey is one of the gene centres of the vine in the world, with a considerably old and culturally-rooted practice of viticulture (cangi and yağcı, 2012; gülcü et al., 2009). in turkish culture, both fruit and leaves of the vine are used. several turkish traditional food dishes use vine leaves to add richness to the cuisine (gülcü and torçuk, 2016). in particular, vine leaves are used as the main ingredient in sarma (turkish stuffed grape leaves), which is a traditional turkish dish prepared in almost every home in turkey and consumed lovingly by all residents. recent years have witnessed an increased demand for edible vine leaves, and the export of vine leaves has also accelerated. tokat and the aegean region are the leading regions with a wide production of edible vine leaves in turkey (cangi and yağcı, 2012; gülcü, 2010; gülcü et al., 2009). while fresh vine leaves may also be sent directly to the market for sale, vine leaves canned or processed in brine are sold more commonly (cangi et al., 2012; semerci and cangi, 2020). vine leaves differ in their shape, thickness and hairiness, and not all kinds are edible (gülcü et al., 2009). only thin, hairless and unsliced vine leaves are used for edible purposes, as these are preferred by consumers (cangi et  al., 2012; gülcü, 2010). thick, hairy and over-sliced vine leaves, on the other hand, are usually not preferred by consumers, due to which the selection of the variety of vine leaves becomes important. in addition, selecting the right time to harvest the vine leaves is important. if leaf removal is performed at the beginning of vegetation, the vines weaken. on the other hand, if leaf removal is performed in the late period of vegetation, the edible quality of the vine leaves decreases (gülcü and torçuk, 2016; gülcü et al., 2009). brined vine leaf production has been in practice in anatolian lands since ancient times (gülcü et al., 2009). the vine leaves are collected in the spring and washed to remove contaminants. afterwards, the brine prepared using salt, and water is transferred to glass or plastic containers, and the prepared vine leaves are added to this brine for fermentation for 4–5 weeks. in the process, when the brine level in the fermentation containers decreases, more brine is added as required (gülcü, 2010). the most widely used varieties in the production of pickled and canned vine leaves in turkey include narince from tokat, sultani from the aegean region and yapıncak from the thrace region (cangi and yağcı, 2012; cangi et al., 2012; gülcü and torçuk, 2016). the vine leaves obtained from vine rootstocks, such as muscat hamburg, hesapali (müşküle), kober 5 bb, karaerik, and kabuğu yufka, are also used for edible purposes (semerci and cangi, 2020). italian journal of food science, 2023; 35 (3) 61 grape-based traditional foods capacity of 5000 l, are generally used in the distillation stage, and at least a 65% ratio of suma in alcohol is maintained. the next step is the sweetening of turkish rakı by adding sucrose at a concentration of 10 g/l. afterward, turkish rakı is diluted with deionized water and then aged for a minimum of one year. the aged turkish rakı is filtered through special filters and pumped into filling machines. in the final stage, turkish rakı is bottled and labeled after performing the standardization controls. the alcohol content in the final product should be 40% (v/v), while the anethole content should be a minimum of 0.8 g/l (anli and bayram, 2010). the flow chart for the production of turkish rakı is presented in figure 4. the main grape varieties used in the production of turkish rakı are sultani, yapıncak, and dimrit. in recent years, aromatic grape varieties, such as muscat blanc, have also been used in the production of turkish rakı (anli and bayram, 2010). anise (pimpinella anisum l.) is another raw material used in the production currently, turkish rakı is being produced mostly by legal companies, which use modern distillation systems. however, in certain regions in central and southern anatolia, traditional rakı is also produced at home (anli et al., 2007). according to the distillate source, two types of rakı are produced in turkey—one type of rakı is produced using fresh grapes only, while the other type of rakı is produced by adding agricultural product-based ethyl alcohol to the grape suma (bergama, 2017; cabaroglu and yilmaztekin, 2011). the production process of turkish rakı is described ahead. first, the grape suma is produced. the cleaned grapes are pressed and turned into must, which is then fermented to obtain the suma. afterward, the aromatization process is performed to obtain the characteristic taste and smell of turkish rakı. the aromatization step is performed using just 6%–10% anise (pinpinella anisum  l.). the next step is the distillation process. traditional retorts composed of copper, with a maximum raisin, grape or mollasses crushing (grape and raisin) yeast fermentation distillation obtaining the suma aniseed (pimpinella anisum) sucrose raki distillation dilution ageing filtration bottling figure 4. the flow chart for the production of turkish rakı (anli and bayram, 2010). 62 italian journal of food science, 2023; 35 (3) aktop s et al. however, considering the chemical composition and the nutritional value of molasses, it is recognized for exerting positive effects on human health as well. glucose and fructose account for 50–80% of the sugars present in molasses. these are simple sugars, which easily pass into the blood through simple diffusion without the prior requirement for breaking down into simpler molecules, thereby enabling rapid energy production in the human body. the average energy value reported for molasses is 293 kcal per 100 g (batu, 2006; türkben et al., 2016). consequently, molasses are recommended for athletes, children, pregnant women, and individuals who require immediate energy (heshmati et al., 2019; türkben and uylaşer, 2018). molasses rich in fe2+, which is utilised easily in the human metabolism and fulfills 35% of the daily iron demand of the human body. iron content in molasses is in the range of 11.3–20 mg/100 g of molasses (mtech and venkatasubramanian, 2017). in addition, molasses is a good source of potassium, calcium, and magnesium, which are important for several physiological processes, such as blood coagulation and the functioning of the heart muscles. molasses is reported as the best source of calcium after milk and dairy products (batu, 2006; heshmati et al., 2019; uçar, 2015). grape molasses may be classified under different groups based on the production method. according to the most general classification, the following three categories exist—liquid molasses produced using traditional methods, liquid molasses produced using modern methods and white solid molasses (batu, 2006). in addition, according to turkish standards, molasses with a ph value of 5.0–6.0 is considered sweet molasses, while molasses with a ph value of 3.5–5.0 is considered sour molasses (batu, 2020; tosun et al., 2014). the traditional method that is preferred for molasses production in turkey has remained unchanged for a long time. with advancements in technology, modern production methods for molasses have also emerged (batu, 2006). in both traditional method and modern methods, the production of liquid molasses from fresh grapes involves four basic steps: obtaining the must, neutralising the free acidity of the must, filtering the turbidity substances and cooking the must (batu and gök, 2006). in the production of liquid molasses using the traditional method, the grapes are washed with water in plastic crates to remove foreign materials, such as straw, garbage, dust and soil. next, the grapes are crushed by the producers to extract the juice (batu, 2006). this process is conducted in cans composed of wood or concrete. another method is to extract the grape juices by squeezing the grapes packed into sacks using a press that is referred to as “vise” (batu, 2006, 2020). afterward, the coarse residues in the must are filtered, followed by the addition of the molasses soil which is a white soil type of turkish rakı. it is used for achieving the characteristic aroma of rakı. the amount of anise used in the production of rakı varies between 6 and 10% depending on various factors, such as the quality of the anise used in rakı production, the rakı variety and the aporak (mixture of the first and final products). anise has several health benefits, such as pain relieving, respiratory facilitation and bloating relief (bergama, 2017; koçarslan, 2002). according to the turkish food codex communiqué on distilled alcoholic beverages (communiqué no: 2016/55), turkish rakı must contain at least 0.8 g/l of anethole (tfc, 2017a). trans-anethole is a phenol ether that confers anise its characteristic taste and odour and constitutes 80–90% of the essential oil (anli et al., 2007). non-fermented grape-based traditional foods non-fermented grape-based traditional foods are as popular as the other kinds of grape-based foods in turkey. the main non-fermented grape-based foods are molasses, pestil, raisins, walnut sausage (köme), köfter, grape tarhana, verjuice, bulama and must. the communities in the regions involved in viticulture in turkey processed the produced grapes in different ways, such as by using drying, cooking, and distillation. non-fermented grapebased foods, thus, became included in the daily diet of these people. in addition, it facilitated the emergence of turkey as a gastronomic tourism host for several tourists. molasses molasses is a traditional turkish food produced in both liquid and solid forms from sugary fruits, such as grapes, mulberries, beets, apples, and figs. molasses is high in energy value and rich in minerals (yildiz et al., 2015). the production of molasses in turkey dates back to ancient times (heshmati et al., 2019; tosun et al., 2014). grape molasses produced in turkey are named according to the place of their origin. while almost every region in turkey that has vineyards produces molasses, the provinces that are known for quality production include tokat (particularly zile), eskişehir, kastamonu, balıkesir, hatay, afyonkarahisar, gaziantep, kırşehir and kayseri. the molasses produced in these regions are considered high-quality in terms of textural and sensory properties, such as taste, smell, colour and consistency. molasses may also contain 5-(hydroxymethyl) furfural (hmf), iron, zinc and certain other metals at concentrations above the limits (batu, 2006; uçar, 2015; yildiz et al., 2015) recommended by the turkish food codex communiqué on grape molasses (communiqué no: 2017/8), which may lead to adverse effects on human health (batu and gök, 2006; tfc, 2017b). italian journal of food science, 2023; 35 (3) 63 grape-based traditional foods consumption during the winter months (batu et al., 2007; yildiz, 2013; yildiz and boyraci, 2020). pestil is obtained by laying the wet fruit puree on a cloth placed on a flat surface and allowing the moisture to dry until the desired product texture is obtained. the drying is performed either under the sun or by using hot air dryers. the low moisture content in pestil ensures a long shelf life and no requirement for cold storage. normal cool and dry conditions are sufficient for the preservation of pestil (kara and küçüköner, 2019; nakilcioğlutaş et al., 2018). fruits such as grapes, mulberries, plums, apricots, apples, and figs are generally used as raw materials for the production of pestil (kamiloglu and capanoglu, 2014; özaltın and çağındı, 2018; yildiz, 2013). however, grapes are the most widely used fruit in the production of pestil (batu et al., 2007). white sugar, edible starch, seasoning, and additives may also be added during the production of pestil (kara and küçüköner, 2019; nakilcioğlutaş et al., 2018). the composition and the production steps of pestil vary with the geographical region. in the gümüşhane province of turkey, pestil production uses milk, honey and wheat flour, unlike pestil production in the other regions. in addition, gümüşhane pestil has containing 50–90% lime and used to clarify the must in molasses production to the must to reduce the free acidity of the must to the ph of 6.0–6.5. this mixture is then heated at 50–60°c, and during this cooking process, foams that are referred to as kef form on the surface of the must (batu and gök, 2006). these foams are removed using a ladle, after which the cooking process is continued until a temperature of 100–110°c is reached. the continuation of cooking is performed under continuous mixing to ensure no caramelization of the molasses. in the traditional method, open boilers with a wider width and a small depth are generally preferred for the cooking of the must, which enables rapid evaporation of the water from the must (batu, 2006, 2020; tosun et al., 2014). this reduces the cooking time and limits the unwanted browning of the must. finally, the molasses is filled in glass jars or plastic or lacquered tin cans for sale (batu, 2006). the flow chart for the production of liquid molasses using the traditional method is presented in figure 5. pestil pestil is a traditional food with high nutritional value, prepared in various regions of turkey, usually for raw material (grape) washing crushing and pressing must obtain addition of molasses soil hold the dark (de-acidification and clarification phase) removal of the upper clear part by boiling in the open boiler (~65-68o brix) packaging figure 5. the flow chart for the production of liquid molasses using the traditional method (türkben and uylaşer, 2018). 64 italian journal of food science, 2023; 35 (3) aktop s et al. the seeds of which have been removed, to remove the acidity of the must. after another round of filtration, a portion of the filtered must is turned into a slurry by adding starch or flour. the remaining must is boiled for 30 min, following which the starch and flour slurry is added to this boiled must. the mixture is then allowed to boil further until the desired consistency is reached. this hot mixture is referred to as “herle.” finally, this herle is poured onto cloths in a thin layer and allowed to dry under the sun. if desired, the pestil is cut into different sizes and packed after sprinkling starch between the cut pieces (batu et al., 2007; özaltın and çağındı, 2018; yildiz and boyraci, 2020). the flow chart for the production of pestil is presented in figure 6. while the sun-drying method is a simple and low-cost method, it has certain disadvantages, such as greater time consumption compared to the industrial drying methods. another disadvantage of the sun-drying method is that the pestil remains uncovered during drying in the sun, which increases the chances of contamination (kara and küçüköner, 2019; köprüalan et al., 2019). in the production of pestil, a non-enzymatic browning reaction may be observed during the drying process and storage. factors such as ph, drying temperature, drying duration, product composition, and water activity affect this non-enzymatic browning (kaymul, 2021). in order to prevent this browning reaction, citric acid and ascorbic acid may be added to the pestil. the reaction may also be prevented by adding lemon juice to the pestil (kara and küçüköner, 2019). in the drying process, the humidity of the environment and the air circulation affect the quality of the pestil (yildiz and boyraci, 2020). in the cases where the temperature is low and the ambient humidity is high, the drying rate decreases, which supports the growth of unwanted microorganisms in the pestil. therefore, convective hot air dryers, which provide rapid drying and microbiological quality, are often preferred in pestil production (kara and küçüköner, 2019; kıralan and gündoğdu, 2021). raisin turkey is the world’s largest producer and exporter of seedless raisins, accounting for approximately 40–45% of the world’s raisin exports. approximately two-thirds of the grapes produced in turkey have seeds, while onethird of the grapes is seedless (mtrt, 2019). the production of seedless raisins in turkey is concentrated in the aegean region, particularly in manisa, akhisar, salihli, turgutlu, çal, kemalpaşa, menemen and çivril (şüyün, 2020). in terms of the vineyard area and the yield, the a soft and shiny texture. owing to these unique features, gümüşhane pestil has received the “geographical registration certificate (grc)” from the turkish patent institute (kaya and kahyaoglu, 2005; yildiz and boyraci, 2020). the dry matter ratio in pestil is generally above 85%, and sugars constitute a significant proportion of this dry matter. in addition, pestil contains minerals and vitamins that play important roles in several metabolic processes in the human body. therefore, pestil is considered a high-energy food (batu et al., 2007; kara and küçüköner, 2019). it is reported that 100 g of grape pestil provides 321.5–356.4 kcal of energy to the human body (cagindi and otles, 2005). the minerals present in pestil include calcium, iron, potassium, magnesium, and phosphorus, while vitamins such as niacin, riboflavin, and thiamine have been reported (batu et al., 2007; nakilcioğlu-taş et al., 2018). since the iron present in pestil is easily absorbed in the human body, a certain portion of the daily iron requirement may be met by consuming pestil. according to a study, the mineral concentrations in pestil were: iron, 0.3–2.8 mg/100 g; calcium, 25.2–36.0 mg/100 g; potassium, 62.0–553.6 mg/100 g (cagindi and otles, 2005). the ca/p ratio is important in child nutrition, and the recommended value is 1.2–2. the ca/p ratio reported for grapes, which are used as raw materials for pestil production, varies between 2 and 2.7 (batu et al., 2007). pestil is also a good source of dietary fiber as it is produced from fruits with high fiber content. in addition, pestil is a good source of carbohydrates and energy due to the presence of starch in it (kara and küçüköner, 2019; özaltın and çağındı, 2018; yildiz and boyraci, 2020). consequently, pestil is preferred by individuals who require energy. on the other hand, those with diabetes and obesity are advised to limit their consumption of pestil. furthermore, the glucose present in pestil exerts positive effects on the physical and mental performance of humans. the brain uses glucose as an energy source. since glucose increases insulin secretion, the former is involved in the crossing of tryptophan across the blood–brain barrier as well as in the synthesis of serotonin. moreover, glucose decreases the consumption rate of glycogen, which is important for athletes as it increases their endurance time (batu et al., 2007). currently, small businesses produce pestil using the traditional method, while modern methods are also used for pestil production. in turkey, pestil is generally produced using traditional methods, the production stages of which are described ahead. first, the grapes are washed, selected, and pressed using a press. the grape juice produced is filtered using a cloth. next, the molasses soil (containing 70% calcium carbonate) is added to the must, italian journal of food science, 2023; 35 (3) 65 grape-based traditional foods grape molasses dilution (brix adjustment) 3/4 part is seperated boiling process (until obrix 40) 1/4 part is seperated addition: (carob flour and starch) mixing mixing and cooking (until the final obrix is 40) obtaining the herle and pouring it on the cloth drying packaging figure 6. flow chart for pestil production (nakilcioğlu-taş et al., 2018). most important production regions in turkey after the aegean region are the mediterranean region and southeastern anatolia. sultani, besni, and corinth (currants) are considered dried grapes (kop, 2021; mtrt, 2019). the principle underlying the drying method is to evaporate the water content present in the food, followed by the removal of the produced steam away from the product surface (alçay et al., 2015). this causes the water activity (aw) to fall below the levels at which microorganisms grow, which ensures that the food is preserved for a longer duration without spoiling (uysal-seçkin and taşeri, 2015). in addition, since the volume of the product is reduced due to water loss, transportation and storage also become easier (alçay et al., 2015). however, certain risks are associated with the drying of grapes. for instance, when the grapes have longer harvested and drying durations, the risk of rains in late august is encountered. in addition, the higher relative humidity around september is a risk factor encountered in the drying process conducted in the aegean region. this higher humidity reduces the drying rate of the grapes and causes the grapes to remain in the exhibition for a longer duration (akdeniz, 2011). raisin is considered an important dried fruit due to its high nutritional value. raisin is a good source of minerals, such as iron, potassium and calcium, in addition to containing b group vitamins and 70% digestible fructose. raisin also serves as a high-fibre food and is rich in antioxidants, and these properties allow the use of raisins in 66 italian journal of food science, 2023; 35 (3) aktop s et al. crates for storage (akdeniz, 2011). the raisin is stored in a moisture-free, cool and dark environment. according to the turkish seedless raisins standards (ts 3411), the maximum final moisture content allowed for the raisin is 16% (tsi, 2011). walnut sausage (köme) walnut sausage is a grape-based traditional food produced in almost every region in turkey. walnut sausage production is a culture belonging to anatolia, and while it remains unknown when this production began, it is estimated to date back several years. according to historical records, walnut sausages were produced during the ottoman empire, although it was not traded. walnut sausage is rich in minerals and vitamins and is also a good source of energy. in turkey, walnut sausage is recognized by different names in different regions, such as köme, molasses sausage, bandırma, and orcik (erdoğan et al., 2003). the production of walnut sausage involves different steps, which are described ahead. first, the dried walnuts are broken and separated from their shells, following which they are strung on a rope using a needle. next, the walnuts hung on the rope are dipped into the pre-prepared herle in groups. the walnuts dipped in herle are placed in a large and wide pot for a certain period, after which the walnuts with herle are arranged on sticks and these sticks are placed on a wheeled bench. afterward, the walnuts with herle are dried in a greenhouse or in rooms with a drying system. the duration of this step is not constant but depends on the ambient conditions. traditionally, the first drying process is terminated if a cracking sound is heard when the walnut sausage is twisted. after these stages, another dipping process is performed, following which all the remaining processes are repeated. this cycle is repeated four times (bayram, 2018; kalkışım and özdemir, 2012). figure 7 presents the flow chart for the production of walnut sausage (köme). several factors affect the walnut sausage production process, and the process usually completes in nearly 9 days. accordingly, the drying process is an important step in the entire process. herle thickness, air temperature, air flow, humidity, and the laying shape are important in the drying process. if the drying process is not performed properly, the deterioration of walnut sausage starts earlier. on the other hand, a higher quality product is obtained because of the desired-level drying of walnut sausages of appropriate thickness. the cracking of walnut sausages is a quality problem. a few reasons for this problem are the excessive thickness of the herle, the addition of excessive flour, and insufficient drying (kalkışım and özdemir, 2012). finally, molding can also be seen the treatment of cancer and prevention of other diseases such as constipation (çağlayan, 2018). at the beginning of the drying process, the sugar content in the grapes is approximately 20%. however, by the end of the drying process, the sugar content increases to approximately 85% (i̇şçi and altındişli, 2016). because the relative humidity is high under adverse meteorological conditions, the water mobility in grapes is reversible. therefore, in an environment with high relative humidity, raisins do not lose water, and rather the moisture from the atmosphere is transferred to raisins. this prolongs the drying duration for grapes and renders the grapes unprotected against microorganisms. in addition, the browning reaction may continue as the increase in the sugar content in raisins with increasing humidity decreases and the enzyme activity is prolonged (akdeniz, 2011; dumanoğlu, 2021). polyphenol oxidase is the main enzyme responsible for the browning reactions in grapes (çağlayan, 2018). among the various drying processes used in turkey and other nations is the use of sodium hydroxide (naoh) as a dipping solution. another process allows to obtain green raisins by dipping grape berries before drying in various dipping solutions inside dark drying rooms (akdeniz, 2011). contrary to these applications, another process allows to obtain brown raisins through direct laying and drying without the use of any dipping solution. in order to obtain visually appreciated brown raisins, bleaching processes with sulfur may be applied additionally (i̇nan, 2012). in the dipping process applied to raisins, the aim is to remove the wax layer on the grape to allow for the raisin to appear brighter and increase the drying speed by approximately 2–3 times (i̇şçi and altındişli, 2016). an example of this situation is as follows: under normal conditions, the grapes dry in nearly 14 days without dipping, while the dipped grapes dry in just 7–8 days (akdeniz, 2011; otağ, 2015). after the dipping process, the bunches of grapes are brought to floor-type exhibitions or high-system wire exhibitions (şen, 2014). the most suitable conditions for the drying of grapes would be available when there is sunny, cloudless and breezy weather. in floor-type exhibitions, the drying process requires nearly 10 days, depending on the air temperature. in high-system wire exhibitions, the drying process is completed in 15–20 days (otağ, 2015). on the other hand, under less sunny, cloudy, and no-breeze conditions, the duration required for the drying of grapes is longer. the raisin obtained from the exhibitions is separated from their bunches and large garbage. finally, the raisins are passed through the scattering machines and filled into sacks or plastic italian journal of food science, 2023; 35 (3) 67 grape-based traditional foods in addition to molasses, flour and starch, egg and molasses soil may be used in köfter production. the use of  molasses soil in köfter production removes the acidity  of the grape juice. molasses soil contains a high amount of calcium carbonate, which ensures the collapse of colloids that reach the isoelectric point (bilişli, 2013). in general, type 550 bread wheat flour and wheat starch are preferred in the production of köfter. it is reported that köfter prepared using flour is harder compared to the one prepared with starch (becerikli and başoğlu, 2018). another ingredient used in the production of köfter is egg (yolcu, 2018). the flow chart for traditional köfter production is presented in figure 8. since köfter is a grape-based product, it is rich in both minerals and phenolic compounds. köfter is a good in walnut sausage because of using of moldy walnuts (erdoğan et al., 2003). köfter köfter is a grape-based traditional food prepared using water, molasses, and starch in turkey (yildiz et al., 2015). köfter is a food with high nutritional value, due to which it is consumed widely in turkey, particularly in the central anatolia region and in the winter season. nevşehir is a prominent province for köfter production in turkey. köfter production is usually conducted in september-october when the grapes ripen. the commercial production of köfter, which is usually produced at home using traditional methods, is also prevalent (becerikli and başoğlu, 2018; goksel et al., 2013). stringing walnuts on strings preparing herle 1. dipping 1. drying packaging 3. drying 2. drying 4. drying 3. dipping 2. dipping 4. dipping figure 7. the flow chart for the production of walnut sausage (köme) (kalkışım and özdemir, 2012). water and molasses mixture (1:1) add: corn starch heating with stirrer (85 oc) cutting with different shape drying spread on mold (köfter mix) figure 8. the flow chart for traditional köfter production (goksel et al., 2013). 68 italian journal of food science, 2023; 35 (3) aktop s et al. source of minerals such as calcium, potassium, iron, magnesium, phosphorus and sodium. according to a study, the mineral contents in köfter samples were as follows: potassium, 7037.00–10247.67 mg/kg; calcium, 1116.00–1480.00 mg/kg; iron, 6.02–9.47 mg/kg (becerikli and başoğlu, 2018). no standard for köfter is used in turkey. however, according to the turkish food codex communiqué on grape molasses (communiqué no: 2017/8), molasses with a ph of 5.0–6.0 is considered sweet molasses (tfc, 2017b). according to a study, the dry matter content in köfter samples was 79.17–82.17 g/100 g, while the ph value was 5.44–5.73 (becerikli and başoğlu, 2018). accordingly, köfter is also considered a sweet grape-based food. several factors affect the quality of köfter. molasses composition, starch type and concentration, and the concentration of other components are a few such factors. in addition, the drying temperature and duration used during the production of köfter may affect the quality of köfter (goksel et al., 2013; yildiz et al., 2015). non-enzymatic browning may occur during food processing, producing compounds with toxic effects, such as melanoidins and 5-hydroxymethylfurfural (hmf). hmf may be produced due to the drying temperature applied during the production of köfter and because of the sugar content in köfter. therefore, hmf concentration could be used as a quality criterion for köfter (goksel et al., 2013). grape tarhana tarhana is a traditional turkish food prepared by kneading wheat flour with yogurt, salt, pepper, and onion, followed by leaving the dough for fermentation, drying, and grinding (aytunç and özsisli, 2020). different varieties of tarhana have emerged due to variations in tarhana production and different raw materials used in different regions of turkey. a few of these varieties are cranberry tarhana (bolu), uşak tarhana (uşak), maraş tarhana (kahramanmaraş), thrace-style fresh tarhana (tekirdağkırklareli-edirne), beyşehir tarhana (konya) and göce (top) tarhana (isparta) (altundağ et al., 2020; sormaz et al., 2019). in regard to the tarhana variety which uses grape (tokat), while the production process used is similar to the production of traditional tarhana, the obtained product differs in several aspects from the other tarhana varieties. while the other tarhana varieties are generally consumed as soup, grape tarhana is consumed as dessert. in addition, no fermentation step is performed in the production process of grape tarhana as in the production of other tarhana varieties (altundağ et al., 2020; coşkun, 2014; sormaz et al., 2019). grape tarhana is a sweet and turkish delight-like traditional food, and its production has been prevalent in tokat for several years (köse and çelik, 2017). grape tarhana is consumed with walnuts, particularly in winter. grape tarhana has a cumbersome production process and is prepared traditionally at home (cangi et al., 2011; kaya et al., 2009). in the preparation of grape tarhana, grape must and molasses soil are used. first, the grape must is clarified and boiled for a while (coşkun, 2014). next, fine bulgur (cracked) wheat (1/8 or 1/9) is added to the must and boiled, until the mixture reaches the desired consistency (kaya et al., 2009; köse and çelik, 2017). afterward, the mixture is poured into trays and allowed to cool and solidify (coşkun, 2014). the cooled mixture attains the form of the molds on the trays, following which the drying process is applied (cangi et al., 2011; sormaz et al., 2019). the drying process is completed in 6–10 days in cool weather and 2–5 days in hot weather (coşkun, 2014). raw material (grape) adding: molasses soil obtaining of the must boiling and holding molding and cooling adding: fine bulgar (cracked) wheat and cooking boiling settling cutting and slicing drying packaging figure 9. the flow chart for the production of grape tarhana (cangi et al., 2011). italian journal of food science, 2023; 35 (3) 69 grape-based traditional foods in turkey the production of verjuice is generally performed using traditional methods and commercial production is not common. however, the production of verjuice with traditional methods does not involve the use of aseptic conditions. this renders the traditionally prepared verjuice vulnerable to microbial contaminants, causing this verjuice to have a short shelf life. in addition, a rapid fermentation event occurs in the verjuice contaminated by wild yeast and molds, which renders it difficult to consume (ergezer et al., 2018). in the production of verjuice, the yediveren variety of unripe grapes is particularly preferred (öztürk, 2020). the different steps followed during the production of verjuice using traditional methods are described ahead. koruk bunches are placed in a plastic tub and crushed using a mallet. sugar is then added to the crushed koruk in a ratio of 1:2, and the mixture is left undisturbed for 1 h. afterward, boiled water is added to the mixture, followed by filtration of the mixture using a filter. the resulting filtrate is collected in a container, warm water is added to it, and the solution in filtered. this process is continued until the desired verjuice concentration is reached. the resulting filtrate is referred to as natural verjuice (ergezer et al., 2018). bulama bulama is a kind of solid molasses produced in the thrace region of turkey and cities such as bursa, balıkesir, gaziantep and ankara. the thrace region has an important potential in terms of both vineyard area width and grape yield. since approximately 69% of the vineyards in the region are in tekirdağ, tekirdağ bulama is particularly prominent among the breakfast products (gülcü, 2010; gülcü and demirci, 2009). monosaccharides constitute 80% of the sugar content in bulama. therefore, due to the easy movement of these monosaccharides in bulama through the digestive system, bulama consumption provides a rapid energy supply to the body. in addition, bulama is a good source of minerals such as iron, potassium, calcium, and magnesium (gülcü and demirci, 2009; yıldırım, 2008). bulama is usually produced using traditional methods, although the content and the preparation steps of bulama vary with the region. the molasses produced in turkey has several names, such as bulama, ağda, zile, çalma and masara (keleş et al., 2019). currently, bulama is being produced under home conditions, only by families interested in viticulture, and is sold and traded in the local markets established by the local producers in the region. bulama is mostly produced in the center of tekirdağ and in şarköy and malkara regions where viticulture is intense (gülcü and demirci, 2009). the steps of the traditional preparation the flow chart for the production of grape tarhana is presented in figure 9. according to a study, the dry matter content in grape tarhana samples ranged between 69.74% and 78.11% (yıldız et al., 2010). according to another study, the samples of grape tarhana had ph values varying between 4.45 and 6.01 (kaya et al., 2009). grape tarhana is considered a healthy dessert as it is a grape-based product and has a high-calorie and nutritional content (altundağ et al., 2020). therefore, this traditional food is preferred by people who require energy. the tarhana produced in gaziantep is quite similar to the tokat grape tarhana. the production stages of the  tarhana produced in gaziantep are described ahead. the grape juice obtained by pressing the grapes is boiled in cauldrons referred to as “mahsere.” next, fine bulgur (cracked) wheat is added to the grape juice in a ratio of 1/10, followed by boiling. the mixture is then poured into containers and allowed to cool and solidify, followed by cutting into pieces. finally, the cut pieces of the product are dried under the sun (cangi et al., 2011; kaya et al., 2009). verjuice koruk (vitis vinifera l.) is the name assigned to the immature grape. verjuice is a traditional sour-tasting beverage obtained by squeezing koruk (unripe grapes). the beverage is consumed directly in turkey and is also used as a flavouring in salads, meals, and appetizers across the world, particularly in the mediterranean nations (ergezer et al., 2018; öztürk, 2020). no standard exists for the production of verjuice, due to which the verjuice produced in each region is different. moreover, the shelf life of verjuice is also limited. the pasteurization process may be applied to extend the shelf life of verjuice. in addition, antimicrobial additives may be added to verjuice to the extent permitted by the relevant legislation. the addition of potassium sorbate as an antimicrobial agent to verjuice is permitted by country legislation (hayoglu et al., 2009). verjuice is a beverage with a low ph value. according to a study, the ph values of verjuice varied between 2.74 and 2.94 (ergezer et al., 2018). in addition, because the raw material of verjuice is unripe grapes, it is rich in phenolic content. the consumption of verjuice as food provides several health benefits, such as positive effects on the digestive system (due to which it is used in the treatment of ulcers), cholesterol and blood pressure lowering effects, etc. (de matos et al., 2017; shojaee-aliabadi et al., 2013). 70 italian journal of food science, 2023; 35 (3) aktop s et al. lab is usually isolated from grape must. the different lab species isolated from the must include l. brevis, l. plantarum, oenococcus oeni, l. hilgardii and leuconostoc mesenteroides (tokatlı et al., 2012). the different must production stages are described ahead. the grapes are harvested in september or october and washed to remove foreign materials such as straw, garbage, dust, and soil. afterward, the grapes are passed through a suitable press and crushed to extract their juices. the extracted grape juice is filtered through a filter, and molasses soil (white soil containing 70% caco3) is added to the filtrate. the purpose of using molasses soil at this stage is to reduce acidity and clarify the must (batu and gök, 2006; batu et al., 2007). in the next step, the must is boiled for 3–5 min. when foaming begins in the must, the boiling process is terminated, and the foam on the surface is removed. finally, the must is removed from the boiler and separated from the sediment (molasses soil) settled at the bottom. when all processes are completed, the must is allowed to rest until it acquires its unique colour. the desired ph value of the final product is 7.6, and the desired dry matter ratio is 30–35%. the brix value in must should be 20. the must may be produced from fresh grapes as well as from raisins. this ensures that must production continues regardless of the season (batu et al., 2007). conclusions grape fruit has been cultivated and processed into various products since ancient times. owing to its high nutritional value and chemical composition, grape fruit serves as a good source of energy and may be used in the treatment of various diseases. turkey is one of the world’s leading nations in terms of grape cultivation. the grapes produced in turkey are consumed as fresh fruit or processed into grape-based foods. hardaliye, molasses, pestil, grape tarhana, wine, turkish rakı, raisins, must, brined vine leaf, verjuice, unripe grape pickle, bulama, köfter and walnut sausage are a few examples of the grape-based foods produced and consumed in turkey. these foods are produced using different methods, such as fermentation, cooking, and distillation. in general, the traditional grapebased foods produced in turkey are meant for domestic consumption and are prepared using traditional methods. however, considering the export potential of foods, such as wine and raisins, it is possible to increase the economic value of these products to higher levels through r&d (research-development) and marketing activities. conflicts of interest the authors express no conflicts of interest associated with this work. of bulama in tekirdağ are described ahead. grape must is obtained by pressing the grapes, and molasses soil is added to the must (batu, 2006). the mixture is then heated to 70–80 °c to allow for coagulation. in another boiler, water and gypsophila l. root are boiled for 10–15 min, and the bitter juice of the plant is discarded. water is added to the remaining gypsophila l. in the boiler, followed by boiling until half of the water evaporates. the remaining gypsophila l. juice is collected in a container. this process is repeated 3 times in total. afterwards, the grape must and the gypsophila l. juice are mixed and boiled after adding sugar. this process is continued until the mixture reaches the consistency of molasses. in a separate bowl, gypsophila l. foam is prepared through whisking. the foam is then added to the molasses and the whisking is continued, allowing the molasses to harden and become lighter in colour. when the desired colour and consistency are achieved, the heating process is terminated, and the bulama is obtained (gülcü and demirci, 2009). according to a study conducted by gülcü and demirci (2009), the water-soluble dry matter content in the bulama samples was approximately 80.06% and the average ph value of the bulama samples was 4.22. accordingly, these samples were classified as sour molasses. according to the turkish food codex communiqué on grape molasses (communiqué no: 2017/8), molasses with ph values of 3.5–5.0 are classified as sour molasses (tfc, 2017b). bulama is a good source of minerals as the raw material used in bulama production is grape. according to a study, the average mineral contents in the bulama samples were as follows: calcium, 561 mg/ kg; sodium, 498.4 mg/kg; magnesium, 190.4 mg/kg; iron, 20.8 mg/kg (gülcü and demirci, 2009). must must is a traditional beverage obtained by boiling grape juice. must is also used in the production of other grapebased foods, such as pestil, walnut sausage, and molasses (batu et al., 2007). several factors may directly affect the must quality, such as the soil features of the vineyard, geographical structure, climatic conditions, soil-water relationship, and sunshine duration (bahar et al., 2018). all these factors, which are directly related to the quality of the must or wine, are evaluated within the term “terroir” (bekar, 2016). in addition, it is important to select a quality raw material during the production of must. in the grape ripening period, the sugar content increases, while the acidity decreases. it is reported that the brix value of the grapes to be processed for the must during harvest should be 22–24, and the titration acidity value in terms of tartaric acid should be 5–6 g/l (gucer et al., 2021). italian journal of food science, 2023; 35 (3) 71 grape-based traditional foods institute of brewing 113(3): 302–309. https://doi.org/10.1002/ j.2050-0416.2007.tb00290.x anonymous, 2021. english map of turkey regions. available at: https ://upload.w ikime dia .org/w ikip e dia/commons/c/c8/ turkey_regions_map-en.svg (accessed 4 september 2021). arici, m. and coskun, f., 2001. hardaliye: fermented grape juice as a traditional turkish beverage. food microbiology 18: 417–421. https://doi.org/10.1006/fmic.2001.0413 aşkın, b., 2018. farklı sıcaklıkların hardaliyenin depolama stabilitesi üzerine etkisi. harran journal of agricultural food science 23(1): 13–21. https://doi.org/10.29050/harranziraat.409434 aydoğdu, h., yıldırım, ş., halkman, a.k. and 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https://ayk.gov.tr/wp-content/uploads/2015/01/u%c3%87ar-asl%c4%b1-geleneksel-t%c3%9crk-tadi-pekmez.pdf� https://ayk.gov.tr/wp-content/uploads/2015/01/u%c3%87ar-asl%c4%b1-geleneksel-t%c3%9crk-tadi-pekmez.pdf� https://doi.org/10.5505/pajes� https://doi.org/10.29133/yyutbd.453960� https://doi.org/10.3906/tar-1301-41� https://doi.org/10.3906/tar-1301-41� https://doi.org/10.1007/s12355-020-00832-z� https://doi.org/10.1007/s12355-020-00832-z� https://doi.org/10.1515/ijfe-2014-0313� https://doi.org/10.1002/mnfr.200900323� https://doi.org/10.34189/tfd.24.01.003� https://doi.org/10.34189/tfd.24.01.003� https://www.dunya.com/saglik/zahmetli-ve-degerli-kuru-uzum-ozel-haberi-435086� https://www.dunya.com/saglik/zahmetli-ve-degerli-kuru-uzum-ozel-haberi-435086� https://doi.org/10.1016/b978-0-12-815270-6.00015-3� https://doi.org/10.1016/b978-0-12-815270-6.00015-3� https://www.resmigazete.gov.tr/eskiler/2017/03/20170321-11.htm� https://www.resmigazete.gov.tr/eskiler/2017/03/20170321-11.htm� https://www.resmigazete.gov.tr/eskiler/2017/06/20170930-24.htm� https://www.resmigazete.gov.tr/eskiler/2017/06/20170930-24.htm� https://doi.org/10.1080/19393210.2013.833298� https://doi.org/10.1080/19393210.2013.833298� https://intweb.tse.org.tr/standard/standard/standardara.aspx� https://intweb.tse.org.tr/standard/standard/standardara.aspx� https://doi.org/10.1501/tarimbil_0000001392� _hlk139376596 _goback ijfs#1913_bozza ital. j. food sci., vol. 32, 2020 970 paper changes in lipid content with roasting temperature of large yellow croaker (larimichthys crocea) roe l. zhang1, m. zhang1, x. yang*2, l. chen1,3,4, w. cheng1,3,4 and p. liang*1,3,4 1college of food science, fujian agriculture and forestry university, fuzhou 350002, pr china 2key laboratory of aquatic product processing, ministry of agriculture and rural affairs; national r&d center for aquatic product processing; south china sea fisheries research institute, chinese academy of fishery sciences, guangzhou 510300, pr china 3engineering research centre of fujian-taiwan special marine food processing and nutrition ministry of education, fuzhou 350002, pr china 4key laboratory of marine biotechnology of fujian province, fuzhou 350002, pr china *corresponding author: yxqgd@163.com and liangpeng137@sina.com abstract this study aims to clarify the changes with roasting temperature in the lipid content of ready-to-eat large yellow croaker (larimichthys crocea) roe product. almost all the lipid class/species showed the same trend with the increasing temperature. except for some minor differences, the relative amounts of lipids decreased with temperature increase from 0°c (control group, raw roe) to 100°c; increased with further temperature increase to 120°c, at which the amount was maximum; and then decreased with further temperature increase to 180°c. finally, 120°c was selected as the optimal processing temperature, which may result in a better appearance and a high lipid quality, indicating its potential application value. this study also enhances the understanding of lipid profile in fish roe and demonstrates the applicability of the lipidomic method in aquatic food production. keywords: larimichthys crocea roe, phospholipid molecular species, lipid content, roasting temperature ital. j. food sci., vol. 32, 2020 971 1. introduction fish roe is known for its high nutritional value. its products have been consumed as caviar (from sturgeon) and caviar substitutes (from other species); other product forms include whole skeins and formulations with oils and cheese bases, in salted or smoked forms, and the international and domestic markets of these products continue to increase (bledsoe et al., 2003). in recent years, the valorization of roe has received much attention because of its gigantic yield and health benefits. some reports have shown that fish roe is available at lower prices. it can be incorporated in various food preparations to combat protein malnutrition and is an interesting source for supplementing the human diet with marine lipids (mahmoud et al., 2008; balaswamy et al., 2009; saliu et al., 2019). in addition, the valorization of roe by encouraging professional exploitation (for fillets, caviar, or nutritional supplement production) can also lower the pressure on the aquatic ecosystem (saliu et al., 2017). the large yellow croaker (larimichthys crocea) is a major commercial marine fish in the south of china, with a total production of approximately 180,000 tons in 2017 (chen et al., 2018). however, its roe is usually large and has an unattractive appearance. during the processing of l. crocea, its roe is a major by-product, which is usually discarded as waste. this is an important commercial loss and an environmental problem from the fish industry. the l. crocea roe has been verified to contain large amounts of n-3 polyunsaturated fatty acids (pufas), mainly eicosapentaenoic acids (epa, c20:5 n-3) and docosahexaenoic acids (dha, c22:6 n-3) as known, which can help prevent the incidence of coronary heart diseases, inflammatory and autoimmune disorders, and cancers (wang et al., 2008; rosa et al., 2012; ozogul et al., 2007). the predominant phospholipids (pls) in l. crocea roe are phosphatidylcholine (pc) and phosphatidylethanolamine (pe), as determined in our previous study (liang et al., 2017a), which corresponds with other reports on fish roe (hayashi et al., 1999; shirai et al., 2006). phospholipids are major polar lipid components, and they serve as building blocks for cell membranes and have important physiological and biological functions in almost all known living beings (burri et al., 2012; suzumura, 2005). the diversity of pl molecule species can be ascribed to the number of carbons and double bonds in the fatty acid moiety and the moiety locations on the glycerol backbone with one headgroup, which are the sn-1 and/or sn-2 position and sn-3 position of pls. marine pls have also been confirmed capable of reducing inflammatory reactions (deutsch, 2007) and preventing colon cancer growth induced by chemicals in vitro (hossain et al., 2009). moreover, our previous study identified that the pls with docosahexaenoic acid (dha) from the l. crocea roe had beneficial effects on the lipid metabolism of hyperlipidemic mice (liang et al., 2017b). the changes in the pls and other lipid components can be affected by the storage time and freezing/cooking temperatures, which causes autoxidation, hydrolytic decomposition, lipid browning, lipid-protein copolymerization reactions, and lipolysis or enzymatic degradation in the food products (igene et al., 1981; lee et al., 1976; wang et al., 2011). additionally, the pl loss can be due to different mechanisms of heat transfer, which cause cell rupture (mondy et al., 1977). furthermore, the pl quantity can affect the flavor and nutritional quality in the food matrix (de lima et al., 2008). however, the changes in the total lipid content and pl molecular species with high temperatures are still not clear for the l. crocea roe. recently, the shotgun lipidomic approach was developed to replace the traditional methods (i.e., thin-layer chromatography) for monitoring the molecular compositions and ital. j. food sci., vol. 32, 2020 972 abundances of individual lipid species from unfractionated lipid extracts more rapidly and with higher sensitivity (wang et al., 2011). for the comprehensive analysis of lipid structures, the developed technology of reversed-phase ultra-performance liquid chromatography (uplc) coupled with electrospray ionization–quadrupole-time-of-flight– mass spectrometry (uplc-esi-q-tof-ms) possesses superior separation ability, higher resolution, greater sensitivity, and faster speed (wang et al., 2011; basconcillo et al., 2009; laaksonen et al., 2006; yan et al., 2010; zhao et al., 2014). herein, we assumed that the changes in pls and other lipid components can be confirmed via shotgun lipidomics. this study aimed to identify the relative changes in lipid content, especially the pl molecular species, with the roasting temperature in the ready-to-eat l. crocea roe product and determine the appropriate roasting temperature that can keep the best lipid compositions of the roe. the study clarifies the value of the l. crocea roe products and broadens the comprehensive utilization of the roe. moreover, the application of lipidomics on the roasting processing of aquatic foods is shown to be beneficial for evaluating the changes in lipid compositions. this study can also promote the lipidomics method application in aquatic food production. 2. materials and methods 2.1. materials and reagents the l. crocea roe was provided by fujian yuehai aquatic food ltd. (ningde city, fujian province). the roe was mixed and kept under refrigeration (0-4°c) for less than 24 h before analysis in the lab of aquatic food products processing at fujian agriculture and forestry university. in total, 10 lipid standards pc (17:0), lysophosphatidylcholine (lpc; 15:0/0:0), phosphatidylglycerol (pg; 15:0/15:0), pc (15:0/15:0), pe (15:0/15:0), sphingomyelin (sm; d18:1/17:0), phosphatidylserine (ps; 17:0/17:0), ceramides (cer; d18:1/17:0), diacylglycerol (dg; 17:0/0:0/17:0), and triglyceride (tg; 15:0/15:0/15:0) were purchased from avanti polar lipids (alabaster, alabama, us). high-performance liquid chromatography (hplc)-grade isopropanol (ipa) and methanol were purchased from merck (darmstadt, germany). other hplc-grade compounds, acetonitrile (acn), formic acid, ammonium formate, leucine-enkephalin, and sodium formate, were purchased from thermo fisher scientific (shanghai, china). 2.2. sample preparation the fresh l. crocea roe was first powderized. this was achieved by drying using a vacuum freeze-dryer (true ten industrial co., ltd. taichung, taiwan) and filtering through an 80mesh sieve. then, a certain amount of water (1:2 w/w) was added into the l. crocea roe powder. the mixture was evenly stirred and moved onto a plate. a specific shape (1 × 4 × 4 cm) of the mixture was cut after extrusion and molding. afterward, the mixture was moved into a commercial microwave oven (newsail ns-x4, henan, china). the roasting temperatures were 100, 120, 140, 160, and 180°c, and the roasting time was 20 min. finally, the finished ready-to-eat product was prepared. after the product was cooled, it was vacuum-packaged and stored in a freezer (-20°c). ital. j. food sci., vol. 32, 2020 973 the finished product was added to 1.4 ml of ipa in a 2 ml centrifuge tube, vortex-mixed for 1 min, and sonicated for 10 min. the samples were kept in a freezer (-20°c) for 1 h and then freeze-centrifuged at 14,000 g for 10 min. the supernatant was collected, and 1 ml was filtered into uplc vials through a 0.22 μm organic filter membrane. the samples were kept in a freezer (-20°c) for later analysis. 2.3. pl molecular species analysis via uplc-q-tof-ms 2.3.1 uplc parameters the uplc system was equipped with a c18csh column (1 × 50 mm, 1.7 µm; waters ltd., elstree, u.k.). the mass spectrometry (ms) method of the xevo g2-s q-tof (waters ltd., manchester, u.k.) was implemented to improve the isotopic distribution and mass accuracy and to reduce the high ion intensities. in total, 2 μl of the samples was injected onto a c18csh column at 55°c. the mobile-phase flow rate was set as 400 µl/min. the mobile phases were as follows: (a) acn/h2o (60%/40%), including 10 mm ammonium formate and 0.1% formic acid; (b) ipa/acn (90%/10%), including 10 mm ammonium formate and 0.1% formic acid. the gradient profile was as presented in table 1. table 1. the gradient profile of uplc. 2.3.2 q-tof-ms parameters for both positive-ion and negative-ion modes, the ms parameters were as follows: capillary voltage of 3 kv, cone voltage of 25 v, esi source temperature of 120°c, desolvation temperature of 500°c, desolvation gas flow of 800 l/h, and cone gas flow of 50 l/h. the mass spectra were acquired over m/z 50 to 2000. leucine enkephalin (m/z 556.2771 in esi+, m/z 554.2615 in esi−) was continuously infused at 30 μl/min and used as the lock mass. ital. j. food sci., vol. 32, 2020 974 2.4. statistical analysis all analyses were conducted in triplicate, and the results were indicated as mean ± standard deviation. the means and standard deviations were calculated using the spss statistical software (version 19.0, spss). the software was used to perform a one-way analysis of variance and tukey’s honest significant difference test at a 95% confidence level (p<0.05) to identify differences among groups. a statistical t-test model was applied for comparative analysis involving different groups. masslynx software version 4.1 was used for the ms data acquisition and analysis. all lipid profile data were first standardized and normalized and then subjected to principal component analysis using simca-p 13.0. heatmaps of lipids data were created using the r software. 3. results and discussion 3.1. effect of roasting temperature on l. crocea roe appearance consumers usually assess the quality of a food product by its appearance, which is also important to evaluate how well a product is cooked. fig. 1 shows the appearances of the prepared l. crocea roe product under different roasting temperatures. the appearance changed gradually (yellow-brown-deep brown) with increase in the temperature, from 0°c (control group) to 180°c. a similar finding has been reported for soybeans (yoshida et al., 2003). nakamura et al. (2011) reported that the color change during grilling consisted of four steps: (1) protein denaturation, (2) water evaporation, (3) browning reaction, and (4) carbonization reaction. according to matsuda et al. (2013), fish fillets began to darken during grilling when the temperature was close to 150°c under radiant (far-infrared radiation) heating. in the present study, the l. crocea roe product started to darken at 140°c and exhibited an attractive appearance at 120°c. meanwhile, cyclic compounds (e.g., some aldehydes, pyrazines) can be formed to release a fragrant odor during roasting. the brown pigments from the l. crocea roe product could also degrade under very high temperatures. the lipids inside may deteriorate through hydrolysis and oxidation (fritsch, 1981). furthermore, pes have been reported to be related to lipid browning deterioration, which may cause a brown color (lee et al., 1976). the pe in the l. crocea roe could be responsible for the browning at high temperatures through hydrolysis and oxidation. figure 1. appearances of the l. crocea roe at different roasting temperatures. ital. j. food sci., vol. 32, 2020 975 3.2. lipid contents of l. crocea roe at different roasting temperatures the lipid profile was identified and separated using uplc-esi-q-tof-ms. more analysis details can be seen in our previous paper (liang et al., 2018). the species of 167 pcs, 105 pes, 17 pis, 26 pgs, 78 pss, 55 pas, 12 cls, 17 sms, 27 cers, 10 mgs, 181 dgs, and 248 tgs were detected in the l. crocea roe product at different roasting temperatures. 3.2.1 effects of roasting temperature on relative content of pl fig. 2 displays the relative content variations of different lipid classes at different roasting temperatures. almost all lipid classes exhibited the same trend. generally, their quantities decreased with temperature increase from 0 to 100°c, increased with further temperature increase to 120°c, at which the amounts were largest; and reduced again with temperature increase from 120°c to 180°c; however, pi, pg, ps, and cl exhibited some minor differences. pi and pg increased with temperature increase from 0 to 100 to 120°c. pi remained constant from 100°c to 160°c, with the largest amount at 160°c, and it decreased slightly from 160°c to 180°c. however, pg decreased gradually with temperature increase from 120°c to 180°c. ps and cl remained stable from 0 to 100°c, and the rest exhibited the same trend. figure 2. effects of roasting temperature on the relative content of pls in l. crocea roe. note: the same letters in the same column means that no significant difference exists between the amounts at the significance level of p<0.05, and vice versa. the variable importance in projection (vip) values were adopted for the selected lipid classes that changed significantly. the vip method is usually used to identify the variables (vip > 1) in the orthogonal projections to latent structures discriminant analysis. according ital. j. food sci., vol. 32, 2020 976 to their vip values (vip > 1), 56 pcs, 24 pes, 17 pss, 6 cls, 5sms, 4 pas, 3 pgs, and 3 pis were selected for analysis among the detected lipid molecular species. all lipid classes except pi showed the highest value at 120°c. vujasinovic et al. (2012) determined that the total pl content increased from 0 to 130°c (0, 90, 110, and 130°c) in roasted pumpkin oil. clark et al. (1991) reported that a high phosphorus content was obtained through the preheating process at 130 °c in crude soybean oil extracted from fine flour. they suggested that pls had better solubility in the hot oil, and pls may be released from cell membranes. however, to the best of our knowledge, this study is the first to identify 120 °c as the temperature at which the highest concentration of most lipid classes was found. further research is needed to obtain the underlying reason for this. perhaps, this temperature allowed the release of more lipids, which could not be extracted using the abovementioned method. apart from the temperature of 120°c, the pl class gradually declined from 0 to 180°c, except for pi, which agrees with the report on safflower seeds (lee et al., 2004), in which temperatures of 0, 140, 160, and 180°c were considered using an electric oven. with increasing temperature, pc, pe, and pa were found to decrease, while pi increased. in the current study, the pl decomposition or formation through a reaction with protein or carbohydrate may explain the pl reduction after the roasting treatment (yoshida et al., 2005). abou-gharbia et al. (2000) determined that pc, pi, pe, and ps in sesame oil decreased with roasting treatment (200°c) and steaming (100°c), while pa and lpc increased. in this study, pi exhibited a distinctive trend, increasing with temperature increase from 0100°c; this may be because it had the highest saturated fatty acid content among the pls of the l. crocea roe product. the decrease in pe may be related to the pi increase; that is, pe transformed to pi with the increasing temperature (lee et al., 2004). pc can be obtained via subsequent methylation of the amine by s-adenosyl methionine from pe. sm is the only lipid belonging to the sphingolipids class and the pl class. it was formed via the transfer of phosphorylcholine from pc to cer via sm synthase (merrill, 2011). therefore, it is possible that some pes were transferred to pcs and that some pcs were transferred to sms. similarly, ps is formed when pa reacts with serine, and pe is formed when pa reacts with ethanolamine (ambrosewicz-walacik et al., 2015). however, it is not clear if the reaction can occur in the l. crocea roe product at high temperatures. further study is needed to explore this in more detail. however, the compositions of each pl species are different in different food matrices; for example, marine pls contain more n-3 pufas. normally, the pls, which contained more unsaturated fatty acids, were easily oxidized. a considerable loss has been detected for the pl molecular species containing more than four double bonds (yoshida et al., 2001a). we analyzed the relative content changes in the pcs with vip of more than four, and the results are illustrated in fig. 3. the relative contents of these nine pcs displayed the same change trend as the pc total amount in fig. 2. almost all the nine analyzed pcs contained unsaturated fatty acids in their sn-2 positions except for one pc (0:0/16:0), and six of them consisted of c20:5 omega-3 and c22:6 omega-3, which influenced the omega-3/omega-6 ratio, meaning that they were easily oxidized in the oven. similarly, the pes, pgs, and pss with the variable of vip > 1 all contained an unsaturated fatty acid at their sn-1, sn-2 positions, or both. ital. j. food sci., vol. 32, 2020 977 figure 3. effects of roasting temperatures on the relative content of pcs in the l. crocea roe. note: the same letters in the same column means that no significant difference exists between the amounts at the significance level of p<0.05, and vice versa. 3.2.2 effect of roasting temperature on the relative contents of other lipids a characteristic pattern of tgs exists in almost every type of oil or other food matrices. the pattern is determined by the abundances of different tg molecular species. yoshida et al. (2001b) and cossignani et al. (1998) determined that the tg fraction decreased, while dg and mg increased over time in the microwave roasting for sunflower and olive oil. in this study, 181 dgs, 248 tgs, 10 mgs, and 27 cers were detected in the l. crocea roe at different roasting temperatures. according to fig. 4, the relative contents of tgs, dgs, and mgs decreased with temperature increase from 0 to 100°c, which corresponds to the findings of previous studies, but the values increased again with temperature increase from 100 to 120°c and reached the highest point at 120°c, which is not mentioned in the other reports. yoshida et al. (2001b) only tested the tg loss at 98, 137, 164, and 172°c; they did not know if the tgs changed at 120°c in sunflower oil; however, the tg content at 137°c was less than that at 172 °c, which disagrees with this study results. in this study, the dgs slightly increased with temperature increase from 140 to 180°c, possibly due to the tg decomposition; however, the mgs decreased, which may be because the temperature was still insufficient for the tg and dg decomposition to mgs. this inference needs further verification. cer remained stable from 0 to 100°c, and the rest exhibited the same trend. ital. j. food sci., vol. 32, 2020 978 figure 4. effects of roasting temperature on the relative amount of glycerol lipids (gls) and cer in l. crocea roe. note: the same letters in the same column means that there is no significant difference between the amounts at the significance level of p < 0.05, and vice versa. according to their vip values (vip>1), 4 cers, 84 tgs, 31 dgs, and 2 mgs were detected in the lipid profile. however, for further analysis, three tgs were selected based on their vip values (vip>3); the compounds were tg (16:0/16:0/18:1), tg (16:0/16:1/16:1), and tg (16:0/16:1/18:1) with 53, 51, and 53 carbon numbers (cn), respectively. they all contained an unsaturated fatty acid linked at the sn-3-position. yoshida et al. (2001a) reported that an unsaturated fatty acid linked at the sn-2-position of glycerol moiety in tgs helped to keep the moiety more stable than the same unsaturated fatty acid at the sn-1 or sn-3 positions. however, this finding disagrees with the results by liu et al. (2017), who studied tgs with 51–56 cn, which were stable than tgs with 26-48 cn. 3.2.3 heatmap analysis of the lipid profile data a heatmap was adopted to better interpret the qualitative information of lipidomics datasets using the r software (fig. 5). the lipid molecular species in the heatmap were selected according to a combination of multidimensional and one-dimensional analyses. the changes appeared between every two neighboring groups based on the vip value and p-value in the student’s t-test (vip>1, p<0.05). green color denotes increase, and red denotes decrease. from fig. 5, all the selected lipid molecular species followed the same trends in figs. 2 and 3. pcs were the molecular species that changed the most between two neighboring groups. ital. j. food sci., vol. 32, 2020 979 figure 5. heatmaps of the lipidomics dataset profiles. note: red color represents an increase, and green represents a decrease. ck-1, ck-2, and ck-3 represent the first, second, and third analyses conducted using the control group, respectively; n100-1, n100-2, and n1003 represent the first, second, and third analyses conducted using the group at 100 °c, respectively; the rest mark number is similar. 4. conclusions the l. crocea roe is a valuable byproduct that contains high amounts of valuable epa and dha. however, it is large and has an unattractive appearance. discarding this roe as waste would be an important commercial loss and an environmental problem from the fish industry. this study investigated the further processing of the roe to make it more acceptable to consumers. the roe was roasted under different temperatures to determine the lipid amount changes. the relative amounts of almost all lipid classes were highest at 120°c, except for pi; meanwhile, the temperature of 120°c allowed to obtain a practically sterilized product, stable at room temperature if well packaged. no previous study has ital. j. food sci., vol. 32, 2020 980 mentioned this point before. perhaps, this temperature is the best for processing without a significant loss of valuable pls, as the obtained product is both of good quality and readyto-eat. moreover, this study clarifies the changes in lipid classes, especially pls molecular species in the l. crocea roe, with temperature. using the lipidomics method for analysis, the study also demonstrates the value of the method in the fish industry. acknowledgments this work was supported by the national natural science foundation of china (grant no. 31801465) and the outstanding young scientific research talent program of fujian agriculture and forestry university (grant no. xjq201808). this research was also funded by the national key r&d program of china (grant no. 2018yfd0901001-04) and the fund of key laboratory of aquatic product processing, ministry of agriculture, china (grant no.nyjg201604). abbreviations phospholipid: pl phosphatidylcholine: pc phosphatidylethanolamine: pe phosphatidylinositol: pi phosphatidylglycerol: pg 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33 (sp1): 12–23 p u b l i c a t i o n s codon the occurrence of aflatoxin m1 in industrial and traditional fermented milk: a systematic review study camilla de souza1, amin mousavi khaneghah2*, carlos augusto fernandes oliveira3* 1department of veterinary medicine, school of animal science and food engineering, university of são paulo, pirassununga, sp, brazil; 2department of food science, school of food engineering, state university of campinas, campinas, sp, brazil; 3department of food engineering, school of animal science and food engineering, university of são paulo, pirassununga, sp, brazil *corresponding authors: amin mousavi khaneghah, department of food science, school of food engineering, state university of campinas, campinas, sp, brazil. email: mousavi@unicamp.br; carlos augusto fernandes oliveira, department of food engineering, school of animal science and food engineering, university of são paulo. av. duque de caxias norte, 225, cep 13635-900, pirassununga, sp, brazil. email: carlosaf@usp.br received: 28 august 2020; accepted: 12 december 2020; published: 9 february 2021 © 2021 codon publications open access review abstract aflatoxin m1 (afm1) is a toxic secreted into the milk of animals fed with diets contaminated by aflatoxin b1, which can cause some adverse health effects in humans. the occurrence of afm1 in dairy products varies based on several factors, including the fermentation process. in this article, the published citations from january 2000 to october 2020 regarding the afm1 occurrence in industrial and traditional fermented milk were systemically reviewed. according to the findings, a reducing trend in the afm1 contamination of fermented milk was observed over the years, mainly in traditional products. despite this trend, further control measures besides the preventative approaches are needed to deal with the high levels of afm1 in fermented milk. keywords: afm1; yogurt; fermented milk; occurrence; contamination; food safety; traditional dairy products introduction aflatoxins are toxic, secondary metabolites synthesized by some fungi species in the genus aspergillus, mainly those belonging to the species a. flavus, a. nomius, and a. parasiticus (ismaiel et al., 2020). aflatoxins are considered the most important mycotoxins, given their carcinogenic and hepatotoxic effects on animals and humans (bhat et al., 2010). among several types of aflatoxins, the most frequent ones found as natural contaminants of foodstuffs are aflatoxins b1 (afb1), b2 (afb2), g1 (afg1), and g2 (afg2) (nejad et al., 2019). while afb1 possesses the highest toxicity, this toxin is also classified as a group 1 carcinogen by the international agency for research on cancer (2002). in addition, afm1 and afm2 are produced by hepatic biotransformation of afb1 and afb2, respectively, and maybe shed through the urine and milk of animals (campagnollo et al., 2016; imamura et al., 2015). milk and milk products have high nutritional and biological value, contributing to a balanced diet for human beings. among dairy products, fermented milk is important, as it is consumed by a wide range of people, from infants to elders (barukcic et al., 2018). some fermented milk has, in their composition, probiotics that lead to improved digestibility, besides some other health promoting factors, such as bioactive peptides and bacteriocins (black, 2011). as afm1 is highly stable through pasteurization, ultra-high temperature processing, and mailto:mousavi@unicamp.br mailto:carlosaf@usp.br italian journal of food science, 2021; 33 (sp1) 13 aflatoxin m 1 occurrence in fermented milk fermentation or mesophilic cultures (kuboka et al., 2019). kefir is a dairy product rich in vitamins, essential amino acids, and minerals, made by fermenting the kefir grains (gamba et al., 2016). kefir is the most common probiotic product consumed in europe and is associated with beneficial health effects related to homeostasis balance (otles and cagindi, 2003). doogh is an iranian fermented product made from yogurt added with potable water, sodium chloride, and probiotic cultures (kiani et  al., 2018). while several studies have been dedicated for evaluating the afm1 levels in milk and other dairy products (fallah, 2010; kim et al., 2011; rahmani et al., 2018), no systematical review was conducted to summarize the findings. therefore, the current investigation was undertaken to systematically review the literature published in the last 20 years regarding the prevalence of afm1 in industrial and traditional fermented milk globally. literature search a systematic literature search was conducted among some international databases such as pubmed, science direct, and google scholar (as gray literature) using the following key terms: “aflatoxins” or “aflatoxin m1” or “mycotoxins” and “occurrence” or “contamination” or “prevalence” or “incidence” or “fermented milk”  or “dairy products” or “cultured dairy” or “yogurt” or “kefir.” all relevant articles published from january 2000 to october 2020 that investigated the prevalence of afm1 in fermented milk were retrieved and screened for eligibility. in addition, the reference lists of other processing methods used in dairy production, the toxin may be found not only in processed milk but also in dairy products (jalili and scotter, 2015). yogurt and other fermented milk products are typically manufactured by fermentation of lactic acid in milk, both traditional and industrialized products with different levels of afm1, given the range of ph values and fermentation conditions (govaris et al., 2002). however, studies related to afm1 contents in these fermented products are scarce and controversial (campagnollo et al., 2016; mahmood fashandi et al., 2018; mousavi khaneghah et al., 2017). figure 1 presents an overview processing steps of fermented milk and some relevant points regarding the afm1 contamination of these products. when fermented milk is produced using milk contaminated with afm1, the mycotoxins are not eliminated at once, as they are resistant to most processing steps (behfar et al., 2012). therefore, to safeguard human health, maximum limits of afm1 residues recommended in most countries range from 0 to 1.0 μg/l of milk (iqbal et al., 2015). in the european union (eu), the tolerable limit for afm1 in milk is no more than 0.05 μg/l (european commission, 2006), while in the united states and brazil, a maximum level of 0.5 μg/l is accepted (agência nacional de vigilância sanitária, 2011; food and drug administration, 2000). besides yogurts, other fermented products are also susceptible to afm1 contamination, including traditional ones, such as lala, kefir, and doogh. lala is traditional african fermented milk produced by natural afm1 in naturally contaminated milks starter culture flavor, fruits raw milk • standardization • homogenization • additions (sugar, powder milk) heat treatment inoculation incubation cooling pre-treatments packaging cold storage cooling negligible degradation of afm1 decontamination effects during fermentation : • binding of afm1 to microbial cells • afm1 degradation by microbial enzymes • changes in the afm1-casein by low ph figure 1. general processing flow chart of fermented milk and relevant steps regarding the aflatoxin m 1 (afm 1 ) contamination during manufacture (in italic). 14 italian journal of food science, 2021; 33 (sp1) de souza c et al. in milk in the african continent (muaz et al., 2021). in addition to the climatic conditions that favor fungal growth in several geographic areas in africa, the lack of effective regulation of aflatoxins in the food chain and the low public awareness of this risk are among important factors that contribute to high prevalence of aflatoxins in african countries (wild et al., 2015). in egypt, 63% of the yogurt samples exceed the eu’s afm1 levels (aiad and aboelmakarem, 2013). the mean prevalence of afm1 in egyptian yogurt samples was higher in the winter than in the summer. coherently, higher afm1 levels in milk samples have also been reported in the winter season in different countries (bilandzic et al., 2014; de roma et al., 2017; fallah, 2010; ruangwises and ruangwises, 2010). the reasons for such a higher prevalence of afm1 in milk and fermented products during the winter are not well established but may involve higher consumption of afb1contaminated feed by dairy cows during this period, as well as differences in the feed storage and diet composition, and rainfall effects (fallah, 2010; hajmohammadi et al., 2020). after incubation of lactobacillus acidophilus and bifidobacterium lactis into the fermented products, a decrease in mycotoxin prevalence was observed at the end of the storage period (ibrahim et al., 2016). in this regard, the percentages of egyptian zabadi yogurt samples exceeding the european limits in 2016 and 2017 were 12.5 and 18.7, respectively. however, these prevalence data were lower in this product than in milk and cheese, mainly in the winter (ismaiel et al., 2020). in nigeria, 20 included articles were also manually searched to identify other suitable studies. after excluding unsuitable articles due to irrelevant content, 150 full texts of potentially eligible articles were downloaded. then, the downloaded citations were examined for inclusion and criteria of final eligibility. inclusion criteria were: (1) availability of full-text article, (2) original cross-sectional research studies (not reviews), (3)  reporting of afm1 prevalence among fermented, milk-based products, (4) indicating an accurate analytical method, and (5) published in the english in order to avoid any mistake during translation from other languages. the citations that did not meet these criteria were excluded. a total of 100 articles were excluded based on the abovementioned exclusion criteria according to prisma, as detailed in figure 2. finally, 50 articles that fulfilled the inclusion criteria were included in this review. the occurrence of aflatoxin m 1 in fermented milk table 1 presents the worldwide prevalence of afm1 in yogurt and other fermented milk products during the last 20 years. several studies reported a high prevalence of afm1 in yogurt and other traditional products in african countries. this is consistent with the high prevalence of afb1 reported in feedstuff used for dairy cows and afm1 literature search in databases [science direct (n = 50), pubmed (n = 40) and google scholar (n =60)] titles and abstracts reviewed: (n = 150) articles assessed for eligibility: (n = 50) excluded: • no original data (review, book, thesis or workshop) (n = 60) • studies on other mycotoxins or other related dairy products (n = 10) • only analytical method development, or insufficient method description, or comparison of different analytical methods (n = 30) final included articles: (n = 50) /data (n = 72) id en tif ic at io n s cr ee ni ng e lig ib ili ty in cl ud ed figure 2. flow chart describing the search and selection of articles evaluated in the study. italian journal of food science, 2021; 33 (sp1) 15 aflatoxin m 1 occurrence in fermented milk ta bl e 1. o cc ur re nc e of a fla to xi n m 1 i n yo gu rt a nd o th er fe rm en te d m ilk s re po rt ed in th e la st 2 0 ye ar s. c ou nt ry ty pe o f pr od uc t s am pl es an al yz ed (n ) po si tiv e sa m pl es lo d (n g/ kg o r l) c on ce nt ra tio n (n g/ kg o r l) a na ly tic al m et ho d r ef er en ce n % r an ge m ea n a fri ca : e gy pt yo gu rt a 30 8 26 5 11 .4 0– 98 .8 0 28 .4 1 e li s a a ia d an d a bo el m ak ar em (2 01 3) e gy pt (w in te r) yo gu rt 24 12 50 n r 56 .6 0– 84 .1 4 64 .6 8 h p lc ib ra hi m e t a l. (2 01 6) e gy pt (s um m er ) yo gu rt 24 12 50 n r 31 .4 6– 66 .0 5 39 .1 3 h p lc ib ra hi m e t a l. (2 01 6) e gy pt (2 01 6 pr od uc tio n) yo gu rt z ab ad y 32 4 12 50 13 0– 24 0 18 5 h p lc is m ai el e t a l. (2 02 0) e gy pt (2 01 7 pr od uc tio n) yo gu rt z ab ad y 32 6 19 50 10 0– 17 0 13 0 h p lc is m ai el e t a l. (2 02 0) n ig er ia yo gu rt 2 10 10 1 58 3. 5– 64 7. 0 0. 62 µ g/ l h p lc a nt ho ny e t a l. (2 01 6) b ur un di yo gu rt 6 6 10 0 n r 8, 20 0– 63 ,2 00 33 ,5 00 e li s a u do m ku n et a l. (2 01 8) k en ya yo gu rt 8 3 37 n r < lo d — 69 0 n r e li s a la ng at e t a l. (2 01 6) k en ya yo gu rt 21 12 57 2 17 –1 ,1 00 13 4 e li s a li nd ah l e t a l. (2 01 8) k en ya la la 27 8 30 2 12 –1 60 48 e li s a li nd ah l e t a l. (2 01 8) k en ya yo gu rt 17 13 77 2 26 –2 70 96 e li s a li nd ah l e t a l. (2 01 8) k en ya la la 8 5 63 2 10 –3 40 11 1 e li s a li nd ah l e t a l. (2 01 8) k en ya yo gu rt n r n r n r 5 n r 37 9. 3 e li s a k ub ok a et a l. (2 01 9) k en ya la la n r n r n r 5 n r 37 9. 3 e li s a k ub ok a et a l. (2 01 9) c on go r ep ub lic yo gu rt 2 3 67 n r 4, 80 0– 26 ,0 00 16 ,1 00 e li s a u do m ku n et a l. (2 01 8) a m er ic as : b ra zi l yo gu rt 53 47 72 3 10 –5 29 n r h p lc ih a et a l. (2 01 1) b ra si l yo gu rt 3 3 10 0 3 75 .1 –1 12 .9 94 h p lc ih a et a l. (2 01 3) a si a: q at ar yo gu rt 21 16 76 n r 4. 16 –3 8. 21 31 .3 2 e li s a h as sa n et a l. (2 01 8) c hi na yo gu rt 27 15 55 5 4. 0– 47 .0 17 .2 h p lc g uo e t a l. (2 01 9) c hi na yo gu rt n r n r n r 0. 6 n r n r e li s a zh ou e t a l. (2 01 9) s ou th k or ea yo gu rt 55 15 27 20 20 –1 50 51 h p lc yo on e t a l. (2 01 6) s ou th k or ea yo gu rt 60 50 83 2 3– 17 2 29 e li s a k im e t a l. (2 00 0) (c on tin ue s) 16 italian journal of food science, 2021; 33 (sp1) de souza c et al. ta bl e 1. c on tin ue d c ou nt ry ty pe o f pr od uc t s am pl es an al yz ed (n ) po si tiv e sa m pl es lo d (n g/ kg o r l) c on ce nt ra tio n (n g/ kg o r l) a na ly tic al m et ho d r ef er en ce n % r an ge m ea n ira n p as te ur iz ed y og ur t 40 40 10 0 n r 2. 1– 61 .7 15 .1 e li s a b ar je st eh e t a l. (2 01 0) ira n yo gu rt 10 10 10 0 n r 7– 53 25 e li s a b ar je st eh e t a l. (2 01 0) ira n yo gu rt 68 45 66 12 15 –1 19 32 h p lc fa lla h (2 01 0) ira n tr ad iti on al y og ur t 60 14 23 12 .5 15 –3 6 17 h p lc fa lla h et a l. (2 01 1) ira n in du st ria l y og ur t 61 30 49 12 .5 15 –1 02 26 h p lc fa lla h et a l. (2 01 1) ira n tr ad iti on al d oo gh 65 9 14 12 .5 13 –2 9 n r h p lc fa lla h et a l. (2 01 1) ira n in du st ria l d oo gh 71 16 22 .5 12 .5 13 –5 3 n r h p lc fa lla h et a l. (2 01 1) ira n yo gu rt 60 48 80 10 19 .7 –3 19 .4 13 0. 5 e li s a r ah im i ( 20 12 ) ira n yo gu rt 60 59 98 n r 6. 2– 87 51 .7 e li s a is sa za de h et a l. (2 01 2) ira n yo gu rt 13 13 10 0 n r 5– 36 13 .5 e li s a a ra st e t a l. (2 01 2) ira n yo gu rt 40 14 35 n r 11 .4 –1 15 .8 13 0. 5 e li s a n ilc hi an a nd r ah im i ( 20 12 ) ira n yo gu rt 80 77 96 5 < lo d — 10 0 29 .1 e li s a m as on e t a l. (2 01 5) ira n yo gu rt 42 10 24 1 6. 3– 21 .3 15 .1 e li s a b ah ra m i e t a l. (2 01 5) ira n d oo gh 44 6 14 n r 7. 0– 12 .1 9. 0 e li s a b ah ra m i e t a l. (2 01 5) ira n yo gu rt 90 90 10 0 n r 5. 0– 83 .0 32 .1 e li s a n ik ba kh t e t a l. (2 01 6) ira n yo gu rt 18 15 83 n r 7. 8– 12 .1 10 .3 e li s a s oh ra bi a nd g ha ra hk ol i ( 20 16 ) ira q yo gu rt 32 32 10 0 n r 0. 16 –4 2. 74 16 .9 2 e li s a n aj im a nd j as im (2 01 4) ira q tr ad iti on al y og ur t 20 15 75 n r 22 .2 –1 72 .9 10 3. 9 h p lc m os sa w ei e t a l. (2 01 6) ira q yo gu rt 20 10 50 n r 30 .5 -1 07 .4 58 .3 7 h p lc m os sa w ei e t a l. (2 01 6) k uw ai t yo gu rt 2 1 50 10 n r n r h p lc iv as ta va e t a l. (2 00 1) le ba no n yo gu rt 64 21 33 5 5– 50 n r e li s a e l k ho ur y et a l. (2 01 0) le ba no n yo gu rt n r n r 72 n r n r 24 .5 5 e li s a h as sa n an d k as sa ify (2 01 4) le ba no n yo gu rt 28 18 64 3. 2 15 –5 45 91 h p lc d ao u et a l. (2 02 0) le ba no n s tra in ed y og ur t 27 24 89 3. 2 37 –1 ,8 43 20 1 h p lc d ao u et a l. (2 02 0) le ba no n yo gu rt a yr an 9 8 89 3. 2 20 –3 15 24 2 h p lc d ao u et a l. (2 02 0) m al ay si a yo gu rt 5 2 40 2 7. 5– 31 25 .4 e li s a n ad ira e t a l. (2 01 6) p ak is ta n yo gu rt 96 59 61 4 4. 0– 61 5. 8 90 .4 h p lc iq ba l a nd a si (2 01 3) p ak is ta n (w in te r) yo gu rt 51 13 25 4 n r 53 h p lc iq ba l e t a l. (2 01 3) p ak is ta n (s um m er ) yo gu rt 45 8 18 4 n r 19 h p lc iq ba l e t a l. (2 01 3) p ak is ta n (w in te r) p la in y og ur t 36 15 42 0. 4 n r 63 .6 h p lc iq ba l e t a l. (2 01 7) p ak is ta n (w in te r) fl av or ed y og ur t 30 17 57 0. 4 n r 50 .5 h p lc iq ba l e t a l. (2 01 7) p aq ui st ão (s um m er ) p la in y og ur t 30 11 37 0. 4 n r 59 .6 h p lc iq ba l e t a l. (2 01 7) p ak is ta n (s um m er ) fl av or ed y og ur t 25 10 40 0. 4 n r 45 .3 h p lc iq ba l e t a l. (2 01 7) ta iw an yo gu rt 24 3 12 5 7– 44 n r h p lc li n et a l. (2 00 4) tu rk ey yo gu rt 40 32 80 50 61 .6 1– 36 5. 64 n r e li s a g ur ba y et a l. (2 00 6) tu rk ey p la in y og ur t 10 4 68 65 n r 1– 10 0 n r e li s a a kk ay a et a l. (2 00 6) tu rk ey yo gu rt w ith fr ui ts 21 7 33 n r 1– 10 0 n r e li s a a kk ay a et a l. (2 00 6) tu rk ey yo gu rt 52 29 56 n r 1– 15 0 n r e li s a a kk ay a et a l. (2 00 6) tu rk ey yo gu rt 80 70 87 5 10 –4 75 66 .1 e li s a a ta se ve r e t a l. (2 01 1) tu rk ey yo gu rt a yr an 80 72 90 5 6– 26 4 36 .5 e li s a a ta se ve r e t a l. (2 01 1) tu rk ey yo gu rt 50 10 20 2 40 .6 2– 72 .0 4 55 .2 8 e li s a te m am og ul la ri an d k an ic i ( 20 14 ) tu rk ey yo gu rt 19 17 89 10 0 16 –1 07 .2 47 .9 2 h p lc s ar ic a et a l. (2 01 4) tu rk ey yo gu rt 60 2 3 5 24 –2 8 n r h p lc s ah in e t a l. (2 01 6) tu rk ey yo gu rt a yr an 60 1 2 5 n r 5 h p lc s ah in e t a l. (2 01 6) e ur op e: ita ly yo gu rt 12 0 73 61 1 1. 0– 32 .2 9. 1 h p lc g al va no e t a l. (2 00 1) po rt ug al p la in y og ur t 48 2 4 10 43 .0 –4 5. 0 44 .0 h p lc m ar tin s an d m ar tin s (2 00 4) po rt ug al yo gu rt w ith fr ui ts 48 16 33 10 19 .0 –9 8. 0 51 .1 2 h p lc m ar tin s an d m ar tin s (2 00 4) s er bi a fe rm en te d m ilk s 30 2 n r n r 6 25 –5 00 19 0 e li s a k es ki c et a l. (2 01 6) s pa in yo gu rt 72 2 3 25 n r 38 .3 4 e li s a c an os an ch o et a l. (2 01 0) s pa in yo gu rt 6 2 33 25 n r 21 .6 e li s a c an os an ch o et a l. (2 01 5) a w ith ou t a ny fu rt he r d es ig na tio n, th e te rm “y og ur t” ap pl ie s to in du st ria l p ro du ct s. e li s a : e nz ym elin ke d im m un os or be nt a ss ay ; h p lc : h ig hpe rfo rm an ce li qu id c hr om at og ra ph y; l c -m s /m s : l iq ui d ch ro m at og ra ph y co up le d to ta nd em m as s sp ec tro m et ry . n r : n ot re po rt ed . italian journal of food science, 2021; 33 (sp1) 17 aflatoxin m 1 occurrence in fermented milk ta bl e 1. c on tin ue d c ou nt ry ty pe o f pr od uc t s am pl es an al yz ed (n ) po si tiv e sa m pl es lo d (n g/ kg o r l) c on ce nt ra tio n (n g/ kg o r l) a na ly tic al m et ho d r ef er en ce n % r an ge m ea n ira n p as te ur iz ed y og ur t 40 40 10 0 n r 2. 1– 61 .7 15 .1 e li s a b ar je st eh e t a l. (2 01 0) ira n yo gu rt 10 10 10 0 n r 7– 53 25 e li s a b ar je st eh e t a l. (2 01 0) ira n yo gu rt 68 45 66 12 15 –1 19 32 h p lc fa lla h (2 01 0) ira n tr ad iti on al y og ur t 60 14 23 12 .5 15 –3 6 17 h p lc fa lla h et a l. (2 01 1) ira n in du st ria l y og ur t 61 30 49 12 .5 15 –1 02 26 h p lc fa lla h et a l. (2 01 1) ira n tr ad iti on al d oo gh 65 9 14 12 .5 13 –2 9 n r h p lc fa lla h et a l. (2 01 1) ira n in du st ria l d oo gh 71 16 22 .5 12 .5 13 –5 3 n r h p lc fa lla h et a l. (2 01 1) ira n yo gu rt 60 48 80 10 19 .7 –3 19 .4 13 0. 5 e li s a r ah im i ( 20 12 ) ira n yo gu rt 60 59 98 n r 6. 2– 87 51 .7 e li s a is sa za de h et a l. (2 01 2) ira n yo gu rt 13 13 10 0 n r 5– 36 13 .5 e li s a a ra st e t a l. (2 01 2) ira n yo gu rt 40 14 35 n r 11 .4 –1 15 .8 13 0. 5 e li s a n ilc hi an a nd r ah im i ( 20 12 ) ira n yo gu rt 80 77 96 5 < lo d — 10 0 29 .1 e li s a m as on e t a l. (2 01 5) ira n yo gu rt 42 10 24 1 6. 3– 21 .3 15 .1 e li s a b ah ra m i e t a l. (2 01 5) ira n d oo gh 44 6 14 n r 7. 0– 12 .1 9. 0 e li s a b ah ra m i e t a l. (2 01 5) ira n yo gu rt 90 90 10 0 n r 5. 0– 83 .0 32 .1 e li s a n ik ba kh t e t a l. (2 01 6) ira n yo gu rt 18 15 83 n r 7. 8– 12 .1 10 .3 e li s a s oh ra bi a nd g ha ra hk ol i ( 20 16 ) ira q yo gu rt 32 32 10 0 n r 0. 16 –4 2. 74 16 .9 2 e li s a n aj im a nd j as im (2 01 4) ira q tr ad iti on al y og ur t 20 15 75 n r 22 .2 –1 72 .9 10 3. 9 h p lc m os sa w ei e t a l. (2 01 6) ira q yo gu rt 20 10 50 n r 30 .5 -1 07 .4 58 .3 7 h p lc m os sa w ei e t a l. (2 01 6) k uw ai t yo gu rt 2 1 50 10 n r n r h p lc iv as ta va e t a l. (2 00 1) le ba no n yo gu rt 64 21 33 5 5– 50 n r e li s a e l k ho ur y et a l. (2 01 0) le ba no n yo gu rt n r n r 72 n r n r 24 .5 5 e li s a h as sa n an d k as sa ify (2 01 4) le ba no n yo gu rt 28 18 64 3. 2 15 –5 45 91 h p lc d ao u et a l. (2 02 0) le ba no n s tra in ed y og ur t 27 24 89 3. 2 37 –1 ,8 43 20 1 h p lc d ao u et a l. (2 02 0) le ba no n yo gu rt a yr an 9 8 89 3. 2 20 –3 15 24 2 h p lc d ao u et a l. (2 02 0) m al ay si a yo gu rt 5 2 40 2 7. 5– 31 25 .4 e li s a n ad ira e t a l. (2 01 6) p ak is ta n yo gu rt 96 59 61 4 4. 0– 61 5. 8 90 .4 h p lc iq ba l a nd a si (2 01 3) p ak is ta n (w in te r) yo gu rt 51 13 25 4 n r 53 h p lc iq ba l e t a l. (2 01 3) p ak is ta n (s um m er ) yo gu rt 45 8 18 4 n r 19 h p lc iq ba l e t a l. (2 01 3) p ak is ta n (w in te r) p la in y og ur t 36 15 42 0. 4 n r 63 .6 h p lc iq ba l e t a l. (2 01 7) p ak is ta n (w in te r) fl av or ed y og ur t 30 17 57 0. 4 n r 50 .5 h p lc iq ba l e t a l. (2 01 7) p aq ui st ão (s um m er ) p la in y og ur t 30 11 37 0. 4 n r 59 .6 h p lc iq ba l e t a l. (2 01 7) p ak is ta n (s um m er ) fl av or ed y og ur t 25 10 40 0. 4 n r 45 .3 h p lc iq ba l e t a l. (2 01 7) ta iw an yo gu rt 24 3 12 5 7– 44 n r h p lc li n et a l. (2 00 4) tu rk ey yo gu rt 40 32 80 50 61 .6 1– 36 5. 64 n r e li s a g ur ba y et a l. (2 00 6) tu rk ey p la in y og ur t 10 4 68 65 n r 1– 10 0 n r e li s a a kk ay a et a l. (2 00 6) tu rk ey yo gu rt w ith fr ui ts 21 7 33 n r 1– 10 0 n r e li s a a kk ay a et a l. (2 00 6) tu rk ey yo gu rt 52 29 56 n r 1– 15 0 n r e li s a a kk ay a et a l. (2 00 6) tu rk ey yo gu rt 80 70 87 5 10 –4 75 66 .1 e li s a a ta se ve r e t a l. (2 01 1) tu rk ey yo gu rt a yr an 80 72 90 5 6– 26 4 36 .5 e li s a a ta se ve r e t a l. (2 01 1) tu rk ey yo gu rt 50 10 20 2 40 .6 2– 72 .0 4 55 .2 8 e li s a te m am og ul la ri an d k an ic i ( 20 14 ) tu rk ey yo gu rt 19 17 89 10 0 16 –1 07 .2 47 .9 2 h p lc s ar ic a et a l. (2 01 4) tu rk ey yo gu rt 60 2 3 5 24 –2 8 n r h p lc s ah in e t a l. (2 01 6) tu rk ey yo gu rt a yr an 60 1 2 5 n r 5 h p lc s ah in e t a l. (2 01 6) e ur op e: ita ly yo gu rt 12 0 73 61 1 1. 0– 32 .2 9. 1 h p lc g al va no e t a l. (2 00 1) po rt ug al p la in y og ur t 48 2 4 10 43 .0 –4 5. 0 44 .0 h p lc m ar tin s an d m ar tin s (2 00 4) po rt ug al yo gu rt w ith fr ui ts 48 16 33 10 19 .0 –9 8. 0 51 .1 2 h p lc m ar tin s an d m ar tin s (2 00 4) s er bi a fe rm en te d m ilk s 30 2 n r n r 6 25 –5 00 19 0 e li s a k es ki c et a l. (2 01 6) s pa in yo gu rt 72 2 3 25 n r 38 .3 4 e li s a c an os an ch o et a l. (2 01 0) s pa in yo gu rt 6 2 33 25 n r 21 .6 e li s a c an os an ch o et a l. (2 01 5) a w ith ou t a ny fu rt he r d es ig na tio n, th e te rm “y og ur t” ap pl ie s to in du st ria l p ro du ct s. e li s a : e nz ym elin ke d im m un os or be nt a ss ay ; h p lc : h ig hpe rfo rm an ce li qu id c hr om at og ra ph y; l c -m s /m s : l iq ui d ch ro m at og ra ph y co up le d to ta nd em m as s sp ec tro m et ry . n r : n ot re po rt ed . 18 italian journal of food science, 2021; 33 (sp1) de souza c et al. countries (table 1). in qatar, the incidence of afm1 in yogurts was analyzed using an immunoenzymatic assay (elisa); 76.1% of the samples were positive. however, none of them showed contamination levels above the eu maximum limits, posing no public health threats in this country (hassan et al., 2018). afm1 prevalence in yogurts produced with buffalo milk in different dairy factories in southern china were evaluated, and none of the samples had levels greater than the limit of 500 ng/ kg determined in the country(guo et al., 2019). another study carried out with cow milk showed afm1 levels inside the limit determined by this country and the eu (zhou et al., 2019). in south korea, 27.27% of the yogurt samples showed afm1, but none of them was above the limit determined by the korean ministry of food and drug safety (0.5 μg/kg) (kim-soo et al., 2016). however, in a previous study, 83% of yogurt samples were contaminated by this toxin (kim et al., 2000). in the mazandaran province of iran, 100% of the pasteurized yogurt and local yogurt samples were contaminated with afm1 (barjesteh et al., 2010). however, in another study in iran, 20.6% of yogurt samples were contaminated with levels above the limits determined by the local regulations (0.05 µg/l) and were greater in the winter than the summer (fallah, 2010). moreover, samples of traditional and industrial yogurt and doogh were evaluated, and the afm1 incidences in both these industrial products were greater in the autumn and winter than in traditional ones. as for doogh samples, the contamination levels were low, and no significant seasonal effect was observed (fallah et al., 2011). seasonal factors may influence the presence of the toxin in these products, as some studies observed higher levels of contamination in milk samples in the autumn and winter compared with summer and spring (kamkar, 2005). these variations may be related to the procedures during processing, degree of milk contamination, type of yogurt, fermentation conditions, geographic regions, season, country, and analytical methods used to detect these toxins (di guan et al., 2011). in general, yogurts have shown lower contamination levels with afm1 than cheese (rabie et al., 2019), as the fermentation process contributes to reducing the concentration of afm1 because of low ph and the production of fermentation-related byproducts such as organic acids, including lactic acid, among other factors (campagnollo et al., 2016). in milk, afm1 binds to casein, and the modifications on its structure caused by ph reduction during fermentation may lead to changes in this bound (govaris et al., 2002). however, the exact mechanisms involved in the mycotoxin decontamination during the fermentation process are not entirely understood. several experimental data indicate that aflatoxin reduction in fermented products occurs through its binding to the cell wall components of starter cultures, as reviewed by muaz et al. (2021), or through degradation samples of yogurt were analyzed, and 10% were contaminated with afm1 (anthony et al., 2016). in a study carried out in nairobi, the capital of kenya, afm1 was analyzed in samples of fermented milk and yogurt, and contamination was observed in levels above 0.05 μg/l (langat et al., 2016). in dagoretti and the westland area belonging to nairobi, 77 and 57% of lala and yogurt, respectively, contained detectable levels of afm1 (lindahl et al., 2018). in nairobi, a study with pasteurized yogurt and lala revealed that all samples had afm1 above the detection limit (5ng/kg). after undergoing an additional experimental fermentation, both products showed a significant reduction in afm1 prevalence (kuboka et al., 2019). the prevalence of afm1 in yogurt and milk samples was evaluated in burundi in the republic of the congo, and 29% of them showed levels much higher than the limits recommended by the eu (udomkun et al., 2018). few studies considering the prevalence of afm1 in fermented milk produced in the americas and european countries were conducted (table 1). in brazil, 95% of the samples of yogurt or dairy-based drinks from the ribeirão preto region were contaminated with afm1 (iha et al., 2011). interestingly, while the naturally contaminated yogurts from were incubated for 12 h, there was a reduction about 6% in the toxin levels (iha et al., 2013). the fermentation process in yogurts contributes to reducing the concentration of afm1 due to factors such as low ph, production of organic acids, and the presence of bacteria that synthesize lactic acid and other byproducts of fermentation (govaris et al., 2002). as for the european countries, afm1 was detected in the cataluña region of spain among 2.8% of the samples analyzed, the only one region that showed contamination above that determined by the eu (cano-sancho et al., 2010). in another study, however, 33% of yogurt samples from the same spanish region were contaminated with afm1, with none of them exceeding the european limits (cano-sancho et al., 2015). in samples of yogurt from italian supermarkets analyzed in 1996, 61% showed levels of afm1, but similar to spain, none of them exceeded the limits determined by the eu (galvano et al., 2001). in a study carried out in portugal, 4.2% of the samples of plain yogurt and 33.3% of the samples of strawberry yogurt were contaminated with this toxin (martins and martins, 2004). in serbia, the mean concentrations of afm1 in dairy products and fermented dairy drinks in 2015 were 0.018 and 0.019 µg/kg, respectively, with 5.86 and 2.64% of the samples exceeding the limits determined by the eu. it was also observed that the toxin levels were more significant in the winter and autumn in both products (keskic et al., 2016). the majority of data describing the prevalence of afm1 in fermented milk were provided by studies in asian italian journal of food science, 2021; 33 (sp1) 19 aflatoxin m 1 occurrence in fermented milk recommended by the eu. strained yogurt, popularly consumed by the lebanese population, showed 88.9% contaminated samples, with 81.5% above the eu acceptable limits. the authors suggested that these findings may be due to low-quality powdered milk in the production, leading to high levels of contamination in the final product. as for the yogurt drink ayran, 88.9% of the samples were positive, with 44.4% above the eu recommended limits (daou et al., 2020). in malaysia, 40% of the yogurt samples collected in january 2014 were contaminated with afm1, although none of them was above the limits determined by the eu (nadira et al., 2017). a study carried out in the winter and summer in pakistan showed that 37 and 29% of the samples of yogurt, respectively, were contaminated with this toxin, and were above the country limits (0.05 μg/l) (iqbal et al., 2013). in punjab, a province of pakistan, 47% of the yogurt samples were above the legal limits (iqbal and asi, 2013). corroborating these findings, another study carried out in the winter and summer showed that plain yogurt and flavored yogurt samples were contaminated with afm1 by 20 and 16%, respectively, and were above the levels determined by the eu during the summer. in the winter, 27.7 and 40%, respectively, were above the eu limits, posing a considerable threat to the population’s health (iqbal et al., 2017). in taiwan, 12.5% of the samples of yogurt beverages were contaminated with afm1 but at low levels (lin et al., 2004). on the other hand, in ankara, turkey’s capital, 32% of the yogurt samples showed afm1 levels above the country’s limit (gurbay et al., 2006). also, in turkey, 11.53% of the yogurt samples, 9.52% of fruit yogurt samples, and 21.15% of strained yogurt samples showed afm1 levels greater than those allowed by the existing regulations in the country (50 ng/kg) (akkaya et al., 2006). corroborating this finding, 20% of the yogurt samples evaluated in other studies showed contamination levels above the acceptable limits by turkish food codex (2008) (50 ng/kg) (atasever et al., 2011; temamogullari and kanici, 2014). another study in ankara showed that 89.5% of the yogurt samples were contaminated with afm1. only 5 were above the limit determined by the local regulations (sarica et al., 2015). on the other hand, in turkey, only two yogurt samples and one sample of ayran showed afm1, but the levels were below the eu limits (sahin et al., 2016). concluding remarks several studies regarding the prevalence of afm1 in industrial and traditional fermented milk were conducted worldwide in the past 20 years, indicating of the toxins by microbial enzymes into less toxic substances (guo et al., 2020). the most studied bacteria with practical afm1-binding abilities are lactic acid bacteria belonging to the genus levilactobacillus spp. (former lactobacillus sp.) such as l. rhamnosus and l. plantarum (sadiq et al., 2019). regarding bio-detoxification, several species in the genera pseudomonas, rhodococcus, streptomyces, bacillus, and pleurotus have been reported to be capable of degrading aflatoxins (guo et al., 2020). however, these bacterial species are not allowed to be used as starter cultures in fermented foods. the combination of fermentation with some emerging technologies, such as ultrasound, ohmic heating, and cold plasma, has been proposed, aiming at improving aflatoxin’s detoxification (gavahian et al., 2021). in shahr-e kord, iran, afm1 was detected in 35% of the yogurt samples, but not above the eu’s acceptable limit (nilchian and rahimi, 2012). in gilan, another province of iran, 63.33% of the yogurt samples were above the eu limits (issazadeh et al., 2012). in central iran, yogurt samples showed mean afm1 contamination levels of 13.55 ng/kg (arast et al., 2012). in isfahan, 80% of the yogurt samples were contaminated with this toxin, and 5% of them were above the limit determined by the eu (rahimi, 2014). in traditional iranian yogurts, these toxin levels were more significant than in industrialized products (mason et al., 2015). still, in iran, aflatoxins levels were evaluated in yogurt and doogh samples, with 23.8 and 13.6%, respectively, yielding positive results (bahrami et  al., 2016). however, in iran, 100% of the yogurt samples collected in 2014 were contaminated, with 22.22% above the afm1 limits determined by the eu (nikbakht et al., 2016). on the other hand, 83.3% of the yogurt samples were positive for afm1 in another study, although none of them was above the limits determined by the institute of standards and industrial research of iran (50 ng/l) (sohrabi and gharahkoli, 2016). in bagdad, the capital of iraq, 100% of the yogurt samples from supermarkets were contaminated with afm1 (jasim and najim, 2014). a study carried out with local and imported yogurts in iraq found that 75 and 50%, respectively, of the samples contained afm1 (al-mossawei et  al., 2016). in kuwait, one sample out of two yogurt samples produced in a local farm was contaminated with afm1 (ivastava et al., 2001). in lebanon, 32.81% of the samples analyzed showed the presence of afm1, with 6.25% of them exceeding the limits determined by the eu (el khoury et al., 2011). still, in lebanon, 72% of the yogurt samples analyzed showed afm1, with 13% above the recommended limits (hassan and kassaify, 2014). in another study carried out in lebanon with different yogurt types, it was observed that 64.3% of the samples were positive for afm1, and 35.7% were above the limits 20 italian journal of food science, 2021; 33 (sp1) de souza c et al. turkish journal of veterinary and animal sciences 35(1): 59–62. available at: https://journals.tubitak.gov.tr/veterinary/issues/ vet-11-35-1/vet-35-1-8-0906-96.pdf bahrami, r., shahbazi, y. and nikousefat, z., 2016. aflatoxin m1 in milk and traditional dairy products from west part of iran: occurrence and seasonal variation with an emphasis on risk assessment of human exposure. food control 62: 250–256. https://doi.org/10.1016/j.foodcont.2015.10.039 barjesteh, m.h., azizi, i.g. and noshfar, e., 2010. occurrence of aflatoxin m1 in pasteurized and local yogurt in mazandaran province (northern iran) using elisa. global veterinaria 4(5): 459–462. available at: http://idosi.org/gv/gv4(5)10/7.pdf barukčić, i., bilandžić, n., markov, k., jakopović, k.l. and božanić,  r., 2018. reduction in aflatoxin m1 concentration during production and storage of selected fermented milks. international journal of dairy technology 71(3): 734–740. https://doi.org/10.1111/1471-0307.12490 behfar, a., khorasgani, z.n., alemzadeh, z., goudarzi, m., ebrahimi, r. and tarhani, n., 2012. determination of aflatoxin m1 levels in produced pasteurized milk in ahvaz city by using hplc. jundishapur journal of natural pharmaceutical products 7(2): 80–84. https://doi.org/10.5812/jjnpp.4707 bhat, r., rai, r.v. and karim, a., 2010. mycotoxins in food and feed: present status and future concerns. comprehensive reviews in food science and food safety 9(1): 57–81. https://doi. org/10.1111/j.1541-4337.2009.00094.x bilandzic, n., bozic, d., dokic, m., sedak, m., kolanovic, b.s., varenina, i., tankovic, s. and cvetnic, z., 2014. seasonal effect on aflatoxin m1 contamination in raw and uht milk from croatia. food control 40: 260–264. https://doi.org/10.1016/j. foodcont.2013.12.002 black, e.p., 2011. dairy—fermented products. in: heldman, d.r., wheeler, m.b. and hoover, d.g. (ed.), encyclopedia of biotechnology in agriculture and food. publishing taylor and francis, new york, pp. 195–199. campagnollo, f.b., ganev, k.c., khaneghah, a.m., portella, j., cruz, a.g., granato, d., corassin, c.h., oliveira, c.a.f. and sant’ana, a.s., 2016. the occurrence and effect of unit operations for dairy products processing on the fate of aflatoxin m1: a review. food control 68: 310–329. https://doi.org/10.1016/j. foodcont.2016.04.007 cano-sancho, g., marin, s., ramos, a.j., peris-vicente, j. and sanchis, v., 2010. occurrence of aflatoxin m1 and exposure assessment in catalonia (spain). revista iberoamericana de micología 27(3): 130–135. https://doi.org/10.1016/j. riam.2010.05.003 cano-sancho, g., perelló, g., nadal, m. and domingo, j.l., 2015. comparison of the nutritional composition and the concentrations of various contaminants in branded and private label yogurts. journal of food composition and analysis 42: 71–77. https://doi.org/10.1016/j.jfca.2015.03.008 daou, r., afif, c., joubrane, k., khabbaz, l.r., maroun, r., ismail,  a. and el khoury, a., 2020. occurrence of aflatoxin m1 in raw, pasteurized, uht cows’ milk, and dairy products in lebanon. food control 111: 5–29. https://doi.org/10.1016/j. foodcont.2019.107055 high frequencies of positive samples at low levels of contamination among different industrial and traditional fermented milk products. a decreasing trend in the contamination of fermented milk products was observed over the years, mainly in traditional products. however, afm1 contamination in fermented milk at levels higher than the recommended tolerable limits was reported in african and asian countries. continuous monitoring and controlling actions from both manufacturers and regulatory bodies are essential to reduce the afm1 contamination levels in industrial and traditional fermented milk. further studies to improve fermentation performance to reduce the afm1 contents in contaminated milk and other similar products are 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https://doi.org/10.3168/jds.2012-6184� _goback _enref_183 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (2): 89–97 issn 1120-1770 online, doi 10.15586/ijfs.v34i2.2181 89 fatty acid profile of functional emulsion-based food products containing conventional and unconventional ingredients almas mukhametov1*, laura mamayeva1, moldir yerbulekova1, gulsim aitkhozhayeva2 1department of technology and safety of food products, kazakh national agrarian research university, 8 abai avenue, almaty, kazakhstan; 2department of land resources and cadastre, kazakh national agrarian research university, almaty, kazakhstan *corresponding author: almas mukhametov, department of technology and safety of food products, kazakh national agrarian research university, 8 abai avenue, almaty a05d5f7, kazakhstan. email: almmukhametov@rambler.ru received: 9 february 2022; accepted: 26 may 2022; published: 16 june 2022 © 2022 codon publications open access paper abstract the creation of new emulsion-based food products has great potential for the food industry at the present stage of its development. the purpose of the paper is to explore the physical and chemical characteristics (content of fatty acids and tocopherols) of nine mixtures (blends) of conventional and unconventional vegetable oils with regard to the changes in the peroxide values of the oil blends stored under different temperatures for different periods. the study was conducted in 2020 in almaty (kazakhstan). nine vegetable oil blends were prepared by mixing conventional and unconventional ingredients. each of the resulting blends was tested in 30 replicates for the content of fatty acids (oleic, linoleic, and linolenic acids) and tocopherols. the blends were stored at 10°c and 20°c. samples were taken to determine peroxide values. the results were compared to the control (refined sunflower oil). in all nine blends, the optimal ratio of the evaluated fatty acids and the optimal concentration of tocopherols were confirmed. after 6 months of storage, the peroxide values of blend no. 1 stored at 10°c and 20°c were 3 and 6, respectively. blend no. 2 stored for the same period at the same temperatures demonstrated the respective peroxide values of 2.5 and 4.5. for blend no. 3, the respective values obtained were 2.5 and 5.5, and for blend no. 4, the respective values were 3.0 and 6.5. the most drastic changes were observed in blend no. 5, with respective peroxide values of 2.5 and 7.2. the respective peroxide values of blend no. 6 were 3.7 and 5.5, blend no. 7, 3.5 and 7.0, blend no. 8, 4.0 and 6.5, and blend no. 9, .5 and 5.5. all in all, the peroxide values of the nine tested blends were significantly lower than those of the control (p ≤ 0.05–0.01). the proposed nine blends can be used as food additives exhibiting biological activities. after 6 months of storage, the minimal changes in the peroxide values were observed in blend nos. 2 and 3, while the maximum changes were reported for blend nos. 5, 7, and 8. in the future, an investigation of the therapeutic effects of the obtained blends should be undertaken, with a focus on possible adverse heating-induced changes in some components (flaxseed oil). keywords: blends; food additives; peroxide value; storage temperature conditions introduction functional foods make up a significant part of modern diet. these data are supported by the global trend toward consumption of functional foods. this trend is predominantly observed in the developed countries. for example, in the united states, the increase in the consumption of special-purpose fat products exceeds the overall food production growth. the use of functional food products is primarily promoted by the studies concerning the structure of modern diet. people want to benefit from the declared properties of functional foods, which include 90 italian journal of food science, 2022; 34 (2) mukhametov a et al. its derivatives inhibit  platelet aggregation, reduce the levels of cholesterol, blood pressure, and the amount of low-density lipoproteins that are harmful to the body. interestingly, the smallest quantities of linolenic acid are found in the body of infants and senior people. thus, fatty acids and phospholipids are one of the most important compounds required by the human body for forming cell membranes and maintaining the normal functioning of the immune system. in this regard, it is very important to look for new, unconventional ingredients that may be incorporated into emulsion-based food products (safonov, 2022). unfortunately, there are very few papers covering the topic, and this fact determines the relevance of this study. most of the available papers have considered generally the effect produced by a single unconventional ingredient. this paper describes nine different blends containing unconventional ingredients. the authors assumed that the physical and chemical properties of the experimental blends would be at least non-inferior to those of conventional emulsion-based food products (with shelf life being equivalent or longer). the purpose of the study was to explore the physical and chemical properties of new blends of refined vegetable oils that could be used in the production of functional emulsion-based food products, subject to the shelf life of obtained blends. the objectives of the study include the following: (a) to prepare the blends of refined vegetable oils for producing functional emulsion-based food products; (b) to explore their physical and chemical properties (fatty acid profile and tocopherols); and (c) to determine the shelf life of the obtained blends. materials and methods methods the study was conducted in 2020 in almaty (kazakhstan). the formulations presented in this paper used both vegetable oils obtained from conventional sources (sunflower, soybeans, and corn; refined oils were used) and unconventional vegetable oils. unconventional oils were extracted by oil seed pressing and purified by separation of plant residues. refined vegetable oils derived from conventional sources were mixed with different amounts of unconventional oils. thus, the mixtures were obtained that could be suitable for the preparation of oil-in-water emulsions. the blends intended for fat emulsions were prepared using the ingredients shown in table 1. the content of fatty acids in the blends and oils under investigation was determined according to the gosudarstvennyy standart (gost) 30418-96 (vegetable stabilization of metabolic processes in the body and maintaining the normal functions of the immune, nervous, and endocrine systems (truzzi et al., 2021). among modern functional fatty products, a focus of the food industry is on the development of mayonnaise, various fat pastes, and creams (khudzaifi et al., 2019). inevitably, manufacturers introduce into their products some additives that are typical for certain regions of the planet, taking into  account the preferences of the local population. plant and animal products are often combined. a variety of sources are available due to various flavors rather than spices, as the latter are added only in small quantities. fat and water-soluble vitamins and dietary fibers contribute to the taste and texture of the finished fatty products (marchetti et al., 2019). the mixtures obtained in this experiment can be used as an alternative to sunflower or olive oil, which are widely used globally. one important and urgent task to be undertaken in the nearest future is to reduce the calorie content of functional food products that are being developed. of note, the average dietary intake of fat is 35% of total calories. therefore, the fat content of food is often reduced so that it falls within the range of 5–25%. this determines the relevance of developing new formulations of functional food products containing fat emulsions. the ultimate goal of developing functional emulsion-based food products is to make them appropriate for consumption by people of different age groups. in order to work properly, the human body needs fatty acids (in particular, linoleic, arachidonic, and linolenic acids). in most cases, this need is met in the diet by including various vegetable oils made from conventional forage crops (sunflower, corn, soybeans, and olive; cerceau et al., 2020). however, in addition to the conventional vegetable oils, oils are also extracted from unconventional crops, which have not been widely used in the food industry. this refers to the oils obtained from the processing of cereals, fruits, seeds, and nuts. wheat germ oil and oils extracted from other cereals are of special importance. these unconventional oils are rich in linolenic, hexaenoic, pentaenoic, and other fatty acids, which are vital for the human body (vargas jentzsch et al., 2018). the optimal ratio of ω-6 and ω-3 fatty acids that constitutes the oil is 10:1. the role of fatty acids (e.g., linoleic and linolenic acids) is crucial, since they form important components of cell membranes, participate in hormone synthesis, regulate cellular metabolic processes, maintain normal blood pressure, remove excess cholesterol from the body, and improve the elasticity of blood vessels. since these acids are not produced in the human body (bulat and volkov, 2016), they are considered essential (juliani et al., 2006). as for arachidonic acid, its synthesis involves linoleic acid and vitamin b6. an insufficient intake of these acids from food sources triggers cardiovascular diseases and activates pathological processes in cell membranes. linoleic acid and italian journal of food science, 2022; 34 (2) 91 fatty acid profile of functional emulsion-based food products content of fatty acids and other components). for each comparison, the sample size of 30 was used. differences were identified using student’s t-test. the level of significance was set at p ≤ 0.05. results the studied blends were found to contain different amounts of fatty acids. in blend no. 1, soybean and flaxseed oils were 0.5 times inferior to sunflower oil in terms of oleic acid content (p ≤ 0.05), and flaxseed oil was twice inferior to soybean and sunflower oils (p ≤ 0.01), the content of linolenic acid in flaxseed oil was seven times higher than in soybean oil (p ≤ 0.001) and more than 100 times higher than in sunflower oil (p ≤ 0.0001). soybean oil had a slightly higher content of tocopherols as compared to the other two oils (table 2). in blend no. 2, the content of oleic acid in sunflower oil was 0.5 times higher than in pumpkin seed oil (p ≤ 0.05), with no significant differences in the content of linoleic acid (p ≥ 0.05). pumpkin seed oil had 16 times higher content of linolenic acid (p ≤ 0.001). there were more tocopherols in sunflower oil (p ≤ 0.05). in blend no. 3, sea buckthorn oil had the highest content of oleic acid. the levels of oleic acid determined in sea buckthorn oil were 1.5–2.0 times higher than in sunflower and corn oils (p ≤ 0.05). corn and sunflower oils had the highest content of linoleic acid as compared to the other oils used in the blend (p ≤ 0.05). corn oil had the highest content of linolenic acid (p ≤ 0.01). the content of tocopherols in sunflower oil was twice lower than in sea buckthorn and corn oils (р ≤ 0.05). in blend no. 4, oleic acid was found in abundance in sea buckthorn oil, with twice less of it found in sunflower oil (р ≤ 0.05), and four times less of it found in wheat germ and barley oils (р ≤ 0.01). in terms of the content of linoleic acid, wheat germ and barley oils were not inferior oils. method for determination of fatty acid content). for this, the capillary column gas chromatography method was applied. the researchers used polar columns zebron zb-50 with the column length of 30 m and internal diameter of 0.32 mm. the peaks were identified by comparing the chromatograms obtained for the analyzed blends and a mixture of fatty acid methyl esters as standards. graphs of the peaks observed were plotted separately for saturated and unsaturated fatty acids. to build all straight lines, the investigators proceeded from the fact that 2–3 points were required for saturated fatty acids and only 1–2 points for unsaturated ones. the volume of each sample was 1 mm3 of hexane solution containing fatty acid methyl esters. study design the study consisted of two parts. in the first part, the researchers assessed the fatty acid profiles of vegetable oils (i.e., measured the levels of oleic, linoleic, and linolenic acids) and quantified the content of tocopherol. the second part of the study highlighted the changes in the stability of the tested oil blends observed at different time points of the storage period (time interval from the beginning of the experiment to 6 months of the experiment). the peroxide value was chosen as the tested parameter. the storage temperatures were 10°c and 20°c. these temperature regimes were convenient for comparing the stability of the experimental blends at different time points of the storage period. the study followed international standards applicable to scientific research. it intended to develop effective blends of vegetable oils using conventional and nonconventional ingredients for their subsequent testing and use asemulsion-based food products. statistical analysis in order to analyze statistical differences, the statistica program (version 10) was used. arithmetic mean values were calculated for each of the tested parameters (the table 1. composition of the vegetable oil blends under investigation. blend no. oils used for blend recipe blend ratio of oils amount of oils as related to the total volume of fat emulsion 1 sunflower, soybean, and flaxseed oils 20:15:10 45 2 sunflower and pumpkin seeds oils 35:15 50 3 sunflower, corn, and sea buckthorn oils 30:15:10 55 4 sunflower, wheat germ, barley, and sea buckthorn oils 5:10:5:10 30 5 sunflower, millet seed, walnut, and apricot kernel oils 10:15:5:5 35 6 sunflower, rapeseed, tomato seed, oat, and plum kernel oils 8:8:8:8:8 40 7 sunflower, sesame, lupine, rye, and flaxseed oil 10:5:5:5:5 45 8 sunflower, mustard, cherry, buckwheat, and rice oils 10:10:10:10:10 50 9 olive, wheat germ, barley germ, rosehip, and chestnut oils 15:15:15:5:5 55 92 italian journal of food science, 2022; 34 (2) mukhametov a et al. walnut oil exceeded by 2.5 times that determined in apricot oil (р ≤ 0.05) and by 1.5 times than that determined in millet seed and sunflower oils (р ≤ 0.05). maximum content of linolenic acid was observed in walnut oil, followed by millet seed oil (р ≤ 0.05), but 7–20 times less linolenic acid was found in the rest of oils (р ≤ 0.001). all oils exhibited lower content of tocopherols as compared to sunflower oil (p ≤ 0.05). in blend no. 6, oleic acid accounted for 72% of the fatty acid composition of plum oil. there was 1.5–2.0 times less oleic acid in rest of the oils added to the blend to sunflower oil (p ≥ 0.05), with the levels of linoleic acid in wheat germ and barley oils being twice higher than those in sea buckthorn oil (p ≤ 0.05). wheat and barley oils contained 8–10 times more linolenic acid than found in sunflower oil (p ≤ 0.01) and 40–50 times more linolenic acid than in sea buckthorn oil (p ≤ 0.0001). sea buckthorn oil contained twice more tocopherols than found in the rest of oils making up the blend (p ≤ 0.05). in blend no. 5, apricot oil contained twice more oleic acid than in the other oils (р ≤ 0.05). the content of linoleic acid in walnut oil was 83%. the levels of linoleic acid in table 2. content of fatty acids and tocopherols in oils used for the blends under investigation. blend no. oils used for the blend recipe oleic acid (%) linoleic acid (%) linolenic acid (%) tocopherols (mg) 1 soybean oil 25 52 8 160 flaxseed oil 28 20 57 113 sunflower oil 36 56 0.5 116 2 sunflower oil 36 56 0.5 116 pumpkin seed oil 26 55 8 86 3 sea buckthorn oil 63 38 0.1 250 sunflower oil 36 56 0.5 116 corn oil 48 56 0.8 247 4 sea buckthorn oil 63 38 0.1 250 sunflower oil 36 56 0.5 116 wheat germ oil 14 59 4.5 140 barley oil 16 56 5.6 100 5 sunflower oil 36 55 0.5 116 millet seed oil 27 67 10 96 walnut oil 35 83 15 52 apricot kernel oil 79 32 1.5 78 6 sunflower oil 36 55 0.5 116 rapeseed oil 44 42 11 55 tomato oil 30 62 2.5 127 oat oil 41 43 2 75 plum kernel oil 72 25 0.5 130 7 sunflower oil 36 55 0.5 116 sesame oil 48 55 3 144 lupine oil 55 23 8 2 rye oil 17 68 12 91 flaxseed oil 28 20 57 113 8 sunflower oil 36 55 0.5 116 mustard oil 31 24 18 110 cherry oil 50 42 10 10 buckwheat oil 40 39 4 50 rice oil 43 53 4 110 9 olive oil 80 22 2.5 90 wheat germ oil 13 65 5.5 480 chestnut oil 72 30 2 78 rosehip oil 55 30 2 260 barley oil 16 58 7 98 italian journal of food science, 2022; 34 (2) 93 fatty acid profile of functional emulsion-based food products oil (р ≤ 0.05), and four times less tocopherols were quantified in the rest of oils making up the blend (р ≤ 0.01). the qualitative parameters of oil mixtures are shown in figures 1–5. in each of these figures, the curves corresponding to numbers 3 and 4 reflect the changes observed in the control sample (refined sunflower oil) stored at 20°c and 10°c. the analysis demonstrated that the peroxide values of all nine oil blends prepared by the researches increased insignificantly even after 3 months of storage. following 6 months of storage, the maximum peroxide value obtained was 7.2 mmol of active oxygen (½ o) per kg of oil blend. this variable was identified for blend no. 5. the results of statistical analysis of changes in peroxide values are presented in table 3. after 6 months of storage, the peroxide values of blend no. 1 stored at 10°c and 20°c were 3 and 6, respectively (figure 1a). blend no. 2 stored for the same period under the same temperature conditions demonstrated the peroxide values of 2.5 and 4.5, respectively (figure  1b). for blend no. 3, the respective values obtained were 2.5 and 5.5 (figure 2a), for blend no. 4, the respective values were 3.0 and 6.5 (figure 2b). the changes were more drastic in blend no. 5, with respective peroxide values of 2.5 and 7.2 (figure 3a). the respective peroxide values (р ≤ 0.05). linoleic acid was found in abundance in tomato oil, thrice less abundant in plum oil (p ≤ 0.01), and 1.5 times less abundant in the rest of the analyzed oils. the maximum content of linolenic acid was reported in rapeseed oil (11%), with its content being 5–20 times lower in the other oils making up the blend (p ≤ 0.01). the content of tocopherols in plum and tomato oils was slightly higher than in sunflower oil (p ≤ 0.05). oat and rapeseed oils contained twice less tocopherols than found in plum and tomato oils (p ≤ 0.05). in blend no. 7, the maximum amount of oleic acid was observed in lupine oil, 0.5–1.5 times less oleic acid was found in sesame, sunflower, and flaxseed oils (р ≤ 0.05), and the minimum amount of oleic acid was observed in rye oil (р ≤ 0.01). the content of linoleic acid was maximum in rye, sesame, and sunflower oils, with twice less of it detected in lupine and flaxseed oils (р ≤ 0.05). flaxseed oil was richest in linolenic acid. the content of linolenic acid was 5 times lower in rye oil (p ≤ 0.01), 7 times lower in lupine oil (≤ 0.01), and 20 times lower in sesame oil (p ≤ 0.001). the minimum content of linolenic acid was reported for sunflower oil (p ≤ 0.0001) in comparison to its content in flaxseed oil. tocopherols were found in abundance in sesame oil, with the minimum levels observed in lupine oil (p ≤ 0.00001). in blend no. 8, the highest content of oleic acid was the characteristic for cherry oil, while the other oils contained 0.3–0.5 times less oleic acid (р ≤ 0.05). linoleic acid was in abundance in sunflower and rice oils. the other oils making up the blend were 0.5 times (buckwheat and cherry oils) and twice (mustard oil) inferior to sunflower and rice oils in terms of the content of linoleic acid (p ≤ 0.05). levels of linolenic acid were maximum in mustard oil, twice lower in cherry oil (р ≤ 0.05), and four times lower in buckwheat and rice oils (р ≤ 0.01). the minimum content of linolenic acid was observed in sunflower oil (p ≤ 0.001). the content of tocopherols was maximum in rice, sunflower, and mustard oils, twice lower in buckwheat oil (р ≤ 0.05), and 10 times lower in cherry oil (р ≤ 0.01). in blend no. 9, the highest content of oleic acid was reported for olive oil. slightly lower level of oleic acid was revealed in chestnut oil (р ≤ 0.05), 1.5 times less oleic acid was detected in rosehip oil (р ≤ 0.05), and seven times less in barley and wheat germ oils (p ≤ 0.01). the maximum amount of linoleic acid was found in wheat germ and barley oils and the minimum amount was found in chestnut, rosehip, and olive oils (р ≤ 0.05). maximum linolenic acid was found in wheat germ and barley oils, with the other oils being twice inferior to wheat germ and barley oils regarding content of linolenic acid (p ≤ 0.05). the maximum content of tocopherols was documented for wheat germ oil, twice less tocopherols were quantified in rosehip figure 1. peroxide values of (a) blend no. 1, and (b) blend no. 2. note: curves 3 and 4 reflect the values obtained for the control sample. curves 1 and 2 correspond to the experimental blends stored at 10°c and 20°c, respectively. p e ro xi d e v a lu e ( 1 /2 o m m o l/ kg ) p e ro xi d e v a lu e ( 1 /2 o m m o l/ kg ) storage period (months) 632 54 10 9 8 7 6 5 4 3 2 1 0 65432 storage period (months) 10 0 1 2 3 10 9 8 7 6 5 4 3 2 1 0 3 4 1 2 2 3 4 1 (a) (b) 94 italian journal of food science, 2022; 34 (2) mukhametov a et al. of blend no. 6 were 3.7 and 5.5 (figure 3b), for blend no. 7 3.5 and 7.0 (figure 4a), for blend no. 8 4.0 and 6.5 (figure 4b), and for blend no. 9 4.5 and 5.5 (figure 5). in all cases, the peroxide values of analyzed blends were significantly lower than the peroxide values of control (p ≤ 0.05). discussion the results presented in this article indicate that the proposed blends of different vegetable oils have a much longer shelf life as compared to the control sample of sunflower oil. this is confirmed by the fact that peroxide values of the prepared oil blends kept under different storage conditions increased by 0.5–2.0 times slower than the peroxide value of the control sample. a range of theoretical and practical studies established that blending of conventional and unconventional vegetable oils not only increases their shelf life but also provides the optimal balance of the three main fatty acids required for functioning of the body: oleic, linoleic, and linolenic acids (samburova et al., 2022). kinetic dependencies signaled that the proposed blends have a longer shelf life figure 2. peroxide values of (a) blend no. 3, and (b) blend no. 4. note: curves 3 and 4 reflect the values obtained for the control sample. curves 1 and 2 correspond to the experimental blends stored at 10°c and 20°c, respectively. figure 3. peroxide values of (a) blend no. 5, and (b) blend no. 6. note: curves 3 and 4 reflect the values obtained for the control sample. curves 1 and 2 correspond to the experimental blends stored at 10°c and 20°c, respectively. 0 storage period (months) p e ro xi d e v a lu e ( 1 /2 o m m o l/ kg ) p e ro xi d e v a lu e ( 1 /2 o m m o l/ kg ) storage period (months) 1 2 3 4 5 6 0 1 32 4 5 6 10 9 8 7 6 5 4 3 2 1 0 0 2 3 4 5 6 7 8 9 10 1 3 4 2 1 1 2 3 4 (a) (a) (b) (b) 10 9 8 7 6 5 4 3 2 1 0 10 9 8 7 6 5 4 3 2 1 0 6543210 p e ro xi d e v a lu e ( 1 /2 o m m o l/ kg ) p e ro xi d e v a lu e ( 1 /2 o m m o l/ kg ) storage period (months) storage period (months) 210 3 3 3 1 1 2 2 4 4 4 5 6 and are less susceptible to oxidative processes than the control sample. vegetable oils have been studied quite well. they are characterized by the basic ratio of fatty acids (papotti et  al., 2015). however, most nutritionists favor the use olive and flaxseed oils. corn, sunflower, and cottonseed oils are claimed to have benefits if used from time to time (marchetti et al., 2019; popescu et al., 2015). ingredients that are typically present in oil mixtures (blends) are sunflower, soy, and flax (vigli et al., 2003). all of these were used in the proposed nine blends. it must be remembered that some reports support the use of flaxseed oil blends in a cold form, since flaxseed oil gives off a rather specific smell when exposed to heat (ventsova and safonov, 2021). hence, the content of flaxseed oil in a blend should not exceed 5% (marchetti et al., 2019). the blends offered by different studies meet the above requirement and are recommended for consumption in a cold form. some authors have observed that flaxseed oil is instable (raveau et al., 2020). they claim that additional protective measures must be taken to compensate for the rancidity of flaxseed oil. nevertheless, stability of the oil blends used in this experiment has been demonstrated. italian journal of food science, 2022; 34 (2) 95 fatty acid profile of functional emulsion-based food products in addition, oils extracted from unconventional sources, such as pumpkin, watermelon, amaranth, and wheat, are being used more extensively. these types of oils have not only high nutritional value but also an apparent therapeutic effect (orsavova et al., 2015). moreover, there is a gradual increase in the production volumes of camelina and hemp oils, both rich in linolenic acid. the blends described in the article organically combine three essential fatty acids. quite often, blends are made figure 5. peroxide value of blend no. 9. note: curves 3 and 4 reflect the values obtained for the control sample. curves 1 and 2 correspond to the experimental blend stored at 10°c and 20°c, respectively. ta bl e 3. c ha ng es in p er ox id e va lu es c al cu la te d fo r th e ni ne b le nd s an d co nt ro l s am pl e st or ed fo r di ff er en t p er io ds u nd er d if fe re nt te m pe ra tu re s. b le nd no . a t t he be gi nn in g of th e ex pe ri m en t c on tr ol e xp er im en t s ig ni fic an ce of d if fe re nc es fo r 3m on th st or ag e c on tr ol e xp er im en t s ig ni fic an ce of di ff er en ce s fo r 6m on th st or ag e a ft er 3 m on th s of st or ag e at 1 0° c a ft er 3 m on th s of st or ag e at 2 0° c a ft er 3 m on th s of st or ag e at 1 0° c a ft er 3 m on th s of s to rag e at 20 °c a ft er 6 m on th s of st or ag e at 10 °c a ft er 6 m on th s of st or ag e at 2 0° c a ft er 6 m on th s of st or ag e at 1 0° c a ft er 6 m on th s of st or ag e at 2 0° c 1 1 3 4 1. 1 1. 6 0. 05 8 10 2 5. 8 0. 05 2 1. 5 3 4 1. 2 2 0. 05 8 10 2. 5 4. 5 0. 05 3 1 3 4 1. 1 2. 2 0. 05 8 10 2. 5 5. 8 0. 05 4 2 3 4 2. 2 2. 8 0. 05 8 10 3 6. 9 0. 05 5 2 3 4 2. 2 2. 7 0. 05 8 10 3 7 0. 05 6 3 3 4 3. 2 3. 5 0. 05 8 10 3. 7 5. 5 0. 05 7 2. 5 3 4 2. 7 2. 9 0. 05 8 10 3. 5 7 0. 05 8 3 3 4 3. 1 3. 7 0. 05 8 10 3. 5 6. 5 0. 05 9 3 3 4 3. 1 3. 2 0. 05 8 10 3. 9 5. 9 0. 05 figure 4. peroxide values of (a) blend no. 7, and (b) blend no. 8. note: curves 3 and 4 reflect the values obtained for the control sample. curves 1 and 2 correspond to the experimental blends stored at 10°c and 20°c, respectively. p e ro xi d e v a lu e ( 1 /2 o m m o l/ kg ) p e ro xi d e v a lu e ( 1 /2 o m m o l/ kg ) storage period (months) storage period (months) 10 9 8 7 6 5 4 3 2 1 0 10 9 8 7 6 5 4 3 2 1 0 3 4 2 1 3 4 2 1 0 1 2 3 4 5 60 6 5 4 321 (a) (b) 10 9 8 7 6 5 4 3 2 1 0 0 1 2 3 4 5 6 p e ro xi d e v a lu e ( 1 /2 o m m o l/ kg ) storage period (months) 3 4 2 1 96 italian journal of food science, 2022; 34 (2) mukhametov a et al. optimal storage conditions and shelf life were determined based on the kinetics of oxidative processes occurring in the developed blends of vegetable oils and emulsion-based food products. acknowledgments this article is written in the framework of the scientific work on “development of the technology of fat products with a balanced fatty acid composition” irn ap08053397. the authors express their gratitude to the anonymous reviewer for the recommendations and comments that improved the quality of the article. conflict of interest the authors declared no potential conflict of interest with respect to the research, authorship, and/or publication of this article. funding the authors received no financial support for the research, authorship, and/or publication of this article. data availability data will be available on request. references bulat p.v. and volkov k.n. 2016. detonation jet engine. part ii— construction features. int j environ sci educ. 11(12): 5020–5033. cerceau c.i., barbosa l.c., alvarenga e.s., maltha c.r. and ismail f.m. 2020. 1h-nmr and gc for detection of adulteration in commercial essential oils of cymbopogon ssp. phytochem anal. 31(1): 88–97. https://doi.org/10.1002/pca.2869 juliani h.r., kapteyn j., jones d., koroch a.r., wang m., charles d. and simon j.e. 2006. application of near-infrared spectroscopy in quality control and determination of adulteration of african 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considered in this paper. it should be mentioned that wheat germ oil is added to blends only in small amounts ranging from 1% to 5%, as it is quite expensive. an effective marketing approach is labeling of therapeutic effects of wheat germ oil used as a blend component. some blends contain pumpkin seed oil and other oils of unconventional origin (such as sea buckthorn, apricot, and hazelnut oils; vigli et al., 2003). most of the mentioned unconventional vegetable oils presented in this paper are components of the analyzed blends. limitations of this study are associated with the smallscale production of the proposed unconventional oils. this means that production of blends based on these types of oils cannot be started and boosted immediately. further research is required focusing mainly on the development of methods of production increase and investigation of therapeutic effects of unconventional vegetable oils. at this stage, the proposed blends can be used as biologically active food additives. conclusions following an analysis of the obtained graphical models of dependencies, the researchers revealed that all nine oil blends demonstrated a slight increase in peroxide value after being stored for 3 months. following 6 months of storage, the maximum peroxide value was confirmed for blend no. 5 (7.2 mmol of active oxygen/kg of oil blend). the obtained results established that the quality of experimental blends improved as compared to sunflower oil. these findings justify the use of these blends as a source of fats in the production of emulsion-based food products. owing to theoretical and experimental studies, scientific basis has been provided for obtaining the fatty phase of emulsion-based food products by using blends having optimal amounts of conventional and unconventional vegetable oils. this approach allows enriching the fatty acid composition of emulsion-based food products. appropriate criteria have been formulated to evaluate the effects of fatty acid 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ciobotă v. 2018. adulteration of clove essential oil: detection using a handheld raman spectrometer. flavour fragr j. 33(2): 184–190. https:// doi.org/10.1002/ffj.3438 ventsova i. and safonov v. 2021. biochemical screening of lipid peroxidation and antioxidant protection in imported cows during adaptation. adv anim vet sci. 9(8): 1203–1210. http://doi.org/ 10.17582/journal.aavs/2021/9.8.1203.1210 vigli g., philippidis a., spyros a. and dais p. 2003. classification of edible oils by employing 31p and 1h nmr spectroscopy in combination with multivariate statistical analysis. a proposal for the detection of seed oil adulteration in virgin olive oils. j agric food chem. 51(19): 5715–5722. https://doi.org/10.1021/jf030100z. contribution to dietary energy intake and dependence of cardiovascular mortality on dietary intake of fatty acids. int j mol sci. 16(6): 12871–12890. https://doi.org/10.3390/ijms160612871. papotti g., bertelli d., graziosi r., maietti a., tedeschi p., marchetti  a. and plessi m. 2015. traditional balsamic vinegar and balsamic vinegar of modena analyzed by nuclear magnetic resonance spectroscopy coupled with multivariate data analysis. lwt—food sci tech. 60(2): 1017–1024. https://doi. org/10.1016/j.lwt.2014.10.042 popescu r., costinel d., dinca o.r., marinescu a., stefanescu i. and ionete r.e. 2015. discrimination of vegetable oils using nmr spectroscopy and chemometrics. food control. 48: 84–90. https://doi.org/10.1016/j.foodcont.2014.04.046 raveau r., fontaine j. and lounès-hadj sahraoui a. 2020. essential oils as potential alternative bio-control products against plant pathogens and weeds: a review. foods. 9(3): 365. https://doi. org/10.3390/foods9030365 rueda a., seiquer i., olalla m., giménez r., lara l. and cabreravique c. 2014. characterization of fatty acid profile of argan oil and other edible vegetable oils by gas chromatography and discriminant analysis. j chem. 2014: 843908. https://doi. org/10.1155/2014/843908 safonov v. 2022. comparison of lpo-aos indices and biochemical composition of animal blood in biogeochemical provinces with 108 issn 1120-1770 online, doi 10.15586/ijfs.v33i2.1976 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (2): 108–115 generally preserve their biological activity after exposure to heat (jablonski and bohach, 1997; jørgensen et al., 2005). the presence of s. aureus in raw milk before processing is a concern because different physical and chemical production techniques are applied during processing and ripening of milk products to prevent growth of this pathogen and production of enterotoxins. nevertheless, if one of these limiting factors fails, there is a risk of accumulation of staphylococcal enterotoxins (jørgensen et al., 2005). thus, it is important to control growth of s. aureus in raw milk and raw milk products. in order to ensure milk safety and prolong milk’s shelf life, while also improving its sensorial characteristics, the dairy industry is developing minimum processing techniques (cava et al., 2007). it has been suggested p u b l i c a t i o n s codon anti-staphylococcal effect of cinnamaldehyde in milk milijana babic1†, milica glisic1†, jasna djordjevic1, nemanja zdravkovic2, radoslava savic-radovanovic1, milan baltic1, marija boskovic cabrol†1* 1department of food hygiene and technology, faculty of veterinary medicine, university of belgrade, bulevar oslobodjenja, belgrade, serbia; 2department of bacteriology and parasitology, scientific institute of veterinary medicine of serbia, vojvodetoze, belgrade, serbia †these authors contributed equally to the work. *corresponding author: marija boskovic cabrol, research associate, department of food hygiene and technology, faculty of veterinary medicine, university of belgrade, bulevar oslobodjenja 18, 11000 belgrade, serbia. email: marijaboskovic116@gmail.com received: 28 october 2020; accepted: 13 may 2021; published: 12 june 2021 © 2021 codon publications open access paper abstract the survival of staphylococcus aureus in inoculated (105 colony forming units [cfu]/ml) 3.2% and 0.5% fat ultrahigh temperature-pasteurized milk samples containing 0%, 0.05%, or 0.1% cinnamaldehyde stored at 4°c or 10°c was evaluated within 15 days. s. aureus populations reached 7.92 (0.5% fat) and 7.95 (3.2% fat) log cfu/ml in control milk samples stored at 10°c, while in milk sample stored at 4°c, s. aureus counts remained almost unchanged. at the end of the study, the number of this pathogen decreased by 1.52–4.04 log  cfu/ml in milk treated with cinnamaldehyde. the greatest anti-staphylococcal effect was achieved in low-fat milk at 10°c and treated with 0.1% cinnamaldehyde. keywords: antibacterial activity, cinnamaldehyde, fat, milk safety, staphylococcus aureus introduction milk and milk products, being highly nutritious foods, are excellent media for the growth of many spoilage and pathogenic microorganisms (noël et al., 2016), including staphylococcus aureus. this pathogen commonly exists in dairy production plants (xing et al., 2016), and it is one of the most important causative infective agents of clinical and subclinical mastitis in dairy cattle (nam et al., 2011; basanisi et al., 2017). s. aureus presents an important public health burden since it is one of the major pathogens responsible for food intoxication (jans et al., 2017). in spite of the fact that pasteurization kills s. aureus, it has little effect on thermostable enterotoxins, which mailto:marijaboskovic116@gmail.com italian journal of food science, 2021; 33 (2) 109 anti-staphylococcal effect of cinnamaldehyde in milk were bought from a local supermarket. cinnamaldehyde (ca) (98% purity) was purchased from carl roth, germany and stored at 4°c prior to use. s. aureus was obtained from the american type culture collection (atcc 25923). determination of minimum inhibitory concentration the minimum inhibitory concentration (mic) of cinnamaldehyde was determined in a non-milk matrix using sterile u-bottom 96-well microplates. the bacterial inoculum density was set to 0.5 on the mcfarland scale, then further diluted 10 times in sterile saline; 5 μl of this suspension was inoculated into 0.1 ml of cation-adjusted mueller–hinton broth (camhb; becton, dickinson and company, sparks, usa) to reach a final s. aureus atcc 25923 inoculum of 5 × 104 colony forming units (cfu)/ well. cinnamaldehyde was diluted in dimethyl sulfoxide (serva, heidelberg, germany) and added to camhb in the levels of 2560–1.25 μg/ml by two-fold dilution in 96-well microtitre plates. after inoculation, plates were incubated for 24 h at 37°c. the mic was the lowest concentration of cinnamaldehyde that did not show any visual growth of s. aureus after macroscopic evaluation, and it was expressed in μg/ml (clinical and laboratory standards institute [clsi], 2006). the plates were prepared in triplicate. sample preparation and storage conditions milk containing 0.5% or 3.2% fat was analyzed for s. aureus to confirm the absence of this pathogen. approximately 5 log cfu/ml of s. aureus was inoculated into s. aureus-free milk containing 0.5% or 3.2% milk fat. the concentration of the inoculum was verified by the standard plate count method and determined as 5.55–5.60 log cfu/ml. to study the survival of s. aureus in milk, different concentrations of cinnamaldehyde (0.05% and 0.1%) were added to milk samples with 0.5% (reduced fat) and 3.2% (whole milk) milk fat, whereas controls were without cinnamaldehyde but were inoculated with s. aureus. the selection of these concentrations of cinnamaldehyde was based on previous sensory evaluations (babic et al., 2019). after addition of cinnamaldehyde, all milk samples were divided into halves and stored in sterile glass bottles at 4°c and 10°c for 15 days. this temperature of 10°c was selected as an abuse temperature. the milk samples are described in table 1. microbiological and ph analysis all milk samples were examined on storage days 0, 3, 6, 9, 12, and 15. for bacterial enumeration, 25 ml of milk that addition of plant extracts, including cinnamon, can enhance microbiological safety, and it positively affects the sensory attributes of processed dairy products and milk-based desserts such as rice pudding and vanilla cream pudding (tayel et al., 2015; lianou et al., 2018). when added to butter, cinnamon (3%) lowered microbial growth during storage and exhibited antioxidant activity, thus retarding the spoilage of butter by positively influencing its sensorial characteristics (vidanagamage et al., 2016). thus, cinnamon could be successfully incorporated in butter as a natural preservative instead of synthetic preservatives. cinnamon contains 85.3–90.5% cinnamaldehyde (doyle and stephens, 2019). together with eugenol, isoeugenol, vanillin, and safrole, cinnamaldehyde is one of the best studied phenylpropenes (nazzaro et al., 2013). transcinnamaldehyde exhibits a wide range of beneficial effects, including antibacterial, antifungal, antioxidant, anti-inflammatory, anti-diabetic, neuroprotective, and antitumor (masghati and ghoreishi, 2018; doyle and stephens, 2019), while cis-cinnamaldehyde, the geometrical isomer of trans-cinnamaldehyde, exhibits antifungal properties (doyle and stephens, 2019). essential oil (eo) of cinnamon has found application in food industry because of its various components, including cinnamaldehyde, a major ingredient of cinnamon bark oil (masghati and ghoreishi, 2018). most essential oils and their components, including trans-cinnamaldehyde, are generally recognized as safe (gras) and accepted by consumers (burt, 2004). owing to their antibacterial and antioxidant properties, essential oils can be used as potential natural preservatives in different foods, including flavored drinks (cava et al., 2007). flavored milk has increased in popularity in recent years; nevertheless, there are few data available in literature about the effect of adding essential oils directly to milk before cheese-making (licon et al., 2020). the focus of the present study was to determine whether trans-cinnamaldehyde could be a potential natural antibacterial agent in milk, hence ultra-high temperature (uht)-pasteurized milk was used as a matrix to eliminate any possible interactions with the microbiota normally present in raw milk. the aims of the study were to: (1) evaluate the anti-staphylococcal effect of different concentrations of cinnamaldehyde (0.05% and 0.1%) on s. aureus in milk; and (2) determine the influence of different fat contents (0.5% and 3.2% milk fat) and different storage temperatures on survival of s. aureus in milk. materials and methods trans-cinnamaldehyde and s. aureus culture, uhtpasteurized milk samples containing 0.5% and 3.2% fat 110 italian journal of food science, 2021; 33 (2) babic m et al. table 1. experimental design. medium cinnamaldehyde temperature milk samples milk containing 0.5% fat with s. aureus 0% 4°c 1. milk containing 0.5% fat with s. aureus and without cinnamaldehyde stored at 4°c. 0.05% 2. milk containing 0.5% fat with s. aureus and 0.05% cinnamaldehyde stored at 4°c. 0.1% 3. milk containing 0.5% fat with s. aureus and 0.1% cinnamaldehyde stored at 4°c. 0% 10°c 4. milk containing 0.5% fat with s. aureus and without cinnamaldehyde stored at 10°c. 0.05% 5. milk containing 0.5% fat with s. aureus and 0.05% cinnamaldehyde stored at 10°c. 0.1% 6. milk containing 0.5% fat with s. aureus and 0.1% cinnamaldehyde stored at 10°c. milk containing 3.2% fat with s. aureus 0% 4°c 7. milk containing 3.2% fat with s. aureus and without cinnamaldehyde stored at 4°c. 0.05% 8. milk containing 3.2% fat with s. aureus and 0.05% cinnamaldehyde stored at 4°c. 0.1% 9. milk containing 3.2% fat with s. aureus and 0.1% cinnamaldehyde stored at 4°c. 0% 10°c 10. milk containing 3.2% fat with s. aureus and without cinnamaldehyde stored at 10°c. 0.05% 11. milk containing 3.2% fat with s. aureus and 0.05% cinnamaldehyde stored at 10°c. 0.1% 12. milk containing 3.2% fat with s. aureus and 0.1% cinnamaldehyde stored at 10°c. was transferred into a sterile stomacher bag and 225 ml of buffered peptone water (bpw; merck, germany) was added. the contents of each bag were homogenized in a stomacher blender (stomacher 400 circulator, seward, uk) for 2 min. serial decimal dilutions were prepared and 0.1 ml of appropriately diluted suspension was plated on baird parker agar (oxoid cm 275, basingstoke, hampshire, uk) with egg yolk tellurite emulsion (oxoid cm 275, basingstoke, hampshire, uk) and incubated at 37°c for 24 h according to en iso 6888-1 (international organization for standardization [iso], 1999). the number of colonies was counted, and results were recorded as colony forming units per milliliter. the ph of milk samples was measured using a portable ph meter (testo 205; testo ag, lenzkirch, germany). the ph meter was calibrated with standard buffer solutions of ph 4.0 and 7.0 prior to use. statistical analysis six randomized milk samples from each group were analyzed on each examination day. number of microorganisms were transformed into logarithms (log) before statistical analysis. statistical analysis of the results was conducted using the spss 20.0 software (ibm, chicago, il, usa). the s. aureus counts were expressed as mean ± standard deviation. a three-way anova analysis was used to investigate factor effects (concentrations of cinnamaldehyde, temperature, and fat%) and interactions among them on log-transformed s. aureus counts. statistical differences between examined groups were determined by tukey’s post hoc multiple comparisons test. p < 0.05 was considered statistically significant. results and discussion anti-staphylococcal effect of cinnamaldehyde in milk during storage the mic of cinnamaldehyde against s. aureus was 160 μg/ml, showing that cinnamaldehyde was able to inhibit growth of this pathogen at low concentrations in the non-milk matrix used. alves et al. (2016) reported a cinnamaldehyde mic of 100 µg/ml against s. aureus, in agreement with the result of the present study. nevertheless, in spite of the good antibacterial effect in vitro, hydrophobic essential oil constituents are impaired by interactions with food matrix components, hence higher concentrations are needed to achieve the same antibacterial effect in food (hyldgaard et al., 2012). thus, in the present study, approximately 4and 9-fold higher concentrations (0.05% and 0.1%) of cinnamaldehyde than the obtained mic were added to milk samples. significant (p < 0.05) antibacterial activity against s. aureus was found in milk samples at the cinnamaldehyde concentrations used (0.05% and 0.1%) when compared with the controls without cinnamaldehyde (table 2). initial s. aureus counts ranged from 5.55 to 5.60 log cfu/ml. on day 0, s. aureus counts were significantly (p < 0.05) higher in controls than in milk samples with cinnamaldehyde at 4°c and at 10°c, indicating the immediate antibacterial effect of cinnamaldehyde. regardless of fat content, in control milk samples without cinnamaldehyde stored at 4°c, with the exception of a slight decrease observed on day 3, the s. aureus populations remained almost unchanged for 15 days compared with the initial populations in milk samples. nevertheless, at 10°c, s. aureus counts increased to approximately 7.92 italian journal of food science, 2021; 33 (2) 111 anti-staphylococcal effect of cinnamaldehyde in milk (3.2% milk fat with 0.05% cinnamaldehyde at 10°c), and 2.96  log  cfu/ml (3.2% milk fat with 0.1% cinnamaldehyde at 10°c). the anti-staphylococcal effect of cinnamaldehyde found in the present study was in agreement with previous reports. alves et al. (2016) reported that growth of s. aureus was inhibited by the combination of nisin and cinnamaldehyde in pasteurized 3% fat milk stored at 4°c for 6 days. the mechanism of cinnamaldehyde’s antibacterial action is known and well described. the antibacterial activity of cinnamaldehyde is attributed to a free hydroxyl group (nazzaro et al., 2013). cui et al. (2016) reported that after treating s. aureus with cinnamon essential oil, cell membrane injury and leakage of intracellular material were observed. loss of atp and dna were detected because of bacterial cell membrane damage. some reports indicate that cinnamaldehyde inhibits the membrane-bound atpase activity (usta et al., 2003; gill and holley, 2004). di pasqua et al. (2006) found that trans-cinnamaldehyde causes changes in the composition of fatty acid and large increase in the proportion of saturated fatty acids in membrane phospholipids. shen et al. (2015) evaluated the effect of cinnamaldehyde on inner membrane permeability of s. aureus by measuring β-galactosidase activity. the authors found that β-galactosidase activity increased with increase in cinnamaldehyde concentration, leading to the conclusion that effects on membranes are dosedependent. in our previous pilot study (babic log cfu/ml (0.5% milk fat) and 7.95 log cfu/ml (3.2% milk fat) by the end of storage (day 15) in milk samples without cinnamaldehyde. growth of s. aureus is possible at temperatures above 8°c at optimum ph values ranging between 6.0 and 7.0 (valero et al., 2009). in all milk groups studied, the ph was within the optimal range (figure 1) and enabled s. aureus to grow and survive at the utilized storage temperatures. in contrast, s. aureus counts decreased during 15 days’ storage in all milk samples with added cinnamaldehyde. the decrease was less pronounced during the first 3 days of storage, and during this time no significant differences (p > 0.05) in s. aureus numbers were recorded between milk samples stored at 4°c and those stored at 10°c. from day 6 until the end of storage period (day 15), significantly greater s. aureus decrease (p < 0.05) was recorded in milk samples with added cinnamaldehyde stored at 10°c than in comparable milk samples stored at 4°c. at the end of the study, in milk samples treated with cinnamaldehyde, s. aureus numbers had decreased by 1.61 log cfu/ml (0.5% milk fat with 0.05% cinnamaldehyde at 4°c), 2.45  log  cfu/ml (0.5% milk fat with 0.1% cinnamaldehyde at 4°c), 1.52  log  cfu/ ml (3.2% milk fat with 0.05% cinnamaldehyde at 4°c), 1.82  log  cfu/ml (3.2% milk fat with 0.1% cinnamaldehyde at 4°c), 3.1  log  cfu/ml (0.5% milk fat with 0.05% cinnamaldehyde at 10°c), 4.04  log  cfu/ml (0.5% milk fat with 0.1% cinnamaldehyde at 10°c), 2.34 log cfu/ml table 2. s. aureus counts (log cfu/ml) in milk with and without added cinnamaldehyde (ca), stored at 4°c and 10°c (mean ± sd), and the significance of interactions between cinnamaldehyde, storage temperature, and milk fat. ca concentration temperature fat days 0 3 6 9 12 15 0% 4°c 0.5% 5.56 ± 0.06a 5.43 ± 0.10ac 5.46 ± 0.06a 5.48 ± 0.04a 5.57 ± 0.11a 5.56 ± 0.05a 3.2% 5.60 ± 0.07a 5.45 ± 0.09a 5.49 ± 0.10a 5.50 ± 0.06a 5.64 ± 0.08a 5.63 ± 0.06a 10°c 0.5% 5.55 ± 0.08a 6.98 ± 0.10b 7.21 ± 0.05b 7.49 ± 0.07b 7.47 ± 0.07b 7.92 ± 0.07b 3.2% 5.57 ± 0.09a 7.00 ± 0.16b 7.23 ± 0.15b 7.43 ± 0.07b 7.45 ± 0.08b 7.95 ± 0.10b 0.05% 4°c 0.5% 5.40 ± 0.06b 5.32 ± 0.07adce 5.15 ± 0.07c 4.62 ± 0.09c 4.44 ± 0.05c 3.95 ± 0.09c 3.2% 5.32 ± 0.07bc 5.23 ± 0.04def 5.11 ± 0.08c 4.89 ± 0.07d 4.28 ± 0.06d 4.08 ± 0.07c 10°c 0.5% 5.21 ± 0.05cd 5.26 ± 0.05cdef 4.51 ± 0.05d 3.81 ± 0.05e 3.04 ± 0.07e 2.45 ± 0.07d 3.2% 5.34 ± 0.07bc 5.30 ± 0.10adef 4.87 ± 0.08eg 4.00 ± 0.13f 3.90 ± 0.08f 3.23 ± 0.07e 0.1% 4°c 0.5% 5.28 ± 0.07bc 5.20 ± 0.08ef 4.94 ± 0.06g 4.46 ± 0.08g 4.10 ± 0.09g 3.11 ± 0.07e 3.2% 5.26 ± 0.06bc 5.18 ± 0.05ef 5.04 ± 0.08cg 4.56 ± 0.06cg 4.26 ± 0.08d 3.78 ± 0.07f 10°c 0.5% 5.11 ± 0.08d 5.14 ± 0.06f 4.08 ± 0.07f 3.62 ± 0.08h 2.51 ± 0.06h 1.51 ± 0.04g 3.2% 5.27 ± 0.06bc 5.25 ± 0.07ef 4.48 ± 0.08d 3.18 ± 0.09i 3.11 ± 0.07e 2.61 ± 0.10h conc. ca × *temp. ns ** ** ** ** ** conc. ca × fat% ns ns ** ** ** ** conc. ca × temp. × fat% * ns ** ** ** ** a–idifferent superscript letters in the same column, p < 0.05. ns: not significant. *p < 0.05; **p < 0.001. 112 italian journal of food science, 2021; 33 (2) babic m et al. 6.59 6.56 6.55 6.54 6.53 6.56 6.55 6.54 6.53 6.56 6.55 6.52 6.59 6.55 6.58 6.57 6.56 6.54 6.52 6.55 6.54 6.43 0% ca (0.5% fat milk) 0 6.3 6.35 6.4 6.45ph 6.5 6.55 6.6 3 6 day 10ºc 9 12 15 0.1% ca (0.5% fat milk) 0.5% ca (3.2% fat milk) 0.5% ca (0.5% fat milk) 0% ca (3.2% fat milk) 0.1% ca (3.2% fat milk) 6.38 6.34 6.32 6.42 6.41 6.36 6.33 figure 1. ph of milk stored at 10°c. et al., 2019), we found that the antibacterial effect of cinnamaldehyde was dependent on its concentration in 1.5% fat milk inoculated with 103 cfu/ml s. aureus stored at 4°c for 12 days. the same observation was made in the present study, showing significantly (p < 0.05) higher inhibition with 0.1% than 0.05% cinnamaldehyde used, but only for milk samples stored at the same temperature. it is supposed that essential oils are more effective when added at higher concentrations because after interactions with food matrix components (e.g. proteins and fats), more of the essential oil remains to interact with the bacterial cells (hyldgaard et al., 2012; boskovic et al., 2017). one of the most important findings of the present study was the greater bacteriostatic effect of cinnamaldehyde at higher temperature. significantly greater s. aureus decrease (p < 0.05) was recorded in milk samples with cinnamaldehyde stored at 10°c than in milk samples with same concentration of cinnamaldehyde stored at 4°c. with the expected exception of day 0, the interactions of storage temperature and cinnamaldehyde concentration (p = 0.001; factorial anova) on s. aureus counts (p = 0.207; factorial anova) were statistically significant. at 4°c, 0.05% cinnamaldehyde decreased the number of s. aureus to 3.95 log cfu/ml in low-fat milk and to 4.08 log cfu/ml in whole milk, while at 10°c, this concentration of cinnamaldehyde decreased s. aureus counts to 2.45 log cfu/ml in low-fat milk and to 3.23 log cfu/ml in whole milk. when added at higher concentration (0.1%), cinnamaldehyde reduced the initial s. aureus population to 3.11 log cfu/ml in low-fat milk and to 3.78 log cfu/ml in whole milk in samples stored at 4°c, while a significantly lower number (p < 0.05) of s. aureus was recorded in milk samples treated with the same concentration of cinnamaldehyde and stored at 10°c (1.51  log cfu/ml in low-fat milk and 2.61 log cfu/ml in whole milk). one possible explanation for this temperature-dependent antibacterial effect of cinnamaldehyde is that bacteria are metabolically more active at higher temperatures. consequently, growth and death rates are higher at higher temperature (smith-palmer et al., 1998; yuste and fung, 2003; guler and seker, 2009). in addition, the lower growth rate of bacteria at lower temperatures can make them less susceptible to antimicrobials (martinsen et al., 1992). also, at lower temperatures, essential oils have lower diffusion rates, and this reduces the efficiency of their antibacterial activity (wojtys and jankowski, 2004; leja et al., 2019). even a small change in temperature causes significant changes in the efficiency of their action, which is why the doses of essential oils must be significantly higher at lower temperatures (leja et al., 2019). these effects are in agreement with the results of present study. in milk samples with the same amount of fat, the anti-staphylococcal effect of 0.05% cinnamaldehyde was significantly (p < 0.05) more pronounced at 10°c than the effect of 0.1% cinnamaldehyde at 4°c (table 2). in addition, smith-palmer et al. (1998) reported that the target site of cinnamon essential oil can change, and oil penetration to the interior of the cell can be reduced due to alterations in membranes at lower temperatures. higher antibacterial italian journal of food science, 2021; 33 (2) 113 anti-staphylococcal effect of cinnamaldehyde in milk however, when milk without cinnamaldehyde was stored at 10°c, decline in ph was observed regardless of the fat content. in fact, on day 3, the ph of milk without cinnamaldehyde stored at 10°c (figure 1) decreased slightly compared with the initial ph values, but from day 6, significant (p < 0.05) ph declines were measured. the ph of this milk kept on decreasing throughout the 15 days’ storage, reaching ph values of 5.32 (0.5% milk fat) and 5.33 (3.2% milk fat). growth of s. aureus in these milk samples (table 2) matched decline in ph. under aerobic conditions, s. aureus can ferment milk sugar and lactose, creating acids responsible for the storage-induced decline in milk ph (medveďová and valík, 2012). the ph of milk samples with cinnamaldehyde added and stored at 10 °c did not significantly differ between each other. conclusion these results indicate that it could be possible to use cinnamaldehyde as a natural anti-staphylococcal agent in milk beverages. s. aureus numbers in milk were affected by cinnamaldehyde in a dose-dependent manner. cinnamaldehyde showed a greater antibacterial effect against s. aureus in low-fat milk than in whole milk. temperature had a strong effect on the anti-staphylococcal effect of cinnamaldehyde; hence, the lower concentration of cinnamaldehyde in milk stored at 10°c tended to have a better anti-staphylococcal effect than the higher concentration of cinnamaldehyde in milk stored at 4°c. nevertheless, even if the results of our study are promising, and if flavored milk is becoming increasingly popular, further investigations are required to determine the antibacterial effectiveness of cinnamaldehyde in raw milk and dairy products and to conduct sensory analysis of final products. acknowledgments the study was supported by the ministry of education, science and technological development of the republic of serbia (contract numbers 451-03-68/2020-14/200143 and 451-03-9/2021-14/200143). conflicts of interest the authors have no conflicts of interest for this article. references alves f.c., barbosa l.n., andrade b.f., albano m., furtado f.b., pereira a.f.m., rall w.l.m. and júnior a.f. 2016. inhibitory activities of the lantibioticnisin combined with phenolic compounds against staphylococcus aureus and listeria activities of essential oils at higher temperatures have been reported previously. guler and seker (2009) reported that the effect of cinnamon on bacilluscereus  reductions in uht-pasteurized milk during 28 days was significantly lower when milk samples were stored at 4°c than at 25°c. yuste and fung (2003) found that addition of 0.3% cinnamon in apple juice was more effective against s. aureus during storage at higher temperatures. the initial contamination was lower (4.34–4.37 log cfu/ml) than in the present study, and counts decreased below detection limits in apple juice stored at 20°c after only 1 day, but it took 7 days of storage at 5°c to obtain the same results. in the present study, the greatest anti-staphylococcal effect was achieved in low-fat milk stored at 10°c and treated with 0.1% cinnamaldehyde, as s. aureus numbers were reduced by more than 4 log cfu/ml. moreover, the effect of interaction between cinnamaldehyde concentration and fat content in milk on s. aureus numbers was significant (p < 0.0001) from day 6 until the end of storage, while for the first 3 days no interaction was observed (day 0, p = 0.423; day 3, p = 0.370; factorial anova). at the end of storage, significant differences (p < 0.05) between the s. aureus counts in whole milk (3.2%) and low-fat milk (0.5%) were found for the same concentrations of cinnamaldehyde. however, no significant differences (p > 0.05) in s. aureus counts were found between milk samples stored at the same temperatures without cinnamaldehyde, regardless of content of milk fat. thus, cinnamaldehyde was more effective in inhibiting the pathogen in low-fat milk than in high-fat milk, which is also consistent with literature. cava-roda et al. 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vol. 27 2015 407 opinion paper italy on the spotlight: expo milan 2015 and italian journal of food science p. fantozzia*, m.f. cabonib, t. gallina toschib, v. gerbic, a. hidalgod, v. lavellid, g. perrettia, p. pittiae, c. pompeid, k. rantsiouc, l. rollec, m. sinigagliaf and b. zanonig a*department of agricultural, food and environmental sciences (dsaaa), university of perugia, via s. costanzo n.c.n., 06126 perugia, italy bdepartment of agricultural and food sciences, alma mater studiorum, university of bologna, p.zza goidanich 60, 47521 cesena (fc), italy cdepartment of agricultural, forestry and food sciences, university of torino, largo paolo braccini 2, 10095 grugliasco (to), italy ddepartment of food, environmental and nutritional sciences (defens), university of milan, via celoria 2, 20133 milan, italy efaculty of bioscience and technology for food, agriculture and environment, university of teramo, mosciano sant’angelo (te), italy fdepartment of the sciences of agriculture, food and environment, university of foggia, via napoli 25, 71122 foggia, italy gdepartment of agricultural, food and forestry systems management (gesaaf), university of florence, via donizetti 6, 50144 florence, italy *corresponding author: paolo.fantozzi@unipg.it the year 2015 will certainly be remembered as the year of the universal exposition (expo) hosted in milan, italy, focusing on a hot theme in the current scenario: “feeding the planet, energy for life”. this event has drawn a wide international attention towards italy as a country with peculiar and valuable food traditions, thus strengthening its reputation as “gastronomic capital of the world” rich in protected designation of origin products (pdos) and characterised by a longstanding food culture. expo 2015 has been a global platform to showcase a vast range of food traditions, as well as a unique opportunity to appreciate different cultures through food practices, thus reflecting on the values that bring people together. expo 2015 was a chance for scientists, regulators, industry representatives and buyers, to meet and share cutting-edge knowledge and opinions, and to start fruitful collaborations and networks. meetings and conferences scheduled within the expo 2015 calendar encompassed various themes, with particular focus on sustainability keywords: expo 2015, feeding the planet, food safety, food security, nutrition, shelf life, biodiversity mailto:paolo.fantozzi%40unipg.it?subject= 408 ital. j. food sci., vol. 27 2015 of the agri-food system, conservation of biodiversity, protection of environment and its natural resources, such as water, land and soil. emerging agri-food technologies were also recognised as essential for understanding natural phenomena, monitoring production factors, tracing food products, managing emergency and crisis, ultimately leading to improvement of well-being and prosperity worldwide. scientists involved in the fields of agriculture, environment and food processing are challenged by these emerging themes with the task to advance their studies in order to have an impact on education, training and future policies of the agri-food sector. the italian journal of food science has been deeply involved, in the last two years, in the diffusion of new ideas and exchange of knowledge on the topics of expo 2015. a primary expo 2015 subject was product sustainability, i.e. new and alternative methods of agricultural production aimed to reduce the environmental impact of food production. accordingly, lo giudice et al. (2013) proposed different approaches to lessen the production impact of sicilian blood oranges on the environment through the application of the life cycle assessment (lca) methodology, while giordano et al. (2013) studied the influence of cropping conditions on volatile fingerprint and mechanical properties of muscat blanc (vitis vinifera l.) grapes grown in the north-west mountain region of italy. another relevant expo 2015 topic was nutrition and biodiversity, in the awareness that lesser-known species rich in bioactive compounds can play an important role in human health. thus, the research presented by jurikova et al. (2014) tackled at the same time sustainability, nutrition and biodiversity, assaying the antioxidant properties of selected cultivars of interspecific crosses of rowan (sorbus aucuparia l.), a thrifty species with high frost resistance. on the other hand, tripaldi et al. (2014) combined tradition and new consumer requirements by evaluating traditional and reduced dry salting methods of an italian valuable pdo product, pecorino romano cheese. the development of new technologies in food science was fostered by mura et al. (2014), which focused on the advances of nanotechnology in food and animal science, as this emerging multidisciplinary field can potentially improve the nutritional value, quality and shelf life of several food products, leading to crucial advantages in terms of consumer safety. the concept of food safety was approached by yasar and boselli (2015) in a survey on perception and awareness of european union food safety policy by different socio-demographic groups of consumers. ijfs strives to promote the advancement of food science: therefore, we would like to acknowledge all the authors for their contribution and the reviewers for their valuable advice. references giordano m., zecca o., belviso s., reinotti m., gerbi v. and rolle l. 2013. volatile fingerprint and physico-mechanical properties of ‘muscat blanc’ grapes grown in mountain area: a first evidence of the influence of water regimes. ital. j. food sci. 25:329. jurikova t., sochor j., mlcek j., balla s., klejdus b., baron m., ercisli s. and ozturk yilmaz s. 2014. polyphenolic profile of interspecific crosses of rowan (sorbus aucuparia l.). ital. j. food sci. 26:317. lo giudice a., mbohwa c., clasadonte m.t. and ingrao c. 2013. environmental assessment of the citrus fruit production in sicily using lca. ital. j. food sci. 25:202. mura s., carta d., roggero p.p., cheli f. and greppi g.f. 2014. nanotechnology and its applications in food and animal science ital. j. food sci. 26:91. tripaldi c., palocci g., fiori m., longo l., f fuselli., catillo g. and addis m. 2014. effect of reduced dry salting on the characteristics of pdo pecorino romano cheese. ital. j. food sci. 26:134. yasar s. and boselli e. 2015. perception and awareness of the european union food safety framework. ital. j. food sci. 27:126. paper 298 ital. j. food sci., vol. 27 2015 keywords: chicken burger, inulin, quality, vegetable oil, wheat fiber quality characteristics of chicken burgers enriched with vegetable oils, inulin and wheat fiber a. cegiełkaa*, m. chmiela, e. krajewska-kamińskab and e. hać-szymańczukc awarsaw university of life sciences, sggw, faculty of food sciences, department of food technology, division of meat technology, 159c nowoursynowska street, 02-787 warsaw, poland bwarsaw university of life sciences, sggw, analytical centre, 8 ciszewskiego street, 02-786 warsaw, poland cwarsaw university of life sciences, sggw, faculty of food sciences, department of biotechnology, microbiology and food evaluation, division of biotechnology and microbiology, 159c nowoursynowska street, 02-787 warsaw, poland *corresponding author: email: aneta_cegielka@sggw.pl abstract the aim of the study was to modify the composition of chicken burgers in terms of nutritional value by substitution of 20% of pork jowl with a mixture of rapeseed oil and linseed oil, and addition of inulin (1%) or wheat fiber (3%). substitution of pork jowl with vegetable oils resulted in significant increase in polyunsaturated fatty acids, and rosemary extract retarded the oxidation process of lipids. addition of wheat fiber was helpful in maintaining the thermal processing yield and texture of burgers. microbiological quality of vacuum packed burgers subjected to 21-day storage at +4°c±1 and -20°c±1 was satisfactory. ital. j. food sci., vol. 27 2015 299 introduction despite the constant dissemination of knowledge in the field of proper nutrition, consumers do not always consider it when choosing foods. results of research over a composition of a daily diet of the average polish consumer indicated, among others, that the consumption structure of fatty acids and the level of intake of fiber were not consistent with nutritional recommendations (dybkowska et al., 2004; radzymińska et al., 2005). therefore, in recent years scientists and manufacturers have taken actions towards reformulation of various food products aimed at improving their nutritional value (waszkowiak et. al., 2001; kowalski and pyrcz, 2009). since meat products provide considerable amounts of fat to the diet (givens et al., 2006), practical strategies of modifying their nutritional value include enrichment with polyunsaturated fatty acids (pufa) (jiménez-colmenero, 2007; pyrcz et al., 2007; valencia et al. 2006; özvural and vural, 2008). fatty acid (fa) composition of meat products may be changed by introducing of vegetable or fish oil into the composition of formula or by replacing some animal fatty raw material with vegetable oil. however, the substitution of the animal fatty raw material with vegetable oil may have negative effect on the technological quality and sensory desirability of the product, among others, the increase of thermal loss, the acceleration of fatty acid oxidation process (nitsch, 2007; andrés et al., 2009; decker and park, 2010). in order to prevent adverse changes in quality of meat products prepared with vegetable oil, addition of other ingredients of natural origin may be applied. potential deterioration of structure or sensory attributes of such products could be avoided by using both vegetable oil and fiber preparation (vural et al., 2004; javidipour et al., 2005). the effective method for retardation of the fa oxidation of meat products enriched with unsaturated fatty acids is the addition of antioxidants of natural origin, such as plant extracts (georgantelis et al., 2007; forell et al., 2010). recently, ready-to-eat meat products have grown in popularity with polish consumers (stangierski and kijowski, 2002; górskawarsewicz, 2007). therefore, the main objective of the present study was to develop a popular in poland ready-to-eat meat product, which is chicken burger, with improved nutritional value. launching such a product into market would facilitate composing a quotidian diet without necessity of changing eating habits or giving up favourite meals. this work includes determination of the effect of 20% substitution of pork jowl with a mixture of vegetable oils (rapeseed oil and linseed oil in mass ratio 7 to 3) and addition of inulin (1%) or wheat fiber (3%) on physical, chemical, and microbiological of chicken burgers. materials and methods materials raw materials: chilled chicken thigh meat and pork jowl, were collected from the local meat processing plant (karczew near warsaw, poland). pork jowl (about 10 kg) was purchased once, then coarse ground in a laboratory grinder mesko wn60 (mesko, skarżysko-kamienna, poland) equipped with a plate with three kidney-shaped orifices. the ground jowl was divided into four lots, which were vacuum packed and stored at -20°c±1 until further use. chicken meat (about 4 kg) was purchased prior to the each replication of experiment. fiber preparations were obtained from the manufacturers: inulin orafti® hpx from beneo-orafti ltd. (tienen, belgium) and wheat fiber vitacel wf400® from j. rettenmeier & söhne gmbh + co. (rosenberg, germany). cold pressed unrefined vegetable oils: rapeseed oil and linseed oil, and spices were obtained from the local supermarket. about 24 h prior to the production of chicken burgers, inulin gel was prepared: 1 part of inulin powder was dissolved in 3 parts of water using an electric blender braun multiquick 7 (braun gmbh, kronberg, germany). the solution was heated to boiling. heating was continued until a clear solution was obtained. the inulin solution was chilled at the room temperature for 60 min, then placed in a laboratory refrigerator (4°c±1). a mixture of vegetable oils was used in the production process of burgers in the form of an emulsion with soy protein. the emulsion was prepared directly before the onset of production of burgers. rapeseed oil and linseed oil were used in a mass ratio 7 to 3, to prepare the mixture of oils. soy protein isolate spi 733 (solae ™, st. louis, mo, usa) was rehydrated (1 part of dry preparation: 4 parts of water) using water provided in the composition of formula. to obtain the emulsion the mixture of oils was mixed with hydrated soy protein using the electric blender (braun multiquick 7) at low speed. the mass ratio of oils, rapeseed and linseed oil, was adopted on the basis of literature data on the nutritional properties of oils and the applicability of them as ingredients in meat preparations, as well as own calculation (kunachowicz et al., 2005; mińkowski et al., 2010). the calculation suggested that the content of polyunsaturated fatty acids (pufa) in chicken burgers, as a result of modification of the recipe composition, should not be less than 1.5 g per 100 g of product. 300 ital. j. food sci., vol. 27 2015 fig. 1 thiobarbituric acid reactive substances (tbars) values of chicken burgers formulated with different combinations of pork jowl, vegetable oils and dietary fiber preparations, during 21 days of storage at +4°c±1 (a) and at -20°c±1 (b). for product description see table 1. a-cmeans in the same figure (a, b) without a common lowercase letter differ significantly (p < 0.05) – influence of recipe composition of burgers (product formula) on tbars value of burgers stored in different periods. a-dmeans in the same figure (a, b) without a common lowercase letter differ significantly (p < 0.05) – influence of storage time on tbars value of burgers of each formula. table 1 composition of chicken burgers containing different combinations of pork jowl, vegetable oils, and dietary fiber preparation. ingredient product formulaa pc po poi pow chicken thigh meat (%) 85.0 85.0 85.0 85.0 pork jowl (%) 15.0 12.0 12.0 12.0 mixture of rapeseed and linseed oil (%) 3.0 3.0 3.0 total raw materials (%) 100.0 100.0 100.0 100.0 waterb (%) 10.0 10.0 10.0 10.0 sodium chloridec (%) 1.8 1.8 1.8 1.8 soy protein isolatec (%) 1.5 1.5 1.5 1.5 black pepperc (%) 0.3 0.3 0.3 0.3 rosemary extractc (%) 0.03 0.03 0.03 inulinc (%) 1.0 wheat fiberc (%) 3.0 aproduct formula: pc control burgers; po, burgers formulated with substitution of 20% of pork jowl by mixture of vegetable oils; poi burgers formulated with substitution of 20% of pork jowl by mixture of vegetable oils, and added inulin; pow burgers formulated with substitution of 20% of pork jowl by mixture of vegetable oils, and added wheat fiber. bin relation to the mass of chicken meat and pork jowl (total raw materials). cin relation to the mass of chicken meat, pork jowl and water. ital. j. food sci., vol. 27 2015 301 chicken burger preparation four formulas of chicken burgers with different combinations of pork jowl, vegetable oils, and dietary fiber preparation (pc, po, poi, pow) were prepared (table 1). the level of substitution of pork jowl with the mixture of vegetable oils and the addition level of inulin or wheat fiber were adopted on the basis of previous studies results (cegiełka and pęczkowska, 2008; cegiełka, 2011). before the production of burgers, pork jowl was thawed (4°c±1, 12 h). chicken meat and pork jowl were ground using a laboratory grinder mesko wn60 equipped with a plate having 5 mm diameter orifices. meat batters were prepared in laboratory mixers kenwood km 070 (kenwood ltd., havant, england). after mixing of chicken meat with nacl (about 5 min) fatty raw materials were added: pork jowl only (pc) or pork jowl and emulsion of oils with soy protein isolate (po, poi, pow). rosemary extract flavour guard p gin:601331 (chr. hansen a/s, hørsholm, denmark) was added to batters containing oils. after the next 5 min, other ingredients were added: black pepper, hydrated soy protein isolate (pc), and – depending on the product formula – inulin gel (poi) or wheat fiber (pow). mixing was continued until a homogenous distribution of all the ingredients was obtained (about 10 min). burgers (100 g±1) were formed using a hamburger mould (about 10.0 cm diameter and 1.0 cm high) and placed in laboratory refrigerator (-28°c±2) for 30 min, in order to maintain the shape. burgers were cooked in a commercial electric grill (unox s.p.a., vigodarzere-padova, italy) preheated to reach the temperature of 200°c. cooking was continued until the internal temperature of burger reached 72°c. the temperature of burgers was monitored using a portable skewer digital thermometer hi 98804 (hanna instruments, woonsocket, ri, usa). the burgers were then cooled at room temperature (about 30 min) over absorbent paper. after cooling, chicken burgers of each formula were divided into two lots: the first one was left in the refrigerator at 4°c±1 until next day (about 24 h), and the second one was devoted to storage research. the procedure was replicated four times. storage conditions before the storage chicken burgers of each formula were vacuum packed in bags in lots of four and then stored at +4°c±1 and -20°c±1 for a maximum of 21 days. yield after thermal processing yield after thermal processing of chicken burgers was determined by weight, after cooking and chilling the products to about 4°c, in relation to the weight of raw burgers. chemical analysis chemical analyzes were carried out on cooked and chilled (4°c±1, 24 h) chicken burgers. content of moisture, protein, total fat, salt, and ash was determined using analytical techniques according to aoac (1990). all analyzes were done in 2 replications. analysis of texture measurements of texture were conducted on cooked and chilled (4°c±1, 24 h) chicken burgers. the measurements were taken using the universal testing machine zwicki 1120 (zwick gmbh & co., ulm, germany) equipped with the warner-bratzler blade. shear force (n), the maximum value of the force registered during movement of the blade through the sample, was estimated at the speed of cross-head of 50 mm/ min. burger samples were prepared by cutting the products into cuboid-shaped pieces (9 mm high, 30 mm wide and 90 mm long). five replicates were measured from five burger samples of each formula. fatty acid composition fatty acid (fa) composition was determined in cooked and chilled (4°c±1, 24 h) chicken burgers and in chicken burgers stored at +4°c±1 and -20°c±1 for 21 days. to determine the contents of fa the lipid extracts of the burgers were analyzed by gas chromatography. procedure proposed by folch et al. (1957) was used for lipid extraction from the sample. fatty acid methyl esters (fame) were obtained according to method of morrison and smith (1964). chromatographic analyzes of fame were performed using an agilent 7890a gc system gas chromatograph (agilent technologies, santa clara, ca, usa) equipped with a split-spiltless injector and a flame ionization detector, using a fused silica capillary column rt®-2330 (0.25 mm internal diameter and 105 m long; restek corp., bellefonte, pa, usa). the mobile phase consisted of helium at a flow of 1.2 ml/min. the fames were identified by comparing their retention times with fames of the reference standards (supleco 37 component fame mix; sigma-aldrich, st. louis, mo, usa). quantification of fa was done by determining the surface areas of their peaks. all analyzes were done in 2 duplicates. lipid oxidation lipid oxidation was assessed in cooked and chilled (4°c±1, 24 h) chicken burgers and in chicken burgers stored at +4°c±1 and -20°c±1 302 ital. j. food sci., vol. 27 2015 for 7, 14 and 21 days. the 2-thiobarbituric acid (tba) test was carried out in each sample in duplicate. thiobarbituric acid reactive substances (tbars) values were determined by an extraction method according to the procedure of shahidi (1990). a constant coefficient of 2.34 was employed for converting the absorbance units to tbars values, which were expressed as mg malondialdehyde/kg sample (mg mad/kg). microbiological analysis microbiological analyzes were carried out in cooked and chilled (4°c±1, 24 h) chicken burgers and in chicken burgers stored at +4°c±1 and -20°c±1 for 7, 14 and 21 days. the analyzes were conducted in analytical center of warsaw university of life sciences sggw (warsaw, poland) in conditions accordant to requirements of pn-en iso 7218:2008 standard (pcs, 2008). the microbiological culture media were prepared according to pkn-cen iso/ts 11133-1:2009 standard (pcs, 2009). the preparation of test samples for microbiological analyzes, initial suspension and decimal dilutions was carried out according to pn-en iso 6887-2:2005 (pcs, 2005b). for quantitative analyzes, 10 g of the sample from central part of burger was collected. next, the first decimal dilution was performed by dosing physiological solution with peptone according to pn-en iso 6887-1:2000 (pcs, 2000). determination of total bacteria count (tbc) was conducted according to pn-en iso 4833:2004+ap1:2005 standard (pcs, 2005a) using pca culture medium (plate count agar) of bio-rad company (bio-rad laboratories, inc., herkules, ca, usa). determination of coliform bacteria was conducted according to pn-iso 8432:2007 standard (pcs, 2007) using vrbl medium (violet red bile lactose agar; bio-rad) and bgbbl (bile green brilliant lactose broth; bio-rad). the presence of salmonella ssp. in 25 g of product was determined according to pn-en iso 6579:2003 standard (pcs, 2003) using mkttn selective media (müller-kauffman’s medium with tetrathionate and novobiocin), rvs (medium acc. to rappaportvassilliads with soya), xld (xylose lysine deoxycholate) and hektoen of bio-rad company. the colonies typical for salmonella ssp. and suspicious colonies were confirmed using api 20e biochemical tests of biomérieux company (biomérieux sp. z o.o., warsaw, poland). statistical analyses microbiological data was analyzed using statistica 6.0 (statsoft inc., tulsa, okla., u.s.a.). all the other data was analyzed using statgraphics plus 4.1. (stsc inc., rocville, md, u.s.a.) by means of the one-way anova test. differences between burger formulas were tested by the tukey hsd test. pearson’s correlation coefficients (r) were calculated to determine the linear correlation between chosen quality attributes of chicken burgers. results and discussion yield after thermal processing, chemical composition and texture yield after thermal processing of chicken burgers ranged from 82.0 to 88.4% and was not affected (p > 0.05) by applied modifications of the composition of formula (table 2). the results obtained in this study are in agreement with those obtained by andrés et al. (2009) who showed that an introduction of squid oil into the composition of formula of frankfurters instead of beef tallow did not affect thermal loss of the product. pyrcz et al. (2007), lópez-lópez et al. (2009), and youssef and barbut (2011) proved, in turn, that thermal loss of scalded sausages increased as the result of replacement of some animal fat with vegetable oil. decrease in processing yield of meat products enriched with table 2 processing yield, chemical composition, and shear force of chicken burgers formulated with different combinations of pork jowl, vegetable oils, and dietary fiber preparation. characteristic product formula1 pc po poi pow processing yield (%) 88.4±3.9a 84.5±3.6a 82.0±2.8a 87.7±4.6a moisture (%) 62.3±0.2a 62.8±0.7a 62.3±0.1a 62.6±0.6a protein (%) 18.0±0.5a 18.4±0.6a 18.7±0.1a 18.3±0.1a fat (%) 14.5±0.6a 13.3±1.7a 13.0±1.3a 13.7±0.8a chlorides (%) 2.3±0.1a 2.3±0.1a 2.2±0.1a 2.3±0.1a ash (%) 2.7±0.1a 2.7±0.2a 2.7±0.2a 2.8±0.2a shear force (n) 31.1±2.6b 23.7±1.6a 21.9±2.5a 29.9±1.1b 1product formula: see table 1. a, bmeans within a raw without a common lowercase letter differ significantly (p < 0.05). ital. j. food sci., vol. 27 2015 303 oil may be counteracted like in this study by an application of oil in form of an emulsion with hydrated vegetable protein (youssef and barbut, 2011) or combined addition of oil and fiber preparation (vural et al., 2004; javidipour et al., 2005). chemical composition of chicken burgers formulated with different combinations of pork jowl, vegetable oils, and dietary fiber preparation is shown in table 2. the content of any of the analyzed chemical component of burgers was not differentiated significantly (p > 0.05) by the applied modifications the composition of formula. slightly lower fat content in burgers prepared with a contribution of vegetable oils (po, poi, pow), when compared to control product (pc), could have been caused by poorer oil maintenance in protein matrix of the product, and as a consequence its loss during thermal treatment. the results obtained in this study are in agreement with those presented by kayaardi and gök (2003), muguerza et al. (2003), pelser et al. (2007) and cáceres et al. (2008) who also showed that replacement of some animal fatty raw material with oil did not exert any influence on the chemical composition of scalded sausages and raw fermented sausages. in contrast, garmiene et al. (2007), and lópezlópez et al. (2009) found in studies on frankfurters and scalded sausages, respectively, that substitution of some animal fatty raw material with oil resulted in a significant increase in water content and decrease in protein content in these meat products. enrichment of ready-to-eat meat products with wheat fiber: beef burgers (cegiełka and bonderski, 2010) and poultry burgers (cegiełka and pęczkowska, 2008), did not differentiate the chemical composition of these products when compared to their counterparts prepared without the fiber. it was also shown that the application of inulin did not affect the chemical composition of turkey meat balls (ergönül et al., 2009). mean values of shear force measured in chicken burgers ranged from 21.9 n to 31.1 n (table 2). measurements of shear force of chicken burgers revealed that the texture of products was impacted (p < 0.05) by the applied modifications the composition of formula (table 2). it was found that substitution of 20% of pork jowl with vegetable oils (po) or application of both oils and inulin (poi) resulted in a significant (p < 0.05) decrease of shear force when compared to the control product (pc). the product enriched with oils and wheat fiber (pow) was characterized by a comparable (p > 0.05) shear force to the control product (pc). in contrast to the results of this study, instrumental measurements of texture of scalded sausages showed that substitution of some animal fat with vegetable oil significantly decreased hardness of these products (ambrosiadis et al., 1996; pyrcz et al., 2007; özvural and vural, 2008). however, in studies on raw sausages it was reported that the deterioration of texture of sausages prepared with oil could be counteracted by addition of dietary fiber preparation (vural et al., 2004; javidipour et al., 2005). some literature findings suggest that dietary fiber preparations could help to obtain the desired texture of ready-to-eat meat products. it was found that the addition of wheat fiber increased the shear force of poultry burgers (cegiełka and pęczkowska, 2008) and beef burgers (cegiełka and bonderski, 2010). in other studies ergönül et al. (2009) showed that inulin addition did not affect significantly the instrumental hardness of turkey meat balls. the above mentioned products, however, did not contain vegetable oil in the composition of formula. fatty acid composition the share of main fa in the overall fa pool of chicken burgers is shown in tables 3, 4 and 5. the results obtained showed that substitution of 20% of pork jowl with a mixture of vegetable oils did not totally changed fatty acid profile of chicken burgers, but improved nutritional value of them in terms of the share of saturated and polyunsaturated fatty acids (sfa and pufa). products enriched with vegetable oils, irrespectively of an addition of fiber preparation (po, poi, pow), contained significantly (p < 0.05) less saturated fatty acids (sfa) than the control product (pc; table 3). in burgers of all the formulas, palmitic acid (c16:0) and stearic acid (c18:0) were present in the highest amounts among sfa, and their contents were significantly (p < 0.05) higher in the pc product when compared to burgers prepared with vegetable oils. introduction of a mixture of vegetable oils into the composition of formula of chicken burgers did not significantly (p > 0.05) increase the share of monounsaturated fatty acids (mufa) in the overall fa pool (table 4), but po, poi, and pow products contained significantly (p < 0.05) more pufa, including nutritionally valuable pufa n-3, when compared to the pc product (table 5). among mufa, oleic acid (c18:1 n-9) was predominant is the products of all the formulas. in burgers prepared with oils, significantly lower (p < 0.05) amounts of myristoleic (c14:1) and elaidic acid (c18:1t) were found when compared to the pc product. the content of polyunsaturated fatty acids (pufa) in chicken burgers with oils (po, poi, pow) was higher than 2.5 g per 100 g of product. irrespectively of the burger formula, the highest share in pufa pool had linoleic acid (la; c18:2 n-6). the la content in burgers was not significantly (p > 0.05) differentiated by application of vegetable oils. chicken burgers of all the formulas contained relatively high amounts of linolenic (c18:3 n-3), arachidonic (c20:4 n-6) 304 ital. j. food sci., vol. 27 2015 table 3 sfa of chicken burgers (g/100 g total fa) formulated with different combinations of pork jowl, vegetable oils, and dietary fiber preparations, with different storage conditions, during 21 days of storage. fa/fa group storage conditions product formula1 pc po poi pow capric c10:0 +4°c±1, 24 h 0.081aa 0.070aa 0.046aa 0.062aa +4°c±1, 21 d 0.083aa 0.073aa 0.044aa 0.060aa -20°c±1, 21d 0.079aa 0.066aa 0.041aa 0.076aa lauric c12:0 +4°c±1, 24 h 0.648aa 0.562aa 0.522aa 0.512aa +4°c±1, 21 d 0.643aa 0.545aa 0.512aa 0.511aa -20°c±1, 21d 0.643aa 0.531aa 0.476aa 0.516aa myristic c14:0 +4°c±1, 24 h 1.466ba 1.212aa 1.185aa 1.152aa +4°c±1, 21 d 1.367aba 1.218aba 1.188aa 1.154aa -20°c±1, 21d 1.469ba 1.206aba 1.163aa 1.158aa palmitic c16:0 +4°c±1, 24 h 21.576ba 18.350aa 18.114aa 17.638aa +4°c±1, 21 d 21.740ba 18.550aa 18.244aa 17.868aa -20°c±1, 21d 21.733ba 18.579aa 18.392aa 17.915aa stearic c18:0 +4°c±1, 24 h 8.633ba 7.352aa 7.351aa 7.023aa +4°c±1, 21 d 8.672ba 7.395aa 7.334aa 7.112aa -20°c±1, 21d 8.656ba 7.537aa 7.604aa 7.256aa arachidic c20:0 +4°c±1, 24 h 0.130aa 0.179ba 0.179ba 0.193ba +4°c±1, 21 d 0.129aa 0.180ba 0.177ba 0.184ba -20°c±1, 21d 0.129aa 0.181ba 0.182ba 0.195ba behenic c22:0 +4°c±1, 24 h nd2 0.073aa 0.076aa 0.085aa +4°c±1, 21 d nd2 0.069aa 0.076aa 0.081aa -20°c±1, 21d nd2 0.073aa 0.079aa 0.085aa sfa +4°c±1, 24 h 32.944ba 28.142aa 27.827aa 27.007aa +4°c±1, 21 d 33.128ba 28.378aa 27.930aa 27.315aa -20°c±1, 21d 33.120ba 28.518aa 28.294aa 27.533aa 1product formula: see table 1. 2nd not detected (the content of the fa was lower than 0.05 g/100 g of total fa). abcmeans within a row without a common lowercase letter differ significantly (p < 0.05) – influence of product formula on fa content in burgers stored in different conditions. ameans within a column with a common uppercase letter do not differ significantly (p < 0.05) – influence of storage conditions on fa content in burgers of different formula. table 4 mufa of chicken burgers (g/100 g total fa) formulated with different combinations of pork jowl, vegetable oils, and dietary fiber preparations, with different storage conditions, during 21 days of storage. fa/fa group storage conditions product formula1 pc po poi pow myrictoleic c14:1 +4°c±1, 24 h 0.133ba 0.114aa 0.113aa 0.115aa +4°c±1, 21 d 0.132ba 0.108aa 0.112aa 0.114aa -20°c±1, 21d 0.135ba 0.108aa 0.112aa 0.113aa palmitoleic c16:1 +4°c±1, 24 h 3.456aa 2.874aa 2.824aa 2.876aa +4°c±1, 21 d 4.438bca 2.860aa 2.800aa 2.853aa -20°c±1, 21d 3.446ca 2.821aa 2.870aba 2.847aa elaidic c18:1t +4°c±1, 24 h 0.372ba 0.282aba 0.288aba 0.271aa +4°c±1, 21 d 0.542ba 0.311aa 0.325aa 0.305aa -20°c±1, 21d 0.455aba 0.306aa 0.317aa 0.306aa oleic c18:1 (n-9) +4°c±1, 24 h 40.237aba 40.837aba 39.712aa 42.830ba +4°c±1, 21 d 40.429aa 41.096aa 39.810aa 40.911aa -20°c±1, 21d 40.313aa 40.953aa 39.499aa 40.779aa eicosanoic c20:1 +4°c±1, 24 h 0.734aa 0.770aa 0.744aa 0.773aa +4°c±1, 21 d 0.731aa 0.774aa 0.741aa 0.779aa -20°c±1, 21d 0.731aa 0.778aa 0.747aa 0.767aa eruic c22:1 +4°c±1, 24 h 0.054aa 0.084aa 0.055aa 0.061aa +4°c±1, 21 d 0.045ba 0.018aa 0.017aa 0.017aa -20°c±1, 21d 0.038ba 0.019aa 0.039ba 0.019aa mufa +4°c±1, 24 h 45.959aa 45.960aa 44.720aa 47.926aa +4°c±1, 21 d 48.297aa 48.234aa 46.749aa 48.066aa -20°c±1, 21d 48.143aa 48.113aa 46.544aa 47.705aa 1product formula: see table 1. 2nd not detected (the content of the fa was lower than 0.05 g/100 g of total fa). abcmeans within a row without a common lowercase letter differ significantly (p < 0.05) – influence of product formula on fa content in burgers stored in different conditions. ameans within a column with a common uppercase letter do not differ significantly (p < 0.05) – influence of storage conditions on fa content in burgers of different formula. ital. j. food sci., vol. 27 2015 305 and eicosatrienoic acid (c20:3 n-3). the significant (p < 0.05) increase in the share of pufa in overall fa pool in po, poi, and pow products when compared to pc product was mainly determined by an increased content of linolenic acid. the presence of valuable nutritionally longchain pufa n-3 acids: eicosapentaenoic (epa) and docosahexaenoic acid (dha), was not observed in the products prepared with oils. this was possibly due to the fact that the share of vegetable oils in the recipe composition of burgers was relatively low. the ratio of pufa to sfa and the ratio of pufa n-6 to pufa n-3 are often used in nutritional characteristics of lipids in food. the valtable 5 pufa of chicken burgers (g/100 g total fa) formulated with different combinations of pork jowl, vegetable oils, and dietary fiber preparations, with different storage conditions, during 21 days of storage. fa/fa group storage conditions product formula1 pc po poi pow linoleic c18:2 (n-6) +4oc±1, 24 h 13.341aa 14.939aa 15.508aa 15.555aa +4oc±1, 21 d 13.054aa 14.730aa 15.433aa 15.400aa -20oc±1, 21d 13.274aa 14.700aa 15.403aa 15.515aa γlinolenic c18:3 (n-6) +4oc±1, 24 h 0.081aa 0.072aa 0.072aa 0.068aa +4oc±1, 21 d 0.077aa 0.071aa 0.072aa 0.068aa -20oc±1, 21d 0.080aa 0.070aa 0.069aa 0.071aa linolenic c18:3 (n-3) +4oc±1, 24 h 1.631aa 5.353ba 6.410ba 6.092ba +4oc±1, 21 d 1.599aa 5.248ba 6.480ba 6.932ba -20oc±1, 21d 1.587aa 5.211ba 6.362ba 6.015ba eicosadienoic c20:2 (n-6) +4oc±1, 24 h 0.327aa 0.291aa 0.275aa 0.280aa +4oc±1, 21 d 0.324aa 0.290aa 0.271aa 0.280aa -20oc±1, 21d 0.324aa 0.296aa 0.289aa 0.285aa eicosatrienoic c20:3 (n-6) +4oc±1, 24 h 0.110aa 0.103aa 0.101aa 0.098aa +4oc±1, 21 d 0.112aa 0.099aa 0.096aa 0.098aa -20oc±1, 21d 0.114aa 0.104aa 0.104aa 0.101aa eicosatrienoic c20:3 (n-3) +4oc±1, 24 h 0.158aa 0.155aa 0.134aa 0.140aa +4oc±1, 21 d 0.140aa 0.115aa 0.107aa 0.110aa -20oc±1, 21d 0.124aa 0.115aa 0.106aa 0.107aa arachidonic c20:4 (n-6) +4oc±1, 24 h 0.198aa 0.212aa 0.202aa 0.195aa +4oc±1, 21 d 0.196aa 0.173aa 0.179aa 0.172aa -20oc±1, 21d 0.216aa 0.226aa 0.218aa 0.179aa eicosapentaenoic (epa) c20:5 (n-3) +4oc±1, 24 h nd2 nd2 nd2 nd2 +4oc±1, 21 d nd2 nd2 nd2 nd2 -20oc±1, 21d nd2 nd2 nd2 nd2 docosahexaenoic (dha) c22:6 (n-3) +4oc±1, 24 h nd2 nd2 nd2 nd2 +4oc±1, 21 d nd2 nd2 nd2 nd2 -20oc±1, 21d nd2 nd2 nd2 nd2 pufas +4oc±1, 24 h 15.862aa 21.125ba 22.701ba 22.427ba +4oc±1, 21 d 15.501aa 20.726bca 22.639ca 22.058ca -20oc±1, 21d 15.720aa 20.722bca 22.569ca 22.271ca 1product formula: see table 1. 2nd not detected (the content of the fa was lower than 0.05 g/100 g of total fa). abcmeans within a row without a common lowercase letter differ significantly (p < 0.05) – influence of product formula on fa content in burgers stored in different conditions. ameans within a column with a common uppercase letter do not differ significantly (p < 0.05) – influence of storage conditions on fa content in burgers of different formula. ues of these ratios for control burgers (pc) were 0.48 and 7.91, respectively (table 6). the introduction of mixture of vegetable oils into the formula composition of burgers resulted in significant (p < 0.05) changes in the value of both ratios. for the po, poi, and pow burgers the ratios of pufa to sfa ranged from 0.75 to 0.83, and the ratios of pufa n-6 to pufa n-3 varied between 2.47 and 2.84. the significant (p < 0.05) increase in the pufa to sfa ratio, and decrease in the pufa n-6 to pufa n-3 ratio in burgers formulated with oils when compared to the control product was the positive effect indicating improvement of the nutritional value of fat in these products. 306 ital. j. food sci., vol. 27 2015 regardless of the temperature of 21-day storage no significant (p > 0.05) changes in the content of any fa were found in any of the burgers. the results obtained confirm the thesis put forward by jiménez-colmenero (2007), who based on the literature data reported that substitution of some animal fatty raw material with oil was an effective method of improvement of fa composition in a wide range of meat products. usefulness of linseed oil and rapeseed oil in improvement to nutritional value of lipids in meat products, expressed by increased contribution of ufa and pufa n-3, was confirmed by garmiene et al. (2007), makała and jerzewska (2008) in scalded sausages, and by pelser et al. (2007) in fermented sausages. it has been also found that the fa composition of meat products may be modified by the application of olive oil (kayaardi and gök, 2003), soybean oil (muguerza et al., 2001), or mixture of vegetable oils (özvural and vural, 2008; lopéz-lopéz et al., 2009). mixture of oils was also used for an improvement in nutritional value of lipids in ready-to-eat meat products: beef burgers (forell et al., 2010) and pork patties (lee et al., 2006). lipid oxidation changes in tbars value in chicken burgers subjected to storage at the temperature of +4°c±1 and -20°c±1 are presented in figs. 1a and 2b, respectively. the highest tbars values were observed in control burgers (pc), irrespectively of the storage temperature and time. significantly lower (p < 0.05) tbars values were observed in burgers prepared with vegetable oils (po, poi, pow), which meant inhibition of the oxidation process of lipids when compared to the pc product. it should be noted that enrichment of burgers with oils was accompanied by addition of rosemary extract, which was intended to protect fa against oxidation. the results obtained confirmed that, irrespectively of product formula, freezing was better method of storage than refrigerating. although lowering the temperature of the storage from +4 to -20 degrees did not stop completely the oxidation process of fa in burgers, it was inhibited significantly. when compared to the results presented by other authors (fernández-lópez et al., 2005; pietrzak and myron, 2008; forell et al., 2010), the tbars values in chicken burgers were relatively low, both after manufacturing (24 h) and 21 days of storage. incorporation of oil into the formula composition of meat product may influence the oxidative stability of lipids in the product. according to kayaardi and gök (2003), the adverse changes of lipids in beef sausage were caused by the partial replacement of beef tallow with olive oil. in turn, muguerza et al. (2003), and pelser et al. (2007) reported that replacement of some animal fatty raw material with vegetable oil in fermented sausages did not intensify the adverse changes in lipids, such as oxidation and hydrolysis. makała and jerzewska (2008) also found that the quality of frankfurters enriched with linseed oil, in terms of lipids oxidative changes, was satisfactory even after 8-week of refrigerating storage. in order to extend the storage stability of chicken burgers with enriched oils, an antitable 6 proportions of pufa : sfa and pufa n-6 : pufa n-3 in chicken burgers formulated with different combinations of pork jowl, vegetable oils, and dietary fiber preparations, with different storage conditions, during 21 days of storage. fa group storage conditions product formula1 pc po poi pow pufa n-6 +4°c±1, 24 h 14.056aa 15.617aa 16.157aa 16.196aa +4°c±1, 21 d 13.762aa 15.363aa 16.052aa 16.018aa -20°c±1, 21 d 14.010aa 15.397aa 16.082aa 16.160aa pufa n-3 +4°c±1, 24 h 1.806aa 5.508ba 6.544ba 6.231ba +4°c±1, 21 d 1.739aa 5.364ba 6.587ba 6.041ba -20°c±1, 21 d 1.711aa 5.362ba 6.487ba 6.122ba pufa : sfa +4°c±1, 24 h 0.48aa 0.75ba 0.82ba 0.83ba +4°c±1, 21 d 0.47aa 0.73bca 0.81ca 0.81ca -20°c±1, 21 d 0.48aba 0.73bca 0.80ca 0.81ca pufa n-6 : pufa n-3 +4°c±1, 24 h 7.91ba 2.84aa 2.4747aa 2.60aa +4°c±1, 21 d 7.91ba 2.86aa 2.44aa 2.65aa -20°c±1, 21 d 8.19ba 2.89aa 2.48aa 2.64aa 1product formula: see table 1. abcmeans within a row without a common lowercase letter differ significantly (p < 0.05) – influence of product formula on fa content in burgers stored in different conditions. ameans within a column with a common uppercase letter do not differ significantly (p < 0.05) – influence of storage conditions on fa content in burgers of different formula. ital. j. food sci., vol. 27 2015 307 oxidant additive of natural origin, which was rosemary extract, was used. the effectiveness of this component in the inhibition of lipid oxidation had already been confirmed in studies on ready-to-eat meat products (nissen et al., 2004; fernández-lópez et al., 2005; georgantelis et al., 2007; forell et al., 2010; kong et al., 2010). microbiological analysis the changes in tbc in chicken burgers stored at the temperature of +4°c±1 and -20°c±1 are shown in figs. 2a and 2b, respectively. it was found that 24 h after preparing, tbc in burgers was as follows: 2.54 log cfu/g for pc product, 2.73 log cfu/g for poi product, 2.78 log cfu/g for po product, and 2.88 log cfu/g for pow product, and was not significantly (p < 0.05) differentiated by the applied modifications of the composition of formula. after the 21-day storage at +4oc±1, the tbc increased to the level of: 5.32 log cfu/g for poi product, 5.61 log cfu/g for pow product, 7.40 log cfu/g for po product, and 8.21 log cfu/g for pc product. the increase of tbc during the whole period of storage was statistically significant (p < 0.05) only in the pc and po product. after the 21-day storage at -20oc±1 the tbc increased to the level of: 2.54 log cfu/g for poi product, 2.65 log cfu/g for pow product, 2.79 log cfu/g for po product, and 2.80 log cfu/g for pc product. the tbc of any of the frozen products was not significantly (p < 0.05) differentiated during the whole period of storage. for chicken burgers of each formula the tbc was significantly (p < 0.05) higher in the refrigerated product than in the frozen one after 21 days of storage (results not showed). the presence of salmonella ssp. was not found in chicken burgers, and the number of coliform bacteria was lower than 10 cfu/g during the whole storage period, regardless of the product formula and storage conditions (temperature and time). the results obtained proved that the microbiological quality of chicken burgers of all the four formulas fulfilled the requirements mentioned fig. 2 total bacteria count of chicken burgers formulated with different combinations of pork jowl, vegetable oils and dietary fiber preparations, during 21 days of storage in refrigerator (a) or freezer (b). for product description see table 1. 308 ital. j. food sci., vol. 27 2015 in ec regulation (2007) with respect to salmonella ssp. despite the fact that the regulation does not require determination of coliform bacteria nor total bacteria count in ready-to-eat meat products from poultry meat, it should be noticed that they may influence both health safety and shelf-life of these products. the results obtained are in agreement with these obtained andrés et al. (2009) who showed that microbiological quality of poultry frankfurters containing squid oil instead of beef tallow was not significantly differentiated when compared to the control product. similarly, lopéz-lopéz et al. (2009), on the basis of determination of total bacteria count and lactic bacteria count, found that microbiological quality of pork frankfurters enriched with olive or algae oil did not differ significantly during storage. tbc in frankfurters after manufacturing ranged depending on product formula from 2.64 to 4.18 log cfu/g, and was comparable to the results obtained in the present study. conclusions after summarizing the results of this study it was found that 20% substitution of pork jowl with a mixture of vegetable oils in the composition of formula of chicken burgers resulted in an improvement in nutritional quality in terms of fa composition. chicken burgers enriched with oils contained significantly less sfa and more pufa, including nutritionally valuable pufa n-3, than the control product, what means the improvement in nutritional value of lipids in these products. the oxidation process of lipids in products containing vegetable oils could be retarded significantly by the addition of 0.03% of rosemary extract. the results of measurements of the shear force of burgers indicated that addition of 3% of wheat fiber to product prepared with the mixture of vegetable oils as the 20% substitute of pork jowl counteracted the changes in texture. microbiological quality of vacuum-packed burgers subjected to 21-day storage at the temperature of +4°c±1 and -20°c±1 was satisfactory. acknowledgements this work was financially supported by the polish ministry of science and higher education in 2009-2011 (grant no n n312 210936). references ambrosiadis j., kyriakos p.v. and georgakis s.a. 1996. physical, chemical and sensory characteristics of cooked meat emulsion style products containing vegetable oils. int. j. food sci. & technol. 31: 189-194. andrés s.c., zaritzky n.e. and califano a.n. 2009. innovations in the development of healthier chicken sausages formulated with different lipid sources. poultry sci. 88: 1755-1764. aoac. association of official analytical chemists. 1990. official methods of analysis. 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online, doi 10.15586/ijfs.v34i1.2086 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (1): 124–131 p u b l i c a t i o n s codon effects of different concentrations of pineapple core extract and maceration process on free-range chicken meat quality norhayati hussain1,2*, choy hew weng1, nursabrina munawar1,3 1department of food technology, faculty of food science and technology, universiti putra malaysia, 43000, serdang, selangor, malaysia; 2halal products research institute, putra infoport, 43400 upm serdang, selangor, malaysia; 3alliance of research and innovation for food (arif), universiti teknologi mara, cawangan negeri sembilan, kampus kuala pilah, 72000 kuala pilah, negeri sembilan, malaysia *corresponding author: hussain n, department of food technology, faculty of food science and technology, universiti putra malaysia, 43000, serdang, selangor, malaysia. email: aryatihussain@upm.edu.my received: 10 june 2021; accepted: 18 october 2021; published: 26 february 2022 © 2022 codon publications open access paper abstract the tenderisation effects of pineapple (josephine variety) core extracts on the quality of free-range chicken meat using different maceration concentrations (30%, 50%, and 100%) and duration (15, 25, and 35 min) were analysed. texture profile analysis, colour, ph, bromelain content, and microbiological analyses of the macerated meat samples were assessed. broiler breast meat macerated with core extract (100%, 35 min) showed 86% reduction in hardness and the ph decreased from 5.87 to 4.99. the pineapple core extract has great potential as a meat tenderiser thus reduces the agriculture by-product and converts it into natural food ingredients. keywords: bromelain content; broiler breast; maceration; meat quality; pineapple core extract; tenderisation process introduction increased export capacity and processing of pineapple are estimated to reach rm320 billion. in malaysia, the pineapple plantation covering 13,433 hectares of farmland with a yield of about 32.37 tonnes of pineapple per hectare produce a total volume of 434,811 metric tonnes (nor mazila, 2020). this mass production generates a substantial by-product consisting of residual pulp, leaf, stem, peels, cores, crowns high in sugar, pectin (insoluble fibre), crude fibre, and proteins. the increasing volume of waste is detrimental to health because pineapple waste takes quite a long time to degrade and attract pests, leading to an increased risk of various dangerous diseases. thus, there is a need to convert this core into a value-added commodity. it consists of a sizeable amount of antioxidant property, sugar, fibre, vitamin c, protein, phenolic compound, carotenoids, and flavonoids (hanafi and abdullah, 2008). pineapple core waste contains a high amount of bromelain enzyme, widely used in the food industry for tenderising meat (janhvi et al., 2016), chill proofing beer (ketnawa and rawdkuen, 2011), leather tanning process, in latex manufacturing (christner et al.,1992), skincare products (frank and schulze, 2010), and pharmaceuticals (bhattacharyya, 2008). bromelain acts on meat by breaking down the collagen fibres and shows hydrolytic activity on the connective tissue, leading to the tenderisation of meat. the bromelain action on meat is affected by various factors such as ph, water-holding capacity, moisture content, and concentration (janhvi et al., 2016). a method to tenderise the tough meat is essential to increase its acceptability. according to gok and bor (2016), the typical way to tenderise meat is by the maceration technique. the maceration time may vary according to the type of marinade used to overtenderise the meat surface, leading to undesirable “mushy” meat (han et al., 2009). mailto:aryatihussain@upm.edu.my italian journal of food science, 2022; 34 (1) 125 effects of different concentrations of pineapple core extract the mixture was vortexed immediately and incubated at 37°c for 10 min. the reaction was stopped by the addition of 5 ml of 1% trichloroacetic acid. the reaction mixture was filtered and the absorbance of the filtrate was measured at 280 nm using a spectrophotometer. using tyrosine as a standard, concentrations of 50 μg/ml, 100 μg/ml, 150 μg/ml, 200 μg/ml, and 250 μg/ml were prepared, and their absorbance read at 280 nm. a standard curve of tyrosine absorbance (y axis) against tyrosine concentration (x axis) was plotted, refer to equation (1): et eb activity (cdu / ml) concentration of standard l es vr tyrosine df , tr − = − × × (1) where cdu = casein digestion unit et = absorbance of enzyme sample df = dilution factor eb = absorbance of enzyme blank vr = reaction volume es = absorbance of standard l-tyrosine tr = reaction time browning inhibition in pineapple core extract juice a low browning inhibition value indicates a high level of browning in the core, as described by kim et al. (2005) the absorbance was read at 420 nm for every 45 min using a uv-vis spectrophotometer (uv-1900, shimadzu, japan). the browning inhibition percentages were calculated using equation (2): inhibition (%) = [(afblank – aiblank) – (afsample – aisample) × 100]/ ablank, (2) where afsample is the final absorption of the sample afblank is the final absorption of the control aiblank is the initial absorption of the control maceration process for tenderisation effect in broiler breast meat the broiler cuts from the breast part were selected and purchased from the local market in pudu, selangor, malaysia. the breast meat was chosen, as it displays a firmer, more rigid, and thicker texture than the other therefore, this study aims to determine the effect of using different concentration core extracts and maceration durations on free-range chicken meat quality. according to the united states department of agriculture (usda), free-range-chicken means that the chicken has full access and freedom to roam outdoors, outside of their pens, at any given time. the pineapple core by product of josephine in this study could be considered as a potential meat tenderiser for a broiler chicken quality, commonly associated with tough and rigid texture among local people. materials and methods preparation of pineapple core extract josephine pineapple was purchased from the new seng kee (nsk) hypermarket located at jalan peel, kuala lumpur, malaysia. the basis of selection was the size (1.2–1.5 kg), firmness (c3 maturation stage), and skin colour (yellow/orange on two thirds), with no secretion from the skin. these parameters were measured visually using the naked eye (yuris and lee, 2014). the pineapples were washed thoroughly using tap water, cored, and then weighed (mettler toledo, us). the pineapples’ cores were extracted out and subjected to the juicing process using a juicer (panasonic, malaysia), filtered using muslin cloth with a mesh size of 2 mm, and stored in a sterile bottle. the yield after the extraction of 20 cores was about 1000 ml of extract. the extracted juice was kept at 4°c before the maceration treatment. proximate analysis, ph, and total soluble solid analysis of pineapple juice core extract following proximate analysis, ash, moisture content, and crude fibre were determined by using the standard aoac method 2006. the ph value was recorded using a digital ph meter (pt-15, sartorius, germany). total soluble solids (tss) in the core extract were determined using a handheld analog refractometer (0-32°) (atago, japan), and the results were expressed as per cent soluble solids (° brix) bromelain content analysis the enzyme activity of fresh pineapple core and macerated broiler breast meat was determined according to the casein digestion method and tyrosine standard (edmund and isaac, 2018). the assay mixture contained 5 ml of freshly prepared 1% casein, which was pre-warmed at 37°c, to be used as substrate and 1 ml of the freshly prepared solubilised bromelain was added in the mixture. 126 italian journal of food science, 2022; 34 (1) hussain n et al. of pinpoint size. the parameter used to count the colonies: regular plates (25–250 counts), plates with more than 250 colonies for all dilutions (too many to count), and plates with less than 25 colonies for all dilutions (too low to count). statistical analysis all results were expressed as the mean ± sd. the results obtained were analysed with a two-way anova using minitab version 17 statistical package to determine if there was any significant difference between maceration duration and concentration of the core extract with regard to meat quality. turkey’s method was used to determine which pair is significantly different from each other. differences were significant when p < 0.05. results and discussion pineapple core extract characteristics the proximate analysis of pineapple core extract revealed the moisture content, ash content, protein content, crude fibre, ph, tss, and enzyme properties (i.e., browning inhibition and bromelain activity) of the josephine variety of pineapple core extract, as shown in table 1. the ph of macerated broiler breast meat the ph values of the control and macerated meat samples are shown in table 2. it can be observed that the ph value of the control meat samples was the highest compared to the treated meat samples. the treated meat samples showed a significant decrease in ph value for all concentrations when the maceration duration increased. however, the ph value significantly increased when the concentration of the core extract decreased. it can be suggested that the lowest ph in broiler breast meat broiler chicken meat parts (debora et al., 2017). the visible fats and connective tissue were removed before maceration. the maceration procedure of the meat was performed, as described by bhaskar et al. (2007). meats were manually cut into uniform size, approximately 2 × 2 × 2 cm before further analysis. the meat and pineapple core extract ratio was maintained as 1:1 (meat: marinade) (gok and bor, 2016). the broiler meat chunks were macerated in the core extract for 15, 25, and 35 min at three different concentrations: 100% (1:0), which contains 250 ml of the whole core extract/without dilution; 50% (1:1), which contains 125 ml of the core extract and 125  ml of distilled water; and 30% (1:2), which contains 75 ml of the core extract diluted with 175 ml of distilled water, respectively, to obtain the maximum effect of up to 250  ml of the total volume. meat samples were macerated at room temperature (27 ± 3°c) and placed in a zip lock bag during the maceration process. fresh meat samples macerated using distilled water was used as a control. texture profile analysis of macerated broiler breast meat texture profile analysis was carried out using the texture analyser (stable micro system, uk) with a flat-ended cylindrical probe. the test samples were compressed to 50% of their original height with the setting of 1.0 mm/s, 4.2 mm/s, and 5.0 mm/s for pre-set speed, test speed, and post-test speed, respectively. the textural parameters of the meat were hardness and chewiness (nadzirah et al., 2016). all the analyses were performed in triplicate at room temperature of 27°c, and the mean and standard deviation (sd) were calculated. colour analysis colour measurement was done on the surface of the macerated meat samples by using chroma meter (cr410, konica minolta, japan). the illuminant d65 (representing typical daylight) was used during analysis. the l* (lightness), a* (redness), b* (yellowness) values of meat samples were measured and calculated. the average value of three meat samples for each maceration duration and concentration (triplicates) were used for statistical analysis (nadzirah et al., 2016). microbiological analysis the meat samples’ microbial load after each maceration duration was measured using the total plate count method (aoac 966.23). after solidifying the nutrient agar (merk, darmstadt, germany), the plates were inverted and incubated for 48 h at 37°c. the entire colony-forming units (cfu) were counted, including those table 1. physicochemical properties of pineapple core extract. analysis josephine pineapple core extract value moisture content (%) 89.67 ± 0.24 ash content (%) 0.75 ± 0.04 protein content (%) 19.68 ± 0.09 crude fibre (%) 1.74 ± 0.14 ph 3.83 ± 0.01 total soluble solid (°brix) 9.60 ± 0.00 browning inhibition (%) 4.18 ± 0.25 bromelain activity (cdu/ml) 152.01 ± 1.54 italian journal of food science, 2022; 34 (1) 127 effects of different concentrations of pineapple core extract to 56.87  gdu/ml. it can be suggested that the bromelain activity was affected by the percentage of the concentrated core extract. pineapple core, which has been regarded as a significant waste from pineapple production, was reported to have more bromelain content than other residue parts (banerjee et al., 2020). these findings agreed with the findings of this study where the higher the concentrations of the pineapple core extract in the macerated meat, the higher the bromelain activity. hence, with the increased maceration duration, higher tenderisation affects the meat, as shown in table 4. the bromelain intervention has been proven by its potential to disrupt the muscle microstructure and cause myofibril protein degradation (bhat et al., 2018). texture profile analysis texture profile analysis measured hardness and chewiness properties in meat to reflect the effect of bromelain enzyme as a natural meat tenderiser. the macerated meat’s hardness and chewiness had significantly reduced (p < 0.05) with the extract’s increased duration and concentration (table 4). the longer maceration duration in meat reduced the hardness (3766.50 to 1044.20  n) and chewiness (4012.95 to 726.65 n) of the macerated meat. these findings align with the study carried out by ketnawa and rawdkuen (2011). they reported that by increasing the concentration of the bromelain extracted from the pineapple peel, a continuous decrease of hardness was found in marinated beef, chicken, and squid samples. a previous study by daniela et al. (2012) observed that an increase in the meat treatments’ action time leads to a significant increase in rigidity index value, reflecting the degree of tenderness of the meat. based on the results, chicken meat macerated with 100% josephine pineapple core extract for 35 min was significantly (p < 0.05) softer in texture than chicken meat subjected to other treatment concentrations and maceration durations. it shows the lowest value of hardness and chewiness compared to the other treatments. ketnawa and rawdkuen (2011) reported that the increase in treated meat’s tenderness was due to proteolysis enzyme action on myofibrillar protein by bromelain. the myofibrillar protein breakdown generates small peptides or protein with low molecular weight, thus increasing the meat samples’ tenderness. bille and taapopi (2008) also found that bromelain’s action in denaturing the protein and breaking down the collagen, muscle fibre, and tissue resulted in increased meat tenderness in their study samples of goat meat (back, ribs, and rear limbs). the samples were marinated with bromelain extract powder and citric marinade to tenderise for 10 min at room temperature. rawdkuen and benjakul (2012) also reported that the enzymes increased the collagen solubility, and this is influenced by the acidic ph (3.83 ± 0.01) of the core extract. these findings are similar to those of mohamad afifi et al. (2018), who reported that the lowest ph (5.61) of the beef cut sample is most likely due to the ph of the core extract and maceration time. according to ketnawa and rawduken (2011), pineapple juice, which contains bromelain, will hydrolyse the muscle, thus releasing amino acid to reduce the meat’s ph. these findings were supported by manohar et al. (2016) who reported that an increase in the pineapple extract concentration decreases the treated boneless meat samples’ ph, thus becoming more acidic. maryana et al. (2018) reported that any ph range treatments from 4 to 5 could decrease meat texture’s hardness. any ph between the isoelectric point of myofibrillar protein reduced the capacity to bind water. burke and monahan (2003) also reported a significant reduction in ph from 5.7 to 3.1 of bovine muscle strips marinated with citrus juice compared to the untreated samples. thus, the meat’s acidity can be used as an indicator to detect the soft meat texture. the lower the ph value, the greater the meat tenderisation effect (manohar et al., 2016). the broiler breast meat macerated in 100% concentration of josephine pineapple core extract for 35 min had the lowest ph value and significant tenderness compared to other treatments. bromelain content of the macerated broiler breast meat the bromelain activity of the diluted core extract and  the macerated meat’s bromelain activity are shown in table  3. it can be observed that when the concentration of the core extract decreased from 100% to 30%, the bromelain activity also decreased from 151.06 cdu/ml table 2. ph value of macerated broiler breast meat at different concentrations and durations. treatments (%) maceration duration (min) 15 min 25 min 35 min control (dw) 5.87 ± 0.02aa 5.87 ± 0.02aa 5.87 ± 0.02aa josephine (30) 5.65 ± 0.01aba 5.50 ± 0.01bb 5.54 ± 0.01bb josephine (50) 5.33 ± 0.01ba 5.20 ± 0.02cb 5.12 ± 0.01cc josephine (100) 5.30 ± 0.01ba 5.16 ± 0.01cb 4.99 ± 0.01dc values are expressed as mean ± standard deviation (n = 3). mean with different superscript capital letters within column are significantly different (p < 0.05). mean with different superscript lower case letters within row are significantly different (p < 0.05). notes: 100%, 50%, and 30%, concentration of the core extract; dw, distilled water; 15, 25, and 35 min, maceration duration. 128 italian journal of food science, 2022; 34 (1) hussain n et al. table 3. bromelain content of the diluted pineapple core extract, macerated broiler breast meat at different concentrations and durations. concentration (%) bromelain activity (cdu/ml) diluted pineapple extract maceration duration (min) 15 min 25 min 35 min control (dw) na 0.00 ± 0.00aa 0.00 ± 0.00aa 0.00 ± 0.00aa josephine (30) 56.87 ± 1.26a 8.95 ± 0.01ba 12.93 ± 0.01bb 17.58 ± 0.01bbc josephine (50) 79.29 ± 1.01b 12.28 ± 0.01ca 20.67 ± 0.02cb 29.49 ± 0.01cc josephine (100) 151.06 ± 1.11c 18.30 ± 0.01da 26.47 ± 0.01db 39.19 ± 0.01dc values are expressed as mean ± standard deviation (n = 3). mean with different superscript capital letters within column are significantly different (p < 0.05). mean with different superscript lower case letters within row are significantly different (p < 0.05). notes: 100%, 50%, and 30%, concentration of the core extract; dw, distilled water; 15 , 25 , and 35 min, maceration duration; na, not available. promoted the structural alteration through the process of collagens cross-link. meat colour analysis the control and treated meat samples’ colour parameters for 15, 25, and 35 min are shown in table 5 (l*, a*, and b*value). the results show that the maceration process significantly affected the l* and b* values of the broiler breast samples. the lightness (l*value) increased as the maceration duration increased but decreased when the core extract concentration decreased compared to the control. according to kim et al. (2012), the meat colour influenced meat quality; hence, they were affected by marination. the colour of the meat is related to muscle pigments, myoglobin, and haemoglobin. however, meat’s discolouration is affected by pigment conditions (amount and chemical state). the entire breast muscle, commonly discoloured as breast muscle, comprises a large portion of the weight (~5%), so it is more sensitive, contributing to discolouration and the meat’s light appearance. therefore, small changes in colour on the breast part is more noticeable than in other parts. serdaroglu et  al. (2007) reported that the increase in lightness is due to the swell of the muscle protein and light reflection altered at low ph and ionic strength, thus forming the lighter colour. according to wismer-pedersen (1959), it is widely accepted that variations in muscle structure may affect light reflectance or light scattering. the extent of denaturation of the muscle proteins differs in ordinary and pale coloured meat. the b*value decreased with increased maceration duration. however, when the concentration of the core extract decreased, the b*value increased. meanwhile, the a*value of the control and treated meat samples shows no significant difference at all concentrations used during the first 15 min. a similar pattern of a*value was also reported by serdaroglu et al. (2007) in which the turkey breast was marinated in grapefruit juice (50% and 100%) and citric acid (0.05  m, 0.1 m, and 0.2 m). the a*value (redness) is related to the concentration of myoglobin and myoglobin denaturation level (francis and clydesdale, 2008; vaudagna et al., 2008). the acid treatment appeared to enhance myoglobin’s conversion to metmyoglobin, which results in lower colour intensity. table 7 shows the colour difference (δe*) of macerated meat in different core extract concentrations at 15, 25, and 35 min. the highest colour difference was found in meat samples macerated in 100% concentration of josephine core extracts for 35 min. according to francis and clydesdale (2008), when colour differences (δe*) exceed the value of 3, the meat’s colour change is detectable to the human eye. microbiological analysis of the macerated broiler breast meat according to table 6, the total microbial count decreased as the maceration duration increased for all treatments. however, when the concentration of the core extract decreased, the total microbial count increased. in this study, the total microbial count was below 7 log cfu/g after 35 min of maceration. in contrast, the highest microbial counts were observed as early as 15 min of maceration using 30% concentration of core extract (4.34 log cfu/g) regardless of the control sample. according to hong et al. (2013), this might be due to the sample’s initial microbial contamination because bacterial counts in fresh meat are generally less than 3 log cfu/g. jeong et al. (2018) also reported that citrus juice such as pineapple juice has an antimicrobial function, causes denaturation of microorganisms, and affects the water-holding capacity. the ph reduction caused by this italian journal of food science, 2022; 34 (1) 129 effects of different concentrations of pineapple core extract ta bl e 4. h ar dn es s an d ch ew in es s va lu e of th e m ac er at ed b ro ile r br ea st m ea t a t d if fe re nt c on ce nt ra tio ns a nd d ur at io ns . tr ea tm en ts (% ) te xt ur e pr ofi le a na ly si s (t pa ) h ar dn es s (n ) c he w in es s (n ) 15 m in 25 m in 35 m in 15 m in 25 m in 35 m in c on tr ol (d w ) 72 92 .3 0 ± 99 .4 8a a 72 92 .3 0 ± 99 .4 8a a 72 92 .3 0 ± 99 .4 8a a 40 12 .9 5 ± 96 .7 5a a 40 12 .9 5 ± 96 .7 5a a 40 12 .9 5 ± 96 .7 5a a jo se ph in e (3 0) 37 66 .5 0 ± 84 .4 0b a 27 55 .6 0 ± 10 4. 47 b b 27 41 .8 0 ± 91 .7 8b b 17 11 .8 7 ± 10 3. 79 b a 12 81 .1 0 ± 85 .9 4b b 80 0. 15 ± 9 6. 87 b c jo se ph in e (5 0) 25 52 .4 0 ± 92 .2 5c a 22 72 .4 0 ± 91 .9 7c b 17 07 .9 0 ± 85 .7 8c c 15 94 .0 6 ± 87 .2 5c a 11 46 .8 3 ± 10 1. 79 c b 73 3. 46 ± 9 7. 18 b c c jo se ph in e (1 00 ) 22 24 .9 0 ± 94 .5 7d a 18 06 .1 0 ± 89 .0 5d b 10 44 .2 0 ± 10 6. 95 d c 11 07 .2 2 ± 87 .7 9d a 10 74 .9 0 ± 91 .8 2c d ab 72 6. 65 ± 9 4. 85 c b va lu es a re e xp re ss ed a s m ea n ± st an da rd d ev ia tio n (n = 3 ). m ea n w ith d iff er en t s up er sc rip t c ap ita l l et te rs w ith in c ol um n ar e si gn ifi ca nt ly d iff er en t ( p < 0 .0 5) . m ea n w ith d iff er en t s up er sc rip t l ow er c as e le tte rs w ith in th e ro w a re s ig ni fic an tly d iff er en t ( p < 0 .0 5) . n ot es : 1 00 % , 5 0% , a nd 3 0% , c on ce nt ra tio n of th e co re e xt ra ct ; d w , d is til le d w at er ; 1 5, 2 5 an d 35 m in , m ac er at io n du ra tio n. ta bl e 5. c ol ou r (l *, a *, b *v al ue ) o f m ac er at ed b ro ile r br ea st m ea t a t d if fe re nt c on ce nt ra tio ns a nd d ur at io ns . tr ea tm en ts (% ) c ol ou r a na ly si s l* va lu e (li gh tn es s) a* va lu e (r ed ne ss ) b* va lu e (y el lo w ne ss ) 15 m in 25 m in 35 m in 15 m in 25 m in 35 m in 15 m in 25 m in 35 m in c on tr ol (d w ) 40 .4 9 ± 0. 40 a a 40 .4 9 ± 0. 40 a a 40 .4 9 ± 0. 40 a a 8. 08 ± 0 .3 0a a 8. 08 ± 0 .3 0a a 8. 08 ± 0 .3 0a a 12 .5 9 ± 0. 25 a a 12 .5 9 ± 0. 25 a a 12 .5 9 ± 0. 25 a a jo se ph in e (3 0) 43 .4 4 ± 0. 35 a a 50 .6 1 ± 0. 94 b b 51 .9 8 ± 0. 49 b bc 8. 05 ± 0 .1 7a a 8. 01 ± 0 .0 9a a 7. 95 ± 0 .3 0b b 12 .4 7 ± 0. 11 a a 11 .7 3 ± 0. 42 b a 11 .6 4 ± 0. 17 b b jo se ph in e (5 0) 46 .6 7 ± 0. 81 b a 52 .6 0 ± 0. 30 c b 55 .1 1 ± 0. 28 c bc 8. 05 ± 0 .0 4a a 7. 31 ± 0 .0 8b b 7. 25 ± 0 .1 2c b 12 .3 1 ± 0. 15 a a 11 .5 2 ± 0. 07 b ab 10 .8 7 ± 0. 24 c b jo se ph in e (1 00 ) 50 .0 1 ± 0. 73 c a 54 .9 9 ± 0. 41 d b 58 .3 5 ± 0. 25 d c 7. 32 ± 0 .0 8b a 6. 75 ± 0 .1 6c b 6. 64 ± 0 .0 7d c 12 .0 5 ± 0. 51 b a 11 .5 2 ± 0. 31 b ab 10 .3 3 ± 0. 32 c d b va lu es a re e xp re ss ed a s m ea n ± st an da rd d ev ia tio n (n = 3 ). m ea n w ith d iff er en t s up er sc rip t c ap ita l l et te rs w ith in c ol um n an d su pe rs cr ip t l ow er c as e le tte rs w ith in ro w a re s ig ni fic an tly d iff er en t ( p < 0 .0 5) . 130 italian journal of food science, 2022; 34 (1) hussain n et al. citrus extract was the primary factor that affected the reduction of microorganisms. according to alvarado and mckee (2017), most microorganisms slowed their growth in an acidic environment. this statement is also supported by kotzekidou et al. (2008), which stated that pineapple extract could suppress the growth of escherichia coli o157:h7 edl-933. the pineapple extract contains active substances such as terpenoids and phenolic compounds, and these compounds attach to the bacterial membrane and deplete the metabolic energy of bacteria. our study found that samples macerated in 100% concentration of the core extract for 35 min had the lowest microbial count (3.98 log cfu/g). conclusions the maceration technique using 100% concentration of the core extract (josephine variety) for 35 min shows the most significant meat tenderisation effect compared to other concentrations and durations used in this study. the hardness and chewiness of the broiler breast meat were reduced. pineapple core extract is applicable as a meat tenderiser in the food industry, which may increase the demand for local pineapple core. acknowledgements the authors would like to thank the faculty and the personnel of food science and technology, universiti putra malaysia (upm), for their assistance in completing the study. author contributions statement c.h.w. conducted the experiment, c.h.w. and n. h. analysed the findings, and c.h.w., n. h., and n. s.m. wrote and edited the manuscript. table 6. total microbial count of macerated broiler breast meat at different concentrations and durations. concentration (%) microbiological load (total microbial count log cfu/g) maceration duration (min) 15 min 25 min 35 min control (dw) 4.35 ± 0.03 4.35 ± 0.03 4.35 ± 0.03 josephine (30) 4.34 ± 0.06 4.18 ± 0.02 4.10 ± 0.02 josephine (50) 4.26 ± 0.04 4.16 ± 0.03 4.03 ± 0.05 josephine (100) 4.07 ± 0.02 4.03 ± 0.06 3.98 ± 0.03 values are expressed as mean ± standard deviation (n = 3). notes: 100%, 50%, and 30%, concentration of the core extract; dw, distilled water; 15, 25 and 35 min, maceration duration. additional information the authors declare no conflict of interest. references abdullah & hanafi, m. 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(2016). tenderization of meat using bromelain from pineapple extract. institute. journal. phramaceutical. science. review. reources, 39(1), 81–85. jeong, k.j., hyeonbin, o., so, y.s. and young, s.k., 2018. effect of different marination condition on quality, microbiology properties, and sensory characteristic of pork ham cooked by the sous-vide method. korean journal for food science of animal resource 38(3): 506. ketnawa, s. and rawdkuen, s., 2011. application of bromelain extract for muscle foods tenderisation. food nutrition sciences 2: 393. https://doi.org/10.4236/fns.2011.25055 kim, d.j., lee, d.h., lee, y.g., park, d.w., kim, g.d., jung, e.y., et al., 2012. the relationship between carcass colorgrade and instrumental values. journal of agriculture life scienc 46: 133. kim, m.j., kim, c.y., & park, i. 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35 (3): 17–43 issn 1120-1770 online, doi 10.15586/ijfs.v35i3.2298 17 p u b l i c a t i o n s codon content of selected polyphenolic substances in parts of grapevine lenka jurasova1†*, tunde jurikova2†, mojmir baron1, jiri sochor1 1department of viticulture and enology, faculty of horticulture, mendel university in brno, lednice, czech republic; 2institute for teacher training, faculty of central european studies, constantine the philosopher university in nitra, nitra, slovakia †these authors shared first authorship. *corresponding author: lenka jurasova, department of viticulture and enology, faculty of horticulture, mendel university in brno, valticka 337, 691 44 lednice, czech republic. email: xjurasov@mendelu.cz received: 7 november 2022; accepted: 30 march 2023; published: 18 july 2023 © 2023 codon publications open access review abstract this review provides an overview of the variety of occurrences, content, extraction and health effects of selected polyphenolic compounds associated with different parts of grapevine (seeds, peel, pulp and stems). the review provides a brief characterisation of grape parts, the content of polyphenolic compounds and their extraction together with their graphical forms of presentation and diversity as determined by different studies. the content of individual polyphenolic compounds differed with studies. effects of different factors were evident in both growing style and geographical location of vineyards as well as extraction methods and analytical conditions. keywords: grapevine, peel, polyphenolic compound, seed, skin, stem introduction the aim of the review was to summarize the occurrence, distribution and determination of polyphenolic compounds in different parts of grapes. the review also demonstrated variations in the values of polyphenolic compounds with respect to the methods used as well as variations within varieties. the polyphenolic compounds were selected based on the literature. among the most studied polyphenolic compounds in grapes are catechin, epicatechin gallate, quercetin, resveratrol and gallic acid (ga). owing to its simple structure among other factors, ga represents the most commonly used standard for determining total polyphenolic compounds. the aim of creating a comparative table was to focus on the current literature. the analytical methods used were listed for each compound. however, in the case of some less usual analytes, older sources (from 2003 onwards) were also used. for the purpose of clarity and to emphasise the diversity of individual results, all mass concentrations were converted to a common unit of µg/g. for determining concentration in solution, the units were changed to µg/ml. moreover, this review considered polyphenols in accordance to their distribution in grapes reflecting their health benefits. polyphenolic compounds found in grapes, their distribution and health benefits polyphenols are a class of compounds comprising one or more phenolic hydroxyl groups bonded to at least one aromatic ring (di lorenzo et al., 2021). the majority of polyphenolic compounds are generated from phenylpropanoid and phenylpropanoid acetate pathway and represent 40% of organic carbon in plants. classification of phenolic compounds is based on different approaches according mailto:xjurasov@mendelu.cz 18 italian journal of food science, 2023; 35 (3) jurasova l et al. stilbenes, tannins, lignans, lignins, monolignols, anthocyanins, isoflavones, chalcones, naphthoquinones and anthraquinones, and diarylheptanoids. as observed, phenol sorting is not dogmatic, and it depends, in particular, on the purpose of dividing them (brenes et al., 2016). the distribution of polyphenols within a grape bunch is different as shown in figures 1–4. the content, abundance and distribution of polyphenolic compounds in grapevines are highly dependent on the geographical and climatic conditions, grape variety, cultivation processes and the stage of ripeness. however, there is no doubt that vitis vinifera is one of the most important sources of polyphenolic substances, especially wine industry wastes (grape skin, stems and seeds), which represent 20% of the weight of processed grapes (teixeira et al., 2018) and therefore are a matter of growing interest and emphasis for farmers. as mentioned above, the concentration of polyphenols varies not only between plant species but also between plant parts. thus, in the following sections, seeds, peels, to the functional group bound to phenol or the number of phenolic units found in the molecule, and this differs mainly according to studies. the easiest division of phenolic compounds is into flavonoids, the most studied group, and non-flavonoids. nowadays, phenolic compounds are classified into groups and subgroups based on the number of phenolic rings and structural elements attached to the rings (butterfield, castegna, lauderback,  & drake et al., 2002). according to this approach, the major classes represent phenolics, flavonoids, stilbenes and lignans (pietta et al., 1998). the group of flavonoids, as the most abundant class in grapes or wine, can be represented by flavanols, flavones, flavanonols and anthocyanins (nollet and gutierrez-uribe, 2018; teixeira et al., 2018; tsimogiannis and oreopoulou, 2019). depending upon products (nollet and gutierrez-uribe, 2018; teixeira et al., 2018; tsimogiannis and oreopoulou, 2019), flavanols, flavones, flavanonols and anthocyanins belong to a group of flavonoids, and are the most abundant polyphenols present in grapes or wine and related products. other explanation divided polyphenolic into the following sub-classes: coumarins, furanocoumarins cabernet sauvignon v3 cabernet sauvignon v2 cabernet sauvignon v1 cabernet sauvignon (sb) cabernet sauvignon (sa) frankovka (sa) frankovka (sb) prokupac (sb) prokupac (sa) merlot (sb) merlot (sa) black tamjanika (sb) black tamjanika (sa) zăcinak (sb) zăcinak (sa) gamay (sb) gamay (sa) župljanka (sb) župljanka (sa) bagrina (sb) bagrina (sa) chardonnay (sb) chardonnay (sa) 0 4 8 12 16 20 24 figure 1. graph shows the values of total polyphenols by using folin’s reagent in seeds. values are expressed in milligram of ga equivalent to per gram of seeds, except for silva et al. (2018), where the equivalent is epicatechin gallate. the same colour shows the measurements of one research group. next to each bar is the description indicating the variety used for measurements. sa: solvent a; acetoh: acidified aqueous ethanol; sb: solvent b; chcit: choline chloride:citric acid; i: irrigation; lr: leaf removal. light blue (castro-lopez et al., 2019), purple (dabetić et al., 2020), green (chorti et al., 2016), yellow (dinis et al., 2020), grey (radovanović et al., 2019), orange (silva et al., 2018) and dark blue (pantelić et al., 2016). italian journal of food science, 2023; 35 (3) 19 content of selected polyphenolic substances in parts of grapevine pathogens. other phenols have a mechanical function and are pollinator attractants, adsorb ultraviolet (uv) rays and reduce the growth of surrounding competitive plants. polyphenols could have a negative allelopathic effect if released from the leaves, roots or decomposing plant tissues. to compete with surrounding plants for water, sunlight and minerals, plants release phenols that may inhibit the growth of adjacent plants. this effect could possibly be utilised in the production of genetically modified plants that produce compounds with allelopathic effects to eradicate weeds (crozier et al., 2008; novak et al., 2008; taiz et al., 2015). polyphenolics played an important role in overcoming the challenges of waterto-land transition. as an example, the evolution of phenylpropanoid pathway in primitive terrestrial plants and algae helped them to adapt to exposition to uv radiation even before transition to land. this pathway leads to the production of more than 5,000 compounds called flavonoids. the land ecosystem means decline of water pulp and stems are described separately. the most abundant group of polyphenols found in grapes are flavanols, represented by simple monomers of catechin and epicatechin gallate, oligomeric proanthocyanidins (opcs; 2–5 units), and condensed tannins (polymers of more than five phenolic units), mainly present in the pulp (crozier et al., 2008). the most abundant form of flavanol group is catechin, mainly found in grape skin and seeds, and traces of monomers or dimers are discovered in grape pulp. the phenolic hydroxyl group is relatively acidic compared with other hydroxyl groups because of its bond to the aromatic ring, causing deprotonation of the oxygen substituent and stabilisation of the complex. it causes reactivity and determines phenols as the building blocks of polymers, such as lignins or suberins, as well as their involvement in the production of wide spectra of compounds in plants. owing to their chemical variety, polyphenols have different functions in plants. many of them are involved in providing defence against herbivores and figure 2. graph shows the values of total polyphenols by using folin’s reagent in grape peel. values are expressed in milligram of ga equivalent to per gram of peel. the same colour shows the measurements of one research group. below each bar is the description indicating the variety used for measurements. sa: solvent a; acetoh: acidified aqueous ethanol; sb: solvent b; chcit: choline chloride:citric acid; p1-10: samples from piranshahr city; s1-10: samples from sardasht city. purple (dabetić et al., 2020), light blue (castro-lopez et al., 2019), pink (singha and das, 2015), black(ni et al., 2017), dark blue (radovanović  et  al., 2019), light green (khoshamad et al., 2020), dark green (chorti et al., 2016), yellow (dinis et al., 2020), grey (radovanović et al., 2019), red (baiano and terracone, 2012) and orange (silva et al., 2018) agiorgitiko i and lr agiorgitiko lr agiorgitiko irrigation agiorgitiko cerceal white vranac merlot touriga nacional preto martinho pinot gris chardonnay welschriesling sauvignon blanc petra riesling prokupac pinot noir shiraz sangiovese cabernet franc merlot cabernet sauvignon 0 50 100 150 200 250 300 350 400 tpc (mg/g) 20 italian journal of food science, 2023; 35 (3) jurasova l et al. cabernet sauvig.. cabernet sauvig.. frankovka (sb) frankovka (sa) prokupac (sb) prokupac (sa) merlot (sb) merlot (sa) black.. black.. zăcinak (sb) zăcinak (sa) gamay (sb) gamay (sa) župljanka (sb) župljanka (sa) bagrina (sb) bagrina (sa) chardonnay (sb) chardonnay (sa) cabernet.. cabernet.. cabernet.. red globe kichmich chorni flame seedles thompson seedles kyoho grape pinot gris chardonnay welschriesling sauvignon blanc petra riesling prokupac pinot noir shiraz sangiovese cabernet franc merlot cabernet.. s10 s9 s8 s7 s6 s5 s4 s3 s2 s1 p10 p1 p2 p3 p4 p5 p6 p7 p8 p9 0 5 10 15 figure 2. continued. italian journal of food science, 2023; 35 (3) 21 content of selected polyphenolic substances in parts of grapevine agiorgitiko irrigation and lr agiorgitiko leaf removal (lr) agiorgitiko irrigation agiorgitiko cerceal white vranac merlot thompson seedless beogradska basemena touriga nacional preto martinho 0 15 30 45 60 75 tpc(mg/g) figure 2. continued. sources, resulting in the formation of a group of substances called suberins. these are polymers of phenolic (hydrophilic) and aliphatic (hydrophobic) groups important in forming periderm in the root and bark because they provide a hydrophobic barrier and prevent water loss. suberins are the main component of the cork and provide physical barrier against pests. lignin is a trimer from monolignol monomers that toughen cellulose fibres in specialised cell wall structures in tracheids and vessels. these structures allow the carrying of plant’s weight on land, which is not required in the aquatic environment, and transportation of water and minerals from the roots to all plant tissues. lignin formation helped plants to overcome the pull of gravity and compete for sunlight with other plants (novak et al., 2008; taiz et al., 2015). last but not least, in addition to many changes, challenges and problems, plants were exposed to a new spectrun of pathogens and herbivores. to deal with this challenge, different subgroups of phenolics that mediate chemical defence, and thus have antiherbivorous, insecticidal and allelopathic (interaction) effects and prevent the spread of fungi and bacteria, were evolved. owing to their toxicity, phenolic metabolites do not occur in the form of free aglycones but are conjugated with cell wall components or generate conjugated glycosides (gl). in addition to the toxic effects, phenols are responsible for the colour, aroma, taste and antioxidative properties of plant organs (war et al., 2012). grapes and their products have displayed a wide range of utilisation. grape by-products have been used for feeding agriculturally important animals with different results. the feeding of chickens by grape pomace and seed extracts led to improvement in growth in a study conducted by liu et al. (2014). on the other hand, no significant increase in the growth was observed in chickens fed with grape seed extracts (gse) and grape pomace (brenes et al., 2016; chamorro et al., 2015). a positive boost in growth could be explained due to various reasons. gse inhibits growth of the pathogen causing coccidiosis oxidative stress (wang et al., 2008), reduces meat lipid oxidation (brenes et al., 2016; iqbal et al., 2015) and increases the abundance of polyunsaturated fatty acids in poultry meat (chamorro et al., 2015). other studies showed a significant effect on pigs’ metabolism after the feeding of grape marc meal or grape pomace. intake of grape by-products improved nitrogen metabolism and growth, modified fatty acid patterns in subcutaneous fat (yan and kim, 2011), and increased feed conversion ratio and antimicrobial effect on escherichia coli in the faeces of weaned pigs after feeding with grape by-product (fiesel et al., 2014). in contrast, no effect on the growth of rabbits was observed (nicodemus et al., 2007; tortuero et al., 1994). moreover, in the study conducted by ferreira et al. (1996), feeding rabbits with grape pomace led to a decrease in feed conversion ratio. no change in the production or composition of milk was observed in case of dairy cows fed with grape pomace or marc (eleonora et al., 2014; hansen and nielsen, 2004). however, moate et al. (2014) described an increase of monosaturated fatty acids, polysaturated fatty acids and linoleic acid in milk after feeding cows with ensiled or dried grape marc. feeding with grape marc decreased weight gain in beef cattle (manterola et al., 1997), and the presence of lignin, tannin and fibre decreased nutrients’ digestibility in fatting lambs after feeding them with grape pomace (eleonora et al., 2014). in contrast, abarghuei et al. (2010) showed a positive effect on retained nitrogen and ruminal parameter in sheep fed with grape pomace. numerous experiments confirmed the use of grape by-products as a nutrient for farm animals. different effects of feeding grape by-products are caused due to different production processes used in wineries, affecting the quality of grape by-products, and also variations among cultivars (gierus et al., 2020; milder et al., 2005). 22 italian journal of food science, 2023; 35 (3) jurasova l et al. cerceal white thompson seedless beogradska besemena s10 s9 s8 s7 s6 s5 s4 s3 s2 s1 p10 p1 p2 p3 p4 p5 p6 p7 p8 p9 0 2 4 6 8 10 12 14 tpc [mg/g] red globe pinot gris chardonnay welschriesling sauvignon blanc petra riesling prokupac pinot noir shiraz sangiovese cabernet franc merlot cabernet sauvignon kichmich chorni flame seedles thompson seedles kyoho grape 0 100 200 300 400 500 600 700 tpc [mg/g] figure 3. graph shows the values of total polyphenols by using folin’s reagent in grape pulp. values are expressed in milligram or microgram of ga equivalent to per gram of pulp. the same colour shows the measurements of one research group. below each bar is the description indicating the variety used for measurements. p1-10: samples from piranshahr city; s1-10: samples from sardasht city. yellow (dinis et al., 2020), red (baiano and terracone, 2012), green (khoshamad et al., 2020), pink (singha and das, 2015), black (ni et al., 2017) and blue (pantelic et al., 2016). italian journal of food science, 2023; 35 (3) 23 content of selected polyphenolic substances in parts of grapevine vranac merlot moscatel viosinho fernão pires malvasia fina rabigato austrian vine slovak vine white vine red vine touriga nacional preto martinho 0 25 50 75 100 125 150 75 200 225 250 tpc (mg/g) figure 4. graph shows the values of total polyphenols by using folin’s reagent in grape stems. values are expressed in milligram of ga equivalent to per gram of stems, except for silva et al. (2018), where the equivalent is epicatechin gallate. the same colour shows the measurements of one research group. below each bar is a description indicating the variety used for measurements. grey (radovanović et al., 2019), green (leal et al., 2020), yellow (hanušovský et al., 2020), blue (domínguez-perles et al., 2014) and orange (silva et al., 2018). polyphenolic compounds of grape seeds, distribution, extraction and health potential grape seeds are pear shaped with a trigone transverse section. the seeds are composed of a cuticle, an epidermis and two integuments going around the albumen and the embryo (cadot et al., 2006). development of the seed and fruit are related. their maximum fresh weight (fw) occurs during colouring of berries, while maximum dry seed weight coincides with maximum berry weight (ristic and iland, 2005). colour of seeds varies from green to dark brown during maturation (kennedy et al., 2000). change of colour, along with hardening, is concomitant with the oxidation of phenolics and maturing of the bunch (cadot et al., 2006). grape seeds are composed of fibre (40%), volatile oils (16%), protein (11%), polyphenolic compounds (7%) and other substances (sugars, minerals etc.; de campos et al., 2008). within the grapes, soluble phenols are distributed unevenly, with dominant representation in seeds (70%), 28–35% found in skins and, in spite of their large volume, the least presence of 10% is found in pulp. supported by various studies (cheng et al., 2012; makris et al., 2007), total polyphenols are maximum in seeds among different analysed grape components in different vine varieties. catechin and epicatechin gallate are the most abundant phenolic compounds present in the seeds and stems. within flavonols, the main representative phenol is rutin (cheng et al., 2012; makris et al., 2007). presence of different phenols is affected by genetic diversity between varieties, regions, light intensity, soil compositions, climatic and agronomic conditions, ripening stages, processes of extraction and storage conditions (de la cerda-carrasco et al., 2015; jordão et al., 2001; nassiriasl and hosseinzadeh, 2009). presence of the most abundant oligomers is different between plant compositions. the major oligomer found in the seeds, according to different studies, is procyanidin b2. on the other hand, procyanidin b1 is reported to be the dominant oligomer in the skin and bunch stems (dwyer et al., 2014; topalovic and mikulic-petkovsek, 2010; vujasinović et al., 2021). despite differences, the studies showed the highest content of polphenols in the following order: the highest concentration was in the seeds followed by the berry skins and must (mustum or young wine) (boso et al., 2019). antioxidant activity of grapes was confirmed by 1,1-diphenyl-2-picrylhydrazyl (dpph) and 2,2’azino bis(3-ethylbenzothiazoline-6-sulfonic acid) (abts) assays, where the highest was found in seed extracts, followed by stem and skin extracts. the higher antioxidant capacity of the seeds may correlate with its higher total phenolic content (tpc), supported by previous results that phenols, dominantly found in the seeds, possess antioxidant activity. 24 italian journal of food science, 2023; 35 (3) jurasova l et al. surprisingly, a negative correlation between the content of phenolics and ec50 describing antioxidant activity was proven by wen et al. (2016), suggested that among phenolics, antioxidant activity is the combined effect of phenolic compounds, sterols and vitamin e. on the other hand, a negative relationship was observed between the quantity of tocols and the content in total phenol (vujasinović et al., 2021). extraction of grape polyphenols in the seeds is dependant upon two conditions, dissolution of concrete polyphenolic compounds in the plant material matrix, and their diffusion in the external solvent medium. ethanol as an extracting solvent was shown to be an efficient method of extracting polyphenol. nawaz et al. (2006) studied different conditions of extraction of polyphenols from grape seeds using 50% ethanol and 50% water as solvents. when compared with a ga standard, extraction of grape seed polyphenols with a 0.2-g/ml solid to liquid ratio, double-stage extraction, and 0.22-m pore size membrane seem to be the most optimal conditions (polyphenols represents 11.4% of the total seeds weight). in respect of concrete polyphenols, it mostly depends on the applied method. as an example, monomeric procyanidins are found in large amounts in grape seeds, but the quantity of extraction is low due to their low water solubility. therefore, methods utilising solvents with lower polarity (soxhlet, supercritical fluid extraction (sfe) and ethanol-assisted extraction) are preferred to increase extractability (colibaba et al., 2015). in their findings, the choice of solvent used for extraction and the effect of extraction were supported by the teixeira group, who tested three different solvents and three different methods for extraction. specifically, soxhlet, ultrasound extraction, and maceration were performed, and methanol, ethanol and, acetone with different polarities were used as solvents, with the highest tpc using 70% acetone, followed by 70% ethanol extract and 70% methanol extract. however, comparable results were obtained with different extraction methods (teixeira et al., 2014). extraction efficiency does not only depend on the selection of the solvent but also on the extracted phenolic compound. the utilisation of ethyl acetate for extraction showed a large recovery effect on flavonols, whereas methanol was the preferred solvent for flavan-3-ol (catechin, epicatechin gallate and epigalocatechin) extraction. owing to the low permeability of tissues to non-polar aprotic solvents, addition of water to the solvent increases process efficiency. as an example, the efficiency of proanthocyanidin extraction was significantly increased by adding water to acetone or ethyl acetate. moreover, the type of analysed material influences measured concentrations because of the relation between the number and weight of seeds, and weight and stage of berry ripening. for example, the highest content of catechin is at grape veraison; then the concentrations decrease until near maturity (downey et al., 2003; freitas and glories, 1999; kennedy et al., 2000). also, decline of monomeric flavanols is more rapid than oligomers because of increasing polymerisation during maturity. many positive effects of polyphenolic compounds extracted from seeds have been described for human health, especially their ability to decrease the occurrence of heart disease. polyphenols mostly contribute to lower the oxidation levels of low-density lipoprotein cholesterol and blood pressure, enhance the functioning of endothelium, reduce inflammation and platelet aggregation, and decrease cell senescence by inhibiting novel proteins activating this process (dohadwala and vita, 2009; moreno et al., 2003; shi et al., 2003). grape seed extracts, through their inhibition of enzymes lipoprotein lipase and pancreatic lipase involved in fat metabolism, could be utilised as a dietary supplement to limit fat absorption and fat accumulation in tissues (bagchi et al., 2000). intake of grape extracts by mice resulted in reducing myocardial injury and myocardial ischemiareperfusion and decrease of superoxide anion production as well as platelet adhesion and aggregation (bagchi et al., 2000; karthikeyan et al., 2007; olas et al., 2008). in addition, procyanidins from gses have demonstrated inhibition of thrombus formation in mice after oral and intravenous administration (sano et al., 2005). moreover, anastasiadi et al. (2012) proved that extracts having abundance of flavonoids and its derivatives from grape seeds, along with the occurrence of phenolic acids, stilbenes and flavonoids from grape stems, possess antimicrobial properties. the antimicrobial activities of several non-flavonoid phenolic compounds from wine were tested, and vanillic and ga exhibited inhibitory effects towards k. pneumoniae and e. coliand (vaquero et al., 2007). it has been demonstrated that extracts from red grape seeds inhibit the growth of important human pathogens, such as e. coli, candida albicans, listeria monocytogenes and salmonella typhimurium. moreover, proanthocyanidins are indicated as dominant components in the protective prevention of inflammation mediated through the reduction of faecalibacterium prausnitzii in the intestinal lumen, leading to the blockade of inflammatory response cascade in the gut. in the central nervous system of mice, the beneficial effects of gse to modulate lipid peroxidation and oxidative damage of dna bases were observed (balu et al., 2006; feng et al., 2005; teixeira et al., 2014). for the above-mentioned reasons, a growing interest is observed in the use of grape seeds, and because the italian journal of food science, 2023; 35 (3) 25 content of selected polyphenolic substances in parts of grapevine table 1. comparison of concentrations of selected major polyphenolic compounds presented in grapevine seeds according to studies. analyte concentration (µg/g or % w/w*) analytical method concentration in studies gallic acid 10,580–105,000 hplc/dad dry weight cotea et al. (2018) 300–6,700 hplc/uv-vis residue silva et al. (2018) 3,130–3,210 hplc/dad dry weight radovanović et al. (2019) 745–2,450 hplc/dad dry weight dabetić et al. (2020) 310–270 hplc/dad extract aybastier et al. 2018 0.02–2.06* hplc/dad extract nakamura et al. 2003 210–1,250 hplc/pda dry weight bucić-kojić et al. (2009) 4,000 hplc/dad/qms extract chamorro et al. (2012) catechin 820 hplc/dad dry weight cotea et al. (2018) 7,700–17,200 hplc/uv-vis residue silva et al. (2018) 456.66–823.90 hplc/ms/qtof dry weight boso et al. (2019) 7,620–8,080 hplc/dad dry weight radovanović et al. (2019) 2,911–15,587 hplc/dad dry weight dabetić et al. (2020) 1,360 hplc/dad extract aybastier et al. (2018) 1.03–4.93* hplc/dad extract nakamura et al. (2003) 1,790–6,640 hplc/pda dry weight bucić-kojić et al. (2009) 8,000 hplc/dad/qms extract chamorro et al. (2012) 674–1,418 hplc/dad dry weight iacopini et al. (2008) epicatechin gallate 11,200–25,500 hplc/uv-vis residue silva et al. (2018) 429.86–445.20 hplc/ms/qtof dry weight boso et al. (2019) 10,340–10,600 hplc/dad dry weight radovanović et al. (2019) 948–6,269 hplc/dad dry weight dabetić et al. (2020) 790–6,200 hplc/pda dry weight bucić-kojić et al. (2009) 8,000 hplc/dad/qms extract chamorro et al. (2012) 472–2,057 hplc/dad dry weight iacopini et al. (2008) trans-resveratrol 3,940–4,990 hplc/dad dry weight cotea et al. (2018) 7.11–37.93 hplc/dad dry weight silva et al. (2018) ferulic acid 11,210–16,290 hplc/dad dry weight cotea et al. (2018) 3,100–11,800 hplc/uv-vis residue silva et al. (2018) procyanidin b1 420–1,410 hplc/dad dry weight cotea et al. (2018) 97.75–106.27 hplc/ms/qtof dry weight boso et al. (2019) 0.7–1.73* hplc/dad extract nakamura et al. (2003) 6,000 hplc/dad/qms extract chamorro et al. (2012) procyanidin b2 310–670 hplc/dad dry weight cotea et al. (2018) 141.92–149.00 hplc/ms/qtof dry weight boso et al. (2019) 7,600–7,860 hplc/dad dry weight radovanović et al. (2019) 0.66–1.54* hplc/dad extract nakamura et al. (2003) 450–5,670 hplc/pda dry weight bucić-kojić et al. (2009) 5,000 hplc/dad/qms extract chamorro et al. (2012) vanillic acid 5,360–13,020 hplc/dad dry weight cotea et al. (2018) 500–1,500 hplc/uv-vis residue silva et al. (2018) (continues) 26 italian journal of food science, 2023; 35 (3) jurasova l et al. table 1. continued. analyte concentration (µg/g or % w/w*) analytical method concentration in studies epicatechin gallate 13,880–15,500 hplc-dad dry weight radovanović et al. (2019) 20–80 hplc/pda dry weight bucić-kojić et al. (2009) 2,000 hplc/dad/qms extract chamorro et al. (2012) rutin 6.74–9.37 hplc/dad dry weight szabó et al. (2021) hplc: high-performance liquid chromatography; dad: diode-array detection; pda: photodiode array; qms: quadrupole mass spectrometry; ms: mass spectrometry; qtof: quadrupole time of flight. highest phenolic content is found in seeds, production of grape seed oil (gso) is increasing. consumption of gso is reported to be beneficial to health, especially because of its high content of phenolic compounds, unsaturated fatty acids, pigments, tocopherols and low cholesterol. in spite of relatively small presence of the mentioned compounds, gso has been determined as a food supplement with considerably positive health effects. gso and its components show health protective effects from antiradical, antioxidant and vitamin activity to a high metabolic value in the body (assumpção et al., 2016; fernandes et al., 2013). bioactive anticancer, antimutagenic and anti-lipid effects and reduction in the risk of cardiovascular diseases qualify gso to be used as a supplement in food and pharmaceutical industries (garavaglia et al., 2016; shinagawa et al., 2015). comparing gso of red and white grapes, on average, higher content of phenolic compounds was found in gso from red varieties, with the highest content found in the hamburg variety (336.3 ± 4.8 μg/g) in accordance with the study conducted by vujasinović et al. (2021), declaring higher content of phenols in gse. figure 1 shows that in spite of using the same analytical method (values of total polyphenols), the results of individual studies vary widely. table 1 shows the content of individual polyphenolic components monitored in grape seeds, depending on the analytical method used. the results of individual studies are different. these differences are due to the choice of the solvent, the standard used, different laboratory conditions, different maturity conditions, and characteristics of different varieties from different locations. an overview of the most abundant polyphenolic compounds in grape seeds—their content and extraction gallic acid gallic acid, as one of the most representative phenolic compounds, was analysed by cotea et al. (2018) using different extraction methods, attaining more than the 10th of dry weight (dw) in the fetească neagră variety. the content of ga varies from 10.58 mg/g of dw using soxhlet extraction to 105 mg/g of dw using the subcritical water extraction method. the use of 75% ethanol for extraction is more effective in comparison to water. additionally, higher pressure (15 bar) was significantly more effective than using 3-bar pressure. bucić-kojić et al. (2009), chamorro et al. (2012), dabetić et al. (2020) and radovanović et al. (2019) measured significantly lower concentrations of ga related to dry weight. radovanović et al. (2019) reported concentrations of 3.21 and 3.13 mg of ga in g of dw extracted ultrasonically for 1 h with 40 ml of solvent system consisting of methanol:acetone:water:acetic acid mixture in the ratio of 30:42:27.5:0.5. dabetić et al. (2020) described the impact of using different solvents for extraction—acidified aqueous ethanol (aae), green solvent (gs) and choline chloride:citric acid (chcit). aybastıer et al. (2018) investigated the effect of addition of 10 m hcl in a methanol:water extraction composition with no significant differences. nakamura et al. (2003) compared concentrations of ga in different health foods containing gse, and obtained different concentrations of ga. bucić-kojić et al. (2009) discovered that 50% ethanol was more efficient for ga extraction than 70% or 96% strength. catechin a high range of concentration, that is, 7,700–17,200 μg/g in residue was found by silva et al. (2018). dabetić et al. (2020) tested extraction with gs and compared it with aae. it was observed that for each variety, the extraction reagents were effective in different manners. the lowest concentrations were found for the gammay variety, where it was 3,020 μg/g for gs and 2,911 μg/g for aae, with lower but statistically insignificant difference. on the other hand, higher concentrations were obtained for the zupljanka cultivar using aae solvent (15,587 μg/g) compared with gs (10,197 μg/g). these values were statistically significant. radovanović et al.( 2019), who used ultrasonic extraction (1 h, 40 ml of solvent consisting of methanol:acetone:water:acetic acid in a ratio of 30:42:27.5:0.5), discovered the concentration of 7,620 μg/g and 8,080 μg/g for the merlot and vranac varieties. italian journal of food science, 2023; 35 (3) 27 content of selected polyphenolic substances in parts of grapevine table 2. comparison of concentrations of selected analytes in grapevine peel according to studies. analyte concentration (µg/g or % w/w**) analytical method concentration in studies resveratrol 6–255 hplc/dad dry weight iacopini et al. (2008) 4,700–8,400 hplc/uv-vis residue silva et al. (2018) 9.7 hplc/pda fresh weight ni et al. (2017) 21.5–174 hplc/dad dry weight chafer et al. (2005) 9.2–29.8 hplc/pda dry weight farhadi et al. (2016) 5.64–13.42 uhplc/dad/msms frozen sample pantelić et al. (2016) rutin 403–1,690 hplc/dad dry weight iacopini et al. (2008) 140–150 hplc/dad dry weight adovanović et al. (2019) 9,800–27,000 hplc/uv-vis residue silva et al. (2018) 15.1–54.4 hplc/dad dry weight chafer et al. (2005) 208–298 hplc/pda dry weight farhadi et al. (2016) 0.88–38.97 uhplc/dad/msms frozen sample pantelić et al. (2016) quercetin 2.9–10.07 hplc/dad dry weight iacopini et al. (2008) 40–50 hplc/dad dry weight radovanović et al. (2019) 72.1–254.7 hplc/dad dry weight chafer et al. (2005) 306–405 hplc/pda dry weight farhadi et al. (2016) 0.57–121.94 uhplc/dad/msms frozen sample pantelić et al. (2016) gallic acid 600–800 hplc/uv-vis residue silva et al. (2018) 1,360–1,400 hplc/dad dry weight radovanović et al. (2019) </=1,200 hplc/dad dry weight teixeira et al. (2014) 122–319 hplc/pda dry weight farhadi et al. (2016) 2.34–8.76 uhplc/dad/msms dry weight pantelić et al. (2016) epicatechin gallate 12,000–23,500 hplc/uv-vis residue silva et al. (2018) nd hplc/dad dry weight radovanović et al. (2019) 28–263 hplc/pda/msms fresh weight rusjan and mikulic-petkovsek (2017) 1.26–4 hplc/ms/qtof dry weight boso et al. (2019) 91.5–233.6 hplc/dad dry weight chafer et al. (2005) 232–482 hplc/pda dry weight farhadi et al. (2016) 2.95–3.56 uhplc/dad/msms frozen sample pantelić et al. (2016) 32–5,219 hplc/dad dry weight dabetić et al. (2020) protocatechic acid 2,300–7,200 hplc/uv-vis residue silva et al. (2018) 1.5–2.4 hplc/fd fresh weight teixeira et al. (2014) 0.4–0.55 uhplc/dad/msms frozen sample pantelić et al. (2016) 65–1,663 hplc/dad dry weight dabetić et al. (2020) catechin 4.83–19.65 hplc-ms-qtof dry weight boso et al. (2019) 48–178 hplc/pda/msms fresh weight rusjan and mikulic-petkovsek (2017) 1,890–2,020 hplc/dad dry weight radovanović et al. (2019) 29,800–55,800 hplc/uv-vis residue silva et al. (2018) 536–897 hplc/dad dry weight chafer et al. (2005) 567–945 hplc/pda dry weight liu et al. (2018) (continues) 28 italian journal of food science, 2023; 35 (3) jurasova l et al. table 2. continued. analyte concentration (µg/g or % w/w**) analytical method concentration in studies 3,27–7,47 uhplc/dad/msms frozen sample pantelić et al. (2016) 17–307 hplc/dad dry weight dabetić et al. (2020) myricetin gl 80–90 hplc/dad dry weight radovanović et al. (2019) 7.7–46.64 uhplc/dad/msms frozen sample pantelić et al. (2016) epigallocatechin gallate 1.9–2.42 uhplc/dad/msms frozen sample pantelić et al. (2016) hplc: high-performance liquid chromatography; uhplc: ultra high-performance liquid chromatography; dad: diode-array detection; pda: photodiode array; qms: quadrupole mass spectrometry; ms: mass spec-trometry; qtof: quadrupole time of flight; fd: fluorescence detection; gl: glucoside. bucić-kojić et al. (2009) measured catechin concentration for the frankovka variety. the authors applied three different concentrations of etoh as an extraction solvent and obtained results in the range of 1,790–6,640 μg/g, with the 50% etoh in water being the most effective concentration. aybastıer et al. (2018) measured catechin in gse (1,360 μg/g) and acid-hydrolysed gse, where it was not observed. iacopini et al. (2008), who studied more than seven varieties and clones, measured catechin in the range of 674–1,418 μg/g dw. chamorro et al. (2012) used double extraction method with methanol–water and acetone– water combination, and obtained a concentration of around 8,000 μg/g of gse. different double extraction method was used by boso et al. (2019) for the mencía (457.66 μg/g) and albariño (823.9 μg/g) varieties. a similar concentration of 820 μg/g dw was obtained through sfe by cotea et al. (2018). nakamura et al. (2003) obtained 1.03–4.93% w/w of catechin in gse. epicatechin gallate silva et al. ( 2018) measured concentration of epicatechin gallate in extract residue of the preto martinho and touriga nacional varieties (25.5 mg/g and 11.2 mg/g, respectively). comparing abundance of epicatechin gallate in dry weight, radovanović et al. (2019) reported relatively high concentrations in the merlot (10.34 mg/g) and vranac (10.60 mg/g) varieties using ultrasonic extraction. contrarily, boso et al. (2019) reported concentrations of 0.445 mg/g in albariño and 0.430 mg/g in mencía varieties using double extraction method with methanol/ water/formic acid (ratio: 50:49:1, v/v/v) and 20 ml acetone/water (ratio: 70:30, v/v). in the study conducted by dabetić et al. (2020), usage of aae for extracting epicatechin gallate led to higher efficiency, compared with the application of gs chcit. bucić-kojić et al. (2009) compared different concentrations of ethanol for extracting epicatechin gallate with the highest efficiency by using 50% ethanol at 80°c (0.62 mg/g of dw). iacopini et al. (2008) reported variance in concentration of epicatechin gallate among different varieties, with the highest concentration found in the canaiolo variety. chamorro et al. (2012) measured concentration, reaching 8 mg/g of dw and described the effect of heat treatment on antioxidant activity and polyphenolic content of grape pomace and gse. silva et al. (2018) reported relatively high concentrations of epicatechin gallate in the gse residue of the preto martinho (25.5 mg/g) and touriga nacional (11.2 mg/g) varieties. epicatechin gallate relatively high concentrations of epicatechin gallate were measured after extraction with methanol:acetone: water:acetic acid concoction (ratio: 30:42:27.5:0.5) in the merlot and vranac varieties (radovanović et al., 2019). contrarily, use of different strengths of ethanol solution (96%, 70%, 50%) reported a low effective extraction concentration of 2 mg/g of dw of epicatechin gallate in the frankovka variety (bucić-kojić et al., 2009; chamorro et al., 2012) using a mixture of methanol/water (50:50, v/v, ph = 2), followed by acetone/water (70:30, v/v) extraction. procyanidin b1 chamorro et al. (2012) measured 6,000 μg/g of procyanidin b1 in gse. cotea et al. (2018) tested subcritical water extraction with different solvents and pressures using soxhlet extraction and supercritical fluid extraction. the last was found to be the most effective method and provided a result of 1,410 μg/g dw. results of other extraction methods were from 420 to 640 μg/g dw. boso et al. (2019) studied concentration of procyanidin b1 in both albariño and mencía varieties, with respective results of 97.75 μg/g and 106.27 μg/g of dw. nakamura et al. (2003) measured the concentration of 0.7–1.73% by volume in gse. procyanidin b2 compared with other studies, which related concentration to dry weight, radovanović et al. (2019) measured the italian journal of food science, 2023; 35 (3) 29 content of selected polyphenolic substances in parts of grapevine highest concentration of 7,860 μg/g. cotea et al. (2018), who tested various extraction methods, obtained results in the range of 310–730 μg/g dw in the fetească neagră variety. bucić-kojić et al. (2009) also optimised the extraction process and used three levels of ethanol concentration to obtain results from 450 to 5,670 μg/g dw, reaching the highest concentration when 50% ethanol was used. a similar high result was obtained by chamorro et al. (2012), who determined procyanidin b2 in approximately 5,000 μg/mg of extract. nakamura et al. (2003) reported a concentration of 0.66–1.54 in volume percentage of gse. boso et al. (2019) carried out double extraction method of seeds for both albariño and mencía varieties and obtained the results of 141.92 μg/g and 149.00 μg/g dw. as mentioned above, cotea et al. (2018) tested different types of extraction methods and, in addition to the already described analytes, studied resveratrol (3,940– 4,990 μg/g dw), ferrulic acid (11,210–16,290 μg/g dw) and vanilic acid (5,360–11,220 μg/g dw). resveratrol has been also studied by szabó et al. (2021) in the pinot noir, cabernet sauvignon, syrah and blue portugal varieties, arriving at concentrations ranging from 7.11 μg/g to 37.93 μg/g. in this wide range of analytes, the authors also studied rutin, typical of grapevine seeds, for which the obtained concentration was 6.74–9.37 μg/g. silva et al. (2018) measured the concentration of vanillic acid in the residue seeds of the preto martinho (500 μg/g of residue) and touriga nacional (1,500 μg/g of residue) varieties. the concentration of preto martinho was close to the results reported by cotea et al. (2018). polyphenolic compounds in peels and pulp of grapes— distribution, extraction and effects on health content of polyphenols in grape peels and pulp is generally lower than in seeds and bunch stems. consequently, the abundance of analytes has been less investigated. resveratrol, catechin and epicatechin gallate, procyanidin derivates, hydroxycinnamic acids, flavonols, stilbenes, anthocyanins and ga are the most abundant polyphenols found in grape peels and pulp. as in the case of seeds, the content of these compounds with the ability to neutralise free radicals is highly influenced by the cultivation and genotype of the grape variety (boso et al., 2019). pantelić et al. (2016) determined hydroxybenzoic acid to be the most abundant polyphenolic compounds present in grape pulp; unequally minor quantities of the flavan 3-ols from peels of red and white varieties were also obtained. from the flavanol group, epigallocatechin gallate was only found in grape pulp. as demonstrated by different studies, flavanols are the most abundant group of phenols found in grapes peel (di lecce et al., 2014; georgiev et al., 2014; pantelić et al., 2016). from this group, rutin and quercetin were found in all peel samples in the study conducted by pantelić et al. (2016). in analysing the peels of white varieties, quercetin was the only flavonol with the highest concentration, except in the chardonnay and pinot gris varieties, where concentration of rutin was predominant. additionally, quercetin had the highest concentration in peels of red varieties, with the exclusion of cabernet franc and pinot noir, where concentration of myricetin (myr) was predominant. these findings align with the results reported by castillo-muñoz et al. (2007), who reported myricetin as a typical flavanol of red grapes and wines. comparing white and red varieties, the contents of flavan-3-ols in peels were of the same order between the groups, supported by the literature (montealegre et al., 2006; pantelić et al., 2016; peña-neira et al., 2004). in contrast, cook and samman (1996) reported that, within the flavonoids, flavan-3-ols were most abundantly found in peels and seeds and in similar concentrations, and anthocyanins were the predominant group of phenols in grape peels. khoshamad et al. (2020) showed significant differences in total polyphenols, flavonoids and anthocyanins content in grape pulp and peels of 20 wild grape varieties. in different studies, the tpc of grape berries varied widely (figures 1–4) because of environmental and genetic factors influencing the composition of grapes. hassanpour et al. (2011) also proved that tpc, as well as total anthocyanins and flavonoids, was affected by climate status in all studied varieties of grapes. the flavonoid content was higher in the varieties grown in the area with a high average annual temperature and low average annual humidity. in addition to the effect of climate, variation in tpc and anthocyanins within the varieties depended on soil composition and genetic factors. antioxidant dpph assay and fluorescence recovery after photo bleaching method proved that antioxidant activity was higher in peels than in pulp, correlating with its tpc and results of the study conducted by guo et al. (2003). correlation between tpc and radical-scavenging activities (rsa) was confirmed by analysing grape seeds and skins and was different between red and white varieties. on the other hand, no correlation was discovered between tpc and rsa in pulp extracts (pantelić et al., 2016). numerous extraction techniques are applied for the recovery of polyphenolic compounds from grape pulp and skin, including solid-liquid extraction, ultrasound-, microwave-, and enzyme-assisted extractions, etc. (tomaz et al.et al., 2019). solid-liquid extraction is the most utilized method for the recovery of phenols from grape pulp. methanol (meoh), 30 italian journal of food science, 2023; 35 (3) jurasova l et al. table 3. comparison of concentrations of selected polyphenolic componds in grapevine pulp according to studies. analyte concentration (µg/g or µg/ml*) analytical method concentration in studies resveratrol <0.1 hplc/pda fresh weight ni et al. (2017) 9.2–28.7 hplc/pda dry weight farhadi et al. (2016) nd uhplc/dad/msms dry weight pantelić et al. (2016) traces hplc/dad palomino et al. (2000) 0.00–4.29 hplc/dad dry weight marshall et al. (2012) nd hplc/dad dry weight wongnarat and srihanam (2017) 11.14–39.75 hplc/pda dry weight özcan et al. (2017) gallic acid 109–192 hplc/pda dry weight farhadi et al. (2016) 0.38–0.74 uhplc/dad/msms frozen sample pantelić et al. (2016) 0.219–2.262 hplc/dad fresh weight liu et al. (2018) 0.07–0.17 hplc/dad fresh weight topalovic and mikulic-petkovsek (2010) 370–440 hplc/dad dry weight wongnarat and srihanam (2017) 125.54–567.47 hplc/pda dry weight özcan et al. (2017) catechin 364–514 hplc/pda dry weight farhadi et al. (2016) nd uhplc/dad/msms dry weight pantelić et al. (2016) 2.39–3.74 hplc/dad fresh weight liu et al. (2018) 70–80 hplc/dad dry weight topalovic and mikulic-petkovsek (2010) 0.05–0.151 uv/vis ivanova et al. (2010) 31.39–760.081 hplc/pda dry weight özcan et al. (2017) epicatechin gallate 149–234 hplc/pda dry weight farhadi et al. (2016) nd uhplc/dad/msms dry weight pantelić et al. (2016) 0.630–2.464 hplc/dad fresh weight liu et al. (2018) 1.02–6.17 hplc/dad fresh weight topalovic and mikulic-petkovsek (2010) <50–350 hplc/dad fresh weight nile et al. (2013) 50 hplc/dad dry weight wongnarat and srihanam (2017) caffeic acid nd hplc/pda dry weight farhadi et al. (2016) 0.301–1.488 hplc/dad fresh weight liu et al. (2018) 100<<500 hplc/dad fresh weight nile et al. (2013) 30–40 hplc/dad dry weight wongnarat and srihanam (2017) 7.72–162.78 hplc/pda dry weight özcan et al. (2017) rutin 77–178 hplc/pda dry weight farhadi et al. (2016) 0.11–0.13 uhplc/dad/msms frozen sample pantelić et al. (2016) 1.267–8.074 hplc/dad fresh weight liu et al. (2018) traces hplc/dad palomino et al. (2000) 2–10 hplc/dad dry weight wongnarat and srihanam (2017) 9.46–169.26 hplc/pda dry weight özcan et al. (2017) quercetin 87–198 hplc/pda dry weight farhadi et al. (2016) nd hplc/dad dry weight pantelić et al. (2016) nd hplc/dad palomino et al. (2000) 0.00 hplc/dad dry weight marshall et al. (2012) <50–450 hplc/dad fresh weight nile et al. (2013) (continues) italian journal of food science, 2023; 35 (3) 31 content of selected polyphenolic substances in parts of grapevine table 3. continued. analyte concentration (µg/g or µg/ml*) analytical method concentration in studies 4 hplc/dad dry weight wongnarat and srihanam (2017) 35.65–131.37 hplc/pda dry weight özcan et al. (2017) protocatechuic acid 0.08–0.12 uhplc/dad/msms frozen sample pantelić et al. (2016) 0.143–0.371 hplc/dad fresh weight liu et al. (2018) epigallocatechin gallate 0.38–0.46 uhplc/dad/msms frozen sample pantelić et al. (2016) myricetin 0.00 hplc/dad dry weight marshall et al. (2012) 10 hplc/dad dry weight wongnarat and srihanam (2017) 1determination of total catechin content. hplc: high-performance liquid chromatography; uhplc: ultra high-performance liquid chromatography; dad: diode-array detection; pda: photodiode array; qms: quadrupole mass spectrometry; ms: mass spectrometry; qtof: quadrupole time of flight; fd: fluorescence detection. ethanol (etoh), acetone (ace), ethyl acetate (etac) and their aqueous solutions are the most frequently used extraction solvents for the recovery of polyphenolics from grapes  (rusjan and mikulic-petkovsek, 2017; yilmaz and toledo, 2004). enzyme-assisted extraction (eae) has been mostly recommended for the recovery of polyphenolic compounds from grape skin. in recent years, enzyme-assisted extraction has become popular because of its low cost, excellent efficiency, and environment-friendly approach (tomaz et al., 2019). in figures 2 and 3, we observe, as in figure 1, large variability in the values of total polyphenols in both pulp and peels. this variability is probably due to, as in the case of total polyphenols in seeds, interference of the folin method, different laboratory conditions and the the variety of grapes examined. however, we generally observe lower concentrations in pulp and peels than in seeds. the most abundant polyphenols in peels and pulp and their extraction resveratrol silva et al. (2018) found high concentration of resveratrol in preto martinho and touriga nacional peel residues (4,700–8,400 μg/g). charef et al. (2005) used supercritical fluid extraction for the tinto mazueleo, cabernet, boval, merlot and tempranillo varieties and obtained results in the range of 21.5–174 μg/g dw, with tempranillo being the most concentrated sample. iacopini et al. (2008) also examined different varieties and observed a similar range of concentration (6–255 μg/g dw). farhadi et al. (2016) applied ultrasonic extraction with hcl and meoh for the six varieties grown in west azerbaijan, and determined concentrations of 9.2–29.8 μg/g dw. pantelić et  al. (2016) studied 13 grape varieties for the concentration of resveratrol. they obtained results in the range of 5.64–13.42 μg/g in frozen samples, and procupac was the variety with the highest resveratrol content. ni et al. (2017) gauged a 9.7 μg/g fw concentration from the skin of kyoho grapes. rutin iacopini et al. (2008) reported relatively high concentration of rutin after a 4-h ethanol:water:hcl 0.12 m solution (ratio: 70:29:1, v/v/v) extraction from the merlot, sangiovese, cabernet sauvignon, canaiolo, colorino, foglia tonda and montepulciano varieties (0.403–1.69 mg/g of dw). farhadi et al. (2016) reported lower concentrations of rutin in dried grape seeds after ultrasonic extraction with methanol–hcl (99:1, v/v). radovanović et al. (2019) reported even lower concentrations of rutin in the merlot and vranac varieties, also using ultrasonic extraction but with a different mixture. supercritical fluid extraction is not effective for the extraction of rutin in the tinto mozueleo, cabernet, boval, merlot and tempranillo varieties (chafer et al., 2005). pantelić et al. (2016) reported significantly different concentrations of rutin in the skins of 13 serbian varieties (in the range of 0.88–38.97 μg/g of fw), with the highest concentration observed in the sangiovese variety. silva et al. (2018) reported concentration of rutin in the extract residues of the preto martinho (9.8 mg/g) and touriga nacional (27 mg/g) varieties. quercetin farhadi et al. (2016) studied concentration of quercetin in six different varieties, ranging from 306 to 405 μg/g dw, finding no major differences between them. chafer et al. (2005) studied the tinto mazueleo, cabernet, boval, merlot and tempranillo varieties, and discovered the highest concentration of quercetin in merlot (254.7 μg/g dw) and the lowest in cabernet (72.1 μg/g dw). pantelić et al. (2016) found the highest concentration in shiraz (121.94 μg/g fw) and the lowest in chardonnay (0.57  μg/g  fw), but overall there was no trend towards higher quercetin concentration in blue varieties. samples 32 italian journal of food science, 2023; 35 (3) jurasova l et al. frozen weight basis), with a concentration of 3.65 μg/g fw for the petra variety. catechin silva et al. (2018) extracted grape skin with a water:ethanol solution (ratio: 50:50) and determined concentration of catechin per 29.8–55.8 mg/g of residue. radovanović et al. (2019) studied the merlot and vranac varieties and found concentrations of 1,890 μg/g and 2,020 μg/g dw by using ultrasonic extraction method. this type of extraction was also used by farhadi et al. (2016), who assessed concentrations between 567–945 μg/g dw in five native grape cultivars of west azerbaijan province. a comparable scope of concentrations was obtained by measurements made by chafer et al. (2005; 536–897 μg/g dw), who used supercritical fluid extraction process. dabetić et al. (2020) studied 10 grape varieties and found the highest concentration of catechin (307 μg/g dw) in the skin of frankovka. rusjan and mikulic-petkovsek (2017) studied double reasonable maturation and differences in grape composition, compared with the control, and found catechin concentration in the range of 48–178 μg/g fw. pantelić et al. (2016) used extraction with methanol acidified with 0.1% hcl and obtained a result of 3.27–7.47 μg/g in frozen mass. of the 13 varieties studied, merlot had the highest catechin content. boso et al. (2019) studied the albariño and mencía varieties using double extraction method (methanol/water/formic acid and acetone/water), and found concentrations of 4.83 μg/g and 19.65 μg/g dw. protocatechic acid teixeira et al. (2014) reported concentrations of protocatechic acid (1.5–2.4 µg/g fw) in the skins of red varieties of vitis vinifera l. pantelić et al. (2016) observed lower concentrations in the range of 0.4–0.55 µg/g fw, with the highest concentration in sauvignon blanc. concentrations of protocatechic acid in the study conducted by dabetić et al. (2020) were in a wide range of 65–1,663 µg/g dw, with the highest significant concentration found in the začinak variety with chcit extraction. silva et al. (2018) reported a concentration of 7.2 mg/g in preto martinho and 2.3 mg/g in touriga nacional grape skin extract residues. myricetin radovanović et al. (2019) discovered myricetin gl in grape skin only in the concentration of 80–90 µg/g dw, but it was not observed in other grape wastes. of the 13 varieties studied, myricetin was not discovered in the chardonnay variety. contrarily, the highest concentration was reported in the sangiovese variety (46.64 µg/g fw) (pantelić et al., 2016). epigallocatechin gallate was discovered in lower concentration in the range of 1.9–2.42 µg/g fw in 13 varieties studied (pantelić et al., 2016). of merlot and vranac skins were ultrasonically extracted for 1 h with 40 ml of solvent consisting of methanol: acetone:water:acetic acid (ratio: 30:42:27.5:0.5), with the concentration of 40 μg/g and 50 μg/g dw (radovanović et al., 2019). iacopini et al. (2008) studied polyphenols in red grapes and found quercetin concentration in the range of 2.9–10.07 μg/g dw. gallic acid radovanović et al. (2019) discovered similar concentrations of ga in the merlot (1.36 mg/g dw) and vranac (1.40  mg/g dw) varieties. farhadi et al. (2016) reported ga concentration of 1.2 mg/g dw in different white varieties. use of methanol:hcl solution (ratio: 99:1, v/v) for the extraction of ga did not lead to high concentrations of ga (0.122–0.319 mg/g dw) in different varieties (muscat, hosseini, graha shira, ag shani, graha shani and ghara ghandome). moreover, silva et al. (2018) observed relatively low concentrations of ga from extract residue in comparison with other analytes in preto martinho and touriga nacional. pantelić et al. (2016) used methanol containing 0.1% hcl for extraction, reporting the concentration of ga in the range of 2.34–8.76 mg/kg of frozen samples, with the highest concentration discovered in the cabernet franc variety. epicatechin gallate the highest concentration of epicatechin gallate was discovered in skin residue (12–23.5 mg/g). water:ethanol solution (ratio: 50:50) was used for extraction (silva et al., 2018). dabetić et al. (2020) found a wide range of concentrations in 10 different varieties, from 32 to 5,219 μg/g dw, with the highest being in cabernet sauvignon. despite the fact that rusjan and mikulic-petkovsek (2017) calculated the concentration of the fresh weight of merlot trigger, the authors arrived at relatively high values compared with other varieties, that is, 28–263 μg/g. chafer et al. (2005) applied supercritical fluid extraction method to the tinto mazueleo, cabernet, boval, merlot and tempranillo varieties, and obtained results in the range of 91.5–233.6 μg/g dw, with merlot having the highest concentration of epicatechin gallate, with the same as quercetin. farhadi et al. (2016) also studied a larger number of samples for extraction; their results indicated that the lowest measured concentration corresponded to the highest measured concentration of the previous author, that is, 232 μg/g dw; their highest measured concentration was 482 μg/g dw. however, these results could be compared superficially only, because the sample treatment procedures differed. in their research, boso et al. (2019) examined two portuguese varieties, albariño and mencía, and measured concentrations of 1.26 μg/g and 4 μg/g dw by double extraction method. pantelić et  al. (2016) had a set of peels of 13 different varieties and obtained the results in the range of 2.95–3.65 μg/g (on a italian journal of food science, 2023; 35 (3) 33 content of selected polyphenolic substances in parts of grapevine gallic acid compared with grape stems, skins and seed pulp are characterised by a small amount of polyphenols, often under the detection limit. wongnarat and srihanam (2017) used triple methanol extraction and discovered slightly higher concentration in white grape pulps. özcan et al. (2017) observed ga as one of the most abundant polyphenols in grape pulps of the razaki, müşküle and cardinal varieties. farhadi et al. (2016) performed ultrasonic extraction of pulp polyphenols using hydrochloric acid in methanol as an extraction solvent, and discovered ga concentration ranging from 109 μg/g fw in the muscat variety to 192 μg/g fw in the ghara shani variety. of the 30 analysed varieties, ga was found to be the most abundant polyphenolic compound in grape pulp, with the highest concentration discovered in the pearl black grape variety (2.262 μg/g fw) (liu et al., 2018). topalovic and mikulic-petkovsek (2010) analysed concentrations of ga in pulps using the maturation process, with peak concentration found in the earlier stages and constant values in the following maturation stages. pantelić et al. (2016) confirmed the high levels of ga in the pulp of different grape varieties, with the highest concentration in the pinot noir variety (0.74 μg/g fw). resveratrol as reported by different studies, the concentration of resveratrol in grape pulp is a compound balanced around discovered limit values. resveratrol was not found in the selected white and blue varieties (wongnarat and srihanam, 2017). palomino et al. (2000) discovered only traces of this compound, and ni et al. (2017) noticed a concentration of <0.1 μg/g fw in the kyoho grape variety. marshall et al. (2012) discovered a concentration of 4.29 μg/g dw in the janet variety, but resveratrol was not found in the majority of varieties examined. farhadi et al. (2016) measured concentrations in the range of 9.2– 28.7 μg/g dw using ultrasonic extraction, and özcan et al. (2017) reported concentrations between 11.14–39.75 μg/g dw, with the highest concentration in the razaki variety. catechin as in the case of other analytes, high concentrations of catechin were observed after ultrasonic extraction, the highest in the ghara shani variety (514 μg/g dw) (farhadi et al., 2016). özcan et al. (2017) observed significantly different concentrations of catechin in grape pulp differing in variety and harvest time. wongnarat and srihanam (2017) found similar concentrations between white and blue varieties in the range of 70–80 μg/g dw. topalovic and mikulic-petkovsek (2010) observed concentrations in the range of 2.39–3.74 μg/g fw during different stages of ripening. ivanova et al. (2010) reported concentrations of total catechin using the spectrometric method, extracting twice for 15 min with 10-ml acetone: water solution (ratio: 80:20, v/v) containing hcl (ratio: 0.1:10, v/v). epicatechin gallate nile et al. (2013) studied epicatechin gallate in 20 grape cultivars, and the range of concentration determined was around 50–350 μg/g in the lyophilised sample. of the six examined varieties, epicatechin gallate was less abundant than catechin, with the highest concentration in the ghara shani variety (234 μg/g dw; farhadi et al., 2016). wongnarat and srihanam (2017) discovered the same concentration of 50 μg/g dw in both white and blue varieties using the triple methanol extraction method. of the 30 varieties examined, the highest concentration of epicatechin gallate was found in the black grape variety, that is, 2.464 μg/g fw (liu et al., 2018). topalovic and mikulicpetkovsek (2010) also found micrograms of epicatechin gallate in 1 g of fresh sample and observed significantly increased synthesis of epicatechin gallate in samples collected in the early stages of ripening. pantelić et al. (2016) did not discover epicatechin gallate in the pulp because either it was not present or was at a concentration below the detection limit. rutin liu et al. (2018) used ultrasonication to gain pulp extracts and measure rutin in six varieties, with results in the range of 77–178 μg/g dw. özcan et al. (2017) extracted rutin with a mixture of methanol, water and formic acid from three varieties, and discovered its concentration between 9.46 μg/g and 169.26 μg/g dw, with the highest concentration in the razaki variety. surprisingly, wongnarat and srihanam (2017), and liu et al. (2018) discovered similar concentrations. wongnarat and srihanam (2017), who studied one white and one blue variety and used triple extraction method, discovered a concentration of 2–10 μg/g dw. liu et al. (2018) converted the concentration of this analyte to frozen weight and accessed a concentration of 1.267–8.074 μg/g, also using three extractions. pantelić et al. (2016) assessed a narrow range of concentrations of 0.11–0.13 μg/g fw in 13 varieties (cabernet sauvignon, merlot, cabernet franc, sangiovese, shiraz, pinot noir, prokupac, riesling, petra, sauvignon blanc, welschriesling, chardonnay and pinot gris). trace amount of rutin in grape flesh was found by palomino et al. (2000). quercetin nile et al. (2013) studied 20 grape cultivars for quercetin concentration, and the range was less than 50–450 μg/g of lyophilised sample. farhadi et al. (2016) measured quercetin in the pulp of five native west azerbaijan varieties and one international variety (muscat alexandera), with the results ranging from 87 to 198 μg/g dw. the highest value was found in the ghara shira variety. as mentioned 34 italian journal of food science, 2023; 35 (3) jurasova l et al. above, the authors used ultrasonic extraction. özcan et  al. (2017) studied the effect of harvest time on physicochemical properties and bioactive compounds, and in the case of rutin discovered the range of 35.65–131.37 μg/g dw in the pulp of three different varieties. the concentration of 4 μg/g dw was found in the white variety by wongnarat and srihanam (2017). the authors used the triple extraction of pulp from one red and one white variety. quercetin was not observed in the pulp of the red variety because it was either not present in this variety or its concentration was under the detection limit. the same reasons could have led to the results reported by other studies (marshall et al., 2012; palomino et al., 2000; pantelic et al., 2016). liu et al. (2018) studied 30 different grape varieties and discovered protocatechuic acid in 11 samples in the range of 0.143–0.371 μg/g fw. the authors used six-fold extraction. the first two extractions were carried out with tetrahydrofuran. the residue was mixed with acidified methanol for two times. the residue was then hydrolysed, and fatty acids were removed with n-hexane. the remainder was extracted twice with a 5-ml mixture of diethyl ether and ethyl acetate. pantelić et al. (2016) used a slightly simpler extraction method, a triple extraction with methanol acidified with hcl. their results appeared to be lower than the results of liu et al. (2018), but this was not stated with certainty because they related the concentration to the weight of the frozen sample. in 13 varieties studied, pantelić et  al. (2016) measured protocatechuic acid in the range of 0.08– 0.12 μg/g. the highest value belonged to cabernet franc. the authors also measured epigallocatechin gallate (0.38– 0.46 μg/g in the frozen sample), with the highest value being for the petra variety; myricetin was not detected in any of the studied varieties. marshall et al. (2012) also did not find myricetin in several clones of muscadines. wongnarat and srihanam (2017), who used triple methanol extraction, observed 10 μg/g dw of myricetin. polyphenols in stems—characteristics, distribution and effect on health grape stems represent 1.4–7.0% of the raw material and 25% of the winemaking by-products, and polyphenols represent 5.8% of dry weight. the grape stem is defined by the abundance of crude fibre and protein, nitrogen free extracts and antioxidant properties. water content in grape stems varies in the range of 55–80%, depending on the grape variety, and the dry matter contains 71% of alcohol-insoluble residues. no differences were reported between red and white varieties (gonzález-centeno et al., 2012; prusova et al., 2020). polyphenols content was studied in grape seeds, skins, must and wines. polyphenols are stored in every part, including stems, except the berry. however, many studies have proved that stem extract contains lower amount of polyphenols and showed lower antioxidant activity, compared with the other parts of the vine (castillo-muñoz et  al., 2007; gonzalez-centeno et al., 2012; nassiri-asl and hosseinzadeh, 2016). wenzel et al. (2015) observed higher tpc, expressed as the amount of ga per dry sample, and related antioxidant activity in seed extracts in comparison with stem and peel extracts, compared with the study conducted by hanušovský et al. (2020). hanušovský et al. (2020) studied samples of three varieties—zweigelt red-skinned, pinot blanc white-skinned and green veltliner whiteskinned, obtained from six different locations (nitra and vienna wine regions). it was discovered that stem tpc was higher than that of other parts of a bunch or the grape pomace in samples from both regions. also, grape stem extracts possessed the highest antioxidant activity. in addition, a higher abundance of lignin increased this property. the steam also confirmed the influence of different grape varieties as well as extraction methods on tpc along with the effect of temperature on extraction (hanušovský et al., 2020; wenzel et al., 2015). the study conducted on flavanol content in the albariño variety showed the greatest abundance of flavanols in bunch stems, with lower quantity in grape peels, must and wine. in the stem bunch, analyses confirmed catechin as the dominant flavanol and high concentration of procyanidin b1 and its derivates (boso et al., 2019). this observation correlates with the results of püssa et al. (2006), who observed a significantly higher content of polyphenols in bunch stems of red grapes, compared with white varieties. moreover, the analysis also determined higher content in anthocyanins in the bunch stems of red varieties. on the other hand, a study conducted by gonzalezcenteno et al. (2012) on the bunch stems of red (cabernet sauvignon, callet, manto negro, syrah and tempranillo) and white (chardonnay, macabeu, parellada and prebsal blanc) varieties did not show typical differences in flavanol content. therefore, it could be argued that tpc is affected more by other factors than the classification of grapes being white or red. this needs to be supported by the studies observing different cultivating factors on the same varieties (boso et al., 2019). the study on the composition of polyphenols in grape stems showed high levels of flavan-3-ols, hydroxycinnamic acids, flavonols (monomeric and oligomeric) and stilbenes (cao and ito, 2003; karvela et al., 2009). (an investigation on factors affecting recovery of antioxidant phenolics and anthocyanins from red grape). several studies confirmed italian journal of food science, 2023; 35 (3) 35 content of selected polyphenolic substances in parts of grapevine table 4. comparison of concentrations of selected analytes in grapevine stems according to studies. analyte concentration (µg/g or µg/ml*) analytical method concentration in studies quercetin >70 hplc/uv-vis extract esparza et al. (2020) 8–38 hplc/dad dry weight jiménez-moreno et al. (2019) 2–21 hplc/ms/ms dry weight anastasiadi et al. (2012) 13–108 hplc/dad extract esparza et al. (2020) 120–140 hplc/dad dry weight radovanović et al. (2019) 0.041–0.215* hplc/dad extract prusova et al. (2020) gallic acid >150 hplc/uv-vis extract esparza et al. (2020) 43–310 hplc/dad dry weight jiménez-moreno et al. (2019) 70–469 hplc/ms/ms dry weight anastasiadi et al. (2012) 10,500–11,500 hplc/uv-vis residue silva et al. (2018) 32,960 hplc/dad dry extract apostolou et al. (2013) 1,430–1,580 hplc/dad dry weight radovanović et al. (2019) 120–1,290 hplc/dad extract esparza et al. (2020) 0.822–4.015 hplc/dad extract prusova et al. (2020) catechin 225–710 hplc/dad dry weight jiménez-moreno et al. (2019) 385–1,858 hplc/ms/ms dry weight anastasiadi et al. (2012) 900–3,500 hplc/dad extract esparza et al. (2020) 29,300–38,700 hplc/uv-vis residue silva et al. (2018) 157.57–1201.00 hplc/ms-qtof dry weight boso et al. (2019) 2,310–2,550 hplc-dad dry weight radovanović et al. (2019) 18.398–78.930* hplc/dad extract prusova et al. (2020) epicatechin gallate 12.3–189 hplc/ms/ms dry weight anastasiadi et al. (2012) 1.742–33.589* hplc/dad extract prusova et al. (2020) 7.04 hplc/ms-qtof dry weight boso et al. (2019) 2,460–2,600 hplc/dad dry weight radovanović et al. (2019) 15,500 hplc/uv-vis of residue silva et al. (2018) resveratrol 10–370 hplc/dad extract esparza et al. (2020) 74–266 hplc/ms/ms dry weight anastasiadi et al. (2012) >250 hplc/uv-vis extract esparza et al. (2020) 21–162 hplc/dad dry weight jiménez-moreno et al. (2019) 2,150–25,410 hplc/uv-vis dried extract sahpazidou et al. (2014) quercetin-3-glucoside 240–1,500 hplc/dad extract esparza et al. (2021) 54.1–137 hplc/ms/ms dry weight anastasiadi et al. (2012) >800 hplc/uv-vis extract esparza et al. (2020) 96–485 hplc/dad dry weight jiménez-moreno et al. (2019) 1.783–11.158* hplc/dad extract prusova et al. (2020) e-viniferin 150–690 hplc/dad extract esparza et al. (2021) >500 hplc/uv-vis extract esparza et al. (2020) 91–310 hplc/dad dry weight jiménez-moreno et al. (2019) 167–499 hplc/ms/ms dry weight anastasiadi et al. (2012) 170–760 hplc/dad dry weight leal et al. (2020) hplc: high-performance liquid chromatography; uhplc: ultra high-performance liquid chromatography; dad: diode-array detection; pda: photodiode array; qms: quadrupole mass spectrometry; ms: mass spec-trometry; qtof: quadrupole time of flight; fd: fluorescence detection. 36 italian journal of food science, 2023; 35 (3) jurasova l et al. trans-caftaric acid as the phenol with highest concentration in both white and red varieties (anastasiadi et  al., 2012; apostolou et al., 2013). the analysis of flavanols in the stems of red varieties showed the presence of diverse flavonols, with a high abundance of quercetin derivatives and the highest content of quercetin-3-o-glucuronide (negro et al., 2003; souquet et al., 2000). in addition, quercetin-3-o glucoside, quercetin-3-o-galactoside and quercetin 3-o-rutinoside were the most abundantly discovered flavanols (apostolou et al., 2013; souquet et al., 2000). comparison of red and white variety stems showed similar characterisation of flavanols, but with much higher flavanol content in red varieties. catechin is the most abundant of all the flavan-3-ols, in both white and red varieties, with the highest concentration in grape stems, followed by skins and seeds. within white varieties, the analysis also showed that catechins are the most concentrated flavan-3-ols in all tissues, with the highest quantity found in grape stems. analysis of stilbenes in grape residues from red varieties confirmed the presence of their derivatives in stems, seeds, pomaces and leaves. contrarily, stilbenes have been found only in skins and stems in white varieties (anastasiadi et al., 2012; apostolou et al., 2013; di lecce et al., 2014; rockenbach et al., 2011). nowadays, grape stems as a winemaking by-product often represent undervalued material and a waste, a problem for the environment. the rich content of bioactive compounds potentiates grape stems as a prospective material for the introduction of added-value products. grape stems are widely used for the production of alcoholic beverages, dietary fibre, plant protein supplements, animal feed and fertilisers (arvanitoyannis et al., 2006), but bioactive compositions remain poorly defined. the transformation of agro-food wastes into products with added value has caught the attention of the food and pharmaceutical sectors (martins et al., 2011). owing to the presence of proanthocyanidins, grape stems and grape clusters are a source of compounds causing excessive astringent taste and influencing organoleptic properties of wine. therefore, they are removed before the vinification process, but the usage of this waste is being discussed intensively. the usage of grape waste as a material source for food production could lead to a replacement of intake of synthetic antioxidants with adverse effects. however, bioactive compounds contained in the vine and their impact on the human and animal health has to be investigated in detail. the chemical composition of grape stems, along with grape variety and growing conditions, strongly influence extraction processes. domínguez-perles et al. (2014) compared the conditions to increase the effectiveness of phenol extraction as determined by response surface methodology. performing experiments on grape stems of greek varieties, lower extraction temperature led to a 34% increase in extracted phenols. compared with pomace and the whole bunch, abts showed significantly higher (p < 0.01) antioxidant activity in grape stems. on the other hand, in slovak samples, dpph assay did not show significant differences in the antioxidant activities of grape by-products. in slovakia and austria, grape stems had significantly fewer (p < 0.01) proteins in comparison with grape pomace and bunch. compared with grape pomace and bunch, tpc analysis showed significantly (p < 0.01) higher content of grape stem in the samples of both countries. the comparison of slovak and austrian wine by-products was characterised by similar nutrition content, condensed tannins and tpc as well as antioxidant activity. gouvinhas et al. (2020) described the effect of climate and altitude on the production of phenols in grape stems. the authors discovered increased numbers of phenols, orthodiphenols and flavonoids in the grape stems cultivated in low altitude areas (lower corgo sub-region). this region is characterised by stressful vine conditions and represented by heavy rains caused by the atlantic ocean and thermal stress. plants respond to stress by synthesising secondary metabolites, including phenols. the impact of thermal stress on these metabolites in the vine was evident during the 2017 and 2018 seasons. the results demonstrated that altitude was a determining factor for the content of polyphenolics. though fluctuating levels of phenols in stems were observed, this by-product is a potential source of phenols. moreover, as in grape seed extracts, antimicrobial activity was observed in grape stem extracts against gastrointestinal tract bacteria s. aureus and e. faecalis. additionally, anastasiadi et al. (2012) described the antimicrobial activity of grape extracts caused by the high abundance of flavonoids, phenolic acids and stilbenes in stems along with flavonoids and their derivatives in seeds. this graph visually shows the least heterogeneous results in terms of percentage values. the rationale could be that the amount of phenolic compounds in the trefoil is least burdened by grapevine species. however, as mentioned above, the folin method is highly burdened by interference, so we cannot prove this claim. moreover, there are still noticeable differences over 100 mg/g. italian journal of food science, 2023; 35 (3) 37 content of selected polyphenolic substances in parts of grapevine catechin concentration was measured by anastasiadi et al. (2012; 385–1,858 µg/g), karvela et al. (2009; 900– 3,500 µg/g), radovanović (2019; 2,310–2,550 µg/g) and boso et al. (2019; 1,201 µg/g). the extraction method and solvent used differed depending on the study. for example, a mixture of methanol, water and some organic acid was applied, or a mixture of ethanol and water. boso et al. (2019) used a two-step extraction method, but their results were still comparable with others (1,201 µg/g of catechin in stems from red variety). however, boso et al. (2019) had measured a concentration of 157.57 µg/g dw in the stems of the white variety. close to this value were the results of jiménez-moreno et al. (2019), who used different ethanol concentrations for extraction and found results in the range from 225–710 µg/g dw. considering these results, we concluded that the extraction method was not a strong factor in the comparison of results. the lowest concentration was found by prusova et al. (2020) in fresh weight. epicatechin gallate the highest concentration of epicatechin gallate was determined by silva et al. (2018; 15.5 mg/g), possibly because of assessing extract residue. this was followed by an epicatechin gallate concentration of 2.6 mg/g by radovanović et al. (2019); these authors used ultrasonic extraction with a solvent consisting of methanol, acetone, water and acetic acid. the respective concentration of epicatechin gallate discovered by boso et al. (2019), prusova et al. (2020), and anastasiadi et al. (2012) was 12.3–189 µg/g dw, 1.742–33.589 mg/l of extract and 7.04 µg/g dw. resveratrol anastasiadi et al. (2012) and jiménez-moreno et al. (2019) reported the concentration of resveratrol as 74–266 µg/g and 21–162 µg/g dw, respectively. jiménezmoreno et al. (2019) investigated the influence of three process parameters on extraction: ethanol concentration, extraction temperature and solid/solvent ratio, observing a wide variety of results of all analytes. the most effective extraction used 50% ethanol as solvent, a temperature of 40°c and a 1:50 solid/solvent ratio. esparza et al. (2020, 2021) analysed the concentration of resveratrol directly from grape stem extract with comparable results. comparing the assyrtiko, mavrotragano, voidomato and muscat varieties, sahpazidou et al. (2014) reported the lowest concentration in the white variety assyrtiko (2,150 µg/g of extract), and the highest abundance in the red variety voidomato (25,410 µg/g of extract). e-viniferin in contrast with other analytes, the value of ε-viniferin was found to be the same throughout different studies (esparza et al., 2020, 2021; jiménez-moreno et al., 2019). analysing dry weight, the abundance of ε-viniferin was in the range of 91 µg/g dw of stem powder of the mazuleo the most abundant polyphenols in grape stems and their effect on health gallic acid the highest amount of ga in grape stems was reported by apostolou et al. (2013), that is, 32,960 µg/g of dry extract. the mazuelo variety was studied by jiménezmoreno et al. (2019), whose results were in the range of 43–310 µg/g dw. they used solvent extraction with five levels of ethanol concentration, two ratios of solid and solvent, and two levels of extraction temperature. this could be the reason for the high scatter of results. ga in mazuelo stem extracts was also studied by esparza et al. (2020). the authors showed the result of a measured concentration higher than 150 µg/mg, which matched their results of 120–1,290 µg/g of extract from different spanish varieties, including mazuelo (esparza et al., 2020). ordinarily, the same results were measured in mandilaria, mavrotragano, voidomatis, asyrtiko, athiri and aidani in the range 70–469 µg/g dw. quite higher results were discovered by radovanović et al. (2019) using extraction with meoh/h2o/hcl (1,430–1,580 µg/g dw) and anastasiadi et al. (2012) (10.5–11.5 mg/g of residue). the lowest concentration of 0.822–4.005 µg/ml of extract was measured by prusova et al. (2020). the reason for different results could be the extraction method. moreover, prusova et al (2020) worked with fresh material. as mentioned above, stems contain up to 80% water. low concentration of ga in the range of 0.013–0.024 µg/g dw in grape stems was discovered by teixeira et al. (2018). esparza et al. (2020) reported a quercetin concentration of >0.07 µg/ml of extract from dried powdered stems, and observed the impact of light and temperature on the stability of phenolic compounds during storage. prusova et al. (2020) measured a concentration of 0.041–0.215 µg/ml of quercetin extract from fresh stems of 10–12-year-old vines. relatively small concentrations were due to its calculation of analyte in diluted stem extract. contrarily, radovanović et al. (2019) anastasiadi et al. (2012) and jiménez-moreno et al. (2019) reported quercetin concentrations of 8–38 µg/g, 2–21 µg/g and 120–140 µg/g of dw, respectively, and the highest concentrations were observed in the merlot and vranac varieties. vines were cultured in serbia, and dried, milled, and phenols were extracted using ultrasound-assisted extraction with a mixture of methanol:acetone: water:acetic acid (ratio: 30:42:27.5:0.5) (radovanović et al., 2019). catechin the highest catechin concentration was measured by silva et al. (2018) in dry extract residue (29.3–38.7 mg/g). 38 italian journal of food science, 2023; 35 (3) jurasova l et al. determine the most studied analytes. we observed variations in the determination of total polyphenolic compounds in figures 1–4. these variations could be due to the use of different solvents or the strong influence of interferences, such as carbohydrates, in the method used. however, it is clear that the higher proportion of phenolic compounds is found in seeds and stems, compared with lower proportions in the fruit peel. in general, the pulp of the berry contains the least proportion of phenolic compounds. funding this paper was supported by the project “study of polyphenolics compounds in wines and parts vines” iga-zf/2021-si2009, and by cz.02.1.01/0.0/0.0/ l6_0l7/0002334 research infrastructure for young scientists, co-financed by operational programme research, development and education. references abarghuei, m.j., rouzbehan, y. and alipour, d. 2010. the influence of the grape pomace on the ruminal parameters of sheep. livest sci. 132(1–3): 73–79. https://doi.org/10.1016/j.livsci.2010.05.002 anastasiadi, m., pratsinis, h., kletsas, d., skaltsounis, a.-l. and haroutounian, s.a. 2012. grape stem extracts: polyphenolic content and assessment of their in vitro antioxidant properties. food sci tech (lwt). 48(2): 316–322. https://doi.org/10.1016/j. lwt.2012.04.006 apostolou, a., stagos, d., galitsiou, e., spyrou, a., haroutounian, s., portesis, n., et al. 2013. assessment of polyphenolic content, antioxidant activity, protection against ros-induced dna damage and anticancer activity of vitis vinifera stem extracts. food chem toxicol. 61: 60–68. https://doi.org/10.1016/j.fct.2013.01.029 arvanitoyannis, i.s., ladas, d. and mavromatis, a. 2006. potential uses and applications of treated wine waste: a review. int j food sci tech. 41(5): 475–487. https://doi. org/10.1111/j.1365-2621.2005.01111.x assumpção, c.f., nunes, i.l., mendonça, t.a., bortolin, r.c., jablonski, a., flôres, s.h. et al. 2016. bioactive compounds and stability of organic and conventional vitis labrusca grape seed oils. j am oil chem soc. 93(1): 115–124. https://doi. org/10.1007/s11746-015-2742-0 aybastier, o., dawbaa, s. and demir, c. 2018. investigation of antioxidant ability of grape seeds extract to prevent oxidatively induced dna damage by gas chromatography-tandem mass spectrometry. j chromatogr b anal tech biomed life sci. 1072, 328–335. https://doi.org/10.1016/j.jchromb.2017.11.044 bagchi, d., bagchi, m., stohs, s.j., das, d.k., ray, s.d., kuszynski,  c.a., et al. 2000. free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention. toxicology. 148(2–3): 187–197. https://doi. org/10.1016/s0300-483x(00)00210-9 variety to 310 µg/g dw using different extraction methods (jiménez-moreno et al., 2019). esparza et al. 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seed proanthocyanidin extract in broiler chickens: effect on chicken coccidiosis and antioxidant status. poult sci. 87(11): 2273–2280. https://doi.org/10.3382/ ps.2008-00077 war, a.r., paulraj, m.g., ahmad, t., buhroo, a.a., hussain, b., ignacimuthu, s., et al. 2012. mechanisms of plant defense https://doi.org/10.4161/psb.21663� https://doi.org/10.1007/s13197-016-2286-9� https://doi.org/10.1007/s13197-016-2286-9� https://doi.org/10.1002/fsn3.246� https://doi.org/10.1002/fsn3.246� https://doi.org/10.13005/ojc/330112� https://doi.org/10.13005/ojc/330112� https://doi.org/10.5713/ajas.2011.11189� https://doi.org/10.5713/ajas.2011.11189� https://doi.org/10.1021/jf030117h� https://doi.org/10.1021/jf030117h� https://doi.org/10.1016/0377-8401(94)90107-4� https://doi.org/10.1016/0377-8401(94)90107-4� https://doi.org/10.1016/b978-0-12-813768-0.00026-8� https://doi.org/10.1016/b978-0-12-813768-0.00026-8� https://doi.org/10.1016/j.foodcont.2005.08.010� https://doi.org/10.1016/j.foodcont.2005.08.010� https://doi.org/10.3989/gya.0222201� https://doi.org/10.3382/ps.2008-00077� https://doi.org/10.3382/ps.2008-00077� _hlk127354910 _hlk127198813 _hlk126935913 _hlk126935922 _hlk120804126 _hlk127204634 _hlk120805341 _hlk127367701 _hlk126935987 _hlk120804975 p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33isp1.1995 43 change in the lifestyle of consumers, climate changes, and the accompanying rapid changes in food systems. international trade means that unsafe food can be distributed widely (pinu, 2016; who, 2020). recently, many food-borne disease outbreaks in the world, the most p u b l i c a t i o n s codon dnafoil, a novel technology for the rapid detection of food pathogens: preliminary validation on salmonella and listeria monocytogenes aly farag el sheikha1,2,3,4,5 1college of bioscience and bioengineering, jiangxi agricultural university, 1101 zhimin road, nanchang 330045, china; 2school of nutrition sciences, faculty of health sciences, university of ottawa, 25 university private ottawa, on k1n 6n5, canada; 3bioengineering and technological research centre for edible and medicinal fungi, jiangxi agricultural university, 1101 zhimin road, nanchang 330045, china; 4jiangxi key laboratory for conservation and utilization of fungal resources, jiangxi agricultural university, 1101 zhimin road, nanchang 330045, china; 5department of food science and technology, faculty of agriculture, minufiya university, 32511 shibin el kom, minufiya government, egypt *corresponding author: aly farag el sheikha, college of bioscience and bioengineering, jiangxi agricultural university, 1101 zhimin road, nanchang 330045, jiangxi, china. email: elsheikha_aly@yahoo.com received: 1 january 2021; accepted: 4 april 2021; published: 19 april 2021 © 2021 codon publications open access original article abstract over the past decades, several tools have been developed for food pathogen detection, including proteomics, metabolomics, immunological, biosensor, and nucleic acid-based approaches. although these techniques are reliable and precise, they are time-consuming, technically challenging, and costly. hence, it is necessary to develop rapid techniques for food pathogen detection, which can be performed at the household level. dnafoil mechanism is a portable, completely self-administered, on-site dna test that does not need expensive instruments or settings to confirm food pathogen detection in as little as 30 min. dnafoil was used successfully for detecting food contamination and adulteration with pork derivatives (down to 0.1%) and vegetal components (down to 0.01%), respectively. in this study, initial validation experiments of dnafoil were investigated to detect listeria monocytogenes and salmonella contamination. to confirm the specificity of the proposed method toward salmonella, 18 different salmonella strains, 6 non-salmonella bacteria, and 2 fungi were investigated; also, in the case of listeria monocytogenes, five bacterial strains, two fungi, and listeria monocytogenes were investigated. the results stated that the swiss decode salmonella and l. monocytogenes solutions can detect as few as 1 and 10 copies of dna per microliter, respectively. the results also showed that the accuracy of our method ranges between 92 and 100%, while the precision value ranged between 88 and 100%. in terms of quality control applicability, dnafoil salmonella and listeria monocytogenes reactions could be visually detected with the naked eye using a lateral flow strip, which could be used for in-place quality control during manufacturing and also can be used for more lab tests. in terms of cost, dnafoil is usually much cheaper than the traditional detection methods. therefore, dnafoil is proposed as a promising and universal detection technology for food pathogens. keywords: dnafoil technology; food pathogen detection techniques; food safety; foodborne diseases; health and economics threats introduction it is well-known that food safety is affected by many factors and variables, including, for example, globalization of food trade, population increase in the world, italian journal of food science, 2021; 33 (sp1): 43–54 mailto:elsheikha_aly@yahoo.com 44 italian journal of food science, 2021; 33 (sp1) el sheikha af salmonella tops the list of foodborne pathogens, with a treatment cost equivalent to $3.6 billion, followed by listeria monocytogenes, which equals $2.8 billion, and then escherichia coli with a value of $271 million (usda ers, 2014). this is in addition to the cost of recalls of the products as well (tyco integrated security, 2012). hence, there is an urgent need to develop simple, sensitive, specific, robust, reliable, inexpensive, and rapid techniques for food pathogen detection, can perform at the household level and ensure food safety. these requirements comply with new, portable, completely self-administered, on-site dna test technology called “dnafoil technology,” which does not need expensive equipment or laboratory settings to get the final results in as little as 30 min (el sheikha, 2019). additionally, dnafoil technique has proven to be effective in: • detecting food contamination through its ability to detect pork contamination in beef as lower as 0.1% (meat and livestock australia limited “mla”, 2018); • detecting of food adulteration through its ability to detect the adulteration of milk products by vegetal materials as lower as 0.01% (aronoff et al., 2018). through five steps (figure 1), it can be clearly understood as to how this technique works. sample preparation prominent of which were due to several microbial species, for example, listeria monocytogenes, escherichia coli, campylobacter jejuni, salmonella sp., shigella sp., have proven that food safety is under severe threat from food pathogens (bintsis, 2017; chlebicz and śliżewska, 2018; faour-klingbeil and todd, 2020). unsafe food containing food pathogens, that is, bacteria, viruses, parasites, or fungi, can cause different diseases ranging from diarrhea to cancers (dwivedi and jaykus, 2011; food safety education program, 2016; who, 2020). centers for disease control and prevention (cdc) estimates that each year 48 million people get sick from foodborne diseases, 128,000 are hospitalized, and 3000 die. foodborne pathogens cause diseases and deaths in all populations, particularly in groups at risk such as infants, children, elderly, and immunocompromised persons (cdc, 2020; fda, 2021; who, 2020, 2021). the most common microorganisms responsible for the major foodborne illnesses are shown in table 1. in addition to the severe health risks caused by foodborne illnesses, they may also threaten international trade and cause significant economic losses. this has been confirmed by the reports received from the usda’s economic research service. these reports indicated that foodborne illnesses cost the united states more than $15.6 billion. table 1. the most common microorganisms responsible for the major foodborne illnesses. foodborne illness or toxin associated microorganism health risks most population group(s) affected reference listeriosis listeria monocytogenes meningitis, mild illness in pregnant women, in babies (miscarriage, stillbirth, premature birth, potentially fatal infection after birth) pregnant women, newborns, the elderly, immuno-compromised individuals buchanan et al. (2017), mayo clinic (2020a) salmonellosis salmonella spp. typhoid fever, inflammatory bowel disease, stomach or bowel disorders all groups bintsis (2017), mayo clinic (2019) shigellosis shigella spp. dehydration, seizures, rectal prolapse, hemolytic uremic syndrome, toxic megacolon, reactive arthritis, bloodstream infections (bacteremia) malnourished children, immuno-compromised individuals, the elderly ncbi (2017), mayo clinic (2020b) campylobacteriosis campylobacter spp. mild to severe diarrhea, bloody diarrhea, stomach pain, cramps, nausea and/or vomiting, fever, muscle pain all groups bintsis (2017), ontario ministry of health and long-term care (2020) botulism clostridium botulinum breathing problems, trouble swallowing, muscle weakness, slurred speech, headache, nausea all groups bintsis (2017), rasettiescargueil (2020) toxoplasmosis toxoplasma gondii headache, seizures, lung problems, severe eye infections, e.g., inflammation of retina, enlarged liver and spleen all groups, especially babies, immunocompromised individuals efsa and ecdc (2016), mayo clinic (2020c) yersiniosis yersinia spp. fever, abdominal pain, diarrhea (which is often bloody) all groups, especially children, adults efsa and ecdc (2016), ontario ministry of health and long-term care (2018) amoebiasis entamoeba histolytica bowel perforation, gastrointestinal bleeding, stricture formation, intussusception, peritonitis, empyema all groups ncbi (2016), park (2015) italian journal of food science, 2021; 33 (sp1) 45 dnafoil is a promising detection technology anatum, salmonella newport, salmonella thyphimurium, salmonella arizonae iiib, salmonella saintpaul, salmonella hadar, salmonella enteritidis abony); (ii) for exclusivity (listeria monocytogenes, bacillus subtilis, enterococcus faecalis, escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, aspergillus brasiliensis, candida albicans). secondly, the strains tested for inclusivity and exclusivity toward listeria monocytogenes: (i) for inclusivity (listeria monocytogenes); (ii) for exclusivity (bacillus subtilis, enterococcus faecalis, escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, aspergillus brasiliensis, candida albicans). all experiments involving living strains were conducted under bsl2 conditions at the independent microbiology lab in couternon, france. sample preparation, dna extraction, and amplification stages dnafoil mechanism is depicted in figure 1. steps 1 and 2 show the dna extraction, lysing, neutralizing, and stabilizing processes of 200 μl of culture using a barrel without the need to use spin-columns and centrifuges. briefly, bacteria cells are broken by an alkaline solution that contains chaotropic salts. this allows dna to be released in the solution. the alkaline ph of the solution is not compatible with downstream dna amplification; therefore, a neutralization/stabilization step is added, and it consists of buffering the ph with a second solution, which also provides monovalent salts that facilitate the dna amplification reaction. amplification stage of dna target is started via step 3; one drop of extracted dna is transferred to the reaction tube and is incubated in a water bath at 65°c. then, using the specific primers and enzymes, the dna targets will amplify and make multiple copies without using thermos-cycling and cold chain. end-point assays after 30 min of incubation at 65°c, dnafoil strips were dipped into the reaction tube (detection step; 4th step). migration by lateral-flow caused a positive band’s appearance due to colloidal gold concentrating on the dna capture line without using electrophoresis and staining (detection step; 5th step). real-time confirmatory assays for real-time assays, 1 μl of extract was combined with 24 μl of salmonella and listeria monocytogenes reactions mix and incubated at 65°c. fluorescence (fam channel) was monitored every 20 s, and the fluorescence signal was plotted over time. without the need to be pre-enriched before analysis is considered the main obstacle in most methods, but the enrichment remains essential for the revival of stressed or injured cells (cossarizza et al., 2017; valderrama et al., 2016). but, through the dnafoil mechanism, the sample preparation and dna extraction stages were completed in a single step without the need to use spin-columns and centrifuges. for the amplification stage, cross contamination is one of the difficulties faced by the commercially available kits used to detect food pathogens, that is, salmonella and listeria monocytogenes (baraketi et al., 2018). in contrast to what happened using the dnafoil technique, it is obvious that the dna target amplification is done in one pot master mix without requiring trained staff, using thermos-cycling and cold chain. for the final stage (dna detection stage), there are several problems generated from dna electrophoresis and staining such as it being time-consuming, gel preparation, smearing, mutagenicity, toxicity, lower efficiency, etc. (drabik et al., 2016; hall, 2020). dnafoil as a detection method during the final stage provides the test strip material, which allows for transport by a capillary force of the target dna through the detection surface, allowing the target to hybridize specifically to their complementary capture sequences (target dna fragments are captured in a band). conjugation of micrometer-sized beads to dna permits the results to be visualized by the naked eye (visible color reaction), enabling immediate, simple to interpret, cost-efficient, and on-site detection, while eliminating the need for advanced expensive instrumentation (el sheikha, 2019). hence, the aim of this study was to investigate initial validation experiments of the dnafoil technique to detect food pathogens, that is, salmonella and listeria monocytogenes. materials and methods reference materials crude bacterial dna extracts were purchased from the culture collection of switzerland (ccos), and experiments were conducted at the swiss decode labs in renens, switzerland. live strains tested were procured from the pasteur institute (france). the strains tested were divided into two groups one for inclusivity and the other for exclusivity toward salmonella and listeria monocytogenes as follows. firstly, the strains were tested for inclusivity and exclusivity toward salmonella: (i)  for inclusivity (salmonella montevideo, salmonella heidelberg, salmonella mbandaka, salmonella enteritidis, salmonella agona, salmonella indiana, salmonella infantis, salmonella arizonae iiia, salmonella senfentberg, salmonella cerro, salmonella virchow, salmonella 46 italian journal of food science, 2021; 33 (sp1) el sheikha af dna extraction stage “without the need to use spin-columns and centrifuges” amplification stage “without using thermos-cycling & cold chain” detection stage “without using electrophoresis & staining” as 1st step of dna extraction: get 200 µl of culture you want to test for dna extraction using barrel transfer one drop of extracted dna to the reaction tube and incubate it for 30 min in a water bath at 65°c, then use specific primers & enzymes after 30 min of incubation, dnafoil strip was dipped into reaction tube & migration by lateral-flow was started positive band means presence of solmenella’s dna or listeria monocytogenes’s dna as 2nd step of dna extraction: mix hot and cold water in the container provided with the test kit postive negative figure 1. procedural diagram for the mechanism of food pathogen detection using dnafoil technology. source: adapted from el sheikha (2019). reproduced with permission of elsevier. statistical analysis all data were presented as the mean value ± standard deviation (sd) of independent experiments on various days. performance metrics accuracy (%) results from the experimental specificity (figure 3a and b) were used to calculate the method accuracy using the following equations: ( ) ( ) accuracy (%) at 10 min tp tn 100 tp tn fp fn × + + = + + (1) ( ) ( ) ( ) tp tn accuracy % at 15 min 100 tp tn fp fn + = × + + + (2) ( ) ( ) ( ) tp tn accuracy % at 20 min 100 tp tn fp fn + = × + + + (3) where tp, fn, fp, and tn represent the number of true positives, false negatives, false positives, and true negatives, respectively. precision (%) results from the experimental specificity (figure 3a and b) were used to calculate the method accuracy using the following equations: ( ) ( ) tp precision % at 10 min 100 tp fp = × + (4) ( ) tpprecision % at 15 min 100 (tp fp) = × + (5) ( ) tpprecision % at 20 min 100 (tp fp) = × + (6) where tp, fn, fp, and tn represent the number of true positives, false negatives, false positives, and true negatives, respectively. results inclusivity and exclusivity to confirm the specificity of our method toward salmonella, 18 different salmonella strains, 6 non salmonella bacteria, and 2 fungi were investigated; also, in the case of listeria monocytogenes, five bacterial strains, two fungi, and listeria monocytogenes were investigated. independent microbiology lab prepared cultures containing 108 cfu/ml. swiss decode analyzed the broth media in a blinded manner. the amplification time for each strain is reported in figure 2a and 2b. the standard amplification time used for our dnafoil kit is 30 min. samples for which the amplification signal was not detected after 30 min were considered as negative (no presence of salmonella or listeria monocytogenes). swiss decode salmonella solution positively identified the 18 salmonella strains after 10 to 13 min (figure 2a). italian journal of food science, 2021; 33 (sp1) 47 dnafoil is a promising detection technology s. m on te vi de o s. h ei de lb er g s. m ba nd ak a s. e nt er iti di s s. a go na s. in di an a s. in fa nt is s. a riz on ae il la s. s en fe nt be rg s. c er ro s. v irc ho w s. a na tu m s. n ew po rt s. th yp hi m ur iu m s. a riz on ae il lb s. s t p au l s. h ad ar s. e nt er iti di s ab on y li st er ia m on oc yt og en si s ba ci llu s su bt ilis en te ro co cc us fa ec al is es ch er ic hi a co li ps eu do m on as a er ug in os a st ap hy lo co cc us a ur eu s as pe rg illu s br as ilie ns is c an di da a lb ic an s 0 5 10 a m pl ifi ca tio n ti m e (m in ) 15 20 25 30 not detected strains li st er ia m on oc yt og en es ba ci llu s su bt ilis en te ro co cc us fa ec al is es ch er ic hi a co li ps eu do m on as a er ug in os st ap hy lo co cc us a ur eu s as pe rg illu s br as ilie ns is c an di da a lb ic an s0 5 10 15 20 25 30 not detected strains a m pl ifi ca tio n ti m e (m in ) (a) (b) figure 2. amplification time—selectivity experiment. (a) different cultures of salmonella (in white), non-salmonella bacteria (in black), and fungus (in gray) were prepared at an independent microbiology lab. (b) listeria monocytogenes (in white), different bacterial cultures (in black), and fungus (in gray) were prepared at an independent microbiology lab. broth media (200 μl) was withdrawn from cultures containing 108 cfu/ml. bacteria were lysed according to the standard dnafoil method. dna detection was performed with a real-time assay according to the swiss decode protocol. data represent mean ± sd, n = 3. 48 italian journal of food science, 2021; 33 (sp1) el sheikha af 0 0 500 h2o h2o 1/10 1/10 1/100 1/100 1/1000 1/1000 1/10000 1/10000 1/100000 1/100000 1000 1500 2000 2500 3000 3500 4000 4500 5000 3 6 9 12 15 time (min) fl uo re sc en ce 18 21 24 27 30 0 0 500 h2o h2o 1/10 1/10 1/100 1/100 1/1000 1/1000 1/10000 1/10000 1000 1500 2000 2500 3000 3500 4000 4500 5000 3 6 9 12 15 time (min) fl uo re sc en ce 18 21 24 27 30 figure 3. amplification time-sensitivity experiment. ten times serial dilution of crude dna extracts from salmonella enterica and listeria monocytogenes were diluted in 10 mm tris ph 8.0 or dnafoil lysis buffer. one microliter of the lysis solution was taken to run a real-time assay. (a) we can observe that the time needed for the detection of salmonella increases as the amount of dna present in the sample decreases. however, the detection time is well below the 30 min used as our standard amplification time. (b) we can observe that the time needed for the detection of listeria monocytogenes increases as the amount of dna present in the sample decreases. however, the detection time is well below the 30 min used as our standard amplification time. (a) (b) italian journal of food science, 2021; 33 (sp1) 49 dnafoil is a promising detection technology enrichment) followed by biochemical tests (metabolic fingerprinting), molecular tests (typically pcr [polymerase chain reaction]), or mass spectrometry (adzitey et al., 2011; corry et al., 2003; ellis et al., 2019) to confirm that the isolate is indeed the pathogen of interest. the gold-standard-based methods have the advantages of being inexpensive, detecting only viable pathogens, and yielding isolates that can further be studied (adzitey and huda, 2011; engberg et al., 2000). however, they are cumbersome, relatively slow, and less efficient (foddai and grant, 2020; jasson et al., 2010; keramas et al., 2004; li and zhu, 2017; myint et al., 2006). regarding the biochemical and mass spectrometry methods, they are rapid, sensitive, and accurate techniques that involve the analysis of entire microbial cells or their extracts (beale et al., 2014; cevallos-cevallos et al., 2011; singh et al., 2011; singhal et al., 2015; toscano et al., 2018; wu et al., 2016; yang et al., 2015). however, they are laborintensive, costly, and the reliance on existing spectral databases of the mass fingerprints of known microbes makes mass spectrometry techniques incapable of identifying new species (anderson et al., 2012; el sheikha and hu, 2020; jadhav et al., 2018; mirmajlessi et al., 2015; reta et al., 2020). molecular techniques have the advantage of being other non-salmonella strain samples were still negative after 30 min. our method also allowed salmonella’s identification in broth media samples where salmonella was mixed with several other non-salmonella strains (data not shown). regarding the listeria monocytogenes, swiss decode l.  monocytogenes solution positively identified the listeria monocytogenes strains after 11 min (figure 2b). the other strain samples were still negative after 30 min. limit of detection (lod) with serial dilutions to determine the sensitivity of our method, a serial dilution of crude bacterial dna extracts was analyzed. crude extracts for salmonella enterica subspc. enterica and listeria monocytogenes were obtained from ccos. crude extracts containing 105 cfu/μl were serially diluted 1:10 either in 10 mm tris ph 8.0 or in our dnafoil lysis buffer. the reactions were analyzed in duplicate by real-time assays (figure 3a and 3b). the results were similar for both dilution methods (data not shown). accuracy and precision (%) accuracy and precision (%) are calculated to measure the performance of our method to identify salmonella and listeria monocytogenes. these results are shown in figure 3a and 3b. the results also showed that the accuracy of our method ranges between 92 and 100%, while the precision value ranged between 88 and 100%. point-of-need detection with lateral flow as real-time thermocyclers may not be present at the point-of-need (i.e., factory), we verified if the dnafoil salmonella and listeria monocytogenes reactions could be visually detected with the naked eye using a lateral flow strip. serial dilutions of salmonella enterica and listeria monocytogenes extracts were prepared and amplified as before. after 30 min of amplification at 65°c, the results were confirmed with dnafoil strips (figure 4a and 4b). discussion the detection of foodborne pathogens has historically been culture-, or conventional-, or cultural-, or goldstandard-based methods, which were used since the inception of microbiological sampling (adzitey and huda, 2010, 2011; bhunia, 2014). these methods mainly involve enrichment (pre-enrichment and/or selective s al m on el la s am pl e (1 /1 00 00 0) water water listeria monocytogenes (1/10000) (b) (a) figure 4. strips results. (a) amplified samples from salmonella dilution 1/100,000 were applied on dnafoil strips (triplicate). a few minutes later, we could see bands appearing. the band on the left confirmed the presence of salmonella, whereas the band on the right is the positive control of the strips. (b) amplified samples from listeria monocytogenes dilution 1/10,000 were applied on dnafoil strips. four minutes later, we could see bands appearing. the band on the left confirmed the presence of listeria monocytogenes, whereas the band on the right is the positive control of the strips. 50 italian journal of food science, 2021; 33 (sp1) el sheikha af purity in 30 min, without lab equipment, technicians, or scientists. the final report provided by meat and livestock australia limited (mla) (2018) illustrated that the dnafoil kit is able to detect pork contamination in beef as lower as 0.1%. the present study illustrated that the dnafoil is a fast-detection technique of salmonella and listeria monocytogenes that can get the final results in as little as 30 min. in addition, the results of this study stated that the swiss decode salmonella and l. monocytogenes solutions can detect as few as 1 and 10 copies of dna per micro liter, respectively. however, the commercially available kits used to detect food pathogens, that is salmonella and listeria monocytogenes, which are based on nucleic acid for detection, are characterized by reliability, high specificity, and sensitivity; they are limited by the difficulties of: • differentiating the viable cells from nonculturable cells; • the primers’ design. moreover, these kits require trained staff to avoid cross-contamination (baraketi et al., 2018). table 2 evaluates the commercial kits used to detect salmonella and listeria monocytogenes. in terms of performance metrics (accuracy, precision %), the results show promising performance, to be used for salmonella and listeria monocytogenes detection. the dnafoil technology is efficient in terms of cost and quality control applicability in terms of cost. dnafoil is available to the partners (academic and industry) that accede to the early access program. the fee to enter the early access program is rapid, less laborious, more sensitive, specific, and efficient, compared to the conventional method (keramas et al., 2004; magistrado et al., 2001). nonetheless, certain components/compounds in foods such as fats, lipids, and salts, enrichment media, or dna extraction solution can inhibit the sensitivity of pcr-based methods (el sheikha, 2010; rossen et al., 1992; wilson, 1997). to overcome the limitations of traditional methods used for pathogen detection from the side and from the other side to meet industrial and commercial food needs, there is an urgent need for fast, sensitive, accurate, and more efficient detection methods in terms of saving time, labor, and preventing human errors (baraketi et al., 2018; law et al., 2015; mandal et al., 2011; rajapaksha et al., 2019). hence, the importance of answering the principal question, namely, why is dnafoil technology proposed to detect food pathogens? this question is the hypothesis on which the research idea was based, and which the results of this study approved and provided the answers as follows: dnafoil is a fast, accurate, precise, sensitive, and reliable technique as a new technology that needs assessment, the “realtime” amplification technology (real-time pcr) is used to evaluate the efficacy and accuracy of dnafoil technology (el sheikha, 2019). aronoff et al. (2018) reported that the efficiency of dnafoil kit used to detect the vegetal material in milk products (dnafoil uniplant) was confirmed using real-time pcr assays. the same authors concluded that the dnafoil uniplant kit provides a quick and reliable method to validate product content with less than 1% adulteration of any product, confirming identity and table 2. commercially available kits based on nucleic acid methods for the detection of foodborne pathogens.* pathogen commercially available kits sensitivity sample matrix company salmonella sp. bax® system standard pcr assays for salmonella 104 cfu/ml, after enrichment poultry, dairy, fruits, vegetables, bakery products, pet food, environmental samples hygiena bax® system real-time pcr assay for salmonella 104 cfu/ml, after enrichment meat, poultry, dairy, fruits, vegetables, bakery products, pet food, environmental samples hygiena genequence® for salmonella 1–5 cfu/25 g food and environmental samples hygiena listeria monocytogenes bax® system pcr assay for l. monocytogenes 105 cfu/ml, after enrichment variety of food types hygiena bax® system pcr assay for l. monocytogenes 24e 104 cfu/ml, after enrichment dairy, meat, fish, vegetables, environmentals hygiena bax® system real-time pcr assay for l. monocytogenes 104 cfu/ml, after enrichment dairy, ready-to-eat meat, seafood, vegetables, environmental samples hygiena genequence® for l. monocytogenes 1–5 cfu/26 g food and environmental samples neogen *source: baraketi et al. (2018). licensed under creative commons attribution 3.0. pcr, polymerase chain reaction. italian journal of food science, 2021; 33 (sp1) 51 dnafoil is a promising detection technology although dna-based techniques have proven to be the best detection tools in food pathogen detection, at the industrial level its practical application has to go a long way (el sheikha et al., 2018; zhao et al., 2014). hence, the demand for a novel, rapid, easy, potent, and universal technology for food pathogen detection is still an urgent need. therefore, it is hoped that the dnafoil technology could be a powerful tool that meets all of the requirements for food pathogen detection and its applications either at the household or industrial level. as a future trend, more applications are recommended for dnafoil technology as a food pathogen detection tool on different food matrices. moreover, the dnafoil test kits for salmonella and listeria monocytogenes are qualified for further validation using iso16140. compliance with ethical standards acknowledgments this research was supported by the research start-up fund from jiangxi agricultural university, china that granted to prof. aly el sheikah (fund n° 9232307245). conflict of interest the author declares that there is no conflict of interest. references adzitey, f. and huda, n., 2010. listeria monocytogenes in foods: incidences and possible control measures. african journal of microbiology research 4: 2848–2855. available at: http://www. academicjournals.org/ajmr adzitey, f. and huda, n., 2011. campylobacter in poultry: incidences and possible control measures. research journal of microbiology 6: 182–192. https://doi.org/10.3923/jm.2011.182.192 adzitey, f., huda, n. and gulam, r., 2011. comparison of media for the isolation of salmonella (xld and rambach) and listeria species (aloa and palcam) in naturally contaminated duck samples. internet journal of food safety 13: 20–25. anderson, n.w., buchan, b.w., riebe, k.m., parsons, l.n., gnacinski, s. and ledeboer, n.a., 2012. effects of solidmedium type on routine identification of bacterial isolates by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry. journal of clinical microbiology 50: 1008–1013. https://doi.org/10.1128/jcm.05209-11 aronoff, r., vernez, i., rotman, n. and rando, g., 2018. detection of milk adulteration using dnafoil uniplant. application note: v0.3 available at: https://www.dropbox.com/s/ho5ich3fpzy5k0s/ application_note_uniplant_1dec17.docx?dl=0. accessed 27 january 2021. €  990 and the program makes access free of charge for a kit of 5 tests. the final cost per test is negotiable, and it is usually much cheaper than the traditional detection methods (aronoff et al., 2018; lüdin et al., 2018). in terms of quality control applicability. the dnafoil output gives a (±) answer, for example, while using dnafoil pork test kit, a positive result indicates the presence of porcine dna in the sample tested. this is enough for an inspector to take instant action. the strips with the results can be stored as evidence to prove that adequate controls are in place during manufacturing. in the case of litigations, the swiss federal lab concluded that dna can be easily extracted from the strip, and such dna can be used for more lab tests (lüdin et al., 2018). conclusions, remarks, and future trends in this study, the developed dnafoil salmonella and listeria monocytogenes reactions correctly identify a wide range of salmonella strains and also listeria monocytogenes, among other bacterial strains. these reactions are specific and sensitive, with a virtual limit of 1 and 10 cfu detection per reaction for salmonella and listeria monocytogenes, respectively. in terms of accuracy and precision, the results show promising performance in which the accuracy ranged between 92 and 100% and the precision ranged between 88 and 100%. real-time pcr may not be present at the point-of-need (i.e., factory); 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34 (1): 59–66 issn 1120-1770 online, doi 10.15586/ijfs.v34i1.2161 59 p u b l i c a t i o n s codon quality of frozen stored flavoured olive oils giacomo squeo1*, graziana difonzo1, antonietta baiano2, roccangelo silletti1, antonella pasqualone1, carmine summo1, and francesco caponio1 1department of soil plant and food sciences, university of bari “aldo moro”, via amendola, bari, italy; 2dipartimento di scienze agrarie, alimenti, risorse naturali e ingegneria (dafne), university of foggia, via napoli, foggia, italy *corresponding author: giacomo squeo, department of soil plant and food sciences, university of bari “aldo moro”, via amendola, bari, italy. email: giacomo.squeo@uniba.it received: 2 december 2021; accepted: 11 january 2022; published: 27 january 2022 © 2022 codon publications open access paper abstract the purpose of the current research was to study the effects of 6-month frozen storage on the quality parameters and the phenolic profiles of flavoured olive oils (foo) produced by co-malaxation or infusion using basil, chilli, or chilli–garlic as flavouring ingredients. the results demonstrated that during frozen storage, foos underwent degradative processes that caused a progressive depletion of phenolic compounds and the rising of oxidative and hydrolytic markers. a clear interaction appeared between storage time, flavouring ingredient and flavouring technique. infusion caused a greater quality loss than co-malaxation, and in basil flavoured oils the drawbacks of infusion were greater than in other flavoured oils. the impact of flavouring method on the phenolic profiles of oil became more evident at the end of the storage period. it was confirmed that oleocanthal is less affected by storage in freezing conditions than other secoiridoids. keywords: freezing; infusion; malaxation; phenolic compounds; spices; virgin olive oil introduction aromatic plants have been used since ancient times as food flavouring ingredients as well as in pharmaceutical, cosmetic and perfumery because of the presence of essential oils. several biological activities, including antimicrobial and antioxidant properties, are assigned to these plants and derived oils or to some of their other constituents (ayadi et al., 2009; ijaz hussain et al., 2008). along the mediterranean basin, spices and herbs have been historically used in association with olive oil, as it represented the main source of fats in the area, to improve its nutritional and organoleptic properties (baiano et al., 2010). over the time, the importance gained by high-quality virgin olive oils (voo) has been the driving force for a renewed interest towards flavoured olive oils (foo), which have been rediscovered as interesting products with unique characteristics as well as these being increasingly appreciated by consumers. flavouring represents a strategy to increase the use of olive oil among non-traditional consumers for adding value to its organoleptic and good health properties (perestrelo et al., 2017). in this perspective, foos represent an important source of income for producers and sellers. oil flavouring is performed by different techniques such as: (i) infusion (akçar and gümüşkesen 2011; ayadi et al., 2009; baiano et al., 2016; caporaso et al., 2013; damechki et al., 2001; issaoui et al., 2011; sousa et al., 2015); (ii)  ultrasound-assisted maceration (veillet et  al., 2010) and (iii) direct malaxation of olive paste with spices (baiano et al., 2009; caponio et al., 2016). the last mentioned technique does not require the implementation of additional equipment in the olive oil mills, and is time-saving without the disadvantages of the other cited methods. in fact, it is reported that ultrasound assisted technology could determine the off-flavour onset because of the oxidative oil degradation (chemat et al., 2004), while infusion is time-consuming and could mailto:giacomo.squeo@uniba.it 60 italian journal of food science, 2022; 34 (1) squeo g et al. italy). olives were processed by a continuous extraction process. after crushing, the olive paste was malaxed for 30 min at 26°c without any flavouring ingredient to produce the control oil (ctr). co-malaxed (m) foos were produced by direct addition of dried basil (b), dried chili (c), and a combination of dried chili and garlic (cg) during malaxation (30 min at 26°c). the amount of spices used were 5%, 20% and 20% + 10% w/w with respect to the amount of olive processed by addition of dried basil, dried chili, and dried chili and garlic, respectively. to produce foos by infusion, the same extraction process as for the control oil was followed. then the spices were left in infusion (with daily stirring) at 15–18°c for 15 days in the case of oils processed by dried basil and for 7 days in the case of chili and dried chili and garlic. the optimal amount of each flavouring ingredient was identified in preliminary trials based on the best sensory result. both ctr and foos were stored under freezing conditions (–18°c) and sampled after 3 (t3) and 6 (t6) months. three independent trials were carried out for each flavouring method, starting from the same olive oil batch. routine analyses free fatty acid (ffa), peroxide value (pv), and spectrophotometric indices (k232, k270 and δk) were determined following the analytical methods described by the european community regulation (eec) regulation 2568/91 and subsequent integrations and amendments (european commission, 1991). extraction of phenolic fraction and determination of phenolic profile the extraction of phenolic compounds and the quantification of total phenolic content (tpc) were carried out according to the procedure described in zago et  al. (2019). tpc was expressed as milligrams of gallic acid equivalents per kilogram of oil. the high-performance liquid chromatography (hplc) analysis of phenolic extracts was carried out according to baiano et al. (2009). the stationary phase was a nova-pack c18 analytical column (150 × 3.9 mm i.d.) with a particle size of 4 μm (waters, milford, ma). the mobile phases were (a) water:acetic acid (98:2, v/v) and (b) methanol:acetonitrile (1:1, v/v) at a constant flow rate of 1 ml/min, according to the following gradient programs: 0–30 min 100% mobile phase (a); 30–45 min, 70% mobile phase (); 45–55  min, 50% mobile phase (a); 55–65 min, 40% mobile phase (a); and 65–75 min, 0% mobile phase (a). phenolic compounds were quantified according to the method of the internal standard considering the response factors, and compromise  the quality of olive oil. previous research conducted by the present authors (caponio et al., 2016) highlighted that malaxation was more effective in extracting phenolic compounds, with a significantly lower level of hydrolysis of secoiridoids than that occurring with infusion. moreover, foos produced by infusion showed lower antioxidant activity and higher extent of oxidative degradation. volatile compounds, in generally, were not significantly influenced by the adopted flavouring method. clodoveo et al. (2016) reported that an ultrasound treatment of olive paste mixed with herbs before malaxation determined a higher level of total phenols as well as tyrosol, hydroxytyrosol and oleuropein derivatives in flavoured oils. yilmazer et al. (2016) reported that only the amount of spices influenced the partition of target compounds in foos whereas temperature and time of malaxation did not exercise significant effects. quality of foos is also affected by storage (ayadi et al., 2009; baiano et al., 2009; gambacorta et al., 2007; issaoui et al., 2011). during storage, foos undergo the same degradative processes that commonly affect the quality of voos, which are, in turn, dependent on light, oxygen, temperature, water, metals and antioxidants (choe and min, 2006). effects of light, oxygen, metals and water could be controlled by using proper packaging materials and process settings during the extraction process (e.g. settings of the vertical centrifuge, filtration etc.). on the other hand, temperature could be easily managed during storage. although low-temperature storage could slow down the rate of degradative phenomena, studies on the quality of voos stored at low or freezing temperature reported contradictory results, especially considering the fate of phenolic compounds (cerretani et al., 2005; li et al., 2014; mousavi et al., 2021; mulinacci et al., 2013). however, a study conducted on the subject has highlighted that foos stored under freezing conditions and the interactions between storage time and flavouring method and ingredients have been poorly investigated. in the light of these considerations, our work was aimed at studying changes in the quality features of three different foos (basil, chilli, and chilli and garlic) obtained by applying two flavouring methods (co-malaxation and infusion) during 6 months of frozen storage in comparison to unflavoured stored voos. material and methods sampling a blend of olives (olea europaea l.) from peranzana, coratina and ogliarola cvs (50%, 30% and 20% w/w, respectively) was used for the experimental trials carried out at a local olive mill (olearia clemente, manfredonia, italian journal of food science, 2022; 34 (1) 61 quality of frozen stored flavoured olive oils inc.  ma) by using the pls_toolbox (eigenvector research inc., usa). results and discussion the effect of storage on foos has been studied by several authors (ayadi et al., 2009; baiano et al., 2009; gambacorta et al., 2007; issaoui et al., 2011) but without considering the frozen storage. taking into consideration the frozen storage, the first aim of the present research was to highlight the effect of freezing on quality parameters, the polar compounds and the tpc of foos sampled for 3 and 6 months after production. table 1 shows the significance of the differences in the quality characteristics of t3 and t6 samples with respect to t0. after 3 months, the effect of storage on the characteristics of foos was different according to the flavouring method. in fact, in the case of infusion (i-oils), a significant increase in the value of several quality parameters (ffa, pv, spectrophotometric constants and polar compounds) was observed with respect to t0 although with some differences linked to the spice. notably, the tpc always decreased significantly. on the other hand, after 3-month frozen storage, the characteristics of foos from co-malaxation (m-oils) were less affected. only in the case of co-malaxation with chilli–garlic as flavouring identification was carried out comparing peak retention period and spectra with those of pure standards. determination of polar compounds polar compounds (pcs) were first separated by silica gel column chromatography, according to the association of official analytical chemists (aoac) method no. 982.27. the efficacy of the separation was checked by thin layer chromatography (tlc) as recommended by the aoac. the polar compounds, recovered in tetrahydrofuran (thf), were then analysed by high-performance size exclusion chromatography (hpsec) following the conditions reported by makhlouf et al. (2021). data elaboration the paired t-test was used to compare the characteristics of oils after 3and 6-month storage with respect to the time of production. the statistical analysis was carried out with minitab 17 (minitab inc., state college, pa) at a significance level of 0.05. principal component analysis (pca) was carried out on the autoscaled data matrix and used as a multivariate tool for data exploration. pca was carried out in matlab environment (the mathworks table 1. statistical significance, expressed as p-value, of the differences in the characteristics of the samples between t3 or t6 and t0. ffa pv k232 k270 δk tagp ox-tag dag pc tpc t3 vs. t0 ctr 0.99 0.01↑ 0.54 0.86 0.18 0.05↑ 0.01↑ 0.01↑ 0.00↑ 0.01↓ i-b 0.31 0.00↑ 0.41 0.00↑ 0.19 0.48 0.00↑ 0.05↑ 0.01↑ 0.00↓ i-c 0.01↑ 0.01↑ 0.29 0.13 0.10 0.01↑ 0.03↑ 0.26 0.05↑ 0.01↓ i-cg 0.11 0.00↑ 0.07 0.03↑ 0.01↑ 0.35 0.07 0.04↑ 0.03↑ 0.04↓ m-b 0.21 0.20 0.30 0.16 0.06 0.90 0.07 0.16 0.13 0.02↓ m-c 0.95 0.65 0.19 0.21 0.30 0.16 0.05 0.29 1.00 0.03↓ m-cg 0.87 0.13 0.51 0.09 0.45 0.63 0.03↑ 0.11 0.09 0.00↓ t6 vs. t0 ctr 0.16 0.24 0.63 0.01↓ 0.42 0.06 0.00↑ 0.11 0.02↑ 0.00↓ i-b 0.67 0.00↑ 0.20 0.08 0.39 0.60 0.00↑ 0.01↑ 0.02↑ 0.00↓ i-c 0.36 0.06 0.01↑ 0.02↑ 0.68 0.25 0.01↑ 0.25 0.02↑ 0.00↓ i-cg 0.07 0.27 0.05↑ 0.02↑ 0.07 0.48 0.02↑ 0.10 0.05 0.00↓ m-b 0.73 0.01↓ 0.07 0.26 0.00↑ 0.68 0.01↑ 0.00↑ 0.02↑ 0.06 m-c 0.02↑ 0.13 0.02↑ 0.07 0.02↑ 0.11 0.01↑ 0.26 0.60 0.00↓ m-cg 0.02↑ 0.04↓ 0.19 0.07 0.42 0.70 0.02↑ 0.05↓ 0.05↑ 0.00↓ ctr: unflavoured oils (control); i: infusion; m: co-malaxation; b: basil; c: chilli; cg: chilli and garlic; ffa: free fatty acids; pv: peroxide value; tagp: triacylglycerol oligopolymers; ox-tag: oxidated triacylglycerols; dag: diacylglycerols; pc: total polar compounds; tpc: total phenolic content. significant differences (p ≤ 0.05) are highlighted in bold. up arrows and down arrows indicate respective significant increase and decrease with respect to t0. 62 italian journal of food science, 2022; 34 (1) squeo g et al. ingredient (m-cg), a significant increase was observed in oxidated triacylglycerols (ox-tag). however, even in this case, the tpc was always negatively affected by storage. at t6, main differences with the oils at production were the significant increase in polar compounds (in particular, ox-tag) and depletion of tpc. other differences were highlighted in the ffa, pv, and spectrophotometric constants. the paired t-test afforded to point out differences in characteristics of oils because of storage. however, it did not show the magnitude of these effects and also did not allow considering the complexity of the evolution of features of oils during freeze storage. for this, the data were explored by pca, and the results are reported in figure 1. the score plot in figure 1a allows discovering the relative effects of flavouring methods, storage and different flavouring agents. first, it clearly appears that the basil figure 1. (a) score plot and (b) loading plot of pca carried out on quality indices, polar compounds, and total phenolic content of stored flavoured (b, c and cg) and unflavoured (ctr) oils. i: infusion; m: co-malaxation; x: no aromatization; b: basil; c: chilli; cg: chilli and garlic. (a) (b) italian journal of food science, 2022; 34 (1) 63 quality of frozen stored flavoured olive oils brought higher amounts of phenolic alcohols at each time point. on the other hand, in cand cg-oil, effect of flavouring technique appeared more clearly after 6 months when co-malaxation and infusion affected an increase in 3,4-dhpea-eda, p-hpea-eda and 3,4-dhpea-ea. an increment in simple phenols (such as tyrosol and hydroxytyrosol) was due to progressive hydrolysis from complex secoiridoids (bendini et al., 2007) and was observed by mulinacci and co-workers (2013) in extra virgin olive oils (evoo) stored for 9 months at room temperature. our findings suggest that this could happen even during frozen storage, although a critical role was played by other factors (kind of spice and flavouring method). the same could be stated for more complex phenols, such as 3,4-dhpea-eda and p-hpea-eda, that in several cases increased after 6-month storage. on the other hand, the observed decrements could be explained in different manners. on the one hand, it could be due to the unavoidable oxidation occurring even at low temperatures. on the other hand, as supposed by cerretani et al. (2005), decrement in phenols after freezing was linked to change in the physical state of oil and the consequent solubilisation of phenols into residual water phase. mousavi et al. (2021) reported that secoiridoids (mostly 3,4-dhpea-eda, 3,4-dhpea-ea and p-hpea-ea) decreased during frozen storage, while (p-hpea-eda) was the most stable phenol. the findings of the present research are in partial agreement with these. in fact, p-hpea-eda was significantly reduced only in the case of m-cg oils, while 3,4-dhpea-eda, 3,4-dhpea-ea and p-hpea-ea were much more affected by storage in freezing conditions, particularly during the first 3 months (table 2). conclusion to the best of authors’ knowledge, this is the first research centring the effect of storage under frozen conditions on the quality features of foos, together with the possible interaction with flavouring methods and flavouring agents. low temperatures during storage could slow down degradation, thus safeguarding the quality of foos. during storage, the samples had a significant decrease in total phenolic compounds and an increase in some hydrolytic and oxidative markers. the aromatization by infusion was the worse due to significant increase in the values of ffa, pv, spectrophotometric constants and polar compounds. also, an interaction with the kind of spice was pointed out. in fact, flavouring with basil by infusion had maximum effect on the quality of the product not only at the time of production but also foos had different characteristics from others. in fact, already at t0, b-oils clustered apart from other samples. it was also observed that technology had a stronger effect than frozen storage on characteristics of b-oils. in fact, b-oils from infusion at t3 and t6 clustered together and showed higher positive scores on pc1 linked to oxidative markers (i.e. pv, k232, k270, ox-tag and polar compounds) as observed in the corresponding loading plot (figure 1b). on the other hand, cand cg-oil followed an evolution similar to that of control samples during storage, indicating a less influence of aromatisation on their chemical characteristics. with time, these samples moved progressively from the negative quadrant of the plot to the positive quadrant because of tpc depletion and increase in hydrolytic and oxidative markers (figure 1b). changes in quality parameters because of flavouring practices are well known and observed by several authors (baiano et al., 2009, 2016; caponio et al., 2016; clodoveo et al., 2016; gambacorta et al., 2007; sacchi et al., 2017; sousa et al., 2015). overall, the results demonstrated that during frozen storage foos underwent degradation that caused progressive depletion of phenolic compounds and increase in oxidative and hydrolytic markers. however, a clear interaction emerged between the storage and the kind of flavouring ingredient and flavouring technique. aromatisation effect was also observed on the phenolic profile of voo (baiano et al., 2016; caponio et al., 2016; sacchi et al., 2017). table 2 shows the significance of differences in the phenolic profile of oils at t3 and t6 with respect to t0, while figure 2 reports the results of pca. in all, 13 phenolic compounds were detected belonging to the common classes found in voos (bendini et al., 2007). it is observed in table 2 that the most important differences in the phenolic profiles of oils at t3 with respect to production was the increase in hydroxytyrosol acetate (3,4-dhpea-ac, in all samples) and decrease of almost all the other compounds. this trend could be easily noted by looking at pca results (figure 2). in spite of some differences linked to spice and flavouring technique, the samples at t3 depicted similar characteristics. after 6-month storage, all the oils had significant higher values of 3,4-dhpea-ac. phenolic alcohols (3,4-dhpea and p-hpea) were significantly higher in some cases (i-c, i-cg and m-c) as also secoiridoids such as decarboxymethyl oleuropein aglycon (3,4-dhpea-eda), oleocanthal (p-hpea-eda) and oleuropein aglycon (3,4-dhpea-ea) (i-b, i-cg, m-b and m-c). on average, cand cg-oil had the higher amount of complex secoiridoids at t6. pca results helped in understanding the interaction of storage, flavouring technique and spice on the phenolic profiles. in b-oils, an important effect of flavouring technique was observed with infusion that 64 italian journal of food science, 2022; 34 (1) squeo g et al. ta bl e 2. s ta tis tic al s ig ni fic an ce , e xp re ss ed a s pva lu e, o f di ff er en ce s in th e ph en ol ic p ro fil es o f th e sa m pl es b et w ee n t3 o r t6 a nd t 0. 3, 4d h p e a ph p e a va ni lli c ac . va ni lli n c um ar ic a c. 3, 4d h p e a -a c fe ru lic a c. 3, 4d h p e a -e d a ph p e a -a c ph p e a -e d a p in or es in ol 3, 4d h p e a -e a ph p e a -e a t3 v s. t 0 c tr 0. 01 ↓ 0. 00 ↓ 0. 00 ↑ 0. 83 0. 17 0. 01 ↑ 0. 26 0. 00 ↑ 0. 10 0. 03 ↑ 0. 03 ↓ 0. 36 0. 13 i-b 0. 00 ↓ 0. 00 ↓ 0. 23 0. 06 0. 11 0. 00 ↑ 0. 10 0. 00 ↑ 0. 05 ↓ 0. 00 ↑ 0. 05 ↓ 0. 18 0. 02 ↓ i-c 0. 53 0. 79 0. 51 0. 54 0. 02 ↓ 0. 00 ↑ 0. 05 ↓ 0. 02 ↓ 0. 00 ↓ 0. 08 0. 03 ↓ 0. 00 ↓ 0. 03 ↓ i-c g 0. 06 0. 05 ↓ 0. 33 0. 05 0. 00 ↓ 0. 03 ↑ 0. 01 ↓ 0. 00 ↓ 0. 00 ↓ 0. 94 0. 01 ↓ 0. 04 ↓ 0. 00 ↓ m -b 0. 47 0. 23 0. 25 0. 92 0. 07 0. 01 ↑ 0. 01 ↓ 0. 00 ↓ 0. 03 ↓ 0. 44 0. 24 0. 05 ↓ 0. 00 ↓ m -c 0. 02 ↓ 0. 77 0. 85 0. 71 0. 01 ↓ 0. 02 ↑ 0. 07 0. 11 0. 02 ↓ 0. 58 0. 01 ↓ 0. 01 ↓ 0. 00 ↓ m -c g 0. 09 0. 01 ↓ 0. 18 0. 25 0. 01 ↓ 0. 01 ↑ 0. 55 0. 00 ↓ 0. 00 ↓ 0. 01 ↓ 0. 01 ↓ 0. 01 ↓ 0. 01 ↓ t6 v s. t 0 c tr 0. 00 ↓ 0. 00 ↓ 0. 09 0. 01 ↑ 0. 73 0. 01 ↑ 0. 48 0. 01 ↑ 0. 13 0. 00 ↑ 0. 07 0. 18 0. 08 i-b 0. 00 ↓ 0. 00 ↓ 0. 83 0. 09 0. 13 0. 00 ↑ 0. 96 0. 00 ↑ 0. 00 ↑ 0. 00 ↑ 0. 01 ↓ 0. 18 0. 01 ↓ i-c 0. 02 ↓ 0. 04 ↑ 0. 08 0. 07 0. 01 ↓ 0. 02 ↑ 0. 04 ↓ 0. 52 0. 33 0. 08 0. 05 0. 08 0. 01 ↓ i-c g 0. 01 ↑ 0. 01 ↑ 0. 01 ↑ 0. 03 ↑ 0. 01 ↓ 0. 01 ↑ 0. 12 0. 00 ↑ 0. 05 ↓ 0. 00 ↑ 0. 01 ↑ 0. 04 ↑ 0. 02 ↓ m -b 0. 03 ↓ 0. 77 0. 14 0. 81 0. 14 0. 01 ↑ 0. 24 0. 30 0. 00 ↑ 0. 03 ↑ 0. 29 0. 03 ↑ 0. 01 ↓ m -c 0. 03 ↑ 0. 03 ↑ 0. 01 ↑ 0. 52 0. 07 0. 00 ↑ 0. 11 0. 00 ↑ 0. 26 0. 01 ↑ 0. 03 ↑ 0. 01 ↑ 0. 82 m -c g 0. 03 ↓ 0. 00 ↓ 0. 80 0. 02 ↑ 0. 07 0. 04 ↑ 0. 68 0. 00 ↓ 0. 01 ↓ 0. 01 ↓ 0. 52 0. 36 0. 05 ↓ c tr : u nfl av ou re d oi ls (c on tro l); i: in fu si on ; m : c om al ax at io n; b : b as il; c : c hi lli ; c g : c hi lli a nd g ar lic ; 3 ,4 -d h p e a : h yd ro xy ty ro so l; ph p e a : t yr os ol ; 3 ,4 -d h p e a -a c : h yd ro xy ty ro so l a ce ta te ; 3 ,4 -d h p e a -e d a : de ca rb ox ym et hy l o le ur op ei n ag ly co n; p -h p e a -a c : t yr os ol a ce ta te ; p -h p e a -e d a : d ec ar bo xy m et hy l l ig st ro si de a gl yc on ; 3 ,4 -d h p e a -e a : o le ur op ei n ag ly co n; p -h p e a -e a : l ig st ro si de a gl yc on . s ig ni fic an t d iff er en ce s (p ≤ 0 .0 5) a re h ig hl ig ht ed in b ol d. u p ar ro w s an d do w n ar ro w s in di ca te re sp ec tiv e si gn ifi ca nt in cr ea se a nd d ec re as e w ith re sp ec t t o t0 . italian journal of food science, 2022; 34 (1) 65 quality of frozen stored flavoured olive oils figure 2. (a) score plot and (b) loading plot of pca carried out on the phenolic profile of stored flavoured (b, c and cg) and unflavoured (ctr) oils. i: infusion; m: co-malaxation; x: no aromatization; b: basil; c: chilli; cg: chilli and garlic. 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salgado 2 1 department of agribusiness, engineering federal fluminense, university, avenida dos trabalhadores, 420, zip code 27225-250, volta redonda, rio de janeiro, brazil 2 department of food technology, institute of technology, federal rural university of rio de janeiro, zip code 23890-971, seropédica, rio de janeiro, brazil *corresponding author: nathaliarm@vm.uff.br abstract this work aimed to evaluate the antimicrobial effectiveness of a biodegradable composite, cellulose acetate film with the incorporation of various combinations of cinnamon, oregano and sweet fennel essential oils (eos) against escherichia coli, staphylococcus aureus and penicillium spp. moreover, thickness, the mechanical and water vapor barrier properties of these films were evaluated. the films with the incorporation of pure oregano eo and the combination oregano + cinnamon eos presented the best results against all microorganisms tested. the water vapor transmission rate of the film was reduced with the addition of the eos and the thickness of the film increased. the eo increased the flexibility of the films, and the film with pure oregano eo was the most resistant, showed 30,9 n of puncture strength. the results suggest that the eos incorporated as natural antimicrobial agents can potentially be used in food packaging, reducing the risk of microbial contamination of food. keywords: active packaging, cinnamon, food packaging, food safety, oregano, sweet fennel   ital. j. food sci., vol 28, 2016 249 1. introduction microorganisms may be responsible for a reduction in food quality, reducing shelf-life and causing economic losses. in addition, contaminated food can cause toxic infections. microbial counts on fresh or processed solid or semi-solid foods are usually higher on the surface of these foods; therefore these surfaces require effective protection (melo, 2003). thus, the development of materials that can serve as a protective barrier for food associated with other techniques, such as selection and control of raw materials, use of good manufacturing practices, and the use of additives that have been appropriately tested are of fundamental importance to ensure food safety, being able to provide consumers with guarantee of quality and safe food for consumption (devlieghere et al., 2004; mastromatteo et al., 2010). the materials used for the production of active packaging include different biopolymer matrices, such as proteins, lipids and polysaccharides (mchugh et al., 1994; chinnan and park, 1995; rojas-grau et al., 2007; atarés et al., 2010a). such packaging must be environmentally acceptable, suitable for a wide range of food products and allow the incorporation of additives, including antimicrobial agents (rossi-márquez et al., 2009; shojaee-aliabadi et al., 2013). the use of active antimicrobial materials for food packaging suggest that the product may have lower concentrations of preservatives or even be free of it, since the packaging with its additives will carry out this function. thus, the consumer will have access to more natural products especially if the preservative used is also a natural product, such an essential oil (naidu, 2000). essential oils (eos) can be used in the development of materials for antimicrobial active packaging because they have an excellent potential to act as a natural microbiological barrier (lambert et al., 2001; abdollahzadeh et al., 2014). several eo have been tested, such as: oregano, cinnamon, thyme, clove, satureja hortensis, lemongrass, ginger, anise, turmeric, guava leaf, nutmeg and lime (rojas-grau et al., 2007; rossimárquez et al., 2009; atarés et al., 2010a; matan, 2012; manso et al., 2011). eos are liquid, volatile, and rich in phenolic compounds. they can be synthesized by aromatic plants as their secondary metabolites (kaul et al., 2003; rodríguez et al., 2007). eos have been widely used for their antibacterial, fungicidal, antiparasitic, insecticidal and medicinal applications (bakkali et al., 2008). in addition, eos are approved for direct contact with food because they are considered to be gras (generally recognized as safe) by the food and drug administration (fda) (burt, 2004). eos (each containing several components) when mixed in various combinations, may produce a synergic effect. this synergistic effect can increase the antimicrobial activity (romano et al., 2009; gibriel et al., 2013). antimicrobial active packaging must meet certain requirements such as: be effective against a large spectrum of microorganisms, show efficiency at low concentrations, not cause undesirable changes in the sensory characteristics of the product, and must be in accordance with the current legislation (soares and gonçalves, 2008). the analysis of antimicrobial properties is important for predicting the behavior of antimicrobial films in food systems. thus, this study aimed to evaluate the in vitro antimicrobial activity of cellulose acetate film incorporated with essential oils of cinnamon, sweet fennel and oregano, that are natural antimicrobial agents, against bacteria (escherichia coli and staphylococcus aureus) and fungus (penicillium spp). the thickness, the mechanical and water vapor barrier proprieties in this cellulose acetate film were also analyzed for characterization them.   ital. j. food sci., vol 28, 2016 250 2. materials and methods 2.1. essential oils three eos of plants were used: oregano, sweet fennel and cinnamom. the oils were acquired commercially from ®ferquima (ferquima indústria e comércio ltda, vargem grande paulista, são paulo-br). in table 1 the common name, plant variety, major components, and part of the plant material used for the extraction of the chosen eos are presented. all selected eos were produced by distillation and kept at room temperature in an amber glass bottle. table 1: description of common name, plant variety, major components and part plant used to obtaining essential oils according to the supplier. common name plant variety major components part plant cinnamon cinnamomum zeylanicum blume cinnamaldehyde leaves sweet fennel foeniculum vulgare var. dulce (mill.) batt. & trab. anetolhole seeds oregano origanum vulgare (l.) carvacrol leaves 2.2. microorganisms the microorganisms evaluated were the bacteria: escherichia coli (atcc 1122) (gramnegative), staphylococcus aureus (atcc 6538) (grampositive) and the fungus penicillium spp. the microbial cultures were obtained from the american type culture collection (atcc) in the universidade federal de viçosa, minas gerais, brazil. bacterial cultures and fungus were pre-activated in brain heart infusion broth (bhi). bacteria were grown on plate count agar (pca) at 35°c/48 h and fungus was grown on potato dextrose agar (pda) at 25°c/5 days. microbial cultures were maintained at refrigeration temperatures as stock culture, in order to obtain freshly cultured microbial suspension. this microbial suspension, following, was used to microbial activity assay by disc diffusion method. 2.3. preparation of active films cellulose acetate matrix films were obtained by casting according to melo (2003) with same modifications. film-forming solutions were prepared by dissolving cellulose acetate (10% w/v) in acetone. essential oils that were either pure or in combination (1:1 or 1:1:1) were added to the filmogenic solutions at a concentration of 50% w/v. six different films were developed: 1) film incorporated with oregano + sweet fennel + cinnamon eos; 2) film incorporated with oregano + sweet fennel eos, 3) film incorporated with oregano + cinnamon eos; 4) film incorporated with sweet fennel+ cinnamon eos; 5) film incorporated with only oregano eo; and 6) negative control, film without any eos. preliminary analysis by disk diffusion method using individually the oregano, sweet fennel and cinnamon eo, presented effective microbial activity for these oils, highlighting the oregano eo (data no shown).   ital. j. food sci., vol 28, 2016 251 the filmogenic solutions were cast on clean, flat glass plates with a glass rod to a predetermined height. the films were dried at temperature room and then removed from the glass plates. 2.4. in vitro antimicrobial activity of the active films the agar disk diffusion method was used to determine the antimicrobial activity (clsi, 2003). microbial suspensions were prepared and adjusted in brain heart infusion broth (bhi). inoculums of the bacteria were 100 µl of a suspension containing approximately 104 colony-forming units (cfu) per ml for bacteria, or 104 spores/ml for the fungus. the inoculums were spread on the surface of mueller-hinton agar plates. then a sterile film disk (10 mm diameter) incorporated with the eo or eos under test was placed in the center of the agar plate. a film disk without any eos was used as negative control (espitia, 2009). the 6 films were previously exposed to uv light (prodicil, 110v, 254nm) for 15 minutes for sterilization. the plates with bacteria were then incubated at 35±2°c for 48h, while plates with fungus were incubated at 25±2°c for 5 days. following incubation, the total diameter of the inhibition zones (colony-free perimeter) was measured with a caliper. the data were expressed as inhibition zones (cm), including the disk area. each experimented was done in triplicate. 2.5. film characterization 2.5.1. thickness the films thickness was measured with a digital micrometer (mitutoyo -japan). the mean thickness was calculated from five measurements. 2.6. water vapor transmission rate (wvtr) the wvtr of films was measured gravimetrically, using the astm e96-95 standard test method. diffusion cells containing anhydrous calcium chloride desiccant (0% rh) were sealed by the test film. the cells were stored in desiccators, each at a constant rh (75%) with a saturated salt solution and temperature of 25°c. the cells were weighed at 24 h intervals (over a 5 day period). transported water vapor was determined from the weight gain of the diffusion cell at a steady state of transfer. the slope of the weight versus time plot (day) was divided by the effective film area (m2) to obtain the wvtr using the equation below. at least 3 replicates were produced for each film type. wvtr= δw a where wvtr is the water vapor transmission rate (g/d m2) and a is the area of the exposed film surface (m2). 2.7. mechanical analysis puncture tests were performed to evaluate the mechanical strength of the film using a texture analyzer (ta.xt plus, stable micro systems haslemere, england). for the puncture test, film discs (6.4 cm diameter) were fixed to a support with a circular opening   ital. j. food sci., vol 28, 2016 252 of 10 mm in diameter and 15 mm in depth. a cylindrical probe of 5 mm diameter was moved perpendicularly to the film surface at a constant speed of 1 mm/s until it passed through the film. the force-deformation curves were obtained and force (n) and deformation (mm) values at the puncture point were recorded to represent the puncture strength (n) and deformation (mm) of the film. ten replications were performed for the mechanical tests. 2.8. statistical analyses the experiment was carried out in a completely randomized design. all analyses were conducted in three replicates in triplicate. differences between the results of the inhibition zones of the essential oils were evaluated by analysis of variance (anova) using the ftest. the tukey test was used for the antimicrobial analyze and the duncan test was applied to films characterization. differences were considered significant when p < 0.05, using the sas® software 9.0 (sas institute inc., nc, usa). 3. results and discussion 3.1. evaluation antimicrobial of active films incorporated with essential oils antimicrobial efficiency was observed in all formulations of the active film incorporated with eo. it was different of the control film (without eo), that not showed antimicrobial activity. the inhibition zones microbial of several films incorporated with oes against the tested microorganisms are shown in table 2. table 2: inhibition zones (cm) formed by actives films incorporate with essential oils against bacterial strains e. coli and s. aureus and fungus penicillium spp., analyzed at an ideal temperature for the microorganisms to develop: bacteria, 35 ± 2ºc/48 h and fungus 25 ± 2ºc/5 days. active films mean diameter of inhibition zones (cm)+ penicillium spp e. coli s. aureus oregano pure 2.67a 0.99ab 3.75a oregano + cinnamon 2.74a 1.14a 2.7b oregano + sweet fennel 1.36b 0.50c 2.08b oregano + cinnamon+ sweet fennel 1.50b 0.73bc 1.11c cinnamon + sweet fennel 1.15b 0.03d 0.4c *in each column, different superscript letters are significantly different (p< 0.05), by tukey test. + +no includes disc diameter (10 mm). e. coli bacteria was more resistant than s. aureus and penicillium spp. to the treatments with active films in this work. the lower antimicrobial activity against gram negative bacteria, i.e. e.coli, can be attributed to the fact that they are in general more resistant due to the external lipopolysaccharide wall surrounding the peptidoglycan cell wall (zinoviadou et al., 2009). in relation to inhibition of the s. aureus microorganism, the active film with oregano eo was the most efficient (p <0.05), followed by 2 different films: film incorporated with   ital. j. food sci., vol 28, 2016 253 oregano + cinnamon eos and the film incorporated with oregano + sweet fennel eos. there were no differences (p>0.05) found for these two latter films (table 2). the film with combination oregano + cinnamon + sweet fennel eos and the film with combination sweet fennel + cinnamon eos were both statistically similar (p> 0.05). they showed lower activity than the other films against the microorganism s. aureus (table 2). the films incorporated with oregano + cinnamon eos and the film incorporated with oregano eo alone proved to be the most efficient against the microorganism e. coli. the film with the combination of cinnamon + sweet fennel eos was the least effective (table 2) against this bacteria. fungus penicillium spp. was most sensitive to the film incorporated with oregano eo alone and to the film with combination of oregano + cinnamon eo (table 2). the films into oregano and the film with combination cinnamon + oregano presented the highest antimicrobial activity against bacteria and fungus tested . in the work of ye et al (2013) was showed that the active components, cinnamaldehyde and carvacrol expressed high antibacterial activities both to e. coli and s. aures, besides of have synergistic antimicrobial activities for the bacterias. the efficiency of the oregano eo against some microorganisms has already been reported by pereira et al. (2008), pesavento et al. (2015) and martucci et al. (2015). positive results were also observed by emiroglu et al. (2010), when a combination of thyme eo with oregano eo was incorporated into soy-based films. these authors showed an in vitro antimicrobial effect against s. aureus and e. coli 0157: h7. according to preliminary studies (data no shown), the inhibitory effect of oregano eo and combinations of eos when incorporated into the cellulose acetate films was lower than that found in pure oregano eo and combinations of eos. the possible causes that would explain this result could be a partial loss of volatile compounds during film preparation. it may also be due to dilution of the eos in the filmogenic solutions. various tests with eos incorporated in films have obtained results that indicate a real potential to use such films in the food industry for packaging. emiroglu et al. (2010) evaluated the effect of a soy edible film incorporated with natural antimicrobial, thyme and oregano eos on fresh ground beef patties; while oussalah et al. (2007) used the alginate based film incorporated with winter savory, oregano and cinnamon eo to wrap ham and bologna. 3.2. film characterization 3.2.1. thickness and water vapor transmission rate (wvtr) adding eos causes an increase in the thickness of the films. there was a difference (p>0.05) between the thicknesses of the control film (without eo) and the active films incorporated with eo. however, the thickness of the different films after incorporating the eos was similar, ranging from 29 to 44mm, as shown in table 3. the water resistance of the films improved with the incorporation of the eos, i.e., there was a reduction in the wvtr. the wvtr of the films with eos presented a significant difference (p< 0.05) when compared to the control films (without eo). however, the wvtr films with eos (table 3) was same. films incorporated with eos showed a better barrier against water vapor. they had a lower permeability, and therefore are indicated for moisture control for moisture sensitive foods as they would give better protection. the hydrophobic nature of eos could potentially increase the hydrophobicity of cellulose acetate film. studies on the incorporation of lipids as eos into films have shown improvements in their water vapor barrier properties (zinoviadou et al., 2009; bahram et al., 2012).   ital. j. food sci., vol 28, 2016 254 table 3: water vapor transmission rate (wvtr) and thickness of differents active films incorporate with essential oils. active films thickness (µm) wvtr (g.m-2d-1) negative control (without eo) 29.0ª ± 1.6 259.61ª ± 69.46 oregano pure 42.8b ± 3.2 162.92b ± 27.23 oregano + cinnamon 38.5b ± 1.0 175.61b ± 14.54 oregano + sweet fennel 44.3b ± 4.7 192.10b ± 1.95 oregano + cinnamon + sweet fennel 42.8b ± 3.2 197.86b ± 7.71 cinnamon + sweet fennel 40.0 b± 0.4 152.80b ± 37.35 *in each column, different superscript letters are significantly different (p< 0.05), by duncan test. 3.3 mechanical analysis the kind of eos and its concentration affect the mechanical properties of films differently. the magnitude of such differences is dependent on the added compound (shojaeealiabadi et al., 2014). puncture tests were performed to evaluate the mechanical strength of the film. table 4 shows the influence of eo incorporations on the mechanical properties of cellulose acetate films. the presence of eo droplets in the film matrix affected both puncture strength and deformation at the break point of the films. table 4: mechanical analysis of different active films incorporate with essential oils puncture tests: puncture strength (ps) and deformation at breaking point (db). active films db (mm) ps (n) negative control (without eo) 2.71ª ± 0.76 23.42ª ± 1.24 oregano pure 3.09b ± 0.38 30.94b ± 6.28 oregano + cinnamon 4.18c ± 0.71 26.53ª ± 1.87 oregano + sweet fennel 3.28bd± 0.19 24.37ª ± 0.29 oregano + cinnamon + sweet fennel 3.98c ± 0.51 24.65ª ± 0.01 cinnamon + sweet fennel 3.58d ± 0.11 18.07c ± 6.59 *in each column, different superscript letters are significantly different (p< 0.05), by duncan test. the puncture strength (ps) ranged from 18.1 to 30.9 n, for films for combinations of cinnamon + sweet fennel eos and pure oregano eo, respectively. the film with pure oregano eo was the most resistant, thus, it had a greater force at breaking point. films incorporated with a combination of oregano + cinnamon + sweet fennel eos and the oregano + cinnamon eos were the most elastic, i.e. they had a higher deformation at the breaking point, while the control film presented the smallest deformation at breaking point (table 4). therefore, the presence of eos in film matrices, makes them more elastic, resulting in more stretchable films, depending on the eo concentrations (shojaee   ital. j. food sci., vol 28, 2016 255 aliabadi et al., 2013). it occurs because, the eo acted as a plasticizer and increased the flexibility of the polymer chains (atef et al., 2015). the kind and amount of phenolic compounds present in oregano, cinnamon and sweet feenel eos cause different binding and interactions between cellulose acetate molecules and essential oil (lee et al., 2015). as resulted, the films containing these eo had different mechanical properties. the work reported by atarés et al. (2010a) who conducted tests using scanning electron microscopy and surface morphology in addition to other tests, observed that films with ginger eo was less resistant and less elastic than those with cinnamon eo. atarés et al. (2010b) evaluated the use of cinnamon and ginger eos in low proportions (less than 1:0.1 ratio of lipid to protein) in sodium caseinate based films. the authors observed that this quantity did not cause any impact on the mechanical properties and only caused a small impact on the water vapor permeability of the film. when agents are incorporated into packaging materials, polymeric matrix physical and mechanical properties are changed, there are modifications specific to each combination of antimicrobial polymer. consequently when a new active packaging material is developed, it is born with new specific characteristics (soares and gonçalves, 2008). 4. conclusions essential oils can be recognized as an appropriate natural food preservative, and may be used to replace the synthetic preservatives. films incorporated with pure oregano eo and the combination of oregano + cinnamon eos appear as an alternative to extend the commercial validity of foods. therefore, there is the possibility to replace the synthetic chemical preservatives direct used in foods for antimicrobial active packaging incorporated with eos for food preservation. active packaging based on cellulose acetate incorporated with eos besides boosting food safety, maintaining their quality and safety, contributes to reducing the environmental impact of traditional packaging because it is based on renewable sources of raw materials. the technology of active materials is an emerging and promising area of research and it can confer multiple benefits for a wide range of products. however, more research needs to be conducted for different applications in order to evaluate the technological, economic and safety potentials. acknowledgements this work was supported by coordenação de aperfeiçoamento de pessoal de nível superior (capes), fundação de amparo à pesquisa do estado do rio de janeiro (faperj), conselho nacional de desenvolvimento científico e tecnológico (cnpq), pró-reitoria de pesquisa, pós-graduação e 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of honey a. romano1, m. cuenca 1, s. makhoul2, f. biasioli2, l. martinello3, a. fugatti3 and m. scampicchio1* 1free university of bolzano, faculty of science and technology, piazza università 1, 39100 bolzano, italy 2department of food quality and nutrition, research and innovation centre, fondazione edmund mach (fem), via e. mach 1, 38010 san michele all’adige (tn), italy 3servizio veterinario dell’azienda sanitaria dell’alto adige, via laura conti 4, 39100, bolzano, italy *corresponding author. tel.: +39 0471017210; fax: +39 0471017009 e-mail address: matteo.scampicchio@unibz.it abstract authentication is a major research theme in food analysis. electronic noses (e-noses) represent an effective tool for food authentication and among others for the determination of food origin. in this work we compare the performance of two e-noses (metal-oxide-sensor based vs mass-spectrometry based) in the determination of geographic origin of honey. we analyzed 14 honey samples from south tyrol, an italian alpine region, which were compared with other 12 commercial samples from diverse european origins. both e-noses afforded 85% of correct identifications. mass spectrometry provided a deep analytical insight, thanks to the possibility to determine mass peaks with good accuracy. keywords: south tyrol italian honeys, geographic origin, ptr-tof-ms, e-nose   ital. j. food sci., vol 28, 2016 327 1. introduction gas-chromatographic methods represent the benchmark for food volatile analysis. in spite of its robustness and analytical power, gas-chromatographic analysis, being a separationbased technique, is time-consuming and has a low analytical throughput. an alternative analytical approach is based on the employment of electronic noses (e-noses). in the enose, an array of electrochemical sensors (gardner, 1999) or a mass spectrometer (pérez pavón et al., 2006) provide a fingerprint of the headspace of a given sample. typically an e-nose, trained using samples of known origin, can be employed to recognize and predict sample identity on the basis of a specific fingerprint. unlike gc-ms, the enose provides little information as to the actual composition of the sample headspace; on the other hand e-noses are generally easy to use, they provide a high analytical throughput and they are relatively inexpensive. proton transfer reaction-mass spectrometry (ptr-ms), similarly to an ms-based e-nose, performs a rapid and direct analysis of the headspace of the sample. unlike in ms-based enoses the use of a soft ionization approach allows to minimize fragmentation, thus increasing the informational content of the mass spectral fingerprint (hansel et al., 1995). the coupling of ptr-ms to time-of-flight (tof) mass analyzers has further enhanced the performance of the technique, allowing for high mass and time resolution. ptr-tof-ms has already been employed in the determination of food origin, with applications, among others, on cheese (galle et al., 2011), ham (del pulgar et al., 2011) and coffee (yener et al., 2015). honey is traditionally consumed and appreciated worldwide, mainly because of its organoleptic properties and nutritional value. even though the main constituents of honey are sugar and water, a great variety of aroma compounds can also be encountered. gas chromatography-mass spectrometry (gc-ms) was often employed to describe the composition of honey headspace, and up to 400 distinct vocs were reported is a single honey type (guyot et al., 1998). the two main factors affecting the quality of honey are its botanical and geographical origin. the botanical origin of the nectar and plant secretions is a major source of aroma compounds and aroma precursors but geographical origin, through the influence of soil and climate, also plays an important role. several gcms studies were carried out with the aim to study and predict the botanical and/or geographical origin of honeys (cuevas-glory et al., 2007; kaškonienė and venskutonis, 2010). this approach mostly applies in the case of honeys deriving from a single plant species (unifloral honeys) and researchers have claimed the discovery of plant specific-markers (castro-vázquez et al., 2006; jerković et al., 2006; verzera et al., 2014; jerković and kuś, 2014); these most often include terpenes, norisoprenoids, nitriles or phenolic compounds and their derivatives (manyi-loh et al., 2011a). less frequently, putative markers of geographic origins have also been detected (radovic et al., 2001). e-noses have been effectively employed in discriminating honeys from different botanical and/or geographical origins (benedetti et al., 2004; dymerski et al., 2014; zakaria et al., 2011). sometimes the e-nose was coupled to another technique, such as fourier transform-infra red (ft-ir) spectroscopy (subari et al., 2012) or electronic tongue (buratti et al., 2004). the coupling of analytical techniques based on different physicalchemical principles and the “merged” dataset thus generated often allowed for an enhancement in discrimination capability. recently, ptr-ms coupled to a quadrupole mass analyzer was employed in the classification of honeys having different botanical origins (kuś and van ruth, 2015): the technique was not always able to perform a correct classification, affording an average prediction accuracy of 77%. in another recent paper (schuhfried et al., 2016), a ptr-ms   ital. j. food sci., vol 28, 2016 328 instrument using a time-of-flight (tof) mass analyzer was employed in the headspace analysis of 70 mono-floral honeys of diverse origins. the higher mass resolution of the tof detector, along with the use of multivariate classification techniques, provided 90-100% correct predictions based on botanical origin. the assessment of food typicality represents an issue of major relevance to the food industry and a challenging task from an analytical point of view. the objective of the present work is thus twofold: (i) find new analytical tools for the valorization of local food production and (ii) compare the performance of two e-noses based on different physicalchemical principles. quite interestingly, the two instruments provide similar performance, with advantages and drawbacks on both sides: the mos-based e-nose is more portable and low-cost but it does not provide information as to sample headspace composition, whereas the ms-based instrument gives a fairly detailed analytical insight. 2. materials and methods 2.1 honey samples honey commercial samples were provided by the servizio veterinario dell’azienda sanitaria dell’alto adige (bolzano, italy). the sample set consisted of 26 honeys, out of which 14 originating from south tyrol and 12 from other european countries (namely italy, romania, spain, germany and czech republic). from the point of view of botanical origins, the sample set was rather heterogeneous, including multi-flower and forest samples as well as monofloral honeys (namely from acacia, chestnut, dandelion, lime and eucalyptus). the year of production of the honeys was 2013. all samplings were performed from the same jar, typically containing 250-500 g of sample. for a more detailed description of the sample set please refer to table 1, supplementary material. 2.2 ptr-tof-ms headspace measurements were performed using a commercial ptr-tof 8000 instrument (ionicon analytik gmbh, innsbruck, austria). the instrumental conditions in the drift tube were the following: drift voltage 550 v, drift temperature 110°c, drift pressure 2.33 mbar affording an e/n value of 140 townsend (1 td = 10-17 v*cm2). sampling was performed with a flow rate of 40 sccm using a heated (110°c) peek transfer line. measurements were performed in an automated fashion by means of a multipurpose gc automatic sampler (gerstel gmbh, mulheim am ruhr, germany). the analytical method was mutuated from a previously validated method for coffee powder headspace analysis (yener et al., 2014), with some minor adaptations (not shown). honey aliquots (1.0 g) were transferred into 40-ml glass screw-capped vials, suitable for volatile analysis. all measurements were performed in triplicate. the measurement order was randomized to avoid possible systematic memory effects. all vials were incubated at 40°c for 30 min before ptr-ms analysis. each sample was measured for 30 s, at an acquisition rate of one mass spectrum per second. data processing of tof spectra included dead time correction, internal calibration and peak extraction steps performed according to a procedure described elsewhere (cappellin et al., 2010). in this case this allowed to reach a mass accuracy better than 0.001 th, which is sufficient for sum formula determination. the baseline of the mass spectra was removed after averaging the whole measurement and peak detection and peak area extraction was performed by using modified gaussian to fit the data (cappellin et al., 2011). to determine the concentrations of volatile compounds in ppbv = nl of voc l-1 headspace the formulas described by lindinger and jordan   ital. j. food sci., vol 28, 2016 329 (1998) were used by assuming a constant reaction rate coefficient (kr=2×10−9 cm3/s) for h3o+ as a primary ion. the ptr-tof-ms dataset was submitted to an initial step of filtration based upon concentration. the selection of mass peaks exceeding an arbitrary threshold has often proven to be an effective empirical approach to improve the discrimination ability of ptrms data (aprea et al., 2015). a concentration threshold arbitrarily set at 1 ppbv, was applied. when peaks having an estimated concentration higher than 1 ppbv were selected, a subset of 55 peaks was generated. for the purpose of multivariate analysis, the mass spectral fingerprint obtained for every sample was normalized by the corresponding total emission. normalization has already been proven useful (yener et al., 2015), generally allowing to compensate variations in total emission, at the same time preserving the mass spectral fingerprint typical of each sample or sample class. 2.3 electronic nose analyses were performed with a pen3 e-nose (airsense analytics, schwerin, germany). the instrument has ten metal oxide semiconductor (mos) sensors displaying different specificity profiles (table 2). the e-nose was equipped with an automated sampling device (headspace sampler hss32 from airsense analytics). the analytical procedure for e-nose honey analysis was validated in a previous work (zuluaga et al., 2011). honey samples were measured directly, with no prior dilution. one-gram aliquots were transferred into 10-ml vials, suited for volatile compound analysis. samples were equilibrated for 20 minutes at 40°c and then analyzed. the e-nose program was based upon measurement cycles of 150 seconds, separated by 450 seconds of sensor flushing with clean air. inlet flow was set at 400 ml/min. e-nose sensor specificities, as stated by the producer, are reported in table 2. table 1: main characteristics of the honey samples used in the study. sample designation geographic origin botanical origin h01 south tyrol acacia h02 south tyrol forest h03 south tyrol forest h04 south tyrol multiflower h05 south tyrol forest h06 south tyrol mixed (forest/flower) h07 south tyrol multiflower h08 south tyrol multiflower h09 south tyrol multiflower h10 south tyrol chestnut h11 south tyrol multiflower h12 south tyrol multiflower h13 south tyrol multiflower   ital. j. food sci., vol 28, 2016 330 table 2: e-nose sensor specificities, as declared by the manufacturer. 2.4. software all data analysis was performed using matlab (statsoft, natick, ma) and r software (the r foundation for statistical computing, vienna, austria). h14 south tyrol multiflower h15 south tyrol multiflower h16 south tyrol dandelion h17 south tyrol multiflower h18 south tyrol multiflower h19 italy chestnut h20 italy chestnut h21 italy lime h22 italy, romania acacia h23 italy, spain, romania forest h24 italy eucalyptus h25 eu multiflower h26 germany multiflower h27 czech republic multiflower h28 czech republic multiflower h29 czech republic forest sensor specificity s1 aromatic compounds s2 broad range, very sensitive, reacts with nitrogen oxides s3 ammonia, aromatic compounds s4 mainly hydrogen, selectively (breath gases) s5 alkenes and less polar aromatic compounds s6 methane, broad range s7 sulfur compounds, terpenes, limonene and pyridine s8 alcohols, broad range s9 sulfur organic compounds s10 reacts on high concentrations (>100ppm), sometimes very selective (methane)   ital. j. food sci., vol 28, 2016 331 2.5. statistical analysis all results are to be intended as means of triplicate measurements. for principal component analysis (pca), all variables were normalized using the respective standard deviations. linear discriminant analysis (lda) was carried out with the aid of the r package mass (venables, ripley, and venables 2002). lda models were cross-validated by “leave-one-out” method. 3. results and discussions 3.1. classification of honey samples based on metal oxide sensors in this study, the headspace of 26 honey samples was analyzed with a portable electronic nose. such device employed an array of ten electronic gas sensors (metal oxide sensors) able to detect and distinguish headspace volatiles via a pattern-recognition algorithm. typical signals generated by the e-nose with honey samples are shown in fig. 1. figure 1: typical e-nose profile obtained on a honey sample. signals were averaged between 80 and 100 seconds, as indicated by the shaded area. the responses from each sensor during 120 s allowed to extract two data: the maximum signal and a plateau value (recorded arbitrarily between 80 and 100 s of measurement). based on a preliminary data analysis (results not shown), the two data sets displayed a high degree of covariance; the plateau values, which displayed better repeatability, where thus used in subsequent analysis, whereas maximum values were discarded. fig. 2 shows the score plot of the principal component analysis (pca). pca is an unsupervised pattern recognition tool very useful to plot in a reduced dimensional space (i.e. generally, the first two principal components are sufficient to explain most of the variance contained in the original dataset), observe any potential similarities between the samples and, in case, identify the most important variables responsible for such similarities. in detail, the score values of the first two principal components (accounting for the 91% of the total   ital. j. food sci., vol 28, 2016 332 variability) allow to establish a relatively good separation between samples from south tyrol from those of other origins, mostly obtained through the employment of component 2. looking at the loading values (fig. 2), the observed discrimination capacity is mainly explained by just 5 metal oxide sensors, and namely s2, s6, s7, s8 and s9. the higher response obtained with sensor s8 in honeys of various european origins is in agreement with mass spectrometric data, showing a higher ethanol content (paragraph 3.2). sensors s7 and s9, which are specific for sulfur compounds, show more intense signals in south tyrol honeys. instead, sulfur compounds do not seem to be important for honey discrimination by ptr-ms, thus suggesting that the analytical responses provided by the two e-noses are somewhat complementary. figure 2: principal component analysis of the autoscaled data obtained by e-nose (● = south tyrol, ○ = other). alphanumeric codes correspond to e-nose sensors. 3.2. classification of honey samples based on ptr-tof-ms we next investigated the potential enhancement offered by a more advanced electronic nose based on mass spectrometry. here we used an on-line proton-transfer-reaction mass spectrometry based on hydronium ions as ion source reagents directly connected to an analyzing time-of-flight mass spectrometer system (ptr-tof-ms). fig. 3 shows a typical mass spectrum obtained for a honey sample, in the range 15-215 th of mass-to-charge ratio (m/z). the high mass resolution provided by the time-of-flight mass analyzer enabled the detection of more than 204 mass peaks. upon filtering based on average concentration a subset of 55 mass peaks, having concentrations higher than 1ppbv, was selected and employed in further analyses.   ital. j. food sci., vol 28, 2016 333 figure 3: typical mass spectrum obtained on a honey sample. the position of some selected mass peaks is highlighted by the corresponding nominal masses. pca was next used to reduce the dataset dimensionality, observe sample similarities and highlight the most important mass fragments (fig. 4). the score values of the first two principal components (accounting for the 53% of the total variability) allow visualize a partial separation between the samples from south tyrol respect those from other origin. separation was achieved thanks to both principal components 1 and 2. the visual inspection of principal component higher than two did not provide an improvement in discrimination ability. to understand which mass fragment is responsible for the observed clustering of the samples, the loading values were then analyzed; a subset of 10 mass peaks was defined (fig. 4), referring to the variables whose loadings showed the highest absolute values for either principal component. figure 4: principal component analysis of the autoscaled data obtained by ptr-tof-ms (● = south tyrol, ○ = other). alphanumeric codes correspond to mass-to-charge ratios as measured by ptr-tof-ms.   ital. j. food sci., vol 28, 2016 334 the mass spectrometric data obtained by means of the ptr-tof has a resolution of 4000 dm/m or higher. this, after calibration, typically allows for the determination of masses up to the third decimal digit, eventually ensuring the assignment of a sum formula to most mass peaks. the cross-matching of mass spectral data with published databases of honey volatiles (kaškonienė and venskutonis 2010; manyi-loh, ndip, and clarke 2011b; wolski et al. 2006) and fragmentation patterns of pure compounds (aprea et al. 2007; buhr, van ruth, and delahunty 2002; demarcke et al. 2009) allowed to tentatively assign some of the detected mass peaks to known constituents of the headspace of honey. all these compounds are well representative of factors having a key impact in affecting honey quality and characteristics such as floral origin, oxidation, fermentation etc. mass peaks m/z 84.082 (with a fragment at m/z 70.066), m/z 81.034 and m/z 111.044 were tentatively attributed to an n-heterocycles and carbonyls which, even though not necessarily reported in honey, are known as maillard reaction intermediates and might participate in non-enzymatic browning reactions that take part in the oxidative alteration of many food products, including honey (nursten 2005). mass peak m/z 107.049 (along with fragment at m/z 79.054 and 13c isotopologue at m/z 108.053) was tentatively assigned to benzaldehyde, a compound previously reported in some unifloral honeys (moreira and de maria 2005) and associated to almond and burnt sugar sensory notes (acree and arn 2004). other relevant mass peaks could be assigned to well-known fermentation products already detected in honey (wolski et al. 2006). they namely were ethanol (m/z 47.048 and fragment at m/z 29.040), methyl-acetate and methyl-formate (m/z 75.044), and acetoin, butyric acid, butyrolactone and ethyl-acetate (m/z 89.060 and fragment m/z 71.049). 3.3. comparison of the e-noses two classification models based on linear discriminant analysis (lda) were next build up on the basis of the most important variables selected by pca for e-nose based on mos sensors and ptr-tof-ms, respectively. lda is a supervised pattern recognition tool especially developed for qualitative classification problems. when the signal from the selected mos sensors were used, the resulting model afforded correct identifications for 82% and 87% of south tyrol honeys and samples of other origin, respectively, with an overall 85% of correct identifications. instead, when the model was built with the selected fragments from ptr-tos-ms, then, the resulting classification model was able to correctly identify 92% and 78% of honeys samples from south tyrol and other origin, respectively, with an overall 85% of correct identifications. further detail about the discrimination is provided in the supplementary material (table 3). the results show that a portable e-nose, once validated, may have classification performance similar or even better than that achievable with instruments based on high resolution mass spectrometry. contrarily to what expected, the higher amount of fragments detected by the high resolution mass spectrometers do not result with a higher capacity to discriminate samples. apparently, the capacity of discriminating the samples is hindered by an increased noise or uncertainty around the signal of individual fragments. table 3: lda confusion matrices, as obtained using e-nose and ptr-tof-ms (left and right, respectively). o ri gi na l model based upon mos-based e-nose model based upon ptr-tof-ms predicted predicted other south-tyrol other south-tyrol other 10 2 11 1 south-tyrol 2 12 3 11   ital. j. food sci., vol 28, 2016 335 4. conclusions this research work presents an unprecedented analytical approach based on two different types of electronic nose. this approach was employed to address the question of the typicality of honeys from south tyrol (italy). ptr-tof-ms, with its high mass resolution, allowed for a rapid, yet thorough characterization of the honey headspace, permitting to pinpoint several candidate aromatic markers. the mos-based electronic nose provided a cost effective solution to the same problem, being more portable and less expensive than the latter instrument. the sample set was of limited size (26 honeys) and the work is thus intended to be a preliminary study. botanical diversity, which is known to play a major role on headspace composition, was probably underestimated, and the corroboration of these first results by means of a more extended survey is indeed advisable. the work also addresses the strategic theme of the typicality of food products issued from a small alpine region (south tyrol). the work demonstrates how the development of novel analytical approaches can enable researchers and institutions to validate the typicality of regional products, representing an undoubted source of added value to all local productions. acknowledgements we thank the province of bolzano for financial support (landesregierung mittels beschluss nr. 1472, 07.10.2013). references acree t.e. and heinrich arn. 2004. “flavornet.” http://www.flavornet.org/. aprea e., biasioli f., märk t.d. and gasperi f. 2007. ptr-ms study of esters in water and water/ethanol solutions: fragmentation patterns and partition coefficients.” international journal of mass spectrometry 262 (1-2): 114-21. aprea e., romano a., betta e., 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ijfs#339_zanoni bozza   ital. j. food sci., vol 28, 2016 352 short communication the case of the 2014 crop season in tuscany: a survey of the effect of the olive fruit fly attack l. cecchi1, m. migliorini1, c. cherubini1, s. trapani2 and b. zanoni2* 1promofirenze, special agency of the florence chamber of commerce laboratorio chimico merceologico unit, via orcagna 70, 50121 florence, italy 2department of agricultural, food and forestry systems management (gesaaf) food science and technology and microbiology section, university of florence, via donizetti 6, 50144 florence, italy *corresponding author. tel.: +39 055 2755507; fax: 055 2755500 e-mail address: bruno.zanoni@unifi.it abstract in this work we compare the chemical composition of olive fruits (cv moraiolo) and the sensory and chemical quality of olive oils extracted in two crop seasons: the 2013 crop season, characterized by a very minor attack by the bactrocera oleae, and the 2014 crop season, characterized by one of the strongest attacks of bactrocera oleae in the last decades. results show that during the 2014 crop season the pulp/stone ratio, moisture, phenolic content, oil content, and sugar content were lower than in the 2013 crop season. moreover, the olive oils from the 2014 crop season were characterized both by higher free acidity values, peroxide value and k232, and by lower values of antioxidants (phenolic compounds and tocopherols). the olive oils from the 2013 crop season did not present any defects, while among those from the 2014 crop season, 41% were defective and did not fall into the extra virgin olive oil category. keywords: extra virgin olive oil, moraiolo cultivar, olive degradation, olive fly, olive oil quality   ital. j. food sci., vol 28, 2016 353 1. introduction in recent decades, extra virgin olive oil (evoo) has been attracting the interest of the scientific community, also due to its well-recognized nutraceutical and sensory properties (cecchi et al., 2015). these properties go to define the quality of evoo and are affected by various factors, one of which is the olive fruits’ health status (migliorini et al., 2012). one of the main risk factors for the health of olive fruits is related to the attack of pathogens and phytophagy, the most dangerous aggressor being the olive fly, bactrocera oleae (perovic et al., 2007; wang et al., 2009; wang et al., 2013). in most crop seasons in the inland areas of tuscany, attacks by the olive fly are scarce, so the effect on evoo quality is low. instead, in some crop seasons, olive fly attacks can affect up to 100% of the olive fruits in some areas, with very heavy consequences on the sensory and nutritional properties of evoo. various parameters can be used to make a quantitative assessment of olive fly attacks. among these parameters, active infestation only accounts for the stages of development of the larvae up to the second age and is used to establish the threshold beyond which a chemical treatment is justified (this threshold is 10% of active infestation). the total infestation takes into account all the types of infestation caused by olive fly and, therefore, can be better correlated to the variations in the chemical parameters of the olive fruits (caleca et al., 2007). in fact, the main risk factors for the fruits’ state of health are those that cause the rupture of the skin. the main consequences of such a rupture are: (i) exposure to oxygen of the olive fruits’ chemical constituents; evaporation of water from the fruit no longer protected by the outer waxy layer; (ii) alteration of the metabolic processes of the fruit; (iii) attacks from exogenous and endogenous enzymes. each of these phenomena affects the olive fruits’ chemical composition, which can compromise the quality parameters of the evoos (servili et al., 2007), as also stated in studies conducted on oils obtained by milling different mixtures of healthy and damaged olives on a laboratory scale (koprivnjak et al., 2010; pereira et al., 2004; mraicha et al., 2010) or on oils from partially damaged olives in different industrial mills (gomez-caravaca et al., 2008). the decrease in the oil quality mainly depends on: i) the kind of infestation, ii) the percentage of damaged fruits, iii) the cultivar, and iv) the fruits’ stage of development (gucci et al., 2012). the 2014 crop season was characterized by a very intense attack by the olive fly and, in tuscany, almost all the olive plantations were totally damaged. one of the main reasons for this attack was the many climatic anomalies during the fall 2013 fall 2014 period (lamma, 2013; lamma, 2014a; lamma, 2014b; lamma, 2015). the main goal of this study was to represent the chemical composition of the olive fruits and the extracted evoos in light of the olive fruit fly attack in the 2014 crop season in tuscany. 2. materials and methods 2.1. data relating to olive fly infestation data relating to active infestation and total infestation were collected from the azienda agricola buonamici organic farm (fiesole, florence, italy) by agroambiente (2014), a tuscany regional government website which collects data on olive infestation.   ital. j. food sci., vol 28, 2016 354 2.2 olive fruit samples during the 2013 and 2014 crop seasons 10 olive trees of both the frantoio and moraiolo cvs were selected from the azienda agricola buonamici organic farm (fiesole, florence, italy) and fattoria altomena (pelago, florence, italy). olive fruit samples, of approximately 500 g, were collected from these plants on a weekly basis: from september 8 to november 24 in 2013, and from september 1 to october 27 in 2014. all the samples were analyzed as soon as they were delivered to the laboratory. 2.3 oil samples the oil samples consisted of olive oils produced in tuscany, mainly in inland areas, which were analyzed by the laboratorio chimico merceologico (promofirenze, special agency of the florence chamber of commerce, florence, italy) in the 2013 and 2014 seasons. the oils were from the same farms in the two crop seasons, with the exception of the farms which did not harvest the olives in 2014 because they had been completely ruined by the olive fly. 2.4 chemical analyses on the olive fruits the total phenolic content, water content and oil content in the olive fruit samples were evaluated as previously reported (cecchi et al., 2013). one hundred olive fruits were weighed and pitted to calculate the pulp/stone ratio. the stones were weighed, and the pulp weight was calculated as the difference between the weight of the whole olive fruit and the weight of the stones. the pulp/stone ratio was calculated by dividing the pulp weight by the stone weight. to measure the sugar content of the olive fruits, eight grams of olive paste were cold extracted (6 ± 2°c) with distilled water in a 200 ml flask for 2 hours. the content of the flask was filtered through paper and 10 ml of the solution obtained were diluted with water in a 20 ml flask. the sugar content was analyzed with an enzymatic method using the chemwell automatic analyzer (awareness technology, chemwell 9210, palm city, fl). three enzymatic kits were used to measure respectively (i) the sum of two monosaccharide contents, namely glucose and fructose; (ii) the disaccharide sucrose content; and (iii) the polyol mannitol content. all the kits were purchased from rbiopharm (darmstadt, germany). measurements were performed by means of external calibration standards: fructose and glucose (purity > 99%, sigma aldrich srl, milan, italy), and mannitol (purity > 98%, sigma aldrich srl, milan, italy). the results provided by the instrument were expressed in g/l; they were also converted into sugar content on a dry matter basis (g/kg dm) as the average of two readings carried out for each sample. the sucrose contents were determined by multiplying the difference between the sum of glucose, fructose, and sucrose contents, and the sum of glucose and fructose contents, by 0.95. 2.5 chemical and sensory analyses on the olive oils the free acidity, peroxide number, and uv spectrophotometric indices (k232, k270, ∆k) of the oil samples were determined according to the analytical methods (eec reg. 2568/1991). the tocopherols of the olive oil samples were determined according to the iso 9936:2006/corr. 1:2008 analytical method (iso 9936/2008), while the total phenolic compounds were quantified according to the coi/t.20/doc. no. 29 analytical method (coi/t.20/doc.29, 2009).   ital. j. food sci., vol 28, 2016 355 sensory evaluation of the olive oil samples was performed according to eec 2568/91 regulations and the following amendments (eec reg. 2568/1991). the form used for the sensory evaluation was developed according to the eec 2568/91 regulation (eec reg. 2568/1991) and the coi/t.20/doc. no 15/rev. 8 february 2015 document (coi/t.20/doc. 15/ rev.7., 2015). 2.6 statistical analyses the f-test was used for each parameter to test if the variances in the data from the two crop seasons were unequal. statistical significance was determined using microsoft excel statistical software (t-test: two-sample assuming unequal variances and using two-tailed distribution). results of p < 0.05 were deemed as differences. 3. results and discussions table 1 compares levels of active and total olive fly infestation in the 2013 and 2014 crop seasons in tuscany. as shown, in the 2013 crop season, the active infestation never exceeded three attacks per 100 olives, remaining below the threshold for chemical treatment (10 attacks per 100 olive fruits). instead, in the 2014 crop season, at the end of july, the active infestation had already passed this threshold. regarding the total infestation, it remained below six attacks per 100 olives throughout the 2013 crop season, while during the 2014 crop season it exceeded 100 attacks per 100 olive fruits. to understand the effect of the attack on the olive fruits’ state of health and on the quality of the extracted evoo, it is enough to observe that at the peak of attack in the 2014 crop season, there was an active infestation of approximately 40% while the total infestation even exceeded 100%; this suggests that at least 60% of the fruits had an infestation due to the presence of exit holes or larvae older than the second age, with an obvious detrimental effect on the quality of the fruit itself. table 1. levels of active and total olive fly infestation in the 2013 and 2014 crop seasons. data are shown as number of attacks per 100 olive fruits. active infestation total infestation active infestation total infestation jul 17 4 4 jul 23 0 0 6 6 jul 31 0 0 12 12 aug 07 0 0 15 16 aug 14 0 0 5 18 aug 20 0 0 9 22 aug 27 1 2 13 30 sep 04 1 2 20 47 sep 11 1 2 26 72 sep 18 3 5 32 79 sep 25 3 3 36 88 oct 02 3 6 40 95 oct 09 2 3 42 111 2013 2014   ital. j. food sci., vol 28, 2016 356 many factors seem to have contributed to the intense olive fly attack in tuscany in the 2014 crop season. first, the 2013 crop season was a year of vegetative charge, as a consequence, in some cases the harvest was not completed; while the 2014 crop season was a year of vegetative discharge. second, tuscany was affected by major climatic anomalies during the period of fall 2013 fall 2014. the mild winter temperatures meant that the flies could overwinter. therefore, they were able to lay their eggs on the olive fruits remaining on the ground, especially in the olive groves where the harvest in 2013 had not been completed; in addition, the june "heat wave" limited the olives’ productivity, due to the concomitant start of the fruit set. finally, the mild temperatures combined with the high summer rainfall created the perfect precondition for a wide and rapid spread of the fly from generation to generation (lamma, 2014b). figures 1 and 2 show the chemical content trends during ripening of the cv moraiolo olive fruits from azienda agricola buonamici in the two crop seasons. the data regarding the cv frantoio olives and the olives from fattoria altomena gave the same conclusions, so they are not shown in this short communication. in the 2014 crop season the pulp/stone ratio did not increase during the ripening period, in contrast to what was observed in 2013 (fig. 1). this may be due to several effects of the olive fly attack, such as the modification or impairment of the growth processes of the fruit and decrease in pulp owing to the trophic effect caused by the parasite. the natural consequence of the lower contribution of the pulp to the total weight of the olive fruit was a lower moisture content in the olives (fig. 1). the moisture contents were comparable until september 22, when the pulp/stone ratio in the two olive oil seasons was not very different. after that date, the moisture in the 2014 crop season plummeted dramatically to values of up to 30% less than in 2013. the sugar content of the olives was also affected by the olive fly attack (fig. 2). in the 2013 crop season we can observe a sugar content of 40-45 g/kg of dry matter at the optimal ripening time (migliorini et al., 2011; cecchi et al., 2013). instead, in the 2014 crop season (october 22) the sugar content was already less than 10-15 g/kg of the dry matter. this drastic reduction may be due to both to the parasite compromising the biochemical maturation process and the evident loss of pulp. a similar effect was seen for the oil content of the olive fruits (fig. 2) and, consequently, the theoretical yields in the mill. in october we observed a break in the biochemical phenomenon of oil accumulation, together with a removal of pulp due to the fly’s trophic activity. finally, the phenolic content of the 2014 crop season was always lower than that of the 2013 crop season, reaching a difference of 35% at the end of october (fig. 2). this was probably due to the combined effect of the fly attack and the high water availability (pannelli et al., 1994) in the 2014 crop season.   ital. j. food sci., vol 28, 2016 357 figure 1: pulp/stone ratio and moisture content trends in cv moraiolo olive fruits during ripening in the 2013 and 2014 crop seasons. 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 25 -a ug 1se p 8se p 15 -s ep 22 -s ep 29 -s ep 6o ct 13 -o ct 20 -o ct 27 -o ct 3n ov 10 -n ov 17 -n ov 24 -n ov pulp/stone ra tio (g/g) pulp/stone ratio 2013 2014 30 35 40 45 50 55 60 65 70 25 -a ug 1se p 8se p 15 -s ep 22 -s ep 29 -s ep 6o ct 13 -o ct 20 -o ct 27 -o ct 3n ov 10 -n ov 17 -n ov 24 -n ov moisture % moisture 2013 2014   ital. j. food sci., vol 28, 2016 358 figure 2. the chemical content trends in cv moraiolo olive fruits during ripening in the 2013 and 2014 crop seasons. 0 10 20 30 40 50 60 70 25 /0 8/ 20 14 01 /0 9/ 20 14 08 /0 9/ 20 14 15 /0 9/ 20 14 22 /0 9/ 20 14 29 /0 9/ 20 14 06 /1 0/ 20 14 13 /1 0/ 20 14 20 /1 0/ 20 14 27 /1 0/ 20 14 03 /1 1/ 20 14 10 /1 1/ 20 14 17 /1 1/ 20 14 24 /1 1/ 20 14 suga r content (g/kg dry ma tter) sugar content 2013 2014 100 150 200 250 300 350 400 450 500 550 600 25 /0 8/ 20 14 01 /0 9/ 20 14 08 /0 9/ 20 14 15 /0 9/ 20 14 22 /0 9/ 20 14 29 /0 9/ 20 14 06 /1 0/ 20 14 13 /1 0/ 20 14 20 /1 0/ 20 14 27 /1 0/ 20 14 03 /1 1/ 20 14 10 /1 1/ 20 14 17 /1 1/ 20 14 24 /1 1/ 20 14 oil content (g/kg dry ma tter) oil content 2013 2014 10000 20000 30000 40000 50000 60000 70000 80000 90000 100000 25 /0 8/ 20 14 01 /0 9/ 20 14 08 /0 9/ 20 14 15 /0 9/ 20 14 22 /0 9/ 20 14 29 /0 9/ 20 14 06 /1 0/ 20 14 13 /1 0/ 20 14 20 /1 0/ 20 14 27 /1 0/ 20 14 03 /1 1/ 20 14 10 /1 1/ 20 14 17 /1 1/ 20 14 24 /1 1/ 20 14 phenolic content (mg/kg dry ma tter) phenolic content 2013 2014   ital. j. food sci., vol 28, 2016 359 the olive fly attack had a strong negative impact on the oil quality. table 2 shows the results of the parameters used to describe oil quality. in the 2014 crop season, the olive fly attack, causing the oil to come into contact with the water in the olive fruits and then with the exogenous and endogenous lipases, may have induced the hydrolytic degradation of the triglycerides, with a clear increase in free acidity. consequently, in the 2014 crop season, as much as 16% of the analyzed oils was found to be outside the legal limits which oil must respect to be classified as extra virgin (eec reg. 2568/1991), while in the 2013 crop season only one oil sample was found to be outside the legal limit of 0.80% free acidity. the attack of the olive fly may have caused the fatty acids’ exposure to strong oxidative conditions; hence, a sharp increase in both the peroxide number and the value of k232 occurred. table 2. results of the parameters used to describe oil quality in the 2013 and 2014 crop seasons. for the mean value of each parameter, columns with letters indicate significant differences at p < 0.05. regarding the antioxidant components of the oil, the tocopherols were also compromised by the infestation, while the decrease in phenolic content was not statistically significant, confirming the unclear correlation between the % of fly attack and the phenolic content reported previously (gomez-caravaca et al. 2008). regarding the oil sensory analysis, during the 2013 crop season all of the oil samples were free from defects, so they fell into the extra virgin olive oil category. in the 2014 crop season, instead, due to the major olive fly attack, 41% of the analyzed oils were defective, so they did not fall into the extra virgin olive oil category. among these, 29% could be categorized as virgin olive oils, while the remaining 12% fell into the "lampante" olive oil category. it also has to be taken into account that several farms did not harvest the completely ruined olives in 2014. this probably caused an underestimation of the negative effects of the olive fly attack on the oils from this crop season in tuscany. in conclusion, this research confirmed that the olive fruit fly is a strong risk factor for the quality of olive oil. a strong infestation by the olive fly causes a significant degradation in the chemical properties of the olive fruits and, as a result, poor oil quality. free acidity peroxide number tocopherol content phenolic content k232 k270 δk (%) (meqo2/kg) (mg/kg) (mgtyr/kg) number of samples 356 354 202 212 235 235 235 mean value 0.21 a 5.2 a 277 a 372 a 1.74 a 0.13 a 0.000 a maximum value 0.91 19.8 616 817 2.30 0.20 0.007 minimum value 0.11 1.4 173 122 1.24 0.08 0.001 standard deviation 0.08 2.08 67 128 0.15 0.02 0.001 number of samples 221 212 35 52 76 76 76 mean value 0.51 b 7.8 b 202 b 330 a 1.90 b 0.16 b 0.004 b maximum value 4.21 29.2 352 637 2.62 0.53 0.053 minimum value 0.11 2.6 120 53 1.56 0.08 0.000 standard deviation 0.39 3.5 49 140 0.22 0.07 0.008 2013 crop season 2014 crop season   ital. j. food sci., vol 28, 2016 360 references caleca v., rizzo r., battaglia i. and palumbo-piccionello m. 2007. tests on the effectiveness of mass trapping by eco-trap (vyoril) in the control of bactrocera oleae (gmelin) in organic farming. int. prot. olive crops. iobc/wprs bull. 30 (9): 139. 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(diptera tephritidae) in the district of bar in montenegro. int. prot. olive crops. iobc/wprs bull. 30 (9): 147. servili m., esposto s., urbani s., lazovic b., adakalic m., perovic t., hrncic s., pucci c., spanedda a.f., terrosi a. and perri e. 2007. spinosad treatment for bactrocera oleae (gmel.) control and olive oil quality in the montenegrin cv žutica. int. prot. olive crops. iobc/wprs bull. 30 (9): 155. wang x.g., johnson m.w., danae k.m. and opp s. 2009. combined effects of heat stress and food supply on flight performance of olive fruit fly. annals ent. soc. am. 102: 727. wang x.g., levy k., nadel h., johnson m.w., blanchet a., argov y., pickett c.h. and danae k.m. 2013. overwintering survival of olive fruit fly and two introduced parasitoids in california. environm. ent. 42: 467. web references agroambiente. 2014. http://agroambiente.info.arsia.toscana.it/arsia/arsia14 lamma. 2013. http://www.lamma.rete.toscana.it/news/autunno-2013-toscana-tra-i-pi%c3%b9-caldi-dal-dopoguerra   ital. j. food sci., vol 28, 2016 361 lamma. 2014a. http://www.lamma.rete.toscana.it/news/linverno-senza-freddo lamma. 2014b. http://www.lamma.rete.toscana.it/news/estate-2014-temperature-anni-80-e-molta-pioggia lamma. 2015. http://www.lamma.rete.toscana.it/news/clima-2014-toscana-anomalie-record-e-curiosit%c3%a0 paper received june 16, 2015 accepted november 26, 2015 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (1): 33–43 issn 1120-1770 online, doi 10.15586/ijfs.v34i1.2140 33 the behavior of apricot kernel oil body and proteins during in vitro gastric and intestinal digestion aslı kancabas kilinc, sibel karakaya* food engineering department, faculty of engineering, ege university, izmir, turkey *corresponding author: sibel karakaya, food engineering department, faculty of engineering, ege university, izmir, turkey. email: sibel.karakaya@ege.edu.tr received: 7 october 2021; accepted: 13 december 2021; published: 25 january 2022 © 2022 codon publications open access paper abstract in this study, an apricot kernel milk suspension was prepared, for which hacıhaliloğlu-type raw and roasted apricot kernels were used. protein, total lipid, and ash contents of apricot kernels and apricot kernel milk were determined. palmitic acid, oleic acid, and linoleic acid were considered as major fatty acids according to gas chromatography. during in vitro gastrointestinal digestion, the proteins of raw and roasted apricot kernel milk were hydrolyzed and pepsin-resistant proteins were determined. no difference was recorded between the pancreatic lipase penetration in the oil–water interface of oil bodies of both milk types. keywords: fatty acids; oil body; protein hydrolysis; vegetable milk; vegetable protein; waste by-products introduction currently, owing to the increasing global population and rising environmental concerns, demand for the sustainability of food and agriculture systems has been intensifying. the global consumption of meat and dairy products is projected to increase by ∼173% and 158%, respectively, from 2010 to 2050 (food and agriculture organization of the united nations [fao], 2011). in particular, considerable concerns for the production and consumption of animal protein in terms of environmental effects, such as increasing greenhouse emissions and water consumption, have caused increased interest in producing alternate sources of proteins, such as vegetable proteins, which are already present in the human diet (fasolin et  al., 2019). moreover, the current intention of consumers to include additional plant-based proteins in their daily diet requires greater knowledge about using alternative protein sources and their impact on the human health. the by-products of alternative protein sources and food wastes have considerable potential to be included in the human diet, especially those rich in protein, vitamins, minerals, fibers, oils, and bioactive compounds (coman et al., 2019; morais et al., 2015). apricot (prunus armeniaca l.) kernels, a valuable by-product obtained from the processing of apricot plants, can have the potential to be used as a functional food ingredient to increase product differentiation and accomplishment of consumer requirements. the total production of apricots in the world is 4.3 million tons per year, and turkey itself produces ∼985,000 tons (turkey ministry of agricultural and forestry, 2020). turkey produces 10–12% of fresh apricot and 60–65% of dried apricot in the world; moreover, ∼10–20% of fresh apricot and 80–85% of dried apricot are exported throughout the world (sığırcı et al., 2015). moreover, the apricot kernel is rich in phytochemicals, vitamins, minerals, unsaturated fatty acids (oleic acid and linoleic acid), α-, γ-, δ-tocopherols, and fibers (al juhaimi et al., 2018; fratianni et al., 2018). lipids found in apricot kernel are in the form of oil body, in which triglyceride particles are 34 italian journal of food science, 2022; 34 (1) aslı kancabas kilinc and sibel karakaya in recent years, rather than dairy products, plant-based food alternatives, such as vegetable milk extracted from soy, almond, rice, and oats, have increased. the beverage obtained from apricot kernel could be a good alternative. generally, studies related to apricot kernel as a functional food ingredient were based on foods added with apricot kernel. elkot et al. (2017) produced stirred yogurt supplemented with apricot kernel powder and reported an increase in the protein content of yogurt. eyidemir and hayta (2009) incorporated apricot kernel flour into the noodle and indicated the increased contents of protein, lipid, and ash in the noodle. to our knowledge, the possibility of extracting vegetable milk from apricot kernel has not been studied to date. in addition, the physicochemical characteristics of oil bodies and proteins of apricot kernel and their behavior during in vitro gastrointestinal digestion have not been explored. as most apricot kernels are waste by-products, converting them into value-added products would be beneficial in terms of both waste utilization and human health. therefore, in order to explore certain physicochemical properties of oil bodies and proteins, protein profile and protein hydrolysis during in vitro gastrointestinal digestion are substantial. the objectives of this study are to: characterize certain physicochemical properties of oil bodies and proteins of apricot kernel milk, understand their behavior during in vitro gastrointestinal digestion, evaluate the effect of microstructure on the physicochemical properties of oil bodies, evaluate it as an alternative vegetable protein source, and, finally, investigate the effect of roasting on all these properties. materials and methods samples and reagents apricot kernels (hacıhaliloğlu-type) were supplied by apricot research and application centre, inonu university, malatya, turkey. following chemicals were purchased from sigma aldrich corporation (st. louis, mo, usa): bradford reactive (b6916), bile acid (b8631), pepsin (p7000), pancreatin (p7545), glycine (g8898), bromphenol blue (b0126), coomassie brilliant blue r (27816), sodium dodecyl sulfate (l4390), marker for sodium dodecyl sulfate– polyacrylamide gel electrophoresis (sds-page; s8445), and fatty acids methyl esters standard (47885-u). miniprotean tgx precast gels (12%) were purchased from bio-rad (new york, usa). all the chemicals were of analytical grade. coated with a stabilizing layer of phospholipids and proteins (gallier et al., 2017). the oil body is a lipid-storage compartment in plant seeds and its structure is similar to that of emulsions. in most oil seeds, oil bodies have a diameter of 0.6–2 µm, while it could be 10–20 µm in some fruits (dave et al., 2019). owing to easy extraction, high recovery yield, and safe use in food products, plant derived oil bodies have received considerable attention (abdullah and zhang, 2020). several studies related to oil bodies from different sources have focused on their physicochemical characteristics such as droplet size, zeta (ζ) potential, emulsifying properties, and their microstructures (dave et al., 2019; ishii et al., 2017; yan et  al., 2016). however, few studies have investigated the behavior of oil bodies from different sources, including almond oil bodies, bovine milk fat globules, and sunflower oilseed bodies during in vitro digestion (gallier et  al., 2012, 2013; mandalari et al., 2008; wang et al., 2020; white et al., 2008). certain studies have focused on the occurrence, structure, and allergenicity of oil bodies (barre et al., 2018), the possibility of oil bodies derived from a plant source with unique facilities that could be used as an alternative to milk fat globules (mantzouridou et al., 2019), the extraction, isolation, and characterization of oil bodies (zderic et al., 2016), and the effects of food processing, such as roasting, sterilization, and high pressure homogenization, on oil bodies (yan et al., 2016; zaaboul et al., 2019). protein contents determined in various types of apricot kernels are reported as 15.1–24.2% (gezer et al., 2011). moreover, proteins determined in apricot kernels contain high levels of tyrosine, cysteine, methionine, aspartic acid, glutamic acid, serine, proline, and alanine (elkot et al., 2017). owing to this valuable composition, especially protein and fat contents, the apricot kernel could be considered as a functional food or functional food ingredient. however, for the food industry, only a small amount of apricot kernel is consumed as nuts and a large amount is a waste by-product. therefore, in spite of having a considerable industrial potential, lack of systematic collection and utilization has led to the lack of exploitation of this valuable product. the primary concern is amygdalin, a cyanogenic glycoside, contained in the kernels and seeds of apples, apricots, almonds, cherries, plums, and peaches (mirzaei and razei, 2019). karsavuran et al. (2015) reported that the amount of amygdalin in sweet hacıhaliloğlu-type apricot seeds was 0.25 mg/g and the predicted amount of hydrogen cyanide was 0.014 mg/g. with this amygdalin level, approximately 800-g seed would have to be ingested to cause poisoning of a child weighing 20 kg. however, this amount that causes poisoning in a child weighing 20 kg was very low for bitter apricot seeds (i.e., for paviot-type, 4.48-g seeds; for alyanak-type, 6.29-g seeds; and for ninfa-type apricot, 12.87-g seeds). italian journal of food science, 2022; 34 (1) 35 behavior of apricot kernel oil body and proteins during in vitro digestion analysis of fatty acid the major fatty acids of apricot kernel were quantified using 7820a gc gas chromatography system (agilent technologies, santa clara, ca, usa) with a flame ionization detector (fid) and conventional injector. a fused silica capillary column was used (j&w db-23, 30-m × 0.25-mm inner diameter, 0.25-µm film thickness; agilent technologies). methyl esters were prepared using the official method ce 2-66 of american oil chemists’ society (aocs). the procedure described by wu et al. (2011) was applied with slight modifications, and the injection volume was 1 µl. the temperature in both injector and detector was 250°c. the temperature program was started at 45°c, held for 4 min, increased to 175°c at a rate of 15°c/min, held for 15 min, then increased to 215oc at a rate of 4oc/5 min and held for 10 min. supelco 37 component fatty acid methyl esters (fame) mixture was used for identifying fatty acids. total titratable acid release the rate of free fatty acid release was monitored using a ph-stat titration unit (automatic potentiometric titrator at-510; kem, kyoto, japan). naoh solution, 0.1 m, was used to maintain ph at 7.0 for 2 h at 37°c. samples obtained from gastric digestion were subjected to ph-stat titration to determine release of free fatty acid. an excessive amount of pancreatic lipase was added to sample at the end of gastric phase to obtain the lipase activity of 2,000 u/ml as recommended by mat et al. (2016). measurement of zeta potential the zeta potentials of apricot kernel milk, roasted apricot kernel milk, and digested samples were measured using malvern zeta sizer nano zs. the apricot kernel milk was diluted 100-fold with purified water, gastric digested samples were diluted with citrate buffer (ph = 2.5), and intestinal digested samples were diluted to 100-fold with phosphate buffer (ph = 7.2) according to the procedure proposed by gallier and singh (2012). optical microscopy the images of the microstructures of oil bodies of milk and digested samples were taken with motorized light microscope psaron floptik (hpts 150, aiv labs, ankara, turkey) equipped with a controlled vehicular access (cva) camera using a digital image. a drop of the preparation of apricot kernel milk for preparing apricot kernel milk, 250 g of apricot kernel was soaked overnight in 1-l ultrapure water at room temperature. the mixture was subjected to agitation with a blender, and filtered using a cheesecloth. the supernatants were stored at -20°c until analyzed. the amount of kernel in the final product was 20 g/100 ml. roasting apricot kernels were roasted in an oven for 10 min at 170°c, and the roasting process was selected as it was generally applied to nuts. chemical composition the protein contents of apricot kernel and both kinds of milk were determined by dumas method using leco-fp 528 analyzer. the protein contents of digested samples were determined using the bradford (1976) method, and bovine serum albumin (bsa) was used as a standard. the total lipid contents of apricot kernel and apricot kernel milk were detected using the methods proposed by folch et al. (1957) and bligh and dyer (1959), respectively. in vitro gastrointestinal digestion the samples were subjected to in vitro gastrointestinal digestion (minekus et al., 2014). accordingly, simulated salivary fluid (ssf), simulated gastric fluid (sgf), and simulated intestinal fluid (sif) were prepared daily from stock solutions. in this method, the activities of enzymes were determined as per the protocol described by brodkorb et al. (2019). pepsin and pancreatin were used in gastric digestion and intestinal digestion, respectively. pefabloc was used to stop enzymatic activity. protein profile the protein profiles of milk from roasted and unroasted apricot kernels and aliquots obtained from in vitro gastric and gastrointestinal digestions were determined using sds-page. then the samples were diluted to a protein concentration of 1 mg/ml and mixed with sample buffer solution in a 1:1 ratio. the samples were maintained at 95°c for 10 min and then loaded on gels (12% mini-protean® tgxtm precast gels) as 40 µl for gastric samples and 20 µl for intestinal samples. moreover, electrophoretic separations were conducted at 150 v using a running buffer, and gels were stained with coomassie brilliant blue r-250 solution (50% ethanol and 10% glacial acetic acid). 36 italian journal of food science, 2022; 34 (1) aslı kancabas kilinc and sibel karakaya sample was placed on a microscope slide, covered with a cover glass, and detected with a 10× objective lens. statistical analysis parallel proximate analysis was conducted in triplicate. data were analyzed using anova (spss for windows version 18.0) and comparisons between mean values were performed using a t-test. differences between mean values were considered significant at p < 0.05. results and discussion chemical composition of apricot kernel and apricot kernel milk table 1 lists protein, total lipid, and ash contents of apricot kernel and apricot kernel milk. protein, total lipid, and ash contents of apricot kernel were higher than that of apricot kernel milk (p < 0.05). different results were reported for protein (15.1%–24.2%) and total lipid contents (27.7–66.7%) (alpaslan and hayta, 2006; gezer et al., 2011). differences in the chemical compositions of apricot kernels were due to their physical characteristics such as kernel weight and length (femenia et al., 1995). proteins hydrolyze during in vitro gastrointestinal digestion there were 10 protein bands in the sds-page profile of samples; their molecular weight (mw) was between 97 kda and 6.5 kda (figure 1a). in the unroasted form, three protein bands whose molecular weight was 97, 66, and 66–55 kda might correspond to storage proteins, specifically nutrient reservoir protein (96 kda), prunin 1 (63 kda), and prunin 2 (53 kda) as reported by ghorab et al. (2018). moreover, the authors indicated that the protein bands with molecular weight of 45, 36, and 29 kda could be hexosyltransferase (43 kda), hydroxyisourate hydrolyze activity (37 kda), and ribosomal protein s18 (31 kda), respectively. in the roasted form, the disappearance of two protein bands with molecular weight of 97 and table 1. the proximate composition of apricot kernel and apricot kernel milk. apricot kernel roasted apricot kernel apricot kernel milk roasted apricot kernel milk protein (%) 21.65 ± 0.08a 22.98 ± 0.98a 3.78 ± 0.72b 3.97 ± 0.81b total lipid (%) 26.22 ± 1.40c 25.87 ± 5.67c 7.74 ± 3.79d 6.53 ± 2.45d ash (%) 2.55 ± 0.20e 2.36 ± 0.51e 0.15 ± 0.07f 0.11 ± 0.1f values are mean ± standard deviation. different letters in the same row indicate statistical significance at p < 0.05. 14.2 kda might be attributed to the breakdown of these proteins during roasting. the sds-page patterns of in vitro gastric digestion of apricot kernel milk (figure 1b) demonstrated that there were no differences in the protein bands of the samples obtained from gastric chyme at different time intervals. this result indicated that pepsin was not able to hydrolyze proteins with molecular weight of 97 and 66 kda. protein band with mw = 20 kda in the sds-page profile of gastric digested apricot kernel milk but not in the undigested apricot kernel milk might be a sign of the breakdown of proteins with mw > 20 kda. a similar sds-page profile was obtained for in vitro gastric digestion of roasted samples (figure 1c). intestinal enzyme pancreatin contains proteases with a molecular weight of 23.8–64 kda, amylase with a molecular weight of 51–55.4 kda, lipase with a molecular weight of 45–50 kda, chymotrypsin with a molecular weight of 25 kda, trypsin with a molecular weight of 24 kda, and ribonuclease with a molecular weight of 13.7–14.7 kda. protein bands with molecular weight of 45, 24, and ∼13 kda were present in the enzymes of intestinal digestive system (figures 1d and e). four protein bands (with mw = 45, 29, 20, and 6.5 kda) were observed in apricot kernel milk whereas seven protein bands (with mw = 45, 36-37, 29, 24, ∼22, 6.5, and <6.5 kda) were observed in roasted apricot kernel milk during intestinal digestion (figure 1e). the fact that protein bands with mw > 45 kda present in the sds-page profile of the gastric digestion system did not appear in the first 15 min of intestinal digestion indicated that these proteins were hydrolyzed in the first 15 min of intestinal digestion. as reported by beisson et al. (2001), for almond oleosins, the 6.5-kda protein fraction that was not hydrolyzed during intestinal digestion as a function of time might be the central hydrophobic domain of apricot kernel oleosins. proteins with mw > 45 kda were completely hydrolyzed at the end of intestinal digestion. the disappearance of bands with mw > 45 kda present in the sds-page profile of gastric digestion established the hydrolysis of proteins with a higher molecular weight during intestinal digestion. the observation of fractions with molecular weight of 45–6.5 kda could be attributed to the hydrolysis of proteins with a higher molecular weight. italian journal of food science, 2022; 34 (1) 37 behavior of apricot kernel oil body and proteins during in vitro digestion high amounts of oleic acid and linoleic acid and a low amount of palmitic acid were determined in the apricot kernel oil (fratianni et al., 2018; mattheus et al., 2016). total titratable acid release no lag phase was observed during release of total titratable acid from in vitro gastric digested apricot kernel milk oil bodies (figure 3). this indicated that pancreatic lipase easily penetrated the oil-water interface. an early and rapid release of fatty acids from oil bodies was identified in the first 10 min of lipolysis. after 10 min, there was a reduction in the lipolysis rate, although acid release continued slowly to reach a value of 76.5 μm for apricot kernel milk and 85.5 µm for roasted apricot kernel milk at the end of 2-h digestion. during the first 30 min, the rate of free fatty acid release increased rapidly as the oil–water interface was easily accessible by pancreatic lipase. the rate of release of free fatty acid slowed down in a study conducted by gallier and singh (2012), three primary almond protein bands (20-42 kda) determined in tricine sds-page were hydrolyzed in the first 15 min of gastric digestion. the acidic polypeptides were hydrolyzed into smaller peptides in the first 2 min of intestinal digestion and the hydrolysis of all peptides was completed after 5 min of intestinal digestion. different results obtained in these two studies could be related to ph = 1.5 of gastric chyme used in the study conducted by gallier and singh (2012) and difference between the sources of proteins (almond kernel versus apricot kernel) used in both studies. composition of fatty acid the oil and milk extracted from apricot kernels were primarily composed of fatty acids such as palmitic acid, oleic acid, and linoleic acid (figure 2). fatty acid profiles of both kernels were identical (figures 2a and b). similarly, figure 1. sds-page patterns. (a) apricot kernel milk; (b) in vitro gastric digests of apricot kernel milk; (c) in vitro gastric digests of roasted apricot kernel milk; (d) in vitro intestinal digests of apricot kernel milk; and (e) in vitro intestinal digests of roasted apricot kernel milk. (a) (b) (c) (d) (e) 38 italian journal of food science, 2022; 34 (1) aslı kancabas kilinc and sibel karakaya fatty acids of almond oil bodies after in vitro intestinal digestion. moreover, rapid increase in the release rate of fatty acid in the first 25 min of intestinal digestion was also identified. measurement of zeta potential it is important to measure the zeta potential and surface charge of emulsified particles on the stability of emulsions. the power of electrostatic impulsion has a significant contribution to prevent flocculation and coalescence (çelebi, 2009). change in the net surface charge on the surface of native apricot kernel bodies was ∼0 mv. the zeta potential of oil bodies is governed by the active component oleosins present on the surface. the zeta potentials of apricot kernel milk and roasted apricot kernel milk were monitored during 2 h of gastric digestion and 2 h of intestinal digestion (figure 4). the because the interfacial saturation of lipolytic products slowly solubilized into micelles. unlike our result, gallier and singh (2012) observed a lag phase of 2 min, indicating the prevention of lipolysis by the membranes of oil bodies of almond, and the release of 133-µmol/ml free figure 3. total titratable acid release from gastric digested apricot kernel milk oil bodies during 120 min of intestinal digestion. figure 2. gas chromatograms of apricot kernels. (a) raw apricot kernel; (b) roasted apricot kernel; (c) apricot kernel milk; (d) roasted apricot kernel milk. (a) (b) (c) (d) italian journal of food science, 2022; 34 (1) 39 behavior of apricot kernel oil body and proteins during in vitro digestion although no research has been conducted on the zeta potential of apricot kernel milk, few studies are determined on the oil bodies of almond, pumpkin seed, soybean, and coconut milk. gallier and singh (2012) reported that the charge of native almond oil body was highly negative (-30 mv). at the start of in vitro gastric digestion, the zeta potential of almond oil body was highly positive and continued to remain positive during 60 min of gastric digestion. along with the in vitro intestinal digestion of almond milk, the zeta potentials of samples were negatively charged. dave et  al. (2019) reported that the zeta potential of oil bodies of coconut milk measured at ph 6.1 was –13 ± 1 mv. at ph 7.5, the zeta potential of oil bodies in washed cream and that of purified oil bodies were –32.6 and -33.8 mv, respectively. the zeta potentials of oil bodies of pumpkin seeds were different at different ph values and salt concentrations. an increase in the concentration of salt up to 10 mm at ph 3 increased the positive charge of zeta potential, which decreased with change in the concentration of salt. however, at ph 7.4, increase in salt concentration from 10 to 100 mm induced negative potentials. ishii et al. (2017) studied the interfacial and emulsifying properties of two types of oil bodies extracted from soybean seeds at different ph values. the zeta potentials of purified oil bodies (ob) and crude oil bodies (obc), including storage and other minor proteins, were –14.2 and –39.0 mv, respectively. this indicated that there was a strong electrostatic repulsion between crude oil bodies than between purified oil bodies. optical microscopy spherical and variable size of oil bodies were observed for apricot kernel milk and roasted apricot kernel milk (figures 5a and b). the size of oil bodies of apricot kernel milk was greater than that of roasted apricot kernel milk. unlike oil bodies of apricot kernel milk, roasting caused aggregation of oil bodies (figure 5b, 0 min). zaaboul et al. (2019) reported that roasted peanut oil bodies were larger compared to raw peanut oil bodies. the flocculation started within the 15 min of gastric digestion of both samples (figures 5a and b). large aggregates were still present after 90 min of gastric digestion of apricot kernel milk, but disruption in aggregates commenced in roasted apricot kernel milk at 90 min of gastric digestion. this result is supported by the increased zeta potential of roasted apricot kernel milk after 90 min of gastric digestion. the flocculation probably occurred due to the destabilization of oil bodies during hydrolysis of interfacial proteins by pepsin. a similar result indicated that the oil bodies of almond flocculated under low ph conditions (ph 1.5) during gastric digestion (gallier and singh, 2012). moreover, a liquid crystalline phase observed zeta potential of apricot kernel milk was ∼0 mv at the beginning of in vitro gastric digestion; moreover, it was increased to +7.17 mv in the 45th min of gastric digestion. at the end of gastric digestion, the zeta potential of apricot kernel milk was +3.68 mv. the positive value of zeta potential indicated that electrostatic repulsion in protein-stabilized oil droplets decreased and caused flocculation because of the low ph value of gastric environment. during in vitro gastric digestion, the zeta potential of roasted apricot kernel milk was different from that of apricot kernel milk. the zeta potential of roasted sample did not change during 0–90 min of in vitro gastric digestion and was approximately +3.0 mv (figure 6). moreover, zeta potential was –14.47 mv in 120 min of in vitro gastric digestion. low zeta potential (0–±5) indicated that the solution or dispersion might have rapidly coagulated or flocculated. therefore, the flocculation continued during 90 min of gastric digestion of roasted apricot kernel milk, but solubilization occurred after this point. however, the low positively charged zeta potential of apricot kernel milk did not change during 120 min of gastric digestion. similarly, makkhun et al. (2015) reported that the emulsion droplets located on the oil bodies of sunflower remained positively charged for 2 h of in vitro gastric digestion. the zeta potential of apricot kernel milk became negative at the very beginning (0–15 min) of intestinal digestion. moreover, the zeta potential of apricot kernel milk was -14.45 mv during the 15 min of intestinal digestion and increased slowly and reached –13.2 mv at the end of intestinal digestion. during intestinal digestion, the zeta potential of roasted apricot kernel milk remained practically the same ranging from –16.23 to –18.2 mv. although there was an insignificant difference between the zeta potentials of apricot kernel milk and roasted apricot kernel milk, a slightly higher zeta potential of roasted samples could be related to the more extensive association of bile salts with the surface of oil bodies in roasted apricot kernel milk than that in apricot kernel milk, as reported by makkhun et al. (2015) for emulsion droplets located on the oil bodies of sunflower. figure 4. the zeta-potentials of in vitro gastric and intestinal digested samples. 40 italian journal of food science, 2022; 34 (1) aslı kancabas kilinc and sibel karakaya surrounding lipid droplets at 90 min of in vitro gastric digestion (figure 5b) could be attributed to the presence of insoluble calcium long-chain fatty acid soaps precipitated around oil bodies as reported by gallier and singh (2012) in the case of almond oil bodies at 30 min of intestinal digestion. in the 30th min of intestinal digestion, although large aggregates were still present, marginally disaggregated oil bodies were observed. this disaggregation was probably due to adding bile salts, and pancreatic lipase that they caused to the solubilization of lipolytic products into the mixed micelles formed of bile salts. this solubilization-induced disaggregation of oil bodies after 60 min of intestinal digestion is shown in figure 6. gallier and singh (2012) reported similar behavior of almond oil bodies, revealing the disruption of aggregates after 30 min of intestinal digestion. few oil bodies of different sizes were still present at the end of intestinal digestion of apricot kernel milk. however, disaggregation did not occur during 120 min of intestinal digestion of roasted apricot kernel milk (figure 6b). 0 min (a) (b) 30 min 90 min 15 min 60 min 120 min figure 5. (a) optical microscopic images of apricot kernel milk and (b) roasted apricot kernel milk digests obtained at different time intervals of in vitro gastric digestion. 0 min 60 min 30 min 90 min 120 min figure 6. (a) optical microscopic images of apricot kernel milk and (b) roasted apricot kernel milk digests obtained at different time intervals of in vitro intestinal digestion. 0 min (a) 30 min 60 min 90 min 120 min italian journal of food science, 2022; 34 (1) 41 behavior of apricot kernel oil body and proteins during in vitro digestion future research must be conducted on the sensory properties of apricot kernel milk. acknowledgments we are grateful to ege university corporate development planning and monitoring coordination unit and directorate of library and documentation for their support in editing and proofreading of this study. disclosure statement the authors declare no conflicts of interest. funding this work was supported by the ege university scientific research project coordination (grant nos. 8295, 13-muh-015). references abdullah, w. and zhang h. 2020. recent advances in the composition, extraction and food applications of plant-derived oleosomes. trends food sci technol. 106:322–332. https://doi. org/10.1016/j.tifs.2020.10.029 al juhaimi f., özcan m.m., ghafoor k., and babiker e.e. 2018. the effect of microwave roasting on bioactive compounds, antioxidant activity and fatty acid composition of apricot kernel and oils. food chem. 243:414–419. https://doi.org/10.1016/j. foodchem.2017.09.100 alpaslan m. and hayta m. 2006. apricot kernel: physical and chemical properties. j am oil chem soc. 83(5):469–471. https://doi. org/10.1007/s11746-006-1228-5 barre a., simplicien m., cassan g., benoist h., and rougé p. 2018. oil bodies (oleosomes): occurrence, structure, allergenicity. rev fr allergol. 58:574–580. https://doi.org/10.1016/j. reval.2018.10.005 beisson f., ferté n., voultoury r., and arondel v. 2001. large scale purification of an almond oleosin using an organic solvent procedure. plant physiol biochem. 39(7–8):623–630. https://doi. org/10.1016/s0981-9428(01)01275-x bligh e.g. and dyer w.j. 1959. a rapid method of total lipid extraction and purification. can j biochem physiol. 37(8): 911– 917. https://doi.org/10.1139/o59-099 bradford m.m. 1976. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal biochem. 72(1–2):248–254. https:// doi.org/10.1016/0003-2697(76)90527-3 brodkorb a., egger l., alminger m., alvito p., assunção r., ballance s., bohn t., bourlieu-lacanal c., boutrou r., carrière f., clemente a., corredig m., dupont d., dufour c., edwards c., conclusion roasting seems to cause aggregation of oil bodies compared to unroasted apricot kernel milk oil bodies. the proteins of apricot kernel milk and roasted apricot kernel milk were hydrolyzed to a greater or lesser extent during in vitro gastrointestinal digestion. however, pepsin resistant proteins were determined in both samples and the flocculation of oil bodies was observed. no difference was recorded between the pancreatic lipase penetration in the oil-water interface of oil bodies of both milk types. moreover, a few oil bodies of different sizes were still present at the end of intestinal digestion of apricot kernel milk. however, disaggregation did not occur during 120 min of intestinal digestion of roasted apricot kernel milk. in addition, 250 ml of apricot kernel milk contains 50-g kernel corresponding to 12.5-mg amygdalin, which is considerably lower than the amount (200 mg) that causes poisoning in a child weighing 20 kg, as reported by karsavuran et al. 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structural and physicochemical changes in almond milk during in vitro gastric digestion: impact p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33i1.1951 52 p u b l i c a t i o n s codon sensory properties of iodine-biofortified potatoes maria piochia*, emma chiavarob, angelo cichellic, luisa torria, lorenzo cerretanid auniversity of gastronomic sciences, piazza vittorio emanuele, bra (cn), italy; bdepartment of food and drug, university of parma, parma, italy; cdepartment of medical, oral and biotechnological science, university “g. d’annunzio” of chietipescara, chieti scalo (ch), italy; ds.a.l.p.a. roseto, roseto degli abruzzi, teramo (te), italy *corresponding author: maria piochi, university of gastronomic sciences, piazza vittorio emanuele, bra (cn), italy. email: m.piochi@unisg.it received: 28 august 2020; accepted: 12 december 2020; published: 01 february 2021 © 2021 codon publications open access paper abstract the present study assessed the sensory impact of potatoes biofortification with iodine and the stability of iodine during six months of storage. four biofortified cultivars (cupido, marabel, orchestra and universa) and their controls (non-biofortified) were evaluated. descriptive analysis was applied with a panel to describe the sensory properties, and triangle tests were applied with consumers to evaluate perceivable differences between controls and respective biofortified samples at the end of shelf life. iodine content was quantified on raw potatoes for three periods of storage. descriptive analysis showed some differences between controls and iodine-biofortified samples, especially in texture (hardness). however, consumers did not significantly discriminate fortified from unfortified samples. iodine was stable during storage in all varieties. orchestra cultivar showed the highest iodine content, while universa the lowest. keywords: biofortification, descriptive analysis, essential micronutrient, functional food, potato, triangle test introduction iodine is an essential micronutrient consumed with diet, and is necessary for the biosynthesis of thyroid hormones which regulate metabolic functions (trumpff et al., 2013). the daily recommended iodine intake ranges from 90 to 250 μg, depending on several factors, among others, age and physiological status (e.g. childhood, adultness, pregnancy/lactation) (zimmermann et al., 2012). inadequate iodine intake is currently one of the main micronutrient deficiencies worldwide, leading to a spectrum of clinical and social issues called ‘iodine deficiency disorders’ (gonzali et al., 2017), and iodine deficiencies still represent a severe problem in certain geographic areas (khattak et al., 2017). iodine biofortification in vegetables is a promising strategy to increase iodine intake and overcome iodine deficiencies in human diet. however, the main critical issues in biofortification of iodine vegetables rely on the stability of the mineral during storage and the potential sensory modifications, which can compromise acceptability of fortified food. there are different strategies to address increase in iodine such as mineral supplementation, food fortification and biofortification of crops (comandini et al., 2013). biofortification is applied to increase the bioavailable nutrient content of the edible portion of crop plants (hotz, 2013). biofortification of plant foods can be obtained by the following three ways: agronomic biofortification, conventional plant breeding of selected cultivars, and genetic engineering (carvalho and vasconcelos, 2013). the aim of the agronomical approach is to increase micronutrients through mineral fertilisers and/or through the improvement of soil mineral elements’ mobilisation (saltzman et al., 2013). recently, many authors have developed and tested specific agronomical practices to increase iodine content in different vegetables and crops, such as potato and tomato (caffagni et al., 2011; zanirato italian journal of food science, 2021; 33 (1): 52–60 mailto:m.piochi@unisg.it 53 italian journal of food science, 2021; 33 (1) m. piochi et al. which was potentially the most critical moment, since the product was not fresh. materials and methods growing and processing of potato cultivars potatoes (solanum tuberosum) are grown in a limited zone of emilia-romagna region in italy, and these were kindly donated by pizzoli s.p.a. (budrio, italy). four international cultivars were chosen for the biofortification process: cupido, marabel, orchestra and universa. these cultivars were chosen because of their maximum usage to produce fresh potatoes for global commercial level. for each cultivar, control and biofortified samples were obtained from the same field of growth to minimise any variability factor other than biofortification process. one batch was analysed for each cultivar. in text, letter ‘c’ is used to identify controls, while letter ‘b’ is used for biofortified samples. in all, eight samples were evaluated (cupido_c, marabel_c, orchestra_c, universa_c, cupido_b, marabel_b, orchestra_b and universa_b). iodized potatoes were obtained by means of a patented agronomic procedure (zanirato and mayerle, 2009) through foliar fertilisation realised during the growing season. the harvest was done three weeks after the iodine treatment. potatoes were stored under conventional industrial storage conditions: in a plastic box put in a warehouse designed for potato storage in absence of light, and in ventilated and conditioned atmosphere at 8°c and 80% of relative humidity during all storage period. no sprout inhibitors were used during storage. a  period of six months was chosen as a shelf life, since it is a reasonable/representative storage time for commercial potatoes in controlled conditions before they are packaged and distributed immediately. sensory evaluation all sensory tests were performed after six months of storage. participants freely joined the sensory activities and written informed consent was obtained from all participants before inclusion in the tests. the study was in conformity with the declaration of helsinki. prior to sensory evaluations, assessors received both verbal and written instructions regarding the evaluation procedures. preparation of samples whole unpeeled potatoes were washed in tap water and cooked for 80 min in a steam oven (chef top, unox s.p.a., padova) at 100°c and 100% of relative humidity. after cooking, whole potatoes were cooled at a room temperature of 20°c for 2 h. approximately 30 min prior to sensory evaluations, potatoes were cut in cubes with side and mayerle, 2009); barley and wheat (caffagni et al., 2011); spinach and lettuce (smoleń et  al., 2014; weng et al., 2013); cabbage, coriander, cucumber, eggplant, long cowpea and hot pepper (weng et al., 2013); carrot and onion (zanirato and mayerle, 2009). as a general rule, food fortification should not alter the stability, colour or flavour of the vehicle food (dwyer et al., 2015). iodine salts may theoretically be involved in colour or oxidative reactions, since iodide is a strong reducing agent and iodate is a strong oxidizing agent (winger et al., 2008). since the modification of the sensory properties of food can negatively affect consumers’ hedonic responses, it is necessary to maintain an adequate level of acceptance when dealing with fortified foods. in fact, in spite of certain consumers that compromise on taste when consuming healthy foods (verbeke, 2006), often functional foods are characterised by critical sensory properties which are more disliked by consumers compared to conventional foods ( dal bello et al., 2017; torri et al., 2016). the effect of iodine on food sensory quality has been studied by many authors (greis et al., 2018; west and koning, 1995). on biofortified vegetables, particularly considering potatoes, studies were performed regarding the process of biofortification (caffagni et al., 2011; zanirato and mayerle, 2009), the stability of iodine during cooking ( caffagni et al., 2012; comandini et al., 2013) and the bioavailability of iodine (tonacchera et al., 2013). however, to the present authors’ best knowledge, no study has explored the stability of iodine during storage in biofortified potatoes and its effect on sensorial properties. therefore, the aim of the present study was two-fold: (1) to evaluate the effects of iodine biofortification on sensory properties by describing the sensory properties of controls and biofortified samples (descriptive analysis [da]) and by verifying whether biofortified cultivars were discriminated from non-fortified controls (triangle test); and (2) to evaluate the chemical stability of iodine in raw biofortified potatoes during storage (iodine analysis). since prior results showed that iodine is stable during domestic cooking, including baking (comandini et al., 2013), the analysis of iodine was done directly on raw potatoes to establish the following: (1) the starting natural quantity of iodine in each cultivar; (2) the effect of iodine biofortification on each cultivar immediately after harvesting (storage time at which potatoes are extremely fresh) and (3) stability at the end of six months (storage time corresponding to end of shelf life, the moment at which the potatoes are the least fresh). sensory analyses were conducted at the end of shelf life (i.e. six months), italian journal of food science, 2021; 33 (1) 54 sensory properties of iodine-biofortified potatoes characteristic(s) that differ. triangle tests were performed following iso 4120:2004 (iso 2004). four triangle tests were performed in total (12 samples). the evaluation session included two subsets, each comprising two triads. each triad had three codified samples, two of which were identical, and one was the odd sample. assessors must identify the odd sample. within each triad, the comparison was between the biofortified sample and the correspondent control. samples were served according to a randomised design (abb, baa, aab, bba, aba and bab). a group of 46 subjects were involved (68% females, aged 24–50 years). instructions required the assessors to taste the samples according to the provided order (from left to right), and to select the sample that they perceived as different from the other two within each triad. assessors were asked to provide an answer even if they were not sure. re-tasting was permitted. no time limitation was imposed on assessors. the presentation order of the triads was randomized across subjects. all evaluations were conducted between 12:00 noon and 2:00 pm. chemical analysis reagents high purity grade solvents were used for iodine extraction. tetramethylammonium hydroxide solution (tmah, 1 m) was bought from sigma aldrich (st. louis, mo), and hydrogen peroxide solution (h2o2 30% m/m) was from carlo erba (arese, mi, italy). ion exchange water (18 m ω) was obtained from millipore direct q5 system (millipore co., bedford, ma). iodine analysis iodine analysis was performed on controls and treated potatoes at three different periods: immediately after harvest (t0), after three months (t3) and after six months of of 15 mm and put on white plastic plates codified with a random three-digit code. each container was closed with a plastic lid. water was provided as palate cleanser at the start and between successive samples. the same preparation procedure was used in both sensory tests. descriptive analysis sensory profiles of eight potato samples were determined by the sensory panel of astra laboratory (imola, italy). test room was designed and managed following the iso 8589:2007 (iso 2007). the panel comprised eight trained judges (60% females, aged 27–45 years). the sensory panel was selected, trained and continuously monitored following the iso 8586-1:2012 (iso 2012). the panel evaluated the eight potato samples with an internally adapted procedure to establish a sensory profile developed following the iso 13299:2016 (iso 2016). the sensory profile approach is referred to in the text as descriptive analysis. a list of six attributes (two for tastes, one for flavour, and three for texture) was finally selected by the panel. attributes, each of which was defined by a specific definition, were evaluated on nine-point scale (table 1). for the purpose of tasting attribute, assessors were required to taste the sample, swallow and evaluate attributes for taste and flavour. then, panellists were required to cut the sample with a knife to re-taste it and to evaluate texture properties. samples were presented to panellists monadically and served in single-use, white plastic containers codified with a random three-digit code. a 1-min break was enforced between samples, when panellists rinsed their mouth with water. the descriptive analysis was conducted in two replicates. each descriptive evaluation session lasted for approximately 1.5 h. triangle tests the triangle test approach was chosen to find any difference between samples without specifying the sensory table 1. sensory attributes and definitions used by the trained panel in descriptive analysis to describe iodine-biofortified potatoes and controls. sensory modality attribute definition scale taste  sweet the perception of sweet taste on the tongue 1 = very low; 5 = moderate; 9 = very intense salty the perception of salty taste on the tongue 1 = very low; 5 = moderate; 9 = very intense flavour typical potato flavour   the presence of an overall flavour typical for cooked potato perceived after swallowing 1 = very low; 5 = moderate; 9 = very intense texture hardness the resistance of flesh to knife cutting and mastication (to the force impressed by teeth at the first bite) 1 = soft; 5 = neither soft nor hard; 9 = hard moistness the presence of liquid in the cut surface and perception of suiciness in the mouth during mastication 1 = dry; 5 = neither dry nor wet; 9 = wet granulation the presence of granules with a certain dimension, perceived during mastication 1 = coarse; 5 = neither coarse nor fine; 9 = fine 55 italian journal of food science, 2021; 33 (1) m. piochi et al. results and discussion sensory properties sensory profiles the sensory effects of addition of iodine to food have been described in previous papers (greis et al., 2018; west and koning, 1995). however, to the best of present authors’ knowledge, this is the first study reporting on the systematic description (done with a trained panel) of the sensory profiles obtained from biofortified potatoes baked in an oven. the two-way mixed anova models indicated a significant effect of the sample (p < 0.05) for all attributes except for salty (f = 2.04, p = 0.06). mean intensity values obtained from replicates of significant attributes were submitted for pca. two principal components were extracted, which accounted for 82.5% of the total data variance, with pc 1 accounting for 58.1% and pc 2 for 24.4% variance (figure 1a). the first component (pc 1) was positively associated with two textural attributes: moistness and granularity, and sweetness, and negatively associated with the typical potato flavour. granulation, moistness and sweetness were highly correlated to each other. the second component (pc 2) was positively associated with hardness, and negatively associated with granularity. the two universa samples (universa_b and universa_c) were highly and positively associated with pc 1, thereby showing a positive correlation with attributes having a positive loading on this component. instead, marabel (marabel_b and marabel_c), cupido (cupido_c and cupido_b) and orchestra_b samples had a negative correlation on pc 1, thereby associated with a high intensity of typical potato flavour. however, cupido_b, cupido_c, marabel_c, orchestra_c and universa_c had a negative score on pc 2. the bootstrap hulls plot showed samples with their confidence areas (figure 1b). some overlapping was observed in terms of perceptive properties, especially between marabel_c and cupido_b and between cupido_c and marabel_b, suggesting perceptive similarities between these two cultivars. instead, universa_b was clearly the most diverse sample which did not show perceptive overlaps with other samples. the sensory profiles of the four cultivars comparing the control samples with the related biofortified samples at t6 are shown in figure 2. observed modifications in the sensory properties may have been induced by the iodine treatment in field, which was done in the last period of growth of tubers (about 2 weeks before harvest). in general, a few differences were found between controls and biofortified samples, and the sensory modifications observed from the t-test were small, similar to a recent storage (t6). t3 and t6 were measured only for a group of biofortified samples. a representative quantity of fresh potatoes was homogenised. about 0.5 g of sample was weighed directly in pyrex test tubes. iodine was extracted by adding 6 ml of tmah solution (0.25 m) and 2 ml of h2o2 (30%) followed by microwave mineralisation (mars express 5, cem srl, cologno al serio, italy). the extract was diluted with ultrapure water, centrifuged and filtered following the procedure previously developed (comandini et al., 2013). iodine content was determined with an inductively coupled plasma mass spectrometer (icp-ms) (agilent, palo alto, ca) using the following parameters: rf power 1550 w, and argon flow rates of 1.05 l/min and 0.2 l/min, respectively, for carrier gas and make-up gas. instrument calibration was performed by employing iodine standards of up to a concentration of 100 mg/l in diluted tmah solutions. iodine analysis was done in triplicate. statistical analysis the effect of samples on the perceived intensity of descriptors from panel was estimated using two-way mixed analysis of variance (anova) models (random factor: judge; fixed factor: sample; model without interactions) separately conducted on each variable (sensory descriptor) considering eight products. all anova models were followed by tukey hsd test (p < 0.05). a  principal component analysis (pca) was conducted on the mean intensity ratings of significant attributes obtained from a two-way mixed anova, with the option of bootstrap hulls, which permitted to visualise the confidence areas of each sample. t-tests (p<0.05) were used to estimate differences between the mean values obtained by the panel for both control samples and means obtained by the relative biofortified samples. for the triangle test, the following parameters were defined for 46 assessors: an α-risk of 0.10, a β-risk of 0.30 and a pd of 0.20. the mean value of the three replicates was used for iodine analysis. values of iodine are expressed in the text as mean and standard error of the mean for each storage time. a one-way anova was conducted to estimate the effect of cultivar on the initial iodine content after harvesting among control samples (fixed factor: cultivar). the effect of iodine treatment within each cultivar was estimated after harvesting by four t-tests (p < 0.05), each comparing the content of iodine in the biofortified sample versus the related control sample. to assess the combined effect of the cultivar and the storage time on the final iodine content, a two-way fixed anova was conducted (fixed factors: cultivar, and time of storage; model with interactions) considering 12 samples (four biofortified cultivars × three storage periods). analyses were conducted with xlstat 2019.1.1 (addinsoft, boston, usa; package sensory). italian journal of food science, 2021; 33 (1) 56 sensory properties of iodine-biofortified potatoes hardness moistness granularity sweet typical potato flavour universa-b marabel-c cupido-b orchestra-c marabel-b universa-c cupido-b orchestra-b –4 –3 –2 –1 0 1 2 3 4 –4 –3 –2 –1 0 1 2 3 4 p c 2 ( 24 .4 % ) pc 1 (58.1%) bi-plot (pc 1 and pc 2: 82.5%) universa-b marabel-c cupido-b orchestra-c marabel-b universa-c cupido-c orchestra-b –4 –3 –2 –1 0 1 2 3 4 –4 –3 –2 –1 0 1 2 3 4 p c 2 (2 4. 4% ) pc 1 (58.1%) bootstrap hulls (pc 1 and pc 2: 82.5%) (a) (b) figure 1. bi-plot (1a) and bootstrap hulls plot (1b) from principal component analysis (pca) of significant attributes used in descriptive analysis to describe eight samples of potatoes, including four controls (cupido_c; marabel_c; orchestra_c; and universa_c) and four biofortified samples (cupido_b; marabel_b; orchestra_b; and universa_b). study which assessed the sensory modifications induced by addition of iodine in wheat bread, sausages and pickled cucumbers (greis et al., 2018). cupido was the only cultivar that did not show any significant differences in the comparison of profiles (figure 2a). the typical potato flavour and sweetness were not affected by the treatment of iodine in any variety. the intensity of descriptor ‘typical potato flavour’ was in agreement with a previous study, showing that the addiction of salt containing 400-mg iodine from potassium iodide or iodate did not affect the flavour of boiled potatoes and boiled rice (west and koning, 1995). moreover, iodine content of up to 100 mg/kg did not affect the sensory attributes of pickle (greis et al., 2018). on the contrary, in the current study, the biofortification seems to influence the texture, with a general increase in the perceived hardness, which significantly increased in three out of four biofortified samples (marabel, orchestra and universa). the two other textural attributes (moistness and granularity) varied depending on the cultivar. in marabel, the biofortified sample (marabel_b) showed a significant decrease (p = 0.03) in granularity and a significant increase in hardness (p = 0.02) (figure 2b). in biofortified orchestra, the hardness significantly increased (p ≤ 0.01), while the perceived saltiness decreased (p = 0.03) (figure 2c). in biofortified universa (figure 2d), both hardness (p ≤ 0.001) and moistness (p ≤ 0.01) increased significantly. in general, the texture of potatoes is determined by several mutually dependent factors (jarén et al., 2016). the consistent increase in hardness found in the current study in biofortified samples may have been linked to the modification induced by the iodine within the starch structure. in fact, it is known that iodine may be bounded within the helical v-amylose component (yu et al., 1996), and this modification in the structure can translate into a microscopical increase in the perception of hardness. a few existing studies on the effect of iodine on texture seems inconsistent across different food products. greis and colleagues (2018) found the largest deviation from the reference for tenderness for sausages but no difference in hardness for iodized and non-iodized pickles. meat samples (sausages) containing a high quantity of iodine were tenderer (greis et al., 2018), probably because of the variation in their water-holding capacity (whc). iodate appeared to soften vegetable pickles in brine during storage (12 days) (amr and jabay, 2004), which goes opposite to our results. taken together, these results suggest that a few sensory modifications induced by the iodine biofortification strongly depend on the type of food matrix used. 57 italian journal of food science, 2021; 33 (1) m. piochi et al. 1 3 5 7 9 sweet salty typical potato flavour hardness moistness granularity cupido cupido-b cupido-c 1 3 5 7 9 sweet salty typical potato flavour hardness* moistness granularity* marabel marabel-b marabel-c(a) (b) 1 3 5 7 9 sweet salty* typical potato flavour hardness** moistness granularity orchestra orchestra-b orchestra-c 1 3 5 7 9 sweet salty typical potato flavour hardness*** moistness** granularity universa universa-b universa-c (c) (d) figure 2. sensory profiles of the control samples (continuous lines) and biofortified samples (dashed lines) for the cultivars: (a) cupido, (b) marabel, (c) orchestra and (d) universa. asterisks indicate significant different mean values from t-tests for the considered attribute (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). overall sensory difference results of triangle tests for the four cultivars are shown in table 2. no significantly perceivable differences were found between any control sample and its respective biofortified sample at t6, thus indicating that the biofortification was not perceived in the population of subjects. the fact that no significant differences were found from triangle tests between treated samples and control samples suggested that differences found from descriptive analysis were minor, and therefore it was unlike that naive consumers may have noticed them. the two sensory techniques gave some slightly different outputs because descriptive analysis was conducted by trained assessors, while triangle tests with naive consumers. apart from the different expertise of the two groups, the approach was fully different. triangle test is an overall difference test (meilgaard et al., 2007b), while descriptive analysis is an approach that requires an analytical evaluation of each descriptor (meilgaard et al., 2007a). iodine content and stability table 3 shows the iodine content after harvest (t0) in controls, and that in biofortified cultivars at three storage periods (t0, t3 and t6). the amount of iodine present in control samples (not treated) was derived from the environment elements (soil, water and air) italian journal of food science, 2021; 33 (1) 58 sensory properties of iodine-biofortified potatoes the four cultivars showed different accumulation efficiencies in agreement with a previous study, which observed that the accumulation efficiency varied not only across potatoes (caffagni et al., 2012) but also for different amounts of iodine provided (caffagni et al., 2011). the increased ratio (the ratio of amount of iodine in biofortified and control samples) was approximately 18 (for cupido), 10 (for marabel) and 8 times more (for orchestra and universa). therefore, cupido showed the highest increased ratio. these differences in accumulation efficiency across varieties could be derived from different responses of cultivars to iodine treatment. since the iodine behaviour in a soil–plant system is very complex due to the high number of factors involved (fuge and johnson, 2015), different varieties of plants absorb iodine differently. the two-way anova showed a significant strong effect of the biofortified cultivar (f = 23.352, p < 0.001) on the final iodine content but no effect of the storage time (f  = 3.088, p = 0.064) nor of the interaction of cultivar and storage time (cultivar × storage time; f = 0.471, p = 0.823) (data not shown). the lack of effect of storage (osterc et al., 2011). at t0, orchestra_c had the significantly highest iodine content (6.7 ± 0.3 µg/100 g) compared to the other control samples, suggesting that this cultivar is naturally richer in iodine. in general, standard errors associated with the iodine analysis were high. this was because the sample preparative phase of this method (extraction) suffers from an intrinsic variability of sub-samples (replicates) that can greatly vary despite a proper homogenisation of the matrix (e.g. for the water content of single tuber, for different accumulations of microand macro-components in tuber etc.), thus resulting in high variations in standard errors. as expected, t-test confirmed that the iodine content significantly (p ≤ 0.01) and strongly increased in all biofortified samples, independent of the initial iodine content (table 3). this suggests that the agronomic treatment was effective in fresh potatoes, confirming results of other authors ( caffagni et al., 2011, 2012; zanirato and mayerle, 2009). table 2. results of triangle tests conducted at t6 to estimate significant differences between biofortified samples and their respective controls. each row shows the results of each cultivar from comparison between the biofortified samples and the relative control samples. cultivar assessors (n) critical number of correct responses correct responses given (n) p d   p-value  cupido 44 20 18 0.11 0.18 marabel 44 20 19 0.15 0.11 orchestra 47 21 20 0.14 0.12 universa 46 20 17 0.05 0.35 note: p d is the maximum proportion of assessors being able to detect a difference between products. table 3. iodine content (µg/100 g) in controls (‘c’) and relative iodine-biofortified samples (‘b’) at three different periods of storage (after harvesting = t0, after three months = t3 and after six months of storage = t6). cultivar  samples storage time t0* t3 t6 cupido  cupido_c  2.2 ± 0.3 cupido_b 40.1 ± 3.9a,b,c,d* 36.4 ± 3.9a,b,c,d 37.0 ± 3.9a,b,c,d marabel  marabel_c 3.2 ± 0.3 marabel_b 32.1 ± 3.9b,c,d,e* 24.5 ± 3.9c,d,e 22.9 ± 3.9c,d,e orchesta  orchestra_c 6.7 ± 0.3     orchestra_b 52.4 ± 3.9a* 42.4 ± 3.9a,b,c 48.7 ± 3.9a,b universa  universa_c 2.8 ± 0.3 universa_b 23.7 ± 3.9c,d,e* 20.9 ± 3.9d,e 15.7 ± 3.9e values are mean ± standard error of three measurements. *mean values of biofortified samples significantly differ from mean values of respective control samples at t0 from t-test (p ≤ 0.01). different letters in mean values across the 12 biofortified samples indicate significantly different iodine contents from two-way anova followed by tuckey’s hsd test (p < 0.05). 59 italian journal of food science, 2021; 33 (1) m. piochi et al. kindly donating potato samples and the astra laboratory (faenza, italy). author contributions all authors contributed to the study’s conception and design. m. piochi undertook the analyses and wrote the paper. all authors critically contributed and commented on the previous versions of the manuscript. all authors read and approved the final manuscript. compliance with ethical standards the research was conducted on human volunteers, and it was in conformity with the declaration of helsinki. written informed consent was obtained from all participants before inclusion in the sensory tests. conflict of interest the authors declare that they have no conflict of interest. vegetables were supplied free of charge with no obligation between researchers and suppliers. references amr, a. and jabay, omar a. 2004. effect of salt iodization on the quality of pickle vegetable. food, agriculture & environment, 2(2), pp. 151–156. https://doi.org/10.1234/4.2004.184 caffagni, a., arru, l., meriggi, p., milc, j., perata, p. and pecchioni, n. 2011. iodine fortification plant screening process and accumulation in tomato fruits and potato tubers. communications in soil science and plant analysis 42(6):706–718. https://doi.org/10.10 80/00103624.2011.550372. caffagni, a., pecchioni, n., meriggi, p., bucci, v., sabatini, e., acciarri, n., ciriaci, t. et al. 2012. iodine uptake and distribution in horticultural and fruit tree species. italian journal of agronomy 7(3):229–236. https://doi.org/10.4081/ija.2012.e32. carvalho, susana m.p. and vasconcelos, marta w. 2013. producing more with less: strategies and novel technologies for plant-based food biofortification. food research international 54(1):961– 971. https://doi.org/10.1016/j.foodres.2012.12.021. comandini, p., cerretani, l., rinaldi, m., cichelli, a. and chiavaro, e. 2013. stability of iodine during cooking: investigation on biofortified and not fortified vegetables. international journal of food sciences and nutrition 64(7):857–861. https:// doi.org/10.3109/09637486.2013.798270. dal bello, b., torri, l., piochi, m., bertolino, m. and zeppa, g. 2017. fresh cheese as a vehicle for polyunsaturated fatty acids integration: effect on physico-chemical, microbiological and sensory characteristics. international journal of food sciences and nutrition 68(7):800–810. https://doi.org/10.1080/09637486.2017.1301891. time indicated that the content of iodine is substantially stabile during the considered time, in agreement with a previous study (caffagni et al., 2012), which noted no significant effect of storage time on iodine content in potato tubers. the lack of effect of interaction (cultivar × storage time) indicated similar trends in iodine contents over time across the considered cultivars. instead, the strong effect of cultivar indicated a difference in the total iodine amount across the biofortified cultivars, as shown in table 3. after six months of storage, among the biofortified samples, orchestra_b had significantly the highest amount of iodine (48.7 ± 3.9 µg/100 g), not significantly different from cupido_b (37.0 ± 3.9 µg/100 g), followed by marabel_b (22.9 ± 3.9 µg/100 g) and universa_b (15.7 ± 3.9 µg/100 g), which did not differ significantly from each other. orchestra cultivar seemed to be the most suited variety for iodine biofortification, while universa cultivar is the least suited because of its lowest accumulation efficiency (according to the iodine increased ratio after treatment) and the lowest final content of iodine after six months. conclusions on the one hand, the data obtained from the present work confirmed the effectiveness of the agronomical practice of iodine biofortification in potatoes. iodine content was stable for six months under the conventional storage conditions in all cultivars. on the other hand, this study extended the knowledge by providing a systematic description of the sensory effects of iodine-biofortified potatoes, considering four different potato cultivars (cupido, marabel, orchestra and universa). the descriptive analysis highlighted a few differences between biofortified and control samples, mainly in texture (hardness), probably because of modifications in the starch structure of potatoes induced by iodine. however, differences were small and were not detected by consumers in triangle tests. therefore, iodine biofortification seems to be a suitable and promising technique for potatoes, since it does not compromise the sensory properties and it allows to develop functional vegetables (that is products with improved nutritional characteristics), which could be successful by consumers. a further development of the study could be the exploration of consumers’ perception related to liking of biofortified samples, targeting specific consumers (e.g. consumers with specific nutritional needs). acknowledgement the authors gratefully acknowledge the pizzoli s.p.a. company (budrio, italy) for useful collaboration and for 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https://doi.org/10.1201/b16452-7 osterc, a., stibilj, v. and raspor, p. 2011. iodine in the environment. in encyclopedia of environmental health, editor-in-chief: j.o. nriagu. elsevier b.v. pp. 280–287. https://doi.org/10.1016/ b978-0-444-52272-6.00705-4. https://doi.org/10.1016/j.gfs.2012.12.003� https://doi.org/10.1016/j.gfs.2012.12.003� https://doi.org/10.1016/j.scienta.2013.11.011� https://doi.org/10.1016/j.scienta.2013.11.011� https://doi.org/10.1210/jc.2012-3509� https://doi.org/10.1210/jc.2012-3509� https://doi.org/10.3168/jds.2015-9922� https://doi.org/10.3168/jds.2015-9922� https://doi.org/10.1016/j.jtemb.2013.01.002� https://doi.org/10.1016/j.foodqual.2005.03.003� https://doi.org/10.1007/s11434-013-5709-2� https://doi.org/10.1016/j.tifs.2007.08.002� https://doi.org/10.1016/j.tifs.2007.08.002� https://doi.org/10.1016/0008-6215(96)00159-0� https://doi.org/10.1016/0008-6215(96)00159-0� https://doi.org/10.1385/bter� https://doi.org/10.1385/bter� https://doi.org/10.1111/j.1753-4887.2012.00528.x� https://doi.org/10.1111/j.1753-4887.2012.00528.x� https://doi.org/10.3945/an.114.007443� https://doi.org/10.1016/j.apgeochem.2015.09.013� https://doi.org/10.1016/j.apgeochem.2015.09.013� https://doi.org/10.1016/j.copbio.2016.10.004� https://doi.org/10.1016/j.lwt.2018.04.009� https://doi.org/10.1016/j.lwt.2018.04.009� https://doi.org/10.1016/b978-0-12-375083-9.00025-8� https://doi.org/10.1016/b978-0-12-375083-9.00025-8� https://doi.org/10.1016/b978-0-12-800002-1.00019-4� https://doi.org/10.1016/j.je.2016.04.003� https://doi.org/10.2320/materia.46.171� https://doi.org/10.2320/materia.46.171� https://doi.org/10.1201/b16452-7� https://doi.org/10.1016/b978-0-444-52272-6.00705-4� https://doi.org/10.1016/b978-0-444-52272-6.00705-4� _hlk49446111 _hlk49446193 _hlk49446397 _hlk16002733 _hlk16235883 paper ital. j. food sci., vol. 27 2015 375 keywords: fruit, grape must, health, juice, sensory analysis effect of the addition of fruit juices on grape must for natural beverage production l. chiusano1,2 *, m. c. cravero1, d. borsa1, c. tsolakis1, g. zeppa2 and v. gerbi2 1crea consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria centro di ricerca per l’enologia, via pietro micca 35, 14100 asti, italia 2università degli studi di torino, dipartimento di scienze agrarie, forestali e alimentari, l.go p. braccini 2, 10095 grugliasco, torino, italy *corresponding author: tel. + 39 0141 433814, fax + 39 0141 436829, e-mail: mariacarla.cravero@entecra.it abstract the consumer attention for products with healthy properties is increased in time, and fruit juices, for their ease of consumption, can satisfy this demand providing them bioactive compounds. the grape juice has numerous health benefits demonstrated by several studies such as, among other, the antioxidant activities and the positive functions of their phenolic compounds. this work is aimed at blending grape and others fruits in a new fruit juice made only with natural ingredients of local production. the grape juice (cv barbera) has substituted water and its percentage was fixed (70%). it was mixed with apple (cv golden delicious), pear (cv williams) and peach (cv red haven) juices to obtain 25 different prototypes. in each of these at least two fruit juices were present and added in a percentage variable from 0 to 25%, with a step of 5%. the objectives of this study were to check the feasibility of the mixing process and the evaluation of the samples overall pleasantness. other sensory aspects of samples were also evaluated by consumers with a jar (just-aboutright) structured scale. the results didn’t reveal particular technological problems regarding the blending process. the brix mean value of the samples was about 15.3, with a significant reduction compared to that of the grape juice (about 19). the ph mean value of the samples (3.44) was significantly higher than that of the grape juice (3.36). the titrable acidity and the antioxidant capacity mean value of the samples was, namely, 6.22 g l-1 and 535.18 mg l-1. the penalty analysis of the liking test pointed out the importance of the persistence in mouth. the overall pleasantness was significantly (p≤0,01) positively correlated with the °brix/acid ratio (r=0.54) and samples with the highest percentage of pear juice were generally preferred. 376 ital. j. food sci., vol. 27 2015 introduction fruit consumption has a positive impact on health (o’neil et al., 2011) and, including also vegetables, five are their daily servings (fsa, 2010), though this advice is generally ignored (wootton-beard and ryan, 2011). in this regard, the “dietary guidelines for americans” consider the 100% fruit juice as alternative to whole fruit (usda, 2010). indeed, fruit juices in general are deemed as one of the main sources of bioactive compounds for diet (rodríguez-roque et al., 2014). even if the link between weight and sweetened beverages, including fruit juice 100%, must to be taken into account, referring to these latters, there is no consistent association (o’neil et al., 2011) and, actually, these have demonstrated to improve nutrient adequacy among children and adolescents of 2-18-year-olds (o’neil et al., 2012). also grape has proved to have numerous health benefits, such as antioxidant activity and the functions of flavonoid compounds (vislocky and fernandez, 2010; wootton-beard and ryan, 2011). grape-based products may prevent cardiovascular deseases, decrease oxidative stress and protect against atherosclerosis. results from animal models suggest that especially purple grape juice more effectively improves blood lipids (vislocky and fernandez, 2010). from a organoleptic and sensory perspective, grape juice is characterised by a high concentration of sugars and acids, a low ph and, generally, a very poor odour/ aroma. thus, grape juice has a high-energy value, which reduces the nutritional, while its high acidity and low odour/aroma intensity can reduce consumer preference. ojeda et al. (2009) highlighted the too high sugar content of the pure grape juice and, for this reason, it is important to reach a right sugar/acids balance to develop appreciable grape juice. to reach this result it is necessary to use the optimal grape variety and/or mixing it with other fruit. the blending, indeed helps to improve flavour, taste, and nutritive value and it reduces the cost of production, improves storability and inhibits microbial growth (bhardwaj and pandey, 2011). as reported by bates and morris (2001), the reasons for producing blends are many and all attributable to adjust and improve acceptability. the aim of this work was to develop an innovative concept of fruit juice obtained by mixing grape with other fruit juices to reduce its sugar concentration, acidity and to improve its olfactory profile. the tested fruit juices (peach, pear and apple) were chosen based on their appreciation by consumers, low acidity and sugar content, high antioxidant activity and high odour/aroma intensity. the use of grape must would also help to reduce the wine surplus that, currently, amounts to, approximately, 30 million hectolitres world wide (ramos et al., 2012; aylward, 2012). materials and methods juice production the grape juice (cv barbera) was provided by terre dei santi (castelnuovo don bosco, asti, italy), while the other fruit juices were provided by valter valle farm (san damiano d’asti, asti, italy). the apple, pear and peach juices were obtained from the golden delicious, williams and red haven cultivars, respectively. for juice production, fruits were directly pressed, and the juice was filtered and stored at +1°c until use. because the aim of this study was to develop a new grape-based juice, the percentage of grape juice was fixed (70%) and the other fruit juices were added in percentages from 0 to 25%, with a step of 5% (table 1). table 1 experimental plan of blending. sample code barbera juice (%) fruit juices (%) pear peach apple s-1 70 0 5 25 s-2 70 0 10 20 s-3 70 0 15 15 s-4 70 0 20 10 s-5 70 0 25 5 s-6 70 5 0 25 s-7 70 5 5 20 s-8 70 5 10 15 s-9 70 5 15 10 s-10 70 5 20 5 s-11 70 5 25 0 s-12 70 10 0 20 s-13 70 10 5 15 s-14 70 10 10 10 s-15 70 10 15 5 s-16 70 10 20 0 s-17 70 15 0 15 s-18 70 15 5 10 s-19 70 15 10 5 s-20 70 15 15 0 s-21 70 20 0 10 s-22 70 20 5 5 s-23 70 20 10 0 s-24 70 25 0 5 s-25 70 25 5 0 this ratio was defined taking into account that, generally, in a fruit juice, the fruit/water ratio is approximately 35:65 (fügel et al., 2005) and in this study water was replaced by grape juice. because for each beverage at least two fruit juices must be present, a total of 25 mixed juices were obtained. the prototypes were then bottled, pasteurised (105°c, 25 min) and stored at ambient temperature. ital. j. food sci., vol. 27 2015 377 three replicates for each of the 25 recipes are been prepared. reagents folin-ciocalteu reagent, sodium hydroxide, glucose, fructose, phosphoric acid, methanol, sulphuric acid, caesium chloride, tartaric, malic and citric acids were purchased from sigmaaldrich (milano, italy). ultrapure water was obtained from a milli-q gradient a10 instrument (millipore corporation, billerica, usa). analyses density, extract, ph, sulphur dioxide, titrable acidity, total sugars, glucose, fructose, ashes and potassium of grape must were determined in accordance with the commission regulation (eec) no. 2676/90 of 17 september 1990, while tartaric, malic and citric acids were determined by hplc (cane, 1990). the polyphenolic composition of the grape must and fruit juices (total polyphenols, anthocyanin and flavonoid contents) was determined by spectrophotometry (di stefano et al., 1989). the glucose, fructose, total sugars, ashes, titrable acidity, ph, tartaric acid, malic acid, citric acid and potassium of the fruit juices and beverages were determined in accordance with italian standard methods (dm 03/02/1989). the fruit juice antioxidant capacity, expressed as vitamin-c equivalent amount or veac index, was determined according to kim et al. (2002). the colour was measured using a konica minolta spectrophotometer cm-5 (minolta corp, osaka, japan) in the cielab colour system with a d65 illuminant. the parameters measured were l* (whiteness or brightness/darkness), a* (redness/greenness) and b* (yellowness/blueness). each sample was evaluated in a 40-ml cuvette (1-cm thickness). all evaluations were performed in triplicate. liking test as reported by mammasse and schlich (2014), literature recommend a range from 50 to 100 consumers in hedonic tests and generally no replication are needed. taking into account this and the limited quantity of samples, the liking test was executed once by recruiting 50 consumers (22 males and 28 females, aged 26-65 years). they have received an invitation and voluntarily have participated to the tests. all tests were conducted individually, and social interaction was not permitted. the test was performed inside an air-conditioned meeting room with white light. the temperature was approximately 21 °c, and the relative humidity was approximately 50%. tests were performed from 11 a.m. over 5 days. for each session, five experimental beverage samples (approximately 30 ml each) were presented in a completely randomised and balanced order. the samples were offered to the consumers in coded plastic cups. natural bottled water was provided to each participant for palate cleansing. to decrease fatigue, there was a 5 minutes break between each sample. during each break, the consumers rinsed their mouths with water. all beverages were evaluated for specific parameters by consumers on a just-about-right (jar) structured scale, and then the consumers were asked to express the overall pleasantness of each product. for jar evaluation, consumers rated the samples on a 5-point jar scale (1 = much too low, 2 = a little too low, 3 = just about right-jar, 4 = a little too much, and 5 = much too much) for five sensory parameters: colour, odour, aroma, sweet taste and persistence in the mouth. for the overall pleasantness evaluation, a segment of known length (100 mm), limited to the extremes of two adjectives of opposite meaning (bipolar scale) was used. consumers were asked to mark the line that corresponded to their degree of overall pleasantness. the data were collected on a paper card. according to pagès et al. (2014), the 5 jar variables were reduced to 3 for data evaluation: “not enough” (by grouping the “much too low” and “a little too low” responses), “jar” and “too much” (by grouping the “much too much” and “a little too much” responses). this grouping of variables leads to simpler analyses, and it allows for obtaining more stable results because non-jar categories are associated with higher frequencies (pagès et al., 2014). statistical analysis compositional data and overall pleasantness were examined by one-way analysis of variance (anova) with tukey’s test (p≤0.05) as a multiple range test with xlstat 2011 (addinsoft sarl, california, usa) and then used for a principal component analysis, also performed with xlstat 2011 (addinsoft sarl, california, usa). the °brix/acid ratio and the overall pleasantness were subjected to pearson’s test (r). the jar data were subjected to a penalty analysis with xlstat-mx 2014.2.07 (addinsoft sarl, california, usa). results and discussion compositional aspects the compositions of grape and fruit juices used for beverage production are reported in table 2, while the composition of the obtained beverages are reported in tables 3 and 4. as highlighted by morales-de la pena et al. (2010), the overall quality of a fruit juice 378 ital. j. food sci., vol. 27 2015 is evaluated by a few parameters such as soluble solids, ph and acidity. the grape juice displayed a total soluble solids content of 19 °brix with approximately 170 g l-1 of sugars, while the peach, apple and pear juices exhibited 11.5, 11.0 and 13.5 °brix, respectively. the mean value of °brix for new beverages was approximately 15.3, with a significant reduction with respect to grape juice, approximately 19. the obtained value is similar to that of a fruit juice (gunathilake et al., 2014) and ideal for the formulation of nutraceutical food beverages (saravanan and aradhya, 2011a). the content of fructose in apple juice (approximately 62 g l-1) was higher than that reported by wu et al. (2007) but lower than that reported by will et al. (2008) and markowski et al. (2009). additionally, the fructose content of pear juice (approximately 75 g l-1) was higher than that reported by colaric et al. (2006). acidity is one of the most important quality parameters for fruit juices (bhardwaj and pandey, 2011), as confirmed by al bittar et al. (2013), who included this factor in the sensory analysis of an innovative grape juice enriched in polyphenols. nevertheless, liu et al. (2006) highlighted that: “high acidity has a negative influence on the palatability of table grapes, as well as the suitability for wines”. the value of the total acidity, expressed as tartaric acid, of the grape juice used in this study (6.26 g l-1) is comparable to that reported for juices made with different grape cultivars (marsellés-fontanet et al., 2013; liu et al., 2006; soyer et al., 2003). the main organic acid in grape is tartaric acid, which has a pk 1 of 3.04, followed by malic acid, which has a pk 1 of 3.40 (liu et al., 2006). the table 2 composition of grape and fruit juices used for beverages production. data are expressed as mean ± sd. fruit juices grape juice pear peach apple glucose (g l-1) 19.08±0.2 39.78±0.2 20.41±0.2 glucose (g l-1) 86.94 ±0.2 fructose (g l-1) 75.43±0.4 41.59±0.5 61.94±0.2 fructose (g l-1) 94.82 ±0.09 ashes (g kg-1) 2.6±0.03 4.3±0.02 2.7±0.02 ashes (g l-1) 3.4 ±0.1 potassium (mg kg-1) 1720±0.2 3542±6 1540±0.4 potassium (mg kg-1) 1223±5 °brix 13.5±0.4 11.5±0.3 11±0.2 °brix 19±0.3 total acidity (g l-1) 4.3±0.2 4.85±0.3 4.13±0.1 total acidity (g l-1) 6.26±0.04 ph 3.73±0.03 3.79±0.01 3.76±0.01 ph 3.36±0.05 tartaric acid (g l-1) 0.281±0.01 0.233±0.02 0.26±0.03 tartaric acid (g l-1) 2.73±0.02 malic acid (g l-1) 0.312±0.03 0.892±0.02 1.028±0.01 malic acid (g l-1) 2.35±0.03 citric acid (g l-1) 0.588±0.03 0.788±0.04 nd citric acid (g l-1) 0.1 ±0.01 polyphenols (mg kg-1) 126.7±5 81.9±4 96.5±4 polyphenols (mg l-1) 446 ±6 density (g l-1) 1.07715 ±0.0004 extract (g l-1) 206.6±0.4 free sulphur dioxide (mg l-1) nd total sulphur dioxide (mg l-1) 11.2±0.4 anthocyanins (mg l-1) 228 ±1.74 (nd – not determined). grape juice had a tartaric acid content of 2.73 g l-1, similar to juice reported by liu et al. (2006). the ph value also plays an important role in the preparation of beverages (bhardwaj and pandey, 2011). the blending process here studied is aimed to increase the ph value of grape must (3.36). our obtained results indicated that the addition of fruit juice with ph values of 3.79 (peach), 3.76 (apple) and 3.73 (pear) increased the ph of grape juice so that it reached a mean value of 3.44 in the prepared beverages. in his research on the properties of fruit juices used for functional beverages, gunathilake et al. (2014) reported a ph of 3.60 for apple juice, while andrés et al. (2014) in their evaluation of the bioactive compounds in non-fermented beverages highlighted that the ph ranged between 3.20 and 4.01, in agreement with saarela et al. (2011). typically, the ph values of fruit juices are below 4, or even 3, depending on the fruits used. the amount of organic acids in the fruit juices depended on the cultivar: apple displayed the highest amount of malic acid, with a content of 1.028 g l-1, while pear juice had the highest citric acid content (0.588 g l-1). aguilar-rosas et al. (2007) reported a malic acid content below 0.35 g l-1 for the same cultivar, whereas buron-moles et al. (2014) reported a malic acid content of 1.4 g l-1. for beverages, the most abundant organic acid was malic acid, with a mean content of 2.84 g l-1, while the mean tartaric acid amount in these samples was 2.39 g l-1. for this compound, the concentration was similar among all of the beverages because the same quantity of grape must was used and because the quantity of tartaric acid is very low for fruit juice. the highest values were ital. j. food sci., vol. 27 2015 379 t a b le 3 c o m p o si ti o n o f sa m p le s o b ta in ed b y m ix in g g ra p e ju ic e a n d f ru it j u ic es o f p ea r, p ea ch a n d a p p le a n d r es u lt s o f a n o va w it h t u k ey ’s t es t. d a ta a re e x p re ss ed a s m ea n ± s d . f o r sa m p le s co d e se e t a b le 1 . v a lu es i n e a ch c o lu m n h a vi n g d if fe re n t le tt er s a re s ig n ifi ca n tl y d if fe re n t a t p < 0 .0 5 . sa m pl e co de ex tra ct °b rix g lu co se fr uc to se ac id ity ph o rg an ic a ci ds (g l -1 ) as he s po ta ss iu m °b rix / (g l -1 ) (g l -1 ) (g l -1 ) (g l -1 ) (g k g1 ) (m g kg -1 ) ac id ra tio ta rta ric m al ic ci tri c s1 16 7.8 5± 0. 21 15 .7 5± 0. 35 b 66 .1 9± 0. 01 d ef 80 .3 ±0 .7 1 cd 6. 00 ±0 .0 0 j 3. 44 ±0 .0 1 fg h 2. 52 ±0 .0 0 ab 3. 45 ±0 .0 0 ab 0. 55 ±0 .0 3 cd ef g 2. 6± 0. 01 b c 12 43 ±1 .6 8 bc de fg 26 .2 5 s2 17 7.9 0± 0. 00 17 .0 0± 0. 00 a 71 .4 0± 1.4 1 ab cd 85 .1 0± 1.5 6 bc 6. 15 ±0 .0 0 h 3. 45 ±0 .0 1 cd ef g 2. 51 ±0 .0 0 ab 3. 32 ±0 .0 9 ab 0. 73 ±0 .0 2 de fg 2. 7± 0. 02 b c 12 98 ±4 .1 5 ab cd ef 27 .6 4 s3 18 1.8 0± 0. 00 17 .0 0± 0. 00 a 75 .5 3± 0. 62 a b 87 .4 4± 0. 55 a b 6. 49 ± 0. 06 e f 3. 43 ±0 .0 1g hi 2. 69 ±0 .0 3 ab 3. 54 ±0 .0 5 a 0. 94 ±0 .0 4 ab cd e 2. 8± 0. 01 b c 13 85 ±9 .7 2 ab c 26 .1 9 s4 18 3. 10 ±0 .0 0 17 .0 0± 0. 00 a 76 .1 8± 0. 81 a 85 .9 5± 0. 78 a bc 6. 75 ±0 .0 0 c 3. 45 ±0 .0 0 de fg 2. 76 ±0 .3 4 a 3. 60 ±0 .2 6 a 1.3 2± 0. 30 a b 3. 1± 0. 01 a bc 14 24 ±2 .6 4 a b 25 .1 9 s5 18 1.4 5± 0. 21 17 .2 0± 0. 00 a 75 .4 7± 0. 18 a b 83 .0 1± 0. 09 b c 6. 94 ±0 .0 0 b 3. 44 ±0 .0 1 fg h 2. 66 ±0 .1 8 ab 3. 47 ±0 .2 0 ab 0. 28 ±0 .0 2 fg 3. 2± 0. 01 a b 14 64 ±2 .5 1 a 24 .7 8 s6 17 6. 25 ±0 .2 1 16 .8 0± 0. 00 a 68 .4 5± 0. 64 c de 85 .7 8± 0. 82 b c 7.2 0± 0. 00 a 3. 43 ±0 .0 0 gh i 2. 47 ±0 .0 1a b 3. 22 ±0 .0 2 ab c 0. 18 ±0 .0 0 g 2. 6± 0. 01 c 11 61 ±5 .6 6 cd ef g 23 .3 3 s7 17 4. 30 ±0 .0 0 16 .8 0± 0. 00 a 70 .3 9± 1.4 2 bc d 85 .0 2± 1.5 6 bc 6. 45 ±0 .0 0 f 3. 45 ±0 .0 1 de fg 2. 51 ±0 .0 4 ab 3. 21 ±0 .0 6 ab c 0. 20 ±0 .0 2 g 2. 7± 0. 01 b c 12 33 ±0 .6 9 bc de fg 26 .0 5 s8 17 8. 05 ±0 .2 1 16 .9 0± 0. 14 a 73 .6 2± 3. 69 a bc 87 .1 8± 2. 48 a b 6. 60 ±0 .0 0 d 3. 45 ±0 .0 1 de fg 2. 37 ±0 .1 8 ab 3. 18 ±0 .0 1 ab cd 0. 22 ±0 .0 3 g 3. 2± 0. 00 a b 12 75 ±0 .6 5 ab cd ef 25 .6 1 s9 18 0. 50 ±0 .0 0 16 .8 ±0 .0 0 a 73 .3 3± 1.8 2 ab c 86 .1 5± 0. 62 a b 6. 51 ±0 .0 3 e 3. 43 ±0 .0 1 gh i 2. 64 ±0 .0 5 ab 3. 30 ±0 .0 7 ab 1.1 8± 0. 00 a b 3. 1± 0. 01 a bc 13 04 ±0 .1 6 ab cd ef 25 .8 1 s10 18 1.7 0± 0. 14 17 .0 0± 0. 00 a 75 .7 0± 0. 82 a b 86 .5 4± 0. 41 a b 6. 64 ±0 .0 0 d 3. 40 ±0 .0 0 j 2. 47 ±0 .1 3 ab 3. 22 ±0 .0 1 ab c 0. 27 ±0 .0 3 fg 3. 2± 0. 03 a b 13 70 ±3 .0 8 ab cd 25 .6 0 s11 18 2. 10 ±2 .2 3 17 .0 0± 0. 00 a 76 .3 3± 1.1 4 a 84 .1 2± 1.2 0 bc 6. 75 ±0 .0 0 c 3. 40 ±0 .0 0 j 2. 60 ±0 .0 0 ab 3. 09 ±0 .0 9 ab cd e 0. 21 ±0 .0 0 g 3. 5± 0. 01 a 13 28 ±5 .8 3 ab cd e 25 .1 9 s12 14 8. 60 ±0 .0 0 14 .6 5± 0. 21 c 51 .4 6± 0. 10 g 75 .8 7± 0. 57 d e 5. 55 ± 0. 00 o 3. 39 ±0 .0 0 j 2. 30 ±0 .0 4 ab 2. 87 ±0 .0 4 bc de f 0. 35 ±0 .2 7 ef g 2. 6± 0. 01 c 10 59 ±9 .9 2 g 26 .4 0 s13 13 4. 00 ±0 .0 0 13 .1 0± 0. 14 e 45 .2 1± 0. 16 h i 68 .3 1± 0. 29 fg 5. 70 ± 0. 00 m 3. 48 ±0 .0 1 ab c 2. 21 ±0 .0 4 ab 2. 60 ±0 .0 6 cd ef g 0. 79 ±0 .1 4 bc de fg 2. 6± 0. 01 c 11 56 ±9 .7 2 ef g 22 .9 8 s14 13 2. 90 ±0 .0 0 13 .0 0± 0. 00 e 45 .6 8± 0. 25 h i 66 .8 6± 0. 44 g 5. 63 ±0 .0 0 n 3. 50 ±0 .0 0 a 2. 38 ±0 .0 9 ab 2. 65 ±0 .0 8 cd ef g 0. 93 ±0 .0 7 ab cd e 2. 8± 0. 01 b c 11 57 ±1 .5 9 ef g 23 .0 9 s15 13 6. 00 ±0 .0 0 13 .1 0± 0. 14 e 48 .3 3± 0. 66 g hi 67 .9 2± 0. 91 fg 5. 93 ±0 .0 0 k 3. 48 ±0 .0 1 ab c 2. 33 ±0 .0 4 ab 2. 62 ±0 .0 7 cd ef g 1.1 4± 0. 06 a bc 3± 0. 00 a bc 12 22 ±3 .0 8 bc de fg 22 .0 9 s16 13 8. 50 ±0 .1 4 13 .2 0± 0. 00 e 50 .7 6± 0. 04 g h 68 .9 2± 1.0 2 fg 6. 00 ±0 .0 0 j 3. 48 ±0 .0 0 ab 2. 29 ±0 .0 3 ab 2. 59 ±0 .0 3 cd ef g 1.5 1± 0. 20 a 3. 1± 0. 01 a bc 12 45 ±3 .9 4 bc de fg 22 .0 0 s17 13 4. 60 ±0 .1 4 13 .2 0± 0. 00 e 44 .5 1± 0. 35 i 70 .6 3± 0. 42 e fg 5. 55 ±0 .0 0 o 3. 46 ±0 .0 0 bc de f 2. 27 ±0 .0 2 ab 2. 66 ±0 .1 3 de fg 0. 72 ±0 .0 3 bc de fg 2. 6± 0. 03 b c 11 18 ±1 .5 7 ef g 23 .7 8 s18 13 5. 9± 0. 14 13 .2 0± 0. 00 e 45 .6 9± 0. 09 h i 69 .9 0± 0. 37 fg 5. 55 ±0 .0 0 o 3. 47 ±0 .0 0 bc d 2. 19 ±0 .0 2 ab 2. 38 ±0 .0 1 fg 0. 85 ±0 .0 4 b cd ef 2. 6± 0. 03 b c 11 21 ±0 .4 9 ef g 23 .7 8 s19 13 8. 75 ±0 .2 1 13 .3 0± 0. 14 e 46 .9 8± 0. 12 g hi 69 .4 1± 0. 31 fg 6. 08 ±0 .0 0 i 3. 47 ±0 .0 1 bc de 2. 11 ±0 .1 2 ab 2. 25 ±0 .1 3 g 1.0 0± 0. 02 a bc 2. 9± 0. 01 a bc 11 71 ±3 .6 6 de fg 21 .8 8 s20 13 9. 55 ±0 .2 1 13 .2 0± 0. 00 e 47 .2 65 ±0 .3 2 gh i 67 .7 7± 0. 11 fg 6. 08 ±0 .0 0 i 3. 46 ± 0. 00 b cd ef 2. 02 ±0 .2 9 b 2. 08 ±0 .3 6 g 1.0 6± 0. 10 a bc 3± 0. 01 a bc 12 01 ±3 .6 4 cd ef g 21 .7 1 s21 14 1.1 0± 0 .0 0 13 .8 0± 0. 00 d 45 .3 8± 0. 10 h i 72 .9 2± 0. 30 e f 5. 78 ±0 .0 0 l 3. 44 ±0 .0 0 ef gh 2. 04 ±0 .1 8 b 2. 08 ±0 .1 8 g 0. 76 ±0 .2 1 bc de fg 2. 7± 0. 03 b c 10 98 ±1 .9 6 fg 23 .8 8 s22 16 8. 20 ±0 .0 0 15 .8 0± 0. 00 b 60 .8 6± 1.2 1 f 82 .5 4± 1.0 5 bc 6. 15 ±0 .0 0 h 3. 44 ±0 .0 0 ef gh 2. 33 ±0 .0 4 ab 2. 51 ±0 .0 9 ef g 0. 72 ±0 .0 4 bc de fg 2. 8± 0. 01 b c 11 22 ±1 0. 78 e fg 25 .6 9 s23 16 9. 80 ±0 .0 0 15 .8 0± 0. 00 b 62 .9 7± 0. 71 e f 82 .5 8± 0. 52 b c 6. 3± 0. 00 g 3. 42 ±0 .0 1 hi j 2. 40 ±0 .0 0 ab 2. 49 ±0 .1 0 ef g 1.1 9± 0. 05 a b 3± 0. 01 a bc 12 42 ±2 .3 9 bc de fg 25 .0 8 s24 17 2. 25 ±0 .2 1 16 .1 0± 0 .1 4 b 62 .6 4± 0. 49 f 85 .6 5± 0. 63 b c 6. 30 ±0 .0 0 g 3. 44 ±0 .0 0 ef gh 2. 37 ±0 .0 3 ab 2. 41 ±0 .0 2 fg 1.1 2± 0. 17 a bc 3± 0. 01 a bc 11 95 ±0 .9 9 cd ef g 25 .5 6 s25 17 6. 50 ±0 .1 4 17 .0 0± 0. 00 a 70 .3 4± 4. 38 b cd 91 .74 ±5 .4 1 a 6. 49 ±0 .0 0 ef 3. 41 ±0 .0 1 ij 2. 32 ±0 .0 8 ab 2. 35 ±0 .0 6 fg 0. 21 ±0 .0 5 g 2. 9± 0. 02 a bc 12 51 ±8 .6 8 ab cd ef g 26 .1 9 found for beverages s-3 and s-4, which contained higher quantities of apple juice. the brix/acid ratio (table 3) is an important parameter usually used to control fruit quality. in this study a positive correlation (r = 0.54, p≤0.01.) resulted between it and the overall pleasantness in accordance with jayasena and cameron (2007). these authors reported that the °brix/acid ratio compared with the °brix alone demonstrated a higher degree of association with the consumer acceptability and it appeared a very useful maturity indicator. the peach juice exhibited the highest potassium content. this characteristic determined an increase in the content of this important component in the beverages containing high percentages of peach, e.g., sample s-5. the lowest value was determined for sample s-12, which was obtained without peach juice. the total polyphenols content ranged between 265.5 mg l-1 for beverage s-5 and 407 mg l-1 for beverage s-24, with a mean value of 359.30 mg l-1. according to the total polyphenol contents of fruit juices, higher values were exhibited by beverages with high percentages of pear juice. the same beverage also displayed some of the highest values for the flavonoid content (627.5 mg l-1 , the highest) and antioxidant capacity (585 mg l-1 , the second highest one). for this parameter, the result for s-24 was similar to that of beverage s-1 (593 mg l-1). the lowest value for the antioxidant capacity (464.41 mg l-1) was displayed by beverage s-5. these results highlighted that the most interesting findings were obtained with a high quantity of apple or pear juice in the beverage, while a high content of peach juice led to a reduction of this parameter. the anova and tukey’s test performed for each parameter of the beverages displayed high variability among all samples and strictly corre380 ital. j. food sci., vol. 27 2015 table 4 polyphenol composition (phen total polyphenols; tai anthocyanins; tfi flavonoids; vceac antioxidant capacity) and cielab values of samples obtained by mixing grape juice and fruit juices of pear, peach and apple and results of anova analysis with tukey’s test. data are expressed as mean ± sd. for sample code see table 1. values in each column having different letters are significantly different at p<0.05. sample phen tai tfi vceac l*(d65) a*(d65) b*(d65) code (mg l-1) (mg l-1) (mg l-1) (mg l-1) s-1 384.5±4.95 cd 84±1.41 abc 540.5±44.55 cdefgh 593.83±10.40 a 68.14±2.86 ab 32.18±0.46 11.20±0.43 s-2 389.5±2.12 bcd 84.5±2.12 abc 478±4.24 ghijkl 578.38±1.04 ab 64.75±0 .01 b 33.9±0.10 11.675±0.15 s-3 366±1.41 efg 82±2.82 abcd 407.5±20.51 l 526.17±4.16 abcd 66.28±0.45 ab 33.56±0.19 11.36±0.26 s-4 362±0.7 gh 82.5±1.06 abc 497±4.95 fghijk 468.09±20.28 cd 66.63±0.19 ab 32.72±0.51 10.52±0.27 s-5 265.5±6.36 o 80±0.00 abcde 447±2.83 ijkl 464.41±10.40 d 66.65±1.98 ab 34.84±1.72 11.23±0.30 s-6 402.5±4.95 ab 88.5± 0.71 a 612.5±0.71 abc 534.26±48.87 abcd 64.89±0.25 b 32.94±0.40 11.31±0.28 s-7 405.5±3.54 a 88±0.00 ab 576±1.41 abcd 476.91±7.28 cd 64.13±0.93 b 32.75±0.07 10.89±0.01 s-8 385.5±2.12 cd 80.5±0.71 abcde 546.5±7.78 bcdefg 492.35±47.83 bcd 63.85±1.52 b 33.52±2.33 10.97±1.19 s-9 380.5±2.12 de 81.5±0.71 abcd 545±5.66 cdefg 530.59±29.12 abcd 65.63±1.25 ab 33.85±0.76 11.46±0.59 s-10 362.5±2.12 fgh 80.5±0.71 abcde 517±1.41 defghi 480.59±10.40 cd 66.21±3.21 ab 34.4±2.77 11.25±1.00 s-11 338.5±4.95 ijk 79.5±0.71 abcde 490.5±.71 fghijk 537.94±18.72 abcd 67.96±2.23 ab 33.8±1.33 10.9±0.72 s-12 377±1.41 def 82±0.00 abcd 581±1.41 abcd 577.65±0.00 ab 66.62±0.40 ab 33.49±1.94 9.45±0 .47 s-13 334±0.00 jkl 67.5±0.71 gh 515.5±4.95 defghi 551.18±0.00 abcd 67.5±2.21 ab 31.66±0.46 9.81±0.01 s-14 325±4.24 klm 63.5±2.12 h 493.5±4.95 fghijk 510±2.08 abcd 68.51±1.62 ab 32.66±1.77 10.04±0.04 s-15 314.5±0.71 mn 68.5±2.12 gh 430±15.56 kl 506.32±11.44 abcd 70.09±0.14 ab 30.79±1.81 9.77±0.95 s-16 309.5±0.71 n 65.5±0 .71 gh 436±9.90 jkl 510.74±15.60 abcd 74.03±4.72 a 31.21±1.16 9.26±1.27 s-17 350±4.24 hi 72±1.41 efgh 552.5±6.36 bcdef 583.53±12.48 a 66.6±0.62 ab 32.86±1.14 11.09±0.09 s-18 342±1.42 ij 69.5±2.12 fgh 514±2.83 defghi 551.17±33.28 abcd 68±2.18 ab 33.46±1.28 10.93±0.60 s-19 333±1.14 jkl 67±0.00 gh 503±0.00 efghij 540.88±6.24 abcd 69.48±1.60 ab 32.71±1.05 10.76±0.69 s-20 321±1.41 lmn 64.5±2.12 gh 469±8.49 hijkl 551.18±29.12 abcd 67.82±2.71 ab 33.63±2.10 9.95±0.09 s-21 365±4.24 fg 68±0.00 gh 571±1.41 abcde 546.76±12.48 abcd 67.49±0.49 ab 32.6±1.43 10.56±1.41 s-22 395.5±0.71 abc 80±7.07 abcde 618±49.5 ab 576.91±7.28 ab 63.95±1.63 b 34.42±0.86 11.88±0.30 s-23 376±2.83 defg 73±1.41 defg 556±0.00 abcdef 548.24±0.00 abcd 70.73±5.76 ab 32.09±1.22 10.05±1.86 s-24 407±7.07 a 78±1.41 cdef 627.5±4.95 a 585±37.43 a 65.57±1.23 ab 32.27±0.23 11.68±0.23 s-25 390.5±7.78 bcd 79±5.66 bcde 576±46.67 abcd 556.32±1.04 abcd 66.51±0.28 ab 32.82±0.91 10.89±0.59 lated with the composition of each single fruit juice and the different percentages used for beverage production. in fact, there were no differences between the beverages for the cielab parameters a* and b* only, and this is due to the high grape must percentage used. sensory aspects concerning the overall pleasantness, the anova highlighted significant differences among the 25 experimental beverages (table 5). even if the content of grape juice was kept constant in all of the beverages at 70%, the different percentages of other fruit juices can influence the acceptability. the most appreciated samples (s-22, s-24 and s-23) had the highest pear juice percentages, while the least appreciated (s-14, s-12, s-1 and s-16) had the lowest pear juice concentrations. the least appreciated was beverage s-14, which was obtained with a mix of apple, pear and peach juices at the same percentage (10%). penalty analysis was used because with this test it is possible to identify the sensory attributes that have the largest influence on consumer liking and provides directions for product reformulation (ares et al., 2014) and also allows one to determine if a specific product attribute is “just about right” (taylor, 2013). penalty analysis combines jar variables and overall liking tests to find correlations between a decrease in consumer acceptance and attributes not at the jar level. this analysis, based on multiple comparisons, is aimed to identify and determine if the rankings on the jar scale are related to significantly different results in the liking scores for each sensory attribute studied on the jar scale. this can be achieved by evaluating the mean decrease in overall liking versus percentage of not-jar variables (i.e., the low percentage of not-jar evaluation determines a low mean decrease in overall liking). when some not-jar categories receive at least 20% (pareto principle) responses for an attribute, this becomes a candidate for penalty analysis. penalty analysis uses the 20% cut-off theory on the percentage of not-jar consumers based on the pareto principle (i.e., the pareto principle recognises that “80% of effects occur from 20% of causes” or the 80-20 rule) and signifies several common occurrences in everyday phenomena. therefore, the 20% cut-off is used as a general rule for penalty analysis (narayanan et al., 2014). in fig. 1 are reported the jar scores for each parameter used in the beverage evaluation. the colour was judged “just right” by 50% of the consumers, odour by 37%, aroma by 38% and persistence in the mouth by 39%. in general, the “jar” value was chosen by a higher number of assessors: the higher frequency was highlighted by the “a little too low” valital. j. food sci., vol. 27 2015 381 table 5 mean values of overall pleasantness and results of anova and tuckey’s test. data are expressed as the mean ±sd. values with different letters are significantly different at p<0.05. sample code fruit juices overall tuckey test pleasantness (p< 0.05) pear (%) peach (%) apple (%) s-22 20 5 5 56.98 a s-24 25 0 5 55.3 ab s-23 20 10 0 55.14 ab s-8 5 10 15 54.18 abc s-9 5 15 10 54.08 abc s-2 0 10 20 54.04 abc s-25 25 5 0 53.07 abcd s-5 0 25 5 53.36 abcde s-11 5 25 0 52.36 abcdef s-3 0 15 15 50.82 abcdefg s-4 0 20 10 50.56 abcdefg s-6 5 0 25 50.54 abcdefg s-7 5 5 20 49.72 abcdefg s-10 5 20 5 48.9 abcdefg s-21 20 0 10 48.38 abcdefg s-15 10 15 5 46.36 abcdefgh s-20 15 15 0 45.7 bcdefgh s-17 15 0 15 43.8 cdefgh s-18 15 5 10 42.9 defgh s-19 15 10 5 42.68 efgh s-13 10 5 15 42.6 efgh s-16 10 20 0 42.46 fgh s-1 0 5 25 41.96 fgh s-12 10 0 20 40.86 gh s-14 10 10 10 36.36 h fig. 1 distribution of jar scores for each sensory attribute evaluated. ue for only the odour. fig. 1 also demonstrates that the “much too much” and “much too low”, although they may affect the overall pleasantness, do not weigh significantly on it because of their low frequency in the responses of consumers. the variables can then be grouped into two main groups with “a little too much” or “a little too low”. the first group corresponds to “much too much”, while the second corresponds to “not enough” for the parameters of 382 ital. j. food sci., vol. 27 2015 colour, aroma, sweet taste and persistence in the mouth. in fig. 2 are displayed the distribution of frequency and then their effect on the mean drop in overall pleasantness. sweet taste, aroma and persistence in mouth exhibited a higher effect on the overall pleasantness if classified as “not enough”. also important for determining the overall pleasantness was the odour, if classified as “not enough”. fig. 2 penalty analysis from jar data. not-jar data with a frequency <20% of total responses are not considered significant. when the sensory parameters were classified as “too much”, they had less impact on the overall pleasantness. a principal component analysis was also performed to highlight the correlation between chemical-physical parameters and overall pleasantness. the first two components explained 72.82% of the variance (fig. 3). the first component explained 50.74% of the variance and was mainly correlated with the tofig. 3 distribution on plane defined by the first two components of chemical-physical parameters, overall pleasantness and beverage samples. ital. j. food sci., vol. 27 2015 383 tal soluble solids, glucose and fructose contents, which corresponded to 12.1, 11.8 and 11.2%, respectively, of the total variance explained by this axis. the second component that explained 22.08% of the total variance is associated with the flavonoids, total polyphenols and antioxidant capacity, accounting for 20.7, 16.7 and 13.5%, respectively, of the total variance explained by this axis. the overall pleasantness was positively correlated with the contents of the total soluble solids and fructose and negatively correlated with the ph, citric acid content and l*. because the overall pleasantness is located in the upper right graph quadrant, all of the beverages placed in the same quadrant are the most appreciated. in particular, the highest appreciation was found for the s-22, s-24 and s-23 samples, as also demonstrated by table 5. the less appreciated samples are on the lower left side of the graph. they can be grouped into two groups: s-16, s-15, s-14, s-20 and s-19 in the lower left quadrant of the pca graph and s-13, s-18, s-17, s-21 and s-12 in the upper left quadrant. the first group exhibited a more transparent colour, with a high value for l* and higher ph and citric acid contents. the second group demonstrated a low overall pleasantness but a high antioxidant capacity. in this group, it must be highlighted that beverage s-14, with the same percentages of fruit juice (10-10-10), had the lowest appreciation and the highest ph. conclusions one of the first internationally accepted descriptions of functional food has been provided by diplock et al. (1999) according to which: “a food product can be considered functional if together with the basic nutritional impact it has beneficial effects on one or more functions of the human organism...”. taking this into account, and also of the scientific evidence regarding the benefits of the products based on grapes to human health, the results obtained in this study have shown that these experimental fruit juices have functional characteristics. additionally, as reported by bhardwaj and pandey (2011), it may be concluded that the formulation of mixed beverages can satisfy consumer tastes and preferences. in particular, the overall pleasantness results indicate a tendency of consumers to prefer samples with the highest percentage of pear juice, followed by samples containing mixtures of peach and apple juices. acknowledgements this study was funded by regione piemonte through the “regional operational programme” regional competitiveness and employment – f.e.s.r. 2007/2013. the authors thank all of the consumers and the technical operator, mrs. maria rosa lottero cra-eno, for her collaboration in the liking test. references aguilar-rosas s.f., ballinas-casarrubias m.l., nevarezmoorillon g.v., martin-belloso o. and ortega-rivas e. 2007. thermal and pulsed electric fields pasteurization of apple juice: effects on physicochemical properties and flavour compounds. j. food eng. 83: 41. al bittar s., périno-issartier s., dangles o. and chemat f. 2013. an innovative grape juice enriched in polyphenols by microwave-assisted extraction. food chem. 141: 3268. andrés v., villanueva m.j., mateos-aparicio i. and tenorio m.d. 2014. colour, bioactive compounds and antioxidant capacity of mixed beverages based on fruit juices with milk or soya. j. food nutr. res. 1 (53): 71. ares g., dauber c., fernández e., giménez a. and varela p. 2014. penalty analysis based on cata questions to identify drivers of liking and directions for product reformulation. food qual. prefer. 32: 65. aylward d. 2012. demarcation: a dynamic methodology for quality grading within the australian wine industry. int. j. qual. innov. 2:18. bates r.p. and morris j.r. 2001. juices and beverages blends. ch. 9. in: “principles and practices of small and medium scale fruit juice processing”. bates r.p., crandall p.g. and morris j.r.. 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35 (2): 71–79 issn 1120-1770 online, doi 10.15586/ijfs.v35i2.2327 71 p u b l i c a t i o n s codon effect of traditional household processing techniques on phenolic compounds, antioxidants activity and γ-aminobutyric acid of cowpea (vigna unguiculata) pods fatima ali alghamdi1, amro b. hassan2, nora abdullah alfaris1, jozaa zaidan altamimi1* 1department of physical sport science, college of education, princess nourah bint abdulrahman university, riyadh, kingdom of saudi arabia; 2department of food science and nutrition, college of food and agricultural sciences, king saud university, riyadh, kingdom of saudi arabia *corresponding author: jozaa zaidan  altamimi, department of physical sport science, college of education, princess nourah bint abdulrahman university, p o. box 84428, riyadh 11671, kingdom of saudi arabia. email: jzaltamimi@pnu.edu.sa received: 20 january 2023; accepted: 2 may 2023; published: 16 may 2023 © 2023 codon publications open access original article abstract the influence of common home processing methods was investigated on the color characteristics, phenolic component, antioxidant activity (2,2-diphenyl-1-picrylhydrazyl [dpph] and ferric-reducing antioxidant power [frap] activity) and the levels of γ-aminobutyric acid (gaba) in cowpea pods. the processing methods significantly increased the total phenolic content, total flavonoid content and the activity of antioxidant compounds (dpph and frap). the gaba content sharply decreased in pods after boiling and drying treatment. however, it significantly increased after fermentation. the fermented cowpea pods displayed the highest content of phenolics, flavonoids, gaba and anthocyanin as well as antioxidant activity. hence, these traditional domestic preparation methods could be recommended for the food industry. keywords: antioxidant activity, cowpea, gaba, phytochemical, traditional processing introduction cowpea (vigna unguiculata) belongs to the genus vigna, subfamily faboideae of the pea family leguminosae or fabaceae. cowpea is native to sub-saharan africa and is widely grown and produced there as well as in other semi-arid tropical and subtropical areas (brink and belay, 2006). it consists of two cultivar types: one has long pods used as vegetables, and the other is used for fodder (morris and li wang, 2018). however, different parts of the plant, such as seeds, green pods and leaves, can be used (lazaridi et al., 2017), but it is commonly consumed as dried seeds. according to iqbal et al. (2006), cowpea is high in proteins, fiber, minerals and vitamins. according to affrifah et al. (2022), cowpea is rich in indispensable amino acids. furthermore, it has been observed that cowpea seeds contain a high quantity of phenolic acids and flavonoids as bioactive compounds with high antioxidant activity. by preventing the onset of numerous diseases, such as atherosclerosis, cancer and other chronic human diseases, phenolic and antioxidant substances are vital and crucial compounds for human health (awika and duodu, 2017). as stated formally, the compounds of phenolic nature and those with antioxidant qualities in the lipophilic fraction of 56 vegetables are higher than those in the hydrophilic fraction. hence, cowpea was considered as one of the vegetable samples with a higher antioxidant activity of 17.01 µmol fe (ii) g fresh weight (fw) and a phenolic content of 8.28 mg gallic acid equivalent (gae)/g (deng et al., 2013). however, cowpea (raw) seeds contain many biofunctional, non-nutrient mailto:jzaltamimi@pnu.edu.sa 72 italian journal of food science, 2023; 35 (2) alghamdi fa et al. inedible part was removed, and stored for the subsequent processing. processing methods traditional household methods (blanching, drying and fermentation) were used to process cowpea pods. boiling of the pods was done traditionally by following the conventional household method used in albaha. the raw cowpea pods were boiled in hot water at 1:2 (w/v) ratio for 45 min over medium heat. following boiling, the water was decanted and the boiled pods were ground and stored in plastic containers at 4°c until further use. a portion of the boiled pods was dried in an oven at 55°c for 9 h before being ground into a fine powder and passed through a 0.5-mm sieve. the dried cowpea flour was kept at 4°c until further use. the cowpea was naturally fermented by adding water in a 1:3 (w/v) ratio and incubated at 37°c for 24 h. raw pods were made from unprocessed seeds (as a control). color measurement a colorimeter (chroma meter cr 400; brand minolta, japan) was used to measure the color of raw and processed samples. a standard white reflector plate was used to calibrate the equipment. the following parameters were measured: l* (lightness), a*, either negative (green) or positive (red), and b*, either negative (blue) or positive (yellow). the following equation was used to calculate the total color difference (∆e). 2 2 2e ( l) ( a) ( b) .∆ = ∆ + ∆ + ∆ anthocyanin content determination according to the method described by egbuna et al. (2018), the anthocyanin content of control and processed cowpea samples was measured. flour, 4 g, was added to 20 ml of methanolic (70%) solution, and the samples were allowed to extraction for 48 h at room temperature. a little above 1 ml of the extract was divided into two test tubes (a1 and a2), each of which contained 1 ml of ethanol hcl (0.01%) solution. next, 10 ml of 2% hcl (ph 0.8) was added to test tube a1, and 10 ml of citric buffer (0.2-m na2hpo4 and 0.1-m citric acid; ph 3.5) was added to test tube a2. for even mixing, both test tubes were rotated at 40 rpm for 2 min. at 520 nm, absorbance was finally measured with 70% methanol used as a blank. the following equation was used to determine the anthocyanin content (ac, μg cyanidin/g): (aa ab) mw df cf1 cf2 ac , lε − × × × × = × compounds, such as phytic acid, trypsin and chymotrypsin enzyme inhibitors, classified as antinutritional factors. accordingly, different traditional and innovative processing methods of cowpea seeds were recommended to enhance their nutritional quality. these methods utilized whole cowpea seeds, flour and processed meals (affrifah et al., 2022). in general, processing methods were observed to significantly enhance the capacity of phytochemical (anthocyanin, flavonoid and phenolic) and antioxidant compounds in seeds. barros et al. (2017) investigated changes in antioxidant capacity during cooking of different cowpea cultivars. the authors stated that the total phenolic and antioxidant activity was decreased after cooking (boiling) grains of cowpea cultivars, which could be due to the migration of these compounds through leaching in cooking broth. the fermentation process increased the digestibility of proteins, amino acids and essential dietary minerals in seeds as well as their bioavailability (hassan et al., 2021). according to kapravelou et al. (2020), natural fermentation significantly improved the nutritional value of cowpea seeds and the digestibility of their essential amino acids. traditional food preparation methods, such as blanching, drying and fermentation, significantly improved nutritional value of cowpea seeds (affrifah et al., 2022; barros et al., 2017). according to the general authority for statistics (2019) bulletins, cowpea grows in different regions of saudi arabia, such as albaha, najran and alqssim (ministry of environment, water, and agriculture, 2019). cowpea is considered as one of the typical traditional foods of these areas. it is commonly consumed in albaha (a southern province of saudi arabia) as boiled green pods. however, in other parts, cowpea meals are prepared in different traditional ways. despite several investigations evaluating the nutritional value of cowpea, the evaluation of phytochemical compounds, antioxidant activity and γaminobutyric acid (gaba) as affected by traditional household methods, particularly blanching, drying and fermentation, has not been investigated. therefore, determining the impact of conventional preparation methods (such as boiling, followed by drying and fermentation) on the color, total phenolic content (tpc), total flavonoid content (tfc), gaba and anthocyanin contents, and the antioxidant activity of cowpea was the aim of this study. materials and methods samples collection and preparation local saudi cowpea cultivar harvested in 2022 was obtained from a local market of the albaha region (south of saudi arabia). the pods were cleaned, italian journal of food science, 2023; 35 (2) 73 evaluation of nutritional values and traditional preparation methods of cowpea where aa = absorbance at 2% hcl solution, ab = absorbance at citric buffer solution, mw = molecular weight of cyanidin-3-glucoside (449 g/mol), df = dilution factor (20 ml/4 g), cf1 = 106 (µg/g), cf2 = 1 l/1,000 ml, ε  = molar extinction coefficient of cyanidin-3-glucoside (26,900 l/mol.cm) and l = path length (1 cm). determination of gaba content the technique first described by zhang et al. (2014) was used to measure the amount of gaba in cowpea pods before and after processing. deionized water (15 ml) was used to extract around 2 g of each sample, which were then centrifuged for 15 min at 10,000 rpm. using a syringe filter (0.45 mm), the supernatant was filtered, and 0.5 ml of the filtered sample was combined with 0.2-m borate buffer at ph 9.0, 6% phenol and 9% naclo (0.4 ml). the mixture was heated for 10 min, then cooled to the emergence of blue color. spectrophotometry at a wavelength of 645 nm was used to quantify the samples of gaba. various gaba concentrations were used to create a calibration curve (r2 = 0.994). preparation of extracts the raw and processed cowpea samples were shaken at room temperature for 24 h after being combined with methanol at a ratio of 1:2 (w/v) according to the procedure described by talhaoui et al. (2014). filter paper (whatman no. 1) was used to filter the mixture. leftovers were washed twice with methanol. the extracted samples were then dried with a rotary evaporator and conserved for further investigation. determining total phenolic contents the folin–ciocalteu (f-c) assay was used to quantify the samples’ tpc following the procedure adopted by waterhouse (2002). briefly, 1.58 ml of water and 100 µl of folin–ciocalteu reagent were added to an aliquot (20  µl) of dried extract methanolic solution (ratio 1:10 w/v). then, the mixture was thoroughly stirred for 10  min and 300 µl of 20% sodium carbonate solution was added. the mixture was incubated for 2 h at 20°c. the absorption was measured at 765 nm relative to a blank solution using an ultraviolet (uv) spectrophotometer. the calibration curve was created using different amounts of gallic acid dissolved in pure methanol (r2  = 0.99743). the amount of total phenol in each gram of dried samples was calculated as milligrams of gae (mg gae/g dry weight [dw]). determining total flavonoid contents according to the colorimetric test (kim et al., 2003), tfc of cowpea extracts was calculated. sodium nitrite (5%) solution, 300 µl, was added to 300 µl of aluminum chloride (10%) and 1 ml of methanolic extract. the mixture was incubated at a temperature of 25°c for 5 min. then 2 ml of sodium hydroxide (1 mol/l) was added to the mixture. the reaction mixture’s volume was immediately increased to 10 ml with distilled water and was vortexed completely. the solution’s absorbance was measured at 510 nm. different concentrations of quercetin were used to create a calibration curve (r2 = 0.9761). the tfc was calculated as milligram of catechin equivalent per gram (mg qe/g dw) of dry weight sample. determination of antioxidant activity radical scavenging activity in order to calculate the antioxidant activity of sample extracts, 2,2-diphenyl-1-picrylhydrazyl (dpph) radicals were scavenged in raw and processed cowpea extract samples by following the method described by chang et al. (2001). the extracts and deionized water (as a control) were incubated in 0.1 ml of 50-mm tris-hcl buffer (ph 7.4) for 30 min at room temperature. then using an uv-visible (uv-vis pd-303 uv) spectrophotometer, the absorbance was determined at 517 nm. trolox equivalent per gram of material (mg te/g) was computed and used to express dpph scavenging. determination of ferric-reducing antioxidant power (frap) the frap of sample extracts was determined following benzie and devaki’s (2017) approach. the methanol extract (0.5 ml, diluted for 10 times) was combined with 2-ml frap solution, followed by adding 10 ml of water; the mixture was allowed to settle down for 20  min. finally, using an uv-vis spectrophotometer to test absorbance at 593 nm against a blank, the result was reported as mg te/g. statistical analysis the data were expressed as mean of three determinations. one-way anova was used to evaluate the data, and significant differences (p < 0.05) were determined using the least significant difference (lsd). using the xlstat software’s pca algorithms, multivariate analysis was carried out as described by vidal et al. (2020). using the xlstat program, the linear partial least squares regression (pls) test verified the most effective conventional processing approach, according to tenenhaus et al. (2005). 74 italian journal of food science, 2023; 35 (2) alghamdi fa et al. the anthocyanin content of cowpea pods was 3.69 mg/g. the content of anthocyanin increased significantly (p  > 0.05) with each processing technique. the content of anthocyanin was similar in blanched and fermented samples (4.35 and 4.50 mg/g, respectively). however, in dried samples, it was significantly higher, at 9.95 mg/g (table 2). in control samples, the gaba content was 2.16 mg/g. both blanching and drying processes caused a significant (p < 0.05) reduction in the gaba content of cowpea pods by 0.90 mg/g and 0.65 mg/g, respectively. however, fermentation significantly increased the content of gaba to 4.50 mg/g. gaba, responsible for significant health functions, is an inhibitory neurotransmitter in humans and animals (komatsuzaki et al., 2007). generally, in plants, gaba concentration varies from 2 nmol/g to 700 nmol/g. however, owing to its extensive bioactivities, a rising awareness is observed about its availability in foods (poojary et al., 2017). hence, several processing techniques have been performed to improve gaba content in foods. elbaloula and hassan (2022) stated that germination of sorghum significantly enhanced gaba concentration in results and discussion influence of traditional processing on cowpea color the effects of various home processing techniques on the coordinates of cowpea color (l*, a* and b*) and the overall difference in color (δe) of cowpea pods are shown in table 1. in general, blanching, drying and fermentation significantly (p < 0.05) caused a change in color parameters, compared to raw samples. a higher increase was observed in blanched and fermented samples, particularly at a* and b* values. blanching and fermentation had the respective maximum values for l* (49.19 and 49.61), compared to dried samples (39.49). interestingly, a* and b* values followed the same manner as that followed by the l* value. the blanched and fermented samples had the highest values than the dried samples. additionally, the total color difference (δe) was significantly different (p < 0.05) in processed pods. δe was 8.52, 4.27 and 9.82 in blanched, dried and fermented pods, respectively. a significant increase in δe was mainly due to the increased changes in l*, a* and b* values in blanched and fermented pods, compared to dried pods. in this study, cowpea’s traditional household cooking methods, particularly drying, were significantly influenced by its lightness, redness and yellowness. for instance, the l* and b* values of dried samples were considerably less than that of control and other treated samples. these results were also reflected in the dried samples’ δe values, compared to different cooking techniques. it is possible that the observed increase in δe in fermented and blanched cowpea meal resulted from water replacing intercellular air and cellular material seeping into water because of ruptured cell membranes (turkmen et al., 2006). influence of traditional processing on cowpea anthocyanin and gaba contents the anthocyanin and gaba contents of control and processed cowpea samples are reported in table 2. table 1. effect of traditional processing methods on the color of cowpea pods. samples l* a* b* ∆e raw 41.40 ± 1.212b 1.97 ± 0.643a 25.95 ± 0.964b 0.0 ± 0.000d blanched 49.19 ± 1.149a 1.35 ± 0.111a 29.35 ± 1.271a 8.52 ± 0.125b dried 39.49 ± 2.357c 1.37 ± 0.110a 22.18 ± 1.422c 4.27 ± 0.020c fermented 49.61 ± 0.566a -2.01 ± 0.448b 29.64 ± 1.163a 9.82 ± 0.060a f-test ** ** ** ** lsd 0.05 2.772 0.7532 2.290 0.132 l: lightness; ∆e: total color difference. values are mean (±sd) of triplicate samples. mean values in the same column without superscripts are not significantly (p < 0.05) different; ns: no significant difference at (p < 0.05) as assessed by least significant difference (lsd). table 2. effect of traditional processing methods on anthocyanin and gaba contents of cowpea pods. samples anthocyanin (mg/g) gaba (mg/g) raw 3.69 ± 0.214c 2.16 ± 0.05b blanched 4.35 ± 0.547b 0.90 ± 0.01c dried 9.95 ± 0.040a 0.65 ± 0.09d fermented 4.50 ± 0.053b 4.50 ± 0.02a f-test ** ** lsd 0.05 0.557 0.103 gaba: γ-aminobutyric acid. values are means (±sd) of triplicate samples. mean values in the same column without superscripts are not significantly (p < 0.05) different; ns: no significant difference at (p < 0.05) as assessed by least significant difference (lsd). italian journal of food science, 2023; 35 (2) 75 evaluation of nutritional values and traditional preparation methods of cowpea sorghum grain, particularly under different salt concentrations. moreover, fermentation was also detected as a positive method for enhancing gaba concentration in food (liao et al., 2013; seo et al., 2013). these statements were in agreement with our findings, which stated that higher values of gaba were noted in fermented cow pea samples. anthocyanin was also affected by the traditional cooking methods of cowpea. it significantly increased in processed cowpea samples. further, drying of cowpea almost increased the content of anthocyanin by 100%, compared to other household preparation methods. influence of traditional processing on tpc of cowpea the impact of conventional home approach on tpc (mg gae/g) and tfc (mg qe/g) is shown in figures 1a and 1b. the tpc of raw cowpea pods was 5.75 mg gae/g. it was evident that tpc of samples increased due to typical processing techniques. tpc increased significantly (p < 0.05) during drying and fermentation, reaching 8.86 mg gae/g and 7.56 mg gae/g, respectively (figure 1a). similarly, the traditional methods of preparation also significantly affected the tfc of cowpea pods (p < 0.05). prior to processing, the tfc was 5.72 mg qe/g (figure 1b). balancing of pods caused a slight increment in tfc to 6.09 mg qe/g. however, sharp increase in tfc was observed when the pods were dried and fermented (p < 0.05), with the respective values of 27.61 mg qe/g and 35.89 mg qe/g (figure 1b). increase in the tpc of processed samples could be due to bound phenolic compounds that were released and then collected by extending the time of the drying process, making them more extractable. yadav et al. (2018) observed a similar result. the tpc was increased after 10.0 t p c ( m g g a e /g ) t f c ( m g q e /g ) (a) (b) raw dried ferme.. 8.0 b b a a 6.0 4.0 2.0 0.0 40.0 raw dried ferme.. 35.0 30.0 c c b a 25.0 15.0 20.0 10.0 5.0 0.0 figure 1. effect of traditional processing methods on the (a) total phenolic content (tpc, mg gae/g) and (b) total flavonoid content (tfc, mg qe/g) of cowpea. values are mean (±sd) of triplicate samples. values with the same letters are not significantly different (p > 0.05) as assessed by least significant difference (lsd). using autoclaving and microwave treatment for 30 min, compared to 15 min. the tpc values of raw pods reported in this study were lower than those (8.28 mg gae/g) observed by deng et  al. (2013). conversely, obtained results did not agree with the findings of barros et al. (2017), who reported that the phenolic compounds were reduced after cooking (boiling) cowpea grains cultivars because of migration of soluble phenolic compounds through leaching in cooking broth. however, variation in tpc could be attributed to different factors associated with cowpea cultivars, maturity at harvest, environmental conditions, solvent type, assay procedure and interaction of phenolic compounds with other food contents (marathe et al., 2011). on the other hand, increase in tpc in fermented samples was caused by microbial enzymes that break down cell walls to release bioactive compounds (hassan et al., 2021). regarding tfc, the findings of this study differed from the results of yadav et al. (2018), who observed that both boiling and fermentation had a negative effect on tfc. instead, the authors attributed reduction to the leaching of flavonoids to cooking water and flavonoid consumption by growth microorganisms during fermentation. nevertheless, the traditional household processing of cowpea meal significantly enhanced both tpc and tfc, particularly during drying and fermentation. hence, both processing methods could be recommended for the food industry. influence of traditional processing on antioxidant activity of cowpea figures 2a and 2b illustrate how the antioxidant activity of unprocessed and processed cowpea pods was 76 italian journal of food science, 2023; 35 (2) alghamdi fa et al. household preparation methods of cowpea on color, phytochemical compounds, antioxidant activity and gaba content (figure 3). the plot showed that the first two principal components accounted for 48.01% of total variance. principal component (pc1) showed 61.15% variation and pc2 accounted for 22.86% of total variation. the pca factor showed that tpc, tfc, dpph, frap, anthocyanin and color differences (∆e) were significantly associated for with pc1. in contrast, gaba was highly correlated with pc2. however, pca displayed a strong positive correlation between tpc, tfc, dpph, frap, anthocyanin, ∆e and gaba with fermented cowpea pods. these interpretations stated that fermentation improved the accumulation of health-promoting phytochemicals, antioxidant activity and gaba of cowpea pods. the partial least squares (pls) regression test analysis described the interactive impact of traditional preparation methods (x variables) on the stated factors (y variables) of cowpea samples (figure 4). referring to the pls model, fermented cowpea pods showed a positive validation score for tpc, tfc, dpph, frap, anthocyanin content, ∆e and gaba content, reflected as the most valid scores. this validation revealed that traditional fermentation could significantly improve the health-promoting metabolites of traditional cowpea meals. conclusions cowpea pods contain a high amount of phenolic compounds with high antioxidant activity and gaba content. significant improvement was observed in tpc, tfc, antioxidant activity (dpph and frap), and anthocyanin and gaba contents during the traditional processing of cowpea, especially fermentation. as a result, evaluated in terms of frap and their capacity to scavenge free radicals. the impact of conventional blanching, drying and spontaneous fermentation on the dpph scavenging activity of cowpea pods is shown in figure  2a. untreated pods had a dpph of 2.5 mg te/g. it was shown that the processing approach increased the dpph activity of pods considerably (p < 0.05). maximum dpph activity was observed in dried (4.92 mg te/g) and fermented (4.86 mg te/g) samples. figure 2b displays the frap values of raw and processed pods. the procedure increased the frap of pods significantly (p < 0.05), as observed in the figure. it was 5.44 mg te/g before processing but increased dramatically after blanching (8.88 mg te/g), drying (10.11 mg te/g) and fermentation (10.61 mg te/g) of cowpea samples. in general, the findings of this study demonstrated significant differences (p < 0.05) in dpph and frap assays between raw and processed cowpea pod samples, particularly during fermentation. therefore, improving the antioxidant activity of fermented cowpea pods could increase both tpc and tfc. moreover, during fermentation, aglycone release from hydrolyzed phenolic glycosides by microbial enzymes contributed to increased antioxidant activity. furthermore, fermentation encouraged the structural collapse of plant cell walls, releasing numerous antioxidant complexes (hur et al., 2014). similarly, adetuyi and ibrahim (2014) discovered that the antioxidant activity was significantly increased in okra seeds after 48-h fermentation. multivariate analysis the principle component of analysis (pca) biplot revealed an apparent clustering of the effect of traditional 6.0 (a) (b) raw blanched blanched dried ferme.. 5.0 d p p h s ca ve ng in g (m g tr ol ox /g ) f r pa (m g tr ol ox /g ) c b a a 4.0 3.0 2.0 1.0 0.0 12.0 raw dried fermen.. 10.0 b a a a 8.0 4.0 6.0 2.0 0.0 figure 2. effect of traditional processing methods on the (a) dpph scavenging assay (mg trolox/g) and (b) ferric reducing power assay (frab, mg trolox/g) of cowpea samples. values are mean (±sd) of triplicate samples. values with the same letters are not significantly different (p > 0.05) as assessed by least significant difference (lsd). italian journal of food science, 2023; 35 (2) 77 evaluation of nutritional values and traditional preparation methods of cowpea biplot (axes f1 and f2: 84.01%) f 2 (2 2. 86 % ) gaba δe fermented blanched tfc frab dpph tpc dried raw –4 –4 –3 –3 –2 –2 –1 –1 f1 (61.15%) active variable active observations 0 0 1 1 2 2 3 3 4 anthocyanin figure 3. the principle component of analysis (pca) of tpc, tfc, dpph, frab, anthocyanin content, gaba and ∆e of raw, blanched, fermented and dried cowpea pods. correlations on axes t1 and t2 t2 tpc dpph frab anthocyanin dried tfc δeblanched fermented gaba raw –1 –1 –0.75 –0.75 0.75 1 –0.5 –0.5 0.5–0.25 0.250 t1 x y active validation –0.25 0.25 0.75 1 0.5 0 figure 4. the partial least squares (pls) regression test analysis of tpc, tfc, dpph, frab, anthocyanin content, ∆e and gaba content of raw, blanched, fermented and dried cowpea samples. 78 italian journal of food science, 2023; 35 (2) alghamdi fa et al. these traditional methods, particularly fermentation, could be considered for use in the food industry as a traditional method for enriching food with secondary metabolism compounds and antioxidants. data availability statement the data used to support the findings of this study are included in the article. acknowledgements this research was supported by princess nourah bint abdulrahman university researchers supporting project (pnursp2023r43), princess nourah bint abdulrahman university, riyadh, kingdom of saudi arabia. conflict of interest the authors declared no conflict of interest. author contributions fatima ali alghamdi: data curation, visualization. amro b. hassan: methodology, writing review and editing. nora abdullah alfaris: data curation and review and editing last draft. jozaa zaidan altamiami soft ware and funding acquisition. all authors have read and agreed to the published version of the manuscript. references adetuyi f.o. and ibrahim t.a. 2014. effect of fermentation time on the phenolic, flavonoid and vitamin c contents and antioxidant activities of okra (abelmoschus esculentus) seeds. nigerian food j (nifoj). 32:128–137. https://doi.org/10.1016/ s0189-7241(15)30128-4 affrifah n.s., phillips r.d. and saalia f.k. 2022. cowpeas: nutritional profile, processing methods and products—a review. legume sci. 4(3):e131. https://doi.org/10.1002/leg3.131 awika j.m. and duodu k.g. 2017. bioactive polyphenols and peptides in cowpea (vigna unguiculata) and their health promoting properties: a review. j funct foods. 38:686–697. https://doi. org/10.1016/j.jff.2016.12.002 barros n.v.a, rocha m.d.m., glória m.b.a., araújo m.a.m. and moreira-araújo r.s. 2017. effect of cooking on the bioactive compounds and antioxidant activity in grains cowpea cultivars. ciência agronômica. 48:824–831. https://doi.org/10.5935/ 1806-6690.20170097 benzie i. and devaki m. 2017. the ferric reducing/antioxidant power (frap) assay for non-enzymatic antioxidant capacity: concepts, procedures, limitations and applications: recent trends and applications. in: apak, r. capanoglu, e. and shahidi, f. 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34 (2): 1–8 issn 1120-1770 online, doi 10.15586/ijfs.v34i2.2137 1 p u b l i c a t i o n s codon a novel cascade approach to extract bioactive compounds from officinal herbs yubin ding1, ksenia morozova1, matteo scampicchio1*, angelo morini2, massimiliano ferrari2 1faculty of science and technology, free university of bozen-bolzano, bolzano, italy; 2naturalsalus srl., bolzano, italy *corresponding author: matteo scampicchio, faculty of science and technology, free university of bozen-bolzano, piazza università 1, 39100 bolzano, italy. email: matteo.scampicchio@unibz.it received: 5 october 2021; accepted: 20 january 2022; published: 1 april 2022 © 2022 codon publications open access paper abstract this research aims to compare a novel cascade extraction method with a conventional solid–liquid extraction method, both assisted by ultrasounds. the cascade extraction method consists of a sequential series of extractions performed with the same hydroalcoholic solvent, which is reused from one herb to the other. in practice, a hydroalcoholic solution is firstly used to extract one botanical herb. the resulting extract is then reused for the extraction of a second herb. the process is repeated as many times as the number of herbs composing the final formulation. the main advantage of this approach is firstly the lower need of solvents compared with the individual extraction procedures, where a fresh solvent is needed on each extraction step. furthermore, extracts of the two methods (solid liquid vs cascade extraction) were characterized with several antioxidant assays (dpph, orac, and frap) and total phenolic content (tpc). the results show that the solid–liquid extraction method achieves similar yields to total phenols and similar tac in comparison to the extracts obtained by the cascade extraction method. also, the hplc analysis of the extracts showed that both methods lead to similar chromatographic profiles either when analyzed by an electrochemical detector (coularray) or by a spectrometric diode array detector (dad). however, our findings support the idea that the cascade extraction method obtains extracts richer of minor peaks, showing a more complex bioactive profile. such results could be explained considering that the solvent used during the series of cascade extractions was enriched not only by antioxidants but also by plant surfactants, like saponins, which increase the solvent solubility. overall, this research shows that the cascade extraction method can not only provide officinal herb extracts with similar phenolic yield and antioxidant capacity than conventional solid–liquid extraction but also with a more complex bioactive profile compared to traditional solid–liquid extraction and with a minor consumption of solvents. keywords: antioxidant ability; cascade extraction; herbal tincture; hplc introduction herbal extracts are hydroalcoholic preparations typically obtained by maceration or percolation (karioti et al., 2011). after extraction with a hydroalcoholic solution, the resulting extracts contain a complex profile of bioactives that can be used as supplements for human health. several studies report the beneficial effect of extracts obtained from botanicals. for example, according to dos reis et al., the extract of ptychopetalum olacoides, which contains tannins, flavonoids, and terpenoids, is used as a remedy for problems concerning the central nervous system (dos reis and mendes, 2018). also, the extract prepared with turnera diffusa has anxiolytic and antidepressant effects (ana maría et al., 2019). however, the composition in bioactives depends on a proper extraction technique. the most traditional way to prepare extracts is the classical maceration of dry herbs with a hydroalcoholic mailto:matteo.scampicchio@unibz.it 2 italian journal of food science, 2022; 34 (2) ding y et al. herb. the procedure continues in a series of extractions, where the extract of each step becomes the solvent for the subsequent step. thanks to such an innovative extraction procedure, during the reutilizations of the solvent, and through the wise selection of the herbal sequences, the hydroalcoholic solvent is progressively enriched not only with the main characteristic bioactives from each botanical herb but also with natural modifiers, which fine-tune the viscosity, ph, and polarity of the initial hydroalcoholic solvent. this, ultimately, will enhance its solvation capacity, leading to final extracts with richer bioactives profile, higher yields, and less solvent consumption. thus, in this work, the extraction efficiency of a traditional solid–liquid extraction method is compared with the proposed cascade extraction approach. total phenolic content (tpc) together with total antioxidant capacity (dpph, frap, and orac) of both approaches were compared. high performance liquid chromatography (hplc) coupled with diode array detector (dad) and coulometric array detector (coularray) were used to analyze the bioactive compounds extracted with both approaches. material and methods reagents ethanol, folin–ciocalteau reagent, sodium carbonate, gallic acid, 2,5,7,8-tetramethylchromane-2carboxylic acid (trolox), 2,2-diphenyl-1-picrylhydrazyl (dpph), sodium acetate, acetic acid, ferric chloride, hydrochloric acid, disodium phosphate, monosodium phosphate, sodium hydroxide, fluorescein, 2,2’-azobis(2amidinopropane) dihydrochloride (aaph), hplc grade ammonium formate, formic acid, and acetonitrile were obtained from sigma-aldrich (usa). 2,4,6-tris(2pyridyl)-s-triazine (98%) was purchased from alfa aesar (usa). the milli-q water was purified by sartorius arium® mini (germany). samples four varieties of officinal herbs, namely, ptychopetalum olacoides benth. (stem), turnera aphrodisiaca willd. (leaf ), schisandra chinensis baill. ( fruit), and poligonum multiflorum thunb. (root), were kindly provided by naturalsalus® and preprocessed based on their standardized protocols. in brief, the dried herbs were grounded with a laboratory mill (perkinelmer, laboratory mill 3100, germany) and sieved (retsch, as 200, germen) to reach a uniform size (<250 µm). the powder was then preserved at ambient temperature before extraction. solution, in a solid-to-liquid ratio of 1:–5 or 1:–10. with slight modifications, other parent techniques have been developed, like percolation, hydrodistillation, boiling with reflux, and soxhlet (alara et al., 2018). unfortunately, such techniques consume large amounts of organic solvents with high purity, and have limited extraction yields and low selectivity. moreover, they need long extraction times, which may cause the thermal degradation of some sensitive bioactives (rostagno et al., 2010). recently, the efficiency of the process has been greatly improved by implementing innovative technologies, such as ultrasound-assisted extraction (uae), supercritical fluid extraction (sfe), and microwave-assisted extraction (mae) (reverchon and de marco, 2006; valachovic et al., 2001; xia et al., 2011). when traditional extraction is assisted with such techniques, the duration of the extraction process is greatly reduced (i.e., a few hours), maximizing the yield of the extracted bioactives and limiting thermal degradation problems (valachovic et al., 2001; vinatoru et al., 2017). despite such improvements, the extraction process still has some limitations. the most evident is that many lipophilic bioactives present in herbs (like carotenoids or terpenes) are scarcely soluble in hydroalcoholic solvents. this clearly limits the potential quality, potency, and effectiveness of the resulting extracts. for this purpose, some recent researches have tried to overcome the low solvation capacity of pure hydroalcoholic solvents by the addition of ionic liquids (han et al., 2011), lime juice (cheok et al., 2013), diethylamines (choi et al., 2000), and saponins. through the careful use of some additives, important solvent properties like viscosity, ph, or polarity can be fine-tuned as required. this, ultimately, could increase the extraction yield of bioactive compounds. a further important limitation on the current use of traditional extraction techniques is the high solvent consumption. generally, for each volume of dry herbs, 5–10 volumes of fresh hydroalcoholic solvent are needed (council of europe, 2019). although such high solvent to sample ratio is intended to maximize the yield of extractable compounds, it represents a relevant manufacturing cost, a high energy demand, and an environmental concern (abubakar et al., 2015). accordingly, this work aims to propose a simple but ingenious cascade extraction protocol, which reuses the same initial hydroalcoholic solvent in a series of extraction cycles. in practice, the procedure starts with a hydroalcoholic solvent, which is used for the first extraction of one selected botanical herb. then, the resulting extract is recycled for the extraction of a second selected botanical italian journal of food science, 2022; 34 (2) 3 a novel cascade approach total phenol content the tpc analysis was based on margraf et al. with small modifications (margraf et al., 2015). briefly, 110 μl of distilled water, 10 μl of sample or gallic acid standard, 10 μl of folin–ciocalteu reagent, and 10 μl of saturated sodium carbonate solution were added to a 96-well microplate. the microplate was incubated for 120 min at 25°c. finally, the absorbance of each well was measured at λ = 765 nm (tecan, infinite m nano+, switzerland). from the absorbance measurements, the total phenol content was expressed as gallic acid equivalent (gae) based on a calibration curve, built with a series of gallic acid standards. antioxidant assays dpph assay the free radical scavenging activity was measured with 2,2-diphenyl-1-picrylhydrazyl stable radical (dpph·). 160 µl of dpph working solution (125 µm) was added to 40 µl of trolox standards or herbal extracts into a 96-well microplate. the mixture was left to stand for 30 min in the dark at room temperature. the absorbance was measured at 515 nm and the result was expressed based on the trolox equivalent (mishra et al., 2012). frap assay the ferric reducing antioxidant power (frap) assay was conducted based on previous research by benzie extraction extractions were performed with an ultrasound reactor (steel®, digital ultrasonic generator, italy). briefly, 10 g of dry herbal powder was mixed with 100 ml of solvent (water: ethanol = 1:1, v/v) and extracted for 40  min at 40°c. ultrasound power was set to 60% (345 w, 38 khz). after extraction, the resulting solution was filtered (ptfe 0.45 µm, pall corporation, usa) and centrifuged (10,000 rpm for 10 min at 20°c, sl 16r centrifuge, thermo scientific, usa). the resultant supernatant was collected and stored at −80°c. cascade extraction consists of using the extract (obtained from an extraction step) as the solvent for the next step. during each step, fresh hydroalcoholic solvent (typically less than 10% of the initial volume) could be added to replace the solvent evaporated and trapped in the herb matrix during the operation, and maintain a fixed initial sample to solvent ratio (1:10). the experimental conditions of solid–liquid ratio, extraction time, temperature, and ultrasound power were applied equally to each extraction step modified from the previous officinal herb extraction conducted by rodríguez-pérez et al. in 2015 (see figure 1). the optimized sequence used in this work is the following: ptychopetalum, turnera, schisandra, and poligonum. for comparison, individual extraction was also performed, similar to cascade extraction, which compensated for the lost solution (100 ml) before performing the analysis. dried officinal herb powder 10 g ultrasound assisted extraction (40°c x 40 min) ultrasound assisted extraction (40°c x 40 min) ultrasound assisted extraction (40°c x 40 min) centrifugation 10.000 rpm x 10 min at 20°c and remove the residual centrifugation 10.000 rpm x 10 min at 20°c and remove the residual centrifugation 10.000 rpm x 10 min at 20°c and remove the residual bring to volume with the solvent bring to volume with the solvent bring to volume with the solvent 100 ml (x 2) 100 ml hydroalcholic solvent dried officinal herb powder 10 g figure 1. the process of cascade extraction used in the current research. 4 italian journal of food science, 2022; 34 (2) ding y et al. ratio) or their mix in comparison with the proposed cascade method. among the individual herbs, the poligonum (10 g in 100 ml of fresh solvent) showed the highest tpc (27.3 ± 3.3 mm), while the ptychopetalum (10 g in 100 ml of fresh solvent) showed the lowest (3.49 ± 0.20 mm). the combination of the herb extracts (each obtained with a sample to solvent ratio of 1:10) resulted in a total phenol content of 59.1 ± 7.3 mm of gallic acid equivalent. concerning the extracts obtained with the proposed cascade extraction, the tpc was 57.0 ± 1.7 mm. such a result is not significantly different from the mix of individual extracts (t-test, p < 0.05). furthermore, it is important to highlight that, although the cascade extraction used the same amount of dry herbs as the traditional maceration method (10 g of each botanical), the volume of hydroalcoholic solvent needed was about one fourth. these findings suggest that the cascade extraction method is able to extract similar amount of phenolic compounds than the maceration method, but with less solvents. the antioxidant assays the results of antioxidant assays, including dpph, frap, and orac, are presented in table 1. in general, those individual sample extracts that showed higher tpc also had higher antioxidant capacity. in detail, the turnera and poligonum showed the highest antioxidant capacity in all the antioxidant assays. on the contrary, the ptychopetalum and schisandra had lower antioxidant capacity. what is striking here is that the extracts obtained with the cascade extraction method have orac, frap, and dpph assay values that are not significantly different (p < 0.05) from those obtained by mixing the individual extracts. overall, these results confirm those obtained by total phenol content (vide supra). the hplc-coularray analysis figure 2 shows the chromatogram obtained by hplc coupled with an electrochemical array detector (coularray) of the extracts. the electrochemical detector is useful for providing quantitative and qualitative information of each compound eluted from the chromatographic column (haque et al., 2021). first, the analysis of the peak current values obtained at different applied potentials can be used to determine the redox potential of each eluted species. furthermore, the peak heights obtained at very high oxidative potentials (i.e., +800 mv vs ref. electrode) can also be used to express the total amount of antioxidant present in the sample, i.e., the total antioxidant capacity (hicks et al., 2017). from figure 2, the herbs poligonum and turnera showed higher peak heights than schisandra and ptychopetalum. and strain with modifications (benzie and strain, 1996). 180 µl of frap reagent (ph = 3.6 acetate buffer, 20 mm ferric chloride, 40 mm tptz in 40 mm hydrochloric acid in 10:1:1 ratio) was added to 20 µl of trolox standard or herbal extracts. after incubation for 1 h at 25°c, the absorbance at 593 nm was recorded and the result was expressed as µmol te/g dw. orac assay the oxygen radical absorbance capacity (orac) assay was performed using the method modified from prior et  al. (2003). phosphate buffer (75 mm, ph 7.0) was used to dilute all the solutions, including fluorescein (3.19 μm), aaph solution (111 mm), trolox, and herbal extracts. 30 μl of sample or trolox standard was premixed with 75 μl of fluorescein and incubated at 37°c for 5 min before adding freshly prepared 75 μl of aaph in a 96-well microplate. the fluorescence signal was monitored every minute, during 2 h reaction time with excitation wavelength at 485 nm and emission wavelength at 535 nm. the antioxidant capacity was calculated based on the area under the fluorescence decay curve and expressed as μmol te/g dw. hplc analysis the sample extracts were analyzed with agilent 1260 infinity hplc system with a binary pump, an autosampler with the temperature control system, a dad (agilent technology, usa), and a 16 channel coularray detector (thermofisher scientific, usa). a kinetex biphenyl column (100 × 2.1 mm, 2.6 µm particle size with a pre column, phenomenex, usa) was applied for the separation of compounds at constant 30°c. the separation was conducted with 6.5 mm ammonium formate in milli-q water with 0.1% formic acid as phase a, and 6.5  mm ammonium formate in acetonitrile with 0.1% formic acid as phase b. 5 μl of the sample was injected with a constant flow rate of 0.3 ml·min−1. all the samples were previously loaded into the autosampler and kept at 5°c before analysis. the elution gradient was 98% of phase a from 0–8 min, then down to 40% at 20 min, to 5% a at 27 min, kept constant for 2 min, and the final equilibration was 98% a at 31 min. the dad detector was set at 280 nm and the coularray was set from 50 to 800 mv, with 50mv increment through the 16 electrodes (vs. pd electrode). results the total phenolic content table 1 presents the toc of the extracts obtained by using the traditional maceration process, either of individual herbs (obtained under the same sample to solvent italian journal of food science, 2022; 34 (2) 5 a novel cascade approach table 1. the tpc, dpph, frap, and orac results of individual herb sample extracts and those from cascade extraction. tpc (mm gae) dpph (mm te) frap (mm te) orac (mm te) (1) ptychopetalum 3.49 ± 0.20 0.37 ± 0.08 1.48 ± 0.20 12.01 ± 0.80 (2) turnera 22.2 ± 3.4 13.44 ± 0.58 11.98 ± 0.86 51.7 ± 1.2 (3) schisandra 6.02 ± 0.41 3.00 ± 0.78 5.10 ± 0.58 22.2 ± 2.1 (4) poligonum 27.3 ± 3.3 13.8 ± 1.2 11.43 ± 0.04 36.57 ± 0.51 mix of individualsa 59.1 ± 7.3 30.6 ± 2.6 30.0 ± 1.7 122.5 ± 4.6 cascade extraction 57.0 ± 1.7 28.5 ± 3.9 30.5 ± 5.0 123.0 ± 1.7 athe accumulation of four individual herb sample extracts. figure 2. the hplc-coularray result of the individual and cascade sample extracts (potential poised at +800 mv versus palladium electrode). this finding correlated with the values of total phenol content and total antioxidant capacity determined previously. meanwhile, the chromatograms confirmed that the cascade extraction was able to extract all the main redox compounds present in the individual herbs, although with less solvents. hplc-dad analysis the hplc-dad analysis was performed to compare the bioactive compound profile of individual and cascade extraction at 280 nm. figure 3 shows the chromatograms of all individual herbs and that of the extract obtained by cascade extraction. the results show that the sum of the main peaks observed in the individual herbs is also present in the cascade extraction. this finding confirms the results obtained by the hplc-coularray and supports the conclusion that both extraction methods, i.e., traditional maceration versus cascade extraction, provide extracts with similar composition. however, through a more detailed analysis of the hplcdad chromatogram, it is possible to observe that the extracts obtained from the cascade method present a more complex profile. with such extracts, several minor compounds (see figure 4) were observed as small peaks along the chromatographic trace. it is important to highlight that such peaks were not present in the hplcdad of the individual extracts. for instance, the peaks observed at a retention time of around 8.5 min (peaks a, b, and c) and at 13.5 min (peak d) were only present in the extracts obtained by the cascade method, while they were absent in the chromatograms of the individual 6 italian journal of food science, 2022; 34 (2) ding y et al. with their adsorption to the solid retentate, which occurs between one extraction and the other. however, figure 3 also shows that some other peaks are bigger in the cascade extract than in the individual extracts. this observation may also be explained considering that the cascade extraction generates solvents that are progressively richer not only of phenols but also of natural modifiers, which modify the solvating power of the solvent. for this purpose, several studies have shown that the botanicals used in this study are naturally rich of surfactants, like saponins, which act as solvent modifiers and could be useful to increase the extraction of certain bioactive compounds (peng et al., 2018; walthelm et al., 2001). in particular, one of the main benefits of using a solvent naturally enriched by saponins is that the solvent viscosity is reduced, the surface tension is lowered, and micelles are generated. overall, such effects may explain the enhanced solvating power of a solvent enriched by natural saponins (vinarov et al., 2018). several extraction methods have been developed focusing on solvent saving during the extraction process. the most traditional and widely applied method is soxhlet extraction. this method continuously recycles the solvent by a series of evaporation/condensation cycles, while accumulating the extracted bioactives in a boiling flask (patel et al., 2019). in recent years, other continuous and batch extraction techniques have been developed (poirot et al., 2007). among other methods, accelerated solvent extraction (richter et al., 1996) and randall extraction (thiex et al., 2003) are very promising for their solvent herbs. this finding suggests that the cascade extraction was able to increase the recovery of more minor bioactives than the maceration method of individual herbs. meanwhile, figure 3 shows that the sample extract obtained by the cascade extraction method results in a chromatogram with a lower content of some peaks, if compared with the samples obtained from the single extraction methods. this is clearly observable in the chromatogram of the first herb, ptychopetalum. such loss of certain phenolic compounds can be explained figure 3. the hplc-dad analysis of individual and cascade extraction at 280 nm. figure 4. the hplc-dad analysis of individual and cascade extraction at certain time 8–9.2 min and 13.1 to 14.1 min. italian journal of food science, 2022; 34 (2) 7 a novel cascade approach benzie, i.f.f. and strain, j.j., 1996. the ferric reducing ability of plasma (frap) as a measure of “antioxidant power”: the frap assay. analytical biochemistry 239(1): 70–76. https://doi. org/10.1006/abio.1996.0292 cheok, c.y., chin, n.l., yusof, y.a., talib, r.a. and law, c.l., 2013. anthocyanin recovery from mangosteen (garcinia mangostana  l.) hull using lime juice acidified aqueous methanol solvent extraction. food science and technology research 19(6): 971–978. https://doi.org/10.3136/fstr.19.971 choi, y.h., kim, j., kim, j.y., joung, s.n., yoo, k.p. and chang, y.s., 2000. modifier effects on supercritical co2 extraction efficiency of cephalotaxine from cephalotaxus wilsoniana leaves. archives of pharmacal research 23(2): 163–166. https://doi.org/10.1007/ bf02975507 council of europe, 2019. european pharmacopoeia 10.0. dos reis, l.f. and mendes, f.r., 2018. ptychopetalum olacoides benth. pp. 401–411. https://doi.org/10.1007/978-94-024-1552-0_36 han, d., zhu, t. and row, k.h., 2011. ultrasonic extraction of phenolic compounds from laminaria japonica aresch using ionic liquid as extraction solvent. bulletin of the korean chemical society 32(7): 2212–2216. https://doi.org/10.5012/ bkcs.2011.32.7.2212 haque, m.a., morozova, k., ferrentino, g. and scampicchio, m., 2021. electrochemical methods to evaluate the antioxidant activity and capacity of foods: a review. electroanalysis 33(6): 1419–1435. https://doi.org/10.1002/elan.202060600 hicks, m.b., salituro, l., mangion, i., schafer, w., xiang, r., gong,  x. and welch, c.j., 2017. assessment of coulometric array electrochemical detection coupled with hplc-uv for the absolute quantitation of pharmaceuticals. the analyst 142(3): 525–536. https://doi.org/10.1039/c6an02432g karioti, a., fani, e., vincieri, f.f. and bilia, a.r., 2011. analysis and stability of the constituents of curcuma longa and harpagophytum procumbens tinctures by hplc-dad and hplc-esi-ms. journal of pharmaceutical and biomedical analysis 55(3): 479–486. https://doi.org/10.1016/j.jpba.2011.02.029 margraf, t., karnopp, a.r., rosso, n.d. and granato, d., 2015. comparison between folin-ciocalteu and prussian blue assays to estimate the total phenolic content of juices and teas using 96-well microplates. journal of food science 80(11): c2397– c2403. https://doi.org/10.1111/1750-3841.13077 mishra, k., ojha, h. and chaudhury, n.k., 2012. estimation of antiradical properties of antioxidants using dpphassay: a critical review and results. food chemistry 130(4): 1036–1043. https:// doi.org/10.1016/j.foodchem.2011.07.127 patel, k., panchal, n. and ingle, p., 2019. review of extraction techniques extraction methods: microwave, ultrasonic, pressurized fluid, soxhlet extraction, etc. international journal of advanced research in chemical science 6(3): 6–21. https://doi. org/10.20431/2349-0403.0603002 peng, s., li, z., zou, l., liu, w., liu, c. and mcclements, d.j., 2018. improving curcumin solubility and bioavailability by encapsulation in saponin-coated curcumin nanoparticles prepared using a simple ph-driven loading method. food and function 9(3): 1829–1839. https://doi.org/10.1039/c7fo01814b saving attributes. however, in comparison with the abovementioned extraction technologies, the cascade extraction approach proposed in this research is still more advantageous. for instance, to prepare an extract composed of four individual botanicals, the single extraction method needs a total of 400 ml of solvent for the extraction of 40  g of herbs. on the contrary, the cascade extraction needs only 140 ml of solvent in total. such solvent reduction is a unique feature of the cascade extraction approach that cannot be simply achieved with other techniques. conclusion this research proposes an innovative extraction method, called cascade extraction, that presents some advantages compared to the traditional solid–liquid extraction (i.e., maceration), especially when the final desired extract is a mixture of several botanical herbs. although the overall extraction efficiency (determined by tpc, dpph, frap, and orac assays, and hplc analysis coupled with the electrochemical detector) of the 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food science and technology, sabzevar branch, islamic azad university, sabzevar, iran *corresponding author: amir hossein elhamirad, associate professor, department of food science and technology, sabzevar branch, islamic azad university, sabzevar, iran. email: elhamirad1974@gmail.com received: 25 june 2022; accepted: 18 april 2023; published: 2 may 2023 © 2023 codon publications open access original article abstract plant extracts play an important role in improving the quality properties and enhancing the shelf life of food products. in the present study, different concentrations of green tea (0–2%), white tea (0–2%), and ginger (0 to 1.5%) extracts were used to formulate a sponge cake. response surface methodology was used to investigate cake quality attributes. for each response, a second-order polynomial model with high coefficient of determination (r2; >0.95) was developed using multiple linear regression analysis. the overall optimum area with high stability was found at a combined level of green tea (1.44%), white tea (1.71%), and ginger (0.75%) extracts. keywords: ginger, response surface methodology, sponge cake, tea introduction consumers’ demand for healthier food products that prevent nutrition-related diseases and improve physical and mental well-being has led to the accelerated growth of functional foods market. a large number of potentially active nutrients and their multifunctional properties make them perfect candidates for the production of health-promoting food and supplements (pinto et  al. 2014; siro et  al. 2008). among different food systems, baked products provide an excellent opportunity to incorporate food-grade fractions. hence, nowadays the bakery industry seeks to improve the health attributes of their products by using functional ingredients that in some way can lead to healthier food products. in the bakery industry, cakes have become popular and a type of popular snack globally. sponge cake is a cake based on flour (usually wheat flour), sugar, and eggs, and is sometimes leavened with baking powder. the quality of cakes depends on many factors, such as the ingredients used for batter preparation, aeration of batters, and process conditions (pinto et al. 2014). functional food products are novel products enriched with various ingredients, such as live microorganisms (probiotics), nutrients, dietary fiber, phytochemicals, and other substances. these ingredients show a possible health-enhancing or disease-preventing values and are utilized at a safe and sufficient concentration to achieve intended benefits (temple 2022). the antioxidant properties of polyphenols found in green tea are the main health-promoting factors of this plant (sinija and mishra 2008; timoshenkova et al. 2020). white tea is a nonfermented brewage made from young tea leaves or norse tea roots and is a rich source of antioxidants, such as catechins, compared to green tea (hajiaghaalipour et  al. 2015). owing to the lack of fermentation in white tea, compared to black and oolong tea, the polyphenolic compounds in this tea are higher in concentration (gramza-michałowska et al. 2016). ginger (zingiber officinale roscoe) has been used as a spice in various foods for thousands of years. ginger is 34 italian journal of food science, 2023; 35 (2) pourzafar h et al. ethanol in the extract was removed with re300 rotary evaporator at 40°c, and the process was continued until the extract reached brix70%. the resulting extracts were refrigerated for experiments. phenolic content, antioxidant activity (2,2-diphenyl 1-picryhydrazyl  [dpph] radical scavenging capacity [rdsc]), and moisture content of the white tea extract were 53.47 ± 2.41 mg gallic acid equivalent (gae)/g dry weight of tea, 67.02 ± 3.81%, and 14.1 ± 1.03%, respectively. these values for green tea extract were 47.2 ± 2.11 mg gae/g dry weight of tea, 58.27 ± 1.89%, and 13.6 ± 0.6%, respectively. the moisture content of ginger extract was 10.7 ± 0.81%, its total phenolic content was 24.67 ± 0.91 mg gae/g dry weight of ginger, and its antioxidant activity was 33.4 ± 1.11%. preparing sponge cake for preparing cake batter (750 g), whole eggs and sugar were whipped in a mixer (shmb-300, sapaor, china) for 10 min, followed by addition of water, skimmed milk, and vanilla. then, flour was sieved along with baking powder with or without green tea, white tea, and ginger extracts (concentrations of these extracts are given in table 1) and added gradually to the cream and mixed slightly using a plastic spoon. oil was added to the recipe and mixed gently to obtain cake batter. finally, equal amount of this batter (50 g) was poured in nonstick cup muffin pans and baked for 20 min in an oven at 180–190°c (800-memmert, germany). sponge cakes were allowed to cool at room temperature. these were taken out from muffin pans with care and placed in plastic zipper bags. the bags with contents were stored in a dry and cool environment prior to analyses. pretreatment showed that adding more than 1.5% of ginger extract (i.e., 2%) caused an unpleasant spicy taste in sponge cakes. therefore, 1.5% of ginger extract is the maximum level considered for tasty sponge cakes. physicochemical analyses sponge cakes were analyzed by pursuing the following methods described by the american association for clinical chemistry (aacc, 2000): moisture considered as a dietary supplement by the us food and drug administration (fda) and is a part of herbal medicines listed in the world health organization (who) monograph. the spicy flavor of ginger is due to important compounds, such as volatile essential oils and nonvolatile oleoresins (shukla and singh 2007). (hollyer et al. 2002) demonstrated the effects of ginger on the stomach and intestines. the pharmacopeias of england, thailand, iran, and china mention ginger as an effective plant in the treatment of nausea and vomiting during pregnancy (keating and chez 2002; moradi lake, taleb, and saidi 2008). various studies have reported the influence of adding natural ingredients on the functional properties of cakes (luo et al. 2020; indriani et al. 2020; lenka, kumari, and pradhan 2020; taglieri et  al. 2022). however, according to our knowledge, no study has been conducted about the fortification of sponge cake by green and white tea and ginger extracts. therefore, the aim of this study was to determine the effect of green tea, white tea, and ginger extracts on the physicochemical and sensory properties of sponge cake, and to find the optimal point of different concentrations of these compounds in producing cake using response surface methodology (rsm). materials and methods materials ingredients used for preparing samples included wheat flour (protein content: 11.18 ± 0.68%; fiber: 2.3 ± 0.10%; fat: 0.69 ± 0.05% moisture: 12.78 ± 1.03%; and ash: 0.58 ± 0.03; sahar company, tehran, iran), white sugar (sugar noor company, sepahan, isfahan, iran), skimmed milk powder (0/1 fat; pegah company, tehran, iran), fresh ginger rhizomes (supermarket, kashan, iran), green tea and white tea (refah company, lahijan, iran), baking powder (behfar company, gilan, iran), canola oil (ladan company, tehran, iran), fresh eggs (zardeh talaei company, tehran, iran), and tap water. all the chemicals used in this study were of analytical grade and purchased from chemical suppliers. extraction process green tea, white tea, and ginger extracts were prepared by the maceration method. the plant samples were dried and powdered using an electric grinder to increase contact surface. powder of each plant was mixed with 70% ethanol as a solvent. for this purpose, 10 g of plant sample powder was poured into an extract container. then, 100-ml ethanol 70% was added gradually. the obtained extract was filtered with whatman paper no. 1, the table 1. independent variable values of the process and their corresponding levels. independent variables symbols –1 0 1+ green tea extract (% w/w) a 0 1 2 white tea extract (% w/w) b 0 1 2 ginger extract (% w/w) c 0 0/75 1/5 italian journal of food science, 2023; 35 (2) 35 response surface methodology for optimizing and producing food formulations of desirable properties replications at the central point to estimate experimental error (table 2). results and discussion fat content statistical models presenting fat, moisture, protein content, total phenolic content, acidity, peroxide value, and sensory properties as a function of dependent variables are shown in table 3, where a, b, and c are (method 44-15a), ash (08-01), protein (method 46-13), and fat (method 30-25). determination of total phenolic content (tpc) total phenolic content of each extract was determined by the folin–ciocalteu micro-method. briefly, in a 50-ml volumetric flask, 1 ml of standard solution of gallic acid, 6 ml of methanol, 2.5 ml of folin–ciocalteu reagent, and 5 ml of 7.5% na2co3 were added, making the final volume by adding purified water. the solutions were stored overnight and the spectrophotometric analysis was performed at λ = 765 nm (wpa s2000, uk). the total phenolic content of the samples and canola oil was expressed as gae using the following linear equation based on the calibration curve: y = 0.008x + 0.029; r2 = 0.094, where y is the absorbance at 760 nm and x is concentration in gae (μg/ml) (capannesi et al. 2000; javanmardi et al. 2003). sensory evaluation cakes were evaluated for their sensory characteristics by 12 semi-trained panelists (six females and males each, aged 20–40 years) using a 5-point hedonic rating test. consumer acceptance testing was done using a successive-category scale to score the overall acceptance of the product. samples were coded with three random digits and placed in disposable colorless plates for presenting to panelists (stone and sidel 2004). panelists evaluated the sensory properties of cake samples on a 5-point scale, where 1, 2, 3, 4, and 5 represented “disliked very much,” “disliked moderately,” “neither liked nor disliked,” “liked moderately,” and “liked very much”, respectively (serin and sayar 2016). all analyses were performed in triplicate. statistical analysis in this study, the microsoft office 2021 software was used to draw tables and charts and minitab version 16 was used to analyze data and draw optimization charts. the data were presented in the form of mean ± standard deviation of three replications. independent variables in actual and coded forms are presented in table 1. deviation from mean at 95% interval was applied to determine differences in results. the response surface methodology with central composite design (ccd) was used to design experiments to investigate the effects of independent parameters (variables), including levels of green tea, white tea and ginger extracts. a central composite design was chosen to optimize process variables at two levels with nine components, including six table 2. statistical design of central composite design. run green tea extract (w/w%) white tea extract (w/w%) ginger extract (w/w%) 1 1 2 1.5 2 2 1 1.5 3 1 1 0.75 4 0 1 1.5 5 1 0 0 6 1 0 1.5 7 0 2 0.75 8 1 1 0.75 9 1 2 0 10 1 1 0.75 11 0 1 0 12 2 1 0 13 2 2 0.75 14 1 1 0.75 15 0 0 0.75 16 1 1 0.75 17 2 0 0.75 table 3. variance analysis (anova) of response surface model for fat content. source df f value p value (prob > f) significant model 9 6.99 0.0058 (p < 0.05) green tea extract (w/w%) (a) 1 28.26 0.0007 white tea extract (w/w%) (b) 1 10.64 0.0115 ginger extract (w/w%)(c) 1 0.0679 0.8010 ab 1 0.0925 0.7687 ac 1 1.97 0.1978 bc 1 2.22 0.1746 a2 1 7.22 0.0276 b2 1 11.84 0.0088 c2 1 1.59 0.2427 36 italian journal of food science, 2023; 35 (2) pourzafar h et al. revealed that the source of changes had no significant effect on the moisture content (p > 0.05).these results were in agreement with senanayake et  al. (2019), who reported that the addition of coconut oil meal and sesame oil meal phenolic extracts had a insignificant effect on the moisture content of cake samples (senanayake et  al. 2019). moreover, ma et  al. (2020) found that the addition of vine extract did not affect the moisture content of bread crumb. the values of r2 and r2-adjusted were 73.17% and 61.74%, respectively, which indicated moderate fit of the model to the experimental data (table 4). table 4. variance analysis (anova) of response surface models for various parameters. parameters model r2 r2-adj r2 pred fat (%) y = 10.6 + 0.056a + 0.034b + 0.04a2 + 0.051b2 73.17 61.74 62.0 moisture (%) y = 9.00+ 0.036a + 0.012b + 0.02a2 + 0.032b2 73.17 61.74 70.12 acidity (%) y= 0.203 – 0.020 a – 0.194b 88.67 75.93 68.32 peroxide value (meq o 2 /kg) y = 0.89 – 0.11a – 0.09b + 0.08 ab + 0.03 a2 97.88 95.48 21.30 total phenol (mg/g) y=153.18 + 22.77 a + 25.01b + 10.65 c –3.28 ab – 3.62 ac – 4.34bc + 3.38 a2 99.66 99.28 59.44 color score y= 40.78 – 0.13 a – 0.13 b – 0.1c2 91.21 81.32 60.22 appearance score y=4.81 – 0.11a – 0.109b – 0.09 c2 90.82 80.50 72.00 flavor score y = 4.75 – 0.1 a – 0.08 b 68.39 61.61 10.72 overall acceptance score y = 4.75 – 0.092 a – 0.07b 85.22 82.05 17.11 a, b, and c are respective concentrations of green tea, white tea, and ginger extract. respective concentrations of green tea, white tea, and ginger extracts. table 3 shows that the effects of green tea extract (a and a2) and white tea extract (b and b2) on fat content were significant (p < 0.05); however, the effect of other parameters on fat content was not significant (p > 0.05). the maximum significance was observed with green tea extract. the correlation between the predicted values and the actual data can be used to evaluate the fitness of the response surface model (table 4). therefore, the model can be used to predict the fat content. according to the coefficient of determination (r2), the proposed model showed a good fit for the obtained results. the model accuracy with r2 and r2-adjusted parameters is also reported in anova tables. the models were checked using a numerical method, including r2, which provided a measure of how well future outcomes are likely to be predicted by the model. in general, a lack of fit test for the model describes variation in the data around the fitted model (trinh and kang 2010). according to figure 1, the optimal concentrations of green tea (1%), white tea (1%), and ginger extracts (0.8%) were determined as significant levels of fat content in the treatments. as the levels of extracts increased, the fat content also increased, and the green tea extract had the maximum effect. figure 1 shows good fit of the experimental data with the predicted data. green and white tea extracts might contain carotenoids and other fat-soluble compounds that significantly affected the fat content of the product (yuanita et al. 2022). moisture content table 5 shows the results of the anova quadratic model using the rsm for the moisture content. the results figure 1. effect of green tea and white tea extracts on the fat content of cake (the concentration of ginger extract was considered constant: 0.75%). italian journal of food science, 2023; 35 (2) 37 response surface methodology for optimizing and producing food formulations of desirable properties according to table 4, r2 and r2adjusted were appropriate, but the model was not fully capable of predicting the data with the test results of the acidity (table 4). peroxide value according to table 7, green (a) and white tea extracts (b), the interactions of these extracts (ab) and the square of green tea extract (a2) had a significant effect on peroxide value changes (p < 0.05), among which, the effect of the green tea extract was more significant than other acidity according to table 6, green tea and white tea extracts had a more significant effect on acidity changes with respect to fand p-values. non-fit was also considered insignificant. according to figure 2, with increasing concentrations of green tea and white tea extracts, the acidity of the product decreased significantly, while the ginger extract had no significant effect on acidity. of all the extracts, green tea extract had a more significant effect on acidity, but figure 2 shows a poor fit of the predicted experimental data. increase in acidity with increasing concentration of extracts could be due to the acidic nature of these compounds (kailasapathy 2006). figure 2. effect of green tea and white tea extracts and ginger concentration on the acidity content of cake (the concentration of ginger extract was considered constant: 0.75%). table 5. variance analysis (anova) of response surface model for moisture content. source df f value p value (prob > f) significant model 9 0.1943 0.9878 p > 0.05 green tea extract (w/w%) (a) 1 0.0571 0.8172 white tea extract (w/w%) (b) 1 0.2017 0.6653 ginger extract (w/w%) (c) 1 0.0034 0.9553 ab 1 1.24 0.2979 ac 1 0.0229 0.8835 bc 1 0.0333 0.8598 a2 1 0.0117 0.9165 b2 1 0.0202 0.8904 c2 1 0.0905 0.7712 table 6. variance analysis (anova) of response surface model for acidity. source df f value p value (prob > f) significant model 9 6.96 0.0059 p < 0.05 green tea extract (w/w%) (a) 1 29.16 0.0006 white tea extract (w/w%) (b) 1 25.78 0.0010 ginger extract (w/w%) (c) 1 0.0595 0.8134 ab 1 0.0131 0.9119 ac 1 0.3479 0.5716 bc 1 1.12 0.3204 a2 1 2.16 0.1799 b2 1 2.16 0.1799 c2 1 1.54 0.2504 table 7. variance analysis (anova) of response surface model for peroxide value. source df f value p value (prob > f) significant model 9 40.88 <0.0001 p < 0.05 green tea extract (w/w%) (a) 1 116.30 <0.0001 white tea extract (w/w%) (b) 1 77.47 <0.0001 ginger extract (w/w%) (c) 1 4.15 0.0760 ab 1 31.31 0.0005 ac 1 0.0000 0.9955 bc 1 1.03 0.3409 a2 1 6.50 0.0342 b2 1 3.55 0.0963 c2 1 2.40 0.1596 38 italian journal of food science, 2023; 35 (2) pourzafar h et al. as expected, the total phenolic content increased with increase in the concentration of extracts. as mentioned in the “extraction process” of the “materials and methods” section, the total phenolic content of white tea and green tea extracts was higher than that of ginger extract. hence, the highest r2 was associated with green tea and white tea extracts. as shown in figure 4, the model had a high fit for experimental data. the results were in agreement with the studies conducted on the effect of various extracts on the phenolic compounds of bakery products (timoshenkova et al. 2020; horanni and engelhardt 2013; izzreen and noriham 2011). the phenolic content and the antioxidant activity of plant extracts could be influenced by plant growth conditions, extracts on the peroxide value. the fand p-values represent this result well (table 7). r2 and r2-adjusted for the peroxide value were significant, which indicated optimal fit of the model to the experimental data (table 4). therefore, the model can be used to predict peroxide value changes. according to r2 and r2-adjusted, the proposed model corresponds to the obtained results. according to figure 4, the correlation between predicted and actual data on green tea extract was higher than white tea and ginger extract. with increasing the concentration of all three extracts, the peroxide value decreased(figure 3). this result was consistent with the findings of khaki et  al. (2009), who investigated the effect of chamomile extract on the quality properties of the cake. however, it was not consistent with the results of (izzreen and noriham 2011). the antioxidant effect of the extracts could be related to their phenolic compounds (khoshnoudi-nia, sharif, and jafari 2020). in some studies, a good linear relationship was reported between antioxidant activity and total phenols present in many plants (sharifi and khoshnoudinia 2022; pourshoaib, ghatrami, and shamekhi 2022; timoshenkova et al. 2020). total phenolic content most of the changes, including concentrations of all extracts and their interactions, indicated a good fit for data in a quadratic model (p < 0.05). the most significant effect was observed for green tea extract (table 8). as shown in table 4, r2 and r2-adjusted demonstrated good fit of both predicted and experimental data. table 8. variance analysis (anova) of response surface model or total phenols. source df f value p value (prob > f) significant model 9 261.27 <0.0001 p < 0.05 green tea extract (w/w%) (a) 1 700.06 <0.0001 white tea extract (w/w%) (b) 1 845.03 <0.0001 ginger extract (w/w%) (c) 1 153.37 <0.0001 ab 1 7.72 0.0240 ac 1 9.38 0.0155 bc 1 13.49 0.0063 a2 1 0.0000 0.9968 b2 1 0.7145 0.4225 c2 1 7.88 0.0230 figure 3. effect of green tea, white tea, and ginger extracts on the peroxide value of cake (the concentration of ginger extract was considered constant: 0.75%). figure 4. effect of green tea, white tea, and ginger extracts on the total phenol content of cake (the concentration of ginger extract was considered constant: 0.75%). italian journal of food science, 2023; 35 (2) 39 response surface methodology for optimizing and producing food formulations of desirable properties results were in agreement with those described by previous studies (senanayake et al. 2018; timoshenkova et al. 2020). various factors affected the crust color of cakes, such as moisture of the cake crust, intensity of millard reactions, and presence of color compounds in cake formulation. however, the ingredients used in cake formulation mainly affected color of the cake crumb (ajila, leelavathi, and rao 2008). appearance score the linear effect of three extracts and the square of ginger extract significantly affected appearance scores of the product (p < 0.05; table 10). as observed in table 4, r2 and r2-adjusted values for the appearance were high, which indicated a significant fit for the experimental data (p < 0.05). this indicated that the model had a good fit for the experimental data. appearance scores decreased with the increasing concentrations of three extracts. the panelists reported that the size of the crumb porous was smaller in fortified samples with extracts, compared to the control sample. studies reported a reduction trend in specific volume after addition of phenolic compounds during cake formulation because of disruption in gluten network and reduced gas-retention capacity (mahmoudi et  al. 2020; taglieri et  al. 2022; usman et  al. 2020; kırbaş, kumcuoglu, and tavman 2019; lavelli and corti 2011). this could be related to the presence of hydroxyl groups in phenolic compounds that may directly bond with proteins and other compositions of wheat flour and affect the texture properties of bakery products (arı akın et al. 2021; elkatry et al. 2022; sabet ghadam et al. 2022; climatic conditions, and extraction methods (onacikgür and zbikowska 2022; khoshnoudi-nia, sharif, and jafari 2020). phenolic compounds are volatile and semi-volatile polymers that are sensitive to environmental conditions, and deprivations during preparation and storage are almost unavoidable (khoshnoudi-nia, sharif, and jafari 2020). therefore, encapsulation of plant extracts can increase and prolong their effectiveness by protecting bioactive compounds (mahmoudi et al. 2020). sensory evaluation color score change in color indicates chemical changes in the food during thermal processes, such as maillard reaction or non-enzymatic browning, browning, caramelization or browning of sugar, frying, and drying (edwards 2007). interaction between extract compounds and other cake ingredients during the preparation and storage periods affect maillard or caramelization reactions (gularte et al. 2012; kırbaş, kumcuoglu, and tavman 2019). the color of extracts can also influence the color of bakery products (timoshenkova et al. 2020).  as shown in table 9, the linear effects of green tea and white tea extracts, besides the square of ginger extract, on color scores were significant (p < 0.05). as observed in table 4, r2 and r2-adjusted were high and the model had a good fit for the experimental data. the results showed decrease in color scores by increasing the concentration of extracts. the lightness of the cake crumb decreased with addition of extracts because of the darker color of extracts, compared to wheat flour. the table 9. variance analysis (anova) of response surface model for color score. source df f value p value (prob > f) significant model 9 9.22 0.0023 p < 0.05 green tea extract (w/w%) (a) 1 32.37 0.0005 white tea extract (w/w%) (b) 1 32.37 0.0005 ginger extract (w/w%) (c) 1 1.26 0.2950 ab 1 0.7201 0.4208 ac 1 1.23 0.3004 bc 1 1.23 0.3004 a2 1 0.3421 0.5747 b2 1 0.3421 0.5747 c2 1 10.71 0.0113 table 10. variance analysis (anova) of response surface model for appearance score. source df f value p value (prob > f) significant model 9 8.80 0.0027 p < 0.05 green tea extract (w/w%) (a) 1 30.12 0.0006 white tea extract (w/w%) (b) 1 28.16 0.0007 ginger extract (w/w%) (c) 1 3.00 0.1214 ab 1 1.61 0.2402 ac 1 0.6854 0.4317 bc 1 0.0444 0.8383 a2 1 0.2244 0.6484 b2 1 2.30 0.1680 c2 1 10.51 0.0118 40 italian journal of food science, 2023; 35 (2) pourzafar h et al. (o'neill, rebane, and lester 2004). therefore, its prediction is less accurate than other parameters. overall acceptance score green tea and white tea extracts had significant effects on overall acceptance scores (p < 0.05; table 12), while ginger extract had no significant effect on overall acceptance scores (p > 0.05). as observed in table 4, r2 and the r2-adjusted for overall acceptance scores were high and indicated optimal fit of the model with the experimental data. optimization the rsm evaluated the effects of green tea, white tea, and ginger extracts’ concentrations, and interactions between the extracts, to optimize the formulation of provided functional sponge cake. the optimal values of variables were obtained by the direct search method and numerical analyses based on the criterion of desirability functions (table 13). optimized treatments were 1.44% for green tea extract, 1.71% for white tea extract, and 0.75% for ginger extract. validation of statistical model was determined by performing the confirmation run. to confirm the reliability of the response surface shahidi et  al. 2020). probably, for this interaction, the firmness of samples containing a higher concentration of extract was more than other samples. as observed in table 4, the model presented based on the concentration of green tea, white tea, and the ginger square extracts revealed good appearance score of the cake sample (r2adj = 80.5, and rpred = 72.00). flavor score the green tea and white tea extracts significantly affects the flavor scores (p < 0.05; table 11). the flavor scores decreased with the increasing concentration of extracts. however, ginger extract did not have a significant effect on flavor scores of cake because of its lower concentration and compatibility of its taste with cake’s flavor (bakery consumers are familiar with ginger taste). white and green tea extracts significantly affected the cake flavor. the panelists felt a slightly bitter and grassy taste in the samples containing a higher concentration of green tea and white tea extracts. these tastes could be related to the phenolic compounds of the extracts (elkatry et  al. 2022). according to table 4, r2 and the r2-adjusted were very low, indicating that the model did not fit well with the experimental data. scores given to the flavor depends on personal taste and eating habits table 12. variance analysis (anova) of response surface model for overall acceptance score. source df f value p value (prob > f) significant model 3 26.90 <0.0001 p < 0.05 green tea extract (w/w%) (a) 1 43.51 <0.0001 white tea extract (w/w%) (b) 1 28.57 0.0001 ginger extract (w/w%) (c) 1 0.0381 0.8480 table 13. optimization criteria used in this study. name goal lower limit upper limit lower weight upper weight importance a: green tea extract within range 0 2 1 1 3 b: white tea extract within range 0 2 1 1 3 c: ginger extract within range 0 1.5 1 1 3 moisture within range 9.83 12.09 1 1 3 acidity within range 0.12 0.29 1 1 3 peroxide value minimize 1.039 2.998 1 1 5 fat minimize 2.04 3.95 1 1 3 total phenols maximize 14.408 194.638 1 1 5 overall acceptance maximize 3 4.9 1 1 5 table 11. variance analysis (anova) of response surface model for flavor score. source df f value p value (prob >f) significant model 3 10.09 0.0008 p < 0.05 green tea extract (w/w%) (a) 1 14.93 0.0017 white tea extract (w/w%) (b) 1 9.11 0.0092 ginger extract (w/w%) (c) 1 1.43 0.2513 italian journal of food science, 2023; 35 (2) 41 response surface methodology for optimizing and producing food formulations of desirable properties conflict of interest the authors declared that they had no conflict of interest. references ajila, c.m., leelavathi, k.u.j.s. and prasada rao, u.j.s. 2008. improvement of dietary fiber content and antioxidant properties in soft dough biscuits with the incorporation of mango peel powder. j cereal sci. 48(2): 319–326. https://doi.org/10.1016/j. jcs.2007.10.001 american association for clinical 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g. pérez-flores1,2*, elizabeth contreras-lópez2, emmanuel pérez-escalante2, aldahir alberto hernández-hernández3 1área académica de enfermería, instituto de ciencias de la salud, universidad autónoma del estado de hidalgo, circuito ex hacienda la concepción s/n, carretera pachuca-actopan, san agustín tlaxiaca, hidalgo, méxico; 2área académica de química, instituto de ciencias básicas e ingeniería, universidad autónoma del estado de hidalgo, carretera pachuca-tulancingo, mineral de la reforma, hidalgo, méxico; 3área académica de ingeniería agroindustrial e ingeniería en alimentos, instituto de ciencias agropecuarias, universidad autónoma del estado de hidalgo, avenida universidad km. 1 s/n exhacienda aquetzalpa, tulancingo de bravo, hidalgo, méxico *corresponding author: jesús g. pérez-flores, área académica de química, instituto de ciencias básicas e ingeniería, universidad autónoma del estado de hidalgo, carretera pachuca-tulancingo km 4.5, 42184 mineral de la reforma, hidalgo, méxico email: jesus_perez@uaeh.edu.mx received: 18 december 2022; accepted: 8 may 2023; published: 25 may 2023 © 2023 codon publications open access original article abstract rapid color degradation during processing and storage is a limitation when using strawberry puree (sp). this work aimed to use image analysis coupled with two machine learning algorithms: ordinary least squares (ols) and artificial neural networks (anns), to predict anthocyanin content (ac) in frozen sp during its storage at –18°c for 120 days. when applying the ols regression model, unsatisfactory ac prediction values were obtained due to multicollinearity. in contrast, a good prediction of ac using anns model was observed by comparing ac in sp predicted by the model versus the experimentally obtained values (coefficient of determination, r2 = 0.977). keywords: anthocyanin content, color measurement, image analysis, machine learning, strawberry puree introduction strawberry (fragaria × ananassa, duch.) fruit contains nutritional compounds, such as sugars, proteins, dietary fibers, vitamins, and minerals; bioactive compounds, such as ascorbic acid, carotenoids, flavonoids, folates; and phenolic compounds, such as anthocyanins, most of which are natural antioxidants and contribute to the high nutritional quality of the fruit (hosseinifarahi et al., 2020; liu et  al., 2018; teribia et  al., 2021). strawberries are non-climacteric fruits, so they must be harvested almost at maturity to guarantee its highest quality in terms of flavor, color, and consistency (mancini et al., 2020). strawberry is one of the most commonly consumed berries, fresh and processed, as a concentrate, juice, or puree in the formulation of different products. in the food industry, strawberry puree (sp) is used to prepare red-colored products with a tasty flavor, such as fruit preparations, ice creams, and smoothies. however, the use of sp in these products is limited by its rapid color degradation during processing and storage (teribia et  al., 2021). this loss of red color is attributed to the degradation of anthocyanins and enzymatic and nonenzymatic browning reactions. in addition, color stability depends on many factors, such as temperature, water activity, light, oxygen, ph, and ascorbic acid (da silva simão et al., 2022). change in color during storage is a quality parameter with the most significant impact on the shelf life of fruit-based products (buvé et al., 2018), because it plays an essential role in influencing the sensory and hedonic expectations of consumers. therefore, change in color can lead to product rejection, even on the shelves of the market (da silva simão et  al., 2022). besides this, studying the relationships between color and pigments is equally important. italian journal of food science, 2023; 35 (2) 89 content of anthocyanin in frozen strawberry puree using 3-dimensional (3d) cielab color space (also referred as l* a* b* coordinates, it covers the entire range of human color perception), to promote its use as a quality control tool by producers, scientists, and food technologists interested in a possible commercial application. materials and methods preparation and processing of strawberry puree strawberries were obtained locally from la central de abastos de la ciudad de pachuca, pachuca de soto, hidalgo, méxico. after strawberries were washed and disinfected, the calyx and leaves were removed and  the fruit was chopped. small pieces were mashed and blended for 1 min at 15,000 rpm in a domestic osterizer blender (oster, mexico). the resulting puree was passed through a 500-μm stainless steel sieve to remove seeds (under atmospheric conditions). the puree prepared was pasteurized in 100-ml glass jars (55-mm diameter and 75-mm height) at 90°c for 15 min. pasteurized purees were cooled with water at 25°c. this process assured the puree’s microbial stability (marszałek et  al., 2015). samples of pasteurized purees (100 g) were manually filled, under aseptic conditions, into small polypropylene containers (180 ml) with some headspace left in containers. finally, samples were stored at –18°c for 120 days, determining ac and color every 5 days. determination of ac the ac of sp was determined in triplicate with a modified ph differential method described by zheng et al. (2011). results were expressed as milligram cyanidin-3-glucoside equivalents per liter of puree (mg l–1). total titratable acidity (tta) the tta of sp was determined using an acid–base titration method. fruit puree (1 ml) and distilled water (50 ml) were added in an erlenmeyer flask. then, a few drops of phenolphthalein were added, and the sample was titrated with aqueous naoh 0.1 mol l–1 to attain a ph 8.1. total acid contents were calculated as gram of citric acid in 100 g of sample and were presented as a mean of triplicate analyses (kafkas et al., 2007). color measurement by image analysis image acquisition system the image acquisition system used to determine color changes in sp during storage consisted of an illumination recently, image analysis has gained interest for its simplicity, reliability, low cost, and speed of analysis to assess food quality, in addition to the fact that it does not require reagents. many properties can be extracted from an image, for example, color, pixels values distribution, statistical greatness, and frequency domain measures (kato et  al., 2019). color space extraction from food matrices image has been previously reported by barbin et  al. (2016), ulrici et al. (2012), and valous et al. (2009), providing a whole idea of the product instead of color measurement of a single point or a reduced area, such as the one spotted by a colorimeter (barbin et al., 2016). hence, the implementation of a computer vision system (cvs) for predicting anthocyanin content (ac) in sp during storage constitutes a nondestructive and low-cost quality control tool that allows making decisions about rotation, applications, or processing conditions of sp when used as an ingredient in the preparation of other food products. computer vision, also called artificial vision, is one of the branches of artificial intelligence (ai) and is responsible for understanding in detail the visual data, similar to human optical systems, to make decisions using other branches of artificial intelligence, such as machine learning or deep learning (xu et  al., 2021). machine learning and deep learning can carry out the tasks of recognition, prediction, or classification, in which they make decisions after a trained and evaluated computational model based on a dataset (barbin et  al., 2016; santos pereira et al., 2018). therefore, a computer vision system is based on the following stages: (1) image acquisition, (2) image segmentation, (3) image feature extraction and selection, and (4) image classification, object detection, or feature prediction using machine learning or deep learning methods (contreras-lópez et  al., 2022; lopes et  al., 2019; oliveira et al., 2021). the basis of deep learning is artificial neural networks (anns). anns are advanced fitting and pattern recognition algorithms that allow users to extract complex relationships among nonlinear variables. as the training progresses, the neural network-based model learns the unknown dynamics of the process. this advantage makes anns very appealing computational tools in applications with little or incomplete understanding of the problem, although experimental measurements are readily available (kazi et  al., 2021). further, the application of multivariate statistical methods, such as multiple linear regression (mrl), to single out color parameters to correlate them with the pigment content in strawberries has been reported as well (amoriello et  al., 2022; hernanz et  al., 2008). therefore, this work aimed to use image analysis to predict ac in frozen sp during its storage, verifying the effectiveness of two machine learning algorithms—ordinary least squares (ols) and anns algorithms—to build models that allow predicting ac 90 italian journal of food science, 2023; 35 (2) garcía-curiel l et al. artificial neural networks approach data preprocessing standardization or z-score normalization on the cielab color space dataset was computed using equation 2, which subdivides data points in terms of standard deviation away from the mean value of the distribution, as follows: z x x s i� � , (2) where z is the result of normalization value, xi is the ith data point, x is the sample’s mean value, and s is the sample’s standard deviation (prihanditya and alamsyah, 2020). anns model a multilayer perceptron (mlp) is a feed-forward anns model that consists of (1) an input layer with nodes representing independent variables, (2) an output layer with nodes representing dependent variables, that is, what is being modeled, and (3) one or more hidden layers containing nodes to help capture nonlinearity in the data (pilkington et al., 2014). in these feedforward networks, the levenberg–marquardt algorithm, which is an iterative algorithm, achieves error minimization. the whole data are randomly split into training and testing groups. the training set is used to train the network whereas the test set is used to evaluate the network’s performance after training (amoriello et al., 2022). the anns model used in this study was based on a multilayer perceptron and was developed and trained with python programming language (v3.8.5) using anaconda (v4.13.0), a free and open-source distribution for managing python libraries. the development environment configuration consists of multiple packages and libraries: keras (v2.3.1) runs on top of the machine learning platform tensorflow (v2.9.1). additionally, other python packages and libraries were used, such as numpy (v1.23.1), scipy (v1.7.3), matplotlib (v3.5.1), pandas (v1.4.3), seaborn (v0.12.1), and scikitlearn (v1.1.1). finally, code was developed on a python notebook using the visual studio code software (v1.70.0) as an integrated development environment (ide). the number of artificial neurons in input and output layers was defined as a function of dependent and independent variables, respectively. in contrast, the number of hidden layers and the number of artificial neurons required in each hidden layer were determined by trial and error to minimize the deviation of predictions from experimental results. chamber, a charge-coupled device (ccd) digital camera, and a personal computer (pc). all were constructed, configured, and calibrated according to the research conducted by contreras-lópez et al. (2022). image analysis red (r), green (g), and blue (b) (rgb) analysis of digital images was carried out using the imagej software (1.53 k; https://imagej.nih.gov/ij/index.html), an open-source program from the national institutes of health and the laboratory for optical and computational sciences, to precisely confirm color changes in samples. image processing was carried through the plugins/analyze/rgb measure menu, and average values were obtained for r, g, and b. subsequently, these values were transformed to cielab color space coordinates, as indicated in the research conducted by wu and sun (2013). coordinates were expressed as l* describing lightness (l* = 0 for black, and 100 for white), a* or redness for intensity in green–red (a* < 0 for green and >0 for red), and b* or yellowness describing intensity in blue–yellow (b* < 0 for blue and >0 for yellow), representing rectangular chromaticity coordinates. subsequently, the overall color difference (δe), hue angle or color angle (h*), and chroma or color saturation (c*) were calculated as reported in the literature (udomkun et al., 2017; wu and sun, 2013). multiple linear regression model the ols regression model was applied to determine how the cielab coordinates were related to ac. the ols regression model is a simple machine learning algorithm and was defined as follows (mollalo et al., 2020): y xi i i� � �� � �0 , (1) where yi is the dependent variable (ac), β0 is the intercept, β is the vector of regression coefficients, xi is the vector of selected explanatory variables (independent variables: l*, a*, and b*), and εi is a random error term. ols optimizes regression coefficient (β) by minimizing the sum of squared prediction errors. prediction performance was evaluated using r2. the numerical tests were performed using python’s statsmodels library (v0.13.2). a correlation matrix heatmap was carried out using python’s seaborn library (v0.12.1) to represent visual correlations between independent and dependent variables. in this study, the ols regression model was trained on 70% of the dataset and tested on the remaining 30% (train_test_split random state = 100 in python’s scikit-learn [v1.1.3]). italian journal of food science, 2023; 35 (2) 91 content of anthocyanin in frozen strawberry puree generally, this change becomes more evident when δe > 5 (contreras-lópez et al., 2022). loss of bright red color in sp stored at freezing temperatures (–18°c) could be associated with the degradation of phenolic compounds and anthocyanins, which can occur at different temperatures (zhang et  al., 2019). in fact, in this work, a decrease in ac was observed during storage (figure 1g). similarly, it was reported that using refrigerated temperatures (4°c) during processing did not improve the color and anthocyanin stability of sp (teribia et  al., 2021). in addition, a decrease in the concentration of the total phenol content was observed in sp at freezing temperatures, but after 9 months of storage (obradović et  al., 2020). in addition, phenolic compounds in strawberries, such as pelargonidin, ellagic acid, p-coumaric acid, quercetin, and kaempferol derivatives, are very unstable during freezing process because of microbial enzymes and nonenzymatic oxidation (aaby et al., 2007; oszmiański et al., 2009). therefore, the storage temperature is an important factor in extending the shelf-life of strawberries (lv et  al., 2022) and derived products, such as sp. moreover, anthocyanins positively correlated with antioxidant activity (zhang et al., 2019). another factor that influences the stability of phenolic compounds is vitamin c, which decreases when the storage temperature or storage time increases compared to whole strawberries. in addition, oxidation of ascorbic acid affects the loss of flavylium pigmentation in anthocyanins (howard et  al., 2014; stan et al., 2016). on the other hand, figure 1h shows an increase in titratable acidity (gram of citric acid per each 100 g of sp), which is associated with the ripeness stage of strawberries and color changes during freezing storage (galoburda et al., 2014; stan et al., 2016). prediction of anthocyanin content in strawberry purees ordinary least squares regression model the ols regression model presented an r2 of 0.928. this indicated that the model was capable of explaining the variability of 92.80% observed in the ac of sp during storage at –18°c. this was a statistical measure of how well the regression line approximated experimental data points. the adjusted r2 reflected model complexity and was considered a more accurate measure of model performance (adjusted r2 = 0.917). on the other hand, model’s p-value was significant (3.84 × 10–12); so, the coefficients were different from 0 and could predict the dependent variable (ac). summary statistics of the ols regression model that described the relationship between after defining the layers, the input data were divided into a training dataset (70% of the input data) and a testing dataset (30%). the anns model was compiled and trained for 2000 epochs and optimized using root mean square propagation (rmsprop) as an optimization algorithm with a learning rate of 0.01, using a random seed of 0 (random_state = 0). the mean square error (mse) was used as a network performance index (loss function), and the mean absolute error (mae) was used as an evaluation metric. in the validation_split parameter, a fraction of 30% of the training data was put aside to monitor training performance. finally, to use anns model, the calculated weights must be available for later use in other applications. the strategy used here was to export trained model in a python pickle file (.pkl) using the python library joblib (v0.13.2). this study used mae and mse as performance parameters to compare ols and anns models (bilgili and sahin, 2010). results and discussion color parameters and anthocyanin content figure 1 shows the evolution of the physicochemical quality attributes of sp samples during frozen storage. the results show a decrease of l* values from 33.760 on day 0 to 19.640 on day 120 (figure 1a), meaning that the fruit developed darker color during storage (caner et al., 2008). hue (h*) is an angular value representing a dominant wavelength (athira et  al., 2019) so that it characterizes color modifications: 0° (or 360°) is defined for red, 90° for yellow, 180° for green, and 270° for blue color (scalisi et  al., 2022). it is observed in figures 1b and 1c that values of a* and b* decreased with increase in storage time; decrease in h* values ranged from 0.641° on day 0 to 0.568° on day 105; however, a slight increase was observed after 105 days (figure 1d). these results implied that the sample maintained its red color during storage. decrease in c* values ranged from 48.388 on day 0 to 34.058 on day 120 (figure 1e), together with decrease in a*, which explained decrease in the redness of sp during storage. this is because chromaticity is a measure that moves from the center of the cielab color space system (c* = 0 = gray) to the direction of puree colors (c* = 100); higher values of c* indicate higher purity or color intensity (contreras-lópez et  al., 2022). finally, δe showed total increase in color during the test period, with value of 0.336 on day 5 to 20.121 on day 120 (figure  1f). 92 italian journal of food science, 2023; 35 (2) garcía-curiel l et al. figure 1. scatter plots of changes in the physicochemical quality attributes of strawberry puree (sp) stored at –18°c for 120 days. (a) lightness, (b) redness, (c) yellowness, (d) hue angle, (e) chroma or color saturation, (f) the overall color difference, (g) anthocyanin content (ac), and (h) total titratable acidity. (a) (c) (e) (g) (b) (d) (f) (h) italian journal of food science, 2023; 35 (2) 93 content of anthocyanin in frozen strawberry puree anns model different ann configurations were developed and compared to determine anns model with a better fitting architecture (input-hidden-output layers and artificial neural number). the anns model built presented the following hyperparameters and activation functions: three artificial neurons in the input layer that checked with l*, a*, and b* coordinates of cielab color space; the input layer was fully connected to the first hidden layer that consisted of 10 artificial neurons applying the activation function as rectified linear unit (relu). the second hidden layer consisted of eight neurons with the same activation function as relu. the last layer, the output layer, received values from the second hidden layer and transformed them into output values to model the ac of sp. the activation function was used to compute the predicted output of each neuron in each layer by using inputs, weights, and biases. in the output layer, activation function was not used. the model summary was printed to identify full-fledged parameters with training and testing. a total of 137 parameters were acquired, including trainable as well as zero non-trainable parameters. once the model was adjusted through 2000 epochs, the loss percentage decreased more slowly. it halted after the 1000th epoch, as observed about the training history in figure 2c, up to the 1999th epoch, the readings were: loss (mse): 0.1805 and validation loss (validation mse): 0.8541, the plot suggests that anns model has a good fit on the problem starting at 1000th epoch; when the mse value no longer decreases, an optimal number of training cycles were reached (rogiers et  al., 2012). in contrast, it is observed in figure 2d that mae values always descended over the epochs, leading to higher accuracy. conversely, the validation mae values had a slight upward trend after 875th epoch. this suggested that anns model was overfitting (palkovits, 2020), which could be attributed to the fact that few data were available. it was observed that the model stopped learning at 1500th epoch. up to 1999th epoch, the readings were mae: 0.2598 and validation mae: 0.8724. the cielab color space coordinates and ac in sp are shown in table 1. according to equation 1, values of β and xi are the following vectors: [0.9323, 0.7076, 0.2024] t and [l*, a*, b*]t, respectively. regression coefficients computed for each independent variable represent the strength and type (positive or negative) of relationship between independent and dependent variables. the statistical significance of coefficients associated with each independent variable is assessed by t-test. model’s coefficients with small p values are important. the associated variables are effective predictors (lukawska-matuszewska and urbański, 2014). finally, one of the methods for determining the presence of multicollinearity is the variance inflation factor (vif). the vif indicates how much the variance of a coefficient associated with explanatory variable increases because of the linear dependence between independent variables. the variables related to high vif values are usually eliminated from the model. vif above 5 or 10 indicates high multicollinearity between independent variables (lukawska-matuszewska and urbański, 2014; wagle et  al., 2017), resulting in less reliable statistical inferences. this could explain why the p values of coefficients of independent variables are not significant (p  >  0.05). these results coincide with what was observed in the correlation heatmap (figure 2a). in this correlation plot, each numerical variable represented a column, and rows showed relationship between each pair of variables. the color-coding of cells made it easy to identify visually the strength relationships (linear and nonlinear) between variables. all relationships between variables presented r2 > 0.880. generally, a pearson correlation coefficient greater than 0.800 indicates the presence of multicollinearity (lev et al., 2022). finally, ac values predicted by the ols regression model versus the true (experimental) ac values showed a linear relationship, obtaining r2 = 0.928 (figure 2b). still, owing to the unsatisfactory results of ols regression model, a second attempt at prediction was accomplished using anns to assess whether it performed better predicting ac. table 1. summary statistics of the ols regression model on selected variables in modeling anthocyanin content in strawberry purees stored at –18°c. variable coefficient standard error t-statistic p-value vif† intercept –41.9350 13.5577 –3.0931 0.0055 l* 0.9323 0.7825 1.1914 0.2468 69.7646 a* 0.7076 0.5613 1.2605 0.2213 17.4823 b* 0.2024 0.7647 0.2646 0.7939 28.8329 †variance inflation factors (vif) for independent variables. 94 italian journal of food science, 2023; 35 (2) garcía-curiel l et al. the use of ols regression modeling during the prediction of ac. table 2 compares mae and mse values for training and test stages of ols and anns models. these results finally, ac in sp, as predicted by anns model, was compared to the experimentally obtained values. to test the model’s suitability, the predicted and actual results were plotted in figure 2e. an r2 of 0.977 illustrated a good agreement between two sets of results, much better than figure 2. results of the ols regression model and training of anns model. (a) correlation heatmap. (b) scatter plot of the predicted vs experimental data of ac achieved by the ols regression model. (c) training history using the mean square error as a loss function. (d) mean absolute error as an evaluation metric. (e) scatter plot (predicted vs true) of the training data of ac achieved by anns model. the optimal regression line between estimated and measured values is shown in (b) and (d). (c) (e) (b)(a) (d) italian journal of food science, 2023; 35 (2) 95 content of anthocyanin in frozen strawberry puree could be correlated with color change and ripeness of the fruit. acknowledgments the authors thank the sistema nacional de investigadores (sni-conacyt) and the universidad autónoma del estado de hidalgo. conflicts of interest the authors declared no conflict of interest for this paper. author contributions l. garcía-curiel: writing original draft, writing review & editing; j. g. pérez-flores: conceptualization, visualization, software, supervision; e. contreraslópez: investigation, resources; e. pérez-escalante: formal analysis; a. a. hernández-hernández: writing review & editing. references aaby k., wrolstad r.e., ekeberg d. and skrede g. 2007. polyphenol composition and antioxidant activity in strawberry purees; impact of achene level and storage. j agric food chem. 55(13): 5156–5166. https://doi.org/10.1021/jf070467u amoriello t., ciccoritti r. and ferrante p. 2022. 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during storage. j. food eng. 216:42–51. https://doi.org/10.1016/j.jfoodeng.2017. 08.002 caner c., aday m.s. and demir m. 2008. extending the quality of fresh strawberries by equilibrium modified atmosphere packaging. demonstrated that the anns modeling approach provided more accurate results than the ols regression model because both mae and mse values were small. this indicated that the best agreement between experimental and predicted ac values was obtained with anns model (bilgili and sahin, 2010; tosun et al., 2016). the predictions were made with 30% of the training data in each case. it was observed that anns model better agreed with the experimental ac values (true values). however, both models could be improved by training with a larger amount of data. therefore, selecting an adequate model for evaluating the relationship between cielab color space and ac was essential for predictions that are more accurate. finally, the use of machine learning models combined with image analysis constituted a novel, robust, cheap, and easy to implement potential tool to predict changes in the ac of sp during frozen storage as well as during storage, processing, and distribution stages. it could also be useful for analyzing other fruit-based products in which the factor with the most significant influence on shelf life is changes in visual aspects, such as color change. conclusion the present study highlights that image analysis and machine learning models represent a promising, nondestructive, less time-consuming, and inexpensive tool for rapidly monitoring quality attributes of sp during frozen storage. in the future research, the best machine learning model to implement a computer vision system could be determined that fruit cultivators and food technologists could use successfully for different purposes throughout the supply chain, such as the assessment of color changes during food processing, ripening, and degradation. prediction of ac in sp during storage is an 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detection of red skin defect of raw hams. innov food sci emerg technol. 16:417–426. https://doi.org/10.1016/j.ifset.2012.09.008 p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33isp1.2059 117 p u b l i c a t i o n s codon effect of natural antioxidants and vegetable fiber on quality properties of fish sausage produced from silver carp (hypophthalmichthys molitrix) ebrahim mahdavi1, peiman ariaii2,* 1department of food science & technology, nour branch, islamic azad university, nour, iran; 2department of food science & technology, ayatolla amoli branch, islamic azad university, amol, iran *corresponding author: peiman ariaii, department of food science & technology, ayatolla amoli branch, islamic azad university, amol, iran, email: p.aryaye@yahoo.com received: 21 april 2021; accepted: 27 july 2021; published: 23 august 2021 © 2021 codon publications open access paper abstract in this study, production of low-fat, nitrite-free sausage based on silver carp surimi was performed. in order to replace oils, inulin fiber (if) was used; natural preservatives such as grape pomace extract (ge) and nisin (ni) were used as nitrite replacements with modified atmosphere packaging (map: 70% co2 + 30% n2). for this purpose; five treatments including, t1: control, t2: control + map, t3: if 5% + map, t4: if5% + ge0.5% + map, and t5: if5% + ge0.5% + ni 0.5% + map, were prepared. the physicochemical and texture properties of sausage at the beginning of storage and chemicals (peroxide value, ph, and color index) and microbial index (total count and psychrotrophic bacteria) during 42 days’ storage in the refrigerator (4 ± 1°c) were evaluated. the results showed that the use of natural preservatives had no effect on the physicochemical properties of the sausage (p > 0.05), but the fi had a positive effect on the texture characteristics (the firmness increased and elasticity decreased), increased moisture and ash, and reduced the fat content of sausages (p < 0.05). all in all, the best results among the treatment containing natural preservative was t5, in all microbial and chemical tests there was no significant difference with t1 (p > 0.05). all in all, a functional product with properties such as fiber and natural antioxidants, low fat and nitrite-free can be produced. keywords: fat replacement; inulin fiber; nitrite; plant extracts; nisin; fish sausage introduction seafood is a valuable source of protein for humans and plays an important role in healthy nutrition. fish have a very high nutritional value and provide most of the essential nutrients for humans, but what makes fish a specifically important food item is the presence of fatty acids. the human body is not capable of producing some unsaturated fatty acids such as omega-3 (kwasek et al., 2020). hypophthalmichthys molitrix is one of the warm water fish species that is widely cultivated in many countries of the world for its rapid growth, high feed conversion ratio, and good nutritional value (fan et al., 2008; stepien et al., 2019). despite intermuscular bones and unpleasant odor (mud), this fish, like other fish, contain high-quality protein and unsaturated fatty acids. to solve the abovementioned problems, as well as produce high value-added products from silver carp, it needs to be processed. on the other hand, with a glance at people’s livelihoods, mechanical life, and lack of time to prepare food, the idea of producing or supplying ready-to-eat or semi-prepared food, such as fish paste, fish ball, fish cake, fish sausage, and burger from seafood, seems to be a good solution (fan et al., 2008; ozpolat and patir, 2015). nitrite is used to prevent spoilage (undesirable physicochemical and biological changes) in sausage and ham (from production to consumption). nitrate and nitrite italian journal of food science, 2021; 33 (sp1): 117–126 mailto:p.aryaye@yahoo.com 118 italian journal of food science, 2021; 33 (sp1) mahdavi e and peiman a so far, no study has been done on sausages with these preservations along with the map. overall, according to the cases stated in this study, we evaluated the effect of natural antioxidants as a nitrite replacement and the use of fat substitutes to reduce the use of oil in sausage formulation produced by silver carp surimi. map was also used for packaging of samples in different treatments. material and methods raw materials at first, 30 kg of silver carp were caught from the farms and transported to the laboratory in ice boxes, followed by washing and removing the skin. after this step, the underlying meat was removed without contact with the viscera and then it was ground first through a 10 mm plate and then through a 5 mm plate in a meat grinder (mkg1300p, panasonic, japan). all chemicals used were purchased by merck germany and were of an analytical grade. preparation of treatments to prepare the surimi, minced meat was washed with drinking water at a temperature below 10°c for 10 min in three stages. rinsing was performed in 0.2% brine in the third step. surimi obtained during experiments was stored at refrigerator temperature (shabanpour et al., 2017). the sausages contained crushed ice 10%, liquid sunflower oil 10%, extenders and binders 7%, nacl 1.5%, spice mixture 1%, monosodium glutamate 0.1%, polyphosphate 0.3%, and 70% mince. sausage was prepared by mixing the ingredients in sequence in a food processor. the mix was stuffed into five-layer synthetic casings and cooked. diameter and length of sausages were 25 mm and 20 cm, respectively. all fish sausages were packed separately in nine labeled polyethylene zipbags and stored at 4°c prior to the measurements. inulin fiber (if) was added to the primary sausage formulation as fat substitutes. in addition, a ranking test previously performed comparing sausage samples with if at different concentrations showed significantly lower acceptability of the samples incorporating 6% or 7% of them when compared to the rest (5% or lower) (data not shown). after these, if at 5% was chosen as optimal for the following study. fat decreased by 5%. also, extracts of orange peel, grape pomace, and ni in concentration of 0.5% (concentration approved by sensory evaluators unchanged in taste of fish sausage) were added separately and combined as a substitute for nitrite and then, the same steps were taken similar to control treatment and finally the treatments were packed into three multilayer flexible are preservatives that give a good pink color (hernándezhernández et al., 2009). due to the potential dangers of using synthetic preservatives such as nitrite and consumers’ interest in using healthy and natural products, food industry researchers favor the use of natural antioxidants in food products. the use of natural preservatives, such as extracts, powders, and essential oils of plants, to increase the shelf-life of meat products is a promising new technology, were . the active ingredients found in plants have antimicrobial and antioxidant properties (hernández-hernández et al., 2009; odunayo olatunde and benjakul, 2018). the following are some of the compounds that have antioxidant and antimicrobial activity as nitrite substitutes. grape pomace is one of the waste products (5000 tons a year) produced annually in juice factories, a rich source of several valuable compounds such as citric acid, tartrate, dietary fiber, and phenolic compounds. anthocyanin (malvidin and penonidine), flavonol (quercetin and meristine), acetylbene, and phenolic acids are the major phenolic compounds, and flavan-3-l, catechin and epi-catechin, and gallic acid are the predominant phenolic compounds in the pulp (drosou et al., 2015). also, due to its red color, it seems to have a pink effect on the sausage. nisin (ni) is a biphasic bacteriocin polypeptide containing 34 amino acids, produced by specific strains of lactococcus lactis subsp. lactis. due to its antimicrobial properties and low toxicity to humans, it has long been regarded as a gras1 food preservative in the food industry. ni is known in two forms, a and z, that are similar in structure and antimicrobial activity and differ in their amino acid in position 27; in type a, histidine and the z variant is asparagine (biswaro et al., 2018). modified atmosphere packaging (map) is used in the storage of fresh (nonfrozen) foods or at least the process. the use of vacuum or modified atmosphere or high co2 packaging is easily feasible for processed meats, but high levels of co2 will have negative effects on product quality, especially texture changes and increased blood loss (hematian et al., 2012). inulin is a nondigestible carbohydrate containing natural fructooligosaccharides (fos) and has dietary fiber properties that are of great interest for its consumption due to its specific health and technological properties. inulin in the mouth creates a fatty mouth feel. the characteristic of inulin as an imitative lipid relates to its ability to bind to water molecules and form a gel-like network (menegas et al., 2013). 1generally recognized as safe. italian journal of food science, 2021; 33 (sp1) 119 fish sausage fortified with natural preservatives ph value five grams of each sample were added to 45 ml of distilled water and placed in a mixer for 30 s. then, the ph of the samples was measured with a digital ph meter calibrated to ph four and seven standards (valipour et al., 2017). color test the color of the sausage was measured using the hunterlab colorflex colorimeter (hunter lab inc). the color test results include three hunter indices a*, b*, l*, where l* is a light symbol which is black (0) and white (100), a* is a green-to-red symbol, –a is green, and +a is red and b* are blue to yellow symbol, which + b is yellow and –b is blue. the experiment was performed in triplicates (choi et al., 2009). microbial analyses ten grams of each sample were mixed and homogenized with 90 ml sterile sodium chloride solution and the required dilutions were prepared. one milliliter of each dilution was used for culture by pour plate method. total count and psychrotrophic bacteria were counted on plate count agar at 37°c for 2 days and 7°c for 10 days. the results were reported as log cfu2/g (javadian et al., 2017). mold and yeast test dilution of the sample was first prepared in peptone water broth, then transferred to a plate containing drbc3 medium. plates were aerobically incubated at 25°c for 5 days (isiri, 2008) inoculation and enumeration of clostridium botulinum to ham samples approximately 108 cfu/ml of clostridium botulinum were added to the ham samples. the samples were then massaged to ensure complete mixing of the bacterium with the hams and placed in specially filled coatings in the baking chamber. at least 10 hams were considered for each treatment. all treatments were packed in zippered nylon bags and stored at refrigerator temperature (4 ± 1°c) during the experiment. for bacterial count at each sampling time, 1 g of sample was mixed with 9 ml of physiological serum and suspended for half an hour. depending on the sample, the dilutions range from 102 to 104. one milliliter of diluted sample was poured into the petri dish and then 10–15 ml of sc agar medium 2colony-forming unit 3dichloran rose bengal agar pouches (three and four layers) under modified atmosphere (map: 70% co2 + 30% n2) (rahmanifarah et al., 2013). the treatments were kept at refrigerator temperature (4 ± 1°c) for 42 days. on 0, 7, 14, 28, 35, and 42 days of storage, three sausages from each section were randomly selected and tested to determine qualitative parameters (physicochemical and microbiological). proximate factors and texture analysis were examined at zero time. all experiments were performed with three replications. in total, five treatments were studied. treatment 1: control treatment (no additives) treatment 2: control treatment + map treatment 3: 5% oil, 5% if + map treatment 4: 5% oil, 5% if + 0.5% grape pomace extract (ge) + map treatment 5: 5% oil, 5% if + 0.5% ge+ 0.5% ni + map proximate composition moisture of samples using oven at 105°c, amount of protein by the kjeldahl method, amount of fat using petroleum ether solvent (boiling point of 40–60°c) by soxhlet extraction, and ash of samples using electric furnace at 550°c were measured (aoac, 2005). cooking loss test thirty grams of samples were stuffed into screw top test tubes and were heated in a steam bath at 70°c for 30 min. the cooked samples were quickly immersed in cool water for 10 min. cooking loss was determined by weighing individual sample before and after cooking, and the difference was expressed as a percentage of the original weight (choi et al., 2009). texture analysis to measure the texture of the sausage, the cubic pieces were cut in 1 × 1 × 1 cm3 dimensions and subjected to compression test by a texture analyzer with a flat probe profile of 40 × 40 mm and a load of 10 kg. the force required to compress the samples to 70% of their initial height was measured at a constant rate of 200 mm/min (vural, 2003). chemical analyses peroxide value peroxide value of the samples was determined according to the pearson method (bagheri et al., 2016). results were expressed in meq oxygen kg–1 lipids. 120 italian journal of food science, 2021; 33 (sp1) mahdavi e and peiman a at 44–47°c was added to the petri dish and thoroughly mixed. after solidification of the medium, about 10 ml of the same medium was poured into the petri dish. plates were then placed in an anaerobic jar and incubated at 37°c for 22 h. at the end of incubation, all plates containing less than 150 colonies were selected and the black colonies on each plate indicating the probability of c. botulinum were counted (isiri, 1994). statistical analysis all experiments were performed in a completely randomized design as triplicates and the result was reported as mean ± sd. statistical analysis of treatments was performed by anova using spss software. significant mean differences were determined by the duncan test at 0.05 level and charts were plotted using microsoft excel software. results and discussion proximate factors the results of moisture content (table 1) in different treatments showed that replacement of oil with if increased moisture content (p < 0.05). but, two treatments that used natural preservatives had no significant effect on moisture content (p > 0.05). the lowest values were observed in control treatments (1 and 2) and the highest moisture content was observed in treatments 3, 4, and 5 (treatments containing if). this is due to the high water absorption properties of the fiber and also because the fiber improves the stability of the emulsion (choi et al., 2010). hydrocolloids are also very important factors in bonding with water and keeping it in the product. so, the increase in moisture after adding fiber is obvious (sahin et al, 2005). results of fat content (table 1) showed that replacement of oil with if reduced fat levels. the highest fat content was observed in control (1 and 2) treatments and the lowest fat content was observed in treatments 3, 4, and 5 (treatments containing if) (p < 0.05). menegas et al. (2013) by replacing if in chicken sausage fermented with corn oil (45% fat) produced a low-fat sausage with 25% fat. results of protein content (table 1) in different sausage treatments showed that replacement of oil with if had no significant effect on protein content. all treatments had no significant difference (p > 0.05). menegas et al. (2013) reported that the use of if in fermented chicken sausage had no significant effect on protein content. results of ash content (table 1) in different sausage treatments showed that replacement of oil with if increased the ash content. the lowest values were observed in control treatments (1 and 2) and the highest amounts of ash were observed in treatments 3, 4, and 5 (treatments containing if). increasing ash by adding if may be due to the fact that if has significant amounts of ash (p < 0.05). cegiełka and tambor (2012) reported that adding if to polish chicken burger increased burger ash content. texture analysis according to the results (figure 1a), replacement of oil with inulin increased the firmness values. the lowest values were observed in control treatments (1 and 2) and the highest values were observed in treatments 4 and 5 (treatments containing if) (p < 0.05). adding fiber seems to increase the ability of the protein to bind to meat. thus, it creates a firmer and coherent texture and prevents deformation of the sausage (álvarez et al., 2012). by definition, elasticity refers to the rate of return of the sample to its original state after removal of the deformation force. elasticity is inversely related to the degree of firmness of the treatment. in fact, the formation of a stable network structure increases the elasticity (zhou et al., 2010). according to the results (figure 1b), the highest values were observed in control treatments (1 and 2) and the lowest values were observed in treatments 3, 4, and 5 table 1. proximate factors of different treatments of sausage. treatment/proximate factors moisture (%) fat (%) protein (%) ash (%) 1 61.94 ± 0.10b 19.64 ± 0.37a 18.29 ± 0.32a 1.73 ± 0.02b 2 62.00 ± 0.43b 19.78 ± 0.40a 17.94 ± 0.43a 1.78 ± 0.02b 3 67.89 ± 0.36a 14.70 ± 0.40b 18.17 ± 0.32a 2.09 ± 0.02a 4 67.75 ± 0.43a 14.95 ± 0.30b 18.18 ± 0.32a 2.11 ± 0.03a 5 67.54 ± 0.44a 15.11 ± 0.14b 17.91 ± 0.31a 2.03 ± 0.03a a,b,cdifferent small letters in the same column represents significant difference (p < 0.05) (t1: control, t2: control +map, t3: fi 5% +map, t4: fi 5% +ge 0.5%+ map, t5: fi 5% +ge 0.5% +n 0.5%+ map). italian journal of food science, 2021; 33 (sp1) 121 fish sausage fortified with natural preservatives peroxide value according to the results of the present study (figure 3a), the peroxide value increased in all treatments with increasing time (p < 0.05); the increase of peroxide value in meat products was also reported by other researchers (javadian et al., 2017; valipour et al., 2017). the use of map slowed the increase in peroxide value, so that the lowest values of peroxide were observed in the nitrite-containing treatments with the modified atmosphere (p < 0.05). the high level of carbon dioxide used in map prevents the growth of aerobic bacteria. the highest level of carbon dioxide in map has been reported up to 50% (silbande et al., 2018). the lowest values were observed on all days of treatment after treatment 2, in treatments 1 and 5 (p < 0.05). the lower peroxide value in the inulin treatment is due to the lower fat content in these treatments and thus to the reduction of oxidative spoilage. also, using two preservatives together more effectively decreased the peroxide value. plant extracts contain phenolic compounds. polyphenols are capable of trapping free radicals, especially proxy radicals, which are one of the key intermediate chain reactants, thereby terminating the cycle of oxidative spoilage reactions. thus, the effect of treatments containing extracts had a higher effect on increasing the peroxide value (fidan et  al., 2019). the reason for the lower peroxide value after addition of ni can be attributed to the effect of bacteriocin on the decrease in lipolytic bacteria (such as pseudomonas species) (dehbandi et al., 2014). dehbandi et al. (2014) also reported that the use of ni slowed the peroxide value in the kilka fish surimi during storage. the acceptable level of peroxide in meat for human consumption is 5 (yanar, 2007). in relation to sausage at the (treatments containing if). amina et al. (2014) examined the effect of adding 5%, 10%, and 15% apple waste fiber to meat nugget texture. apple fiber increased texture firmness and reduced nugget elasticity. cooking loss cooking sausage efficiency depends on cooking temperature, cooking time (kim and chin, 2007), ingredients, and fat content in the product (huang et al., 2005). the results of cooking loss (figure 2) in the present study showed that with increasing time the values of cooking loss increased in all treatments. replacing the oil with if reduced the cooking loss. the highest values were observed in control (1 and 2) on all days of storage and the lowest values were observed in treatment  3 (treatments containing if) (p < 0.05). loss of cooking is associated with the amount of fat and water-holding capacity (eldemery, 2010). adding dietary fiber to meat products appears to improve water-holding capacity and improve emulsion, thereby increasing cooking efficiency (choi et  al., 2010). various researchers have suggested this as the capacity to bind to water in dietary fiber that improves tissue properties (choi et al., 2010, 2015). eldemery (2010) also reported that adding orange fiber to meat burger formulation decreased burger cooking loss and with increasing concentration, lower cooking loss were observed. c 1 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 2 4 6 8 10 12 (a) (b) 2 3 treatment f irm ne ss ( kg ) e la st ic ity ( cm ) 4 5 1 2 3 treatment 4 5 c b a a a a b b b figure 1. texture analysis of different treatments of sausage [firmness (a) elasticity (b)] (t1: control, t2: control +map, t3: fi 5% +map, t4: fi 5% +ge 0.5%+ map, t5: fi 5% +ge 0.5% +n 0.5%+ map). 1 0 1.5 2 2.5 3 3.5 4 7 14 21 storage time (day) c oo ki ng lo ss ( % ) 28 35 42 2 3 4 5 figure 2. cooking loss of different treatments during storage) t1: control, t2: control +map, t3: fi 5% +map, t4: fi 5% +ge 0.5%+ map, t5: fi 5% +ge 0.5% +n 0.5%+ map). 122 italian journal of food science, 2021; 33 (sp1) mahdavi e and peiman a 1 0 0 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 1 2 3 4 5 6 7 (a) (b) 7 14 21 storage time (day) p v ( m eq /k g) ph 28 35 42 0 7 14 21 storage time (day) 28 35 42 2 3 4 5 1 2 3 4 5 figure 3. changes in peroxide value (pv) (a) and ph (b) of different treatments during storage (t1: control, t2: control +map, t3: fi 5% +map, t4: fi 5% +ge 0.5%+ map, t5: fi 5% +ge 0.5% +n 0.5%+ map). end of the storage period, the peroxide value was at an acceptable level in all samples except treatments 3 and 4. ph value the ph value of food affects microbial flora composition. in fish, gram-negative and proteolytic microorganisms are dominant. in many foods of animal origin, such as poultry, meat, fish, and dairy products, ph reduction occurs during storage due to chemical changes such as post-slaughter changes in meat or processing. if the food storage time is increased and the microorganisms proliferate, the ph will rise and will be contaminated with the infection and food born microorganisms. results of sausage ph values (figure 3b) in the present study showed that ph values first decreased and then increased. the initial decrease in ph may be due to the activity of lactic acid bacteria and acidification of the environment. but, the increase in ph after storage time is due to the natural pattern of spoilage of meat products that is related to the activity of spoilage by microorganisms (kim et al., 2011). the use of modified atmosphere slowed the ph value. high levels of carbon dioxide reduce bacterial growth and result in slowing the increase in ph compared to the control treatment. overall, the lowest ph values were observed in nitrite + modified atmosphere treatment. using two preservatives more effectively decreased the ph value. overall, the lowest values were observed on all days of treatment after treatment 2 in treatments 1, 5 (p < 0.05). the latter treatments had no significant difference (p > 0.05). the low ph of the sausages treated with the extract is due to its antioxidant properties and the phenolic compounds present in the extract that can protect the sausages from the function of the internal proteases and thus inhibit protein breakdown and production of amines (fan et al., 2008). the mechanism of ni is due to its antibacterial properties and reduction in the capacity of bacteria for oxidative deamination of nonprotein nitrogen compounds (such as ammonia and trimethylamine), thereby reducing the ph-increasing process (dehbandi et al., 2014). color test the color index l indicates the brightness symbol (black to white). so, more the l, lighter the meat. the color index a is the indicator of the color change from green to red. the color index b represents the color change from blue to yellow. results of color index showed that replacement of nitrite with preservative reduced color index l and a, and increased color index b. sodium nitrate and nitrite are used to create a bright red (pink) and prevent turbidity of meat products, as well as antimicrobial preservatives, to create a special flavor. so, it seems natural to replace nitrite with other compounds and change its color. according to the results of using modified atmosphere, the color index l decreases (figure 4a). overall, the highest values were observed at the end of the storage period after the control treatments in treatment 5. there was no significant difference between the two latter treatments (p < 0.05). this indicates that the oxidation process is decreased. in fact, the brightness values of the sausage samples are correlated with the peroxide values. as the number of peroxides increases, the brightness decreases and the samples darken. in fact, it can be stated that the use of three preservatives, prevents oxidation of pigments and act as a chemical preservative such as nitrite. the color index a (figure 4b) also decreased in all treatments. generally, one of the causes of oxidation in meat products is the presence of compounds such as myoglobin and hemoglobin, which in the presence of metals, such as iron, act as peroxidants. thus, it is one of the factors affecting the oxidation of oxymyoglobin (light red) to metmyoglobin (brown color) during the storage of meat products. therefore, as a result of redox oxidation, color index a is reduced (jin et al., 2007). italian journal of food science, 2021; 33 (sp1) 123 fish sausage fortified with natural preservatives 1 0 35 37 39 41 43 45 47 (a) (b) (c) 7 14 21 storage time (day) c ol or in de x (l ) c ol or in de x (a ) c ol or in de x (b ) 28 35 42 0 5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 5.5 6 6.5 7 7.5 8 7 14 21 storage time (day) 28 35 42 0 7 14 21 storage time (day) 28 35 42 2 3 4 5 1 2 3 4 5 1 2 3 4 5 figure 4. changes in color index l, b, and a (a, b, and c, respectively) of different treatments during storage (t1: control, t2: control +map, t3: fi 5% +map, t4: fi 5% +ge 0.5%+ map, t5: fi 5% +ge 0.5% +n 0.5%+ map). 1 0 0 1 2 3 4 5 6 7 8 7 14 21 storage time (day) t v c ( lo g c f u /g ) 0 1 2 3 4 5 6 7 8 p t c ( lo g c f u /g ) 0 1 2 3 4 5 6 7 8 9 c lo st rid iu m b ot ul in iu m ( lo g c f u /g ) 28 35 42 0 7 14 21 storage time (day) 28 35 42 0 7 14 21 storage time (day) 28 35 42 2 3 4 5 1 2 3 4 5 1 2 3 4 5 (a) (b) figure 5. changes in total viable count (tvc) (a), psychrotrophic bacteria count (ptc) (b), and clostridium botulinum (c) of different treatments during storage (t1: control, t2: control +map, t3: fi 5% +map, t4: fi 5% +ge 0.5%+ map, t5: fi 5% +ge 0.5% +n 0.5%+ map). the color index b (figure 4c) decreased and increased during the storage period, which was consistent with the results of giatrakou et al. (2010), who also reported that yellow index changes in meat products did not have a specific pattern and can change during storage by product type, formulation, and form of packaging. total and psychrotrophic bacteria the results of total count bacteria (tvc) (figure 5a) and psychotrophic bacteria (ptc) (figure 5b) were somewhat consistent (p < 0.05) and were increased during storage time (p < 0.05). map decreased the growth of tvc and ptc so that the lowest values were observed in nitrite + modified atmosphere treatment (treatment 2). gases used in modified atmosphere, such as carbon dioxide, have antimicrobial activity, and their mechanism is to dissolve in the water content of the food and produce carbonic acid, which enters the cell membrane of the microorganism and after ionization disrupts intracellular electrical balance and ultimately causes bacterial death (hematian et al., 2012). use of two preservatives together more effectively slowed down the growth of tvc. the lowest values were observed on all days of treatment 2 and then in treatment 1 and treatment 5 (p < 0.05), and similar results were observed with ptc bacteria, indicating a positive effect of the two preservatives as a substitute for nitrite in sausages. the lower tvc and ptc in the extract-containing treatments can be 124 italian journal of food science, 2021; 33 (sp1) mahdavi e and peiman a chemical and microbial properties to conventional sausage treatment (containing nitrite). among the treatments containing natural preservatives, the best results were observed in treatment 5 (5% oil, 5% if + 0.5% ge + 0.5% ni + map). based on the results, a functional product with properties, such as fiber and natural antioxidants, low fat, and nitrite-free, can be produced. references álvarez, d., xiong, y.l., castillo, m., payne, f.a. and garrido, m.d., 2012. textural and viscoelastic properties of pork frankfurters containing canola–olive oils, rice bran, and walnut. meat science 92: 8–15. 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s., 2004. essentialoils: their antibacterial propertied and potential application in foods – a review. international food mashinicrobiology 94(3): 223–253. https://doi.org/10.1016/j. ijfoodmicro.2004.03.022 cegiełka, a. and tambor, a., 2012. effect of inulin on the physical, chemical and sensory quality attributes of polish chicken burgers. journal of food research 1: 161–178. https://doi. org/10.5539/jfr.v1n1p169 choi, y.s., choi, j.h., han, d.j., hack-youn, y., lee, m.a., jeong, j.y., et al. 2010. effects of replacing pork back fat with vegetable oils and rice bran fiber on the quality of reduced-fat frankfurters. meat science 84: 557–563. https://doi.org/10.1016/j. meatsci.2009.10.012 choi, y.s., choi, j.h., han, d.j., kim, h.y., lee, m.a., kim, h.y., et al. 2009. characteristics of low-fat meat emulsion systems with pork fat replaced by vegetable oils and rice bran fiber. meat science 82: 266–271. https://doi.org/10.1016/j. meatsci.2009.01.019 choi, y., kim, h.-w., hwang, k.e., song, d.-h., jeong, t.-j., kim,  t.-b., et al. 2015. effects of fat levels and rice bran fiber on the chemical, textural, and sensory properties of frankfurters. food science and biotechnology 24(2): 489–495. https://doi. org/10.1007/s10068-015-0064-5 dehbandi, a., motallebi, a.a., razavilar, v. and pourgholam, r., 2014. effect of nicin z on some of spoilage chemical and due to phenolic compounds such as cineol. phenolic compounds in plant extracts destroy the microorganisms and cause liposaccharides to exit and increase the permeability of the cytoplasmic membrane to atp4. atp release leads to the termination of cell energy storage and cell death (burt, 2004). ni causes a gap in the plasma membrane and the cytoplasmic components secreted in the surrounding plasma space, and the ability of the compounds to transfer to the plasma membrane is impaired, and after atp hydrolysis, cell death occurs. the anionic lipid present in the membrane is an active site for ni binding (li et al., 2016). in the present study, no mold or yeast was observed. clostridium botulinum clostridia are bacteria that occur in the environment and on the flora of the intestines of humans and animals (wagner et al., 2006). results of c. botulinum (figure 5c) showed that with increasing time, c. botulinum decreased in all treatments. according to the results of the map, the process of growth of c. botulinum was slowed so that on day 14 the lowest value of the bacterium was observed in control + map treatment (t2). the use of the preservative effectively inhibited the bacterium as no treatment was observed on day 21 due to the antimicrobial activity of the extracts. phenolic compounds in plant extracts destroy microorganisms, resulting in the release of liposaccharides and increased permeability of the cytoplasmic membrane to atp. atp withdrawal results in the depletion of cell energy storage and cell death (burt, 2004). c. botulinum is a gram-positive bacterium, and the fact that ni is effective against gram-positive bacteria but not effective against gram-negative bacteria has been well established. ni is specifically active against vegetative cells and heat-resistant spores of bacillus, clostridium, and listeria monocytogenes. the mechanism of ni antimicrobial activity against the vegetative walls of cells is binding to the cytoplasmic membrane. ni is a cationic antimicrobial that binds to electrons with a negative charge through electrostatic bonding. after binding, ni enters the membrane and creates a temporary small cavity. this process causes the rapid release of ions, amino acids, and cellular atp (vongsawasdi et al., 2012). conclusion the results of the present study showed that addition of if had a positive effect on a physicochemical and texture of sausages. also, the use of two natural preservatives combined with modified atmosphere had almost similar 4adenosine triphosphate https://doi.org/10.1016/j.meatsci.2012.03.012� https://doi.org/10.1002/fsn3.275� 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2istituto zooprofilattico sperimentale del lazio e toscana, roma, italy *corresponding author: giuliano palocci, consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria, centro di ricerca zootecnia e acquacoltura, monterotondo, italy. email: giuliano.palocci@crea.gov.it received: 10 march 2021; accepted: 6 november 2021; published: 17 november 2021 © 2021 codon publications open access paper abstract in the production of some traditional cheeses from vegetable rennet, raw extracts of cynara cardunculus flowers are used as the coagulant. during the preparation of this rennet, there are many factors that can influence its coagulation activity. we studied the flowers of cynara cardunculus var. altilis to evaluate the effects of some of these factors: ripening stage of the flower at harvest, type of drying, part of the flower subjected to drying, toasting of the pistils, and maceration time of the pistils. the results show that it is possible to improve the coagulation activity of the traditional preparation of cynara cardunculus flowers through some practices such as the rapid drying of the flowers/pistils at a controlled temperature, the toasting treatment of the pistils carried out after the slow drying of the flowers, and the extension of the extraction time to 24 h. keywords: clotting activity; cynara cardunculus; drying process; maceration time introduction the use of vegetable rennet in cheese production is limited to a few cheeses. the excessive proteolytic nature of plant coagulants can negatively affect the cheese-making process and favor a reduction in the cheese yield and the presence of some defects in flavor and texture. this may have limited the diffusion of vegetable rennet (lo piero et al., 2002). an exception to this general rule is represented by the aqueous extract of cynara cardunculus flowers, which is the most common vegetable rennet in the mediterranean region (barros et al., 2001). the milk-clotting activity of this plant extract has been known for centuries and has been successfully used to produce cheeses from ovine and caprine milk (silva et al., 2003), which are highly appreciated. these cheeses are widespread mainly in spain and portugal, where in many cases only the raw extract of dried cynara cardunculus flowers is used as a coagulant (almeida and simões, 2018). furthermore, some of them (ibores, flor de guia, la serena, torta del casar, azeitao, castelo branco, evora, nisa, serpa, serra da estrela) have been recognized as protected designation of origin (pdo), as evidence of their close link with origin area and traditional practices of cheese-making. in italy, only a few traditional ovine cheeses are made with vegetable rennet (caciofiore, casoperuto). cheeses produced with cynara cardunculus differ from those produced with animal rennet due to their softer texture, more intense odor, and flavor, including the bitter taste (alavi and momen, 2020; barbosa et al., 1981). it has been observed that the formation of bitter taste peptides is caused by a strong proteolytic activity of thistle enzymes (agboola et al., 2004; alavi and momen, italian journal of food science, 2021; 33 (4): 57–66 mailto:giuliano.palocci@crea.gov.it 58 italian journal of food science, 2021; 33 (4) tripaldi c et al. thistle aspartic proteases cut the same peptide bond as chymosin, their proteolytic activity is more extensive. according to conceição et al. (2018), cardosins reveal a more intense secondary proteolytic action on cheese αs and β-casein than other coagulants, with impact on the cheeses’ biochemical and sensory properties. in addition, three cyprosins from dried flowers of cynara cardunculus, which were isolated, purified, and characterized by heimgartner et al. (1990), have milk-clotting activity. generally, vegetable rennet enzymes have been obtained by aqueous extraction of various plant organs, such as flowers, seeds, roots, and leaves. there are several different ways to prepare aqueous extracts of plant material (sousa and malcata, 1996). in one method, dried whole or crushed cardoon flowers are soaked in water at room temperature for a variable time. then, the filtrate is collected, and this crude extract is used to coagulate milk (roseiro et al., 2003). an alternative method of extraction is grinding the dried flowers with crude kitchen salt, laying the paste on a cotton cloth, and solubilizing the enzymes by percolation with warm milk (sousa and malcata, 2002). the crude extract can also be further purified to obtain partially purified enzyme or pure enzyme depending upon the degree of purification (shah et al., 2014). milk-clotting proteases have also been produced by in vitro techniques (shah et al., 2014). the activity of the plant extract (almeida and simões, 2018) depends on numerous factors, such as the thistle flower ecotype, the part of the flower used, its stage of maturity, the drying time, the final moisture content, the ph of the buffer, the salt concentration of the buffer, and the homogenization time (correia et al., 2016; folgado and abranches, 2020; guiné et al., 2016; sousa and malcata, 1996). in particular, the different profiles of aspartic proteases found among flowers of different genotypes further increase the variability of the flower extracts and their clotting time (folgado and abranches, 2020). this results in cardoon extract preparations that may vary in terms of clotting and proteolytic activities, and may influence the yield and final characteristics of cheese (sousa and malcata, 2002). for all these reasons, the objective of this paper was to evaluate the effects of some factors influencing the milk clotting activity of cardoon rennet to reduce its variability. two kinds of drying methods for cardoon flowers were compared: the slow drying method adopted by a farm and an experimental fast drying method. second, the effects of the stage of the flower on harvesting and the part of the flower subjected to drying were estimated. finally, the effect of the extraction time of the crude extract was assessed in all samples. 2020). in sheep and goat cheeses produced with cynara cardunculus extracts, a slightly bitter taste was detected (conceição et al., 2018; roseiro et al., 2003). cheeses made with cow’s milk tend to develop a bitter taste (alavi and momen, 2020; barbosa et al., 1981). it was found that ovine caseins are less likely than bovine caseins to form hydrophobic bitter peptides following proteolytic action (pelissier and manchon, 1976). according to macedo et al. (1996), the bitter taste of the ovine cheese could be due to the formation of several peptides identifiable in the digests of isolated bovine alfa-s and beta-casein from ovine milk. the concentration of bitter peptides (those with a molecular size of 165–6500 g·mol−1) was the lowest in ovine cheese made from calf rennet; however, cheese made from cardoon coagulant was perceived to be less bitter by a sensory panel (agboola et al., 2004). cynara cardunculus flowers produce cardosins and cyprosins, aspartic proteases that accumulate in mature flowers; in fact, the concentration of the aspartic proteases in the fresh flower is lower (cordeiro et al., 1994). to date, nine different aspartic proteases have been found at the protein level: six cardosins and three cyprosins (folgado and abranches, 2020). cardosins a and b were extracted from the stigmae and stylets of dried flowers of cynara cardunculus (silva et  al., 2003). cardosins a and b are among the aspartic proteases that are found in many varieties of plant species. aspartic proteases are involved in protein degradation during the plant development process, protein storage mechanisms, responses to stress and pathogens, reproduction, and plant senescence (cordeiro et  al., 1994; gonzález-rábadea et al., 2011; pissarra et al., 2007). cardosin b is more proteolytic than cardosin a. in terms of activity and specificity, cardosin a is similar to chymosin as it cleaves the same peptide bond (phe105-met106) of κ-casein. cardosin b is similar to pepsin; in ovine caseins, as1-casein is cleaved by cardosin b at bonds leu156-asp157 and trp164-tyr165, whereas β-casein is cleaved at peptide bonds leu127-thr128, leu165-ser166, and leu190-tyr191 (macedo et al., 1993; silva and malcata, 1999; silva et al., 2006; veríssimo et al., 1995). cardosin a is often highlighted as being more suitable for promoting milk clotting due to its higher specificity and lower proteolytic activity than cardosin b. however, the amount of cardosin a needed for milk clotting is 10-fold higher than that of cardosin b, i.e., the specific activity of cardosin a is lower than that of cardosin b (silva et al., 2003). according to some authors (silva and malcata, 1998, 1999; silva et al., 2003), although italian journal of food science, 2021; 33 (4) 59 effects of the drying method for flowers collected from the same farm both in the pre-ripening phase (not fully violet) and in the maturity stage (fully violet). all samples were subjected to fast drying of both the whole flowers and the pistils. in the first case, the pistils were separated after drying; in the second case, they were separated from the flower before drying and immediately after being collected. fast drying was carried out at a temperature of 35°c with a dryer prototype, which was created by “evoluzione natura®” company for withering, drying, dehydration, and sanitizing of plant material. the system was based on controlled forced ventilation, dehumidification, and emission of ultraviolet radiation. water activity (aw) was measured before, during, and at the end of drying by aqualab 4te meter group, inc., usa. the experimental drying lasted about 60 h for the whole flowers and about 12 h for the pistils. during the preliminary tests, it was found that these were the times required to reach an average aw of less than 0.500. in the end, all pistils were vacuum-packed and kept at room temperature. rennet extraction to prepare the raw extracts, 2.5 g of pistils was soaked in 100 ml of tap water. the amount of pistils was the same as that used on the farm to prepare a rennet solution for traditional vegetable rennet cheese. the pistils were kept in tap water for three maceration times: 2, 15, and 24 h at a temperature of 15°c. in total, 12 raw extracts were prepared for each sample and were analyzed in duplicate for a total of 24 raw extracts. at the end of the extraction, the extracts were filtered, and the ph was determined. the samples were then frozen at −20 °c and maintained until analysis. milk coagulation properties the milk clotting properties were determined by the zannoni and annibaldi (1981) method using the materials and methods plant materials the flowers of cynara cardunculus var. altilis were harvested from plants grown on a farm near rome (agricoltura nuova cooperative; rome, italy) in 2019. on this farm, for some years, vegetable rennet has been produced from cynara cardunculus. this rennet, used to produce certain sheep farm cheeses, is prepared as illustrated in figure 1. the flowers of cynara cardunculus var. altilis, collected in the maturity stage, were dried in a cool and dry room for approximately 3 weeks (slow drying method), and then the pistils were separated from the flowers and subjected to heat treatment at 120°c for 15 min (toasting treatment). samples of flowers of cynara cardunculus var. altilis intended for the experimental trial (figure 2) were full ripening whole flowers slow drying separating pistils from dried flowers toasting treatment figure 1. method used by the farm for the preparation of rennet from cynara cardunculus flowers. full ripening pre-ripening fast drying of whole flowers separating pistils from dried flowers full ripening pre-ripening fast drying of pistils seperating pistils from dried flowers figure 2. experimental design for the preparation of rennet from cynara cardunculus flowers. 60 italian journal of food science, 2021; 33 (4) tripaldi c et al. where y is the dependent variable; μ is the overall mean; t represents the treatments; and e is the residual error. statistical analysis was performed using the general linear model (glm) procedure in sas software (sas institute, 2011) version 9.3. the level of significance was set at p < 0.05. the effect of aw on clotting time was analyzed. the relationship between these factors was shown by performing the boxplot procedure (sas software) on the mean data of groups. in fact, the aw in the experimental method was used as a threshold value (below 0.500) to fix the end of drying. in the farm, the end of drying carried out in a cool and dry room is determined by time (3 weeks) and the average final aw values were higher than 0.500. results table 1 compares the effects of the two drying methods on the coagulation properties of the crude extracts. for these two samples, we started from fully ripened whole flowers being submitted to the farm method (slow drying) and then to the experimental method (fast drying). after both kind of drying, the pistils were separated from the other parts of the flower and only in the farm method the pistils were toasted. then, to prepare the raw extracts, the toasted pistils and pistils from the fast dried flowers were used. all the crude extracts were subjected to the three maceration times and the data shown in the tables are the results of all the maceration times. the raw extracts prepared with farm and experimental methods reach the lowest value of curd firmness after 60 min from the addition of the rennet and the highest at 90 min. the samples produced by the experimental method showed a lower curd firming time (11.89 vs 13.88 min) and higher curd firmness at 90 min after rennet addition (44.18 vs 41.27 mm). in general, better rennet capability is characterized by a brief clotting and curd firming time and an elevated curd firmness. the fast-drying rennet, named experimental method, had a better clotting ability than the rennet produced by using the slow drying method. in table 2, we evaluated the clotting activity of samples subjected to slow drying. both samples were from fully mature, whole flowers. after the flowers were dried, the pistils were separated. the pistils of the sample prepared according to the farm method were toasted, while the pistils of the second sample were not toasted, in order to evaluate the specific effect of the toasting treatment. formagraph instrument (maspres, firenze, italy). measurement is based on the movement of small pendulums immersed in linearly oscillating samples of milk. minute forces are applied to the pendulums because of the formation of a gel in the moving milk sample. the result is the registration of the coagulation properties of the milk (mcmahon and brown, 1982). the formagraph parameters are milk clotting time (r,  min), curd firming time (k20, min), and curd firmness (a, mm). the parameter r measures the time from the addition of the rennet to the milk up to the point where the baseline starts to increase in width (bittante, 2011). repeatability values within laboratories of milk clotting time were 96% (duranti et al., 2003). the parameter k20 is the interval from the start of gel development until an oscillation width of 20 mm is attained. the curd firmness is the width of the graph at a definite time from rennet addition (bittante, 2011). this last parameter is measured 30 min after rennet addition or at twice the clotting time (a2r) (delacroix-buchet et al., 1994). to better estimate the curd firmness of our samples, which coagulate more slowly than those using animal rennet (liburdi et al., 2019), we measured this parameter at 60, 75, and 90 min (a60, a75, a90) after adding the rennet. the amount of pistils (50 g/100 l of milk) used to evaluate the milk clotting activity was the same as used on the farm dairy to prepare rennet solution to produce traditional vegetable rennet cheese. for each batch of samples analysis, two reference commercial coagulants were used. first: animal rennet (hansen standard 160  imcu/ml, 80% chymosin and 20% pepsin) used according to formagraph method (200 µl of 1.6% rennet solution for 10 ml of milk). second: commercial vegetable rennet (galium, prodor) used according to the dose indicated on the label (100 g of rennet/100 l of milk). all samples were analyzed in duplicate. standardized milk powder used as substrate was prepared according to iso 23058 idf 199: 2006. then, 110 g of low-heat, low-fat, and spray-dried milk powder was added to 1 l of cacl2 solution (0.5 g/l). the final ph of substrate was approximately 6.5. the temperature during pre-heating and analysis of the milk samples was 35 °c. statistical analysis the effects of the drying method: slow method applied in farm and experimental fast method, toasting treatment applied in farm, ripening phase, part of the flower, and maceration time were analyzed using the following linear model: y = μ + t + e italian journal of food science, 2021; 33 (4) 61 effects of the drying method for flowers 22.42  mm), 75 (38.79 vs 33.16 mm and 39.66 vs 32.28 mm), and 90 min after rennet addition (45.15 vs 41.31 mm and 46.14 vs 40.33 mm). the effects of the maceration times of the raw extracts at 2, 15, and 24 h are shown in table 4. the evaluation of the maceration time was carried out on all the samples and included all the treatments (pre-ripening and ripening phase, whole flowers and pistils, farm and experimental drying, toasting or not). for all parameters, the results obtained at the three maceration times were significantly different. the best coagulation aptitude was obtained after 24 h of maceration: clotting and curd firming times of crude extracts were lower, and curd firmness at 60, 75, and 90 min was higher. ph values decreased with increasing extraction time. the correlation coefficients among the formagraph parameters were analyzed for all samples. we found that all correlation coefficients were significant (p < 0.0001): +0.70 between r and k20; −0.73, −0.86, −0.90 between table 1. effect of drying method on milk coagulation properties. method of drying farm method experimental method no. of samples 24 24 r (min) 40.19 42.83 k20 (min) 13.88a 11.89b a60 (mm) 25.37 26.09 a75 (mm) 35.79 37.41 a90 (mm) 41.27b 44.18a different letters within the same row indicate a significant difference (p < 0.05). table 2. effect of toasting treatment included in the farm method on milk coagulation properties. toasting treatment yes no no. of samples 24 24 r (min) 40.19b 59.53a k20 (min) 13.88 14.62 a60 (mm) 25.37a 13.26b a75 (mm) 35.79a 21.02b a90 (mm) 41.27a 32.96b different letters within the same row indicate a significant difference (p < 0.05). table 3. effect of ripening phase and part of the flower submitted to experimental fast drying on milk coagulation properties. experimental fast-drying method ripening phase part of the flower pre full whole pistils no. of samples 48 48 48 48 r (min) 46.85a 39.96b 47.03a 39.77b k20 (min) 12.24a 11.11b 12.73a 10.62b a60 (mm) 22.50b 30.47a 22.42b 30.55a a75 (mm) 33.16b 38.79a 32.28b 39.66a a90 (mm) 41.31b 45.15a 40.33b 46.14a different letters within the same row indicate a significant difference (p < 0.05). the characteristics evaluated were significantly different for the two types of treatments, except for the curd firming time. the coagulation time was reduced by 32% (40.19 vs 59.53 min) in the toasted samples. the curd firmness measured after 60, 75, and 90 min was significantly higher in flowers subjected to the toasting treatment (25.37 vs 13.26 mm; 35.79 vs 21.02 mm; 41.27 vs 32.96 mm). therefore, according to our results, toasting of the pistils after traditional slow drying of the flowers improves the coagulation characteristics of the thistle rennet. table 3 shows the effects on the crude extract characteristics of both the ripening phase and the part of flower in the samples subjected to experimental fast-drying. significantly, lower milk coagulation times were achieved with flowers in the complete ripening phase compared to pre-ripened flowers and using directly dried pistils compared to whole dried flowers (39.96 vs 46.85 min and 39.77 vs 47.03 min, respectively). the firming time values had the same tendency as the clotting time values and were lower in the complete phase and in the pistils (11.11 vs 12.24 min and 10.62 vs 12.73 min). in the pistils and in complete phase samples, the higher curd firmness values were found: 60 (30.47 vs 22.50 mm and 30.55 vs table 4. effect of maceration time of the crude extracts on milk coagulation properties. hours 2 h 15 h 24 h no. of samples 64 64 64 ph 5.97a 5.76ab 5.66b r (min) 48.71a 44.82b 40.67c k20 13.62a 12.11b 11.03c a60 (mm) 22.10b 25.05ab 28.17a a75 (mm) 29.27c 34.13b 39.11a a90 (mm) 37.20c 41.80b 45.80a different letters within the same row indicate a significant difference (p < 0.05). 62 italian journal of food science, 2021; 33 (4) tripaldi c et al. these samples came from whole flowers collected at a pre-ripening stage and quickly dried (pr-wf-fd) (aw = 0.623) and from whole flowers slowly dried and not toasted (fr-wf-sd-nt) (0.557). the other samples had lower aw and faster clotting times. these included fr-wf-sd-t slowly dried and toasted samples (0.457); pr-p-fd pistils quickly dried from premature (0.401) and fr-p-fd mature flowers (0.324); and fr-wf-fd whole flowers dried quickly and collected when fully ripe (0.323). discussion according to the classification of the milk coagulation properties with animal rennet by the method of zannoni and annibaldi (1981), good-quality cow milk has a clotting time between 11:30 and 18:00 min. the results obtained from the raw extracts showed very long milk coagulation times: from 40.19 min with the farm method to 42.83 min with the experimental method, compared to 14.52 min with reference animal rennet. regarding curd firmness, 90 min after adding the rennet (a90), it ranged from 41.27 mm (farm method) to 44.18 mm (experimental method). these values are comparable k20 and a60, a75, a90, respectively; −0.94, −0.95, −0.88 between r and a60, a75, a90, respectively. formagraph parameters from raw extracts were highly correlated, i.e., clotting time and curd firming time were positively correlated, and both were negatively correlated with curd firmness. in raw extracts and standard rennet, when the coagulation time is longer, the time necessary to firm the curd is extended. the greater the curd firmness, the more the milk coagulates in a short time. the coagulation activity is good when the coagulation time is shorter, and the curd consistency is greater. similarly, the correlation coefficients (p < 0.0001) found for cow milk coagulated with animal rennet (mariani et al., 1997) were +0.58 between r and k20; −0.80 between k20 and a30; and −0.89 between r and a30. all samples during formagraph analysis were compared with the reference coagulants, i.e., animal and commercial vegetable rennet whose average data were, respectively: r 14.52 and 11.38 min; k20 2.79 and 2.23 min; a30 44.70 and 50.30 mm; a2r 44.52 and 44.20. figure 3 shows the box plot of the distribution of clotting time according to aw classes. we observed that the two samples with higher aw had higher coagulation time. 70 60 50 40 30 fr-wf-fd fr-p-fd pr-p-fd aw c lo tt in g tim e (m in ) pr-wf-fd f prob > f > .0001 38.95 fr-wf-sd-t fr-wf-sd-nt figure 3. box plot of distribution of clotting time according to a w classes. fr: full ripening; wf: whole flower; fd: fast drying (experimental method); p: pistils; pr: pre-ripened; sd: slow dried; t: toasted; nt: not toasted. italian journal of food science, 2021; 33 (4) 63 effects of the drying method for flowers the senescence process (cordeiro et al., 1994). clotting times decreased as flower development progressed due to increased enzymatic activities in the extracts (cordeiro et al., 1994). according to martins et al. (1996), the drying time of thistle flowers subjected to the traditional method gave the following results. the aw decreased from fresh (aw 0.866) to medially dried (1 day): 0.674 and dried (30 days): 0.592. better clotting activity was observed in medially dried flowers on a wet and dry matter basis. in contrast, ordiales et al. (2012) investigated the milk-clotting activity of cynara cardunculus at three ripening stages: opening of flower, flower fully open, and flower beginning to dry out. milk clotting activities of aqueous extracts after maceration for 1 and 24 h did not vary significantly according to the ripening stages. on the other hand, cynara cardunculus, including its variety altilis (ramos et al., 2014), is a source of phenolic compounds. the content of phenolic compounds was highest in the early stages of maturation and decreased as the maturity stage progressed (mandim et al., 2020). phenolic compounds could have a role in the clotting activity of flowers. protein–polyphenol interactions were reported to modify the functional properties of foods (yildirim-elikoglu and erdem, 2018). phenolic compounds are easily oxidized to form pigments. these pigments attach to proteins, including native enzymes, leading to inactivation of these enzymes (barros et al., 2001). the interactions between casein micelles and polyphenols decrease the enzymatic gelation properties at both the first and second stages of the renneting process (haratifar and corredig, 2014). considering that the crude extract is often contaminated by other floral compounds, such as phenolic compounds (conceição et al., 2018), it can be assumed that the best performance of mature flowers could be due to both the greater content of proteases and to the lower content of polyphenols. among the different treatments used to prepare vegetable rennet from cynara cardunculus flowers, the toasting treatment was very effective at improving clotting activity. some hypotheses can be made to explain this positive effect. in a study (wang et al., 2008) on bitter gourd, it was shown that aspartic protease was activated by heating treatment. only few aps of the plant have been functionally characterized. for most aps of the plant, a definitive role was not assigned (wang et al., 2008). the results obtained with the different maceration times indicate that the longer the extraction time, the greater the coagulating activity of the raw extract. the same trend was observed in cynara cardunculus flowers by with those obtained 30 min after the addition of the animal rennet (44.70 mm). these results suggest that, by coagulating cow’s milk with raw extracts prepared both with the farm and experimental methods, it is possible to obtain curd firmness similar to that obtained with animal rennet by extending the total coagulation time by 1 h. similar results were found by liburdi et al. (2019) for samples of cow’s milk coagulated with cynara cardunculus and analyzed by formagraph: coagulation time of 42.30 min and firmness of the curd after 60 min equal to 29.45 mm. in cheese from animal rennet, the parameters of milk clotting activity are related to cheese yield; it was demonstrated that a faster coagulation time and firmer curd were positively correlated with cheese yield (aleandri et al., 1989; johnson et al., 2001; ng-kwai-hang et al., 1989; okigbo et al., 1985; pretto et al., 2012). moreover, the reduction in coagulation time is an undoubted advantage, as it contributes to decreasing the duration of the process. in a recent paper, ben amira et al. (2017) studied the technological properties of milk gels produced by chymosin and wild cardoon rennet (c. cardunculus var. sylvestris). higher curd firmness, similar to chymosin values, was obtained following the optimization of extraction conditions of wild cardoon rennet. it would be interesting to evaluate the effects of different coagulating activities of the raw extracts of cynara cardunculus on the yield and characteristics of the cheese. regardless, the effects of any changes in the preparation of vegetable rennet on the characteristics of traditional cheese should not be overlooked. the results of the correlation between the aw of the flowers/pistils and the clotting time suggest that the aw should be less than 0.45 so that the clotting time is less than 45 min. in traditional practice, which always starts from fully ripe flowers, the addition of toasting of the pistils, which follows the slow drying of the flowers, is very effective in reducing the aw and the clotting time. if fast drying is performed, the factors that favor the reduction of aw are the collection of flowers at the fully ripe stage and the direct drying of the pistils. the best result, in terms of clotting time, was obtained from the pistils obtained from the ripe flowers and dried directly. however, separation of the pistils from the flowers before drying is time-consuming, which means higher costs to produce vegetable rennet. in the flowers, the quantity of enzyme increased during development and was mainly present in the purple parts of styles and corollas. the maximum activity of enzymes observed in mature flowers may indicate involvement in 64 italian journal of food science, 2021; 33 (4) tripaldi c et al. barbosa, m., corradini, c. and battistotti, b., 1981. cheese-making experiments carried out on some italian cheeses with vegetable rennet from cardo (cynara cardunculus l.). scienza tecnica lattiero casearia 32: 203–221. barros, r.m., ferreira, c.a., silva, s.v. and malcata, f.x., 2001. quantitative studies on the enzymatic hydrolysis of milk proteins brought about by cardosins precipitated by ammonium sulfate. enzyme microbiology technology 29: 541–547. https:// doi.org/10.1016/s0141-0229(01)00431-8 ben amira, a., makhlouf, i., flaviu petrut, r., francis, f., bauwens, j., attia, h., besbes, s. and blecker, c., 2017. effect of extraction ph on techno-functional properties of crude extracts from wild cardoon (cynara cardunculus l.) flowers. food chemistry 225: 258–266. https://doi.org/10.1016/j.foodchem.2017.01.040 bittante, g., 2011. modeling rennet coagulation time and curd firmness of milk. journal dairy science 94: 5821–5832. https://doi. org/10.3168/jds.2011-4514 conceição, c., martins, p., alvarenga, n., dias, j., lamy, e., garrido, l., gomes, s., freiras, s., belo, a., brás, t., paulino, a. and duarte, m.f., 2018. cynara cardunculus: use in cheesemaking and pharmaceutical applications. in: koca, n. (ed.) technological approaches for novel applications in dairy processing. intech open, vol. 1, pp. 73–107. cordeiro, m.c., salome, m.p. and brodelius, p.e., 1994. tissuespecific expression of multiple forms of cyprosin (aspartic proteinase) in flowers of cynara cardunculus. physiology plant 92: 645–653. https://doi.org/10.1111/j.1399-3054.1994.tb03035.x correia, p., vítor, a., tenreiro, m., correia, a.c., madanelo, j. and guiné, r.p.f., 2016. effect of different thistle flower ecotypes as milk-clotting in serra da estrela cheese. nutrition food science 46: 458–475. https://doi.org/10.1108/nfs-12-2015-0157 delacroix-buchet, a., barillet, f. and lagriffoul, g., 1994. caractérisation de l’aptitude fromagère des laits de brebis lacaune à l’aide d’un formagraph. lait 74: 173–186. https://doi. org/10.1051/lait:1994315 duranti, e., bolla, p., caroli, a., chiofalo, l., di stasio, l., fortina, r., martini, m., piccolo, v. and zullo, a., 2003. problems concerning ovine milk clotting aptitude. italian journal animal science 2: 89–95. https://doi.org/10.4081/ijas.2003.89 folgado, a. and abranches, r., 2020. plant aspartic proteases for industrial applications: thistle get better. plants (basel) 9: 147– 156. https://doi.org/10.3390/plants9020147 gonzález-rábadea, n., badillo-corona, j.a., aranda-barradas, j.s. and oliver-salvador, m.d.c., 2011. production of plant proteases in vivo and in vitro – a review. biotechnology advances 29: 983– 996. https://doi.org/10.1016/j.biotechadv.2011.08.017 guiné, r.p.f., tenreiro, m.i.c. and correia, a.c., 2016. analysis of factors influencing the physical, chemical and sensorial properties of serra da estrela cheeses. food measure 10: 643–657. https://doi.org/10.1007/s11694-016-9348-6 haratifar, s. and corredig, m., 2014. interactions between tea catechins and casein micelles and their impact on renneting functionality. food chemistry 143: 27–32. https://doi.org/10.1016/j. foodchem.2013.07.092 heimgartner, u., pietrzak, m., geertsen, r., brodelius, p., da silva figueiredo, a.c. and pais, m.s.s., 1990. purification and ordiales et al. (2012), who reported better results after 24 h than after 1 h of maceration. ben amira et al. (2017), studying a model based on four variables to optimize the extraction conditions of cynara cardunculus, found that the best extraction time was 50 min compared to 145 and 240 min. conclusions the results indicate that it is possible to improve the coagulating activity of the crude extract of cynara cardunculus by modifying the rennet preparation process. the most effective innovations compared to the traditional process were the toasting treatment following the slow drying of the flowers and the fast drying of the flowers/pistils at a controlled temperature. extending the extraction time can further improve clotting activity. during the production of typical cheeses, it is necessary to evaluate whether innovations in the preparation of vegetal rennet can lead to changes in the characteristics of the cheese. acknowledgements we would like to thank agricoltura nuova cooperative, rome, italy, for their kind cooperation and for providing the samples of plant materials. funding this 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and polyphenols: binding mechanisms, related https://doi.org/10.1080/87559129.2017.1377225� https://doi.org/10.1080/87559129.2017.1377225� https://doi.org/10.1007/bf00129389� https://doi.org/10.1016/j.foodchem.2007.10.085� _hlk51236996 _goback _hlk34315912 _hlk83294295 _hlk83291308 _hlk83197767 _hlk83200164 _hlk51238083 _hlk51156447 _hlk38639073 _hlk82075860 _hlk82075819 _hlk51155946 _hlk45707717 _hlk82075965 _hlk51156497 _hlk51156541 _hlk82076878 _hlk82077842 _hlk51156775 _hlk51156975 _hlk45709383 _hlk39152211 _hlk82077597 _hlk82083991 _hlk34734945 _hlk82096473 _hlk83303348 _hlk83303434 _hlk82099414 _hlk57709923 _hlk47533643 _hlk47089746 _hlk50036974 _hlk83215999 _hlk51157666 _hlk63181801 _hlk83721666 _hlk83729063 _hlk83725668 _hlk83730695 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (2): 21–27 issn 1120-1770 online, doi 10.15586/ijfs.v34i2.2178 21 p u b l i c a t i o n s codon marker-assisted selection of dairy cows for β-casein gene a2 variant carla sebastiani1†, chiara arcangeli1†, martina torricelli1*, marcella ciullo1, nicoletta d’avino1, giulia cinti2, stefano fisichella1, massimo biagetti1 1istituto zooprofilattico sperimentale dell’umbria e delle marche-togo rosati (izsum), perugia, italy; 2r&d cooperlat, società cooperativa agricola, jesi, ancona, italy †these authors contributed equally. *corresponding author: martina torricelli, istituto zooprofilattico sperimentale dell’umbria e delle marche-togo rosati (izsum), via salvemini 1, 06126 perugia, italy. email: m.torricelli@izsum.it; giulia cinti, r&d cooperlat, società cooperativa agricola, via piandelmedico 74, 60035 jesi (ancona), italy. email: g.cinti@trevalli.cooperlat.it received: 25 january 2022; accepted: 25 february 2022; published: 2 april 2022 © 2022 codon publications open access paper abstract many studies highlighted potential associations of β-casein a1 with specific human diseases and a minor digestibility of milk, due to the bioactive peptide β-casomorphin 7 (bcm-7) release during digestion. conversely, the ancestral β-casein a2 variant seems to be a favorable trait because it is not associated with bmc-7 release. the aim of this work was to evaluate frequencies of β-casein variants in offspring of previously genotyped cows inseminated with a2 homozygous semen. the frequency of the a2/a2 animals has almost doubled from 37 to 69%. these are encouraging results with the perspective of reaching the goal of producing a2 milk. keywords: β-casein; bovine; marker-assisted selection (mas); milk; polymorphisms; variants introduction marker-assisted selection (mas) is a methodology that allows for the selection of important genetic animal traits in the population of interest by exploiting the genetic information at specific markers. advancement in genomics made easier the identification of markers which could be ultimately utilized in mas. in particular, the genome wide association study (gwas) approach, initially used in human genetics research to associate genetic variations with particular diseases, is of special interest (raina et al., 2020). the method is based on scanning the genome of many different individuals for genetic markers that can be used to predict the presence of a disease in the population under study. more recently, gwas has been applied to the field of domestic animal breeding and genetics, and many genetic markers affecting important economical traits have been described (du et al., 2021; sharma et al., 2015). cattle breeding programs, consisting of marker-assisted selection (mas), has been applied to the selection of dairy cows for the presence of β-casein a2 variant, known to confer better digestibility to milk (duartevazquez et  al., 2017; kaminski et al., 2007; park et al., 2021). milk and dairy products are among the main components of the food tradition of many countries and they play a fundamental role in human health due to their valuable nutritional properties. in spite of various sources of milk being available on the market, bovine milk represents the most consumed variety of milk worldwide (faye and konuspayeva, 2012). it contains essential nutrients such as proteins with high biological value, lipids, carbohydrates (mainly lactose), minerals (calcium, phosphorus, zinc, and magnesium), and vitamins (i.e., b2, b12, d, and  a) (jenness et al., 1979; muehlhoff et al., 2013). regarding milk protein fraction, it is composed by soluble mailto:m.torricelli@izsum.it mailto:g.cinti@trevalli.cooperlat.it 22 italian journal of food science, 2022; 34 (2) sebastiani c et al. in the last two decades, much attention has been paid to the a1 and a2 β-casein content of milk, due to their suggested role in human health. a1 and a2 variants differ from each other at the gene level for a point mutation, which causes an amino acid change. particularly, at position 67 of the protein chain, a histidine in the a1 variant is replaced by a proline in the a2 variant. because of this difference in the protein sequence, the β-casein variants a1 and a2 are differently processed during digestion. actually, digestive enzymes perform a proteolytic cleavage at position 67 of the β-casein chain only when a histidine is present generating a seven amino acids peptide named β-casomorphin 7 (bcm-7), while cleavage is prevented by the presence of a proline at the same position. other variants are characterized by a proline at position 67 (a3, e, d, i, and h) and could exhibit the same behavior of the a2 variant as well as other variants, with a histidine at the same position (b, c, f, and g), and could behave as the a1 variant resulting in the formation of the β-casomorphin 7 (bcm-7) (bodnar et al., 2018). bmc-7 is a bioactive peptide with strong opioid activity and an oxidant effect (efsa scientific report 2009; kay et al., 2021) and its release has been related to the alteration of the physiology of different organ systems. in particular, several studies demonstrated the correlation with the onset of various human pathological conditions, such as heart disease, sudden infant death syndrome, milk intolerance, and also with the aggravation of symptoms associated with schizophrenia, autism, and type  1 diabetes (caroli et al., 2009; cieslinska et al., 2015; kaminski et al., 2007; kay et al., 2021; mclachlan, 2001; pal et al., 2015; reichelt et al., 2012). in more detail, it proteins, also known as whey proteins, and by insoluble proteins, that are caseins, which represent about 80% of the total bovine milk proteins. these are subdivided into four groups, αs1, αs2, β, and k, encoded respectively by the csn1s1, csn1s2, csn2, and csn3 genes, all located on chromosome 6 (rijnkels, 2002). in spite of high milk consumption all over the world, some people experience digestive disorders following the intake of milk and dairy products, because of lactose malabsorption or digestive difficulties due to other dairy components such as β-casein, which represents about 36% of milk protein content (milan et al., 2020). specifically, some studies have shown a correlation between human health and some β-casein variants (kay et al., 2021; thiruvengadam et al., 2021). indeed, bos taurus csn2 gene harbors many nucleotide substitutions leading to the formation of 12 protein variants (a1, a2, a3, b, c, d, e, f, g, h1, h2, and i), seven of which (a1, a2, a3, b, c, i, and e) have been identified mainly in european cattle breeds (barroso et al., 1999; daniloski et al., 2021; hohmann et al., 2021; massella et al., 2017) (table 1). among the dairy cattle breeds, a1 and the ancestral a2 variants are the most common while b and i variants are generally less frequent with some variability depending on the breed (farrel et al., 2004). other variants, such as a3 and c, are rarely found (farrel et al., 2004; massella et al., 2017) and others are related to specific breeds or geographic areas, as it happens for the e and f variants that have been found with a very low frequency, only in the italian piedmontese breed and in animals reared in the emilia-romagna region (northern italy), respectively (massella et al., 2017; voglino et al., 1972). table 1. differences in the amino acid sequence of β-casein variants. β-casein variant amino acid position 36 37 67 72 88 93 106 122 138 a2* glu (e) glu (e) pro (p) gln (q) leu (l) met (m) his (h) ser (s) pro (p) a1* his (h) a3* gln (q) b* his (h) arg (r) c* lys (k) his (h) e* lys (k) i* leu (l) d f his (h) leu (l) g his (h) leu (l) h1 ile (i) h2 glu (e) leu (l) glu (e) in bold: amino acid variations with respect to the a2 ancestral variant. arg: arginine; gln: glutamine; glu: glutamic acid; his: histidine; ile: isoleucine; leu: leucine; lys: lysine; met: methionine; pro: proline; ser: serine; *allele variants detected in european cattle breeds. italian journal of food science, 2022; 34 (2) 23 marker-assisted selection of dairy cows has been reported in the literature that bmc-7, binding to µ receptor in the gastrointestinal (gi) tract, may alter gut microbiota, can increase inflammatory response, and may induce mucin production, thus triggering lactose intolerance-like symptoms. moreover, bmc-7 may promote oxidative stress, may deregulate insulin metabolism, and can modulate dna methylation reactions affecting neurodevelopment (kay et al., 2021). however, the european food safety authority (efsa) in 2009 carried out a meta-analysis of data present in the literature, releasing a scientific report that supports the absence of a cause–effect relationship between the consumption of milk containing the a1 variant and the etiology of the aforementioned diseases, so further studies are necessary in this field (efsa scientific report, 2009). therefore, the discussion on the adverse human health effects of the β-casein variant a1 remains still open also because other studies demonstrated that milk obtained from a2 homozygous cows seems to be more digestible than milk containing β-casein a1 (deth et al., 2016; he et al., 2017; ramakrishnan et al., 2020). this effect could be traced back to the increase in the rate of gastrointestinal transit of a2 milk and to the lack of the pro-inflammatory effect instead associated with a1 milk consumption (brooke-taylor et al., 2017; kay et al., 2021). in addition, a2 milk consumption increases the natural production of glutathione (gsh), which is reported to be a key antioxidant, widely recognized for its association with many health benefits. the consumption of a2 milk induces a twofold increase of blood gsh levels compared to the levels derived from conventional milk intake (deth et al., 2016). interestingly, human breast milk, which is recommended by the world health organization (who) as the exclusive food for newborn feeding, is characterized by the presence of a β-casein protein carrying a proline residue in position 67 which is therefore very similar to the bovine a2 variant (kay et al., 2021). as a consequence, in many countries, including australia, the united kingdom, the united states, new zealand, the netherlands, china and more recently also italy, a2 cow’s milk has been made commercially available, and it is widely recommended for people who suffer from milk-intolerance and for newborns who need formulas more soft to their digestive system (brooke-taylor et al., 2017). considering this scenario, this study focused on the planning and execution of a breeding program based on the mas selection of the β-casein a2 variant, in farms located in central italy providing milk for an important drinking milk producing plant. a2 heterozygous and homozygous cows, previously genotyped (sebastiani et  al., 2020), were artificially inseminated with semen from bulls homozygous for the a2 variant, and their offspring have been likewise analyzed in order to identify a2/a2 animals for a2 milk production. materials and methods sampling a total of 1452 italian holstein friesian cows, reared in farms located in central italy and previously genotyped (sebastiani et al., 2020), were subjected to artificial insemination with semen from a2/a2 selected bulls (co.s.a.p.a.m. soc. coop., lodi, italy; abs italia srl, cremona, italy; inseme spa, modena, italy). among these, 640 were a2 homozygous and 812 were a2 carriers (a1/a2, a2/b, a2/i) animals. from the pregnant cows, 534 heifers were born. from these animals, whole blood samples were collected in tubes containing ethylenediaminetetra-acetic acid (edta) as an anticoagulant and stored at −20 °c until genetic analysis. samples were taken in a single withdrawal, simultaneously with the mandatory periodic tests required by italian national health programs and during breeders’ voluntary health controls. dna extraction and sequencing genomic dna was extracted using high pure pcr template preparation kit (roche life science, mannheim, germany) according to the manufacturer’s instructions. pcr reactions of both exons 6 and 7 were performed as previously described (sebastiani et al., 2020). amplicons were analyzed through 2% agarose gel electrophoresis containing midori green advanced dna stain (nippon genetics europe gmbh, düren, germany). pcr products were purified with qiaquick® pcr purification kit (qiagen, hilden, germany) and sequenced in both directions using brilliantdyetm terminator cycle sequencing kit v3.1 (nimagen bv, nijmegen, netherlands) according to the manufacturer’s instructions. sequencing reactions were analyzed in a 3500 genetic analyzer (applied biosystems; thermo fisher scientific inc.). the obtained nucleotide sequences were aligned to the bovine β-casein gene (accession number x14711.1) using the clustalw tool of the bioedit v7.2.5 software (hall, 1999). electropherograms were analyzed at each investigated mutation point to identify peaks in heterozygosity. in particular, polymorphisms at positions 36  and 37 of exon 6 and at positions 67, 72, 88, 93, 106, 122, and 138 of exon 7 were analyzed to discriminate the different β-casein variants. statistical analysis allele and genotype frequencies were directly calculated dividing the number of copies of each allele and genotype 24 italian journal of food science, 2022; 34 (2) sebastiani c et al. by the total alleles and by the total individuals, respectively. furthermore, the hardy–weinberg (hw) equilibrium was verified using chi-square test (p < 0.05) by the r studio software (r core team, 2020). results in this study, 1452 already genotyped dams (sebastiani et al., 2020), at least carrier of the a2 allele (a2 heterozygous and homozygous animals), were artificially inseminated using commercial semen from a2 homozygous bulls in order to increase a2 variant and a2/a2 genotype frequencies in the female progeny. from inseminated cows that got pregnant, 534 heifers were born. among these, 238 derived from a2 homozygous parents were analyzed to confirm the a2/a2 genotype as they have to be used for the production of a2 certified milk. the remaining 296 heifers, born from a2 heterozygous dams, were analyzed in order to define their genotype and separate the a2 homozygous ones in the herds with the same purpose. here, we report the results of the analysis carried out on this offspring in terms of allele and genotype frequencies of the different csn2 gene variants. no deviation of hw equilibrium was observed at the considered polymorphic sites. in the female offspring population obtained by the artificial insemination of a2 heterozygous and homozygous cows, sequencing analysis of the csn2 gene pcr products highlighted the presence of four β-casein variants (a1, a2, b, and i) and four genotypes, (a2/a2, a1/a2, a2/b, a2/i) as shown in table 2. after the application of the mas on the herds participating in the project, the frequencies of a2 and i alleles, both characterized by a proline in position 67, increased from 60.65 to 84.46% table 2. allele and genotype frequencies (%) in the examined animals, before (dams) and after (heifers) mas selection. allele allele frequency (%) genotype genotype frequency (%) dams heifers dams heifers a2 60.65 84.46 a2/a2 36.96 68.91 a1 30.39 10.86 a1/a2 35.79 21.72 b 5.68 1.40 a1/a1 9.88 / i 3.10 3.28 a2/b 7.55 2.81 a3 0.15 / a2/i 3.83 6.55 c 0.03 / a1/b 3.07 / a1/i 2.03 / b/i 0.25 / b/b 0.18 / a2/a3 0.12 / a3/b 0.12 / a1/a3 0.06 / a1/c 0.06 / and from 3.10 to 3.28%, respectively, in the progeny compared to dams. at the same time, the frequencies of the unfavorable a1 and b alleles decreased from 30.39 to 10.86% and from 5.68 to 1.40%, respectively. a3 and c alleles and their related genotypes, that were present with low frequencies in the population of dams, were not further found in the offspring. regarding genotypes, the most interesting result concerned the about twofold increase of a2/a2 frequency from 36.96 to 68.91%. a similarly intriguing result, derived from the mas application, was the consistent reduction of the frequency of a1/a2 animals, from 35.79 to 21.72%. the frequencies of the other a2 heterozygous genotypes found in the progeny, that is a2/i and a2/b, varied from 7.55 to 2.81% and from 3.83 to 6.55% (table 2, figure 1). interestingly, since the i variant should behave in the same manner as a2 allel frequencies dams a1 a2 b i heifers 90 (a) (b) 80 70 60 50 40% 30 20 10 0 70 60 50 40 % 30 20 10 0 genotype frequencies dams a1/a2 a2/a2 a2/b a2/i heifers figure 1. graphics of the allele (a) and genotype (b) frequencies variation (%) in the examined dams and heifers after mas selection. italian journal of food science, 2022; 34 (2) 25 marker-assisted selection of dairy cows variant in term of bmc-7 formation, a2/i animals could at least be used for the production of a more digestible milk, even though they could not contribute to the marketing of a certified a2 milk. in conclusion, the first generation from the genotyped dams produced an extra number of 368 a2/a2 cows in addition to the a2 homozygous cows in reproductive age still present in the herds. so, both groups of animals may be used for reproduction and for the related a2 drinking milk production. discussion the data presented here were derived from a larger research project whose purpose was primarily to evaluate the frequencies of the β-casein csn2 gene alleles in italian holstein friesian dairy cattle reared in farms supplying milk to an important milk plant in central italy. in this study, we wanted to evaluate the increase of the a2 allele and a2/a2 genotype frequencies, in the female progeny of dams, previously genotyped and fertilized with a2/a2 semen in a selection program based on mas. the final goal was the production of a2 cow’s milk, due to its supposed association with health benefits for humans. in fact, the presence in milk of the a2 isoform of β-casein is increasingly considered a desirable characteristic, because it confers greater digestibility to milk. this could allow milk intake even by people who suffer from lactose intolerance-like symptoms despite not being really lactose intolerant (park et al., 2021). the italian holstein friesian breed is not among those breeds characterized by the highest frequency of the a2/a2 genotype, but it has a sufficiently high frequency to allow an effective genetic selection of this trait (canavesi, 2016). production of a2 milk can be accomplished following a mas scheme, which is based on selecting animal carriers of specific polymorphisms that characterize the a2 variant. during the course of a mas-based selection plan, aimed at obtaining a consistent number of a2 homozygous cows in the herd, milk coming from a2/a2 animals can be collected separately and directed to a2 milk commercialization. this approach requires organizing, logistical, and management efforts of cow sheds and milking barns, which however is rewarded by an economic gain in the sale of a type of milk with beneficial properties for human health. in the last years, the commercialization of a2 milk has conquered large market portions in many non-european countries, while in europe and in italy it still remains a niche product (brooke-taylor et al., 2017). actually, few medium/large italian dairy industries have taken this route commercializing certified a2 milk, whose distribution is slowly spreading, starting to generate the interest of consumers. the data reported here are very encouraging because they confirm the expected genetic improvement in the farms analyzed in this survey. the use in the future of certified a2/a2 sexed semen could help to accelerate the increase in the number of female animals to be used for a2 milk production. conclusions in recent decades, advances in assisted reproductive technologies, animal molecular genetics, and statistical analysis applied to animal genetic improvement helped to maximize the genetic gain in livestock breeding worldwide. in the past, cows produced milk whose β-casein protein was represented only by a2 isoform, considered to be ancestral, but over time changes in the genetic heritage led to the occurrence of other variants (farrel et al., 2004). cows have thus acquired the ability to produce milk with different β-casein isoforms, in particular a1 and a2. nowadays, β-casein a2 milk can be considered “a return to the origins” because it comes from selected cows that produce only the ancestral β-casein a2 protein. it has to be noted that in addition to the indexes evaluated for the selection of the best breeding bulls regarding traits related to morphology, health, and productivity, information about milk quality, such as β-casein genotype, has also been included. in conclusion, the valorization of this genetic trait and production of a2 milk could be advantageous for the whole drinking milk chain, from producers to consumers. author contributions s.f., g.c., and m.b. conceptualized the study; c.s. and m.b. formulated the study; c.a. and c.s. did the formal analysis; c.a., m.t., m.c., and c.s. were in charge of investigation; g.c., s.f., and n.d. arranged the resources; c.a. and c.s. prepared the original draft; m.b., c.s., m.t., c.a, and g.c. reviewed and edited the manuscript; m.b. and c.s. supervised the study; m.b., g.c., and s.f. 26 italian journal of food science, 2022; 34 (2) sebastiani c et al. a randomized, cross-over clinical trial. nutr. j. 15(1): 82–87. https://doi:10.1186/s12937-016-0201-x du, l., duan, x., an, b., chang, t., liang, m., xu, l., zhang, l., li, j., guangxin, e. and gao, h., 2021. genome-wide association study based on random regression model reveals candidate genes associated with longitudinal data in chinese simmental beef cattle. animals. 11(9): 2524. https://doi.org/10.3390/ ani11092524 duarte-vázquez, m.á., garcía-ugalde, c., villegas-gutiérrez, l.m., garcía-almendárez, b.e. and rosado, j.l., 2017. production of cow’s milk free from beta-casein a1 and its application in the manufacturing of specialized foods for early infant nutrition. foods. 6(7): 50. https://doi:10.3390/foods6070050 farrel, h.m., 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https://doi.org/10.5713/ajas.14.0715� https://doi.org/10.1016/j.foodchem.2020.127765� https://doi.org/10.1016/j.foodchem.2020.127765� https://doi.org/10.1111/j.1365-2052.1972.tb01233.x� https://doi.org/10.1111/j.1365-2052.1972.tb01233.x� https://doi.org/10.3390/nu7095339� https://doi.org/10.3390/nu7095339� https://doi.org/10.3390/dairy2020017 https://doi.org/10.1007/s11033-020-05919-0� https://doi.org/10.1007/s11033-020-05919-0� https://doi.org/10.3390/nu12123855 https://doi.org/10.3402/mehd.v23i0.18958� ole_link13 ole_link14 ole_link15 paper 282 ital. j. food sci., vol. 27 2015 keywords: antibiotic resistance; lactobacillus; gastrointestinal tolerance; plasmid; probiotic antibiotic susceptibility of potentially probiotic lactobacillus strains junhua hana, dahuan chena, shanshan lic, xingfeng lia, wen-wen zhoud, bolin zhangb,*, yingmin jiaa,* acollege of biological science and engineering, hebei university of science and technology, shijiazhuang, heibei, 050018, china bschool of biological science and biotechnology, beijing forestry university, beijing, 100083, china c dongcheng district center for disease control and prevention, beijing, 100009, china dschool of biosystems engineering and food science, zhejiang university, hangzhou zhejiang, 310058, china *corresponding authors: yingmin jia, tel. +86 311 81668010, jjyymm0311@163.com bolin zhang, tel. +86 10 62338221, zhangbolin888@163.com abstract susceptibility of 29 lactobacilli to 13 antibiotics was assayed by paper disc diffusion method. plasmids and gastrointestinal tolerance were detected. the relationship between plasmids and antibiotic resistance was discussed. the results showed that all of the strains were resistant to bacitracin, polymyxin b, kanamycin, and nalidixic acid. many strains were relatively sensitive to chloramphenicol and tetracycline. six strains contained plasmids and showed good gastrointestinal tolerance. β-lactam resistance gene blr was found in the plasmid of l. plantarum cicc 23180 by pcr. the study will be helpful to promote the safety evaluation and development of potentially probiotic lactic acid bacteria. mailto:zhangbolin888@163.com ital. j. food sci., vol. 27 2015 283 1. introduction due to the claimed benefits, lactobacillus bacteria are widely used in food, feed, medical and health related fields. many lactic acid bacteria (lab), such as streptococcus thermophilus and lactobacillus delbruekii subsp. bulgaricus, have been used safely for a long history. they are agreed to be secure and do not have the possibility of pathogenic. currently, new beneficial bacteria are being developed continuously and will enter the market. however, the security of these new strains has caused great concern. evaluation of antibiotic sensitivity is an important part of safety assessment. now, overuse of antibiotics has become a serious social problem. this led to the emergence of a large number of antibiotic-resistant strains. once the resistance-related factors are tranferred to other microorganisms, especially pathogens via food carrier, it will cause tremendous problems. the evolution of antibioticresistant foodborne pathogens has been widely reported (threlfall et al., 2000; walsh et al., 2008; white et al., 2002). moreover, the resistance and resistance-related genes of bifidobacterium, lactobacillus and pediococcus strains to different antibiotics were studied systematically (hummel et al., 2007; huys et al., 2004; maria et al., 2007). the tetm gene transfer of tetracycline resistance in lactobacillus plantarum among strains was reported by niamh et al. (2010). in this study, 29 lactobacillus strains isolated from the food environment with potentially probiotic effects (jin et al., 2009; li et al., 2009; liu et al., 2011; sun et al., 2009; zhao et al., 2013) were used. these strains were assayed for susceptibility to 13 antibiotics by agar disc diffusion method. furthermore, some strains with higher resistance were analysed for the presence of plasmids. then, the tolerance of the plasmidcontaining strains under simulated gastrointestinal conditions was investigated. by plasmid elimination and pcr, the relationship between the plasmid profiles and resistance patterns of the strains was explored. this will provide a reference for the safety evaluation method and also will be helpful to improve the evaluation system of probiotics. 2. materials and methods 2.1 bacterial strains and cultivation 29  lactobacillus strains  used in the study were listed in table  1. lactobacillus strains were cultured in mrs (de man, rogosa, and sharpe) medium at 37°c for 18h under anaerobic condition. quality control strain recommended by clinical and laboratory standards institute (clsi) in the antibiotic  sensitivity test was  e. coli atcc25922 purchased from the institute of microbiology, chinese academy of sciences. the e. coli atcc25922 was activated and cultivated in lb medium (yeast extract 5 g/l, tryptone 10 g/l, nacl 10 g/l) at 37°c. 2.2 testing of antibiotic susceptibility 13 kinds of antibiotics paper discs were purchased from the national institute for the control of pharmaceutical and biological products (table 2), each piece with a diameter of 6.5 mm. the quality  was fully complied with  the  who criteria. antibiotic susceptibility was semi-quantitatively determined with k-b method by antibiotic paper disc diffusion referring to the clsi as described by charteris et al. (1998a). briefly, 1.0 ml lactobacillus suspension (approximately 1.5×108 cfu/ml) was added to sterile petri dish with diameter of 90 mm, and then mixed with a 15 ml mh (muller hinton, mh) agar (beef extract powder 6g/l, casein actable 1 source of the tested strains for antibiotic susceptibility test. species source (original number) lactobacillus plantarum cicca 23124 (l11), cicc 23131 (b31), cicc 23135 (b37), cicc 22195 (c35), cicc 23166 (zj1), cicc 23138 (c8-1), cicc 23180 (ch8) lactobacillus rhamnosus cicc 23119 (1132), cicc 22175 (ll), atccb 7469, cicc 22151 (lk-mt), cicc 22173 (r11) lactobacillus salivarius cicc 23182 (ch-10) lactobacillus acidophilus cicc 22162 (ch-2) lactobacillus casei cicc 23184 (y5-2b) lactobacillus helveticus cicc 22154 (llb) lactobacillus pentosus cicc 23116 (sn23), cicc 22161 (lp-4), cicc 22160 (lp-5), cicc 22159 (lp-b), cicc 22156 (ind-3), cicc 22157 (lp-a) lactobacillus paralimentarius cicc 22148 (412), cicc 22149 (413) lactobacillus delbrueckii cicc 22153 (lb), cicc 22163 (lc) lactobacillus paracasei cicc 22165 (5m1), cicc 22167 (5m7), cicc 23183 (d-400) acicc, china center of industrial culture collection. batcc, american type culture collection. 284 ital. j. food sci., vol. 27 2015 ids hydrolysate  17.5 g/l,  soluble  starch,  1.5 g/l, agar 17 g/l, ph 7.3±0.1) until the medium solidified. the antibiotic paper discs were pasted closely onto the solidified medium with sterile tweezers after 5min at room temperature. three discs were pasted in each dish. the distance was more than 24 mm of each disc center and more than 15 mm from disc edge to the inner edge of dish. next, the dishes were placed at room temperature for 1.5 h and then incubated at 37°c. after 24 h, the inhibition zone diameter was measured around the antibiotic disc with vernier caliper and recorded. for one tested strain, each antibiotic disc was done 3 times. the inhibition zone diameter was averaged standard sensitive strain of e. coli atcc25922 was used as the control. the operation was the same as the above. the antibiotic susceptibility of the tested strains was evaluated according to the clsi criteria (table 2). 2.3 plasmid dna extraction 10 ml of lactobacillus suspension cultured overnight was centrifugated at 10000 rpm for 5 min. then the precipitation was suspended with 500 μl of lysozyme solution (10 mg/ml). the mixture was placed in a water bath for 45 min at 37°c. then plasmid dna of lactobacilli strains was extracted and purified with dna extraction and purification kit of tiangen biotech (beijing) co., ltd. plasmid dna was observed by agarose gel electrophoresis. antibiotic susceptibility and plasmid stability were tested after cultivated 30 generations at 37°c in mrs medium according to the above methods. table 2 the content of antibiotic paper discs and criterion for judgement. antibiotics content/disc inhibition zone diameter (mm) rc i s vancomycin 30 μg ≤9 10-11 ≥12 penicillin g 10 u ≤14 15-17 ≥18 ampicillin 10 μg ≤21 22-28 ≥29 bacitracin 0.04 u ≤10 10-12 ≥12 cephalothin 30 μg ≤14 15-17 ≥18 streptomycin 10 μg ≤11 12-14 ≥15 kanamycin 30 μg ≤13 14-17 ≥18 tetracycline 30 μg ≤11 12-14 ≥15 chloramphenicol 30 μg ≤12 13-17 ≥18 gentamicin 10 μg ≤12 13-14 ≥15 nalidixic acid 30 μg ≤13 14-18 ≥19 multi-polymyxin b 300 μg ≤11 12-14 ≥15 rifampicin 5 μg ≤16 17-19 ≥20 cnote: r-resistant; s-susceptible; i-intermediate. 2.4 gastrointestinal tolerability test  in order to explore the application safety in  human, the gastrointestinal  tolerability of those lactic acid bacteria containing the plasmids were tested. for acid tolerance test, lactobacillus cells were harvested by centrifugation at 6000 rpm for 15 min, washed twice with 0.01 mol/l pbs, ph 7.2 after cultured for 18 h at 37°c in mrs broth, and then suspended in 20 ml sterile saline (0.85%, w/v) adjusted to ph 2.5 with sterile hydrochloric acid. for bile tolerance test, the modified method of lee et al. (1999) was referred to test bile tolerance. the lactobacillus cells were centrifuged (6000 rpm, 15 min) after cultivated for 18 h at 37°c in mrs broth and suspended in 20 ml sterile saline (0.85 %, w/v) supplemented with 0.3% (w/v) bile salts (taurocholate, sigma) at ph 6.8. for pepsin and trypsin tolerance test, lactobacillus cells were centrifuged (6000 rpm, 15 min) after cultivated for 18 h at 37°c in mrs broth, then suspended in 20 ml sterile simulated gastric and pancretic juices. fresh simulated gastric and pancreatic juices were prepared daily according to charteris et al (1998b). pepsin (sigma) was added into the simulated gastric juice with a final concentration of 5 mg/ml. then the ph was adjusted to 2.5 with sterile hydrochloric acid. trypsin (sigma) was added into the simulated pancreatic juices with a final concentration of 10 mg/ml. then ph was adjusted to 8.0 with 0.1 m naoh. all of the tolerability detection, the initial bacterial counts were adjusted to about 108 cfu/ ml and were checked by viable count determination on mrs agar. for the tolerance assay, the bacterial suspensions were incubated and counted at 37°c for 0,1,2,3,4,5,6 h, respectively. all tests were repeated three times to estimate the standard error. 2.5 detection of antibiotic resistance genes part of the antibiotic-resistant genes of those lactic acid bacteria containing both plasmids and high tolerance were investigated. the β-lactam resistance-related gene sequence of blr, ecp1569, nps-1 and the chloromycetin resistancerelated gene sequence of cmla, cat, cmla1 in plasmids were found in national center for biotechnology information (ncbi). the primers were designed and synthesized by beijing sunbiotech co., ltd (table 3). the pcr programmes were performed with the plasmid template of the tested strains according to the following procedures: initial heating at 94°c for 4 min was followed by 34 cycles of the following sequence: 94°c for 30 s, 72°c for 1 min, and 72°c for 1 min. final extension took place at 72°c for 7 min. the amplification products were separated ital. j. food sci., vol. 27 2015 285 by conventional 1.0% (w/v) agarose gel electrophoresis  (100v, 4°c) in tae (tris-acetate-edta) buffer and visualised by ethidium bromide staining. the target fragment was recovered and sequenced by takara biotechnology (dalian, china) co., ltd. the resistance-related gene of plasmid was determined by comparison with the known fragment. 3. results and discussion 3.1 antibiotic susceptibility antibiotic susceptibility of the tested strains was evaluated according to the anti-microbial  drug sensitivity standard of clsi criteria. the sensitivity of the tested lactobacillus to 13 kinds of antibiotics was shown in table 4. the tested 29 strains were generally resisitant to multi-polymyxin b, bacitracin, kanamycin, nalidixic acid, and were mostly sensitive to chloramphenicol and tetracycline. the same species of lactobacillus generally had similar resistance patterns. but there was species specificity such as the different antibiotic sensitivity in l. plantarum, l. rhamnosus, and l. pentosus. moreover, the antibiotic-resistant level of different strains is also different. antibiotic resistance of the foodborne lactic acid bacteria had heen reported in the 1980s. the researchers generally believed that the resistance was a result of the long evolution and it was generally endogenous resistance and obtained resistance (zeng et al., 2004). so, the resistant lactic acid bacteria of natural or isolated from human intestinal can indirectly reflect the habitat of used antibiotic. it can be seen  from table 5, the  29  strains s h o w e d   d i f f e r e n t p a t t e r n s o f r e s i s t table 3 the primers of the resistance genes in the experiment. gene sequence of the primer annealing temperature fragment size blr--up 5’-cgtcttattgaattaacaggttgg -3’ 53°c 125 bp blr--down 5’-cacgaagccatgttgtgttc -3’ ecp-1569--up 5’-caatcaacagagatgtgggctg-3’ 57°c 155 bp ecp-1569--down 5’-gtaccgtagtactctgttcaggtgg-3’ nps-1--up 5’-tcattcttctggcctgtagc-3’ 54°c 782 bp nps-1--down 5’-ggcgataccgctcagttac-3’ cmla--up 5’-caaggagatggtttcgtgcg-3’ 56°c 551 bp cmla--down 5’-catgcccaaacctagaaacgc-3’ cat--up 5’-ggcatttcagtcagttgctc-3’ 55°c 530 bp cat--down 5’-tggaagccatcacaaacg-3’ cmla1--up 5’-gctgaagccaagctgagac-3’ 56°c 492 bp cmla1--down 5’-ctacgttgtggcgtcaatg-3’ ance  to  13  kinds of antibiotics. to bacitracin, polymyxin b, kanamycin and nalidixic acid, the resistance rate of the 29 tested strains was 100%. to β-lactam and aminoglycosides, the resistance  percentage  was 20.7%-37.9% and 86.2%-100%, respectively. all of the 29 strains were mostly sensitive to  chloramphenicol and tetracycline. among of the tested antibiotics, the nalidixic acid and polymyxin b can inhibite dna synthesis and interfer cell membrane formation, respectively. the resistance of lactobacillus to these kinds of antibiotics may be due to the thicker cell wall of gram-positive bacteria. while the tested strains showed different sensitivity to the antibiotics, such as streptomycin, kanamycin, tetracycline, chloramphenicol, gentamicin with protein synthesis inhibitition effect. most lactobacillus strains showed resistance to those antibiotics against gram-negative bacteria, for example, streptomycin, gentamicin, kanamycin. this was consistent with report of zhang et al (2007). 3.2 plasmid dna extraction of antibiotics-resistant lactobacillus strains 16 cicc strains with relatively strong antibiotic resistance were screened for plasmid extraction. as can be seen from fig. 1, among these strains, only cicc 23180, 22161, 22175, 22157, 23124, and 22154 contained plasmids. l. plantarum cicc 23180 showed 6 plasmid dna bands and there is one band greater than 23 kb. l. pentosus cicc 22157 showed two plasmid dna bands of 10 kb and 5 kb, respectively. l. rhamnosus cicc 22175 and l. plantarum cicc 23124 contained respectively 2 and 4 of plasmid dna bands and both of the two strains contained a 10 kb plasmid. l. helveticus cicc 286 ital. j. food sci., vol. 27 2015 t a b le 4 t h e se n si ti vi ty r es u lt s o f 2 9 l a ct o b a ci ll u s s tr a in s to 1 3 a n ti b io ti cs . l. pla nta rum l. pe nto su s l. rha mn os us l. sa liv ari us l. ac ido ph ilu s l. ca se i l. he lve tic us l. pa rac as ei l. de lbr ue ck ii l. pa ral im en tar ius ci cc ci cc ci cc ci cc ci cc ci cc ci cc ci cc ci cc ci cc ci cc ci cc ci cc ci cc ci cc at cc ci cc ci cc ci cc ci cc ci cc ci cc ci cc ci cc ci cc ci cc ci cc ci cc ci cc 23 16 6 23 13 1 23 18 0 23 13 5 23 12 4 23 13 8 22 19 5 22 16 1 22 15 7 22 16 0 22 15 9 22 15 6 23 11 6 22 17 5 23 11 9 74 69 22 15 1 22 17 3 23 18 2 22 16 2 23 18 4 22 15 4 22 16 5 22 16 7 23 18 3 22 15 3 22 16 3 22 14 8 22 14 9 va nc om yc in r r r r r r r r r r r r r r r r r r r s r r r r r s s r r pe nic illin g r r s r r r r s r i r s s i r r i i s s s s s s s s s s r ce ph alo thi n r r r r s s s i r i i s s r s i i i i i s s s s s s s i i ba cit rac in r r r r r r r r r r r r r r r r r r r r r r r r r r r r r am pic illin r i i i i i i i i r i r i r i i r i r i i r i i r i i r r mu ltip oly my sin b r r r r r r r r r r r r r r r r r r r r r r r r r r r r r str ep tom yc in r r r r r r r r r r r r r r r r i r r r r r r r r r i r r ka na my cin r r r r r r r r r r r r r r r r r r r r r r r r r r r r r tet rac yc lin e r i s r s i i i s s s s i r s s s s s s s s s s s s s s s ch lor am ph en ico l i s r s s s s r s i i i i r s i i i i s i i s s s s s i s ge nta mi cin r r r r r r r r r r r r r r r r i r r r r r r r s s i r r na lid ixic ac id r r r r r r r r r r r r r r r r r r r r r r r r r r r r r rifa mp icin r r r i r r s r i r i s s i s s s s r r s s s s s s s s r table 5 the percentage of the antibiotic resistance of 29 lactobacillus strains. antibiotics quantity percentage of resistant strains of resistance (%) vancomycin 26 89.7 penicillin g 11 37.9 cephalothin 6 20.7 bacitracin 29 100 ampicillin 10 34.5 multi-polymyxin b 29 100 streptomycin 27 93.1 kanamycin 29 100 tetracycline 3 10.3 chloramphenicol 3 10.3 gentamicin 25 86.2 nalidixic acid 29 100 rifampicin 10 34.5 fig. 1 the plasmids in lactobacillus (1.cicc 23180, 2.cicc 22161, 3.cicc 22175, 4.cicc 22157, 5.cicc 23124, 6.cicc 22154. m. λhindiii marker). 22154 showed only one plasmid dna band of about 10 kb. lactic acid bacteria generally contain plasmids. the plasmid size was usually 1.9 kb-84.8 kb. most of the plasmid was less than 20 kb (wang and lee, 1997). in the culture process from generation to generation, many plasmids might disappear from the bacterial cell, but most of the plasmids were stable. in the study, the plasmids of the above six strains and the antibiotic susceptivity showed no changes after cultivated 30 generations. 3.3 gastrointestinal tolerability resistance to gastrointestinal stress is very important for one strain to play the potential probiotic function (guglielmotti et al., 2007). if the strains have a high tolerance to gastrointestinal stress, it will have the chance to  survive and play the probiotic effects in the gastrointestinal environment. the tolerance of the selected six strains to low ital. j. food sci., vol. 27 2015 287 ph, bile salt, pepsin and trypsin is presented in fig. 2. as shown in fig. 2a, the viable counts of l. pentosus cicc 22161 strain reduced to below 106 cfu/ml after 4 h and 1.32 × 104 cfu/ ml  after  6 h. however, the viable numbers of l. helveticus cicc 22154,  l. pentosus cicc 22157,  l. plantarum cicc 23124,  23180 and l. rhamnosus cicc 22175 were still more than 106 cfu/ml after 6 h in the gastric acid of ph 2.5. thus, these five strains showed higher tolerance in acid environment.  for bile tolerance, except the l. pentosus cicc 22161, the viable counts of the other five strains were still more than 106 cfu/ml after 6 h in the medium containing bile salt (fig. 2b). however, the viable cells of l. pentosus cicc 22161 had decreased to 2.0×106 cfu/ml within 3 h. and it declined to only 1.8 × 104 after 6 h. for pepsin tolerance, among of six strains, the viable cells of l. pentosus cicc 22161 and l. helveticus cicc 22154 decreased significantly in 6 h and it is less than 104 cfu/ml and 106 cfu/ml after 6 h exposure to 5 mg/ml pepsin solution (ph 2.5), respectively (fig. 2c). for trypsin tolerance, as can be seen in fig. 2d, the viable counts of the tested six strains still remained at 106 cfu/ml or more after 6 h exposure to 10 mg/ml trypsin solution (ph 8.0). 3.4 detection of resistance genes according to the above results, except strain l. pentosus cicc 22161 and l. helveticus cicc 22154, the tested strains may be able to survive in the simulated gastrointestinal environment. however, if the above strains contain antibiotics-resistant plasmids, there is the possibility of resistance transfer to other bacteria, especially pathogenic bacteria. it will be a potential hazard to human health and be a serious social problem. so, the plasmid-determined resistant gene should be checked firstly before subsequent utilization. after 0.02% sds combined with heat treatment of the four strains (cicc 22175, 22157, 23124, 23180), only the plasmids of l. plantarum cicc 23180 were removed and the resistance to cephalothin and chloromycetin disappeared simultaneously (unpublished results). so, the primers of β-lactam resistance-relatfig. 2 the viable counts of strains ccicc 22154, 22175, 22161, 22157, 23180 and 23124 in the gastrointestinal environment after 6 hs at 37°c. a: ph 2.5; b: 3 mg/ml bile; c: 5mg/ml pepsin; d: 10 mg/ml trypsin. 288 ital. j. food sci., vol. 27 2015 ed genes including blr, ecp-1569 and nps-1 as well as chloromycetin resistance-related genes including cmla, cat and cmla1 were designed. the plasmid-determined resistant genes of l. plantarum cicc 23180 were detected by pcr. as shown in fig. 3, the plasmid of l. plantarum cicc 23180 contained β lactam resistance gene blr, excluding other resistant genes. blr gene encodes beta-lactamase, which can hydrolyze β-lactam ring and then make the β-lactam antibiotic inactivation. this is probably the main reason of the bacteria resistant to β-lactam antibiotics. in the present study, the successful amplification of blr gene in l. plantarum cicc 23180 indicated that its cefalotin fig. 3 the pcr result in the genome and plasmid of cicc 23180. m. marker; 1. blr; 2. ecp-1569; 3. nps; 4. cmla; 5. cat; 6. cmla1; 7.control. uct types. and studies have shown that more genes associated with antibiotic resistance are located in plasmids and transposons (doucet et al., 1992; mayya et al., 2011). but unlike the chromosome dna, both plasmids and transposons can provide the possibility of transferability for resistance genes between bacteria. pier et al. (2003) proved the high transferability of plasmid pcf10 that encodes tetracycline resistance from enterococcus faecalis og1rf to enterococcus faecalis bf3098c during cheese and sausage fermentation. joanna et al. (2008) reported the transferability of erythromycin resistant plasmid (pamβ1) from lactococcus lactis sh4174 to lactococcus lactis bu2-60. a similar study also indicated that the transferability of tetracycline resistance in e. italicus lmg 22195 from fermented milk (miriam et al., 2010). so, the assessment of antibiotic resistant of potentially probiotic lactic acid bacteria used in food industry, especially the resistance-related genes and the transferability are very necessary. we can also say that,  exploring the probiotic property and safety of  lactic acid bacteria are equally important. 4. conclusions the tested 29 strains of potential probiotic lactobacillus showed different resistance to antibiotics.  those  resistant strains containing both plasmids and high tolerance to gastrointestinal condition may cause food safety problems. so these strains need to be re-assessed carefully. the study found that the plasmid of l. plantarum cicc 23180 exactly carried the cephalothin-related gene blr. however, the transferibility of the resistance-related gene remains to be further studied. this study provides a reference in investigating the relationships between antibiotic resistance spectrum and the plasmids and evaluating the safety of probiotics. acknowledgements this work was supported by the science and technology research youth fund project (2010240) and the natural science foundation of hebei province (c2013208161, c2010000863). authors also thank national high-tech project (“863 plan”, no. 2011aa100902) from chinese ministry of science and technology for part fund. references charteris w.p., kelly p.m., morelli l., collins j.k. 1998a. antibiotic susceptibility of potentially probiotic lactobacillus species. journal of food protection. 61: 1636. charteris w.p., nelly p.m., morelli l., collins j.k. 1998b. development of an in vitro methodology to determine the transit tolerance of potentially probiotic lactobacillus and bifidobacterium species in the upper human gastrointestinal tract. journal of applied bacteriology. 84: 759. doucet p.f., trieu c.p., andremont a., courvalin p. 1992. resistance may be due to the effect of the betalactamase to β-lactam antibiotics. while in the study, the genes of cat, cmla and cmla1 were not detected in the plasmids of l. plantarum cicc 23180. however, l. plantarum cicc 23180 strain was resistant to chloramphenicol. at the same time, plasmid elimination and escherichia coli transformant test showed that chloramphenicol resistance-related genes should be present in plasmid dna of l. plantarum cicc 23180 (unpublished results). therefore, the plasmid of the cicc 23180 strain may contain other genes encoding chloramphenicol resistance. in recent years, more studies have been done on antibiotic resistance of probiotics. it was shown that the antibiotic resistance was variable, species-dependent and related to the prodital. j. food sci., vol. 27 2015 289 conjugal transfer of plasmid dna from enterococcus faecalis to escherichia coli in digestive tracts of gnotobiotic mice. antimicrobial agents and chemotherapy. 36(2): 502. franz c.m.a.p., hummel a.p., holzapfel w.h. 2005. problems related to the safety assessment of lactic acid bacteria starter cultures and probiotics. mitteilungen aus lebensmitteluntersuchung und hygiene. 96: 39. guglielmotti d.m., marco´ m.b., golowczyb m., treinherimer j.a., quiberoni a.l. 2007. probiotic potential of lactobacillus delbrueckii strains and their phage resistant mutants. international dairy journal. 17: 916. hummel a., holzapfel w.h., franz c.m. 2007. characterisation and transfer of antibiotic resistance genes from enterococci isolated from food. systematic and applied microbiology. 30: 1. huys g., d’haen k.d., collard j.m., swings j. 2004. prevalence and molecular characterization of tetracycline resistance in enterococcus isolates from food. applied and environmental microbiology. 70: 1555. jin s., zhang g.l., ji d.d., zhang b.l. 2009. study on lactic acid bacteria on inhibiting mutagenic and carcinogenic substances. science and technology of food industry. 30(12): 165. joanna l., louise f., aine m., niamh t., susanne s., bodil j., hilko van der voet, sigrid r.a., declan b., henk a., karen a.k., andrea w., jacek b. 2008. a standardized conjugation protocol to asses antibiotic resistance transfer between lactococcus species. international journal of food microbiology. 127: 172. klare i.,  konstabel c., werner g.,  huys g., vankerckhoven v., kahlmeter g., hildebrandt b., sibylle müllerbertling s,. wolfgang w.w., goossens h. 2007. antimicrobial susceptibilities of lactobacillus, pediococcus and lactococcus human isolates and cultures intended for probiotic or nutritional use. journal of antimicrobiology chemotherapy. 59: 900. lee y.k., nomoto k., salminen s., gorbach s.l. 2009. selection and maintenance of probiotic microorganisms. in: lee, y.k. and salminen, s. (2nd, ed.), handbook of probiotics. john wiley & sons, new york, pp 177-188. li sh.y., li .pf., shi j.h., lei sh.ch., zhang y.y., zhang k.p. 2008. isolations of the bifidobacterium from cows and their resistance characteristics to given antibacterial drugs. dairy industry china. 1: 1. li ch., wang s., zhan h.n., zhao h.f., pei j.w., zhang b.l. 2009. roles of lactobacillus paralimentarius 412 in sourdough fermentation. food and fermentation industries. 35(5): 99. liu y.q., zhou f., zhao h.f., zhan h.n., zhang b.l. 2011. factors affecting the production of folic acid by lactic acid bacteria. china dairy industry. 39(3): 10. maria r.d., monica m., bruno b. 2007. antibiotic resistance of lactic acid bacteria and bifidobacterium spp. isolated from dairy and pharmaceutical products. international journal of food microbiology. 115: 35. mayya p., zhosephine g., sofia m. 2011. tn5045, a novel integron-containing antibiotic and chromate resistance transposon isolated from a permafrost bacterium. research in microbiology. 162: 337. miriam z., geert h., giorgio g. 2010. molecular basis and transferability of tetracycline resistance in enterococcus italicus lmg 22195 from fermented milk. international journal of food microbiology. 142: 234. niamh t., declan b., se´amus f. 2010. characterisation and transferability of antibiotic resistance genes from lactic acid bacteria isolated from irish pork and beef abattoirs. research in microbiology. 161(2): 127. pier s.c., daniela c., simona g. 2003 gene transfer of vancomycin and tetracycline resistances among enterococcus faecalis during cheese and sausage fermentations. international journal of food microbiology. 88: 315. sun x.q., zhang x.l., wang s., zhang b.l. 2009. optimized production and application of eps by lactobacillus pentosus strains in fermented milks. journal of dairy science and technology. 5: 212. threlfall e.j., ward l.r., frost j.a., willshaw g.a. 2000. the emergence and spread of antibiotic resistance in food-borne bacteria. international journal of food microbiology. 62: 1. wang t.f., lee b.h. 1997. plasmids in lactobacillus. critical reviews in biotechnology. 17(3): 227. hite d.g., zhao s., simjee s., wagner d.d., mcdermott p.f. 2002. antimicrobial resistance of foodboe pathogens. microbes and infection. 4: 405. zeng h.y., qin l.k., jiang p. 2004. development review on acquired antibiotic resistance in lactic acid bacteria from food. food science. 25(12): 189. zhang z.y., liu c., guo x.k. 2007. research progress of antibiotics resistance in lactic acid bacteria. chinese journal of microecology. 19(5): 478. zhao h.f., zhou f., qi y.q., dziugan p., bai f.l., walczak p., zhang b.l. 2013. screening of lactobacillus strains for their ability to bind benzo(a)pyrene and the mechanism of the process. food and chemical toxicology. 59: 67. paper received january 16, 2014 accepted august 25, 2014 http://med.wanfangdata.com.cn/paper/periodicalinfo.aspx?periodicalid=n2008epst0010808 http://med.wanfangdata.com.cn/paper/periodicalinfo.aspx?periodicalid=n2008epst0010808 http://jac.oxfordjournals.org/search?author1=ingo+klare&sortspec=date&submit=submit http://jac.oxfordjournals.org/search?author1=carola+konstabel&sortspec=date&submit=submit http://jac.oxfordjournals.org/search?author1=guido+werner&sortspec=date&submit=submit http://jac.oxfordjournals.org/search?author1=geert+huys&sortspec=date&submit=submit http://jac.oxfordjournals.org/search?author1=vanessa+vankerckhoven&sortspec=date&submit=submit http://jac.oxfordjournals.org/search?author1=vanessa+vankerckhoven&sortspec=date&submit=submit http://jac.oxfordjournals.org/search?author1=gunnar+kahlmeter&sortspec=date&submit=submit http://jac.oxfordjournals.org/search?author1=bianca+hildebrandt&sortspec=date&submit=submit http://jac.oxfordjournals.org/search?author1=sibylle+m%c3%bcller-bertling&sortspec=date&submit=submit http://jac.oxfordjournals.org/search?author1=sibylle+m%c3%bcller-bertling&sortspec=date&submit=submit http://jac.oxfordjournals.org/search?author1=wolfgang+witte&sortspec=date&submit=submit http://jac.oxfordjournals.org/search?author1=herman+goossens&sortspec=date&submit=submit http://www.cabdirect.org:80/search.html?q=au%3a%22lee+yuan+kun%22 http://www.cabdirect.org:80/search.html?q=au%3a%22nomoto%2c+k.%22 http://www.cabdirect.org:80/search.html?q=au%3a%22salminen%2c+s.%22 http://www.cabdirect.org:80/search.html?q=au%3a%22gorbach%2c+s.+l.%22 http://www.ncbi.nlm.nih.gov/pubmed?term=zhao h%5bauthor%5d&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed?term=zhou f%5bauthor%5d&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed?term=qi y%5bauthor%5d&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed?term=dziugan p%5bauthor%5d&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed?term=bai f%5bauthor%5d&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed?term=walczak p%5bauthor%5d&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed?term=walczak p%5bauthor%5d&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed?term=zhang b%5bauthor%5d&cauthor=true&cauthor_uid=23747815 http://www.ncbi.nlm.nih.gov/pubmed/23747815/ paper 424 ital. j. food sci., vol. 27 2015 keywords: antioxidant capacity, bioactive compounds, buckwheat groats, rhizopus oligosporus, solid-state fermentation effect of solid-state fermentation with rhizopus oligosporus on bioactive compounds and antioxidant capacity of raw and roasted buckwheat groats wronkowska małgorzata*, honke joanna and piskuła mariusz konrad department of chemistry and biodynamics of food, division of food science, institute of animal reproduction and food research of polish academy of sciences, tuwima 10, 10-748 olsztyn, poland *corresponding author: m.wronkowska@pan.olsztyn.pl abstract the effect of solid-state fermentation with rhizopus oligosporus on the changes in the total phenolic compounds, rutin, vitamin b and c, tocopherol, phytic acid and antioxidant capacity of raw and roasted buckwheat groats was studied. the roasted groats contained reduced level of studied bioactive compounds as compared to raw groats. in this study was evidenced that the solidstate fermentation with rhizopus oligosporus enhanced water soluble vitamins (thiamine, pyridoxine and l-ascorbic acid) as well as tocopherols contents. in contrast the decrease of the inositol hexaphosphate, phenolic compounds, the rutin content and antioxidant capacity determined by acl and abts methods was noticed. mailto:m.wronkowska%40pan.olsztyn.pl?subject= ital. j. food sci., vol. 27 2015 425 introduction globally, there is a growing interest in buckwheat products as healthy foods. the major producers of buckwheat are china, russia, ukraine and kazakhstan, but it is also cultivated in slovenia, poland, hungary, brazil and austria (bonafaccia et al., 2003). rutin (quercetin-3-rutinoside) is the main buckwheat flavonoid, which poses antioxidant, anti-inflammatory and anticarcinogenic properties. buckwheat is also rich in other antioxidant compounds such as phenolic acids, tocopherols, reduced glutathione, inositol phosphates and melatonin (wijngaard and arendt, 2006). this pseudocereal is characterised also in a high content of thiamine (vitamin b1) and riboflavin (vitamin b2), proteins with a well balance amino acid composition, including a high lysine content, phytosterols, soluble carbohydrates, d-chiro-inositol, fagopyritols and thiamin-binding proteins. its fatty acid composition is superior to that of cereal grains, with typically 80% unsaturated fatty acids, including more than 40% of linoleic acid, an essential polyunsaturated fatty acid (steadman et al., 2001). raw and roasted buckwheat groats are particularly popular in central and eastern europe. roasted buckwheat groats are usually served like rice after cooking, while raw buckwheat groat or flour is used as a substitute for wheat flour in products for people with allergy to gluten and can be a valuable ingredient in diets or food products for coeliac patients (wronkowska et al., 2010). roasting affects the chemical composition and functional properties of buckwheat groats. the reduction of parent antioxidants as well as the formation of maillard reaction products after roasting was observed (zielińska et al., 2007a). tempeh, or “tempe”, if we use authentic indonesian spelling, a traditional product originating from indonesia, is usually made from soybeans. the traditional tempeh process involves soaking and cooking, cooling and dehulling of the beans, followed by 20-30 hours of solid-state fermentation with rhizopus oligosporus. tempeh products have high protein contents of 4050% and can be serve as tasty protein complements to starchy staples, and can substitute meat or fish (nout and rombouts, 1990). the solid-state fermentation with rhizopus oligosporus has several beneficial effects, for example enhances antioxidant properties such as free-radical scavenging activity. handoyo et al. (2006) used fermentation with r. oligosporus to produce a fermented buckwheat flour that not only had a higher content of amino acids and minerals than non-fermented buckwheat samples, but also a lower content of allergic proteins. however, the production of tempeh-like products from buckwheat groats themselves has not been investigated. therefore, in order to promote the utilization of raw or roasted buckwheat groats as well as to create a new type of healthy food, the effects of solid-state fermentation with rhizopus oligosporus on the changes in the total phenolic compounds, rutin, vitamin b and c, tocopherol, phytic acid and antioxidant capacity of raw and roasted buckwheat groats was addressed in this study. materials and methods chemicals acetonitrile and methanol (hplc-grade) were provided by merck (darmstad, germany). rutin (quercetin-3-rutinoside), l-ascorbic acid, 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts), 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), thiamine, riboflavin, pyridoxine, takadiastase from aspergillus oryzae (ec no 232588-1) and inositol hexaphosphoric acid (dodecasodium salt) from corn were purchased from sigma (sigma chemical co., st. louis, mo, u.s.a.). other reagents of reagent-grade quality were from poch, gliwice, poland. water was purified with a milli-q-system (milipore, bedford, usa). all solutions prepared for hplc were passed through a 0.45 µm nylon filter before use. solid-state fermentation (ssf) the raw (b) and roasted common (fagopyrum esculentum moench) buckwheat groats (rb) were purchased from a local healthy food shop (olsztyn, poland). the ssf was performed according to the modified method of handoyo et al. (2006). briefly, 50 grams of raw or roasted buckwheat groats were cooked with 200 ml of deionized water for 5 min at 100ºc. after cooling to room temperature, the excess of water was discarded. the drained samples were then inoculated with rhizopus oligosporus nrrl 2710 (approx. 104 spores/g, northern regional research laboratory, peoria, illinois, usa) and incubated at 37ºc for 24 h. the fermented raw groats (ssf-b) and fermented roasted groats (ssf-rb), as well as unfermented materials, were lyophilized using a labconco co. (kansas city, missouri, usa) laboratory freeze-dryer at a pressure of 13 pa at -60°c for 24 h. sample preparation for measurement of rutin, total phenolic compounds and antioxidant capacity the lyophilized buckwheat samples were extracted in triplicate with 80% aqueous methanol (1/10; w/v) for 2 h, with shaking at 37oc (1400rpm, comfort r, eppendorf, germany). samples were then centrifuged at 2600xg at 4oc for 15 min in a beckman gs-15r centri426 ital. j. food sci., vol. 27 2015 fuge (beckman instruments, inc., palo alto, cl, usa). the samples were preserved at -20oc prior to further analysis. preparation of hydrophilic and lipophilic extracts for the measurement of antioxidant capacity by photo-induced chemiluminescence (pcl) assay for the hydrophilic extract, about 100 mg of lyophilized buckwheat samples were extracted for 3 min with 1 ml of hplc-grade water using a genie-2 type vortex (scientific industries, usa). next, samples were centrifuged for 5 min at 4°c, at 16100xg (5415 r, eppendorf, germany) and the fresh supernatants were directly used to determine the antioxidant capacity formed by water-soluble antioxidants (acw). for the lipophilic extracts, about 100 mg of lyophilized buckwheat samples were extracted for 3 min with a mixture of 200 µl of n-hexane and 800 µl of methanol using a genie-2 type vortex (scientific industries, usa). next, samples were centrifuged for 5 min at 4°c, at 16100xg (5415 r, eppendorf, germany) and the fresh supernatants were directly used to determine the antioxidant capacity formed by lipid-soluble antioxidants (acl). determination of total phenolic compounds the content of total phenolic compounds (tpc) was determined according to shahidi and naczk (1995). buckwheat extracts (0.25 ml) were mixed with 0.25 ml of folin-ciocalteu reagent (previously diluted with water, 1:1 v/v) and 0.5 ml of na 2 co 3 solution, and 4 ml of water. the mixture was allowed to stand at room temperature for 25 min and then centrifuged at 2000xg for 10 min. the absorbance of the supernatant was measured at 725 nm using a spectrophotometer (uv-160 1pc, shimadzu, japan). the data were calculated as rutin equivalents. determination of rutin content by hplc rutin content was analyzed in a hplc system (shimadzu, kyoto, japan) comprising two pumps (lc-10 ad), a uv detector (spd-10a) set at 330 nm, an autosampler set for 5 μl injection (sil-10 advp), a column oven (cto-10 asvp), and a system controller (sil-10 advp) according to method described by zielińska et al. (2010). determination of thiamine (b1) and riboflavin (b2) by hplc to determine the content of thiamine and riboflavin, the modified method of prodanov et al. (1997) was used. the 500 mg of lyophilized buckwheat were extracted by acid hydrolysis with 15 ml of 0.3 m hcl in autoclave (15 min at 120°c) and, after cooling, the ph was adjusted to 5-5.4 using 4 m sodium acetate. then 5 ml of an aqueous solution of taka-diastase enzyme (100 u/mg) was added to the samples and incubated for 3 h at 45°c, in a water bath with shaking (120rpm, hs-b20, ika labortechnik, germany). after enzymatic hydrolysis, the extracts were filtered by whatman no. 40 filters and water was added to complete the volume to 25 ml. the samples were preserved at -20oc prior to hplc analysis. determination of pyridoxine (b6) and l-ascorbic acid by hplc analysis of pyridoxine and l-ascorbic acid content was made by the modified method of esteve et al. (1997). approximately 100 mg of lyophilized buckwheat samples were added to 1 ml of 5% aqueous solution of metaphosphoric acid. the samples were extracted in triplicate, and then centrifuged at 12000xg for 10 min at 4oc (gs-15 r beckman instruments, inc., palo alto, cl, usa). the supernatant were mixed with 100 µl of dithiotreitol (dtt), incubated for 1 h (without light) and then centrifuged at 13000xg for 5 min at 4oc. the samples were preserved at -20oc prior to further hplc analysis. determination of tocopherol content tocopherol (α-t, β-t, γ-t, δ-t) was extracted with methanol (0.5 g of sample/7 ml). after evaporation, extracts were redissolved in nhexane. the hplc analysis was run with a shimadzu system (lichrospher® si-60, 5-µm particle size, 4 x 250-mm column) according to the method described by pegg and amarowicz (2009). the tocopherols contents were calculated from the peak areas using standard curves of tocopherols. determination of inositol hexaphosphates and its lower forms the analysis of the content of tri-, tetra-, penta-, and hexaphosphate inositols was made according to the method of honke et al. (1999). inositol hexaphosphate was determined as follows: exactly 0.5 g of the buckwheat samples were extracted with 20 ml 0.5 m hcl for 5 h using a bm1 magnetic stirrer (ika, staufen, germany). the extract was centrifuged at 3500xg for 40 min (centrifuge mpw-360, factory of precise mechanics, warsaw, poland) and the supernatants were decanted, frozen overnight (-20°c), thawed at room temperature and recentrifuged at 3500xg for 40 min. the supernatants (15 ml) were evaporated under reduced pressure to dryness at 40°c and dissolved in 15 ml of 0.025 m hcl. the samples were transferred to minicolumns filled with dowex ag 1-x8 resin, from which the inositol phosphates were eluted using 2 m hcl (5 x 4 ml). after removal of the solital. j. food sci., vol. 27 2015 427 vent by evaporation with an air stream, the dry residue was dissolved in a mobile phase. then the samples were analysed by hplc. the inositol hexaphosphates contents were calculated from the peak areas using standard curves of inositol hexaphosphates. measurement of the antioxidant capacity of buckwheat products against abts+• and o 2 -• the antioxidant capacities of the 80% aqueous methanol extracts from lyophilized buckwheat samples were determined against abts+• radical cation using a spectrophotometric assay. the abts+• radical cation was prepared by mixing abts+• stock solution (7 mm in water) with 2.45 mm potassium persulfate. this mixture remained for 12-24 h until the reaction was complete and the absorbance was stable. antioxidant capacity was determined following the procedure described by re et al. (1999) with a minor modification. the abts+• solution was diluted with 80% methanol to an absorbance of 0.700 ± 0.020 at 734 nm. for the photometric assay, 1.48 ml of the abts+• solution and 20 µl of the buckwheat extracts or trolox standards were mixed and measured immediately and again after 6 min at 30°c and 734 nm using a spectrophotometer (uv-160 1pc, shimadzu, kyoto, japan). appropriate solvent blanks were run in each assay. the antioxidant capacity was calculated on the basis of percentage inhibition of absorbance at 734 nm using a trolox standard curve and was expressed as μmol trolox/g of dry matter (d.m.). the photo-induced chemiluminescence assay (pcl), carried out according to the method of popov and lewin (1999), was used to measure the antioxidant capacity of unfermented and fermented groat extracts against superoxide anion radicals (o 2 −•), which were generated from luminol, a photosensitizer, under exposure to uv light. the antioxidant capacity of buckwheat water or methanol extracts were determined using acw (antioxidative capacity in water-soluble substances) and acl (antioxidative capacity in lipid-soluble substances) kits (analytik jena, leipzig, germany) reported in details by zielińska et al. (2010). antioxidant capacity was expressed in terms of μmol trolox/g d.m. statistical analysis the measurement were performed in triplicate for each of two independent fermentation batches. the data are the mean results with the standard deviation. the effects of the two parameters, type of product (p) and fermentation process (f) or their interactions (p x f) were tested using a two-way anova (statistica, ver. 7.1, usa). fisher’s least significant difference test at a significance level of p<0.05 was performed for post-hoc comparison. results and discussion total phenolic compounds (tpc) and rutin (ru) contents table 1 shows total phenolic compounds (tpc) and rutin (ru) contents of unfermented and fermented raw and roasted buckwheat groats. raw buckwheat groats (7.2 mg ru equiv/g d.m.) was almost two times richer in phenolic compounds table 1 the phenolic, vitamin b1, b2, b6, c and α-, γand δ-tocopherol contents of ssf raw and roasted buckwheat groats. b ssf-b rb ssf-rb anova p f p x f the phenolic contents rutin [µg/g d.m.] 205.84±0.94a 164.54±24.59b 158.84±3.42b 142.09±1.81b ns *** *** total phenolic compounds 7.19±0.84a 5.15±0.23b 3.54±0.02c 3.41±0.16c *** *** *** [mg ru equiv/g d.m.] content of vitamin b1, b2, b6 and c thiamine (b1) [µg/g d.m.] 9.70±0.29b 10.91±0.50a 5.41±0.13d 7.56±0.29c *** *** *** riboflavin (b2) [µg/g d.m.] 1.65±0.12a 1.70±0.08a 1.29±0.09b 1.18±0.01c *** *** *** pyridoxin (b6) [mg/g d.m.] 0.12±0.01b 0.33±0.01a 0.11±0.02b 0.12±0.01b *** *** *** l-ascorbic acid [mg/g d.m.] 0.05±0.01c 0.11±0.02a 0.05±0.01c 0.09±0.01b *** *** *** content of α-, γand δ-tocopherol α-tocopherol 0.73±0.06b 1.10±0.12a 0.27±0.17c 0.64±0.07b *** *** *** γ-tocopherol 106.20±1.97b 126.88±3.18a 60.40±0.59d 95.08±1.01c *** *** *** δ-tocopherol 2.93±0.30b 3.45±0.28a 3.78±0.32a 2.10±0.07c ns *** *** control buckwheat groats: raw (b) and roasted (rb); fermented buckwheat groats: raw (ssf-b) and roasted (ssf-rb). data expressed as mean±standard deviation (n=6). different letters within the same line indicate statistically significant differences at p<0.05 in nir fisher test (for interactions pxf). *** significant effects by kind of products (p), fermentation process (f) or their interactions (pxf) at p<0.05; ns, not significant. 428 ital. j. food sci., vol. 27 2015 than the roasted groat (3.5 mg ru equiv/g d.m.). our results confirms results of zielińska et al. (2007b). ssf led to a statistically significant decrease in the rutin content of raw groats (by 20%) but did not affect the rutin content of roasted groats. performed two-way anova analysis showed that the content of rutin was closely associated with the used ssf process. it is well known that buckwheat groats contain mainly rutin and a little amounts of isovitexin, depending on the cultivar and growth conditions (wijngaard and arendt, 2006). isovitexin was not detected in our buckwheat samples. the rutin content of raw buckwheat groats was 205.8 µg/g d.m. and 23% lower (p<0.05) for roasted groats. it is connected with the hydro-thermal processes used for roasting. dietrich-szostak and oleszek (1999) found that rutin content in buckwheat groat was affected by temperature and heating time adversely. wang et al. (2011) found the reduction of rutin content after the fermentation of peanut flour with the different strains of lactic acid bacteria. ssf led to a statistically significant decrease in tpc for raw groats (by 28%), but did not affect the tpc of ssf roasted groats. dordevic et al. (2010) showed that the fermentation of buckwheat by s. cerevisiae and l. rhamnosus caused the increasing of tpc compared to nonfermented samples. dueñas et al. (2012) found significant increase of phenolic acid content in the soybean fermented with different microorganisms (aspergillus oryzae, rhizopus oryzae and bacillus subtilis). vitamins b and c contents the contents of vitamins b1, b2, b6 and c before and after fermentation of raw and roasted buckwheat groats are presented in table 1. the raw and roasted groats used for fermentation had different thiamine and riboflavin contents, but the levels of pyridoxine and l-ascorbic acid were similar. the level of vitamin c in whole buckwheat flour ranges from 3.9 to 7.3 mg/ l00 g, our results are similar to those presented by other authors (wijngaard and arendt, 2006). the concentrations of b1 and b2 in the raw groats were 80 and 28%, higher, respectively, than those of roasted groats. this finding is in accordance with reports regarding the presence of vitamins b in buckwheat (wijngaard and arendt, 2006). the ssf of raw groats caused a statistically significant increase of thiamine, pyridoxine and l-ascorbic acid (p<0.05) compared to the control samples. the pyridoxine content increased almost three-fold, the l-ascorbic acid content more than two-fold, whereas the thiamine content by 12%. fermentation did not change the riboflavin content of raw groats. ssf of roasted groats caused the increasing of thiamine content by 40%, the l-ascorbic acid content almost doubled, the pyridoxine contents did not change and riboflavin content decreased. the increase of the contents of some b-group vitamins obtained in this study is similar to those of previous reported by other authors. the increases in thiamine content that we obtained after fermentation of the raw and roasted buckwheat groats are in contrast to results reported for ssf of other substrates. nout and rombouts (1990) found that tempeh fermentation of soya caused the increase of content of vitamins, except for thiamine for which decrease of the content was observed. fadahunsi (2009) showed the effect of rhizopus oligosporus on the vitamin content in the flour obtained from bambara nut. after the 24 h fermentation this author found the significant reduction in the thiamine content, while riboflavin, folacin, niacin and biotin content increased significantly. in traditional turkish fermented wheat-flour-yoghurt mixture (tarhana) ekinci (2005) found no significant differences in thiamine and pyridoxine contents with the increase of fermentation period. while, he observed significant increases of riboflavin, niacin, pantothenic acid, ascorbic acid and folic acid during the fermentation. tocopherol content table 1 shows the tocopherol content in raw and roasted buckwheat groats before and after fermentation. in buckwheat, γ-tocopherol is the major tocopherol homologue and the β-form is present in only trace amounts. in our study α-t, γ-t, δ-t were found in the raw and fermented buckwheat materials, whilst β-t was not detected. the raw groats had almost threeand twofold higher contents of α-t and γ-t, respectively, compared to the roasted groats. gemrot et al. (2006) found for gourd seeds the decrease of tocopherol content under the influence of roasting process. in the literature data there are no information concerning the tocopherol level in fermented buckwheat groats. the ssf of raw and roasted groats caused a statistically significant increase of the α-t and γ-t. the content of δ-tocopherol was significantly associated with the used fermentation process, but not with the type of the product. in contrast to results obtained in this study the literature data showed the decrease of tocopherols content under the influence of fermentation. for soya fermented with a. oryzae decreased of α-t content was observed by esaki et al. (1994), but they do not observed a modification of tocopherol content for soya fermented by b. subtilis or r. oligosporus. denter et al. (1998) found that in soybean tempeh the tocopherols content slightly decreased as a consequence of fermentation with 14 varieties of rhizopus studied. ital. j. food sci., vol. 27 2015 429 contents of inositol hexaphosphate and its lower forms table 2 shows the contents of inositol hexaphosphate (ip-6) and its lower forms (ip5, ip-4 and ip-3) in raw and roasted buckwheat groasts before and after fermentation. phytic acid may be classified as a prohealthy or antinutritional compound, depending on its action. it can form an iron chelate that inhibits iron-mediated oxidative reactions and limits site specific dna damage. phytic acid inhibits tumor growth by suppressing the formation of damaging hydroxide free radicals and other reactive oxygen species (vucenik and shamsuddiny, 2003). the raw groats contained 18.3 mg/g d.m. of ip-6. after roasting, a decrease by 43% was noted. this finding was in accordance with the previously reported by other authors (zieliński et al., 2006). in our study, ip-5, ip-4 or ip-3 were not found in raw groats, but they were detected after roasting. after 24 h fermentation with rhizopus oligosporus, the formation of ip-3, ip-4, and ip-5 was noted in fermented raw groats. moreover, ssf caused the significant increase of ip-3 and ip-4 in fermented roasted groats (table 2). these findings were related to the threeand two-fold decreased content of ip-6 in fermented raw and roasted groats. fermentation, steaming and extrusion cooking were identified as processes causing the degradation of ip-6 to the lower forms (zieliński et al., 2006). egounlety and aworh (2003) showed that fermentation with r. oligosporus reduced the phytic content by 30.7% in soybean, 32.6% in cowpea and 29.1% in ground bean. antioxidant capacity in this study, the antioxidant capacity of raw and roasted groats before and after fermentation was measured against the 2,2’-azinobis-(3ethylbenzothiazoline-6-sulfonate) radical cation (abts+•) and by the photo-induced chemiluminescence assay (pcl) against the superoxide anion radical (o 2 -•). the pcl method can be conducted by two different protocols, acw and acl, which allowed for measurement of antioxidant capacity of the waterand lipid-soluble components, respectively. finally, it was possible to calculate the total antoxidant capacity as a sum of acw and acl values. the results are compiled in table 3. the antioxidant capacity of raw groats against abts+• and o 2 -•, expressed as acw+acl, was 34 and 20% higher compared to the roasted groats. it should be also noted that lipophilic antioxidants (acl) were the main contributor (up to 80%) to the total antioxidant capacity of raw and roasted groats. zielińska et al. (2007a) observed decrease by 27% of abts value for buckwheat groats after using roasting process. also zhang et al. (2010) found that the scavenging activity of tartary buckwheat flour against o 2 -• was reduced by roasting. the ssf of raw and roasted groats caused statistically significant decreases (p<0.05), by 32 and 15%, respectively, of antioxidant capacity measured against abts+•. also decrease of total antioxidant capacity evaluated by pcl (acw+acl) was noticed under the influence of ssf. similar finding were presented by berghofer et al. (1998) for the fermented product obtained from faba bean, soybean and oat. in our study lipophilic antioxidants were significantly reduced after fermentation of raw and roasted groats (by 35 and 13%). on the other hand, these lipophilic antioxidants highly contributed, up to 75%, to the total antioxidant capacity of the both fermented buckwheat products. the observed decrease in antioxidant capacity of fermented raw and roasted groats could be connected with the increasing of water soluble table 2 content of inositol phosphates of ssf buckwheat groats. sample inositol phosphates [mg/g d.m.] ip-3 ip-4 ip-5 ip-6 b nd nd nd 18.33±0.24a ssf-b 0.33±0.04b 1.65±0.15b 2.01±0.18b 5.65±0.19c rb 0.29±0.01b 2.22±0.07a 3.80±0.08a 10.51±0.07b ssf-rb 0.44±0.05a 1.86±0.18b 3.62±0.09a 5.66±0.10c anova effects p *** *** *** *** f *** *** *** *** p x f *** *** *** *** control buckwheat groats: raw (b) and roasted (rb); fermented buckwheat groats: raw (ssf-b) and roasted (ssf-rb). data expressed as mean±standard deviation (n=6). different letters within the same column indicate statistically significant differences at p<0.05 in nir fisher test (for interactions pxf). *** significant effects by kind of products (p), fermentation process (f) or their interactions (pxf) at p<0.05; ns, not significant; nd, non detected. 430 ital. j. food sci., vol. 27 2015 antioxidants (e.g. vitamins b, l-ascorbic acid) and lipid-soluble antioxidant (e.g. increased tocoperols level) and decreasing of the phenolic compounds, including rutin. in our study, the total content of vitamins b was positively correlated with the antioxidant capacity of buckwheat groats before and after fermentation, when evaluated by abts test (r = 0.43) and by acw assay (r = 0.86). the level of ascorbic acid was also positively correlated with antioxidant capacity, as measured by acw assay (r = 0.74). moreover, a very high correlation was calculated between rutin contents and antioxidant capacity, as determined by the abts test (r = 0.99). the same observation was made in relation to tpc contents and values provided by abts. phenolic compounds, including rutin, could be extracted by medium used for the acl assay since a high correlation was also noted between ru and tpc vs acl (r = 0.90 and r = 0.77, respectively). in our study, no correlation existed between total tocopherols and acl values, what could indicate that this group of compounds has no impact on the formation of antioxidant capacity of non-fermented and fermented buckwheat groats, both raw and roasted. therefore, the antioxidant capacity of fermented groats clearly depended on the antioxidant activity of vitamins b, vitamin c and rutin. it is a known that vitamins b have little or no antioxidant activity (gliszczyńskaświgło, 2006). on the other hand, the antioxidant activity of rutin provided by abts and pcl acl assay (1.16±0.02 and 1.38±0.07 mmol trolox, respectively) as reported by zielińska et al. (2010), was higher than antioxidant activity of l-ascorbic acid (1.05±0.02 mmol trolox by abts and 1.0 mmol trolox in plc acw) (re et al., 1999). therefore, the noted decrease of the antioxidant capacity evaluated by abts and pcl assay in ssf raw and roasted groats was mainly related to the decreased rutin content and could not be ameliorated by increased content of both vitamins b and c. general remarks two-way anova used for statistical analysis of the obtained data indicated that both analysed parameters: type of product (raw and roasted), fermentation process (fermented and unfermented) and their interactions significantly influenced on the obtained results. only in the case of rutin and δ-tocopherol content, the type of product had not significant effect. also principal component analysis (pca) was performed on the covariance matrix of the samples with no rotation (data not showed). two principal components were extracted (pc1 and pc2) and together explained 83.66% of the total variance. the pc1 was differentiated by almost all investigated compounds except rutin, ip-5, ip6, δ-tocopherol and antioxidant capacity determined by acl and abts methods. conclusions solid-state fermentation with rhizopus oligosporus was used to obtain tempeh-type products from raw and roasted buckwheat groats. the used ssf enhanced water soluble vitamins (thiamine, pyridoxine and l-ascorbic acid), as well as and α-, δand γ-tocopherol contents. after fermentation, a decrease in total phenolic compounds as well as rutin contents was observed. these changes had an impact on abts•+ radical cation and superoxide anion radical (o 2 −•) scavenging activity of fermented raw and roasted buckwheat groats. based on the correlation studies and knowledge on the antioxidant activtable 3 the antioxidant capacity of ssf buckwheat groats provided by abts and pcl assay. sample antioxidant capacity (μmol trolox/g d.m.) against abts+• against o 2 -• acw acl acw + acl b 22.93±1.24a 3.97±0.59b 17.32±0.59a 21.29±0.11 ssf-b 15.68±0.94b 4.57±0.59a 11.18±0.59d 15.75±0.12 rb 15.15±2.34b 3.76±0.59c 13.19±0.59b 16.95±0.74 ssf-rb 12.82±0.51c 3.84±0.59c 11.50±0.59c 15.34±0.20 anova effects p *** *** *** *** f *** *** *** *** p x f *** *** *** *** control buckwheat groats: raw (b) and roasted (rb); fermented buckwheat groats: raw (ssf-b) and roasted (ssf-rb). data expressed as mean±standard deviation (n=6). different letters within the same column indicate statistically significant differences at p<0.05 in nir fisher test (for interactions pxf). *** significant effects by kind of products (p), fermentation process (f) or their interactions (pxf) at p<0.05; ns, not significant. ital. j. food sci., vol. 27 2015 431 ity of analysed bioactive components it should be noted that rutin content was the main factor responsible for the antioxidant capacity of fermented products. on the other hand, the fermented products were rich source of vitamins b and c and therefore ssf with rhizopus oligosporus can be recommended for production of tempeh-like functional buckwheat-based foods with reduced antinutritional factor. acknowledgments this research was supported by statutory found from the department of chemistry and biodynamics of food of the institute of animal reproduction of the polish academy of sciences in olsztyn, poland. the authors thank phd agnieszka ornatowska for technical help in the analysis of bioactive compounds. the authors declares that they have no conflict of interest. references berghofer e., grzeskowiak b., mundigler n., sentall w.b. and walcak j. 1998. antioxidative properties of faba bean, soybean-and oat tempeh. international journal of food science and nutrition 49: 45-54. bonafaccia g., marocchini m. and kreft i. 2003. composition and technological properties of the flour and bran from common and tartary buckwheat. food chemistry 80: 9-15. denter j., rehm h.-j. and bisping b. 1998. changes in the contents of fat-soluble vitamins and provitamins during tempe fermentation. international journal of food microbiology 45: 129-134. dietrich-szostak d. and oleszek w. 1999. effect of processing on the flavonoids content in buckwheat (fagopyrum esculentum moench) grain. journal of agriculture and food chemistry 47(10): 4384-4387. dordević t.m., šiler-marinković s.s. and dimitrijević-branković s.i. 2010. effect of fermentation on antioxidant properties of some cereals and pseudo cereals. food chemistry 119: 957-963 dueñas m., hernández t., robredo s., lamparski g., estrella i. and muñoz r. 2012. bioactive phenolic compounds of soybean (glycine max cv. merit): modifi cations by different microbiological fermentations. polish journal of food and nutrition sciences 62, 241-250. egounlety m. and aworh o.c. 2003. effect of soaking, dehulling, cooking and fermentation with rhizopus oligosporus on the oligosaccharides, trypsin inhibitor, phytic acid and tannins of soybean (glycine max merr.), cowpea (vigna unguiculata l. walp) and groundbean (macrotyloma geocarpa harms). journal of food engineering 56: 249-254. ekinci r. 2005. the effect of fermentation and drying on the water-soluble vitamin content of tarhana, a traditional turkish cereal food. food chemistry 90: 127-132. esaki h., onozaki h. and osawa t. 1994. food phytochemicals for cancer prevention. in: huang m.t., osawa t., ho c.t., rosen r.t. 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cereal chemistry 83(4): 391-401. wronkowska m., haros m. and soral-śmietana m. 2013. effect of starch substitution by buckwheat flour on glutenfree bread quality. food and bioprocess technology 6: 1820-1827. wronkowska m., zielińska d., szawara-nowak d., troszyńska a. and soral-śmietana m. 2010. antioxidative and reducing capacity, macroelements content and sensorial properties of buckwheat-enhanced gluten-free bread. international journal of food science and technology 45: 1993-2000. zhang m., chen h., li j., pei y. and liang y. 2010. antioxidant properties of tartary buckwheat extracts as affected by different thermal processing methods. lwtfood science and technology 43: 181-185. zielińska d., szawara-nowak d. and michalska, a. 2007a. antioxidant capacity of thermally-treated buckwheat. polish journal of food nutrition sciences 57: 465-470. zielińska d., szawara-nowak d. and zieliński h. 2007b. comparison of spectrophotometric and electrochemical methods for the evaluation of the antioxidant capacity of buckwheat products after hydrothermal treatment. journal of agricultural and food chemistry 55, 6124-6131. zielińska d., szawara-nowak d. and zieliński h. 2010. determination of the antioxidant activity of rutin and its contribution to the antioxidant capacity of diversified buckwheat origin material by updated analytical strategies. polish journal of food and nutrition sciences 60(4): 315-321. zieliński h., michalska a., piskuła m.k. and kozłowska h. 2006. antioxidants in thermally treated buckwheat groats. molecular nutrition and food research 50: 824-832. paper received july 16, 2014 accepted december 1,2014 http://informahealthcare.com/action/dosearch?action=runsearch&type=advanced&result=true&prevsearch=%2bauthorsfield%3a%28berghofer%2c+e.%29 http://informahealthcare.com/action/dosearch?action=runsearch&type=advanced&result=true&prevsearch=%2bauthorsfield%3a%28grzeskowiak%2c+b.%29 http://informahealthcare.com/action/dosearch?action=runsearch&type=advanced&result=true&prevsearch=%2bauthorsfield%3a%28mundigler%2c+n.%29 http://informahealthcare.com/action/dosearch?action=runsearch&type=advanced&result=true&prevsearch=%2bauthorsfield%3a%28sentall%2c+w.+b.%29 http://informahealthcare.com/action/dosearch?action=runsearch&type=advanced&result=true&prevsearch=%2bauthorsfield%3a%28sentall%2c+w.+b.%29 http://informahealthcare.com/action/dosearch?action=runsearch&type=advanced&result=true&prevsearch=%2bauthorsfield%3a%28walcak%2c+j.%29 ijfs#304_boselli_bozza   ital. j. food sci., vol 28, 2016 190 review the herbaceous character of wines m. mozzona, s. savinia, e. bosellia* and j.h. thorngateb adepartment of agricultural, food, and environmental sciences, università politecnica delle marche, via brecce bianche 60131, ancona, italy bconstellation brands, inc., 1178 galleron road, saint helena, ca 94574, usa *corresponding author. tel.: +39 0712204923 e-mail address: e.boselli@univpm.it abstract the herbaceous (vegetative) character of wine can be sensorially pleasant or not depending on different circumstances. this character can be specific to a particular grape cultivar (an endogenous varietal character) or produced during the winemaking procedure, from either an endogenous or exogenous cause. several volatile wine components containing oxygen (aldehydes and alcohols with 6 carbon atoms), nitrogen (alkylmethoxypyrazines), and selected sulfur compounds (thiols, sulfides, disulfides) are reviewed and described. the effects of micro-oxygenation and storage in wood on the herbaceous post-fermentative character are discussed. finally, the preventive tools and winemaking technologies used to reduce or to control this sensory character are described. keywords: green leaf volatiles, herbaceous, methoxypyrazines, thiols, vegetative, wine aroma   ital. j. food sci., vol 28, 2016 191 1. introduction the aroma complexity of wine is the result of several different factors (amerine and roessler, 1976; rapp, 1998). it consists of many hundred volatile compounds of various origin, which are present at concentrations ranging from fractions of ng/l up to mg/l and are able to interact with the olfactory epithelium to generate a sensory perception (robinson et al., 2014a,b). many of these odorous substances derive directly from the grapes (rapp, 1998) and their formation is affected by both the grape metabolism and the viticultural ecosystem. other impactful odorant compounds originate from the prefermentation biochemistry occurring during grape crushing and maceration. the ‘fermentative aromas’ (rapp and mandery, 1986) originate from the metabolism of the microorganisms responsible for the alcoholic and malolactic fermentations. the chemical reactions occurring during the finishing and ageing of the wines also play a crucial role in the final bouquet of wine. moreover, the effect of the addition of chemicals and adjuncts, the characteristics of the cooperage used for the processing and storage and the closure systems are also determining factors. the fascination exercised by wine on the consumer is due in large part to the complexity of these orthonasal cues and to the retronasal perceptions evoked at the first sip. due to the huge variability of odor notes, the volatiles are the components that often best define the parameters of quality and typicality of wine. moreover, some aroma components are robust markers of identity, playing an important role in the protection of local products from commercial frauds (boselli et al., 2008). a focus on ‘varietal winemaking’ is becoming increasingly important for countries characterized by an extremely variegated viticulture in terms of pedo-climatic conditions and cultivated genotypes which results in a large number of wines with specific geographical determination (e.g., over 400 in italy). occasionally, wines may express excessive sensations of herbaceous (‘vegetative’) offflavor, resulting in a reduction in fruit intensity and detrimentally impacting palate structure. consumers rarely appreciate wines with pronounced herbaceous flavors; these products are described as possessing immature character, lacking refinement and elegance, and as such are not sought after by the modern consumer. in order to reduce or avoid these olfactory qualities in wines it is of critical importance to know the origins and causes of these characters. this paper reviews the chemical compounds known to be responsible for the herbaceous character of wines and reports on the preventive tools and winemaking technologies used to minimize or control ‘herbaceousness’. the herbaceous aroma in wines is described in reference to the genesis of the molecules responsible for the olfactory perception. these components can be ascribed to three different chemical classes: alkylmethoxypyrazines (endogenous varietal and exogenous herbaceous character) aldehydes and alcohols with 6 carbon atoms (pre-fermentative herbaceous character) selected low molecular weight sulphur compounds (herbaceous character in fermentation) the contribution of any of these compounds to the final aroma depends on their concentration, their perception threshold and the extent to which they are modulated by interactions with both volatile and non-volatile components of wine.   ital. j. food sci., vol 28, 2016 192 2. herbaceous character due to methoxypyrazines 2.1. endogenous (varietal) herbaceous character the varietal herbaceous character is usually described as ‘green pepper’, or ‘tomato leaf’ and is mainly derived from a group of nitrogen-containing compounds, the alkylmethoxypyrazines, which were first discovered in bell peppers (buttery et al., 1969) and are present in green plant tissues, including grapes (bayonove et al., 1975). methoxypyrazines are used by insects and plants in chemical defense or, conversely, they may act in insects as pheromones. the alkylmethoxypyrazines (mp) are chemical compounds based on the heterocyclic aromatic structure of pyrazine, originating from the metabolism of amino acids (fig. 1). r= ch2ch3, etmp r= chch3ch2ch3, sbmp r= ch(ch3)2, ipmp r= ch2ch(ch3)2, ibmp figure 1: chemical structure of alkylmethoxypyrazines. etmp: 3-ethyl-2-methoxypyrazine; sbmp: 3-secbutyl-2-methoxypyrazine; ipmp: 3-isopropyl-2-methoxypyrazine; ibmp: 3-isobutyl-2-methoxypyrazine. the main mp found in grapes, musts and wines are 3-ethyl-2-methoxypyrazine (etmp); 3sec-butyl-2-methoxypyrazine (sbmp); 3-isopropyl-2-methoxypyrazine (ipmp); and 3isobutyl-2-methoxypyrazine (ibmp). the nature of the alkyl radical largely determines the olfactory perceptions of these compounds (sala et al., 2004) as summarized in table 1. table 1: odor descriptors and olfactory threshold in water (ng/l) of the main alkylmethoxypyrazines (mp) according to sala et al. (2004). alkylmethoxypyrazines odor description threshold etmp raw potato, earthy, green, bell pepper 400-425 sbmp green, ivy leaves, bell pepper 1-2 ipmp earthy, cooked asparagus, green pepper 2 ibmp earthy, green, bell pepper, musty 0.5-2 their aroma is generally described as ‘bell pepper’, ‘leafy’, ‘ivy leaves’, ‘vegetable’, ‘peas’, ‘asparagus’, ‘ginseng’, ‘roasted’, ‘musty’ and ‘raw potatoes’. mp represents a narrow, delineated group of extremely powerful odorants characterized by extremely low sensory perception thresholds (e.g., 1-2 ng/l in distilled water; kotseridis et al., 1999). ibmp is typically the most predominant mp in grape and wine and, as such, is a major contributor to the vegetative character (sala et al., 2004; allen n n r och3   ital. j. food sci., vol 28, 2016 193 et al., 1991; lacey et al., 1991). this vegetative character is most commonly, although not exclusively, associated with sauvignon blanc, cabernet sauvignon and other bordeaux varietals (allen et al., 1991). ipmp may also be present in certain grapes and thus found in the derived wine as a varietal character. however, it is the major exogenous contributor to the pool of methoxypyrazines because it is the most abundant and potent of the mp released by ladybugs, insects of the family coccinellidae that can be accidentally incorporated with grapes during harvest operations (botezatu et al., 2012), as reported in the next section. different pathways leading to the mp responsible for the herbaceous character of wines have been proposed. one utilizes the amino acid leucine (for ibmp) or valine (for ipmp) and an unknown 1,2-dicarbonyl compound leading to the formation of 3-alkyl-2hydroxypyrazine (hp). the final steps of these pathways involve the methylation of hp to form the associated mp, catalyzed by an o-methyltransferase enzyme (dunlevy et al., 2010; dunlevy et al., 2013). a very wide range of mp levels can be found in grapes due to changes in their concentration throughout the ripening process. although the content of both ipmp and ibmp increases during early berry development, ipmp was found to decrease before veraison, whereas ibmp decreased rapidly after veraison in cabernet sauvignon grapes (hashizume and samuta, 1999). similarly, mp levels were found to decrease progressively and rapidly with grape maturity both in cabernet sauvignon and merlot (roujou de boubee et al., 2000). in sauvignon blanc grapes, lacey et al. (1991) found the same trend, although allen et al. (1991) found an increase in ibmp during ripening. a positive linear relationship was found between the content of ibmp in grapes at different ripening stages and the derived wines from the bordeaux region (produced from merlot, cabernet franc and cabernet sauvignon grapes) (kotseridis et al., 1999). ryona et al. (2010) hypothesized that the ibmp formation during ripening was due to the methylation of 3-isobutyl-2-hydroxypyrazine (ibhp) to ibmp. therefore, ibhp is a key intermediate in both the formation and degradation of ibmp (hashizume et al., 2001; harris et al., 2012) although previous studies attributed this decrease to the photolability of ibmp (hashizume and samuta, 1999; ryona et al., 2008). further research is necessary in order to better understand the formation of these compounds and control their concentrations in wines. the excessive green bell pepper aroma found in red wines containing ibmp is generally considered unfavorable to wine quality. however, the presence of this compound at low levels is often noted to augment the quality of certain wines obtained from red varieties (e.g., cabernet franc, cabernet sauvignon, carmenere, merlot) or from white varieties (sauvignon blanc, semillon) by adding to the intrinsic flavor complexity of these varietals (allen et al., 1995; belancic and agosin, 2007). the presence of ibmp can be a positive quality factor when it is not dominant but is in balance and complemented by other herbaceous and fruity aromas (sala et al., 2004). the content of mp in the wine depends primarily on grape composition (roujou de boubee et al., 2002). viticultural factors may also influence the mp content in grapes and wines. understanding the mechanisms that influence the formation of wine volatile compounds through grape growing, winemaking and storage is important in developing strategies for the production of wines with specific sensory attributes that appeal to target markets (robinson et al., 2014b). ibmp concentrations were found to be significantly negatively correlated with the number of buds per vine (chapman et al., 2004). the levels of mp can also be controlled in the vineyard by either extending the maturation or by defoliation (sala et al., 2004). high correlations between the decrease of malic acid and the decrease of ibmp as the grapes ripened, irrespective of other cultural and climatic variables, has been reported (roujou   ital. j. food sci., vol 28, 2016 194 de boubee et al., 2000), indicating that ibmp levels could serve as markers indicative of poor ripening. in general, light-exposed clusters are reported to have reduced ibmp levels at harvest, although there is some ambiguity in the literature (ryona et al., 2008). light exposure seems to have two opposing effects on the concentration of mp in grapes: (a) by promoting the biological formation of mp in immature grapes, and (b) by reducing the mp in ripening grapes (whether by photodegradation or initiating demethylation) (hashizume and samuta, 1999; harris et al., 2012; heymann et al., 1986). the actual content of mp in grapes would result from the total sum of these two processes. light exposure has previously been shown to affect other wine constituents, such as superior alcohols, phenols and esters (d’auria et al., 2003), dimethylsulfide (marais and pool, 1980), terpenes and norisoprenoids (calo' et al., 1996). cluster exposure to sunlight will inhibit ibmp accumulation pre-veraison (ryona et al., 2008) and several studies have reported that pre-veraison cluster light exposure will result in decreased ibmp levels at harvest (dunlevy et al., 2010; ryona et al., 2008). marais and pool (1980) reported a decrease of the ibmp levels in sauvignon blanc by up to 50% in lightexposed berries across several sites in south africa studied over two years, and they showed a reduction in ibmp at both veraison and harvest. thus, the incident light conditions can affect the ibmp levels in the final wine. the effect of the light exposure has been linked to the number of leaf layers (sala et al., 2004). scheiner et al. (2010) evaluated the effects of basal leaf removal timing and severity on ibmp concentration in grape berries. the complete leaf removal significantly reduced the ibmp concentrations and consequently the herbaceous character. early leaf removal reduced the ibmp concentrations more than later removal. another factor that can affect ibmp concentrations is the vine water status. sala et al. (2005) reported that irrigation increased the ibmp content significantly; similar results have been reported by other authors (mendez-costabel et al., 2014a). rainfall during the growing season may result in higher ibmp concentrations in the grapes at harvest, due to increased and prolonged vegetative growth (roujou de boubee et al., 2000). lack of rainfall has been shown to decrease perceptual green characters in wine (mendezcostabel et al., 2014b). in summary, all of these studies demonstrate the complex relationship between viticultural practices and varietal aroma. it is difficult to predict the final wine aroma because of the multiple compounds and pathways involved. 2.2. exogenous herbaceous character lady beetles (coleoptera: coccinellidae) have been identified as a second source of high mp content in wine. this off-flavor has been termed ‘ladybug taint’ (lbt) (pickering et al., 2005). lbt is a wine defect caused by the inadvertent incorporation of ladybeetles into the wine during the winemaking process. harmonia axyridis, also known as the multicolored asian ladybeetle (malb), and coccinella septempunctata (seven spots) are the species considered responsible for causing the taint in many wine regions in europe and in the united states (botezatu et al., 2012; kogel et al., 2015; galvan et al., 2008). coccinellidae can emit a potent mix of odor-active compounds that may serve various behavioral functions, including defense, aggregation and mate-attraction (al abassi et al., 1998; cudjoe et al., 2005). the mp composition varies among species, although ipmp is the major exogenous contributor to lbt in wines. botezatu et al. (2012) reported that coccinellidae could release dimethyl-, isopropyl-, sec-butyland isobutyl-2methoxypyrazine into wine at concentrations that negatively impact product quality when the ladybeetles are incorporated with grapes during the harvest operations. pickering et al. (2005) showed that the inclusion of harmonia axyridis during fermentation of white and   ital. j. food sci., vol 28, 2016 195 red musts significantly modified the wine aroma and affected the sensory properties. white wines acquired a peanut, bell pepper and asparagus flavor, while in red wines peanut, asparagus, bell pepper, earthy and herbaceous flavors predominated. the same authors observed a correlation between the decrease of the positive fruit, floral aroma and the increase of the number of beetles added both in white and red wines. further research is needed to investigate treatments aimed at reducing the impact of these beetles and manipulating their concentration in wines for specific commercial targets. 2.3. techniques mitigating the herbaceous character due to methoxypyrazines the increase of the mp content happens primarily on the first day after destemming and crushing of the grapes, before the start of the alcoholic fermentation. it is possible that the duration of maceration may influence the levels of mp in the final wines (sala et al., 2005). various strategies have been investigated to reduce the levels of mp in the finished product. the spinning cone column is a modern, fast and efficient technology for extracting volatile components, such as ethanol to obtain low-alcohol wines, at high speed and low temperature (pickering, 2000). it was first applied by dairies in new zealand to remove grassy pyrazine flavors from whey and it has been suggested that this method could be adapted to remove mp from wine (schmitt, 2014). other strategies have focused on methods to reduce mp in finished white and red wines without altering the concentrations of other volatiles. the levels of ipmp (as well as other volatiles) were lowered by activated charcoal addition to white wine. treatment with oak chips reduced the intensity of ladybug taint in both white and red wines, while other applications generally had no effect on white wine and limited effect on red wines (pickering et al., 2006). the treatment of grape juice and must with silicone (a non-polar sorbent) prior to fermentation was found to reduce mp without altering the concentrations of the main fermentationderived compounds. therefore, it was used as a strategy for reducing all the mp without affecting the other positive volatile compounds (ryona et al., 2012). the exposure of wine to oxygen during and after bottling is a crucial interval affecting the wine aroma compounds. the closure and the packaging can effect post-bottling modifications with resultant changes in mp concentrations during aging. all types of closure reduced significantly mp concentrations in wine (silva et al., 2011). the greatest decrease in the mp content was evident in multilayer aseptic packages, due to the higher sorption capacity and gas permeability of this material (blake et al., 2009). light and storage temperature did not influence significantly the mp decomposition in wines during a 12-month ageing period (blake et al., 2010). thermovinification (or hot maceration) is a process used to increase productivity, modify the aroma profile and reduce the risk of microbial contamination. it has the potential to reduce ipmp in malb infested grapes (pickering et al., 2008). heating of coccinellidaeaffected grape must (riesling and pinot noir) prior to fermentation as a possible remedial intervention resulted in a moderate decrease of all mp (kogel et al., 2015). an alternative approach is the flash-détente technique, also reported in the literature as flash vacuum expansion. in this technique, plant materials are heated quickly (60-90°c) and then exposed to high vacuum (20-50 hpa) (brat et al., 2000). flash-détente has been also applied to crushed and destemmed grapes which were heated at 80-85°c, submitted at 20-50 hpa and then cooled down to 30°c (ageron et al., 1995; moutounet and escudier, 2000). the heating of the must followed by sudden expansion and instantaneous evaporation of water explosively ruptures the cellular tissue structure resulting in the extraction of pigments, phenols, aroma compounds and other dry matter present in the cells. initially, this technique was used to promote the release of   ital. j. food sci., vol 28, 2016 196 anthocyanins and tannin (he et al., 2012; escudier et al., 1998); however, its ability to remove deleterious volatiles was soon recognized. according to vinsonneau et al. (2006) the flash-détente technique can improve the quality of red wines aged for 9 months; the most significant decrease of the herbaceous character was observed for cabernet sauvignon and cabernet franc. it must be noted that the sequential application of a heating treatment and of high vacuum is not a selective approach and contributes to the evaporation of both positive and undesired components, including mp (jones and gnekow, 2011). for this reason, it is also possible to remove aldehydes and alcohols with 6 carbon atoms, which are pre-fermentative herbaceous aroma compounds formed during destemming (escudier, 2001; razungles, 2010; favarel, 2013) as discussed in the next section. 3. pre-fermentative herbaceous character aldehydes and alcohols with 6 carbon atoms (c6) are volatile, odorous molecules which can contribute to the herbaceous aroma in wine. they are mainly generated through the enzymatic breakdown of c18 polyunsaturated fatty acids contained in plant membranes. their concentrations in must can be in the order of several hundreds of µg/l (moreno and peinado, 2012) or even more than 13000 µg/l (ferreira et al., 2000), with very variable odour thresholds (400-8000 µg/l) (guth, 1997). their levels depend on several factors, including the grape variety and ripeness, treatments prior to fermentation, and temperature/duration of contact with the skins. therefore, they can be considered prefermentative volatile compounds. their cut-grass-like aroma is the characteristic odor of freshly damaged green leaves; therefore, these compounds are often referred as green leaf volatiles (glv) (harsch et al., 2013). the c6 alcohols frequently found in grapes include hexanol, (z)-3-hexenol and (e)-2-hexenol. as well as (e)-2-hexenol, (e)-3-hexenol may also be found in wine at levels of µg/l (gigot et al., 2010). the c6 aldehydes commonly identified in grapes are hexanal and (e)-2-hexenal; also c7 compounds such as heptanal, and (e)-2-heptenal (kalua and boss, 2008) have been found, but at lower concentration with respect to c6 aldehydes. at low concentrations (generally less than 0.5 mg/l threshold), these c6 volatiles compounds contribute positively to the overall aroma of the wine. however, at higher levels they lead to unwanted organoleptic notes such as herbaceous, green fruit, and crushed leaf and may also impart a bitter flavor (d’onofrio, 2011; ferrandino et al., 2012). these c6 compounds may be present in free volatile form or in bound form, as glycosides (cabrita et al., 2006; di stefano et al., 1998; lanati et al., 2000; sefton, 1998). the c6 alcohols were identified only as free volatile compounds in casorzo malvasia and schierano malvasia, while in the aromatic candia malvasia they were present both as free and glycosylated forms, although the former predominated (borsa et al., 2008). several studies were conducted in order to distinguish grape varieties on the basis of their c6 compounds composition. rapp et al. (1993) have shown that the content of (e)-3hexenol and its isomer (z)-3-hexenol are the most important analytical parameters to discriminate monovarietal wines of riesling, müller-thurgau, kerner, scheurebe, ehrenfelser and bacchus. also, moret et al. (1984), references these two compounds among the significant parameters discriminating venetian white wines. more recently, oliveira et al. (2006) showed that (e)-3-hexenol/(z)-3-hexenol ratio clearly discriminates loureiro wines from those of alvarinho, avesso and trajadura from portugal. moreover, 1-hexanol/(e)-3-hexenol and 1-hexanol/(z)-3-hexenol ratios may also be able to discriminate vinhos verdes monovarietal wines (north of portugal).   ital. j. food sci., vol 28, 2016 197 aldehydes are the major c6 compounds in ripe grapes of riesling, while in cabernet sauvignon c6 alcohols prevail (kalua and boss, 2010). analyzing the evolution of c6 volatile compounds during berry development of cabernet sauvignon, it was found that aldehydes increased significantly during the middle development stage but then decreased throughout the late development stage. the alcohols were present throughout the growth of berry, but reached significant levels only towards the end of ripening (kalua and boss, 2010). the greater increase of the alcohols over the aldehydes across the course of ripening is a positive aspect because alcohols have a higher perception threshold than the aldehydes, so the resulting herbaceous character is less pronounced (d’onofrio, 2011). the c6 aldehydes and alcohols derive from the oxidation of grape polyunsaturated fatty acids such as oleic acid (c18:1ω9), linoleic acid (c18:2ω6) and linolenic acid (c18:3ω3) initiated by the lipoxygenase pathway when the berries are crushed. four enzymatic activities are sequentially involved in this pathway. first, an acyl-hydrolase frees the fatty acids with 18 carbon atoms from membrane lipids. next, a lipoxygenase catalyzes the fixation of oxygen. the peroxides obtained are then split into c6 aldehydes (drawert et al., 1966). some of these may be reduced to their corresponding alcohols by an alcohol dehydrogenase in the grape (crouzet, 1986). the c6 compounds increase from veraison to harvest, with the greatest concentration expressed at harvest (kalua and boss, 2010; yang et al., 2009). for the neutral cultivar nebbiolo grown in piedmont (italy), the pre-fermentative volatile compounds present in highest concentration were hexanal and e-2-hexenal (ferrandino et al., 2012). the greater concentration of e-2-hexenal compared to z-3-hexenal could be explained by an increased activity of 3z,2e-enal-isomerase, which catalyzes the reaction from z-3-hexenal to e-2-hexenal (kalua and boss, 2010). in addition, the low concentration of these two aldehydes in the pre-veraison berries could be explained by an increased activity of the alcohol dehydrogenase that converts these aldehydes to the corresponding alcohols. during ripening, the concentration of aldehydes increases. therefore, some c6 compounds that contribute to the grape aroma may be produced pre-veraison, rather than post-veraison. the evolution of (e)-2-hexen-1-ol and (e)-2-hexenal to form thiols with tropical scents, such as 3-mercaptohexan-1-ol (3mh) and 3-mercaptohexyl acetate (3mha), has been recently confirmed during fermentation (harsch et al., 2013). this pathway will further reduce the perceptual herbaceousness, both by reducing the c6 pool as well as by formation of the ‘fruity’ thiols which will serve to mask herbaceousness. 3.1. avoiding the pre-fermentative herbaceous character: from the harvest to the winemaking process the overall perceptual herbaceous character is related to the ripening stage of the grapes, but can arise in the must due to forceful mechanical action on the clusters. therefore, it is important to consider the level of ripeness, the type of harvest, the transport, the crushing, the time and temperature of maceration, the type and the degree of pressing, the type of pumps, the degree of oxygenation and the use of enological adjuvants with regards to herbaceous expression. high temperature pretreatment of must immediately following crushing (“hot break”) was shown to result in fiveto six-fold higher concentrations of (e)2-hexenal in concord grape juice as compared to conventional hot press, especially with immature fruit, resulting in persistence of green aromas in the juice. in contrast, depectinization, pressing, and pasteurization decreased the content of (e)-2-hexenal of the juice of concord grapes due to the reduction to (e)-2-hexenol (iyer et al., 2010).   ital. j. food sci., vol 28, 2016 198 3.2.1. the harvest harvest timing is an important factor in minimizing/preventing the formation of herbaceous compounds. additionally, accumulation of the c6 alcohols and aldehydes derived from the enzymatic oxidation of linoleic and linolenic acid can be reduced by limiting mechanical operations starting with the harvest. mechanical harvesting increases herbaceous character due to uncontrolled enzymatic oxidation (nardin et al., 2006); consequently, those grapes must be pressed quickly. in contrast, manual harvesting reduces the mechanical stress on the clusters with concomitant lower losses plus lower contamination with leaves, earth, water and other foreign substances. while mechanical harvesting certainly speeds up the overall process of winemaking, it comes at the expense of the mechanical stresses on the cluster and the acceleration of the oxidation reactions. 3.2.2. destemming and crushing destemming (separating and removing the stalks from the grape berries) and crushing (breaking the grape in order to release the juice and some pulp material) are the next mechanical processes involved in winemaking. the influence of crushing on the herbaceous aroma in must and in the corresponding wine is well established (ribereaugayon et al., 2005; hashizume and samuta, 1997). usually, destemming and crushing are performed by a single machine, a crusher-destemmer or destemmer-crusher, so-named in relation to the succession of the two processing stages. the destemmercrusher first removes the stalks and then breaks open the grapes, thus avoiding the contact of the expressed juice with the stems. in contrast, in the crusher-destemmer undesired flavors can potentially be extracted from the stalks, leading to an increase of herbaceous aromas and bitter tastes. furthermore, the presence of stalks reduces the intensity of the red wine color due to the absorption of anthocyanins (nardin et al., 2006). it is important to use equipment that reduces mechanical stresses, such as excessive pressure, abrasions and lacerations, in order to reduce the enzymatic oxidation activity on fatty acids. in this way, the polyphenol and potassium extraction from the stalks is also minimized. the use of inert gases or vacuum systems in concert with the crushing and pressing of the clusters is a very popular approach to remove oxygen and slow down the oxidation of the must and increase the formation of volatile ‘fruity’ esters and glutathione (boselli et al., 2010; di lecce et al., 2013). in the case of the wines produced by carbonic maceration, crushing is completely skipped and the tank is filled with whole clusters by means of a conveyor belt that minimizes the damage to the plant tissues. the herbaceous and bitter flavors may arise in some varieties as the stalks are not separated and removed in this process. although wines produced by using carbonic maceration can be obtained by grapes harvested prematurely, the aromatic profiles of such wines do not exhibit herbaceous characters but instead show pleasant fruity notes (etaio et al., 2011). this phenomenon is explained by the limited production of c6 aldehydes and alcohols due to both the lack of oxygen in the tank and the lack of crushing of the grapes. 3.2.3 pre-fermentative maceration the enzyme that catalyzes the peroxides scission to aldehydes is associated with the membranes, consequently the aldehyde levels are proportional to the intensity of maceration of these solid parts. to limit their concentration in red wines, it is important to control the maceration temperature, not to employ long pre-fermentative maceration and to continually monitor the maceration stage.   ital. j. food sci., vol 28, 2016 199 4. herbaceous character in fermentation sulfur is present in many organic compounds within the grape such as amino acids, peptides, proteins, vitamins and co-enzymes and may also result from sulfur spraying (kwasniewski et al., 2014). volatile sulfur compounds (vsc) may then originate from yeast metabolism during the alcoholic fermentation. these molecules are characterized by a strong aromatic impact at low concentrations due to very low odor thresholds (mestres et al., 2000). vsc can be distinguished based on their functional groups (thiols, sulfides, disulfides etc.). however, there is another classification based upon the boiling point (b.p.) of 3methylthiopropanol (b.p. 90°c): light sulfur compounds show a b.p. lower than 90°c, whereas heavy sulfur compounds have a b.p. higher than 90°c (mestres et al., 2002). the light sulfur compounds such as hydrogen sulfide, ethanethiol and methanethiol arise from the degradation of the sulfur-carbon bond of methionine or cysteine derivatives (landaud et al., 2008). they usually have an unpleasant effect on the quality of the wine, are characterized by off-flavors of ‘rotten egg’, ‘sewer’, ‘cooked vegetable’ or ‘cooked cabbage’, ‘onion’, ‘garlic’ or ‘rubber’ and show high volatility and low perception thresholds (µg/l) (mestres et al., 2000; goniak and noble, 1987). dimethyl sulfide at low concentration is considered less undesirable since it shows aromas of ‘cooked corn’ and ‘asparagus’ (bell and henschke, 2005). hydrogen sulfide, ethanethiol and methanethiol are present in reduced wines and, due to their low boiling points, can be volatilized by racking and aeration (moreira et al., 2002). in table 2, the sensory properties of light sulfur compounds are listed according to different authors and their boiling point is reported (ribereau-gayon et al., 2005; landaud et al., 2008; benkwitz et al., 2012). for a review of the lower molecular weight sulfur compounds see mestres et al. (2000). table 2: sensory properties and boiling point of light sulfur compounds (c0-c2) according to ribereaugayon et al., 2005; landaud et al., 2008; benkwitz et al., 2012. light sulfur compounds chemical formula flavor note perception threshold (µg/kg) boiling point (°c) probable precursor hydrogen sulfide h2s rotten egg 10-80 -61 cysteine, sulfate, sulfite methanethiol ch3sh cooked cabbage, onion 0.3 6 methionine ethanethiol ch3ch2sh onion, rubber 1.1 35 unknown dimethyl sulfide ch3sch3 cooked corn, asparagus 25 35 cysteine other volatile sulfur compounds include the higher molecular weight grape-derived thiols, such as 3mh, 3mha, 4-mercapto-4-methyl-pentan-2-one (4mmp), and 4-mercapto4-methyl-pentan-2-ol (4mmpoh), all of which contribute positively to the varietal aroma of wine and are thus referred to as varietal sulfur compounds (bell and henschke, 2005). the interaction of methoxypyrazines with varietal thiols is still unclear (wyngaard et al., 2014). campo et al. (2005) showed that the fruity character is related to the presence of   ital. j. food sci., vol 28, 2016 200 varietal thiols (3mha) and is negatively affected by the presence of methoxypyrazine. the vegetative aroma due to ibmp, such as ‘bell pepper’ has been shown to be slightly masked by the fruity aroma, as berry flavorings were added to a neutral red wine containing added bell pepper (hein et al., 2009). as mentioned earlier, the volatile thiols also have an important sensory role due to their biosynthesisis metabolizing c6 compounds (c6-thiol precursors); roland et al. (2010) demonstrated that (e)-2-hexen-1-ol played a key role in the formation of the glutathionylated conjugate 3-s-glutathionylhexan-1-ol (g3mh). this conversion diminishes the perceptual herbaceousness by reducing the c6 pool while concomitantly increasing the masking pool. harsch et al. (2013) found that the addition of sodium hydrosulfide (nash) to grape juice greatly increased the formation of 3mh and 3mha from (e)-2-hexen-1-ol and (e)-2-hexenal precursors during the fermentation of sauvignon blanc musts under commercial winemaking conditions. while these two precursors exhibit a thiol-forming potential during fermentation, (e)-2-hexenal and (e)-2-hexen-1-ol are quickly catabolized in the very early stages following yeast addition, whereas the synthesis of h2s (the thiol source) by yeasts begins after the conversion of sugars. this timing problem may be responsible for the low content of 3mh and 3mha in finished sauvignon blanc wines in spite of the high potential for their formation. 4.1. oxidation of volatile thiols oxygen plays an important role in controlling the sensory defects associated with sulfur compounds. micro-oxygenation can reduce undesired sulfur compounds, such as methanethiol and ethanethiol, to levels below their odor threshold, with no significant increase in dimethyl disulfide (gomez-plaza et al., 2011). this may be explained by the role of quinones as oxygen oxidizes trans-caftaric acid and other phenolic compounds and leads to the formation of the corresponding o-quinones (vidal and aagaard, 2008; waterhouse and laurie, 2006). furthermore, glutathione plays an important role during the oxidation of white must, forming the 2-s-glutathionyl-caftaric acid or grape reaction product (grp) which is very common in wines (boselli et al., 2006). this derivative is not oxidized by polyphenol oxidase and does not modify the color of the must (du toit et al., 2007; roland et al., 2010). pre-fermentative operations could provide a positive contribution to the aroma profile of white wine, such as sauvignon blanc, by increasing the g3mh precursor. at this stage of winemaking, the cysteinylated and glutathionylated precursors of varietal thiols are not oxidizable because of the chemical stability of the thioether bond under oxidative conditions (roland et al., 2011). varietal thiols, however, are highly reactive compounds and their concentrations can decrease during storage (herbst-johnstone et al., 2011). oxygen and iron can oxidize these compounds into their disulfide forms. in addition, they can participate in chemical reactions with the products of phenolic oxidation (o-quinones) catalyzed by transition metals (e.g., iron and copper) (herbst-johnstone et al., 2011; nikolantonaki et al., 2010; nikolantonaki and waterhouse, 2012). 5. control of the herbaceous character after the alcoholic fermentation the aging stage of a wine is a delicate technological phase linked to a variety of chemicalphysical modifications that help define the final organoleptic characteristics. microoxygenation is a technique that may be used during this stage, employing a controlled   ital. j. food sci., vol 28, 2016 201 flow of oxygen in tanks to simulate the complex reactions that occur during wine maturation in barrel (celotti and zucchetto, 2003). this treatment results in many changes to the organoleptic qualities of the wine; these changes have been empirically separated into two main phases (moutounet et al., 2001): structuring: this takes place from the end of the alcoholic fermentation to the beginning of malolactic fermentation (mlf). in this phase, oxygen is consumed by polymerization reactions, which increase the aggressiveness and intensity of tannins on the palate. at the same time a decline in the aromatic intensity and complexity of the wine is observed. the changes in the chemical composition of wine caused by mlf are complex and often involve an enhancement of the fruity aroma and ‘dairy’ note and a decrease of the herbaceous character (liu, 2002). harmonization: in this phase the intensity and the aromatic complexity increases, whereas the herbaceous character decreases. furthermore, there is an increase in the color intensity, especially the purple hue. it is important to end the micro-oxygenation at the second stage in order to prevent excessive oxidation of the wine components leading to off-flavors. it is critical to prevent violent oxygenation during either stage to avoid generalized, uncontrolled oxidation. the sensory changes occurring in red wines exposed to different micro-oxygenation regimens and with different woods have been described (celotti and zucchetto, 2003; moutounet et al., 2001). it was emphasized how the controlled application of oxygen, as well as the wood contact in barrels, may decrease the astringency and the herbaceous characters of wine. barrel-ageing of wines is costly and laborious and is thus not suitable for all wines and wine styles. micro-oxygenation is an affordable alternative for accelerating the stabilization of color and phenolic compounds and for improving the sensory quality of red wines (llaudy et al., 2006; parish et al., 2000). oxygen addition led to an increase of the fruity and floral notes, whereas the herbaceous character significantly decreased also in cencibel red wines (cejudo-bastante et al., 2011). however, hernandez-orte et al. (2009) reported a strong dependence of the effects of microxygenation on the grape variety (cabernet sauvignon or tempranillo), the use of barrels or steels vats and the period of maturation of the wines. sensory analysis was used to follow important perceptual sensations, such as astringency and herbaceousness, which are modified by controlled oxygenation. the herbaceous character showed a fairly rapid decrease during initial micro-oxygenation; however, for longer treatment times there was a general increase in the herbaceous note. the best results were obtained with up to 2 months of treatment of micro-oxygenation without the addition of tannin. the same studies (celotti and zucchetto, 2003; moutounet et al., 2001) highlighted the difficulty in accurately determining the oxygen dosage and the appropriate treatment times. however, it was demonstrated that the application of sensory analysis in concert with careful analytical control of the process allowed the optimal time for terminating the treatment to be decided. 6. conclusions the herbaceous character of wines is of important concern to the wine scientist, to the winemaker and finally to the wine consumer. different classes of compounds with very different chemical structures can contribute to this particular sensory feature of wines. alkylmethoxypyrazines, for instance, are key contributors to vegetativeness in sauvignon blanc, as are some selected volatile thiols. the herbaceous character can be specific to a particular grape cultivar (an endogenous varietal character) or produced during the winemaking procedure, from either an endogenous or an exogenous cause. the positive or   ital. j. food sci., vol 28, 2016 202 negative (off-flavor) attributes associated with the herbaceous character depend not only on the chemical composition (qualitatively and quantitatively) of the headspace of wine and/or the wine itself, but also on the odor activity values of the different volatile components as determined 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technology, universitas padjadjaran, indonesia 2department of food industrial technology, universitas padjadjaran, indonesia *corresponding author: bambang nurhadi, faculty of agricultural industrial technology, universitas padjadjaran, indonesia. email: bambang.nurhadi@unpad.ac.id received: 11 august 2021; accepted: 21 december 2021; published: 8 february 2022 © 2022 codon publications open access paper abstract virgin coconut oil (vco) has many health benefits; however, drinking of vco directly is still uncommon. in order to overcome this problem, microencapsulation can be one of the solutions. unfortunately, emulsion is an unstable system and rapidly separates into two layers. therefore, in this study, we carried out the explanatory research of microencapsulation process with descriptive analysis. it comprised two emulsion treatments, using homogenization method, and three drying techniques, to determine the effect of pickering emulsion with microcrystalline cellulose (mcc) and different drying techniques on the characteristics of vco powder (before drying: creaming index and emulsion droplet size; and after drying: drying yield, color intensity, moisture content, particle morphology, microencapsulation efficiency, peroxide value, rehydration particle size, and dissolving time). the results demonstrated that all emulsion treatments did not depict any emulsion instability up to 21 days of storage, and the obtained vco powders had different characteristics. the highest microencapsulation efficiency was 33.49 ± 1.59%, obtained from the emulsion using tween 80 and mcc by spray drying, and the lowest peroxide value was 0.464 ± 0.084 meq o2/kg, obtained from the emulsion using tween 80 and mcc by vacuum drying. the future application of this study is expected to produce vco powder that can improve the ease handling of vco and also commercialize for being used as a non-dairy creamer. keywords: drying techniques, microcrystalline cellulose, microencapsulation, pickering emulsion, virgin coconut oil introduction virgin coconut oil (vco) is well known for being rich in ingredients that are suitable as functional foods. vco contains about 90% saturated fatty acids and about 10% unsaturated fatty acids (nurhasanah et al., 2019). there are lots of medium chain triglycerides (mcts) in these saturated fatty acids consisting of caprylic acid (c8:0), capric acid (c10:0) and lauric acid (c12:0). these mcts are ‘unique’ due to their physicochemical properties that have shorter chains (6–12 hydrocarbons) and smaller molecular weight compared to long-chain triglycerides (lct) (13–21 hydrocarbons), making them absorbed and hydrolyzed faster in the body, resulting in rapidly oxidized and producing higher energy (nurhasanah et  al., 2019; parrish, 2017). vco, compared to copra oil, increases high-density lipoprotein (hdl) and lowers low-density lipoprotein (ldl) significantly, and does not cause coronary heart disease because of the absence of trans fatty acids. besides the above-mentioned major components, there are also tocopherols, β-carotenes and other phenolic compounds, which are the minor components found in almost all types of vegetable oils, including vco that has better antioxidant capacity than bleached, refined and deodorized coconut oil. thus, vco lowers cholesterol level in the human body, increases 68 italian journal of food science, 2022; 34 (1) nurhadi b et al. been used often in food processing compared to cellulose nanocrystals (cnc) because cnc is more expensive and both of them could stabilize the emulsion system. the emulsion formation was carried out in two treatments using homogenization method. mcc was added directly to the coarse emulsion because it stabilizes the emulsion system without the help of surfactants (costa et al., 2019). besides mcc, tween 80 with the hydrophile–lipophile balance (hlb) value of 15 was also used as an additional surfactant to mcc because the combination of these emulsifiers could increase the stability of emulsion for a longer period compared to using the surfactant alone (hu et al., 2015). maltodextrin was used as a glass maker in this study. maltodextrin has rapid dispersion, high water solubility and low viscosity, strong flavor-binding ability, and is able to form an amorphous glass matrix network to protect the core material so as to facilitate drying process and maintain vco stability against oxygen (klinjapo and krasaekoopt, 2018; muhamad et al., 2018). however, since maltodextrin exhibits poor emulsifying capacity, it is usually combined with other ingredients to maintain emulsion stability (klinjapo and krasaekoopt, 2018). drying process of emulsion commenced after the emulsion system was formed. spray drying is one of the drying techniques that is widely used in the food industry (rajabi et al., 2015). this technique uses continuous high temperature and pressure to produce the final product in the form of a dry powder with a very short contact time between the hot air and the product; therefore, it is safe to dry the vco that contains bioactive components (anandharamakrishnan and ishwarya, 2015; rajabi et al., 2015). apart from spray drying, freeze drying and vacuum drying were also used in this study. freeze drying uses the principle of freezing at low temperature and ice crystal sublimation so that bioactive components of vco are not damaged (liu et al., 2008). vacuum drying uses a low-pressure drying technique (usually lower than the atmosphere pressure), in which the heat required for the drying process is lower and the drying process is faster than a typical dryer so that the bioactive components present in vco can be maintained (bazyma and kutovoy, 2005; parikh, 2015). these three drying techniques have different drying principles and have their own advantages and do not cause any damage to the bioactive components of vco. hence, these three drying techniques were selected for the drying process used to encapsulate vco. thus, in this study pickering emulsion mechanism was studied to determine the effect of pickering emulsion with mcc and different drying techniques on the characteristics of vco powder (measurement before drying: creaming index and emulsion droplet size, and measurement after drying: drying yield, color intensity, particle morphology, moisture content, microencapsulation efficiency, peroxide value, rehydration particle size, endurance, accelerates recovery rate, activates anti-aging hormones, and prevents cancer, obesity, dementia, heart attack and other diseases associated with premature aging (hee et al., 2017). these are the reasons for using vco for emulsion. unfortunately, the limited use of vco in food products is due to its liquid form, which complicates the transportation and storage process. it solidifies at room temperature with ease under its melting point (patil et al., 2016). in addition, only a few people consume vco directly because of people’s perception that it is difficult to accept oil consumption (hee et  al., 2015). therefore, it is necessary to process vco into a more convenient form for general consumption and its wide application in food products while protecting its bioactive components. microencapsulation is a method used to solve problems by giving a new perception to the public. microencapsulation masks the oily taste in the mouth so that it does not leave the unpleasant taste in throat, extends the shelf life to reduce loss during distribution and storage, leads to easy handling, and increases the solubility and bioavailability of bioactive components (nedovic et al., 2011). the process of microencapsulation consists of two stages, which are mixing the core material with the coating material to form an emulsion, followed by drying the formed emulsion (amaral et al., 2019). in this study, mechanism of pickering emulsion was used for emulsion formation. pickering emulsion has many advantages, especially it being surfactant-free, having high stability against coalescence, and ostwald ripening (díaz et al., 2020). high stability against coalescence is a major benefit of stabilization using solid particles, with solid particles partially wetted by each of the liquid phases, that is oil and water phases (partial dual wettability) so that the particles are absorbed at the oil and water interface to form a mechanical barrier, which reduces the tendency of flocculation (xie et al., 2019). pickering emulsion also has advantages in terms of its use in smaller quantities, efficiency, easy of using, good oil retention in dry powder, difficult to extract the surface oil, and not susceptible to environmental changes (bai et al., 2018). that is why, in recent times pickering emulsion has been widely developed and applied to products, especially to dry products. microcrystalline cellulose (mcc) is the solid particle used for pickering emulsion in this study. mcc has the advantages of having natural ingredients, biocompatibility and biodegradability (alavi, 2019), and has important functions in preventing emulsion instability (costa et al., 2019). also, mcc in the emulsion system has the advantage of being able to withstand to high temperatures so that the emulsion system is more stable when exposed to heat during the drying process. in addition, mcc has italian journal of food science, 2022; 34 (1) 69 characteristics of virgin coconut oil emulsion powder was obtained with this concentration based on the studies conducted by (gomes et al. (2018) and vicente et al. (2018). vco at 40% of total solid (w/w) was added gradually to the mixture while stirring. according to conducted studies, the best microencapsulation efficiency is obtained when the concentration of oil is 30% of total solid (w/w). the higher the concentration of oil, the lower the microencapsulation efficiency obtained (alcântara et al., 2019). in this study, vco at 40% of the total solid (w/w) was used with the help of pickering emulsion as the novelty of the study (high oil concentration) because pickering emulsion can increase emulsion stability for a longer period. the emulsion formed was then homogenized using a rotor-stator homogenizer (dlab d500, china) (14,000 rpm, 5 min). after this, the emulsion was poured gradually into the mcc and maltodextrin dispersion with mechanical stirring using a magnetic stirrer for 5 min followed by homogenization using the same homogenizer as used before. for the second emulsion treatment, vco at 40% of the total solid (w/w) was added gradually to the mcc and maltodextrin dispersion with mechanical stirring by using a magnetic stirrer for 5 min followed by homogenization using the same homogenizer. the schematic of vco pickering emulsion using the combination of tween 80 and mcc or using mcc alone is shown in figure 1. it is observed in figure 1, how the tween 80 and mcc solid particles arrange themselves around the vco droplet to enhance the stabilization of emulsion. the emulsion formed was kept in a glass beaker and stored overnight at room temperature (25 ± 3°c) before drying using the three techniques, spray drying, freeze drying and vacuum drying. characterization of emulsions stability of emulsions the emulsion formed was immediately transferred to a test tube, closed and stored at 25 ± 3°c. monitoring the stability of emulsion was carried out every week for 21 and dissolving time). for oxidation measurement, we chose peroxide value because oxidation reaction occurs in stages, where hydroperoxide is formed as a result of primary oxidation, while other techniques (anisidin and thiobarbituric acid [tba] values) are used to measure the rancidity of the secondary or further oxidation. if the primary oxidation is bad, then it is possible that the results of the follow-up test would be bad as well. in addition, there is only a small amount of unsaturated fatty acid in vco and because of limited time (30 days of storage), we only used peroxide value for oxidation measurement in this study. materials and methods materials virgin coconut oil was obtained from bali coconut, a local market in badung, north kuta, bali, indonesia. tween 80 was obtained from croda, united kingdom. vivapur mcg 811 f mcc was obtained from jrs pharma, germany. maltodextrin with a dextrose equivalent (de) of 18 to 20 was obtained from qin huang dao lihua starch co. ltd., qin huang dao, china. nhexane, hydrochloric acid, ammonium thiocyanate, barium chloride dihydrate, iron (iii) sulfate heptahydrate, iron (iii) chloride, methanol, chloroform, isooctane and 2propanol were pro-analyst chemicals and obtained from supelco, merck kgaa, darmstadt, germany. preparation of emulsions the total solid was set at 30% (w/w). the dispersion of maltodextrin and mcc in water was conducted according to the procedure described by iijima and takeo (2000) and lim and roos (2017) with some modifications. maltodextrin with de 18–20 was dissolved in distilled water at 55°c under heating and mechanical stirring by using a magnetic stirrer up to complete dissolution. the heating process was continued up to 70°c. then, mcc at 5% of vco (w/w) was added gradually to the maltodextrin dispersion at 70°c with stirring using a magnetic stirrer up to complete dissolution. the mcc and maltodextrin dispersion was then cooled at room temperature to 45 ± 3°c before the core material was added into it. emulsion was formed using a homogenization method according to the procedure described by alcântara et al. (2019) and xu et al. (2016) with some modifications. for the first emulsion treatment, tween 80 at 1% of vco (w/w) was mixed into distilled water at 25 ± 3°c under mechanical stirring by using a magnetic stirrer until it was thoroughly mixed. tween 80 at 1% of vco (w/w) was used in this study because good emulsion stability figure 1. schematic of vco pickering emulsion using the combination of tween 80 and mcc, or mcc alone. 70 italian journal of food science, 2022; 34 (1) nurhadi b et al. and kunz (2011) with some modifications. the emulsion was kept in a glass beaker and stored overnight at room temperature (25 ± 3°c) and transferred to a plastic container for the initial freezing process at −50°c for 24 h using a freezer (thermo scientific hera freeze basic, united states). after the freezing process, a freeze dryer (christ alpha 1-4 ld plus, germany) was used for the freeze-drying process with temperature kept at −50°c and pressure at 0.001 mbar for 48 h. the freeze-dried products were then milled in a mortar and pestle, and sieved with a 40-mesh tyler sieve to obtain a fine powder. all freeze-dried vco powders were kept at 4°c while waiting for ‘day zero analysis’, followed by storage tests at room temperature (25 ± 3°c). vacuum drying process the vacuum drying process also was carried out in duplicate according to the procedure described by nurhadi and roos (2017) with some modifications. the emulsion was kept in a glass beaker and stored overnight at room temperature (25 ± 3°c), and transferred to a silicone pan for drying using a vacuum oven (vwr scientific, united states) with temperature and vacuum pressure set at 60°c and 25 inhg, respectively, for 6 h. the vacuum-dried products were then milled in a mortar and pestle, and sieved with a 40-mesh tyler sieve to obtain fine powder. all vacuum-dried vco powders were then kept at 4°c while waiting for ‘day zero analysis’, followed by storage tests at room temperature (25 ± 3°c). powder analysis drying yield the drying yield (%) is the ratio between the weight of the microcapsules obtained from the drying process (g) and the total solid of emulsion which consists of vco, tween 80, mcc, and maltodextrin (g). the drying yield was calculated using equation (3) (zhong et al., 2009): drying yield % m m c t � � � �100%, (3) where mc is the weight of microcapsules obtained from the drying process (g), and mt is the total solid of the emulsion (g). color analysis color intensity was determined as described by yam and papadakis (2004) using a spectrophotometer (konica minolta cm-5 spectrophotometer, japan). this measurement was performed on day zero and every 15 days during days of storage right after its preparation. the height of the cream formed during storage was calculated using the creaming index (ci) as described by equation (1) (akhtar et al., 2014): creaming index h h %,c t � �100 (1) where hc is the height of the cream (cm) and ht is the height of the initial emulsion (cm) droplet size of emulsions the droplet size of emulsion was determined by using the laser diffraction particle size analyzer (ls 13 320, beckman coulter, united states). the measurement was carried out immediately after the emulsion was formed. the droplet size of the emulsion was expressed as the average size, and the calculation of polydispersity index (pdi) was exercised using equation (2) (hirschle et al., 2016): pdi d � � � � � � � � 2 , (2) where σ is the standard deviation of the particle size distribution (µm), and d is the mean diameter of the particle (µm). spray drying process the emulsion was kept in a glass beaker and stored overnight at room temperature (25 ± 3°c); it was diluted with distilled water for as much as 50% of the total emulsion under stirring. the spray drying process was carried out in duplicate according to the procedure described by sansone et al. (2011) with some modifications. a mini spray dryer (buchi b-290, flawil-switzerland) was used for the spray drying process with inlet temperature adjusted at 150 ± 3°c, outlet temperature maintained at 70 ± 3°c, aspirator was set at 85%, pump was set at 25%, and the actual flow rate was maintained at 5 ml/min; the mixture was stirred gently while the feed was pumped to spray dryer to maintain homogeneity. the dried products were collected at collection chamber after the drying process was completed and sieved with a 40-mesh tyler sieve to obtain a fine powder. all the spray-dried vco powders were then kept at 4°c while waiting for ‘day zero analysis’, followed by storage tests at room temperature (25 ± 3°c). freeze-drying process the freeze-drying process also was carried out in duplicate according to the procedure described by anwar italian journal of food science, 2022; 34 (1) 71 characteristics of virgin coconut oil emulsion powder the oil was dried and put into hull to be extracted with soxhlet. meanwhile, 2-g freeze-dried powder was weighed and put into hull and immediately extracted with soxhlet. the extraction process was carried out with a multi-heater extraction rack (buchi b-810, switzerland) for 3 h using n-hexane solvent. then the oil extract was dried in an oven at 105°c for 30 min to obtain a constant weight. the total oil was calculated using equation (7): o % w w w %,t � � � � �2 1 100 (7) where w is the weight of the sample (g), w1 is the constant weight of empty flask (g) and w2 is the constant weight of extract + flask (g). surface oil (os) or non-encapsulated oil is determined by the washing method using n-hexane as described by lim and roos (2017). this measurement was conducted on day zero and every 15 days during the 30-day storage at room temperature (25 ± 3°c). the sample was weighed as 2 g on a funnel having a filter paper. the sample was washed for four times by using 10-ml n-hexane for each wash. the beaker containing n-hexane and extracted surface oil was then left for two days for evaporation of n-hexane. then the beaker containing surface oil and remaining hexane was dried using an oven at 105°c for 30 min to obtain a constant weight. the calculation of surface oil was carried out by using equation (8): o % w w w %,s ( ) � � �2 1 100 (8) where w is the weight of the sample (g), w1 is the constant weight of beaker (g) and w2 is the constant weight of surface oil + beaker (g). the microencapsulation efficiency was calculated by comparing the encapsulated oil (total oil surface oil) and the total oil from the sample as described by anwar and kunz (2011). the microencapsulation efficiency was calculated by using equation (9): microencapsulation efficiency % o o o %,t s t ( ) � �� � �100 (9) where ot is the amount of total oil (%) and os is the amount of surface oil (%). peroxide value peroxide indicates oil damage because of oxidation, which is characterized by the appearance of rancid odor in the products. the peroxide value of the sample was measured according to the official methods of international organization for standardization (iso) and international the 30-day storage at room temperature (25 ± 3°c). before analysis, the instrument was calibrated using cm-a210 white calibration plate (japan). then the samples were captured and the results obtained were in the form of l* indicating the degree of lightness to darkness, a* indicating the degree of redness to greenness and b* indicating the degree of yellowness to blueness. hue angle (h*), which indicates the color of vco powder, was calculated using equation (4), and chroma (c*) value, which indicates the saturation of vco powder, was calculated using equation (5) as described by kuck and noreña (2016): h tan b a * * *� � � �� � � �� �1 , (4) c a b* * *� �[( ) ( ) ] ./2 2 1 2 (5) morphology of vco powder the morphology of vco powder was observed by using a scanning electron microscope (sem), tabletop microscope hitachi tm 3000 (hitachi hightechnologies corporation, tokyo, japan). the sample was placed on a double-sided tape attached to the specimen stub. the specimen stub was then measured at appropriate height and put at a specimen stage. the observation mode was set, and the morphological structure of the particle was observed on computer screen. moisture content the moisture content was measured using the gravimetric method according to official method of association of official analytical chemistry (aoac, 2005b), with removal of water vapor by convective heat transfer using an oven at 105°c for 3 h. the moisture content was measured in duplicate and the calculation of moisture content was performed on a wet basis using equation (6): moisture content %wb w w w w � � � � � �1 2 1 100 ( ) % (6) where w is the constant weight of empty dish (g), w1 is the weight of sample (g) and w2 is the constant weight of dried sample + dish (g). microencapsulation efficiency total oil (ot) is the overall oil in a microcapsule and comprises encapsulated oil (oe) and surface oil (os). total oil was determined by using the soxhlet extraction method according to the official method described by aoac (2005a). sprayand vacuum-dried powders were weighed as 2 g and hydrolyzed. filter paper containing 72 italian journal of food science, 2022; 34 (1) nurhadi b et al. pdi d � � � � � � � � 2 (10) where σ is the standard deviation of particle size distribution (µm), and d is the mean diameter of particle (µm). dissolving time dissolving time was measured as described by tinay and ismail (1985). distilled water, 50 ml, was added to 1 g of sample and stirred using a magnetic stirrer at a speed of 892 rpm with a 3-mm × 7-mm magnetic bar at 25 ± 3°c. the time taken for all solid particles to dissolve completely in the solvent was recorded as the dissolving time. results and discussion characterization of emulsions creaming index emulsion instability usually occurs if the emulsion is stored for a long period. the major cause of emulsion instability is creaming due to gravitational separation, resulting in the separation of emulsion system into two layers. however, the pickering emulsion system is more stable because it requires more energy to remove solid particles from the emulsion system (ahsan et al., 2020). as observed in table 1, all vco emulsions did not depict creaming or other types of instability (creaming index = 0%) up to 21 days of storage. this means that pickering emulsion with mcc, with or without tween 80 can maintain the stability of emulsion system for up to 21 days. in the mechanism of pickering emulsion, mcc is absorbed onto the vco droplet surface, thus covering the droplet surface by forming a thick layer which acts as a barrier to prevent flocculation between vco droplets (xu et al., 2016). apart from this, addition of mcc in high concentrations also result in the presence of mcc in continuous phase, which increases the viscosity to make a gel-like continuous phase (wei et al., 2019; zhang et al., 2017a, 2017b). this happened in this study because the mcc used was dairy federation (idf) (2006), and the oil extraction process from vco powder was carried out as described by partanen et al,(2008) with several modifications. this measurement was carried out on day zero and every 15 days during the 30-day storage at room temperature (25 ± 3°c). the sample was weighed as 0.5 g in a test tube and 0.5ml distilled water was added to it, and homogenized with a vortex mixer for 30 sec. pipette 400-µl sample solution into a microcentrifuge tube and 1.5-ml isooctane:isopropanol (2:1 ratio) solution was added to it. the solution was centrifuged using a microcentrifuge (thermo scientific sorvall legend micro 17r centrifuge, united kingdom) (5,000 rpm, 5 min, 25°c). the upper phase (supernatant) was separated for analysis. the extraction process was continued for up to three times of extraction using the same solvent and centrifuge. a 100-µl aliquot of the extraction was moved to a test tube and 9.8 ml of chloroform:methanol (7:3 ratio) solution was added to it. for color formation, 50 µl of fe2+ solution (filtrate from the mixture of barium chloride solution [0.2 g bacl2 · 2h2o was dissolved in 25-ml distilled water] and iron (iii) sulfate solution [0.25 g feso4 · 7h2o was dissolved in 25-ml distilled water]) and 50 µl of 30% ammonium thiocyanate solution (7.5 g dissolved in 25-ml distilled water) were added to samples. the samples were homogenized using a vortex mixer and incubated in dark for 10 min. the absorbance was measured by using a spectrophotometer (vis7220g/uv-9200 ray leigh spectrophotometer, beijing, china) at 500 nm, and the peroxide value was calculated by using a standard curve of fe3+ (0–5 ppm). particle size of rehydrated vco powder the particle size of rehydrated vco powder was determined using the laser diffraction particle size analyzer (ls 13 320, beckman coulter, united states). for rehydrated microcapsules, 50 ml of distilled water was added to 1 g of sample and stirred by using a magnetic stirrer at a speed of 892 rpm with a 3-mm × 7-mm magnetic bar at 25 ± 3°c until dissolved completely. the measurement was carried out immediately after the rehydration process. the particle size of vco powder was expressed as the average size, and the pdi was calculated using equation (10) (hirschle et al., 2016): table 1. the characteristics of vco pickering emulsion with mcc. sample creaming index (%) droplet size (µm) pdiday 0 day 7 day 14 day 21 vco emulsion with tween 80 and mcc 0 0 0 0 1.3620 ± 0.0193 0.2024 ± 0.0103 vco emulsion with mcc 0 0 0 0 1.4678 ± 0.0163 0.1691 ± 0.0119 values are mean ± sd of duplicate determination. pdi: polydispersity index. italian journal of food science, 2022; 34 (1) 73 characteristics of virgin coconut oil emulsion powder those produced by using surfactants or solid particles only. this has also been proved by pichot et al. (2010) and raman and aichele (2020). the pdi value demonstrates an overview of the distribution data of particles of different sizes in an emulsion system. pdi is a scale and has no dimensions, with the pdi value are ranging from <0.05 (rarely found) to a maximum of 1.0 (vicente et al., 2018). according to das and chaudhury (2011), a pdi value of >0.30 indicates that the particles are no longer homogeneous. according to mcclements (2005), a pdi value of >0.50 indicates a very heterogeneous and wide distribution of particle size or very polydispersity, and a pdi value of 1.0 indicates that the sample has the possibility of containing large particles or aggregates which would eventually form a sediment (vicente et al., 2018). as observed in table 1, both emulsion systems had a pdi < 0.30, which means they had a homogeneous particle size distribution. powder analysis drying yield the vco powder has different drying yield values depending on the microencapsulation process and coating materials. low drying yield can occur during the drying process because of stickiness in drying chamber, milling and sieving tools, or spill during the drying, milling and sieving processes (nurhadi et al., 2012). as observed in table 2, spray drying resulted in the lowest drying yield followed by vacuum drying, and the highest drying yield was obtained by freeze drying with the same ratio of oil and coating materials. spray drying had the lowest drying yield because of high probability of microencapsules sticking to the drying chamber (yanuwar et al., 2007). furthermore, the relatively low inlet temperature of spray drying used in this study (150°c) with the aim of maintaining its bioactive components might have contributed to low drying yield (tonon et al., 2008). vacuum drying had a slightly higher drying yield than spray drying. this was because the vacuum process absorbed water of the sample so that the sample would be sprayed in the oven, resulting in a low drying yield. meanwhile, freeze drying had the highest drying yield because almost all solids of the freeze-dried emulsion could be used. freeze-drying process is through water freezing and ice sublimation so that it never causes any product loss. similar results were observed by román et al. (2020), as the drying yield of rice bran oil powder produced from freeze-drying technique was higher than spray drying technique (96.9% vs. 50.8%). from the viewpoint of different emulsion treatments, the drying yield obtained from vco emulsion with tween 80 and mcc a colloidal type vivapur mcg 811 f mcc containing 11.30–18.80% carboxymethyl cellulose (cmc). presence of cmc made droplets in the continuous phase unable to move freely because of the formation of a three-dimensional (3d) network between mcc and water that bound vco droplets tightly to prevent phase separation (ahsan et al., 2020; xu et al., 2016). this mechanism was in accordance with the terminal velocity equation of stokes' law because emulsion stability is directly proportional to viscosity (mcclements, 2005). similar results were observed by xu et al. (2016), where the stability of emulsion system made with low molecular surfactants was increased by mcc adsorption on oil droplet surface. kargar et al. (2012) and xu et al. (2016) also had a similar result, where the addition of mcc to an o/w emulsion without surfactant produced a good emulsion stability. the vco emulsion prepared with mcc alone had a higher viscosity than the vco emulsion prepared with tween 80 and mcc. the use of surfactant (tween 80) resulted in the –oh group from tween 80 binding to the –oh group from water so that it can reduce interaction between mcc particles and water to form a 3d network, resulting in decreased emulsion viscosity (raman and aichele, 2020; vashisth et al., 2010). although the viscosity was different, both emulsions had a good stability of up to 21 days. therefore, pickering emulsion with mcc, with or without tween 80, could maintain the stability of vco emulsion system for a longer period. droplet size of emulsions the stability of an emulsion system also depends on the size of droplet, where the size of droplet is influenced by the homogenization process (time and speed), and the type and concentration of emulsifier (hadnadev et al., 2013). as observed in table 1, the vco emulsion with tween 80 and mcc had a smaller droplet size. this phenomenon occurred due to the process by which surfactant and solid particles reached the oil–water interface during the emulsification process. at the beginning of the process, the droplet size would be reduced drastically so that the interface area in the emulsion system increased rapidly. the low molecular surfactant (tween 80) would gather quickly at the oil–water interface and reduced its surface tension, thereby allowing a more efficient breakdown of oil droplets during the emulsification process, resulting in smaller droplet size of emulsion. besides, the presence of this surfactant could temporarily stabilize the emulsion system (pichot et al., 2010; raman and aichele, 2020). furthermore, the added solid particles (mcc) would coat all the available oil–water interfaces and significantly reduce the coalescence. this mechanism of stabilization of mixed emulsifier system produced smaller emulsion droplets than 74 italian journal of food science, 2022; 34 (1) nurhadi b et al. freeze drying. however, lower l* value was obtained from vacuum-dried powder. similar results were observed by horszwald et al. (2013) and michalska and lech (2018). the long-term use of heat in the vacuum drying process allowed the product to be oxidized and could even be burnt due to caramelization reaction (nonenzymatic browning). meanwhile, the spray drying process used high temperatures in a very short time, so it did not significantly affect the characteristics of vco powder. similarly, the freeze-drying process also did not use heat so that the original color of the product could be maintained. however, all vco powders obtained from all was lower than that obtained from vco emulsion only with mcc. low viscosity from the emulsion system caused low drying yield, and vice versa (sarungallo et al., 2019). this result was in accordance with the literature, where the emulsion system with tween 80 and mcc had a lower viscosity, resulting in a lower drying yield. color analysis according to table 3, the high degrees of lightness to darkness (l* value) in vco powder resulted from spray and table 2. characteristics of vco powder from pickering emulsion with mcc and different drying techniques. sample drying yield (%) moisture content (% wb) rehydrated particle size (µm) pdi dissolving time (sec) a 44.98 ± 6.78% 3.83 ± 0.01% 1.5017 ± 0.0099 0.1358 ± 0.0029 108.50 ± 16.26 b 94.03 ± 2.83% 3.51 ± 0.47% 1.4820 ± 0.0139 0.1423 ± 0.0040 78.75 ± 2.47 c 48.98 ± 4.11% 2.19 ± 0.56% 1.5203 ± 0.0155 0.1339 ± 0.0088 156.50 ± 3.54 d 53.86 ± 4.98% 3.49 ± 0.24% 1.5460 ± 0.0104 0.1153 ± 0.0017 188.25 ± 3.18 e 96.43 ± 1.71% 4.74 ± 1.38% 1.5197 ± 0.0025 0.1304 ± 0.0019 101.50 ± 5.66 f 55.66 ± 6.25% 3.42 ± 1.52% 1.5493 ± 0.0064 0.1106 ± 0.0013 258.20 ± 12.73 values are mean ± sd of duplicate determination. pdi: polydispersity index; wb = wet basis. a = vco + tween 80 + mcc by spray drying; b = vco + tween 80 + mcc by freeze drying; c = vco + tween 80 + mcc by vacuum drying; d = vco + mcc by spray drying; e = vco + mcc by freeze drying; f = vco + mcc by vacuum drying. table 3. the l*, a*, b*, hue and chroma values of vco powder from pickering emulsion with mcc and different drying techniques during storage. sample l* a* b* hue chroma a day 0 92.46 ± 1.17 −0.30 ± 0.10 3.29 ± 1.13 95.28 ± 0.15 3.30 ± 1.13 day 15 91.78 ± 1.39 −0.26 ± 0.02 2.91 ± 1.07 95.40 ± 1.56 2.92 ± 1.07 day 30 91.85 ± 1.75 −0.24 ± 0.04 3.01 ± 0.56 94.56 ± 0.04 3.02 ± 0.56 b day 0 93.32 ± 0.17 −0.14 ± 0.09 1.83 ± 0.47 94.96 ± 4.02 1.84 ± 0.46 day 15 93.83 ± 0.34 −0.17 ± 0.09 2.01 ± 0.17 94.82 ± 3.00 2.02 ± 0.16 day 30 93.72 ± 0.42 −0.16 ± 0.10 2.03 ± 0.02 94.37 ± 2.81 2.04 ± 0.01 c day 0 79.64 ± 0.22 −1.45 ± 0.44 10.21 ± 3.26 98.12 ± 0.13 10.31 ± 3.29 day 15 80.22 ± 0.22 −1.21 ± 0.39 8.31 ± 2.36 98.23 ± 0.30 8.39 ± 2.39 day 30 79.14 ± 0.70 −1.24 ± 0.36 2.33 ± 0.40 117.78 ± 2.91 2.64 ± 0.53 d day 0 95.17 ± 0.46 −0.12 ± 0.07 2.48 ± 0.56 92.91 ± 2.28 2.48 ± 0.55 day 15 94.60 ± 0.15 −0.09 ± 0.09 2.74 ± 0.49 92.09 ± 2.30 2.74 ± 0.49 day 30 94.22 ± 0.11 −0.09 ± 0.08 2.62 ± 0.45 92.16 ± 2.22 2.62 ± 0.44 e day 0 93.54 ± 0.26 −0.18 ± 0.03 1.82 ± 0.15 95.63 ± 1.45 1.83 ± 0.14 day 15 93.19 ± 0.29 −0.18 ± 0.04 1.78 ± 0.15 95.70 ± 1.84 1.79 ± 0.15 day 30 92.84 ± 0.17 −0.18 ± 0.04 1.66 ± 0.01 96.12 ± 1.31 1.66 ± 0.01 f day 0 86.77 ± 1.76 −0.84 ± 0.47 7.49 ± 1.26 96.18 ± 2.50 7.54 ± 1.30 day 15 85.99 ± 1.70 −0.74 ± 0.39 7.63 ± 0.73 95.39 ± 2.38 7.66 ± 0.76 day 30 85.05 ± 2.05 −0.69 ± 0.43 7.23 ± 0.24 95.41 ± 3.23 7.27 ± 0.28 *values are mean ± sd of duplicate determination. a = vco + tween 80 + mcc by spray drying; b = vco + tween 80 + mcc by freeze drying; c = vco + tween 80 + mcc by vacuum drying; d = vco + mcc by spray drying; e = vco + mcc by freeze drying; f = vco + mcc by vacuum drying. italian journal of food science, 2022; 34 (1) 75 characteristics of virgin coconut oil emulsion powder using tween 80 and mcc also had lower l* and a* values but a higher b* value because of the yellowish color of tween 80 that affected the lightness and the color of vco powder. the color or shade (hue angle value) of vco powders ranged from 92.09 to 98.23, confirming that the sample was tended yellow in hue, with the highest hue angle value found in sample c (vacuum-dried vco powder from the emulsion using tween 80 and mcc). all vco powders had stable hue angle values, except sample c, and increased up to 117.78 ± 2.91 on the 30th day of storage because of decrease in b* value. the sprayand freeze-dried vco powders had a low saturation (chroma value) compared to vacuum-dried vco powder, especially sample c, and were stable for up to 30 days of storage. meanwhile, the chroma value of sample c decreased steadily until the 30th day of storage, resulting in a more faded color. overall, vco powders obtained from all drying techniques had a white to slightly yellowish hue. however, vco powder obtained from spray drying (samples a and d) and freeze drying (samples b and e) had a slightly yellowish and a little bit of greenish in white hue but still having a bright coloration. meanwhile, the vco powder obtained from vacuum drying tended to be more yellowish and greenish than the powders obtained from other two drying techniques because of the long vacuum drying process. morphology of vco powder as observed in figure 2, the spray-dried vco powders (samples a and d) had a specific particle morphology, which was spherical in shape with a smooth surface drying techniques had white to light cream color, which met the commercial standards of consumer acceptance based on sni 4444:2009 (badan standarisasi nasional, 2009). all samples of vco powder produced by different drying techniques had a slightly greenish color with different degrees of redness to greenness (a* value) but still close to zero, which is neutral, except for vacuum-dried vco powder with a more greenish color. the lengthy process of vacuum drying resulted in the formation of green pigments from colorless precursors present in vco. the pigments were not from the chlorophyll group, and as described by joslyn and sano (1956), the specific compound responsible for the green pigment formed in garlic maceration tissue was not yet known. all vco powders also had a positive degrees of yellowness to blueness (b* value), which means that powders tended to have a yellowish color. vacuum-dried vco powder had the highest b* value, because of tween 80 and mcc or mcc alone. these results were in accordance with the results obtained by nurhadi et al. (2012), where the vacuum-dried honey powder had the highest b* value because of the long-term heating effect of drying process. after 30 days of storage, there was a decrease in the b* value of vacuum-dried vco powder, especially in sample c, caused by a greenish pigment formed due to lengthy vacuum drying process. during storage, the greenish pigment turned brown due to exposure to oxygen and would gradually turn yellowish and finally to a light cream color, as described by joslyn and sano (1956). the vco powder obtained from pickering emulsion with mcc and different drying techniques demonstrated no significant difference in lightness after 30 days of storage. in addition, vco powder obtained from an emulsion figure 2. morphology of vco powder from pickering emulsion with mcc and different drying techniques. a = vco + tween 80 + mcc by spray drying; b = vco + tween 80 + mcc by freeze drying; c = vco + tween 80 + mcc by vacuum drying; d = vco + mcc by spray drying; e = vco + mcc by freeze drying; f = vco + mcc by vacuum drying. (a) 76 italian journal of food science, 2022; 34 (1) nurhadi b et al. figure 2. (continued) (b) (c) (d) (e) italian journal of food science, 2022; 34 (1) 77 characteristics of virgin coconut oil emulsion powder high porosity of this freeze-dried vco powder made it suitable for instant powder drink because it caused rapid dissolution in solvent, but the dissolution was just not uniform because of the nonuniform shape compared to spherical-shaped particles’ dissolution. samples c and f, which were vacuum-dried vco powders, had an angular and nonuniform morphology with large cavities on their surfaces. similar result was observed by mutlu et al. (2020). the presence of cavities in the particles was caused by the suction/vacuum process of water vapor from the heated material to produce dry products with large hollows (reis, 2014). this particle morphology was almost the same as that of freeze-dried particles but not porous and also had a high surface oil, so it was very oily resulting in low wettability and solubility, making it unsuitable for instant powder drinks. moisture content the vco powders had different moisture contents depending on the drying techniques and ranged from 2.19% to 4.74% (table 2). the moisture content obtained was under 5%, which means all of the vco powders were still accepted in the food industry (díaz et al., 2020). the freeze-dried vco powder from emulsion with only mcc had the highest moisture content because of its porous structure, so it was more hygroscopic with a higher adsorption capacity than other dried samples (gong et al., 2007). the freeze-drying process that used temperatures lower than −40°c also resulted in the obstruction of mass transfer and sublimation process because of rapid freezing of outer layer pores, thereby increasing the retention of moisture in microcapsules. similar results were observed by kuck and noreña (2016), where the grape skin phenolic extract powder produced by freeze drying had a higher water content than spray drying. and no pores but some dents caused by rapid water loss during the initial drying process (ré, 1998; román et al., 2020; wilkowska et al., 2017). the smooth surface of spray-dried particles was caused by the atomization process of spray drying (pasrija et al., 2015). similar results have been reported by several studies using different coating materials, such as modified starch (melgosa et  al., 2019), pea protein (moreno et al., 2016), various biopolymers (gallardo et al., 2013), a combination of maltodextrin and pea protein isolate (román et al., 2020) or a combination of maltodextrin and soy protein isolate (carneiro et al., 2013). in addition, there were also small particles adhered to the surface of spray-dried particles that could be the oil non-capsulized by the matrix, thus inducing other particles to stick to the surface and form a cluster (román et al., 2020). these spherical-shaped particles were perfect for microencapsulation with bioactive components perfectly protected inside the coating agent. likewise, spray-dried vco powder had a more regular and uniform shape and size so that the solubility would be more uniform. samples b and e, which were freeze-dried vco powders, had a wrinkled sponge-like morphology with an irregular surface and nonuniform shape, and very light and high porosity. therefore, these particles had a large surface area. the presence of pores and wrinkles in the particles because of the usage of mcc fastened water removal from the material (dhar et al., 2012) as well as the fast sublimation process of ice crystals on the matrix during freeze drying, resulting in the formation of pores which previously were ice crystals (smrdel et al., 2008). similar particle morphology has been reported by other studies using different coating materials, such as sodium alginate in anthocyanin extract microcapsules (zhang et  al., 2020), a combination of various polymers in fish oil microcapsules (anwar and kunz, 2011), and a combination of maltodextrin and pectin in the microcapsules of moringa stenopetala leaf extract (dadi et al., 2020). figure 2. (continued) (f) 78 italian journal of food science, 2022; 34 (1) nurhadi b et al. effect of material interactions, and the microencapsulation techniques (dadi et al., 2020). in figure 3, all vco powders had a low microencapsulation efficiency, which is <50%. the low microencapsulation efficiency is caused by the high concentration of oil (40%), so there would be a lack of coating material to encapsulate oil, thus resulting in high surface oil and low encapsulated oil (frascareli et al., 2012). similar results were also demonstrated in the study conducted by mcnamee et al. (1998) on encapsulation of soybean oil with gum arabic, where the encapsulation efficiency was reduced from 100% to 48% with oil and gum arabic ratio increasing from 0.25 to 5.00. several other studies have also demonstrated similar results (ahn et al., 2008; alcântara et al., 2019). the freeze-dried vco powder from both emulsion treatments had the lowest microencapsulation efficiency because of high surface oil on particles. the freezing process induced demulsification in the emulsion system because some droplets froze first and the volume of frozen droplets increased, resulting in collisions between droplets, thus damaging water and emulsifier layers that covered the droplets. many droplets froze together to form larger ice domains due to coalescence, and also some droplets froze because of partial contact with other ice, which is commonly referred to as ‘partial coalescence’. the coalescence of particles produced an even larger particle, which at a certain particle size triggered phase separation resulting in high non-encapsulated oil the vacuum-dried vco powder from emulsion using tween 80 and mcc had the lowest moisture content caused by vacuum conditions of drying that reduced the heat required to evaporate water so that water evaporation occurred quickly at low pressure (piwińska et  al., 2015). similar results of the moisture content were also obtained by nurhadi et al. (2012), where the vacuum dried honey powder had a lower moisture content than spray-dried honey powder. vco powders obtained from emulsions with mcc only (samples d and f) had a higher moisture content. this was due to the higher viscosity of emulsion, resulting in decreased water diffusion during the drying process because the total solid concentration was more than 20% (frascareli et al., 2012). in addition, use of tween 80 also produced vco powder with a low moisture content because of foam formation in the emulsion system (mayasari et al., 2019). the foam layer dried faster than the liquid under the same drying conditions. as a result, the drying process was completed quickly and produced microcapsules with a lower moisture content (kamsiati, 2006). microencapsulation efficiency microencapsulation efficiency is defined as the ratio between encapsulated oil and total oil contained in particles (tonon et al., 2012). the microencapsulation efficiency depends on several factors, such as the ratio of core material to coating material, chemical properties, figure 3. microencapsulation efficiency of vco powder from pickering emulsion with mcc and different drying techniques during storage. a = vco + tween 80 + mcc by spray drying; b = vco + tween 80 + mcc by freeze drying; c = vco + tween 80 + mcc by vacuum drying; d = vco + mcc by spray drying; e = vco + mcc by freeze drying; f = vco + mcc by vacuum drying italian journal of food science, 2022; 34 (1) 79 characteristics of virgin coconut oil emulsion powder the microencapsulation efficiency of vco powder demonstrated decrease on the 15th and 30th day of storage, which means that more oil was diffused from matrix, resulting in high surface oil. similar results were observed by esparza et al. (2020), where the microencapsulation efficiency of hempseed oil powder stabilized with nanocrystalline cellulose pickering emulsion decreased after 30 days of storage. peroxide value peroxide is an indicator to determine the level of oxidation of fats and oil products. the peroxide value of all vco powders depicted a higher value compared to the characterization result of vco, which was only 0.052 meq o2/kg. this indicated that exposure to light, oxygen and heat during the microencapsulation process could facilitate oxidation (mubarak, 2017). however, increase in the peroxide value was still below the maximum limit of the quality requirements of vco peroxide value, which is 2.0 meq o2/kg based on sni 01-7381-2008 (badan standardisasi nasional, 2008), which established that vco powder was still safe for consumption. a slight increase in the peroxide value of vco powder was due to a small amount of unsaturated fatty acids (about 10%) that we discovered in vco, which could be oxidized easily. in figure 4, the spray-dried vco powder (samples a and d) demonstrated the highest peroxide value, and the in freeze-dried samples (lin et al., 2007). similar results were obtained by anwar and kunz (2011) in microencapsulationof fish oil using freeze drying. the highest microencapsulation efficiency was obtained by spray drying in the emulsion using tween 80 and mcc (sample a), with a microencapsulation efficiency of 33.49 ± 1.59%. as observed in figure 2, particles produced by the spray-drying technique had no cracks and pores in their microstructure, so they would have a good microencapsulation efficiency (pereira et al., 2019). besides this, using tween 80 in the emulsion system resulted in a smaller emulsion droplet size, which would result in a higher microencapsulation efficiency. similar results were observed by soottitantawat et al. (2005), and also supported by shamaei et al. (2017), where there was no correlation between microencapsulation efficiency and microparticle size (or emulsion droplet size). in addition to spray drying, freezeand vacuum-dried vco powders obtained from the emulsion with mcc only had a higher microencapsulation efficiency. this may be affected by the viscosity of emulsion, where using tween 80 resulted in a lower emulsion viscosity, but the emulsion with mcc only had a higher viscosity. thus, a stronger matrix was formed in the emulsion using mcc only that helped to prevent the diffusion/migration of oil from matrix to produce microcapsules with higher encapsulated oil (sarungallo et al., 2019). figure 4. peroxide value of vco powder from pickering emulsion with mcc and different drying techniques during storage. a = vco + tween 80 + mcc by spray drying; b = vco + tween 80 + mcc by freeze drying; c = vco + tween 80 + mcc by vacuum drying; d = vco + mcc by spray drying; e = vco + mcc by freeze drying; f = vco + mcc by vacuum drying. 80 italian journal of food science, 2022; 34 (1) nurhadi b et al. to high degradation rate, resulting in secondary oxidation products in the form of aldehyde and ketone compounds (ketaren, 1986). some of the main aldehyde compounds produced from secondary oxidation were n-alkanal, trans-2-alkenes, 4-hydroxy-trans-2-alkenes and malondialdehyde (chaijan and panpipat, 2017). these aldehyde compounds would undergo an oxidation process to form carboxylic acids, which belong to the free fatty acid group. hydrogen present in this carboxylic acid would form fatty acid radicals that react with oxygen to again produce peroxides during storage (widodo et al., 2020). this increased the peroxide value of sample f on the 30th day of storage. vco powder obtained from emulsion with tween 80 and mcc (samples a–c) also demonstrated a higher increase in peroxide value on the 15th day of storage. this could be due to the use of tween 80, which belonged to the triglyceride group, allowing the fatty acid components of tween 80 to be oxidized during storage, resulting in a higher peroxide value compared to the nontween 80 samples. particle size of rehydrated vco powder as observed in table 2, all rehydrated vco powders had a slight increase in the particle size compared with their emulsions’ droplet size but not different significantly (table 1). these results indicated that the emulsion systems had the possibility of slight coalescence because of heating and freezing during the microencapsulation process. all rehydrated vco powders had the same pdi value, which was still below 0.30, indicating that there hydrated dried particles were homogeneous. however, the rehydrated freeze-dried vco powder from both emulsions depicted higher pdi values than other dried samples with the same emulsion treatment. these phenomena occurred due to the greater coalescence process during freezing than heating so that a higher pdi was obtained (lin et al., 2007). dissolving time dissolving time is the time required for all particles to dissolve homogeneously in solvent through stirring (pereira et al., 2019). the dissolving time is one of the parameters of microcapsule rehydration properties and is important for producing instant powder drinks. the lower the dissolving time, the better it would be for instant powder drink. as presented in table 2, the dissolving time of vco powders was in the range of 78.75–258.50 sec, while the dissolving time of microencapsulated pink vacuum-dried vco powder depicted the lowest peroxide value on day zero. exposure to high temperatures of heat during the spray drying process could be the reason for accelerated oxidation, resulting in high peroxide formation in the sample (anwar and kunz, 2011). the vacuum process helped to minimize the oxygen that was contacted with the sample, so it prevented peroxide formation. however, peroxide values from all drying techniques did not differ significantly because basically vco only contained a small amount of unsaturated fatty acids (about 10%), which could be easily oxidized (nurhasanah et al., 2019). during storage, the peroxide value of vco powder first depicted an increase on the 15th day then a decrease on the 30th day. similar trends were obtained in different vco powder samples. these results indicated that the oxidation would gradually increase to reach a peak, then it would decrease. however, the increase in peroxide value on the 15th day tended to be higher in vacuum dried vco powder, and the lowest increase was observed in spray-dried vco powder. previous study conducted by anwar and kunz (2011) established that spray-dried powder tended to be unstable due to the presence of pro-oxidants at high temperatures, which could result in rapid peroxide decomposition after its formation. therefore, only a slight increase in peroxide value during the 15 days of storage was observed compared to other dried samples. the stability of vco powder during storage was also influenced by several other factors, such as particle morphology and surface oil (tolun et al., 2016). vco powders obtained from freeze drying (samples b and e) and vacuum drying (samples c and f) had an irregular morphology. as a result, the surface area was larger, so that it was more likely to be exposed to oxidation than fine particles (pereira et al., 2019; tolun et al., 2016). in addition, the freezeand vacuum-dried vco powders had high porosity and large cavities. these conditions facilitated the diffusion of oxygen on particles’ surface and even the particles in surface, so that oxygen easily decomposed the non-encapsulated oil (anwar and kunz, 2011). moreover, the freezeand vacuum-dried vco powders had a higher surface oil than the spray-dried vco powder. therefore, this could be one of the reasons for high increase in peroxide value on the 15th day of storage because surface oil tended to be oxidized more easily (condori et al., 2011). a different trend was observed in sample e of vco powder, where its peroxide value depicted a downward trend up to the 30th day of storage. sample f of vco powder also depicted a decrease in peroxide value on the 15th day, and then an increase occurred on the 30th day of storage. decrease in peroxide value during storage was because only a few new peroxides were formed compared italian journal of food science, 2022; 34 (1) 81 characteristics of virgin coconut oil emulsion powder drying produced vco powder with the lowest drying yield (44.98–53.86%), had bright and white spherical shape particles, smooth surface and no pores, but with some dents on the particle surface. it has the highest microencapsulation efficiency (33.49 ± 1.59%) and peroxide value on day zero (0.558 ± 0.058 meq o2/kg) with not too slow dissolving time. freeze drying produced vco powder with the highest drying yield (94.03–96.43%), had bright and white wrinkled sponge-like particles with an irregular and very porous surfaces. it had the smallest rehydrated particle size (1.4820 ± 0.0139 µm) and the fastest dissolving time in solvent (78.75 ± 2.47 sec), with the highest peroxide value on the 30th day of storage (0.578 ± 0.088 meq o2/kg). vacuum drying produced vco powder with a more yellowish color with angular shape particles with large cavities on the surface. it had the lowest moisture content (2.19 ± 0.56%), the highest peroxide value on the 15th day of storage (0.615 ± 0.065meq o2/kg), and the longest dissolving time (258.20 ± 12.73 sec). based on the results, spray drying was the best technique to produce vco powder with good characteristics compared to other techniques; hence, sample a (vco emulsion using tween 80 and mcc with spray drying) had no creaming index up to 21 days of storage, had small size of emulsion droplet, and bright and white color vco powder. it had spherical shape particles, high microencapsulation efficiency and not too slow dissolving time. therefore, spray drying technique is worth applying in various applications of microencapsulation than other two drying techniques. overall, the characteristics of vco powders obtained in this study were quite good, but had low microencapsulation efficiency. further research is required to study and increase the concentration of solid emulsifier and to replace mcc with cnc with a lower concentration but providing better stability and higher microencapsulation efficiency to vco powder so that it could be used as a non-dairy creamer in accordance with quality requirements and consumer requirements. references ahn, j.-h., kim, y.-p., lee, y.-m., seo, e.-m., lee, k.-w., & kim, h.-s. 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(2019) was in the range of 329–351 sec. relatively fast dissolving time of vco powders in water is one of the important requirements of the food industry, because it can expand the possible use of vco in food products that are insoluble in water in their original form (pereira et al., 2019). the dissolving time is influenced by different structural characteristics of vco powders related to matrix and drying conditions (farias and ratti, 2009; rahman and perera, 2007). freeze-dried vco powder (samples b and e) had the minimum dissolving time compared to other drying principles whereas vacuum-dried vco powder had the longest dissolving time. this was due to the high porosity of freeze-dried vco powder, as depicted in figure 2, so that it had the shortest dissolving time because particle porosity also played an important role in powder rehydration (farias and ratti, 2009; fernandes et al., 2013; stapley, 2008). similar results were observed by karthik and anandharamakrishnan (2013). the spraydried vco powder had a longer dissolving time than freeze-dried vco powder but faster than vacuum-dried vco powder. this was in line with particle morphology, because the spray-dried vco powder had nonporous particles compared to freeze-dried vco powder, but it also had a larger surface area than vacuum-dried vco powder, so that its dissolving time was in between freeze and vacuum-dried vco powders. meanwhile, the vacuum-dried vco powder, with a sticky characteristic, had the smallest surface area among all vco powders obtained from different drying techniques. it tended to stick together due to wetness of surface oil, affecting the wettability of particles, resulting in the longest dissolving time in samples c and f. vco powders from emulsion with tween 80 and mcc (samples a–c) had a faster dissolving time because of tween 80, which formed foam in the emulsion system, resulting in a porous microcapsule (ngamwonglumlert and devahastin, 2017). the high porous structure resulted in high solvent penetration of particles, hence shorter time required to dissolve all particles homogeneously in the solvent (farias and ratti, 2009). conclusion the pickering emulsion method (with or without tween 80) could be used to produce vco powders of good characteristics, which, of course, were also affected by drying techniques. usage of mcc resulted in thicker emulsion system. however, addition of tween 80 decreased emulsion viscosity (but both emulsion treatments had a good stability of up to 21 days of storage) and resulted in a emulsion of smaller droplet size than without addition of tween 80. so did the drying techniques. spray 82 italian journal of food science, 2022; 34 (1) nurhadi b et al. costa, medronho, filipe, mira, lindman, edlund, & norgren. 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(2017). structural, physicochemical and biological properties of spray-dried wine powders. food chemistry, 228, 77–84. https:// doi.org/10.1016/j.foodchem.2017.01.115 paper 336 ital. j. food sci., vol. 27 2015 keywords: foodborne pathogens, bacillus cereus, escherichia coli, proteolytic enzymes, 16srrna prevalence and antibiotic resistance of food borne bacterial contamination in some egyptian food samy selim1,2*, mona warrad3, el fatih el dowma1 and mohamed abdel aziz2 1department of clinical laboratory sciences, college of applied medical sciences, aljouf university, sakaka, p.o 2014, saudi arabia 2botany department, faculty of science, suez canal university, ismailia, p.o. 41522, egypt 3department of medical laboratory sciences, college of applied medical science in al qurait, aljouf university, al qurait, saudi arabia *corresponding author: sadomm2003@yahoo.com abstract this study was undertaken to investigate the prevalence and antibiotic resistance of food borne bacterial contamination in some egyptian food. total viable bacteria and total coliform bacteria were isolated from different sources of food; carbohydrates (bread, flour and basbousa), vegetables (outer and inner tissues of potato and outer and inner tissues of cucumber) and proteins (minced meat, cheese and milk). the study resulted in maximum value of total viable bacteria found in outer tissue of potato 68x104±1.0, while the minimum value found in inner tissues of potato and cucumber. the study resulted in total coliform was maximum value in minced meat 6.4x103±0.3. basbousa and inner tissue of potato and cucumber were free from coliforms. the ability of isolates to producing proteolytic enzymes was tested, we found that 326 isolate (63.92%) from all isolates had this ability, thus we selected most 2 potent proteolytic isolates. the two isolates were identified as bacillus cereus and escherichia coli. the identification confirmed by microlog 34.20 system and 16srrna for two isolates and the same result was founded. sensitivity tested for the most potent proteolytic species to 12 of the most commonly used antibiotics in the egyptian pharmacy. the results showed that all species were sensitive to most of antibiotics, except b. cereus which was strongly susceptible to azteronam and ceftazidim. the data showed that raw meat, cooked food products, and raw milk were most commonly contaminated with foodborne pathogens and many pathogens were resistant to different antibiotics. the study provided useful information for assessment of the possible risk posed to consumers, which has significant public health impact. mailto:sadomm2003%40yahoo.com?subject= ital. j. food sci., vol. 27 2015 337 introduction it has been estimated that as many as 30% of people in industrialized countries suffer from a food borne disease each year and in 2000 at least two million people died from diarrheal disease worldwide (who, 2002a). foods are not only of nutritional value to those who consume them but often are ideal culture media for microbial growth, chemical reactions that cause offensive and sensory changes in foods are mediated by bacteria that use food as carbon and energy source. some of the major bacterial genera which cause food born infection and intoxication (pundir and jain, 2011). contamination of food can affect a large number of populations. about 2.5 million people die every year from water born diseases. more than 40% of total population of indonesia & 60% in thailand suffered from gastroenteritis per year. a total of 32.7% outbreak was involved with restaurant catering bakery products (nazir and islam, 2007). fruits and vegetables carry microbial flora while passing from the farm to the table. the produce is exposed to potential microbial contamination at every step including cultivation, harvesting, transporting packing, storage and selling to the final consumer (fda, 2000). sources of environmental microbial contamination include raw materials, processing equipment, manufacturing activities, sanitation and maintenance practices, workers, waste, animal and insect pests, and microbial growth niches embedded in equipment and in structural components of the building. the survival and growth of microorganisms in a food-processing environment may lead to contamination of the finished product that may, in turn, result in a reduction of microbiological safety and quality. most food plants have locations that can promote the growth of pathogens and spoilage microorganisms that may be transferred directly on to product or carried into additional niches. the origins of these growth habitats are mainly unhygienic design, construction, and maintenance and repair activities that prevent easy cleaning and disinfection. the presence of water and nutrients (food product) is required to form a microbial growth niche and the chemical composition of the food and conditions of water activity, ph, temperature, etc., will select the “normal” organisms that can grow there. microbial growth niches may be established when water is used to clean dry processing environments not designed for wet cleaning and not all points in the equipment are promptly and completely dried (jay, 1996, tesfaye et al., 2011). vanderzant and splittstoesser, (1992) demonstrated that the microbial growth on equipment for processing perishable foods is governed mainly by the ecology of the food, the process, packing room temperature, presence of food residue on the equipment, and efficacy of cleaning and disinfection. recontamination of a biocidaly treated food may increase the risk of foodborne illness if the food is not heated to destroy pathogens before consumption. perishable foods that do not receive a biocidal treatment in the final container may be decontaminated by spoilage microorganisms before packing (vanderzant and splittstoesser, 1992, haileselassie et al., 2013). the research project will deal with investigate the prevalence and antibiotic resistance of bacillus cereus, escherichia coli contamination in some egyptian food. 2. materials and methods 2.1. food samples all food samples were collected from mansoura city, egypt. they collected from different shops, super market, groceries and butchers. ten samples were taken from each of bread, flour, basbousa, potatoes, cucumber, minced meat, cheese and milk. 2.2. preparation of samples twenty five grams of each of the following samples bread, flour, basbousa, outer tissue and inner tissue of potatoes and cucumber, minced meat, cheese and ten ml of raw milk (unboiled/ unpasteurized) was homogenized in 225 ml sterile physiological saline solution (0.85% nacl) in 500 ml conical flask using a plender for 1-2 minutes, then decimal dilutions were prepared. 2.3. isolation of bacteria one ml of appropriate dilution was inoculated on both of nutrient agar medium and macconkey agar medium; the plates were incubated aerobically for 24h at37oc. total viable bacteria (t.v.b) were enumerated on nutrient agar medium using pour plate technique. total coliform (t.c) bacteria were counted on macconkey agar medium by using pour plate technique also. the plates were incubated aerobically for 24h at 37oc. 2.4. purification after the incubation period (24h), the growing colonies were enumerated for counting. after counting a sterile wire loop was used to pick the isolate from the plate and was streaked on freshly prepared nutrient agar medium then inoculated for 24h at 37oc in order to get pure culture. the growing colonies were purified and examined by using cultured morphological appearance and gram reaction. 338 ital. j. food sci., vol. 27 2015 2.5. proteolytic assay proteolytic activity was carried out according to casein pholine method (ramalakshmi et al., 2012). culture media was centrifugated at 7200 rpm for 10 min and supernatant was used as enzyme source. however, 1% casein (in 0.1 m phosphate buffer and ph 7.0) was used as substrate. 1 ml each of enzyme and substrate was incubated at 50°c for 60 min. the reaction was terminated by adding 3 ml of trichloroacetic acid (tca). one unit of protease activity was defined as the increase of 0.1 unit optic density at 1 h incubation period. then it was centrifuged at 5000 rpm for 15 min. from this, 0.5 ml of supernatant was taken, to this 2.5 ml of 0.5m sodium carbonate was added, mixed well and incubated for 20 min. then it was added with 0.5 ml of folin phenol reagent and the absorbance was read at 660 nm using spectrophotometer. the amount of protease produced was estimated and expressed in microgram of tyrosine released under standard assay conditions. based on the tyrosine released the protease activity was expressed in microgram of tyrosine released by 1 ml of enzyme in 30 minutes at 300c on tyrosine equivalent. 2.6. identification of bacterial isolates 2.6.1. conventional methods the appearance of cultures, cell morphological characteristics and physiological characteristics of the purified selected identified isolates were studied. media and reagent were prepared according to standard and procedures as described by (macfaddin, 1980). the identification was carried out by traditional characters and biochemical tests for isolates according to (krieg et al., 1994) and confirmed out by biolog micolog 34.20 system for most potent proteolytic bacterial species. characterization of the most potent isolates were completed and confirmed by biolog microlog 34.20 system at the unit of identification of microorganisms and biological control unit of agriculture reasearch center, giza, egypt. 2.6.2. molecular method the polymerase chain reaction (pcr) methods based on 16s rrna gene for identification of isolates were used. genomic dna was extracted and purified by using qiagen kit (qiagen company). the purity was assessed from the a260/a280 ratios: cultures of bacteria were streaked on tryptic soy agar medium and incubated at 37°c for 24 h. a single colony of each pathogen was grown in (lb) broth medium in erlenmeyer flask and incubated at 37°c for 24 h. culture was harvested by centrifugation at 4°c for 10 min, dna was extracted from pellets according qiagen kit instructions. full length 16s rrna (1500 bp) were amplified from isolates by pcr using universal forward primer 518f    (5’–ccagcagccgcggtaatacg-3’) and 800r (5’-taccagggtatctaatcc-3’). optimum conditions (denaturation 94-1 min, annealing 63-45 s and extension 72-2 min, 35 cycles). amplified 16s rrna was purified from 0.8% melting point agarose gel. bands obtained from pcr product were eluted and purify by (qiagen elution kit) pcr instructions, dna band desired was excised from ethidium bromide stained agarose gel with a razor blade, transferred to ependorf tube. dna was sequenced directly using specific primer with concentration 20 pmol macrogen sequencing company, korea. 2.7. antibiotic susceptibility test 2.7.1. antibiotic disks antimicrobial susceptibility profile of identified bacterial species, bacillus cereus and e.coli against different antibiotics ampicillin, aztreonam, cefadroxil, ceftazidime, chloramphenicol, ciprofloxacin, erythromycin, imipenem, neomycin, norfloxacin, streptomycin and vancomycin were studied. the antibiotic discs used in this research were purchased from oxoid ltd., england. 2.7.2. disc diffusion agar method antibiotic susceptibility test for the bacterial isolates was carried out by disc diffusion technique according to baure et al., (1966). the technique was done by inoculation of pure colonies of the tested organism into 5 ml of sterile nutrient broth and incubation at 37°c for 24h. then 0.1ml of bacterial suspension (0.5 mcfarland turbidity) was spreading by sterile swabs on nutrient agar plates. duplicate plates were prepared for the strain. antibiotic discs were applied to the surface of plates at constant distances. the plates were incubated at 37°c for 24h. at the end of incubation period zones of inhibition were measured as (mm). the entire diameter of the zone was measured including the diameter of the disc. the end point of the reading was taken as complete inhibition of the growth to the naked eye. our (+++) or (++++) indicate high inhibitory effect (large diameter of clearing zone) and (-) indicate no inhibitory effect (good growth). 2.8. statistical analysis the variations between experiments were estimated by standard deviations, and statistical significance of changes was estimated by student’s t-test. only the probability p ≤ 5% was regarded as indicative of statistical significance. ital. j. food sci., vol. 27 2015 339 3. results 3.1. isolation of total viable bacteria from different types of food all growing isolates were enumerated, collected, purified and tabulated. all growing isolates were collected from investigated types of food. table 1 includes the isolates numbers and sources of collected isolates. total viable bacterial counts (tvb) and total coliform bacterial count (tcb) of three main groups of food were tabulated in table 4. in carbohydrates, the highest count 40.5x103cfu/gm was recorded in bread. the total viable bacterial count in flour was 35.3x103cfu/gm. the lowest count 1.1x103 cfu/gm was recorded in basbousa. both fig. 1 and table 4 show these results. the count of total coliform bacteria (tcb) was 5.4x103 and 5.3x103 cfu/gm in flour and bread respectively. this group of bacteria (tcb) was not recorded in basbousa (table 1). the counts of t.v.b were 68x104 and 52.7x104 cfu /ml in the epidermis of both of potato and cucumber respectively. while the count was reduced to a lowest count 1.0x102 cfu /ml of the inner tissues of both potato and cucumber. it table 1 counts of total viable bacteria (t.v.b) and total coliform (t.c.b) & log10 cfu/ ml for samples of food collected from al-mansoura city, egypt. t.c.b t.v.b types of food counts log 10 counts log 10 (cfu/ml) (cfu/ml) i-carbohydrates bread 40.5x103± 0.5 4.61 5.3x103± 0.4 3.72 flour 35.3x103± 0.9 4.55 5.4x103± 0.4 3.73 basbousa 1.1x103± 0.1 3.03 0.0 0.0 ii-vegetables potato outer tissue 68x104± 1.0 5.83 3.2x103± 0.3 3.51 inner tissues 0.1x103± 0.1 1.98 0.0 0.0 cucumber outer tissue 52.7x104± 2.5 5.72 1.8x103± 0.2 3.26 inner tissues 0.1x103± 0.0 2.0 0.0 0.0 iii-proteins minced meat 28x103± 3.5 4.44 6.4x103± 0.3 3.80 cheese 42.7x104± 2.3 5.63 2.1x103± 0.2 3.32 milk 39.7x104± 0.6 5.60 2.33x103± 2.5 3.37 all values are the means of triplicate plate. fig. 1 the percentage of proteolytic and non-proteolytic isolates from different kinds of food is 340 ital. j. food sci., vol. 27 2015 is important to notice that tcb was not recorded in inner tissues of potato and cucumber. the counts of tcb were 3.2x103 and 1.8x103 cfu/ ml from the outer tissue of potato and cucumber respectively. the total viable bacterial count in milk was 39.7x104cfu/ml while the tcb was 2.33x103 cfu/ml. from table 1 we notice that the highest count of total viable bacteria was 42.7x104 in cheese. and also the lowest count of tcb was 2.1x103cfu/ml in cheese. from table 4, the count of t.v.b was 28x103 cfu/ml, while the count of tcb was 6.4x103 cfu/ml. 3.2. screening test for detection of most potent proteolytic bacterial species fig. 1 showed the potency of proteolytic activities of all purified isolates. the proteolytic isolates were 326 isolates (63.92%), while the nonproteolytic isolates were 184 isolates (36.08%). it also shows that the largest clearing zone was 21 mm in case of isolates 62 and 412 (fig. 2). this indicates that these isolates were the most potent of proteolytic activity. 3.3. presumptive and confirmation identification the cultural study, morphological appear ance, gram reaction and physiological characteristics of four most potent proteolytic isolates table 2 morphological and biochemical features of strains 62 and 412. character and biochemical tests code of isolate 62 412 growth aerobic or facultative anaerobic facultative anaerobic morphology of colony colony smooth, convex, circular and creamy in color colony smooth, convex, circular and creamy in color gram stain + cell shape straight rods, arranged in paires straight rods, arranged singly or in pairs motility motile motile flagella arrangement peritrichous peritrichous oxidase -gelatine hydrolysis + d-glucose, acid production + + d-glucose, gas production + nitrate reduction + + catalase production + + oxidationfermentation f f voges-proskaure nt arginine dihydrolase nt acid production: l-arabinose + lactose nt + maltose nt + trehalose nt + d-mannitol + d-xylose + indole production nt + methyle red nt + citrate (simmons) nt h 2 s production nt nt= not tested for this isolate because of results of another biochemical tests, f= fermentative. fig. 2 example of proteolytic activity of isolated species. were studied according to krieg et al. 1994. these results were tabulated in table 2. the presumptive identification of the two most potent proteolytic activities. isolate numbers 62 and 410 were identified as bacillus cereus and escherichia coli respectively. the most potent bacterial isolates were confirmed by using biolog microlog 34.20 system for identification. 16s rrna gene bands which were detect by specific primer at 1500 bp. the 16s rrna sequences for two isolates were blasted with genebank sequence ital. j. food sci., vol. 27 2015 341 database (table 3) and found closet to the same isolates identified by conventional methods. 3.4. antibiotic susceptibility test of different antibiotics the antibiotic susceptibility test was obtained on the bacterial isolates by using 12 different antibiotics by disc diffusion method (table 4). bacillus cereus was resistant (non susceptible) to aztreonam and ceftazidime. escherichia coli (gram negative bacteria) were resistant (non susceptible) to vancomycin. while gram positive organisms bacillus cereus was non susceptible (resistant) to vancomycine. bacillus cereus was resistant to ampicillin, while escherichia coli was susceptible (sensitive) to ampicillin. bacillus cereus was resistant to ampicillin while escherichia coli was non susceptible (sensitive) to ampicillin. the studied bacterial species both gram positive and gram negative were sensetive to 8 antibiotics (chloramphenicol, cephadroxil, erythromycin, norfloxacin, imipenem, neomycin, ciproloxacin and streptomycin). escherichia coli was sensitive to three more (ampicillin, aztreonam and ceftazidime) i.e. it was sensitive to 11 antibiotics, thus escherichia coli was the most sensitive bacterial species. bacillus cereus was sensitive to one more vancomycin and ampicillin respectively i.e. each of them was sensitive to 9 antibiotics. the smallest inhibition zone was 8mm which was recorded in escherichia coli due to the effect of erythromycin and ceftazidime respectively. table 4 antimicrobial susceptibility profile of studied microorganisms against different antibiotics expressed as diameter of clearing zones. antibiotics (conc.) bacillus escherichia cereus coli ampicillin am10 0 10 aztreonam atm30 0 17 cefadroxil cfr30 22 16 ceftazidime caz30 0 8 chloramphenicol c30 23 24 ciprofloxacin cip5 20 20 erythromycin e15 23 13 imipenem ipm10 37 25 neomycin n30 20 17 norfloxacin nor10 21 20 streptomycin s10 20 15 vancomycin va30 17 0 table 3 blast analysis of 16s rrna sequences of the representative isolates. closest validly described species identities isolate description accession number match total % similarity 62 bacillus cereus km007-1 kf055368 481 485 99 412 escherichia coli xuzhou21 cp001925 1465 1469 99 4. discussion the bacterial count is considered an index of quality that gives an idea about the hygienic measures during processing and helps in the determination of keeping quality of the product (aberle et al., 2001). this work will emphasize on the counts and characteristics of bacterial genera that is considered to be important in healthy foods giving an attention to their classification and identification. the main scope of this work is to count the different bacterial species found in the different food sources and study the antibiotic susceptibility patterns of these bacterial species which are isolated from al-mansoura city, egypt. bacterial counts of foods includes t.v.b and t.c.b are similar with the results of pradnya and sonali (2008) who found the counts of t.v.b and t.c.b were in range 9-10 log cfu/ml for local open market in india. all isolated bacterial species are common components of the bacterial flora of mammals, birds, insects’ reptiles and are commonly found in soil, on plants, water and foods as normal flora (gilmore et al., 2013). these groups of bacteria were isolated by pradnya and sonoli, 2008; nazir and islam, 2007; easa, 2010; olufemi and akinyera, 2011; kudjawu et al., 2011. presence of escherichia coli and t.c.b in food usually indicates lack of hygiena in handling and post process contamination, therefore escherichia coli & t.c.b enumeration are used as food quality parameter (gonzalz et al., 2003). the present study was initiated by collection of food samples. all isolates were selected and purified and initial morphologically identified (cocci 46.7% and rods 53.3%) and (gram’s stain as gram positive rods & cocci 60.3% and gram negative rods 39.7%). for some species, the range is wide and the growth occurs in a variety of substrates (as a true for coliform bacteria) but for others (e.g. many of pathogens) can grow in limited kinds of substrates. thus, the bacteria found in food differ according to their ability of utilization of energy. the foods that are most often involved in staphylococcal food poisoning differ widely from one country to another (bennett and lancette, 1995). the present study was concerned with isolation of the bacterial content of different food samples, which collected from open markets in al-mansoura city, egypt. all isolates were selected, purified and initial morphological342 ital. j. food sci., vol. 27 2015 ly identified by shape and gram stain as 46.7% cocci and 53.3% rods and gram positive cocci and rods 60.3% and gram negative rods 39.7%. 16s rrna gene sequencing will continue to be the gold standard for the identification of bacteria, and the automation of the technique could enable it to be used routinely in clinical microbiology laboratories, as a replacement of the traditional phenotypic tests. modern technologies have made it possible to construct a high density of oligonucleotide arrays on a chip with oligonucleotides representing the 16s rrna gene sequence of various bacteria. such a design will facilitate automation of the annealing and detection of the pcr products of 16s rrna gene amplification, and hence routine identification of most clinical isolates will be possible. the use of 16s rrna gene sequencing has several advantages. first, the turnaround time is short. because amplification of the 16srrna gene takes only four to six hours, and the annealing and detection of pcr products takes only another few hours, theoretically the identification can be completed within one day. second, it can be used for slow growing bacteria, unlike most commercially available kits that are based on phenotypic tests that require the detection of growth of the organism in the presence of certain specific substrates, and hence the slow growing bacteria are usually ‘‘unidentified’’ when the growth control shows a negative result. third, the problem of ‘‘unidentifiable strains’’ will be overcome and there would be minimal misidentification – the identification of a clinical strain is clearly defined by the number of base differences between it and the existing species. fourth, oligonucleotides representing all bacterial species, including those rarely encountered clinically, can be included in the array, making it easy to identify the rare species. lastly, such a technique will be applicable not only to pyogenic bacteria, but also to other organisms such as mycobacteria (el-hadedy and abu el-nour, 2012). antibiotic resistance (gram positive and gram negative bacterial species) from food sources are important and serious problem in clinical field (el-aidy, 2007). the antibiotic sensitivity against bacteria is assayed by disc-diffusion method and in our study as well (selim, 2011; selim et al., 2012; 2013)). in this study, the antibiotics susceptibility patterns of potent proteolytic bacterial species (bacillus cereus and escherichia coli) against 12 different antibiotics were investigated. antibiotics include ampicillin (am), aztreonam (atm), cefadroxil (cfr), ceftazidime (caz), chloramphenicol (c), ciprofloxacin (cip), erythromycin (e), imipenem (ipm), neomycin (n), norfloxacin (nor), streptomycin (s) and vancomycin (va). in this study bacillus cereus was resistant to 3 antibiotics (ampicillin, azactam and cerazdime). beta-lactam antibiotics also bind to inhibit the action of other cytoplasmic proteins that had a role in peptidoglycan synthesis and turn over (abigal and dixe, 1994). transpeptidation reactions that cross links the peptide side chain of polysaccharide peptidoglycan back bone. transpeptidase and other proteins were called “penicillin binding protein”. the net result of betalactam binding to this protein was to stimulate endogenous enzyme that degrade peptidoglycan (abigal and dixe, 1994). the inhibitory effect of vancomycin was similar to the effect of beta-lactam antibiotic. our results showed that bacillus cereus and escherichia coli were sensitive to cefadroxil, chloramphenicol, ciprofloxacine, erythromycin, imipenem, neomycin, norfloxacin and streptomycin. neomycin, aminoglycoside antibiotic; erythromycin, as macrolid antibiotic have the ability to bind to the 50s or 30s ribosomal subunit (inhibit protein synthesis). also, inhibitory effect of ciprofloxacin and norfloxacin, as quinolones antibiotic, may be due to having the ability to inhibit bacteria by interfering with their ability to make dna with diverse targets dna gyrase, this inhibition effect leads to preventation of multiply of bacteria. the present results showed that all tested escherichia coli strains were resistant to vancomycin antibiotic, while srinivasona et al., (2007) who found that all tested escherichia coli strains were resistant to two or more antimicrobial used in veterinary medicine. bacillus cereus was resistant to aztreonam and ceftazidime. antibiotic resistance can be categorized in three types: natural or intrinsic resistance; mutational resistance and extrachromosomal or aquired resistance. the resistance of isolates to beta-lactam antibiotic may be due to drug inactivation: i.e. ampc cephalosporinase (beta lactamase enzyme that open the beta-lactam (ring) as an intrinsic resistance. target site modification (i.e. change in pbpspenicillin binding proteins-) as mutational resistance represented in drug inactivation (abigail and dixie, 1994). moreover the resistance of isolates to aminoglycoside antibiotic and erythromycin macrolides antibiotics may be due to inaccessibility of the target as an intrinsic resistance, reduced permeability or uptake as mutational resistance and aquired resistance represented in drug activation (diab et al., 2002; 2004). the resistance of isolates to furadantine antibiotic may be due to chromosomal or plasmide mediated and inhibition of nitrofuran reductase. also the resistance of isolates to fluoroquinolones antibiotics may be due to reduced permeability or uptake as mutational resistance (fange et al., 2009). the mode of action of ciprofloxacin and norfloxacine as a quinolones antibiotic as accumulated with the explanation of fange et al., (2009). in our present study, four studied bacterial species which isolated from food sources were investigated to all selected antibiotics. most of the selected antibiotics represent the following classes ; beta-lactam, aminoglycoside, macrolide and quinlones. total bacterial counts is conital. j. food sci., vol. 27 2015 343 sidered an index of quality which gives an idea about the higienic measures during processing and help in the determination of keeping quality of the product (aberle et al., 2001). in this study, the identified bacterial species were bacillus cereus (13.72%) and escherichia coli (18.3). these results had a strong support of many researches. these bacterial species are common components of the microbial flora community. generally, the methods of production, transportation, handling and sale of food entirely unhygienic and entirely depend on the traditional system, such system could pose favorable environment for bacterial contamination. the existences of these bacterial species which isolated in different food sources. these results are agreement with fda, 2000. moreover the studied food sources are considered as reservoir for some pathogenic bacteria (manges et al., 2006). the bacillus species are of the soil origin and may contaminate bread through the raw material and bakery requirements used. bacillus cereus is 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association, washington, dc., 1219. who (world health organization) 2002a. food safety and foodborne illness. world health organization fact sheet, geneva, 237. paper ital. j. food sci., vol. 27 2015 357 keywords: camel milk, kefir, grain, traditional method, chemical properties, microbial properties, flavor profile analysis kefirs manufactured from camel (camelus dramedarius) milk and cow milk: comparison of some chemical and microbial properties g. kavas department of dairy technology, faculty of agriculture, ege university, 35100 i̇zmir, turkey *corresponding author: tel. +90 232 3111420, email: gokhan.kavas@ege.edu.tr abstract this study examined the production possibilities of kefir from fresh camel milk fermented with grain. the findings were then compared with kefir manufactured from cow’s milk. cow’s milk was fermented with 2.5% grains. the 1% (v/w) glucose enriched camel’s milk was fermented with 10% grains and left in an incubator at 25°c. physical-chemical and sensorial analyses of the kefir samples were measured on day one (18 hours) of storage and microbiological analyses were measured on days one, three and five. some physical-chemical parameters were found to be higher in camel milk and its kefir than in cow milk and its kefir, some were found to be close and some were found to be lower. addition of 1% glucose and 10% grains to the camel milk affected the titration acidity and viscosity of kefir to significant levels. the kefir produced from camel milk was perceived as sourer, whereas its other properties were found to be close to those of cow milk. the cholesterol levels of camel milk and its kefir were detected to be higher when compared to those of cow milk and its kefir, but the cholesterol level decreased in both examples after the production of kefir. in terms of the composition of fatty acids, it was determined that sfa and the small, medium chain fatty acids ratio was low in camel milk and its kefir, but mufa and the long chain fatty acids ratio was high. pufa ratio was high in camel milk but low in its kefir. in microbiological analysis, yeast levels increased in kefir samples with the lactobacillus ssp. strains, and the increase in the number of yeasts was higher than in the cow milk kefir. in kefir samples, lactobacillus ssp. strains increased on day one and three of storage, but diminished after day three. 358 ital. j. food sci., vol. 27 2015 introduction kefir is a dairy product that has been produced for years in eastern europe and mongolia, before spreading to caucasia (gaware et al., 2011). kefir is produced by adding specific amounts of the kefir grain (traditional method) or the modified culture (industrial method) manufactured from this grain (pogačić et al., 2013) into the milk of various animals. ethyl alcohol and lactic acid fermentations are developed together during the product formation, thus causing it to taste somewhat acidic. kefir grains are off-white and slightly yellowish, irregular in shape and with a circumference taken up by polysaccharide matrixes that (jianzhong et al., 2009) compose 25% of the dry weight soluble in water (pogačić et al., 2013), and a diameter of 0.3cm -2 cm (beshkova et al., 2002). homofermentative lactobacilli make up 65-80% of the flora. in the grain flora, homofermentative and heterofermentative lactic acid streptococci make up 20%, and lactose-fermentative and non-fermentative yeasts make up a further 5%. the percentage of acetic acid bacteria (in production with grain) is relatively small (irigoyen et al., 2005). species of the microorganisms in the grain, their proportion to each other and their numbers change according to the origin of the grain and conditions of use (ferreira et al., 2010). today, kefir is regarded as a fermented dairy beverage that is anti-bacterial and antiinflammatory (lopitz-otsoa et al., 2006), anti-tumoral (shiomi et al., 1982), anti-apoptotic (matsuu et al., 2003), anti-allergic (umeda et al., 2005), anti-oxidant, and anti-mutagenic (liu et al., 2005). it also lowers systolic and diastolic blood pressure and bad cholesterol (agerbaek et al., 1995), adjusts lactose dyspepsia (hertzler and clancy, 2003), and contains bioactive peptides, exopolysaccharides and their bacteriosis, and has a strong probiotic effect on human health (rattray and o’connell, 2011). camel milk differs from the milk of other ruminant animals in its composition and physiological properties. camel milk is rich in long chain fatty acids, but contains low amounts of short chain fatty acids (gorban and izzeldin 1999). vitamins a, b2, e, c and minerals ca, na, k, zn, mg and fe are far more abundant in camel’s milk than in cow’s milk. lactose intolerant people (elhatmi et al., 2007) can consume camel milk. due to camel milk not containing β-lactoglobulin and some casein derivatives, it is similar to human milk with its hypoallergenic (shabo et al., 2005), immunoglobulin content. it is anti-diabetic effective (hamad et al., 2011) and because it contains more peptidoglycan recognition proteins (pgrp) and natural protective proteins than other ruminant milk, it has an antimicrobial and antiviral effect (el-agamy et al., 1992). not to mention that camel milk is anti-carcinogenic and anti-hypertensive (hamad et al., 2011) and renoprotective, it reinforces immunity, increases metabolism and muscle mass, is bone-forming and also has therapeutic effects on some diseases such as hepatitis b, autism (laila et al., 2013) and tuberculosis (agrawal et al., 2004). today, we know that yoghurt, probiotic yoghurt (attia et al., 2001; el-agamy et al., 1992), stabilizer augmented yoghurt (mulior et al., 2013) and many mild cheeses whose clotting is poor due to enzymatic coagulation (mehaia, 1993; ramet, 1987) can be produced from camel milk. our research utilized kefir that was manufactured from camel (camelus dramedarius) milk (cam). kefir made from cow’s milk (com) was used as the control sample. the physicalchemical properties in raw milks were analyzed, along with these properties in the cow milk kefir (comk) and camel milk kefir (camk) samples. sensorial tests were conducted as of the eighteenth hour of day one and microbiological analyses were made on day one, three and five of storage. materials and methods camel milk, kefir grain camel (camelus dramedarius) milk was procured from a native camel farm in denizli sarayköy (turkey). cow milk and kefir grain were procured from the department of dairy technology pilot dairy plant, ege university agricultural faculty. kefir production in this study, kefir was produced from camel and cow milk using the traditional (grain) method as shown in fig. 1. physical-chemical analyses in the raw milk and kefir samples, dry matter (binder ed-53 germany) and ash (protherm pfl 110/6 turkey) were calculated via a gravimeter, fat with the gerber method, ph value of the titration acidity in terms of lactic acid with a ss-3 zeromatic (beckman instruments inc., california, usa) brand ph meter, protein with the kjeldahl method (aoac, 1990), lactose value with an atago polax x 2l (japan) model polarimeter (horwitz, 1965), viscosity value with a brookfield digital viscometer, model dv-ii+pro (usa) model viscometer as cp [at speed 180 mpa, between 15.7% and 67.7% torque]. determining the fatty acid composition in samples and preparation of fat extraction and fatty acid methyl esters each homogenized sample was extracted using the gerber method, thus fat was obtained ital. j. food sci., vol. 27 2015 359 fig. 1 kefir production with the traditional (grain) method. ** the glucose and grain ratio added to camel milk and cow milk was determined as a result of preliminary study. ***physical-chemical and sensorial analyses were measured at day one (18 hours) of storage and microbiological analyses were measured at days one, three and five. (iso 11870:2009 idf 152:2009) and fatty acid methyl esters were prepared pursuant to aocs (2009), after which they were examined in the gas chromatograph (gc). [chromatography is a supelco sp-2380 fused silica capillary column (60 m 0.25 mm i.d., 0.2 mm film thickness; supelco inc., bellefonte, pa, usa) and flame ionization detector hewlett-packard gc (model 6890). injection volume was 1 µl. gc furnace temperature was set to reach 220°c from 100°c when 4°c/minute. injector and detector temperature was 300°c, carrier gas was helium and the flow rate was 1 ml/min]. determining the cholesterol level in samples in samples, the cholesterol level was analyzed according to the findings of ossa et al., (1995); and then examined by gas chromatography (gc). [chromatography was a hp-5 silica capillary column (25 m 0.32 mm i.d., 0.52 mm film thickness; hewlett-packard, usa) and fid 360 ital. j. food sci., vol. 27 2015 (flame ionization detector) hewlett-packard gc (model 6890). injection volume was 1µl. gc furnace temperature was set to 300°c, injector temperature to 280°c, colon temperature to 270°c for 15 minutes. carrier gas was helium and the flow rate was 1.5 ml/min. microbiological analyses for counting lactobacillus ssp., de mann rogosa sharpe (mrs) agara (merck darmstadt, germany) (sharpe et al., 1966) was used. lactococcus ssp. was cultured on m l7 agara (merck darmstadt, germany) (terzaghi and sandine, 1975). for yeast, glucose-salt agara (anonymous, 1990) plantation was done. isolation and census of lactic acid bacteria were conducted according to idf standard 149 a (1997) and idf standard 163 (1992). yeasts were incubated at 25°c for three to five days. after this period, colonies that had developed in petri dishes were counted as cfu/ml on days one, three and five of storage. sensory evaluation the sensory evaluation was made by a panel of nine individuals who evaluated kefir samples in terms of consistency and flavor on a scale from 1 to 5. for altuğ and elmaci (2011), the method of flavor profile analysis was utilized. statistics two different milk and two different kefir samples were analyzed in three parallels and two repetitions. spss statistics analysis software (ibm spss statistics) was used. data that gained importance were analyzed using the variance analysis anova based on the duncan multiple comparison test on a p<0.01 level. results and conclusions in the cam sample, compared to the com sample, fat and lactose values were found to be table 1 physical-chemical properties of raw camel milk, raw cow milk and of kefirs produced from these milks. analyses milk samples kefir samples cam com camk comk dry matter 12.73±0.12a 12.80±0.09b 11.10±0.02c 10.70±0.03d fat (%) 3.60±0.08a 3.50±0.06b 3.20±0.02c 3.30±0.01d titratable acidity 0.127±0.02a 0.132±0.02b 0.92±0.01c 0.81±0.02d ph 6.46±0.32a 6.44±0.27b 4.10±0.10c 4.55±0.14d protein (%) 3.05±0.03a 3.21±0.03b 2.82±0.03c 3.09±0.03d ash (%) 2.932±0.10a 1.461±0.09b 1.423±0.05c 1.068±0.05d lactose (%) 6.22±0.05a 6.20±0.51b 3.45±0.07c 3.54±0.02d a, b, c, d: the differences between the values in the same rows are statistically significant (p < 0.01). higher; there was twice as much ash and similar ph and dry matter values. in addition, protein, titration acidity in terms of lactic acid (table 1) and viscosity values were detected to be lower. in the research, it was determined that results regarding the ph value 6.46 ph, fat (3.60%), protein (3.05%), lactose (6.22%), total dry matter (12.73%), ash (2.932%) and titratable acidity in terms of lactic acid (0.12%) of camel milk were within the boundaries found in the literature, and that the lactose and ash levels were higher in our studies (fao, 1982; el-amin and wilcox, 1992). ph values of kefir samples were determined to correspond with the turkish food codex (2009/25) and wszolek et al. (2006). lactic acid was detected to be higher in the camk sample (0.92%la) than in the comk sample (0.81%la), whereas for viscosity, it was vice versa. with the addition of 1% (v/w) glucose into the cam sample, simulating the development of lactic acid bacteria, we aimed to increase the titration acidity and viscosity. to this end, the effect that the addition of 1% glucose (v/w) and 10% grain into the cam sample has on titration acidity and viscosity was found to be significant (p<0.01), and lactic acid and viscosity were detected to have increased. however, viscosity in the camk sample was lower than the comk sample. in addition, the glucose and grain ratio added to camel milk was determined as a result of preliminary study. in that preliminary study, some portion of the cam sample was inoculated with yeast grains in ratios of 2.5%, 5%, 7.5% and 10% without the addition of glucose, while the other portion of the sample had the same process with the addition of glucose. afterwards, viscosity values and titration acidity were detected in kefir samples, and viscosity values are given in tables 2 and 3. grain being added into camel milk by 2.5% did not have a major effect, and since the 2.5% grain addition into the comk sample provided the desired viscosity value, other grain ratios were not tested. all in all, with the 1% glucose (v/w) and 10% grain addition into camel milk, a four times greater increase was reached in viscosity than in the one with just the grain addition, and the titration acidity diminished from ital. j. food sci., vol. 27 2015 361 4.78 ph (in the sample with grain addition only) to 4.10 ph. results correspond with the literature (lewis, 1986). in this study, problems that might occur in fermentation were associated with low viscosity value obtained from the camk sample, lower serum protein content of camel milk than cow milk (farah, 1996), poor interaction between denature serum proteins of camel milk and k-casein, lack of β-lactoglobulin from serum proteins and different derivatives of β-casein, low amounts of catable 2 viscosity values of the kefir samples produced by injecting grain in certain amounts in cam with/without glucose addition and the ones produced from com without glucose addition (cp). grain ratio (%) camk (gra) camk (gra+g) comk (gra)   viscosity (cp) viscosity (cp) viscosity (cp)  2.5 5.21aa 5.87 ab 111.475c 5 5.46ba 8.44 bb nt 7.5 7.12ca 18.81 cb nt 10 9.28da 37.18 db nt nt:not-tested ; gra: grain ;g+gra: glucose +grain a, b, c, d, e: the differences between the values in the same column are statistically significant (p < 0.01). a, b, c, d: the differences between the values in the same rows are statistically significant (p < 0.01). sein and its derivatives (laleye et al., 2008) and the anti-bacterial effect of camel milk (hashim and khalil, 2004). many factors (content of protein and denature serum proteins, casein ratio and its content, interactions between denature serum proteins and k-casein) may affect viscosity (puvanenthiran et al., 2002). based on the ph change, these factors might also affect the micelle surface area and size, micelle content and water binding capability in casein micelles (anema and klostmeyer, 1996; dalgleish and table 3 fatty acid compositions of kefir samples made from camel/cow milk (g/100g) name of fatty acid methyl ester (g/100g) and formula of molecule com comk cam camk butyric acid methyl ester (c4:0) 0.064 0.043 nd nd caproic acid methyl ester (c6:0) 0.264 0.422 0.140 0.121 caprylic acid methyl ester (c8:0) 0.037 0.024 0.003 0.002 capric acid methyl ester (c10:0) 0.085 0.061 0.004 0.003 undecanoic acid methyl ester (c11:0) 0.007 0.009 nd nd lauric acid methyl ester (c12:0) 0.127 0.101 0.021 0.021 tridecanoic acid methyl ester (c13:0) 0.002 0.017 0.005 0.004 myristic acid methyl ester (c14:0) 0.386 0.303 0.321 0.309 myristoleic acid methyl ester (c14:1) 0.051 0.034 0.050 0.050 pentadecanoic acid methyl ester (c15:0) 0.021 0.013 0.029 0.029 cis-10pentadecanoic acid methyl ester (c15:1) 0.012 0.036 0.013 0.010 palmitic acid methyl ester (c16:0) 0.901 0.740 1.049 0.967 palmitoleic acid methyl ester (c16:1) 0.063 0.027 0.292 0.268 heptadecanoic (margaric) acid methyl ester (c17:0) 0.015 0.011 0.020 0.018 cis-10-heptadecanoic acid methyl ester (c17:1) 0.016 0.074 0.024 0.019 stearic acid methyl ester (c18:0) 0.430 0.361 0.507 0.457 oleic acid methyl ester (c18:1n9c) 0.840 0.581 0.843 0.715 linoleic acid methyl ester (c18:2 n6c) 0.088 0.072 0.107 0.099 γ-linolenic acid methyl ester (c18:3 n6) 0.034 0.177 0.053 0.036 arachidic acid methyl ester (c20:0) 0.028 0.017 0.028 0.023 cis-11eicosenoic acid methyl ester (c20:1) nd nd 0.013 0.010 cis-11,14-eicosadienoic acid methyl ester (c20:2) nd nd 0.009 0.005 arachidonic acid methyl ester (c20:4n6) 0.012 0.067 0.028 0.017 behenic acid methyl ester (c22:0) 0.016 0.094 0.023 0.009 other fatty acids nd 0.017 0.017 0.007 short-chain fatty acids (4-6c) 0.33 0.46 0.14 0.12 medium-chain fatty acids (8-12c) 0.26 0.20 0.03 0.03 long-chain fatty acids (>12c) 2.92 2.62 3.41 3.05 saturated fatty acids (sfa) 2.38 2.22 2.15 1.96 monounsaturated fatty acids (mufa) 0.98 0.75 1.24 1.07 polyunsaturated fatty acids (pufa) 0.13 0.32 0.20 0.16 nd: non-detected. 362 ital. j. food sci., vol. 27 2015 law, 1988, 1989). the drop in ph causes the interaction between serum proteins and case micelles and the viscosity to increase (anema et al., 2004). however, in the current study it was determined that based on the drop in ph (4.10 ph) in the camk sample, the viscosity value was lower than in the comk samples. this is thought to result from the effect of one or more of the parameters given above (anema and li, 2003) also, the camel milk is low in casein and serum proteins and the composition of these. it was found that the milk type in kefir samples has an important effect on titration acidity, ph and viscosity; the effect of titration acidity on viscosity is vital as well (p<0.01). protein, lactose and fat in the camk sample were found to be higher than in the comk sample, whereas dry matter and ash were found to be lower. in general, effect of the milk type on dry matter, fat, protein and lactose was established as significant, as well as the effect of glucose addition into milk and grain ratio on titration acidity (p<0.01). cam and com, and fatty acid compositions of kefirs produced from these milks are given in table 3. in cam and camk samples, it was determined that the short (c4:0-c6:0) and medium (c8:0-c12:0) chain fatty acids ratio, as well as the saturated fatty acids (sfa) ratio were lower than in com and comk samples, but the long chain fatty acids ratio and the monounsaturated fatty acids (mufa) ratio were higher. it was also established that the ratio of polyunsaturated fatty acids (pufa) in the cam sample was higher than the com sample; however, its ratio in the camk sample (0.16 g/100 g) was lower than in the comk sample (0.32 g/100 g). thus, the conclusion: camel milk and its kefir contain some fatty acids that affect our health positively in terms of fatty acid composition in higher amounts than cow milk and its kefir. the camel milk fatty acid composition changes pursuant to the species and the diet of that specific camel, as well as its region and the climate of that region (chilliard et al., 2000; konuspayeva et al., 2008). the results we obtained from this research were similar to the results of other researchers (agrawal et al., 2004; shamsia, 2009). in the cam, com, camk and comk samples, cholesterol levels were different and an important relationship between the milk type and the cholesterol level was detected (p<0.01) (table 4). along with this, it was determined in the research that the cholesterol level decreased after production of kefir by using different milks, and the effect that kefir production has on the drop in cholesterol levels was regarded as significant (p<0.01). according to some researchers (gorban and izzeldin, 1999; goudjil et al., 2003; konuspayeva et al., 2008; sieber, 2005), cholesterol level of kefir production was higher than cow milk, but it was also found to be lower according to some other researchers (alabdulkarim, 2012; agrawal et al., 2004). initially, in the grain, lactobacillus ssp. strains were found to be as 1.93 x107 cfu/ml, lactococcus ssp. strains as 5.54 x107 cfu/ml and yeast as 1.68x106 cfu/ml. in the study, lactobacillus ssp. strains (fig. 2a) and yeast levels (fig. 2b) increased in both kefir samples throughout the storage process. in addition, the increase in the lactobacillus ssp. strains in the camk sample was found to be higher. lactococcus ssp. strains (fig. 2c) were detected to have increased in kefir samples at day one and three of storage, but to have decreased after day three. levels of lactobacillus ssp. strains in kefir samples were close to one another at the inception of storage, but the one in the camk sample took the lead after day one of storage. lactobacillus ssp. strains in the camk sample at days one, three and five of storage increased, in comparison with the starting level, respectively at levels of 0.99 cfu/ml, 1.71 cfu/ml and 2.59 cfu/ml. however, in the comk sample, it was respectively: 0.91 cfu/ ml, 1.28 cfu/ml and 2.18 cfu/ml. lactococcus ssp. strains in the comk sample at days one and three of storage increased, in comparison with the starting level, respectively at levels of 0.05 cfu/ml and 1.02 cfu/ml. however, this lessened in day five by 0.41 cfu/ml compared to day three of storage. development of lactococcus ssp. strains in the camk sample was the same as the comk sample at day one; however, its increase after day one of storage was lower than the one in the comk sample. in the camk sample, an increase respectively at levels 0.04 cfu/ml and 0.8 cfu/ml was detected in day one and three of storage in comparison with the starting level, whereas after day five, a decrease took place. this decrease was ten times more than the comk sample. yeast level increased in both kefir samples throughout the storage process, but the one in the comk sample was approximately three times higher than the camk’s. generally, it was concluded that microorganism levels in camk and comk samples in storage were above the minimum values set forth by “turkish food codex, communiqué on fermented milks” (turkish food codex, communiqué no: 2009/25). table 4 cholesterol levels of kefirs samples made from camel/cow milk (mg/100g). cholesterol levels (mg/100g) com comk 14.60aa 7.97ab cam camk 21.28ba 18.24bb a, b, c, d, e: the differences between the values in the same column are statistically significant (p < 0.01). a, b, c, d: the differences between the values in the same rows are statistically significant (p < 0.01). ital. j. food sci., vol. 27 2015 363 fig. 2 lactobacillus ssp. (a), yeast (b) and lactococcus ssp. (c) levels in kefirs produced from camel and cow milks. research helped discover an important relationship between the milk type and the storage period on microorganism levels. in addition, the effect that glucose has on microorganism development was established as significant (p<0.01). in the camk sample produced with the addition of 1% glucose, increase in the lactobacillus ssp. strains was higher than in the comk sample, but this was vice versa for the increase in the yeast level. progress of the increase (lactobacillus ssp., yeast), also the decrease (lactococcus ssp.) in the microorganism levels in the camk sample after day three of storage show parallels with the comk sample (koroleva et al., 1978; koroleva and bavina, 1978; oner et al., 2010). in the research, microbial flora in the camk sample went through a different development. this result corresponds with the literature data regarding other fermented dairy products of camel milk (abutarboush, 1996; attia et al., 2001; jumah et al., 2001; abdel rahman et al., 2009). in addition, the research pinpointed that the usage of grain in producing kefir from camel milk was more effective (abu-tarboush, 1996; abdel rahman et al., 2009; mehaia, 1993). in the flavor profile evaluation of kefir samples, panelists determined that sour, sweet, salty, bitter, fermented milk, cream, greasy, cheesy, sharp, gas, alcohol, metallic and burnt milk flavor densities were perceived differently between the camk and comk samples (fig. 3). in the flavor profile evaluation, it was detected that the camk sample was sourer, cheesier and had a sharper aroma than the comk sample. consistency and appearance in the camk sample were detected to be lower than the comk sample. in general, the camk sample was appreciated by the panelists and was defined as “sourer” than the comk sample. fig. 3 flavor profile evaluation of the camk and comk samples. 364 ital. j. food sci., vol. 27 2015 conclusions in the current research, an increase was obtained in the viscosity value in the kefir produced by adding 1% (v/w) glucose and 10% grain into camel milk. dry matter, ash and titratable acidity in the camel milk kefirs were higher than in cow milk kefirs; whereas fat, ph, protein and lactose values were lower. cholesterol level of camel milk and its kefir product was found to be higher than that of cow milk and its kefir. along with this, it was detected in this study that proportion of camel milk and its kefir to cow milk and its kefir in terms of sfa is low. however, it is high in terms of mufa. the pufa ratio of camel milk is high compared to cow milk. however, the pufa ratio in the camel milk kefir was defined to be lower than the one in the cow milk kefir. lastly, it was also confirmed in the study that some compounds, which have positive effects upon metabolism in camel milk and its kefir, have a higher impact than on the cow milk kefir’s metabolism. references abdel rahman i̇.a., dirar, h.a. and osman m.a. 2009. microbiological and biochemical changes and sensory evaluation of camel milk fermented by selected bacterial starter cultures. afr. j. food sci. 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(ed.) fermented milks. blackwell science ltd, oxford, pp. 174-198. paper received september 4, 2014 accepted november 5, 2014 98 issn 1120-1770 online, doi 10.15586/ijfs.v33i4.2099 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (4): 98–106 effect of different plants’ aromatic essential oils on frozen awassi lamb meat’s chemical and physical characteristics ahmed s.a. al-obaidi1, ayad b. mahmood2, zaid k. khidhir2,*, hemn g. zahir2, ziyad t. al-doori3, hiewa othman dyary4,* 1department of animal production, college of agriculture, university of diyala, iraq; 2department of animal sciences, college of agricultural engineering sciences, university of sulaimani, iraq; 3department of public health, college of veterinary medicine, university of tikrit, iraq; 4department of basic sciences, college of veterinary medicine, university of sulaimani, sulaymaniyah, iraq *corresponding authors: zaid k. khidhir, department of animal sciences, college of agricultural engineering sciences, university of sulaimani, iraq. email: zaid.khzir@univsul.edu.iq; hiewa othman dyary, department of basic sciences, college of veterinary medicine, university of sulaimani, sulaymaniyah, iraq. email: dyary.othman@univsul.edu.iq received: 10 july 2021; accepted: 2 november 2021; published: 8 december 2021 © 2021 codon publications open access paper abstract the effect of drenching awassi lambs with three aromatic essential oils from sage (salvia officinalis l.), clove (syzygium aromaticum l.), and laurel (laurus nobilis l.) was investigated on meat chemical and physical characteristics, and oxidative and deterioration measurements. twenty-four awassi lambs, five to six months old, were divided into four groups. a concentrated diet was provided to the lambs at a rate of 3% of the body weight. the treatments were as follows: t1 was served as the untreated control, while t2, t3, and t4 were drenched with oils of sage, clove, and laurel, respectively. drenching was carried out using water-soluble capsules containing 500 mg oil/capsule/day. treatments lasted 90 days. at the end of the treatment period, the animals were fasted overnight and slaughtered. the carcasses were cleaned and kept at 4°c for 24 h. the longissimus dorsi (ld) muscle was then separated and preserved in a plastic bag for three preservation periods: no freezing and 30 days and 60 days freezing at −18°c. several physical, fat, and protein stability analyses of meat were done after the preservation periods. the results indicated no significant effect of drenching awassi lambs with different aromatic essential oils on the meat’s physical and chemical characteristics. however, these oils, especially clove oil, affected fat and protein stability with increasing preservation period by freezing. keywords: awassi; essential oil; laurus nobilis l.; meat; salvia officinalis l.; syzygium aromaticum l. introduction meat is classified as a perishable commodity due to its high moisture content and nutrient availability, making it suitable for microbial growth (kumar et al., 2015, 2017). meat contamination might occur at different stages, starting from the field and ending with the preparation for consumption, including slaughter, transportation, handling, storage, processing, marketing, and consumer’s handling (niyonzima et  al., 2015). treatments used to keep meat from contamination and spoilage vary with each stage, including heating, refrigeration, hydrostatic pressure, packaging, ionizing radiation, chemical preservatives, salts, and bioactive compounds. selecting appropriate treatments to maintain the meat and meat products’ hygiene depends on several factors (chen et al., 2012). fats’ oxidation and destruction by free radicals in cell membranes are natural processes affecting membrane italian journal of food science, 2021; 33 (4) 99 effect of different plants’ aromatic essential oils shelf-life without affecting the meat’s quality and taste is important. therefore, this research aimed to investigate the effects of three aromatic essential oils from sage (salvia officinalis l.), clove (syzygium aromaticum l.), and laurel (laurus nobilis l.) on meat chemical and physical characteristics, and oxidative and deterioration measurements after different freezing periods. materials and methods extraction of volatile oil dried seeds of clove (syzygium aromaticum l.) and green leaves of sage (salvia officinalis l.) and laurel (laurus nobilis l.) were collected from local markets in sulaymaniyah. the plants’ identities were confirmed at the department of horticulture, faculty of agricultural sciences, university of sulaimani, where voucher specimens were deposited. the plants were dried in a freeze dryer and ground in a laboratory grinder. a hot oil extraction technique was used to extract oils. then, water was added at a ratio of 5:1, and the mixture was subjected to hydro-distillation for 3 h using a clevenger-type apparatus (clevenger, 1928). the volatile oil content was calculated as a relative percentage (v/w). later, the essential oil was extracted from the milled sample by the hydro-distillation method using the clevenger set in 1000 ml distilled water and refrigerated until use (ranjitha and vijiyalakshmi, 2014). animals and treatments twenty-four awassi lambs, 5–6 months old, with an average weight of 28.4 kg, were divided according to weight into four treatment groups. the lambs were raised in individual cages and acclimatized for 2 weeks, followed by a treatment period lasting 90 days. during the experimental period, the lambs were fed a concentrated diet consisting of 35% wheat flour, 40% barley, 12% wheat bran, 10% soybean meal, 2.9% salt and limestone, and 0.1% premix. the energy content was 2791 cal/kg, and the protein content was 13.75/kg. feed was provided to the lambs at 3% of their body weight. the lambs were subjected to weekly weight measurements, and the amount of feed was adjusted according to weight change. the first group of lambs (t1) served as a control without drenching. groups t2, t3, and t4 were drenched with sage oil, clove oil, and laurel oil, respectively. the drenching process was carried out using plastic syringes attached to a rubber tube. each animal was given 0.5 ml of the extracted oil daily. after the treatment period, the animals were fasted overnight and slaughtered. the carcasses were cleaned and transport and functions. cell membrane phospholipids are rapidly affected by the oxidation process closely related to the fatty acids’ saturation level. free radicals react with these fatty acids and produce hydroperoxides that decompose to volatile aromatic compounds such as alkanes and aldehydes. these toxic substances affect animal products’ nutritional value (aminzare et  al., 2019), safety for consumption, quality of meat, organoleptic characteristics, and storage period (fernandes et al., 2018). controlling free radicals’ biological damage has recently become a topic of interest to researchers. synthetic antioxidants such as butylated hydroxyanisole (bha) and butylated hydroxytoluene (bht) have been used commercially to prevent or reduce lipid peroxidation’s unwanted effects. however, the demand to find or use plant-based natural compounds has recently increased, with consumers trying to protect meat and meat products from oxidation, spoilage, and pathogens (aminzare et  al., 2019; veneziani et  al., 2017). natural antioxidants play an essential role in this field, protecting food from spoilage and maintaining public health. essential oils are natural sources rich in antioxidants that inhibit free radicals such as phenolic compounds. these oils preserve animal fatty tissue and protect animal products from off-flavor and reactive oxygen resulting from oxidation of polyunsaturated fatty acids (nitiema et  al., 2012). bioactive compounds such as essential oils improve food quality and protect consumers from the adverse effects of oxidative stress and microbial spoilage (simitzis and deligeorgis, 2011). essential oils are complex mixtures of many ingredients and are mainly composed of terpenoids with low molecular weight aliphatic hydrocarbons such as aromatic aldehydes and phenols (dorman and deans, 2000). they have antimicrobial activities, as many studies have confirmed the antimicrobial effects of these oils when used with different foods, which extends the shelf life by reducing spoilage (calo et  al., 2015). for example, adding 0.25% cassia oil to refrigerated fresh chicken sausages increases the shelf life by 5–6 days and lowers the microbial count (sharma et al., 2017). in another study, it was found that wrapping chicken meat burgers with edible film incorporated with 0.10% oregano and 0.15% thyme essential oils increased shelf life up to 30 days (soni et al., 2018). feed processing with antioxidants such as essential oils is one of the easiest ways to deliver these antioxidants to membrane phospholipids, reducing the oxidation from free radicals and protecting animal products such as meat from fat oxidation (fasolato et al., 2015). therefore, this method is considered the easiest and the most effective compared to the treatment of postmortem meat. there is a lack of sufficient research on using essential oils with awassi sheep, the most common breed raised in iraq. fresh meat or meat refrigerated for 2–3 days is used for cooking. finding a method to naturally increase the 100 italian journal of food science, 2021; 33 (4) al-obaidi asa et al. linear increase (p ≤ 0.05) in thaw loss was observed for all oils with increasing preservation periods of up to 60 days (table 1). no significant cooking loss was observed in the treated groups for all preservation periods, while a significant decrease was diagnosed with increasing preservation periods. table 2 illustrates the effect of different oils and freeze-preservation periods on tba, ffa, and tvn values of the ld muscle. the results indicated a decreasing effect of oil treatments on tba, ffa, and tvn values in all treatment groups. group t3 drenched with clove oil recorded the lowest values than the rest of the oil drenching treatments and the control for all preservation periods. data also recorded a significant linear increase in tba, ffa, and tvn values for all treatments, increasing the preservation period to 60 days. values are presented as mean ± sem. different lowercase letters indicate significant differences between means within columns, while different uppercase letters indicate significant differences between means within rows (p ≤ 0.05). tba, thiobarbituric acid; tvn, total volatile nitrogen; ffa, free fatty acids. figure 1 shows the effect of different oil treatments and freeze-preservation periods on the ld muscles’ chemical composition. there was no significant effect of oil treatments and preservation periods on the muscles’ chemical components, despite decreasing moisture content and increased protein and fat content for all treatments with increasing preservation period. discussion maintaining meat’s ultimate ph is very important because of its relationship to meat quality, color, and shelf life. parvar et  al. (2018) stated that feed additives, such as essential oils, had no significant effect on meat’s final ph 24 h postmortem. smeti et  al. (2018) noted no significant effect of rosemary essential oils on lamb meat’s final ph. moreover, de oliveira monteschio et al. (2017) reported no significant effect of feeding clove and rosemary essential oils on final meat ph. these results agree with the results achieved in this research (table 1). the results also agreed with rivaroli et al. (2020) and ornaghi et al. (2020), who indicated that the average ph of ld is between 5.5 and 5.8. this value is influenced by chilling and the stress to which the animal is subjected before slaughter. the research data indicated that the meat ph of the animals was within the normal limits, indicating the animals were calm when slaughtered and the carcasses were cooled well. microorganisms and meat enzymes cause proteolysis, producing organic sulfides, ammonia, and amines, which raise the ph (muela kept for 24 h at 4°c. afterward, the longissimus dorsi (ld) muscle was separated, divided into several parts as appropriate, and each part was preserved in a plastic bag. three preservation periods were used: zero freezing (p1), and 30-day freezing (p2) and 60-day freezing (p3) at −18°c. physical measurements several physical measurements were recorded on meat samples for each storage period, including ph, water holding capacity (whc) (dolatowski and stasiak, 1998), thaw loss, and cooking loss (purchas and barton, 2012). chemical measurements several chemical measurements were also made on the stored meat samples at the end of each storage period, including meat chemical composition (moisture, protein, ether extract, and ash) (horwitz and latimer, 2005), thiobarbituric acid (tba) (gheisari et al., 2010), total volatile nitrogen (tvn) (pearson and muslemuddin, 1971), and free fatty acids (ffa) (pearson and dustson, 1985). statistical analysis sas statistical analysis program was used to determine the effect of oil drenching and storage period on the studied measurements. duncan’s test was used to analyze the data to determine the effect of oil drenching and storage period on the studied measurements. probability values of ≤0.05 were considered statistically significant. results there was no significant effect of oil treatments on the ld muscle ph in all preservation periods. in contrast, a significant increase in ph values (p ≤ 0.05) occurred for most treatments with increasing freezing periods (table 1). also, different oils did not affect the ld muscle whc according to the control for all preservation periods, while a significant decrease in whc was observed for most treatments with increasing freezing periods. values are presented as mean ± sem. different lowercase letters indicate significant differences between means within columns, while different uppercase letters indicate significant differences between means within rows (p ≤ 0.05). whc, water holding capacity. no significant effects on thaw loss were observed for different oils and preservation periods, while a significant italian journal of food science, 2021; 33 (4) 101 effect of different plants’ aromatic essential oils ta bl e 1. e ff ec t o f di ff er en t o ils a nd fr ee ze -p re se rv at io n pe ri od s on lo ng is si m us d or si m us cl es ’ p h , w h c , t ha w la ss , a nd c oo ki ng lo ss . p ar am et er ph w h c (% ) th aw lo ss (% ) c oo ki ng lo ss (% ) p er io ds g ro up s n o fr ee zi ng (p 1) 30 -d ay fr ee zi ng (p 2) 60 -d ay fr ee zi ng (p 3) n o fr ee zi ng (p 1) 30 -d ay fr ee zi ng (p 2) 60 -d ay fr ee zi ng (p 3) 30 -d ay fr ee zi ng (p 2) 60 -d ay fr ee zi ng (p 3) n o fr ee zi ng (p 1) 30 -d ay fr ee zi ng (p 2) 60 -d ay fr ee zi ng (p 3) c on tro l ( t1 ) ab 5. 72 ± 0 .0 4 aa b 5. 80 ± 0 .0 5 aa 5. 91 ± 0 .0 2 aa 34 .5 2 ± 0. 74 aa b 34 .3 2 ± 0. 35 ab 33 .8 5 ± 0. 96 ab 3. 89 ± 0 .0 4 aa 4. 49 ± 0 .0 8 aa 34 .7 5 ± 0. 24 aa 34 .1 6 ± 0. 30 ab 33 .4 1 ± 0. 08 s ag e oi l ( t2 ) ab 5. 58 ± 0 .0 6 aa b 5. 77 ± 0 .0 3 aa 5. 95 ± 0 .0 2 aa 34 .3 3 ± 0. 96 aa b 34 .1 0 ± 0. 96 ab 33 .5 6 ± 0. 74 ab 3. 75 ± 0 .0 4 aa 4. 10 ± 0 .0 3 a a 34 .5 3 ± 0. 14 aa b 33 .9 5 ± 0. 15 ab 33 .1 0 ± 0. 08 c lo ve o il (t 3) ab 5. 40 ± 0 .0 6 aa 5. 71 ± 0 .1 2 aa 5. 78 ± 0 .0 5 aa 34 .6 3 ± 1. 00 aa 34 .5 3 ± 0. 96 aa 33 .7 5 ± 0. 96 ab 3. 48 ± 0 .0 3 aa 4. 28 ± 0 .0 7 aa 34 .4 9 ± 0. 18 aa 34 .1 0 ± 0. 08 ab 33 .3 8 ± 0. 08 la ur el o il (t 4) aa 5. 63 ± 0 .0 4 aa 5. 81 ± 0 .3 1 aa 5. 79 ± 0 .3 5 aa 34 .5 5 ± 0. 74 ab 34 .2 2 ± 0. 74 ac 33 .8 5 ± 0. 96 ab 3. 62 ± 0 .1 0 aa 4. 37 ± 0 .0 1 aa 34 .6 9 ± 0. 07 aa b 34 .2 2 ± 0. 10 ab 33 .6 5 ± 0. 05 102 italian journal of food science, 2021; 33 (4) al-obaidi asa et al. this decrease may be due to the myofibril proteins shrinkage resulting from the presence of ice crystals that cause damage to muscle cells and denatured proteins, which increases in size with increasing storage period (ripoll et al., 2012). et  al., 2010). this research agreed with the results that indicated increasing ph values corresponding to an increased storage period. increase in ph value with increasing storage period of oil treatments was not at the same level in the control group. the increase in control treatment ph value may have resulted from proteolysis, reduced by the effect of oils and their active substances. the antimicrobial activity of essential oils is due to the presence of hydroxyl groups. this antimicrobial activity is mediated by several mechanisms, including influence on the cytoplasmic membrane and active transport (sharma et al., 2020). whc is one of the most important measurements that determine meat quality and other characteristics. it is affected by many postmortem factors, including the extent of ph decline, proteolysis, and others (hufflonergan and lonergan, 2005). the results obtained in this research (table 1) agree with rivaroli et  al. (2020), who indicated no significant effect of different essential oils on meat whc. ripoll et  al. (2012) confirmed that whc is greatly affected by meat ph. the average ph values of the treated lambs’ meat may explain the absence of differences in whc values between these treatments. the results also agree with muela et  al. (2015), who observed a decrease in whc with an increased freezing period. the degradation of meat protein and its decreased ability to hold water may explain this outcome. there is a positive relationship between whc and ph, as lactic acid production leads to a decrease in ph. as the ph reaches the isoelectric point of meat proteins (especially myosin), the excess charges that bind the protein with water are minimal, resulting in decreased whc. with an increase in the ph and its rise above the isoelectric point of proteins, the negative or positive charges that are not associated with the protein increase, enabling the proteins to bind more water molecules, resulting in increased whc. in the case of freezing (as in this research), although the ph increased as a result of proteolysis, a decrease in whc occurred. table 2. effect of different oils and freezing periods on longissimus dorsi muscles’ tba, ffa, and tvn. parameter tba mg malonaldehyde/kg ffa (%) tvn mg n/100 g periods groups no freezing (p1) 30-day freezing (p2) 60-day freezing (p3) no freezing (p1) 30-day freezing (p2) 60-day freezing (p3) no freezing (p1) 30-day freezing (p2) 60-day freezing (p3) control (t1) ac 0.96 ± 0.01 ab 1.35 ± 0.01 aa 1.58 ± 0.02 ac 0.90 ± 0.31 ab 1.19 ± 0.71 aa 1.42 ± 0.28 ac 8.92 ± 0.05 ab 10.87 ± 0.18 aa 12.15 ± 0.39 sage oil (t2) bc 0.66 ± 0.01 bb 1.06 ± 0.08 ba 1.34 ± 0.03 bcc 0.66 ± 0.25 cb 0.84 ± 0.36 ca 0.98 ± 0.21 abc 7.89 ± 0.04 bb 9.45 ± 0.16 ba 10.94 ± 0.58 clove oil (t3) db 0.47 ± 0.02 da 0.77 ± 0.02 da 0.76 ± 0.08 cc 0.51 ± 0.17 db 0.68 ± 0.85 da 0.82 ± 0.34 cb 5.24 ± 0.59 da 6.75 ± 0.19 da 7.22 ± 0.43 laurel oil (t4) cc 0.56 ± 0.01 cb 0.92 ± 0.01 ca 1.08 ± 0.01 abc 0.82 ± 0.20 bb 0.96 ± 0.43 ba 1.21 ± 0.22 bc 7.04 ± 0.19 cb 8.17 ± 0.23 ca 9.32 ± 0.30 figure 1. effect of different oil treatments on the percentages of a. moisture, b. ash, c. ether extract, and d. protein content of the treated lambs’ longissimus dorsi muscles after different preservation periods. italian journal of food science, 2021; 33 (4) 103 effect of different plants’ aromatic essential oils in table 2 agreed with ozogul et al. (2017), who observed a significant decrease in ffa for the different nano oil treatments and all preservation periods, compared with the control treatment. the results also indicated that a decrease in ffa is due to additives’ ability to inhibit lipolytic bacteria growth (maqsood et  al., 2015; rahman et al., 2020). several hypotheses explain the essential oils and phenolic compounds’ antimicrobial activity, but they are not yet proven (kalogianni et al., 2020). olatunde and benjakul (2018) reported that essential oils could destroy bacteria by interacting with bacterial cell wall proteins. this interaction increases membranes’ permeability and leaking of cytoplasmic structures and potassium ions, causing cell death (bajpai et al., 2008). at the same time, other researchers reported that the decrease in ph and increase of phenols increased essential oils’ hydrophobicity, favoring their attachment to pathogen’s lipid cell membranes and increasing antimicrobial activity (gutierrez et  al., 2009). due to their hydrophobic properties, phenolic compounds are bound with microbial membrane lipids causing their disruption (devi et  al., 2010; trombetta et  al., 2005). this disruption leads to intracellular compounds efflux, protein functional dysregulation, and cell death (devi et al., 2010). tvn is a product of meat and nonprotein nitrogenous substance degradation and a measure of meat deterioration. degradation of nitrogenous substances is caused by microbial and endogenous proteolytic enzyme activity. this study’s results agreed with saleh et  al. (2021), who reported decreased tvn values by feed additives, and ozogul et  al. (2017), who indicated a decrease in tvn following different nano oil treatments and storage times. the maximum permissible meat tvn content is 150 mg/kg. research reports the antimicrobial effectiveness of phenolic compounds, which possibly destabilizes the bacterial cell membrane (pisoschi et al., 2018), leading to permanent damage of the cell membrane and intracellular organelles and bacterial internal enzyme inhibition causing cell death. besides, phenolic compounds’ antioxidant efficacy may increase proteins’ stability and reduce tvn by reducing radicals (moroney et  al., 2013). our results confirmed increases in tvn values with increasing storage period. custódio et  al. (2018) indicated that tvn increases due to protein degradation by the internal meat enzymes and microorganisms’ action. this study’s results revealed no significant effect of essential oils as feed additives on ld chemical composition. other studies also reported similar results (ranucci et al., 2019; rivaroli et al., 2020). a decrease in moisture content with increasing storage time might occur due to protein denaturation, lack of whc, and decomposition by microorganisms. this decrease in moisture leads to increased dry matter (protein ratios, ether extract, and ash) content (al-rubeii et al., 2009; sharma et al., 2015). this study indicated no significant effect of treatment with essential oils in cooking loss, which agrees with previous studies (de oliveira monteschio et  al., 2017; smeti et al., 2018). the results confirmed thawing losses with increasing preservation period and indicated a rise in total thaw loss with increased freezing time. these results coincide with previous studies (muela et al., 2015; ornaghi et al., 2020). freezing increases thaw loss due to muscle membranes’ mechanical damage by ice crystals, which leads to increased loss. membrane damage may lead to increased protein denaturation and decreased whc. also, prolonged freezing causes increased water loss in meat by ice crystals damaging the cell membranes (lu et al., 2019). fat and protein oxidations affect meat quality and are related to meat deterioration. tba level is an indicator of the extent of lipid oxidation through malondialdehyde concentration and color intensity measurement. a tba content of 0.6–2.0 mg malondialdehyde/kg is considered to be within normal limits (falowo et  al., 2014). the results revealed that the different essential oils decreased tba levels. these results agreed with parvar et al. (2018) and ranucci et al. (2019). the oxidative indicator’s decrease is because the essential oils are absorbed into the circulatory system ingestion, then distributed to muscles and the rest of the tissues (velasco and williams, 2011). phenolic compounds are considered antioxidants due to their ability to inhibit free radicals by incorporating them into the aromatic ring (maqsood et  al., 2014). nieto et  al. (2010) stated that feed additives allow antioxidants such as essential oils to get into tissues and cellular membranes, protecting them from oxidative stress by reactive oxygen. this process has proven to be more effective than treating meat with antioxidants postmortem (kumar et al., 2018). the even distribution of antioxidants within the tissues and cells ensures the effectiveness of these compounds against fat and protein oxidation. the results in table 2 also agreed with that reported by politeo et  al. (2006), who ranked 12 essential oils in the descending order according to their capacity as antioxidants, including the oils used in this research. they showed that the antioxidant efficacy of clove oil was the highest. other studies recorded an increase in tba relative to the increased storage time (ranucci et al., 2019; rivaroli et al., 2020). o’sullivan et al. (2004) stated that increased storage time leads to increased lipid oxidation, breakdown of peroxides, and increased secondary compounds resulting from oxidation, represented by malondialdehyde. ffa is formed from fat and oil hydrolysis and is considered a measure of degradation by microorganisms and lipolytic enzymes (rahman et al., 2015). the ffa results 104 italian journal of food science, 2021; 33 (4) al-obaidi asa et al. dorman, h.j. and deans, s.g., 2000. antimicrobial agents from plants: antibacterial activity of plant volatile oils. j appl microbiol. 88: 308–316. https://doi.org/10.1046/j.1365-2672. 2000.00969.x falowo, a.b., fayemi, p.o. and muchenje, v., 2014. natural antioxidants against lipid-protein oxidative deterioration in meat and meat products: a review. food res int. 64: 171–181. https://doi. org/10.1016/j.foodres.2014.06.022 fasolato, l., cardazzo, b., balzan, s., carraro, l., taticchi, a., montemurro, f. and novelli, e., 2015. minimum bactericidal concentration of phenols extracted from oil vegetation water on spoilers, starters and food-borne bacteria. ital j food saf. 4: 4519. https://doi.org/4519.10.4081/ijfs.2015.4519 fernandes, r.d.p.p., trindade, m.a. and de melo, m.p., 2018. natural antioxidants and food applications: healthy perspectives. in: alternative and replacement foods. elsevier, pp. 31–64. gheisari, h.r., møller, j.k.s., adamsen, c.e. and skibsted, l.h., 2010. sodium chloride or heme protein induced lipid oxidation in raw, minced chicken meat and beef. czech j food sci. 28: 364–375. https://doi.org/10.17221/182/2009-cjfs govaris, a., botsoglou, e., florou-paneri, p., moulas, a. and papageorgiou, g., 2005. dietary supplementation of oregano essential oil and–tocopheryl acetate on microbial growth and lipid oxidation of turkey breast fillets during storage. int j poult sci. 4: 969–975. https://doi.org/10.3923/ijps.2005.969.975 gutierrez, j., barry-ryan, c. and bourke, p., 2009. antimicrobial activity of plant essential oils using food model media: efficacy, synergistic potential and interactions with food components. food microbiol. 26: 142–150. https://doi.org/10.1016/j. fm.2008.10.008 horwitz, w. and latimer, g.w., 2005. official methods of analysis of aoac international. gaithersburg, md: aoac international. huff-lonergan, e. and lonergan, s.m., 2005. mechanisms of water-holding capacity of meat: the role of postmortem biochemical and structural changes. meat sci. 71: 194–204. https:// doi.org/10.1016/j.meatsci.2005.04.022 kalogianni, a.i., lazou, t., bossis, i. and gelasakis, a.i., 2020. natural phenolic compounds for the control of oxidation, bacterial spoilage, and foodborne pathogens in meat. foods. 9: 794. https://doi.org/10.3390/foods9060794 kumar, s., mendiratta, s., agrawal, r.k., sharma, h. and kumar, r., 2017. quality evaluation of mutton nuggets incorporated with optimized level of flaxseed flour. nutr. food sci. 47: 67–77. https://doi.org/10.1108/nfs-07-2015-0081 kumar, s., mendiratta, s., agrawal, r.k., sharma, h. and singh, b., 2018. antioxidant and antimicrobial properties of mutton nuggets incorporated with blends of essential oils. j food sci technol. 55: 821–832. https://doi.org/10.1007/s13197-017 3009-6 kumar, a., mendiratta, s., sen, a., kandeepan, g., talukder, s., sharma, h., soni, a., irshad, a. and kumar, s., 2015. preparation and storage stability of meat spread developed from spent hens. vet world. 8: 651. https://doi.org/10.14202/ vetworld.2015.651-655 lu, x., zhang, y., zhu, l., luo, x. and hopkins, d.l., 2019. effect of superchilled storage on shelf life and quality characteristics of conclusion the results of this research indicated no significant effect of drenching awassi lambs with different aromatic essential oils of sage, clove, and laurel at a concentration of 500 mg/head/day on the physical and chemical characteristics of meat. however, these oils’ effect was positive on fat oxidation and protein stability, increasing the preservation period, 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issn 1120-1770 online, doi 10.15586/ijfs.v34i4.2246 11 p u b l i c a t i o n s codon perceptions of spanish consumers towards novel lamb burgers enriched with natural antioxidants and healthy fatty acids andres horrillo1, carlos díaz-caro2, eva crespo-cebada1, david tejerina3, francisco javier mesías1*, antonio rodríguez-ledesma1, susana garcía-torres3 1research institute on agricultural resources, universidad de extremadura – ctra, badajoz, spain; 2faculty of business, finance and tourism, universidad de extremadura, cáceres, spain; 3meat quality area, cicytex junta de extremadura, badajoz, spain *corresponding author: francisco javier mesías, research institute on agricultural resources, universidad de extremadura – ctra, cáceres s/n, 06071 badajoz, spain. email: fjmesias@unex.es received: 17 june 2022; accepted: 23 october 2022; published: 15 november 2022 © 2022 codon publications open access paper abstract this paper explores the perceptions of spanish consumers on the use of natural antioxidants and healthy fatty acids in novel formulations of lamb burgers to improve their nutritional content and shelf life. focus groups have been used to obtain information on burger consumption and purchase, as well as on the opinions about the use of natural ingredients. results highlight the importance consumers place on the presence of ingredients that help improve product quality, and their acceptance to pay a premium. the use of cherry or pecan nut is a valid option, provided that consumer trust is promoted through adequate information policies. keywords: focus group; lamb meat; natural ingredients; novel formulation; processed meat introduction in recent years, trends in meat consumption have been changing, both in terms of levels of consumption and type of product, with red and processed meats declining due to consumers’ awareness on health and the environment (godfray et al., 2018). in fact, a clear example is the lamb consumption, which has decreased considerably both in spain and the european union (rabadán et al., 2020), with significant losses for the production industry. this fact, together with the changes in the lifestyle of the population in the last years—consumers demanding more fast or ready to eat food—has forced the industry to develop strategies in order to diversify their products in a way such that they become more attractive to the consumer, as is the case of burgers. burgers are extensively consumed and a popular meal worldwide due to its convenience, low cost and pleasant flavour. in spain alone, the consumption of burgers exceeds 3 million units per week (mapa, 2018). in spite of the numbers, burgers are perceived as unhealthy food, on account of their association with red meat (binnie et al., 2014), and because of their content of saturated fats and lack of fibre, which relate to the prevalence of cardiovascular disease, colorectal cancer and obesity (spencer et al., 2005). also recently some initiatives have attempted to improve these unhealthy features of burgers. thus, several studies have examined the addition of antioxidants to meat products in order to minimise oxidation processes and to maintain their properties during storage or to improve their health benefits. the addition of some by-products of tomato (garcía et al., 2009), grape and olives (andrés et al., 2017; sáyago-ayerdi et al., 2009), essential oils (pateiro et al., 2018), cinnamon bark oil (hussain et al., 2021) or even spices such as oregano (vergara et al., 2020) has been analysed. the addition of fruit or vegetable by-products to burgers, in addition to providing mailto:fjmesias@unex.es 12 italian journal of food science, 2022; 34 (4) horrillo a et al. might not be willing—or able—to be totally honest when answering complex questions on their perceptions or attitudes towards certain foods or technologies, as they may be reluctant to share their opinions, unfamiliar with the subject under study or simply lack confidence to answer (donoghue, 2010; eldesouky and mesias, 2014). therefore, qualitative research has been considered as a valid approach for this particular research study as it is a flexible and adaptable type of research and is suitable to find out the nature of a problem or identify alternative actions (guerrero et al., 2009; stewart et al., 1994). given its potential, qualitative research has been broadly applied in the food sector, with applications in the study of attitudes towards food safety (behrens et al., 2010), ready-to-eat foods (vidal et al., 2013) or consumer perception towards imported fruits (vaca and mesías, 2014). amongst the numerous qualitative research techniques available, we decided to use focus groups as it is one of the most popular methods for the preliminary stages of a research, as is the case of this study (eldesouky and mesias, 2014), also being adequate for research studies related to the development and application of new products or services (horrillo et al., 2020). this technique is based on group dynamics with a moderator whose role is to promote discussion by the exchange of opinions amongst the participants and has certain advantages against other more structured research approaches using questionnaires, as it allows and promotes more freedom to speak amongst the participants (gaspar et al., 2016). taking into account this context, the purpose of this paper is to explore consumer perceptions on the use of natural ingredients (cherry and pecan nuts) in burgers made of merina lamb (autochthonous breed from spain), with the purpose of improving their nutritional properties and preservation. various topics were analysed, such as burger consumption and purchasing habits, the factors that influence burger purchasing, the influence of origin, species and breed on the burger purchase decision and the opinion on enhanced and processed meat products. methodology focus groups a focus group should include 6–12 participants (malhotra and birks, 2006) in order to run smoothly, because less than 6 people would make it hard for group dynamics to take place, whilst discussion flow in a group exceeding 12 participants may be hard as it would be difficult to have the opinion of the less talkative, and the moderator may encounter difficulties to conduct the discussions around the topic of research. antioxidant compounds, adds dietary fibre to the product, and therefore, madane et al. (2019) considered that they have dual properties. lamb meat from the autochthonous breed merina is considered a traditional product with good sensory attributes and higher n-3 concentration levels (linares et al., 2007, 2008), and its production is associated, to a large extent, to the sustainable use of the dehesa1 ecosystem (gaspar et al., 2008). the addition of natural ingredients, such as cherry (prunus avium l. var. pico negro) as an important source of antioxidants or pecan nuts (carya illinoinensis (wangenh), k. koch, var. osage) as a source of healthy fat [rich in mono(mufa) and polyunsaturated fatty acids (pufa)], both also containing dietary fibre, might in principle result in a healthier and an attractive product for the consumer. however, generally, they have a negative attitude towards healthier meat products due to unfamiliarity and perception of overprocessing, although plant-based ingredients together with fat and salt reduction show specific conditions under which consumers’ acceptance would be possible (barone et al., 2021). on the other hand, lang (2020) examined the nature of consumer response to blending plant-based ingredients (mushrooms) to traditional meat-based foods and the individual lifestyle and motivational differences that influence this response. from the viewpoint of producers and manufacturers, these new products such as lamb burgers with natural ingredients such as cherries and pecan nuts could fill a niche in the market and be totally acceptable both in technological and nutritional terms. consumers, however, might not share this perception as they tend to be reluctant to try new products and technologies, on account of their concern about the potential risks and the doubts about the possible benefits involved, but also as a sign of neophobia, that is, a common negative reaction to all things new (frewer et al., 2011). this is one of the reasons why the stakeholders would benefit from understanding consumer perceptions towards these burgers with natural ingredients such as cherries and pecan nuts. nevertheless, this is not a simple task, as the number of factors affecting consumer perception towards food is numerous, for example, health, food culture and traditions, which make it hard to use traditional quantitative research approaches. specifically, in the case of mutton, hastie et al. (2022) found that familiarity and previous experience with the product were significant predictors of consumer appreciation and overall taste. besides, consumers 1the dehesa is an agroforestry system located in the sw of the iberian peninsula, used for livestock-range farming characterised by its mix of pasture and evergreen oak stands. italian journal of food science, 2022; 34 (4) 13 consumer perceptions on the use of natural ingredients in lamb burgers it is also recommended for the groups to represent the sociodemographic diversity of the population under study, although on occasions it may be advisable to use homogeneous groups (horrillo et al., 2020). the venue where the discussion groups are held is also relevant, because an informal and relaxed atmosphere helps participants forget that they are being watched and interviewed (gaspar et al., 2016). taking into account all the above parameters, four focus groups were held for the purpose of this research study between march and july 2020, which were attended by a total of 40 participants (9–12 people per session). these sessions were held in three big cities in the region of extremadura (southwestern spain), to include population diversity. a public relations company was commissioned for the selection of the participants and moderation of the discussions. participants were selected by convenience sampling, which is a habitual technique in qualitative research (eldesouky and mesias, 2014), with the main selection criteria being willingness to participate in the study and to be meat consumers. as the sample was not meant to be representative of the population, due to its size and the qualitative nature of the study, efforts were made to include a variety of people according to the sociodemographic and purchasing characteristics of the participants, which could enrich the discussion. table 1 shows the breakdown by gender, age and highest level of studies completed by the participants involved in the sessions. focus groups development the sessions began with the explanation of the research project under which the activity was being performed in order to provide some background to the participants. subsequently, the participants were asked to introduce themselves, as a way to break the ice, and the discussions followed according to the script shown in table 2, where the moderator would be introducing the topics for discussion and encourage participants to provide their opinions. when the topic proposed by the moderator had been dealt with, he would continue the session by introducing a new topic to discuss. as shown in table 2, on conclusion of each session, participants were offered a taste of the lamb burger that was enhanced with cherry (prunus avium l. var. pico negro) and pecan nuts [carya illinoinensis (wangenh), k. koch, var. osage], following which they were asked to provide their feedbacks. the research was conducted in compliance with the university of extremadura bioethics and biosecurity committee regulations regarding studies with human participants. all participants provided their written informed consents after reading an informative document with details on the purposes of the study, the methods used for data collection, the audio recording and data confidentiality. the sessions were videoand audio recorded for subsequent analysis. each session lasted an average of approximately 120 min in total. on conclusion of each session, the attendees were given a gift that valued 5–10 euros as a thank you for their help. data analysis the video and audio recordings of the four sessions were transcribed and anonymised for subsequent analysis. the table 1. gender, age and highest level of studies completed. variable % gender women 57.5 men 42.5 age <30 years old 15.0 31–44 years old 25.0 45–65 years old 50.0 >66 years old 10.0 level of study primary education 22.5 secondary education 20.0 university studies 57.5 table 2. script followed in the focus groups. processed meat products frequency of consumption of burgers and settings types of burgers consumed types of packaging purchasing factors effects of origin, species and breed on the purchase decision types of burgers purchased according to animal species and processing method importance of species or breed, type of farm production and origin of the meat product organoleptic properties enhanced meat products opinion on natural ingredients or additives effects of enhanced burgers on organoleptic properties willingness to pay for enhanced products tasting session and discussion about lamb burgers enhanced with cherry (prunus avium l. var. pico negro) and pecan nuts [carya illinoinensis (wangenh) k. koch, var. osage] 14 italian journal of food science, 2022; 34 (4) horrillo a et al. geographical indication (pgi) ‘merino lamb.’ lamb legs and breasts were deboned, chopped, and the meat was minced. the minced meat was distributed into two batches. each batch was mixed in an automatic mixer (mainca pm70, spain), for 5 min, adding salt (1% w/w) and white ground pepper (0.2%). the ingredients were added to each batch and mixed: freeze-dried cherry (6%) and pecan nuts (10%). burgers were prepared (100 g/10 cm diameter) using a conventional burger maker (mainca mh/ma, spain). burgers were packaged using vacuum skin film, which preserves and extends shelf life compared to other types of packaging (kameník et al., 2014). for packaging purposes, a packaging machine (ulma® smart 300) was used, and the samples were packaged in trays (p 1520sw30e, faerchs), made of co-extruded polyamide or polyethylene (pp/evoh/pe eost) and wrapped in film (vstm, cryovac®). burgers were then frozen until the tasting test was carried out during the focus group. quality trait analysis in order to understand the differences of the nutritive and antioxidant content in the final quality of the two products (lamb burgers with cherries or pecan nuts natural ingredients) versus the control lamb burgers (table 3), the nutritive and antioxidant compositions were determined, which showed that the addition of pecans improved the natural antioxidants content (α and γ -tocopherols), mufa and pufa and fibre, with respect to the cherry lamb burger, which was richer transcriptions of each session were compared and analysed in order to find which aspects were relevant in all the sessions. the analysis of the data collected was performed using content analysis (stewart and shamsasani, 2014). content analysis is a research technique that seeks to obtain valid and replicable inferences from texts in order to reduce the amount of input materials (flick, 2009). for the purposes of this paper, the information was initially processed and organised by common topics, grouping under each topic all the ideas or concepts that were mentioned repeatedly during all the sessions. all the terms and their meanings were taken into account for the analysis, with the comments provided for each question being later analysed with the purpose of identifying similarities and differences. with the purpose of improving the robustness of the results, analyst triangulation was applied, a procedure frequently used in qualitative research (eldesouky et al., 2015; horrillo et al., 2020). finally, the frequency of mention of each category or concept was calculated as a way of showing its relative importance, as those items that generally receive the highest frequency of mention are assumed to be the most relevant ones. figure 1 summarises the methodology carried out in this research. preparation of samples burgers were made with the meat obtained from legs and breasts of merino lambs raised under the protected design of the focus group sessions processed meat products enhanced processed meat products selections by means of convenience sampling (9–12 per session) burger tasting session content transcription effect on the purchase decision moderator script design of the focus group sessions participant selection videos final report 4 focus group 40 participants analysis of the results content by topics development of the focus group sessions figure 1. methodological procedure followed in the research. italian journal of food science, 2022; 34 (4) 15 consumer perceptions on the use of natural ingredients in lamb burgers tasting session prior to the tasting process, the participants were informed about the products to be tasted—type of product, origin of raw material, as well as information about the ingredients of the burgers—and they were warned about possible allergens in order to rule out potential participants. they were also showed fresh and skinpacked burgers, as found at the point of sale, with the aim of further leading the discussion to the type of packaging. burgers were thawed for 24 h under refrigeration (4 ± 1°c) before each focus group session. during the development of each session, burgers were cooked in an adjoining room on two electric grills (one for burger with cherries and other for burger with pecan nuts) until an internal cooking temperature of 72–75°c was achieved, measured using a digital food thermometer. cooked burgers were cut into four pieces and placed on different identified trays according to the type of burgers, and were presented at the same time to the participants. finally, each participant tasted a piece of each type of burger, and the debate continued under the guidance of the moderator. in phenols content and total antioxidant activity. the moisture, protein and ash content were determined according to aoac standards (2003). the fat content was determined using the method developed by folch et al. (1957). tocopherols (α and γ) were quantified according to the method proposed by cayuela et al. (2003). the total phenolic compound was determined according to singleton and rossi (1965), and the total antioxidant activity was determined following the method described by cano et al. (1998). phenol content was quantified using the method described by singleton and rossi (1965). the fatty acid profile was determined according to ortiz et al. (2020a), and the indexes to measure the nutritional quality of the fatty acids were estimated: saturated fatty acids (sfa), mufa, pufa, n-6/n-3 and the atherogenic [% ai = 100 × (c12:0 + 4 × c14:0 + c16:0)/(mufa+ n − 3 pufa + n − 6 pufa)] and thrombogenic indexes {% ti = 100 × (c14:0 + c16:0 + c18:0)/[(0.5 × mufa + 0.5 × n − 3 pufa + 3 × n − 6 pufa + (n − 3 pufa/n − 6 pufa)]} (rios-mera et al., 2021). table 3. nutritive, antioxidant and fatty acids profiles of lamb burgers with natural ingredients added. lamb burgers (n = 8) with cherry with pecan nut control nutritional composition (mean ± standard deviation) protein content (%) 15.08 ± 2.71b 17.63 ± 0.86a 16.64 ± 1.27a,b * fat content (%) 12.59 ± 1.03b 16.26 ± 0.46a 14.68 ± 2.50a *** moisture (%) 62.07 ± 2.78a 57.08 ± 1.24b 62.24 ± 2.77a *** ash content (%) 3.92 ± 0.38a,b 3.61 ± 0.13b 4.34 ± 0.45a ** calorific power (kcal/g) 6.8 ± 0.02c 7.28 ± 0.24a 6.9 ± 0.03b *** dietary fibre (%) 0.04 ± 0.01b 0.52 ± 0.15a – *** nutritional quality of fatty acids indices sfa 46.61 ± 0.48a 35.39 ± 1.17b 46.55 ± 0.26a *** mufa 49.24 ± 0.47b 55.41 ± 0.67a 49.50 ± 0.27b *** pufa 4.15 ± 0.18b 9.20 ± 0.54a 3.94 ± 0.13b *** n6/n3 0.82 ± 0.43b 12.38 ± 0.56a 8.68 ± 0.45b *** % ai 0.46 ± 0.02b 0.72 ± 0.01a 0.71 ± 0.02a *** % ti 0.62 ± 0.04b 1.22 ± 0.01a 1.20 ± 0.03a *** antioxidant composition α-tocopherol (µg/g) 3.82 ± 1.32 4.57 ± 0.71 4.21 ± 0.80 ns γ-tocopherol (µg/g) 0.09 ± 0.04b 18.64 ± 2.28a 0.16 ± 0.07b *** phenols content (mg of gallic acid/g of meat) 3.21 ± 0.20a 1.49 ± 0.14b 0.82 ± 0.07c *** total antioxidant activity 2.19 ± 0.22a 0.71 ± 0.05b 0.66 ± 0.04b *** ai, atherogenic index; mufa, sum of monounsaturated fatty acids; pufa, sum of polyunsaturated fatty acid; ti, thrombogenic index; sfa, sum of saturated fatty acids. ns, not significant; p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. a, b, c, different letters in the same row indicate significant differences for different burger formulations (with cherry, with pecan nuts and control) according to tukey’s hsd test. 16 italian journal of food science, 2022; 34 (4) horrillo a et al. another aspect that raised many comments was price, which is considered as one of the most relevant factors in the purchase decision of consumers (9%), together with comments stating that participants bought minced meat to make burgers at the butcher’s shop (11%). in the same way, the type of packaging is an aspect that is highly mentioned, mainly in terms of lack of awareness of the skin packaging they were presented in, as well as a dislike for the plastic packaging they are presented with. and finally, with regard to the frequency of consumption, participants appeared to state that they did not usually eat burgers, and they believed that the population that mostly ate them was young people. effects of origin, species and breed on the purchase decision table 5 displays the mentions made by the participants relating to the effects of origin, species and breed on the burger purchase decision. results processed meat products table 4 shows the results obtained from the opinions of the consumers on the frequency of consumption and the commercial format of the burgers. the most commented categories were ‘types of burgers consumed’ (35%) and ‘purchasing factors’ (23%). more specifically, there were a large number of comments regarding the types of burgers consumed, with comments relating to home-made burgers consumption obtaining 9% of the responses, followed by ‘difference between home-made and industrially made burgers’ receiving 7% of the responses. format and origin are the least frequent comments together with ‘easy to cook’ and ‘processed meat does not help make sales.’ table 4. frequency of consumption and commercial formats of burgers (% of mention by the participants). category subcategory % mention burger frequency of consumption not habitually consumed 9 consumed by young people 9 types of burgers consumed need of easy-to-cook food 4 the name ‘processed meat’ does not help sales 4 difference between home-made and industrially made burgers 7 home-made consumers burgers (healthier option and trying to make them in different ways) 9 need to highlight that burger are natural products 4 not used to these lamb formats, but to more traditional ones (chop, leg) 4 the burger format may lead to more sales of lamb, as it is more popular with younger people 3 setting home consumption 6 restaurant consumption 4 packaging type aware of skin packaging 3 unaware of skin packaging 8 disliked the plastic packaging presented 4 purchasing factors price is a determinant for purchase 9 occasional consumption of lamb burgers as opposed to other types of burgers 2 usually purchased packaged burgers 1 usually purchased butcher’s burgers (or minced meat to make burgers) 11 table 5. effects of origin, species and breed on the burger purchase decision (% of mention by the participants). category subcategory % mention type of burger purchased distinction between an industrial or home-made burger 1 pork burger 3 beef burger 15 there are no lamb or goat meat products 1 chicken burgers 6 mixed meat burger 6 importance of the species or breed they look at the species before they buy 20 they do look at the breed before they buy 8 they do not look at the breed before they buy 5 organoleptic properties of the product the organoleptic properties of lamb are very particular 5 tasting meats of other species in order to accept their consumption 3 place of origin they look at the place of origin of the meat 11 they do not look at the place of origin of the meat 5 greater preference if the burger is made with meat from extremadura 6 type of farming production they look at the ways lambs are produced 4 italian journal of food science, 2022; 34 (4) 17 consumer perceptions on the use of natural ingredients in lamb burgers as shown in table 5, the comment that received most mentions (20%) referred to consumers who take into account the species before they buy the meat. following this comment was the mention relating to the purchase of beef burgers, although this category included a vast variety of answers from the participants, from the purchase of single meat–type burgers to mixed meat–type burgers. the category ’organoleptic properties of the product’ received less variety of comments, probably due to the participants’ lack of awareness. the category ‘place of origin’ was mentioned in a relevant number of times, which shows how important this factor is in the purchase decision, with a greater number of participants looking at the place of origin of the meat during purchase. finally, the type of production and, specifically, the lamb production process were mentioned in 4% of the comments. enhanced processed meat products table 6 shows the opinion of participants on their awareness and willingness to pay for enhanced processed meat products. the participants stated their awareness of enhanced processed meat products with a frequency of mention above 27%. they associated these types of products mainly with burgers and sausages. generally speaking, most of the participants recognised that they do not pay attention to the origin of the ingredients of the enhanced products they buy or consume (23%). additionally, the results also reveal some degree of uncertainty about them, with some participants associating products with additives with unhealthy food. the comments from table 6 also reveal concerns about misleading advertising (2%), as little is explained about the use of ingredients in food (2%) and the preference for natural ingredients (4%). regarding the organoleptic properties, the participants explained that on many occasions the enhancement of these products can mask the original flavour of the food. such techniques may also bring certain benefits by improving the flavour, taste, smell, aroma, texture and external appearance—mainly colour—when the final purpose is to increase product sales. on the other hand, in terms of the potential health benefits of the enhanced meat products, the participants only stated that its consumption is motivated by taste rather than the health benefits they provide. one of the most discussed categories amongst participants was the selling price of the enhanced product, with comments such as: these products require a more complex process, and for this reason they must have a higher price: male, 35 years old, butcher; table 6. awareness and willingness to pay for enhanced processed meat products (% of mentions by participants). category subcategory % mention awareness awareness of the product 27 ingredients do not look at the origin of the ingredients 23 do look at the origin of the ingredients 5 prefer natural ingredients 4 necessary explanation of the ingredients 2 not healthy (reluctant to term ‘additive’) 10 there is misleading publicity on additives 2 organoleptic and healthy improvements vary the original flavour of the product 2 consumption motivated by taste and not by the benefits for health 6 they improve the properties of the product 2 settings recreational consumption, outside the home 6 willingness to pay yes, they are willing to pay for additive-enhanced products 6 yes, they are willing to pay for products without additives 2 more information on product additives would improve sales 2 do not understand the price premium of products with additives (in extremadura) 1 if they are better in quality, a price premium is to be expected: male, 38 hospitality business; lamb meat has always been more expensive than pork or chicken and is to be expected that their products will be more expensive too: male, 65 years old, retired. six percent of the participants were willing to pay a price premium for these types of products, whereas others preferred to rather pay for products without additives. another mention was the addition of more information about the additives and ingredients, which would increase customer trust and market sales. the participants also preferred to consume the product outside their homes, in restaurants and bars, during their leisure time. lamb burger enhanced with cherry and pecan nut: commercialisation, sale and acceptance table 7 shows the findings from the discussion about lamb burgers enhanced with natural ingredients (pecan 18 italian journal of food science, 2022; 34 (4) horrillo a et al. agreed that they should be sold in specialist restaurants (14%) and specialist shops (18%), and not in supermarkets (16%). they mentioned that they saw this product as a gourmet option (21%) and not as an everyday product that is easy to find in butchers’ or supermarkets. and finally, figure 2 shows the consumer perceptions following a tasting session of lamb burgers enhanced with cherry or pecan nuts. a comment was made by all the participants relating to the nice flavour that the added components contributed to the meat. also, the addition of cherries seemed to have a higher acceptance amongst both participants who liked lamb and those who didn’t, in the latter group because it covered the lamb flavour. in general, these burgers, with the incorporation of cherries or pecan nuts, were highly accepted. this opinion was reinforced by the natural characteristics and the local origin of these natural ingredients. finally, the participants were willing to pay a price premium for the product, on account of both nutritional and functional improvements. discussion an aspect that is highly valued by the participants in terms of burger consumption is the way in which they are prepared. they especially appreciate that the burger is healthy and made with butcher’s meat. this seems to agree with the findings from other consumer evaluation studies with similar methods on traditional food products (pieniak et al., 2013) and also with specific studies carried out on burgers (arbaiza et al., 2020; mccormick et al. 2003; viana et al., 2014). in terms of the setting for burger consumption, the participants were more inclined to consume them at home than in restaurants, as is the case with other research studies carried out on burgers (atalaya et al., 2018). another aspect that was frequently mentioned is the price, which is seen as one of the most relevant aspects by participants, and this is in line with other papers dealing with food-purchase decisions (eldesouky et al., 2020; groves, 2001). in terms of packaging, most participants stated that they were not aware of the skin-pack format. in this sense, some research studies have proven that the type of packaging is usually a relevant attribute in the purchase decision (heide and olsen, 2017; ortiz et al., 2020b; silayoi and speece, 2007), and therefore a higher degree of consumer awareness of the benefits of the packaging could improve acceptance of the product (lindh et al., 2013). out of the factors affecting the purchase decision, the importance that consumers place on species is high. on the other hand, meat consumption continues to decline table 7. participant opinions on the specific case of the lamb burger enhanced with cherry and pecan nut (% of mentions by participants). category subcategory % mention opinion on enhanced lamb burgers reluctance towards lamb (it is expensive, it has bad press— cholesterol—and strong flavour) 9 differences between the flavour of meat from grazing sheep and from sheep fed on fodder 3 young people should eat more lamb 3 overcome the cultural barrier of lamb consumption 3 natural ingredients differentiate whether the added product (enhanced) improves preservation of the meat or improves the nutritional values of burgers 1 the fact that the ingredients come from products or brands with recognition in the area is valued 4 potential place of purchase and/ or promotion food fairs 1 media 3 gourmet product (+quality) 21 it is difficult to find lamb for mincing in the butcher’s 3 not available on the shelves of large supermarkets 16 tasting counters in large supermarkets 4 butchers’ 3 specialist restaurants 14 specialised shops 18 price change of the format of the suckling lamb to a larger one in order to obtain more meat from the carcass 34 reduce production costs 1 nuts and cherries). as the table shows, the attendees mention that there is a certain degree of reluctance in the population towards consumption of lamb. this meat is seen to have a strong flavour that young consumers are not used to. in addition to this, it has received a negative press for being a red meat. it has been classified as one of the types of meat that is responsible for increasing blood cholesterol levels. nevertheless, it has also received some positive comments by some attendees, who considered it to be a healthy meat produced in a natural environment (especially in pasture-based systems). in terms of the place where these products should be promoted and/or commercialised, the participants clearly italian journal of food science, 2022; 34 (4) 19 consumer perceptions on the use of natural ingredients in lamb burgers paying a price premium to obtain better nutritional/ functional properties (21.4%) cherry masks the flavour of lamb. higher acceptance (22.9%)tasty (38.6%) additives are well accepted if they contribute an added value, they are natural, local and of recognised brands (10%) figure 2. perceptions obtained following the tasting session of an enhanced burger with cherries or pecans. in recent years, especially fresh meat, and more specifically lamb in europe (mandolesi et al., 2020). although there has been an increase in the consumption of processed meat (mapa, 2019), with beef products being more popular than lamb. this fact coincides with the number of mentions made about the purchase of beef burgers in comparison to other types of burgers. this trend towards beef meat can be connected to the perception that these are healthier meats (grunert et al., 2004) in comparison to other meats. origin is another relevant factor in the purchase decision, as some research studies have stated (rabadán et al., 2021; sama et al., 2018). our findings also show that most participants gave importance on the origin of the lamb meat, in line with the findings obtained by bernabéu and tendero (2005) who found that the second most valued attribute for regular lamb consumers was the origin of the product, just after external appearance. on the other hand, the improvement of the technological properties of enhanced processed meat products is currently a line of research that is in full development (cózar et al., 2018; câmara et al. 2020). in this sense, the prevention of oxidation of the proteins and lipids becomes essential for the preservation of the shelf life of meat products, with additives becoming necessary to attain this. the opinions revealed in this study specifically showed a general dislike of additives in a wide variety of processed products. many participants did not distinguish between artificial and natural additives and agreed that the ‘products with additives’ tend to be less healthy. nevertheless, they also gave positive scores to natural ingredients and to having an appropriate explanation of the added product on the label. in parallel, various studies on consumer preferences and behaviour have also been conducted for these products (guerrero et al., 2017; viana et al., 2014). specifically, polizer rocha et  al. (2018) assessed consumer perceptions of frankfurt sausages with various attributes, with the findings being in line with this study, where these enhanced processed products are habitual in consumers’ diets. also, pires et  al. (2019) assessed the consumer perceptions of mortadella with reduced sodium content and/or enhanced with omega-3, focusing on the health aspect of the products as a solid reason for consumers’ purchase behaviour. with regards to the willingness to pay a price premium for enhanced processed products, there was generally a greater percentage of positive comments, although this trend was opposed by other mentions that were made in relation to the willingness to pay for artificial additive-free products. these results are in line with the research studies such as that of martínez michel et al. (2011) on consumer preference for chicken meat. in that study, the consumers preferred to choose additive-free and added flavour–free natural products, although certain communities (young people) were willing to pay for chicken with differentiated added properties (light, low in sodium, etc.). the importance of the label in the commercialisation of meat products is a relevant topic under discussion (eldesouky et al., 2020; janssen et al., 2016; picardy et al., 20 italian journal of food science, 2022; 34 (4) horrillo a et al. the ‘peculiar’ flavour of lamb can attract certain burger consumers who seek different aromas for occasional meals, or look for products of specific breeds (linares et al., 2012). some participants also mentioned the difficulty in finding minced lamb meat in butchers’ shops to make homemade burgers. the main reason is the small lamb formats slaughtered in spain, where the usual carcasses being produced are below 11–12 kg in weight (bernabéu and tendero, 2005; font et al., 2006). these carcasses provide very low number of trimmings and low-quality meat that could be used as by-products to make minced meat. the deboning process is more demanding compared to beef, and this would increase the price of the final product. cózar and vergara (2018) made lamb burgers from the manchego breed using high and low commercially valued cuts such as leg, neck and flank, although they did not study the impact of the deboning process on the final price. as mentioned above, label information about the properties and origin of the product has been described as essential for the marketing of meat products (bernués et al., 2003; fontet al., 2011). for new food products it is necessary to implement an adequate technological process, providing a balance between the market needs (reduced oxidation, packaging that can extend the shelf life, etc.) and the adequate additive or ingredient concentrations in order to prevent a change in the flavour of the product (cózar et al., 2018). however, it would also be necessary to implement an adequate promotion policy and diversify the range of lamb meat products on the markets (linares et al., 2020). nevertheless, promoting this product through advertising may not be sufficient to attract potential buyers. a label marking that ‘cherry’ has been used, for example, may suggest that the final product has acquired some sweet aromas or flavours that may not be accepted by the consumers, and this may compromise the purchase. it would therefore be necessary to carefully assess how the information on the label needs to be provided. conclusions meat consumers are clearly interested in eating quality and healthy meats. this is especially the case with meats, such as lamb, which are perceived as ‘red meat’ and therefore as unhealthy. however, when meat is consumed in the form of burgers, this makes it possible to add certain products that could improve the nutritional profile of the meat. this makes it necessary to know consumers’ views in order to promote new products enriched with natural ingredients (cherry and pecans) that could fill a niche in the lamb market. the use of focus groups has 2020; van loo et al., 2014), with a variety of results being found. in this study, the participants mentioned the need to have more information of the specific ingredients on the label, a fact that would reassure consumers. in the eyes of the buyer, the label is a sign of quality as much as meat colour at the time of purchase (rabadán et al., 2020), even for consumers who are familiar with the product (grunert et al., 2004). these findings are in line with those of solomando et al. (2021), who studied the sensory properties of boiled and dry-cured sausages enhanced with microcapsules of fish oil, and highlighted the effects of the information provided on the label. in this case, such information increased the scores obtained in the tests and questions about the intention of purchase, given that the provision of precise nutritional data on the label and statements on the nutritional values and health properties are information that is easily understood by consumers without the need to have precise knowledge on nutrition (feunekes et al., 2008; van kleef et al., 2008). lamb consumption amongst the spanish population is low in comparison with other types of meat (mapa, 2019). this meat is traditionally associated with special occasions or festivities (blasco, 2017) and its consumption is particularly low amongst the youth population (bernabéu and tendero, 2005). lamb is not a popular meat for many reasons, amongst which the main reasons being strong flavour and little presence in the home diet (alcalde et al., 2012). the focus group participants highlighted, aside from price, the fact that lamb recipes are much more complex than recipes made with other meats such as chicken and pork. the latter meats are currently the most popular (alcalde et al., 2012). the attendees of the sessions did not disapprove of the presence of natural ingredients in the meat, but what they found more interesting is the fact that such ingredient could add benefits to lamb, which is considered to be a positive aspect from the point of view of quality, health or meat preservation, and always under the premise of using healthy products (fernandes et al., 2017; florowski et al., 2019). in this context, it seems possible to develop burgers enhanced with vegetal ingredients with the purpose of making them healthier. urruzola et al., (2018) concluded that there is a market niche for the introduction of a burger enhanced with vegetable ingredients in its formulation and that it would be recommendable for these properties to appear on the label so that potential buyers can be aware of the benefits it has (bernués et al., 2003). fat and fatty acids profile of meat are factors that are responsible for the flavour of the meat, which is mainly determined by the animal species and their feeding habits (erasmus et al., 2016). some attendees mentioned that italian journal of food science, 2022; 34 (4) 21 consumer perceptions on the use of natural ingredients in lamb burgers atalaya, j.a., francia, j.m., ipanaque, c., gutierrez, m.f. and garcía,  h., 2018. hamburguesas vegetarianas veggisima. lima, peru: universidad san ignacio de loyola. barone, a.m., banovic, m., asioli, d., wallace, e., ruiz-capillas, c. and grasso, s., 2021. the usual suspect: how to co-create healthier meat products. food research international. 143: 110304. https://doi.org/10.1016/j.foodres.2021.110304 behrens, j.h., barcellos, m.n., frewer, l.j., nunes, t.p., franco, b.d.g.m., destro, m.t., et al. 2010. consumer purchase habits and views on food safety: a brazilian study. food control. 21(7): 963–969. https://doi.org/10.1016/j.foodcont.2009.07.018 bernabéu, r. and tendero, a., 2005. preference structure for lamb meat consumers. a spanish case study. meat science. 71(3): 464–470. https://doi.org/10.1016/j.meatsci.2005.04.027 bernués, a., olaizola, a. and corcoran, k., 2003. extrinsic attributes of red meat as indicators of quality in europe: an application for market segmentation. 14: 265–276. binnie, m.a., barlow, k., johnson, v. and harrison, c., 2014. red meats: time for a paradigm shift in dietary advice. meat science. 98(3): 445–451. https://doi.org/10.1016/j.meatsci.2014.06.024 blasco, m., sanudo, c., balado, j. and campo, m.m., 2017. actitudes de compra y consumo de carne de cordero. estudio comparativo de consumidores en zaragoza y castellón. proceedings of the xvii jornadas sobre producción animal, aula dei campus, zaragoza, spain. pp. 729–731. câmara, a. k. f. i., okuro, p. k., da cunha, r. l., herrero, a. m., ruiz-capillas, c., and pollonio, m. a. r. (2020). chia (salvia hispanica l.) mucilage as a new fat substitute in emulsified meat products: technological, physicochemical, and rheological characterization. lwt, 125, 109193. cano, a., hernández-ruíz, j., garcía-cánovas, f., acosta, m. and arnao, m.b., 1998. an end-point method for estimation of the total antioxidant activity in plant material. phytochemical analysis. 9(4): 196–202. https://doi.org/10.1002/(sici)10991565(199807/08)9:4<196::aid-pca395>3.0.co;2-w cayuela, j.m., garrido, m.d., bañón, s.j. and ros, j.m., 2003. simultaneous hplc analysis of α-tocopherol and cholesterol in fresh pig meat. journal of agricultural and food chemistry. 51(5): 1120–1124. https://doi.org/10.1021/jf020754s cózar, a., rubio, n. and vergara, h., 2018. combined effect of the spice and the packaging method on lamb burgers shelflife made with high value cuts. cyta—journal of food. 16(1): 544–552. https://doi.org/10.1080/19476337.2018.1431310 cózar, a. and vergara, h., 2018. lamb burgers made with low and high value cuts: effect of the spice added and the packaging method on shelf life. cyta—journal of food. 16(1): 1115–1124. https://doi.org/10.1080/19476337.2018.1537303 donoghue, s., 2010. projective techniques in consumer research. journal of family ecology and consumer sciences/tydskrif vir gesinsekologie en verbruikerswetenskappe. 28(1): 47–53. https://doi.org/10.4314/jfecs.v28i1.52784 eldesouky, a. and mesias, f., 2014. an insight into the influence of packaging and presentation format on consumer purchasing attitudes towards cheese: a qualitative study. spanish journal of agricultural research. 12(2): 305–312. https://doi.org/10.5424/ sjar/2014122-5520 proved to be an interesting tool for this purpose, due to their exploratory and unstructured nature. in this study, two of the most relevant aspects for the participants were the origin of the meat and the use of quality natural ingredients from the region. however, this information should be clearly displayed on the label of the meat products so that consumers can have it available at the time of purchase. however, consumer knowledge and understanding of food labelling should also be studied in order to effectively design these tools. thus, these burgers could become an attractive option for a segment of the population that seeks to consume more natural and healthy foods, because the participants also stated that they would be willing to pay a premium for improved lamb burgers compared to traditional ones. however, the fact that the focus groups indicated that burgers are mostly consumed at home raises the issue of addressing the promotion of these products primarily to retailers rather than to restaurants and burger bars. the addition of cherries or pecan nuts to burgers, natural ingredients perceived by consumers as healthy and nutritious, can contribute to discovering new market niches that allow the development of the lamb meat industry. in addition, it can help promote the consumption of this type 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https://doi.org/10.1016/j.lwt.2014.05.009� https://doi.org/10.1016/j.foodqual.2012.08.005� https://doi.org/10.1111/ijfs.14932� https://doi.org/10.1016/j.amepre.2005.01.009� https://doi.org/10.1016/j.amepre.2005.01.009� https://doi.org/10.1016/s0022-3182(12)80832-6� https://doi.org/10.26461/15.03� https://doi.org/10.12706/itea.2014.006� https://doi.org/10.12706/itea.2014.006� _goback _hlk65063513 _hlk62651763 _hlk62651792 _hlk111916811 98 issn 1120-1770 online, doi 10.15586/ijfs.v35i2.2269 p u b l i c a t i o n s codon italian journal of food science, 2023; 35 (2): 98–114 p u b l i c a t i o n s codon a pre-competitive research on frozen gnocchi: quality evaluation and market survey antonio stasi and antonietta baiano* dipartimento di scienze agrarie, alimenti, risorse naturali e ingegneria (dafne), university of foggia, via napoli, foggia, italy *corresponding author: a. baiano, dipartimento di scienze agrarie, alimenti, risorse naturali e ingegneria (dafne), university of foggia, via napoli, 25-71122 foggia, italy. email: antonietta.baiano@unifg.it received: 31 august 2022; accepted: 3 june 2023; published: 20 june 2023 © 2023 codon publications open access original article abstract this research was aimed to evaluate quality and safety of five formulations of gnocchi, namely, traditional, tomato basil, pumpkin, spinach, with cauliflower pieces, and stuffed with cheese, to be marketed in a frozen state. an online survey was performed to assess the consumer desires and willingness to buy these new products in order to assess consumer preferences and targets and verify whether fresh and frozen gnocchi could become market substitutes or complements. the results highlighted that frozen gnocchi were a good product under different points of view and characteristics of an ideal consumer. keywords: consumer preference; fresh pasta; online survey; quality; willingness to pay introduction gnocchi, a typical italian dish prepared with potatoes, are widely known globally. this italian name is derived from the term ‘nocchio’, which refers to knots in wood, or from the term ‘nocca’, which means ‘knuckle’. both these terms describe the classic shape of gnocchi. the preparation of the basic recipe is very simple, as it involves mixing and kneading of potatoes, previously boiled, peeled, and mashed with water, potato starch and flour (generally soft wheat, but sometimes rice flour, in the case of gluten-free products). potato flakes can be used as an alternative to fresh potatoes. the obtained dough is then sectioned into small pieces of variable shapes and sizes. as for pasta, gnocchi are prepared by cooking in salted-boiling water and then dressed with various sauces depending on the type of gnocchi and recipe used. cooking is very quick, and the optimal cooking point is the rise of gnocchi on the surface of the boiling water. in the absence of a specific legislation, gnocchi are covered within the regulatory framework for fresh and stabilized pasta outlined by law no. 580 of 1967 (ministry of agriculture, food sovereignty and forestry, 1967), subsequently amended by dpr n. 187 of 2001 (ministry of agriculture, food sovereignty and forestry, 2001) and dpr n. 41 of 2013 (ministry of agriculture, food sovereignty and forestry, 2013). in the past, gnocchi were mainly prepared at domestic and artisan levels whereas, nowadays, they are also produced at industrial level. the consumption of gnocchi is widespread in italy, france and germany, although markets, such as those of the united kingdom, spain and eastern european countries, are also growing. few data on the production and consumption of gnocchi are available. in 2013, italy produced 11,5000 tons of gnocchi against 27,000 tons in france, and exported almost half of its production (storchi, 2014). the italian production of packaged gnocchi is constantly growing, passing from €65 million to over €165 million in 1995–2017. in 2017, 20% of italian gnocchi were exported to extra-eu countries for sale on the shelves, as the product is stable at room temperature. the remaining 80% of exports comprised mailto:antonietta.baiano@unifg.it italian journal of food science, 2023; 35 (2) 99 a pre-competitive research on frozen gnocchi frozen state. successively, an online survey was performed to assess the consumer desire and willingness to buy new products. more than 500 interviews were conducted, and a gnocchi preferences database was set up for analysis. the purpose of this market analysis was to verify whether frozen gnocchi could be substituted for other types of gnocchi, and whether frozen gnocchi had the same target. the research also focussed on both traditional and flavoured gnocchi to verify whether consumer desire and willingness to buy frozen version are affected by the presence of other ingredients in the formulations. the analysis was carried out by hypothesizing a system of simultaneous equations of gnocchi purchase behaviour, in which consumers are willing to purchase different types of frozen gnocchi. materials and methods gnocchi production six types of gnocchi were considered: traditional gnocchi; tomato-basil gnocchi; pumpkin gnocchi; spinach gnocchi; gnocchi with cauliflower pieces; and cheese-stuffed gnocchi. owing to their formulations, all types of gnocchi, except those stuffed with cheese, were gluten-free. for each formulation, a preliminary work was carried out to develop the best recipe, considering both production of dough capable of mechanical formulation and ability to meet the needs of a wide range of consumers by avoiding strong tastes, application of preservatives, and using ingredients referred to the mediterranean diet. the optimized formulations are reported in table 1. production was carried out in an industrial plant located in apulia region (southern italy). according to the production process, hot water, flour and other ingredients were mixed and kneaded (condor pps 130, san giovanni lupatoto, italy). successively, the dough was transferred to forming machine (m-agnelli a/250, bussero, italy). in the case of cheese-stuffed gnocchi, formulation was combined with cheese extrusion (sandore nf 80, zanè, italy). gnocchi were then transported to freezing tunnel (pugnale, milano, italy) by a conveyor belt (bonfiglioli bn63b4, bologna, italy) where products achieved a temperature of -18°c within 7 min. three batches, each of about 100 kg, were produced for each type of gnocchi. the weight of each dumpling was 3.5±0.3 g. sampling for sampling, five aliquots, each of 500 g, were taken from each batch. each aliquot was submitted to physical, chemical, and microbiological analyses. refrigerated product, which was done to european markets (bonetto, 2018). owing to its small size, the market of gnocchi has interesting growth margins because of product and process innovation. according to the available statistical reports (expert market research, 2022), the global market of gnocchi is driven by the growing requirement of convenience foods and is expected to further increase in 2022–2027 at a compound annual growth rate (cagr) of 4.50%, particularly in north america, europe, the asia pacific, latin america, the middle east and africa. in addition, according to the gnocchi regulatory framework, producers are free to modify shapes and ingredients, thus creating a variety of formulations. considering that the current sale channels are essentially limited to fresh gnocchi (prepared for consumption within a few hours of production), refrigerated product (with a shelf life of 80–120 days, reduced to 60 days in the case of stuffed gnocchi, at temperatures of 4–8° c), and stable product at room temperature (shelf life of 180 day in a cool and dry place), an interesting market niche to explore is that of frozen product, which would allow a further extension of shelf life in the absence of preservatives. in fact, gnocchi are currently sold in a frozen state almost exclusively as ready-to-eat meals or as a semi-finished product to companies that deal in ready-to-eat meals. innovation must be accompanied with a careful control of the effects of product/process changes made on the safety and quality characteristics of new products as well as on consumer demands. the safety of gnocchi is generally related to its microbial population (chunfeng et al. 2005; del torre et al., 2004), while their quality is strongly related to their physico-chemical (mainly water activity [aw], texture and ph), nutritional, and sensory properties (alessandrini et al., 2010; bercot et al., 2014; liu et al., 2016). in addition, studies on consumer preferences and willingness to buy/pay are useful tools to forecast the success of any product on market (barcelon et al., 2014; defrancesco et al., 2017). previous studies on innovation arrived at in gnocchi production generally referred to changes in some ingredients and their effects of product’s quality. in this context, it is worth mentioning the addition of orange-fleshed sweet potato (da silva et al., 2016), mix of tilapia and tuna (coradini et al., 2019), sea water (santagata et al., 2021), red rice, or buckwheat (cappa et al., 2021). process innovation was less considered: an example was the production of frozen gluten-free gnocchi through turbo cooking (cappa et al., 2017). the present research aimed to evaluate quality and safety of new formulations of gnocchi that can be marketed in a 100 italian journal of food science, 2023; 35 (2) stasi a. and baiano a. table 1. formulations of gnocchi. ingredients (%) types of gnocchi traditional tomato-basil pumpkin spinach with cauliflowers stuffed with cheese water 52 50 50 52 20 50 cauliflower – – – – 40 – wheat flour – – – – – 30 corn flour 14 – – – – – potato flakes 13 10 15 15 – 8 potato starch 14 18 18 18 23 11 corn starch 6 14 14 13 – – tapioca – – – – 16 – salt 1 2 1 1 1 0.93 tomato – 3 – – – – basil – 2 – – – – pumpkin – – 1 – – – spinach – – – 1 – – safflower – – – – – 0.07 turmeric – – 1 – – – paprika – 1 – – – – physical and chemical analyses gnocchi samples were submitted to the following analyses immediately after freezing: • moisture content, expressed as moisture percentage, measured through a thermo balance (sartorius ma35m-230n, goettingen, germania) at 130°c until a constant weight was reached. • water activity, aw, determined by using a testo 650 water activity system (testo, milan, italy). • ph, through a si analytics lab845 ph-meter (wheilheim, germany). all analyses were repeated for at least five times for each aliquot. microbiological analyses firstly, immediately after preparation, all types of gnocchi were examined. for this, 10 g of gnocchi was withdrawn from each aliquot, diluted with 90 g of buffered peptone water and homogenized for 2 min in a stomacher lab blender model 400 (seward medical, england). decimal dilutions (from 10-1 to 10-6) were prepared and 0.1 ml of each dilution was spread on the surface of the following medium supplied by hyserve gmbh & co. kg (uffing, germany): • plates of compact dry total count, and incubated at 30°c for 48 h to enumerate total aerobic bacteria (iso  4833-1; international organization for standardization (iso), 2013). • plates of compact dry total coliforms, and incubated at 37°c for 24 h to enumerate total coliforms (iso 4832; iso, 2006). • plates of compact dry yeasts and moulds, and incubated at 30°c for 72 h to quantify yeasts and moulds (iso 21527-2; iso, 2008). in order to evaluate the evolution of microbiological quality of gnocchi under different storage conditions, additional analyses were performed on three of the new formulations, namely, traditional gnocchi, gnocchi with cauliflower pieces and cheese-stuffed gnocchi, because of their consumer target, not homogeneous (inhomogeneous) structure, and animal origin, respectively. such analyses were performed on samples stored under the following time–temperature pairs: just frozen gnocchi, 0  day/–18±2°c (t0); and the following thermal abuse conditions: 40 days/–12±2°c (t1), 10 days/–6±2°c (t2) and 2 days/8±2°c (t3). gnocchi were analysed for total aerobic bacteria (iso 4833-1; iso, 2013), total coliforms (iso 4832; iso. 2006), sulphite-reducing clostridia (iso 15213; iso, 2003), coagulase-positive staphylococci (uni en iso 6888-1; uni italian standardization, 2018), yeasts and moulds (iso 21527-2; iso, 2008), escherichia coli (iso 16649-2; iso, 2001), listeria monocytogenes (iso 11290-1; iso, 2017a), salmonella ssp. (iso 65791; iso, 2017b), and bacillus cereus (mi 016/12 rev. 2; google.it, 2013). in addition, traditional gnocchi were also analysed for salmonella ssp. (iso 6579-1; iso, 2017) whereas gnocchi with with cauliflower pieces and italian journal of food science, 2023; 35 (2) 101 a pre-competitive research on frozen gnocchi spring  water. the sensory sessions were conducted in a sensory room equipped with eight booths according to the iso standard 8589 (iso, 2007). the temperature was kept at 24±2°c. humidity was in the range of 70–85%. market survey a survey of six gnocchi formulations (traditional, tomato basil, pumpkin, spinach, with cauliflower pieces, and cheese-stuffed) was conducted on the italian market. a questionnaire comprising 19 questions was prepared and distributed through an open access online document that gathered 532 responses. the first part of the questionnaire contained 11 detailed questions on socio demographic characteristics of respondents and family components (place of residence, age, gender, education level, employment, household composition, household income, and food expenditure). the remaining eight questions concerned food habits and methods of choice of gnocchi (frequency of consumption, preferred types of gnocchi in terms of formulation, packaging and storage, and selling prices). the questionnaire was structured with open, closed, and multiple-choice questions as well as with preference lists and scales. the survey was conducted online on a convenience sample. the sampling strategy followed the snow-ball effect strategy by multiple sharing of the link on messaging app and social media. statistical analysis mean values and standard deviations were calculated using the excel software v. 14.0.0 for mac. the analysis of variance (anova) of the experimental data was performed using the statistical package statistica for windows v. 8.0. (statsoft inc., tulsa, ok). anova and the tukey hsd test (p < 0.05) were applied to determine statistically significant differences among samples. concerning the market survey, first the respondent characteristics were analysed using descriptive statistics. then, an econometric model was set up to estimate the impact of the following: socio-demographic variables of respondents, their food consumption habits, and respondents’ declared preferences about gnocchi for the price they were willing to pay. the econometric strategy was based on a system of simultaneous equations, in which each model represented a certain type of gnocchi considered for analysis. the strategy was coherent with the choice context of specific product, as one could choose at the same time to buy one or more types of gnocchi: pij = f(zi, hi, ri), (1) cheese-stuffed gnocchi samples were also analysed for pseudomonas ssp. (iso/ts 11059; iso, 2009b). the analyses were performed in duplicate for each aliquot, and the results were expressed as cfu/g. nutritional analysis the proximate analysis of just-frozen traditional gnocchi, gnocchi with cauliflower pieces and cheese-stuffed gnocchi was performed. the following parameters, expressed per 100 g of gnocchi were evaluated: energy (as kj and kcal; eu reg 1169; european union [eu], 2011); carbohydrate, total fats and saturated fatty acids (in grams; istisan 34; istituto superiore di sanita [istisan], 1996); proteins (in grams, iso 1871; iso, 2009a); fibre (in grams; iso 5498; iso, 1981); sodium chloride (in grams; mi 019/14 rev 3, 2017 and eu reg. n. 1169; european union, 2011), moisture (as %; istisan 34; istisan, 1996), and ash (in grams; uni en iso 2171; uni italian standardization, 2010). on cauliflower samples, the following additional analyses were performed: calcium, iron, potassium (as mg/kg; iso 21424; iso, 2018b), vitamin a (as μg/100 g; istisan 34; istisan, 1996), vitamin c (as mg/100 g; istisan 34; istisan, 1996), and vitamin d (μg/100 g; iso 20636; iso, 2018a). the analyses were performed in duplicate for each aliquot. sensory analysis the sensory evaluation of traditional gnocchi, gnocchi with cauliflower pieces, and cheese-stuffed gnocchi was performed by a panel consisting of 11 highly trained judges (with >100 h of descriptive training and >1000 h of testing experience). gnocchi samples were cooked in unsalted boiling water. two cooking periods were considered: the period corresponding to the emersion of gnocchi on water surface (optimal cooking time), and the period obtained by prolonging cooking for another 20  s to evaluate the effects of overcooking. panellists were asked to evaluate five parameters, namely, visual appearance, odour, taste, texture and stickiness, on a 5-point scale (from 0 to 4, with 2.5 as the lower limit for considering the product as acceptable) anchored with extremely low and extremely high parameters related to the perception of stimuli. the sensory procedures were replicated thrice. samples were coded with random 3-digit numbers and served monodically. to balance out any possible order-effect, the order of presentation was randomized for each panellist and each session, and the samples were evaluated using a completely randomized design. in order to reduce carry-over effects, a 1-min break was provided between samples, during which panellists were required to rinse their mouths thoroughly with 102 italian journal of food science, 2023; 35 (2) stasi a. and baiano a. (ministry of agriculture, food sovereignty and forestry, 2001) for fresh pasta (0.92 ≥ aw ≥ 0.97), with the exception of those referred to traditional gnocchi, in which water activity was slightly lower but above the limit value required for the growth of selected pathogens (0.85; food and drug administration (fda), 2011. as can be inferred from data, moisture content and water activity values were not correlated. in particular, traditional gnocchi had one of the higher moisture contents and the lowest water activity. their formulation strongly differed from other samples for their high content of corn flour, known for its high sorption capacity (ahmed and islam, 2018), which is, in turn, related to lower amylose content (around 29%), compared with those with potato starch (~54%) and tapioca (36%) (horstmann et al., 2016). in agreement with biduski et al. (2018), starch samples with high amylose content have lower water absorption because of the greater stiffness of hydrogel structure that resists swelling. the average ph (table 2) was in the range 4.62–5.95, with the lowest value detected in tomato-basil gnocchi because of the acids contained in characterizing ingredients, and the highest ph measured in cheese-stuffed gnocchi mainly because of the contribution of the stuffed ingredient. all the samples showed ph values above the lower limit required for the growth of pathogens; however, because of the temperature range required for growth of pathogen, freezing guaranteed to limit their development (food and drug administration [fda], 2019). microbiological profiles of gnocchi in spite of gnocchi being an excellent growth substrate for microorganisms, especially in the case of storage under improper conditions, this was the first study to include a comprehensive microbiological characterization of gnocchi. in fact, previous studies were limited to the enumeration of total bacteria and moulds during refrigerated storage and the growth and survival of specific microorganisms, such as listeria monocytogenes or staphylococccus aureus (hu et al., 2017; szymczak and dąbrowski, 2015; wu et al., 2018). where p is the price that the ith consumer is explicitly willing to pay for the jth type of gnocchi; z is the socio-demographic characteristics of the ith consumer; h is the food consumption habits of the ith consumer; r is the general preferences about gnocchi of the ith consumer. for the estimation of the model, stata 14.0 was used to run a seemingly, unrelated, regression (sure) method developed by zellner (1992). the method allowed estimating more models simultaneously and considering model error dependence. such a need, which was not considered in standard regression model, was linked to the simultaneity of the choice, and in our case to the simultaneity of the purchase of different types of gnocchi. results and discussion physico-chemical characteristics of optimized formulations as shown in table 2, the lowest moisture content was found in cheese-stuffed samples of gnocchi. the dough of this sample had added water comparable with those of other formulations (with the exception of gnocchi with pieces of cauliflower) and had a cheese filling having a water content of around 37% and representing 20% of the total mass of the product. the highest percentage of moisture was estimated in gnocchi with pieces of cauliflower, whose formulation consistently differed from those of other samples. in fact, although the amount of added water was significantly lower than that of other formulations, a higher water intake derived from cauliflower (around 92–94%) and the amount of dry ingredients was lower than that in other formulations. water activity is a suitable tool for predicting the growth of microorganisms. the average water activity values (table 2) were in the range established by dpr n. 187 table 2. physico-chemical characteristics of gnocchi. parameters types of gnocchi traditional tomato-basil pumpkin spinach with cauliflowers stuffed with cheese moisture (%) 55.7±0.6b,c 50.6±2.3b 52.1±3.1b,c 50.7±3.6b 56.7±1.2c 44.2±0.3a a w 0.90±0.01a 0.92±0.01a,b 0.93±0.01b 0.92±0.02a,b 0.93±0.01b 0.95±0.01c ph 5.10±0.11b 4.62±0.22a 5.40±0.10c 5.42±0.09c 5.72±0.11d 5.95±0.12e different letters indicate significant differences among types of gnocchi (tukey test, p < 0.05). italian journal of food science, 2023; 35 (2) 103 a pre-competitive research on frozen gnocchi t2 storage conditions had the highest concentration of total coliforms. these data were in agreement with the literature, as some strains, such as pseudomonas fragii, grow at temperatures as low as –6°c and pseudomonas fluorescens grow at temperatures as low as –4°c (feiner, 2006). these data highlighted the detrimental effects of storage temperatures higher than –18°c. in any case, based on the recommendation no. 011 issued by the italian frozen food institute (iias, 2006), which provides useful information for determining minimum storage period for frozen food marketed in italy, the experimental gnocchi could be labelled with a shelflife of 18 months if stored under freezing conditions. nutritional value of gnocchi based on the nutritional information (table 5), gnocchi were low in calories, supplying from 141 kcal/100  g (gnocchi with pieces of cauliflower) to 185 kcal/100 g (cheese-stuffed gnocchi). obviously, these products are intended to represent the first course, providing high intake of carbohydrate but small amounts of proteins and lipids (just a little more in cheese-stuffed gnocchi). as a result, they had a medium glycemic index (around 68; johnson-greene, 2020). gnocchi with cauliflower pieces had a good mineral content contributed by fresh vegetable, while the intake of both fatand watersoluble vitamins was negligible. the sodium/nacl content of cheese-stuffed gnocchi was almost double, compared with the other two types of gnocchi. probably because, beyond the salt added to the formulation, more sodium comes from the other ingredients used in the production of the gnocchi. the goal of reducing salt intake is pursued globally by all institutions dealing with public health. for example, according to the ‘salt reduction targets for 2017’ published by the public health england (phe, 2017), pasta should contain no more that 0.88-g salt or 350-mg sodium, 0.5-g salt per 100 g. in this perspective, the experimental gnocchi must be labelled with the recommendation of not adding salt to the cooking water with only limited amount of seasoning. regarding the microbial profiles of just prepared gnocchi, the formulation containing pieces of cauliflower showed the highest mesophilic load (together with those stuffed with cheese) and total coliforms (table 3). according to the collected data, a modification to washing procedures was suggested in the case of gnocchi with cauliflower. owing to the peculiar conformation of cauliflower inflorescence, water was difficult to reach its various parts, hence a washing extension and/or the use of floating water could be useful to improve its hygienic quality. traditional gnocchi generally showed the lowest microbial load, although their total aerobic bacteria were 1 logarithmic cycle higher than those detected by hu et al. (2017). the presence of yeasts and moulds was negligible in all the samples. in order to better understand the evolution of microbial profile under specific storage conditions, additional analyses were performed on traditional gnocchi, gnocchi with cauliflower pieces, and cheese-stuffed gnocchi; the results are reported in table 4. individual and interactive effects of the type of gnocchi and the storage conditions on the concentration of various microbial groups were analysed. results showed that pathogens were below the detection limit in all the samples to prove product’s safety and compliance with the safety criteria established by eu reg. n. 1441 (european union, 2007), which relates to the microbiological criteria applicable to food products. surveys performed by other researchers highlighted the presence of listeria monocytogenes in refrigerated shrimp salads-contained gnocchi (burnett et al., 2005). however, a high presence of moulds and yeasts was detected in traditional gnocchi whereas a high total bacterial load and occurrence of pseudomonas spp. were observed in cheese-stuffed gnocchi. concerning the effects of storage conditions, the combination t1 (40 days/–12±2 °c) was the most disadvantageous conditions for total bacterial load and occurrence of yeasts and pseudomonas spp., while the combination t2 resulted in a greater presence of total coliforms. concerning the interactive effects of the type of gnocchi and the storage conditions, those stuffed with cheese and subjected to t1 storage conditions showed the highest levels of total bacterial load, yeasts, and pseudomonas spp., while those subjected to table 3. microbial loads (cfu/g) of just prepared gnocchi. microorganisms types of gnocchi traditional tomato-basil pumpkin spinach with cauliflowers stuffed with cheese total aerobic bacteria 6×103a 3.8×104c 1.6×104b 1×104b 4.2×104c,d 4.9×104d total coliforms 6.3×102a 1×103b 5.5×102a 6.6×102a 2.6×103c 7.7×102a yeasts 102a 3×102b 4×102b,c 102a 5×102c 3×102b moulds 102a 102a 102a 102a <102a 102a different letters indicate significant differences among types of gnocchi (tukey test, p < 0.05). 104 italian journal of food science, 2023; 35 (2) stasi a. and baiano a. table 4. microbial loads (cfu/g) of gnocchi stored in different conditions. microorganisms gnocchi t0 (0 days; –18°c) t1 (40 days; –12±2°c) t2 (10 days; –6±2°c) t3 (2 days; 8±2°c) total aerobic bacteria traditional 1.3×102 2×102 5.3×102 5.8×103 cauliflower pieces 4.4×102 8×103 6.5×103 2.2×103 stuffed with cheese 1.6×104 1.2×105 105 9.6×104 total coliforms traditional <10 <10 <10 <10 cauliflower pieces <102 6×102 5×102 6×102 stuffed with cheese 1.6×102 103 1.6×103 1.2×103 yeasts traditional <4×102 <4×102 <4×102 <6×102 cauliflower pieces <102 <4×102 <4×102 <102 stuffed with cheese <102 <102 <102 <102 moulds traditional 8×103 2.6×103 <102 3×103 cauliflower pieces <102 < 4×102 <102 <102 stuffed with cheese <102 <102 <102 <102 salmonella spp. traditional absent in 25 g absent in 25 g absent in 25 g absent in 25 g cauliflower pieces n.d. n.d. n.d. n.d. stuffed with cheese n.d. n.d. n.d. n.d. escherichia coli traditional <10 <10 <10 <10 cauliflower pieces <10 <10 <10 <10 stuffed with cheese <10 <10 <10 <10 listeria monocytogenes traditional absent in 25 g absent in 25 g absent in 25 g absent in 25 g cauliflower pieces absent in 25 g absent in 25 g absent in 25 g absent in 25 g stuffed with cheese absent in 25 g absent in 25 g absent in 25 g absent in 25 g bacillus cereus traditional <102 <102 <102 <102 cauliflower pieces <102 <102 <102 <102 stuffed with cheese <102 <102 <102 <102 coagulase+staphylococci traditional <102 <102 <102 <102 cauliflower pieces <102 <102 <102 <102 stuffed with cheese <102 <102 <102 <102 sulphite-reducing clostridia traditional <10 <10 <10 <10 cauliflower pieces <10 <10 <10 <10 stuffed with cheese <10 <10 <10 <10 pseudomonas spp. traditional n.d. n.d. n.d. n.d. cauliflower pieces <102 3.6×103 6×102 <4×102 stuffed with cheese 3×103 3.1×104 2×104 104 n.d.: not determined. sensory evaluation of gnocchi the results of sensory evaluations (table 6) highlighted that all the parameters were evaluated as more than acceptable. the highest scores were generally assigned to sample odour. the extension of cooking period exerted negative effects on taste and, in higher extents, on texture and stickiness that sometimes were considered less than acceptable. cheese-stuffed gnocchi received significantly higher scores for taste, texture, and stickiness, probably because of the presence of wheat flour in their formulations and the consequent formation of a gluten network that was able to improve their textural properties. online survey: profiles, behaviors and attitudes of respondents table 7 describes the profile of participants of online survey. most of the respondents were males (60%) in the age groups of 18–30 years (56%), 31–40 years (10%), italian journal of food science, 2023; 35 (2) 105 a pre-competitive research on frozen gnocchi table 5. nutritional information on gnocchi. types of gnocchi traditional with cauliflowers stuffed with cheese energy kj 645a 598a 785b kcal 152a 141a 185b carbohydrates 34.71b 31.60a 36.76c sugars 0.06b <lqa 0.92c fructose glucose 0.07a 0.13b 0.68c lactose <lqa <lqa <lqa maltose 0.07a 0.07a 4.18b saccharose <lqa <lqa <lqa fats 0.23a 0.47b 0.95c saturated fatty acids <0.10a <0.10a 0.32b fiber 2.02b 2.02b 1.06a proteins 1.79a 1.61a 6.87b sodium 0.72a 0.72a 1.11b nacl 1.40a 1.79b 2.77c ash 1.76a 1.85a 3.14b minerals ca n.d. 332.71 n.d. fe n.d. 2.31 n.d. k n.d. 1016.33 n.d. vitamins a n.d. <0.5 n.d. c n.d. <lq n.d. d n.d. <0.5 n.d. different letters indicate significant differences among types of gnocchi (tukey test, p < 0.05). lq: quantification limit. the values are expressed in g/100 g, with the exception of those referred to ca, fe, and k, which are expressed in mg/kg, and those referred to vitamins a, c, and d, expressed in μg/100 g, mg/100 g, and μg/100 g, respectively. n.d.: not determined. table 6. results of sensory analysis. types of gnocchi visual appearance odour taste texture stickiness c.t. c.t.+20 s c.t. c.t.+20 s c.t. c.t.+20 s c.t. c.t.+20 s c.t. c.t.+20 s traditional 3.8±0.8a,a 3.4±0.7a,a 3.9±0.6a,a 3.7±0.7a,a 3.7±0.5b,a 3.1±0.5a,a 3.5±0.5b,a 2.8±0.5a,a 3.1±0.4b,a 2.7±0.5a,a with cauliflower pieces 3.5±0.7a,a 3.1±0.7a,a 3.8±0.4a,a 3.7±0.5a,a 3.7±0.6b,a 3.2±0.4a,a 3.4±0.5b,a 2.7±0.5a,a 3.1±0.3b,a 2.6±0.5a,a stuffed with cheese 3.5±0.8a,a 3.4±0.7a,a 3.9±0.5a,a 3.7±0.5a,a 4.0±0b,b 3.7±0.2a,b 3.7±0.6b,a 3.1±0.7a,b 3.2±0.4b,a 2.9±0.5a,b different lowercase letters indicate significant differences among the cooking times (tukey test, p < 0.05). different uppercase letters indicate significant differences among types of gnocchi (tukey test, p < 0.05). 41–50 years (20%) and 51–60 years (11%). the education level of the participants was high, since about 18% stated to have a specialization or a phd, and 44% declared to be graduates (with bachelor’s or master’s degree). concerning employment, about 58% of respondents stated to have a job, while 10% were unemployed. the number of student respondents was also significant (~28%). in all, 237 of the 532 respondents indicated their specific occupation. the respondents mainly comprised employees (22%), freelancers (14%), teachers (11%), university professors (10%), catering operators (6%), entrepreneurs (5%), workers (5%), and those employed in the food quality control sector (5%). concerning the family size, 38% of participants’ families consisted of four individuals, while around 3% stated as having a larger family (>5 members). the incidence of families comprising one or two persons (~11% and 18%, respectively) was interesting. the average number of family members was 3.3. concerning the place of residence, most of the respondents were urbanites, with ~43% residing in cities of 50,001–200,000 inhabitants, or in a town with 5000– 20,000 inhabitants (~33%). regarding the geographical origin of participants, most of them lived in puglia (73.3%) and lombardia (10.5%), followed by emilia-romagna (2.6%), lazio and piemonte (2.4%), abruzzo (1.9%), toscana (1.7%), veneto (1.3%), campania (0.9%), basilicata (0.8%), marche (0.6%), sicilia (0.4%), and calabria, molise, liguria, sardegna, trentino-alto-adige, and umbria (0.2% each). the gross annual income of respondents was €15,000– 28,000 (~40%) and €28,000–55,000 (~27%); 46% of the respondents spent €50–100 on food per week. most of the respondents were involved in food purchase, but with some differences: 19.4% of the respondents were 106 italian journal of food science, 2023; 35 (2) stasi a. and baiano a. table 7. profile of respondents (n = 532). variable % gender males 60.0 females 40.0 age (years) 18–30 55.8 31–40 9.6 41–50 20.3 51–60 10.9 61–70 2.4 71–80 0.6 >80 0.4 education level compulsory school 4.9 high school diploma 33.4 bachelor’s degree 19.0 master’s degree 25.0 specialization/phd 17.7 occupation student 27.6 housewife 2.6 employed 57.7 retired 2.1 unemployed 10.0 number of family members 1 10.7 2 18.2 3 17.5 4 37.8 5 13.0 >5 2.8 number of inhabitants of the city in which the respondent lives <5000 2.4 5000–20,000 32.9 20,001–50,000 10.6 50,001–20,0000 43.2 200,001–500,000 6.8 >500,000 4.1 gross annual household income (€) 0–15,000 20.5 15,001–28,000 40.2 28,001–55,000 27.5 55,001–75,000 7.1 >75,000 4.7 average weekly expenditure on food purchases (€) <50 19.4 50–100 46.4 (continued) table 7. continued variable % 101–150 21.0 151–200 6.6 >200 1.9 he/she doesn’t know 4.7 who makes food purchases in the respondent family only the respondent 19.4 the respondent but also other family members 67.9 the respondent but only upon instructions of other family members 8.4 the respondent doesn’t make food purchases in his/her family 4.3 presence of any food intolerance, unwelcomed foods, and digestion difficulties in the respondent family yes 67.1 no 32.3 no reply 0.6 the only family member to make food purchase; 67.9% of them alternated with other members; 8.5% indulged in making purchases if instructed by other family members; and only 4.3% never indulged in food purchase. about 67% of the respondents declared as having food intolerance or digestive difficulties or simply aversions to certain foods in case of one or more family members. intolerance, allergies, and/or aversions were for categories of milk derivatives, cereal derivatives, fishery products and meat. many respondents stated as having more than one allergy/intolerance/aversion. regarding eating habits, 82% of the respondents declared to rarely consume gnocchi, while only 1% consumed gnocchi for several times a week (figure 1a). these data confirmed that gnocchi were a niche product and, therefore, product innovation could help to reach further market segments. about 80% of the respondents preferred refrigerated gnocchi and only 3% declared a preference for frozen product (figure 1b). according to these data, growth margins for the frozen product appeared to be high. among the gnocchi choice parameters (figure 1c), ingredients and visual appearance were fundamental or very important. cost and brand were considered moderately important (47.2% and 41.4%, respectively). calories generally never invoke concerns, as gnocchi are rarely eaten, although they have medium-to-low energy content. a typical consumer of gnocchi is traditional eater, loyal to classic shapes, mainly the traditional and wavy ones (figure 2). the most popular packaging size was 250 g (45.7%) and 500 g (49.2%), probably because these were more italian journal of food science, 2023; 35 (2) 107 a pre-competitive research on frozen gnocchi figure 1. online survey on gnocchi: (a) frequency of consumption (%); (b) preference for preservation method (%); and (c) importance of some parameters of choice (%). frozen gnocchi 45 35 25 15 5 50 40 30 20 10 0 10,9 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 16,5 24,8 30,3 33,6 33,5 31,6 36,8 47,2 41,4 22,4 19,0 28,6 14,8 17,3 17,7 13,0 10,0 26,5 10,3 80,5 3,0 6,4 5,8 3,6 3,4 9,0 8,3 never gnocchi stable at room temperature refrigerated gnocchi ingredients calories visual appearance price brand not important very important (a) (b) (c) extremely importantnot very important moderately important rarely once a week more than once a week 4,9 82,0 6,2 0,9 108 italian journal of food science, 2023; 35 (2) stasi a. and baiano a. suitable to the mentioned number of family members (figure  3a). bags were the most appreciated type of packaging, especially in a transparent version, perhaps because they occupied less space (figure 3b). trays were appreciated only if completely transparent, although their strength better protected from mechanical damages, especially compression forces. the packaging transparency correlated substantially with the importance of visual appearance in the choice of gnocchi. the respondents considered traditional gnocchi as very pleasant (figure 4a) and were less enthusiastic for the formulations containing other ingredients. gnocchi with cauliflowers or stuffed with cheese were rated as unpleasant by increased proportion of respondents. these data evaluated consumers as traditionalists. the price per (a) (b) (c) (d) (e) (f) 0 50 100 150 200 250 300 350 288 234 105 90 35 19 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% traditio... wavy shape round shape alternative shape romana’ gnocchisunken wavy shape figure 2. online survey on gnocchi: preferences regarding shape: (a) classic shape; (b) round shape; (c) wavy shape; (d) sunken wavy shape; (e) ‘romana’ shape; and (f) alternative shape. kilogram that respondents were willing to pay was <€2.50/kg and €2.50–4/kg, with a predominance who would be willing to pay <€2.50/kg for traditional gnocchi and gnocchi with cauliflower pieces, and €2.50/kg–4/kg for other types (figure 4b). last but not least, the sure model was estimated. the objective of the model was to identify significant variables affecting willingness to purchase and pay for different types of frozen gnocchi. as shown in table 8, the sure model outperformed well and presented a discrete number of statistically significant variables interpreting consumers’ preference and behavior. selection of the variables shown in table 8 is defined by a step-wise procedure, which led to a selection of 10 italian journal of food science, 2023; 35 (2) 109 a pre-competitive research on frozen gnocchi figure 3. online survey on gnocchi: preferences regarding (a) size (%); and (b) type of packaging (possibility to express more than one preference). significant regressors. in detail, the results highlighted the following relations: • imp_price: represents the level of preference of consumers for low-price products. the mean coefficient, below –0.10, indicated the inverse relationship with the price that consumers are willing to pay, which confirms coherence with the economic theory of demand. • imp_ingr: the importance of ingredients table in the label when choosing gnocchi. this variable shows a positive coefficient of about 0.1 average, indicating a direct relation with willingness to purchase and pay. frozen gnocchi are preferred by people caring about ingredient information provided on the label. • gnocchi_amb: this variable indicates the level of substitution between frozen gnocchi and those at room temperature (such as pasta). the negative value of the coefficient indicates the level of substitution between two products. in fact, the more the consumers prefer room-temperature gnocchi, the less they are willing to purchase frozen gnocchi. • gnocchi_hab: it indicates consumer habits of eating gnocchi. this variable is measured in terms of frequency of consumption of gnocchi on a scale of 1–5. a negative value of coefficient indicates that consumers that are more frequent have weaker preferences for frozen gnocchi. maybe, they prefer fresh ones. • intolerant: when a family member presents intolerance, such as gluten, gnocchi could be preferred to pasta. in fact, in these cases, consumers are willing to purchase and pay more. • income: a positive sign for this variable confirms that the higher the income, the higher the willingness to pay for frozen gnocchi. following the economic theory, if income and consumption have positive relationship, then the good is perceived as of normal quality whereas if there is an inverse relationship, the good is perceived as of inferior quality. therefore, frozen gnocchi are not perceived as low-quality products. • no_family: results of the analysis show that the number of family members in a household has a positive relationship with the price consumers are willing to pay for frozen gnocchi. such result could be an interesting one, because bigger families could, indeed, prefer frozen gnocchi, compared to fresh ones, as they could be stored in fridge for a longer period. • gender: results highlight another important variable for the identification of the target. male consumers are 300 0 5 10 15 20 25 30 35 40 45 50 45,7 49,2 3,0 2,1 250g 500g 750g 1kg non-transparent tray with transparent closing film 63 transparent tray with non-transparent closing film 93 totally transparent tray 177 140not transparent plasting bag transparent plasting bag 262 250200150100500 (a) (b) 110 italian journal of food science, 2023; 35 (2) stasi a. and baiano a. <2.50 €/kg 2.50–4 €/kg 5.51–7 €/kg 7.01–9,5 €/kg >9,5 €/kg4.01–5,50 €/kg 60 50 40 30 20 10 0 43,4 traditional pumpkin spinach tomato-basil with cauliflower pieces stuffed with cheese 40,0 11,1 25,6 51,3 16,7 4,9 1,3 28,8 49,4 14,3 6,4 0,9 34,8 44,7 14,3 5,1 0,9 44,0 38,2 12,8 3,9 1,1 28,8 42,7 18,4 7,9 1,9 4,3 0,8 (a) (b) 80 70 60 50 40 30 20 10 0 74,2 2,4 1,5 3,9 35,2 32,9 19,4 6,8 5,8 29,9 38,7 20,1 6,2 5,1 31,0 35,5 19,7 8,5 5,3 11,7 28,228,9 16,7 14,5 traditional pumpkin spinach tomato-basil with cauliflower pieces stuffed with cheese very pleasant moderately pleasing neither pleasant nor unpleasant moderately unpleasant very unpleasant 28,0 30,8 17,7 12,011,5 17,9 <2.50 €/kg 2.50–4 €/kg 5.51–7 €/kg 7.01–9,5 €/kg >9,5 €/kg4.01–5,50 €/kg 60 50 40 30 20 10 0 43,4 traditional pumpkin spinach tomato-basil with cauliflower pieces stuffed with cheese 40,0 11,1 25,6 51,3 16,7 4,9 1,3 28,8 49,4 14,3 6,4 0,9 34,8 44,7 14,3 5,1 0,9 44,0 38,2 12,8 3,9 1,1 28,8 42,7 18,4 7,9 1,9 4,3 0,8 (a) (b) 80 70 60 50 40 30 20 10 0 74,2 2,4 1,5 3,9 35,2 32,9 19,4 6,8 5,8 29,9 38,7 20,1 6,2 5,1 31,0 35,5 19,7 8,5 5,3 11,7 28,228,9 16,7 14,5 traditional pumpkin spinach tomato-basil with cauliflower pieces stuffed with cheese very pleasant moderately pleasing neither pleasant nor unpleasant moderately unpleasant very unpleasant 28,0 30,8 17,7 12,011,5 17,9 figure 4. online survey on gnocchi preferences: (a) regarding formulations; and (b) distribution (%) of respondents in relation to the price they are willing to pay for various formulations. willing to pay more for frozen gnocchi, compared to female consumers. • age: young consumers are less willing to pay and buy frozen gnocchi, compared to elderly ones. • dim_city: it indicates the dimension of the town/city of consumers. results of this estimation show that people living in bigger cities and metropolitan areas would prefer frozen gnocchi. minimal differences emerged on comparison of results among different types of gnocchi. the most important is about household with intolerant people. they would mostly purchase traditional gnocchi over other varieties with flavors and condiments (spinach, cheese, pumpkin, etc.). such a result could be related not only to intolerance but to the presence of more allergies. the following variables never significantly affect the respondents’ willingness to pay: • geographical origin: there is no geographical influence, perhaps because gnocchi are known and (b) italian journal of food science, 2023; 35 (2) 111 a pre-competitive research on frozen gnocchi table 8. results of seemingly unrelated regression estimating preferences of frozen gnocchi consumers. variables p_traditional p_punp p_spinach p_tomato p_cheese imp_price –0.139** –0.145** –0.103* –0.128** –0.158** [0.045] [0.046] [0.046] [0.045] [0.053] imp_ingr 0.125** 0.123** 0.120** 0.125** 0.088+ [0.039] [0.040] [0.040] [0.039] [0.046] gnocchi_amb –0.269+ –0.13 –0.058 –0.175 –0.23 [0.139] [0.143] [0.144] [0.141] [0.164] gnocchi_hab –0.266* –0.253* –0.055 –0.151 –0.264* [0.114] [0.117] [0.118] [0.115] [0.134] intollerant 0.260* 0.12 0.04 0.051 0.049 [0.110] [0.113] [0.114] [0.111] [0.130] income 0.220** 0.210** 0.210** 0.161** 0.266** [0.053] [0.054] [0.055] [0.053] [0.062] no_family 0.090* 0.065 0.100* 0.088* 0.036 [0.042] [0.043] [0.043] [0.042] [0.049] gender –0.284** –0.215* –0.298** –0.342** –0.262* [0.107] [0.109] [0.111] [0.108] [0.126] age 0.084+ 0.128** 0.047 0.098* 0.061 [0.044] [0.045] [0.046] [0.044] [0.052] dim_city –0.061 –0.046 –0.074+ –0.037 –0.008 [0.041] [0.042] [0.042] [0.041] [0.048] constant 2.984** 2.899** 2.673** 2.624** 3.279**   [0.339] [0.347] [0.351] [0.342] [0.400] observations 532 532 532 532 532 r2 0.131 0.118 0.089 0.1 0.092 the standard error values are shown in square brackets. levels of significance of the variables: ** p<0.01; * p<0.05; +p<0.1. r2: coefficients of determination consumed in a homogeneous manner throughout the regions. • brand: respondents pay more attention to intrinsic quality than to brand appeal. • visual aspect: appearance of the product does not represent an important variable regarding the willingness to pay, as it affects in advance the decision to buy the product. • energy intake. • average expenditure for food purchases: gnocchi are consumed infrequently; therefore, as they do not significantly affect expenditure, even consumers that spend less on food afford to buy them. • subject responsible for family purchases. • employment status. • level of education. conclusion based on microbiological criteria, it is possible to express a positive evaluation on the hygienic–sanitary suitability of frozen gnocchi, although, also considering their physicochemical characteristics, strict compliance with cold chain and avoidance of thermal abuse are required. the nutritional characteristics make gnocchi an excellent first course, although cheese-stuffed gnocchi also guarantee a certain amount of protein. sensory evaluation highlights the good quality of the product if evaluated at an optimal cooking period, after which deterioration of parameters, such as texture and stickiness, takes place. the stuffed-cheese gnocchi obtained the highest sensory scores. finally, concerning the new product’s sale potential, the estimation of the model allowed identifying the ideal target: the frozen gnocchi presented as traditional or prepared with other ingredients. frozen gnocchi are perceived as ‘normal’ good product and not a lowquality food. the market survey has clearly highlighted the profile of a traditionalist consumer regarding the formulation (preference for the ‘base’ preparation), shape (classic and wave) and packaging (transparent). 112 italian journal of food science, 2023; 35 (2) stasi a. and baiano a. cappa c., franchi r., bogo v. and lucisano m. 2017. cooking behavior of frozen gluten-free potato-based pasta (gnocchi) obtained through turbo cooking technology. lwt – food sci technol. 84: 464–470. https://doi.org/10.1016/j.lwt.2017.06.004 cappa c., laureati m., casiraghi m.c., erba d., vezzani m., lucisano m., et al. 2021. effects of red rice or buckwheat addition on nutritional, technological, and sensory quality of potato-based pasta. foods. 10: 91. https://doi.org/10.3390/foods10010091 chunfeng j., yingmin j., yaping g., gao m. and baozhong s. 2005. effect of irradiation on microbiological safety of chilled cooked dumpling. acta agricul nucl sin. 19(5): 367–370. coradini m.f., da cunha alexandre a.a., lewandowski v., de castro p.l., lucca braccini g. and de souza m.l.r. 2019. chemical, microbiology and sensorial composition of potato gnocchi with the inclusion of dehydrated mix of tilapia and tuna. ceppa bull. (curitiba). 37(2): 49–58. da silva e.m.m., rossini a.f. and de carvalho j.l.v. 2016. quality evaluation of gnocchi pasta prepared with orange-fleshed sweet potato. biosci j uberlândia, 32(1): 81–88. https://doi. org/10.14393/bj-v32n1a2016-29598 defrancesco e., perito m.a., bozzolan i., cei l. and stefan g. 2017. consumers’ preferences for health related and environmental friendly food attributes of italian pasta. proc syst dyn innov food netw. 2017, 252–256. del torre m., stecchini m.l., braconnier a. and peck m.w. 2004. prevalence of clostridium species and behaviour of clostridium botulinum in gnocchi, a repfed of italian origin. int j food microbiol. 96(2): 115–131. https://doi.org/10.1016/j. ijfoodmicro.2004.01.004 european union (eu). 2007. commission regulation (ec) n. 1441/2007 amending regulation (ec) n. 2073/2005 on microbiological criteria for foodstuffs. off j eur union. l322, 12–29. european union. 2011. regulation (eu) no. 1169/2011 of the european parliament and of the council of 25 october 2011 on the provision of food information to consumers. off j eur union. l304, 18–63. expert market research. 2022. global gnocchi market report. available at: https://www.expertmarketresearch.com/reports/ gnocchi-market. accessed 19 july 2022. feiner g. 2006. meat products handbook. practical science and technology. woodhead series in food science, technology and nutrition. woodhead, sawston, cambridge, pp. 574–594. https://doi.org/10.1533/9781845691721.3.574 food and drug administration (fda). 2011. chapter 14: pathogenic bacteria growth and toxin formation as a result of inadequate drying. available at: https://www.fda.gov/files/food/published/ fish-and-fishery-products-hazards-and-controls-guidancechapter-14-download.pdf. accessed 19 july 2022. food and drug administration (fda). 2019. hazard analysis and risk-based preventive controls for human food: draft guidance for industry. available at: https://www.fda.gov/files/food/published/draft-guidance-for-industry--hazard-analysis-andrisk-based-preventive-controls-for-human-food---potentialhazards-for-foods-and-processes-%28appendix-1%29.pdf. accessed 19 july 2022. the consumer does not appear to spend a large amount, regardless of the product type. in any case, the price that respondents are willing to pay increases with the importance attributed to product’s ingredients, in the presence of food intolerances/allergies/repulsions, with increase in income, number of family members and age. the willingness to pay decreases with the importance that respondents attribute to the price increases as well as increase in the frequency of consumption. males appear more likely to pay than females. the ideal consumer is a city male of middle age, with high income and a big family. according to the results obtained, innovation is possible, but it must be ‘mild’ and, while there is a realistic opportunity for the industry to produce and sell 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https://doi.org/10.1080/01621459.1962.10480664� https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/604338/salt_reduction_targets_for_2017.pdf� https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/604338/salt_reduction_targets_for_2017.pdf� https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/604338/salt_reduction_targets_for_2017.pdf� https://doi.org/10.3390/foods10112585� https://doi.org/10.1111/1750-3841.12816� 14 issn 1120-1770 online, doi 10.15586/ijfs.v33i3.2048 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (3): 14–24 p u b l i c a t i o n s codon food purchasing, preservation, and eating behavior during covid-19 pandemic: a consumer analysis sibel bolek sağlık bilimleri üniversitesi, university of health sciences, i̇stanbul, turkey *corresponding author: sibel bolek, sağlık bilimleri üniversitesi, university of health sciences, i̇stanbul, turkey. email: sibel.bolek@sbu.edu.tr submitted: 7 april 2021; accepted: 21 august 2021; published: 11 september 2021 © 2021 codon publications open access paper abstract due to the highly infectious virus known as covid-19 impacting the lives of the populace, more than any other event in recent memory, there is a pandemic in the world. in order to determine food purchasing behavior and eating habits, food preservation techniques and source of knowledge about covid-19, 992 consumers living in i̇stanbul, the most populous city in turkey, were surveyed. the questionnaire was disseminated to participants via an online platform. thirty questions, including the demographics of participants, changes in purchasing behavior, knowledge, and attitudes about food preservation techniques, changes in eating habits, and source of knowledge about covid-19, were asked. the results of this study surveyed that covid-19 has changed food purchasing and eating habits of turkish consumers significantly (p < 0.05). during the survey in late march of 2020 and late december of 2020, about 65% of respondents have tried to consume more food that boost the immune system and 58% of the respondents have been more willing to buy fresh products. consumers have greatly adopted preserving of food stuffs by freezing during quarantine days. this survey revealed that the effective use of media tools could increase awareness and lead to behavioral changes that can reduce the spread of covid-19, especially in consumers aged over 65 years. keywords: covid-19; eating habits; food purchasing habits; media introduction covid-19 pandemic is one of the greatest challenges that the world has faced without prior preparation. covid-19 is much more than a health crisis. it is also an inevitable economic crisis because of the sudden decline in economic activities. the pandemic is giving rise to substantial changes in the social habits of the people throughout the world, which will leave deep scars. moreover, the covid-19 pandemic has caused apprehensions about threats to food security (hirvonen et al., 2021). one of the reasons why covid-19 pandemic has led to significant changes is the uncertainty about what will happen in the near future, as well as food purchasing, food preservation, and consumption behavior of people because of the lockdown and social isolation directives. during the pandemic, people are compelled to stay at home and to go outside only to meet the most urgent needs such as purchasing food. hence, covid-19 has changed consumers’ life and spending habits (criteo coronavirus survey, 2020). one of the major problems that covid-19 has indicated is the debate on food insecurity faced by majority of the population. covid-19 caused a critical weakness in the us food supply system (chenarides et al., 2021). it has threatened the accessibility of food by effecting food costs and infrastructure such as public transit access, distribution, shortages of certain products, and changes in food assistance (niles et al., 2020). this statement has revealed the significance of food security in times of crises and shocks. mailto:sibel.bolek@sbu.edu.tr italian journal of food science, 2021; 33 (3) 15 effects of covid-19 on food habits of the present study could be a standard in food security, health care, and other service settings during covid-19 and beyond. food retailers and distributors may consider increasing their capacity to cope with temporary excess demand by investing in capital and labor resources. literature review many studies have been conducted to explain changes in dietary and food purchasing habits during the covid19 pandemic. moreover, several studies have been performed to determine effects of covid-19 pandemic on food supply chains and consumer panic buying behaviors (hobbs, 2020). grashuis et al. (2020) investigated the grocery shopping preferences during the covid-19 pandemic. their results revealed that covid-19 caused significant changes in grocery shopping preferences. when covid-19 is spreading at an increasing rate, consumers are not usually willing to shop at grocery stores. ben hassen et al. (2020) investigated impact of covid19 on food behavior and consumption in qatar. their results indicated that consumers adopted healthier diets and increased the consumption of domestic food because of food safety concerns. chang and meyerhoefer (2021) surveyed the effects of covid-19 on online food shopping services. according to their results, covid-19 pandemic caused a significant increase in online food shopping in taiwan. celik and dane (2020) surveyed the effects of covid-19 pandemic outbreak on food consumption preferences. their survey revealed that the first food choice of consumers shifted from meat and bakery to fruits and vegetables. marty et al. (2021) surveyed nutritional value of diet and food choice motives during the covid-19 pandemic in france. their results revealed that consumers’ awareness of the importance of sustainable food choices significantly increased. however, to the best of the author’s knowledge, no data with respect to the real food preservation habits of the population during covid -19 are available, so far. the present study aimed to analyze both changes in food purchasing and preservation habits which help food authorities to take the necessary precautions during pandemic situations such as covid-19. survey design with feedback from key state-level agencies as well as reviews of relevant literature, a survey was developed by observing consumer food behavior to evaluate the changes in food purchasing and preservation behavior of consumers in i̇stanbul, the most populous city in turkey. the questionnaire was pilot tested on 20 comparable consumers for clarity and validity, and necessary adjustments were done. data were collected by a specific the covid-19 pandemic holds several implications for canadian food supply chains such as maintaining and enhancing supply chain resilience (hobbs, 2020). the pandemic is evidently challenging the whole food chain system. one of the major concerns shared by all food companies is preserving the health of workers and the provision of sufficient workforce because of those who do not want to work due to sickness or the fear of coronavirus. food security is also related to access of consumers to food rather than food availability during lockdown (gundersen et al., 2021). chenarides et al. (2021) reported that food prices are a key determinant of food insecurity. on the other hand, consumers are also expected to take additional measures to protect their health. news about the importance of a strong immune system in the fight against covid-19 prompted consumers to purchase foods that strengthen the immune system. food achievement patterns were also substantially altered in comparison to pre-covid levels (restrepo et al., 2021). understanding the covid-19 effect behind restriction policies is also substantial (aday and aday, 2020). changes in the food purchasing behavior varied by age, gender, and education (ali et al., 2021; wang et al., 2020). investigating changes in food purchasing, preservation, and consumption habit is critical to both understand how habits of consumers change and adapt during lockdown and to ensure beneficial guidance in emergency management efforts. creating shock-resistant food systems requires collective action through the entire agrifood chain (bakalis et al., 2020). in particular, at the start of the pandemic crisis, consumer demand for food has soared and some store shelves have been emptied due to over-buying of basic products. examining for food insecurity and providing resources may decrease shortand long-term results, such as potential long-term effects on health outcomes related to duration of household food insecurity and higher health care expenditures associated with food insecurity. each country should realize the seriousness of the situation and sometimes tighten or relax measures according to the spread of the pandemic. therefore, the aim of this survey was to understand how a pandemic such as the covid-19 influences the food purchasing and preservation behavior. it was hypothesized that food choice, preservation motives, and nutritional quality of diet changed during covid19 outbreak. this survey aimed to help address consumer awareness, knowledge and, attitudes about food preservation techniques creating influential food safety and nutrition communications that would help policy makers, educators, communities, health professionals, companies, and others to best understand the most important issues for consumers to adjust their policies and strategies to the current pandemic circumstances. the results 16 italian journal of food science, 2021; 33 (3) sibel bolek respondents had their monthly average income above 15,000 tl, which can be considered as a very good salary according to turkish life standards. changes in the purchasing behavior of consumers changes in the purchasing behavior of consumers are given in table 2. most of the consumers (83 ± 0.15%) have changed shopping habits since the covid-19 pandemic started (p < 0.05). forty percent (40 ± 0.08%) of the consumers have adopted online purchasing, whereas 60 ± 0.06% of the consumers have adopted in-store food purchasing. consumers from all demographics, but especially from the age group below 65 years, have adopted online food purchasing (p < 0.05). the citizens aged below 65 years have been restricted to venture out of their homes shortly after the first case has been seen in turkey. as grocery shopping remains a necessity during the pandemic, many people have questions about how to shop safely. grocery stores have imposed more and more safety rules such as metering consumers and requiring consumers to wear masks. due to the news that the virus can live on boxes and cans, consumers have gotten fearful and anxious when shopping at physical grocery stores. it seems that online food purchasing is likely to continue long after the pandemic. scacchi et al. (2021) reported that the covid-19 pandemic caused adoption of online grocery purchase among italian consumers, suggesting a modern and low-risk shopping method. questionnaire. the questionnaire was carefully designed to minimize its influences on consumers and to achieve accurate information that will reflect the consumers’ own attitudes. the questionnaire was disseminated to participants via an online platform. the survey lasted from late march to late december of 2020. no direct interactions, such as face-to-face interviews, were carried out. consumers were asked about their food purchasing, food preservation, and consumption behavior during the covid-19 pandemic. the data were collected by interviewing 992 randomly selected consumers. questionnaire design is highlighted in the following sections: (i) demographics of respondents, (ii) changes in purchasing behavior of consumers, (iii) knowledge and attitudes of consumers about food preservation techniques, (iv) changes in eating habits, and (v) source of knowledge about covid-19 and food. the sections were designed to determine the changes in food purchasing, preservation, and dietary habits as well as to reveal information sources of consumers during the covid-19 pandemic. ethical aspects the respondent information required an individual and anonymous completion of the questionnaire. consumers could quit at any time. it was not possible to link the specific answers to individuals. statistical analysis data were subjected to analysis of variance (anova) by the general linear model (glm) procedure of sas statistical programme (sas, 1999). all the means were compared by duncan’s multiple range test at the level of p < 0.05. results respondents’ characteristics the demographic properties of consumers are given in table 1. the age of the youngest consumer was 18, whereas the oldest was 79 years old. thirty percent of the consumers were under 20 years of age, 39% were between the ages of 20 and 65 years, and 37% were over 65 years. forty-seven percent of the consumers were male. thirty percent of the consumers were university graduates, 44% were high school graduates, and 26% were elementary school graduates. monthly average income of 40% of the participants was between 2400 and 4000 tl (turkish lira). in the twelfth month of 2020, 1 dollar was roughly 7.45 tl, and 1 euro was roughly 8.90 tl. only 3% of the table 1. socioeconomic and demographic characteristics of the participants. age <20 30% 20–65 39% >65 37% gender female 53% male 47% education elementary 26% high school 44% university 30% monthly incomea <2400 tlb 20% 2400–4000 tl 40% 4000–7500 tl 32% 7500–15,000 tl 21% >15,000 tl 3% anational average gross income per person was 11,019 dollars in turkey in 2020. btl: turkish lira, 1 euro = 8.90 tl (december, 2020). italian journal of food science, 2021; 33 (3) 17 effects of covid-19 on food habits no statistically significant difference (p > 0.05). głąbska et al. (2020) reported that the covid-19 pandemic has increased the importance of health and weight control of polish adolescents. hygiene and sanitation are imperatives that must be strictly followed in the food industry. food handlers should implement hygiene practices accurately to protect the consumers from foodborne diseases. the food safety measures such as cleaning of surfaces and utensils, frequent hand-washing, and cooking food to the right temperature are already implemented to prevent foodborne illnesses. there is every reason to believe that the existing effective hygiene and sanitation measures are as effective on covid-19 as on other microbiological risks. however, as seen in table 2, 55 ± 0.09% of the consumers are not sure whether consuming the food they purchase is safe during covid-19 (p < 0.05). when the results were evaluated in terms of age groups, it was seen that there was no statistically significant difference (p > 0.05). food businesses should implement additional hygiene and sanitation measures, based on risk, all the more in the case an employee tests positive for covid-19. ellison et al. (2021) surveyed a panel of 1370 u.s. households during the covid-19 pandemic. the results of their study revealed that the covid-19 pandemic has substantially changed what is “normal” globally, touching all aspects of life, including food purchasing and acquisition preferences. knowledge and attitudes of consumers about food preservation techniques during the covid-19 pandemic appropriate food preservation has allowed for less trips to the grocery store and less time in public places during the covid-19 pandemic. as seen in table 3, freezing has been the most preferred food preservation method. sixty-six percent (66 ± 0.08%) of the consumers have frozen their foods during the covid-19 pandemic (p < 0.05). freezing is a preferred method since it eliminates the need for purchasing additional equipment and utensils that are required for drying and canning. most of the respondents (95%) who have adopted freezing to a great extent during the pandemic were aged below 20 years (p < 0.05). this may be due to the lack of time for young consumers. however, elderly people have also adopted drying and canning. turkish people have had refrigerators in their houses for the past 40 years, so the consumers over 65 years are experienced in drying and canning that are ancient food preservation techniques before the advent of refrigerators. as seen in table 3, only 20 ± 0.06% of the consumers have felt it necessary to wash or remove packaging before at the beginning of the covid-19 pandemic, because of market uncertainties, some of the major agricultural producing nations implemented export restrictions. they mainly focused on staple crops that are of high importance for food security including maize, wheat, and rice. moreover, consumers who believed that they would suffer from food shortages due to covid-19 stockpiled food in their homes. shelves were emptied quickly in markets. however, as seen in table 2, 90 ± 0.07 of the consumers have not experienced food product shortages. health professionals are recommending people consider taking some foods for strengthening their immune system daily as the coronavirus lockdown continues. as seen in table 2, only %10 ± 0.05 of the consumers has purchased food products which they would not have purchased if it was not for covid-19. the risk of covid-19 cross-contamination to food is very low (gov.uk., 2020). however, since covid-19 is a respiratory disease (butler and barrientos, 2020), the possibility of the respiratory tract becoming infected when chewing contaminated food cannot be completely ruled out. as seen in table 2, sixty-six percent (66 ± 0.11%) of the consumers purchased more packaged food since the covid-19 pandemic started (p < 0.05). since people are encouraged to wash foodstuff purchased from supermarkets, which is far more easily done when the product is plastic-wrapped, plastic packaging has played an important role in food safety during lockdown (packaging europa, 2020). consumers from all demographics, but especially those aged more than 65 years (p < 0.05), have had a more favorable opinion of the healthfulness of packaged foods. at the same time, 27 ± 0.08% of the respondents said that they have not changed their perceptions on the healthfulness of packaged foods. healthy diet has a great importance for health, particularly in times when the immune system might need to fight back (kau et al., 2011). sustaining a healthy lifestyle and nutritious diet is extremely important during the covid-19 pandemic. avoiding nutrient deficiencies helps ensure that all nutrients required for immune cell triggering, interaction, differentiation, or functional expression are available as needed. fruits and vegetables are increasingly being consumed thanks to their properties of nutrition and freshness, which are much appreciated. moreover, freshwater fish and seafood are popular among consumers since their perception is associated with healthy, high-quality products. as seen in table 2, %58 ± 0.12 of the consumers have been more willing to buy fresh products since the covid-19 pandemic started (p < 0.05). when the results were evaluated in terms of age groups, it was seen that there was 18 italian journal of food science, 2021; 33 (3) sibel bolek table 2. changes in the purchasing behavior of consumers. results obtained for different age groups overall <20 20–65 >65 i have changed shopping habits since the covid-19 pandemic started. a) yes 83 ± 0.15a 82 ± 0.08a 85 ± 0.12a 83 ± 0.09a b) no, never 17 ± 0.12b 18 ± 0.07b 15 ± 0.12b 17 ± 0.07b i have adopted…………....food purchasing since the covid-19 pandemic started (fill in the blank). a) online 40 ± 0.08b 51 ± 0.04a 53 ± 0.12a 15 ± 0.08b b) in store 60 ± 0.06a 49 ± 0.05a 47 ± 0.12b 85 ± 0.09a i have experienced food product shortages at stores from which i am trying to buy. a) yes 22 ± 0.07b 20 ± 0.06b 27 ± 0.12b 18 ± 0.10b b) no, never 78 ± 0.10a 80 ± 0.08a 73 ± 0.13a 82 ± 0.10a i have purchased food products which i wouldn’t have done if it wasn’t for covid-19. a) yes 10 ± 0.05b 5 ± 0.06b 2 ± 0.08b 22 ± 0.11b b) no, never 90 ± 0.07a 95 ± 0.06a 98 ± 0.07a 78 ± 0.14a i have purchased more packaged food since the covid-19 pandemic started. a) yes 66 ± 0.11a 70 ± 0.12a 65 ± 0.10a 63 ± 0.15a b) no, never 34 ± 0.10b 30 ± 0.13b 35 ± 0.12b 37 ± 0.15b i have had a more favorable opinion of the healthfulness of packaged foods a) yes 73 ± 0.05a 63 ± 0.11a 72 ± 0.11a 85 ± 0.06a b) no, never 27 ± 0.08b 37 ± 0.10b 28 ± 0.11b 15 ± 0.07b i have been more willing to buy fresh products since the covid-19 pandemic started yes 58 ± 0.12a 55 ± 0.05a 57 ± 0.10a 62 ± 0.09a no 42 ± 0.12b 45 ± 0.06b 43 ± 0.10b 38 ± 0.08b i am very confident that the food i am purchasing is safe to consume. yes 21 ± 0.14b 31 ± 0.05b 23 ± 0.12b 20 ± 0.08b,c no 24 ± 0.10b 20 ± 0.04c 25 ± 0.11b 24 ± 0.07b i am not sure 55 ± 0.09a 49 ± 0.04a 52 ± 0.12a 56 ± 0.14a the values are expressed as the mean ± sd, and different superscript letters show significant differences (p < 0.05). putting food items in the freezer since the start of the covid-19 pandemic. while it is possible for a person to contact covid-19 from touching an infected surface or object and then touching his or her face, there is currently no evidence to support the idea that the virus can be spread by food packaging (csis, 2020). a majority of the turkish people (76 ± 0.05%) seem to understand this issue well. when the results were evaluated in terms of age groups, it was seen that there was no statistically significant difference (p > 0.05). freezing retards the growth of microorganisms and enzymes that cause food spoilage. however, freezing has very little impact on the infectivity of foodborne enteric viruses (bozkurt et al., 2020; nasheri et al., 2019). only a few studies have been performed on the effect of freezing on coronavirus infectivity. lamarre and talbot (1989) showed that the infectious titer of human coronavirus did not cause any significant reduction when subjected to 25 cycles of thawing and freezing. as shown in table 3, 44% of the turkish consumers think that freezing the foods kills the covid-19 virus. when the results were evaluated in terms of age groups, majority of all age groups have no idea about this question (p < 0.05). according to the recent study by pastorino et al. (2020), the 92°c and 15 min protocol was able to totally inactivate the virus (>6 log10 decrease). as given in table 3, 77% of the turkish consumers agree that cooked meat is not a great risk for the consumer in terms of covid-19. fifty-five percent (55%) of the turkish consumers do not agree that covid-19 can spread through dairy products. however, 20% of them have no idea about this issue. italian journal of food science, 2021; 33 (3) 19 effects of covid-19 on food habits table 3. knowledge and attitudes of consumers about food preservation techniques during the covid-19 pandemic. results obtained for different age groups overall <20 20–65 >65 which food preservation technique have you adopted most during covid-19 quarantine days? a) drying 21 ± 0.05b 3 ± 0.02b 27 ± 0.09b 33 ± 0.04b b) freezing 66 ± 0.08a 95 ± 0.05a 61 ± 0.10a 42 ± 0.05a c) canning 13 ± 0.07c 2 ± 0.01b 12 ± 0.08c 25 ± 0.04c i have needed to wash or remove packaging before putting in the freezer since the covid-19 pandemic started. a) yes 20 ± 0.06b 22 ± 0.09b 31 ± 0.07b 18 ± 0.04b b) no, never 76 ± 0.05a 78 ± 0.10a 69 ± 0.08a 82 ± 0.05a freezing the foods kills the covid-19 virus. i absolutely agree 18 ± 0.04c 12 ± 0.02c 23 ± 0.07b 18 ± 0.03c i agree 26 ± 0.03b 23 ± 0.04b 25 ± 0.06b 30 ± 0.03b i have no idea 39 ± 0.05a 41 ± 0.04a 32 ± 0.06a 45 ± 0.04a i do not agree 10 ± 0.06d 14 ± 0.02c 12 ± 0.03c 4 ± 0.08d i absolutely disagree 7 ± 0.05d 10 ± 0.03c 8 ± 0.09d 3 ± 0.06d cooked meat is not a great risk for the consumer in terms of covid-19. i absolutely agree 42 ± 0.07a 41 ± 0.05a 45 ± 0.12a 40 ± 0.12a i agree 35 ± 0.06b 35 ± 0.04b 38 ± 0.06b 33 ± 0.08b i have no idea 12 ± 0.06c 10 ± 0.03c 7 ± 0.06c 20 ± 0.06c i do not agree 8 ± 0.05d 11 ± 0.04c 11 ± 0.04c 5 ± 0.04d i absolutely disagree 3 ± 0.05e 3 ± 0.02d 3 ± 0.04d 2 ± 0.05e the covid-19 can spread through dairy products i absolutely agree 11 ± 0.05e 8 ± 0.07c,d 9 ± 0.03d 17 ± 0.02b i agree 13 ± 0.04d 12 ± 0.07c 14 ± 0.04c 13 ± 0.04b,c i have no idea 20 ± 0.03b 20 ± 0.06b 10 ± 0.07d 30 ± 0.06a i do not agree 17 ± 0.02c 20 ± 0.04b 22 ± 0.06b 10 ± 0.04c i absolutely disagree 38 ± 0.04a 40 ± 0.02a 45 ± 0.06a 30 ± 0.05a the values are expressed as the mean ± sd, and different superscript letters show significant differences (p < 0.05). the results of this section indicated that much more education is needed on food and covid-19. changes in eating habits of consumers during the covid-19 pandemic since many nutritional deficiencies may cause immune dysfunction resulting in increased susceptibility to infectious diseases, special attention should be given to promote immune function to enhance viral resistance among the populace (khayyatzadeh, 2020). poor nutritional quality diet is one of the main risk factors for noncommunicable diseases (marty et al., 2021). as shown in table 4, 65 ± 0.15% of the respondents have tried to consume more food that boost the immune system, and 62 ± 0.09% of the consumers have eaten more often since the covid-19 pandemic started (p < 0.05). when the results were evaluated in terms of age groups, it was seen that there was no statistically significant difference (p > 0.05). antioxidants cause an increase in the number of t-cell subsets, lymphocyte response to mitogen, interleukin-2 production, potentiated natural killer cell activity, and the response to influenza virus vaccine compared with placebo (chandra, 1992). many studies have stated that fruits and vegetables that supply micronutrients can support immune function thanks to their high antioxidant activity and their rich vitamin c, vitamin e, and beta-carotene content. sixty-four percent (64 ± 0.04%) of the respondents have tried to eat more fresh fruit and vegetable since the covid-19 pandemic started (table  4). turkish consumers have had a proper eating habit regarding fruit and vegetable consumption since the covid-19 pandemic started. the increase in the consumption of dairy products of consumers was investigated (table 4). fifty-two percent (52 ± 0.07%) of the respondents have tried to consume more dairy products since the covid-19 pandemic started. dairy products are a great source of energy, protein, and 20 italian journal of food science, 2021; 33 (3) sibel bolek table 4. changes in eating habits. results obtained for different age groups overall <20 20–65 >65 i have tried to consume more food that boosts the immune system since the covid-19 pandemic started. a) yes 65 ± 0.15a 60 ± 0.16a 70 ± 0.09a 65 ± 0.10a b) no 45 ± 0.16b 40 ± 0.18b 30 ± 0.08b 35 ± 0.09b i have eaten…………………since the covid19 pandemic started. (fill in the blank) a) less often 38 ± 0.08b 46 ± 0.10b 33 ± 0.14b 36 ± 0.09b b) more often 62 ± 0.09a 54 ± 0.07a 67 ± 0.12a 64 ± 0.08a i have tried to consume more fresh fruit and vegetable since the covid-19 pandemic started a) yes 64 ± 0.04a 50 ± 0.02a 71 ± 0.14a 70 ± 0.05a b) no 36 ± 0.07b 50 ± 0.03a 29 ± 0.12b 30 ± 0.06b i have tried to consume more dairy products since the covid-19 pandemic started. a) yes 52 ± 0.07a 42 ± 0.06b 53 ± 0.05a 56 ± 0.04a b) no 48 ± 0.08b 48 ± 0.04a 47 ± 0.03b 44 ± 0.04b i have tried to consume more wholegrain since the covid-19 pandemic started a) yes 35 ± 0.10b 30 ± 0.06b 39 ± 0.05a 37 ± 0.04b b) no 65 ± 0.09a 55 ± 0.07a 41 ± 0.05a 48 ± 0.04a i have tried to consume less red meat since the covid-19 pandemic started a) yes 49 ± 0.02a 50 ± 0.06a 45 ± 0.14b 51 ± 0.07a b) no 51 ± 0.03a 50 ± 0.06a 55 ± 0.12a 49 ± 0.07a i have tried to consume more fish and seafood since the covid-19 pandemic started a) yes 53 ± 0.06a 45 ± 0.03b 55 ± 0.11b 58 ± 0.06a,b b) no 47 ± 0.05b 75 ± 0.04a 68 ± 0.11a 65 ± 0.04a i have tried to consume more home cooked meal since the covid-19 pandemic started. a) yes 77 ± 0.08a 61 ± 0.05a 85 ± 0.08a 85 ± 0.11a b) no 23 ± 0.07b 39 ± 0.06b 15 ± 0.07b 15 ± 0.12b i have taken vitamin supplement since the covid-19 pandemic started a) yes 51 ± 0.04a 32 ± 0.07b 41 ± 0.06b 54 ± 0.08a b) no, never 49 ± 0.05a 68 ± 0.08a 59 ± 0.04a 46 ± 0.09b the values are expressed as the mean ± sd, and different superscript letters show significant differences (p < 0.05). micronutrients, including riboflavin, calcium, selenium, magnesium, and vitamins b5 and b12 (gaucheron, 2011). therefore, it is very healthy to increase the trend towards the consumption of dairy products. whole-grain food intake has a protective effect against oxidative stress, inflammatory and pathology of infectious origin (jacobs et al., 2007). since micronutrients are generally present in higher concentrations in the outer part of the grain, refined flours which comprise only starchy endosperm are lower in micronutrients than whole grains. as seen in table 4, only 35 ± 0.10% of the respondents have tried to consume more wholegrain since the covid-19 pandemic started. in turkey, the most consumed types of bread are traditional white bread, which comprise only starchy endosperm. this explains why 65% of consumers answered “no” to the question. increasing consumption of red meat, especially in its processed forms, has negative health effects (richi et al., 2015). however, 51% of the turkish consumers have not tried to consume less red meat since the covid-19 pandemic started. the average red meat consumption in 2019 was 13.6 kg per capita which is less than the developed countries (özen et al., 2019). this rate, which is already low in turkey, has not changed much with the advent of the covid-19 pandemic. when the results were evaluated in terms of age groups, it was seen that there was no statistically significant difference (p > 0.05). fish and other sea foods are a rich source of high protein. they are low in calories, total fat, and saturated fat. they contain numerous vitamins and minerals. as seen in table 4, 53 ± 0.06% of the respondents have tried to consume more fish and seafood since the covid-19 pandemic started. italian journal of food science, 2021; 33 (3) 21 effects of covid-19 on food habits fifty-nine percent of the consumers think that the daily news on tv is extremely reliable or reliable, whereas 36% of the consumers think that daily news on tv is unreliable or extremely unreliable. majority of the participants (87%) aged over 65 years rely on daily news on tv (p < 0.05). it was revealed that the “age” criterion significantly affected the ratio of trusting the daily news in turkey (p < 0.05). newspapers carry information about economy, politics, sports, entertainment, business, trade, industry, and commerce. many professors and scientists from universities have written articles in newspapers about covid-19, and they have given advice about hygiene precautions since the covid-19 pandemic started. as seen in table  5, majority (81%) of the respondents think that the newspapers are extremely reliable or reliable. kiousis (2001) showed that newspapers were regarded as more credible than the internet and television. hence, newspapers have the very important duty of raising public awareness. eighty-four percent of the consumers from all demographics stated that scientific journals are extremely reliable or reliable, whereas only 3% of the consumers think they are unreliable or extremely unreliable. consumers think that newspapers are as reliable as scientific journals (p > 0.05). eighty-four percent of the consumers from all demographics indicated that university scientists are extremely reliable or reliable. these results revealed that scientists are one of the primary sources of trusted information and they are very important for public education about covid-19. health professionals have a very important role in improving access and quality of health care for the population. they have been at the front line of the covid-19 outbreak response and they have been exposed to hazards that put them at risk of infection. as shown in table 5, 58% of the participants rely on them. magazines and talk shows have a wide audience in turkey and sometimes scientists are invited to these programs. however, only 10 ± 0.07% of the consumers have found them extremely reliable since the covid-19 pandemic started. conclusion the results of this survey revealed that the covid-19 pandemic has caused substantial changes in food purchasing behavior and eating habits of turkish consumers. consumers from all demographics, but especially from the age group below 65 years, have adopted online food seventy-seven percent (77 ± 0.08%) of the consumers have tried to consume more home-cooked meal since the covid-19 pandemic started. in turkey, during the quarantine days, people started to share their home-made meals and healthy diets through social media. moreover, people have tried to bake bakery products such as bread, muffin pida. then, they have shared photographs of them with “stay at home” hashtag in social media. when the results were evaluated in terms of age groups, consumers over 65 years have paid more attention to eating healthy than other age groups (p < 0.05). older people appear to be at a higher risk of developing more serious complications of covid-19 disease (cdc, 2020). although younger people appear generally to be at lower risk (jordan et al., 2020), majority of the consumers aged below 25 years have also paid more attention to eating healthy. it seems that consumers will want to continue maximizing their health in order to boost their immunity and reduce vulnerability to disease long after the pandemic. marinković and lazarević (2021) surveyed eating habits and consumer food shopping behavior in serbia during the covid-19 pandemic. their study revealed that eating and purchasing habits of consumers were altered significantly during the covid-19 pandemic. source of knowledge about covid-19 and food consumers have received a variety of information about covid-19 and food from the websites, televisions, newspapers, scientific journals, university scientists, health professionals, talk shows, and magazines. many professors and scientists from universities have been invited to tv programs to share their opinions and studies on covid-19. moreover, they have given several advices through newspapers. furthermore, the minister of health of the republic of turkey have often shared decisions and advises of the scientific committee of covid-19 through tv and social media. the source of information is of crucial importance. when people do not trust the source of information, they generally are not willing to change their behaviors (bolek, 2020). it was investigated whether consumers found media tools reliable during the covid-19 outbreak. fifty-nine percent of the participants have opined that publications and websites of the government give extremely reliable information about the covid-19 pandemic (table 5). when the results were evaluated in terms of age groups, this ratio was relatively low (39%) for the participants aged below 25 years (p < 0.05). however, majority of the consumers (75%) aged over 65 years rely on websites and government publications for information on covid-19. 22 italian journal of food science, 2021; 33 (3) sibel bolek table 5. source of knowledge about covid-19 and food. results obtained for different age groups overall <20 20–65 >65 websites and publications of government a) extremely reliable 26 ± 0.02b 16 ± 0.05d,c 28 ± 0.11b 35 ± 0.04b b) reliable 33 ± 0.04a 23 ± 0.02b 36 ± 0.14a 40 ± 0.04a c) unreliable 26 ± 0.05b 35 ± 0.03a 25 ± 0.14b,c 18 ± 0.04c d) extremely unreliable 11 ± 0.03c 20 ± 0.07c 8 ± 0.12c 5 ± 0.04d e) no idea 4 ± 0.04d 6 ± 0.02d 3 ± 0.12c,d 2 ± 0.04d,e daily news on television a) extremely reliable 30 ± 0.05a 22 ± 0.04b,c 27 ± 0.08b 40 ± 0.09a b) reliable 31 ± 0.04a 23 ± 0.07b,c 32 ± 0.04a 37 ± 0.10a c) unreliable 22 ± 0.08b 31 ± 0.06a 23 ± 0.05c 12 ± 0.08b d) extremely unreliable 14 ± 0.07c 24 ± 0.03b 14 ± 0.04d 5 ± 0.08c e) no idea 3 ± 0.06d 0 ± 0.04 4 ± 0.03e 6 ± 0.06c television and radio programs a) extremely reliable 29 ± 0.04a 25 ± 0.04b 30 ± 0.04a 33 ± 0.04a,b b) reliable 27 ± 0.04a 20 ± 0.04c 27 ± 0.04a,b 35 ± 0.04a c) unreliable 25 ± 0.04a,b 33 ± 0.04a 22 ± 0.04b 20 ± 0.04c d) extremely unreliable 14 ± 0.04c 20 ± 0.04c 16 ± 0.04c 7 ± 0.04d e) no idea 3 ± 0.04d 2 ± 0.04d 5 ± 0.04d 3 ± 0.04d,e newspapers a) extremely reliable 55 ± 0.04a 54 ± 0.04a 50 ± 0.04a 60 ± 0.04a b) reliable 26 ± 0.04b 21 ± 0.04b 27 ± 0.04b 29 ± 0.04b c) unreliable 8 ± 0.04c 6 ± 0.04c,d 16 ± 0.04c 3 ± 0.04c,d d) extremely unreliable 5 ± 0.04d 9 ± 0.04c 4 ± 0.04d 3 ± 0.04c,d e) no idea 6 ± 0.04c,d 10 ± 0.04c 3 ± 0.04d 5 ± 0.04c scientific journals a) extremely reliable 70 ± 0.14a 69 ± 0.05a 72 ± 0.11a 70 ± 0.10a b) reliable 14 ± 0.12b 28 ± 0.04b 23 ± 0.12b 20 ± 0.08b c) unreliable 2 ± 0.10c 1 ± 0.04c 2 ± 0.08c 3 ± 0.08c d) extremely unreliable 1 ± 0.12c 0 ± 0.03 1 ± 0.05c 2 ± 0.06c e) no idea 3 ± 0.10c 2 ± 0.03c 2 ± 0.05c 5 ± 0.06c university scientists a) extremely reliable 43 ± 0.14a 38 ± 0.14b 42 ± 0.09a 50 ± 0.12a b) reliable 41 ± 0.12a,b 44 ± 0.12a 38 ± 0.10b 40 ± 0.12b c) unreliable 10 ± 0.08c 8 ± 0.13c 18 ± 0.09c 4 ± 0.13c d) extremely unreliable 3 ± 0.16d 6 ± 0.13c,d 1 ± 0.07d 2 ± 0.13c,d e) no idea 3 ± 0.16d 4 ± 0.10d 1 ± 0.07d 4 ± 0.14c health professionals a) extremely reliable 28 ± 0.07a,b 21 ± 0.07b 28 ± 0.07b 35 ± 0.06a b) reliable 30 ± 0.08a 29 ± 0.04a 32 ± 0.08a 30 ± 0.05b c) unreliable 18 ± 0.04c 20 ± 0.05b 18 ± 0.05c 15 ± 0.04c d) extremely unreliable 11 ± 0.04d,e 15 ± 0.03c 12 ± 0.05d 5 ± 0.03d e) no idea 13 ± 0.05d 15 ± 0.02c 10 ± 0.06d,e 15 ± 0.03c talk shows and magazines a) extremely reliable 10 ± 0.07c 15 ± 0.05c 3 ± 0.06e 12 ± 0.04c b) reliable 31 ± 0.06a 29 ± 0.05b 24 ± 0.06b,c 39 ± 0.04a c) unreliable 33 ± 0.04a 37 ± 0.04a 35 ± 0.08a 28 ± 0.04b d) extremely unreliable 15 ± 0.05b 9 ± 0.04d 26 ± 0.05b 9 ± 0.04c,d e) no idea 11 ± 0.05c 10 ± 0.02d 12 ± 0.04d 12± 0.04c the values are expressed as the mean ± sd, and different superscript letters show significant differences (p < 0.05). italian journal of food science, 2021; 33 (3) 23 effects of covid-19 on food habits review. critical reviews in food science and nutrition, 61(1), 116–138. https://doi.org/10.1080/10408398.2020.1719383 butler, m.j. and barrientos, r.m., 2020. the impact of nutrition on covid-19 susceptibility and long-term consequences. brain, behavior, and immunity, 87, 53–54. https://doi.org/10.1016/j. bbi.2020.04.040 celik, b. and dane, s., 2020. the effects of covid-19 pandemic outbreak on food consumption preferences and their causes. journal of research in medical and dental science 8(3): 169–173. center for disease control and prevention (cdc), 2020. coronavirus disease 2019 (covid-19). available at: https:// www.cdc.gov/coronavirus/2019-ncov/need-extra-precautions/ older-adults.html center for strategic and international studies (csis), 2020. covid-19 and food security. available at: https://www.csis.org/programs/ global-food-security-program/covid-19-and-food-security chandra, r.k., 1992. effect of vitamin and trace-element supplementation on immune responses and infection in elderly subjects. lancet 340: 1124. https://doi. org/10.1016/0140-6736(92)93151-c chang, h.h. and meyerhoefer, c.d., 2021. covid-19 and the demand for online food shopping services: empirical evidence from taiwan. american journal of agricultural economics 103(2): 448–465. https://doi.org/10.1111/ajae.12170 chenarides, l., manfredo, m. and richards, t.j., 2021. covid-19 and food supply chains. applied economic perspectives and policy 43(1): 270–279. https://doi.org/10.1002/aepp.13085 criteo coronavirus survey, 2020. coronavirus consumer trends: consumer electronics, pet supplies, and more. resource document. available at: https://www.criteo.com/insights/ coronavirus-consumer-trends/ ellison, b., mcfadden, b., rickard, b.j. and wilson, n.l., 2021. examining food purchase behavior and food values during the covid-19 pandemic. applied economic perspectives and policy 43(1): 58–72. https://doi.org/10.1002/aepp.13118 gaucheron, f., 2011. milk and dairy products: a unique micronutrient combination. journal of the american college of nutrition 30(suppl 5): 400s–409s. https://doi.org/10.1080/07315724.201 1.10719983 głąbska, d., skolmowska, d. and guzek, d., 2020. populationbased study of the changes in the food choice determinants of secondary school students: polish adolescents’ covid-19 experience (place-19) study. nutrients 12(9): 2640. https://doi. org/10.3390/nu12092640 gov.uk. (2020). guidance for consumers on coronavirus (covid-19) and food. available at: https://www.gov.uk/government/publications/ guidance-for-consumers-on-coronavirus-covid-19-and-food/ guidance-for-consumers-on-coronavirus-covid-19-and-food grashuis, j., skevas, t. and segovia, m.s., 2020. grocery shopping preferences during the covid-19 pandemic. sustainability 12(13): 5369. https://doi.org/10.3390/su12135369 gundersen, c., hake, m., dewey, a. and engelhard, e., 2021. food insecurity during covid-19. applied economic perspectives and policy 43(1): 153–161. https://doi.org/10.1002/aepp.13100 hirvonen, k., de brauw, a. and abate, g.t., 2021. food consumption and food security during the covid-19 pandemic in addis purchasing. majority of the respondents have been more than willing to buy fresh products since the covid-19 pandemic started. consumers have adopted the practice of preserving their foods by freezing during quarantine days. moreover, the covid-19 pandemic has caused many changes in the eating habits of turkish consumers. sixty-five percent of the consumers have tried to consume more food that boosts the immune system since the covid-19 pandemic started. furthermore, they have had a proper eating habit regarding fruit and vegetable consumption. when the results were evaluated in terms of age groups, consumers over 65 years paid more attention to eating healthy than other age groups (p < 0.05). on the other hand, 77% of the consumers have tried to consume more home-cooked meal since the covid-19 pandemic started. furthermore, turkish consumers relied on media tools to a great extent to obtain information about covid-19. data obtained from the study indicated that much more education is needed on food and covid-19. media tools should be used effectively to communicate accurate information during the covid19 pandemic. future research should investigate how the pandemic affects long-term disparities in food and nutrition for disadvantaged populations and how the 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_hlk57399323 _hlk63000025 _hlk40904838 _hlk40906736 _hlk40721020 _hlk40719414 _hlk77428270 _hlk40385282 _hlk75294755 _hlk77424762 _hlk40537756 _hlk40474764 78 issn 1120-1770 online, doi 10.15586/ijfs.v33isp2.2053 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (sp2): 78–91 p u b l i c a t i o n s codon biodecontamination of milk and dairy products by probiotics: boon for bane razieh sadat mirmahdi1, alaleh zoghi2, fatemeh mohammadi1, kianoush khosravi-darani2*, shima jazaiery3, reza mohammadi4, yasir rehman5 1student research committee, department of food science and technology, national nutrition and food technology research institute, faculty of nutrition science and food technology, shahid beheshti university of medical sciences, tehran, iran; 2department of food science and technology, national nutrition and food technology; 3department of nutrition, school of public health, iran university of medical sciences, tehran, iran; 4department of food science and technology, school of nutrition sciences and food technology, research center for environmental determinants of health (rcedh), health institute, kermanshah university of medical sciences, kermanshah, iran; 5department of life sciences, school of science, university of management and technology, lahore, pakistan *corresponding author: kianoush khosravi-darani, prof. food biotechnology. shahrake gharb, farahzadi blv., hafesi st. no7, tehran iran, p. o. box: 19395-4741, tehran, iran. email: k.khosravi@sbmu.ac.ir and kiankh@yahoo.com received: 17 april 2021; accepted: 31 may 2021; published: 1 july 2021 © 2021 codon publications open access review article abstract in recent decades, “contamination of the environment, food, and feed by different contaminants such as heavy metals and toxins is increasing due to industrial life.” commercial milk and milk products can be contaminated with heavy metals and mycotoxins. biosorption is a low-cost method and has good potential for decontamination. in dairy products, “various starters, especially probiotics, can be used as biosorbants, while microorganisms are able to bind to heavy metals and toxins and decrease their bioavailability and hazards in the human body.” in this article, the key role of dairy starters and probiotics in the decontamination of toxins and heavy metals, and the best probiotics for decontamination of aflatoxins and heavy metals has been reviewed. after a quick glance at introducing dairy products and the main risks in association with the intake of some hazardous materials from dairy products, the application of biological systems is mentioned. then, the article is focused on the role of beneficial microorganisms as the last chance to decrease the risk of exposure to toxins and heavy metals in dairy products. this review can be helpful for biotechnologists and scientists who have challenges about the existence of heavy metals and toxins in milk and dairy products, and help them to find the best method to decrease the content of the usual contaminants. keywords: aflatoxins; biosorption; decontamination; heavy metals; dairy products introduction the world health organization (who) defines food safety as, “approaches and methods for certifying the manufacture, maintenance, distribution and utilization of food happen in an assured system.” however, some people defined safe food as food without any contamination (el sheikha, 2015). heavy metals naturally exist in the environment. industrial activities can increase their content in air and soil, leading to phytotoxicity of plants (asati et al., 2016; yang et al., 2018). milk and dairy products have an important role in the human food chain, especially children’s food; so, contamination of dairy products by toxins and heavy metals is one of the most important issues that can negatively impact consumers’ health. milk and dairy mailto:k.khosravi@sbmu.ac.ir mailto:kiankh@yahoo.com italian journal of food science, 2021; 33 (sp2) 79 biodecontamination of milk and dairy products dairy products is aflatoxin m1 (afm1). afm1 is a metabolite of aflatoxin b1 (afb1) after ingestion of contaminated feed (afb1) by livestock. about 0.3 to 6.2% of afb1 (abdelmotilib et al., 2018) can be bio-transformed into afm1 (4-hydroxyafb1) and can excrete into milk and urine (iha et al., 2013; karazhiyan et al., 2016). afm1 is carcinogenic and toxicogenic, and can resist pasteurization and sterilization processes (gonçalves et al., 2020). afm1 compared with afb1 is approximately 10 times less mutagenic, genotoxic, and toxigenic. its carcinogenic effects are displayed in different kinds of species (elsanhoty et al., 2014). afm1 can also cause gene mutation, dna damage, cell transformation in mammalian cells, and chromosomal anomalies. food and drug administration (fda) and the european commission recommended that the maximum permissible limits of afm1 in milk are 0.5 μg/kg and 0.05 μg/kg, respectively (commission, 2006; fda, 2019) it is reported that mycotoxins in milk and dairy products, which can be produced by different kinds of fungi are: aflatoxins (by aspergillus), compactin (by penicillium), cyclopaldic acid (by penicillium), and patulin (by penicillium) (el sheikha, 2019). many reports have investigated regarding contamination of milk by heavy metals and toxins all over the world. according to tables 1 and 2, which present some of the above reports, the amount of lead in iraq, brazil, china, spain, and italy was more than the maximum permissible products can be contaminated with heavy metals under certain conditions through contamination of water and animal feed with environmental contaminants such as metal and cement smelters, sewage effluents, and industrial waste. heavy metals’ accumulation in milk can easily enter the human body and be dangerous for consumer’s health (abedi et al., 2020). dairy product contamination (heavy metal and aflatoxin) is very common all over the world (ziarati et al., 2018). heavy metals’ toxicity occurs in levels of about 1.0–10 mg/l; however, lead and cadmium could have a toxic effect in 1–100 μg/l (alkorta et al., 2004). for example, different levels of exposure to cadmium could cause renal dysfunction, hepatic injury, and lung damage (miura et  al., 2017; naidoo et al., 2019; zhang et al., 2014). arsenic poisoning can cause death through disorder in essential metabolic enzymes (khairul et al., 2017). maximum permissible limits of heavy metal contents in milk (considered by international dairy federation) are 2.6 µg/kg for cadmium, 10 µg/kg for copper, 20 µg/kg for lead, and 328 µg/kg for zinc (malhat et al., 2012). aflatoxins directly (through eating contaminated food) and indirectly (primary contaminated products such as milk of contaminated livestock) can enter into the human body by the use of contaminated dairy products. aflatoxins can cause negative effects on human health, such as liver or kidney cancer and chronic intoxications (karazhiyan et al., 2016). the most common aflatoxin in table 1. some important data about milk contamination to heavy metals (from 2014 to 2021). country contamination concentration reference egypt pb cd 0.044–0.751 mg/l 0.008–0.179 mg/l meshref et al., 2014 serbia pb cd 54.3–95.2 lg/kg 2.13–4.82 lg/kg suturović et al., 2014 iraq pb 32 µg/l alani and al-azzawi, 2015 pakistan pb cd 0.014 mg/kg 0.001 mg/kg ismail et al., 2015 bangladesh pb cd 0.2 mg/l 0.073 mg/l muhib et al., 2016 iran pb cd 14.0 µg/kg 1.11 µg/kg shahbazi et al., 2016 brazil pb 2.12–37.36μg/l de oliveira et al., 2017 mexico pb as 0.03 mg/kg 0.12 mg/kg castro-gonzález et al., 2018 poland pb cd 5.24 μg/l 0.15 μg/l halagarda et al., 2018 turkey pb cd as 0.0055 mg/l 0.088 mg/l 0.002 mg/l seğmenoğlu and baydan, 2021 as: arsenic, cd: cadmium, pb: lead 80 italian journal of food science, 2021; 33 (sp2) mirmahdi rs et al. table 2. some important data about milk contamination to mycotoxins in world from 2014 to 2021. country contamination concentration reference croatia afm 1 0.003–1.135 μg/l bilandžić et al., 2014 china afm 1 oa zea α-zea 80.4 ng/kg 56.7 ng/kg 14.9 ng/kg 24.3 ng/kg huang et al., 2014 serbia afm 1 0.01–1.2 μg/kg kos et al., 2014 iran afm 1 > 0.05 μg/l fallah et al., 2015 macedonia afm 1 408.1 ng/l dimitrieska-stojković et al., 2016 pakistan afm 1 >2610 ng/l aslam et al., 2016 argentina afm 1 293 ng/l michlig et al., 2016 bosnia and herzegovina afm 1 60 ng/l bilandžić et al., 2016 italy afm 1 52 ng/l de roma et al., 2017 tanzania afm 1 0.627 ng/ml karczmarczyk et al., 2017 malaysia afm 1 144 ng/l shuib et al., 2017 kosovo afm 1 83 ng/l camaj et al., 2018 el salvador afm 1 approximately 100 ng/l peña-rodas et al., 2018 turkey afm 1 78.69 ng/l eker et al., 2019 ethiopia afm 1 207 ng/l zakaria et al., 2019 kenya afm 1 4563 ng/l kuboka et al., 2019 brazil afm 1 45.18 ng/l venâncio et al., 2019 ecuador afm 1 0.0774 μg/kg puga-torres et al., 2020 spain afm 1 0.009–1.36 μg/kg rodríguez-blanco et al., 2020 india afm 1 1116 ng/l sharma et al., 2020 morocco afm 1 4.46 ± 14.09 ng/l mannani et al., 2021 malawi afm 1 0.551 μg/l njombwa et al., 2021 spain afm 1 afb 1 12.6 ng/kg 0.61 μg/kg bervis et al., 2021 afm 1 : aflatoxin m 1, afb 1 : aflatoxin b 1, oa: ochratoxin a, zea: zearalenone, α-zea: α-zearalenone. limits. also, afm1 in china and india, and cadmium in poland and spain, were higher than permissible limits. this information confirms the importance of decontamination in milk and dairy products. there are different methods for the decontamination of dairy products, such as physical, chemical (reverse osmosis, ion exchange, freeze concentration, and evaporation) (patterson and minear, 2013), and biological methods (using different biomaterials such as bacteria and yeasts biomass, plants, and seaweeds) (abdelmotilib et al., 2018; hashim and chu, 2004; hayat et al., 2017; satyapal et al., 2016; sulaymon et al., 2013; vishnoi et al., 2014). adsorption is one of the most important decontamination strategies in dairy products (giovati et al., 2015; massoud et al., 2019; milanowski et al., 2017; porova et  al., 2014). there are different biosorbents, such as “algae, plants, yeasts, fungi, and bacteria,” for the bioremoval of toxins and metals in fermented dairy products (e.g., kefir, kumis, yogurt, and doogh). probiotic bacteria can also be used for this purpose. fermented dairy products are very popular, and they have a perfect taste (el sheikha et al., 2018; yerlikaya, 2014). probiotics can reduce contamination (heavy metals and aflatoxins) in fermented dairy products (zoghi et al., 2014). they are widely used for bioremoval of toxins (massoud et al., 2018; zoghi et al., 2017, 2019) as well as heavy metals (arsenic, mercury, lead, and cadmium) (hadiani et al., 2018, 2019; khosravi-darani et al., 2019), heterocyclic aromatic amines (khosravi-darani et al., 2019; sarlak, 2020), and even pesticides (wochner et al., 2018). in this article, reports about the influence of adding starters and probiotics into the formulation of dairy products on the bioremoval of contaminations such as toxins and heavy metals are reviewed. starters and probiotics in dairy products food fermentation by microorganisms is one of the most economic and widely practiced methods for improving italian journal of food science, 2021; 33 (sp2) 81 biodecontamination of milk and dairy products via the manufacture of lactic acid and/or the sprinkling of secondary metabolites in the product matrix (ryan et  al., 2015). different starters have been used for producing various dairy products all around the world. some of these products and their starters are mentioned in table 3. texture, flavor, and functionality, and also for enhancing the shelf life of food products (ray et al., 2014; salque et  al., 2013). the fermentation process can be carried out with starter cultures to certify consistency in commercial products by using familiar microorganisms with favorable traits, such as a high amount of acidification table 3. some fermented dairy products and related starters. fermented dairy products country/region of origin starters reference acidophilus milk — lactobacillus acidophilus, lactobacillus casei, bifidobacterium bifidum, lactobacillus bulgaricus, streptococcus thermophilus raftaniamiri et al., 2010 buttermilk egypt and ethiopia (cultured buttermilk) lactic acid bacteria (e.g., lactococcus, lactobacillus, streptococcus, and leuconostocs) el sheikha and montet, 2014; kumar et al., 2015 cheese — (cheddar cheese) lactic acid bacteria starter culture (lactococcus lactis ssp. lactis, lactococcus lactis ssp. cremoris, and streptococcus salivarius spp. thermophilus) ferreira and viljoen, 2003 matzoon armenia lactic acid bacteria macori and cotter, 2018 leben arab world (leben from camel milk) lactococcus lactis, lactobacillus pentosus, lactobacillus plantarum, lactobacillus brevis, and pediococcus pentosaceus fguiri et al., 2013 kishk arab world freeze-dried yogurt starter culture tamime et al., 2000 kumis central asia turkic countries central asia lactococcus lactis subsp. lactis, streptococcus thermophilus, lactobacillus delbrueckii subsp. bulgaricus, lactobacillus helveticus, lactobacillus casei subsp. pseudoplantarum, and lactobacillus brevis kluyveromyces marxianus var. lactis, saccharomyces cerevisiae, candida inconspicua, and candida maris simova et al., 2002 ymer denmark streptococcus lactis, streptococcus diacetilaclis., streptococcus cremoris, and leuconostoc citrovorum poulsen, 1970 kefir estonia, hungary, greece, latvia, romania, slovakia, bosnia and herzegovina lactobacilli lactococcus acetic acid bacteria and yeast garrote et al., 2001 dahi india lactobacillus case or lactobacillus acidophilus yadav et al., 2005 mishti doi india streptococcus salivarius ssp. thermophiles, lactobacillus acidophilus, lactobacillus delbrueckii ssp. bulgaricus, lactobacillus acidophilus, lactococcus lactis ssp. lactis, saccharomyces cerevisiae gupta et al., 2000 matsoni georgia lactobacillus streptococcus, kluyveromyces marxianus, candida famata, saccharomyces cerevisiae, lodderomyces elongisporus, kluyveromyces lactis bokulich et al., 2015 wara africa lactobacillus sp., leuconostoc sp., pediococuus sp., lactococcus sp., yeasts el sheikha and montet, 2014 biruni sudan lactic acid bacteria el sheikha and montet, 2014 mish sudan and egypt lactic acid bacteria el sheikha and montet, 2014 rob sudan lactic acid bacteria el sheikha and montet, 2014 doogh iran lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus casei, bifidobacterium lactis sarlak et al., 2017 yogurt serbia streptococcus thermophilus and lactobacillus bulgaricus elsanhoty et al., 2014 clabber united states starters like kefir dyomina et al., 2017 82 italian journal of food science, 2021; 33 (sp2) mirmahdi rs et al. fao (2001) defined probiotics as, “viable microorganisms, that while ingested in sufficient amounts, exert health benefits on the host (fao/who, 2001).” the main beneficial effects of probiotics on human health include mucosal immunity support, decreasing lactose intolerance, preventing respiratory infections or diarrheas, feasible hypocholesterolemia effects, prevention of intestinal pathogens, inhibition of colon cancer or inflammatory bowel disease (sanders et al., 2014; yu et al., 2015). the application of microorganisms, especially probiotics, recently has been investigated for their potential to heavy metals and aflatoxins reduction (zoghi et al., 2014). most species known as probiotic bacteria are bifidobacterium (b.), lactobacillus (l.), bacillus, and yeast saccharomyces (s.) cerevisiae, and some strains of escherichia (e.) coli. a practical taxonomy of nonpathogenic, fermentative, and nontoxigenic probiotic bacteria is lactic acid bacteria (lab), which are used widely in food industries (zoghi et al., 2017). lab usually have gram-positive cell walls, and peptidoglycan is their main cell wall structural component; teichoic acid, lipoteichoic acid, some neutral polysaccharides, and a proteinous s-layer are their minor components (zoghi et al., 2014). toxins’ bioremoval in milk and dairy products in recent decades, several scientific studies have been done regarding decontamination in dairy products, especially the biological decontamination method. some of these researches are mentioned in table 4. el khoury et al. (2011) investigated the application of lab including l. bulgaricus and streptococcus thermophiles on the reduction of afm1. they showed that using lab is a potential method to decrease afm1 with the higher efficiency of l. bulgaricus compared to streptococcus thermophiles. they also mentioned that the level of afm1, which is bound by lab, enhanced with increasing the time of inoculation (el khoury et al., 2011). the binding ability of yogurt cultures was different. it is suggested that the difference in the binding ability of lab is attributed to the difference in their cell-wall structure (sarimehmetoğlu and küplülü, 2004). in addition to lab, using s. cerevisiae is considered as an effective way for microbial detoxification (karazhiyan et al., 2016). a systematic review by campagnollo et al. (2020) focused on parameters influencing the binding process of afm1 by yeast. the overall binding level of yeast was reported as 52.05%, in which the lowest binding capacity was related to the yeast extract peptone and the highest binding was associated with the ruminal fluid. also, different factors, including temperature, yeast, ph, and the type of aflatoxin, have been mentioned as the major parameters in the process of decontamination (campagnollo et al., 2020). moreover, the effect of different treated s. cerevisiae, including heat, acid, and ultrasound treated, on the binding with afm1 was assessed by karazhiyan et al. (2016). among all treated yeasts, acid treatment had the most positive impact on yeast cells for improving their binding ability to aflatoxins which can be attributed to the release of monomers from polysaccharides under acidic conditions and their further changes into aldehydes after breaking down of glycosides linkages. after acid treatment, heat-treated yeasts showed the highest binding ability due to protein denaturation and maillard reaction product formation, which caused an increase in the permeability of cell walls. comparison between viable and unviable yeasts (heat, acid, and ultrasound treated) exhibited higher efficiency of unviable cells, which indicates that such treatments increase the binding capacity of yeasts (karazhiyan et al., 2016). in a study performed by taheur et al. (2017), a novel strategy for the reduction of mycotoxins using kefir grains was examined. the results showed that kefir microorganism grains could adsorb 82 to 100% of afb1, zearalenone, and ochratoxin a after cultivation in milk. the main strains that were able to adsorb mycotoxins were l. kefiri, kazachstania servazzii, and acetobacter syzygii. the l. kefiri kflm3 was found to be the most active strain with an adsorption rate of 80 to 100% of the mycotoxins, and k. servazzii kfgy7 was found to retain higher mycotoxin than others after the desorption experiments. as a result, kefir consumption can assist in diminishing gastrointestinal absorption of mycotoxins and their toxic effects (taheur et al., 2017). heavy metals’ bioremoval in milk and dairy products in table 5, investigations regarding heavy metal bioremoval in milk and dairy products are illustrated. in two different studies by massoud et al. (2019, 2020a), application of s. cerevisiae to reduce the concentrations of lead and cadmium in milk was examined. the optimization process was also performed considering three factors including contact time, concentrations of biomass, and initial content of heavy metals (massoud et al., 2019, 2020a). generally, the rate of removal of heavy metals increased with an increase in the biomass, contact time, and concentration of heavy metals. they concluded that optimized conditions for lead removal were obtained after 4 days (at the end of storage time) with the content of 22×108 cfu/ml of yeast and 70 μg/l of lead in milk (massoud et al., 2019). similarly, the optimized process for cadmium bioremoval was achieved after 4 days with 80 μg/l of cadmium and 30×108 cfu/ml of s. cerevisiae italian journal of food science, 2021; 33 (sp2) 83 biodecontamination of milk and dairy products table 4. aflatoxin decontamination in milk and dairy products. product microorganism removal w/w% contaminant conditions reference milk lactobacillus rhamnosus (milk whey medium) 46.0% afb 1 optimal condition: 60 min in ph 3.0 bovo et al., 2014 milk kefir starters 1. l. acidophilus, bifidobacterium, & streptococcus thermophiles (thermophilic lactic culture) 2. lactococcus lactis subsp. cremoris, leuconostoc, lactococcus lactis subsp. lactis biovar diacetylactis, lactococcus lactis, subsp. lactis, 3. debaryomyces hansenii, kluyveromyces marxianus subsp. marxianus., yeast pool, lactic acid bacteria pool full kefir starters 11.67–34.66% yeast pool 65.33–68.89% lab pool 65% afm 1 toxin concentration: 150, 200, and 250 ng/l temperature: 4 °c time: 7 days kamyar and movassaghghazani, 2017 milk lactobacillus helveticus 85% afm 1 time: 60 min ismail et al., 2017 milk saccharomyces cerevisiae 81.3% afm 1 time: 48 h foroughi et al., 2018 yogurt a: s. thermophilus & l. bulgaricus b: 50% s. thermophilus & l. bulgaricus 50% l. planetarum c: 50% s. thermophilus and l. bulgaricus, 50% l. acidophilus treatment b: highest reduction 31.5–87.8% afm 1 temperature: 5°c, storage time: 1, 3, 5, and 7 days elsanhoty et al., 2014 yoghurt lactobacillus acidophilus 90% afm 1 108 cfu/ ml, initial concentration of afm 1 :0.1, 0.5, 0.75 μg/l adibpour et al., 2016 yoghurt saccharomyces cerevisiae 76.46% afm 1 aflatoxin m 1: 100, 500, and 750 g/ m l , in 1, 7, 14, and 21 days, yeast treatments: heat, acid, and ultrasound karazhiyan et al., 2016 yoghurt lactobacillus plantarum, bifidobacterium animalis, bifidobacterium bifidum yogurt starters and b. bifidum, b. animalis (60.8%), yogurt starters and l. plantarum, b. bifidum 55.1%) afm 1 storage time: 1 or 10 days sevim et al., 2019 yogurt l. plantarum, b. animalis, & b. bifidum, l. plantarum 49–60% afm 1 contact time: 4 h temperature: 42°c sevim et al., 2019 kefir lactobacillus casei & kefir starter 88.17% afm 1 aflatoxin m 1 500 pg, kefir starters 2, 4, 6, 8, 10%, l. casei: 0.1, 0.3, 0.5, 0.7, 0.9 % in 48 h sani et al., 2014 kefir kefir-grains 96.8% afg 1 toxin concentration 5, 10, 15, 20, 25 ng/g, kefir grain:5, 10, 20, 10, 25%, in 0, 2, 4, 6, 8 h, at 20, 30, 40, 50, 60°c ansari et al., 2015 (continues) 84 italian journal of food science, 2021; 33 (sp2) mirmahdi rs et al. table 4. continued product microorganism removal w/w% contaminant conditions reference kefir kefir grains: lactobacillus kefiri, kazachstania servazzii, acetobacter syzygii 82–100% afb 1, zea, oa 1 μg/ml mycotoxin, kefir grains 10% w/v in 24 at 25°c taheur et al., 2017 uht skim milk lactic acid bacteria (lactobacillus rhamnosus, lactobacillus delbrueckii spp. bulgaricus, bifidobacterium lactis), saccharomyces cerevisiae lab pool (30 min): 11.5 ±2.3% lab (60 min): 11.7 ± 4.4%, saccharomyces: (30 min), 90.3 ± 0.3%, saccharomyces: 60 min, 92.7 ± 0.7% afm 1 0.5 ng afm 1 ml−1, lab pool: 1010 cells ml−1 yeast: 109 cells ml−1 contact time: 30 min or 60 min corassin et al., 2013 fermented milk drink lactobacillus casei shirota afb 1 -lys reduction: 82.37% serum afb 1 lysine adduct 4-week intervention phases, (a): probiotic drinks 2 twice a day (b): placebo for 6, 8, or 10 weeks redzwan et al., 2016 doogh lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus casei, , bifidobacterium lactis day 28, lactobacillus acidophilus: 98.8 ± 1.3% afm 1 0.500 ppb toxin, 1,14, or 28 days at 5 °c, l. acidophilus 9 log cfu/ml sarlak et al., 2017 ergo fermented milk l. plantarum lactobacillus acidophilus, lactobacillus brevis, lactobacillus casei subsp. casei, lactobacillus helveticus, streptococcus faecalis, streptococcus thermophiles, leuconostoc mesenteroides, subsp. cremoris 57.33% 54.04% afm 1 time: 1–5 days temperature: 25°c shigute and washe, 2018 afm 1 : aflatoxin m 1, afb 1 : aflatoxin b 1, oa: ochratoxin a, zea: zearalenone, afg 1 : aflatoxin g 1 . table 5. heavy metals decontamination in milk and dairy products. product microorganism contaminant removal% w/w conditions reference milk saccharomyces cerevisiae pb 70% opt. at 22×108 cfu inoculation of yeast, lead content 70 μg/l massoud et al., 2019 kefir lactococcus lactis, kluyveromyces marxianus, co-culture ni, cu, cd, pb, fe 81.53%, 73.45%, 79.48%, 68.53%, 58.17% time: 10 days cherni et al., 2020 milk saccharomyces cerevisiae cd 70% cadmium content in milk 80 μg/l, 30×108 cfu saccharomyces cerevisiae, storage time the 4th day, masoud et al., 2020 milk lactobacillus acidophilus pb cd 80% 75% 1 × 1012 cfu of l. acidophilus, in 4 days with the initial pollution of 100 µg/l. massoud et al., 2020b milk saccharomyces cerevisiae hg 70% contact time: 30 days, initial concentration of hg: 80 µg/l and biomass dosage 22 × 108 cfu massoud et al., 2021 lead: pb, nickel: ni, copper: cu, cadmium: cd, iron: fe, mercury: hg. italian journal of food science, 2021; 33 (sp2) 85 biodecontamination of milk and dairy products (massoud et al., 2020a). therefore, they have introduced applying s. cerevisiae as a novel and useful technology for the bioremoval of heavy metals from foodstuff (massoud et al., 2019, 2020a) different treatments, such as caustic, ethanol, acidic, and heat, can enhance the biosorption of heavy metals by microorganisms. in a study by yekta göksungur et al. (2005), “potential of baker’s yeast in bioremoval of cadmium and lead with 3 pretreatments (caustic, heat and ethanol)” was examined. ethanol-treated yeast strains could remove the most content of metals and it can be explained by improving the availability of yeast binding sites and maybe enhancing the metals accessibility (göksungur et al., 2005). mechanisms of bioremoval and stability of complexes (probiotics/starters-heavy metal/toxin) afm1 and other toxins are accumulated in milk and dairy products because they are able to bind to milk protein components such as casein (dyomina et al., 2017; granados-chinchilla, 2016; sarlak et al., 2017). therefore, numerous investigations have been focused on the removal of toxins using microorganisms, such as lab (dyomina et al., 2017; sarlak et al., 2017). although the mechanism of bioremoval of toxins and heavy metals by lab was not well known until now, it is proposed that toxins are highly linked by cell wall components of microorganisms and are not metabolically degraded (zoghi et al., 2014). yeast and lab are used widely to reduce toxins and metal ions. as both viable and dead cells are capable of adsorbing toxins, it is sensible to conclude that the removal of toxins is by adhesion to the components of microorganism’s cell wall relative to covalent binding, as reviewed by shetty et al. (2006) (shetty and jespersen, 2006). it is indicated that mannan components of the s. cerevisiae cell wall play an important role in toxin binding (devegowda et al., 1996). generally, the cell wall proteins of s. cerevisiae are bound to β-1,3-glucans by covalent linkage by β-1,6-glucan chains (shetty and jespersen, 2006). apart from this, the major part of the lab cell is made up of peptidoglycan, which contains teichoic and lipoteichoic acids. also, a proteinous s-layer and neutral polysaccharides as components of the lab cell wall have been recognized and reviewed by lahtinen et al. (2004). a study by yiannikouris et al. (2004) indicated the interactions between zearalenone and β-d-glucans, in which β-1,3 d-glucan chains constitute a stable helical link with zearalenone and stabilized by β-1,6 d-glucan chains (yiannikouris et al., 2004). in order to investigate the mechanism of binding of aflatoxins to l. rhamnosus it is indicated that carbohydrates in the cell wall are predominantly responsible for binding to aflatoxins. in samples treated by urea, it is shown that hydrophobic interactions play a significant role in binding, and treatment by nacl and cacl2 showed that electrostatic interactions played a minor role (haskard et al., 2000). also, it is stated that afm1 is bound to lab cell wall components by weak noncovalent interactions. the difference in the binding ability among different microorganisms is attributed to the cell wall and cell envelope structures (el khoury et al., 2011). similarly, turbic et al. (2002) mentioned that the different binding ability of lab highly depended on the strain of the microorganisms (turbic et al., 2002). another study associated with the mechanism of biosorption illustrated that nonviable cells, including heat and acid-treated cells, produced complexes with higher stability, which means better access of groups in treated cells rather than viable ones. this phenomenon emphasizes that the viability of cells is not an important factor for the binding ability of cells (haskard et al., 2001). furthermore, it is shown that acids might be capable of breaking amine binding in peptides and proteins, which leads to the production of peptides and even amino acids, and consequently, more accessible aflatoxin binding sites will be available (el-nezami et al., 2002). similarly, it is noted that hydrophobic interactions are highly expected in lab, which is treated by acid because acid treatment leads to denaturation of proteins and enhanced hydrophobic binding sites (haskard et al., 2000). moreover, the mechanisms of bioremoval could be influenced by various factors including types of microorganisms or even the status of biomass (living or nonliving microorganism), chemical properties of toxic materials, and environmental factors, such as temperature as well as ph (javanbakht et al., 2014). for more illustration, javanbakht et al. (2014) investigated the mechanism of removal of heavy metals by microorganisms. they suggested that two different types of pathways are involved in biosorption, which depends on cell metabolism and is divided into metabolism-dependent and metabolism-independent groups. the first pathway only occurs in viable cells through the transformation of metals across the cell wall. the second mechanism is involved in the physicochemical interaction between metals and functional groups of cell surface such as physical adsorption and ion exchange without depending on the cell metabolisms (javanbakht et al., 2014). to investigate the stability of complexes, haskard et al. (2001) evaluated the stability of 12 complexes between 86 italian journal of food science, 2021; 33 (sp2) mirmahdi rs et al. in  vivo and in vitro conditions. also, more experiments should be done for finding optimum conditions for special starters in special dairy products for better decontamination. declarations funding this research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors. conflicts of interest/competing interests the authors declare that they have no conflicts of interest. availability of data and material not applicable. code availability not applicable. authors’ contributions rm was involved in writing and original draft preparation; az was responsible for writing, review, and editing; kkd was concerned with conceptualization and supervision; fm was involved in writing and editing; and sj, rm, and yr were responsible for review and editing. references abdelmotilib, n.m., hamad, g.m., elderea, h.b., salem, e.g. and el sohaimy, s.a., 2018. aflatoxin m1 reduction in milk by a novel combination of probiotic bacterial and yeast strains. european journal of nutrition & food safety 8(2):83–99. https://doi. org/10.9734/ejnfs/2018/39486 abedi, a.-s., nasseri, e., esfarjani, f., mohammadi-nasrabadi,  f., moosavi, m.h. and hoseini, h., 2020. a systematic review and meta-analysis of lead and cadmium concentrations in cow milk in iran and human health risk assessment. environmental science and pollution research 27: 10147–10159. https://doi. org/10.1007/s11356-020-07989-w adibpour, n., soleimanian-zad, s., sarabi-jamab, m. and tajalli, f., 2016. effect of storage time and concentration of aflatoxin m1 on toxin binding capacity of l. acidophilus in fermented milk product. journal of agricultural science and technology 18: 1209–1220. available from: http://jast.modares.ac.ir/article-23-4462-en.html lab and afb1 considering both viable and nonviable cells and concluded that 71% of afb1 remained bound, indicating the high stability of the complexes. also, they showed that nonviable cells retained a higher amount of afb1, as mentioned above (haskard et al., 2001). based on their results, the stability of complexes depends upon three factors including strain, treatment type, and environmental conditions. fazeli et al. (2009) conducted a study to investigate the effect of strains, including l.  casei, l. plantarum, and l. fermentum, on the reduction of afb1 and concluded that all the strains were able to remove afb1, although l. casei was found to be a stronger binder of afb1 rather than other bacteria (fazeli et al., 2009). a study by zoghi et al. (2020) showed that adsorption of patulin by lab can be reversible in simulated gastrointestinal conditions. the reversibility of binding between lab and patulin can be explained by the sense of noncovalent electrostatic bonds (van der waals and hydrogen bonds) (zoghi et al., 2020). similarly, in another study, the adsorption of afb1, zearalenone, and ochratoxin a by kefir grains in simulated gastrointestinal ph was reversible. in ph 3, further amounts of toxins were released (taheur et al., 2017). moreover, reduction of afb1 from a gastrointestinal model by several cells, including l. rhamnosus, l. plantarum, and l. acidophilus, were examined by motameny et al. (2012), and they concluded that l. plantarum was the most active cell (motameny et al., 2012). conclusions aflatoxins and heavy metals frequently contaminate milk and dairy products at different levels. in the food industry, controlling aflatoxin and heavy metal levels in dairy products is a challenge for researchers. according to the recent studies summarized in this review, it is revealed that using different microorganisms (such as probiotics) in different dairy products could result in the removal of toxins and heavy metals by creating bonds between contaminants and these microorganisms. using the starters in fermented dairy products can be helpful in the decontamination of toxins and heavy metals. according to this review, l. bulgaricus, kefir grains, l. acidophilus, and l. rhamnosus could be useful for decreasing afm1 and other toxins in milk and dairy products. also, for decontamination of heavy metals, kefir grains had the best ability for the bioremoval of different metals. 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the liquid sourdough preparation aniello falciano1*, annalisa romano1,2, blanca e. garcía almendárez3, carlos regalado-gónzalez3, prospero di pierro1,2* and paolo masi1,2 1department of agricultural sciences, university of naples federico ii, naples, italy; 2centre for food innovation and development in the food industry, university of naples federico ii, naples, italy; 3facultad de química, departamento de investigación y posgrado en alimentos, universidad autónoma de querétaro, querétaro, mexico *corresponding authors: aniello falciano, department of agricultural sciences, university of naples federico ii, portici 80055, naples, italy. email: aniello.falciano@unina.it; prospero di pierro, department of agricultural sciences, university of naples federico ii, portici 80055, naples, italy; centre for food innovation and development in the food industry, university of naples federico ii, portici 80055, naples, italy. email: prospero.dipierro@unina.it received: 20 april 2022; accepted: 29 august 2022; published: 7 october 2022 © 2022 codon publications open access paper abstract the aim of this work was to investigate the effect of refreshments on the growth of endogenous microorganisms during liquid sourdough preparation by using an italian and mexican wheat flours and its effects on the physicochemical properties (ph, total titratable acidity, water activity, moisture content and reducing sugars). the liquid sourdoughs were prepared (dy 200) and incubated for 6 days at 20°c. the sourdoughs were refreshed every day and compared with the not-refreshed ones. preliminary results showed that in the early stages of the microbial growth process, their population was greater in the sourdough made from the mexican wheat flour than that of the italian one. however, after 6 days, the microbial population was not significantly different in refreshed or not-refreshed samples for both sourdoughs (italian and mexican). similarly, physicochemical properties did not show significant differences. keywords: backslopping; leavening agent; sourdough; spontaneous fermentation introduction the art of baking is a very ancient technology. the beer foam was initially used for leavening of bread by ancient egyptians, which was then replaced by sourdough (carnevali et al., 2007); in fact the sourdough fermentation is one of the oldest cereal fermentations known by mankind. sourdough is a mixture of wheat and/or rye flour and water, possibly with added salt, fermented by spontaneous lactic acid bacteria (lab) and yeasts from the flour and environment. the microbial ecosystem varies from one sourdough to another depending on the geographical position, which determines its acidifying and leavening capability. the microbial community makes the dough metabolically active and can be reactivated and optimised in time through consecutive refreshments (or re-buildings, replenishments, backslopping) (corsetti and settanni, 2007). the term ‘refreshment’ deals with the technique by which a dough made of flour, water, and sometimes other ingredients ferments spontaneously, and it is subsequently added as an inoculum to start the fermentation of a new mixture of flour and water or other ingredients. the sourdough fermentation is a process with very complex mechanisms (hammes and gänzle, 1998; thiele et al., 2002), and during fermentation carbohydrates and flour proteins undergo biochemical changes due to the action of microbial and indigenous enzymes (spicher, 1983). the rate and magnitude of these changes greatly affect the sourdough properties and ultimately the quality of the final baked product (arendt et al., 2007). many mailto:aniello.falciano@unina.it mailto:prospero.dipierro@unina.it 100 italian journal of food science, 2022; 34 (3) falciano a et al. 72% and fibres 2% (la molisana, campobasso, italy). the average moisture content of both flour types was 13%. chemicals the following were used for the study: plate count agar (pca), potato dextrose agar (pda) (bd, franklin lakes, nj, usa), nacl, naoh, 3,5-dinitrosalicylic acid, sodium potassium tartrate, d-glucose. all chemicals used were of analytical grade, purchased from sigma–aldrich (st. louis mo, usa). preparation of sourdoughs four types of liquid sourdough were prepared, two for each type of flour (mexican and italian flour). the liquid sourdough was prepared by mixing 500 g of flour with 500 ml of distilled water. the ingredients were mixed in a spiral mixer (grilletta im5, famag s.r.l, milano, italy) for 10 min at speed 1, and the sourdoughs were fermented at 25°c ± 1 for 6 days. the samples were remixed every day for 5 min, and one sample for each type of flour was refreshed by removing 200 g of dough that was replaced with 100 g of flour plus 100 ml of distilled water. the aliquots of samples, taken each day before remixing, were used for the following experiments. table 1 shows the different samples prepared. determination of microbial populations serial dilutions of liquid sourdough samples in 0.85 % nacl solution were used for determining the microbial count using the following media: pca for estimation of total aerobic mesophilic bacteria and pda containing 14 mg/l of tartaric acid, 50 mg/l of chloramphenicol, and 50 mg/l of rose bengal for yeasts and other fungi. exactly, 1 ml of appropriate dilutions was pour plated in triplicate. counts of total aerobic mesophilic bacteria were obtained after 48 h of incubation at 37°c, while the count of yeast and other fungi were obtained after 5 days of incubation at 30°c (ben omar and ampe, 2000). all values were performed by counting on a colony counter. results were calculated as the means of three determinations ± standard deviation . intrinsic properties of sourdough depend on the metabolic activities of its resident lab: lactic fermentation, proteolysis and synthesis of volatile compounds, production of anti-mold, and antiropiness are amongst the most important activities during the fermentation of sourdough (gobbetti et al., 1999; hammes and gänzle, 1998). the fermentation of natural yeast consequently improves the dough properties, such as improving the volume, texture, flavour and nutritional value of bread; delaying the staling process of bread, and protecting bread from mold and bacterial spoilage (de vuyst and vancanneyt, 2007). in fact nowadays, its application is on the rise, and sourdough is used in the production of a variety of products such as bread, pizza, cakes and crackers, as the improved quality of sourdough bakery products became an important marketing tool (de vuyst and gänzle, 2005). because fermentation can be performed as firm dough or as a liquid suspension of flour in water, sourdoughs can vary in its consistency. the ratio of flour and water is called the dough yield (dy) and is defined as: dy = (flour weight + water weight) × 100/flour weight. following this approach, wheat sourdough with dy 160 is firm dough, while dy 200 is liquid sourdough (decock and cappelle, 2005). the liquid fermentation system is preferred by industries due to the following technological and analytical advantages: (1) ease of management and reproducibility under operating conditions; (2) easier control of fermentation parameters (e.g. temperature, ph, dough yield), and addition of nutrients (e.g. vitamins, peptides, carbohydrates) to condition microbial performance; (3) greater suitability to deal with microbial metabolism to obtain an optimal organoleptic profile; (4) greater suitability of application as natural starter without changes to the current bread formulations; and (5) increased suitability for use with different technologies to produce various baked goods (carnevali et al., 2007). this work was carried out to investigate the effect of refreshments on the growth of endogenous microorganisms during the preparation of liquid sourdough (dy 200) incubated for 6 days using wheat flours from two different geographical locations (italian and mexican flour), and their effects on physicochemical properties, such as ph, total titratable acidity (tta), water activity (aw), moisture content and reducing sugars. materials and methods materials for liquid sourdough preparation, two types of commercial wheat soft flour ‘00’ were used. the first flour type, mexican flour, had a protein content of 11.1%, fat 2.2%, carbohydrates 71.6% and fibres 2.1% (san antonio, tres estrellas, toluca, méxico). the second one was the italian flour, with a protein content of 11%, fat 2%, carbohydrates table 1. different samples of liquid sourdough. dmnr dmr dinr dir sourdough not refreshed, prepared with mexican flour sourdough refreshed, prepared with mexican flour sourdough not refreshed, prepared with italian flour sourdough refreshed prepared, with italian flour italian journal of food science, 2022; 34 (3) 101 effects of refreshment on liquid sourdough preparation determination of ph, titratable acidity, moisture content, water activity and reducing sugars the values of ph were determined using a ph meter equipped with an immersion probe, calibrated using standard solutions at ph 7.00, 4.01 and 10.00. after calibration, the electrode was rinsed with distilled water, dried and immersed in the sample. total titratable acidity was measured in 10 g sample, which was homogenised with 90 ml of distilled water for 3 min in a stomacher apparatus (seward, london, uk) and expressed as the amount (ml) of 0.1 m naoh needed to achieve a ph of 8.3 (ercolini et al., 2013). the moisture content using the thermobalance (xm 50 precisa, biltek, esenler, istanbul, turkey) was calculated using the following equation 1: − = × (mi mf ) moisture content (%) i )m( 100 (1) mi – fresh weight, g mf – dry weight, g the values of water activity (aw) were determined by aqua-lab instrument (cx-2, decagon devices, pullman, wa, usa), calibrated with saturated kcl (aw = 0.984) standard. the determination was carried out by preparing a homogeneous sample of the product. the value was detected in balanced conditions and read directly on the screen. reducing sugars were determined using dns assay (wood et al., 2012). dns reagent contain 3,5-dinitrosalicylicacid (10 g/l), sodium potassium tartrate (30 g/l) and naoh (16 g/l) and is stored in darkness at room temperature. d-glucose calibration curves were created covering appropriate ranges as described in the relevant sections. each reaction contained 50 µl of sample and 1 ml of dns reagent (1:20, sample:dns reagent). the resulting solutions were heated in a thermocycler (biometra t-gradient, germany) at 100°c for 1 min, and held for 2 min at 20°c to cool, and analysed using a spectrophotometer (genesys 10uv, thermo scientific, waltham, ma, usa) at 540 nm. results and discussion the microbial population of the sourdoughs was enumerated using two different culture media: pca for estimation of total aerobic mesophilic bacteria and pda for yeasts and other fungi. figure 1 shows the growth of aerobic mesophilic bacteria during the 6 days of incubation. the initial concentration of bacteria was higher in 2 4 6 8 10 21 3 time (days) a er ob ic m es op hi le s (l og u f c /g ) 4 5 6 figure 1. growth of total aerobic mesophilic bacteria (log ufc/g) of the different sourdoughs, with pca method. (○): dmr, (●): dmnr, (△): dir, (▲): dinr. each value is represented as mean ± sd (n = 3). sourdoughs made with mexican flour (4 log ufc/g) than in sourdoughs made with italian flour (3.2 log ufc/g). in mexican sourdoughs, refreshed or not, growth was intense and reached almost stationary phase in the first 3 days of fermentation; on the other hand, the italian sourdoughs reached stationary phase after 5 days, probably due to lower initial population than mexican sourdoughs. the growth of yeasts during the 6 days of incubation (figure 2) showed a growing trend similar to bacteria; in this case, the initial concentration of yeasts was higher in sourdoughs made with mexican flour (4.2 log ufc/g) than in sourdoughs made with italian flour (3.8 log ufc/g). 21 3 time (days) 4 5 6 2 4 6 8 10 ye as t ( lo g u f c /g ) figure 2. growth of yeast and other fungi (log ufc/g) of the different sourdoughs, with pda method. (○): dmr, (●): dmnr, (△): dir, (▲): dinr. each value is represented as mean ± sd (n = 3). 102 italian journal of food science, 2022; 34 (3) falciano a et al. initially, the microbial population of the sourdough represents that of the flour. each microbial group did not generally exceed 5 log ufc/g. during the time, lab and yeasts become more adapted to the environmental conditions of the sourdough, to the point of dominating the mature sourdough. similar studies state that the population ranged from 6 to 9 log ufc /g and 5 to 8 log ufc /g, respectively (minervini et al., 2012). figures 3 and 4 show the results for ph and tta. the initial ph values in mexican and italian sourdoughs were 5.9 and 5.6, respectively, while the tta was 0.8 ml and 0.1m naoh in each. during fermentation, the physicochemical parameters change, mainly due to the microbial metabolism (paramithiotis et al., 2014). the ph values decreased after 6 days of incubation to 3.7 both for mexican and italian sourdoughs. similar ph values were also found by vrancken et al., (2011). generally, the ph values between 3.5 and 4.3 are considered as an index of well-developed sourdough fermentation (gobbetti and gänzle, 2012). however, in the mexican sourdoughs, the ph decreased quickly after 3 days of incubation with respect to the italian sourdoughs that showed a gradual trend. no differences were observed between the ph values of refreshed or not-refreshed sourdoughs. these results are in accordance with the bacterial growth, and their produced metabolites such as lactic acid (maifreni et al., 2004). in fact, tta values increased in both mexican and italian sourdoughs, with higher values in the mexican one due to the higher bacterial population at the beginning. after 6 days of incubation the not-refreshed sourdoughs showed higher values of tta than those refreshed for both flours. this behaviour can be related to the refreshment procedure that can act as a dilution factor on the sourdough. 21 3 time (days) 4 5 6 3 4 5 6 7 ph figure 3. evolution of ph of the different sourdoughs during 6 days of incubation. (○): dmr, (●): dmnr, (△): dir, (▲): dinr. each value is represented as mean ± sd (n = 3). 21 3 time (days) 4 5 6 0 2 4 6 8 m l n ao h , 1 m figure 4. evolution of tta of the different sourdoughs during 6 days of incubation.(○): dmr, (●): dmnr, (△): dir, (▲): dinr. each value is represented as mean ± sd (n = 3). 30 45 40 35 55 60 50 m oi st ur e (% ) 21 3 time (days) 4 5 6 figure 5. evolution of moisture content (%) of the different sourdoughs during 6 days of incubation (○): dmr, (●): dmnr, (△): dir, (▲): dinr. each value is represented as mean ± sd (n = 3). figures 5 and 6 show the moisture content (%) and aw values. in each sourdough, there are no significant differences in moisture content and aw values during the 6 days of incubation both in the refreshed and notrefreshed sourdoughs. these results confirm that both the incubation and refreshment did not affect the aqueous environment in the sourdoughs, preserving the favourable condition for microbial growth (tecante, 2019). minervini et al. (2014) stated that aw values between 0.96 and 0.98 do not limit the growth of most microorganisms. figure 7 shows the results of reducing sugar content during the fermentation. as shown during incubation, the reducing sugars increased linearly reaching its maximum concentration in each sourdough after 4 days, which can be related to the amylolytic activity of bacteria (tecante, 2019). also in this case, the values show italian journal of food science, 2022; 34 (3) 103 effects of refreshment on liquid sourdough preparation the physicochemical properties of refreshed or not refreshed sourdoughs. in summary, daily refreshment is not necessary during the first 6 days of liquid sourdough preparation. acknowledgments this research was funded by the miur (prin 2017 –2017sftx3ythe neapolitan pizza: processing, distribution, innovation and environmental aspects), the mexican agency for international cooperation (amexcid) and the italian ministries of foreign affairs and international cooperation (maeci) (cooperazione italia/messico, 2018–20; pgr-2020, cup: e68d20000670001). references arendt, e.k., ryan, l.a. and dal bello, f. 2007. impact of sourdough on the texture of bread.  food microbiology.  24(2): 165–174. https://doi.org/10.1016/j.fm.2006.07.011 ben omar, n. and ampe, f. 2000. microbial community dynamics during production of the mexican fermented maize dough pozol. applied and environmental microbiology. 66(9): 3664– 3673. https://doi.org/10.1128/aem.66.9.3664-3673.2000 carnevali, p., ciati, r., leporati, a. and paese, m. 2007. liquid sourdough fermentation: industrial application perspectives. food microbiology. 24(2): 150–154. https://doi.org/10.1016/j. fm.2006.07.009 corsetti, a. and settanni, l. 2007. lactobacilli in sourdough fermentation. food research international. 40(5): 539–558. https:// doi.org/10.1016/j.foodres.2006.11.001 de vuyst, l. and gänzle, m. 2005. second international symposium on sourdough: from fundamentals to applications. trends in food science & technology. 1(16): 2–3. https://doi. org/10.1016/2fj.tifs.2004.08.003 de vuyst, l. and vancanneyt, m. 2007. biodiversity and identification of sourdough lactic acid bacteria. food microbiology. 24(2): 120–127. https://doi.org/10.1016/j.fm.2006.07.005 decock, p. and cappelle, s. 2005. bread technology and sourdough technology. trends in food science & technology. 16(1–3): 113–120. https://doi.org/10.1016/j.tifs.2004.04.012 ercolini, d., pontonio, e., de filippis, f., minervini, f., la storia, a., gobbetti, m. and di cagno, r. 2013. microbial ecology dynamics during rye and wheat sourdough preparation. applied and environmental microbiology. 79(24): 7827–7836. https://doi. org/10.1128/aem.02955-13 gobbetti, m., de angelis, m., arnaut, p., tossut, p., corsetti, a. and lavermicocca, p. 1999. pentosani aggiunti nella panificazione: fermentazioni di pentosi derivate da batteri lattici a lievitazione naturale. microbiologia degli alimenti. 16(4): 409–418. gobbetti, m. and gänzle, m. (eds.). handbook on sourdough biotechnology. springer science & business media, new york, ny, usa, 97–99 pp. greater reducing sugars in mexican than in italian sourdoughs, probably due to higher initial microbial population. moreover, the differences in reducing sugar content observed in the refreshed and not-refreshed sourdoughs could be related to the sourdough refreshment, where there is increased polysaccharides concentration, due to fresh flour addition. conclusions these results showed that in the early stages of microbial growth, the microbial population was greater in the sourdough made from the mexican wheat flour than the italian one, due to different geographic environments. however, after 6 days of incubation, the microbial populations were not significantly different in both types of sourdoughs, either refreshed or not refreshed. in addition, there were no significant differences in 21 0.960 0.965 0.970 0.975 0.980 0.985 0.990 0.995 3 time (days) aw 4 5 6 figure 6. evolution of water activity of the different sourdoughs during 6 days of incubation (○): dmr, (●): dmnr, (△): dir, (▲): dinr. each value is represented as mean ± sd (n = 3). 21 3 time (days) 4 5 6 0 20 30 10 40 50 g su ga r/ k g figure 7. evolution of reducing sugars (g/kg). (○): dmr, (●): dmnr, (△): dir, (▲): dinr. each value is represented as mean ± sd (n = 3). https://doi.org/10.1016/j.fm.2006.07.011� https://doi.org/10.1128/aem.66.9.3664-3673.2000� https://doi.org/10.1016/j.fm.2006.07.009� https://doi.org/10.1016/j.fm.2006.07.009� https://doi.org/10.1016/j.foodres.2006.11.001� https://doi.org/10.1016/j.foodres.2006.11.001� https://doi.org/10.1016/2fj.tifs.2004.08.003� https://doi.org/10.1016/2fj.tifs.2004.08.003� https://doi.org/10.1016/j.fm.2006.07.005� https://doi.org/10.1016/j.tifs.2004.04.012� https://doi.org/10.1128/aem.02955-13� https://doi.org/10.1128/aem.02955-13� 104 italian journal of food science, 2022; 34 (3) falciano a et al. spicher, g. 1983. baked goods. in: rehm j.h., reed g. (eds.) biotechnology. verlag chemie, weinheim, germany, 1–80 pp. tecante, a. 2019. chemical and rheological description of pozol dough fermentation inoculated with streptococcus infantarius subsp. infantarius 25124 and lactobacillus plantarum a6. international journal of biotechnology & bioengineering. 5: 1–9. thiele, c., gänzle, m.g. and vogel, r.f. 2002. contribution of sourdough lactobacilli, yeast, and cereal enzymes to the generation of amino acids in dough relevant for bread flavor. cereal chemistry. 79(1): 45–51. https://doi.org/10.1094/cchem.2002.79.1.45 vrancken, g., rimaux, t., weckx, s., leroy, f. and de vuyst, l. 2011. influence of temperature and backslopping time on the microbiota of a type i propagated laboratory wheat sourdough fermentation. applied and environmental microbiology. 77(8): 2716–2726. https://doi.org/10.1128/aem.02470-10 wood, i.p., elliston, a., ryden, p., bancroft, i., roberts, i.n. and waldron, k.w. 2012. rapid quantification of reducing sugars in biomass hydrolysates: improving the speed and precision of the dinitrosalicylic acid assay. biomass and bioenergy. 44: 117–121. https://doi.org/10.1016/j.biombioe.2012.05.003 hammes, w.p. and gänzle, m.g. 1998. sourdough breads and related products. in: wood, b.j.b. (ed.) microbiology of fermented foods. blackie academic and profesional, london, 199 pp. maifreni, m., marino, m. and conte, l. 2004. lactic acid fermentation of brassica rapa: chemical and microbial evaluation of a typical italian product (brovada). european food research and technology. 218(5): 469–473. https://doi.org/10.1007/ s00217-004-0877-6 minervini, f., de angelis, m., di cagno, r. and gobbetti, m. 2014. ecological parameters influencing microbial diversity and stability of traditional sourdough. international journal of food microbiology. 171: 136–146. https://doi.org/10.1016/j. ijfoodmicro.2013.11.021 minervini, f., lattanzi, a., de angelis, m., di cagno, r. and gobbetti, m. 2012. influence of artisan bakery-or laboratory propagated sourdoughs on the diversity of lactic acid bacterium and yeast microbiotas. applied and environmental microbiology. 78(15): 5328–5340. https://doi.org/10.1128/ aem.00572-12 paramithiotis, s., doulgeraki, a.i., karahasani, a. and drosinos, e.h. 2014. microbial population dynamics during spontaneous fermentation of asparagus officinalis l. young sprouts. european food research and technology. 239(2): 297–304. https://doi. org/10.1007/s00217-014-2222-z https://doi.org/10.1094/cchem.2002.79.1.45� https://doi.org/10.1128/aem.02470-10� https://doi.org/10.1016/j.biombioe.2012.05.003� https://doi.org/10.1007/s00217-004-0877-6� https://doi.org/10.1007/s00217-004-0877-6� https://doi.org/10.1016/j.ijfoodmicro.2013.11.021� https://doi.org/10.1016/j.ijfoodmicro.2013.11.021� https://doi.org/10.1128/aem.00572-12� https://doi.org/10.1128/aem.00572-12� https://doi.org/10.1007/s00217-014-2222-z� https://doi.org/10.1007/s00217-014-2222-z� _hlk102765487 _hlk102765646 _hlk102765739 _hlk95397563 _hlk95732414 _hlk102838608 _hlk95465441 _hlk95745474 _hlk95494001 _hlk95494585 _hlk95724743 _hlk95727038 #228_tirloni_bozza ! ital. j. food sci., vol 28, 2016 612 paper evaluation of the in vitro antimicrobial activity of mixtures of lactobacillus sakei and lactobacillus curvatus isolated from argentine meat and their effect on vacuum-packaged beef s. stella, c. bernardi, p. cattaneo, f.m. colombo and e. tirloni* department of health, animal science and food safety, università degli studi di milano, via celoria 10, 20133, milano, italy *corresponding author. tel.: +39 0250317855; fax: +39 0250317870 e-mail address: erica.tirloni@unimi.it abstract a l. sakei and a l. curvatus-based mixtures exerted an antagonistic activity in vitro against 12 different spoilage and pathogenic bacteria. no activity was exerted by the cell-free supernatants. the addition of the two mixtures to sliced vacuum-packaged beef showed a better capability of l. sakei to adapt to meat substrate. after 60 days, total viable count (5.8 vs 7.4 log cfu/g), gram-negative bacteria (2.5 vs 6.4 log cfu/g) and enterobacteriaceae (2.3 vs 4.4 log cfu/g) were significantly lower in l. sakei-inoculated samples if compared with control ones; tvc and enterobacteriaceae were also significantly lower in l. curvatus-inoculated samples than control ones. the addition of mixtures gave no significant effects on meat ph and colour. the use of high dosage of viable cells could be suggestible in order to exert an early conditioning of meat environment. keywords: lactobacillus sakei, lactobacillus curvatus, biopreservation, vacuum-packed beef, antimicrobial activity ! ital. j. food sci., vol 28, 2016 613 1. introduction meat preservation is a hard race against spoilage and potential pathogenic microorganisms and restriction methods need to be applied in order to reduce their growth and prolong the shelf-life. recently, alternative technologies for the decontamination of meat products have been developed and implemented such as bioprotective cultures, natural antimicrobials, gamma, electron and x-ray irradiation, ozone, active packaging, high hydrostatic pressure, ohmic heating and steam pasteurization among the others (devlieghere et al., 2004; aymerich et al., 2008; zhou et al., 2010; loretz et al., 2011). all the alternative technologies effort to be mild: their combination, as in the hurdle theory proposed by leistner (2000), may improve their efficacy against pathogens and spoilage microorganisms, without modifying the sensorial qualities of the products. in chilled vacuum-packaged raw meat, the oxygen source is restricted determining a selective effect on the microbial population; the main spoilage microorganisms associated with these type of food result as psychrotrophic, both gram-positive bacteria, mainly lactic acid bacteria (lab) (lactobacillus spp., leuconostoc spp., carnobacterium spp.) and brochothrix thermosphacta, and gram-negative, mainly represented by enterobacteriaceae (shaw and harding, 1984; holzapfel, 1998; labadie, 1999; nychas and drosinos, 2000; fontana et al., 2006; ercolini et al., 2009; pennacchia et al., 2011). in vacuum packaged meat, the natural lab population increases during storage, becoming the predominant microflora: in particular, at chilling temperatures, lab are able to exert antagonistic actions towards the growth of spoilage and pathogenic microorganisms in beef, pork, poultry and fish (katla et al., 2002; yamazaki et al., 2003; castellano et al., 2008). in the last years, lab have received great consideration as bioprotective cultures, leading to the discovery and characterization of several antimicrobial peptides (mainly bacteriocins, organic acids, carbon dioxide, ethanol, hydrogen peroxide and diacetyl), whose activity is well known (vignolo et al., 2000; cleveland et al., 2001; castellano and vignolo, 2006; aymerich et al., 2008; dortu et al., 2008; ravyts et al., 2008). their action is also due to the lowering of food ph and to the competition for nutrients (vandenbergh, 1993). different studies indicated that, during the storage, a gradual selection of lab population occurs in the meat ecosystems, leading to the predominance of few lactobacillus species (vignolo et al., 2010; 2012). l. sakei and l. curvatus have been observed as the most widespread species in vacuum-packaged beef (yost and nattress, 2002; fontana et al., 2006; stella et al., 2013). previous studies underlined the abilities of these two species as bioprotective cultures for meat, and their application to vacuum-packaged argentine beef has already been described (castellano and vignolo, 2006; castellano et al., 2008). their mechanism of action is expressed through the ability to produce not only bacteriocins but even organic acids. moreover the good adaption to meat environment of l. curvatus and l. sakei was already proved, showing an important competitiveness in this substrate and an efficient use as an extra hurdle to minimize the risk of listeriosis in different muscle foods (schillinger et al., 1991; hugas, 1998; castellano and vignolo, 2006; fadda et al., 2008). in a previous work 73 lactobacilli were isolated from 8 lots of vacuum-packaged bovine rump hearts imported in italy from argentina, submitted to random amplified dnapolymerase chain reaction and identified, showing a prevalence of lactobacillus sakei (56 strains grouped in 18 different clusters) and lactobacillus curvatus (8 strains grouped in 6 different clusters) (stella et al., 2013). ! ital. j. food sci., vol 28, 2016 614 one strain from each of the most representative clusters obtained of l. sakei (≥5 strains) and l. curvatus (≥2 strains), for a total 6 l. sakei and 2 l. curvatus strains, were chosen. two specific mixtures were prepared (one l. sakei-based mixture and one l. curvatus-based mixture) and evaluated in vitro for their antimicrobial activity against spoilage and potential pathogenic microorganisms. moreover, the effect of the addition of the two mixtures to sliced vacuum-packaged beef was investigated, considering microbiological and physical-chemical parameters 2. materials and methods 2.1. preparation of lactobacillus strains and spoilage and potential pathogenic bacteria all l. sakei and l. curvatus strains were stored in cryovials (microbanktm, pro-lab diagnostics, richmond hill, canada) at -70°c until the use. for each strain, a loop of the frozen culture was transferred to a test tube containing 10 ml of mrs broth (oxoid, basingstoke, uk) and incubated overnight at 30°c in jars (anaerojar, oxoid) with anaerobiosis generators (anaerogen, oxoid). all the strains were re-inoculated into cooled mrs broth tubes and the initial absorbance (540 nm) (shimadzu, uv1601, mccormick place, chicago, il, usa) was measured. all the tubes were incubated at 15°c and the absorbance was measured after 24 and 48 h. precultures were collected in exponential growth phase, defined as a change of absorbance of 0.05-0.2 at 540 nm. if necessary, the cultures were diluted before preparing the mixture in order to obtain the similar od (optical density). two specific mixtures were prepared (l. sakei-based mixture of strains n. 3, 42, 55, 77, 106 and 111 and l. curvatus-based mixture of strains n. 25 and 65) adding the same aliquot of broth of each strain. a selection of 12 spoilage or pathogenic microorganisms was used as target strains for the test: escherichia coli atcc 25922, escherichia coli 0157:h7 dsm 13526, proteus vulgaris atcc 8427, salmonella typhimurium atcc 14028, serratia marcescens atcc 14756, yersinia enterocolitica atcc 23715, pseudomonas aeruginosa atcc 27853, pseudomonas fluorescens atcc 13525, pseudomonas putida atcc 49128, listeria monocytogenes atcc 7644, listeria innocua atcc 33090 and staphylococcus aureus atcc 6538. each strain, stored in cryovials at -70°c until the use, was subcultured aerobically overnight at 37°c (30°c for p. fluorescens and p. putida) in 10 ml tsb tubes (triptyc soy broth, oxoid). all the strains were re-inoculated into cooled tsb tubes and the initial absorbance was detected. all the tubes were incubated at 15°c and the absorbance was measured after 24 and 48 h. precultures were collected as reported above. 2.2. antimicrobial activity test for the evaluation of the antimicrobial activity, each mixture, prepared as reported above, was inoculated into mrs broth tubes and incubated at 30°c for 48 h in anaerobiosis. after incubation, each of the two broths was spotted by a sterile swab (carlo erba, rodano, i) onto the surface of mrs agar plates, subsequently incubated for 48 h at 30°c in an anaerobic jar. for each spoilage or pathogenic microorganism, 0.2 ml of bacterial suspension were added to a 5 ml share of semisolid agar (bhi, brain heart infusion broth, oxoid + agar 0.7%), maintained in a water bath (45°c) and then poured over the mrs plates previously spotted with each mixture. to avoid the dispersion of lactobacilli from the spot into bhi, a little amount (3-4 drops) of the inoculated semisolid medium was firstly distributed by a sterile pasteur pipette (carlo erba) on the surface of the spot; after solidification (about 3 minutes at room temperature), the remaining bhi was poured ! ital. j. food sci., vol 28, 2016 615 on the plates. after aerobic incubation at 37°c (30°c for p. fluorescens and p. putida) for 24 h, the plates were checked. a clear zone around the lactobacillus spot indicated the inhibition of the target microorganisms. the tests were conducted in triplicate. 2.3. antimicrobial activity of cell-free supernatants against spoilage and potential pathogenic microorganisms in order to determine if the inhibition was due to the production of antagonistic compounds, the cell-free supernatants of the cultured mixtures were tested against the same bacteria. the mixtures were subcultured in mrs broth as described above. after 48 h of incubation, an aliquot of each culture was centrifuged at 7700 rpm for 10 min. for each broth, ph was measured by a ph meter (amel instrument, 334-b, milan, i): three independent measurements were performed on each sample. the supernatants obtained were subsequently filtered by 0.2 "m filters (sacco, cadorago, i) and maintained at 4°c. each of the 12 target strains was inoculated into 10 ml tsb tubes and prepared as described in section 2.2; 1 ml of inoculated tsb was then transferred into 20 ml flasks of tryptic soy agar (oxoid), maintained in a water bath at 45°c, carefully mixed and poured in sterile petri plates. once the medium was solidified, blank discs (oxoid) were dipped with the supernatant of each mixture and placed onto the plates, subsequently incubated at 37°c (30°c for p. fluorescens and p. putida) for 24 h. clear zones around the discs were recorded. finally, in order to evaluate if the eventual inhibition was due to the production of organic acids, the ph of cell-free supernatants were adjusted to 6.5 with naoh (1 n) (sigma aldrich, st. luis, usa) and the same test was repeated. all the tests were performed in triplicate. 2.4. preparation and inoculation of vacuum-packaged meat slices two bovine rump hearts were sliced in a commercial cutting plant. from each meat cut, a total of 42 slices (1-cm thick, 50 g of weight) were obtained and inserted into individual sterile plastic bags, with a diffusion coefficient of 6/14 cm3 m-2 atm-1 24 h-1 to oxygen at 25°c and 75% relative humidity (cryovac, elmwood park, nj). the 42 slices obtained from each rump heart were divided into two series (each series including 21 discs) inoculated as follows: • cls (control samples l. sakei), inoculated with 0.5 ml of sterile saline solution; • ls (l. sakei), inoculated with 0.5 ml of a mixture of the six strains of l. sakei (final concentration of 5 log cfu g-1); and • clc (control samples l. curvatus), inoculated with 0.5 ml of sterile saline solution; • lc (l. curvatus), inoculated with 0.5 ml of a mixture of the two strains of l. curvatus (final concentration of 5 log cfu g-1). a loop of the frozen culture of each strain was transferred to a test tube containing 10 ml of mrs broth (oxoid) and incubated overnight at 30°c in jars (anaerojar, oxoid) with anaerobiosis generators. all the strains were re-inoculated into cooled mrs broth tubes and the initial absorbance (540 nm) was detected. all the tubes were incubated at 15°c and the absorbance was measured after 24 and 48 h. precultures were collected in exponential growth phase. the bacterial cells were pelleted by centrifugation at 7700 rpm for 10 min at 4°c and washed twice in 10 ml of 0.1 m phosphate buffered saline (pbs) with ph 7.0. cell density of each strain was determined by microscopy (1000x) (meiji techno america, ! ital. j. food sci., vol 28, 2016 616 usa). an average value from 10 randomly picked fields of view was considered. as needed, precultures were diluted in 0.85% nacl solution to obtain 5 log cfu ml-1 suspensions prior to inoculate the products. l. sakei-based mixture of strains n. 3, 42, 55, 77, 106 and 111 and l. curvatus-based mixture of strains n. 25 and 65 were finally prepared adding the same aliquot of each strain at a final concentration nearly of 5 log cfu ml-1 (each l. sakei strains has a final concentration of 4.22 log cfu ml-1, each l. curvatus strains has a final concentration of 4.70 log cfu ml-1). after inoculation, the plastic bags were submitted to a vacuum pump (final vacuum of 99%), sealed using a packaging machine (orved vm 16, musile di piave, i) and immediately stored at 4°c. samples were submitted in triplicate to analyses after inoculation (t0) and after 10 (t10), 20 (t20), 30 (t30), 40 (t40), 50 (t50) and 60 (t60) days of storage. 2.5. microbiological analyses 10 g of each sample were diluted in physiological saline (0.85% nacl) with 0.1% peptone and homogenized in a stomacher for 60 s (seward stomacher 400 blender mixer homogenizer, international pbi, milano, it). serial 10-fold dilutions were prepared and the following parameters were evaluated: total viable count (tvc) was performed on plate count agar (pca, biogenetics, ponte san nicolò, i) (iso 4833:2003) and incubated at 30°c for 48h; lactobacilli were enumerated on mrs agar (oxoid) (iso 15214:1998) incubated at 30°c for 48h in anaerobiosis, gram-negative bacteria were enumerated on tryptone soy agar (oxoid) supplemented with 10 ui ml-1 of penicillin g (oxoid) (tsap) and incubated at 30°c for 48h; the number of enterobacteriaceae was determined on violet red bile glucose agar (vrbga, biogenetics) according to the iso 21528-2:2004 method. 2.6. determination of ph and colour parameters evaluation at each sampling time, ph was measured by a ph meter (mod. xs ph6, ghiaroni &c., buccinasco, italy): three independent measurements were performed on each trimmed sample diluted 1:5 with distilled water; means were then calculated. the surface colour of the meat was assessed 45 min after opening the packages, in order to allow blooming (deoxymyoglobin oxygenation) on six randomly chosen spots of each sample surface using a minolta cr-200 chromameter (minolta, osaka, j). l* (lightness), a* (“red” index) and b* (“yellow” index) parameters were determined. chroma was calculated as a2+b2, the hue angle (h) was calculated as h = arctan (b*⁄a*), where h = 0 for red hue and h = 90 for yellowish hue. total colour differences (∆e*) between treated and control samples were calculated as: √(l1*-l2*)2 + (a1*-a2*)2 + (b1*-b2*)2. a ∆e* more than 2.3 means a variation hardly perceptible to the human eye, while ∆e* more than 3.0 a variation well perceptible to the human eye. 2.7. statistical analysis the experimental data from inhibition halos were analyzed by a two-way univariate analysis of variance, performed with mixed procedure of sas software (sas inst. inc., cary, nc, 2006) in order to in order to test mean inhibition halos size differences for the comparisons of interest at lactobacillus mixtures by target strains levels. data from meat inoculation tests were also analyzed by a two-way univariate analysis of variance using the same sas procedure to test response variable mean differences at the levels of interest of lactobacillus mixtures by time. for all statistical evaluations, threshold ! ital. j. food sci., vol 28, 2016 617 levels of p ≤ 0.05 and p ≤ 0.01 were considered for significance. a two-way multivariate analysis of variance was also performed on color parameters, considering the vector of values l*, a*, b* as the response variable; glm procedure of sas software was used. 3. results and discussions 3.1. antimicrobial activity against spoilage and potential pathogenic microorganisms the mean rays of the inhibition halos obtained from antimicrobial evaluation of l. sakei mixture and l. curvatus mixture are reported in table 1. the two mixtures exerted an antagonistic activity, producing evident halos against all the 12 target strains tested (66.7% of the halos induced by l. curvatus mixture and 52.8% of halos produced by l. sakei mixture were >10 mm). generally, l. curvatus mixture resulted significantly more effective if compared to l. sakei mixture (p = 0.0383), showing also a higher prevalence of halos > 20 mm (19.4% of the plates inoculated with the l. curvatus mixture vs 5.5% of those inoculated with l. sakei mixture). considering the different target strains, l. curvatus mixture produced significantly wider halos against y. enterocolitica (p = 0.0383) and p. aeruginosa (p = 0.0325). if we consider the results of the target strains clustered in homogenous categories, it is evident that the most sensitive belonged to the genus pseudomonas, whose components produced significantly higher halos if compared with enterobacteriaceae (p < 0.0001), listeria spp. (p = 0.0004) and staphylococcus aureus (p = 0.0117), according to the results obtained by moore et al. (2006) and tirloni et al. (2014) who underlined that most of the species of pseudomonas fail to grow under acid conditions. in particular, p. fluorescens resulted to be the most susceptible among the 12 target strains tested as significantly wider halos were observed if compared with all the other strains (p < 0.01). secondly, p. putida resulted to be significantly more susceptible if compared to e. coli o157:h7 (p = 0.0357), e. coli (p = 0.0215), l. innocua (p = 0.0200), l. monocytogenes (p = 0.0411), p. vulgaris (p = 0.0084), s. marcescens (p = 0.0003) and s. typhimurium (p = 0.0215). finally p. aeruginosa produced significantly wider halos if compared to s. marcescens (p = 0.0185). moreover, enterobacteriaceae showed a high variability in susceptibility with differences among the various species due to the many interspecific and intraspecific differences among the bacteria tested (liu et al., 2013). serratia marcescens was by far the most resistant target strain, and it showed significantly smaller halos if compared to y. enterocolitica (p = 0.0116), the most sensitive of enterobacteriaceae. many authors highlighted the presence of an evident antagonistic activity of lab against listeria monocytogenes, microorganism typically related to vacuum-packaged meat products (jones et al., 2008; awisheh and ibrahim, 2009). even in our study, both l. monocytogenes and l. innocua, showed the production of modest halos (between 9.7 and 14 mm). considering the cell-free supernatants and the ph-adjusted supernatants, no activity was recorded for all the target strains tested, highlighting that the antagonistic effect originates probably from the nutrient competitive exclusion while the involvement of extracellular compounds was not detected in the species considered in this test. the mechanism of the antibacterial activity of lactobacillus strains usually appears to be multifactorial: the wellknown production of bacteriocins by l. sakei and l. curvatus strains, reported in many previous studies (castellano and vignolo, 2006; castellano et al., 2008; 2010) was not confirmed in our research. ! ital. j. food sci., vol 28, 2016 618 table 1: halos (in mm) expressed as a mean of three replication induced by lactobacillus curvatus mixture and lactobacillus sakei mixture against spoilage or pathogenic target microorganisms. target strains l. curvatus mixture l. sakei mixture escherichia coli atcc 25922 8.3±1.5 13.3±3.8 escherichia coli o157:h7 dsm 13526 14.0±2.6 10.0±2.0 proteus vulgaris atcc 8427 10.3±1.2 7.3±1.5 salmonella typhimurium atcc 14028 13.3±4.9 8.3±2.9 serratia marcescens atcc 14756 2.3±1.2 2.7±1.2 yersinia enterocolitica atcc 23715 19.7±3.1a 13.7±1.5b pseudomonas aeruginosa atcc 27853 21.3±3.5a 10.0±5.0b pseudomonas fluorescens atcc 13525 56.7±23.1 21.7±13.5 pseudomonas putida atcc 49128 30.0±34.7 17.3±2.5 listeria monocytogenes atcc 7644 14.0±3.6 10.7±1.5 listeria innocua atcc 33090 11.7±5.7 9.7±2.3 staphylococcus aureus atcc 6538 13.7±1.5 12.7±1.5 values are expressed as mean ± standard deviation. different lower-case letters are pointing out significant difference ( p<0.05) at the levels of interest of experimental mixtures by target strains. the potential antagonistic activity of lab towards spoilage microorganisms is strongly favored by the packaging technique: actually, vacuum or modified atmosphere packaging are the main methods for distribution and commercialization of fresh meat. these systems help the extension of shelf-life limiting the replication of enterobacteriaceae and pseudomonas spp., bacterial communities that often dominate aerobic spoilage of fresh meat at temperatures between -1 and 25°c. dominating microflora composed by lab contribute to the quality of meat thanks to their carbohydrate and protein catabolism. as underlined also from our results, the inhibitory properties of lab, are not only related to the production of organic acids (mainly lactic and acetic) or other compounds (organic acids, bacteriocins, hydrogen peroxide e.g.) but bioprotective actions are also due to the competition for nutrients. in fact, apart from the metabolic activity, starter cultures occupy vital niches, thereby discouraging the colonization of undesired microorganisms. in this context, lab interact with other microorganisms and with the environment, increasing their biomass at a rate that depends on the physical and chemical characteristics of the substrates: in chilled fresh meat, competition for a growth-limiting substrate such as glucose and oxygen interaction among species occurs (gill, 1976). in this context, the prevalence of certain species will be determined by their relative initial level, affinity for the substrates, substrate availability as well as the relative growth rate of the competing species at different temperatures (castellano et al., 2008). as a matter of fact, in order to obtain an important growth inhibition of the target strains, the presence of high loads of live and metabolically active cells, is fundamental. ! ital. j. food sci., vol 28, 2016 619 3.2. inoculation of vacuum-packaged meat slices lab cultures, and in particular l. sakei and l. curvatus, have been often studied for the application to food with good results thanks to the inhibition of pathogens and spoilage microorganisms and with the aim to extend the shelf-life of raw meat without important changes in its sensorial properties (castellano and vignolo, 2006). in this study, the antagonistic activity observed in vitro was also revealed on meat (fig. 1). considering the global effect of the application of l. sakei mixture to meat during the whole trial, tvc resulted significantly lower in treated samples (ls) if compared with the control ones (cls) (p=0.0089), reaching at the end of the trial the loads of 5.8±0.4 and 7.4±0.5 log cfu g-1, respectively. the addition of l. sakei mixture resulted, since the beginning of the trial, in a constantly higher level of lab in ls samples if compared with cls (p < 0.0001). in particular, in ls samples, lab reached the plateau level between 8 and 8.5 log cfu g-1 after only 20 days from the beginning of the experiment. in cls samples, the lab naturally present on the slices showed a rapid increase from the beginning until t20; afterwards, they reached a plateau level between 6.6 and 7.4 log cfu g-1. considering gram-negative bacteria for the whole period, ls samples values resulted to be significantly lower than cls ones (p = 0.0029). moreover, enterobacteriaceae resulted to be significantly lower in ls samples considering the whole trial (p < 0.0001). in particular ls samples showed a very stable trend (ls t0=2.3±0.5 vs t60=2.3±0.5 log cfu g-1), while in cls samples, enterobacteriaceae reached a value of 4.4±1.9 log cfu g-1 at t60; anyway such level of contamination is not generally associated to evident sensorial spoilage of raw meats. considering the effect obtained from the application of l. curvatus mixture to meat in the whole experimental period, tvc resulted significantly lower in treated samples (lc) than in control (clc) ones (p = 0.0013). the addition of l. curvatus mixture resulted, since the beginning of the trial, in a constantly higher level of lab in lc samples if compared with clc (p < 0.0001). in particular, in lc samples, lab reached the plateau level between 7.9 and 8.4 log cfu g-1 after only 20 days from the beginning of the experiment, according with ls results. in clc samples, the lab naturally present in the product, characterized in this case by a higher load if compared with cls (3.5±0.6 log cfu g-1 at t0), showed a rapid increase from t20; afterwards, they reached a plateau level between 6.1 and 7.5 log cfu g-1. considering gram-negative bacteria, the loads resulted to be quite comparable between lc and clc samples until t20 and then very highly fluctuant data were obtained; considering the whole period no significant differences were recorded (p = 0.3325). enterobacteriaceae resulted to be constantly lower in lc samples until t60, showing a general significant difference (p = 0.0225): in particular they showed a very stable trend for the whole study (lc t0=2.3±0.6 vs t60=3.0±1.5 log cfu g-1). in clc samples, enterobacteriaceae showed an increasing trend since the beginning of the trial, even if not reaching the threshold level of 5 log cfu g-1 (cls t0=2.5±0.8 vs t60=4.0±1.8 log cfu g-1). the effect of the inoculation with l. sakei mixture resulted generally more evident than the treatment with l. curvatus mixture, suggesting a better capability to adapt to vacuum packaged meat substrate. the better adaptation of l. sakei mixture (ls) confirmed the preponderance of l. sakei in long shelf-life vacuum packaged meat lab population, as highlighted in the previous study (stella et al., 2013). ! ital. j. food sci., vol 28, 2016 620 figure 1: results of total viable count (tvc), lactic acid bacteria (lab), gram-negative bacteria and enterobacteriaceae. cls= control samples for lactobacillus sakei; ls= samples inoculated a mixture of the six strains of lactobacillus sakei; clc= control samples for lactobacillus curvatus; lc= samples inoculated with a mixture of the two strains of lactobacillus curvatus. ! ital. j. food sci., vol 28, 2016 621 in any case, the capability of both of the two lab mixtures to inhibit the growth of spoilage bacteria, clearly demonstrated in vitro, was also confirmed on meat substrate, also if it resulted more limited. this could be explained by the different growth rates and competitiveness of the cultures if applied to a complex matrix like meat: the adaptation to a substrate depends especially on the metabolic activity of cultures, which occupy vital niches, thus discouraging colonisation of undesired microorganisms. generally, an antagonistic effect was detected both for l. sakei and l. curvatus treatments: despite the promising current knowledge and laboratory studies, lab strains often suffer from a limited effectiveness in foods; among the others, the main factors involved are the poor adaptation to food environment, the inactivation of antimicrobial compounds through proteolytic enzymes or the binding to food ingredients and the ph buffering action (holzapfel et al., 1995). in our case, the production of organic acids by the cultures inoculated on meat is supposable, even if their activity could be limited by the buffering capacity of meat. the metabolic activity of lab population of vacuum packaged meat could be deduced by the slight reduction of ph of meat during the trial, with a 0.25 and 0.39 decrease in meat treated with the two mixtures (ls: t0 = 5.48 vs t60 = 5.23; lc: t0 = 5.80 vs t60 = 5.51), very close to the decrease observed (0.30-0.44) in control samples (cls: t0 = 5.71 vs t60 = 5.27; clc: t0 = 5.75 vs t60 = 5.45). 3.3. determination of colour parameters the addition of lab mixtures did not result in an evident modification of meat colour. the multivariate analysis of the data evidenced some significant differences in whole meat colour between inoculated and control samples both for l. curvatus (t10 and t40) and l. sakei (t10, t30, t40 and t60). in any case, the analysis of the single colour parameters (table 2) did not give univocal results, with few significant differences but without any clear trend. however, the application of lab cultures did not negatively affect meat colour for the whole period considered. during the storage the hue value, a form of data reduction involving both a* and b*, and plottable in cylindrical coordinates when chroma and l* are known, is the main parameter used to attest the colour display life: in this study an increase of hue values between t0 and t10 was detected, afterwards they reached a stability level, without pointing out any significant difference among the series. chroma, also termed saturation index, used as an indicator of the loss of colour saturation, was characterized by a slight reduction during the whole period and did not show important differences between treated and control samples. in three sampling times (t10, t20 and t50) a perceptible total colour difference (expressed as ∆e) between ls samples and cls samples was not detected (∆e<2.3), in two sampling times (t0 and t30) this difference was acceptable (2.3<∆e<3), while in 2 sampling times (t40 and t60) the samples got ∆e >3, highlighting very strong, perceivable differences in meat colour (table 2). considering lc and clc, in four sampling times (t0, t20, t30 and t50) a perceptible total colour differences between ls samples and cls samples (expressed as ∆e) was not detected, in four sampling times (t0, t20, t30 and t50) this difference was acceptable (2.3<∆e<3), while just only in one sampling time (t40) the samples got ∆e >3. ! ital. j. food sci., vol 28, 2016 622 table 2: values of l*, a*, b* and hue angle values measured on ls, cls, lc and clc samples. samples t0 t10 t20 t30 t40 t50 t60 ls l* 44.5±1.7 44.2±1.5 43.1±2.1 42.5±5.5 43.7±2.8b 44.8±2.0 44.8±3.0a a* 23.1±2.2 20.8±3.6 20.1±1.2 19.7±1.6b 20.8±1.1a 16.9±2.1 16.9±1.1b b* 15.8±1.9 12.8±4.1b 13.3±1.0 13.0±1.0b 14.5±0.3 13.2±0.9 13.4±1.2 hue-angle 34.41 40.82 42.06 41.40 44.56 41.69 42.25 chroma 27.95 19.50 19.88 19.67 20.71 19.76 19.94 cls l * 44.6±3.5 43.5±1.9 41.7±4.3 44.4±1.9 51.0±1.3a 45.7±1.9 40.9±5.4b a* 20.9±1.6 21.7±0.8 19.8±1.1 23.1±3.8a 17.6±3.2b 17.4±1.5 20.0±1.2a b* 14.0±1.5 14.9±0.8a 12.4±2.1 15.0±2.2a 14.9±1.2 13.7±1.0 13.7±1.3 hue-angle 33.88 45.17 39.96 45.51 45.25 42.84 42.95 chroma 25.14 20.93 19.25 21.06 20.96 20.13 20.16 δe (ls-cls) 2.83 2.23 1.68 2.73 7.38 1.03 3.89 lc l* 39.7±3.5 42.0±1.6 44.8±3.8 47.2±3.1 46.3±1.8 42.5±1.4 45.4±6.0 a* 22.3±2.4 22.8±2.4 20.6±1.4 19.6±1.7 19.0±2.0b 19.3±1.9 21.1±1.8 b* 13.5±1.1 15.2±2.3a 14.8±1.1 14.5±1.3 13.1±2.2b 14.3±0.6 15.4±1.1 hue-angle 31.12 45.78 45.02 44.55 41.55 44.12 46.15 chroma 26.09 21.16 20.88 20.71 19.72 20.56 21.30 clc l * 40.7±3.2 42.0±4.9 44.5±1.8 48.2±3.5 43.8±3.1 42.8±3.6 48.2±4.4 a* 23.0±2.7 20.9±1.3 19.8±1.5 20.2±0.9 22.5±1.6a 21.0±4.0 20.1±1.2 b* 14.8±2.7 12.4±3.2b 13.4±1.4 14.5±1.2 15.1±1.6a 15.0±1.7 15.2±1.1 hue-angle 32.66 39.96 42.20 44.45 45.61 45.38 45.77 chroma 27.34 19.25 19.92 20.67 21.10 21.01 21.16 δe (lc-clc) 1.73 2.80 1.44 1.06 3.27 0.70 2.77 values are expressed as mean ± standard deviation. cls= control samples for lactobacillus sakei; ls= samples inoculated a mixture of the six strains of lactobacillus sakei; clc= control samples for lactobacillus curvatus; lc= samples inoculated with a mixture of the two strains of lactobacillus curvatus. different lower-case or upper-case letters are pointing out significant difference, respectively at p<0.05 or p<0.01 , at the levels of interest of lactobacillus mixtures by time. 4. conclusions historically l. sakei and l. curvatus have been recognized for their useful role in food biopreservation by contrasting the growth of spoilage and pathogenic microorganisms without the production of sensorial changes. l. sakei mixture and especially l. curvatus mixture tested in this work showed promising antimicrobial activity in vitro against a wide number of spoilage and pathogenic bacteria. no activity was recorded in the supernatants and in the ph adjusted supernatant, for all the target strains tested, highlighting that the antagonistic effect originates probably from the nutrient competitive exclusion. moreover, the effect of the addition of the two mixtures to sliced vacuum-packaged beef was investigated: the high loads detected on meat, that is a lesser inhibiting effect of the two bacteria on meat than that demonstrated in vitro, could be related to the slighter competitiveness of the cultures if applied to a complex substrate and to the buffering ! ital. j. food sci., vol 28, 2016 623 capacity of meat, which decreased the potential action of organic acids. the use of higher dosage of lab cultures could be suggested as an effective mean to determine an early conditioning of meat environment, in order to 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j. food sci., vol 28, 2016 536 short communication status of benzoic acid amount during processing from yoghurt to its by-product drink (doogh) zahra esfandiari1*, mohammad saraji2, roya alsadat madani1 and elham jahanmard1 1 department of research and development, department of food and drug, isfahan university of medical sciences, isfahan, iran 2 department of chemistry, faculty of chemistry, isfahan university of technology, isfahan, iran *corresponding author: research_esfandiary@mui.ac.ir abstract the addition of benzoic acid is forbidden in by-product drink of yoghurt "doogh" in iran. however, this preservative can be naturally found in milk and its products. a total of 24 and 48 samples of yoghurt and doogh were analyzed by hplc method to assess the natural and permitted amount of benzoic acid. all samples of yoghurt and doogh contained benzoic acid in mean amount of 1.5-5.0 mg/kg and 0.8-4.7 mg/ l, respectively. these findings showed that the amount of 6 mg/l can be defined for benzoic acid as admissible limit for doogh in iran. keywords: benzoic acid, yoghurt, doogh, admissible limit, hplc, iran   ital. j. food sci., vol 28, 2016 537 1. introduction doogh is a traditional fermented drink which is widely used in asia and produced from salt, set yoghurt and water (zamani mazdeh et al., 2014). this product is known with different names like "ayran" in turkey and "lassi" in india (yildiz et al., 2012; hingmire et al., 2009). similar to other acidified drinks, one of the main problems with doogh is microbial contamination leading to the spoilage and safety reduction of this product (elvira et al., 2014; sospedra et al., 2012). for this purpose, the antimicrobial preservatives such as benzoic acid and/or its salts are usually added to doogh to impart many advantages including increasing their shelf life, but the unpleasant effects of these additives have been reported in some surveys (zengin et al., 2011; kamankesh et al., 2013; zamani mazdeh et al., 2014). the use of chemical additives in different countries is restricted by the specific policies adopted. in iran, the scientific panel on the food additives in " food and drug administration: fda" and "institute of standards and industrial research of iran: isiri" has prohibited the use of benzoic acid in the dairy products. the existence of benzoic acid in those products leads to fine payment by the producers and suspension of the production licenses (isiri, 2008). recent evidences suggest the natural occurring of benzoic acid in yoghurt and its transfer to diluted and salted formulation prepared thereof (doogh) (esfandiari et al., 2013; zamani mazdeh et al., 2014; amirpour et al., 2015). this can cause the misinterpretation of the inspected results regarding the existence of the natural or added benzoic acid to doogh. in fact, the use of benzoic acid is not allowed in the dairy products in iran even though the presence of the naturally-occurring benzoic acid in the fermented dairy products has been reported by many workers (sieber et al., 1995; iammarino et al., 2011; hornickova et al., 2014). the source of benzoic acid can be hippuric acid as the natural compound in milk that changes to benzoic acid through the fermentation of the lactic acid bacteria in yoghurt (sieber et al., 1999). to author's knowledge, there have been no controlled study which determines the natural amount of benzoic acid in doogh. therefore, this project was undertaken to assess the level of benzoic acid as the natural and permitted value in doogh. these findings could help fda and isiri to reassess the status of the enacted rules for benzoic acid in doogh and resolve the legislative issue in iran. 2. materials and methods 2.1. samples set yoghurt samples (n=24) were taken from three commercial brands of the dairy processing plants (a, b and c; for each 4 samples) and small scale brands (d, e and f; for each 4 samples) with high sale in isfahan, iran. a total of 12 doogh samples were manually prepared by mixing 0.7, 40 and 59.3 g of salt (nacl), yoghurt sampled in the previous stage (for each brand 2 samples) and water, respectively. a sum of 36 doogh samples from three aforementioned brands (a, b and c; for each 12 samples) were purchased from the local food stores located in isfahan, iran. all samples were kept at 4º c before the examination would be begun. the analysis was performed in two replicates to determine the mean of the measurements for benzoic acid. 2.2. instrumentation and quantification a hewlett–packard 1090-ii liquid chromatograph (now agilent, waldbronn, germany) equipped with a diode array detector was used. the system was equipped with a   ital. j. food sci., vol 28, 2016 538 rheodyne 7725i injector with a 20-µl loop. the separations were carried out at the room temperature on a 3-µm, 150 × 3 mm i.d. hector-a c18 reversed-phase column (rstech co., south korea) was preceded by a guard column (4 ×4 mm, 5 µm) of the same packing material from merck (darmstadt, germany). the separation was performed isocratically using a mobile phase consisting of hplc grade methanol (40%, v/v) and 0.25 mm ammonium acetate aqueous solution (60%, v/v at ph = 4.5). the mobile phase flow rate was 0.4 ml/min and the detection was performed at 230 nm. the stock standard solution of benzoic acid (99%, merck) was prepared in methanol at 1000 mg/l concentration level. more diluted standard solutions were prepared in the pure water at the concentrations ranging from 1 to 50 mg/l. pure water was prepared by overseas equipment & services water purification system (ok, usa). estimation of benzoic acid amount was performed according to a previous reported method (guarino et al., 2011). a 2-g yoghurt sample was mixed with 5 ml methanol/water (35/65, v/v). for doogh analysis, 3 ml of the sample was mixed with 2 ml methanol. the mixture was ultrasonicated for 5 min. it was then heated at 50 ºc for 5 min and centrifuged in 2000 rpm (5 min). the clear supernatant solution was injected into hplc. 3. results and discussions the results corresponding to benzoic acid level in the industrial (a, b and c) and small scale brands (d, e and f) yoghurt samples are listed in table 1. the mean values of benzoic acid were in the range of 1.5-2.9 and 3.6-5.0 mg/kg in the samples collected from three yoghurt processing plants and small scale brands, respectively. our findings imply to the presence of benzoic acid in all samples of yoghurt. the surveys on the presence of benzoic acid in all yoghurt samples in various studies, performed by mihyar et al. (1999), el zeiny (2009), yildiz et al. (2012), cakir and cagri-mehmetoglu (2013) and amirpour et al. (2015), revealed that the ranges found were higher than those described in the current study. mihyar et al. (1999) showed that the content of benzoic acid was in the range of 10.6 to 1998.8 mg/kg of labaneh yoghurt in amman. el-zeiny (2009) also pointed out high concentration level of benzoic acid in the amount of 921 mg/kg in yoghurt ice dressing in saudi arabia. furthermore, the amount of benzoic acid is stated from 9.36 to 26.21 in all 25 yoghurt samples gathered from five cities in turkey (yildiz et al., 2012). in another study in turkey, a mean of 35.2 mg/kg of benzoic acid found in all 21 yoghurt (cakir and cagri-mehmetoglu, 2013). in a recent experiment by amirpour et al., (2015) in iran, the mean amount of 29.3±8.6 mg/kg of benzoic acid detected in all yoghurt samples collected from four different brands of the dairy processing plants. in contrast, the detection of benzoic acid was reported in 80% of yoghurt samples in turkey (koyuncu and uylaser, 2009). additionally, benzoic acid was not observed in yoghurt samples examined in spain, brazil and china (gonzalez et al., 1998; tfouni and toledo, 2002; wang et al., 2006). table 1 illustrates the benzoic acid value in the manually manufactured doogh. benzoic acid was found in the range of 0.8-2.2 and 2.6-4.0 mg/kg in doogh made from the yoghurt collected from the dairy processing plants and small scale brands, respectively. since doogh is a diluted and salted formulation prepared from yoghurt, processing yoghurt to doogh leads to a decrease in the benzoic acid content with amount of around 60-75% as table 1 shows the amount of benzoic acid was higher in yoghurt and doogh samples collected from the small scale brands. a probable explanation could be related to the lack   ital. j. food sci., vol 28, 2016 539 of controlling the quality criteria in the laboratory and proper hygiene practices as observed by the researchers. table 1: mean of benzoic acid concentration (mg/kg) in yoghurt and manually manufactured doogh (mg/l). sample brand yoghurt a (mean±sd) manually manufactured dooghb (mean±sd) a 2.9±0.13 2.2±0.34 b 1.7±0.14 1.2±0.02 c 1.5±0.10 0.80±0.10 d 4.0±0.39 2.6±0.34 e 3.6±0.12 2.6±0.21 f 5.0±0.21 4.0±0.25 astandard deviation calculated for 4 samples of yoghurt. bstandard deviation calculated for 2 samples of manually manufactured doogh. benzoic acid concentrations quantified in the industrially made doogh are revealed in table 2. the results demonstrated all samples presented benzoic acid with the levels ranging between 3 and 5.6 mg/l. the presence of benzoic acid in our study was near agreement with (yildiz et al., 2012 and esfandiari et al., 2013) that reported the content of benzoic acid in ayran and doogh samples in turkey and iran ranged from 1.54 to 16.57 and 0.94 to 9.77 mg/ l, respectively. akbari-adergani et al. (2013) and zamani mazdeh et al., (2014) pointed out the high concentration of benzoic acid with the mean of 195.9 and 21.3 in 27 and 130 doogh samples in iran. in similar study in iran, benzoic acid content in doogh samples purchased from four dairy brands including a, b, c and d were 22.2, 20.0, 19.4 and 21.5 mg/kg, respectively (amirpour et al., 2015). table 2: benzoic acid (mg/ l) level in the investigated doogh. sample brand concentration range mean±sd (n=12) a 3.9-4.2 4.0±0.1 b 3.0-5.2 4.3±0.7 c 4.0-5.6 4.7±0.5 the amount of benzoic acid in the industrially manufactured doogh was higher than manually ones. whereas manually manufactured doogh was prepared under the laboratory conditions and the industrially manufactured doogh was purchased from the local stores, it seems that the storage, handling and hygiene conditions in the dairy processing plants is plausible justification for this status. therefore, this part of the food chain requires continuous monitoring for good hygienic practice in the milk processing (smith, 2003). totally, the reason for the difference in the amount of benzoic acid in yoghurt and doogh of the present study and others is not clear but it may be attributed to the several factors including the feed of the milkproducing animal, the season of milking, the breeding   ital. j. food sci., vol 28, 2016 540 conditions, the content of hippuric acid in the raw milk, samples size, type of commercial lactic acid bacteria starter, processing technique, storage condition and type of yoghurt analyzed as mentioned in other studies (sieber et al., 1999; garmiene et al., 2008; qi et al., 2009; hornickova et al., 2014; javanmardi et al., 2015). the low level of benzoic acid found in the manually and industrially manufactured doogh indicates that this compound is as the indigenous constituent transferring from yoghurt to doogh. regarding with the codex standard act, the permitted amounts of benzoic acid as preservative used in the fermented milk drink is 300 mg/kg (codex, 2003). therefore the amount of benzoic acid detected in present study should not affect the public health. to sum up, the permissible amount of benzoic acid could define less than 6 mg/l in doogh. below this value, samples can be considered "acceptable" in iran because such a low concentration could originate from the natural endogenous formation of benzoic acid and not from the fraudulent addition. 4. conclusions an investigation of 72 yoghurt and doogh samples was carried out to define the natural occurring amount of benzoic acid. in the current study, it was found that all yoghurt and doogh analyzed contained benzoic acid in low level at less than 6 mg/l. this amount can be a permitted amount without having any harmful effect on the human health and considered as the admissible level for benzoic acid in doogh in the national act by iranian supervision authorities. moreover, it is suggested further studies on the continuous monitoring and the measuring of benzoic acid content in milk and its products to declare the amount of this compound on the labels of packaging. the inserted data on the labels of packaging can be useful for the exposure estimation of the consumers with this compound. acknowledgements sincere thanks are forward to research council of isfahan university of medical sciences for supporting the research project number 292227. references akbari-adergani b., eskandari s. and bahremand n. 2013. determination of sodium benzoate and potassium sorbate in "doogh" samples in post market surveillance in iran 2012. journal of chemical health risks 3:65-71. amirpour m., arman a., yolmeh a., akbari azam m. and moradi-khatoonabadi z. 2015. sodium benzoate and potassium sorbate preservatives in food stuffs in iran. food additives and contaminants: part b 8:142-148. cakir r. and cagri-mehmetoglu a. 2013. sorbic and benzoic acid in non-preservative-added food products in turkey. food additives and contaminants: part b 6: 47-54. codex standard. 2003. "codex standard for fermented milks. http:// www.codexalimentarius.org/input/download/standards/cxs_243e.pdf. accessed 26 july 2015. elvira l., duran c.m., urrejola. j. and espinosa f.r.m. 2014. detection of microbial contamination in fruit juices using non-invasive ultrasound. food control 40:145-150. el-zieny m. 2009. gc-ms analysis of benzoate and sorbate in saudi dairy and food products with estimation of daily exposure. journal of food technology 7:127-134. esfandiari z., badiey m., mahmoodian p., sarhangpour r., yazdani e. and mirlohi m. 2013. simultaneous determination of sodium benzoate, potassium sorbate and natamycin content in iranian yoghurt drink (doogh) and the associated risk of their intake through doogh consumption. iranian journal of public health 42:915-920.   ital. j. food sci., vol 28, 2016 541 garmiene g., salomskiene j., jasutiene i., macioniene i. and miliauskiene i. 2010. production of benzoic acid by lactic acid bacteria from lactobacillus, lactococcus and streptococcus genera in milk. milchwissenschaft 65:295-298. gonzalez m., gallego m. and valcarcel m. 1998. simultaneous gas chromatographic determination of food preservatives following solid-phase extraction. journal of chromatography a 823:321-329. guarino c., fuselli f., la mantia a. and longo l. 2011. development of an rp-hplc method for the simultaneous determination of benzoic acid, sorbic acid, natamycin and lysozyme in hard and pasta filata cheeses. food chemistry 127:1294-1299. hingmire s.r., lembhe a.f., zanjad p.n., pawar v.d. and machewad g.m. 2009. production and quality evaluation of instant lassi. international journal of dairy technology 62:80-84. hornickova s., dragounova h., hejtmankova k., michlova t. and hejtmankova k. 2014. production of benzoic acid in feremented goat's and sheep's milk. scientia agriculturae bohemica 4: 247-253. iammarino m., taranto a.d., palermo c. and muscarella m. 2011. survey of benzoic acid in cheeses: contribution to estimation of an admissible maximum limit. food additives and contaminants: part b 4:231-237. isiri. 2008. "institute of standards and industrial research of iran" 2th revision. no. 2453. doogh-specification and test method. javanmardi f., nemati m., ansarin m. and rafie s.a. 2015. benzoic and sorbic acid in soft drink, milk, ketchup sauce and bread by dispersive liquid-liquid microextraction coupled with hplc. food additives and contaminants: part b 8:32-39. kamankesh m., mohammadi a., modarres tehrani z., ferdowsi r. and hosseini h. 2013. dispersive liquid-liquid microextraction followed by high-performance liquid chromatography for determination of benzoate and sorbate in yogurt drinks and method optimization by central composite design. talanta 109: 46-51. koyuncu n. and uylaser v. 2009. benzoic acid and sorbic acid levels in some dairy products consumed in turkey. asian journal of chemistry 21: 4901-4908. mihyar g.f., yousif a.k. and yamani m.i. 1999. determination of benzoic and sorbic acids in labaneh by highperformance liquid chromatography. journal of food composition and analysis 12:53-61. qi p., hong h., liang x. and liu d. 2009. assessment of benzoic acid levels in milk in china. food control 20:414-418. sieber r., bütikofer u. and bosset j.o. 1995. benzoic acid as a natural compound in cultured dairy products and cheese. international dairy journal 5:227-246. sieber r., bütikofer u. and bosset j.o. 1995. benzoic acid as a natural compound in cultured dairy products and cheese. international dairy journal 5:227-246. smith g. 2003 “dairy processing, improving quality”. woodhead publishing limited, crc press, new york washington, dc. sospedra i., rubert j., soriano j.m. and manes j. 2012. incidence of microorganisms from fresh orange juice processed by squeezing machines. food control 23:282-285. tfouni s.a.v. and toledo m.c.f. 2002. determination of benzoic and sorbic acids in brazilian food. food control 13:117123. wang l., zhang x., wang y. and wang w. 2006. simultaneous determination of preservatives in soft drinks, yogurts and sauces by a novel solid-phase extraction element and thermal desorption-gas chromatography. analytica chimica acta 577:62-67. yildiz a., erdogan s., saydut a. and hamamci c. 2012. high-performance liquid chromatography analysis and assessment of benzoic acid in yogurt, ayran, and cheese in turkey. food analytical methods 5:591-595. zamani mazdeh f., esmaeili aftabdari f., moradi-khatoonabadi z., shaneshin m., torabi p., shams ardekani m.r. and hajimahmoodi m. 2014. sodium benzoate and potassium sorbate preservatives in iranian doogh. food additives and contaminants: part b 7:115-119. zengin n., yuzbasioglu d., unal f. and aksoy h. 2011. the evaluation of the genotoxicity of two food preservatives: sodium benzoate and potassium benzoate. food and chemical toxicology 49:763-769. paper received november 5, 2016 accepted april 11, 2016 ijfs#203_benjakul_bozza   ital. j. food sci., vol 28, 2016 480 paper effect of some amino acids on yield and characteristics of pacific white shrimp treated with alkaline soaking solution p. kingwascharapong and s. benjakul* department of food technology, faculty of agro-industry, prince of songkla university, hat yai, songkhla 90112, thailand *corresponding author. tel.: +66 74286334; fax: 66 74558866 e-mail address: soottawat.b@psu.ac.th abstract effects of alkaline soaking solution (ass), 0.75% naoh containing 2.5% nacl, ph 11.5, containing different amino acids at various concentrations on yield and characteristics of pacific white shrimp (litopenaeus vannamei) were studied. the lowest cooking loss with the highest cooking yield was obtained for the sample treated with ass containing 3% glutamic acid (p<0.05). when shrimp were treated with ass having 3% monosodium glutamate (msg) with the same mole equivalent to glutamic acid, the higher weight gain but slightly lower cooking yield were obtained (p<0.05). ass containing 3% msg had no effect on color and shear force of cooked shrimp. keywords: glutamic acid, monosodium glutamate, amino acids, alkaline, treatment, pacific white shrimp   ital. j. food sci., vol 28, 2016 481 1. introduction nowadays, pacific white shrimp (litopenaeus vannamei) is one of important species having a high market demand worldwide due to its appealed appearance, taste, flavor and texture (manheem, 2013). thailand is well known as the world largest shrimp producer, manufacturer and exporter. in 2013, shrimp was exported from thailand with the value of 28,617 million bahts (the customs department, 2013). shrimp processing, especially freezing, can lead to the denaturation or aggregation of proteins (carnetro et al., 2013). these changes can result in drip loss and sensorial changes in the product (gonçalves and ribeiro, 2009). furthermore, cooking also causes the changes in quality attributes, mainly by affecting textural and physicochemical properties and inducing weight loss (carnetro et al., 2013). the addition of water binding agent is therefore required in order to retain the quality of shrimp during processing. phosphate and bicarbonate have been widely used as water binding agents, which can increase water uptake and lower cooking loss (rattanasatherin et al., 2008; carnetro et al., 2013 and chatarasuwan et al., 2011). bicarbonate treatment could increase the weight gain of shrimp, mainly due to the repulsion between protein molecules mediated by its alkaline ph (chatarasuwan et al., 2011). due to the strict regulation of the uses of phosphate and bicarbonate in shrimp and shrimp products, an alternative additive with the property equivalent to both phosphate and bicarbonate are currently gaining attention for shrimp processing industry. some amino acids were reported to retard protein denaturation and retain protein functionality of frozen fish muscle (campo-deano et al., 2009; zhou et al., 2006). owing to the hydrophilic nature of some amino acids, their uses along with alkaline treatment could show the synergism in water uptake or water binding of shrimp muscle. amino acid with different side chains, pi and molecular properties might exhibit varying efficacy as processing aid for shrimp processing. monosodium glutamate (msg) is the sodium salt of glutamic acid. it has been used widely as a flavor enhancer with umami taste. since it is readily soluble in water, it can be used with ease for shrimp treatment and yield the product with improved properties. however, no information regarding the uses of amino acids and their salts as the processing aid for quality improvement of shrimp exists. the objective of this study was to investigate the impact of amino acids in conjunction with alkaline treatment on yield and characteristics of pacific white shrimp. 2. materials and methods 2.1. collection and preparation of shrimp pacific white shrimp (litopenaeus vannamei) (55-60 shrimp/kg) were purchased from a local market in hat yai, songkhla province, thailand. shrimp with storage time less than 6 h after capture were stored in the insulated box containing ice using a shrimp/ice ratio of 1:2 (w/w). the samples were transported to the department of food technology, prince of songkla university within 2 h. upon arrival, shrimp were cleaned using tap water. shrimp were peeled and deveined manually. prepared shrimp were placed in polyethylene bag and stored in ice until use.   ital. j. food sci., vol 28, 2016 482 2.2 effects of amino acids at various concentrations in alkaline soaking solution on weight gain, cooking loss and cooking yield of shrimp 2.2.1 preparation of treated shrimp shrimp (peeled and deveined) samples were mixed with alkaline soaking solution (ass; 0.75% naoh containing 2.5% nacl, ph 11.5) in the presence of glycine, glutamic acid or arginine at levels of 1, 2 and 3% (w/v) using the shrimp/solution ratio of 1:2 (w/v). the mixtures were stirred gently for 30 min at 4 °c and allowed to stand at 4 °c for 30 min. after treatment, the shrimp samples were placed on the plastic screen for 5 min (4 °c) to drain off solution. sample soaked in 2.5% nacl containing 3% mixed phosphates (sodium tripolyphosphate+tetrasodium pyrophosphate; 1:2, w/w) (manheem, 2013) and those treated with 2.5% nacl containing 0.75% naoh (ph 11.5) were used as the positive controls. those without soaking were used as the control. all shrimp samples without and with different treatments were divided into two portions. the first portion was used as raw shrimp and another portion was subjected to cooking. to prepare cooked shrimp, the samples were subjected to steaming until the core temperature of the second segment of shrimp reached 85°c. the samples were cooled rapidly in iced water for 1 min, and drained on a screen for 5 min at 4 °c. both raw and cooked shrimp samples were weighed and the weight gain, cooking yield and cooking loss were calculated. 2.2.2. analyses 2.2.2.1 determination of weight gain weight gain was determined by weighing the shrimps before and after soaking in the solutions. weight gain was calculated as follows: weight gain (%) = [(b-a)/a] × 100 where: a = initial weight (before soaking) b = weight after soaking, followed by draining 2.2.2.2 determination of cooking loss and cooking yield cooking loss and cooking yield were measured by weighing the shrimps before and after heating according to method of manheem et al. (2012). cooking yield and cooking loss were calculated by the following equations: cooking loss (%) = [(b-c)/b] × 100 cooking yield (%) = (c/a) × 100 where: a = initial weight (without soaking and steaming) b = weight after soaking, followed by draining c = weight after steaming, followed by cooling in iced water   ital. j. food sci., vol 28, 2016 483 2.3 effect of glutamic acid at various concentrations in ass adjusted to different phs on shrimp treatment shrimp (peeled and deveined) samples were mixed with ass (0.75% naoh containing 2.5% nacl) in the presence of glutamic acid at various concentrations (1, 2, and 3%) having ph 7 and 11.5. to adjust the ph of ass, 6 m and 2 m hcl was used. shrimp samples were treated with solutions and analysed for weight gain, cooking loss and cooking yield as described previously. 2.4 comparative study of glutamic acid and monosodium glutamate in ass on shrimp treatment 2.4.1. preparation of treated shrimp shrimp (peeled and deveined) samples were mixed with ass (ph 11.5) in the presence of glutamic acid or monosodium glutamate (msg) at various concentrations (1, 2, and 3%) with ph of 11.5. msg was used at the same mole equivalent to glutamic acid. samples were treated as mentioned above. both raw and cooked shrimp were analysed for weight gain, cooking loss and cooking yield. additional analyses were performed as follows: 2.4.2. determination of color color of raw and cooked shrimp were determined and expressed as l* (lightness), a* (greenness/ redness) and b* (yellowness/ blueness). the second segment of shrimp was subjected for measurement using a hunterlab colorimeter (hunter associates laboratory, inc., reston, virginia, usa) using a cie lab scale (young and whittle, 1985). 2.4.3. determination of shear force shear force of raw and cooked shrimp was measured using the ta-xt2i texture analyzer (stable micro systems, surrey, england) equipped with a warner-bratzler shear apparatus (brauer et al., 2003). the operating parameters consisted of a cross head speed of 10 mm/s and a 25 kg load cell. the shear force, perpendicular to the axis of the second segment muscle fibers, was measured. 2.4.4. determination of protein pattern of soaking solutions after being soaked with shrimp, the resulting soaking solutions were subjected to sdspage to determine the patterns of proteins leached out into solutions. sds-page was performed using 10% running and 4% stacking gels as described by leammli (1970). soaking solution (20 ml) was mixed with 10 ml of 10% (w/v) sds solution. the mixture was then homogenized at 11,000 rpm for 1 min. the homogenate was incubated at 85 °c for 1 h to dissolve total proteins. the sample was then centrifuged at 7,500 xg for 15 min to remove undissolved debris using a microcentrifuge (mik-ro20), hettich zentrifugan, tuttlingen, germany). protein concentration of the supernatant was determined by the biuret method (robinson and hogden, 1940). sample (10 µg protein) was loaded onto the gel consisting of 4% stacking gel and 10% separating gel. separation was performed by electrophoresis apparatus (mini-protein iii, bio-rad, usa) using 30 ma. protein was fixed and stained for 3 h in 1.25% coomassie brilliant blue r-250 in 40% methanol and 10% glacial acetic acid. gels were destained for 15 min with destaining   ital. j. food sci., vol 28, 2016 484 solution i (50% methanol and 7.5% glacial acetic acid) and with the destaining solution ii (5% methanol and 7.5% gracial acetic acid) for 3 h. wide range molecular weight standards were used and the molecular weight of protein was estimated. 2.4.5. sensory evaluation cooked samples were subjected to sensory analysis. the samples were evaluated by 30 panelists from the department of food technology with the age of 25-35, using the 9-point hedonic scale, where 9 = like extremely; 7= like moderately; 5 = neither like or not dislike; 3 = dislike moderately; 1 = dislike extremely (meilgaard et al., 1990). panelists were acquainted with shrimp consumption and had no allergies to shrimp. all panelists were asked to evaluate for color, appearance, flavor, taste, texture and overall likeness. samples were presented in the plates coded with three-digit random numbers. 2.5. statistical analyses a completely randomized design (crd) was used for the whole experiments. experiments were run in triplicate using different lots of shrimp samples. data were subjected to analysis of variance and mean comparison was carried out using duncan’s multiple range test. statistical analyses were performed using the statistical package for social science (spss 11.0 for window, spss inc., chicago, il, usa). 3. results and discussion 3.1 weight gain, cooking loss and cooking yield of pacific white shrimp treated with ass containing amino acids at various concentrations weight gain of pacific white shrimp treated with ass (ph 11.5) in the presence of amino acids at different concentrations (1, 2 and 3%) is shown in fig. 1a. weight gain of the treated shrimp increased, when the concentrations of amino acids in ass increased (p<0.05), except those treated with ass containing glutamic acid, in which weight gain decreased with increasing concentration (p<0.05). amongst all amino acids, glutamic acid showed the higher increasing effect on weight gain at low level (1%). under the alkaline condition, carboxyl groups, both at α-carbon and γ-carbon, were deprotonated and coo– became dominant. those negatively charged residues might penetrate into the swollen muscle along with ass. some coo– groups of glutamic acid could interact with positively charged domain of proteins via ionic interaction, while the rest of coo– groups were able to bind with water. as a result, the water could be retained in the muscle after treatment. nevertheless, in the presence of an excessive glutamic acid, those coo– groups in the solution (aqueous phase) more likely competed with muscle proteins in binding with water. as a consequence, the less water was retained in the shrimp muscle as indicated by the lowered weight gain, when the level of glutamic acid in ass was higher than 1%. glycine has been known as the smallest amino acid and has h atom as the side chain. due to its small molecule, it could migrate easily into shrimp muscle and subsequently interact with water by hydrogen bonding via side chains within muscle. additionally, carboxyl groups at α-carbon of glycine, which was deprotonated under the alkaline condition, could interact with water via ionic interaction. consequently, water could be more imbibed, particularly when the level of glycine increased. this was reflected by the increased weight gain of shrimp after being treated with glycine at higher concentrations.   ital. j. food sci., vol 28, 2016 485 for shrimp treated with ass containing arginine, weight gain increased as the levels of arginine increased (p<0.05). at ph 11.5, some carboxyl groups of arginine (pi=10.76) became deprotonated. those groups could interact with muscle proteins and simultaneously bound with water. it was noteworthy that ph of solution (11.5) was close to pi of arginine. therefore, coo– group of arginine in ass was present at low intensity. owing to the lower abundance of coo– group in ass containing arginine, water was less bound with proteins, in comparison with that containing glutamic acid. wolfenden et al. (1981) found that hydration potential of arginine was high at ph 7. at neutral ph, nh2 group mostly became protonated, in which nh3+ was formed. those positively charged groups effectively bound with water. however, in the present study, the ph of solution was 11.5, which was above pi (10.76). as a result, nh3+ was not present in ass, and nh2 group at the side chain became abundant. arginine is also reported frequently to form hydrogen bonds with other side chains and water molecule (borders et al., 1994). thus, charge of amino acid under the alkaline condition and the way those amino acids interacted with water and proteins in muscle more likely affected weight gain of treated shrimp. differences in weight gain among all treatments were plausibly governed by the differences in water binding or water holding capacity of different amino acids under the alkaline conditions. it was found that weight gain of shrimp treated with ass containing 3% arginine was higher than those treated with mixed phosphates and other samples (p<0.05). it is noted that treatment of shrimp with ass containing 1% glycine, 1% arginine or 3% glutamic acid led to the lower weight gain, compared with ass without amino acids. thus, amino acids in alkaline solution had varying impact on weight gain of shrimp after treatments. cooking loss and cooking yield of shrimp samples treated with ass containing different amino acids at various concentrations are shown in figs. 1b and 1c, respectively. cooking loss of shrimp decreased when the concentration of glutamic acid in ass increased (p<0.05). however, no differences in cooking yield were noticeable, when glycine at different levels was used (p>0.05). overall, when shrimp samples were treated with ass in the presence of all amino acids, the decrease in cooking loss was obtained, in comparison with those treated with only ass (p<0.05). the weight loss was in the descending order in samples treated with ass containing glycine, arginine and glutamic acid, respectively. shrimp treated using ass comprising 3% glutamic acid had the lower cooking loss than that treated with m-p (p<0.05). generally, the lowest cooking loss was found with the sample treated with ass containing 3% glutamic acid (p<0.05). when heat was applied, denaturation and coagulation of proteins were augmented, which in turn lowered water-holding capacity. moreover, the increased protein-protein interaction was obtained (niamnuy et al., 2008), leading to the enhanced release of water from shrimp muscle. after cooking, the cooking yield of shrimp with different treatments varied (fig. 1c), in which water molecules might be bound with amino acids or proteins in different fashions. the highest cooking yield was obtained with shrimp treated with ass containing glutamic acid, compared with other treatments (p<0.05). it was suggested that glutamic acid had the potential to bind water in shrimp muscle, when heat was applied. the efficacy in water holding during heating was dependent on concentrations used (p<0.05). however, the concentration of glycine had no effect on cooking yield of shrimp (p>0.05). efficiency in increasing cooking yield was in the descending order: glutamic acid, arginine and glycine. the negatively charged residues, especially carboxyl group at α-carbon and γ-carbon of glutamic acid are strongly hydrated (collins et al., 2007). the two amino acids that have the highest water-binding ability are aspartic acid and glutamic acid (low et al., 1978). an   ital. j. food sci., vol 28, 2016 486 ionic side chain of aspartic acid, glutamic acid and lysine has been claimed to bind 4-7 water molecules (zayas, 1997). figure 1: weight gain (a), cooking loss (b) and cooking yield (c) of pacific white shrimp treated with 0.75% naoh containing 2.5% nacl (ph 11.5) in the presence of amino acids at different concentrations. note: m-p: solution containing 2.5% nacl and 3% mixed phosphates (tetrasodium pyrophosphate and sodium tripolyphosphate, 2:1, (w/w)), ass: 0.75% naoh containing 2.5% nacl (ph 11.5), gly: glycine glu: glutamic acid, arg: arginine. different lowercase letters on the bars indicate significant differences (p<0.05). bars represent the standard deviation (n=3). 0 2 4 6 8 10 12 14 16 w ei gh t g ai n (% ) e cd c d d d f d b c a a b (a) 0 5 10 15 20 25 30 35 40 45 50 c oo ki ng lo ss (% ) h c g f f f c b a cd e de (b) 0 10 20 30 40 50 60 70 80 90 100 c oo ki ng y ie ld (% ) a b c c c d e f f g g h (c)   ital. j. food sci., vol 28, 2016 487 when comparing with the sample treated with ass alone, all samples treated with amino acids showed the increased cooking yield (p<0.05). only sample treated with ass containing 3% glutamic acid had the higher cooking yield than that treated with m-p (p<0.05). the sample treated with ass having 2% glutamic acid had similar cooking yield to those treated with mixed phosphate (p>0.05). therefore, glutamic acid in ass was shown to play a vital role in increasing the cooking yield by holding water in muscle during heating. 3.2 weight gain, cooking loss, cooking yield and physical properties of pacific white shrimp treated with ass containing glutamic acid at various concentrations and phs weight gain, cooking loss and cooking yield of pacific white shrimp samples after soaking in ass in the presence of glutamic acid at various concentrations with different phs (7.0 and 11.5) are shown in fig. 2. weight gain and cooking yield of shrimp samples soaked in ass, ph 11.5 were higher than those treated with ass at ph 7.0 (p<0.05), for all concentrations of glutamic acid used. at ph above pi or very alkaline ph, proteins had more negative charge, in which protein molecules repulsed each other, resulting in the swollen muscle structure (chantarasuwan et al., 2011b). as a consequence, water could be more uptaken into shrimp (chantarasuwan et al., 2011b). simultaneously, glutamic acid could be taken into the muscle along with nacl. glutamic acid might favor the water binding via its coogroup. weight gain was slightly decreased as the concentrations of glutamic acid used increased (p<0.05), regardless of ph. generally, weight gain of all samples was lower than that of samples treated with ass and with m-p (p<0.05), except that treated with ass containing 1% glutamic acid (ph 11.5) that showed the similar weight gain (p>0.05). among pacific white shrimp treated with sodium carbonate and sodium bicarbonate at various phs (5.5-11.5), the highest weight gain and cooking yield were observed in those treated at ph 11.5 (chantarasuwan et al., 2011b). therefore, ph of soaking solution was the prime factor determining weight gain of shrimp. cooking loss (fig. 2b) of shrimp decreased, when the concentration of glutamic acid in ass increased (p<0.05), irrespective of ph used. lower cooking loss was found as ass had ph of 11.5, compared with ph 7.0 (p<0.05). the decrease in cooking loss was in accordance with the increase in cooking yield. the lowest cooking loss was observed in shrimp soaked with ass containing 3% glutamic acid (ph 11.5) (p<0.05). glutamic acid could provide negative charge to muscle, thereby enhancing the water binding capacity of muscle proteins. bound water was held tightly during heating as indicated by the lowered cooking loss and increased cooking yield. aaslyng et al. (2003) suggested that a higher cooking loss was found in the sample with the low ph, whereas high water holding capacity was achieved at medium and high ph. when comparing the cooking loss of shrimp treated with mixed phosphates (m-p), the sample treated with ass containing glutamic acid at levels of 2 or 3% (ph 11.5) had the lower cooking loss (p<0.05). this reflected the high efficiency of ass containing glutamic acid at a sufficient amount in lowering the cooking loss. the augmented repulsion of muscle compartment also allowed glutamic acid with the high negative charge to penetrate into muscle and bind more water, thus resulting in the lowered cooking loss. in the presence of glutamic acid at 2 and 3%, no differences in cooking yield were obtained, compared with the sample treated with ass alone (p<0.05), when ph of soaking solution was 7.0 (fig. 2c). on the other hand, cooking yield of samples treated with ass containing glutamic acid at all levels were higher than that of sample treated with ass alone, when ph was 11.5 (p<0.05). the result indicated the paramount role of ph in water holding capacity of muscle when heating was implemented. as the shrimp were treated   ital. j. food sci., vol 28, 2016 488 with ass (ph 11.5) containing glutamic acid at levels higher than 1%, similar cooking yield was obtained, compared to that found in the sample treated with mixed phosphates (p<0.05). therefore, ass containing glutamic acid showed the potential in improving the cooking yield, and its efficacy was comparable to mixed phosphates. figure 2: weight gain (a), cooking loss (b) and cooking yield (c) of pacific white shrimp treated with 0.75% naoh containing 2.5% nacl with phs 7.0 and 11.5 in the presence of glutamic acid at various concentrations. note: m-p: solution containing 2.5% nacl and 3% mixed phosphates (tetrasodium pyrophosphate and sodium tripolyphosphate, 2:1, (w/w)), ass: 0.75% naoh containing 2.5% nacl (ph 11.5), glu: glutamic acid. different lowercase letters on the bars indicate significant differences (p<0.05). bars represent the standard deviation (n=3). 0 2 4 6 8 10 12 14 16 18 w ei gh t g ai n (% )     ph 7 ph 11.5 a c b cd de b f e (a) 0 5 10 15 20 25 30 35 40 45 c oo ki ng lo ss (% ) d a e b c f g c f     ph 7 ph 11.5 (b) 0 15 30 45 60 75 90 105 c oo ki ng y ie ld (% ) de d c c b e c f a     ph 7 ph 11.5 (c)   ital. j. food sci., vol 28, 2016 489 3.3 comparative study of ass containing glutamic acid and msg on shrimp treatment glutamic acid with high efficacy in increasing cooking yield was selected as the potential additive in ass. however, glutamic acid had low solubility. conversely, msg, a salt form, was cheaper and soluble with ease in water. msg was used at the same mole equivalent to glutamic acid for shrimp treatment. 3.3.1 weight gain, cooking loss and cooking yield weight gain of shrimp treated with ass containing glutamic acid at the levels of 1%, 2% and 3% or msg at the levels of 1%, 2% and 3% mole equivalent of glutamic acid is shown in fig. 3a. sample treated with ass containing 1 or 2% msg had similar weight gain to that treated with mixed phosphate (positive control) (p>0.05). it was postulated that the negatively charged glutamic acids from msg were able to bind tightly with water molecule via ionic interaction within protein compartment, leading to the increased water holding in shrimp muscle. it was found that msg showed higher ability in water holding than glutamic acid as indicated by higher weight gain. for cooking loss (fig. 3b), shrimp treated with ass containing msg had the higher cooking loss than those treated with glutamic acid counterpart, particularly at levels of 2 and 3% (p<0.05). the cooking loss was lower in samples treated with ass comprising both glutamic acid and msg at higher concentrations (p<0.05). for samples treated with mixed phosphate and ass alone, the cooking losses of 19.19% and 31.07%, respectively were observed. the cooking loss of 13.76-19.62% was obtained for sample treated with ass having glutamic acid, while cooking loss of 17.51-20.41% was found for the sample treated with ass containing msg. during cooking, muscle proteins underwent denaturation to a higher extent, while the amount of water retained in shrimp meat decreased with coincidental increase in fat and protein contents (manheem et al., 2012; benjakul et al., 2008). the result suggested that shrimp muscle could retain more water when glutamic acid and msg were incorporated in ass. however, glutamic acid showed the slightly higher ability in lowering cooking loss of shrimp, compared with msg, especially at level of 2-3%. for cooking yield (fig. 3c), the opposite results were observed, in comparison with cooking loss. the lowest cooking yield was observed in the control (without treatment). cooking yield of treated shrimp increased, when the concentrations of both glutamic acid and msg increased (p<0.05). similar cooking yield was found in shrimp treated with ass containing 2% glutamic acid or 3% msg (p>0.05). it was found that shrimp treated with ass containing 2 or 3% glutamic acid or 3% msg had the higher cooking yield than that of sample treated with mixed phosphates (p<0.05). since msg was more soluble and cheaper than glutamic acid, it was selected for treatment of shrimp. the appropriate concentration of msg in ass was 3%. 3.3.2 shear force shear force of raw and cooked pacific white shrimp treated with ass (ph 11.5) in the presence of glutamic acid and msg at different levels (1%, 2% and 3%) is presented in table 1. for raw shrimp, all treatments had no impact on shear force (p>0.05). it was noted that shrimp treated with ass containing glutamic acid and msg at all levels (1-3%) had similar shear force (p>0.05). all samples treated with ass containing either glutamic acid or msg had the similar shear force to those treated with mixed phosphates (p>0.05), except those treated with ass containing 3% glutamic acid, which showed the lower shear force (p<0.05). generally, shrimp tended to have the non-significant decrease in shear   ital. j. food sci., vol 28, 2016 490 force after treatment, particularly with increasing concentrations of glutamic acid and msg in ass used for treatment. when proteins imbibed water within their structure, the muscle had the lower resistance to the force applied. figure 3: weight gain (a), cooking loss (b) and cooking yield (c) of pacific white shrimp treated with 0.75% naoh containing 2.5% nacl (ph 11.5) in the presence of glutamic acid and msg at various concentrations. note: m-p: solution containing 2.5% nacl and 3% mixed phosphates (tetrasodium pyrophosphate and sodium tripolyphosphate, 2:1, (w/w)), ass: 0.75% naoh containing 2.5% nacl (ph 11.5), glu: glutamic acid. msg: monosodium glutamate. different lowercase letters on the bars indicate significant differences (p<0.05). bars represent the standard deviation (n=3). 0 10 20 30 40 50 60 70 80 90 100 c oo ki ng y ie ld (% ) a b cd c d d e e f (c) 0 5 10 15 20 25 30 35 40 45 c oo ki ng lo ss (% ) a b b c c cd d e f (b) 0 2 4 6 8 10 12 w ei gh t g ai n (% ) a b c c c d e e (a)   ital. j. food sci., vol 28, 2016 491 table 1: shear force of raw and cooked of pacific white shrimp treated with 0.75% naoh (ph 11.5) containing 2.5% nacl in the presence of glutamic acid and msg at various concentrations. treatments shear force (g) raw cooked no treatment 1970±177 �,abc 2194±326 b m-p 2057±212 bc 1578±341 a ass 1890±416 abc 2031±345 b ass+1% msg 2127±219 c 1549±130 a ass+2% msg 1995±284 abc 1525±334 a ass+3% msg 1794±275 abc 1472±83 a ass+1% glu 1972±329 abc 1555±205 a ass+2% glu 1665±253 ab 1515±126 a ass+3% glu 1618±243 a 1449±118 a �mean±sd (n=3). note: m-p: solution containing 2.5% nacl and 3% mixed phosphates (tetrasodium pyrophosphate and sodium tripolyphosphate, 2:1, (w/w)), ass: 0.75% naoh containing 2.5% nacl (ph 11.5), glu: glutamic acid. msg: monosodium glutamate. different lowercase superscripts in the same column indicate significant differences (p<0.05). for cooked shrimp, lower shear force was observed in all treated samples, in comparison with those without treatment and those treated with ass alone (p<0.05). nevertheless, all samples had the similar shear force, compared to those treated with mixed phosphates (p>0.05). when heat was applied, protein denaturation and aggregation took place. those phenomena resulted in the toughness as well as high resistance to force. no differences in shear force were found among shrimp treated with ass containing glutamic acid and msg at different concentrations used (p>0.05). myofibrillar proteins with increased negative charge favored the repulsion of polypeptide chains, which resulted in the swelling of muscle and became less resistant to shear force applied. when the concentration of glutamic acid or msg in ass increased, non-significant decrease in shear force was obtained (p>0.05). therefore, treatment of shrimp using ass containing glutamic acid or msg did not had the negative impact on textural property and their shear force was comparable to that of shrimp treated with mixed phosphates. 3.3.3 color color parameters (l*, a* and b*) of raw and cooked shrimp treated with ass (ph 11.5) in the presence of glutamic acid or msg at different concentrations are shown in table 2. for raw shrimp, l* value increased when concentrations of msg in ass increased (3%) (p<0.05). the a* value generally increased as the concentrations of both glutamic acid and msg in ass increased (p<0.05). basically, a* value indicates the reddish color. conversely, no changes in b* value were observed as the level of glutamic acid or msg in ass increased (p<0.05). thus, raw shrimp turned to be slightly reddish as the levels of glutamic acid or msg in ass increased. appearance of the product plays a significant role in maintaining high consumer acceptance (bono et al., 2012). glutamic acid or msg in ass might induce the change of proteins associated with carotenoid, named carotenoprotein. this led to the enhanced exposure of free carotenoids, especially astaxanthin, as evidenced by more reddish color. astaxanthin was reported as the major   ital. j. food sci., vol 28, 2016 492 pigment in shrimp meat (niamnuy et al. 2008). after cooking, no difference in color was observed among shrimp treated with ass containing glutamic acid or msg at all levels (p>0.05). it was obvious that l*, a* and b*-values of cooked shrimp increased, in comparison with raw counterparts. the increase in intensity of red color alter cooking is caused by muscle protein denaturation and the release of carotenoid pigment bound to the protein (carotenoproteins) (latscha 1989; niamnuy et al. 2008). the a*and b*values of samples treated with ass containing 2 or 3% glutamic acid were lower, compared with the control (no treatment). during soaking, some proteins including carotenoproteins were partially solubilized or leached out. as the results, less pigments were retained in the meat. coincidentally, the soaking solution was more reddish in color as the glutamic acid or msg concentrations in ass increased (data not shown). there was no difference in all color parameters between cooked shrimp treated with ass containing glutamic acid or msg at all levels and those treated with mixed phosphates. table 2: color of raw and cooked of pacific white shrimp treated with 0.75% naoh (ph 11.5) containing 2.5% nacl in the presence of glutamic acid and msg at various concentrations. samples treatments l* a* b* raw no treatment 46.46±1.58 ab -1.64±1.05 a 0.12±1.91 cd m-p 46.33±3.43 ab -1.69±0.36 a -3.23±1.79 a ass 44.96±2.10 a -1.66±0.34 a -2.01±1.61 ab ass+1% msg 45.42±2.58 a 0.27±0.96 b -1.13±0.48 bc ass+2% msg 46.81±2.10 ab 1.28±1.13 bc -0.88±1.28 bc ass+3% msg 48.87±2.51 b 2.30±1.15 d 0.16±1.96 cd ass+1% glu 45.22±2.39 a 0.89±1.51 b -0.02±1.63 cd ass+2% glu 46.09±0.88 a 2.01±0.74 cd 0.08±1.95 cd ass+3% glu 46.35±1.22 ab 2.77±0.87 d 1.19±2.71 d cooked no treatment 70.02±4.04 c 13.18±4.40 d 16.34±3.98 b m-p 65.50±4.30 ab 8.55±2.45 ab 12.37±4.63 a ass 69.35±5.37 bc 11.85±4.02 cd 13.39±3.20 ab ass+1% msg 65.09±2.40 a 11.17±2.77 abcd 14.34±3.86 ab ass+2% msg 64.77±3.95 a 8.86±1.31 ab 11.28±3.63 a ass+3% msg 63.17±4.55 a 8.27±1.94 a 10.92±3.18 a ass+1% glu 65.50±3.75 ab 11.32±1.91 bcd 15.64±3.06 ab ass+2% glu 64.94±2.38 a 9.69±1.60 abc 14.40±3.13 ab ass+3% glu 64.44±2.05 a 9.04±1.30 abc 11.78±2.18 a �mean±sd (n=3). note: m-p: solution containing 2.5% nacl and 3% mixed phosphates (tetrasodium pyrophosphate and sodium tripolyphosphate, 2:1, (w/w)), ass: 0.75% naoh containing 2.5% nacl (ph 11.5), glu: glutamic acid. msg: monosodium glutamate. different lowercase superscripts in the same column under the same state of sample indicate significant differences (p<0.05). 3.3.4 protein patterns of soaking solutions protein patterns of soaking solutions after shrimp treatments are shown in fig. 4. band intensity of myosin heavy chain (mhc) and actin slightly increased as concentration of both glutamic acid and msg in ass increased. the result suggested that more mhc and actin were solubilized and leached out to solution to a higher extent when glutamic acid and msg levels in ass increased. the increase in mhc band intensity in soaking   ital. j. food sci., vol 28, 2016 493 solutions was in agreement with the higher cooking yields (fig. 3c). protein extraction and dissociation of myofibrillar proteins were mainly due to the ionic effect and ph alteration (bendall, 1954). apart from mhc and actin, protein with mw of 25.6 and 18.2 kda also increased with increasing levels of glutamic acid and msg in ass. chantarasuwan et al. (2011) reported that pacific white shrimp soaked in sodium bicarbonate and sodium carbonate solution had the increase in band intensity of mhc as ph of soaking solution increased. protein patterns of ass and ass with mixed phosphates after shrimp soaking were similar, in which actin and protein with mw of 18.2 kda were dominant. after those proteins were leached out from shrimp muscle, the soaking solution could be more penetrated to looser muscle compartment and retained inside the muscle as indicated by higher weight gain and cooking yield. figure 4: protein patterns of soaking solution (0.75% naoh and 2.5% nacl, ph 11.5) in the presence of glutamic acid and msg solutions at different concentrations after soaking with shrimp. m, standard marker; m-p: solution containing 2.5% nacl and 3% mixed phosphates (tetrasodium pyrophosphate and sodium tripolyphosphate, 2:1 (w/w)), ass: 0.75% naoh containing 2.5% nacl (ph 11.5), msg: monosodium glutamate, mhc: myosin heavy chain. numbers indicate the concentration of glu or msg (%). 3.4 sensory properties of cooked shrimp treated with alkaline soaking solution containing msg sensory properties of cooked shrimp without and with different treatments are shown in table 3. the lowest likeness score for all attributes tested except flavor was found in the control sample (without treatment) (p<0.05). during heating, the shrinkage of muscle of shrimp occurred, resulting in more compact in microstructure of shrimp with less juiciness. among all samples, ass +3%msg sample showed the highest score for all attributes except for flavor that showed the lower score than the control and m-p sample m   1 %   m -p   2 %   3 %   1 %   2 %   3 %   a ss   ass+ glu ass+ms g mhc   actin   25.6 kda   18.2 kda 12.6 kda 97   30   66   20.1   45.5   14.4   k da     ital. j. food sci., vol 28, 2016 494 (p<0.05). this might be associated with fishy odor/flavor developed in of ass +3%msg sample. msg is sodium salt of glutamic acid and provides a flavoring function similar to naturally occurring free glutamate in foods (yamaguchi and ninomiya, 2000). monosodium l-glutamate (msg) has been used as a flavor enhancer since 1908, when it was identified as the source of umami taste (pleasant savory taste) (imada et al., 2014). since ass +3%msg samples had the high water holding capacity, more water was retained in shrimp meat as shown by a high cooking yield (fig. 3). decreased toughness with high juiciness contributed to the higher likeness score for texture and appearance. for color likeness, the highest score was observed for ass +3%msg sample (p<0.05). treatment of shrimp using 0.75% naoh containing 2.5% nacl and 3% msg (ass+3%msg) rendered the resulting cooked shrimp with the highest overall likeness score (p<0.05). however, the score was similar to that of shrimp treated with mixed phosphates (p>0.05). table 3: likeness score of cooked pacific white shrimp with different treatments. attributes no treatment m-p ass ass+3% msg appearance 5.50±1.25 �,a 7.76±0.66 c 7.00±0.98 b 8.00±0.74 c color 6.03±1.56 a 7.76±0.66 c 7.07±1.01 b 7.87±0.82 c flavor 7.10±1.03 b 7.31±1.13 b 6.80±1.19 ab 6.40±1.57 a texture 5.57±2.10 a 7.55±0.81 bc 7.00±1.74 b 7.80±0.85 c taste 5.80±1.85 a 7.83±0.73 b 7.73±0.78 b 8.13±0.73 b overall 5.77±1.45 a 7.76±0.85 bc 7.40±0.81 b 8.07±0.69 c �mean±sd (n=3). note: m-p: solution containing 2.5% nacl and 3% mixed phosphates (tetrasodium pyrophosphate and sodium tripolyphosphate, 2:1 (w/w)), ass: 0.75% naoh containing 2.5% nacl (ph 11.5), ass+3% msg: 0.75% naoh containing 2.5% nacl (ph 11.5) in the presence of 3% monosodium glutamate. different lowercase superscripts in the same row indicate significant differences (p<0.05). 4. conclusion amino acids in ass had the pronounced impact on quality improvement of pacific white shrimp. glutamic acid showed the marked effect on increasing cooking yield, while arginine could increase weight gain effectively. ph of ass played a paramount role in water uptake and lowering the cooking loss of treated shrimp. msg, the water soluble salt, showed the comparable impact on quality improvement to glutamic acid. glutamic acid or msg at higher level in ass enhanced the solubility of mhc and actin, facilitating the migration of soaking solution and water holding capacity of shrimp muscle. cooked shrimp treated with ass containing msg had the increased overall likeness score, but showed less score in flavor associated with the slightly fishy odor. the use of ass containing 3% msg was recommended as the promising soaking solution for shrimp treatment. acknowledgement this work was supported by national research council of thailand and prince of songkla university, thailand. the trf distinguished research professor grant was also acknowledged for the financial support.   ital. j. food sci., vol 28, 2016 495 references aaslyng d.m., bejerholm c., ertbjerg p., bertram c.h. and andersen j.h. 2003. cooking loss and juiciness of pork in relation to 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35 (1): 26–40 nutrigenomics: linking food to genome asma jabeen1, geetika malik1, javid iqbal mir1, rozy rasool2* 1central institute of temperate horticulture, indian council of agricultural research, srinagar, kashmir, india; 2department of entomology, punjab agricultural university, ludhiana, punjab, india *corresponding author: rozy rasool, punjab agricultural university, ludhiana, punjab, india. email: roziarasool24@ gmail.com received: 25 july 2022; accepted: 28 november 2022; published: 26 january 2023 © 2023 codon publications open access review abstract nutrigenomics has an undoubtedly immeasurable potential for revamping human health. it has become an important regimen due to its consequential role in medical and nutritional sciences. it is an interdisciplinary science that amalgamates the information from physiology, pathology, genetics, molecular biology, and nutrition to establish the effects of ingested nutrients on expression and regulation of genes. the ultimate aim of nutrigenomics is to ascertain the nutritional requirement of an individual in accordance with genetic makeup. moreover, it aims to purvey treatment in the management of certain ailments having a dietary role based on individual’s genomic profile. therefore, vast research in the field of nutritional genomics is a dire need to make people aware regarding their health and diet relationship. here, we have given an overview of nutrigenomics coupled with novel technologies to produce utilitarian information for health professionals and researchers by divulging certain properties that interfere with the genomic machinery. keywords: gene; interaction; micronutrients; nutrigenomics; nutrition introduction although our connection with food is complicated and ever-evolving, our desire for it is fundamental (grayson, 2010). influence of diet and environment on the health of an organism is not a new concept (emilien and hollis, 2017). nature alone cannot contribute to molecular mechanism that eventually governs the health of a human being (mead, 2007). the exogenous environmental factors that also include dietary nutrients and the genetic makeup of an individual play an important role in expression of a phenotypic trait via the central dogma of biology (reddy et al., 2017). over the 20th century, nutritional scientists focused on the discovery, use, and administration of vitamins and minerals in order to treat the deficiency diseases such as scurvy in the case of vitamin c deficiency; colon cancer, heart disease, and immune dysfunction due to vitamin e deficiency; colon cancer, heart disease, and immune dysfunction due to folic acid deficiency; kwashiorkor and marasmus due to protein deficiency; nerve problem and memory loss due to deficiency of niacin (dwivedi et al., 2014; mitra et al., 2005). malnutrition has repeatedly been shown to be common among hospitalized patients in modern developed societies. we usually mention that in the developed countries such as the usa, uk, spain, the netherlands, and canada, due to a wave of non-communicable diseases, there has been a shift from infectious disease predominance to communicable disease epidemic. so, the center of interest of nutritional science and modern medicine shifted to investigation on optimization and maintenance of homeostasis at all levels, namely, cellular, tissue, organ, and at whole body (afshin et al., 2019; mohan et  al.,  2007; raj et  al.,  2007). this requires the basic understanding of the effect of nutrients at molecular level, involving nutrient-related interactions at the genome, proteome, and metabolome levels of individuals, which led to the shifting of nutrition research from italian journal of food science, 2023; 35 (1) 27 nutrigenomics makeup or genomic profile, development of new drugs would become the gold standard (brand et  al., 2021). a renowned biologist and director professor yvonne dragon, division of systems toxicology, believes that new biological understanding will come from familiarity of genes, proteins, and metabolites to fight common diseases (kumar, 2007a). role of food in disease prevention and management in recent years, food has become the central dogma for nutritionists in establishing interaction between lifestyle associated metabolic diseases. reconsideration of “food as medicine” by general public, scientific developments in health sector, and demand for disease-combating diet has opened new area for intervention in health-related issues, which allows individuals to improve their health and prevent diseases (ohlhorst et  al., 2013). dietary fibers have a protective effect against bowel cancer (kelley et  al., 2007). growth of colonic tumors in both invitro and in-vivo systems is inhibited by consumption of fish oil which is rich in omega-3 fatty acids (syvertsen et al., 2007). fruits and vegetables rich in bioactive components can prevent carcinogenesis by several mechanisms such as blocking metabolic activation through increasing detoxification. detoxification enzymes such as flavonoids, phenols, isothiocyanates, allyl sulfur compounds, indoles, and selenium can be modulated on consuming plant foods (fuller, 1992; mitall and garg, 1992). intake of proper diet with sufficient minerals and vitamins that are involved in regulatory and enzymatic processes reduces the risk of cancer. the deficiency of these micronutrients may lead to abnormalities. for example, zinc and folate are involved in dna repairing process. further natural compounds from plant source such as apigenin (celery, parsley), curcumin (turmeric), epigallocatechin-3-gallate (green tea), resveratrol (red grape, peanuts, and berries), genistein (soybean), and allyl sulfur (garlic) have been reported to affect the cell cycle by different mechanisms (farouque et al., 2006). personalized nutrition nutrigenomics contribute in the designing of optimized dietary intervention strategies to restore and improve metabolic homeostasis, improve health and wellbeing, and prevent diet-related diseases. however, personalized diets that could be uniquely tailored according to the specific demands of a given individual considering his/her genetic background, lifestyle, and history are also included here. the speed of penetration of this new science in daily life is expected to come from both the diversity and complexity of food and food practices and the physiology to molecular biology and genetics, giving rise to a new approach of nutrigenomics (neeha and kinth, 2013). earlier, nutrigenomics was defined as “the effect of nutrients on the expression of an individual’s genetic makeup” (mead, 2007; riscuta, 2016). recently, the definition was broadened to understand the influence of nutrients on the genome, transcriptome, proteome, and metabolome (ronteltap et  al., 2009). it involves characterizing the gene products, their physiological functions, and interactions (neeha and kinth, 2013). it also analyzes the effects on gene expression due to the bioactive food components (nutrients and non-nutrients) (alagawany et al., 2022; koziołkiewicz, 2011). bioactive food components act as dietary signals (chadwick, 2004) and from a nutrigenomics point of view, cellular sensor systems such as peroxisome proliferated activated receptor γ (pparγ) and retinoid x receptors (rxrs) recognize these dietary signals which influences the expression of genes, synthesis of proteins, and production of metabolites (amin et al., 2012). nutrigenomics seeks to understand how nutrition influences homeostasis due to these dietary signatures (müller and kersten, 2003). it also aims to understand the mechanisms that influence the genetic susceptibilities (van ommen and stierum, 2002) by identifying the genes that sway the risk of diseases related to diet on a genome-wide scale. origin of nutrigenomics with the advancement in science and technology, particularly after the completion of the human genome project, scientists appeared skeptical and started questioning like (1) are gene expressions and metabolic responses the outcome of interaction between genotype and environment (including nutrient)? (2) will metabolic processes sway the individual in response to gene expression? (3)  how does nutrient–gene interaction lead to the prescription of exact diets (cozzolino and cominett, 2013)? to answer the above questions, a new subject in the form of nutrigenomics came into existence (dauncey, 2012). the term “nutrigenomics” was first described by peregrin (2001) and subsequently appeared in a review published in the journal current opinion in biotechnology (van ommen and stierum, 2002). there are several problems with our health system. firstly, we actually treat people after they get sick, neither do much to keep them healthy nor understand what’s going on while they are healthy; and secondly, the one size fits all approach, that is, giving people the same treatment even though they are all individuals and may respond differently. human genetics so far has largely focused on explicit genetic diseases with possible genetic basis. there have been extremely limited therapeutic benefits, apart from some diagnostic applications. based on an individual’s genetic 28 italian journal of food science, 2023; 35 (1) jabeen a et al. nutrients behave as transcription factors after interacting with a receptor, which binds tightly to dna and intensely induces gene expression. for instance, as in the case of lac operon, which is by default turned off if lactose is not present in a cell (e.g., bacterial cell) (siddique et  al., 2009). the laci inhibitor gene binds with promoter region and as a result will not allow rna polymerase to transcribe, and hence no transcription of dna takes place. however, if lactose is present in the medium, it will interact and change the structural conformation of laci, thus losing its capacity to attach with the promoter. rna polymerase easily binds with the promoter to transcribe the structural genes (lacy, laca, lacz) to produce proteins such as β-galactosidase, permease, and transacetylase, respectively. these proteins help in the uptake and breakdown of lactose to release energy. 2. dna susceptibility to transcriptional machinery and regulation of chromatin structure nutrients can alter the structure of dna by a process known as epigenetics (park et  al., 2017). epigenetics denotes continual dna configurational changes without change in sequence that results in the alteration of expression of genetic material (table 1). the sustained effects of epigenetic mechanism are mediated by chromatin remodelling and methylation of dna (liu and qian, 2011). a methyl group is added to cytosine base to repress gene activity, or tails of histones are attached with combination of different molecules that alters activity of the dna wrapped around them and ultimately gene expression (dna methylation) (fuks, 2005). similarly active chromatin, that is, non-methylated cytosine and acetylated histones indicate that gene is active and hence transcription is possible. on the other hand, silent chromatin, that is, methylated cytosine and non-acetylated histones indicate that gene is silent and hence transcription is inhibited. the folic acid– rich diet influences dna methylation during folic acid metabolism and can switch off some important genes (colson et  al., 2017; crider et  al., 2012). dolinoy et  al. (2006) in an experiment reported the role of folate-rich diet in mother mouse, wherein methylated iap sequences of the offspring genome and the silencing of agouti gene resulted in normal brown color of child coat, whereas, folate-deficient diet resulted in yellow coat color, obesity, and tumors. 3. prevention of dna damage cytokinesis-blocked lymphocyte (cbmncyt) with micronucleus assay is commonly used with peripheral blood lymphocytes and is presently the best biomarker for nutritional genomic studies of dna damage. the extent of chromosomal dna damage is complexity of our metabolic systems. our diet is omnivorous and consists of a variety of plants, animals and their derived products, water, as well as fungi, yeasts, and a diversity of bacteria. in the past, many food compounds were dismissed because no obvious nutritional roles were known for them. most foods are a vastly complex and synergistic or non-synergistic mix of several nutrients and many other components, the importance of which is being unveiled step by step. we are exposed to these complex mixtures throughout life, and while our biochemical processes extract energy, building blocks, and regulators from food to enable us to grow and function properly they are, in addition, dependent on other factors, including physical activity, feeling and emotion, and social and economic factors (palou, 2007). nutrigenomics and related terms the nutrient requirements of different individuals differ due to considerable heterogeneity in the sequence of the human genome (ramos-lopez et  al., 2017). nutrigenetics explained this genetic difference as the key basis for diverse response to the diet among different individuals (elsamanoudy et  al., 2016; farhud and yeganeh, 2010). these differences may not be at whole gene level but rather can be at single nucleotide polymorphisms level (irimie et  al., 2019). thus, both nutrigenetics and nutrigenomics are two sides of a same coin (figure 1). it holds much promise for providing better nutritional advice by identifying the dietetic components having both detrimental and beneficial health effects. it also determines the necessity of individual nutritional requirement based on genetic makeup and understands the association between diet and chronic diseases (sales et al., 2014). this can occur in three ways: 1. regulation of transcription factors and transcription process figure 1. mechanisms of influence of nutrients on gene expression. nutrient absorption nutrient utilization food/nutrient tolerance nutrient requirement food/nutrient genetic variation n u tr ig e n e ti c s n u trig e n o m ic s genome evolution/selection genome mutation rate in-vitro genome viability genome programming gene expression italian journal of food science, 2023; 35 (1) 29 nutrigenomics a biomarker for gene amplification (figures 2 and 3). similarly, fenech (2012) also reported that dna damage can be caused due to deficiency or excess of nutrition and that the effects are of the same magnitude as that of many common environmental toxicants. diet-gene regulation/nutrients and gene expression nutrigenomics illustrates the influence of nutrients on gene or protein expression that is regulated at specific time under particular environment and also generates response that results from simultaneous functioning of gene or protein networks in regulation of related human disease or disorder. in recent time, there is more interest in nutrigenomics research to understand the effects of nutritious food and medication on metabolic disorders measured by the occurrence of micronuclei in lymphocytes. the accumulation of micronuclei in the cbmncyt assay is particularly sensitive to nutritional deficiency or excess. mead (2007) reported the association of micronuclei with nutrition and noted in studies that even moderate folate deficiency within the physiological range in cultured lymphocytes caused as much dna damage as 10 times the annual allowed limit of exposure to x-rays. basically, micronuclei originate from chromosomes that fail to engage the spindle during nuclear division or from acentric chromosome fragments and act as biomarkers of chromosome loss or chromosome breakage, respectively. by using a cytokinesis-blocking agent (cytochalasin-b), once-divided cells that can express this damage are identified as binucleated cells. within these binucleated cells, it is also possible to measure nucleoplasmic bridges, which arise from dicentric chromosomes and nuclear buds (nbuds), table 1. key plants elements with epigenetic modifications. sl. no. key plants bioactive element epigenetic functions reference(s) 1. apples phloretin demethylation, reexpression tsgs, hdac inhibition orlikova et al., 2012; paluszczak et al., 2010 2. broccoli isothiocyanates chromatin remodeling, activation of p21 gene, induces dna methylation and gene reactivation (cyclin d2) ho et al., 2009; hsu et al., 2011; myzak et al., 2004 3. cashew nuts anacardic acid hati, dna repair balasubramanyam et al., 2003; link et al., 2010 4. fenugreek rhaponticin dna repair and gene reactivation wani and kumar 2016 5. citrus hesperidin dnmt inhibitor fang et al., 2007 6. cinnamon coumaric acid dnmt inhibitor, increases p53 acetylation olaharski et al., 2005; stavinoha and vattem 2015 7. coffee caffeic acid dnmt inhibitor lee and zhu 2006 8. garlic allyl mercaptan histone acetylation, hdac inhibitor nian et al., 2008 9. grapes resveratrol dnmt inhibitor, induces dna demethylation and gene reactivation howitz et al., 2003; paluszczak et al., 2010; stefanska et al., 2010 10. soyabean genistein dnmt inhibitor, decreases hdac activities, increases acetylation at tsg bosviel et al., 2012; fang et al., 2005; majid et al., 2008 11. tea epigallocatechin gallate (egcg) dnmt inhibitor fang et al., 2003; lee et al., 2005 12. tomatoes lycopene induces dna demethylation and gene reactivation (gstp1, rarbeta, hin-1) king-batoon et al., 2008 13. red pepper capsaicin dnmt inhibitor srinivasan 2013 14. turmeric curcumin dnmt inhibitor marcu et al., 2006; shu et al., 2011 15. ginger gingerol reduces platelet aggregation and ldl atherogenic modifications tchombe et al., 2012 16. mango, blackberry gallic acid hati choi et al., 2009b 17. frequently found in fruits and vegetables quercetin dnmti, induces p16 promoter, demethylation and gene expression howitz et al., 2003; tan et al., 2009 18. red cabbage, red onion cyaniding dnmti paluszczak et al., 2010 19. found in walnuts, many other fruits and vegetables myricetin dnmti fang et al., 2007; lee et al., 2005; paluszczak et al., 2010 30 italian journal of food science, 2023; 35 (1) jabeen a et al. carbohydrates increase the expression of chrebp (carbohydrate response element-binding protein), which regulates lipid and glucose metabolism at the transcription level, thereby contributing to the development of metabolic diseases (lizuka, 2017). the chrebp plays a significant role in glucose intolerance, fatty liver, and dyslipidemia in humans (benhamed et al., 2012). effect of lipids on gene expression profound effects of fat on gene expression led to alteration in growth, differentiation of cells, and metabolism. lipids activate various transcription factors such as ppars, srebp-1, lxrs, hnf4, and fxr (table 2) responsible for lipogenesis, glucose metabolism, and so on. (georgiadi and kersten, 2012). however, ppar (peroxisome proliferator–activated receptor), an important transcriptional factor that is copiously expressed in enterocytes, mediates the effects of dietary lipids on gene expression (bordoni et al., 2021). besides regulating various functions such as intestinal nutrient transport and metabolism, intestinal cholesterol flux, intestinal motility, and lessening oxidative stress, ppar acts as a master regulator of fatty acid catabolism. so, ppar is therapeutically important for patients suffering from inflammatory bowel disease (reen et  al., 2015). the nutritional effect of these fatty acids verily depends on the level of saturation. the ω-3 polyunsaturated fatty acids as anti-inflammatory agents interact with genes involved in cancer and various inflammations, hence proving their role in nutrigenetics (deckelbaum et  al., 2006). they also have a significant effect on cardiac arrhythmia (brouwer et  al., 2002). similarly, oleic and linoleic fatty acids decrease plasma levels of low-density lipoprotein and cholesterol (sacks and katan, 2002). effect of proteins on gene expression protein kinase c (pkc), calcium ion (ca2+), and k+ channel proteins influence the normal level of insulin secretion by increasing atp to adp ratio through glucose metabolism and closes k+ atp channel, leading to depolarization of β-cells (szymczak-pajor and sliwinska, 2019). fekete and brown (2007) reported that protein rich diets result in the moderation of total body fat by causing a shortage of mrna which is essential for fatty acid synthase gene expression in the adipocytes. similarly, a low input of essential amino acids triggers reduction in synthesis and also alters the functions of norepinephrine and camp by means of a protein called chop (or c/ebp, also known as a stress-responsive transcription factor) (zhang et  al., 2002). it attaches with associated with lifestyle transition. dietary interventions are supposed to play essential role in maintenance of an individual’s well-being and immunity transformation. effect of carbohydrates on gene expression the simplest monosaccharide (glucose) in nature provides a best model of how living organisms deal effectively by developing regulatory mechanisms with a varying level of nutrients supply (lee et  al., 2008). glucose is the primary physiological stimulus in the β-cells of pancreas for the regulation of insulin synthesis and secretion (yamashita et  al., 2001). furthermore, expression of genes encoding lipogenic and glycolytic enzymes, for example, acetyl-coa carboxylase in liver is induced by glucose in presence of insulin (henquin, 2000). thus, it plays a key role in transcriptional regulation, though insulin and glucagon were long known as critical in regulating gene expression. (sanhueza and valenzuela, 2012). unbalanced diets increase the risk of developing chronic diseases by altering nutrient–gene interactions (fukasawa et  al., 2010). diets rich in excess figure 2. expression of micronuclei and nucleoplasmic bridges during nuclear division. figure 3. biomarkers scored in the cytokinesis-block micronucleus cytome assay. micronucleus formation nucleoplasmic bridge formation c y t o c h a l a s in -b b l o c k c y t o k in e s is b l o c k bnc with nbud, gene ampli cation bnc with mn, chromosome breakage or loss bnc with npb, dicentric chromosome apoptosis necrosis g 1 ,s,g 2 g 0genotoxic italian journal of food science, 2023; 35 (1) 31 nutrigenomics 1. transcriptomics the study of expression of genes at the mrna level is known as transcriptomics. there are several methods to contour gene expression, however, dna micro array is the most popular one. dna microarray technology has successfully measured the changes in genetic expression by evaluating the interactions between diet and genes. in this technique, mrna transcripts extracted from tissues are subjected to create complementary labeled strand of dna (cdna) through reverse transcriptase-in vitro transcription (rt-ivt). the labeled cdna is hybridized with a probe (complimentary strand) speckled on a glass or nylon substrate (array) containing a set of genes of known sequences. confocal fluorescent scanner is used to quantify the color intensity, and by examining the resulting image, raw data on gene expression is extracted. using specific software, raw data is analyzed to determine upand downregulation of genes with respect to control (figure 4). data obtained from microarray can be used by an investigator to establish which genes are expressed differently, that too at mrna level and thus may be able to design better treatment strategies (reecy et al., 2006). in comparison to the microarray technology, rna sequencing technology (rna-seq), a novel transcriptomics method has proven tremendous analytical capability for gene expression studies. rna sequencing technology holds great potential for studying nutrient–gene interactions and provides a comprehensive understanding of rna expression at gene expression enhancers and gets activated by means of stressful stimuli such as dna damage, cellular growth arrest, and hypoxia (ren et al., 2017). effect of micronutrients on gene expression micronutrients play a vital role in maintaining genome stability (table 3) by regulating dna synthesis, methylation, and repair (fenech, 2012). savini et al. (2016) and surendran et  al. (2018) communicated that identification of indispensable micronutrient-rich foods for dna replication and repair or for the prevention of genome damaging events is vital for maintaining proper health and metabolism in individuals. the amount of micronutrients that appear to be protective against genome damage varies greatly between foods (fenech, 2005, 2008), as well as between individuals, due to taste preferences (diószegi et  al., 2019; garcia-bailo et  al., 2009) or cultural or religious constraints (el-sohemy et  al., 2007). therefore, careful choices are need of the hour to formulate patterns of diet that are optimum for maintenance of genome health. tools of nutrigenomics new opportunities in the field of science are now possible with the discovery of various “omics” technologies discussed below, to aid in our understanding of the role and impact of various nutrients (nutrition) on individual’s health. table 2. transcription-factor pathways mediating nutrient–gene interactions. nutrients compounds transcription factors reference(s) carbohydrates glucose usfs, srebps, chrebp müller and kersten 2003; uyeda et al., 2002 fats cholesterol ppars, srebps, lxrs, hnf4, fxr müller and kersten 2003; edwards et al., 2000; horton et al., 2002 proteins amino acids c/ebps geiger et al., 2016 micronutrients: vitamin a rar, rxr müller and kersten, 2003; rahman et al., 2020; anitha et al., 2021vitamins vitamin d vdr vitamin k pxr calcium calcineurin/nf-ats schuster 2006; rahman et al., 2020 minerals iron irp1, irp2 zinc mtf1 other food compounds flavonoids er, nfκb, ap1 er, nfκb, ap1 er, nfκb, ap1 schuster 2006; baldwin 2019 xenobiotics car, pxr ap1, activating protein1; car, constitutively active receptor; c/ebp-caat, enhancer binding protein; chrebp, carbohydrate-responsive elementbinding protein; er, estrogen receptor; fxr, farnesoid x receptor; hnf, hepatocyte nuclear factor; irp, iron regulatory protein; lxr, liver x receptor; mtf1, metal-responsive transcription factor 1; nfκb, nuclear factor κb; nfat, nuclear factor of activated t cells; ppar, peroxisome proliferatedactivated receptor; pxr, pregnane x receptor; rar, retinoic acid receptor; rxr, retinoid x receptor; srebp, sterol-responsive element-binding protein; usf, upstream stimulatory factor; vdr, vitamin d receptor. 32 italian journal of food science, 2023; 35 (1) jabeen a et al. of bioinformatics data. this huge data gathering has a significant advantage in terms of speeding up our knowledge acquisition, which aids nutritionists in harnessing the molecular mechanisms of nutrition to improve production efficiency (wang et al., 2017) 2. proteomics proteome refers to the total set of proteins expressed in a given cell. proteomics studies are related to expression levels, structure of proteins, and biochemical activity. thus, it addresses the questions of nutritional bioefficacy and identifies as well as quantifies bioactive proteins and peptides. in addition to techniques such as western blot, enzyme-linked immunosorbent assay (elisa), immune histochemical staining, and mass spectrometry, it also uses protein specific biomarkers for rapid disease diagnoses (klopfleisch et  al., 2010). however, major platforms for proteomic studies of biomedical topics are elisa and ms-based protein assays. nowadays, malditof or tof-ms is an emerging technology for reliable and rapid diagnosis and microbial identification. microbiologists have reported using maldi-tof intracellular level in response to dietary interventions (hasan et al., 2019). the rna sequencing technique or workflow can be described in three main steps: tissue examination in the lab, bioinformatics analysis of sequence data, and biological interpretation table 3. role of micronutrients in maintaining genomic stability. sl. no. micronutrients role in genomic stability consequences of deficiency food uptake for remediation reference(s) 1. vitamin c and e prevention of dna and lipid oxidation breaking of dna strands, oxidative dna lesions and lipid peroxide adducts on dna tomato, brussels sprout, drumstick leaves, kale, chilli, coriander leaves krajcovicovákudlácková et al., 2006; sorensen et al., 2001 2. vitamin d antioxidant activity by increasing the level of glutathione in normal cells, induction of apoptosis in cancerous cells breaking of dna strands, chromosome breaks and oxidative dna lesions green vegetables, onion, chow chow schafer and cockfield 2019; pizzino et al., 2017 3. folate and vitamins b2, b6 and b12 methylation of dna, synthesis of dtmp uracil misincorporation in dna and dna hypomethylation beet root, potato, pepper, turnip, mushroom, garlic, cauliflower thomas and fenech, 2009 4. vitamin b3 (niacin) required as substrate for poly (adp-ribose) polymerase, which is required for cleavage and rejoining of dna and telomere impairment of dna repair, chromosome breaks, mutagen sensitivity carrot, turnip, celery, mushroom, beans halliday et al., 2010 5. zinc required as cofactor for cu/ zn superoxide dismutase, dna replication, zn finger proteins increased dna damage and chromosomal breakage. spinach, broccoli lewis et al., 2005; yan et al., 2008 6. iron required as component of ribonucleotide reductase and mitochondrial cytochromes reduced dna repair, increased tendency for oxidative damage to mitochondrial dna amaranthus, spinach, cabbage, carrot, beans zhang, 2014; canniatti-brazaca and germano, 2011 7. magnesium cofactor for various dna polymerases, required in nucleotide excision repair, essential for microtubule polymerization reduced fidelity of dna replication, dna repair and chromosomal segregation, survival of genomically aberrant cells green leafy vegetables hartwig, 2001 8. calcium plays an important role in chromosome segregation, apoptosis reduced dna replication and repair, survival of aberrant cells agathi, curry leaves henneke et al., 2017 figure 4. schematic representation of micro array technology. sample cell line cdna labelled image acquisition dataarray hybridization tissue/blood rt-ivt labellingmrna italian journal of food science, 2023; 35 (1) 33 nutrigenomics genetic and nutritional counseling or delivering nutrigenomics services major barriers in offering nutrigenomics services to the public include lack of proper education in nutrition, genetics, and practitioners (hudson et  al., 2007). most practitioners in the healthcare sector do not have enough expertise in molecular testing and clinical genetics as well as lack sufficient knowledge in nutritional science, which is required to infer nutrigenomics tests (murray adn botkin, 1995; roosan et  al., 2022). it is mostly true for physicians, who are usually hesitant to provide advice in nutrition-related matters to patients due to lack of sufficient confidence in their nutrition knowledge (kolasa, 2005). therefore, practitioners must know how to understand and communicate sensitive information during counseling to patients on nutrigenomics (kurzenhauser and hertwig, 2006). besides, sufficient knowledge about nutrition is required to advice patients about modification in their diets as well as how to quantify risk, to design test outcomes, and to deal with false negative or positive test results (marteau, 1999), as these are highly susceptible to false impression, even by healthcare professionals. nutrigenomics products nutrigenomics encourages different perspective on foods, healthy, quick to consume and digest, and easy to carry (ashwell, 2004). according to this, the nutrigenomics tools contribute to the search for novel, functional, bioactive compounds intended to have effects like drugs, but without being categorized as drugs (kruger and mann, 2003). functional foods these are very important groups of food having some important bioactive ingredients linked with growth, development, and prevention from diseases (utkina and karagodin, 2021). see table 4. in japan, nearly 350 food items have been accepted as functional, with each having specified health claims such as for high cholesterol, hypertension, diabetes, and hypotension (htun et al., 2017). the trends in the development of functional foods include certain minerals, vitamins, and antioxidants with reduced calorie content, lower fat content or with healthier fat content as well as having low glycemic index (garcia-casal, 2007). phytosterols, phytoestrogens, fructooligosaccarides, omega-3 fatty acids, and polyphenols are among others (nijveldt et al., 2001). because the inception of agriculture and livestock, genetics has been applied in food for improving the edible plant’s genome. either due to no load or less pesticides, these are considered as healthier and safer (alavanja et  al., 2004). some ms for a variety of purposes, including detection of antibiotic resistance, biological warfare agents, and blood and urinary tract pathogens, and in epidemiological studies (weng et al., 2021). 3. metabolomics the study which involves chemical processes in the metabolites is called metabolomics. various techniques including nuclear magnetic resonance (nmr) spectroscopy, gas chromatography-mass spectrometry (gc-ms), and high performance liquid chromatography (hplc) analyze the whole metabolism. these techniques also reflect the behavior of different patterns of genes to contribute to the knowledge of how food containing over or under nutrients or secondary metabolites, which can affect the health or illness of an individual (nielsen and el-sohemy, 2012). predominantly the two methods, namely, nmr and mass spectrometry are used for conducting metabolic studies to identify, quantify, display, and generate profiles of a number of metabolites with more robustness and sensitivity in a body fluid sample. the profiles are then run through monitors to generate data for interpretation (kumar, 2007b). potential issues associated with delivering nutrigenomics nutrigenomics offers important health benefits for individuals; however, it raises certain legal, ethical, and social issues mainly with respect to nutritional and lifestyle advice as well as how public may access nutrigenetic tests, as discussed below. genetic tests and managing nutrigenomics information nutrigenomics approach like other genome-based science requires personal genetic information (vineis and christiani, 2004). due to the personal nature of genetic information, it is important that the autonomy of a person regarding the decisions based on the information have to be respected (horne and vohl, 2020). this indicates that the information provided for testing purposes or research must be given without any fear or manipulation (stevenson, 1999). consent to genetic testing is usually linked with prospects of privacy, which involve liberty from unnecessary tests, and the ability to withhold information from third parties, namely, bio banks and electronic repositories, particularly in which samples are stored (anonymous, 2006). as per professional obligations, the genetic information is expected to remain a top secret between patients and healthcare professionals (joffe and herholdt, 2020). 34 italian journal of food science, 2023; 35 (1) jabeen a et al. dietary supplements diet is an important factor in cancer etiology and prevention. ayurvedic medicine prescribes many plant-based medicines for the treatment of cancer. turmeric has shown to be a potent antioxidant and antiinflammatory agent, with additional promise as a chemopreventive agent. many chronic diseases are polygenic and result from interaction between genes and environmental factors. dietary intervention based on nutritional requirement, nutritional status, and genotype (i.e., “individualized nutrition”) can be used for the prevention, control, or treatment of chronic diseases such as cvds, metabolic syndromes, and cancer (afman and müller, 2006). these disorders are partly mediated by chronic exposure to certain food components. for example, the association between the amount of calories (jenkins et al., 1998), the levels and types of vitamins, fat, and carbohydrates with atherosclerosis, diabetes, obesity, cancer, hypertension, and other chronic diseases has been demonstrated (kumar, 2007a). hca-sx or super citrimax, a novel derivative of hca (dried fruit rind of garcinia cambogia, also known as malabar tamarind), is a unique source of (−)hydroxycitric acid (hca). it is safe when taken orally and is bioavailable in the human plasma. under the experimental conditions, roy et al. (2004) demonstrated that hca-sx supplement has been observed to be conditionally effective organizations (environmental) still accuse that these foods act as poison for one’s health and as well as for the environment. however, so far there is no scientific evidence indicating any risks to the health of consumers (vidal, 2009). transgenic foods throughout the world, several transgenic foods have been commercialized (table 5), mostly in australia, china, the united states of america, and canada. the known ones are bt corn, having endotoxin derived from bacillus thuringiensis, which kills the larvae of stem borer insects and soybeans resistant to herbicide glyphosate (broderick et al., 2006). nutraceuticals nutraceuticals comprising “any nontoxic food extract supplement that has scientifically proven health benefits for both disease treatment and prevention” (defelice, 1995) provide protection against disorders, for example, cancer, obesity, high blood pressure, cardiovascular diseases (cvds), gastrointestinal tract disorder, type ii diabetes, inflammation, microbial, viral, and parasitic infections, psychotic diseases, spasmodic disorders, and ulcers. (abbasi et al., 2015). table 5. reported sanative effects of foods in different diseases. sl. no. foods diseases references 1. garlic, ginger, turmeric cloves cardiovascular and bone diseases rastogi et al., 2017 2. white brinjal, fenugreek, pomegranate type 2 diabetes khan et al., 2009 3. saffron, turmeric, basil obesity alappat et al., 2010 4. onion, mint, garlic, fenugreek neurodegenerative diseases and protection against oxidative damage to red blood cells hwang et al., 2009; kaviarasan et al., 2004 5. cardamom, cinnamon hypertension davis and yokoyama, 2011 6. black pepper, bay leaf gastrointestinal diseases speroni et al., 2011 7. mustard bladder cancer bhattacharya et al., 2010 table 4. transgenic crops approved for commercial use. sl. no. products altered trait company year trade name 1. tomato thicker skin and altered pectin content zeneca/petoseed 1995 2. potato ama1gene (improved protein content) monsanto company 2010 protato 3. squash resistant to viruses ashgrow seeds 1995 freedom ii 4. rape seed altered fatty acid composition calgene inc. 1995 laurical 5. papaya resistant to viruses cornell university 1997 6. corn bt cry protein monsanto company 1996 yieldgard 7. cauliflower introgression of ‘or’ gene (β-carotene rich) cornell university 8. soybean resistant to weedicide monsanto company 1995 roundup ready italian journal of food science, 2023; 35 (1) 35 nutrigenomics alavanja, m., hoppin, j. and kamel, f., 2004. health effects of chronic pesticide exposure: cancer and neurotoxicity. annual review of public health. 25: 155–197. https://doi.org/10.1146/ annurev.publhealth.25.101802.123020 amin, t., mahapatra, h., bhat, s.v. and gulleria, s.p.s., 2012. application of nutrigenomics in food industry: a review. indian journal of horticulture. 2(3–4): 54–59. anitha, a., viswambharan, v., thanseem, i., iype, m., parakkal, r., surendran, s.p., et  al., 2021. vitamins and cognition: a nutrigenomics perspective. current nutrition and food science. 17(4): 348–336. https://doi.org/10.2174/1573401316999200901180443 anonymous, 2006. united states government accountability office nutrigenetic testing: tests purchased from four websites mislead consumers. washington: us gao. pp. 1–27. ashwell, m., 2004. concepts on functional food. washington, dc: international life science institute (ilsi), ilsi press. 48 p. balasubramanyam, k., swaminathan, v., ranganathan, a. and kundu, t.k., 2003. small molecule modulators of histone acetyltransferase p300. journal of biological chemistry. 278(21): 19134–19140. https://doi.org/10.1074/jbc.m301580200 baldwin, w.s., 2019. phase 0 of the xenobiotic response: nuclear receptors and other transcription factors as a first step in protection from xenobiotics. nuclear receptor research. 6: 101447. https://doi.org/10.32527/2019/101447 benhamed, f., denechaud, p.d., lemoine, m., robichon, c., moldes, m., bertrand-michel, j., et al., 2012.the lipogenic transcription factor chrebp dissociates hepatic steatosis from insulin resistance in mice and humans. journal of clinical investigation. 122(6): 2176–2194. https://doi.org/10.1172/ jci41636 bhattacharya, a., li, y., wade, k.l., paonessa, j.d., fahey, j.w. and zhang, y., 2010. allyl isothiocyanate rich mustard seed powder inhibits bladder cancer growth and muscle invasion. carcinogenesis. 31(12): 2105–2110. https://doi.org/10.1093/ carcin/bgq202 bordoni, l., petracci, i., zhao, f., min, w., pierella, e., assmann, t.s., et al., 2021. nutrigenomics of dietary lipids. antioxidants. 10(7): 994. https://doi.org/10.3390/antiox10070994 bosviel, r., dumollard, e., déchelotte, p., bignon, y.j. and bernardgallon, d., 2012. can soy phytoestrogens decrease dna methylation in brca1 and brca2 oncosuppressorgenes in breast cancer? omics. 16(5): 235–244. https://doi.org/10.1089/ omi.2011.0105 brand, a., evangelatos, n. and özdemir, v., 2021. placebogenomics: a new concept and tool for personalized medicine and public health. omics. 25(2): 76–78. https://doi.org/10.1089/omi. 2020.0221 broderick, n., raffa, k.f. and handelsman, j., 2006. midgut bacteria required for bacillus thuringiensis insecticidal activity. proceedings of the national academy of sciences. 103(41): 15196–15199. https://doi.org/10.1073/pnas.0604865103 brouwer, i.a., zock, p.l., van amelsvoort, l.g.p.m., katan, m.b. and schouten, e.g., 2002. association between n-3 fatty acid status in blood and electrocardiographic predictors of arrhythmia risk in healthy volunteers. american journal of cardiology. 89(5): 629–631. https://doi.org/10.1016/s0002-9149(01)02314-1 in weight management and lowered abdominal fat leptin expression in experimental animals as well as in humans. in 2008, lau et al. showed that supplementation of hca and niacin-bound chromium (iii) is safe and efficacious for weight loss. conclusions and future perspectives the accurate and specific knowledge of dietary requirements might be useful to mitigate or prevent chronic diseases and also helps to understand the influence of nutrition on homeostasis, metabolic pathways and develop dietary-interventional strategies. nutrigenomics laid the foundation for understanding variability in preferences, requirements, and responses to diet by humans. it acts as a new tool for nutritional research by targeting the specific gene through nutritional manipulation to alleviate the problems or to get desired performance associated with health and production. this approach would provide longand short-term benefits to individual’s health by initiating the diet-specific new diagnostic tests against the unfavorable responses to diet, by identification of specific populations with specific nutrient requirements. in future, scientists will constantly contribute to develop new innovations in nutrigenomics research, which will certainly update our basic understanding of nutrient–gene relationship with the help of molecular technologies, especially next generation sequencing. therefore, future researches 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https://doi.org/10.1002/9783527626588.ch25 utkina, a.s. and karagodin, v.p., 2021. nutrigenomics as a tool for optimizing the composition of specialized food products by the efficiency criterion. iop conference series: earth and environmental science. 677: 042050. https://doi.org/10.1088/ 1755-1315/677/4/042050 uyeda, k., yamashita, h. and kawaguchi, t., 2002. carbohydrate responsive element binding protein (chrebp): a key regulator of glucose metabolism and fat storage. biochemical pharmacology. 63(12): 2075–2080. https://doi.org/10.1016/ s0006-2952(02)01012-2 van ommen, b. and stierum, r., 2002. nutrigenomics: exploiting systems biology in the nutrition and health arena. current 150 issn 1120-1770 online, doi 10.15586/ijfs.v33isp1.2092 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (sp1): 150–162 p u b l i c a t i o n s codon cooking methods affect eating quality, bio-functional components, antinutritional compounds and sensory attributes of selected vegetables nusrat maqbool1, sajad ahmad sofi1, hilal a. makroo1, shabir a. mir2, darakshan majid1* and b.n. dar1* 1department of food technology, islamic university of science & technology, india; 2department of food science & technology, government college for women, m. a. road, srinagar, jammu and kashmir, india *corresponding authors: b.n. dar, department of food technology, islamic university of science & technology, india. email: darnabi@gmail.com. darakshan majid, department of food technology, islamic university of science & technology, india. email: syed.darakshan@gmail.com received: 19 june 2021; accepted: 10 october 2021; published: 5 november 2021 © 2021 codon publications open access paper abstract the study aimed to evaluate the effect of boiling, air-frying and microwave methods of cooking on the phytochemical and antinutritional activity of some vegetables. total phenolic content was the highest in kale (9.70 mg gae/g) using air frying and in carrot using microwave (9.15 mg gae/g) and boiling (5.16 mg gae/g) methods. the cooking of vegetables for 15 min of air frying depicted a significant increase in total flavonoids. oxalate content in vegetables were significantly reduced by air frying, tannins by boiling and saponin in microwave cooking. a significant decrease in oxalate content was observed in kale by air frying and boiling methods, in carrot by microwave cooking, and reduction in tannins in tomatoes by air frying and boiling methods. keywords: antinutrients, beans, cooking, kale, phytochemicals, spinach introduction in recent years, consumption of food with bioactive ingredients with nutraceutical potential has increased in consumer diet. vegetables are one of the natural foods associated with numerous healthy ingredients and are recommended as a part of a healthy lifestyle for end users. vegetables, consumed as raw, cooked or in processed form, are parts of fresh plants which provide nutrition to humans (groch, 2008). consumption of vegetables in the human diet is associated with health benefits because of immunoregulatory and antioxidant properties to fight inflammatory, allergic, atherogenic, microbial, thrombotic, cardiovascular diseases, cancer and diabetes (rashmi and negi, 2020). vegetables are a rich source of fibers, vitamins, minerals and phenolic components (septembre-malaterreb et al., 2018). the presence of bioactive nutrients in vegetables is associated with health benefits to fight chronic diseases (eliassen et al., 2012; pojer et al., 2013). phytochemicals, such as flavonols, isoflavones, phenolic acids, flavones, and carotenoids, present in vegetables have demonstrated antioxidant, anti-inflammatory and antitumor effects (septembremalaterreb et al., 2018). besides phytochemicals, there are secondary metabolites such as antinutrients with a detrimental effect on nutrients and sensory properties of vegetables. antinutrients most commonly present in vegetables are lectins, tannins, phytic acids, saponins, phytates, glucosinolates, cyanogens and phytoalexins, with reduced bioavailability of essential components and inhibition of digestive enzyme activity (septembremalaterreb et al., 2018; kaur et al., 2015). kale, a leafy and nutritionally important vegetable of the family brassica, is consumed in different parts of the world (singh et al., 2009). kale is a good source of ascorbic acid, carotenoids, flavonoids and polyphenol-rich compounds with health-promoting phytochemicals having reduced mailto:darnabi@gmail.com mailto:syed.darakshan@gmail.com italian journal of food science, 2021; 33 (sp1) 151 cooking methods affect eating quality, bio-functional components, antinutritional compounds and sensory attributes the phytochemical and antioxidant activity of cooked vegetables. the present study proposes to use optimum time by applying boiling, air frying and microwave methods of cooking, and their effect on phytochemical and antioxidant activity of kale, spinach, beans, carrot and tomatoes. material and methods materials fresh kale (brassica oleracea var. acephala l.), spinach (spinacia oleracea l.), beans (phaseolus vulgaris l.), carrot (daucus carota l.) and tomatoes (solanum lycopersicum l.) were purchased from the vegetable market of kashmir valley. after proper removal of post-harvest nonedible parts, the edibles were soaked in clean water (25°c, ph 7), dried and diced into uniform slices. the dried vegetables were subsequently divided into four parts and treated with boiling, air frying and microwave cooking method. cooking treatments boiling: vegetable samples (100 g) were boiled at 100°c in a stainless steel pan with 150-ml distilled water for 2, 4 and 6 min until it becomes tender. air frying: vegetable samples (100 g) were fried at 200°c in an air fryer with 5 ml of hot refined oil for 7.5, 10 and 15 min. microwave: vegetable samples (100 g) were micro-cooked in 150 ml of water at 800-w power for 1, 2.5 and 5 min. the cooking was followed by an afterward tempering period of a few minutes at room temperature. the tempered samples were subjected to homogenization in a blender. the homogenized samples were packed in polyethene pouches, sealed and stored at -20°c for further analysis. proximate analysis the proximate composition of vegetable samples was determined according to standard procedures of the association of official analytical chemists (aoac, 2002) for moisture (method 925.09), fat (method 920.39), protein (method 955.04), crude fiber (method 962.09) and ash content (method 991.43). phytochemical and antioxidant activity of vegetables preparation of extract homogenized vegetable samples, 2 g, were extracted with 80% methanol (50 ml). then centrifugation of risk of heart disease, cancers and diabetes (chenard et al., 2015; ferioli et al., 2013; korus and lisiewska, 2011). spinach is a nutritious leafy vegetable containing a good amount of vitamins, dietary fiber, minerals and polyunsaturated fatty acid, a therapeutic ingredient used against arthritis, obesity, cancer, osteoporosis and anemia (bassey and khan, 2015; patricia et al., 2014). beans belong to the family fabaceae and are rich in proteins, starch, dietary fiber, minerals and vitamins (siddiq et al., 2010). beans have been associated with many health benefits because of dense and rich ingredients such as flavonoids, phenolic acids and isoflavones (aparicio-fernandez et al., 2005; granito et al., 2008; lin et al., 2008; chung et al., 2008). carrots are one of the most important vegetables with a potentially rich source of carbohydrates, minerals and carotenoids with anticarcinogenic, antioxidants, and immune-boosting properties (fiedor and burda, 2014; tanaka et al., 2012). tomato is a globally cultivated fleshy vegetable with dense and rich nutritive components, including vitamins, minerals and phytochemicals that impart health benefits (frusciante et al., 2007). tomato usage in the diet has been related to the reduction of inflammatory risk processes, cancer, cardiovascular disease, hypertension, diabetes and obesity (adams et al., 2011. vegetables with health beneficial ingredients are primarily consumed after processing using different cooking methods with aim of enhancing palatability, digestibility and taste. cooking treatment induces physical and chemical changes, structure, nutritional quality and texture of vegetables (rehman et al., 2003; tepe and ekinci, 2021). it also induces cell wall permeability and increases the extraction of phytochemicals and reduction in ant nutrients (rodrıguez-amaya, 1999). a significant loss of vitamins and phytochemicals has been observed during the cooking of vegetables that varies with different cooking methods (lin and chang, 2005). different cooking methods, such as boiling, microwave, steaming, griddled, frying, and baking, affect the structural and nutritional profile of vegetables (fabbrin and crosby, 2016; zhan and hamauzu, 2004). oven cooking, air frying, boiling, steaming, microwave treatment, steaming and vacuum cooking are different methods of cooking used for different vegetables to enhance their nutritional profile, bioactive potential, reduce the antinutritional ability and improve sensory acceptability (fratianni et al., 2021; nartea et al., 2021; rana et al., 2021; rinaldi et al., 2021; salamatullah et al., 2021; sun and ling, 2021). domestic methods of cooking, such as boiling, air frying and microwave cooking, have increased with an added interest in 152 italian journal of food science, 2021; 33 (sp1) maqbool n et al. minor modifications. cooked vegetable sample (1 g) was treated with 75 ml of 3-m sulfuric acid, stirred constantly for 1 h, followed by filtering through whatman filter paper no. 1; 25-ml filtrate aliquot was titrated with potassium permanganate (kmno4 0.05 m) solution till faint pink color appeared as endpoint. titration with 1 ml of 0.05-m kmno4 is related to 2.2 mg of oxalate (chinma and igyor, 2007) and expressed as mg/g of oxalate. tannin content the tannin content in vegetables was analyzed according to the standard procedure of aoac (2002) with minor modifications. the cooked vegetable sample (3  g) was extracted with distilled water for 4 h and filtered for tannin analysis. tannin extract amounting to 25 ml was treated with 25 ml of indigo solution. the mixture was diluted with 750 ml of distilled water and titrated with 0.1-n kmno4 solution until the blue color of the solution changed to green color. finally, tannin content in samples was calculated using the following equation: tanin (mg/g) = (v – v0) × 0.004157 × 250 × 100/g × 25, (2) where v and vo are volume of 0.1-n kmno4 solution for the titration of sample and blank (without the sample), respectively; 0.004157 is the tannin factor. saponin content saponin content in cooked vegetables was determined by the method described by obadoni and ochuko (2001). cooked vegetables (3 g) were mixed with 20% aqueous ethanol (30 ml) with constant stirring for 3 h on a hot plate. the sample of ethanol mixture was filtered and concentrated, followed by the addition of 10-ml diethyl ether in a separating funnel with vigorous shaking. the separated aqueous part was used and treated with n-butanol (20 ml). the n-butanol extract was washed twice with 5% aqueous nacl (10 ml). the remaining solution was heated in a water bath. finally, the concentrated sample formed after evaporation was dried on a dry bath to a constant weight. the saponin content was expressed using the following equation: 2 1 w w saponin (mg/g) 100, w − = × (3) where w1 is the weight of the evaporating disk, w2 is the weight of the disk and sample and w is the weight of the sample. sensory analysis of cooked vegetables the sensory evaluation of cooked vegetables was performed by 15 semi-trained panellists having the basic knowledge of sensory analysis, nutritional interest and samples was performed at 2,200 rpm for 15 min at room temperature. the decanted supernatant was stored at 4°c for determining total phenolic content, total flavonoid content and antioxidant activity of vegetable samples. total phenol content the total phenolic content of vegetable samples was determined according to the method demonstrated by singleton et al. (1999). a sample extract of 0.2 ml was treated with folin ciocalteu reagent (1 ml). after 3 min, the reaction mixture was added with 20% sodium carbonate solution (0.80 ml) and stirred properly and incubated for 2 h at ambient temperature. the absorbance value of the reagent mixture was measured at a wavelength of 725 nm using an ultraviolet-visible spectroscopy (uv/vis) (uv-1700 spectrophotometer, japan). the results were expressed as mg gae/g using gallic acid as standard. total flavonoid content the total flavonoid content of cooked vegetables was measured by the method described by ebrahimzadeh et al. (2008). a sample extract of 2 ml was mixed with 10% aluminium chloride (100 μl), followed by the addition of 1 n potassium acetate (100 μl). the reagent mixture was diluted with 2.8 ml of distilled water. the sample mixture was incubated at room temperature for 30 min and absorbance was measured at 415 nm. the total flavonoid content was expressed as quercetin equivalent (mg qe/g of sample) using quercetin as standard. dpph (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity the dpph free radical assay was used to determine the antioxidant activity of cooked vegetables as per the method described by mulla and ahmed (2019). sample extract, 100 µl, was used for antioxidant activity and was added with 1 ml of 0.01% dpph reagent. the sample mixture was stirred properly and incubated for 30 min in the dark. absorbance was measured at 517 nm after 30 min using a spectrophotometer. the antioxidant activity using free radical scavenging was measured as follows: − = × abs control abs sample antioxidant acitivity (%) 100 abs control (1) where abs control = absorbance of control at 30 min, and abs sample = absorbance of sample solution at 30 min. antinutritional component analysis of cooked vegetables oxalate content the oxalate content of cooked vegetables was determined using the method described by ghoora et al. (2020) with italian journal of food science, 2021; 33 (sp1) 153 cooking methods affect eating quality, bio-functional components, antinutritional compounds and sensory attributes carbohydrate content in selected fresh vegetables varied from 2.05 to 8.97 g/100 g, with the highest reported in beans (8.97 g/100 g) and the lowest in kale (2.05 g/100 g). variations in the nutrient composition of selected vegetables could be due to different agro-climatic conditions (singh et al., 2001). results of the nutritional composition of fresh vegetables demonstrated similar results as reported by naz et al. (2018) and rani and fernando (2016). effect of cooking methods on phytochemicals of vegetables total phenolic content boiling, air frying and microwave cooking methods depicted increase in the level of total phenolic content of selected vegetables (figure 1). among raw vegetables, total phenolic content was reported the highest in tomatoes (2.03 mg gae/g), followed by carrot (1.97 mg gae/g), beans (1.69 mg gae/g), spinach (1.19 mg gae/g) and the lowest in kale (1.13 mg gae/g). boiling time from 2 to 8 min significantly increased the total phenolic content from 4.01 to 5.00 mg gae/g for kale, 3.32 to 4.30 mg gae/g for spinach, 2.07 to 4.01 mg gae/g for beans, 1.76 to 5.60 mg gae/g for carrot and 2.99 to 3.63 mg gae/g for tomatoes. the air frying of selected vegetables from 7.5 to 15 min reported a significant increase in total phenolic content. increase in total phenolic content was reported in: kale from 1.60 to 9.70 mg gae/g, spinach from 4.84 to 7.47 mg gae/g, beans from 6.14 to 8.04 mg gae/g, carrot from 2.82 to 5.12 mg gae/g and tomatoes from 5.31 to 6.58 mg gae/g. microwave cooking also demonstrated increase in total phenolic content with respect to processing time (1, 2.5, and 5 min). the total phenolic content of selected vegetables demonstrated an increasing trend with increase in processing time from 1 to 5 min in microwave cooking. increase in the total phenolic content of selected vegetables in 1 to 5 min was reported in: kale from 4.31 to 7.78 mg gae/g, spinach from 4.45 to 7.24 mg gae/g, beans from 5.20 to 7.17 mg gae/g, carrot from 2.77 to 9.15 mg gae/g willingness to participate. the assessment was exercised for color, flavor, taste texture and overall acceptability score using a 5-point hedonic rating scale to generate the following measurable sensory scores: excellent for a score of 5, good for a score of 4, average for a score of 3, fair for a score of 2 and poor for a score of 1. statistical analysis the experimental data were expressed as mean and standard deviation in triplicate using spss version 16. phytochemical and antinutritional analyses were performed using one-way anova, followed by duncan’s multiple range tests at a 5% level of significance. results and discussion proximate composition of fresh vegetables the nutrition analysis of fresh vegetables selected for different cooking methods is presented in table 1. the selected vegetables differed significantly in nutritional composition. the moisture content of fresh vegetables ranged from 84 to 83 g/100 g. tomatoes depicted the highest moisture content (91.60 g/100 g), followed by carrot (89.30 g/100 g), kale (88.60 g/100 g), beans (81.80 g/100 g) and the lowest in spinach (81.97 g/100 g). the selected fresh vegetables were low in protein content, ranging from 0.86 to 3.93 g/100 g. beans reported the highest protein content of 3.93 g/100 g and the lowest was determined in carrot (0.86 g/100 g). low fat content was reported in all vegetables, ranging from 0.20 to 0.60 g/100 g. fiber content in fresh vegetables ranged from 1 to 10 g/100 g, and higher content was observed in spinach (10.00 g/100 g), followed by beans (5.00 g/100 g), kale (4.00 g/100 g), carrot (3.00 g/100 g) and the lowest in tomatoes (1.00 g/100 g). ash content in selected fresh vegetables was the highest in spinach (2.00 g/100 g), followed by kale (1.60 g/100 g), carrot (1 g/100 g), tomatoes (0.80 g/100 g) and the lowest in beans (0.10 g/100 g). table 1. proximate composition of vegetables. proximate composition (g/100 g) kale spinach beans carrot tomatoes moisture 88.60 ± 0.10 81.97 ± 1.00 81.80 ± 0.01 89.30 ± 0.10 91.60 ± 1.00 ash 1.60 ± 0.02 2.00 ± 0.10 0.10 ± 0.69 1.00 ± 0.15 0.80 ± 0.01 fat 0.60 ± 0.01 0.63 ± 0.01 0.20 ± 0.39 0.20 ± 0.39 0.40 ± 0.01 protein 3.15 ± 0.02 2.97 ± 0.01 3.93 ± 0.02 0.87 ± 0.01 0.96 ± 0.01 crude fiber 4.00 ± 0.10 10.00 ± 0.10 5.00 ± 0.10 3.10 ± 0.10 1.00 ± 0.15 carbohydrate 2.05 ± 0.02 2.43 ± 0.01 8.97 ± 0.01 5.53 ± 0.02 5.24 ± 0.02 154 italian journal of food science, 2021; 33 (sp1) maqbool n et al. in kale, spinach and beans than the boiling and microwave methods of cooking. selected vegetables cooked for 7.5, 10 and 15 min by air frying method depicted a similar trend of increased flavonoid content. the air frying method with an optimized duration of 7.5–15 min reported an increase in flavonoid content from: 1.35 to 2.41 mg qe/g for kale, 0.96 to 2.23 mg qe/g for spinach, 0.38 to 1.42 mg qe/g for beans, 0.04 to 0.19 mg qe/g for carrot and 0.25 to 0.31 mg qe/g for tomatoes. microwave cooking for a short duration of 1, 2.5 and 5 min also depicted a similar trend of increased flavonoid content in kale, spinach, beans, carrot and tomatoes (figure 2). in microwave cooking, at a processing time of 5 min, kale had the highest flavonoid content (0.97 qe mg/g), followed by spinach (0.95 qe mg/g), tomatoes (0.79 qe mg/g), carrot (0.52 qe mg/g) and the lowest in beans (0.44 qe mg/g). a significant increase in the flavonoid content of selected vegetables by different cooking methods is attributed to extracted flavonoid compounds of vegetable matrix and changes in the structure of cell walls of vegetables during cooking (wachtel-galor et al., 2008). variations in the flavonoid content of selected vegetables because of different cooking methods and processing time could be due to differences in the extraction of flavonoids in vegetables, heating medium and duration of cooking. similar results of increased total flavonoid content in vegetables were reported by geetha et al. (2018), mazzeo et al. (2011) and saikia and mahanta (2013). antioxidant activity the reported antioxidant activity of fresh vegetables was the highest in tomatoes (25.90%), followed by carrot (14.81%), beans (11.11%), kale (7.40%) and the lowest in and tomatoes from 3.79 to 6.63 mg gae/g. use of different cooking methods indicated significant improvement in phenolic content of selected vegetables as compared to raw vegetables; however, the use of microwave as a cooking medium significantly increased the total phenolic content of selected vegetables than boiling and air frying methods. the increasing trend of total phenolic content in selected vegetables was due to the release of polyphenols from intracellular complexes during cooking, inactivation of polyphenol oxidase (ppo) enzyme (oboh, 2005), release of bioactive compounds because of the breakdown of cell walls (ferracane et al., 2018), and increase in free flavonols (stewart et al., 2000). similar findings were also reported in cooked vegetables by geetha et al. (2018), hossain et al. (2017) and saikia and mahanta (2013). total flavonoid content the total flavonoid content of selected vegetables is evidenced in figure 2. fresh vegetables contained 0.05–0.10 mg qe/g of total flavonoids, and the highest flavonoids were reported in spinach and the lowest in tomatoes and kale. boiling, air frying and microwave cooking methods reported an increasing trend in total flavonoids content in selected vegetables. the boiling method of cooking with respect to the duration of 2, 6 and 8 min significantly increased the total flavonoids content of vegetables. with increasing boiling time from 2 to 6 min, flavonoids content in: kale was increased from 0.63 to 1.33 mg qe/g, spinach from 0.15 to 0.78 mg qe/g, beans from 0.12 to 0.33 mg qe/g, carrot from 0.04 to 0.27 mg qe/g and tomatoes from 0.38 to 0.61 mg qe/g. the air frying method of cooking demonstrated a significant increase in flavonoid content 10 9 8 7 6 5 4 3 2 0 4 t p c ( m g g a e ) 8 time (min) air-frying microwave boiling 12 16 0 2 4 time (min) 6 80 1 2 3 time (min) 4 5 1 0 10 9 8 7 6 5 4 3 2 1 0 10 9 8 7 6 5 4 3 2 1 0 figure 1. effect of different cooking methods on the phenolic content (mg gae/g) of kale (⬜), spinach (○), beans (△), carrot (▽) and tomatoes (◊). italian journal of food science, 2021; 33 (sp1) 155 cooking methods affect eating quality, bio-functional components, antinutritional compounds and sensory attributes effect of cooking methods on antinutritional components of vegetables oxalate content boiling, air frying and microwave cooking evidenced a decrease in the level of oxalate content in selected vegetables as compared to fresh ones (figure 4). the highest oxalate values in raw vegetables were observed in tomatoes (6.60 mg/g), followed by spinach (5.72 mg/g), beans (3.96 mg/g), kale (1.58 mg/g) and the lowest in carrot (1.41 mg/g). cooking of selected vegetables by boiling significantly reduced oxalate content with respect to the optimized processing time of 2, 6 and 8 min. reduction in oxalate content was observed in 2–8 min of boiling, varying from 0.88 to 0.22 mg/g for kale, 3.10 to 1.44 mg/g for spinach, 4.10 to 2.42 mg/g for beans, 4.10 to 2.42 mg/g for carrot and 5.50 to 4.40 mg/g for tomatoes. air frying with optimized processing time (7.5, 10 and 15 min) also decreased the oxalate content of selected vegetables. reduction in oxalate content at 15 min of processing was observed for kale (0.20%), 1.32 mg/g for spinach, 0.44 mg/g for beans, 0.46 mg/g for carrot and 1.54 mg/g for tomatoes. the microwave cooking method at a processing time of 1–5 minutes reduced oxalate content from: 0.77 to 0.44 mg/g in kale, 2.42 to 0.88 mg/g in spinach, 4.62 to 2.86 mg/g in beans, 0.98 to 0.32 mg/g in carrot and 6.38 to 4.35 mg/g in tomatoes. in general, cooking methods reduced the oxalate content of vegetables as compared to raw vegetables; however, air-frying and boiling presented significantly higher reductions than microwave cooking. the significant reduction in oxalate content in selected vegetables could be due to leaching spinach (3.70%) (figure 3). the boiling method significantly increased the antioxidant activity of selected vegetables with respect to the optimized cooking time of 2, 6 and 8 min. the boiling method of cooking for 2–8 min reported a significant increase in the antioxidant activity of kale (18.50–75.10 %), spinach (32.90–85.96%), beans (27.03–51.85%), carrot (42.22–83.70%) and tomatoes (38.51–97.20%). antioxidant activity in selected vegetables differed significantly with respect to optimized time (7.5, 10 and 15 min) in the air frying method of cooking. antioxidant activity significantly increased from: 7.40% to 28.14% for kale, 37.03% to 55.10% for spinach, 27.40% to 43.70% for beans, 51.80% to 64.40% for carrot, and 35.88% to 67.93% for tomatoes. the microwave method of cooking established a similar trend of increased antioxidant activity in selected vegetables with respect to optimized processing time (1, 2.5 and 5 min) as compared to fresh vegetables. the optimized cooking time of 5 min demonstrated higher antioxidant activity, with the highest of 92.50% reported in carrot, followed by 91.40% for beans, 79.25% for tomatoes, 62.50% for spinach and 54.07% for kale. an increase in the antioxidant activity of selected vegetables because of different cooking methods could be due to the maillard reaction (saikia and mahanta, 2013), secondary metabolites (tayebeh et al., 2021), the release of antioxidant compounds from disrupted cell walls, inhibition of oxidative enzymes and reduced oxidation of antioxidants (morales and babel, 2002), and increased phytochemicals during cooking (zeb et al., 2018). similar findings were reported by agamy (2016), hossain et al. (2017), and mirzaei et al. (2014). f la vo no id ( m g q e /g ) 0.0 0 4 8 time (min) time (min) time (min) 12 16 0 1 2 3 4 0 2 4 6 85 0.5 1.0 1.5 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 air-frying microwave boiling figure 2. effect of different cooking methods on the flavonoid content (mg qe/g) of kale (⬜), spinach (○), beans (△), carrot (▽) and tomatoes (◊). 156 italian journal of food science, 2021; 33 (sp1) maqbool n et al. 100 90 80 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 0 4 8 time (min) a nt io xi da nt a ct iv ity d p p h ( % ) 12 16 0 1 2 time (min) 3 4 5 0 2 4 time (min) 6 8 air-frying microwave boiling figure 3. effect of different cooking methods on the antioxidant activity of kale (⬜), spinach (○), beans (△), carrot (▽) and tomatoes (◊). 7 6 5 4 3 2 0 4 o xa la te c on te nt ( m g/ g) 8 time (min) 12 16 1 0 7 6 5 4 3 2 1 0 7 6 5 4 3 2 1 0 0 1 2 3 time (min) 4 5 0 2 4 time (min) 6 8 air-frying microwave boiling figure 4. effect of different cooking methods on the oxalate content of kale (⬜), spinach (○), beans (△), carrot (▽) and tomatoes (◊). out of soluble oxalate from disrupted tissues during the cooking process (catherwood et al., 2007; yadav and sehgal, 2003). reduction in oxalate content in cooked vegetables was also observed by agiriga and siwela (2018), akhtar et al. (2014) and hemmige et al (2017). tannin content cooking of vegetables by boiling, air frying and microwave methods significantly reduced (p < 0.05) the tannin content as compared to fresh vegetables (figure 5). the tannin content in fresh vegetables ranged from 6.30 to 15.4 mg/g. the highest tannin content was observed in beans (15.4 mg/g), followed by carrot (10.2 mg/g), spinach (9.20 mg/g), tomatoes (8.30 mg/g) and kale (6.30 mg/g). the tannin content in cooked vegetables reduced significantly with respect to the optimized processing time for boiling (2, 4 and 6 min), air frying (7.5, 10 and 15 min) and microwave cooking (1, 2.5 and 5 min). the reduced tannin content in selected vegetables at 15 min of boiling was observed as 0.30 mg/g for kale, 1.20 mg/g for spinach, 1.20 mg/g for beans, 1.0 mg/g for carrot and 0.1 mg/g for tomatoes. however, air frying for 15 min and italian journal of food science, 2021; 33 (sp1) 157 cooking methods affect eating quality, bio-functional components, antinutritional compounds and sensory attributes 16 14 12 10 8 6 4 ta nn in c on te nt ( m g/ g) 2 0 16 14 12 10 8 6 4 2 0 16 14 12 10 8 6 4 2 0 0 4 8 time (min) 12 16 0 1 2 3 time (min) 4 5 0 2 4 time (min) 6 8 air-frying microwave boiling figure 5. effect of different cooking methods on the tannin content of kale (⬜), spinach (○), beans (△), carrot (▽) and tomatoes (◊). microwave for 5 min, respectively, recorded 2.0 mg/g and 0.1 mg/g for kale, 1.30 mg/g and 4.10 mg/g for spinach, 2.60 mg/g and 1.20 mg/g for beans, 1.50 mg/g and 1.20 mg/g for carrot and 1.0 mg/g and 1.50 mg/g for tomatoes. the reduction of tannins in cooked vegetables could be related to the thermal degradation of tannins during the process of cooking (miglio et al, 2008). similar results in vegetables were also reported by alajaji and el-adawy (2006) and hemmige et al. (2017). saponin content cooking vegetables by boiling, air frying and microwave methods significantly reduced the saponin content of vegetables as compared to fresh ones (figure 6). saponin content in fresh vegetables ranged from 19.0 to 32.10 mg/g. the highest saponin content in raw vegetables was observed in spinach (32.10 mg/g), followed by kale (24.50 mg/g), beans (22.30 mg/g), tomatoes (20.30 mg/g) and carrot (19.0 mg/g). the saponin content of cooked 35 30 25 20 15 10 0 4 s ap on in c on te nt ( m g/ g) 8 time (min) 12 16 5 0 35 30 25 20 15 10 5 0 35 30 25 20 15 10 5 0 0 1 2 3 time (min) 4 5 0 2 4 time (min) 6 8 air-frying microwave boiling figure 6. effect of different cooking methods on saponin content of kale (⬜), spinach (○), beans (△), carrot (▽) and tomatoes (◊). 158 italian journal of food science, 2021; 33 (sp1) maqbool n et al. b1 5 4 3 2 1 0 kale b2 b3 a1 a2a3 m1 m2 m3 b1 5 4 3 2 1 0 spinash b2 b3 a1 a2a3 m1 m2 m3 beans b1 5 4 3 2 1 0 b2 b3 a1 a2a3 m1 m2 m3 b1 5 4 3 2 1 0 tomatto b2 b3 a1 a2a3 m1 m2 m3 b1 5 4 3 2 1 0 carrot b2 b3 a1 a2a3 m1 m2 m3 color flavor taste texture over all acceptability figure 7. effect of different cooking methods on the sensory evaluation of vegetables. a1, a2 and a3 are air frying method of cooking vegetables for 7.5, 10 and 15 min; b1, b2 and b3 are boiling method of cooking vegetables for 2, 6 and 8 min; and m1, m2 and m3 are microwave method of cooking vegetables for 1, 2.5 and 5 min. italian journal of food science, 2021; 33 (sp1) 159 cooking methods affect eating quality, bio-functional components, antinutritional compounds and sensory attributes 2006). the sensory scores of cooked vegetables by boiling, air frying and microwave cooking were in the acceptable range, with a high recommendation of processes for preparing semi-processed vegetables with good consumer acceptance. similar results of sensory evaluation of cooked vegetables have been reported by şengul et al. (2014), soomro et al. (2018) and sultana et al. (2008). conclusion vegetables are a rich source of vitamins and phytochemicals, and their consumption in daily diet, besides providing nutrition, helps to fight chronic diseases. cooked vegetables such as kale, spinach, beans, carrot and tomatoes are a rich source of phytochemicals and antioxidants as compared to fresh ones, which are lower in antinutrients. the cooking of vegetables by boiling, air frying and microwave methods manifests significant improvement in bioactive components. cooking vegetables significantly increases their phytochemical and antioxidant activity. boiling, air frying and microwave cooking of vegetables significantly decrease their antinutritional components, thus decreasing the bioavailability of nutrients in vegetables. the stated methods of cooking of selected vegetables increased phenolic content and flavonoids but reduced saponin, tannin and oxalate contents. phytochemicals had a positive relationship with the cooking time for all three cooking methods. consumer acceptability of cooked vegetables was highest for the air frying method of cooking as compared to boiling and microwave cooking. references adams g.g., imran s., wang s., mohammad a., kok s., gray d.a., channell g.a., moris g.a. and harding s.e. 2011. the hypoglycaemic effect of pumpkins as anti-diabetic and functional medicines. food res. int. 44:862–867. https://doi.org/10.1016/j. foodres.2011.03.016 agamy n.f. 2016. effect of boiling and microwave cooking on some antioxidant compounds in highly consumed vegetables in egypt. cent eur j oper res. 2(2):76–84. agiriga a.n. and siwela m. 2018. effect of thermal processing on the nutritional, antinutrient, and in vitro antioxidant profile of monodoramyristica (gaertn.) dunal seeds. prev nutr food sci. 23(3):235–244. https://doi.org/10.3746/pnf.2018.23.3.235 akhtar m.s., israr b., nighat bhatty n. and ali a. 2014. effect of cooking on soluble and insoluble oxalate contents in selected pakistani vegetables and beans. int j food prop. 14(1):241–249. https://doi.org/10.1080/10942910903326056 alajaji s.a. and el-adawy a. a. 2006. nutritional composition of chickpea (cicer arietinum l.) as affected by microwave cooking and other traditional cooking methods. food chem. 19(8):806– 812. https://doi.org/10.1016/j.jfca.2006.03.015 vegetables is significantly reduced with boiling, air frying and microwave cooking. boiling significantly reduced saponin content with an increase in cooking time from 2 to 6 min. reduction in saponin content at 6 min of processing time in cooked vegetables was 16.20 mg/g for kale, 20.60 mg/g for spinach, 9.10 mg/g for beans, 7.30 mg/g for carrot and 3.30 mg/g for tomatoes. in air frying, saponin content reduced significantly at the processing time of 7.5, 10 and 15 min. reduction in saponin content of cooked vegetables at 15 min of air frying was 15.20 mg/g for kale, 13.2 mg/g for spinach, 1.10 mg/g for beans, 7.30 mg/g for carrot and 9.20 mg/g for tomatoes. microwave cooking also decreased saponin content with an optimized cooking time of 1, 2.5 and 5 min. a higher level of reduction in the saponin content of cooked vegetables at 5 min of processing was as follows: 7.70 mg/g for kale, 14.50 mg/g for spinach, 4.70 mg/g for beans, 1.30 mg/g for carrot and 2.0 mg/g for tomatoes. however, microwave and air frying demonstrated a significantly higher reduction in saponin content in cooked vegetables than by the boiling method. reduction of saponin content could be related to thermal degradation associated with heat supplied to cooked vegetables. this was also observed by alajaji and el-adawy (2006), badifu and okeke (1992), hemmige et al. 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(fazeli, et al., 2007, behnia, 2008, sharafzadeh and alizadeh, 2012). probiotics are used in dairy products as well as in food supplements and in agriculture as feed additives because of their beneficial health effects (nagpal et al., 2012). according to the currently international fao/who definition (2001), probiotics are “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. probiotics improve the health of both animals and humans through the improvement of its intestinal health by the regulation of microflora, stimulation and development of the immune system, synthesizing and enhancing the bioavailability of nutrients, reducing symptoms of lactose intolerance and reducing the risk of certain other diseases (kumar et al., 2011; nagpal et al., 2012). at present, probiotics are introduced as suitable replacing of antibiotics in order to confront with pathogens in animals and human being, and consumption of probiotic food products and medicines have considerable vogue. several investigations suggested that using probiotic is associated with reducing the risk of antibiotic-associated diarrhea (32). the foods which contain probiotic bacteria are put in the group of special products, and according to the dairy products international federation (idf) recommendation, these probiotic products should have a minimum concentration of 106 cfu/g probiotic bacteria and consumer a total of some 108 to 109 probiotic microorganisms should be consumed daily for the suitable probiotic effects (15). the most commonly important probiotics belong to lactobacillus and bifidobacterium genus. there are many well-characterized strains of lactobacilli and bifidobacteria available for human use (hussain et al., 2009; kechagia et al., 2012). some of the beneficial properties of probiotics are anticarcinogenic, immunologic enhancement, antiatherogenic, cholesterol-lowering, anti-obesity and antidiabetic characteristics (nagpal et al., 2012). kephir (kefir) is a viscous, highly acidic beverage produced from cow, goat, sheep or mare milks. the fermentation is initiated by “kefir grains” (clusters of yeast and bacteria), which are added to raw, pasteurized or uht-treated milk (sarkar, 2007; ribeiro and ribeiro, 2010). kefir contains high contents of thiamine, riboflavin, pantothenic acid, vitamin c, protein and minerals; hence, kefir has both therapeutic and nutritional attributes (sarkar, 2007). kefir also has greater amounts of threonine, serine, alanine and lysine than milk (sarkar, 2007). kefir also has medical effects in order to treat hypertension, body skin fineness, stress and depression, cholesterolemia and arthritis (xiao et al., 2003; ninane el al., 2005). aflatoxins are naturally occurring mycotoxins that are produced by some species of fungi like aspergillus flavus and aspergillus parasiticus. aflatoxin m1 (afm1) is one of the metabolites of aflatoxin b1 that is excreted into milk when lactating animals are given feed with aflatoxin contaminated food (10). contamination of milk or milk products with afm1 is considered as a potential risk for public health (11, iarc, 2002). international agency for research on cancer (irac) classified afm1 as a group 2b agent (possibly carcinogenic to humans) and exposing to chronic aflatoxin causes acute liver problems, mutation and liver cancer (behfar et al., 2012). aflatoxin m1 is relatively stable during pasteurization, sterilization and preparation of dairy products (fallah,   ital. j. food sci., vol 28, 2016 519 2010). therefore, industries sustain irretrievable economic losses, if it is not controlled, and its potential risks for human health especially for children (elkhoury et al., 2011). the aim of this research was to produce kefir and reviewing thymus extract effects in combined with lactobacillus acidophilus and bifidobacterium bifidum on the afm1 concentration in kefir drink. 2. materials and methods 2.1. chemicals and instrumentation lyophilized lactobacillus acidophilus and bifidobacterium bifidum (chr hansen company, denmark) were used as probiotic bacteria. the pasteurized low fat milk (1.5% fat) and kefir grain (iran) were used to produce kefir drink. thymus extracts (zard band company, iran) (were prepared at 3 different concentrations including 2, 4, 6 g/l. afm1 was procured from merk company, iran) and 200 ppb concentration was used to add the kefir drink as follow explanation. 2.2. producing kefir drink in order to produce kefir drink, 1 gram kefir grain was added to 1 litter pasteurized low fat milk (1.5% fat), and this was incubated at 38°c and acidity measurements were performed at different times until reaching 42°c (standard and industrial search of iran). at least this product was kept in refrigerator at 4°c, subsequently (moatter and shams kashani, 1378; iran standard and industry research institute, 1385). 2.3. adding aflatoxin m1 and thymus extracts to produced kefir ten ml of produced kefir was poured equally in 4 tubes and 10 ml of contaminated milk with 200 ppb of afm1 was added to each tube. finally, different doses of thymus extract (2, 4, 6 g/l) were added to the samples (otles and cagindi, 2003; marhamatizadeh et al., 2011; marhamatizadeh et al., 2012). 2.4. adding aflatoxin m1, thymus extract and lactobacillus acidophilus or bifidobacterium bifidum to kefir four g/l fixed thymus with 1 × 108, 3 × 108 and 6 × 108 bifiodobacterium bifidum or lactobacillus acidophilus were poured in 4 tubes and 10 ml of contaminated milk by 200 ppb of afm1 was added to them, subsequently (otles and cagindi, 2005; marhamatizadeh et al., 2011; marhamatizadeh et al., 2012). finally, all samples were kept into the incubator for 24 hours at 24°c. after 24 hours, the coagulation was separated from liquid by using a cloth filter, and remained liquid was incubated at 14°c for 24 hours. it was kept for 48 hours in refrigerator at 4°c and all samples were analyzed with elisa reader (europroxima company), subsequently. 2.5. elisa test samples were centrifuged at 2,000 rpm for 10 min at 7°c and the upper oil layer was removed. then, 100 ml of these samples were used for elisa test. elisa reader device in three repetitions and distinguished three optical densities for every sample was done. and every of three optical densities were put in excel program (collected by europroxima   ital. j. food sci., vol 28, 2016 520 company) and three concentrations were got that the average of these three shows afm1 reminder in every samples. 2.6. statistical analyses the data was analyzed by using kruskal-wallis test in a meaningful surface at p< 0.05 by spss software (spss for windows, version 20, spss inc, chicago, il, usa). 3. results 3.1. evaluating primary milk contamination with afm1 the results showed that primary milk contains 32 ppb afm1 and then 200 ppb of afm1 were added to milk that totally afm1 becomes 232 ppb. table 1 shows the changes of aflatoxin level in the present of thymus extracts, bifidobacterium bifidum and lactobacillus acidophilus bacteria in various groups. table 1: comparison of afm1 concentration in the presence of bifidobacterium bifidum and lactobacillus acidophilus bacteria and thyme extracts. sample tubes added aflatoxin (ppb) afm1 value added in the form of (ppb) initial aflatoxin (ppb) afm1 value primary the remaining amount of aflatoxin (ppb) afm1 value reduced aflatoxin (ppb) afm1value decreased (ppb) reduced aflatoxin (%) afm1value decreased (%) 40 g kefir 200 232 174.3 57.7 24.87 2 g/l thyme extract 200 232 154.6 77.4 33.36 4 g/l thyme extract 200 232 155.9 76.1 32.80 6 g/l thyme extract 200 232 162 69.8 30 4 g thyme extract and 1×108 bifidobacterium bifidum 200 232 169.5 62.5 26.93 4 g/l thyme extrac and 3×108 bifidobacterium bifidum 200 232 175.3 56.7 24.43 4 g/l thyme extract and 6×108 bifidobacterium bifidum 200 232 148.3 83.7 36.07 4 g/l thymus extract and 1×108 lactobacillus acidophilus 200 232 125.8 106.2 45.77 4 g/l thymus extrac and 3×108 lactobacillus acidophilus 200 232 186.1 45.9 19.78 4 g/l thymus extracts and 6×108 lactobacillus acidophilus 200 232 169.1 63 27.15 3.2. evaluating kefir containing thymus in detecting afm1 the results indicated in table 1 showed that thymus extracts in kefir caused afm1 reduction largely. the reduction percent of afm1 in the kefir samples containing different concentrations of thymus extracts 2, 4 and 6 gr/l were 33.36%, 32.80% and 30%,   ital. j. food sci., vol 28, 2016 521 respectively. the most reduction of afm1 was detected by 2 gr of thymus and thymus extract with 6 g/l concentration showed the least percent of afm1 reduction. 3.3. evaluating probiotic kefir containing thymus and lactobacillus acidophilus in afm1 reduction the data in table 1 shows that fixed quantity of thymus (4 gr/l) with different amount of lactobacillus acidophilus declined aflatoxin. reduction of afm1 in the presence of thymus extract and different value of lactobacillus acidophilus 1×108, 3×108 and 6×108 were 45.77%, 19.78% and 27.15, respectively. the most decrease of aflatoxin was detected with 1×108 level of lactobacillus acidophilus and the amount 3×108 of bacteria has the least reduction of aflatoxin. 3.4. evaluating probiotic kefir containing thymus and bifidobacterium bifidum in deleting afm1 the results showed that bifidobacterium bifidum bacteria decrease the amount of afm1 in the samples. the most decline percent of afm1 in the presence of 6×108 of bifidobacterium bifidum occurred with 36.07%; and 1×108 and 3×108 of bacteria with 26.93% and 24.43%, respectively, had the least afm1 reduction. 4. conclusions the aim of the present study was the evaluation of the effect of thymus extract and lactobacillus acidophilus and bifidobacterium bifidum bacteria on the reduction of aflatoxin amount in kefir beverage. furthermore, the possibility of producing a new probiotic food based on kefir and thymus was assessed. the results of present study indicated that thymus extract has anti-aflatoxin activity and capability for aflatoxin reduction. the thymus extract in combination with probiotic bacteria has the more ability for aflatoxin decline. the results of the current research showed that thymus extract with 2 gr/l concentration in combination with 6×108 bifidobacterium bifidum have the most effect in reduction of afm1 and the strongest antiaflatoxin activity was shown by the thymus extract with 4 gr/l concentration in combination with 1×108 lactobacillus acidophilus. our results are agreed with other studies (otles and cagindi, 2003; lee et al., 2003; tratnik et al., 2006). probiotic foods have been produced in order to treat intestinal infections as well as genital diseases. in three decades ago, commercial probiotic products have been supplied to the world market grew. kefir is a fermented dairy product which originates from the caucasus mountains in eastern europe (tratnik et al., 2006). kefir is the most popular probiotic product in europe. kefir has beneficial effects in healing and homeostasis due to its vitamins, minerals and essential amino acids (otles and cagindi, 2003). the vitamin content of kefir affects both type of milk and microbiological flora (sarkar, 2007, arslan 2014). aflatoxins are a group of fungal secondary metabolites which are recognized as being of economic and health importance (10). afb1 is currently of great interest due to their toxic, carcinogenic and mutagenic nature on human and animal health (11). a number of studies reported that many micro-organisms, including bacteria, yeasts, moulds, actinomycetes and algae are able to remove or degrade small amounts of aflatoxin in foods and feeds (lee et al., 2003).   ital. j. food sci., vol 28, 2016 522 several lactic acid bacteria strains have shown different capabilities for binding afm1 in solutions and in milk (haskard, et al., 2001). the various studies have reported that certain lactobacilli and bifidobacteria are capable of removing afb1 from liquid solutions by binding the toxin (peltonen et al., 2001; haskard et al., 2001). some researchers have suggested that aflatoxin binds predominantly to polysaccharides and peptidoglycans of the bacterial cell wall (lahtinen et al., 2004). lopez et al., (2003) showed that saccharomyces yeast reduced 90% afm1 in the milk. powders and extracts of many herbs, plants and spices have been reported to inhibit the production of aflatoxin (paranagama et al., 2003). a recent study showed that the essential oils of t. daenensis and thymus spp. (elam ecotype) flowers exhibited antibacterial activities against l. monocytogenes from chicken meat (ghasemi pirbalouti et al., 2009). lixandru et al., (2010) evaluated antimicrobial activity of some plant essential oils against bacterial and fungal species. the results showed thymus, coriander and basil oils proved the best antibacterial activity, while thymus and spearmint oils better inhibited the fungal species. thymus vulgaris extracts with 0.01 and 1% 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el-denshary, nabila s. hassan, fathia manaa and mosaad a. abdel-wahha. 2012. antioxidant and hepatorenoprotective effects of thyme vulgaris extract in rats during aflatoxicosis. global journal of pharmacology 6(2):106-117. naqhdi badi h. and makizade tafti m. 1382. a reviewing on thymus. pharmautical plants. no. 7. ninane v., berben g., romne j.m. and oger r. 2005. variability of the microbial abundance of kefir grain starter cultivated in partially controlled conditions. biotechnol. agron. soc. environ. 9(3):191-194. otles s. and cagindi o. 2003. kefir: a probiotic dairy-composition, nutritional and therapeutic aspects. pakistan journal of nutrition 2:54-59. doi:10.3923/pjn.2003.54.59 paranagama p.a., abeysekera k.h.t., abeywickrama k.p. and nugaliyadde l. 2003. fungicidal and anti-aflatoxigenic effects of the essential oil of cymbopogon citratus (dc.) staps. 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potential of kefir as a dietetic beverage. a review. british journal of nutrition 109:280-290. sassahara m., pontes netto d. and yanaka e.k. 2005. aflatoxin occurrence in foodstuff supplied to dairy cattle and aflatoxin m1 in raw milk in the north of paraná state. food chem. toxicol. 43(6):981-4. seher arslan. a review: chemical, microbiological and nutritional characteristics of kefir. 2014. cyta. journal of food 2015, 13(3):340-345. http://dx.doi.org/10.1080/19476337.981588. seyed mohammad nabavi, anna marchese, morteza izadi, valeria curti, maria daglia and seyed fazel nabavi .2015. plants belonging to the genus thymus as antibacterial agents: from farm to pharmacy. food chemistry 173:339-347. some medicinal plants cultivated in iran. shahram sharafzadeh and omid alizadeh. 2012. journal of applied pharmaceutical science 2(1):134-137. tratnik l., bozanic r., herceg z. and drgalic i. 2006. the quality of plain and supplemented kefir from goat’s and cow’s milk. international journal of dairy technology 59:40-46. doi:10.1111/j.1471-0307.2006.00236.x udagawa s. 2005. fungal spoilage of foods and its risk assessment. nippon ishinkin gakkai zasshi 46(1):11-5. unusan n. 2006. occurrence of aflatoxin m1 in uht milk in turkey. food chem toxicol. 44(11):1897-900. xiao j.z., kondo s., takahashi n., miyaji k., oshida k., hiramatsu a., iwatsuki k., kokubo s. and hosono a. 2003. effects of milk products fermented by bifidobacterium longum on blood lipids in rats and healthy adult male volunteers. journal of dairy science 86:2452-2461. paper received august 6, 2015 accepted april 11, 2016 ijfs#260_yamaner_bozza   ital. j. food sci., vol 28, 2016 208 paper efficacy of neutralised electrolysed water and mild heat against foodborne pathogens isolated from ficus carica c. yamanera*, m. ayvaza, r. konakb, n. tanb, i. kosoglub and a. dimogloc aadnan menderes university, department of agricultural biotechnology, south campus, aydin, turkey berbeyli fig research station, aydin, turkey cgebze technical university, department of environmental engineering, gebze, kocaeli, turkey *corresponding author. tel.: +90 2567727148; fax: +90 2567727233 e-mail address: cigdemileri80@gmail.com abstract problems with microorganism toxins in dried fig exports are becoming very important. chlorine-based sanitizers are effective way of controlling microorganisms, but on the other hand their reaction with natural organic and inorganic matter may potentially form carcinogenic products. therefore, different sanitizers for the disinfection of food and food contact surfaces are required as an alternative to chlorine-based sanitizers. some earlier studies revealed that neutralised electrolysed water (new) may be a potential substitute for cleaning and sanitizing agents in packaged products. in order to make a contribution to solve toxins problems, the antibacterial and antifungal effect of neutralised electrolysed water (new) on the foodborne pathogens were evaluated in this study. spores of aspergillus flavus and penicillium expansum were isolated from the surface of fig fruits. escherichia coli and bacillus cereus known to occur on the surface of figs were also evaluated. vegetative cells and spores of bacterium and fungi were exposed to five different concentrations of new (100, 75, 25, 5 and 1%) at three different temperatures (22, 50 and 70°c) for 1, 3 and 5 min. according to the results, at 22°c, 1% neutralised electrolysed water exposure for 1 min effectively decreased the number of vegetative cells of e. coli and b. cereus by approximately 8.5 log cfu/ml and 6.3 log cfu/ml, respectively. at 50°c, 5% neutralised electrolysed water exposure for 1 min significantly reduced the a. flavus and p. expansum spore numbers by 5.54 log cfu/ml and 7 log cfu/ml, respectively. with the effect of mild temperature (22-50°c), low chlorine neutralised electrolysed water (9.22 mg/l 33.85 mg/l available chlorine concentrations) showed a strong antibacterial and antifungal activity against foodborne pathogens. as a conclusion, neutralised electrolysed water can be used widely as a sanitizer in fig enterprises, instead of high cost chlorine based disinfectants. keywords: aspergillus flavus, bacillus cereus, penicillium expansum, sanitizer   ital. j. food sci., vol 28, 2016 209 1. introduction figs (ficus carica l.) are an economically important and highly valued fruit with high contents of fibre, minerals and polyphenols. they are mainly grown in the aegean region of turkey. with the yearly production of 275,002 tons, turkey ranks the first in the world in fig production and exportation (tuik, 2012). turkey owns the 26% of the world’s fig production and 36% of the world’s fig exportation (çalişkan and polat, 2012). thirty percent of figs produced in turkey are marketed as fresh figs, while the remaining fruit is consumed as dried figs. due to the high content of carbohydrate and water activities of semi-dried figs on the tree and fallen figs collected from the soil, figs can be considered a good substrate for mycotoxin formation. water activities for semi-dried on the tree, fallen figs collected from the soil, figs taken from the drying stage and those from warehouses have been reported as 0.880.94; 0.76-0.87; 0.70-0.80 and 0.69-0.73aw, respectively (karbancioglu-guler and heperkan, 2009). the most common toxigenic fungi reported in turkish dried figs are aspergillus section nigri, fusarium spp., and aspergillus section flavi penicillium spp.; their toxin types are aflatoxin, citrinin, fumonisin, patulin, and ochratoxin a (heperkan, 2006). figs are usually contaminated with escherichia coli, bacillus cereus and bacillus cereus spores. the numbers of these microorganisms on dried figs were reported to vary from a few hundreds to thousands per gram of fruit (frazier and westhoff, 1988; akbas and ozdemir, 2008). poor storage conditions such as damp environments and high storage temperatures may cause bacterial growth to reach to a level of about 107-108 cfu/g for dried figs. the main purpose of all methods applied for food preserving is to prevent or limit microbial and enzymatic changes in foods. it was estimated that food-borne diseases cause approximately 76 million illnesses, 325,000 hospitalisations, and 5,000 deaths in the united states each year (mead et al., 1999). according to the environmental protection agency (epa) (2001), chlorine-based sanitizers are effective way of controlling microorganisms, but on the other hand their reaction with natural organic and inorganic matter may potentially form carcinogenic products (fawell, 2000; allende et al., 2009). many of these products may cause cancer and generates reproductive and developmental problems in laboratory animals. therefore, different sanitizers for the disinfection of food and food contact surfaces are required as an alternative to chlorine-based sanitizers. electrolysed water generation technology was initially developed in russia (mamadzhanov et al., 1974), and then modified in the 1980s by japanese food industry researchers (hati et al., 2012). in this technology, by using tap water and salt, system generates two stream simultaneously, one is alkaline and the other is acidic, with the characteristics of cleaning and sanitising solutions, respectively. researchers evaluated the effectiveness of these solutions as a sanitizing and cleaning agent. studies revealed that they can be a potential effective substitute for cleaning and sanitizing agents in packaged products (iwasawa et al., 1993; hayashibaraet al., 1994). the electrolysed water acidic solution is reported to be highly effective in deactivating the e. coli, s. enteritides, s. aureus, c. albicans and l. monocytogenes strains (venkitanarayanan, 1999; ileri et al., 2006). the disinfection mechanism of the acidic electrolysed water can be attributed to several factors, including the destruction of bacterial protective barriers, the increase in membrane permeability, the leakage of cellular inclusions, and the decrease in activities of some key enzymes (zeng et al., 2010). however, the potential application of strong acid electrolyzed water (saew) is limited because of its low ph (≤ 2.7). at this low ph, dissolved cl2 gas can be rapidly lost due to volatilization, decreasing the bactericidal activity of the solution by the time (len et al., 2000) and may affect human health and the environment adversely. moreover, the high   ital. j. food sci., vol 28, 2016 210 acidity of strong acid electrolyzed water may cause the corrosion of equipment and consequently limit its practical application. in japan, strong acid electrolyzed water (ph 2.5±0.2; 20-60 mg/l available chlorine concentrations (acc)) and slightly acidic electrolysed water (ph 5.0-6.5; 10-30 mg/l acc) usage in food products is authorised by the japanese ministry of health and welfare (zacharia et al., 2010). recently, slightly acidic or neutralised electrolysed water has been of interest, as it minimises human health and safety risks from cl2 degassing, reduces corrosion of surfaces, and limits phototoxic side effects (guentzel et al., 2008). in spite of these advantages, neutralised electrolysed water in vitro inactivation of different food pathogens has not been intensively studied. our previous study is among the very few preliminary one aimed to unveil the bactericidal effectiveness of acid electrolysed water against different food spoilage and pathogenic microorganisms (ileri et al., 2006). in turkish enterprises, routine fig processing as follows; initially dried figs are washed with salty (3-6% nacl) high-temperature water (60-80ºc) and then processed further and finally packaged. the presence of salt increases the heat resistance of spores due to reduced water activity leading to spore dehydration (juneja and sofos, 2002). while this treatment may not be enough to decrease the microbial population of figs. thus it was suggested that neutralised electrolysed water was used instead of salty and high temperature water in fig enterprises to the optimum temperature and the optimum concentration of new under laboratory conditions. therefore, the effectiveness of new on spores of aspergillus flavus and penicillium expansum isolated on the surface of fig fruits, vegetative cells of escherichia coli atcc 35218 and bacillus cereus atcc 11778 known to occur on the surface of figs and bacillus cereus atcc 11778 spores was evaluated under controlled conditions. 2. materials and methods 2.1. bacterial cultures and the preparation of inocula e. coli atcc 35218 and b. cereus atcc 11778 were plated on nutrient agar (na) and incubated at 37°c for 24 h. after incubation, e. coli atcc 35218 and b. cereus atcc 11778 suspensions were prepared by transferring several colonies to a 10 ml of nacl solution (0.9%, w/v) with the sterile inoculation loop and vortexed using a thermal mixer. the 1 ml aliquots of undiluted and diluted cultures were transferred to a cuvette, and optical densities of the solutions at 600 nm were measured. aliquots of the samples were also used to perform serial dilutions. dilutions were plated on na plates and incubated at 37°c overnight. colonies were counted to determine viable bacterial cell counts (cfu/ml) at each dilution ratio, and then a standard curve was prepared by using absorbance and viable cell count. 2.2. preparation of spore solution of b. cereus b. cereus atcc 11778 was grown on nutrient agar (difco, becton dickinson, detroit, mi, usa) containing 500 ppm bacto dextrose and 3 ppm manganese sulphate for 7 days at 30°c. after this incubation, sporulation was determined by the schaeffer-fulton method (schaeffer and fulton, 1933). each agar plate with spores was rinsed twice with approximately 10 ml of sterile-distilled water while each plate was revolved on a   ital. j. food sci., vol 28, 2016 211 turntable, and the agar surface was gently agitated with a glass spreader to facilitate spore removal or the harvesting of the spores. the accumulated spore suspensions were washed four times by centrifugation at 4000 rpm for 20 min and re-suspended in sterile nacl solution (0.9%, w/v). tween 80 (0.05%, v/v) (sigma, st. louis, mo, usa) was added to spore suspensions in order to minimise the clumping of spores and improve the accuracy of spore counts. all transfers, inoculations and harvesting for the preparation of spore suspensions were performed under laminar flow conditions. the spore counts were enumerated by direct microscopic counting on a thoma slide. for verification of the number of spores in the suspension, the spore suspension was first heatshocked at 80°c for 10 min to kill vegetative cells. next, the number of viable spores was estimated by spread-plating 0.1 ml of spore suspension on nutrient agar at 30°c for 24 h. the spore suspension was stored at 4°c until use (akbas and ozdemi̇r, 2008). 2.3. preparation of spore solution of aspergillus flavus and penicillium expansum aspergillus flavus was isolated from fig fruits in the laboratory of adnan menderes university. a. flavus was cultured on potato dextrose agar plates (merck) at 25°c for one week. after a portion (about 15 ml) of 0.1 m dibasic sodium phosphate adjusted with 0.05 m citric acid monohydrate of ph 7.0 (pc) with 0.005% (v/v) tween 80 was poured onto the plate, spores were suspended in pc buffer by rubbing gently with a glass rod. clumped spores in pc buffer were scattered by sonication for 2 min. the suspension was filtered through eight layers of sterile cheese cloth to remove hyphae. the spore concentration of the suspension was estimated with a haemocytometer. the suspension was diluted with pc buffer to obtain a concentration of 107 cells per ml (fuji̇kawa and itoh, 1996). penicillium expansum was isolated and identified from fig fruits at the microbiology laboratory of adnan menderes university. spore suspensions were prepared on petri dishes for 1 week with pda and 50 mg/l of streptomycin. after one week of incubation at 25°c, spores were collected and suspended in sterile ringer's solution. after filtering through eight layers of sterile cheese-cloth, spores were counted and brought to a final concentration of 107 cell per ml (spadaro et al., 2002). 2.4. preparation of treatment solutions new was obtained by electrolysis of a mixture of nacl (20 g/l) and tap water using stel10h-120-01 generator (stel 10h120-01, russia) at 40.0 v, 9.0 a and produced at a rate of 250 ml/22 sec. a cylindrical electrochemical cell for processing solutions comprised an inner, hollow, tubular anode, an outer, cylindrical cathode, and a permeable, tubular, ceramic diaphragm that was arranged between the anode and cathode and divided the inter-electrode space into anode and cathode chambers so that a working section of the cell was formed. there was a hole connecting the internal chamber with the channel for the liquid withdrawal. the internal chamber was connected by the annular channel to the horizontal channel used for feeding liquid subjected to the treatment. the new dilutions were prepared by using sterile tap water at rates of 100% (undiluted), 75%, 25%, 5% and 1%, while deionised water (dw) alone was used as control. analytical indices (orr, ph and acc) of the treated solutions were measured immediately after new preparation and before each bactericidal and fungicidal experiment. the ph was measured with a ph meter (hi 2211-02, hanna, usa), and orp was measured with an orp meter (hi98120, hanna, usa). the ph meter was calibrated using commercial   ital. j. food sci., vol 28, 2016 212 standard buffers at ph 4.0 and 7.0 (merck ltd., germany). the acc was measured on the basis of the iodometric method reported by dychdala (1983). 2.5. in vitro inactivation of microorganisms by new in vitro inactivation experiments for their disinfection potential against vegetative cells of e. coli atcc 35218, b. cereus atcc 11778 and spores of a. flavus, p. expensum, and b. cereus were carried out at 22, 50 and 70°c for 1, 3, and 5 min. the bacteria and spore suspensions were prepared as described above. different concentrations of the new solutions were prepared with sterile tap water. before use, each new was diluted and placed in a water bath at 22, 50 and 70°c to reach the desired temperature before inoculation. one millilitre of each vegetative cell and spore suspension was separately added to 9 ml of different concentrations of new (1, 5, 25, 75, and 100%, v/v) and sterile dw. after inoculation, 1 ml aliquots were periodically collected at the end of incubation time (1, 3, and 5 min) at 22, 50 and 70°c, and transferred into the membrane filter funnel containing sterile dw (50 ml). the samples were drawn completely through the filter. the funnel was rinsed with sterile dw (150 ml). the membrane filter was removed from the funnel and placed into the prepared petri dish. agar plates were incubated at the proper temperature (bacterium and fungi, 35±2 and 30°c, respectively) and for the appropriate time period (bacterium and fungi, 24 and 72 h, respectively). 3. results and discussions 3.1. physicochemical properties of tested solutions one of the first applications of electrolysed water was the sterilisation of medical instruments in hospitals; it has since been used in the medical, dental, agriculture, dairy and food industries. based on the literature review carried out as part of this study, it was found that applications in the food industry have been the most frequently researched. applications of electrolysed sanitising solutions for microbial inactivation in fresh lettuce have been reported by park et al. (2001), koseki et al. (2003), delaquis et al. (2004) and ongeng et al. (2006). it was also used for the disinfection of tomatoes (bari et al., 2003), spinach (rahman et al., 2010), cucumbers (koseki et al., 2004) and strawberries (kosekî et al., 2003; udompijitkul et al., 2007). to provide ease of use in fig enterprises, tap water was used for the preparation of different concentrations of new. the orp, ph and acc for the treated solutions are shown in figure 1. the ph, orp and acc for five different concentrations of new solution (100, 75, 25, 5 and 1%) used in this study were ph 7.45-7.84, 900-807 mv and 712.6-9.2 mg/l, respectively. differences between the ph and orp of different concentrations of eas were very small, but the difference between acc was significant. the dw was used as a control solution in this study; it had a ph of 7.58, an orp of 353 mv, and no available chlorine was detected.   ital. j. food sci., vol 28, 2016 213 figure 1: physicochemical properties of tested solutions. 3.2. antimicrobial effects of different dilutions of new on pure cultures of e. coli, b. cereus and spores of b. cereus, a. flavus and p. expansum for vegetative cells, very rapid killing was observed. at all of the concentrations tested, new reduced bacterial numbers to the detection limit shown below within 1 in at 22°c (figs. 2, 3), corresponding to a log10 reduction factor of 8.5 and 6.3 (within 1 min) for e. coli and b. cereus, respectively. 0   100   200   300   400   500   600   700   800   900   1000   0   2   4   6   8   10   12   14   16   18   20   100   75   25   5   1   o r p  ( m v )/  a cc  ( pp m )   ph /e c   (m s)   concentrations  (%)   ph   ec  (ms)   orp  (mv)   concentrations (%) 100 75 25 5 1 ph 7,5 7,5 7,6 7,7 7,8 ec (ms) 17,7 14 6,12 1,8 0,7 orp (mv) 900 894 877 840 807 acc (ppm) 712,6 530,2 174,7 33,9 9,2   ital. j. food sci., vol 28, 2016 214 figure 2: the effectiveness of different concentrations of new against e.coli atcc 35218 vs treatment time and temperature. at 50°c, the 1 min treatment of control group of b. cereus achieved the 0.6 log10 cfu/ml reduction of their population. however, under the same conditions, a pure culture of b. cereus vegetative cells using 1% new (available chlorine of 9.22 mg/l) was reduced by 6.6 log10 cfu/ml (8.06 log10 cfu/ml to 1.5 log10 cfu/ml). the decrease in the control group depends on temperature and was found to be 1.6 log10 cfu/ml at the same temperature for 5 min (fig. 3). figure 3: the effectiveness of different concentrations of new against b. cereus atcc 11778 vs treatment time and temperature. 0   1   2   3   4   5   6   7   8   9   10   22°c   50°c   70°c   22°c   50°c   70°c   22°c   50°c     70°c   22°c   50°c   70°c   0   1   3   5   s ur vi vi ng b ac te ria l p op ul at io n (lo g 1 0 cf u/ m l) treatment time (min.) and temperature 100%   75%   25%   5%   1%   control   0   1   2   3   4   5   6   7   8   9   22°c   50°c   70°c   22°c   50°c   70°c   22°c   50°c     70°c   22°c   50°c   70°c   0   1   3   5   s ur vi vi ng b ac te ria l p op ul at io n (lo g 1 0 cf u/ m l) treatment time (min.) and temperature 100%   75%   25%   5%   1%   control     ital. j. food sci., vol 28, 2016 215 at 70°c, a reduction of 7.2 and 7.92 log10 cfu/ml in the control group was observed for 1 and 5 min, respectively (fig. 3). issa-zacharia et al. (2010) reported that saew (ph 5.6, 23 mg/l acc; and 940 mv orp) effectively reduced the populations of e. coli, s.aureus and salmonella spp. by 5.1, 4.8, and 5.2 log10 cfu/ml, respectively. kim et al. (2000) observed that b. cereus was more resistant to treatment than e. coli o157:h7 and only 3 log reductions were achieved after 10 s of rox eo water treatment. kiura et al. (2002) showed that reduction of the population of b. subtilis by 3.5 log10 cfu/ml was achieved by 5 min of acid electrolysed water (aew) treatment (ph 2.32, 4.95 mg/l acc). the effect of ozonation was investigated to reduce e. coli, b. cereus and b. cereus spores in dried figs. b. cereus spores, e. coli and b. cereus counts were decreased by 3.5 log numbers at 1.0 ppm ozone concentration for 360 min ozone treatment (akbas and ozdemir, 2008). the effectiveness of different concentrations of new was evaluated against spores of a. flavus and p. expansum using varying treatment times and temperatures in vitro (figs. 4 and 5). it was observed that spores were more highly resistant to the activity of new than vegetative cells. at 22°c, the treatment of spores of a. flavus for 1 min using new (25%, 174.7 mg/l) resulted in a reduction of 5.83 log10 cfu/ml. under the same conditions, the reduction of spores of p. expansum was 6.04 log10 cfu/ ml. antimicrobial effectiveness of new associated with contact temperature increased up to 50°c, but the control group was not affected by this increase in temperature. at 50°c, the numbers of spores of a. flavus and p. expansum were reduced by more than 5.54-7 log10 cfu/ml within 1 min of exposure to new (33.9 mg/l acc) (figs. 4 and 5). figure 4: the effectiveness of different concentrations of new against spores of a. flavus vs treatment time and temperature. 0   1   2   3   4   5   6   7   8   22°c   50°c   70°c   22°c   50°c   70°c   22°c   50°c     70°c   22°c   50°c   70°c   0   1   3   5   s ur vi vi ng s po re s po pu la tio n (lo g 1 0 cf u/ m l) treatment time (min.) and temperature 100%   75%   25%   5%   1%   control     ital. j. food sci., vol 28, 2016 216 figure 5: the effectiveness of different concentrations of new against spores of p. expansum vs treatment time and temperature. at 70°c, populations of the control groups decreased to undetectable levels. therefore, the effectiveness of new was not determined at this temperature (figs. 4 and 5). chlorine at levels of 100 to 200 ppm is currently recommended for the control of postharvest pathogen spores in dump tanks and other recirculating water systems (willet et al., 1989). however, the use of chlorine has disadvantages, such as the corrosion of metal equipment, reliance on manual monitoring of chlorine concentrations, sensitivity to organic load, effectiveness within a narrow ph range, and the formation of harmful chlorinated byproducts (roberts and reymond, 1994). eaw is an alternative to chlorine and chlorine compounds; it has the lowest amount of free chlorine and a very wide ph range. therefore, it has been reported that eaw can be effectively used for the control of postharvest pathogen spores (okull and laborde, 2004; al-haq and sugi̇yama, 2004; koseki et al., 2004). in their studies on a laboratory scale, audenaert et al. (2012) demonstrated that electrolysed oxidising water (eow) has the potential to control fusarium spp. in wheat grains. it was observed that b. cereus endospores were substantially more resistant to the activity of new and temperature than fungi spores. control groups of b. cereus spores were not affected by high temperature (70°c), but reduction of b. cereus spores exposed to new was increased at the same temperature. at 22°c, decreases in viable spores were initially rapid with a more than 5 log10 cfu/ml reduction observed after 1 min of exposure to undiluted new (100%). under the same conditions, log reduction of 4.77 log10 cfu/ml was obtained when the concentration of new was decreased from 100% to 75%. a longer exposure time was more effective in reducing the spore germination. after 3 min of exposure to 100, 75 and 25%, log reductions of 6.26 log10 cfu/ml, 4.84 log10 cfu/ml and 4.3 log10 cfu/ml, respectively, were observed. at 22°c and 100% new, the number of viable spores dropped below the experimental detection limit within 5 min, which corresponds to the log reduction factor of 7.04. after 5 min of exposure to 75% new, 1.86 log10 cfu/ml viable spores were still observed (fig. 6). it was determined that new, which has a low concentration and low content of acc, was 0   1   2   3   4   5   6   7   8   22°c   50°c   70°c   22°c   50°c   70°c   22°c   50°c     70°c   22°c   50°c   70°c   0   1   3   5   s ur vi vi ng s po re s po pu la tio n (lo g 1 0 cf u/ m l)   treatment time (min.) and temperature 100%   75%   25%   5%   1%   control     ital. j. food sci., vol 28, 2016 217 more effective against spores of b. cereus at high temperatures. at 70°c, 5 log10 cfu/ml reductions were achieved after 1 min of 5% new treatment (fig. 6). robinson et al. (2010) reported that, at the concentration of 99%, analytes produced a log reduction factor of greater than five in viable b. atrophaeus endospores within 90 s, and at concentrations of 10% or lower, the sporicidal effect was not statistically significant. figure 6: the effectiveness of different concentrations of new against spores of b. cereus atcc 11778 vs treatment time and temperature. physicochemical properties and bactericidal activity of new and aew under four different storage conditions were investigated by cui et al. (2009). they reported that new with a near-neutral ph (ph 6.5) and low orp value (550 mv) had a bactericidal activity (ph <3.0, orp > 1000 mv) similar to aew. different researchers have reported similar results to those from the study of cui et al. (2009) (marais and brozel, 1999; kiura et al., 2002; robinson et al., 2010). they observed that available chlorine is one of the main contributing factors for the antimicrobial activity. our results also indicated that the concentration of free chlorine in eow was more substantial for the microbial growth reduction than ph. 4. conclusions the new solution was synthesised as the result of the electrochemical activation. the solution of high reaction ability is in the meta-stable state and contains strong oxidants such as clo2, hclo, clo., cl., ho.2, o3, h2o2, h3o+, cl2o etc. it should be noted that somatic cells of animals and humans have a system of enzymatic protection against the action of the peroxidation factors mentioned. microbial cells, especially anaerobic ones, are deprived of this protection. this fact determines the high selectivity of the new action on the micro flora. the findings of this study demonstrated that the bactericidal efficacy was retained against vegetative cells at dilutions as low as 1% and against bacteria and fungi spores at dilutions as low as 5% new. also, 5% new with a ph of 7.7, acc of 33.9 mg/l 0   1   2   3   4   5   6   7   8   9   22°c   50°c   70°c   22°c   50°c   70°c   22°c   50°c     70°c   22°c   50°c   70°c   0   1   3   5   s ur vi vi ng s po re s po pu la tio n (lo g 1 0 cf u/ m l) treatment time (min.) and temperature 100%   75%   25%   5%   1%   control     ital. j. food sci., vol 28, 2016 218 and orp value of 840 mv can effectively inactivate pure cultures of spores of b. cereus and fungi in vitro. in addition, as a result of being non-corrosive, more stable for storage, inexpensive, and posing less potential health hazards to workers due to a lack of cl2 degassing, 5% new has more potential for application in fig enterprises than aew (acidic and includes high available chlorine) and chlorine-based disinfectants, which have high associated costs. acknowledgements this research was supported by this research was 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management of postharvest diseases and disorders of apples, pears, and cherries. post-harvest pomol newsl 7: 1-4. zeng x., tang w., ye g., ouyang t., tian l., ni y. and li p. 2010. studies on disinfection mechanism of electrolyzed oxidizing water on e. coli and staphylococcus aureus. j. food sci. 75 (5): 253-260. paper received september 11, 2015 accepted december 10, 2015 paper ital. j. food sci., vol. 27 2015 443 keywords: dry-ripened venison sausage, olive oil, acceptance test, preference test, quantitative descriptive sensory analysis effect of partial replacement of pork meat with olive oil on the sensory quality of dry-ripened venison sausage m.c. utrilla1,3, a. garcía ruiz2,3 and a. soriano1,3* 1department of analytical chemistry and food technology, faculty of sciences and technologies chemistries, university of castilla-la mancha, avda. camilo josé cela s/n, 13071 ciudad real, spain 2department of analytical chemistry and food technology, school of engineers agronomist, university of castilla-la mancha, ronda de calatrava 7, 13071 ciudad real, spain 3regional institute for applied scientific research (irica), university of castilla-la mancha, avda. camilo josé cela s/n, 13071 ciudad real, spain *corresponding author: tel. +34 926295300, fax +34 926295318, email: almudena.soriano@uclm.es abstract six assays of low-fat venison salchichon were produced using varying proportions of olive oil to replace the traditional pork meat added. the control contained 75% lean venison and 25% pork meat; in the other assays, 15, 25, 35, 45 and 55% of the pork meat was replaced by olive oil. samples were evaluated by quantitative descriptive sensory analysis and consumer testing. descriptive sensory analysis revealed significant differences for most of the attributes studied. the replacement of 35% or more of pork meat by olive oil, prompted a decrease in odour intensity, spicy odour, hardness and an increase of fat mouthfeel, together with the olive oil perception. by contrast, the replacement of 25% of pork meat by olive oil yielded a salchichon not greatly different to the control. consumers accepted all assays, but preferred those in which no more than 25% of the pork meat was replaced by olive oil. from a sensory standpoint, therefore, it is recommended that the replacement of pork meat by olive oil in this product should not exceed 25%. mailto:almudena.soriano%40uclm.es?subject= 444 ital. j. food sci., vol. 27 2015 introduction though the production of venison in spain is high, its economic value is relatively low because it is considered to be simply a by-product of hunting, oriented to obtain flashy awards. the autonomous community of castilla-la mancha is also the main venison exporter in spain, accounting the 80% of the total exportation, being germany is primary destination. despite the large venison production, its consumption in the region – an indeed in spain generally – is fairly limited; venison is mainly consumed in certain rural areas and some restaurants. cynegetic venison is a highly nutritive meat, characterized by a high protein and heme iron content and a low presence of subcutaneous and intramuscular fat (zomborszky et al., 1996; hoffman and wiklund, 2006). in addition, this meat has distinctive organoleptic properties, differing from those of other meats, such as its intense and attractive red colour, tenderness and variety of flavours, reflecting the fact that deers are raised in the wild and feed on natural pastures. a wide range of ripened products is obtained from hunted deer, including cecina (dry-ripened meat), and dry fermented sausages, as chorizo and salchichon. these are generally labelled “gourmet products” in the international market. in the production of venison chorizo and salchichon, a certain amount of pork meat has to be added to lean venison in order to ensure gradual drying, acceptable tenderness and the development of their distinctive flavour. however, consumer interests are based on low-fat foods rich in unsaturated fatty acids to get a healthy diet. on the other hand, olive oil is a staple of the mediterranean diet, and its main source of fat. it is remarkable for its characteristic fatty acid composition, and particularly for its high oleic acid content, ranging between 55 and 83% (codex stan 33-1981). virgin olive oil is a natural juice that can be consumed unrefined, thus retaining its original composition; this makes it a prime source of mainly-antioxidant micronutrients, including phenol compounds, vitamin e, carotenes and squalene (owen et al., 2000). several investigations have been carried out on the partial replacement of pork meat by olive oil in pork and/or beef dry sausages (bloukas et al., 1997; muguerza et al., 2001, 2002, 2003; severini et al., 2003; kayaardi and gök, 2003; del nobile et al., 2009; beriain et al., 2011); however, there is not studies to address the use of olive oil in making venison salchichon. the aim of this study was to elaborate cynegetic venison salchichon with the highest percentage of replacement of pork meat by olive oil, that allow to maintain sensory characteristic of the traditional salchichon sausage. it is also hoped to increase the venison products consumption and subsequently to raise the economic value of cynegetic venison. materials and methods raw materials lean venison was obtained from hind legs of male deer (cervus elaphus) obtained during the 2009-2010 hunting season on two neighbouring reserves in ciudad real (central spain). vegetation in the two reserves was very similar, comprising pine forests, woodlands and scrub. a total of 67.5 kg of venison was used. pork meat was obtained from castrated male pigs (progeny of a pietrain male x dalain female cross) raised intensively and slaughtered at the age of seven months. a total of 16 kg was used. extra virgin olive oil was produced at an oil-mill in ciudad real from cornicabra olives harvested in 20082009. a total of 3.5 l were used. the soy protein concentrate used, arcontms, is practically tasteless and guarantees high protein solubility. its chemical composition was: ≤ 6% moisture, ≥ 72% protein, ≤ 3% fat and 20% fibre. finally, a commercial salchichon formula (salchichón casero 933, manufacturas ceylan s.l., valencia, spain) was used, comprising salt, spices, lactose, saccharose, polyphosphates (e-450i, ii), sodium ascorbate (e-301) and potassium nitrate (e-252). venison salchichon production six assays of venison salchichon were made taking into account the findings of a previous study aimed at reducing the pork meat content of this product (utrilla et al., 2014). all assays contained 75% lean venison. the original 25% pork meat was partially replaced by 0% (control), 15, 25, 35, 45 and 55% extra virgin olive oil, in assays 1 to 6, respectively. olive oil was added to the salchichon in the form of an organogel obtained by emulsifying olive oil with soy protein concentrate (arcontms) and mineral water, at a ratio of 10:1:8, respectively (table 1). venison and pork meat were minced separately in an unger w-98 mincer (andher, camtable 1 relative percentages of lean venison, pork meat and organogel (olive oil + water + soy protein) in different assays of venison salchichon. assays lean venison (%) pork meat (%) organogel (%) 1 75 25.00 0.00 2 75 21.25 3.75 3 75 18.75 6.25 4 75 16.25 8.75 5 75 13.75 11.25 6 75 11.25 13.75 ital. j. food sci., vol. 27 2015 445 po de criptana, spain) with an 8 mm plate. venison was then mixed with on the appropriate proportion of pork meat and the olive oil as organogel (to join a total of 15 kg), and the commercial salchichon mixture (salchichón casero 933, manufacturas ceylan s.l., valencia, spain). an amount of 33 g mixture/kg, previously dissolved in 1 l of cold mineral water, was used. all ingredients were minced in an av-80 vacuum mixer (andher, campo de criptana, spain). the mixture was covered with a cotton cloth and left to settle for 20 h at 0º c, in order to the whole mass could involve the spices and additives. it was then fed through an h52 pas hydraulic piston-based sausage stuffer connected to a vae-10 vacuum system (andher, campo de criptana, spain), into synthetic collagen casings, with a 38-40 mm diameter. horseshoe-shaped salchichon sausages were then tied off at 60 cm intervals. salchichon sausages were maintained at 20°-22ºc and a relative humidity of 60% for 2 h, and finally cured at 11°-12ºc and a relative humidity of 75% for 28 days, in a zanotti curing chamber (grupo momplet, valencia, spain). after ripening, salchichon sausages were vacuum-packed and stored at 8°-10ºc for 45 days until its evaluation. two replicates of the six assays of salchichon sausages were done. samples to determine physicochemical characteristics (moisture, fat and protein content) in salchichon, the casing was removed and the entire content was ground in a domestic blender. all analyses were carried out in duplicate in two sausages of each replicate. the samples for sensory analysis were presented to the panellists and consumers in 3 mm thick slices without skin, at 20°-22º c (room temperature) and tagged (number-letter-number). samples were presented randomized at each tasting session in order to minimise any effects due to order of presentation. unsalted crackers and mineral water were provided to the panellist and consumers to cleanse the mouth between samples. the trained panel evaluated three samples per session, however consumers tasted the six assays of sausages in the same session. the trained panel evaluated each sample in duplicated. physicochemical composition moisture content was measured in accordance with the standard iso r-1442 (1973). total fat was extracted with petroleum ether (40°60º) following the standard iso r-1443 (1973). total nitrogen was measured using the kjeldahl method (method 16245, aoac 1980). protein nitrogen content was obtained by multiplying total nitrogen by a factor of 6.25. quantitative descriptive sensory analysis the quantitative descriptive sensory analysis was carried out in a tasting room equipped in accordance with une-en iso 8589:2010, by a 9 member sensory panel (6 women, 3 men, ages 2552 years) with previous experience in fermented sausages. three training sessions were held, employing three different commercial venison salchichon sausages, elaborated with cinegetic venison and pork meat. the qualification of the panel members was based on reproducibility verification and concordance between the tasters. attributes intensities were rate on non-structured scales of 10 cm and in accordance with une-iso 4121:2006. all the scales were anchored at the extremes with the terms “weak” and “very intense,” except for the colour intensity scales in which the colour was indicated at the extremes. the visual attributes evaluated were: amount of fat (fat particles), fat colour (0=white; 10=yellow) and lean colour (0=pink; 10=black). the odour attributes studied were: black pepper, spices, cured and olive oil odour as well as odour intensity. the attributes that defined the texture profile of the samples were: hardness (strength to breakdown the product), juiciness (amount of juice released during chewing), chewiness (attribute related to the cohesiveness of the product) and fat mouthfeel (attribute related to the perception of the fat quantity of the product). finally, the taste attributes (including retronasal perceptions) evaluated were the following: intensity of the taste, salty, pungent (nasal and oral mucosa irritation), pepper and olive oil taste and intensity of the aftertaste. consumer tests the consumer tests were carried out in a tasting room equipped in accordance with une-en iso 8589:2010. an untrained group of 44 habitual consumers of pork salchichon participated in the study, 15 men aged between of 22 and 45 (mean age 31) and 29 women aged between of 21 and 52 (mean age 30). consumers were recruited from students, staff and faculty of the food science and technology area of the university of castilla-la mancha. consumers were instructed to express their evaluation for overall acceptability considering the external appearance, odour, taste and texture of the slices. in the same session they evaluated their acceptance and preference of the six assays of samples. acceptance test to grade the acceptability of each sample, consumers used a non-structured or linear hedonic scale of 10 cm, anchored at either end by the phrases “strongly like” (left end) and “strongly dislike” (right end), enabling consumers to mark the point which best represented their satisfaction with the sample. 446 ital. j. food sci., vol. 27 2015 preference test a hedonic ranking test was used (une-iso 8587:2010), whereby each consumer was presented with a sample from each assay and asked to order the samples by degree of preference, giving 1 point to the least preferred and 6 to the most preferred. statistical analysis one-way anova was performed to study the influence of the olive oil amount in physicochemical parameters, and attributes evaluated by the quantitative descriptive sensory analysis and by the acceptance test. when the interaction was significant, the means were compared using the student-newman-keuls test. the friedman test was performed to check the significance of differences between consumer preferences. when significant differences were found, the fisher’s least significant difference (lsd) test was calculated following standard une-iso 8587:2010, to compare the means. all statistical procedures were carried out using spss 19.0 statistical software for windows xp (spss, inc., chicago, il, usa). results and discussion physicochemical composition results (mean±standard deviations) for moisture, fat and protein content of six salchichon assays at the end of ripening are shown in table 2. moisture values at the end of ripening were between 30.4-32.1 g/100 g, similar to those reported in other dry sausages varieties containing olive oil (bloukas et al., 1997; muguerza et al., 2002; beriain et al., 2011). significant differences in fat content were found between assays, directly reflected the varying proportion of olive oil, whose fat content is higher than that of pork meat. values at the end of ripening (21.1-40.2 g/100 g dm) were lower than those reported in pork and or beef sausages made with a partial replacement of pork backfat with olive oil (41.4-59.0 g/100 g dm) (bloukas et al., 1997; muguerza et al., 2001; muguerza et al., 2002; beriain et al., 2011), probably because the lean venison and pork meat used contains less fat than the raw materials used in those studies. on the other hand, significant inter assay differences in protein content were recorded at end of ripening process. the higher the olive-oil content, the lower the protein nitrogen content, as it was expected a significant inverse correlation being recorded between the two parameters (r=-0.948; p<0.001). protein nitrogen values at the end of ripening (43.6-59.6 g/100 g dm) were higher than those reported by other authors (25.8-44.7 g/100 g dm) (bloukas et al., 1997; muguerza et al., 2001; muguerza et al., 2002; beriain et al., 2011), since venison salchichon had a lower fat content than those made with pork and/or beef. quantitative descriptive sensory analysis significant inter-assay differences were found for all the sensory attributes studied, except for fat colour, salty taste and taste intensity. therefore, in all assays, the fat were white, presented scores ranged between 0.89 and 1.63 (0=white; 10=yellow), the salty taste was considered medium, approached 5.0, while taste intensity was judged to be moderate-to-high, scoring between 6.86 and 7.60. mean scores (± standard deviation) assigned by the tasting panel for each of the visual attributes studied are shown in table 3. assays table 2 physicochemical parameters (means±standard deviations) of venison salchichon with different percentage replacement of pork meat by olive oil. assay 1 assay 2 assay 3 assay 4 assay 5 assay 6 moisture (g/100 g) 31.69±2.29 32.07±1.64 31.15±0.46 30.79±0.73 32.02±0.26 30.40±0.09 fat (g/100 g dm) 19.04a±2.14 21.11a±0.06 29.91b±0.67 36.29c±1.09 37.88c,d±0.52 40.24d±1.58 protein (g/100 g dm) 59.57d±1.54 56.58c,d±1.97 54.19c±0.62 49.76b±1.39 48.06b±0.82 43.62a±0.55 different superscripts (a,b,c,d) in the same row denote significant differences (p<0.05). assay 1 (0% replacement); assay 2 (15% replacement); assay 3 (25% replacement); assay 4 (35% replacement); assay 5 (45% replacement); assay 6 (55% replacement). dm: dry matter. table 3 visual attributes (means±standard deviations) of venison salchichon with different percentage replacement of pork meat by olive oil. assay 1 assay 2 assay 3 assay 4 assay 5 assay 6 amount of fat 6.07c±0.93 6.00c±0.61 5.91c±0.75 4.65b±0.82 4.52b±0.88 3.28a±0.74 fat colour 0.89±0.64 1.16±0.46 1.31±0.52 1.27±0.67 1.63±0.90 1.46±0.68 lean colour 7.44b±0.50 6.81b±0.95 6.56b±0.70 7.41b±1.06 7.16b±0.89 5.84a±0.90 different superscripts (a,b,c) in the same row denote significant differences (p<0.05). assay 1 (0% replacement); assay 2 (15% replacement); assay 3 (25% replacement); assay 4 (35% replacement); assay 5 (45% replacement); assay 6 (55% replacement). ital. j. food sci., vol. 27 2015 447 made without or with smaller amounts of olive oil (assays 1, 2 and 3) displayed larger amounts of visible fat; as the proportion of olive oil increased, the amount of visible fat declined. lean venison was dark brown in all except assay 6, where it was pinker. similar findings were reported by muguerza et al. (2001) in a study of pamplona-style chorizo made with lean pork (75%) and pork meat (25%) partially replaced by olive oil (0, 10, 15, 20, 25 and 30%) and in a later study (muguerza et al., 2002) of salchichon made with lean pork, lean beef and varying proportions of pork backfat (10, 20 and 30%) partially replaced by olive oil (0 and 20%). these authors recorded significant inter-assay differences in colour, noting that colour intensity decreased as the amount of olive oil increased. by contrast, beriain et al. (2011), in another study of pamplona-style chorizo in which pork backfat was partially replaced by olive oil emulsified with alginate, found no significant difference between appearance profiles. however, in that study 71.4% of the tasting panel expressed a preference for chorizo in which 50% of pork backfat had been replaced by olive oil, on the grounds that its appearance was more appealing than that of the control, due to its effective imitation of the rice-grain effect typical of this type of chorizo (boe, 1980). mean scores (± standard deviation) assigned by the tasting panel for odour attributes are shown in table 4. assays containing the lowest amounts of olive oil (assays 1, 2 and 3) were judged to display the greatest odour intensity (7.83-8.04). the black pepper odour characteristic of salchichon weakened as the proportion of olive oil increased, being most intense in assays 1 and 2 (5.09-5.15). spicy and cured odour also decreased with increasing proportions of olive oil; the lowest intensity for both attributes (2.44 and 4.16, respectively) was recorded in assay 6 (55% replacement). finally, olive oil odour was identified from assay 3 onwards, becoming more intense as the proportion of olive oil increased. muguerza et al. (2002) also reported greater oil odour intensity with rising proportions of olive oil in place of pork backfat. scores for the attributes defining the texture profile are shown in table 5. the replacement of 35% or a high amount of pork meat by olive oil (assays 4, 5 and 6) was considered to give rise to excessive softness (hardness scores below 5). the control scored highest for chewiness (6.17), whereas the assays containing varying proportions of olive oil received scores of around 5. assays 1 and 2 (0 and 15% substitution) scored lowest for juiciness (4.63-5.18), the remainder received scores of between 6 and 6.8 points. finally, fat mouthfeel increased with higher proportions of olive oil, scores rising from 4.86 (assay 1) to 7.34 (assay 6); the panellist judged the mouthfeel of assays 5 and 6 (7.02 and 7.34, respectively) to be over-fatty. so, addition of olive oil to venison salchichon prompted an increase in juiciness and fat mouthfeel and a decrease in both hardness and chewiness. similar findings were reported by muguerza et al. (2001) who noted that types containing larger amounts of olive oil were too soft, although they recorded no difference in juiciness among types. bloukas et al. (1997), in a study of salchichon made with lean pork, lean beef and pork backfat partially replaced by olive oil, with or without added soy protein, table 4 odour attributes (means±standard deviations) of venison salchichon with different percentage replacement of pork meat by olive oil. assay 1 assay 2 assay 3 assay 4 assay 5 assay 6 odour intensity 7.95b±0.64 8.04b±0.58 7.83b±0.74 7.05a±0.87 6.58a±0.74 6.85a±0.62 black pepper odour 5.15c±0.63 5.09c±0.62 3.74b±0.75 3.64b±0.63 3.30b±0.75 2.33a±0.74 spicy odour 4.99d±0.77 4.35c,d±0.58 4.45c,d±0.78 3.96c±0.99 3.30b±0.86 2.44a±0.88 cured odour 6.08c±0.87 6.79c±0.81 5.97c±0.54 6.13c±0.65 4.91b±1.06 4.16a±0.88 olive oil odour 0.00a±0.00 0.00a±0.00 2.56b±0.78 3.33c±0.81 4.04d±1.20 6.01e±1.01 different superscripts (a,b,c,d,e) in the same row denote significant differences (p<0.05). assay 1 (0% replacement); assay 2 (15% replacement); assay 3 (25% replacement); assay 4 (35% replacement); assay 5 (45% replacement); assay 6 (55% replacement). table 5 texture attributes (means±standard deviations) of venison salchichon with different percentage replacement of pork meat by olive oil. assay 1 assay 2 assay 3 assay 4 assay 5 assay 6 hardness 6.05d±0.66 5.74d±0.80 4.91c±0.55 4.03b±0.79 3.12a±0.54 2.79a±0.91 juiciness 4.63b±0.61 5.18b±0.79 6.05a±0.77 6.80a±0.83 6.68a±0.57 6.03a±1.15 chewiness 6.17b±0.40 4.95a±0.42 5.20a±0.53 5.00a±0.00 5.00a±0.00 5.07a±0.27 fat mouthfeel 4.86a±0.75 4.88a±0.70 6.06b±0.86 6.64b,c±0.88 7.02c±0.98 7.34c±0.98 different superscripts (a,b,c,d) in the same row denote significant differences (p<0.05). assay 1 (0% replacement); assay 2 (15% replacement); assay 3 (25% replacement); assay 4 (35% replacement); assay 5 (45% replacement); assay 6 (55% replacement). 448 ital. j. food sci., vol. 27 2015 found significant differences as a function of the way oil was added: salchichon to which oil was added in liquid form was judged to be too soft, and obtained lower scores for odour and flavour intensity, whereas scores for salchichon made with olive oil combined with soy protein were similar to those of controls. finally, scores for taste attributes are shown in table 6. significant inter-assay differences were recorded for most taste-related attributes. olive oil taste was identified from assay 3 (25%) onwards, increasing as a function of the proportion of added olive oil, to a maximum score of 6.96 for assay 6 (55% substitution). spicy taste decreased with rising proportions of olive oil, which masked the black pepper taste characteristic of salchichon. by contrast, beriain et al. (2011) found no significant difference in taste between pamplona-style chorizo types made with and without olive oil. to summarise, variation in the percentage replacement of pork meat by olive oil had a marked influence on the results of descriptive sensory analysis. the replacement of 35% or more, of pork meat by olive oil, resulted in a reduction of the fat particles visibility. it also prompted a decrease in odour intensity, spicy odour, hardness and an increase of fat mouthfeel, together with the olive oil perception (odour and taste). by contrast, the replacement of 25% of pork meat by olive oil yielded a salchichon not greatly different in appearance, texture, odour and taste to the control. table 6 taste attributes (means±standard deviations) of venison salchichon with different percentage replacement of pork meat by olive oil. assay 1 assay 2 assay 3 assay 4 assay 5 assay 6 taste intensity 7.26±0.78 7.06±0.78 7.50±0.84 7.50±0.78 7.60±0.93 6.86±0.91 salty taste 5.07±0.27 5.00±0.00 5.00±0.00 5.00±0.00 5.00±0.00 5.04±0.13 pungent taste 2.86c±0.80 2.69b,c±0.78 1.93b±0.90 2.00b±0.81 2.11b±0.79 0.93a±0.48 pepper taste 4.06c±0.67 3.15b±0.90 2.66b±0.78 2.71b±0.72 2.58b±0.70 1.53a±0.74 olive oil taste 0.00a±0.00 0.00a±0.00 3.04b±0.78 3.51b±0.90 4.63c±0.82 6.96d±1.25 aftertaste intensity 7.42b±0.76 7.20b±0.73 8.02b±0.59 7.49b±0.85 7.59b±0.79 6.49a±0.94 different superscripts (a,b,c,d) in the same row denote significant differences (p<0.05). assay 1 (0% replacement); assay 2 (15% replacement); assay 3 (25% replacement); assay 4 (35% replacement); assay 5 (45% replacement); assay 6 (55% replacement). table 7 means and standard deviations of the scores obtained for different assays of venison salchichon with different percentage replacement of pork meat by olive oil in the consumers acceptance test. assay 1 assay 2 assay 3 assay 4 assay 5 assay 6 appearance 6.86b±1.96 7.01b±1.47 7.25b±1.71 6.63a,b±1.83 6.47a,b±1.67 5.84a±2.27 odour 7.60b±1.40 7.20b±1.46 7.10b±1.87 6.18a±2.39 6.06a±1.99 5.63a±2.43 taste 6.42±1.78 6.33±1.67 6.29±2.02 5.88±2.19 5.58±2.13 5.27±2.43 texture 5.91±2.09 6.30±2.06 6.39±2.19 5.93±2.26 5.83±2.40 5.31±2.43 overall acceptance 6.34±1.84 6.47±1.93 6.58±1.86 5.91±2.26 5.75±2.30 5.42±2.34 different superscripts (a,b) in any row denote significant differences (p<0.05). assay 1 (0% replacement); assay 2 (15% replacement); assay 3 (25% replacement); assay 4 (35% replacement); assay 5 (45% replacement); assay 6 (55% replacement). consumer tests acceptance test the scores awarded by the consumers for different assays of venison salchichon sausage with different percentage of olive oil added are shown in table 7. from these results, it can be concluded that all the samples were accepted because the average score was above 5.0 (satisfaction threshold). significant inter-assay differences were recorded for appearance and odour, but not for the rest of the attributes studied. assays 1, 2 and 3 received the highest scores for odour (7.10-7.60). therefore, the replacement of up to 25% of pork meat by olive oil yields a product as acceptable to the consumer, in terms of all sensory parameters, as controls containing no olive oil. preference test consumers ordered samples by degree of preference, giving 1 point to the least preferred and 6 to the most preferred (table 8). the preference order was assay 3 > assay 2 > assay 1 > assay 4 > assay 5 > assay 6. the friedman test showed significant differences (p<0.05) between assays. after having applied the fischer method to calculate the least significant difference (lsd), it can be affirmed that the samples from assay 6 were significantly different from the rest as the least preferred. on the other hand, the samples from assays 4 and 5 were not significantly different from each ital. j. food sci., vol. 27 2015 449 other. the samples from assays 1, 2 and 3 were not significantly different from each other as the most preferred by consumers. the samples from assays 1, 2 and 3 were the most preferred mainly for six reasons (good flavour, proper texture, pleasant odour, good aspect and attractive colour). among the reasons for preferring the sausage from assay 6 the least, 36.4% of consumers thought the texture were not right and 31.8% highlighted the bad flavour. they also stressed the bad aspect, disagreeable odour and unattractive colour. conclusions in sensory terms, low-fat venison salchichon in which equal or more than 35% of pork meat had been replaced by olive oil presented a lower acceptation than products containing less olive oil. its appearance was deemed less favourable due to poorer visibility of fat particles, the texture was regarded as over-soft, and the mouthfeel was considered excessively fatty. there was also a decrease in odour quality and intensity, as well as in cured and spicy odour, together with an unfavourable taste and odour of olive oil. the trained panel found that replacement of 25% of pork meat by olive oil yielded a salchichon not greatly different to the traditional product. consumers also preferred salchichon in which no more than 25% of the pork meat had been replaced by olive oil, largely because of its good taste and acceptable texture. acknowledgements the authors are grateful to the department of education and science of castilla-la mancha regional council for the award of a pre-doctoral grant, and to the university of castilla-la mancha for financing this study. references aoac. 1980. method 16245. determination of total nitrogen and protein nitrogen. in “official methods of analysis” 13th ed. association of official analytical chemists, washington dc, usa. beriain m.j., gómez i., petri e., insausti k. and sarriés m.v. 2011. the effects of olive oil emulsified alginate on the physico-chemical, sensory, microbial, and fatty acid profiles of low-salt, inulinenriched sausages. meat sci. 88: 189. bloukas j.g., paneras e.d. and fournitzis g.c. 1997. effect of replacing pork back fat with olive oil on processing and quality characteristics of fermented sausages. meat sci. 45: 133. boe (boletín oficial del estado). 1980. orden de 7 de febrero de 1980, norma de calidad para los productos cárnicos, embutidos crudos curados, en el mercado interior. boe 21 march 1980. codex stan 33-1981. standard for olive oils and olive pomace oils. available in: http://www.codexalimentarius.org. del nobile m.a., conte a., incoronato a.l., panza o., sevi a. and marino r. 2009. new strategies for reducing the pork back-fat content in typical italian salami. meat sci. 81: 263. hoffman l.c. and wiklund e. 2006. game and venison-meat for the modern consumer. meat sci. 74: 197. iso method 1442. 1973. determination of moisture content. in “international standards meat and meat products”. international organization for standarization, geneva, switzerland. iso method 1443. 1973. determination of total fat content. in “international standards meat and meat products”. international organization for standarization, geneva, switzerland. kayaardi s. and gök v. 2003. effect of replacing beef fat with olive oil on quality characteristics of turkish soudjouk (sucuk). meat sci. 66: 249. muguerza e., gimeno o., ansorena d., bloukas j.g. and astiasarán i. 2001. effect of replacing pork back fat with preemulsified olive oil on lipid fraction and sensory quality of chorizo de pamplona, a traditional spanish fermented sausage. meat sci. 59: 251. muguerza e., fista g., ansorena d., astiasarán i. and bloukas j.g. 2002. effect of fat level and partial replacement of pork back fat with olive oil on processing and quality characteristics of fermented sausages. meat sci. 61: 397. muguerza e., ansorena d., bloukas j.g. and astiasarán i. 2003. effect of fat level and parcial replacement of pork back fat with olive oil on the lipid oxidation and volatile compounds of greek dry fermented sausages. j. food sci. 68: 1531. owen r.w., mier w., giacosa a., hule w.e., spiegelhalder b. and bartsch h. 2000. phenolic compounds and squalene in olive oils: the concentration and antioxidant potential of total phenols, simple phenols, secoroids, lignans and squalene. food chem. toxicol. 38: 647. severini c., de pilli t. and baiano a. 2003. partial substitution of pork back fat with extra-virgin olive oil in salami products: effects on chemical, physical and sensorial quality. meat sci. 64: 323. une-iso 4121:2006. 2006. análisis sensorial. directrices para la utilización de escalas de respuestas cuantitativas. in: “análisis sensorial” 2ª ed. p. 195. aenor, madrid, españa. une-en iso 8589:2010. 2010. guía general para el diseño de salas de cata. in “análisis sensorial” 2ª ed. p. 53. aenor, madrid, españa. une-iso 8587:2010. 2010. análisis sensorial. metodología. ordenación. in “análisis sensorial” 2ª ed. p. 239. aenor, madrid, españa. utrilla m.c., garcía ruiz a. and soriano a. 2014. effect of partial reduction of pork meat on the physicochemical and sensory quality of dry ripened sausages: development of a healthy venison salchichon. meat sci. 98: 785. zomborszky z., szentmihály g., sarudi i., horn p. and szabó c.s. 1996. nutrient composition of muscles in deer and boar. j. food sci. 61: 625. paper received july 16, 2014 accepted january 24, 2015 table 8 total scores obtained for different assays of venison salchichon with different percentage replacement of pork meat by olive oil in the consumers preference test. assay 1 assay 2 assay 3 assay 4 assay 5 assay 6 total scores 169 172 183 148 144 108 assay 1 (0% replacement); assay 2 (15% replacement); assay 3 (25% replacement); assay 4 (35% replacement); assay 5 (45% replacement); assay 6 (55% replacement). paper ital. j. food sci., vol. 27 2015 409 keywords: consumer test, fat content, quantitative descriptive sensory analysis, venison salchichon sausage determination of the optimal fat amount in dry-ripened venison sausage m.c. utrilla1,3, a. soriano1,3* and a. garcía ruiz2,3 1department of analytical chemistry and food technology, faculty of sciences and technologies chemistries, university of castilla-la mancha, avda. camilo josé cela s/n, 13071 ciudad real, spain 2department of analytical chemistry and food technology, school of engineers agronomist, university of castilla-la mancha, ronda de calatrava 7, 13071 ciudad real, spain 3regional institute for applied scientific research (irica), university of castilla-la mancha, avda. camilo josé cela s/n, 13071 ciudad real, spain *corresponding author: tel. +34 926 295300, fax +34 926 295318, email: almudena.soriano@uclm.es abstract six types of salchichon sausage were made using cynegetic venison lean and different amounts of pork meat (40, 30, 25, 20, 15 and 10%) in order to choose the lowest fat content without decreasing the sensory quality of the traditional salchichon sausage. all samples were evaluated using quantitative descriptive sensory analysis, finding significant differences; as the amount of pork meat increased, sausages exhibited a lighter colour and an intense spice and cured odour, as well as being juicier and easier to chew. furthermore, consumer tests were carried out. all types of sausages were accepted by consumers (scores > 5.5 for all attributes) finding significant differences in the preference test. mailto:almudena.soriano@uclm.es 410 ital. j. food sci., vol. 27 2015 introduction the production of cynegetic deer in spain is high, accounting 148195 animals hunted in 2011 (magrama, 2014); however, its economic value is relatively low because it is considered to be simply a by-product of hunting, oriented to obtain flashy awards. in the autonomous community of castilla-la mancha (central spain), the area designated for hunting spans more than 7 million hectares of which almost 2 million are designated for big game hunting, mainly venison and wild boar (jccm, 2010). castillala mancha is also the main venison exporter in spain, constituting 80% of the total exportation, being germany its primary destination. despite the large venison production, its consumption is fairly limited in the region and in spain generally. venison is mainly consumed in certain rural areas and restaurants. cynegetic venison is a highly nutritive food characterized by a high protein and heme iron content, and a low presence of subcutaneous and intramuscular fat (zomborszky et al., 1996; hoffman and wiklund, 2006). in addition, this meat has specific organoleptic properties that differ from other species such as its intense and attractive red colour, tenderness and variety of flavours (soriano et al., 2009), reflecting the fact that the animals live in the wilderness and nourish on naturally occurring feed. a wide range of cured products are obtained from cinegetic deer meat, including cecina (drycured meat), and dry fermented sausages, as chorizo and salchichon. these are generally labelled “gourmet products” in the international market. to make venison salchichon, an appropriate amount of pork fat has to be added to obtain gradual drying as well as sensory characteristics such as juiciness, tenderness and flavor. in the traditionally produced cynegetic venison salchichon, the fat content is around 4050% (habitual practices of local manufacturers in castilla-la mancha region). today, this fat amount is considered excessive in terms of the who recommendations for a healthy diet (who, 2004), which suggest the consumption of lowfat foods. these recommendations are followed by meat manufacturers that are producing meat products containing smaller amounts of fat. on the other hand, an excessive amount of fat can cause an excessive oxidation of the lipids or rancidity (soriano et al., 2010). several studies have been carried out to characterise the physicochemical and sensory quality of venison (stevenson et al., 1992; peña et al., 1993; zomborszky et al., 1996; wiklund et al., 2001 and 2003). however, very few studies have been reported on microbiological, physicochemical and sensory characteristics of dry sausages made with venison (vioque et al., 2003; soriano et al., 2006; garcía ruiz et al., 2010; soriano et al., 2010; utrilla et al., 2014). until now, no study has focused on the reduction of fat in cured sausages made with meat from cynegetic species. however, several scientific studies have been carried out to reduce the fat content in dry-ripened sausages made with pork and/ or beef (papadima and bloukas, 1999; mendoza et al., 2001; muguerza et al., 2002; liaros et al., 2009; olivares et al., 2010; olivares et al., 2011), foal and pork meat (lorenzo and franco, 2012). the objective of this study was to obtain a healthier venison salchichon with the lowest fat content that at the same time maintains sensory characteristics of the traditional salchichon. material and methods raw materials lean venison (cervus elaphus) was obtained from hind legs of male deer obtained during the 2008-2009 hunting season on three neighbouring reserves in ciudad real (central spain). vegetation in the three reserves was very similar, comprising pine forests, woodlands and scrub. a total of 69 kg of venison was used for each replicate of the experiment. pork meat with a high fat content was obtained from castrated male pigs (progeny of a pietrain male x dalain female cross) raised intensively and slaughtered at the age of seven months. a total of 21 kg of pork meat was used for each replicate of the experiment. a commercial salchichon formula (ceylamix salchichón casero 933, manufacturas ceylan s.l., valencia, spain) was used, comprising salt, spices, lactose, saccharose, polyphosphates (e-450i, ii), sodium ascorbate (e-301) and potassium nitrate (e-252). cynegetic venison salchichon sausage production six types of venison salchichon were made, each containing a different proportion of pork meat (40, 30, 25, 20, 15 and 10%) and lean venison (60, 70, 75, 80, 85 and 90%, respectively). types were labelled from type 1 (40% pork meat and 60% lean venison) to type 6 (10% pork meat and 90% lean venison) (table 1). venison and pork meat were minced separately in an unger table 1 percentages of raw meats used to elaborate the different types of cynegetic venison salchichon. type venison (%) pork meat (%) 1 60 40 2 70 30 3 75 25 4 80 20 5 85 15 6 90 10 ital. j. food sci., vol. 27 2015 411 w-98 mincer (andher, campo de criptana, spain) with an 8 mm plate. venison was then mixed with the appropriate proportion of pork meat and the ceylamix commercial formula (33 g/kg mixture) previously dissolved in 1 l of cold mineral water, in an av-80 vacuum mixer (andher, campo de criptana, spain). the mixture was covered with a cotton cloth, and left to settle for 20 h at 0ºc, in order to the whole mass could get the spices and additives. it was then fed through an h52 pas hydraulic piston-based sausage stuffer connected to a vae-10 vacuum system (andher, campo de criptana, spain), into synthetic collagen skins, with a 38-40 mm diameter. horseshoeshaped salchichon sausages were then tied off at 60 cm intervals. average weight of sausages was 737 g. salchichon sausages were maintained at 20-22ºc and a relative humidity of 60% for 2 h, and finally ripened at 11°-12ºc and the relative humidity of 75% for 28 days, in a zanotti curing chamber (grupo momplet, valencia, spain). after ripening, salchichon sausages were vacuumpacked and stored at 8-10ºc for 45 days until its evaluation. the six types were made in duplicate, so the same experiment was repeated on two different dates, in order to maximize reproducibility of the results. quantitative descriptive sensory analysis the quantitative descriptive sensory analysis was carried out in a tasting room that was equipped in accordance with une-en iso 8589:2010. judges the evaluation of the samples was carried out by a panel test formed by 9 panellists (6 women, 3 men, ages 25-52 years). the panel was previously trained in the evaluation of the attributes and scales employing different commercial venison salchichon. the qualification of the panel members is based on reproducibility verification and concordance between the tasters. attributes a focus group was organized to discuss and choose the most apropiate attributes. the sensory evaluation of the attributes was carried out using non-structured scales of 10 cm and in accordance with une-iso 4121:2006. all the scales were anchored at the extremes with the terms “weak” and “very intense,” except for the colour intensity scales in which the colour was indicated at the extremes. the visual attributes evaluated were: amount of fat, fat colour (0=white; 10=yellow) and lean colour (0=light pink; 10=black). the colour scales used were photographs of different types of venison salchichon sausages. the olfactory attributes studied were: black pepper, spices and cured odour as well as odour intensity. the attributes that defined the texture profile of the samples were: hardness, juiciness, chewiness and fat mouthfeel. finally, taste attributes evaluated were the following: intensity of the taste, salty and pepper taste and intensity of the aftertaste. data collection was organized on paper. preparation of the samples the samples were presented to the panellists in 3 mm thick slices without skin, at 20°-22ºc (room temperature) and tagged (number-letter-number). three samples were evaluated per sesión at a time. unsalted crackers and mineral water were provided to the panellists to cleanse the mouth between samples. samples were presented in all possible orders at each tasting session in order to minimise any effects due to order of presentation. all samples were evaluated in duplicate. consumer tests the consumer tests were carried out in a tasting room equipped in accordance with une-en iso 8589:2010. consumers a group of 138 habitual consumers of salchichon sausage was used: 42 men aged between 20 and 49, and 96 women aged between 19 and 54. consumers were recruited from students, professors and staff of the food science and technology area of the university of castilla-la mancha. preparation of the samples samples were presented at 20°-22ºc (room temperature) in 3-mm slices, without skin, using a 3-character alphanumeric code. samples were presented in all possible orders at each tasting session in order to minimise any effects due to order of presentation. unsalted crackers and mineral water were provided to the panellists to cleanse the mouth between samples. consumers were instructed to carry out their evaluation for overall acceptability considering the cross section external appearance, odour, taste and texture of the slices. in one session, consumers evaluated the six samples corresponding to the six different types of venison salchichon sausage. acceptance test to grade the acceptability of each sample, consumers used a non-structured or linear hedonic scale of 10 cm, anchored at either end by the phrases “strongly like” and “strongly dislike”, enabling consumers to mark the point which best represented their satisfaction with the sample. attributes were evaluated in the order: odour, 412 ital. j. food sci., vol. 27 2015 aspect, texture, taste and overall acceptance. data collection was organized on paper. preference test a hedonic ranking test was used (une-iso 8587:2010), whereby each consumer was presented with a sample from each type and asked to order the samples by degree of preference, giving 1 point to the least preferred and 6 to the most preferred. statistical analysis one-way analysis of variance (anova) was performed to study the influence of the amount of fat in the attributes evaluated in the quantitative descriptive sensory analysis and the acceptance test. when the interaction was significant, the averages were compared using the studentnewman-keuls test. friedman’s (non-parametric) test was performed, following standard une-iso 8587:2010, to check the significance of differences between consumer preferences, and differences between particular sample means were analysed according the fisher’s least significance difference (lsd). all statistical procedures were carried out using the spss 19.0 statistical software package for windows xp (spss, inc, chicago, il, usa) with updating rights (license uclm 7876875). results and discussion quantitative descriptive sensory analysis visual attributes of the cynegetic venison salchichon sausage with different amount of pork meat added are shown in table 2. significant differences were found for the three studied attributes. the types with the highest fat content exhibited the pinkest colored lean while the types with the lowest fat content were dark brown. the fat colour in all samples was white except for samples bellowing type 6 (10% fat), which exhibited a more yellowish colour. this was possibly influenced by the darker colour of the lean. furthermore, all samples showed an amount of visible fat that was directly proportional to the pork meat added during the elaboration. attributes that defined the olfactory profile are shown in table 3. significant differences were found for all studied attributes. samples with the highest amount of fat, types 1, 2 and 3 (4025%), exhibited a higher intensity of odour (7.68.0) and a more pronounced spice, black pepper and cured odour in comparison to types 4, 5 and 6 (20-10%) which despite having elevated odour intensity (6.8-7.1) exhibited less intensity in all of the attributes studied. these results do not coincide with those obtained by mendoza et al. (2001), which did not find significant differences in the intensity of odour, obtaining scores between 6.5-7.5 (in a scale of 1-10) with regard to the fat content (6.3, 12.5 and 25% of fat). it should be noted that the authors obtained scores slightly lower than those obtained in this study, possibly due to the higher olfactory intensity of venison in comparison to beef and pork. on the other hand, odour intensity, spice and ripened odour presented similar scores than those obtained by garcía ruiz et al. (2010) in a study about sensory properties of venison sausages made with 50% venison lean and 50% pork meat. table 4 organizes the scores awarded by the panellists for the attributes that defined the texture profile of the cynegetic venison salchichon sausage with different quantities of pork meat added. samples bellowing type 1 exhibited the lowest table 2 visual attributes (means ± standard deviations) of the cynegetic venison salchichon with different pork meat added. type 1 type 2 type 3 type 4 type 5 type 6 amount of fat 7.19a±0.78 6.58b±0.93 5.95c±0.96 5.07d±0.92 3.86e±0.77 2.32f±0.59 fat colour 2.03a±0.73 2.28a±0.73 2.26a±0.85 2.23a±0.67 1.98a±0.65 2.85b±1.06 lean colour 3.12a±0.60 4.36b±1.10 3.92b±0.85 6.02c±1.07 5.76c±0.84 7.24d±1.06 different superscripts (a, b, c, d, e, f) in the same row denote significant differences (p<0.05). type 1 (40% pork meat); type 2 (30% pork meat); type 3 (25% pork meat); type 4 (20% pork meat); type 5 (15% pork meat); type 6 (10% pork meat). table 3 odour attributes (means ± standard deviations) of the cynegetic venison salchichon with different pork meat added. type 1 type 2 type 3 type 4 type 5 type 6 odour intensity 7.96a±0.72 7.67a±0.57 7.62a±0.64 6.78b±0.55 6.98b±0.98 7.10b±1.20 black pepper odour 6.29a±0.74 6.07a±1.06 5.90a±0.72 5.02b±1.29 5.09b±1.48 4.86b±1.93 spices odour 6.60a±0.73 6.33a±1.03 6.33a±0.87 5.32b±1.66 5.35b±0.93 5.45b±1.20 cured odour 7.12a±0.75 6.76a±0.91 6.81a±0.96 6.01b±1.71 5.67b±1.40 5.96b±0.98 different superscripts (a, b) in the same row denote significant differences (p<0.05). type 1 (40% pork meat); type 2 (30% pork meat); type 3 (25% pork meat); type 4 (20% pork meat); type 5 (15% pork meat); type 6 (10% pork meat). ital. j. food sci., vol. 27 2015 413 hardness (4.59). samples from types 2-6 showed values between 5.16-5.61, indicating a good texture for those sausages. therefore, the reduction in the addition of pork meat to levels below 30% did not negatively affect this attribute. however, samples from types 5 and 6 presented the lowest juiciness and were more difficult to chew. so the addition of pork meat below 20% to venison salchichon negatively influenced those attributes. moreover, as the amount of fat increased the juiciness and the fat mouthfeel also increased, highlighting that samples from types 2 and 3 presented similar values for those attributes. the scores for both attributes were more different between type 1 with more fat content (6.5 for juiciness and 6.4 for fat in the mouth) and type 6 with less fat content (3.7 for juiciness and 1.8 for fat in the mouth). other varieties of dry-fermented sausages evaluated by a trained panel, showed higher scores for texture attributes (mainly hardness and juiceness) as the fat level increased (papadima and bloukas, 1999; liaros et al., 1999; mendoza et al., 2001; lorenzo and franco, 2012). in addition, the values found in this study for juiciness, chewiness and fat mouthfeel were similar to those reported by garcía ruiz et al. (2010) in venison sausages (50% lean venison-50% pork meat). table 5 shows the means and standard deviations of the taste attributes of the different types of cynegetic venison salchichon sausages. significant differences were not found for the salty and black pepper taste attributes; all of the samples exhibited a proper salty taste with scores ranging between 5.0 and 5.3 and a pepper taste with scores from 4.2 to 4.9. on the other hand, significant differences were found for the taste intensity which was slightly lower for the types with the least fat content (5 and 6), and for the aftertaste intensity which was lower for type 6, with the lowest fat content, owing to the fact that lipids experiment lipolysis and lipid oxidation during the curing process that contributes the flavour (samelis et al., 1993). mendoza et al. (2001) also found significant differences in the taste intensity of the dry sausages in accordance with the fat content. the samples with the lowest fat content (6.5% and 12.5%) received lower scores ranging between 5.5 and 5.9 while the samples with the highest fat content (25%) received a score of 7.3. these scores are slightly lower than those obtained in this study (6.5-7.8) possibly due to the intense taste of cynegetic venison and its special organoleptics properties. garcía ruiz et al. (2010) found in its study of venison sausages elaborated using 50% lean venison and 50% pork meat, scores of 7.77 for taste intensity, 5.10 for salty taste and 7.38 for aftertaste intensity. in summary, results obtained in the quantitative descriptive sensory analysis were highly influenced by the fat content of the samples even though all samples were accepted by the tasting panel (whitout sensory defects). these results do not coincide with those obtained by muguerza et al. (2002) which determined that the cured sausages made with 10% pork backfat were unacceptable from a sensory point of view because of its winkled surface and excessive hardness. consumer tests acceptance test the scores awarded by the consumers for different types of cynegetic venison salchichon sausage with different pork meat added are shown in table 6. from these results, it can be concluded that all the samples were accepted table 4 texture attributes (means ± standard deviations) of the cynegetic venison salchichon with different pork meat added. type 1 type 2 type 3 type 4 type 5 type 6 hardness 4.59a±0.71 5.16b±0.72 5.19b±0.88 5.61b±0.84 5.27b±0.74 5.39b±1.11 juiciness 6.55a±0.86 5.82b±0.56 6.09b±0.60 4.98c±1.03 4.18d±0.98 3.72e±1.13 chewiness 4.99a±0.44 4.96a±0.23 5.26a±0.67 5.12a±0.52 6.07b±0.86 6.28b±0.47 fat mouthfeel 6.43a±0.85 5.81b±1.23 5.76b±1.11 4.46c±1.16 3.32d±0.78 1.80e±0.97 different superscripts (a,b,c,d,e) in the same row denote significant differences (p<0.05). type 1 (40% pork meat); type 2 (30% pork meat); type 3 (25% pork meat); type 4 (20% pork meat); type 5 (15% pork meat); type 6 (10% pork meat). table 5 taste attributes (means ± standard deviations) of the cynegetic venison salchichon with different pork meat added. type 1 type 2 type 3 type 4 type 5 type 6 taste intensity 7.46a±0.84 7.32a±0.66 7.76a±0.86 7.50a±0.84 6.71b±0.77 6.52b±1.00 salty taste 5.10±0.31 5.09±0.67 5.33±0.62 5.14±0.36 5.04±0.57 5.04±0.54 pepper taste 4.90±1.05 4.23±0.59 4.84±1.09 4.74±1.19 4.27±1.17 4.69±1.21 aftertaste intensity 7.38a±0.86 7.26a±0.45 7.68a±0.89 7.55a±0.90 7.27a±0.77 6.67b±1.00 different superscripts (a,b) in the same row denote significant differences (p<0.05). type 1 (40% pork meat); type 2 (30% pork meat); type 3 (25% pork meat); type 4 (20% pork meat); type 5 (15% pork meat); type 6 (10% pork meat). 414 ital. j. food sci., vol. 27 2015 table 6 means and standard deviations of the scores obtained for different types of cynegetic venison salchichon in the acceptance test. type 1 type 2 type 3 type 4 type 5 type 6 aspect 7.18a±2.04 6.84a±1.89 7.19a±1.79 5.80b±2.16 5.91b±2.01 5.47b±2.28 odour 7.39a±1.80 6.66b±2.08 7.25a±1.75 6.22b,c±2.06 6.47b,c±2.09 6.01c±2.23 taste 7.12a±1.80 6.82a±1.90 7.14a±1.78 6.32b±2.07 6.08b±2.25 5.94b±2.17 texture 7.22a±1.96 6.86a,b±1.82 7.14a±1.94 6.36b,c±2.12 6.34b,c±2.19 6.22c±2.06 overall acceptance 7.16a±1.93 6.93a,b±1.85 7.20a±1.81 6.48b,c±2.00 6.11c±2.21 5.90d±2.12 different superscripts (a,b,c,d) in any row denote significant differences (p<0.05). type 1 (40% pork meat); type 2 (30% pork meat); type 3 (25% pork meat); type 4 (20% pork meat); type 5 (15% pork meat); type 6 (10% pork meat). because the average score was above 5.0 (satisfaction threshold). the consumers found significant differences for all the attributes studied. samples from types 1, 2 and 3 were awarded higher scores for aspect (6.8-7.2), taste (6.8-7.1) and overall acceptance (6.9-7.2). the score for odour was also higher for types 1 and 3. an addition of fat between 25% and 40% to elaborate cynegetic venison salchichon sausage therefore appears to provide, at least in the opinion of the habitual sausage consumers, better organoleptics characteristics than a lower addition. olivares et al. (2011) found high consumer acceptability for aroma and overall quality in dryripened pork sausages elaborated with 20% and 30% pork meat than those with 10% pork meat. preference test after having applied the fischer method to calculate the least significant difference (lsd), the consumer preference scores for each of the samples yielded the following order of preference: type 3 > type 1 > type 2 > type 4 > type 5 > type 6. the samples achieving the greatest consumer preference were those from types 1, 2 and 3, scores differing significantly from those awarded to types 4 and 5, which achieved a lower degree of consumer preference; type 6 samples received the lowest scores. samples from types 1, 2 and 3 were preferred over the rest for six reasons: good flavour, appropriate texture, pleasant odour, acceptable fat content, good appearance and attractive colour; type 6 was the least preferred, due to poor flavour and inappropriate texture. the results obtained from the preference test coincide with those of the acceptance test leading the authors to conclude that the quantity of fat added to venison salchichon sausage should be at least 25% to achieve a good sensory quality similar to that of the traditional product. conclusions the results obtained in the quantitative descriptive sensory analysis and the consumer tests perfectly coincide revealing that, from a sensory point of view, using 25% of pork meat and 75% of venison lean is enough. such a quantity of fat assures proper texture for this type of product helping to attain a satisfactory odour, taste and appearance for the consumer as well as similar attributes to those of traditionally made salchichon sausage with a higher fat content. acknowledgements the authors are grateful to the department of education and science of castilla-la mancha regional council for the award of a pre-doctoral grant, and to the university of castilla-la mancha for financing this study. references garcía ruiz a., mariscal c., gonzález viñas m.a. and soriano a. 2010. influence of hunting-season stage and ripening conditions on microbiological, physicochemical and sensory characteristics of venison (cervus elaphus) chorizo sausages. ital. j. food sci. 22: 386-394. hoffman l.c. and wiklund e. 2006. game and venisonmeat for the modern consumer. meat sci. 74: 197-208. jccm gobierno de castilla-la mancha. 2010. la caza en castilla-la mancha. in: medio rural. planes, programas y campañas. (http://www.jccm.es). liaros n.g., katsanidis e. and bloukas j.g. 2009. effect of the ripening time under vacuum and packaging film permeability on processing and quality characteristics of lowfat fermented sausages. meat sci. 83: 589-598. mendoza e., garcía m.l., casas c. and selgas m.d. 2001. inulin as fat substitute in low fat, dry fermented sausages. meat sci. 57: 387−393. ministerio de agricultura, alimentación y medio ambiente (magrama). 2014. avance del anuario de estadística del magrama 2013. available on: http://www.magrama.gob.es muguerza e., fista g., ansorena d., astiasaran i. and bloukas j.g. 2002. effect of fat level and partial replacement of pork backfat with olive oil on processing and quality characteristics of fermented sausages. meat sci. 61: 397-404. olivares a., navarro j.l., salvador a. and flores m. 2010. sensory acceptability of slow fermented sausages based on fat content and ripening time. meat sci. 86: 251-257. olivares a., navarro j.l. and flores m. 2011. effect of fat content on aroma generation during processing of dry fermented sausages. meat sci. 87: 264-273. papadima s.n. and bloukas j.g. 1999. effect of fat level and storage conditions on quality characteristics of traditional greek sausages. meat sci. 51: 103-113. ital. j. food sci., vol. 27 2015 415 paper received july 10, 2014 accepted november 12, 2014 peña f., domenech v. and molera m. 1993. composición de la canal de ciervas (cervus elaphus) de sierra morena. periodo estival. archivos de zootecnia 42: 115-124. samelis j., aggelis g. and metaxopoulos j. 1993. lipolytic and microbial changes during the natural fermentation and ripening of greek dry sausages. meat sci. 35: 371-385. stevenson j.m., seman d.l. and littlejohnl r.p. 1992. seasonal variation in venison quality of mature farmed red deer stags in new zealand. j. anim. sci. 70: 1389-1396. soriano a., cruz b., gómez l., mariscal c. and garcía ruiz a. 2006. proteolysis, physicochemical characteristics and free fatty acid composition of dry sausages made with deer (cervus elaphus) or wild boar (sus scrofa) meat: a preliminary study. food chem. 96: 173-184. soriano a., sánchez-migallón b., utrilla m.c., villaseñor p.j., montoro v., vicente j. and garcía ruíz a. 2009. influencia del tiempo de eviscerado y de desollado de la canal del ciervo cinegético sobre los parámetros físico-químicos, microbiológicos y sensoriales de su carne. proceedings of v congreso nacional de ciencia y tecnología de los alimentos, murcia, españa. soriano a., mariscal c., utrilla m.c. and garcía ruiz a. 2010. free fatty acids and lipid oxidation in venison chorizo sausages made at different stages of the hunting season and under different ripening conditions. ital. j. food sci. 22: 275-284. une-iso 4121:2006. 2006. análisis sensorial. directrices para la utilización de escalas de respuestas cuantitativas. in: análisis sensorial. 2ª edición. aenor. madrid. españa. pp. 195-209. une-en iso 8589:2010. 2010. guía general para el diseño 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2001. fatty acid composition of m. longissimus lumborum, ultimate muscle ph values and carcass parameters in reindeer (rangifer tarandus tarandus l) grazed on natural pasture or fed a commercial feed mixture. meat sci. 58: 293-298. world health organisation (who). 2004. draft global strategy on diet, physical activity and health. in: http://www. who.int/es. zomborszky z., szentmihályi g., sarudi i., horn p. and szabo c.s. 1996. nutrient composition of muscles in deer and boar. j. food sci. 61: 625-635. http://www.who.int/es http://www.who.int/es ijfs#413_rustioni_bozza   ital. j. food sci., vol 28, 2016 510 paper grape seed ripening evaluation by ortho-diphenol quantification l. rustioni* and o. failla dipartimento di scienze agrarie e ambientali produzione, territorio, agroenergia, università degli studi di milano, via celoria 20133, milano, italy *corresponding author: laura.rustioni@unimi.it abstract two millennia of viticulture recognize the seed browning importance on tannin ripening and, thus, on grape and wine quality. this color change was recently attributed to phenolic oxidations. however an objective chemical index able to quantify the oxidation status of seed tannins was missing, probably due to the heterogeneous oxidation polymerizations. this work suggests the adoption of the ortho-diphenol quantification as indication of the tannin ripening process, because ortho-dihydroxylated substitutions are highly susceptible to oxidation. the method proposed is based on the ortho-diphenol characteristic complexation with molybdenum. different cultivars (merlot, pinot noir, croatina, aladasturi, alexandrouli, odjaleshi and tavkveri) were studied during three vintages in oltrepò pavese, italy. the color darkening correlated with the ortho-diphenol decrease. we believe this index could find useful applications in viticulture, supporting harvesting time decisions. keywords: vitis vinifera, tannins, seed browning, viticulture, wine quality   ital. j. food sci., vol 28, 2016 511 1. introduction ”… naturalis autem maturitas est, si cum expresseris vinacea, quae acinis celantur, iam infuscata et nonnulla praeter modum nigra fuerint. nam colorem nulla res vinaceis potest adferre nisi naturae maturitas …”. in his treatise “de re rustica”, columella (4-70 a.d.) suggested to use the seed darkening as the best grape ripening index. in 2000, kennedy, matthews and waterhouse described the seed color change during berry development, and, in 2005, ristic and illand, published a color chart to define the grape seed ripening. two millennia of grapevines cultivation confirmed the importance of grape seed color. also winegrowers are aware of the importance of this character: traditionally they check the seed color to evaluate the grape ripening status. despite the increased knowledge in grape chemistry and physiology, the visual observation of the seed color is still one of the best available methods to evaluate the grape phenolic ripening. this method has some disadvantages: i) it is a subjective evaluation; ii) seeds have not an homogeneous color; iii) considering the color chart published by ristic and illand (2005), it is not easy to discriminate the different brown tonality, especially in the last phenological steps (the most important for ripening estimation). for decades, a number of researchers focused their attention on phenolic content and polymeric subunit composition (kennedy et al., 2000) or extractability during winemaking (rolle et al., 2013). they payed attention to the phenolic concentration, overlooking this evident physiological color change. it should be noted that rolle et al. (2013) proposed an interesting acoustic method based on the physiological seed hardening during ripening, but the instruments required for this analysis are not widely diffused among grape and wine analytical laboratories. traditionally, winemakers consider seed lignification as the main responsible of this color variation. in their belief, lignin deposition would act as a barrier against tannins extraction, resulting also in a decrease in the detectable total phenolics (ribereaugayon et al., 1998). however, the grape seed hardening is due to the lignification of the inner layers of the outer integument (ristic and iland, 2005), whereas nearly all of the soluble seed phenolics are localized in the thin-walled cells between the epidermis and the inner lignified layers (adams, 2006). adams (2006) also suggests that the seed browning characteristic of fruit ripening is the result of tannins and flavan-3-ols oxidation. also kennedy et al. (2000) proposed seed polyphenol oxidation as the best explanation for their ripening study results. pilati et al. (2007) described a rapid accumulation of h2o2 and an activation of the ros scavenging enzymes at veraison. thus, the hypothesis of a characteristic oxidative burst during ripening seems to be agreed among researchers. in wine industries, tannins play a fundamental role, affecting the product quality in terms of astringency, body and bitterness. their organoleptic feature change during ripening. at the moment, winegrowers usually assess the seed tannin ripening status by: i) a subjective visual color evaluation; and/or ii) a subjective organoleptic estimation by tasting; and/or iii) an approximate relationship between the anthocyanin accumulation or total phenolic content and the seed tannins evolution. thus, an objective and accessible method to describe seed tannin ripening is still missing. the aim of this work is to develop an index representative of the oxidative status of the seed phenolic compounds. 2. materials and methods grapevines were all cultivated in the same germplasm collection located in oltrepò pavese (lombardy region, northern italy) already described in rustioni et al. (2013). plant material was collected during 3 growing seasons: 2009, 2010 and 2011. in the first   ital. j. food sci., vol 28, 2016 512 experimental year, merlot and pinot noir grapes were studied. in 2010, also croatina (a local cultivar) was analyzed, together with merlot and pinot noir. in 2011, other 4 cultivars were included in the study: aladasturi, alexandrouli, odjaleshi and tavkveri (all of them are georgian varieties). the list of cultivars and sampling dates is reported in si 1. all samples were collected in 3 biological replications and the fresh seeds were extracted. a number of berries per replication were analyzed (depending on the seed number) as shown in si 1. berries were weighed and seeds were separated, counted, weighed and the color class was attributed following ristic and illand (2005). seeds were then extracted in 25 ml of methanol for 20 hours, and, then, in other 25 ml of methanol for 4 hours. all the extractions were performed in dark conditions and under continuous shaking. finally the two extracts were mixed and kept at -20°c until analysis. within 3 months samples were analyzed following the method proposed by maestro durán et al. (1991). two solutions were prepared: sol. a (water:ethanol 50:50) and sol. b (5% sodium molybdate in sol. a). 10 ml of diluted sample were added by 2 ml of sol. a (blank) and with sol. b (reacted) for 15 minutes. the reacted sample was then read against the blank at 370 nm by a jasco 7800 spectrophotometer (jasco, mary’s court, easton, maryland). dilutions were set up to optimize the absorbance range. a calibration curve was obtained by using standard caffeic acid solutions. samples were also reacted with the folin ciocalteu solution to quantify the total phenolic content following di stefano, cravero and gentilini (1989). data were statistically analyzed using the spss® statistical software (version pasw statistics 19, spss inc, chicago, illinois). 3. results and conclusions the list of samples analyzed, together with some general information, are reported in si1. the number of berries considered depended on the expected number of seeds of each cultivar. generally, in 50 ml of methanol, about 20-50 seeds were extracted. however, taking into account the seeds and berries number and weights, it was possible to elaborate the data concerning total phenolic content and ortho-diphenols concentrations in relation to the sampled grapes (e.g.: mg/seed; mg/kg of grapes). information concerning the number of seeds per berry as well as their phenolic content could be a useful support for winemaking technique optimization (maceration timing, seeds separation, aging expectations …) (table 1). these data could also be interesting for phenotypic characterization (rustioni et al., 2014). however the important environmental effect on these parameters should not be forgotten. as an example, in our experimental conditions, pinot noir generally had a high number of seeds/berry, and these seeds had a high phenolic concentration. in the opposite situation we found odjaleshi. nevertheless, intermediate characteristics were also recorded (e.g.: aladasturi had a lot of seeds but they were not particularly concentrated in phenolic compounds). seed browning was studied following the method proposed by ristic and illand (2005). fig. 1 reports the obtained data. in fig. 1a, each point represents the average value of the three biological replications: the seed color level generally increased during berry development. in fig. 1b each dataset (cultivar and year) is reported in a different color. it appears that the trend was consistent in all the varieties. nevertheless, an important variability among the samples was recorded. the explanation could be found in the vintage and cultivar diversity (phenological shifts) as well as in the ripening degree variability among seeds.   ital. j. food sci., vol 28, 2016 513 table 1: cultivar characteristics: seed phenolic content and number of seeds per berry. cultivar average standard deviation minimum maximum seed phenolic content mg/seed pinot noir 1.520 0.698 0.373 2.922 merlot 1.022 0.519 0.220 2.311 croatina 1.002 0.583 0.287 2.617 tavkveri 0.746 0.402 0.206 1.606 alexandrouli 0.596 0.144 0.347 0.844 aladasturi 0.505 0.319 0.226 1.358 odjaleshi 0.422 0.233 0.179 1.139 number of seeds per berry aladasturi 3.2 0.2 2.9 3.5 pinot noir 2.3 0.4 1.6 3.3 tavkveri 2.2 0.6 1.3 3.4 alexandrouli 1.8 0.2 1.4 2.2 merlot 1.7 0.3 1.1 2.8 croatina 1.6 0.3 1.1 2.9 odjaleshi 1.5 0.2 1.1 2.1 figure 1: seed color change during the growing season. in figure 1a each point represents the average value of the three biological replications. in fig. 1b each dataset (cultivar and year) is labeled by a different symbol. the obtained regression lines are classified depending on the sampling year (2009: light grey; 2010: dark grey; 2011: black). a similar variability was found in the ortho-diphenol content decreasing trends during berry development (fig. 2). also in this case we attribute this heterogeneity to vintage, cultivar and seed variability. however, we observed a consistent decrease in the orthodiphenol concentration in seeds (fig. 2b), and we propose to adopt this value as an indicator of the grape phenolic ripening status. recently, the central role of phenolic   ital. j. food sci., vol 28, 2016 514 oxidation in seed browning process has been underlined (kennedy et al., 2000; ristic and iland, 2005; adams, 2006; pilati et al., 2007). figure 2: ortho-diphenol concentration decrease during the growing season. in fig. 2a: each point represents the average value of the three biological replications. in fig. 2b each dataset (cultivar and year) is labeled by a different symbol. the obtained regression lines are classified depending on the sampling year (2009: light grey; 2010: dark grey; 2011: black). however, in our knowledge, an “in deep” assay able to quantify tannin oxidation does not exist: via-radical polymerizations produce heterogeneous products due to the high reactivity of the instable reaction intermediates. ortho-diphenol could be easily oxidized because the resulting phenoxyl semi quinone radical is stabilized by a second oxygen atom, while meta-diphenol are less susceptible to oxidation (waterhouse and laurie, 2006). thus, the catechol (ortho-diphenol) moieties are probably the most oxidizable groups in flavan-3-ols and proanthocyanidins. for that reason, it is possible to correlate the decrease in ortho-diphenol concentration to the oxidation processes characteristic of seed ripening. a number of studies have been carried out to clarify the role of anthocyanins-metal complexes on fruits and foods colors, and the importance of the orthodiphenol substitutions are well known (kondo et al., 1992; boulton 2001). of course, complex solutions such as grape and wine could encourage further studies considering multiway interactions (rustioni, 2015). nevertheless, in our knowledge, considering non pigmented phenolics, any paper reports interferences in the ortho-dihydroxylated quantification through molybdenum complexation by copigments. fig. 3 reports the correlation between the ortho-diphenol quantification and the seed color level (ristic and iland, 2005). the data dispersion should be attributed to the fundamentally different approaches of the tested methods. nevertheless, the trend clearly appears: a decrease in ortho-diphenol content corresponds to the browning process characteristic of seed ripening. the majority of the tannin synthesis occur before veraison. however, winegrowers are aware of the quality impact produced by the phenolic evolution during fruit ripening. traditionally they use a visual inspection of the seed browning as signal of the phenolic ripening status, which results from proanthocyanidin oxidation. thus, the method proposed by ristic et al. (2005) improved this qualitative evaluation. however, this technique is limited by the color inhomogeneity and by the subjectivity of the records.   ital. j. food sci., vol 28, 2016 515 figure 3: correlation between ortho-diphenol concentration and seed color. thus, the aim of the present work is the production of an objective chemical index able to describe the tannin seed ripening. it does not require exclusive equipment and it is easy and fast to achieve. for these reasons we hope it will be a useful and practical support for the grape and wine industry. moreover, this index could be adopted by researchers to characterize fruit quality in relation to treatments or vineyard managements. references adams d.o. 2006. phenolics and ripening in grape berries. am. j. enol. vitic. 57(3):249-256. boulton r. 2001. the copigmentation of anthocyanins and its role in the color of red wine: a critical review. am. j. enol. vitic. 52(2):67-87. columella l.g.m. 4-70 a.d. de re rustica, liber undecimus. di stefano r., cravero m.c. and gentilini n. 1989. metodi per lo studio dei polifenoli dei vini. l’enotecnico maggio: 8389. kennedy j.a., matthews m. and waterhouse a.l. 2000. changes in grape seed polyphenols during fruit ripening. phytochemistry 55:77-85. kennedy j.a., matthews m.a. and waterhouse a.l. 2000. changes in grape seed polyphenols during fruit ripening. phytochemistry 55(1):77-85. kondo t., yoshida k., nakagawa a., kawai t., tamura h. and goto t. 1992. structural basis of blue colour development in flower petals from commelina communis. nature 358:515-518. maestro durán r., borja padilla r., martín martín a., fiestas ros de ursinos j.a. and alba mendoza j. 1991. biodegradación de los compuestos fenólicos presentes en el alpechin. grasa y aceites 42(4):271-276. pilati s., perazzolli m., malossini a., cestaro a., demattè l., fontana p., dal ri a., viola r., velasco r. and moser c. 2007. genome-wide transcriptional analysis of grapevine berry ripening reveals a set of genes similarly modulated during three seasons and the occurance of an oxidative burst at véraison. bmc genomics 8: 428. url http://www.biomedcentral.com/1471-2164/8/428 ribereau-gayon p., glories y., maujean a. and dubourdieu d. 1998. trattato di enologia ii chimica del vino stabilizzazione trattamenti. edagricole 141-204. ristic r. and iland p.g. 2005. relationships between seed and berry development of vitis vinifera l. cv shiraz: developmental changes in seed morphology and phenolic composition. australian journal of grape and wine research 11:43-58.   ital. j. food sci., vol 28, 2016 516 rolle l., giacosa s., torchio f., perenzoni d., rìo segade s., gerbi v. and mattivi f. 2013. use of instrumental acoustic parameters of winegrape seeds as possible predictors of extractable phenolic compounds. j. agric. food chem. 61:87528764 rustioni l. 2015. the effect of copper ions on color in extracts of cabernet sauvignon and sangiovese skins under wine-like conditions. south african journal of enology and viticulture 36(3):389-392. rustioni l., basilico r., fiori s., leoni a., maghradze d. and failla o. 2013. grape colour phenotyping: development of a method based on the reflectance spectrum. phytochemical analysis 24(5):453-459. rustioni l., maghradze d., popescu c.f., cola g., abashidze e., aroutiounian r., brazão j., coletti s., cornea v., dejeu l., dinu d., eiras dias j. e., fiori s., goryslavets s., ibáñez j., kocsis l., lorenzini f., maletic e., mamasakhlisashvili l., margaryan k., mdinaradze i., memetova e., montemayor m.i., muñoz-organero g., nemeth g., nikolaou n., raimondi s., risovanna v., sakaveli f., savin g., savvides s., schneider a., schwander f., spring j.l., pastore g., preiner d., ujmajuridze l., zioziou e., maul e., bacilieri r. and failla o. 2014. first results of the european grapevine collections' collaborative network: validation of a standard eno-carpological phenotyping method. vitis 53:219-226. waterhouse a.l. and laurie v.f. 2006. oxidation of wine phenolics: a critical evaluation and hypotheses. am. j. enol. vitic. 57(3):306-313. paper received january 21, 2016 accepted march 7, 2016 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (sp1) 1–11 issn 1120-1770 online, doi 10.15586/ijfs.v33isp1.1961 1 p u b l i c a t i o n s codon bacterial conjugated linoleic acid bio-fortification of synbiotic yogurts using propionibacterium freudenreichii as adjunct culture omid zaheda, kianoush khosravi-daranib*,  amir mohammad mortazavianb, abdorreza mohammadib astudent research committee, department of food science and technology, national nutrition and food technology research institute, faculty of nutrition science and food technology, shahid beheshti university of medical sciences, tehran, iran; bdepartment of food science and technology, national nutrition and food technology research institute, faculty of nutrition science and food technology, shahid beheshti university of medical sciences, tehran, iran *corresponding author: k. khosravi darani, (prof. of food biotechnology) national nutrition and food technology research institute, faculty of nutrition sciences and food technology, shahid beheshti university of medical science, p.o. box: 19395-4741, tehran, iran. email: k.khosravi@sbmu.ac.ir and kiankh@yahoo.com received: 20 september 2020; accepted: 28 december 2020; published: 6 january 2021 © 2021 codon publications open access paper abstract in this study, propionibacterium freudenreichii was used for in situ production of conjugated linoleic acid (cla) in yogurt. firstly, effects of process variables, including strain type, percentage of milk fat, percentage of inoculum, quantity of sunflower oil, concentration of inulin, temperature of fermentation and time of storage at 4°c, on production of cla by propionibacterium freudenreichii were investigated using screening method of the plackett– burman design. then optimisation of cla production process was conducted using three major factors of milk fat percentage, inulin concentration and storage time at 4°c using central composite design. analysis of variance established that the models were highly significant (p ≤ 0.05). the model demonstrated that the production of cla was affected by these three factors. optimised cla production by propionibacterium freudenreichii ssp. shermanii in yogurts was achieved after 17 days of storage at 4°c in skim-milk containing 1.75% (w/w) fat and 2.25% (w/v) inulin as prebiotic. reconfirmation test established that at the highlighted optimum conditions, the highest concentration of produced cla was 6.4 mg g–1 lipid in yogurt, which is a 256% increase in total cla production, compared with control samples. results demonstrated that propionibacterium freudenreichii ssp. shermanii not only leads to production of synbiotic yogurts containing inulin but also increases cla production in yogurts. keywords: conjugated linoleic acid, probiotic, propionibacterium freudenreichii, yogurt introduction in addition to nutritional and sensory characteristics of food products, health beneficial aspects are other important criteria for consumers to choose food products. one of the best manners to receive essential nutrients with minimum side effects is enrichment of food products (grunert, 2005). functional foods play important roles in this area as tendency to consume functional foods has increased recently. such characteristics are found in a new group of products called synbiotics, which contain probiotics and prebiotics simultaneously (holzapfel and schillinger, 2002). various food products are established as probiotic carriers, of which fermented dairy products, such as yogurt and cheese, include the largest proportion in research and marketing (pandey and mishra, 2015). propionic acid bacteria (pab) are widely applied as beneficial probiotic bacteria in several food technologies mailto:k.khosravi@sbmu.ac.ir mailto:kiankh@yahoo.com 2 italian journal of food science, 2021; 33 (sp1) omid zahed et al. 2006; kim, 2003; ross et al., 2010), inoculum size (yang et al., 2017), ph-value (cousin et al., 2016), incubation and fermentation temperatures (khan et al., 2011), added prebiotics (ogawa et al., 2001), la-rich sources (xu et al., 2004), dissolved oxygen (kim et al., 2000) and storage time at 4°c (akalin et al., 2007). therefore, optimisation of conditions is critical for the growth and production of cla by pab (khodaiyan et al., 2008). the aim of this study was to investigate factors affecting cla production in yogurts by p. freudenreichii ssp. freudenreichii and ssp. shermanii using the plackett–burman design (pbd). in addition, effect of variables (bacterial strains, milk fat concentration, inoculum percentage, prebiotic (inulin) concentration, sunflower oil quantity, fermentation temperature and storage time at 4°c) on production of cla was investigated. to optimise the most important affecting factors, response surface methodology (rsm) design was used in yogurts containing p. freudenreichii ssp. shermanii for production of cla. materials and methods materials skim-milk powder and 40% (w/w) fat cream were kindly gifted by pak dairy, tehran, iran. inulin powder with an average degree of polymerisation of ≥25 was provided by ava salamat javid, tehran, iran. sunflower oil (margarine foods, tehran, iran) was purchased from supermarkets. cla standard was purchased from sigma, st. louis, mo, usa. all analytical reagents and chemicals were purchased from merck, darmstadt, germany. all solvents used were of analytical or high performance liquid chromatography (hplc) grade. preparation of cultures a commercial yogurt starter culture (yoflex express 1.0) containing streptococcus thermophilus (st) and l.  delbrueckii ssp. bulgaricus (lb) was selected because of the mild acid-production activity of pab used in this study. the yoflex express 1.0 was purchased from chr. hansen, horsholm, denmark, and used based on manufacturer’s recommendations. commercial starter culture (ps-4) containing p. freudenreichii ssp. shermanii was purchased from chr. hansen, horsholm, denmark. cultures were obtained in freeze-dried (dvs) form and stored at –18°c. the pab (ps-4) was weighed to prepare an initial count of 8 log colony-forming unit (cfu) ml–1. pre-cultures were prepared by dissolving each culture in 60 ml of sterilised skim-milk and activating them at 42°c for 20 min before use. the p. freudenreichii ssp. freudenreichii ptcc no. 1674 was provided by the research and (zárate et al., 2011) because of their ability to produce important metabolites, for instance, propionic acid (van wyk et al., 2018), folate (rad et al., 2016), vitamins b2, b7, b12 and k ( abou ayana et al., 2016; zárate, 2012) and bacteriocins (ahmadi et al., 2015) are used in industrial and commercial scales ( farhadi et al., 2012; kouya et al., 2008). use of pab in production of dairy products such as yogurt increases product viscosity through the production of exopolysaccharides and inhibits growth of undesirable microorganisms in the product through the production of propionic acid and bacteriocins. this increases shelf life of the product. in addition, growth of pab does not interfere with the growth of lactic acid bacteria (lab) in dairy products ( ekinci and gurel, 2008; gorret et al., 2001). conjugated linoleic acid (cla) is another valuable metabolite produced by pab in culture media (van wyk et al., 2018; yang et al., 2017). in fact, cla is a fatty acid naturally found in milk fats and dairy products such as yogurt, butter and cheese (van wyk et al., 2018). the compound belongs to a group of omega-6 fatty acids, and is a geometric isomer of linoleic acid (la; yang et al., 2017). beneficial properties of cla include preventing increase of body fats (corbo et al., 2014), anti-carcinogenesis properties (colon, prostate, skin and breast cancers) (masso-welch et al., 2004), antioxidant properties (zárate, 2012), lowering of blood serum cholesterol (hernandez, 2013), anti-inflammation properties (olson et al., 2017), anti-diabetic properties (balci yuce et al., 2017) and regulation of the system. daily intake of 3 g of cla is recommended to prevent cancers; however, the cla content of dairy products is only 0.5–9.9 mg g–1 of fats (zárate, 2012). commercially, most of cla is produced through the chemical isomerisation of la, in which harmful by-products are produced as well. in the chemical production method, various isomers of cla are produced (ogawa et al., 2001). studies have verified that c9t11-cla, t9t11-cla and t10c12-cla isomers prevent diseases in the human body and include medical uses (yang et al., 2017). dairy pab has the potential to convert unsaturated fatty acids cis-9 cis-12 la (c9c12-18:2) to cis9-trans-11 (c9t11-18:2), trans-10-cis-12 (t10c1218:2) and trans-9-cis-11(t9c11-18:2) conjugate isomers (hennessy et al., 2012). thus, it is possible to produce dairy products with high cla levels by developing products fermented by pab, which produces increased cla levels by converting la present in milk to cla. environmental and growth factors greatly affect cla production in dairy products (yang et al., 2017). several studies have been conducted on the effects of process variables on microbial production of cla, including probiotic strains (lactobacillus sp., bifidobacterium sp., propionibacterium (p.) sp., leuconostoc sp., lactococcus sp., enterococcus sp. and pediococcus sp.) (fukuda et al., italian journal of food science, 2021; 33 (sp1) 3 bacterial conjugated linoleic acid bio-fortification of synbiotic yogurts the mrs agar was acidified to ph 5.4 using acetic acid. sodium lactate agar was used for selective enumeration of pab (tharmaraj and shah, 2003). the incubation temperature for l. delbrueckii ssp. bulgaricus, s. thermophilus and p. freudenreichii ssp. shermanii, respectively, were 45°c for 72 h, 37°c for 24 h and 30°c for 5–7 days under anaerobic conditions using gas generating pack a (merck, darmstadt, germany), except for s. thermophilus. lipid extraction and cla analysis extraction of cla was conducted based on the method by lin et al. (1999), in which yogurt was mixed with chloroform–methanol in a ratio of 2:1 (v/v), and was refrigerated and centrifuged for 6 min at 4,500 × g. the organic phase layer was collected and dehydrated with 0.3 g of sodium sulphate and stored in refrigerator for 24 h. the middle phase was separated from sodium sulphate using decantation and used in experiments. to remove the organic solvents (chloroform-methanol), rotary evaporator was used to dry off. thereafter, in order to saponify fatty acids 1 ml solution of 1n sodium hydroxide in methanol was added into the solution and then it was incubated at 100°c for 15 min. then hydrochloric acid solution in methanol was added to methylate present fatty acid, and the mixture was incubated at 60°c for 20 min using water bath. at this stage, 2 ml of distilled water was added and homogenised for 15 min using vortex mixer to release methyl esters from methanol, followed by formation of polar bonds between methanol and water. then n-hexane was added and homogenised to transfer methyl esters from aqueous phase to organic phase. after removing aqueous phase, anhydrous sodium sulphate was mixed with organic phase and 1 µl of this mixture was injected into gas chromatographic columns (capillary bp10; philips scientific model 4410, uk) fitted with a flame ionisation detector. the column was 25 m in length and 0.22 mm in diameter with a thickness of 0.25 µm. the initial temperature of the column was 150°c with 1-min holding time, injection temperature was 250°c, final temperature was 230°c with 10-min duration and a temperature ramp of 5°c in 1 min. in this study, the total quantity of cla (mg g–1 lipid) was reported as the sum of the production of two isomers (c9t11-18:2 and t10c12-18:2). experimental design this study was conducted progressively at three levels step by step. as mentioned previously, different factors might affect bio-production of cla in yogurt samples by pab. the first optimisation step included identification of variables with significant effects on cla production by pab using pbd. after identification of effective and technology department of ministry of sciences (persian type culture collection), tehran, iran. the strain was sub-cultured in sodium lactate medium (slm) containing 1% (v/v) sodium lactate syrup, casein peptone 10 g l–1 and yeast extract 10 g l–1 at 30°c under micro-aerobic conditions (grinstead and barefoot, 1992) milk preparation after preparing of reconstituted milk with 13% (w/v) of commercial skim-milk powder in distilled water (dw), the milk was pasteurised at 90°c for 30 min and cooled in an ice bath to temperature below 35°c to prevent possible heat shocks to probiotic bacteria. for preparing various percentages of milk fats, pearson square method was used. fermentation in this study, values of independent variables in yogurt samples were calculated based on the design of experiments (pbd and rsm) at each stage. after inoculation, yogurt samples were transferred into 100-ml polypropylene cups, and milks were incubated at 30–43°c (based on the design of experiments) using laboratory oven until a ph of 4.6 was reached. ph values of yogurt samples were determined with a ph meter 605 (methrohm ag, herisau, switzerland). then samples were quickly cooled using ice bath and stored at 4°c. three yogurt samples were prepared to verify the model and compare productions of cla by p. freudenreichii ssp. shermanii. the control yogurt, which contained traditional yogurt starter cultures (st and lb) only, was not supplemented with p. freudenreichii ssp. shermanii (ps4) and prebiotics (inulin). other samples (yc and ps4) included yogurt starter culture and p. freudenreichii ssp. shermanii, and in the third yogurt sample (yc, ps4 and inulin), p. freudenreichii ssp. shermanii was added in addition to traditional starter cultures and 2.27% (w/v) inulin. fat content of milk in all three yogurt samples was 1.75% (w/w). analyses were conducted after an overnight storage of yogurt samples and after 7, 16 and 21 days of storage at 4°c. count of viable bacteria cell count of the starter cultures (st and lb) and probiotics (pab) was conducted in duplicate after incubation time. yogurt samples (1 ml) were added to 9 ml of 0.15% (w/v) sterile peptone water (merck, germany) and viable bacteria were counted as formed colonies using the pour plate method. lb and st were plated in mrs agar and m17 agar (merck, germany) (dave and shah, 1996). 4 italian journal of food science, 2021; 33 (sp1) omid zahed et al. significant variables in cla production, effective factors identified at three levels were optimised using central composite design (ccd) under rsm designations. moreover, the best conditions for independent variables in cla production by pab were provided and the quantities of cla production in pab yogurt samples were compared with those in control yogurt samples, which only contained starter cultures (yoflex express 1.0). plackett–burman design effective factors and their levels were selected based on the literature review. the selected variables, including media compositions (e.g. strain type, milk fat percentage (mfp), inoculum percentage, sunflower oil quantity and inulin concentration) and environmental factors (e.g. incubation and fermentation temperatures and storage time at 4°c), are shown in table 1. high levels (+) and low levels (–) represent two different levels of independent variables. rsm design the rsm is a set of statistical techniques for designing experiments, constructing models, assessing effects of factors and searching for the optimal conditions of the factors for optimal responses. in general, rsm is a great tool for optimising conditions when several factors are involved in production of a product (cousin et al., 2016; grinstead and barefoot,1992; khodaiyan et al., 2008). a combination of factors that produces a specific optimal response can be identified using design factor and rsm (khodaiyan et al., 2008). for additional accurate predictions on the optimum conditions of cla bioproduction and to minimise the number of test sets, ccd under rsm was designed. in this study, all factors were used at three levels (table 2). experimental ranges of the three significant variables for ccd trials are shown in table 2. statistical analysis statistical analysis of the results was conducted using minitab statistical software v.16 (minitab, usa), and response surface plots were drawn. data were statistically treated using analysis of variance (anova). all data were presented as the mean value ± standard deviation (sd) of independent experiments on various days. in general, p ≤ 0.05 was established statistically significant. results and discussion selection of the most important affecting factors using the plackett–burman design the primary purpose of screening experiments is to select important major effects from less important ones. ta bl e 1. p ro ce ss v ar ia bl es , s el ec te d le ve ls a nd e ig ht tr ia ls o f th e p la ck et t– b ur m an n de si gn to s tu dy th e im pa ct o f se ve n fa ct or s (a nd fi nd in g m ai n va ri ab le s) o n m ic ro bi al p ro du ct io n of c la in sy nb io tic y og ur t. r un in de pe nd en t v ar ia bl es r es po ns e a s tr ai ns b m ilk fa t % (w /w ) c in ul in % (w /v ) d s un flo w er oi l ( g l– 1 ) e in oc ul um si ze (% ) f te m pe ra tu re (° c ) g st or ag e tim e (d ay s) ci s9, tr an s 11 c la m g g– 1 lip id tr an s10 , ci s12 c la m g g– 1 lip id e xp er im en ta l to ta l c la m g g– 1 lip id p re di ct ed to ta l c la m g g– 1 lip id 1 p ff ** 1 0 0. 1 1 43 14 4. 1 ± 0. 11 0. 2 ± 0. 13 4. 3 ± 0. 14 4. 3 2 p ff 3 0 0 2 30 14 4. 4 ± 0. 17 n d 4. 4 ± 0. 17 4. 4 3 p ff 3 2 0 1 43 1 4. 6 ± 0. 09 0. 8 ± 0. 05 5. 4 ± 0. 07 5. 3 4 p fs * 3 2 0. 1 1 30 14 4. 5 ±0 .1 1 0. 2 ±0 .1 4 4. 7 ± 0. 14 4. 7 5 p ff 1 2 0. 1 2 30 1 4. 6 ± 0. 14 1. 1 ± 0. 12 5. 7 ± 0. 05 5. 6 6 p fs 3 0 0. 1 2 43 1 4. 8 ± 0. 10 n d § 4. 8 ± 0. 10 4. 8 7 p fs 1 2 0 2 43 14 5 ± 0. 09 n d 5. 0 ± 0. 09 4. 9 8 p fs 1 0 0 1 30 1 5. 5 ± 0. 07 n d 5. 5 ± 0. 07 5. 4 * p fs : p ro pi on ib ac te riu m fr eu de nr ei ch ii ss p. s he rm an ii (p s 4) (c od e1) . ** p ff : p ro pi on ib ac te riu m fr eu de nr ei ch ii ss p. fr eu de nr ei ch ii. § n d : t he a m ou nt w as le ss th an d et ec tio n lim it. c la : c on ju ga te d lin ol ei c ac id . italian journal of food science, 2021; 33 (sp1) 5 bacterial conjugated linoleic acid bio-fortification of synbiotic yogurts table 2. main process variables, range and 17 trials of central composite design to study the impact of main and interaction effects on optimisation of microbial production of cla in synbiotic yogurt. run x1 – milk fat % (w/w) x2 – inulin % (w/v) x3 – storage time (days) cis-9,trans-11 cla (mg g–1 lipid) trans-10,cis-12 cla (mg g–1 lipid ) total cla (mg g–1 lipid ) 1 1.00 1 1 4.2 nd 4.2 2 3.50 1 1 4 0.1 4.1 3 1.00 3 1 4.1 nd* 4.1 4 3.50 3 1 3.9 0.1 4.0 5 1.00 1 21 5 0.3 5.3 6 3.50 1 21 4.9 0.2 5.1 7 1.00 3 21 4.9 0.9 5.8 8 3.50 3 21 4.7 0.5 5.2 9 1.00 2 11 5.2 0.4 5.6 10 3.50 2 11 4.9 0.4 5.3 11 2.25 1 11 4.8 0.6 5.4 12 2.25 3 11 5.1 0.4 5.5 13 2.25 2 1 4.4 nd 4.4 14 2.25 2 21 5.2 0.6 5.8 15 2.25 2 11 5.2 0.8 6.0 16 2.25 2 11 5.4 0.5 5.9 17 2.25 2 11 5.1 0.7 5.8 *nd: the amount was less than detection limit. cla: conjugated linoleic acid. in this study, student’s t-test was conducted to demonstrate significance of each factor (t-value = coefficient/ standard error (sb)) (khosravi-darani and zoghi, 2008). the tabulated t-value (degree of freedom = 6) at p ≤ 0.05 was 1.94. each variable linked to t-value higher than the tabulated t-value (1.94 for p ≤ 0.05) was significant. table 3 refers to statistical calculations of cla production in yogurt samples by pab. results established that mfp, prebiotic (inulin) concentration and storage time at 4°c were significant due to their t-values being higher than 1.94. based on table 3, addition of 2% (w/v) inulin to yogurts increased the production of cla. this increase might be due to the prebiotic role of inulin, which was an important factor in growth and maintenance of probiotics and caused longer survival of p. freudenreichii during the storage period at 4°c as well as greater production of cla in yogurt. mohanty et al. (2018) reported that prebiotics, especially inulin, were good candidates of functional foods. salem et al. (2007) demonstrated that addition of 1% inulin to dairy cheese promoted growth and longer survival of existing strains. in another study done by effat et al. (2019), it was reported that addition of 1–3% prebiotics, such as inulin, to milk increased survival and viability of the probiotic propionibacterium strains. table 3 shows that storage of yogurt containing p. freudenreichii at 4°c for 14 days increased the production of cla. the pab may adapt and survive at acidic ph of 2 (van wyk et al., 2018). owing to the fact that yogurt samples containing p. freudenreichii had ph higher than 2, p. freudenreichii was able to grow and produce cla during the storage time. akalin et al. (2007) reported increase in cla production in yogurts during storage for 28 days. in addition, results in table 3 indicate that yogurt samples containing 1% fat (w/w) with p. freudenreichii increased cla production. biohydrogenation pathway is also a mechanism for the formation of cla in yogurts (ha et al., 1989). in order to convert la to cla in this pathway, la isomerase plays an important role. starter cultures, such as pab, did not affect cla formation without presence of la. increase in the proportion of milk fat and la in yoghurt with p. freudenreichii increased production of cla. kishino et al. (2002) found that lactobacillus plantarum aku 1009a could produce high content of cla (3.88 mg ml–1) in nutrient media with 0.06% (w/v) la. khosravi-darani et al. (2014) reported that cla content in probiotic yogurts containing pab increased by 40% from average 8.01 mg g–1 fat in non-treated yogurts to 11.03 mg g–1 fat in probiotic yogurts containing grape seed oil as a source of la. optimisation of cla production using response surface methodology after selecting the most important affecting factors, central composite design and rsm method were used to 6 italian journal of food science, 2021; 33 (sp1) omid zahed et al. table 3. statistical data for analysis of variance of cla production in yogurt by pab.a factors coefficient t-value a (strains) –0.025 –0.35 b (milk fat (%) w/w) –0.150 –2.14 c (inulin (%) w/v) 0.225 3.14 d (sunflower oil, g/l) –0.100 –1.42 e (inoculum size, %) 0 0 f (temperature, °c) –0.1000 –1.42 g (storage time, days) 0.375 5.28 aa 0 = 4.9 (mean of experimental cla), standard error, s b = 0.07, estimated error, s2 e = 0.04, tabulated t-value (degree of freedom 6) at p ≤ 0.05 is 1.94. cla: conjugated linoleic acid; pab: propionic acid bacteria. optimise the three factors (mfp, prebiotic concentration and storage time at 4°c). design matrix for these factors in optimisation sets is described in table 2. results of rsm in the form of anova are provided in table 4. p < 0.05 demonstrates that the model terms are significant. the anova results established that quadratic regression for the production of cla by p. freudenreichii ssp. shermanii in yogurt models was significant. the lack-offit test was insignificant (p = 0.314) and only 1.8% of the total variations were not explained by the model (r2 = 98.2%). the quadratic model was based on eq. (1): y = 2.626 + 0.741x1 + 1.001x2 + 0.187x3 – 0.155 (x1) 2 – 0.242 (x2) 2 – 0.005 (x3) 2 – 0.04x1x2 – 0.006x1x3 + 0.01x2x3, (1) where y, x1, x2 and x3 were equivalent experimental response, mfp, inulin concentration and storage time at 4°c, respectively. effects of various levels of variables on cla production in yogurts by p. freudenreichii ssp. shermanii can be achieved using eq. (1). based on t-test and p-value, table 4 shows that mfp, inulin concentration and storage time at 4°c significantly affected production of cla, while the three affecting factors were not significant (p ≤ 0.05). effects of inulin and milk fat percentage on cla production figure 1 shows the effects of mfp, concentration of inulin and storage time at 4 °c in yogurt on production of cla by p. freudenriechii in surface plots. in surface plot, response is plotted for two independent variables at a time, while other variables are fixed. quantities of fat and free la in milk and presence of inulin play important roles in survival of probiotic bacteria such as pab as well as production of cla in yogurts ( akalin et al., 2007; xu et table 4. analysis of variance results for cla production in yogurt by p. freudenreichii ssp. shermanii. source of variation degree of freedom sum of squares mean square p regression 9 7.839 0.871 0.000 linear 3 4.290 0.769 0.000 square 3 3.404 1.256 0.000 interaction 3 0.145 0.048 0.157 lack of fit 5 0.123 0.024 0.314 pure error 2 0.020 0.010 total 16 7.982 factors degree of freedom coefficient estimate standard error p intercept 1 2.626 0.391 0.000 x 1 1 0.741 0.270 0.029 x 2 1 1.001 0.368 0.030 x 3 1 0.187 0.023 0.000 x 1 2 1 –0.155 0.055 0.027 x 2 2 1 –0.242 0.087 0.027 x 3 2 1 –0.005 0.000 0.000 x 1 x 2 1 –0.040 0.040 0.356 x 1 x 3 1 –0.006 0.004 0.182 x 2 x 3 1 0.010 0.005 0.088 cla: conjugated linoleic acid; pab: propionic acid bacteria. al., 2005). figure 1a shows that increase in mfp up to 2.1% (w/w) increased production of cla in yogurt by p. freudenreichii ssp. shermanii; however, production of cla decreased at higher fat proportions. results were similar to those established by wang et al. (2007), who reported that the maximum production of cla (78.8 µg ml–1) was produced by p. freudenreichii ssp. shermanii in mrs media containing 12 mg ml–1 of sunflower oil as a source of la. however, production of cla decreased at higher concentrations of sunflower oil. wang et al. (2007) demonstrated that at 9.6 mg ml–1 sunflower oil in slm media, 73.9 µg ml–1 cla was produced by p. freudenriechii. again, the concentration of cla decreased significantly when concentration of oil was higher than 9.6 mg ml–1. nieman (1954) reported that free fatty acids disrupted permeability of cytoplasmic membranes in gram-positive bacteria and negatively affected the production of cla. wang et al. (2007) reported antibacterial activity of la. other studies have demonstrated that free fatty acids have negative and inhibitory effects on production of cla by bacteria such as lactobacillus plantarum, p.  freudenreichii and lactobacillus spp. (alonso et al., 2003; lin, 2000; lin et al., 1999). as shown in fig. 1a, production of cla by p. freudenreichii ssp. shermanii in yogurts increased with increase in the concentration of inulin to nearly 2% (w/v). increase in concentration italian journal of food science, 2021; 33 (sp1) 7 bacterial conjugated linoleic acid bio-fortification of synbiotic yogurts of inulin by 2% (w/v) or more decreased production of cla. the figure also shows that high concentrations of inulin had negative effects and decreased the production of cla. addition of high concentrations of inulin to yogurts favoured further survival of yogurt starter culture bacteria, resulting in greater decrease in yogurt ph. at lower ph, probiotic bacteria, such as pab, have less ability to grow and function and hence cla production decreases by these bacteria. results of this study are similar to the results of a study done by effat et al.( 2019), who reported that increasing inulin concentration in yogurts from 3% to 5% decreased survival rate of probiotic bacteria. in another study performed by akalin et al. (2007), significant increase in cla levels was reported when fructooligosaccharides (fos) were added to yogurts and a 2.90-fold increase was observed in total cla production in yogurts manufactured with 2% fos using bifidobacterium animalis. effects of yogurt storage time at 4°c and mfp on cla production figure 1b shows that at a constant mfp, production of cla in yogurts increased with increasing storage time at 4°c. increase in the concentration of cla continued until day 16 of storage of yogurt at 4°c, and then concentration of cla decreased mildly. studies have been conducted on the effects of yogurt storage time at 4°c on cla production by different probiotics with various results. the results obtained by boylston and beitz (2002) indicated no significant change in yogurts’ cla content during storage for 7 days. in another study, shantha et al. (1995) also showed stability in yogurts’ c9t11-cla isomer concentration at refrigerated storage for 42 days. in a study done by akalin et al. (2007), relative decrease was reported in the concentration of c9t11-cla isomer after 28 days. the major reason for decrease in yogurts’ cla concentration at storage time included oxidative 3 5.2 2 5.4 5.6 1 5.8 (a) 2 1 3 cl a m g/ g inu lin % (w /v) milk fat % (w/w) 20 4.5 10 5.0 5.5 1 6.5 (b) 2 03 cl a m g/ g milk fat % (w/w) st or ag e t im e(d ay ) figure 1. surface plot of interactive effect on cla production in yogurt by p. freudenreichii ssp. shermanii. (a) effect of inulin and milk fat percentage; (b) effect of storage time of yogurt at 4°c and milk fat percentage. milk fat 3.50 [1.7576] 1.0 inulin c 3.0 [2.2727] 1.0 storage 21.0 [16.9596] 1.0 figure 2. optimisation plot of cla production in yogurt by p. freudenreichii ssp. shermanii. reactions that caused destruction of conjugated double bond system. figure 2 points the best conditions for the production of cla in yogurts by p. freudenreichii ssp. shermanii. the best values for the three variables of mfp (x1), inulin concentration (x2) and storage time at 4°c (x3) included 1.75% (w/w), 2.27% (w/v) and 17 days, respectively; the highest cla production by p. freudenreichii ssp. shermanii was seen in yogurts containing inulin (x2). verification of the model to verify the model, yogurt samples were prepared under optimal conditions of mfp (1.75% w/w), inulin concentration (2.27% w/v) and storage time at 4°c (~17 days) in three replicates, and the quantity of cla in yogurts containing p. freudenreichii ssp. shermanii under optimal conditions was compared with two other yogurt samples from section 2.4. the highest quantity of cla included 6.4 ± 0.2 mg g–1 lipid. model and regression didn’t establish significant lack of fit between experiments and 8 italian journal of food science, 2021; 33 (sp1) omid zahed et al. shows that on early days of storage of yogurt samples (up to day 6), no significant differences were seen in the production of cla by p. freudenreichii ssp. shermanii for yogurt samples (with or without inulin), with 189% and 191% increase in production of cla, respectively, compared with control yogurt samples. on day 16 of storage, quantity of cla in yogurts containing inulin reached to 6.4 mg g–1 lipid, increasing by 256%, compared with control samples. for yogurts without inulin, this increase was 239%. in yogurt samples containing p. freudenreichii and inulin, decreased concentration of cla was observed after day 16, similar to the results of optimisation shown in fig. 2. as previously stated, oxidative reactions that destroyed conjugated double bond systems were the major reasons for decrease in cla concentration. microbiological viable count analysis bacterial count results of the three yogurt samples produced using the co-culture method during 3 weeks are compared with each other in table 5. viable counts of s. thermophilus in control yogurt samples without inulin during 21 days of storage decreased from 9.41 log cfu  ml–1 on day 1 to 8.70 log cfu ml–1 on day 21. in this yogurt sample, a decrease in l. delbrueckii ssp. bulgaricus was seen from 8.19 log cfu ml–1 to 5.87 log cfu ml–1, which was much higher for l. delbrueckii ssp. bulgaricus than for s. thermophilus in all samples. low storage temperatures and over acidification have been reported for this decrease (ekinci and gurel, 2008). predicted values of cla production by p. freudenreichii ssp. shermanii in yogurts (6.70 mg g–1 lipid; fig. 3). as seen in fig. 3, the highest production rate of cla occurred in yogurt samples containing p. freudenreichii ssp. shermanii, compared with control yogurt within the first 24 h of storage. this production rate of cla was equal to 4.9 and 4.5 mg g–1 lipid, respectively, for the yogurt samples of p. freudenreichii ssp. shermanii and inulin and those without inulin, while this value of cla was 2.6 mg g–1 lipid for control yogurts. similar results were reported in a study done by wang et al. (2007), which resulted in the highest production of cla in three culture media of slm, mrs and skim-milk at 24 h. figure 3 0 2 4 6 8 0 4 8 12 16 20 24 c la p ro du ct io n (m g g– 1 l ip id ) storage days figure 3. cla production during storage of yogurt samples at 4°c. yogurts: yc (), yc + ps4 (), yc + ps4 + inulin (). table 5. viable cell count of starter cultures in fermented skim-milk during 21 days of storage at 4°c.a viable count storage time (days) yc (log cfu ml–1) yc + ps4 (log cfu ml–1) yc + ps4 + inolin (log cfu ml–1) streptococcus thermophilus 1 9.41 ± 0.02 9.43 ± 0.02 9.35 ± 0.04 7 9.08 ± 0.03 9.42 ± 0.04 9.19 ± 0.07 16 9.04 ± 0.06 9.31 ± 0.01 9.27 ± 0.02 21 8.70 ± 0.05 8.76 ± 0.01 8.64 ± 0.1 lactobacillus delbrueckii ssp. bulgaricus 1 8.19 ± 0.08 8.04 ± 0.04 8.32 ± 0.05 7 7.12 ± 0.09 7.12 ± 0.09 8.02 ± 0.09 16 6.43 ± 0.03 7.08 ± 0.06 7.07 ± 0.01 21 5.87 ± 0.04 6.19 ± 0.02 6.08 ± 0.05 p. freudenreichii ssp. shermanii 1 — 9.18 ± 0.04 9.32 ± 0.5 7 — 7.97 ± 0.01 9.05 ± 0.03 16 — 6.16 ± 0.05 8.00 ± 0.01 21 — 5.98 ± 0.08 6.33 ± 0.09 amean ± standard deviation (sd). yc: yogurt starter culture containing streptococcus thermophiles and lactobacillus delbrueckii ssp. bulgaricus. yc + ps4: yogurt starter culture containing streptococcus thermophiles, lactobacillus delbrueckii ssp. bulgaricus and p. freudenreichii ssp. shermanii. yc + ps4 + inulin: yogurt starter culture containing streptococcus thermophiles, l. delbrueckii ssp. bulgaricus, p. freudenreichii ssp. shermanii and 2.25% inulin added to yogurt sample. cfu: colony-forming unit. italian journal of food science, 2021; 33 (sp1) 9 bacterial conjugated linoleic acid bio-fortification of synbiotic yogurts compliance with ethical standards the authors do not have any kind of interests. research does not involve human participants and/or animals. informed consent is not applicable. funding this study is related to the project no.1397/75698 from student research committee, shahid beheshti university of medical sciences, tehran, iran. we also appreciate the “student research committee” and “research & technology chancellor” in shahid beheshti university of medical sciences for their financial support of this study. references abou ayana i.a., el-deeb a.m. and ibrahim a.e. 2016. research article using of dairy propionibacteria as bio-preservative in kareish cheese. international journal of dairy science 11: 114– 123. https://doi.org/10.3923/ijds.2016.114.123 ahmadi n., khosravi-darani k., zarean-shahraki s., mortazavian m. and mashayekh s. 2015. fed-batch fermentation for propionic, acetic and lactic acid production. oriental journal of chemistry 31: 581–590. https://doi.org/10.13005/ojc/310174 akalın a., tokuşoğlu ö., gönç s. and aycan ş. 2007. occurrence of conjugated linoleic acid in probiotic yoghurts supplemented with fructooligosaccharide. intnational dairy journal 17: 1089– 1095. https://doi.org/10.1016/j.idairyj.2007.02.005 alonso l., cuesta e. and gilliland s. 2003. production 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https://doi.org/10.1016/j. foodres.2005.07.007 corbo m.r., bevilacqua a., petruzzi l., casanova f.p. and sinigaglia  m. 2014. functional beverages: the emerging side of functional foods: commercial trends, research, and health implications. comprehensive reviews in food science and food safety 13: 1192–1206. https://doi.org/10.1111/1541-4337.12109 similar results are reported from other studies ( ekinci and gurel, 2008; güler-akin and akin, 2007; ranadheera et al., 2012). results presented in table 5 indicate that a similar decrease was observed in the viable count of s. thermophiles and l. delbrueckii ssp. bulgaricus during 21 days of storage in yogurt containing p. freudenreichii ssp. shermanii (yc and ps4). this decrease was less pronounced for l. delbrueckii ssp. bulgaricus. results demonstrated that addition of p. freudenreichii ssp. shermanii to yogurt samples did not affect negatively yogurt starter cultures (st and lb). comparison of yogurt samples with and without inulin demonstrated that presence of prebiotics, such as inulin, could significantly affect the count number of p. freudenreichii ssp. shermanii. as shown in table 5 for yogurts containing inulin (yc, ps4 and inulin), number of p. freudenreichii ssp. shermanii after 16 days of storage was 8.00 log cfu ml–1. in yogurts without inulin, this value was 6.16 log cfu ml–1. other studies (capela et al., 2006; oliveira et al., 2009) have reported positive effects of prebiotics, such as polydextrose, oligofructose and maltodextrin, on the survival of probiotics. conclusions in this study, p. freudenreichii ssp. shermanii was used with traditional yogurt starter cultures (st and lb) to enrich and produce cla in yogurts. results from pbd design demonstrated that three factors of mfp, inulin concentration and storage time at 4°c significantly affected cla production in yogurts by p. freudenreichii ssp. shermanii using rsm design and optimising conditions of the three highlighted factors. the highest 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https://doi.org/10.1016/j.lwt.2014.12.003� https://doi.org/10.1016/j.lwt.2014.12.003� https://doi.org/10.1007/s13594-016-0286-1� https://doi.org/10.1007/s13594-016-0286-1� https://doi.org/10.1016/j.foodchem.2012.06.025� https://doi.org/10.1016/j.foodchem.2012.06.025� https://doi.org/10.1016/j.idairyj.2009.12.003� https://doi.org/10.1016/j.idairyj.2009.12.003� _goback #291_rokaityte_bozza ital. j. food sci., vol 29, 2017 253 paper effect of lactic acid and bioactive component mixtures on the quality of minced pork meat a. rokaityte*1, g. zaborskiene1.2, v. baliukoniene1, i. macioniene2 and a. stimbirys1 1lithuanian university of health sciences, veterinary academy, department of food safety and quality, tilzes st. 18, lt-47181, kaunas, lithuania 2food institute, kaunas university of technology, radvilenu st. 19, kaunas lt-51180, lithuania *corresponding author: anita.rokaityte@lsmuni.lt abstract the objective of this study was to investigate the effects of mixtures of lactic acid (la), thymol (th), linalool (ln) and dihydroquercetin (dhq) on the quality of minced pork meat during 7 days of storage at +4 °c temperature. dhq+la+ln, dhq+la and la exhibited the greatest antibacterial effect on the agar well diffusion assay and resulted in the best sensory evaluation. samples treated with dhq+la had a statistically significant effect on the total bacterial count and showed the best antibacterial effect on the e. coli count. however, the reducing effect on the total amount of biogenic amines was not significant in all cases of treatment. keywords: dihydroquercetin, lactic acid, linalool, minced meat ital. j. food sci., vol 29, 2017 254 1. introduction the production of safe and high quality meat and meat products along with recent consumer demand for all-natural and clean-label products is challenging. a significant level of meat product spoilage takes place every year at different levels of the production chain including preparation, storage, and distribution (dinesh and jayasena, 2013). many synthetic additives have been used over the years for preserving fresh meat and meat products and to extend the period of refrigerated storage (somolinos et al., 2010). synthetic additives have been accused of having some carcinogenic and toxic properties. however, untreated products and natural foods may be more susceptible to the growth of food-borne pathogens than conventional food versions (jay et al., 2005). the most important food-borne pathogenic bacteria that have survived and grow in these products include escherichia coli, staphylococcus aureus, salmonella spp., listeria monocytogenes, and bacillus spp. (oroojalian et al., 2010). these bacteria cause a great proportion of foodborne outbreaks in different foods (warriner and namvar, 2009). in this context, natural alternatives are attracting interest as food preservatives in order to ensure the safety of food (mariutti et al., 2011). one of the traditional ways of controlling microbial growth in these products, thus improving safety and delaying spoilage, is the addition of essential oils (eos) (dinesh and jayasena, 2013). the composition and structure, as well as the functional groups of the oils, play an important role in determining their antimicrobial activity. usually, compounds with phenolic groups are the most effective. eos may be applied as part of a hurdle system to achieve preservative action (mastromatteo et al., 2009). as an example, lower levels of eos can be combined with existing and novel preservation technologies including low temperature and acidity, modified atmosphere packaging (map), high hydrostatic pressure, preservatives (e.g., nitrite, nisin, etc.) and low-dose irradiation. a series of preservative hurdles is established by these combined processes, which in turn improves the microbial stability and sensory quality of meat and meat products (al-reza, 2010; skandamis and nychas, 2001; zhou et al., 2010). although good antimicrobial activities were observed for many eos, some limitations have also been identified in the application of eos in meat and meat products. the interaction of some eos with food ingredients and structure may decrease their effectiveness (skandamis and nychas, 2000). additionally, the markedly reduced activity of eos may result in food systems such as meat and meat products when compared to in vitro results. this may be attributed to the presence of fats, carbohydrates, proteins, and salts in such systems. it is difficult to maintain consistent quality because the composition of an individual eo can vary due to several factors including the time of harvesting, variety, the part of the plant used, and method of extraction (hyldgaard et al., 2012). in addition, hyldgaard et al. (2012) reported that the antimicrobial potency of eo constituents depends on ph, temperature (rattanachaikunsopon and phumkhachorn, 2010), and the level of microbial contamination (somolinos et al., 2010). further, the use of eos as preservatives in food has been limited, as they are required in high concentrations in order to achieve the sufficient antimicrobial activity (hyldgaard et al., 2012). lower concentrations of eos can be combined with other antimicrobial compounds and/or other preservative technologies to obtain a synergistic effect without compromising antimicrobial activities (nguefack et al., 2012). weak organic acids are one of the several primary agents used to control microorganisms in both fermented and nonfermented foods (buchanan et al., 2002). for example, lactic acid, citric acid and acetic acid are either naturally produced or added to food or marinades to achieve food safety and meet quality requirements (mani-lόpez et al., 2012). lactic acid has shown antimicrobial activities against many pathogenic organisms ital. j. food sci., vol 29, 2017 255 because of it is abilities to reduce ph level, exert feedback inhibition and interfere with proton transfer across cell membranes (davidson et al., 2005). dihydroquercetin (also known as taxifolin) is a member of a group of flavonoids (vladimirov et al., 2009). satisfactorily pure dihydroquercetin may be extracted from siberian larch (larix sibirica ledeb). it is also found in the açaí palm, in milk thistle seeds and in small quantities in red onion. dihydroquercetin has a positive effect on human health, as it prevents the accumulation of free radicals (trouillas et al., 2004), influences the physical properties of lipids in biological membranes, ameliorates cerebral ischemiareperfusion injury (wang et al., 2006) and activates the formation of collagen fibres (tarahovsky et al., 2007). the application of dihydroquercetin is quite widely distributed in the production of different categories of products. in general, dihydroquercetin can be used as a natural antioxidant and antimicrobial activities additive in the food industry (wang et al., 2011). the objective of the study was to investigate the antimicrobial effects of bioactive components (lactic acid, linalool, dihydroquercetin) used in combination on microorganisms mostly found in pork minced meat and on the sensory properties and formation of biogenic amines. 2. materials and methods 2.1. preparation of bioactive component solutions for the antibacterial properties analyses powdered concentrate of dhq (99.4%), extracted from siberian larch (larix sibirica ledeb) and produced by the company flavit ltd, pushtino (russia) was used. dhq was diluted into 35 °c distilled water to make 10 ml of 0.024% (w/v) dhq aqueous solution. la (50.0%), th (99.5%) and ln (97.0%) were purchased from sigma-aldrich chemie gmbh (steinheim, germany) and kept at 4 °c. all of the solutions (each of them 10 ml) were made on the day of the research: 0.5% (w/v) la aqueous solution, 0.003% (w/v) th aqueous solution and 0.003% (w/v) ln aqueous solution. 2.2. antimicrobial assay of bioactive components the agar well diffusion method was used to determine the antimicrobial activity of la (0.5%) and bioactive components (ln 0.03%, th 0.03% and dhq 0.024%). reference strain cultures of conditionally pathogenic esherichia coli atcc 25922 and pathogenic bacteria such as staphylococcus aureus atcc 25923, salmonella typhimurium atcc 13076, bacillus cereus atcc 11778, and listeria monocytogenes atcc 19111 were used in this experiment. bacteria cultures were kept in a “viabank” (medical wire & equipment, jk) system at minus 72 °c. during the tests, the examined cultures were pre-cultivated on the plate count agar (pca, liofilchem, italy) slants for 18-24 h under the optimal temperature (30-37 °c). after cultivation, the bacterial culture was washed with a sterile physiological solution and cell suspensions were prepared according to the procedure of mcfarland no 0.5 (approx. 1.5 ×108 cfu/ml). one millilitre of bacterial cell suspension was added to 100 ml of the melted pca cooled to 45 °c. the prepared mixture of bacteria cell suspension and the medium was mixed and 10 ml was poured into each of 90 mm petri dish. fifty microlitres of the tested bioactive components was poured into wells of 8 mm diameter made in the hardened agar. the antimicrobial activity was assessed following 24 h of incubation at 30 ºc or 37 ºc by measuring the diameter of the inhibition zone around the wells (mm). the ital. j. food sci., vol 29, 2017 256 strains were considered as exhibiting no antimicrobial activity if clear zones around the wells were not revealed. 2.3. meat samples meat of pork carcasses from 1-year-old pig, after 48 h since postmortem were purchased from a local establishment in kedainiai, lithuania. the meat was trimmed of all exterior fat and connective tissue. the samples were transported to the laboratory while being kept at 4 °c and minced with a sterilized meat mincer to 3 mm in size. the minced meat samples were divided into 4 groups (4x0.5 kg) considering different treatments with bioactive substances. the minced meat samples were weighed and packed using a multivac r230 model 542 packaging machine (multivac, wolfertschwenden, germany). the samples were packaged under atmospheric air without the use of any gas composition. the samples were stored in the dark under refrigeration conditions (+ 4 °c) for 7 days. the samples were named as follows: (i) dhq (0.024%) + la (0.5%) + ln (0.003%), (ii) dhq (0.024%) + la (0.5%), (iii) untreated control group. analyses of microorganisms, ph and biogenic amines were carried out on the 1st, 3rd, 5th and 7th day of storage. the whole experiment was replicated three times and the results are displayed as the mean values. 2.4. microbial analysis 2.4.1 detection of total aerobic bacterial count samples of 10 g were taken at random for each sample and aseptically weighed into a sterile stomacher bag with 90 ml of sterile 0.1% (w/v) buffered peptone water (ref 611014, liofilchem, italy) and homogenized for 1 min in a model 400 stomacher (seward medical, london, uk). 1.0 ml was seeded onto plate count agar (ref 610040, liofilchem, italy) and incubated at 30 ºc for 72 hours. 2.4.2 detection of escherichia coli samples of 10 g were homogenized with 90 ml of sterile buffered peptone water 0.1% (w/v). 0.1 ml of solution was plated using the pour plate method on tryptone bile xglucuronide medium agar (ref 4021562, biolife, italy) and incubated at 37 ºc for 24 hours. 2.4.3 detection of salmonella samples of 25 g were homogenized with 225 ml of buffered peptone water 0.1% (w/v) and incubated at 37 °c for 24 h. after incubation, 1.0 ml of the pre-enrichment culture was transferred into 9.0 ml of tetrathionate broth and incubated at 42 °c for 24 h. the enrichment culture was streaked onto xlt4 (difco) agar plates and incubated for 24 h at 37 °c. presumptive salmonella colonies were confirmed by using api 20e (biomérieux 20100). agglutination tests were done with salmonella polyvalent o and h antisera (mast diagnostics, uk). 2.4.4 detection of listeria monocytogenes samples of 25 g were homogenized with 225 ml of listeria enrichment broth (merck), incubated at 30 °c for 24 h. after incubation, 0.1 ml was plated in duplicate on palcam ital. j. food sci., vol 29, 2017 257 selective listeria agar (merck). the presence of l. monocytogenes was determined after incubation of the plates at 37 °c for 24 h. up to five colonies were selected for serological and biochemical confirmation using a listeria latex test (oxoid) and an api assay (biomérieux sa, marcy i’ etoile, france), respectively. 2.4.5 detection of staphylococcus aureus samples of 25 g were homogenized with 225 ml of buffered peptone water and seeded onto baird-parker rpf agar (biolife, milan, italy) and incubated aerobically at 35 °c for 24 and 48 h. microbiological data were transformed into logarithms of the number of colony forming units (cfu/g). 2.5. determination of biogenic amines a reversed-phase high-performance liquid chromatography method was used for the quantitative analysis of the biogenic amines – tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine. biogenic amines were extracted from a homogenized sample with 0.4 mol/l perchloric acid. the derivatization of samples was carried out using the modificated methodology of ben-gigirey et al. (2000). the extract was derivatised for 45 min by a dansyl chloride (5-dimethylaminonaphtalene1-sulfonylchloride) solution in acetone at 40 oc. the samples were filtered through a 0.45 μm membrane filter (millipore co., bedford, ma, usa) and 10 μl was injected into a chromatographic system (aligent 1200 series, germany). an analysis was performed using a lichro column cart® 95 125-4. carrier phase – eluents: b – acetonitrile, a – ammonium acetate 0.1mol/l. the analysis lasted 28 min, changing the content of eluents during the first 19 min from 50% of b to 90% of b (from 50% of a to 10% of a respectively), then leaving the content constant for 1 min – 90% of b (10% 99 of a); later, to ensure the isolation of materials for another analysis, eluent with a composition of 50% of b and 50% of a was added to the chamber for 8 minutes. a flow rate of 0.9 ml/min was maintained during analysis, with the column set at 40°c. uv detection was observed at 254 nm. biogenic amines were identified by comparing the retention time of each amine in the chamber with the retention time of the respective reference material. the internal standard method of calculating the peak area for the defined amount of reference material was used to perform quantitative analysis. the limit of detection is between 0.02-0.1 μg/ml for different biogenic amines. 2.6. ph value the ph of all samples was measured according to the standard method for determination of meat ph: lst iso 2917:2002. the average ph of the sample was determined. ph measurements were carried out using a pp-15 ph-meter (sartorius professional meter for ph measurement, germany). 2.7. acceptability evaluation a ten-member panel was used to evaluate sensory taste, odour, and the overall acceptability attributes of the minced pork meat treated with la and bioactive components. before evaluation, the treated minced pork samples were wrapped in aluminium foil individually and cooked in a steam-cooker (multigourmet fs20, braun, germany) for 30 min. each sample was served warm in dishes coded with 3-digit random ital. j. food sci., vol 29, 2017 258 numbers and presented in individual booths to each panellist for evaluation. the panellists were required to rinse with water before tasting each sample. a 9-point hedonic scale was used to score the sensory attributes, where 1=dislike extremely, 9=like extremely, while the limit of acceptability was 5=neither like nor dislike. the sensory evaluation was accomplished at 2 day intervals up to the end of refrigerated storage at +4 °c. 2.8. statistical analysis of the data data were statistically analysed using spss 20.0 software (spss inc., chicago, illinois, usa). differences between dates were evaluated by the analysis of variance method (oneway anova) with a significant level of p≤0.05 (draper and smith, 1998). multiple comparisons were estimated by the fishers least significant difference method and the dunnett’s test was applied when the control group was present. the student’s t-test was used to determine the average values of indicators, standard deviations (sd) and linear correlations. the correlation was considered reliable when p<0.05. 3. results and discussion the antimicrobial activity of the bioactive components was determined against common food-borne pathogenic bacteria (escherichia coli, staphylococcus aureus, salmonella spp., listeria monocytogenes, bacillus spp.). the evaluation of antibacterial activity was done using the agar well diffusion method. the results suggest that the bioactive components exhibited different antimicrobial activity. la (0.5%) and it is mixtures with other bioactive components (ln 0.03%, th 0.03% and dhq 0.024%) had an antimicrobial effect against all tested bacteria. the resulting inhibition zone diameters ranged from 10.5±0.2 mm to 21.5±0.2 mm. a significantly higher inhibitory effect on the e. coli reference strain was reported with the dhq+la mixture (the inhibition zone diameter was 14.3±0.2 mm) (p≤0.05). on the s. aureus reference strain a significantly higher inhibitory effect was reported with the la solution (the inhibition zone diameter was 15.8±0.2) (p≤0.05). the dhq+la+ln mixture showed a higher antimicrobial activity on the s. typhimurium reference strain compared to the other bioactive components (the inhibition zone diameter was 13.0±0.0 mm) (p≥0.05). the ln and th solution did not affect all the microbes examined except b. cereus (the inhibition zone diameter was 9.0±0.0 mm) (p≥0.05). moreover, the dhq solution (0.024%) did not inhibit the growth of all the bacteria tested (table 1). the acceptability evaluation scores of the minced pork meat treated with bioactive components during the 3 days of refrigerated storage at +4 °c are shown in fig. 1. the odour of the minced pork meat treated with bioactive components dhq+la+ln, dhq+la and ln, assessed by the panellists, was significantly higher (p≤0.05) compared to the control. the taste and overall acceptability of the minced pork meat treated with dhq+la+ln were scored significantly higher (p≤0.05) than the control. the scores of taste, odour and the overall acceptability of the minced pork meat treated with dhq+la+th as compared to the control were significantly lower (p≤0.05). therefore, further studies have been carried out with dhq+la+th, th, ln and dhq because these bioactive components have lower acceptability scores for minced pork meat and antimicrobial activity using the agar well diffusion method. the bioactive components that exhibited the greatest antibacterial effect in the agar well diffusion assay and had the best acceptability evaluation (dhq+la+ln, dhq+la and la) were further tested for their microbiological and chemical attributes. ital. j. food sci., vol 29, 2017 259 significant differences were observed between the ph values of the control meat and all treated samples after 5 and 7 days of storage (p≤0.05). besides, there were significant differences in ph among la and dhq+la+ln, dhq+la treated samples after 5 days of storage (p≤0.05) (fig. 2). table 1. antimicrobial activity of bioactive components against the reference strains. target microorganisms zone of inhibition, mm bioactive components la ln th dhq dhq+la+ln dhq+la+th dhq+la e. coli atcc 25922 12.5±0.1 0.0±0.0 0.0±0.0 0.0±0.0 12.5±0.0 12.8±0.2 14.3±0.2* s. aureus atcc 25923 15.8±0.2* 0.0±0.0 0.0±0.0 0.0±0.0 15.5±0.1 10.5±0.2 12.5±0.2 s. typhimurium atcc 13076 12.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 13.0±0.0 12.0±0.1 12.5±0.2 l. monocytogenes atcc 19111 20.5±0.1 0.0±0.0 0.0±0.0 0.0±0.0 20.5±0.2 19.3±0.2 21.0±0.1 b. cereus atcc 11778 15.5±0.1 9.00±0.0 9.0±0.0 0.0±0.0 15.5±0.1 15.5±0.1 21.5±0.2 * the mean difference is significant at the 0.05 level. la – lactic acid 0.5%; ln – linalool 0.03%; th – thymol 0.03%; dhq – dihydroquercetin 0.024% figure 1. acceptability evaluation of minced pork meat treated with bioactive components during 3 days of storage at +4 °c. the results presented in this study showed the effect of dhq and la on the total aerobic bacterial count (fig. 3). all combinations had effects on the total aerobic bacterial count compared to the control sample after 7 days of storage (p≤0.05). 0 1 2 3 4 5 6 7 8 9 dhq+la+ln dhq+la+th dhq+la la th ln dhq c taste odor overall acceptability ital. j. food sci., vol 29, 2017 260 figure 2. variation of the mean ph values of minced pork meat during 7 days of storage at +4 °c. figure 3. variation of the total aerobic bacterial count in minced pork meat during 7 days of storage at +4 °c. salmonella spp., l. monocytogenes and st. aureus were not detected in any of the minced pork meat samples during the 7 days period. therefore, we could not assess the antibacterial effect of the mixtures. la, used in a mixture with ln and dhq, statistically significantly reduced the e. coli count. in addition, the mixture of la and dhq was distinguished by a strong bactericidal activity against e. coli. dimitriević et al. (2007) noted that the anti-listerial effect of essential oils (thymus vulgaris and rosmarinus officinalis) was noticeably increased using it with la. the same synergistic effect was reported by naveena et al. (2006), who found that the combination of syzygium aromaticum essential oil and la provided a decrease in the psychrotrophic and coliform counts of buffalo meat. studies on the antibacterial mechanism of the phenolic compounds found in essential oils focused on their effects on the cellular membrane, changing it is structure and permeability. lin et al. (2004) state that the damage to the cell membrane might explain the observed effects, since phenolics could cause sublethal injury to cell membranes, causing disruption of the proton motive force due to loss of h+-atpase. this could make bacteria more susceptible to an acidic environment. moreover, at low ph, the 5,0 5,2 5,4 5,6 5,8 6,0 6,2 24 h 3 d 5 d 7 d ph dhq+la+ln dhq+la la c 6,0 6,5 7,0 7,5 8,0 8,5 9,0 24 h 3 d 5 d 7 d l og 10 cf u/ g dhq+ln+la dhq+la la c ital. j. food sci., vol 29, 2017 261 hydrophobicity of an essential oil increases, enabling it to more easily dissolve in the lipids of the cell membranes of target bacteria. figure 4. variation of the e. coli count in minced pork meat during 7 days of storage at +4 °c. the large correlation between the ph and total bacterial count was observed in samples treated with dhq+la+ln (r=0.648, p≤0.05) and dhq+la (r=0.692, p≤0.05) during the 7 day period (table 2). table 2. correlation coefficients r between the ph and average values of biogenic amines, e. coli counts and the total bacterial count during 7 days storage at +4 °c. samples correlation coefficients parameters ph tbc e. coli tba dhq+la+ln ph 1.00 0.648* -0.070 -0.178 tbc1 0.648* 1.00 0.078 -0.230 e. coli -0.070 0.078 1.00 0.149 tba2 -0.178 -0.230 0.149 1.00 dhq+la ph 1.00 0.692* -0.497* -0.457 tbc 0.692* 1.00 -0.474 -0.282 e. coli -0.497* -0.474 1.00 0.484 tba -0.457 -0.282 0.484 1.00 la ph 1.00 0.578 -0.033 -0.539* tbc 0.578 1.00 -0.539 -0.374 e. coli -0.033 -0.539 1.00 0.364 tba -0.539* -0.374 0.364 1.00 control ph 1.00 -0.348 -0.136 -0.326 tbc -0.348 1.00 -0.440 -0.336 e. coli -0.136 -0.440 1.00 -0.397 tba -0.326 -0.336 -0.397 1.00 * the mean difference is significant at the 0.05 level. 1tbc total bacterial count; 2tba total amount of biogenic amines. la – lactic acid 0.5%; ln – linalool 0.03%; th – thymol 0.03%; dhq – dihydroquercetin 0.024% 0,0 0,5 1,0 1,5 2,0 2,5 3,0 24 h 3 d 5 d 7 d l og 10 cf u/ g dhq+la+ln dhq+la ital. j. food sci., vol 29, 2017 262 a medium negative correlation between the ph and e. coli count was found in samples treated with dhq+la (r=-0.497, p≤0.05). however, we did not find correlations between the biogenic amine contents and total bacterial count. the capability to form biogenic amines is generally considered a strain specific characteristic rather than a species property. it is thus difficult to find precise correlations between the biogenic amine contents and total bacterial count (standarová et al., 2008). the formation of biogenic amines was intensive during the first 3 days of storage (table 3). table 3. variation of the biogenic amines (mg/kg) in minced pork meat during 7 days of storage at +4 °c. biogenic amines samples days of storage 24 h 3 d 5 d 7 d tryptamine dhq+la+ln 14.39±0.86 13.71±2.41 6.47±0.52 3.47±0.39 dhq+la 17.17±0.57 10.04±1.97 5.53±0.18 1.88±0.43 la 18.68±1.25 10.47±1.03 3.83±0.24 1.64±0.16 control 14.16±0.91 11.90±2.36 5.90±0.47 2.73±0.38 phenylethylamine dhq+la+ln 63.61±5.22 205.93±4.68 57.46±1.20 17.68±1.87 dhq+la 57.85±4.72 148.36±7.42 6.23±0.73* 2.94±0.61* la 78.27±2.18 159.28±8.13 38.42±1.54 16.23±1.13 control 113.65±4.56 168.64±5.61 37.38±0.93 16.66±1.58 putrescine dhq+la+ln 11.64±1.39 34.58±3.64 45.78±1.58 65.57±2.34 dhq+la 16.52±1.94 29.43±2.18 38.54±1.12 43.18±3.50 la 17.60±2.40 23.09±3.05 35.67±1.39 44.61±1.92 control 16.52±1.28 27.89±4.24 36.85±0.91 42.10±2.48 cadaverine dhq+la+ln 23.05±3.72 40.92±3.29 51.99±4.38 77.71±4.66 dhq+la 19.21±2.15 32.24±2.65 45.52±2.57 51.56±2.51 la 22.45±1.39 39.86±3.07 48.63±3.11 60.34±3.68 control 34.76±3.60 44.45±2.91 46.06±2.98 48.78±3.04 histamine dhq+la+ln 23.54±1.49 18.17±1.38 6.61±0.76 2.47±0.54 dhq+la 21.88±2.08 16.49±1.60 4.18±0.35 1.82±0.21 la 19.84±1.67 14.33±0.92 3.07±0.64 1.26±0.35 control 24.32±0.68 13.01±1.77 5.65±0.51 1.87±0.72 tyramine dhq+la+ln 47.55±1.24 55.81±7.38 93.54±3.44 132.43±5.79 dhq+la 52.48±0.92 67.05±5.06 87.48±2.76 115.97±4.17 la 34.37±2.48 41.88±8.17 84.07±2.95 145.41±6.82 control 38.64±1.61 49.35±6.98 66.65±1.73 108.24±3.48 spermidine dhq+la+ln 38.71±4.37 22.30±1.26 12.54±1.65 10.09±0.24 dhq+la 48.96±1. 31 35.79±2.71 10.85±1.89 7.26±0.84 la 32.58±3.64 28.14±1.93 9.68±0.71 9.61±0.37 control 48.76±2.52 38.80±2.09 12.91±1.38 7.48±0.21 spermine dhq+la+ln 41.16±1.81 40.49±1.57 21.00±0.94 7.71±0.97 dhq+la 54.03±3.77 48.06±1.30 18.04±2.48 5.43±0.60 la 51.38±2.26 42.18±2.61 17.68±1.57 5.89±2.34 control 48.19±3.49 48.67±1.55 18.85±1.90 3.97±0.91 total biogenic amines dhq+la+ln 263.65±6.58 431.91±5.46 295.39±4.82 317.13±5.07 dhq+la 288.10±4.61 387.46±7.14 216.37±5.66 230.04±4.60 la 275.17±5.14 359.23±6.62 241.05±4.92 284.99±4.75 control 339.00±4.62 402.71±5.57 230.25±3.24 231.83±5.68 * the mean difference is significant at the 0.05 level la – lactic acid 0.5%; ln – linalool 0.03%; th – thymol 0.03%; dhq – dihydroquercetin 0.024% ital. j. food sci., vol 29, 2017 263 the total amount of biogenic amines decreased from the 3rd to 5th days of the experiment in all cases of treatments. a significant lower amounts of phenylethylamine was found between dhq+la and all cases of samples between 5 and 7 days of storage (p≤0.05). after 5 days of storage, the control meat sample was fully unacceptable and the degration of amines began, with a noticeable smell of ammonia. in the cases of the other treatments, the ba increased after 5 days of storage. moreover, in a lower ph environment, bacteria are strongly encouraged to produce the amino acid decarboxylase as a part of their defence mechanism against acidity (karovičová and kohajdová, 2005; teodorovic et al., 1994). therefore, the acidic conditions caused here by the addition of organic acids to minced meat may have not only reduced but also encouraged the production of ba. besides this, the same raw material can lead to different amine levels depending on the presence of decarboxylating microorganisms, either derived from environmental contamination and the conditions supporting their growth and activity (stadnik and dolatowski, 2010). the type and amount of ba detected in the minced pork meat was far below the level that can cause a health risk during the 7 days of storage. 4. conclusions the mixture of 0.024% dhq and 0.5% la solutions exhibited the greatest antibacterial effect on minced pork meat. a significant negative correlation between the ph and e. coli count was only found in samples treated with dhq+la (r=-0.497, p≤0.05). however, the dhq+la and dhq+la+ln mixtures could be used by the food industry as a natural barrier to control the growth of pathogens and natural spoilage microflora, while reducing the formation of biogenic amines in minced pork, thus providing a balance between sensory acceptability and antimicrobial efficacy. our results encourage further research, focused on the application of la and bioactive component mixtures in low doses to control the growth of the microorganisms mostly found in selected foods, particularly meat products. references al-reza s.m., rahman a., lee j., & kang s.c. 2010. potential roles of essential oil and organic extracts of zizyphus jujuba in inhibiting food-borne pathogens. food chem. 119: 981-986. ben-gigirey b., vieites baptista de sousa j., villa t., barros-velázquez j. 2000. characterization of biogenic amineproducing stenotrophomonas maltophilia strains isolated from white muscle of fresh and frozen albacore tuna. int. j. food microbiol. 57:19-31. buchanan r.l., whiting r.c., golden m.h. 2002. modeling acid inactivation of foodborne microorganisms. in: juneja, v.k., sofos, j.n. 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biomed. sci. 13:127-141. ital. j. food sci., vol 29, 2017 265 wang y., zu y., long j., fu y., li s., zhang d., li j., wink m., efferth t. 2011. enzymatic water extraction of taxifolin from wood sawdust of larix gmelini (rupr.) rupr. and evaluation of its antioxidant activity. food chem. 126:1178-1185. warriner k. and namvar a. 2009. what is the hysteria with listeria? trends in food science & technology. 20: 245-254. zhou g. h., xu x. l., & liu y. 2010. preservation technologies for fresh meat a review. meat sci. 86:119-128. paper received october 11, 2015 accepted december 9, 2016 paper ital. j. food sci., vol. 27 2015 513 keywords: bush okra, common okra, mineral content, recommended dietary allowance, sticky sauce adequacy of mineral contents of raw and plain sticky sauce of common and bush okra moyib oluwasayo kehinde*1, oladapo francis olumide1, moyib folake ramat2 and 1banjoko oluwakemi o. 1 department of chemical sciences, tai solarin university of education, pmb 2118, ijebu-ode, nigeria 2 department of agricultural sciences, tai solarin university of education, pmb 2118, ijebu-ode, nigeria *corresponding author: tel. +234 8188 464 072, email: kmoyib@hotmail.com, moyibok@tasued.edu.ng abstract in nigeria, common okra (abelmoschus esculentus l.) and bush okra (corchorus olitorius l.) are popular mucilage vegetables used as sticky sauce for easy consumption of starchy staples. both raw vegetables and sticky sauce of common as well as bush okra were estimated for their potential in the provision of daily dietary allowance of important minerals. modified methods of the association of official analytical chemists (aoac) were used to estimate the assessed minerals. the results showed that the raw and sticky sauce of assessed common and bush okra contained appreciable levels and essential minerals, but are not adequate to meet recommended dietary allowance, except for fe and cu. comparatively, the two species of okra varied significantly in their mineral content of the raw and plain sauce. there was also a negative effect of cooking on the mineral contents, which reduced significantly to an average of 30% on a dry weight basis. therefore, the two vegetables, either as a fresh or sticky sauce, require additional sources of p, k, na, mg, ca, mn, and zn to meet recommended dietary allowance. furthermore, dried mucilage sauce, though, could be an appreciable post harvest management and storage but not without a loss of about one-third mineral content in the process. however, the sauce of common okra and bush okra are good sources for any of the assessed mineral restricted diets. mailto:kmoyib@hotmail.com 514 ital. j. food sci., vol. 27 2015 introduction vegetables are nutritious foods that provide sufficient amount of nutrients needed for normal body functions, maintenance and reproduction. also, their intake in different combination is essential for the maintenance of healthy life and normal body function (rumeza et al., 2006). vegetables have a lot of health benefits, which include reduced cancers, diabetes and cardiovascular diseases (cox et al., 2000). leafy vegetables are major sources of nutrients in rural areas where they contribute substantially to protein, mineral, vitamins, fiber and other nutrients which are usually in short supply in daily diets. besides their use as food, they also add flavor, variety, taste, color and aesthetic appeal to what would otherwise be a monotonous diet (mepba et al., 2002). thousands of leafy vegetables abound in nigeria and are used primarily as food and medicine. these vegetables are diverse in species from different families and orders, and many of which have specific regional or local area of domestication (fafunso and bassir, 1987; mepba et al., 2007). however, they have crossed regional or local barriers through migration and exchange of goods. presently, they are being cultivated throughout the country but are concentrated in their domesticated regions or localities. as a consequence, they bear different local names from one region or locality to the other in order of increasing distance. for example, bush okra in the southern part of nigeria, is known as “ewedu” in lagos and ogun state, whereas, it is called, “ooyo” in osun and oyo state. leafy vegetables are seasonal and in abundance shortly after the rainy seasons but become scarce during the dry season. they are sold in many nigerian markets to meet daily demand as an important complement of staple dishes (fafunso and bassir, 1987; mepba et al., 2007). two of the abundant vegetables in nigeria are common okra (abelmoschus esculentus) and bush okra (corchorus olitorius); common okra is popularly grown at every nooks and crannies of nigeria while bush okra is concentrated in southwest nigeria. common okra and bush okra are largely cultivated by both men and women for domestic and commercial purposes. common okra, abelmoschus esculentus (l.) moench (synonyms, hibiscus esculentus l.), belongs to the family of malvaceae, and is known as lady’s finger in english. it was believed to have originated in south-east asia and have spread widely in tropical, subtropical and warm temperate regions, but is particularly popular in west africa (hamon and sloten, 1995). on the other hand, bush okra (corchorus olitorius l.) belongs to the family of tiliaceae, its english name is jew’s mallow. genetic diversity points to africa as its first centre of origin (singh, 1976). at present, corchorus olitorius has widely spread all over the tropics and probably occurs in all countries of tropical africa (edmonds, 1990). it is a leading leaf vegetable in many african countries such as côte d’ivoire, benin, cameroon, and nigeria (schippers, 2000). common and bush okra are mucilaginous vegetable, both the fruit and leaves of these popular vegetables are used as food in nigeria. their young immature fruits are important vegetable, consumed cooked or fried. in nigeria, they are usually boiled in water to make slimy sticky soups and sauces. the young leaves of common okra are commonly used as spinach and sometimes as cattle feed (burkill, 1997) while that of bush okra are more valued as cooked slimy sticky sauce, compared to common okra fruit. in nigeria, sticky sauces from these two vegetables are found suitable for easy consumption of starchy balls made from cassava, yam or millet (akoroda, 1988). the very small fruits of common okra can fetch a higher price, being of prime quality while jew’s mallow is a high quality leafy vegetable in market value, consumers’ preference and nutritional value. the two vegetables are grown under rain fed conditions, the immature fruits of common okra and fresh leaves of bush okra can be conserved by drying, whole or chopped, or by pickling and sell as dried ground for the preparation of this slimy sauce during the dry season. common and bush okra mucilage is suitable for medical and industrial applications. common okra mucilage has been used as a plasma replacement or a blood volume expander and its leaves are sometimes used as basis for poultices, as an emollient and to treat dysuria. okra mucilage is added as size to glaze paper and is also used in confectionery. roasted common okra seeds are used in some areas as a substitute for coffee. tests conducted in china suggest that an alcohol extract of abelmoschus leaves can eliminate oxygen free radicals, alleviate renal tubular-interstitial diseases, improve renal function and reduce proteinuria (tomoda et al., 1980). on the other hand, jute mallow has been the most widely used packaging fibre for more than 100 years because of its strength and durability, low production costs, ease of manufacturing and availability in large and uniform quantities. in kenya, the root scrapings of jew’s mallow are used to treat toothache; in congo, the root decoction is a tonic and leafy twigs is used against heart troubles; in tanzania, an infusion of the leaves is taken against constipation; while in nigeria, the seeds are used as a purgative and febrifuge (edmonds, 1990; burkill, 2000). fresh common okra can be transported quite easily in bulk and kept for a few days without much loss of quality. however, jew’s mallow ital. j. food sci., vol. 27 2015 515 leaves cannot be kept long. mostly, the product is sold on the harvest day, and it is constantly kept wet. if cooled to 20°c it can be kept for about 1 week, in cold storage for several weeks. if the leaves are dried and pounded to powder, the product can be stored for at least half a year (akoroda, 1988). since, both vegetables are prepared as a sticky slim sauce by boiling in water and common okra is considered more stable in quality, then it is assumed that a lesser proportion of its nutrients would be lost through the boiling process when compared to jew’s jute. therefore, the present study was designed to estimate the mineral content of raw and sticky sauce of common and bush okra for recommended daily dietary intake and to compare mineral nutrient loss during preparation of common okra and bush okra sauce. the study will enhance knowledge on boiled and dried mucilage as a means of post harvest handling and storage for maintaining mineral constituents. materials and methods samples collection and preparation indigenous fresh green moderate size of common okra fruits and leafy bush okra samples were purchased from three retailers, as three replicates, in three major markets at ijebu-ode (new market, oke-aje and ita-ale). the samples were transported in properly labeled polythene bags to the chemistry laboratory, tai solarin university of education, ijagun, ijebu-ode, ogun state nigeria. twenty grams of wholesome samples were handpicked from each of the common okra and bush okra samples, destalked, rinsed thrice in deionized water, drained, chopped into smaller pieces, and divided into two portions. preparation of raw vegetables the first portions were air dried in an air-circulating oven at 65°c for 5hrs for bush okra samples while common okra was dried for 10 hrs. the dried samples were ground in a sample attrition mill (model no. ed-5), sieved into 4mm particle sizes, kept at 4°c and labeled as r (raw) samples. preparation of plain sticky sauce samples the second portions were further chopped into smaller pieces and boiled for 5 min to slim sticky sauce as usually prepared as complement to stew for consumption of starchy staples in the southwest of nigeria but without salt and then air dried at 55°c until constant weight was assumed, ground and sieved into 4mm particle sizes and labeled s (sauce) samples. assay methods all chemical analyses were carried out in triplicates using modified methods of the association of official analytical chemists (aoac, 2005). a gram of 4mm particle size of common and bush okra samples were combust at 500°c for 5 hrs in a cool muffle furnace and left overnight to cool to room temperature, the residue was weighed as ash content and kept at 4°c for mineral analysis. elemental analysis a half-gram ash of each sample was, subsequently, digested in 2.5 ml selenium/h 2 so 4 mixture (3.5 g se/1l h 2 so 4 ) at 200°c. the residue was re-suspended in selenium/h 2 so 4 mixture; na, mg, ca, and k were determined using jenway digital flame photometer (pfp7 model), phosphorous by the vanodo-molybdate method, while fe, mn, cu, and zn were determined using buck scientific atomic absorption spectrophotometer (buck 210vgp model). statistical analysis estimation of precision was measured using the statistical analysis system software package [19]. detection of variation between vegetables, raw and cooked, and purchased markets for minerals were based on analysis of variance (anova) using general linear model (glm) and useful relationships between minerals were estimated using pearson correlation analysis, in the same sas software package. results mineral levels in raw and sticky sauce of common okra and bush okra samples of nigerian common okra and bush okra by different retailers were obtained from three different major local markets in ijebuode and were assessed for nine macro and micro minerals in both raw and sticky sauce. the precision measures the nine assessed minerals, across all samples of raw and sticky sauce of common and bush okra as estimated in sas provided in table 1. in overall samples, the decreasing order of abundance of minerals were as follows: k>na>ca>p>fe>mg>zn>mn>cu, with the following mean values on a dry weight basis, 376.03±25.37 mg/100g, 250.2±8.58 mg/100g, 176.73±19.88 mg/100g, 82.11±7.47 mg/100g, 4.23±0.44 mg/100g, 2.91±0.33 mg/100g, 2.39±0.26 mg/100g, 0.88±0.12 mg/100g and 0.75±0.08 mg/100g, respectively. the results indicate the abundance of macro minerals over micro minerals but with a fall in mg. on a clos516 ital. j. food sci., vol. 27 2015 er observation made from the visualized bar graphs, levels of minerals were higher in both raw and sticky sauce of bush okra than common okra except cu for raw, mn and fe for cooked. levels of all the minerals in the two vegetables were reduced upon boiling (fig. 1). based on simple calculations of the estimates obtained in sas, common okra lost more of na, k, mg, and cu on cooking than bush okra while the latter lost more of p, ca and mn than the former but both lost almost the same amount of fe and zn. the minimum estimated loss in common okra on cooking and drying was 8.12% for ca and the maximum was 50.49% for fe with a mean loss of 30.91% while for bush okra, the loss ranged from 4.78% for na to 58.10% for mn with a mean loss of 30.63%. the difference in the overall loss in minerals from the two vegetables could be considered small and insignificant. therefore, one third of the minerals are lost in the cooking and drying process when packaged as a post harvest handling or table 1 descriptive statistics between the raw and boiled common okra and bush okra in ijebu-ode. variable mean sd se± cv range minimum maximum n mg/100g raw-common okra sodium 258.77 2.5 1.02 0.97 7 255.1 262.1 9 potassium 351.32 2.4 0.98 0.68 6.5 347.9 354.4 9 calcium 95.42 2.28 0.93 2.39 5.7 92.4 98.1 9 phosphorus 60.32 2.3 0.94 3.82 5.9 57.2 63.1 9 iron 4.24 0.99 0.4 23.25 2.21 3.1 5.31 9 zinc 2.67 0.99 0.4 36.93 2.23 1.52 3.75 9 magnesium 2.48 0.72 0.3 29.12 1.62 1.74 3.36 9 manganese 0.87 0.5 0.2 57.4 1.08 0.42 1.5 9 copper 1.05 0.21 0.08 19.76 0.48 0.78 1.26 9 sauce-common okra sodium 223.17 2.32 0.95 1.04 6 220 226 9 potassium 235.17 2.79 1.14 1.19 8 231 239 9 calcium 87.67 2.25 0.92 2.57 5.2 84.9 90.1 9 phosphorus 47.94 2.32 0.95 4.85 6.05 44.75 50.8 9 iron 2.1 0.99 0.4 46.96 2.23 0.95 3.18 9 zinc 1.56 0.99 0.4 63.08 2.28 0.39 2.67 9 magnesium 1.32 0.72 0.3 54.64 1.62 0.58 2.2 9 manganese 0.63 0.5 0.2 79.17 1.1 0.17 1.27 9 copper 0.67 0.21 0.08 31.11 0.47 0.4 0.87 9 raw-bush okra sodium 278.17 2.79 1.14 1 8 274 282 9 potassium 487.52 2.4 0.98 0.49 6.5 484.1 490.6 9 calcium 289.62 2.33 0.95 0.8 6.1 286.4 292.5 9 phosphorus 127.27 2.25 0.92 1.77 5.2 124.5 129.7 9 iron 7 1 0.41 14.21 2.46 5.74 8.2 9 zinc 3.39 0.99 0.4 29.13 2.31 2.2 4.51 9 magnesium 4.99 0.72 0.3 14.48 1.62 4.25 5.87 9 manganese 1.43 0.5 0.2 35.12 1.11 0.96 2.07 9 copper 0.7 0.19 0.09 27.27 0.48 0.38 0.86 9 sauce-bush okra sodium 264.87 2.46 1 0.93 6.8 261.3 268.1 9 potassium 457.57 2.61 1.06 0.57 7.4 453.7 461.1 9 calcium 234.92 2.58 1.05 1.1 7.3 231.1 238.4 9 phosphorus 96.94 2.32 0.95 2.4 6.05 93.75 99.8 9 iron 3.55 0.99 0.4 27.77 2.23 2.4 4.63 9 zinc 1.98 0.99 0.4 49.75 2.3 0.8 3.1 9 magnesium 2.8 0.72 0.3 25.84 1.63 2.05 3.68 9 manganese 0.6 0.5 0.2 83.83 1.11 0.13 1.24 9 copper 0.46 0.21 0.08 44.88 0.46 0.2 0.66 9 sd, standard deviation; se, standard error; cv, coefficient of variation. ital. j. food sci., vol. 27 2015 517 fig. 1 trends of minerals levels in raw and sauce of common and bush okra on dry weight basis. raw_al, raw common okra; sauce_al, common okra sauce; raw_co, raw bush okra; sauce_co, bush okra sauce. fig. 2 proportion of mineral loss by common okra and bush okra upon cooking at 100°c for 5 min okro, common okra; olitorius, bush okra. management for later use as slimy sauce. the trend in mineral loss per vegetable in cooking is provided in fig. 2. interaction of vegetable type, purchased market and cooking on mineral content analysis of variance (anova) based on general linear model (glm) in the same sas package, in overall, showed a very large variation in all the assessed minerals (p<0.0005) but less variation was observed for cu (p<0.005) and zn (p< 0.05) while mn was not significantly varied. the variations observed in the nutrients were highly contributed by the differences in the vegetable type and raw/cooking (processing). however, only raw/cooking contributes to the variation observed in zn. manganese, though, not generally significant but was significantly varied between raw or cooked vegetable. therefore, there was a significant difference between common okra and bush okra for na, p, k, ca, mg, fe, and cu but no significance difference was observed between the two vegetables for zn and mn. the results also showed that cooking has a significantly negative effect on the levels of all the minerals assessed, that is, the levels of minerals reduced on cooking. effect of market was also tested for only the three micro minerals; zn, mn and cu were significantly varied while the rest were not. duncan multiple grouping provides a visual variation of the nutrients between the vegetables, raw/boiled, and markets, based on the significant difference between their means, by assigning them into groups using letters a, b and c. a detailed anova measures and duncan grouping were also given in table 2. 518 ital. j. food sci., vol. 27 2015 relationship among minerals pearson correlation analysis was used to identify useful associations among the nutrients. the matrix generated (table 3) revealed that na was strongly significantly correlated with all the other macro minerals (p<0.0005) and fe but slightly with zn and mn. therefore, as the level of na increases, the level of all p, ca, k, mg, fe, zn and mn also increases and vice-versa. potassium and calcium were independently correlated with all the macro minerals and fe but both were insignificant with the rest of micro minerals. therefore, as levels of k and ca increases, level of other macro minerals and fe increases. a strong positive relationship was observed among fe, zn and mg (p<0.005). manganese on the other hand was correlated with only na and p while cu had no relationship with any of the minerals. the correlation matrix also showed that the levels of the minerals were higher in bush okra than common okra except cu that was inversely correlated while zn and mn were insignificant. in addition, the levels of minerals were observed to be inversely correlated with cooking except that of p, k and ca. therefore, table 2 effect of species type, cooking and purchased market on mineral levels based on glm-anova. model species raw/sauce market mean cv ss r2 pr>f pr>f ss pr>f ss pr>f ss macro-mineral mg/100g na 256.24 2.58 9187.47 0.91 *** *** 5599.81 *** 3586.81 ns 101.3 k 382.89 6.20 224919 0.95 *** *** 192890 **** 32017 ns 101.3 p 83.12 6.5 22901 0.97 *** *** 20168 *** 2734 ns 101.3 ca 176.9 7.83 180737 0.98 *** *** 174882 *** 5850 ns 101.3 mg 2.9 26.78 40.70 0.77 *** *** 23.32 *** 16.88 ns 10.45 micro-minerals fe 4.22 24.39 73.62 0.77 *** ** 26.65 *** 46.96 ns 19.41 cu 0.70 30.27 1.04 0.53 ** ** 0.55 * 0.50 ** 0.85 mn 0.88 59.64 2.12 0.27 ns ns 0.40 * 1.71 *** 5.02 zn 2.4 41.22 11.4 0.37 * ns 1.94 ** 9.45 *** 19.41 duncan grouping na k ca p mg mn fe zn cu by species commonokra b b b b b a b a a bush okra a a a a a a a a b by process raw a a a a a a a a a sauce b b b b b b b b b ns, not significant; *, p<0.05; **, p<0.005; ***, p<0.0005, ss, sum of square. means with the same letter are not significantly different. table 3 correlation matrix among the minerals based on pearson model. veg_c is degree of common consumption (okra<ewedu), proc_c, raw to cooking vice versa. proc_c na k ca p fe zn mg mn cu veg_c ns 0.74*** 0.90*** 0.97*** 0.92*** 0.50* ns 0.65** ns -0.46* proc_c 1 -0.58** ns ns ns -0.68*** -0.53** -0.54** -0.46* -0..56** na 1 0.95*** 0.79*** 0.86*** 0.74*** 0.45* 0.77*** 0.42* ns k 1 0.91*** 0.93*** 0.68*** ns 0.76*** ns ns ca 1 0.98*** 0.63** ns 0.76*** ns ns p 1 0.72*** ns 0.82*** 0.43* ns fe 1 0.89*** 0.97*** ns ns zn 1 0.83*** ns ns mg 1 ns ns mn 1 ns ns, not significant; *, p<0.05; **, p<0.005; ***, p<0.0005. ital. j. food sci., vol. 27 2015 519 cooking reduces the level of these minerals in the assessed vegetables, thereby supporting the results obtained from anova. discussion adequacy of the minerals in common and bush okra the adequacy of mineral content for estimated average requirements (ear) and recommended daily allowance / adequate intake (rda/ai) in both the raw and sticky sauce of common and bush okra per hundred grams were judged using dris (dris, 2004). the mean levels of potassium (235.51 487.52 mg/100g) and sodium (223.17 278.17 mg/100g) estimated in the raw and sticky sauce of both common okra and bush okra were too low to meet the rda/ai for k (4700 mg) and na (1500 mg). therefore, the content of sodium in both the raw and sauce of common bush (223.2 258.8 mg/100g) and bush okra (264.9 278.1 mg/100g) is considered safe due to health impairments such as increased urinary calcium loss and hypertension that could result from excess intake of sodium (wardlaw and kessel, 2002). since, cooking reduces na level, then, common okra and bush okra sauces are good in restricted na diets. neither the raw nor the sauce of the two vegetables could also provide the daily ear of calcium (800 mg), although bush okra could provide about 30% estimated average requirement (800 mg) while common bush okra could only provide about 11%. therefore, additional supplementary sources of ca such as fortified cereal and condensed cow’s milk (usda, 2010) were required in the walnut diets. the magnesium level, either in the raw or sauce of the two vegetables (0.67 4.99 mg/100g) were too low for any mg provision in the body when compared with the daily ear of 330 mg. so, the phosphorus content of the two vegetables (48 127 mg/100 g) was also inadequate to provide for 580 mg ear. estimated average requirements for mn is yet to be evaluated but the rda/ai is 2.3 mg and each of the raw vegetable and sauce could provide an approximately proportion of 25% of the allowance except for 1.43 mg obtained in raw bush okra that could provide about 62%. both the raw of common okra (1.05 mg/100 g) and bush okra (0.7 mg/100 g) had an adequate ear content of cu (700 ug) while their sauce had a proportion of 96 and 66%, respectively. however, raw common okra contains cu level that is slightly higher than the 900 ug rda but lower than tolerable upper limit level of 10000 ug, [20], which is reduced upon cooking by 36% (0.67 g) and therefore posed, no health hazard. none of the vegetables, either raw or cooked sauce could meet the daily average requirement and allowance for zn intake of 9.4 and 11 mg, respectively. only the raw bush okra met the estimated average requirement of fe (6 mg), which is 77.7% of daily allowance intake (8 mg) but was reduced upon cooking by 34%, providing about 76.5% ear while raw common okra could provide for 70.1% ear, which was also reduced upon cooking by 30%. in overall, plain sauce of these vegetables contains appreciable levels of minerals assessed but did not meet the daily dietary allowance of these minerals except for cu and fe. therefore, the sauce should be taken to complement carbohydrate based meal or soup supplement that contains additional sources of minerals or should be made into a proper soup with top sources of minerals for provision of adequate daily dietary intake of these minerals for health implications. interaction of species, cooking and market on mineral levels boiling was observed to reduce all the mineral contents in both the common and bush okra with a variation between the two okra vegetables, common okra lost more in macro minerals than bush okra (except in ca and k) while bush okra lost more of micro minerals. by simple estimation, based on average, about one-third of the mineral content was lost on dry weight basis, when considered for use as a mucilage sticky sauce after boiling. similar loss due to boiling was reported in both common and bush okra and many other vegetables (mepba et al., 2002; auta et al., 2011; aye, 2012; yakubu et al., 2012; ilelaboye et al., 2013). there are also reports on species differences for loss in minerals as presently observed (mepba et al., 2002, ilelaboye et al., 2013; shahnaz et al., 2003). varying degree of mineral loss due to cooking / boiling were reported in similar studies, a proportion of 6 to 30% mineral loss (ilelaboye et al., 2013), 25 to 55% (aye, 2012) and 30 to 54% (yakubu et al., 2003). in addition, shahnaz et al. (2003) reported a pronounced effect of peeling combined with cooking on mineral loss of some vegetables (14 to 74%). the mineral loss in the present study ranged between 4.0%, for na in common okra, to 58%, for mn in bush okra, which is comparable to the previous reported losses. the differences in ranges reported could be largely due to differences in species type, region, analytical methods, varying degree of cooking time and temperature. whatsoever, there is loss of minerals due to either boiling or cooking, which is as a result of mineral leaches from the vegetables into the cooking water during the process. most of the proteins that contain the inorganic elements such as prosthetic groups are denatured during the process with loss of these minerals into the boiling water. market effect was limited to three micro minerals, mn, cu and zn which could be attributed to diverse agricultural practices from different 520 ital. j. food sci., vol. 27 2015 sources, soil fertility, environments and use of fertilizers. similar useful associations obtained among the minerals have earlier been observed in food crops. the strong association obtained between fe and zn in the present study corroborates that of similar studies in plants (beebe et al., 2000; silva et al., 2012). conclusions the study detected appreciable levels of p, na, mg, k, ca, fe, mn, cu, and zn in both common and bush okra per 100g which were reduced upon cooking at 90-100oc for 5 min. the two species differed widely in their levels of mineral content in both raw vegetable and sticky sauce. they also lost specific mineral on cooking at different proportion but had an overall equal proportion of a mean percent loss. there was also an influence of market differences on cu mn and zn. dried mucilage as a postharvest management for later use such as sticky sauce reduced the mineral content by a mean proportion of about 30%. plain soup of these vegetables either fresh or after storage requires additional sources of these minerals, except fe and cu, to meet recommended daily intake but are good sources for na and any of these minerals restricted diets. references akoroda m.o. 1988. cultivation of jute (corchorus olitorius) for edible leaf in nigeria. tropical agriculture 65(4): 297-299. aoac. 2005. “official methods of analysis”. 18th ed. association of official analytical chemists. washington d. c. auta r., james s.a., auta t. and sofa e.m. 2011. nutritive value and phytochemical composition of processed solanum incanum (bitter garden egg). science world journal 6(3): 1-6. aye p.a. 2012. effect of processing on the nutritive characteristics, anti-nutritional factors and functional properties of cnidoscolus aconitifolius leaves (iyana ipaja). am. j. food. nutr. 2(4): 89-95. beebe s., gonzalez a.v. and rengifo j. 2000. research on trace minerals in the common bean. food nutr. bull. 21, 387-391. burkill h.m. 1997. the useful plants of west tropical africa. 2nd ed. vol. 4, families m-r. royal botanic gardens, kew, united kingdom. 969 pp. burkill h.m. 2000. the useful plants of west tropical africa. 2nd ed. vol. 5, families s–z, addenda. royal botanic gardens, kew, united kingdom. 686 pp. cox b.d., wlichelow m.j. and prevost a.t. 2000. seasonal consumption of salad vegetables and fresh fruit in relation to the development of cardiovascular disease and cancer. public health nutr. 3: 19-29. denton l. 1997. a review of corchorus olitorius l. in nigeria. in: schippers r.r. and budd l. 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(editor). evolution of crop plants. longman, london, united kingdom. pp. 290-291. tomoda m., shimada k., saito y. and sugi m. 1980. plant mucilages. 26. isolation and structural features of a mucilage, ‘okra-mucilage f’, from the immature fruits of abelmoschus esculentus. chemical and pharmaceutical bulletin. 28: 2933-2940. usda. 2010. national nutrient database for standard reference, sr23. 2010 wardlaw g.m. and kessel m.w. 2002. in perspectives in nutrition. 5th edition, mcgraw-hill companies inc. pp 418-464. yakubu n., amuzat a.o. and hamza r.u. 2012. effect of processing methods on the nutritional contents of bitter leaf (vernonia amygdalina). am. j. food. nutr. 2(1): 26-30. paper received october 23, 2014 accepted december 4, 2014 http://www.iom.edu/global/news%20announcements/~/media/files/activity%20files/nutrition/dris/dri_summary_listing.pdf http://www.iom.edu/global/news%20announcements/~/media/files/activity%20files/nutrition/dris/dri_summary_listing.pdf paper 468 ital. j. food sci., vol. 27 2015 keywords: active packaging, antioxidant, biodegradable polymer, poly (butylene adipate co-terephthalate), thermoplastic starch development of an active biodegradable film containing tocopherol and avocado peel extract j.c.f. fidelis1, a.r.g. monteiro1*, m.r.s. scapim1, c.c.f. monteiro2, d.r. morais3, t. claus3, j.v. visentainer3 and f. yamashita4 1state university of maringa, department of food engineering, av colombo, 5790 bloco 13 cep 87030-900 maringá, pr, brazil 2state university of maringa, department of design. rua dom pedro ii, 598, cep 87200-055, cianorte, pr, brazil 3state university of maringa, department of chemistry av colombo, 5790 bloco 13 cep 87030-900 maringá, pr, brazil 4state university of londrina, department of food science. rod. celso garcia cid, km 380, p.o.box 10.011 cep 86057-970londrina, pr, brazil *corresponding author: argmonteiro@uem.br abstract thermoplastic starch (tps) films and poly(butylene adipate co-terephthalate) (pbat) (60/40 m/m) containing toco-70 (tocopherol/soybean oil 70/30 m/m) and avocado peel extract (exa) were produced using blown film extrusion. the formulations of the 5 films (fc/f1/f2/f3 and f4) were established through mixture design with constraints maintaining constant pbat and tps proportion, and varying the antioxidant concentrations. adding antioxidants reduced the water vapour permeability (kw) of the films, with formulation f2 presenting higher decrease in relation to fc, 77.8%. the presence of exa improved the mechanical properties of the films. the production of the films was determined to be viable after they presented good processability in a pilot extruder, as well as mechanical properties appropriate to production and utilization in industry. the presence of exa and toco 70 provided the films with antioxidant activity; their application as active packaging requires further studies. http://argmonteiro%40uem.br ital. j. food sci., vol. 27 2015 469 1. introduction the fundamental function of food packaging is to delay the deterioration of the product in all physical, chemical, microbiological, sensory and nutritional aspects, ensuring the quality of the product until it is consumed (restuccia et al., 2010). the oxidation of food sensitive substances form hydroperoxides that decompose into aldehydes, ketones, alcohols, acids, esters or hydrocarbons, altering the color, texture, smell and taste, as well as reducing the shelf life (mcclements and decker, 2000). active packaging interacts with the food to increase its shelf life, and materials containing antioxidants are a promising alternatives used to develop new active packaging (byun et al., 2010; pereira de abreu et al., 2012; pereira et al., 2013). tocopherol is a liposoluble antioxidant that is widely applied in food as a primary antioxidant. tocopherol is highly stable when applied in films of polyethylene and polypropylene, even under severe extrusion conditions. (al-malaika et al., 1999; al-malaika et al, 2001). recent studies regarding films elaborated from biodegradable polymers with tocopherol have generated positive results concerning antioxidant migration when applied to food (byun et al., 2010; graciano-verdugo et al., 2010; lópez et al., 2011; hwang et al., 2013; martins et al., 2012). several active principles originating from plants have been applied as anti-oxidants (dicastilho et al., 2011; pereira de abreu et al., 2012; rubilar et al., 2013), such as the avocado; the avocado is an oleaginous fruit native of tropical america (mexico) that is rich in phenol derivatives. only 69% of this fruit are edible, the remaining is constituted of stone and skin. the use of the skin to extract antioxidants may add value to the residue and minimize environmental issues. (wang et al., 2010) studied the antioxidant capacity of peels and seeds in various avocado species and concluded that these residues also contain large quantities of phenol compounds, among other antioxidants. rodríguez-carpena et al. (2011) used avocado peel and seed extracts to inhibit lipid oxidation in meat products. active packaging can be manufactured using ecofriendly materials, such as biodegradable polymers. starch has been used as an alternative to produce biodegradable films, but the materials present a low mechanical strength and are hygroscopic (mali et al, 2005; ren et al., 2009; scapim, 2009). the conversion of starch into thermoplastic starch (tps) involves heating and shearing with a plasticizer agent, such as glycerol. the materials produced from blends of thermoplastic starch and other biodegradable polymers may be used to develop low-cost biodegradable packaging with better mechanical and barrier properties (brandelero et al., 2012; olivato et al., 2011; ren et al., 2009; scapim, 2009). poly(butylene adipate co-terephthalate) (pbat) is a biodegradable polyester with mechanical properties similar to the those of polyethylene films with resistance to fat and humidity and temperature variations (basf, 2013). previous studies have studied biodegradable materials produced using blends of thermoplastic starch and pbat (brandelero et al., 2012; olivato et al., 2011; ren et al., 2009; scapim, 2009). the objective of this study was to characterize and assess the mechanical and structural properties of biodegradable films composed of thermoplastic starch, pbat and antioxidants (tocopherol and bark extract avocado) seeking better interaction among these compounds in order to suggest further studies on the application of these films in active packaging. 2. materials and methods 2.1. materials the film production utilized native cassava starch (indemil com. ind. ltda, brasil), glycerol (synth p.a, brasil), poly(butylene adipate co-terephthalate) (ecoflex®, basf chemical company, brasil) and toco-70 (tocopherols solution/soybean oils 70/30 m/m) (danisco, brasil). the avocado fruits (persea americana mill) were utilized at an intermediate maturity stage as moderately tender and green peel derived from são paulo state after being purchased in maringá pr. 2.2. production and characterization of avocado peel extract the peels were separated from fruits, cut into 2-cm squares, dried in a greenhouse at 60°c for 24 h, ground in an analytical mil and sieved. the extracts were prepared according to santos et al. (2011) with some modifications. the previously dried and ground peels were soaked in ethanol at 95% v/v (proportion 1/10 m/v) with stirring for 4 h. the solution was vacuum filtered and concentrated via rotary evaporation at 40°c. the dry extracts (exa) were stored in amber flasks at -18°c, under nitrogen atmosphere (n 2 ) until the analyses. the compounds in exa were characterized and quantified using ultra-high performance liquid chromatography coupled to electrospray ionization mass spectrometry in negative ion mode (uplc-esi(-)-ms) (acquity uplctqd, waters, eua). approximately 10.0 mg of dry exa and 1.0 mg of standard in hplc grade methanol were injected (3 µl) into uplc-esi()-ms equipped with an acquity column uplc beh c18 (2,1 x 50 mm) with 1.7 µm particles at 30°c. a gradient elution at 0.20 ml/min with solvent a (formic acid at 0.1%, in milli-q water) and b (methanol) was used with the following 470 ital. j. food sci., vol. 27 2015 method: from 95% of a and 5% of b to 100% b in 8.00 min, and the system was stabilized at 10.00 min. the spectra were acquired under the following conditions: capillary and cone stress of -3000 v and -30 v, respectively, with a source temperature of 150°c and a temperature of desolvation of 350°c. 2.3. preparation of the biodegradable films we produced 5 different types of biodegradable films, where the film control (fc) was used as a reference and was composed of starch/ glycerol/pbat the proportions 42/18/40 (w/w/w). in the other films concentrations toco-70 and exa were determined using a mixture design with constraints (table 1), were proportion of starch/glycerol/pbat was maintained at 42/18/40 (m/m/m). the results were analyzed using the statistica 7.0 software (statsoft, eua). the films were processed in the technology laboratory of food science and technology department of state university of londrina (uel brazil). the antioxidant compounds were mixed with glycerol. initially, the pellets were produced in a pilot twin screw extruder (bgm, model d-20, brazil) equipped with screws measuring 20 mm in diameter and 680 mm in length. the speed of the screws was set at 100 rpm, and the temperature profile in all five heating zones was 90/120/120/120/120°c. the film was produced in a pilot single-screw extruder (bgm, model el-25, brazil) equipped with a screw measuring 250 mm in diameter. the speed was maintained at 35 rpm, and the temperature profile in all four heating zones and in the matrix for balloon formation was 90/120/120/130/130°c. 2.4. biodegradable films 2.4.1. thickness (δ) and density (ρ) the thickness (δ) of the films was determined using a digital micrometer (0.001 mm resolution, mitutoyo, japan). twelve random points on each film formulation were assessed. for the density (ρ), the average masses of 10 square samples (25 mm x 25 mm) of film were calculated after being conditioned in desiccators with anhydrous calcium chloride for 20 days. 2.4.2. water vapour permeability (kw) the films were conditioned at 64±2% rh and 25±2°c for 72 h. the film samples were fixed to a circular opening of a permeation cell (area of 60 mm2) with silicone grease. the interior of the cell was filled with magnesium chloride saturated solution (33% rh) and was stored at 25±2°c in a desiccator that contained sodium nitrate saturated solution (64% rh) to maintain a 31% rh gradient across the film. the sample were weighed every 3 h during 72 h of testing time. the changes in the weight of the cell or mass gain (m) were plotted as a function of time (t). the slope of each line was calculated using linear regression, and the water vapour permeation ratio (kwr) was obtained using equation (1): kwr = (m/t).(1/a) where m/t is the angular coefficient of the curve and a is the sample permeation area. the kw was calculated as equation (2): kw= kwr.st/sp(rh 1 -rh 2 ) where st is the mean sample thickness (m), sp is the water vapour saturation pressure at the assay temperature (pa), rh1 is the relative humidity of the desiccator and rh2 is the relative humidity in the interior of the permeation cell. the tests were conducted in triplicate. 2.4.3. sorption isotherms the sorption isotherms of the films were determined according to the methodology described by scapim (2009) using the following relative humidities at 25°c: 11.3, 33, 43.2, 52.9, 64.5, 75.3, 84.3 and 90.2%. the isotherms were modeled according to the guggenheim-anderson-de boer (gab) model (equation 3) using the quasi-newton method in statistica 7.0. x w = where x w (g of water/g of dry matter) is the relative humidity of balance, m 0 is the water content in the monolayer (g of water/g of solids), a w is the water activity, and c and k are constants from the gab model that represent the sorption heat in the first layer and sorption heat of the multilayer. table 1 formulation of biodegradable films according to mixture design and the constraints. films component (m/m/m) film formulation starch + glycerol exa toco-70 + pbat x 1 x 2 x 3 fc 1.000 f1 0.994 0.006 f2 0.992 0.008 f3 0.986 0.006 0.008 f4 0.993 0.003 0.004 ° x 1 + x 2 + x 3 = 1 ital. j. food sci., vol. 27 2015 471 2.4.4. determination of solubility (β) and diffusion (dw) coefficients the method used to calculate the solubility coefficient (β) was proposed by larotonda et al. (2005) using the first order derivative of the gab. this model is correlated with the water activity and water vapor pressure (p s ) at 25°c according to equation 4. where β(g/g.pa)is the solubility coefficient, c, k, and m 0 are the gab model parameters and ps (pa) is the water vapor pressure at 25°c. the a w was the average of the relative humidity gradient used in kw(g/m.pa.day) (item 2.4.2). the coefficient of water vapor diffusion (dw) was calculated according to equation 5 using the values of β and kw, as determined in section 2.4.2, where ρ is the film density: pva dw= kw/ρ.β ρ.β 2.4.5. mechanical properties the mechanical properties (ultimate tensile strength and elongation at rupture) were assessed according to astm d 882-91 (astm, 1996) using a texturometer (stable micro systems, model ta.tx2i, england) with a 30-mm distance between the clutches and a traction speed of 500 mm/min. the samples were 80 mm long and 6 mm wide; they were conditioned under 64.5% rh at 25°c for seven days before the analyses. for each formulation, 10 repetitions were performed. 2.4.6 microstructures analysis the analyses were conducted using scanning electron microscope (shimadzu, model ss-550n, japan) based on the methodology descripted by scapim (2009). the samples were immersed in liquid nitrogen and fractured with the aid of tweezers before being conditioned in a desiccator with calcium chloride to remove any humidity for three weeks. the samples were subsequently overlaid with two golden covers through a metallizer (shimadzu ic-50 ion coater, japan). after preparation, the samples were visualized through electronic scanning microscope to have its fracture area surface analyzed. 2.4.7. antioxidant capacity of avocado peel extract and films the analyses were carried out according to the method described by serpen et al., (2012). sample of 10.0 mg of avocado peel extract and each film formulation was reacted with 5 ml of 2.2-diphenyl-1picrilhidrazil (dpph•) in ethanol/water (50/50, v/v) over 1, 2, 3 and 4 hours under magnetic stirring and protection from light. afterward, the blend was centrifuged for 1 min at 300-400 rpm (8 g) and the absorbance of the supernatant was measured through a spectrophotometer (thermoscientific, model genesys 10 uv, usa) at 525 nm. methanolic solutions of (±)-6-hydroxy-2,5,7,8-tetramethylcromano-2-carboxylic acid (trolox) in different concentrations were used to obtain the calibration curve for 5 ml (y = 29.448x + 5.4989, r² > 0.99) with the oxidant capacity expressed in µmol of trolox equivalents (et)/g for each film formulation. 3. results and discussion 3.1. antioxidant compounds and antioxidant capacity of avocado peel extract. the following antioxidant compounds were identified and measured in the avocado peel extract: citric acid (133±10 µg/100 g extract), catechin hydrate (82±3 µg/100 g extract), malic acid (75±2 µg/100 g extract), epicatechin (62±3 µg/100 g extract) and tartaric acid (11.8±0.1 µg/100 g extract). prior studies have revealed positive results for active packaging developed using plant extracts with compositions similar to exa (dicastilho et al., 2011; lópez et al., 2011; pereira de abreu et al., 2012). the antioxidant capacities of exa and toco70 were 188±8 mmol et/g o and 162±8mmol et/g, respectively. wang et al. (2010) assessed different avocado species and concluded that both the seed and peel contain copious quantities of phenolic compounds and high antioxidant capacities. the authors found variations from 38 to 189.8 µmol of et/g in peels from different species; the peel contributes 38% of the antioxidant capacity of the entire fruit. therefore, the extraction concentrated the relevant compounds, presenting an antioxidant capacity more than a thousand times higher than that reported by (wang et al., 2010). 3.2. films characterization 3.2.1. sorption isotherms the films all exhibited practically constant water sorption until 60% ur, and above 60% 472 ital. j. food sci., vol. 27 2015 an increase in sorption occurred (fig. 1) due to the hydrophilic nature of the starch. according to talja et al. (2008), for relative humidities above 60%, a replacement of starch-starch and starch-glycerol interactions with starchwater and water-glycerol interactions may occur, justifying the increase in water sorption from the humidity. adding toco-70 and exa decreased the hydrophilicity of the films under ur above 75% while formulations f2, f3, f4 and f1 presented reductions of 34, 21, 11 and 10%, respectively, concerning water sorption in the control film (fc). the formulation containing only toco-70 (f2) presented the lowest water sorption in high relative humidities most likely due to the compatibility between the tocopherol and the soybean oil present in the compound with the starch and pbat blend (brandelero et al., 2012). the gab model was satisfactorily adjusted to the experimental isothermal data, and the determination coefficients (r²) varied from 0.89 to 0.99 (table 2). the constant values of sorption for the multilayer (k) are correlated with the isothermal behavior in relative humidity above 65% (fig. 1); specifically, the higher the water sorption for the films in that area are, the higher the k values are. 3.2.2. water vapour permeability (kw), the coefficient of solubility (β), the coefficient of diffusion (dw), the thickness (δ) and the density (ρ) the films containing antioxidants presented values for the water vapour permeability (kw), the coefficient of solubility (β) and the coefficient of diffusion (dw) lower than for the control film (fc) (table 3) due to the hydrophobic nature of exa and toco-70. film f2 contained only toco-70 and presented the lowest hydrophobicity, with a decrease of 77.8% in relation to the control formulation (cf). considering that the hydrophilicity of the cassava starch is a major obstacle for its application in packages, the addition of antioxidants may also contribute to improve this characteristic (avérous and boquillon, 2004; brandelero et al., 2011; olivato et al., 2011; ren et al., 2009). according to the model generated using the mixture design (r2=0.83) that was correlated with the kw in accordance with the film components (fig. 2), the films containing only one type of antioxidant present a reduced kw that has proven to be more effective than the blends. this behavior may be verified using the positive interaction model +5.x 2 .x 3 ; specifically, when the film contains both antioxidants (x 2 = avocado extract; x 3 = toco-70), an increase in kw occurs. brandelero et al. (2012) verified that the presence of soybean oil in the atp/pbat blends facilitated an interaction between the carboxyl group of the fatty acids and the pbat, as well as film compaction promoted by the starch, which explains why formulation f2 presented the lowest β e dw coefficients. both exa and toco-70 acted as compatibilizers, helping polymers interact and enabling the formation of a more compact structure that gives the molecules less fig. 1 water sorption isotherms of biodegradable films at 25 °c. table 2 parameters for the gab equation at 25°c for the biodegradable films. parameters films formulations for gab model fc f1 f2 f3 f4 m 0 0.0348 0.0370 0.0394 0.0399 0.0465 c 168.52 159.57 150.49 163.361 139.76 k 1.02 0.97 0.86 0.91 0.87 r² 0.97 0.99 0.89 0.94 0.96 table 3 water vapour permeability (kw), thickness (δ), density (ρ), coefficient of solubility (β) and coefficient of diffusion (dw) for the biodegradable films. formulation kw (x10-6) δ ρ β (x10-5) dw (x106) (g/m.pa.day) (µm) (g/cm³) (g/g.pa) (m²/day) fc 14.51±0.47 196±37 1.278 4.50 0.25 f1 6.75±0.46 149±19 1.475 4.12 0.11 f2 3.21±0.52 146±13 1.165 3.24 0.08 f3 6.03±0.81 130±17 1.279 3.69 0.13 f4 7.53±0.98 161±17 1.153 3.95 0.17 ital. j. food sci., vol. 27 2015 473 space to move, as shown by the coefficient of diffusion. these additives also provide lower hydrophilicity, as the coefficient of solubility indicates. possibly, such improvement in the interaction of polymers promoted by the presence of antioxidants contributed to the lower thickness of the films since all of the formulations with an antioxidant presented reduced thickness compared with the control formulation, fc, with higher mean value, 196±37µm, and f3 with the lower mean value, 130 ±17µm. 3.2.3. mechanical properties according to the model generated using the mixture design (r2=0.77) to correlate the mechanical properties with the film components, the avocado extract (exa) had a larger effect than toco-70 concerning the tensile strength (fig. 3) and the elongation of the films (fig. 4). according to olivato et al. (2012), both citric and malic acids promote reticulation (crosslinking) between thermoplastic starch fig. 2 permeability of water vapour of films according to starch/pbat, exa and toco-70 contents. fig. 3 tensile strength of the films according to starch/ pbat, exa and toco-70 contents. fig. 4 film elongation at break according to starch/pbat, exa and toco-70. and pbat, making the films resistant toward traction. exa contains both acids in its composition, improving the mechanical properties of the films. 3.2.4. antioxidant capacity of biodegradable films according to the model generated while using the mixture design (r2=0.98) and correlating the antioxidant capacity with the film components, the effect of the avocado peel extract (exa) on the antioxidant capacity of the films was higher than that of tocopherol (toco70) (fig. 5). the antioxidant capacity of the exa extract and the toco-70 solution are similar, however, the films containing exa presented a higher antioxidant capacity compared to the films containing toco-70, most likely due to the higher thermal stability of the exa. 474 ital. j. food sci., vol. 27 2015 3.2.5. desirability function as observed in the analyses of kw the presence of toco-70 provided better result when compared with formulation fc, with 77.8% decrease. however, by assessing the contour surfaces of the mechanical properties and the antioxidant capacity, it is possible to observe the occurrence of a tendency to have these characteristics improved when exa is more present than toco70. the desirability function indicated that the formulation that maximizes the ultimate tensile strength, elongation at rupture, and the antioxidant capacity while minimizes the permeability to water vapor (kw) is a film whose composition should consist of 98.95% starch + pbat + glycerol, 0.556% exa, and 0.494% toco-70. after these results, further studies are going carry out with the same composition defined by desirability function. 3.2.6 microstructure in the surface and film fracture micrographs neither pores, non-gelatinized granular neither starch nor phase separation were detected, thereby indicating good interactions between the components of the films for all formulations. 4. conclusions the production of biodegradable films composed of thermoplastic starch, pbat and antifig. 5 antioxidant capacity of the films according to starch/ pbat, exa and toco-70 contents. oxidants (tocopherol and avocado peel extract) is viable since the films presented good processability in a pilot extruder, as well as the appropriate mechanical properties for industrial production and applications. the presence of exa provided the films with antioxidant activity enabling their application as active packaging. it is required to conduct further studies to test the application of this blend of polymers and antioxidants in biodegradable and active packaging for products rich in lipids, such as hamburguers, nuggets, peanuts and soy, to verify the occurrence 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w., bostic r.t., and gu l. 2010. antioxidant capacities, procyanidins and pigments in avocados of different strains and cultivars. food chemistry 122(4), 1193-1198. http://dx.doi.org/10.1016/j.foodchem.2010.03.114. paper received october 23, 2014 accepted february 2, 2015 #342_symoniuk_bozza ital. j. food sci., vol 29, 2017 276 paper kinetics parameters of refined and cold-pressed rapeseed oils after oxidation by rancimat e. symoniuk *, k. ratusz and k. krygier adepartment of food technology, warsaw university of life sciences (sggw), nowoursynowska street 166, 02-787 warsaw, poland *corresponding author. tel.: +48 225937525; fax: +48 225937527 e-mail address: edyta_popis@sggw.pl abstract this works summarizes results of quality assessment of five labelled as refined and five labelled as cold-pressed rapeseed oils. the analyzed oils were characterized by a good quality that meet requirements of the codex alimentarius standard. oxidative stability of the oils was determined by rancimat test at five temperatures (90, 100, 110, 120, 130 °c). the oxidation rate constant (k) was observed to increase along with increasing process temperature. the arrhenius equation and active complex theory were used to compute the exponential factor (z), activation energy (ea), reaction rate constant at various temperatures (k) as well as enthalpy (∆h++) and entropy (∆s++) of the oxidation reaction of the analyzed oils. the ea, ∆h++and ∆s++ values ranged from 76.43 to 82.44 kj/mol, from 72.12 to 79.26 kj/mol and from –48.35 to –68.45 j/molk for the refined oils as well from 74.24 to 77.56 kj/mol, from 71.04 to 75.47 kj/mol and from –56.40 to –69.99 j/molk for the cold-pressed oils, respectively. keywords: oxidation kinetics, oxidative stability, rancimat, rapeseed oil ital. j. food sci., vol 29, 2017 277 1. introduction today, rapeseed (brassica napsus l.) oil is produced mainly from double zero “00” cultivar. double zero cultivar compared to first rapeseed cultivars is characterised by low content of eruic acid (0-2%), whose decomposition products are harmful to human health and glucosinolate (8-15 μm/g of seeds), which did not allow for full use of the protein contained in the seeds. rapeseed are used mainly for edible oil production, but also as an important feedstock for biodiesel production. rapeseed may be manufactured with the coldor hot-pressing method or via extraction and purified by refining. the refined oil, also referred to as universal cooking oil, is the most popular on the market and used for various culinary purposes. however, in last years has been observed a growing interest in the cold-pressed oil. compared to crude oil, the refined oil contains less free fatty acids, phosphoand glycolipids and metal ions, it has also a brighter color and higher oxidative stability. a negative effect of refining is an increase in the number of trans isomers and a decrease in the content of natural antioxidants, e.g. tocopherols (čmolik et al., 2000; tasan and demirci, 2003; karabulut et al., 2005). one of the key reactions proceeding in edible oils during storage and heat treatment is oxidation. the oxidation products formed contribute to the development of unpleasant odor, to deterioration of oil quality and nutritive value, and may also induce toxic effects on a human body (jacobsen and nielsen, 2008; matthäus et al., 2010; shahidi and zhong, 2010). the process of oxidation is linked with the notion of oxidative stability, namely resistance to oxidation under specified conditions. lipid oxidation may be analyzed with various methods. the most reliable one is a shelf test that consists in placing a sample of oil at a room temperature and periodical measurements of the peroxide and anisidine values (ratusz et al., 2002; velasco et al., 2003; farhoosh, 2007; kowalska et al., 2014). owing to a long duration of this test (a few months), an increasing interest is expressed today in the so-called accelerated tests: they are significantly faster, more precise and unbiased (ostrowska – ligęza et al., 2010). today, the following accelerated tests are found applicable for the evaluation of the oxidative stability of fresh edible oils: electron spin resonance (esr), magnetic spectroscopy (nmr), furier transform (ft), active oxygen method (aom) and differential scanning calorimetry (dsc) (wanasundara et al., 1995; andersen and skibsted, 2002; rohn and krohn, 2005). however, the most popular method of edible and nonedible oil stability assessment in the metrohm rancimat (pawar et al., 2013). the method of oxidative stability evaluation in the rancimat test consists in the measurement of the concentration of volatile products of oxidation, e.g. short-chain fatty acids formed as a result of accelerated oxidation of the sample. the accelerated oxidation of fat in a reaction vessel is induced by sample heating and blowing through with air. the volatile products of oxidation formed in this process are blown away to water in a measuring vessel, the electrode is immersed in. the electrode allows measuring conductivity of water with oxidation reaction products dissolving in it. a rapid increase of conductivity is tantamount to the formation of secondary products of oxidation that are well soluble in water. results of conductivity measurements in time are presented in the form of a graph. the time preceding the process of accelerated oxidation, the moment of sudden increase of conductivity, is a measure of sample resistance to oxidation and is referred to as inductive time, induction time or oxidative stability index (osi) (matthäus, 1996; anwar et al., 2003; jain and sharma, 2011). many kinetic parameters of the oxidation process may be determined based on results of the rancimat test. they may be applied to differentiate the origin of oils and to identify differences or similarities between them. these parameters may be very useful in predicting oxidative stability of oils during their heat treatment, storage and distribution ital. j. food sci., vol 29, 2017 278 (tan et al., 2001). many studies were conducted to determine kinetic parameters of edible oils, however they mainly concerned refined oils, whereas investigations are missing that would address a comparison of oxidation kinetics of oils produced with various methods. considering the above, the objective of this study was to determine and compare kinetic parameters of refined and cold-pressed rapeseed oils. 2. materials and methods 2.1. chemical reagents all the solvents and chemical reagents used in the study were of analytical grade and were purchased from poch s.a. (gliwice, poland). the supelco 37 component fatty acid methyl esters (fame) mix was from sigma aldrich gmbh (schnelldorf, germany). deionized water (0.05 μs) was obtained using an hlp smart 2000 apparatus (hydrolab, poland). 2.2. material the experimental material included ten oils labelled as rapeseed oils: five refined (r) and five cold-pressed (c). the oils were purchased on the local market within their shelf-life. the cold-pressed oils were packed in bottles made of dark-green glass, whereas the refined ones were in ethylene polyterephthalate (pet) bottles. once purchased, the oils were transported to the laboratory and subjected to analyses. 2.3. chemical analysis the physicochemical quality of oils was determined based on the lipid values. the extent of hydrolytic changes in the analyzed oils, expressed as the acid value (av), was determined according to the aocs cd 3d-63 (aocs, 2000). the content of peroxides, expressed as the peroxide value (pv), was determined according to the aocs 965.33 (aocs, 1999). the extent of oxidative changes, expressed as the anisidine value (anv), was determined according to the aocs cd 18-90 (aocs, 2002). the total oxidation index (totox) was computed based on results achieved for the peroxide value and anisidine value (totox = 2pv + anv). fatty acid composition of the analyzed oils was determined using gas chromatography according to the aocs ce 1h-05 (aocs, 2005) with small modifications. methyl esters of fatty acids were prepared according to the aocs 2-66 (aocs, 1997). determinations were conducted in a hewlett – packard model 5890 ii gas chromatograph (agilent technologies, avondale, pa, usa) with a flame ionization detector, equipped in a supelcowax 10 column (30 m x 0.25 mm x 0.25 μm). injection temperature was 240 °c, and injection was at 1:25. assay conditions were as follows: helium flow rate 1ml/min and oven temperature 240 oc. detector’s temperature was set at 240 oc. peaks were identified by comparing their retention times with that of feme mix standard. 2.4. rancimat test oxidative stability oxidative stability of the oils was determined using a rancimat 743 metrohm apparatus (herisau, switzerland). the rancimat test was conducted on 2.50 ± 0.01 g samples of oils that were oxidized at five temperatures: 90 oc, 100 oc, 110 oc, 120 oc and 130 oc, at a ital. j. food sci., vol 29, 2017 279 constant air flow of 20 l/h. the volatile products formed from the oxidation reaction were soluble in 0.06 l of deionized water. eight samples of oils were placed in the apparatus and analyzed simultaneously. the samples were placed at random. the induction times were recorded automatically by the apparatus’ software and taken as the break point of the plotted curves with the accuracy of 0.01 h. 2.5. kinetic analysis based on the results obtained for the oxidative stability of the oils assayed at different temperatures, graphs were plotted and regression lines were determined according to the following equations: ln(τrancimat) = a(t) + b (1) ln(τrancimat) = a(1/t) + b (2) activation energy ea (kj/mol) of the oxidation reaction, exponential factor z (h-1) and oxidation reaction rate constants at different temperatures were determined for individual rapeseed oils using the arrhenius equation, according to a method by kowalski et al. (2004): 𝑘 = ze!!"/!" (3) values of reaction enthalpy (∆h++) and entropy (∆s++) were calculated based on the equation derived from the activated complex theory: ln(k/t) = ln(k!/h) + (δs/r) – (δh/rt) (4) 2.6. statistical analysis all rancimat measurements were conducted in three replications for each oil sample, then results were averaged and presented in tables. data were subject to analysis of variance (one-way anova). statistical differences were determined with the tukey’s multiple comparison test at a significance level of p = 0.05. pearson’s linear correlations were calculated at the p<0.05 level. all statistical analyses were performed using the statistica 10.0 software (2010, statsoft, tulsa, ok, usa). 3. results and discussion results of the evaluation of the initial quality of the analyzed oils were collected and presented in table 1. the refined oils were characterized by lower acid value (0.09 – 0.27 mg koh/ g oil) and peroxide value (0.97 – 2.02 meq o2/ kg oil) compared to the coldpressed oils. in turn, they showed higher anisidine values (1.10 – 2.42), which may be due to high temperatures applied during one of the stages of the refining process – deodorization, that cause the formation of secondary products of oxidation (čmolik and pokorńy, 2000). similar values of the above indices were reported for refined rapeseed oils by ratusz et al. (2005). ital. j. food sci., vol 29, 2017 280 the cold-pressed oils were characterized by higher acid value (1.03 – 1.47 mg koh/ g oil) and peroxide value (2.12 – 8.28 meq o2/ kg oil). in addition, they exhibited a higher degree of oxidation expressed as totox index (4.75 – 17.25) compared to the refined oils (3.04 – 5.56). the peroxide values of the cold-pressed oils differed statistically significantly. great differences in the value of this index were also determined by matthäus and brühl (2003). higher values of these quality indices are linked with the fact that the coldpressed oils are subjected only to mechanical treatment (filtration, centrifugation) and not to the complete refining process (degumming, deacidification, bleaching, deodorization) as in the case of the refined oils. higher contents of free fatty acids and primary oxidation products depend on the quality of raw materials used for oil manufacturing. the anisidine values of the cold-pressed oils ranged from 0.18 to 0.69. values of particular lipid indices of the analyzed refined and cold-pressed rapeseed oils did not exceed the values recommended in codex alimentarius standard (2013). according to this standard, the threshold value of the acid value reaches 0.6 for refined oils and 4.0 mg koh/ g oil for cold-pressed oils, whereas that of the peroxide value reaches 10 and 15 meq o2/ kg oil, respectively. table 1. characteristics of the initial quality of the analyzed rapeseed oils. oil av (mg koh/g) pv (meq o2/kg) anv (absorbance x 1000) totox r1 0.13±0.01ab 2.02±0.03b 1.32±0.05e 5.36±0.16d r2 0.27±0.03c 0.98±0.02a 1.61±0.04f 3.57±0.10b r3 0.25±0.02bc 1.02±0.03a 1.54±0.05f 3.58±0.16b r4 0.09±0.03a 0.97±0.04a 1.10±0.03d 3.04±0.07a r5 0.19±0.01abc 1.57±0.05c 2.42±0.02g 5.56±0.11d ca* 0.6 10.0 c1 1.25±0.02e 4.38±0.02e 0.31±0.05ab 9.07±0.02f c2 1.03±0.03d 5.85±0.02f 0.18±0.04ab 11.88±0.02g c3 1.09±0.04d 2.12±0.03b 0.51±0.02bc 4.75±0.11c c4 1.47±0.04f 3.24±0.03d 0.57±0.03b 7.05±0.04e c5 1.27±0.01e 8.28±0.03g 0.69±0.02b 17.25±0.05h ca** 4.0 15.0 a,b,c,d,e,f,g,h mean values in columns are statistically significantly different at a significance level of p=0.05 *threshold values for refined oils according to codex alimentarius (2013) **threshold values for cold-pressed oils according to codex alimentarius (2013) the composition of selected fatty acids of the analyzed oil was summarized in table 2. the investigated rapeseed oils were characterized by a low content of saturated fatty acids (6.49 – 8.48 %). as indicated by literature data, the fatty acid composition of rapeseed oils is predominated by monounsaturated fatty acids, including mainly oleic acid, which was also confirmed in our study (59.31 – 63.22 %). the analyzed oils were characterized by ca. 30% content of polyunsaturated fatty acids. the fatty acid composition of the analyzed oils was typical of rapeseed oil (roszkowska et al., 2014). differences in fatty acid composition of refined and cold-pressed oils were small and depended, most of all, on the raw materials used to produce the oils (ramos et al., 2009). oxidative stability of the investigated oils was examined with rancimat test at different temperatures and respective results were presented in table 3. ital. j. food sci., vol 29, 2017 281 table 2. fatty acid composition of the analyzed rapeseed oils [%]. fatty acid oil sample r1 r2 r3 r4 r5 c1 c2 c3 c4 c5 palmitic (16:0) 4.85±0.03b 4.54±0.02a 4.95±0.03b 4.61±0.02a 5.89±0.01e 5.00±0.01cd 5.07±0.01d 5.89±0.02e 5.85±0.02e 4.98±0.02cd palmitoleic (16:1) 0.23±0.01ab 0.24±0.01ab 0.22±0.01a 0.25±0.02ab 0.28±0.01ab 0.28±0.01ab 0.30±0.01b 0.28±0.02ab 0.26±0.01ab 0.29±0.01ab stearic (18:0) 1.65±0.02c 1.53±0.02ab 1.45±0.01a 1.52±0.00ab 2.13±0.00e 1.68±0.01c 1.51±0.01ab 1.54±0.00b 1.92±0.02d 1.56±0.01b oleic (18:1) 62.14±0.04 b 62.87±0.05c 63.22±0.08c 63.21±0.06c 59.34±0.08a 61.15±0.05b 62.16±0.07b 61.26±0.05b 59.31±0.04a 62.21±0.09b linoleic n-6 (18:2) 19.72±0.02 c 19.26±0.02a 19.10±0.04a 19.15±0.03a 19.15±0.02a 19.69±0.04c 19.54±0.04b 19.14±0.03a 19.22±0.03a 19.58±0.03bcd linolenic n-3 (18:3) 9.12±0.01 a 9.56±0.02b 9.23±0.03a 9.43±0.04b 10.92±0.02d 9.75±0.03c 9.57±0.03b 10.15±0.02d 11.25±0.03e 9.73±0.02c arachidonic (20:0) 0.48±0.01 de 0.42±0.00b 0.52±0.00f 0.50±0.00ef 0.46±0.00cd 0.37±0.00a 0.45±0.00c 0.45±0.01c 0.47±0.00cd 0.39±0.00a eicosenoic (20:1) 1.21±0.01cd 1.26±0.02ce 1.31±0.01e 1.27±0.01ce 1.02±0.00ab 1.06±0.01b 1.18±0.02c 0.96±0.01a 0.98±0.00a 1.05±0.01b other 0.60bc 0.32ab 0.10a 0.16a 0.81cd 1.02d 0.22a 0.32ab 0.74c 0.21a σ sfa 6.98b 6.49a 6.92b 6.63a 8.48d 7.05b 7.03b 7.89c 8.24d 6.93b σ mufa 63.58c 64.37d 64.65d 64.63d 60.64a 62.49b 63.64c 62.50b 60.55a 63.55c σ pufa 28.84c 28.82c 28.33a 28.58b 30.07f 29.44e 29.11d 29.29de 30.47g 29.31de a,b,c,d,e,f,g mean values in columns are statistically significantly different at a significance level of p=0.05. table 3. induction time [h] of the analyzed rapeseed oils at 90, 100, 110, 120, 130 °c in rancimat test. temperature oil sample t[k] t[°c] r1 r2 r3 r4 r5 c1 c2 c3 c4 c5 induction time [h] 403 130 3.02±0.15b 2.65±0.17ab 2.42±0.11ab 2.63±0.16ab 2.95±0.12b 1.93±0.10a 1.92±0.12a 2.28±0.15ab 2.09±0.16a 1.89±0.10a 393 120 5.72±0.20g 5.08±0.13efg 4.56±0.14cde 4.98±0.12def 5.65±0.14fg 3.77±0.06ab 3.54±0.12b 4.27±0.011bcd 3.87±0.10abc 3.46±0.11a 383 110 11.36±0.21e 10.48±0.19cde 9.97±0.16c 10.35±0.22cd 11.25±0.18de 7.28±0.11ab 6.66±0.15a 7.94±0.18b 7.57±0.21ab 7.19±0.14ab 373 100 22.21±0.31e 21.32±0.24de 20.76±0.25d 21.26±0.21de 21.63±0.30de 13.56±0.24ab 12.99±0.22a 16.03±0.27c 14.80±0.25bc 13.56±0.23ab 363 90 42.55±0.28f 40.89±0.22e 40.78±0.22e 40.78±0.22e 41.12±0.25e 26.21±0.27b 25.18±0.25b 33.46±0.22d 28.57±0.21c 23.67±0.26a a,b,c,d,e,f,g mean values in columns are statistically significantly different at a significance level of p=0.05. ital. j. food sci., vol 29, 2017 282 the induction time of rapeseed oil oxidation recorded by rancimat depends on process temperature. stability of the oils was decreasing along with temperature increase. the highest oxidative stability of the analyzed rapeseed oils was determined at a temperature of 90 °c, whereas the lowest one – at 130 °c. compared to the cold-pressed oils, the refined oils were characterized by longer induction times in the rancimat test at all temperatures. rancimat is the most commonly used method to determinate the oxidative stability of oils for edible and nonedible purpose (anwar et al., 2007; casal, 2010; farhoosh, 2010; giuffrè, 2016; giuffrè, 2016a, giuffrè, 2016b). the requirements for “rapeseed fuel for diesel engines” were published in the official din standard where oxidation stability at 110 °c is one of the most important parameters and the value has to be less than 6h (din 51605, 2010).our paper shows that the oxidative stability of analyzed rapeseed oils at 110oc varied, and ranged from 9.97– 11.36 h for refined oils, and from 6.66 to 7.94 for coldpressed oils. in the case of edible oils the most commonly used temperature of measuring the oxidative stability for refined oil is 120 °c, but in the case of cold-pressed oils is 100 °c. the induction time of analyzed refined rapeseed oils at 120 °c were between 4.56 to 5.72 h. on the other hand, cold-pressed rapeseed oils were characterized by induction time at 100 °c between 12.99 16.03 h. the induction times were higher than those presented by szterk et al. (2010) in their study of cold-pressed rapeseed – (7.07 h), camelina (6.12 h), primrose (6.14 h), amaranth (6.14 h). the obtained induction times were also lower than those for hazelnut oil (22.44 h) in ciemniewska-żytkiewicz et al. (2014). oil oxidation at oxygen (air) excess is the first order reaction. therefore, kinetic analysis may be carried out for the oil oxidative transformation constant (kowalski et al., 2004). based on the determined induction times (table 3) and arrhenius equation (3), the following kinetic parameters were calculated acc. to the method by kowalski et al. (2004): activation energy of oxidation reaction ea, pre-exponential factor z, reaction rate constants k for particular temperatures of the rancimat test, as well as enthalpy ∆h++ and entropy ∆s++ of oxidation activation. correlations between logarithm induction time τrancimat and temperature and the reverse of temperature were determined for each type of oil (figs. 1 and 2). the character of these correlations was linear with a correlation coefficient of r>0.99 and they may be determined using equations 1 and 2. figure 1. semi-logarithmic correlation between lnτrancimat and (1/t) for oxidation of refined rapeseed oils. 0,20 0,40 0,60 0,80 1,00 1,20 1,40 1,60 1,80 2,4 2,5 2,6 2,7 2,8 ln τr an ci m at [1/temperature (k)] * 1000 r1 r2 r3 r4 r5 ital. j. food sci., vol 29, 2017 283 figure 2. semi-logarithmic correlation between lnτrancimat and (1/t) for oxidation of cold-pressed rapeseed oils. table 4 presents values of particular kinetic parameters of rapeseed oil oxidation. the refined rapeseed oils were characterized by generally higher values of activation energy (ea) – a minimal energy of molecules needed to initiate the oxidation reaction, compared to the cold-pressed oils. the ea values of oxidation reaction ranged from 76.43 to 82.44 kj/mol for the refined oils as well as from 74.24 to 77.56 kj/mol for the cold-pressed oils. the smaller load of energy required to initiate the oxidation reaction in the case of the cold-pressed oils may depend on many factors like, e.g., fatty acid composition, presence of endogenous antioxidants and prooxidants, e.g. primary and secondary products of oxidation, and metal ions. the cold-pressed oils were characterized by a higher totox indicator than the refined ones (table 1), which may affect a reduction in activation energy. the ea values obtained for the refined oils in this study were lower than the values reported by kowalski et al. (2004) – 85.3 kj/mol and by farhoosh et al. (2008) – 89.94 kj/mol, and similar to the value achieved by ciemieniewska–żytkiewicz et al. (2014) – 80.99 kj/mol which fitted within the range obtained in our study. differences in the values of this kinetic parameter between investigations of various authors may result from the biological variability of the raw material (ripening stage of a variety) (adhvaryu et al., 2000). in the case of the analyzed rapeseed oils, the value of a reaction rate constant k was increasing along with increasing temperature of oxidation. the rate of oxidation reaction was the highest at a temperature of 130 °c. the cold-pressed oils were characterized by a higher value of oxidation reaction rate constant at all temperatures compared to the refined oils. the same dependency was noted by kowalski et al. (2004) and farhoosh et al. (2008) for rapeseed oils as well as by ostrowska–ligęza et al. (2010) for olive oil and by ciemieniewska-żytkiewicz et al. (2014) for hazel nuts. the oxidation rate constant of the cold-pressed oil was 2-3-fold higher than that of the refined oils despite small differences in the values of activation energy. it may be due to the fact that the cold-pressed oils contain more pro-oxidants that are removed from the refined oils in the refining process. the prediction of oil oxidation rate at low temperatures is limited based on data obtained. the course of oxidation process of oils differs at low and high temperatures (tan et al., 2001). 0,20 0,40 0,60 0,80 1,00 1,20 1,40 1,60 2,4 2,5 2,6 2,7 2,8 ln τ r an ci m at [1/temperature (k)] * 1000 c1 c2 c3 c4 c5 ital. j. food sci., vol 29, 2017 284 table 4. kinetics parameters of refined and cold-pressed rapeseed oils oxidation. parameter rancimat calculation based on induction time r1 r2 r3 r4 r5 c1 c2 c3 c4 c5 equation 1 a 0.0289 0.0300 0.0310 0.0301 0.0287 0.0282 0.028 0.0291 0.0285 0.0279 b 4.2293 4.3176 4.3990 4.3265 4.2030 3.9593 3.9145 4.122 4.022 3.9033 r2 0.9999 0.9996 0.9989 0.9994 0.9999 0.9999 0.9997 0.9985 0.9998 0.9981 equation 2 a 4.2210 4.3855 4.5279 4.5279 4.1975 4.1283 4.0998 4.26 4.1778 4.0775 b 9.9824 10.4480 10.8460 10.8460 9.9301 9.9344 9.8809 10.21 10.036 9.8208 r2 0.9990 0.9985 0.9979 0.9979 0.9985 0.9984 0.9998 0.9999 0.9995 0.9956 ea [kj/mol] 76.85 79.85 82.44 82.44 76.43 75.17 74.65 77.56 76.07 74.24 z† 9.6x109 2.81x1010 7.01x1010 7.01x1010 8.51x109 8.60x109 7.60x109 1.61x1010 1.09x1010 6.62x109 k† at 130 oc 0.7611 0.5538 0.5438 0.5438 0.7233 1.5670 1.6167 1.0435 1.5178 1.5896 k† at 120 oc 0.4246 0.3020 0.2908 0.2908 0.4048 0.8858 0.7828 0.7962 0.8522 0.9049 k† at 110 oc 0.2297 0.1596 0.1505 0.1505 0.2198 0.4861 0.4295 0.4286 0.4642 0.5002 k† at 100 oc 0.1203 0.0815 0.0752 0.0752 0.1155 0.2583 0.2282 0.2232 0.2448 0.2679 k† at 90 oc 0.0608 0.0401 0.0361 0.0361 0.0586 0.1325 0.1171 0.1121 0.1246 0.1386 δh++ [kj/mol] 73.67 76.67 79.23 79.26 72.12 71.98 71.46 74.38 75.47 71.04 δs++[j/molk] -64.42 -55.84 -48.43 -48.35 -68.45 -67.47 -69.60 -62.33 -56.40 -69.99 † z and k from rancimat in h-1 ital. j. food sci., vol 29, 2017 285 in addition, temperature increase is linked with increased solubility of oxygen – an increase in temperature by 10oc causes oxygen solubility increase by 25% (robertson, 2000). enthalpy and entropy were computed based on the active complex theory and results of linear regression from tables 4. a high correlation (r > 0.99) is indicative of a very good fit and characterizes the effect of temperature on lipid oxidation using the active complex theory. the ∆h++ value determined for the analyzed refined rapeseed oils ranged from 72.12 to 79.26 kj/mol, and that for the cold pressed oils from 71.04 to 75.47 kj/mol. in turn, the ∆s++ value ranged from -48.35 to -68.45 j/molk for refined oils and from -56.40 to -69.99 j/molk for cold-pressed oils. kowalski et al. (2004) obtained ∆h++ and ∆s++ values at 82.0 kj/mol and – 52.7j/molk for refined rapeseed oil, whereas farhoosh et al. (2008) at 86.79 kj/mol and 112.99 j/molk, respectively. the negative ∆s++ values indicate that the active complexes are more ordered than molecules of reagents. higher negative values point to a lesser probability of the formation of an active complex and to a slower rate of lipid oxidation (farhoosh et al., 2008). results of our study confirm that the above statement is true for the samples of both refined and cold-pressed rapeseed oils. 4. conclusions in summary, the oxidative stability of the refined rapeseed oils determined with the rancimat test is higher than that of the cold-pressed rapeseed oils. in turn, the coldpressed oils are characterized by higher values of oxidation rate constant than the refined oils. in addition, they show slightly lower values of activation energy – ea, which is linked, among other things, with a higher extent of oxidation of a fresh sample (a higher content of primary oxidation products) that accelerates the process of oxidation. results obtained in 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codon italian journal of food science, 2022; 34 (2): 67–73 issn 1120-1770 online, doi 10.15586/ijfs.v34i2.2202 67 p u b l i c a t i o n s codon proximate analysis of lipid composition in moroccan truffles and desert truffles fatima henkrar1*, lahsen khabar2* 1plant biotechnology and physiology laboratory, faculty of sciences, university mohammed v-rabat, morocco; 2botanical, mycology and environment laboratory, faculty of sciences, university mohammed v-rabat, morocco *corresponding authors: fatima henkrar, plant biotechnology and physiology laboratory, faculty of sciences, university mohammed v-rabat, morocco. email: f.henkrar@um5r.ac.ma; lahsen khabar, botanical, mycology and environment laboratory, faculty of sciences, university mohammed v-rabat, morocco. email: l.khabar@um5r.ac.ma received: 18 march 2022; accepted: 17 may 2022; published: 10 june 2022 © 2022 codon publications open access paper abstract lipid composition in truffle is essential for nutraceutical and medicinal purposes. currently, there is no data regarding the lipid content in moroccan truffles. therefore, we determined the fatty acid and sterol composition of six moroccan truffles and desert truffles. the gas chromatography analysis revealed the predominance of fatty palmitic, oleic and linoleic acids. the prominent sterol components were brassicasterol and ergosterol. besides, the sterol analysis discriminated between the tuber and terfezia truffles. these differences seem to be exploitable at a taxonomic level. this is a preliminary report disclosing the fatty acid and sterol composition of moroccan truffles, indicating the potential use of lipids analysis, especially sterol analysis, as biomarker for truffles distinction. keywords: desert truffles, discrimination, fatty acid, gas chromatography, moroccan truffle, sterol introduction the truffles are edible ascocarp of hypogenous ascomycetes fungi that grow underground (khabar et al., 2001; lee et al., 2020). the term ‘desert truffles’ is used to describe truffles growing particularly in arid and semi-arid areas (khabar et al., 2001; morte et al., 2021), such as morocco, algeria, tunisia, egypt, south africa, saudi arabia, iraq, syria and kuwait (khabar, 2014; lee et al., 2020). the genera found abundantly in those areas are terfezia and tirmania. besides, other genera exist as well, namely delastria and picoa (khabar, 2014). in mediterranean countries, especially in north africa, the truffles are harvested in abundance and known as ‘terfass,’ also called ‘kame,’ ‘kholassi,’ ‘zoubaïdi,’ ‘truffles of the deserts’ and ‘truffles of the sands’ because of their development in sandy soil (khabar, 2014). the determination of lipid composition in truffles is essential both for lipid analysis as well as for nutraceutical and medicinal purposes. the truffles contain only 4–9% by dry weight of total lipids (tang et al., 2011). fatty acids and phytosterols are the main lipid compounds in truffle fruiting bodies, which are well known for their potential human benefits. several studies through global chromatographic analysis demonstrated that desert truffles are rich in fatty acids, both saturated and unsaturated that have many positive effects on health (akyüz, 2013; al-shabibi et al., 1982; bokhary et al., 1989; doğan and aydın, 2013; veeraraghavan et al., 2021). for example, in terfezia boudieri from tunisia, hamza et al. (2016) reported that essential fatty acids, like linoleic and oleic acids, account for 76% of the total fat content. linoleic acid or omega-6 is an essential fatty acid and one of the most aromatic compounds in most truffle species (lee et al., 2020), which has protective and antioxidative effects beneficial for human health (sokoła-wysoczańska et al., 2018). while, oleic acid, a bioactive compound, has the aptitude in reducing cholesterol levels (lee et al., 2020). another example of terfezia claveryi from saudi arabia, which is closely related to t. boudieri (dahham et al., 2018), was found to have arachidic, myristic, palmitic, behenic, pentadecanoic, stearic, heneicosanoic, nonadecanoic and margaric acids 68 italian journal of food science, 2022; 34 (2) henkrar f and khabar l collected directly from their natural environments and transported to the laboratory. under the fume hood, the samples were surface sterilised with ethanol, peeled and then fragmented by hand. several pieces were taken from the glebe and stored in pillboxes at −64°c. alternatively, other samples were sun-dried for 2 months before being stored at −64°c. the different steps of extraction, separation and analysis of lipids were released at the laboratory of mycology, phytopathology and environment of the littoral france university, following the method of fontaine et al. (2001). extraction of total lipids the extraction was performed with approximately 20–40 mg of freeze-dried material (pieces of truffle glebe). the solvent used for extraction was a mixture of dichloromethane and methanol (2:1 v/v) with 0.05% bht (butylated hydroxytoluene; sigma) as antioxidant. the freeze-dried fungal material was first ground in 40 ml of the solvent using ultra-turrax homogenizer. the first extractions were performed in the dark to preserve the ergosterol, a photosensitive sterol. the extraction of total lipids was carried out under reflux (1 h at 70°c) with some pieces of pumice stone. after filtration, the lipid extract was recovered under nitrogen blowdown and rotary evaporator at 60°c. this step was repeated thrice. separation of fatty acids and total sterols by saponification the crude lipid extract was used to separate fatty acids and total sterols by saponification. the crude extract was saponified under reflux (1 h at 90°c) in 2 ml of 6% (w/v) methanolic potash and some pieces of pumice. after cooling, two successive cold extractions with hexane were performed. the first extraction permitted the as saturated fatty acids along with unsaturated fatty acids (palmitoleic, oleic, erucic and linoleic) (bokhary et al., 1989). the lipid composition of desert truffles depends highly on the species as well as growing environments. for instance, t. boudieri from saudi arabia was rich in pentadecanoic, margaric, stearic and arachidic acids (bokhary et al., 1989). whereas, the same species from turkey contained mainly oleic, linoleic, linolenic, palmitic, palmitoleic, stearic and behenic acids (akyüz, 2013). the most identified phytosterols in truffle reports were brassicasterol and ergosterol. harki et al. (1996) reported that the prominent components identified in tuber melanosporum were ergosterol and brassicasterol, accounting for about 90% of total sterols. as well, the major sterol components in the tuber ascocarps were brassicasterol and ergosterol, accounting for about 17–64% and 25–67% of total sterols, respectively (tang et al., 2012). in terfezia truffles, brassicasterol levels were 98% of the total sterols, while ergosterol was present in lower amounts (tejedor-calvo et al., 2021). other phytosterols such as beta-sitosterols, stigmasterol and campestanol were also present in low contents (dahham et al., 2018). the six species included in this study were natives of morocco. terfezia arenaria, commonly called ‘pink terfess of mamora,’ was harvested from acidic soil, in semi-arid climate under helianthemum guttatum. it is an appreciated edible fungus, detected by the ‘mark’ method (khabar, 2014) unlike delastria rosea, known as ‘bitter terfess of taida’ due to its bitter flavour. it was collected under pinus pinaster var. atlantica and pinus halepensis in mamora forest between november and december (khabar, 2014). similarly, tuber oligospermum was also collected under p. pinaster var. atlantica in mamora forest, starting from december until april. the ascocarp of t. boudieri originated from bouaarfa region, collected from limestone soil, under arid and sub-saharan climate, and terfezia leptoderma was obtained from mamora forest from the acidic soil under h. guttatum at the beginn ing of february until may. tuber asa, commonly called ‘terfass male of terfass’ because of its hard consistency, collected as well under h. guttatum on acidic soil of mamora forest, towards the end of february at the same time as t. leptoderma (khabar, 2014). the aims of this work were 1) to determine the lipid profile of the six moroccan truffle species, 2) to determine the relation between the genus, species and lipid composition of truffles, and 3) to determine whether the lipid profile can be used as a tool for species or genus distinction. materials and methods fungus material six moroccan truffle species were used in this experiment. the ascocarps of different species (table 1) were table 1. the name and location of six moroccan truffle species used in this study. species name location terfezia leptoderma (1) mamora forest under helianthemum t. leptoderma (2) mamora forest under pinus pinaster terfezia arenaria mamora forest terfezia boudieri bouaarfa tuber asa mamora forest tuber oligospermum mamora forest under p. pinaster delastria rosea mamora forest under p. pinaster italian journal of food science, 2022; 34 (2) 69 lipid composition of moroccan truffles statistical analysis the data were analysed using r studio software. the means and standard deviations were calculated. the pairwise comparisons among means were performed using two-way anova and tukey hsd test. to indicate significant differences, we used the multcompletters4() function from the multcompview package. results and discussions the objective of this work was to define the nature and proportion of fatty acids and sterols in six species of moroccan truffles and to determine if this lipid profile could be used as a classification tool to discriminate between species or genus. this study was conducted for the first time on moroccan truffles, disclosing distinctly the fatty acid and sterol components of truffles and desert truffles grown in morocco. fatty acid composition the fatty acid composition of moroccan truffles has not been reported previously, and studies on fatty acid content of other truffles are scarce. the first and most reported studies on fatty acid composition were focused on tirmania pinoyi, tirmania nivea, t. boudieri, t. claveryi and picoa lefebvrei from saudi arabia (bokhary et al., 1989; bokhary and parvez, 1995) and t. claveryi from iraq (al-shabibi et al., 1982). recently, other studies appeared on fatty acid composition of t. boudieri from iraq (dahham et al., 2018), t. boudieri from turkey (hamza et al., 2016), t. claveryi and picoa juniperi from spain (murcia et al., 2003) as well as t. nivea from libya (shah et al., 2020). the chromatographic analysis results for the identification of fatty acids compositions are presented in (table 2 and figure 1). seven fatty acids were detected in the six truffles species used in this experiment; four saturated fatty acids [palmitic (c16:0), stearic (c18:0), arachidic (c20:0) and behenic (c22:0)], and three unsaturated fatty acids [palmitoleic (c16:1), oleic (c18:1) and linoleic (c18:2)]. bokhary et al. (1989) reported that palmitic, stearic, oleic and linoleic acids were predominant in t. nivea and t. boudieri which originated from saudi arabia. as well, the turkish t. boudieri was also rich in behenic, palmitic, palmitoleic, stearic, oleic, linoleic and linolenic acids (akyüz, 2013), which agree with our results and particularly with t. boudieri. furthermore, a recent study on fatty acid composition in tuber maculatum, tuber aestivum/uncinatum, tuber borchii, t. melanosporum and t. nivea revealed the dominance of palmitic, stearic, oleic and linoleic acids followed recovery of unsaponifiable fraction (sterols), while the second one enabled the retrieval of saponifiable fraction (fatty acid). to perform the first cold extraction, one volume of distilled water was added to the cooled saponified extract, followed by six volumes of hexane. this mixture was then vigorously stirred for 1 min with a vortex. after decantation, the organic phase (upper layer), which contains the unsaponifiable elements, was taken out and dehydrated with anhydrous sodium sulfate. this step was repeated three times, and the recovered extract was concentrated in a rotary evaporator at 50°c. for the fatty acid recuperation, the aqueous phase was acidified to ph 1 with 1 m hcl to liberate them from their saline combination. afterwards, the acidified phase was extracted by performing three extractions with hexane. the concentration of these extracts was done under nitrogen. fatty acid analysis the fatty acids were solubilise in 1 ml boron trifluoride–methanol (14% w/v). the methylation reaction was carried out for 2 min at 90°c in a water bath and then stopped by immersing the tubes in ice. after addition of 1 ml of distilled water and 2 ml of hexane, the tubes were vortexed for 30 s. the organic phase (upper phase) was taken out and dehydrated by adding anhydrous sodium sulfate. this step was repeated thrice. the methylated fatty acids were purified on silica gel of tlc (20 × 20 cm, type silicagel f 254, merck) with a solvent system composed of diethyl ether/hexane (10/90; v/v). the fatty acid spots were detected by primuline and eluted in about 1 ml of dichloromethane. thereafter, the fatty acids were immediately taken up in 25–100 µl of hexane and injected into the gas chromatograph. fatty acids were identified by comparing their relative retention times with internal standard such as methyl c 17:0 (methyl margarate) as well as other known standards (alltech). sterol analysis to obtain a better separation in gas chromatography, sterols were acetylated either for 12 h at room temperature or 2 h at 60°c by the mixture of toluene/acetic anhydride/ pyridine (1/2/1; v/v/v). sterol acetates were purified on silica gel thin layer with dichloromethane as migration solvent. cholesterol acetate (1 mg/ml) was used as a control to localise acetylated products after spraying with 0.01% (w/v) primuline solution. the acetylated sterols were taken up in 25–100 µl of hexane and injected into the chromatograph. the sterol acetates were identified by comparing their relative retention times against an internal standard, cholesterol in alcohol form (non-acetylated) along with brassicasterol and other known acetylated standards. 70 italian journal of food science, 2022; 34 (2) henkrar f and khabar l for d. rosea where the opposite was true; the level of oleic acid was higher than linoleic acid. the same results were also reported by hamza et al. (2016). the t. boudieri was characterised by its higher content of linoleic acid (54.10%) compared to oleic and palmitic acids that represented 22 and 20.40%, respectively (hamza et al., 2016). linoleic acid level was considerably high in tuber compared to terfezia species. the rate of oleic acid was, on the other hand, slightly lower in tuber genus compared to terfezia genus. this remark goes with tejedor-calvo et al. (2021), who reported that linoleic acid content in tuber brumal and t. melanosporum reached 78.3 and by traces of polyunsaturated fatty acids (shah et al., 2020). the main fatty acids detected were palmitic, oleic and linoleic acids. the other fatty acids were also present but at lower levels. similar findings were also reported in various species of terfezia and tuber (hamza et al., 2016; tejedor-calvo et al., 2021). our results demonstrated that the rate of palmitic acid (c16:0) is appreciably equal in all the species studied. we could also notice that the linoleic acid level was generally higher compared to the oleic acid (c18:1) level in all the species studied except figure 1. fatty acid composition of the six moroccan truffle species used in this study. table 2. fatty acid composition of the six moroccan truffle species through gas–liquid chromatography analysis (percentage of dry weight of the lipid fraction). species c 16:0 c 16:1 c 18:0 c 18:1 c 18:2 c 20:0 c 22:0 terfezia leptoderma (1) 16.080 ± 0.936a 0.913 ± 0.372a 1.600 ± 0.185a 27.530 ± 0.598a 48.513 ± 1.179a 4.570 ± 0.589a 0.790 ± 0.147a t. leptoderma (2) 17.456 ± 2.167a 1.733 ± 0.221a 1.026 ± 0.047a 26.106 ± 0.740a 48.633 ± 1.075a 5.753 ± 0.247a 0.803 ± 0.100a terfezia arenaria 21.470 ± 1.822b 1.066 ± 0.133a 3.253 ± 0.206a 27.020 ± 0.727a 38.803 ± 0.525b 8.563 ± 0.577b 0.900 ± 0.095a terfezia boudieri 20.823 ± 1.019b 0.830 ± 0.303a 2.433 ± 0.508a 28.333 ± 1.028a 42.770 ± 1.127c 4.580 ± 0.545a 0.710 ± 0.156a tuber asa 16.470 ± 1.483a 0.940 ± 0.096a 1.810 ± 0.259a 15.983 ± 0.879b 61.186 ± 0.315d 4.773 ± 0.396a 0.740 ± 0.070a tuber oligospermum 16.670 ± 0.138a 1.090 ± 0.389a 2.023 ± 0.200a 20.593 ± 0.450c 54.926 ± 1.601e 4.720 ± 0.500a 0.780 ± 0.105a delastria rosea 12.420 ± 1.574c 1.230 ± 0.166a 1.263 ± 0.112a 60.160 ± 0.916d 19.836 ± 1.237f 4.503 ± 0.678a 0.830 ± 0.052a data shown as mean ± standard deviation (n = 3). different superscript letters in the same column indicate a statistically significant difference (p < 0.05). italian journal of food science, 2022; 34 (2) 71 lipid composition of moroccan truffles sterol composition the sterol composition of moroccan truffles has never been reported before. the first report on sterol was that by weete et al. (1985), which mentioned about both terfezia and tuber genera. further, other studies principally on tuber species including t. melanosporum (harki et al., 1996; sancholle et al., 1988), tuber magnatum, t. melanosporum, t. aestivum, tuber albidum and tuber indicum were released (sommer et al., 2020). four sterols (brassicasterol, ergosterol and lanosterol) were identified in the ascocarps of the examined truffle species (table 3 and figure 2). furthermore, the main 61.12%, respectively, compared to t. leptoderma and t. arenaria which noticed only 51.3 and 30.9%, respectively. the fatty acid results of moroccan truffles and desert truffles proved their richness in unsaturated and healthy fatty acids such as linoleic acid, suggesting their equivalent culinary value compared to european truffles. regarding fatty acid discrimination, it seems that these criteria could not differentiate clearly between the species of the two genera of tuber and terfezia. indeed, the ratio of linoleic acid or oleic acid was approximately equal between the different species of the two genera. nevertheless, the fatty acid composition could distinguish between delastria and other two genera. figure 2. sterol composition of the six moroccan truffle species. table 3. sterol composition of the six moroccan truffle species through gel permeation chromatography analysis. sterols brassicasterols ergosterols lanosterol (1.31) n.i. (1.42) terfezia leptoderma (1) 96.870 ± 1.260a 3.016 ± 0.621a 0 ± 0a n.d. t. leptoderma (2) 92.410 ± 2.606a 8.153 ± 0.143a 0 ± 0a n.d. terfezia arenaria 96.833 ± 1.045a 0 ± 0b 2.003 ± 0.532a n.d. terfezia boudieri 97.190 ± 2.416a 0 ± 0b 3.110 ± 0.298a n.d. tuber asa 40.526 ± 2.377b 23.196 ± 0.718c 17.470 ± 0.856b 12.220 ± 2.186b tuber oligospermum 46.006 ± 2.001c 21.470 ± 1.990c 16.223 ± 4.441b 17.286 ± 1.997b delastria rosea 21.343 ± 1.770d 42.9633 ± 0.621d 22.826 ± 3.471c 12.886 ± 1.806b data shown as mean ± standard deviation (n = 3). lanosterol and n.i. compounds are reported with their retention time (in minutes) between brackets. different superscript letters in the same column indicate a statistically significant difference (p < 0.05). n.i., not identified; n.d., not detected. 72 italian journal of food science, 2022; 34 (2) henkrar f and khabar l demonstrating the richness of moroccan truffles in essential unsaturated fatty acid, such as linoleic acid. there was a slight difference between tuber and terfezia species in fatty acid component, which is not sufficient to differentiate between them. however, sterol analysis distinguished between these two genera. hence, a comparison of their sterol composition with reported data seems to be plausible for tuber and terfezia distinction. finally, a deeper study on other nutrient compounds and bioactive molecules of moroccan truffles as well as their antioxidant evaluation is predetermined to improve their edible and culinary interest through their health benefits. references akyüz, m., 2013. nutritive value, flavonoid content and radical scavenging activity of the truffle (terfezia boudieri chatin). journal of soil science and plant nutrition. 13:143–151. al-shabibi, m.m.a., toma, s.j. and haddad, b.a., 1982. studies on iraqi truffles. i. proximate analysis and characterization of lipids. canadian institute of food technology journal. 15:200–202. https://doi.org/10.4067/s0718-95162013005000013 bokhary, h.a. and parvez, s., 1995. studies on the chemical composition of the ascomycete fungus phaeangium lefebvrei pat. journal of king saud university. 7:215–224. bokhary, h.a., suleiman, a.a. and basalah, m.o., 1989. the fatty acid components of the desert truffle ‘al kamah’ of saudi arabia. journal of food protection. 52(9):668–669. https://doi.org/ 10.4315/0362-028x-52.9.668 dahham, s.s., al-rawi, s.s., ibrahim, a.h., aman, s.a.m. and amin, m.s.a.m., 2018. antioxidant, anticancer, apoptosis properties and chemical composition of black truffle terfezia claveryi. saudi journal of biological sciences. 25:1524–1534. https:// doi.org/10.1016/j.sjbs.2016.01.031 doğan, h.h. and aydın, s., 2013. determination of antimicrobial effect, antioxidant activity and phenolic contents of desert truffle in turkey. african journal of traditional, complementary and alternative medicines. 10:52–58. fontaine, j., grandmougin-ferjani, a., hartmann, m.a. and sancholle, m., 2001. sterol biosynthesis by the arbuscular mycorrhizal fungus glomus intraradices. lipids. 36:1357–1363. https://doi.org/10.1007/s11745-001-0852-z hamza, a., zouari, n., zouari, s., jdir, h., zaidi, s., gtari, m. and neffati, m., 2016. nutraceutical potential, antioxidant and antibacterial activities of terfezia boudieri chatin, a wild edible desert truffle from tunisia arid zone. arabian journal of chemistry. 9:383–389. https://doi.org/10.1016/j.arabjc.2013.06.015 harki, e., klaebe, a., talou, t. and dargent, r., 1996. identification and quantification of tuber melanosporum vitt. sterols. steroids. 61:609–612. https://doi.org/10.1016/s0039-128x(96)00121-3 khabar, l., 2014. mediterranean basin: north africa. ch.10. in: kagan-zur, v., roth-bejerano, n., sitrit, y. and morte, a. (eds.) desert truffles: phylogeny, physiology, distribution and domestication. springer, berlin, heidelberg, pp. 143–158. sterols were ergosterol and brassicasterol with the highest percentage in all the examined species. similar results were disclosed in black truffle, where the sterol composition of t. melanosporum was analysed and ergosterol along with brassicasterol were identified as the major components (90%) (harki et al., 1996). as well, the examination of tuber sinense, t. aestivum, t. indicum, tuber himalayense and t. borchii revealed the predominance of brassicasterol and ergosterol, with 17–64% and 25–67% of total sterols, respectively (tang et al., 2012). besides, the ratio of ergosterol to brassicasterol changes according to the genera studied. in terfezia species, brassicasterol was the main sterol identified, accounting for 92–97% of the total sterols, affirming the results of weete et al. (1985), who reported that brassicasterol levels were 98% of total sterols in terfezia truffles, while ergosterol was registered at very low amounts (0–8%). on the other hand, in tuber species (tuber asa and tuber oligospermum) and d. rosea, the ergosterol represented a considerable amount compared to terfezia species, accounting for 23, 21 and 43% of the sterols, respectively. these species also contained 40, 46 and 21% of brassicasterol, respectively. a recent study by tejedor-calvo et al. (2022) demonstrated that ergosterol and brassicasterol were the two main sterols in t. claveryi and t. aestivum ascocarps, with differences in ergosterol to brassicasterol ratio depending on the ascocarp genus. lanosterol was also detected in tuber species, as well as in d. rosea, in considerable quantities, accounting for 16 and 23% of the sterols, respectively. while in terfezia, they were either totally absent or present in very small quantity (approximately 2–3%). the high amount of brassicasterol in terfezia will increase the quality interest of the moroccan genera, knowing that brassicasterol has several health benefits, such as antioxidative activity and anti-infective properties. finally, terfezia genus was distinguished by the high brassicasterol content, while the tuber genera and delastria were characterised by the equivalent amount of ergosterol and brassicasterol. hence, sterol analysis proved their importance to highlight differences between species and to separate the tuber from terfezia truffles collected in morocco. these differences seem to be exploitable at the taxonomic level. conclusion the lipid composition and concentration were highly influenced by truffle speciation and their growing area. this was the first report of lipid composition of moroccan truffles, divulging the fatty acid and sterol compositions of six species of truffles and desert truffles and italian journal of food science, 2022; 34 (2) 73 lipid composition of moroccan truffles sterol fingerprints. journal of agricultural and food chemistry. 68(49):14393–14401. https://doi.org/10.1021/acs.jafc.0c06011 tang, y., li, h.-m. and tang, y.-j., 2012. comparison of sterol composition between tuber fermentation mycelia and natural fruiting bodies. food chemistry. 132(3):1207–1213. https://doi. org/10.1016/j.foodchem.2011.11.077 tang, y., li, y.-y., li, h.-m., wan, d.-j. and tang, y.-j., 2011. comparison of lipid content and fatty acid composition between tuber fermentation mycelia and natural fruiting bodies. journal of agricultural and food chemistry. 59:4736–4742. https://doi. org/10.1021/jf200141s tejedor-calvo, e., amara, k., reis, f.s., barros, l., martins, a., calhelha, r.c., venturini, m.e., blanco, d., redondo, d., marco, p. and ferreira, i.c.f.r., 2021. chemical composition and evaluation of antioxidant, antimicrobial and antiproliferative activities of tuber and terfezia truffles. food research international. 140:110071. https://doi.org/10.1016/j.foodres.2020.110071 tejedor-calvo, e., garcía-barreda, s., sánchez, s., morte, a., siles-sánchez, m.d.n., soler-rivas, c., santoyo, s. and marco, p., 2022. application of pressurized liquid extractions to obtain bioactive compounds from tuber aestivum and terfezia claveryi. foods. 11(3):298. https://doi.org/10.3390/ foods11030298 veeraraghavan, v.p., hussain, s., balakrishna, j.p., dhawale, l., kullappan, m., ambrose, j.m. and mohan, s.k., 2021. a comprehensive and critical review on ethnopharmacological importance of desert truffles: terfezia claveryi, terfezia boudieri, and tirmania nivea. food reviews international. 0:1–20. https://doi. org/10.1080/87559129.2021.1889581 weete, j.d., kulifaj, m., montant, c., nes, w.r. and sancholle, m., 1985. distribution of sterols in fungi. ii. brassicasterol in tuber and terfezia species. canadian journal of microbiology. 31: 1127–1130. https://doi.org/10.1139/m85-212 khabar, l., najim, l., janex-favre, m. and paraguey-leduc, a., 2001. contribution à l’étude de la flore mycologique du maroc. les truffes marocaines. bulletin de la société mycologique de france. 117:213–229. lee, h., nam, k., zahra, z. and farooqi, m.q.u., 2020. potentials of truffles in nutritional and medicinal applications: a review. fungal biology biotechnology. 7(9):1–17. https://doi.org/10.1186/ s40694-020-00097-x morte, a., kagan-zur, v., navarro-ródenas, a. and sitrit, y., 2021. cultivation of desert truffles—a crop suitable for arid and semi-arid zones. agronomy. 11:1462. https://doi.org/10.3390/ agronomy11081462 murcia, m.a., martínez-tomé, m., vera, a., morte, a., gutierrez, a., honrubia, m. and jiménez, a.m., 2003. effect of industrial processing on desert truffles terfezia claveryi chatin and picoa juniperi vittadini): proximate composition and fatty acids. journal of the science of food and agriculture. 83:535–541. https://doi.org/10.1002/jsfa.1397 sancholle, m., weete, j.d., kulifaj, m. and montant, c., 1988. changes in lipid composition during ascocarp development of the truffle, tuber melanosporum. mycologia. 80:900–903. https://doi.org/10.2307/3807580 shah, n.n., hokkanen, s., pastinen o., eljamil, a. and shamekh, s., 2020. a study on the fatty acid composition of lipids in truffles selected from europe and africa. 3 biotech. 10:415. https://doi. org/10.1007/s13205-020-02414-y sokoła-wysoczańska, e., wysoczański, t., wagner, j, czyż, k., bodkowski, r., lochyński, s. and patkowska-sokoła, b., 2018. polyunsaturated fatty acids and their potential therapeutic role in cardiovascular system disorders—a review. nutrients. 10(10): 1561. https://doi.org/10.3390/nu10101561 sommer, k., krauß, s. and vetter, w., 2020. differentiation of european and chinese truffle (tuber sp.) species by means of p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (3): 25–34 issn 1120-1770 online, doi 10.15586/ijfs.v34i3.2241 25 p u b l i c a t i o n s codon nutritious elderly diet: pigmented rice-porridge from shear-heat milling process chawakwan nitikornwarakul1*, rodjanawan wangpradid1, chirat sirimuangmoon2, somwang lekjing1, akihiro nishioka3, tomonori koda3 1faculty of innovative agriculture and fishery establishment project, prince of songkla university surat thani campus, surat thani, thailand; 2school of agro-industry, mae fah luang university, chiang rai, thailand; 3graduate school of organic materials science, yamagata university, yonezawa, yamagata, japan *corresponding author: chawakwan nitikornwarakul, department of food technology, faculty of innovative agriculture and fishery establishment project, prince of songkla university surat thani campus, surat thani, thailand email: chukwan.t@psu.ac.th received: 8 june 2022; accepted: 12 july 2022; published: 6 august 2022 © 2022 codon publications open access original article abstract the objective of this study was to develop nutritious elderly diet formulations. each formulation contained three components, pigmented rice flours from shear-heat milling, freeze-dried protein bouillon cube, and sesame oil. the final products were tested with 100 panelists and monitored for quality changes (physical, chemical, microbial, and sensory properties) for 6 months. the three developed formulations provided a suitable ratio of the main nutrients and adequate energy. the products were highly accepted (95%) and easy to swallow (score 4.2/5), indicating a high potential for launching in the elderly market. the products can retain their quality and achieve microbiology safety during storage. keywords: elderly diet; pigmented rice; modified flour; shear and heat milling; rice porridge; consumer test introduction the societal transitions due to aging populations are of imminent concern in many countries. it is estimated that by 2030, 70 million of the global population will be aged 60 years or older, and, by 2050, the age group of over 65-year-olds is predicted to be 88.5 million (chernoff, 2016). these trends indicate that a growing population segment is at a high risk of disability and morbidity. the elderly population will become a major concern to be addressed in national policies on social, economic, and especially health aspects (risonar et al., 2009). suitable nutrition is a key factor contributing to the good health of the elderly. when people age, there are substantial changes in the body, especially in the digestive system, such as a reduced ability to chew, swallow, and digest, due to problems with the teeth and gastrointestinal alterations (gallego et al., 2022; leslie and hankey, 2015), making consuming food more difficult. reduction of total caloric intake can induce frailty of the body, which can result in vulnerability and functional decline (clegg and williams, 2018; lammes et al., 2012). such cases are susceptible to malnutrition and sarcopenia. the design of elderly food products is essential, and they need to provide sufficient energy and nutrients to meet the maintenance needs and are presented in forms that facilitate consumption. rice, the main carbohydrate resource consumed by almost half of the world’s population, is rich in many nutrients that are beneficial to the body (jukanti et al., 2020). however, most of the valuable micronutrients, fatty acids, and fiber are removed during dehulling (bhattacharya, 2017; park et al., 2018). the nutritional quality of white rice is therefore dramatically below that of pigmented rice. the pigmented rice varieties are mainly black, red, or dark purple, and their coloring is in the outer aleurone layer that contains flavones, mailto:chukwan.t@psu.ac.th 26 italian journal of food science, 2022; 34 (3) nitikornwarakul c et al. forms that facilitate consumption, and be acceptable to the target consumers. materials and methods raw materials five commercial pigmented rice varieties, namely, riceberry rice (mah boonkrongtm), sangyod rice (chang thongtm), hom nil rice (green nichtm), munpoo rice (nature zonetm), and black sticky rice (be herbtm) were obtained from a local department store. eggs, salmon, seaweed, pumpkins, and carrots were purchased from a local department store in surat thani province, thailand. preparation of rice flour the pigmented rice flour was obtained using a shm process according to the method described by kanke et  al. (2021) with some modifications. a commercial milling machine (kgw-g015, west co. ltd., japan) was applied as the shear and heat milling machine (shmm) at yamagata university, yonezawa campus. the machine consists of two metal mortars (90 mm diameter) and a ring heater. the gap between the upper mortar and the lower mortar was adjusted to 10 μm. during milling, the lower mortar was rotated at 180 rpm, while the upper mortar connected to the heater was heated to 120˚c. preparation of bouillon cube salmon was washed with potable water before cutting into small pieces. the egg was prepared as an omelet before being added to the soup base. the step of making the soup began with adding diced carrots, pumpkins, and corn into boiling water seasoned with seaweed. then, the protein parts (omelet and salmon) were added and boiled until all ingredients were completely cooked. the soup was kept at room temperature before being weighed into portions according to the calculated formulation and was then frozen at −20˚c. the frozen bouillon cube was then dried for 72 h using a freeze dryer (christ, beta 1–8 lscplus, osterode am harz, germany). preparation of elderly diet formulations the current study proposes three elderly diet formulations generated at three caloric concentrations, namely, 380, 450, and 530 kcal per portion. the energy per portion was set based on the daily energy requirement at 1,500, 1,800, and 2,100 kcal (jansen et al., 2017). tannin, phenolics, sterols, tocols, γ-oryzanols, amino acids, and essential oils (mbanjo et al., 2020). many studies have reported on the functional properties of pigmented rice, such as anti-atherosclerosis, anti-allergic, anti-diabetic, anti-inflammatory, anticancer, and antitumor activities (boue et al., 2016; ling et al., 2002; nam et al., 2005; punvittayagul et al., 2014). thai pigmented rice varieties, such as riceberry rice, sangyod rice, hom nil rice, munpoo rice, and black sticky rice, have gained a lot of interest due to growing consumer demand for health-promoting food products (ratseewo et al., 2019). the big challenge of applying pigmented rice on a diet for the elderly is the hard texture of the outer layer of rice grains that makes them difficult to chew and digest. katsuno et al. (2010) proposed a new method for rice processing using a shear and heat milling (shm) machine. the process is a physical treatment applying mechanical shear, and heat during the milling. this promising technology can successfully modify the gelatinization properties of rice flour, resulting in amorphous rice starch without the addition of water during processing. the advantages of shm over other methods are safety, simplicity, saving time, and low operating cost. the current study appears to be the first to apply this shm technique to modify pigmented rice. rice porridge is a popular dish in many asian countries, especially in china, korea, taiwan, japan, and thailand. it is prepared by adding water to rice at volume proportions of 6–7 folds water to rice, and then boiling until complete gelatinization of starch is achieved (rhim et al., 2011). the dish is particularly favored by the elderly due to the ease of chewing. various rice porridges include ingredients like chicken, pork, fish, egg, seaweed, and vegetables, providing many options to the consumers. however, the rice portion is mostly made from white rice, which has fewer nutrients than pigmented rice. moreover, the instant rice-porridge products available in the market are generally rich in carbohydrates but provide insufficient protein and fat. the energy requirements of individuals differ and can be defined as the amount of energy needed to match energy expenditure, which is the sum of basal metabolic rate (bmr); thermic effect of food (tef); and energy for physical activity. in the elderly, energy expenditure decreases with age resulting from a decreased resting energy expenditure and physical activity (ritz, 2001). there are limited products that are specially made for the elderly group. therefore, the objective of this research was to develop nutritious elderly diet formulations with different energy levels that can provide sufficient energy and nutrients to meet the requirements, be presented in italian journal of food science, 2022; 34 (3) 27 nutritious elderly diet dislike, 4 = like slightly, and 5 = like very much) was performed to rate the appearance, color, flavor, taste, ease of swallowing, and overall acceptability of the rice porridge product. the packaging design was also judged on a similar scale for appearance, attractiveness, and overall acceptance. eer (k cal/day) = 662 – (9.53 × age[yr]) + pa × ((15.91 × weight[kg]) + (539.6 × height[m])) (1) eer (k cal/day) = 354 – (6.91 × age[yr]) + pa × ((9.36 × weight[kg]) + (726 × height[m])) (2) shelf life evaluation physical analysis rehydration was performed by adding hot water into the diet formulations in the ratio of 200 ml: 50 g, stirring with a spoon, and observing for 5 min. chemical analysis the moisture content of the rice flour samples and bouillon cubes was obtained using a moisture analyzer (sartorius moisture analyzer, ma 37, göttingen, germany). a water activity meter (aqualab, cx3te, usa) was applied for measuring the water activity (aw) of the samples. microbial analysis the samples were monitored for aerobic microorganisms and yeast and mold every month for 6 months. for total aerobic microorganisms, plate count agar (pca) (himedia, mumbai, india) was used to enumerate the colonies. for yeast and mold counts, potato dextrose agar (pda) (himedia, india) with 0.01% chloramphenicol was used. colonies were counted and are reported as cfu/g. sensory evaluations sensory evaluation performed by 35 untrained panelists (aged between 20 and 50 years) was organized monthly during product storage. the porridge formulations were labeled with three-digit codes and randomly served to the panelists. sensory evaluation using the 9-point hedonic scale (the maximum for “extremely liked” and the minimum for “extremely disliked”) was evaluated on proximate analysis the main ingredients and porridge formulations were analyzed for the proximate composition of carbohydrate, protein, fat, ash, and moisture contents, following aoac (1984) methods. to determine protein and fat content, the kjeldahl and soxhlet methods were used, respectively. the water content of each sample was determined using a moisture analyzer (sartorius moisture analyzer, ma 37, göttingen, germany). to analyze the ash content, the sample was dried at 550°c in a muffle furnace until a white or light gray ash resulted. the total carbohydrate content of the sample was obtained using the following equation: total carbohydrate (%) = 100 −[moisture (%) + crude protein (%) + crude lipid (%) + total ash (%)] total energy caloric contents of the main ingredients and porridge formulations were determined using a bomb calorimeter (leco, ac 500, usa). one gram of grounded sample was pressed to a pellet. then it was analyzed for gross energy using the bomb calorimeter, which measures the amount of a sample’s combustion heat generated under an oxygen atmosphere in a closed vessel. consumer testing the consumer acceptance test of the developed porridge formulations and packaging designs in this study was evaluated by 100 panelists aged between 60 and 90 years, including both sexes. the panelists were interviewed to collect personal information on gender, age, health status, eating behavior, daily activities, choice of food, and purchase behavior. the estimated energy requirement (eer) was calculated according to the method suggested by the institute of medicine (2005) using equation [1] for males, equation [2] for females, and pa as indicated in table 1. following calculation, a suitable recipe was selected accordingly. sensory evaluation using the 5-point hedonic scale (1  = dislike very much, 2 = dislike slightly, 3 = neither like nor table 1. the physical activity (pa) coefficients used to determine energy needs. group physical activity coefficient sedentary (pal 1.0–1.39) low active (pal 1.4–1.59) active (pal 1.6–1.89) very active (pal 1.9–2.5) males 1.0 1.13 1.26 1.42 females 1.0 1.16 1.31 1.56 28 italian journal of food science, 2022; 34 (3) nitikornwarakul c et al. (2022) revealed natural omega-3 polyunsaturated fats such as eicosapentaenoic acid (epa; 20:5 n-3), docosapentaenoic acid (dpa; 22:5 n-3), and docosahexaenoic acid (dha; 22:6 n-3) in salmon. moreover, high content of epa + dha at 10.29–26.92 mg/g was reported in salmon (tan et al., 2020). other ingredients such as pumpkin, corn, carrot, and seaweed were added to the formulations because of their abundance in vitamins and minerals (maqbool et al., 2021; onwude et al., 2017). according to the results, these ingredients had ash contents of 7.98, 6.62, 3.75, and 2.76%, respectively. apart from the main ingredients presented in table 2, sesame oil was included in a separate package to provide energy from fat as well as a nice flavor. the elderly diet formulations were generated at three caloric concentrations, which were 380, 450, and 530 kcal per portion. the energy per portion was defined based on the daily energy requirement at 1,500, 1,800 and 2,100 kcal according to the elderly diet study suggested by jansen et al. (2017). the diet formulations were generated regarding the macronutrients requirement for the elderly; carbohydrate 55–60%, protein 10–35%, and fat 30% of total energy intake (chernoff, 2016). a deficit of essential nutrients has become a common finding in elderly groups (chernoff, 2016). the research carried out by risonar et al. (2009) also revealed that the protein and fat intakes of the studied elderly group were only 24–51% of the recommended daily levels. a properly developed diet, therefore, needs to have adequate macronutrients and energy. the three formulations were calculated and constructed based on the proximate compositions of all ingredients, to provide suitable amounts of carbohydrate, protein, and fat. the developed elderly diet with three main components, namely, mixed pigmented rice flour from the shm process, freeze-dried bouillon cube, and sesame oil is shown in figure 1. the results from proximate analysis and total energy of the elderly diet formulations are presented in table 3. the three formulations appearance, color, flavor, taste, ease of swallowing, and overall liking. statistical analysis the data from the consumer acceptance test of the products and packaging design were assessed for analysis of variance (anova). the other experimental data from proximate, physical, chemical, microbial, and sensory analyses are presented as means of three replications with standard deviation. statistical analysis was performed using anova and duncan’s multiple range test to compare the means at p < 0.05 required for significance. spss v22 for windows (ibm, armonk, ny, usa) was used for all analyses of variance. results and discussion the ingredients for developing an elderly diet in this research were exquisitely selected based on their health benefits. the proximate compositions of the main ingredients are presented in table 2. the mixture of five pigmented rice flours was a good source of carbohydrates and rich in dietary fiber, which is in agreement with prior studies (sati and singh, 2019; seechamnanturakit et al., 2018). furthermore, many studies have reported this ingredient as a source of bioactive compounds such as anthocyanin, proanthocyanidins, phenolic acids, flavonoids, vitamin e, and minerals (chen et al., 2016; yodmanee et al., 2011). egg and salmon contained 13.20 and 16.98% protein, respectively. rehault-godbert et  al. (2019) and reksten et al. (2022) also reported similar findings. the chosen ingredients, especially salmon, egg, and pigmented rice flour also provide good levels of fat content. food with polyunsaturated and monounsaturated fats is recommended for elderly people, while cholesterol sources should be avoided to reduce the risk for coronary heart disease (chernoff, 2016). nechev et  al. table 2. the proximate compositions of ingredients in an elderly diet formulation. ingredient nutritional content (%) carbohydrate protein fat ash total dietary fiber mixed pigmented rice flour 74.78 ± 0.95a 9.68 ± 0.07c 3.37 ± 0.96b 1.67 ± 0.09e 1.52 ± 0.04b egg 0.07 ± 1.50d 13.20 ± 0.22b 3.74 ± 0.09b 0.63 ± 0.01f 0.61 ± 0.08f salmon 0.45 ± 1.11d 16.98 ± 0.27a 9.39 ± 0.49a 0.38 ± 0.15f 0.99 ± 0.07d seaweed 0.08 ± 0.90d 6.90 ± 0.14d 0.63 ± 0.01d 2.76 ± 0.21d 1.62 ± 0.04b pumpkin 1.71 ± 0.42d 2.8 ± 0.09e 1.85 ± 0.10c 7.98 ± 0.48a 1.27 ± 0.07c carrot 4.53 ± 0.93c 0.97 ± 0.02g 0.15 ± 0.07d 3.75 ± 0.96c 0.52 ± 0.03f corn 11.43 ± 0.79b 2.32 ± 0.05f 1.80 ± 1.04c 6.62 ± 0.30b 0.86 ± 0.03e n = 3. values are presented as mean ± standard deviation. values with the same superscript within the same column are not significantly different (p ≥ 0.05). italian journal of food science, 2022; 34 (3) 29 nutritious elderly diet range of 60–69 years, which accounted for 67%. in the age ranges of 70–79 years and 80–90 years, there were 23 and 10 subjects, respectively. the panelists were interviewed for personal information, eating behavior, physical activities, gender, age, occupation, number of meals per day, and daily activities. after obtaining the information, the total energy need was estimated and the suitable formulation was served to each individual. the results of consumer acceptance are summarized in tables 4 and 5. according to the score on the product’s visual quality (appearance and color), the panelists rated it as like slightly (score 3.8–4.0). according to the hedonic scores after testing the product, all porridge formulations had well accepted flavor, taste, and texture with an overall acceptability score of 4 out of 5 (slightly liked). moreover, the panel perceived this as an easily swallowed product (score 4.7–4.8/5), which is the highlight of this elderly diet. the packaging design of each formulation provided complete product information (name of the product, ingredient list, nutrition table, and allergen information) presented in a nice design (figure 1). the hedonic scores on appearance, attractiveness, and overall acceptance of the packaging designs had their means in the range of 4.8–4.9, which translates into “like very much.” the developed elderly diet formulations were monitored for physical, chemical, microbiological, and sensorial quality for 6 months at ambient storage. the porridge products can be easily prepared within 2–3 min by adding hot water. the water uptake ability was constant during the 6 months. the moisture content and water activity of the products (figure 2) increased over the storage period, had similar percentages of the main nutrients; carbohydrate (45%), protein (17%), and fat (22%), which are fit for elderly body requirements (berner, 2019; lecerf, 2019). the difference among the formulations is the amount of each ingredient and the total energy per portion, in which the analytical calories of formulations i, ii, and iii were 379, 453, and 528 kcal/portion, respectively. according to the survey study on eating behavior, the result interestingly demonstrated that the consumers over 65 years were being more careful to eat healthy than other younger groups (bolek, 2021). in general, instant porridge products available in the market are usually abundant in carbohydrates but cannot supply enough protein and fat (akonor et  al., 2021; mahgoub et al., 2020). in addition, the current research on the elderly diet has focused on the development of food ingredients, snacks, or beverages, e.g., a low-fat bologna-type meat product (reyes-padilla et al., 2018), a high-protein yoghurt (kersiene et al., 2020), and a ready to drink product from riceberry (jaroenwanit et al., 2021). there is limited study on a complete meal product that is specifically designed for the elderly. the new porridge formulations in this study were created to respond to this demand. all three formulations have suitable proportions of carbohydrates, protein, and fat, and are presented in a form that is convenient to prepare and consume. in the current study, the consumer acceptance test of the developed elderly diet (formulations i, ii, and iii) and their packaging designs (figure 1) involved 100 panelists aged between 60 and 90 years. the panel group was 68% female and 32% male. the majority were in the age figure 1. components of elderly diet (a), elderly product from pigmented rice (b), packaging design of the elderly diet products formulation i (380 kcal) (c), formulation ii (450 kcal) (d), and formulation iii (530 kcal) (e). (a) (b) (c) (d) (e) 30 italian journal of food science, 2022; 34 (3) nitikornwarakul c et al. table 3. the proximate compositions and total energy of elderly diet formulations. nutritional content (%) formulation i ii iii carbohydratens 45.35 ± 0.09 45.05 ± 0.12 45.00 ± 0.06 proteinns 16.77 ± 0.24 17.03 ± 0.36 17.31 ± 0.31 fatns 22.64 ± 0.18 22.29 ± 0.21 21.84 ± 0.41 moisture contentns 5.59 ± 0.25 6.06 ± 0.32 6.15 ± 0.29 ashns 5.32 ± 0.34 5.37 ± 0.33 5.34 ± 0.47 total dietary fiberns 4.33 ± 0.07 4.20 ± 0.10 4.36 ± 0.05 total energy (kcal) 379 ± 0.2 453 ± 0.2 529 ± 0.1 values are presented as mean ± standard deviation. ns = values are not significantly different in the same row (p ≥ 0.05). table 4. the consumer rating of each elderly product on a 5-point hedonic scale. formulation attribute appearancens colorns flavorns tastens texturens ease of swallowingns overall acceptabilityns i 4.03 ± 0.81 4.00 ± 0.61 3.91 ± 0.72 3.73 ± 1.04 3.82 ± 0.78 4.70 ± 0.47 3.88 ± 0.89 ii 3.82 ± 0.67 4.06 ± 0.78 3.65 ± 0.85 3.59 ± 1.08 3.82 ± 0.67 4.76 ± 0.43 3.85 ± 0.78 iii 3.82 ± 0.77 3.79 ± 0.74 3.82 ± 0.73 3.64 ± 0.96 3.94 ± 0.75 4.76 ± 0.44 3.85 ± 0.80 values are presented as mean ± standard deviation. ns = values are not significantly different in the same column (p ≥ 0.05). table 5. the consumer rating of product’s package on a 5-point hedonic scale. formulation attribute appearancens attractivens overall acceptabilityns i 4.88 ± 0.33 4.88 ± 0.33 4.91 ± 0.29 ii 4.82 ± 0.39 4.82 ± 0.39 4.88 ± 0.33 iii 4.88 ± 0.33 4.91 ± 0.29 4.94 ± 0.24 values are presented as mean ± standard deviation. ns = values are not significantly different in the same column (p ≥ 0.05). but the values still complied with dry product regulations (laullen, 2018; prabhakar and mallika, 2014). the moisture content of mixed rice flour and bouillon cube increased significantly, indicating moisture uptake from the atmosphere inside the packaging and the environment outside through diffusion of vapors. this increasing trend is related to the high relative humidity of the air. so moisture is constantly absorbed from the environment to the product (techakanon and tangrujiwatanachai, 2019). khan et al. (2014) reported the same finding in an instant wheat porridge mix product. instant soup mix products also had increased moisture during storage for 6 months (dhiman et al., 2017). the water activity of the product is a principal factor affecting product stability and its shelf life. the same increasing trend was also observed in water activity. however, each component of the porridge products had aw below 0.5 throughout the storage period, indicating a low tendency of microbial growth. accordingly, no microbials (aerobic bacteria and yeast, and mold) were detected in the samples, confirming product safety for up to 6 months of storage at ambient temperature (results not shown). table 6 shows the sensory quality results of the three porridge formulations for 6 months. the shelf life of a food product containing fat is often limited by the development of rancidity, which consumers find unpleasant (heinio et al., 2016). although salmon in the bouillon cube contained a good amount of lipids (9.39%), the lower water activity after freeze-drying (0.17–0.29) can help prevent lipid oxidation (sun et al., 2002). more than 80% of the fatty acids in sesame oil are unsaturated, e.g., oleic, linoleic, palmitic, stearic, and linolenic acids (mujtaba et al., 2020). however, it is reported to be very resistant to oxidation rancidity due to natural antioxidants such as sesamin, sesamol, sesamolin, and tocopherols (aslami et al., 2018; hegde, 2012). therefore, pigmented rice flour was the key ingredient that could provoke a rancid smell as perceived by the panelists. this finding is in agreement with the study of colored rice reported by paiva et al. (2014). the hedonic scores on flavor attributes of formulations i, ii, and iii significantly decreased after storage for 5, 3, and 3 months, respectively. chen et al. (2019) described that dehulling to produce brown rice disrupts the outer bran layers, allowing lipase and lipoxygenases in the pericarp and aleurone layers to be in contact with lipids, and thus trigger lipid oxidation. one other factor contributing to rancidity was the atmosphere in the packaging, which had oxygen participating in the hydrolysis and oxidation reactions of lipids (klaykruayat et al., 2020). although changes in the flavor of the product were detectable, the panelists still rated the flavor attribute of the three formulations as like italian journal of food science, 2022; 34 (3) 31 nutritious elderly diet mix pigmented rice flour formulation i m oi st ur e co nt en t ( % ) 8.0 (a) (b) (c) (d) 7.0 cd e de i i i g gh e f ef d cd cd cdbcbc bc ab ab ab a k h j i g f f f e e e de de cd c c b b b a a bc h c cdecd c cde c b a a a a a a a a j j i i hi ghi ghi fgh efg efgefg efgdef def bcd bcd ab a abc cde fg ab abab 6.0 5.0 4.0 3.0 2.0 1.0 0.0 0 1 2 3 storage time (month) 4 5 6 mix pigmented rice flour formulation ii mix pigmented rice flour formulation iii mix pigmented rice flour formulation i a w 0.00 0.05 0.15 0.10 0.25 0.20 0.35 0.30 0.45 0.40 a w 0.00 0.05 0.15 0.10 0.25 0.20 0.35 0.30 0.45 0.50 0.40 0 1 2 3 storage time (month) 4 5 6 mix pigmented rice flour formulation ii mix pigmented rice flour formulation iii bouillon cube formulation i m oi st ur e co nt en t ( % ) 8.0 9.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 0 1 2 3 storage time (month) 4 5 6 0 1 2 3 storage time (month) 4 5 6 bouillon cube formulation ii bouillon cube formulation iii bouillon cube formulation i bouillon cube formulation ii bouillon cube formulation iii figure 2. the moisture content of each component (a). mixed pigmented rice flour and (b). bouillon cube and the a w of each component (c). mixed pigmented rice flour and (d). bouillon cube in three formulations of elderly product during 6 months of storage. different letters above bars indicate significant differences (p < 0.05). slightly at the end of the storage period (average score of 5.6). during the study, the visual characters of the products (appearance and color attributes) were rated as 5.7– 6.6 (like slightly – like moderately). the scores for taste were higher throughout the storage, 7–7.9. similar to the consumer acceptance test, the panel liked the products for their ease of swallowing with a high score of 8 out of 9 (like very much). the testers rated the porridge formulations for the elderly as overall “moderately liked to liked very much” until the end of the tested storage period. conclusion the developed elderly diet was provided in three components (mixed pigmented rice flour, freeze-dried bouillon cube, and sesame oil) and had a nutritional balance of 45% carbohydrate, 17% protein, and 22% fat. three formulations were generated at three caloric concentrations and provided 379, 453, and 528 kcal per portion. according to the consumer acceptance test, the newly developed diet was well accepted by 100 panels, with an overall acceptance score of 4 out of 5 for product and 5 out of 5 for packaging design. this showed high potential for launching to target the elderly product market with its key properties being easy and rapid preparation and ease of swallowing. the product can retain its quality for at least 6 months with only a minor change in odor. the elderly diet formulations were moderately liked in terms of overall acceptance and achieved microbiological standards during 6 months of storage at ambient temperature. to improve the product’s qualities and prolong the shelf life (reduce moisture uptake and unpleasant flavor development), the selection of suitable packaging materials and packing conditions are suggested for future study. conflict of interest the authors declare no conflict of interest. 32 italian journal of food science, 2022; 34 (3) nitikornwarakul c et al. table 6. the sensory evaluations of elderly product formulation i, ii, and iii during 6 months of storage on a 9-point hedonic scale. attribute storage time (month) 0 1 2 3 4 5 6 formulation i appearance 5.7 ± 1.0b 6.0 ± 0.6a 5.9 ± 0.7a 5.9 ± 0.5a 5.9 ± 0.5a 5.9 ± 0.4a 5.9 ± 0.5a color 6.1 ± 0.9a 6.0 ± 0.8a 5.9 ± 0.7a 6.0 ± 0.4a 5.9 ± 0.3a 5.9 ± 0.4a 5.9 ± 0.2a flavor 6.8 ± 0.4a 6.8 ± 0.4a 6.6 ± 0.5a 6.6 ± 0.4a 6.3 ± 0.4ab 5.8 ± 0.5b 5.6 ± 0.3c taste 7.2 ± 0.9a 7.1 ± 0.9a 7.2 ± 0.8a 7.1 ± 0.5a 7.1 ± 0.5a 7.1 ± 0.5a 7.1 ± 0.4a ease of swallowing 7.8 ± 0.8a 8.0 ± 0.8a 8.0 ± 0.8a 8.0 ± 0.6a 8.0 ± 0.7a 8.0 ± 0.5a 8.0 ± 0.5a overall liking 7.3 ± 0.8b 7.4 ± 0.7b 7.9 ± 0.8a 7.5 ± 0.5a 7.4 ± 0.5a 7.4 ± 0.4a 7.2 ± 0.7a formulation ii appearance 6.6 ± 0.8b 6.6 ± 0.8a 6.7 ± 0.7a 5.9 ± 0.4c 5.9 ± 0.5c 5.9 ± 0.4c 5.9 ± 0.5c color 6.2 ± 0.8a 6.3 ± 0.7a 6.4 ± 0.6a 6.0 ± 0.4b 5.9 ± 0.3b 5.9 ± 0.4b 5.9 ± 0.6b flavor 7.2 ± 0.8a 7.4 ± 1.0a 7.6 ± 0.9a 6.6 ± 0.5b 6.3 ± 0.4b 5.8 ± 0.5c 5.6 ± 0.3c taste 7.7 ± 0.9a 7.7 ± 0.9a 7.9 ± 0.9a 7.1 ± 0.5b 7.1 ± 0.5b 7.1 ± 0.5b 7.1 ± 0.4b ease of swallowing 7.9 ± 0.7a 7.9 ± 0.6a 7.8 ± 0.7a 8.0 ± 0.6a 8.0 ± 0.7a 8.0 ± 0.5a 8.0 ± 0.5a overall liking 8.0 ± 0.6a 8.1 ± 0.6a 8.0 ± 0.6a 7.5 ± 0.5b 7.4 ± 0.5b 7.3 ± 0.3b 7.2 ± 0.7c formulation iii appearance 6.4 ± 1.4a 6.2 ± 1.1b 6.0 ± 1.1b 5.9 ± 0.3c 5.9 ± 0.6c 5.9 ± 0.4c 5.9 ± 0.9c color 6.2 ± 1.0a 6.1 ± 0.9a 6.3 ± 0.9a 6.0 ± 0.8b 6.0 ± 0.7b 5.9 ± 0.6b 5.9 ± 0.6b flavor 7.4 ± 0.8a 7.5 ± 0.8a 7.6 ± 0.9a 6.6 ± 0.5b 6.2 ± 0.6bc 5.6 ± 0.3c 5.6 ± 0.6c taste 7.4 ± 1.0a 7.5 ± 0.9a 7.7 ± 0.9a 7.1 ± 0.4b 7.1 ± 0.4b 7.0 ± 0.5b 7.0 ± 0.9b ease of swallowing 7.9 ± 1.1a 7.8 ± 1.0a 7.9 ± 0.8a 8.0 ± 0.3a 8.0 ± 0.4a 7.9 ± 0.4a 7.9 ± 0.3a overall liking 7.4 ± 0.9b 7.6 ± 0.9a 7.6 ± 0.7a 7.5 ± 0.5ab 7.5 ± 0.6b 7.3 ± 0.6b 7.3 ± 0.4b values are presented as mean ± standard deviation. values with the same superscript within the same roll are not significantly different (p ≥ 0.05). acknowledgments this research was funded by prince of songkla university, hatyai campus (project grant no. sit6202029s). the authors would like to thank assoc. prof. seppo karrila for his kind support. furthermore, the authors are thankful to the food innovation and product development (fipd) laboratory for the provided lab space and equipment support. references akonor, p.t., atter, a., owusu, m., ampah, j., andoh-odoom,  a., overa, r., et al. 2021. anchovy powder enrichment in brown rice-based instant cereal: a process optimization study using response surface methodology (rsm). food science and nutrition. 9: 4485–4497. https://doi.org/10.1002/fsn3.2424 aoac, 1984. official methods of analysis. 14th ed. association of official analytical chemists, arlington, tx. aslami, f., iqbal, s., nasir, m., anjum, a.a., swan, p. and sweazea,  k., 2018. effect of hydrogenated fat replacement with white sesame seed oil on physical chemical and nutritional properties of cookies. italian journal of food science. 30: 13–25. berner, y.n., 2019. nutrition in the elderly. in: ferranti, p., berry, e.m. and anderson, j.r. 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_hlk108514997 _hlk94624831 _hlk109884037 paper ital. j. food sci., vol. 27 2015 385 keywords: kiwi, kiwi puree powder, freeze drying, maltodextrin, vitamin c freeze drying of kiwi (actinidia deliciosa) puree and the powder properties gülşah çalişkan, kadriye ergün* and s. nur dirim department of food engineering, ege university, bornova, 35100, izmir, turkey *corresponding author: kadriye_ergun555@hotmail.com abstract in this study, it was intended to investigate the production of freeze dried kiwi (actinidia deliciosa) puree in the form of powder that can be used as a natural alternative to synthetic additives used in food products such as pudding, instant tea, and sauces for improving their flavour. in order to obtain the powder product, kiwi puree as plain and with maltodextrin (dextrose equivalence of 10-12, as 10 % by weight) addition were freeze dried. drying behaviour of plain kiwi puree and kiwi puree with md were explained by logarithmic model (r2=0.994, rmse=0.024, χ2=0.0008) and wang and singh model (r2=0.999, rmse=0.012, χ2=0.0002), respectively. the effective moisture diffusivity (d eff ) value was calculated as 7.3x10−10 m2/s and it was observed that it was not affected by the addition of md. the vitamin c content of fresh kiwi fruit was evaluated as 66.3 mg/100 g kiwi and there was a loss of 17.1% for plain and 19.8% for md containing powders respectively after freeze drying. it was also observed that, the addition of maltodextrin decreased cohesiveness, on the other hand, increased bulk and tapped densities, average time values for wettability and solubility, and glass transition temperature of the powder products. mailto:kadriye_ergun555%40hotmail.com?subject= 386 ital. j. food sci., vol. 27 2015 introduction kiwi fruit contains high amounts of vitamins (vitamin c (100-400 mg vitamin c/100 g), a, b 2 , and e), minerals (calcium, iron, copper, phosphorus, magnesium, and potassium), carotenoids (beta carotene, lutein, and xanthophyll), phenolic compounds (flavonoids and anthocyanins) and antioxidant compounds (cassano et al., 2006). kiwi fruit is being processed to obtain juice, frozen food, wine, jam, marmalade, and canned and dried slices. drying might be a suitable technique to prolong the shelf life of kiwi, which is susceptible for microbial spoilage and softening due to its high moisture content. fruit juices, purees and powders are being marketed due to an increased demand for ready-to-eat foods. in addition, powder products, with a long-term ambient shelf life and microbiological stability can reduce the transportation, and storage costs as well (jinapong et al., 2008). thus, alternatives to conventional processing technologies are being explored to produce better quality products. due to high content of vitamin c, it is essential to protect vitamin c during drying of kiwi (kaya et al., 2010). freeze drying is an important process for the protection of sensitive compounds such as vitamin c, phenolic compounds, biological activity, appearance, color, texture, aroma, and nutritional values of foods which compensates its high operating costs for drying of foods (zea et al., 2013; wang et al., 2006). in addition, fernandes et al. (2011) reported that for producing whole fruit powder, drying fruits at low temperature and reduced pressure with low amounts of carrier is apparently the best alternate. because, there exist some difficulties for drying of food extracts, juices, and purees because of the stickiness problems resulted by low glass transition temperatures of their components such as sugars and organic acids. in order to prevent problems in drying and obtaining powder products with acceptable properties, the drying aids that have high t g is to be used. the use of drying agents such as gum arabic, maltodextrin, whey protein, sucrose etc. improves the drying process, and leads to an effective drying (nadeem et al., 2011). numerous studies were carried out with freeze drying of foods which contain sensitive compounds such as carrot (lin et al., 1998), pumpkin (que et al., 2008), kiwi (ergün, 2012) mango (shofian et al., 2011) pineapple (marques et al., 2011), papaya (shofian et al., 2011; marques et al., 2011) and guava (wang et al., 2006). several researchers studied on drying of kiwi fruits such as convective, microwave, vacuum microwave, and freeze drying (kaya et al., 2010; ergün, 2012; doymaz et al., 2009; kiranoudis et al., 1997) methods. describing dehydration kinetics is important in the design and optimisation of drying processes (simal et al., 2005). thin layer drying models, generally means to dry as one layer of sample which provide uniform temperature assumption and suitable for lumped parameter models, are important in mathematical modelling of drying. although, models depend on the process conditions, they are practical and provide sufficiently good results (erbay and icier, 2009). the properties of food powders such as bulk density, hygroscopicity, degree of caking, dispersibility, wettability, solubility, particle size, and size distribution are useful for design, and control of processing, handling, storage operations, and product quality control. properties of powder products are usually studied in two groups such as particle properties (particle size, shape, distribution, density and morphological properties), and bulk properties (bulk density, wettability, solubility, porosity, cohesiveness, and flowability). in this study it was intended to investigate the production of freeze dried kiwi (actinidia deliciosa) puree in the form of powder that can be used as a natural alternative to synthetic additive used in food products such as pudding, instant tea, and sauces for improving their flavour. also, an alternative product with the advantages of high nutritional value, long durability, easiness for usage in dry mixture formulations, being portable easily, and a healthier food additive for the consumers consumption will be obtained. in addition to the mentioned purposes: it was also aimed to determine the drying behaviour of kiwi puree (pure and with 10% md) during freeze drying and the effect of maltodextrin addition and the properties of the powder product. material and methods the fresh kiwi fruits were obtained from a local supermarket in izmir, turkey. they were peeled and grounded into puree by using a home type blender (tefal smart, mb450141, turkey). in order to obtain the puree with maltodextrin addition, maltodextrin (md) with dextrose equivalence (de) value of 10-12 (as chemical industry and commerce limited company, turkey) was added directly to puree in suitable amounts (10% by weight). freeze drying the freeze drying experiments were performed in a pilot scale freeze dryer (armfield, ft 33 vacuum freeze drier, england). prior to drying kiwi puree was frozen in a layer of 3 mm in the petri dishes at 40ºc in an air blast freezer (frigoscandia, helsinborg, sweden) for two hours, then freeze dried under vacuum (13.33 pa absolute pressure), at 48ºc condenser temperature. the temperature of the heating plate was set to 30ºc, which was constant during the drying process. the powder was obtained by grindital. j. food sci., vol. 27 2015 387 ing the dried material, obtained as pellets of diameter of petri size, in a blender (tefal smart, mb450141, turkey), and powder was stored in glass jars in the dark at 20±1ºc until further tests were carried out. physical and chemical analyses the moisture content of kiwi puree and freeze dried kiwi puree powders (kpp) were determined according to aoac (2000). for this process, each experiment for increasing time periods was carried out with new samples of equal mass, and moisture loss was determined gravimetrically by using a digital balance with 0.01 precision (ohaus ar2140, usa). moisture ratio was calculated according to equation (1). (1) where the m t , m 0 and m e are the moisture content at any time, initial, and equilibrium moisture content (kg water/ kg dry matter), respectively. drying data was fitted to ten well-known thin layer drying models (lewis, page, modified page i, henderson and pabis, logarithmic (asymptotic), midilli, modified midilli, two-term, two-term exponential, and wang and singh) (erbay and icier, 2009). nonlinear regression analysis was used to evaluate the parameters of the selected model by using statistical software spss 16.0 (spss inc., usa). the goodness of fit was determined using the coefficient of determination (r2), root mean square error (rmse), and the reduced chi-square (χ2) that can be described by the equations given by erbay and icier, 2009. where mr exp,i and mr pre,i is the experimental, and predicted moisture ratio at observation i; n is number of the experimental data points, and n is number of constants in model. the effective moisture diffusivity (d eff ) of freeze dried kiwi slices were calculated by fick’s diffusion model (eq. 2). (2) where t is the time (s), d eff is the effective diffusivity (m2/s) and l is the thickness of samples (m). for long drying times, a limiting case of eq. (3) is obtained, and expressed in a logarithmic form; (3) the effective diffusivity was calculated by plotting experimental moisture ratio in logarithmic form versus drying time. from eq.(3), a plot of ln mr versus drying time gives a straight line with a slope of: (4) water activity was measured by using testoag 400, germany, water activity measurement device. the ph values of kiwi puree and the powders were measured using a ph meter (inolab wtw ph 720, germany) directly and after dissolving the powder in deionised water (1 g/1 g) respectively. the color values (l*, a*, and b* values) of fresh kiwi fruits, and the powders were measured with minolta cr-400 colorimeter, japan, calibrated with white standard plate three times and results as the average of three measurements were expressed in accordance with the cie lab. system. the l* value, is a measure of lightness which ranges between 0 and 100. increases in a* value in positive, and negative scales correspond to increases in red or green color, respectively. the b* value represents color ranging from yellow (+) to blue (-). the vitamin c content of fresh kiwi fruits was determined according to hişil (2007). freeze dried powders were rehydrated to the initial moisture content prior to the analysis. the indication principle of vitamin c value is based on extraction with 10% oxalic acid afterwards adding of 2,6-dichlorophenolindophenol solution. the absorbance was measured at 518 nm by a varian cary 50 uv/vis spectrophotometer. glass transition temperature glass transition temperature of the powder samples was determined by a differential scanning calorimeter (ta instruments, q10, usa) equipped with a thermal analysis station. an empty sealed aluminum pan was used as a reference in each test. nitrogen gas at a flow rate of 50 ml/min was used as the purge gas to avoid water condensation around the samples. about ten milligrams of kiwi sample was sealed in aluminum pans and cooled from room temperature to -40°c at 10°c/min for formation of glassy state in kiwi sample and equilibrated for 10 min. the heating rate was 10°c/min and the temperature range varied between -40 and 120°c, depending on sample moisture content. dsc thermograms, presenting the heat flow (w/g) and temperature relationship were used to analyze the thermal transitions in samples during heating and cooling. ta instruments universal analysis software was used to analyze the onset, mid and end points of the glass transition. the glass transition temperature (tg) was calculated as the average of the onset and end point values. thermo gravimetric analysis thermo gravimetric analysis (tga) was carried out by perkin elmer diamond tg/dta (canada) under nitrogen flow. the assay con388 ital. j. food sci., vol. 27 2015 ditions were as follows: isotherm at 30 °c and heating from 30.00°c to 1000.00°c at 10.00°c/ min. five milligrams of equilibrated samples was introduced into the apparatus and the measurements were plotted during the heating. scanning electron microscope (sem) the morphology of the powder samples, prepared by placing the powders on aluminium stubs using a double-sided adhesive tape and then coating with gold, were examined with a scanning electron microscope (semphillips xl30s feg, eindhoven, netherlands) operating at 5kv accelerating voltage. analysis of the powder properties for the determination of bulk density, the method explained by jinapong et al. (2008) was used. the average wettability and solubility times of freeze dried kiwi puree powders were determined by using the method explained by gong et al. (2008) and goula and adamopoulos (2008), respectively. flowability and cohesiveness values of the powders were evaluated in terms of carr index (ci) and hausner ratio (hr), respectively. both ci and hr were calculated from the bulk (ρ bulk ), and tapped (ρ tapped ) densities of the powder as shown below eqs. (5) and (6), respectively. (5) (6) statistical analysis data were analyzed by using statistical software spss 16.0 (spss inc., usa). the data were subjected to analysis of variance (anova), and duncan’s multiple range test (α=0.05) to determine the difference between means. the drying experiments were replicated twice and all the analyses were triplicated. results and discussion results of physical and chemical analyses kiwi is harvested through a long season. however, due to its high moisture content, storage period and its direct use in food compositions are limited and this makes necessary the drying to obtain pure, minimally processed, decreased in volume and easy to use form of the kiwi. the results of the experimental study showed that, it was possible to dry the fresh kiwi puree under the freeze drying condition. in order to improve the drying process, to see the effect of maltodextrin addition and to obtain a more stable powder, maltodextrin was used as a drying aid. the amount of md to be used to prevent quality losses during drying and to obtain powder which has almost the same properties with fresh kiwi was determined by the preliminary tests. for this purpose, md with amounts of 5, 10, 15, and 20% of the puree weight were added to the fresh kiwi puree. the addition of md as 5% of the puree weight was not suitable since there was no decrease in the drying time of kiwi puree. for the md amounts being more than 10 %, the powders lost their quality characteristics such as specific color, vitamin c content etc. similar results were observed by quek et al. (2007). it was reported that after addition of the 10% md watermelon powders lost their redorange color. therefore, as a result of the preliminary tests, the concentration of md in the puree necessary for successful drying and powder production was determined as %10 of the puree weight. zea et al. (2013) reported that powder obtained by freeze drying of guava and pitaya pulp was found to be very hygroscopic and difficult to compact. in order to minimize this problem the researchers added 10% maltodextrin to guava and pitaya mash. the drying behaviour of the freeze drying process was determined from the mass loss in samples of known initial moisture content. for the drying process, the total drying time was determined to be nine and ten hours respectively for the samples of kiwi puree, and kiwi puree with maltodextrin until getting constant weight of the samples. similar results were obtained by marques and freire (2005) in their freeze drying study on pulps of tropical fruits as ten to thirteen hours. the average values of the experimental results of the analysis applied on fresh kiwi puree and freeze dried powders are given in table 1. the initial moisture content of kiwi puree was found to be as 81.19 % (wet basis, wb), and this result was consistent with kaya et al. (2010) (81% wb). the final moisture content of kiwi powder is 9.55 % (wb) after removal of 88.24% of water. for the sample with md, 94.31% of water was removed where the initial dry matter content of the sample was higher than the plain sample due to maltodextrin addition and the amount of water to be removed at the same drying time decreased. the residual moisture in the powder decreased, and the moisture content of the sample with md was found to be 56% lower than the plain sample, and this differences between samples was found to be statistically significant (p<0.05). the moisture ratio were calculated by using the determined moisture content values and the data were fitted to ten thin layer drying models (lewis, page, modified page i, henderson and pabis, logarithmic, midilli, modified midilli, two-term, two-term exponential, and wang and singh). the coefficient of correlation ital. j. food sci., vol. 27 2015 389 table 1 the physical and chemical properties of kiwi puree and freeze dried kiwi puree powders. properties fresh kiwi puree freeze dried kiwi kiwi puree the freeze dried kiwi puree powder with md puree powder with md moisture content (% wb) 81.19 ±0.02b 9.55±0.64r 73.82±0.04a 4.20±0.05p water activity 0.98 ±0.01b 0.28±0.03r 0.96±0.01a 0.22±0.01p ph 3.16±0.01a 3.37±0.01p 3.38±0.01a 3.60±0.02r color l* 47.37±0.35a 77.93±0.53p 48.84±0.34a 78.12±0.44p a* -0.67±0.24b 1.16±0.09r -0.74±0.08a -6.53±0.12 p b* 17.5±0.29a 21.77±0.17p 17.85±0.18a 22.08±0.11p vitamin c (mg/100g, wb) 66.3±0.28b 54.97±0.13r 51.07±0.09a 40.95±0.51p a-b different letters in the same row indicate significant difference between averages of puree and puree with md at p<0.05. p-r different letters in the same row indicate significant difference between averages of powder and powder with md at p<0.05. (r2) was accepted one of the primary criterion for selecting the best model to define the freeze drying curves of kiwi puree powders. for freeze drying process of kiwi puree the highest r2 value (0.994), and the lowest rmse (0.02459), and χ2 (0.00083) values were obtained from logarithmic model (fig. 1). however, for freeze drying of kiwi puree with md the best fit was obtained from wang and singh model (r2=0.999, rmse=0.012, χ2=0.0002) (fig. 2). in the literature, the convective drying characteristics of kiwi slices were explained with two term exponential (kaya et al., 2010), page (ceylan et al., 2007; simal et al., 2005), and henderson and pabis (doymaz, 2009) models. the effective moisture diffusivity (d eff ) of freeze dried kiwi puree and pure with md were evaluated as 7.3x10−10 m2/s. the difference between calculated values was 0.002x10−10 m2/s and this was not considered to be effective. kaya et al. (2010) reported that the effective moisture diffusivity values of kiwi slices which were dried under different drying conditions (air velocity, temperature, and relative humidity) varied between 0.589 and 6.574 x10−10 m2/s. simal et al. (2005) reported that the effective moisture diffusivity of hot air dried kiwi slices (30-90°c) ranged between 3.00 and 17.21 x10−10 m2/s. the d eff value of kiwi powder was found to be similar to the d eff value (7.13x 10−10 m2/s) of kiwi slices which were dried at 50 °c hot air temperature (simal et al., 2005). the effective moisture diffusivity values in foods are in the range of 10−12 to 10−6 m2/s. water activity is considered as one of the most important quality factors especially for long term storage and also it is related to moisture content, and responsible for biochemical reactions. the values of water activity under 0.6 is generally considered as microbiologicalfig. 1 experimental and computed moisture ratio values obtained by selected models for pure kiwi puree powder (r2≥0.993). 390 ital. j. food sci., vol. 27 2015 ly stable (quek, 2007) and between 0.20, and 0.40 ensure the stability of the product against browning, and hydrolitical reactions, lipid oxidation, auto-oxidation, and enzymatic activity (amrques et al., 2007). the water activity values of freeze dried kiwi puree powders (plain powder and powder with md) were found to be as 0.287, and 0.225, respectively. in literature water activity values around 0.28 was also expressed for freeze dried guava and pitaya powders with 10% md (zea et al., 2013). drying process and addition of md showed the significant effects on the water activity of freeze dried kiwi puree powders (p<0.05). the ph value of kiwi puree was measured as 3.16. souflerosa et al. (2001) reported that the ph value of kiwi ranges between 3 and 4, due to the content of including the acids such as gluconic, galacturonic, oxalic, succinic, fumar ic, oxcaloacetic, and p-coumaric acids. harder et al. (2009) and arroqui et al. (2004) measured the ph value of kiwi nectar and puree as 3.50 and 3.41, respectively. the ph values of powders (kiwi puree powder and powder with md) were found to be as 3.37 and 3.60, respectively. results showed that the drying process and addition of md caused a significant increase in the ph value of powders (p<0.05). the increase in the ph values was found as 6.65% and 6.51% for plain and md containing powders, respectively. this increase was comparable with the increase in 3.64% in freeze drying of guava concentrate mahendran (2010) and the reason for the increase can be explained with the loss of some acidic compounds during drying. color of the dried products is an important quality factor, which reflects the sensory attractiveness, and the quality of the powders (quek et al., 2007). thus, the color of the processed products should ideally remain unchanged after production. the color values (l*, a* and b*) of kiwi puree were measured as 47.37, -0.67, and 17.5 respectively. these values are quite different than the measurements of ancos (1999) reporting the color values (l*, a*, and b*) of kiwi puree 36.01, -12.35 and 23.03, respectively and this shows the differences between the cultivars and the storage time after harvest. the variation of color values for plain and md containing samples depending on the drying time were shown in figs. 3 and 4, respectively. as shown in fig. 3, the l*, b* and a* values of freeze dried kiwi puree powder increased throughout the drying period and reached the final values as 77.93, 1.16, and 21.77, respectively. chopda and barrett (2001) reported that the increase in l* (brightness), a* (redness) and b* (yellowness) values following production of guava puree powder was most likely a result of non-enzymatic browning during freeze drying which produced a darker product. the addition of md in freeze drying, increased the l* (78.12), and b* (22.08) values, but decreased a* value (-6.53) (table 1). results showed that, drying process increased the brightness values of samples (p<0.05); addition of md caused superior bright color but it was not found to be statistically significant (p>0.05). the same effect was also observed for yellow-blue (b*) value. nevertheless, both drying process, and addition of md showed a significant effect on the green-red (a*) value of the samples (p<0.05). for the determination of vitamin c, freeze dried powders were rehydrated to the initial moisture content prior to the analysis to obtain comparable results. the vitamin c content of kiwi was found fig. 2 experimental and computed moisture ratio values obtained by selected models for kiwi puree powder with md (r2≥0.994). ital. j. food sci., vol. 27 2015 391 fig. 3 the variation of color values of plain samples depending on the drying time. to be as 66.3±0.28 mg/ 100 g (wb) kiwi. the freeze drying process caused a significant (17.1%) decrease on the vitamin c content of kiwi powder (p<0.05). the vitamin c loss during drying is similar to losses of 18.8% (mahendran, 2010) and 16% (marques et al. 2006) during freeze drying of some other fruit concentrate and pulps. also, the addition of md caused an insignificant loss in the vitamin c content (19.82%) (p>0.05). this decrease may occur due to the dilution effect. exposure to heat, light, oxygen and metals may also lead to vitamin c losses. lin et al. (1998) did not observed significant loss of vitamin c in freeze-dried carrots. the vitamin c losses can be due to not only the freeze drying, but also by the operations before drying such as cutting, slicing and freezing. therefore, grinding process, prepafig. 4 the variation of the color values of samples with md depending on the drying time. ration of maltodextrin and kiwi puree blend may cause more vitamin c losses for the kiwi puree. marques et al. (2011) reported that the vitamin c losses for freeze dried fruits are considerably smaller when compared the vitamin c losses caused to others drying methods due to the low temperatures, and to the use of vacuum in the process. glass transition temperature in order to have safety storage, and stability of powders, the powders should be kept below glass transition temperature (t g ). so the t g value of kiwi powders was determined. kiwi powder exhibited well defined t g (average -18°c) represented by an endothermic change in the base 392 ital. j. food sci., vol. 27 2015 line (fig. 5). moisture content and water activity are the main factors affecting t g of materials. however, in the consideration of food materials with similar moisture content and water activity values, the high acid and sugar content may decrease the t g value. the increases in t g values of kiwi puree powders with carriers possibly due to the addition of carriers, and the lower moisture content of carrier-incorporated powders. t g of kiwi puree powders with md (t g average -5°c) was found to be higher (fig. 6). silva et al. (2006) reported that, addition of 30% md (w/w, de20) increased tg of freeze dried camufig. 5 dsc thermogram for freeze dried kiwi puree powders. camu pulp from -58.8°c to -40.1°c for the moisture content values between 0.2 to 0.5 (g dry solid/ g sample). after this value, t g increased rapidly with decreasing moisture content. in their study, mosquera et al. (2010) observed an increase in tg with the addition of md and this increase was slightly more where md with low de was used. thermo gravimetric analysis the results of the analysis of the samples of kiwi puree powders by tga are shown in fig. 6 dsc thermogram for freeze dried kiwi puree powders with md. ital. j. food sci., vol. 27 2015 393 the figs. 7 and 8. these spectra determine the changes of weight in relation to change of temperature that the samples experiment when exposed to heating from room temperature to 1000°c. tga spectra showed that the loss of matter began around 50°c for both samples but the kinetics of thermal decomposition is differfig. 7 the variation of the weight of freeze dried kiwi puree powder with respect to time. ent for them. at 100°c, the sample with 10% md lost around 6.5% of its own weight, but the sample that was dried without md lost around 8.5% of its own weight. their components were considerably stable until 150°c because the loss of matter is not significant. however, between 100 and 220°c, reactions such as maillard’s fig. 8 the variation of the weight of freeze dried kiwi puree powder with md with respect to time. 394 ital. j. food sci., vol. 27 2015 reaction or the condensation between phenolic acids and proteins may occur. as of 150°c, the loss of matter is significant, and the phenomena are exothermic for all samples. scanning electron microscope (sem) selected images from the sem microstructure analysis of the freeze dried kiwi puree powders were shown in fig. 9 (a and b). the microstructures of freeze-dried kiwi powder had a skeletal-like structure with void spaces previously occupied by ice prior to freeze drying. this is because the absence of liquid phase in the material during freeze drying process suppressed the transfer of liquid water to the surface and the ice was converted to vapor without passing the liquid state (krokida and maroulis, 1997). micrographs revealed that powder particles of all fig. 9 scanning electron micrographs of freeze dried plain (a) and md containing (b) kiwi puree powder at 500x magnification. powders were irregular in shape. irregular shape of powder particles may due to the fibrous and porous nature of the kiwi fruit powders since powder was prepared from whole fruits (zea et al., 2013). powder properties the powder properties of freeze dried kiwi puree powders are given in table 2. the tapped and bulk densities of freeze dried kiwi puree powder were found to be as 0.257 and 0.161 g/ml, and the addition of md significantly increased the tapped and bulk densities of powder (0.416 and 0.316 g/ml) (p<0.05). marques et al. (2006) reported that, apparent density of the studied pulps has presented a linear relationship with moisture content where the apparent densities of fruit pulps decreased linearly with moisture table 2 the powder properties of freeze dried kiwi puree powders. powder properties freeze dried kiwi puree powder freeze dried kiwi puree powder with md tapped density (g/ml) 0.26±0.01a 0.42±0.02b bulk density (g/ml) 0.16±0.01a 0.32±0.01b solubility (s) 26±3a 290 ±48b wettability (s) 78.5±2a 186 ±0.71b flowability (ci) 38±3b (bad) 24.04±2.87a (fair) cohesiveness (hr) 1.60±0.08b (high) 1.32± 0.05a (intermediate) a-bdifferent letters in the same row indicate significant difference between averages at p<0.05. ital. j. food sci., vol. 27 2015 395 content (dry basis) during freeze drying and the real density increased. the researchers reported that the remaining solids after moisture removal have higher densities than water and the overall solid density tends to increase as moisture is removed. mahendran (2010) dried the guava concentrate with different drying methods (freeze drying, tunnel drying and spray drying with the 30, 40, 50 and 60% concentrations of md) and the bulk density of guava powders were measured as 0.63 g/ml; 0.69 g/ml and 0.61, 0.60, 0.57 and 0.54 g/ml, respectively. in this study, on the contrary of the results given by mahendran (2010) the bulk density increased with the addition of the md. lower density of the dried product is recommended to increase its attractiveness for consumers (durance and wang, 2002). the average solubility time of the freeze dried kiwi puree powder was found to be as 26 seconds. the reason for the addition of md was to improve the drying process and at the same time maltodextrin is highly soluble in the water to be used as a carrier. however, addition of maltodextrin caused a significant increase in the average solubility time of the powder (290s) (p<0.05). in a study by mahendran (2010) guava concentrate was dried with spray, tunnel, and freeze driers and the freeze dried guava powder was found highly soluble (96%) compared with the other drying methods. the solubility of the powder is related with moisture content, particle size, and chemical conversions in the material (goula and adamopoulos, 2008). wettability is the ability of the powder particles to overcome the surface tension between themselves, and water. wettability depends on particle size, density, porosity, surface tension, surface area, and surface activity of particle. besides the effects of physical properties, the chemical composition of the powders also influences wettability depending on the content of fats, proteins, and carbohydrates on their surface (fang et al., 2008). also, goula and adamapoulos (2008) reported that the residual moisture content of the powder affects the bulk density, wettability, flowability, and cohesiveness. the residual moisture content of powders is significantly affected the operational conditions, and carrier concentrations. the average wettability time of freeze dried kiwi powder was found to be as 78.5 seconds. addition of md caused a significant increase in the average wettability time as 186s (p<0.05). flow difficulties and caking are common problems in industries producing food powders. the flowability and cohesiveness properties of kiwi powders in terms of carr index and hausner ratio were evaluated. the classification of powder flowability based on carr index (ci) is very good (<15), good (15-20), fair (20-35), bad (3545), and very bad (>45). the powder cohesiveness based on hausner ratio (hr) is classified as low (<1.2), intermediate (1.2-1.4), and high (>1.4) (jinapong et al., 2008). kiwi powder with higher moisture content showed bad flowability (37.15±3.15) and high (1.59±0.08) cohesiveness. however, addition of md caused a significant decrease in cohesiveness (1.29), and significant increase in flowability (22.36) behaviours of powder (p<0.05). the kiwi powder containing md with low moisture content showed superior flow properties compared to kiwi powder. conclusions the present work describes the possibility of producing kiwi puree powder by freeze drying, and the changes in some physicochemical and powder properties of powders which were affected by drying process and addition of md. the results showed that freeze drying can satisfactorily be applied for drying of kiwi puree to obtain powders that can be used as an ingredient which have high vitamin c content for flavoring and improving nutritional value purposes. the possible uses of this dried product as a food supplement with valuable constituents of kiwi fruits and storage test might be studied in future projects. references ancos b. 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(2007). the physicochemical properties of spray-dried watermelon powders. chemical engineering and processing 46: 386-92. wang z.l., finlay w.h., peppler m.s. and sweeney l.g. (2006). powder formation by atmospheric spray-freezedrying. powder technology 170: 45-52. zea l.p., yusof y.a., aziz m.g., ling c.n. and amin n.a.m. (2013). compressibility and dissolution characteristics of mixed fruit tablets made from guava and pitaya fruit powders. powder technology 247: 112-119. paper received march 25, 2014 accepted december 1, 2014 48 issn 1120-1770 online, doi 10.15586/ijfs.v34i3.2236 p u b l i c a t i o n s codon p u b l i c a t i o n s codon valorisation of date fruits by-products for the production of biopolymer polyhydroxybutyrate (phb) using the bacterial strain bacillus paramycoides zohra chekroud1*, leila djerrab2, amer rouabhia3, mohamed abdesselem dems4, imane atailia5, faiçal djazy2, mustapha adnane smadi6 1research laboratory of interactions, biodiversity, ecosystems and biotechnology, lribeb university, skikda, algeria; 2laboratory of research on the physical chemistry of surfaces and interfaces lrpcsi university, skikda, algeria; 3research laboratory of biological treatment of residues, department of chemical engineering and food technology, institute of vitivinicultural and agri-food research (ivagro), university of cadiz, cadiz, spain; 4bioinformatics and biostatistics unit (bibsu), national center for biotechnology, constantine, algeria; 5superior school of food sciences and agro food industries, el harrach, algeria; 6laboratory of animals biotechnologies, national center for biotechnology, constantine, algeria *corresponding author: zohra chekroud, research laboratory of interactions, biodiversity, ecosystems and biotechnology lribeb. university of 20th august 1955-skikda. b.p.26, el hadeik road 2100 skikda, algeria. email: chekroudzohra@yahoo.fr received: 20 may 2022; accepted: 27 july 2022; published: 23 august 2022 © 2022 codon publications open access paper abstract the aim of this research was to study the valorization of the date fruit by-product by conversion of date syrup into biopolymer polyhydroxybutyrate (phb), based on the metabolic capacity of the bacterial strain bacillus paramycoides to accumulate phb from date syrup. total reducing sugars in date syrup was essayed using 3',5-dinitrosalicylic acid (dns) and hplc methods. bacillus paramycoides was isolated from soil of the botanic garden of skikda university, algeria. the accumulated phb was extracted using chloroform. it was quantified as crotonic acid in concentrated sulfuric acid (h2so4) by spectrophotometry at 300nm. date syrup is characterized by high levels of total sugars (79.66 g/l) with 31.86 g/l of total reducing sugars. phb accumulation reached its maximum (104.3 ug/ml) after 96 h of incubation at ph 7 and temperature 37°c using tryptophane as the nitrogen source and acid pretreated syrup at a concentration of 8%. hplc analysis on aminex hpx-87h showed that the produced phb from date syrup is characterized by a chromatogram peak with a retention time at 22.5 min. keywords: bacillus paramycoides; bioprocess; date syrup; polyhydroxybutyrate (phb); valorization introduction date palm phoenix dactylifera is a tropical and subtropical tree (chandrasekaran and bahkali, 2013). it is the main crop of algerian saharan agriculture with an average production estimated at 420.290 million tons (bouguedoura et  al., 2015). this production is however accompanied by a substantial loss of large amounts of date during the postharvest processes (abbés et  al., 2011; nancib et  al., 2015). due to their soft texture, the lost dates known as date by-products are not edible and are often discarded (chandrasekaran and bahkali, 2013). they are mainly used for animal feed (majzoobi et al., 2020). fermentation processes employing microoganisms is the most common italian journal of food science, 2022; 34 (3): 48–58 10.15586/ijfs mailto:chekroudzohra@yahoo.fr italian journal of food science, 2022; 34 (3) 49 valorisation of date fruits by-products method for biosynthesis of value products using date products and wastes as raw materials. many eco-friendly products could be derived from date by-products such as organic acids, enzymes, amino acids, biomass (chandrasekaran and bahkali, 2013), bioethanol (ahmad et al., 2021), and biopolymers. omar et al. (2001) used date molasses for the production of polyhydroxybutyrate (phb) using the bacterial strain bacillus megaterium. hence, petrochemical polymers led to severe crisis of the environment with detrimental impact on the ecosystems (mohapatra et al., 2020), and there has been an increasing demand to produce eco-friendly biopolymers synthesized by microbes and plants from inexpensive and renewable sources (narayanan et  al., 2020) like agro-food wastes. phb has attracted much attention in recent years due to its varied properties (thermoplastic and elastomeric), biocompatibility, and biodegradability (keshavarz and roy, 2010). it was reported that phb could be synthesized by many gram-positive and gram-negative bacteria as intracellular carbon and energy reserve material under nitrogen and phosphorus limiting conditions and surplus of carbon source (anderson and dawes, 1990). the main constraint in phb production is accounted to the cost of raw materials, thus the use of cheap carbon sources like agro food wastes cloud be highly significant (singh et al., 2013). the goal of this research was to study a bioprocess technology for the valorization of date fruit by-products to a biodegradable biopolymer and to provide an alternative solution for non renewable fossil resources. the bioprocess is based on the capacity of the bacterial strain bacillus paramycoides to accumulate phb from date syrup. material and methods preparation of date syrup date fruits of the variety “deglet nour” with poor commercial quality were collected during the month of september 2020, from a private factory in the south of algeria specialized in the exportation of dates. the fruits were pitted, washed with distilled water, and cut into small pieces. they were added to hot water at a ratio of 1/2.5 (weight/volume) (chniti et al., 2017). the obtained juice was filtered through a gauze and hand pressed. the juice was then boiled at 70°c for 30 min (el-nagga and abd el-tawab, 2012) until obtaining a concentrated thick, dark syrup. the final crude syrup was stored in sterilized dark bottles at room temperature. treatment of date syrup the obtained crude syrup underwent two types of treatment: centrifugation and hydrolytic treatment. a quantity of 15g of syrup was weighed and diluted in 100 ml of sterile distilled water (ashraf et al., 2015). the obtained solution was centrifuged at 3500 g for 15 min and the supernatant was recovered and filtered using whatman filter paper no.1. for hydrolytic treatment, the obtained filtered supernatant was hydrolyzed by adding 5 ml of 5m or 1.5 n sulfuric acid or 3m hcl to 100 ml of the solution. the whole concoction was incubated at 90°c in water bath for 1h. date syrup was then centrifuged at 3000 g for 15 min (kundu et  al., 1984) to eliminate the formed debris. finally, ph was adjusted to 7 using 1m naoh. at the end of the treatment two phases were formed; a clear black supernatant and a brown pellet. the supernatant was recovered to be used later. crude, centrifuged, and acid hydrolyzed syrups were sterilized by tyndallization to avoid thermal degradation of sugars. physico-chemical characteristics of date syrup ph, alkalinity, and total solids of the obtained syrup were determined according to standard methods (tallon et al., 2005). ph was measured directly using a calibrated ph meter (crison glp21) after agitation of the sample. the alkalinity was determined by diluting 1ml of the sample into 50 ml of distilled water. the solution was then titrated by h2so4 until the ph 4.3. total solids were measured after drying the syrup at 110 ± 5°c. total sugars (t sug) with sucrose and reducing sugars were essayed by an hplc equipped with a refractive index detector and a shodex column (sh1011, 8.0 × 300 nm). total reducing sugars (trs) after date syrup acid treatment was essayed using 3',5-dinitrosalicylic acid (dns) method (gusakov et al., 2011). a volume of 25 ul of dns was firstly added to 25 ul of the diluted sample (20 ul of sample with 925 ul distilled water). the obtained solution was heated for 10 min at 105ºc and cooled for 5 min. distilled water (250 ul) was added to the cold solution and absorbance was read at 540nm against a blank of distilled water. the concentration of reducing sugars was determined from a calibration curve previously prepared from different concentrations of glucose (0.25–5 g/l). primary screening of the bacterial strains producing phb the bacterial strain producing phb from date syrup was isolated from the soil of the botanic garden of skikda university, algeria. pure bacterial colonies isolated from 1 g of soil using the technique of serial dilutions were inoculated on mineral salt medium (msm) agar medium (sharma et  al., 2007) added with 2% glucose as carbon source and incubated at 37°c for 48h. colonies having the capacity to grow on the msm agar medium with glucose were added to ethanolic solution with 3% sudan black b (c29h24n6 224-087 segma) for 30 min and those 50 italian journal of food science, 2022; 34 (3) chekroud z et al. giving dark blue color were considered as positive for phb production (mohd zahari et al., 2012). secondary screening of the bacterial strains producing phb to select the best strain producing phb, the colonies showing positive sudan black staining were re-cultured in msm liquid medium (sharma et  al., 2007) with 2% of glucose. a bacterial inoculum of each strain was prepared by inoculating a loop of the bacterial colony into 100 ml of nutrient broth in 250 ml conical flasks. the inoculated flasks were incubated for 24 h at 37°c with an agitation rate of 150rpm. cells were then collected by centrifugation at 10,000 g for 15 min at 4°c, washed aseptically by sterile distilled water, and resuspended in 250 ml erlenmeyer flasks containing 100 ml of liquid msm mineral medium added with 2% glucose and incubated at 37°c for 72h. the strain showing the best yield of phb accumulation was chosen to test its capacity to accumulate phb using 2% of centrifuged date syrup instead of 2% glucose in msm solid medium. sudan black staining was used to confirm phb production from date syrup. the produced phb bacterial strain from date syrup was then identified on the basis of its 16srrna partial sequence by comparing consensus sequences to a database library of known 16srrna gene sequences in genbank (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) by multiple sequence alignment. optimization of physico-chemical phb production conditions to test the effect of physico-chemical factors on phb production and bacterial cell growth, a bacterial inoculum was firstly prepared as previously described. bacterial cells were recovered by centrifugation at 10,000 g for 15 min at 4°c and washed aseptically with sterile distilled water. they were resuspended in msm liquid medium with 2% of centrifuged date syrup. ph of the medium was adjusted to 7 and the cultures were incubated in a rotary incubator at 37°c and an agitation rate of 150rpm during 24, 48, 72, and 96 h. the effect of aeration was tested by augmenting the agitation rate from 150 rpm to 200 rpm and 300 rpm. the ph was tested at 3, 7, and 8 by adding naoh (2n) or hcl (2n). the cells growth and bioaccumulation of phb from date syrup were also tested at 30°c, 37°c, and 44°c. the nitrogen source in the msm medium (nh4cl) was substituted by ammonium sulfate, yeast extract, beef extract, and peptone, one at a time. the effect of syrup treatment was tested by replacing centrifuged date syrup with crude syrup and acid hydrolyzed syrup with 5m and 1.5 n sulfuric acid or with 3m hcl. finally, the effect of syrup concentration was tested by adding 2, 4, 6, and 8% of 5m h2so4 hydrolyzed date syrup to the medium, one at a time. the experiments were conducted in triplicates. extraction of phb from bacterial cells the technique of boiling chloroform was used. the bacterial cells accumulating phb under different conditions were centrifuged at 4000 g for 10 min. the cells were resuspended in an equivalent amount of 4% nacl and incubated for 1 h at 37°c. the cells pellet was washed with acetone, ethanol, and distilled sterile water to eliminate undesirable elements. the solution was recentrifuged and the supernatant was discarded. the polymer granules were dissolved in boiling chloroform, which was then allowed to evaporate (adwitiya et  al., 2009) to obtain pure phb. quantification of phb the extracted phb was quantified using an uv spectrophotometric analysis. the chloroform extracted phb was converted to crotonic acid. ten milliliters of concentrated sulfuric acid (98%) was added to the chloroform dissolved phb for 15 min. the solution was left for cooling. phb was determined quantitatively as crotonic acid by measuring the absorbance at 300 nm in a uv spectrophotometer using h2so4 solution as blank. standard solution of pure crotonic acid was prepared at different concentrations (0.1–2.0 ug/ml). the quantity of accumulated phb expressed as microgram per milliliter of bacterial cells (ug/ml) was measured by comparing the absorbance of phb converted to crotonic acid with the absorbance of the standard pure crotonic acid concentrations (elsayed et al., 2013). cells growth ten milliliters of the culture medium containing bacterial cells accumulating phb was centrifuged at 10,000 g for 10 min. the supernatant was discarded and the bacterial pellet was washed twice with sterile distilled water. the cells pellet was then scrubbed to a weighing pan and dried at 100°c for 48h. the dried cells were diluted to an appropriate concentration and the absorbance was measured at 600nm. cells’ dry matter was determined according to a standard curve (naheed et  al., 2012) previously generated from cells dry matter concentrations (0.1–1ug/ml). it was expressed as cdw ug/ml. the yield of phb accumulation was calculated as percentage of phb content (ug/ml) per cells dry matter (ug/ml). http://www.ncbi.nlm.nih.gov/blast/blast.cgi italian journal of food science, 2022; 34 (3) 51 valorisation of date fruits by-products identification of phb using aminex hpx-85x. hplc analysis to confirm the synthesis of phb polymers, samples containing phb were analyzed by using an hplc (lachrom elite vwr-hitachi). samples were eluted with 0.014 n h2so4 at a flow rate of 0.7 ml min-1 from an aminex hpx-87h ion exclusion organic acid analysis column (c18 4.6x250) (torrance, ca, usa) preceded by an ion exclusion guard column of aminex hpx-85x. hplc. the produced phb was measured as crotonic acid dissolved in concentrated h2so4. crotonic acid and pure phb digested into crotonic acid using concentrated h2so4 were used as standards. statistical analysis all the experiments were conducted in triplicates. the results expressed as mean ± standard error were analyzed by one way anova analysis of variance (one-way anova), followed by pairwise comparisons using the fisher’s least significant difference (lsd) post hoc test. the statistical significance was considered at p < 0.05. data analysis was carried out using statistica 10 software (statsoft, inc.). results date syrup characterization date syrup extracted from “deglet nour” was characterized by high levels of total sugars (t sug) (79.66g/l) and a total reducing sugars (trs) rate of 31.86 g/l (table 1). trs rate increased to 78.86 g/l,72.96g/l, and 72.2 g/l after treatment of date syrup with 5mh2so4, 3mhcl, and 1.5 m h2so4, respectively (table 1). screening of the bacterium producing phb from date syrup a total of 20 bacterial strains were isolated from the soil of the botanic garden of skikda university. the preliminary screening of phb producing strains was further identified by a sudan black method. nine isolates (bg1-bg9) showed a black-blue color when stained with sudan black b (figure 1), which indicates that they are positive phb producing. the quantitative screening in msm liquid medium with 2% of glucose as carbon source revealed that the highest quantity of phb was accumulated by the strain bg5 (95.67 ug/ml) followed by the strain bg2 (83.92 ug/ml) after 72h of incubation at 37°c (figure 2). the strain showing the highest level of phb accumulation was tested for its table 1. physico-chemical characteristics of date syrup. parameters ph ts(g/k) ts% alkalinity g/l t sug content sucrose content trs content before date syrup acid treatment trs content after date syrup acid treatment (g/l) date syrup (g/l) (g/l) (g/l) 5m h 2 so 4 3m hcl 1.5 h 2 so 4 5.63 713.8 79 15.8 79.66 47.8 31.86 78.86 72.96 72.2 ts: total solids; ts%: percentage of total solids; t sug: total sugars; trs: total reducing sugars before date syrup acid pretreatment; 5m h 2 so 4 : total reducing sugars after date syrup pretreatment with 5m h 2 so 4 ; 3m hcl: total reducing sugars after date syrup pretreatment with 3m hcl, 1.5m h 2 so 4 : total reducing sugars after date syrup pretreatment with 1.5 m h 2 so 4 figure 1. screening of some bacterial isolates on msm solid medium with date syrup as carbon source using sudan black staining. (a) before staining, (b) after staining (greenblue colonies). 1:bg1; 2:bg2; 3:bg3; 4:bg4; 5:bg5; 6:bg6; 7:bg7;8:bg8; 9:bg9; bg1-bg9: the nine isolated bacterial strains showing positive phb production using glucose as carbon source. (a) (b) 52 italian journal of food science, 2022; 34 (3) chekroud z et al. ability to accumulate phb from date syrup instead of glucose. sudan black staining confirmed the capacity of the strain bg5 to produce phb from date syrup. bg5 was identified on the basis of its partial 16srrna sequencing by comparing consensus sequences to a database library of known 16srrna gene sequences in genbank (http://www.ncbi.nlm.nih.gov/blast/ blast.cgi). analysis showed high identity of bg5 with bacillus paramycoides mccc 1a04098, which has a partial 16s rrna sequence ncbi assessing number nr_157734.1. effect of incubation period on phb production the results demonstrated in figure 3 revealed that phb yield was directly proportional with the incubation period. it increased gradually from 22.18% after 24h of incubation to 26.36% after 72h with a slight increase after 96h (26.65%), where it reached its maximum with a maximum phb accumulation (64.14 ug/ml) and a maximum cell growth (240.69 ug/ml). effect of aeration rate the aeration was monitored by agitation of the bacterial cultures. our results (figure 4) revealed that the phb yield increased with the increase in the agitation rate from 150 rpm to 300 rpm. the maximum yield (36.22%) with a maximum phb production (80.17 ug/ml) and a maximum cell growth (221.35 ug/ml) were recorded at 300 rpm. effect of ph the rate of phb production was tested in acid (ph 3), alkaline (ph 8), and neutral (ph 7) media. it was observed that the best rate of phb accumulation (27.61%) was obtained at ph 7 with a phb quantity of 75.66 ug/ml and a biomass of 274 ug/ml. it decreased significantly beyond ph 7 (figure 5). figure 4. effect of aeration on phb accumulation. results are calculated as mean of tri-triplicates ± standard error; different letters indicate significant differences using anova test followed by the post-hoc fisher test (lsd) at p < 0.05. p = 0.000 for phb content, p = 0.001 for cdw. figure 2. phb content in the nine isolated bacterial strains. results are expressed as mean of tri-replicates±standard error. bg1, bg2, bg3, bg4, bg5, bg6, bg7, bg8, and bg9: the nine isolated bacterial strains showing positive phb productionusing glucose as carbon source. figure 3. the effect of the incubation period on phb accumulation. results are calculated as mean of tri-triplicates ± standard error; different letters indicate significant differences using anova test followed by the post-hoc fisher test (lsd) at p < 0.05. p = 0.000 for both phb and cdw. figure 5. effect of ph on phb accumulation. results are calculated as mean of tri-triplicates ± standard error; different letters indicate significant differences using anova test followed by the post-hoc fisher test (lsd) at p < 0.05. p = 0.0001 for both phb content and cdw. http://www.ncbi.nlm.nih.gov/blast/blast.cgi http://www.ncbi.nlm.nih.gov/blast/blast.cgi italian journal of food science, 2022; 34 (3) 53 valorisation of date fruits by-products effect of temperature phb synthesis increased from 21.67% at 30°c to 28.39% at 37°c. it decreased significantly at 44°c (9.40%). maximum phb quantity and maximum cell biomass were recorded at 37°c (76.85 ug/ml and 270.67 ug/ml, respectively) (figure 6). effect of various nitrogen sources using tryptophane as a nitrogen source with date syrup gave the best phb content (63.13 ug/ml) and cells biomass (226.33 ug/ml) followed by peptone (50.61 ug/ml phb and 175.33 ug/ml cdw). there was, however, a decrease in cells biomass growth and phb accumulation when we used inorganic nitrogen source (nh4)2so4 (44.25 ug/ml phb and 153.67 ug/ml cdw) (figure 7). effect of date syrup treatment the effect of crude, centrifuged, and hydrolyzed date syrup was tested. it was noted that hydrolyzed date syrup with 5mh2so4 gave the best yield of phb production (34.32%) with maximum phb content (89.35 ug/ml) and maximum cells growth (260.33 ug/ml). there was however a significant decrease in phb yield with crude syrup (11.06%) where the phb content decreased to 27.68 ug/ml and the cells growth to 250.33 ug/ml (figure 8). effect of date syrup concentration the effect of syrup concentration on phb accumulation by bacillus paramycoides is demonstrated in figure 9. the phb yield was directly proportional with the concentration of date syrup. it increased c a b e d d a c b e 0 5 10 15 20 25 30 35 40 0 50 100 150 200 250 300 1. 5 m h 2s o 4 5 m h 2s o 4 3m h c l c ru de d at e sy ru p c en tr if ug ed sy ru p % p h b type of treatment phb cdw % phb ph b (u g/ m l ) c d w (u g/ m l ) d e b c a e d b c a 0 5 10 15 20 25 30 35 0 50 100 150 200 250 (nh4)2so4 meat extract peptone yeast extract tryptophane ) l m/gu( w d c) l m/gu( b hp phb cdw % phb % ph b nitrogen source figure 6. effect of temperature on phb accumulation. results are calculated as mean of tri-triplicates ± standard error; different letters indicate significant differences using anova test followed by the post-hoc fisher test (lsd) at p < 0.05. p = 0.0001 for both phb content and cdw. figure 8. effect of date syrup treatment on phb accumulation. results are calculated as mean of tri-triplicates ± standard error; different letters indicate significant differences using anova test followed by the post-hoc fisher test (lsd) at p < 0.05. p = 0.000 for both phb content and cdw. figure 7. effect of nitrogen sources on phb accumulation. results are calculated as mean of tri-triplicates ± standard error; different letters indicate significant differences using anova test followed by the post-hoc fisher test (lsd) at p < 0.05. p = 0.000 for both phb content and cdw. figure 9. effect of date syrup concentration on phb accumulation. results are calculated as mean of tri-triplicates ± standard error; different letters indicate significant differences using anova test followed by the post-hoc fisher test (lsd) at p < 0.05. p = 0.000 for both phb content and cdw. 54 italian journal of food science, 2022; 34 (3) chekroud z et al. crotonic acid (figure 10c) revealed that the three chromatograms share the same peak with a retention time of 22.5 min. this confirms the synthesis of phb by bacillus paramycoides using date syrup as the carbon source. discussion the agro-food industry produces large amounts of wastes. many strategies have been established in europe and other countries of the world for the valorization of food waste streams and the recovery of biomolecules (baiano, 2014). in this study, an isolated bacterial strain from the botanic garden of skikda university (bg5) showing the best phb accumulation using 2% of glucose (95.67 ug/ml) (figure 2) was confirmed by sudan black staining to accumulate phb using 2% of date syrup, and it was identified as bacillus paramycoides. many bacterial strains have been confirmed to accumulate phb from agro wastes like date syrup (mostafa et  al., 2020), banana peel, sugarcane bagasse, corn cob, and teff (eragrostistef) straw (getachew and woldesenbet, 2016). a wide range of phb producer bacillus species are recorded in the literature (mohapatra et  al., 2017). bacillus pumilus, bacillus megaterium, and bacillus subtilis have been reported to produce phb from from 26.06% at 2% of date syrup to 32.62% at 8%. the increase of phb yield was accompanied with an increase of the phb content and cell growth. we obtained 104.3 ug/ml of phb and 319.73 ug/ml of cells’ dry matter at 8% of syrup. the physico-chemical conditions were monitored in triplicates. results were expressed as mean± standard error. variance analysis (anova) showed that there was a very high significant difference between all the conditions (pr < 0.05). statistically, the multiple comparisons of the means performed with the lsd post hoc test indicated that the highest amount of phb production was obtained after 96h of incubation at 37°c and ph 7, using tryptophane as nitrogen source and 8% of hydrolyzed syrup by 5m h2so4 as the carbon source. hplc analysis of produced phb the phb produced by bacillus paramycoides using date syrup was identified by an hplc on aminex hpx-87h technique. the obtained chromatograms of re-crystallized crotonic acid (figure 10a), pure phb converted to crotonic acid (figure 10b), and phb produced by bacillus paramycoides from date syrup converted to figure 10. aminex-hplc analysis of phb. (a) standard crotonic acid (with retention time of 22.5min), (b) standard pure phb (with retention time of 22.5 min), (c) produced phb from date syrup (with retention time of 22.5min). (c) (b)(a) italian journal of food science, 2022; 34 (3) 55 valorisation of date fruits by-products agro-food wastes (singh et  al., 2013; vu et  al., 2021; werlang et  al., 2021). the bacterial aptitude for utilizing complex carbon sources like agro-food wastes varies according to the material biochemical composition and the enzymes involved by the bacterium (belal, 2013). the date syrup used in our approach contains high levels of sugars (79.66 g/l) with 31.86 g/l of reducing sugars (table 1), which makes it a promising source for phb production since bacteria accumulates phb under nutrient stress conditions and surplus of carbon source (blunt et al., 2018). the optimization of fermentation parameters is among the strategies now used to improve phb production (gurieff and lant, 2007). the yield of phb accumulation reached its maximum after 96h of incubation (26.65%). the best incubation period for phb accumulation depends on the bacterial strain. the increase in the phb content (64.14 ug/ml) and cells growth (240.7 ug/ml) at this incubation period (figure 3) is in accordance with the results recorded by gomaa (2014) and mostafa et al. (2020). some authors however reported that the best incubation periods are 48h (thapa et al., 2018) and 72 h (singh et al., 2013). agitation is an important factor for phb production. it helps in mixing the oxygen, the heat and the nutrients and facilitates the distribution of air in the nutrient broth so that it increases the liquid-gaz contact area (mantzouridou et  al., 2002). this research revealed that increasing the agitation rate to 300 rpm increased the cells growth (221.35 ug/ml) and the phb content (80.17 ug/ml) (figure 4). slow agitation rate leads to an increase in the viscosity of the culture medium and a reduction of the mass transfer (bandaiphet and prasertsan, 2006). ph of the medium is a crucial factor for the activity of the polymerase enzyme responsible of phb production (bhagowati et al., 2015). it is clear that ph 7 is more favorable for phb production where optimum phb accumulation (75.66 ug/ml) and optimum cells growth (274 ug/ml) were obtained (figure 5). many authors have reported that ph 7 is the best ph for phb accumulation using different bacterial strains (mostafa et  al., 2020; singh et  al., 2013). phb synthesis decreased at acidic and alkaline ph. the incubation temperature had a significant effect on phb synthesis; this is due to the polymerase enzyme involved in the phb polymerization, highly affected by the temperature changes—mainly the high temperatures (getachew and woldesenbet, 2016). the maximum phb yield with a maximum cell growth and phb accumulation were achieved at 37°c (figure 6). aly et  al. (2013) reported the same results, using the bacterial strain bacillus cereus. singh et  al. (2013), however, recorded that maximum phb production by bacillus subtilis was obtained at 40°c. the nitrogen source affects phb accumulation and bacterial cells growth. bacteria accumulate phb under stress conditions of nitrogen (hungund et  al., 2013). in our study, organic sources mainly tryptophane enhanced phb accumulation by bacillus paramycoidesand cells growth (63.31 ug/ml and 226.33 ug/ml, respectively) in comparison with inorganic sources (nh4)2so4 (figure 7). this may be explained by the fact that organic sources are considered as precursors of amino acids and bacterial growth factors (patel et al., 2017). ammonium sulfate was reported by kritika et al. (2016) and singh et al. (2013) to be the best nitrogen source for phb accumulation high contents of phb (89.35 ug/ml) and cells biomass (260.33 ug/ml) were achieved using acid hydrolyzed date syrup in comparison with centrifuged and crude syrup (figure 8). this is due to the low levels of simple sugars in non treated syrup in comparison with acid-treated syrup (mcadam et al., 2020). sucrose hydrolysis increased with the increase in sulfuric acid concentration (sen et  al., 2019). the total rate of reducing sugars increased from 31.86 g/l to 78.86 g/l, 72.96 g/l, and 72.2 g/l after treatment of date syrup with 5mh2so4, 3mhcl, and 1.5 m h2so4, respectively (table 1). the same results were reported by gomaa (2014) where acid preatreated molasses enhanced phb accumulation in comparison with untreated molasses. the carbon source is the major factor affecting phb production costs (aljuraifani et al., 2019; gomaa, 2014). phb production and cells growth are directly proportional to the syrup concentration (figure 9). they reached their maximums at 8% (104.3 ug/ml and 319.73 ug/ml, respectively). this is due to the high content of sugar in concentrated date syrup. the use of the renewable resources depends on the nature of the complex material and the hydrolytic capacity of the bacterium enzymes (belal, 2013). gabr (2018) revealed that the best levels of phb were accumulated by bacillus sp. at 8% of date syrup. therefore, the selection of an economically cost-effective carbon source is the key determining the final product market costs (mcadam et  al., 2020). date syrup by-products are abundant and inexpensive sources for phb production that may decrease the costs of bioplastic production. phb is traditionally identified using hplc on aminex hpx-87h technique (karr et  al., 1983). the technique is based on the conversion of phb into crotonic acid by concentrated sulfiric acid. crotonic acid is measured in samples containing from 0.01 to 14 ug of phb. crotonic acid and pure phb were used as standards. chromatograms with the same peaks at the retention time 22.5 min were obtained (figure 10), with crotonic acid, pure phb, and produced phb using bacillus paramycoides and date syrup as carbon source which 56 italian journal of food science, 2022; 34 (3) chekroud z et al. confirms the conversion of date syrup into phb by bacillus paramycoides. conclusion date syrup, which is an inexpensive by-product, is a promising source for the production of phb biopolymer. the latest is successefully produced using a bacterial strain isolated from soil, bacillus paramycoides, and date syrup as carbon source. the best contents of phb were reached after 96 h of incubation at 37°c, an agitation rate of 300 rpm, and ph 7 using acid preatreted date syrup at a concentration of 8%. furthemore, more investigations are required to characterize the phb chemical structure to determine the possibility of producing phb at industrial levels from date syrup. acknowledgement the authors are grateful to mr nasser zaydi, the chief of the hygiene laboratory of the state of skikda, the engineers of the laboratory of animals biotechnologies, national center for biotechnology, constantine, algeria, pr. luis isidoro romero garcia, dr. kaoutar aboudi, and miss hind zine el abidine from the faculty of sciences, the university of cadiz , spain. authors contribution all the authors contributed in the conception and design of the study. bacterial isolation and phb optimization conditions were performed by leila djerrab and zohra chekroud. amer rouabhia conducted hplc analysis. mohamed abdesselem dems realized the statistical analysis. mustapha adnane smadi provided necessary material and helped in realizing spectrophotometric analysis. imane attailia helped in the manuscript redaction. faiçal djazy provided some chemical products. the first draft of the manuscript was written by zohra chekroud and leila djerrab. all the authors read and approved the final manuscript. conflict of interest there is no conflict of interest to declare. references abbès, f., bouaziz, m.a., blecker, c., masmoudi, m., attia,h. and besbes, s., 2011. date syrup effect of hydrolytic enzymes (pectinase/cellulase) on physico-chemical characteristics, sensory and functional properties. lebensmittel-wissenschaft & technologie 44(8):1827–1834. https://doi.10.1016/j.lwt.2011.03.020 adwitiya, p., ashwini, p., avinash, a.k., badri, r., kajal, d., vomsi, p. and srividya, s., 2009. mutagenesis of bacillus thuringiensis iam 12077 to increase the production of poly backslash betahydroxybutyrate (phb). turkish journal of biology 33(3): 225–223. https://doi.org/10.3906/biy-0808-10 aljuraifani, a.a., berekaa, m.m. and ghazwani, a.a., 2019. bacterial biopolymer (polyhydroxyalkanoate) production from low-cost sustainable sources. microbiology open 8(6):e00755. https://doi.org/10.1002/mbo3.755 ahmad, a., naqvi, a., jaskani, m.j.,waseem, m.,ali, e.,khan, i.a., manzoor, m.f., siddeeg, a. and aadil, r.m., 2021. efficient utilization of date palm waste for the bioethanol production through saccharomyces cerevisiae strain. food sciences & nutrition 9(4):2066–2074. https://doi.org/10.1002/fsn3.2175 aly, m.m., albureikan, m., el rabey, h. and kabli, s.a., 2013. effects of culture conditions on growth and poly –beta-hydroxybutyric acid production by bacillus cereus mm7 isolated from soil samples from saudi arabia. life sciences journal 10(4):1884–1891. anderson, a.j. and dawes, e.a., 1990. occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. microbiology review 54(4):450–472. https://doi. org/10.1128/mr.54.4.450-472.1990 ashraf, s., ali, s. and ul-haq, i., 2015. acidic pre-treatment of sugarcane molasses for citric acid production by aspergillus niger ng-4. international journal of current microbiology and applied sciences 4(6):584–595. baiano, a., 2014. recovery of biomolecules from food wastes: a review. molecules 19(9):14821–14842. https://doi.org/10.3390/ molecules190914821 bandaiphet, c. and prasertsan, p., 2006. effect of aeration and agitation rates and scale-up on oxygen transfer coefficient, kla in exopolysaccharide production from enterobacter cloacae wd7. carbohydrate polymers 66(2):216–228. https://doi.org/10.1016/ j.carbpol.2006.03.004 belal, e.b., 2013. production ofpolyβ -hydroxybutyric acid (phb) by rhizobium elti and pseudomonas stutzeri. current research journal of biological sciences 5(6):273–284. https://doi. org/10.19026/crjbs.5.5429 bhagowati, p., pradhan, s., dash, h.r. and das, s., 2015. production, optimization and characterization of polyhydroxybutyrate, a biodegradable plastic by bacillus spp. biosciences biotechnology and biochemistry 79(9):1454–1463. https://doi.org/10.1080/091 68451.2015.1034651 blunt, w., levin, d.b. and cicek, n., 2018. bioreactor operating strategies for improved polyhydroxyalkanoate (pha) productivity. polymers 10(11):1197. https://doi.org/10.3390/polym10111197 bouguedoura, n., bennaceur, m., babahani, s. and benziouche, s.e., 2015. date palm status and perspective in algeria. in: elkhayri, j.a., jain, s.m. and johnson, d.v. 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n s codon italian journal of food science, 2023; 35 (3): 99–114 issn 1120-1770 online, doi 10.15586/ijfs.v35i3.2302 99 p u b l i c a t i o n s codon quality and stability of different seafood products treated with high hydrostatic pressure (hpp) intended for raw consumption ana cristina de aguiar saldanha pinheiro1, silvia tappi1,2*, giacomo braschi1,2, jessica genovese1, francesca patrignani1,2, pietro rocculi1,2 1department of agricultural and food science, alma mater studiorum, university of bologna, campus of food science, piazza goidanich 60, cesena (fc), italy; 2interdepartmental centre for agri-food industrial research, alma mater studiorum, university of bologna, campus of food science, via ravennate 933, cesena (fc), italy *corresponding author: silvia tappi, department of agricultural and food science, alma mater studiorum, university of bologna, campus of food science, piazza goidanich 60, cesena (fc), italy email: silvia.tappi2@unibo.it received: 15 november 2022; accepted: 23 june 2023; published: 23 august 2023 © 2023 codon publications open access paper abstract the consumption of raw fish has rapidly increased in recent years, but being a highly perishable product, it is characterised by a very short microbiological shelf life. high hydrostatic pressure (hpp) processing is a non thermal technology has emerged recently as a promising alternative to thermal processing for food pasteurization capable of maintaining fresh-like characteristics and nutritional value. however, the induced changes in product quality should be assessed carefully. the present research aimed to investigate the effect of hpp on different seafood products, namely grey mullet, tiger prawn and rose shrimp, intended for raw consumption. three pressure levels (400, 500 and 600 mpa) were applied for 10 min. during refrigerated storage, microbiological quality, chemical parameters, colour and texture and fat oxidation were analysed. results showed that the application of lower pressure was able to inactivate e. coli, pseudomonas and/or positive coagulase staphylococci; however, they were able to recover during storage. in addition, the application of 600-mpa pressure extended the microbiological shelf life by up to 30 days. for all samples, general whitening occurred while the texture was affected in a different way for the three considered species. fat oxidation was only minimally affected and remained quite low during storage. keywords: grey mullet, microbiological inactivation, rose shrimp, shelf life, tiger prawn introduction the consumption of raw fish has rapidly increased in recent years, also in the areas where it was not a traditional habit, because of not only changes in food taste but also to the adoption of culinary traditions of other countries. sushi and sashimi which are typically oriental specialities are becoming increasingly popular in european countries as well. moreover, the use of low-temperature cooking and processing, such as cold smoking, is spreading fast (brutti et al., 2010). these new habits have increased microbiological risks for fish product consumption. moreover, seafood products are highly perishable, their microbiological shelf life is very short and, in order to sell a fish product to be consumed raw, a strategy to increase its shelf life could increase its marketability. therefore, a non-thermal technology able to reduce microbial load is highly necessary. high-pressure processing (hpp) is a non-thermal technology that has recently emerged as a promising alternative to thermal processing for food pasteurization mailto:silvia.tappi2@unibo.it 100 italian journal of food science, 2023; 35 (3) pinheiro acas et al. european union (eu) legislation requires seafood products to be frozen for at least 24 h before raw consumption, hpp was applied to frozen-thawed products. three pressure levels were applied, and microbiological quality, safety, chemical parameters, colour and texture were analysed during the refrigerated storage. materials and methods preparation of fish samples grey mullet (mugil cephalus) striped prawn (melicertus kerathurus) and deep-water rose shrimp (parapenaeus longirostris) were fished in the adriatic sea. products were fast-frozen in an industrial blast chiller at a temperature of -18°c and kept for 24 h at economia del mare (cesenatico, italy). thawing was carried out at 4°c for 16  h; then the seafood samples were subjected to mechanical deboning and shell removal. flesh was manually cut into pieces and packaged in polyethylene terephthalate (pet) trays (vr11-6t-t1, technopress srl., fc, italy) containing six mono-portions of 15–20 g approximately, each sealed under vacuum with a high barrier pet film (thickness 40 µm; oxygen transmission rate [otr] 60 cm3/m2/24 h/bar; 4°c, 0% rh; cryovac sealed air corp., nj, usa). hpp treatment the vacuum-packed samples were subjected to hpp treatment performed by hpp italia s.r.l (parma, italy). the samples were placed in a 350-l chamber, filled with water and subjected to 400-, 500or 600-mpa pressure for 10 min. an untreated sample was used as a control. the samples were coded using initial of the species (m for grey mullet, p for striped prawn and s for rose shrimp) and 0-, 400-, 500and 600-mpa pressure was applied. for each treatment, 24 packages were prepared. storage after treatment, the samples were stored in the laboratories of the campus of food science of the university of bologna at 2 ± 1°c. during storage, the samples were subjected to analytical determinations after 0, 1, 6, 9, 14, 21, 28 and 35 days. storage duration was determined for each sample based on the results of microbiological analysis, considering the end of the shelf life when reaching a microbial load of 6 log cfu/g referred to total mesophilic bacteria. three different packages were used for each hpp treatment at each storage period. capable of maintaining fresh-like characteristics and nutritional value. application of pressure higher than 300 mpa for a few minutes at room temperature has shown to significantly reduce the initial microbial load in many fish species (truong et al., 2015). the extent of microbial inactivation depends not only on treatment parameters, such as pressure level, holding time and temperature, but also on the characteristics of microflora in the product (truong et al., 2015). the inactivation of microorganisms by hpp is the result of a combination of factors, including changes in cell membranes, cell walls, proteins and enzyme-mediated cellular functions (campus, 2010). cell membranes are the primary site of pressure-induced damage, with consequent alterations of cell permeability, transport systems, loss of osmotic responsiveness, organelle disruption and inability to maintain intracellular ph. in general, gram-negative bacteria and cells in the growth phase are more sensitive than gram-positive bacteria and cells in the stationary phase. nevertheless, investigations have shown that cell disruption is more dependent on the geometry of the bacteria, rather than gram-type. for example, morphological changes in the rod-shaped escherichia coli and pseudomonas aeruginosa were observed whereas staphylococcus aureus (cocci) was more resistant to pressure. however, in complex matrices such as food, the desired effect on microbial inactivation may also produce physical and biochemical changes, which may affect the product properties negatively. the denaturation of proteins in fish muscle could cause significant changes of important parameters for consumer acceptability. in particular, the application of high pressure is known to lead to a cooked appearance (matser et al., 2000), which could be specifically detrimental in products intended for raw consumption. moreover, the effect of protein structure on enzymatic activity can lead to variations in the textural properties of seafood products not only after the treatment but also during refrigerated storage. the effect of hpp has been studied in a variety of fish and seafood matrices, but results are very variable depending on process parameters as well as specific product characteristics (truong et al., 2015). in a product aimed for raw consumption, microbiological quality is of paramount importance throughout the shelf life; however, the effect of hpp on quality could lead to undesirable changes. the aim of the present research was to investigate the effect of hpp treatment on different types of seafood products, that is, grey mullet, tiger prawn and rose shrimp, intended for raw consumption. because the italian journal of food science, 2023; 35 (3) 101 effect of high hydrostatic pressure on different seafood products for each sample and storage time, an average of at least 15 measurements was calculated. texture the texture was evaluated with a texture analyser model ta.hdi 500 (stable micro systems, godalming, uk) equipped with a 25-kg load cell. briefly, 15 g of each sample was inserted in a cylindrical cup (diameter of 3  cm) and a piston was used to compress the sample at 1 mm/s up to 50% of its height. the pressure was held for 60 s. maximum force was considered as hardness (force, f1 [newton, n]) and force at the end of the compression was considered as index of resistance to compression (force f2 [n]). the texture was evaluated with at least six replicates for each sample. peroxide value (pv) lipids were extracted from 25 g of sample with a method described by bligh and dyer (1959). the peroxide value was determined by the ferrothiocyanate method (chapman and mackay, 1949). results were expressed as the amount of oxygen (o2) per kilogram of fat/oil (meq o2/kg fat/oil). the analysis was performed with at least triplicates for each sample. statistical analysis the significance of differences was tested by the analysis of variance (anova) using tukey’s honestly significant difference (hsd) test as a post-hoc test (p < 0.05). statistical analysis was conducted with statistica 8.0 for windows. results and discussion microbial inactivation the effects of hpp treatments (400, 500 and 600 mpa), compared to the untreated controls, on the microbiological quality of packaged grey mullet, striped prawn and rose shrimp are reported in tables 1, 2 and 3, respectively. in all tested conditions, salmonella spp. and listeria monocytogenes were not detected during the shelf life of the considered products. in addition, coagulase positive staphylococci were not found in untreated and hpp-treated samples of grey mullets and striped prawn. in general, the application of hpp treatments increased the microbiological shelf life of the considered products, and the inactivation effect became more severe with increased pressure. the microbiological threshold to define product shelf life was fixed analytical determinations microbiological analyses microbiological analyses were performed on untreated grey mullet, striped prawn and red shrimp, and the samples were treated with 400-, 500and 600-mpa pressure. immediately after hpp treatments for the stated storage period, all the samples were investigated for the presence of salmonella spp. and listeria monocytogenes according to en iso 6579-1:2017/a1:2020 and iso 11290-1:2017. microbial groups considered in this research were total mesophilic bacteria (tmb), lactobacillus spp., pseudomonas spp., sulphite-reducing anaerobic bacteria, total coliforms, e. coli, and coagulase positive staphylococci. in all, 10 g of samples were serially diluted using physiological saline solution (0.9% nacl), and appropriate inoculum was included or spread in different selective culture media, such as plate count agar (pca; oxoidthermofisher, milan, italy) for tmb; de man, rogosa and sharpe agar (mrs; oxoid-thermofisher) supplemented with cycloheximide (0.2% p/v) for lactobacillus spp.; selective pseudomonas agar base (pab; oxoid-thermofisher) for pseudomonas spp.; reinforced clostridial agar (rca; oxoid-thermofisher) for sulphite-reducing anaerobic bacteria, violet red bile agar (vrba; oxoid-thermofisher) supplemented with 4-methylumbelliferyl-β-d-glucuronide (mug; oxoid-thermofisher) for total coliforms and e. coli, respectively; and baird parker agar (bp) for coagulase positive staphylococci. plates were incubated for 24/48 h at 30°c for pseudomonas spp. (pa) and 37°c for lactobacilli, sulphite-reducing anaerobic bacteria, total coliform, e. coli and coagulase positive staphylococci. sulphite-reducing anaerobic bacteria were incubated under anaerobic conditions using a gas generating kit (oxoid-thermofisher). ph values ph values of the samples homogenised for 60 s with an ultraturrax (t-25, ika, germany) with distilled water (sample–water ratio: 1:2 w/w) were assessed with a ph meter (crison, barcellona). the analysis was performed at least in triplicate for each sample. colour colour parameters lightness (l*), redness (a*) and yellowness (b*) were measured with a spectrophotocolorimeter model colorflex™ (hunterlab, reston, va, us). the tristimulus l*, a*, b* measurement mode (international commission on illumination [cie], 1976) was used. the hue angle (h°) was calculated as follows: 1 btan ah 360 2π ∗ − ∗ = ×� 102 italian journal of food science, 2023; 35 (3) pinheiro acas et al. table 1. evolution of microbial cell loads (log cfu/g) of total mesophilic count (tmc), lactobacillus spp., pseudomonas spp., total coliforms, sulphite-reducing anaerobic bacteria (ab), and e. coli during the refrigerated storage of packaged grey mullet (mugil cephalus) flesh with applied high hydrostatic pressure (hpp) treatments (400, 500 and 600 mpa).     mullet log cfu/g m-0 m-400 m-500 m-600 days of storage 0 d tmc 4.67 ± 0.43 <1** <1 <1 lactobacillus spp. 3.18 ± 0.33 <1 <1 <1 sulphite-reducing ab 4.20 ± 0.54 <1 <1 <1 pseudomonas spp. 4.45 ± 0.22 <1 <1 <1 total coliforms 2.56 ± 0.38 <1 <1 <1 e. coli 1.12 ± 0.10 <1 <1 <1 2 d tmc 5.14 ± 0.44 <1 <1 <1 lactobacillus spp. 3.30 ± 0.29 <1 <1 <1 sulphite-educing ab 5.15 ± 0.51 <1 <1 <1 pseudomonas spp. 5.20 ± 0.45 <1 <1 <1 total coliforms 1.80 ± 0.12 <1 <1 <1 e. coli  1.22 ± 0.10  <1 <1 <1 6 d tmc 6.22 ± 0.52 <1 <1 <1 lactobacillus spp. 4.00 ± 0.28 <1 <1 <1 sulphite-reducing ab 5.54 ± 0.46 <1 <1 <1 pseudomonas spp. 5.27 ± 0.35 <1 <1 <1 total coliforms 1.85 ± 0.16 <1 <1 <1 e. coli 1.50 ± 0.20 <1 <1 <1 12 d tmc -* 5.67 ± 0.31 <1 <1 lactobacillus spp. <1 <1 <1 sulphite-reducing ab <1 <1 <1 pseudomonas spp. 2.46 ± 0.29 <1 <1 total coliforms <1 <1 <1 e. coli    <1 <1 <1 19 d tmc 7.52a ± 0.43 4.22b ± 0.41 2.20c ± 0.23 lactobacillus spp. 1.06 ± 0.32 <1 <1 sulphite-reducing ab <1 <1 <1 pseudomonas spp. 5.11 ± 0.29 <1 <1 total coliforms <1 <1 <1 e. coli   <1 <1 <1 32 d tmc 5.55a ± 0.42 3.10b ± 0.33 lactobacillus spp. <1 <1 sulphite-reducing ab <1 <1 pseudomonas spp. <1 <1 total coliforms <1 <1 e. coli   <1 <1 *not analysed because the microbiological threshold fixed at 6 log cfu/g was reached in the previous time of analysis. **under detection limit. in the same row, values with different superscript letters are significantly different (p < 0.05). italian journal of food science, 2023; 35 (3) 103 effect of high hydrostatic pressure on different seafood products as an increased yield of the shucking process of bivalves and crustaceans (patterson, 2014). in general, according to the literature, hpp between 150 mpa and 450 mpa is commonly applied on fish samples (perez-won et al., 2020) because higher pressure, aimed to increase microbial inactivation, is generally associated with significant changes in physicochemical, texture and sensory properties of products, such as increase in discoloration, cooked appearance or lipid oxidation (truong et al., 2015). however, the data resulting from this research evidenced that hpp treatment at 400 mpa is not able to increase significantly the shelf life of the considered products. although this level of pressure seemed adequate to inactivate e. coli during the shelf life of the considered fish samples, other microbial groups, involved in spoilage and safety issue, such as pseudomonas or positive coagulase staphylococci, were able to retrieve during storage, highlighting the critical issue of viable but not culturable (vbnc) cells. although the efficiency of microbial inactivation is influenced by various factors, such as food matrix characteristics and food processing parameters, the physiological diversity within a microbial population also has to be taken into consideration, especially in the validation of the effectiveness of a treatment on a specific food product (patrignani et al., 2019). moreover, a further consideration is required in relation to the biogenic amines, potentially present in these types of products. in fact, although histamine was not detected, the application of 400–600-mpa hpp appears to reduce several microbial groups that are able to produce biogenic amines, such as lactic acid bacteria and coliforms, reducing the risk of production of these indicators of food safety and quality indices (borges et al. 2023). pseudomonas spp., strict aerobic bacteria, whose growth decreased in vacuum, was able to recover in grey mullet and striped prawn samples treated at 400 mpa during their shelf life. pseudomonas spp., also having a psychrotrophic behaviour and able to produce specific h2s off-flavours, could have a negative impact to produce specific proteases, which could potentially affect the textural properties of food matrix. ph value the initial ph values measured in grey mullet, tiger prawn and rose shrimp samples according to hpp treatment and during storage are reported in table 4. the initial values for the untreated samples were 6.4 ± 0.1, 7.2 ± 0.01, 7.6 ± 0.03 for grey mullet, tiger prawn and rose shrimp, respectively. while ph values for grey mullet were consistent with the literature (tsogas et al., 2019), for prawn and shrimp, these values were slightly higher, compared to literature (bindu et al., 2013; kaur et al. 2013). at the attaining of 6 log cfu/g for total mesophilic count (tmc) even if, according to regulation 2073/2005, other important microbiological criteria, such as cell load of escherichia coli and positive coagulase staphylococci, were considered in the data discussion. more specifically, the detected microbiological data for grey mullets, untreated and in relation to the pressure applied and storage period are reported in table 1. as shown, the control sample was spoiled within 6 days of storage at 2°c, reaching a cell load of total mesophilic bacteria of 6.22 log cfu/g with a corresponding level of total coliforms of 1.8 log cfu/g. in contrast, application of 400, 500 and 600 mpa pressure prolonged product shelf life to 12, 19 and 32 days, respectively. the application of hpp treatments ranging between 400 mpa and 600 mpa decreased the cell loads of e. coli, compared to the untreated sample, under the detection limit of 1 log cfu/g. pseudomonas spp. was strongly affected by the level of applied hpp since cell load of 2.4 cfu/g were detected at 400-mpa pressure after 12 days of treatment to further increase on the 19th day of storage. in all the other treated samples, and for each time of sampling considered, pseudomonas spp. was always under the detection limit (<1 log cfu/g). regarding untreated striped prawn (table 2), the total mesophilic bacteria reached the threshold level of 6.0 log cfu/g after 6 days of refrigerated storage, while the application of hpp pressure of 500 mpa and 600 mpa determined a significant increase in the product’s shelf life to 19 days and 32 days, respectively, and the cell load of e. coli was always under the detection limit. however, for the sample treated at 500-mpa pressure, after 19 days, lactobacilli and pseudomonas spp. were able to recover and reach the respective cell load of 4.49 log cfu/g and 5.72 log cfu/g, different from the samples treated at 600 mpa, where these microbial groups were found to be under the detection limit. applying a pressure level of 400 mpa resulted in a shelf life of about 12 days. similar trends were observed in the shrimp products (table 3), where the application of a pressure of 600 mpa allowed the shelf-life threshold to reach after 28 days. in contrast, the samples treated with 500-mpa pressure reached the spoilage threshold after 21 days. interestingly, 400-mpa pressure was not able to inactivate completely the coagulase-positive staphylococci, which were able to recover but only after reaching the shelf-life threshold. the rationale for the use of hpp for fish and fish products is based on its ability to inactivate pathogenic and spoilage microorganisms and microbial enzymes, resulting in an increased shelf life (de alba et al., 2019) as well 104 italian journal of food science, 2023; 35 (3) pinheiro acas et al. table 2. evolution of microbial cell loads (log cfu/g) of total mesophilic count (tmc), lactobacillus spp., pseudomonas spp., total coliforms, sulphite-reducing anaerobic bacteria (ab), and e. coli during the refrigerated storage of packaged striped prawn with applied high hydrostatic pressure (hpp) treatments (400, 500 and 600 mpa). striped prawn log cfu/g p-0 p-400 p-500 p-600 days of storage 0 d tmc 4.65a ± 0.44 2.45b ± 0.43 <1 <1 lactobacillus spp. 3.95 ± 0.29 <1** <1 <1 sulphite-reducing ab 3.83 ± 0.39 <1 <1 <1 pseudomonas spp. 4.63 ± 0.56 <1 <1 <1 total coliforms 4.60a ± 0.48 1.30b ± 0.33 <1 <1 e. coli 2.10 ± 0.33 <1 <1 <1 2 d tmc 4.50a ± 0.46 2.96b ± 0.43 <1 <1 lactobacillus spp. 3.50 ± 0.36 <1 <1 <1 sulphite-reducing ab 3.20 ± 0.51 <1 <1 <1 pseudomonas spp. 5.25 ± 0.48 <1 <1 <1 total coliforms 4.75 ± 0.49 <1 <1 <1 e. coli 2.80 ± 0.15 <1 <1 <1 6 d tmc 6.30a ± 0.52 2.88b ± 0.55 <1 <1 lactobacillus spp. 5.82 ± 0.42 <1 <1 <1 sulphite reducing ab 4.42 ± 0.49 <1 <1 <1 pseudomonas spp. 5.35 ± 0.35 <1 <1 <1 total coliforms 3.75 ± 0.46 <1 <1 <1 e. coli 2.95 ± 0.15 <1 <1 <1 12 d tmc 7.20a ± 0.39 4.61b ± 0.23 2.00c ± 0.15 1.50c ± 0.50 lactobacillus spp. 6.80a ± 0.55 5.12b ± 0.34 <1 <1 sulphite reducing ab 5.84 ± 0.51 <1 <1 <1 pseudomonas spp. 5.30a ± 0.45 3.88b ± 0.39 <1 <1 total coliforms 3.72a ± 0.43 1.48b ± 0.46 <1 <1 e. coli 3.20 ± 0.23 <1 <1 <1 19 d tmc 8.35a ± 0.48 6.18b ± 0.35 4.80c ± 0.39 lactobacillus spp. 6.56a ± 0.30 4.59b ± 0.33 <1 sulphite-reducing ab <1 <1 <1 pseudomonas spp. 7.83a ± 0.37 5.72b ± 0.47 <1 total coliforms 1.95 ± 0.34 <1 <1 e. coli <1 <1 <1 32 d tmc 6.51 ± 0.39 lactobacillus spp. <1 sulphite-reducing ab <1 pseudomonas spp. <1 total coliforms <1 e. coli <1 *not analysed because the microbiological threshold fixed at 6 log cfu/g was reached in the previous time of analysis. **under detection limit. in the same row, values with different superscript letters are significantly different (p < 0.05). italian journal of food science, 2023; 35 (3) 105 effect of high hydrostatic pressure on different seafood products table 3. evolution of microbial cell loads (log cfu/g) of total mesophilic count (tmc), lactobacillus spp., pseudomonas spp., total coliforms, sulphite-reducing anaerobic bacteria (ab), e. coli and positive coagulase (pc) staphilococci during the refrigerated storage of packaged rose shrimp in relation to the applied high hydrostatic pressure (hpp) treatments (400, 500 and 600 mpa).   rose shrimp   log cfu/g s-0 s-400 s-500 s-600 days of storage 0 d tmc 5.04a ± 0.47 4.46a,b ± 0.56 3.78b,c ± 0.53 3.34c ± 0.45 lactobacillus spp. 5.34 ± 0.44 <1** <1 <1 sulphite-reducing ab 4.53a ± 0.51 2.48b ± 0.35 2.38b ± 0.43 <1 pseudomonas spp. 5.16 ± 0.37 <1 <1 <1 total coliforms 3.53 ± 0.46 <1 <1 <1 e. coli 2.26 ± 0.45 <1 <1 <1 pc staphilococci 2.20 ± 0.20 <2 <2 <2 7 d tmc 6.48a ± 0.49 5.18b ± 0.46 4.35b,c ± 0.39 3.63c ± 0.47 lactobacillus spp. 6.05 ± 0.55 <1 <1 <2 sulphite-reducing ab 6.08 ± 0.36 <2 <2 <1 pseudomonas spp. 5.32 ± 0.42 <1 <1 <1 total coliforms  4.70 ± 0.20 <1 <1 <1 e. coli  3.60 ± 0.15 <1 <1 <1 pc staphilococci 3.40 ± 0.25 <2 <2 <2 14 d tmc -* 8.15a ± 0.35 4.31b ± 0.51 4.20b ± 0.56 lactobacillus spp. <2 <1 <1 sulphite-reducing ab <2 <2 <1 pseudomonas spp. 3.56 ± 0.44 <1 <1 total coliforms <1 <1 <1 e. coli   <1 <1 <1 pc staphilococci   2.60 ± 0.41 <2 <2 21 d tmc 5.64 ± 0.42 3.6 ± 0.41 lactobacillus spp. <1 <1 sulphite-reducing ab <2 <1 pseudomonas spp. <1 <1 total coliforms <1 <1 e. coli   <1 <1 pc staphilococci   <2 <2 28 d tmc 8.43a ± 0.37 5.46b ± 0.43 lactobacillus spp. <1 <1 sulphite-reducing ab 4.32 ± 0.33 <1 pseudomonas spp. <1 <1 total coliforms <1 <1 e. coli   <1 <1 pc staphilococci   <2 <2 35 d tmc 7.49 ± 0.39 lactobacillus spp. <1 sulphite-reducing ab <1 pseudomonas spp. <1 total coliforms <1 e. coli <1 pc staphilococci <2 *not analysed because the microbiological threshold fixed at 6 log cfu/g was reached in the previous time of analysis **under detection limit. in the same row, values with different superscript letters are significantly different (p < 0.05). 106 italian journal of food science, 2023; 35 (3) pinheiro acas et al. table 4. ph and peroxide values (pv; meq o 2 /kg fat) measured in hpp-treated seafood samples during refrigerated storage. storage time (days) 1 6 9 14 21 28 35 sample ph m-0 6.41 ± 0.13a 6.34 ± 0.03b 6.46 ± 0.05a 6.23 ± 0.03b m-400 6.44 ± 0.06a 6.55 ± 0.06a 6.52 ± 0.04a 6.47 ± 0.02a 6.34 ± 0.07c m-500 6.44 ± 0.01a 6.50 ± 0.04a 6.45 ± 0.01a 6.46 ± 0.01a 6.51 ± 0.04a 6.31 ± 0.10a 6.51 ± 0.01a m-600 6.46 ± 0.01a 6.50 ± 0.03a 6.41 ± 0.02a 6.45 ± 0.01a 6.41 ± 0.03b 6.40 ± 0.03a 6.51 ± 0.01a p-0 7.18 ± 0.01b 7.20± 0.01c 7.54 ± 0.12a 7.44 ± 0.06b p-400 7.45 ± 0.01a 7.29 ± 0.03b 7.27 ± 0.01b 7.25 ± 0.01c 7.06 ± 0.01c p-500 7.40 ± 0.01a 7.45 ± 0.01a 7.32 ± 0.01b 7.24 ± 0.03c 7.47 ± 0.02a 7.27 ± 0.04b 7.17 ± 0.01b p-600 7.50 ± 0.02a 7.44 ± 0.08a 7.26 ± 0.02b 7.54 ± 0.02a 7.36 ± 0.03b 7.36 ± 0.01a 7.37 ± 0.01a s-0 7.59 ± 0.04b 7.68 ± 0.08a 7.73 ± 0.03a s-400 7.65 ± 0.05a 7.44 ± 0.03b 7.47 ± 0.03b 7.46 ± 0.01a s-500 7.55 ± 0.11b 7.52 ± 0.02b 7.50 ± 0.01b 7.39 ± 0.03a 7.37 ± 0.01b s-600 7.74 ± 0.02a 7.61 ± 0.01ab 7.48 ± 0.01b 7.51 ± 0.04a 7.48 ± 0.01a 7.41 ± 0.02 sample pv m-0 0.88 ± 0.09b 0.55 ± 0.08b 1.40 ± 0.05a 0.33 ± 0.10b m-400 1.00 ± 0.06b 0.68 ± 0.10b 0.77 ± 0.06b 1.50 ± 0.11a 1.52 ± 0.07a m-500 0.99 ± 0.05b 0.90 ± 0.14a 1.00 ± 0.22ab 1.25 ± 0.11a 1.10 ± 0.06a 1.51 ± 0.14a 0.79 ± 0.04b m-600 1.28 ± 0.05a 0.92 ± 0.07a 0.92 ± 0.05b 1.14 ± 0.05a 1.57 ± 0.10a 1.06 ± 0.17a 1.75 ± 0.15a p-0 1.88 ± 0.03ab 1.36 ± 0.15b 1.56 ± 0.16b p-400 2.03 ± 0.02a 2.03 ± 0.11a 1.37 ± 0.02c 1.30 ± 0.14a 1.30 ± 0.14a p-500 1.54 ± 0.14b 2.5 ± 0.07a 2.68 ± 0.41a 1.57 ± 0.11a 1.92 ± 0.31a 1.89 ± 0.07a 1.78 ± 0.2a p-600 1.52 ± 0.11b 0.70 ± 0.07c 1.96 ± 0.11b 1.44 ± 0.16a 1.82 ± 0.12a 0.89 ± 0.03b 1.66 ± 0.12a s-0 0.91 ± 0.14a 0.43 ± 0.02b 0.59 ± 0.01b s-400 0.63 ± 0.12a 0.82 ± 0.13a 0.59 ± 0.02b 0.68 ± 0.11a s-500 0.66 ± 0.08a 0.86 ± 0.04a 0.82 ± 0.05a 0.72 ± 0.01a 0.62 ± 0.04a s-600 0.98 ± 0.10a 0.67 ± 0.03ab 0.85 ± 0.08a 0.57 ± 0.13a 0.49 ± 0.02a 0.65 ± 0.04 different superscript letters indicate significant differences (p < 0.05) between samples of the same specie at the same storage time. in the present study, despite no difference being observed between grey mullet samples just after treatment, after 6 days, ph was slightly higher in hpptreated samples, compared to the untreated sample. during storage, a slight decrease was observed for m-0 sample, while for the samples subjected to high pressure, values showed little variability until the end of the storage period. on the other hand, for tiger prawn, all samples showed higher ph values of about 7.2–7.5 after hpp, compared to the control, without differences in relation to different pressure levels applied. however, while in the case of control sample, an increase in ph was observed during storage, treated samples showed an opposite trend (figure s1). in sample p-400, ph values decreased by about 0.4 points, while in samples p-500 and p-600, they showed little variability. in rose shrimp, only sample s-600 showed a significant increase in ph just after treatment, compared to the untreated sample. during storage, the control sample showed a slight increase, while for all treated samples, ph values decreased progressively. increase in ph after pressurization of fish and seafood tissues was observed in literature (angsupanich and ledward, 1998; bindu et al., 2013; briones-labarca et al., 2012; kaur et al., 2013) and was explained with the induction of protein unfolding by pressure and the following ionisation of denatured proteins. italian journal of food science, 2023; 35 (3) 107 effect of high hydrostatic pressure on different seafood products significant differences were observed for h° values between the control and the pressure-treated samples just after treatment. however, while for grey mullet, h° value increased due to hpp, for shrimp and prawn, the values of this chromatic parameter decreased. hue angle is calculated using both red and yellow indexes; in all samples a* was decreased remarkably, while changes in b* were higher in shrimp and prawn, compared to grey mullet. according to the literature, variation in colour during storage could depend on degradation of myofibrillar proteins and disorganisation of myofibrils caused by enzymatic and non-enzymatic reactions as well as on the possible oxidation of pigments (yagiz et al., 2007). hence, change in colour occurring during storage in seafood products subjected to high pressures could differ significantly depending on the species and the adopted treatment conditions. in the present study, the final effect for all samples was general whitening and occurrence of a cooked appearance, which is typical for muscle foods subjected to pressurization (figure s2). considering that visual quality is a very important parameter for consumer acceptability, and that these products are intended for raw consumption, the cooked on the other side, the evolution of ph during storage could be attributed mainly to the activity of spoilage microorganisms that produced both basic (e.g. ammonia, trimethylamine and other biogenic amines) and acidic (e.g. lactic acid in the case of lactobacillus spp.) compounds. color values for luminosity (l*) and hue angle (h°) of the three seafood species are reported in figure 1. luminosity of the flesh was significantly increased by about 20 units straight for the three considered species after hpp treatment. this effect was largely observed in many fish species and attributed to protein denaturation. considering that for each investigated species there was no significant difference in the values of the treated products just after the treatment as a function of the different pressure levels adopted, it was assumed that protein denaturation occurred to a similar extent in all samples for hpp of 400–600 mpa. during storage, all l* values were very close to the initial values for the control and the pressure-treated samples. 30 40 50 60 70 80 30 40 50 60 70 80 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 20 25 30 35 40 45 50 55 60 60 65 70 75 80 85 90 95 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 ho l* ho l* l* l* ho h o time (d) time (d) time (d) m-0 m-400 m-500 m-600 p-0 p-400 p-500 p-600 s0 s4 s5 s6 (a) (b) (c) (d) (e) (f) figure 1. colour coordinates of luminosity (l*) and hue angle (h°) of (a and b) mullet, (c and d) shrimp and (e and f) rose shrimp. different letters indicate significant differences between samples (p < 0.05). 108 italian journal of food science, 2023; 35 (3) pinheiro acas et al. influenced both myofibrillar proteins and connective tissues, bringing about myofibrillar fragility and gaping. a similar behaviour could be observed for sample m-400, but with a more gradual decrease of both values. indeed, after 9 days, the resistance to compression was significantly higher, compared to the control, possibly because of a partial enzymatic denaturation caused by pressure. however, samples m-500 and m-600 showed quite constant values for both parameters for all storage periods with fewer differences between them. in striped prawn (figures 2c and 2d), a slight but significant reduction of hardness was observed for the 500 and 600-mpa treatments, while the resistance parameter was reduced only for 600-mpa pressure. during storage, hardness showed a fluctuating behaviour but tended to increase, compared to the initial value for samples p-0, p-400 and p-500, while it remained practically unchanged for sample p-600. resistance was shown to increase during storage only in samples p-0 and p-400, although the storage was shorter due to microbiological spoilage. on the contrary, in rose shrimp (figures 2e and 2f), hardness was not influenced by any pressure applied, while the resistance to compression was found significantly higher after 600-mpa treatment. during storage, both parameters showed a decreasing trend for all samples. the values were highly variable and very few significant changes were observed. appearance could represent a problem that could be addressed probably by a marketing and/or informative strategy. texture texture is a very important parameter for appreciation of seafood products. even if, in general, the texture profile analysis (tpa) test is carried out on fish fillets, considering the specific characteristics of the investigated product, in the present study compression, followed by the application of steady pressure, was applied. results of the two parameters analysed, that are, hardness (f1) and index of resistance to compression (f2) (figure s3), are reported in figure 2 for all the three considered species. in the present work, the observed effect was different for the three considered species. for grey mullet (figures 2a and 2b), a reduction in the initial hardness was observed for higher applied pressures (500 and 600 mpa) just after the treatment. moreover, the increasing pressure decreased resistance to compression of the tissues in proportion to the applied pressure level (figure 2b). for both parameters, in the control sample, a decrease was observed during storage, as reported by chéret et al. (2005) for sea bass. the softening of the tissue during refrigerated storage could be attributed to enzymatic activity of proteases that 0 20 40 60 80 100 0 10 20 30 40 50 60 0 20 40 60 80 100 120 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 0 10 20 30 40 50 60 70 80 90 100 r es is ta nc e to c om pr es si on ( n ) r es is ta nc e to c om pr es si on ( n ) r es is ta nc e to c om pr es si on ( n ) h ar dn es s (n ) h ar dn es s (n ) h ar dn es s (n ) time (d) time (d) time (d) 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 m-0 m-400 m-500 m-600 (a) (b) (c) (d) (e) (f) p-0 p-400 p-500 p-600 s0 s4 s5 s6 figure 2. texture parameters of hardness (n) and resistance to compression (n) of (a and b) mullet, (c and d) shrimp and (e and f) rose shrimp. different letters indicate significant differences between samples (p < 0.05). italian journal of food science, 2023; 35 (3) 109 effect of high hydrostatic pressure on different seafood products low level of pressure of about 100 mpa applied at 10°c for 5 min showed to increase enzymatic activity of calpain in sea bass muscles (chéret et al., 2005), while teixeira et al. (2013) observed that rate of pressurization could also play a role in the activation of the same enzyme in sea bass fillets. in general, increase in pressure level and holding period was shown to inactivate enzymes. however, although seafood muscles were shown to be more sensitive to pressure compared to bovine muscles, the food matrix could strongly affect enzymes (truong et al., 2015). the application of pressure caused variation in protein structure that could lead to the breakdown of cell membrane and release of proteolytic enzymes in the cytoplasm, favouring their denaturation. lipid oxidation table 4 shows peroxide values measured in the three considered species subjected to hpp treatment during refrigerated storage. a slight but significant increase in peroxide values was observed after 600-mpa treatment in grey mullet samples. however, while no difference was observed in rose shrimp samples, lower values were measured in prawn samples treated at higher pressures. moreover, peroxide values remained very low for all the storage periods considered (below 1.75, 2.7 and 0.98 meq o2/kg fat for grey mullet, prawn and shrimp, respectively). in general, an enhancement of lipid oxidation was observed in the literature for many seafood products, particularly when pressures above 300 mpa were applied (truong et al., 2015). this increase was mainly attributed to the presence of haemoglobin and myoglobin containing iron in their structure that was released because of pressurization, and could promote oxidation. however, yagiz et al. (2009) established that 300-mpa treatment helped to inhibit lipid oxidation of atlantic salmon, compared to the untreated sample and the sample subjected to 150-mpa pressure during storage. the authors suggested that perturbation to changes to cell membrane structure caused by pressure made phospholipids less susceptible to oxidation even in a fish that was considered fatty. moreover, presence of astaxanthin (a carotenoid) was believed to have a powerful antioxidant effect. studies related to the effect of pressure on the activity of enzymes responsible for lipid oxidation are still scarce. the reduced oxidation level showed by the evolution of its primary index in the present study was due to different reasons. firstly, the considered seafood species were characterised by a low content of fat (about 2.5, 0.8 and 1.1 g/100 g for grey mullet, prawn and shrimp, respectively) and were not susceptible to lipid oxidation. secondly, we assumed that further reduction in lipid these results are in contrast with increase in hardness measured after applying high pressure, as observed by bindu et al. (2013) in indian white prawn and by jantakoson et al. (2012) and kaur et al. (2013) in black tiger shrimp. moreover, the two considered parameters between two crustaceans showed different behaviour during storage. an increasing tendency was observed in prawn, while in shrimp, values decreased progressively for the considered period. decrease in hardness during storage, attributed to the effect of proteolytic enzymes, was also observed by kaur et al. (2013). however, values of hardness in sample p-600 and resistance in samples p-500 and p-600 were mostly constant. this effect could be explained by a partial inactivation of such enzymes. in general, an increase in hardness after hpp treatment was observed by many authors on different fish species, such as rainbow trout and mahi mahi (yagiz et al., 2007), cod (angsupanich and ledward, 1998), tuna (zare, 2004). this was explained by the unfolding of actin and sarcoplasmic proteins and the formation of new hydrogen bonded networks and by an increase in protein– protein interactions and bond formation. on the other hand, briones-labarca et al. (2012) found no differences in red abalone treated with up to 500-mpa hpp, compared to the control. chéret et al. (2005) observed a softening effect after subjecting sea bass to a pressure of up to 300 mpa, while no change in hardness was observed with 400and 500-mpa treatment. besides modification to myofibrillar proteins, hpp also promoted ph values and modification to hydrogen and hydrophobic bonds that resulted in changes to the structural characteristics of proteins. moreover, an effect on collagen and the connective tissue of red abalone was observed by briones-labarca et al. (2012) through scanning electric microscope. this confirmed a significant change in the microstructure of the flesh upon application of high pressure. hence, the effect of hpp on fish texture was due to modifications to water bonding and holding capacity, activity of enzymes, such as proteases, that could be inhibited or enhanced, and structural modification of myofibrillar and sarcoplasmic protein. the results obtained in the present study confirmed that the effect on texture strictly depended on the considered species of the raw material and the specific tissue structure. moreover, the effect observed during storage probably depended on the possible inactivation of proteolytic enzymes, which again probably was matrix-dependent. the effect of hpp on proteolytic enzymes in different species of fishes was studied by different authors, but results appeared to vary depending on seafood species, pressure level, holding period, and type and structure of the enzyme. 110 italian journal of food science, 2023; 35 (3) pinheiro acas et al. environments as well as high mortality rate. in addition, the spore-forming quality of c. botulinum fosters its survival in water sediments and fish, certainly influenced by geographical location, feeding habits of the fish species, types of product samples, and the detection method used (aleksandr et al., 2015). then again, the data emphasized, following the hpp treatment, a shift in microbial populations responsible for reaching the threshold level. in addition, this aspect requires further investigation to understand product spoilage patterns. author contributions conceptualization: s.t; p.r.; methodology: s.t., f.p., p.r.; formal analysis: a.c.d.a.s.p., g.b.; investigation: a.c.d.a.s.p., g.b.; resources: f.p., p.r.; data curation: a.c.d.a.s.p., j.g., g.b.; writing-original draft: a.c.d.a.s.p., g.b.; writing review & editing: s.t., j.g., f.p., p.r.; supervision: s.t, f.p.; project administration: p.r.; funding acquisition: f.p., p.r. references aleksandr n., margarita t., inga e., olga v., vadims b. and aivars b. 2015. major food borne pathogens in fish and fish product: a review.  ann microbiol.  66(1): 1–15. https://doi.org/10.1007/ s13213-015-1102-5 angsupanich k. and ledward d.a. 1998. high pressure treatment effects on cod (gadus morhua) muscle. food chem. 63(1): 39–50. https://doi.org/10.1016/s0308-8146(97)00234-3 bindu j., ginson j., kamalakanth c.k., asha k.k and srinivasa gopal t.k. 2013. physico-chemical changes in high pressure treated indian white prawn (fenneropenaeus indicus) during chill storage. innov food sci emerg technol. 17: 37–42. https:// doi.org/10.1016/j.ifset.2012.10.003 bligh e.g. and dyer w.j. 1959. a rapid method of total lipid extraction and purification. can j biochem physiol. 37(8): 911– 17. https://doi.org/10.1139/o59-099 borges a.f., cózar a., patarata l., gama l.t., alfaia c.m., fernandes  m.j., et al. 2020s. effect of high hydrostatic pressure challenge on biogenic amines, microbiota, and sensory profile in traditional poultryand pork-based semidried fermented sausage.  j food sci.  85(4): 1256–1264. https://doi. org/10.1111/1750-3841.15101 briones-labarca v., perez-won m., zamarca m., aguileraradic  j.m. and tabilo-munizaga g. 2012. effects of high hydrostatic pressure on microstructure, texture, colour and biochemical changes of red abalone (haliotis rufecens) during cold storage time. innov food sci emerg technol. 13: 42–50. https:// doi.org/10.1016/j.ifset.2011.09.002 brutti a., rovere p., cavallero s., d’amelio s., danesi p. and arcangeli g. 2010. inactivation of anisakis simplex larvae in raw fish using high hydrostatic pressure treatments. food cont. 21(3): 331–333. https://doi.org/10.1016/j.foodcont.2009.05.013 oxidation could have been due to modification to cell membranes induced by pressure. also, a very low presence of oxygen in packages would have surely inhibited oxidative reactions during storage. conclusions the investigation of the effect of hpp treatment on different types of considered seafood products, intended for raw consumption, highlighted a significant increase in microbiological shelf life at the highest applied pressure levels (500 and 600 mpa) for all the considered species. even if lower pressure (400 mpa) appeared adequate to inactivate e. coli, pseudomonas, and/or positive coagulase staphylococci, they were recovered during fish product storage, stressing the issue of viable but non culturable cells (vbnc). on the other side, the application of 600  mpa allowed to extend the microbiological shelf life by up to around 30 days. in terms of visual quality, the final effect for all samples was general whitening and occurrence of a cooked appearance, typical for muscle foods subjected to pressurization. the texture response was strictly dependent on the considered species of raw material and specific tissue structure. fat oxidation was minimally affected and remained quite low during storage. further studies are in progress in our laboratories to better clarify the physico-chemical and biological causes of detected differences through microstructural assessment and their overall sensorial impact. moreover, the effect of hpp on the production of biogenic amines is also under evaluation in order to clarify the effectiveness of the proposed technology on the safety aspect. however, considering the effect on microbiological shelf life, for all the three considered species, attaining a pressure of over 500 mpa appears necessary, particularly because the effect on quality did not show changes at higher pressures. it is important to emphasize that in the optic of the commercialisation of hpp-treated seafood intended for raw consumption, a specific marketing/informative strategy is required, evidencing to the final consumer the important advantages of this non-thermal technology in terms of nutritional and sensorial food properties. moreover, in addition to l. monocytogenes and salmonella pathogens, the effect of hpp process must be considered against vibrio spp. and clostridium botulinum, especially in vacuum packaging, because these pathogens are responsible for human diseases through the consumption of contaminated fish and fish products. these pathogens have a wide distribution in aquatic https://doi.org/10.1007/s13213-015-1102-5� https://doi.org/10.1007/s13213-015-1102-5� https://doi.org/10.1016/s0308-8146(97)00234-3� https://doi.org/10.1016/j.ifset.2012.10.003� https://doi.org/10.1016/j.ifset.2012.10.003� https://doi.org/10.1139/o59-099� https://doi.org/10.1111/1750-3841.15101� https://doi.org/10.1111/1750-3841.15101� https://doi.org/10.1016/j.ifset.2011.09.002� https://doi.org/10.1016/j.ifset.2011.09.002� https://doi.org/10.1016/j.foodcont.2009.05.013� italian journal of food science, 2023; 35 (3) 111 effect of high hydrostatic pressure on different seafood products perez-won m., lemus-mondaca r., herrera-lavados c., reyes j.e., roco t., palma-acevedo a. and 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frozen/refrigerated storage. j food proc preserv. 43(8): e14009. https://doi.org/10.1111/jfpp.14009 yagiz y., kristinsson h.g., balaban m.o. and marshall m.r. 2007. effect of high pressure treatment on the quality of rainbow trout (oncorhynchus mykiss) and mahi mahi (coryphaena hippurus). j food sci. 72(9): 509–515. https://doi. org/10.1111/j.1750-3841.2007.00560.x yagiz y., kristinsson h.g., balaban m.o., welt b.a., ralat m. and marshall m.r. 2009. effect of high pressure processing and cooking treatment on the quality of atlantic salmon. food chem. 116(4): 828–835. https://doi.org/10.1016/j.foodchem.2009.03.029 zare z. 2004. high pressure processing of fresh tuna fish and its effects on shelf life. high pressure processing of fish. mcgill university, quebec, canada. https://api.semanticscholar.org/ corpusid:99272819 campus m. 2010. high pressure processing of meat, meat products and seafood. food eng rev. 2(4): 256–273. https://doi. org/10.1007/s12393-010-9028-y chapman r.a. and mackay k. 1949. the estimation of peroxides in fats and oils by the ferric thiocyanate method. j am oil chem soc. 26(7): 360–363. https://doi.org/10.1007/bf02651444 chéret r., chapleau n., delbarre-ladrat c., verrez-bagnis  v. and lamballerie m.d. 2005. effects of high pressure on texture and microstructure of sea bass (dicentrarchus labrax l.) fillets. j food sci. 70(8): e477–e483. https://doi. org/10.1111/j.1365-2621.2005.tb11518.x de alba m., pérez-andrés j.m., harrison s.m, brunton n.p., burgess c.m. and tiwari b.k. 2019. high pressure processing on microbial inactivation, quality parameters and nutritional quality indices of mackerel fillets. innov food sci emerg technol. 55: 80–87. https://doi.org/10.1016/j.ifset.2019.05.010 jantakoson t., kijroongrojana k. and benjakul s. 2012. effect of high pressure and heat treatments on black tiger shrimp (penaeus monodon fabricius) muscle protein. int aquatic res. 4(1): 1–12. https://doi.org/10.1186/2008-6970-4-19 kaur b.p., kaushik n., rao 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(ultra) high pressure homogenization potential on the shelf life and functionality of kiwifruit juice. front microbiol. 10(feb). https://doi.org/10.3389/ fmicb.2019.00246 patterson m.f. 2014. food technologies: high pressure processing. in: encyclopedia of food safety, vol. 3. academic press, cambridge, ma, pp. 196–201. https://doi.org/10.3390/foods9030273� https://doi.org/10.1021/jf3049643� https://doi.org/10.1007/s12393-014-9084-9� https://doi.org/10.1111/jfpp.14009� https://doi.org/10.1111/j.1750-3841.2007.00560.x� https://doi.org/10.1111/j.1750-3841.2007.00560.x� https://doi.org/10.1016/j.foodchem.2009.03.029� https://api.semanticscholar.org/corpusid:99272819� https://api.semanticscholar.org/corpusid:99272819� https://doi.org/10.1007/s12393-010-9028-y� https://doi.org/10.1007/s12393-010-9028-y� https://doi.org/10.1007/bf02651444� https://doi.org/10.1111/j.1365-2621.2005.tb11518.x� https://doi.org/10.1111/j.1365-2621.2005.tb11518.x� https://doi.org/10.1016/j.ifset.2019.05.010� https://doi.org/10.1186/2008-6970-4-19� https://doi.org/10.1007/s11947-012-0870-1.matser� https://doi.org/10.1007/s11947-012-0870-1.matser� https://doi.org/10.3389/fmicb.2019.00246� https://doi.org/10.3389/fmicb.2019.00246� 112 italian journal of food science, 2023; 35 (3) pinheiro acas et al. 6.0 6.2 6.4 6.6 6.8 7.0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 7.0 7.2 7.4 7.6 7.8 8.0 7 7.2 7.4 7.6 7.8 8 m-0 m-400 m-500 m-600 p-0 p-400 p-500 p-600 s-0 s-400 s-500 s-600 time (days) ph ph ph (a) (b) (c) figure s1. ph measured in hpp-treated seafood samples during refrigerated storage. supplementary italian journal of food science, 2023; 35 (3) 113 effect of high hydrostatic pressure on different seafood products m 0m 0 p 0p 0 s 0s 0 m 400m 400 p 400p 400 s 400s 400 m 500m 500 p 500p 500 s 500s 500 m 600m 600 p 600p 600 s 600s 600 figure s2. digital images of grey mullet (m), striped prawn (p) and rose shrimp (s) samples treated at 400-, 500and 600-mpa pressure, compared to the untreated samples. 114 italian journal of food science, 2023; 35 (3) pinheiro acas et al. 0 20 40 60 80 0 5 10 15 20 25 30 35 f1 f2 f or ce ( n ) time (s) figure s3. results of the analysed hardness (f1) and index of resistance to compression (f2). _hlk56781082 _hlk510604571 _hlk510602300 _hlk137473821 p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (2): 9–20 issn 1120-1770 online, doi 10.15586/ijfs.v34i2.2141 9 p u b l i c a t i o n s codon effect of high-moisture extrusion on soy meat analog: study on its morphological and physiochemical properties monirul islam1,2, yatao huang1, md. serajul islam3, ningyu lei1, lei shan1, bei fan1, litao tong1, fengzhong wang1* 1institute of food science and technology, chinese academy of agricultural sciences/key laboratory of agro-products processing, ministry of agriculture and rural affairs, beijing, people’s republic of china; 2rural development academy (rda), bogura, bangladesh; 3state key laboratory of food science and technology, jiangnan university, wuxi, people’s republic of china *corresponding author: fengzhong wang, institute of food science and technology, chinese academy of agricultural sciences, beijing, 100193, people’s republic of china. email: wangfengzhong@sina.com received: 10 october 2021; accepted: 7 december 2021; published: 2 april 2022 © 2022 codon publications open access paper abstract there has been a growing interest in meat analog, microstructure characteristics, and anti-nutritional content obtained from soybean. high-moisture extrusion parameters are the input extruder of moisture content (>40%) that get the advantages of lower energy input. thermo-mechanical treatment has a considerable influence on structural properties of soy-based meat analog. texturized soy proteins can substitute meat products while providing a high-protein food ingredient which can be consumed directly as meat analogs. therefore, this review aims to the effect on soybean of micro-structural and physicochemical properties of meat analogs by high-moisture extrusion. thus, further studies are required concerning a large-scale meat products with purify protein structure. keywords: soy meat analog; high-moisture extrusion; allergenic protein; anti-nutritional factors; textural properties introduction soybean (glycine max), which has its origin in east asia (especially in china and japan), belongs to the legume family (kader et al., 2017). it is an excellent alternative source of proteins, complex carbohydrates, polyunsaturated fatty acids, soluble fibers, and isoflavones. nowadays, it’s grown worldwide as an edible bean (peluso et al., 2014). wang et al. (2020) reported that 60 varieties were used for the product development by 2018; 398 million tons of soybeans were produced worldwide, with 61% for oilseed production. in addition, salgado and donadopestana (2011) found that 90% of the total soybean production in the world is from the united states of america (usa), brazil, south america, and northwestern europe. moreover, jooyandeh (2011) reported soybean oil composition that is 15% saturated, 61% polyunsaturated, and 24% monounsaturated fats. furthermore, fiala (2008) described that by 2030, the increase in the population, industrialization, and urbanization will also increase the plant meat demand by about 72%. golbitz and jordan (2006) found that soybeans typically contain 35–40% proteins with well-balanced amino acid composition, 30% carbohydrate, 15–20% fat, 10–30% moisture content as well as fiber, calcium (ca), iron (fe), zinc (zn), and vitamin b complex. the nutritional composition of soybean/legume is presented in table 1. in addition, soybean contains some minor compounds, such as lecithin, isoflavones, bio-peptides, and others, that are effective against chronic cancer, cardiovascular diseases, and type ii diabetes (dixit et al., 2011; singh, 2010; singh et al., 2008). soybean protein is suitable for people, who lack plant protein, for nutritional value and mailto:wangfengzhong@sina.com 10 italian journal of food science, 2022; 34 (2) islam m et al. table 1. nutritional composition of plant based protein, quorn protein and microbial proteins legume. protein source protein content mineral content references legume protein legume seed (20–30%) – (riascos et al., 2010) – soy protein (concentrate): potassium (2.4%), phosphorus (0.9%), calcium (0.4%), magnesium (0.3), iron (0.02%), zinc (0.005%) (singh et al., 2008) oilseed protein safflower seed (13–17%) – (asgar et al., 2010) cottonseed (23%) – rapeseed (25%) – gourd seed calcium (0.055%), magnesium (0.720%), potassium (0.965%), iron (0.021%), zinc (0.016%) (olaofe et al., 1994) pumpkin seed calcium (0.072%), magnesium (0.778%), potassium (1.127%), iron (0.027%), zinc (0.017%) (olaofe et al., 1994) cereal protein maize (8.8–11.9%) – (orcutt et al., 2006; riaz, 2004) wheat (8–17.5%) – oats (8.7–16%) – rice (7–10%) – barley (7–14.6%) – rye (7–14%) – quorn protein mycoprotein 11% sodium (mg) 5.0 cholesterol (g) 0 iron (mg) 0.5 zinc (mg) 9.0 selenium (µg) 20 (finnigan et al., 2019) bacterial protein bacillus cereus (ram horn) 68% bacillus licheniformis (potato starch processing waste) 38% corynobacterium ammoniagenes (glucose + fructose) 61% escherichia coli (ram horn) 66% corynobacterium ammoniagenes (glucose + fructose) 61% cupriavidus necator (synthetic growth medium) 40–46% – (ritala et al., 2017) good health effects on calcium metabolism and lowering cholesterol (shih et al., 2016). moreover, soy products, such as soy flour, soy milk/powder, soy protein isolate (spi), soy protein hydrolysates (guo et al., 2018; he et al., 2019; simmons et al., 2012), could be used as food additives and nutraceutical ingredients. meat alternatives could be classified as plant-based (soy, gluten, pea, etc.), cell-based (vitro or cultured meat), and fermentation-based (mycoproteins) (sha & xiong, 2020). in recent years, plant-based meat has been developed for meeting consumer demands, exponentially grown market, and the sustainability of future food supply (sha & xiong, 2020). the plant-based meat market’s expansion is predicted to increase from $4.6 billion in 2018 to $85 billion in 2030, and as a milestone by 2026, it would achieve $30.9 billion (sha & xiong, 2020). among the several mechanistic techniques for the texturization of plant-based meat, extrusion is the most often applied one (dekkers et al., 2018). plant proteins represent a safe, sustainable, and practical non-pharmacological approach for lowering cholesterol. recently, the most famous attributes of plant-based protein, that is soybean protein, is their health benefits linked to the prevention and treatment of many chronic diseases. regular consumption of soy products reduces one’s risk for chronic diseases such as cancer, heart disease, and stroke (jooyandeh, 2011). moreover, plant-based meat analog consumption is also beneficial in protecting against heart disease, lowering blood cholesterol; reducing the risk of cancer, and increasing bone mass (joshi & kumar, 2015). furthermore, recent studies have well established that the plant-based food and beverage help italian journal of food science, 2022; 34 (2) 11 effect of high-moisture extrusion plant-based meat analogs meat analog is also called meat substitute, faux meat, mock meat, or imitation meat. analog can be defined as the compound of the same structure, such as texture, but slightly different in composition. there are two stages in the production of conventional meat analogs: preparation of emulsion and formation of its chunk. typically, the emulsion is primed by mixing, chopping, and emulsifying proteins, fats, salts, and other inclusions to form a protein matrix. meat analogs can be produced at low moisture (<35%) using a single screw extruder or at high moisture (>50%) using a twin-screw extruder (lin et al., 2000). moreover, riaz (2004) described that meat analog could be formed into strips, sheets, patties, disks, and other shapes. it can absorb water at least three times its weight when cooked in boiling water for at least 15 min. the fibrous, anisotropic structure of meat analog products contributes to the meat-like feel and sensory view (elzerman et al., 2013). table 2 presents summaries of the comparison between the plant-based and animal-based alternatives to meat. meat analogs look like textured meat and are healthy (cholesterol-free) and low-cost. in addition, mycoprotein meat analog, originated from fungus, is used as a healthy food alternative for its high-protein in the improvement or management of immune system, have potential antimicrobial effects, helps in reducing risk of cardiovascular and gastrointestinal diseases with improved physiological functions, decreases risk of low bone mass as well as very high levels of antioxidant activity (paul et al., 2019). in addition, high-moisture extrusion (hme), over 40% of moisture contents during processing, has the enormous advantage of lesser energy input, lower waste discharge, higher efficiency, and more excellent value of texturized products. therefore, it is lately considered as the better choice for developing plant protein-based meat substitutes (zhang et al., 2019). for texture optimization, exogenous polysaccharides are one of the functional primary additives used in food industries. at the time when proteins are denatured during the extrusion process, the dormant reactive sites of the interior proteins would become available, and the structure proteins would become flexible, which permits the protein-polysaccharide interactions. polysaccharides could be used as crosslinker to alter the conformation of proteins, interact with proteins by cross-linking to protein side chains through maillard reaction, and form a complex structure of protein (caillard et al., 2010). table 2. a comparison among cultured meat, plant-based meat, and animal-based meat. dimension cultured meat plant-based meat animal-based meat references background information origins, history, and technical operation idea articulated around 1930. since 2000s, research into animal-cell culturing to produce meat. available in the market for several decades, made from concentrated proteinsoy, wheat, pea by extrusion or coagulation. traditional in china, japan, research on cell cultures for various production purposes since early 2000s. (bryant & barnett, 2018; sharma et al., 2015; smetana et al., 2015) nutritional value identical of regular meat. amino-acid profile nutritionally to meat. rich in protein but extraction needed due to indigestible walls. consumption and production rates and patterns not available in the market (optimists predict market introduction by 2019). established. modest, relatively stable market share, increase since by 2010. products from extracted protein. position as meat alternative plant meats are more sustainable and animal friendly than animal-based meat. functional equivalent to meat. potentially proteinrich source for human consumption. products rising. current practice and situation technological proof of principle in 2013. ongoing research and development. use of purified ingredients, increased efforts to improve meat similarity. growing and processing under research, have many challenges. (berghout et al., 2015; dekkers et al., 2018; osen et al., 2014; van der weele et al., 2019) lifestyle and consumption proper meat produced without animal suffering. established as vegan alternatives replacing meat. novel: green product color and market profile. supporters and opposition attracts venture capitalists and entrepreneurs in silicon valley, animal welfare organizations, and other innovators. emerging interest from meat industry. mix of alternative retail sector, experiments with hybrids of meat also startups and meat companies. pioneer companies and scientists, no consolidated coalition. (aiking, 2011; aiking et al., 2006; alexandratos & bruinsma, 2012) 12 italian journal of food science, 2022; 34 (2) islam m et al. and low-fat content, and quality texture. the primary function of meat analogs is to replace the animal meat in the human diet. the consumers of soybean-based meat analogs include not only vegetarians but also the non-vegetarians who want to reduce their meat consumption for health reasons. for example, soybean meat analogs contain protein (50 to 95%, dry matter), and soy protein is primarily used as the protein ingredient (chen et al., 2010). samard et al. (2019) demonstrated the high-moisture meat analogs are coupled with a higher integrity index and they have the stability of springiness as well as cutting strength than low-moisture meat substitute which is produced using the similar formula and screw-speed. the mechanical approaches summarized to make plant-based meat analogs are displayed in table 3. high-moisture extrusion of soy meat the hme approach can be used to impart a specific texture to proteins such as soy-proteins, whey-proteins, pea-protein, or wheat-gluten (pietsch et al., 2017; wolz et al., 2016). hme is a crucial technology for transforming plant-based protein into palatable meat-like products. during the hme process, the input extruder of moisture content is more than 40%, which results in the advantages of lower energy input and higher quality of the texturized products (zhang et al., 2019). chen et al. (2010) described that when moisture content of soy meat is increased from 28 to 60% (wet base), the indicator residence time and specific mechanical energy (sme) are significantly decreased. in addition, when cooking temperature is increased from 140 to 150°c, the in-line viscosity at die and sme reduced considerably, particularly at lower moisture contents (lmc). most studies found the effects of processing parameters were done for low to moderate moisture processing. still, these parameters can significantly affect protein structure during high-moisture processing (palanisamy et al., 2019). due to its desirable texture and nutritional value, soy protein can be used to make soy protein products by the extrusion process (wu et  al., 2019). for the traditional extrusion methods, extrusion at higher moisture conditions would reduce viscous dissipation at the lower extrusion temperature. still, these changes would be expected during high-moisture extrusion conditions (macdonald et al., 2009). zahari et al. (2020) reported the chosen parameters including concentration (0, 20, 40, 60%) of hemp protein concentrate (hpc), target moisture content (65, 70, 75%), temperature (40–120°c), and screw speed (300–800 rpm), whereas, spi was extruded with 500 rpm at 70 and 75% of target moisture content. to summarize some literature, extrusion parameters and selected formulations of high-moisture meat analogs are shown in table 4. macdonald et al. (2009) also reported that high-moisture extrusion was useful to generate high-quality protein foods. mechanical treatment is more effective during the high-moisture extrusion process for other plant proteins when compared with wheat gluten. therefore, the influence of hme processing on the change of protein-protein interactions is used to form the anisotropic structures of spis (chen et al., 2011; fang et al., 2014). die-cutting die-cutting is a critical technology used to bring out the indentation of die-cutting of the surface to retouch the processing equipment, where this technique is a complex core unit. the die-cutting indentation position must be precise to cause simultaneously, and the complete platform stress must assure that the place of two active faces in contacts top and bottom to be mutually parallel. shen et al. (2012) reported the speed of 7000 rh-1, travelling schedule 61 mm, cap board located 80°, the cam midpoint distance was 279 mm in a stand die-cutting machine. for instance, “the follower maximum pivot angle is 20°, main cam swing follower 240-mm long, roller radius 15  mm, vice-cam swing follower 240-mm long, roller radius 15 mm, the base circle initial radius is 60 mm. moreover, the cam angle for outer dwell is 110°, the cam angle for inner dwell is 10°, work travel angle of follower is 150°, return travel angle of follower is 90°, allowed pressure angle of actuating travel is 35° allowed pressure angle of return travel is 60°, and allowable curvature radius of the real contour line of cam is 3 mm in a platform die-cutting machine (shen et al., 2012). table 3. overview of mechanistic techniques to make plant-based meat analogs. technique starting material equipment type product process key technology references bottom-up strategy – – structure/ structural element – length scale anisotropy (post, 2012) wet spinning protein isolate, coagulation bath wet spinning setup: barrel, spinning nozzle, water bath, winding device fibers – micrometer (post, 2012) italian journal of food science, 2022; 34 (2) 13 effect of high-moisture extrusion the oxidation of polyunsaturated fatty acids, and also may also be responsible for turning into volatile off-flavors. characteristics of soy meat microstructure scanning electron microscopy (sem), light microscopy (lm), fourier transform infrared spectroscopy (ft-ir), as well as differential scanning calorimeter (dsc) are usually used to obtain more detailed information on the protein network microstructure of the meat analog. the micro-extraction technique is an environment-friendly procedure due to the reduction of polluting solvents and sample volume. for instance, the micro-extraction approach increases the extraction yield and diminishes the sample equilibrium time (barzegar et al., 2019). micro-extraction includes several performances such as single drop micro-extraction (sdme), dispersive liquid–liquid micro-extraction (dllme) and hollow fiber micro-extraction (hflpme). preece et al. (2017) reported “the cotyledon-cells protein bodies found in size range from 2.4 to 13.5 μm when used sem approach without sample-hydration.” lakemond et al. (2000) described two significant storage proteins that compose 60–80% of the total soybean protein: the β-conglycinin and glycinin. soybean protein is a complex mixture containing various table 4. extrusion-parameters involved for screening and selected formulations of high-moisture meat analogs (hmma). formulation speed of screw targeted moisture content (%) temperature (oc) (zone 1–2–3–4) visual appearance color references 0% hpc and 100% soy spi 500 70 40–60–80–100 light color, compact zahari et al. (2020)500 75 light color, compact 20% hpc and 80% soy spi 800 65 40–60–80–100 compact zahari et al. (2020)800 70 compact 800 75 less compact 600 75 less compact 600 80 – 400 75 less compact 300 75 less compact 40% hpc and 60% soy spi 800 65 40–60–80–100 compact, dark spot zahari et al. (2020)800 70 compact 800 75 pale, less compact 60% hpc and 40% soy spi 800 60 40–60–80–100 – zahari et al. (2020)800 62.5 – 800 65 less compact 800 70 less compact 800 75 came out foamy 800 65 60–80–100–120 less compact hpc: hemp protein concentrate; spi: soy protein isolate. die pressure the viscosity of the molten blend may be attributed to the decrease in pressure with the increase in temperature. the growth in feed moisture and die temperature then affects the mass thickness over the extruder and decreases the die-pressure value. zhang et al. (2020a) found that the coefficient variation of the die pressure and die temperature was 26.71 and 1.74%. in addition, the measured die pressure in extrusion cooking of apple meat blend ranged from 9 to 16 mpa. the negative coefficient of the first-order term of temperature, screw speed, and moisture content indicated that die pressure increased with temperature decrease (singha & muthukumarappan, 2017). high-pressure homogenization high-pressure homogenization (hph) is an applicable unit operation based on cavitation to improve processed soybean materials’ extraction yields. debruyne (2006) demonstrated that homogenization could cause a negative effect on separation efficiency. in addition, investigations may require evaluating the scalability of this promising result obtained by using a lab-scale homogenizer. denaturation of the lipoxygenase occurred of enzyme high-temperature treatment may also catalyze 14 italian journal of food science, 2022; 34 (2) islam m et al. tehrani (2014) found that adding soy-flour, flour of splitpea, and wheat-starch could improve low-fat hamburger’s texture properties by reducing shrinkage. for example, smith et al. (1976) proved that the presence of textured soy protein was associated with the substantial reduction of shrinkage in the blended ground. the transversal cutting strength of texturized vegetable protein (tvp) and meat samples was slightly higher than their longitudinal cutting strength. it is stated that the cutting strength in parallel and vertical directions of extrudates could indicate the texturization degree or fibrous-structure formation (fang et al., 2014; gu & ryu, 2017). when sme is decreased, there is an increase in instrumental chewiness and hardness. as a result, instrumental chewiness and hardness of meat analogs increased with decreasing spc-wg ratio (fiorentini et al., 2020). additionally, fang et al. (2014) reported that the instrumental hardness and chewiness of texturized soybean proteins increased more than 22 and 17%, respectively. day and swanson (2013) described that high chewiness in meat analogs corresponded with low sme values, indicative of low-melt viscosity. to summarize the literature, common soy meat analogs available in the market are presented in table 5, which were both prominently reduced due to the denaturation of protein molecules. anti-nutritional factors a wide variety of anti-nutritional substances are available in most of the potential and alternative plant-derived nutrient sources. metabolic products arising in living systems may be defined as anti-nutrient substances that affect health or food production by themselves or through their food utilization. anti-nutritional substances could be usually divided into four groups: (1) factors that affect mineral utilization, including gossypol pigments, phytates (hexa-phosphates of myo-inositol), oxalates and glucosinolates, (2) factors that affect protein utilization and digestion, including lectins, protease inhibitors and tannins, (3) anti-vitamins, (4) miscellaneous substances for example, mycotoxins, cyanogens, mimosine, alkaloids, nitrate, phytoestrogens, photosensitizing agents, and saponins. besides, ma et al. (2020) recounted those anti-nutritional compounds that reduced the bio-availability of the essential nutrients or energy in the diet. reducing the content of the anti-nutritional factors (anfs) can efficiently improve the use of soy nutrients. based on protein content, products from soy-protein are divided into three broad categories: (1) soy flour and grits (50% protein on a moisture-free basis); (2) soy protein concentrates (70% protein), and (3) isolated soy proteins (90% protein) reported by singh et al. (2008). in addition, thadavathi et  al. (2019) reported that the high proteins, and each of them has unusual denaturation temperatures. glycinin and β-conglycinin are two main storage proteins, and their denaturation temperatures were 68 and 86°c, correspondingly (peng et al., 2016). throughout the low moisture extrusion, the ingredients can undergo structural changes caused by high temperature and shear. it can also affect the product’s characteristics, such as microstructure and expansion (beck et al., 2018). dsc, tg and dtg measurements the differential scanning calorimetry (dsc) analysis can be conducted with thermal-analysis-systems model q-200. non-isothermal degradation can be measured by using thermogravimetric/differential-thermal-analysis (tg/dta). the thermal analysis techniques such as thermogravimetric-analysis (tga) can provide information on thermal stability, including thermal-degradation of protein films. zhang et al. (2020b) observed that the mixtures pass through extruder from the mixing-zone to the melting-zone for peanut protein/exogenous polysaccharide mixture, the endothermic-peaks of arachin and conarachin were both prominently reduced due to the denaturation of protein-molecules. in the mixing zone, 2% ws (wheat starch) could cause a significant decrease in the thermal transition peak temperature (tp) value and a significant increase in the enthalpy changes (δh) value of the conarachin. as a result, it indicated that 2% ws accelerated the thermal transition of conarachin and the energy required to open the increased molecule. guo et al. (2012) described that exogenous polysaccharides had no significant effect on the δh value of arachin, due to the relatively tight structure of arachin. in addition, zhang et al. (2020b) found that the exogenous polysaccharides had enhanced the protein-lipid interaction, when 0.1% ca or 2% ws was added. moreover, the exogenous polysaccharides could decrease, particularly when 2% ws was added, and the value significantly reduced from 310 to 302°c. textural properties there are two textural properties as the transverse (t) and directions cutting force in longitudinal (l) are positively correlated with the carrageenan (icgn) concentration in the meat analog sample. the cutting force can also be interpreted as an indirect indicator of texturization and hardness (palanisamy et al., 2018). the elasticity differences between the raw samples containing 0.75, 1.5, and 3% icgn were not significant, while no significant differences between all icgn added cooked samples could be detected. shahiri tabarestani and mazaheri italian journal of food science, 2022; 34 (2) 15 effect of high-moisture extrusion protein vegetable-based foods were used as an alternative for meat products which was enormously advantageous for the increasing human population in the world, and it could address the environmental concerns as well as the health considerations from meat consumption. moreover, aijie et al. (2014) reported that the anfs decreased during germination, which was attributed to the activation of several enzymes in the seed. the summarized literature on anti-nutrients and anfs of soybean are presented in table 6. allergenic protein there are many kinds of allergenic compounds and anf’s present in soybean. according to the report of food allergens, almost 2% of adults and 5–8% of children have food allergic reactions associated with the consumption of soybean or soybean derived food products (john et al., 2017). additionally, gagnon et al. (2010) reported that food allergies are a big concern for health in most countries. in addition, allergic reactions to food affect 4–6% of children and 1.5% of adults (uguz et al., 2005). there are different allergenic proteins present in soybeans. however, soybean proteins allergic reactions are mostly transient and non-life-threatening and are usually outgrown after 3 years of age. it seems to be tolerant within 2–5 years after the initial diagnosis. soybean allergenic proteins can be detecting to soybean allergens. in recent years, researches about allergenic soybean proteins detection have been rapidly expanded. currently using two most approaches table 5. common soybean meat analogs existing in market. name of product introduction/first reported main ingredients/origin characteristics/remarks references tofu china pressed soy curd prepared from coagulated soy. most widely recognized meat alternatives, blind taste, can impart flavor by smoking/marinating. (sadler, 2004) tivall 1997 soy-based fibrous vegetable protein. simulate meat muscle, provide a different eating textures to other soy formats. (sadler, 2004) tempeh 1851 in indonesia fermented soy-based cake. controlled fermentation of soy leads, similar shape to burger patties. (kumar et al., 2017; malav et al., 2015) grillers original burgers extruded vegetable protein burgers. veggie goodness, soy protein concentrate, water for hydration. (kyriakopoulou et al., 2019) schnitze rehydrated soy protein products. – (kyriakopoulou et al., 2019) table 6. comparison of the soybean and other grains substitute of anti-nutritional factors and fractions. plant substitutes anti-nutritional factors references soybean meal protease inhibitors, saponins, anti-vitamins , phytic acid, lectins, phytoestrogens and allergens. (francis et al., 2001)rapeseed meal rapeseed meal, protease-inhibitors, phytic acid glucosinolates and tannins. pea seed meal pea seed protease-inhibitors, tannins, lectins, cyanogens and phytic acid. sesame meal sesame phytic acid and protease inhibitors. cottonseed meal cottonseed meal phytic-acid, phyto-estrogens, gossypol and anti-vitamins. various grains grains fractions arabinoxylans β-glucans cellulose soybean soluble – – – (choct, 1997) insoluble – – 4.4% wheat soluble 1.8% 0.4% – insoluble 6.3% 0.4% 2% barley soluble 0.8% 3.6% – insoluble 7.1% 0.7% 3.9% maize soluble 0.1% – – insoluble 5.1% – 2% sorghum soluble 0.1% 0.1% – insoluble 2% 0.1% 2.2% 16 italian journal of food science, 2022; 34 (2) islam m et al. additives, such as starch or lipid, probably have a positive or negative effect on forming the desired texture. furthermore, asgar et al. (2010) reported that other types of legumes and oilseeds could also be used as protein-rich materials to develop a variety of high-moisture meat analogs, which could contribute to alternative protein sources. market potential alternate plant proteins in contemporary years, plant-based proteins have received increasing attention as good substitutes for animal-based proteins. the important reasons for the increasing acceptability of plant-based protein are the low cost and fibrous texture (echeverria-jaramillo et al., 2021). plant-based protein’s growing trend is setting out to increase the number of vegetarians or meat avoiders (aschemann-witzel et al., 2021). proteins, in particular, plant-based proteins are more important in the face of future challenges, ensuing from unceasing population growth and the unevenness between malnutrition and overweight/obesity (mittermeier-kleßinger et al., 2021). time trends for the development of alternative protein ingredients are demonstrated in table 7. the key drivers of market growth include: (1) consumers concern over food safety in relation to animal products; (2) growth in the number of vegetarians, meat avoiders, and meat reducers; (3) meat eaters seeking more variety in their diet; (4) growing interest in healthy eating which includes incorporating more plant-based foods into the diet. conclusion this review concluded that high-pressure extrusion is an effective technology that can potentially be used to produce natural food products in which heat treatment will be reduced. this enhances the hydrophobic interactions and increases the visible viscosity to stabilize the newly formed conformation of the texturized effects. plant proteins have some physiologically active components such as protease phytosterols, inhibitors saponins, and isoflavones. the thermo-mechanical treatment affects the microstructural changes and the rheological properties in spc during high-moisture extrusion processing. therefore, the importance of soybean meat analog products as an alternative soy meat source that should be explored along with studies to clarify the underlying effects of the quality and increasing acceptability of plant-based protein are the low cost and fibrous texture. future study are required on protein-extraction yield and purification which could be predicted well using another mechanistic model developed as well as on flavor and texture meat analog products. like enzyme linked immunosorbent assays (elisa) and immunoblotting, also using by radio allegro-sorbent test inhibition, enzyme allego-sorbent test, polymerase chain reaction, mass spectrometry, high-performance liquid chromatography (wang et al., 2014). elisa is a powerful tool for detecting proteins, elisa such as sandwich elisa, competitive elisa and indirect elisa are widely used to determine soybean proteins in food products (wang et al., 2014). immunoblotting is a powerful research tool which can indicate molecular mass and immunoreactivity of allergenic proteins. beardslee (2000) found soybean allergenic proteins glycinin g1 acidic chain and a 22 kda g2 glycinin by using immunoblotting. sensory evaluation the mouthfeel and fibrousness are the same as meat, which are the primary sensory properties to consider when consuming the meat alternatives. the addition of iot-carrageenan (icgn) concentration is essential to increase the fibrousness of the product because of its denser structure. palanisamy et al. (2018) observed extra 2.25 to 3% icgn can increase elasticity which could be detected significantly with the overall scores’ acceptance ranged from 1.73 to 2.49%. the icgn concentration was positively correlated with the fibrousness; the hardness could also be influenced by the preference (palanisamy et  al., 2018). cheftel et al. (1992) reported that texturization with hme is entirely different from other protein texturization processes such as manufacturing cheese curds, sausages, tofu, in which fiber was formed by extrusion cooking or by spinning. in addition, proteins would be plasticized in the heating chamber during the extrusion process of texturizing a long cooling die at the end. the process could be optimized by varying the temperature, moisture, pressure, and shear. health characteristics of soybean regular consumption of soy products can reduce the risk of chronic diseases such as cancer, stroke, heart disease, and type 2 diabetes (jooyandeh, 2011). soy-based foods also provide beneficial health compounds, including vitamins, minerals, fiber, and flavonoids. in addition, various clinical trials have investigated the potential of soybean and soybean products to protect against the risk of chronic diseases. furthermore, scheiber et al. 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was carried out to detect the concentration of fatty acid in female and male specimens of commercially important giant red shrimp (aristaeomorpha foliacea) obtained from (including 20 male shrimps and also 20 female shrimp) mediterranean sea. in fatty acid composition, the saturated fatty acid fraction was dominant, followed by polyunsaturated fatty acid and monounsaturated fatty acid for both sexes. the analyses indicated that pufas, and the mufas content were higher in female shrimp than in those of males and they were statistically significant differences in fatty acid profile between females and males (p<0.05). mailto:ilkanali%40yahoo.com?subject= ital. j. food sci., vol. 27 2015 433 introduction the giant red shrimp (aristaeomorpha foliacea risso, 1827) belongs to the family aristeidae, which includes other important species such as the blue and red shrimps (aristeus antennatus risso, 1816) and the scarlet shrimp (plesiopenaeus edwarsianus johnson, 1868) (ragonese et al., 1997). a. foliacea is widely distributed in the eastern and western atlantic, indian ocean and western pacific, in the waters of japan, australia, new zealand and in the mediterranean sea. in the mediterranean sea, the species inhabits muddy bottoms of the continental slope approximately between 100 and 1200 m depth. this species plays an important role in the overall biomass of the mediterranean sea and represent an important commercial resource among the other shrimp species since 1959 (d’onghia et al., 1998; desantis et al., 2003; fernandez et al., 2011). due to its economic relevance, recently there are many studies on this species from mediterranean sea and there has been a considerable amount of research on nutritional value of various species of shrimp. however there is not any data on the nutritional and fatty acids composition of a. foliacea. seafoods are important source of nutrients in the human diet. crustaceans such as shrimps have high nutritive value, are low in fat, especially saturated fatty acids; contain high amount of polyunsaturated fatty acids (omega-3 and omega 6) (oksuz et al., 2009; tag el-din et al., 2009; turan et al., 2011; shalini et al., 2013). these fatty acids could not be synthesised by the human body and must be obtained through the diet are crucial for normal brain structure and function (alasalvar et al., 2002; richardson, 2003). in addition to this, these fatty acids have great importance to humans for prevention of coronary artery diseases, diabetes, hypertension and cancer (visentainer et al., 2007; cengiz et al., 2012). the levels of these fatty acids are low in many modern diets, particularly those in which highly processed foods predominate. the omega-3 polyunsaturated fatty acids (pufas) that the human needs (epa and dha) are found in appreciable quantities only in oily fish, seafood, aquatic invertebrates and algae (richardson, 2003; gökce et al., 2011). therefore, this study was carried out to determine the nutritive value and fatty acid content of giant red shrimp collected from the mediterranean sea of turkey. materials and methods collection and preparation of samples the samples were caught by bottom trawlers between 450 and 500 m of depth, during in 2013 from mediterranean sea of turkey (36˚ 22’ 707”n 24˚ 25’ 941” e /36˚ 14’ 919” n34˚ 19’ 163” e). immediately, after collection, shrimps were stored in a container, preserved in crushed ice and transferred to the laboratory, where the heads, shells and intestines were separated and placed in labeled polyethylene bags respectively and stored at -20°c until processing for analysis. for each season, 20 female and 20 male samples of a. foliacea were obtained by random sub-sampling. fatty acid analysis the samples were transported with dry ice to the accredited industrial services laboratory of turkey/istanbul. the methyl esters of fatty acids of samples were prepared according to iupac methods ii. d. 19 (1979). the analyses were carried out by using a perkin elmer autosystem. xl gas chromotography and flame ionization detector (fid) equipment and a supelco 2330 fused silica capillary column (30 m x 0.25 mm x 0.20 μm film thickness) for determining the fatty acid composition. data analysis for data analysis independent samples t-test was used to identify significant differences in fatty acid concentration. statistical significance was defined at p<0.05. the mean values were obtained from 3 experiments and reported as means±sd (dinçer and aydin, 2014). results and conclusions table 1 shows mean weights (g) of female and male species of shrimp (aristaeomorpha foliacea) obtained from mediterranean sea. table 1 the nutritional value and mean weights (g) of female and male shrimps. parameter female male weight (g) 29.96a±6.53 12.26b±2.07 protein (%) 15.72a±1.02 18.0b±0.80 lipid (%) 0.72a±0.11 0.51b±0.13 different letters (a,b) in the same row represent significant statistical differences (p<0.05). the mean weight for female shrimps was found to be higher than the mean weights for male shrimps. similar results were reported by yilmaz and yilmaz (2007) for penaeus semisulcatus collected from mediterranean sea of turkey and also by turkmen (2012) and cevik et al. (2008) for penaeus kerathurus and parapenaeus longirostris respectively. our findings are consistent with prior research. the levels of protein and lipid vary depending upon season, age, maturity, sex, water temperature, spawning cycle and availability of food, types of diet and 434 ital. j. food sci., vol. 27 2015 feeding system of organism (oksuz et al., 2009; turan et al., 2011; rosli et al., 2012). in a study on crangon crangon protein and lipid content were 18.47 and 0.95% respectively (turan et al., 2011). saglik and imre (1997) determined total lipid as 0.93% for parapenaeus longirostris and 0.58% for penaeus semisulcatus. yanar et al. (2011) found that protein and lipid of penaeus semisulcatus ranged between 22.76-23.53% and 0.76-1.44% respectively. fatima et al. (2012) reported that lipid in the muscle tissue of fenneropenaeus penicillatus varied from 0.92 to 1.0% and of f. merguiensis from 0.87 to 0.98%. protein and lipid were also reported as 20% and 1.1% for parapenaeus longirostris and 14.2% and 2.6% for plesionika martia by oksuz et al. (2009). dincer and aydin (2014) determined that protein and lipid of metapenaeus affinis ranged between 18.4-19.1% and 1.07-1.30% respectively. in the present study the content of protein and lipid were identified as slightly lower than those reported previously for some shrimp species. the main reason for this is thought to be related to variation in seasonal feeding habits (different types of diet and feeding system) and habitats. in the study, the protein content for male shrimps was found to be higher than the protein content for female shrimps whereas the lipid content was found to be lower in male shrimp (p<0.05). similar results were reported by dincer and aydin (2014) for female and male species of metapenaeus affinis. the ratios of pufa/sfa and n-6/n-3 and the fatty acid compositions of the investigated shrimp are presented in table 2. the fatty acids analyzed were grouped as saturated fatty acids (sfas), monounsaturated fatty acids (mufas) and polyunsaturated fatty acids (pufas). in the present study, in both groups, sfa was the highest followed by pufa and mufa. these results were in agreement with that obtained by turan et al. (2011) who reported highest levels of sfa followed by pufa and mufa for brown shrimp (crangon crangon) from sinop region, black sea. similar results were also reported by ouraji et al. (2011) for wild indian white shrimps (fenneropenaeus indicus); by oksuz et al. (2009) for rose shrimp (parapenaeus longirostris) and red shrimp (plesionika martia); by yanar et al. (2011) for penaeus semisulcatus; by fatima et al. (2012) for fenneropenaeus merguiensis and f. penicillatus and by dincer and aydin (2014) for metapenaeus affinis. according to the results, c16:0 (palmitic acid) and c18:0 (stearic acid) were the main saturated fatty acids in both shrimp species. in both sexes, the predominant monounsaturated fatty acids were found as c18:1 (oleic acid). the principal acids in pufa group were eicosapentaenoic acid (c20:5, epa), docosahexaenoic acid (c22:6, dha) and linoleic acid (c18:2) for female and male shrimp species. these results agree with studies on fatty acids found in other shrimp species (oksuz et al., 2009; tag eldin et al., 2009; saglik and imre 1997; ouraji et al., 2011; turan et al., 2011; yanar et al., 2011; fatima et al., 2012). in the present study, the rate of sfas, pufas and mufas were determined as 43.69%, 29.33% and 24.37% for female shrimps and as 47.15%, 25.41% and 17.34% for male shrimps respectively. however, different percentage compositions of fatty acids obtained from various species and subspecies of sea and freshwater shrimps were also reported by several authors. these differences among species might be associated with the different characteristics of the shrimp species (karuppasamy et al., 2013). in a study, turan et al. (2011) reported sfa, mufa and pufa rates in browncolor shrimp at 33.04, 22.17 and 29% respectively. oksuz et al. (2009) reported the mufa rate in p. longirostris and p. martia at 26.09% and 34.47% respectively. ouraji (2011) reported the rate of sfa in wild white indian shrimp and its cultured specimen at 32.88 and 33.79% respectively. emami et al. (2014) reported that the rate of sfa in penaeus vannamei at 37.26%, in penaeus semisulcatus at 49.12% and the rate of mufa in p. vannamei at 24.9%, in p. semisulcatus at 33.76% and the pufa in p. vannamei at 37.84%, in p. semisulcatus at 16.9% respectable 2 fatty acid composition of female (f) and male (m) shrimps. parameters female (%) male (%) c6:0 1.23a±0.03 2.70b±0.01 c8:0 0.11±0.01 n.d c14:0 3.43a±0.03 2.16b±0.10 c16:0 27.59a±1.20 27.29a±0.60 c18:0 11.10a±0.04 14.37b±0.75 c24:0 0.23a±0.04 0.63b±0.03 ∑sfa 43.69a 47.15b c16:1 2.69a±0.20 1.66b±0.23 c18:1 21.68a±0.70 15.68b±1.00 ∑mufa 24.37a 17.34b c18:2n6 6.26a±0.02 4.41b±0.60 c20:3n3 0.11±0.01 n.d c20:5n3(epa) 13.36a±0.50 11.47b±0.60 c22:6n3(dha) 9.60a±0.50 9.53a±0.05 ∑pufa 29.33a 25.41b pufa/sfa 0.67 0.54 ∑n3 23.07 21.00 ∑n6 6.26 4.41 n6/n3 0.27 0.21 unidentified 2.61 10.1 n.d.: below detection limit; data are expressed as mean±sd of triplicate measurements. different letters (a,b) in the same row represent significant statistical differences (p<0.05). ital. j. food sci., vol. 27 2015 435 tively. the results obtained in this study showed slightly similarity to the findings of the mentioned researchers. this difference may be due to geographical variation, seasonal conditions and different types of diet and feeding system. fatty acid content is also influenced by species, maturity period, size and age of shrimp. the indices of pufa/sfa and n-6/n-3 ratios were widely used to evaluate the nutritional value of fat for human consumption. according to some nutritional recommendations the pufa/sfa ratio in human diets should be above 0.45 and, within the pufa, the n-6/n-3 ratio should not exceed 4.0 (alfaia et al., 2010. in the present study the pufa/sfa and n-6/n-3 ratios of a. foliacea (for both female and male shrimps) were within the range reported for human diets. it could be demonstrated that the giant red shrimp (a. foliacea) is a desirable item in human diet when the levels of n3/n6 and pufa/sfa ratios were considered. comparison of fatty acid composition between two sexes the fatty acid compositions of female shrimp species found to be 43.69% saturated (sfas), 29.33% polyunsaturated acids (pufas) and 24.37% monounsaturated (mufas) whereas the fatty acid compositions of male shrimp consist of 47.15% saturated (sfas), 25.41% polyunsaturated acids (pufas) and 17.34% monounsaturated (mufas). among these the highest concentrations of sfas (47.15%) were detected in male shrimp species while the highest concentrations of pufas (29.33%) and mufas (24.37%) were detected in female shrimp. there is a significant difference between the sfa, pufa and mufa profiles in both sexes (p<0.05). similar results were reported for female and male species of metapenaeus affinis by dincer and aydin (2014) and by eskandari et al. (2014) for female and male species of m. affinis. based on results, the amount of palmitic acid (c16:0) for female shrimp (27.59%) was almost the same as in male shrimp species (27.29%) (p>0.05), while the amount of oleic acid (c18:1) (21.68%) was higher than those in male shrimp (15.68%) (p<0.05). the present study also showed that the amount of docosahexaenoic acid (c22:6, dha) of female shrimp (9.60%) are almost the same as in male shrimp species (9.53%) (p>0.05) whereas the levels of eicosapentaenoic acid (c20:5, epa) and linoleic acid (c18:2) were higher than those in male shrimp (p<0.05). in a study, dincer and aydin (2014) reported that the epa content of male metapenaeus affinis was lower than female m. affinis. the ratio of pufa to sfa (0.54) and n-6 to n-3 (0.21) for the male shrimp was found to be lower than those in female shrimp. although both shrimps were subjected to the same sea water and climate conditions, there were naturally some differences between them, in terms of their size, sex and quantity of lipid. in conclusion, from a nutritional point of view, both male and female giant red shrimps demonstrated acceptable quality; in particular, the female giant red shrimps had the highest levels of pufas, and the mufas content. both sexes are low in fat and are considered to belong to a low fat class group. further investigations are required to obtain more information about this species. acknowledgments the authors are grateful to assist. prof. dr. yusuf kenan bayhan for providing the shrimp samples for the study. references alfaia c.m.m., alves s.p., lopes a.f., fernandes m.j.e., costa a.s.h., fontes c.m.g.a., castro m.l.f., bessa r.j.b. and prates j.a.m. 2010. effect of cooking methods on fatty acids, conjugated isomers of linoleic acid and nutritional quality of beef intramuscular fat. meat sci. 84: 769. alasalvar c, taylor k.d.a., zubcov e., shahidi f. and alexis m. 2002. differentation of cultured and wild sea bass (dicentrarchus labrax): total lipid content, fatty acid and trace mineral composition. food chem. 79: 145. cengiz e.i., ünlü e., bashan m., satar a. and uysal e. 2012. effects of seasonal variations on the fatty acid composition of total lipid, phospholipid and triacylglycerol in the dorsal muscle of mesopotamian catfish (silurus triostegus, heckel, 1843) in tigris river (turkey) turk. j. fish. aquat. sci. 12: 33. çevik f., bayhan y.k. and derici o. 2008. metal concentrations in the muscle of male and female shrimp (parapenaeus longirostris, lucas, 1846) collected from marmara sea and their relationships with season. asian j. chem. 20 (3): 2229. desantis s., labate m, cirillo f., labate g.m., maiorano p. and onghia g.d. 2003. testicular activity and sperm glycoproteins in giant red shrimp aristaeomorpha foliacea. j. northw atl. fish. sci. 31: 205. dinçer m.t. and aydın i. 2014. proximate composition and mineral and fatty acid profiles of male and female jinga shrimps (metapenaeus affinis, h. milne edwards, 1837) turk j. vet. anim. sci. 38: 445. d’onghia g., maiorano p., matarrese a. and tursi a. 1998. distribution biology and population dynamics of aristaeomorpha foliacea (risso, 1827) (decapoda, natantia, aristeidae) in the north-western ionian sea (mediterranean sea). crustaceana. 71(5): 518. emami s.m., matinfar a., kamali a. and soltani m. 2014. fatty acid and amino ccid composition of marine (penaeus semisulcatus) and farmed (penaeus vannamei) shrimp species from bushehr. iran. j. appl. environ. biol. sci. 4(4): 262. eskandari s., bitaab m.a., abtahi b., ghaffari f., namin m.m. and khorjestan s.m. 2014. determination of fatty acids composition in persian gulf shrimp, metapenaeus affinis. j. paramedical sci. 5 (2): 212. fatima h., ayub z., siddiqui g. and ali s.a. 2012. fatty acid composition of two candidate species of aquaculture, fenneropenaeus merguiensis and f. penicillatus (crustacea: decapoda) in pakistan. pakistan j. zool. 44(4): 969. fernàndez m.v., maltagliati f., pannacciulli f.g. and roldàn m.i. 2011. analysis of genetic variability in aristaeomorpha foliacea (crustacea, aristeidae) using dna-issr (inter simple sequence repeats) markers. c. r. biologies. 334: 705. gökçe m.a., tasbozan o., tabakoglu s.s., çelik m., ozcan f. and basusta a. 2011. proximate composition and fat436 ital. j. food sci., vol. 27 2015 ty acid profile of shabbout (barbus grypus, heckel, 1843) caught from the ataturk dam lake, turkey. j. food agric. environ. 9 (2): 148. i.u.p.a.c. 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ital. j. food sci. 23: 1. yılmaz a.b. and yılmaz l. 2007. influences of sex and seasons on levels of heavy metals in tissues of green tiger shrimp (penaeus semisulcatus de hann, 1844). food chem. 101: 1664. paper received october 14, 2014 accepted january 7, 2015 paper ital. j. food sci., vol. 27 2015 345 keywords: bread, flour, potato starch, wheat bread quality substituted by potato starch instead of wheat flour f. nemar1*, a. dilmi bouras1, m. koiche1, n-e. assal2, a. mezaini1 and j. prodhomme2 1laboratory of natural local bio-resources, department of biology, faculty of science, hassiba benbouali university, chlef, bp. 151, chlef 02000, algeria 2lactamel group, sidi bel abbes, algeria *corresponding author: tel. +213 559 932507, email: fawzia.nemar@gmail.com, f.nemar@univ-chlef.dz abstract wheat bread constitutes the most regularly consumed food in the world, the international market for wheat undergoes strong pressure and prices are unceasingly increasing. the aim of this study is to substitute wheat flour by potato starch in bread preparation. mixtures flours were characterized for composition, damaged starch, and alveograph properties. according to the results of alveograph parameters, they decrease with the rate of incorporation of potato starch. this decrease can be corrected by adding vital gluten. the results of physicochemical analysis showed a decrease in protein levels, an increase in moisture content (about 2%) and carbohydrates levels due to the composition of potato starch. however, sensory analysis (p ≤ 0.05) showed that the addition 80% of potato starch leads to bread with better characteristics: taste, colour and odour, based on that, it is highly advisable as an ingredient in the standard preparation of wheat bread. 346 ital. j. food sci., vol. 27 2015 introduction bread is an important component of the algerian diet, while wheat production in the country is insufficient. therefore, substantial quantities of this cereal must be imported every year. making bread by partial substitution of wheat is not a new idea and it is worthwhile to reveal some of the efforts made in the past to make bread from local materials, such as cereal flour or root starches. potato occupies the fourth place in the world list of food crops, after wheat, rice and corn, with an annual world production of approximately 300 million mg (cip, 2008). the country now produces enough potatoes and its price is also within affordable limits of average people. so potato appears to be one of the most promising substitutes in bread making in order to help reduce dependence on wheat flour. potato starch is an important raw material in the food industry because its properties and their proportions vary according to the environment and genotypes of potato (vasanthan et al., 1999; kaur et al., 2002; zaidul et al., 2007). potato starch is largely used in food and non-food fields (paper, cardboard, textiles, mining, drilling, adhesives, etc.). originally, it was produced for baking by adding it to cereal flour (roussel et al., 1996; singh et al., 2003). the addition of modest amounts of potato starch helps preserve the freshness of bread and it also confers a distinctive character and a pleasant flavor (yanez et al., 1981; willard and hix, 1987). the aim of this work is to evaluate the possibility of substituting wheat flour for high percentages of potato starch in bread making process and to evaluate the physical, chemical, nutritional and sensory properties of the produced bread. material and methods raw material algerian wheat flour was obtained from nekhla mill, algeria. ingredients like sugar, salt, instant active dry yeast and shortening were purchased from the local market, while potato starch was obtained from michel come, rambouillet, france. methods physical and chemical composition moisture content the moisture content of samples was determined according to the aacc official methods 46-30, where a sample of 5 g is weighed and placed in a moisture dish. the sample is warmed to 130°c in an air oven during 2 hours; then the residue is cooled to room temperature and weighed (afnor, 1991). ash content it was determined according to the aacc official methods 08-01 (aacc, 1995). where a sample of 3-5 g is weighed and placed in an ash cup, then the sample is heated at 900°c in an ash oven until complete combustion of the organic matter, and the residue is cooled to room temperature and then weighed (aacc, 1995). protein content protein content is determined by the kjeldahl distillation method (by analyzing total nitrogen contents). two grams of dry sample are weighed and placed with hot concentrated sulfuric acid. the ammonia liberated from the resulting ammonium sulphate, after adding sodium hydroxide was distilled into 1 m boric acid then titrated with 0.1 m hcl. the nitrogen value estimated was multiplied by 5.7 (protein factor) to obtain the value of crud protein. this is expressed as the percentage of dry sample mass (aacc, 1995). fat content according to ugrinovits et al. (2004) the crude fats were determined by the soxhlet method. they are extracted from 10 g of each sample using a soxhlet apparatus with low boiling point petroleum ether (40-60°c) as solvent. a rotaryevaporator was used to evaporate the solvent after each extraction. falling number test the level of enzyme activity was measured by the falling number test (standard method aacc 56-81b), and this is to evaluate the α-amylase activity of the flour by measuring the consistency of the gelatinized starch. seven grams of the sample is weighed and combined with 25 ml of distilled water in a glass falling number tube with a stirrer and shaken to form a slurry. then, it is placed in the falling number instrument (aacc, 1995). alveograph characteristics  an amount of 250 g of the sample with salty solution was mixed in the alveograph mixer. after 8 minutes of kneading, the passage was opened and the extraction began. the dough patty was cut as soon as it arrived at a mark on the extraction plate. the dough patty was rolled and cut with the cutter and then was placed in the oven of the dough pieces at 25.5°c. after 28 minutes, each dough patty was inflated with air and its individual characteristics (p, l, w) were measured (aacc, 1995). ital. j. food sci., vol. 27 2015 347 w: the work of the deformation energy (baking strength); l: the length of the curve (extensibility); p: maximum height (tenacity); p / l: ratio curve configuration. bread making  the conventional straight-dough method for pan bread was performed according to the procedure developed by aacc. the formula used to make bread is given in table 1. the level of substitution of wheat flour by potato starch was 80%. to make bread, the dry ingredients were manually mixed and then added to a mix containing water. the components were thoroughly kneaded with the mixer for 5 min at low speed. the mixing speed was then changed to high speed for 5 min. the dough was divided into pieces of 100 g, rounded by hand and allowed to relax for 25 min. the dough was moulded then panned and fermented for 90 min at 30°c in a fermentation cabin. gas retention during fermentation was evaluated using an indicator of growth containing 25 g of dough which was subjected to fermentation in the same conditions as the dough. the bread was baked at 220°c/20 min in an electric oven. subsequently, the baked bread samples were then depanned and cooled to evaluate their external and internal properties by a 1h at room temperature, packed in polyethylene bags used for further analyses. bread evaluation loaves were organoleptically evaluated for their external and internal properties by a jury of twenty tasters. the method of 5 point score (in a hedonistic qualification scale) was used (amerine et al., 1973). the panel members were asked to score for crust colour, crumb colour, texture, flavour and overall acceptability. statistical analysis in this study, all experiments were performed in triplicate. statistical analysis was performed using xlstat program to compare the results. the level of significance was considered at p ≤ 0.05. results and discussion physical and chemical composition the result of proximate composition analysis of wheat flour and potato starch is as shown in table 2. the wheat flour protein content used in this study was about 10%, this result is similar to that reported by lindahl and eliasson (1992). according to ugrinovits et al. (2004) the strength of the flour is partially determined by its wet gluten content. the wet gluten content of potato starch is about 1.72% where as wet gluten content of wheat flour is 30.08%, which is a normal level, potato starch contained lower proteins (trace) and higher carbohydrate than wheat flour. however this value of wet gluten, added to the vital gluten in the mixture allows compensating for the deficit of protein potato starch (32.77%). the results of baking test show that potato starch alone is not enough to produce bread, and the same result was found for the mixture with high level. it might be due to the value of gluten that is lower. therefore, it is necessary to add a percentage of vital gluten. however, the value of the falling number of the table 1bread mix formula. formula wheat flour 500 g salt 10 g sugar 5 g yeast 10 g water 300 ml dough improver 0.1 g table 2 physical and chemical characteristics of mixtures. parameter\product 100% 100% s.p 20% f 80% s.p wh.g g moisture (%) 15.80±0.026 17.80±0.035 17.60±0.011 16.21±0.015 mineral (ash) (%) 0.53±0.025 0.18±0.02 0.19±0.0152 0.27±0.01 wet gluten (%) 30.10±0.155 / 1.72±0.092 32.77±0.196 falling number (s) 312 220 181 126 protein content (%) 10 trace / / fat content (%) 0.9 trace / / f: wheat flour; s.p: potato starch; wh.g: without gluten; g: with gluten. 348 ital. j. food sci., vol. 27 2015 mix is lower (220) than that of wheat flour and also lower than the optimal standards for bread which is 200 to 300 seconds (godon and loisel, 1997). this might be due to the decreased resistance of potato starch to enzymatic degradation. also, the fact that the gelatinization temperature is lower than that of wheat flour can be considered as another reason. alveograph characteristics the results of alveograph test, summarized in table 3, make it possible to predict baker quality of flour. this test is an interesting practice which is very appreciated by professionals of the second transformation, due to the fact that it reflects through alveographic parameters measured the ability of flour to be managed according to its baking strength for a specific purpose (roussel and chiron, 2002). the alveograph parameters of wheat flour and mixtures (with 80% of potato starch) showed (table 3), that overpressure (p), a measure of dough tenacity, which is an indicator of gas retention by the dough as indicated by wang et al. (2002), varied from 58 to 150 mm. the measure of alveograph dough extensibility (l), ranged from 45 to 116 mm. the values for curve configuration ratio, indicating the configuration ratio of the alveograph curve, varied from 1.09 to 2.25. the index of swelling (g) varied from 6.9 to 19.10 cm and the baking strength representing the energy necessary to inflate the dough bubble to the point of rupture ranged from 10 to 210*10-4 j. these differences of results are due to the addition of gluten which controls these parameters, where the p value increases to 99mm h 2 o. it is higher than the limit of 80 mm. these results show that the composite potato starch and wheat flour lead to dough which is less resistant to deformation and low extensibility for rate incorporation of 80%. mixed baking test in order to know the influence of the substitution of wheat flour by potato starch (20/80), several tests were carried out in the laboratory and other tests at the bakery with the assistance of a french expert in bakery (j. prodhomme). the experimental baking studies showed that the concentration of 80% potato starch with the addition of gluten did not affect the handling of dough except for some defects of extensibility during shaping. this similarity in results is due to the role of the added gluten; the essential element for baking (especially during kneading and shaping), which plays a very significant role in increasing the uptake of water and the resistance of the dough. a significant criterion observed during almost all stages of baking, is the stickiness of the dough. in a similar vein, the properties of gas retention within the composite dough are followed by measurement of the volume of the dough during fermentation using the indicator of growth containing 25g of dough subjected to fermentation under the same conditions as the loaves of bread. the results obtained are highly significant (p ≤ 0.05), they show that the pastes incorporate up to 80 % of potato starch, which experience less raising during fermentation, but remain comparable with those obtained with 100 % of wheat flour (fig. 1). external and internal aspects of breads obtained are shown in figs. 2 and 3. the incorporation of potato starch at levels of 80% gives breads with optimal characteristics. the breads resulting from the potato starch have a good appearance, presenting regular and smooth crusts similar to the breads resulting from wheat flour. as for the coloration of the crust, bread with potato starch presents a less dark coloring compared to bread with wheat flour. dupin et al. (1992) and boyacioglu and d’appolonia (1994) showed that the dark coloration of bread is influenced by the rise of the rate of both damaged starch and total sugars present in flour, which were highest in starch potato. concerning the appearance of the crumb related to (fig. 2), bread has aired cells, badly dispersed and not homogeneous. that can be explained by the irregular distribution or the incorporation of α-amylases. as for the appearance of crumbs (fig. 3), bread has aired cells, poorly dispersed and not homogeneous. again, that can be explained by the bad distribution or the incorporation of α-amylases. table 3 rheological characteristics of the flours (with and without addition of vital gluten). rheological characteristics rate of incorporation of the potato starch 0% 80% without gluten 80% with gluten alveographic measurements p (mm) 81 20 99 g (cm) 19.1 06.7 14.8 p/l 01.09 06.68 02.25 w (10-4 j) 210 10 193 ital. j. food sci., vol. 27 2015 349 bread evaluation the approximate composition of bread made from potato starch and wheat flour is shown in table 4. the control bread obtained shows a protein rate of 10.5 %, a rate 60.4% of sugar of and 0.9 % of fat. these values are similar to the values given by cabrol (2006). however, bread prepared containing potato starch 80% present a reduction in proteins fig. 1 influence incorporation of potato starch on the profiles of gas retention. fig. 2 appearance of the crust of potato starch bread (80%). fig. 3 appearance of the crumb of potato starch bread (80%). (6.87%) and fat (0.37%). this result might be due to the composition of potato starch that, in fact, is rich in sugar and low in fat and proteins. the sensory quality statistics reveal that potato starch influences the crumb of bread (fig. 4). bread with potato starch 80% acquires a very white coloration, and therefore receives the highest score compared to control bread. meanwhile, the texture of bread is more developed than that of the control bread. however, we observed that there is significant difference (p ≤ 0.05) in the p value therefore stating the sensory characteristics of potato starch bread 80% are not affected. conclusions the aim of this study was to analyze samples of bread at 80% of potato starch and compared with control bread produced from wheat flour under the same conditions for their nutritional, physicochemical and sensory characteristics. the formulation of our bread was made as follows: potato starch, wheat flour, gluten, yeast, salt and a dough improver. the results show that the loaves can be prepared by potato starch even at high percentage (80%) and gluten. breads obtained by this formula were nutritionally, physically, chemically and at the table 4 nutritional composition of breads. products humidity % glucids (%) protein (%) fat (%) fibre (%) wheat bread 28.20 60.4 10.5 0.9 potato starch bread 31.33 61.36 6.87 0.37 0.07 fig. 4 evaluation of some characters of quality of the potato starch bread 80% and wheat bread prepared in bakery. 350 ital. j. food sci., vol. 27 2015 sensorial level comparable to the control bread. a high percentage of consumers said they saw no difference. these results support the partial substitution of wheat flour by potato starch in wheatbased food products to minimize costs. future studies are needed to investigate the pasting properties of mixtures of wheat flour and potato starch (by rva), to determine the interaction between wheat flour and potato starch and also to interpret their rheological properties by differential scanning calorimeter and rheometer. acknowledgements financial support was received from the lactamel group, sidi bel abbes, algeria. references aacc. 1995. “approved methods of the aacc”. 9th ed. american association of cereal chemists, paul, minnesota. afnor.1991. “recueil de normes-contrôle de la qualité des produits alimentaires: céréales et produits céréaliers” 3rd ed. association française de normalisation, paris. amerine m.a., pangborn rm. and roseller eb.1973. principles of sensory evaluation of food. new york and london: academic press. boyacioglu m.h. and d’appolonia b.l. 1994. characterization and utilization of durum wheat for breadmaking. study of flour blends and various additives. j. cereal chemistry, 71(1): 28-34. cabrol c. 2006. observatoire du pain [en ligne]. la composition nutritionnelle des pains français. disponible sur: <http://www.observatoiredupain.fr/default. asp?idr=110985 >. cip. 2008. “the international year of the potato, ipc”. <http://www.cipotato.org>. dupin h., cuq j.l., malewiak m.i., leynaud-rouaud c. and berthier a.m. 1992. alimentation et nutrition humaines. ed. esf éditeur, paris. p56, 745-747. godon b. and loisel w. 1997.guide pratique d’analyses dans les industries des céréales. technologie et documentation. paris. p 819. liu c.y., shepherd k.w. and rathjen a.j. 1996. improvement of durum wheat pastamaking and breadmaking qualities. j. cereal chemistry, 73:155-166. lindahl l. and eliasson a.c. 1992. a comparison of some rheological properties of durum and wheat flour doughs. j. cereal chemistry, 69: 30-34. lovedeep k., singh n. and sodhi n.s .2002. some properties of potatoes and their starches ii. morphological, thermal and rheological properties of starches. j. food chemistry, 79: 183-192. roussel p. and chiron h. 2002. les pains français : évolution, qualité, production.2, france : mae-erti editeurs. isbn/2-84601-693-3. roussel p., robert y. and crosnier j.c. 1996. la pomme de terre. paris, france. inra. isbn 2-7380-0676-0. singh j., sing n., kaur l., sodhi n.s. and gill b.s. 2003. morphological, thermal and rheological properties of starches from different botanical sources. j. food chemistry, 81(2): 219-231. ugrinovits m.s., arrigoni e., dossenbach a., haberli g., hanich h., schwerzenbach j., richemont l., rychener m., thormann h. and stalder u. 2004. céréales, produits de l’industrie meunière, prémélanges pour four, mélanges de farines instantanées. ch. 14. in: “manuel suisse des denrées alimentaires”. ed msda. vasanthan t., bergthaller w., driedger d., yeung j. and sporns p. 1999. starch from alberta potatoes: wet-isolation and some physicochemical properties. j. food research international, 32: 355-365. wang j., rosell c.m. and barber c.b. 2002. effect of the addition of different fibres on wheat dough performance and bread quality. food chemistry, 79: 221-226. willard m. j. and hix v.m. 1987. potato flour. in: “potato processing”. w. f. talburt and o. smith (4th ed.), pp. 665681. new york: van nostrand reinhold. yadav a.r., guha m., reddy s.y., tharanathan r.n. and ramteke r.s. 2007. physical properties of acetylated and enzyme-modified potato and sweet potato flours. j. food science, 72(5): e249-e253. yanez e., ballester d., wuth h., orrego w., galtas v. and estay s. 1981. potato flour as partial replacement of wheat flour in bread: baking studies and nutritional value of bread containing graded levels of potato flour. j. food technology, 16: 291-298. zaidul i.s.m., yamauchi h., kim s.j., hashimoto n. and noda t. 2007. rva study of mixtures of wheat flour and potato starches with different phosphorus contents. j. food chemistry, 102(4): 1105-1111. paper received february 25, 2014 accepted october 30, 2014 #316_dreassi_bozza ital. j. food sci., vol 29, 2017 90 paper phenolic compounds , carotenoids and antioxidant activity in five tomato (lycopersicon esculentum mill.) cultivars a. zanfini1, g.g. franchi2, p. massarelli2, g. corbini1 and e. dreassi*1 1dipartimento di biotecnologie, chimica e farmacia, università degli studi di siena, via a. moro 2, 53100 siena, italy 2dipartimento di scienze mediche, chirurgiche e neuroscienze, università degli studi di siena, strada delle scotte 6, 53100 siena, italy *corresponding author. elena.dreassi@unisi.it abstract in this study we examined the antioxidants content (polyphenols and carotenoids) and the total hydrophilic (haa) and lipophilic (laa) antioxidant activity of red (cv. shiren and red pear), yellow (cv. yellow pear-shaped), pale yellow (cv. snowball) and black (cv. black trifele) ripe tomato fruits. for each studied cultivars, the haa was higher than the laa. the correlation between antioxidants (polyphenols and carotenoids) and teac values (haa and laa) was also estimated and the only significative correlation was obtained between rutin and teac-haa. statistical analysis shows significative differences in antioxidants content (especially for lycopene and β-carotene) between the analyzed tomato cultivars. keywords: abts, antioxidants, β-carotene, haa, lla, lycopene, rutin, teac, tomato ital. j. food sci., vol 29, 2017 91 1. introduction the nutraceutical quality of tomato fruits is related to their antioxidant and antitumoral properties. the habitual consumption of tomatoes has been associated with decreased risk of chronic degenerative diseases including certain types of cancer and heart diseases (lavelli et al., 2000; giovannucci, 1999, 2002; rao and agarwal, 1998). epidemiological findings confirmed that the beneficial health effects are due to the presence of bioactive molecules such as carotenoids, particularly lycopene, and polyphenolic compounds, particularly flavonoids. lycopene has been considered the most efficient for quenching singlet oxygen (di mascio et al., 1989). its protective role in human health has been well described in numerous papers which showed that the dietary intake of lycopene is associated with a decreased risk of prostate cancer and degenerative diseases (giovannucci, 1999, 2002; rao and agarwal, 1998, 1999). in human diet, tomatoes and tomato products are the predominant sources of lycopene. the lycopene contents and the qualitative and quantitative composition of tomato fruits depend on cultivar, ripening stage, climatic conditions, etc (abushita et al., 2000; zanfini et al., 2007). many authors have focalized their research on the variations in the carotenoid profile in relation to different cultivars (abushita et al., 1997, 2000; leonardi et al., 2000; zanfini et al., 2007). similar studies focused their attention on the polyphenolic compounds such as flavonoids and hydroxycinnamic acids. the phenolic fraction of tomato fruits contains quercetin, naringenin, rutin and chlorogenic acid as the main compounds (clifford, 1999; hertog et al., 1992; justesen et al., 1998). their quantitative changes in relation to cultivar, season and country of origin were also investigated and the ability of these compounds as scavengers of peroxyl radicals has been well described (halliwell, 1999; stewart et al., 2000). the antioxidant capacity of tomato fruits has been widely investigated and a clear influence of the genetic factors (type of cultivar) has been found. the tomato cultivars characterized by a high carotenoid contents showed the highest antioxidant activity and a strong correlation between the antioxidant power and lycopene content was also found (raffo et al., 2002, 2006; zanfini et al., 2010). today, especially in the mediterranean area, black, white or yellow tomato fruits are commonly present in local markets and used as fresh products for salads or for culinary preparations. the aim of the present study was to examine the polyphenolic fraction, the lycopene and β-carotene contents and the hydrophylic and lipophilic antioxidant activities of red, yellow, pale yellow and black tomato fruits. this work is justified by the fact that the color of fruits and vegetables can be an index of the antioxidant potential and may be assist in the selection of food consumption (chávez-mendoza et al., 2015). we analyzed five different cultivars: two red cultivars, the cv. read pear which is characterized by a typical pear shaped fruit and the more commercial cv. shiren which is a conventional cherry type tomato; the cv. yellow pear-shaped with yellow pear shaped fruits; the cv. snowball with pale yellow fruits and the black cultivar cv. black trifele. 2. materials and methods 2.1. reagents and standards all solvents used were of hplc grade from bhd (poole, england). the β-carotene, lycopene, vanillin, kaempferol-3-o-glucoside, naringin, naringenin, and the acids gallic, protocatechuic, vanillic, chlorogenic (4-cqa), caffeic, benzoic and cinnamic were purchased from sigma-aldrich chemie gmbh (steinheim, germany); lutein, quercetin ital. j. food sci., vol 29, 2017 92 and rutin standard from icn biochemical inc. (ohio, usa); folin-ciocalteau reagent, magnesium oxide and silica gel 60 (0.063-0.200 mm) from merck (darmstadt, germany); βapo-8'-carotenal and abts from fluka chemie (buchs, switzwrland); trolox from hoffman la roche aldrich chem. co. (saint louis, mo, usa). 2.2. plant material and tomato sampling different tomato cultivars were investigated: lycopersicon esculentum mill. cv. red pear, cv. snowball, cv. black trifele and cv. yellow pear-shaped. seeds were from graines baumaux mirecourt france and from baker creek heirloom seeds co., mansfield (mo), usa. the commercial cherry type cv. shiren was also analyzed. seeds were allowed to germinate under glass in february-march 2014, then plants were transplanted and cultivated in open air nearby siena (southern tuscany, italy, 43°15'11.8"n 11°36'11.5"e) under the climatic conditions typical of the mediterranean area. figure 1 shows for illustration purpose the colors of various tomato cultivar and below are listed their morphological characteristics. figure 1. sample of various tomatoes fruits used in the analysis: (a) cv. shiren; (b) cv. red pear; (c) cv. yellow pear-shaped; (d) cv. snowball and (e) cv. black trifele. yellow pear-shaped (or yellow pear): plants are indeterminate and hardy. they produce bright yellow, pear-shaped cherry tomatoes with a sweet, mild flavor. they ripe in 75-80 days and are 25-50 mm long. red pear (or red pear-shaped) is almost analogous, with bright red fruits. snowball (or white beauty) is a cultivar introduced in north america in the mid-1800s. plants are indeterminated and yield very heavy crops of 140 up to 220-340 g, 65-75 mm, yellow-white, somewhat flattened round tomatoes with very mild sweet flavors. they ripe in 80-85 days. skin and flesh are pale yellow to parchment or creamy white, with a pink blush on the blossom end. black trifele (or japanese black trifele) is, despite the name, a cultivar of russian origin. pear-shaped fruit has green-streaked shoulders, deepening to a burnished mahogany and finally to a darkened, nearly black base. they ripe in 80-85 days, reach 65-75 mm long and wide, 170 g weight. shiren is a more recently obtained cultivar. plants are indeterminate, compact and particularly suitable for full-sun or greenhouse cultivation. the plant is very resistant to many viruses and produces a very elegant “fishbone” cluster with long shelf life fruits. fruits are ital. j. food sci., vol 29, 2017 93 cherry-like globose in shape, with a diameter of 35 mm and a weight of 10-20 g; they ripe in 55-68 days. full ripe (4.5-5.5°brix) and healthy fruits harvested during july and august 2014 were analyzed for antioxidant content as well as for hydrophilic (haa) and lipophilic antioxidant activity (laa). for the analysis ten tomatoes were sampled from four different representative plants and combined into one sample which is analysed as reported below. the ripeness was determined both visually and by determination of ° brix after chopped and centrifuged an aliquot of the various tomato samples with exacta optech gmbh (munchen, germany) refractometer. 2.3. lc–uv-ms analysis of hydrophilic extracts lc-ms system consisted of an agilent 1100 series system (agilent technologies, palo alto, ca) including a vacuum solvent degassing unit, a binary high-pressure gradient pump, an uv detector and a 1100 msd model vl benchtop mass spectrometer with api-es interface. nitrogen was used as nebulizer gas and drying gas (350°c). the nebulizer gas, the drying gas, the capillary voltage, and the vaporizer temperature were set at 40 psi, 9 l/min, 3000 v and 350°c, respectively and fragmentor 70 ev. the lc-esi-ms determination was performed by operating the msd in both positive and negative ion mode. mass spectra were acquired over the scan range m/z 50-1500 using a step size of 0.1 µ. the chromatographic separation was performed using a pursuit c18 (3 µm, 5x2.0 mm) (varian) column. the sample was injected (20 µl) after filtration. the separation was performed by using linear gradient elution for 60 min with a mobile phase of 0.2% (v/v) formic acid in water and acetonitrile (from 90:10 to 30:70 v/v in 60 min) at the flow rate of 1.5 ml/min. after the chromatographic separation an aliquot of the eluent (400 µl/min) was directed to msd for spectra analysis. the ms analysis was used for the identification of the main compounds present in the polyphenolic fraction. their identification was obtained for comparison with the retention time and with the fragmentation pattern of the relative standards. the uv-vis analysis was used for quantitative purposes. the quantitative analysis was performed for the main identified compounds using calibration curves obtained by injecting the standard solutions at various concentrations. all analyses were run in triplicates and results were expressed with the standard deviation of the means. 2.4. lycopene, β-carotene and lutein analysis carotenoids and xanthophylls were extracted using a procedure previously published with small variations (setiawan et al., 2001). a sample of 5 g of homogenized fresh tomatoes was extracted using 10 ml thf in presence of 0.01% butylated hydroxytoluene (bht) and internal standard (β-apo-8'-carotenal). this extraction was performed twice. the organic fractions were collected and evaporated to dryness under nitrogen. the residue was dissolved in chloroform, appropriately diluted with the mobile phase mixture (methanol: acetonitrile: dichloromethane 50:48:2), then filtered (0.45 mm minisart srp 4 filter, sartorius, germany) and analyzed by using hplc. lc 410 series perkin-elmer apparatus (norwalk, connecticut, usa) equipped with a uv/vis lc295 perkin-elmer detector set at 290 nm and totalchrom ver.6.3.1 software were used (perkin elmer). chromatographic separation was done using a reversed-phase lichrospher 100 rp 18 column (5 µm, 125 x 4.6 mm) (merck). elution was carried out using a mixture of methanol: acetonitrile: dichloromethane (50:48:2) at a flow rate of 1.0 ml/min. quantitative analysis of lutein, lycopene and β-carotene was based on the internal standard method. three replications were carried out to examine each sample. ital. j. food sci., vol 29, 2017 94 2.5. lipophilic and hydrophilic extracts and their antioxidant activities determination total antioxidant activity was measured both, for hydrophilic and lipophilic extracts of the various tomato cultivars. in brief, 5 g of fresh tomato sample was extracted with 10 ml of ch2cl2 and then centrifuged at 1620 g for 10 min. the extraction was performed twice and the supernatant fractions were collected and evaporated to dryness under nitrogen. the dried residue was dissolved in 3 ml of ch2cl2 and analyzed for the determination of lipophilic antioxidant activity (laa). the pellet was extracted with 10 ml of 60% methanol in milli-q water. the samples were then sonicated with sonorex rk103h apparatus (bandelin electronic, berlin, germany) for 10 min and centrifuged at 1620 g for 10 min. the supernatant was then transferred to a new tube and used for determination of hydrophilic antioxidant activity (haa) and polyphenol content. the antioxidant activity was measured using abts radical cation (abts+) decolorization assay (re et al., 1999). in brief, 1 ml of the abts solution was added to different volumes of the lipophilic or hydrophilic extract (20, 40 or 60 µl) and diluted at a final volume of 2 ml using ethanol. the solution was vortexed for 10 s and the decolourization produced by the presence of antioxidants was measured at 751 nm (uv/visible lambda 2 spectrophotometer, perkin-elmer, norwalk, connecticut, usa), 10 min after initial mixing. the teac assay is a standard method used for antioxidant activity assessment of vegetables with numerous advantages such as reproducibility, simplicity and a good estimate of the antioxidant activity of pure compounds and complex matrices (thaipong et al., 2006). trolox was used to prepare the standard curve and the activity was reported as equivalent millimolar trolox related to fresh weight (mm teac/100 g fw). the laa and haa were measured in triplicates for each extract. 2.6. statistical evaluation of data all analyses were run in triplicates and results were expressed with the standard deviation of the means. a one-way analysis of variance (anova) was performed to test the significance of the observed differences (using stata 9.0, statacorp lp). data were analyzed considering the tomato cultivar as experimental factor and when the observed differences were significant (p≤0.05) the mean values were then compared by bonferroni’s multiple comparison test. 3. results and discussion 3.1. polyphenolic fraction analysis 3.1.1 identification of the main polyphenolic compounds by lc-uv-ms in the present study 13 different polyphenolic compounds were detected and identified in the extracted samples. the identified compounds belonging to five groups of phenolic compounds (benzoic acids, hydroxycinnamic acids, phenolic aldehydes, flavonols and flavanones) are listed in table 1. identification of the chromatographic peaks was made by comparison of mass spectra with those provided by commercial standards and by comparing their retention times in correlation to ms fragmentation patterns. ital. j. food sci., vol 29, 2017 95 table 1. list of polyphenolic compounds identified in tomato samples. compound retention time (min) [m+h] + [m+h]identification (m/z) 1 5.1 --169 gallic acid 2 9.5 155 153 protocatechuic acid 3 19.7 169 167 vanillic acid 4 20.4 355 353 chlorogenic acid 5 21.3 181 179 caffeic acid 6 23.4 153 151 vanillin 7 28.4 123 121 benzoic acid 8 30.1 611 609 rutin 9 32.6 287 285 kaempferol-3-o-glucoside 10 33.1 581 579 naringin 11 41.1 149 147 cinnamic acid 12 44.7 303 301 quercetin 13 47.1 273 271 naringenin under the experimental conditions used, the most intensive signals detected for the main compounds was the pseudomolecular ion ([m+h]+ or [m-h]-). some benzoic acid derivates or phenolic acids were identified. gallic acid comes out at rt 5.1 min, giving the characteristic molecular ion at 169 m/z. the acids p-hydroxybenzoic acid, vanillic acid, protocatechuic acid were also identified by their retention times, their pseudomolecular ion ([m+h]+ or [m-h]-) and mass fragmentations in comparison with standard solutions. a comparison with the ms spectra described in literature was also carried out (guash-jané et al., 2004; tian et al., 2005). hydroxycinnamic acids were also identified. the presence of chlorogenic acid and caffeic acid was detected in all the analyzed samples. the phenolic aldehyde vanillin was also detected. flavonols were mainly characterized by quercetin (303 m/z) and rutin (611 m/z). the presence of kaempferol-3-o-glucoside (449 m/z) was also detected. flavanones were mainly represented by naringenin (273 m/z). comparison of total ion content (tic) and uv (289 nm) chromatograms obtained from the lc-uv-ms analysis of the various cultivars revealed similar profiles (same compounds were observed even if in different quantity). additionally, we found that the profiles obtained from the analysis of the various cultivars were essentially identical with some qualitative differences detected for minor components (unknown compounds). 3.1.2 quantitative analysis of the main identified compounds the main polyphenolic components identified were quantified as previously reported and the most abundant polyphenols detected in tomato cultivars are reported in table 2. the most abundant were chlorogenic acid, rutin and quercetin. the determination of phenolic compounds showed that snowball and black trifele cultivars contained the lowest concentration of antioxidant compounds, while the cultivar that possessed the highest concentration, especially of rutin (40.18 µg/g of fw), was shiren. these observations are perfectly in agreement with haa values showed below. quantitative determination of phenolic compounds showed that contents were in agreement with the few analytical data reported on red, purple or yellow cultivars ital. j. food sci., vol 29, 2017 96 (martínez-valverde et al., 2002; vallverdú-queralt et al., 2011; garcíavalverde et al., 2013; choi et al., 2014; raiola et al. 2016). table 2. mean content (µg/g of fw)±sd of the main polyphenolic compounds in the analyzed tomato cultivars. tomato cultivar red pear snowball black trifele yellow pear shiren gallic acid 1.03±0.06c 1.09±0.08c 0.04±0.01b nd a 0.62±0.02d chlorogenic acid 12.53±1.09b 15.08±1.03b 9.26±0.83a 17.54±1.26b 8.86±0.06a caffeic acid 0.69±0.02c 0.47±0.05b 0.01±0.01a 0.03±0.01a 1.32±0.09d rutin 14.46±1.11c 9.96±0.95a 12.40±0.92b 26.32±1.86c 40.18±2.45d kaempferol-3-oglucoside 0.13±0.01 a 0.10±0.02a 0.13±0.01a 0.54±0.02c 0.20±0.01b naringin 0.12±0.01b 0.06±0.01a 0.23±0.01c 0.23±0.01c 0.50±0.02d quercetin 23.91±1.16d 16.92±1.23c 8.01±0.75b 15.34±1.14c 0.15±0.01a naringenin 3.63±0.27b 0.54±0.04a 10.14±1.01c 5.36±0.43b 10.21±0.92c different letters in the rows represent statistically significant differences (p>0.05). nd = not detected. 3.2. lutein, lycopene and β-carotene analysis we carried out a quantitative analysis of lutein, lycopene and β-carotene in the different investigated tomato cultivars. all samples exhibited a similar chromatographic profile, showing that the two carotenoids lycopene and β-carotene were the main component of the lipophilic extracts for the cultivars with red fruits. the compounds were identified by comparing the retention time with those obtained from the reference standard injections. the quantitative data were obtained using the internal standard method and are presented in table 3. lycopene content ranged from 1.02 µg/g (recorded for yellow pear tomatoes) to 184.42 µg/g of fresh weight (recorded for shiren tomatoes), while β-carotene ranged from 2.60 µg/g (recorded for yellow pear tomatoes) to 64.81 µg/g (recorded for shiren tomatoes). additionally, the quantitative data showed that the yellow pear cultivar had a β-carotene content more abundant than the lycopene content. the two investigated carotenoids were not detected in the snow ball cultivar in which only lutein was detected. lutein was also very abundant in black trifele cultivar. table 3. principal carotenoids (lycopene and β-carotene) and xantophylls (lutein) content of various tomatoes cultivars. tomato cultivar mg/g of fresh fruit lutein lycopene β-carotene red pear 0.18±0.02b 42.18±2.31e 12.4±0.12d snowball 0.08±0.01a nda nda black trifele 1.12±0.04d 60.9±1.92c 8.42±1.66c yellow pear 0.20±0.02b 1.02±0.28b 2.60±0.84b shiren 0.21±0.02bc 184.42±2.61d 64.81±0.37e different letters in the columns represent statistically significant differences (p>0.05). nd = not detected. ital. j. food sci., vol 29, 2017 97 from the results of table 3 emerged, that there is a significant difference in the ability of tomatoes to synthesize carotenoids in relation to cultivar. the cultivar with the highest content of carotenoids was shiren, on the contrary, the yellow pear cultivar had low ability in carotenoids production. characterization of carotenoid content of yellow tomatoes and the differences with the red ones dates back to the 50s (jenkins and mackinney, 1955) and many authors have determined significant influence of cultivars on the content of tomato carotenoids (abushita et al., 2000; binoy et al., 2004; choi et al. 2014; ilahy et al., 2011; raiola et al. 2016; zanfini et al. 2007). our results are in accord with data reported by these authors and demonstrate that lutein, lycopene and β-carotene content is significantly related with the cultivar and the color of the tomatoes as earlier reported by other authors (li et al., 2013). 3.3. antioxidant activity the antioxidant activity of tomato lipophilic (laa) and hydrophilic (haa) extracts was also performed using the trolox equivalent antioxidant capacity (teac) assay. for each studied cultivar, the haa was higher than the laa and the shiren type tomatoes, characterized by a high carotenoid and total phenolic contents, showed the highest antioxidant activity (table 4). table 4. teac values (µm trolox/100 g fw) measured for each investingated cultivar. tomato cultivar haa teac laateac red pear 502.8±52.3a 75.4±2.0c snow ball 472.8±48.7a 47.7±1.3a black trifele 499.2±60.3a 121.7±0.9d yellow pear 643.5±54.1ab 58.3±1.0b shiren 706.0±19.3b 131.0±2.1e different letters in the columns represent statistically significant differences (p>0.05). statistical analysis shows significative differences in antioxidants activity only for laa between the analyzed tomato cultivars. the correlation between the content of the various antioxidant (independent variable) and the teac value (dependent variable) was also estimated. the only significative correlation was obtained between rutin and teac haa (r2 = 0.982, p<0.05). 4. conclusions this study has confirmed the important role played by cultivar in determining the antioxidant potential of fresh raw tomatoes. results show that phenolic compounds but especially carotenoids are responsible for the differences among tomatoes in accord with the cultivar. in effect the values of haa and laa reflect the 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and sies h. 1989. lycopene as the most efficient biological carotenoid singlet oxygen quencher. arch. biochem. biophys. 274:532-538. giovannucci e. 2002. a review of epidemiologic studies of tomatoes, lycopene, and prostate cancer. exp. biol. med. 227:852-859. giovannucci e. 1999. tomatoes, tomato based products, lycopene, and cancer: review of epidemiological literature. j. nat. cancer inst. 91:313. guasch-jané m.r., ibern-gómez m., andrés-lacueva c., jáuregui o. and lamuela-raventós r.m. 2004. liquid chromatography with mass spectrometry in tandem mode applied for the identification of wine markers in residues from ancient egyptian vessels. anal. chem. 76:1672-1677. halliwell b. 1999. how to characterize biological antioxidants. free rad. res. commun. 9:1-32. hertog m.g.l, hollman p.c.h. and katan m.b. 1992. content of potentially anticarcinogenic flavonoids of 28 vegetables and 9 fruits commonly consumed in the netherland. j. agric. food. chem. 40:2379-2383. ilahy r., hdider c., lenucci m.s., tlili i. and dalessandro g. 2011. phytochemical composition and antioxidant activity of high-lycopene tomato (solanum lycopersicum l.) cultivars grown in southern italy. sci. hortic. 127:255-261. justesen u., knuthsen p. and leth t. 1998. quantitative analysis of flavonols, flavones, and flavanones in fruits, vegetables and beverages by high-performance liquid chromatography with photo-diode array and mass spectrometric detection. j. chromatogr. a 799:101-110. lavelli v., peri c. and rizzolo a. 2000. antioxidant activity of tomato products as studied by model reactions using xanthine oxidase, myeloperoxidase, and copperinduced lipid peroxidation. j. agric. food chem. 48:1442-1448. leonardi c., ambrosino p., esposito f. and fogliano v. 2000. antioxidative activity and carotenoid and tomatine contents in different typologies of fresh consumption tomatoes. j. agric. food chem. 48, 4723-4727. raffo a., la malfa g., fogliano v., maiani g. and quaglia g. 2006. seasonal variations in antioxidant components of cherry tomatoes (lycopersicon esculentum cv. naomi f1). j. food comp. anal. 19:11-19. raffo a., leonardi c., fogliano v., ambrosino p., salucci m., gennaro l., bugianesi r., giuffrida f. and quaglia g. 2002. nutritional value of cherry tomatoes (lycopersicon esculentum cv. naomi f1) harvested at different ripening stages. j. agric. food chem. 50,:6550-6556. rao a.v. and agarwal s. 1998. bioavailability and in vivo antioxidant properties of lycopene from tomato products and their possible role in the prevention of cancer. nutr. cancer 31:199-203. rao a.v. and agarwal s. 1999. role of lycopene as antioxidant carotenoid in the prevention of chronic diseases: a review. nutr. res. 19:305-323. re r., pellegrini n., proteggente a., pannala a., yang m. and rice-evans c. 1999. antioxidant activity applying an improved abts radical cation decolorization assay. free radic. biol. med. 26:1231-1237. setiawan b., sulaeman a., giraud d. and driskell j. 2001. carotenoid content of selected indonesian fruits. j. food comp. anal. 14:169-176. ital. j. food sci., vol 29, 2017 99 stewart j., bozonnet s., mullen w., jenkins g.i., lean m.e.j. and crozier a. 2000. occurrence of flavonols in tomatoes and tomato-based products. j. agric. food chem. 48:2663-2669. thaipong k., boonprakob u., crosby k., cisneros-zevallos l. and hawkins byrne d. 2006. comparison of abts, dpph, frap, and orac assays for estimating antioxidant activity from guava fruit extracts. j. food comp. anal. 19, 669–675. tian s., nakamura k., cui t. and kayahara h. 2005. high-performance liquid chromatographic determination of phenolic compounds in rice. j. chromatogr. a 1063:121-128. zanfini a., corbini g., la rosa c. and dreassi e. 2010. antioxidant activity of tomato lipophilic extracts and interactions between carotenoids and α-tocopherol in synthetic mixtures. lwt food sci. technol. 43:67-72. zanfini a., dreassi e., la rosa c., d’addario c. and corti p. 2007. quantitative variations of the main carotenoids in italian tomatoes in relation to geographic location, harvest time, varieties and ripening stage. ital. j. food sci. 19:181-190. paper received november 4, 2015 accepted may 30, 2016 #429_sagan_bozza ! ital. j. food sci., vol 28, 2016 697 paper the effect of infrared radiation in modifying nutritional and mechanical properties of grass pea seeds a. sagan*1, d. andrejko1, t. jaśkiewicz1, b. ślaska-grzywna1, m. szmigielski1, a. masłowski1 and w. żukiewicz-sobczak2 1department of biological bases of food and feed technologies, university of life sciences in lublin, głęboka 28, 20-612 lublin, poland 2university college of social sciences in lublin, zamojska 47, 20-102 lublin, poland *corresponding author. agnieszka.sagan@up.lublin.pl abstract the aim of this work was to evaluate the effect of exposure of grass pea seeds to ir (infrared) radiation applied at varying time intervals on their mechanical properties, trypsin inhibitor activity, and reactive lysine content. grass pea seeds cv. derek were exposed to ir radiation at 180°c for 30, 60, 90, 120, and 180 s, respectively. compared with that of raw seeds, ir heating of grass pea seeds reduced trypsin inhibitor activity. reactive lysine proved to be relatively stable in the applied heating conditions. in addition, the process led to the reduction in the value of breaking load required for the destruction of a single seed, which may facilitate further processing, for example, flaking. therefore, ir heating can be used in processing grass pea seeds. keywords: grass pea, infrared radiation, reactive lysine, breaking load ! ital. j. food sci., vol 28, 2016 698 1. introduction grass pea (lathyrus sativus l.) is one of the oldest legumes cultivated by man. currently, it is still an important nutritional component in arabic countries, the caucasus, and the indian subcontinent. the advent of cultivation of this species in poland is related to tartar settlers who relocated to the podlasie region in the 17th century. however, nowadays, it can be found only in limited regional cultures. the recent increasing interest in unconventional food products of plant origin also involves grass pea. the nutritional value of grass pea seeds is primarily associated with their relatively high protein content (24-36%), accompanied by a very rich amino acid composition (milczak et al., 2001). the significant amount of oleic, linoleic, and linolenic acids in fat, which is similar to the composition and utility of soybean oil, contributes to the high dietary value of the seeds (grela et. al., 2010). it should also be noted that they contain non-starchy polysaccharides (ca. 5%), which can stimulate bifidobacteria in the digestive tract, enriching the organism with b-group vitamins, with a beneficial effect in absorbing calcium and reducing both the content of putrefactive bacteria and cholesterol absorption (tomomatsu, 1994). grass pea seeds are suitable for human consumption and can be used to manufacture the feed and the production of the so-called “plant casein” (dziamba, 1997). however, the usability of grass pea seeds may be limited by bioactive components with anti-nutritional activity, such as protease inhibitors, tannins, or neurotoxins. the negative effect of anti-nutrients found in the seeds of leguminous plants, including grass pea, can be minimized through thermal processing (grela et al., 2001; szmigielski and matyka, 2004). it seems highly advisable to assess the effect of infrared (ir) radiation on the seeds, including both modifications of the chemical composition and accompanying changes in the mechanical properties. special attention should be paid to lysine because thermal processing may reduce its reactivity, thus limiting the availability of this essential amino acid (sagan and jaśkiewicz, 2011). lysine is involved in building proteins and producing hormones, enzymes, and antibodies. lysine is needed for proper bone formation in children, as it helps in absorbing calcium (sarwar et al., 2012). the aim of the present work was to evaluate the effect of exposure of grass pea seeds to ir radiation, at varying time intervals, on the mechanical properties and selected nutritional components of grass pea seeds. 2. materials and methods the research material was seeds of grass pea (lathyrus sativus l.) cv. derek. the material harvested in 2011 was provided by “spójnia” hodowla i nasiennictwo ogrodnicze (breeding and horticultural company) in nochowo, poland. the seed moisture content was 11±0.2%. table 1 presents the geometric data and the basic physical properties of grass pea seeds. table 1: basic physical properties of grass pea seeds (cv. derek) (± standard deviation). mass of 1000 seeds (g) mean size of seeds (mm) angle of tipping over plate (°) bulk density (kg·m -3) bulk density tapped (kg·m-3) 116.3±2.49 5.90±0.03 18.3±1.15 810.66±23.09 896.13±10.99 ! ital. j. food sci., vol 28, 2016 699 six 500-g samples of grass pea seeds were prepared. five of them were exposed to ir radiation at 180°c (the temperature adopted for the process was the temperature of the seed surface exposed to heating) for 30, 60, 90, 120, and 180 s, respectively. processing was carried out using a laboratory apparatus, which was designed and constructed especially for this purpose, with a wavelength emitted λ = 2.5 3.0 %m (andrejko et al., 2011). by applying uniaxial compression of individual seeds between two parallel plates, mechanical properties were evaluated using a dynamometer (instron 4302, instron ltd., high wycombe, uk). individual seeds of grass pea were placed with their seed leaves parallel to the surface of the fixed plate and then they were compressed with the help of the upper plate. the speed of the compression plate shift applied during the test had a constant value of 10 mm/min. force required for destructing the structure of a single seed was determined. the values of maximal forces were read out using specialist computer software (instron 12) provided by the instron company. the result was reported as the arithmetic mean of 50 replications. protein, fat, ash, and raw fiber of the grass pea seeds were determined following aoac 984.13, 920.39, 942.05, 962.09, respectively (aoac, 1990). amino acids were determined by ion-exchange chromatography, following pn-en iso 13903 (2006). prior to the analysis, the sample was hydrolyzed with 6 m hcl at 105°c for 24 h. next, automatic amino acid analyzer aaa 400 was used to determine amino acids (ingos, praha, czech republic). sulfur-containing amino acids were determined after oxidation to methionine sulfone and cysteic acid with performic acid (prepared by mixing 30% hydrogen peroxide and 99.9% formic acid in a ratio of 1:9) at 0°c for 16 h. tryptophan was determined after alkaline hydrolysis with a saturated barium hydroxide solution at 105°c for 20 h. according to the procedure of oser (1951), essential amino acid index (eaa index) was calculated taking into account the ratio of eaa in the test protein relative to their respective amounts in the whole egg protein. the fatty acid composition was determined in conformity with pn-en iso 5508 (1996) and pn-en iso 5509 (2001). the extracted fat was saponified with a 0.5 m methanolic sodium hydroxide solution at 85-95°c for 10 min. fatty acid salts were esterified with anhydrous methanol using bf3 as a catalyst at 100°c for 10 min. the methyl esters of the fatty acids were analyzed using a gas chromatograph with a flame ionization detector (varian 450-gc, middelburg, the netherlands) on 30 m long column select biodiesel for fame (agilent technologies, santa clara, usa). based on the study by kakade et al. (1974), trypsin inhibitor activity (tia) was determined. the analytical evaluation of tia is based on the estimation of the part of trypsin activity that is blocked by a buffered extract containing trypsin inhibitors obtained from a seed sample and takes place under conditions ensuring the maximum activity of this enzyme. n-α-benzoyl-dlarginine-p-nitroanilide (bapa), a synthetic substrate, was used. the reaction between trypsin and bapa yields yellow p-nitroaniline whose concentration is registered at 410 nm. the calculation of the result is based on a diagnostic reaction between the standard bapa solution and the standard trypsin solution. the absorbance of this solution corresponds to 40 conventional trypsin units (tu). extracts obtained from the tested samples containing a trypsin inhibitor characterized by tia reduce the activity of this enzyme, which is reflected in a proportional decrease in the quantity of produced pnitroaniline and reduced absorbance of the analyzed solution converted into trypsin units inhibited (tui) per 1 mg dry seed mass (tui·mgdm−1). reactive lysine was determined by using the hplc technique according to ramírez-jiménez et al. (2004). the method consists in creating a colored complex of ε-dnp-lysine in the reaction with dinitrofluorobenzene (dnfb), and thereafter, hydrolyzing the sample and determining the content of ε-dnp-lysine using a liquid chromatograph with uv-vis spectroflow 773 detector (kratos analytical, manchester, uk). reducing sugars were extracted from the sample with 40% ethanol for 1 h and determined with the luff-schoorl method (pn-r! ital. j. food sci., vol 28, 2016 700 64784, 1994). the method is based on the reduction of copper salt from the luff solution (copper citrate with sodium carbonate) by reducing sugars contained in the extract. the reaction was carried out at a boiling point for 10 min. after cooling, the excess of copper ions was reduced with hydrogen iodide generated after the addition of 30% ki solution and 6 m h2so4. liberated iodine was titrated with a 0.1 m sodium thiosulfate solution using starch as an indicator near the end point. all chemical analyses were performed in three replications. the results of the determination of the tia of reactive lysine and reducing sugars in the grass pea seeds were subjected to the analysis of variance (anova). the significance of differences between the mean values was verified using tukey’s test, with p<0.05. calculations were done using statistica 8.0 software. 3. results and discussions the value of the physical parameters determined for the grass pea seeds (table 1) were similar to those presented by other authors (altunas and karadag, 2006). the mass of 1000 seeds (116.3±2.49 g) was similar to the average quoted by dziamba (1997). seed resistance to compression plays an important role in many technological processes. thus, an attempt has been made to determine the changes caused by the thermal activity of ir radiation in the mechanical resistance of the grass pea seeds. the data presented in fig. 1 show that the thermal effect of ir radiation on the grass pea seeds led to a decrease in breaking load, in accordance with the exposure time. the highest values of breaking load were registered for seeds that had not been thermally processed (1.019 kn), while heating the seeds for even a short period of 30 s with ir radiation reduced the value of breaking the load to 0.885 kn. furthermore, increasing the time of the seed exposure to ir radiation led to a reduction in the measured values. the lowest value, i.e., 0.463 kn, was obtained from the seeds subjected to heating for 180 s. the dependence presented in fig. 1 has a nearly linear character; hence, it was described by means of the first-degree regression equation. the good correspondence of the experimental data and the equation is confirmed by the high value (close to 1) of the determination coefficient r2. f = -0.0031t + 0.9967 r2 = 0.975 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 0 20 40 60 80 100 120 140 160 180 200 t (s) f ( kn ) figure 1: effect of heating time (t) with infrared radiation on the value of breaking load (f) of the grass pea seeds. ! ital. j. food sci., vol 28, 2016 701 the dependencies obtained are not characteristic of one type of material only, namely, grass pea seeds. on the basis of the results recorded previously (andrejko et al., 2011), it was concluded that the values of the breaking load causing destruction of wheat cv. zawisza were lower following ir radiation. fasina et al. (2001) recorded the similar observations as follows: after the thermal processing of beans, peas, and lentils, reduced forces destroying the seeds were noted and were found to be different for each individual material, despite the same conditions of processing. the content of the chemical components in the grass pea seeds (table 2) was similar to the values quoted in the available sources (grela et al., 2010; smulikowska et al., 2008; tamburino et al., 2012). the analyzed seeds contained 24.35% of protein with a high nutritional value, resulting from their amino acid composition (table 3). table 2: chemical composition of grass pea seeds (± standard deviation). basic components (%) protein 24.35±0.30 fat 0.88±0.18 ash 2.62±0.08 raw fiber 5.16±0.42 fatty acids (% of fa total) c 16:0 9.09±0.31 c 18:0 4.41±0.22 c 18:1 12.87±0.42 c 18:2 57.30±1.02 c 18:3 14.53±0.21 sfa 14.87 mufa 13.09 pufa 72.09 n-3 14.42 n-6 57.41 pufa n-6/n-3 3.98 table 3: content of amino acids in grass pea seeds (± standard deviation). amino acids (g·kg−1) phenylalanine 10.7±0.35 tryptophan 4.2±0.05 methionine 2.0±0.03 threonine 9.3±0.10 leucine 16.3±0.11 isoleucine 10.1±0.10 valine 11.2±0.18 lysine 15.7±0.20 eaai1 72.0 1essential amino acid index. ! ital. j. food sci., vol 28, 2016 702 among the essential amino acids, lysine and leucine were the most abundant, which is consistent with the results obtained for an italian variety (tamburino et al., 2012). the content of the essential amino acids in the grass pea seeds was similar to soybean, except for methionine, which was definitely less abundant in grass pea than in soybean. the index of essential amino acids, eaai, amounting to 72.0, remained within the range of the values obtained for polish varieties of bean seeds (sagan and jaśkiewicz, 2011). the fatty acid profile in the analyzed seeds was similar to that quoted by other authors (grela et al., 2010). the high share of pufa in the sum of fatty acids is notable, as well as the high ratio of n-6:n-3. the high level of polyunsaturated fatty acids is extremely important in preventing cardiovascular disease. the content of trypsin inhibitors was at a level of 37.09±1.41 tui/mg of dry mass of raw seeds. grela et al. (2001) and szmigielski and matyka (2004) noted a lower activity of trypsin inhibitors in raw grass pea seeds (19.64 and 23.13 tui/mg of dry mass). heating grass pea seeds with ir radiation led to a reduced tia, which becomes more significant with the longer time of processing. statistically significant changes were already observed after 60 s exposure of the seeds to ir radiation (table 4). after 60 s of radiation, the tia was 90% of its value in the raw samples and was only 41% after 180 s. this confirms some previous analyzes, which revealed that thermal processing reduces the content of antinutrients in the seeds of grass pea. the reduction in the activity of trypsin inhibitors is extremely important because of their anti-nutritional effects. these inhibitors block trypsin, which leads to the inhibition of some processes, thereby restricting the use of the protein and reducing proteolysis in the gastrointestinal tract; in turn, this may result in the inhibition of the growth of the organism (sarwar et al., 2012). table 4: trypsin inhibitor activity, content of reactive lysine, and reducing sugars in grass pea seeds (± standard deviation). time of processing (s) trypsin inhibitors activity (tui·mgdm -1) reactive lysine (g·kgdm -1) reducing sugars (g·kgdm -1) 0 37.09a±1.41 15.21a±0.53 7.00a±0.54 30 35.93a±0.89 15.18a±0.25 6.89a±0.42 60 33.49b±1.39 15.20a±0.04 6.72ab±0.39 90 27.57c±0.96 14.84ab±0.76 5.95ab±0.25 120 22.80d±0.68 14.86ab±0.42 5.71b±0.14 180 15.34e±0.74 13.95b±0.14 5.65b±0.32 a,b,cstatistically significant differences in columns (p<0.05). in order to determine the influence of the processing procedures on the nutritional value of the food, the content of reactive lysine may be used as an indicator. exposing grass pea seeds to high temperature may pose a risk of lowering the nutritional value of protein, for example, by reducing the content of reactive lysine, which at higher temperatures reacts with other native compounds. this amino acid becomes inaccessible for the digestive processes after blocking the free ε-amino group. in this process, the susceptibility of other leguminous seeds occurring during the thermal processing was also observed by sagan and jaśkiewicz (2011), and žilić et al. (2006), among others. reactive lysine in the analyzed seeds was relatively resistant to the ir radiation. statistically significant losses of this compound were noted only as a result of the 180 s exposure to the ir radiation (table 4). the retention of reactive lysine in the sample heated in this manner amounted to 92%, ! ital. j. food sci., vol 28, 2016 703 and it was higher in comparison with soybean seeds of serbian varieties subjected to heating with ir radiation at a temperature of 150°c and flaking for 2-3 min (retention after the process: 79% and 55%) (žilić et al., 2006). the results may confirm earlier studies on the influence of thermal processes on the content of reactive lysine in bean seeds (sagan and jaśkiewicz, 2011), which suggest that prolonged heating may lead to a reduction in the content of this amino acid. the content of reducing sugars exceeded the sum of glucose and fructose determined by piotrowicz-cieślak et al. (2008) in the seeds of derek and krab lines and was lower than that in ethiopian varieties (urga et al., 1995). a significant reduction was noted in the content of reducing sugars after 120 and 180 s of heating (table 4). after this period, the content of reducing sugars in the grass pea seeds was 81% of the initial amount. this may result from the reaction of these compounds with other components occurring at higher temperature, for example, with amino acids (maillard’s reactions), including reactive lysine (naranjo et al., 1998). 4. conclusions ir heating of grass pea seeds resulted in a decreased tia, compared with that of raw seeds. reactive lysine proved to be relatively stable in the applied heating conditions. in addition, the process reduced the value of breaking load required for destructing a single seed. this may facilitate further processing, for example, flaking. therefore, ir heating can be applied in processing of grass pea seeds. references altunas e. and karadag y. 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accessions of grass pea (lathyrus sativus l.) grown in europe and nutritional traits of their seeds. genet. resour. crop evol. 57:693. grela e.r., studziński t. and matras j. 2001. antinuritional factors in seed of lathyrus sativus cultivated in poland. lathyrus lathyrism newsl. 2:101. kakade m.l., rackis j.j., mcghee j.e. and puski g. 1974. determination of trypsin inhibitor activity of soy products: a collaborative analysis of an improved procedure. cereal chem. 51:376. milczak m., pędziński m., mnichowska h., szwed-urbaś k. and rybiński w. 2001. creative breeding of grasspea (lathyrus sativus l.,) in poland. lathyrus lathyrism newsl. 2:18-23. naranjo g.b., malec l. and vigo m.s. 1998. reducing sugars effect on available lysine loss of casein by moderate heat treatment. food chem. 62(3):309. oser b.l. 1951. method for integrating essential amino acid content in the nutritional evaluation of protein. j. am. diet. 27:396. ! ital. j. food sci., vol 28, 2016 704 piotrowicz-cieślak a.i., 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1994. health effects of oligosaccharides. food technol. 10:61. urga k., fite a. and kebede b. 1995. nutritional and nutritional factors of grass pea (lathyrus sativus) germplasms. bul. chem. soc. ethiopia. 9(1):9. žilić s., božović i., savić s., and šobajić s. 2006. heat processing of soybean kernel and its effect on lysine availability and protein solubility. cent. eur. j. biol. 1(4):572. paper received february 10, 2016 accepted may 10, 2016 p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33i4.2079 21 p u b l i c a t i o n s codon local food consumption during the covid-19 pandemic zohra ghali-zinoubi1,2 1college of administrative and financial sciences, saudi electronic university, riyadh, saudi arabia; 2higher institute of management of tunis, university of tunis, le bardo, tunisia *corresponding author: zohra ghali-zinoubi, college of administrative and financial sciences, saudi electronic university, riyadh, saudi arabia & higher institute of management of tunis., university of tunis., le bardo, tunisia. email: zohragh@yahoo.fr received: 30 may 2021; accepted: 6 october 2021; published: 5 november 2021 © 2021 codon publications open access paper abstract the influence of intrinsic quality, health consciousness, environmental awareness, local support, and proximity of process on consumers’ intention to consume local food during the covid-19 pandemic was tested, with food availability as a moderator. online survey results were analyzed using a two-step structural equation modelling (sem). health consciousness was the major reason for consuming local food. intrinsic quality and proximity of process were also significant drivers. local support and environmental awareness have little impact on the intention to purchase local food. this study contributes to knowledge regarding the main factors driving local food consumption during a health crisis, providing directions. keywords: consumer behavior; covid-19 pandemic; local food; purchase intention; tunisia introduction the coronavirus disease of 2019 (covid-19) pandemic has dramatically upset people’s everyday lives, brought about a global recession that the world had not witnessed since the second world war, and created a health crisis around the world (smith and wesselbaum, 2020). the food sector and its stakeholders are particularly in the spotlight (ben hassen et al., 2021a; galanakis, 2020) because food is essential for human survival, and therefore it cannot be placed under lockdown. indeed, the emergence of covid-19 has significantly affected the global food systems at several levels, i.e., for producers, distributors, and consumers (cranfield, 2020; qi et al., 2020; xie et al., 2020). notably, both consumption patterns and consumers’ behavior changed as the pandemic progressed (celimli and adanacioglu, 2021; eger et al., 2021; yuen et al., 2020). in this context, what should be eaten has become one of the main concerns for customers who seek to improve their immunity and increase their food security. in april 2020, worldwide google searches for “food delivery” and “local food” reached an all-time high (shveda, 2020). thus, the crisis forced people to reexamine the sources of their groceries and the mode through which they procured food. in a globalized era, the distribution of food from the producer to geographically dispersed consumers relies on a large and complex supply chain. the coronavirus crisis has disrupted this supply chain and caused gaps in food delivery. moreover, the lockdowns instituted in response to the covid-19 pandemic have led to shortages of labor, interruptions of logistics, and inconsistency in the demand for and supply of food (pantano et al., 2020; rizou et al., 2020). this has resulted in empty supermarket shelves, food wastage, and hindered food procurement due to interruptions in exports and imports (aday and aday, 2020). local food systems can alleviate this growing threat of the global food system crisis (memery et al., 2015; peterson, 2013; pressman et al., 2020). the existing literature names several reasons for the increased demand for local food providers during the covid19 italian journal of food science, 2021; 33 (4): 21–32 mailto:zohragh@yahoo.fr 22 italian journal of food science, 2021; 33 (4) ghali-zinoubi z wheat (over 431 million us dollars), sugar ($204 million), soybeans ($162 million), vegetable oils ($160 million), corn ($145 million), and barley ($134 million). with the emergence of the covid-19 pandemic and the breakdown, social distinction, and other restrictive measures taken in tunisia and across the globe, the food supply faced several issues, while the food availability on the local market became a vital concern (ben hassen et al., 2021a; kirkm and kirkin, 2020; pantano et al., 2020; sheth, 2021). in this context of global breakdown and interruption of international transport, local food became the single viable alternative. indeed, several studies conducted during the covid-19 pandemic confirmed the growth of local food consumption amidst the pandemic (ben hassen et al., 2021b; celimli and adanacioglu, 2021; duda-chodak et al., 2020; loiseau et  al., 2020; mesić et al., 2020). however, to the best of our knowledge, no studies have examined the factors driving this growth. furthermore, from a marketing perspective, there are no studies focusing on the behavior of tunisian consumers toward local food during the covid-19 pandemic. the current study was conducted in the aforementioned context to fill this gap. it examines motivations for local food consumption during the covid-19 pandemic. the main objective of this paper was to examine the main motivations for local food consumption during the covid-19 pandemic and the moderating role of food availability. this paper begins with a literature review, from which five hypotheses are derived. next, the research methodology was developed. the discussion of the results and their implications are presented in the last section, which also includes the study’s limitations and directions for future research. literature review what is local food? the concept of “local food” has been the focus of several studies in the literature (bazzani and canavari, 2017; bentsen and pedersen, 2020; meyerding et al., 2019; picha et al., 2017). however, there is still no agreedupon definition of this concept (memery et al., 2015; skallerud and wien, 2019). for instance, several studies have used an objective approach, defining local food in relation to the geographic distance that the food travels from the producer to the final customer. in other words, the term “local food” has been used to describe food systems or short supply chains wherein the food is produced in areas close to where the consumers live (meyerding et al., 2019; skallerud and wien, 2019). however, this approach has been criticized because pandemic. first, during a health crisis, the most crucial issue for any consumer is to consume healthy, nutritious, and safe food, such as local food (arsil et al., 2018; skallerud and wien, 2019). second, during the pandemic, consumers have become more interested in what they consume and from where they source them (severo et al., 2020; sheth, 2020). the intrinsic quality of food matters more than ever (ben hassen et al., 2020). local food is consumed quickly (aprile et al., 2016; loiseau et al., 2020). this ensures that it is fresher and more nutritious, with stronger immunity-boosting properties (ozturk and akoglu, 2020). third, the distribution chain of local food is transparent, short, safe, and fair, because it is produced in the same geographic zone (aprile et al., 2016; roy et al., 2019; toukabri and ghali-zinoubi, 2020). this reassures consumers and leads them to perceive the reduced risk of product contamination during its journey from the producer to the final consumer. fourth, shorter distances between producers of local food and consumers are associated with reduced carbon impacts and waste production. moreover, local production stimulates accountability among farmers, meaning that they will be more likely to engage in environmentally friendly practices (mesić et al., 2020). therefore, local foods are perceived as both sustainable and environmentally friendly products (ozturk and akoglu, 2020; rousseau and deschacht, 2020). fifth, consuming locally available foods during times of crisis is a positive behavior that supports local producers and distributors, while consequently making consumers aware of their responsibility (jribi et al., 2020; mesić et al., 2020). however, the consumption of local food still depends on availability (sowers et al., 2019). this is because local food increases customer trust, in addition to being accessible and easy to find (kumar and kashyap, 2018, verma, 2020), thereby benefiting the retailer (steinhart et al., 2013). tunisia is a mediterranean country located in north africa. agriculture plays a vital role in its economy, and 10.1% of its gross domestic product (gdp) in 2019 came from agricultural products. according to the moderator intelligence report (mir) of 2020, agricultural produce constitutes almost 6% of the total tunisian exports. in 2019, the vegetable production of this country exceeded 3.0 million metric tons, constituted by 545 metric tons of citrus, 288.7 metric tons of dates, and 130 metric tons of tomatoes. this country is the second-biggest exporter of organic foods (olive, date, vegetables, vines, orange, apple, etc.) in africa with almost 80% of its production (mir, 2020). however, tunisia does not have food sovereignty, despite the important and varied local food production. it has had a trade deficit in agriculture production for several years (almayed, 2019). for example, in 2020, tunisia imported food for over 2.56 billion us dollars while its exports amounted only to 1.95 billion us dollars (salah, 2021). its leading agriculture imports are italian journal of food science, 2021; 33 (4) 23 local food consumption health consciousness health consciousness is the degree to which issues related to personal health are of concern to an individual (pu et al., 2020). it also refers to the degree to which health-related actions are integrated into a person’s everyday activities (janetius and krithik, 2020). customers with high health consciousness are more likely to engage in healthy habits and take proactive measures to protect their health (mesić et al., 2020; pu et al., 2020). in addition, people have improved their health consciousness during the pandemic. local food is characterized by having a short distribution chain, freshness, and nutritional value, and by being harvested at the optimal stage (roy et al., 2019). the consumption of local food during the coronavirus pandemic has grown because it is considered safer and healthier (xie et al., 2020). based on the arguments above, we hypothesize that: h2. during the covid-19 pandemic, health consciousness positively influences consumers’ intention to purchase local food. proximity to the production process proximity to production refers to sharing knowledge of the internal functioning of trade (quality, origin, the production process, and the distribution chain) with customers (bergadaà and del bucchia, 2009). in other words, proximity to the production is based on formal and informal exchanges of relevant and timely information to meet wishes and expectations of the actors involved in a transaction (anderson and narus, 1990). this proximity dimension shows customers the transparency of the manufacturing process and the useful, sanitary, and nutritious qualities of the product (merle and piotrowski, 2012). in geographically dispersed food systems, production and distribution chains are complex, large, and include several intermediaries (loiseau et al., 2020; roy et al., 2019; todorovic et al., 2018). this means that the processes of production and distribution are not transparent to customers. thus, easy access to the source of production leads to greater food security (mazieres and gauthier, 2015; toukabri and ghali-zinoubi, 2020). consequently, proximity to the production, which focuses on the relationship between the producer and consumer, becomes important to reassure customers, especially as safety and nutritious attributes of products are regarded (loiseau et al., 2020; skallerud and wien, 2019). local food chains have been found to meet these requirements (todorovic et al., 2018). during covid-19, customers have become more concerned with health and the environment. short and transparent production, distribution processes, and smaller supply chain have become essential criteria to consumers may struggle to accurately assess the distance between producers and markets (the places of purchase), especially at higher levels of congruence. a perceptual approach has been developed as an alternative. it considers food as “local” based on the consumer perception, in other words it is local if the consumer subjectively believes it to be so. this approach is more versatile but has issues regarding reliability, especially when neighbors living on the same street estimate the same product differently. in this context, bazzani and canavari (2017, p. 514) suggested that the meaning of “local” should be expressed in terms of connection to a geographical area rather than in terms of food miles. considering the advantages and disadvantages of both approaches, we will use the definition developed by the industry of global distribution (igd) (2005, p. 3), where “local food must be grown or produced within 30 miles of where the buyer lives.” motivation for local food consumption intrinsic quality the existent literature in marketing shows that intrinsic quality is an ambiguous and multidimensional concept. (fandos and flavian, 2006). it is among the most important criteria that consumers use to evaluate food products (memery et al., 2015). the literature distinguishes between extrinsic and intrinsic quality. intrinsic attributes consist of an objective quality assessment, are intangible, specific to every product, and cannot be altered without modifying the nature of the product (fandos and flavian, 2006). several studies have found that consumers perceive local food as having superior intrinsic quality in terms of freshness, taste, naturalness, nutrition, health, and safety (mesić et al., 2020; meyerding et al., 2019; roy et al., 2019). however, local food only presents these attributes if purchased from local producers, especially in their production season (ozturk and akoglu, 2020). during the covid-19 pandemic, strengthening immunity has become a major concern for consumers. local foods meet these objectives for consumers because they consider them free from preservatives and chemicals, perceiving them as natural and wholesome. such attributes are believed to provide health benefits (memery et al., 2015). therefore, intrinsic quality is important motivation for consumers to purchase local food during the pandemic (mesić et al., 2020; shveda, 2020). given the above information, the first hypothesis of the study is as follows: h1. during the covid-19 pandemic, intrinsic quality positively influences consumers’ intention to purchase local food. 24 italian journal of food science, 2021; 33 (4) ghali-zinoubi z chain is associated with fewer negative environmental impacts (e.g., carbon emissions and other pollution forms), once there is less packaging and no need for shipping facilities, packing facilities, or refrigeration. similarly, jribi et al. (2020) identified a positive impact of covid-19 on tunisian consumer awareness in terms of attitudes and behaviors toward food waste. therefore, we posit the following: h5. during the covid-19 pandemic, environmental awareness positively influences consumers’ intention to purchase local food. moderating role of food availability food availability is a proxy of the service level provided to end customers (lovel et al., 2005; verma, 2020). consumers have a positive perception of local food if it is accessible and available (steinhart et al., 2013). the higher the availability of local food in the market, the higher is the familiarity of the consumer with its characteristics and, consequently, the perception of its values (toukabri and ghali-zinoubi, 2020). therefore, product availability increases the intention to purchase it. during the covid-19 pandemic period, which was characterized by a general lockdown and restrictions on international transport, the demand for local food grew prominently (duda-chodak et al., 2020; loiseau et al., 2020; mesić et al., 2020). food availability becomes crucial for local retailers to satisfy the increasing demand of their customers. this is because local food became the single viable alternative available for customers in several countries during the covid-19 pandemic. consumers consume local food to protect their health, environment, and the local community (memery et al., 2015). consequently, the following hypotheses are proposed: h6. during the covid-19 pandemic, local food availability strengthens the relationship between the purchase intention of the consumer and its predictors. h6.a. during the covid-19 pandemic, local food availability strengthens the relationship between intrinsic quality and purchase intention. h6b. during the covid-19 pandemic, local food availability strengthens the relationship between health consciousness and purchase intention. h6c. during the covid-19 pandemic, local food availability strengthens the relationship between proximity to the production process and purchase intention. reassure customers, minimize contamination risks, and increase food security (hobbs, 2020; mesić et al., 2020). therefore, local food has fostered increasing interest among customers, and its consumption has grown prominently (shveda, 2020). thus, we hypothesize that: h3. during the covid-19 pandemic, proximity to the production process positively influences consumers’ intention to purchase local food. local support consumption of local food has empathic, social, and local loyalty motivations (skallerud and wien, 2019). in fact, individuals consume local food to help local producers compete with national producers and imports (mesić et al., 2020; meyerding et al., 2019). it is a moral obligation (peterson, 2013) and behavior in favor of the local community and retailers (memery et al., 2015). an increase in local food consumption revitalizes the economy and plays a role in reducing the environmental damage that occurs while shipping food (e.g., carbon emissions) (ozturk and akoglu, 2020). during the coronavirus pandemic, local restaurants and stores were temporary or extendedly closed. however, the “shop local” movement also increased during such times. several studies have stated that consumers are more aware of the impact of the covid-19 crisis on local suppliers, retailers, and the wider community (severo et al., 2020). to support them and reduce the impact of the crisis on their activities, consumers are more motivated to purchase local food. this has been translated into favorable purchasing intention (mesić et al., 2020). based on this argument, we hypothesize that: h4. during the covid-19 pandemic, local support positively influences consumers’ intention to purchase local food. environmental awareness according to the world health organization (2020) report, the covid-19 pandemic has highlighted that maintaining sanitation and hygienic conditions, providing safe water, and reducing air pollution are all crucial measures for protecting human health during any infectious disease outbreak. rousseau and deschacht (2020) analyzed the online search behavior of 20 european countries and found that public awareness of nature and the environment increased during the covid-19 pandemic. this is because protecting the environment is an important factor in reducing the risk of infection (severo et al., 2020). consuming local food is a way to reduce environmental pollution and improve food safety, as it reduces the risk of contamination associated with a long supply chain, in addition to being more sustainable (ozturk and akoglu, 2020). overall, a short distribution italian journal of food science, 2021; 33 (4) 25 local food consumption data collection the research context we chose was the country of tunisia. the survey forms were made available online, using google forms. they were distributed mainly using email and social media networks (facebook, linkedin, whatsapp, and instagram). the survey questionnaire was distributed using the convenience sampling approach. this speedy, easy, and low-cost technique does not allow the generalization of results (yadav, 2016). however, it has provided pertinent and reliable findings in the context of food consumption studies (ghali-zinoubi, 2020, 2021). a pre-study, including 23 surveys that were distributed to our family members and colleagues, was conducted to check the clarity and understandability of the survey. no revisions were made to the structure and content of the survey because respondents did not report any ambiguity or difficulty in understanding the questions. the data were collected from may 03, 2020, to september 29, 2020. at the beginning of the survey, we provided examples of local food such as vegetables (potatoes, onions, spinach, parsley…), fruits (orange, apple, figs, peaches, apricots, etc.), eggs, meals, milk, and olive oil. they are the main local food produced in the different regions of tunisia. in the end, 287 responses were returned out of 430 distributed questionnaires. however, we considered only 272 responses after eliminating incomplete or inappropriate (all answers were similar) responses. table 1 summarizes the demographic profile of the sample. h6d. during the covid-19 pandemic, local food availability strengthens the relationship between local support and purchase intention. h6e. during the covid-19 pandemic, local food availability strengthens the relationship between the environmental awareness and purchase intention. materials and methods survey design the survey was designed using references from local food literature. the variable health consciousness was measured using dutta-bergman’s (2004) five-item scale adapted to the coronavirus context (pu et al., 2020). environmental awareness was measured using severo et al.’s (2020) six-item scale. local support was measured using megicks et al.’s (2012) scale, which included three items. proximity to the production was measured by adapting the mazieres and gautier (2015) scale. intrinsic quality was measured using six items adapted from megicks et al. (2012). purchase intention was measured using the two-item scale of lee et al. (2010), and food availability was measured using the four-item scale of arsil et al. (2018). to adapt these measurement scales to the context of the coronavirus pandemic, the prefix “during the covid-19 pandemic” was added to the different items as following pu et al. (2020). all items were rated on a five-point likert scale ranging from strongly agree to strongly disagree. intrinsic quality local support h6e h6d h6c h6b h6a enviromental awerness food availability h5 h4 h3 h2 h1 purchase intention health consciousness proximity of the production figure 1. proposed conceptual model. 26 italian journal of food science, 2021; 33 (4) ghali-zinoubi z table 1. demographic description of the sample. variables/criteria n % variables/criteria n % gender education female 158 58.08 elementary and middle school 23 8.45 male 114 41.92 high school 54 19.85 age graduate 122 44.85 18–35 68 25 postgraduate 60 22.05 36–59 156 57.35 phd 13 4.78 older than 60 main household purchaser myself other members both 48 113 102 57 17.65 41.54 37.5 20.96 household monthly income (td)* less than 1000 from 1001 to 1500 more than 1500 61 139 72 22.42 51.10 26.47 *td: tunisian dinar. 1 td = 0.36 us $ in june 2021. results and discussion data were analyzed using the sem procedure. this method is considered appropriate as it examines simultaneous dependency among relationships (severo et al., 2020, p. 6). to test the hypotheses of dependent relationships, both measurement and structural models will be assessed. measurement model the reliability, and convergent and discriminant validities were tested using a confirmatory factor analysis (cfa). the internal consistency of the survey items was measured using cronbach’s alpha (α). the values ranged from 0.789 to 0.887, which exceeded the threshold of 0.7 established by hair et al. (2013). the composite reliability (cr) scores ranged from 0.743 to 0.888, exceeding the threshold established by bagozzi et al. (1998). therefore, the reliability of the scales of all constructs of the conceptual model was confirmed. the convergent validity was tested through the factor loading and the average variance extracted (ave). the factor loading of all items was above 0.6 (ranging from 0.623 to 0.889), meeting chin et al.’s (1997) criterion. in addition, the ave value ranged from 0.611 to 0.785, which was within the acceptable limit of 0.5 (hair et al., 2013). hence, convergent validity was confirmed for every construct. these results are summarized in table 2. the discriminant validity was also assessed by measuring the square root of ave of each construct (bagozzi and yi, 1988). the results showed that they were higher than its correlation value (as shown in table 3). this criterion was met for every construct. thus, the discriminant validity is confirmed. structural model goodness of fit indices the goodness of fit indices of the conceptual model was assessed using a structural model. the sem results showed that the absolute, incremental, and parsimonious index values (x2 = 206.429, x2/df = 2.788, fi = 0.912; tli  = 0.892; cfi = 0.911; ifi = 0.912; rmsea = 0.04) were well above the recommended thresholds (bagozzi and yi, 1988). therefore, the conceptual model presents a good fit. hypothesis testing and discussion test of direct impact to test the hypotheses, we examined β-values (the association between the independent and dependent constructs), t-values, and p-values. the path coefficients indicated that “intrinsic quality” and “health consciousness” had a positive significant influence on purchase intention (β = 0.428, t-value = 17.334; p < 0.01 and β = 0.449, t-value = 22.267; p < 0.01, respectively). therefore, h1 and h2 were supported. these findings were in line with previous findings which showed that the health and the personal well-being of consumers are their main motivations for purchasing local food (birch et al., 2020; memery et al., 2015). this interest in maintaining health becomes more important in the era of covid19 (duda-chodak et al., 2020; janetius and krithika, 2020). furthermore, the “proximity to the production” also had a positive and significant influence on purchase intention (β = 0.388, t-value = 24.584; p < 0.05). italian journal of food science, 2021; 33 (4) 27 local food consumption table 2. measurement model: reliability and convergent validities. measurement items factor loadings t-values cronbach’s α intrinsic quality (iq): cr = 0.888; ave = 0.733 iq1. during the covid-19 pandemic, i buy local food because it is free from preservatives. 0.834 24.734** 0.827 iq2. during the covid-19 pandemic, i buy local food because it is free from chemicals. 0.823 33.754** 0.823 iq3. during the covid-19 pandemic, i buy local food because it is natural. 0.788 34.842** 0.823 iq4. during the covid-19 pandemic, i buy local food because it is wholesome. 0.887 31.783** 0.851 iq5. during the covid-19 pandemic, i buy local food because it has a good appearance. 0.754 26.647* 0.827 iq6. during the covid-19 pandemic, i buy local food produce because it lasts longer. 0.742 24.752** 0.816 health consciousness (hc): cr = 0.821; ave = 0.785 hc1. during the covid-19 pandemic, i feel that living life in the best possible health is very important to me. 0.823 36.754** 0.887 hc2. during the covid-19 pandemic, eating right, exercising, and taking preventive measures will keep me healthy for life. 0.814 36.184** 0.827 hc3. during the covid-19 pandemic, my health depends on how well i take care of myself. 0.889 13.762** 0.876 hc4. during the covid-19 pandemic, i actively try to prevent disease and illness. 0.837 24.818** 0.828 hc5. during the covid19-19 pandemic, i do everything i can to stay healthy. 0.847 19.528* 0.856 environmental awareness (ea): cr = 0.789; ave = 0.611 ea1. the covid-19 pandemic has made me increase the separation of organic and recyclable waste. 0.778 23.766* 0.803 ea2. the covid-19 pandemic has caused me to reduce water consumption further, as this is a finite environmental resource. 0.792 27.337* 0.789 ea3. the covid-19 pandemic made me worry even more about the natural resources for future generations. 0.763 36.547** 0.823 ea4. the covid-19 pandemic made you realize the reduction in air pollution. 0.811 17.831* 0.811 ea5. the covid-19 pandemic made me realize, even more, the environmental impact caused on the planet. 0.801 22.781** 0.793 ea6. the covid-19 pandemic has increased my environmental awareness. 0.788 24.972* 0.801 local support (ls): cr = 0.816; ave = 0.743 ls1. during the covid-19 pandemic, i buy local food to support local producers. 0.623 28.627** 0.844 ls2. during the covid-19 pandemic, i buy local food to support local retailers. 0.688 26.738** 0.822 ls3. during the covid-19 pandemic, i buy local food to support the local community. 0.724 24.343* 0.773 proximity of process (pp): cr = 0.833; ave = 0.713 pp1. during the covid19 pandemic, i am interested to know very well the rules of production and distribution of the local foods. 0.854 28.972** 0.851 pp2. during the covid-19 pandemic, i am interested in being very familiar with the production methods used by the producers who produce this local food. 0.836 27.384** 0.802 pp3. during the covid-19 pandemic, i am interested to know as to who the producers of this local food are. 9.812 31.827** 0.826 purchase intention (pi): cr = 0.743; ave = 0.776 pi1. during the covid-19 pandemic, i am willing to buy local food while shopping. 0.845 18.993** 0.881 pi2. during the covid-19 pandemic, i will make an effort to buy local food in the near future. 0.844 21.366** 0.823 availability: cr = 0.811; ave = 0.783 av1. during the covid-19 pandemic, local food is available. 0.833 17.529** 0.842 av2. during the covid-19 pandemic, local food is easier to find. 0.833 12.333** 0.827 av3. during the covid-19 pandemic, local food has cheaper price. 0.853 21.023** 0.852 av.4. during the covid-19 pandemic, local food provides an assurance of product origin. 0.845 21.333** 0.783 note: **p < 0.01. *p < 0.05. 28 italian journal of food science, 2021; 33 (4) ghali-zinoubi z table 3. discriminant validity test. latent variables iq hc ea ls pp pi av iq 0.856 hc 0.534** 0.886 ea 0.675** 0.373** 0.781 ls 0.531** 0.462** 0.422** 0.861 pp 0.674* 0.561** 0.372* 0.467** 0.844 pi 0.554** 0.333** 0.323* 0.556** 0.324* 0.880 av 0.621** 0.234** 0.341** 0.433 0.233 0.33** 0.793 mean (s.d) 4.266 (0.4316) 4.333 (4.363) 4.233 (0.466) 4.666 (0.522) 4.666 (0.492) 4.301 (0.449) 4.001 (0.692) notes: **p < 0.01; *p < 0.05. s.d: standard deviation. the square roots of aves are the numbers written in bold on the diagonal. numbers below the diagonal represent constructs’ correlations. hence, h3 was supported. this finding was in line with toukabri and ghali-zinoubi (2020). the common point among the three predictors was that they expressed self interest (“what is good for me”) by focusing on concerns over health, safety, and well-being, which translated into self-centered motivations (birch et al., 2018). during the covid-19 pandemic, this interest in health and safety gained additional attention (pu et al., 2020). the strong association between self-centered motivations and behavioral intentions is justified by the fact that local food meets health and safety requirements (birch et al., 2018; mesić et al., 2020; meyerding et al., 2019). the influence of “local support” on purchase intention was positive and significant (β = 0.111, t-value = 10.233; p < 0.05). therefore, h4 was supported. this finding was in line with memery et al. (2015) and arsil et al. (2018). the construct “environmental awareness” had a positive influence on purchase intention, but the relation was weak (β = 0.043, t-value = 1.633; p > 0.05). therefore, h5 was not supported. these two variables expressed a “do good” for the environment and wider community, which translated into altruistic motivations (what is good for “us”). the findings of this study showed a weak impact of the altruistic motivations compared with self-centered ones, although local support was a significant driver of intention of purchasing local food. likewise, rousseau and deschacht (2020) analyzed online search behavior in the european union and found that the covid-19 crisis did not affect public awareness of environmental issues. birch et al. (2018) also reported a stronger impact of self-centeredness than of altruism in the context of local food consumption. however, severo et al. (2020) conducted a study in portugal and brazil and found that the pandemic was instrumental in people’s behavioral change, increasing environmental awareness and sense of social responsibility. these results are summarized in table 4. test of the moderating role of local food availability the moderating role of food availability (table 5) was significant for the relationships between intrinsic quality and purchase intention, health consciousness and purchase intention, and proximity to the production and purchase intention. however, it was weak in the relationship between local support and purchase intention, and environmental awareness and purchase intention. these results show that regardless of food availability, the table 4. hypothesis testing. path β-values t-statistics relationship h1 iq → pi 0.428 17.334** supported h2 hc → pi 0.449 22.267** supported h3 pp → pi 0.388 24.584* supported h4 ls → pi 0.111 10.233* supported h5 ea → pi 0.043 1.633 not supported note: **p < 0.01. *p < 0.05, iq: intrinsic quality; hc: health consciousness, pp: proximity of process, ls: local support, ea: environmental awareness, pi: purchase intention. table 5. hypothesis testing. path β-values t-statistics relationship h6a iq * av → pi 0.223 6.333* supported h6b hc * av → pi 0.154 3.667** supported h6c pp * av → pi 0.111 11.628* supported h6d ls * av → pi 0.033 1.333 not supported h6e ea * av → pi 0.004 1.066 not supported note: **p < 0.01. *p < 0.05; iq: intrinsic quality; hc: health consciousness, pp: proximity of process, ls: local support, ea: environmental awareness, pi: purchase intention, av: availability. italian journal of food science, 2021; 33 (4) 29 local food consumption purchase intention during the covid-19 pandemic. the findings indicated that the pandemic strengthened the relationships between self-centered motivations and purchase intention. however, it was not significant for altruistic relationships. this means that during the covid-19 pandemic, local food, if available, is mainly purchased for health protection, accessibility, and its high quality. managerial implications the findings of this study showed that during the coronavirus pandemic, consumers purchased local food because of its superior intrinsic quality, health benefits, and accessibility. practitioners would be wise to focus their marketing campaigns considering these features to boost the consumption of their products during, as well as after, the pandemic. appropriate packaging, effective labeling strategies, and coherent branding promoting these benefits of local foods should be implemented to attract customers and increase their local food consumption. moreover, advertising should highlight known features, such as superior intrinsic quality, supporting local food, and healthy ingredients. marketers of local food should communicate slogans such as “proudly eating local food” or “tastier, healthier, more nutritious and safer food.” local retailers may also extend their support to local producers by providing details pertaining to the local source of produce and enhancing the transparency of their supply chain. moreover, local food producers and the wider community can act as a unique platform to improve sales of existing food products and invent new products both during and after the current health crisis. governments should also play an active role to make local food more available by supporting local farmers with the required infrastructure, providing seeds and other financial aids. furthermore, a suitable program should be added to local curricula to educate the next generation of consumers about the importance of local products. local support has been found to have a significant impact on consumer intention to purchase local food. several scholars have argued that covid-19 has disrupted and changed consumer habits, lifestyles, and long-term behaviors (naja and hamadeh, 2020; picha et al., 2017; pressman et al., 2020; qi et al., 2020). the pandemic brought the community closer together and highlighted the need to address social inequalities. marketers should emphasize the importance of supporting local businesses and the wider community while building branding messages and communication strategies that focus on the attributes we presented. such messages should inform and educate the public about the economic, environmental, and social benefits to be consumer was more self-centered than altruistic during the covid-19 pandemic. in other words, the consumer intent to purchase local food is mainly due to its good quality and health benefits, irrespective of its availability. these results are in line with toukabri and ghali-zinoubi (2020). conclusion, implications, and limitations the findings indicate that, during the covid-19 pandemic, health consciousness is the highest motive for consuming locally. intrinsic quality and proximity to the production chain are also significant drivers of local food consumption. however, local support and environmental awareness have little impact on the intention to purchase local food. food availability plays an important moderating role in three relationships (health consciousness/ purchase intention, intrinsic quality/purchase intention, and proximity to the production/purchase intention). many people have become more concerned with what they eat and from where they purchase their food. these findings also have several theoretical and managerial implications. theoretical implications this study enhanced the theoretical background of local and healthy food. first, several studies have argued that local food consumption is more important than ever, during the covid-19 pandemic (ben hassen et al., 2021b; cranfield, 2020; duda-chodak et al., 2020; naja and hamadeh, 2020; rizou et al., 2020). other studies have argued that this growth is because local food is safe, healthy, and accessible (aprile et al., 2016; arsil et al., 2018; meyerding et al., 2019; ozturk and akoglu, 2020). however, this study tested more predictors of local food consumption, such as proximity to the production and local support. then, it may be considered among the first to test these predictors in the context of the covid-19 pandemic. second, this paper examined the impact of both self-centered and altruistic motivators on the intention to purchase local food. the findings showed that self-centered motivations are important drivers for local food consumption while the effect of altruistic motivations is still too weak. the study was developed in the context of a developing country (tunisia), where environmental consciousness is still in its early stage (ghali-zinoubi, 2021). these findings are in contrast to those of several other studies which were conducted in developed countries and argued that both motivators are important (rousseau and deschacht, 2020; skallerud and wien, 2019). third, this study is among the few studies that examine the moderating role of food availability 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convenience sampling. this method is speedy, easy, and low cost; however, it hinders generalizability and replicability of results. second, this study was developed in a single country. cross-cultural studies would increase the inclusiveness of the research. third, in this study, the motivations for purchasing local food were limited to five. however, several other motivations can be studied, such as short supply chains, local identities, ethical identities, past uses, and interest in traceability. fourth, in this study, the focus was on the main drivers of local food purchase intention while the barriers were not studied. future research should examine barriers that could inhibit consumers from purchasing local food, including high price, availability, and diversity. finally, the covid-19 issue has taught us that individual action yields benefits that are multiplied when implemented on a global scale. we can master the crisis and flatten the curve of its progression if we work 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https://doi.org/10.1097/nt.0000000000000415� https://doi.org/10.3390/ijerph17165693� https://doi.org/10.3390/ijerph17165693� https://doi.org/10.3390/ijerph17197106� https://doi.org/10.3390/ijerph17197106� https://doi.org/10.1016/j.tifs.2020.06.008� https://doi.org/10.1007/s10640-020-00445-w� https://doi.org/10.1080/15378020.2019.1600891� https://www.statista.com/statistics/1190806/import-and-export-of-agriculture-and-food-in-tunisia/� https://www.statista.com/statistics/1190806/import-and-export-of-agriculture-and-food-in-tunisia/� _hlk84242349 _hlk84016988 ole_link3370 ole_link3371 ole_link3373 ole_link3374 _hlk84002929 _hlk84024530 _hlk21757629 _hlk21758357 p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33i4.2107 11 p u b l i c a t i o n s codon paulownia tomentosa flower polysaccharide as an effective immunopotentiator to enhance immune responses for newcastle disease vaccine in mice xiaolan chen1*, junjie jin2, fuxing hao1, haifeng yang1, hongxiang sun3, chunmao jiang1 1jiangsu agri-animal husbandry vocational college, taizhou, china; 2wenzhou vocational college of science and technology, wenzhou, china; 3zhejiang university, hangzhou, china *corresponding author: xiaolan chen, jiangsu agri-animal husbandry vocational college, taizhou, jiangsu province, china. email: cxl7972563@163.com received: 23 july 2021; accepted: 27 september 2021; published: 2 november 2021 © 2021 codon publications open access paper abstract to investigate the immunomodulatory activity and explore the mechanism of paulownia tomentosa flower polysaccharides (ptfp). ptfp was orally administrated to mice for seven successive days before and after newcastle disease vaccination. the results demonstrated that compared with the vaccine control (vc) group, ptfp enhanced the inhibition of hemagglutination assay antibody titers, promoted the antigen-specific immunoglobulin (ig)g, igg1, igg2a, and igg2b antibodies responses, enhanced proliferation of spleen t and b lymphocytes, increased the secretions of interferon-γ and interleukin-10 cytokines of spleen lymphocytes, and promoted the activation of natural killer cells. therefore, ptfp, as an effective immunopotentiator, could induce a mixed t-helper (th)1 and th2 immune responses and an innate immune response. keywords: immune responses, immunopotentiator, newcastle disease vaccine, paulownia tomentosa flower polysaccharides introduction in recent years, polysaccharides from chinese medicinal herbs have drawn more attention because of their effective immune enhancement, favorable safety, and excellent biocompatibility (sun et al., 2018; tang et al., 2019; chen et al., 2020). various polysaccharides, such as astragalus polysaccharide, lentinan, angelica sinensis polysaccharide, and lycium barbarum polysaccharide, have been proved to possess potent immunomodulatory activity (su et al., 2014; wang et al., 2016; sun et al., 2018; chen et al., 2020; ren et al., 2021). the paulownia tomentosa (p. tomentosa), as chinese herbal medicine, has been widely used to treat stomach disorders, diarrhea, gonorrhea, erysipelas, hypertension, enteritis, tonsillitis, bronchitis, and dysentery (dai et al., 2015; liu et al., 2017; lee et al., 2018; wang et al., 2019). recent researches have proved that p. tomentosa possesses various pharmacological activities, such as antibacterial, anti-inflammatory, antiphlogistic, antitussive, antiasthmatic, immunomodulatory, antioxidant, antiviral, and anticholinesterase activities (dai et al., 2015; liu et al., 2017; wang et al., 2019). p. tomentosa flower polysaccharides (ptfp), which are extracted from the p. tomentosa flower, are the main water-soluble component of this flower. it has been reported that ptfp could serve as a new immunopotentiator to enhance humoral and cellular immune responses (wang et al., 2019). newcastle disease (nd), one of the most contagious and devastating diseases in the poultry industry around the world, is caused by an avian paramyxovirus type 1 serotype of the genus avulavirus in the paramyxoviridae family (zhai et al., 2011(a); yuan et al., 2020; chen et al., 2021). this disease reduced the production of eggs, led to respiratory and central nervous infections, death italian journal of food science, 2021; 33 (4): 11–20 mailto:cxl7972563@163.com 12 italian journal of food science, 2021; 33 (4) chen x et al. (il)-10 enzyme-linked immunosorbent assay (elisa) kits were obtained from boster biological technology co., ltd (china). all other regents and chemicals were of analytical grade. preparation of ptfp ptfp was prepared by water decoction and ethanol precipitation as previously described (yang et al., 2019). in brief, dried cultured p. tomentosa flowers were extracted twice with boiling water for 2 hours and 1 hour, respectively. after filtration, the merged decoction was then condensed, and the suspension was precipitated with 95% ethanol four times for a total of 12 hours. the solution was then centrifuged and concentrated to a specific volume, dried under reduced pressure at 60°. the carbohydrate concentration (%) of the total ptfp was 48 compared with d-glucose, and the luteolin and apigenin concentration (%) of the total ptfp was 3.12 and 4.35, respectively. cells and animals human leukemia cell line k562 was obtained from shanghai institute of cell biology, chinese academy of sciences. institute of cancer research mice (5–6 weeks, 18–22 g, male and female) were purchased from shanghai slake laboratory animal co., ltd and housed in zhejiang university. the mice were maintained under pathogen-free conditions and acclimatized for 7 days before experiments. the animal experiments were conducted at the zhejiang university. all animal experiments were conducted in compliance with the guide for the care and use of laboratory animals and approved by the animal welfare and ethics committee, zhejiang university (january 16, 2019, authorization number is no.18227). all the animals were anesthetized with ether, the blood samples were drawn from the eyes, and the mice were killed by the cervical dislocation method. method experiment design the mice were randomly divided into five groups: blank control (bc), vaccine control (vc), ptfp-low dose (ptfp-l), ptfp-medium dose (ptfp-m), and ptfphigh dose (ptfp-h) groups with 12 animals per group. the experimental procedure is represented as a schematic illustration in figure 1. the mice (except the bc group) were subcutaneously immunized with 0.1ml nd vaccine (106.0 eid50/0.1ml) on day 4 and boosted with the same dose on day 18. the mice in ptfp groups were continuously orally administrated with different doses of ptfp (30, 60, and 120 mg/kg) for 7 days (from day 1 to day 7), once a day. in addition, the oral administration of poultry, and caused an immense economic loss in the poultry industry ( ma et al., 2019; yuan et al., 2020). vaccination is the most effective and productive approach to prevent and control the spread of nd (yang et al., 2020). the commercial vaccines available were live or inactivated virus-based vaccines. so to enhance immune responses for the nd vaccine, immunopotentiators or adjuvants were commonly required (zhai et al., 2011(b); ma et al., 2019; yuan et al., 2020 ). the immunomodulatory activity of ptfp was determined in our previous study by orally administrating it into the nd-vaccinated chickens. . the in vivo experiment results demonstrated that ptfp could improve lymphocyte proliferation, increase antibody response, and enhance the secretion of interferon (ifn)-γ, indicating that ptfp has the potential to improve immune responses in nd-vaccinated chickens(yang et al., 2019). however, the mechanism of ptfp enhancing immune responses for the nd vaccine is still unknown. hence, to further investigate the immunomodulatory activity and mechanism of ptfp for improving the nd vaccine response. mice used as the model animal were orally administrated with the immunopotentiator ptfp. later they were immunized with nd-vaccine twice at an interval of 14 days. after the second dose of vaccination, inhibition of hemagglutination assay (iha) titers against nd and antigen-specific immunoglobulin (ig)g and isotypes (igg1, igg2a, and igg2b) antibodies were determined. meanwhile, for further immune responses evaluation, spleen lymphocytes proliferation, together with cytokines, and natural killer (nk) cells activity were measured. materials and methods materials dried-cultured p. tomentosa flower were obtained from bozhou guoxin pharmaceutical co., ltd (anhui, china). attenuated ndvaccine (lasota strain, no. 1170121) was purchased from guangxi liyuan biotechnology co., ltd (china). fetal bovine serum was obtained from gibco (carlsbad, ca). roswell park memorial institute (rpmi)1640 medium was purchased from hyclone, logan, ut. concanavalin a (con a) and lipopolysaccharide (lps) were obtained from sigma-aldrich (st. louis, mo). the antigen and positive control sera used for the nd virus (ndv)-specific iha was purchased from qingdao yebio biological technology co., ltd (china). horse radish peroxidase (hrp)-conjugated rabbit anti-mouse igg antibody was purchased from sigma-aldrich. hrpconjugated goat anti-mouse igg1, igg2a, and igg2b antibodies were obtained from southern biotechnology associates (birmingham, al). ifn-γ and interleukin italian journal of food science, 2021; 33 (4) 13 paulownia tomentosa flower polysaccharide as an effective immunopotentiator to enhance immune responses stop the reaction. the optical density (od) was measured at 492 nm using a bio-rad 680 elisa reader (bio-rad, hercules, ca). determination of spleen lymphocytes proliferation at 14 days after boosting immunization, splenocytes were harvested from the mice (wang et al., 2016; kumar et al., 2017; huang et al.,2020; lu et al.,2020; yu et al., 2020; gan et al., 2021). briefly, the mice were sacrificed by cervical dislocation, and the spleens from different groups were aseptically separated. later, they were crushed and passed through a 200-mesh sterile cell strainer, and the red blood cells were separated using cells lysis solution. the collected spleen lymphocytes (5×106 cells/ml) were seeded into a 96-well plate with stimulated ndv antigen (0.0625 hau/ml), con a (final concentration 5 μg/ml), or lps (final concentration 10 μg/ml), respectively. the cells incubated with rpmi-1640 medium were used as the negative control. after cultivation for 44 hours at 37° in 5% carbon-di-oxide atmosphere, 50 μl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (mtt; 2 mg/ml) was added and further cultured for 4 hours at 37°. subsequently, the supernatant was removed, and 150  μl dimethyl sulfoxide (dmso) was added. the absorbance at 570 nm (a570) was measured by using a microplate reader. the stimulation index (si) was calculated as the ratio of absorbance values of nd antigen, con a, or lps stimulated cells to untreated cells (negative control group) as shown in equation (1). 570 570 a (stimulated experimental group) si a (negative control group) = (1) determination of spleen lymphocytes cytokines by elisa the spleen lymphocytes (5×106 cells/ml) from different groups were incubated in 24-well plates and restimulated with ndv antigen (0.0625 hau/ml). after incubation at 37° for 72 hours, the productions of cytokines inf-γ and il-10 were detected in the supernatants collected after performing elisa according to the manufacturer’s instructions. determination of nk cells activity the nk cells activity was determined as previously described (xu et al., 2019; zhang et al., 2020). in brief, the spleen lymphocytes (1×107 cells/ml)) of mice from procedure was performed again for 7 days from day 15 to day 21. the mice in bc and vc groups were administrated with the same volume of physiological saline orally. about 14 days after the boost vaccination (at day 32), the mice were sacrificed, serum samples were collected, and spleens were harvested for subsequent immunological tests. inhibition of hemagglutination assay (iha) iha titers against the ndv in the serum samples were carried out according to the iha procedure as previously described (wu et al., 2012; wang et al., 2013; liu et al., 2014; zhang et al., 2014). briefly, serum samples of different groups were collected at 14 days after boosting immunization and were heat-inactivated at 56° for 30 minutes. the serum samples (25 μl) were serially two-fold diluted using phosphate buffer saline (pbs) in a v-shaped microtiter plate. later 25 μl ndv antigen (4 hemagglutinating units (hau) was added and incubated for 30 minutes at room temperature. here, pbs was used as the negative control. finally, 25 μl of 1% chicken erythrocyte suspension was added, and the samples were reincubated for 30 minutes at room temperature. the iha titer was expressed as the reciprocal value of the highest serum dilution, which completely inhibited the hemagglutination of chicken erythrocytes. determination of antigen-specific antibodies in serum ndv-specific igg and its isotypes (igg1, igg2a, and igg2b) antibodies in the serum were measured on day 14 after boosting vaccination by elisa (wu et al., 2012; sun et al., 2020). briefly, 96 well elisa plates were coated with 100 μl ndv antigen (0.5 hau/ml, carbonate solution, ph = 9.6) per well at 4° overnight. after washing thrice with pbs containing 0.5% tween 20, elisa plates were blocked with pbs containing 1% bovine serum albumin and incubated for 2 hours at 37°. then, the plates were washed thrice, and 100 μl of serially diluted serum samples were added and reincubated for 2 hours at 37°. after rewashing thrice, the hrp-conjugated anti-mouse igg (1:8000 diluted), igg1 (1:6000 diluted), igg2a (1:4000 diluted), or igg2b (1:4000 diluted) antibodies were added into plates and incubated for 2 hours at 37°. then elisa plates were washed thrice, and 100 μl of tetramethylbenzidine was added to the plates. after incubation for 15 minutes, 50 μl of 2 m sulphuric acid was added to d4 prime vaccination d1 d7 d15 d21 d32 d4 boost vaccination oral administration oral administration serum spleen 14 days after boost vaccination figure 1. the schematic illustration of the vaccination and treatment schedule of the experiment. 14 italian journal of food science, 2021; 33 (4) chen x et al. (p < 0.05). iha antibody titers in ptfp-m group were higher than that of ptfp-l and ptfp-h groups and were not detected in the bc group (with no vaccination), whose data is not shown. the results demonstrated that ptfp, as an immunopotentiator, could promote iha antibody responses in mice against nd-vaccine. antigen-specific igg and their isotypes (igg1, igg2a, and igg2b) responses the antigen-specific igg and isotypes (igg1, igg2a, and igg2b) antibodies were determined by elisa to further evaluate the humoral responses induced by ptfp in mice immunized with nd-vaccine. as shown in figure 3a, ptfp in all groups significantly induced stronger antigen-specific igg titers than the vc group (p < 0.05). igg titers in the ptfp-m group were higher than the ptfp-l and ptfp-h groups. the results of igg titers were consistent with iha titers results (figure 2). the levels of antigen-specific igg isotypes (igg1, igg2a, and igg2b) in different groups were determined to investigate the effects of ptfp on t-helper (th)1 or th2 immune responses. the igg1 antibody was associated with a th2-biased immune response. as shown in figure 3b, igg1 antibody titers in all the ptfp groups were significantly promoted compared with the bc group (p < 0.05). the levels of igg1 antibody induced by the ptfp-m group were higher than the ptfp-l and ptfp-h groups. the results of igg2a and igg2b (th1-associated) antibodies titers are shown in figures 3c and 3d, respectively. igg2a and igg2b titers in all ptfp groups markedly increased (especially igg2b) than the bc group. moreover, igg2a and igg2b titers were induced by the ptfp-m group were higher than that in ptfp-l and ptfp-h groups. the results of igg isotypes (igg1, igg2a, and igg2b) in different groups had a similar trend to igg titers (figure 3a). in addition, nd-specific igg and its isotypic (igg1, igg2a, and igg2b) antibodies were not detected in the bc group (with no vaccination), and the data were not shown. spleen lymphocytes proliferation to further investigate the effects of ptfp on cellular immune responses in mice against the nd-vaccine, spleen lymphocytes from mice were collected at 14 days after the boost vaccination, and the lymphocytes proliferation was stimulated with ndvantigen, con a, or lps and determined. as shown in figure 4a, after incubation with ndv antigen for 44 hours, lymphocytes proliferation in the ptfp groups (ptfp-l, ptfp-m, and ptfp-h groups) was significantly promoted compared with the vc groups (p < 0.05). the lymphocyte proliferation in the ptfp-m group was higher than the ptfp-l and ptfp-h groups. the result of lymphocytes proliferation with con a-stimulation is shown in figure 4b. the lymphocytes proliferation in the ptfp-l, ptfp-m, and ptfp-h different groups were collected as the effector cells, and the human leukemia k562 cells (2×105 cells/ml) were used as the target cells. about 100 μl of spleen lymphocytes were added into a 96-well plate with k562 cells (100 μl) in the ratio of 50:1 (effector cells: target cells) and incubated for 20 hours at 37°. the mtt method was used to measure the cell viability of nk cells wherein 50 μl of mtt (2 mg/ml) was added to the plates and incubated for 4 hours. then the supernatant was removed, dmso was added, and the absorbance was determined at 570 nm by a microplate reader. the cell viability of nk cells was calculated according to equation (2). t s e t a570 (a570 a570 ) nk cells viability= ×100% a570 − − (2) where a570t: absorbance value of target cells control; a570s: absorbance value of samples; and a570e: absorbance value of effector cells control. data analysis all data were expressed as the mean ± standard deviation (sd). the statistical significance of differences was assessed by analysis of variance and tukey’s multiple comparisons. a probability value of p < 0.05 was considered statistically significant. results antibody responses iha antibody titers figure 2 shows the significant increase in the iha antibody titers in ptfp groups compared with the vc group 8 vc ptfp-l ptfp-m ptfp-h 6 4 2 0 vc pt fp -l pt fp -m pt fp -h ih a ti te rs ( lo g2 ) figure 2. effect of ptfp on iha antibody response in the mice immunized with nd vaccine. the iha antibody titers (log2) in serum from mice of different groups at 14 days after the boost immunization were determined by iha assay. the values were presented as mean ± sd (n =12). significant differences with the vc group were designated as *p < 0.05 and **p < 0.01. iha, inhibition of hemagglutination assay; vc, vaccine control; ptfp, paulownia tomentosa flower polysaccharides; ptfp-l, ptfp-low dose; ptfp-m, ptfp-medium dose; ptfp-h, ptfphigh dose. italian journal of food science, 2021; 33 (4) 15 paulownia tomentosa flower polysaccharide as an effective immunopotentiator to enhance immune responses vc pt fp -l pt fp -m pt fp -h vc pt fp -l pt fp -m pt fp -h vc pt fp -l pt fp -m pt fp -h vc pt fp -l pt fp -m pt fp -h 6 (a) (b) (c) (d) 5 3 4 2 1 0 4 6 8 2 0 a nt i-n d v ig g ti te rs ( lo g2 ) 8 6 4 2 0a nt i-n d v ig g 1 tit er s (lo g2 ) a nt i-n d v ig g 2a ti te rs ( lo g2 ) 4 6 8 2 0a nt i-n d v ig g 2a ti te rs ( lo g2 ) figure 3. effects of ptfp on antigen-specific antibodies responses in the mice immunized with nd vaccine. the antigen-specific igg (a) and isotypes igg1 (b), igg2a (c), and igg2b (d) titers in the serum were measured by an indirect elisa at 14 days after the secondary immunization. the values are presented as mean ± sd (n = 12). significant differences with vc group were designated as *p < 0.05, **p < 0.01 and ***p < 0.001. nd, newcastle disease; ndv, nd-vaccine; vc, vaccine control; ptfp, paulownia tomentosa flower polysaccharides; ptfp-l, ptfp-low dose; ptfp-m, ptfp-medium dose; ptfp-h, ptfphigh dose. groups, especially the ptfp-m group, were improved compared with the vc group (p < 0.05). the trend of lymphocytes proliferation with lps stimulation was similar to con a stimulation (figure 4c). all ptfp groups significantly promoted higher lymphocytes proliferation than the vc group (p < 0.05). the effect of lymphocytes proliferation with lps stimulation in the ptfp-m group was better than those in the ptfp-l and ptfp-h groups. spleen lymphocytes cytokines spleen lymphocytes from immunized mice were collected and incubated with the ndv antigen for 72 hours 14 days after the second vaccination. the secretion of cytokines ifn-γ and il-10 were measured, and the results are shown in figures 5 a and 5b. the levels of ifn-γ in ptfp-l, ptfp-m, and ptfp-h groups were significantly increased compared with the vc group (p < 0.05). the ifn-γ in ptfp-m groups was higher than that in the ptfp-l and ptfp-h groups. the il-10 expressions in all ptfp groups were significantly higher than that in the vc group (p < 0.05), and il-10 levels in ptfp-l and ptfp-m groups were higher vs. ptfp-h group. nk cells activity to further investigate the effect of ptep on immunological enhancement, the cytotoxic activity of nk cells against human leukemia k562 cells was determined. the nk cells activity of splenocytes significantly increased in mice orally administrated with ptfp compared with the vc group (figure 6) and was more strongly induced in ptfp-l and ptfp-m groups vs. the ptfp-h group. discussion recently, natural polysaccharides have been considered as novel immunopotentiators or immunomodulators because of their potent immune enhancement and low toxicity (sun et al., 2018; zeng et al., 2019; zhao et al., 2020;). numerous studies have demonstrated that polysaccharides extracted from chinese medicinal herbs could enhance immune responses and promote the efficiency of vaccines by oral administration ( xie et al., 2012; feng et al., 2013; tang et al., 2019; zhang et al., 2020). in our previous study, ptfp, extracted from p. tomentosa flower, had been proved to possess the immunomodulatory activity and enhance immune responses for orally administered nd-vaccine in chickens (yang et al., 2019). wang et al. (2019) have reported that ptfp as a new immunopotentiator or adjuvant could enhance humoral and cellular responses in chickens by injection administration. however, the mechanism of ptfp promoting 16 italian journal of food science, 2021; 33 (4) chen x et al. vcbc pt fp -l pt fp -m pt fp -h vcbc pt fp -l pt fp -m pt fp -h 8 stimulated with ndv(a) (b) (c) stimulated with con a 6 4 2 0 s tim ul at io n in de x vcbc pt fp -l pt fp -m pt fp -h stimulated with lps 5 4 3 2 1 0 s tim ul at io n in de x 5 4 3 2 1 0 s tim ul at io n in de x figure 4. effects of ptfp on spleen lymphocytes proliferation in the mice immunized with nd-vaccine. splenocyte proliferation was detected by the mtt method after stimulation with nd antigen (a), con a (b), or lps (c) for 44 hours. the values are presented as mean ± sd (n = 12). significant differences with the vc group were designated as *p < 0.05, **p < 0.01, and ***p < 0.001. nd, newcastle disease; ndv, nd-vaccine; lps, lipopolysaccharide; con a, concanavalin a; bc, blank control; vc, vaccine control; ptfp, paulownia tomentosa flower polysaccharides; ptfp-l, ptfp-low dose; ptfp-m, ptfp-medium dose; ptfp-h, ptfp-high dose. vcbc pt fp -l pt fp -m pt fp -h vcbc pt fp -l pt fp -m pt fp -h (a) 4 3 2 1 0i f n -γ c on ce nt ra tio n (p g/ m l) (b) 0 50 100 150 200 250 il -1 0 c on ce nt ra tio n (p g/ m l) figure 5. effects of ptfp on spleen lymphocytes cytokines in the mice immunized with nd vaccine. the spleen lymphocytes from immunized mice were incubated with nd antigen for 72 hours. the ifn-γ (a) and il-10 (b) cytokines in supernatants of spleen lymphocytes were determined by elisa. the values are presented as mean ± sd (n = 12). significant differences with vc group were designated as *p < 0.05, **p < 0.01, and ***p < 0.001. ifn, interferon; il, interleukin; bc, blank control; vc, vaccine control; ptfp, paulownia tomentosa flower polysaccharides; ptfp-l, ptfp-low dose; ptfp-m, ptfp-medium dose; ptfp-h, ptfphigh dose. italian journal of food science, 2021; 33 (4) 17 paulownia tomentosa flower polysaccharide as an effective immunopotentiator to enhance immune responses the stimulation of lymphocytes proliferation indicates the capacity of effective t and b lymphocytes immunity and hence are commonly used as an indicator to reflect the state of cellular immunity (yang et al., 2008; feng et al., 2013; huang et al., 2013). con a and lps were cooperated to stimulate t and b lymphocytes proliferation, respectively (yang et al., 2008; wang et al., 2016). in figure 4, the ptfp markedly increased the lymphocytes proliferation with the stimulation of ndv antigen, con a, or lps vs. the vc group. the result indicated that ptfp could effectively promote the ndv antigen-stimulated lymphocytes proliferation response, con a-stimulated t lymphocytes proliferation, and lpsstimulated b lymphocytes proliferation. th1 lymphocytes mainly secreted ifn-γ, il-2, and il-12 cytokines, whereas the th2 cells predominantly produced il-4, il-6, and il-10 cytokines (liu et al., 2009). to further investigate the cellular immune response induced by ptfp, the spleen lymphocytes cytokines ifn-γ and il-10 were measured. ifn-γ, one of the main cytokines representing cellular immunity, could promote the antibody isotype switching to igg2a and improve the differentiation and proliferation of th1 cells (zhang et al., 2020; coutant et al., 2017). il-10, a th2-type cytokine, is considered an anti-inflammatory cytokine that regulates immune response (courant et al., 2017). the ptfp both significantly improved the secretion of ifn-γ and il-10 cytokines when compared with the bc and vc groups (figure 5a,b), indicating that ptfp both enhanced the th1 and th2 immune responses, which was consistent with the result of igg1, igg2a, and igg2b antibodies (figure 3b–d). nk cells (the cytolytic effector lymphocytes of innate immunity) could recognize and eliminate virus-infected cells and tumor cells and are crucial in the innate immune system (park et al., 2017; xie et al., 2019). they act as a defense line against viral infections, and the activation of these cells plays a crucial role in regulating immune responses (park et al., 2017; xu et al., 2019). in figure 6, compared with the vc group, ptfp significantly enhanced the nk cells activity by increasing the lysing of human leukemia k562 cells, indicating that ptfp with oral administration could markedly promote the activation of nk cells and improve immune responses. based on the results presented above, we investigated the effects of ptfp by oral administration on the mice immunized with nd vaccine and explained the mechanism of ptfp influences on the immune responses. first, the oral administration of ptfp enhanced the nd virus-specific iha titers (figure 2), increased the levels of anti-ndv igg antibody titers (figure 2a), and thereby improved the humoral immune response for the nd vaccine. meanwhile, the ptfp promoted the productions of anti-ndv igg1(th2-type), igg2a, and igg2b (th1-type) vcbc pt fp -l pt fp -m pt fp -h 40 30 20 10 0 n k c el ls a ct iv ity ( % ) figure 6. effect of ptfp on nk cell activity in the splenocytes from the mice immunized with nd vaccine. splenocytes from different groups were collected 14 days after the second immunization, and the nk cells activity was measured by mtt assay. the values are presented as mean ± sd (n = 12). significant differences with the vc group were designated as *p < 0.05, **p < 0.01, and ***p < 0.001. nk, natural killer; bc, blank control; vc, vaccine control; ptfp, paulownia tomentosa flower polysaccharides; ptfp-l, ptfp-low dose; ptfp-m, ptfp-medium dose; ptfp-h, ptfp-high dose. and regulating immune responses for nd vaccine by oral administration was still unexplored. therefore, to further evaluate the immunomodulatory activity and investigate the mechanism of ptfp for nd-vaccine, mice were used as the model animal. different doses of ptfp were orally administrated to it, and they were later immunized with nd vaccine. ndv-specific antibody immune responses play an important and indispensable role in protecting the body from nd virus infection. the iha was performed to determine the levels of specific antibodies against the hemagglutinin-neuraminidase protein (yuan et al., 2020). compared with the vc group, the mice orally administered with ptfp, especially with the ptfp-m dose, significantly increased the nd virus-specific iha titers (figure 2), indicating that ptfp with oral administration enhanced the antibody responses against the nd vaccine. in addition, the ptfp significantly promoted higher anti-ndv igg antibody titers than the vc group (figure 3a), which was consistent with the results of iha in figure 2. to further investigate the th1 and th2 immune responses induced by ptfp, antigen-specific igg1, igg2a, and igg2b antibodies were determined. igg1 antibody is associated with th2-type immune, whereas igg2a and igg2b antibodies are associated with th1-biased immune response (feng et al., 2013). the ptfp promoted the productions of th2-biased igg1 antibody and igg2a and igg2b antibodies (th1-biased) compared with the vc group (figures 3b–d). these results suggested that ptfp induced a strong humoral immune response and elicited a mixed th1 and th2 response. 18 italian journal of food science, 2021; 33 (4) chen x et al. qinglan project of jiangsu province (2021), key research and development projects in zhejiang province and anhui province (2020c02032, s202104g01020071), taizhou science and technology supporting agriculture project (tn202006), jiangsu agri-animal husbandry vocational college project (nsfpt201828), and animal medicine science and technology innovation team (nsf2021tc02). references chen x., han w., wang g., zhao x. 2020. application prospect of polysaccharides in the development of anti-novel coronavirus drugs and vaccines. int. j. biol. macromol.164:331–343. https:// doi.org/10.1016/j.ijbiomac.2020.07.106 chen x., 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lower than those in the ptfp-m group. no serious adverse effects of ptfp were observed after administration. it is necessary to design more groups to narrow the concentration gap between groups to find the best dose and study the side effects of ptfp in mice shortly. the cost of p. tomentosa flower is cheap, and the production process of ptfp is also simple. ptfp, as a new-type immunopotentiator, could play a significant role and have great developing space in poultry breeding. conclusion in this study, to further investigate the immunomodulatory activity and explore the mechanism of ptfp for the nd vaccine, the mice were used as the model animals. different doses of ptfp were orally administered to mice, and the nd vaccine was subcutaneously injected into them. our finding demonstrated that orally administered ptfp enhanced the antigen-specific antibody immune responses and induced mixed th1 and th2 immune responses. meanwhile, the ptfp also increased the proliferation of t and b 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https://doi.org/10.2174/1389557519666190913151632 https://doi.org/10.2174/1389557519666190913151632 https://doi.org/10.1016/bs.pmbts.2019.03.003 https://doi.org/10.3382/ps.2011-01433 https://doi.org/10.3382/ps.2011-01433 https://doi.org/10.1016/j.vaccine.2011.04.097 https://doi.org/10.1016/j.vaccine.2011.04.097 ole_link20 ole_link21 ole_link19 ole_link23 ole_link22 ole_link16 ole_link15 ole_link9 ole_link7 ole_link6 ole_link1 ole_link2 ole_link13 ole_link12 ole_link14 ole_link17 ole_link18 _hlk69118852 ole_link67 _enref_2 _enref_13 _enref_10 _enref_32 _enref_24 _enref_26 _enref_35 _enref_27 _enref_7 _enref_8 _enref_36 _enref_20 _enref_25 _enref_14 _enref_38 _enref_5 _enref_6 _enref_3 _enref_22 paper 290 ital. j. food sci., vol. 27 2015 keywords: meat quality, postmortem ageing, sensory panel, tenderness, veal calves effect of ageing time in vacuum package on veal longissimus dorsi and biceps femoris physical and sensory traits g. baldi*, s. ratti, c. e. m. bernardi, v. dell’orto, c. corino, r. compiani and c. a. sgoifo rossi department of health, animal science and food safety, university of milan, via g. celoria 10, 20133 milan, italy *corresponding author: gianluca.baldi@unimi.it abstract study evaluated the effects of vacuum ageing (2, 4, 6, 8, 10, 12, 16 days) on veal loin (longissimus dorsi; ld) and silverside (biceps femoris; bf) physical and sensory characteristics. ageing did not affect cooking loss, increased ld ph and l*, a* and b* in both muscles. shear force (sf) decreased until day 6 in ld and day 10 in bf. aroma, flavor and taste were not affected, while texture traits were improved. sf was negative correlated with tenderness and juiciness and positive correlated with bf fibrousness and stringy sensation. ageing improved texture properties without altering other sensory traits. mailto:gianluca.baldi%40unimi.it?subject= ital. j. food sci., vol. 27 2015 291 introduction tenderness is one of the main factors affecting consumer’s preference (reicks et al., 2011). since in eu veal calves are slaughtered at no more than 8 months of age (eu regulation 1234/2007) consumers expect a tender meat from them. therefore, ensuring a tender product is a critical aspect for veal producers and retailers, because tenderness is closely related to consumer’s satisfaction and they are also willing to pay more for tender meat (dransfield et al., 1998; feuz et al., 2004; alfnes et al., 2005). post mortem ageing improves meat tenderness due to the proteolysis of myofibrillar, structural and connective proteins starting from the onset of post-mortem phase (kemp et al., 2010; nishimura, 2010; ouali et al., 2013). nowadays meat cuts are extensively stored vacuum packaged, a practice that does not significantly affect veal aroma, color, appearance, flavor and texture traits when compared to traditional bone-in carcass ageing (ngapo and gariépy, 2006). in light of the different degrees of tenderness and tenderization rates among muscles (rhee et al., 2004), this technique can allow to maximize tenderization through the different duration of postmortem ageing, based on specific muscle or commercial cut characteristics. since veal is not commonly aged in commercial practice, it is necessary to evaluate the effects of long term chilling storage not only on meat tenderness, but also on physical and sensory properties that can affect it. for example, color is an important aspect for veal quality, so preserving veal appearance is essential. however, in some studies prolonged ageing has led to development of off-flavor in beef (spanier, 1997). the aim of this study was to evaluate the effect of postmortem ageing time in vacuum package at refrigeration temperature on physical and sensory parameters of veal loin, (m. longissimus dorsi; ld) and silverside (m. biceps femoris; bf), frozen after ageing and then thawed before quality evaluation, in order to simulate a typical consumer habit (jeremiah, 1996). the first cut was selected due to its economic significance, while the second one because its recognition as a less tender hindquarter beef cut when cooked with dry-heat cooking methods (sullivan and calkins, 2011), failing thus consumers’ expectation for tenderness. material and methods two (2) days post mortem, 8 right loin (ld muscle from 6th rib to the 6th lumbar vertebrae) and silverside (bf muscle) whole primal cuts were collected from the carcasses of 8 male milkfed holstein veal calves. calves were similar in age (231±16 d) and sourced from the same farm, being fed the same diet and slaughtered on the same day. cold carcass weight (163.50±15 kg), conformation (seurop conformation score: r), fatness score (european fatness score 1-5: 2) and serum lactate (54.52±1.32 mg/dl) were similar. the serum lactate was determined using blood samples collected during exsanguination by the central laboratory of the veterinary hospital of the university of milan using a commercial kit (sentinel diagnostics, milan, italy). this evaluation was aimed to assess differences in individual animal stress level, which can impair meat tenderness (gruber et al., 2010). after collection, each muscle was divided in 8 subsamples and each of them was then vacuum packaged. subsamples were assigned to one of the seven different postmortem ageing treatments: 2, 4, 6, 8, 10, 12 and 16 days randomized, while the remaining one was used to determine chemical composition. subsamples distribution between treatments was done ensuring that each portion of the muscle was equally represented in every ageing time, as reported by mandell et al. (2001). all subamples were kept at 0°c until the end of the established ageing period before being frozen at -20°c, as done by campo et al. (2000) and mandell et al. (2001). prior to measurement, subsamples were thawed for 24 hours at 4°c and from each subsample a 1.50 cm steak was removed for sensory evaluation, while the remaining part was used for physical and chemical analysis. physical and chemical analysis chemical composition (dry matter, ether extract, crude proteins and ash) was determined, according to aoac (2000), on designated samples trimmed from the external fat and connective tissue, and homogenized for 30 seconds. on each subsample subjected to different aging time, a fresh cut surface was created removing a slice perpendicular to the fiber axis and, after blooming for 60 minutes in a dark room at 4°c, its color was assessed by a cr-300 chroma meter device (minolta camera, co., osaka, japan) calibrated on the cie l*a*b* (cie, 1976) color space (calibration plate 21533131 y 93.4 x 0.3456 y 0.3321, minolta cameras). the chroma meter had an 8-mm measuring area, was set in d-65 lighting, and an average of 10 repetitions was recorded as the value for each sample. ph was measured with a portable ph-meter (hi 98150, hanna instruments inc., woonsocket, ri, usa) on a homogenate prepared by grinding the slice removed to create a fresh cut surface and mixing it with deionized water. cooking loss was determined, as described by honikel (1988), as the weight lost after cooking in water bath, until core temperature attained 75°c (monitored with a temperature meter hanna instruments hi98840) and 24 hours of storage at 4°c. before being weighed after cooking, samples were blotted dried. the difference between preand post-cooking weights was used to calculate the percentage lost during cooking (cooking loss). after cooking loss deter292 ital. j. food sci., vol. 27 2015 mination, from 2.5 cm thick cooked samples six cylindrical cores (1.27 cm in diameter), parallel to fiber orientation, were obtained and used for shear force (sf) evaluation with a warner-bratzler shear force texture analyzer (model 4466; instron corp., canton, ma). peak force (kg/cm2) was recorded for each core and the average of six values per sample was used for the statistical analysis. sensory analysis steaks of bf and ld samples were cut to 1.5 cm thick and cooked for 60 seconds at the greatest power (200°c) on double-plated grills, before being cut into 1.5 cm cubes. core temperature was monitored with a thermocouple (pentronic ab,198 gunnebobruk, sweden) and it was not allowed to exceed 68°c. sensory evaluation was performed by 10 expert and trained judges (uni en iso 13299:2010), confident with meat sensory evaluation, on each sample aged for 2, 4, 8, 10 and 16 days. three cubes per samples were presented on white plastic plates to each panelist, which during training and sampling, had access to unlimited water and unsalted crackers and each sample was identified by 3-digit codes. judges were trained in two tasting session with the aim to allow them to find and familiarized with sensory descriptors relative to veal aroma, taste, flavor and texture. at each judge was asked to evaluate the intensity of each attribute by assigning a score between 1 (absence of the sensation) and 9 (extremely intense sensation). descriptors (table 1) includes the main beef sensory parameters and some of the defects that could affect vacuum packaged aged meat. statistical analysis statistical analysis was performed using sas® 9.3 (sas institute inc., 2012 cary nc) software. data from the physical analysis was analyzed by one-way anova, considering post mortem ageing time as the main effect. data from sensory profile were analyzed by three-way anova considering the effects of judge, replications and ageing time and their interactions. least square means were compared according to f test, with the level of significance set at p≤0.05. pearson correlation analysis was also performed to evaluate the relationship between sf and sensory texture characteristics. results and conclusions physical and chemical characteristics the average chemical composition of ld and bf muscles is summarized in table 2. results are consistent with data reported for lean veal meat in some national food composition databases (denmark: national food institute, 2009; usa: united states department of agriculture, 2011). although a significant effect (p=0.05) of ageing time on ld ph was found (table 3), it increase only from 2 to 8 days of ageing, while no significant differences were evident since the day 4 of ageing. regarding bf, its ph was not affected by ageing time. this last data is consistent with other studies that found no differences in veal ph during 7 (revilla et al., 2006) or 14 days of ageing (oliete et al., 2006), and the review by table 1 descriptors, definitions and standards for sensory analysis. attribute definition aroma veal aroma associated with cooked veal loin metallic aroma associated with blood or rare meat off flavor aroma associated with meat at the end of shelf life taste salty salty taste sweet sweet taste flavor veal flavor associated with cooked veal loin metallic flavor associated with blood or rare meat off flavor flavor associated with meat at the end of shelf life texture tender the force needed to masticate the meat ready for swallowing (chewing 5 times) fibrous presence of fibers during swallowing juicy the degree of juice released while chewing the meat stingy production of a large quantity of saliva for swallowing table 2 average chemical composition of the muscles sampled for the trial (least square means±sd). trait longissimus dorsi biceps femoris moisture, g kg-1 751.72±5.12 754.91±3.32 ash, g kg-1 12.34±1.04 11.72±0.65 crude protein, g kg-1 211.50±3.22 212.24±3.96 ether extract, g kg-1 24.52±3.24 21.20±2.47 ital. j. food sci., vol. 27 2015 293 table 3 effect of ageing time on veal ld and bf physical traits (least square means±sem). ageing time 2 d 4 d 6 d 8 d 10 d 12 d 16 d p ph ld 5.54±0.04 a 5.62±0.04 ab 5.63±0.04 ab 5.70±0.04 b 5.68±0.04 b 5.71±0.04 b 5.70±0.04 b 0.05 bf 5.60±0.04 5.59±0.04 5.63±0.04 5.63±0.04 5.68±0.04 5.68±0.04 5.67±0.04 ns cooking loss, % ld 25.41±0.53 26.16±0.53 25.72±0.53 25.96±0.53 25.98±0.53 25.79±0.53 25.66±0.53 ns bf 28.81±0.11 28.55±0.11 29.87±0.11 28.81±0.11 29.85±0.11 27.62±0.11 28.77±0.11 ns l* ld 48.20±0.68 a 50.32±0.47 b 51.00±0.54 bc 52.91±0.93 cd 52.80±0.90 cd 52.49±0.86 cd 53.08±0.90 d ≤0.01 bf 48.51±0.53 a 50.46±0.43 b 50.71±0.52 b 52.74±0.75 c 53.04±0.55 c 53.12±0.56 c 53.88±0.55 c ≤0.01 a* ld 9.71±0.47a 10.27±0.34 a 12.82±0.40b 12.49±0.68 b 12.52±0.66 b 12.57±0.63 b 12.53±0.66 b ≤0.01 bf 10.85±0.25 a 11.27±0.20 a 13.70±0.25b 14.86±0.34 b 14.87±0.27 b 14.11±0.25 b 14.03±0.26 b ≤0.01 b* ld 9.86±0.25 a 10.52±0.17 b 11.97±0.20c 12.22±0.34 c 12.41±0.33 c 12.73±0.31 c 12.87±0.33 c ≤0.01 bf 10.58±0.25 a 11.23±0.20 b 12.46±0.25c 13.62±0.34 d 13.65±0.27 d 13.39±0.25 d 13.22±0.26 d ≤0.01 sf, kg ld 2.89±0.15 a 2.59±0.11 ab 2.42±0.16 bc 2.21±0.15 c 2.12±0.18 c 2.09±0.21 c 2.05±0.17 c ≤0.01 bf 2.89±0.13 a 2.73±0.09 ab 2.65±0.13 ab 2.45±0.13 bc 2.22±0.11 cd 2.19±0.11 cd 1.96±0.11 d ≤0.01 a,b,c,d in the same row indicates significant differences between the different ageing times. ngapo and garyépi (2006), that suggests postmortem ageing did not increase veal ph. however, although the reported slight differences in ld ph values across post mortem times, it fell within the normal range (5.40-5.70) for both muscles. cooking loss (table 3) was not affected by ageing time in either muscle. findings are in agreement with other works (klont et al. 2000; mandell et al. 2001), even if is difficult to make a comparison with the previous study due to the different cooking methods and endpoint temperatures to between studies. however, for both muscles, results of the present study are intermediate between the cooking loss values reported by the previous authors (19.1-38.2%). regarding color parameters (table 3), ageing increased lightness (l*), redness (a*) and yellowness (b*) in both muscles (p≤0.01). this concurs with mandell et al. (2001), which suggested color parameters tended to increase only during the first week of ageing, before becoming stable. insausti et al. (1999) also found l* to increase during vacuum storage in longissimus dorsi of young cattle. lightness increasing can be attributed, as reported by klont et al. (2000), to the increasing of meat light scatter properties due to post mortem protein denaturation and degradation. oliete et al. (2006) found an increasing of a* and b* measured 1 hour after blooming in vacuum packaged veal and young cattle longissimus dorsi. these studies attributed the increasing in redness to the faster blooming of aged meat. indeed, the more meat is aged the faster it blooms because of the reduced activity of enzyme that compete for oxygen with mb. the rising of yellowness was, instead, attributed the increasing of metmyoglobin formation during storage time. a lightness increasing can exert a positive effect on veal appearance, while an improvement of redness can represent a negative factor. indeed, in several studies on veal carcasses, a decreasing in lightness and an increasing in redness moving from lightest to darkest veal was reported, while b* was not related to color score (denoyelle and berny, 1999; hulsegge et al. 2001; lagoda et al. 2002; vandoni and sgoifo rossi, 2009). the magnitude of l*, a* and b* increasing, higher than those found in the study of mandell et al. (2001), could be promoted by the combination of freezing and thawing and blooming time, this latter not applied by mandell et al. (2001), that can have exacerbated the impact of ageing on 294 ital. j. food sci., vol. 27 2015 meat color stability. indeed, freezing and thawing promote myoglobin denaturation, increase susceptibility to oxidation and reduce the activity of metmyoglobin reducing enzymes. these effects, coupled with the loss of nadh (cofactor of these enzymes) in the exudate, reduce meat color and oxidative stability as reviewed by leygonie et al. (2012). post mortem ageing of ld reduced sf (p≤0.01), but there were no further improvements in tenderness after 8 days of ageing (table 3). these findings are consistent with mandel et al. (2001), who reported decreases in sf for ld and semimembranosus muscles comparing veal aged for 2 days with veal aged for at least 7 days, but the same study found no differences in sf for ageing periods beyond 7 days. furthermore, revilla et al. (2006) also found a reduction in sf loin during 7 days of ageing. eight days of ageing was needed to significantly decrease sf values for 2 days aged bf; there was no further improvement in sf values until bf was aged for 16 days. this slower tenderization rate of bf compared to ld agrees with data relative to this muscle collected from lean beef carcasses graded as usda quality grade select (gruber et al., 2006). sensory analysis the f values of ageing time for aroma, taste, flavor and texture parameters of ld and bf sensory profile are reported in table 4 and table 5, respectively. results indicated that postmortem ageing time affected (p ≤0.01) sensory texture of both ld and bf. in particular, postmortem ageing improved ld sensory tenderness (p≤0.01). tenderness was higher at day 4 days in comparison with day 2, and at day 16 in comparison to day 4 (tab. 4). increasing ageing from 2 to 4 days also improved juiciness (p≤0.01) and reduced stringy sensation (p≤0.01). improvements in eating quality associated with ageing were also perceived for fibrousness, with significant reduction (p≤0.01), starting from the 8th days post mortem. these results are consistent with mandell et al. (2001), which found an increase in perceived ld tenderness comparing samples aged 2 days with the average of the values recorded for samples aged 7 and 14 days, while no significant difference was detected increasing ageing period from 7 to 14 days. bf sensory analysis (table 5), showed that perceived tenderness significantly increased with ageing (p≤0.01) from days 2 and 4 to day 8, and also between 8 to 16 days post mortem. juiciness was improved (p≤0.01) from 2 to 8 days of ageing, but no further. fibrousness was also reduced (p≤0.01) from 2 to 8 days and from 8 to 16 days of ageing and stringy sensation decreased (p≤0.01) from 2 to 4 days and from 4 to 10 days of ageing, but no further. the improvement of perceived tenderness and juiciness, as well as reductions in fibrousness and stringy rankings, are common when sensory panels evaluate the effects of postmortem ageing on beef palatability attributes (jeremiah and gibson, 2003; miller et al. 1997 and table 4 effect of ageing time on ld sensory profile (least square means). descriptors f value 2 d 4 d 8 d 10 d 16 d sem p aroma veal 1.47 6.98 6.77 6.62 6.92 6.65 0.20 n.s. metallic 1.32 4.18 4.29 4.59 4.17 4.87 0.32 n.s. off flavor 3.02 2.33 2.15 2.52 2.37 2.87 0.22 n.s. taste sweet 1.14 4.67 5.27 5.07 5.20 5.38 0.32 n.s. salty 1.47 3.81 4.32 3.83 3.68 4.22 0.30 n.s. flavor veal 0.89 6.62 6.82 6.50 6.82 6.95 0.24 n.s. metallic 1.56 4.12 4.61 4.34 3.92 4.77 0.32 n.s. off flavor 1.64 2.97 2.21 2.96 2.88 3.08 0.31 n.s. texture tender 16.21 4.92 a 6.35 b 6.77 bc 6.88 bc 6.94 c 0.23 ≤0.01 juicy 10.82 4.75 a 5.79 b 5.95 b 6.34 b 6.33 b 0.26 ≤0.01 fibrous 4.94 5.05 a 3.95 b 3.87 b 3.70 b 3.57 b 0.30 ≤0.01 stringy 3.89 4.68 a 3.96 ab 3.62 b 3.32 b 3.34 b 0.29 ≤0.01 a,b,c in the same row indicates significant differences between the different ageing times. ital. j. food sci., vol. 27 2015 295 campo et al. 1999). in the latest study a multivariate approach (principal component analysis) was used to differentiate aged from unaged meat. this indicated that aged meat was characterized by tenderness and juiciness sensation, while unaged meat was characterized by fibrousness and residue (similar to stringy sensation) ones. in the present study post mortem ageing did not affect aroma, flavor and taste for both muscle. this disagrees with mandell et al. (2001), where meat flavor was improved by ageing veal more than 7 days. there was a low incidence for the panel detecting undesirable palatability attributes such as metallic aroma and flavor and off flavor. this concurs with jeremiah and gibson (2003), that found low levels of off flavor and metallic aroma/flavor attributes in beef, regardless of post mortem ageing time. furthermore, the same authors, did not find differences in off or metallic aroma and salt and metallic flavor prolonging ageing time until 28 days. the lack of effect of ageing time on negative sensory descriptors is an important outcome, as some past studies have reported increases in undesirable flavor and aroma defects for beef after prolonged ageing (spanier et al. 1997 and monsón et al. 2005). correlation between sf and texture sensory traits based on pearson correlation coefficients to examine the relationship of sensory texture characteristics and sf for both muscles, there was a negative relationship between sf and sensory tenderness (r=-0.67; p≤0.01 and r=-0.83; p≤0.001 for ld and bf respectively) and juiciness (r=-0.53; p≤0.05 and r=-0.72; p≤0.01 for ld and bf respectively). the negative correlation between sf and sensory tenderness is in agreement to the findings of shackelford et al. (1999) in beef and monteiro et al. (2013) in veal. positive correlations were found between sf and fibrousness (r= 0.78; p≤0.01) as well as sf and stringy sensation (r= 0.78; p≤0.01) for bf. these findings agrees with the positive correlation between fibrousness and sf found by caine et al. (2003) and the negative correlation between sf and juiciness found by monteiro et al. (2013). the lack of relationship between sf with stringy and fibrousness rankings for ld muscle in respect to bf muscle could be explained by the lower collagen content of ld relative to bf (rhee et al. 2004). indeed, fibrousness and stringy sensation were lower in ld muscle and the lower detectability could have been at the basis of the lack of significant interaction. our results indicate that postmortem ageing under vacuum conditions improved the instrumental and sensory tenderness rankings for veal m. longissimus dorsi and m. biceps femoris, without any negative effects on the main meat sensory traits such as aroma, flavor, taste and juiciness measured after frozen storage and thawing. ageing, coupled with freezing and thawing, have, however, reduced oxidative stability in both muscles, table 5 effect of ageing time on bf sensory profile (least square means). descriptors f value 2 d 4 d 8 d 10 d 16 d sem p aroma veal 1.00 6.69 6.60 6.80 7.04 6.93 0.18 n.s. metallic 1.50 4.15 4.43 4.31 3.81 3.71 0.29 n.s. off flavor 0.19 2.31 2.40 2.42 2.35 2.26 0.19 n.s. taste sweet 0.74 5.00 5.12 5.34 5.41 5.31 0.27 n.s. salty 2.27 3.73 3.84 4.33 3.88 4.33 0.28 n.s. flavor veal 1.47 6.38 6.48 6.67 6.94 6.52 0.20 n.s. metallic 2.47 4.04 4.78 4.44 4.25 4.36 0.29 n.s. off flavor 1.54 3.04 3.00 2.58 3.27 2.29 0.22 n.s. texture tender 15.43 3.80 a 4.52 a 5.65 b 6.38 bc 6.64 c 0.31 ≤0.01 juicy 5.83 4.27 a 4.57 ab 5.13 b 5.80 b 5.80 b 0.29 ≤0.01 fibrous 9.88 6.58 a 5.34 b 4.78 bc 4.15 cd 3.76 d 0.33 ≤0.01 stringy 21.37 6.64 a 5.81 ab 5.00 bc 4.41 cd 3.39 d 0.30 ≤0.01 a,b,c,d in the same row indicates significant differences between the different ageing times. 296 ital. j. food sci., vol. 27 2015 without affecting other veal technological properties as cooking loss and ph. there were different postmortem tenderization trends for each muscle evaluated in the study. the improvements in ld tenderness and related sensory traits occurred mainly during the first week of postmortem ageing, while in bf postmortem ageing effects were also evident until the tenth day. at these experimental conditions, a minimum period of 4 days for ld muscle and 8 days for bf muscle was necessary to obtain a perceivable tenderizing effect. a prolonged ageing, for at least one week for veal ld and two weeks for veal bf can be applied for frozen veal, mainly destined for ho.re.ca market, in which product appearance is a secondary trait, while tenderness is the primary goal. vacuum ageing could be also apply for fresh veal market, considering indeed its potentially lower impact than that emerged in this study on oxidative stability, as veal will not undergone to freezing and thawing process before being prepared for retail exposition. acknowledgements authors age grateful to vercelli s.p.a. 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t.l. and koohmaraie m. 1999. evaluation of slice shear force as an objective method of assessing beef longissimus tenderness. j. anim. sci. 77:2693. p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33isp1.2088 137 p u b l i c a t i o n s codon underutilized horse chestnut (aesculus indica) flour and its utilization for the development of gluten-free pasta syed insha rafiq1, khalid muzaffar2, syed mansha rafiq3, dc saxena1*, bn dar2 1department of food engineering and technology, sliet, longowal, punjab, india; 2department of food technology, iust, awantipora, india; 3department of food science & technology, niftem kundli, sonepat, haryana, india *corresponding authors: dc saxena, department of food engineering and technology, sliet, longowal, punjab india. email: dcsaxena@yahoo.com; bn dar, department of food technology, iust, awantipora, india. email: darnabi@gmail.com received: 11 june 2021; accepted: 18 september 2021; published: 7 october 2021 © 2021 codon publications open access paper abstract there has been a growing demand for the production of gluten-free products due to increased occurrence of celiac disease. thus, different research groups have been investigating the use of various available materials for the development of these functional products to fulfill customer’s needs. horse chestnut (aesculus indica) seeds are underutilized, low-cost, and gluten-free, found in hilly areas of the himalayan region of kashmir valley, india. to determine their potential as an alternative to conventional food grains, an investigation was conducted to determine the physicochemical, functional, pasting, and thermal properties of horse chestnut (hcn) flour and its compatibility for the development of gluten-free pasta. hcn flour comprised 73.34% carbohydrate, 11.36% protein, 6.34% crude fiber, 3.27% fat, 3.16% ash, 3.15g/g oil absorption capacity, and 4.65% water absorption index. hcn flour showed 505 cp peak, 354 cp trough, 151 cp breakdown, 472 cp final viscosity, and 66.05°c pasting temperature. transition temperatures (onset, peak, and conclusion) and enthalpy change (δh) were 60.12°c, 69.90°c, 81.53°c, and 10.56 j/g, respectively. pasta prepared from hcn flour using guar gum (0, 0.5, and 1%) was analyzed for color, cooking qualities, and textural and sensory analysis. the present results showed that hcn flour possesses good nutritional quality and has properties comparable to conventional wheat flour. therefore, hcnflour-based pasta can act as a nutritious alternative to conventional gluten-free pasta and add variety to the diet of people suffering from celiac disease. keywords: celiac disease; cooking; horse chestnut; nonconventional; nutritional; pasta introduction horse chestnut (aesculus indica) is abundantly found in hilly areas of the himalayan region of kashmir valley, india. the seeds of the tree are harvested during october and november. horse chestnut (hcn) seeds are mainly consumed by wild animals and many seeds are wasted. the seeds are kept in running water to reduce the bitterness attributed to the presence of anti-nutrients and then added in powdered form to wheat flour for making halwa (porridge) and chapattis, a traditional flatbread (rajeasekaran and joginder, 2009). hcn seeds can be used as nutritional supplements because they are nutritionally good and have a distinctive composition of carbohydrate, crude protein, fat, and ash. oleic acid, linoleic acid, palmitic acid, linolenic acid, arachidic acid, and myristic acid are the major fatty acids present in the hcn seed. the chief minerals present in hcn include potassium, phosphorus, calcium, sulfur, iron, copper, zinc, and manganese (majeed et al., 2010). the seeds italian journal of food science, 2021; 33 (sp1): 137–149 mailto:dcsaxena@yahoo.com mailto:darnabi@gmail.com 138 italian journal of food science, 2021; 33 (sp1) rafiq si et al. improve their textural characteristics (lorenzo et  al., 2018; palavecino et al., 2017). to improve the quality attributes like mouthfeel, texture, consumer acceptability, and shelf-life of gluten-free products, diverse research has been done using alternative components like hydrocolloids, emulsifiers, starches, and nongluten proteins (capriles et al., 2016; kumar et al., 2019). the worldwide market for gluten-free food products increased up to 83% with an estimated annual growth rate of 12.3% for pasta products until 2022 (chauvin, 2019). gluten-free products are not only consumed by celiac patients but also by other individuals as well (bresciani et al., 2021). researchers have been motivated by united nations for the agenda 2030 to research healthy formulations for bakery and pasta products and the use of functional agro-industrial by-products for fortification in foods (simonato, 2021). because of its good nutritional profile and gluten-free nature, hcn flour may represent a potential nonconventional alternative to conventional flours. only limited information is available regarding hcn flour and there are no reports about the production of gluten-free pasta from hcn four. therefore, it is essential to characterize the flour to explore possible utilization in food industry and to optimize the ingredients for the development of quality pasta from hcn flour. so, in the present study, characterization of hcn flour with respect to physicochemical, functional, thermal, and pasting properties, and development and characterization of pasta were carried out. materials and methods raw materials fully matured horse chestnut seeds were obtained from horse chestnut trees located at anantnag, jammu & kashmir, india, cleaned and stored at 5°c till used. hard wheat was procured from punjab agriculture university (ludhiana). the chemicals and reagents were procured from sigma-aldrich. flour preparation horse chestnut flour was prepared with slight modification to the method of singh et al. (2013). the kernels obtained after manual dehulling of hcn seeds were sliced and then dried in a tray dryer at 60°c. the slices before drying were treated to reduce the anti-nutritional factors in the previous work of rafiq et al. (2016). the dried seed slices were ground in a laboratory-type grinder to obtain hcn flour. both wheat and hcn flours were sieved using a 60-mesh sieve. hcn and wheat flour were then analyzed for various properties. also have several medicinal uses like in the treatment of fever, viral diseases, skin infections, and cardiovascular disorders (kaur et al., 2011). hcn seeds are rich in polyphenols, flavonoids, fiber, and essential minerals (majeed et al., 2010). pasta, a traditional delicacy, is a popular staple food used throughout the world because of its low cost, longer shelf life, and ease of preparation. commonly, wheat flour is used in the preparation of pasta (kaur et al., 2012). pasta food products are produced by extruding feed mixture in an extruder equipped with different dyes that determine the shape of the final product. the pasta products are then dried and packed. it has been recognized as an ancient means of nourishment and versatile food from nutritive and gastronomic perspectives (fuad and prabhasankar, 2010). pasta is considered a superior medium for the incorporation of nutrients as per the reports of food and drug administration and world health organization (pasqualone et al., 2016). the selection of flour for pasta development depends on water absorption and water solubility indexes and the acceptability and cooking parameters of the developed pasta (rudra et al., 2020). the behavior of starch and protein in pasta during cooking is such that starch swells and solubilizes but protein gets coagulated during cooking (hager et al., 2012). best-quality pasta has acceptable firmness with low solid loss, and stickiness (phongthai et al., 2017). gluten as the structure builder promotes viscosity, elasticity, extensibility, and cohesiveness in pasta products (lazaridou et al., 2007). gluten helps to maintain the shape of bakery products and pasta due to the extensibility and elastic dough behavior with resilience and textural properties acceptable to consumers (phongthai et al., 2017). however, due to the amplified dominance of celiac disease and other health issues such as gluten allergy, nonceliac gluten sensitivity, and other gluten-related disorders, demand for gluten-free pasta products has increased (dib et al., 2018; ozgoren and yapar, 2019). to avoid these disorders, grains other than wheat may be used, either individually or in combined formulation, to replace gluten in food products. the only possible treatment for individuals having celiac disease is to consume foods devoid of gluten (witczak et al., 2017). however, gluten-free products have low nutritional quality, and defective textural and sensory property due to crumbly and fragile dough formation (cai et al., 2016). hence, to counterbalance the absence of gluten, fortification with gums, proteins, and different flours are considered (foschia et al., 2017; padalino et al., 2016). to replace gluten and to maintain the dough structure and texture of the final product, various types of protein and hydrocolloids are used in gluten-free products (foschia et al., 2017). hydrocolloids, such as xanthan gum, guar gum, hydroxypropyl methylcellulose, and carboxymethyl cellulose used as an addictive in gluten free products can italian journal of food science, 2021; 33 (sp1) 139 underutilized horse chestnut (aesculus indica) flour and its utilization sample was weighed to calculate oac as shown in the below equation: weight of residue oac (g / g) sample weight = emulsion properties the emulsion activity and stability of samples were calculated by using the method of du et al. (2014). sample (3.5  g) and water (50 ml) were agitated for 30 s using the high setting homogenizer (yorco, india). afterward, groundnut oil (25 ml) was added and then again homogenized for 30 s, followed by another addition of 25 ml groundnut oil and then homogenized for 90 s. the emulsion obtained was then centrifuged for 5 min at 1100 × g. emulsion activity (%) volume of emulsified layer emu emulsion volume before centrifugation = × after calculating the emulsion activity, emulsion stability was evaluated by heating the flour emulsion samples (15  min at 85ºc), then cooling, followed by centrifugation for 5 min at 1100 × g. the percent emulsion activity that remained after heating can be expressed as the emulsion stability. foaming properties the foaming properties, viz., foaming capacity (fc) and foaming stability (fs), were observed with minor modifications to the method followed by jia et al. (2021). sample (3 g) and distilled water (50 ml) were mixed, and the dispersion was homogenized for 3–5 min. the mixture was then instantly poured into the measuring cylinder and the increase in volume percent upon whipping was calculated. the percentage of volume increase was the foaming capacity and the change in the foam volume after 1 h of storage was recorded as the foaming stability. fc and fs were calculated using the below mentioned formulas: 2 1 2 3 2 1 v v fc(%) 100 v v fs(%) 100 v v − = × = × − where, v1 = volume prior to whipping (ml), v2 = foam volume after whipping (ml), and v3 = foam volume (ml) after holding least gelation concentration (lgc) the method of khan and saini (2016) was used to determine the gelation properties of the flour samples. two physicochemical characteristics of hcn flour color analysis flour color analysis was done in triplicates, with color flex spectro-colorimeter (d-25, ruston, usa), and color values (l*, a*, and b*) were determined. proximate analysis standard methods were employed to analyze the flour samples for moisture, protein, ash, fiber, and fat content (aoac, 2006), while the carbohydrate content was determined using the difference method. bulk density an accurately weighed sample was poured into a graduated cylinder. tapping during the filling was done for uniform packing. bulk density was calculated using the below equation: 3 3 weight of the sample (g) bulk density (g / cm ) volume of sample (cm ) = true density the method followed by raigar and mishra (2015) was used to determine true density. an accurately weighed flour sample (1 g) was taken and poured into a burette filled with toluene. the equation given below was used to calculate true density: 3 3 weight of flour sample (g) true density (g / cm ) rise in toluene level (cm ) = functional properties water absorption capacity water absorption capacity (wac) of the flour was determined per vázquez-luna et al. (2019) with slight modifications. approximately, 5 g of the sample was mixed with 70 ml of water for 1 min and then held at room temperature for 30 min, followed by centrifugation (3000 × g for 15 min). after centrifugation, the sediment obtained was weighed and wac was calculated: weight of se dim ent wac(g / g) weight of flour sample = oil absorption capacity the method followed by khan and saini (2016) was used to determine the oil absorption capacity (oac) of the sample. five grams of flour and 75 ml of oil were mixed and agitated for 30 min and then centrifuged for 10 min at 3000 × g. afterward, oil was drained while the retained 140 italian journal of food science, 2021; 33 (sp1) rafiq si et al. observed by squeezing the pasta between glass plates. the weight of the cooked pasta after being drained for 2  min was taken as the cooked weight (g). the cooking loss was calculated by evaporation of cooking water to dryness at 110°c overnight. water uptake water uptake is the difference between the weight of cooked and uncooked samples, and is expressed as uncooked pasta weight percentage (çabuk and yilmaz, 2020). the investigation indicated the absorption of water during the cooking process. cooking loss the method followed by teterycz et al. (2020) was employed for cooking loss determination. sample (10 g) was cooked in boiling water (300 ml) and then cooking water was dried overnight (105°c). the residue left after drying was weighed and then cooking loss was calculated. texture for determination of texture, the samples were cooked till optimum cooking time, and then drained with 10 min rest time. four pasta strands were taken and placed under the compression plate of pasta firmness/stickiness rig. the resulting force-time curve was used to determine firmness, cohesiveness, springiness, adhesiveness, chewiness, and gumminess. sensory evaluation panel comprising 12 trained members have done the sensory evaluation of pasta samples; evaluated the attributes like firmness, slipperiness, chewiness, surface adhesiveness, appearance; and determined the total score for each pasta sample. perceived intensities were scored on a 5-point scale. statistical analysis the experimental data was statistically analyzed by oneway anova using triplicate readings for all experiments except sensory evaluation where 12 observations were averaged. results and discussion physiochemical properties of hcn flour the desired color characteristics for the flour are low chroma (a*) value and high lightness (l*) value to meet the consumer preference. the color parameters and nutritional composition of hcn and wheat flour are given in table 1. results showed that the l* value of the flours was almost similar; however, significant difference percent, 4, 6, 8, 10, 12, 14, 16, 18, and 20% (w/v) of flour suspensions in 5 ml of water were boiled for 1 h, cooled in cold water, and kept at 4°c for 2 h. pasting characteristics rapid visco analyzer (tecmaster, warriewood, australia) was used for the determination of the pasting profile. a programmed heating and cooling cycle (taking sample 3 g, 14% mb; and distilled water 25 ml) was used for generating the viscosity profile. experimental conditions include holding the samples for 1 min at 50°c, heating to 95°c at 12°c/min cooling rate, holding for 2.5 min at 95°c), cooling to 95 to 50°c at 12°c/min heating rate, and then holding for 2 min at 50°c. thermal properties a differential scanning calorimeter (dsc821, mettler toledo, switzerland) was involved in determining thermal properties of the sample. the samples were prepared with flour (3mg, dwb) and water (7 µl), sealed, and kept undisturbed at room temperature for 1 h. the sample was heated at 20–120°c at a rate of 10°c/min. pasta preparation different pasta formulations (control sample (durum wheat); native sample (hcn flour + 0% gum); pasta formulation containing hcn flour and 0.5 and 1% guar gum, respectively) were mixed using hobart benchtop mixer (5kpm50, usa) for 1 min at low speed followed by conditioning for 5 min with distilled water (30 ml). the sample was fed to a single screw extruder (model: dolly la monferrina italy) of 7 mm die diameter. pasta was then transferred to cabinet drier for 4 h set at 60°c, reducing the moisture content to 8%, and then packed in high-density polyethylene pouches. characterization of pasta color parameters color parameters of pasta were determined using a color spectrophotometer (cm-3600d, konica minolta, bremen, germany). color analysis was done in terms of l*, a*, and b* values. cooking properties cooking time was evaluated by using the aacc (2000) method. pasta sample (10 g) was cooked in water (300 ml). the optimum time of cooking is the point of disappearance of the inner white core of pasta and was italian journal of food science, 2021; 33 (sp1) 141 underutilized horse chestnut (aesculus indica) flour and its utilization table 1. physicochemical characteristics. physicochemical horse chestnut flour wheat flour color l* 92.07 ± 0.49a 92.85 ± 0.28a a* 3.47 ± 0.21a 2.03 ± 0.07b b* 13.70 ± 0.26a 9.69 ± 0.15b moisture content (%) 9.71 ± 1.23b 11.35 ± 0.54a protein (%) 7.78 ± 1.19b 11.36 ± 1.10a crude fat (%) 3.27 ± 0.39a 1.40 ± 0.57b crude fiber (%) 6.34 ± 0.22a 1.67 ± 0.14b ash (%) 3.16 ± 0.05a 0.88 ± 0.03b carbohydrate (%) 69.74b 73.34a energy value (cal/100g) 327.51b 346.84a bulk density (g/ml) 0.64 ± 0.12a 0.51 ± 0.10a true density (g/ml) 1.25 ± 0.24a 1.42 ± 0.12a results presented are mean values and the superscripts significantly differ in row (p < 0.05). was observed in a* and b* values. this variation in the color parameters may be ascribed to a difference in the natural pigment content inherent to hcn and wheat flours depending on the origin of the plant (drakos et al., 2017). nutritional composition of hcn and wheat flour also varied significantly (table 1). moisture content of hcn flour was low (9.71%) compared to wheat flour (11.35%). similarly, protein and carbohydrate content of hcn flour was lower than wheat flour. however, hcn flour showed significantly higher values for ash, fat, and crude fiber content than wheat flour. high ash content of hcn flour could be related to high mineral content of the hcn seeds being rich in nitrogen, potassium, phosphorus, sulfur, calcium, iron, etc. (majeed et al., 2010). the bulk density did not differ significantly in both the flours (table 1). similar results were observed for the true density of hcn flour (1.25 g/ml) and wheat flour (1.42 g/ml). functional properties of hcn flour water absorption capacity wac describes the interaction of flour and water under a limited quantity of water. the wac of flour plays a significant part in food research as it affects other determining properties of food material. hcn flour showed significantly lower values of wac, 2.21 g/g as compared to wheat flour, 4.65 g/g (table 2). hydrophilic behavior and gel-forming ability of starch, and protein quality are the determining factors that affect the wac of flour (al-farga et al., 2016). higher number of hydrophilic components in flour is responsible for its elevated wac (njintang et al., 2008). the variation in the wac table 2. functional characteristics. properties hcn flour wheat flour wac (g/g) 2.22 ± 0.12b 4.65 ± 0.08a oac (g/g) 3.24 ± 0.15a 1.10 ± 0.05b foaming capacity (%) 20.2 ± 1.17b 31.40 ± 0.71a foaming stability (%) 22.32 ± 2.41b 54.05 ± 5.52a emulsion activity (%) 58.69 ± 0.31b 61.01 ± 1.33a emulsion stability (%) 62 ± 0.27b 68.32 ± 0.12a wac, water absorption capacity; oac, oil absorption capacity. results presented are mean values and the superscripts significantly differ in row (p < 0.05). behavior of the flours could be related to difference in the composition of the flour. oil absorption capacity oac capacity is an important property of the flour, which defines its application in various food formulations and determines its capacity to entrap oil which affects flavor retention and mouthfeel of the developed product (khattab and arntfield, 2009). oac of flour is important for new product development and storage stability. significantly higher values of oac of 3.15 g/g in hcn flour than 1.10 g/g in wheat flour was observed (table 2). interaction of nonpolar side chains present in the flour with oil through hydrocarbon side chains affect their oac (adebowale and lawal, 2004). flour particle size, starch content, and occurrence of nonpolar amino acids are other factors that affect the oac of flour (chau et al., 1997; ofori et al., 2020). also, the protein type and content contribute to oil-retaining properties of the foods (ravi and sushelamma, 2005). foaming properties the foaming properties are essential parameters for the formulation of food products. results related to foaming capacity and stability of hcn, and wheat flour is given in table 2. carbohydrate and protein content play an important role in determining the foaming properties of flour (sreerama et al., 2012). formation of interfacial layer by proteins is responsible for foaming, keeps air bubbles in suspension, and slows down coalescence rate (adebowale and lawal, 2004). hcn flour and wheat flour showed foaming capacity of 20.20 and 31.40%, respectively. low-protein and high-fat content of hcn flour is responsible for lower foaming capacity of hcn flour (siddiq et al., 2010). formation of the protein-fat complex may influence the foaming properties. foam stability for horse chestnut flour (22.32%) was also lower than wheat flour (54.05%). good foam stability is most likely associated with soluble proteins’ surface activity 142 italian journal of food science, 2021; 33 (sp1) rafiq si et al. to swelling that is responsible for the low pt of horse chestnut flour. the pasting temperature has also been related to the amount of protein present. shevkani et al. (2015) have reported high pasting temperature with higher amount of protein in rice flour. peak viscosity (pv) which indicates the degree of granule swelling was 505 cp for hcn flour and 1215 cp for wheat flour. the low peak viscosity could be due to high fat content in hcn flour that produces more fluid system and subsequently low viscosity. trough viscosity was 354 cp (hcn flour) and 1005 cp (wheat flour) and depicts a decline following pv owing to swollen starch granules disruption upon high temperature and shear. breakdown viscosity was 151 cp for hcn flour and 210 cp for wheat flour. the low breakdown viscosity indicates higher resistance to shearing at high temperature. final viscosity (fv) was 472 cp for hcn flour and 1560 cp for wheat flour. besides this, setback viscosity (sbv) of hcn flour (118 cp) was considerably lower than that of wheat flour (301 cp). the important factors that affect setback and final viscosities are polymerization or reordering of leached amylose and linear amylopectin (abu et al., 2006). the difference in pasting parameters of the flours could be linked to the occurrence of components such as proteins, minerals, lipids, etc., which can bind with starch, thereby affecting the pasting parameters of the flour (ocheme et al., 2018). the protein and lipid components of the flour affect the pasting properties through amylose–protein and amylose– lipid interactions (yu et al., 2012). due to the protective effect of protein molecules on the integrity of starch granules, viscosity of corn starch paste decreased (singh et al., 2014). difference in the viscosity of wheat flour pastes is correlated to variation in the proportion of polymeric or monomeric proteins among wheat flours (singh et al., 2016). thermal properties of hcn flour thermal properties are related to gelatinization behavior described as granular disruption, loss of in continuous water phase while the presence of highly ordered globular proteins in the flour which resist surface denaturation could be related to low foamability (du et al., 2014). emulsion properties the ability of protein to form a stable emulsion is known as emulsion activity, and it gives information about the ability of protein to occupy the interfacial layer of oil– water emulsion while the emulsion stability is related to the strength of an emulsion to stress, provided by the proteins (singh et al., 2010). emulsion capacity and stability values of hcn and wheat flour are significantly different (table 2). emulsion activity and stability of hcn flour was lower (58.69 and 62%) than wheat flour (61.01 and 67.87%), which might be related to the difference in quality and content of protein. proteins having several polar side chains make them more hydrophilic, thereby increasing the protein–water interactions, and thus affecting their emulsification properties (yan et al., 2020). the high emulsion stability depends on the globular nature of proteins in the flour. the emulsifying properties of different foods vary due to varied composition and processing conditions and the capacity of protein to form and stabilize emulsions (adebowale et al., 2005). least gelation concentration lgc is expressed as the quantity of flour required per volume to obtain a gel. results related to lgc of flour samples are presented in table 3. flour which has lower value of lgc has greater ability to form gels and low concentration of flour is required for gel formation. higher concentration (6 g/100 ml) of hcn flour was required for initial gel formation than wheat flour (4 g/100 ml). a firm gel was obtained when using 12% of hcn flour and 10% of wheat. the difference in the gel-forming ability among various types of flours is related to different ratios of components like carbohydrates, proteins, and lipids (drakos et al., 2017). the lower lgc value of wheat flour might be related to high protein and starch content, which are responsible for gel formation (joshi et al., 2015). pasting characteristics pasting profile of wheat and hcn flour is depicted in figure 1. pasting properties play a significant part in the selection of raw materials for industrial use as a thickener, binder, etc. rigidity of starch granules which affects swelling of starch granules and leaching of amylose in solution determine the pasting properties of the flour (leewatchararongjaroen and anuntagool, 2016). compared to wheat flour (pasting temperature: 83.36°c), the pasting temperature of hcn flour was low (66.05°c). it could be the starch in hcn flour that is less resistant table 3. least gelation concentration. concentration hcn gel wheat gel 2% no no 4% no weak 6% weak weak 8% weak weak 10% weak firm 12% firm firm 14% firm firm 16% firm hard 18 % hard hard 20% hard hard italian journal of food science, 2021; 33 (sp1) 143 underutilized horse chestnut (aesculus indica) flour and its utilization double helical and crystalline structure of starch (rincón-londoño et al., 2016). table 4 shows the results related to onset, peak, and conclusion temperatures, and enthalpy of gelatinization (δh) of hcn and wheat flour. transition temperatures for hcn flour were to = 60.12, tp = 69.90, and tc = 81.53 ºc) while for wheat flour transition temperatures were high (to = 78.32, tp = 81.12, and tc = 86.34°c). starch granule size, their form and distribution, and the bonding between micellar network of the granule and other flour components are responsible for variation in transition temperatures of flours (xu et al., 2019). enthalpy of gelatinization of 10.56 j/g was observed for hcn flour while for wheat flour, it was 13.21 j/g. variation in the shape of starch granules, and proportion of large and small granules are the factors that affect the enthalpy of gelatinization (kaur and singh, 2005). the decreased gelatinization enthalpy can also be due to the disruption of crystalline regions upon depolymerization of amylose and breakdown of amylopectin. color characteristics of pasta customer acceptance depends on the color of the product and the most desirable attribute of pasta quality is the bright yellow color (petitot et al., 2010). color values (l*, a*, and b*) of raw and cooked horse chestnut pasta, and control were significantly different (table 5). the l* value for horse chestnut pasta was less than the control pasta sample. the differences in color parameters between 1600 1500 1400 1300 1200 1100 1000 v is co ci st y 900 800 700 600 500 400 300 200 100 0 0 1 2 3 4 5 6 7 time (min) 8 9 10 11 12 13 14 15 wheat hcn flour 45 200 250 300 350 400 450 500 550 s pe ed ( rp m )600 650 700 750 800 850 900 950 te m pe ra tu re ( ˚c ) 50 55 60 65 70 75 80 85 90 95 figure 1. pasting profile of wheat and hcn flour. table 4. thermal characteristics. parameters horse chestnut flour wheat flour onset temp. (°c) 60.12 ± 0.11b 78.32 ± 0.98a peak temp. (°c) 69.90 ± 0.23b 81.12 ± 0.12a conclusion temp. (°c) 81.53 ± 0.17b 86.34 ± 0.18a δh (j/g) 10.56 ± 0.41b 13.21 ± 0.63a results presented are mean values and the superscripts significantly differ in row (p < 0.05). control and hcn pasta samples could be related to the inherent color of the flour and the pigments present. the addition of gum in pasta formulation at different levels showed no significant effect on pasta color. similar effect of hydrocolloids on color characteristics of developed noodles was observed by shere et al. (2020) color values of cooked and uncooked pasta were significantly different which could be related to the leaching of pigments on cooking. cooking characteristics of pasta the cooking characteristics of pasta are influenced by numerous phenomena taking place during cooking, viz., hydration, starch gelatinization, and interaction with nonstarchy constituents. cooking parameters for the pasta samples including cooked weight, water uptake, solid loss, and cooking time are given in table 6. the 144 italian journal of food science, 2021; 33 (sp1) rafiq si et al. table 5. color characteristics of raw and cooked pasta sample raw pasta cooked pasta l* a* b* l* a* b* a 75.40 ± 2.05a 1.30 ± 0.34b 20.73 ± 1.2 a 70.60 ± 1.68a 0.04 ± 0.01a 19.34 ± 0.76a b 62.85 ± 1.36b 1.49 ± 0.40a 18.09 ± 1.83b 55.84 ± 1.46b 0.04 ± 0.03a 16.10 ± 0.70b c 61.41 ± 1.90b 1.35 ± 0.32b 17.49 ± 1.31c 56.17 ± 2.01b 0.03 ± 0.03a 15.50 ± 0.72c d 61.82 ± 1.41b 1.38 ± 0.29ab 18.88 ± 0.53b 56.65 ± 1.61b 0.02 ± 0.02a 15.20 ± 1.61c results presented are mean values and the superscripts significantly differ in column (p < 0.05). sample a: control (durum wheat); sample b: hcn seed flour; sample c: hcn seed flour + 0.5% gum addition; sample d: hcn seed flour + 1% gum addition. table 6. cooking properties of pasta. parameters sample (g) cooking time (min) cooked wt. (g/100g) solid loss (g/100g) water uptake (g/100g) sample a 5 5.1 ± 0.20a 13.49 ± 0.86a 6.9 ± 0.42c 137 ± 7.21a sample b 5 4.2 ± 0.25bc 8.57 ± 0.12c 16.8 ± 1.43a 115 ± 2.65c sample c 5 4.5 ± 0.40ac 10.30 ± 0.90b 10.6 ± 0.52b 125 ± 3.61bc sample d 5 4.8 ± 0.56ab 12.39 ± 0.38a 8.13 ± 0.45c 132 ± 6.56ab sample a: control durum wheat; sample b: native horse chestnut flour; sample c: 0.5% gum addition; sample d: 1% gum addition. results presented are mean values and the superscripts significantly differ in column (p < 0.05) (n = 6). cooking time, cooked weight, and water uptake increased linearly (p < 0.05) with gum addition in pasta formulation. results observed are in accordance with findings of sozer et al. (2007). the enhancement in water uptake with the addition of gum might be related to the accessibility of carboxyl and hydroxyl groups in gum structure, thereby increasing the water uptake of the pasta sample (gull et al., 2018). in addition, higher water uptake occurs during extended cooking time, as extra water diffuses and interacts with both protein and starch matrices. besides this, increased affinity for water occurs because of a surge in polar amino-acid groups during cooking owing to protein denaturation (alonso et al., 2000), leading to subsequent increase in water uptake for pasta. the cooked weight of hcn pasta samples increased from 9.57/5 g to 12.39g/5 g with an increase in the concentration of gum used. the rise in cooked weight may be related to water-binding and water-holding capacity of the gum used for the preparation of pasta (widelska et al., 2019). cooking loss is regarded a vital indicator of pasta quality. cooking loss of pasta samples significantly decreased (p < 0.05) with an increase in the addition of gum concentration from 0 to 1% in the pasta formulation. the decrease in cooking loss might be due to the addition of guar gum, which improved the protein network, formed a matrix with the proteins where starch granules get entrenched, and thus decreased the tendency of solid loss (chauhan et al., 2017; martin-esparza et al., 2018). hcn pasta made with 1% gum showed comparable cooking parameters to that of the control sample. for good-quality pasta, an acceptable cooking loss is around 8% (dick and youngs, 1988). hence, the hcn-flour-based pasta containing 1% gum has a cooking loss in the acceptable range and could be considered as good quality. a desirable firmness and cooking resistance, low cooking loss, and stickiness are characteristics of high-quality pasta (lucisano et al., 2012). studies have shown lowest cooking loss with improved physical and textural properties of gluten-free pasta prepared with 0.6% of xanthan gum concentration (milde et al., 2020). kamsiati and herawati (2019) observed cooking loss of 5.060% and hardness of 27.530n in cassava macaroni with guar gum addition of 0.25%. textural properties of pasta the textural assessment of pasta is an important criterion to estimate its quality and acceptance by consumers (milde et al., 2020). textural attributes of the developed pasta are presented in table 7. the use of gum showed significant effects (p < 0.05) on the various textural attributes of the tested pasta samples. firmness of the native hcn pasta sample (5.44n) was lower than the control sample (2.307n). moreover, with the addition of gum, the firmness of hcn pasta samples increased from 2.307n to 4.297n, which might be related to an increase in the binding between protein and starch (duda et al., 2019). adhesiveness of hcn pasta (native) was higher than control pasta while adhesiveness of hcn pasta prepared with 1% gum addition was comparable with control. pasta adhesiveness decreased significantly (p < 0.05) with increased gum concentration in the pasta formulation. the adhesiveness is associated with the amount of starch italian journal of food science, 2021; 33 (sp1) 145 underutilized horse chestnut (aesculus indica) flour and its utilization table 7. textural properties of cooked pasta. parameters sample a sample b sample c sample d hardness (n) 5.44 ± 0.63a 2.307 ± 0.10d 3.139 ± 0.22c 4.297 ± 0.42b adhesiveness (ns) −0.390 ± 0.02c −0.202 ± 0.07a −0.354 ± −0.27b −0.398 ± 0.09c cohesion 0.699 ± 0.07a 0.470 ± 0.02c 0.584 ± 0.07b 0.695 ± 0.08a springiness % 85.572 ± 2.34a 53.558 ± 1.92b 84.577 ± 3.45a 88.060 ± 3.71a gumminess 387.654 ± 4.23a 110.460 ± 3.65d 187.047 ± 4.27c 304.454 ± 2.81b chewiness 331.724 ± 4.45a 59.160 ± 3.45d 158.199 ± 2.45c 268.101 ± 4.45b sample a: control durum wheat; sample b: native horse chestnut flour; sample c: 0.5% gum addition; sample d: 1% gum addition. results presented are mean values and the superscripts significantly differ in row (p < 0.05) (n = 6). table 8. sensory evaluation of pasta. parameters sample a sample b sample c sample d slipperiness 3.67a ± 0.82 1.83c ± 0.98 3.00b ± 1.10 3.53a ± 1.75 firmness 4.00a ± 1.26 1.50c ± 0.84 2.50b ± 1.22 3.33a ± 0.82 chewiness 3.50a ± 1.97 2.67b ± 1.52 3.00a ± 1.51 3.57a ± 1.26 surface adhesiveness 3.50a ± 1.22 1.13c ± 1.03 2.50b ± 1.52 3.40a ± 1.02 appearance 4.33a ± 0.82 2.67c ± 1.51 3.17b ± 1.60 4.00a ± 1.67 overall sensory score 4.5a ± 0.26 1.63c ± 0.13 2.94b ± 0.10 4.43a ± 0.17 sample a: control durum wheat; sample b: native horse chestnut flour; sample c: 0.5% gum addition; sample d: 1% gum addition. mean ± s.d. with different superscripts in a row differ significantly (p < 0.05) (n = 12). and gelatinization of starch. besides, addition of gum results in the development of continuous protein network, which reduces pasta adhesiveness. padalino et  al. (2013) and widelska et al. (2019) also observed that the hydrocolloid addition to the gluten-free pasta formulation led to the decrease in adhesiveness. other textural attributes including cohesiveness, springiness, gumminess, and chewiness also increased with the addition of gum in pasta formulation. mirhosseini et al. (2015) observed a similar kind of improvement in textural attributes with the addition of polysaccharide gums to gluten-free pasta formulations. hcn-flour-based pasta made with 1 % gum show comparable textural properties with that of the control sample. sensory quality of pasta the prepared pasta samples were evaluated for appearance, firmness, chewiness, surface adhesiveness, and slipperiness by sensory panellists using a 5-point scale. the mean values of sensory attributes for pasta samples are shown in table 8. sensory score of native hcn pasta (sample b) was significantly low compared to control sample (sample a), which could be related to the difference in composition and properties of their corresponding flours (bolarinwa and oyesiji, 2021). among the hcn-flour-based pasta samples, sample b (0% gum) showed a minimum score for sensory attributes (appearance, firmness, chewiness, slipperiness, and adhesiveness) while sample d (1% gum) showed the highest score. no significant difference was observed in the sensory parameters of sample a (control) and sample d. overall, sensory score of sample d was comparable with that of the control sample. enhancement in the textural properties of pasta with the addition of gums in pasta formulation led to an improvement in sensory quality. shere et al. (2020) also observed that gum addition enhanced the overall sensory quality of the developed instant noodles. conclusions the results provide an insight about the physicochemical, functional, pasting, and thermal properties of hcn flour, and the quality characteristics of pasta made from hcn flour. the results showed significant difference between the various properties of wheat and hcn flour. difference in the ratio of flour constituents (protein, starch, etc.) could be the reason for the difference in properties of the two flours. cooking, textural, and sensory characteristics of hcn-flour-based pasta samples showed that good-quality pasta comparable with commercial 146 italian journal of food science, 2021; 33 (sp1) rafiq si et al. sensory attributes. heliyon 7(1): e06052. https://doi.org/10.1016/j. heliyon.2021.e06052 bresciani, a., giordano, d., vanara, f., blandino, m. and marti, a., 2021. high-amylose corn in gluten-free pasta: strategies to deliver nutritional benefits ensuring 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quality characteristics of glutenfree pasta. journal of food measurement and characterization 11: 1188–1195. https://doi.org/10.1007/ s11694-017-9495-4 chau, c.f., cheung, p.c. and wong, y.s., 1997. functional properties of protein concentrates from three chinese indigenous legume seeds. journal of agricultural and food chemistry. 45(7):2500–2503. https://doi.org/10.1021/jf970047c chauvin, e.a., 2019. health and wellness series—nutrition and women’s health global analysis report. agriculture and agri-food canada. available at: http://www.agr.gc.ca/eng/ industrymarkets-and-trade/international-agri-food-market intelligence/reports/health-and-wellnessseries-nutrition-andwomen-s-health/?id=1546614914143. dib, a., wójtowicz, a., benatallah, l., zidoune, m.n., mitrus, m. and sujak, a., 2018. optimization of rice-field bean gluten-free pasta improved by the addition of hydrothermally treated rice flour. italian journal of food science 30(2): 226–248 dick, j.w. and youngs, v.l., 1988. 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thi minh chau nguyen1, mohsen gavahian2,*, pi-jen tsai2,* 1international master’s degree program in food science, international college, national pingtung university of science and technology, 1, shuefu road, neipu, pingtung 91201, taiwan; 2department of food science, agriculture college, national pingtung university of science and technology, 1, shuefu road, neipu, pingtung 91201, taiwan *corresponding authors: mohsen gavahian, national pingtung university of science and technology, taiwan, email: mohsengavahian@yahoo.com; pi-jen tsai; national pingtung university of science and technology, taiwan, email: pijen@mail.npust.edu.tw received: 28 august 2020; accepted: 12 december 2020; published: 11 february 2021 © 2021 codon publications open access paper abstract although phytochemical contents of gac fruit have been extensively analyzed, information about the bioactive compounds and valorization of gac leaves is limited. in this study, gac (momordica cochinchinensis spreng.) leaves at different maturity stages (young: yl, mature: ml and old: ol leaves) were extracted during a 20 min of 150-w sonication process. color, phytochemicals, antioxidant activity, and inhibitory effects against carbohydrate-hydrolyzing enzymes were assessed by colorimetric, high-performance liquid chromatography, and spectrophotometric methods, respectively. results indicated a decrease in l* (lightness) and an increase in a* (greenness–redness) during maturation of leaves. the yl extract had the highest contents of phytochemicals with 4897.01 (mg gallic acid equivalent [gae] per 100 gram dried weight [dw]), total phenolics, 592.81 (mg quercetin [qe]/100 g dw), total flavonoids, 34.77% α-amylase inhibitory activity, and 40.21% α-glucosidase inhibitory activity. myricetin (43%), vitexin (22%), and esculetin (11%) were the major bioactive compounds detected in yl extract. also, the superoxide dismutase (sod)-like capacity of the extract decreased from 11,599.96 to 3,999.63 u/g dw during the transformation of yl to ol. extract of gac leaves was found to be a potential ingredient for food preservation and supplementation that could reduce postprandial hyperglycemia. keywords: carbohydrate-hydrolyzing enzymes inhibitory activity; emerging processing technologies; gac (momordica cochinchinensis spreng.) leaves; hyperglycemia treatment; ultrasonic-assisted extraction introduction ultrasound-assisted extraction is an innovative technique used widely for extracting bioactive compounds from plant materials (munekata et al., 2020). through cavitation phenomena of bubbles, ultrasound-assisted extraction can increase the permeability of plant cell walls, enhance the contact surface area between solvent and samples, and release more phenolic compounds (moreira et al., 2019). this simple and low-cost technique has been suggested to be an alternative to the conventional extraction method, as it can save extraction time and enhance extraction efficiency (guglielmetti et  al., 2017; i̇şçimen et al., 2018; karadag et al., 2019; surin et al., 2020). this technology is used to extract gac peel and other plant material (chuyen et al., 2020). starch hydrolysis because of the activities of α-amylase in the salivary pancreas and glands combined with α-glucosidase in the small intestine is the main reason of blood glucose. inhibiting the functioning of these enzymes could reduce postprandial hyperglycemia. hence, many mailto:mohsengavahian@yahoo.com mailto:pijen@mail.npust.edu.tw italian journal of food science, 2021; 33 (sp1) 35 ultrasound-assisted extraction of gac leaves materials and methods plant materials the fresh gac leaves were collected in pingtung county, taiwan in november 2019. the identity of the plant species (m. cochinchinensis spreng.) was confirmed as described by experts in the literature. these leaves were divided into three different stages of maturity (yl: young; ml: mature and ol: old leaves) according to visual color and size as described in the literature (angmo et al., 2019). the average width and length of leaves were measured using a ruler. besides, the color values of leaves (l*: lightness; a*: greenness– redness and b*: blueness–yellowness) were measured using a colorimeter (ze 2000, nippon denshoku, japan). then, leaves were dried at 45°c for 48 h using the oven drying method (ov-100, precision oven, hipoint, taiwan) until the final water activity of samples reached 0.32 (roukas and kotzekidou, 2020). dried leaves were ground into powder using a pulverizing machine (rtn08, rt, taiwan), sieved by a 0.2–0.5-mm mesh screen to maintain constant particle size, packaged in airtight polyethylene films (0.04-mm thickness) and kept at –4°c until analysis (schematic representation is presented in figure 1). chemicals standards of phenolic and flavonoid compounds: 4-nitrophenyl α-d-galactopyranoside (pnpg), 3,5dinitrosalicylic acid (dns), α-amylase from bacillus sp., intestinal acetone powder from rat, folin & ciocalteu’s phenol reagent, l-methionine, riboflavin and nitrotetrazolium blue chloride (nbt) were purchased from sigma-aldrich, usa. aluminum chloride hex hydrate, dimethyl sulfoxide (dmso), potassium acetate, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium sodium tartrate-4-hydrate, sodium acetate, sodium carbonate, sodium hydroxide and sodium hydrogen phosphate were purchased from j.t. baker, avantor, pa, usa. methanol was obtained from aencore chemical pvt. ltd., surrey hills, australia. extraction procedure a mixture of gac leaves powder (1 g) and 50% aqueous ethanol (40 ml) was extracted using a 150-w ultrasonic bath (dc150, delta, taiwan) for 20 min based on preliminary studies. the temperature was monitored by a laboratory thermometer and adjusted at 25°c. these extraction conditions were defined according to a previous study with some modifications (tian et al., 2019). the extract was then centrifuged at 10,000 rpm for 20 min, filtered with 90-mm filter paper and kept at -20°c until further analysis. of the medicines prescribed for type ii diabetes contain bioactive compounds with carbohydrate-hydrolyzing enzymes inhibitory activity (adekola et al., 2017; özcan, 2020). usually, synthetic medicines taken for diabetes (e.g., voglibose, acarbose and miglitol) are good competitive inhibitors of α-glucosidase and α-amylase, hence inhibit patient’s ability to break down complex carbohydrates into glucose. however, some side effects, including diarrhea, flatulence and abdominal discomfort, have been reported for the long-term prescription of these medicines (mogole et al., 2020). besides, there is a trend for replacing synthetic medicines with natural ones among the health-conscious consumers. therefore, there is a growing market for natural medicines such as bioactive compounds extracted from plants. moreover, some plant extracts have been suggested as a suitable source of carbohydrate hydrolase inhibitors with probably limited or no side effects (veiga et al., 2020). for example, dioscorea polystachya and morus alba have been demonstrated for their high α-amylase and α-glucosidase inhibitory potential, respectively; and the blends of pineapple, apple and ginger as a beverage can be used as a dietary supplement to prevent diabetes mellitus because of their hypoglycemic effects (ademosun et al., 2020; ng and see, 2019). the food and pharmaceutical industries are exploring the possibility of developing new anti-diabetics from other plant materials to satisfy market demand. belonging to the cucurbitaceae family, gac (momordica cochinchinensis spreng.) is a perennial plant distributed widely in many regions of the world, including northeastern australia as well as south-east and south asia (chuyen et al., 2014). gac fruit was used as a natural colorant in cuisines and was believed to improve human vision. previous studies have demonstrated that high contents of carotenoids, lycopene and medicinal components (e.g., saponins and triterpenoids) are found in fruit, aril and seeds of gac, respectively (abdulqader et al., 2018; chuyen et al., 2014; kha et al., 2013; yu et al., 2017). besides, gac leaves were consumed in traditional medicine for various purposes such as curing fever, back pain, inflammation, wart and hemorrhoids. however, information about its bioactive components and biological effects is limited. a previous study had explored carbohydrolytic enzyme inhibitory, anti-inflammatory and antioxidant abilities of other species of momordica, that is, m. dioica, m. charantia, m. charantia var. muricata (nagarani et al., 2014). however, to the best of our knowledge, there is no published paper in the scientific literature that has examined the bioactive content and possible biological effects of gac leaves (m. cochinchinensis spreng.). the objectives of this research were to analyze the phytochemicals available in gac leaves and to explore their best maturity stages that could yield an extract with the highest α-amylase and α-glucosidase inhibitory activities. 36 italian journal of food science, 2021; 33 (sp1) nguyen tmc et al. identification of phenolic and flavonoid compounds using high performance liquid chromatography the sample extract was filtered using a 0.45-µm syringe filter (ptfe013n045i, puretech syringe filter, taiwan) before injecting it into the high-performance liquid chromatography (hplc) system (l-7100, hitachi, japan). the injected extract (20 µl) was then separated chromatographically on a mightysil rp-18 column (250 mm × 4.6 mm, 5 µm) (dobrinčić et al., 2020). the mobile phases were 0.25% formic acid, 2% methanol in ultrapure water (solvent b) and 100% methanol grade (solvent c) with a flow rate of 1 ml/min. the chromatographic peaks in extracts were identified and measured by collating their uv-visible spectra (280 nm) and retention period with those of external standards. the content of each polyphenol was presented as milligram per 100 g of dried weight sample (mg/100 g dw). determination of α-amylase inhibitory activity α-amylase inhibitory activity was determined based on a previous report with slight modifications (shori, 2020). briefly, the solvent of extract was first removed by a vacuum evaporator, the residue was then dissolved by 2-ml 50% dmso. next, a mixture of this dmso extract (100  µl) and 0.5 u/ml α-amylase solution (100 µl) was transferred in a tube and incubated at 37°c for 5 min. next, 100 µl of starch solution (1%) was added and re-incubated for 5 min at 37°c. the reaction ended using chemical analysis of extracts determination of total phenolics content total phenolics content (tpc) was evaluated according to the folin–ciocalteu assay with minor modifications (singleton and rossi, 1965). a mixture of leaves extract (0.1  ml), 50% folin–ciocalteu reagent (0.1 ml) and 2% sodium carbonate (2 ml) was incubated at about 25°c in a dark place for 30 min. the absorbance of reactive mixture was recorded with an ultraviolet spectrophotometer (u-200 spectrophotometer, hitachi, japan) at 760-nm wavelength. the tpc was measured using gallic acid calibration curve and presented as milligram gallic acid equivalent (gae) per 100 g dried weight (dw) of sample (mg gae/100 g dw). determination of total flavonoids content total flavonoids content (tfc) of gac leaves was determined using a colorimetric method of aluminum chloride (alcl3) with slight modifications (nguyen et al., 2020). an aliquot of 250 µl of extracts was mixed well with 750  µl of 95% ethanol, 50 µl of 10% aluminum chloride, 50 µl of 1 m potassium acetate and 1.4 ml of distilled water in a test tube. the mixture was left for 30 min at room temperature in dark before recording at 415 nm using an ultraviolet spectrophotometer. the tfc was represented as milligram quercetin equivalent per 100 g dried weight sample (mg qe/100 g dw) through a qe calibration curve. fresh leaves extract sample ultrasonic-assisted extraction sample powder y m o grinding seperate into three stages of maturity oven drying (45˚c) figure 1. schematic representation of the protocol employed to prepare gac leaves extract. yl: young leaf; ml: mature leaf; ol: old leaf. italian journal of food science, 2021; 33 (sp1) 37 ultrasound-assisted extraction of gac leaves 2.4 × 10-6 m riboflavin was added before transferring the test tubes into a light box and incubated for 15 min. the blue formazan formed was read at 560 nm. the control (buffer instead of sample) and background (riboflavin replacement buffer) were also determined. the sodlike activity (percentage of scavenging ability) was first defined by equation 3, then measured through the sod standard curve and represented as unit per gram dried weight sample (u/g dw). c s bg c a (a a ) scavenging ability (%) 100%, a − − = × (3) where ac is the control absorbance, as is the sample extract absorbance and abg is the background absorbance. besides, the relationship between maturity level of gac leaves and the sod-like activity of the extract was explored using different equations to develop a mathematical model for the prediction of antioxidant activity of extract as a function of degree of maturity. statistical analysis the results were presented as mean ± standard deviation (sd) (n = 3). the significant difference between samples was compared by duncan’s multiple-range tests (p < 0.05) by one-way analysis of variance (anova) using ibm spss statistics version 22. the correlation between phenolic compounds and the ability to inhibit carbohydrate hydrolyzed enzymes was determined using pearson’s correlation analysis with two-tailed test. results and discussion physical properties of gac leaves at different maturity stages average size at different maturity stages of gac leaves, the average size (width and length) was measured as represented in table 1. 1 ml of dns color reagent, then incubated for 5 min in a boiling water bath and cooled down. the absorbance was read at a wavelength of 540 nm. the background (replacing the starch by ph 6.9 buffer) and control (50% dmso instead of sample extract) were also prepared by the same procedure. the α-amylase inhibitory activity was calculated according to equation 1: c s bg c a (a a ) -amylase ia (%) 100% a α − − = × (1) where ia is inhibitory activity inhibitory activity, ac is the control absorbance, as is the sample extract absorbance and abg is the background absorbance. determination of α-glucosidase inhibitory activity α-glucosidase inhibitory activity was determined according to a previous method with partial modifications (dos santos pereira et al., 2020). before pre-incubated for 5 min at 37°c, a mixture of dmso extract (5 µl) and α-glucosidase solution (10 µl) was put into a 96-well microplate. then, 0.01-m p-nitrophenyl α-d-galactopyranoside (10 µl) was added in each well and incubated for 20 min at 37°c. the mixture reaction was stopped by adding 200-µl 0.1-m sodium carbonate. the absorbance was recorded at a wavelength of 405 nm using a microplate reader (amr-100, clubio, taiwan). control without sample, and background without pnpg were also determined. ability to inhibit α-glucosidase was calculated according to equation 2: c s bg c a (a a ) -glu cos idase ia (%) 100%, a α − − = × (2) where ia is inhibitory activity inhibitory activity, ac is the control absorbance, as is the sample extract absorbance and abg is the background absorbance. determination of superoxide dismutase-like activity superoxide dismutase (sod)-like capacity was evaluated following the previous procedure with slight modifications (cheng et al., 2015). a volume of 2.95 ml of 1.67 × 10-4 m nbt and 0.01-m l-methionine was added into each tube containing 50 µl of extract sample; 3 µl of table 1. average size and color values of gac leaves at different maturity stages. average size color values width (cm) length (cm) l* a* b* yl 7.07 ± 0.51c 9.63 ± 0.70c 44.89 ± 0.08a -7.44 ± 0.06c 18.08 ± 0.09b ml 14.67 ± 1.06b 15.40 ± 0.70b 42.76 ± 0.26b -6.58 ± 0.10b 18.37 ± 0.13a ol 16.37 ± 0.45a 17.57 ± 0.31a 37.95 ± 0.24c -6.44 ± 0.02a 16.14 ± 0.09c the values are presented as mean ± standard deviation (sd) (n = 3). a–cvalues in columns with different letters indicate the significant difference (p < 0.05). yl: young leaf; ml: mature leaf; ol: old leaf. l*: lightness; a*: greenness-–redness; b*: blueness-yellowness. 38 italian journal of food science, 2021; 33 (sp1) nguyen tmc et al. phytochemicals in different maturing stages the phytochemicals obtained from yl, ml and ol extracts are listed in table 3. in yl extract, myricetin, vitexin and esculetin accounted for nearly 43%, 22% and 11%, respectively, of all the individual compounds detected, which were approximately 4.15, 10.62 and 2.36 times higher than that in ol extract (figure 2). this indicated that these compounds were reduced with the aging of plant. similarly, chlorogenic acid and vanillic acid, respectively, decreased from 339.74 and 228.56 mg/100 g dw in yl extract to 56.53 and 11.63 mg/100 g dw in ol extract. in addition, catechin, caffeic acid and quercetin reduced by leaf senescence couldn’t be detected in ol extract. this observation suggested that because of the aging process, there were notable differences in the amounts of components at different maturing stages of gac leaves, and this was demonstrated in the guava leaf extract (angmo et al., 2019). results demonstrated that extract of gac leaves contained several phenolic and flavonoid compounds that are found in the leaves of momordica species (nagarani et al., 2014). for example, gallic acid, chlorogenic acid, ferulic acid, quercetin, ellagic acid and catechin were identified in these momordica leaves extract; the major components of m. charantia var muricata and m. charantia leaves were chlorogenic acid and quercetin, while m. dioica had rutin and ellagic acid. some compounds (including catechin, vanillic acid, epicatechin, chlorogenic acid and myricetin), in a previous study, have established inhibitory ability against α-amylase and α-glucosidase thru ic50 value with the following respective values: catechin, 8.41 mm and 13.4  μm; vanillic acid, 27.89 mm and 277.38 μm; epicatechin, 7.34 mm and 11.75 μm; chlorogenic acid, 11.57  mm and 231.80  μm; and myricetin, 1.19 mm and 2.73 μm (tan et al., 2017). containing the highest levels of these compounds in the extract, yl has clearly demonstrated a strong inhibitory ability toward α-amylase and α-glucosidase. in addition, ol demonstrated a significant increase in ellagic acid and morin with 2.36 and 1.88 times higher than that in yl, respectively; and 3.06 and it was observed that ol had the highest values of width and length (16.37 and 17.57 cm, respectively) as compared to those of ml and yl. this suggests that the leaf size increases with development of plant and reaches the maximum limit at old stage. similar results were demonstrated at three different maturity stages of beet leaves (angmo et al., 2019). color values the color values of gac leaves at different maturity stages were evaluated using l*, a* and b* parameters as presented in table 1. with significant difference in samples, following color value ranges were obtained: lightness l* (37.95–44.89), yellowness b* (16.14–18.08) and greenness a* (-7.44–-6.44). the decrease in l* and increase in a* from yl to ol indicated that the green of gac leaves was becoming darker and browner with leaf maturation. this suggested that the plant growth could affect pigment components and color parameters in different parts of the plant. similar observations were reported in a previous study (patsilinakos et al., 2018). total phenolics content and total flavonoids content total phenolics content and total flavonoids content of gac leaves at three maturing stages extracted by ultrasound-assisted extraction treatment are presented in table 2. according to the results, the extract obtained from yl had the highest tpc and tfc values (4897.01 mg gae/100 g dw and 592.81 mg qe/100 g dw, respectively) followed by that of ml and ol. this observation indicated that the secondary metabolites (phenolics and flavonoids) are probably reduced by oxidative stress during plant growth and aging. previous studies have established a similar trend in coffee arabica l. leaves, the highest content of total phenolics was found in the extract of young leaves (ngamsuk et al., 2019). however, the tfc expressed a slight difference in trend: it increased significantly (p  < 0.05) from mature to old stage after decreasing from young to mature stage, that is, some new flavonoid compounds were synthesized in smaller quantities in old stage. table 2. phytochemicals content and carbohydrate hydrolyzing enzymes inhibitory activity at different maturity stages. maturing stage tpc (mg gae/100 g dw) tfc (mg qe/100 g dw) α-amylase inhibitory activity (%) α-glucosidase inhibitory activity (%) sod-like activity (scavenging ability %) yl 4897.01 ± 98.74a 592.81 ± 18.87a 34.77 ± 1.71a 40.21 ± 1.55a 24.29 ± 1.00a ml 4318.08 ± 261.32b 365.69 ± 14.67c 32.18 ± 1.14b 29.17 ± 2.34b 16.27 ± 1.21b ol 3329.28 ± 223.70c 487.73 ± 18.64b 29.98 ± 0.55b 41.82 ± 0.29a 10.15 ± 1.71c the results are presented as mean ± standard deviation (sd) (n = 3). a–cin the same column, different letters express remarkable difference between samples (p < 0.05), duncan’s test. yl: young leaf; ml: mature leaf; ol: old leaf; tpc: total phenolic content; tfc: total flavonoid content; gae: gallic acid equivalent; dw: dry weight; qe: quercetin. italian journal of food science, 2021; 33 (sp1) 39 ultrasound-assisted extraction of gac leaves table 3. concentrations of total phenolic and flavonoid compounds at different maturity stages. compounds chemical formula ol (mg/100 g dw) ml (mg/100 g dw) yl (mg/100 g dw) esculetin c 9 h 6 o 4 227.41 ± 39.28cb 320.02 ± 61.72bb 537.29 ± 46.95ca catechin c 15 h 14 o 6 nd 25.08 ± 3.64eb 66.14 ± 3.07gha chlorogenic acid c 16 h 18 o 9 56.53 ± 5.07fgc 151.55 ± 21.57db 339.74 ± 41.25da vanillic acid c 8 h 8 o 4 11.63 ± 0.67hc 39.22 ± 9.50db 228.56 ± 11.85ea caffeic acid c 9 h 8 o 4 nd 1.42 ± 0.16eb 2.24 ± 0.50ha epicatechin c 15 h 14 o 6 28.34 ± 1.99ghb 23.25 ± 4.09eb 46.06 ± 5.00gha p-coumaric acid c 9 h 8 o 3 22.26 ± 2.85ghb 24.29 ± 2.45eb 51.97 ± 1.82gha sinapic acid c 11 h 12 o 5 10.77 ± 0.92hc 31.93 ± 3.26eb 88.16 ± 11.01fga ferulic acid c 10 h 10 o 4 10.61 ± 1.75hc 26.23 ± 2.00eb 62.56 ± 4.11gha vitexin c 21 h 20 o 10 104.91 ± 23.36dec 247.20 ± 17.31cb 1114.31 ± 135.69ba ethyl 3,4-dihydroxybenzoate c 9 h 10 o 4 71.91 ± 11.33ef nd nd rutin c 27 h 30 o 16 16.70 ± 1.90ha 5.17 ± 0.40eb 1.90 ± 0.35hc resveratrol c 14 h 12 o 3 nd nd 1.74 ± 0.36h ellagic acid c 14 h 6 o 8 364.71 ± 48.92ba 118.85 ± 9.96dc 154.51 ± 4.99fb myricetin c 15 h 10 o 8 530.73 ± 58.48ac 968.44 ± 95.50ab 2203.48 ± 118.71aa morin c 15 h 10 o 7 138.26 ± 19.50da 32.45 ± 6.98ec 73.64 ± 4.17ghb cinnamic acid c 9 h 8 o 2 4.38 ± 0.57hc 14.26 ± 2.61eb 17.98 ± 0.46gha quercetin c 15 h 10 o 7 nd 11.19 ± 2.49eb 52.53 ± 3.62gha hesperidin c 28 h 34 o 15 0.63 ± 0.16h nd nd the values are presented as mean ± standard deviation (sd) (n = 3). a–hvalues in columns with different lowercase letters indicate the significant difference (p < 0.05). a–cvalues in rows with different uppercase letters indicate the significant difference (p < 0.05). nd: not detected. yl: young leaf; ml: mature leaf; ol: old leaf; dw: dry weight. 14% 12% 20% 23% 16% 14% 12% 47% 33% 3% 6% 7% 3% 7% 7% 11% 22% 43% myricetin vitexin esculetin chlorogenic acid ellagic acid others figure 2. main components of gac leaves extract as affected by maturity stages. yl (young leaf), ml (mature leaf), and ol (old leaf) are presented in the inner, middle, and outer graphs, respectively. 40 italian journal of food science, 2021; 33 (sp1) nguyen tmc et al. reduction and oxidation into h2o2 and o2 (cheng et al., 2015; stephenie et al., 2020). sod activity at different maturity stages of gac leaves is presented in table 2. the data ranged from 10.15% to 24.29% of free radical scavenging activity of sod (equivalent to 3999.63–11599.96 u/g dw), which presented the same result with m. charantia linn. leaf extract (22%) but lower than that of m. charantia var. muricata and m. dioica leaf extracts, that is, 35% and 51%, respectively (nagarani et al., 2014). besides, the highest sod level was achieved from yl extract (11599.96 u/g dw), followed by ml and ol (7289.70 and 3999.63 u/g dw, respectively) (figure 3) (p < 0.05). this demonstrates a similar trend with the content of phytochemicals and carbohydrate hydrolyzing enzyme inhibitory activity. the high correlation between them has been proved in a previous study (shimada et al., 2020). for example, a previous study explained the association between anti-glucosidase ability and sod-like activity (correlation coefficient: 0.72), and between total soluble phenolic content and sod-like activity (correlation coefficient: 0.71). according to figure 3, the sod scavenging activity of gac leaves extract depended on the degree of maturity, which declined with the aging of leaves. a polynomial model was developed, which well predicted (r2 = 1) the changes in the sod scavenging activity of gac leaf extract (equation 4): y = 675.87x2 – 6337.9x + 17, (4) where y is the sod scavenging activity of the extract in u/g dw and x2 is the degree of maturity of gac leaves (yl = 1, ml = 2 and ol = 3). conclusion for the first time, bioactive compounds of gac leaves were extracted and analyzed whereas previous research 4.26 times higher than that in ml, respectively. with a low ic50 value (morin: 32 μm, ellagic acid: 18.4 μg/ml) (assefa et al., 2020; proença et al., 2017), the increase of these two compounds may explain a higher inhibition of α-glucosidase in ol extract. according to the results, yl samples present the best maturing stage of gac leaves for obtaining the highest concentration of bioactive compounds and the strongest inhibition of carbohydrate-hydrolyzing enzymes for further experiments. effects on carbohydrate-hydrolyzing enzymes’ inhibitory activity for the starch hydrolysis enzymes’ inhibitory activity, the effects of maturing stages of gac leaves presented the same tendency with the content of phytochemicals (table 2). the higher α-amylase and α-glucosidase inhibitory abilities belonged to yl extract (34.77 and 40.20%, respectively), which is probably because of higher concentrations of most phenolic and flavonoid components in yl. it has been explained previously, how flavonoid compounds could contribute in lowering the blood sugar and what are the mechanisms involved in their anti diabetic properties (al-ishaq et al., 2019). the results demonstrated that gac leaves contain bioactive compounds that could lower the blood sugar, and the concentration of these compounds varies depending on the maturity stage of gac leaves. this indicates the importance of effective compounds in lowering the blood sugar level, and this study established that such an extract and its effective components could be established as possible ingredients for developing nutritional supplements. the results of the present study were similar to those reported for extracts obtained from the leaves of momordica charantia linn and momordica charanti var. muricata (with an approximate α-amylase inhibition of 45% and α-glucosidase inhibition of 40%, respectively) (nagarani et al., 2014). besides, previous research done on ceylon cinnamon leaves demonstrated a similar trend, that is, higher concentrations of polyphenolic compounds were correlated with stronger anti-amylase activity (abeysekera et al., 2019). however, the anti-glucosidase activity obtained from yl and ol extracts was almost the same in spite of significant difference in tpc and tfc, suggesting that several new compounds (e.g., ethyl 3,4-dihydroxybenzoate and hesperidin) were synthesized in the old stage of gac leaves. effects on sod scavenging activity superoxide dismutase, an important antioxidant enzyme in animals and plants, is a free radical scavenger that transformed reactive oxygen species formed during yl 0 2000 4000 6000 8000 10000 12000 14000 11600 ± 536 * # 7290 ± 651 4000 ± 919 ml gac leaves samples s o d s ca ve ng in g ac tiv ity ( u /g d w ) ol figure 3. superoxide dismutase (sod) scavenging activity of the extract as affected by maturation degree of gac leaves. yl: young leaf; ml: mature leaf; ol: old leaf. italian journal of food science, 2021; 33 (sp1) 41 ultrasound-assisted extraction of gac leaves cheng, c.-w., chen, l.-y., chou, c.-w. and liang, j.-y. 2015. investigations of riboflavin photolysis via coloured light in the nitro blue tetrazolium assay for superoxide dismutase activity. journal of photochemistry and photobiology b: biology 148: 262–267. https://doi.org/10.1016/j.jphotobiol.2015.04.028 chuyen, h.v., nguyen, m.h., roach, p.d., golding, j.b. and parks,  s.e., 2014. gac fruit (momordica cochinchinensis 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https://doi.org/10.3390/foods8090389� https://doi.org/10.1088/1757-899x/736/2/022063� https://doi.org/10.1088/1757-899x/736/2/022063� https://doi.org/10.1016/j.jsps.2019.11.010� https://doi.org/10.1016/j.foodchem.2018.06.013� https://doi.org/10.1080/14756366.2017.1368503� https://doi.org/10.1007/s11356-020-07928-9� paper ital. j. food sci., vol. 28 2016 57 keywords: e. coli o157:h7, environmental stress, leafy green vegetables, rocket leaves, inoculation method recovery and behaviour of stressed escherichia coli o157:h7 cells on rocket leaf surfaces inoculated by different methods anas a. al-nabulsi1*, amin n. olaimat2, tareq m. osaili1, heba m. obaidat1, ziad w. jaradat3, reyad r. shaker4 and richard a. holley5 1department of nutrition and food technology, faculty of agriculture, jordan university of science and technology, irbid, jordan 2department of clinical nutrition and dietetics, faculty of allied health sciences, hashemite university, zarqa, jordan 3department of biological and genetic engineering, jordan university of science and technology, irbid, jordan 4department of clinical nutrition and dietetics, university of sharjah, sharjah, united arab emirates 5department of food science, faculty of agriculture and food science, university of manitoba, winnipeg, canada *corresponding author: tel. +962 02 7201000, fax +962 02 7201078, email: anas_nabulsi@just.edu.jo abstract e. coli o157:h7 is an emerging public health concern worldwide because of its low infectious dose and ability to survive under adverse conditions. tests were conducted to determine the ability of unstressed e. coli o157:h7 cells or those stressed by acid, cold, salt exposure or starvation to survive on the surfaces of rocket leaves after contamination by three methods (dip, spray or spot inoculation) and following storage at 10 or 25ºc. e. coli o157:h7 numbers recovered from rocket leaves contaminated by the different techniques were in the order of dip > spot > spray inoculation. numbers of stressed e. coli o157:h7 recovered after inoculation by all three methods increased significantly over 7d storage at 10 or 25ºc, while unstressed e. coli o157:h7 only grew following dip inoculation. exposure to adverse environmental conditions may increase the risk of e. coli o157:h7 survival and spread on leafy green vegetables. mailto:anas_nabulsi%40just.edu.jo?subject= 58 ital. j. food sci., vol. 28 2016 introduction escherichia coli o157:h7 is a facultatively anaerobic, gram-negative rod-shaped enteric bacterium which produces shiga toxins 1 and/or 2 as important virulence factors and emerged as a foodborne pathogen in 1982. e. coli o157:h7 infection presents with numerous symptoms including abdominal pain, watery and bloody diarrhea, vomiting, mild fever, sometimes leading to hemorrhagic colitis and the hemolytic uremic syndrome (hus) with renal tubular damage (who, 2011). scallan et al. (2011) and thomas et al. (2013) reported that e. coli o157:h7 is associated with 63,153 and 12,827 foodborne illnesses every year in the us and canada, respectively. e. coli o157:h7 illness outbreaks have been associated with a variety of foods including ground beef, spinach, lettuce, radishes, vegetable sprouts, fermented sausages, unpasteurized fruit juices, apple cider and raw milk (chauret, 2011). annually, fresh produce is responsible for 45.9% of foodborne illnesses, 38.1% of hospitalizations and 22.9% of deaths caused by contaminated food in the us (painter et al., 2013). furthermore, e. coli o157:h7 has been associated with repeated illness outbreaks resulting from the consumption leafy vegetables including lettuce, spinach, parsley and rocket leaves (olaimat and holley, 2012; nygard et al., 2008). in recent years contamination of raw or minimally processed food products such as fresh produce with e. coli o157:h7 has become a concern worldwide because of its low infectious dose and ability to survive for long periods in the environment (chauret, 2011; delaquis et al., 2007). this contamination may occur on the surface of leafy vegetables due to the transfer of the pathogen from soil or water (delaquis et al., 2007). survival of e. coli o157:h7 on these surfaces is affected by several factors including nutrient availability, competition with indigenous microflora, uv radiation and relative humidity (brandl, 2006). fresh produce can be contaminated with foodborne pathogens during preor post-harvest processes (olaimat and holley, 2012). agricultural production and post-harvest environments may exert a variety of stresses on pathogens, which may affect their survival on the final product. in response, strategies have been developed by exposed pathogens to reduce the impact of this stress, including formation of aggregates in protective niches, localization in biofilms, and internalization within plant tissues. bacteria have also been shown to respond to these stresses through genetic and/or physiological means including stress adaptation, development of cross-protection mechanisms, conversion to a viable but non-culturable (vbnc) state, through heterogeneous phenotypic expression, and by sheer genetic diversity (dinu et al., 2009). studies have shown that ability of e. coli o157:h7 to exhibit responses to sub-lethal environmental stresses, which may enable its survival under more severe conditions, enhance its resistance to subsequent processing conditions, and/ or enhance its virulence. therefore, understanding the effects of environmental stress caused by acid, cold, starvation and abnormal osmotic conditions on the survival of e. coli o157:h7 is important in order to assess and minimize the risk of foodborne illnesses caused by this organism (chung et al., 2006). it has been reported that damaged produce supports the growth of foodborne pathogens, however, intact vegetables like lettuce, tomatoes, endive, carrots, cabbage, asparagus, broccoli and cauliflower may also permit the growth or survival of pathogens (olaimat and holley, 2012). several studies have investigated growth and survival of e. coli o157:h7 on the surfaces of leafy green vegetables including lettuce (lang et al., 2004b; markland et al., 2013; mcevoy et al., 2009; chang and fang, 2007), parsley (islam et al., 2004; lang et al., 2004b), spinach (markland et al., 2013; luo et al., 2009), basil (markland et al., 2013) and thale cress (cooley et al., 2003). however, little information is available on the survival of stressed e. coli o157:h7 cells on the surfaces of leafy greens (mcevoy et al., 2009). since produce contamination may occur during preor post-harvest activities, different techniques have been used to reproduce contamination that may occur commercially. spot inoculation, where a volume of inoculum containing a known cell density is applied at several locations on produce surfaces, can represent contamination that may occur from contact with soil, workers’ hands, or equipment surfaces. dip inoculation can represent contamination that may occur from run-off as well as irrigation, flume water use and water immersion which are common among industry practices. additionally, spray inoculation can represent contamination that may result from aerosols (beuchat et al., 2003). given the physical differences among each of the ways produce can become accidentally or deliberately contaminated, it became of interest to determine whether the experimental method used for fresh produce inoculation could influence inoculated pathogen survival or growth. thus, the objective of the current study was to compare the recovery of unstressed or acid-, cold-, starvedand saltstressed e. coli o157:h7 cells from the surfaces of rocket leaves inoculated by different methods and stored at 10° or 25ºc. materials and methods preparation of bacterial strains and inocula four clinical isolates of e. coli o157:h7 (00:0304, 02:0627, 02:0628, and 02:3581) now in the department of nutrition and food technology, jordan university of science and technology culture collection were stored individually at -80ºc ital. j. food sci., vol. 28 2016 59 in trypticase soy broth (tsb; oxoid ltd., basingstoke, uk) containing 20% (vol/vol) glycerol (sigma-aldrich, st. louis, mo). frozen stock cultures were activated by transferring one loopful from each culture to 10 ml of tsb and incubating at 37ºc for 24 h. the strains were streaked on sorbitol macconkey agar (smac) plates and stored at 4ºc. one colony was transferred to 10 ml tsb and incubated at 37ºc for 24 h. equal volumes of each strain were mixed to prepare an e. coli o157:h7 cocktail which was centrifuged at 4,500 rpm for 20 min, the supernatant fluid was removed and the pellet was washed with sterile deionized water and then transferred to 10 ml of sterile deionized water. this suspension was diluted in sterile deionized water to achieve 108 cfu/ml. preparation of stressed e. coli o157:h7 acid-stressed cells were prepared by transferring a loopful of each strain to 10 ml of tsb containing 10 g/l glucose and incubated at 37ºc for 18 h where ~ 9 log cfu/ml was reached at a final ph 4.9 ± 0.1 (al-nabulsi et al., 2014; leenanon and drake, 2001). equal volumes of each strain were mixed in sterile tubes to prepare a cocktail mixture containing equal numbers of each strain. salt-stressed cells were prepared by transferring a loopful from each strain into 10 ml of tsb supplemented with 0.65 m nacl and incubated at 37ºc for 18 h where ~ 9 log cfu/ml was reached. a cocktail was prepared containing equal numbers of each strain as described above and resuspended in 10 ml sterile deionized water (al-nabulsi et al., 2014; hajmeer et al., 2006). cold-stressed cells were prepared by inoculating a loopful of each strain into 10 ml of tsb at 37ºc for 18 h where ~ 9 log cfu/ml was reached. a cocktail was prepared containing equal numbers of each strain as described above, was resuspended in 10 ml tsb and incubated for 7 d at 5ºc (al-nabulsi et al., 2014; leenanon and drake, 2001). starved cells were prepared by inoculating a loopful of each strain into 10 ml of tsb which was incubated at 37ºc for 18 h. a cocktail was prepared containing equal numbers of each strain as described above in saline solution (0.85% nacl, ph 6.6) and incubated further for 48 h at 37ºc (al-nabulsi et al., 2014; leenanon and drake, 2001). inoculation of leaf surfaces by spot, spray or dip methods rocket leaves were purchased from a supermarket in irbid, jordan on the day of each experiment. damaged leaves were removed; intact leaves were washed with tap water and dried using a salad spinner. unstressed and stressed e. coli o157:h7 cells were used to inoculate the rocket leaves to obtain an inoculum level of 7.0 log cfu/ leaf. the following procedures were used for inoculation of rocket leaves: for spot inoculation, 50 µl cell suspension was added at different places on the surface of each leaf; for dip inoculation leaves were dipped in 100 ml of inocula prepared as described above for 1 min, and for spray inoculation 50 µl of inocula was sprayed on each leaf using a gas chromatography sample syringe connected to a nitrogen gas supply at 2 psi. inoculated leaves were placed in a biosafety cabinet for 2 h to dry. after that, the leaves were incubated at 4ºc for 22 h to allow to e. coli o157:h7 cells to attach to the leaf surfaces (lang et al., 2004), and samples were stored at 10 or 25ºc for 7 d. microbiological analysis the inoculated leaves were analyzed at 0.5, 1, 3, and 7 d after storage at 10 or 25ºc. the leaves were transferred to a sterile stomacher bag, treated in a stomacher (easy mix, aes laboratoire, france) for 2 min, serially diluted in 0.1 % peptone water and plated on sorbitol macconkey agar supplemented with 0.05 mg/l cefixime and 2.5 mg/l potassium tellurite (ct smac). the solidified ct smac (20 ml) had been overlaid with 10 ml tsa (thin agar layer format) to facilitate the growth of injured cells. inoculated plates were incubated at 37ºc for 18-24 h. statistical analysis data presented are means of three experiments with two replicates for each experiment (n=6). values were analyzed by spss software, version 19 (ibm inc., armonk, ny) using a univariate general linear model. differences were considered significant at p ≤ 0.05. results behaviour of unstressed e. coli o157:h7 recovered from leaf surfaces inoculated by spot, dip and spray methods the initial number of e. coli o157:h7 applied to each leaf was ~ 7.0 log cfu/leaf by each of the three methods. however, significantly higher numbers of e. coli o157:h7 cells were recovered from the dip-inoculated rocket leaves (7.10 log cfu/leaf) compared to the spotor spray-inoculated leaves (6.35-6.71 log cfu/leaf). further, e. coli o157:h7 numbers recovered from the dip-inoculated leaves significantly increased and by 7 d reached 8.20 or 8.37 log cfu/leaf at 10 or 25°c, respectively. the spray and spot methods did not perform differently from each other, and numbers of the pathogen present on sprayand spot-inoculated leaves also increased during storage; however, changes (0.07-0.4 log cfu/leaf) were significantly smaller than with dip-inoculated samples (1.1-1.3 log cfu/leaf) (table 1). 60 ital. j. food sci., vol. 28 2016 behaviour of acid-stressed e. coli o157:h7 recovered from leaf surfaces inoculated by spot, dip and spray methods the numbers of acid-stressed e. coli o157:h7 recovered from rocket leaves differed with the three methods and were ranked in the order of dip > spray > spot inoculation, although the numbers recovered following all inoculation methods were similar (p > 0.05). during storage, e. coli o157:h7 numbers recovered from dip-, spotor spray-inoculated rocket leaves significantly increased, and increases by 7 d were 0.9 to 1.3 log cfu/leaf at 10°c and 1.2 to 1.6 log cfu/leaf at 25°c. meanwhile, the highest e coli o157:h7 numbers present by 7 d storage at both temperatures were on rocket leaves inoculated by dipping (8.51-8.78 log cfu /leaf) and by spraying (7.79-7.93 log cfu /leaf) (table 2). behaviour of cold-stressed e. coli o157:h7 recovered from leaf surfaces inoculated by spot, dip and spray methods e. coli o157:h7 numbers recovered from the dip-inoculated leaves were significantly higher (7.53 log cfu/leaf) than those recovered from either the spot-inoculated (6.65 log cfu/leaf) or spray-inoculated leaves (6.48 log cfu/leaf). during storage, e. coli o157:h7 numbers recovered from rocket leaves, regardless of the inoculation method used, significantly increased (0.8-1.29 log cfu/leaf) at 10 and 25ºc by 7 d (table 3). behaviour of starvation-stressed e. coli o157:h7 recovered from leaf surfaces inoculated by spot, dip and spray methods as with cold-stressed cells, numbers of starvation-stressed e. coli o157:h7 recovered from the dip-inoculated rocket leaves were significantly higher (7.50 log cfu/leaf) than those recovered from the spot(6.64 log cfu/leaf) or spray-inoculated leaves (6.46 log cfu/leaf). during storage at 10 or 25ºc, the numbers of e. coli o157:h7 cells recovered from rocket leaves inoculated using the three methods remained constant for 1 day, but after that there was a significant increase in their numbers (0.7-1.1 log cfu/leaf). spot and spray inoculation methods had the same effect on e. coli o157:h7 numbers during storage; however dip inoculation enabled higher recoveries from the leaves at all storage intervals (table 4). table 1 viable count of unstressed e. coli o157:h7 cells on the surface of rocket leaves stored at 10° or 25ºc after inoculation by three methods. inoculation method 10ºc 25ºc day dip spot spray dip spot spray 0 7.10±0.75ab 6.71±0.41abab 6.35±0.51aa 7.10±0.75ab 6.71±0.41abab 6.35±0.51aa 0.5 7.95±0.08bb 6.74±0.52aba 6.43±0.21aa 7.10±0.44ac 6.71±0.32ab 6.24±0.29aa 1 7.98±0.08bc 7.19±0.58ab 6.43±0.20aa 7.67±0.59ab 7.21±0.62aa 6.35±0.76aa 3 7.92±0.85bb 6.50±0.74ba 6.59±0.83aa 7.72±0.08bb 6.81±0.61aa 6.73±0.46aa 7 8.20±0.44bb 6.78±0.48aba 6.72±0.63aa 8.37±0.11bb 6.89±0.46aa 6.78±0.54aa values in the same row at each temperature with the same uppercase letter are not significantly different (p ≥ 0.05). values in the same column with the same lowercase letter are not significantly different (p ≥ 0.05). table 2 viable count of acid-stressed e. coli o157:h7 cells on the surface of rocket leaves stored at 10° or 25ºc after inoculation by three methods. inoculation method 10ºc 25ºc day dip spot spray dip spot spray 0 7.23±0.47aa 6.49±0.75aa 6.75±0.84aa 7.23±0.47aa 6.49±0.75aa 6.75±0.84aa 0.5 7.81±0.39bb 6.76±0.26aa 6.89±0.51aa 8.09±0.34ab 7.12±0.24aa 7.16±0.58aa 1 8.07±0.12bc 6.94±0.72aba 7.54±0.51bb 8.27±0.36abb 7.47±0.13aba 7.59±0.16aba 3 8.12±0.08bb 7.42±0.41ba 7.66±0.49bab 8.50±0.18bcc 7.49±0.24aba 7.82±0.2bb 7 8.51±0.04cc 7.36±0.25ba 7.79±0.11bb 8.87±0.07cc 7.71±0.07ba 7.93±0.04bb values in the same row at each temperature with the same uppercase letter are not significantly different (p ≥0.05). values in the same column with the same lowercase letter are not significantly different (p ≥ 0.05). ital. j. food sci., vol. 28 2016 61 table 3 viable count of cold-stressed e. coli o157:h7 cells on the surface of rocket leaves stored at 10° or 25ºc after inoculation by three methods. inoculation method 10ºc 25ºc day dip spot spray dip spot spray 0 7.53±0.16ab 6.65±0.32aa 6.48±0.25aa 7.53±0.16ab 6.65±0.32aa 6.48±0.25aa 0.5 7.99±0.12bb 7.24±0.13ba 7.01±0.29bca 7.53±0.51ab 6.90±0.57aa 6.76±0.48aa 1 7.81±0.68abb 7.23±0.15ba 6.69±0.66aba 7.86±0.10bb 7.06±0.57aba 6.97±0.51aba 3 8.15±0.06bcb 7.39±0.04bca 7.34±0.03ca 8.35±0.04bc 7.52±0.06bcb 7.41±0.09bca 7 8.43±0.09cb 7.45±0.08ca 7.38±0.09ca 8.39±0.05bc 7.94±0.07cb 7.62±0.07ca values in the same row at each temperature with the same uppercase letter are not significantly different (p ≥ 0.05). values in the same column with the same lowercase letter are not significantly different (p ≥ 0.05). table 4 viable count of starvation-stressed e. coli o157:h7 cells on the surface of rocket leaves stored at 10° or 25ºc after inoculation by three methods. inoculation method 10ºc 25ºc day dip spot spray dip spot spray 0 7.50±0.19ab 6.64±0.30aa 6.46±0.27aa 7.50±0.19ab 6.64±.30aa 6.46±0.27aa 0.5 7.50±0.47ab 6.74±0.56aa 6.77±0.73aba 7.86±0.52ab 7.01±0.59aa 6.79±0.50aa 1 7.81±0.38ab 6.96±0.53aba 6.79±0.50aba 8.11±0.47abb 7.08±0.39aa 6.93±0.51aa 3 8.14±0.06bc 7.43±0.05cb 7.31±0.07ba 8.32±0.11bcb 7.54±0.07ba 7.44±.0.06ba 7 8.46±0.12bb 7.36±0.06bca 7.21±0.43ba 8.51±0.06cb 7.54±0.07ba 7.54±0.08ba values in the same row at each temperature with the same uppercase letter are not significantly different (p ≥ 0.05). values in the same column with the same lowercase letter are not significantly different (p ≥ 0.05). table 5 viable count of salt-stressed e. coli o157:h7 cells on the surface of rocket leaves stored at 10° or 25ºc after inoculation by three methods. inoculation method 10ºc 25ºc day dip spot spray dip spot spray 0 7.10±0.91aa 6.94±0.38aa 6.45±0.26aa 7.10±0.81aa 6.94±0.38aa 6.45±0.26aa 0.5 7.81±0.37bb 6.86±0.49aa 6.82±0.55aba 7.86±0.52ab 6.99±0.60aa 6.81±0.52aa 1 7.86±0.38bcb 6.88±0.53aa 6.88±0.50aba 8.08±0.49ab 7.09±0.57aa 6.97±0.50aa 3 7.89±0.40bcb 7.02±0.50aa 6.94±0.52aba 8.13±0.51ab 7.20±0.53aa 7.30±0.52aa 7 8.44±0.07cb 7.36±0.15aa 7.31±0.17ba 8.32±0.11bb 7.38±0.44aa 7.45±0.06ba values in the same row at each temperature with the same uppercase letter are not significantly different (p ≥ 0.05). values in the same column with the same lowercase letter are not significantly different (p ≥ 0.05). behaviour of salt-stressed e. coli o157:h7 recovered from leaf surfaces inoculated by spot, dip and spray methods numbers of salt-stressed e. coli o157:h7 were also higher (p < 0.05) on dip-inoculated leaves than those of other treatments. their numbers significantly increased on sprayor dip-inoculated leaves, and by 7 d reached 7.31 and 8.44 log cfu/leaf, respectively, at 10ºc and 7.45 and 8.32 log cfu/leaf, respectively, at 25ºc. however, there was no change in the numbers of e. coli o157:h7 recovered from spot-inoculated rocket leaves (p > 0.05). increases during storage were 0.4 log cfu/leaf on samples spot-inoculated, but numbers on leaves from the other treatments increased 1.0 1.3 log cfu/leaf at both temperatures (table 5). 62 ital. j. food sci., vol. 28 2016 discussion different inoculation methods have been used experimentally to contaminate fresh produce in studies of the survival or inactivation of pathogens (al-nabulsi et al., 2014; lang et al., 2004 a,b; singh et al., 2002). however, it is possible that the method chosen for inoculation may affect pathogen behaviours (survival, growth, injury, or death). in the present study three inoculation techniques (dip, spot and spray) were used and there was variability in the number of e. coli o157:h7 present on the leaves contaminated. it was found that dipping yielded larger numbers of unstressed or stressed e. coli o157:h7 cells on rocket leaves. this may have been because of the greater exposure of leaf surfaces, including cut surfaces where some cells could have been internalized. these results are similar to those obtained by lang et al. (2004a) who showed that higher numbers of e. coli o157:h7 and salmonella were recovered from dip-inoculated tomatoes compared to those spotor spray–inoculated. in another study, lang et al. (2004b) observed that applying the cell suspension to the surface of lettuce by dipping enhanced the internalization of bacteria at the cut edge and via stomata which can facilitate bacterial access to internal leaf tissue. the results of the current study also indicated that bacterial numbers recovered from spot-inoculated leaves were not significantly different from those recovered from those that were spray-inoculated. similarly, lang et al. (2004b) found that the number of e. coli o157:h7 and salmonella recovered from lettuce when inoculated by spot or spray methods were similar. however, they recommended using the spot method as the standard for inoculation in studying the efficacy of sanitizers against pathogenic bacteria. singh et al. (2002) found that some sanitizers (thyme oil, aqueous chlorine dioxide, ozonated water) were less effective against e. coli o157:h7 on lettuce inoculated by dipping or sprinkling than by the spot or drop method. it should be noted that even when fresh produce was washed and sanitized using chemical agents such as chlorine, only a 1-2 log microbial reduction was achieved (olaimat and holley, 2012). unstressed e. coli o157:h7 cells were able to grow when inoculated by dipping at 10 or 25ºc, but cells inoculated by spraying or spotting survived without significant change in numbers at both temperatures over 7 d storage. these results are similar to those reported by chang and fang (2007) who found that e. coli o157:h7 numbers on lettuce increased by 2.7 log cfu/g at 22ºc, although they decreased by 1.4 log cfu/g at 4ºc. francis and o’beirne (2001) also reported that e. coli o157:h7 numbers increased by up to 2.5 log cfu/g after 12 d on spot-inoculated shredded lettuce, coleslaw and soybean sprouts at 8ºc. luo et al. (2010) found that storage of spray-inoculated lettuce at 5ºc allowed e. coli o157:h7 to survive, but its growth was limited. at 12ºc there was more than a 2.0 log cfu/g increase in e. coli o157:h7 numbers after 3 d storage. in contrast, markland et al. (2013) did not detect e. coli o157:h7 cells after 4 d on basil plants that were spray irrigated. the behaviour of microorganisms in food products depends on the interaction of intrinsic and extrinsic factors such as temperature, ph, and water activity. bacteria may encounter sub-lethal stresses in variety of food products, particularly minimally processed food such as fresh produce. responses of bacteria to these stresses may enhance their survival under more severe conditions, enhance their resistance to subsequent processing conditions and perhaps enhance virulence. thus, understanding the effects of environmental stress on the behaviour of pathogens is important in order to assess and minimize the risk of foodborne illness (chung et al., 2006). in e. coli o157:h7 exposure to stress can initiate several mechanisms to minimize the effects of the challenge. for example, the rpos gene regulates expression of > 50 proteins that are involved in the general stress response. also, heat and cold shock genes can play a major role in the level of expression of the response by stressed e. coli o157:h7. these mechanisms facilitate adaptation of e. coli o157:h7 to environmental change and increase its survival (chauret 2011). in the current study, numbers of stressed e. coli o157:h7 recovered by the three different methods increased significantly at 10° and 25ºc. in contrast, mcevoy et al. (2009) found that the behaviour of cold-stressed e. coli o157:h7 was similar to that of unstressed cells on fresh iceberg lettuce where the cold-stressed and unstressed cells grew significantly at 30ºc, but survived without changes in their numbers at the non-permissive growth temperature of 5ºc after 8 d. several factors are likely to affect growth and survival of e. coli o157:h7 on fresh produce including its type (ph, surface smoothness, porosity, nutrient availability), physiological state, moisture, storage temperature > 7ºc, and the identity of the bacterial strain. it was of interest from the present work that stressed cells of a 4 strain e. coli o157:h7 cocktail on rocket leaves were able to increase in numbers during a week of storage at 10º and 25ºc to similar extents, but unstressed cells did not. in conclusion, it appears that the method used for bacterial inoculation of produce leaves can influence the levels of e. coli o157:h7 recovered from treated samples. the greatest uptake of cells from the inoculum occurred when leaves were dipped. thus the importance of controlling the quality of water used in produce plant flumes and for produce immersion becomes apparent. spot and spray inoculation yielded lower but similar levels of contamination. thus produce contact with unclean equipment surfaces, ital. j. food sci., vol. 28 2016 63 handling of produce in an unsanitary manner by employees and the occurrence of aerosols during processing can increase the bacterial burden that is likely to occur on the final product. most importantly, when cells stressed by acidic ph, cold, starvation or salt exposure were inoculated on rocket leaves, cells were able to grow slowly at both 10º and 25ºc, whereas unstressed cells did not increase in numbers during 7 d storage. this unanticipated feature of e. coli o157:h7 may enhance its ability to be spread through shipments of produce during distribution, increasing risks associated with this foodborne pathogen. acknowledgments the authors thank the deanship of scientific research at jordan university of science and technology (grant 282/2011). references al-nabulsi a.a., osaili t.m., obaidat h.m., shaker r.r., awaisheh s.s. and holley r.a. 2014. inactivation of stressed escherichia coli o157:h7 cells on the surfaces of rocket salad leaves by chlorine and peroxyacetic acid. j. food prot. 77: 32-39. beuchat l.r., farber j.m., garrett m., harris l.j., parsih m.e., suslow t.v. and busta f.f. 2003. standardization of a method to determine the efficacy of sanitizers in inactivating human pathogenic microorganisms on raw fruits and vegetables. compr. rev. food sci. food safety 6: 174-178. brandl m.t. 2006. fitness of human enteric pathogens on plants and implications for food safety. ann. rev. phytopathol. 44: 367-392. chang j.m. and fang t.j. 2007. survival of escherichia coli o157:h7 and salmonella enterica serovars typhimurium in iceberg lettuce and the antimicrobial effect of rice vinegar 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zoonoses public health 60: 563-71. mcevoy j.l., luo y., zhou b., feng h. and conway w.s. 2009. potential of escherichia coli o157:h7 to grow on field-cored lettuce as impacted by postharvest storage time and temperature. int. j. food microbiol. 128: 506509. nygard k., lassen j., vold l., andersson y., fisher i. and lofdahl s. 2008. outbreak of salmonella thompson infections linked to imported rucola lettuce. foodborne pathog. dis. 5: 165-173. olaimat a.n. and holley r.a. 2012. factors influencing the microbial safety of fresh produce: a review. food microbiol. 32: 1-19. painter j.a., hoekstra r.m., ayers t., tauxe r.v., braden c.r., angulo f.j. and griffin p.m. 2013. attribution of foodborne illnesses, hospitalizations, and deaths to food commodities by using outbreak data, united states, 19982008. emerg. infect. dis. 19: 407-415. scallan e., hoekstra r.m., angulo f.j., tauxe r.v., widdowson m.a., roy s.l., jones j.l. and griffin p.m.. 2011. foodborne illness acquired in the united states— major pathogens. emerg. infect. dis. 17: 7-15. singh n., singh r.k., bhunia a.k. and stroshine r.l. 2002. efficacy of chlorine dioxide, ozone, and thyme essential oil or a sequential washing in killing escherichia coli o157:h7 on lettuce and baby carrots. lwt food sci. techn. 35: 720-729. thomas m.k., murray r., flockhart l., pintar k., pollari f., fazil a., nesbitt a. and marshall b. 2013. estimates of the burden of foodborne illness in canada for 30 specified pathogens and unspecified agents, circa 2006. foodborne pathog. dis. 10: 639-648. world health organization (who). 2011. enterohaemorrhagic escherichia coli (ehec), fact sheet n°125. available at: http://www.who.int/mediacentre/factsheets/fs125/en/ (accessed on may 30, 2014). paper received june 23, 2014 accepted april 15, 2015 http://www.who.int/mediacentre/factsheets/fs125/en paper ital. j. food sci., vol. 27 2015 487 keywords: sensory analysis, parmigiano-reggiano, grated cheese, compliance score relationship between sensory results and compliance scores in grated parmigiano-reggiano cheese m. zannoni*1 and e.a. hunter2 1organismo controllo qualità produzioni regolamentate, via ferruccio ferrari 6, 42124 reggio emilia, italy 2independent statistician, 23 st.ninians terrace morning side, eh105pw, edinburgh, scotland, united kingdom *corresponding author: tel. +39 0522 934266, fax +39 0522 564500, email: mariozan@ocqpr.it abstract the regulations for protected designation of origin require a certification body to verify compliance with the provisions of the product specification. the grated parmigiano-reggiano is evaluated with a scorecard containing 21 quantitative descriptors and 4 qualitative evaluations of compliance with the regulations. to better understand the relationship between sensory compliance and quantitative descriptors we have tested 24 samples of grated parmigiano-reggiano. correlations and partial least squares gave us a better understanding of compliance evaluation. the work allowed us to define the most important descriptors in relationship with compliance and showed that it is possible to predict compliance values using descriptor values. 488 ital. j. food sci., vol. 27 2015 introduction worldwide the generic term used to describe products attributed to a defined region is products with a geographic indication (gi) as for example darjeeling tea in india, tequila spirit in mexico or napa valley wines in usa. within the european union (eu), protected geographical indication (pgi) is applied to products whose characteristics originate mainly, but not exclusively, from a particular region (e.g. scottish farmed salmon, bayonne ham, turron de alicante), whereas protected designation of origin (pdo) have proven characteristics resulting solely from the region of production (i.e. parma ham, isigny butter, valencia rice). cheese is the most important pdo product. the european regulations on quality schemes for agricultural products and foodstuffs number 2081 of 1992, number 506 of 2006 and the latest number 1151 of 2012 provide for product specifications to include organoleptic characteristics. in the eu database door, the so called “single document” for each pgi and pdo product gives general information about the characteristics of the product and it’s processing (european commission door, 2013). these regulations require a certification body to verify compliance with the provisions of the product specification (official journal of the european union, 2012). since an organoleptic specification is included in the product description, compliance indicates agreement with the registered specification. testing for compliance with the registered organoleptic specification is different from sensory quality control. the latter is less demanding since production conditions are tightly controlled within a food processing plant and consequently food attributes are relatively stable. gi is always connected to the artisanal process with the product being produced in many small scale plants with different conditions of production i.e. source of raw material, climatic condition, production equipment, operation of this equipment, packaging of the product, management of the product prior to distribution, etc. such heterogeneity makes for problems in defining the characteristic in the official production standard, hence the official sensory definitions tend to be general and thus not precise. sensory properties define the distinctiveness of the product at the moment of consumption. for gis many different sensory characteristics have to be measured in order to allow both control of the process and certification. within europe, there is no unanimity in how sensory properties should be evaluated; in most cases there is expert evaluation that is not easily translated into sensory analysis. when a sensory panel is employed there are many different approaches to panel composition, the scorecard and to the presentation of the results of the data analysis. the compliance evaluation of a gi has something in common with quality control judgments made by product experts on the conformity with pre-defined sensory characteristics. compliance is an assessment of how “typical” the product is of the gi. this is different from quality as perceived by consumers. a product can be compliant with the specification (‘typical’ of the gi) but might be considered poor quality by consumers who do not know and appreciate the qualities of this gi. on the other hand a product could be found to be of good quality by consumers but not compliant with the specification of the gi. sensory evaluation of cheeses is also employed to evaluate compliance with pre-established sensory specifications. thus it is used both in quality control (in cheese producing companies) and in scoring compliance (for gis). the most common system of organoleptic evaluation by a control body is that using the traditional method of forming an overall quality score by summing up the scores on number of characteristics (bodyfelt, 1988). another system of quality scoring of some attributes (appearance, consistency, odour/flavour) is detailed by the norm iso 22935-3/idf 99-3 “milk and milk products – sensory analysis” part 3 (international standard organisation 2009, kraggerud et al., 2012). a further method with some differences from the above is used for the spanish pdo cheese idiazabal which uses a scorecard with 8 parameters (4 appearance, 1 texture, 1 odour, 1 taste and 1 aftertaste) each with a compliance score and hence a total compliance score (pérez-elortondo, 2007). for this cheese compliance is determined with a decision tree. yet another system of quality scoring for compliance is used by the italian parmigiano-reggiano pdo cheese. the scorecard (garavaldi et al., 2010) contains attributes for compliance as well as quantitative descriptive attributes (qda). a compliance score is derived for each of 4 properties (appearance, odour, taste and texture). the italian asiago pdo cheese is evaluated with a 6-attributes (colour, “holes”, sweet, salty, sour, bitter) qda scorecard with quality ranges (zannoni and marangon, 2011), i.e. should the intensity of the perceived attribute be out of the range specified for any one of the characteristics then the product is not compliant. availability of funds, technical support, the interests of producers and market requirements are all factors, which affect the method chosen to score compliance. there is no general agreement on how to tackle this most important problem; the choice of approach is currently determined by the specific requirements of each gi. the only nation where there is uniformity in ital. j. food sci., vol. 27 2015 489 sensory evaluation of gis is france. the institute for the designation of origin (inao, 2008) has given guidance notes for the organization and operation of sensory panels for all pdo/ pgi products. in practice for french appellation d’origine protegée (aop) pdo cheeses a common system of evaluation is a total compliance score (cantal, 2011) (salers, 2011) (picodon, 2008) (abondance, 2010) (fourme d’ambert, 2008) derived from the sum of compliance scores of 3 parameters (appearance, texture and taste). from those examples, it is evident that there are four types of methods to evaluate the compliance of gi with their specifications. the most common is to give a total score indicating the deviance from pre-established sensory specification (cantal, 2011) (salers, 2011) (picodon, 2008) (abondance, 2010) (fourme d’ambert, 2008) as indicated by the iso norm 22935-3. another method employs a quality score for every parameter (pérez-elortondo et al., 2007). a less common method (zannoni and marangon, 2011) employs a qda scorecard with quality (or compliance) ranges for every attribute. a fourth model (garavaldi et al., 2010) employs a compliance score for visual, odour, texture and taste together with a qda with 24 descriptors. parmigiano-reggiano is one of the most popular italian cheeses, with protected designation of origin (pdo) from 1954 (from 1996 in eu). the most common end use of this cheese is grated over pasta. the increasing success in foreign markets and customer demand for convenience has led to an increasing proportion of the cheese being grated before being packaged in a modified atmosphere prior to distribution. the grated cheese comes from parmigianoreggiano pdo wheels, which are cut into large pieces, grated by a grating machine and then transferred by a conveyor belt to a packing machine. the process of grating/packaging lasts only few minutes. the quality of the final products depends not only on the quality of the original cheese, but also on the operation of the grating process (fig. 1). for the product to be marketed as grated parmigiano-reggiano cheese, it must be certified by the official control body, organismo controllo qualità produzioni regolamentate (ocqpr), for compliance with the parmigiano-reggiano regulations. these state that in the grated form the product must keep the characteristics of the original cheese (zannoni, 2007). the sensory analysis used by the certification body, for assessing the compliance of grated parmigiano-reggiano has been used since 2002. the scorecard for grated parmigiano-reggiano has been evaluated in a previous paper (zannoni and hunter, 2013). bearing in mind that there is no unanimity on how the compliance evaluation of gis is carried out, this paper contributes to knowledge of the relationship between compliance and quantitative descriptors, using grated parmigiano-reggiano cheese as an example. materials and methods samples the regulations for parmigiano-reggiano cheese state that the cheese can be grated only in a plant, located in the production area of the cheese, operating as prescribed by the regulations. moreover the producer has to be authorized and regulated by the control body ocqpr. the minimum age of the product is 12 months but a maximum age is not defined. twenty four samples of grated parmigianoreggiano, each from a different processor, were collected. four hundred grams for each sample were collected in the production plant in four 100g bags under modified atmosphere. sample preparation each of 24 samples was divided in two parts (sub-samples of two 100g bags) for sensory analysis; the products were identified with letters a – x. thus 48 sub-samples were evaluated by the panel. in each tasting session four sub-samples were evaluated. order of presentation for each assessor was defined by sets of 4x4 latin squares. samples were refrigerated to between 2 and 8°c and their temperature was raised to 13°c temperature during the evening before tasting. the samples were served at room temperature raising the tasting temperature of the samples to approximately 16°c. each panellist was served with 20 g sub-sub-samples on a plastic petri dish. scorecard for evaluating the sensory compliance of pdo fig. 1 determinants of grated parmigiano-reggiano quality. 490 ital. j. food sci., vol. 27 2015 parmigiano-reggiano grated cheese with the regulations, the designated control body organismo controllo qualità produzioni regolamentate (ocqpr), uses a “mixed” scorecard, with both descriptive and compliance scores (zannoni and hunter, 2013). the quantitative descriptive part of the scorecard has 21 descriptors; a 1-7 scale for 5 attributes (colour intensity, odour intensity, aroma/taste intensity, particle size and degree of solubility). for the 16 descriptors connected to defects a 1-4 scale is used because it is more acceptable to the panellists. in additable 1 correlations of each variable of each modality with the appropriate compliance variable. a) variables of colour modality correlated with compliance visual variable. variable mean corr prob colour intensity 3.19 0.04 ns brown 0.07 -0.44 0.014 lemon yellow 0.09 0.19 ns other colours 0.08 -0.45 0.013 b) variables of appearance modality correlated with compliance visual variable. variable mean corr prob particles size 3.29 -0.05 ns large grains 0.31 -0.84 <.001 long threads 0.49 -0.47 0.009 c) variables of odour modality correlated with compliance odour variable. variable mean corr prob odour intensity 3.56 0.06 ns rancid 0.53 -0.57 0.001 rind 0.76 -0.39 0.028 sour 0.23 -0.16 ns d) variables of texture modality correlated with compliance texture variable. variable mean corr prob degree of solubility 3.90 0.92 <.001 dryness 0.83 -0.81 <.001 rind particles 0.62 -0.72 <.001 sandy 0.63 -0.61 0.001 e) variables of aroma/taste modality correlated with compliance aroma/taste variable. variable mean corr prob aroma/taste intensity 4.31 0.37 0.036 salty 0.60 0.11 ns pungent 0.77 -0.04 ns sour 0.70 -0.07 ns rancid 0.89 -0.63 <.001 rind 1.11 -0.66 <.001 tion the scorecard has an additional 4 qualitative evaluations of compliance for visual, odour, texture and aroma (“back of the nose” odour)/ taste using a 1-7 scale with 1 minimum and 7 maximum score. panel the nine panellist were aged between 34 and 69 years and had from 5 to 18 years experience of sensory analysis of parmigiano-reggiano cheese. statistical data analysis the univariate (one variable at a time) analysis of this data is fully described in zannoni and hunter (2013). the starting point for the analysis described in this paper is the 24 sample means from this analysis. the scorecard presents 4 modalities: visual (colour plus appearance), odour, texture and aroma/taste with a compliance score. for each of these modalities the individual variables have been correlated with the relevant compliance score (table 1a-e). note that for colour (but not appearance), odour, texture and aroma/taste there is an intensity measurement followed by some descriptors related to defects. the next step in the analysis is to “predict” each of the four compliance scores from all the other sensory data. for each of the four compliance scores there are twenty one possible explanatory factors and yet only twenty four sample values. in such circumstances multiple regression analysis is known to be problematic. one solution to this problem is to use principal components regression (pcr). first the explanatory data is summarised by the principal components scores on the much smaller number of principal dimensions (typically 2, 3 or 4), which summarise the data, and these scores are then used in the regression instead of the initial data. the multiplier for each variable of the initial data can be obtained using the regression coefficients plus the loadings of the initial data on the relevant dimension. however, we have chosen to use partial least squares (pls) regression, which has many similarities with pcr. pls (martens and naes, 1989) is an iterative technique and has been found to produce more effective prediction equations in most circumstances. the number of (pls) dimensions (1, 2, 3....) was determined by the computationally intensive technique of cross-validation. a value of predictive residual error sum of squares (press) was calculated by taking each unit of data in turn and forming a prediction equation from the remaining units of data using 1, 2, .... dimensions. ostens (ostens, 1988) test of significance was used to judge how many pls diital. j. food sci., vol. 27 2015 491 table 2a predicting each of the compliance variables in turn from the total sensory data. compliance variable visual odour texture aroma/taste mean 4.96 4.55 4.68 4.38 no of pls dimensions 2 1 3 2 % variance accounted for 76.7 28.4 91.4 73.6 press predictive error sum of square 2.858 2.125 1.708 2.229 standard error of prediction 0.345 0.298 0.267 0.305 table 2b predictive equations for the compliance variables from pls, the predicted compliance scores are calculated using the equation: predicted score = const + coeff1 * colour intensity (colour) + ……+ coeff n* descriptor n …+ coeff 21* rind (aroma taste) (n = 1….21). visual odour texture ar./taste n const %y const %y const %y const %y 4.58 76.7 4.42 28.4 3.58 91.4 3.61 73.6 colour coeff %x coeff %x coeff %x coeff %x 1 colour intensity 0.021 0.9 0.012 1.3 -0.02 50.3 0.029 1.9 2 brown -0.024 15.4 -0.005 16.3 -0.022 22.3 -0.017 14.2 3 lemon yellow 0.019 1.2 -0.002 0.0 -0.011 7.6 -0.014 8.1 4 other colours -0.013 11.5 -0.001 6.4 -0.002 19.4 -0.004 5.9 appearance 5 particles size -0.066 10.3 0.013 14.7 -0.052 82.2 0.032 15.0 6 large grains -0.401 67.3 -0.025 20.8 -0.229 70.4 -0.117 21.6 7 long threads -0.213 44.0 -0.026 29.0 -0.134 37.1 -0.137 37.1 odour 8 odour intensity -0.010 5.3 0.002 1.4 -0.037 21.0 -0.014 1.7 9 rancid -0.044 14.6 -0.028 21.4 -0.055 18.0 -0.121 20.6 10 rind -0.142 51.9 -0.024 64.6 -0.171 60.3 -0.106 62.5 11 sour -0.039 0.4 -0.01 0.6 -0.030 10.7 -0.005 0.8 texture 12 solubility 0.184 53.2 0.046 61.2 0.391 84.3 0.226 76.6 13 dryness -0.101 43.0 -0.021 56.3 -0.248 83.2 -0.116 68.9 14 rind particles 0.016 93.6 -0.035 56.7 0.004 99.6 0.010 99.1 15 sandy 0.059 28.0 -0.017 45.8 -0.117 55.7 -0.096 50.9 aroma_taste 16 aroma/taste intensity 0.059 9.7 0.013 6.5 0.114 26.6 0.058 15.6 17 salty 0.021 5.6 0.002 0.6 0.047 24.5 0.019 2.0 18 pungent -0.002 3.2 -0.008 0.5 0.057 9.0 -0.006 2.9 19 sour -0.001 1.3 -0.008 0.3 0.037 3.5 -0.012 0.3 20 rancid -0.038 23.6 -0.032 35.4 -0.092 25.5 -0.146 35.0 21 rind 0.016 93.6 -0.035 56.7 0.004 99.6 0.010 99.1 mensions (table 2a) were required. once the number of dimensions were determined the predictive equations were found (table 2b). the genstat (vsn international) statistical package was used. results in previous work (zannoni and hunter, 2013) the scorecard was evaluated by fitting a mixed model to each attribute and by using generalised procrustes analysis for each modality. the results showed good discrimination between samples and good agreement between assessors. correlations the correlations of descriptors scores for each sample with the corresponding compliance score e.g. odour intensity, rancid, rind, sour correlated with odour compliance. table 1 shows that 13 out of the 21 descriptors 492 ital. j. food sci., vol. 27 2015 were significantly correlated with the relevant compliance variable. the only significant positive correlation is that of solubility. negative correlations were found, in descriptors (with a 4-points scale) considered defects: large grains, long threads, rancid odour, rind odour, dry, rind particles, sandy, rancid aroma, rind aroma. it is interesting to note that some of these descriptors large grains, long threads, rind particles, rind aroma are influenced by processing conditions. large grains and long threads are determined by the grating conditions i.e. type of grating surface, pressure applied. rind particles and rind aroma are determined by the percent of rind in the cheese used for grating. partial least squares (pls) pls has been used to predict each of the compliance variables in turn from all the sensory data. the percent variance explained for the pls correlations showed very good results for texture 91.4% (with 3 dimensions) and for visual and aroma/taste (76.7 and 73.6 % respectively, both with 2 dimensions). the results for texture probably occurred because this modality is very strongly positively correlated with solubility and strongly negatively correlated with dryness, rind particles and sandy. the rather disappointing results for odour (28.4% of variance explained) showed the difficulties the panel had with this modality. fig. 2 a,b,c,d : pls: relationship between actual and predicted compliance data for each modality (visual, odour, texture, aroma). letters represents the samples. a) b) c) d) ital. j. food sci., vol. 27 2015 493 the equations for predicting each of the compliance scores from all the individual descriptors allows the importance of every descriptor to be better judged. correlations among the descriptors not directly related to a compliance score is logical because when we run a pca with all the data the 4 compliances show their relationships being grouped together in an area opposed to that of descriptors considered defects which are grouped together even though they belong to different modalities. this makes sense because in cheese the presence of one defect always involves other defects. for instance if we find rind odour this is an odour defect but also a texture one (i.e. low solubility) and of aroma/taste (rind aroma and often rancid). in predicting the visual compliance the most important predictors are rind aroma and the presence of rind particles in texture. both are related to large grains which is the visual descriptor correlated with the presence of rind in the grated cheese. colour is of minimal importance in this prediction. the most important predictor of odour compliance by far was rind odour followed by other rind descriptors in other modalities. in case of texture compliance we have the contribution of an important very large positive correlation (solubility) and three substantial negative correlations (dryness, rind particles, sandy) to the prediction. the aroma/taste compliance is predicted mostly by the descriptor rind in aroma/taste, texture and odour. this value seems to depend slightly more than the others on the quantity of cheese rind present in the samples. the fig. 2 shows relationships between actual and predicted compliance. it is evident that three of the four modalities are closely predicted. conclusions in evaluating compliance of a pdo/pgi, it is clear that compliance cannot be separated from the descriptors. it is also true that compliance could be the results of the interaction of many sensory perceptions, not all of which are present in the scorecard. nevertheless the scorecard has been refined by long experience in order to find the most important perceptions connected to the definition of the desired quality of the product. pls has allowed a better understanding of compliance evaluation of grated parmigiano-reggiano cheese and has confirmed the usefulness of the scorecard for official control. three important points come out from this work: the most important descriptors in relationship with compliance were found to be concerned with the presence of rind in samples. the regulations allow a maximum of 18 % of rind by weight in grated cheese. the sensory control has been shown to be effective in finding rind in the product. an increase in the amount of rind causes a decrease in the sensory quality with clear disadvantages for consumers. it is possible to satisfactorily predict compliance values using descriptor values with the exception of odour. the compliances results expressed in a numeric manner are a practical way to show the sensory quality of this pdo cheese thus allowing its employment by the official certification body. another important finding of this work is the importance of descriptor solubility (“positive” descriptor) in the quality assessment of a product that is normally spread over warm pasta. negative descriptors were, mainly descriptors connected to an excess of rind: large grains, rind odour, presence of rind particles and rind aroma. extra cheese ring is readily available in plants for vacuum packed sliced parmigiano-reggiano because the ring flat parts of the wheel are removed before slicing the wheels into 200 or 300 g pieces. this work showed that one of the most important quality problem influencing the compliance score was the addition of extra quantities of cheese ring not belonging to the original wheels. references bodyfelt f.w., tobias j. and trout g.m. 1988. the sensory evaluation of dairy products, avi book, new york london melbourne agincourt. comitè interprofessionnel du fromages du cantal, plan d’inspection aoc cantal. 2011. http://www.aop-cantal. com/sites/all/files/pi%20aoc%20cantal%20+%20gt%20 24-10-11_0.pdf. accessed 15 march 2013. comitè interprofessionnel des fromages du cantal, plan d’inspection aoc salers. 2011. http://www.aop-salers.com/ sites/www.aop-salers.com/files/fichier/pi%20salers%20 +%20gt%2024-10-11.pdf. accessed 15 march 2013. european commission, agriculture and rural development, door. http.//ec.europa.eu/agriculture/quality/door/ list/html. accessed 12 march 2013. galwey n.w. 2006. introduction to mixed modelling: beyond regression and analysis of variance. john wiley & sons, chichester, uk. garavaldi a., zannoni m., giussani b., roncoroni s., galassi l. and turrini m. 2010. scheda sensoriale per il parmigiano-reggiano: scelta dei descrittori e messa a punto del profilo, scienza e tecnica lattiero-casearia, 61, 367-379. inao, directive du conseil des agréments et contrôle inaodir-2008 – 02, http://www.inao.gouv.fr/repository/editeur/pdf/directives/inao-dir2008-02.pdf. accessed 15 march 2013. international standard organisation iso 22935-3/idf 99-3 (2009) milk and milk products – sensory analysis – part 3: guidance on a method for evaluation of compliance 494 ital. j. food sci., vol. 27 2015 with product specifications for sensory properties by scoring, iso and idf. kraggerud h., solem s. and abrahmsen r.k. 2012. quality scoring. a tool for sensory evaluation of cheese?, food quality and preference, 26:221-230. martens h. and naes t. 1989. multivariate calibration. john wiley, chichester, uk. osten d.w. 1988. selection of optimal regression models via cross-validation. j. chemometrics, 2, 39-48. official journal of the european union 2012, 55, 14 december 2012, 1-29. pérez-elortondo f.j., ojeda m., albisu m., salmeròn j., etayo i. and molina m. 2007. food quality certification: an approach for development of accredited sensory evaluation methods. food quality and preference, 18: 425. syndicat du picodon aop, plan de contrôle aoc picodon. 2008. http://www.picodon-aop.fr/images/contenu/syndicat/ picodon_plancontrole2008.pdf accessed 15 march 2013. syndicat interprofessionnel de la fourme d’ambert. 2008. http://www.fourme-ambert.com/fromage-aop/qualite-aopgarantie/controle-qualite-de-la-fourme-d-ambert/les-criteres-de-degustation accessed 15 march 2013. syndicat interprofessionnel du fromage d’abondance, plan de contrôle aoc abondance (2010) http://www.fromageabondance.fr/pdf/plan-controle.pdf accessed 15 march 2013. zannoni m .(2007) l’analisi sensoriale nel controllo del formaggio parmigiano-reggiano dop, in: l’analisi sensoriale nel controllo dei prodotti a denominazione d’origine, reggio emilia, 6 novembre 2007, dipartimento controllo qualità p.r., reggio emilia, italy. zannoni m. and hunter e.a. 2013. evaluation of a sensory scorecard for grated parmigiano-reggiano cheese, italian journal of food science, 25, 23-34. zannoni m. and marangon a. 2011. l’introduzione dell’analisi sensoriale nel comparto dell’asiago. in: cavella s. and di monaco r. (eds.), terzo convegno della società italiana di scienze sensoriali, portici (na), 1-2 dicembre 2011, cues, salerno, italy. paper received january 20, 2015 accepted march 28, 2015 paper ital. j. food sci., vol. 27 2015 521 keywords: cheese preference, children food preference, colour preference, gender, neophobia, nutrition education children preferences of coloured fresh cheese prepared during an educational laboratory f. tesini*1, m. laureati2, r. palagano1, m. mandrioli1, e. pagliarini2 and t. gallina toschi1,3 1distal, department of agricultural and food sciences, alma mater studiorum university of bologna, p.zza goidanich 60, 47521 cesena, fc, italy 2department of food, environmental and nutritional sciences (defens), university of milan, via celoria 2, 20133milan, italy 3ciri, department of agricultural and food sciences, interdepartmental centre for industrial research in agri-food, alma mater studiorum university of bologna, p.zza goidanich 60, 47521 cesena, fc, italy *corresponding author: federica.tesini@unibo.it abstract choices among young consumers are mainly driven by food preferences; in particular, a connection between appearance and acceptance of food has been highlighted, together with a general lack of knowledge of food processing. for these reasons, educational activities are important to increase scientific knowledge and awareness. the cheese-making educational laboratory described herein involved children, adolescents, and their parents/teachers in the preparation of fresh and naturally-coloured cheeses. at the end of the activity, both the colour preference and possible relation between preference and colour of cheese prepared were investigated administering a short questionnaire. 522 ital. j. food sci., vol. 27 2015 introduction food preference has a fundamental role in driving consumer’s choices and habits, especially in children (cooke, 2007; nicklaus et al., 2004, laureati et al., 2015a). therefore, the number of food products specifically developed for children is growing and this market is acquiring increasing importance (issanchou, 2015). consequently, the youngest consumers are frequently involved in research and development programs, since their food habits will influence choices as they grow older and because their preferences, even if partly driven by advertisements (ustjanauskas et al., 2014), seem to be strictly related to the sensory aspects of food (pagliarini et al., 2005; mustonen and tuorila, 2010). in 1994, moskowitz (moskowits, 1994) highlighted how a visually pleasing product tends to be more appreciated by children, as visual attributes seem to be, among different sensory characteristics, those that mainly determine its success (kildegaard et al., 2011; topcu, 2015). indeed, children tend to create an “ideal picture” of each food product that can be related to their own idea of “good”; this picture represent a sort of reference point that can be used to dislike products whose appearance is not close to their expectation (mustonen and tuorila, 2009). this phenomenon, called food neophobia, is defined as the fear of eating new or unfamiliar food (pliner and hobden 1992) and is related to both the quality and variety of diet (laureati et al., 2015b). nutritional education and food-related diseases prevention activities can be used for children and teenagers in order to: i) reduce the impact of poor food habits on health, and ii) avoid food neophobia, mainly responsible for refusing consumption of fruits and vegetables (wardle et al., 2003). moreover, a lack of awareness of procedural knowledge, both in terms of food processing and nutritional values, must be taken into account (worsley, 2002), highlighting the link between traditional food preparation and sensory properties (laureati et al., 2006). nowadays, food education is mainly based on the social-cognitive theory (sct) (bandura and adams 1977), which incorporates the interaction of personal, environmental and behavioural factors. according to this theory, principles that are highly influential in establishing changing food behaviour in children are both models and repeated exposure. among the models, those that have been found to be effective in children include cartoon characters, peers, mothers, unfamiliar adults and teachers; moreover, children seem to be influenced more by the behaviour of multiple rather than single models (lowe et al., 2004). furthermore, according to zajonc’s “mere exposure” theory (zajonc, 1968), repeated exposure to a specific food increases the liking and consumption of that food (cooke et al., 2011; wardle et al., 2003b) through a mechanism that is believed to be a “learned safety” behaviour (kalat and rozin, 1973). according to this theory, repeated ingestion of an unfamiliar food without negative consequences leads to increased acceptance of that food. several educational interventions for food have investigated all these factors, reporting promising results, especially for consumption of fruits and vegetables (evans et al., 2012; laureati et al., 2014; reverdy et al., 2008). both principles of imitation and repeated exposure characterise food laboratories aimed at involving children in food preparation, thus familiarising them with food ingredients and technologies. furthermore, it has been reported that the involvement of children in food preparation can contribute to increasing their acceptance for that food, according to the principle of repeated exposure (chu et al., 2012). for all these reasons, food educational interventions can play a relevant role in driving choice, interest and preference of children through better awareness of both sensory characteristics and technological process of traditional food products (mustonen and tuorila, 2010). the educational cheese-making activity reported herein, called cheese making in one hour, was conceived by a committee of referees as part of the festival della scienza 2014 (which took place in genoa, italy, from october 24 to november 2, 2014) and is considered as a “teaching tool” in order to increase scientific information about the cheese-making process. this practical laboratory took the form of a family entertainment during which children and parents or teachers could share principles of traditional cheese making, spending some family or educational time away from daily work, but related to increased knowledge of food science. the activity involved children, teenagers and adults (age from 6 to 87 years) in the traditional preparation of a fresh “primo sale like” cheese. cheeses were naturally coloured (using turmeric, rocket and beetroot juice), and in order to keep the attention of participants, each was actively involved in the process by preparing his/ her personal small cheese. at the end of the activity, both the colour preference and possible relation between the colour of cheese prepared and preference were investigated by administration of a short and simple questionnaire. materials and methods the cheese-making activity involved 738 participants (395 females and 343 males) with an age between 5 and 87 years and divided in groups of no more than 30 individuals. both families and school classes could participate in the activity by asking the festival staff to make ital. j. food sci., vol. 27 2015 523 fig. 1 children at the cheesemaking educational laboratory, during collection of a yellow-curd into a dedicated shaper. table 1 natural dyes and their amounts are reported together with the final colour of each cheese and its visual aspect. cheese colour dye amount and appearance (g/l of milk) yellow turmeric 5 pink beetroot juice 20 white and green rocket 20 a reservation; given the popularity of the festival, mainly represented by children and partly by parents/teachers, a large number of participants were enrolled. each participant had an active role in the laboratory, giving his/her contribution to one of the 4 phases of cheese making: 1) adding starter, rennet and natural flavour to milk; 2) curd cutting and synaeresis; 3) curd breaking and purge; 4) cheese shaping. during the festival, the laboratory was repeated at least 3 times a day by 4 previously-trained scientific facilitators. due to the fact that the cheese-making laboratory should last only one hour (related to the timing of the festival), one pot (bartscher gmbh, germany) was dedicated to each of the 4 main phases; in this way, participants could move from one pot to the next instead of proceeding step-by-step using a single pot such that each of the phases could be observed in a short length of time. cheese-making phases 1) starter, rennet and milk natural flavouring fresh whole cow milk was heated to 36° c and 17 g/l of natural whole yoghurt was added as a starter. in order to produce coloured cheeses, different natural dyes were added (rocket, turmeric and beetroot juice) as reported in table 1. while turmeric and beetroot juice were added to the milk 30 minutes after the addition of starter, together with 0.5 ml/l milk of liquid rennet (80% chymosin, 20% pepsin, provided by graco), rocket was added during the last step of cheese shaping. 2) curd cutting and synaeresis after enzymatic coagulation, due to the addition of liquid curd, the gel obtained was cut into 4 × 4 cm squares, and 30 minutes later participants could observe the phenomena of curd synaeresis. 3) curd breaking and purge the squares of curd were broken into pieces of approximately 1.5 cm; during this phase, rocket was added (20 g/l milk) for preparation of green cheese. 4) cheese shaping finally, the small pieces of curd were collected in dedicated shaper (60 g cheese shaper in polypropylene and polyethylene, tecnolatte srl, italy) by each participant as shown in fig. 1. 524 ital. j. food sci., vol. 27 2015 5) questionnaire at the end of the cheese-making laboratory, each participant was administered a dedicated and simplified questionnaire. the questionnaire contained a first part regarding personal information: age and gender. next, each participant was asked to provide information on: i) which colour of cheese he/she preferred and ii) which one he/she prepared. the filled questionnaires were collected in three dedicated paper boxes, one for each cheese colour. statistical analysis preference data were analysed according to chi square test (p>0.05). statistical analysis of data was carried out using the sas/stat statistical software package version 9.3.1. (sas institute inc., cary, usa). results the cheese-making laboratory was visited by 1900 participants. many questionnaires were incomplete, while some participants preferred to not give their opinion. data from 738 participants was collected considering the gender dimension and 5 age subgroups (1: 5-7 years old, 77 participants; 2: 8-10 years old, 142 participants; 3: 11-13 years old, 182 participants; 4: 14-18 years old, 203 participants; 5: >18 years old, 134 participants); subsequently, the visual preference for one of the three coloured cheeses was considered, as reported in table 2. data were divided in the 5 subgroups reported above, selected mainly according to different stages of cognitive development (guinard, 2001). in addition, different food preferences and consumption behaviour observed in these age groups confirmed that this type of age subdivision is relevant (laureati et al., 2015). considering colour preference, regardless of age, the majority of participants preferred the white and green cheese (333 vs. 243 yellow and 162 pink; chi square=59.49; p<0.0001), which could be considered as the most “traditional” because it is present on the italian market, rather than pink or yellow cheeses, and probably seen for the first time during the laboratory activity by the most of the respondents. as reported in table 2, sorting by age, it could be seen that the youngest participants (between 5 and 7 years old) chose the pink cheese as their favourite, while next oldest age subgroup (8 to 10 years) preferred yellow. subjects from 11 years old and older chose the white and green cheese as their favourite, as did the overall population. this result may be associated with both the attractive effect of colours on the children and to the tendency of the youngest, as observed by moskowitz (moskowitz, 1994), to prefer food products with a pleasing appearance. starting from the age of 11, children started to prefer, in accordance with adults, the white cheese mixed with rocket. moreover, the youngest children’s preference can be explained by food neophobia, which is particularly high for fruits and vegetables, reaching its highest level between 2 and 6 years (pelchat and pliner, 1995; pliner, 1994; pliner and loewen, 1997; laureati et al., 2015c). it then tends to decrease when children move towards adolescence (addessi et al., 2005), finally becoming relatively stable in adulthood, probably because of an increased number of food experiences (cooke and wardle, 2005). on the other hand, the preference of older respondents for a white cheese enriched with a vegetable (rocket), may be linked to a low acceptance of adults for an innovative food, while its dislike among the younger interviewees could be related to the visual presence of rocket inside cheese (russel and worsley, 2008; laureati et al., 2015). it has already been demonstrated that traditional food products have been always recognised by consumers as linked to a specific geographical origin and highest sensory quality (guerrero et al., 2009); as a consequence, this cheese was probably considered much more similar to product already on the italian market compared to the pink and yellow ones. table 2 non-parametric statistical analysis of data to identify significant differences in preference of cheese colour by age. data were analysed using a chi square test. interviewees were subgrouped by age: 5-7, 8-10, 11-13, 14-18, >18 years. preferred cheese age subgroup (years) yellow pink white and green total chi square df p value 5-7 27 35 15 77 7.9 2 0.02 8-10 69 28 45 142 17.9 2 0.00 11−13 59 34 89 182 25.0 2 0.00 14-18 57 44 102 65 27.4 2 0.00 >18 31 21 82 134 47.9 2 0.00 all 243 162 333 738 59.5 2 0.00 ital. j. food sci., vol. 27 2015 525 sorting by gender (table 3), no differences in preferences were seen regarding the white and green cheese, except for the oldest group (>18 years). a clear gender-related difference was, however, found for the pink and the yellow cheeses: the former seemed to be much more appreciated by females (all age classes except 14−18 years, but only if p<0.10 is considered as marginally significant), while the latter was favoured among males (especially from 8-10, 14-18). in order to determine if this could influence the visual preference of participants, they were also questioned about the colour of the cheese they prepared; however, a significant association between cheese preferred and colour preference table 3 non-parametric statistical analysis of data to identify significant differences in preference of cheese colour by gender and age. data were analysed using a chi square test. f= female; m=male; df=degrees of freedom. interviewees were subgrouped by age: 5-7, 8-10, 11-13, 14-18, >18 years. prefered cheese gender age subgroup (years) 5-7 8-10 11-13 14-18 >18 total yellow f 10 24 31 18 25 108 m 17 45 28 39 6 135 total 27 69 59 57 31 243 chi square 1.8 6.4 0.2 7.7 11.6 3.0 df 1 1 1 1 1 1 pvalue 0.2 0.0 0.7 0.0 0.0 0.1 pink f 29 23 22 23 18 115 m 6 5 12 21 3 47 total 35 28 34 44 21 162 chi square 15.1 11.6 2.9 0.1 10.7 28.5 df 1 1 1 1 1 1 p value 0.0 0.0 0.1 0.8 0.0 0.0 white and green f 4 20 47 47 54 172 m 11 25 42 55 28 161 total 15 45 89 102 82 333 chi square 3.3 0.6 0.3 0.6 8.2 0.4 df 1 1 1 1 1 1 p value 0.1 0.5 0.6 0.4 0.0 0.5 for one of the three coloured cheeses was not seen, even if some significant differences were seen among the youngest consumers (table 4). in particular, when sorting by age, the subgroup from 8 to 10 years who prepared the yellow cheese more frequently preferred this cheese over the others (26 of 52, chi square=7.5 p=0.02). likewise, the subgroup from 5 to 7 years who prepared the pink cheese preferred it more frequently than others (12 of 21 choices, chi square=7.1 p=0.03). however, the older participants did not show a consistent trend of preferred cheese over than prepared. data from literature suggest that 7−8 exposures are needed to produce a learning effect table 4 non-parametric statistical analysis of data to assess if the colour of sample prepared could affect the overall colour preference. data were analysed using chi square test. f= female; m=male; df=degrees of freedom. interviewees were subgrouped by age: 5-7, 8-10, 11-13, 14-18, >18 years. prepared chesee age subgroup preferred cheese (years) yellow pink white and green total chi square df p value yellow 5-7 15 14 8 37 2.3 2 0.31 8-10 26 10 16 52 7.5 2 0.02 11-13 21 9 37 67 17.7 2 0.00 14-18 18 17 30 65 4.8 2 0.09 >18 14 9 39 62 25 2 0.00 pink 5-7 7 12 2 21 7.1 2 0.03 8-10 19 10 9 38 4.8 2 0.09 11-13 9 12 18 39 3.2 2 0.20 14-18 22 16 37 75 9.4 2 0.01 >18 6 6 19 31 10.9 2 0.00 white and green 5-7 5 9 5 19 1.7 2 0.43 8-10 24 8 20 52 8 2 0.02 11-13 29 13 34 76 9.5 2 0.01 14-18 17 11 35 63 14.9 2 0.00 >18 11 6 24 41 12.6 2 0.00 526 ital. j. food sci., vol. 27 2015 that can influence consumer appreciation regarding a product (maier et al., 2007). thus, it might well be that the single exposure during the cheese-making educational activity was not enough to influence children choice. conclusions the results of this study highlight how visual preference for a food, in terms of colour, changes during different stages of life. indeed, the data demonstrates how children can be influenced by food appearance and how the aspect of a product can be related to its acceptance, especially among younger individuals; in fact, the youngest participants tended to prefer intensively coloured cheese vs. the white and green version, probably because of the presence of rocket. moreover, the cheese-making laboratory was considered to be a useful tool in order to catch and keep participants’ attention, involving them in a practical activity while sharing educational and scientific information related both to food processing (fresh cheese production) and sensory (visual) aspects. aknowledgements the activity described in this work was made possible by the kind permission of 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lactic acid bacteria, xi-gua-mian, fermented watermelon, taiwan isolation and characterization of lactic acid bacteria from xi-gua-mian (fermented watermelon), a traditional fermented food in taiwan yi-sheng chena,*, hui-chung wua, chi-rong yua, zih-yin chena, yi-chen lua and fujitoshi yanagidab adepartment of biotechnology, ming chuan university, no. 5 de-ming road, gui-shan, taoyuan 333, taiwan b the institute of enology and viticulture, yamanashi university, 1-13-1 kitashin, kofu, yamanashi 400-0005, japan *corresponding author: tel. +886 33507001 ext. 3540, fax +886 33593878, email: yisheng@mail.mcu.edu.tw abstract young watermelon fruit was peeled and pickled for fermentation to produce a unique fermented food named xi-gua-mian (fermented watermelon) in taiwan. in this study, we investigated the lab microflora in xi-gua-mian. a total of 176 lab isolates were identified; 118 cultures were isolated from the xi-gua-mian sample collected from three different farmers markets and 58 from six young watermelon fruit samples. these isolates were characterized phenotypically and then divided into seven groups (a to g) by restriction fragment length polymorphism analysis, sequencing of 16s ribosomal dna and other genotypic analysis. lactobacillus plantarum was the most abundant lab found in xi-gua-mian samples collected in southern taiwan, tainan city and pediococcus pentosaceus was the most abundant lab in northern taiwan, taoyuan county. we found that lab stains are similar in samples collected in the same geographic region but significant variations were observed between samples collected among different regions. on the other hand, a greater lab diversity was observed in the young watermelon fruit samples. in addition, 10 lactococcus lactis subsp. lactis showed inhibitory activity against the indicator strain l. sakei subsp. sakei jcm 1157t. this is the first report describing the distribution and varieties of lab existing in the xi-gua-mian and the young watermelon fruits. mailto:yisheng@mail.mcu.edu.tw 10 ital. j. food sci., vol. 28 2016 introduction watermelon (citrullus lanatus) is a popular fruit in taiwan. the farming area dedicated to watermelon production in taiwan is reported to be largest among all fruits (lin et al., 2009). to have a better harvest, surplus fruits are eliminated and only one fruit is retained for every stock in the early phase of watermelon cultivation. in taiwan, farmers use the eliminated young watermelon fruits to produce a unique fermented food named xi-gua-mian (fermented watermelon). these immature watermelon fruits are peeled, cut, mixed with salt (nacl) and then placed in a bucket. salt is added to a final concentration of approximately 3-6%, and the bucket is sealed with heavy stones placed on the top of the cover. this process usually continues for 3 days and then the exuded water is drained. the bucket is sealed again with heavy stones and the fermentation process continues for at least 2 weeks at room temperature. because of the contribution of the lactic acid bacteria (lab), it has a special sour and sweet flavor. xi-gua-mian is usually applied as a seasoning for various pork, seafood and poultry dishes in order to add a slightly acidic taste. although the product is very popular, it has not been studied in detail. lactic acid bacteria (lab) has been frequently found in various taiwanese fermented foods such as yan-tsai-shin (fermented broccoli stems), yan-jiang (fermented ginger), jiang-sun (fermented bamboo shoot), suan-tsai (fermented mustard), dochi (fermented black beans), jiang-gua (fermented cucumbers), yan-dong-gua (fermented wax gourd) and pobuzihi (fermented cummingcordia) (chang et al., 2011; chen et al., 2006a, 2006b, 2010, 2012, 2013a, 2013b; lan et al., 2009). in these cited studies, various lab species, such as enterococcus faecium, lactobacillus plantarum, lactococcus lactis subsp. lactis, weissella cibaria and w. paramesenteroides, were frequently found in the taiwanese fermented products. however, there has been very little research reported on lab distribution in fermented watermelon (xi-gua-mian). one important attribute of lab is the bacteriocin-producing abilities to inhibit food spoilage bacteria and many lab strains isolated from the taiwanese fermented foods were found to produce various bacteriocins. some bacteriocins produced by these strains were further identified as novel bacteriocins in the later studies such as enterocin tw21, weissellicin l and enterocin t (chang et al., 2013; chen et al., 2013c; liang et al., 2013). the objectives of this study were to isolate lab from the xi-gua-mian, to identify the isolates to the species level and to detect the antibacteri16s rdna rflp groups a b c d e f g sample no. location ph salt con. (g/l) viable acid-producing cells (log cfu/ml)α lactic acid (g/l) l. p la nt ar um l. p en to su s p. p en to sa ce us lc . l ac tis s ub sp . l ac tis le u. m es en te ro id es w . p ar am es en te ro id es e. c as sl ifl av us fermented watermelon s1 tainan 4.6 3.8 7.36±0.18 35.5 30 8 s2 tainan 3.9 3.8 6.77±0.17 95.0 36 4 s3 taoyuan 4.1 6.0 8.00±0.05 80.0 40 fresh watermelon w1 hualien – – 1.84±0.09 – 7 (1β) 1 2 w2 hualien – – 3.77±0.01 – 2 1 7 (7) 2 1 w3 hualien – – 3.25±0.03 – 3 1 3 (2) 1 2 w4 tainan – – 3.04±0.06 – 10 w5 tainan – – 3.51±0.08 – 12 w6 chiayi – – 1.48±0.03 – 3 total 71 14 40 20 4 3 24 α the data are expressed as the mean±sd (n=3). β number of blis-producing strains. abbreviations: l., lactobacillus; p., pediococcus; lc., lactococcus; leu., leuconostoc; w., weissella; e., enterococcus. table 1 analysis results and characteristics of isolates. ital. j. food sci., vol. 28 2016 11 al activities of the isolates. our results provide an example to understand the rich resources of lab strains in the traditional taiwanese fermented food. materials and methods xi-gua-mian and the young watermelon fruit samples a total of 3 xi-gua-mian samples (s1-s3) were collected at three traditional farmers markets located in tainan city and taoyuan county (table 1, fig. 1a). in addition, six young watermelon fruit samples (w1-w6, approximately 8-15 cm in size) were collected from hualien county, tainan city and chiayi county (table 1, fig. 1b). samples were stored at 4°c and analyzed within 24 h of acquisition from the markets and the watermelon fields. the salt concentration and ph of xi-gua-mian juice was measured by using a model sk-5s salt meter (sato keiryoki, tokyo, japan) and a model b-112 compact ph meter (horiba, kyoto, japan), respectively. lactic acid in each xi-gua-mian samples was determined with a d-/l-lactic acid test kit (r-biopharm ag, darmstadt, germany), according to the manufacturer’s instructions. isolation of lab an initial analysis results showed that the xigua-mian samples s1 and s2 contained 3.8 % nacl and sample s3 contained 6 % (table 1). therefore, mrs agar (difcotm lactobacilli mrs broth; sparks, md, usa) containing 3 % nacl was used for the isolation of lab from xi-gua-mian samples s1 and s2. on the other hand, mrs agar containing 6 % nacl was used for isolation from sample s3 and mrs agar without adding nacl was used for isolation from young watermelon fruit samples. to distinguish acid-producing bacteria from other bacteria, 1% caco 3 was added to the mrs agar, and only colonies with a clear zone around them were selected (kozaki et al., 1992). 0.5 g of crushed young watermelon fruit samples, and 0.5-ml aliquot of each xigua-mian juice samples were taken for lab isolation. the isolation procedures of lab were performed according to the methods described by chen et al. (2013a). rflp and sequence analysis of 16s rdna rflp and sequence analysis of 16s rdna were used to classify and identify the bacterial isolates. a colony pcr method described by sheu et al. (2000) was performed in this study. pcr reactions were carried out using a genomics taq gene amplification pcr kit (genomics, taipei, taiwan) and performed on a gene amp pcr system 9700 (perkinelmer corp., boston, ma, usa) under the following conditions: 95°c for 3 min, 30 cycles of 95°c for 30 s, 60°c for 30 s and 72°c for 90 s, a final extension of 72°c for 10 min, and completion at 4°c (chen et al., 2013b). 16s rdna gene was amplified using the 16s rdna universal primers 27f (5’agagttt gatcctggctcag -3’) and 1492r (5’ggttaccttgttacgactt-3’) (chen et al., 2013b). rflp analysis of 16s rdna was also performed, as described by chen et al., (2013b). in this study, three restriction enzymes, accii (cg/cg), haeiii (gg/cc) and alui (ag/ct) (chen et al., 2013b), were mainly used for grouping. for sequence analysis of 16s rdna, the pcr products were purified and then sequenced with the following primer: 5´-gtcaattcctttgagttt-3´ (920r). sequence homologies were examined by comparing the obtained sequences with those in the dna data bank of japan (ddbj; http://www. ddbj.nig.ac.jp/) using blast. differentiation of lactobacillus plantarum, l. pentosus, and l. paraplantarum a multiplex pcr assay with reca gene-derived primers was performed using the methods and conditions described by torriani et al. (2001). fig. 1 pictures of (a) xi-gua-mian and (b) young watermelon fruit. http://www.ddbj.nig.ac.jp http://www.ddbj.nig.ac.jp 12 ital. j. food sci., vol. 28 2016 differentiation of leuconostoc mesenteroides and leu. pseudomesenteroides a rapid identification method described by jang et al. (2003) was used to distinguish leuconostoc mesenteroides and leu. pseudomesenteroides isolates. briefly, a pcr product of the isolate was amplified by using leuconostoc-specific primers and then digested by using the restriction enzyme tsp509i (/aatt) (jang et al., 2003). restriction fragments were visualized on a 2% agarose gel in 1× tae. differentiation of w. paramesenteroides and w. hellenica in this study, isolate which showed high sequence homology to w. paramesenteroides and w. hellenica was further confirmed by using the restriction enzyme hhai (gcg/c) described by chen et al. (2012). effect of nacl on growth of isolates effect of nacl on growth of isolates was assessed, as described by kozaki et al. (1992), by testing isolates for growth in mrs broth containing 0, 3 and 6% nacl. detection of antibacterial activity the agar well diffusion method as described by srionnual et al. (2007) was used to detect and determine the antibacterial activities of isolates. lactobacillus sakei subsp. sakei jcm 1157t was used as the indicator strain in this study. antibacterial activity was further confirmed by ph adjustment and proteinase k treatment (srionnual et al., 2007). to determine whether nisin is the antibacterial substance, a pcr assay with the nisin-specific pcr primers, nisl: 5’-cgagcataataaacggc-3’ and nisr: 5’-ggatagtatccatgtct gaac-3’, described by villani et al. (2001), were used for amplification in this study. in addition, a nisin z producing strain, lc. lactis subsp. lactis c101910 (yanagida et al., 2006) was used as the positive control and a non-blis (bacteriocin-like inhibitory substance) producing strain was used as the negative control. results in the xi-gua-mian samples collected from different markets, analyses of xi-gua-mian juice revealed different salt concentrations from 3.8 to 6.0% and lactic acid concentrations from 35.5 to 95.0 g/l (table 1). the average number of viable acid-producing cells was 7.36±0.18, 6.77±0.17 and 8.00±0.05 log cfu/ml from the xi-gua-mian samples s1, s2 and s3, respectively (table 1). the detailed analysis values of each sample are shown in table 1 and a total of 118 acid-producing bacteria were isolated from these samples. on the other hand, a total of 58 acid-producing bacteria were isolated from the young watermelon fruit samples. the number of viable acid producing cells on the six different young watermelon fruit samples was listed in table 1. the total 176 isolates were initially divided into six groups (r1-r6) according to cell morphology and the results of the 16s rdna rflp analysis. of these isolated strains, 85 were placed in group r1, 40 in group r2, 20 in group r3, 4 in group r4, 3 in group r5, and 24 in group r6, according to rflp patterns observed following digestion of their dna with accii, haeiii, and alui. to identify the isolates, representative strains were randomly selected from each group, and 16s rdna sequencing analysis was performed. the results identified group r1 isolates as lactobacillus plantarum-related species, group r2 as pediococcus pentosaceus, group r3 as lactococcus lactis subsp. lactis, group r4 as leuconostoc mesenteroides, group r5 as weissella paramesenteroides, and group r6 as enterococcus cassliflavus. the identification of group r1 isolates was further verified using a multiplex pcr assay with reca gene-derived primers (torriani et al., 2001). an expected amplification band located at 318 bp and one at 218 bp (fig. 2, lane 1 and 2) was respectively obtained from 71 and 14 isolates. seventy-one isolates were therefore identified as l. plantarum and re-classified into group a. the remaining 14 isolates were identified as l. pentosus and re-classified into group b. all 4 isolates in group r4 were confirmed as leu. mesenteroides based on tsp509i digested fragments of the pcr product of leuconostocspecific primers and re-classified into group e (jang et al., 2003) (fig. 2, lane 3; table 1). isolates in group r5 were further verified based on hha i digested fragments of their 16s pcr product (chen et al., 2012). all 3 strains were identified as w. paramesenteroides and re-classified into group f (fig. 2, lane 4; table 1). following the re-classification of groups r1, r4 and r5, isolates in the remaining groups were also reclassified with a new code. the detailed distributions of lab species are shown in table 1. effect of nacl on growth of all 176 isolates was estimated. all p. pentosaceus, e. cassliflavus, l. plantarum, l. pentosus, w. paramesenteroides and lc. lactis subsp. lactis isolates grew well in mrs broth containing 0, 3 and 6 % nacl except leu. mesenteroides isolates. growth of leu. mesenteroides isolates was observed neither in 3 nor 6 % nacl mrs broth. ten isolated lc. lactis subsp. lactis strains showed antibacterial activity against l. sakei subsp. sakei jcm 1157t (table 1). the blis produced by all 10 strains maintained their antibacterial activities after neutralization (ph 6.5) ital. j. food sci., vol. 28 2016 13 but lost their antibacterial activities completely after treatment with proteinase k. in addition, nisin-specific primers were used to amplify a pcr fragment and identify the blis from these 10 strains. an expected amplification band located at 320 bp (fig. 2, lane 5) was obtained from all lc. lactis subsp. lactis isolates and the nisin z producing strain, lc. lactis subsp. lactis c101910 (yanagida et al., 2006). no amplification band was observed from the negative control strain. discussion in this study, lab diversity in xi-gua-mian samples collected from different farmers markets and young watermelon fruits were studied. the final concentration of lactic acid and low ph values determined in the xi-gua-mian samples suggested that lab contributed to the aroma and flavor development in xi-gua-mian. the experimental data were treated according to critical values of student’s t-test. the viable acid-producing cell numbers between xigua-mian and fresh watermelon was significantly different (p<0.0002). we also found that the viable acid-producing cell numbers within geographical areas were different, but the statistical difference (standard deviation within xigua-mian group and fresh watermelon group was 0.61 and 0.93, respectively) was less than that between xi-gua-mian and fresh watermelon groups (4.56). in addition, halotolerance of all isolates were assessed. all isolated strains grew well in mrs broth containing 3% and 6 % nacl except leu. mesenteroides isolates. presence of nacl in xigua-mian and isolation medium therefore might limited the growth of leu. mesenteroides. presence of leu. mesenteroides was only observed in fresh watermelon fruits but not in xi-gua-mian. it is therefore considered that salt concentration has effect on diversity of lab in the xigua-mian. similar influence of nacl concentrations on diversity of lab in fermented foods has also been found in our previous studies (chen et al., 2006a; 2006b). compared to the isolation results of xi-guamian, fewer viable acid-producing cell number were observed from the young watermelon fruit samples. it is presumably because the raw material always presents a lower number or microorganisms or the absence of salt in substratum used for the isolation from young watermelon fruit samples does not allow the selection of lab. as in the case of xi-gua-mian samples, lab stains are similar in samples collected in the same geographic region and diversities were observed between samples collected among different regions in the young watermelon fruit samples (table 1). different climate conditions were considered as the main factor, which may affect the distribution of lab. although xi-gua-mian samples s1 and s2 were collected at different traditional farmers markets located in tainan city, l. plantarum and l. pentosus were the most abundant lab found in these two samples (table 1). different to the isolation results obtained in the tainan city, p. pentosaceus was the most abundant lab found in the sample collected in taoyuan county (table 1). geographically, tainan city is located in southern part of taiwan that belongs to the tropics, while taoyuan county is in northern subtropical regions. it is therefore considered that regional factors, such as climate conditions, raw materials for fermentation and fermentation methods, may affect the distribution of lab. lactobacillus plantarum has been identified elsewhere as one of the most abundant lab found in several taiwanese fermented vegetables such as fermented bamboo shoots (jiangsun), fermented cucumbers (jiang-gua), fermented broccoli stems (yan-tsai-shin) and fermented cummingcordia (pobuzihi) (chen et al., 2010b, 2012, 2013a, 2013b). as well as l. plantarum, p. pentosaceus also have been previously found fig. 2 16s rdna rflp patterns of accii, haeiii and alui digests from groups a to g. lane m, size marker; a, accii digested patterns; h, haeiii digested patterns; u, alui digested patterns; 1, amplification products obtained from the reca multiplex assay of l. plantarum isolates; 2, amplification products obtained from the reca multiplex assay of l. pentosus isolates; 3, tsp509i digested patterns of leuconostoc-specific pcr products from group e isolates; 4, hhai digested patterns of group f isolates; 5, pcr products using nisin-specific primers. 14 ital. j. food sci., vol. 28 2016 as the most abundant lab in the fermented mustard (suan-tsai) (chen et al., 2006a). in addition, l. plantarum was found both in the partial samples of xi-gua-mian and the young watermelon fruits. it is therefore considered that l. plantarum found in xi-gua-mian may originate from the young watermelon fruits. to clarify these points, advanced analysis on more xigua-mian and young watermelon fruit samples will be necessary in the future. the results of the antibacterial activity assay indicated that total 10 lc. lactis subsp. lactis isolates showed inhibitory activities against l. sakei subsp. sakei jcm 1157t. complete inactivation of these blis produced by all 10 strains were observed after treating the cell-free supernatant with proteinase k, which indicates the proteinaceous nature of the active agents. when amplified with nisin-specific primers, the amplification band located at 320 bp indicated the existence of nisin-producing genes and blis from these 10 lc. lactis subsp. lactis could be nisin-related variants (villani et al., 2001; zendo et al., 2003). however, detailed information such as heat stability, their effect on enzymes, inhibition spectra, accurate molecular mass and amino acid sequences were not established in the current study. although lab have been widely found in various fresh fruits, vegetables and various plant pickles, little information on the diversity of lab associated with fermented watermelon or young watermelon fruits was obtained from previous studies. future studies in our laboratory will characterize and identify the nisin-like blis, and we anticipate that the blis of lab will be useful as food preservatives. the authors also hope that the results of this study can offer useful information for the improvement of xi-guamian production. acknowledgments the authors would like to thank the national science council taiwan for financially supporting this study under contract no. most 103-2313-b-130-002 and most 104-2320b-130-001 granted to yi-sheng chen. references chang s.y., chen y.s., pan s.f., lee y.s., chang c.h., chang c.h., yu b. and wu h.c. 2013. enterocin tw21, a novel bacteriocin from dochi-isolated enterococcus faecium d081821. j. appl. microbiol. 115: 673-678. chang c.h., chen y.s. and yanagida f. 2011. isolation and characterization of lactic acid bacteria from yan-jiang (fermented ginger), a traditional fermented food in taiwan. j. sci. food agric. 91: 1746-1750. chen y.s., liou m.s., ji s.h., yu c.r., pan s.f. and yanagida f. 2013a. isolation and characterization of lactic acid bacteria from yan-tsai-shin (fermented broccoli stems), a traditional fermented food in taiwan. j. appl. microbiol. 115: 125-132. chen y.s., wu h.c., liu c.h., chen h.c. and yanagida f. 2010. isolation and characterization of lactic acid bacteria from jiang-sun (fermented bamboo shoots), a traditional fermented food in taiwan. j. sci. food agric. 90: 1977-1982. chen y.s., wu h.c., lo h.y., lin w.c., hsu w.h., lin c.w., lin p.y. and yanagida f. 2012. isolation and characterisation of lactic acid bacteria from jiang-gua (fermented cucumbers), a traditional fermented food in taiwan. j. sci. food agric. 92: 2069–2075. chen y.s., wu h.c., wang c.m., lin c.c., chen y.t., jhong y.j. and yanagida f. 2013b. isolation and characterization of lactic acid bacteria from pobuzihi (fermented cummingcordia), a traditional fermented food in taiwan. folia microbiol. 58: 103-109. chen y.s., yanagida f. and hsu j.s. 2006a. isolation and characterization of lactic acid bacteria from suan-tsai (fermented mustard), a traditional fermented food in taiwan. j appl microbiol 101: 125-130. chen y.s., yanagida f. and hsu j.s. 2006b. isolation and characterization of lactic acid bacteria from dochi (fermented black beans), a traditional fermented food in taiwan. lett. appl. microbiol. 43: 229-235. chen y.s., yu c.r., ji s.h., liou m.s., leong k.h., pan s.f., wu h.c., lin y.h., yu b. and yanagida f. 2013c. enterocin t, a novel class iia bacteriocin produced by enterococcus sp. 812. arch. microbiol. 115: 125-132. jang j., kim b., lee j. and han h. 2003. a rapid method for identification of typical leuconostoc species by 16s rdna pcr-rflp analysis. j. microbiol. methods 55: 295-302. kozaki m., uchimura t. and okada s. 1992. experimental manual of lactic acid bacteria. asakurasyoten, tokyo, japan, pp. 29-72. lan w.t., chen y.s. and yanagida f. 2009. isolation and characterization of lactic acid bacteria from yan-donggua (fermented wax gourd), a traditional fermented food in taiwan. j. biosci. bioeng. 108: 484-487. leong k.h., chen y.s., lin y.h., pan s.f., yu b., wu h.c. and yanagida f. 2013. weissellicin l, a novel bacteriocin from sian-sianzih-isolated weissella hellenica 4-7. j. appl. microbiol. 115:70-76. lin y.h., chen k.s., liou t.d., huang j.w. and chang p.f.l. 2009. development of a molecular method for rapid differentiation of watermelon lines resistant to fusarium oxysporum f. sp. niveum. bot. stu. 50: 273-280. sheu d.s., wang y.t. and lee c.y. 2000. rapid detection of polyhydroxyalkanoate-accumulating bacteria isolated from the environment by colony pcr. microbiology 146: 2019-2025. srionnual s., yanagida f., lin l.h., hsiao k.n. and chen y.s. 2007. weissellicin 110, a newly discovered bacteriocin from weissella cibaria 110, isolated from plaa-som, a fermented fish product from thailand. appl. environ. microbiol. 73: 2247-2250. torriani s., felis g.e. and dellaglio f. 2001. differentiation of lactobacillus plantarum, l. pentosus, and l. paraplantarum by reca gene sequence analysis and multiplex pcr assay with reca gene-derived primers. appl. environ. microbiol. 67: 3450-3454. villani f., aponte m., blaiotta g., mauriello g., pepe o. and moschetti g. 2001. detection and characterization of a bacteriocin, garviecin l1-5, produced by lactococcus garvieae isolated from raw cow’s milk. j. appl. microbiol. 90: 430-439. yanagida f., chen y.s., srionnual s. and shinohara t. 2006. bacteriocin from lactococcus lactis subsp. lactis c101910 isolated from lake water. jpn. j. lactic acid bact. 17: 51-56. zendo t., fukao m., ueda k., higuchi t., nakayama j. and sonomoto k. 2003. identification of the lantibiotic nisin q, a new natural nisin variant produced by lactococcus lactis 61-14 isolated from a river in japan. biosci. biotechnol. biochem. 67:1616-1619. paper received september 9, 2014 accepted february 19, 2015 ijfs#245_tezel_bozza   ital. j. food sci., vol 28, 2016 271 paper effect of temperature, gum concentration and gum ratio on creep-recovery behaviour of carboxymethyl celluloseguar gum mixtures: modeling with rsm and ann sibel uzuner1, g. bengusu tezel2* and nese k. cakmak3 1department of food engineering, faculty of engineering and architecture, abant izzet baysal university, 14280, bolu, turkey 2*department of chemical engineering, middle east technical university, 06800, ankara, turkey 3department of chemical engineering, faculty of engineering, cumhuriyet university, 58140, sivas, turkey *corresponding author. tel.: +90 3122104391; fax: +90 3122102600 e-mail address: bengusu@metu.edu.tr abstract in the present study, the effects of temperature (15, 25, 35°c), gum concentration (1.5, 2.0 and 2.5 %) and gum ratio (25:75, 50:50, 75:25) on the creep-recovery rheological properties of carboxymethyl cellulose (cmc)-guar gum (gg) mixtures were investigated. within the studied range of experimental design, recovery index (∆j) value of cmc-gg gum solution was analyzed based on the design factors. experimental recovery index responses were modelled using rsm (r2= 0.9711) and ann (r2= 0.9829). keywords: artificial intelligence, creep and recovery test, response surface methodologyyrheological properties   ital. j. food sci., vol 28, 2016 272 1. introduction gums are widely used in food industry to stabilize emulsion, prevent ice recrystallization (kayacier and dogan, 2006). the use of two or more gums is a very widespread practice in the food industry due to synergistic interaction between two or more different gums (kayacier and dogan, 2006). guar gum (gg) is used as an economical thickening and stabilizing agents in the food industry and is often combined with xanthan, locust bean gum (lbg), or carboxymethyl cellulose (cmc) to increase synergistic changes in viscosity or gelling behaviour (prado et al., 2005). in generally, gum or gum combination is used in coating, drug delivery and food additives such as dressings, sauces, cereal products, fruit filling, confectionary products and frozen and baked products (pai et al., 2002). synergistic interactions of mixed gums have an attractive in the food industry due to reducing production costs and improving product quality and mouth feel (mao and rwei, 2006; norziah et al., 2006). the determination of viscoelastic properties of product is very important for prediction of product stability in many processing, transportation and storage (steffe, 1996). to determine viscoelastic properties of the samples, creep-recovery test is a very crucial method for providing knowledge about the internal structure of material (dolz et al., 2008). several studies have been conducted on creep of food materials for evaluating in emulsions (dolz et al., 2008; yılmaz et al., 2012), salad dressing (zhang et al., 2008), milk (bayarri et al., 2009), tomato juice (augusto et al., 2013), instant pudding (dogan et al., 2014). dogan et al. (2014) also evaluated the effect of four different gums (carrageenan, alginate, guar and xanthan gum) and their different combinations on creep-recovery properties of the pudding samples. foods are very complex systems due to possible interactive effects of factors such as temperature, gum type, gum concentration etc. to reduce the number of extensive experimentation and learn about complex interactive effects, modeling and optimization is an inevitable stage for a process (bas and boyacı, 2007; mingzhi et al., 2009). therefore, optimization of the mixed gum formulation is important for both economical and rheological characterization of product (dogan et al., 2014). for this purpose, response surface methodology (rsm) is the mostly used statistical methods in optimization of rheological properties parameters. however, rsm does not adequately explain nonlinear models (ross, 1999). to overcome this problem, nonlinear models based artificial intelligence models such as artificial neural networks (ann) can be considered. ann has been successfully applied in modeling of creep-recovery behavior for used in formulation of instant pudding (dogan et al., 2014), deformation of the grape molasses (toker and dogan, 2013) and construction of predictive models in meat emulsion (yılmaz, 2012). there are many studies have been conducted on the synergistic behavior of gum mixtures like carrageenan-locust bean gum (tako and nakamura, 1986), xanthan-locust bean gum (copetti et al., 1997), xanthan-modified guar gum (pai and khan, 2002), cmcarabic gum (garcia-abuin et al., 2010). however, there is a limited number of studies in the literature using creep and recovery test in order to evaluate and optimize gum combinations. to the best of our knowledge, no published literature was found on the effect of gum concentration, temperature and gum ratio on mixed gum (cmc-gg) optimization by response surface methodology approach based on creep-recovery parameters of the gum combination. the aim of the this study was to observe the effects of gum concentration, temperature and gum ratio and their interaction terms on the creep-recovery characteristics of cmc-gg mixture samples and then to perform desirability function to determine optimum percentage deformation of mixture by considering several factors simultaneously.   ital. j. food sci., vol 28, 2016 273 2. materials and methods 2.1. materials polymer powders of guar gum (gg) and sodium carboxylmethyl cellulose (cmc) (sigmaaldrich corp, st louis, mo), with nominal molecular weight of 700,000 g/mol were kindly provided by dr. kerim yapici, nanotechnology engineering, sivas, turkey. 2.2. preparation of gum solutions the cmc and gg powders were dissolved in distilled water seperately at 25°c for 6 h using a magnetic stirrer with gentle shaking in order to prepare 1.5, 2 and 2.5% (w/v) stock solutions of cmc and gg. the cmc stock solution was thoroughly mixed with gg solution at different volume ratios, cmc:gg, such as 75:25; 50:50 and 25:75 respectively. 2.3. rheological measurements creep and recovery tests of the samples measurements were carried out by a stress controlled rheometer (malvern kinexus pro, uk) fitted by a cone-and-plate system. cone and plate geometries are well suited for studying viscoelastic (stress growth, creep and dynamic) properties of foods. diameter and angle of the cone were chosen 50 mm and 1°, respectively. the gap between the cone-and-plate was fixed at 0.05 mm for all measurements. a peltier plate assembly was used for temperature controlling purpose during the measurements with ±0.1°c precision. firstly amplitude sweep over a stress was performed under at a constant frequency of 1 hz frequency at 25°c to find linear viscoelastic region (lvr). then, stress sweep tests were conducted between 0.1 and 10 hz to determine lvr. creep – recovery tests were carried out under the constant shear stress of 0.05 pa (in lvr) at three different temperatures (15, 25, and 35°c) to measure the deformation of mixtures. as known, creeprecovery tests include two parts, one of which is creep phase in which 0.05 pa. constant stress was applied for 120 s and the compliance values were recorded as a function of time. in the second part, which is recovery part, the applied stress was removed and then compliance values were also obtained as a function of time for 120 s. in creep-recovery test, the stress response of samples under constant stress in a total time of 240 s was measured. each measurement was replicated two times. the effect of gum concentration, temperature and gum ratio on the creep-recovery characteristics data of cmc-gg mixed gel was examined. recovery index or recovery percentage (∆j) is used to quantify the viscoelastic response of a materials as given in eq. (1). the following equation as proposed by dolz et al. (2008) was used to calculate the percentage deformation of each gum mixtures. (120) (240) 100 (120) j j j j − δ = (1) in this equation j(120) and j(240) refer to the values of the compliance at the final creep phase and at the final recovery phase, respectively. 2.4. experimental design and response surface methodology (rsm) one of the models to describe chemical processes in analytical methods is quadratic model   ital. j. food sci., vol 28, 2016 274 in comparison with linear and cubic equation. the design consists of three replicated center points. the individual and combined effects of temperature, gum concentration and gum ratio on recovery percentage were studied using box–behnken response surface method (box and behnken, 1960) (bbd) using minitab 16.0 (minitab inc. state college, pa, usa) (table 1). table 1. factors and their coded levels of bbd. independent variable representation low (-1) center point (0) high (+1) temperature (°c) x1 15 25 35 gum concentration (%) x2 1.5 2.0 2.5 mixed gum ratio x3 25:75 50:50 75:25 all creep and recovery tests were conducted in duplicate. the collected data were fit to a quadratic mathematical model expressed as: y = b0 + b1x1 + b2x2 + b3x3 + b12x1x2 +b13x1x3 + b23x2x3 + b11x12 + b22x22 + b33x32+e where y is the response (∆j, % ), b’s are regression coefficients. the independent variables; temperature, gum concentration and gum ratio were coded as x1, x2, and x3, respectively. the analysis of variance (anova) and regression analysis were performed to define the coefficients of the predictive model and significant terms using minitab 16.0 (minitab inc. state college, pa, usa). the optimum conditions for maximizing the apparent viscosity was determined using response optimizer tool in minitab 16.0 (minitab inc. state college, pa, usa). 2.5. model validation the constructed model was verified by additional trials at the optimal rheological properties of cmc-gg gum mixtures carried out in triplicate at the design conditions. 2.6. artificial neural network (ann) modeling the input variables used in the ann models were temperature (x1), gum concentration (x2), and gum ratio (x3). the ann model was generated as output variable recovery index (∆j, y1). several anns with different numbers of hidden layer neurons was developed for describing creep behavior of gum mixtures. input data were randomized into three sets: learning, validation and testing. usually 30% of data are used for testing and remaining 70% for training and validation (mehdizadeh and movaghernejad, 2011). the experimental data including 30 data points was divided so that 70% was used to train the model, 15% was used to validate the model and 15% was used to test the generalization ability of the model. activation function of hidden layer was “logsig” and the one in output layer was “purelin” as shown in equations (2-3). xe1 1 logsig(x) −+ = (2)   ital. j. food sci., vol 28, 2016 275 xe1 1 logsig(x) −+ = (3) training of the network was performed with the function of “trainlm”, that updates weight and bias values according to levenberg-marquardt optimization. in learning of network “learngd” as adaption learning function was used. the maximum training epochs were 1000, and mean square error was 0.0001. the other parameters of neural network were taken as defaults of neural network toolbox, matlab r2011a. the performance of the ann was statistically measured by the mean squared error (mse), average relative deviation (ard) and the coefficient of determination (r2). these statistics were used to assess the performance of model predictions compared to the actual data. the r2 represents how well the approximated function predicts the response versus just using the response mean. values closest to 1 are the best. the mse is a representation of the difference between the predicted and actual values and gives a sense of how close the predicted values are to the observed values in the units of those values. lower values of mse are best and it was calculated using the formula in equation 4. the ard represents the percent error. lower values are best and it was calculated using the formula in equation 5. ( ) n xx mse 2n 1i iexp,ipre,∑ = − = (4) ard = 100 n xpre,i − xexp,i( ) xexp,ii=1 ∑ (5) where xpre,i is the predicted output from observation i, xexp,i is the experimental (target) output from observation i, is the average value of experimental output, and n is the total number of data. 2.7. selection of the best model to compare the constructed models and decide on the best one among them, performances of the models was measured using root mean square error (rmse), mean absolute error (mae), and coefficient of determination (r2), which are common comparison tools in modeling. a statistical difference measure test was also carried out to evaluate the performance of the model by calculating root mean square error (rmse) and mean absolute error (mae) values as follows: ( ) 5.0 1 2 exp,, 1 ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ −= ∑ = n i iipred xxn rmse (6) ∑ = −= n i iipred xxn mae 1 exp,, 1 (7) x   ital. j. food sci., vol 28, 2016 276 where xexp is the experimental value and xpred is the predicted value of recovery index (∆j, %) value, n is the number of data. 2.8. statistical analysis statistical analyses were accomplished using minitab 16.0 to test the significance of different rheological properties of mixed gums. the pairwise comparisons were made by tukey’s test with a significance level of 0.05. 3. results and discussion 3.1. effect of concentration, ratio and temperature on the creep behaviour of gum mixtures fig. 1a-c shows graphical representation of creep and recovery experimental results for corresponding to all the mixed gum studied at 15, 25, 35°c respectively, in a time interval between 0 and 240 s. following the application of stress, 0.05 pa, deformation occured and deformation was recorded as function of time, j=f(t), during the rheological creep and recovery measurements. at time, t=120, j(120) corresponds maximum deformation value. in fig. 1c, j(120) values are nearly 7 pa-1 for 2.5% 50:50 at 35°c. on the other hand, it has reached to 2.8 pa-1 at 15°c for the same level as seen in fig. 1a. this implies that j(120) values gets higher value due to weaking internal structure of gum mixtures and thus higher deformability and more softening structure at 35°c. sozer (2009) also reported that high compliance value shows weak structure of material. a) t=15°c   ital. j. food sci., vol 28, 2016 277 b) t=25°c c)t=35°c fig. 1. creep and recovery plot of gum mixtures for different temperature levels a) t=15°c b) t=25°c c)t=35°c.   ital. j. food sci., vol 28, 2016 278 cmc and gg is used as thickener and stabilizer in food processing. during food processing, temperature takes critical role due to affected rheological properties of food systems. upon increasing temperature, it causes a change deformation structure of the gum mixtures. gum mixture structure is weakened with increase of temperature. creep compliance value, j(t), increases with temperature due to the related with the softness of the material structure (hayta and schofield, 2005). these data also show that the recovery index in all gum mixtures is positively correlated with temperature at the fixed gum concentration and ratio (fig. 2b-c). the drop in viscoelasticity at low temperature level comes from strong or stiffer structure of gum mixtures. on the other hand, there is no significant change for the effect of gum ratios on the recovery indexes for the fixed gum concentration at 15°c (p>0.05). a) b)   ital. j. food sci., vol 28, 2016 279 c) fig. 2. response surface showing the effect of a) gum concentration vs gum ratio, b) temperature vs gum ratio, c) temperature vs gum concentration on rheological properties of gum mixtures. as cmc-gg mixtures concentration increased, recovery index values get higher and viscoelastic response of the mixtures is pronounced (fig.2a). it is resulted from intermolecular interaction between cmc-gg mixture depended strongly concentration of the mixture. for 2.5% (w/v) gum mixture showed higher compliance, j(t), values indicating higher deformation than concentration of 1.5 and 2% (w/v) for all temperatures as depicted in figs. 1a-c. recovery index, calculated as in eqn. 1, has changed with gum ratio for 1.5% (w/v) and 2% (w/v) gum mixtures. when the mixed gum has 2% 50:50, designated as center model points, lower deformation is significantly achieved in fig. 1a and b. conversely, the strongest synergistic effect on recovery indexes has been observed when changed with mixed ratio (25:75 and 75:25) tabulated as in fig. 1b. it is associated with increased elastic nature of gum mixture especially in 25:75 gum ratio in higher presence of gg at all temperature levels. guar gum which highly extend in gum mixture entangles with network structure and binds with cmc. furthermore, guar gum more dominates the structural arrangement in higher gum mixture indicating that is highly elastic nature and recovery indexes attains to 87.50% at 25°c shown in table 2. the results also suggested that hydrophilic intrinsinc interaction between cmc-gg (tipvarakarnkoon and senge, 2008) which leads to the increasing softening gum structure. therefore, viscoelastic properties of the mixtures in the aqueous medium are enhanced. even in lower concentration of gum mixture, significant synergetic effect was also observed for 1.5% 25:75 and 75:25 mixtures in fig. 1b. it could be related that lower degree entanglement of gum mixture at lower concentration permits to the more elastic effect contribution on mixture nature at 25°c.   ital. j. food sci., vol 28, 2016 280 table 2. experimental measured values according to box-behnken response surface method (rsm) versus predicted values of rsm and ann models for recovery index (%). temperature (oc) (x1) gum conc. (%) (x2) mixed gum ratio (x3) recovery index (%) (y1) experimental rsm pred. ann pred. 35 1.5 50:50 77.24±3.04 bc 77.90 76.45 25 2.0 50:50 61.52±1.89 f 60.84 61.36 15 1.5 50:50 58.80±2.45 f 60.25 58.75 15 2.0 25:75 68.44±0.73 de 68.24 68.00 35 2.0 25:75 81.68±1.48 ab 81.86 80.71 25 2.5 75:25 77.91±0.30 bc 78.96 77.09 15 2.5 50:50 72.08±0.06 cd 71.42 71.49 25 1.5 25:75 78.11±0.60 bc 77.05 77.29 35 2.5 50:50 82.48±1.97 ab 81.01 81.48 35 2.0 75:25 76.00±0.86 bc 76.63 75.26 25 2.0 50:50 59.96±2.93 f 60.84 59.86 25 2.0 50:50 61.06±1.27 f 60.84 60.91 15 2.0 75:25 63.64±3.52 ef 63.01 63.39 25 2.5 25:75 87.50±0.16 a 88.56 86.30 25 1.5 75:25 77.27±3.05 bc 76.19 76.48 data within the same column with different superscript letter are significantly different (p <0.05). when higher presence of cmc (75:25) at the 25°c, recovery index value (77.91%) gets smaller value as compared value at 25:75 ratio revealing that more cmc addition led a smaller viscoleastic deformation of mixture and viscous behaviour of mixture increase at the 25°c. hence, viscous flow deformation gains to importance for the higher cmc content of solution and viscous properties of mixing also increase at 35°c as seen in fig. 1c, maximum compliance value of 1.5% 50:50 of solution is nearly same than 2.0% 75:25 of solution. it suggests also that strong viscoelastic effect of gg plays major role in determining the direction of synergetic effect of mixed gums at 35°c. for comprehensive understanding of interactions between concentration, mixing ratio, temperature study on optimization of creep properties of cmc-gg mixtures was achieved to get most resistant and stable product during the efficient food product design. 3.2. identification of optimized rheological characteristics of carboxymethyl celluloseguar gum mixtures conditions through response surface modeling rsm is a frequently used technique for modeling and determining optimal process conditions. estimation of response surface analysis of experimental results for prediction of recovery index value (∆j) in gum mixtures based on temperature (°c) (x1), gum concentration (%, w/v) (x2) and gum ratio (v/v) (x3) per bbd (table 2), were further elucidated to identify the best process conditions in the ranges tested. the highest recovery index of about 87.50% was obtained at 25°c, 2.5% gum concentration, and 25:75 of gum ratio, whereas the poorest conditions were 15°c, 1.5% gum concentration, and 50:50 gum ratio, giving about 58.80% of recovery index (table 2). second-order polynomial equations (quadratic model) were established to identify the relationship between recovery index and three dependent variables: temperature (°c) (x1),   ital. j. food sci., vol 28, 2016 281 gum concentration (%, w/v) (x2) and gum ratio (v/v) (x3). table 3 shows the effect of process variables and interactions on recovery index. table 3. anova table for bbd model. term coefficient t value p value intercept 60.84 76.69 0.000 block -0.576 -1.623 0.121 temperature (oc) (x1) 6.805 14.01 0.000 gum conc. (%) (x2) 3.569 7.347 0.000 mixed gum ratio (x3) -2.612 -5.376 0.000 x1* x1 2.024 2.830 0.011 x2* x2 9.780 13.68 0.000 x3* x3 9.570 13.38 0.000 x1* x2 -2.013 -2.929 0.009 x1 *x3 -0.220 -0.320 0.752 x2* x3 -2.186 -3.182 0.005 r-sq = 0.9711%; r-sq(adj) = 0.9581 %; r-sq(pred) = 0.9299 %. another run with excluded insignificant terms according to table 4 expressed by eqn. 8: y=60.84+6.81x1+3.57x2-2.61x3-2.01 x1 x2 -2.19 x2 x3 +2.02x12 +9.78 x22+9.57 x32 (8) where y is predicted recovery index (∆j, %). equation 8 was found fairly adequate to represent the data with r2 of 0.97. the insignificant lack of fit for recovery percentage was (p = 0.46 >0.05), which also proved that the model fit the experimental data well. table 4. significance of term coefficients for bbd; a: temperature (oc); b: gum concentration (%); c: mixed gum ratio. term coefficient standard error coefficient t value p value constant 60.84 0.775 78.47 0.000 a 6.805 0.475 14.33 0.000 b 3.569 0.475 7.518 0.000 c -2.612 0.475 -5.501 0.000 a*b -2.013 0.672 -2.997 0.007 b*c -2.186 0.672 -3.256 0.004 a2 2.024 0.699 2.896 0.009 b2 9.780 0.699 13.994 0.000 c2 9.570 0.699 13.693 0.000 all factors showed significant effects (p<0.05) on recovery percentage and the interactions between temperature-temperature, concentration–concentration, ratio-ratio interactions, temperature–concentration and concentration-ratio showed significant effects (table 4). the most important factors determining the recovery index were temperature with highest   ital. j. food sci., vol 28, 2016 282 coefficient (6.81), which indicates that it is the most dominant factor influencing the overall recovery index value followed by gum concentration (3.57) and gum ratio (2.61) (table 4). estimation of recovery index at varying process variables (temperature, gum concentration, and gum ratio) are represented in response surfaces plots shown in fig. 2ac. fig. 2a shows the effect of gum concentration and gum ratio on the recovery index at mid-point temperature of 25°c. the recovery index of the gum -mixtures was strongly influenced by both variables. a decrease in gum ratio from 75:25 to 25:75 gave the highest recovery indexes of 87.50 % at about 25°c and 2.5 % gum concentration was observed (fig. 2a). gum ratio had the most negative effect on the recovery index, meaning that the increase in gum ratio caused decrease in deformation when stress was applied to gum mixture. on the other hand, the effect of temperature and gum ratio on recovery indexes is shown in fig. 2b, where gum concentration was set at 2.0 % (w/v) as the center point. a linear increase in recovery indexes was observed as temperature increased (fig. 2b). similarly, the positive effect of temperature was observed in fig. 2c, showing the effect of temperature and gum concentration on recovery indexes. therefore, to determine the optimal treatment conditions, the response optimizer tool in minitab® 16.1 (minitab inc.) was used. optimization was performed regarding maximum recovery index value since low recovery index shows that mixed gums had higher resistance or lower deformation when stress is applied. higher resistance of the sample against stress is important for product quality during handling and storage (dogan et al., 2014). conditions for obtaining optimum recovery percentage of 95.38%, as predicted by the model, were found to be 35°c and 2.5 (%, w/v) and 50:50 gum ratio. these conditions were experimentally tested in triplicate to validate the models predictive ability. a recovery percentage of 90.34±2.79% was obtained and there was no significant difference between the experimental and model predicted values (p>0.05). 3.3. predictive modeling of creep behaviour using ann 3.3.1. effect of architecture and topology on neural network selection of network topology in ann modeling is the key issue. several parameters such as the number of hidden layers, the number of neurons, the transfer function, the epochs and learning rate affected the network topology. the number of hidden neurons is one of the most important parameters of ann modeling. a high number of neurons performs satisfactorily for training data but may fail for testing data (over-fitting), while a few hidden neurons cause unsatisfactory convergence (under-fitting) (tokatli et al., 2009). in this study, the number of neurons in the hidden layer was chosen by a trial and error method, varying the neurons from 3 to 12. several ann models with different network topologies were trained, tested, and their performance was evaluated to select the best network topology. the r2, rmse, and ard statistics from training and testing data for different ann topologies is summarized in table 5. table 5 shows that the 3-9-1 topology was chosen as the best topology (fig. 3) with the minimum mse and ard, and maximum r2 values. in case of training data set, the coefficient of determination (r2) and ard values were 0.9889 and 0.003%, respectively, whereas in testing data set, r2 was 0.9866 and ard was 0.009% (table 5). ann with 3-9-1 topology was also performed (r2 values of 0.9889, 0.9784, 0.9866 and mse values of 1.8478, 6.3565, 4.5024 in training, testing and checking (validation) periods, respectively) (table 6).   ital. j. food sci., vol 28, 2016 283 table 5. performance of different artificial neural network (ann) model in estimation of recovery index (%). network r2 mse ard (%) training testing training testing training testing 3:3:1 0.9820 0.6355 2.49 145.79 0.005 0.080 3:4:1 0.9942 0.9364 0.88 11.50 0.0003 0.004 3:5:1 0.9859 0.9664 2.17 4.24 0.002 0.039 3:6:1 0.9321 0.9091 12.85 26.97 0.006 0.025 3:7:1 0.9889 0.8964 1.85 4.46 0.002 0.002 3:8:1 0.9806 0.9445 3.38 10.67 0.0005 0.016 3:9:1 0.9889 0.9866 1.85 4.50 0.003 0.009 3:10:1 0.9926 0.9692 1.27 6.21 0.005 0.043 3:11:1 0.9910 0.9888 1.30 11.69 0.002 0.037 3:12:1 0.9970 0.9372 4.47 12.07 0.004 0.031 fig. 3. schematic representation of ann to simulate the rheological properties of gum mixtures. table 6. performance of neural network model for prediction of recovery index (∆j) values of gum mixture statistical parameter training testing validating overall recovery index (%) r 2 0.9889 0.9784 0.9866 0.9829 mse 1.8478 6.3565 4.5024 3.0417 3.4 comparison of rsm and ann models fig. 4 shows the plot of predicted recovery index by rsm and ann against the experimental values. the ann model exhibited a better relationship between experimental and predicted recovery index values than the rsm model. the results showed that the ann predictions were closer to experimental values than those of rsm.   ital. j. food sci., vol 28, 2016 284 though both the models based on rsm and ann performed well and offered stable response in prediction; the ann based approach was better in predicting and fitting than the rsm model for testing the data. thus, the accuracy of ann model was higher and has better fitted data than the rsm model. fig. 4. verification predicted values versus actual experimental values of rsm and ann models for rheological properties of gum mixtures. 4. conclusions detailed creep and recovery viscoelastic measurements at contant 0.05 pa revealed that recovery index (∆j) remarkably changed according to temperature, gum concentration and gum ratio. elastic or viscous effects contribution on the gum mixture structure affected the creep properties of cmc-gg mixtures. within the studied range of experimental design, higher content of gg of mixed gum solution increased ∆j value at all levels. in addition to mixed gum ratio, synergisim effect of gum mixtures was observed especially at 25°c. on the other hand, deformation properties of gum mixtures significantly changed with temperature. furthermore, optimization and modeling of gum mixtures are taken into consideration to obtain both best quality of product and economical point of the view. experimental recovery index responses were optimized using prediction models such as response surface methodology (rsm) and artificial neural network (ann) models. several ann topologies were evaluated in order to predict creep test for gum mixtures. these two methods were also compared for their predictive abilities. the best fit model was identified in ann model with the highest coefficient of determination (r2) and lowest mse values. therefore, ann can be considered as a prediction tool for rheological properties of gums mixtures.   ital. j. food sci., vol 28, 2016 285 acknowledgements the authors would like to thank dr. kerim yapıcı at the cumhuriyet university nanotechnology engineering laboratory, sivas, turkey, for providing with stress controlled rheometer and all materials used in the experiments. references augusto p.e.d., ibarz a. and cristianini m. 2013. effect of high pressure homogenization (hph) on the rheological properties of tomato juice: creep and recovery behaviours. food res int. 54: 176. bas ̧d. and boyaci i.h. 2007. modeling and optimization ii: comparison of estimation capabilities of response 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ambiente mediterraneo, cantina sperimentale di barletta, via vittorio veneto 26, 76121 barletta, italy *corresponding author: tel.+39 088 352 1346, fax +39 088 352 8955, email: serafino.suriano@entecra.it abstract a study has been carried out to determine the effects of lactic acid bacteria inoculation time on the fundamental components, procyanidins and biogenic amines content of aglianico wines produced in apulia region. three different malolactic fermentation (mlf) techniques were compared: the co-inoculation, the sequential inoculation, and the traditional technique (spontaneous mlf). in the co-inoculation technique there was a delayed start and a late finish of the alcoholic fermentation. the colour intensity of the wine obtained with a spontaneous mlf was higher both at racking and after 12 months. significant changes in content of flavan-3-ols were found in wines made with different mlf managements. the levels of catechin monomers ((+)-catechin, (-)-epicatechin, (-)-epicatechin-o-gallate) and procyanidin oligomers (b1-b4, and trimer c1) were lower in the co-inoculation wine. in the wine produced with a spontaneous mlf, the content of biogenic amines was significantly higher compared to the other two wines. ital. j. food sci., vol. 27 2015 311 introduction the most important microbial activity that is responsible for the conversion of must into wine is the alcoholic fermentation (af), which is carried out by saccharomyces yeasts. the malolactic fermentation (mlf) typically follows alcoholic fermentation and is carried out by indigenous lactic acid bacteria (lab) or induced by inoculation with selected bacterial starters. it is a decarboxylation of l-malic acid, a dicarboxylic acid, with formation of a monocarboxylic acid, the l-lactic acid and carbon dioxide, which is catalyzed by malolactic enzymes which are nadp dependent and require divalent cations such as manganese or magnesium ions (vincenzini et al., 2005). the mlf causes a significant evolution of wine and produces remarkable changes in its phenolic composition and sensorial characteristics (costello et al., 2012; cabrita et al., 2008; lòpez et al., 2011; suriano et al., 2012). in addition to the reduction of the acidity of the wine, mlf increases the aromatic complexity and smoothness (versari et al., 1999; costantini et al., 2009; lopez et al., 2011). generally the mlf is favoured in red wines, in novello wines, in white wines aged in barrique, or in some sparkling base wines (cavazza et al., 2003). on the other hand, this fermentation produces a small amount of acetic acid and sometimes may also generate unpleasant odours, bitter-tasting compounds or substances that may be dangerous to consumers’ health, such as biogenic amines or precursors of ethyl carbamate (lonvaud-funel, 1999). it has been verified by analysis that the concentration of biogenic amines in wine at the end af is always quite low, while increases after mlf (gafner, 2005). moreover, it was found that wines which undergo spontaneous mlf often have higher biogenic amine concentration than those in which the mlf is conducted by select malolactic bacteria (cerruti et al., 1987; masquè et al., 2008). biogenic amines are synthesized by microorganism through decarboxylation of amino acids. between the main biogenic amines in wines there are tyramine, histamine, putrescine, cadaverine and phenylethylamine, synthesized by the decarboxylation of the amino acids tyrosine, histidine, ornithine, lysine and phenylalanine. these compound can cause adverse physiological reactions in susceptible individuals. histamine can cause headaches, allergies, diarrhoea, palpitations and vomiting (stockley, 2004; bodmer et al., 1999), while tyramine is strongly vasoconstrictive (silla-santos, 1996). these effects may be enhanced by alcohol, which prevents the organism’s detoxifying mechanisms from working properly and by the presence of other amines such as putrescine and cadaverine (landete et al., 2005), both associated with poor sanitary quality of grapes (leitao et al., 2005) and responsible for major sensory defects in wines (lehtonen, 1996). usually, the lab used for mlf belong to the oenococcus oeni species, anyway, it is possible to also find other bacteria of the lactobacillus, leuconostoc and pediococcus species (dicks et al., 1995). however, even for the most resistant bacteria the conditions found in wine are close to the limits of survival, so that the transformation of 4-5 g/l of malic acid may requires even 1520 days (cavazza et al., 2003). several times, this process may take several months, may occur in some barrels and tanks but not in others and may be responsible for the occurrence of problems related to indigenous lab species carrying out the mlf (lonvaud-funel, 2001) which may cause a range of undesirable changes to wine sensory properties, altered wine colour, and may even lead to the generation of biogenic amines (davis et al., 1985). such a long time can be especially critical for those wines (such as novello wines) that must be processed and placed on the market in a short time, and moreover could be a risk since in the season in which the mlf takes place there may be sudden temperature drops which may determine an arrest of the process until the next spring. there are advantages subsequent to an early and fast mlf such as: a more efficient utilization of fermentation tank in the busy postharvest period, thus a decrease of energetic costs resulting in optimization of the winemaking process; moreover it is possible a decrease of the microbiological risks reducing the growth of undesired microorganism and also allows an early commercialization of wines (jussier et al. 2006). it is therefore of fundamental importance a correct management of mlf. in this paper the influence of inoculation of lactic bacteria on changes occurring on the polyphenolic characteristics, colour, biogenic amines and proanthocyanidin in aglianico red wines was investigated by comparing the techniques of co-inoculation and sequential inoculation to a spontaneous mlf. materials and methods experimental design and winemaking this research was conducted during the 2012 harvest on aglianico grape variety, grown in a vineyard trained on espalier training system with guyot pruning and cultivated according to the principles of organic viticulture. concerning the different possibilities of mlf management, in the le.vin.sud company of cerignola (foggia, southern italy) were carried out three experimental tests in order to evaluate the influence of the timing of lactic bacteria inoculation, comparing the technique of co-inoculation (inoculation of bacteria 24 hours after the yeast 312 ital. j. food sci., vol. 27 2015 inoculation), sequential inoculation (at the end of the af) and the traditional technique without inoculation of any lab, i.e. a spontaneous mlf, which was favoured by acting on certain oenological practices, as further explained. the aglianico grapes were first destemmed and crushed, subsequently the mass of must and pomace was mixed, homogeneized and introduced in three different steel tank. from each steel tank, 3 x 100 kg (in triplicate) of must with pomace was utilized for each of the three winemaking techniques adopted, with the aim to determine the repeatability of the differences among the compared treatments. the different batches of must and pomace were subjected to the following winemaking protocols: co-inoculation or simultaneous inoculation of lab (sim). after crushing and destemming of grapes 40 mg/l of so 2 was added. after two hours, saccharomyces cerevisiae strain lalvin r7 (lallemand inc, castel d’avezzano-verona – italia) previously hydrated in water for 15 min at 38 °c was inoculated in the must (20 g/hl, about 6 x 106 cfu/ml.). after 24 hours a lactic bacterial culture of lactobacillus plantarum v22tm (lallemand inc, verona-italy) was inoculated. the inoculation rate was 1g/hl (2 x 107 cfu/ml) must/wine prior re-hydrated in chlorine free water at 20°c for 15 min. the alcoholic fermentation took place under controlled temperature by cooling the mass if the temperature exceeded the threshold of 26°c. sequential inoculation post alcoholic fermentation of lab (paf). the only difference from the previous protocol was the time of addition of the bacteria. the lactic bacteria were added at racking, which was performed at the end of the alcoholic fermentation (10 day pomace contact). the doses of yeast and bacteria employed were the same. after the inoculation of bacteria, at a dosage of 20 g/hl opti’malo plus bacterial nutrient (lallemand inc, verona –italy) were added at wine in according to the manufacturers instructions. spontaneous mlf (control). this mlf process was used as a comparison test for the others processes. after crushing and destemming of grapes were added about 40 mg/l of so2, and then 20 g/l of previously hydrated saccharomyces cerevisiae (lalvin r7) yeast were inoculated. also for this thesis, at racking/post alcoholic fermentation were added 20 g/hl of opti’malo plus bacterial nutrient. all the vinification were carried out at 26°c ± 1. during the fermentative pomace contact period (10 days in all vinifications) the cap was pumping over three times a day and the temperature and must density were recorded. at the end of this period, all wines were pressed at 2 bars, racked with no added sulphur dioxide for encourage mlf (in control and paf) and stored at 25°c. after mlf, the wines were racked again and 20 mg/l sulphur dioxide was added. the wines were cold stabilised (-4°c) for 1 month and then bottled without filtration. all analyses were made in triplicate at racking and after 6 months in the bottle (12 months after racking). af was monitored by ethanol production and sugar depletion. mlf was monitored by l-malic acid degradation and l-lactic acid production. af and mlf were considered complete when residual sugars were less than 2.5 g/l and l-malic acid was less than 0.12 g/l. wine composition total acidity, volatile acidity, reducing sugars, ph, total so 2 , alcohol and total dry extract were all determined on wine according to eec regulation 2676/90. chemicals and reference compounds standards, including trans-caffeoyl-tartar ic acid, trans-p-coumaroyl-tartaric acid, caffeic acid, ferulic acid, p-coumaric acid, quercetin, myricetin, kaempferol, were supplied by sigma aldrich. while standard of (+)-catechin, (-)-epicatechin, procyanidin b1, procyanidin b2, epigallocatechin, epigallocatechin gallate were supplied by extrasynthese. the purities of the standards were all over 95%. all the solvents (methanol, acetonitrile, ethyl acetate) were hplc grade. all the solutions were obtained with distilled deionised water using carlo erba reagents. spectrophotometric analysis phenolic compounds were determined by spectrophotometric methods (di stefano et al., 1989; di stefano et al., 1997) using a uv/ vis mod lambda 25 double beam spectrophotometer (perkin elmer s.p.a.). the total anthocyanins index was expressed as malvidin 3-glucoside and calculate by the following expression: e maxvis x 16.17 x d (d=dilutions). the monomeric anthocyanins after separation and absorption on a c18 set pak cartridge were eluted with 5 ml of acetonitrile and then diluted with hydrochloric ethanol and calculated by: e maxvis x 16.17 x d (d=dilutions). the total polyphenols index expressed as (+)-catechin was measured by: e1cm, 75 0nm x 186.5 x d (d=dilutions). the total flavonoids index was expressed as (+)-catechin and calculate with the graphic method of di stefano (1989). the flavanols reactive to vanillin (flavonols vanillin assay) were expressed as (+)-catechin = δe x 290.8 x d (δe=absorbance difference between tests with and without vanillin; d=dilution). the proanthocyanidin content was determined after hot acid hydrolysis (bate-smith reaction) using a ferrous salt (feso 4 ) as catalyst and expressed as cyanidin chloride. colour intensity and hue were estimated by measuring absorbance at 420, 520 and 620 nm according to eu regulation 1990. ital. j. food sci., vol. 27 2015 313 hplc analysis the fixed acids of wine (tartaric, malic, lactic, citric and shikimic acids) were determined by an hplc isocratic elution (hplc 1100 series agilent technologies) with a phenomenex synergi 4u hydro-rp 80a (250x4.60 mm, 4 micron) with guard column, a mobile phase of phosphoric acid 10-3 m, 0.7 ml/min flow rate, 25°c and a uv detector set at 210 nm (cane, 1990). for flavans determination, the wine was separated into two fractions containing, respectively, individual catechins and oligomeric proanthocyanidins, using a c18 1g sep-pak cartridge as described by sun et al. (1999). about 5 ml of wine was adjusted to ph 7 and then filtered through a sep-pak cartridge preconditioned with h 2 o. elution was carried on with 10 ml of h 2 o to eliminate phenolic acids. after drying the cartridges with n 2 , elution was carried out with 15ml of ethyl acetate to elute catechins and oligomeric proanthocyanidins (f i + f ii). each fraction was evaporated to dryness and dissolved in methanol, followed by hplc analysis. a thermo ods rp-c18 hypersil 200x2.1 (5 μm) column with a guard column was used for flavans analysis. two ml of each extracted fraction were filtered on a 0.45 μm nylon membrane and immediately inject according to squadrito’s method (2007). separation was carried out at 30°c, the flow rate was 0.25 ml/ min and the injection volume 10 μl. the detection was set at 280 nm, using phosphoric acid 10-3 m (solvent a) and acetonitrile (solvent b). the gradient elution program was: from 91 to 86% a in ten minutes; from 86 to 82% a in ten minutes; from 82 to 60% a in ten minutes; from 60 to 40% a in five minutes; from 40 to 91% a in five minutes; equilibration time of five minutes. the peaks identification was performed comparing the retention times and absorption spectra of pure compounds (supplied from extrasynthese) and were found analogues to values reported in the literature (baoshan et al., 1998; ricardo et al., 1991). the determination of biogenic amines (ba) in wine was carried out by hplc/fld. a hewlett-packard (agilent technologies palo alto, ca, usa) 1100 series hplc instrument was used, with a fluorescence detector set at excitation and emission wavelengths of 340 and 450 nm, respectively. the samples were subjected to an automatic pre-column derivatization procedure using o-phthalaldehyde (opa reagent, agilent technologies, palo alto, ca, usa). all separations were performed on a 200 x 4.6 mm, 5-µm alltima c18 column (alltech, deerfield, il, usa), protected by a 7.5x4.6 mm guard cartridge of the same type. samples were injected into the column after being filtered through a 0.2 mm rc filter (schleicher and schuell, keen, nh, usa). the two eluents used as mobile phases were sodium acetate 50 mm (ph 7.2)/thf (96:4) v/v (eluent a) and methanol (eluent b). the elution gradient programme followed the method described by nicolini (2003). from a stock solution of 200 mg/l containing agmatine, cadaverine, phenylethylamine, histamine, putrescine, and tyramine (standards purchased by sigma-aldrich) in methanol, four diluted solution were prepared and injected: 2.5, 5.0, 10.0, 20.0 mg/l. quantification of the ba was performed with an internal standard of 10mm of norvaline solution. statistical analysis multivariate statistical analysis was per formed using r statistical software (r core team (2013), r foundation for statistical computing, vienna, austria). chemical analyses were repeated three times for each sample and the data are presented as mean ± sd. the one way analysis of variance (anova), and duncan multiple comparison test to measure variation between treatments at a probability level of p<0.05 were performed. results and discussion wines composition the musts collected from the steel tanks had the following chemical/physical characteristics: control (spontaneous mlf) 210 g/l of reducing sugars, ph 3.30 and total acidity 6.40 g/l; sim: 205 g/l of reducing sugars, ph 3.27 and total acidity 6.52 g/l; paf: 214 g/l of reducing sugars, ph 3.35 and total acidity 6.24 g/l. the winemaking process began on the 12th of october with the crushing and destemming of grapes and the yeasts inoculation for all the three experimental processes. the kinetics of af and malic acid degradation are reported in figs. 1 and 2 respectively. in table 1 it is reported the time required for the af and the mfl for each winemaking. the duration of the fermentation process was identical for the paf and the control (both 8 days), while it was longer for the sim (about 10 days). however, all alcoholic fermentations were regular and complete. lab in the sim were able to perform mlf in 23-24 days from the beginning of winemaking. the wine obtained by sequential inoculation (paf) carried out the degradation of malic acid in 40-41 days from the beginning of the winemaking. instead, the wine under went a spontaneous mlf, despite the absence of added lab, has finished the mlf after 57 days from the beginning of the vinification. therefore, the wine obtained by the sim technique has finished the mlf 33-34 days before of control wine. this data is important since time is a key factor from an economic, techni314 ital. j. food sci., vol. 27 2015 fig. 1 kinetics of alcoholic fermentation. fig. 2 time course of malic acid degradation from the start of alcoholic fermentation. table 1 time required to complete af and mlf. treatment time for af time for malic acid degradation vinification time (days) (days after bacterial inoculation) † af + mlf (days) ‡ co-inoculation (sim) 10±0 23±1 24±1 sequential inoculation (paf) 8±0 33±4 41±4 spontaneous mlf (control) 8±0 na 57±4 † mlf was considered complete when malic acid concentration was below 0.12 g/l. ‡vinification time is the time from destemming/crushing to completion of af and mlf. cal and practical point of view, for a good or ganizational management of the winery. table 2 shows the results of the chemical/physical analysis of wines at the end of alcoholic fermentation (racking) and 12 months after racking. differences were observed in the alcohol content, acidic profile, ph and total acidity. the sim wine after alcoholic fermentation showed obvious signs of the beginning of mlf. indeed, the malic acid content (1.44 ital. j. food sci., vol. 27 2015 315 g/l) was less than control (1.61 g/l) and paf (1.64 g/l) wines. in sim wine the partial transformation of malic acid has produced a certain amount of lactic acid already at racking. moreover, sim wine showed a lower total acidity and a higher ph compared to control and paf wines. this was mainly due to the transformation of a diprotic acid (malic acid) with two acidic functional groups into a monoprotic acid (lactic acid) with only one acidic functional group, with a corresponding decrease in acidity and an increase of ph. it was observed a difference in the alcohol content of wines, in particular the sim wine showed the lowest alcohol content. probably, since the sugar content of the must subjected to the sim process was slightly smaller thus less alcohol was produced. another possible explanation is linked to the lactic acid bacteria that could have used part of the reducing sugars, in addition to malic acid, as nutrients for their metabolism. this not only may have influenced the alcohol content of wine, but furthermore had furnished an increased energy for the cellular development of the bacteria resulting in the production of more volatile compounds and greater amounts of acetic acid, as it was found in sim wine. indeed, the volatile acidity expressed as acetic acid was slightly higher in the sim wine than in the other two wines. both possibilities may have contributed to the lower alcoholic contable 2 wines composition after af (racking) and after mlf (12 months after racking). at racking 12 months after racking control sim paf control sim paf alcohol (vol. %) x 12.39 ab 12.06 b 12.48 a 12.40 a 12.10 b 12.42 a s 0.08 0.07 0.08 0.12 0.08 0.09 residual sugars (g/l) x 2.40 a 2.40 a 2.40 a 2.35 b 2.42 a 2.45 a s 0.18 0.15 0.20 0.22 0.25 0.20 total dry extract (g/l) x 30.50 a 29.40 b 30.50 a 30.20 a 29.80 b 30.40 a s 2.50 1.80 2.10 2.08 2.18 2.10 ph x 3.35 b 3.53 a 3.36 b 3.45 b 3.61 a 3.44 b s 0.02 0.03 0.02 0.03 0.02 0.02 total acidity (g/l) x 7.65 a 6.15 b 7.50 ab 5.63 a 5.03 c 5.10 b s 0.38 0.42 0.45 0.35 0.40 0.39 volatile acidity (mg/l) x 0.54 b 0.60 a 0.56 b 0.55 b 0.60 a 0.50 c s 0.03 0.02 0.03 0.07 0.08 0.06 total so2 (mg/l x 24.05 a 22.10 b 22.04 b 32.10 b 58.10 a 30.10 c s 4.10 3.80 3.50 2.90 3.50 2.80 tartaric acid x 3.04 b 2.90 c 3.20 a 2.91 a 2.86 ab 2.62 b s 0.28 0.22 0.18 0.4 0.38 0.25 l-malic acid (g/l) x 1.61 ab 1.44 b 1.64 a 0.12 a 0.05 b 0.06 b s 0.09 0.10 0.12 0.01 0.00 0.00 l-lactic acid (g/l) x 0.10 b 0.60 a 0.12 b 1.45 b 1.52 a 1.41 b s 0.00 0.01 0.00 0.08 0.06 0.05 citric acid (g/l) x 0.25 b 0.27 ab 0.28 a 0.23 b 0.30 a 0.25 ab s 0.02 0.02 0.04 0.01 0.02 0.02 x, mean of three replicates; s, standard deviation. mean values followed by the same letter in a row are not significantly different at the 0.05 level of significance. tent, as it is confirmed by the findings of some other authors which have observed a delay of alcoholic fermentation and a use of the sugars of must by lab (lafon-lafourcade et al., 1983). after one year of storage, the sim wine still has a lower acid strength, represented by a higher ph and a lower total acidity compared to the other wines. the content of sulfur dioxide, in order to favour the mlf especially in the control, has been deliberately kept low. there were no significant differences in respect of tartaric acid and citric acid. polyphenolic composition and wine colour table 3 shows the polyphenolic composition and chromatic characteristics of wines after alcoholic fermentation and 12 months after racking. the effects of different mlf starts showed a marked change in the content of polyphenols in sim wine already at the end of the af. indeed, the index of total polyphenols, the total flavonoids, the total and monomeric anthocyanins contents are higher in the sim wine, with variations ranging from 5 to 17%, than in the other wines. the differences in tannins (proanthocyanidins and flavans reacting with vanillin) content between wines were not significant. after 12 months from racking, all the wines had finished the mlf thus a natural reduction of the polyphenolic compounds (total flavonoids, flavans, and an316 ital. j. food sci., vol. 27 2015 thocyanins) was observed. anyway, the total anthocyanins were still slightly higher in the sim wine compared to control and considerably higher compared to paf wine. from the other side, the content of proanthocyanidins (high molecular weight tannins) was significantly higher in the control than in the other wines. therefore, an early mlf as for the sim and paf processes, causes greater losses over time of higher molecular weight tannins (proanthocyanidins). the increase in ph observed at racking for sim wine has promoted the polymerization processes of high molecular weight tannins leading to a partial precipitation and thus their removal by pouring operations. some authors (caroline and eveline, 2011; costello et al., 2012) in tests of management of mlf have observed similar effects in respect of these compounds. it is well known that mlf can reduce the colour intensity in red wines due to numerous factors associated with the mlf (burns  et al., 2013). indeed, the aglianico wines obtained with the sim process, already at the end of alcoholic fermentation (table 3), showed a colour intensity of 8.84 which was significantly lower than paf and control wines. usually, this index is positively correlated to the anthocyanin content, in this case, considered that in sim wine there was an increase in ph mostly due to the partial mlf, despite a slightly higher anthocyanin content respect to the other wines, table 3 phenolic composition and chromatic characteristics of aglianico wines after af (racking) and after mlf (12 months after racking). at racking 12 months after racking control sim paf control sim paf total phenols (mg/l) x 1812 b 2062 a 1831 b 1674 b 1793 a 1652 ab s 66 66 37 49 52 41 total flavonoids (mg/l) x 1982 c 2388 a 2008 b 1466 b 1585 a 1583 a s 73 74 45 42 44 36 vanillin index (mg/l) v x 1026 a 1013 b 1017 b 829 b 935 a 659 c s 38 39 23 19 33 21 proanthocyanidins (mg/l) l x 2489 a 2490 a 2398 b 2135 a 1693 b 1533 c s 71 77 59 67 55 38 total anthocyanins (mg/l) x 321 ab 338 a 315 b 223 b 242 a 169 c s 15 16 19 12 12 15 monomeric anthocyanins (mg/l) x 232 ab 251 a 227 b 90 b 110 a 92 ab s 12 9 12 4 6 8 d.o. 420 nm (p.o. 1cm) x 3.49 a 2.82 c 3.06 b 2.84 a 2.54 b 2.47 ab s 0.07 0.07 0.06 0.06 0.05 0.05 d.o. 520 nm (p.o. 1cm) x 7.16 a 5.17 c 6.23 b 4.03 a 3.69 b 3.39 c s 0.20 0.16 0.18 0.10 0.01 0.01 d.o. 620 nm (p.o. 1cm) x 1.06 a 0.85 c 0.94 b 0.75 c 0.78 b 0.99 a s 0.03 0.02 0.03 0.02 0.03 0.04 colour intensity (p.o. 1cm) x 11.71 a 8.84 c 10.23 b 7.62 a 7.01 b 6.85 c s 0.20 0.10 0.14 0.10 0.12 0.08 tint (e420/e520) x 0.49 0.54 0.49 0.70 0.69 0.73 v/l index x 0.41 0.41 0.44 0.39 0.55 0.43 x, mean of three replicates; s, standard deviation. mean values followed by the same letter in a row are not significantly different at the 0.05 level of significance. a change in the balance of the ph-dependent anthocyanin pigments has determined a certain loss of the red colour. the colour reduction may also be due to the precipitation of the free anthocyanins molecules with polysaccharides and potassium bitartrate. twelve months after racking, the differences in colour intensity, although reduced, remained quite significant and the control wine still showed the highest value. also the 420 nm and 520 nm absorbances were higher in the control wine, while the hue did not show significant differences between wines. the composition of monomeric catechins and oligomeric procyanidins is shown in table 4. a common feature to all wines is the predominance of the (+)-catechin among all monomeric flavanols. among dimeric procyanidins b2 and b4 are present in greater quantities. the trimeric procyanidins were detected in all wines but in small quantities. at racking, the sim wine differed from the other two wines because of the lowest content of almost all the flavan-3-ols, with the exception of a few gallic acid esters such as epicatechin gallate, epigallocatechin gallate and procyanidin b2 gallate. less difference were found between the control and paf. after one year from racking, all wines showed a reduction in the content of (+)-catechin, (-)-epicatechin, epigallocatechin, procyanidin b1, b3 and b4, while there was a general increase of procyanidin b2, gallic acid esters and trimeric procyital. j. food sci., vol. 27 2015 317 anidins c1 and t2. the sim wine confirmed a lower content of almost all forms of monomeric catechins and oligomeric procyanidins, while the paf showed concentrations that were even higher than control wine. also in this case, a faster mlf in sim and paf from the early stages of racking had caused a lower acidic strength, resulting in a loss of these compounds. table 5 concentration (mg/l) of biogenic amines in aglianico wines. at racking 12 months after racking control sim paf control sim paf histamine x 2.78 a 2.44 c 2.65 b 3.53 a 0.24 ab 0.20 b s 0.74 0.22 0.39 0.92 0.02 0.02 agmatine x 1.45 b 1.54 a 1.57 a 1.56 c 2.41 b 2.93 a s 0.46 0.38 0.40 0.55 0.63 0.74 putrescine x 3.74 a 3.32 b 3.66 ab 10.51 a 8.48 c 9.54 b s 0.82 0.67 0.74 1.86 1.59 1.48 tyramine x 0.70 b 0.72 ab 0.74 a 0.76 b 1.40 a 0.60 c s 0.08 0.09 0.06 0.09 0.10 0.03 cadaverine x 1.42 ab 1.35 b 1.44 a 1.60 a 1.59 a 1.16 b s 0.36 0.42 0.40 0.39 0.28 0.29 phenylethylamine x 0.29 ab 0.36 a 0.27 b 0.42 b 0.60 a 0.58 ab s 0.05 0.06 0.06 0.06 0.07 0.06 total biogenic amines x 10.38 9.73 10.33 18.38 14.72 15.01 x, mean of three replicates; s, standard deviation. mean values followed by the same letter in a row are not significantly different at the 0.05 level of significance. table 4 concentration (mg/l) of monomeric catechins and oligomeric procyanidins in aglianico wines. at racking 12 months after racking control sim paf control sim paf (+)-catechin x 40.36 ab 36.7 c 41.34 a 35.06 b 28.73 c 39.68 a s 1.74 1.83 1.88 1.25 1.44 1.32 (−)-epicatechin x 25.61 b 19.24 c 27.22 a 21.98 b 16.96 c 24.59 a s 1.21 0.71 1.29 1.12 1.09 1.15 procyanidin b1 x 17.60 a 13.30 b 17.42 ab 12.09 a 7.76 c 10.39 b s 0.45 0.80 0.73 0.71 0.74 0.83 procyanidin b2 x 37.40 a 29.60 b 36.40 ab 39.06 b 33.18 c 40.95 a s 1.75 1.67 1.98 1.44 1.56 1.22 procyanidin b3 x 10.80 a 5.81 b 10.30 ab 7.52 b 4.95 c 8.84 a s 0.78 0.46 1.04 0.34 0.46 0.27 procyanidin b4 x 30.73 b 27.55 c 32.60 a 28.26 b 24.77 c 32.59 a s 1.75 1.85 1.78 1.88 1.36 1.33 procyanidin b2 gallate x 22.28 c 23.10 b 25.20 a 32.36 a 26.33 c 27.39 b s 0.97 0.85 1.04 0.98 1.44 1.65 epicatechin gallate x 3.74 b 4.30 a 3.31 c 5.73 b 4.55 c 7.12 a s 0.24 0.88 0.37 0.54 0.74 0.67 gallocatechin x 5.21 a 3.40 c 4.30 b 5.62 a 2.48 c 5.04 b s 0.24 0.23 0.27 0.38 0.55 0.29 epigallocatechin x 4.33 ab 2.41 b 4.73 a 3.99 a 3.08 b 3.48 ab s 0.37 0.46 0.63 0.74 0.74 0.74 epigallocatechin gallate x 1.35 c 3.30 a 2.37 b 1.24 c 4.45 a 4.01 b s 0.08 0.08 0.04 0.04 0.03 0.04 trimer t2 x 6.53 c 6.95 b 8.64 a 7.67 b 8.44 a 8.22 ab s 0.72 0.83 6.78 0.34 0.46 0.48 trimer c1 x 7.33 a 5.39 b 7.11 a 8.17 b 6.70 c 9.28 a s 0.77 0.71 0.84 0.66 0.53 0.79 x, mean of three replicates; s, standard deviation. mean values followed by the same letter in a row are not significantly different at the 0.05 level of significance. biogenic amines composition table 5 shows the concentrations of biogenic amines in aglianico wines. the average concentration of total amines at racking differs slightly between thesis submitted at different management of mlf, ranging from 9.73 mg/l in sim wine to 10.38 mg/l in control wine. af318 ital. j. food sci., vol. 27 2015 tion in almost all the amines investigated with respect to the control (spontaneous mlf). after 12 months from racking, the average total content of biogenic amines was lower in the wine underwent sequential inoculation compared to the co-inoculation. acknowledgments the authors thank apulia region for the financial support in the regional development program 2007/2013, axis i improvement of competitiveness in agricultural and forestry sectors, integrated projects of the production chain measure 124. authors also thank le.vin.sud company for grapes and for support in vinification. references abrahamse c.e. and bartowsky e.j. 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mlf (18.38 mg/l) was higher than co-inoculation (14.72 mg/l) and sequential inoculation (15.01 mg/l) wines. in percentage the increase was 77.0% in control, 51.3% in sim and 45.3% in paf wine. this higher percentage in the control wine could be ascribed to a release of amino acids as a consequence of yeast lysis during af and to the proliferation of labs with carboxylase activity during spontaneous mlf. putrescine was the most abundant amine, with control showing the highest amount at racking (3.74 mg/l before mfl) and also 12 months after racking (10.51 mg/l). histamine, which is thought to be the cause of various adverse reactions to wines (taylor et al., 1989; wantke et al., 1996), after 12 months increased its concentration in the wine produced with spontaneous mlf (control) wine and vice versa decreased in sim and paf wines. indeed, after 12 months histamine was almost absent in wines subjected to inoculation of lab (0.24 mg/l in sim and 0.20 mg/l in paf), while the control wine showed an higher value (3.53 mg/l) which was anyway very similar to values reported by other authors (izquierdo-cañas et al., 2008; pramateftaki et al., 2006). probably the histidine decarboxylase activity was present and active in wine produced with spontaneous mlf for the whole period of aging, instead was absent or not active in wines subjected to inoculation of lab. conclusion the simultaneous inoculation with yeast and bacteria (sim) has reduced the duration of mlf of about 33 days compared to the wine obtained without the addition of any lab (control). the simultaneous inoculation already at the end of the af showed evident signs of the onset of mlf. after 12 months from racking, there was a weakening of differences in phenolic compounds content, but the wine underwent a spontaneous mlf (control) remained more colourful and more rich in high molecular weight tannins. concerning the content of monomeric catechins and oligomeric procyanidins it was observed the predominance of (+)-catechin among the monomeric flavanols and the procyanidin b2 among the dimeric procyanidins. these compounds were lower in the co-inoculation wine compared to the other two wines. the different mlf managements led to a different evolution in the content of biogenic amines. the simultaneous (sim) and the sequential (paf) inoculation of lactic acid bacteria for the mlf led to a significant reducital. j. food sci., vol. 27 2015 319 reclassify leuconostoc oenos as oenococcus oeni [corrig.] gen. nov., comb. nov.. int. j. syst. bacteriol. 45:395-397. di stefano r., cravero m.c. and gentilini n. 1989. metodi per lo studio dei polifenoli dei vini. l’enotecnico, 25(5):83-89. di stefano r., ummarino i. and gentilini n. 1997. alcuni aspetti del controllo di qualità nel campo enologico. lo stato di combinazione degli antociani. ann. istit. sperim. enol. asti: 105-121. gafner j. 2005. stabilità biologica e amine biogene. vinidea. net, rivista internet tecnica del vino. n. 2/1. izquierdo-cañas p.m., garcía-romero e., gómez-alonso s., gonzález m.f. and palop-herreros m.l. 2008. amino acids and biogenic amines during spontaneous malolactic fermentation in tempranillo red wines. j. food compos. anal. 21(8):731-735. lafon-lafourcade s., carre e. and ribéreau-gayon p. 1983. occurrence of lactic acid bacteria during the different stages of vinification and conservation of wines. appl. environ. microbiol. 46:874-880. landete j.m., ferrer s., polo l. and pardo i. 2005. biogenic amines in wines from three spanish regions. j.  agric. food chem. 53:1119-1124. leitao m., marques a. p. and san romao m. v. 2005. a survey of biogenic amines in commercial portuguese wines. food control 16:199-204. lonvaud-funel a. 1999. lactic acid bacteria in the quality improvement and depreciation of wine. anton, leeuw, 67:317-331. lonvaud-funel a. 2001. biogenic amines in wines: role of lactic acid bacteria. fems microbiol. lett. 199:9-13. lòpez r., lòpez-alfaro i., gutièrrez a. r., tenorio c., carijo p., gonzàles-arenzana l. and santamarìa p. 2011. malolactic fermentation of tempranillo wine: contribution of the lactic acid bacteria inoculation to sensory quality and chemical composition. int. j. food science tech. 46:166-174. masqué m.c., romero s.v., rico s., elórduy x., puig a., capdevila f., suárez c., heras j.m. and palacios a.t. 2008. coinoculo di lieviti e batteri: effetti sulla qualità e sulle amine biogene. www.infowine.com, rivista internet di viticoltura ed enologia, n. 8/2. nicolini g., larcher r., and bertoldi d. 2003. indagine sul tenore di ammine libere in mosti d’uve di varietà autoctone. riv.viti. enol. 1:15-27. pramateftaki p.v., metafa m., kallithraka s. and lanaridis p. 2006. evolution of malolactic bacteria and biogenic amines during spontaneous malolactic fermentations in a greek winery. lett. in appl. microbiol. 43:155-160. ricardo da silva j.m., bourzeix m., cheynie v. and moutounet m. 1991. procyanidin composition of chardonnay, mauzac and grenache blanc grapes. vitis 30:245252. silla-santos m.h. 1996. biogenic amines: their importance in foods. int. j. food microbiol. 29:213-231. squadrito m., corona o., ansaldi g. and di stefano r. 2007. relazione fra i percorsi biosintetici degli hcta, dei flavonoli e degli antociani nella buccia dell’uva. riv. vit. enol. 3:59-70. sun b.s., pinto t., leandro m.c., ricardo-da-silva j.m. and spranger i. 1999. transfer of catechins and proanthocyanidins from solid parts of the grape cluster into wine. am. j. enol. vitic. 50(2):179-183. suriano s., ceci g. and tamborra p. 2012. impact of different winemaking techniques on polyphenolic compounds of nero di troia wine. italian food & beverage technology 70:5-15. stockley c.s. 2004. can histamine in wine cause adverse reactions for consume. australian and new zealand grapegrower and winemaker 485(77):79-82. taylor s.l., stratton j.e. and nordlee j.a. 1989. histamine poisoning (scombroid fish poisoning): an allergy-like intoxication. clinical intoxication. 27(4-5):225-240. versari a., parpinello g.p. and cattaneo m. 1999. leuconostoc oenos and malolactic fermentation in wine: a review. j. ind. microbiol. biotechnol. 23:447-455. wantke f., hemmer w., haglmüller t., götz m.,·and jarisch r. 1996. histamine in wine. int. arch. allergy immunol. 110:397-400. zùniga m., pardo i. and ferrer s. 1993. an improved medium for distinguishing between homofermentative and heterofermentative lactic acid bacteria. international journal of food microbiology, 18: 37-42. paper received july 11,2014, accepted september 23, 2014 p u b l i c a t i o n s codon italian journal of food science, 2023; 35 (2): 13–21 issn 1120-1770 online, doi 10.15586/ijfs.v35i2.2290 13 p u b l i c a t i o n s codon pretreatment of kaempferol-3-o-rutinoside protects h9c2 cells against lps-induced inflammation through the ampk/sirt1 pathway yao-yao ma1, xiao-ni zhao1, lan zhou1, sheng-nan li2, juan bai2, ling-li shi2, fang hua2*, peng zhou1* 1department of integrated traditional chinese and western medicine, anhui university of chinese medicine, anhui, p.r. china; 2school of pharmacy, anhui xinhua university, anhui, p.r. china *corresponding authors: fang hua, school of pharmacy, anhui xinhua university, anhui, p.r. china. email: happyhf6941@sina.com; peng zhou, department of integrated traditional chinese and western medicine, anhui university of chinese medicine, anhui, p.r. china. email: zhoupeng@ahtcm.edu.cn received: 14 october 2022; accepted: 17 february 2023; published: 17 april 2023 © 2023 codon publications open access original article abstract kaempferol-3-o-rutinoside (kr) is a flavonoid glycoside derived from traditional chinese medicine, plants, and tea, and has good myocardial protection. this study focuses on the mechanism of myocardial protection with kr and whether myocardial protection can be achieved by activating the adenosine monophosphate-activated protein kinase–silent mating-type information regulation 2 homolog 1 (sirtuin) (ampk/sirt1) signal pathway. molecular docking technique was used to predict the binding affinity of kr to ampk. the inflammatory injury model of h9c2 cells was established by lipopolysaccharide (lps) induction. h9c2 cell proliferation was detected by cell counting kit-8 assay. the apoptosis rate was measured by flow cytometry. levels of interlukin (il)-1β, il-6, and tumor necrosis factor-α (tnf-α) were determined by enzyme-linked-immunosorbent serologic assay. ampk and sirt1 expression levels were quantified through reverse transcription-polymerase chain reaction and western blotting assay. results indicated that kr has a certain binding affinity with ampk. kr could increase the growth of h9c2 inhibited by lps, reduce apoptosis rate, and reverse the elevated levels of il-1β, il-6, and tnf-α. furthermore, kr could suppress the expression levels of ampk and sirt1, and ampk-specific inhibitor (compound c) could significantly counteract the activation of kr, indicating that its anti-inflammatory effect is achieved by regulating the ampk/sirt1 signaling pathway. keywords: ampk/sirt1 signaling pathway, inflammation, kaempferol-3-o-rutinoside, myocardial protection introduction at present, the incidence of cardiovascular diseases (cvd) and accounted mortality are on the rise in china. cvd mortality ranks first among all causes, accounting for more than 40% of the disease deaths in china (zhao et al., 2019). chronic heart failure (chf) is a common cardiovascular disease and a serious end-stage of various heart diseases, which seriously threatens the health of patients (d’alessandra et al., 2020). ventricular remodeling (vr) after acute myocardial infarction (ami) is the pathophysiological basis for the development of chf (zhao et al., 2021). the myocardium in the infarcted area becomes thinner and dilated, while the myocardium in the non-infarcted area becomes hypertrophic and elongated, leading to the enlargement and deformation of the ventricular cavity, resulting in decrease of ventricular systolic function (schumacher et al., 2019). the resulting vr extends through the whole course of the disease and is an important risk factor for long-term death. therefore, to explore how to prevent or reverse vr after ami and delay or inhibit the pathophysiological process of ami after chf is an inevitable choice to effectively improve the prognosis of ami patients and prevent and treat chf. mailto:happyhf6941@sina.com mailto:zhoupeng@ahtcm.edu.cn 14 italian journal of food science, 2023; 35 (2) ma y-y et al. materials and methods chemicals and materials the purity of kr is more than 98% (desite biotechnology co. ltd., chengdu, china). ampk-specific inhibitor (compound c) was obtained from abmole bioscience inc. (usa). lipopolysaccharide (lps, cat. no. l8880) was obtained from solarbio science & technology co. ltd. (beijing, china). fetal bovine serum (fbs, bl201a) and dulbecco’s modified eagle medium (dmem, bl304a) were purchased from biosharp (china). penicillin–streptomycin solution was purchased from shandong sparkjade biotechnology co. ltd. (china). mouse tumor necrosis factor-α (tnf-α; mm-0180r1), interlukin (il)-1β (mm-0047r1), il-6 (mm-0190r1) and enzyme-linked-immunosorbent serologic assay (elisa) kits were bought from meimian (jiangsu, china). antip-ampkα (af3423) and anti-sirt1 (df6033) were purchased from affinity biosciences (melbourne, australia). anti-glyceraldehyde 3-phosphate dehydrogenase (gapdh, 380626) was purchased from zen-bio science (chengdu, china). trizol (g3013), sybr (g3326), and annexin v-fitc/pi cell apoptosis detection kit (g1511) were purchased from servicevbio biotechnology co. ltd. (wuhan, china). molecular docking the cb-dock molecular docking platform was used to calculate center and size of the target cavity, and ligand was uploaded for molecular docking. protein data bank (pdb) formats of ampk (pdb code: 4cff) (xiao et al., 2013) and kr or a-769662 in spatial data file (sdf) format were input to cb-dock for molecular docking (liu et al., 2020). in vitro experiment cell culture and treatment dmem containing 10% fbs, 1% penicillin–streptomycin in dmem were used to culture h9c2 cells at 37°c and 5% co2. h9c2 cells in logarithmic growth phase were trypsinized with 0.25% and seeded in six-well plates. for pharmacodynamic study, h9c2 cells were divided into the following three groups: control group, model group (10-μg·ml-1 lps), and kr group (25-μm kr). h9c2 cells were pre-incubated with kr for 12 h. then, h9c2 cells were stimulated with 10-μg·ml-1 lps for 6 h (fanghua). to further investigate the effect of kr on ampk, h9c2 cells were divided into the following five groups: control group, model group, kr group, compound c group (10-μm compound c), and kr+compound c group (25-μm kr + 10-μm compound c). h9c2 cells were adenosine monophosphate-activated protein kinase (ampk) is an evolutionarily conserved serine/threonine protein kinase, known as the “cellular energy regulator.” it plays an important role in maintaining cell and energy balance (herzig and shaw, 2018). ampk exists in the form of α, β, and γ, and is directly activated by interaction between sestrin1 and thr 172, which have been demonstrated in cardiomyocytes (yuan et al., 2022). when activated, ampk maintains cell energy balance, turning on the productive catabolic pathway while shutting down the energy-consuming synthetic pathway, which contributes to cell survival and cardiovascular system function when cells are hypoxic (wu and zou, 2020; zhao et al., 2022). studies have shown that normal energy metabolism of the myocardium is the material basis for maintaining the stability of cardiac internal environment and myocardial systolic function. after myocardial ischemia, the substrate of myocardial tissue energy supply is transformed from fatty acid β oxidation to anaerobic glycolysis of glucose, leading to the accumulation of free fatty acid (ffa) and lactic acid and insufficient myocardial energy supply (wang et al., 2019). therefore, it is possible to improve the activity of ampk and promote its anti inflammatory effect to achieve anti-vr after ami. kaempferol-3-o-rutinoside (kr) is a flavonoid glycoside extracted from traditional chinese medicine, plants, and tea. a previous study has found that lu´an guapian tea has the highest content of kr (bai et al., 2017). evidence has found that kr is beneficial for human health, including anti-diabetic activity, and cerebral protective, hepatoprotective, anti-cancer, and cardioprotective effects (hua et al., 2018; li et al., 2021; wang et al., 2015, 2021; yu et al., 2021). the outstanding effect of kr is that it has a good protective effect on myocardial cells. previous studies have found that kr improves hemodynamics and cardiac function, reduces myocardial fibrosis and cardiomyocyte apoptosis, and decreases excessive release of inflammatory factors by inhibiting nuclear factor kappa b–oligomerization domain-like receptor family pyrin domain-containing 3–caspase-1 (nf-κb/nlrp3/caspase-1) signaling pathway. the myocardial protective effect of kr is related to its regulation of nf-κb/nlrp3/caspase-1 signaling pathway (hua et al., 2022). however, the activation of nf-κb is regulated by the upstream ampk–silent mating type information regulation 2 homolog 1 (sirtuin) (sirt1) signaling pathway, which leads to myocardial cell damage (chen et al., 2018). it is speculated that kr may be one of the important pathways to prevent and treat myocardial cell injury by regulating the inflammatory response of ampk/sirt1 signal pathway. in this study, molecular docking was used to predict the target ampk of kr. it was verified by in vitro experiments, which were used to explore the role of kr in vitro underlying molecular mechanisms. italian journal of food science, 2023; 35 (2) 15 kaempferol-3-o-rutinoside pretreatment protects h9c2 cells against lps-induced inflammation via ampk/sirt1 pathway reaction system on ice in rnase free tube: the dose of rna was determined by using eppendorf biophotometer plus (od 1.8–2.0), μl 5*reaction mix, 3-μl supreme enzyme mix, and diethyl pyrocarbonate (depc)-treated water was replenished to 20 μl. after mixing, the program was run as follows: at 25°c for 10 min, random primers were paired with rna templates to remove genomic dna, and at 55°c for 15 min, reverse transcription reaction and double-stranded dna (dsdnase) were rapidly inactivated. reverse transcriptase was inactivated at 85°c for 5 min, and the final complementary dna (cdna) product was obtained. rt-pcr was performed using the sybr green pcr kit. all pcr reactions were performed up to 10-µl volume using the roche lightcycler 96. the amplification procedure includes the following: pre-denaturing at 95°c for 5 min, then denaturing at 95°c for 15 s, annealing at 60°c for 60 s, and performing pcr under 40 cycles. the melting curve is from 60°c to 95°c. mrna expression levels of ampk and sirt1 were analyzed using 2-δδcq method, and were normalized to gapdh. the primer sequences used are shown in table 1. results molecular docking results molecular docking results showed that kr had a certain binding activity with ampk (table 2 and figure 1). vina score of kr was significantly higher than its ligand (sbi0206965). the docking results were further verified by in vitro experiments. kr promoted h9c2 cells proliferation under lps treatment h9c2 cells were cultured with kr at 25 μm for 12 h. cck8 assay confirmed that kr significantly increased lps-induced h9c2 cell survival (p < 0.01). kr appreciably increased the growth of h9c2 inhibited by lps (figure 2). pre-incubated with kr, compound c, or kr+compound c for 12 h, and stimulated with lps for 6 h. detection of h9c2 cells proliferation by cell counting kit-8 (cck-8) assay h9c2 cells were seeded into 96-well plates at a density of 3 × 103 cells/well. the cells were incubated with 10% cck-8 (sparkjade, 10 μl) solution at 37°c for 1 h. after mixing, optical density (od) at 450 nm was measured with labserv k3 microplate reader. detection of apoptosis by flow cytometry cell culture supernatants were collected, digested with ethylenediaminetetraacetic acid (edta)-free trypsin, and centrifuged. after obtaining h9c2 cells, washed twice with phosphate-buffered saline (pbs) solution, and harvested and dissociated into single-cell suspensions. a 100-μl cell suspension was added with 5-μl annexin v-fitc and 5-μl propidium iodide (pi), mixed and incubated for 10 min at room temperature in the dark (tang et al., 2022). then, 400-μl staining buffer was added prior to detection by beckman dxflex flow cytometer (beckman, usa) and analyzed using flowjo v10. detection of il-1β, il-6, and tnf-α by elisa h9c2 cells were seeded in a 6-well plate (5 × 104 cells/ well) and incubated at 37°c and 5% co2. with appropriate intervention, the supernatant was collected by centrifugation at 1,000 g for 10 min. il-1β, il-6, and tnf-α were detected by elisa. detection of protein expression by western blotting assay the bicinchoninic acid assay (bca) kit was used to determine the concentration of protein. proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (sds-page). after lysis of h9c2 cells in lysate, the supernatant was removed after centrifugation, 4x loading buffer was added, and the protein was fully denatured through boiling. the proteins were separated on 10% polyacrylamide gel and transferred to a polyvinylidene fluoride (pvdf) membrane; then blocked in 5% skimmed milk powder for 2 h and incubated overnight at 4°c. specific proteins were identified using antibodies against p-ampk (1:1,000), sirt1 (1:500), and gapdh (1:5,000). the membranes were incubated for 2 h the next day with horseradish peroxidase (hrp)-conjugated secondary antibodies corresponding to primary antibody. electrogenerated chemiluminescence (ecl) (biosharp, bl520b) was used to develop and fix, and gel imager (tanon 5200 imaging system, china). the experiment was repeated thrice. image j was used to analyze gray values of the bands. detection of messenger rna (mrna) expression by reverse transcription-polymerase chain reaction (rt-pcr) after kr treatment, defrost template rna and reagent on ice, mix, and centrifuge the solution. preparation of table 1. primers used in rt-qpcr. primers sequence (5′ → 3′) ampk forward 5′-gccgagaagcagaagcacgac-3′ reverse 5′-gcttgcccaccttcactttccc-3′ sirt1 forward 5′-agtaacagtgacagtggcacatgc-3′ reverse 5′-cctccgtcagctccagatcctc-3′ gapdh forward 5′-aagagggatgctgcccttac-3′ reverse 5′-atccgttcacaccgaccttc-3′ 16 italian journal of food science, 2023; 35 (2) ma y-y et al. table 2. docking results of sbi-0206965 and kr with ampk. compound vina score cavity score center (x, y, and z) size (x, y, and z) sbi-0206965 –7.0 459 49, –43, 34 23, 23, 23 kr –8.5 459 49, –43, 34 26, 26, 26 (a) (b) (c) (d) figure 1. docking results of sbi-0206965 and kr with ampk. (a) 3d complex of sbi-0206965–ampk. (b) amino acid binding of sbi-0206965–ampk. (c) 3d complex of kr-ampk. (d) amino acid binding of kr to ampk. kr: kaempferol-3-o-rutinoside. kr reduced the apoptosis rate of h9c2 cells induced by lps compared to the control group, the apoptosis rate was significantly higher in the model group (p < 0.001). in contrast, kr can significantly reduce the apoptosis rate (p < 0.001) (figure 3). kr reduced the overexpression of inflammatory cytokines levels of il-1β, il-6, and tnf-α were increased significantly in the model group, compared to the control group (p < 0.05 and <0.01). kr could reverse the elevated levels of il-1β, il-6, and tnf-α (p < 0.05 and <0.01) (figure 4). kr could inhibit the overexpression of inflammatory cytokines. c el l v ia bi lit y (% ) 0.0 10µg/mllps kr 25µm 0.5 1.0 1.5 figure 2. kaempferol-3-o-rutinoside resists the proliferation of h9c2 cells inhibited by lps. the values are expressed as mean ± sd (n = 3). **p < 0.01 vs the model group. italian journal of food science, 2023; 35 (2) 17 kaempferol-3-o-rutinoside pretreatment protects h9c2 cells against lps-induced inflammation via ampk/sirt1 pathway control (a) (b) q1105 104 104 106 102 103 102 fitc p i p i 101 100 100 0.94 q4 95.0 q2 3.14 q3 0.91 q1105 104 104 106 102 103 102 fitc fitc p i 101 100 100 0.79 q4 80.2 q2 18.1 q3 0.96 model q1105 104 104 106 102 103 102 fitc p i 101 100 100 0.55 q4 95.8 q2 3.24 q3 0.40 kr a po pt os is r at e( % ) 0 10µg/mllps kr 25µm 5 10 15 20 figure 3. kaempferol-3-o-rutinoside reduces the apoptosis rate of h9c2 cells induced by lps. (a) the apoptosis rate is measured by flow cytometry. (b) statistical chart of apoptosis rate. the values are expressed as mean ± sd (n = 3). ***p < 0.001 vs the model group. il -1 β (p g/ m l) 0 (10µg/ml)lps kr (25µm) 10 20 30 40 50 il -6 ( pg /m l) 0 (10µg/ml)lps kr (25µm) 50 100 150 200 t n f -α ( pg /m l) 0 (10µg/ml)lps kr (25µm) 200 100 400 300 500(a) (b) (c) figure 4. kaempferol-3-o-rutinoside reduced the overexpression of inflammatory cytokines. (a) il-1β, (b) il-6, and (c) tnf-α. the values are expressed as mean ± sd (n = 6). *p < 0.05, **p < 0.01 vs the model group. kr increased the mrna expression levels of ampk and sirt1 compared to the control group, the mrna expression levels of ampk and sirt1 decreased considerably in the model group (p < 0.01). compared to the model group, kr was able to reverse mrna overexpression (p < 0.01) (figure 5). the results pointed out that the protective effect of kr on lps-induced myocardial cell damage was related to the activation of ampk/sirt1 signaling 18 italian journal of food science, 2023; 35 (2) ma y-y et al. il-6, and tnf-α (sun et al., 2021). inflammatory response plays an important role in the occurrence and development of vr. therefore, it is very important to treat the over release of inflammatory factors against vr after ami. ampk system is a novel anti-inflammatory signaling pathway, connecting with sirt1 (sirtuin), p53, foxo3α, and p300 to control the occurrence and development of inflammation (chen et al., 2022). therefore, ampk activity can be improved to promote its anti-inflammatory effect and achieve the purpose of anti-vr after ami. sirt1 is a class of highly conserved protein deacetylases that catalyzes the deacetylation of histone and non-histone lysine residues, and is associated with important life activities, such as cell survival, proliferation, aging, apoptosis, cell metabolism, and energy restriction (wu, 2021). sirt1 exists in the nucleus and cytoplasm and plays an anti-inflammatory role, which is considered a potential therapeutic target for cvd (chen et al., 2020). ampk promotes the transcription and activity of nicotinamide (nam) phosphoribosyltransferase (nampt) synthesized by nicotinamide adenine dinucleotide+ (nad+), increases the level of [nad+]/ [nadph], and mediates the activation of sirt1, thus playing an anti-inflammatory function (hurley et al., 2021). sirt1 then interacts with the rela/p65 subunit of the nf-κb complex, which could deacetylate nf-κb p65 subunit lys310 to inhibit nf-κb transcriptional activity (deng et al., 2021). in short, the anti-inflammatory mechanism of vr after ami is closely related to the ampk/sirt1/ nf-κb signaling pathway, that is, activation of ampk may up-regulate sirt1 expression, inhibit the activity of nf-κb inflammatory proteins, and down-regulate the expression of downstream inflammatory factors, thereby inhibiting the inflammatory response. kaempferol-3-o-rutinoside has a good cardiovascular protective effect, so it is important to further study r el at iv e a m p k m r n a le ve ls (2 –δ δ cq ) 0.0 (10µg/ml)lps kr (25µm) 0.5 1.0 1.5(a) r el at iv e s ir t 1 m r n a le ve ls (2 –δ δ cq ) 0.0 (10µg/ml)lps kr (25µm) 0.5 1.0 1.5(b) figure 5. kaempferol-3-o-rutinoside increased the mrna expression levels of (a) ampk and (b) sirt1. the values are expressed as mean ± sd (n = 3). **p < 0.01 vs the model group. pathway. next, the mechanism was verified by using ampk-specific inhibitors. effects of kr and compound c on the protein expression level of ampk/sirt1 signal pathway protein expression levels of ampk and sirt1 in the model group decreased considerably, compared to the control group (p < 0.05 and <0.01). after treatment with kr, the expression levels of p-ampk and sirt1 was augmented (p < 0.05 and <0.01). after the intervention of compound c, the expression levels of ampk and sirt1 were returned to the original level. after the combined application of kr and compound c, compound c could significantly counteract the activation of kr (p < 0.05) (figure 6). therefore, kr could play a role in myocardial protection by activating the ampk/sirt1 signal pathway. effects of kr and compound c on the mrna expression level of ampk/sirt1 signal pathway the mrna expression levels of ampk and sirt1 in the model group was consistent with the protein expression. after kr treatment, the mrna expression levels of p-ampk and sirt1 were increased (p < 0.01). after combined application of kr and compound c, the effect of compound c on mrna expression was consistent with those of protein, both of which have the effect of resisting kr (p < 0.01) (figure 7). discussion an acute inflammatory response occurs in the short term after ami, and inflammatory cytokines, including il-1β, italian journal of food science, 2023; 35 (2) 19 kaempferol-3-o-rutinoside pretreatment protects h9c2 cells against lps-induced inflammation via ampk/sirt1 pathway pa m p k /g a p d h e xp re ss io ns (r el at iv e pr ot ei n le ve l) 0.0 lps kr compound c lps kr25 compound c gapdh p-ampk 0.5 1.0 1.5 s ir t 1/ g a p d h e xp re ss io ns (r el at iv e pr ot ei n le ve l) 0.0 lps kr compound c lps kr25 compound c gapdh sirt1 0.5 1.0 1.5 (c) (d) (a) (b) figure 6. effects of kaempferol-3-o-rutinoside (kr) and compound c on the proteins of ampk/sirt1 signal pathway. (a) picture of western spots of p-ampk, (b) p-ampk. (c) picture of western spots of sirt1, (d) sirt1. the values are expressed as mean ± sd (n = 3), **p < 0.01, *p < 0.05 vs the model group; **p < 0.01 vs the kr group. r el at iv e a m p k m r n a le ve ls (2 –δ δ cq ) 0.0 lps kr compound c 0.5 1.0 1.5(a) r el at iv e s ir t 1 m r n a le ve ls (2 –δ δ cq ) 0.0 lps kr compound c 0.5 1.0 1.5(b) figure 7. effects of kaempferol-3-o-rutinoside (kr) and compound c on the proteins of ampk/sirt1 signal pathway. (a) p-ampk, (b) sirt1. the values are expressed as mean ± sd (n = 3). **p < 0.01 vs the model group; **p < 0.01 vs the kr group. 20 italian journal of food science, 2023; 35 (2) ma y-y et al. bai, and ling-li shi analyzed the data. the manuscript was written by yao-yao ma. all authors approved the final version of the paper. conflict of interest the authors stated that they had no conflict of interest to declare. data availability the data that supported the findings of this study are available from the corresponding authors upon reasonable request. acknowledgment this study was supported by the outstanding youth scientific research project of anhui universities (2022ah030158), natural science foundation of the anhui higher education institutions of china (kj2021a1167), and the open fund of state key laboratory of tea plant biology and utilization (skltof20210112). references bai w.x., wang c., wang y.j., zheng w.j., wang w., wan x.c., et  al. 2017. novel acylated flavonol 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org/10.1016/j.mad.2020.111215 d’alessandra y., chiesa m., carena m.c., beltrami a.p., rizzo p., buzzetti m., et al. 2020. differential role of circulating micrornas to track progression and pre-symptomatic stage of chronic heart failure: a pilot study. biomedicines. 8(12): 597. https://doi. org/10.3390/biomedicines8120597 its mechanism. kr has a protective effect on the tnfhuman  umbilical vein  endothelial cells (huvec) injury model established in vitro, and its mechanism is related to its anti-inflammatory and endothelial cell protection (yu et al., 2021). previous research has demonstrated that kr could decrease the protein expression levels of tlr4, myd88, and nf-κb in lps-induced h9c2 cell injury, indicating that it inhibits inflammatory response through tlr4/myd88/nf-κb signaling pathway (hua et al., 2021). our latest finding has indicated that kr could decrease il-1β level and inhibited the expression levels of nf-κb, nlrp3, asc, caspase-1 p20, gsdmd, and il-1β, suggesting that the protective effect of kr was related to regulating the nf-κb/nlrp3/caspase-1 signaling pathway (hua et al., 2022). thus, our studies found that kr is closely related to nf-κb. therefore, it is of great significance to explore whether kr elucidates its myocardial protective effect by regulating the upstream signaling pathway of nf-κb. in this study, molecular docking results showed that kr could bind to ampk, and its therapeutic effect and correlation with the ampk/sirt1 signaling pathway was further studied by in vitro experiments. the in vitro results have shown that kr could reduce apoptosis rate, reverse the elevated levels of il-1β, il-6, and tnf-α, and suppress the expression levels of ampk and sirt1, indicating that its anti-inflammatory effect could be achieved by regulating ampk/sirt1 signaling pathway. conclusion in this study, the molecular docking prediction combined with in vitro validation was performed to study the mechanism of myocardial protection with kr. the protective mechanism of kr on the myocardium was further improved. however, a limitation of this study is that the mechanism of action is still not exhaustive. in the future study, gene silencing and gene overexpression of ampk or sirt1 must be added to explore the mechanism of kr. acknowledgments this study was supported by the open fund of state key laboratory of tea plant biology and utilization (skltof20210112), and natural science foundation of the anhui higher education institutions of china (kj2021a1167). author contributions fang hua and peng zhou designed the study and revised the manuscript. yao-yao ma, xiao-ni zhao, and lan zhou carried out the experiments. sheng-nan li, juan https://doi.org/10.1021/acs.jafc.7b00239� https://doi.org/10.1155/2022/7655142� https://doi.org/10.1155/2022/7655142� https://doi.org/10.1016/j.metabol.2018.03.004� https://doi.org/10.1016/j.metabol.2018.03.004� https://doi.org/10.1016/j.mad.2020.111215� https://doi.org/10.1016/j.mad.2020.111215� 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ole_link38 ole_link71 p u b l i c a t i o n s codon italian journal of food science, 2023; 35 (1): 79–90 issn 1120-1770 online, doi 10.15586/ijfs.v35i1.2332 79 p u b l i c a t i o n s codon use of salicylic acid during cultivation of plants as a strategy to improve its metabolite profile and beneficial health effects humberto ramos-sotelo, marely g. figueroa-pérez* agricultura sustentable y protegida. universidad tecnológica de culiacán, culiacán, méxico *corresponding author: marely g. figueroa-pérez, culiacán-imala, km 2, los ángeles, 80014 culiacán rosales, sinaloa. email: marely_100@hotmail.com received: 2 february 2023; accepted: 13 february 2023; published: 9 march 2023 © 2023 codon publications open access review abstract chemical elicitors in plants during cultivation have been applied in soil, hydroponic solutions, or sprayed on the leaves to induce physiological changes and stimulate the production of bioactive compounds. salicylic acid (sa) is a phenolic compound present in plants with multiple functions, including stimulus of plant growth and induction of plant defense responses under conditions of stress. recently, the use of sa as elicitor has generated much interest, due to the growing number of studies demonstrating its positive effects in fruits, vegetables, and herbs on the induction of phytochemicals, mainly phenolic compounds, alkaloids, saponins, carotenoids, among others. the health benefits of plant materials treated with sa are mainly their antioxidant capacities determined by the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (abts) and 2,2-diphenyl-1-picrilhidrazil (dpph) assays and anti-inflammatory properties determined in vitro, as well as hypoglycemic and hypolipidemic properties and renal protection evaluated by in vivo studies. therefore, the exogenous application of sa during cultivation of different plants could be an alternative to increase their economic value and could be the basis for designing standardized procedures in the production of bioactive compounds. keywords: antioxidant capacity; bioactive compounds; elicitation; health; salicylic acid introduction secondary metabolites of plants comprise a large group of compounds that are synthesized during their growth and do not play any role in development, photosynthesis, or reproduction. however, these compounds are crucial in several important processes, such as plant defense against environmental stresses or attack by pathogens (cohen and kennedy, 2010). plants are an important source of secondary metabolites, which have been used for the production of drugs, due to their beneficial health properties, which are associated with a protective effect against oxidative processes (baenas et al., 2014). plants growing under unfavorable environments, such as water deficit, extreme heat or cold, oxygen deficiency, among others, result in an accelerated expression of some genes related to the synthesis of secondary metabolites. this effect has been associated with the generation of reactive oxygen species (ros) during environmental stress (jenks, 2007). it has been shown that similar effects can be produced intentionally during the cultivation of plants to improve their content of bioactive metabolites (baenas et al., 2014). optimizing the bioactive compounds’ composition of plant food would be a cost-effective strategy for enhancing nutrition and preventing diseases among the population. moreover, this approach could improve the economic value of some medicinal plants (singh and dwivedi, 2018). one of the most used strategies for this purpose is the exogenous application of elicitors. mailto:marely_100@hotmail.com 80 italian journal of food science, 2023; 35 (1) ramos-sotelo h et al. and others. for some alkaloids, the nitrogen atom is not in a carbon ring (bribi, 2018). most alkaloids are derived from amino acids, including tryptophan, phenylalanine, lysine, tyrosine, histidine, and ornithine. some nonamino acid compounds such as terpenoids, purine nucleotide, and polyketide are also precursors of these compounds (ziegler and facchini, 2007). glucosinolates are thioglucosides with a common structure, characterized by side chain (r) with different aliphatic, aromatic, and heteroaromatic carbon skeletons, all derived from amino acids by a process of long chain elongation, hydroxylation, or oxidation (vig et al., 2009). terpenes constitute the largest class of natural products, and these are extensively used in the industrial sector. the most important biological terpenes include carotenoids and phytosterols. carotenoids are a group of plant pigments responsible for bright red, yellow, and orange hues in many fruits and vegetables, including alphacarotene and beta-carotene, lutein, lycopene, β-cryptoxanthin, and zeaxanthin, among others (singh and sharma, 2015). on the other hand, phenolic compounds include a large group that can be classified according to the number of phenol rings that they contain; phenolic acids, stilbenes, lignans, flavonoids (flavones, flavonols, flavanones, flavanols, anthocyanins, chalcones, dihydrochalcones, anthocyanins, and isoflavones), and tannins. phenolic acids are constituted chemically at least by one aromatic ring, which has one hydrogen atom substituted by a hydroxyl group. these metabolites are divided in two groups: hydroxybenzoic acids and hydroxycinnamic acids (vuolo et al., 2019). stilbenes are a group of phenylpropanoid-derived compounds characterized by a 1,2-diphenylethylene backbone (c6-c2-c6). the lignans are a group of polyphenols comprising a large variety of individual structures, mostly consisting of two phenylpropanoids c6–c3 linked by a bond between the central atoms of the respective side chains (position 8 or β), also called β-β’ bond (haminiuk et al., 2012). flavonoids have a c6–c3–c6 general structural backbone in which two c6 units (ring a and ring b) consisting of two phenyl rings contain a heterocyclic pyrane ring (c). due to the hydroxylation pattern and variations in the chromane ring (ring c), flavonoids are divided into different sub-groups. chalcones, though lacking the heterocyclic ring c, are still categorized as members of the flavonoid family (tsao, 2010). flavanols or flavan-3-ols are often commonly called catechins. these compounds and epicatechin can form polymers, which are often referred to as proanthocyanidins. these are traditionally considered to be condensed tannins and the hydrolysable tannins are gallotannin or tannic acid (haminiuk et al., 2012). many of these compounds are naturally synthesized by plants in response to attack of pathogens (baenas et al., 2014). among the elicitors mostly studied in recent years is salicylic acid (sa), an endogenous regulator of growth in plants that controls several physiological processes, such as systemic defense signaling against biotic and abiotic stress. several studies have shown that exogenous application of sa also generates different changes in plant physiological processes and reactions, such as prevention of ethylene production (khan et al., 2013), increase in the growth parameters (khandaker et al., 2011), and modulation of the bioactive metabolite synthesis (ananieva et al., 2004). in this review, we will discuss the studies conducted in the last decades regarding the exogenous application of sa during the cultivation of plants and the effect of this treatment on bioactive metabolites of the crop. it describes the broad variety of plant secondary metabolites induced by sa, such as phenolic compounds, terpenoids, alkaloids, among others, and their health beneficial properties, which can provide an overview of the possible fields of application of the sa-elicited plant foods. finally, we conclude our review by providing suggestions that can be applied during plant cultivation to increase the production of specific secondary metabolites. classification and synthesis of bioactive compounds plant-based foods have generated great interest in research in recent years, in addition to providing macronutrients and micronutrients. these are a rich source of bioactive compounds, which, although not being classified as nutrients, or considered essential for human health, have an important beneficial impact on some diseases (guerriero et al., 2018). in nature, there are three large groups of bioactive compounds, which include nitrogenous and sulfur (s) substances, terpenoid compounds, and the bioactive widely studied, the phenolic compounds (cohen and kennedy, 2010). nitrogen (n) and sulfur are the main plant nutrients and serve as constituents of proteins and several other important organic compounds that exert biological activities (such as alkaloids and glycosinolates). furthermore, these compounds control yield and quality of plants (ibrahim et al., 2012). alkaloids are n-containing organic compounds that often contain one or more rings of carbon atoms, where nitrogen atoms are usually located and whose position of those in the carbon ring varies with different alkaloids; pyrrolidine, pyridine, quinoline, indole, steroidal, diterpenoid, italian journal of food science, 2023; 35 (1) 81 use of salicylic acid during cultivation the term elicitor originally included only molecules capable of inducing the synthesis of phytoalexins; however, nowadays, this concept is used for all compounds that induce any type of plant defense. elicitors can be classified as biotic or abiotic, physical or chemical, and depending on their origin and molecular structure. biotic elicitors include lipopolysaccharides, oligosaccharides, and polysaccharides, such as pectin and cellulose, chitosan, chitin and glucans, galacturonides, some proteins including cellulase, cryptogein, glycoproteins, oligandrin and pectolyase, as well as other compounds of complex composition, such as fungal spores, mycelia cell wall, and microbial cell wall. on the other hand, abiotic elicitors can be chemicals, for example, acetic acid, benzothiadiazole, silicon, ethanol, ethene, hydrogen peroxide, inorganic salts, and metal ions or physical as an altered gas composition, chilling, co2, drought, extreme temperatures, high pressure, high or low osmolarity, uv irradiation, saline stress, wounding and ozone, among others. furthermore, some plant hormones acts as elicitors, either when they are produced by the plant in response to some pathogen attack or when they are applied exogenously in low concentrations to the plants during cultivation. examples of these compounds are jasmonic acid, methyl jasmonate, ethylene, cytokinin, gibberellin ga3 methyl salicylate, and sa (baenas et al., 2014). salicylic acid sa is a phenolic compound, consisting of a ring linked to a hydroxyl and a carboxyl group. this acid regulates physiological functions in plants, when it is exogenously regarding phenolic compounds, these are basically derived of the malonic and shikimic acid pathways, whose precursors are derived from glycolysis and the pentose phosphate pathway, resulting in the production of different compounds, such as simple flavonoids, phenolic acids, tannins, coumarins, and anthocyanins. nitrogen-containing compounds, such as alkaloids, glucosinolates, and cyanogenic glycosides, are synthesized from amino acids like tryptophan, tyrosine, and tryptophan, also derived of the shikimic acid pathway. terpenoids are units of isoprene, synthesized from acetyl coa or 3-phosphoglycerate (cohen and kennedy, 2010) (figure 1). classification of elicitors in plants secondary metabolites are ubiquitous in plants and serve different purposes; for example, they provide the blue color to blueberries and the red color to blood-oranges. they also contribute to the bitterness of grapefruits, to the astringency of unripe persimmon, and the texture of red wines (vuolo et al., 2019). during plant growth, these phytochemicals are synthesized as a defense mechanism; therefore, when plants are exposed to an adverse situation, such as pathogens attack, ultraviolet (uv) radiation, drought, heavy metals, nutrient deficiency, increased soil salinity, and other types of environmental stresses, the synthesis rate of secondary metabolites increases (figure  2). these produce changes in the phytochemical profile after the stress situation, and these changes depend on several factors, such as the type and intensity of the stress, the plant species, and the type of metabolites involved (kulbat, 2016). figure 1. general outline of biosynthetic pathways of secondary metabolites in plants. primary carbon metabolism erytrose-4phosphate acetyl coa 3-phosphoglycerate shikimic acid pathway aromatic amino acids mevalonic acid pathway phenolic compounds (flavonoids, coumarins, lignin, tannins) nitrogen containig secondary metabolites (alkaloids, glucosionates, cyanogenic glycosides) terpenoid compounds (isoprene, saponins, mono, di and triterpenes, carotenoids) methylerythritol 4-phosphate pathway malonic acid pathway 82 italian journal of food science, 2023; 35 (1) ramos-sotelo h et al. precursors: in the cytoplasm starting from phenylalanine by the phenylpropanoid pathway and in the chloroplast through the isochorismate pathway (gondor et al., 2016) (figure 3). phenylalanine ammonia-lyase (pal) is the enzyme that initiates the phenylpropanoid pathway by transforming phenylalanine into trans-cinnamic acid and nh3 through a nonoxidative deamination. transcinnamic acid participates as a precursor of diverse phenolic compounds biosynthesis, such as lignin, lignans, and flavonoids. furthermore, some studies indicate that sa is also synthesized from phenylalanine, via trans-cinnamic acid, which is then converted into sa via two intermediates: ortho coumaric acid or benzoic acid, depending on the plant species (dempsey et al., 2011). plants can use three biosynthetic routes to produce benzoic acid: a β-oxidative and a nonoxidative route from cinnamoyl co-a and a nonoxidative route from trans-cinnamic acid (zhang et al., 2014). regarding the isochorismate pathway, it is known that chorismate is synthesized in the plastid, and is then converted into isochorismate by an isochorismate synthase (ics). after that, an amino acid conjugation of isochorismate, followed by a enzymatic conversion or a spontaneous decomposition results in the synthesis of sa and the gene responsible for this conversion is pbs3 (lefevere et al., 2020). due to the importance of both pal and ics in sa accumulation, it is possible that the pal and ics routes are integrated by a metabolic or regulatory grid in the biosynthesis of this hormone. the recently identified genes pbs3 and eps1 have demonstrated to be important in applied, plays an important role in the germination of seeds, either by inhibiting germination or increasing seed vigor, depending on the concentration. recent studies also suggest that sa is a regulator of photosynthesis, it controls chloroplast and leaf structure, stomatal closure, chlorophyll and carotenoid accumulation, and the activity of important enzymes related to photosynthesis, such as rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and carbonic anhydrase. in addition, sa participates in the regulation of the alternative oxidase (aox) route both in thermogenic and nonthermogenic plants through the induction of gene expression. aox combines ubiquinol oxidation with the reduction of molecular oxygen to produce water in a reaction that is not sensible to inhibitors of the cytochrome oxidase pathway, which allows the control of atp synthesis to keep the homeostasis and regulate the growth of plants. these growth-promoting effects of sa in plants are also related to the increase in photosynthesis, to changes in the hormonal status, transpiration, and stomatal conductance (lefevere et al., 2020). other studies have demonstrated that sa is involved in the regulation of flowering by interacting with components of the photoperiod pathway through a co-independent branch. sa is also involved in the regulation of senescence, which is characterized by a decrease in the photosynthetic activity, a loss of antioxidant capacity, and therefore higher levels of ros (dempsey et al., 2011). sa is widely distributed in plants at different basal levels depending on plant species. sa is synthesized by two different compartmentalized routes with different figure 2. production of bioactive metabolites in plants under environmental stress factors. uv radiation chemical priming agents pathogen and herbivore attack cold heat drought heavy metals salinity nutrient deficiency italian journal of food science, 2023; 35 (1) 83 use of salicylic acid during cultivation the pathogen-induced sa production and to encode enzymes that catalyze reactions involved in the synthesis of sa (dempsey et al., 2011). pathtways induced by sa as elicitor sa has been intensively studied in recent years due to its function as an endogenous signal mediating local and systemic plant defense responses against pathogens (aftab et al., 2010). in addition, it has also been demonstrated that sa is involved in the plant response to different types of abiotic stresses such as drought, chilling, heavy metal toxicity, heat, and osmotic stress, playing a crucial role in the regulation of physiological and biochemical processes in plants (vicente and plasencia, 2011). it has been demonstrated in different plant species that exogenous application of sa induces synthesis and accumulation of antioxidant compounds. the molecular mechanism of elicitation by sa is complex and depends on the concentration applied, the growth stage and nutritional uptake by plants, environmental conditions, etc. sa regulates the pal enzyme activity, which catalyzes key biosynthetic reactions to produce secondary metabolites that act as a defense mechanism against environmental stresses (puthusseri et al., 2012). signal recognition of sa is mediated by receptors and binding sites located on the plasma membrane, such as npr1, npr3, and npr4, which activate a complex cascade of events that is initiated by inhibiting the activity of catalase, the enzyme responsible for breaking down hydrogen peroxide in water and oxygen. this produces an increase in the h2o2 levels, which produces an elevation in oh radicals that generate an oxidative stress status in cells, which in turn initiates a series of signaling cascades that involve the activation of mitogen-activated protein kinases (mapks) and g proteins, which leads to an increase in the production of secondary metabolites (khalil et al., 2018). calcium flux also participates as signaling in plant cells after sa exogen application (rodas-junco et al., 2013). in unstimulated cells, ca2+ concentrations in cytosol are maintained at lower levels; when plants are attacked by a pathogen, their abscisic acid levels increase in response to the attack, which activates calcium channels, causing a ca2+ influx into cytosol. it is known that exogenous sa also produces an increase in abscisic acid levels in the plant, which causes the same effect as a pathogen attack, increasing the calcium concentrations in the cytosol. this ca2+ influx is involved in diverse physiological and cellular processes, associated with ca2+-binding proteins, calcium-dependent kinases (cdpks), phospholipases, and through secondary messengers such as inositol 1,4,5triphosphate (ip3) and diacylglycerol (dag). cdpks trigger diverse signaling cascades to coordinate cellular processes such as regulation of the oxidative stress, hormonal signaling, and gene expression (herrera-vásquez et al., 2015). furthermore, studies have shown that g-proteins play an important role in stimulating ion channels, phospholipases a, c, and d, ros generation, and apoptosis. g-protein activation stimulates the accumulation of camp, ip3 and dag, which triggers the activation of pka and pkc. this causes the phosphorylation of mapks, resulting in gene expression that leads to several enzymatic reactions involved in secondary metabolite production (rodas-junco et al., 2015; ruelland et al., 2014) (figure 4). figure 3. biosynthesis of sa in plants. pal: phenylalanine ammonia-lyase; pbs3: proteasome subunit beta type-3; eps1: er-retained pma1 suppressing; ics: isochorismate synthase. salicylic acid chorismate cinnamate isochorismate benzoate phenylalanine salicylic acid salicylic acid phenylpropanoid pathway isochorismate pathway pal ics pbs3 eps1 pbs3/eps1 84 italian journal of food science, 2023; 35 (1) ramos-sotelo h et al. sa and bioactive compounds there are a large number of studies that demonstrate the beneficial health properties of secondary metabolites in plants, which vary depending on the compound’s structure and concentration. it has been reported that some nitrogen compounds, such as alkaloids, among which are choline, trigonelline, sitsirikine, and others, exert several pharmacological effects, including anti-inflammatory, anti-diarrheal, anti-cancer activities, and therapeutic potential for hypertension, hyperlipemia, diabetes, cardiovascular system, and central nervous system diseases (qian et al., 2017). sulfur substances predominate in some vegetables of the cabbage family, onions, garlic, etc. garlic and onion contain sulfur compounds, such as allicin (2-propene1thiolsulfinate), thiosulfinates, diallyl disulfide, and s-alk(en)yl-l-cysteine sulfoxides, which have been associated with health properties such as antioxidants, antibacterial, anti-inflammatories, and inhibitors of the proliferation of human tumor cells (higuchi et al., 2003). many terpenoids have biological activities, particularly against certain cancers and eye disease (johnson, 2002). on the other hand, phytosterols, mainly sitosterol, stigmasterol, and campesterol, are associated with the reduction of risk of coronary heart disease by improving ldl cholesterol concentrations. they also have anti-cancerous properties and are immune system modulators (chawla and goel, 2014). phenolic compounds have shown various beneficial effects on human health, such as anti-aging, anti-inflammatory, antioxidant, and anti-proliferative activities. so they have been pointed out as an alternative to improve the incidence of certain chronic diseases, such as diabetes, cancer, and cardiovascular diseases, through the control of oxidative stress (lin et al., 2016). there are an increasing number of studies that evaluate the exogenous application of sa to different plant species and the effects on its content of bioactive compounds (table 1). the foliar application of sa has increased the total phenolic compounds and total flavonoids on solanum lycopersicum (javanmardi and akbari, 2016), amaranthus tricolor l (khandaker et al., 2011), ocimum basilicum (gharib, 2007), achillea millefolium (gorni and pacheco, 2016), and mentha pipperita (figueroaperez et al., 2014, 2015, 2018). figure 4. general mechanism induced by elicitation with sa. mapks: mitogen-activated protein kinases, camp: cyclic adenosine monophosphate, ip 3 : inositol 1,4,5-triphosphate, dag: diacylglycerol, and pka and pkc: protein kinase a and c. abscisic acid g-protien activation mapks activation mapks activation oxidative stress secondary metabolism gene expression transcriptional reprogramming citoplasmatic acidification phospholipases a,c and d camp, ip3 and dag accumulation pka and pkc activation enzymetic reactions catalase h2o2 oh o2 p h2o receptors npr1, npr3/npr4 h+ ca2+ cu2+ fe2+ ca2+ ca2+ ca2+ k+/cl– salicyclic acid italian journal of food science, 2023; 35 (1) 85 use of salicylic acid during cultivation on the other hand, it has been shown that sa elicitation could improve other bioactive compounds. for example, saponins and alkaloids have been detected in mentha pipperita (figueroa-pérez et al., 2018). isatis tinctoria l. hairy root cultures treated with sa increased the alkaloid content (qing-yan et al., 2019). sa applied to root cultures of stemona curtisii significantly improved the production of the alkaloids, namely, oxyprotostemonine, stemocurtisine, and stemocurtisinol (chotikadachanarong et al., 2011). it has been found that foliar application of 2 mm sa in peppermint plants increased their phytosterols contents, such scholine, trigonelline and vinblastine (figueroaperez et al., 2015). carotenoid content, mainly crocin, was highly improved in crocus sativus plants after sa application (tajik et al., 2015). likewise, foliar application of sa in yarrow (achillea millefolium l.) plants resulted in linear increases in chlorophyll content, as well as a high production of essential oils. furthermore, addition of sa to the medium of alternanthera tenella leaves cultured in vitro induced an increase in betacyanins of the leaves (gorni and pacheco, 2016). the molecular response mechanism underlying sa elicitation involves the upregulation of some genes related to the alkaloids and flavonoids synthesis, such as aromatic amino acid decarboxylase (aadc), yucca monooxygenase (yucca), 4-coumarate coenzyme a ligase (4cl), chalcone synthase (chs), chalcone isomerase (chi), and flavonoid 3′-hydroxylase (f3′h). specifically, yucca gene exhibits the greatest transcriptional abundance for the maximal alkaloid production in isatis tinctoria l. hairy root cultures after sa elicitation, which suggested that this gene might be more sensitive and key for inducing alkaloid biosynthesis (qing-yan et al., 2019). health benefits of licited plants with sa there are several studies on the effect of elicitation with sa on the metabolite content of plants. the main effects with health benefits determined in these plants have been related to their antioxidant capacities. it has been shown that the application of salicylic acid 1 mm during cultivation of saffron (crocus sativus l.) increased the crocin content (a carotenoid responsible for the color of flowers) and improves the antioxidant activity of stigmas.(tajik et al., 2015). furthermore, elicitation with 300 μm sa in plant cell suspension cultures of thevetia peruviana increased the antioxidant capacity by 1.66-fold determined by the abts assay, compared to the control culture (mendoza et al., 2018). blanch et al. (2020) found that foliar application of sa (100 mg/l) during cultivation of vitis vinifera cv syrah plants, significantly increased (3-fold) the antioxidant capacity of the fruits, this effect was associated to increases of some phenolic compounds, such as myricetin, trans-resveratrol and phenolic acids, mainlygallic, chlorogenic, caffeic and trans-ferulic acids. furthermore, it has been shown that 12 mm sa applied postharvest to kinnow mandarin under cold storage increased twofold the total antioxidant capacity of the fruits determined by dpph test. sa treatment also improved the activity of the antioxidant enzymes catalase, peroxidase, and superoxide dismutase. these enzymes help to scavenge free radicals in the fruit, which can damage the cells under stress. these effects were related to phenolic compounds and ascorbic acid content (haider et al., 2020). lee et al. (2013) showed that sa-treated aloe vera adventitious roots cultured on ms liquid media decreased the anti-inflammatory activity in uvb-treated mouse skin cells, suppressing the activity of cox-2, nf-kb, and ap-1. foliar spraying of 2 mm sa in ammi visnaga potentiated the radical scavenging activity of plant extracts using dpph assay and these effects were higher for drought stressed aerial parts sprayed with 2 mm sa. the cytotoxic activity of extracts of ammi visnaga were evaluated against different cell lines as liver cancer (hepg2), breast cancer (mcf-7), lung cancer (a549), and colon cancer (caco2), and the major effect was observed for the methanolic extracts of the fruits, roots, aerial parts, and umbels against mcf7 cell line and fruits for hepg2 cell line (osama, 2019). it also has been reported that application of 2 mm sa during 60 min to centella asiatica (l.) leaves effectively inhibited nitric oxide (no) production in lps-stimulated raw 264.7 macrophage cells, related to the reduction in the transcription level expression of inos in a dose-dependent manner, which suggests that elicitation with sa increased anti-inflammatory activity in centella asiatica leaves (buraphaka and putalun, 2020). on the other hand, it has been demonstrated that the administration of infusion prepared with 2 mm sa-treated peppermint to diabetic rats for 4 weeks, decreased serum glucose (up to 25%) and increased serum insulin levels (up to 75%) as compared to diabetic controls. furthermore, these infusions prevented oxidative damage on pancreas β-cells, improved serum lipid profile, and decreased hepatic damage in diabetic rats (figueroa-perez et al., 2015). also, they decreased the renal accumulation of 14 inflammation-related proteins, associated with glomerular hypertrophy, tubular damage, expansion of mesangial matrix, and cell death in diabetic rats (figueroa-pérez et al., 2018). in addition, it has been 86 italian journal of food science, 2023; 35 (1) ramos-sotelo h et al. ta bl e 1. b io ac tiv e m et ab ol ite s’ c on te nt o f fo od p la nt s in r es po ns e to th e ap pl ic at io n of s al ic yl ic a ci d du ri ng it s cu lti va tio n. n o. p la nt s pe ci es d os e an d m od e of a pp lic at io n of s a ta rg et c om po un ds a nd in cr ea se r ef er en ce 1 to m at o (s ol an um ly co pe rs ic um ) fo lia r a pp lic at io n at 4 50 m g/ l, 3 w ee ks a fte r f ru iti ng u nd er gr ee nh ou se c on di tio ns to ta l p he no lic c om po un ds (2 .1 -fo ld ); to ta l fl av on oi ds (1 .2 -fo ld ); vi ta m in c (2 .8 -fo ld ) (j av an m ar di a nd a kb ar i, 20 16 ) 2 c hi ne se c hi ve (a lli um tu be ro su m ) fo lia r a pp lic at io n of 5 00 a nd 1 50 μ m s a c hl or op hy ll, p he no ls a nd fl av on oi ds , v ita m in c , a nd v ol at ile co m po ne nt s (w an g et a l., 2 02 2) 3 (c or ia nd ru m s at iv um ) s up pl em en ta tio n w ith 2 25 m g/ l s a o n th e gr ow th m ed iu m d ur in g 30 d ay s g al lic , b en zo ic , f er ul ic a nd 3 -o c af fe oy lq ui ni c ac id s, q ue re ce tin 3o -r ut in oo si de , a nd g lu cr on id e an d ka em pf er ol -3 -o -r ut in os id e (k dh im e t a l., 2 02 0) 4 r ed a m ar an th (a m ar an th us tri co lo r l .) fo lia r a pp lic at io n at 1 0− 5 m u nd er g re en ho us e co nd iti on s 1 w ee k af te r s ow in g to ta l p he no lic c om po un ds (1 .2 -fo ld ); be ta cy an in s (1 .3 -fo ld ); ch lo ro ph yl l ( 1. 3fo ld ) (k ha nd ak er e t a l., 2 01 1) 5 s w ee t b as il (o ci m um ba si lic um l .) fo lia r a pp lic at io n at 1 m m to 1 -m on th o ld p la nt s un de r c on tro lle d en vi ro nm en ta l c on di tio ns to ta l fl av on oi ds (1 .9 -fo ld ); to ta l p he no lic c om po un ds (1 .3 -fo ld ); to ta l fl av an ol s (1 .7 -fo ld ) (k ar al ija a nd p ar ić , 2 01 7) 6 pe pp er m in t ( m en th a pi pe rit a) fo lia r a pp lic at io n at 0 .5 a nd 2 m m , t w o do se s 45 a nd 6 0 da ys af te r p la nt in g r os m ar in ic a ci d (1 .7 -fo ld ); he sp er id in (1 .5 -fo ld ); ga lla to ca te ch in ga lla te (2 .8 -fo ld ); qu er ce tin (1 .6 -fo ld ); se rja ni c ac id 3β -a ra bi no py ra no si de (9 -fo ld ); st ig m as te ry l 3 βd -g lu co py ra no si de (2 -fo ld ); tri go ne lli ne (1 .8 -fo ld ) (f ig ue ro a pe re z et a l., 2 01 4, 20 15 ) 7 b ro cc ol i ( b ra ss ic a ol er ac ea e) d ai ly e xo ge no us s pr ay in g at 1 00 µ m to 7 -d ay -o ld s pr ou ts o n da ys 3, 5 a nd 7 in do le g lu co si no la te (1 .3 -fo ld ) (p ér ez -b al ib re a et a l., 2 01 1) 8 ya rr ow (a ch ill ea m ill ef ol iu m ) fo lia r a pp lic at io n at 0 .5 m m 2 0 da ys a fte r t ra ns pl an tin g th e se ed lin gs c hl or op hy ll (1 .6 -fo ld ); es se nt ia l o ils (2 -fo ld ); to ta l p he no lic co m po un ds (1 .5 -fo ld ) (g or ni a nd p ac he co , 2 01 6) 9 c ha m om ile (m at ric ar ia ch am om ill a) fo lia r a pp lic at io n at 7 m m o n 6w ee kol d pl an ts in s ta ge o f le af ro se tte h er ni ar in (2 .4 -fo ld ); z) a nd (e )2βdgl uc op yr an os yl ox y4m et ho xy ci nn am ic (1 .8 -fo ld ) (d uč ai ov á et a l., 2 01 3) 10 s t j oh n’ sw or t ( h yp er ic um pe rfo ra tu m ) fo lia r a pp lic at io n at 2 m m in a s in gl e do se a nd h ar ve st ed 7 d ay s la te r to ta l p he no lic c om po un ds (1 .9 -fo ld ); ul ig in os in b (1 .6 -fo ld ) (d e m at os n un es e t a l., 20 14 ) 11 m ar jo ra m (o rig an um m aj or an a) fo lia r a pp lic at io n at 1 m m o n 2m on th o ld p la nt s, a t t w o do se s: af te r 7 5 da ys a fte r s ow in g an d 1 w ee k la te r c hl or op hy ll (1 .4 -fo ld ); pr ol in e (1 .6 -fo ld ); m ic ro el em en ts c on te nt (1 .2 -2 -fo ld ) (g ha rib e t a l., 2 00 7) 12 g in ge r ( zi ng ib er o ffi ci na le r os co e) fo lia r a pp lic at io n at 1 m m a t t he s ec on d le af s ta ge o nc e a w ee k fo r 4 w ee ks m yr ic et in (2 -fo ld ); fis et in (2 -fo ld ); m or ie n (1 .6 -fo ld ); an th oc ya ni n (2 -fo ld ) (g ha se m za de h et a l., 2 01 2) 13 s ca rle t s ag e (s al vi a co cc in ea ) fo lia r a pp lic at io n at 1 m m s a th re e tim es , e ve ry 7 d ay s to p la nt s gr ow in g un de r s al t s tre ss c on di tio ns to ta l p he no lic c om po un ds (1 .2 -fo ld ); to ta l c ar ot en oi ds (1 .4 -fo ld ) (g rz es zc zu k et a l., 2 01 8) 14 th ym e (t hy m us v ul ga ris ) fo lia r a pp lic at io n at 3 m m e ve ry 2 1 da ys fo r 2 m on th s to 1 -m on th ol d pl an ts to ta l fl av on oi ds (2 -fo ld ); to ta l p he no lic c om po un ds (2 -fo ld ); ka em pf er ol -3 -g lu co si de (2 -fo ld ); ap ig en in 7 -o -g lu cu ro ni de (2 -fo ld ); ch lo ro ge ni c ac id (1 .6 -fo ld ); ro sm ar in ic a ci d (1 .3 -fo ld ); er io ci tri n (2 -fo ld ); di hy dr oq ue rc et in (2 .3 -fo ld ) (k ha lil e t a l., 2 01 8) 15 s w ee t w or m w oo d (a rt em is ia an nu a l. ) tw en ty -o ne -d ay -o ld p la nt s gr ow in g in a h yd ro po ni c so lu tio n su pp le m en te d w ith 1 00 µ m s a fo r 5 d ay s c ar ot en oi ds (1 .2 -fo ld ); ar te m is in in (1 4fo ld ); di hy dr o ar te m is in ic ac id (5 -fo ld ) (k um ar i e t a l., 2 01 8) 16 a lo e ve ra (a sp ho de lo id ea e) th irt yfiv eda yol d ad ve nt iti ou s ro ot s gr ow in g in a h yd ro po ni c so lu tio n su pp le m en te d w ith 2 m m s a fo r 7 d ay s a lo e em od in (1 1fo ld ); ch ry so ph an ol (1 3fo ld ) (l ee e t a l., 2 01 3) (c on tin ue s) italian journal of food science, 2023; 35 (1) 87 use of salicylic acid during cultivation shown that hypolipidemic properties of common beans sprouts can be significantly improved by elicitation with 1 and 2 mm sa, which decreased tag intestinal absorption in rats fed with a high fat and fructose (hff) diet and supplemented with bean sprouts (10%), this beneficial effect was associated to an increase in hesperidin and soysaponin-i contents of elicited sprouts (mendozasanchez et al., 2019). future trends research oriented to the production of functional foods has been growing over the last years due to the increasing interest of people to consume natural products. bioactive metabolites extracted from medicinal plants have a great therapeutic value for which they are used all over the world. the food industry continues to look for ingredients with nutritional and nutraceutical properties, to develop functional products with elevated health beneficial properties (singh and dwivedi, 2018). however, in many cases, the cultivation conditions of the raw material used to produce nutraceutical foods are not controlled, which generate variations in its bioactive metabolite content, and therefore, its health beneficial properties. furthermore, in some cases, the plant has a low potential for chemical synthesis of these compounds; thus, it is important to establish a regulation of natural products used in the food industry and generate strategies to produce quality nutraceutical foods (baenas et al., 2014). sa as an elicitor may be a complementary tool to breeding programs, production management, or genetic engineering applications. the controlled short-time elicitation with sa at low doses, during the cultivation of some medicinal plants, can be used by the producers to obtain healthier products with enhanced bioactive metabolites content. also, these preharvest treatments with sa can be of great interest for the pharmaceutical industry as tools to enhance the extractable yields of specific active compounds in plants with medicinal properties. understanding how a plant changes its content of bioactive metabolites in response to a specific sa treatment would increase the economic value of medicinal plants and could be the basis for designing standardized procedures that generate high-quality nutraceutical foods. on the other hand, it would be of great interest for the evaluation of nutraceutical properties of sa-elicited plants, including biological studies, to demonstrate the potential to produce safe and valuable nonpharmacological alternatives for human health through this strategy, which may provide a new approach for disease prevention and treatment.ta bl e 1. c on tin ue d. n o. p la nt s pe ci es d os e an d m od e of a pp lic at io n of s a ta rg et c om po un ds a nd in cr ea se r ef er en ce 17 c an ol a (b ra ss ic a na pu s l. ) o ne -w ee kol d se ed lin gs g ro w in g in n ut rie nt s ol ut io n su pp le m en te d w ith 5 µ m s a c hl or op hy ll a (2 -fo ld ) (m on ire h et a l., 2 01 1) 18 q ui no a (c he no po di um qu in oa ) fo lia r a pp lic at io n at 4 00 m g/ l du rin g ve ge ta tiv e gr ow th a t 4 5 an d 60 d ay s af te r s ow in g c hl or op hy ll a (1 .5 -fo ld ) a nd b (2 .5 -fo ld ); to ta l c ar ot en oi ds (2 .4 fo ld ); to ta l p he no lic c om po un ds (1 .5 -fo ld ) (a bd a lla h et a l., 2 01 5) 19 a rt em is in in (a rt em is ia a nn ua l) fo lia r a pp lic at io n at 1 m m a t 1 0da y in te rv al s st ar tin g fro m 3 0 da ys a fte r p la nt in g an d en di ng a t d ay 9 0 a rt em is in in (1 .5 -fo ld ); to ta l c hl or op hy lls (1 .3 -fo ld ); to ta l ca ro te no id s (1 .2 -fo ld ) (a fta b et a l., 2 01 0) 20 w he at (t rit ic um a es tiv um ) 0. 5 m m s a w as a dd ed to th e hy dr op on ic s ol ut io n of 2 -w ee kol d pl an ts a nd c ol le ct ed 7 d ay s la te r q ue rc et in (9 -fo ld ); m yr ic et in (4 .8 -fo ld ); ru tin (1 .9 -fo ld ) (g on do r e t a l., 2 01 6) 21 b er ga m ot (m on ar da d id ym a) fo lia r a pp lic at io n of s al ic yl ic a ci d at a c on ce nt ra tio n of 1 m m h yd ro xy ci nn am ic a ci ds , fl av on oi ds , a nd p he no lic c om po un ds (s kr yp ni k et a l., 2 02 2) 22 c en te lla a si at ic a (l .) 2 m m s a w as a dd ed to le av es fo r 4 0 m in tr ite rp en oi ds a ro un d 13fo ld (a si at ic a ci d, m ad ec as si c ac id , as ia tic os id e, a nd m ad ec as so si de ) (b ur ap ha ka a nd p ut al un , 20 20 ) 23 a lo e ve ra (a sp ho de lo id ea e) 4 m m s a w as a dd ed to th e nu tri tiv e so lu tio n of 3 -w ee kol d pl an ts an d co lle ct ed 1 4 da ys la te r a lo e em od in (5 .6 -fo ld ); ch ry so ph an ol (1 2fo ld ) (l ee e t a l., 2 01 3) 24 n ia ga ra r os ad a (v iti s la br us ca ) g ra pe fo lia r a pp lic at io n of 1 a nd 2 m m ol l −1 s a in th e pr eh ar ve st pe rio d r ut in , c ya ni di n3, 5di gl uc os id e an d 3o -g ly co si di c de lp hi ni di n (g om es e t a l., 2 02 1) 25 fl am e gr ap es (v iti s vi ni fe ra ) s ix a pp lic at io ns o f 0. 25 , 1 , a nd 2 m m o f s a in th e ve ra is on s ta ge . p he no lic c om po un ds , a nt ho cy an in s, a nd fl av on oi ds (v az qu ez e t a l., 2 02 2) 88 italian journal of food science, 2023; 35 (1) ramos-sotelo h et al. conclusion the controlled use of elicitors of sa as preharvest treatment of some medicinal plants could be an effective strategy to obtain tailored foods with enhanced health-promoting compounds. exogenous application of sa to fruits, vegetables, and herbs during cultivation produces significant increases in the content of bioactive metabolites, such as phenolic compounds, alkaloids, saponins, vitamins, carotenoids, among others. these changes in the plant could improve its health beneficial properties, such as antioxidant, antidiabetic, anticancer, and antiobesogenic, among others. however, since the effects of these elicitors depend on many factors, including the plant species, it is important to conduct studies oriented to elucidate the specific effect of sa on the plant of interest, to establish protocols that result in the controlled production of bioactive compounds. references abd allah, m.m.s., el-bassiouny, h.m.s., elewa, t.a.e. and el-sebai, t.n., 2015. effect of salicylic acid and benzoic acid on growth, yield and some biochemical aspects of quinoa plant grown in sandy soil. international journal of chemtech research 8(12): 216–225. https://sphinxsai.com/2015/ch_vol8_ no12/1/(216-225)v8n12ct.pdf aftab, t., khan, m.m.a., idrees, m., naeem, m. and moinuddin. 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_hlk44420993 _30j0zll #436_carballo_bozza ital. j. food sci., vol 29, 2017 123 paper sensory properties and physico-chemical changes in the biceps femoris muscle during processing of dry-cured ham from celta pigs. effects of cross-breeding with duroc and landrace pigs r. bermúdez1, d. franco1, j. carballo*2 and j.m. lorenzo1 1centro tecnológico de la carne de galicia, rúa galicia nº 4, parque tecnológico de galicia, san cibrán das viñas, 32900 ourense, spain 2área de tecnología de los alimentos, facultad de ciencias de ourense, universidad de vigo, 32004 ourense, spain *corresponding author. tel.: +34 988387052; fax: +34 988387001 e-mail address: carbatec@uvigo.es abstract we investigated how cross-breeding celta pigs with landrace and duroc pigs affected the physico-chemical properties and sensory characteristics of the biceps femoris muscle during the manufacturing of dry-cured ham. the intramuscular fat (imf) content was significantly (p<0.001) affected by cross-breed: the imf content of hams from the duroc x celta crosses (13.92%) was higher than that of hams from pure-bred celta pigs (8.03%), and the imf content of hams from the landrace x celta crosses was intermediate (12.27%). instrumental colour parameters were also slightly affected by cross-breed: hams from cross-bred pigs were yellower (cie b*-value) and lighter (cie l*-value) than hams from the pure-bred celta pigs. at the end of the process, shear force did not differ significantly (p>0.05) between groups, although the values were lowest in hams from duroc x celta crosses. in sensory analysis, panellists described hams from cross-bred pigs as of softer texture (p<0.01) and juicier (p<0.01) than hams from the pure-bred celta pigs. keywords: biceps femoris muscle, crossbreeding, dry-cured ham, physico-chemical characteristics, sensory properties ital. j. food sci., vol 29, 2017 124 1. introduction the celta breed was the typical breed of pig raised on farms in galicia (nw spain) until the middle of the 20th century, when it underwent a strong decrease in numbers until near disappearance (< 200 head) due to the introduction of improved breeds and crosses (gómez and lorenzo, 2013). however, in recent years native pig breeds have become highly appreciated for their rusticity and also for the quality of their meat products. drycured meat products have an important added value for the pig industry, and production of celta pigs is mainly focused on obtaining raw meat to manufacture products such as dry-cured ham (bermúdez et al., 2012), dry-cured “lacón” (lorenzo et al., 2014) and sausages (gómez and lorenzo, 2013). dry-cured meat products represent a large proportion of the meat products on the european market, especially in the mediterranean countries, and dry-cured ham is recognised as a high quality product of increasing economic importance (jiménezcolmenero et al., 2010). however, the quality of the end product is closely linked to the characteristics of the raw material, especially those related to the degree of marbling and the fatty acid composition, which in turn depend on factors such as breed, slaughter age and finishing feed (bermúdez et al., 2012; garcía-gonzález et al., 2008). in this regard, franci et al. (2007) concluded that breed had a marked effect on the physicochemical and sensory properties of tuscan dry-cured ham. current pig breeding schemes in europe are based on a backcross or on a three-four cross. in spain, the most common cross is one that uses different breeds such as landrace, large white, pietrain and belgian landrace. during the last few years, the duroc breed has been introduced by national breeders to supply dry-cured meat producers who prefer hams with higher levels of intramuscular fat (gou et al., 1995). one of the options most often used to improve the productive parameters of the celta pig is to cross this breed with the duroc or landrace breed (franco et al., 2014). franco et al. (2014) concluded that cross-breeding with landrace and duroc lines affected the carcass characteristics and meat quality. therefore, the landrace and duroc genotypes may affect the physico-chemical and sensorial properties of celta dry-cured ham. the aim of this study was to evaluate how cross-breeding celta pigs with landrace and duroc pigs affects the physico-chemical and sensorial properties of the bicep femoris muscle throughout the manufacturing of drycured ham. 2. materials and methods 2.1. animals and management a total of 52 pigs (26 entire females [ef] and 26 castrated males [cm]) were divided into three groups according to genotype: 16 pure-bred celta pigs (c), 20 landrace x celta (c × l) cross-bred pigs, and 16 duroc x celta (c × d) cross-bred pigs. the pigs in each group were balanced for gender (half males/half females), they were born at around the same time and the live weight at birth of all pigs was similar. all of the celta pigs were registered in the record of births in the farm stud book. animals were reared all together in an outdoor system by porco celta (a pig farming co-operative operating in the province of lugo, galicia, nw spain). the pigs were fed ad libitum a commercial diet (17% protein, 2.4% fat and 3250 kcal/kg metabolic energy) and were provided free access to water. as the pigs were reared in a natural environment, part of the diet was obtained from natural vegetation. campsite style huts and trees in the area provided shade. the pigs were reared following the recommendations of the legislation for pig welfare and protection ital. j. food sci., vol 29, 2017 125 (council directive 2008/120/ec, 2009). the pigs were slaughtered when they reached live weights of 167.30±11.65, 168.90±14.81 and 165.43±7.54 kg (p>0.05) for the c, c×l and c×d groups respectively; these weights were achieved at an age of around 12 months for c group, and at around 10 months for the c×l and c×d groups. the animals were transported to the abattoir the day before slaughter. the pigs from different groups were not mixed at any time, and various measures were taken to minimize stress. pigs were slaughtered in an accredited abattoir (matadero municipal de sarria, lugo, spain) and were stunned using carbon dioxide, according to the specifications outlined in the spanish legislation. 2.2. samples ham pieces were obtained after refrigerating the carcasses for 24 h at 4 °c. a total of 90 ham pieces (30 randomly chosen from each group) were used in the study. raw pieces were dry salted with an excess of coarse salt (approximately 0.5 kg/kg of ham). a pile was formed by alternating layers of ham pieces and salt. the pieces were thus totally covered with salt. ham pieces were salted for 11 days in a salting room at 2-5 °c and 90-95% relative humidity. after the salting stage, the pieces were removed from the pile and then brushed, washed and transferred to a post-salting room where they were held for 120 days at 3-6 °c and around 85-90% relative humidity. after the post-salting stage, the pieces were ripened for 115 days in a room where the temperature was gradually increased to 30 ºc and the relative humidity was gradually decreased to 40% for adequate drying of the thighs. the hams were then left to mature for a further 11 months (“bodega” step) in a chamber at 12-24 ºc and 70-80% relative humidity. samples were taken from the fresh pieces, and at the following times: end of the salting stage, end of the post-salting period, end of the drying-ripening stage and after 165 and 330 days of the “bodega” step. at each stage, a total of five ham pieces from each group were randomly collected and analyzed. hams were transported to the laboratory under refrigerated conditions (< 4 °c) for analysis. once in the laboratory, the entire pieces were skinned, boned, and the biceps femoris (bf) muscles were obtained. the muscle samples were vacuum packed and stored at -30 ºc for no more than four weeks until analysis. 2.3. determination of ph, water activity and colour parameters the ph of the samples was measured using a digital ph-meter (thermo orion 710 a+, cambridgeshire, uk) with a penetrating probe. water activity was assessed using a water activity meter fast-lab (gbx, romans-sur-isère cédex, france), previously calibrated with sodium chloride and potassium sulphate solutions. colour measurements were made with a cm-600d colorimeter (minolta chroma meter measuring head, osaka, japan). briefly, at each stage of the process the muscle from each ham piece was cut and, after a blooming period of 30 min, the colour of the slices was measured three times. the cielab colour space lightness (l*), redness (a*) and yellowness (b*) were determined. before each series of measurements, the instrument was calibrated with a white ceramic tile. 2.4. determination of the chemical composition moisture, fat, ash and protein (kjeldahl n x 6.25) were quantified according to the respective iso recommended standards 1442:1997 (iso, 1997), 1443:1973 (iso, 1973), 936:1998 (iso, 1998) and 937:1978 (iso, 1978). total chlorides were quantified according to the charpentier-volhard method (iso standard 1841-1:1996) (iso, 1996). ital. j. food sci., vol 29, 2017 126 2.5. warner-bratzler (wb) test a texture analyzer ta-xt2 (stable micro systems ltd., godalming, surrey, uk) was used to perform warner-bratzler (wb) test. the samples for wb shear test were obtained by cutting pieces of approximately 1 x 1 x 2.5 cm (height x width x length). the pieces were cut across the fibers with a wb shear blade with a triangular slot cutting edge (1 mm thickness) at a crosshead speed of 3.33 mm/s. maximum shear force, shear firmness and total work required to cut the samples were determined. 2.6. assessment of lipid oxidation lipid stability was evaluated using the method proposed by vyncke (1975) with the modifications described by lorenzo and carballo (2016). briefly, a meat sample (2 g) was dispersed in 5% trichloroacetic acid (10 ml) and processed in an ultra-turrax homogenizer (ika t25 basic, staufen, germany) for 2 min. the homogenate was maintained at -10 °c for 10 min and centrifuged at 2360 x g for 10 min. the supernatant was filtered through a whatman no. 1 filter paper. the filtrate (5 ml) was reacted with a 0.02 m tba solution (5 ml) and incubated in a water bath at 96 °c for 40 min. the absorbance was measured at 532 nm. thiobarbituric acid reactive substance (tbars) values were derived from a standard curve for the quantification of malondialdehyde (mda), constructed using known concentrations of 1,1-3,3 tetraetoxipropane. the tbars values were expressed as mg mda/kg sample. 2.7. sensory analysis at the end of the manufacturing process (after 330 days of the bodega step), samples from the biceps femoris muscle of the five pieces of each pig group were analysed by sensory evaluation. the sensory evaluation was conducted by eight panellists from the meat technology centre of galicia. therefore, forty qualifications were obtained for each attribute in each pig group. the panellists received training (for four months) in the attributes and the scale to be used according to the method proposed by the iso regulations (iso, 2012). the panellists carried out the sensory evaluations in individual cubicles, according to the iso regulations (iso, 2007). the panellists were given water at the beginning of the session and between tasting samples to clean the palate and remove residual flavours. the samples were individually labelled with random three-digit numbers. ten sensory traits of the muscle, grouped according to appearance (lightness, colour of lean meat, fat yellowness and marbling), odour (intensity, rancidity and cured), taste (saltiness), and texture of the lean meat (hardness and juiciness) were assessed according to the methodology proposed by the iso regulations (iso, 1991; iso, 1994; iso, 2006). the intensity of each attribute was expressed on a structured scale ranging from 0 (very low) to 9 (very high). the normality of data was checked using the shapiro-wilk’s normality test. 2.8. statistical analysis analysis of variance (anova) was applied to all variables considered in the study by using ibm spss statistics 19.0 software (ibm corporation, somers, ny, usa). the least squares means (lsm) were separated using duncan's t-test. a significance level of p<0.05 was used in all lsm tests. cross-breed and ripening time were included as fixed effects in the model, to study physico-chemical and sensorial properties of the bf muscle. ital. j. food sci., vol 29, 2017 127 the model used was expressed as follows: yij = μ + ci + rj + εij where yij represents the observed values of the dependent variables, μ is the overall mean, ci is the effect of cross-breed, rj is the effect of ripening time, and εij is the residual random error associated with the observation. pearson´s linear correlation coefficients were determined for the different variables with the above-mentioned statistical software package. 3. results and discussion 3.1. influence of cross-breed on physico-chemical changes during the manufacturing of dry-cured ham the effects of cross-breed on the ph, water activity and chemical composition (moisture, intramuscular fat, protein, ash and chlorides) of bf muscle throughout the manufacturing process of dry-cured ham from celta pig are summarized in table 1. the mean ph (at 24 h) of samples from duroc x celta cross-bred pigs was significantly higher (p<0.01) than the mean ph of the samples from the other pigs. this is consistent with findings reported by alonso et al. (2009), who noted a relatively high ph of meat from duroc crosses, and could be related to a lower glycogen content or to a higher buffer capacity in the duroc x celta pig muscle. significant differences (p<0.001) in ph values were observed during the manufacturing of dry-cured ham. an increase in the final ph relative to the ph of raw pieces (from 5.59, 5.82 and 5.69 to 6.00, 5.92 and 5.90 for respectively c, c×d and c×l) was observed. the final ph values were similar to those reported by other authors for different types of ham, such as iberian (martín et al., 1998) and serrano ham (gou et al., 1995), but lower than those reported for teruel p.d.o. dry-cured hams matured for 20 months (cilla et al., 2005). the increase in ph values throughout the manufacturing process may be related to the release of low-weight nitrogen molecules and ammonia ascribed to endogenous and exogenous proteolytic enzyme activities (virgili et al., 2007). the average moisture content in raw pieces was similar across groups, although the lowest values corresponded to samples from the celta x landrace cross-bred pigs (73.69%, 73.48% and 73.16% for respectively c, c×d and c×l). the water content decreased during the post-salting stage (around 4-5%) owing to the osmotic effect produced by the salt covering the entire surface of the hams. at subsequent stages (drying-ripening and “bodega”) the decrease is due to dehydration process. the water loss during the dryingripening and “bodega” stages of hams from pure-bred celta pigs was significantly (p<0.05) higher than in hams from the cross-bred pigs (around 5-6%, table 1). in terms of water loss, the dehydration was more intense during the “bodega” stage due to the duration of this stage and the environmental conditions (higher t and lower rh) in the storage chamber. the moisture content was significantly (p<0.001) higher in hams from the cross-bred pigs than in those from pure-bred celta pigs (52.36%, 58.30%, and 58.93% for respectively c, c×d and c×l). this outcome is not consistent with findings reported by carrapiso and garcía (2005), who did not observe any significant effect (p>0.05) of cross-breed on the moisture content of iberian ham. moisture contents were positively correlated with instrumental colour attributes of cie l*-values (r = 0.722, p<0.01), cie a*values (r = 0.633, p<0.01) and cie b*-values (r = 0.555, p<0.01) and negatively correlated with ph values (r = -0.666, p<0.01). similarly, water activity decreased gradually throughout the manufacturing process (table 1). ital. j. food sci., vol 29, 2017 128 the decrease in water activity can be attributed to salt diffusion and the intense dehydration that the pieces undergo during the drying-ripening stage. the aw values were positively correlated with moisture content (r = 0.918, p<0.01) and negatively correlated with salt content (r = -0.882, p<0.01). in addition, aw was also correlated with shear force (wb test) (r = -0.285, p<0.05). the imf content of fresh pieces from the duroc x celta cross-bred pigs (13.92%) was significantly higher (p<0.001) than in hams from pure-bred celta pigs (8.03%), while the levels were intermediate in hams from the landrace x celta cross-bred pigs (12.27%). this finding is consistent with those reported by alonso et al. (2009) and latorre et al. (2003), who observed a higher imf content in meat from duroc cross-bred pigs, as duroc pigs are generally fatter than the other breeds. however, other authors (carrapiso and garcía, 2005; tejeda et al., 2002) observed that the duroc line did not modify the imf content in crosses with the iberian pig. finally, franci et al. (2007) found that the imf content was higher in hams from cinta senese pigs than in hams from large white pigs, and the imf contents of hams from the crosses between these breeds were intermediate. data on imf contents are of particular interest because of the influence of this parameter on essential quality traits. imf has a clear effect on quality of meat as it reduces the shear force during chewing, making separation of muscle fibres easier, thus improving the sensations of juiciness and tenderness (lawrie, 1998). the contribution of imf to juiciness is particularly important in dry-cured ham because of the strong dehydration of the product during the ripening process (ventanas et al., 2005). juiciness has been indicated to be the main trait influencing the overall quality of iberian dry-cured ham (ruiz et al., 2002) and imf has been closely associated with this parameter (ruiz carrascal et al., 2000). the significantly higher imf contents in the bf muscle of hams from duroc x celta and landrace x celta cross-bred pigs than in those from pure-bred celta pigs may affect the acceptability of the final product. total chloride content (expressed as g/100 g of dry matter) increased significantly (p<0.001) during the salting and post-salting stages as a result of diffusion of salt throughout the whole pieces. during the next steps, the concentration of total chlorides in bf muscle continued to increase until the end of the process (table 1). in this type of muscle (internal muscle), the differences between groups may be due to intrinsic heterogeneity, because the connective tissue, skin, fat, bones, etc. act as barriers to nacl diffusion. the bf muscle, which is covered by subcutaneous fat and also contains intermuscular fat, is one of the muscles with the lowest concentration of sodium chloride during the first stages of the process; the concentration depends on thickness of subcutaneous fat. the final mean contents (15.87%, 15.14% and 16.59% of dry matter for respectively c, c×d and c×l) were within the range of values (13-20% of dry matter) reported by other authors (buscailhon et al., 1994; gou et al., 1995) for dry-cured hams. however, these nacl contents were higher than those reported by some authors for iberian ham (mariscal et al., 2004; martín et al., 1998), serrano ham (mariscal et al., 2004), and italian ham (møller et al., 2003) (9.29-11.4% of dry matter). the colour of dry-cured ham is one of the most important characteristics of appearance (ruiz et al., 2002) and it is assumed to influence the consumer’s choice of sliced ham in the supermarket. the influence of ripening time and cross-breed on colorimetric parameters is shown in table 2. the luminosity (cie l*-value) values decreased throughout the whole process. lightness is related to the thin aqueous layer on the muscle surface, and the cie l*-value depends on the movement of moisture (dehydration) towards the surface. ital. j. food sci., vol 29, 2017 129 table 1. effect of cross-breeding on the changes of ph, water activity and proximate composition of bicep femoris muscle during the manufacture of dry-cured celta ham. results expressed as means±standard deviation of values from five samples in each group and sampling point. fresh piece after salting after post-salting after drying-ripening “bodega” stage sem significance time first point second point ph c 5.59±0.06a,1 5.63±0.06a,1 5.76±0.03b 5.90±0.05c 5.86±0.05c,1 6.00±0.05d,2 0.03 *** c×d 5.82±0.09a,2 5.77±0.05a,2 5.85±0.06a,b 5.98±0.10c,d 6.04±0.04d,2 5.92±0.028b,c,1 0.02 *** c×l 5.69±0.09a,1 5.59±0.11a,1 5.80±0.09b 5.98±0.07c 5.98±0.05c,2 5.90±0.02b,c,1 0.03 *** sig. genotype ** ** n.s. n.s. *** *** aw c 0.98±0.00e,2 0.96±0.00d,2 0.96±0.01d 0.94±0.01c 0.90±0.01b,1 0.87±0.02a,1 0.01 *** c×d 0.96±0.00d,1 0.99±0.01e,3 0.94±0.01c 0.95±0.01c 0.92±0.01b,2 0.90±0.01a,2 0.01 *** c×l 0.99±0.00d,3 0.95±0.01c,1 0.96±0.01c 0.95±0.01c 0.92±0.01b,2 0.90±0.01a,2 0.01 *** sig. genotype *** *** n.s. n.s. *** * moisture (%) c 73.69±0.81e 72.46±0.46e,2 68.05±0.59d 65.36±0.93c 56.56±1.60b,1 52.36±3.09a,1 1.47 *** c×d 73.48±0.58f 71.29±0.80e,1 67.82±0.46d 65.67±1.36c 61.01±1.17b,2 58.30±1.01a,2 1.00 *** c×l 73.16±0.58f 70.63±0.61e,1 67.29±1.34d 65.34±0.99c 61.97±1.13b,2 58.93±1.19a,2 0.92 *** sig. genotype n.s. ** n.s. n.s. *** *** imf (% dry matter) c 8.03±0.24a,b,1 7.74±0.09a,1 8.28±0.53b,1 8.02±0.34a,b,1 8.03±0.28a,b,1 7.83±0.42a,b,1 0.07 n.s. c×d 13.92±1.29b,c,d,3 15.21±2.68d,2 14.86±1.97c,d,3 12.78±0.40a,b,c,3 11.07±0.96a,2 12.43±1.45a,b,2 0.38 ** c×l 12.27±1.05b,2 13.94±0.94c,2 10.17±0.69a,2 11.39±0.50b,2 10.20±0.37a,2 11.95±0.37b,2 0.27 *** sig. genotype *** *** *** *** *** *** protein (% dry matter) c 85.04±1.15e,2 83.40±1.16d,3 75.86±1.36c 73.32±0.92b,2 71.00±1.13a,2 71.49±1.34a,2 1.09 *** c×d 80.93±2.35e,1 76.70±1.31d,1 71.33±2.15c,1,2 68.44±1.96b,1 69.02±1.01b,1 65.11±0.69a,1 1.03 *** c×l 81.12±0.81f,1 78.78±1.33e,2 74.99±0.79d,2 71.73±0.72c,2 70.32±1.00b,1,2 66.59±1.37a,1 0.94 *** sig. genotype ** *** *** *** * *** ash (% dry matter) c 4.59±0.18a,1,2 8.28±0.71b,2 14.86±0.95c,3 16.60±1.26d 19.80±1.06e,2 19.68±1.11e 1.09 *** c×d 4.33±0.14a,1 6.09±0.76b,1 11.49±1.17c,1 15.98±0.86d 17.11±1.17d,1 18.32±0.85e 1.02 *** c×l 4.85±0.31a,2 5.92±0.69b,1 12.82±0.65c,2 15.95±0.47d 18.22±0.96e,1 19.80±1.29f 1.08 *** sig. genotype * *** *** n.s. ** n.s. ital. j. food sci., vol 29, 2017 130 chlorides (% dry matter) c 0.51±0.19a 3.62±1.10b 11.74±1.87c,2 13.02±1.79c 16.15±1.14d,2 15.87±1.81d 1.16 *** c×d 0.34±0.41a 3.24±0.74b 8.18±1.06c,1 13.09±1.14d 12.20±2.04d,1 15.14±0.80e 1.02 *** c×l 0.69±0.20a 2.85±0.51b 9.57±0.60c,1 14.35±0.56d 14.45±1.18d,2 16.59±1.52e 1.13 *** sig. genotype n.s. n.s. ** n.s. ** n.s. a–fmeans in the same row (corresponding to the same genotype and parameter) not followed by a common letter are significantly different (p<0.05; duncan test) (differences among sampling points). 1-3means in the same column and parameter not followed by a common number are significantly different (p<0.05; duncan test) (differences among genotypes). significance: n.s.: not significant; * (p<0.05); ** (p<0.01); *** (p<0.001). sem is the standard error of the mean. table 2. effect of cross-breeding on the changes of colour parameters of bicep femoris muscle during the manufacture of dry-cured celta ham. results expressed as means±standard deviation of values from five samples in each group and sampling point. fresh piece after salting after post-salting after drying-ripening “bodega” stage sem sig. first point second point time lightness (l*) c 48.90±2.39d 47.84±2.28d,1 40.35±1.45b,1 43.06±1.19c,1 39.73±1.46a,b,1 37.12±2.51a,1 0.89 *** c×d 50.59±1.72c 50.17±2.44c,2 45.58±2.00a,b,2 47.53±1.67b,2 46.14±1.14b,2 43.65±0.96a,2 0.64 *** c×l 47.97±1.57d 47.03±2.46d,1 41.22±1.25a,1 45.03±1.74c,d,1 44.23±2.26b,c,2 41.87±1.27a,b,2 0.60 *** sig. genotype n.s. * *** ** *** *** redness (a*) c 18.14±0.27c,2 18.16±1.98c,2 13.48±1.17b 13.05±0.86a,b,1,2 13.82±1.63b,2 11.52±0.98a 0.52 *** c×d 13.43±1.68b,c,1 13.97±0.27c,1 13.32±1.13b,c 12.01±0.80a,1 12.30±0.87a,b,1,2 11.02±0.68a 0.31 *** c×l 16.69±1.89b,2 16.44±2.17b,1,2 13.53±0.97a 13.59±1.45a,2 11.60±1.14a,1 11.18±1.31a 0.49 *** sig. genotype *** * n.s. n.s. * n.s. yellowness (b*) c 13.13±1.17b,2 13.00±1.66b 7.55±0.91a,1 7.65±0.79a,1 7.15±0.97a,1 8.63±0.80a,1 0.52 *** c×d 11.35±1.40b,1 12.24±0.79c 12.24±1.54b,c,3 9.71±1.14a,2 11.13±0.64a,b,2 11.04±0.81a,b,2 0.27 *** c×l 12.96±0.99c,1,2 13.30±0.62d 9.83±0.67a,b,2 8.94±0.51a,2 10.39±0.87b,2 10.09±0.67b,2 0.37 *** sig. genotype n.s. n.s. *** ** *** ** a-dmeans in the same row (corresponding to the same genotype and parameter) not followed by a common letter are significantly different (p<0.05; duncan test) (differences among sampling points). 1-3means in the same column and parameter not followed by a common number are significantly different (p<0.05; duncan test) (differences among genotypes). significance: n.s.: not significant; * (p<0.05); ** (p<0.01); *** (p<0.001). sem is the standard error of the mean. ital. j. food sci., vol 29, 2017 131 the decrease in cie l*-values may be related to decrease in moisture content (r = 0.722, p<0.01). to this regard, sanabria et al. (2004) observed that moisture loss increased the concentrations of pigments (e.g. myoglobin) and led to a reduction in cie l*-values. on the other hand, cie l*-values also decreased as salt concentration increased (r = -0.741, p<0.01). a similar trend in cie l*-values was reported by marušić et al. (2011) and pérez-palacios et al. (2011) for dry-cured ham. however, cilla et al. (2005) did not report any significant changes in the colour parameters, except for the bf muscle redness index in dry-cured teruel p.d.o hams matured for different lengths of time (between 12 and 26 months). relative to cie a*-values, a decrease in redness was observed until the end of the process; this may be due to the decrease in moisture content (r = 0.633, p<0.01) and an increase in salt content (r = -0.644, p<0.01). the final values were within the range of those described by sanabria et al. (2004) in iberian ham, and very similar to those reported by marušić et al. (2011) for istrian ham and by cilla et al. (2005) for teruel ham. finally, the yellowness values decreased as the processing time increased, and the decrease was more pronounced during the post-salting stage (table 2). the decrease in cie b*-values may also be due to a decrease in moisture content (r = 0.555, p<0.01) and an increase in salt content (r = -0.696, p<0.01). a similar pattern was reported by sanabria et al. (2004) for iberian ham. the final values of this parameter were also similar to those reported by other authors for different varieties of ham (cilla et al., 2005; marušić et al., 2011; pérez-palacios et al., 2011). the instrumental colour parameters were also slightly affected by cross-breed (table 2), as hams from the cross-bred pigs were yellower (cie b* value) and lighter (cie l* value) than those from pure-bred celta pigs. thus, the higher lightness (cie l*) values in dry-cured hams from crosses between celta pigs and both duroc and landrace pigs may be related to the higher imf content. this is consistent with the findings of ramírez and cava (2008), who reported a positive correlation between cie l*-value and imf content in iberian ham. in the present study, cie l*-value was positively correlated with the imf content (r = 0.535, p<0.01). the changes in tbars values during the manufacturing of dry-cured ham are shown in fig. 1. the tbars values increased significantly (p<0.001) during the salting and postsalting periods, reaching the highest values after the drying-ripening period in hams from c and c×l groups, and after the post-salting stage in hams from the celta x duroc crossbred pigs. the increase in malondialdehyde contents during the post-salting and dryingripening stages may be related to the pro-oxidant action of metallic ions present as impurities in the salt used in the curing process. the tbars values were positively correlated with nacl content (r = 0.542, p<0.01). from these maximum values, a significant decrease (p<0.001) was observed until the end of the process, reaching final average values of 1.33, 1.07 and 1.00 mg mda/kg of muscle for c, c×d and c×l respectively. the decrease in tbars values was associated with the advanced reactions of secondary lipid oxidation products with protein residues, especially for conditions of low water activity to yield oxidatively modified proteins (kikugawa et al., 1991). increased tbars values in dry-cured ham during the first stages of production followed by a decrease toward the final stages have previously been reported for iberian ham (andrés et al., 2004) and parma ham (koutina et al., 2012). at the end of the process, there were no significant differences (p>0.05) between groups, although the fat of hams from purebred celta pigs tended to be more oxidized. this finding seems contravene previous observations, since lipid oxidation is usually positively related to fat content (jo et al., 1999). however, the highest fat unsaturation of celta pig compared to most of the other pig breeds (franco et al., 2006) could be responsible for the highest oxidation of fat in hams from pure celta breed. ital. j. food sci., vol 29, 2017 132 figure 1. effect of cross-breed on the changes in tbars index of bicep femoris muscle during the manufacture of dry-cured celta ham. plotted values are means and standard deviations from five samples in each group and sampling point. as = after salting, aps = after post-salting, adr = after drying-ripening, bs1 = after bodega stage 1, bs2 = after bodega stage 2. a-d means in the same genotype not followed by a common letter are significantly different (p<0.05) (differences among processing steps). 1-2 means in the same processing step not followed by a common number are significantly different (p<0.05) (differences among genotypes). the final mean value (1.13±0.17 mg mda/ kg muscle) was two times higher than values obtained in istrian ham (marušić et al., 2011), iberian ham (andrés et al., 2004) and teruel p.d.o. ham (cilla et al., 2006). the changes in maximum shear force during the manufacturing of dry-cured hams are shown in fig. 2. shear force increased significantly (p<0.001) during the process. the final mean value of shear force (3.58 kg/cm2) was lower than those obtained by soriano pérez (2001) and franci et al. (2007), who reported values of 6.84 kg/cm2 and 20.9 kg/cm2 respectively. a similar trend was observed by serra et al. (2005), who suggested a negative non-linear relationship between hardness and water content. in the present study, moisture content was negatively correlated with shear force (r = -0.541, p<0.01). in addition, texture has previously been linked to imf content (ruiz-ramírez et al., 2005); this is consistent with our observations as imf content was negatively correlated with shear force (r = -0.756, p<0.01). monin et al. (1997) also included other variable in the drycured ham process, such as level of proteolysis, because they observed that changes in hardness depended on both water content and protein state. these authors found that muscles initially became harder during the early processing stages, because of the decrease in protein solubility and water content, and they then became softer in texture as proteolysis occurred. at the end of process, shear force did not differ significantly (p>0.05) between groups, although the lowest values were found in hams from landrace x celta cross-bred pigs (see fig. 2). this finding is consistent with those reported by franci et al. (2007) who observed that shear force values were higher in pure-bred cinta senese pigs than in cinta senese x large white cross-bred pigs. ital. j. food sci., vol 29, 2017 133 figure 2. effect of cross-breed on the changes in maximum shear force of bicep femoris muscle during the manufacture of dry-cured celta ham. plotted values are means and standard deviations from five samples in each group and sampling point. as = after salting, aps = after post-salting, adr = after drying-ripening, bs1 = after bodega stage 1, bs2 = after bodega stage 2. a-e means in the same genotype not followed by a common letter are significantly different (p<0.05) (differences among processing steps). 1-3 means in the same processing step not followed by a common number are significantly different (p<0.05) (differences among genotypes). 3.2. influence of cross-breed on sensory characteristics ten descriptors were evaluated in the sensory analysis of the dry-cured ham (fig. 3). within appearance, lightness and lean redness scores were similar for the dry-cured hams from all three groups of pigs considered, although the lightness values were highest for samples from cross-bred pigs, while the redness of the lean meat was highest in samples from pure-bred celta pigs. instrumental and sensory results of lean meat colour indicate that discrimination between celta and crosses with landrace and duroc pigs is difficult. this confirms the results obtained in other comparisons between local breeds and crossbreeds (iberian compared with iberian×duroc) by carrapiso et al. (2003), who concluded that instrumental colour measurement of the lean portion of dry-cured ham is not particularly useful for assessing differences perceived by panellists. however, marbling scores differed significantly between groups and were highest in ham samples from cross-bred pigs (1.5, 5.6 and 5.4, p<0.001 for c, c×d and c×l respectively). these outcomes are consistent with data reported for instrumental colour determination and imf and moisture contents. thus, the cie l* values were positively correlated with imf content (r = 0.751, p<0.01), marbling (r = 0.516, p<0.01) and moisture content (r = 0.617, p<0.01). these findings are consistent with those reported by carrapiso and garcía (2008) and ramírez and cava (2008), who also obtained a significant correlation between the cie l* value and marbling and suggested that the colour of iberian ham is more strongly influenced by fat distribution than by the chemical imf content of the muscle. the odour (intensity, rancidity and cured) traits were not significantly (p>0.05) affected by cross-breed, although odour intensity was highest in samples from pure-bred celta pigs (7.8, 7.3, 7.2, p>0.05 for c, c×d and c×l respectively). similarly, the imf content was negatively correlated with odour intensity (r = -0.543; p<0.01) as reported by ramírez ital. j. food sci., vol 29, 2017 134 and cava (2008), who observed a close relationship between imf/marbling, aroma and odour intensity. on the other hand, salty taste was not significantly (p>0.05) affected by cross-breed, although slightly higher values were obtained in ham samples from the crosses (5.3, 5.7 and 5.5 for c, c×d and c×l respectively). figure 3. effect of cross-breed on the sensory characteristics of dry-cured celta ham at the end of the manufacturing process. plotted value for each attribute and pig group is the mean of forty evaluations. finally, panellists observed significant between-group differences in texture traits that were consistent with those detected by instrumental methods (see fig. 2). hardness scores were correlated with wb shear force (r = 0.574, p<0.01). panellists described hams from the cross-bred pigs as softer in texture (p<0.01) and juicier (p<0.01) than hams from purebred celta pigs (figure 3). these results are consistent with those reported by ramírez and cava (2008), who also observed a positive correlation between hardness scores and tpa hardness and wbsf. differences in sensory textural attributes can be caused by several factors: (i) the final ph of fresh meat, (ii) the imf content and (iii) the moisture content (ramírez and cava, 2008). increasing the imf content reduces the force required to chew the meat, by easing separation of muscle fibres, and causes an enhanced perception of meat tenderness (essén-gustavson et al., 1994). thus, better sensory texture parameters have been reported in iberian hams with higher imf content, with significant positive correlations between marbling and juiciness (ramírez and cava, 2008). in the present study, juiciness scores were correlated with imf content (r = 0.529, p<0.01), marbling (r = 0.585; p<0.01) and moisture content (r = 0.527, p<0.01). 4. conclusions the physico-chemical and sensory properties of dry-cured ham from celta pig can be improved by crossing this breed with others. hams from celta x duroc and celta x landrace cross-bred pigs had more intramuscular fat than hams from pure-bred celta ital. j. food sci., vol 29, 2017 135 pigs. the results of the wb test also suggest an effect of cross-breed, as hams from celta x duroc and celta x landrace cross-bred pigs were of softer texture than hams from purebred celta pigs. sensorial analysis demonstrated that hams from celta pure breed were harder and less juicy than those from crosses. although the two crosses improve the quality of hams, overall considering the texture and sensory traits it seems that the crossing with the duroc genotype has the better improvement results. acknowledgements 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bordini c. 2007. changes of free amino acids and biogenic amines during extended ageing of italian dry-cured ham. lwt-food sci. technol. 40(5):871. http://dx.doi.org/10.1016/j.lwt.2006.03.024. vyncke w. 1975. evaluation of the direct thiobarbituric acid extraction method for determining oxidative rancidity in mackerel (scomber scombrus l). fette seifen anstrichmittel 77(6):239. http://dx.doi.org/10.1002/lipi.19750770610. paper received february 22, 2016 accepted september 20, 2016 paper 450 ital. j. food sci., vol. 27 2015 keywords: egg yolk proteins, cucurbita ficifolia protease, hydrolysis, antioxidant, ace-inhibitory activity, immunostimulating activity biological activity of egg-yolk protein by-product hydrolysates obtained with the use of non-commercial plant protease a. zambrowicz*, e. eckert, m. pokora, a. dąbrowska, m. szołtysik, ł. bobak, t. trziszka and j. chrzanowska wrocław university of environmental and life sciences, department of animal product technology and quality management, chełmońskiego 37/41, 51-630 wrocław, poland *corresponding author: tel. +48 71 320 7773, email: aleksandra.zambrowicz@up.wroc.pl abstract enzymatic hydrolysis leads to improved functional and biological properties of protein by-products, which can be further used as nutraceuticals and protein ingredients for food applications. the present study evaluated ace-inhibitory, antioxidant and immunostimulating activities in hydrolysates of egg-yolk protein by-product (yp), generated during industrial process of delipidation of yolk. the protein substrate was hydrolyzed using non-commercial protease from asian pumpkin (cucurbita ficifolia). the reaction was conducted in 0.1 m tris-hcl buffer (ph 8.0) at temperature of 37°c for 4 hours using different enzyme doses (100-1000 u/mg of substrate). the protein degradation was monitored by the determination of the degree of hydrolysis (dh), release of free amino groups (fag) and by rp-hplc. in the obtained hydrolysates we also evaluated biological activities. it was shown that the highest dh of substrate (46.6%) was obtained after 4h of reaction at the highest amount of enzyme. this hydrolysate exhibited antioxidant activity, including ferric ion reducing (frap) (56.41 μg fe2+/mg), ferric ion chelating (695.76 μg fe2+/mg) and dpph free radical scavenging (0.89 μmol trolox eq /mg) as well as ace-inhibitory (ic 50 =837.75 μg/ml) activities. the research showed improved biological properties of enzymatically modified yp by-product. ital. j. food sci., vol. 27 2015 451 introduction nowadays, the identification of bioactive food components, which can provide health benefits is one of the objectives of scientific research worldwide. special attention is given to bioactive peptides due to their role in the prevention of numerous diseases (sharma and rana, 2011). these peptides, released via the enzymatic hydrolysis of food proteins reveal numerous biological activities: antioxidant, antihypertensive, antimicrobial, antidiabetic, opioid and immunostimulating. these may have positive effects the cardiovascular, nervous, immune or digestive systems of the body (mine and kovaks-nolan, 2006; chay pak ting et al., 2011; yu et al., 2011; pokora et al., 2014). the egg is recognized as a very valuable source of proteins for human nutrition, as well as proteins, which may be precursors of peptides with biological activity (mine and kovaks-nolan, 2006; yu et al., 2011; zhipeng et al., 2011). ace-inhibitory peptides are one of the best characterized peptides derived from eggs. the hydrolysis of ovalbumin, the main protein of egg white, conducted by gastrointestinal enzymes, results in the release of several ace-inhibitory peptides (miguel et al., 2004; miguel et al., 2007). the effectiveness of these peptides was validated in tests in vivo conducted on spontaneously hypertensive rats (miguel et al., 2007). antihypertensive activity was also demonstrated by some peptides released from egg-white protein treated by alcalase (liu et al., 2010; zhipeng et al., 2011). as a result of peptide purification from those hydrolysates, ace-inhibitory peptides: rvpsl and qiglf were obtained (liu et al., 2010; zhipeng et al., 2011). it was also shown that egg is a rich source of proteins in which sequence numerous antioxidant peptides are encrypted. phosphopeptides derived from egg phosvitin having molecular masses 1-3 kda exert a strong ability to inhibit the oxidation of linoleic acid, to scavenge dpph free radicals and to chelate iron ions (ii) (xu et al., 2007). egg-yolk hydrolysates composed of peptides with a molecular weight lower than 1 kda obtained with the use of proteinase from bacillus ssp., also exhibited antioxidant capacities. superoxide-scavenging activity and suppression of discoloration by β-carotene have also been observed (sakanaka and tachibana, 2006). egg yolk peptides obtained during alkalase and protease n digestion of delipidated egg yolk proteins were found to boost the systemic antioxidant status in the blood by increasing the gsh concentration in red blood cells (young, fan and mine, 2010). it has been demonstrated that the consumption of egg yolk protein hydrolysates with antioxidative properties leads to the inhibition of tumor cell proliferation in the colon (ishikawa et al., 2009). some peptides derived from egg proteins can act as immune modulators and may be used as nutraceuticals for the prevention or treatment of lifestyle dependent diseases. immunomodulatory peptides may exhibit anti-inflammatory activity by decreasing the production of pro-inflammatory cytokines (mattsby-baltzer et al., 1996; cross and gill, 2000; mine and kovacs nolan, 2006). egg yolk peptides significantly reduce pro-inflammatory cytokine, il-8, in the caco-2 cell line (young and mine, 2010). furthermore, immunostimulatory activity, assayed as the ability to enhance the capacity of phagocytic cells in mice, was present in ovalbumin hydrolysates prepared by gastrointestinal enzymes (biziulevičius et al., 2005). bioactive peptides can be also released from protein by-products generated during isolation of biologically active substances naturally occurring in egg. one such protein waste is a by-product of lysozyme and cystatin extraction from hen egg white by ethanol method (sokołowska et al., 2007). our previous studies showed that this by-product, which itself exhibits poor functional properties, can be a rich source of ace-inhibitory and antioxidative peptides (pokora et al., 2013; 2014; zambrowicz et al., 2013). attention is also drawn to egg yolk as a source of substances, which may find wide application in the prevention and treatment of various medical conditions. egg yolk is mainly used for the extraction of valuable phospholipids such as lecithin, which is more valuable than plant-derived lecithin due to the specific chemical composition. the main by-products of this process are partially denatured and defatted egg yolk proteins in the form of insoluble granule fractions (siepka et al., 2010). the preparation of bioactive peptides by enzymatic hydrolysis of proteinaceous by-products could become an interesting method of waste disposal if the process was costeffective. therefore cheap and effective enzymes for this process are preferred. plant serine protease isolated from cucurbita ficifolia pulp used in this study exhibits strong proteolytic properties and is a relatively cheap proteolytic enzyme (illanes et al., 1985; curotto et al., 1988). the aim of this study was the enzymatic hydrolysis of a by-product of egg yolk phospholipid isolation, in order to obtain hydrolysates with antioxidant, ace-inhibitory and immunostimulatory activities. materials and methods substrate eggs from 40-45 weeks old lohman brown laying hens (housed in a bedding system) were stored at 4°c for 1 week. the eggs were automatically broken and their macroscopic parts were separated on an industrial scale. phospholipids were extracted from the egg yolks (siepka et al., 452 ital. j. food sci., vol. 27 2015 2010).  defatted granules, a by-product of phospholipid extraction from the egg yolk, were lyophilized and stored frozen until used. enzyme non-commercially available protease from c. ficifolia was isolated according to the procedure described by dryjański and wilusz (1990). serine protease was obtained by extraction of the homogenized pumpkin pulp separated from the solids by centrifugation (5000 g, 20 min, 4°c). to the supernatant, ammonium sulfate was added to 50% saturation, and allowed to stand for 24 hours, and then centrifuged (9600 rev/min, 30 min). the resulting precipitate (the enzyme preparation) was desalted by dialysis for 12 hours using distilled water (4°c), and then 0.02 m of phosphate buffer at ph 6.0. determination of proteolytic activity of protease from c. ficifolia proteolytic activity was determined by reaction with 1% casein as a substrate (bdh, ltd., england) at ph 8.3 (kunitz, 1945). the substrate with the enzyme was incubated for 10 min at 37°c. the reaction was stopped by the addition of 5% trichloroacetic acid (tca). the samples were then centrifuged, and the absorbance of supernatants were measured at λ=280 nm. one unit of enzymatic activity (u) was defined as the amount of enzyme giving an increase in absorbance of 0.1 at 280 nm under reaction conditions. determination of protein content total protein content (n x 6.25) in insoluble substrate was determined using the kjeldahl method. protein content in hydrolysates and peptide fractions was determined by the method of lowry et al. (1951). enzymatic hydrolysis yp hydrolysis was carried out according to a modified method of zambrowicz et al. (2013a). 1% substrate suspension in 0.1 m tris-hcl buffer (ph 8.0) was hydrolyzed at 37o c for 4 hours using c. ficifolia protease at doses of 100, 200, 400 and 1000 u of active enzyme applied on 1 mg of yp substrate. the reaction was ended by heating the mixture at 100ºc for 15 min. the hydrolysates were cooled, centrifuged (5500 g, 10 min, 10°c), then the supernatants were lyophilized and stored at 4°c until used. the degree of hydrolysis the degree of hydrolysis (dh %) was determined as the percentage ratio of protein soluble in 10% trichloroacetic acid (tca) to total protein (spellman, 2003). tca was added to the hydrolysates (1:1) and after 1 h of incubation at 4°c the samples were centrifuged (4500 g, 15 min, 20°c). the concentration of the trichloroacetic acid-soluble product in the supernatant was measured spectrophotometrically and calculated from the following equation: dh (%) = (mg soluble protein after hydrolysis / ml ÷ mg soluble protein before hydrolysis/ml) × 100% the content of free amino acid groups the content of free amino acid groups (fag) (μmol/g) was determined by using trinitrobenzene sulfonic acid (tnbs, sigma) according to a modified method by kuchroo et al. (1983). reversed-phase high-performance liquid chromatography peptide profiles of hydrolysates were monitored by reversed-phase high-performance liquid chromatography (rp-hplc). separation was performed using a zorbax xdb-c 18 agilent column (1.8 mm × 50 mm). the operation conditions were as follows: injection volume: 50 μl; mobile phase a – 0.1% tfa in water; mobile phase b – 0.1% tfa in acetonitrile, column temperature: 30°c. flow rate: 1ml/ min. analysis time and gradient conditions can be found in drawings. the absorbance of eluent was monitored at λ=230 nm. determination of ace-inhibitory activity ace (ec 3.4.15.1) inhibitory activity was measured spectrophotometrically according to the method described by miguel et al. (2004) with some modifications. a hydrolysate solution (40 µl) mixed with a hippuryl-his-leu (hhl) substrate solution (5 mmol/l in 100 mmol/l potassium phosphate containing 300 mmol/l sodium chloride, ph 8.3) was preincubated at 37°c for 5 min, and the reaction was initiated by adding 20 µl (2 mu) of ace solution, and then incubated for 30 min at the same temperature. the enzymatic reaction was terminated by the addition of 150 µl of 1 m hcl. the liberated hippuric acid was extracted using 1 ml of ethyl acetate and vigorously shaking, 750 µl of the upper layer was transferred into a test tube and evaporated under vacuum. the hippuric acid left in the tubes was re-dissolved in 800 µl of distilled water. the content of hippuric acid was determined spectrophotometrically at λ=228 nm. all samples were tested in 3 replications. inhibition activity was calculated using the following equation: inhibitory activity (%) = = ((ac – as) / (ac – ab)) ×100 ital. j. food sci., vol. 27 2015 453 where ac is the absorbance of the buffer (control), as is the absorbance of the reaction mixture (sample), ab is the absorbance when the stop solution was added before the reaction occurred (blank). the ic 50 value was defined as the concentration of peptides in µg/ml required to reduce 50% of ace activity, which was determined by analysis of ace inhibition (%) versus peptide concentration. determination of antioxidant activity as the ability to scavenge of dpph free radicals antioxidant activity was determined by a modified method of yen and chen (1995) as the ability to scavenge of dpph (2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl) free radicals in an aqueous solution of peptides. absorbance measurements were made at λ=517 nm after 30 min incubation. the antioxidant activity of the analyzed peptides was determined on the basis of the standard curve prepared for trolox equivalent. determination of antioxidant activity by frap method antioxidant activity was determined as the ability to reduce the oxidation of iron fe(iii) to fe(ii) ions in a reaction with tptz (2,3,5-triphenyltetrazoliumchloride). the absorbance was measured at λ=593 nm. the concentration of fe2+ ions was determined on the basis of the standard curve for known feso 4 solutions (benzie and strain, 1996). determination of iron fe(ii) ion chelation chelation of iron ions was determined by colorimetric measurement of the quantity of fe(ii) not bound to the peptides in a reaction mixture with ferrozine (3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate) (xu et al., 2007). absorbance measurement was made at λ=562 nm. the ability to chelate iron ions was determined on the basis of the standard curve for a fecl 2 solution. determination of immunostimulatory activity immunostimulatory activity of the cytokine secretion in human whole blood was determined at the department of immunochemistry, institute of immunology and experimental therapy, polish academy of sciences in wrocław (poland). cytokine secretion was induced according to the procedure described by inglot et al. (1996). blood samples from at least 10 donors were collected in syringes containing sodium heparin. within 1 h after collection, the blood was diluted  10-times  with rpmi 1640 medium supplemented with penicillin/streptomycin, l-glutamine and 2% fetal bovine serum. 1 ml portions of the cell suspension were distributed in two 24-well flat-bottomed tissue culture plates. to the cell suspension of whole human blood (1 ml sample) the hydrolysates were added at 1.0, 10 and 100 μg. as a reference, the positive lipopolysaccharide inducer of e. coli at a concentration of 4 mg/ml was used. control wells containing non-treated  blood cell samples were used to measure the spontaneous production of cytokines (negative control). the plates were incubated for 22 h at 37°c in a 5% co 2 atmosphere. after incubation, the plates were centrifuged at 200 g for 15 min at room temperature. the supernatants were collected and used for determination of the cytokines. il-6 and il-10 were determined by microplate enzyme-linked  immunosorbent assay using commercially available sets from becton dickinson (franklin lakes, nj, usa) according to the procedure recommended by the manufacturer. statistical analysis all experiments were carried out in triplicates. the data obtained were subjected to multi-factor variance analysis (anova), followed by the duncan’s multiple range test to determine the significant difference between sample at p<0.05 level using the statistica v.9.0. the results of immunostimulating activity were considered significant by a non-parametric wilcoxon test at p≤0.05 (*) and 0.05≤ p≤ 0.1 (**) versus control (untreated cells). results and discussion enzymatic hydrolysis egg yolk protein preparation (yp), as a by-product of lecithin extraction, was treated by a non-commercial serine protease isolated from c. ficifolia in order to evaluate antioxidant, ace-inhibitory and immunostimulatory properties. the progress of hydrolysis was monitored by determining the degree of hydrolysis (dh) (%) (fig. 1), the increase in the concentration of free amino groups (fag) (fig. 2), and by rp-hplc peptide profile analysis (fig. 3). dh depended on the enzyme dose and reaction time. dh increased slowly in the first 0.5 h, followed by a faster rate of increase up to 4 h, indicating that the maximum cleavage of proteins occurred in the last hour of hydrolysis. dissimilar kinetics of protein substrate degradation with various proteolytic enzymes has been observed by other authors (otte et al., 1998; zambrowicz et al., 2012). typically, enzymatic hydrolysis is most extensive during the first 30 minutes and then slows down, indicating a maximum of protein degradation in the first hour of hydrolysis. 454 ital. j. food sci., vol. 27 2015 fig. 1 effect of time on the degree of hydrolysis (dh) during hydrolysis of yp by protease from c. ficifolia. a-c means followed by the same letter do not differ significantly (p≤0.05). fig. 2 effect of time on the free amino groups contents (fag) during hydrolysis of yp by protease from c. ficifolia. fig. 3 peptide profiles (rp-hplc) of yp protein hydrolysates obtained after using: a. 100 u/mg, b. 200 u/mg, c. 400 u/ mg and d. 1000 u/mg of protease from c. ficifolia. probably, the hydrolysis process with the use of protease from c. ficifolia proceeded according to the “one-by-one” mechanism. during the initial stage of the reaction (determining the overall level of hydrolysis) it is necessary to partially unfold native protein molecules. as a result, the protein loses its stability, more peptide bonds are exposed on the outside of the molecule (intermediate products) and the enzyme has access to the hydrolyzed peptide bond. in a further step, intermediate products are very rapidly degraded to small peptides (kunst, 2003). the use of the lowest dose of enzyme (100 u/ mg) resulted in a nearly 15% dh of yp. the increase of the dose of c. ficifolia proteinase to 400 u/mg did not exert any significant impact on dh rate, whereas the addition of the enzyme at 1000 u/mg resulted in a dh value of more than 46% after 4 hours of digestion. analysis of the fag concentration of the obtained hydrolysates confirmed the above results. the greatest increase in concentration was observed during the long time of hydrolysis. the application of enzyme at doses from 100 to 400 u/mg resulted in similar levels of increases of fag from 2255.20 to 2325.74 µm gly/g. the most intensive increase of fag (4525.1 µm gly/g) occurred during the 4-hour reaction using 1000 u/mg of protease. ital. j. food sci., vol. 27 2015 455 protein-peptide profiles obtained by rp-hplc technique demonstrate the extent of yp hydrolysis (fig. 3). they were identified with both a longer retention time (3-7 min) specific for the more hydrophobic peptides, and peaks with a short retention time (0.5-1 min) typical for hydrophilic peptides. the wide distribution of degradation products indicates that each of the hydrolysates is composed of peptides with different hydrophobic properties, which may impact the biological activity of the obtained hydrolysates. the results described above indicate that serine protease from c. ficifolia is an effective enzyme in hydrolyzing egg yolk protein by-product. previously, the proteolytic properties of this enzyme had been tested on casein, a protein from corn gluten, and egg white proteins (illanes et al., 1985; curotto et al., 1989; pokora et al., 2014). the biological activities of yp enzymatic hydrolysates the antioxidant activity of yp hydrolysates was studied in terms of the scavenging effect on dpph radicals, ferric reducing power (frap), and iron chelating activity (table 1). the enzymatic treatment of yp leads to an increase in dpph free radical scavenging activity in the hydrolysates. elias et al. (2008) explained that the antioxidant activity of the hydrolysates/peptides is the result of the proteolytic action of the enzyme. the specific amino-acid sequence of peptides and their changed physical properties allow exposing the amino acid residues and their action as electron donors. as a result of these reactions, the peptides combine with radicals and form stable complexes, which inhibit oxidation processes. the various yp hydrolysates showed different potencies in scavenging dpph radicals. the results indicated no direct relationship between dh and the values of dpph free radical scavenging activity. the highest dpph scavenging potency was shown by the hydrolysate obtained after 4 hours degradation with an enzyme dose of 1000 u/mg (0.89 μmol trolox eq /mg) (table 1). the significant level of dpph free radical scavenging activity (0.63 μmol trolox eq /mg) was also observed for the hydrolysate obtained with the use of 200 u/mg protease after 0.5 hour digestion. in previous works, yp protein by-product was treated with pepsin and neutrase leading to final hydrolysates with dh values: 45.3% and 27.6%, respectively (zambrowicz et al., 2014; pokora et al., 2013). peptic hydrolysate and hydrolysate obtained by neutrase showed dpph free radical scavenging activity values: 0.5 troloxeq/ mg and 0.44 µm trolox eq /mg, respectively (zambrowicz et al., 2014; pokora et al., 2013). it may be explained that the potency of yp-hydrolysates to scavenge dpph free radical depends more on the specificity of the enzyme than on the degree of hydrolysis (dh). our results also indicate that yp is a better source for peptides exhibiting dpph scavenging activity than other protein waste, such as by-products of lysozyme and cystatin isolation from egg white. egg white protein by-product hydrolysates obtained with trypsin and neutrase exhibited free radical scavenging activity up to 0.21 and 0.17 µmol troloxeq /mg, respectively (zambrowicz et al., 2013). previously, serine protease c. ficifolia was used by dąbrowska et al. (2013) to evaluate the antioxidant activity of bovine casein. casein hydrolysates possessed a different ability to scavenge of dpph radicals (from 0.06 to 2.21 µmol trolox eq /mg), depending on enzyme dose and reaction time. however, the dpph scavenging potency of many of them was at the same level as the yp hydrolysates. in most of the hydrolysates obtained with different doses of protease, the ferric reducing ability was increasing gradually with the time of hydrolysis. the only exception was the 0.5 h hydrolysate obtained with the enzyme dose of 200 u/mg, which possessed significantly higher ferric reducing activity than the products obtained during long time of degradation (more than 30 minutes). a maximum value of this activity reached 56.41 μg fe2+/mg, for the 4 h hydrolysate obtained with the participation of 1000 u/mg of the enzyme (table 1). the ferric reducing activity of the 4h hydrolysate increased with the increased doses of the enzyme. the application of protease at 100, 400 and 1000 u/ mg resulted in hydrolysates 3.44, 5.0 and 5.47 times more potent than yp, respectively. on the other hand, hydrolysates exerted more than 3 times lower ferric reducing activity in comparison to the yp hydrolysate prepared with neutrase (177.35 μg fe2+ /mg) (pokora et al., 2013). this results gives an indication that unconventional protease from c. ficifolia is characterized by a lesser ability to release peptides with ferric reducing activity from yp than commercially available neutrase. an increase in chelating activity was also observed as a result of progress in hydrolysis. the highest chelating activity was obtained in hydrolysates with dh above 35 %. yp degraded with participation of 1000 u/mg protease during 3 and 4 hour reactions exhibited ferrous ion chelating activity at 692.49 and 695.76 μg fe2+/mg, respectively (table 1). significant ferric chelating power was also shown by hydrolysates in which dh ranged from 15% to 20%. similar results were obtained by torres-fuentes et al. (2011), who analyzed the antioxidant properties of plant protein hydrolysates in terms of their ability to complex iron ions. numerous antihypertensive peptides (eg. ovokinin, ovokinin 2-7, radhp, ypi, dlin) derived from egg white proteins by enzymatic hydrolysis have been characterized (miguel et al., 2004; liu et al., 2010). interest has been aroused in ace inhibitory peptides generated from egg yolk proteins, because they have not been described as a 456 ital. j. food sci., vol. 27 2015 source of peptide inhibitors of ace as much. the hydrolysates obtained in this work exhibited various abilities to inhibit the ace enzyme (table 1). the hydrolysates of dh lower than 10% did not exert any ace inhibitory activity. the most active inhibitor of ace (ic 50 =467.5 μg/ml) was the hydrolysate obtained by an enzyme dose of 1000 u/mg after 4-hours digestion. whole egg yolk in native form as a potential source of ace-inhibitory peptides was tested by you and wu (2011). the level of this activity (ic 50 ) for hydrolysates prepared with the use of gastrointenstinal (pepsin, pancreatin) and microbial (thermolysin, alkalase) proteases ranged from 133.4 µg/ml to 210.2 µg/ml (you and wu, 2011). such significant differences in the level of ace-inhibitory activity may result from the fact that in the present study we used a by-product, denatured protein of the yolk granular fraction. denaturation has a significant impact on the physico-chemical and biological properties of proteins. the results indicate that protein by-product obtained from the isolation of phospholipids from hen egg yolk may be a better source of ace-inhibitory peptides than other protein byproduct from the isolation of cystatin and lysozyme from egg white. the peptic hydrolysate (dh: 38.3%) of this protein preparation exhibited an activity of ic 50 =643.1 μg/ml (zambrowicz et al., 2013). recently, we indicated that serine protease from c. ficifolia may be effective in the conversion of this protein by-product to a value added product with ace-inhibitory activitable 1 biological activities of yp hydrolysates obtained with using serine protease from c. ficifolia. all data were expressed as mean values (mean±sd, n=3). values sharing the same letter at the same enzyme dose and test group are not significantly different at p<0.05. enzyme dose time of hydrolysis dpph scavenging ferric reducing ferrous ion-chelating ace inhibitory [u/mg] activity ability (frap) activity activity [ic 50 ] [µm trolox eq /mg] [µg fe2+/mg] [µg fe2+/mg] [µg/ml] 0 substrate 0.15±0.01a 10.3±0.16a 376.2±18.80a nda 100 0.5 0.28±0.03a 33.26±1.38a 497.10±8.22b ndc 1 0.32±0.03b 36.44±1.62b 466.26±3.89c ndc 3 0.18±0.02c 48.78±1.20c 474.25±4.01d 968.5±17.25b 4 0.42±0.02b 35.40±0.67d 574.38±13.67a 837.75±15.25a 200 0.5 0.63±0.02a 46.14±1.33a 460.72±11.74b ndc 1 0.20±0.03c 35.53±1.21a 507.59±2.88d ndc 3 0.28±0.01d 37.11±0.73b 553.75±13.79c 890.75±11.25b 4 0.27±0.01b 44.68±1.66b 626.16±6.56a 777.0±14.25a 400 0.5 0.20±0.03ab 38.91±1.41a 468.70±12.07d ndc 1 0.21±0.03a 36.77±1.20a 513.73±3.81c ndc 3 0.20±0.04a 35.44±1.30ab 573.56±3.77b 803.12±10.25b 4 0.26±0.02a 51.46±0.78ab 625.86±7.11a 718.75±4.5a 1000 0.5 0.22±0.02a 21.71±1.37d 642.32±2.83d 657.75±4.5a 1 0.25±0.02c 27.78±1.75c 654.79±5.78b 650.0±4.5a 3 0.43±0.03b 43.33±1.06b 692.49±1.86c 554.75±7.75a 4 0.89±0.03a 56.41±1.13a 695.76±14.91a 467.5±6.0a ty (pokora et al., 2014). the 50% inhibition of ace was obtained with the presence of 9071.7 μg/ml of the hydrolysate. studies have shown a high linear correlation between dpph free radical scavenging activity and immune activity with a positive correlation coefficient of 0.96 (he et al., 2014). therefore hydrolysates with the highest dpph free radical scavenging potency were also evaluated in terms of their immunostimulatory properties. it was assessed as the results of cytokines il-10 and il-6 induction by hydrolysates in whole human blood cell cultures (ex vivo) (fig. 4). 4-hour hydrolysate obtained with the use of 1000 u/ mg of c. ficifolia protease appeared to be slight inducer. the use of 100 µg/ ml of protease resulted in a low increase the concentration of il6, which reached the value: 3.05 ng/ ml (fig. 4 a). however, the results were not statistically significant compared to the positive control (lp). extremely different cytokine inducing activity was exerted by the yolkin, naturally occurring in egg yolk. yolkin is a mixture consisting of several peptides of an apparent molecular weight of 1 to 35 kda, produced as a result of vitellogenin ii hydrolysis by cathepsins during the formation of an egg. its constituent peptides were found to be efficient inducers of il-1β, il-6 and il-10 secretion. a complex at a concentration of 100 µg/ml showed almost the same activity as the lps-treated control in stimulating cytokine production. (polanowski et al., 2013). the biological activity of enzymatic hydrolysates ital. j. food sci., vol. 27 2015 457 is determined by the protein sequence (type and location of amino acid residues) as well as by the specifity of the enzyme (clemente, 2000; park et al., 2001). these two crucial factors are responsible for the disparate level of immunostimulatory activity of yp-hydrolysates. conclusions the effect of enzymatic modification of an egg yolk protein preparation (yp), obtained as a byproduct of phospholipid extraction, on its biological properties were evaluated. the hydrolysis process of yp was performed with the use of noncommercial serine protease from c. ficifolia. the most effective degradation of yp was noticed under the conditions: enzyme dose 1000 u/ mg and duration 4 h, when a significant degree of hydrolysis (46.6%) was obtained. enzymatic hydrolysis of yp provided hydrolysates/ peptides exhibiting antioxidant and ace-inhibitory activities.  the yp hydrolysates showed significant antioxidant and degree of hydrolysis-dependent ace-inhibitory activity. the 4-hour hydrolysate obtained with the highest amount of enzyme (1000 u/mg) showed the highest biological activity among the tested hydrolysates. it exhibited ferric ion reducing potential (frap) (56.41 μg fe2+/mg), ferric ion chelating activity (695.76 μg fe2+/mg), dpph free radical scavenging activity (0.89 μmol trolox eq /mg) and aceinhibitory (467.5 μg/ml) activity. novel biological effects of egg-yolk protein by-product hydrolysates was shown. acknowledgments the project “innovative technologies in the production of biopreparations based on new generation eggs,” innovative economy operational programme priority 1.3.1, thematic area “bio”, co-financed by the european union through the european regional development fund within the innovative economy operational programme, 2007-2013. this project was supported by the wroclaw centre of biotechnology programme, the leading national research centre 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e. and trziszka t. 2013a. evaluation of the aceinhibitory activity of egg white proteins degraded with pepsin. pol. j. food nutr. sci. 63(2): 103-108. zhipeng y., wenzhu z., jingbo l., jing l. and feng c. 2011. qiglf, a novel angiotensin i-converting enzyme-inhibitory peptide from egg white protein. j. sci. food agric. 6: 921-926. paper received july 30, 2014 accepted january 24, 2015 44 issn 1120-1770 online, doi 10.15586/ijfs.v35i2.2248 p u b l i c a t i o n s codon italian journal of food science, 2023; 35 (2): 44–53 peptide extraction from silver carp (hypophthalmichthys molitrix) scales via enzymatic hydrolysis and membrane filtration xiao-yan zu1,2, ya-qi huang1, ya-jing zhao1,3,guang-quan xiong1,2, tao liao1,2, hai-lan li1,2* 1institute of agro-products processing and nuclear agricultural technology, hubei academy of agricultural sciences, wuhan, china; 2key laboratory of agricultural products cold chain logistics, ministry of agriculture and rural affairs, wuhan, china; 3college of petrochemical technology, lanzhou university of technology, lanzhou, china *corresponding author: hai-ian li, institute of agro-products processing and nuclear agricultural technology, hubei academy of agricultural sciences, no. 5 nanhu avenue, hongshan district, wuhan 430064, hubei province, china. email: hl.li@hotmail.com received: 22 june 2022; accepted: 29 december 2022; published: 3 may 2023 © 2023 codon publications open access paper abstract in this work, peptides were extracted from silver carp (sc) scales via protease hydrolysis and separated using two membranes (m1 and m2). the results revealed that the water: sc scale ratios of 50.6 ml/g, alkaline protease 1 (ap1) dose of 2313.6 u/ml, and ph of 8.14 were the optimal hydrolysis conditions, and the peptide yield reached 88.77 ± 0.32%. the optimal conditions of peptide separation were clarified: the operating pressure of the m1 (m2) was 0.25 (0.4) mpa, the liquid temperature was 30°c, and the operation time was 65 min. in this case, the permeability of the m1 (m2) reached 91.73 ± 96% (79.83 ± 7.23%), and the average molecular weight of the peptides was 758 da (576 da). compared with m1 peptides, m2 peptides contained more acidic and aromatic amino acids exhibiting free-radical scavenging and tyrosinase inhibition properties. it might provide a way to utilize sc scales as a promising material to produce bioactive peptides. keywords: membrane filtration; peptide extraction; response surface methodology; silver carp scales introduction silver carp (sc), hypophthalmichthys molitrix, a freshwater fish, has the highest aquaculture production in china. in 2020, the sc aquaculture output reached 3.81 million tons and was 2.30 times that of tilapia (oreochromis mossambicus, 1.65 million tons) (china fishery statistical yearbook, 2020). in the last decade, only a small part of sc scales has been used to produce local characteristic food products, while most of them are processed into inexpensive feed and even discarded as garbage, which possess serious threats to biological resources (kraiem et al., 2015). sc scales are rich in biomolecules, and other advantages include low price, small size, easy processing through enzymolysis, and low production cost (xu et al., 2021). therefore, there is a tendency to replace tilapia scales with those of sc scales to produce fish scale peptides by inland processing companies. previous studies on fish scale peptides have mainly focused on the structure of fish scales and the physicochemical properties of their peptides. for instance, okuda et al. (2009) reported that the hydroxyapatite content of fish scales reached 19.31%, which could directly affect the extraction rate of the peptides. chen et al. (2016) clarified that the preparation of collagen from fish scales through acid extraction resulted in a substantial loss of collagen. the use of enzymatic extraction of peptides does not introduce foreign substances, ensuring the purity of the product (liu et al., 2012). recent studies have mainly focused on biological functions and action pathways, such as fish scales peptides and glycated italian journal of food science, 2023; 35 (2) 45 peptide extraction from silver carp preparation of peptides to remove impurities from the sc scales, 4% (v/v) hcl was added at a ratio of 10:1 (ml/g, v/w). an ultrasonic cleaner (kq3200db, kunshan ultrasonic instrument co., ltd., china) with 90 w was used to assist decalcification for 2 h. the decalcified scales were washed repeatedly with flowing water and put into a blast drying oven (dhg-9203a, shanghai yiheng scientific instrument co., ltd., china) at 50°c for 18 h. after the procedure, transparent, decalcified sc scales were obtained. proteases were then used to hydrolyze the sc scales. membrane filtration was carried out by using laboratory membrane separation equipment (lng-nf-101, shanghai langi membrane separation equipment engineering co., ltd., china). finally, white peptide powder was obtained by freeze-drying. selection of proteases five grams of decalcified scales were put into distilled water. at the appropriate temperature and ph conditions, five proteases (ap1, ap2, ap3, np, and fp) were used to hydrolyze the sc scales, with all proteases at the same enzyme activity of 4800 u/ml. the enzymatic hydrolysis conditions were as follows: liquid to solid ratio of 10:1 (v/w), enzyme content of 2 µl, and a hydrolysis time of 4.5 h. to obtain the supernatants, the inactivated liquid was centrifuged at 2775 g for 20 min. the peptide yield of the supernatant was used as the indicator of the enzymatic hydrolysis effect for selecting protease. enzymatic hydrolysis optimization based on response surface methodology after screening protease, the yield of peptide was used as the index of enzymatic hydrolysis effect to conduct single-factor experiment. the effects of water to sc scale ratios (10–60 ml/g), protease dose (0.6–1.6 µl/ml), and ph value (7.5–10.0) on peptide yield were carefully studied. the factors of fish scale mass, enzymatic hydrolysis time, and temperature remained at 1 g, 4 h, and 50°c, respectively. combined with the results of the single-factor experiments and considering the peptide yield as the response value, the box–behnken design of rsm was employed to conduct a three-factor and three-level experiment for further optimization of the hydrolysis conditions. three-dimensional response surface and contour maps were generated to analyze the contributions of the factor to the response value as well as the interaction among factors (davidovich-pinhas et al., 2015). peptides conjugated with xylose via maillard reaction (fsp-mrps), which could protect against alcohol induced liver damage. fsp-mrps significantly reduce the elevated activities of serum aspartate aminotransferase and alanine aminotransferase, decrease the elevated levels of hepatic malondialdehyde and triglycerides, and inhibit the decrease of hepatic superoxide dismutase, catalase, and glutathione peroxidase caused by alcohol induced liver damage (chen et al., 2020). peptides derived from crimson snapper scales possess excellent free-radical scavenging activities in vitro, and red tilapia scales can be used as a starting material for producing antioxidant peptides (cai et al., 2021; sierra et al., 2021). to the best of our knowledge, these studies rarely involved peptide extraction and separation processes, which are very important in controlling product qualities and production costs and are urgently needed in industrial production. extraction optimization of peptides usually refers to the degree of hydrolysis (dh). the higher the dh, the more complete the enzymatic hydrolysis, but this does not mean that the peptide has high biological activity (gbogouri et al., 2004). in industry, the yield of peptides with specific molecular weights (i.e., target peptides) is the key indicator for measuring the production efficiency and the quality of products. in this study, considering peptide yield as an indicator, peptides of sc scales (scsps) were extracted by protease hydrolysis subjected to response surface methodology (rsm). the scsps were then separated and purified through membrane filtration, followed by analysis of the amino acid (aa) composition by ion-exchange chromatography with ninhydrin postcolumn derivatization. this work provides reference data for further utilization of sc scales and the efficient production of peptides. materials and methods materials sc scales were purchased from liangzihu aquatic products processing (wuhan, china). alkaline protease 1 (ap1, 2,400,000 u/ml, liquid) and neutral protease (np, 1600 au/g, powder) were purchased from genencor bioengineering (jiangsu, china). alkaline protease 2 (ap2, 2400 u/mg, powder) and flavor protease (fp, 500 u/mg, powder) were provided by novozymes biotechnology (jining, china). alkaline protease 3 (ap3, 200 u/mg, powder) was acquired from yuanye biotechnology (shanghai, china). the filtration membranes were supplied by langji (shanghai, china). all reagents in the experiments were of analytical grade. 46 italian journal of food science, 2023; 35 (2) zu x-y et al. liquid. membrane flux is expressed by the following formula (2): j v t m l/m h a 2 m ( )� � � (2) where, v (l) is the filtrate volume, t (h) is the operating time, and am (m 2) is the membrane filtration area. two groups of 5 l scsp solutions (ph 7.5, concentration 12.2 mg/ml) were prepared, and filtrates were collected through the m1 and m2 under their optimal operating pressures. the membranes were washed repeatedly with deionized water, and the eluate was collected and combined with the filtrates. the total volume of filtrates was then determined. according to the law of material balance, the permeability of peptides (p, %) was calculated by the below formula (3): p c v c v ( )% %� � � �1 1 0 0 100 (3) where, c1 (mg/ml) is the mass concentration of the filtrate, v1 (ml) is the filtrate volume, c0 (mg/ml) is the initial mass concentration of the solution, and v0 (ml) is the initial volume of the solution. determination of peptide yield peptide yield can be characterized by the biuret method via the colors of the complexing reactant between the peptide bond and copper sulfate. it was determined by the method with minor modifications (zhang et al., 2019). a standard solution of 10 mg/ml bovine serum albumin was diluted to different concentrations and mixed with 4 ml of biuret reagent. after reacting for 30 min at room temperature, the absorbance value of the solution was measured at 540 nm with distilled water as the blank control. drawing a standard curve, the regression equation was obtained as follows: y = 0.0549  x + 0.002 (r2 = 0.9992 in the concentration range of 0–10  mg/ml), where y (a) is the absorbance and x (mg/ml) is the concentration of bovine serum protein. the scsp sample was added to a 15% (w/v) trichloroacetic acid solution with a volume ratio of 1:1 for precipitating proteins. after vortexing, the mixture was reacted for 10 min and then centrifuged at 2775 g for 20 min. the supernatant was mixed with 4 ml of biuret reagent, left to react for 30 min at room temperature, and its absorbance was measured at 540 nm using a double beam uv spectrophotometer (uh5300, hitachi co., ltd., japan). the corresponding concentration was obtained by comparison with the standard curve, and the yield of peptide (y, %) was calculated according to equation (4). y c f v m (%) � � � �1 100% (4) as shown in table 1, the three variables and their respective ranges were selected on the basis of single-factor tests. a quadratic model is given in the following formula (atukuri et al., 2019): y b b x b x b x xi i ii i i k i k ij i i i j k j� � � � � �� � � �� �0 2 11 1 � (1) where, y is the response function; b0 is the center point of the system; ε is the random error; bi, bii, and bij represent the coefficients of the linear, quadratic, and interactive effects, respectively; and xi, xi 2, and xixj represent the linear, quadratic, and interactive effects of the independent variables (water:sc scale, ap1 dose, and ph value), respectively. peptides isolated via membrane filtration a total of 3 l scsp samples (12.2 mg/ml) were prepared. polyethersulfone (pes) resin with a filtration area of 0.24 m2 was used as the nf membrane. in the experiment, a 5000 da membrane (m1) was operated to remove proteases and macromolecular impurities from the samples. filtrates with molecular weights less than 5000 da were further separated through a 3000 da membrane (m2). then, the 3000–5000 da filtrates were marked as m1 peptides, and filtrates < 3000 da were marked as m2 peptides. the effects of operating pressure (0.10–0.45 mpa), operating time (5–80 min), and operating temperature (10– 40°c) were analyzed for membrane flux. in the operation time experiment, starting from the first drop of filtrate in the measuring cylinder, samples were taken every 5 min. in other experiments, sampling was concluded after 5 min of operation. determination of membrane flux and peptide transmittance membrane flux (jm, l/[m 2•h]) refers to the volume of filtrate that passes through the membrane per unit time and unit membrane area under certain operating conditions. this is usually used to measure the permeation rate and capacity of membrane elements to the material table 1. process factors and levels. variables code unit coded variable levels −1 0 1 water:sc scale a ml/g 45 50 55 ap1 dose b µl/ml 0.9 1.0 1.1 ph c 7.5 8.0 8.5 italian journal of food science, 2023; 35 (2) 47 peptide extraction from silver carp (anova) with duncan’s multiple range test was used to examine significant (at p values of 0.05) differences between comparable treatments. one-way anova is used to study the difference of the average value of the dependent variable among different groups under different levels of a single factor. data were analyzed by rsm using design-expert statistical software (dx 10.0.4, statease inc, usa) to optimize the enzymatic hydrolysis conditions. all experiments were performed with three independent replicates, and the means ± standard deviations (sd) of the replicates were reported. results and discussion different proteases affect the yield of silver carp scale peptides results are expressed as mean ± sd from triplicate determinations. different lowercase letters show a significant (p < 0.05) difference among the time groups, and different capital letters show a significant (p < 0.05) difference among the protease groups. as shown in table 2, ap resulted in higher yields of peptides during the entire enzymolysis process than np and fp. in addition to hydrolyzing peptide bonds, ap has ser active sites that can also hydrolyze ester bonds and amide bonds, as well as promote transesterification and transpeptidation (puri et al., 2002). in the case of aps, the scsp yield in the ap1 and ap3 groups was significantly higher (p < 0.05) than that in the ap2 group during the entire enzymatic hydrolysis process, indicating that the hydrolyzing ability of ap relates to its state and that higher enzyme activity can be observed in the liquid state than in the solid state. compared with ap3, ap1 led to a superior yield of scsps most of the time, where the yield of scsps reached a maximum of 31.27 ± 1.22%. the scsp yields where, c1 (mg/ml) is the peptide concentration in the test solution, f is the dilution ratio, v (ml) is the total volume of test liquid, and m (mg) is the total mass of the sample. determination of peptide molecular weight the molecular weight of the peptides was determined by gel permeation chromatography (gpc, waters 2690 d, waters co., usa). the chromatographic conditions (jia et al., 2010) with some modifications were as follows: using an aqueous-phase separation column, the flow rate was 0.6 ml/min, the mobile phase was 0.1 m nano3 aqueous solution, and the buffer solution was sodium acetate solution at ph 2.7. here, a waters 2410 refractive index detector was used, and both the column and detector temperatures were kept at 313 k. the molecular weight of the peptide was determined by using polyethylene oxide as the standard. amino acid analysis the aa compositions were analyzed by an aa analyzer (l-8900, hitachi co., japan) according to the method reported by matsuo et al. (2019). the enzymatic hydrolysate and peptides were dissolved in 0.02 m hcl solution, and the suspension was filtered through a 0.45 µm filter. then, the samples were sent to the food quality supervision, inspection, and testing center of the ministry of agriculture (wuhan, china) for aa analysis. the samples were analyzed by ninhydrin postcolumn derivatization through an aa analyzer equipped with a c18 column (4.6 × 150 mm, 5 µm, waters co., usa). the aa contents were calculated using standard aas. statistical analysis data were analyzed via spss 20.0 statistical analysis system (spss inc., usa). one-way analysis of variance table 2. effect of different proteases on the yield of peptides. time (min) peptide yield (%) ap1 ap2 ap3 np fp 30 26.99 ± 0.14da 26.06 ± 0.89da 23.36 ± 1.10ca 17.14 ± 1.87ba 11.93 ± 1.83aa 60 31.27 ± 1.22db 28.84 ± 1.61cb 27.23 ± 0.90cb 24.62 ± 0.50bb 17.85 ± 1.39ab 90 31.90 ± 0.22dbc 30.77 ± 1.39dbc 28.02 ± 0.98cbc 26.25 ± 1.18bbc 19.98 ± 0.03ac 120 32.15 ± 0.39cbcd 31.22 ± 1.04cc 28.49 ± 0.82bbc 27.89 ± 0.18bcd 21.16 ± 0.25acd 150 32.67 ± 0.58dcd 30.94 ± 1.02cc 28.58 ± 1.13bbc 28.24 ± 0.65bde 22.01 ± 0.56ade 180 32.54 ± 0.52cbcd 31.52 ± 0.91cc 29.29 ± 0.75bc 29.48 ± 0.57bde 22.00 ± 0.65ade 210 32.45 ± 0.74cbcd 31.39 ± 0.77cc 28.85 ± 1.28bbc 29.45 ± 1.05bde 22.40 ± 1.10ade 240 33.29 ± 0.66cd 31.98 ± 0.79cc 28.48 ± 0.50bbc 29.44 ± 1.59bde 22.52 ± 0.51ade 270 33.46 ± 0.96dd 31.78 ± 1.40dc 28.53 ± 0.90cbc 30.15 ± 0.79be 23.50 ± 0.59ae 48 italian journal of food science, 2023; 35 (2) zu x-y et al. optimization of enzymatic hydrolysis conditions by response surface methodology response surface design and regression model variance analysis as shown in table 3, the highest yield of scsps reached 90.7 ± 4.50% under the following conditions: ph = 8.0, ratio of liquid to material = 50 ml/g, and ap1 amount = 1.0 µl/ml. the yield results had a high error and were further verified through anova. the results of anova are shown in table 4. the regression equation was obtained as follows: y = 88.16 + 0.39 a − 0.26 b + 2.40 c − 3.25 ab − 3.32 ac − 4.27 bc − 2.61 a2 − 2.56 b2 − 6.33 c2. for the model, p = 0.0023 < 0.01, indicated that the model was highly significant. the lack-of-fit phase p = 0.1606 > 0.05 indicated that the data were reliable and that the fitting degree was good. high correlation coefficients of r2 and r2adj revealed that there were good correlations between each single factor (a, b, c) and the response value of y (scsp yield) and could explain 93.41% of the test results. the f and p values (prob > f) represent the influence of every factor on the response value (ikoma et al., 2003). as shown in the results, the contributions of the three single factors to the peptide yield were in the order of ph, water:sc scale ratio, and ap1 dose. the primary term c, the interaction terms ab and ac, and the quadratic terms a2 and b2 all significantly affected the peptide yield at the p < 0.05 level, and both the interaction terms bc and the show little improvement by prolonging the enzymolysis time beyond 60 min. thus, ap1 and a 60 min hydrolysis time were selected for subsequent experiments. single-factor experiments from figure 1a, the yield of scsp increased with increasing water:sc scale ratio. at a ratio of 50 ml/g, the peptide yield was 86.30 ± 4.55%, which was significantly (p < 0.05) higher than that of the lower-ratio groups. no changes were observed among the 50 and 60 ml/g groups. per figures 1b and 1c, the scsp yield increased initially and then decreased with a further increase in ap1 dose and ph value. when the ap1 dose was 1 µl/ml, the scsp yield reached 87.97 ± 2.03% and was significantly (p < 0.05) higher than that of the other groups. when the ph value was 8.0, the peptide yield reached a maximum of 88.23 ± 1.25%, which was significantly higher than that of the ph 8.5–10.0 groups (p  <  0.05). thus, a ratio of 50 ml/g, an ap1 dose of 1 µl/ml, and a ph value of 8.0 were implemented in the subsequent experiments. notably, the effect of ph on the enzymatic hydrolysis reaction is mainly manifested in changing the spatial conformation and active site of the enzyme molecule (anwar and saleemuddin, 1998). therefore, insufficient or excess ph will cause enzyme denaturation and change the specific binding of the enzyme molecule to the substrate, thus reducing the enzymatic hydrolysis effect and peptide yield. figure 1. effects of different water: sc scale values (a); ap1 doses (b); and ph values (c) on peptide yield. the different lowercase letters show significant (p < 0.05) differences among the columns. (a) (c) (b) italian journal of food science, 2023; 35 (2) 49 peptide extraction from silver carp was steep, and the interaction between them showed a strong effect on the yield of peptides (p = 0.0051 < 0.01). in figure 2, the contour map under the interaction of each factor shows an oval shape and suggests the significant interaction of each factor, which is consistent with the results of anova of the regression model. the contour density along the ph value was higher than that along the ap1 dose or water:sc scale ratio, indicating that ph value had the most significant effect on the peptide yield. this result is consistent with the data in table  4 (p  =  0.0155 < 0.05). in addition, there was a synergistic effect among the three factors of ap1 dose, ph, and water:sc scale ratio. the synergy of these factors significantly (p < 0.05 or p < 0.01) affected the yield of peptides. for example, the single factor ap1 dose showed little effect on the yield of scsps, whereas the interaction of ap1 dose with ph significantly affected scsp yield (p = 0.0051 < 0.01). method validation the optimal process parameters were obtained by design-expert 10.0.4 software. when the water:sc scale ratio was 50.613 ml/g, the ap1 dose was 0.964 µl/ ml, and the ph value was 8.140. the highest peptide yield was 88.57%. thus, after optimization, the parameters water:sc scale ratio of 50.6 ml/g, an ap1 dose of 0.96 µl/ml, and a ph of 8.14 were chosen. the experiment was conducted in triplicate, and the peptide yield was 88.77 ± 0.32%. there was no significant difference between the obtained scsp yield and the predicted value of the regression equation, indicating that the model was valid. table 4. response surface quadratic model anova and regression coefficient estimation. source sum of squares df mean square f p prob > f model 452 9 50.22 11.03 0.0023 significant a-water:sc scale 1.2 1 1.20 0.26 0.6234 b-ap1 dose 0.55 1 0.55 0.12 0.7381 c-ph 46.08 1 46.08 10.12 0.0155 ab 42.25 1 42.25 9.28 0.0187 ac 44.22 1 44.22 9.71 0.0169 bc 73.10 1 73.10 16.05 0.0051 a2 28.57 1 28.57 6.27 0.0407 b2 27.49 1 27.49 6.04 0.0437 c2 168.71 1 168.71 37.05 0.0005 residual 31.88 7 4.55 lack of fit 21.99 3 7.33 2.96 0.1606 not significant pure error 9.89 4 2.47 cor total 483.88 16 r2 = 0.9341 r2 adj = 0.8494 cv = 2.58% table 3. independent variables and response value. run no. a water:sc scale (ml/g) b ap1 dose (µl/ml) c ph y peptide yield (%) 1 50 1.1 7.5 79.1 ± 2.33 2 45 0.9 8.0 79.3 ± 5.04 3 45 1.0 7.5 72.7 ± 3.21 4 55 1.1 8.0 80.2 ± 3.50 5 55 1.0 8.5 79.1 ± 2.41 6 45 1.1 8.0 87.8 ± 5.58 7 50 1.0 8.0 87.6 ± 5.18 8 50 1.1 8.5 76.4 ± 2.51 9 50 1.0 8.0 87.0 ± 7.05 10 55 1.0 7.5 82.0 ± 4.75 11 50 1.0 8.0 88.6 ± 2.01 12 55 0.9 8.0 84.7 ± 2.55 13 50 1.0 8.0 90.7 ± 4.50 14 50 0.9 7.5 73.6 ± 5.83 15 50 0.9 8.5 88.0 ± 3.23 16 45 1.0 8.5 83.1 ± 4.50 17 50 1.0 8.0 86.9 ± 5.05 quadratic term c2 significantly affected the peptide yield at the p < 0.01 level. analysis of the response surface plot according to the interaction results from table 3 and figure 2, with increasing ap1 dose, water:sc scale ratio, and ph value, the scsp yield initially increased and then decreased. among them, the ph and ap1 dose surface 50 italian journal of food science, 2023; 35 (2) zu x-y et al. increasing pressure when the two membrane pressures (figure 3) were greater than 0.25 and 0.4 mpa for m1 and m2, respectively. this is because the concentration of solute molecules in the membrane surface area is high, resulting in more macromolecule solutes accumulating on the surface of the membrane to form a gel layer (fugère et al., 2005). when the pressure is too high, the thickening of the dense gel layer produces strong resistance to the liquid material, thereby counteracting the driving force generated by the pressure and reducing the flux of the membrane. membrane flux and peptide permeability of each component as shown in figure 3a, with increasing operating pressure, the membrane flux increased gradually and then plateaued. in general, the internal and external pressure difference forms turbulence on the membrane surface, which increases the tangential shear force of the filtrates, thus leading to the increase of the flow rate and permeability per surface unit membrane (nghiem et al., 2005). however, the membrane flux remained unchanged with figure 2. three-dimensional response surface plots (a, c, and e) and contour maps (b, d, and f) for the interactions of enzymatic hydrolysis conditions. (a) (c) (e) (b) (d) (f) figure 3. effects of different operating pressures (a), temperatures (b), and times (c) on the membrane flux and peptide permeability of each component (d). (b) (d) (a) (c) italian journal of food science, 2023; 35 (2) 51 peptide extraction from silver carp of 100–576 da accounted for 100% of the peptides. this observation was related to the damping effect of the membrane material in the filtration process, where most of the interception was of water-soluble, small-molecule substances. analysis of amino acids of different components as presented in table 6, m1 peptides contained more hydrophobic aas and basic aas, while m2 peptides contained more acidic (glu, asp) and aromatic aas (phe, try), as well as his, val, and iso. the proportions of ala, gly, and phe in m2 peptides were enhanced by 94.79, 860.60, and 17.95%, respectively, compared with those in the enzymatic hydrolysate. numerous studies have shown that fish scales and skin peptides have antioxidant effects. the smaller the molecular weight, the more obvious is the antioxidant function, based on gastrointestinal absorption (ngo et al., 2010; wu et al., 2015). these functions may be mainly related to their aa compositions (gómez-guillén et al., 2011). studies have revealed that acidic aas are weakly acidic due to them containing carboxylates that can dissociate h+ and have a strong promoting effect on the scavenging of dpph and h2o2 free radicals and the reduction of iron (hernández-ledesma et al., 2010). the electron-dense side-chain groups contribute to the antioxidant properties of a peptide when there are relatively high number of try and his residues in the peptide (udenigwe and aluko, 2011). in addition, schurink et al. (2007) noted that phe and val can inhibit tyrosinase. ishikawa et al. (2007) reported that ala, gly, l-iso, and l-leu play a positive role in inhibiting melanin synthesis. based on the above studies, it was speculated that the m2 peptides from sc scales may have a strong free-radical scavenging effect and melanin inhibitory effect. conclusion referring to the peptide yield as an indicator, ap1 showed the best enzymatic hydrolysis effect on sc scales. according to theoretical and experimental results, a water:sc scale ratio of 50.6 ml/g, ap1 dose of 2313.6 u/ ml, and a ph of 8.14 were determined to be the optimal process parameters, and the peptide yield was as high as shown in figure 3b, membrane flux increased linearly with gradual increase in the operating temperature. the higher the temperature, the lower is the viscosity and greater the thermal movement of solute molecules, which facilitates the transport of solutes across the membrane and increases the filtrate permeability per unit membrane area (lau and ismail, 2009). when there is excess temperature, the solute molecules accumulate on the membrane surface and membrane pores over time, reduce the mass transfer coefficient, and cause irreversible damage to the membrane elements (benítez et al., 2009). in addition, pes was used as the membrane element material with a limit temperature of 40°c in this experiment. herein, although a high temperature was beneficial to the increase in membrane flux, we still recommend that the temperature does not exceed 35°c during operation. in terms of the operating time data shown in figure 3c, the membrane flux decreased slowly in the early stage and decreased rapidly in the intermediate stage. after 65 min, the membrane flux tended to be stable, probably because a large number of macromolecular solutes were deposited on the membrane surface, forming a permeable multilayer cake (kaya and dayanir, 2020). small-molecule solutes and solvent molecules can enter the membrane pores. under pressure, solutes with large diameters are blocked by the filter cake layer and enter the boundary layer into a circulation system (choobar et  al., 2019). thus, after 65 min, the material–liquid boundary layer, filter cake layer, and inside of the membrane pores reached dynamic equilibrium. the calculated peptide permeability results are shown in figure 3d. the obtained peptides could penetrate through the m1 and m2 membranes, and the permeability was as high as 91.73 ± 6.96% and 79.83 ± 7.23%, respectively. determination of peptide molecular weight by gel permeation chromatography as shown in table 5, the average molecular weight (mw) of the m1 peptides was 758 da, accounting for 92.32% of the peptides. the mw of the m2 peptides was 576 da, accounting for 79.16% of the peptides, and a range table 5. molecular weight of filter samples. sample peak mn mw mp polymolecularity area % m1 peptide main peak 499 758 850 1.382463 92.32 minor peak 67 71 69 1.062646 7.68 m2 peptide main peak 441 576 497 1.304956 79.16 minor peak 88 100 119 1.135745 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as 88.77 ± 0.32%. in membrane separation, the optimal operating pressure, temperature, and time for m1 were 0.25 mpa (0.4 mpa for m2), 35°c, and 65 min, respectively. under these conditions, the permeability of scsps through the m1 (m2) was as high as 91.73 ± 6.96% (79.83 ± 7.23%). the molecular weight of m1 (m2) peptides was 797 da (576 da), accounting for 92.32% (79.16%) of the total peptides. m1 peptides contained more hydrophobic and basic aas, while m2 peptides contained more acidic aas, which can scavenge free radicals and inhibit tyrosinases and melanin. this study will provide support for the further utilization of sc scales. acknowledgments the authors appreciate prof. xiuqin kong of lanzhou university of technology for her support in this work. funding the study was supported by the national key research and development program of china (2019yfd0902000), which is an environmental-friendly project. table 6. amino acid compositions of different components. amino acid proportion (%) enzymatic hydrolysate m1 peptides m2 peptides asp 0.59 ± 0.01 0.72 ± 0.05 thr 0.25 ± 0.00 0.55 ± 0.00 ser 0.47 ± 0.03 1.76 ± 0.02 glu 1.71 ± 0.03 1.69 ± 0.08 pro 0.34 ± 0.00 ala 1.73 ± 0.03 4.55 ± 0.08 3.37 ± 0.02 gly 0.33 ± 0.03 1.49 ± 0.02 3.17 ± 0.06 cys 5.99 ± 0.66 19.15 ± 1.05 1.59 ± 0.05 val 2.74 ± 0.15 2.32 ± 0.04 met 8.15 ± 0.48 14.88 ± 1.31 6.47 ± 0.04 ile 0.57 ± 0.00 2.61 ± 0.03 phe 13.15 ± 1.67 10.57 ± 1.45 15.51 ± 1.12 orn 5.59 ± 0.08 lys 11.98 ± 1.09 9.76 ± 1.11 7.29 ± 0.46 his 0.14 ± 0.00 trp 49.82 ± 3.29 31.54 ± 2.07 46.55 ± 1.41 arg 3.12 ± 0.55 4.82 ± 0.56 2.00 ± 0.00 hydrophobic aas 24.18 ± 1.17 34.67 ± 1.23 25.40 ± 0.83 basic aas 15.08 ± 1.01 14.58 ± 0.82 9.43 ± 0.45 acidic aas 1.71 ± 0.03 0.59 ± 0.01 2.41 ± 0.06 aromatic aas 62.97 ± 2.28 42.45 ± 1.43 62.06 ± 1.22 italian journal of food science, 2023; 35 (2) 53 peptide extraction from silver carp environmental science and technology. 39: 7698–705. https://doi.org/10.1021/es0507665 ngo, d.h., qian, z.j., ryu, 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of animal science and technology, china agricultural university, beijing 100193,china *corresponding author: tel. +86 10 62733799, fax +86 10 62829099, email: renlp@cau.edu.cn abstract the study aims to investigate the effects of garlic extracts on color, lipid oxidation, and oxidative breakdown products in raw ground beef during refrigerated storage. the two treatments were: control group (c, with no addition) and experiment group (d, 50 mg garlic extracts added to 100 g beef). adding garlic extracts significant increased a* value (p a ≤ 0.05), and significant decreased tbars and pv values (p a ≤ 0.05). the ph and –sh value of d group had a decreasing tendency (p a =0.0522) and an increasing tendency (p a =0.0636) respectively compared to c group. garlic extracts protected phospholipids, fatty acids and polypeptides from oxidation. the results indicate that garlic extracts have the antioxidant activity, helping maintain the meat color, inhibiting lipid oxidation and protein degradation of raw ground beef during refrigerated storage. mailto:renlp%40cau.edu.cn?subject= 140 ital. j. food sci., vol. 28 2016 introduction lipid oxidation is one of the primary mechanisms of quality deterioration in meat and meat products. the adverse changes in quality are manifested in flavor, color, texture and nutritive value, and the possible production of toxic compounds (claudia et al., 2014). beef and its products are rich in protein and lipids, which make them most suitable for consumer. however, beef contains high level of unsaturated fatty acids which are prone to oxidation (tichivangana and morrissey, 1985). to prevent or reduce lipid oxidation, butylated hydroxytoluene (bht), butylated hydroxyanisole (bha), tertiary butylhydroquinone (tbhq), trihydroxybutyrophenone (thbp), propyl gallate (pg), nordihydroguaiaretic acid (ndga) and ethoxyquin have been applied in meat products (trindadea et al. 2014). however, their application had been restricted because of their potential health risks and toxicity (sun and fukuhara, 1997). natural antioxidants can be used as alternatives to the synthetic antioxidants because of their safety and equivalent or greater effect on inhibition of lipid oxidation (andrew et al., 2014). garlic (allium sativum) has been a favorite additive to enhance the flavor of food as well as herbal medicine for many years in various cultures. it is well known that garlic has antimicrobial, antiprotozoal, antimutagenic, antiplatelet and antihyperlipidemic properties. garlic holds a unique position for therapeutic potential due to the antioxidant activity by scavenging reactive oxygen species (ros), enhancing the cellular antioxidant enzymes, and increasing glutathione in the cells. numerous studies have demonstrated that garlic exhibits cardioprotective (rahman and lowe, 2006), liver-protective (wang et al., 1998), beneficial effects in diseases such as ischemic-reperfusion arrhythmias and infarction (rietz et al., 1993), ischemic heart disease (arora et al., 1981), hypertension (foushee et al., 1982), hyperlipidemia (ernst et al., 1985), as well as prevent the processes of cancer (tanaka et al., 2006) and aging (li et al., 2012). a number of researchers reported that garlic and different garlic extracts have antioxidant activity contributed by organosulfur compounds, flavonoids and phenolic compounds (borek, 2001; otunola and afolayan, 2013). rahman et al. (2012) researched antioxidant properties of raw garlic extract using the 2, 2-diphenyl1-picrylhydrazyl (dpph) scavenging methods. cao et al. (2013) had studied the effects of garlic on quality and shelf life of stewed-pork during refrigerated storage. however, less attention has been assigned to garlic extracts used as an antioxidant in beef, especially in identifying the oxidative breakdown products so far. the objective of the present study was to determine the effect of garlic extracts on ph, color stability, lipid oxidation, and oxidative breakdown products of raw ground beef during refrigerated storage in order to provide a scientific basis for using garlic extracts as natural antioxidants to maintain the meat quality, extend shelf-life and prevent economic loss. materials and methods materials garlic extracts (10:1 of garlic: garlic extracts, three percent allicin content) was purchased from yuanshen bio-tech ltd. (xian, china). glutathione were obtained from sigma aldrich inc. (st. louis, mo, usa). ethanol, chloroform, methanol, ammonium thiocyanate, iron (ii) chloride, trichloroacetic acid (tca), thiobarbituric acid (tba), 5, 5'-dithiobis (2-nitrobenzoic acid) (dtnb), cumene hydroperoxide, tetraethoxypropane and other reagent were ‘analar’ grade from china medicine (group) beijing chemical reagent corporation (beijing, china). water for ultra-performance liquid chromatography quadrupole time of flight (uplc-qtof) was purified with a milli-q gradient a10 system (millipore, beijing, china). formic acid and acetonitrile were hplc-grade (fisher scientific, new jersey, usa). three simmental crossed cattle (620±15kg, 18 months) were slaughtered at a local commercial abattoir (jinweifuren co. ltd., beijing, china). transport, slaughtering, or invasive procedures on live animals involving in this study were handled in strict accordance to the guidelines approved by the animal welfare committee of china agricultural university (permit number: dk1008). after ageing at 4°c for 72 h, the longissimus dorsi (ld) were excised from 12th and 13th rib of left half side carcass. the muscles were minced twice through a 5 mm plate of meat mincer (model jys-a800, joyoung, china) after removing the connective tissue and visible fat. the contents of moisture, protein and fat of the ground beef were 73.14%, 22.26%, and 2.83% respectively. treatments the meat sample of each cattle was formed into two patties (100 g portions) using a meat former and assigned to the following two treatments:: control group (c, 100 g ground beef with no addition) and experiment group (d, 50 mg garlic extracts added to 100 g ground beef). to eliminate the influence of microorganism and ensure thorough mixed, garlic extracts dissolved in 10 ml of a distilled water and ethanol mixture (1:1, v/v) and then mixed with the muscles of the experiment group. the same volume of distilled water and ethanol mixture (with no added ingredients) was added to the control group. ital. j. food sci., vol. 28 2016 141 meat samples were put into a constant temperature incubator (mjx-320, jiangnan, ningbo, china) at 4 °c for 13 days. meat samples (three replicates) were collected in 1.5 ml centrifuge tube at storage times of 1, 3, 5, 7, 10, 13 days and were frozen rapidly in liquid nitrogen for subsequent analysis. analysis of ph and color a ph meter (ph spear, eutech instruments, usa) was used to measure the ph of the ground beef. the color of the ground beef was determined by portable colorimeter (cr400/410, minolta, japan). the specifications of the colorimeter are light source: pulsed xenon lamp; illuminant: c, d65; illumination area: φ8/φ11; inter instrument agreement: δe*ab within 0.6; repeatability: within δe*ab0.07 standard deviation. the color results were calculated based on l*, a*, b* (lightness, redness, and yellowness respectively) in the cielab space. a white plate (cie l*= 97.83, a*=-0.43, b*=1.98) was used for calibration. analysis of lipid oxidation lipid oxidation was evaluated by the peroxide value (pv) and thiobarbituric acid reactive substances (tbars) according to the method of richards et al. (2003) (richards and dett mann, 2003) and free thiol groups (-sh) was analyzed following the method proposed by egelandsdal et al. (2011) with minor modifications. determination of pv: approximately 0.3 g of sample was homogenized for 30 s with 5 ml of cold chloroform/methanol (1:1) in a 50 ml glass tube using a refiner (fj-200, jintan, china). subsequently, the glass tube was rinsed for 30 s again with 5 ml of cold chloroform/ methanol (1:1). the homogenate and wash solution were then combined in a 25 ml glass test tube. adding 3.08 ml of 0.5% nacl then mixed for 10 s with a vortex (g560e, scientific industries inc., new york, usa). next, the mixture was centrifuged at 1821g (dl-6000b, shanghai anting, china) for 6 min at 4°c. two milliliters of the lower chloroform layer was removed and transferred to a tube by a glass syringe before 1.3 ml chloroform/methanol (1:1) was added to the 2 ml sample. then 25 μl of 3.94 mol/l ammonium thiocyanate and 25 μl of 18 mmol/l iron (ii) chloride were added to the tube, mixing thoroughly after each addition. finally, the sample was incubated at room temperature for 20 min and the absorbance at 500 nm was measured using a spectrophotometer (uv1102, shanghai tianmei, china). a standard curve was constructed using cumene hydroperoxide and the pv in the sample was expressed as µmol/kg. determination of tbars: mixing 50% trichloroacetic acid (tca) with 1.3 % thiobarbituric acid (tba) on the day of use and then heating to 65°c. approximately 0.12 g of the muscle sample was added to 1.2 ml the tca tba mixture and mixed via a vortex (g560e, scientific industries inc., new york, usa) and then heated at 45°c for 60 min using a vapor-bathing constant temperature vibrator (hq45, chinese academy of sciences, wuhan, china) before the sample was centrifuged at 12,000 g for 5 min (mikro 200r, hettich, germany). the absorbance of the supernatants at 532 nm was determined. a standard curve was prepared using tetraethoxypropane, and the concentration of tbars in the samples was expressed as µmol/kg. determination of -sh: approximately 0.1 g of the muscle sample was weighed into a 10 ml centrifuge tube then 1700 μl phosphate-buffered saline (ph=8.2) and 100 μl dtnb were added. the mixture was heated at 45 °c away from light for 60 min using a vapor-bathing constant temperature vibrator (mjx-320, jingbo jiangnan, china). eight milliliters of methanol was added to the tube which was vor texed for 30 s before centrifuging at 1821g for 20 min at 4°c. subsequently, 1 ml supernate was transferred to a new centrifuge tube, diluted with 3 ml methanol and then the absorbance was measured at 412 nm. glutathione was used for generating a standard curve and concentration of -sh in samples was expressed as mmol/kg. analysis of oxidative breakdown products sample preparation: 50 mg muscle sample from each treatment at 13 days was weighed into a 2 ml eppendorf tube. the first step: 1.5 ml cold water/methanol (1:1) and 0.5 g 1 mm zirmil ceramic beads were added to the tube and then mixed thoroughly using a tissue homogenizer (precellys 24, bertin, france) for two cycles at 6500 hz, 40 s for each cycle. the mixture was centrifuged at 12,000 g (mikro 200r) for 10 min at 4°c. supernate (400 μl) was collected in a new eppendorf tube and stored at 4°c for further use. the second step: the residual sediment from step one was extracted by 1.5 ml cold chloroform/methanol (3:1) again with the same procedure of homogenization and centrifugation and the same volume of supernate collected in the eppendorf tube. the mixed samples from two steps, were then concentrated in a centrifugal concentration meter (zls-1, herexi, hunan, china). water/methanol (9:1; 120 μl) was used to dissolve the samples with vortex oscillation for 40 s. supernate (100 μl) was collected in a lining tube for subsequent uplcqtof analysis. uplc analysis: the substances in the 6 μl extracted sample were separated using a uplc system (acquity uplc/xevo g2 q tof, waters) after being loaded onto a high-resolution and high142 ital. j. food sci., vol. 28 2016 performance uplc beh c18 column (1.7 μm, 2.1 mm × 50 mm, waters). the column temperature was 50 °c and the flow rate was 0.3 ml/ min with solvent a (water + 0.1 % formic acid) and solvent b (acetonitrile + 0.1% formic acid). the elution program consist of 98:2 (a: b) for 1 min, increasing gradually to 100 % b by 16 min, then 100 % b held for 2 min, a ramp to 98:2 (a: b) within 10 s, and returned to 98:2 (a: b) for 2 min (re-equilibration of the column) before the loading of the next sample. q-tof ms conditions: the separated components from the uplc were subsequently analyzed by quadrupole time of flight mass spectrometry (q-tof ms) equipment (acquity uplc/ xevo g2 q tof, waters) operated using a negative electrode for electrospray ionization. the settings were as following: data acquired in a full scan mode (mass: charge ratio (m/z) 50-1200) at a rate of 2 spectra/s; capillary and sampling cone voltages at 2500 and 35 v, respectively; desolvation temperature at 350°c; desolvation gas flow at 720 l/h; cone gas flow at 50 l/h; source temperature at 105°c. all gas paths used nitrogen and data were collected using masslynx 4.1 data management software (waters, usa). the final data was expressed by the id number, relative m/z values, retention times and ion intensity. statistical analysis this experiment was assigned two factors with two treatments and seven storage time levels.s. the mixed model procedure of sas 9.0 (sas institute inc.) was used to analyze the effects of garlic extracts and storage time. the following statistical model was used for analysis: y ik = μ + α i + β k + (αβ) ik + e ik where y ik is an observed value for tbars, pv, -sh, ph, l*, a*, and b*, taken from sample receiving treatment i at time k;μis the overall mean; α i is the fixed effect of treatment i; β k is the fixed effect of time k; (α β) ik is the interaction between treatment and storage time; and e ik is the residual value. the variation tendency of ph and color, lipid oxidation with different storage times was analyzed by the contrast model of sas 9.0. the means that were significantly different were analyzed using the t-test and different storage times were analyzed using duncan’s multiple comparison tests. a level of p ≤ 0.05 was considered significant and 0.05<p < 0.1 was considered tendency. differences in oxidative breakdown products among treatments were analyzed with principal component analysis (pca) and orthogonal partial least squares-discriminant analysis (oplsda) using simca-p software (umetrics, ume, sweden). the different oxidative breakdown products between two treatments were analyzed in terms of ion intensity using the t-test. metabolites that met the criterion of p ≤ 0.05 of the t-test and a fold change > 1.5 were selected as different oxidative breakdown products whose formulae were searched in metlin database. table 1 the effects of garlic extracts and storage time on physical meat quality, lipid oxidation in raw ground beef during refrigerated storage. items tbars pv -sh ph l* a* b* treatments c 22.41a 46.56a 6.62 5.86 51.22 13.50b 14.07b d 19.59b 30.22b 7.10 5.85 51.35 14.49a 14.59a sem 0.6121 9.7158 0.3015 0.0027 0.2020 0.2086 0.1422 times 1 10.25c 23.78b 7.16ab 5.91a 51.05b 18.05a 14.71a 3 13.50b 28.94b 8.14a 5.84b 50.13c 15.68b 13.52b 5 26.92a 42.11ab 7.23ab 5.84b 51.96a 15.44b 14.76a 7 24.20a 69.32a 7.12ab 5.83b 51.99a 13.98c 15.02a 10 26.07a 44.82ab 6.03bc 5.82c 51.01b 10.18d 13.55b 13 25.08a 21.39b 5.45c 5.91a 51.57ab 10.65d 14.43a sem 0.6121 9.7158 0.3015 0.0027 0.2020 0.2086 0.1422 p a <0.001 0.0500 0.0636 0.0522 0.4248 <0.001 0.0001 p b l <0.001 0.5139 <.0001 0.1356 0.0123 <0.001 0.5835 q <0.001 0.0085 0.0238 <.0001 0.0852 0.9871 0.8118 p ab 0.0383 0.9684 0.1200 0.0035 0.5571 0.0034 0.0152 means in the same column with different superscripts are significantly different (p <0.05).c-control group, d-garlic extracts group. p a is the p value of treatments; p b is the p value of storage times; l stands for the linear effects of storage times; q stands for the quadratic effects of storage times. p ab is the p value interaction between treatments and storage times. ital. j. food sci., vol. 28 2016 143 results the effects of garlic extracts and storage time on physical meat quality, lipid oxidation in raw ground beef during refrigerated storage according to table 1, l* and a* value presented an increasing and decreasing linear change respectively (p b l<0.05), tbars, pv, -sh, and ph had a quadratic change over storage time (p b q<0.05). more details, tbars value significant increased in the first five storage days (p b <0.05), and then no significant changes in the residual storage time. pv and -sh value were firstly increased and then decreased with the highest value at seventh day and third day respectively of storage time. on the contrary, ph was firstly decreased and then increased with the lowest value at tenth day of storage time, ranging from 5.82 to 5.91. however, storage time had neither linear nor quadratic effect on b* value in raw ground beef during refrigerated storage. as shown in table 1, adding garlic extracts could significant increased (p a ≤ 0.05) a* and b* values. the ph of d group had a decreasing tendency compared to c group (p a =0.0522). there was no significantly different on l* value between treatments (p a =0.4248). tbars and pv values of d group were significant lower (p a ≤ 0.05), and –sh tended to increase (p a =0.0636) than c group in raw ground beef during refrigerated storage. because table 1 could not show the effects of garlic extracts on every storage time, the significant indicators a*, pv, and tbars values (p a ≤ 0.05) influenced by garlic extracts were further analyzed on every storage time. fig. 1 showed that a* value was significant increased following the addition of garlic extracts on days 3, 7 and 13 during refrigerated storage relative to the control(p < 0.05). adding garlic extracts had a tendency to reduce the pv values on days 5 and 13 (0.05<p < 0.1), and sigfig. 1 effects of garlic extract on a* value at different storage time of raw ground beef. fig. 2 effects of garlic extract on pv at different storage time of raw ground beef. fig. 3 effects of garlic extract on tbars at different storage time of raw ground beef. nificantly reduced the pv value on 10 days of storage time as compared to control (p < 0.05) (fig. 2). fig. 3 showed that a decreasing tendency of tbars value in e group was found compared to c group at the storage time 3, 7, and 10 days (0.05<p < 0.1). the tbars value was significantly (p < 0.05) lower in d group than c group at the end of storage time. effects of garlic extract on oxidative breakdown products pca score plot of different treatments at the 13 days storage time of raw ground beef was shown in fig. 4. these results show an easily visible separation of two different groups c and d marked with black and red colors. fig. 5 showed the loading scores plot from opls-da between c group and d group. each numbered point represented one breakdown product, which the further away from the center, the more likely that breakdown products between two groups are different. 144 ital. j. food sci., vol. 28 2016 fig. 5 loading scores plot from opls-da between c group and d group at the end of storage time in raw ground beef. fig. 4 pca score plot of different treatments at the end of storage time in raw ground beef. according to the results from opls-da analysis and original data, the different breakdown products were confirmed between c and d group and shown in table 2. there were total nine different breakdown products significantly higher in d group than c group (p < 0.05) and could been divided into three categories, including phospholipids, fatty acids and polypeptides. pc was belongs to phospholipids. fatty acids contained malic acid, 16-hydroxy-9e-hexadecenoic acid and 9-hydroxy-10e-octadecen-12-ynoic acid. polypeptides were made up with gln-ileasn-leu, arg-pro-lys-arg, met-his-gln –asn, thr-lys-lys-thr, and gly-arg-cys. ital. j. food sci., vol. 28 2016 145 discussions ph and color the ph of fresh meat is an important indicator to determine the freshness of meat, which can be influenced by the accumulation of organic acids and amines during refrigerated storage. organic acids, produced by gram positive bacteria and released by the decomposition of lipids oxidative, decrease the ph of meats, whereas the rise of ph was due to the decomposition of alkaline ammonia, which induced by gram negative bacteria (lefebvre et al., 1994). the ph value with a quadratic change over storage time in this study because the result of the interaction of these two effects. the results ph value was inconsistent with cao et al. (2013) found that all the samples gradually increased in stewedpork during storage at 4°c for 12 days (cao et al., 2013). the reason of different results was mainly caused by adding ethanol to eliminate the influence of microorganism in the present study. the color of foods is one of the major determinants of its appeal to consumers and consequently, sales of the product. the color of meat and meat products is influenced by the percentage of red appeared deoxymyoglobin, metmyoglobin and oxymyoglobin in muscle, which would predispose the meat to a faster browning rate (hunt et al., 1999). the result of decreased a* value over storage time in the present study highly agreed with fernandez-lopez et al. (2005) (fernandez-lopez et al., 2005) mainly was caused by the formation of metmyoglobin during storage. several authors have studied the effect of different antioxidants on the color of meat and meat products and have reported that the antioxidants could retard the decrease of a * values during storage (fernandez-lopez et al., 2005; sanchez-escalante et al., 2001; kim et al., 2013). in agreement with these studies, garlic extracts also could retard the decrease of a * values compared to the control group during storage. garlic extracts could stabilize the redness in raw ground beef during refrigerated storage due to its antioxidant properties preventing the oxidation of oxymyoglobin. lipid oxidation lipid oxidation is a critical and undesirable phenomenon for meat and meat productions since the undesirable rancid off-flavours and potentially toxic products, leading to the qualitative deterioration. peroxides are the primary products of lipid oxidation which is generated by oxygen attacking on the double bond in fatty acids. therefore, peroxide value (pv) is usually as an indicator to clarify the extent of oxidation (mottram, 1998). peroxide is very reactive and actually degraded into secondary oxidation products during the storage of lipid-containing foods (juntachote et al., 2007). therefore, pv value increased and thereafter decreased during storage in present study was caused by the peroxide formation and degradation. similar results of pv fluctuated over storage time were reported by teets et al (2008) (teets and were, 2008). the tbars value is an index of secondary lipid oxidation and can be used to monitor lipid oxidation in meat samples during storage (qin et al., 2013). the secondary lipid oxidation products mainly aldehydes (or carbonyls), which contribute to off-flavors in oxidized meat and meat products, result from the degradation of lipid hydroperoxides formed during the oxidation process of polyunsaturated fatty acids(fernandez et al., 1997). xie et al. (2012) reported that the tbars value was increased with the storage time at 4°c for 3 days in beef of five cattle breeds (xie et al., 2012). the results of this study showed that a similar trend, which the tbars value significant increased in the first five storage days. cao et al (2013) found that stewed-pork treated with extracts of ginger, onion, garlic had lower pv and tbars value compared to the control. park et al. (2010) reported that all extractable 2 different breakdown products between c and d treatments at the end of storage time (n=3). products formula [m-h] calculated [m-h] observed masserror p fold change pc(15:1(9z)/0:0) c 23 h 46 no 7 p 478.2939 478.2930 1.88 0.0099 +∞ malic acid c 4 h 6 o 5 133.0142 133.0142 0 <0.001 +∞ 16-hydroxy-9e-hexadecenoic acid c 16 h 30 o 3 269.2122 269.2119 1.11 0.0034 10.54 9-hydroxy-10e-octadecen-12-ynoic acid c 18 h 30 o 3 293.2122 293.2110 4.09 0.0166 2.35 glnile-asn-leu c 21 h 38 n 6 o 7 485.2729 485.2773 9.07 0.0047 +∞ arg-pro-lys-arg c 23 h 45 n 11 o 5 554.3532 554.3462 12.63 0.0094 +∞ met-his-gln-asn c 20 h 32 n 8 o 7 s 527.2042 527.2073 5.88 0.0159 +∞ thr-lys-lys-thr c 20 h 40 n 6 o 7 475.2886 475.2886 0 0.0196 +∞ gly-arg-cys c 11 h 22 n 6 o 4 s 1 333.1351 333.1349 0.60 0.0494 +∞ [m-h]-, protonated molecular ion; mass error expressed as ppm. fold change, which was based on the original data, was defined as the fold difference in the observed concentrations between c and d groups. a positive number for a fold change indicates that the value of d group was greater than the c group, whereas the opposite is indicated by a negative number. 146 ital. j. food sci., vol. 28 2016 tion solvents of garlic could reduce the tbars value in fresh pork patties during refrigerated storage (park and chin, 2010). sallam et al (2004) observed that garlic extracts had a marked effect in reducing pv and tbars values of chicken sausage (sallam et al., 2004). in agreement with earlier studies, the same results of decreased pv and tbars values in the present study indicate that garlic extracts are effective at delaying lipid peroxidation in raw ground beef during refrigerated storage. according to yin et al. (2003), four garlic-derived organosulfur compounds, diallyl sulfide (das), diallyl disulfide (dads), s -ethyl cysteine (sec), nacetyl cysteine (nac), significantly delayed oxymyoglobin and lipid oxidations in ground beef (yin and cheng, 2003). otunola et al. (2013) found that garlic extracts have high flavonoids and phenolics contents and high antioxidant activities (otunola and afolayan, 2013). therefore, the ability of garlic extracts to inhibit lipid oxidation is probably related to their antioxidant activity contribute to organosulfur compounds, flavonoids and phenolics compounds. free thiol (-sh) was used to stabilize primary oxidation products (sista et al., 2000). the increase of -sh value at the first three storage days was probably caused by oxidized glutathione (gssh) being transformed into glutathione (gsh) under the action of glutathione reductase. on the contrast, transforming hydrogen peroxide into water under the action of glutathion peroxidase results in the decrease of –sh value after three days during storage time. the decrease –sh value in beef over storage time was observed by sullivana et al. (2012) (zakryswaliwander et al., 2012). gsh is considered an abundant antioxidant within the cell and is essential for regulation of intracellular redox status (izigov et al., 2011). allicin is formed by alliin enzymatically modified under the action of alliinase in garlic (okada et al., 2005). according to the study of kim et al. (1997), allicin is the main antioxidative component of freshly crushed garlic cloves (kim et al., 1997). horev azaria et al. (2009) reported that allicin could directly raising glutathione content in the cell and indirectly increasing glutathione by allicin derivatives such as s-allylmercaptoglu tathione and s-allylmerc aptocysteine (horev-azaria et al., 2009). therefore, the existence of allicin could explain the results that adding gailic extracts increase the –sh value. breakdown products in the present study, garlic extracts could protect the phospholipids, unsaturated fatty acids and polypeptides from oxidation. phospholipids, which constitute the lipid bilayer defining the outer confines of a cell, are the primary substrates for lipid oxidation and membrane components in close contact with the catalysts of lipid oxidation, which are located in the aqueous phase of the muscle cell (pulfer and murphy, 2003). skeletal muscle was susceptible to oxidative due to the membrane lipid systems that were high in unsaturated fatty acids (chan and decker, 1994) (chan and decker, 1994). xie et al. (2012) found that higher unsaturated fatty acids content in qinchuan cattle lead to more easily lipid oxidation (xie et al., 2012). in addition, gruen et al. (2001) revealed that increase of oxidation can enhance protein degradation (grune et al., 2001). therefore, the results indicate that garlic extracts could help to stabilize the muscle membrane and reduce the degradation of fat and protein due to its antioxidant. conclusions the results of this study clearly revealed that the effects of garlic extract on color, lipid oxidation and oxidative breakdown products of raw ground beef during refrigerated storage. the results of higher a * value and lower pv, tbars values indicate that garlic extracts have the antioxidant activity, helping maintain the beef color, inhibiting lipid oxidation. in addition, our experiments provide new and important information regarding the effects of garlic extracts on inhibiting lipid oxidation and protein degradation through protecting phospholipids, unsaturated fatty acids and polypeptides in raw ground beef during refrigerated storage. overall, garlic extracts could be used as natural antioxidants to 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ground beef. meat sci. 63: 23-28. zakrys-waliwander p.i., o’sullivan m.g, o’neill e.e. and kerry j.p. 2012. the effects of high oxygen modified atmosphere packaging on protein oxidation of bovine m. longissimus dorsi muscle during chilled storage. food chem. 131: 527-532. paper received october 24, 2014 accepted june 25, 205 92 issn 1120-1770 online, doi 10.15586/ijfs.v33isp1.2065 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (sp1): 92–102 et al., 2021; heshmati et al., 2019). they pose carcinogenic, mutagenic, immunosuppressive, and teratogenic consequences. the most common afs are aflatoxin b1 (afb1), b2 (afb2), g1 (afg1), and g2 (afg2) (heshmati et al., 2017). afb1 is the most carcinogenic type for humans and animals (mokhtarian et al., 2020). the international agency for research on cancer (iarc) classified afb1 as a group 1 carcinogen (elzupir et al., 2010). aflatoxins production may occur during harvesting, transportation, storage, or on the farm. afs are very stable chemical compounds resistant to heat and food processes (cui et al., 2020). they can contaminate various foods, including cereal, dairy products, oilseeds, p u b l i c a t i o n s codon the prevalence and risk assessment of aflatoxin in sesame-based products ali heshmati1, mina khorshidi1*, amin mousavi khaneghah2* 1department of nutrition and food safety, school of medicine, nutrition health research center, hamadan university of medical sciences, hamadan, iran; 2department of food science and nutrition, faculty of food engineering, university of campinas (unicamp), são paulo, brazil *corresponding author: mina khorshidi, department of nutrition and food safety, school of medicine, nutrition health research center, hamadan university of medical sciences, hamadan, iran. email: mkhorshidi2992@gmail.com; mousavi khaneghah, department of food science and nutrition, faculty of food engineering, university of campinas (unicamp), são paulo, brazil. email: mousavi@unicamp.br received: 9 may 2021; accepted: 4 june 2021; published: 5 july 2021 © 2021 codon publications open access paper abstract the contamination of aflatoxins (afs) in 120 samples of sesame seeds, tahini, and tahini halva collected from iran’s market were evaluated. the exposed risk due to ingestion of aflatoxin b1 (afb1) via their consumption was estimated with the aid of the monte carlo simulation (mcs). the highest prevalence of af (55%) was associated with sesame seed samples, followed by tahini (45%) and tahini halva (32.5%). the afb1 concentration in sesame seeds, tahini, and tahini halva was in the ranges of 0.21–12.35, 0.23–5.81, and 0.27–3.56 μg/kg, respectively. the concentration of the total aflatoxin (taf) in 7 (17.5%), 8 (20%), and 2 (5%) samples of sesame seeds, tahini, and tahini halva, respectively, was below the limit of european regulations (4 µg/kg), while the levels of afb1 in 10 (25%), 7 (17.5%), and 6 (15%) samples of sesame seeds, tahini, and tahini halva, respectively, were higher than the european regulations (2 µg/kg). as the percentile 50 and 95 of margin of exposure (moe) with afb1 for sesame seed, tahini, and tahini halva was more than 10,000, it could conclude the intake of aflatoxin through the consumption of mentioned products did pose a not remarkable cancer risk for adults. keywords: mycotoxin; contamination; risk assessment; traditional products; sesame based introduction today, humans pay considerable attention to food safety and contaminants (milicevic et al., 2021; rapa et al., 2021; xinyu et al., 2020). mycotoxins produced by fungi are among the most important food contaminants and have a negative impact on public health, food safety, and the national economy of many countries, especially developing countries (batrinou et al., 2020; grumi et al., 2020). the most critical factors in food contamination with mycotoxins are moisture, intrinsic properties and nutrients, long shelf life and ph, and high-water activity (wang et al., 2018). aflatoxins (afs) are produced by aspergillus fungi, especially a. flavus and a. parasitics and rarely by a. nomius (de souza mailto:mkhorshidi2992@gmail.com mailto:mousavi@unicamp.br italian journal of food science, 2021; 33 (sp1) 93 prevalence and risk assessment of aflatoxin in sesame-based products prevalence of afs in sesame seeds and their products was demonstrated by previous studies (anthony et  al., 2014; apeh et al., 2016; asadi et al., 2011; esan et al., 2020; fapohunda et al., 2018; hosseininia et al., 2014; kollia et al., 2016; li et al., 2009; reddy et al., 2011; sabry et al., 2016; sebaei et al., 2020; sirhan et al., 2014; tabata, 2007; torlak and akan, 2013; var et al., 2007). no studies have been performed to evaluate mycotoxin contamination in tahini and tahini halva samples available in iran’s market. therefore, the current study aimed to determine the level of afs in sesame seed, tahini, and tahini halva, and halva consumed in western iran. furthermore, the exposed risk due to ingestion of afb1 via their consumption was estimated with the aid of the monte carlo simulation (mcs). material and methods materials acetonitrile, hno3 (65%), phosphate buffer solution (pbs), methanol, potassium bromide (kbr), were purchased from merck (darmstadt, germany). the af standards were purchased from sigma-aldrich (st. louis, mo, usa) sample collection samples (n = 120; 40 fresh sesame seed, 40 tahini, and 40 tahini halva) were collected from the local market in hamadan province, iran, from april 2020 until august 2020. samples were stored in a refrigerator (4°c) until analysis. chemical properties moisture content (%) of sesame seed, tahini, and tahini halva was determined by drying in an oven at 100°c. the protein and fat contents of samples were determined by the kjeldahl method and soxhlet extraction, respectively (zebib et al., 2015). extracted fat acidity was measured by titration by sodium hydroxide (0.01°n) in the presence of phenolphthalein as an indicator. extract and cleanup of af the method applied for the extraction and cleanup of af was similar to the previous one with slight modifications (torlak and akan, 2013). samples were wholly powdered and mixed. fifty grams of ground sample was weighed and transferred into a 250 ml flask. then, 150 ml of a mixture of water and methanol (30:70; v/v) was added and placed spices, and nuts (javanmardi et al., 2020; khaneghah et al., 2018; mozaffari nejad et al., 2020). due to the high toxicity of afs, exposure to these contaminations could threaten human health. the committee on food additives of joint fao/who (jecfa) recommends that the presence of mycotoxins in meals be minimized to reduce the potential risk (di sanzo et al., 2018). therefore, in order to control afs in food products, some regulations were established in many countries; the measurement and monitoring of afs in food products are crucial (sebaei et al., 2020). with the scientific name of sesamum indicum l., sesame seed belongs to the pedaliaceae family and is one of the oldest and most momentous oilseeds globally (lee et al., 2020). it contains 58%–44% oil, 25%–25% protein, 13.5% carbohydrates, and 5% ash (kollia et al., 2016). it is also a significant source of dietary fiber and micronutrients such as minerals, including calcium, phosphate, iron, potassium, vitamins such as e, thiamine, and niacin, lignans tocopherols, and phytosterols (elleuch et al., 2011; yao et al., 2021). sesame seeds have antioxidant, anti-inflammatory, anti-fungal, anti-viral, and natural antibacterial effects (dravie et al., 2020). sesame seeds are consumed in different forms in the world (namiki, 2007). it is widely used in the iranian food industry as an ingredient in confectionery products, bread, and pastries. therefore, ensuring the mycotoxicological quality of sesame is very important (asadi et al., 2011; eghbaljoogharehgheshlaghi et al., 2020; elleuch et al., 2011; kollia et al., 2016). tahini is made by grinding and roasting sesame seeds known in the middle east (gholami et al., 2020; sebaei et al., 2020). tahini halva, also known as halva, helva, halawi, halawh, is produced by mixing tahini with sugar, citric or tartaric acid, and saponaria officinalis root extract (öğütcü et al., 2017; osaili et al., 2018a; var et  al., 2007). consumption of tahini halva has increased due to its excellent nutritional value and health properties in other countries, including the united states and european countries, iran, turkey, saudi arabia, iraq, greece, jordan, bulgaria, and bosnia and herzegovina (anilakumar et al., 2010). tahini halva consists of about 45% tahini, 45%–55% sugar, 2% ash, and 2.5% moisture and is consumed with bread in breakfast and dinner (osaili et al., 2018b). when sesame seeds were stored improperly, they can be contaminated with mycotoxins, especially afs (anthony et al., 2014). therefore, the eu sets limits for afb1 and taf in sesame and its products as 2 and 4 μg/kg, respectively. in contrast, the limit of these mycotoxins, according to the iranian institute of standards and industrial research (isiri), was 5 and 15 μg /kg (inso, 2020). the 94 italian journal of food science, 2021; 33 (sp1) heshmati a et al. on a shaker and stirred for 10 min. then, it was filtered through filter paper of whatman no. 2. twenty milliliters of filtered solutions were collected and transferred into a 100 ml flask, and 40 ml of distilled water was added and mixed for 5 min and filtered. for the cleanup of afs, 15 ml of filtrate was transferred through the immunoaffinity column at a flow rate of 2–3 drops/s. further, the column was washed three times with 10 ml distilled water at the same flow rate. the elution of afs was performed by acetonitrile (1 ml). the eluate was gathered in a vial, and its volume was reached 2 ml with acetonitrile. then, 100 µl of eluted afs was injected into a high-performance liquid chromatography (hplc) instrument. analysis of afs the quantification of afs in sesame products was carried out by hplc (knauer-germany), equipped with a smartline pump, fluorescence detector, reverse phased c18 (150 mm × 4.6 mm i.d and 5 μm particle size). in the fluorescence detector, an excitation wavelength of 333 nm and emission wavelength of 430 nm was applied for afs determination. the mobile phase was water/methanol/ acetonitrile (6:102:94, v/v/v) and contained 100 mg kbr and 100 μl hno3. then, it is diverted into hplc in the isocratic method with a flow rate of 0.8 ml/min. the column temperature of hplc was maintained at 40°c. the validation of afs analysis method the linearity, accuracy, repeatability, limit of detection (lod), and limit of quantification (loq) were determined (heshmati et al., 2017). risk assessment for risk assessment of afb1 intake through sesames seed, tahini, and tahini halva, the probabilistic approach was considered and estimated daily intake (edi). the following equations are used to calculate the margin of exposure (moe): edi (ng/kg bw/day) = the concentration of afb1 × average daily consumption (kg)/average body weight (kg) per capita consumption of sesame in iran is 3 g/day (eghbaljoo-gharehgheshlaghi et al., 2020). in this study, 70 kg is considered as the mean body weight for an adult in iran. moe = benchmark dose lower confidence limit 10% (bmdl10)/edi where, bmdl10 is the lowest dose that is 95% certain to cause no more than 10% cancer prevalence. the efsa panel on contaminants in the food chain (contam) suggested 400 ng/kg bw/day for bmdl10 reference value (chain et al., 2020). a moe of 10,000 or larger has little concern for public health, while a moe of less than 10,000 shows a potential danger for consumers (heshmati et al., 2017). edi and moe were estimated by mcs using crystal ball software (version 11.1.2.3 oracle). statistical analysis for statistical analysis, the spss software version 20 (ibm, pasw statistics, usa) was applied. the mean and standard deviation of afs levels in different samples were determined. one sample t-test was used to compare the mean of afb1 and taf with the allowable limit. the significant level was considered p < 0.05. one-way anova and tukey’s test were applied to determine the significant difference of moisture, fat, protein content, and extracted fat acidity levels of these samples. result and discussion the chemical properties of samples the moisture, fat, protein, and extracted fat acidity amount of sesame seed, tahini, and tahini halva samples are shown in table 1. sesame seed had a higher moisture, fat, and protein value than tahini and tahini halva samples. method validation the lod of afs for sesame seeds, tahini, and tahini halva samples ranged from 0.04 to 0.08, 0.05 to 0.09, and 0.07 to 0.08 µg/kg, respectively while loq for them ranged from 0.13 to 0.25, 0.18 to 0.31, and 0.21 to 0.27 µg/kg, respectively (table 2). moreover, the recovery range for afs of sesame seeds, tahini, and tahini halva samples was 82.15–96.23, 77.36–95.63, and 83.65–98.65, respectively (table 3). the determination coefficients (r2 > 0.992) of the table 1. chemical proprieties of sesame and related products. parameters sesame seed tahini tahini halva moisture (%) 6.87 ± 0.38a 1.15 ± 0.07c 2.5 ± 0.06b fat (%) 52.31 ± 2.89a 49.13 ± 2.12b 28.45 ± 2.32c protein (%) 22.34 ± 1.87a 20.67 ± 1.76b 9.87 ± 0.75c extracted fat acidity (%, in oleic acid) 0.82 ± 0.09a 0.90 ± 0.10a 0.76 ± 0.07a the different superscript letters within each row indicated significant differences (p < 0.05). italian journal of food science, 2021; 33 (sp1) 95 prevalence and risk assessment of aflatoxin in sesame-based products table 3. recovery of aflatoxin from sesame and related products. aflatoxin type spiked level (µg/kg) recovery ± rsd (%) sesame seed tahini tahini halva afb 1 0.5 82.15 ± 4.51 84.56 ± 10.36 87.65 ± 12.74 2 87.63 ± 8.56 88.32 ± 13.08 90.23 ± 15.32 5 85.25 ± 3.65 87.32 ± 8.98 85.32 ± 14.56 afb 2 0.5 90.23 ± 10.23 79.65 ± 4.56 94.36 ± 12.02 1.5 92.35 ± 12.32 82.36 ± 7.25 98.65 ± 4.08 3 95.63 ± 15.36 77.36 ± 14.97 94.82 ± 6.23 afg 1 0.5 89.69 ± 12.31 83.65 ± 4.56 88.63 ± 4.51 1.5 92.34 ± 8.96 86.53 ± 45.23 87.56 ± 14.23 3 94.23 ± 14.85 82.03 ± 12.78 83.65 ± 14.32 afg 2 0.5 90.23 ± 11.57 90.23 ± 15.63 89.32 ± 4.36 1.5 96.23 ± 8.69 95.63 ± 10.56 90.56 ± 8.69 3 94.23 ± 10.23 92.53 ± 8.02 95.36 ± 10.26 af: aflatoxin; rsd: relative standard deviation table 2. validated parameters for aflatoxin analysis in sesame and related products. aflatoxin type equation of calibration curve range of linearity (ng/ml) r2 sesame seed tahini tahini halva lod loq lod loq lod loq afb 1 y = 13562.23x + 456.03 0.15–25 0.998 0.06 0.21 0.07 0.23 0.08 0.27 afb 2 y =10623.15x – 4856.45 0.25–20 0.997 0.08 0.25 0.09 0.31 0.07 0.22 afg 1 y = 20354.67x + 120.09 0.16–20 0.996 0.06 0.22 0.05 0.18 0.08 0.26 afg 2 y = 14600.18x – 809.53 0.12–25 0.992 0.04 0.13 0.06 0.19 0.07 0.21 lod and loq in µg/kg. af: aflatoxin; lod: limit of detection; loq: limit of quantification. regression equations showed acceptable linearity, and good recovery was obtained for spike samples and was similar to values reported in previous studies (heshmati et al., 2017). the findings obtained during method validation were conformed with accepted criteria (aoac international, 2002). the prevalence of afs in sesame seeds in this study, 40 samples of sesame seeds were analyzed for afs. sesame seeds had the highest prevalence (55%) of total afs among the three analyzed samples. the detection rates of afs were higher in sesame seed samples than in the tahini and tahini halva. afb1, afb2, afg1, and afg2 were detected in 19 (47.5%), 5 (12.5%), 6 (15%), and 5 (12.5%) of 40 sesame seeds samples, respectively (table  4). afb1 was the most abundant af, and its level varied from 0.21 to 12.35 μg/kg. in addition, 10 and 6 samples contained afb1 more than the accepted limit according to european (2 µg/kg) and iranian standard (5 µg/kg), respectively, and the total afs content of samples was lower than the permitted level in iran (15 mg/kg). there are several reports of af contamination in sesame seeds (table 5). the different levels of total afs and afb1 have been reported in previous similar studies. for example, anthony et al. (2014) reported that 8 (26.67%) out of 30 sesame samples studied in nigeria were contaminated with afb1 at levels above the limit of european regulations (anthony et al., 2014). esan et al. (2020) surveyed the contamination of total afs in sesame seeds of nigeria. they demonstrated that the positive samples contaminated with total afs ranged from 0.29 to 88.5  µg/kg (esan et al., 2020). in another study, kollia et al. (2016) investigated 30 samples of sesame seeds from the greek market. they observed that the amount of afb1 in eight samples exceeded the limit of european regulations (kollia et al., 2016). in an investigation, among 28 samples of sesame products from egypt by sabry et al. (2016), a higher prevalence of afb1 and afg1 than other mycotoxins in the range of 60%–100% and 33.33%–100%, respectively, were reported. the mean range of afb1 and afg1 in different provinces was 18.63–66.79 and 14.88–51.47 g/kg, respectively (sabry et al., 2016). 96 italian journal of food science, 2021; 33 (sp1) heshmati a et al. (2020) in egypt, mean afb1 in 16 samples of branded tahini and 101 samples of local tahini reported 0.10 ± 0.24 μg/kg and 13 ± 19.3 μg/kg, respectively (sebaei et al., 2020). li et al. (2009) reported that 19% and 32% of 100 of the sesame paste (tahini) samples studied in china contained afb1 at levels higher than the chinese regulation (5 μg/kg) and european regulations (li et al., 2009). in addition, torlak and akan (2013) studied afs contamination in 104 samples of tahini from the anatolia region of turkey. the mean of afb1 and total afs of samples was 0.93 ± 0.62 μg/kg and 1.17 ± 0.55 μg, respectively, which both were lower than the turkish standard (afb1: 5 μg/kg and taf: 10 μg/kg) and our study. these authors indicated that the roasting process applied in tahini production does not eliminate afs (torlak and akan, 2013). for the traditional production of tahini, sesame is roasted for 2 h at a temperature of 100°c to 150°c (torlak and akan, 2013). afs are resistant to heat and are difficult to eliminate at insufficient temperatures. in general, afs are eliminated at 237°c to 306°c. in foods heated, important parameters determining the reduction of af include table 4. the contamination status of aflatoxin of sesame and related products. aflatoxin type contamination status sesame seed tahini tahini halva afb 1 contamination level (µg/kg) no. of contaminated samples 19 (47.5) 14 (35) 11 (27.5) mean ± sd (µg/kg) 1.67 ± 0.45 0.85 ± 0.24 0.55 ± 0.17 0.15–2 9 (22.5) 7 (17.5) 5 (12.5) 2–5 4 (10) 4 (10) 6 (15) >5 6 (15) 3 (7.5) 0 range (µg/kg) 0.21–12.35 0.23–5.81 0.27–3.56 afb 2 no. of contaminated samples 5 (12.5) 3 (7.5) 4 (7.5) mean ± sd (µg/kg) 0.13 ± 0.06 0.1 ± 0.06 0.07 ± 0.03 range (µg/kg) 0.25–1.62 0.31–1.75 0.22–0.92 afg 1 no. of contaminated samples 6 (15) 4 (10) 2 (5) mean ± sd (µg/kg) 0.10 ± 0.05 0.07 ± 0.04 0.04 ± 0.03 range (µg/kg) 0.22–1.41 0.18–1.32 0.26–1.02 afg 2 no. of contaminated samples 5 (12.5) 5 (12.5) 4 (10) mean ± sd (µg/kg) 0.05 ± 0.02 0.08 ± 0.04 0.06 ± 0.03 range (µg/kg) 0.13–0.85 0.19–1.02 0.21–0.74 taf contamination level (µg/kg) no. of contaminated samples 22 (55) 18 (45) 13 (32.5) mean ± sd (µg/kg) 1.95 ± 0.48 1.10 ± 0.30 0.72 ± 0.22 >4 15 (37.5) 14 (35) 11 (27.5) 4–15 7 (17.5) 8 (20) 2 (5) >15 0 0 0 af: aflatoxin. the prevalence of afs in tahini a lower rate of af contamination was observed in tahini than that in sesame seed. eighteen (45%) of tahini samples contained total afs. the highest prevalence of afs in tahini halva was afb1 (35%), followed by afg2 (12.5%), afg1 (10%), and afb2 (7.5%). it was observed that the afb1 value of seven samples was higher than the limit of european regulations (2 µg /kg) while only three samples had afb1 contamination higher than the recommended standard of iran (5 µg/kg). also, 18 samples contained total af with an average value of 1.10 ± 0.30 µg/kg, among which the concentration of eight samples was higher than the limit of european regulations (4 µg/kg). it seems that washing and peeling of sesame seeds before tahini preparation could reduce the amount of afs. before tahini preparation, sesame seeds were roasted. the previous studies indicated that roasting operation could cause afs degradation (emadi et al., 2021; yazdanpanah et al., 2005). the prevalence of afs in tahini was reported in other studies. for example, in a study performed by sebaei et al. italian journal of food science, 2021; 33 (sp1) 97 prevalence and risk assessment of aflatoxin in sesame-based products related to the dilution effect of other products, including sugar, emulsifier, in the formulation. few studies have been performed to evaluate afs in tahini halva. var et al. (2007) investigated afb1 contamination in 34 samples of helva in turkey. no afb1 was found in helva samples. it seemed the discrepancies in afs contamination prevalence in different studies could be due to differences in sampling geographical areas and storage conditions and the afs measuring method. risk assessment of afb1 intake findings of percentile 50 (as the median of the population) of edi of afb1 calculated using the mcs approach showed the lowest (0.02 ng/kg bw/day) and highest (0.05 ng/kg bw/day) of edi in sesame seed and tahini halva, respectively (figure 1). the percentile 95 of edi of afb1 through sesame seed, tahini, and tahini halva was 0.09, 0.05, and 0.03 ng/kg bw/day, respectively. as shown in figure 2, the percentile 95 of moe with afb1 through sesame seed, tahini, and tahini halva was calculated as 25,485, 50,092, and 77,114, respectively. because data table 5. the contamination status of aflatoxin of sesame and related products. country sample type no of samples positive n (%) method mycotoxin range (µg/kg) mean ± sd (μg/kg) reference iran sesame seeds 269 50% hplc taf 1.43 ± 4.38 hosseininia et al. 2014 iran sesame seeds 269 50% hplc afb 1 1.25 ± 3.7 hosseininia et al. 2014 egyptian tahini (brand) 16 hplc afb 1 0.10 ± 0.2 sebaei et al. 2020 egyptian tahini (local) 101 hplc afb 1 13 ± 19.3 sebaei et al. 2020 china sesame paste 100 37 (37%) lc taf 0.54–56.89 6.75 li et al. 2009 china sesame paste 100 37 (37%) lc afb 1 0.39–20.45 4.31 li et al. 2009 iran sesame seeds 182 33 (18.1%) lc afb 1 1.62 ± 1.32 asadi et al. 2011 malaysian sesame seeds 8 7 (87.5%) elisa afb 1 0.5–1.82 0.9 reddy et al. 2011 nigeria sesame seeds 60 12 lcms/ms taf 0.29–88.5 16.9 esan et al. 2020 nigeria sesame seeds 60 12 lcms/ms afb 1 0.29–79.3 14.8 esan et al. 2020 nigeria sesame seeds 30 26.66 hplc afb 1 14.71–140.90 69.72 ± 41.68 anthony et al. 2014 turkey tahini 104 14.42 hplc afb 1 0.93 ± 0.62 torlak and akan 2013 turkey tahini 104 15.38 hplc taf 1.17 ± 0.55 torlak and akan 2013 turkey tahini helva 34 0 tlc afb 1 <1 <1 var et al. 2007 egyptian sesame seeds 28 88.89 hplc afb 1 33.66 ± 1.35 sabry et al. 2016 fct, abuja, nigeria sesame seeds 24 13 lcms/ms afb 1 3.6 fapohunda et al. 2018 nigeria sesame seeds 46 23 (50%) tlc afb 1 0.79–37.25 apeh et al. 2016 japan sesame seeds 47 5 hptlc afb 1 0.6–2.4 tabata 2007 jordan sesame seeds 46 2 hplc taf 100–1280 sirhan et al. 2014 greek sesame seeds 30 77.60% hplc afb 1 2.0 ng afb1 g-1 kollia et al. 2016 moisture content, heating temperature, and nutrient media. it has been reported that roasting dried wheat at 150°c for 30 min, green coffee beans at 150°c to 180°c for 10 to 15 min, and pistachio nuts at 150°c for 30 min will reduce af levels to 50%, 42.2%–55/9%, and 63%, respectively (pankaj et al., 2018). the prevalence of afs in tahini halva the results showed that tahini halva samples contained four types of af, i.e., afb1, afb2, afg2, and afg2, with mean values of 0.55 ± 0.17, 0.07 ± 0.03, 0.04 ± 0.03, and 0.06 ± 0.03 μg/kg, respectively (table 4). except for afb2, the detection rate of afb1, afg2, and afg2 was lower in tahini halva samples than in the sesame seeds and tahini samples. the average total afs of tahini halva samples was 0.72 ± 0.22 µg/kg. according to iranian standards, the concentration of total afs and afb1 was not higher than the allowable limit. in contrast, the total afs level of two samples was higher than the limit of european regulations (4 μg/kg). also, in six samples, the concentration of afb1 was higher than the limit of european regulations (2 µg/kg). the lower level of afs in tahini halva could be 98 italian journal of food science, 2021; 33 (sp1) heshmati a et al. sesame seed 1.00 edi 0.90 0.80 0.70 0.60 c um ul at iv e pr ob ab ili ty 0.50 0.40 0.30 0.20 0.10 0.00 0.00 0.01 0.02 0.03 0.04 0.05 ng/kg bw/day 0.06 0.07 0.08 0.09 0.10 0.11 mean = 0.05 95% = 0.09 tahini 1.00 edi 0.90 0.80 0.70 0.60 c um ul at iv e pr ob ab ili ty 0.50 0.40 0.30 0.20 0.10 0.00 0.00 0.01 0.02 ng/kg bw/day 0.03 0.04 0.05 0.06 mean = 0.03 95% = 0.05 tahini halva 1.00 edi 0.90 0.80 0.70 0.60 c um ul at iv e pr ob ab ili ty 0.50 0.40 0.30 0.20 0.10 0.00 0.00 0.01 0.02 ng/kg bw/day 0.03 0.04 mean = 0.02 95% = 0.03 figure 1. cumulative probability plot of edi of afb1 through sesame seed, tahini, and tahini halva consumption. italian journal of food science, 2021; 33 (sp1) 99 prevalence and risk assessment of aflatoxin in sesame-based products sesame seed 1.00 moe 0.90 0.80 0.70 0.60 c um ul at iv e pr ob ab ili ty 0.50 0.40 0.30 0.20 0.10 0.00 4000 8000 12000 16000 20000 24000 28000 32000 36000 40000 44000 48000 mean = 11121 95% = 25485 tahini 1.00 moe 0.90 0.80 0.70 0.60 c um ul at iv e pr ob ab ili ty 0.50 0.40 0.30 0.20 0.10 0.00 10000 20000 30000 40000 50000 60000 70000 80000 90000 100000 110000 mean = 21713 95% = 50092 tahini halva 1.00 moe 0.90 0.80 0.70 0.60 c um ul at iv e pr ob ab ili ty 0.50 0.40 0.30 0.20 0.10 0.00 20000 40000 60000 80000 100000 120000 140000 mean = 33716 95% = 77114 figure 2. cumulative probability plot of moe of afb 1 through sesame seed, tahini, and tahini halva consumption. 100 italian journal of food science, 2021; 33 (sp1) heshmati a et al. regarding the percentile 50 and 95 of moe was more than 10,000 (heshmati et al., 2017), it could be concluded that afb1 intake through sesame seed, tahini, and tahini halva consumption has no remarkable cancer risk for adults. conclusions the results of this study indicated the high prevalence of afs in sesame seed (55%), tahini (45%), and tahini halva (32.5%) samples. in addition, the levels of taf in 7 (17.5%), 8 (20%), and 2 (5%) samples of sesame seeds, tahini, and tahini halva exceeded the limit of european regulations (4 µg/kg), respectively. furthermore, 10 (25%), 7 (17.5%), and 6 (15%) samples of sesame seeds, tahini, and tahini halva contained afb1 more than the limit of european regulations (2 µg/kg), respectively. however, risk assessment indicated that the intake of af 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worapong usawakesmanee1, supachai pisuchpen2, nicha khatcharin1, chanonkarn rujirapong1 1centre of excellence in functional foods and gastronomy, faculty of agro-industry prince of songkla university, hat yai, songkhla, 90110, thailand; 2centre of excellence in bio-based materials and packaging innovation, faculty of agro-industry prince of songkla university, hat yai, songkhla, 90110, thailand *corresponding author: sunisa siripongvutikorn, centre of excellence in functional foods and gastronomy, faculty of agro-industry, prince of songkla university, hat yai, songkhla, 90110, thailand. email: sunisa.s@psu.ac.th received: 8 march 2023; accepted: 19 june 2023; published: 10 july 2023 © 2023 codon publications open access original article abstract liang or gnetum gnemon var. tenerum, an indigenous southern vegetable has recently attracted increasing interest due to its high nutritional value, creamy taste and lack of smell. the leaves with or without stem are washed with chlorinated water at 100 ppm for 15 min, stored at 4°c and investigated for physiochemical, chemical and sensory evaluations over time. total phenolic and flavonoid contents were higher in treatments with stems (p <  0.05). washing significantly increased moisture content and water activity (aw) in all treatments (p < 0.05). in addition, washing resulted in significantly higher dpph and abts activity (p < 0.05). however, washing and stem detachment had no effect on sensory and physicochemical qualities. the sensory score of the 8-days stored sample was comparable to the fresh one (day 0). keywords: antioxidant; gnetum gnemon; preparation; quality; stem; washing introduction liang or gnetum gnemon var. tenerum, a common signature southern vegetable, has the potential to become a new economic plant with less or free from pesticide residue. generally, liang is grown in backyards or as a fence plant by people in southern thailand. liang has a creamy, umami taste with less green flavour and is often grown as an intercrop between various economic plants such as rubber, palm oil, durian and orchard plants to maximise the use of space and increase income. the tenerum shrub variety is grown in thailand, whereas in malaysia and indonesia, the gnemon variety is grown as a tree (anisong et al., 2022). preliminary tests showed that the tenerum variety produced leaves containing essential amino acids with high health benefits including antioxidant, antidiabetic, anti-inflammatory, anti-breast cancer and gut microbiota–enhancing effects (suksanga et al., 2022) due to high protein and phytochemical compounds such as chlorophyll, beta-carotene, phenolic compounds, flavonoids and dietary fibre, both soluble and insoluble. after harvesting, the leaves are usually bunched with rubber bands or packaged in open bags for transport to local or fresh markets (figure 1), while in supermarkets, packaging in sealed bags is usually applied (figure  2). to serve the new generation of people who live busy lives, ready-to-cook or minimal process ingredients are required to meet their needs. in supermarkets, ready-tocook leafy vegetables are usually packaged as leaves without stems. liang leaves are eaten as a fresh vegetable or as a side dish with spicy foods. the leaves are also cooked and used in recipes for various menus (suksanga et al., 2022). liang 2 italian journal of food science, 2023; 35 (3) siripongvutikorn s et al. to the liberation of substrates and enzymes from damaged or injured plant cells (leveau and lindow, 2001). injured leaves lead to lower quality and shortened product shelf life (ariffin et al., 2017). physically damaged cells or wounds enhance both organic and inorganic nutrient release that accelerates microbial growth and chemical reactions (aruscavage et al., 2008, 2010). washing also increases moisture content, aw level as well as physical damage due to excessive forces during washing and draining (fao and who, 2003; mulaosmanovic et al., 2021). however, no scientific information is available on the preparation (washing and stem detachment) of leafy vegetables, particularly liang. this is of significant interest for a new s-curve for liang because of its consumer palatability, low chemical or pesticide content and high nutritional value. therefore, quality changes in liang due to the preparation process represent is of utter importance. this study focused on the effect of stem detachment and washing on the physical, chemical and sensory qualities and antioxidant activity to develop a proper method for liang leaf preparation in current commercial markets. materials and methods leaf preparation and sampling young liang or gnetum gnemon var. tenerum leaves (pae-salat) were collected from a farm. to avoid injury from weight and dense packing, 2 kg of bulky liang was packed into a low-density polyethylene bag (ldpe) and sent to the laboratory within 24 h, as shown in figure 4. tropical leafy vegetables are usually stored above 4°c to avoid chilling injury. the sample was checked for visual damage, and old, torn and rotten leaves were removed before the stem was detached following hygienic practice by wearing sanitation gloves. leaves with and without stems  were soaked in chlorinated water at 100 ppm for 15  min, washed twice with running tap water to remove the chlorine residue and drained in a basket for 10 min with a controlled thickness of leaves overlay at not more than 1 cm. the liang leaves were divided into four groups as follows: no washing with stem (nws), no washing without stem (nwns), washing with stem (ws) and washing without stem (wns) (see figure 5). leaves are typically washed before cooking to ensure hygiene and safety (figure 3). gardeners and merchants generally spray water on vegetables or soak them to remove dirt. this reduces plant temperature and controls weight loss. low temperature and appropriate humidity (60–70%) could slow down the leaf deterioration rate after harvesting (de frias et al., 2018). however, each preparation process causes cumulative physical damage (mulaosmanovic et al., 2021), leading to chemical and microbiological spoilage (ariffin et al., 2017). wounds also increase biochemical and chemical reactions owing figure 1. flowchart of vegetable preparation from farm to market. figure 2. liang leaves bunched with a rubber band (a) and sealed in a plastic bag (b). (a) (b) figure 3. flowchart of the conventional washing process. figure 4. liang leaves received from the farm (a) liang leaves packed in ldpe and (b) liang leaves. (a) (b) farm vegetables spray or soak with water bunch with rubber band or pack in bag market wash under running tap or soak in water leaves remove rotten, stem and old stage drain in basket italian journal of food science, 2023; 35 (3) 3 quality changes of liang leaves during storage laboratory inc., virginia, usa) as described by lee et al. (2022) and expressed as δe, as shown in equation 2. � � � �e l a b� � �( ) ( ) ( )2 2 2 where, δe = colour difference between standard (fresh produce) δl = difference between lightness (l*) standard (fresh produce) δa = difference between redness-greenness (a*) standard (fresh produce) δb = difference between yellowness-blueness (b*) standard (fresh produce) ph, moisture content and a w the ph, moisture content and aw were measured using a ph meter (sartoriussartorius ag, docu-ph+ meter, goettingen, germany), oven method (aoac, 2000), and an aw analyser (aqualab pre., decagon devices inc., washington, usa) at predetermined times. brix value brix values were measured using a refractometer (atago, pen refractometer, tokyo, japan) (thakulla et al., 2021). chlorophyll content chlorophyll content was measured by the colorimetric method at 400–700 nm, as described in aoac methods 940.03 662 and 646 (aoac, 2000) for chlorophyll a and b, respectively. finally, all four groups were stored at 4°c for 8 days, as presented in figure 6. on days 0, 4, 5, 6, 7 and 8, samples were collected for physical, chemical, quality and sensory evaluation. physiochemical and chemical quality determination colour change colour changes were determined by cie l*, a* and b* using a colorimeter (colorflex ez, hunter associates figure 5. the groups of liang leaves used in this study including (a) no washing with stem (nws); (b) no washing without stem (nwns); (c) washing with stem (ws) and (d) washing without stem (wns). (a) (c) (d) (b) figure 6. flowchart of liang leaves preparation. no washing no washing draining washing washing liang leaves no stem detachment stem detachment no stem detachment stem detachment remove rotten, old and torn storage 4 italian journal of food science, 2023; 35 (3) siripongvutikorn s et al. incubated in the dark for 30 min at 30°c. finally, the absorbance of the mixture was measured at 517 nm and reported as µg gallic acid equivalent/g dw using gallic acid as the standard at a concentration of 0.5–3.5 µg/ml (r2 = 0.9959). abts radical scavenging activity 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (abts) assay was determined as described by arnao et  al. (2001). abts radical was generated by incubating 7.4 mm of abts solution in dark at 30°c for 12 h. the radical solution was then diluted to obtain an absorbance of 1.1 ± 0.02 at 734 nm. then, 20 µl of sample extract was mixed with 280 µl of radical solution and kept in the dark for 2 h at 30°c. the absorbance of the mixture was measured at 734 nm and reported as mg gallic acid equivalent g dw using gallic acid as the standard at a concentration of 5–30 µg/ml (r2 = 0.998). ferric reducing antioxidant power (frap) assay the ferric–reducing antioxidant power (frap) assay was determined using the method of benzie and strain (1996). a freshly prepared frap solution containing 300 mm acetate buffer ph 3.6, 10 mm tptz (2, 4, 6tripyridyl-s-triazine) in 40 mm hcl and 20 mm fecl3·6h2o (ratio 10:1:1) was warmed at 37°c for 30 min. next, 15 µl of the sample extract was mixed with 285 µl of frap solution and incubated for 30 min at 37°c. the absorbance of the mixture was measured at 593 nm and reported as µg gallic acid equivalent/g dw using gallic acid as the standard at a concentration of 6–30 µg/ml (r2 = 0.9981). sensory evaluation before the sensory evaluation, all samples were steamed for 3 min, and eight attributes including appearance, colour, texture, odour, flavour, taste, overall acceptability and consumer acceptance were evaluated using a 9-point hedonic scale by 50 untrained panelists. statistical analysis all quality parameters, except for sensory tests, were assessed using a completely randomized design (crd), whereas the sensorial score was determined using a randomized complete block design (rcbd). differences in mean values were tested using anova with specific differences between groups or treatments assessed by tukey’s test. fibre content fibre content was determined as described in aoac method 2009.01. total phenolic content, total flavonoid content and antioxidant activity sample preparation and extraction sample was extracted using the method described by srisook et al. (2021) with some modifications, such as ethanol 90% for 24 h instead of 95% ethanol for 5 days. liang leaves and 90% ethanol (v/v) at a ratio of 1:10 were mixed and stirred in the dark at 25°c for 24 h. the mixture was then separated by vacuum suction using a buchner funnel and centrifuged at 4°c for 15 min at 12,000 rpm. an evaporator was used to vaporise the ethanol and to obtain a concentrated sample. total phenolic content (tpc) determination tpc was determined using the method described by singleton and rossi (1965) with some modifications. briefly, 20 µl of sample extract was added to 96-well plates followed by 100 µl of 10% folin reagent (v/v). after incubation in the dark at 30°c for 6 min, 7.5% na2co3 (anhydrous) (w/v) was added, and the mixture was incubated for another 30 min. the absorbance was measured at 765 nm using a microplate reader (varioskan lux, thermo scientific, singapore). tpc content was reported as mg gallic acid equivalent/g dw using gallic acid as the standard at a concentration of 50–170 µg/ml (r2 = 0.999). total flavonoid content (tfc) determination tfc was determined using the method described by ha et al. (2020) with some modifications. briefly, 100 µl of sample extract was mixed with 100 µl 2% alcl3·6h2o (w/v) and incubated in the dark at 30°c for 60 min. the absorbance of the mixture was then measured at 420 nm and reported as mg quercetin equivalent/g dw using quercetin as the standard at a concentration of 10–50 µg/ml (r2 = 0.9963). dpph radical scavenging activity 2,2-diphenyl-1-picryl hydrazyl (dpph) radical scavenging activity was determined using the method described by brand-williams et al. (1995) with some modifications. first, 100 µl of sample extract was mixed with 100 µl 0.2 mm dpph in 95% ethanol. the sample was italian journal of food science, 2023; 35 (3) 5 quality changes of liang leaves during storage brix value the brix value is a measure of the soluble solids content of a solution (zoecklein et al., 2010). brix values of all treatments increased at fourth day of storage (figure 9). an increase in brix value indicates an increase in water-soluble substances such as sugar, soluble fibre, amino acids, salt and organic acids or a decrease in water in the solution system such as evaporation and respiration (kusumiyati et al., 2020). therefore, the increase in brix value was related to the higher water-soluble solids retained in the leaf samples. the unwashed samples (nws, nwns) exhibited higher brix values than the washed samples (ws, wns) during storage for 5–8 days. washing increased the water uptake of the samples. brix values gradually decreased after storage for 4 days because of the higher utilisation of soluble solids particularly sugar, weak acids and minerals from microbial growth and biochemical reactions over time. decrease in brix value by microbial utilisation of soluble solids is also observed in yogurt, wort and beer (adadi et al., 2017; kim and han, 2019). the sharp increase in brix values on eighth day of storage indicated an increase in soluble solid compounds due to a softening process by microbial and biochemical autolyses. results revealed that the utilisation and production of such compounds occurred naturally, in parallel, as a result of the reaction. therefore, when the former was lower than the latter, the increment increased rather than decreasing. results and discussion physicochemical changes moisture and a w moisture content of all no washing treatments (nws and nwns) decreased at fourth day of storage, whereas the moisture content of all washing treatments (ws and wns) increased and was significantly higher than that of the no washing treatments (figure 7). at fourth day of storage, the aw of the no washing treatments (nws and nwns) remained constant, whereas the aw of the washing treatments (ws and wns) increased (figure 8). throughout the study, the moisture content of wns treatments was significantly higher than ws treatments because stem detachment in wns treatments resulted in an increased surface area, particularly at the end of the petiole, leading to higher water uptake and weight retention. using ldpe plastic bags also prevented moisture loss, with moisture content reducing slightly until reaching equilibrium during 5 days of storage. aw of ws and wns were higher than those of nws and nwns, indicating the effect of picking up water, as described in the moisture content determination. thus, washing resulted in an increase in aw and moisture content. results showed the drawbacks of washing in terms of an increase in aw, an important parameter for microbial spoilage and biochemical reactions which lead to faster product deterioration. figure 7. moisture content of liang or gnetum gnemon var. tenerum leaves after storage at 4°c for 8 days. different uppercase letters indicate significant differences within the same treatment group. different lowercase letters indicate significant differences between treatments within each day (p < 0.05). nws means no washing with the stem; nwns means no washing without the stem; ws means washing with the stem; wns means washing without the stem. 6 italian journal of food science, 2023; 35 (3) siripongvutikorn s et al. in buffering capacity. it is recognised that a buffer can resist changes in ph if it is sufficient to bind with added protons and the starting ph of the solution (bobulescu, 2020). while acidic compounds can reduce ph by providing hydrogen ions (h+), basic compounds increase ph by providing hydroxide ions (oh–) (bartee et al., nd). consequently, ph gradually increased until day 7 of storage. at eighth day of storage, a decrease in the ph value the initial ph of the fresh leaf samples was 6.20 ± 0.03 (figure 10), which is similar to the ph of approximately 6 as reported by anisong et al. (2022). at the fourth day of storage, the ph of all treatments significantly decreased, indicating three features including reduction of basic compounds, increase in acid and decrease figure 8. the water activity of liang or gnetum gnemon var. tenerum leaves after storage at 4°c for 8 days. different uppercase letters indicate significant differences within the same treatment group. different lowercase letters indicate significant differences between treatments within each day (p < 0.05). nws means no washing with the stem; nwns means no washing without the stem; ws means washing with the stem; wns means washing without the stem. figure 9. brix values of liang or gnetum gnemon var. tenerum leaves after storage at 4°c for 8 days. different uppercase letters indicate significant differences within the same treatment group. different lowercase letters indicate significant differences between treatments within each day (p < 0.05). nws means no washing with the stem; nwns means no washing without the stem; ws means washing with the stem; wns means washing without the stem. italian journal of food science, 2023; 35 (3) 7 quality changes of liang leaves during storage fibre content the fibre content of liang leaves is shown in figure 11. the total dietary fibre content was approximately 31% (dw), which was slightly lower than the 36.3% dw as reported by anisong et al. (2022). the fibre content remained constant during storage. the experimental ph of all treatments was observed. the reduction in ph in all treatments resulted in an unpleasant organic smell that was similar to the smell of sweet, fermented sticky rice (khao mak), which may be due to fermentation by yeast and lactic acid bacteria (mongkontanawat and lertnimitmongkol, 2015) as well as anaerobic respiration (toro and manuel, 2015). figure 10. ph of liang or gnetum gnemon var. tenerum leaves after storage at 4°c for 8 days. different uppercase letters indicate significant differences within the same treatment group. different lowercase letters indicate significant differences between treatments within each day (p < 0.05). nws means no washing with the stem; nwns means no washing without the stem; ws means washing with the stem; wns means washing without the stem. figure 11. fibre of liang or gnetum gnemon var. tenerum leaves after storage at 4°c for 8 days. different uppercase letters indicate significant differences within the same treatment group. different uppercase letters indicate significant differences within the same treatment group. different lowercase letters indicate significant differences between treatments within each day (p < 0.05). nws means no washing with the stem; nwns means no washing without the stem; ws means washing with the stem; wns means washing without the stem. 8 italian journal of food science, 2023; 35 (3) siripongvutikorn s et al. confirmed the oscillation of chlorophyll content in rocket leaves stored in dark at 4°c with a relative humidity of 65 ± 4.5% for 2 days, depending on the day–night cycle (circadian regulation). interestingly, the chlorophyll content of liang leaves in this study was higher than kale, which is considered as a high chlorophyll vegetable, at 136.18– 172.10 mg/100 g (lal, 2014). these results indicated that liang leaves could be used as an alternative source of chlorophyll. the results for total chlorophyll (chl a/b) revealed that the chlorophyll a (chl a) content in all treatments was higher than that of chlorophyll b (chl b). the chl a/b ratio in this study was 1.2, which is lower than that of most plant types (generally higher than 2) including trees, shrubs, herbs, conifers, broad-leaves, evergreen and deciduous (li et al., 2018). the low chl a/b ratio was due to oxidative stress, as chl a is more prone to oxidative damage than chl b (kasajima, 2019). the chl a/b ratio of healthy rice leaves was recorded at 3.5, and decreased to 1.5 after subjection to oxidative stress (kasajima, 2019). shade plants generally produce more chlorophyll to increase photosynthesis efficiency because of the absorption of blue light in a low-light environment (beneragana and goto, 2010). this result was supported by herrera et al. (2022), who reported that plants respond to shade by increasing the production of light-harvesting complexes by increasing chl b. shade-tolerant plants respond to low-light environments in diverse ways than normal plants by decreasing the chl a/b ratio. farmers usually plant liang as an intercrop between rubber trees, and the plants adapt to the shaded environment by increasing the total chlorophyll content and chl b, leading to a low chl a/b ratio. tpc, tfc and antioxidant activity tpc and tfc phenolic compounds are produced by chloroplasts to protect cells from damage caused by reactive oxygen species (ros), a by-product of photosynthesis (zhang et  al., 2018). leaves are major photosynthetic organs, and green-leaf plants contain an abundance of phenolic compounds (zhang et al., 2018). the tpc of liang leaves at day 0 was 4.32 mg/gae dw lower than malaysia liang leaves which were 8.70 mg/gae dw as reported by wazir et al. (2011). the tpc and tfc values of liang leaves are presented in figures 13 and 14, respectively. fluctuations in the tpc and tfc in each treatment were observed during storage. the results in this experiment concurred with tpc values in other vegetables and fruits after storage at 4°c, indicating that active plants or plant parts try to remain homeostatic for survival until the end of life (hubert et al., 2017; kim, 2015). cold storage results corresponded to the fibre content of walnuts stored at 4°c (zhang et al., 2017). during storage, the texture became tougher but the fibre content remained the same. therefore, the total fibre content was not a good parameter for determining quality changes in liang leaves, with soluble and insoluble fibres being better options. details on parameters such as reducing and non-reducing sugar and cellulose contents require further investigation. colour change δe values of liang leaves are shown in figure 12. the colour of the adaxial and abaxial surfaces and ground leaves in all treatments significantly changed during storage. after storage for 4 days, δe of the upper side of the treatments without stem (nwns and wns) was higher than that of treatments with stem (nws and ws). δe of the adaxial surface of the wns treatment was the highest, followed by that of ws, indicating that washing had a greater effect on δe at 7 and 8 days of storage. excess water or high moisture content and mechanical injury induced by washing and stem detachment led to biochemical reactions and increased microbial functions (mulaosmanovic et al., 2021). interestingly, δe values of the lower side of the stem detachment treatments (nwns and wns) were significantly higher than those of stem treatments (ws and nws). washing also resulted in a higher δe than no washing, suggesting that washing and stem detachment played significant roles in leaf colour. the δe values of ground ws and wns gradually increased, while those of the others remained constant during storage. the highest δe in the ground sample was found in the nws treatment, while the adaxial and abaxial surfaces under the wns treatment had the highest δe, indicating that grinding affected the δe value of the leaves. the difference between the δe values of the nonground sample (adaxial and abaxial surfaces) and the ground sample was caused by the water after the samples were ground. therefore, the colour quality of stored leaves could be preserved using ground samples. chlorophyll content at fourth day of storage, changes in chlorophyll content in the nws treatment remained constant but not in the other treatments (nwns, nws and ws). see table 1. chlorophyll content in the nws treatment decreased, whereas it increased in the ws treatment at eighth day of storage. the oscillation of chlorophyll content in this study may be due to the plant cell metabolism during storage (mei et al., 2022). larrinaga et al. (2019) italian journal of food science, 2023; 35 (3) 9 quality changes of liang leaves during storage figure 12. colour difference (δe) of adaxial surface (a), abaxial surface (b) and ground leaves (c) of gnetum gnemon var. tenerum leaves after storage at temperature 4oc for 8 days. different uppercase letters indicate significant differences within the same treatment group. different lowercase letters indicate significant differences between treatments within each day (p < 0.05). nws means no washing with the stem; nwns means no washing without the stem; ws means washing with the stem; wns means washing without the stem. (a) (b) (c) 10 italian journal of food science, 2023; 35 (3) siripongvutikorn s et al. of wounds, requiring more energy and a curing agent to treat the damaged cells. the tpc content of all treatments increased or remained constant compared with that of the control (day 0). the tfc content of the nws and ws treatments was equal to that of the control (day 0), whereas nwns (second bar) and wns (last bar) during storage for 8 days were lower on day 0, indicating a loss of nutrition supply from the stems. detaching the stems can be linked induced phenolic production (polyphenolic phytoalexins) (hubert et al., 2017). a decrease in tpc and tfc during 8 days of storage with stem detachment (nwns and wns) indicated a reduced availability of nutrients to produce phenolic compounds, in contrast to treatments with stems (nws and ws) which provided nutrients from the stems to the leaves. an increase in phenolic compounds production in wounded, stressed plants was observed in sweet potato roots (dovene et al., 2019). stem detachment can also injure plant cells as a result table 1. total chlorophyll (chl a+b), chlorophyll a (chl a), chlorophyll b (chl b) and the ratio of chlorophyll a to b (chl a/b) of liang or gnetum gnemon var. tenerum leaves after storage at 4°c for 8 days. time treatment chl a + b (mg/g dw) chl a (mg/g dw) chl b (mg/g dw) chl a/b d0 nws 226.28 ± 22.25aba 114.55 ± 7.46ba 101.53 ± 6.71ba 1.13 ± 0.10aa nwns 226.28 ± 22.25aa 114.55 ± 7.46aa 101.53 ± 6.71aa 1.13 ± 0.10aa ws 226.28 ± 22.25aa 114.55 ± 7.46aa 101.53 ± 6.71aa 1.13 ± 0.10ba wns 226.28 ± 22.25aa 114.55 ± 7.46ba 101.53 ± 6.71aa 1.13 ± 0.10aa d4 nws 254.46 ± 9.96aa 138.41 ± 5.48aa 116.05 ± 4.49aa 1.19 ± 0.00ac nwns 188.16 ± 2.60bb 103.61 ± 1.35abb 84.55 ± 1.26bb 1.23 ± 0.00aab ws 172.60 ± 10.57bb 95.01 ± 6.28bb 77.60 ± 4.29bb 1.22 ± 0.01abb wns 177.26 ± 1.47bb 98.31 ± 1.00cb 78.95 ± 0.47bb 1.25 ± 0.01aa d8 nws 200.07 ± 2.55bb 110.67 ± 1.38bb 89.40 ± 1.19cb 1.24 ± 0.00ab nwns 181.50 ± 5.16bc 101.41 ± 2.82abc 80.10 ± 2.45bc 1.27 ± 0.02aa ws 177.03 ± 6.56bc 99.44 ± 4.01abc 77.59 ± 2.56bc 1.28 ± 0.01aa wns 229.74 ± 5.79aa 126.56 ± 3.41aa 103.18 ± 2.40aa 1.23 ± 0.01ab remarks: nws means no washing with the stem; nwns means no washing without the stem; ws means washing with the stem; wns means washing without the stem. different uppercase letters indicate significant differences within the same treatment group. different lowercase letters indicate significant differences between treatments within each day (p < 0.05). figure 13. total phenolic content of liang or gnetum gnemon var. tenerum leaves after storage at 4°c for 8 days. different uppercase letters indicate significant differences within the same treatment group. different lowercase letters indicate significant differences between treatments within each day (p < 0.05). nws: no washing with the stem; nwns: no washing without the stem; ws: washing with the stem; wns: washing without the stem. italian journal of food science, 2023; 35 (3) 11 quality changes of liang leaves during storage antioxidants instead of very short-lived natural radicals such as hydroxyl (ho·), lipid alkyl (l·) and lipid peroxyl (loo·) (munteanu and apetriel, 2021; yeo and shahidi, 2019). ferric ion–reducing antioxidant power (frap) is a method for determining the electron transfer ability of antioxidants by reducing the colourless complex ferric ion (fe3+) to blue ferrous complex (fe2+) in an acidic environment (ph 3.6) (munteanu and apetriel, 2021). generally, the results revealed that ws treatment had the highest antioxidant capacity, followed by nws and wns treatments (figures 15–17). remarkably, nws offered antioxidant capacity comparable to wns treatments, even to the cutting of organs of living things; this not only causes injury but also requires more energy for recovery. antioxidant compounds could be used to alleviate stress and control wounds (comino-sanz et al., 2021). antioxidant activity (dpph, abts and frap) dpph and abts assays are used to stabilize 2,2diphenyl-1-picrylhydrazyl (dpph) or 2,2′azinob is-(3-ethylbenzothiazoline-6-sulfonic acid (abts) to measure the ability of hydrogen and electron transfer of figure 14. total flavonoid content of liang or gnetum gnemon var. tenerum leaves after storage at 4°c for 8 days. different uppercase letters indicate significant differences within the same treatment group. different lowercase letters indicate significant differences between treatments within each day (p < 0.05). nws means no washing with the stem; nwns means no washing without the stem; ws means washing with the stem; wns means washing without the stem. figure 15. dpph of liang or gnetum gnemon var. tenerum leaves after storage at 4°c for 8 days. different uppercase letters indicate significant differences within the same treatment group. different lowercase letters indicate significant differences between treatments within each day (p < 0.05). nws means no washing with the stem; nwns means no washing without the stem; ws means washing with the stem; wns means washing without the stem. 12 italian journal of food science, 2023; 35 (3) siripongvutikorn s et al. capability of antioxidant in aqueous phase. as mentioned above, the frap value indicates the reducing power of the antioxidants to metal ions. the antioxidant activity in plants is generated by phenolic compounds and also other compounds such as vitamin c and pigments such as chlorophyll and carotenoids (sarker et al., 2020), therefore explaining why abts radical scavenging in this study was not well related to phenolic compounds because carotenoid, chlorophyll and vitamin c contained in liang leaves were 3706 µg/100 g dw (anisong  et  al.,  2022), though nws contained lower tpc and tfc. the results indicated that injury from washing induced different groups of phenolic compounds with higher antioxidant ability in liang leaves during storage (pratyusha, 2021). the abts assay exhibited the highest antioxidant capacity, followed by frap and dpph assays. the higher abts value compared to dpph indicated strong polarity by donating electrons and h+. pongsetkul et  al. (2023) also confirmed relation of abts assay and hydrogen donating figure 16. abts of liang or gnetum gnemon var. tenerum leaves after storage at 4°c for 8 days. different uppercase letters indicate significant differences within the same treatment group. different lowercase letters indicate significant differences between treatments within each day (p < 0.05). nws means no washing with the stem; nwns means no washing without the stem; ws means washing with the stem; wns means washing without the stem. figure 17. frap of liang or gnetum gnemon var. tenerum leaves after storage at 4°c for 8 days. different uppercase letters indicate significant differences within the same treatment group. different lowercase letters indicate significant differences between treatments within each day (p < 0.05). nws means no washing with the stem; nwns means no washing without the stem; ws means washing with the stem; wns means washing without the stem. italian journal of food science, 2023; 35 (3) 13 quality changes of liang leaves during storage comparable to those of fresh liang leaves (day 0). the odour, texture and flavour of nws treatments remained stable during the storage period. the sensory scores showed that the panelists accepted all treatments after 8 days of storage, even though there was an unpleasant organic smell starting on day 5 of storage when the bag was opened. however, after cooking, the off-odour of 226.28 ± 22.25 mg/g dw and 2.71–5.25 mg/100 g dw (data in process for publication), respectively. sensory evaluation the sensory qualities of treatments during storage for 8 days at 4°c are shown in table 2. all treatments were table 2. sensory scores of liang or gnetum gnemon var. tenerum leaves after storage at 4°c for 8 days. attributes condition time day 0 day 4 day 5 day 6 day 7 day 8 appearance nws 7.52±0.97aba 7.30±1.13ba 7.79±0.74aa 7.45±0.97aba 7.52±1.00aba 7.79±0.82aa nwns 7.73±0.87aa 7.87±0.86aa 7.57±1.01aa 7.67±0.92aa 7.67±0.88aa 7.57±0.86aa ws 7.40±0.98aa 7.51±0.95aa 7.43±0.98aa 7.60±0.88aa 7.71±0.99aa 7.43±1.07aa wns 7.83±0.79aa 7.63±1.16aa 7.77±0.82aa 7.37±1.25aa 7.57±0.90aa 7.77±0.86aa color nws 7.52±1.09aa 7.36±0.99aa 7.73±0.88aa 7.45±0.90aa 7.45±0.94aa 7.73±0.80aa nwns 7.57±0.94aa 7.77±0.77aa 7.60±0.81aa 7.50±0.94aa 7.43±0.90aa 7.47±0.97aa ws 7.31±0.99aa 7.54±0.89aa 7.54±0.89aa 7.57±0.92aa 7.63±0.97aa 7.66±0.94aa wns 7.63±0.85aa 7.70±1.02aa 7.53±0.94aa 7.30±1.09aa 7.70±0.84aa 7.63±0.89aa odor nws 7.21±1.17aa 7.15±1.12aa 7.28±0.96aa 7.48±0.80aa 7.39±1.06aa 7.45±1.03aa nwns 7.23±0.97aa 7.63±0.89aa 7.20±0.92aa 7.30±0.99aa 7.10±1.24aa 7.23±1.04aa ws 7.26±1.22aa 7.43±1.24aa 7.17±0.89aa 7.17±1.04aa 7.20±0.99aa 6.94±1.21aa wns 7.50±0.97aba 7.53±0.82aa 7.33±0.92abca 7.03±1.25bca 7.17±0.99abca 6.97±1.33ca odor nws 7.21±1.17aa 7.15±1.12aa 7.28±0.96aa 7.48±0.80aa 7.39±1.06aa 7.45±1.03aa nwns 7.23±0.97aa 7.63±0.89aa 7.20±0.92aa 7.30±0.99aa 7.10±1.24aa 7.23±1.04aa ws 7.26±1.22aa 7.43±1.24aa 7.17±0.89aa 7.17±1.04aa 7.20±0.99aa 6.94±1.21aa wns 7.50±0.97aba 7.53±0.82aa 7.33±0.92abca 7.03±1.25bca 7.17±0.99abca 6.97±1.33ca texture nws 7.45±1.23aa 7.00±1.12aa 7.30±1.33aa 7.61±0.90aa 7.15±1.39aa 7.55±1.03aa nwns 7.47±0.82aba 7.67±0.88aa 7.30±0.92aba 7.30±1.09aba 7.00±1.17ba 7.40±0.97aba ws 7.37±1.06aa 740±1.35aa 7.11±1.13aa 7.17±1.20aa 7.29±1.05aa 7.20±1.05aa wns 7.50±0.97aa 7.57±0.94aa 7.47±0.86aa 7.17±1.23aa 7.47±1.14aa 7.10±1.37aa flavor nws 7.00±1.22aa 7.15±1.25aa 7.30±0.98aa 7.45±1.06aa 7.18±1.13aa 7.33±0.96aa nwns 7.23±1.01aa 7.50±1.25aa 7.13±1.43aa 7.10±1.03aa 7.10±1.24aa 7.07±1.26aa ws 7.11±1.21aa 7.23±1.24aa 7.03±1.04aa 7.00±1.06aa 7.20±1.02aa 6.91±1.15aa wns 7.37±1.07aba 7.63±0.81aa 7.30±0.88aba 7.07±1.23aba 7.23±0.94ab 7.03±1.13ba taste nws 7.06±1.14aa 6.91±1.33aa 7.27±0.88aa 7.18±1.33aa 7.12±1.32aa 7.21±1.05aa nwns 7.20±1.00aa 7.40±1.25aa 7.00±1.58aa 7.30±0.99aa 6.80±1.42aa 6.87±1.41aa ws 7.26±1.38aa 7.26±1.34aa 7.06±1.11aa 6.97±1.12aa 7.14±1.26aa 6.91±1.25aa wns 7.37±1.00aa 7.30±1.39aa 7.20±0.89aa 6.97±1.19aa 7.10±0.96aa 7.17±1.12aa overall nws 7.12±1.19aa 6.94±1.25aa 7.15±1.30aa 7.30±1.05aa 7.03±1.26aa 7.55±0.90aa nwns 7.37±0.93aa 7.50±1.20aa 7.07±1.36aa 7.23±1.01aa 6.87±1.36aa 7.13±1.20aa ws 7.17±1.29aa 7.26±1.15aa 7.14±1.00aa 6.97±1.15aa 7.14±1.14aa 7.03±1.12aa wns 7.40±1.04aa 7.57±0.68aa 7.37±0.96aa 7.20±1.27aa 7.30±0.95aa 7.20±1.16aa acceptance nws 75.76fd 87.88cc 81.82dd 90.91bc 75.76ec 93.94aa nwns 90.00bc 93.33ab 86.67cb 83.33dd 93.33aa 86.67cd ws 94.29aa 82.86dd 85.71cc 85.71cc 88.57bb 88.57bc wns 93.33cb 100aa 96.67ba 100.00aa 93.33ca 90.00db remarks: nws means no washing with the stem; 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(2003). conclusions the physicochemical, chemical and sensory qualities of liang leaves after washing with chlorinated water at 100 ppm for 15 min and detaching the stem were determined. washing increased the moisture content and aw in liang leaves and led to lower soluble solid content. stem detachment and washing of liang leaves did not affect the amount of fibre and chlorophyll after storage at 4°c for 8 days. liang leaves without stems contained lower tpc and tfc contents; however, washing increased antioxidant capacity based on the dpph and abts methods, while stem detachment did not cause significant differences. liang leaves stored at 4°c for 5  days produced an acid-like odour during storage, but this unpleasant odour dissipated after steaming. liang leaves maintained acceptable quality when stored at 4°c for at least 8 days, and microbial quality should be further examined in future studies. this study provides a starting point for the future development of commercial ready-to-eat and ready-to-cook liang leaf products. however, postharvest techniques and storage conditions require more detailed investigations to extend the shelf life of liang leaves. acknowledgement this research was supported by the national science, research, and innovation fund (nsrf) and the prince of songkla university (grant no. agr6505112m). we thank the prince of songkhla university and its faculty of agro-industry for the equipments and laboratory support. references aoac, 2000. official methods of analysis, 17th edition. washington dc, usa: association of official analytical chemist. adadi, p., kovaleva, e.g., glukhareva, t. and shatunova, s., 2017. production and analysis of non-traditional beer supplemented with sea buckthorn. agronomy research. 15(5). https://doi.org/ 10.15159/ar.17.060 anisong, n., siripongvutikorn, s., wichienchot, s. and puttarak, p., 2022. a comprehensive review on nutritional contents and functional properties of gnetum gnemon linn. food science and technology. 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consumer fair prices for less pesticide in potato c. serefoglu1* and s. serefoglu2 1ankara development agency, aşağı öveçler mah. 1322. cad. no. 11, 06460 çankaya, ankara, turkey 2the ministry of defence of turkey, 06100, bakanlıklar, ankara, turkey *corresponding author: cserefoglu@gmail.com abstract this study estimates turkish citizens’ willingness to pay (wtp) for reduced pesticides on potatoes. these estimates rely on data collected from 393 persons covering all regions in turkey through an online survey during the period from june 22 july 21, 2014. the average wtp was found to be about tl 1.68 for all observations including zero bids and tl 2.91 excluding zero bids. the results of the probit model show that cosmetic defects, free-pesticide potatoes with insect damages, age, and gender were identified by the model to have significant impacts on the probability of wtp. mailto:cserefoglu%40gmail.com?subject= 108 ital. j. food sci., vol. 28 2016 introduction pesticides are defined by the european commission (ec) (2009) as substances or mixtures of substances including chemical compounds intended for killing, destroying, or mitigating any pest. the use of pesticides has tragically and rapidly increased since 1960’s due to the green revolution (carvalho, 2006). as explained by hoppin et al. (2007), pesticides could cause some respiratory diseases to farmers. similarly, alavanja et al. (2004) stated that indirect exposures which occur by way of drinking water, food or air happen more frequently than direct exposures occurring to individuals who apply pesticides in agriculture. the consumption level of pesticides in turkey increased to 54,000 tonnes in 2002 but during the last decade the level notably decreased to 40,000 tonnes (mfal, 2012). the amount of pesticides used in turkey seems quite low when compared with countries such as germany and france in europe according to the fao statistics. stated and revealed preferences are the methods that are often used to measure the wtp of consumers. as stated by eberle and hayden (1991), each individual`s valuation of a nonmarket good is reflected through a direct questionnaire approach. thus, our research is mainly based on the contingent valuation method (cvm) and food safety issues through the responses which come from an online survey which covers the whole of turkey. the food safety issue plays a crucial role for both policy makers and consumers, with fast dissemination of information through social network. as underlined by rowell (2004), food safety and sustainable food supply are on the agenda of developed countries to develop diets that are fundamentally affordable and health-enhancing. the overall objective of this study is to assess turkish consumers’ attitudes towards purchasing reduced pesticides that are guaranteed not to be risky to human health. the specific objectives are: determine consumers’ attitudes and concerns toward pesticide use in potatoes and ascertain consumers’ willingness to pay higher bid amounts for reduced pesticides in potatoes by ensuring no pesticide residues, and estimate consumers’ mean wtp for reduced pesticides potatoes. there are several reasons why the potato product is chosen. firstly, potato is one of the most consumed vegetables in turkey and its consumption increases yearly even if its price increases, this is according to the data extracted from the database of the turkish statistic institute (turkstat). second, it is a traditional food that has a wide usage with different vegetables. last but not least is the over-use of pesticides used on potatoes and pesticide residues in it (birinci and uzundumlu, 2009; ayaz and yurttagul, 2008). following the introduction, methodology will be covered in detail in section 1. within the framework of the methodology, there is discussion of: sample size, and data analysis covering both questionnaire design and descriptive statistics including the socio-economic characteristics of the respondents and consumer preferences with respect to health risks and why the cvm is used. the second section will comprehensively focus on the econometric results and their interpretations. regarding the econometric results, descriptive statistics and regression analysis will be included, and also, the assumptions made to perform the study is included. the paper ends with a brief conclusion. materials and methods the online survey as mentioned in the previous sections randomly covered all of turkey through the social network. surveys’ results in table 1 clearly demonstrate that the rate of participation in survey in the north east region (ne) is proportionally higher than other regions while some regions such as aegean region (aeg) and the south east region (se) has a lower participation rate considering their population. a high rate of responses in some regions might be explained with a fast spreading of surveys linked with the help of respondents. the analysis was based on applying the cvm that is defined as “any approach to valuation of a commodity which relies upon individual responses to contingent circumstances posited in an artificially structured market” (seller et al., 1985). this method was first proposed by ciriacy-wantrup in 1947 in order to estimate the benefits of the prevention of soil erosion (kontoleon et al., 2005; cameron, 1992). the cvm, which is basically based on a survey-based methodology for eliciting consumers’ valuations of non-market goods and services, has been widely applied by researchers and policy makers in health economics and food safety for several decades and received considerable attention in the literature. it was stated by jean et al. (1995) that benefit estimates that are comparable to estimates from market-based approach can be produced by the cvm. there are a number of studies which have been used in surveys with discrete answers that have been analysed with logit and probit techniques (buzby et al., 1995; akgungor et al., 2001; garming and waibel, 2006; kalogeras et al., 2009). determining sample size the sample size is defined by considering the current turkish population and calculated according to the formula provided by fink (2003): ital. j. food sci., vol. 28 2016 109 where n is the sample size determined, n is the population size, p is level of precision. the sample size is 400 at 95% confidence level and a 5% margin of error. but 393 samples were used after the first elimination due to the incompleteness. survey and data generation before moving further through online survey, the first draft was shared with 10 turkish consumers by using face to face interview method in order that the perspective of a consumer side is truly reflected in the format of questions. after receiving some positive and negative feedback, the questionnaire form was finally rearranged in a short and clearer way as the first draft shared with consumers was found slightly longer and unclear instructions. particularly, open-ended questions were not preferred by these consumers. instead, options were included in some of the questions. also, the answer choices were re-organized according to the consumer`s expectations. following pre-test with turkish consumers, the link to the online survey was shared with turkish consumers via the social networks such as in general e-mails, facebook, linkedin and forums, and in particular regional development agency network covering all turkey for one month as from june 22 until july 21, 2014. the survey mainly comprised of three parts. the first part covered the questions to elicit perceptions that are related to pesticide residues. the consumers were asked about their perceptions of pesticide residues in potatoes as well as the cosmetic defects. the question on cosmetic defects intended to measure whether or not the consumers would be willing to purchase fresh produce with insect damage, such as worm holes or irregular shape of the potatoes. the second part included wtp questions. the survey asked consumers the maximum wtp for reduced pesticide residues in potatoes. socioeconomic questions were inserted in the third part. for simplicity, the survey was designed to simulate consumers’ potato purchasing behaviour of their respective households under alternative prices on reduced pesticides in potatoes. the scenario was built on the consumers that were provided with a label that guarantees that the potatoes were tested and certified that they do not contain pesticide residues harmful to human health by assuming no change in quality. by doing so, we were able to see if the consumer`s wtp is enough to justify these increased costs of production with a reduction in pesticide use. regression models of the cvm probit and logit which are known as non-linear functions of unknown coefficients in literature are widely applied in binary choice models. though both models may give similar results, there are slight differences because of table 1 percentages of survey and turkish population at the level of nuts 1 regions (source: primary data extracted from survey). 110 ital. j. food sci., vol. 28 2016 the tail of observations. amemiya (1981) expressed that the samples with heavier tails are more appropriate for logit models. a similar stance was made by cakmakyapan and goktas (2013). they observed that logit model is generally preferred for large sample sizes (500 and 1000) and probit model is usually for smaller sample sizes. so, probit model will ultimately be employed for estimations because of the sample size. alternatively, tobit model will be applied to measure wtp amounts that are obtained through single bounded dichotomous questions since the endogenous variable includes zero values. probit model the probit model is defined by wooldridge (2006) as: zn=xnβ+un. where β is a vector of parameters including the intercept term; xn is a vector of covariates; u is the error term which either has the standard logistic distribution or the standard normal distribution. in either case, u is symmetrically distributed about zero. zn is the unobservable amount that respondents are willing to pay for the reduced pesticides in potatoes. wtpi is the observed dichotomous variable stating whether the individual pays or not. it can be defined as follow: wtpn=0 if wtpn*≤0 wtpn=1 if wtpn*>0 as it is indicated by wooldridge (2006), the main goal in binary responses is to explain the effects of x on the response that follows the probability p(y=1|x). p(wtp=1|x)=p(wtpn*>0|x)=p[e>-(β0+xβ|x]= =1-g[-(β0+xβ0]=g(β0+xβ). the direction of the effect of xj on e(wtp*|x)= β0+xβ and on e(wtp|x)=p(y=1|x)=g(β0+x β) is similar to each other. it is not possible to apply ols due to the nonlinear nature of e(y|x). maximum likelihood methods thus must be used in order to estimate limited dependent variable models. the maximum likelihood can be written as follows (wooldridge, 2006); ƒ(wtp|xi;β)=[g(xiβ)]y[1-g(xiβ)]1-y, wtp-0,1, it can easily be seen that when y=1 results in g(x, β) and when y=0, we get 1g(xiβ). the function of log likelihood for observation is a function of the parameters and the data (xi, yi) li(β)=wtpilog[g(xiβ)]+(1-wtpi)log[1-g(xiβ)]. tobit model the general formulation of the tobit model can be expressed in the following way (greene, 2000; wooldridge, 2006); wtpn* =xiβ+ui. wtp=0 if yn* ≤0. wtp=wtp* if wtpn*<0 e[wtpn*|xnβ] is xn`β. wherefore, the nth individual, xn is a vector of explanatory variables, ui is a random disturbance term, and β is a parameter vector common for each individual. by assuming the random error is independent and normally distributed among respondents, the expected wtp for an observation drawn at random from the population is e[wtp|xn]= ϕ(xn`β/σ)+ xn`β+σλn) where ϕ (xn`β/σ)/φ(xn`β/σ); where ϕ represents the normal distribution function and σ represents the standard deviation. moreover, the expected value of wtp for observations above zero, which will be called e(wtp*), is simply xβ plus the expected value of the truncated normal error terms. the expected wtp can be expressed as e(wtp)= ϕ(xβ/σ)e(wtp*) wooldridge (2006) points out that the function of the tobit model which is based on maximum likelihood estimation can be shown as: ln l (β, σ)= (wtpn=0) ln[1-g(xn β/σ)]+(wtpn>0)ln{(1/σ)g [(wtpn-xn β)/σ]} where g(.) is the standard normal cumulative distribution function; g(.) is the standard normal density function; and σ refers the standard deviation of the error term. by maximising the log-likelihood function, the tobit estimator is obtained. results and conclusions as indicated in table 2, 63.10 % (248) of the 393 respondents that were considered in the study are males, and 36.90 % (145) are females, which represents all of turkey. it is also shown that 54.20 % (213) of the surveyed respondents are 31-45 years old, followed by individuals of 18-30 and 46-64 years old, representing 38.17 % (150) and 7.38 % (29) of the sample respectively. the educational attainment of the respondents is in favour of higher level of education, 53.94 % (212) acquired a university degree followed by 42.24 % (166) of post graduate degree. when comparing the above figures with ital. j. food sci., vol. 28 2016 111 the data of turkstat as in table 3, our sample has higher income and education levels, and a higher percentage of males. regarding working status, a great majority of respondents (70.74 %) are employed in the public sector, while only 18.32 % and 4.33 % of the respondents work in the private sector and are unemployed respectively. taking into consideration income level of respondents, it was found that the middle income group was overwhelmingly predominant. respondents from low, medium and high income level consisted of roughly 12 %, 66 % and 32 % respectively. the average size of the household of respondents is 3 individuals per household and their age distribution reflected 31-45 years old. table 2 characteristics of the sample. sample size:393 freq. % gender 393 100 male 248 63,10 female 145 36.90 age 393 100 18-30 150 38.17 31-45 213 54.20 46-64 29 7.38 >64 1 0.25 employment status 393 100 public sector 278 70.74 private sector 72 18.32 retired 5 1.27 unemployed 17 4.33 housewife 5 1.27 student 13 3.31 ngo 3 0.76 education 393 100 pri&high school 15 3.82 graduate 212 53.94 post graduate 166 42.24 household size 393 100 1 person 47 11.96 2 people 63 16.03 3 people 123 31.30 4 people 107 27.23 >4 people 53 13.49 monthly income (1 tl=£0,28) 393 100 849 tl or less 16 4.07 850 tl – 1449 tl 29 7.38 1500 tl – 2149 tl 43 10.94 2150 tl – 2799 tl 69 17.56 2800 tl – 3449 tl 44 11.20 3500 tl – 4149 tl 64 16.28 4150 tl or more 128 32.57 place of residence during the first 15 years of life 393 100 city or suburb 251 63.87 small town 96 24.43 village 46 11.70 table 3 comparison of sample sociodemographics versus turkey’s population. sociodemographies sample turkey’s population* female (%) 36.9 49.8 household size 3.1 3.7 graduates (%) 96.2 12.0 median income (tl) 3150 1838 median age 40 31 *elaborated from data extracted from turkstat. table 4 fundamentally indicates the basic preferences stated by turkish consumers for pesticides and food safety issues. survey results showed that approximately 75 % of respondents have no idea about the pesticides and their harmful effects whereas only 20 % indicated limited knowledge about pesticides. respondents aged 46-64 showed a higher degree of knowledge about pesticides. a great majority of those having pesticide knowledge specified mass media as a source of knowledge on pesticides. when a cross check question about the pesticides in potatoes was later asked, more than 50 % of respondents again indicated no idea about it; while 32 % of those have an opinion of “there are pesticide, hormone and other chemicals that are harmful for health”. regular shapes of potatoes are predominantly remarked by respondents (around 56 % of respondents). a similar viewpoint comes from another question to observe how cosmetic defects are important for individuals. more than 86 % of respondents pointed out that they are not willing to pay for potatoes with insect damages even though they are pesticide-free produce. this finding might be interpreted that for those who are willing to pay more for pesticidefree products, suppliers should ensure that they can be provided with satisfactory quality standards. ott and maligaya (1989) quoted in weaver et al. (1992) found that 88 % of the respondents would be unwilling to accept those defects. apart from cosmetic defects, independent science-based advice is one of the most important critical issues in food safety in the european union. european food safety authority (efsa) as an independent body is responsible for carrying out risk assessment from risk management (efsa, 2014). conflict of interest inevitably appears when the same institutions both control and monitor the same findings. this is a crucial issue for turkey as well. therefore, a question was asked to observe the respondents’ opinions on “who should carry out food safety control?”. the least frequent responses for this question are municipalities and public agents with roughly 4% and 12% respectively. the majority, 37%, of respondents preferred having an independent laboratory certification for more fair and transparent food safety control. 112 ital. j. food sci., vol. 28 2016 table 4 pesticide concerns and purchasing preferences of turkish consumers. source of concern freq. % remember a serious incident 1 1.05 heard concern expressed over one or more of mass media 48 50.53 heard concern expressed by ngo`s 4 4.21 heard concern expressed by public agents 7 7.37 other 35 36.84 opinion about the pesticides in potatoes freq. % there is no pesticide, hormone and other chemicals 17 4.33 there are pesticide, hormone and other chemicals, but residues are not risky for health 33 8.40 there are pesticide, hormone and other chemicals that are harmful for health 127 32.32 no idea 216 54.96 purchasing preferences freq. % no preservative including pesticide and hormones 21 5.34 taste 78 19.85 price 71 18.07 regular shape 220 55.98 brand 3 0.76 purchasing place of potatoes freq. % open-air market 151 38.42 greengrocer 46 11.70 supermarket/hypermarket/shopping centre 174 44.27 villagers 15 3.82 others 7 1.78 importance of cosmetic defects freq. % not important 0 0.00 less important 53 13.49 more important 268 68.19 highly important 72 18.32 food safety control freq. % municipalities 16 4.07 public agents 50 12.72 universities 66 16.79 independent agents 139 37.37 producer unions 13 3.31 consumer unions 109 27.74 based on the data in tables 1, 2, and 4, respondents aged 31 to 45 and having master and phd. degrees were found to be more willing-toaccept insect damage in reduced pesticides in potatoes than those aged 46 and older, and those having non-college and college degrees respectively. males, lower income households and college graduates were found to be less willing to accept cosmetic defects in reduced pesticides in potatoes than were females, high income households and non-college graduates respectively. finally, the survey results show that respondents considering pesticides in potatoes that are harmful for health and having no idea about it were found to be more willing-to-pay than were those considering no harmful pesticides in potatoes and having no idea about pesticides respectively. this matter was comprehensively argued by ravenswaay (1990). she mainly discussed that people with college degrees might be less concerned than those with non-college degrees since reaching knowledge for them is less costly than others. they are, as a result of this deduction, least willing-to-pay for the safe food. additionally, it is possible to make regional comparisons at the level of nuts 1 regions. respondents from south east region (se), middle eastern anatolia (me) and west marmara region (wmar) were found to be less willing to pay for extra payment per kg for pesticide-free potatoes than were other regions, while respondents from ist (istanbul) and east marmara region (emar) which are largely industrialised parts of turkey were found to be more willing to pay for it than were other regions. despite this result, it does not make sense at all to have any correlation between income and willingness to pay for the price increase per kg for potatoes has no major effect on individuals’ incomes. this is supported by bunte et al. (2010). they showed that any reduction in organic prices for some products such as potato has no considerable effect on demand. respondents from se and emar were found ital. j. food sci., vol. 28 2016 113 to be more willing-to-accept insect damage on pesticide-free produce than were other regions and respondents from west black sea (wbs), and aeg are less willing to accept insect damage on reduced pesticides in potatoes than were other regions. variance inflation factor (vif) should not ideally exceed rule of 4, rule of 10 in literature. if it exceeds the rule of thumb, it is regarded as casting doubts on the estimations of regression analysis. as attentively viewed from the results given in table 5, the vif values among independent variables change between 1.02 and 1.38 and mean vif value is 1.14, which has sufficiently concrete evidence that there is no serious multi-collinearity in the model. table 6 exhibits the estimation results provided from the ordered probit model. as is illustrated, cosmetic defects for consumer preferences, free-pesticide potatoes with insect damages, indicating reasons of health for wtp questions, age, and gender were identified by the model to have significant impacts on the probability to wtp while spending the first 15 years in a village was found to negatively impact the probability to wtp. however, income and education were not found to have a significant impact, positively or negatively, on the probability to wtp. being female increases the probability of wtp by 21% as revealed in most of the studies (henson, 1996; gill et al., 2000; loureiro et al., 2002; kontoleon et al., 2005; sundstrom and andersson, 2009). this can easily be explained as women are more sensitive to food safety problems than men. also, those indicating health reasons for wtp question were found to increase the probability to wtp by 43%. on the contrary, kalogeras et al. (2009) found that health aspect does not significantly influence the probability of wtp. similar effects were observed on cosmetic defects and age. considering cosmetic defects as an important feature for their purchasing preferences raises the probability to wtp by 12%. in much the same way, the age of our model had a positive impact (by 10%) on wtp as in most of the studies (misra et al., 1991; kontoleon et al., 2005; dettmann and dimitri, 2010). contrariwise, the age of the consumers were found to have a negative effect table 5 collinearity diagnostic. variable vif 1/vif working condition 1.38 0.72475 income 1.38 0.725141 education 1.15 0.871934 age 1.12 0.893866 insect damage 1.06 0.943308 living in a village 1.06 0.945987 reason for health 1.04 0.96083 harmful pesticides 1.04 0.960983 cosmetic defects 1.02 0.981626 mean vif 1.14 table 6 the probit model. dependent variable: wtp variable coefficient standard error marginal effect standard error constant -2.70513*** 0.739544 knowledge -0.25266 0.180227 -0.09928 0.07116 cosmetic defects 0.319988** 0.128319 0.124498** 0.0499 insect damage 0.41714* 0.223001 0.154209** 0.07671 harmful pesticide 0.242785 0.159825 0.093258 0.06043 reason for health 1.151586*** 0.149467 0.434584*** 0.05094 age 0.267368** 0.122852 0.104025** 0.04781 working condition 0.078549 0.063429 0.030561 0.02469 gender 0.556782*** 0.152852 0.210075*** 0.05503 education level 0.046347 0.36927 0.018112 0.14491 income 0.038651 0.131314 0.015038 0.05109 living in a village -0.39952* 0.219629 -0.15794* 0.0866 ***indicates significance at 1% level, **at 5% level, *at 10% level. probit regression number of obs = 393 lr chi2(11) = 93.65 prob > chi2 = 0.0000 log likelihood = -220.82775 pseudo r2 = 0.1749 166 left-censored observations at pay <=0; 227 uncensored observations; 0 right-censored observations. 114 ital. j. food sci., vol. 28 2016 table 7 the tobit model. dependent variable:mwtp variable coefficient standard error marginal effect standard error constant -5.15815*** 1.344697 knowledge -0.27364 0.328352 -0.1237628 0.14583 cosmetic defects 0.681142*** 0.228425 0.3135761*** 0.10497 insect damage 0.607647 0.373106 0.2955922 0.19146 harmful pesticide 0.361183 0.284776 0.1690033 0.13527 reason for health 2.334047*** 0.288372 0.9972112*** 0.11239 age 0.529826** 0.220929 0.243915** 0.10148 working condition 0.166313 0.112667 0.076565 0.05181 gender 0.903691*** 0.263868 0.4291193*** 0.12871 education level 0.379784 0.655299 0.1673579 0.2761 income 0.028448 0.233872 0.0130964 0.10767 living in a village -0.87384** 0.41634 -0.3699325** 0.16148 ***indicates significance at 1% level, **at 5% level, *at 10% level. tobit regression number of obs = 393 lr chi2(11) = 98.80 prob > chi2 = 0.0000 log likelihood = -627.37228 pseudo r2 = 0.0730 on the wtp for organic potatoes by loureiro and hine (2002) and reduced pesticides in tomatoes by akgungor et al. (2007). additionally, spending the first fifteen years in a village reduces the probability to wtp by 16%, ceteris paribus. the interpretation could be made that those people who spent their first fifteen years in a village might have a lower level of education, thus, less knowledge of pesticide impacts and less sensitiveness to the topic. table 7 summarizes the results of the tobit model concerning their marginal effects. individuals who considered cosmetic defects as important features for potato preferences, who are female, and who were indicating health reasons for wtp questions have higher wtp. to put it in context, considering cosmetic defects as important features for potato preferences raises the wtp amount by tl 0.31, and similarly, being female raises the wtp amount by tl 0,4 respectively, ceteris paribus. respondents who spent their first fifteen years in a village have significantly lower wtp. the mean wtp amount was estimated for the reduced pesticides in potatoes in turkey on the basis of cvm study. the survey covering all of turkey showed that respondents, representing different geographical areas, on an average are willing to pay extra tl2.90 if the zero respondents corresponding to approximately 42% are not included in the models. if it was included, the mean would be extra tl1.67. these absolute numbers can be given in percentages as 48% and 83% price premium for reduced pesticides in potatoes per kg, respectively. the average market price for potato was found as tl 3.50 based on the virtual turkish super-market prices for those dates. the estimations could be likely interpreted that demand for organic food among turkish consumers is growing. in a similar study, gil et al. (2000) presented that spanish consumers living in navarra and madrid would be willing to pay 17 % and 5.6 % more for organic potatoes, respectively. this big gap between turkish and spanish consumers can be explained mainly by the organic markets in turkey that are not sufficiently saturated yet. a similar result was found by akgungor et al. (2007) that turkish consumers would be willing to pay 36% price premium for organic products or certified products. also, weaver et al. (1992) found that 26% of respondents in pennsylvania were willing to pay more than 15% for organic tomatoes. as seen from the values and percentages, there are no extreme prices that are accepted by consumers. this situation was argued by rawenswaay (1990) that consumers would be willing to pay modest amounts to reduce perceived health risks in food. two important caveats can be placed on any discussion drawn from the survey results. first, actual wtp cannot be observed as it is solely based on stated preferences. second is the homogenous distribution of individuals with respect to income and education. in spite of the fact that education and income are found to be significant factors for many wtp studies, no relationship was found in our model. the first one seems more important while income has a minor impact on an individual’s budget as indicated by bunte et al (2010). however, there is no consensus in literature inital. j. food sci., vol. 28 2016 115 dicating a certain effect of education on wtp amount. though dettman and dimitri (2010) found a positive relation between education and wtp for organic products, misra et al. (1991); buzby et al. (1995); thompson and kidwell (1998); borceletti and nardella (2000) and sundstrom and andersson (2009) found a negative relation. it was also affirmed by van ravenswaay (1995) that the people with higher education level may be less concerned about pesticides because they might be better able to reach reliable information. these results might help to affirm why there is no significant impact of education on wtp in our model considering an outstandingly high rate of educated respondents. lastly, survey results show that the respondents overwhelmingly indicate that they have no idea about the level of pesticide residues used in the food. roughly 32% of respondents considered that there are serious pesticide residues in potatoes, which are harmful to human health. an interesting finding from the survey results comes from the question “who should be responsible for controlling and monitoring of residues in food”. approximately 37.4% of respondents were in favour of independent laboratories while only 12.7 % went for public agents as an answer to this question. this clearly demonstrates that there is a high demand from consumers’ side to independent agents for neutral decisions rather than public institutions. as a result, this study stresses the consumer attitudes for pesticides in potatoes by employing cvm and single-bounded probit and tobit models. one of the drawbacks of the survey is based on the stated preferences rather than revealed preferences. the respondents might answer the questions with overestimation if compared with real situations. it would thus be better as a future research agenda to conduct another study in order to observe if similar results were truly provided by respondents. references akgungor s., miran b. and abay c. 2001. consumer willingness to pay for food safety labels in urban turkey. journal of international foodandagribusiness marketing, 12(1):91. akgungor s., miran b. and abay c. 2007. consumer willingness to pay for organic products in 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residues when risks are ambiguous” in: j. caswell (ed.) valuing food safety and nutrition. chap.14:287. van ravenswaay e.v. 1990. “consumer perception of health risks in food” michican state university. weaver r. evans d. and luloff a.e. 1992. pesticide use in tomato production: consumer concerns and willingness-to-pay. agribusiness, vol. 8:131. wooldridge j. 2006. “introductory econometrics” 4th edition. south western. paper received october 10, 2014 accepted may 5, 2015 http://www.tarim.gov.tr ital. j. food sci., vol. 28 2016 117 appendices appendix 1. survey instrument appendix 1.1. hypothetical scenario health risks resulting from pesticide use have made food safety a priority issue on the public policy agenda in developed countries. a research made in u.s showed that pesticide residues were rated a serious risk by 68 of respondents attending in a survey. pesticides can cause many types of health problems in humans. “pesticides have been linked to a wide range of human health hazards, ranging from shortterm impacts such as headaches and nausea to chronic impacts like cancer, reproductive harm, and endocrine disruption (toxic action center1)”. ec directive 2009/128/ec determined the sustainable use of pesticides to reduce health risks resulted from pesticides. therefore, eu countries minimise or ban the use of pesticides for health reasons. turkey as a candidate country for eu membership has to harmonize her own legislations and directives. the amount of pesticide use in turkey has gradually increased since 2009 and it was over 40,000 ton in 2011 according to the data taken by the ministry of food, agriculture and livestock of turkey. particularly, potato is one of the most consumed vegetables which seriously include pesticide residues in turkey. the scenario it is proposed for this survey is a price increase for reduced pesticides in potatoes per kg. the research project is aimed at evaluating your opinion of reduced pesticides in potatoes. reduced pesticides are in general valued for one or more of the following attributes: better taste, food safety, health, freshness, environment preservation and local production. good agricultural practices are “practices that address environmental, economic and social sustainability for on-farm processes, and result in safe and quality food and non-food agricultural products” (fao, 2003). more precisely the main aim of this study is to find out what would persuade you to buy reduced pesticides in potatoes. on this basis the questionnaire tries to find out your opinion of the quality and availability of the reduced pesticides in potatoes in turkey and the price that you would be willing to pay for these reduced pesticides in products. finally, for the purposes of the study you are required to give truthful answers and we recommend that you think carefully about the scenario previously mentioned, your disposable income and health concerns during the questionnaire survey. furthermore, you should notice that this survey is completely anonymous and confidential. however, if you desire a copy of the final study, you should provide an email address so it can be sent to you. appendix 1.2. questionnaire questions about qualifying candidates 1) please indicate your current place of residence. 2) please indicate whether you participate in the decisions regarding the payments in your household. a) yes b) no questions about perceptions for food 3) please indicate whether or not you have an idea regarding level of pesticides and hormones in potatoes, if you indicate choice a, please go question 5. a) no idea b) little information c) sufficient information c) all information in detail 4) please indicate your recalling of pesticide information as related to level of concern for human health. a) remember a serious incident b) heard concern expressed over one or more of mass media c) heard concern expressed by ngo`s d) heard concern expressed by public agents e) other 5) please indicate the most important feature of potato for your purchasing preferences. a) no preservative including pesticide and hormones b) taste c) price d) regular shape e) brand 6) please indicate how cosmetic defects are important for your purchasing preferences in pesticide free products. cosmetic defects refer growth cracks and knobby or irregular growth. a) not important b) less important d) more important c) highly important 7) please indicate if you accept potatoes with insect damage, such as worm holes in pesticide free products. a) yes b) no 8) please indicate your opinion about the pesticides, hormones, and other chemicals for potatoes. a) there is no pesticide, hormone and other chemicals b) there are pesticide, hormone and other chemicals, but residues are not risky for health c) there are pesticide, hormone and other chemicals that are harmful for health d) no idea 9) please indicate what you generally do in order to alleviate your concern over pesticide dangerous in the potatoes. a) nothing b) washing it with plenty of water c) consuming by peeling off it d) cooking e) other (please specify) 10) please indicate whether or not fresh fruit and vegetables are as healthy as it was in the past with respect to health safety. a) never healthy b) still healthy c) better healthy d) no idea questions about willingness-to-pay at this stage, you should consider that the payment vehicle for the reduced pesticide in potato will lead to increases in potato prices if you favour the reduced pesticides in potato. moreover, we strongly recommend you to consider your disposable income, health concerns, and possible positive and negative consequences of the reduced pesticide in potato when making your decision. 11) would you be willing to pay extra 2 tl/per kg for reduced pesticides in potato? if answer is yes, please go to question 12, otherwise go to question 18. a) yes b) no 12) would you be willing to pay extra 2.5 tl/per kg for reduced pesticides in potato? a) yes b) no if answer is no, please go to question 17. 13) would you be willing to pay extra 3 tl/per kg for reduced pesticides in potato? a) yes b) no if answer is no, please go to question 17. 14) would you be willing to pay extra 3.5 tl/per kg for reduced pesticides in potato? a) yes b) no if answer is no, please go to question 17. 15) would you be willing to pay extra 4 tl/per kg for reduced pesticides in potato? a) yes b) no if answer is no, please go to question 17. 16) would you be willing to pay above 4 tl/per kg for reduced pesticides in potato? also please indicate how much you would be willing to pay. a) yes (please specify): tl b) no how much:........................................................................ 1 http://www.toxicsaction.org/problems-and-solutions/pesticides http://www.toxicsaction.org/problems-and-solutions/pesticides 118 ital. j. food sci., vol. 28 2016 17) would you please indicate the reason for the expressed amount? a) more healthy b) a reasonable price for my budget c) more tasty d) protecting environment e) protecting local producers f) other (please specify) questions about social and economic factors 18) regarding your age, which of the following would you select? a) 17 or less b) 18-30 c) 31-45 d) 46-64 e) 65 or more 19) regarding your working condition, which of the following would you select? a) public sector b) private sector c) retired d) unemployed e) housewife f) student g) farmer h) ngo 20) regarding your gender, which of the following would you select? a) male b) female 21) regarding your marital status, which of the following would you select? a) married b) single 22) regarding your family composition, which of the following would you select? a) have children b) do not have children 23) regarding the size of your household, which of the following would you select? a) one person b) two persons c) three persons d) four persons e) more than four persons 24) regarding your education level, which of the following would you select? a) primary school graduate b) secondary school graduate c) high school graduate d) bachelor’s degree graduate e) master’s degree graduate f) ph.d. ’s degree graduate g) other:............................................................................ 25) regarding your monthly income, which of the following would you select? a) 849 tl or less b) 850 tl – 1449 tl c) 1500 tl – 2149 tl d) 2150 tl – 2799 tl e) 2800 tl – 3449 tl f) 3500 tl – 4149 tl g) 4150 tl or more 26) please indicate the place of residence during the first 15 years of life? a) city or suburb b) small town c) farm 27) please indicate the place you are currently living? a) less than 3 years b) 3-5 years c) 6-10 years d) 11-20 years e) more than 20 years 28) please indicate from where do you generally purchase potatoes? a) open-air market b) greengrocer c) supermarket/hypermarket/shopping center d) villagers e) others 29) please indicate your preference about which agent should ideally and fairly be responsible for food safety control? a) municipalities b) public agents c) universities d) independent agents e) producer unions f) consumer unions thank you for your time! appendix 2. summary and descriptions of variables variable obs mean std. dev. min max pay 393 1.676845 1.621334 0 6 bid 393 0.5776081 0.4945699 0 1 knowl 393 0.2417303 0.4286774 0 1 cosm_def 393 3.048346 0.5626138 2 4 insect_dam 393 0.1399491 0.3473765 0 1 harmfulpes 393 0.3231552 0.4682776 0 1 age 393 2.697201 0.6123035 2 5 work_cond 393 1.608142 1.289334 1 8 gender 393 0.3689567 0.4831373 0 1 marital 393 0.4707379 0.4997793 0 1 hav_child 393 0.4274809 0.4953436 0 1 household 393 3.142494 1.197383 1 5 livingincity 393 0.6386768 0.4809963 0 1 livingindist 393 .2442748 0.4302041 0 1 livinvilage 393 0.1170483 0.3218877 0 1 educ 393 0.956743 0.2036944 0 1 income 393 2.223919 0.6273835 1 3 livinvilage 393 0.1170483 0.3218877 0 1 ital. j. food sci., vol. 28 2016 119 appendix 3. multicollinearity analysis k nowl c os m_d ef i ns ect_d am h ar mfulpes r eas onh ealth a ge w or k _c ond g ender e ducdumy i ncome livinvilage k nowl 1.0000 c os m_d ef -0.0380 1.0000 i ns ect_d am 0.2519*** -0.0739 1.0000 h ar mfulpes 0.3469*** -0.0401 0.1604*** 1.0000 r eas onh ealth -0.0888 -0.0306 -0.0828 0.0709 1.0000 a ge 0.1435*** -0.0092 -0.0281 0.0129 -0.0121 1.0000 w or k _c ond 0.0057 0.0016 0.1114** -0.0179 (-)0.1438*** -0.0667 1.0000 g ender -0.0129 (-)0.1127* 0.0716 0.0129 0.0402 (-)0.1647* 0.0853** 1.0000 e ducdumy 0.0032 -0.0262 -0.0224 -0.0135 0.0508 -0.0439 (-)0.327*** 0.0707 1.0000 i ncome -0.0216 -0.0018 (-)0.109* 0.0309 0.0623 0.2566*** (-)0.4558*** (-)0.2059***0.2357*** 1.0000 livinvilage 0.1457*** 0.0955* 0.0356 0.0531 -0.0762 0.1803*** -0.0306 -0.0816 -0.0393 0.0467 1.0000 appendix 4. regression analysis source ss df m s number of obs = 393 f ( 9, 383) 10.79 m odel 14.56733 9 1.61859267 prob > f 0 r esidual 57.46829 383 .150047753 r -squared 0.2022 a dj r -squared 0.1835 t otal 72.03562 392 .183764345 r oot m se 0.38736 k nowl c oef. std. e rr. t p>t [95 % c onf. i nterval] c osm_def -0.01601 .0350985 -0.46 0.649 -0.0850178 0.053002 i nsect_dam 0.233844 .0579889 4.03 0.000 0.1198276 0.34786 h armfulpes 0.290287 .0426197 6.81 0.000 0.2064893 0.374085 r easonh ealth -0.07965 .0416259 -1.91 0.056 -0.1614897 0.002198 a ge 0.100169 .0337963 2.96 0.003 0.0337199 0.166619 w ork_c ond -0.01019 .0178243 -0.57 0.568 -0.0452324 0.024859 e ducdumy 0.06667 .102861 0.65 0.517 -0.1355734 0.268913 i ncome -0.04631 .0366208 -1.26 0.207 -0.1183125 0.025693 livinvilage 0.126524 .062492 2.02 0.044 0.0036537 0.249395 _cons -0.01413 .1961847 -0.07 0.943 -0.3998678 0.3716 appendix 5. covariance matrix of coefficients of regress model e(v ) c osm_d ef i nsect_d am h armfulpes r easonh ealth a ge w ork_c ond e ducdumy i ncome livinvillage _cons c osm_d ef 0.0012319 i nsect_d am 0.0001513 0.00336271 h armfulpes 4.661e -05 -0.00041276 0.00181644 r easonh ealth 3.774e -05 0.0001939 -0.0001506 0.00173271 a ge 3.531e -05 0.00002271 6.14e -06 0.00001231 0.0011422 w ork_c ond 4.64e -07 -0.0000685 8.00e -06 0.00008935 -2.13e -05 0.00031771 e ducdumy 8.484e -05 -0.00015164 0.00011349 -9.37e -06 0.0003194 0.0004571 0.0105804 i ncome -1.32e -06 0.00014854 -5.893e -05 9.25e -06 -0.000321 0.00025825 -0.000478 0.00134108 livinvilage -0.0002205 -0.00013419 -0.0001421 0.00020032 -0.000363 0.00004616 0.000235 7.79e -06 0.0039053 _cons -0.0039642 -0.00104393 -0.0005647 -0.00142089 -0.002716 -0.00152239 -0.010951 -0.0020787 0.0008133 0.0384884 120 ital. j. food sci., vol. 28 2016 appendix 6. correlation matrix of coefficients of regress model e(v ) c osm_d ef i nsect_d am h armfulpes r eason_health a ge w ork_c ond e ducdumy i ncome livinvillage _cons c osm_d ef 1 i nsect_d am 0.0743 1 h armfulpes 0.0312 -0.167 1 r easonh ealth 0.0258 0.0803 -0.0849 1 a ge 0.0298 0.0116 0.0043 0.0087 1 w ork_c ond 0.0007 -0.0663 0.0105 0.1204 -0.0354 1 e ducdumy 0.0235 -0.0254 0.0259 -0.0022 0.0919 0.2493 1 i ncome -0.001 0.0699 -0.0378 0.0061 -0.2594 0.3956 -0.127 1 livinvilage -0.1005 -0.037 -0.0534 0.077 -0.1719 0.0414 0.0366 0.0034 1 _cons -0.5757 -0.0918 -0.0675 -0.174 -0.4096 -0.4354 -0.5427 -0.2893 0.0663 1 appendix 8. statistic values of wtp before and after trimming outlier p ercentiles smallest p ercentiles smallest 1% 0 0 1% 0 0 5% 0 0 5% 0 0 10% 0 0 obs 393 10% 0 0 obs 374 25% 0 0 sum of w gt. 393 25% 0 0 sum of w gt. 374 50% 2 mean 1.676845 50% 2 mean 1.497326 l argest std. d ev. 1.621334 l argest std. d ev. 1.444224 75% 2.5 6 75% 2.5 4 90% 4 6 v ariance 2.628723 90% 4 4 v ariance 2.085784 95% 4 6 skewness 0.424015 95% 4 4 skewness 0.2272442 99% 4 6 k urtosis 2.16069 99% 4 4 k urtosis 1.633528 pay t rimmed data (5% ) pay appendix 7. logit model wtp coef. std. err. knowl -0.4106674 0.3074221 cosm_def 0.5257642** 0.2167355 insect_dam 0.6643928*** 0.3706913 harmfulpes 0.4106739 0.269297 reasonhealth 1.886197*** 0.2523839 age 0.4439607** 0.2078363 work_cond 0.1279345 0.1060791 gender 0.9363477*** 0.2617843 educdumy 0.0939652 0.6196589 income 0.0539704 0.2209946 livinvilage (-)0.681425* 0.3660309 _cons -4.444088 1.238931 number of obs=393 lr chi2 (11)=93.33 prob>chi2=0.0000 pseudo r2=0.1743 ***indicates significance at 1% level, **at 5% level, *at 10% level. paper 416 ital. j. food sci., vol. 27 2015 keywords: rennet paste, chemical and sensory characteristics effect of artisanal rennet paste on the chemical, sensory and microbiological characteristics of traditional goat’s cheese c. tripaldi*, g. palocci, a. garavaldi1, t. bogdanova2 and s. bilei2 consiglio per la ricerca e la sperimentazione in agricoltura, centro di ricerca per la produzione delle carni e il miglioramento genetico, via salaria 31, 00016 monterotondo, roma, italy 1fondazione crpa studi ricerche onlus, corso garibaldi 42, 42100 reggio emilia, italy 2istituto zooprofilattico sperimentale delle regioni lazio e toscana, via appia nuova 1411, 00178 roma, italy *corresponding author: carmela.tripaldi@entecra.it abstract in a study using three replicates, marzolina goat cheese made with artisanal rennet paste from goat kid was compared with cheese made with commercial liquid rennet from calf. samples of fresh cheese were subjected to chemical and microbiological analyses. samples of ripened cheese collected after 50 days of ripening were submitted to chemical and sensory analysis. results of this study show that cheese made with artisanal rennet pastes did not contain pathogenic microorganisms and that this kind of rennet provided the enzymatic content necessary to achieve the typical characteristics of traditional cheeses. mailto:carmela.tripaldi%40entecra.it?subject= ital. j. food sci., vol. 27 2015 417 introduction in recent years, there has been an increased interest in the safety and promotion of cheeses prepared according to local traditional processes. the renewed attention is given to artisanal rennet, which is considered one of the most important factors affecting the character istics of some typical mediterranean cheeses. arps usually originate from lamb or kid abomasa, and farmers directly prepare them for use during cheese making. according to current legislation, the use of arps is allowed after a special derogation from regulation (ec) n. 852/2004 for foods with traditional characteristics (ec reg. n. 2074/2005). to obtain this derogation, it has been necessary to study their safety and hygienic characteristics. results of a number of studies indicate that traditional cheese-making does not compromise health and hygiene (cosseddu and pisanu, 1980; deiana et al., 1980; pisanu and cosseddu, 1982; barzaghi et al., 1997; calandrelli et al., 1997; irigoyen et al., 2001; moatsou et al., 2004; moschopoulou et al., 2007; tripaldi et al., 2012). determining the role and influence of arps on the sensory characteristics of cheeses is also necessary. marzolina is a traditional italian cheese made from the milk of local goat breeds of the latium region, in the centre of italy and from artisanal kid rennet pastes. marzolina is characterised by a weigth of about 150 g and high salt content that preserves the cheese for long periods even in a natural room. arp is traditionally used in the process for marzolina cheese and its particular flavor has been attributed to the use of rennet pastes (addis et al., 2005). however factors such as the time and the effort required to prepare the rennet at the farm, as well as the lower demand for strongly flavoured cheeses, have contributed to the rapid replacement of arp with commercial liquid rennet (clr). the aim of this trial was to study the hygienic and health characteristics of cheese from arp so contributing to the approval of the derogation for using artisanal rennet pastes. moreover this trial was finalised to evaluate the effect of rennet pastes, as compared to liquid rennet, on the chemical, and sensory characteristics of marzolina goat cheese. materials and methods marzolina cheese made with arp from kid was compared with that made with clr from calf. both cheeses were produced by farmers. two kinds of rennet were used and three replicates were performed. the trial was carried out according to the process usually employed by farmers in a small cheese farm. thirty litres of milk for each kind of rennet in each replicate were processed. abomasa were removed from suckling kids slaughtered at the age of 30–45 days and they were submitted to a drying phase preceded by a salting phase. ten abomasa were ground, merged and then utilised in all replicates of the trials. the commercial liquid calf rennet (naturen®), used in the trial was produced by chr. hansen’s (denmark). data specifying proteolytic activity of clr were supplied by chr. hansen’s. enzimatic characteristics of arp were determined according to the following methods. total milk clotting activities of the artisanal rennet pastes were determined according to iso 23058 idf 199: 2006 method known as remcat method. to determine the chymosin and pepsin content, test samples were prepared by dissolving 25 g of rennet paste in 100 g of buffer solution (ch3cooh/ch3coona) at ph 5.5; the samples were centrifuged at 3000 rpm (2189 g, refrigerated centrifuge alc 4237r, alc, milano, italy) for 30 min at 4°c. the supernatant was analysed as described in the international idf standard 110b: 1997. chymosin and pepsin enzymes were expressed as a percentage of the sample’s total milk clotting activity. lipase activity was analysed as described in the food chemical codex (1981). characteristics of both types of rennet are shown in table 1. during the study, raw goat milk was utilised and no starter cultures were added. after coagulation at a temperature below 35°c, the curd was cut, reduced to small granules and then packed in a small cylindrical mould. after about 12 hours from the start of cheese-making, the cheeses were subjected to dry salting and then air-dried in a natural room for one week. finally, they were packaged under vacuum and stored at 4°c. during each experiment, samples of fresh cheese were collected before dry salting. samtable 1 enzymatic activity of artisanal rennet pastes and commercial liquid rennet. rennet g or ml of rennet total imcu × g-1 or ml-1 total imcu on 100 l of milk chymosin (% ru) chymosin imcu ilu × g-1 on 100 l of milk of rennet on 100 l of milk of rennet arp 19 129 2493 95,34 2377 35,76 clr 20 162 3229 80,00 2583 arp: artisanal rennet paste; clr: calf liquid rennet. imcu: international milk clotting unit; ru: rennet unit;ilu: international lipase units. 418 ital. j. food sci., vol. 27 2015 ples of fresh cheese were subjected to chemical analyses, which were conducted in duplicate. analyses consisted of measurements of moisture (idf, 1986), total nitrogen (tn) (fil-idf, 1993), soluble nitrogen (sn) (fil-idf, 1991), fat (fil-idf, 2001), salt (idf, 1988), ash (aoac, 2000), and free fatty acids (ffas). ffas (mmol/ kg) were analysed by capillary gas chromatography (de jong and badings, 1990) and expressed as mmol/kg to assess each individual ffa independent of its molecular weight. samples of fresh cheese were also subjected to microbiological analysis. all samples were subjected to qualitative tests for salmonella, listeria monocytogenes and escherichia coli o157, and to quantitative analyses for l. monocytogenes, sulphite-reducing clostridia, total microbial count at 30°c, coagulase-positive staphylococci, and β-glucuronidase-positive e. coli (tripaldi et al., 2012). samples of cheese ripened for 50 days were also collected and then subjected to chemical and sensory analyses. descriptive sensory analysis was performed to determine the differences in sensory characteristics of the two kinds of cheeses. ten panelists (5 males and 5 females; between 20 and 50 years of age) were selected and trained in accordance with iso 8586-1:1993 e iso 85862:1994 standards. for the laboratory conditions, uni iso 8589 standard was followed. the test was carried out according to uni 10957:2003 standard based on triplicate analysis of each sample. twenty-two descriptors were identified, as follows: 3 visual (color intensity, color omogeneity, rind color), 5 olfactory (smell intensity, stable straw smell, lactic smell, vegetable smell, other smell), 5 basic taste and trigeminal sensations (salty, sweet, sour, bitter, piquant), 6 retro-olfactory (flavour intensity, stable straw flavour, lactic flavour, vegetable flavour, other flavour, persistence), and 3 tactile descriptors (adhesiveness, moisture, firmness). attributes were rated on a continuous scale of values from 0 to 10 (0 = absence of the intensity, maximum intensity = 10). sensory descriptors and their rating scale were defined according to uni 10957:2003 and european guide for the sensory evaluation of hard and semi-hard cheese standard (bérodier e al.1997; lavanchy et al.1994). the glm procedure of sas software (sas institute inc., 2007) was used for statistical analysis of chemical parameters by using the model y ijl = μ + a i + b j + a i ×b j + e ijl where y ijl = qualitative characteristics of the cheese a i = fixed effect of the kind of rennet (i = 1 for arp; i = 2 for clr) b j = fixed effect of days of ripening (j = 1: 1d; j = 2: 50d) e ijl = residual of error data processing of sensory evaluations was carried out according to uni 10957:2003 standard. results and discussion results of proteolytic and lipolytic activity of arps and clr are summarised in table 1. the clotting characteristics were different: 129.38 total international milk clotting units (imcu) × g−1 and 95.34% of chymosin for arp and 162.00 total imcu × ml−1 and 80.00% of chymosin for clr. the latter was subjected to a preliminary test and then added to milk in quantity of 19.93 ml/100 l of milk. this led to coagulation in approximately 60 minutes, the clotting time used by farmers. during the experiment, the addition of rennet to 100 l of milk resulted in 2493 and 3229 total imcu for arp and clr, respectively. chymosin imcu values, 2377 and 2583 per 100 l of milk for arp and clr, respectively, were more similar than that of total imcu. . the lower activity of total clotting of arp could be due to dilution of enzymes caused by the presence of milk in the abomasa used to produce the rennet (pirisi et al., 2007). the high chymosin percentage (95.34 and 80.00) in the rennet paste could be attributed to the completely milk-based diet and/or to complete filling of the stomach with milk when the kid was killed (bustamante et al., 2000; addis et al., 2005). we observed that in our study the kind of diet given to the kid and the status of the abomasum before the slaughtering agreed with the conditions found by other authors (bustamante et al., 2000; addis et al., 2005). the international lipase units (ilu) of the rennet paste, 35.76 ilu × g−1, was similar to the average value (36.18 ilu × g−1) obtained from other samples of rennet paste from the same region (tripaldi et al., 2012). the chemical characteristics of fresh and ripened goat cheese made using arp and clr are reported in table 2. the moisture values were not largely affected by the kind of rennet in both fresh and ripened cheese (69.65 and 42.30% in arp cheese and 69.81 and 40.87% in clr cheese). generally, the cheese moisture depends on the temperature and relative humidity conditions of cheese-making and the ripening conditions (irigoyen et al., 2002); thus, it is difficult to find differences between the two cheeses in which the only change is the type of rennet. we can observe slight differences in protein content between the two kinds of cheeses (10.60 vs 11.13 and 20.98 vs 21.30 in arpand clr-treated fresh cheese and in arpand clr-treated ripened cheese, respectively). the fat content of the two kinds of cheese also differed slightly (13.29 vs 14.03% in arp and clr of fresh cheese and 28.19 vs 30.31% in arpand clr-treated ripened cheese, respectively). on the contrary, santoro and faccia (1998) observed a significant difference in fat content in canestratopugliese cheese ital. j. food sci., vol. 27 2015 419 made with rennet having different characteristics. they attributed this result to the different aggregation states of the casein micelles in the curd. the salt in moisture (s/m) content of samples of fresh cheese was similar (0.79% in arp-treated cheese and 0.82% in clr-treated cheese), while s/m content was higher in arp-treated than in clr-treated ripened cheese (7.40 vs 6.01%; p < 0.05). the difference in salt content in the two kinds of ripened cheese is probably due to manual dry salting, a practice that is subject to large variations. our results show that ripened marzolina cheese has higher salt content in comparison with the majority of ripened cheeses (2.67 in arp-treated cheese and –3.13% in clr-treated cheese), corresponding to about 5% of dry matter. soluble protein as a percentage of total protein was higher in arp-treated fresh cheese than in clr-treated fresh cheese (4.96 vs 3.98%). on the contrary, higher values of this proteolysis index were found in ripened clr-treated cheese in comparison with those obtained in ripened arp-treated cheese (10.11 vs 9.12%). during cheese ripening, the higher salt content of arp relative to the one of clr cheese may influence negatively the proteolytic process, as observed in romano type cheese (guinee and fox, 1984). similar values of soluble n/total n at ph 4.6 were found in protected designation of origin (pdo) sheep cheese canestrato pugliese (corbo et al., 2001) at 1 and 35 days of ripening (5.59-6.78% and 8.65-11.50%, respectively). values of the soluble n/total n at ph 4.6 (7.87%) of the traditional italian cheese piacentinu ennese at 2 months of ripening (fallico et al., 2006) are lower than those of ripened marzolina cheese. differences in all parameters between fresh and ripened samples of both kinds of cheese were observed. with arp and clr, the moisture of ripened cheese significantly decreased compared with that of fresh cheese. the other cheese components protein, fat, s/m and ash increased significantly during cheese ripening as a result of the decrease in moisture. the soluble protein/total protein ratio was significantly higher in ripened cheese as result of increased proteolysis (upadhyay et al., 2004). table 3 shows the individual and total ffa (tffa) content of cheese made with arp compared with cheese made with clr. the tffa content was higher in arp-treated cheese than in clr-treated cheese: 8.94 versus 4.09 mmol× kg-1 and 39.51 versus 36.56 mmol× kg-1 in fresh and ripened cheese, respectively. the difference between fresh and ripened cheese of arpand clr-treated cheeses was significant. short chain free fatty acids (scffas) are the most abundant ffas in arp and clr. similar to tffas, scffa was present at higher levels in arp-treated cheese (5.58 vs 2.06 mmol× kg-1 and 21.46 vs 15.75 mmol× kg-1, in fresh and ripened cheese, respectively). the difference was significant between ripened cheeses made with arp and with clr and between fresh and ripened cheese of both arpand clr-treated cheeses. butyric and capric acids were the most abundant ffas. levels of butyric acid were the highest in both kinds of fresh cheese. levels of capric acid were the highest in both kinds of ripened cheese.. butyric acid levels were significantly higher in fresh or ripened arp cheese than in clr cheese (2.27 vs 0.81 mmol × kg-1and 4.89 vs 3.85 mmol × kg-1, p ≤ 0.05). caproic acid only in ripened cheese was significantly higher in arp-treated cheese than in clr-treated cheese (4.85 vs 3.31 mmol × kg-1). also capric acid was higher in arp ripened cheese than in clr ripened cheese (8.39 vs 6.05 mmol × kg-1, p ≤ 0.06) the content of all individual fatty acids of both groups of ripened cheeses were higher than that of both groups of the fresh ones (p ≤ 0.01). levels of medium-chain ffas (mcffas) and long-chain ffas (lcffas) were higher in fresh cheese made with arp (1.91 vs 1.02 mmol × kg1and 1.44 vs 1.00 mmol ×kg-1 for mcffas and lcffas, respectively) and lower in ripened cheese made with arp (10.58 vs 12.04 mmol × kg-1 and 7.48 vs 8.77 mmol × kg-1, respectively). the diftable 2 composition of fresh and ripened marzolina goat cheese made using artisanal rennet paste (arp) and commercial liquid rennet (clr) at 1 day and 1 month of ripening. fresh cheese ripened cheese p arp + ++ clr + ++ arp + ++ clr + ++ se rennet ripening moisture (%) 69,65 ns a 69,81 ns a 42,30 ns b 40,87 ns b 1,15 ns * protein (%) 10,60 ns b 11,13 ns b 20,98 ns a 21,30 ns a 0,60 ns * soluble protein (% of total protein) 4,96 ns b 3,98 ns b 9,12 ns a 10,11 ns a 0,13 ns * fat (%) 13,28 ns b 14,03 ns b 28,19 ns a 30,31 ns a 0,73 ns * nacl (% of moisture) 0,79 ns b 0,82 ns b 7,40 a a 6,01 b a 0,44 * * ash (%) 1,66 ns b 1,71 ns b 5,09 ns a 4,36 ns a 0,26 ns * arp: artisanal rennet paste; clr: calf liquid rennet. + kind of rennet; ++ ripening time. a, b, *: p<0.05. 420 ital. j. food sci., vol. 27 2015 table 3 free fatty acids (mmol x kg-1) in fresh and ripened marzolina goat cheese made using artisanal rennet paste (arp) and commercial liquid rennet (clr) at 1 day and 1 month of ripening. fresh cheese ripened cheese p arp + ++ clr + ++ arp + ++ clr + ++ se rennet ripening c4:0 2,27 a b 0,81 b b 4,89 a a 3,85 b a 0,33 * ** c6:0 1,28 ns b 0,37 ns b 4,85 a a 3,31 b a 0,31 * ** c8:0 0,60 ns b 0,24 ns b 3,29 ns a 2,50 ns a 0,40 ns ** c10:0 1,42 ns b 0,62 ns b 8,39 ns a 6,05 ns a 0,80 ns ** scffas 5,58 ns b 2,06 ns b 21,46 a a 15,75 b a 1,45 * ** c11:0 0,01 ns b 0,00 ns b 0,04 a a 0,03 b a 0,00 * ** c12:0 0,44 ns b 0,20 ns b 2,42 ns a 1,81 ns a 0,22 ns ** c14:0 0,47 ns b 0,22 ns b 2,75 ns a 3,13 ns a 0,43 ns * c15:0 0,03 ns b 0,02 ns b 0,18 ns a 0,23 ns a 0,03 ns * c16:0 0,94 ns b 0,56 ns b 5,05 ns a 6,69 ns a 0,84 ns * c16:1 0,02 ns b 0,01 ns b 0,13 ns a 0,15 ns a 0,03 ns * mcffas 1,91 ns b 1,02 ns b 10,58 ns a 12,04 ns a 1,55 ns ** c17:0 0,02 ns b 0,01 ns b 0,10 ns a 0,13 ns a 0,02 ns * c18:0 0,45 ns b 0,33 ns b 2,15 ns a 2,73 ns a 0,34 ns * c18:1 0,88 ns b 0,63 ns b 4,57 ns a 5,05 ns a 0,89 ns * c18:2 0,06 ns b 0,03 ns b 0,41 ns a 0,56 ns a 0,10 ns * c18:3 0,03 ns b 0,01 ns b 0,23 ns a 0,30 ns a 0,05 ns * lcffas 1,44 ns b 1,00 ns b 7,48 ns a 8,77 ns a 1,40 ns * tffas 8,94 ns b 4,09 ns b 39,51 ns a 36,56 ns a 4,24 ns ** arp: artisanal rennet paste; clr: calf liquid rennet. + kind of rennet; ++ ripening time. scffas = short chain free fatty acids mcffas = medium chain free fatty acids lcffas = long chain free fatty acids. tffas = total free fatty acids. a, b, *: p<0.05; a, b, **: p<0.01 ference was significant for both groups of ffas only between fresh and aged cheeses. the higher butyric acid content of cheese made with arps compared with cheese made with clr is confirmed by the high specificity of the enzymatic activity of pregastric lipase for scfas, especially butyric acid, esterified to the sn-3 position of triglycerides (pitas and jensen, 1970; kim and lindsay, 1993). fontecha et al. (2006) found higher butyric acid content in spanish goat cheese made with rennet paste compared with cheese made with clr. the higher concentration of capric acid compared with butyric acid in ripened cheeses was observed in other goat cheeses (buffa et al., 2001; poveda and cabezas, 2006; atasoy and turkoglu, 2009). according to buffa et al. (2001), the capric acid content of cheese from goat milk increased during ripening while butyric acid varied slightly from the start to the end of ripening. the small increase in butyric acid content was probably due to its metabolic conversion to aromatic compounds (buffa et al., 2001). sensory attributes of each kind of cheese are shown in fig. 1. mean values of the following sensory descriptors were significantly different fig. 1 sensory profile of ripened marzolina goat cheese made using artisanal rennet 14 pastes (arp) and commercial liquid rennet (clr). ital. j. food sci., vol. 27 2015 421 (p<0.05) in the two kinds of cheese (arp vs clr, respectively): color omogeneity (6.6 vs 7.1), rind color (3.7 vs 4.0), stable straw smell (4.4 vs 4.0), lactic smell (4.2 vs 4.6), other smell (3.3 vs 2.8), salty (4.2 vs 3.6), sweet (2.9 vs 3.2), sour (3.6 vs 3.3), bitter (2.9 vs 2.6), piquant (3.9 vs 3.3), flavour intensity (7.1 vs 6.8), stable straw flavour (4.7 vs 4.5), lactic flavour (4.4 vs 4.8), other flavour (3.8 vs 3.4), persistence (7.2 vs 6.7), adhesiveness (4.3 vs 5.0), moisture (4.1 vs 4.5) and firmness (5.7 vs 4.9). there was no significant interaction between assessor and replicate, suggesting high repeatability of panellist assessment in the three replicates. sample-replicate and sample-panellist interactions were not significant, showing either homogeneity of samples in the three replicates or good agreement among panellist assessments during sensory evaluation. analysis of the eyes and slits in cheese showed a higher number of samples made with arps having these defects compared with those in cheese samples made with clr (82 vs 57). the basic tastes salty, acid, bitter and piquant were more pronounced in cheeses made with arp than in cheeses made with clr. in addition, the smell and flavour of cowshed were more marked in cheese made with arp than in cheese made with clr. its texture was firmer, less tacky and less moist than the latter kind of cheese. the odour and flavour given by lactic acid to cheeses made with clr are more dominant than in cheeses made with arp. generally, the sweet, salty and sour attributes of taste were less pronounced. the texture was tackier, more moist and less firm than that of cheese made with arp. it is noteworthy that basic tastes salty and piquant, which are more pronounced in cheese made with arp than in cheese made with clr, agree with the chemical results. as reported by addis et al. (2005), cheese made with arps has a major amount of butyric acid, which may be responsible for piquancy in cheese (rennet paste has been associated with piquancy or pungency and with characteristic flavours of certain cheeses from the mediterranean basin (anifantakis, 1976; nelson et al., 1977; woo and lindsay, 1984; battistotti and corradini, 1993; barzaghi et al., 1997; calandrelli et al., 1997). in a study on idiazabal cheese (etayo et al., 2006), cheese made with lamb rennet pastes showed higher butyric acid content and received higher scores compared to cheese made with commercial liquid lamb rennet. . a larger number of eyes and slits in cheese made using arps was also observed by ferrandini et al. (2012). this could be attributed to different textural properties (ferrandini et al., 2011) of cheese made with the two kinds of rennet. in fact, results of microbiological analyses carried out on arp used in this study (tripaldi et al., 2012) exclude the presence of microorganisms markers of hygiene characteristics including germs causing microbiological spoilage in cheese. table 4 displays the microbiological characteristics of the cheese. salmonella and l. monocytogenes, pathogens considered as health markers (reg. ce 2073/2005), were undetected by qualitative analyses. β-glucuronidase-positive e. coli and coagulase-positive staphylococci, which include staphylococcus aureus, are considered as hygiene markers (reg ce 2073/2005). the maximum count tolerated for coagulase-positive staphylococci in cheese from raw milk is 105cfu × g-1. samples with more elevated counts must be ta b le 4 m ic ro b io lo g ic a l ch a ra ct er is ti cs o f fr es h m a rz o li n a g o a t ch ee se m a d e u si n g a rt is a n a l re n n et p a st e (a r p ) a n d c o m m er ci a l li q u id r en n et ( c l r ). sa m pl e re pl ic at e sa lm on el la s pp li st er ia m on oc yt og en es e. c ol i e. c ol i c oa gu la se p os iti ve en te ro ba ct er ia ce ae su lp hi te re du ci ng to ta l m es op hi lic (in 2 5 g) (in 2 5 g) (c fu × g -1 ) o :1 57 st ap hy lo co cc i (c fu × g -1 ) cl os tr id ia co un t (in 2 5 g) (c fu × g -1 ) (c fu × g -1 ) (c fu × g -1 ) a r p 1 ne ga tiv e ne ga tiv e <1 0 ne ga tiv e <1 0 <1 0 <2 2. 6x 10 7 c lr 1 ne ga tiv e ne ga tiv e <1 0 ne ga tiv e <1 0 <1 0 <2 3. 0x 10 8 ar p 2 ne ga tiv e ne ga tiv e <1 0 ne ga tiv e <1 0 3. 5x 10 3 <2 9. 3x 10 5 c lr 2 ne ga tiv e ne ga tiv e <1 0 ne ga tiv e <1 0 4. 8x 10 3 <2 1.3 x1 06 ar p 3 ne ga tiv e ne ga tiv e 10 0 ne ga tiv e <1 0 6. 2x 10 3 <2 4. 9x 10 6 c lr 3 ne ga tiv e ne ga tiv e <1 0 ne ga tiv e <1 0 <1 0 <2 1.5 x1 05 422 ital. j. food sci., vol. 27 2015 analysed for staphylococci enterotoxins. counts of coagulase-positive staphylococci in all samples were lower than the detection limit of the method (10 cfu × g-1). the count of β-glucuronidasepositive e. coli was 100 cfu × g-1 in only one sample, but lower than the detection limit in other samples (10 cfu× g-1). qualitative analysis for e. coli o157 in our samples gave negative results. sulphite-reducing clostridia is another group of microbial pathogens considered as hygiene markers, but legislation has not established permissible levels for these in food. their levels in all samples were lower than the detection limit of the method (2 cfu × g-1). the mean total mesophilic count in our samples was 5.6 × 107cfu× g-1. microbiological analyses of the cheese samples confirmed the results obtained during the monitoring of some arps collected in the same region of marzolina production (tripaldi et al., 2012). similar results were obtained in three pdo raw ewe milk cheeses from spain, as manchego, idiazabal and zamorano cheese (etayo et al., 2006), where the hygienic quality of cheeses made with lamb rennet paste is comparable to that of cheeses manufactured with non-paste commercial rennet. another study on idiazabal did not detect e. coli, clostridium, salmonella or l. monocytogenes, and levels for other microorganisms were below the limits of the european legislative standards for cheese manufactured with raw milk (gil et al., 2007). the results of our study show that treatment with arps did not favour the growth of microbial pathogens and that arps provided the enzymatic content necessary to achieve the typical characteristics of traditional cheeses. conclusions butyric acid was the main marker of cheeses made with arps because of the high specificity of enzymatic activity of pre-gastric lipase for butyric acid. results of sensory evaluation show that the piquant flavours as well as the odour and flavour of cowshed were more pronounced in cheeses made with arp, confirming the results for other mediterranean cheeses made with rennet paste. therefore, the use of arps provided the enzymatic content necessary to achieve the typical characteristics of traditional cheeses. at the same time, this kind of rennet did not favour the growth of microbial pathogens in cheese. acknowledgements this work was funded by the agenzia regionale per lo sviluppo e l’innovazione dell’agricultura del lazio, italy (arsial) and to the ruggieri farm from itri (lt) for providing the marzolina cheese samples. references addis m., piredda g., pes m., di salvo r., scintu m.f. and pirisi a. 2005. effect of the use of three different lamb paste rennets on lipolysis of the pdo pecorino romano cheese. int. dairy j. 15: 563. addis m., piredda g. and pirisi a. 2008. the use of lamb paste rennet in the traditional sheep cheese production small rum. res. 79: 2. anifantakis e. 1976. influence d’unepresured’agneausur la qualité du fromagekefalotyri. lait, 56: 76. association of official analytical chemists international. 2000. 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london, 207. woo a.h. and lindsay r.c., 1984. concentration of major free fatty acids and flavour development in italian cheese varieties. j. dairy sci. 67, 9. paper received may 29, 2014 accepted december 1, 2014 p u b l i c a t i o n s codon italian journal of food science, 2023; 35 (1): 1–9 issn 1120-1770 online, doi 10.15586/ijfs.v35i1.2264 1 health benefits of co-supplementing mealworm protein hydrolysate and cranberry fruit extract jae hong park1#, sang in lee2#, woo sung kwon2, sungbo cho3*, in ho kim1* 1department of animal resource and science, dankook university, cheonan, republic of korea; 2department of animal biotechnology, kyungpook national university, sangju, republic of korea; 3school of mongolian medicine, inner mongolia university for nationalities, tongliao, people’s republic of china #these authors contributed equally to this work. *corresponding authors: sungbo cho, school of mongolian medicine, inner mongolia university for nationalities, tongliao 028000, inner mongolia autonomous region, people’s republic of china. email: blue0555@hotmail.com; in ho kim, department of animal resource and science, dankook university, cheonan, 31116, republic of korea. email: inhokim@dankook.ac.kr received: 25 july 2022; accepted: 15 november 2022; published: 2 january 2023 © 2023 codon publications open access paper abstract the demand for valuable protein sources is increasing. the mealworm has been highlighted as a good source of protein. nevertheless, beneficial effects of mealworm such as the antioxidative and/or anti-inflammatory effects are rarely studied. it is well-known that cranberry fruit has a strong antioxidant effect. the biologically active compounds in mealworm and cranberry could boost the antioxidative and/or anti-inflammatory effects. the current study investigated the interactive effects of mealworm protein hydrolysate (mwph) and cranberry fruit extract (cfe) in mammals. we evaluated growth performance, relative organ weight, immune responses, antioxidant enzyme activities, blood properties, and fecal microflora. a 2 × 2 factorial experimental design was used. the co-supplementation of mwph and cfe improved serum glutathione peroxidase. mwph affected a lower serum il-1β and fecal clostridium density. the co-supplementation appeared more effective in terms of good health and potentially the prevention of disease. keywords: cranberry; extracts; immunity; inflammation; mealworm; microbiota introduction demand for protein is expected to increase in the future, owing to the increasing global population. recently, insects have been recognized as the best alternatives to meet protein requirements (van huis, 2013). the edible larvae of the common pest insect tenebrio molitor (yellow mealworm; ymw) distributed worldwide are a good source of protein, fat, vitamins, and minerals (kim et al., 2014). ymws contain high-quality protein (shockley and dossey, 2014), and contain more essential amino acids than soybeans (yi et al., 2013). in addition, they have higher unsaturated fatty acid content than meat, and are relatively rich in vitamin a and iron (rumpold and schlüter, 2013). recent studies have reported that mealworms can partially replace soybean and fish meal as a livestock protein feed source, and can improve the growth and productivity of various animals, including chicken and fish (bovera et al., 2015; ido et al., 2019). hence, ymws are extensively used in livestock feed due to the richness of amino acids and proteins (hong et al., 2020). the ymw is also attracting attention as a useful raw material for the production of physiological active peptides. physiologically active peptides are natural antioxidants, generally defined as a peptide with physiological activity and low molecular weight, which helps in their easy absorption into the body (arihara et al., 2001). their bioactive peptide contents and chitin were known to possess antioxidant and antimicrobial properties (di mattia et al., 2019; matheswaran et al., 2019). nevertheless, the antioxidant and antimicrobial 2 italian journal of food science, 2023; 35 (1) park jh et al. korea). the mwph was prepared as follows: 40 kg defatted mealworms were mixed with 360 l water, and the ph was adjusted to 6.5–7.0 using 1 n naoh. next, 0.01% (w/w) formulations of each of alcalase and flavorzyme were added, and the mixture was hydrolyzed at 50°c for 3 h while stirring at 40 rpm. subsequently, the mixture was heated at 85°c for 30 min to deactivate the enzymes, followed by centrifugation at 152 rpm under 130 l/h flow rate conditions. next, microbial cells were removed by following vibrating membrane separation process, after which ultrafiltration was performed to obtain hydrolysate with molecular weight of 100 kda and 30 kda. the final hydrolysate was mixed with malto-dextrin at 1:1 ratio and freeze-dried at −60°c before use (figure 1). the composition of amino acids in unhydrolyzed mealworm and mwph were analyzed. the cfe was prepared in three different extract methods, namely, hot water, methanol and hcl, and ethanol. the water extraction was carried at 80°c water for 3 h and, secondly, cranberry fruit was extracted by 80% methanol and 0.3% hcl for 5 h. the third method is as follows: 5-fold volume of 70% ethanol was added to 100 g cranberry fruit and extracted for 5 h using a reflux condenser. this extract was concentrated under reduced pressure at 40 ± 1°c and freeze-dried before use (figure 1). animals and experimental design a total of 40 mice (balb/c female inbred, 42 days old) with an initial average body weight of 29.4 ± 0.1 g were purchased from central lab. animal inc. (seoul, korea) and fostered in ventilated cages with a 12 h daylight (06:00–18:00) and 12 h dark cycle. the mice were allowed free access to water and food throughout the study. mice were randomly assigned to one of the four experimental treatments (one replication; 10 mice per treatment). the experimental treatments were arranged in a 2 × 2 factorial design with two levels of mwph (0 and 2%) and two levels of cfe (0 and 400 ppm). cfe was added to the basal diet, while mwph substituted 2% of the casein protein in the basal diet. basal diets were prepared based on the american institution of nutrition (ain-93g) guidelines for the nutritional requirements of mice (table 1). sampling and analysis the amino acid compositions of unhydrolyzed mealworm and mwph were quantified in an amino acid analyzer (sykam s-433 d, sykam gmbh, eresing, germany). the amino acid compositions of the samples were compared with effects of ymws were reported in mostly in vitro studies. a few fish feed research demonstrated the antioxidant enzyme activities in rainbow trout (henry et al., 2018) and olive flounder (jeong et al., 2021). there is a limited study regarding the antioxidant effects of ymw dietary supplementation in mammals. recently, ringseis et al. (2021) reported that the 10% ymw supplementation to growing pigs did not show a significant alternation in the antioxidant enzymes catalase (cat), glutathione peroxidase (gpx), and superoxide dismutase (sod) in the liver and of gpx and sod in gastrocnemius muscle. therefore, it is tempting to boost the antioxidant effect of ymw by co-supplementing with natural antioxidants. cranberries are a group of evergreen dwarf shrubs in the family ericaceae, belonging to the subgenus oxycoccus of the genus vaccinium. fresh cranberries, which are red in color and have a sour and sweet taste, are mainly cultivated in the cool climates of northern united states, canada, and chile. these berries have several biological functions that help prevent chronic disease in humans (rupasinghe et al., 2015). cranberries are also rich in phenolic compounds that exert strong antioxidant effects (cho et al., 2012). cranberry fruit extracts (cfe) contain a good source of polyphenols such as proanthocyanidins, flavanols (vvedenskaya and vorsa, 2004), and quercetin (duthie et al., 2006; zheng and wang, 2003), which is attributed to the strong antioxidant properties. in addition, cranberries are rich in anthocyanins, water-soluble red pigments of the flavonoid family that exhibit anticancer, antioxidant, and anti-aging effects (song et al., 2018). so far, the antioxidant and anti-inflammatory effect of cfe in mammals are also limited. therefore, we hypothesized that the combination of ymw and cranberry as a dietary supplement would improve health in mammals. in the present study, we aimed to verify the in vivo efficacy of a diet supplemented with ymw and cfes either alone or in combination. toward this, the effects of feeding mealworm protein hydrolysate (mwph) and cranberry fruit extract (cfe) on mouse growth, organ weight, cox-2 and nf-kb expression, blood parameters, and fecal microflora were investigated. materials and methods ethical endorsement the experimental protocol and procedures used in this study were approved by the institutional animal care and use committee of dankook university (dk-1-2136). preparations of mwph and cfe the mwph and cfe used in this study were purchased from jeonbuk institute for food-bioindustry (jeonju, italian journal of food science, 2023; 35 (1) 3 health benefits of mwph and cfe the supernatants were aspirated and stored at 4°c until further analysis. serum level of pro-inflammatory cytokines (interleukin-6 [il-6], il-1β, and tumor necrosis factor-α [tnf-α]) were measured using a commercial elisa kit (elisa mak; biolegend, san diego, ca) and absorbance was measured at 450 nm using a spectrophotometer. antioxidant enzyme (superoxide dismutase [sod] and glutamic peroxidase [gpx]) activity was analyzed using specific enzyme assay kits (cayman chemical, michigan, usa). following blood sampling, mice were anesthetized in chambers saturated with isoflurane and euthanized by cardiac puncture. kidneys, spleens,  and livers were harvested carefully for further analysis, and organ weight was calculated as a percentage of live weight. dna extraction the qiaamp dna stool kit (qiagen) was used to extract dna from frozen stool samples according to the manufacturer’s instructions. briefly, the procedure involved lysis of the bacterial cells within the fecal material in asl buffer, adsorption of impurities that inhibit ex reagent, and purification of the dna on a spin column. asl buffer was specially developed to remove inhibitory substances from stool samples. the dna was eluted in a final volume of 200 µl and stored at −20°c. the total volume of each amplification reaction mixture was 25 µl, consisting of 1 × taqman universal pcr master mix (applied biosystems), both primers (each at 300 nm concentration), 200 nm taqman mgb probe, 60 ng purified target dna, and bovine serum albumin at a those of standards (sigma aldrich, st. louis, mo, usa). the chemical compounds of cfes from different extract methods were analyzed by hplc using an agilent 1100 hplc system equipped with quaternary pumps, an auto-sampler, and a diode array detector set at 520 nm. the body weight (bw) of each mouse was measured at the beginning and end of the study. body weight gain (bwg), feed intake (fi), and feed efficiency (fe) were assessed at the end of the study. on day 42, all mice were  placed in metabolic cages and fresh fecal samples were collected. on the same day, blood samples were drawn by using edta-treated tubes, stored at 37°c for 2 h, and centrifuged at 3500 rpm for 15 min at 4°c. then, figure 1. the process of cranberry extraction mealworm protein hydrolysate used in the current research. table 1. composition of the basal diets (on as-fed basis). ingredients, g/kg ymw− ymw+ cfe− cfe+ cfe− cfe+ casein 200 200 180 180 sucrose 100 100 100 100 dextrose 132 132 132 132 corn starch 398 398 398 398 cellulose 50 50 50 50 soybean oil 70 70 70 70 mineral mix 35 35 35 35 vitamin mix 10 10 10 10 l-cystine 3 3 3 3 choline bitartrate 2.5 2.5 2.5 2.5 yellow mealworm – – 20 20 cranberry fruit extract – 0.4 – 0.4 total 1000.0 1000.4 1000.0 1000.4 defatted mealworms 40 kg in 360 l water, ph 6.5–7.0 cranberry extraction by heat by acid ultra�ltration filtration concentration freeze dryer by ethanol 70% ethanoldistrilled water 80°, 3 hrs 25°, 5 hrs 80% methanol +0.3% hcl 25°, 5 hrs 80°c, 40 rpm, 30 mins 0.45 um vibrating membrane separation malto-dextrin at 1:1 ratio molecular weight of 30 kda 0.01% alcalase and flavorzyme, 50°c, 40 rpm, 3 hrs enzyme reaction enzyme deactivation microbial cell removal ultra�ltration non-filtrate filtrate freeze dryer 4 italian journal of food science, 2023; 35 (1) park jh et al. were calculated using the 2−δδct method (livak and schmittgen, 2001). statistical analysis all statistical analyses were performed in a 2 × 2 factorial design using anova tests (sas inst. inc., cary, nc, us). statistical significance was set at p < 0.05. results active compounds and amino acids the quantity of amino acids in unhydrolyzed mealworm and mwph were estimated. both did not contain threonine, histidine, isoleucine, tryptophan, and taurine. the mwph had higher quantity of amino acids compared to unhydrolyzed mealworm (table 4). the quantity of active compounds from cranberry fruits was compared based on the three different extract methods. the ethanol methods showed the highest quantity of active compounds; thus, cfe from ethanol was used for the animal trial. the cfe from ethanol methods final concentration of 0.1 µg/µl (new england biolabs). amplification (2 min at 50°c, 10 min at 95°c, followed by 45 cycles of 15 s at 95°c and 1 min at 60°c) and detection were carried out on an abi prism 7900 sequence detection system (applied biosystems). total bacteria were used as an endogenous control to normalize target gene expression (table 2). quantitative real-time (qrt)-pcr to measure the expression of cox-2 and nf-kb, mice were euthanized at the end of the experiment. kidney samples were frozen in liquid nitrogen and stored at −80°c. total rna was isolated from the kidneys using trizol (invitrogen, carlsbad, ca, usa). for qrt-pcr, total rna (1 µg) was used as a template for cdna synthesis performed using the maxima first-strand cdna synthesis kit (life technologies, carlsbad, ca, usa). sequence-specific primers (table 3) were designed using primer3 (http://frodo.wi.mit.edu/), and the reaction was performed on the cfx real-time pcr system (bio-rad, hercules, ca, usa) under the following conditions: 94°c for 3 min, followed by 40 cycles of 94°c for 30 s, 59–61°c for 30 s, and 72°c for 30 s. target gene expression levels were normalized to that of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase and table 4. the quantity of amino acid from unhydrolyzed mealworm and mealworm protein hydrolysate (mwph) (mg/g dry weight). amino acids unhydrolyzed mealworm mwph essential threonine 0.00 0.00 methionine 7.49 ± 1.26 13.6 ± 1.74 lysine 20.5 ± 1.96 34.6 ± 2.68 histidine 0.00 0.00 isoleucine 0.00 0.00 leucine 0.24 ± 0.06 0.93 ± 0.07 phenylalanine 0.16 ± 0.05 0.54 ± 0.06 tryptophan 0.00 0.00 valine 0.17 ± 0.06 0.57 ± 0.05 nonessential arginine 0.23 ± 0.04 0.63 ± 0.06 taurine 0.00 0.00 the values represent mean ± sem, n = 5. table 2. group-specific primers based on 16s rdna sequences used in this study. target bacteria sequences (5′ – 3′) total bacteria f actcctacgggaggcag r gtattaccgcggctgctg bifidobacterium f cgcgtcyggtgtgaaag r ccccacatccagcatcca lactobacillus f gaggcagcagtagggaatcttc r ggccagttactacctctatccttcttc clostridium f gggggtttcaacacctcc r gcaagggatgtcaagtgt firmicutes f ggagyatgtggtttaattcgaagca r agctgacgacaaccatgcac bacteroidetes f gaaggtcccccacattg r cgckacttggctggttcag table 3. list and sequences of primers used in this study. gene symbol description accession no. forward primer (5′ -> 3′) reverse primer (5′ -> 3′) cox-2 cyclooxygenase-2 nm011198 ctcacgaagg aactcagcac ggattggaac agcaaggatt nf-kb1 nuclear factor of kappa light polypeptide gene enhancer in b cells 1 nm008689 gtctgcctctc tcgtcttcc cagtgggctg tctccagtaa gapdh glyceraldehyde-3-phosphate dehydrogenase nm001289726 cgagacccca ctaacatcaa ggttcacacc catcacaaac italian journal of food science, 2023; 35 (1) 5 health benefits of mwph and cfe was no significant interactive effect of mwph plus cfe supplementation on the expression of cox-2 and nf-kb (table 8). serum cytokine and antioxidant enzyme levels serum il-1β concentration was lower in mice fed the diet containing mwph than in those fed diets lacking mwph (p < 0.05). the gpx concentration in mice fed diets containing mwph or cfe was higher than that in mice fed diets lacking mwph or cfe (p < 0.05). the concentrations of il-6, tnf-α, and sod were not influenced by mwph or cfe supplementation. in addition, there was not an interaction between mwph and cfe supplementation on serum levels of cytokines and antioxidant enzymes (p < 0.05; table 9). fecal bacteria there were no differences in bacterial counts of bifidobacterium, lactobacillus, bacteroidetes, and firmicutes in mice fed diets containing mwph or cfe, but clostridium counts decreased with the supplementation of mwph (p < 0.05). there was no significant interactive effect of mwph and cfe supplementation on fecal bacteria (table 10). contained 80.6 and 2.917 µg/ml of total anthocyanin and proanthocyanin, respectively (table 5). weight gain, feed intake, and feed efficiency after 6 weeks, neither mwphnor cfe-supplemented diets showed a significant difference in bwg, fi, or fe compared to the control. in addition, there was no significant interactive effect of mwph and cfe supplementation on bwg, fi, and fe (table 6). organ size there were no differences in the relative weights of the kidney and liver between mice fed diets with mwph or cfe and those fed unmodified diets. however, the relative weight of the spleen increased with supplementation of mwph (p < 0.05). no interactive effects were observed of mwph and cfe supplementation on the relative organ weights (table 7). cox-2 and nf-kb expression the expression of cox-2 and nf-kb was not influenced by supplementation with mwph or cfe. also, there table 5. chemical compounds of cranberry fruit extracts from different extract methods. compounds unit hot water acid ethanol cyanidin 3-gluciside chloride mg/g 0.000 0.125 ± 0.08 0.000 cyanidin 3-galactoside chloride mg/g 0.226 ± 0.08 0.692 ± 0.12 0.737 ± 0.17 cyanidin chloride mg/g 0.022 ± 0.001 0.030 ± 0.001 0.029 ± 0.001 proanthocyanin µg/ml 0.000 0.000 2.917 ± 0.07 total anthocyanin µg/ml 0.000 0.000 80.56 ± 3.45 total polyphenol µg/ml 5.658 ± 0.35 9.693 ± 0.42 13.14 ± 0.49 the values represent mean ± sem (dry weight), n = 6. table 6. effects of the mwph and cfe supplementation on growth performance in mice1. items mwph − mwph + sem mwph cfe p-value cfe− cfe+ cfe− cfe+ − + − + mwph cfe mwph × cfe initial bw, g 29.3 29.5 29.5 29.3 0.45 29.4 29.4 29.4 29.4 0.888 0.986 0.847 finial bw, g 32.6 33.7 32.3 32.1 0.60 33.2 32.2 32.5 33.0 0.524 0.767 0.634 bwg, g 3.30 4.20 2.80 2.80 0.29 3.80 2.80 3.10 3.50 0.062 0.467 0.464 fi2, g 113 123 126 132 2.63 118 129 120 128 0.031 0.098 0.669 fe3 0.029 0.034 0.022 0.021 0.004 0.032 0.020 0.026 0.028 0.066 0.752 0.505 mwph = mealworm protein hydrolysate, cfe = cranberry fruit extract, + or – = supplemented with or without 2% mwph and 400 ppm cfe, respectively. bwg, body weight gain; fi, feed intake; fe, feed efficiency. 6 italian journal of food science, 2023; 35 (1) park jh et al. table 10. effects of the mwph and cfe supplementation on fecal microbiota in mice1. items mwph − mwph + sem mwph cfe p-value cfe− cfe+ cfe− cfe+ − + − + mwph cfe mwph × cfe bifidobacterium spp., log10 9.12 9.31 9.24 9.32 0.15 9.22 9.28 9.18 9.32 0.328 0.945 0.537 lactobacillus spp., log10 8.25 8.48 7.79 8.20 0.13 8.36 8.00 8.02 8.35 0.205 0.258 0.749 clostridium spp., log10 9.79 9.96 8.36 9.03 0.22 9.88 8.70 9.08 9.50 0.003 0.187 0.414 bacteroidetes, % 48.4 49.4 49.5 49.2 0.32 48.9 49.4 49.0 49.3 0.539 0.664 0.381 firmicutes, % 51.6 50.6 50.5 50.8 0.32 51.1 50.6 51.0 50.7 0.539 0.664 0.381 mwph = mealworm protein hydrolysate, cfe = cranberry fruit extract, + or – = supplemented with or without 2% mwph and 400 ppm cfe, respectively. table 7. effects of the mwph and cfe supplementation on relative organ weight in mice1. items mwph − mwph + sem mwph cfe p-value cfe− cfe+ cfe− cfe+ − + − + mwph cfe mwph × cfe kidney (r), % 0.52 0.54 0.59 0.55 0.009 0.53 0.57 0.55 0.55 0.127 0.812 0.115 kidney (l), % 0.53 0.53 0.61 0.55 0.01 0.53 0.58 0.57 0.54 0.156 0.382 0.266 spleen, % 0.25 0.24 0.27 0.32 0.01 0.25 0.30 0.26 0.28 0.015 0.461 0.109 liver, % 4.09 4.26 4.35 4.24 0.06 4.18 4.29 4.22 4.25 0.373 0.787 0.292 mwph = mealworm protein hydrolysate, cfe = cranberry fruit extract, + or – = supplemented with or without 2% mwph and 400 ppm cfe, respectively. table 9. effects of the mwph and cfe supplementation on serum profiles of mice1. items mwph − mwph + sem mwph cfe p-value cfe− cfe+ cfe− cfe+ − + − + mwph cfe mwph × cfe il-6, pg/ml 130.4 75.73 50.82 46.30 30.3 103.1 48.60 90.6 61.0 0.418 0.657 0.706 il-1β, pg/ml 54.94 65.46 33.67 28.60 4.95 60.20 31.14 44.30 47.03 0.001 0.697 0.276 tnf-α, pg/ml 12.86 9.05 10.46 10.02 0.76 10.95 10.23 11.65 9.53 0.643 0.184 0.286 sod, u/ml 4.50 7.27 8.36 7.62 0.78 5.89 7.99 6.43 7.44 0.199 0.525 0.277 gpx, u/ml 498.9 612.5 936.2 1382 98.1 555.7 1159 717.6 997.4 0.001 0.012 0.103 mwph = mealworm protein hydrolysate, cfe = cranberry fruit extract, and + or – = supplemented with or without 2% mwph and 400 ppm cfe, respectively. il-6, interleukin-6; il-1β, interleukin-1β; tnf-α, tumor necrosis factor-α, sod, superoxide dismutase; gpx, glutathione peroxidase. table 8. effects of the mwph and cfe supplementation on cox2 and nf-kb expression in mice1. items mwph − mwph + sem mwph cfe p-value cfe− cfe+ cfe− cfe+ − + − + mwph cfe mwph × cfe cox-2 1.00 0.88 1.19 1.16 0.13 0.94 1.18 1.10 1.02 0.426 0.803 0.873 nf-κb 1.00 0.60 0.65 0.64 0.11 0.80 0.65 0.83 0.62 0.553 0.422 0.451 mwph = mealworm protein hydrolysate, cfe = cranberry fruit extract, + or – = supplemented with or without 2% mwph and 400 ppm cfe, respectively. cox-2, cyclooxygenase-2; nf-κb, nuclear factor kappa b. italian journal of food science, 2023; 35 (1) 7 health benefits of mwph and cfe amino acids with a low molecular weight that makes it easy to absorb them into the body (kim et al., 2013). previous studies have reported that protein hydrolysate from mealworm larvae (yu et al., 2017) and protaetia brevitarsis larvae (lee et al., 2017) increased antioxidant activity. therefore, in the present study, the increase in gpx activity in mouse blood can be considered as the effect of physiologically active peptides from mwph. cranberries exhibit high antioxidant activity, which is closely related to flavonoid pigments such as anthocyanins, flavonols, and proanthocyanidins (brown and shipley, 2011; feghali et al., 2012). the antioxidant capacity of proanthocyanidins is reportedly reported to be stronger than that of vitamins c and e and catechins (ariga, 2004). in this study, gpx levels were higher in mice fed on cfe, which is likely due to the abundant polyphenolic compounds in cfe. furthermore, cfe can contribute to gut microbiota regulation. the positive effects of polyphenols derived from many plant sources on the gut microbiota have already been investigated (anhê et al., 2017; heyman-lindén et al., 2016; roopchand et al., 2015). however, in our study, cfe did not affect fecal microbiota in mice fed on cfesupplemented diet when compared with mice fed on nonsupplemented diet. in this study, the number of clostridium bacteria was significantly lower in the mice fed on mwph supplemented diet, showing that mwph supplementation inhibits the growth of harmful microorganisms in the intestine. the outer shell of mealworm larvae is mainly composed of brown mealworm larvae that develop an exoskeleton during metamorphosis into pupae. it has been reported that chitin can be used as a source of dietary fiber that provides a favorable environment for the growth of beneficial bacteria in the gastrointestinal tract (hamed et al., 2016; ringø et al., 2012). we therefore expect that chitin contained in mwph may have influenced our results. conclusions combination of mwph and cfe could be advantageous for boosting anti-inflammation via regulating cytokine activation. the combination significantly reduced the expression of il-1 as compared to individual supplements. besides, mwph-supplemented diet improved the immune function and reduced harmful gut bacteria in mice. the effect of cfe supplementation on mice increased levels of antioxidant enzymes in serum. this study provides preliminary data for the further evaluation of dietary supplementation with mwph and cfe. in future studies, it is necessary to investigate the relative effects of these two additives at various feed levels. discussion the study was carried out to investigate the healthy benefits of co-supplementing mwph and cfe in mice. organ weight can be an important indicator of pathology, and measurements of the liver, kidneys, and spleen are recognized as indicators of toxicity and immunity in vivo. since there was no difference in the liver and kidney weights of mice in this study, our data suggest that neither mwph nor cfe had any harmful effects in the mice. however, mwph supplementation increased spleen weight in this study. it is generally known that immunopotentiators increase the weight of the thymus and spleen (chen et al., 2012). since the spleen is largely composed of immune cells, it can be inferred that mwph supplementation increases spleen immune cell. the protein complex nf-κb is involved in inflammatory response regulation, immune modulation, apoptosis, cell proliferation, and epithelial differentiation among other processes. it regulates the expression of various genes and forms the central axis of the intracellular signaling system. the cytokines tnf-α, il-1, il-6, and the inducible enzyme cox-2 are regulated by nf-κb (liu et al., 2017). in a previous in vitro study based on lipopolysaccharide-activated macrophages, mealworm larval protein inhibited the expression of inos and cox-2 proteins in a concentration-dependent manner (seo et al., 2019). however, this effect was not observed in the mice in our study. cytokines are relatively small molecular weight immune proteins that play an important role in cell signaling (arango duque and descoteaux, 2014). cytokines involved in inflammatory processes are classified as either inflammatory or anti-inflammatory. inflammatory cytokines, such as il-1β, il-6, and tnf-α, are involved in immune-related disorders, such as rheumatoid arthritis, atopic dermatitis, and asthma (girodet et al., 2016; sato et al., 1999; turner et al., 2014). anti-inflammatory cytokines such as il-10 and tgf-β protect against these diseases by performing functions such as the induction of b cells proliferation and tissue repair (girodet et al., 2016). in this study, il-1β levels were significantly lower in the mwph supplementation group. these results suggest that mwph exerts anti-inflammatory effects via regulating cytokine expression and may help suppress the conversion to chronic inflammation due to enhanced immune function. antioxidants are known to slow aging by scavenging cellular oxidants and active radicals in the body (kim et al., 2003). several physiologically active peptides act as natural antioxidants and function in hypertension relief, immune regulation, pain relief, and antibacterial action (xing et al., 2021). they are usually composed of 3–20 8 italian journal of food science, 2023; 35 (1) park jh et al. macrophage activation is increased in asthma. american journal of respiratory cell and molecular biology 55(4): 467–475. https://doi.org/10.1165/rcmb.2015-0295oc hamed, i., özogul, f. and regenstein, j.m., 2016. industrial 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https://doi.org/10.1006/ bbrc.1999.1455 seo, m., lee, h.j., lee, j.h., baek, m., kim, i.-w., kim, s.y., hwang,  j.-s. and kim, m., 2019. a study of the anti-inflammatory effect of protein derived from tenebrio molitor larvae. journal of life science 29(8): 854–860. shockley, m. and dossey, a.t., 2014. insects for human consumption. in: mass production of beneficial organisms. elsevier, pp. 617–652. #500_martini_bozza ital. j. food sci., vol 29, 2017 138 paper influence of fat content on quality of cow's milk m. martini*a, i. altomonteb, a. bortoluzzi moroc, c. caneppeled and f. salarib adipartimento di scienze veterinarie, università di pisa, viale delle piagge, 2, pisa (italy), centro interdipartimentale di ricerca nutraceutica e alimentazione per la salute, via del borghetto 80, 56124 pisa, italy bcentro interdipartimentale di ricerche agro-ambientali enrico avanzi, università di pisa, via vecchia di marina 6, 56122 s. piero a grado, pisa, italy ccentro de ciências rurais, departamento de zootecnia, universidade federal de santa maria, bairro camobi, campus universitário, camobi 97105900, santa maria, rs, brasil programa ciência sem fronteiras nº processo: csf-2064/12-0 duniversidade luterana do brasil, campus canoas av. farroupilha, nº 8001. bairro são josé 92425-900 canoas/rs-brasil. programa ciências sem fronteiras n° processo: csf 206948/2012 *corresponding author. mina.martini@unipi.it abstract the aim of the study was to verify whether changes in the percentage of fat in highly selected cows produce variations in the physical structure of the fat and changes in milk composition. individual milk was sampled from 50 cows. fat was evaluated in each individual in order to create two groups of animals with lower and higher percentage. the group with higher fat content showed a significantly larger diameter of the fat globules, less c14:0 and more c16:1. in conclusion the diameter variations observed result in few changes in milk fatty acid composition, thus maintaining a consistent nutritional quality. keywords: cow, fatty acids, milk fat globules, milk quality ital. j. food sci., vol 29, 2017 139 1. introduction the nutraceutical composition of foods consumed daily is becoming increasingly important. particular focus has been on consumers belonging to all age groups especially with regard their food lipid intake, since lipids have been implicated in several diseases such as obesity, insulin resistance and atherosclerosis (olofsson et al., 2009). for these reasons, the number of studies on the physical and chemical structure of fat in several edible products of animal origin have increased (le et al., 2014, martini et al., 2016) cow's milk is consumed on a daily basis from childhood, thus it is important to know and monitor the milk lipid changes that occur naturally, or are induced by different farming techniques. it is thus necessary for consumers to drink throughout their life a guaranteed product from a nutritional and nutraceutical point of view. for the dairy industry it is important to assess how changes in the morphometry of the milk fat globules (mfgs) lead to changes in yields, ripening and the nutritional quality of cheeses (martini et al., 2016). milk lipids are spherical structures and research has shown that the differences related to the diameter and number of mfgs are of interest with regard to the chemical-physical and nutritional properties of milk and lipid digestion and absorption (martini et al., 2013). the secretion of milk fat globule is not yet completely understood. furthermore it is known that their size and distribution vary as a function of factors such as species, breed, parity, stage of lactation and amount of fat secreted (martini et al., 2016). some authors (wiking et al., 2004 and caroll et al., 2006) have found positive relationships between daily fat yield and the average diameter of the mfgs. these findings are probably due to the fact that part of the membrane of the mammary epithelial cell is sacrificed to envelop the globule, then larger mfgs may be secreted to reduce the amount of membrane lost by the cell (argov et al., 2008). some authors have hypothesized also that relationships between fat secretion and mfg size are due to the effects on the mammary metabolism of the energy balance and the availability of nutrients (martini et al., 2013). moreover, the effects of energy balance on the metabolism of the mammary gland are not yet fully known (german, 2011). the purpose of this study is to check whether small changes in fat secretion in highly selected cows, which have been reared in the same conditions, would result in variations in the physical structure of the fat and in changes in the milk composition. 2. materials and methods 2.1. animals and sampling milk from the morning milking of 50 pluriparous friesian cows (151 ± 25 days in milk) was sampled. the cows were reared intensively in a single dairy farm in tuscany (central italy). all the cows were fed with the same diet consisting in a mixed ration formulated according to nrc (2001) requirements for dairy cattle and made up of corn silage, corn meal, alfalfa hay, hay ryegrass, soybean meal 44% pg, integral cotton seed, cane molasses, and commercial complementary feed. all individual milk samples were taken during the same day by in-line milk meters (de laval) and were refrigerated at 4°c before being taken to the laboratory for analysis. 2.2. milk analysis all the fresh samples were analysed in duplicate for total solids (ts), fat, protein, casein content according to aoac methods (2004). fat extraction was performed using hexane and ethanol, according to rose-gottlieb’s method modified by secchiari et al. (2003). ital. j. food sci., vol 29, 2017 140 methyl esters of fatty acids (fame) were obtained after transesterification with sodium methoxide according to christie (1982). the composition of the milk fatty acids was determined by gas chromatography using a perkin elmer auto system (perkin elmer, norwolk, ct, usa) equipped with a flame ionization detector and a capillary column (30 m × 0.25 mm; film thickness 0.25 mm; factorfour varian, middelburg, the netherlands). the helium carrier gas flow rate was 1 ml·min–1. the oven temperature program was as follows: level 1, 50°c held for 2 min, level 2, 50 to 180°c at 2°c·min–1 then held for 20 min, level 3, 180 to 200°c at 1°c·min–1 then held for 15 min, and finally level 4, 200 to 220°c at 1°c·min–1 then held for 30 min. the injector and detector temperatures were set at 270 and 300°c, respectively. individual fas were identified by comparing their retention times with those of the following authenticated standards: fim_fame mix (restek corporation, 110 benner circle, bellefonte, pa 16823) and methyl 9(z), 11(e)-octadecadienoate (matreya llc international dealers & representatives-superchrom s.r.l., via c. menotti 11, milan, 20129 ) in addition, nonadecanoic acid methyl ester (c19:0 restek corporation, 110 benner circle, bellefonte, pa 16823) was used as internal standard to calculate the recovery of the fatty acid methyl esters. the desaturase index was calculated for: cis-9 14:1/14:0, cis-9 16:1/16:0, cis-9 18:1/18:0, following kelsey et al. (2003). 2.3. morphometric analysis of milk fat globules a direct method (martini et al., 2013) was used to determine the diameter (μm) and the number of fat globules per ml of milk in each fresh sample by a fluorescence microscope (leica ortomat microsystem, milan, italy) equipped with a camera (tiesselab, milan, italy) and ts view 2.0 image software. 2.4. statistical analysis the frequency distribution of the total counted and measured mfgs was evaluated and globules were grouped into three size-categories: small globules (sg) with a < 2µm diameter, medium-sized globules (mg) with a diameter from 2 to 5 µm, and large globules (lg) with a > 5 µm diameter. the variability in the fat secreted from each of the 50 cows was evaluated in order to create two groups of animals (groups a and b) secreting lower fat percentages (less that 32 g kg-1, made up of 16 individuals) and higher (more than 37 g kg-1, made up of 12 individuals) respectively, compared to the average value (34 g kg-1). individuals whose fat percentage was between 32 and 37 g kg-1 were excluded. the results were analyzed by anova using jmp software (2002) considering the group (a or b) as the fixed factor. least significance means were compared by t-test. significant differences were considered at p< 0.05. 3. results milk yield, chemical and physical characteristics are reported in table 1. milk yield in the two groups of animals was similar. the higher fat content of group b corresponded to a significantly larger average diameter of globules and to increases in the percentages of the large globules. in group b, 22% of the total number of globules was made up of large globules, whereas in group a, the percentage of large globules was only 12%. this confirms the hypothesis of other authors (martini et al., 2016) who have linked the positive relationship between the fat secreted and the average diameter of the ital. j. food sci., vol 29, 2017 141 mfgs with a balance of the mammary gland cell. in fact, as the secretion of fat is an apocrine mechanism, it involves losses in the membrane, which is sacrificed to envelop the globules. larger mfgs may therefore be secreted to reduce the amount of membrane lost by the cell per unit volume of fat. table 1. milk yield, quality and characteristics of the fat globules in the milk from two groups of cows. group a (fat<32 g kg-1) (n‡=16) group b (fat >37 g kg-1) (n‡=12) average daily milk yield (kg-1) † 29.64±1.65 28.58±1.92 fat (g kg-1) 27.9±0.50** 40.5±0.60** proteins (g kg-1) 30.50±1.00 31.70±1.1 casein (g kg-1) 24.34±0.996 25.08±1.068 casein/fat ratio 0.90±0.039** 0.62±0.045** protein/fat ratio 1.13±0.045** 0.81±0.052** dry matter (g kg-1) 134.4±5.30 133.9±6.20 ash (g kg-1) 6.9±0.10 6.9±0.10 number of milk fat globules (n/ml) 11.82x109±9.53 8.48x109±1.10 milk fat diameter (μm) 2.90±0.18* 3.50±0.21* sg § (%) 38.15±3.35 30.45±3.89 mg ¶ (%) 49.96±3.44 47.54±4.00 lg ††(%) 11.89±3.13* 21.98±3.64* * p<0.05; ** p <0.01 †calculated as kg of milk produced until the day of sampling /days in milk at the day of sampling. ‡ n= number of cows; §sg=small globules with a diameter <2 μm.; ¶ mg=medium globules with a diameter between 2 and 5 μm; ††lg=large globules with a diameter >5 μm. in addition, as previously observed in sheep (martini et al., 2012), subjects with a smaller average diameter (group a) showed a higher number of globules/ml of milk (+39%). from the point of view of the milk quality, total nitrogen did not differ significantly in the two groups of cows, however lower casein/fat and protein/fat ratios were observed in group b. this may be a limiting factor, especially for cheese-making since protein/fat ratio is a critical characteristics for the cheese yield and for the recoveries of fat and water in the cheese (guinee et al., 2007). moreover, some authors have suggested that there is a link between milk protein synthesis and fat (heid and keenan, 2005). cebo et al. (2012) have also reported that genetic polymorphism at the αs1-casein (csn1s1) affects both the structure and composition of the mfgs. on the other hand, the milk of group a showed a lower average diameter of mfgs (p <0.05). it has been observed that milk with smaller sized globules increases cheese yield in emmental production (michalski et al., 2004), and that in sheep a greater diameter of the mfg leads to a worsening of the rheological parameters, due to negative relations with the content of casein and the casein:fat ratio (martini et al., 2016). studies on the variability in the average mfg diameter in friesian cows have shown contrasting results in terms of the relations between diameter and the milk yield and quality. some authors have reported positive correlations between the diameter and the amount of fat (briard-bion et al., 2008), whereas others have not observed any relationship (logan et al., 2014). ital. j. food sci., vol 29, 2017 142 in addition, the diameter of mfgs has been reported to affect milk fatty acid composition (lopez et al., 2011). in our study, the changes in the fatty acid profile (table 2) were limited and concern an increase (p <0.05) in myristic acid in group a (+11% g kg-1 of the total fatty acids), and an increase (p <0.05) in palmitoleic acid in group b (+19% g kg-1 of the total fatty acids). this study highlights that, although statistically significant, small changes (0.6 microns) in the diameter of the milk fat globules in homogeneous animals only have a slight effect on the milk fatty acid profile. table 2. fatty acid composition (g kg-1of total fatty acids), classes of milk fatty acids (%) and desaturase indexes in milk from two groups of cows. group a (fat <32.00 g kg-1) (n†=16) g kg-1 of the total fatty acids group a (fat <32.00 g kg-1) (n†=16) g kg-1 of milk group b (fat>37.00 g kg-1 (n†=12) g kg-1 of the total fatty acids group b (fat>37.00 g kg-1 (n†=12) g kg-1 of milk c4:0 25.88±1.04 0.72±0.029 25.74±0.86 1.04±0.035 c6:0 20.49±0.71 0.57±0.020 19.43±0.85 0.79±0.034 c8:0 12.98±0.48 0.36±0.013 12.2±0.67 0.49±0.027 c10:0 31.07±1.30 0.87±0.036 28.34±0.16 1.15±0.006 c11:0 2.88±1.44 0.08±0.040 2.75±0.17 0.11±0.007 c12:0 36.62±1.45 1.02±0.040 33.34±1.76 1.35±0.071 c13:0 1.36±0.06 0.04±0.002 1.32±0.07 0.05±0.003 c14:0 125.31±3.65* 3.50±0.101* 113.25 ±4.41* 4.59±0.179* c14:1 13.67±0.40 0.38±0.010 13.19±0.49 0.53±0.020 c15:0 13.59±0.42 0.38±0.012 13.28±0. 51 0.54±0.021 c15:1 3.06±0.15 0.09±0.004 3.26±0.18 0.13±0.007 c16:0 346.77±7.75 9.67±0.216 344.19±9.36 13.94±0.379 c16:1 13.13±0.68* 0.37±0.019* 15.58±0.83* 0.63±0.034* c17:0 7.81±0.26 0.22±0.007 8.00±0.31 0.32±0.013 c17:1 2.88±0.23 0.08±0.006 3.17±0.28 0.13±0.011 c18:0 89.55±3.65 2.50±0.102 91.84±4.40 3.72±0.178 c18:1 trans-9 5.52±0. 27 0.15±0.007 5.52±0.33 0.22±0.013 c18:1 trans-11 6.90±0.28 0.19±0.008 7.05±0.34 0.29±0.014 c18:1 cis-9 192.89±9.21 5.38±0.257 203.38±11.12 8.24±0.450 c18:2 trans-9,12 2.31±0. 08 0.06±0.002 2.19±0.10 0.09±0.004 c18:2 cis-9,12 35.64±1.90 0.99±0.053 33.53±2.29 1.36±0.093 c18:3n3 3.52±0.15 0.10±0.004 3.60±0.18 0.15±0.007 c18:3 n6 13.06 ±0.11 0.36±0.003 12.03±0.13 0.49±0.005 c20:0 1.17±0.05 0.03±0.001 1.23±0.01 0.05±0.001 cla cis-9, trans11 5.34±0.11 0.15±0.003 5.17±0.13 0.21±0.005 c20:1 0.57±0.05 0.02±0.001 0.56±0.06 0.02±0.002 c21:0 0.77±0.01 0.02±0.001 0.86±0.01 0.03±0.001 c20:2 0.22±0.03 0.006±0.001 0.17±0.03 0.01±0.001 c20:3n6 1.17±0. 06 0.03±0.002 1.17±0.07 0.05±0.003 c20:4 0.11±0.04 0.003±0.001 0.14±0.05 0.01±0.002 c20:3 n3 0.25±0.03 0.007±0.001 0.21±0.03 0.01±0.001 c22:0 0.73±0.06 0.02±0.002 0.70±0.07 0.03±0.003 c22:1 1.61±0.11 0.04±0.003 1.42±0.13 0.06±0.005 ital. j. food sci., vol 29, 2017 143 c20:5 0.24±0.03 0.007±0.001 0.22±0.04 0.01±0.002 c23:0 0.34±0.02 0.01±0.001 0.33±0.03 0.01±0.001 c22:2 0.34±0.03 0.01±0.001 0.42±0.04 0.02±0.002 c24:0 0.43±0.03 0.01±0.001 0.44±0.04 0.02±0.002 c24:1 0.19±0.06 0.005±0.002 0.30±0.06 0.01±0.002 c22:5 0.77±0.04 0.02±0.001 0.74±0.05 0.03±0.002 c22:6 0.94±0.21 0.03±0.006 0.55±0.25 0.02±0.010 group a (fat <32.00 g kg-1) (n†=16) % of the total fatty acids group a (fat <32.00 g kg-1) (n†=16) g kg-1 of milk group b (fat>37.00 g kg-1 (n†=12) % of the total fatty acids group b (fat>37.00 g kg1 (n†=12) g kg-1 of milk short chain fa‡ (≤c10) 9.04±0.28 2.52±0.078 8.57±0.34 3.47±0.014 medium chain fa (≥c11≤c17) 55.66±1.14 15.53±0.032 55.13±1.37 22.33±0.055 long chain fa (≥c18) 35.29±1.28 9.85±0.036 36.30±1.54 14.70±0.062 saturated fa 70.73±1.11 19.73±0.031 69.72±1.34 28.24±0.054 monounsaturated fa 24.04±1.07 6.71±0.030 25.34±1.21 10.26±0.049 polyunsaturated fa 5.18±0.22 1.45±0.006 4.89±0.49 1.98±0.020 group a (fat <32.00 g kg-1) (n†=16) group b (fat>37.00 g kg-1 (n†=12) unsaturated /saturated fa ratio§ 0.42±0.03 0.44±0.03 n3/n6 ratio¶ 0.06±0.005 0.07±0.006 desaturase c14 index†† 0.10±0.003 0.11±0.004 desaturase c16 index†† 0.04±0.002 0.04±0.003 desaturase c18 index†† 0.32±0.01 0.31±0.01 * p<0.05 †n= number of cows; ‡ fa: fatty acids; § unsaturated /saturated fa ratio: :∑unsaturated fatty acids/:∑saturated fatty acids; ¶n3/n6 ratio:∑n3 fatty acids /∑n6 fatty acids; †† desaturase c14 index: [c14:1]/[c14:1+c14:0].; desaturase c16 index: [c16:1]/[c16:1+c16:0]; desaturase c18 index: [c18:1c9]/[c18:1cis 9+c18:0] 4. conclusions in high-selected dairy breeds significant variations in fat secreted influence the morphometry of the fat globules, casein:fat and protein:fat ratios. however, the range of variation observed in the diameter does not lead to significant changes in the fatty acid composition. thus maintaining a consistent nutritional quality of bovine dairy milk. references aoac. 2004. “official methods of analysis” 20th ed. association of official analytical chemists. gaithersburg. md. argov n., lemay d.g. and german j.b. 2008. milk fat globule structure and function: nanoscience comes to milk production. trends food sci. technol. 19:617. ital. j. food sci., vol 29, 2017 144 carroll s.m., depeters e.j., taylor s.j., rosenberg m., perez-monti h. and capps v.a. 2006. milk composition of holstein, jersey, and brown swiss cows in response to increasing levels of dietary fat. anim. feed sci. technol. 131:451. cebo c., lopez c., henry c., beauvallet c., ménard o., bevilacqua c., bouvier f., caillat h. and martin p. 2012. goat αs1-casein genotype affects milk fat globule physicochemical properties and the composition of the milk fat globule membrane. j. dairy sci. 95:6215. christie w.w. 1982. a simple procedure for rapid transmethylation of glycerolipids and cholesteryl esters. j. lipid res. 23:1072. german j.b. 2011. dietary lipids from an evolutionary perspective: sources, structures and functions. matern. child nutr. 7:2. guinee t.p., mulholland e.o., kelly j. and callaghan d.j.o. 2007. effect of protein-to-fat ratio of milk on the composition, manufacturing efficiency, and yield of cheddar cheese. j. dairy sci. 90:110. heid h.w. and keenan t.w. 2005. intracellular origin and secretion of milk fat globules. eur. j. cell biol. 84:245. jmp. 2002. user’s guide. version 5.0. sas. inst. inc.. cary. nc. usa. kelsey j.a., corl b.a., collier r.j. and bauman d.e. 2003. the effect of breed. parity. and stage of lactation on conjugated linoleic acid (cla) in milk fat from dairy cows. j. dairy sci. 86:2588. le t.t., van camp j., dewettinck k., 2014. milk fat globule membrane material: isolation techniques, health-beneficial properties, and potential applications, in: atta-ur-rahman, editor(s), studies in natural products chemistry, elsevier, 2014, 41:347. lopez c., briard-bion v., ménard o., beaucher e., rousseau f., fauquant j., leconte n. and robert b. 2011. fat globules selected from whole milk according to their size: different compositions and structure of the biomembrane, revealing sphingomyelin-rich domains. food chemistry. 125:355. martini m., salari f. and altomonte i. 2016. the macrostructure of milk lipids: the fat globules. crit. rev. food sci. nutr. 56:1209. martini m., altomonte i. and 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1791:448. secchiari p., antongiovanni m., mele m., serra a., boccioni a., ferruzzi g., paletti f. and petacchi, f. 2003. effect of kind of dietary fat on quality of milk fat from italian friesian cows. livest. prod. sci. 83:43. wiking l., stagsted j., björck l. and nielsen j.h. 2004. milk fat globule size is affected by fat production in dairy cows. int. dairy j. 14:909. paper received april 22, 2016 accepted july 28, 2016 ijfs#38_peano_bozza   ital. j. food sci., vol 28, 2016 376 paper quality indicators for modified atmosphere packaging (map) storage of high-quality european plum (prunus domestica l.) cultivars n.r. giuggiolia, f. sottileb and c. peanoa* adepartment of agricultural, forest and food sciences (disafa), università degli studi di torino, largo paolo braccini 2, 10095 grugliasco, to, italy bdepartment of agricultural and forest sciences, università degli studi di palermo, viale delle scienze 11, 90128 palermo, pa, italy *corresponding author. tel.: +39 0116708646; fax: +39 0116708658 e-mail address: cristiana.peano@unito.it abstract the use of quality indicators is crucial in selling plums in more distant markets and the evaluation of freshness through multiple index is fundamental to evaluate the goodness of the storage technique. in this study we evaluated the quality of two european plums cultivars ('ramasin' and 'ariddo di core' with purple and yellow flesh colour respectively) after modified atmosphere packaging (map) storage, through the selection of the most appropriate indicators. the headspace gas composition, the flesh fruit firmness (fff), the soluble solid content (ssc), the titratable acidity (ta), the colour and the chlorophyll content of plums wrapped with 5 different films (f1, f2, f3, f4 and f5) were evaluated for up 21 days of storage (at 1±1°c and 90-95% relative humidity). for both cultivars, the multilayered films (f1 and f2, 90 and 65 µm respectively) offered better effectiveness over other films. the total chlorophyll concentration, showing a good correlation with the colorimetric parameters of luminance (l*) and chroma (respectively r2=0.92 and r2= 0.96) confirmed, in the case of the ariddo di core cultivar, the results obtained by monitoring other parameters thus highlighting the usefulness of integrating multiple indexes in evaluating the performance of the storage methods used. keywords: plum, film, quality index, chlorophyll, passive atmosphere   ital. j. food sci., vol 28, 2016 377 1. introduction thanks to its adaptive behaviour in different climatic conditions, plums represent one of the most versatile of fruit trees species and its production is considered valuable for the future development of fruit trees sector (blazek, 2007; sottile et al., 2010a). in recent years the recovery and development of high quality local germplasm cultivars, such as the ramasin in piedmont and ariddo di core in sicily, have evidenced a high level of diversity along the italian production of the european plum (prunus domestica l.). these cultivars have been important for local market since years; more recently they are also being coveted by wider markets for the high nutraceutical characteristics of fruits (sottile et al., 2010b). the wide ripening period for such cultivars, combined with the different areas of cultivation, support an extension to the commercial calendar and represent a good opportunity for expanding the market for these fruits. the availability of numerous varieties, together with quality and price indicate the efficiency of the supply chain and distribution system which is today one of the most important sales channels in the area of horticultural fresh products (dean, 2011). the post-harvest management of these fruits appears problematic because plums evidence a cultivar-dependent high perishability, and require specific cares in terms of handling along all the supply chain. if not combined with other storage techniques, low refrigeration temperatures are not sufficient to maintain fruit quality up to the consumer; in fact, prolonged exposure to low temperatures would be responsible for enzymatic browning of the internal tissues and of the formation of skin damages (taylor et al., 1993; abdi et al., 1997). among the different storage techniques such the use of absorbers (sharma et al., 2012) or antagonists of ethylene (singh and singh 2012), the modified and the controlled atmospheres (elzayat and moline, 1995; prange and delong 2006; giuggioli et al., 2008; girgenti et al., 2010; diaz-mula et al., 2011 a, diaz-mula et al., 2011 b, sottile et al., 2013) are well known to have positive effect to improve the shelf life of plums. active map on sanacore and ariddo di core plums was performed with wrapped bulk preserving the quality more than 40 days for local consumption (peano et al., 2010) and different map box liners were used to maintain the shelf life and the quality of 'friar' plums (cantín et al., 2008). a key issue to success is also represented by high uniformity of the fruits as regards quality parameters (crisosto et al., 2004). for this reason the selection and the monitoring of quality indicators is important not only for defining the time for harvesting but also for the maintainance of the commercial value of the product. there are several studies on the evolution of quality parameters during plum postharvesting fruit management (usenik et al., 2008; pérez-marín et al., 2010; sottile et al., 2013;) but the identification of indicators useful to monitor quality along the distribution is still difficult. the pulp firmness and its relative evolution during storage is closely cultivar-dependent and specifically related to the stage of maturity at harvest time (sharma et al., 2012); anyway, if not integrated with other quality indicators, this parameter would be impractical for the fresh fruit market due to the absence of reference classes especially for european plums (valero et al., 2007; usenik et al., 2008). in case of deeply pigmented cultivars, due to an usual early change of the skin colour, the pulp firmness is usually adopted as a ripening indicator (crisosto and kader 2000; sottile et al., 2010 a). generally, for stone fruits, the skin colour is one of the most important harvesting markers; the quantitative and qualitative development of the pigments in the skin is able to characterise the epidermis (chlorophyll, anthocyanins and carotenoids); the development of these pigments is closely related to the biological and physiological stress during the storage of the fruits (merzlyak et al., 1997; abbott 1999). according to previous studies (sharma et al., 2012; valero et al., 2013) in some case the skin colour of purple-flesh plums is not a parameter useful to assess the effectiveness of differing   ital. j. food sci., vol 28, 2016 378 storage treatments. all these aspects are very important in affecting the aesthetic appearance of the product (abbott 1999), while they have several limits and they are not always positively correlated to a correct stage of fruit ripeness (usenik et al., 2008). it has been reported that a total soluble solids content (ssc) ranging from 14 to 16% (westwood 1978) or from 10 to 15% (diaz-mula et al., 2009) determines fruit ready for consumption. however, the aromatic profile of plums, as of most stone fruit species, is even more affected by the total titratable acidity (ta) value than to the sugar content (crisosto et al., 2004; crisosto et al., 2007). many studies (ziosi et al., 2008; infante et al., 2011) have revealed a close correlation between chlorophyll content within the pulp tissues of the stone fruit and the ripening degree of the fruit; this evidence demonstrates that visible uv spectroscopy is a non-destructive technique which could be considered useful for monitoring and characterising the different stages of fruit ripening (zude et al., 2003; ceccarelli et al., 2008). it is therefore evident that the evaluation of the effectiveness of any post-harvest treatment should consider the uniformity of the fruit by including multiple quality indices; this aspect should be more valuable for those cultivars that are often considered minor for the lower diffusion but with a high commercial capacities if new post-harvesting techniques, such as modified atmosphere packaging (map), are developed. on the basis of these considerations, the aim of this work was to evaluate the influence of the different packaging films used for map storage up to 21 days at 1 ±0.5°c of two european plum cultivars characterized by high tasting excellence and differing pigmentation (yellow and purple) also in order to assess the most important quality indices for fresh consumption. 2. materials and methods 2.1. fruit samples two european plum cultivars (prunus domestica l.) were used, both belonging to local italian germplasm and with different pigmentations: the cv. 'ramasin' with a purplecoloured flesh is from the piedmont territory and was harvested in mid-july; the cv. 'ariddo di core' is a yellow-coloured flesh variety from sicily and is harvested in august. both cultivars are characterised by fruits of small size and limited shelf life but with a high tasting quality well recognized by the local consumers. fruits were picked by hand, and selected based on size uniformity and absence of damage. after a refrigeration (2 hours) they were placed in polyethylene terephthalate (pet) trays and transported within 24 hours to the fruit and vegetable warehouse (agrifrutta soc. coop. s.r.l. piedmont, italy) for storage-testing. 2.2. packaging and storage conditions the fruit samples were unwashed previously to be packaged. for both cultivars the sampling unit considered was the flowpack. the pet 0.250 kg trays (l14 x w9.5 x h5) were heat sealed with different films using a taurus 700 model horizontal machine (delphin, italy). the materials used for the different packages were: f1: multilayer produced by co-extrusion of pet, evoh and pe of 90µm, (corapack, italy); f2: multilayer produced by co-extrusion of pet, evoh and pe of 65 µm, (corapack, italy); f3: polypropylene (pp) film, 25µm, (trepack, italy);   ital. j. food sci., vol 28, 2016 379 f4: low density monolayer polyethylene (pe) film, 25 µm, (trepack, italy); f5: non commercial biodegradable film, 25 µm, (novamont, italy); for each package, the control sample (control) is represented by fruits preserved using a polypropylene (pp) macroperforated (6 mm diameter holes) film of 25 µm (trepack, italy). the o2 and co2 transmission rate properties of the films were measured at 23°c and 50% of relative humidity in accordance with astm f 2622-08 and astm f 2476-05 standards (table 1). with the exception of the biodegradable film (f5) whose water permeability value was supplied directly by the manufacturer (147cm3 m-2 24h), tested films resulted within the high water barrier film classification (van tuil, 2000). all fruits were packed under normal atmospheric conditions (0.2 co2 kpa /21.2 o2 kpa). this was performed in order to create passive modified atmosphere packaging (map) during storage conditions through to the synergistic action of the fruit respiratory metabolism and the selectivity of the film to the gases. due the macro hole (6-mm-diameter) the pp film (control) has no changed the atmosphere inside the packages along all the storage time. the fruits were stored for all the period under constant refrigeration conditions based on 1±0.5°c with a relative humidity (rh) level of 90-95% and in the dark. qualitative evaluations were performed at the picking time (0) and after 7, 14 and 21 days of storage. table 1: characteristics of film used for map storage of plum fruits. film gas transmission rate at 23°c and 50% ur cm3/(m2 24h bar) film o2 (astm f2622-08) co2 (astm f2476-05) f1 1572 6111 f2 1572 6111 f3 1456 4616 f4 10990 55360 f5 2276 44494 2.3. headspace composition and qualitative parameters sampling of the gases (o2 and co2) within the headspace of the packaging was performed with a check point ii portable gas analyser (pbi dansensor, italy). three random trays were used for each measurement for a total of 0.750 kg of fruit. in order to avoid any alteration to its internal atmosphere, the air sampled for analysis was fed back into the container using a porous septum (15 mm diameter pbi dansensor, italy) positioned on the surface of the film. instrument calibration was performed after each measurement using a vacuum sample in normal atmosphere (aday and caner, 2011). the value is recorded as kpa and it is the average of three measurements. the weight of each container was measured using an electronic balance (se622, wvr science education, usa) with an accuracy of 0.01 grams, at the harvest and at the end of each storage period. the relative weight loss was expressed as a percentage (%). the fruit flesh firmness (fff) (kg/cm2) was measured by a manual penetrometer (facchini, alfonsine, italy) using a pipette tip with a 7.9-9 mm diameter in accordance with species standards. the skin of the fruit was not removed. each value is the average of two measurements taken from opposite sides of each fruit. the data recorded is the average of   ital. j. food sci., vol 28, 2016 380 30 measurements (three random trays for a total of 0.750 kg of fruit). soluble solids content (ssc) were determined in the juice (from three trays randomly chosen for each treatment) with a digital refractometer atago pr-101 (atago, japan) at 20°c. two readings (30 fruits) were taken on each fruit and averaged; results were expressed as °brix. the titratable acidity (ta) (meq/l) was measured with an automatic titrator (titritino plus 484, metrohm, switzerland); 5 ml of pulp juice were used for each sample (shaken, centrifuged and filtered), diluted in 15 ml of distilled water which was neutralised with sodium hydroxide (naoh) 0.1n. the value is the average of 3 measurements (three random plastic containers for a total of 0.750 kg of fruit). 2.4. colour in this study the colour evolution and total chlorophyll content were monitored only for the yellow-flesh cultivar cv. 'ariddo di core'. colour was measured on the first 15 nonmouldy fruits from each tray (three trays were randomly chosen for each package). the mean of the 30 fruit measurements was used for data analysis. cielab or l*a*b* space was used to describe the color. this color space is device-independent and able to create consistent colors regardless of the device used to acquire the image. l* is the luminance or lightness component, which ranges from 0 to 100, while a* (green to red) and b* (blue to yellow) are two chromatic components, with values varying from –120 to +120 (yam and papakadis, 2004). these values were used to calculate chroma, which indicates the intensity or color saturation, using the following equation: c* = [a*2 + b*2]1/2 (2) hue angle was calculated as follows: h° = arctangent[b*/ a*] (3) where 0° = red-purple, 90° = yellow, 180° = bluish-green, and 270° = blue (mcguire, 1992). 2.5. chlorophyll chlorophyll monitoring was carried out using uv-vis spectrophotometry, a nondestructive analytical, qualitative and quantitative technique that makes use of a spectrophotometer to allow molecule recognition and quantification as a function of spectrum absorption. the uv-vis analyses were performed using a varian cary 500 double beam spectrophotometer equipped with a varian dra-2500 integrating sphere. the background noise was subtracted using the spectralon® as a reference. the spectra were recorded in a range between 350 and 800 nm at a resolution of 3 nm. each uv-vis measurement was made in diffuse reflection (dr) mode positioning the equatorial part of the surface of each fruit (95mm2 area) in line with the reflection sphere. for each sample the chlorophyll concentration was calculated by processing the spectra acquired from each fruit (average of two fruit / 60 fruits / acquisitions) as per the kubelka munk (1931) f(r) function. it was first necessary to establish a calibration equation by means of the direct extraction of chlorophyll from plums exhibiting different degrees of maturity as per the official extraction methodology (aoac, 2006).   ital. j. food sci., vol 28, 2016 381 2.6. statistical analysis all statistics were performed using spss for windows version 20.0. the data obtained were treated with one-way analysis of variance (anova) and the means were separated using the duncan test (p ≤ 0.05). as the sample sizes were identical, it was possible to perform a parametric test for the percentages. 3. results and discussions 3.1. headspace composition and qualitative parameters changes of o2 and co2 (kpa) gases within the package headspaces are shown in figs. 1 and 2 respectively for cv. 'ramasin' and cv. 'ariddo di core'. for both cultivars each packaging film succeeded in changing the initial atmospheric conditions, equal to 0.2 kpa of co2 and 21.2 kpa of o2 maintaining different map conditions up to the end of the storage period (21 days). figure 1: o2 and co2 headspace gas composition of cv. 'ramasin' plums stored in map at 1±0.5°c. figure 2: o2 and co2 headspace gas composition of cv. 'ariddo di core' plums stored in map at 1±0.5°c.   ital. j. food sci., vol 28, 2016 382 in general a decreasing trend in o2 content corresponds to an increase in the internal concentration of co2, product of the respiration of the plums; in this case, the co2 accumulation, at a constant temperature (1±0.5°c), is determined by the interaction of two factors: the permeability of the film and the storage period (exama et al., 1993; varoquaux et al., 2002). during the first 7 days o2 and co2 contents did not differ significantly in both varieties. as the storage goes on, the values increases their differences evidencing the active performance of the different packaging films used. according to what reported in previous map studies on stone fruits (diaz-mula et al., 2011 a, girgenti et al., 2014) the multilayer films (f1 and f2) are able to maintain higher concentrations of co2 within the flow pack when compared with other films (f3, f4, f5) due to higher gas barrier properties (table 1). this condition is maintained by both the cultivars for all the storage time. in particular, after 21 days of storage, co2 ranged between 9.5 and 13.1 kpa for cv. 'ramasin' and between 15.0 kpa and 17.4 kpa of co2 for cv. 'ariddo di core'. with the 'ramasin' cultivar, the highest value is registered with the f1 film, while for the cv. 'ariddo di core' with the f2 film. all other films, from the 7th day of storage evidenced co2 values lower than 5 kpa. for both cultivars the lowest values were obtained with the f4 film (respectively 1.2 kpa for the cv. 'ramasin' and 2.4 kpa for the cv. 'ariddo di core'). throughout the storage period, the cv. 'ariddo di core', under all map conditions, presented higher values of co2 than the cv. 'ramasin', suggesting a greater respiratory metabolism for the fruit of this cultivar. for the cv. 'ramasin', the equilibrium point (13.4 kpa o2 and 13.1 kpa co2) was only reached at the end of the storage period (21 days) with the f1 film, while in the case of the cv. 'ariddo di core' it is reached between the 14th and 21st day with both of the multilayer films (f1 and f2) with values ranging between 10 and 15kpa; this condition is however immediately lost. the weight losses observed (data not shown) increase with the storage time, but the rate for both cultivars is a function of the specific film employed. the control showed the greatest weight loss (3 % of the fresh weight after 21 days) confirming what was observed in previous map studies for stone fruits (sottile et al., 2013; girgenti et al., 2014). all map packages ensure controlled weight loss within a similar range of values (0.5-0.7% for cv. 'ramasin' and 0.6-0.9 % for 'ariddo di core' after 21 days of storage). based on these results, it is not possible to use the weight loss as quality parameter to identify the map film with the best performances. the cv. 'ramasin' (table 2) and cv. 'ariddo di core' (table 3) present very different fff values at harvest (1.1 kg/cm2 and 3.5 kg/cm2 respectively). however both cultivars exhibit a similar evolution for this parameter. in fact, the fff values decreased with time, reaching their lowest values after 21 days of storage. this result is associated with a decrease of pectin polymerisation within the cell tissues and although this trend is common to all packages, fruit stored under normal atmospheric conditions (control) showed a stronger decreasing trend and evidenced values significantly lower at the end of the storage period respect to map storage (0.5 kg/cm2 for cv. 'ramasin' and 1.1kg/cm2 for 'ariddo di core'). as reported in previous studies (sottile et al., 2013) plums stored under map conditions with the highest levels of co2 use to evidence higher pulp firmness; for both cultivars the multilayer films (f1 and f2), ensuring higher values of co2 (figs. 1 and 2), are able to better control pulp firmness decay as compared to other films. in particular, for cv. 'ramasin', the f1 film is significantly different compared to the f2 film, while in the case of the cv. 'ariddo di core', these two films do not exhibit significant differences in terms of performance.   ital. j. food sci., vol 28, 2016 383 table 2: evolution of qualitative characteristics of plums cv. 'ramasin' stored in map at 1±0.5°c. time (days) film 7 14 21 harvest 1.10±0.21 fff (kg/cm2) f1 0.86±0.2a 0.85±0.1a 0.80±0.2a f2 0.89±0.1 a 0.84±0.1a 0.80±0.1b f3 0.77±0.1a 0.70±0.1a 0.78±0.2b f4 0.78±0.1b 0.70±0.1a 0.71±0.1b f5 0.76±0.1b 0.72±0.1a 0.73±0.1b control 0.62±0.1c 0.53±0.1b 0.49±0.2c harvest 16.0±0.7 ssc (°brix) f1 17.0±0.3n.s 17.5±0.3d 17.9±0.5d f2 17.0±0.4 n.s 17.7±0.3d 17.9±0.7d f3 16.9±0.6 n.s 18.2±0.8bc 18.6±0.5bc f4 17.1±0.8 n.s 18.0±0.5c 18.5±0.6c f5 17.1±0.3 n.s 18.3±0.6b 18.8±0.3b control 17.0±0.6 n.s 18.8±0.3a 20.1±0.5a harvest 5.1±0.0 ta (meq/l) f1 4.9±0.0n.s 4.8±0.2a 4.5±0.0a f2 4.9±0.2 n.s 4.8±0.1a 4.5±0.1a f3 5.1±0.1 n.s 4.6±0.3a 4.5±0.0ab f4 5.0±0.1 n.s 4.6±0.1a 4.4±0.0ab f5 5.0±0.1 n.s 4.5±0.1a 4.4±0.0b control 5.0±0.1 n.s 4.2±0.2b 3.7±0.1c results were expressed as means ± standard deviation. values in the column followed by different letters are significantly (p<0.05) different according to duncan’s test. at harvest, the two cultivars present different ssc (16.0° brix and 18.5° brix respectively for cv. 'ramasin' and cv. 'ariddo di core'). after the first 7 days of storage, in spite of the low refrigeration temperatures (1 ±0.5°c), all packages presented an increase in the soluble solids content values in accordance with the observations of guerra and casquero (2008), with the green gage variety and sottile et al., (2013) yellow-fleshcultivars; this trend is evident with continued storage. the increase is related to the concentration of sugars resulting from weight loss of the fruit (loss of water) and also due to the increasing extractability (sucrose inversion) during the increasing of the ripening degree. for both cultivars, over the whole storage period, the control presented a higher soluble solid content compared to the map packaged ones thus confirming the co2 effect during storage and its ability to delay the ripening processes in accordance with díaz-mula et al. (2009). the f1 and f2 films showed statistically significant differences compared to the other map films (f3, f4 and f5) after 14 days of storage in the case of the cv. 'ramasin' (table 2) and after only 7 days of storage for the cv. 'ariddo di core' (table 3) evidencing a better control of the soluble solids content dinamics and maintaining these values close to those measured at harvest.   ital. j. food sci., vol 28, 2016 384 table 3: evolution of qualitative characteristics of plums cv. 'ariddo di core' stored in map at 1±0.5°c. time (days) film 7 14 21 harvest 3.55±0.11 fff (kg/cm2) f1 3.25±0.1 a 3.25±0.3 a 2.39±0.1 a f2 3.25±0.1 a 3.04±0.1 a 2.49±0.2 a f3 2.85±0.2 b 2.50±0.1 b 1.32±0.2 c f4 2.79±0.1 b 2.47±0.1 b 1.35±0.2 c f5 2.87±0.1 b 2.50±0.1 b 1.53±0.2 b control 2.53±0.1 c 2.24±0.4 c 1.04±0.1 d harvest 18.5±0.1 ssc (°brix) f1 18.6±0.1 bc 18.7±0.1 c 19.1±0.2 c f2 18.6±0.1 bc 18.7±0.1 c 19.0±0.1 c f3 18.7±0.1 b 19.1±0.0 b 19.5±0.1 b f4 18.7±0.1 b 19.0±0.1 b 19.5±0.1 b f5 18.7±0.1 b 19.0±0.1 b 19.5±0.1 b control 18.9±0.1 a 19.6±0.1 a 20.0±0.1 a harvest 8.6±0.7 ta (meq/l) f1 5.0±0.1 n.s 4.3±0.0 n.s 4.1±0.1 a f2 5.0±0.1 n.s 4.4±0.1 n.s 4.1±0.0 a f3 4.5±0.0 n.s 4.3±0.0 n.s 3.6±0.0 b f4 4.5±0.1 n.s 4.3±0.0 n.s 3.5±0.0 b f5 4.5±0.0 n.s 4.3±0.1 n.s 3.5±0.0 b control 4.5±0.0 n.s 4.1±0.1 n.s 3.9±0.0 c results were expressed as means ± standard deviation. values in the column followed by different letters are significantly (p<0.05) different according to duncan’s test. over the entire storage period, compared to the values measured at harvest (5.1 meq/l for cv. 'ramasin' and 10.2 meq/l for cv. 'ariddo di core'), the ta diminished for all packages for both cultivars; both for ramasin and ariddo di core cvs., map determined higher ta values compared to the control. the maturity index obtained from the ssc/ta ratio (data not shown) indicates a progressively increase during the whole storage period for both cultivars; for all map packages this value is lower respect to the control, thus confirming the observations reported from previous studies (díaz-mula et al., 2009). the colour variables a* and b* are not independent and their difficult perception to the human eye force to their correlation in order to calculate chroma (c) and hue angle parameters (alcobendas et al., 2012). table 4 reports the colour components l*, chroma and hue angle as measured for the yellow flesh fruit cv. 'ariddo di core'. just after 7 days of storage each film packaging influenced a change in skin colour thus indicating the presence of a maturation process even at low storage temperatures (1±0.5°c). 3.2. colour and chlorophyll monitoring the luminance values (l*) decrease compared to those measured at harvest (46.9) thus indicating a darkening of the tissues as a result of the ripening (table 4). each of the map   ital. j. food sci., vol 28, 2016 385 packages presented no statistically significant differences compared to control as the fruit maintained higher l* values. the multilayer films (f1 and f2), to which correspond higher concentrations of co2 within the flow pack headspace (fig. 2), are more differentiated than the other films presenting significantly higher values of l* over the entire storage period and reaching after 21 days values equal to 42.4. the chroma values follow in a similar way the decreasing trend of l* values. for each film the hue angle values decreased at increasing storage time in accordance with findings reported by díaz-mula et al. (2011 a), but statistically significant lower values (redder fruit) were observed throughout the storage period in respect to control packed fruit. table 4: colorimetric parameters of plums cv. 'ariddo di core' stored in map at 1±0.5°c. time (days) film l* chroma h angle harvest 46.9±0.5a 21.1±0.4a -1.31±0.01d 7 f1 43.7±0.2b 17.0±0.6bc -1.39±0.02c f2 43.7±0.3b 17.1±0.9bc 1.39±0.02c f3 43.6±0.2b 16.9±0.6c -1.42±0.01b f4 43.3±0.3c 16.9±0.8b -1.40±0.01b f5 43.4±0.3c 16.9±0.9c -1.42±0.01b control 42.5±0.3d 15.8±0.9d -1.48±0.03a harvest 46.9±0.5a 21.1±0.4a -1,31±0.01c 14 f1 43.5±0.2b 16.3±0.5b -1.36±0.54bc f2 43.6±0.3b 16.3±0.6b -1.45±0.04ab f3 42.4±0.2d 15.6±0.7c -1.47±0.02a f4 42.5±0.2cd 15.7±0.4c -1.48±0.03a f5 42.5±0.3c 15.6±0.3c -1.48±0.01a control 41.6±0.4e 13.2±0.7d 1.53±0.00a harvest 46.9±0.5a 21.1±0.4a -1.31±0.01c 21 f1 42.4±0.3b 15.6±0.8b -1.47±0.04b f1 42.4±0.5b 15,3±0.8b -1.47±0.02b f1 41.6±0.2c 14.3±0.8c -1.47±0.02b f1 41.7±0.2c 14,6±0.7c -1.48±0.02b f1 41.6±0.4c 14.6±0.6c -1.48±0.02b control 39.5±0.3d 12.6±0.8d -1.55±0.03a results were expressed as means ± standard deviation. values in the column followed by different letters are significantly (p<0.05) different according to duncan’s test. the skin changes from green to yellow color are closely correlated to the chlorophyll degradation process during ripening (abdi et al., 1997; crisosto et al., 2004) as shown in fig. 3. the total pigment content, measured using uv-vis spectrophotometry, decreases progressively throughout the storage period thus confirming evolution of the ripening processes even at low temperatures (zude-sasse et al., 2002; solovchenko et al., 2005). for each quality control all map film tested evidenced to be able to manage the loss of total chlorophyll content maintaining higher values than the control. the high barrier capacity films (f1 and f2) were differentiated already within 7 days of storage thanks to the fruits have maintained the main quality values near to the time of harvest.   ital. j. food sci., vol 28, 2016 386 figure 3: chlorophyll total content (mm) in cv. 'ariddo di core' plums stored in map at 1±0.5°c. the high co2 levels, corresponding to f1 and f2 films, (fig. 2) would be responsible for the proper maintenance of the integrity and fluidity of cell membranes which are indispensable conditions for the stabilisation of the photosynthetic pigment content (pongprasert et al., 2011). the total chlorophyll content showed a good correlation to the colour components l* (fig. 4) and chroma (fig. 5) according to a linear regression index equal to r2= 0.92 and r2= 0.96, respectively. in particular, decreasing values in chlorophyll correspond to a loss of brightness and chromaticity (table 4) associated with the concentration of carotenoids that are responsible for the change in colour from green to yellow (increasing value of a*) of the fruit skin. figure 4: linear regression of total chlorophyll content and the l* colour parameter in cv. 'ariddo di core' plums stored in map at 1±0.5°c.   ital. j. food sci., vol 28, 2016 387 figure 5: linear regression of total chlorophyll content and the chroma colour parameter in cv. 'ariddo di core' plums stored in map at 1±0.5°c. 4. conclusions differents and multiples indexes of quality have been considered to evaluate the goodness of the map technique for the storage of 'ramasin' and 'ariddo di core' plum cultivars. the headspace composition showed how high co2 increments associated with higher barrier capacity films (multilayer) are usually related to a higher fruit quality at the end of the storage period. among the qualitative index considered, for both cultivars, the flesh fruit firmness allowed a better evaluation of the effectiveness of different packaging since the beginning of the storage test. the colour parameter (lightness and chromaticity), linked to the chlorophyll content of the plums, is well known to be particularly appreciated by the consumers and for this it can be suggested to further evaluation of the performance of the tested films for the management of a map storage protocol. results of this research confirm the correlation that exists between colour and the pigments content in organic matrices and more generally within food systems (ramakrishnan and francis, 1973; francis, 1985; watada and abbott, 1975; takahata et al., 1993; ameny and wilson, 1997; chen and tang, 1998; arias et al., 2000; bron et al., 2005; ceccarelli et al., 2008). according to mcguire (1992) the monitoring of the total total chlorophyll content is highly correlated with the colour parameters, then it is important to consider also that it derives from a non-destructive technique such as uv-vis whose numerous advantages are even more interesting when they can be applied to cultivars of excellence and high perishability such as those considered within this study. acknowledgements this work has been carried out as a part of the prin research program quality and safety monitoring of the fruit supply chain: new technologies for post-harvest management, and it was entirely supported by the italian ministry of education, university and research (miur). the authors wish to thank agrifrutta soc.coop.agr. 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25:123. zude-sasse m. 2003. non-destructive prediction of banana fruit quality using vis/nir spectroscopy. fruits 58:135. yam k.l. and papakadis s.e. 2004. a simple digital imaging method for measuring and analysing color of food surfaces. j. food eng. 61:137. paper received january 20, 2015 accepted july 22, 2015 paper 32 ital. j. food sci., vol. 28 2016 keywords: aspergillus flavus, aflatoxin genes, flourometer, molecular markers, genetic diversity molecular characterization of aflatoxigenic aspergilli-contaminated poultry and animal feedstuff samples from the western region of saudi arabia youssuf a. gherbawy*1, 2, yassmin m. shebany1, 2 and helal f.alharthy1 1biological sciences department, faculty of science, taif university, taif, saudi arabia 2botany department, faculty of science, south valley university, qena, egypt *corresponding author: youssufgherbawy@yahoo.com abstract the aflatoxigenic abilities of 64 and 17 isolates of aspergillus flavus and a. parasiticus isolated from poultry and animal feedstuff samples collected from the western region of saudi arabia were studied. thirty-three (51.6%) and 13 (76.5%) isolates of a. flavus and a. parasiticus, respectively, were aflatoxigenic. the ranges of aflatoxins in a. flavus and a. parasiticus isolates were 4.4110 and 143.6-271.3 ppm (µg/g), respectively. a. parasiticus isolates generally produced a greater amount of aflatoxins than a. flavus. a. flavus isolates from poultry, cattle, and camel and cattle feeds produced aflatoxin amounts in the range 5.7-110, 4.4-19.0, and 7.0-28.5 ppm, respectively. from poultry feedstuff samples, a. parasiticus produced aflatoxins in the range 212.5-232.4 ppm. some aflatoxin biosynthesis genes (aflr, omt-1, ver-1, and nor-1) were detected with variable frequencies in all a. flavus and a. parasiticus isolates. the genetic diversity among 64 isolates of a. flavus using internal transcribed spacer sequence results and the amplification of some aflatoxin biosynthesis genes revealed that the investigated isolates showed high heterogeneity. mailto:youssufgherbawy@yahoo.com ital. j. food sci., vol. 28 2016 33 introduction aflatoxin contamination of agricultural commodities has gained global significance as a result of their deleterious effects on human and animal health as well as their importance in international trade. the contamination of foods by aflatoxigenic fungi, particularly in tropical countries, may occur during pre-harvesting, processing, transportation, and storage (ellis et al., 1991; manonmani et al., 2005). regular monitoring of toxigenic mycobiota in agricultural-based feeds and foods is an essential pre-requisite in the development of strategies to control or prevent mycotoxin exposure of feed animals and human population. studies on the prevalence of toxigenic mycobiota of animal/poultry feeds have been regularly and frequently reported, including studies from brazil (oliveira et al., 2006; rosa et al., 2006), argentina (dalcero et al., 1997), nigeria (osho et al., 2007), spain (accensi et al., 2004), and pakistan (saleemi et al., 2010). the polymerase chain reaction (pcr) first described by saki et al. (1985) requires the presence of specific target sequences. when genes involved in the biosynthetic pathway are known, they represent a valuable target for the specific detection of toxigenic fungi. the first researchers to use this approach for the detection of toxigenic fungi were geisen et al. (1996) and shapiro et al. (1996), describing a diagnostic pcr directed against dna sequences in the aflatoxin biosynthetic gene cluster. however, when the genes responsible for mycotoxin production are unknown, other sequences can function as a target. examples are rdna sequences, genes, or anonymous dna marker sequences. geisen (1998) and edwards et al. (2002) reviewed available diagnostic pcrs for toxigenic fungi. the advantages of the pcrbased approach for the detection of toxigenic fungi compared with those of the classical mycological or chemical analysis is mainly the time aspect. for the chemical analysis of mycotoxins in food, elaborated protocols for sample preparation and expensive laboratory equipment are necessary. classical mycological analysis requires the isolation and cultivation of the fungi on different media and at least one week of growth for their reliable identification. in addition, much expertise is required to recognize the species, particularly for the main genera of toxigenic fungi fusarium, penicillium, and aspergillus. in contrast, dna extraction from food samples and raw materials of food can be performed in a few minutes (knoll et al., 2002a). further, the use of modern thermocyclers can reduce analysis time to less than 1 h (knoll et al., 2002b). the aflatoxin biosynthetic pathway involves approximately 25 genes clustered in a 70 kb dna region (yu et al., 2004). a. flavus, a. parasiticus, and other aspergillus section flavi species share nearly identical sequences and conserved gene order in the cluster. in recent years, pcr detection of aflatoxin biosynthetic gene presence or expression has been used as a diagnostic tool for aflatoxigenic fungi in selected food commodities (geisen, 2007; gallo et al., 2012). aflatoxins are regarded as potent hepatocarcinogens and immunosuppressants, and there are reports showing that this group of mycotoxins poses the biggest threat to the poultry and livestock industry through low productivity and death (van egmond, 1989; chukwuka et al., 2010; pedrosa and borutova, 2011). therefore, the potential risks of aflatoxicosis in saudi poultry and livestock must be clearly evaluated in order to ensure prompt legislative action and mitigation of aflatoxin contamination in feed. this study was designed to determine and evaluate the aflatoxin-producing potentials of aspergillus section flavi isolated from poultry and animal feedstuff samples collected from the western region of saudi arabia. furthermore, the isolates were tested for the presence of four of the characterized aflatoxin biosynthetic genes in their genome in relation to aflatoxin production. materials and methods samples sixty-four a. flavus and 17 a. parasiticus isolates were used throughout this investigation. these isolates were retrieved from poultry and animal feedstuff samples collected from the western region of saudi arabia (taif, makkah, and jeddah). the isolates were identified according to their morphological features as well as sequence results of internal transcribed spacer (its) regions. the sequence results were deposited in the genbank. determination of total aflatoxin abilities of aspergillus species isolates the aflatoxin-producing abilities of the isolates were determined by cultivating the fungal strains in czapek yeast extract agar (ben fredj et al., 2009) medium for 5 days at 25±2°c. total aflatoxins were extracted by grinding the moldy agar (20 g) in a waring blender for 5 min with methanol (100 ml) containing 0.5% nacl. the mixture was then filtered through a fluted filter paper (whatman 2v, whatmanplc, middlesex, uk), and the filtrate was diluted (1:4) with water and re-filtered through a glass-fiber filter paper. two milliliters of the glass-fiber filtrate were placed on aflatest® wb sr column (vicam, watertown, ma, usa) and allowed to elute at 1-2  drops/s. the columns were washed twice with 5 ml of water, and aflatoxin was eluted from 34 ital. j. food sci., vol. 28 2016 the column with 1 ml high performance liquid chromatography (hplc)-grade methanol. a bromine developer (1 ml) was added to the methanol extract, and the total aflatoxin concentration was read in a recalibrated vicamseries-4 fluorometer set at 360 nm excitation and 450 nm emissions (lewis et al., 2005). molecular detection of aflatoxin biosynthetic genes in aflatoxigenic species of aspergilli the isolation of dna from mycelia was performed according to the method described by farber et al. (1997). four published primer sets were used for the specific detection of nor1, ver-1, omt-a, and aflr genes (criseo et al., 2008). the 400, 537, 797, and 1032-bp fragments were amplified, respectively. a typical pcr was carried out under the following conditions: 5 μl of genomic dna were used as a template (2 μg/ml), 0.5 u eurotaq polymerase (euroclone, pero-milan, italy), 1 × reaction buffer, 2.5 mm mgcl 2 , 200 μm of each dntp, and 7.5 pmol of each primer, in a total reaction volume of 50 μl. a total of 35 pcr cycles with the following temperature regimen were performed: 95°c, 1 min; 65°c, 30 s; 72°c, 30 s for the first cycle; and 94°c, 30 s; 65°c, 30 s; 72°c, 30 s for the 34 remaining cycles (criseo et al., 2008). pcr products were separated on a 1.3% (wt/vol) agarose gel stained with ethidium bromide. statistical analysis of frequency of aflatoxin biosynthetic genes cluster analysis of data was performed by hierarchical cluster analysis (spss software, spss inc., usa; norusis, 1993). results and discussion total aflatoxin potentials of aspergillus species isolates thirty-three out of 64 (51.6%) and 13 out of 17 (76.5%) of a. flavus and a. parasiticus isolates were aflatoxigenic producers, respectively. the aflatoxin range in a. flavus and a. parasiticus isolates was 4.4-110 and 143.6-271.3 ppm (µg/g), respectively (tables 1 and 2). dutta and das (2001) carried out a groundwork study, in which 256 feed samples collected from different parts of northern india were analyzed for aflatoxigenic strains of a. flavus/parasiticus and for detection of afb1. out of 198 a. flavus and 15 a. parasiticus strains isolated, 76% and 86%, respectively, were found to be toxigenic. razzaghi-abyaneh et al. (2006) surveyed the distribution of aspergillus section flavi in cornfield soils in iran and their results indicated that only 27.5% of a. flavus isolates were aflatoxigenic (b1 or b2 or both), and all the a. parasiticus isolates produced aflatoxins of both b (b1 and b2) and g (g1 and g2) types. pitt (1993) also, reported that a. flavus isolates produced b1 and b2 or both types, while a. parasiticus produced the four aflatoxin types. these results support the present findings indicating that the level of aflatoxin production by a. parasiticus was higher than that by a. flavus isolates (tables 1 and 2). further, koehler et al. (1975) reported that a. parasiticus isolates generally produced a greater amount of aflatoxins than a. flavus. the range of aflatoxin production by a. flavus isolated from poultry, cattle, and camel and cattle was 5.7-110, 4.4-9.0, and 7.0-28.5 ppm, respectively. from poultry feedstuff samples, a. parasiticus produced aflatoxins in the range 212.5-232.4 ppm. in pakistan, saleemi et al. (2010) studied the mycoflora of poultry feed and mycotoxin-producing potential of aspergillus species. they reported that the toxigenic fungi content among aspergillus isolates was 73.58%, and that of aflatoxigenic isolates of a. flavus and a. parasiticus was 83.33% and 85.71%, respectively. further, they recorded that, among toxigenic a. flavus isolates (10/12), six produced four aflatoxins (afb1, afb2, afg1, and afg2), two produced afb1, afb2, and afg1, one produced afb1, afb2, and afg2, and one produced afb1 and afb2. among aflatoxigenic isolates of a. parasiticus (6/7), five produced four aflatoxins (afb1, afb2, afg1, and afg2) while one produced three (afb1, afb2, and afg1). the production range of aflatoxins from four isolates (tuh212, 221, 222, and 225) of a. parasiticus retrieved from cattle feed samples was 143.6-271.3 ppm. further, two isolates (tuht216 and 220) of a. parasiticus isolated from cattle and camel feed samples contained 195.5 and 211.2 ppm of aflatoxins (table 1). among isolates of a. flavus collected from taif samples, tuht53 showed the lowest aflatoxin potential (7.0 ppm) and tuht44 showed the highest (106.8 ppm). isolates tuht185 and tuht180 from feed samples collected from jeddah showed the lowest (5.7 ppm) and highest (33.0 ppm) levels of aflatoxins, respectively (table 1). for a. flavus isolates retrieved from feed samples collected from makkah, tuht117 and tuht121 showed the lowest (5.0 ppm) and the highest (110 ppm) aflatoxin levels, respectively. the results shown in table 2 indicated that, from a. parasiticus isolates, the lowest aflatoxin producer was tuht212 (143.6 ppm), while the highest production was recorded in isolate tuht222 (269.5 ppm). data from different geographic areas demonstrated a great variability in the mycotoxin-producing potential of a. flavus and closely related species (horn and dorner, 1999). these results are in accordance with previous reports showing that these two species have the ability to produce both b and g aflatoxins (pitt and hocking, 1997; kumeda et al., 2003; ghiasian et al., 2004). in alital. j. food sci., vol. 28 2016 35 table 1 total aflatoxins (ppm) and aflatoxigenic genes detected in 64 strains of aspergillus flavus isolates collected from feedstuff samples. strains code source of isolation location total aflr omt-a ver-1 nor-1 afs (ppm) tuht43 poultry taif n.d. + + + + tuht44 poultry taif 106.8 + + + + tuht46 poultry taif 7.8 + + + + tuht47 poultry taif n.d. + + + + tuht53 poultry taif 7.0 + + + + tuht59 poultry taif n.d. + + + tuht63 poultry taif n.d. + + + tuht84 poultry taif n.d. + tuht85 poultry taif n.d. + tuht86 poultry taif 11.0 + + + + tuht87 poultry taif n.d. + + + tuht89 cattle taif n.d. tuht91 cattle taif 10.0 + + + + tuht92 camel & cattle taif n.d. + + tuht93 poultry taif 16.0 + + + + tuht94 poultry taif 28.4 + + + + tuht98 camel & cattle taif n.d. + tuht99 cattle taif 19.0 + + + + tuht100 poultry makkah n.d. + tuht104 poultry makkah n.d. + + + tuht106 poultry makkah n.d. + + tuht107 poultry makkah 8.9 + + + + tuht108 poultry makkah n.d. + + tuht109 poultry makkah 20.0 + + + + tuht110 poultry makkah 12.0 + + + + tuht111 cattle makkah 13.0 + + + + tuht115 camel & cattle makkah n.d. + + + tuht116 cattle makkah 19.0 + + + + tuht117 cattle makkah 4.4 + + + + tuht118 cattle makkah 15.0 + + + + tuht119 camel & cattle makkah n.d. + + tuht120 camel & cattle makkah 28.5 + + + + tuht121 poultry makkah 110.0 + + + + tuht123 poultry makkah n.d. + + tuht124 poultry makkah 6.6 + + + + tuht126 poultry makkah 13.2 + + + + tuht152 poultry makkah n.d. + tuht154 poultry makkah 8.5 + + + + tuht155 poultry makkah n.d. + + tuht156 poultry makkah 12.6 + + + + tuht157 cattle makkah n.d. + tuht158 cattle makkah 7.3 + + + + tuht160 poultry jeddah n.d. + + tuht112 horses jeddah n.d. + + + tuht161 poultry jeddah n.d. + + + + tuht163 poultry jeddah n.d. + + tuht164 poultry jeddah n.d. + + tuht165 camel & cattle jeddah 7.0 + + + + tuht166 camel & cattle jeddah n.d. + + tuht168 horses jeddah 6.1 + + + + tuht172 camel & cattle jeddah n.d. + + tuht173 camel & cattle jeddah n.d. + + + + tuht174 camel & cattle jeddah 7.5 + + + + tuht176 cattle jeddah n.d. + + tuht177 cattle jeddah 14.0 + + + + tuht180 poultry jeddah 33.0 + + + + tuht181 poultry jeddah 21.0 + + + + tuht185 poultry jeddah 5.7 + + + + tuht186 camel & cattle jeddah 8.0 + + + + tuht187 camel & cattle jeddah n.d. + tuht188 camel & cattle jeddah 18.2 + + + + tuht189 camel & cattle jeddah 9.13 + + + + tuht190 camel & cattle jeddah n.d. + + tuht193 camel & cattle jeddah 14.3 + + + + total 33 53 53 42 51 36 ital. j. food sci., vol. 28 2016 table 2 total aflatoxins (ppm) and aflatoxigenic genes detected in aspergillus parasiticus isolates collected from feedstuff samples. strains code source of isolation location total aflr omt-a ver-1 nor-1 afs (ppm) tuht226 cattle taif n.d. + + + tuht227 cattle taif n.d. + + + + tuht228 cattle jeddah n.d. + + tuht229 poultry jeddah n.d. + + + tuht26 poultry taif 212.7 + + + + tuht211 poultry taif 232.4 + + + + tuht212 cattle makkah 143.6 + + + + tuht213 poultry makkah 231.2 + + + + tuht214 poultry makkah 224.4 + + + + tuht215 poultry jeddah 212.5 + + + + tuht216 camel & cattle jeddah 195.4 + + + + tuht219 cattle taif 265.5 + + + + tuht220 camel & cattle taif 211.2 + + + + tuht221 cattle makkah 271.3 + + + + tuht222 cattle makkah 269.5 + + + + tuht223 poultry jeddah 227.2 + + + + tuht225 cattle jeddah 269.7 + + + + total (17 isolates) 13 16 17 15 16 geria, riba et al. (2010) determined the aflatoxin-producing capacity of 150 a. flavus isolates collected from wheat and its derivatives in 2004 and 2006, and the results showed that 72% of the strains produced aflatoxins. these strains produced amounts of afb1 in the range 12.1234.6 mg/g of cya medium. the results of the present study indicate that the aflatoxigenic species of aspergillus vary in their aflatoxin potential according to the substrate and environmental factors. these results are in agreement with those reported by abbas et al. (2005). detection of some of aflatoxin biosynthesis genes in aspergillus species the production of aflatoxin involves a complex biosynthetic pathway consisting of at least 25 genes (yabe et al., 1999; criseo et al., 2001a, bhatnagar et al., 2003; yu et al., 2004; scherm et al., 2005). all of the identified biosynthesis-related genes are located within a 75 kb dna region in both a. parasiticus and a. flavus, and their relative positions in the cluster of both fungal species are similar (yu et al., 2000; ehrlich et al., 2005). pcr was used for the detection of aflatoxigenic aspergilli based on the intermediated enzymes, including norsolorinic acid reductase encoding gene nor-1, the versicolorina dehydrogenase encoding gene ver-1, the sterigmatocystin 0-methyl transferase encoding gene omt-1, and the regulatory gene aflr (erami et al., 2009). representative aflatoxigenic and non-aflatoxigenic a. flavus and a. parasiticus isolates were subjected for detection of aflatoxin biosynthesis genes. detection of aflatoxin biosynthesis genes in a. flavus isolates pcr was applied using four sets of primers for different genes involved in the aflatoxin biosynthetic pathway. bands of fragments of aflr, omt-1, ver-1, and nor-1 genes were visualized at 1032 bp, 797 bp, 537 bp, and 400 bp, respectively (fig. 1). all examined a. flavus isolates yielded different dna banding patterns with a number of bands ranging from zero to four (tables 1 and 2). table 1 outlines the total aflatoxin and aflatoxigenic genes (aflr, omt-a, ver-1, and nor-1) detected in 64 strains of aflatoxigenic and nonaflatoxigenic a. flavus isolates collected from feedstuff samples. a. flavus isolates were represented by 35 isolates from poultry feed samples, 16 from camel and cattle feed, 11 from cattle feed, and two from horse feed. thirty-eight out of 64 (59.4%) a. flavus isolates contained all four aflatoxin biosynthesis genes; among them 21 isolates were retrieved from poultry feedstuff samples, eight from camel and cattle feed, eight from cattle feed, and one from horse feed (table 1). this result is in agreement with criseo et al. (2001a), who used specific pcr-based methods to prove that aflatoxigenic a. flavus isolates always contain the complete gene set. among the 38 isolates tht showed the presence of all four targeted genes, two isolates (tuht43 and 47) were not aflatoxigenic. therefore, this result indicated clearly that the presence of the four tested genes is not a sufficient marker for the differentiation between aflatoxigenic and non-aflatoxigenic isolates. other studies (flaherty and payne, 1997; chang et al., 1999a,b; 2000, cary et al., 2002; takahshi et ital. j. food sci., vol. 28 2016 37 al., 2002; ehrlich et al., 2003) have suggested that regulation of aflatoxin biosynthesis in aspergillus spp. involves a complex pattern of positive and negative acting transcriptional regulatory factors affected by environmental and nutritional parameters. furthermore, the lack of aflatoxin production apparently does not need to be related only to an incomplete pattern obtained in pcr-based detection. different mutations may be responsible for the inactivation of aflatoxin biosynthetic pathway genes in other a. flavus strains (geisen, 1996). six isolates (9.4% of the tested isolates) with three gene amplicons were not aflatoxigenic (table 3). from these, four, one, and one isolates were retrieved from poultry, camel and cattle, and horse feeds, respectively. twelve isolates (18.8% of the tested isolates; six from poultry, five from camel and cattle, and one from cattle), contained two gene amplicons and seven isolates (10.9%) contained one gene amplicon (table 3). on the other hand, one non-aflatoxigenic isolate (tuht89) showed no bands, indicating a deletion of the targeted genes in this isolate. criseo et al. (2001a) proved that non-aflatoxigenic isolates of a. flavus were lacking one, two, three, or four pcr products, indicating that the genes do not exist in these strains or that the primer binding sites changed. further, criseo et al. (2001b) reported that aflatoxin biosynthesis in a. flavus is strongly dependent on the activities of regulatory proteins and enzymes encoded by the four genes aflr, nor-1, ver-1, and omt-a. gherbawy et al. (2012) reported on the presence of a complete set of these genes in seven aflatoxigenic isolates of a. flavus retrieved from date palm. the frequencies of the four aflatoxin biosynthesis genes aflr, omt-a, ver-1, and nor-1, in the tested isolates were 53, 53, 42, and 51, respectively (table 1). criseo et al. (2008) used 134 of non-aflatoxin producing strains of a. flavus isolated from food, feed, and officinal plants to study the different genes involved in the aflatoxin biosynthetic pathway. their results indicated that the nor-1 gene was the most representative (88%) of the four aflatoxin structural assayed genes, followed by ver-1 and omt-a, which were found at the same frequency (70.1%). a lower incidence (61.9%) was observed for aflr. further, criseo et al. (2008) demonstrated that a high number of aflatoxin non-producing strains (61.9%) contain the aflr gene. this could impair the use of aflr to identify aflatoxigenic aspergilli. five out of ten a. flavus isolates were not aflatoxin producers (scherm et al., 2005), table 3 origin and genetic patterns of 64 aflatoxigenic aspergillus flavus isolates collected from feedstuff samples in this study. values in brackets are percentages of the total samples analyzed. sample name no isolates complete three two one zero set bands bands band band poultry 35 21 4 6 4 camel & cattle 16 8 1 5 2 cattle 11 8 1 1 1 horses 2 1 1 total 64 (100) 38 (59.4) 6 (9.4) 12 (18.8) 7 (10.9) 1 (1.6) fig. 1 aflatoxin biosynthesis genes amplifications. lanes 1 7, aspegillus flavus (tuht 43, 44, 47, 91, 168, 188 and 219); lanes 8 13, a. parasiticus (tuht 26, 212, 216, 221, 223 and 227); lanes 14 -16, a. flavus (tuht 87, 104, 115), lane 17 a. parasiticus (tuht 226), lane 18 a. parasiticus (tuh 229); lane 19, a. flavus (tuht 160); lane 20, a. parasiticus (tuht 228); lane 21 & 22, a. flavus (tuht 119 & 176); lane 23, a. flavus (tuht 163); lanes 24; a. flavus (tuht 164); lane 25 a. flavus (tuht84); lane 26 a. flavus (tuht157); lane, 27 negative control and lane 28, positive control. asterisked lanes were non aflatoxigenic isolates. 38 ital. j. food sci., vol. 28 2016 indicating that the frequencies of occurrence of aflr and omt-a, ver-1, and nor-1 genes were 8, 5, 9, and 5, respectively. detection of some of aflatoxin biosynthesis genes in a. parasiticus isolates seventeen a. parasiticus isolates collected from different feedstuff samples from various cities in saudi arabia were examined for the presence of aflatoxin biosynthesis genes using a specific primer set as mentioned above. the results indicated the presence of four bands for aflr, omt-1, ver-1, and nor-1 genes at 1032 bp, 797 bp, 537 bp, and 400 bp, respectively (fig. 1). all aflatoxigenic and non-aflatoxigenic isolates examined yielded different dna banding patterns with the number of bands ranging from 2 to 4 (tables 2 and 4). table 2 shows the total aflatoxin and aflatoxigenic genes detected in a. parasiticus isolates collected from three different feedstuff samples (poultry, camel and cattle, and cattle). thirteen out of 17 a. parasiticus isolates were aflatoxigenic. the frequencies of occurrence of aflr, omt-1, ver-1, and nor-1 genes in a. parasiticus isolates were 16 (94.1%), 17 (100%), 15 (88.2%), and 16 (94.1%), respectively. gherbawy et al. (2014) reported that omt-a was the most prevalent gene in a. flavus and a. parasiticus isolated from chili samples collected from taif city (saudi arabia). further, their results indicated that this gene was recovered from 27 out of 30 a. flavus isolates and two isolates of a. parasiticus, while nor-1, aflr, and ver-1 genes were recovered from 25, 26, and 24 isolates of aflatoxigenic and non-aflatoxigenic isolates of a. flavus. out of seven a. parasiticus isolates collected from poultry feedstuff samples, 6 (85.7%) contained four genes, while two (14.3%) showed the amplicons of three genes. the two a. parasiticus isolates collected from camel and cattle feedstuff samples showed a complete set of the targeted genes (tables 2 and 4). amplification of the four targeted genes in eight a. parasiticus isolates collected from cattle feedstuff samples showed that six (75%) had the four genes and one (12.5%) contained three genes (tables 2 and 4). further, one isolate contained two genes. geisen (1996) reported the presence of the abovementioned genes from two isolates of a. parasiticus. additionally, scherm et al. (2005) indicated the presence of a complete set of genes (aflr, omt-1, ver-1, and nor-1 genes) in three isolates of a. parasiticus. the findings herein showed the presence of four targeted genes in all aflatoxigenic isolates of a. parasiticus and in one (tuht229) non-aflatoxigenic isolate. further, all non-aflatoxigenic isolates were missing one or more of the targeted genes. rashid et al. (2008) studied the presence of aflr, omt-1, ver-1, and nor-1 genes in 35 a. parasiticus isolates from stored wheat grains in pakistan. their results revealed that only one isolate showed the complete set of genes. additionally, omt-1, ver-1, and nor-1 genes appeared in 8, 10, and 13 isolates. deletion of aflr in a. parasiticus abolishes the expression of other aflatoxin pathway genes (cary et al., 2000). finally, the regulation of aflatoxin biosynthesis genes in aspergillus spp. is affected by environmental and nutritional parameters (flaherty and payne, 1997; chang et al., 2000; cary et al., 2002; takahshi et al., 2002; ehrlich et al., 2003). genetic diversity among a. flavus strains isolated from feedstuff samples sixty-four aflatoxigenic and non-aflatoxigenic isolates of a. flavus represented different sources of isolation and different locations were used in this part. using the its region of rrna sequencing results and amplification of some aflatoxin biosynthesis genes, the genetic diversity among those strains was studied. using its sequencing results of 64 isolates of a. flavus, a neighbor joining tree was constructed (fig. 2). the population of a. flavus split into several clades and sub-clades; the bootstrap values for these clades and sub-clades ranged from 1 to 100, indicating a high heterogeneity in this population. further, the clustering system did not correlate with the type of sample or its location. for example, a. flavus isolate tuth157 (isolated from cattle feedstuff sample collected from makkah) clustered together with isolate tuth63 (isolated from poultry feedstuff sample collected from taif) in one sub-clade with a 98 bootstrap value. additionally, isolates tuth154 and tuth193 constituted one sub-clad with a 69 bootstrap value, although the first one was table 4 origin and genetic patterns aflatoxigenic aspergillus parasiticus isolates collected from feedstuff samples. values in brackets are percentages of the total samples analyzed. sample name no isolates complete set three bands two bands poultry 7 6 1 camel & cattle 2 2 cattle 8 6 1 1 total 17 (100) 14 (82.4) 2 (11.8) 1 (5.9) ital. j. food sci., vol. 28 2016 39 fig. 2 phylogenetic tree based on the internal transcribed spacer (its) region of rrna of aflatoxigenic and non aflatoxigenic 64 isolates of aspergillus flavus. the tree was constructed by neighbor-joining algorithm using maximum composite likelihood model. bootstrap factors less than 55 were not shown. the tree was rooted with aspergillus niger [he649376] as the out-group. red rods indicated non aflatoxigenic isolates. isolated from poultry feedstuff samples from makkah and the second from cattle and camel feedstuff samples from jeddah (fig. 2 and table 1). therefore, clustering according to the its sequencing results did not indicate any relationship among the isolate clustering system and their geographical distributions and even the sources of isolation. aflatoxigenic isolates spread all over the constricted phylogentic tree without separation of the clades into toxigen40 ital. j. food sci., vol. 28 2016 fig. 3 the hierarchical cluster analysis using average linkage between groups form aspergillus flavus isolates based on amplification of aflatoxins biosynthesis genes. red bars indicated non aflatoxigenic species. ic and non-toxigenic. for example, aflatoxigenic isolate tuht189 clustered with non-aflatoxigenic isolate tuht187 with a 96 bootstrap value (fig. 2). since the clustering system was based on its sequencing results, with non-functional spacers, there is no correlation between clustering system and toxin production. the present results show that isolates identified as a. flavus had a polyphyletic origin, supporting the genetic heterogeneity of a. flavus as previously demital. j. food sci., vol. 28 2016 41 onstrated by other studies (geiser et al., 1998, 2000; van den broek et al., 2001; chang et al., 2007; goncalves et al., 2012). the genetic diversity among a. flavus isolates was studied using the results of amplification of some aflatoxin biosynthesis genes. the results were subjected to hierarchical cluster analysis using average linkage between groups to construct a dendrogram showing the correlation between the isolates (fig. 3). a. flavus isolates did not follow any rule in their clustering system. for example, aflatoxigenic isolates tuht121 and tuht126 (isolated from a poultry feedstuff sample collected from makkah) and non-aflatoxigenic isolates tuht85 (poultry feedstuff samples from taif) and tuht98 (camel and cattle feeds from taif) were clustered together as shown in fig. 3. on the contrary, tuht112 (horse feeds from jeddah), tuht160 (poultry feeds from jeddah), tuht87 (poultry feeds from taif), and tuht104 (poultry feeds from makkah) were non-aflatoxigenic isolates clustered together (fig. 3). generally, these results indicate that the presence or absence of pcr products for the targeted aflatoxin biosynthesis genes was not correlated 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egmond h.p. 1989. aflatoxinm1: occurrence, toxicity, regulation. in: van egmond h.p. ed. , mycotoxins in dairy products. elsevier, london, pp. 11-15. yabe k., nakamura m. and hamasaki t. 1999. enzymatic formation of g-group aflatoxins and biosynthetic relationship between gand bgroup aflatoxins, appl. environ.microbiol. 65: 3867-3872. yu j., chang p.k. and ehrlich k.c. 2004. clustered pathway genes in aflatoxin biosynthesis. appl. environ. microbiol. 70:1253-1262. yu j., chang p.-k., bhatnagar d. and cleveland r.e. 2000. cloning of a sugar utilization gene cluster in aspergillus parasiticus. biochem. biophys. acta. 1493: 211-214. paper received november 29, 2014 accepted march 3, 2015 http://www.ncbi.nlm.nih.gov/pubmed?term=wheeler ka%5bauthor%5d&cauthor=true&cauthor_uid=8110599 http://www.ncbi.nlm.nih.gov/pubmed?term=tanboon-ek p%5bauthor%5d&cauthor=true&cauthor_uid=8110599 http://www.ncbi.nlm.nih.gov/pubmed/8110599 paper ital. j. food sci., vol. 28 2016 83 keywords: flaxseed, folch method, tocopherols, phenolic antioxidants, fatty acids, gc-ms variation in physico-chemical/analytical characteristics of oil among different flaxseed (linum usittatissimum l.) cultivars nazia yaqoob1,2, ijaz ahmed bhatti1, farooq anwar3,4*, muhammad mushtaq5 and william e. artz6 1department of chemistry, university of agriculture faisalabad, pakistan 2department of chemistry, govt college for women university, faisalabad, pakistan 3department of chemistry, university of sargodha, sargodha, pakistan 4department of pharmaceutical chemistry, college of pharmacy, prince sattam bin abdulaziz university, al-kharj 11942, saudi arabia 5department of chemistry, govt college university lahore, pakistan 6department of food science and human nutrition, university of illinois urbana, champaign, illinois, usa *corresponding author: fqanwar@yahoo.com abstract the present study evaluates and compares the proximate parameters of flaxseed, as well as the physicochemical characteristics of the extracted flaxseed oils of locally grown eight cultivars. the oil, protein, fiber and ash content of the seeds ranged from32.56-39.98%, 16.02-18.50%, 23.3026.88 and 3.20-3.60%, respectively showing considerable variation among cultivars. the quality attributes such as unsaponifiable matter, peroxide value, acid value, para-anisidine value, conjugated dienes and trienes as well as tocopherols content of the tested flaxseed oils varied significantly (p<0.05) among cultivars. the major tocopherol was γ-tocopherol (173.7 to 257.9 mg/l) followed by relatively low quantities of α-tocopherol (8-12 mg/l), while δ-tocopherol was not detected. α-linolenic acid was found to be the principal fatty acid in the range of 44.51 to 54.87%, while the second major fatty acid present in the oils was oleic acid (21.05 to 30.96%). the variation in the characteristics of oils among different cultivars observed during present investigation might be attributed to difference in genetic makeup and harvesting regimes of the flax plants. mailto:fqanwar%40yahoo.com?subject= 84 ital. j. food sci., vol. 28 2016 introduction flax (linum usitatissimum l.) is a multi-purpose and economically important oilseed crop. flaxseeds, which contain approximately 36 to 40% oil, have long been used in human diet and animal feed (toure and xueming, 2010). by virtue of the presence of physiologically active components, which provide health benefits beyond basic nutrition, flaxseed is often grouped as ‘‘functional food’’ (hasler et al., 2000). historically, the oil extracted from flaxseeds, has been used as a basic component in the preparation of paints or polymers, linoleum, varnishes, inks and cosmetics (el-beltagi et al., 2007; zhang et al., 2008; jhala and hall, 2010). however, during the past decade, there has been an increasing interest in the use of flaxseed oil to improve human health status due to its high nutraceutical potential (oomah, 2001; choo et al., 2007). the potential health benefits of flaxseed oil including reduction in serum cholesterol levels and decreased incidence of diabetes, breast and colon cancer can be ascribed to the presence of high-value antioxidants, tocopherols, lignans and essential fatty acids (muir and westcott, 2003; hosseinian et al., 2006; choo et al., 2007; toure and xueming, 2010). flaxseed oil is one of the richest sources of unsaturated fatty acid, especially, linolenic acid (c18:3) with amount in the range of 50-60% of the total fatty acids present (flachowsky et al., 1997). the agronomic conditions, such as the soil characteristics, agro-climatic conditions and the cultivar influence the unsaturated fatty acids composition in flaxseed (duan et al., 2003). moreover, studies indicate that the oil content, fatty acids profile and other physicochemical properties vary in the flaxseed crops grown in different parts of the world (taylor and morrice, 1991; wakjira et al., 2004; hall et al., 2006). traditionally, flaxseed has been grown in the asian subcontinent for its oil; however in pakistan its applications have been limited to industrial uses. the present study mainly focused on the evaluation and comparison of proximate parameters of flaxseed, as well as the physicochemical characteristics (such as the refractive index, density, iodine value, acid value, peroxide value, para-anisidine value etc.) and the composition of tocopherols and fatty acids of flaxseed oil from different flax cultivars (linum usitatissimum l.) indigenous to pakistan to assess their nutritional value. experimental seeds and chemicals/reagents commercially available hybrid varieties (chandni, ls-108, ls-105, ls-99 and ls-29) of flaxseed used in this study were obtained from the ayub agricultural research institute (aari), faisalabad, pakistan and ls-33, ls-31 and ls-13 were obtained from national agriculture research center (narc) in islamabad, pakistan. three different seed samples for each of the flaxseed variety were collected (8×3=24). all the reagents (analytical and hplc grade) used were from merck (darmstadt, germany) or sigma–aldrich (buchs, switzerland). pure standards of tocopherols (α-tocopherol, γ-tocopherol, δ-tocopherol), and fatty acid methyl esters were obtained from sigma chemical co. (st. louis, mo, usa). extraction of oil seeds of different flaxseed cultivars were crushed with a domestic electric grinder. the oil from the seeds was extracted using the folch method (folch et al., 1957). after oil extraction, the solvent was removed under vacuum in a rotary evaporator (eyela, rotary vacuum evaporator n.n. series equipped with an aspirator and a digital water bath sb-651, 33 japan) at 45°c. the extracted oil was then stored in a refrigerator at 4°c until used for analyses. analysis of oil seed residues proximate analyses of oilseed residues (meals), left after oil extraction, were completed according to standard methods. protein contents (n6.25) were determined according to aoac method 954.01 (aoac, 1990), using a kjeldahl apparatus. the fiber contents were determined employing iso method 5983 (iso, 1981). briefly, 2 g of finely ground defatted sample was taken and boiled with 250 ml of 0.255 m h 2 so 4 , followed by the filtration and washing of insoluble residues. the residues were then boiled with 250 ml of 0.313 m naoh, filtered, washed, and dried. the dried residues were weighed and burnt at 600oc using a muffle furnace (eyela, tmf-2100, tokyo, japan) and the loss of mass was determined gravimetrically. ash contents were determined by following iso method 749 (iso, 1977). two grams of meal was carbonized by heating on a gas flame and then ashed in an electric muffle furnace at 600oc, until a constant mass was achieved. physicochemical properties of oil the extracted oils were analyzed for density, refractive index, peroxide value (pv), acid value, iodine value (iv), saponification value, and unsaponifiable matter following aocs methods cc10a-25, cc7-25, cd1-25, cd8-53, f-9a44, cd3-25, and ca61-40, respectively (aocs, 1997). the determinations of conjugated dienes (cd) and conjugated trienes (ct) were made using a hitachi u-2001 spectrophotometer. the oil samples were diluted with isooctane and abital. j. food sci., vol. 28 2016 85 sorbance values recorded at 232 and 268 nm for conjugated dienes (cd) and conjugated trienes (ct), respectively. specific extinctions were determined following the iupac method ii d.23 (iupac, 1987). the para-anisidine value of the flaxseed oil samples was monitored according to iupac method ii d.26 (iupac, 1987). the oil samples diluted with isooctane were reacted with p-anisidine solution (0.25% w/v) in acetic acid for 10 min and the absorbance of the resulting colored solution recorded at 350 nm using a spectrophotometer. tocopherols for tocopherol analysis, an hplc method was adopted from yaqoob et al. (2010), with some modifications. a waters alliance 2695 hplc system equipped with ymc-pack ods am-303, c 18 column (250mm x 4.6mm x 5μm) and agilent series 1050 diode array detector, (uv 295 nm) was used. the temperature of the column was maintained at 30oc. the chromatographic separation was performed by isocratic elution with a mixture of acetonitirile and isopropanol (40:60 v/v) at a flow rate of 1 ml/min (pressure 120 bar). briefly, flaxseed oil (1 g) was accurately weighed into a 5 ml sample vial wrapped in aluminum foil to prevent photo-oxidation. the oil was dissolved in 5 ml acetonitrile before injection. samples were injected into the column through an injection loop (20 μl). tocopherols were identified by comparing the retention times of the unknowns with those of pure standards of α-, γ-, and δ-tocopherols. acquisition of data was made using agilent chem-station software. the samples were prepared and analyzed separately in triplicate. fatty acids profile fatty acid methyl esters (fames) were prepared by iupac standard method 2.301 (iupac, 1987) which involved the trans-esterification of fatty acids with methanol under base –catalyzed conditions. briefly, 0.2 g of oil was placed in 10 ml capped vials; 5ml of redistilled methanol was added followed by the addition of a pellet of koh. the content of the vials were heated at 60oc in a heating mantle until the droplets of fats disappeared. upon cooling, the reaction mixture was gently transferred to a separating funnel. small amount of n-hexane was added. separating funnel was shaken gently. the upper hexane layer was recovered and washed with distilled water. this hexane solution was dried over anhydrous sodium sulfate, filtered and used for gas chromatographic analysis. fames were separated on an agilent 5890 series ii gc fitted with a 7673b auto sampler and a capillary column (30 m × 0.25 mm × 0.25 μm) with a db-wax (film thickness 0.20 μm) stationary phase and a flame ionizing detector (fid). helium was used as carrier gas at a flow rate of 1.5 ml/min. other conditions were as follow: injection volume 1μl, split mode (split ratio1:100), injector temperature, 280ºc, initial oven temperature, 170°c; hold up 2 min, 170240ºc (ramp rate 2ºc/min) hold up 10 min, detector temperature 260 ºc. fames were identified by comparing their relative and absolute retention times with those of authentic standards. the fa composition was reported as a relative percentage of the total peak area. the internal standard used was nonadecanoic acid. all of the quantifications were done by agilent chem-station software. statistical analysis three different seed samples for each of the variety were taken and analyzed individually in triplicate and data reported as mean ± sd (n= 3× 3 =9). an analysis of variance (anova) was performed using minitab 2000 version 16.1 statistical software (minitab inc. state college, pa, usa). significant differences (p<0.05) of means were calculated using duncan’s multiple range tests. results and discussion proximate analysis of seeds the data obtained for the proximate analysis of flaxseeds of eight different cultivars grown in pakistan is presented in table 1. oil contents varied from 33.25 to 38.38% indicating a significant difference among cultivars selected (p<0.05). the variety chandani had the highest oil yield whereas ls-13 contained the lowest. in another study from pakistan, anwar et al., (2013) investigated the oil yield for soxhletextracted flaxseed to be 42.80%; such variation in oil yield may be linked to the different extraction method used. the oil content of pakistani flaxseeds are comparable to those grown in canada, north america and egypt, i.e. 36%, 31.9 to 37.8% and 36-39%, respectively (hettiarachchy et al., 1990; oomah and mazza 1998; el-beltagi et al., 2007). however, the present oil contents from pakistani flaxseed cultivars were lower than those reported for polish flaxseed cultivars, i.e., 41.4% (kozlowska, 1989) but higher than ethiopian flaxseed cultivars, 29.1-35.9% (wakjira et al., 2004). such variations in flaxseed oil content with in the countries might be linked to varietal and agro climatic conditions of the regions. the moisture, crude protein, fiber and ash contents of different cultivars of pakistani flaxseed ranged from 5.98 to 6.22%, 16.02 to 18.50%, 23.30 to 26.80% and 3.21 to 3.60%, respectively. there was no significant difference 86 ital. j. food sci., vol. 28 2016 in moisture content for flaxseeds between different cultivars (p>0.05). however, crude protein, fiber and ash contents were notably different among the cultivars of flaxseeds (p<0.05). depending on the cultivar and growing conditions, flaxseed has been reported to contain an average of 23% to 34% protein, 4% ash and 5% fiber (muir and westcott, 2003). our results are comparable to the previous reports on flaxseed cultivars grown in different regions of the world. protein contents of polish and north american cultivars were reported to be greater than 20%, while canadian cultivars had protein generally less than 20% (oomah and mazza, 1998; choo et al., 2007). crude fiber content in different flaxseed residues has been reported to be in the range of 7-10% (gutierrez et al., 2010). the difference in oil, crude protein, and fiber and ash contents of flax seed of different cultivars might be attributed to differences in growing conditions and genetic makeup of flax plants (oomah and mazza 1993; duan et al., 2003). physico-chemical properties of oil the physico-chemical parameters determined for oils extracted from eight different cultivars of flaxseeds are presented in table 2. the results indicated that the refractive index (40°c) and density (25°c) ranged from 1.4706 to 1.4737 and 0.9278 to 0.929 mg/ml, respectively, with nonsignificant (p>0.05) difference among cultivars our findings are consistent with the previous reports in which the refractive index of flaxseed oil at 20˚c was reported to be 1.475, while the density of flaxseed oil at 25˚c was 0.925 to 0.935 (przybylski, 2005). the density of flaxseed oil is greater than most other vegetable oils, and this might be attributed to the greater content of linolenic acid (green and marshall, 1984). the iodine value, unsaponifiable matter, saponification number and acid value are characteristic for flaxseed oils that contain a large percentage of polyunsaturated fatty acids. the iodine values for the tested oils ranged from 195 to 199 g of i 2 /100 g of oil with non-significant table 1 proximate analysis of pakistani flaxseed (linum usittatissimum. l) cultivars. variety parameters oil content (%) moisture content (%) protein content (%) fiber content (%) ash content (%) chandni 38.38±1.80ab 6.02±0.11a 18.50±0.56ab 26.80±1.22a 3.60±0.12ab ls-108 36.38±1.52de 6.12±0.13ab 18.00±0.45d 26.80±0.98a 3.55±0.11d ls-105 37.01±1.46e 6.22±0.15ab 17.98±0.63cd 26.00±1.06ab 3.50±0.11cd ls-99 38.02±1.60c 6.10±0.13a 17.86±0.48bc 25.5±1.14ab 3.50±0.13bc ls-33 35.30±1.35f 5.99±0.12ab 16.02±0.62a 23.50±0.88b 3.21±0.12a ls-31 34.98±1.47a 5.98±0.10ab 16.62±0.54a 23.3±1.16b 3.26±0.14a ls-29 38.00±1.59d 6.02±0.14a 17.99±0.39e 25.75±1.06ab 3.58±0.14e ls-13 33.25±1.31bc 6.00±0.11b 17.00±0.52ab 23.68±1.14b 3.40±0.16ab values (mean ± sd) are average of triplicate samples of each cultivar, analyzed individually in triplicate (n = 1 x 3 x 3), (p<0.05). different letters in superscript indicate significant differences. table 2 physico-chemical characteristics of oil extracted from pakistani flaxseed (linum usittatissimum. l.) cultivars. parameters varieties chandni ls-108 ls-105 ls-99 ls-33 ls-31 ls-29 ls-13 refractive index (40°c) 1.4729±0.009a 1.4728±0.006a 1.4707±0.005a 1.4737±0.007a 1.4732±0.005a 1.4734±0.006a 1.4706±0.009a 1.4728±0.007a density g/ml (25°c) 0.928±0.18a 0.929±0.12a 0.9278±0.14a 0.928±0.14a 0.928±0.16a 0.929±0.15a 0.928±0.19a 0.9279±0.21a iodine value g 198±3.96a 199±4.26a 195.9±3.24a 201±4.39a 195±3.85a 196±4.96a 197±3.68a 199±3.88a of i/100 g of oil unsap matter (%) 2.20±0.04c 2.60±0.04a 2.20±0.02c 2.00±0.02d 2.4±0.03b 1.96±0.05d 2.0±0.02d 1.80±0.04e saponification value mg 189±2.78a 187±3.25a 185.9±3.70a 186±3.46a 185±4.58a 185.86±3.72a 187±3.94a 184±4.71a of koh/100 g of oil ffa (% as oleic acid) 1.399±0.03d 1.579±0.04c 1.624±0.02bc 1.399±0.03d 1.725±0.05a 1.721±0.04ab 1.447±0.02d 1.732±0.05a peroxide value 1.00±0.02c 1.20±0.03ab 1.18±0.02ab 1.00±0.04c 1.14±0.03b 1.20±0.02a 1.22±0.03ab 1.14±0.05b (meq/kg of oil) 1cm 1% (λ 232) 5.07±0.20ab 4.70±0.15b 4.81±0.28b 4.79±0.19b 4.80±0.22b 4.75±0.14b 5.55±0.22a 4.76±0.18b 1cm 1% (λ 268) 1.80±0.08ab 2.00±0.09a 1.90±0.08a 1.60±0.04cd 1.50±0.05d 1.80±0.07ab 1.70±0.05bc 1.40±0.06bcd para-anisidine value 1.13±0.05c 1.41±0.05ab 1.40±0.06ab 1.29±0.05bc 1.36±0.05ab 1.42±0.06ab 1.48±0.05a 1.32±0.04ab values (mean ± sd) are average of triplicate samples of each cultivar, analyzed individually in triplicate (n = 1 x 3 x 3), (p<0.05). different letters in superscript indicate significant differences. ital. j. food sci., vol. 28 2016 87 (p>0.05) among cultivars. iodine value for flaxseed oil has been reported to vary between 180 to 203 g of i 2 /100g of oil (przybylski, 2005). long et al. (2011) reported iodine value of flaxseed oil to be 162 i 2 /100g. the saponification value and unsaponifiable matter of the tested flaxseed oils ranged from 184 to 189 mg of koh/100g of oil and 1.8 to 2.6%, respectively. saponification values did not differ significantly (p>0.05) whereas the unsaponifiable matter varied significantly within the oils of different cultivars (p<0.05). in previous reports, the percentage of unsaponifiable matter in flaxseed oil was in the range of 0.1 to 1.7% for raw oil, and up to 0.6% for refined flaxseed oil (eskinetal, 1996; choo et al., 2007). teh and birch (2013) reported the unsaponifiable value to be 0.4% for cold pressed flaxseed oil. free fatty acids (ffa) are produced by the hydrolysis of triglycerides (lafontan and langin, 2009). the ffa content of the tested flaxseed oils ranged from 1.40 to 1.73%, as oleic acid. the ffa content varied significantly within different flaxseed cultivars (p<0.05). in a previous report, ffa value for flaxseed oil was reported to be 0.1 to 2.0% (przybylski, 2005). long et al. (2011) reported the ffa in the flaxseed oil extracted by enzymatic extraction and solvent extraction to be 1.5 and 1.1%, respectively. ffa value for the cold pressed flaxseed oil was reported to be 0.75% (teh and birch, 2013). ffa in most of the freshly extracted crude vegetable oils is normally below 1.0%. these hydrolytic products are mainly formed as result of chemical hydrolysis (due to presence of moisture in seeds) or enzymatic hydrolysis. a low value of oil ffa is an indication that the seeds have been preserved under proper storage conditions with good state. the peroxide value for the flaxseed oils of different cultivars ranged from 1.0 to 1.22 meq/ kg of oil that is well below the limit for peroxide value. choo et al., (2007) reported the peroxide value ranging from 0.5 to 2.9meq/kg of coldpressed flaxseed oil sold in new zealand. peroxide value for the enzymatic, solvent extracted flaxseed oils was reported to be 1.2 and 1.0 meq/kg of oil, respectively (long et al. 2011) while that for cold pressed flaxseed oil was reported to be 2.04 meq/kg of oil (teh and birch, 2013). peroxide value is an indicator of primary oxidation products; the extent of these products may range up to 10-15 meq/kg of oil (choo et al., 2007). the p-anisidine value of the oils extracted from different cultivars of flaxseed ranged from 1.13 to 1.48(p<0.05). the values are higher than those investigated by choo et al. (2007) who reported the p-anisidine value to be in the range of 0.36 to 0.4. however, the present values were comparable to the values of enzymatically and solvent extracted flaxseed oil reported previously, 1.2 and 1.0, respectively (long et al., 2011). conjugated dienes and trienes produced as a result of secondary oxidation of polyunsaturated fatty acids can be determined by measuring the absorbance at 232 and 272 nm, respectively (frankel, 2005). the specific extinction at 232 and 272 nm of different flaxseed oils ranged from 4.70 to 5.55 and 1.40 to 2.00, respectively. choo et al., (2007) reported absorbencies at 232 and 270 nm of 1.7 to 2.75 and 0.2 to 0.4, respectively for cold pressed flaxseed oil. teh and birch (2013) reported absorbencies at 232 and 272 nm for the cold pressed flaxseed oil to be 2.02 and 0.02, respectively which is very low as compared to our present results. our results are comparable to those reported previously (reed et al., 2001). tocopherol content the tocopherol contents of the different flaxseed oils are shown in table 3. gamma (γ)tocopherol was the main tocopherol in flaxseed oils, with contribution of approximately 90% of the total tocopherols. the γ-tocopherol content ranged from 173.7 to 257.9 mg/kg of oil and significantly differed in different cultivars (p<0.05). alpha (α) tocopherol was the other tocopherol found in the oils (~10%) while delta tocopherol was not detected. the contents of α tocopherol varied from 39 to 18.7 (mg/kg of oil) showing a significant difference among different cultivars (p<0.05). the difference in the contents of tocopherols might be due to the varying getable 3 tocopherol contents (mg/kg) of oil extracted from pakistani flaxseed (linum usittatissimum. l.) cultivars. tocopherol cultivars chandni ls-108 ls-105 ls-99 ls-33 ls-31 ls-29 ls-13 γ-tocopherol 204.0±5.0bc 217.8±4.8b 179.6±5.8de 173.7±5.5e 201.3±6.2c 192.2±4.9cd 257.9±5.6a 190.8±4.3a α-tocopherol 24.9±0.2e 20.4±0.2f 30.2±0.2c 18.4±0.3g 27.2±0.3d 20.9±0.2f 38.5±0.3a 32.8±0.3b δ-tocopherol nd nd nd nd nd nd nd nd total tocopherols 228.9 238.2 209.8 192.1 228.5 213.1 296.4 223.6 values (mean ± sd) are average of triplicate samples of each cultivar, analyzed individually in triplicate (n = 1 x 3 x 3), (p<0.05). different letters in superscript indicate significant differences. nd = not detected. 88 ital. j. food sci., vol. 28 2016 netic makeup and growing conditions of different cultivars (oomah and mazza, 1997). our results are consistent with previous reports in which gamma tocopherol was reported as the predominant tocopherol in flaxseed oils (green and marshall, 1984; herchi et al. 2011). the γand α-tocopherol content of the pakistani cultivars is considerably greater than that in the flaxseed oil varieties of american, canadian, new zealand and turkish origin which contain an average127, 93, 140 and 146 (mg/kg of oil) of tocopherols, respectively (budinetal.,1995; oomah and mazza, 1997; choo et al., 2007; bozan and temelli, 2008). the tocopherol contents of pakistani flaxseed cultivars were comparable to those reported for egyptian cultivars (210 mg/kg of oil) (el-beltagi et al., 2007). teh and birch (2013)reported the contents of γ-tocopherol to be 370 (mg/kg of oil) in cold pressed flaxseed oil, which is considerably higher the present finding. fatty acid profile the fatty acid profile showed a significant variation in the contents of fatty acids within the oils of different flaxseed cultivars (p<0.05) as shown in table 4. the amount of total unsaturated fatty acids in flaxseed oils of the selected cultivars was observed to be in the range of 88.79 to 89.78% while the amount of total saturated fatty acids ranged from 10.21 to 11.20 % with non-significant (p>0.05) varation among cultivars. one distinct feature of flaxseed oil is the presence of high amount of linolenic acid. in the current study the quantities of linolenic acid were observed to be 44.51 to 54.87%, for different cultivars (p<0.05). the results are comparable to the previous reports for american and egyptian flaxseed varieties with 45 to 52% and 46 to 50% alpha linolenic (ala) acid, respectively (declercq et al., 1992; el-beltagi et al., 2007). for ethiopian flaxseed cultivars the ala contents were 52% (wakjira et al., 2004). however, the ala contents of the pakistani cultivars were less than those reported for the flaxseed cultivars grown in new zealand and canada, i.e. 59.65 and 59%, respectively (hettiarachchy et al., 1990; choo et al., 2007). moreover, bozan and tamelli (2008) reported ala levels to be 56.5 to 61% for flaxseed from turkish origin, i.e. greater than our findings. the trends for fa results in the present study are also in agreement with the reports that with an increase in ala in flaxseed oil, there is a corresponding decrease in oleic acid (choo et al. 2007). the flaxseed cultivar ls-33 had the highest contents of ala (54.87%) and lowest amount of oleic acid (21.05%), while ls-99 contained the lowest amount of alpha linolenic acid (44.51%) and the highest amount of oleic acid (30.96%). overall, the amount of linolenic acid ranged from 44.51 to 54.87%, while that of oleic acid ranged from 21.06 to 30.96% for different cultivars of flaxseed grown in pakistan. conclusions the oil yield considerably varied among the selected flaxseed cultivars. similarly, the significant differences for most of the physico-chemical/analytical characteristics among the tested oils were recorded. such variations in oil quality characteristics might be linked to different genetic makeup of the cultivars as well as to their variable harvesting conditions. overall, the flaxseed cultivar chandi, ls-99 and ls-29 had relatively higher oil yield; the cultivar ns29, ls-108, chandi and ls-33 exhibited greater amount of tocopherols whereas those of ls33, ls-29 and ls-105 were rich in alpha linolenic acid (ala) among others. the findings of this comparative study can be useful for selection of economically and nutritionally important flaxseed cultivars, especially, as ingredient for functional foods and nutraceuticals. acknowledgements authors are thankful to the higher education commission of pakistan for the funding of the project under the indigenous ph.d. 5000 scholarship scheme and international research support initiative program. table 4 fatty acid composition (g/100g of fa) of oil extracted from pakistani flaxseed (linum usittatissimum. l) cultivars. fatty acid (fa) varieties chandni ls-108 ls-105 ls-99 ls-33 ls-31 ls-29 ls-13 c 16:0 5.95±0.07cd 5.94±0.07b 5.8±0.06b 6.14±0.07a 6.11±0.19bc 6.52±0.01bc 6.27±0.01d 6.21±0.03cd c 18:0 4.63±0.04c 4.70±0.12d 4.41±0.03cd 4.97±0.08bc 4.83±0.06ab 4.68±0.06a 4.58±0.01d 4.4±0.04bc c 18:1 (n-9) 30.46±0.63ab 28.86±0.53de 26.25±0.35e 30.96±0.47c 21.05±0.45f 28.33±0.56a 24.09±0.58d 26.75±0.59bc c 18:1 (n-7) 1.69±0.58a 1.64±0.51a 1.61±0.54a 1.53±0.41a 1.46±0.44a 1.83±0.64a 1.69±0.49a 1.8±0.63a c 18:2 (n-6) 11.26±0.04ab 11.18±0.01d 7.63±0.74cd 11.88±0.21bc 11.68±0.8a 10.5±0.01a 10.08±0.1e 9.42±0.13ab c 18:3 (n-3) 46.02±0.10f 47.67±0.11d 54.29±0.44c 44.51±0.12e 54.87±0.01a 48.13±0.14g 53.29±0.02b 51.41±0.08e values (mean ± sd) are average of triplicate samples of each cultivar, analyzed individually in triplicate (n = 1 x 3 x 3), (p<0.05). different letters in superscript indicate significant differences. ital. j. food sci., vol. 28 2016 89 references american oil chemists society (aocs) 1997.official and recommended practices of the american oil chemists society, 5th edn. aocs press, champaign. association of official analytical chemists (aoac) 1990.official methods of analysis of the association of official analytical chemists, 15th edn. aoac inc., virginia. bozan b. and temelli f,. 2008. chemical composition and oxidative stability of flax, safflower and poppy seed and seed oils. bioresour. technol. 99: 6354. budin j.t., breene w.m. and putnam, d.h. 1995. some compositional properties of camelina (camelina sativa l.) seeds and oils. j. am. oil chem. soc. 72: 309. choo w.s., birch j. and dufour j.p. 2007. physicochemical and quality characteristics of cold-pressed flaxseed oils. j. food compos. anal. 20: 202. declercq d.r., daun j.k. and tipples k.h. 1992. in: crop bulletin, canadian grain commission, winnipeg, manitoba, canada no. 202, pp. 1. duan j.k., barthet v.j., chornick t.l. and dugiud s. 2003. structure, composition and variety development of flaxseed. thompson l.u., cunnane s.c. 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ital. j. food sci., vol 28, 2016 579 paper chemical, nutritional, physical and antioxidant properties of pecorino d'abruzzo cheese m. mattera*, a. durazzo, s. nicoli, m.g. di costanzo and p. manzi consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria crea, centro di ricerca alimenti e nutrizione, via ardeatina 546, 00178 roma, italy *corresponding author. tel.: +39 06 514941; fax: +39 06 51494550 e-mail address: maria.mattera@crea.gov.it abstract proximate composition, minerals, cholesterol, fat soluble vitamins, fatty acids (fa), colour and antioxidant properties of pecorino d’abruzzo cheese, manufactured at three different months, were investigated. protein, calcium (p≤0.05) and polyunsaturated fa content was higher in winter samples than in summer, whereas cholesterol and total fat were lower. summer cheese samples showed a lower content in saturated fa (55.4%, p≤0.05) and higher content of monounsaturated fa (38.3%, p≤0.05) than winter samples. colour and ft-ir spectra varied (p≤0.05) seasonally. the dairy products supplied a good level of coverage (%) of some nutrients that are daily required according to italian recommendations. keywords: fatty acids, antiradical activity, cholesterol, minerals, colour parameters, fat soluble vitamins ! ital. j. food sci., vol 28, 2016 580 1. introduction traditional food products, closely linked to a system of historic and cultural traditions and connected to a specific geographical area, have an important role in maintaining and supporting agro-biodiversity as well as sustainability. traditional food products are a good source of nutrients, and the levels thereof can be influenced by a range of parameters related to the environment, as well as to food preparation and processing. generally speaking, pecorino is a term indicating cheeses made of raw or thermised ewes’ milk, among which there are cheeses with a defined geographical origin or labelled as protected designations of origin (pdo) reporting also the specification of the region, e.g., romano, sardo, siciliano. in this study, the name of “pecorino d’abruzzo” cheese indicates an italian traditional food product, recognised by the italian ministry of agriculture, food and forest (mipaaf). this hard or semi-hard cheese, made from ewes’ milk, is a local produce linked to the sheep breeding tradition of the homonymous italian region (abruzzo) and it is processed according to a traditional cheese-making technique in farms located in mountain areas. the quality of a product is expression of its own nutrient composition. the information on the physicochemical and nutritional characteristics of “pecorino d’abruzzo” is scarce or currently not available. in addition, there are many literature references on fatty acid (fa) profile and conjugated linoleic acid (cla) contents applying to pecorino cheese in general, however the information on seasonal variation of other nutrients is very limited. according to some authors, variations in milk and cheese ewe’s composition may occur due to different factors. raynal-ljutovac et al. (2008) reported that protein content for ewes’ milk can be different depending on the stage of lactation, season, age and animal feeding. jaeggi et al. (2005) studied the influence of seasonal changes in ovine milk, in details, on the composition and yield of hard-pressed cheese, and found significant differences in the physico-chemical characteristics of the ovine milk collected at different times of the year (february, may and august), as well as in the cheese produced with it. todaro et al. (2014) also reported that the season greatly affects the quality of milk and cheese, as it is the case of the milk and cheese produced by sheeps in valle del belice, mainly fed at pasture for most of the year. addis et al. (2015) analyzed pecorino cheeses manufactured from march to june and they also found that the month of production affected cheese composition. prandini et al. (2011) reported that ewes’ milk and products thereof are richer in cla than other cheeses (e.g. cows, goats). nudda et al. (2005) reported substantial differences in the fatty acids pattern in ewes’ cheeses from march to june, and explained that the progressive variation of fatty acid profile was due to the temporal variation of the animal diet, in terms of grass matured. the most significant seasonal variations were found by de la fuente et al. (2009) in polyunsaturated fa (in particular cla and linolenic acid) contents, with the highest values occurring in spring and summer and the lowest in winter. based on the above, this research, that falls within the framework of the italian project “terravita” (mipaaf), investigated the difference between chemical, physical and nutritional characteristics of “pecorino d’abruzzo” cheeses manufactured at different months (february, july, december). in particular, macronutrients, minerals (sodium, calcium, potassium, magnesium and phosphorous), vitamin a and e, cholesterol, antioxidant properties and colour (cie l*a*b*) parameters were studied, in order to update available data on composition and nutritional properties of this dairy product and investigate how and/or if these results are influenced by the manufacturing season. finally, a qualitative analysis of the major functional groups was also performed by fourier transformed infrared spectroscopy (ftir) on attenuated total reflectance (atr) to show the fingerprint of these cheeses. ! ital. j. food sci., vol 28, 2016 581 2. materials and methods 2.1. samples traditional pecorino cheeses were supplied by a farm located at anversa degli abruzzi, in the province of l’aquila (italy), at 40 km from the two most important national parks of the region: the majella park and the national park of abruzzo. this organic farm was founded in 1977 and generation by generation followed traditional farming practices, typical of that geographical area, in the production of their products. the pecorino d’abruzzo cheeses samples are made of raw organic ewes’ milk, rennet and salt, and are sold with the label “made with organic”. they contain, apart from water and salt, at least 95% percent organically produced ingredients. besides the manufacturer's name or transformation/distribution company the product shows in the label the code of the national control expressly authorized by the italian ministry of agriculture, food and forestry (mipaaf) (eu regulation, no. 271, 24 march 2010). in this research, pecorino d’abruzzo cheeses, made by ewes’ milk collected in different seasons, were studied: the pec-a and pec-c batches belonged to cheeses manufactured in winter time (february and december, respectively), whereas the pec-b batch was produced in summer (july). for each batch (pec-a, pec-b and pec-c), three different samples were analyzed. regarding the manufacture of pecorino d’abruzzo cheeses, after milking the ewes’ milk is warmed up to 38°c, and natural microflora and lamb rennet paste are added. the milk is left curdling for less than one hour, and then the curd is broken, pressed with hands and fitted into special baskets (‘fiscelle’ in italian) to facilitate whey drainage. afterwards, it is salted and stored into ripening rooms. if the maturing process lasts over one month, the rounds periodically grease with olive oil. after purchase, the cheeses under investigation were sampled in appropriate aliquots and stored at -20°c until analysis. 2.2. chemicals all used reagents were of hplc grade or at least of the highest available purity. standards were obtained from sigma aldrich (st louis, mo, usa) and merck kgaa (darmstadt, germany). ultrapure water, of the grade required for critical laboratory applications, was prepared by an ion exchange system to >18 mω resistivity (elgapurelab ultra, veolia, uk). 2.3. analytical methods 2.3.1 proximate composition water, protein, fat and ash contents were determined according to the italian official methods of cheeses analysis (1986), and total carbohydrates were determined by difference to 100 of macronutrient contents. total energy was calculated using the conversion factors according to regulation (eu) no. 1169/2011. the designation according to firmness characteristics was also calculated by the moisture on fat free basis (mffb %), i.e. percent ratio between weight of moisture in the cheese and difference between total weight of cheese and weight of fat in cheese, in accordance to the codex standard for cheese (codex stan 283-1978, amendments 2006, 2008, 2010, 2013). ! ital. j. food sci., vol 28, 2016 582 2.3.2 mineral calcium, magnesium and phosphorous were determined according to the aoac method (aoac, 2002), which was also applied for potassium and sodium. the samples were analyzed after ashing: 1.0 g of sample was weighed into platinum crucibles and ashed in the furnace at 525c° for 16 hours. calcium, sodium, potassium and magnesium were determined using an atomic absorption spectrometer (perkin elmer model: a. analyst 300, norwalk, ct), while phosphorus was measured at 400 nm by a spectrophotometer (shimadzu model: 1800 tokyo, japan). 2.3.3 fatty acid the extraction and determination of fatty acids were performed according to a modified version of the method by prandini et al. (2007). 2 g of sample were dissolved in ethanol/water (2:1, v/v) and extracted twice with diethyl ether/petroleum ether mixture (1:1 v/v). fatty acid methyl esters (fame) were prepared according to the methods of prandini et al. (2007). the samples were analysed by gc (clarus 500-perkin elmer) equipped with flame ionization detector (fid), using a fused silica capillary column sp 2380 (supelco), 60 m x 0.32 mm x 0.2 µm film thickness and helium carrier gas. the oven temperature was programmed as follows: initial temperature 60°c for 5 min, 5°c/min to 180°c and then 3°c/min to 240°c. the injection and detector temperatures were held at 240°c and 260 °c, respectively. 2.3.4 unsaponifiable fraction α-tocopherol, β-carotene, trans retinol and cholesterol were determined according to panfili et al. (1994). briefly, samples were subjected to alkaline digestion and extracted twice with hexane/ethyl acetate (9:1, v/v). the organic phase was collected and evaporated to dryness. the residue was dissolved in 2 ml mobile phase (2-propanol 1% in n-hexane), injected and analysed by an hplc analytical system alliance (waters model: 2695, milford, ma). in the chromatographic procedure, a phenomenex, kromasil 5µm si 250x4.6 mm, a fluorescence detector (waters model: 2475, milford, ma, usa) and uv/vis detector (waters model: 2487. milford, ma, usa) connected in series were utilized to determine cholesterol (208 nm), β-carotene (450 nm), α-tocopherol (excitation 280 nm, emission 325 nm) and retinol (excitation 325 nm, emission 475 nm). 2.3.5 degree of antioxidant protection (d.a.p.) the dap index was calculated as a molar ratio between antioxidant compounds (ac) and oxidation target (ot), as follows (pizzoferrato et al., 2007): ).(. )(.. ... 1 molesnto molesnca pad n i i ° ° = ∑ = generally, α-tocopherol and β-carotene, when present, are used as ac, while cholesterol content was used as ot molecule. ! ital. j. food sci., vol 28, 2016 583 2.3.6 radical scavenging activity the radical scavenging activity was evaluated on fat-soluble vitamins-rich extracts dissolved in 2-propanol 1% in n-hexane by the dpph assay according to the methods of brand-williams et al. (1995), with appropriate modifications. the radical scavenging activities of these extracts were evaluated towards the stable radical 1,1-diphenyl-2picrylhydrazyl (dpph) as follows: 1.5 ml of the extract was reacted with 1.5 ml of dpph radical (60%m), the decrease in absorbance at 515 nm for 400min was monitored and ethyl acetate was used as blank. the control was prepared using 1.5 ml of 2-propanol 1% in nhexane. the percentage of inhibition of dpph was calculated using the formula: % radical scavenging activity (inhibition of dpph activity) = [(abscontrol-abssample) /abscontrol] *100; where abscontrol is the absorbance of the reaction control and abssample is the absorbance in the presence of the sample measured at 400 min, and then the radical scavenging activity was expressed as µmol α-tocopherol/100g. 2.3.7 colour measurements the cie l, a* and b* measurements were made on grated and sliced samples using an handheld tristimulus colorimeter (konica minolta cr-400, minolta limited, milton keynes, uk) controlled with the software cm-s100w (spectramagic nx) with d65 illuminant and 10° viewing angle, calibrated with a white tile. the following parameters were determined: l* value, that is an indicator of luminosity (the degree of lightness) and varies from black to white; the a* value, indicator of green (negative values) and red (positive values); the b* value, indicator of blue (negative values) and yellow (positive values); hue angle (h) and chroma (c*) colour intensity, which is the distance of a colour from the origin (a*=b*=0) in the a*, b* space. 2.3.8 fourier transform infrared (ftir) analysis the ftir spectra were acquired according to lerma-garcía et al. (2010). different slices of each sample were examined by ft-ir (nicolet is10 thermo fisher scientific, usa) with an atr (attenuated total reflectance) accessory (with diamond crystal). each sample was placed in atr surface and pressed with the clamp to obtain a thin layer. all infrared spectra were recorded (32 scans/sample) in the range between 4000 and 700 cm-1 at a resolution of 4 cm-1, with automatic atmospheric suppression. data were elaborated with omnic 8.2.0.387 (thermo fisher scientific inc., usa) software. 2.3.9 statistical analysis analysis of variance, one-way anova test with the tukey’s post hoc comparison, was used for multiple comparison of mean values of nutrients and parameters regarding cheeses produced at different months (february, july, december). p-values ≤0.05 were considered significant. all statistical analyses were performed using the kaleidagraph 3.6 software (synergy software, reading, pa. usa). 3. results and discussions 3.1. proximate composition in table 1 proximate composition, percentage ratio of fat/protein and moisture/fat free basis (mffb), energy values, ph, mineral contents, calcium (ca), phosphorous (p), sodium ! ital. j. food sci., vol 28, 2016 584 (na), potassium (k), magnesium (mg) and the molar ratio of calcium/phosphorous of traditional pecorino d’abruzzo cheeses are reported. in all samples, the ph values were moderately acid, and a slightly but not significant increase from february to december was observed according to cheese manufacturing season. these ph values were higher when compared with those reported by addis et al. (2015) for pdo pecorino romano cheeses and by jaeggi et al. (2005) for ovine hard-pressed cheese. todaro et al. (2014) found that there were slight changes in milk ph during the year, although it was lower in summer and spring. as shown in table 1, the samples in february (pec-a) exhibited a higher value of water 47.5 g/100g and a lower value of protein, fat and ash content than those produced in july (pec-b) and december (pec-c). table 1: proximate composition (g/100g), percentage ratio of fat/protein and moisture/fat free basis (mffb)°, energy values (kcal/100g and kj/100g)°°, ph, mineral contents (mg/100g) and molar ratio of calcium/phosphorous on a fresh matter (f.m.) and dry matter (d.m.) basis of traditional pecorino d’abruzzo cheeses. samples pec-a pec-b pec-c pec-a pec-b pec-c month of production february july december february july december f.m. (%) d.m. (%) water (g/100) 47.5±0.0a 39.1±0.0b 36.9±0.0c ° mffb (%) 64.2 57.4 54.9 * protein (g/100g) 21.6±0.1c 23.2±0.1b 25.1±0.1a 41.2±0.1a 38.0±0.1c 39.8±0.1b fat (g/100g) 26.1±0.1c 32.0±0.2b 32.8±0.1a 49.6±0.2b 52.5±0.3a 51.9±0.2a ash (g/100g) 3.5±0.0c 4.1±0.0b 4.8±0.0a 6.7±0.1b 6.7±0.0b 7.6±0.1a ** carbohydrates 1.3 1.7 0.4 fat/protein (%) 1.2 1.4 1.3 °° energy kcal (kj) 327 (1355) 387 (1606) 397 (1645) ph 5.4±0.0 5.6±0.1 5.8±0.0 ca (mg/100g) 669.2±5.9b 661.0±4.3b 720.0±5.3a 1273.8±11.2a 1085.0±7.1c 1141.9±8.5b p (mg/100g) 458.6±1.9c 484.9±6.4b 504.0±0.7a 872.9±3.7a 795.9±10.5b 799.3±1.1b na (mg/100g) 580.5±5.1c 840.5±0.7b 1074.5±2.0a 1104.9±9.8c 1379.5±1.2b 1704.0±3.1a k (mg/100g) 84.0±0.2a 80.3±0.1b 76.7±1.1c 159.9±0.4a 131.8±0.1b 121.6±1.7c mg (mg/100g) 41.0±0.4 39.5±0.9 39.2±0.6 78.0±0.7a 64.8±1.6b 62.2±0.9b molar ratio ca/p 1.1±0.0 1.1±0.0 1.1±0.0 data are means ± standard deviation (sd) of triplicate analyses; values in the same row with different superscript letters are significantly different (p≤0.05). °moisture/fat free basis (%): in accordance to the codex standard for cheese (codex stan 283-1978); *proteins: total nitrogen 6.38; **carbohydrates calculated by difference to 100 of macronutrient contents; °°energy calculated using the conversion factors according to regulation (ue) no. 1169/2011. ! ital. j. food sci., vol 28, 2016 585 however mffb ranged between 54% and 69% for all samples, and in terms of firmness it designated samples as firm or semi-hard cheeses. on the other hand, when taking into consideration the ripening times, cheeses can be designated as unripened/fresh (codex stan 283-1978). generally, macronutrient content of commercial pecorino cheeses, as reported by raynal-ljutovac et al. (2008), is very wide. fat and protein contents are reported in literature to range 29.0 to 36/37 g/100g and 25.0 to 28.0 g/ 100g, respectively, in different ewe milk cheeses (pecorino cheeses). in a study of manzi et al. (2007) on different pdo italian cheeses (pecorino romano, pecorino toscano, pecorino sardo), the following ranges were found: 23.8 40.8 g/100g for moisture content, 21.8 30.3 g/100g for protein content 30.8 40.2 g/100g for fat content and 3.4 10.1 g/100g for ash. so, the macronutrients of pecorino d’abruzzo cheeses within this study had lower values than those obtained by manzi et al. (2007) for the analysed pdo italian pecorino cheeses. raynal-ljutovac et al. (2008) reported that the protein content for ewes’ milk can range between 4.75 g/100g and 7.20 g/100g and that the main non-individual factors of protein content variation were the stage of lactation, season, age and feeding. jaeggi et al. (2005) found that the ewe’s milk produced in february had a higher fat and a lower protein content (37.19% and 24.84%, respectively) with a lower casein:fat ratio than milk used in may (35.46%; 26.59%) and august (35.70%; 26.13%); and the same applied to cheeses. moreover, todaro et al. (2014) reported that the monthly levels of fat in milk showed a decreasing trend from january to april, and then an increase until reaching a maximum value in july/august. this result could explain the lowest (p≤0.05) fat content among pecorino d’abruzzo samples in february. however, as reported by todaro et al. (2014), the higher fat content in summer cheese depends on the higher percentage of fat observed in summer ewes’ milk, and this fact is the consequence of milk concentration in late lactation and a high level of dry and fibrous forage in ewes’ diet. addis et al. (2015) analyzed several samples of pecorino cheeses manufactured at different times of the year (from march to june) and found that the cheeses produced in late winter and spring were characterized by a less fat and salt content and a higher protein level with respect to those produced in early summer. the outcomes by addis et al. (2015) and todaro et al. (2014) are in accordance with this study, and probably they could explain the differences observed and also expressed on a dry matter (d.m.) basis, for protein, fat and some mineral (ca, p, k, mg) concentrations (table 1). in particular, as regards mineral content, the samples produced during winter had a major protein and ca (p≤0.05) content than those manufactured in summer. 3.2. minerals the content of ca, na and k was significantly (p≤0.05) different among all samples (table 1), whereas a significant difference (p≤0.05) for p and mg contents was found only among samples of cheese produced in february and in those produced in july and december. on dry matter basis, the samples produced in february exhibited the highest value for minerals, such as ca, p, k and mg (1273.8: 872.9: 159.9: 78.0 mg/100g, respectively) and the lowest value for na (1104.9 mg/100g). raynal-ljutovac et al. (2008) also reported for sheep milk cheeses that mg content is less variable, by showing a mean value of 80 mg/100g d.m. that is higher than what was obtained in this study for pecorino d’abruzzo samples (on average 68.3mg/100 d.m.). however, depending on the ph obtained during the cheese-making process, ca and p content ranged between 661.0 and 720.0 mg/100g and 458.6 to 504.0 mg/100g, respectively. regarding the mineral components, few data are available in literature for ! ital. j. food sci., vol 28, 2016 586 sheep milk cheeses. as for the other investigated nutrients, the data obtained for the mineral composition of pecorino d’abruzzo samples were lower than those reported by literature. for instance, in pdo italian cheeses, such as pecorino romano, pecorino sardo and pecorino toscano, the average ca content was, respectively, 938.5, 940.4 and 860.0 mg/100g, and p content was 634.5, 714.8 and 658.9 mg/100g (manzi et al., 2007). in all samples the percentage ratio of fat/protein and the molar ratio ca/p were always higher than 1.0 (table 1). these results may be easily explained by the high content in fat, protein and minerals of the ewes’ cheeses under investigation that thus resulted a food with high nutritional value. the experimental results of this study matched addis et al. (2015) that reported that ewes’ milk produced in june and july is characterized by a higher fat content, generally not offset by an equal increase in protein content. the fat to protein ratio thus ranges from 1.03 to 1.13 in march/april, whereas in june/july it exceeds 1.20 (ranging from 1.22 to 1.35). 3.3. unsaponifiable fraction in table 2, cholesterol, α-tocopherol, trans-retinol and β-carotene contents, and the degree of antioxidant protection (dap exp-3) in samples of traditional pecorino d’abruzzo cheese are shown. table 2: cholesterol (mg/100g), α-tocopherol, trans-retinol, β-carotene contents (µg/100g) and degree of antioxidant protection (dap exp-3) on a fresh matter (f.m.) and dry matter (d.m.) basis of traditional pecorino d’abruzzo cheeses. samples pec-a pec-b pec-c pec-a pec-b pec-c month of production february july december february july december f.m. (%) d.m. (%) cholesterol 72.9±0.4c 96.1±1.1a 81.1±0.0b 138.7±0.8b 157.7±1.9a 128.6±0.0c α-tocopherol 769.2±4.8c 898.8±2.0b 945.9±5.5a 1464.0±9.2b 1475.3±3.3ab 1500,1±8.8a trans -retinol 308.3±0.7 299.2±18.5 309.8±3.4 586.9±1.4a 491.1±30.4b 491.3±5.4b β-carotene nd* nd* nd* dap (exp-3) 9.5±0.1 8.4±0.1 10.5±0.1 data are means ± standard deviation (sd) of triplicate analyses; values in the same row with different superscript letters are significantly different (p≤0.05); *nd= not detectable; β-carotene detection limit 0.16ng/20 µl injected. as regards cholesterol content, cheese samples exhibited on average a value of 83.4 mg/100g, and pec-b cheeses (summer produces) had on average the highest cholesterol content (96.1 mg/100g). however, the amount of cholesterol is quite variable between winter and summer samples, and the values ranged in accordance with the data obtained by manzi et al. (2007) on different pdo italian pecorino cheeses. the winter samples had significantly a lower cholesterol value than those manufactured with milk produced in summer (p≤0.001), even when expressed on dry matter basis (table 2). the cholesterol content seems therefore to be influenced by the manufacture month. ! ital. j. food sci., vol 28, 2016 587 regarding fat-soluble vitamins, there was a great variability in this study: α-tocopherol ranged between 945.9 and 769.2 µg/100g and trans retinol between 299.2 and 309.8 µg/100g. as expected, the β-carotene was not detectable in all studied cheese samples (noziere et al., 2006). upon comparison of cheeses, pec-c samples (december) showed the highest values for αtocopherol, whereas no significant differences (p≤0.05) were observed for trans retinol. on the contrary, significant (≤0.05) differences for both α-tocopherol and trans retinol were found (table 2) by comparing cheeses produced in february with the other samples on dry matter basis. this result could be explained by literature data. according to manzi et al. (2007), in some pdo italian cheeses, the average α-tocopherol content was 850 µg/100g and the range of variability for trans retinol was very wide: 322.1 to 545.9 µg/100g in pecorino romano, 256.2 to 484.6 µg/100g in pecorino sardo and 337.4 to 602.4 µg/100g in pecorino toscano. calderòn et al. (2007) reported that the concentration of vitamin e in cow milk was linearly related to the experimental amount of feed in the diet, and milk characteristics differed according to season. the vitamin e content in milk was higher during the grazing period (may-september) than during the winter feeding period (february-march), and this fact was linked to the proportion of grazed grass or grass silage in the forage, unlike vitamin a (agabriel et al., 2007). indeed, revilla et al. (2014) investigated the effects of milk origin (i.e., sheep, goat and cow) and season on the vitamin composition of different types of cheese, and found that seasonality had a significant effect on the concentration of vitamin a (i.e. higher during summer and autumn), but not on vitamin e. they also reported that the seasonal changes in animal feeding practices were the main reason for the differences observed. so, in this study, the main chemical characteristics of pecorino d’abruzzo cheeses also seem to be affected by the month of manufacture; there is, in fact, a significant (p≤0.05) effect of seasonal change on cheese composition. the degree of antioxidant protection (dap), that is a parameter to estimate the potential oxidative stability of fat in foods, has been also evaluated (table 2). this parameter is related to farm systems and/or cheese manufacture. as reported by pizzoferrato et al. (2007), it can discriminate products from grazing and zero-grazing feeding systems, and when this value is ≥7.0 the pasture is predominant (grazed herbage exceeds 15% in animals’ total diet). in this study, the dap reached the highest values in winter samples (pec-a and pec-c), although it can be stated that in all samples the values were always ≥7.0. despite several studies on quantification of single antioxidants in cheeses are published in literature, there are only few investigations on antioxidant properties of cheeses (gupta et al., 2009). in this research, the radical scavenging activity of fat soluble vitamin-rich extracts, derived from pecorino d’abruzzo samples, was studied. the dpph assay is a simple, quick and cheap method to evaluate the free radical scavenging activity of compounds and/or samples. this evaluation is related to the structure of the active substances present in lipophilic extracts and their possible interactions. this measurement might hence be considered an index of the antioxidant properties of cheeses. lucas et al. (2006) studied the antioxidant properties in french cheeses, such as abondance, tomme de savoie, cantalet, salers and rocamadour and concluded that the variation of the total antioxidant activity in cheeses was probably due to variations in antioxidants, such as αtocopherol, retinol, β-carotene. dpph values of fat soluble vitamin-rich extracts, obtained from pecorino d’abruzzo cheese, were higher in winter than in summer samples (fig. 1) and that thus reflected the behaviour of dap. ! ital. j. food sci., vol 28, 2016 588 figure 1: radical scavenging activity (µmol α-tocopherol/100g) of fat-soluble vitamins-rich extracts derived from traditional pecorino d’abruzzo cheeses. means followed by different letters are significantly different (p≤0.05). 3.4. fatty acid composition fatty acid (fa) composition of traditional pecorino d’abruzzo cheeses is reported in table 3. the samples produced during winter (december) had the highest percentage of short and medium chain fatty acids (scfas, 5.25% and mcfas 21.59%, respectively, p≤0.05), and consequently of saturated fatty acids (sfas 66.25%). on the contrary, the samples produced during summer (july) had the highest percentage of long chain fatty acids (lcfas 81.99%, p≤0.05) and were the richest in mufa (38.30%), primarily all cis and trans c18:1 (pec-b samples 36.58 g/100g of total fa; p≤0.05). the cheeses produced during winter showed on average a large amount of pufa (8.33% and 7.90% in pec-c and peca, respectively). todaro et al. (2014) explained any increase in mufa, especially oleic acid, with a major content in ewes’ milk produced during summer and, hence in cheeses, as a result of the mobilization of long-chain fa from the body fat deposits of the sheep to balance the energy deficits that they undergo in summer, when the feeding regime may not be sufficient to satisfy their energy needs. de la fuente et al. (2009) reported that in ewes’ milk there were significant seasonal variations in pufa content with higher values in spring and summer than in winter. it is possible that factors, such as season and feeding system, could explain also in this study the differences observed. the period of cheese manufacture seems to affect the type and proportion of fa present in milk and in cheeses as well. during summer, the proportion of scfa decreased, while lcfa increased (table 3). in particular, sfa such as lauric acid (c12:0), myristic acid (c14:0) and palmitic acid (c16:0), that are defined atherogenic, resulted more concentrated (p≤0.05) in the pecorino d’abruzzo cheeses produced in winter. these results matched the data reported by several authors (nudda et al., 2005; todaro et al., 2014; de la fuente et al., 2009). on the contrary, the content of stearic acid (c18:0) was higher in pecorino d’abruzzo produced in summer (15.55 g/100g of fa in pec-b sample; p≤0.05) than in winter cheese samples (pec-c). this result is important because it can be used for the biosynthesis of oleic acid in human metabolism, and differed from the other sfas for the beneficial effect on blood cholesterol level: it lowers ldl-cholesterol without affecting blood concentrations of hdl-cholesterol (hunter et al., 2010). ! ital. j. food sci., vol 28, 2016 589 table 3: fatty acid composition given as mean ± s.d. (g/100g of total fatty acids) of traditional pecorino d’abruzzo cheeses. samples pec-a pec-b pec-c month of production february july december free fatty acids lipid numbers usual names means sd means sd means sd c4:0 butyric 2.25 0.10 1.96 0.04 1.81 0.21 c5:0 0.05 0.00 0.12 0.00 0.15 0.06 c6:0 caproic 1.37ab 0.09 1.23b 0.06 1.61a 0.08 c8:0 caprylic 1.16b 0.09 1.09b 0.05 1.68a 0.05 c10:0 capric 3.35b 0.24 3.12b 0.13 5.61a 0.13 c12:0 lauric 2.18b 0.15 2.04b 0.08 3.34a 0.06 c13:0 0.06 0.01 0.04 0.00 0.06 0.00 c14:0 myristic 8.01b 0.51 6.89b 0.17 10.44a 0.11 c14 :1 c9 myristoleic 0.81a 0.04 0.52b 0.01 0.72a 0.03 c15:0 1.04a 0.05 0.75b 0.01 1.16a 0.02 c15:1 0.37a 0.02 0.24b 0.00 0.25b 0.01 c16:0 palmitic 24.88a 1.02 21.28b 0.04 26.93a 0.25 c16 :1 c9 palmitoleic 1.11a 0.05 0.69b 0.00 0.97a 0.06 c17:0 margaric 0.83 0.03 0.68 0.00 0.85 0.12 c17:1 0.26a 0.01 0.18b 0.00 0.26a 0.02 c18:0 stearic 14.23a 0.67 15.55a 0.19 11.98b 0.33 c18:1 (t+c) 29.28ab 3.25 36.58a 0.32 23.15b 1.62 c18:2, t9 t12 linolelaidic 1.08 0.12 1.57 0.15 1.60 0.27 c18:2, c9 c12 (n-6) linoleic acid (la) 3.28a 0.13 2.15c 0.02 2.69b 0.17 c20:0 arachidic 0.53 0.02 0.45 0.00 0.44 0.02 c18:3 (n-6) g-linolenic acid 0.12bc 0.01 0.08c 0.02 0.19a 0.00 c:18:3 (n-3) a-linolenic acid (ala) 1.41a 0.06 0.82b 0.01 1.61a 0.11 c20:1 gondoic 0.08ab 0.00 0.10a 0.00 0.06b 0.00 c18:2, c9 t11 cla rumenic 1.38b 0.04 1.23b 0.00 1.63a 0.09 c20:2 0.29a 0.01 0.21b 0.00 0.29a 0.02 c22:0 behenic 0.26a 0.01 0.17b 0.00 0.18b 0.01 c20:4 (n-6) arachidonic 0.12ab 0.00 0.08b 0.00 0.14a 0.02 c20:5 (n-3) epa 0.12a 0.00 0.08b 0.00 0.12a 0.01 c22:6 (n-3) dha 0.11a 0.00 0.10a 0.00 0.05b 0.00 ω3/ω6 ratio° 0.47 0.43 0.59 ʃ short chain fatty acids (scfa)* 4.84 0.90 4.40 0.76 5.25 0.78 ʃ medium chain fatty acids (mcfa)** 15.81 2.78 13.61 2.44 21.59 3.81 ʃ long chain fatty acids (lcfa)*** 79.35 8.90 81.99 9.91 73.16 8.13 ʃ saturated fatty acids(sfa) 60.21 55.37 66.25 ʃ monounsaturated fatty acids (mufa) 31.89 38.30 25.42 ʃ polyunsaturated fatty acids (pufa) 7.90 6.33 8.33 fatty acids are expressed in g/100g of total fatty acid (fa). data are means ± standard deviation (sd) of triplicate analyses; different letters in the same row correspond to statistical differences (p≤0.05); °ω3/ω6 ratio of total omega 3 fatty acid (ala; epa; dha) and total omega 6 fatty acid (la; γ-linolenic acid; arachidonic acid) *scfa: c4:0 to c9:0; **mcfa: c10:0 to c15:1; ***lcfa: c16:0 to c22:6. ! ital. j. food sci., vol 28, 2016 590 todaro et al. (2014) reported that the ewes’ cheeses produced in summer were lower in saturated fa and higher in linoleic acid (la), mufa and ω3 pufa than those produced in autumn and spring; these results, except for la and ω3 pufa content, are in accordance with the results obtained within this research study. among essential fatty acids (efas), la (c18:2 cis9, cis12 -ω6) differed significantly (p≤0.05) among samples, and reached higher values in winter cheeses (3.28:2.69 g/100g of total fa in pec-a: pec-c, respectively); a similar behaviour was reported for α-linolenic acid (ala, c18:3, ω3). ala is present in food at lower concentrations than la, and it is an important component in cheeses for healthy nutrition (xu, 2015). indeed, ala and γlinolenic acid significantly increased in cheeses produced during winter: 0.12 and 0.19 g/100g total fa for γ-linolenic acid and 1.41 and 1.61 g/100g total fa for a-linolenic acid in pec-a and pec-c, respectively. several studies (astrup et al., 2011) underline that replacing sfas by pufas in the diet decreases the risk of cardiovascular diseases (cvds) and that dietary short-term intake of a cheese naturally rich in cis-9, trans-11 cla appears to cause favourable biochemical changes of atherosclerotic markers in comparison with commercial cheese (sofi et al., 2010). as regards that, it was important to highlight in the studied cheeses the presence of long chain fatty acids (lc-pufas), such as eicosapentaenoic (c20:5 -epa) acid and docosahexaenoic (c22:6 -dha) acid, especially in the samples produced during late winter (february). however, the values obtained in this study for c20:5 and c22:6 were on average higher than those reported by nudda et al. (2005). conjugated linoleic acid (cla), detected as c18:2, c9 t11 (rumenic acid -ra), got higher value in the samples produced in december (1.63 g/100g total fa pec-c) than in samples produced in july. the importance of cla in food, as shown by in vivo studies on animal models, is linked to positive physiological effects, in terms of reduction of the growth rate of cancer cells, improvement of the immune system, and more in general beneficial effects on health (jahreis et al., 1999; whigham et al., 2000; xu, 2015). recently, more information on the effects of cla on body composition and energetic metabolism are reported by lehnen et al. (2015). the consumption of foods naturally enriched with cla (and not supplemented) during lifetime should contribute to reducing increased adiposity and hence the risk of other diseases related to obesity. ewe milk is the richest in cla, among the milk of other ruminant species (except for goat); it also has the highest content on total trans fa, as well as trans vaccenic acid (va), and this was seasondependent (jahreis et al., 1999). thus, the manufacture time, i.e. season, of pecorino cheese is of paramount importance. apropos of that, nudda et al. (2005) reported substantial differences in the fatty acid pattern in ewes’ cheeses from march to june: a decrease in the cla e va concentration in milk fat as lactation progressed. this was probably due to the availability of pasture feeding and a different fa composition of grass lipids. similarly, as reported by addis et al. (2005), the nutritional value of cheese fat, associated in particular with la, ra and va contents, decreased exactly with the progress of season. however, it is known that other factors, such as the period of lactation (kim et al., 2009), the processing temperature (shantha et al., 1992) or ripening time (prandini et al., 2011; kim et al., 2009) may also affect the total amount of cla in cheeses. as regards the ω3/ω6 ratio was higher in winter cheeses than in summer samples (0.59, 0.47, 0.43 for pec-c, pec-a, pec-b, respectively), while a meta-analysis study on dairy products (palupi et al., 2012) shows that the ω3/ω6 ratio is higher in summer than in winter cheeses. the profile of fatty acid composition confirms the important role of ewes’ dairy products, in particular of pecorino d’abruzzo cheese, as a natural source of pufa and cla in human nutrition, especially when manufactured in winter. ! ital. j. food sci., vol 28, 2016 591 3.5. nutritional information the knowledge of the nutritional supply of some nutrients may help to drive dietary choices among consumers and understand the tight relationship between food and health. it is therefore interesting for a nutrient dense food like pecorino cheese to add nutritional information (table 4) on the level of coverage (%) for both energy and some nutrients for human adults (aged 18-59) according to italian recommendations (sinu, 2014) for daily requirements. table 4: daily coverage requirements (%) for i) calcium, phosphorous and vitamin a, out of population reference intake (pri); ii) vitamin e, out of adequate intake (ai); and iii) sodium, out of suggested dietary target (sdt) for the italian population^, and average content of minerals (ca, p, na), vitamins a and e supplied by one serving (50 g) of traditional pecorino d’abruzzo cheeses (sinu, 2014). ^ adult *calcium: coverage (%) out of pri *phosphorous: coverage (%) out of pri °sodium: coverage (%) out of sdt *vitamin a: coverage (%) out of pri °°vitamin e: coverage (%) out of ai man 21.8 3.4 34.2 34.5 20.8 woman 25.5 3.6 average content for serving (50g) mineral (mg) 341.7 241.3 415.9 vitamin a (μg re) 152.9 vitamin e (mg α-te) 0.4 ^aged 18-59 *the italian population reference intake (pri) is 1000 mg/day for calcium, 700 mg/day for phosphorous; 700%g re/day for men and 600%g re/day for women for vitamin a (%g retinol equivalents); °the suggested dietary target (sdt) for sodium is 2.0 g/day (corresponded to nacl ≤ 5 g/day); °°the adequate intake (ai) for vitamin e (mg α-tocopherol equivalents) is 13mg α-te /day for men and 12mg α-te /day for women. from a nutritional point of view, one serving (50 g) of pecorino d’abruzzo cheese added, on average, only 185 kcal (768 kj) to total daily calories intake (table 1), but it is worth pointing out that 50 g of this cheese (table 4) supply 0.4 g of sodium (1.1 g of sodium chloride), that is about 20.8% of the suggested dietary target (sdt; ≤ 2000 mg/day), and 341.7 mg of calcium, that is, about 34.2% out of the italian population reference intake (pri) for adults (1000 mg/day) daily requirements. in addition, this serving (50 g) supplies 0.44 mg of vitamin e, that cover about 3.4% and 3.6% of daily adequate intake (ai) for respectively men and women, and 152.9 µg of vitamin a, that cover in adults about 21.8% and 25.5% of population reference intake (pri) for men and women, respectively, in accordance with italian recommendations (sinu, 2014). 3.6. colour measurements the colour is the first quality parameter evaluated by consumers and an indicator for severe heat treatments, storage, ripening times, etc.; it could also be related to bioactive ! ital. j. food sci., vol 28, 2016 592 compounds profile. however, few studies have been published on the evaluation of colour parameters in pecorino cheeses. for example, juan et al. (2008) when investigating the effect of high-pressure (hp) treatment at 300mpa on ewes’ milk cheese in real industry conditions found that this treatment induced a colour variation, that is, a decrease in lightness and an increase in yellowness related to the structural changes of cheeses. other authors (rohm and jaros, 1996; buffa et al., 2001) studied the changes in colour parameters of cheeses during ripening time, and several changes were observed: a decrease of l and an increase of a* and b* values. the colour parameters measured in both grated (a) and sliced (b) pecorino d’abruzzo cheeses are shown in table 5. in this study (table 5), all colour parameters presented significant differences (p≤0.05) among samples depending on the production period, that is, either in winter or summer, but they were not actually influenced by the shape (grated or sliced). table 5: colour measuraments of a) grated or b) sliced traditional pecorino d’abruzzo cheeses. samples pec-a pec-b pec-c pec-a pec-b pec-c month of production february july december february july december a) grated b) sliced l* 73.9±0.2a 69.0±0.5c 71.0±0.6b 85.7±1.1a 78.5±0.4c 80.5±0.6b a* -3.3±0.1b -3.3±0.1b -2.9±0.1a -5.9±0.2b -6.5±0.1a -6.1±0.1b b* 14.0±0.2b 17.0±0.7a 13.9±0.2b 18.3±0.5b 23.0±0.8a 17.9±1.1b c* 14.4±0.3b 17.3±0.7a 14.2±0.2c 19.3±0.5b 23.9±0.8a 18.9±1.0b h 103.3±0.1a 101.0±0.5b 101.6±0.2a 107.9±0.1a 105.7±0.5b 108.9±1.0a *d65; data are means ± standard deviation (sd); values in the same row with different superscript letters are significantly different (p≤0.05). in particular, all samples showed significant (p≤0.05) differences for l* (brightness). for cheeses manufactured in summer time (july), the brightness (l*) was lower, whereas the b* (yellowness) and c* (croma) values reached higher values than those reported for the samples manufactured during winter (february and december). these results applied to both grated and sliced samples. in the pecorino d’abruzzo samples produced during winter (december or february), the c* value tended to decrease, thus indicating that the samples became grey or looked less brilliant during this period, and suggesting that cheese might run a major risk of loosing colour in winter. moreover, the hue angle (h), that is a qualitative attribute of colour related to the differences in absorbance at different wavelengths, resulted higher in winter samples than in the cheeses produced in july. indeed, higher h values corresponded to lower b* values, and matched a minor yellowness in the cheeses where the β-carotene was not detectable. these are, nevertheless, preliminary results and need further investigation and more specifically targeted studies. 3.7. qualitative application of ftir-atr technique to pecorino d’abruzzo cheeses cheeses are heterogeneous food products, and hence show qualitative differences related to a different chemical composition, maturation process, seasonality and/or various ripening times. the ftir-atr technique has been widely applied in food science for ! ital. j. food sci., vol 28, 2016 593 different purposes, e.g., determination of the geographical origin of cheeses, of sensory and textural properties, shelf life, as well as classification of cheeses according to the manufacture month and manufacturing technique (lerma-garcía et al., 2010; karoui et al., 2005; koca et al., 2007; vlachos et al., 2006). moreover, it is worth highlighting that mid-infrared spectroscopy allows to monitor specific functional groups and smaller molecules (koca et al., 2007). the ftir-atr spectra (range 4000-700 cm-1) of the studied traditional pecorino d’abruzzo cheese provided a qualitative information by characterization of typical absorption bands (fig. 2). indeed, several significant differences appeared on the typical absorption bands arising from amide (1718 to 1581 cm-1 [nh-i] and 1581 to 1483 cm-1 [nh-ii]), from lipids (1765 to 1718 cm-1 [c=o ester] and 2984 to 2831 [ch3 and ch2]), from -nh (in the region 3300 to 2984 cm-1) and -oh (3300 to 3700 cm-1). figure 2: atr-ir spectra average of traditional pecorino d’abruzzo cheeses. in particular, the ft-ir qualitative study of the pecorino d’abruzzo cheeses under investigation allowed the identification of several significant (p≤0.05) changes in peak intensities in absorption bands of specific functional groups vibrations, that identify aliphatic and carbonyl stretching of acyl chain of fatty acids among samples (table 6). however, the ratios ch3 asym/ch2 asym (2957 cm-1/2920 cm-1) and ch3 sym/ch2 sym (2873cm-1/2851 cm-1) were not significant. moreover, as reported in table 6, the region of the carbonyl groups presents a maximum at 1741 cm-1 (vlachos et al., 2006; rohman et al., 2011). the total amount of formed carbonyls or other secondary oxidation products causes a shoulder at 1728 cm-1 (vlachos et al., 2006) that overlaps the stretching vibration at 1741 cm-1, and thus a broadening of this peak to lower wavenumbers results. no significant differences (p≥0.05) were found among winter and summer samples when peak intensity changes are considered in this spectral area. 4. conclusions results show that the main chemical characteristics of traditional pecorino d’abruzzo cheese samples, manufactured at different times of the year, are affected by the month of pec-a pec-b pec-c 0,02 0,04 0,06 0,08 0,10 0,12 0,14 0,16 0,18 0,20 0,22 0,24 0,26 ab so rb a nc e 1000 1500 2000 2500 3000 3500 4000 wav enumber s ( cm1) ! ital. j. food sci., vol 28, 2016 594 production (season). as expected, in this traditional dairy product there was a great compositional variability: winter samples have a major protein content (p≤0.05) and a lower cholesterol and fat content than those manufactured in summer. table 6: wavelengths and peak intensities of main functional groups that identify aliphatic and carbonyl stretching of acyl chain of fatty acids of traditional pecorino d’abruzzo cheeses. samples pec-a pec-b pec-c month of production february july december functional group wavelength cm-1 peak intensities asym -ch3 2957 0.047±0.01 b 0.050±0.00b 0.058±0.00a asym -ch2 2920 0.073±0.01 b 0.092±0.01ab 0.098±0.02a sym -ch3 2873 0.028±0.00 b 0.032±0.01b 0.037±0.01a sym -ch2 2851 0.046±0.01 a 0.061±0.00ab 0.065±0.01b c=o 1741 0.060±0.02 0.087±0.02 0.090±0.02 oxidation shoulder peak 1728 0.040±0.01 0.046±0.00 0.047±0.01 ch3 and ch2 ratio wavelength cm -1 ratio peak intensities ratio asym/asym 2957/2920 0.663 0.548 0.597 sym/sym 2873/2851 0.614 0.524 0.579 data are result of means ± standard deviation (sd) of at least 15 spectra; values in the same row with different superscript letters are significantly different (p≤0.05). cheeses produced during summer showed a lower content in sfas and a higher content of mufas than those produced in winter, whereas pufas and cla were higher in winter samples. however, the ratio ω3/ω6 of fa was higher in winter cheese samples than in those manufactured in summer. the different fa profiles between winter and summer samples are also confirmed by the differences observed in the peak intensities in specific absorption bands of fatty acids. in addition, the dap parameter, as well as the dpph values found in fat-soluble vitaminsrich extracts derived from pecorino cheeses, reached the highest values in winter samples, whereas the information about the differences of colour, e.g. lower brightness (l*) and major yellowness (b*) in summer samples, can help to improve the physical knowledge of this traditional cheese. as regard the nutritional value, pecorino d’abruzzo cheese exhibited appreciable nutritional properties due to a good level of coverage (%) of some nutrients for human adults (aged 18-59) according to italian recommendations for daily requirements. this work thus contributes to improving the information about the nutritional characteristics of pecorino d’abruzzo, that is currently scarce or not available, as well as to updating the italian food composition data base. ! ital. j. food sci., vol 28, 2016 595 acknowledgements this research was financially supported by mipaaf “terravita” project ((dm 25870/7303 02.12.2011). the authors thank dr. francesca melini for the editing and the linguistic revision of this paper. references addis m., cabiddu a., pinna g., decandia m., piredda g., pirisi a. and molle g. 2005. milk and cheese fatty acid composition in sheep fed mediterranean forages with reference to conjugated linoleic acid cis-9, trans-11. j. dairy sci. 88:3443-3454. addis m., fiori m., riu g., pes m., salvatore e. and pirisi a. 2015. physico-chemical characteristics and acidic profile of pdo pecorino romano cheese: seasonal variation. small ruminant res. 126:73-79. agabriel c., cornu a., journal c., sibra c., grolier p. and martin b. 2007. tanker milk variability according to farm feeding practices: vitamins a and e, carotenoids, color, and terpenoids. j. dairy sci. 90:4884-4896. aoac official methods of analysis of the 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2016 106 issn 1120-1770 online, doi 10.15586/ijfs.v33i1.1962 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (1): 106–115 p u b l i c a t i o n s codon analytical approaches for discriminating native lard from other animal fats nazrim marikkar1*, marcello alinovi2, emma chiavaro2* 1national institute of fundamental studies, hanthana road, kandy, sri lanka; 2department of food and drug, university of parma, parma, italy *corresponding authors: marikkar nazrim, national institute of fundamental studies, hanthana road, kandy, sri lanka. email: nazrim.ma@nifs.ac.lk; emma chiavaro, department of food and drug, university of parma, parma, italy. email: emma.chiavaro@unipr.it received: 25 september 2020; accepted: 27 january 2021; published: 25 february 2021 © 2021 codon publications open access paper abstract establishing the distinguishable characteristics of lard from other common animal fats might be helpful for authentication initiatives in foods and feeds. in this study, fatty acid and triacylglycerol compositions, thermal and spectroscopic characteristics of native lard (nl), respectively, were compared with those of beef tallow (bt), mutton tallow (mt), and chicken fat (cf) by using gas liquid chromatography (glc), high-performance liquid chromatography (hplc), and differential scanning calorimetry (dsc). glc analysis showed that the comparison of the overall fatty acid data might not be suitable for the discrimination of different animal fats, but the use of the principal component analysis and the percent palmitic acid enrichment factor [paef (%)] calculations were useful. hplc analysis showed that nl displayed a tag profile, which was quite different from those of either bt or mt, but appeared to be closely similar to that of cf. results of dsc thermal analysis showed that both melting and crystallization curves of nl were remarkably different from those of other animal fats. keywords: animal fats; fatty acid composition; food authentication; thermal analysis; triacylglycerol composition introduction meat has been the main source of protein for humankind from time immemorial. beef, pork, chicken, and mutton have been the preferred sources of meat for mankind. according to the worldwide meat intake assessment, pork is the most widely consumed meat accounting for over 36%, followed by poultry (35%) and beef (22%) (fao, 2012). the consumption patterns of different animal species across the world might vary according to religious beliefs and cultural preferences. for instance, consumption of pork is not desirable for some segments of the society due to their religious restrictions. either deliberate or accidental mixing of pork with other meat species is a great concern for adherents of islam and orthodox judaism. according to some reports, mixing of pork with other commonly consumed meat types is reported to have occurred due to fraudulent reasons (saeed et al., 1989). hence, a great deal of effort has been made to develop analytical methodologies to detect fraudulent practices related to the presence of pork in food systems. researchers in the past frequently employed deoxyribonucleic-acid-based methodologies to differentiate between different meat species in meat products (aida et al., 2005). lipid-based detection methods, on the other hand, are advantageous since lipid component in food matrices is minimally affected by the processing conditions (che man et al., 2005; marikkar et al., 2005; sawaya et al., 1990b). as considerable amount of literature has been available on the detection of animal fats in food systems (che man et al., 2011; marikkar et al., 2005; nurjuliana et al., 2011; nina naqiyah et al., 2017; rohman and che man, 2010; rohman et al., 2011), it would be highly beneficial to discuss the distinguishable mailto:nazrim.ma@nifs.ac.lk mailto:emma.chiavaro@unipr.it italian journal of food science, 2021; 33 (1) 107 analytical approaches for discriminating native lard determination of fatty acid composition the top hexane layer of the fame solution was injected on an agilent 6890n gas chromatograph (agillent technologies, singapore) equipped with a polar capillary column rtx-5 (0.32 mm internal diameter, 30 m length, and 0.25 mm film thickness; restex corp., bellefonte, pa) and a flame ionization detector (fid). split injection was conducted with a split ratio of 58:1 using nitrogen as a carrier gas at a flow-rate of 1.00 ml/min. the temperature of the column was 50°c (for 1 min), and was programmed to increase to 200°c at 8°c/min. the temperatures of the injector and detector were maintained at 200°c (nina naqiyah et al., 2013). hplc analysis of tag composition the separation of triacylglyceride (tag) components of animal fats was performed on a waters model 510 liquid chromatograph equipped with a merck lichrosphere rp-18 column (5 μm) (12.5 cm × 4 mm i.d.; merck, darmstadt, germany) and a differential refractometer model 410 as a detector (waters associates, milford, ma). each fat sample was loaded in the sample vials after dissolving in chloroform and filtering through a whtaman no. 40 filter paper. the isocratic mobile phase was a mixture of acetone:acetonitrile (63.5:36.5 v/v) and the flow rate was set at 1.5 ml/min. each sample was chromatographed three times, and the data were reported as peak area percentages. the identification of the tag peak of the samples was done in accordance with the tag profiles of animal fats reported previously by marikkar et al. (2002). thermal analysis by dsc thermal analyses of all animal fats were carried out on a mettler toledo differential scanning calorimeter (dsc 823 model, columbus, ohio, usa). data elaboration was performed with a thermal analysis software (stare software, version 9.0x, schwerzenbach, switzerland). nitrogen (99.99% purity) was used as the purge gas at a rate of ~20 ml/min. approximately 4–8 mg of molten sample was placed in a standard dsc aluminum pan and then hermetically sealed. an empty, hermetically sealed dsc aluminum pan was used as the control. the samples were held at 70°c isotherm for 1 min to eliminate the thermal history of the samples, then cooled at 5°c/min to –70°c and held for 1 min. the sample was then brought from –70°c to 70°c at the same rate (marikkar et al., 2001; nina naqiyah et al., 2017). statistical analysis all the analyses were performed in triplicate and the results were expressed as mean value ± standard deviation. data were statistically analyzed by one-way analysis of variance (anova) and tukey’s hsd post hoc test using minitabtm statistical software (minitab® characteristics of native lard (nl) from other common animal fats. hence, the purpose of this article is to explore the literature and experimental studies on nl and other animal fats in terms of compositional and physico-chemical characteristics using different analytical techniques, such as gas liquid chromatography (glc), high performance liquid chromatography (hplc), and differential scanning calorimetry (dsc). materials and methods sampling and reagents samples of animal fats, namely, nl, beef tallow (bt), mutton tallow (mt), and chicken fat (cf) were extracted by rendering adipose tissues of animals collected in triplicate from three slaughter houses located in different locations of sri serdang, malaysia. thirty-six samples were collected and analyzed in this study. all chemicals used in this experiment were of either analytical or hplc grade. fat extraction protocol the rendered fat was squeezed out using double-folded muslin cloth. the removal of impurities was carried out by filtering the melted animal fats through cotton wool. a  small proportion of anhydrous sodium sulfate was added to remove residual moisture, and the samples were then filtered through whatman no. 2 filter papers (marikkar et al., 2001). analytical methodologies preparation of fatty acid methyl esters the determination of fatty acid composition required a derivatization step with methyl ester for all the animal fats using sodium methoxide. preparation of fatty acid methyl esters of animal fat was done according to the association of official agricultural chemists (aoac) method 969.33. a 50-mg portion of animal fat was weighed into a 20-ml test tube (with screw cap). after adding 2 ml of 2n sodium hydroxide in methanol, the sample tube was closed and heated at 80°c for 1 h. after allowing the tube to cool down for a few minutes, 2 ml of 25% borontrifluoride solution in methanol was added. the tube was closed and heated again for 1 h at 80°c. subsequently, 5 ml portions of water and hexane were added into this. the contents of the tube were shaken well and allowed to undergo phase separation. the clear supernatant of the solution was transferred into a 2-ml auto-sampler vial (aoac, 2007). 108 italian journal of food science, 2021; 33 (1) marikkar n et al. the degree of unsaturation could also be considered a parameter for the discrimination of nl from other common animal fats. as shown in table 1, unsaturated/saturated (us/s) fatty acid ratio was found to be useful in discriminating nl from bt and mt, but not nl and cf. this finding was largely in conformity with the results previously reported by rashood et al. (1996). if either overall fatty acids (fa) distribution or degree of unsaturation was considered singularly, differentiating nl from cf might not be feasible. in such situations, applying multivariate data analysis techniques such as pca on the overall fatty acid data would be beneficial. in essence, pca helps to reduce the number of observed variables into a smaller number of variables that accounts for most of the variance in a particular dataset. an outcome of pca would possibly group samples with dissimilar characteristics into different groups. as shown in table  1, fatty acids such as lauric acid (c12:0), myristic acid (c14:0), palmitic acid (c16:0), palmitoleic acid (c16:1), stearic acid (c18:0), oleic acid (c18:1) and linoleic acid (c18:2) were present in all four types of animal fats. henceforth, they can be taken as independent variables to develop a pca model. the score plot shown in figure 1 represents the projection of samples defined by the principal component 1 (pc1, which explains 86% of the dataset variance) and the principal component 2 (pc2 which explains 9% of the dataset variance). according to the score plot, a good group separation was achieved with the pca model, in such a way that nl samples were located in the lower left quadrant, cf in the upper left quadrant, mt in the upper right quadrant, and bt in the lower right quadrant. according to the loading plot shown in figure 2, stearic acid (c18:0), oleic acid (c18:1), and linoleic acid (c18:2) were the most discriminating parameters that influence separation along the pc 1 axis, while palmitic acid (c16:0) and palmitoleic acid (c16:1) were the most discriminating parameter along the pc 2 axis. determination of sn-2 positional distribution of fatty acids within the glycerol chain using the pancreatic lipase hydrolysis method was also adopted as an alternative option to find effective discrimination between nl and other animal fats. this procedure is laborious, as it involves isolation of triacylglycerol using column chromatography followed by preparation of 2-mag by pancreatic lipolysis. calculation of the fatty acid enrichment factor (faef) corresponding to individual fatty acid could be possible based on the fa distribution of 2-mag. as shown in figure 3, faef corresponding to the palmitic acid was quite high for nl (283%) when compared to that of any other animal fat [bt (69%); mt (70%); cf (84%)]. this was because more than 74% of the fatty acids present in 2-mag of nl were saturated fatty acids, of which about 68% was palmitic acid (marikkar release 14.12.0, new york, usa) at α = 0.05. principal component analysis (pca), which is a multivariate statistical method that entails data reconstruction and reduction, was carried out using unscrambler 9.7 (camo, usa) software. results and discussions fatty acid distribution fatty acid composition is one of the important attributes that determine the nature of most natural lipids. in this study, the fatty acid composition of four common animal fats is compared as shown in table 1. in nl and other animal fats, oleic acid (c18:1) has been the most dominant fatty acid. palmitic acid (c16:0), linoleic acid (c18:2), and myristic acid (c14:0) are other fatty acid types that occur in appreciable amounts in nl. other than these, occurrence of fatty acids such as c14:1 and c15:0 was rarely detected in nl, but they were detected in minute amounts in other animal fats. according to haas (2005), the prevalent fatty acids of animal depot lipids are either 16 or 18 carbons in their chain length and are either fully saturated or contain one or two double bonds. investigations by marikkar and yanty (2014) showed that nl was generally composed of higher amounts of unsaturated fatty acids (50.2–74.72%) than saturated fatty acids (33.76–46.08%). the relative variations of individual fatty acids in nl has been mainly attributed to differences in sex and the breed types, climatic conditions due to the diet, and the system of animal rearing (enser, 1995; muriel et al., 2002). according to che man et al. (2005), experimental diets composed by soybean or canola oil could cause an increase in the proportions of unsaturated fatty acids in swine fat. among samples extracted from the different body parts of the animals, the fatty acid composition of nl can also slightly vary. however, in the majority of the cases, oleic acid was always found to be the most predominant fatty acid of nl (enser, 1995). similar to nl, cf was also found to possess high oleic acid (43.94%), followed by palmitic acid (25.39%) (table 1). these values were closely comparable to those reported previously by lee and foglia (2000a). in contrast to both nl and cf, the distribution of fatty acids in bt and mt would be remarkably different. particularly, the stearic acid content of bt and mt might differ from that of nl, which is low in saturated fatty acids. hence, this feature was thought to be helpful to distinguish nl from bt/mt. even though the presence of high stearic acid content is a common characteristic feature of both bt and mt, it may be varied slightly due to the influence of various other factor factors, namely, breed, sex, and nutritional conditions (grompone, 1989; grompone and moyna, 1983; holia and press, 1987; yilmaz and karakaya, 2009). italian journal of food science, 2021; 33 (1) 109 analytical approaches for discriminating native lard table 1. fatty acid compositions (%, peak area) of native lard, chicken fat, beef fat, and mutton fat.1 fatty acids native lard chicken fat beef fat mutton fat c10:0 0.08b ± 0.01 — — 0.29a ± 0.06 c12:0 0.19a ± 0.11 0.64a ± 0.93 0.11a ± 0.05 0.46a ± 0.06 c14:0 2.28b ± 1.10 1.62b ± 0.65 6.15a ± 0.31 6.40a ± 0.49 c15:0 0.05c ± 0.04 — 0.46b ± 0.24 0.76a ± 0.02 c16:0 24.64b ± 1.90 25.39b ± 1.01 31.07a ± 0.78 27.38ab ± 1.22 c16:1 1.07c ± 0.46 5.32a ± 0.48 2.56b ± 0.07 0.52c ± 0.18 c17:0 0.25b ± 0.23 – 0.82b ± 0.62 1.85a ± 0.13 c18:0 11.53c ± 1.67 4.84d ± 0.18 16.53b ± 1.25 30.90a ± 0.50 c18:1 42.62a ± 0.74 43.94a ± 1.77 35.70b ± 1.71 29.82c ± 1.04 c18:2 17.29a ± 3.11 18.26a ± 1.64 6.59b ± 0.61 1.61b ± 0.06 ∑sfa 39.02 32.48 55.15 68.05 ∑usfa 60.98 67.52 44.85 31.95 us/s 1.56 2.08 0.81 0.47 1each fatty acid value in the table represents the mean ± standard deviation of three replicates. means within each row with different superscripts are significantly (p < 0.05) different. pc2 pc1 5 0 –5 –10 –15 result7, x-expl: 86%,9% –10 –5 0 5 10 15 20 scores cf cfcf ld ld ld bt bt mf mfmf bt figure 1. score plot of pca based on fatty acid composition for the discrimination of animal fats. abbreviations: ld, lard; cf, chicken fat; bt, beef tallow; mt, mutton tallow. et al., 2005; rashood et al., 1996). this characteristic feature could be effectively used to differentiate nl from other animal fats. tag distributional pattern comparative tag distributional ratios of different triacylglycerol classes of animal fats are presented in table 2. the tag molecules present in most natural oils and fats could be classified into four subgroupings: trisaturates (sss), monounsaturates (sus/ssu), diunsaturates (suu), and triunsaturates (uuu) by considering their degree of unsaturation. according to table 2, there was hardly any significant (p > 0.05) difference among animal fats with respect to the amount of triunsaturates (uuu), but the amount of trisaturates (sss) was remarkably higher for both bt and mt than those for either nl or cf. this was due to the fact that the major tag molecular species of nl were pll, ool, lpo, opo, ppo, and spo, of which spo, lpo, and opo had become the most dominant tag molecular species (marikkar et al., 2002; yanty et al., 2011). on the other hand, the most commonly found tag molecular species of bt and mt were ssp, sss poo, 110 italian journal of food science, 2021; 33 (1) marikkar n et al. x-loadingspc2 –0.4 –0.6 result1.x-expl: 86%,8% –0.4 –0.2 0 0.2 0.4 0.6 0.8 –0.2 0 0.2 0.4 0.6 0.8 pc1 c18:2c/t c12 c14:0 c18:1c c16:0 c18:0 figure 2. loading plot of pca of the animal fats based on the fatty acid composition. (pc1: , pc2: ). 300 250 200 150 e nr ic hm en t f ac to rs (% ) 50 c14:0 fatty acids c16:0 ld cf bt mt c16:1 c18:0 c18:1 c18:2 c18:3 c20:0 0 figure 3. comparative fatty acid enrichment factors (%) of lard and other common animal fats. abbreviations: ld, native lard; bt, beef tallow; mt, mutton tallow; cf, chicken fat. table 2. comparison of trisaturated (sss), monounsaturated (ssu), diunsaturated (suu), and triunsaturated (uuu) triacylglycerol distribution among different animal fats.1 parameter native lard chicken fat beef fat mutton fat % trisaturated 36.15c ± 0.30 33.99d ± 0.44 63.07a ± 0.65 55.11b ± 0.58 % monounsaturated 44.86b ± 0.26 51.52a ± 0.72 34.62d ± 0.35 41.86c ± 0.51 % diunsaturated 18.22a ± 0.28 13.76b ± 0.55 1.84c ± 0.33 2.11c ± 0.45 % triunsaturated 0.69a ± 0.10 0.75a ± 0.14 0.51a ± 0.07 0.95a ± 0.17 us/s ratio 1.76:1 1.94:1 1:1.71 1:1.23 1each value in the table represents the mean of three replicates. means within each row with different superscripts are significantly (p < 0.05) different. italian journal of food science, 2021; 33 (1) 111 analytical approaches for discriminating native lard ppo, ppp, soo, pso, spp, sso, and ooo (marikkar et al., 2002). some early investigations of the 1960s suggested that nl possessed 38% of ssu, 41% of usu, and very low percentages of sus (1%) and uus (7%) (chacko and perkin, 1965). according to the literature, uuu and suu were the most abundant tag species present in cf (lee and foglia, 2000b). among these, loo, ooo, opo, and pso were found to be predominant in cf (marikkar et al., 2002). some major differences in the tag distribution were found between nl and bt/mt, and those differences can make them easily distinguishable. hence, the us/s ratio for nl (1.76:1) was remarkably different from those of either mt (1:1.23) or bt (1:1.71). on the contrary, the differences in the us/s ratio between nl (1.76:1) and cf (1.94:1) were minor, which could make their discrimination more difficult. in order to discriminate between nl and cf, tag peak ratios such as spo/ lpo, lpo/opo, spo/opo, and ool/spo can be calculated. according to figure 4, nl and cf displayed only slight differences among ratios of spo/lpo, lpo/opo, and spo/opo, but showed a strong difference with respect to the ool/spo ratio. in this case, cf was found to show higher ool/spo ratio than that of nl. dsc thermal profiles the overlay presented in figure 5 shows dsc cooling curves of nl (curve a), bt (curve b), mt (curve c), and cf (curve d). dsc cooling curves are recognized worldwide to provide thermal characteristics of plant oils (tan and che man, 2000), animal body fats (yilmaz and karakaya, 2009), lard (yanty et al, 2011), milk fat (campos et al., 2002), etc. according to the cooling curve of nl (figure 5; curve a), there were two well-separated 0 50 100 150 spo/lpo ld cf lpo/opo peak ratio p ea k ra tio × 1 00 spo/opo ool/spo 200 250 300 350 400 450 450 figure 4. selected tag peak ratio of lard and chicken fat. abbreviations: ld, native lard; cf, chicken fat. and defined sharp exothermic thermal transitions, indicating two steps of crystallization; transition above 0°c was defined as the high-melting group (hmg) while the transition below 0 °c was identified as the low-melting group (lmg). these thermal features are largely in conformity with the results mentioned in previous reports (marikkar et al., 2001; rashood et al., 1996; yanty et al., 2011). however, a few differences observed in the onset temperatures and heat flux profiles could be possible due to either sample to sample variation or variation in the dsc scanning rate. in an early report, campos et al. (2002) demonstrated the effect of different cooling rates on the dsc curve of nl. as illustrated in figure 5, the cooling profiles of other animal fats were more complex heat flux curves involving multiple thermal transitions as compared to that of nl. particularly, peak broadening is a characteristic feature seen in curves b, c, and d, indicating a wider range of melting points of the constituent tag molecules. both bt and mt showed sharp thermal transitions at 29.8 and 26.5°c, and broad crystallization transitions at 5.9 and 4.9°c, respectively. the cooling profile of bt was comparable to that of buffalo body fat reported previously by lambelet and ghanguli (1983). the occurrence of broader melting peaks in the low-temperature range in bt and mt could be due to the difference in the nature of tag distributional pattern in these animal fats as compared to that of nl. the cooling profile of cf represented by curve d, displayed a single sharp peak at 8.9°c and a broad peak at –1.9°c. overall, the thermal events recorded by cf in this study were roughly similar to the one reported previously by lee and foglia (2000b). as these features were quite dissimilar from those of nl, the thermal profile obtained 112 italian journal of food science, 2021; 33 (1) marikkar n et al. a (ld) b (bt) c (mt) d (cf) –75 –50 –25 temperature (ºc) 0 25 50 75 e x o h e a tf lo w (w /g ) e n d o figure 5. dsc cooling thermograms of lard and other common animal fats. abbreviations: ld, native lard; bt, beef tallow; mt, mutton tallow; cf, chicken fat. from this study could provide a basis for differentiating cf from nl. a comparison between the heating profiles of nl and other animal fats is shown in figure 6. as previously seen with cooling curves, the heating curves of nl also had two well-separated endothermic transitions, identified as the lowand high-melting regions (curve a). the low-melting region consisted of a major sharp peak at 1.10°c with a shoulder-peak at -17.2°c, while the high-melting transition was represented by a sharp peak with the maxima at 28.25°c. in the melting profile of cf (curve b), there was substantial overlap between the lowand high-melting transitions. the low-melting region consists of a major peak at 1.85°c with two shoulder peaks at -8.0 and -23.0°c, respectively, while the high-melting region is represented by a broad peak resulting from the overlap of two adjacent peaks appearing at 17.2 and 35.6°c, respectively. in the case of bt and mt, their melting curves were comparably similar to each other, but they were remarkably different from those of nl and cf. in the melting curve of bt, the major peak was located at 12.1°c, with two smaller shoulder peaks at 6.5 and –15.0°c, respectively, representing the low-melting region, while the peak appearing at 47.5°c with a shoulder at 34.1°c represented the high-melting region (figure 6). this curve was comparably similar to the dsc melting profile of bt reported previously by rodriguez et al (2001) and aktas and kaya (2001). the melting profile of mt (figure 6, curve d) also showed four steps of melting transitions. the low-melting region of mt is represented by a major peak at 11.4°c with two smaller shoulder peaks at 4.05 and -15.00°c, respectively, while the hmg fraction had a major peak at 45.10°c with a shoulder peak at 32.65°c. conclusions a comparison between lard and other common animal fats has been made on the basis of fatty acid, tag composition, and dsc thermal profiles. the different nature italian journal of food science, 2021; 33 (1) 113 analytical approaches for discriminating native lard a (ld) e x o e n d o h e a tf lo w (w /g ) b (cf) c (bt) d (mt) –75 –50 –25 0 temperature (ºc) 25 50 75 figure 6. dsc heating thermograms of lard and other common animal fats. abbreviations: ld, native lard; bt, beef tallow; mt, mutton tallow; cf, chicken fat. of esterification of different fa in tag molecules is the primary factor that makes animal fats to differ in their chemical and physical characteristics. fatty acid compositional analysis showed that paef of lard was much higher than those of other common animal fats. major differences between nl and bt/mt were also found on the basis of tag composition. the degree of unsaturation of the tag species in animal fats had a strong influence on the tag separation in the reverse-phase silica column and could help differentiate different animal fats. lard displayed a dsc cooling curve with two distinct exothermic sharp peaks at 4.9 and -16.9°c, respectively, while cooling curves of other animal fats were more complex, involving multiple thermal transitions, resulting in broader peaks. references aoac, 2007. official methods of analysis of the aoac international. 18th ed. association of official analytical chemists, washington, dc. aida, a.a., che man, y.b., wong, m.c.v.l., raha, a.r. and son, r., 2005. detection of raw meat and fat of pig using pcr for halal authentication. meat science 69: 47. https://doi.org/10.1016/j. meatsci.2004.06.020 aktas, n. and kaya, m., 2001. detection of beef body fat and margarine in butterfat by differential scanning calorimetry. journal of thermal analysis and calorimetry 66: 795. https://doi. org/10.1023/a:1013196106365 campos, r., narine, s.s. and marangoni, a.g., 2002. effect of cooling rate on the structure and mechanical properties of milk https://doi.org/10.1016/j.meatsci.2004.06.020� https://doi.org/10.1016/j.meatsci.2004.06.020� https://doi.org/10.1023/a:1013196106365� https://doi.org/10.1023/a:1013196106365� 114 italian journal of food science, 2021; 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33 (1) 115 analytical approaches for discriminating native lard yilmaz, m.t. and karakaya, m., 2009. differential scanning calorimetry analysis of goat fats: comparison of chemical composition and thermal properties. journal of the american oil chemists’ society 86: 877. https://doi.org/10.1007/s11746-009-1420-5 chemical composition. journal of the american oil chemists’ society 77: 143. https://doi.org/10.1007/s11746-000-0024-6 yanty, n.a.m., marikkar, j.m.n., che man, y.b. and long, k., 2011. composition and thermal analysis of lard stearin and lard olein. journal of oleo science 60: 333. https://doi.org/10.5650/ jos.60.333 https://doi.org/10.1007/s11746-009-1420-5� https://doi.org/10.1007/s11746-000-0024-6� https://doi.org/10.5650/jos.60.333� https://doi.org/10.5650/jos.60.333� _hlk51074788 _hlk62030237 paper 366 ital. j. food sci., vol. 27 2015 keywords: gelatin, gel strength, extraction, temperature, swim bladder, yellowfin tuna characteristics of gelatin from swim bladder of yellowfin tuna (thunnus albacores) as influenced by extracting temperatures o. kaewdang1, s. benjakul1*, t. prodpran2, t. kaewmanee3 and h. kishimura4 1department of food technology, faculty of agro-industry, prince of songkla university, hat yai, songkhla 90112, thailand 2department of material product technology, faculty of agro-industry, prince of songkla university, hat yai, songkhla 90112, thailand 3department of food science and nutrition, faculty of science and technology, prince of songkla university, pattani 94000, thailand 4laboratory of marine products and food science, research faculty of fisheries sciences, hokkaido university, hakodate, hokkaido 041-8611, japan *corresponding author: tel. 66 7428 6334, fax 66 7455 8866 email: soottawat.b@psu.ac.th abstract gelatin was extracted from the swim bladder of yellowfin tuna (thunnus albacores) at different temperatures (60, 70 and 80°c) with the extraction yields of 35.6%, 41.1% and 47.3% (dry weight basis), respectively. the α-chains of gelatin decreased with increasing extraction temperatures. similar amino acid compositions were noticeable among all gelatins, in which glycine constituted the major amino acid. imino acids ranged from 169 to 172 residues/1,000 residues. the gel strength of gelatin extracted at lower temperature was higher than that of gelatins extracted at higher temperatures. gelling and melting temperatures for swim bladder gelatin were 11.0715.24 and 20.36-22.33°c, respectively. higher gelling and melting points were observed for gelatin extracted at lower temperatures. microstructure of gel of gelatin extracted at 60°c was finer with smaller voids, compared with others. ftir spectra of obtained gelatins revealed the significant loss of molecular order of the triple-helix. thus, extraction temperatures showed the direct impact on characteristics of gelatin from swim bladder. mailto:soottawat.b@psu.ac.th ital. j. food sci., vol. 27 2015 367 introduction gelatin is a fibrous protein obtained by partial denaturation or hydrolysis of collagen. gelatin represents biopolymer with many applications in food, materials (for edible and biodegradable packaging), cosmetic, pharmaceutical and photographic industries (jellouli et al., 2011). the source, type of collagen and processing conditions have the influence on the properties of the resulting gelatin (kittiphattanabawon et al., 2010). different types of gelatins have varying thermal and rheological properties such as gel strength, melting and gelling temperatures (benjakul et al., 2012). these properties are governed by several factors such as chain length or molecular weight distribution, amino acid composition and hydrophobicity, etc. (gómez-guillén et al., 2002; norziah et al., 2009). commercial gelatins are produced mainly from porcine and bovine skins and bones by alkaline or acidic extraction (benjakul et al., 2009). however, both judaism and islam forbid the consumption of any pork-related products, while hindus do not consume cow-related products. additionally, bovine gelatin has a high risk for bovine spongiform encephalopathy (nagarajan et al., 2012). furthermore, the need to exploit the fish processing byproducts has led to the production of gelatin as an alternative to mammalian counterpart (gómez-guillén et al., 2011). fish gelatin has been extracted mainly from fish skin such as seabass (sinthusamran et al., 2014), cobia (silva et al., 2014) skipjack tuna, dog shark and rohu (shyni et al., 2014) and unicorn leatherjacket (kaewruang et al., 2013), etc. among fish processing industries, canned tuna industry is economically important. tuna including yellowfin, skipjack and tongol have been the important species for canning in thailand with a large volume of raw materials used. approximately two-thirds of the whole fish are utilized and the remainings involving the viscera, head, bone and swim bladder become the byproducts (klomklao et al., 2004). fish swim bladders can be used for production of “isinglass” (weber et al., 2009). recently, kaewdang et al., (2014) reported that alkaline pretreatment was essential for gelatin extraction from yellowfin tuna swim bladder. however, no information on the effect of extracting temperature on characteristics and properties of gelatin has been reported. therefore, the objectives of this investigation were to extract and characterize gelatin from the swim bladder of yellowfin tuna using different extraction temperatures. 2. materials and methods 2.1. chemicals all chemicals were of analytical grade. sodium dodecyl sulphate (sds), coomassie blue r-250 and n,n,n’,n’-tetramethylethylenediamine (temed) were procured from bio-rad laboratories (hercules, ca, usa). high-molecular-weight markers were purchased from ge healthcare uk limited (buckinghamshire, uk). food grade bovine bone gelatin with the bloom strength of 150-250 g was obtained from halagel (thailand) co., ltd., (bangkok, thailand). 2.2. collection and preparation of swim bladder swim bladders of yellowfin tuna (thunnus albacares) were obtained from tropical canning public co., ltd., songkhla, thailand. swim bladders with the length of 8-12 cm were placed in polyethylene bags, inserted in ice using a sample/ice ratio of 1:2 (w/w) and transported to the department of food technology, prince of songkla university, songkhla. upon arrival, swim bladders were washed with distilled water and cut into pieces with the length of approximately 2 cm. the prepared samples were then placed in polyethylene bag and frozen at -20°c. the samples were stored at -20°c until used. the storage time was not longer than 3 months. prior to extraction, frozen swim bladders were thawed using running water until the temperature was 0-2°c 2.3. extraction of gelatin from swim bladder prior to gelatin extraction, swim bladders were pretreated with alkaline solution as per the method of kaewdang et al. (2014). prepared swim bladders were added with the mixed alkaline solution (na 2 co 3 :naoh; 7:3) having the concentration of 4% (w/v) at a ratio of 1:10 (w/v). the mixture was stirred for 12 h at room temperature (28-30°c) using an overhead stirrer model w20.n (ika®-werke gmbh & co.kg, stanfen, germany). the alkaline solution was changed every 6 h. the residues were washed with tap water until a neutral or faintly basic ph was obtained. to extract gelatin, alkali pretreated samples were soaked in distilled water with different temperatures (60, 70 and 80°c) using a swim bladder/water ratio of 1:5 (w/v) in a temperature-controlled water bath (w350, memmert, schwabach, germany) for 24 h with a continuous stirring at a speed of 150 rpm. the mixtures were then filtered using a buchner funnel with a whatman no. 4 filter paper (whatman international, ltd., maidstone, england). the filtrates were freeze-dried using a freezedryer (coolsafe 55, scanlaf a/s, l ynge, denmark). the dry gelatin extracted from swim bladder from yellowfin tuna at 60, 70 and 80 °c was referred to as ‘g60’, ‘g70’ and ‘g80’, respectively. all gelatin samples were weighed, calculated for extraction yield and subjected to analyses. 368 ital. j. food sci., vol. 27 2015 2.4. analyses 2.4.1. yield gelatin yield was calculated by the following equation. weight of dry gelatin (g) x 100 yield (%) = ______________________________________ weight of initial dry swim bladder (g) where the weight of dry swim bladder was calculated by subtracting moisture content determined by aoac (2000) from the initial wet weight. 2.4.2. sds-polyacrylamide gel electrophoresis (sds-page) sds–page was performed by the method of laemmli (1970). samples were dissolved in 5% sds solution. the mixtures were then heated at 85°c for 1 h using a temperature controlled water bath model w350 (memmert, schwabach, germany). the mixtures were centrifuged at 8,500 g for 5 min using a microcentrifuge (mikro20, hettich zentrifugan, 170 germany) to remove undissolved debris. solubilized samples were mixed at 1:1 (v/v) ratio with the sample buffer (0.5 m tris– hcl, ph 6.8, containing 5% sds and 20% glycerol). samples were loaded onto a polyacrylamide gel made of 7.5% separating gel and 4% stacking gel and subjected to electrophoresis at a constant current of 20 ma/gel. after electrophoresis, the gels were stained with 0.05% (w/v) coomassie blue r-250 in 50% (v/v) methanol and 7.5% (v/v) acetic acid for 30 min. finally, they were destained with a mixture of 50% (v/v) methanol and 7.5% (v/v) acetic acid for 30 min and destained again with a mixture of 5% (v/v) methanol and 7.5% (v/v) acetic acid for 1 h. high-molecular-weight protein markers were used to estimate the molecular weight of proteins. 2.4.3. amino acid analysis amino acid composition of gelatin samples was analyzed according to the method of nagarajan et al. (2012) with a slight modification. the samples were hydrolyzed under reduced pressure in 4 m methanesulphonic acid containing 0.2% (v/v) 3-2(2-aminoethyl) indole at 115 °c for 24 h. the hydrolysates were neutralized with 3.5 m naoh and diluted with 0.2 m citrate buffer (ph 2.2). an aliquot of 0.04 ml was applied to an amino acid analyzer (mlc-703; atto co., tokyo, japan). 2.4.4. fourier transform infrared (ftir) spectroscopic analysis ftir spectra of the gelatin samples were obtained using a ftir spectrometer (equinox 55, bruker, ettlingen, germany) equipped with a deuterated l-alanine tri-glycine sulphate (dlatgs) detector. a horizontal attenuated total reflectance accessory (hatr) was mounted into the sample compartment. the internal reflection crystal (pike technologies, madison, wi, usa), made of zinc selenide, had a 45° angle of incidence to the ir beam. spectra were acquired at a resolution of 4 cm-1 and the measurement range was between 400 and 4,000 cm-1 (midir region) at room temperature. automatic signals were collected in 32 scans at a resolution of 4 cm-1 and were ratioed against a background spectrum recorded from the clean empty cell at 25°c. analysis of spectral data was carried out using the opus 3.0 data collection software programme (bruker, ettlingen, germany). 2.4.5. determination of gel strength gelatin gel was prepared by the method of kittiphattanabawon et al. (2010). gelatin was dissolved in distilled water (60 °c) to obtain a final concentration of 6.67% (w/v). the solution was stirred until the gelatin was completely solubilized and then transferred to a cylindrical mold with 3 cm diameter and 2.5 cm height. the solution was incubated at the refrigerated temperature (4°c) for 18 h prior to analysis. the gel strength was determined at 8-10°c using a texture analyzer (stable micro system, surrey, uk) with a load cell of 5 kg and crosshead speed of 1 mm/s. a 1.27 cm diameter flatfaced cylindrical teflon® plunger was used. the maximum force (grams), taken when the plunger had penetrated 4 mm into the gelatin gels, was recorded. 2.4.6. determination of gelling and melting temperatures gelling and melting temperatures of gelatin samples were measured following the method of boran et al. (2010) using a controlled stress rheometer (rheostress rs 75, haake, karlsruhe, germany). the gelatin solution (6.67%, w/v) was prepared in the same manner as described previously. the solution was preheated at 35°c for 30 min. the measuring geometry included a 3.5 cm parallel plate and the gap was set at 1.0 mm. the measurement was performed at a scan rate of 0.5°c/min, frequency of 1 hz, oscillating applied stress of 3 pa during cooling from 35 to 5°c and heating from 5 to 35°c. the gelling and melting temperatures were calculated, where tan δ became 1 or δ was 45°. 2.4.7. microstructure analysis of gelatin gel the microstructure of gelatin gel was visualized using a scanning electron microscopy (sem). gelatin gels having a thickness of 2-3 mm were fixed with 2.5% (v/v) glutaraldehyde in 0.2 ital. j. food sci., vol. 27 2015 369 m phosphate buffer (ph 7.2) for 12 h. the samples were then rinsed with distilled water for 1 h and dehydrated in ethanol with a serial concentration of 25%, 50%, 70%, 80%, 90% and 100% (v/v). dried samples were mounted on a bronze stub and sputter-coated with gold (sputter coater spi-module, west chester, pa, usa). the specimens were observed with a scanning electron microscope (jeol jsm-5800 lv, tokyo, japan) at an acceleration voltage of 20 kv. 2.4.8. determination of color of gelatin gel the color of gelatin gels (6.67% w/v) was measured with a hunter lab colorimeter (color flex, hunter lab inc., reston, va, usa). l*, a* and b* values indicating lightness/brightness, redness/greenness and yellowness/blueness, respectively, were recorded. the colorimeter was warmed for 10 min and calibrated with a white standard. the total difference in color (δe*) was calculated according to the following equation. (gennadios et al., 1996): where δl*, δa* and δb* are the differences between the corresponding color parameter of the sample and that of the white standard (l* = 93.6, a*= -0.94 and b* = 0.40). 2.5. statistical analysis all experiments were run in triplicate, using three different lots of samples. data were subjected to analysis of variance (anova) and mean comparisons were carried out using a duncan’s multiple range test (steel and torrie, 1980). statistical analysis was performed using the statistical package for social sciences (spss for windows: spss inc., chicago, il, usa). 3. results and discussion 3.1. extraction yield yield of gelatin from the swim bladder of yellowfin tuna extracted at various temperatures was different. increasing yield was obtained when the extraction temperatures increased (p < 0.05). yield of 35.6%, 41.1% and 47.3% (on dry weight basis) was found for g60, g70, and g80, respectively. this result was in agreement with kaewruang et al. (2013), duan et al. (2011) and kittiphattanabawon et al. (2010) who reported the increasing yield of gelatin as the extraction temperature increased with higher temperatures, the bondings stabilizing α-chains in the native mother collagen were destroyed to a higher extant. as a consequence, the triple helix structure became amorphous and could be extracted into the medium with ease, leading to the higher yield (sinthusamran et al., 2014). in addition, the higher energy applied could induce thermal hydrolysis of peptide chains, resulting in the formation of shorter peptides. as a result, those small peptides could be easily extracted into water. the yield and characteristics of gelatin are associated with the type of raw material and gelatin extraction process, including the pretreatment process and extraction temperatures. (nagarajan et al., 2012; kittiphattanabawon et al., 2010; montero and gómezguillén, 2000). 3.2. protein patterns protein patterns of gelatin from the swim bladder of yellowfin tuna extracted at different temperatures are shown in fig. 1. the band intensity of α 1 -chain and α 2 -chain decreased with increasing extraction temperature. the decreases in α 1 -chain band intensity were observed in g70 and g80, in comparison with that found in g60. among all gelatin samples, g80 possessed the lowest α-chain band intensity. this might be caused by the degradation induced by the thermal process. therefore, the extraction temperatures played a major role in protein components of resulting gelatin. kittiphattanabawon et al. (2010) reported that the gelatins extracted from the skins of brownbanded bamboo shark and blacktip shark with higher extraction temperature contained more peptides with the mw less than α-chain and the lower proportion of high mw (greater than β-chain) fractions, compared with those obtained from lower temperature extraction. gelatins from splendid squid skin with higher extraction temperatures contained a lower band intensity of the α-chains than those obtained with lower extraction temperature (nagafig. 1 protein patterns of gelatins from the swim bladder of yellowfin tuna extracted at different temperatures. m: high molecular weight markers. g60, g70 and g80 represent gelatin extracted from swim bladder at 60, 70 and 80°c, respectively. 370 ital. j. food sci., vol. 27 2015 rajan et al., 2012). on the other hand, gelatin from skin of unicorn leatherjacket extracted at higher temperature (65-75°c) had α-chain retained at higher level than that extracted at lower temperature (kaewruang et al., 2013). this was due to the thermal inactivation of indigenous proteases in the skin of unicorn leatherjacket at high temperature. generally, gelatins with a higher content of α-chains showed better functional properties including gelling, emulsifying and foaming properties (gómez-guillén et al., 2002). in general, the formation of peptide fragments is associated with lower viscosity, low melting point, low setting point, high setting time, as well as decreased bloom strength of gelatin (muyonga et al., 2004a). the results suggested that g70 and g80, which were extracted at higher temperatures, had the shorter chains as indicated by lower content of α-chain. 3.3. amino acid composition amino acid compositions of gelatins from the swim bladder of yellowfin tuna extracted at different temperatures are shown in table 1. glycine was the predominant amino acid in all gelatin samples, ranging from 305 to 314 residues/1000 residues. this implied that gelatin obtained was derived from its mother collagen. collagen consists of one-third glycine in its molecule (balti et al., 2011). it was noted that g80 had the higher glycine content than g60 and g70. the higher glycine in g80 might be caused by free glycine, which was released to a high extent during extraction at high temperature. alanine (121-122 residues/1000 residues) was found at high content. alanine plays a role in viscoelastic property of gelatin (giménez et al., 2005). low contents of cysteine (1 residues/1000 residues), tyrosine (5-6 residues/1000 residues), histidine (7-8 residues/1000 residues) and hydroxylysine (10 residues/1000 residues) were observed in all gelatin samples. for imino acids, all gelatins contained proline and hydroxyproline contents of 95–99 and 72–74 residues/1000 residues, respectively. regenstein and zhou (2007) reported that glycine, alanine, proline and hydroxyproline are four of the most abundant amino acids in gelatin. the properties of gelatin are largely influenced by the amino acid composition and their molecular weight distribution (gómez-guillén et al., 2009). when comparing the content of imino acids (proline and hydroxyproline), gelatin from swim bladder had the lower imino acid content than those from seabass skin (198-202 residues/1000 residues) (sinthusamran et al., 2014) and from carp skin (188-190 residues/1000 residues) (duan et al., 2011). the imino acid content of fish collagens and gelatins correlates with the water temperature of their normal habitat (foegeding et al., 1996; regenstein and zhou, 2007). it has been known that imino acid content, especially hydroxyproline content, affected functional properties of gelatin, especially gelling property (aewsiri et al., 2008; benjakul et al., 2009). therefore, amino acid composition of gelatin from swim bladder was governed by extraction temperature. 3.4. fourier transform infrared (ftir) spectroscopy ftir spectra of gelatins extracted using different temperatures are shown in fig. 2. ftir spectroscopy has been used as a well-established technique to monitor the functional groups and secondary structure of gelatin (kong and yu, table 1 amino acid compositions of gelatins from the swim bladder of yellowfin tuna extracted at different temperatures. amino acids number of residues/1000 residues g60 g70 g80 alanine 121 121 122 arginine 52 52 53 aspartic acid/asparagine 49 48 46 cysteine 1 1 1 glutamic acid /glutamine 80 80 78 glycine 307 305 314 histidine 7 8 7 isoleucine 14 14 13 leucine 29 30 28 lysine 26 26 26 hydroxylysine 10 10 10 methionine 17 16 16 phenylalanine 16 16 16 hydroxyproline 74 72 73 proline 95 99 99 serine 41 41 40 threonine 30 30 30 tyrosine 6 6 5 total 1000 1000 1000 imino acids 169 171 172 fig. 2 atr-ftir spectra of gelatins from the swim bladder of yellowfin tuna extracted at different temperatures (see fig. 1 caption). ital. j. food sci., vol. 27 2015 371 2007). the absorption bands were situated in the amide region. the absorption in the amide-i region, owing to c=o stretching vibration, is probably the most useful for infrared spectroscopic analysis of the secondary structure of proteins (benjakul et al., 2009). it depends on the hydrogen bonding and the conformation of the protein structure (benjakul et al., 2009; uriartemontoyaetal et al., 2011). g60, g70 and g80 exhibited the amide-i bands at the wavenumbers of 1652.8, 1653.7 and 1652.9 cm-1, respectively. the characteristic absorption bands of g60, g70 and g80 in amide-ii region were noticeable at the wavenumbers of 1544.6, and 1545.5 and 1543.5 cm-1, respectively. amide-ii arises from bending vibration of n-h groups and stretching vibrations of c-n groups. in addition, amideiii was detected at the wavenumbers of 1241.9, 1241.3 and 1240.8 cm-1 for g60, g70 and g80, respectively. the amide-iii represents the combination peaks between c-n stretching vibrations and n-h deformation from amide linkages as well as absorptions arising from wagging vibrations from ch 2 groups from the glycine backbone and proline side-chains (jackson et al., 1995). g80 had the lowest amplitude, whereas g60 exhibited the highest amplitude at amideiii region. this indicated that the greater disorder of molecular structure due to transformation of an α-helical to a random coil structure occurred at higher temperature. these changes were associated with loss of triple-helix state as a result of denaturation of collagen to gelatin (muyonga et al., 2004b). the result reconfirmed the higher degradation of gelatin extracted at higher temperatures. amide-a band, arising from the stretching vibrations of the n-h group, appeared at 3338.3, 3339.1 and 3339.3 cm-1 for g60, g70 and g80, respectively. amide-a represents nh-stretching coupled with hydrogen bonding. normally, a free n-h stretching vibration is found in the range of 3400-3440 cm-1 (muyonga et al., 2004b). when the n-h of a peptide is involved in a hydrogen bond, the position shifts to lower frequencies (doyle et al. 1975). in amide-a region, the lower wavenumber was found in g60, suggesting the hydrogen bonding involvement of n-h in α-chain. on the other hand, the lower wavenumber with the concomitantly higher amplitude of amide-a observed in g80 could be associated with the higher degradation of gelatin and higher free amino groups. the amide b was observed at 3082.1, 3080.9 and 3081.8 cm-1 for g60, g70 and g80, respectively. amide b corresponds to asymmetric stretch vibration of =c-h as well as –nh 3 +. thus, the secondary structure of gelatins obtained from the swim bladder of yellowfin tuna was affected to some degree by extraction temperature. 3.5. gel strength gel strength of gelatin from the swim bladder of yellowfin tuna extracted at different temperatures is presented in fig 3. g60, g70 and g80 had the gel strength of 72, 64 and 51 g, respectively. the difference in gel strength between the samples could be due to the differences in intrinsic characteristics, especially molecular weight distribution. protein degradation resulted in the formation of peptides with shorter chain length, which might show the lower ability to from the junction zone or anneal each other. the longer chains in g60 could undergo aggregation to form gel network more effectively than g70 and g80. as a result, a stronger gel network could be formed as indicated by the higher gel strength. bloom strength of commercial gelatins ranges from 100 to 300, but gelatins with bloom values of 250-260 are fig. 3 gel strength of gelatin from the swim bladder of yellowfin tuna with different temperatures. different uppercase letters on the bars denote significant differences (p< 0.05). bars represent the standard deviations (n = 3). (see fig. 1 caption). table 2 gelling and melting temperatures and gel color of gelatin from the swim bladder of yellowfin tuna extracted at different temperatures. samples melting point gelling point colour (c˚) (c˚) l* a* b* δe* g60 22.33±0.42a 15.24±0.27a 27.98±0.57c -2.07±0.02c 8.21±0.11c 66.09±0.57a g70 22.05±0.45a 14.86±0.24a 42.79±0.47b -0.76±0.10b 16.79±0.24b 53.39±0.42b g80 20.36±0.27b 11.07±0.58b 45.79±0.78a -0.34±0.05a 19.03±0.20a 51.32±0.80c mean ±sd (n = 3). different uppercase superscripts in the same column indicate significant differences (p < 0.05). 372 ital. j. food sci., vol. 27 2015 the most desirable (holzer, 1996). different gel strength was reported for gelatin from skin of different species including splendid squid (85–132 g) (nagarajan et al., 2012), brownbanded bamboo shark and blacktip shark (206–214 g) (kittiphattanabawon et al., 2010) and bigeye snapper (108 g) (binsia et al., 2009). 3.6. gelling and melting temperatures the gelling temperatures of all the gelatin samples were in the range of 11.07-15.24°c (table 2). thermal transitions were monitored by changes in the phase angle (δ) of dissolved gelatins during cooling (35-5°c) and subsequent heating (5-35°c). it was found that g80 had the lowest gelling point (11.07°c) (p < 0.05), while no difference in gelling point were observed between g60 and g70 (p > 0.05). in general, fish gelatin is not able to form gel at room temperature (norland, 1990). it has been known that imino acid content is an essential factor governing gelation of getatin (gilsenan and ross-murphy, 2000). however, the similar amino acid content was observed among all samples (169-172 residues/1000 residues). the result indicated that the gelling temperature was affected by the extraction temperature, more likely related with varying chain length. as a thermoreversible gel, gelatin gel starts melting when the temperature increases above a certain point, which is called the gel melting point (karim and bhat, 2009). the melting temperatures of gelatin gel from swim bladder were in the range of 20.36-22.33°c. g80 had the lowest melting point (20.36°c) (p < 0.05). nevertheless, g60 and g70 showed similar melting points (p > 0.05). typical melting points for fish gelatins ranged from 11 to 28°c (karim and bhat, 2009). gómez-guillén et al. (2002) reported that melting points of cod, hake, sole and megrim were 13.8, 14, 19.4 and 18.8°c, respectively. melting points of red and black tilapia skin gelatins were 22.4 and 28.9°c, respectively (jamilah and harvinder, 2002). there was a relationship between melting point and molecular weight of gelatin. low molecular weight gelatins melt at lower temperature than high molecular weight counterparts (gilsenan and rossmurphy, 2000). the results suggested that lower melting point of g80 was attributed to the lower molecular weight of peptide chains. temperature of the environment also affects the gelling and melting temperatures of gelatin (gudmundsson, 2002). poorer gel strength of g80 (fig. 3) was in accordance with lower gelling and melting points. 3.7. microstructures of gelatin gels the microstructures of gelatin gels from swim bladder with different extraction temperatures are illustrated in fig. 4. in general, the conformation fig. 4 microstructures of gelatin gel from the swim bladder of yellowfin tuna extracted at different temperatures. magnification: 3000 (see fig. 1 caption). ital. j. food sci., vol. 27 2015 373 and chain length of the proteins in gel matrix directly contributed to the gel strength of gelatin (benjakul et al., 2009). gelatin extracted at 60°c showed the finest gel network with small voids. conversely, the coarser networks with the larger voids were found in gel of the gelatin extracted at higher temperatures. the fine gel structure of gelatin extracted at lower temperature was in accordance with the higher gel strength (fig. 3). it has been known that the microstructure of the gel is related to the physical properties. the gelatin gel network was governed by the pretreatment conditions (yang et al., 2008) and gelatin concentration (yang and wang, 2009). gelatin extracted at lower temperatures had the lower degradation, in which proteins with higher chain length were present. as a result, junction zones could be formed to a greater extent. this led to the high aggregation with a strong and ordered network. in the first stage of gel network formation, there is competition between intramolecular folding and intermolecular aggregate formation (yang and wang, 2009). for gelatin extracted at lower temperature, longer chains might undergo aggregation to a higher extent. thus, the arrangement of peptides in the network during gelation as determined by chain length directly affected gel properties of gelatin. 3.8. color color of the gelatin gel from swim bladder with different extraction temperatures expressed as l*, a* and b* is shown in table 2. gel of gelatin extracted at lower temperatures (g60) showed the lower l*-value (lightness) than others (g70 and g80) (p < 0.05). the higher redness (a*-value) and yellowness (b*-value) were found in the latters (p < 0.05). generally, the increases in l*, a* and b*-value of gelatin increased with increasing extraction temperatures. for yellowness (b*value), an increase was observed in all gelatin gels when the extraction temperatures increased (p < 0.05). this might be due to a non-enzymatic browning reaction taken place at the higher temperature, especially when the extraction time increased (ajandouz and puigserver, 1999). among all the gelatin samples, those extracted at a lower temperature (60˚c) showed the highest total difference in the color value (δe*) (66.09) with the lowest lightness (l*-values). these results showed that the extraction temperatures had the impact on color of gelatin extracted from the swim bladder of yellowfin tuna. 4. conclusion swim bladder from yellowfin tuna could be an alternative source of gelatin. gelatin extracted at a higher temperature had the highest extraction yield, but possessed the poorer gel properties. extraction conditions also affected the color of resulting gelatin. the appropriate extraction temperature for gelatin from swim bladder was 60 °c, providing the highest gel strength. acknowledgements the 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species in food grade fish gelatins and isinglass with parv-19 anti-parvalbumin antibodies. j. agric. food chem. 57: 11328. yang h. and wang y. 2009. effects of concentration on nanostructural images and physical properties of gelatin from channel catfish skins. food hydrocolloids 23: 577. yang h., wang y., zhou p. and regenstein j.m. 2008. effects of alkaline and acid pretreatment on the physical properties and nanostructures of the gelatin from channel catfish skins. food hydrocolloids 22: 1541. paper received september 17, 2014 accepted november 7, 2014 #351_alvarruiz_bozza ! ital. j. food sci., vol 28, 2016 639 paper drying kinetics of saffron floral bio-residues a. alvarruiz*a, c. lorenzoa, g.l. alonsoa and j. serrano-díaza,b aescuela técnica superior de ingenieros agrónomos, universidad de castilla-la mancha, campus universitario, 02071 albacete, spain bdepartment of food technology, nutrition and food science, regional campus of international excellence campus mare nostrum, murcia university, 30100 murcia, spain *corresponding author. tel.: +39 967599200; fax: +39 967599238 e-mail address: andres.alvarruiz@uclm.es abstract the kinetics of hot-air drying of saffron floral bio-residues was studied at two air-drying temperatures (70 and 90ºc) and four air-flow rates (2, 4, 6 and 8 m·s-1). no constant-rate drying period was observed during drying. ten thin-layer drying models and the theoretical fick’s diffusion model were fit by non-linear regression to drying data. three statistical parameters, chi-squared, correlation coefficient and relative percentage deviation were used to compare the models. effective moisture diffusivity, calculated using fick’s diffusion model, was in the range of 0.78-1.86 x 10-10 m2·s-1. according to the statistical parameters, three drying models (the logarithmic, two-term and midilli-kucuk models) were equally good to describe the drying curve and fit the data better than the other models. the model constants were independent of air-flow rate. the use of air at 90 ºc decreased drying time in half compared with drying at 70ºc. keywords: saffron, floral waste, drying, thin-layer models ! ital. j. food sci., vol 28, 2016 640 1. introduction saffron (crocus sativus l.) spice is the dehydrated stigma of the flowers of this plant of the iridiaceae family. saffron spice production worldwide is about 250 tons. according to the ministry of agriculture of iran (ghorbani, 2008), the main producer exporter of saffron spice is iran (93.7% of world production in 2005), with an export value of $100 million. other countries, such as india, greece, spain, morocco and italy, are also producers and marketers of saffron spice. spain is noted for producing saffron spice with the highest quality recognized worldwide. moreover, spain is also the leader in its trade, because it processes and re-exports saffron spice produced in other countries (i). stigma is only 7.4% of the fresh weight of a flower (serrano-díaz et al., 2013a). tepals, stamens and styles are also part of the flowers of saffron, but they have traditionally been thrown away. about 173,250 flowers weighing over 68 kg were used in castilla-la mancha (spain) in 2009 to obtain 1 kg of saffron spice. as a result, 63 kg of these floral bioresidues (53 kg of tepals, 9 kg of stamens and 0.5 kg of styles) were generated (serranodíaz et al. 2013b). the introduction of the forced cultivation and mechanization of saffron production (garvi palazón, 1987; gracia et al., 2008) will cause an increase in the production capacity and the concentration of these bio-residues in companies producing saffron spice, as stated in the white book of saffron (ii). this new situation is raising interest in the exploitation of saffron floral bio-residues. many studies have demonstrated the biomedical properties of saffron tepal extracts. moshiri et al. (2006) demonstrated the efficacy of the extracts in the treatment of mild-to-moderate depression. fatehi et al. (2003) showed that they lower blood pressure and reduce the contractions induced by electrical field stimulation. hosseinzadeh and younesi (2002) concluded that they have antinociceptive and anti-inflammatory effects. zheng et al. (2011) found that the saffron stamen and perianth possess significant antifungal, cytotoxic and antioxidant activity. bergoin (2005) extracted and characterized the volatile fraction and colorant compounds from fresh flowers and explored their use for the cosmetic, perfume and fragrance industries. the high phenolic content of the saffron floral bio-residues (serrano-díaz et al., 2014b; nørbæk et al., 2002), their antioxidant properties (serrano-díaz et al., 2012) their adequate nutritional composition (serrano-díaz et al., 2013b) and the absence of cytotoxicity (serrano-díaz et al., 2014a) show that these products could be used as food ingredients with high added value. traditionally, the remains of flowers that are generated in the production of saffron spice have been thrown near the saffron field; deterioration within hours has been observed, even though they were exposed to the sun. this spoilage could be due to their high moisture (serrano-díaz et al., 2013b), which favors microbial attack. as in saffron stigmas, there is a need to dewater the saffron floral bio-residues the same day as the flowers are harvested. the technique used for dehydration of the stigma to produce saffron spice differs by country: sun drying, drying at room temperature in air-ventilated conditions (india, iran and morocco), drying at moderate temperatures (greece and italy) and drying at high temperatures (spain). carmona et al. (2005) characterized the timetemperature profile during the traditional dehydration process in castilla-la mancha (spain) compared with other dehydration processes. del campo et al. (2010) studied the effect of mild temperature during dehydration in the main components responsible for the quality of saffron spice. drying is the most common way to preserve the quality of aromatic and medicinal plants (rocha et al., 2011). hot air dehydration, by itself or combined with infrared radiation, has been successfully used to dry a number of flower commodities, such as marigold flower (siriamornpun et al. 2012), torch ginger (juhari et al., 2012), chrysanthemums, roses (castro et al., 2003), chamomile (borsato et al., 2009), daylily (mao et al., 2006) ! ital. j. food sci., vol 28, 2016 641 and oregano (cesare et al., 2004) among others, while preserving their color, antioxidant properties and/or bioactive compounds. serrano-díaz et al. (2013a) studied the conditions of hot-air drying of saffron floral residues to achieve minimal deterioration of the physicochemical quality of these products and concluded that the best quality was achieved with air at 90 ºc combined with a flow rate of 2, 4 and 6 m s-1, but the kinetics of the drying process of these products have never been studied. the aim of this study was to select and test the best drying model for hot-air dehydration of saffron floral bio-residues and to determine the influence of temperature and air-flow rate on dehydration kinetics. 2. materials and methods 2.1. plant material the floral bio-residues generated by saffron spice production were from the agrícola técnica de manipulación y comercialización s.l. company (minaya, spain) during the 2010-2011 harvest season. the floral bio-residues were collected after separating the stigma from flowers using traditional procedures for the protected designation of origin azafrán de la mancha. the thickness of the different floral tissues was measured with calipers. fresh floral bio-residues were stored at -20ºc. 2.2. hot-air drying hot-air drying was performed in a laboratory-scale hot-air dryer (fig. 1). the dryer was equipped with four 500-w electric resistors, coupled to an automatic temperature (± 0.1ºc) controller. the air was impelled trough the drying bed by a 0.5-cv fan equipped with an automatic air velocity controller (± 0.1 m·s-1). the evolution of the product was monitored by weighing the sample periodically with a mettler (switzerland) pm2000 balance (± 0.01 g) linked to a computer. figure 1: hot air dryer set-up. 1: fan, 2: air velocity meter, 3: electric heater, 4: diversion valve, 5: sample holder, 6: scales. ! ital. j. food sci., vol 28, 2016 642 fresh floral tissues were placed on a cylindrical sample holder (9.2-cm diameter) with a perforated bottom. sample size was kept constant (50 ± 2 g) for each experiment. weight loss was recorded at 5-min intervals, and drying was continued until the moisture difference was lower than 5% (w/w). dry runs were performed at temperatures of 70ºc and 90 ºc, with air-flow of 2 m·s-1, 4 m·s-1, 6 m·s-1 and 8 m·s-1. reynolds numbers, calculated using the slab half-thickness as the characteristic dimension (about 5·10-4 m), were on the order of 50, 100, 150 and 200, respectively. the sample temperature during the drying process was determined with an infrared laser thermometer (range: -33 to +250ºc, accuracy: ± 2ºc). sample moisture level was determined with a halogen lamp moisture balance, model xm120t (cobos, barcelona, spain) at 105 ºc, in triplicate. when moisture loss was less than 0.1% in 180 s, samples were considered to have reached constant mass. nine measurements were obtained for each combination of temperature-air flow. 2.3. mathematical models the moisture ratio (mr) was defined as: !" = !!!!!!!!!! eq. 1 where m is sample moisture at time t, me is equilibrium moisture content, and mo is initial moisture content. all moisture content was determined on a dry basis. because drying experiments were carried out using hot air, with very low relative humidity, the moisture ratio was simplified to !/!!. the experimental data were fit to ten thin-layer drying models and to the theoretical fick’s diffusion model for a slab (table 1). references for the model equations can be found elsewhere (akpinar, 2006). table 1: mathematical drying models. model name model equation exponential !" = exp!(−!") page !" = exp!(−!!!) modified page !" = exp!(−!")! henderson and pabis !" = !!exp!(−!") logarithmic !" = ! exp −!" + ! two term !" = ! exp −!!! + !!!"# −!!! two term exponential !" = ! exp −!" + 1 − ! !!"# −!"# wang and singh !" = 1 + !" + !!! verma !" = ! exp −!" + 1 − ! !!"# −!" midilli-kucuk !" = exp −!!! + !" fick’s diffusion eq (2) ! ital. j. food sci., vol 28, 2016 643 the thin-layer drying models are simple empirical models that give good results when the assumptions needed for developing the analytical solutions to fick’s second law, namely the surface resistance or the geometry, are not truly met. their main drawback is that their parameters lack physical meaning. conversely, rigorous or phenomenological models can give a hint to the mechanism of the underlying process. an innovative approach to mathematical modelling of the drying of eggplant slabs considering the shrinkage effect can be found elsewhere (brasiello et al., 2013; russo et al., 2013). the analytical solution to fick’s second law for a slab, in the case of negligible surface resistance, is (crank, 1975): !" = !!! ! !!!! ! ! !!! exp − !!!! !!!!" !!! eq. 2 where d is the effective water diffusivity (m2·s-1), and l is the half-thickness of the slab. for long drying times, eq. 2 can be simplified by taking only the first term in the summation, leading to the henderson and pabis equation with a theoretical value for the constant a of 8/π2. the data were also fit to eq. 2, and the effective diffusivity was calculated. the number of summation terms was adjusted to ensure that the error in mr was less than 0.1%. 2.4. statistical analysis all non-linear regressions were performed using the solver optimization tool (grg nonlinear method) included in the microsoft excel 2010tm spreadsheet, by minimizing the sum of the square differences between the experimental and calculated moisture ratios. comparison of the goodness of fit for each equation was determined by means of the following parameters: correlation coefficient (r), reduced chi-square (χ2) and mean relative percentage deviation (p): !! = !!!! !"!" − !"!" ! !!! eq. 3 ! % = !""! !"!"!!"!" !"!" ! !!! eq. 4 where mrei and mrci are experimental and predicted moisture ratios, respectively; n is the number of experimental data-points; n is the number of model parameters. multiple linear regressions were performed to determine the influence of temperature and air-flow rate using spss 19.0 for windows (spss inc., chicago, il, usa). 3. results and discussions figure 2 shows that the drying rate was decreasing from the beginning of the drying process, and there was no constant rate period. this pattern suggests that the drying resistance would be inside the product rather than in the outside air layer and is in agreement with the results reported for thin-layer drying of similar products, such as ! ital. j. food sci., vol 28, 2016 644 saffron (akhondi et al., 2011), betel leaves (pin et al., 2009), mint leaves (doymaz, 2006) and spinach leaves (doymaz, 2009). figure 2: drying rate of saffron flowers versus moisture ratio. table 2 shows the results of the statistical parameters obtained for each model. the best model was the one with the highest r-values, the lowest χ2 values and the lowest p values. the two-term model was the best with regard to the r and χ2 values, with all r values higher than 0.99987 and χ2 values lower than 1.15·10-5, but the logarithmic model also gave very good agreement, with r values higher than 0.99986 and χ2 values lower than 1.56·10-5. these two models also had very low p values (<4.8%). the midilli-kucuk model had the lowest p values, all lower than 4.2%, very high r values (> 0.99981) and low χ2 values (< 1.89·10-5). therefore, the three best models were the two-term, logarithmic and midillikucuk models. the verma model also gave a good fit, with r > 0.99924, χ2 < 9.40·10-5 and p < 5.4%. in contrast, the theoretical fick’s diffusion model (eq. 2) gave one of the worst fits, with r > 0.98751, χ2 < 3.30·10-3 and p in the range of 9.7-41.3%, while the henderson and pabis model gave better results than the theoretical model (r > 0.99810, χ2 < 2.09·10-4 and p < 10.8%). the logarithmic model has been reported by several authors as the one that gave the best fit for thin-layer drying of a number of products, such as finger millet (radhika et al., 2011), olive cake (akgun and doymaz, 2005), mint leaves (doymaz, 2006), spinach leaves (doymaz, 2009) and apricots (togrul and pehlivan, 2002). pin et al. (2009) found that the logarithmic model gave the best results for drying betel leaves at 40-60ºc, while the midilli and kucuk model was better for drying at 70ºc. in some instances, the two-term drying model proved to be better than the logarithmic model for drying sultana grapes (yaldiz et al., 2001). other authors have claimed that the midilli and kucuk model was the best thin-layer drying model for drying potato, apple and pumpkin slices (akpinar, 2006) or saffron (akhondi et al., 2011). our results agree with those of previous works and confirm that the two-term, the logarithmic and the midilli-kucuk model are the three best models for drying saffron floral bioresidues. this suggests that the verma model could also be acceptable. ! ital. j. food sci., vol 28, 2016 645 table 2: statistical parameters of the drying models. model temperature re χ2 r p (%) exponential 70 50 1.15e-04 0.99995 10.0 100 1.10e-04 0.99903 4.4 150 1.30e-04 0.99974 7.2 200 3.14e-04 0.99905 13.9 90 50 1.38e-04 0.99935 3.7 100 5.32e-04 0.99920 11.2 150 7.52e-05 0.99935 3.1 200 4.83e-04 0.99917 19.0 page 70 50 1.24e-05 0.99989 3.0 100 1.10e-04 0.99903 4.6 150 2.44e-05 0.99976 2.3 200 1.17e-04 0.99876 7.4 90 50 3.86e-05 0.99960 5.0 100 4.00e-05 0.99964 4.7 150 4.90e-05 0.99946 4.7 200 1.81e-05 0.99982 4.5 modified page 70 50 1.24e-05 0.99989 3.0 100 1.10e-04 0.99903 4.6 150 2.44e-05 0.99976 2.3 200 1.17e-04 0.99876 7.4 90 50 3.86e-05 0.99960 5.0 100 4.00e-05 0.99964 4.7 150 4.90e-05 0.99946 4.7 200 1.81e-05 0.99982 4.5 henderson and pabis 70 50 1.15e-05 0.99991 4.8 100 9.40e-05 0.99924 5.4 150 6.04e-05 0.99945 4.5 200 2.09e-04 0.99810 10.8 90 50 1.62e-05 0.99985 3.7 100 2.00e-06 0.99998 1.2 150 3.28e-05 0.99968 4.2 200 4.03e-07 1.00000 1.1 logarithmic 70 50 1.15e-05 0.99991 4.8 100 1.56e-05 0.99986 2.2 150 1.21e-05 0.99987 2.1 200 3.93e-06 0.99996 1.4 90 50 4.07e-07 1.00000 0.3 100 8.22e-07 0.99999 0.4 150 8.63e-07 0.99999 0.7 200 4.03e-07 1.00000 1.1 two term 70 50 1.15e-05 0.99991 4.8 100 2.84e-06 0.99997 0.7 150 5.95e-06 0.99994 1.0 200 2.31e-06 0.99997 0.9 90 50 1.15e-05 0.99987 2.1 100 1.37e-06 0.99999 0.5 150 8.63e-07 0.99999 0.7 200 4.96e-07 1.00000 1.2 ! ital. j. food sci., vol 28, 2016 646 two term exponential 70 50 1.15e-04 0.99995 10.0 100 1.10e-04 0.99903 4.4 150 1.30e-04 0.99974 7.2 200 3.42e-04 0.99876 12.9 90 50 1.38e-04 0.99935 3.7 100 5.32e-04 0.99920 11.2 150 7.52e-05 0.99935 3.1 200 4.83e-04 0.99917 19.0 wang and singh 70 50 1.60e-03 0.99235 38.7 100 1.23e-03 0.99270 18.4 150 1.99e-03 0.99091 22.7 200 2.94e-03 0.98678 31.0 90 50 1.05e-03 0.99390 23.7 100 4.54e-04 0.99680 16.1 150 1.12e-03 0.99341 21.5 200 1.19e-03 0.99288 42.1 verma 70 50 1.13e-05 0.99991 4.6 100 9.40e-05 0.99924 5.4 150 6.64e-06 0.99993 1.2 200 5.64e-06 0.99994 1.4 90 50 1.62e-05 0.99985 3.6 100 2.53e-06 0.99998 1.4 150 3.28e-05 0.99968 4.2 200 1.42e-06 0.99999 0.8 midilli-kucuk 70 50 1.09e-05 0.99991 4.2 100 1.12e-05 0.99990 1.4 150 1.89e-05 0.99981 2.0 200 8.33e-06 0.99991 2.0 90 50 5.10e-06 0.99995 1.2 100 3.43e-06 0.99997 1.3 150 1.03e-05 0.99988 1.7 200 6.33e-06 0.99993 2.3 fick’s diffusion 70 50 2.55e-03 0.99768 33.2 100 2.43e-03 0.99488 13.0 150 1.26e-03 0.99682 9.7 200 1.41e-03 0.98751 16.6 90 50 1.87e-03 0.99625 20.0 100 3.30e-03 0.99609 27.4 150 1.59e-03 0.99628 15.8 200 2.55e-03 0.99586 41.3 table 3 shows the constants for the four best drying models, together with the values of the effective diffusivity obtained with the solution to fick’s equation (eq. 2). akgun and doymaz (2005), doymaz (2006) and doymaz (2009) calculated the effective diffusivities for the simplified fick’s equation for drying olive cake. note that they calculated deff using the traditional method of computing the slope of ln (mr) versus time by linear regression, while we obtained deff by non-linear regression. our results for deff were in the range of 0.78-0.93 x 10-10 m2·s-1 at 70ºc and 1.55-1.86 x 10-10 m2·s-1 at 90 ºc, which is lower than the results for olive cake at the same temperatures (6.252 x 10-9 m2·s-1 and 7.887 x 10-9 m2·s-1, respectively) or spinach leaves at 70ºc (1.5 x 10-9 m2·s-1). ! ital. j. food sci., vol 28, 2016 647 table 3: constants of selected drying models. model temperature re logarithmic a k c 70 50 1.0527 0.0012 0.0000 100 1.0238 0.0011 0.0311 150 0.9638 0.0011 0.0252 200 0.9676 0.0013 0.0417 90 50 1.0948 0.0023 0.0130 100 1.1547 0.0021 0.0037 150 1.0677 0.0022 0.0198 200 1.1786 0.0025 0.0000 two term a k0 b k1 70 50 0.0059 0.0012 1.0468 0.0012 100 0.4423 0.0018 0.6488 0.0008 150 0.4598 0.0016 0.5523 0.0007 200 0.9275 0.0014 0.0915 0.0002 90 50 0.0072 0.0003 1.0666 0.0021 100 0.0068 0.0005 1.1454 0.0021 150 0.0198 0.0000 1.0678 0.0022 200 0.0051 0.0016 1.1737 0.0025 verma a k g 70 50 1.0587 0.0012 0.0089 100 1.0186 0.0010 0.0380 150 0.3303 0.0006 0.0013 200 0.0399 0.0000 0.0013 90 50 1.0747 0.0021 0.0276 100 1.1576 0.0021 0.0135 150 1.0426 0.0020 0.0283 200 1.2324 0.0026 0.0090 midilli-kucuk a k n b 70 50 1.0229 0.00087 1.0390 5.62 x 10-7 100 1.0871 0.00167 0.9352 3.73 x 10-6 150 0.9729 0.00093 1.0107 6.06 x 10-6 200 1.0076 0.00135 0.9838 1.12 x 10-5 90 50 0.9820 0.00087 1.1307 1.22 x 10-5 100 1.0437 0.00087 1.1274 9.03 x 10-6 150 0.9701 0.00089 1.1232 1.55 x 10-5 200 1.0173 0.00089 1.1472 4.28 x 10-6 fick’s diffusion d (m2/s) 70 50 0.92 x 10-10 100 0.78 x 10-10 150 0.82 x 10-10 200 0.93 x 10-10 90 50 1.66 x 10-10 100 1.55 x 10-10 150 1.61 x 10-10 200 1.86 x 10-10 the model constants were regressed against the drying air temperature and flow rate to determine the influence of these variables (table 4). the model constants that gave non! ital. j. food sci., vol 28, 2016 648 significant regressions (p > 0.05) were omitted from the table. note that the flow rate variable was non-significant for all model constants and does not appear in the equations. table 4: influence of drying temperature on model constants. t: absolute temperature (k). model constant regression equation r2 logarithmic a -1.044+0.006t 0.699 k -1.240+5.64·10-5t 0.969 two term a 7.688-0.022t 0.722 b -8.023+0.026t 0.722 k1 -0.025+7.585·10 -5t 0.907 verma k -0.025+7.565·10-5t 0.853 midilli-kucuk n -1.403+0.007t 0.865 fick’s diffusion d -8.985·10-10+4.044·10-12t 0.959 radhika et al. (2011) developed the relation equations between the constants of the logarithmic model and the drying temperature for the drying of finger millet. their results were in agreement with ours for a and k constants. however, we did not find a significant relationship between c and drying temperature. akpinar (2006) obtained regression equations for the four midilli-kucuk model constants and found a significant influence of both air temperature and air-flow rate. in our results, only the n constant depended significantly on temperature. figure 3 shows the variation with time of the experimental moisture ratio for 70 and 90ºc, together with the prediction lines obtained using the logarithmic, two-term and midillikucuk models. figure 3: experimental moisture ratio during drying time and prediction lines with the logarithmic model at 70ºc (a) and 90ºc (b). ! ital. j. food sci., vol 28, 2016 649 it can be seen that the three model lines overlap almost completely; therefore, the three models describe the data equally well. although a small influence of the variable flow rate on mr variation may be apparent from fig. 3, the effect was not statistically significant. the drying time needed to arrive at mr values below 0.05 at 90ºc was about one half the time needed at 70ºc. 4. conclusions drying rate curves for saffron floral bio-residues did not show a constant-rate drying period, and all the drying occurred during the falling-rate drying period. moisture diffusivity obtained using the theoretical fick’s diffusion model was in the range of 0.781.86 x 10-10 m2 s-1 at 70-90 ºc. the logarithmic, two-term and midilli-kucuk models were the best thin-layer model to fit the drying data, but other models, like the verma model, gave also good agreement to the experimental data. the model constants were independent of air-flow rate. regression equations were obtained to describe the influence of temperature on model constants. increasing the temperature from 70 to 90 ºc halved the drying time. acknowledgements the authors thank the agrícola técnica de manipulación y comercialización s.l company (minaya, spain) for providing the samples. they are also grateful to the consejería de educación y ciencia of the jccm and feder for funding this project (ref.: poic10-0195-984). thanks to marco a. garcía and eulogio lópez for technical assistance. references akgun n.a. and doymaz i. 2005. modelling of olive cake thin layer drying process. j. food eng. 68:455. akhondi e., kazemi a. and maghsoodi v. 2011. determination of suitable thin layer drying curve model for saffron (crocus sativus l.) stigmas in an infrared dryer. sci. iran. 18:1397. akpinar e.k. 2006. determination of suitable thin layer drying curve model for some vegetables and fruits. j. food eng. 73:75. bergoin m. 2005. application du concept de raffinage végétal au safran du quercy (crocus sativus) pour la valorisation intégrée des potentiels aromatiques et colorants. m. sc. thesis, institut national polytechnique de toulouse. borsato a.v., doni-filho l., rakocevic m., cocco l.c. and paglia e.c. 2009. chamomile essential oils extracted from flower heads and recovered water during drying process. j. food process. preserv. 33:500. brasiello a., adiletta g., russo p., crescitelli s., albanese d. and di matteo m. 2013. mathematical modeling of eggplant drying : shrinkage effect. j. food eng. 114:99. carmona m., zalacain a., pardo j.e., lópez e., alvarruiz a. and alonso g. l. 2005. influence of different drying and aging conditions on saffron constituents. j. agric. food chem. 53:3974. castro s.g., madamba p.s. and elepaño a.r. 2003. design, development and testing of a small-scale dryer for flowers and foliage. philippine agric. scient. 86:409. cesare l.f., forni e., viscardi d. and nani r.c. 2004. influence of drying techniques on the volatile phenolic compounds, chlorophyll and colour of oregano (origanum vulgare l. ssp. prismaticum gaudin). ital. j. food sci. 16:165. crank j. 1975. “the mathematics of diffusion”. clarendon press, oxford. del campo c.p., carmona m., maggi l., kanakis c.d., anastasaki e.g., tarantilis p.a., polissiou m.g. and alonso g.l. 2010. effects of mild temperature conditions during dehydration procedures on saffron quality parameters. j. sci. food agric. 90:719. ! 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ital. j. food sci., vol 28, 2016 651 tutuncu m.a. and labuza t.p. 1996. effect of geometry on the effective moisture transfer diffusion coefficient. j. food eng. 30 433. yaldiz o. ertekin c and uzun h.i. 2001. mathematical modelling of thin-layer solar drying of sultana grapes. energy 26: 457. zheng c.j., li l., ma w.h., han t. and qin l. p. 2011. chemical constituents and bioactivities of the liposoluble fraction from different medicinal parts of crocus sativus. pharm. biol. 49:756. urls cited (i) http://comtrade.un.org/db/ (april 22, 2015) (ii) www.europeansaffron.eu/archivos/white%20book%20english.pdf (may 29, 2015) paper received december 5, 2015 accepted june 5, 2016 #268_khan_bozza ital. j. food sci., vol 28, 2016 625 paper the characterization of blossom honeys from two provinces of pakistan k.a. khan*, a.a. al-ghamdi and m.j. ansari 1bee research chair, plant protection department, college of food and agriculture sciences, king saud university, po box 2460, riyadh 11451, saudi arabia *corresponding author. khalidtalpur@hotmail.com abstract this study characterized fifteen blossom honeys collected from eleven locations in the punjab and khyber pakhtunkhwa (kpk) provinces of pakistan. mean values for physicochemical parameters (i.e., moisture, water activity, free acidity, ph, electric conductivity, diastase activity, total acidity, ash, total protein, hydroxymethylfurfural, fructose, glucose, sucrose, maltose, raffinose, reducing sugars, total sugars, and fructose/glucose ratio) were 18.09%, 0.57, 13.52 meq kg-1, 4.27, 414.41µs cm-1, 10.56 dn, 26.80 meq kg-1, 0.15%, 313.30 mg 100 g-1, 15.39 mg kg-1, 35.49%, 30.77%, 4.41%, 2.12%,0.11%, 66.25%, 72.88%, and 1.16, respectively. the sucrose content was slightly high in three honeys. in general, all of the remaining honey samples met the criteria for international honey standards. keywords: chemical analyses, comparison, honeys, qualitative properties ! ital. j. food sci., vol 28, 2016 626 1. introduction honey is a natural sweet material made by honeybees from floral nectar, plant secretion or excretion of insects (which suck the sap from living plant parts), that honeybees collect, modify and intermix with their own particular substances, deposit and leave it in the cells of the comb to ripen and mature (mendes et al., 1998). humans have used honey as a reliable sweetener for centuries. honey holds a particular place in the food and medical industries, and it has been regarded as a highly nutritive food in many civilizations (feás et al., 2010 a). pakistan has diverse landscape, climate and environmental conditions, including sandy beaches, deserts, high mountains and pictorial valleys, each featuring specific vegetation. the country has a great potential for honey production because of its congenial climate conditions and variety of bee flora. punjab, the most populous province of pakistan, has the majority of its bee flora in the northern and central regions, where an ample amount of honey is harvested (izhar-ul-haq et al., 2010). khyber pakhtunkhwa (kpk) also has environmental conditions that are conducive for the growth of high floral biodiversity that contributes to honey production, and its export to the middle east and western countries (mairaj et al., 2008). variation in the composition of honey largely arises from the climate and environmental conditions of an area, its nectar and pollen, and the abilities of beekeepers (white, 1978). the physicochemical properties of honey affect its honey storage, quality, granulation, texture, flavor, and nutritional characteristics as well as its medicinal qualities (iftikhar et al., 2011). the characterization of honey promotes the understanding of its medicinal properties as well as its antibacterial and antioxidant characteristics (adebiyi et al., 2004). most of its physicochemical properties can be used to reveal adulteration; therefore, studies of certain quality parameters are needed to ensure the purity of honey (khan et al., 2006). this study sought to characterize the quality parameters and discriminative properties of 15 different blossom honeys collected from diverse areas of two provinces of pakistan. previous work has mainly focused on a few samples from specific locality. 2. materials and methods all of the chemicals and reagents employed in this study were of analytical grade. fructose, glucose, sucrose, maltose, raffinose, bovine serum albumin, folin & ciocalteu’s phenol reagent, phosphoric acid and 5-(hydroxymethyl) furfural were purchased from sigma-aldrich (st. louis, mo, usa). hiper solv for hplc™, water for high-performance liquid chromatography hplc, was obtained from bdh laboratory supplies (poole, uk). acetonitrile (hplc grade) was acquired from fisher scientific uk ltd. (leicestershire, uk). 2.1. honey samples in this study, fifteen samples of blossom honey were collected from different apiaries of punjab and kpk provinces between march and april 2014. nine samples, i.e., eucalyptus (eucalyptus spp.), sunflower (helianthus annuus), acacia (acacia spp.), mustard (brassica campestris), ziziphus (ziziphus mauritiana), clover (melilotus officinalis), citrus (citrus spp.), currant bush (carissa opaca), and multifloral honeys, were collected from seven locations of punjab, whereas six honey samples, i.e., loquat (eriobotrya japonica), eucalyptus (eucalyptus spp.), ziziphus (ziziphus mauritiana), acacia (acacia spp.), citrus (citrus spp.), and clover ! ital. j. food sci., vol 28, 2016 627 (melilotus officinalis), were collected from four locations of kpk (fig. 1). these samples were brought to the chair of engineer abdullah ahmad buqshan for bee research, plant protection department, college of food and agriculture sciences, king saud university, riyadh, saudi arabia and placed in a refrigerator at 4°c until analysis in may 2014. figure 1: map of pakistan showing the honey collection sites in punjab and khyber pakhtunkhwa. 2.2. moisture content the moisture content (expressed as a percentage) of all honey samples was determined via refractometery using a refractometer (abbe mark ii model 10480, cambridge instruments inc., buffalo, ny, usa) at 20°c according to the guidelines of the international honey commission (ihc) (bogdanov, 2002). 2.3. water activity (aw) water activity in honey samples was measured using a bench-top aqualab cx-2 water activity meter (decagon device, inc., wa, usa) at 20°c (habib et al., 2014). 2.4. electrical conductivity (ec) ec was measured by making a 20% (w/v) honey solution in distilled water according to ihc guidelines (bogdanov, 2002) via an hi-9835 ec/tds/nacl meter (hanna instruments, woonsocket, ri, usa). the measurements were recorded in µs cm-1. ! ital. j. food sci., vol 28, 2016 628 2.5. ash content ash content was measured using a predetermined ec value for the honey samples and by substituting those values into the following formula: x1 = (x2 0.143)/1.743, where x1 denotes the ash content and x2 represents the ec (in ms cm-1) at 20°c (piazza et al., 1991). the ash content is expressed as a percentage. 2.6. ph determination to measure ph, 5 g of honey was mixed in 25 ml of distilled water, and a ph reading was taken using a professional benchtop bp3001 ph meter (trans instruments, singapore). 2.7. acidity (free and total) ten grams of honey was dissolved in 75 ml of co2-free water (micropure uv tankthermo scientific, hungary) in a 250-ml beaker. this solution was titrated with 0.1 m sodium hydroxide solution until the ph reached 8.3. then, the free and total acidity were measured according to ihc guidelines (bogdanov, 2002) and are expressed in meq kg-1. 2.8. diastase activity (da) da was determined according to feás et al. (2010a). five grams of honey was placed in a beaker and dissolved completely in 20 ml of distilled water and 2.5 ml of an acetate buffer solution. this mixture was transferred to a flask containing 1.5 ml of nacl solution. then, 10 ml of this solution was placed in a 50-ml volumetric flask and placed in a thermostatic bath (thermolab industries) at 40°c with a second flask containing 10 ml of starch solution. after 15 min, 5 ml of starch solution was pipetted into the honey solution and mixed. after the first 5 min, 1 ml aliquots from this solution were removed, and 5 ml of iodine solution was added at periodic intervals. the sample absorption was monitored at 660 nm against a water blank in a 1 cm cell using a perkinelmer lambda 25, uv/vis/spectrometer (shelton, ct, usa). da is expressed in gothe units per gram of honey, i.e., the “diastase number” (dn). one gothe unit is the amount of enzyme that converts 0.01 g of starch into a given point in 1 h at 40°c. 2.9. sugar content in this experiment, 5 g of honey were mixed in distilled water; to create a total volume of 100 ml, it was transferred to a 100 ml volumetric flask and adjusted with water. this honey solution was filtered using a 0.55 mm whatman filter paper (whatman international limited, maidstone, uk) and stored in vials at 4°c. the sugar content was determined using an hplc system equipped with a refractive index (ri) detector (perkinelmer series-200a, usa). sugar separation was performed at 85°c in a sugar-pak™ 1 column (6.5×300 mm) manufactured by waters (usa). the hplc pump, column oven, auto sampler, and ri detector were observed using a totalchrom workstation, version 6.3.1 (2006). the mobile phase consisted of 100% hplc-grade water (hiprsolv for hplc, bdh laboratory supplies, poole, uk). the injection volume of the honey samples was adjusted to 1 µl accompanying a flow rate of 0.6 ml min-1. the peaks were recognized by matching respective retention times with those standards. furthermore, honey samples were spiked with standards, to confirm the identity of the chromatographic peaks. the average peak areas of the triplicate injections were used for peak quantification. a calibration curve was generated for each sugar using standard solutions (10–30 mg ml-1). ! ital. j. food sci., vol 28, 2016 629 the honey samples in the crystallized form were liquefied using a water bath (thermolab industries) at 40°c. the sugar results are expressed in g 100 g-1 honey. 2.10. total protein content lowry’s method of protein estimation was used to determine the total protein content of the honey samples. the basic principal in this method was the formation of a copperprotein complex and the reduction of phosphomolybdate and phosphotungstate present in folin & ciocalteu’s reagent to hetero polymolybdenum blue and tungsten blue, respectively. bovine serum albumin (0–100 µg ml-1) was used as a standard to prepare the calibration curve (habib et al., 2014). the results of the protein content were measured in mg 100 g-1. 2.11. hydroxymethylfurfural (hmf) one gram of honey was mixed in 10 ml of acetonitrile:water (1:1) solution. this mixture was homogenized via constant shaking for 10 min and then filtered using a 0.45-µm syringe filter into vials and set for injection into hplc system equipped with series 200 uv/vis detector (perkinelmer series-200a, usa). the injection volume was 10 µl, and the separation was performed using a symmetry® c18 5 µm (3.9 × 150 mm) column manufactured by waters (ireland), maintained at 22°c with a run time of 5 min. the mobile phase was 0.01 n phosphoric acid (86%) and acetonitrile (14%), with a flow rate of 0.2 ml min-1 (chinnici et al., 2003). the hmf found in the honey samples is expressed in mg kg-1. 2.12. statistical analysis all of the tests were conducted in triplicate. spss for windows (version 17, ibm, armonk, ny, usa) was used to analyze the data. the mean ± standard deviation differences among samples were ascertained using a one-way analysis of variance (anova) followed by tukey’s post hoc test. the cluster analysis of 12 parameters (moisture content, free acidity, ph ,ec, diastase activity, total acidity, ash, hmf, fructose, glucose,sucrose, and maltose) was applied to pakistani (punjab and kpk) and mediterranean honey samples in order to determine the similarity among the honeys of different countries. for this analysis, each parameter data were represented by mean value and the fractions of zero were used for the parameters (free acidity, ec, diastase activity, and ash), which were not available (n.a.) for compared honeys. the dendrogram was constructed by ward’s linkage method with euclidean distances. discriminat analysis was performed using past v.3.12 software. 3. results and discussion the physicochemical analyses of honey samples from punjab and kpk provinces are summarized in tables 1 and 2 and their sugar profiles are mentioned in tables 3 and 4. all the data were reported as mean values ± standard deviations. table 5 gives a comparison of relevant published data of honey parameters from some mediterranean countries to the present study. moisture content is one of the factors that determine the shelf life of honey during storage (peŕez–arquillue et al., 1994). it can be as low as 13% or as high as 23%, depending on the source of the honey and the climate conditions (bradbear, 2009). the moisture level ! ital. j. food sci., vol 28, 2016 630 of honey can increase under high humidity permeability and high storage humidity because of its hygroscopic property (özcan and ölmez, 2014). higher moisture content can promote honey fermentation during storage (idris et al., 2011). the moisture content values of all tested honey samples from both provinces varied from 16.32 to 19.91% (tables 1 and 2) and were in acceptable range (≤ 20%) of international quality regulations according to the codex alimentarius commission (2001). during the comparison of different mediterranean honeys, the moisture values of pakistani honeys were not statistically different from each other and from turkish honeys reported previously (özcan and ölmez, 2014). among the six types of compared honeys, the lowest mean moisture content of 17.01% was in italian honeys (truzzi et al., 2014) while the moroccan honeys (aazza et al., 2014) had the highest (18.97%). the variant moisture content might be the result of the diverse prevailing weather conditions across the disparate geographical locations of different countries or the different beekeeping practices that affect the degree of honey maturity. the water activity of analysed honey samples varied from 0.51 to 0.66 (tables 1 and 2). these results were in accordance with arid regions and mexican honeys, with aw ranges of 0.52 to 0.64 (habib et al., 2014) and 0.569 to 0.613 (mondrago´ n-cortez et al., 2013), respectively. the water activity is a key factor that controls food stability by limiting or preventing microbial growth. osmotolerant yeasts can grow under minimal aw conditions of 0.6 (chirife et al., 2006). the mustard honey from punjab, which had the highest moisture content, had the highest aw and might be candidate for fermentation. the presence of organic acids and inorganic ions in honey results in its acidity (terrab et al., 2004). the total acidity of honey samples from both provinces ranged from 15.90 to 39.06 meq kg1 (tables 1 and 2), both of which were acceptable (i.e., below 50 meq kg-1; codex alimentarius commission, 2001). the lowest total acidity was associated with citrus, whereas the highest value was found in multifloral and eucalyptus honeys. the difference in total acidity of honey is due to the harvesting season (habib et al., 2014). the free acidity of the honey samples ranged from 3.97 to 24.77 meq kg-1. the lowest free acidity was observed in clover and loquat honeys of punjab and kpk, respectively, whereas the highest values were found in eucalyptus honey samples of both provinces. the total and free acidity of the analyzed honey samples approximated the values recorded at other geographical locations (habib et al., 2014). a comparison between pakistani and some mediterranean honeys presented a substantial variation (table 5). nectar source, climatic conditions and soil properties of different countries might explain the acidity variation among the honey samples. the ph values of tested honey samples of both provinces ranged from 3.22 to 5.18 (tables 1 and 2). lower ph was in acacia honey, whereas higher values were associated with the multifloral and ziziphus honeys of punjab and kpk, respectively. the ph of honey is related to its storage and microbial growth and is responsible for its changes in texture and stability. the ph value of honey provides clues about its origin (i.e., floral vs. forest, where forest honey typically has a higher ph value; feás et al., 2010 a). the ihc has not described the limit of ph in honey; however, a low ph inhibits microbial contamination (habib et al., 2014). the ph of the honey samples obtained from both provinces matched the values recorded by gulfraz et al. (2011) and rodríguez et al. (2014). honey samples from pakistan showed a significant variation in ph from other mediterranean honeys. ! ital. j. food sci., vol 28, 2016 631 table 1: physicochemical properties of blossom honeys taken from the punjab province of pakistan. s. no . honey type location moisture content (%) water activity (aw) free acidity (meq kg-1) ph ec µs cm-1 diastase activity (dn) total acidity (meq kg-1) ash (%) total protein (mg 100 g-1) hmf (mg kg-1) 1 eucalyptus hassana bdal 18.64±0.04 b 0.59±0.00c 19.62±0.08a 4.31±0.02b 448.59±0.41d 13.56±0.24b 36.19±0.72a 0.17±0.00c 407.41±2.84a 18.40±0.52c 2 sunflower dg khan 18.78±0.03b 0.55±0.01f 17.09±0.09bcd 3.92±0.01e 319.17±0.41f 9.12±0.53cd 29.59±1.42bc 0.10±0.00e 241.72±1.69f 29.16±0.73b 3 acacia rawalpi-ndi 16.32±0.35 e 0.53±0.00g 14.80±0.21d 3.85±0.01f 467.87±0.41c 10.15±0.24c 26.91±0.49cd 0.18±0.00c 346.84±3.61c 9.31±0.24f 4 mustard lillah 19.91±0.42a 0.66±0.00a 15.57±0.14cd 4.34±0.01b 302.28±0.41h 9.26±0.53cd 26.02±0.54d 0.09±0.00e 242.37±2.46f 37.25±0.86a 5 ziziphus lillah 17.88±0.06d 0.56±0.01e 17.05±1.81bcd 5.16±0.02a 569.60±0.41a 16.64±0.24a 28.00±0.76cd 0.24±0.00a 289.40±1.21d 9.10±0.17f 6 clover narang mandi 18.58±0.03 b 0.61±0.00b 3.97±0.21e 4.12±0.01d 367.81±1.60e 8.98±0.53cd 19.02±0.51e 0.13±0.00d 207.07±0.80g 13.19±0.35e 7 citrus salam, sargodha 18.42±0.03 bc 0.58±0.00d 5.07±0.65e 4.29±0.01b 308.03±1.55g 5.73±0.75e 18.92±0.50e 0.09±0.00e 235.77±3.43f 15.48±0.22d 8 currant bush salgiran, murree 17.98±0.08 cd 0.56±0.00ef 17.29±1.49abc 4.21±0.01c 446.56±0.41d 8.72±0.53d 31.50±2.19b 0.17±0.00c 272.18±1.17e 7.43±0.53g 9 multifloral rawalpi-ndi 17.67±0.06 d 0.51±0.00h 18.77±0.46ab 5.18±0.02a 503.53±0.41b 12.90±0.24b 36.59±0.61a 0.21±0.00b 357.83±1.88b 19.38±0.36c all analyses were performed in triplicate, and the mean value ± standard deviation (sd) are reported. mean values in the same column but with different superscript letters differ significantly (p ˃ 0.05). table 2: physicochemical properties of blossom honeys taken from the khyber pakhtunkhwa province of pakistan. s. no. honey type location moisture content (%) water activity (aw) free acidity (meq kg-1) ph ec (µs cm-1) diastase activity (dn) total acidity (meq kg-1) ash (%) total protein (mg 100 g-1) hmf (mg kg-1) 1 loquat harripur 18.04±0.09c 0.59±0.00a 4.37±1.19d 4.19±0.02c 322.11±2.15e 9.51±0.53cd 20.42±2.13bc 0.10±0.00e 391.40±2.12b 11.48±0.31d 2 eucalyptus mardan 17.37±0.05de 0.54±0.01e 24.77±1.58a 4.11±0.01d 439.14±0.41c 11.51±0.24b 39.06±4.47a 0.16±0.00c 377.60±1.08c 23.33±0.43a 3 ziziphus noushehra 17.49±0.07d 0.55±0.00d 12.70±0.25c 5.01±0.01a 591.64±2.36a 14.68±0.24a 25.89±1.54b 0.26±0.00a 270.77±2.65e 6.22±0.16e 4 acacia noushehra 17.21±0.06e 0.57±0.00c 11.35±0.88c 3.22±0.01e 487.53±0.41b 9.43±0.53cd 18.65±0.87c 0.20±0.00b 412.67±3.03a 4.71±0.39f 5 citrus swat 18.29±0.08b 0.58±0.01b 5.25±0.95d 4.27±0.02b 286.45±1.28f 9.75±0.53c 15.90±1.12c 0.08±0.00f 311.36±5.65d 13.98±0.26c 6 clover mardan 19.24±0.08a 0.58±0.01b 17.60±0.55b 4.12±0.03d 357.03±0.41d 8.44±0.53d 33.16±3.30a 0.12±0.00d 262.02±1.15f 19.17±0.44b all analyses were performed in triplicate, and the mean value ± standard deviation (sd) are reported. mean values in the same column but with different superscript letters vary significantly (p ˃ 0.05) ! ital. j. food sci., vol 28, 2016 632 tunisian and italian honeys were statistically similar to average ph values, while the turkish honeys had comparatively high values (table 5). the difference observed in the ph values among the different honey samples might be because of their acidity and mineral concentrations (kamal et al., 2002). the ec results showed variations based on the floral origin of the honey samples from both provinces, and they ranged from 286.45 to 591.64 µs cm-1. citrus honey was associated with less ec, whereas ziziphus honey with more ec (tables 1 and 2). the ec of analysed honey samples matched the values reported by idris et al., (2011). truzzi et al., (2014) reported a low average ec of italian honey samples among the compared mediterranean honeys while the tunisian honeys (boussaid et al., 2014) showed high values (table 5). the ec of honey is correlated with the intensity of its organic acids, mineral salts, and proteins; furthermore, it varies with changes in floral origin and is essential for differentiating the floral origins (habib et al., 2014). the ec values of blossom honeys should be below 800 µs cm-1, whereas honeydew honeys have ec values above 800 µs cm-1 (feás et al., 2010 b). in this study, the ec measurements were below 800 µs cm-1, which suggests that the origin of all the tested honey samples was floral. the ash content is an important parameter to determine floral origin and differentiates nectar honey and honeydew honey (white, 1978). floral honey samples have a lower (≤ 0.6%) ash content than honeydew honeys (≤ 1.2%) (feás et al., 2010 b). the ash content of a honey is primarily due to certain nitrogen compounds, vitamins, minerals, aromatic substances and pigments (mairaj et al., 2008). the ash content of the tested honey samples ranged from 0.08 to 0.26% (tables 1 and 2). the ash content in this study was below 0.6%, which indicates that the tested samples were nectar honeys (codex alimentarius commission, 2001). the average ash content of analysed honeys was compared to some mediterranean honeys. turkish honeys (özcan and ölmez, 2014) had the minimum average ash content while the moroccan (aazza et al., 2014) and tunisian honeys (boussaid et al. 2014) reflected greater values than pakistani honeys (table 5). the differences in the ash content of the tested honey samples might be because of soil type where the nectar plant was located (gómez-díaz et al., 2012), the atmospheric conditions, and plant physiology (kamal et al., 2002). diastase is an enzyme found in honey, and its level changes based on the geography, plant source, and the freshness of honey. da might indicate aging and point out the treated temperature during the processing of honey (fallico et al., 2006). the lowest acceptable da value is 8 on gothe’s scale according to international regulations (codex alimentarius commission, 2001). the range of da in the current study was 5.73 to 16.64 dn. in punjab, the lowest da was found in citrus honey, whereas the highest was in the ziziphus honey (table 1). similarly, the lowest and highest da values were observed in the clover and ziziphus honeys of the kpk, respectively (table 2). the da of the honey samples corroborated previously reported values (feás et al., 2010 a; iftikhar et al., 2011). the da of pakistani honeys was lower than turkish and moroccan honey samples (table 5). the results indicate that the honey samples were natural because their da was within the acceptable range. the total protein content of the honey samples of both provinces ranged from 207.07 to 412.67 mg 100 g-1. these results are similar to the honey samples taken from arid regions, with the protein content ranging from 204.84 to 578.87 mg 100 g-1 (habib et al., 2014). the total protein content of the tested honey samples of both provinces was higher than that of samples from india, where it varied from 48 to 229.3 mg 100 g-1 (saxena et al., 2010). the protein content of honey depends on the presence of the enzymes introduced by honeybees as well as that putatively derived from floral nectar (saxena et al., 2010); therefore, this value varies among the honey samples. ! ital. j. food sci., vol 28, 2016 633 table 3: sugar content (expressed as a percentage) of blossom honeys taken from the punjab province of pakistan. s. no. honey type location fructose (g 100 g-1) glucose (g 100 g-1) sucrose (g 100 g-1) maltose (g 100 g-1) raffinose (g 100 g-1) reducing sugars (g 100 g-1) total sugars (g 100 g-1) f/g ratio 1 eucalyptus hassanabdal 35.71±0.02e 30.79±0.01d 7.23±0.02a 3.15±0.03b 0.111±0.006c 66.50±0.02g 76.99±0.08b 1.16±0.00d 2 sunflower dg khan 37.13±0.05b 32.93±0.02a 2.52±0.02g 1.47±0.03f 0.043±0.002f 70.06±0.07b 74.09±0.04d 1.13±0.00e 3 acacia rawalpindi 35.62±0.03e 32.82±0.08a 1.12±0.04i 2.93±0.05c 0.160±0.010b 68.44±0.05d 72.65±0.13f 1.08±0.01f 4 mustard lillah 34.90±0.02f 32.31±0.03b 4.36±0.01e 0.62±0.08h n.d.* 67.21±0.03f 72.19±0.05g 1.08±0.00f 5 ziziphus lillah 33.65±0.08g 24.75±0.06f 2.76±0.03f 2.08±0.04d 0.064±0.002de 58.41±0.04i 63.30±0.09h 1.36±0.01a 6 clover narang mandi 38.03±0.03a 32.24±0.02b 6.15±0.02c 1.04±0.06g 0.021±0.002g 70.27±0.05a 77.48±0.03a 1.18±0.00c 7 citrus salam sargodha 36.63±0.03d 31.04±0.02c 1.41±0.03h 3.39±0.11a 0.406±0.015a 67.67±0.01e 72.88±0.06f 1.18±0.00c 8 currant bush salgirran murree 36.55±0.01d 29.45±0.03e 6.41±0.01b 1.17±0.04g 0.084±0.002d 66.00±0.02h 73.66±0.14e 1.24±0.00b 9 multifloral rawalpindi 36.93±0.03c 32.35±0.04b 5.18±0.01d 1.69±0.05e 0.052±0.007ef 69.28±0.04c 76.20±0.07c 1.14±0.00e * not detected ; limit of detection (lod) = 1.2 mg 100 g-1 all analyses were performed in triplicate, and the mean value ± standard deviation (sd) are reported. mean values in the same column but with different superscript letters vary significantly (p ˃ 0.05). table 4: sugar content (expressed as a percentage) of blossom honeys taken from the khyber pakhtunkhwa province of pakistan. s. no. honey type location fructose (g 100 g-1) glucose (g 100 g-1) sucrose (g 100 g-1) maltose (g 100 g-1) raffinose (g 100 g-1) reducing sugars (g 100 g-1) total sugars (g 100 g-1) f/g ratio 1 loquat harripur 36.40±0.02b 31.30±0.02d 4.10±0.02c 3.67±0.06a 0.097±0.003b 67.70±0.03b 75.57±0.07b 1.16±0.00b 2 eucalyptus mardan 37.03±0.06a 32.86±0.01a 9.71±0.01a 2.06±0.05d 0.082±0.002cd 69.89±0.07a 81.74±0.05a 1.13±0.00c 3 ziziphus noushehra 32.45±0.04f 25.03±0.06f 4.36±0.02b 1.74±0.07e 0.093±0.002bc 57.48±0.06f 63.67±0.08f 1.30±0.01a 4 acacia noushehra 34.74±0.01d 32.72±0.04b 1.97±0.06e 2.43±0.05c 0.072±0.004d 67.46±0.05c 71.93±0.04d 1.06±0.00e 5 citrus swat 35.51±0.03c 31.58±0.01c 4.01±002d 2.82±0.07b 0.236±0.012a 67.09±0.02d 74.16±0.11c 1.12±0.01c 6 clover mardan 32.99±0.03e 29.92±0.02e 3.97±0.01d 0.96±0.08f 0.050±0.002 e 62.81±0.11e 67.89±0.08e 1.10±0.00d all analyses were performed in triplicate, and the mean ± standard deviations (sd) are reported. mean values in the same column but with different superscript letters vary significantly (p ˃ 0.05). ! ital. j. food sci., vol 28, 2016 634 honey freshness is widely judged based on its hmf content (bacandritsos et al., 2006; corbella and cozzolino, 2006) as fresh honeys are mainly deprived of this compound therefore, hmf content might increase during honey processing and/or aging (habib et al., 2014). the hmf values of the honey samples ranged from 4.71 to 37.25 mg kg-1. the lowest hmf values were in currant bush and acacia honeys, and the highest hmf values were observed in mustard and eucalyptus honeys (tables 1 and 2). the hmf values of the honey samples in this study corroborated those of mairaj et al. (2008), akhter et al. (2010), feás et al. (2010 a), and feás et al. (2010 b). pakistan honey samples presented a substantial variation in average hmf from other mediterranean honeys. italian honeys (truzzi et al., 2014) had the minimum average hmf while the tunisian honeys (boussaid et al., 2014) had maximum values (table 5). hmf values of punjab honeys were statistically similar to turkish honeys (özcan and ölmez, 2014). many factors, including temperature and heat time as well as ph, storage environment, and floral origin, influence the levels of hmf in honey samples; therefore, hmf indicates overheating and poor storage conditions (fallico et al., 2006). long-term heat treatments of honey samples inactivate its natural enzymes, and increased hmf occurs due to fructose degradation (mairaj et al., 2008). all of the analyzed samples from both provinces of pakistan showed hmf values within the acceptable range (< 80 mg kg-1 for tropical or arid regions) according to the international quality regulations of the codex alimentarius commission (2001). honey is primarily composed of sugars (rodríguez et al., 2014). the monosaccharides glucose and fructose are important components of honey while fructose is always the primary sugar, followed by glucose (habib et al., 2014).fructose and glucose were the major sugars in all of the tested honey samples. the amounts of fructose and glucose in the honey samples of both provinces ranged from 32.45 to 38.03 g 100 g-1 and from 24.75 to 32.93 g 100 g-1, respectively, whereas other minor sugars containing sucrose, maltose and raffinose ranged from 1.12 to 9.71 g 100 g-1, 0.62 to 3.67 g 100 g-1 and 0.021 to 0.406 g 100 g-1, respectively (tables 3 and 4). raffinose was not detected in the mustard honey sample from punjab. the total sugar content of the honey samples ranged from 63.30 to 81.74 g 100 g-1. the fructose/glucose (f/g) ratio and the reducing sugars (fructose + glucose) of the honey samples ranged from 1.06 to 1.36 and from 57.48 to 70.27 g 100 g-1, respectively (tables 3 and 4). because glucose is comparatively less soluble in water than fructose, the f/g ratio can most likely be used to evaluate the granulation of honey (anklam, 1998). the results of the current study were in line with the international quality regulations of the codex alimentarius commission (2001). the reducing sugars of all of the tested honey samples were higher than 60%, except for the ziziphus honeys of both provinces. the comparison of examined pakistani honeys with the moroccan and tunisian honeys (based on available data) revealed some variations. the fructose, glucose, sucrose and maltose contents were statistically different in compared honeys (table 5). the average sucrose content of the examined pakistani honeys slightly higher than moroccan and tunisian honeys but overall within the permissible limits (codex alimentarius commission, 2001); however, the clover and currant bush honeys from punjab were slightly high (table 3). early honey harvesting might explain the high sucrose content (azeredo et al., 2003). cluster analysis (ward’s methodeuclidean distances) (fig. 2) data categorized honeys into two main groups. the first group consisted of turkish and italian honeys and the second was comprised of pakistani (punjab and kpk), moroccan and tunisian honeys. it is interesting to note that in the second group, pakistani honeys were quite similar even though these belonged to different provinces (punjab and kpk). in second group, moroccan honeys were close to pakistani honeys while the tunisian honeys were away from the remaining group members. turkish and italian honeys fell in first group and were close to each other. ! ital. j. food sci., vol 28, 2016 635 table 5: comparison of physico-chemical properties of honeys from pakistan (i & ii) and some mediterranean countries (iii-vi). honey origin moisture content (%) free acidity (meq kg-1) ph ec (µs cm-1) diastase activity (dn) total acidity (meq kg-1) ash (%) hmf (mg kg-1) fructose (g 100 g-1) glucose (g 100 g-1) sucrose (g 100 g-1) maltose (g 100 g-1) i punjab, n=9 (mean values of present study) 18.24±0.12ab 14.36±0.54c 4.38±0.02b 414.83±0.67c 10.56±0.43b 28.08±0.86bc 0.15±0.01c 17.63±0.44c 36.13±0.03c 30.96±0.03b 4.13±0.02b 1.95±0.05d ii kpk, n=6 (mean values of present study) 17.94±0.07ab 12.67±0.88c 4.15±0.02c 413.98±1.17c 10.55±0.44b 25.51±2.24cd 0.15±0.00c 13.15±0.32d 34.85±0.03d 30.57±0.02c 4.69±0.03a 2.28±0.06c iii morocco, n=17 (aazza et al., 2014) 18.97±0.76a 22.93±2.73a 3.94±0.06d 500.71±1.66b 12.42±1.01ab 29.66±1.16b 0.36±0.01a 44.80±0.78a 38.47±0.04a 30.76±0.05bc 0.13±0.01d 3.47±0.05a iv tunisia , n=6 (boussaid et al., 2014) 18.71±0.47a n.a. 3.86±0.07d 548.33±1.80a n.a. 22.59±0.92d 0.26±0.02b 20.01±0.37b 36.93±0.10b 33.49±0.19a 1.89±0.07c 2.64±0.03b v italy, n=43 (truzzi et al., 2014) 17.01±0.80b 18.16±1.01b 4.55±0.05a 87.00±0.17d n.a. 21.56±0.82d n.a. 1.67±0.21e n.a. n.a. n.a. n.a. vi turkey, n=8 (özcan and ölmez, 2014) 18.30±0.26ab n.a. 4.24±0.07bc n.a. 13.78±1.43a 34.93±1.65a 0.05±0.01d 18.36±0.50c n.a. n.a. n.a n.a. the data are mean values ± standard deviation (sd). mean values in the same column, but with different superscript letters differ significantly (p ˃ 0.05). na: not available. ital. j. food sci., vol 28, 2016 636 figure 2: dendogram (ward’s methodeuclidean distances) showing the comparison of honeys from different countries. these observations indicated the key role of climate, floral origin and soil properties of a particular region that determine the physicochemical characteristics of the honey. 4. conclusions the present study characterized nine pakistani blossom honey samples from punjab and six from kpk. a comparison was also made to some mediterranean honeys. the results of this study allow us to assess the quality of pakistani honeys and help to establish certain standards. based on the studied quality parameters (i.e., moisture, aw, acidity, ph, electric conductivity, diastase activity, ash, hmf, and sugar content) of the different honey samples from both provinces, pakistani honeys meet international standards. however, the slightly higher sucrose content of certain honey samples and the lower values of reducing sugars in ziziphus honey indicate early harvesting by beekeepers. acknowledgements the project was financially supported by king saud university, vice deanship of research chairs. references aazza s., lyoussi b., antunes d. and miguel m.g. 2014. physicochemical characterization and antioxidant activity of 17 commercial moroccan honeys. int. j. food sci. nutr. 65(4):449. ital. j. food sci., vol 28, 2016 637 adebiyi f.m., akpan i., obiajunwa e.i. and olaniyi h.b. 2004. chemical/physical characterization of nigerian honey. pak. j. nutr. 3(5):278. akhter s., masood s. and shaukatullah. 2010. physicochemical analysis and sensory evaluation of different samples of honey collected from northern areas of pakistan. pak. j. biochem. mol. biol. 43(1):41. anklam e. 1998. a review of the analytical methods to determine the geographical and botanical origin of honey. food chem. 63(4):549. azeredo l. da c., azeredo m.a.a., souza s.r. and dutra v.m.l. 2003. protein contents and physicochemical properties in honey samples of apis mellifera of different floral origins. food chem. 80(2):249. bacandritsos n., sabatini a.g., papanastasiou i. and saitanis c. 2006. physico-chemical characteristics of greek fir honeydew honey from marchalina hellenica (gen.) in comparison to other mediterranean honeydew honeys. ital. j. food sci. 18(1):21-31. bogdanov s. 2002. harmonized methods of the international honey commission. http://www.ihcplatform.net/ihcmethods2009.pdf. 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september 29, 2015 accepted june 1, 2016 #481_yilmaz_bozza ital. j. food sci., vol 29, 2017 186 paper ultrasound-assisted extraction of lycopene and β-carotene from tomato-processing wastes t. yilmaz*1, s. kumcuoglu2 and s. tavman2 1food engineering department, engineering faculty, celal bayar university, muradiye-manisa, turkey 2food engineering department, engineering faculty, ege university, bornova-izmir, turkey *corresponding author. tuncay.yilmaz@cbu.edu.tr abstract ultrasound-assisted extraction (uae) of lycopene and β-carotene from tomato-processing wastes were investigated. hexane:acetone:ethanol (2:1:1 v/v/v) including 0.05% (w/v) butylated hydroxyl toluene (bht) was used as a solvent, with 1:35 w/v solid liquid ratio at 15±5°c. ultrasonic power (50, 65, 90w) was applied in uae for 1-30 min. conventional organic solvent extraction (cose) was applied under the same solvent and temperature conditions for 10-40 min. uae was more effective and required a shorter time than cose. maximum lycopene and β-carotene yields were obtained using 90w ultrasonic power for 30 and 15 min, respectively. keywords: lycopene, β-carotene, ultrasound assisted extraction, tomato processing wastes ital. j. food sci., vol 29, 2017 187 1. introduction the tomato is popular because of its healthy composition and consumption both in raw and processed forms all over the world. the essential role of carotenoids, being a major dietary source of vitamin a, is having remarkable effects on the immune response and intercellular communication (taylor et al., 2000; su et al., 2002; sun et al., 2010). studies show that antioxidant-rich diet reduces or prevents risks of epithelial and prostate cancers, cardiovascular diseases and cataracts (tonucci et al., 1995; arab and steck, 2000; stahl and sies, 2005; capanoglu et al., 2008; lianfu and zelong, 2008). lycopene and β-carotene are used in industry for their wide color range between yellow and red (sabio et al., 2003; alda et al., 2009). however, for their great solubility in oil and fat, both of them are used as a natural colorant in the food, cosmetics, and pharmaceutical industries. β-carotene is used as pro-vitamin a, animal feed, an additive in cosmetics, multivitamin constituent and anti-oxidant (ben-amotz and fishler, 1998). the basic carotenoid in tomato is lycopene, and it is known for its high anti-oxidant capacity. depending on protective properties of lycopene against cancer and oxidants, lycopene is one of the important components used in pharmaceuticals and cosmetic formulations (kumcuoglu et al., 2014; dolatabadi et al., 2016). various amounts and type of wastes at various stages appear during tomato processing. dry pomace consists of 44% seed and 56% pulp and skin (kaur et al., 2008; lavelli and torresani, 2011; dolatabadi et al., 2016). a major part of waste is composed of seed and skin, which contain five times more lycopene than tomato pulp (shi et al., 1999; taylor et al., 2000). since the skin is the major source of lycopene, it must be separated from other parts for better extraction (george et al., 2004; kaur et al., 2008). total lycopene content in tomato pulp and tomato skin varies between 90 to 190 mg/kg and about 120 mg/kg in fresh weight, respectively, while β-carotene is found to be about 3 mg/kg (baysal et al., 2000; alda et al., 2009; poojary and passamonti, 2015; kumcuoglu et al., 2014). besides having high antioxidant activity, lycopene degrades and isomerizes under light irradiation and high temperature treatments (chen et al., 2009). to isolate all-trans-lycopene and avoid isomerization, suitable and fairly controlled conditions should be satisfied. extraction of lycopene is applied by classic solvent extraction (cose) method traditionally (lianfu and zelong, 2008). but in traditional extraction methods, long process time and high amounts of solvent are needed. decreasing the solvent consumption, shortening the extraction time, increasing the extraction yield, and enhancing the quality of extracts can be reached by new methods such as ultrasound-assisted (uae), microwave-assisted, or supercritical extraction (wang and weller, 2006; fantin et al., 2007; wang et al., 2008). sound waves produced by an ultrasonic probe at frequencies greater than human hearing cause a mechanical impact, allowing greater penetration of solvent into the plant body, known as the “sponge effect.” another effect of ultrasonic power is producing highenergy cavitation bubbles containing solvent vapor. these bubbles implode near cell walls causing very high local temperatures, pressure increase and cell wall destruction, which eases mass transfer from cell to solvent and enhances micro-streaming. the combination of these effects intensifies solvent penetration and satisfies sufficient mixing for extracting high amounts of active components. besides easing extraction, ultrasound may also produce free radicals within the cavitation phenomenon such as highly reactive hydroxyl radicals in water-contained solutions (toma et al., 2001; vinatoru, 2001; mason and lorimer, 2002; wang et al., 2008; jerman et al., 2010; sutkar et al., 2010). the aim of this study was to evaluate the effects of time and ultrasonic intensity in uae of lycopene and β-carotene from tomato-processing wastes and to compare the efficacy between uae and cose. ital. j. food sci., vol 29, 2017 188 2. materials and methods 2.1. material tomato wastes used in this study were supplied from a tomato-paste-manufacturing pilot plant (ege university, agricultural faculty izmir, turkey, 2012). the raw material was dried, from 75% to 4.90±2.50 % moisture content, in a vacuum drier at 40oc for 24 h. before extraction process, dried raw material containing 48.80±4.70% skins and % 51.20±3.10 seeds was grounded in a laboratory scale hammer mill (armfield hammer mill, england) and then subjected to sieving. the average particle size of the powder was determined by screen analysis. samples with 286±24 µm average size were used in the extraction process. then, the samples were packed under vacuum and stored at-40oc until the extraction process. 2.2. methods 2.2.1 conventional organic solvent extraction (cose) extraction of lycopene and β-carotene was carried out according to the method of sadler et al. (1990) and modified as described by perkins-veazie et al. (2001). the mixture of solvents hexane: methanol: acetone (2:1:1 v/v), containing 0.05% (w/v) bht (butylated hydroxyl toluene), were used to extract carotenoids from the sample. a 1.14 g dried sample was placed into a 50 ml flask, and a 40 ml solvent mixture was added to satisfy 1:35 (w/v) ratio. then, these flasks were agitated continuously in a shaking water bath. extractions were applied at 15±5oc temperature for 10, 20, 30 and 40 minutes. after the extraction process 15 ml of cold distilled water was added after the extraction process and then suspension was centrifuged by 1000 rpm at 5oc. the solution was then allowed to stand for 5 min for separation of polar and non-polar layers. non-polar phase was used for lycopene and β-carotene determination. filtered non-polar phases were first dried under nitrogen flow and then kept at -40oc in amber bottles as described in previous study (kumcuoglu et al., 2014) for 6 months before subjected to hlpc analyses. 2.2.2 ultrasound assisted extraction (uae) method a high-intensity ultrasonic probe with maximal input power of 400 w and operating frequency of 24 khz (model up400s, dr. hielscher, germany) equipped with a h14 sonotrode (dr. hielscher, germany) was used for the extraction experiments. solvent composition and liquid-solid ratio were the same as applied in cose. ultrasonic probe was immersed 7 cm into the solution from the top of the 150 ml flask. ultrasonic treatments were performed for 1, 2, 5, 10, 15, 20 and 30 min. in this study, ultrasonic power levels and corresponding ultrasonic intensities were 50, 65, 90 w and 32.50, 42.25, 52 w/cm2 respectively. during uae experiments, the temperature of samples was kept at 15±5oc by using indirect cold water circulation system. for lycopene and β-carotene determination, the same procedure was applied as the one implemented in cose. 2.2.3 lycopene and β-carotene determination solvent-free extracts were dissolved in hplc-grade hexane and then analyzed by highperformance liquid chromatography (hplc 1200 agilent technologies/usa) using a ital. j. food sci., vol 29, 2017 189 diode array detector (dad) at 475 nm by applying barba et al. (2006) procedure. all extracts were filtered through a 0.22 µm filter membrane before injection to the hplc column. extracts were diluted with hplc grade hexane to fit the concentrations to the calibration curves. the calibration curves were determined by injecting 20 µl samples of 2.5, 5.0, 7.5, 10 and 20 mg/kg of pure β-carotene (95% synthetic, hplc grade-sigma chemical co.) and samples of 20, 30, 50, 80 and 100 mg/kg of pure lycopene (90-95% from tomato, sigma chemical co.). correlation coefficients of calibration curves were 0.9975 and 0.9979, respectively. c18 column (10 µm, 3x300 cm waters/usa) was used for the separation. the column temperature was 30oc. methanol: acetonitrile (90:10 w/w) was used as a mobile phase at a flow rate of 0.9 ml/min, while 20 µl hexane phase was injected for each sample. at the beginning of analyses of each extract, the column was washed with mobile phase in order to remove pigments other than lycopene and β-carotene, which are not soluble in this polar solvent. identification of carotenoids in the extracts was done by comparing their retention time with the retention time of their standards. results were calculated as mg/kg of dry weight. 2.2.4 statistical analysis all measurements were carried out in triplicate. the results were expressed as the mean value ± the standard deviation. data were subjected to statistical analysis using spss 16.0 (spss inc. chicago, il, usa), and analysis of variance (anova) was applied using generalized linear model to determine the single and multiple effects of the parameters on the conditions by comparing the mean values. all significant differences were reported at p<0.05. duncan’s multiple range test (mrt) was used in post hoc analysis for multiple comparisons. 3. results and discussions 3.1 conventional organic solvent extraction (cose) effect of extraction time for lycopene and β-carotene in cose is given in fig 1. it has been observed that the amount of extracted lycopene and β-carotene increased with time at the beginning of the extraction period. statistical evaluation of the results showed that time was an important factor in extraction of lycopene and β-carotene (p<0.05).there is an increase in yield for 10 min and 20 min extraction periods (p<0.05) while longer extraction periods such as 30 min and 40 min yields were similar (p>0.05) for both lycopene and βcarotene yields. this can be explained by driving force decrease in osmotic balance; as the diffusion of carotenoids from material to the solution in cose takes place slowly so that the osmotic pressure between the inside and the outside of the cell easily reached equilibrium (sun et al., 2011; kumcuoglu et al., 2014). ital. j. food sci., vol 29, 2017 190 figure 1: effect of extraction time for lycopene and β-carotene in cose in this study, the amount of extracted lycopene and β-carotene in dried samples were between 52.2157.19 mg/kg dry weight and between 4.42-4.90 mg/kg, respectively. in previous studies, similar results were found; extracted lycopene values of fresh tomatoes were reported between 8.5 136 mg/kg (lugasi et al., 2003; karakaya, 2007; kumcuoglu et al., 2014). the lycopene values in tomato skin were determined between 25.5 141 mg/kg, and extracted β-carotene values in fresh tomatoes varied between 0.5-9.5 mg/kg, depending on genetic, agronomic, climatic factors and processing conditions (rao et al., 1998; rao and agarwal, 1999; baysal et al., 2000; toor and savage, 2005; bravo et al., 2012). 3.2 ultrasound assisted extraction (uae) the results of the general linear model indicates that extraction time, ultrasonic power and their composite effects are significant for both lycopene and β-carotene yields as given in table 1, and the applied model seems significant to describe extraction for each carotenoid (p<0.05). ital. j. food sci., vol 29, 2017 191 table 1: anova of response for ultrasound-assisted extraction experiments for lycopene and β-carotene ss, sum of squares; df,degrees of freedom, significant effects, α=0.05. table 2: identification and chromatographic data for lycopene and β-carotene after uae. component power (w) extraction time (min) 1 2 5 10 15 20 30 50 28.44ce±1.68 31.72cd±0.69 37.39cc±3.48 43.00cb±2.20 44.20cab±0.03 47.03ca±0.28 48.05ca±0.65 lycopene 65 35.79be±1.43 39.23bd±1.37 57.49bc±5.75 60.02bb±3.73 65.34bab±0.08 62.93ba±0.24 63.00ba±0.56 90 35.89ae±2.52 56.34ad±0.42 67.34ac±3.38 70.07ab±2.20 73.73aab±0.04 75.90aa±0.07 76.87aa±0.33 50 3.46ce±0.16 3.56cd±0.10 4.05cc±0.41 4.15cb±0.13 4.72ca±0.05 5.00ca±0.02 5.24ca±0.07 carotene 65 3.07be±0.10 3.57bd±0.06 4.71bc±0.26 4.99bb±0.08 5.28ba±0.03 5.52ba±0.01 5.66ba±0.06 90 3.92ae±0.08 5.00ad±0.05 5.30ac±0.29 6.01ab±0.20 6.12aa±0.07 5.92aa±0.01 5.41aa±0.03 data are means ± standard deviation. for every compound, different apices (capital letters) in a row indicate significant difference with respect to extraction time; different apices in a column (small letters) indicate significant differences with respect to ultrasonic power. p<0.05, duncan’s multiple range test. lycopene β-carotene source ss df p source ss df p corrected model 14296.275a 20 <0.0001 corrected model 49.510b 20 <0.0001 intercept 179.031.254 1 <0.0001 intercept 1447.072 1 <0.0001 power 6.725.223 2 <0.0001 power 12.436 2 <0.0001 time 6.828.752 6 <0.0001 time 32.242 6 <0.0001 power * time 742.299 12 <0.0001 power * time 4.832 12 <0.0001 error 282.484 42 error 1.359 42 total 193610.013 63 total 1497.941 63 corrected total 14578.759 62 corrected total 50.869 62 a. r squared = .981 (adjusted r squared = .971) b. r squared = .973 (adjusted r squared = .961) ital. j. food sci., vol 29, 2017 192 it was observed that the extraction yield increased exponentially in a few minutes (2 min), later increased gradually (10 min) and then became constant during extraction (table 2). the initial sharp increase in the rate of extraction was due to the large β-carotene and lycopene concentration gradient between the extracting solvent and the cell. later, the concentration gradient decreased as the extraction became difficult thanks to the interior location of cells. similar results were observed for extraction of all-trans-lycopene from red grapefruit and lycopene from tomato-processing wastes (kumcuoglu et al., 2014; xu and pan, 2013). in previous studies, similar results were observed for ultrasonic extraction of all-trans-lycopene, and time was found to be the most important factor affecting extraction yield. it is reported that most of all-trans-lycopene could be extracted during the 1/3 of total extraction period (30 min), and then lycopene degradation and isomerization led to the reduction of lycopene amount due to the side effect of sonication called ultrasonic degradation (wang and weller, 2006; jerman et al., 2010; sun et al., 2010; xu and pan, 2013; kumcuoglu et al., 2014). as the power increased from 50w to 65w, lycopene value increased significantly (p<0.05), while the increase in β-carotene value between 50w and 65w was not significant (p>0.05). but application of 90w had a significant effect on both lycopene and β-carotene contents (p<0.05). cavitation and thermal effects play an important role in uae. with an increase in power, more energy was getting transferred for cavitation, and this resulted in the increase in lycopene and β-carotene yield. at low ultrasonic intensities, thermal effect can be ignored because the heat produced by ultrasound may be completely diffused. as the ultrasonic intensity is further increased, the cavitation effect becomes less important compared to thermal effects during extraction of sensitive products such as carotenoids (soria and villamiel, 2010; sun et al., 2011; eh and teoh, 2012). it was reported that during sonication the extreme physical conditions of temperature and pressure caused carotenoid isomerization (chen et al., 2009). besides enhancing extraction efficiency, high ultrasonic power could cause thermal degradation to thermally sensitive components such as βcarotene (lianfu and zelong, 2008; adekunte et al., 2010). as a result, lycopene content increased with time from 15 min to 30 min at all ranges of ultrasonic power; β-carotene content started to decrease at 90w. this can be explained by sensitivity differences of lycopene and β-carotene in thermal effects. in previous studies, it was shown that lycopene was relatively resistant to thermal degradation compared to other carotenoids such as α-tocopherol and β-carotene (taylor et al., 2000). 4. conclusions in this study, various parameters affecting the cose and uae of lycopene and β-carotene were investigated. maximum lycopene and β-carotene yields were obtained in uae at 90w for 30 min and 90w for 15 min extraction time, respectively. uae extraction yields were significantly higher (p<0.05) than cose for both lycopene and β-carotene yields except when ultrasonic power of 50w was applied. extracted values of β-carotene obtained from 50w treatments at 15±5oc after 5 min extraction were similar to the values from cose at 20oc after 20 min extraction because the effect of heat was still a predominant factor for extraction efficiency of β-carotene at this ultrasonic intensity. the results indicated that uae was more effective and requires shorter time than cose even at lower temperatures. the ultrasound was beneficial for extracting compounds from tomato waste while shortening extraction time and being able to extract heat-sensitive compounds by increasing mass transfer at a lower temperature. ital. j. food sci., vol 29, 2017 193 acknowledgements the authors want to thank ege university, scientific research project (project no: 2009-muh-082) for financial support. references adekunte a.o, tiwari b.k., cullen p.j., scannell a.g.m. and o’donnell c.p. 2010. effect of sonication on colour, ascorbic acid and yeast inactivation in tomato juice. food chem. 122:500. alda l.m., gogoa i., bordean d., gergen i., alda s., moldovan c. et al. 2009 . lycopene content of tomatoes and tomato products. j. agroaliment. process technol. 15:540-542. arab l. and steck s. 2000. lycopene and cardiovascular disease. am. j. clin. nutr. 71:1691. barba a.i.o. and ferna v. 2006. food chemistry application of a uv – vis detection-hplc method for a rapid determination of lycopene and b -carotene in vegetables. food chem. 95:328. baysal t., ersus s. and starmans d.a.j. 2000. supercritical co2 extraction of β-carotene and lycopene from tomato paste waste. j. agric. food chem. 48:5507. ben-amotz a. and fishler r. 1998. analysis of carotenoids with emphasis on 9-cis-carotene in vegetables and fruits commonly consumed in israel. food chem. 62:515. bravo s., garcía-alonso j., martín-pozuelo g., gómez v., santaella m., navarro-gonzález i. et al. 2012. the influence of post-harvest uv-c hormesis on lycopene, β-carotene, and phenolic content and antioxidant activity of breaker tomatoes. food res. int. 49:296. capanoglu e., beekwilder j., boyacioglu d., hall r. and de vos r. 2008. changes in antioxidant and metabolite profiles during production of tomato paste. j. agric. food chem. 56: 964. chen j., shi j., xue s.j. and ma y. 2009. comparison of lycopene stability in waterand oil-based food model systems under thermaland light-irradiation treatments. lwt food sci. technol. 42:740. dolatabadi z., elhami rad a.h., farzaneh v., akhlaghi feizabad s.h., estiri s.h. and bakhshabadi h. 2016. modeling of the lycopene extraction from tomato pulps. food chem. 190:968. eh a.l.s. and teoh s.g. 2012. novel modified ultrasonication technique for the extraction of lycopene from tomatoes. ultrason. sonochem. 19:151. fantin g., fogagnolo m., medici a. and perrone d. 2007. isolation of lycopene from crude tomato extract via selective inclusion in deoxycholic acid. tetrahedron lett. 48:9148. george b., kaur c., khurdiya d.s. and kapoor h.c. 2004. antioxidants in tomato (lycopersium esculentum) as a function of genotype. food chem. 84:45. jerman t., trebše p. and mozetič vodopivec b. 2010. ultrasound-assisted solid liquid extraction (usle) of olive fruit (olea europaea) phenolic compounds. food chem. 123:175. karakaya s. 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35 (1): 106–117 p u b l i c a t i o n s codon marjoram oil attenuates oxidative stress and improves colonic epithelial barrier function in dextran sulfate sodium-induced ulcerative colitis in balb/c mice bouayad di1*, hadria grar1,2, sadia berzou3, omar kheroua1, djamel saidi1,4, hanane kaddouri1 1laboratory of physiology of nutrition and food safety, department of biology, faculty of natural and life sciences, university oran 1 ahmed ben bella, oran, algeria; 2department of biology, faculty of natural and life sciences, university of mostaganem, algeria; 3laboratory of clinical and metabolic nutrition, department of biology, university oran 1 ahmed ben bella, oran, algeria; 4higher school of biological sciences of oran (essbo), oran, algeria *corresponding author: ismahan bouayad debbagh, laboratory of physiology of nutrition and food safety, department of biology, faculty of natural and life sciences, university oran 1 ahmed ben bella, bp 1524 el m’naouer, oran, algeria. email: bouayadd.bio@gmail.com received: 26 december 2022; accepted: 7 february 2023; published: 16 march 2023 © 2023 codon publications open access original article abstract in this study, we explored the effects of marjoram oil (mo) on dextran sulfate sodium (dss)-induced colitis in balb/c mice. marjoram oil was found to significantly reduce the severity of dss-induced colonic inflammation, as reflected by improved disease activity index, prevented colon length shortening, lower histopathological score, decreased myeloperoxidase activity, and reduced interlukin 6 (il-6) levels. moreover, marjoram oil pretreatment enhanced the colonic epithelial integrity by decreasing paracellular permeability. marjoram oil was found to clearly reduce the colonic levels of thiobarbituric acid reactive substances assay and enhance the activity of superoxide dismutase, catalase, and glutathione sulfhydryl content. marjoram oil could exert a protective effect on ulcerative colitis through its anti-inflammatory and antioxidant properties. keywords: dextran sulfate sodium, experimental colitis, origanum majorana, oxidative stress, inflammation, ussing chamber introduction inflammatory bowel disease (ibd) is a long-term gastrointestinal disorder marked by recurrent inflammation and extensive mucosal destruction in the intestine (mijan and lim, 2018). histologically, extensive ulcerated regions with significant neutrophil infiltration and epithelial cell necrosis characterize ulcerative colitis (uc) (wang et al., 2019). although the cause of ibd is unknown, various factors are known to have a role in its development, including breakdown of the epithelial barrier, a decrease in commensal bacteria tolerance, immunologic alterations, oxidative stress, and an increase in inflammatory mediators (liu et al., 2017; goyal et al., 2014). immunological cell infiltration into the gut’s lamina propria, as well as uncontrolled synthesis of pro-inflammatory mediators and reactive oxygen species (ros) in colonic tissues, contributes to the breakdown of intestinal barrier integrity and an increased inflammation of the mucosa (maloy and powrie, 2011). an overproduction of ros causes lipid peroxidation which reduces cellular antioxidant capacity and leads to severe colonic inflammation (tahan et al., 2011). endogenous antioxidants can ordinarily protect the intestinal mucosa from oxidative damage (mandalari et al., 2011). nevertheless, inflammation increases the demand for these antioxidants, resulting in a pro-oxidant– antioxidant imbalance, resulting in mucosal damage (mandalari et al., 2011; zhu and li, 2012).  myeloperoxidase (mpo) activity is another indicator of the severity of ulcerative colitis. in colitis, mpo is widely mailto:bouayadd.bio%40gmail.com?subject= italian journal of food science, 2023; 35 (1) 107 protective effect of marjoram oil on ulcerative colitis in balb/c mice through its anti-inflammatory and antioxidant properties oil of origanum majorana suppressed the development of colon cancer cells through p38 mitogen-activated protein kinase (mapk)-provoked apoptosis in vitro and in vivo (athamneh et al., 2020). to date, no scientific data exist on the effect of origanum majorana on intestinal inflammation. therefore, the purpose of our study was to evaluate the possible protective effect of origanum majorana on dss-provoked colitis in balb/c mice. materials and methods chemicals marjoram oil was obtained from commercial cap pharm (el captain company, egypt). dextran sulfate sodium (40 kda), fluorescein isothiocyanate conjugated dextran (fitc-dextran; catalog number: fd4 [4 kda]), and mouse interlukin 6 (il-6) enzyme-linked-immunosorbent serologic assay (elisa) kit were purchased from sigma aldrich (france). the remaining solvents and chemicals were obtained from sigma chemical co. (st. louis, mo, usa). marjoram oil composition was analyzed using perkin elmer clarus 500 gas chromatograph capillary column hp-5 (30 m × 0.25 mm id, 0.25-µm film thickness), coupled to clarus 500 mass spectrometer (gc-ms). more details about the chemical composition of marjoram oil were reported earlier by bouayad debbagh et al. (2021). experimental animals  male balb/c mice, 8-week old, were kept at the university of oran’s animal facility with a 12-h light–dark cycle at 23 ± 2°c. animals had free access to commercial standard diet and water. all experimental procedures involving animals were approved by the current algerian legislation covering the protection of animals. colitis induction and design of treatment animals were divided into the following four groups, with eight mice in each group: control group (ctrl) received tap water; dss group (dss) received dss 4% (40 kda; sigma-aldrich) in their drinking water ad libitum for 7 days; marjoram oil group (m) received 0.5ml/kg body weight per os (by gavage) of marjoram oil for 14 day; and marjoram oil + dss group (m+dss), in which mice received mo (0.5 ml/kg body weight) orally for 14 day and were exposed to dss 4% for 7 days (day 15–21) (figure 1). used as a marker of oxidative stress and gastrointestinal inflammation (klebanoff et al., 2005). a previous investigation demonstrated that inflammatory cytokine-caused apoptosis was found to have a deleterious impact on the structure of adherent junction and epithelial barrier functions (su et al., 2013). animal models of ibd are suitable for studying the pathogenesis of the disease and developing new drugs. dextran sulfate sodium (dss)-provoked acute colitis is among the most regularly adopted methods (bang and lichtenberger, 2016) because of its great homogeneity, lesions’ reproducibility predominantly in the distal colon, and similarity to clinical ibd (valatas et al., 2013). currently, several therapies, such as nonsteroidal anti-inflammatory medications, corticosteroids, immune-suppressing medications, and biological agents are being investigated (baakhtari et al., 2018; yang et al., 2015). nevertheless, homeostasis and maintenance of intestinal barrier are rarely emphasized in conventional pharmacotherapy, and interest in integrative and complementary medicines is growing (larussa et al., 2017). patients with ibd frequently use complementary and alternative medicines (cams) (cheifetz et al., 2017; lin and cheifetz, 2018), which may include plants or herbal items, probiotics, and prebiotics (cheifetz et al., 2017). thus, in ibd patients, use of cam, particularly herbal medicines, is increasing as a safe anti-oxidant/anti-inflammatory therapeutic option (wan et al., 2014). owing to their high phenolic content, plants belonging to family lamiaceae are studied extensively as natural antioxidant sources (trouillas et al., 2003). marjoram (origanum majorana) is the most important member of family lamiaceae, which includes a wide variety of aromatic herbs and shrubs and volatile oil plants (paterson et al., 2006). polyphenols, such as flavonoids, having several therapeutic properties are found in abundance in origanum majorana (goel and vasudeva, 2015). leaves of origanum majorana are found to exhibit antioxidant (duletic et al., 2018; erenler et al., 2016), antimicrobial (della pepa et al., 2019; guerra-boone et al., 2015), anti-neurodegenerative (della pepa et al., 2019; duletic et al., 2018; erenler et al., 2016; guerra-boone et al., 2015), hepatoprotective (mossa et al., 2013), cardioprotective (ramadan et al., 2013), antiulcer (al-howiriny et al., 2009), anti inflammatory (arranz et al., 2015), and anticancer properties (al benhalilou et al., 2019 dhaheri et al., 2013). origanum majorana has also demonstrated positive effects in acute infectious diarrhoea (makrane et al., 2018). recent studies have demonstrated that essential 108 italian journal of food science, 2023; 35 (1) bouayad di et al. 500-μl buffer solution) were taken for 90 min at 30-min intervals for recording electrophysiological parameters. the concentration of fd-4 was determined using a fluorescence microplate reader with a 520-nm emission wavelength and a 490-nm excitation wavelength (perkinelmer, waltham, ma, usa). the apparent permeability coefficient (papp) was calculated for this period as follows: papp (cm/s × 10−6) = dc/dt × (v/(a × c0)), where dc/dt is the change in serosal concentration during 60–90 min (mol/l/s) period, c0 is the initial marker concentration in mucosal reservoir (mol/l), “v” is the volume of serosal reservoir (cm3), and “a” is the exposed intestinal area in the chamber (cm2). histological assessment of colitis distal colon sections were taken and fixed in phosphate buffered formalin (10%), and then dried and paraffin embedded. for light microscopic examination, these samples were stained with hematoxylin and eosin (h&e). histological damage was determined using the scoring system of stillie and stadnyk (2009). measurement of il-6 level level of il-6 in plasma samples was measured in duplicate using commercially mouse il-6 elisa kit (sigmaaldrich) according to manufacturer’s instructions. myeloperoxidase activity assay the mpo activity is defined as the quantity of enzyme that degraded 1 μmol of h2o2 per minute. it was measured in units per milligram of protein. in this study, the mpo activity was measured in proximal colon clinical colitis severity assessment the disease activity index (dai) was measured daily on the basis of clinical manifestations of colitis (cooper et al., 1993) comprising body weight, faecal bleeding, stool consistency, and diarrhoea (table 1). analysis of colon inflammation the animals were sacrificed after 21 days of treatment. blood was collected and the plasma was kept at −80°c for assays. the colon was excised and washed with saline solution. the length and weight of colon was measured. a 5-mm section of the distal colon was put in freshly prepared 10% (w/v) formaldehyde (ph 7.0), processed, and paraffin-embedded for histopathological evaluation. the mid-distal portion (30–60% region) was used for electrophysiological studies. the remaining colon tissue was kept at −80°c for additional biochemical analysis. ex vivo experiments: ussing chamber assay after sacrifice, distal colon was removed and excised through mesenteric border. colonic tissue samples were immediately placed in ussing chambers as sheets (physiologic instrument, san diego, ca) with an exposed area of 0.2 cm². both sides were filled with ringer solution (5 ml), oxygenated continuously (95% o2 and 5% co2), and maintained at 37°c. the mucosal-to-serosal flux of fitc-dextran with a 4-kda mean molecular mass (fd4) was measured across colonic samples to determine paracellular permeability. colonic paracellular permeability was determined by measuring mucosal-to-serosal flux of fitc-dextran across colonic samples. after a stabilization period of 15–25 min, the mucosal reservoir was injected with fd4 at a concentration of 2.2 mg/ml. for measuring fd4, basolateral samples (500 μl replaced by appropriate figure 1. experimental protocol of the study. tap water control group dss group m + dss group marjoram group tap water tap water days 1 14 21 marjoram oil gavage (0.5 ml/kg) marjoram oil gavage (0.5 ml/kg) dss (4%) dss (4%) italian journal of food science, 2023; 35 (1) 109 protective effect of marjoram oil on ulcerative colitis in balb/c mice through its anti-inflammatory and antioxidant properties catalase (cat) activity was determined according to the method of aebi (1984), in which 20-µl of supernatant was mixed with 1.8 ml of 100 mmol/l phosphate buffer (ph 7.4) and 200-µl h2o2 (50 mm). following a decrease in absorbance at 240 nm, cat activity was measured spectrophotometrically in units/milligram of protein, compared to a standard curve. superoxide dismutase (sod) was determined according to the method described by marklund and marklund (1974). the mixture included 970 µl of tris-hcl (50 mm, ph 8.2) and 15 µl of supernatant. the reaction began by adding 15 µl of pyrogallol (12-mm pyrogallol in 1-mm hcl) to the mixture. a control was prepared from the same mixture by adding 985-µl tris-hcl instead of 15-µl of pyrogallol. change in absorbance was measured during a 5-min period at a wavelength of 325 nm, and variation in absorbance per minute was calculated and expressed per milligram of protein, corresponding to the quantity of enzyme that suppressed auto-oxidation of pyrogallol by 50%. glutathione peroxidase was determined by rodrigues et al. (2002) method, in which 200 µl of tissue homogenate was added to a solution of tris-hcl (0.4 m, ph 7), 0.1-ml sodium azide (10 mm), and 0.2-ml reduced glutathione (1 mm) followed by 0.1 ml of 0.2-mm hydrogen peroxide. after a 10-min incubation period at room temperature, the mixture was centrifuged at 1,200×g for 10 min after adding 0.4 ml of 10% trichloroacetic acid. after this, 0.2 ml of supernatant and 0.1 ml of ellman’s reagent were combined, and the absorbance was measured at 340 nm. reduced glutathione (gsh) levels in colon tissues were assessed according to the method described by sedlak and lindsay (1968). gsh reacts with dithiobis2-nitrobenzoic acid (dtnb) to produce thio-2-nitrobenzoic acid, which has a wavelength of 412 nm. for measuring it, 1 ml of tissue homogenate (100-mg sample diluted with 0.9 ml of 0.12 mmol/l phosphate buffer, ph 7.2) was combined with 800 μl of ice-cold distilled water and 200 μl of trichloroacetic acid (50%) and incubated for 15 min; 400 μl of supernatant was mixed with 800 μl of tris buffer (0.4 mol/l, ph 8.9) and 20-μl dtnb reagent (0.01 mol/l) after centrifugation at 1,200×g for 15 min. after 5 min of incubation, the absorbance of the reaction mixture was measured at 412 nm against a blank reagent. statistical analysis data were presented as mean ± standard error of mean (sem). results were examined by one-way anova followed by fisher’s least significant difference (lsd) test tissue according to lenoir et al. (2011). in a 50-mm phosphate buffer with hexadecyl trimethyl ammonium bromide (0.5%), a section of colon was homogenized in ice with a t25 ultra-turrax system (htab; ika, staufen, germany). the homogenate was sonicated in ice for 30 s prior to centrifugation at 3,200 g for 10 min at 4°c. the supernatants were frozen and thawed thrice prior to being centrifuged for 10 min at 3,200×g at 4°c. in microcuves, 50 μl of supernatants were combined with 1 ml of phosphate buffer (10 mm), 500 μl of 0.22% guaiacol, and 10 μl of h2o2 (0.3 %). after 3 min, absorbance was measured at every minute for 20 min at 470 nm. the study was performed in triplicate. in vivo antioxidant activity homogenization of tissues and quantification of proteins in ice cold water, frozen tissue specimens were weighed (range 100 mg) and homogenized with 0.9-ml potassium phosphate buffer (0.12 mmol/l, ph = 7.4). the samples were centrifuged twice at 3,000×g for 20 min at 4ºc. superoxide dismutase, catalase, glutathione peroxidase (gpx) activities, and reduced glutathione level were determined in all supernatants. protein content was measured according to the method described by lowry et al. (1951). thiobarbituric acid reactive substances assay (tbars) lipid peroxidation was measured by the method of salih et al. (1987), in which 2 g of colon was homogenized in 30 ml of trichloroacetic acid (10%) and 200 µl of butyl hydroxyl toluene. samples were centrifuged for 10 min at 3,500 rpm. thiobarbituric acid (0.02 m), 5 ml, was then added to 5 ml of supernatant and the mixture was heated for 30 min at 100°c. absorbance of the supernatant was read against blank specimen at 532 nm. tbars was expressed in nanomole (nmol) of mda (malondialdehyde) per gram of tissue. table 1. disease activity index (dai) score (cooper et al., 1993) (n = 8). score weight loss stool consistency bleeding 0 none normal no bleeding 1 1–5% – – 2 5–10% loose stools slight bleeding 3 10–15% – – 4 more than 15% watery diarrhoea gross bleeding 110 italian journal of food science, 2023; 35 (1) bouayad di et al. figure 2. marjoram oil attenuated clinical signs of dextran sulfate sodium (dss)-induced colitis in mice. (a) body weight changes *p < 0.05, **p < 0.01 j21 vs j14. (b) scores of disease activity index (dai) in marjoram pre-treated mice and colitic (dss) mice monitored every day. data are expressed as the mean ± sd (n = 6 mice/groups). *p < 0.05 compared with dss. 32 30 28 26 24 22 20 0 1 2 3 4 5 6 7 85 10 15 time (day) days (after dss) b od y w ei gh t ( g) 4 3.5 4.5 3 2 2.5 1.5 1 0.5 0d is ea se a ct iv ity in de x (d a i) 20 control (a) (b) marjoram m+dss dss control marjoram m+dss dss 25 figure 3. (a) colon length. (b) colon weight. (c) colon macroscopic analysis. data are expressed as mean ± sd (n = 6 mice/ groups). ##p < 0.01, ###p < 0.001, compared with the control group; *p < 0.05, **p < 0.01, ***p < 0.001, compared with the dss group. 0 1 2 3 4 5 6 7 8 9 10 11 12cm 5 10 15 c ol on le ng th ( cm ) 0.00 0.5 1.0 1.5 c ol on w ei gh t ( g) co ntr ol ma rjo ra m m+ ds s ds s co ntr ol ma rjo ra m m+ ds s ds s (a) (b) (c) (statistica stat soft, maisons-alfort, france); p < 0.05 was considered as statistically significant. results marjoram oil improved dss-induced colitis mice exposed to dss-induced colitis, as evidenced by severe anorexia, weight loss (figure 2a), reduction in dai score from day 5 until the last experimental day (figure 2b), decreased colon weight (figure 3b), and drab hair color. the length of colon in dss-treated mice was significantly shorter by 43.69% (p < 0.001) than that in mice of both control and marjoram groups (figure  3a), indicating the successful establishment of mice colitis. pre-treatment of colitic mice with marjoram oil showed a reduction in the severity of colitis as evidenced by a decreased weight loss, prevented colon shortening, and reduced dai score on day 8 (p < 0.05). the length of italian journal of food science, 2023; 35 (1) 111 protective effect of marjoram oil on ulcerative colitis in balb/c mice through its anti-inflammatory and antioxidant properties colon was significantly restored (27%) in the (m+dss) group, compared to the dss group (p < 0.001). marjoram oil alleviated dss-induced colonic cellular permeability after dss treatment, fitc-dextran permeation was significantly higher, compared to both control and marjoram groups (p < 0.05). however, mice from the (m+dss) group possessed a lower colonic permeability to fitcdextran (p < 0.05; figure 4a). to determine the functional viability and integrity of colonic epithelium under ex vivo conditions, transepithelial ion conductance measurements were done for 120 min. as illustrated in figure 4b, transepithelial ion conductance in colonic tissues from dss group was considerably (p < 0.05) higher than that of the control group, indicating that colonic permeability to ions was relatively increased in colitic group. in contrast, the (m+dss) group showed a significant recovery (p < 0.01) in transepithelial ion conductance. these results suggested that marjoram oil could decrease paracellular permeability by maintaining colonic epithelial integrity. marjoram oil ameliorated dss-induced histopathological damage representative results and microscopic scores are illustrated in figure 5. the dss-treated mice had severe epithelial damage, including important cellular invasion into the lamina propria and colon mucosa, goblet cell depletion, thickening of the mucosa, and total architecture alteration, leading to a high microscopic damage score (figure 5b). however, pre-treatment of dss-treated mice with marjoram oil reduced infiltration of inflammatory cells with a minimum decrease of epithelial cells, resulting in a significantly reduced microscopic damage score when compared to colitic mice. these results suggested the effective protection rendered by marjoram oil to counter dss-induced colitis. marjoram oil decreased pro-inflammatory cytokine levels as shown in figure 6, induction of colitis by dss produced a marked increase in il-6 level (p < 0.01). however, pre-treatment with marjoram oil significantly reduced the il-6 content in plasma (p < 0.05) when compared to the dss group. marjoram oil reduced colonic myeloperoxidase activity in the dss group, colonic inflammation resulted in an increase in the activity of mpo in comparison to the control group (p < 0.001), reflecting the enhanced neutrophil infiltration that characterized this inflammatory process. however, pre-treatment of dss-treated group with marjoram oil significantly reduced the colonic activity of mpo, compared to colitic animals (p < 0.001, vs the dss group; figure 7). marjoram oil decreased oxidative stress in dss-induced colitis in the dss group, substantial oxidative stress was generated in the colonic mucosa as evidenced by a marked increase in tbars (figure 8) levels and a significant decrease in sod and cat activity in the colon (figures 9a and 9b). gsh content in the colon of the dss-treated mice was relatively lesser than that in the control and marjoram oil groups (p < 0.01; figure 9d). interestingly, marjoram oil reversed the loss of sod figure 4. analysis of basal gut permeability in male balb/c mice. (a) colonic permeability to fitc-dextran (fd4) was measured in ussing chamber. (b) transepithelial ion conductance was measured in the colon. bars indicate mean and sd. n = 6/group. (*p < 0.05, **p < 0.01 compared to dss group; #p < 0.05, ##p < 0.01 compared to control group). co ntr ol ma rjo ra m m+ ds s ds s 0 100 200 300 400 p er m ea nc e cl ea ra nc e (c m /s ec × 1 0– 6 ) 0 10 0 50 100 150 20 30 40 c on du ct an ce ( m s /c m 2 ) control marjoram m+dss dss time (min) (a) (b) b 112 italian journal of food science, 2023; 35 (1) bouayad di et al. figure 5. effects of marjoram oil on histological damage in dss-induced mice colitis. (a) histological appearance of colonic tissue sections (h&e, g×10). (b) changes in histological score. data are expressed as the mean ± sd (n = 6). **p < 0.01 is significantly different from the dss group, and ###p < 0.001 is significantly different from the control group. control marjoram m+dssdss c on tro l ma rjo ra m m+ ds s ds s 5 10 15 h is to lo gi ca l s co re 0 (a) (b) figure 6. effect of marjoram oil on plasma levels of il-6 in mice treated with 4% dss. statistical analysis was performed using one-way anova. values are mean ± sd (n = 6 for each group). *indicates significant differences (*p < 0.05, **p < 0.01) compared with dss group. ##p < 0.01 compared to control. co ntr ol ma rjo ra m m+ ds s ds s 0 100 200 300 400 il -6 ( pg /m l) co ntr ol ma rjo ra m m+ ds s ds s 0 10 20 30 u /m g pr ot ei n figure 7. colonic myeloperoxidase activity. data represent mean ± sd (n = 6 per group), ###p < 0.001 compared with control group; ***p < 0.001 compared with dss group. and cat activity (p < 0.01) and increased gsh content, compared to the dss group. furthermore, colonic levels of tbars were reduced in colitic mice pre-treated with marjoram oil. however, no significant changes in glutathione peroxidase activity were observed in the colonic tissues of different groups (figure 9c). discussion one of the two main types of ibds is ulcerative colitis, increasing rapidly in prevalence globally (kokkinidis et al., 2017). anti-inflammatory medications, including corticosteroids and 5-amino salicylic acid and its derivatives, are frequently used to treat ibd. in contrast, because of the development of additional oxidative stress, these medications are considered as a double-edged sword, as they cause hepatotoxicity and other adverse reactions (uko et al., 2012). for this reason, natural products have garnered substantial research interest and are being investigated extensively. our study explored the preventive effects of marjoram oil as a therapeutic strategy for acute ulcerative colitis caused by dss. in this study, a colitis model was established with mice being fed with 4% dss solution for 7 days. dai score was used as an indicator to evaluate the gravity of gastrointestinal disease in experimental groups. our results italian journal of food science, 2023; 35 (1) 113 protective effect of marjoram oil on ulcerative colitis in balb/c mice through its anti-inflammatory and antioxidant properties figure 8. effect of marjoram oil on the tbars levels in colonic tissue of dss-treated mice (n = 6). values are expressed as mean ± sd and analyzed using one-way analysis of variance followed by anova. (##p < 0.01, compared to control; **p < 0.01 compared with dss group). co ntr ol ma rjo ra m m+ ds s ds s 0 1 2 3 4 t b a r s ( nm ol /g ti ss ue ) figure 9. effect of marjoram oil on (a) catalase, (b) superoxide dismutase, (c) glutathione peroxidise, and (d) total glutathione activities in the colonic tissues of dss-treated mice (n = 6). values are expressed as mean ± sd and analyzed using one way analysis of variance followed by anova. #p < 0.05, ##p < 0.01, ###p < 0.001, compared to control; *p < 0.05, **p < 0.01, ***p < 0.001, compared with the dss group). 0.5 1.0 1.5 c at ( u /m g pr ot ) 0.0 g px ( u /m g pr ot ) 0.00 0.05 0.10 0.15 0.25 0.20 co ntr ol ma rjo ra m m+ ds s ds s co ntr ol ma rjo ra m m+ ds s ds s co ntr ol ma rjo ra m m+ ds s ds s (a) (c) 2 4 6 8 s o d ( u /m g pr ot ) 0 co ntr ol ma rjo ra m m+ ds s ds s (b) 2 4 6 8 10 g s h ( u /m g pr ot ) 0 (d) weight as reported by cooper et al. (1993). higher dai score was related to smaller colon length and elevated histopathological score. dss is a heparinlike polysaccharide. it is considered to cause mucosal damage and inflammation initially through a direct toxic influence on intestinal epithelial cells by disrupting mucosal barrier to cause colitis (perse and cerar, 2012). dss also causes indirect damage because of modifications in resident bacteria (okayasu et al., 1990) followed by the mustering and activation of inflammatory cells and the upregulation of inflammatory mediators, resulting in severe colitis (lewellyn et al., 2017). the clinical symptoms of ibd induced by dss were lessened by marjoram oil. administration of marjoram oil to colitic mice resulted in a considerably reduced symptoms of the disease, such as diarrhoea, bloody stools, and weight loss, which were evidenced by a lower dai score. in accordance with previous results reported by al howiriny et al. (2009), histopathological damage of the gastric mucosa was significantly prevented by administering marjoram extract. ibd is characterized by inflammation that affects the integrity of epithelial barrier (landy et al., 2016). tissue showed that the dss-treated mice displayed abnormal physiological conditions with an increased clinical score (dai), indicating the earlier onset of colitis symptoms, such as diarrhoea, bloody stools, and reduced body 114 italian journal of food science, 2023; 35 (1) bouayad di et al. conductance or its reciprocal transepithelial electrical resistance is considered as a sensitive biomarker of intestinal barrier integrity and its function (catanzaro et al., 2015). changes in transcellular and paracellular ion transport through the epithelium of colonic mucosa, or a disruption in the functional integrity of intercellular tight junctions, could explain an increase in electrical conductance (mlodzik-danielewicz and tyrakowski, 2005). in the present study, dss was found to increase the electrical conductivity and cellular permeability to fitc-dextran. our results were in line with the study conducted by kitajima et al. (1999), which showed that dss enhanced colonic mucosal permeability, resulting in the alteration of inflammatory reactions and mucosal barrier function. indeed, loss of intestinal barrier function in the dss colitis model was related to changes in tight junction protein distribution (uko et al., 2012). the impairment of intestinal barrier function induced by dss in our study was clearly diminished by marjoram oil in colitic mice. this amelioration was related to a decrease in il-6 level, which plays an integral role in developing ibd (helieh et al., 2013; mazur-bialy et al., 2017). our data supported the previously reported anti-inflammatory properties of marjoram oil. arranz et al. (2015) indicated that sabinene hydrate and terpineol in sweet marjoram essential oil inhibited the production of proinflammatory cytokines. in the current study, the status of inflammation in colitic animals was confirmed by an increase in mpo activity, a specific biomarker of inflammation in colonic mucosa (malle et al., 2007). however, pre-treatment with marjoram oil significantly reduced the colonic mpo activity. mpo is highly expressed in neutrophils, but less so in monocytes and certain macrophages (malle et al., 2007). its activity is linearly related to neutrophil infiltration (iba et al., 2003). bhattacharyya et al. (2014) showed that mpo was able to catalyse the generation of ros. when ros formation surpasses the antioxidant capability of cells, cellular macromolecules are destroyed. this oxidative stress is hypothesized to have a role in the development of several human diseases, particularly ulcerative colitis (mekkioui and djerdjouri, 2012; xu et al., 2009). it was reported that dss-treated mice exhibited a significant increase in lipid peroxidation (amirshahrokhi et al., 2011; yan et al., 2015). this was in line with the results of our research, in which tbars concentrations were higher in colitic mice than in other groups. interestingly, pre-treatment with marjoram oil resulted in a considerable reduction of tbars level, demonstrating the antioxidant activity of marjoram oil. this result confirmed the results of our recent study, in which marjoram oil demonstrated a strong in vitro antioxidant power (bouayad debbagh et al., 2021). glutathione sulfhydryl is thought to be the first line of defense against oxidative stress and inflammatory cascades (zhu and li, 2012). gsh is the main reducing agent for colonic epithelial cells; it is frequently reported to diminish during severe dss-induced colitis (oz et al., 2005; wardman et al., 2007). hence, in agreement with the above findings, we discovered lower gsh levels in the colitic group. enzymatic antioxidants are another line of oxidative defense system. in the current study, the activity of cat and sod was reduced in the colonic tissues of colitic group. similar to what was observed in our study and in human ibd patients (mueller et al., 2013), several studies have revealed that when rodents are exposed to dss, enzymatic antioxidants are depleted, suggesting enhanced oxidative stress-mediated colon injury (nishiyama et al., 2012; pandurangan et al., 2015; xing et al., 2013). conclusions our data suggested that marjoram oil lessened dssinduced experimental colitis as evidenced by reduced severity of ulcerative colitis, preventing oxidative damage, reducing neutrophil infiltration, and inhibiting il-6 production. importantly, marjoram oil preserved the intestinal barrier function from damage caused by inflammatory stimuli. acknowledgments this research was supported by the directorate general for scientific research and technological development (dgrsdt, mesrs), algeria. conflict of interest the authors declared no conflict of interest with respect to research, authorship, and/or publication of this article. references aebi, h. 1984. catalase in vitro. methods enzymol. 105:121–126. https://doi.org/10.1016/s0076-6879(84)05016-3 al dhaheri, y., attoub, s., arafat, k., abuqamar. s., viallet, j., saleh, a., al agha, h., eid, a. and iratni, r. 2013. antimetastatic and anti-tumor growth effects of origanum majorana on highly metastatic human breast cancer cells: inhibition of nf_b signaling and reduction of nitric oxide https://doi.org/10.1016/s0076-6879(84)05016-3� italian journal of food science, 2023; 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https://doi.org/10.1152/ajpgi.00004.2013� https://doi.org/10.3748/wjg.v20.i39.14099� https://doi.org/10.3748/wjg.v20.i39.14099� https://doi.org/10.1016/j.bbrc.2019.02.056� https://doi.org/10.1016/j.bbrc.2019.02.056� https://doi.org/10.1016/j.freeradbiomed.2007.06.026� https://doi.org/10.1016/j.intimp.2013.02.008� https://doi.org/10.1016/j.intimp.2013.02.008� https://doi.org/10.1016/j.phymed.2009.02.021� https://doi.org/10.1016/j.phymed.2009.02.021� https://www.ncbi.nlm.nih.gov/pubmed/26884937� https://www.ncbi.nlm.nih.gov/pubmed/26884937� https://dx.doi.org/10.3748%2fwjg.v21.i8.2475� https://dx.doi.org/10.3748%2fwjg.v21.i8.2475� https://doi.org/10.1258/ebm.2011.011358� paper ital. j. food sci., vol. 27 2015 437 keywords: lipolysis, ovine blue cheese, penicillium roqueforti, volatile compounds evaluation of lipolysis and volatile compounds produced by three penicillium roqueforti commercial cultures in a blue-type cheese made from ovine milk e. salvatore*, m. addis, m. pes, m. fiori and a. pirisi agris sardegna, department of animal science, loc. bonassai, 07040 olmedo, ss, italy *corresponding author: tel. +39 079 2842391, fax +39 079 389450, email: esalvatore@agrisricerca.it abstract the aim of this work was to compare the effect of three different penicillium roqueforti commercial cultures (named ps1, ps2 and ps3) on proteolysis, lipolysis and volatile flavour profile of a blue cheese made from ovine milk and lamb paste rennet. proteolytic parameters were not significantly affected by the penicillium roqueforti culture, while cheeses manufactured using ps2 and ps3 cultures showed the higher amount of free fatty acids (ffa) and volatile ffa when compared with ps1 culture after 30 days of ripening. this study can provide important information for obtaining the desired extent of lipolysis in this type of blue cheese. mailto:esalvatore%40agrisricerca.it?subject= 438 ital. j. food sci., vol. 27 2015 introduction blue cheeses represent a cheese variety characterised by the presence of blue or blue-green veins, caused by induced growth of the mould penicillium roqueforti within the cheese matrix. this category includes, among others, pdo (protected designation of origin) or pgi (protected geographical indication) cheeses made from bovine (gorgonzola, italy; danablu, denmark; stilton, united kingdom), and ovine (roquefort, france) milk. the manufacture process of blue mould cheeses has been well described previously (ardö, 2011), but it can vary depending on country or region where cheese is produced. in particular in sardinia island (italy), a small production of ovine blue cheese is manufactured on industrial scale. this cheese is characterized by the use of lamb paste rennet for its production, differently from most of blue cheeses, where the milk coagulation is usually induced by the action of liquid rennet. cheeses produced with paste rennet are characterized, at late ripening stages, by high amounts of free fatty acids due to the presence of lipolytic enzymes (lipases) in the rennet extract (addis et al., 2005; virto et al., 2003). the ovine blue cheese is made following the process described herein. thermised whole ovine milk is inoculated with a penicillium roqueforti culture and a mesophilic starter at 36°c. milk is coagulated using a water solution of lamb paste rennet, and the coagulum is cut into small granules (about 4 mm in size), drained, moved into moulds, dry salted and ripened for 30 days at 10°c and 85% of relative humidity. cheeses are pierced using a stainless steel needle 7 days after production. at the end of ripening (30 days) cheeses are cylindrical in shape (height and diameter around 100 and 200 mm, respectively) and weigh between 2.5 and 3.0 kg. the growth of penicillium roqueforti within the cheese matrix results in a high production of its extracellular enzymes, and consequently in an extensive secondary proteolysis and lipolysis of blue cheeses during ripening (calzada et al., 2013; contarini and toppino, 1995; prieto et al., 1999; 2000). furthermore, blue cheeses are characterised by an high level of flavour compounds produced by lipid, lactose and protein catabolism (ardö, 2011); in particular a large amount of methyl ketones is produced by the β-oxidation of free fatty acids followed by a decarboxylation reaction (qian et al., 2002; voigt et al., 2010). the aim of this work was to compare the effect of three different penicillium roqueforti commercial cultures on proteolysis, lipolysis and volatile flavour profile of sardinian ovine blue cheese after 30 days of ripening, in order to provide useful information to cheese makers about the biochemical effects produced by each culture during ripening of this cheese. materials and methods mould cultures three commercially available p. roqueforti cultures, named ps1 (prb 6 hyp 5 d, danisco deutschland gmbh, niebull, germany), ps2 (pr4, chr. hansen, hørsholm, denmark) and ps3 (pv lyo 10 d, danisco deutschland gmbh, niebull, germany), were separately used to produce blue-type cheeses. more details about the specific properties of each mould culture can be found in the respective product description documents provided by the supplier. mould cultures were dissolved in water and added to 50 l of milk before renneting at a final concentration of 5.0e+6 cfu per l of milk. small-scale cheese-making cheese production was performed at the dairy technology laboratories of agris sardegna (olmedo, italy). whole ovine milk was placed in a staining steel cheese vat, batch-heated at 65°c in 10 min, and quickly cooled down to 36°c (in 5 min). after cooling, a mould culture and a milk starter culture, prepared using a freeze-dried mixed culture (bulk set hm m4 lyo, danisco, deutschland gmbh, niebull, germany; 1 l·100 l-1 of milk), were added to milk. the composition of the milk starter culture was: lactococcus lactis subsp. lactis, lactococcus lactis subsp. cremoris, lactococcus lactis subsp. lactis biovar. diacetylactis, leuconostoc mesenteroides subsp. cremoris. milk was coagulated using a water solution (30 g·100 l-1 of milk) of lamb paste rennet (caglificio manca, thiesi, italy). about 45 min after the addition of rennet, the coagulum was cut into small granules (about 4 mm in size), and the drained curd was moved into moulds and kept at 20-25°c under saturated humidity conditions for 18 h. cheeses were then kept at 10°c for 24 h and 90-95% of relative humidity, dry salted and finally ripened for 30 days at 10°c and 85% of relative humidity. cheeses were pierced using a stainless steel needle 7 days after production. three replicates were carried out for each treatment level, for a total of nine cheese-making trials. cheese composition and nitrogen fractions samples were taken for analysis 1 day after production and after 30 days of ripening. cheeses were analysed for ph (ph meter 420 a, orion, boston, usa), total solids (ts) (iso, 2004), fat (soxhlet method), total nitrogen (tn) (idf, 1993), soluble nitrogen at ph 4.6 (sn), soluble nitrogen in 12% trichloroacetic acid (tca-sn), and soluble nitrogen in 10% phosphotungstic acid (pta-sn) (gripon et al., 1975). ital. j. food sci., vol. 27 2015 439 free fatty acids analysis the free fatty acids content was determined as previously described by addis et al. (2005) on cheeses 1 day after production and after 30 days of ripening. volatile flavour profile analysis volatile compounds were determined by spme (solid-phase microextraction) -gc-fid/ms on cheeses after 30 days of ripening. 0.5 g of freshly grated cheese was placed in a 10 ml vial, hermetically sealed with a seal and thin viton septa. the vials were held at 37°c in a thermostated autosampler (8200 cx varian, walnut creek, ca, usa) for 5 min to reach equilibrium between sample and above headspace prior to spme headspace sampling. a divinylbenzene (dvb)/carboxen (car)/polydimethylsiloxane (pdms) 50/30 µm fiber (supelco inc., bellefonte, pa, usa) was exposed to headspace under constant stirring for 7 min in samples after 30 days of ripening. during headspace sampling, samples were maintained at 37°c, and volatile compounds adsorbed on the fiber were immediately thermally desorbed in the injector port of a 3800cx varian gc (walnut creek, ca, usa) equipped with a 1077 split/ splitless injector (250°c), coupled with a flame ionization detector (fid; 250°c), and a saturn 2000 ion trap mass spectrometer system (ms detector) (walnut creek, varian, ca, usa). volatile compounds were injected in splitless mode in two identical capillary columns (db-wax 30 m, 0.32 mm i.d., 0.25 µm film thickness; j. & w. scientific, folsom, ca, usa) connected one to fid and the other to mass spectrometer. the column was operated with helium (1 ml·min-1, constant flow), and the column temperature was held at 40°c for 3 min, then increased to 200°c at a rate of 4°c·min-1, and finally held at 200°c for 5 min. ms detector was programmed in electron ionization (ei) mode at an ionization voltage of 70 ev in the acquisition range between 20-300 m/z, and at a scan rate of 1 scan/sec. the trap, manifold and transfer line temperature were set to 200°, 80° and 200°c respectively. volatile compounds were identified by comparison of their mass spectral data with those of the nist 98 library (nist, usa), by their linear retention indexes (van den dool and kratz, 1963) and by comparison with authentic standard compounds (when available). statistical analysis statistical treatment of data was performed using the spss statistical package, release 11.5 (spss, chicago, il, usa). data of chemical composition, nitrogen fractions and free fatty acids were examined using a bifactorial anova model with “p. roqueforti culture factor” (pc) and “ripening stage factor” (r) as fixed effects, while lsd test (least significant difference test, p < 0.05) for multiple comparisons was used to separate treatment means. the results of volatile compounds were examined using a monofactorial anova model with “p. roqueforti culture factor” (pc) as fixed effect. results and discussion chemical composition and nitrogen fractions the chemical composition of cheeses 1 day after production and after 30 days of ripening is reported in table 1. gross composition was not significantly affected by the penicillium roqueforti culture at 1 day and after 30 days, whereas it changed significantly (p < 0.05) during the ripening (with the exception of protein content). the values of moisture, fat and protein to total solids ratio after 30 days of ripening were in agreement with data reported by lawlor et al. (2003) for other blue-type cheeses. ph values increased of around 1.4 units for all treatments from day 1 to day 30, probably as consequence of the consumption of lactate and the oxidative formation of nh 3 from amino acids operated by moulds during ripening (cantor et al., 2004). it has been seen, for example, that the ph may increase of around 2 units in danablu during the first 5 weeks of ripening (ardö, 2011). the data reported in table 1 indicate that all proteolytic parameters increased significantly throughout ripening (p < 0.05). the level of ph 4.6-sn (expressed as a percentage of total nitrogen) ranged from 31.03 to 33.97% after 30 days of ripening (table 1), in some agreement with results published for a number of different bluetype cheeses with longer ripening times (from 34% up to 72% of ph 4.6-sn; fernández-salguero et al., 1989; lawlor et al., 2003; voigt et al., 2010). the level of secondary proteolysis is higher in blue cheeses compared to other cheese varieties (cantor et al., 2004); therefore, the raised values at 30 days reported here for tca-sn and pta-sn (both expressed as a percentage of total nitrogen; table 1), compared with the values of ph 4.6-sn, highlighted that the most of the soluble fraction included non-protein substances (fernandez-salguero et al., 1989). free fatty acids analysis table 2 summarises the extent of lipolysis (expressed as mmol of free fatty acids per kg of cheese) of cheeses made with each of the three penicillium roqueforti cultures both 1 day and 30 days after production. the type of penicillium roqueforti culture significantly influenced (p < 0.05) the amount of short-chain (c4:0-c8:0), medium-chain (c10:0c14:0), and long-chain (c16:0-c18:3) ffa. overall, all individual ffa increased significantly (p < 440 ital. j. food sci., vol. 27 2015 table 1 composition and nitrogen fractions of cheeses (ps1, ps2, ps3) 1 day after production and after 30 days of ripening. 1 day 30 days seme f testf ps1 ps2 ps3 ps1 ps2 ps3 pcg rh ph 4.85 4.82 4.83 6.35 6.38 6.05 0.18 ns * moisture (g.100g-1) 49.26 49.63 50.04 42.28 42.97 42.30 0.89 ns * fata 55.02 54.93 54.88 56.15 56.63 55.60 0.26 ns * proteina 38.25 38.02 38.19 37.50 37.67 37.42 0.16 ns ns ph 4.6-snb 8.92 8.84 9.25 31.03 33.97 32.10 2.89 ns * tca-snc 5.40 5.60 5.65 28.28 30.86 29.42 2.93 ns * pta-snd 1.11 1.23 1.49 12.92 13.05 11.85 1.42 ns * aexpressed as a percentage of total solids (% w/w). b,c,dexpressed as a percentage of total nitrogen (% w/w); bsoluble nitrogen at ph 4.6; csoluble nitrogen in 12% trichloroacetic acid (tca); dsoluble nitrogen in 10% phosphotungstic acid (pta). estandard error mean. fsignificant differences: * p < 0.05; ns, no significant differences. gp. roqueforti culture factor. hripening stage factor. 0.05) during ripening, and cheeses manufactured with the cultures named ps2 and ps3 showed the higher amount of each ffa after 30 days of ripening when compared with ps1 culture. three free fatty acids (c4:0, c16:0, c18:1) represented around 60% of total free fatty acids for all treatments both 1 day and 30 days after production. after 30 days of ripening, the content of c4:0, c16:0, and c18:1 ranged on average (depending on the culture tested) from 16.07 to 31.65 mmol. kg-1, from 11.88 to 22.90 mmol.kg-1, and from 17.09 to 32.14 mmol.kg-1 of cheese, respectively. in general, the action of penicillium roqueforti lipases releases higher concentrations of longchain ffa than shortand medium-chain ffa (ardö, 2011). a recent study reported that paltable 2 free fatty acids content (mmol.kg-1 of cheese) in cheeses (ps1, ps2, ps3) 1 day after production and after 30 days of ripening. 1 day 30 days semb f testc ps1 ps2 ps3 ps1 ps2 ps3 pcd re c4:0a 0.47 0.57 0.74 16.07 28.28 31.65 3.37 * * c6:0 0.11 0.14 0.17 3.89 9.46 11.55 1.19 * * c8:0 0.20 0.20 0.20 2.74 5.50 6.85 0.68 * * c10:0 0.15 0.17 0.21 5.83 10.93 13.81 1.39 * * c12:0 0.11 0.11 0.13 2.56 4.64 5.96 0.59 * * c14:0 0.21 0.22 0.23 6.53 9.11 12.68 1.25 * * c16:0 0.46 0.45 0.47 11.88 15.20 22.90 2.24 * * c18:0 0.12 0.11 0.11 2.71 3.51 4.98 0.49 * * c18:1 0.34 0.32 0.31 17.09 27.38 32.14 3.31 * * c18:2 0.07 0.05 0.02 2.62 3.97 4.51 0.47 * * c18:3 0.02 0.02 0.02 2.08 2.85 3.88 0.39 * * tffas 2.26 2.41 2.61 74.00 120.83 150.90 15.16 * * ac4:0, butyric acid; c6:0, caproic acid; c8:0, caprylic acid; c10:0, capric acid; c12:0, lauric acid; c14:0, myristic acid; c16:0, palmitic acid; c18:0, stearic acid; c18:1, oleic acid; c18:2, linoleic acid; c18:3, linolenic acid; tffas, total free fatty acids. bstandard error mean. csignificant differences: * p < 0.05. dp. roqueforti culture factor. eripening stage factor. mitic and oleic acids reached the highest levels of long-chain ffa in a blue cheese (calzada et al., 2013). on the contrary, the values of butyric acid presented in the present study were higher than those found by other authors in different blue cheese varieties (calzada et al., 2013; woo et al., 1984). in particular, ps1, ps2 and ps3 cheeses after 30 days of ripening showed about 1.8-3.6 times higher values of c4:0 when compared with results of calzada et al. (2013), who reported that the content of butyric acid in a blue cheese after 90 days of ripening reached a value of 1.32 mg.g-1 (14.98 mmol.kg-1) of cheese dry matter. the raised values of c4:0 and, in general, of short-chain ffa observed in the present study can be ascribed to the use of lamb paste ital. j. food sci., vol. 27 2015 441 rennet for milk coagulation. lamb paste rennet contains a pregastric lipase, which preferentially hydrolyzes short chain fatty acids (kim ha and lindsay, 1993). furthermore, it is important to point out that even the lipolytic system of penicillium roqueforti can exhibit a selectivity similar to that of the pregastric lipase (kim ha and lindsay, 1993). spme analysis cheeses (ps1, ps2 and ps3) after 30 days of ripening were subjected to volatile flavour profile analysis by spme-gc-fid/ms (table 3). a total of 34 volatile compounds were identified in cheese samples, and among them only some volatile ffa (butyric, pentanoic and hexanoic acids) and some alcohols (2-pentanol, 1-pentanol and phenyl ethyl alcohol) were significantly affected by penicillium roqueforti culture (p < 0.05). ketones and acids represented almost the totality of volatile fraction and resulted as more abundant in all samples (about 70 and 27%, respectively). among ketones, 2-heptanone and 2-nonanone presented the highest values of fid peak area (table 3). these results were in agreement with those reported in literature relating to concentration of ketones in this category of cheeses (ardö, 2011; cantor et al., 2004). the presence of ketones is correlated to the typical flavour of blue cheeses, and they are produced by the β-oxidation of free fatty acids followed by a decarboxylation reaction. table 3 volatile compounds (fid peak area) in cheeses (ps1, ps2, ps3) after 30 days of ripening. lria ps1 ps2 ps3 semb pcc 839 2-propanone 275,452 196,359 179,488 70,535 ns 926 2-butanone 4,681 3,307 3,295 671 ns 1,004 2-pentanone 949,455 706,739 742,756 108,431 ns 1,107 2-hexanone 22,208 12,493 17,289 2,347 ns 1,211 2-heptanone 1,791,884 1,299,484 1,624,590 146,423 ns 1,315 2-octanone 28,394 19,954 30,148 4,275 ns 1,333 3-hydroxy 2-butanone 7,969 8,587 5,481 1,753 ns 1,422 2-nonanone 1,047,296 678,913 1,204,917 187,792 ns 1,481 8-nonen 2-one 78,175 55,547 87,753 12,625 ns 1,631 2-undecanone 13,624 3,499 11,418 3,510 ns 1,690 acetophenone 362 183 295 95 ns ketones 4,219,501 2,985,068 3,907,431 493,937 ns 1,581 propanoic acid 1,805 2,052 2,763 435 ns 1,609 2-methyl propanoic acid 951 1,141 1,400 139 ns 1,671 butyric acid 407,334 1,137,832 957,216 136,292 * 1,712 3-methyl butyric acid 6,539 3,390 4,240 912 ns 1,787 pentanoic acid 2,476 8,945 6,831 1,142 * 1,889 hexanoic acid 314,878 673,581 540,975 63,090 * 1,990 heptanoic acid 1,313 2,965 2,445 363 ns 2,103 octanoic acid 62,326 102,518 81,564 8,519 ns 2,316 decanoic acid 8,299 17,473 11,960 1,935 ns acids 805,921 1,949,897 1,609,395 208,018 * 910 ethyl acetate 293 176 125 36 ns 1,057 ethyl butyrate 5,024 6,800 3,500 1,703 ns 1,256 ethyl hexanoate 9,744 15,343 10,222 2,315 ns 1,459 ethyl octanoate 11,645 28,596 26,947 4,415 ns esters 26,511 50,914 40,794 7,354 ns 945 2-propanol 10,390 2,791 7,434 2,096 ns 1,137 2-pentanol 75,497 24,527 45,705 9,397 * 1,270 1-pentanol 2,115 281 0 368 * 1,344 2-heptanol 90,554 34,925 52,055 14,579 ns 1,538 2-nonanol 8,074 2,898 8,414 2,048 ns 1,575 2,3-butanediol 1,402 2,296 2,664 264 ns 1,950 phenyl ethyl alcohol 236 0 0 30 * 2,047 phenol 323 322 341 31 ns alcohols 188,591 68,368 117,699 26,439 ns 1,301 styrene 2,537 5,351 2,784 940 ns hydrocarbons 2,537 5,351 2,784 940 ns 940 3-methylbutanal 229 403 245 66 ns aldehydes 229 403 245 66 ns totals 5,243,291 5,060,001 5,678,349 398,884 ns alinear retention indexes using a db-wax column. bstandard error mean. cp. roqueforti culture factor, significant differences: * p < 0.05; ns, no significant differences. 442 ital. j. food sci., vol. 27 2015 the reaction is catalysed by enzymes contained both in spores and mycelium of penicillium spp. (qian et al., 2002; voigt et al., 2010). table 3 also highlights that ps1 presented significantly lower values of acids than other cheeses. this was consistent with what discussed above concerning the higher lipolytic activity of ps2 and ps3 compared to ps1 (table 2), and was also probably due to the greater aptitude of ps1 in converting ffa to 2-alkanones. among volatile ffa, butyric acid was the most abundant (50, 58 and 59% of total ffa in ps1, ps2 and ps3, respectively) followed by hexanoic (39, 35 and 34% of total ffa in ps1, ps2 and ps3, respectively) and octanoic (8, 5 and 5% of total ffa in ps1, ps2 and ps3, respectively) acids. the origin of raised values of butyric acid has been discussed in the previous section, while hexanoic and octanoic acids are important flavor compounds of blue cheeses (ardö, 2011). esters are produced by free fatty acids esterification with primary alcohols, and may attenuate the typical pungent flavour of blue cheeses due to the methyl ketones (moio et al., 2000). they represented only 1% on average of totals volatile compounds (table 3); ps2 and ps3 showed higher fid peak area (but without any statistical significance) of esters as a consequence of their higher content of ffa compared to ps1. the strong reducing environment present in ripened cheese favoured the production of 2-alkanols from corresponding 2-alkanones. ps1 showed significantly higher values of 1and 2-pentanol when compared with other samples (p < 0.05), and tended to have the highest levels both of 2-alkanones and 2-alkanols. the parallel evolution of these volatile compounds was previously observed in other blue cheeses (gonzáles de llano et al., 1990). conclusions the results indicated that the ovine blue cheese made in sardinia was more subjected to lipolysis and presented higher amounts of short chain fatty acids when compared to the most known blue cheese varieties. this evolution of lipolysis in the product was also 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woo a.h., kollodge s. and lindsay r.c. 1984. quantification of major free fatty acids in several cheese varieties. j. dairy sci. 67: 874. paper received july 7, 2014 accepted january 19, 2015 44 issn 1120-1770 online, doi 10.15586/ijfs.v34i4.2253 p u b l i c a t i o n s codon effect of seven non-conventional starch rich sources on physico-chemical and sensory characteristics of extruded snacks syed zameer hussain1, rumaisa gaffar1, bazila naseer1*, tahiya qadri1, uzma noor shah2#, monica reshi1 1division of food science and technology, sher-e-kashmir university of agricultural sciences and technology of kashmir, j&k, india; 2department of life sciences, school of sciences, jain university, banglore, india #the author has helped and contributed in revising the manuscript and language editing. *corresponding author: bazila naseer, division of food science and technology, sher-e-kashmir university of agricultural sciences and technology of kashmir, j&k, india. email: sheikhbazila@gmail.com received: 27 june 2022; accepted: 5 december 2022; published: 29 december 2022 © 2022 codon publications open access paper abstract starch-rich foods, such as cereal sources (rice, maize, and barley), are commonly used raw materials for extrusion cooking due to their excellent expansion characteristics. other nonconventional starch sources like green banana, water chestnut, and potato can also be employed for extrusion cooking. the main aim of the study was to evaluate the extrusion behavior and sensory acceptability of nonconventional starch-rich food sources like rice, maize, barley, wheat, water chestnut, potato, and green banana. maize, rice, wheat, potato, water chestnuts, barley, and green banana flour samples were evaluated for various physicochemical, pasting, and morphological properties, and were subjected to extrusion cooking at the moisture content of 15%, screw speed of 300 rpm, and barrel temperature of 125°c. the developed extruded snacks from selected crops were also evaluated for various physicochemical, pasting, and morphological properties. potato flour and green banana flour recorded the highest starch content of 78.27 and 76.61%, respectively. the highest peak viscosity (6025 cp), trough viscosity (2968 cp), breakdown viscosity (3057 cp), pasting temperature (92°c), and minimum peak time (4.67 min) were recorded in the case of green banana flour. the structural assessment of all the flour samples was done through scanning electron microscopy. the highest expansion ratio (5.06), as well as overall acceptability (4.28), was recorded in the case of corn-based snacks. the highest bulk density and hardness were recorded in the case of barley-based snacks. the highest values of water absorption index and water solubility index were recorded in the case of green banana flour–based snacks. keywords: starch rich, green banana, potato, extrusion cooking, scanning electron microscopy, pasting properties, sensory evaluation introduction snacks, being an indispensable part of the modern diet, are an effective carrier to improve the nutrition-based needs of the population. with the growing knowledge about a healthy diet, snacks can readily be consumed to improve overall nutrition. snacks are primarily made from starch-rich materials due to their good puffing and expansion characteristics. extrusion is the most commonly employed multidimensional processing technique for developing snacks (liu and hsieh, 2008). it changes the molecular conformation of starch, which then interacts with other macromolecules by simultaneous application of high temperature and pressure. although starch-based materials like corn, rice, wheat, potato, barley, and water chestnut have been explored for the development of snacks through extrusion either solely or in combination with other food materials (hernandez-díaz et  al., 2007; kaur et  al., 2015); reddy et  al., 2014) the comparative evaluation of these starchbased food materials for the development of snacks has italian journal of food science, 2022; 34 (4): 44–56 italian journal of food science, 2022; 34 (4) 45 utilization of non-conventional starch sources in extrusion cooking physicochemical analysis of flour samples moisture content moisture content was determined by the aoac method 930.04 (aoac, 2005). five grams of the sample was weighed and dried at 60–70°c for 6–8 h, to constant weight. the loss in weight was determined to calculate the percent moisture content. crude protein crude nitrogen was determined by kjeldahl method (aoac, 1995). half a gram of the sample in powdered form was placed in kjeldahl tubes and 5 g of digestion mixture (potassium sulfate + ferrous sulfate + copper sulfate in the ratio of 5:0.5:0.25) was added. after adding 10 ml of concentrated h2so4, the mixture was heated till the color changed to green. then, tubes were cooled and 10 ml of distilled water was added to each sample. then 40–50 ml of naoh (40.00%) was added to it till the color changed to brown. tubes were fitted in assembly, subjected to steam distillation, and ammonia released from the tubes was collected in the flask fitted in assembly containing 25 ml boric acid (4%) and 5 ml red indicator. flask containing boric acid and indicator results in the formation of ammonium borate, which was titrated with 0.1 n hcl till color changed to brown. the resulting nitrogen content was multiplied by a factor of 5.95 to get crude protein content. nitrogen titere value normality of hcl weight of sample (g) (%) � � �14 ��100 (1) crude protein = crude nitrogen × 5.95 (2) crude fiber crude fiber was determined by following a gravimetric procedure of aoac (1995). one gram of sample was subjected to acid hydrolysis with 2.5 n hcl followed by alkali digestion with 0.1 n naoh. the residue obtained was then washed with double distilled water and ignited in a muffle furnace at 600°c for 6 h. at high temperature, the organic matter in the residue got oxidized and inorganic residue was left behind. the difference in the weight before and after was determined to calculate the percentage crude fiber as: crude fiber w (%) � � � w w1 2 100 (3) where w1: weight of crucible with dry residue (g) w2: weight of crucible with ash (g) w: weight of sample (g) not been studied so far. in addition to this, green banana offers a significant potential to be used as a base material for the development of snacks due to its high starch content and excellent nutritional profile (kaur et  al., 2015). india is the second largest producer of bananas (fao stat, 2017) and very limited research is documented on the processing and utilization of green bananas. moreover, the ripening of green banana is a very tedious practice and an appreciable quantity of bananas is wasted during post-harvest handling. thus, there exists a possibility of exploring green banana as a base material for the development of processed products like snacks. therefore, the present study was envisaged to evaluate and compare different starch-based materials for the development of snacks with a broader aim to explore a nonconventional source of starch like green banana for extrusion processing. material and methods raw materials maize (var. c-7), rice (var. jhelum), and wheat (var. shalimar wheat 2) were procured from mountain research center for field crops, khudwani, shere-kashmir university of agricultural science and technology of kashmir (skuast-k), india; potato (var. shalimar potato-1) from the department of vegetable sciences, skuast-k; water chestnuts were harvested from wular lake of kashmir, india, which is asia’s largest lake. barley (a land race of kargil, namely, “naas”) was procured from mountain agriculture research and extension station, kargil, skuast-k and green banana (var grand naine) from the department of fruit science, sher-e-kashmir university of agricultural science and technology of jammu, india. flour preparation maize, rice, wheat, and barley were ground in a laboratory mill (3303, perten, hagersten, sweden). potatoes were washed, peeled, sliced, and dried in a tray drier (nsw-154, s-narang, scientific works new delhi) at 40 ± 5°c. before flour preparation, the usual practice is to dry the whole water chestnuts over traditional chullas (made of mud) for about 10–12 days after which the flour is made from kernels extracted from these water chestnuts. green banana (var grand naine) was peeled and sliced to 0.5 to 1 cm thickness. the slices were dried in a tray drier at 40 ± 5°c. dried potato slices and green banana slices were ground to flour in a lab mill (3303, perten, hagersten, sweden). all the seven flour samples were kept in separate polybags and stored at room temperature for further analysis. 46 italian journal of food science, 2022; 34 (4) hussain sz et al. ready-to-eat snacks. the temperature at the feed zone, compression zone, and die zone were maintained at 60°c, 80°c, and 125°c, respectively, throughout the experiment and the screw speed was kept constant at 300 rpm. earlier, the moisture content of the flour samples was adjusted to 15% through preconditioning. the extruder was equipped with a torque indicator, which showed the percent of torque in proportion to the current drawn by the drive motor. the extruded samples were collected in trays, cooled, and packed in high density polyethylene (hdpe) pouches for further analysis. determination of product responses for developed snacks extruded snacks developed from maize, rice, wheat, potato, water chestnuts, barley, and green banana flour were evaluated for below mentioned parameters. system parameter specific mechanical energy (sme) is the ratio of net mechanical energy input (after no load correction) to mass flow rate and was calculated as per the following equation (pansawat et al., 2008): sme(kwh/kg) actual screwspeed (rpm) rated screwspeed (rpm) tor � � % qque motor power rating (kwh) mass flowrate (kg/h) 100 1000� �% (7) product characteristics bulk density bulk density (bd) was measured using the volumetric displacement method as described by singh et al. (2016). a known weight of the sample (extrudates) was taken into a pre-weighed (w1) measuring cylinder and the weight of the filled cylinder (w2) as well as the volume of the sample (v1) was noted. the bd was expressed using the following equation: bd (ρb) = w2 – w1 ÷ v1 (8) water absorption index (wai) and water solubility index (wsi) wai and wsi were determined as per standard procedure given by singh et  al. (2016). one gram of the sample (w1) was mixed with 10 ml distilled water and kept at ambient temperature for 30 min and centrifuged for 10 min at 2000 rpm. then final weight was taken (w2), water absorption capacity was expressed as percent water bound per gram of the sample. crude fat crude fat analysis was done using soxtec 2045 (pelican india) (aacc, 2000). sample for oil extraction was taken in thimble and put inside an extraction cup. the extraction cups were initially dried in oven at 130°c for 15 min and the weight of empty cups was noted. after cooling, the cups were filled with 70 ml of petroleum ether, which acted as solvent for fat extraction and the thimble containing the sample was placed inside the cup. the extraction cups were then mounted on the heating plate of the instrument and the temperature raised. after attaining the required temperature, the petroleum ether was allowed to boil for 30 min till the fat was dissolved in the petroleum ether. the solvent was then recovered for 20 min. the recovered ether was collected and the fat contained in extraction cups was estimated using the following formula: fat weight of fat (g) weight of sample (g) (%) � �100 (4) ash content standard aacc procedure (aacc, 2000) was followed. five grams of the sample was put in a pre-weighed silica dish, charred on the hot plate, and incinerated in a muffle furnace at a temperature of 550 ± 10°c for about 3 h. the dish was cooled, weighed, and ash content was expressed as percent ash as given below: ash weight of ash (g) weight of sample (g) (%) � �100 (5) carbohydrate content carbohydrate content was estimated by the difference method using the below given equation (aoac, 1995). total carbohydrate content (%) [100 %(protein content fat � � � � � � � � content moisture ash crude fiber)] � � � � � � � � (6) extrusion processing extrusion cooking was carried out in a twin-screw extruder (basic technology pvt. ltd., kolkata, india) with a 2.5 mm barrel diameter and 8:1 length to diameter ratio. the extruder was fitted with a die nozzle of 0.42 mm diameter. the preliminary trials were conducted to determine the best extrusion conditions (i.e., barrel temperature and screw speed) and moisture content of the feed material for the development of italian journal of food science, 2022; 34 (4) 47 utilization of non-conventional starch sources in extrusion cooking breakdown viscosity (peak viscosity – hold viscosity), and set back viscosity (—fvhold viscosity) scanning electron microscopy scanning electron microscopy (sem) was used to study the morphology of the flour samples. the samples were glued onto a sample holder using double-sided cellophane tape and then coated with gold. the coated samples were photographed using a scanning electron microscope (hitachi s-300h-tokyo, japan), at an accelerator potential of 5 kv to visualize the microstructure. statistical analysis statistical analysis of data was conducted using spss software (version 21). all the experiments were carried out in triplicate and data were analyzed using design factorial in completely randomized design (crd). the significance of microwave heating was assessed by one factorial crd at 5% level of significance. a p-value of less than 0.05 was used to designate the statistical significance in all the tested parameters. results and discussion physicochemical analysis of different flour samples table 1 illustrates the proximate composition including the starch content of flour different samples. the moisture content of flour different samples was in the range of 7.56% (for potato flour) to 12.20% (for rice flour). the highest percentage of crude fiber (5.54%) was recorded in barley flour followed by wheat flour (4.51%), whereas the least crude fiber content recorded in corn flour (0.42%) was statistically at par with that of rice flour (0.62%). the highest protein content recorded in the case of barley flour (11.83%) was found to be statistically at par with that of wheat flour (11.79%), whereas the least protein content was recorded in water chestnut flour (2.82%). the highest carbohydrate content (82.04%) recorded in the case of potato flour was found statistically at par with that of green banana flour (81.74%) and water chestnut flour (81.50%). at the same time, the highest starch content (78.27%) recorded in potato flour was statistically at par with that of green banana flour (76.61%); however, the least carbohydrate content (67.39%) and starch content (62.40%) were recorded in the case of barley flour. the highest fat content (3.10%) was recorded in corn flour followed by barley flour (2.05%), whereas the least fat content recorded in rice flour (0.23%) was statistically at par with that of water chestnut flour (0.28%), which was further at par with that of potato flour (0.35%) (table 1). wac ( ) w w % � � �1 2 1 100 w (9) wsi was determined from the amount of dried solids received by evaporating the supernatant from the water absorption index test described above. wsi was expressed as follows: wsi weight of dissolved solids in supernatant weight of dry sol (%) � iids �100 (10) expansion ratio the expansion ratio (er) was calculated as the ratio of extrudate thickness and the die diameter by using a digital vernier caliper of 0.001 mm accuracy (jyothi et  al., 2009). hardness hardness was determined using ta-xt2i texture analyzer by applying a compression force (cf) using a p50 compression probe (50 mm. dia. cylinder aluminum) (singh et al., 2016). sensory evaluation sensory evaluation of the samples was done by semitrained panelists. a panel of 30 semi-trained judges selected from the scientific staff of division of food science and technology, skuast-k, shalimar, carried out the sensory evaluation in the laboratory of food science and technology. noise-free and well illuminated sensory space was used for the test. judges were also acquainted with rating method, specific terminology used, and different sensory characteristics. coded samples were presented to the panelists in plastic cups covered with lids. each coded sample was evaluated thrice by judges in a randomized manner. the panelists were provided with a glass of water to rinse their mouths and were given a 10 min break post each assessment. sensory evaluation was done on a 5-point scale (1-extremely dislike and 5-extremely like) for various attributes (i.e., appearance, color, texture, flavor, and mouthfeel). the overall acceptability of each sample was calculated as the average of scores obtained for selected sensory attributes (naseer et al., 2021). pasting properties pasting properties were determined with a rapid visco analyzer (rva starch tm, new port, scientific warrie wood, australia) in accordance with the methods described by kaur et  al. (2015). the different recorded parameters were pasting temperature (pt), peak time, peak viscosity, hold/trough viscosity (tv) (minimum viscosity at 95°c), final viscosity (fv) (viscosity at 50°c), 48 italian journal of food science, 2022; 34 (4) hussain sz et al. tuber flour gelatinizes at relatively low temperature, with rapid and uniform swelling of granules. higher pt of green banana flour may be attributed to its strong intermolecular forces within the flour matrix due to the closely packed smaller starch granular arrangement (figure 1f). peak viscosity (pv) of flour samples ranged from 362 to 6025 cp (table 2). the lowest pv was recorded for water chestnut flour (362 cp) and the highest for green banana flour (6025 cp), which suggests the possible use of banana flour as a thickener in food application. pv indicates the water holding capacity of flour suspension and is attained before the structural breakdown of swollen starch granules takes place (hussain et  al., 2014). disintegrated starch structure in water chestnut flour (as was evident in figure 1g) restricts swelling of starch granules, which leads to lower pv. as far as product quality is concerned, high pv is always desirable (bhattacharya and corke, 1996). higher starch content (76.61%) of green banana flour together with its higher pt (92°c) may be the possible reason for its high pv. tv of flour samples ranged from 325 to 2968 cp (table 2). the lowest tv was recorded for water chestnut flour (325 cp), whereas the highest was recorded for green banana flour (2968 cp). the high tv of green banana flour suggests its high holding strength during cooling. interaction of starch with the highest ash content was recorded in the case of water chestnut flour (2.38%), which was statistically at par with that of banana flour (2.06%), whereas the least ash content recorded in the case of potato flour (0.43%) was statistically at par with that of rice flour (0.47%) and wheat flour (0.53%). these results are in accordance with the results reported by qamar et  al. (2017) for protein, ash, and carbohydrate contents of corn flour; by onwuka et al. (2015) for fat, moisture, and ash contents of banana flour; by hussain et al. (2019) for protein, fat, and crude fiber contents of barley and water chestnut flour. pasting characteristics of different flour samples pasting profile depicted in table 2 demonstrates the wide range of viscosity parameters for different flour samples. pt), which indicates the onset of rise in viscosity, was found in the range of 60.9–92°c for different flour samples. the lowest pt of 60.9°c was recorded for potato flour and the highest (92°c) for green banana flour (table  2). lower pt of potato flour can be attributed to the tendency of large swollen starch granules to gelatinize, which was evident in the sem micrograph (figure  1a) as well. danbaba et  al. (2014) reported that table 1. physicochemical analysis of different flour samples. parameters flour samples moisture (%) fat (%) ash (%) crude fiber (%) protein (%) starch (%) carbohydrate (%) rice 12.20a ± 1.16 0.23a ± 0.11 0.47a ± 0.02 0.62a ± 0.08 8.03a ± 0.91 73.03a ± 7.21 78.45a ± 4.25 wheat 12.15ba ± 1.15 1.70b ± 0.24 0.53ba ± 0.02 4.51b ± 0.04 11.79b ± 1.35 65.18b ± 4.25 69.32b ± 6.33 corn 10.72c ± 1.08 3.10c ± 0.51 1.69c ± 0.24 0.42ca ± 0.03 8.23c ± 1.02 72.82ca ± 5.12 75.84c ± 6.42 barley 11.95dab ± 1.38 2.05d ± 0.11 1.24d ± 0.17 5.54d ± 0.18 11.83db ± 1.22 62.40d ± 3.42 67.39d ± 7.86 potato 7.56e ± 0.76 0.35e ± 0.02 0.43eab ± 0.03 2.33e ± 0.01 7.29e ± 0.56 78.27e ± 4.56 82.04e ± 5.65 green banana 10.34f ± 1.18 0.62f ± 0.01 2.06f ± 0.20 1.49f ± 0.03 3.75f ± 0.21 76.61fe ± 1.86 81.74fe ± 2.21 water chestnut 9.68g ± 0.84 0.28gae ± 0.01 2.38gf ± 0.07 3.34g ± 0.00 2.82g ± 0.02 70.50gc ± 6.95 81.50gef ± 4.87 data are expressed as mean ± sd; values in the same column with different superscripts are statistically different. table 2. pasting properties of different flour samples. parameters flour samples peak viscosity (cp) trough viscosity (cp) breakdown viscosity (cp) final viscosity (cp) setback viscosity (cp) peak time (min) pasting temperature (°c) rice 2940a ± 17 1727a ± 12.76 1213a ± 17.69 4343a ± 25.51 2616a ± 12.66 5.13a ± 0.07 80.90a ± 2.57 wheat 2106b ± 11.15 1292b ± 11.23 814b ± 20.15 3332b ± 11.01 2040b ± 11.67 5.60b ± 0.05 77.45b ± 1.69 corn 2205c ± 17.15 1329c ± 10.96 876c ± 23.54 3403c ± 16.01 2074c ±17.03 5.64cb ± 0.1 73.28c ± 1.74 barley 2915d ± 24 925d ± 12.58 1990d ± 6.80 3744d ± 8.73 2819d ± 15.01 5.70dbc ± 0.04 85.07d ± 2.28 potato 3817e ± 13.65 2490e ± 11.50 1327e ± 9.84 5474e ± 11.53 2984e ± 8.45 6.87e ± 0.05 60.9e ± 2.81 green banana 6025f ± 22.54 2968f ± 13.15 3057f ± 21.51 3302f ± 12.22 334f ± 5.50 4.67f ± 0.03 92.00f ± 2.79 water chestnut 362g ± 9.01 325g ± 15.27 37g ± 3.05 452g ± 7.63 127g ± 9.01 4.90g ± 0.1 71.65g ± 1.28 data are expressed as mean ± sd; values in the same column with different superscripts are statistically different; cp: centipoise. italian journal of food science, 2022; 34 (4) 49 utilization of non-conventional starch sources in extrusion cooking figure 1. sem micrographs of (a) rice flour, (b) wheat flour, (c) corn flour, (d) barley flour, (e) potato flour, (f) green banana flour, and (g) water chestnut flour. (a) (b) (d) (f) (g) (c) (e) 50 italian journal of food science, 2022; 34 (4) hussain sz et al. starch granules reflect the crystalline and ordered molecular arrangement of rice flour. reddy and bhotmange (2013) also reported the presence of intact crystalline starch granules in basmati rice flour. the microstructure of wheat flour shown in figure 1b depicts a compact structure of irregularly shaped particles of different sizes. massive starch arrangement embedded within the gluten matrix was evident in figure 1b, wherein gluten possibly acted as a cementing material in forming a compact and dense structure. sakhare et  al. (2014) also reported the highly compact packed structure of wheat kernels. the micrograph shown in figure 1c indicates that somewhat spherical shaped large and small starch granules are present in corn flour. loosely packed starch arrangement within protein and lipid matrix was also observed in figure  1c. a large part of the shapeless cracked surface observed in corn flour can be attributed to the presence of soluble starch remnants (haros et al., 2006). some crater-like depressions and eroded starch surfaces were seen in figure 1c, which indicates kernel breakage due to milling. hall and sayre (1970) also reported the presence of crater-like small indentations on polygonal starch structures in pearl corn. the scanning electron micrograph depicted in figure 1d demonstrates that barley flour consists of numerous round and polygonal shaped edged starch granules. the small and large starch granules with smooth surfaces in figure 1d confirm the presence of both aand b-type starch granules in barley flour. densely packed clusters of starch embedded in flour matrix and a small amount of protein adhered to densely packed starch granules were also observed in figure 1d. nair et  al. (2011) also reported the presence of large (a-type) and small (b-type) starch granules surrounded by the protein matrix in barley flour. sullivan et al. (2010) also reported bimodal distribution of starch granules in barley flour. the sem micrograph depicted in figure 1e indicated the presence of oval and polygonal starch granules in potato flour. the small and immature starch granules were seen adhered to the large starch granules (figure  1e). similar observations were reported by horovitz et  al. (2011) for potato starch granules. the sem micrograph depicted in figure 1f indicates that green banana flour consists of elongated, round, and oval-shaped starch granules. a large number of smooth, small, and intact starch granules without any rupture were evident in figure 1f. babu et al. (2014) also reported the presence of oval and elongated smooth starch structures in green banana flour. the micrograph depicted in figure  1g demonstrates that in water chestnut flour starch granules are somewhat irregular and asymmetric in shape with a rough surface. rough granular structures indicated that starch was highly damaged, which can be attributed mainly to hydrothermal pre-conditioning of water chestnuts before flour preparation. the fissures seen on the surface of granules were probably due to the drying of water chestnut kernels before milling (figure 1g). soluble fibers in unripe banana might be responsible for its high tv (mota et  al., 2000). breakdown viscosity (bdv) of flour samples was found in the range of 37 to 3057 cp (table  2). bdv depicts the potential of flour suspension to withstand high temperature under continuous shear conditions (hussain et  al., 2014). high bdv is desirable for the production of snacks (bhattacharya and corke, 1996). water chestnut flour had the lowest breakdown viscosity (37 cp), which can be attributed to its lowest peak viscosity. the highest bdv (3057 cp) recorded for green banana flour indicates its high susceptibility to shear-induced degradation. fv indicates the ability of the flour to form a viscous paste after cooking and cooling and was found in the range of 452–5474 cp. the lowest fv was recorded in the case of water chestnut flour (452 cp) possibly because of its high damaged starch content (figure 1g) which has a low tendency to develop viscous pastes. at the same time, highest fv of potato flour (5474 cp) can be attributed to its highest starch content (78.27%) (table 1) and large-sized starch granules (figure  1e). setback viscosity (sbv), which denotes the tendency of cooked starchy material to re-associate and retrograde upon cooling (hussain et  al., 2014), ranged from 127 to 2984 cp (table 2) for different flour samples. lower sbv (127 cp) recorded for water chestnut flour demonstrates lower syneresis of cooked starch pastes during storage. however, the higher sbv of potato flour (2984 cp) indicates its high retrogradation tendency and ability to form a cohesive gel upon cooling. the peak time of flour samples indicates the time required to reach the peak in viscosity and ease of cooking a particular sample. the peak time of flour samples ranged from 4.67 to 6.87 min (table 2). the minimum peak time of 4.67 min was recorded for green banana flour and the highest (6.87 min) for potato flour. the minimum peak time of banana flour demonstrates its easy cooking ability and can be attributed to its soft and smaller starch granular structure (figure 1f). at the same time, the high peak time of potato flour can be attributed to the high swelling degree of its starch granules due to their large granular structures (figure 1e). the peak time of wheat flour (5.60  min), corn flour (5.64  min), and barley flour (5.70 min) were found to be statistically at par with each other. scanning electron microscopy of different flour samples the microstructure analysis of different flour samples showed a disparity in the starch granular arrangement within the flour matrix, which can be attributed to variation in genotype, climatic conditions as well as processing variability. sem micrograph depicted in figure  1a shows closely packed starch granules of distinct shapes with fused smaller and larger starch granules in rice flour. intact starch granules with some rough surfaces were evident in the sem micrograph of rice flour. these intact italian journal of food science, 2022; 34 (4) 51 utilization of non-conventional starch sources in extrusion cooking effect of different raw material on product characteristics of developed extrudates bulk density and hardness bd is a measure of the degree of expansion undergone by the melt as it exits the extruder (meng et  al., 2010). bd and er are important parameters of extruded snacks as far as consumer acceptability is concerned. lightweight and puffed snacks are preferred by the consumers. table 3 shows that the bd of different extrudates ranged from 51.79 to 79.68 kg/m3. the highest bd was recorded in barley-based snacks (79.68kg/m3), while the lowest bd (51.79 kg/m3) was recorded in the case of cornbased snacks followed by rice-based snacks (55.11 kg/m3) (table 3). these findings demonstrate that out of all types of snacks, barley-based snacks were the most dense, whereas corn-based snacks were lighter followed by ricebased snacks. the higher fiber content of barley flour (5.54%) (table 1) could be the possible reason behind the higher bd of barley-based extrudates and lower bd of cornand rice-based extrudates. similar results have been reported by kirjoranta et al. (2015) for barley-based extrudates and reddy et  al. (2014) for corn-based extrudates. hardness is associated with the expansion and cell structure of the extrudates. the more is the force needed by the probe to penetrate into the extrudate, the higher is the hardness of extrudates (meng et al., 2010). hardness values of different extrudates were significantly (p < 0.05) different and were found in the range of 17.05–51.55 n (table 3). among all types of snacks, the highest hardness value (51.55 n) was recorded in the case of barley-based snacks followed by wheat-based snacks (43.24 n), whereas the least hardness value (17.05n) was recorded for corn-based snacks. several studies reported a highly positive correlation between bd and hardness (altan et  al., 2008; bhattacharya, 1997; hussain et  al., 2017); effect of different raw materials on system parameter of extruded snacks specific mechanical energy sme, the mechanical energy delivered to the unit mass of the material by motor drive in the extruder, measures the work done by the motor per unit mass in the extrusion system (prabhakar et  al., 2017). the mechanical energy facilitates the starch conversion and correlates well with the physical attributes of extrudates such as expansion, density, and texture characteristics (altan et  al., 2008). thus, the higher is the sme, the higher is the degree of starch gelatinization and thus the extrudate expansion (hussain et  al., 2017). table 3 depicts that sme values of extrudates developed from different samples were found in the range of 50.86–85.46 wh/kg (table 3). the highest sme (85.46wh/kg) recorded for potato-flour-based extrudates was statistically at par with that of corn flour–based extrudates (83 wh/kg), and green banana flour–based extrudates (82.39 wh/kg) while the lowest sme (50.86 wh/kg) was recorded for wheat-based extrudates, which were statistically at par with that of barley-based extrudates (52.16 wh/kg). meuser et  al. (1990) reported that sme increases with the increase in starch content of the feeding material. thus, the higher starch content (78.20%) of potato flour was the possible reason for its higher sme. gropper et al. (2002) reported that protein and fat content affect the sme inversely by forming thermoplastic complexes with water, which restricts the fragmentation of starch granules. however, the nonsignificant difference in sme of wheat-based and barley-based snacks viz-a-viz that of potato-, corn-, and green-banana-based snacks demonstrate the nonsignificant effect of protein and fat content on sme in the present study. an almost similar range of sme has also been reported by singh et  al. (2019) for corn-based snacks, and by pansawat et al. (2008) for rice-based extrudates. table 3. system and product responses of extrudates prepared from different flour samples. parameters flour samples sme (wh/kg) bulk density (kg/m3) expansion ratio hardness (n) wai (g/g) wsi (%) rice-based snacks 80.45a ± 0.12 55.11a ± 0.11 4.82a ± 0.02 21.72a ± 0.21 4.24a ± 0.09 29.84a ± 0.05 wheat-based snacks 50.86b ± 0.12 73.20b ± 0.10 2.84b ± 0.05 43.24b ± 0.36 3.47b ± 0.07 21.70b ± 0.08 corn-based snacks 83.00ca ± 0.32 51.79ca ± 0.09 5.06c ± 0.06 17.05c ± 0.11 5.35c ± 0.05 32.68c ± 0.06 barley-based snacks 52.16db ± 0.07 79.68d ± 0.07 1.96d ± 0.03 51.55d ± 0.16 3.07d ± 0.11 19.03d ± 0.06 potato-based snacks 85.46ec ± 0.05 62.51e ± 0.07 3.97e ± 0.01 33.24e ± 0.1 3.73e ± 0.08 27.56e ± 0.08 green-banana-based snacks 82.39face ± 0.08 59.29f ± 0.08 4.11fe ± 0.03 27.84f ± 0.24 5.50f ± 0.15 35.07f ± 0.09 water-chestnut-based snacks 73.25g ± 0.11 68.58g ± 0.08 3.05g ± 0.04 37.61g ± 2.23 3.85g ± 0.05 23.21g ± 0.05 data are expressed as mean ± sd; values in the same column with different superscripts are statistically different; sme: specific mechanical energy; wai: water absorption index; wsi: water solubility index. 52 italian journal of food science, 2022; 34 (4) hussain sz et al. reported that the higher is the starch content of the feed material, the higher will be the wai of extrudates due to greater exposure of hydrophilic groups of starch to water molecules, thereby allowing better moisture penetration into the porous extrudate structure. at the same time, higher protein and fiber content are known to reduce wai due to their dilution effect on starch (singh et  al., 2007). jones et  al. (2000) reported that fiber, starch, and protein have a conjugation effect on wsi. lower starch and higher protein and fiber content affect the extent of gelatinization and dextrinization, which reduces wai and wsi. the highest starch content recorded in the case of potato flour (78.2%) was found statistically at par with that of green banana flour (76.61%), whereas protein content and crude fiber content of potato flour were significantly (p < 0.05) higher than that of green banana flour (table 1), which justifies the highest wai and wsi of green-banana-flour-based extrudates compared to potato-flour-based extrudates as well as other extrudates. likewise, the least wai and wsi of barley-flourbased extrudates were possibly due to the lowest starch content (62.40%), highest protein content (11.83%), and crude fiber content (5.54%) of barley flour compared to other flour samples. similar wai and wsi values were reported by reddy et  al. (2014) for corn-based extrudates; ding et  al. (2005) for rice-based extrudates; hernandez-díaz et al. (2007) for wheat-based extrudates; and gamalth (2008) for banana-based extrudates. sensory evaluation of different flour-based extrudates sensory characteristics of different flour-based extrudates are presented in figure 2. mouthfeel, described as a sensation recognized by the nervous system in the cavity of the mouth (singh et  al., 2019), was found in a range of 3.00–4.35. green-banana-based snacks recorded the highest mouthfeel score (4.35) followed by corn-based snacks (4.10) while the least mouthfeel scores were recorded for riceand barley-based extrudates. the highest mouthfeel score of green-banana-based snacks was possibly due to their fruity taste while the presence of an appreciable amount of soluble sugars in corn (zilic et al., 2011) was the possible reason for the high mouthfeel score of corn-based extrudates. at same the same, the presence of tannic acids in barley (niffenegger, 1964) possibly had a negative effect on the taste of the snacks developed from it, while the bland taste of rice could be the possible reason for the lower mouthfeel score of ricebased extrudates. sharma et al. (2012) also reported that a bitter taste was noticed in barley-based muffins due to the presence of phenolics. the highest visual color score was recorded for rice-based extrudates (4.66) followed by corn-based extrudates (4.20), while green-banana-based snacks recorded the meng et  al., 2010), thus, low-density products naturally offer low hardness. these results are in agreement with the findings of hardness reported by kirjoranta et  al. (2015) for barley-based extrudates; by singh et al. (2016) for corn-based extrudates; and by ding et  al. (2006) for wheat-based extrudates. expansion ratio during extrusion cooking, the sudden drop of pressure at the exit die causes a flash off of material moisture (arhaliass et  al., 2009) because of which air pockets are formed within the sample, which leads to the formation of porous and puffed snacks (yanniotis et  al., 2007). as far as consumer acceptability is concerned, high er is desirable (hussain et  al., 2017). er of snacks developed from different flour samples ranged from 1.96 to 5.06 (table  3). highest er (5.06) was recorded in the case of corn-based snacks followed by rice-based snacks (4.82) and green-banana-based snacks, whereas the lowest er was recorded in the case of barley-based snacks (1.96) (table  3). however, er of green-banana-based snacks (4.11) was statistically at par with that of potato-based snacks (3.97) (table 3). since er and bd are inversely related (meng et  al., 2010), higher fiber content of barley could be the possible reason behind lower er of barley-based snacks viz-a-viz lower fiber content of corn, rice, and green banana flour justifies the higher er of extrudates developed from them. these results were found in accordance with the results of er reported by reddy et  al. (2014) for corn-based snacks; ding et  al. (2005) for rice-based snacks; hernandez-díaz et  al. (2007) for wheat-based extrudates; and gamalth (2008) for banana-based extrudates. wai and wsi wai is a measure of the water holding capacity of starch and gives the weight and volume occupied by starch gel formed upon interaction with water (kaur et  al., 2015) while wsi indicates the extent of polysaccharides leached from starch granules in the presence of excess water and measures the amount of soluble solids present in the extrudates as a result of starch degradation during extrusion cooking (ding et  al., 2005). thus, higher wsi and wai are desirable as far as extrudate quality is concerned (anderson, 1982). both wai and wsi values of different flour-based extrudates were significantly different (p < 0.05) and were found in the range of 3.07–5.50 g/g and 19.03–35.07%, respectively. highest wai (5.50 g/g) as well as wsi (35.07%) was recorded in the case of green-banana-flour-based extrudates, while least wai (3.07g/g) and wsi (19.03%) were recorded for barley-based extrudates. delgado-nieblas et  al. (2014) italian journal of food science, 2022; 34 (4) 53 utilization of non-conventional starch sources in extrusion cooking appearance as well as for texture was recorded by ricebased snacks followed by corn-based extrudates while the least scores were attained by barley-based snacks for both the sensorial parameters. due to the relatively higher starch content and the lower crude fiber content (table 1) of rice and corn flour, the expansion (table 2) was maximum in snacks developed from them compared to other snacks, which could be the reason behind their maximum textural and appearance scores. likewise, the low starch content and the high percentage of crude fiber in barley flour (table 1) limited the expansion of the barley-based snacks, which led to harder snacks with undesirable appearance. overall acceptability of different snacks varied from 2.89 to 4.28 (figure 2e). cornbased snacks were found to be highly acceptable with an overall acceptability score of 4.28 followed by rice (4.23) lowest color scores (2.66) (figure 2b). the comparatively bright and white color of rice-based snacks and the desirable yellow color of corn-based extrudates could be the possible reasons for their high visual scores. a low color score of green banana snacks can be ascribed to their inordinate sugar content (li et  al., 2018), which leads to the formation of brown pigments through the caramelization of sugars during extrusion cooking (adubofuor et  al., 2016). gomes et  al. (2016) also reported an increase in the darker color of bread when the level of green banana flour in the formulation was increased. the scores for appearance and texture of different extrudates varied from 2.83 to 4.70 and 2.73 to 4.56, respectively. appearance implies the visual characteristics of the extruded product including its size as well as texture and shape (singh et al., 2019). the highest score for 0 2 4 6 rice based snacks wheat based snacks corn based snacks barley based snacks potato based snacks green banana based snacks water chestnut based snacks 0 1 2 3 4 5 rice based snacks wheat based snacks corn based snacks barley based snacks potato based snacks green banana based… water chestnut based… 0 1 2 3 4 5 rice based snacks wheat based snacks corn based snacks barley based snacks potato based snacks green banana… water chestnut… 0 1 2 3 4 5 rice based snacks wheat based snacks corn based snacks barley based snacks potato based snacks green banana… water chestnut… 0 1 2 3 4 5 rice based snacks wheat based snacks corn based snacks barley based snackspotato based snacks green banana snacks water chestnut based snacks figure 2. sensory evaluation of extrudates prepared from different flour samples. (a) mouthfeel, (b) color, (c) appearance, (d) texture, and (e) overall acceptability. (a) (b) (c) (d) (e) 54 italian journal of food science, 2022; 34 (4) hussain sz et al. snacks. the outcome of the present study will provide a basic guideline for food processors and researchers in the selection of suitable base materials for the development of extruded snacks. references aacc (american association of cereal chemists), 2000. international approved methods of analysis. 11th ed. aacc international, st. paul, mn. adubofuor, j., amoah, i. and osei-bonsu, i., 2016. sensory and physicochemical properties of pasteurized coconut water from two varieties of coconut. food science and quality management 54: 26–32. altan, a., mccarthy, k.l. and maskan, m., 2008. extrusion cooking of barley flour and process parameter optimization by using response surface methodology. journal of science of food and agriculture 88(9): 1648–1659. https://doi.org/10.1002/jsfa.3262 anderson, r.a. (1982). water absorption and solubility and amylograph characteristics of roll-cooked small grain products. cereal chemistry 59: 265–269. aoac, 1995. official methods of analysis. 15th ed. association of official analytical chemists, arlington, va, pp. 114–320. aoac, 2005. official method of analysis of the association of official analytical chemists. 10th ed. scientific research publishing, washington, dc. arhaliass, a., legrand, j., vauchel, p., fodil-pacha, f., lamer, t. and bouvier, j.m., 2009. the effect of wheat and maize flours properties on the expansion mechanism during extrusion cooking. food bioprocess technology 2(2): 186–193. https://doi.org/ 10.1007/s11947-007-0038-6 babu, a.s., mahalakshmi, m. and parimalavalli, r., 2014. comparative study on properties of banana flour, starch and autoclaved starch. trends in carbohydrate research 6(1): 38–44. bhattacharya, m. and corke, h., 1996. selection of desirable starch pasting properties in wheat for use in white salted or yellow 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effect of extrusion conditions on the physicochemical properties and while the least overall acceptability score was recorded for barley-based snacks (2.89). overall acceptability was determined as the average value of all the sensory characteristics (singh et al., 2019). based on the sensory scale used (1-represents poor, 2-fair, 3-good, 4-very good, and 5-excellent), the inference drawn out of sensory evaluation was that cornand rice-based snacks were rated in the scale of very good to excellent; wheat-, potato-, green-banana-, and water-chestnut-based snacks in the scale of good to very good; and barley-based snacks were rated on the scale of fair to good. conclusion evaluation of viscous behavior of starch through rapid visco analyzer (rva) as well as understanding its structure through sem was deemed essential to predict the extrusion behavior of starch-rich materials. the different starch-rich materials selected in the present study showed diverse pasting profiles, starch structures as well as extrusion behavior. the highest starch content (78.2%), as well as carbohydrate content (82.04%), was recorded in potato flour, which was statistically at par with that of green banana flour. however, the lowest starch content (62.40%) as well as carbohydrate content (67.39%) was recorded in the case of barley flour. the highest peak viscosity (6025 cp), tv (2968 cp), breakdown viscosity (3057 cp), pt (92°c), and minimum peak time (4.67 min) were recorded in the case of banana flour, which was in conformity with its elongated, round, and oval-shaped starch granules revealed through sem (figure 1f) as well as higher values of extrusion characteristics such as wai and wsi. in addition to starch content, it was observed that crude fiber also had a major influence on the physical characteristics of snacks. despite the lower starch content of corn and rice flour than potato and green banana flour, the highest erer, overall acceptability, and lower bd and hardness were 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and south indian parotta. journal of food science and technology 51(12): 4108– 4113. https://doi.org/10.1007/s13197-013-0939-5 sharma, p., gujral, h.s. and singh, b., 2012. anti-oxidant activity of barley as affected by extrusion cooking. food chemistry 1 3 1 : 1 4 0 6 – 1 4 1 3 . h t t p s : / / d o i . o r g / 1 0 . 1 0 1 6 / j . f o o d c h e m . 2011.10.009 paper ital. j. food sci., vol. 27 2015 397 keywords: date fibre, ice cream, elemental composition, nutraceutical ingredient mineral contents and physical, chemical, sensory properties of ice cream enriched with date fibre filiz yangilar erzincan university school of health department of nutrition and dietetics, 24100, erzincan, turkey tel. +90 446 2266669, fax: +90 446 2265862 email: f_yangilar@hotmail.com abstract date samples of amber cultivar straining from medina region (saudi arabia) were analysed for their chemical compositions and physicochemical properties of date fibre in the present study. fibre rich date pieces were found to contain 80.2 g/100 g total dietary fibre, 16.32 g water/g sample water-holding capacity while 9.50 g oil/g sample oil-holding capacity. it can be stated from these results that fibre content of date is a valuable dietary fibre source and used in food production as an ingredient. effects of the addition of date fibres at different concentrations (1, 2, 3 and 4%) were investigated on the physical, chemical, sensory properties and mineral content of ice cream in the present study. it was found that elemental composition of ice cream samples was affected significantly by the addition of date fibre concentrations (p<0.05) and the rates of k, mg and zn especially increased in the samples depending on the content of date fibre while the content of ca and p decreased. it was determined from the sensory results that ice cream sample containing date fibre in the rate of 1 and 2% received the highest score from panellists. 398 ital. j. food sci., vol. 27 2015 introduction dietary fibre as a class of compounds includes a mixture of plant carbohydrate polymers, both oligosaccharides and polysaccharides (cellulose, hemicelluloses, pectic substances, gums, resistant starch, inulin) that may be associated with lignin and other non-carbohydrate components (polyphenols, waxes, saponins, cutin, phytates, resistant protein; elleuch et al., 2011). over the last decades, knowledge on dietary fibre has increased considerably, both in the physiological and analytical areas. health benefits of dietary fibre are associated with bowel function, reduced risk of coronary heart diseases, type 2 diabetes and improved weight maintenance (agostoni et al., 2010; hauner et al., 2012; westenbrink et al., 2012). in addition, dietary fibre can provide a multitude of functional properties when they are incorporated in food systems. several advantages of using fruit fibres in ice cream production are improvement in body due to the fibrous framework and melting properties, reduction of cold impression, reduction of recrystallization causing prolonged shelf-life, and enhancing mixed viscosities allowing freezing at higher overrun, causing no negative effect on the ice crystal sizes, and leading to a more homogenous air-bubble formation (anonymous, 2000; dervisoglu and yazici, 2006). thus, fibre addition contributes to the modification and improvement of the texture, sensory characteristics and shelf-life of food due to their water binding capacity, gel-forming ability, fat mimetic, anti-sticking, anti-clumping, texturising and thickening effects (dello staffolo et al., 2004; gelroth and ranhotra, 2001; thebaudin et al., 1997, chap. 23; soukoulis et al., 2009). there is little data dealing with the study of the functionality of dietary fibre in ice creams (soukoulis et al., 2009). date (phoenix dactylifera l.) provides a good source of dietary fibre content, is also considered to be a commercially important agricultural commodity as well as vital element of the daily diet and a nutritious food in the arabian world (khan et al., 2008; al-farsi and lee, 2008) generally being consumed fresh or processed into various products (singh et al., 2013). annual production rate of date all across the world in 2010 was about 7.91 million tons, which increased in the rate of 6.6% in when compared to 2009 (fao, 2011; ahmed et al., 2013). different varieties of date vary in their chemical composition especially in sugars and dietary fibre (mustafa et al., 1986; ahmed et al., 1995; rahman and al-farsi, 2005; singh et al., 2013). the importance of date in human nutrition comes from its rich composition of carbohydrates (70-80%), salts and minerals, dietary fibres, vitamins, fatty acids, antioxidants, amino acids and protein (el-beltagy et al., 2009; al-shahib and marshall, 2003; el-nagga and abd el–tawab, 2012; al-farsi et al., 2005, 2007; biglari et al., 2008; hong et al., 2006; mansouri et al., 2005; vayalil, 2002; kchaou et al., 2013). in fact, date fruit has been used in traditional medicine as immune system stimulator (puri et al., 2000), and as treatment for various infectious diseases (duke, 1992; martín-sánchez et al., 2013). however, such a valuable nutrient is generally discarded or used in animal feeding. a serious economic loss can be experienced unless such a useful fruit and its products are used in human diet and food since it is rich in bioactive compounds, which can be extracted and used as value added materials. development of new food products using date flesh is the topic of very limited number of studies. the objective of the present study is to characterize and evaluate the functional properties of the us date fibre (df) taking into account the quality and nutritional content of ice cream. materials and methods materials cows’ milk and cream were obtained by the research and application farm of atatürk university. amber dates were purchased from the palm garden in medina city of saudi arabia. sugar, salep and emulsifier (monoand diglycerides) were obtained from local markets. skim milk powder was supplied by pınar dairy products co. (turkey). preparation of date flour date fibre concentrates were extracted from the medina cultivar ‘amber’ as described previously (elleuch et al., 2008). df from whole fruits were extracted in boiling water for 15 min, using a magnetic stirrer. after solubilisation of the sugars (sucrose, glucose and fructose), date fibres and pits were recovered through filtration using a 0.2 mm sieve. the pits were then removed. the fibres were concentrated by successive rinsing (water at 40°c) and filtration until the residue was free of sugar as described. the residues obtained were pressed dried, in oven at 65°c for 24 h and milled in a mill laboratory at 2890 rpm, then at 5000 rpm until they could pass through a 0.2 mm sieve to recover the date fibre concentrate, and stored at -18°c for subsequent physicochemical analyses and incorporation studies. chemical composition moisture content was determined according to the association of official analytical chemists (aoac, 1997) method. ash was analysed by combusting the sample in a muffle furnace at 550°c for 4 h. the residue was dissolved in hno 3 and ital. j. food sci., vol. 27 2015 399 the mineral constituents (ca, k, na, mg, p and fe) were determined using an inductively couple plasma spectrophotometer (perkin-elmer, optima 2100 dv, icp/oes, shelton, ct, usa). the bligh and dyer method (hanson and olley, 1963) was used to determine the lipid content. protein content was determined by micro kjeldahl method (aoac, 1990) and expressed as: % n 2 x6,23. total sugars were extracted through ethanol (80%) (ninio et al., 2003). after centrifugation, the supernatant was collected and the total sugar content was analysed using phenol/ sulphuric acid reagent (dubois et al., 1956). the total phenolic content was analysed according to the folin-ciocalteu method developed by al-farsi et al. (2005). the extract (200 μl) was mixed with 1.5 ml of folin-ciocalteu reagent (previously diluted 10-fold with distilled water) for 5 min at room temperature. 1.5 ml of aqueous sodium bicarbonate (60 g/l) was added and the mixture was vortexed and allowed to stand at room temperature. after 90 min, the absorbance was measured at 725 nm. the total phenol concentration was expressed as the mean ± sd as mg of gallic acid equivalent (mg gae) per 100 g of fresh weight of date for two replicates. aoac enzymatic-gravimetric official method (991.43; aoac, 1995) was used to determine dietary fibres while dry matter content, fat, ash, acidity (ºsh) and ph of ice cream samples were determined as in demirci and gunduz (1994). mineral contents (ca, k, na, p, s, mg, fe, mn, zn, ni) of ice cream samples were determined using an inductively couple plasma spectrophotometer (perkin-elmer, optima 2100 dv, icp/oes, shelton, ct, usa) and following the method described by guler (2007). samples were decomposed in a microwave oven (berghof speed wave mws-2, eningen, germany). for this purpose, about 0.5 g ice cream sample was weighed into the digestion vessels, added concentrated nitric acid (10 ml) and digestion process was realized over each sample at 210°c and under 176 psi pressure for 10 min. after cooling, the carousels were removed from the oven, 30% hydrogen peroxide (2 ml) was added to samples and then second digestion was applied at 195°c and under 95 psi pressure for 5 min. the vessels were immediately closed after the addition of oxidants. at the end of the digestion process, the samples were diluted in with distilled water to an appropriate concentration and filtered through whatman no. 42 filter paper. all diluted digests were eventually analysed using an inductively couple plasma spectrophotometer (icp-oes). water and oil holding capacities, and ph of fibre water and oil holding capacities (whc and ohc) of the fibres were determined according to the methods of mac-connell et al. (1997) and caprez et al. (1986), respectively. whc and ohc values represented the amount of water and oil absorbed per gram of sample, respectively. ph of df was measured using a ph meter (wtw 3401) and following the method described by suntharalingam and ravindran (1993). ice cream manufacture the ice cream samples were prepared in the pilot plant of food engineering department, agriculture faculty, atatürk university. initially, the fat ratio of cows’ milk was adjusted to 6% by adding cream. then, the milk was divided into five equal parts of 2 kg. for each mix, skim milk powder (125 g), sugar (405 g), salep (stabilizer) (16.2 g), emulsifier (monoand di-glycerides) (6.75 g) were added to each mix. then prepared date fibres were added at four different concentrations: 1, 2, 3 and 4% to mixture weight. the mixes were pasteurized at 85°c for 25 min and stored at 4°c for 24 h. then, they were iced in ice cream machinery (-5°c; ugur cooling machineries co., nazilli, turkey) and hardened at -22°c for 24 h and stored at -18°c and used for physical, chemical, mineral and sensory analyses. ice cream samples were produced in duplicate. ice cream analysis physical measurements. overrun was determined using a standard 100 ml cup, according to the equation [(volume of ice cream)-(volume of mix)/volume of mix×100] given by jimenez-florez et al. (1993). first dripping and complete melting times were measured according to guven and karaca (2002) 25 g of tempered samples were left to melt (at room temperature, 20ºc) on a 0.2 cm wire mesh screen above a beaker. first dripping and complete melting times of the samples were accepted to be seconds. the viscosities of the ice cream mixes were determined at 4ºc using a digital brookfield viscometer, model dv-ii (brookfield engineering laboratories, stoughton, ma, usa). before measuring the viscosity, the samples were stirred gently to remove the air from the mixes (akin et al., 2007). the color analyses (l*, a* and b* values) of the ice cream mix were carried out using in minolta colorimeter (chroma meter, cr-200, osaka, japan; anonymous, 1979). the colorimeter was calibrated using a white reference plate before measurements. light source for the colorimeter was standard daylight (c) and the standard observer was 2°. sensory evaluation eight professional panellists from the food engineering department of atatürk university, erzurum, turkey, participated in the study to determine some properties using a score test for flavour, body and texture, color and appearance, 400 ital. j. food sci., vol. 27 2015 resistance to melting and general acceptability. hardened ice cream samples were tested at a serving temperature of -10ºc and scored their sensory characteristics in a scale ranging from 1 (poor) to 9 (excellent). warm water and bread were also provided to the panellists to cleanse their palates between samples. all panellists were non-smokers, had prior testing experience with a variety of dairy products including milk, cheese and ice cream and had previously used flavour profile procedures adapted from roland et al. (1999). statistical analysis all statistical analysis was performed using sas for windows (sas, 1998). analysis of variance was performed using the routine proc anova. significant treatment was separated using duncan’s multiple range test (duzgunes et al., 1987). results and discussion physical and chemical characteristics of date fibre dry matter, fat, acidity (osh) and ph values of milk, skim milk powder and cream used in the production of the ice cream are given in table 1. date and date fibre were analyzed for moisture, ash, fat, total sugars, color, total phenolic content, whc and ohc (table 2). date fibres are rich in protein (9.01 g/100 g). earlier, elleuch et al. (2008) reported 9 g/100 g protein contents for tunisian dates and similar to the present work. presence of high protein content in fruit fibres (11.6-14.4 g/100 g) is reported in the literature (bravo and saura-calixto, 1998; saura-calixto, 1998). in the present study, calcium, sodium, potassium and magnesium contents of date fibre were measured to be 1925, 56.5, 981 and 1807 mg/kg, respectively. ahmed et al. (2013) reported that the sodium content was significantly lower than other minerals (5586 mg/kg); however, the fibres were rich in potassium. the barhee cultivar possessed exceptionally higher amount of potassium (2600 mg/ kg), and the maximum sodium was found in owadi cultivar. these results are significantly different from the reported values for date flesh (elleuch et al., 2008). the variation could originate from the cultivar, and agro-climatic as well as environmental conditions. date contains high proportions of total dietary fibre (80.2 g/100g) similarly to those reported in deglet-nour and allig (two varieties of date) between 88 and 92%, respectively (elleuch et al., 2008). in addition, the contents of dietary fibre in dried apricots, prunes, figs, and raisins were 7.7, 8.0, 12.2, and 5.1 g/100 g, respectively (camire and ougherty, 2003; marlett et al., 1994; vinson, 1999). thus, dates and their by-products serve as good sources of fibre compared with syrups and other fresh and most dried fruits. in addition, these df contents are close to levels measured for df preparations from apple (liberty cultivars) (89.8%), but notably higher than those of other fruit df concentrates reported for grapefruit, lemon, orange, apple and mango (28-78.2%) (figuerola et al., 2005; vergara-valencia et al., 2007), table 1 the gross chemical, physical properties and mineral contents of raw milk, skim milk, cream. analysis milk skim milk powder cream dry matter (%) 11.37 95.17 63.76 fat (%) 3.5 1.00 65.00 ash (%) 0.67 acidity (osh) 5.81 13.98 ph 6.40 4.95 minerals (mg kg-1) ca 1224.00 k 1397.00 mg 91.67 p 869.54 na 327.90 fe 13.56 not determined. table 2 chemical and physical properties of date fibre and date. chemical analysis date date fibre moisture (g/100 g) 13.61±0.11 3.87±0.13 ash (g/100 g) 1.79±0.07 2.06±0.04 ph 6.00±0.21 5.71±0.02 protein (g/100 g) 1.23±0.16 9.01±0.75 fat (g/100 g) 3.41±0.03 0.98±1.21 total sugars 78.20 0 total phenolic contentc 186±2.30 0.73±0.01 total dietary fibre (g/100 g) 8.75±0.96 80.2±1.06 minerals (mg kg-1) ca 23.40±0.51 1925±1.84 k 428±0.14 981±2.04 mg 84.51±0.22 1807±0.82 p 90.19±1.36 1325±0.51 na 17.65±0.12 56.5±0.05 fe 2.03±0.07 24.82±1.36 physical analysis l* 23.8±0.04 61.08±0.05 a* 11.0±0.03 6.35±0.01 b* 8.9±0.07 14.72±0.01 whca 16.32±0.47 ohcb 9.50±0.23 awater holding capacity (g water/g, sample); boil holding capacity (g oil/g, sample); cg/100 g of df concentrates; l* = lightness; a* = redness (+) and blueness (–); b* = yellowness. ital. j. food sci., vol. 27 2015 401 grape skins (54.1–64.6%) (bravo and saura-calixto, 1998; saura-calixto, 1998), citrus peel (57%; chau and huang, 2003), or fibre from lime peels (66.7% and 70.4%; ubando et al., 2005) and mango peel (71%; larrauri et al., 1996). whc was found to be 16.32 (g water/g, sample) in date fibre in the present study. whc in of date fibres was reported to be significantly higher than those of fruit and vegetable fibres (femenia et al., 1997; gan and latiff, 2011; lopez et al., 1996; vergara-valencia et al., 2007; ahmed et al., 2013), but similar to those found in date (15.5 g/g, dry matter) by elleuch et al. (2008). ohc is another functional property of some ingredients used in formulated food. in general, date fibre showed significantly higher ohc (9.5 g oil/g, sample) when compared to other fruit and vegetable derived fibres (gan and latiff, 2011; vergara-valencia et al., 2007). the highest ohc was observed for allig cultivar (9.9 g oil/g sample) followed by elleuch et al. (2008). higher ohc of date fibre indicated that it could be used as an ingredient to stabilize foods with a high percentage of fat (elleuch et al., 2008; ahmed et al., 2013). the mean l*, a* and b* values were found to be 61.08, 6.35 and 14.72, respectively. this could be due, on the one hand, to the wash operations during the extraction and concentration of df and, on the other hand, to the solubility of pigments responsible for the dark units of color. elleuch et al. (2008) reported that l*, a* and b* values were 61.92, 7.11 and 14.85, respectively for allig, which are convenient with the present study. goñi et al. (2009) informed that pp associated with polysaccharides and proteins in cell walls are significant constituents of date fibre. table 2 shows date fibre polyphenol (pp) contents (0.73 g/100 g). physical and chemical characteristics of ice cream samples the results of some physical, chemical analyses and mineral contents of ice cream samples are given in tables 3 and 4. the dry-matter content of control sample was lower than other samples at statistically significant levels (p<0.05). the dry matter rates of ice cream increased with the addition of df concentration. the highest fat and acidity ratios were found to be in control sample (4.63%). ph values of ice table 3 some chemical and physical properties of ice cream samples with date fibre. analysis c df1 df2 df3 df4 moisture (%) 33.15±0.02a 33.32±0.29b 33.49±0.10a 33.63±0.36b 34.05±0.01c ash (%) 0.89±0.01a 0.92±0.01ab 0.95±0.01b 1.06 ±0.02c 1.10±0.01c fat (%) 4.63±1.41d 4.17±0.14c 4.15±0.03c 3.91±0.01b 3.86±0.02a acidity(osh) 8.99±0.00e 6.23±0.02a 6.38±0.01b 6.54±0.01c 6.73±0.02d ph 6.20±0.02e 5.62±0.02d 5.56±0.01c 5.23±0.03b 5.09±0.01a l* 83.33±0.01d 82.26±0.04d 80.27±0.03c 77.64±0.91b 75.45±0.07a a* 1.62±0.05a 2.54±0.04b 2.73±0.01c 3.21±0.04d 3.90±0.01e b* 9.15±0.02a 9.20±0.01a 11.60±0.14b 12.40±0.01c 12.50±0.02c overrun (%) 40.51±0.00e 39.32±0.21d 37.20±0.01c 32.24±0.19b 29.87±0.06a complete melting time (s) 0.43±0.02b 0.50±0.00c 0.46±0.02b 0.35±0.01a 0.38±0.00a mean values followed by different letters in the same row are significantly different (p<0.05). c: control (without date fibre); df1: ice cream with made date fibre 1% (w/w); df2: ice cream with date fibre 2% (w/w); df3: ice cream with date fibre 3% (w/w); df4: ice cream with date fibre 4% (w/w). table 4 elemental composition (mg kg-1) of the ash in ice cream with date fibre. concentrations of minerals c df1 df2 df3 df4 ca 1844.36±12.72e 1623.25±2.82d 1547±2.12c 1481.40±2.12a 1514.06±7.77b k 1669.56±21.20a 1913.06±4.15b 1939.18±1.33bc 2043.46±80.63cd 2135.46±49.95d na 537.68±6.37b 572±0.04c 690±0.70d 528.5±0.70a 573±0.01c p 1100.86±0.01c 1257.05±4.24d 1019±2.12b 1100±0.71c 1006±2.82a s 875.24±1.41a 938.50±17.67b 980±0.70c 1015±7.07d 1103±2.12e mg 159.31±1.39a 161.32±1.15a 164.78±0.72b 171.06±0.12c 183.33±1.59d fe 10.82±0.24a 11.17±0.05b 14.73±0.02c 21.02±0.01d 29.65±0.22e mn 0.32±0.01b 0.35±0.07c 0.26±0.02a 0.30±0.01b 0.40±0.01d zn 57.84±0.86a 70.82±0.95b 84.03±0.89c 91.13±1.36d 94.56±3.93d ni 0.97±0.06a 1.20±0.14b 1.14±0.01ab 1.70±0.01c 1.61±0.01c mean values followed by different letters in the same row are significantly different (p<0.05). c: control (without date fibre); df1: ice cream with made date fibre 1% (w/w); df2: ice cream with date fibre 2% (w/w); df3: ice cream with date fibre 3% (w/w); df4: ice. 402 ital. j. food sci., vol. 27 2015 cream samples were not statistically different maybe due to ph of date (6.00). viscosity is one of the most important properties of an ice-cream mixture since it can result in a desirable body and texture in ice creams. therefore, the measurement of viscosity is important to know the effect of df on the characteristics of ice-cream mixtures. it could be seen in the present study that the addition of df significantly (p<0.05) affected the viscosity behaviour of ice cream samples (fig. 1). viscosity of ice-cream samples increased significantly by adding df (3 and 4%). as shown in fig. 1, the lowest and highest viscosity rates value were obtained in df1 sample the sample with 4% df. the control sample had an average of 5175 viscosity. similar results were reported in grape wine lees added in ice cream by hwang et al. (2009), in frozen yogurt by guven and karaca (2002), in cape gooseberry (physalis peruviana l.) added in ice cream by erkaya et al. (2012) and the citrus fibre added in ice cream mixes by dervisoglu and yazici (2006). ice cream color was affected by the addition of df. the date fibre had a brownish color. ice cream fortified with date fibre had significantly higher a* and b* values and lower l* values compared to the control sample. lightness (l*) values of ice cream samples were closer to each of dietary fibre but with df1 and df2 samples, it was found to be significantly higher than the fig. 1 the obtain of date fibre and production of ice cream. other samples (table 3). all samples had negative a* values and df3 and df4 samples had close but significantly higher values than other samples. increase in the concentration of date fibre contributed to the color values of the samples (p<0.05). the addition of date fibre increased the b* values of all samples. the lowest b* value was obtained in df1 samples while the highest b* was obtained in the df4 samples. dervisoglu and yazici (2006) reported that the addition of citrus fibre increased the color properties similarly to the results of present study. overrun and melting time are associated with the amount of air incorporated during the manufacturing process. these features can define the structure of the end final product since the presence of air gives the ice cream an agreeable light texture and influences the physical properties of melting and hardness of the end product (sofjan and hartel, 2004; cruz et al., 2009; dagdemir, 2011). all ice cream samples had normally lower overrun values (29.87-40.51%) than those reported in literature (80-120%). although the rate of df lowered the overrun values of the ice-cream samples, control samples had higher overrun values than the df added samples (table 3). since the viscosity of ice cream increased in df added samples, it was possible that less air was incorporated in the ice cream mix with df during batch freezing, which resulted in lower overrun than for control (withital. j. food sci., vol. 27 2015 403 out df). the decrease of overrun values for ice creams with df was in agreement with the results indicated in literature (dervisoglu et al., 2005; temiz and yesilsu, 2010). el-samahy et al. (2009) reported that the decrement of overrun in ice cream containing concentrated cactus pear pulp might be attributed to increment of mix’s viscosity that extremely affects whipping rate of mixes. hwang et al. (2009) reported that the overrun values of ice-cream samples decreased significantly when grape wine lees was added. it was found by sun-waterhouse et al. (2013) that overrun rate of ice – cream containing green kiwi fruit was 90.5% and higher than that found in the present study. however, similar results with the present study were found with cape gooseberry (physalis peruviana l.) added in ice cream by erkaya et al. (2012). as can be seen in table 3, the complete melting times of the ice cream samples were significantly longer for df4 samples (0.50 g min-1) and the period got longer as the fibre content increased. this is due possibly to some compounds existent in df4, which have the ability of water absorption. akin et al. (2007) reported that the decrease in melting rate of ice cream with inulin might originate from its ability to reduce the free movement of water molecules. df (3 and 4%) concentration affected the first dripping times positively (fig. 2). results of the present study indicated that the first dripping times were prolonged as the fibre contents increased in the ice cream samples (p<0.05). it was found by dervisoglu and yazici (2006) that citrus fibre samples extended dripping times. these findings were similar to those in the present study. statistically significant differences (p<0.05) were found in terms of major element contents such as ca, k, mg and s between the samples except for mn concentration in all ice cream samples. dairy products are known to be excellent sources of ca, p and mg and supply dietary fibre a significant amount of calcimine, a bioavailable form (mckinley, 2005). addition of date fibre lowered ca content of the samples in the present study (table 4). the highest ca was found to be 1844.36 mg/kg in control samples. mg and s values of ice cream samples increased with the addition of date fibre (p<0.05). increasing k in human diet may provide protection from hypertension in people who are sensitive to high levels of na. the highest rate of s and na was fibre detected in the samples with 4% and 2% df to be 1103 and 690 mg/kg, respectively, while the lowest rates were 875.24 in control and 528.5 mg/kg with 3% df, respectively. elements like fe, zn and mn are classified as micro-nutrients. the addition of df significantly increased fe, zn and mn contents of the ice-cream samples (p<0.05). similar results were reported by erkaya et al. (2012) in cape gooseberry (physalis peruviana l.) added ice cream samples. it can be suggested by considering such a result that date fibre may be a good source to enhance dairy products such as icecream, which is poor in minor elements like fe and zn. wu et al. (2005) reported that zn acts as a non-enzymatic antioxidant, so that its consumption helps to prevent oxidative damage of the cell. the ice cream sample with 4% df had the highest zn content (94.56 mg/kg). sensory evaluations results of the sensory evaluation of the ice cream samples on a scale from 1 (poor) to 9 (excellent) are shown in a radar plot in fig. 3. fortifying ice cream with df had a significant effect on all sensory properties except sweetness. all the fibre-enriched samples received lower scores for total evaluation in terms of sensory characteristics (p<0.05). ice cream enriched with up to fig. 2 viscosity values of ice cream containing date fibre and control. 404 ital. j. food sci., vol. 27 2015 fig. 3 effect of the addition of df on the sensory profile of ice cream. c: control; df1: 1% (w/w) date fibre added; df2: 2% (w/w) date fibre added; df3: 3%(w/w) date fibre added; df4: 4%(w/w) date fibre added. 1 and 2% df had similar mouth feeling, showed resistant to melting and gave general acceptability ratings as control sample. panellists preferred the ice cream samples more to the others. conclusions it can be shown as the results of the present study that fibre of date (especially at 1 and 2%) may be successfully used as a good natural source of nutritive ingredients in ice cream production. the addition of date fibre improved the viscosity, first dripping times, complete melting times and mineral compositions, but had no significant effect on overrun of ice creams. the enrichment of food with date fibres is an effective way to enhance nutritional and physiological aspects and to promote functionality by influencing rheological and thermal properties of the final product. references akın m.b., akın m.s. and kırmacı z. 2007. effects of inulin and sugar levels on the viability of yogurt and probiotic bacteria and the physical and sensory characteristics in probiotic ice-cream. food chem. 104(1): 93-99. al-farsi m., alasalvar c., morris a., barron m. 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characteristics and oxidative stability of bread fortified with encapsulated shrimp oil sirima takeungwongtrakula, soottawat benjakula* and aran h-kittikunb adepartment of food technology, faculty of agro-industry, prince of songkla university, hat yai, songkhla, 90112 thailand bdepartment of industrial biotechnology, faculty of agro-industry, prince of songkla university, hat yai, songkhla, 90112 thailand *corresponding author: tel. +66 74 286334, fax +66 74 558866, email: soottawat.b@psu.ac.th abstract characteristics and oxidative stability of bread fortified with micro-encapsulated shrimp oil (mso) were determined. the addition of mso could improve the loaf volume of bread. chewiness, gumminess and resilience of resulting bread were decreased. bread crust and crumb showed higher redness and yellowness when mso was incorporated (p<0.05). microstructure study revealed that mso remained intact with bread crumbs. the addition of mso up to 3% had no adverse effect on bread quality and sensory acceptability. oxidation took place in bread fortified with 5% mso to a higher extent, compared with those with 1 or 3% mso. therefore, the bread could be fortified with mso up to 3%. mailto:soottawat.b@psu.ac.th ital. j. food sci., vol. 27 2015 477 introduction hepatopancreas, a byproduct generated from the manufacturing of hepatopancreas-free whole shrimp, is the excellent source of lipids with high polyunsaturated fatty acids (pufa) (37.42 g/100g oil) and carotenoids (2.02 mg/g oil). shrimp oil from hepatopancreas contained linoleic acid as the most abundant fatty acid, followed by oleic acid. additionally, shrimp oil also contained pufa including eicosapentaenoic acid (2.15 g/100 g oil) and docosahexaenoic acid (6.20 g/100 g oil) (takeungwongtrakul et al., 2012), (takeungwongtrakul et al., 2014a). the recommended daily intakes for linoleic acid, linolenic acid and long-chain n-3 pufa are 4.4-20 g/d; 1.35-2.2 g/d and 0.16-1.6 g/d, respectively (meyer et al., 2003). nevertheless, oil from hepatopancreas is very susceptible to oxidation, leading to undesirable off-odor (takeungwongtrakul et al., 2012). rancidity is the major drawback for application of shrimp oil. encapsulation of oil under the appropriate condition can be a promising means to extend its shelf-life. encapsulation has appeared as a key technology in delaying or inhibiting oxidation and masking undesirable odor and flavor in the final product. the process involves the conversion of the oil into a free flowing powder, which can be easily handled and used for food fortification. encapsulation can be defined as a process, in which tiny droplets, namely core, are surrounded by wall materials (gallardo et al., 2013). proteins and carbohydrates are frequently used as matrices for micro-encapsulation of lipophilic compounds by spray drying (gharsallaoui et al., 2007). takeungwongtrakul et al. (2014b) reported that the use of whey protein and na-caseinate in combination with glucose syrup as the wall materials could improve encapsulation efficiency of micro-encapsulated shrimp oil more effectively than protein alone and protein in combination with gum arabic or maltodextrin. amongst several encapsulation techniques, spray-drying is the most common micro-encapsulation technology used in food industry due to its low cost, continuous production, ease of industrialization and available equipment (gharsallaoui et al., 2007). the world’s food market is currently focused on foods that provide nutritive values and health benefits to consumers. functional foods are rapidly expanding and draw the great attention (ezhilarasi et al., 2014). fortification of highly nutritive ingredients such as polyunsaturated fatty acid rich oil, etc. is gaining the interest for food industry. the incorporation of micro-encapsulated oil into foods enables the development of new functional foods with minimal impact on the organoleptic properties of the food products (ezhilarasi et al., 2014). wall materials surrounding oil droplets can act as the shield, preventing the oil from oxidation. borneo et al. (2007) fortified micro-encapsulated n-3 fatty acids in cream-filled sandwich cookies without any adverse effect on sensory properties. bread has become popular, especially for the new generation (cleary et al., 2007). the fortification of shrimp oil rich in pufa and astaxanthin in the encapsulated form could increase the nutritive value of bread. as a consequence, the consumers can obtain the active nutrients with the health benefit from the bread. nevertheless, no information regarding the fortification of micro-encapsulated shrimp oil in bread has been reported. the objective of this study was to investigate the effects of micro-encapsulated shrimp oil fortification on the characteristics and sensory property of bread. materials and methods chemicals ethylenediamine tetraacetic acid (edta) was obtained from merck (darmstadt, germany). tannic acid (99.5% purity) was purchased from sigma (st. louis. mo, usa). essential oil (100% purity) from lemon was obtained from botanicessence (bangkok, thailand). sodium caseinate was procured from vicchi enterprise co., ltd. (bangkok, thailand). whey protein concentrate was obtained from i.p.s. international co., ltd. (bangkok, thailand). glucose syrup (dextrose equivalent; 40-43) was purchased from charoenworrakit co., ltd. (samut prakan, thailand). wheat flour, sugar, salt, shortening, milk powder and yeast were procured from a local market in hat yai, songkhla, thailand. collection and preparation of hepatopancreas from pacific white shrimp hepatopancreas of pacific white shrimp (litopenaeus vannamei) with the size of 50-60 shrimp/kg was obtained from the sea wealth frozen food co., ltd., songkhla province, thailand during february and march, 2014. pooled hepatopancreas (3-5 kg) was placed in a polyethylene bag. to maintain the quality of hepatopancreas during transportation, the bag was imbedded in a polystyrene box containing ice with a sample/ice ratio of 1:2 (w/w) and transported to the department of food technology, prince of songkla university, hat yai, songkhla within approximately 2  h. the sample was stored at -18°c until use, but the storage time was no longer than 1 month. prior to oil extraction, hepatopancreas was thawed using running water (25°c) and ground in the presence of liquid nitrogen using a blender (phillips, guangzhou, china) for 30 sec. 478 ital. j. food sci., vol. 27 2015 extraction of oils from hepatopancreas oil was extracted from hepatopancreas following the method of takeungwongtrakul et al. (2014). the prepared hepatopancreas (20 g) was homogenized with 90 ml of cold solvent mixtures (isopropanol: hexane, 50: 50, v/v) (4°c) at the speed of 9500 rpm using an ika labortechnik homogenizer (selangor, malaysia) for 2 min at 4ºc. the extract was filtered using a whatman filter paper no.4 (whatman international ltd., maidstone, england). the residue was extracted with cold solvent mixtures for another two times. the hexane fraction was pooled and repeatedly washed with an equal quantity of 1% nacl in order to separate the phases and remove traces of polar solvents. hexane fraction (approximately 135 ml) was then added with 2-5 g anhydrous sodium sulphate, shaken very well, and decanted into a round-bottom flask through a whatman no. 4 filter paper. the solvent was evaporated at 40ºc using an eyela rotary evaporator n-1000 (tokyo rikakikai, co. ltd, tokyo, japan) and the residual solvent was removed by nitrogen flushing. the obtained oil with the yield of 19.04% (w/w) was used for micro-encapsulation. preparation of shrimp oil-in-water emulsion aqueous stock mixed solution of whey protein concentrate, sodium caseinate and glucose syrup at a ratio of 1: 1: 2 (w/w/w) in deionized water was prepared. the mixture was stirred overnight using a magnetic stirrer at room temperature (28°-30°c) to obtain the homogenous wall material solution as per the method of takeungwongtrakul and benjakul (2014b). shrimp oil was added into the solution at a core/wall material ratio of 1:4 (v/v). the mixtures were homogenized at a speed of 10,000 rpm for 3 min using a homogenizer (model t25 basic, ika labortechnik, selangor, malaysia). the obtained coarse emulsions were then passed through a microfluidics homogeniser (model hc-5000, microfluidizer, newton, ma, usa) at a pressure level of 4,000 psi for four passes. emulsions were added with lemon essential oil + tannic acid + edta. prior to the incorporation, lemon essential oil (200 ppm) was dissolved in shrimp oil, whereas edta (50 ppm) and tannic acid (100 ppm) were dissolved in aqueous stock solution. preparation of micro-encapsulated shrimp oil (mso) the prepared emulsion was subjected to drying using a laboratory scale spray-dryer (labplant ltd., labplant sd-06a, huddersfield, uk) equipped with a 0.5 mm diameter nozzle. the emulsion was fed into the main chamber (215 mm diameter × 500 mm long) through a peristaltic pump. feed flow rate was 8.08 ml/min; drying air flow rate was 4.3 m/s and compressor air pressure was 40.61 psi. air inlet temperature was 180°±2°c. the outlet temperature was controlled at 90°±2°c. the obtained powder referred to as micro-encapsulated shrimp oil (mso) was collected in the amber bottle and capped tightly. mso was determined for the total oil content using the mixture of chloroform and methanol as per the method of shahidi and wanasundara (1995). mso contained 18±1.34% shrimp oil and had 1.06±0.05% moisture content. fortification of mso in bread bread was prepared with the following formulation: wheat flour (500 g), sugar (20 g), salt (8 g), shortening (20 g), milk powder (25 g) and yeast (7  g). flour and other ingredients were mixed and kneaded uniformly, in which water (300 ml) was added during kneading. thereafter, mso was directly added to dough at different levels (0, 1, 3 and 5%, w/w). the dough was kneaded for another 10 min. dough (150 g) was then subjected to bulk fermentation for approximately 1 h at 30ºc and 75% relative humidity, followed by scaling, intermediate proving, moulding and second proving (for about 1-1.25 h). finally, baking was carried out at 220ºc for 20 min in baking oven (yxd-20, guandzhou xinnanfang electro-thermal equipment co., ltd., guandzhou, china). after baking, bread samples were removed from mold and allowed to cool at room temperature. bread incorporated with 5% (w/w) spray dried empty capsules (without the addition of shrimp oil) was used as the control bread. the bread samples were subjected to analyses. characterization of bread fortified with mso loaf volume the volume of bread was determined by sesame seed displacement method after the loaves were cooled to room temperature for approximately 2 h (jiamyangyuen et al., 2005). texture profile analysis texture profile analysis (tpa) was performed using a ta-xt2 texture analyzer (stable micro systems, godalming, surrey, uk) with a cylindrical aluminum probe (35 mm diameter). the samples were sliced (each 2.0  cm thickness) and placed on the instrument’s base. the tests were performed with two compression cycles. texture measurements were performed ten times for each sample and mean values were reported. hardness, springiness, ital. j. food sci., vol. 27 2015 479 cohesiveness, gumminess, chewiness and resilience were calculated from the force-timecurves generated for each sample (gökmen et al., 2011). hardness is expressed as the maximum force for the first compression, which relates to the strength of the samples under penetration. gumminess is defined as the force required to disintegrate a semi solid food before it is ready for swallowing. springiness is a measure of how much the samples structure is broken down by the initial penetration and is calculated as the ratio of the time from the start of the second area up to the second probe reversal over the time between the start of the first area and the first probe reversal. cohesiveness is a measure of the degree of difficulty in breaking down the internal structure of sample. resilience reflects the redeformation capacity of sample tissues after penetration (chang et al., 2012). chewiness is related to the time required for masticating a bread piece prior to swallow, and the low chewing value means easy break of the bread in the mouth (krupa-kozak et al., 2012). color measurement the color of crust and crumb samples were determined using a colorimeter (colorflex, hunter lab reston, va, usa) and reported in the cie system, including l*, a*, b* and δe*, representing lightness, redness/greenness, yellowness/ blueness and total difference of color respectively. δe* was also calculated using the following equation: δe* = [(δl*)2+(δa*)2+(δb*)2]1/2 where δl*, δa* and δb* are the differences between the color parameter of the samples and the color parameters of the white standard (l* = 92.85, a* = 1.20, b* = 0.46). scanning electron microscopy (sem) bread morphology was evaluated by scanning electron microscopy (sem). fresh bread was sliced by a razor blade and mounted on a bronze stub and sputter-coated with gold (sputter coater spi-module, west chester, pa, usa). the specimens were observed using a scanning electron microscope (quanta 400, fei, eindhoven, netherlands) at an acceleration voltage of 15 kv with magnification of 2000×. sensory evaluation sensory evaluation was performed by 30 untrained panelists with ages ranging from 20 to 35 years, who were familiar with the consumption of bread. panelists were asked to evaluate for crust color, crumb color, odor, texture, appearance and overall likeness of bread samples using a nine-point hedonic scale, in which a score of 1 = not like very much, 5 = neither like nor dislike and 9 = like extremely, respectively. panelists were asked to hand-feel the sample for texture. freshly prepared bread was taken randomly for sensory evaluation at day 0 and 3. each bread loaf was cut in half and slices were subsequently cut to a thickness of 2 cm. bread was served in a closed odorless plastic container at room temperature. the samples were labeled with random three-digit codes. the order of presentation of the samples was randomized according to “balance order and carry-over effects design” (carr et al., 1999). changes in volatile compounds in bread during storage bread was placed in polyethylene bag and sealed. packaged bread samples with different treatments were stored at room temperature (28°-30°c). the samples were taken at day 0, 1, 2 and 3 for analyses. volatile compounds in bread was analysed by headspace gc-ms using a solid-phase microextraction gas chromatography mass spectrometry (spme gc-ms) following the method of gökmen et al. (2011) with a slight modification. extraction of volatile compounds by spme fiber to extract volatile compounds, 1 gram of bread slice was placed in a headspace 20 ml -vial (agilent technologies, palo alto, ca, usa). the vial was tightly closed by means of a capper. a carboxen–polydimethylsiloxane solid phase micro-extraction fiber (50/30 µm dvb/car boxen™/ pdms stableflex™) (supelco, bellefonte, pa, usa) was used to adsorb the volatile lipid oxidation compounds released from the sample. the fiber was inserted into the vial and equilibrated at 40 °c for 30 min prior to gc-ms analysis. gc-ms analysis gc-ms analysis was performed in a hp 5890 series ii gas chromatography (gc) coupled with hp 5972 mass-selective detector equipped with a splitless injector and coupled with a quadrupole mass detector (hewlett packard, atlanta, ga, usa). compounds were separated on a hp-innowax capillary column (hewlett packard, atlanta, ga, usa) (30 m±0.25 mm id, with film thickness of 0.25 µm). the gc oven temperature program was: 35ºc for 3 min, followed by an increase of 3ºc/min to 70ºc, then an increase of 10ºc/min to 200ºc, and finally an increase of 15ºc/min to a final temperature of 250ºc and holding for 10 min. helium was employed as a carrier gas with a constant 480 ital. j. food sci., vol. 27 2015 flow of 1 ml/min. the injector was operated in the splitless mode and its temperature was set at 270ºc. transfer line temperature was maintained at 260ºc. the quadrupole mass spectrometer was operated in the electron ionization (ei) mode and source temperature was set at 250ºc. initially, full-scan-mode data was acquired to determine appropriate masses for the later acquisition in scan mode under the following conditions: mass range: 25-500 amu and scan rate: 0.220 s/scan. all analyses were performed with ionization energy of 70 ev, filament emission current at 150 µa, and the electron multiplier voltage at 500 v. analyses of volatile compounds identification of the compounds was done by consulting chemstation library search (wiley 275.l). identification of compounds was performed, based on the retention time and mass spectra in comparison with those of standards from chemstation library search (wiley 275.l). quantification limits were calculated to a signal-to-noise (s/n) ratio of 10. repeatability was evaluated by analysing 3 replicates of each sample. the identified volatile compounds, related to lipid oxidation, including aldehydes, alcohols, ketones, etc., were presented in the term of abundance of each identified compound. statistical analysis all experiments were run in triplicate. all analyses were conducted in five replications. statistical analysis was performed using one-way analysis of variance (anova). mean comparison was carried out using duncan’s multiple range test (steel and torrie, 1960). results and discussion characteristics of bread fortified with mso loaf volume loaf volume of bread fortified with mso at different levels is shown in table 1. incorporation of mso (1-5%) resulted in the increase in loaf volume (p< 0.05). however, similar loaf volume was obtained, regardless of amount of mso added (p > 0.05). loaf volume of bread incorporated with spray dried empty capsule (control bread) was not different from that of bread containing mso at a level of 1% (w/w) (p> 0.05). nevertheless, bread fortified with mso at the levels of 3 and 5% (w/w) had higher loaf volume than the control bread (p< 0.05). spray dried empty capsule, including whey protein concentrate, sodium caseinate and glucose syrup, could increase loaf volume of bread to some degree. those proteins as well as glucose syrup might strengthen the loaf structure via interaction with wheat gluten, in which the bread matrix could hold gas more efficiently. whey proteins demonstrated the ability to increase the loaf volume of the bread (nunes et al., 2009). gluten proteins of wheat flour create unique visco-elastic properties of dough, which allow dough to expand due to the formation of carbon dioxide during fermentation and retain most of this gas inside the dough texture (wehrle et al., 1997). gökmen et al. (2011) and ezhilarasi et al. (2014) reported that increasing amount of micro-encapsulated oil or active compounds decreased loaf volume of bread. encapsulated substances could decrease the concentration of gluten in the formulation and lower the retention of gases during the baking process. therefore, bread fortified with mso with the range of 3-5% (w/w) had table 1 loaf volume and textural at properties of breads incorporated with mso at different levels at day 0 and 3 of storage. storage time mso loaf volume hardness springiness cohesiveness gumminess chewiness resilience (day) (% w/w) (ml) (g) (mm) (g.mm) 0 control 296.06±9.36ba 1362.22±80.23ab 0.90±0.02aa 0.59±0.02aa 759.25±50.08ab 739.70±38.73ab 0.22±0.01ba 0 % 269.25±10.84ca 1020.99±54.21bb 0.91±0.03aa 0.61±0.02aa 670.14±57.86abb 712.87±51.31ab 0.24±0.01aa 1 % 315.22±10.47aba 1022.73±46.70bb 0.91±0.01aa 0.59±0.02aa 622.09±46.49bb 554.13±38.71bb 0.22±0.01ba 3 % 317.67±9.13aa 938.58±65.57bb 0.90±0.02aa 0.60±0.03aa 610.30±42.41bb 480.51±37.57bcb 0.22±0.01ba 5 % 317.47±8.86aa 927.85±69.48bb 0.90±0.03aa 0.60±0.01aa 503.91±42.57ca 466.17±47.67cb 0.22±0.02ba 3 control 290.83±8.96ba 2454.22±84.41aa 0.89±0.02aa 0.50±0.02ab 1146.05±54.24aa 1034.57±56.96aa 0.19±0.01ab 0 % 267.22±11.34ca 2158.52±49.27ba 0.90±0.01aa 0.52±0.02ab 1097.85±39.01aa 979.92±70.76aa 0.19±0.02ab 1 % 312.17±8.86aa 1532.61±58.13ca 0.90±0.02aa 0.52±0.03ab 838.77±44.37ba 759.42±64.20ba 0.19±0.01ab 3 % 313.89±10.37aa 1257.53±77.38da 0.90±0.01aa 0.53±0.01ab 720.33±40.92ca 622.02±59.62ca 0.19±0.01ab 5 % 313.33±5.77aa 1225.42±34.58da 0.90±0.01aa 0.52±0.02ab 672.64±54.40ca 617.68±60.45ca 0.17±0.01ab control = added with 5% (w/w) spray dried empty capsule without the addition of shrimp oil. data are expressed as mean±sd (n=3). lowercase letters in the same column within the same storage time indicate significant difference (p < 0.05). uppercase letters in the same column within the same sample indicate significant difference (p < 0.05). ital. j. food sci., vol. 27 2015 481 the higher loaf volume, in comparison with the control bread. after storage of 3 days at room temperature, in which mold was not detected, no difference in loaf volume was noticeable in comparison with that found at day 0 (p> 0.05). thus, bread structure was not collapsed within 3 days of storage. it was noted that the addition of mso had no influence on the shelf-life of bread. textural properties textural properties of bread samples containing mso at various levels are presented in table 1. the addition of mso generally had the effects on the texture profile of bread. however, mso had no effect on hardness (p> 0.05), irrespective of amount used. it was noted that, the control bread had higher hardness value than others (p< 0.05). the proteins in spray dried empty capsules with smaller size might be distributed more uniformly and strengthened bread structure more efficiently. for gumminess, the addition of mso decreased the value. the decrease was more pronounced as the level of mso increased (p< 0.05). it was found that bread incorporated with 5% mso showed the lowest gumminess, compared with others (p< 0.05). the addition of mso or spray dried empty capsule had no impact on springiness and cohesiveness (p> 0.05). chewiness of bread decreased as the amount of mso in bread increased (p< 0.05). control bread showed similar chewiness to that without mso (p> 0.05). for resilience, bread without mso (0%) showed the higher value than others (p< 0.05). after the storage at room temperature for 3 days, hardness, gumminess and chewiness increased, whilst the cohesiveness and resilience decreased (p< 0.05). nevertheless, no changes in springiness were observed after the storage (p> 0.05). these results indicated that bread staling took place upon storage, probably due to retrogradation (henna-lu and norziah, 2011). therefore, mso addition had the direct impact on textural property to different degrees, depending on the amount of mso incorporated. color color of bread crust was affected by the amount of mso added as shown in table 2. the photographs of bread crust are shown in fig. 1 a. bread crust had the decrease in l*-value, but the increases in a*-, b*and δe*values as the level of mso increased (p< 0.05). amongst all samples, that added with 5% mso showed the lowest l*-value but highest a*-, b*and δe*values (p< 0.05). it was found that bread incorporated with spray dried empty capsule (control) had the lower l*value than others, except for that added with 5% mso. the color of bread crust is mostly attributed to non-enzymatic chemical reactions such as maillard and caramelization reaction that produce colored compounds (formation of the golden yellow color) during bread baking (gökmen et al., 2011). proteins in spray dried empty capsule could serve as the reactant, especially for browning reaction, especially at crust region. gökmen et al. (2011) reported that the particles in the crust region were partially destroyed due to more severe thermal conditions during baking. furthermore, the wall materials also contained some sugars, which more likely underwent caramelization at high temperature. this could contribute to the brown color of bread crust. purlis and salvadori (2009) reported that bread had high browning reaction rates, particularly when caramelization octable 2 color of breads incorporated with mso at different levels at day 0 and 3 of storage. storage time mso crust color crumb color (day) (% w/w) l* a* b* δe* l* a* b* δe* 0 control 56.16±3.89ba 15.20±2.37ba 36.30±1.36aba 54.28±4.32ba 71.08±1.12aa 0.13±0.02da 11.67±0.89da 25.17±1.47ea 0 % 69.13±2.98aa 9.76±1.12ca 32.12±1.07ca 41.91±1.56cda 67.66±1.22bb 0.12±0.02da 11.89±0.60da 28.35±1.19da 1 % 67.88±3.46aa 12.28±1.08ba 35.38±1.06ba 45.42±2.65ca 64.53±1.56cb 2.15±0.41ca 13.72±1.42ca 32.08±1.91ca 3 % 64.95±2.55aa 13.75±1.17ba 35.73±0.43ba 47.77±1.77ca 63.90±2.07cdb 7.19±0.68ba 18.92±0.91ba 35.89±0.76ba 5 % 47.27±1.99ca 20.08±0.52aa 38.67±1.28aa 63.47±1.56aa 60.77±1.48db 11.45±0.51ab 22.59±0.61ab 41.46±0.83aa 3 control 56.68±3.95ba 14.38±2.26ba 36.31±1.42aa 53.69±0.91ba 72.98±1.09aa 0.12±0.01da 12.63±1.76da 23.97±1.36da 0 % 67.58±2.16aa 9.67±0.99ca 32.69±0.92ca 42.77±1.88da 71.99±1.33aa 0.11±0.04da 12.20±0.76da 24.61±1.12cdb 1 % 66.72±2.20aa 11.59±2.17bca 35.72±1.27ba 46.09±1.09ca 70.94±1.69aa 2.27±0.33ca 13.90±1.28ca 26.55±1.19cb 3 % 64.10±3.29aa 13.19±2.04ba 35.96±1.10ba 48.28±1.37ca 66.17±2.02ba 7.58±0.36ba 20.13±0.86ba 34.80±1.39ba 5 % 46.36±0.57ca 20.44±0.69aa 38.54±0.97aa 64.32±.041aa 65.11±1.54ba 13.34±0.50aa 26.12±0.99aa 40.90±0.69aa control = added with 5% (w/w) spray dried empty capsule without the addition of shrimp oil. data are expressed as mean±sd (n=3). lowercase letters in the same column within the same storage time indicate significant difference (p < 0.05). uppercase letters in the same column within the same sample indicate significant difference (p < 0.05). 482 ital. j. food sci., vol. 27 2015 curred. therefore, browning reaction had a pronounced influence on bread color, particularly during bread baking. for bread fortified with mso, the increases in redness (a*value) were more likely due to the orange/red color of mso. shrimp oil contained a high amount of astaxanthin (takeungwongtrakul et al., 2014). as a result, the bread crust turned to be more orange in color, when mso was added, especially at higher levels. thus, the color of bread crust might depend on both non-enzymatic browning reactions and astaxanthin partially released from mso. however, the changes in color of crust were more likely caused by the releases of astaxanthin from mso as evidenced by the more a*-value (redness) in color of resulting bread. when comparing l*-, a*-, b*and δe*values of all bread crust, all bread samples had no change in color after 3 days of storage (p> 0.05). the result suggested that the pigments in mso were stable after 3 days of storage as evidenced by the unchanged color of bread crust. it was presumed that wall material might protect the oxidation of astaxanthin to some degree during the storage. the color of bread crumb was determined (table 2). the levels of mso incorporated in bread were coincidental with the color. the decrease in l*value and increases in a*-, b*and δe* values of bread crumb were found as the level of mso increased (p< 0.05). for color of bread crumb, crumb does not undergo maillard reaction, but is affected by the ingredients in the formula (conforti and davis, 2006). oils from shrimp hepatopancreas were reddish orange in color due to the presence of astaxanthin (takeungwongtrakul et al., 2014). additionally, surface oil and oil released to the surface of mso during bread making could also contribute to color of bread crumb. when mso at a level of 5% fig. 1 the photographs of breads incorporated with mso at different levels. a) bread crust, b) bread crumb. was incorporated, bread crumb had the lowest l*value but highest a*-, b*and δe*values than others (p< 0.05). for control bread (with only spray dried empty capsule), a*and b*values of crumb were not different from those of bread without mso (p> 0.05). spray dried empty capsule was visually white in color without red or yellow color. after 3 days of storage, the control bread had no change in l*value (p> 0.05), whilst other bread had the increase in l*value (p< 0.05). for a*and b*values, all bread crumb had no change in a*-, b*and δe*values (p> 0.05). nevertheless, crumb of bread incorporated with 5% mso had the increases in a*and b*values. it was noted that those with 0 and 1% mso had the decrease in δe*value after 3 days of storage (p< 0.05). the photographs of bread crumb are shown in fig. 1b. during storage, the oil might be released from the wall to some degree. as a result, oil with high content of astaxanthin could contribute to the increase in a*and b*values to some extent. this was obvious for bread fortified with 5% mso. therefore, the addition of mso directly affected the color of both crust and crumb of bread. microstructure sem microphotographs of all bread crumbs incorporated with the different levels of mso are shown in fig. 2. in general, mso was embedded in the crumb of bread, which was constructed by gluten network. these results were consistent with gökmen et al. (2011) who incorporated nano-encapsulated flax seed oil into bread. powders added to dough remained intact in the bread crumb. for the control bread, the bead of spray dried empty capsule was observed throughout the crumb (fig. 2a). howital. j. food sci., vol. 27 2015 483 fig. 2 surface morphology of breads incorporated with mso at different levels. (magnification: ×2000). 484 ital. j. food sci., vol. 27 2015 ever, there was no bead in the bread without mso and spray dried empty capsule (fig. 2b). it was clearly illustrated that the number of bead, representing mso, increased as the level of mso increased. in general, mso were located uniformly in the crumb matrix. those mso could serve as the source of pufa and astaxanthin rich shrimp oil. sensory property crust color, crumb color, texture, appearance, odor and overall likeness scores of all bread samples added with different amounts of mso at day 0 and 3 of storage are shown in table 3. there were no differences in all attributes amongst all bread samples (p> 0.05) at day 0 of storage, except for crumb color and overall likeness scores of bread incorporated with 5% mso, which had the lower score (p< 0.05). the addition of 5% mso to bread had negative effect on crumb color and overall likeness of bread. this was due to the marked increases in a* and b* values of bread crumb (table 2). gökmen et al. (2011) reported that the addition of micro-encapsulated n-3 fatty acids could increase functionality of bread. shrimp oil is one of the important sources of n-3 fatty acids (takeungwongtrakul et al., 2012). thus, mso at 3% (w/w) could be added into bread to improve the nutritive values of bread without the negative effect on sensory property of bread. after 3 days of storage, no differences in all attributes were observed amongst all bread samples (p> 0.05). nevertheless, crumb color, odor and overall likeness of bread added with 5% mso were lower than others (p< 0.05). wall materials could protect the entrapped core by providing a physical barrier against environmental conditions (gallardo et al., 2013). food fortification is good way to induce the general population to consume components, such as n-3 fatty acids, and will add value to food product manufactured by the food industry (borneo et al., 2007). however, the addition of 5% mso might result in the increased free oil, especially at the surface of mso. this led to more free oil, which was susceptible to oxidation. as a consequence, the lower score of odor likeness was found. therefore, mso must be incorporated at the appropriate level to avoid the undesirable attributes of bread. volatile compounds volatile compounds in bread samples added with mso at different levels after 3 days of storage are displayed in table 4. volatile compounds in bread without mso (days 0) were also determined. lipid oxidation generates a number of products, including volatile compounds, which are the major contributors to the rancid off-flavors and off-odors in the food product (ross and smith, 2006). in general, all compounds present in bread without mso at day 0 were lower in abundance than those found after 3 days of storage. nevertheless, 3-methyl-1-butanol, 2-pentyl-furan, heptenal and 2-octen-1-al were also lower in abundance after storage, plausibly due to the volatilization or decomposition. several derivatives of aldehyde, ketone and alcohol can be formed by the oxidation of lipids (varlet et al., 2006). aldehydes are the most prominent volatiles produced during lipid oxidation and have been used to successfully follow lipid oxidation in a number of foods (shahidi and pegg 1994). after the storage, the bread without mso contained new volatile compounds including decanal. the highest amount of lipid oxidation products such as 3-methyl-1-butanol, benzeneethanol, benzaldehyde and 2-methyl-1-propanol table 3 likeness score of breads incorporated with mso at different levels at day 0 and 3 of storage. storage time mso crust color crumb color texture appearance odor overall likeness (day) (% w/w) 0 control 7.04±0.78aa 6.90±1.08aa 6.47±1.17aa 7.00±1.04aa 7.13±0.94aa 7.03±1.00aa 0 % 6.92 ±0.65aa 6.75±1.16aa 6.70±0.76aa 6.79±0.92aa 7.00±0.94aa 6.90±0.82aa 1 % 7.04±0.81aa 6.90±0.90aa 7.03±1.27aa 7.07±1.16aa 6.93±0.82aa 6.83±0.95aa 3 % 7.31±0.89aa 7.10±0.90aa 7.00±0.98aa 6.93±1.00aa 6.97±0.88aa 6.80±0.77aa 5 % 6.67±0.88aa 5.54±1.12ba 6.97±1.37aa 6.63±1.36aa 6.47±0.95aa 5.63±1.12ba 3 control 6.94±0.89aa 6.90±1.24aa 6.04±0.91aa 7.00±0.89aa 6.52±1.23aa 6.44±0.93aa 0 % 6.72±0.96aa 6.74±0.96aa 6.38±1.10aa 7.00±1.21aa 6.58±1.31aa 6.36±0.91aa 1 % 6.83±1.33aa 6.87±1.41aa 6.74±0.90aa 7.06±1.21aa 6.56±1.01aa 6.28±0.96aa 3 % 7.00±0.80aa 7.00±0.91aa 6.70±0.95aa 6.74±1.21aa 5.96±0.75aa 6.14±1.09aa 5 % 6.66±1.14aa 5.43±1.07ba 6.60±1.22aa 6.39±1.36aa 5.16±0.94ba 5.36±0.99ba control = added with 5% (w/w) spray dried empty capsule without the addition of shrimp oil. data are expressed as mean±sd (n=3). lowercase letters in the same column within the same storage time indicate significant difference (p < 0.05). uppercase letters in the same column within the same sample indicate significant difference (p < 0.05). ital. j. food sci., vol. 27 2015 485 was found in bread without mso after 3 days. among all the aldehydic compounds, benzaldehyde was found to be the major aldehyde in bread without mso (0% mso), followed by nonanal and decanal, respectively. additionally, volatile ketones (dihydro-5-pentyl-2(3h)-furanone) and volatile alcohols (3-methyl-1-butanol, benzeneethanol, 2-methyl-1-propanol, 1-hexanol, 1-octen-3-ol and 1-octanol) were also found in bread without mso. maire et al. (2013) reported that flour appeared relatively rich in alcohols (3-methyl-1-butanol, 1-pentanol, 1-hexanol and 1-octen-3-ol). these compounds were also reported by hansen and hansen (1994) in flour with different millings, formed by either lipid oxidation or microorganism metabolism. 1-octen-3-ol is a volatile generated from linoleic acid oxidation in the presence of singlet oxygen (lee and min, 2010). this indicated that lipid oxidation took place in bread without mso. maire et al. (2013) reported that dough preparation seemed to be the crucial step toward lipid oxidation due to enzymes (lipoxygenase and lipase) as well as air inside the dough texture. additionally, auto-oxidation could occur during baking by high temperatures, which promote an accelerated oxidation of ingredients in bread without mso. after storage, the formation of most volatile compounds in bread increased as the amount of mso increased from none to 5%. those compounds included 1-hexanol, nonanal, 1-octen3-ol, 1-octanol, (z)-3-decen-1-ol and benzetable 4 volatile compounds in breads incorporated with mso at different levels after storage of 3 days at 30ºc. compounds peak area (abundance) × 106 control 0% mso 1% mso 3% mso 5% mso 2-methyl-1-propanol nd 424 (309) 414 388 264 3-methyl1-butanol 1350 1383 (1406) 1293 1264 1255 2-pentyl-furan nd nd (128) nd nd nd 3-hydroxy-2-butanone 180 nd nd nd 168 heptenal nd nd (74) nd nd 118 1-hexanol 414 302 (253) 402 426 441 nonanal 296 207 (122) 303 366 414 2-octen-1-al nd nd (33) nd nd nd 1-octen-3-ol 102 89 (63) 133 176 226 decanal 41 40 (nd) 26 78 83 benzaldehyde 347 450 (354) 285 274 477 1-octanol 29 35 (33) 48 52 53 2-octen-1-ol nd nd 21 17 20 2-furanmethanol nd nd nd 152 220 (e)-6-nonen-1-ol 89 nd 102 119 nd (z)3-decen-1-ol 58 nd 59 67 135 benzenemethanol 22 nd nd nd 44 benzeneethanol 795 716 (683) 728 856 1437 3-hydroxy-2-methyl-4h-pyran-4-one nd nd nd nd 50 dihydro-5-pentyl-2(3h)-furanone nd 19 (15) 23 30 20 nd: non-detectable * value in the parenthesis represents the abundance of compound in sample at 0 day. control: added with 5% (w/w) spray dried empty capsule without the addition of shrimp oil. neethanol. however, 3-methyl-1-butanol decreased with increasing mso. this could be due to volatilization. benzaldehyde of sample added with 1 and 3% mso showed the lower abundance than that without mso. benzaldehyde formed might bind with protein matrix of bread. heptenal was also found in the sample added with 5% mso. higher abundance in nonanal and benzaldehyde was observed in the sample incorporated with 5% mso, compared with others after 3 days of storage. volatile compounds in bread added with 5% mso were generally highest in abundance, compared with those added with others. takeungwongtrakul et al. (2014) reported that surface oil content of mso prepared using whey protein concentrate: sodium caseinate: glucose syrup (1: 1: 2, w/w/w) as wall materials was 2.48 % (w/w). bread with higher level of mso incorporated showed the higher amount of surface oil, which was more susceptible to oxidation. as a result, oxidation took place to a higher extent. abundance of volatile compounds in all bread correlated well with the sensory property as shown in table 3, in which bread added with 5% mso had the lowest score of odor likeness. for bread added with 1% or 3% mso and control bread, similar amount of volatile compounds was noticeable and no difference in sensory property of bread was observed (p > 0.05) (table 3). therefore, 3% mso was the appropriate level to fortify in bread without negative effect on quality and acceptability. 486 ital. j. food sci., vol. 27 2015 conclusion mso prepared using whey protein concentrate, sodium caseinate and glucose syrup (1: 1: 2, w/w/w) as wall materials could be fortified in bread product. fortification of mso had impact on the bread loaf volume, color and sensory properties. mso up to 3% could be incorporated into bread to improve the nutritive value without affecting its sensorial properties. the fortified bread was quite stable up to 3 days of storage, in which no marked changes in color occurred and only slight increases in volatiles were obtained. acknowledgement “this work was supported by the higher education research promotion and national research university project of thailand, office of the higher education commission”. the trf distinguished research professor grant and prince of songkla university were also acknowledged. references borneo r., kocer d., ghai g., tepper b. and karwe m. 2007. stability and consumer acceptance of long-chain omega-3 fatty acids (eicosapentaenoic acid, 20: 5, n-3 and docosahexaenoic acid, 22: 6, n-3) in cream-filled sandwich cookies. j. food sci. 72: s049-s054. carr b.t., meilgaard m. 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properties of wheat dough. cereal chem. j. 74:739744. paper received october 30, 2014 accepted february 2, 2015 p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33isp1.2054 103 p u b l i c a t i o n s codon aflatoxin m1 in traditional and industrial pasteurized milk samples from tiran county, isfahan province: a probabilistic health risk assessment khadijeh jafari1, ayub ebadi fathabad2, yadolah fakhri3, maryam shamsaei4, mohammad miri5,*, reza farahmandfar6, amin mousavi khaneghah7,* 1environment research center, research institute for primordial prevention of non-communicable disease, isfahan university of medical sciences, isfahan, iran; 2social determinants of health research center, department of public health, school of health, birjand university of medical sciences, birjand, iran; 3food health research center, hormozgan university of medical sciences, bandar abbas, iran; 4department of desert combating, faculty of natural resources and desert studies, yazd university, yazd, iran; 5non-communicable disease research center, department of environmental health engineering, school of health, sabzevar university of medical sciences, sabzevar, iran; 6department of food science and technology, sari agricultural sciences and natural resources university, sari, iran; 7department of food science and nutrition, faculty of food engineering, university of campinas (unicamp), campinas, são paulo, brazil. *corresponding authors: mohammad miri, non-communicable disease research center, department of environmental health engineering, school of health, sabzevar university of medical sciences, sabzevar, iran. emails: m_miri87@yahoo. com, m_miri87@ssu.ac.ir and amin mousavi khaneghah, department of food science and nutrition, faculty of food engineering, university of campinas (unicamp), campinas, são paulo, brazil. email: mousavi@unicamp.br received: 16 april 2021; accepted: 4 june 2021; published: 13 july 2021 © 2021 codon publications open access paper abstract in this study, the aflatoxin m1 (afm1) concentration in traditional and industrial milk and risk assessment due to afm1 exposure using the monte carlo simulations technique was investigated. the mean concentration of afm1 in traditional and industrial milk samples was 53.00 ± 11.49 and 54.33 ± 12.22 ng/l, respectively, which was higher than european union and codex standards. percentile 95% of hazard quotient (hq) adults and children due to industrial ingestion milk was 1.056 and 4.956, and traditional milk was 1.031 and 5.116, respectively. hazard quotient in all age consumers was higher than 1. therefore, consumers are at a considerable health risk. keywords: aflatoxin m1; industrial and traditional; milk; risk assessment; seasonal variation introduction the exposure of humans to various types of toxic chemical compounds (natural or artificial) may cause a wide range of human health problems, including genital diseases, mental disorders, dysfunction, especially in kidneys and liver, suppression and weakening the immune system, and a variety of cancers (bahrami et al., 2016; khaneghah et al., 2019; khodaei et al., 2020; mir et al., 2021; mousavi khaneghah et al., 2021; rahmani et al., 2018; rastegar et al., 2017; shahbazi et al., 2015; sipos et  al., 2021). among them, the mycotoxins are the secondary metabolites produced by fungi, mainly the aspergillus, fusarium, penicillium, and alternaria genera, causing substantial concerns all over the world over the last few decades (coppa et al., 2021; de souza et al., 2021; ertas et al., 2011; fakhri et al., 2019; mahmood fashandi et al., 2018; roohi et al., 2020). mycotoxins belong to the aflatoxins group (around 300 different mycotoxins) and are produced by three filamentous fungi species called aspergillus parasiticus, aspergillus flavus rarely by aspergillus nominus (campagnollo et  al., 2016; fakhri et al., 2018; heshmati et al., 2019). italian journal of food science, 2021; 33 (sp1): 103–116 mailto:m_miri87@yahoo.com mailto:m_miri87@yahoo.com mailto:m_miri87@ssu.ac.ir mailto:mousavi@unicamp.br 104 italian journal of food science, 2021; 33 (sp1) jafari k et al. was due to the cold season (autumn and winter). they reported that afm1 could be found in dairy products with an overall prevalence percentage of 63.53 and 54.05 based on the type of sample and production process, respectively. a study by ansari et al. (2019) reported that the autumn and winter seasons had shown the highest concentration and contamination (above 50 ng/l). to control and prevent exposure to afm1, more than 60 countries in the world have set the maximum permissible limit for afm1 in dairy products. the codex alimentarius and european union (eu) have determined the 50 ng/kg of afm1 concentration as the maximum amount of afm1 remaining in raw and warmed milk (de souza et al., 2021; khaneghah et al., 2021; mannani et al., 2021). the fao (food and agriculture organization, the united nations) has set the maximum allowable concentration of afm1 to 500 ng/l (cai et al., 2012; cavallarin et al., 2014). also, the iranian institute of standards has declared the concentration of afm1 to 100 ng/l in raw milk samples (hashemi, 2016; inso 5925; khaneghahi abyaneh et al., 2019). the health risk assessment of afm1 exposure due to dairy products and milk consumption is a valuable way to risk and evaluate the liver’s developing cancer (serraino et al., 2019; tsakiris et al., 2013). the afm1 risk assessment is defined as assessing the daily oral intake and hazard quotient (hq). these data and studies can be used for assessing the cancer risk in toxicological studies to estimate the severity and probability of toxin contamination (heshmati et al., 2017, 2019; nabizadeh et al., 2018; oteiza et al., 2017; škrbić et al., 2015). some studies in iran have been done for characterizing the concentration of afm1 in various cities; however, the available evidence on the risk assessment of afm1 exposure is limited in developing countries (e.g., iran) (bahrami et al., 2016; mohajeri et al., 2013). in developing countries, milk and its products are produced with traditional and industrial methods, increasing the risk of exposure to afm1. moreover, traditional livestock, especially in rural areas, can exacerbate this risk (bahrami et  al., 2016). traditional iranian dairy products made from milk are native to some areas of iran. also, in consuming this type of milk, the usual industrial processes of milk preparation do not occur, and boiling milk for consumption is done by individuals in their home. in the present study, samples of traditional milk were cow’s milk, which were purchased from local distributors in the same area (rural regions) that are manufactured in rural households under unacceptable hygiene conditions (bahrami et al., 2016; de souza et al., 2021; kaur et al., 2021). in this regard, the current investigation was undertaken to determine the afm1 level in traditional and different industrial brands available in the markets of tiran aflatoxin b1  (afb1) is the typical type and most toxic one among 18 aflatoxin types. aflatoxin m1 (afm1) is an afb1 hydroxylated metabolite deformed by the enzymes of cytochrome p450 in the liver. this is secreted through the milk glands in lactating cows by afb1, which is consumed through food (marhamatizadeh and goosheh, 2016; škrbić et al., 2014); and about 0.5% to 6% of afb1 is converted into afm1. when afb1 contaminated animal feeds with lactating animals, after 12 to 24 h, afm1 could be detected and reached a maximum after 72 h when the feeding with the contaminated animal feeds is reached (asi et al., 2012; iqbal et al., 2014; mousavi khaneghah et al., 2018). after afb1 intake, about 72 h, the afm1 concentration was decreased to an undetectable level (khaneghah et al., 2018). the ratio of swallowed afb1 to the excreted afm1 has been reported with various percentages in different sources; however, typically, it has been in the range of afb1 averages 1%–2% that varies from day to day, type of animal, and type of milk (bervis et al., 2021; gonçalves et al., 2017; heshmati et al., 2019). milk is a very nutritious food rich in micro and macronutrients, vital to human health evolution and human body growth. therefore, the safety and hygiene of milk play a critical role in human health (bahrami et al., 2016; iqbal et al., 2015). aflatoxin m1 in dairy products and milk is highly resistant to conventional milk processing methods, including pasteurization, ultra-high temperature (uht), and other processing methods (de roma et al., 2017; khaneghah et al., 2021; rahmani et al., 2018; turkoglu and keyvan, 2019). the pasteurization method can reduce the concentration of afm1; however, it cannot eliminate this contaminant (mahmood fashandi et  al., 2018; naeimipour et al., 2018). some adverse health effects such as carcinogenicity, teratogenicity, preventing rna encoding and protein synthesis, brain damage, colon damage, lung, liver, kidney, mutation, immune suppression, and digestive disorders were associated with the consumption of contaminated milk by afm1 (fallah et al., 2016; kaur et al., 2021; pokharel et al., 2021). in order to assess the overall prevalence of afm1 in industrial and traditional milk, several studies have been carrying out. the study by de souza et al. (2021) stated that the afm1 concentration had been reduced in the contaminated milk, which was mainly carried out in traditional products. however, preventative approaches are recommended, and it is necessary to reduce the concentration of aflatoxin. tajik et al. (2016) investigated the afm1 concentration in the west-azerbaijan in the pasteurized milk. their study showed that 77.7% of the samples were contaminated with afm1; and in the pasteurized milk, about 70% of them exceeded the standard value given by european commission (50 ng/l). makhdoumi et al. (2021) showed that the prevalence of afm1 in conventional and industrial dairy products italian journal of food science, 2021; 33 (sp1) 105 aflatoxin m1 in traditional and industrial pasteurized milk samples from tiran county, isfahan province samples were transferred to the laboratory in pre-sterilized stainless steel containers (for traditional) and under dark environment conditions, away from smell and light at 1 to 5°c. sterilized milk samples (industrial samples) and traditional samples were stored at 4°c. samples preparation was conducted to determine afm1 in samples of milk by using enzyme-linked immunosorbent assay (elisa) method according to the instructions given in the manufacturer kit (r-biopharm, darmstadt, germany). traditional and industrial milk (pasteurized) traditional milk means milk that is produced in villages and does not go through the industrial process. in the present study, the meaning of traditional milk is milk produced and marketed in traditional livestock farms in villages around tiran county without any industrial process for its preparation. pasteurized milk is the samples prepared in the dairy industry with mechanized equipment and methods. analysis of aflatoxin m1 by elisa method based on the information recommended by the kit catalog, the limit of detection (lod) was 5 ng/kg, and the analysis area was 50%–150%. the afm1 cross-reactivity in these kits is 100%, and the probability of crossover reaction with aflatoxin g1, g2, b1, and b2 is zero. validation of elisa was carried out by determining recoveries, and the mean variation coefficient (cv) for milk samples spiked with different concentrations of afm1. the mean recovery score in spiked milk samples (5, 10, 20, 40, and 80) was 99.96%, county besides assessing the effects of seasonal changes on the afm1 level in milk. also, to conduct a probabilistic risk assessment of afm1 exposure due to consumption of milk. materials and methods sampling area the present study was conducted in tiran city, isfahan province, iran. the geographical location of the tiran county is at 32°42′12.96″ east and 51°9′ 6.84″ north and 1640 m above the sea level (figure 1). tiran has arid climates, and the annual average rainfall is 116.9  mm (jafari et al., 2018). based on the last census (iran statistical center, 2016), the county population was 71,583, and per capita milk consumption in this province is 90 kg/person/year. moreover, isfahan province, with 1,250,000 tons of milk in 2018, has been the first raw milk producer in iran. sampling and preparation in this study, 156 traditional and industrial milk samples (60 traditional milk (a) and 96 industrial milk samples from 15 brands of b-p) were collected from 15 best selling milk brands during autumn 2017 and winter 2018 (26 samples were taken in each month). sampling was performed randomly from all locations of the milk supplier in the study area. industrial pasteurized milk was randomly obtained from large and busy stores in the city, and traditional milk samples (unheated raw milk) were also obtained from traditional dairy stores in tiran. the tiran decimal degrees 0 0.25 0.5 1 1.5 2 decimal degrees 00.5 1 2 3 4 isfa han isfahan persian gulf caspian sea n s w e n s w e figure 1. location of tiran county in isfahan province and iran (jafari et al., 2018). 106 italian journal of food science, 2021; 33 (sp1) jafari k et al. ir was 0.19 l/day, ef was 350 days, ed was 30 and 6 years for adults and children, respectively, and bw was 70 and 15 kg for adults and children, respectively. at was equal to 10,950 and 2190 days for adults and children, respectively. the rfd was also 0.2 ng/kg per day (xiong et al., 2021). monte carlo simulation (mcs) method the simulation of monte carlo was used to minimize uncertainties in the results. when the point values of a variable were used to assess the exposure risk of pollutants with a population, the probability of interference and error, and finally, the uncertainty in the result is obtained (fakhri et al., 2018; huang et al., 2017; keramati et al., 2018, 2019). crystal ball software (version 11.1.1.1 oracle, inc. united states of america) was used to determine uncertainties with 5000 trails in the mcs. the percentile 95% of hq was selected as a worse scenario for the health risk of consumers. statistical analysis the shapiro–wilk normalization test was performed to verify the data normality. the difference between the concentration of afm1 in the two groups of samples (traditional vs. industrial samples) and seasons was assessed using the mann–whitney test. furthermore, the association between afm1 concentrations and different brands was investigated by the post-hoc test. a p-value of <0.05 was considered as a significance level for all tests. all statistical analyses were carried out using spss21 software. results and discussion afm1 concentration the afm1 concentration in industrial and traditional samples in autumn and winter is shown in figure 2. also, the results of afm1 concentration based on the different seasons and different types of milk are shown in tables 1 and 2. while the afm1 mean concentration in samples of traditional milk produced in autumn and winter is 54.33 ± 11.65 and 53.00 ± 12.22 (ng/l), the overall afm1 mean concentration in traditional and industrial milk samples is 53.00 ± 11.49 and 54.33 ± 11.22 ng/l, respectively (see the table 1). in the industrial milk brands, b and h with a mean concentration of 60.00 (12.64) ng/l had the highest concentration of afm1, and l and o brands with a mean concentration of 48.33 (13.29) ng/l had the lowest afm1 concentration. the total mean of afm1 concentration in samples of milk was 53.33 (11.87) ng/l, which was more than the recommended standard with a cv of 1.2%. according to manufacturer’s guideline, the recovery rate in spiked milk was 95%, with a cv of 15%. also, the detection range of these kits is 50–80 ng/kg. afm1 stock solution (50 mg/l) was obtained by solubilizing the afm1 standard powder (sigma aldrich, germany) in a chloroform/methanol solution with a volume ratio (v/v) of 19:81 and kept at a temperature of –20°c. before analysis, by chloroform:methanol at a 1:1 v/v ratio, the stock solution to obtain different standard solution concentrations was diluted (fallah, 2010; maggira et al., 2021; tajik et al., 2016). afm1 analysis was carried out according to the instructions given in the kit. notably, samples remained fully protected from light (prevention from afm1 inactivation). health risk assessment the health risk of afm1 exposure in the milk samples was calculated according to the united states environmental protection agency (usepa, 2011; hooshfar et al., 2020; kaur et al., 2021). the daily exposure to afm1 by milk was calculated as following: × × × = × m m ing c ir ef ed edi bw at (1) where, edi is the estimated daily intake per day by afm1 consumed in milk (ng/kg/day). cm denotes afm1 concentration in milk (ng/l), and irm is the milk ingestion rate according to liter/day. based on the approximate information available, the amount of milk consumption for people with high consumption was 222 g/day, the average consumption was 174 ml/day, and the minimum consumption was 74 ml/day. however, according to the previous study in iran (march 2011–march 2012), iranians approximately consumed 190 ml or less than one glass of milk/ dairy products per day.) (abyaneh et al., 2019; home society economy politics sports culture international multimedia tourism, 2019; nejad et al., 2019; pérezgregorio et al., 2011), ef is exposure frequency (day/year), ed is exposure duration (according to years), bw is body weight (kg), and at, the averaging time (based on days). the hq was investigated to determine the afm1 non-carcinogenic risk induced by milk intake. if the hq is higher than 1, the risk of liver cancer for the consumer is more (fakhri et al., 2018; kuiper-goodman, 1990; nabizadeh et al., 2018). while hq values less than 1 indicate that milk intake does not cause harmful effects for the consumers (bahrami et al., 2016). the risk index was calculated according to the following equation: edi hq rfd = (2) where rfd is the reference dose of aflatoxin. italian journal of food science, 2021; 33 (sp1) 107 aflatoxin m1 in traditional and industrial pasteurized milk samples from tiran county, isfahan province 39 a b c a fla to xi n m 1 co nc en tr at io n (n g/ l) d e f 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 figure 2. afm1 concentrations in traditional and industrial milk samples based on the season (a: afm1 in traditional milk, autumn; b: afm1 in traditional milk, winter; c: industrial milk, autumn; d: industrial milk, winter; e: total afm1 concentration in industrial samples; f: total afm1 concentration in traditional samples). table 1. afm1 concentration in milk samples based on the brand. brand of milk number afm1 concentration according to number of samples mean concentration ± sd lower than 50 ng/l standard or 50 ng/l higher than 50 ng/l traditional (a) 60 19 13 18 53.66 ± 11.49 b 6 1 1 4 60 ± 12.64 c 12 6 2 4 50 ± 12.06 d 6 3 0 3 51.66 ± 13.29 e 6 3 0 3 55 ± 16.43 f 6 1 3 2 56.66 ± 10.32 g 6 2 2 2 51.66 ± 11.69 h 6 1 1 4 60 ± 12.64 i 6 3 0 3 53.33 ± 15.05 j 6 2 2 2 51.66 ± 11.69 k 6 1 3 2 55 ± 12.24 l 6 4 0 2 48.33 ± 13.29 m 6 2 2 2 51.66 ± 11.69 n 6 2 2 2 50.00 ± 8.94 o 6 4 0 2 48.33 ± 13.29 p 6 1 2 3 57.14 ± 11.12 total 156 55 31 70 53.66 ± 11.49 108 italian journal of food science, 2021; 33 (sp1) jafari k et al. market by using elisa while about 1.8% of uht milk samples and 59.5% of pasteurized milk were above the eu and codex standards (xiong et al., 2018). according to the results shown in table 2, the afm1 mean concentration in traditional and industrial milk samples in autumn was higher than in the winter season, which may be due to storage conditions and increased moisture in autumn, but p-value was not significant (aydemir atasever et al., 2021; makhdoumi et al., 2021; rahmani et al., 2018). in another investigation, the concentration of afm1 in uht milk was significantly lower in spring and winter than in summer and autumn (heshmati and milani, 2010). similar results regarding the afm1 levels in milk and uht from brazil and serbia were reported as higher afm1 concentrations in summer and autumn than spring and winter (de oliveira et al., 2013; tomašević et  al., 2015). in another study, afm1 concentration in raw milk of cattle, sheep, and goat samples collected among autumn and spring were investigated, while no significant correlation in afm1 among seasons was noted (bilandžić et al., 2017). in another study, 119 samples out of 125 powder milk uht and pasteurized milk collected from brazil were contaminated with afm1 in the range of 10 to 200 ng/l and the mean concentration of 31 ng/l (shundo et al., 2009). no significant association regarding afm1 concentration among different seasons was noted (p-value was 0.704 according to mann–whitney test). according to de roma et al. (2017), seasonal changes were considered as an influential factor in the concentration of afm1. also, bahrami et al. (2016) demonstrated that afm1 levels were significantly higher in winter than their corresponding values in summer. therefore, seasonal variation was considered as an effective parameter on the concentration of afm1 (akbar et al., 2019; ansari et al., 2019). moreover, no significant difference in the afm1 level was observed among the traditional and industrial samples (p-value was 0.563 according to the mann–whitney test). however, based on the findings of some similar studies conducted in iran, the level of contamination in the traditional milk samples was notably higher than the collected samples from industrial ounces, which of afm1 in the eu and the codex exceeding the permissible limit (50 ng/l) (bahrami et al., 2016). fifty-five cases from 156 samples had contamination lower than 50 ng/l, 31 samples were 50 ng/l, which complied with the european union’s recommended standards of 50  ng/l (fallah, 2010; fallah et al., 2016), 70 cases had a high contamination rate of afm1 that were higher than the recommended standard of 50 ng/l. overall, the lowest afm1 concentration was 40 and the highest was 70 ng/l. however, this concentration of afm1 was lower than the iranian standard limit (100 ng/l) (khaneghahi abyaneh et al., 2019). in another study, the mean concentration of afm1 in the pasteurized milk was 76.2 ng/l (tajik et al., 2016), which is higher than the results of our study. one of the reasons for the higher level of afm1 in westazerbaijan may be due to geographical conditions, more contamination of animal feed with afb1. so, control and monitoring of milk and local health conditions are crucial. afm1 concentration in traditional milk, surveyed by omeiza gabriel kehinde et al. (2021), that afm1 concentration in the traditional products was the highest concentration compared to industrial products. there was a significant difference between the type of feed, type of dairy herds, and holding capacity of the dairy herds (kehinde et al., 2021). xiong et al. (2021) showed the health risk for child group 2–4 years old that consume contaminated milk with afm1. about 5.3% of the samples were above the 50 ng/l limits, and the afm1 level in uht milk was lower than pasteurized milk. comparing the afm1 concentration in traditional milk and industrial samples were carried out (it is notable, the traditional milk was purchased from rural areas around tiran county and was prepared as well as sold by individuals on private farms. also, boiling and milk preparation was carried out at the buyer’s house by themselves.). moreover, comparing the afm1 concentration in the traditional and industrial samples with the eu, the codex limit, and european union standards (50 ng/l) indicated that 44.9% of samples had a concentration higher than the maximum allowable level. in a study conducted by xiong et al. (2018), the mean concentration of afm1 100 ng/l was assigned as the afm1 concentration in uht and pasteurized samples of milk collected from china’s table 2. afm1 concentration range in the traditional and industrial pasteurized milk samples based on the season. season/month under 50 ng/l (%) 50 ng/l (%) higher than 50 ng/l (%) total samples autumn 7 (26.92) 5 (19.23) 14 (65.38) 26 autumn 10 (38.46) 7 (26.92) 9 (34.61) 26 autumn 10 (38.46) 2 (7.69) 14 (65.38) 26 winter 10 (38.46) 4 (15.38) 12 (46.15) 26 winter 10 (38.46) 5 (19.23) 11 (42.30) 26 winter 8 (30.76) 8 (30.76) 10 (38.46) 26 total 55 (35.25) 31 (19.87) 70 (44.87) 156 italian journal of food science, 2021; 33 (sp1) 109 aflatoxin m1 in traditional and industrial pasteurized milk samples from tiran county, isfahan province kos et al., 2014; milićević et al., 2017b; serraino et al., 2019; tsakiris et al., 2013). the risk assessment of afm1 in milk and dairy products in lebanon measured afm1 concentration in the raw milk, uht, and pasteurized milk was 0.011–0.440, 0.015–7.350, and 0.013–0.219 µg/l, respectively. the health risk assessment for adults with an average body weight of 75.49 kg was at a safe level (hq < 1). according to a study by daou et al. (2020), the milk consumption rate for lebanon was 113.7 g/person/day that was lower than the milk consumption rate in our study (0.19 l/day). this difference in the body weight and daily milk consumption rate can be one of the reasons for the low hq value compared to our study. the afm1 concentration in all brands was safe regarding non-carcinogenic risk due to milk consumption in iranian consumers, which agrees with one of the previously published reports (hooshfar et al., 2020). however, the calculated hq in summer and winter after measuring the afm1 by elisa and hplc techniques were reported as 0.54 and 1.245, 0.88, and 1.45, respectively (bahrami et al., 2016). in another similar investigation performed after afm1 level assessment by elisa method, hq was higher than 1 (milićević et al., 2017a), while the findings of both investigations, as mentioned earlier, demonstrated a potential risk of liver cancer for consumers. afm1 was measured in dairy products from turkey; and in cheese samples, the hq of receiving afm1 was more than 1 (sakin et al., 2018). in a study by rahmani et al. (2018), probabilistic health risk assessment of afm1 in milk samples of the east region was carried out. in many middle east countries, the hq was higher than 1 for children at a considerable risk of cancer, unlike adults (rahmani et al., 2018). the risk of cancer and non-carcinogenic diseases in brazilian children aged 0–5 years was assessed by exposure to afm1 in samples of uht milk, powder milk, and infant formula. the results of the study showed that the range of afm1 was from 150 to 1020 ng/kg and all positive samples exceeded the limit set by the european union, and the number of hepatocellular carcinoma cases associated with afm1 exposure was higher than 0.001 cases per 100,000 people (conteçotto et al., 2021). as in the present study, there was a risk of cancer for children. the study of sharma et al. (2020) showed the presence of afm1 in raw and pasteurized milk samples of india and it is necessary to pay attention to the amount of this mycotoxin. the study that evaluated cancer and non-carcinogenic risk in the infant with an age of below 6 months in iran showed only one of the infant formula milk samples were contaminated with afm1 and did not concern the health risk to consumers (hooshfar et al., 2020). therefore, attention to different methods for detoxification of dairy products, attention to the manner were associated with the non-hygienic condition of livestock storage among a variety of livestock, including cattle, goats, and sheep (bahrami et al., 2016; fallah, 2010; fallah et al., 2016; mohammadi, 2011). moreover, the variety in the type of measurement method, geographical location, weather, seasonal, feeding systems, food storage, and farmland and pasture techniques are among other possible reasons (bahrami et al., 2016; iqbal et al., 2015). in the present study, almost all samples were prepared from the exact geographical location and climate. therefore, the high afm1 in some samples could be correlated with the type of feeding, poor health conditions of forage, and livestock feed (bilandžić et al., 2017; de roma et al., 2017; khaneghah et al., 2021; visciano et al., 2015). however, according to the sampling season, the level of afm1 was higher in winter than in autumn (not significant). health risk assessment percentile 95% of hq in adults and children due to industrial ingestion milk was 1.056 and 4.956 and due to traditional milk was 1.031 and 5.116, respectively. the hq value in both adult and children consumers was higher than 1. therefore, consumers are at a considerable health risk (figures 3 and 4). in a study by bahrami et al. (2015), the health risk assessment of afm1 was assessed by consuming traditional dairy products in the western part of iran. in this study, the average body weight for each adult iranian was considered to be 60 kg. the results showed that the hq was higher than 1 for dairy products in winter (1.24 ng/kg body weight in the elisa method). in summer, the hq was less than 1 for both methods. in this study, it is noteworthy to assess health risk, and the amount of milk and dairy consumption per iranian in 2013 was calculated to be equal to 70 kg (annual statistics of iranian agriculture) (bahrami et al., 2016). in another study in iran, the average body weight of iranian adults was considered to be 70 kg, which can effectively calculate the results. in this study, the mean concentration of afm1 in the traditional cheeses was 139.4 ± 2.4 ng/kg. the afm1 concentration was not higher than 500 ng/kg as the maximum permissible limit. after performing a health risk assessment, the hq was less than 1 (shahbazi et al., 2017; tomašević et al., 2015). assessment of the health risk of afm1 in infant formula milk for infants with 0 to 6 months old, an average weight of 5.7 kg, and the consumption rate of infant formula milk equal 19.4 g/day showed that hq was less than 1 (hooshfar et al., 2020). the difference in results with our study was the difference in the amount of afm1 concentration in milk samples, age, body weight, and milk consumption rate (bahrami et al., 2016; duarte et al., 2013; 110 italian journal of food science, 2021; 33 (sp1) jafari k et al. adult 50 45 40 35 30 25 20 15 10 5 0 0.130 0.414 0.697 hq f re qu en cy 50 45 40 35 30 25 20 15 10 5 0 f re qu en cy 0.981 1.264 1.041 2.258 3.476 hq 4.693 5.910 p 95% = 1.056 children p 95% = 1.056 figure 3. percentile 95 of hq in adults and children due to ingestion industrial pasteurized milk content of afm1. of storage, the lifetime of food intended for feeding livestock and poultry, as well as the need for stricter legal requirements and monitoring legal recommendations are recommended (min et al., 2020). a study that evaluated the prevalence of milk contamination with afm1 worldwide found that most countries had moderate levels of afm1 in milk in recent years, which were lower than eu levels. however, several countries, including pakistan, india, and several countries around sub-saharan africa, have high levels of afm1 that exceeded eu and us standards. therefore, it has been speculated that high levels of afm1 in milk can indicate high levels of afb1 in animal feed, products italian journal of food science, 2021; 33 (sp1) 111 aflatoxin m1 in traditional and industrial pasteurized milk samples from tiran county, isfahan province adult p 95% = 1.031 50 60 40 30 20 10 0 f re qu en cy 0.181 0.441 0.702 0.962 1.222 hq children p 95% = 5.116 50 40 30 35 45 20 15 25 10 5 0 f re qu en cy 1.229 2.408 3.586 4.765 5.943 hq figure 4. percentile 95 of hq in adults and children due to ingestion traditional milk content of afm1. such as corn used to make animals feed may have high levels of afb1, which after consumption can lead to animal and humans health adverse effect (turna and wu, 2021). contamination of goat’s milk with afm1 in brazil showed that in all milk samples, the recommended levels of afm1 in milk were lower than the permissible limit. however, the potential risk of liver cancer and edi levels for afm1 through goat’s milk for 1-year-old children were higher than the tolerable daily intake and estimated values (de matos et al., 2021). the results of the study about the amount and risk of afm1 in pasteurized milk products, esl and uht milk from china in summer and winter 112 italian journal of food science, 2021; 33 (sp1) jafari k et al. references abyaneh, h.k., bahonar, a., noori, n., yazdanpanah, h. and aliabadi, m.h.s., 2019. exposure to aflatoxin m1 through milk consumption in tehran population, iran. iranian journal of pharmaceutical research: ijpr 18(3): 1332–1340. 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fakultesi dergisi 27(1): 34–42. bahrami, r., shahbazi, y. and nikousefat, z., 2016. aflatoxin m1 in milk and traditional dairy products from west part of iran: occurrence and seasonal variation with an emphasis on risk assessment of human exposure. food control 62: 250–256. https://doi.org/10.1016/j.foodcont.2015.10.039 bervis, n., lorán, s., juan, t., carramiñana, j.j., herrera, a., ariño, a. et al., 2021. field monitoring of aflatoxins in feed and milk of high-yielding dairy cows under two feeding systems. toxins 13(3): 201. https://doi.org/10.3390/toxins13030201 bilandžić, n., varenina, i., kolanović, b.s., luburić, d.b., varga, i., želježić, b., et al., 2017. occurrence of aflatoxin m1 in raw cow, goat and sheep milk during spring and autumn in croatia during 2016. toxin reviews 36(4): 290–296. https://doi.org/10.1080/15 569543.2017.1306785 cai, y., lv, j., zhang, w. and zhang, l., 2012. dietary exposure estimates of 16 polycyclic aromatic hydrocarbons (pahs) in xuanwei and fuyuan, counties in a high lung cancer incidence area in china. journal of environmental monitoring 14(3): 886– 892. https://doi.org/10.1039/c2em10807k campagnollo, f.b., ganev, k.c., khaneghah, a.m., portela, j.b., cruz, a.g., granato, d., et al., 2016. the occurrence and effect of unit operations for dairy products processing on the fate of aflatoxin m1: a review. food control 68: 310–329. https://doi. org/10.1016/j.foodcont.2016.04.007 cavallarin, l., antoniazzi, s., giaccone, d., tabacco, e. and borreani, g., 2014. transfer of aflatoxin m1 from milk to ripened cheese in three italian traditional production methods. food control 38: 174–177. https://doi.org/10.1016/j.foodcont.2013.10.008 conteçotto, a.c.t., pante, g.g., castro, j.c., souza, a.a., lini, r.s., romoli, j.c.z., et al., 2021. occurrence, exposure evaluation and risk assessment in child population for aflatoxin m1 in dairy showed that 5.3% of milk samples had a level of afm1 more than the standard limit of 50 ng/kg, which is less contamination compared in these studies. similar to the present study, afm1 concentrations were higher in winter. children aged 2–4 years had the highest risk of exposure to afm1 in milk, which is due to the type of storage and observance of hygienic conditions, especially in winter is necessary (xiong et al., 2021). afm1 exposure through yogurt consumption and the risk of liver cancer were studied in hamedan, iran. although it was found that a high percentage of yogurt samples in iran were contaminated with afm1 content, there was no particular concern about the risk to public health according to european standards (ec) and the iranian institute for standards and industrial research (isiri) (heshmati et al., 2020) conclusions in this study, afm1 concentration in milk samples [traditional (60 samples) and industrial milk (from 15 brands, b–p) was measured. also, the afm1 concentration in two seasons of the year (autumn and winter) was measured. according to the results, approximately 45% of traditional and industrial milk samples were contaminated with afm1 more than the standard level (eu and codex). aflatoxin levels in autumn were higher than in winter, but p-value was not significant. moreover, the amount of afm1 in samples of traditional milk was higher than that of industrial. the non-carcinogenic risk of exposure to afm1 showed that hq was higher than 1 for adults and children, indicated that milk consumers in tiran county are at considerable risk of developing liver cancer-related afm1. recommendation it is also important to enforce strict rules and more supervision in livestock farms and production centers. the most important idea to reduce the exposure of afm1 is to prevent the production of afb1 in the fields, and the best solution is to secure farms, industrial and traditional livestock farms, educate people about the dangers and appropriate ways to store livestock feeds, the type of food that is used, and improving their storage environment to reduce 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food engineering, kınıklı, 20020 denizli, turkey *corresponding author. tel: +90 2582963111; fax +90 2582963262 e-mail address: fisik@pau.edu.tr abstract tomato paste waste materials are rich in bioactive food components, but have a low economic value. in this study, the potential use of tomato pomace in crackers was studied. wheat flour was partially (4%, 8%, 12%) substituted with dried tomato pomace meal. tomato pomace addition caused a significant (p<0.05) increase in protein, ash, dietary fiber (soluble, insoluble, total), minerals (mg, ca, k, p, mn, zn, fe), total phenolics, antioxidant capacity, hunter a and b color values, but decrease in l value. although the samples having 12% tomato pomace had lower scores, the crackers were liked statistically equally by the panelists. keywords: cracker, tomato pomace, total phenolics, dietary fiber, minerals, sensory   ital. j. food sci., vol 28, 2016 526 1. introduction tomato (lycopersicon esculentum l) is the world’s second largest vegetable crop (valencia et al., 2002). worldwide, about 37 million tons of tomatoes are processed in the industry (keskin, 2012). a large part of the world tomato crop is processed into tomato paste, which is used as an ingredient in many products such as soups, sauces, and ketchup (sanchez et al., 2003). the problems of industrial wastes are becoming harder to solve, and much effort will be needed to develop the nutritional and industrial potential of by-products, waste and under-utilized agricultural products (ajayi et al., 2006). tomato industry generates large amounts of by-products, and these by-products representing 10-30% of total processed tomatoes contain tomato seeds, peels, pulp and cores (rahmatnejad et al., 2009). seeds and peels present in tomato pomace (tp) consist of the substances that are rich in nutritional value. it is underlined in some studies that they are rich in biologically active compounds, such as dietary fiber, protein, oil, mineral matters, phenolic compounds and carotenoids (el-adawy and taha, 2001; schieber et al., 2001; sogi et al., 2002; knoblich et al., 2005; calvo et al., 2008). crackers are popular snack foods in human diet (sedej et al., 2011). they are dry, thin and crisp bakery products and the low level of moisture, decreased even further with baking, left no medium for mold growth (han et al., 2010). there are different types of crackers such as; saltiness cracker, soda cracker, sprayed cracker, cream cracker, savory cracker, matzos cracker, water cracker, graham cracker, etc. (yoneya and nip, 2006). the basic ingredients in cracker production are wheat flour, fat (or shortening), salt, leavening agents (yeast, chemical leaveners, or combination), whey powder, sugar and/or glycose syrup (yoneya and nip, 2006; gundogdu sertakan, 2006). crackers are usually produced with soft white flour (kweon et al., 2014). but, the contents of some components in white flour, like amino acids (lysine, tryptophan) and dietary fiber that play an important role in nutrition, are low (elgun and ertugay, 1995). crackers could be an alternative food for the consumption of tomato pomace that is rich in biologically active compounds. in the study, white wheat flour used in soda cracker production was substituted with tomato pomace meal to improve the nutritional and functional properties of crackers and it was aimed to determine the potential use of tomato paste waste material, usually utilized in animal feeding and rich in biologically active components, in human diet. the use of waste materials in human diet could also decrease environmental pollution problem 2. materials and methods 2.1. materials commercial wheat flour (type 650), wheat starch, corn oil, sugar, salt and baking powder (sodium bicarbonate, sodium acid pyrophosphate) were purchased from local markets in denizli, turkey. waste materials of tomato paste production were obtained from honaz paste plant (honaz, denizli, turkey). 2.2. methods 2.2.1. preparation of tomato pomace meals tomato pomace was dried in a cabinet dryer (yücebaş machine analytical equipment industry, izmir, turkey). the dryer was consisted of a centrifugal fan to supply the   ital. j. food sci., vol 28, 2016 527 airflow, an electric heater, and an electronic proportional controller (enda, euc442, istanbul, turkey). the air velocity was kept constant at 0.2 m s-1 during drying at 60°c. the relative humidity of ambient air changed between 19 and 21%. after drying, pomaces were ground with a grinder (toper tks-16s, izmir, turkey) to a particle size of < 1000 µm. 2.2.2. production of crackers soda crackers were prepared according to the procedure of han et al. (2010) with some modifications. formulations presented in table 1 were used to produce crackers. crackers containing tomato pomace meals were prepared by substituting 4, 8 or 12% of flour in the formulae with dried tomato pomace. the preparation of crackers included steps of mixing of dry and liquid ingredients for 3-4 minutes to form a dough (kitchenaid, artisan series, model 5ksm150, usa), resting of dough for 10 minutes, passing of dough through a set of smooth stainless steel rotating drums 3 times (kitchenaid, artisan series, model 5ksm150, usa) by sheeting and laminating the dough, and cutting square shaped crackers from the dough sheet. crackers were baked in an electric oven (özköseoğlu, istanbul, turkey) at 200°c for 10 minutes. after baking, crackers were left in the oven for an additional 2 minutes with the heat off but with forced air circulation. this process simulated the drying and cooling stages of a tunnel-type commercial baking oven. baked crackers were then removed from the oven and allowed to cool down to room temperature. table 1: soda cracker formulations. ingredients (g) control tp4a tp8b tp12c wheat flour 450.0 430.0 410.0 390.0 dried tomato pomace meal 20.0 40.0 60.0 wheat starch 50.0 50.0 50.0 50.0 water 200.0 250.0 250.0 250.0 corn oil 75.0 75.0 75.0 75.0 sugar 17.5 17.5 17.5 17.5 salt 5.5 5.5 5.5 5.5 baking powder 5.0 5.0 5.0 5.0 atp4: 4% of wheat flour was substituted with dried tomato pomace powder, btp8: 8% of wheat flour was substituted with dried tomato pomace powder, ctp12: 12% of wheat flour was substituted with dried tomato pomace powder. 2.2.3. analytical measurements total solids, ash, and oil contents of the samples were determined according to the methods of aoac (1990). crackers were analyzed for their total protein contents by a dumatherm nitrogen-determination system run under the combustion method (gerhardt analytical systems, dumatherm, germany) (anonymous, 2011). dietary fiber contents were determined with the fiber assay kit (megazyme k-tdfr, wicklow, ireland) according to the mes-tris aoac method 991.43 (1995) and aacc method 32-07 (1995). samples were first suspended in the mes-tris buffer and then,   ital. j. food sci., vol 28, 2016 528 digested by heat-stable α-amylase, protease, and amyloglucosidase to remove starch and protein. insoluble dietary fiber was recovered from the enzyme digestate after filtration. soluble dietary fiber in the filtrate was precipitated with ethanol and filtered. all dietary fiber fractions collected were dried at 103±2°c for a night. total dietary fiber content was calculated as the sum of insoluble and soluble dietary fiber contents. all dietary fiber contents were corrected for residual protein, ash, and blank. inductively coupled plasma optical emission spectrometer (icp-oes, perkin elmer, optima 8000, massachusetts, usa) was used to determine the mineral elements (mg, ca, k, p, mn, zn and fe) of wheat flour, tomato pomace and crackers. in the pretreatment stage, digestion of 1g sample was performed using a mixture of hno3:h202 (8:4) in a microwave oven (milestone start d, sorisole, italy) (gopalani et al., 2007; anonymous, 2016). in the first step of digestion, the samples were digested for 15 min until reaching 110°c, and in the second step they were kept at this temperature for 15 min. the optimal operation conditions for icp-oes analysis of mineral matters were as follows: rf power, 1450 w; plasma gas (ar) flow rate, 15 l/min; auxiliary gas (ar) flow rate, 0.2 l/min; nebulizer flow rate, 0.7 l/min; sample flow rate, 1.5 ml/min; delay time, 15s. sensitive wavelengths for mineral identification were obtained from the tables provided by the manufacturer (boss and fredeen, 2004). for the extraction of phenolics, crackers were mixed with 70% (v/v) methanol with a 1:10 (w/v) ratio and homogenized (ika-ultra turrax, staufen, germany). then the mixture was treated in an ultrasonic water bath (elma e 60 h, new jersey, usa) for 10 minutes and shaken in a mechanical orbital shaker (wiseshake sho-1d, wertheim, germany) for 15 minutes. finally, the liquid extract was separated from solids by centrifugation (26,000g for 20 minutes at 4°c, hettich, universal 30 rf, massachusetts, usa). the supernatant was recovered and the extraction step was duplicated for the precipitate. the supernatants of the two steps were mixed and stored at -24oc until total phenolic content and antioxidant activity analyses. folin-ciocalteu method (singleton et al., 1999) was used to determine total phenolic content. 1 ml of extract was mixed with 5 ml of 1:10 (v/v) folin-ciocalteu reagent:water mixture for 5 minutes and 4 ml of 75g/l sodium carbonate (na2co3) was then added. after incubation at room temperature for 2 hours, the absorbance of the reaction mixture was measured by a spectrophotometer (t80 uv/vis spectrometer, pg instruments ltd., leicestershire, united kingdom) at 760 nm. gallic acid (0–100mg/l) was used as a standard to produce the calibration curve and the total phenolic content of extracts was expressed in mg of gallic acid equivalents (gae)/100 g dry matter of wheat flour, tomato pomace meal and crackers. dpph assay was used to determine the antioxidant activity of extracts (thaipong et al., 2006). the stock solution was prepared by dissolving 24 mg dpph with 100ml methanol and then stored at -20°c until needed. the working solution was obtained by mixing 10ml stock solution with 45ml methanol to obtain an absorbance of 1.10± 0.02 units at 515 nm using the spectrophotometer. cracker extracts (150 μl) were mixed with 2850 μl of the dpph solution and allowed to react for 24 h in the dark. then the absorbance was taken at 515 nm. the standard curve was linear between 25 and 800μm trolox. results were expressed in μm te/100 g dry mass. additional dilution was needed if the dpph value measured was over the linear range of the standard curve. 2.2.4. physical and sensory properties of crackers color values (hunter l, a, b) of crackers were determined by a hunter labmini scan xe model colorimeter (reston,va, usa) (anonymous, 1995).   ital. j. food sci., vol 28, 2016 529 in sensory evaluation, a panel of 48 subjects in the department of food engineering (pamukkale university, denizli, turkey) evaluated the sensory properties of soda crackers and assigned scores for color, smell, flavor, crispiness, and overall acceptability on a hedonic scale from 1 (dislike extremely) to 7 (like extremely). the panel consisted of students, staff and faculty members (30 females, 18 males), and 60% of the subjects were between 18 and 25, 27% between 26 to 40 years old, and 13% older than 40 years. the samples were labeled randomly with three-digit numerical codes. during the panel, subjects were instructed to rinse their mouths with water, and eat unsalted crackers before tasting each sample. the panel was performed in partitioned boots equipped with daylight. 2.2.5. statistical analysis “minitab 13 statistical software” was used for the statistical analysis of data. anova (one-way analysis of variance) with tukey’s multiple comparison test was performed to determine significant differences at α=0.05. 3. results and discussion crude protein, crude oil, crude ash, soluble, insoluble and total dietary fiber contents, mineral matters (mg, ca, k, p, mn, zn, fe) and total phenolic compounds contents, antioxidant activity values and color values of wheat flour and tomato pomace meal are given in table 2. results indicated that tomato pomace used in this study is a good source of crude protein, crude oil, dietary fiber, and minerals, and has higher total phenolic compounds and antioxidant capacity values than wheat flour. in the present work, soda cracker was supplemented with tomato pomace meal in order to enrich the nutritional status of the final product. in previous studies, tomato pomace was reported to have a protein content of 16.2719.65%, oil content of 5.85-10.75%, dietary fiber content of 54.79-59.03% and ash content of 3.472-4.046% (alvarado et al., 2001; del valle et al., 2006; isik, 2013). our results were mostly similar to those reported in the literature. the small differences can be due to several factors including climate, geography, geochemistry, agricultural practices like fertilization and genetic composition (toledo and burlingame, 2006). tomato pomace powder had higher a and b values in color than wheat flour, which can be explained by higher carotenoid contents of tomato pomace, especially lycopene (sharma and le maguer, 1996; schieber et al., 2001; knoblich et al., 2005; sikora et al., 2008). lycopene is an important carotenoid which gives red color to tomato, and lycopene content of tomato peel is about 3025 μg/100 g (sharma and le maguer, 1996; sikora et al., 2008). 3.1. effect of tomato pomace addition on the proximate chemical composition of crackers ash, crude protein, soluble, insoluble and total dietary fiber, and mineral contents of soda crackers increased significantly (p < 0.05) with tomato pomace powder addition (table 3). the reason is most likely that tomato pomace meal, which substituted wheat flour in cracker production, had higher crude protein, dietary fiber, crude ash and mineral contents than wheat flour (table 2).   ital. j. food sci., vol 28, 2016 530 table 2: chemical and nutritional properties of wheat flour and tomato pomace meal. parameter wheat floura tomato pomace meal crude protein (%) 10.87 16.31 crude oil (%) 1.66 5.38 total dietary fiber (%) 2.89 59.94 soluble dietary fiber (%) 1.39 4.91 insoluble dietary fiber (%) 1.50 55.03 crude ash (%) 0.480 3.492 mg (ppm) 398.3 2850.6 ca (ppm) 380.4 3625.5 k (ppm) 1950.4 24500.3 p (ppm) 1403.2 4625.1 mn (ppm) 9.5 40.1 zn (ppm) 12.5 41.5 fe (ppm) 19.1 130.5 total phenolic compounds (mg gae/ 100g) 104.12 427.81 total antioxidant activity (µmol te/ 100g) 2.364 80.34 hunter color values l 94.43 54.95 a 0.44 16.12 b 9.20 19.65 aall values are in dry basis. a generous intake of dietary fiber may help to reduce risk for developing diseases such as coronary heart disease, stroke, hypertension, diabetes, obesity and certain gastrointestinal disorders such as constipation, diverticulitis and large bowel cancers (anderson et al., 2016; mudgil and barak, 2013). dietary fiber intake recommendations for adults generally fall in the range of 20 to 35 g/day or 10 to 13 g per 1,000 kcal energy intake (marlett et al., 2002). in this case, by the consumption of 100g of control, tp4, tp8 and tp12 crackers, an adult can take about 6.2%, 17.7%, 22.3% and 28.5% of his daily recommended dietary fiber intake, respectively. crackers containing tomato pomace powder at different ratios provide significantly higher dietary fiber intakes than control cracker for the consumers. minerals are essential for a wide variety of metabolic and physiologic processes in human body. they are useful for many actions in the body like muscle contraction, normal heart rhythm, nerve impulse conduction, oxygen transport, oxidative phosphorylation, enzyme activation, immune functions, antioxidant activity, bone health, and acid base balance of the blood (williams, 2005; saldamlı and sağlam, 2007; lakshmi, 2014). an adequate daily amount of minerals is necessary for optimal functioning.   ital. j. food sci., vol 28, 2016 531 table 3: chemical and nutritional properties of crackers supplemented with various amounts of tomato pomace powder. parameter ca,b tp4 tp8 tp12 crude protein (nx5.7) (%) 7.35±0.16c 7.63±0.08b 7.80±0.01ab 7.82±0.04a crude oil (%) 16.50±1.15a 16.75±0.29a 17.62±0.88a 18.06±0.97a total dietary fiber (%) 1.86±0.29d 5.30±0.55c 6.68±0.43b 8.54±0.58a soluble dietary fiber (%) 1.02±0.49b 1.82±0.19ab 2.01±0.22a 2.50±0.55a insoluble dietary fiber (%) 0.84±0.21d 3.48±0.41c 4.67±0.30b 6.04±0.42a crude ash (%) 1.269±0.192b 1.520±0.151ab 1.554± 0.109ab 1.693±0.120a mg (ppm) 185.0±39.6b 283.9±54.6ab 306.1±21.4ab 400.7±114.7a ca (ppm) 213.9±15.9c 356.2±15.0b 493.9±8.3a 569.9±89.4a k (ppm) 1491.1±109.2d 2108.9±41.9c 2738.7±140.5b 3090.0±132.7a p (ppm) 1871.8±164.6b 2123.1±157.1ab 2250.2±252.8ab 2314.2±147.2a mn (ppm) 9.7±1.7b 12.3±2.4b 14.4±1.6ab 23.0±8.5a zn (ppm) 8.8±2.6b 11.0±3.1b 17.8±2.5a 22.6±2.9a fe (ppm) 8.9±2.4c 12.6±2.5bc 15.8±1.0b 22.1±4.3a total phenolic compounds (mg gae/ 100g) 52.52±6.50d 68.59±3.67c 104.402±6.69b 127.585±9.26a total antioxidant activity (µmol te/ 100g) 7.20±0.97c 7.67±2.12c 13.23±2.11b 18.11±1.59a hunter color values l 68.55±1.82a 63.04±0.26b 57.17±0.40c 53.06±1.14d a 4.15±1.06d 7.28±0.27c 9.67±0.20b 10.87±0.20a b 21.10±0.90b 22.39±0.15a 23.08±0.12a 22.82±0.45a aall values are in dry basis; b different letters within the row across the table show significant differences at α=0.05. in this study, addition of tomato pomace meal to cracker formulation caused increases (p<0.05) in mineral contents (mg, ca, k, p, mn, zn and fe). substitution of wheat flour by tomato pomace powder in crackers at a level of 12% increased mineral levels in crackers between a ratio of 23.6% (for p) and 166.4% (for ca). according to our calculations, an adult can take about 15.45% of k, 5.70% of ca, 10.83% of mg, 28.93% of p, all of mn, 22.60% of zn and 24.55% of fe daily requirements by the consumption of 100g of tp12 cracker. on the other hand, he or she can take about 7.45% of k, 2.14% of ca, 5.00% of mg, 23.39% of p, 44.09% of mn, 8.80% of zn and 9.88% of fe daily requirements by the consumption of 100g of control cracker (baysal, 2007; saldamli and sağlam, 2007). 3.2. effect of tomato pomace addition on total phenolic compounds and total antioxidant ativity values in crackers the results in table 3 show that the substitution of wheat flour by tomato pomace meal powder increased the total phenolics contents and antioxidant activity values of crackers. cracker samples having all levels of tomato pomace powder had significantly (p<0.05)   ital. j. food sci., vol 28, 2016 532 higher total phenolics contents than control crackers. crackers having 8 and 12% of tomato pomace powder had significantly (p<0.05) higher antioxidant activity values than control crackers. the reason for these results is most likely that the tomato pomace meal had a higher total phenolics content and antioxidant activity value than wheat flour (table 2). indeed, tomato skins and seeds, which are the main portion of tomato pomace, include polyphenolic compounds primarily the quercetin, rutin, chlorogenic acid, naringenin and kaempferol (verhoeyen et al., 2002; sikora et al., 2008; navarro-gonzalez et al., 2011; kamiloglu et al., 2013). additionally, tomato skin, which presents the important portion of tomato pomace, is a rich source of lycopene (sharma and le maguer, 1996; schieber et al., 2001; knoblich et al., 2005) and lycopene is a carotenoid which has the highest antioxidant activity in common carotenoids (asicioglu, 2005; kamiloglu et al., 2013). lycopene is the most abundant carotenoid in tomatoes, accounting about 83% of the total pigments present, and is responsible for the bright red color of tomatoes (kamiloglu et al., 2014). tomato skin and seeds also contain other components which have high antioxidant activity primarily β-carotene as a carotenoid and vitamin c (knoblich et al., 2005; sikora et al., 2008; strati and oreopoulou, 2011). 3.3. effect of tomato pomace addition on physical and sensory properties of crackers addition of tomato pomace powder had a decreasing effect on l color value, and increasing effect on a and b color values (table 2). these are mostly due to their higher a and b, and lower l values of tomato pomace than wheat flour (table 2). these higher a and b color values of tp4 and tp8 were also liked more by the panelists in sensory evaluation (table 4). sensory evaluation results of soda crackers are presented in table 4. control crackers and crackers substituted with tomato pomace received similar (p>0.05) scores in color, smell, flavor, crispiness and overall acceptability. although increasing the substitution level of tomato pomace powder to 12% caused some reductions in the scores, this decrease was statistically insignificant. in the sensory evaluation, a number of panelists reported that tp12 crackers had a little bitterness taste (data not shown). it’s thought that this was most likely from a bitter component, named tfi, presented in tomato seeds. tfi is a furostanol saponin and it’s chemical structure was established as 5α-furostane-3β,22,26-triol-3-[o-β-d-glucopyranosyl (1→2)-β-d-glucopyranosyl (1→4)-β-d-galactopyranoside] 26-o-β-d-glucopyranoside by sato and sakamura (1973). table 4: results of sensory evaluation. cracker sample color smell flavor crispiness overall acceptability control 4.48±0.60 4.88±0.48 4.71±0.25 4.81±0.24 4.88±0.24 tp4 5.17±0.23 4.92±0.29 4.71±0.48 4.65±0.30 4.92±0.16 tp8 4.79±0.91 4.77±0.58 4.50±0.57 4.73±0.27 4.69±0.24 tp12 4.15±0.59 4.77±0.31 4.38±0.25 4.56±0.12 4.54±0.53   ital. j. food sci., vol 28, 2016 533 4. conclusions byproducts of tomato processing industries have been known as a good source of biologically active food components, but have a low economical value. in this research, potential use of tomato pomace powder in human diet was studied and successful results were obtained. tomato pomace powder addition increased the crude protein, soluble, insoluble and total dietary fibers, mineral, total phenolic contents and total antioxidant capacity of crackers. crackers having tomato pomace powder had higher a and b color values than control, and colors of tp4 and tp8 had higher scores in sensory analysis in spite of the difference with others was insignificant (p>0.05). panelists liked crackers equally in terms of color, smell, flavor, crispiness, and overall acceptability. but results of sensory evaluation indicated that substitution of wheat flour higher than 12% by tomato pomace powder in the production of soda crackers is not recommended. acknowledgements this work was funded 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(2005). dietary supplements and sports performance: minerals. j. int. soc. sports nutr. 2(1), 43. yoneya t. ve nip w.-k. 2006. cracker manufacture. ch. 23. in: bakery products, science and technology. y.h. hui (ed.), p. 411. blackwell publishing, iowa 50014, usa. paper received may 1, 2016 accepted may 10, 2016 #536_benjakul_bozza ital. j. food sci., vol 29, 2017 145 paper a comparative study of the physicochemical properties and emulsion stability of coconut milk at different maturity stages u. patil1, s. benjakul*1, t. prodpran2, t. senphan3 and n. cheetangdee1 1department of food technology, faculty of agro-industry, prince of songkla university, hat yai, songkhla, 90112, thailand 2department of material product technology, faculty of agro-industry, prince of songkla university, hat yai, songkhla, 90112, thailand 3faculty of engineering and agro-industry, maejo university, sansai, chiangmai, 50290, thailand *corresponding author. tel.: +66 74286334; fax: +66 74558866 e-mail address: soottawat.b@psu.ac.th abstract based on chemical analysis, mature coconut (mc) milk had the highest moisture content (p<0.05), followed by immature coconut (imc) and overlay mature coconut (omc) milk, respectively. omc milk had the highest lipid content while imc milk showed the lowest lipid content (p<0.05). the lowest protein and carbohydrate contents were found in mc milk (p<0.05). cocosin with mw of 55 kda was observed as the major protein in all coconut milks; however, the band intensity slightly decreased with increasing maturity stages. increase in oil droplet size was observed with increasing maturity stages. therefore, maturity stages have an influence on the chemical compositions, properties and emulsion stability of coconut milk. keywords: coconut milk, droplet size, emulsion stability, physicochemical properties, protein pattern ital. j. food sci., vol 29, 2017 146 1. introduction coconuts (cocos nucifera) are extensively used in many traditional foods of the asian and pacific regions (onsaard et al., 2005). coconut milk is commonly used in several cuisines such as curries and desserts (tansakul and chaisawang, 2006). it contains high amounts of medium chain saturated fatty acids (mcfas), especially lauric acid (raghavendra and raghavarao, 2010). lauric acid is converted into a very valuable compound known as monolaurin, which has antiviral and antibacterial properties. the consumption of coconut milk may help to protect the body from infections (debmandal and mandal, 2011). coconut milk is a milky white oil-in-water emulsion extracted from grated coconut meat with or without the addition of water. the emulsion in coconut milk is naturally stabilised by coconut proteins (globulins and albumins), as well as phospholipids (raghavendra and raghavarao, 2011). the major protein (∼65%) in coconut endosperm is an 11s globulin known as cocosin with a molecular weight (mw) of 55 kda (garcia et al., 2005), and is believed to play a significant role in stabilising the coconut milk emulsion (tangsuphoom and coupland, 2008). generally, both intrinsic factors (e.g. protein compositions, etc.) and environmental conditions (e.g. ph, temperatures, etc.) can affect the stability of the coconut milk emulsion (raghavendra and raghavarao, 2010). on the other hand, the instability of the coconut milk emulsion is required for the production of virgin coconut oil (vco). in recent years, coconut milk is immensely used for the extraction of vco. moreover, vco has gained much popularity in the scientific community due to the presence of mcfas, its high degree of saturation and good stability. it can be obtained by breaking the emulsion of coconut milk using different extraction methods (raghavendra and raghavarao, 2010). thus, to maximise the yield of vco, coconut milk emulsion must be destabilised to a high degree, so that oil can be released and separated effectively the quality and stability of coconut milk emulsion could be governed by intrinsic factors, especially at different maturity stages. however, no information exists regarding the influence of maturity stages on the characteristics and emulsion stability of coconut milk. a better understanding of the physicochemical properties and emulsion stability of coconut milk at different maturity stages could be beneficial in the manufacturing of vco with prime quality and high yield. therefore, this comparative study was carried out to evaluate the physicochemical properties and emulsion stability of milk obtained from coconut at three different maturity stages. 2. materials and methods 2.1. chemicals sodium hydroxide, boric acid and nile blue a were purchased from sigma (st. louis. mo, usa). sodium dodecyl sulphate and isooctane were obtained from merck (darmstadt, germany). methanol, ethanol, acetic acid, chloroform, petroleum ether, hydrochloric acid, sulphuric acid, n-hexane and cyclohexane were procured from lab-scan (bangkok, thailand). chemicals for electrophoresis were obtained from biorad (richmond, va, usa) and protein molecular weight marker was procured from ge healthcare (buckinghamshire, uk). ital. j. food sci., vol 29, 2017 147 2.2. preparation of coconut meat and coconut milk coconuts at three different maturity stages including immature coconut (imc) (9-10 months old from pollination), mature coconut (mc) (11-12 months old from pollination) and overlay mature coconut (omc) (14-15 months old from pollination) were purchased from a plantation site in yaring district, pattani province, thailand and transported to the department of food technology, prince of songkla university, hat yai, songkhla. coconuts were subjected to deshelling, paring and removal of water. coconut kernel was collected manually and grated using a rotary wedge cutter machine. to prepare coconut milk, the grated coconut meat was pressed using a hydraulic press machine (model stainless steel hydraulic press a2, sakaya, bangkok, thailand) with a maximum pressure of 10.35 mpa for 2 min. thereafter, coconut milk was collected and analysed. 2.3. proximate analysis of coconut meat and coconut milk coconut meat and coconut milk at three different maturity stages were analysed for moisture, ash, lipid and protein contents according to the method of aoac (aoac, 2000). the protein content was calculated using 6.25 (as the factor) and the carbohydrate content was calculated as the difference from the sum total of the aforementioned proximate analysis components. the values were expressed as g/100 g (wet weight basis). 2.4. colour determination coconut milk colour was measured using a colourimeter (hunterlab, model colourflex, va, usa). the colour was reported as l*, a*, b* values, indicating lightness, redness/greenness and yellowness/blueness, respectively. total difference in colour (∆e*) and the difference in chroma (∆c*) were also calculated using the following equations: δe∗ = (∆𝐿∗)! + (∆𝑎∗)! + (∆𝑏∗)! where ∆l*, ∆a* and ∆b* are the differences between the corresponding colour parameter of the sample and the white standard (l* = 93.55, a* = 0.84, b* = 0.37). δc∗ = 𝐶!"#$%& ∗ − 𝐶!"#$%#&% ∗ where c∗ = (𝑎∗)! + (𝑏∗)! 2.5. ph measurement a digital ph meter (eutech, ph700 thermo scientific, usa) was used to measure the ph values of coconut milk. 2.6. sdspolyacrylamide gel electrophoresis (sds-page) the protein patterns of coconut milk were determined by sds-page, according to the method of laemmli (1970), using 4% staking gel and 12% separating gel. the coconut milk samples (10 ml) were homogenised with 10 ml of 50 g/l sds at a speed of 12,000 rpm for 1 min. the homogenate was heated at 95°c for 1 h, followed by centrifugation at 7000 × g for 10 min at 25°c using a centrifuge (beckman coulter, allegra™ centrifuge, ca, ital. j. food sci., vol 29, 2017 148 usa). the protein concentration of the supernatant was determined by the biuret method (robinson and hogden, 1940), using bovine serum albumin (bsa) as a standard. the prepared samples were mixed with sample buffer containing 2% sds, 10% glycerol and 0.05% bromophenol blue in 0.5 m tris-hcl, and the resulting solution had a ph of 6.8. under reducing condition, β-mercaptoethanol was added to the sample buffer in order to obtain a final concentration of 5% and the mixtures were heated at 95°c for 3 min prior to loading. the prepared mixtures (20 µg protein) were loaded onto the gel. electrophoresis was performed using a vertical gel electrophoresis unit (mini-protein ii; bio-rad laboratories, richmond, va, usa) at a constant voltage of 20 ma/gel. after electrophoresis, the gels were stained with 0.5 g/l coomassie brilliant blue r-250 in 500 ml/l methanol and 75 ml/l acetic acid for 30 min. finally, they were destained with a mixture of 500 ml/l methanol and 75 ml/l acetic acid for 30 min and destained again with a mixture of 50 ml/l methanol and 75 ml/l acetic acid for 1 h. the relative mobility (rf) of proteins was calculated and their molecular weight was estimated from the plot between rf and log (mw) of standards. 2.7. microstructure determination of oil droplets 2.7.1 confocal laser scanning microscopy (clsm) the microstructures of coconut milk samples were examined with a confocal laser scanning microscope (clsm) (model fv300; olympus, tokyo, japan.). the samples were dissolved in nile blue a solution (1:10) and manually stirred until uniformity was obtained. fifty microlitres of sample solutions were smeared on the microscope slide. the clms was operated in the fluorescence mode at excitation and emission wavelengths of 533 and 630 nm, respectively; using a helium neon red laser (hene-r) for lipid analysis. a magnification of 400x was used. 2.7.2 phase contrast microscopy oil droplets in coconut milk were observed under a phase contrast microscope (model ix50; olympus, tokyo, japan) equipped with camera. samples were placed on a glass slide, covered with cover slip and observed at 400x magnification. 2.7.3 determination of particle size the particle size distribution of coconut milk emulsion was determined using a laser particle size analyser (lpsa) (model ls 230, beckman coulter®, fullerton, ca, usa) as per the method of castellani et al. (2006). prior to analysis, the sample (5 ml) was diluted with 1 ml sodium dodecyl sulphate (sds) in order to dissociate flocculated droplets. the surface-weighted mean particle diameter (d32) and the volume-weighted mean particle diameter (d43) of the emulsion droplets were measured. 2.7.4 determination of coalescence and flocculation coconut milk samples were diluted with distilled water in the presence and absence of sds. the coalescence index (ci) and flocculation factor (ff) were calculated using the following equations (intarasirisawat et al., 2014): d43˗sds d43+sds ff= ital. j. food sci., vol 29, 2017 149 (d43+sds,t d43+sds,in) d43+sds,in where d43+sds and d43-sds are the volume-weighted mean particle diameter of the emulsion droplets in the presence and absence of sds, respectively; d43+sds, in and d43+sds, t are the volume-weighted mean particle diameter of the emulsion droplets in the presence of sds at time 0 and the designated storage time (24 h), respectively. determination was conducted at room temperature (28-30°c). 2.7.5 statistical analysis experiments were carried out in triplicate using three different lots of samples. data were subjected to analysis of variance (anova). comparison of means was carried out by duncan’s multiple range test. for paired comparison, t-test was used (steel and torrie, 1980). statistical analysis was performed using the statistical package for social science (spss 11.0 for windows, spss inc., chicago, il, usa). 3. results and discussion 3.1. proximate compositions of coconut meat and milk the proximate compositions of coconut meat and milk at three different maturity stages are shown in table 1. mc meat had the highest moisture content (61.07 g/100 g), followed by imc (53.94 g/100 g) and omc (39.50g/100 g), respectively. a similar trend was observed in coconut milk, in which mc had the highest moisture content (61.55 g/100 g), followed by imc (55.36 g/100 g) and omc (36.59 g/100 g), respectively. the high moisture content of mc meat and milk was more likely due to the absorption of coconut water inside the endosperm, since the beginning of germination. water uptake is an essential step towards germination (bewley and black, 1994). on the other hand, low moisture content was observed in omc meat and milk. the result suggested that the absorbed water in the endosperm was utilised during embryo development (bewley and black, 1994). it was found that there was a general increase of lipid content in coconut meat and milk with increasing maturity. omc meat and milk were found to have the highest lipid content (p<0.05). the lipid content of coconut increased with maturity stage due to the accumulation of lipids in the endosperm (lópez‐villalobos et al., 2001). lower protein content was observed in both mc meat and milk as compared with those of imc and omc (p<0.05). cocosin is a reserve protein found in coconut endosperm and serves as nitrogen source during germination (balasundaresan et al., 2002). the result suggested that proteins could be degraded and utilised at the beginning of germination. since water was utilised during the germination of omc, the proportion of proteins was slightly increased. the ash content of both meat and milk decreased with maturity. the ash contents are indices of the mineral content (obasi et al., 2012). it has been reported that coconut water contains sugars, vitamins, minerals, amino acids and phytohormones(yong et al., 2009). the decrease in ash content in omc meat and milk suggested that minerals are more likely used up during the germination process. a significantly lower carbohydrate content was observed in mc meat and milk (p<0.05). the obtained results are in accordance with that of jeganathan (1970) who found that coconut milk at the mature stage, had a carbohydrate content of 5.5%. white et al. (1989) ci= × 100 ital. j. food sci., vol 29, 2017 150 found that coconut milk at the mature stage is composed predominantly of galactose and arabinose with a small amount of mannose and glucose. balasubramaniam (1976) reported that galactomannans and cellulose are present in the kernel of maturing and matured coconuts, whereas mannans are almost absent from very immature kernel and increased with maturation. endosperm rich nutrients appear to function as a food reservoir for embryo development (balasundaresan et al., 2002). thus, reserved materials, particularly carbohydrates, were degraded and utilised during maturity. the decrease in carbohydrate plausibly led to the increased proportion of lipid in mc, as compared with the imc sample. the results revealed that different maturity stages had marked impact on the chemical composition of coconut meat and milk. table 1. proximate composition of coconut meat and milk at three different stages of maturity. content (g/100 g) coconut meat coconut milk imc mc omc imc mc omc moisture 53.94±0.60b 61.07±1.02a 39.50±0.82c 55.36±0.15b 61.55±0.13a 36.59±1.05c lipid 18.59±0.89b 20.86±0.95b 32.45±0.35a 17.28±1.46c 30.34±0.96b 44.20±0.85a protein 4.79±0.16a 3.95±0.09b 4.45±0.56a 3.35±0.29a 2.90±0.06b 3.34±0.49a ash 1.15±0.02a 1.14±0.04a 1.04±0.03b 1.03±0.05a 1.00±0.04a 0.80±0.03b carbohydrate 21.53±0.98a 13.05±0.95b 22.34±0.85a 22.98±1.21a 4.21±0.93c 15.07±1.63b imc: immature coconut, mc: mature coconut and omc: overlay mature coconut. values are presented as mean±sd (n=3). different lowercase letters in the same row, within the same commodity, indicate significant difference (p<0.05). 3.2. colour of coconut milk l*, a* and b* values of coconut milks at three different stages of maturity are shown in table 2. the coconut milks were milky white in colour as evidenced by high l*-value (lightness). in general, coconut milk is an oil-in-water emulsion, where oil droplets are dispersed in the water phase. light scattering of oil droplets is mostly associated with the white colour of coconut milk. all samples had low a* and b*values, suggesting that deterioration did not occur in all samples. it was observed that ∆e* and ∆c* decreased with increasing maturity stages, where imc had the highest values, followed by mc and omc samples, respectively. the highest l*value was found in the mc sample (p<0.05). the turbidity, cloudiness, or opaque appearance of emulsion is dependent on light scattering which is mediated by the dispersed oil droplets (mcclements, 2002). table 2. colour and ph of coconut milk at three different stages of maturity. samples l* a* b* δe* δc* ph imc 92.90±0.24a 0.10±0.05c 5.09±0.06a 4.83±0.05a 4.09±0.06a 7.00±0.01a mc 94.86±1.83c -0.28±0.04b 4.48±0.12a 4.48±0.13b 3.84±0.12b 6.39±0.02b omc 93.09±0.18b -0.35±0.03a 4.22±0.08b 3.88±0.08c 3.23±0.08c 5.58±0.13c imc: immature coconut, mc: mature coconut and omc: overlay mature coconut. values are presented as mean±sd (n=3). different lowercase letters in the same column indicate a significant difference (p<0.05). ital. j. food sci., vol 29, 2017 151 lightness is not only determined by lipids or oils, but also by the size of oil droplets, which is another prime factor governing the colour, particularly the lightness of coconut milk. some differences in indigenous pigments present in coconut milk of different maturity stages were also presumed. 3.3. ph of coconut milk the phs of freshly prepared coconut milks at three different stages of maturity are shown in table 2. imc milk was found to have a ph of 7.0 while mc milk was slightly acidic in ph (ph 6.39). the lowest ph (5.58) was obtained in omc milk (p<0.05). reserved food materials such as proteins, carbohydrates and lipids provide nourishment to growing embryo (samson et al., 1971). the breakdown of these stored food materials by some enzymes, possibly occurred for embryo development. acidic metabolites or degradation products such as acidic amino acids may contribute to the lowered ph. the ph of 5.58 found in omc milk is close to the isoelectric point of coconut proteins (pi = 4-5) (samson et al., 1971; monera and del rosario, 1982; kwon et al., 1996). therefore, ph may affect the emulsifying properties of proteins in coconut milk, especially those stabilising oil droplets in the aqueous phase. 3.4. electrophoretic patterns of coconut milk proteins the protein patterns of coconut milks at different stages of maturity under reducing and non-reducing conditions are shown in fig. 1. under non-reducing condition, there were six protein bands with mw of 55, 46, 33, 25, 18 and 16 kda. the major protein in coconut endosperm is 11s globulin, which is referred to as cocosin, with mw of 55 kda (garcia et al., 2005). a hexamer (55 kda) consists of acidic (32-34 kda) and basic (22-24 kda) subunits, which are linked by a disulphide bridge (garcia et al., 2005; tangsuphoom and coupland, 2008). some proteins present in the aqueous phase of coconut milk could act as emulsifier to stabilise fat globules (peamprasart and chiewchan, 2006). cocosin plays a prominent role in regulating the stability of coconut milk (tangsuphoom and coupland, 2008). in the present study, smaller bands of proteins with mw higher than 55 kda were found. bands with higher intensity were found in imc as compared with others. in the omc sample, protein bands with mw of 70 and 46 kda almost disappeared, indicating the degradation of proteins during germination. under the reducing condition, several major protein bands with mw of 55, 33, 31, 25, 21, 20, 18 and 16 kda were observed, which are in agreement with previous reports of garcia et al. (2005) and demason and sekhar (1990). under the reducing condition, cocosin dissociated into acidic and basic polypeptides with the coincidental appearance of proteins with mw of 32 and 22 kda. other proteins with mw greater than 55 kda were not found. the occurrence of protein with mw of 20 kda was also observed under reducing conditions. protein with mw of 36 kda was found only in the mc sample. in general, the omc sample showed the lower band intensity of most proteins as compared with others. the result confirmed that the protein composition of coconut milk changed with maturity stages. ital. j. food sci., vol 29, 2017 152 figure 1. sds-page patterns of coconut milk proteins with three different maturity stages. a: low molecular weight standards; b: high molecular weight standards; imc: immature coconut, mc: mature coconut and omc: overlay mature coconut. 3.5. microstructure of oil droplets in coconut milk the microscopic structures of coconut milk emulsions at three different stages of maturity were visualised by confocal laser scanning microscopy (clsm) and phase contrast microscopy (fig. 2). in the same coconut milk sample, similar results were observed when both clsm and phase contrast microscopies were used. clsm generally provides higher clarity and better resolution images of the emulsion microstructure than convention optical microscopy. the observation of the microstructure of the emulsion was facilitated using a fluorescence dye such as nile blue a, in order to label the lipid. however, phase contrast microscopy provides excellent contrast, and a halo is formed even around a small oil droplet. for imc (fig. 2a), smaller oil droplets with uniform shape and size were distributed uniformly in the aqueous phase. an emulsion with the same size of oil droplets is referred to as a monodisperse emulsion, whereas that containing a range of droplet sizes is referred to as a polydisperse emulsion (mcclements, 2004). coconut milk is an oil-in-water emulsion naturally stabilised by coconut proteins (birosel et al., 1963). in the present study, imc had higher protein/lipid ratio as compared with the mc sample (table 1). high protein content can lead to efficient localisation of protein films at the oil-water interphase. thus, this could increase the emulsion stability of coconut milk. moreover, proteins could stabilise the coconut milk emulsion by lowering the interfacial tension between two phases, in which oil droplets are dispersed uniformly throughout the water phase. however, polydisperse emulsion was observed in mc and omc (figs. 2b and c) with a wide range of oil droplet sizes. large sizes of oil droplets were abundantly observed in the omc. in omc, coconut milk contained a high amount of lipid. thus, the present proteins may not be sufficient to stabilise the emulsion. the low ph of omc milk could be another factor enhancing the destabilisation of emulsion, by lowering the repulsion of protein film surrounding the oil droplets. in general, the emulsion was less stable as evidenced by the larger droplets with non-uniform distribution. the results clearly indicated that maturity stages affected oil droplet size. ital. j. food sci., vol 29, 2017 153 figure 2. confocal laser scanning micrographs (i) and phase contrast microscopy (ii) of coconut milk at three different stages of maturity: (a) imc: immature coconut; (b) mc: mature coconut; (c) omc: overlay mature coconut. magnification: 400×. scale bar = 50 μm. ital. j. food sci., vol 29, 2017 154 3.6. particle size distribution particle size distributions expressed as d32 and d43 of coconut milk emulsions at three different stages of maturity are shown in table 3. the d32 increased from 3.38 µm (imc) to 5.48 µm (omc), while d43 increased from 5.29 µm (imc) to 13.38 µm (omc) with increasing maturity stages. coconut milk from omc contained the largest oil droplets (d43 and d32), followed by those from mc and imc. the d32 is related to the average surface area of droplet exposed to the continuous phase per unit volume of emulsion. the smaller d32 indicates higher specific surface area (intarasirisawat et al., 2014). the d43 is the sum of the volume ratio of droplets in each size class multiplied by the mid-point diameter of the size class. the d43 can be used as the index of coalescence and flocculation (hebishy et al., 2013). the proteins in coconut milk are known to function as emulsifier, which stabilises the oil droplets in coconut milk (dionisio, 1963; monera and del rosario, 1982). the largest size of oil droplets in omc can be attributed to the ph of the omc milk, close to pi. as a result, there was a decrease in repulsion between protein films surrounding the oil droplets, thereby facilitating the coalescence. when the repulsive forces dominate, the droplets tend to remain as individual entities (mcclements, 2004) and form a stable emulsion. after 24 h of storage, both d32 and d43 increased (table 3), indicating the coalescence of oil droplets. among all samples, a slight increase in d32 and d43 were observed in the omc sample. the result suggested that the collapse of emulsion in the omc milk was pronounced at the initial time and less coalescence occurred after 24 h. conversely, the emulsion collapsed continuously in the mc and imc samples. table 3. droplet size and stability of coconut milk at three different stages of maturity. samples storage time (h) d32 (µm) d43 (µm) ff ci imc 0 3.38±0.10c 5.29±0.20c 1.18±0.03a 24 4.16±0.40b 8.10±0.10c 0.77±0.04b 53.22±0.59a mc 0 5.07±0.25b 12.31±0.15b 0.90±0.90a 24 5.55±0.60a 14.22±0.21a 0.78±0.03b 15.52±0.89b omc 0 5.48±0.13a 13.38±0.03a 1.26±0.04a 24 5.66±0.26a 13.44±0.01b 1.26±0.01a 0.45±0.50c ff: flocculation factor, ci: coalescence index. imc: immature coconut, mc: mature coconut and omc: overlay mature coconut. values are presented as mean±sd (n=3). different uppercase letters in the same column at the initial storage time (0 h) indicate significant difference (p<0.05). different lowercase letters in the same column after the designated storage time (24 h) indicate significant difference (p<0.05). 3.7. coalescence and flocculation the coalescence index (ci) and flocculation factor (ff) of the coconut milk emulsions were investigated to determine the instability of the emulsion as shown in table 3. emulsions are thermodynamically unstable due to the unfavourable contact between oil and water (fredrick et al., 2010) and their physical structures are likely to change over time by various mechanisms including coalescence and flocculation. in imc, higher ci was observed after 24 h, as compared with the mc and omc samples (p<0.05). on the other ital. j. food sci., vol 29, 2017 155 hand, ff decreased, suggesting that the individual oil droplets assembled to form larger oil droplets as evidenced by the increase in droplet size. the increase in d43 also reconfirmed the assembly of individual droplets into larger flocs (intarasirisawat et al., 2014). the formation of larger oil droplets indicates poor emulsion stability (fredrick et al., 2010). the interactions between oil droplets depend on the quality and quantity of proteins (damodaran, 2005). the proteins in imc were plausibly not effective in stabilising the coconut milk emulsion, especially after the extended storage. however, the lowest rate of ci was observed for omc. this coincided with the lowest rate of changes in d32 and d43 of omc. for omc, initial ph close to pi might not favour the solubility of proteins and as such, it was presumed to have poor emulsifying property. additionally, the partial crystallization of lipid within the oil droplets could be another factor that favours the destabilisation of emulsion by coalescence (rousseau, 2000). nevertheless, the emulsion initially found was slightly altered upon storage time. the results suggested that the oil droplet size and emulsion stability of coconut milk depended on the maturity stages. 4. conclusions coconut milk and meat at three different maturity stages had varying proximate compositions. cocosin with mw of 55kda was predominantly observed in coconut milks, regardless of the maturity stage. polydisperse emulsion was observed in coconut milk at the mature and overlay mature stages, whilst the monodisperse counterpart was found in coconut milk of the immature stage. the stability of coconut milk emulsion depends on intrinsic factors, mainly ph and protein content. thus, the maturity stages had influence on the physicochemical properties and emulsion stability of coconut milk. the present study has provided a better understanding of the impact of maturity stage on the characteristics and emulsion stability of coconut milk, used as the starting material for vco production. omc was more appropriate for the production of vco with higher yield as compared with other stages. acknowledgements this study was supported by thailand's education hub for the southern region of asean countries (teh-ac, 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s. and raghavarao k. 2010. effect of different treatments for the destabilization of coconut milk emulsion.j. food eng. 97:341-347. raghavendra s. and raghavarao k. 2011. aqueous extraction and enzymatic destabilization of coconut milk emulsions.j. am. oil chem. soc. 88:481-487. robinson h.w. and hogden c.g. 1940. the biuret reaction in the determination of serum proteins. 1. a study of the conditions necessary for the production of a stable color which bears a quantitative relationship to the protein concentration.j. biol. chem. 135:707-725. rousseau d. 2000. fat crystals and emulsion stability a review. food res. int. 33:3-14. samson s., cater c. and mattil j. 1971. preparation and characterization of coconut protein isolates.cereal chem. 48:182190. steel r.g. and torrie j.h. 1980. “principles and procedures of statistics”.2nd ed. mcgraw-hill, new york. ital. j. food sci., vol 29, 2017 157 tangsuphoom n. and coupland j. n. 2008. effect of surface-active stabilizers on the microstructure and stability of coconut milk emulsions. food hydrocoll. 22:1233-1242. tansakul a. and chaisawang p. 2006. thermophysical properties of coconut milk. j. food eng. 73:276-280. white a.r., elmore h.w., watson m.b. and gill j.p. 1989. purification and partial characterization of polysaccharides from coconut milk. ann. bot. 64:205-209. yong j.w., ge l., ng y.f. and tan s.n. 2009. the chemical composition and biological properties of coconut (cocos nucifera l.) water.molecules. 14:5144-5164. paper received june 19, 2016 accepted september 3, 2016 #570_prusova_bozza ital. j. food sci., vol 29, 2017 222 paper a study on the content of terpenic compounds in the cultivar ‘moravian muscat’ (vitis vinifera l.) m. baron, j. sochor, b. prusova*, l. tomaskova and m. kumsta department of viticulture and enology, faculty of horticulture, mendel university in brno, valtická 337, cz-691 44 lednice, czech republic *corresponding author. tel.: +420 519367259; fax: +420 519367222 e-mail address: prusova.bozena@email.cz abstract terpenic represent a very interesting group of aromatic substances. they occur in aromatic grapevine cultivars and create muscat and flower aromas. in 2013, their contents as well as contents of other aromatic substances were determined in juices made of the moravian muscat cultivar after 5, 12 and 24 of maceration. pedigree: muscat ottonel x prachtraube. as control, fresh juice analysed immediately after the pressing was used. the aim of this experiment was to find out which terpenic compounds are present in grapes of this cultivar and which length of maceration would be the most suitable for making wine. attention was paid also to levels of monoterpens, phenols, ethyls, alcohols and acids as well as to basic analytical parameters. results of a chemical analysis indicated that an optimum is the maceration of grapes for 24 hours because after this time interval the highest amount of terpenic substances was released. keywords: terpenes, maceration, antioxidant activity, volatile compounds, analytical parameters ital. j. food sci., vol 29, 2017 223 1. introduction terpenic compounds are members of an important group of aromatic compounds and are characterized by floral, muscatel or fruity aromas that are synthesized in berries and stored in their skin. this aroma of a wine depends on its content of volatile compounds, over 680 of which have been identified in wines from some white grape varieties (peinado et al., 2004). although terpenes mainly give off a pleasant aroma some of them may show a negative effect on quality of wine. so, for example, yeast of the genus streptomyces may synthetize sesquiterpenes either on cork or in the barrel. their presence may jeopardize the sensory quality of wine (jackson, 2008). the aroma of young wines is the product of a biochemical and technological sequence. its formation derives from the grapes and juice production (grape de-stemming, crushing, and pressing technology), and is decisively influenced by the fermentation procedure. all of these parameters will determine the complexity of the wine aroma. (tao et al., 2008) its quality and quantity influenced by the cultivar, soil, climate and viticultural practices. (ribéreau-gayon et al., 2006, santiago et al., 2011) the experiment monitored the occurrence of terpenic compounds in juice samples after different periods of maceration. terpenes contribute to some white wines aroma, especially these produced from muscat grapes and others aromatic ones of high terpene contents (gewürtztraminer, traminer, huxel, sylvaner). (dziadas and jeleń, 2010) the variety which was chosen is 'muscat moravsky'. pedigree: muscat ottonel x prachtraube. it was chosen for its good reputation in the south moravia (czech republic). there are many preconcentration techniques to obtain volatile compounds, such a terpenes, subsequently analyzed by gas chromatography. the most widely used are headspace sorptive extraction techniques (hsse), such a spe or spme, purge and trap technique – sample is stripped by inert gas, which is concentrated in sorption column, steam distillation, continuous distillation and extraction. all this preconcentration techniques needs expensive equipment, or their performance needs high temperature, which can lead to terpene cyclization. technique liquid-liquid extraction (lle) used in this experiment is easy to perform, even it use high temperature. for purpose of this experiment, where we compare the length of maceration of similar samples is this sample preparation sufficient. the product of terpene cyclization is αterpinol, so in each sample we can compare its increasing. the aim of this experiment was to found out if (and to which extent) the length of maceration shows an effect on the increase in the content of terpenic and other compounds in produced wine. terpenes, because of their high concentrations and low aroma thresholds, are the principal components responsible for the characteristic aroma of a wine (carballeira lois et al., 2001). 2. materials and methods 2.1. sampling site and procedure grapes used in the experimental part of this study originated from vineyards situated in the locality velké bílovice (wine-growing subregion of velké pavlovice). in this region, the average annual precipitations and temperature are 550 mm and 9.5 °c, respectively. phenological data of the grapes and vine in year 2013: shooting: (bbch05) – 25.4.2013, full antesis: (bbch 65) – 12.6.2013, verasion: (bbch 81) – 9.8.2013, ripening: (bbch 88) – 11.9.2013. ital. j. food sci., vol 29, 2017 224 rootstock cr2 typical for czech republic was used. the czech grape training is modificated german training specially rhone_hessen, nevertheless fruitfull wood is horizontal tying. clasps planting: 1x1.2 mb. high of trunk is 0.75 m. berries was collected random from the top, middle, and bottom of selected clusters. in order to obtain representative sample, colored berries was not favoured over greens. berries were stored in a sealed plastic bags in the refrigerator until processing. 2.2. experimental variants of maceration and processing of samples in this experiment, grapes of the variety moravian muscat' were used. these grapes were harvested on the 11 september 2013. their sugar content was 19 °nm (i.e. 19 kg of natural sugars in 100 liters of juice). harvested grapes were crushed and destalked in a stainless destalking-crushing machine, macerated for 0 (variant 1); 5 (variant 2); 12 (variant 3) and 24 hours (variant 4) at the temperature of 14 °c, and finally pressed. wine was made from each juice sample and used for the estimation of the following parameters: ph and contents of alcohol, titratable acidity and sugars, respectively. in individual wine samples, the content of aromatic compounds was estimated as well. 2.3. estimation of total titratable acidity (eec no 2676/90) the content of total titratable acidity was estimated by titration in an automatic titrator titroline easy (manufacturer si analytics gmbh, germany). titrations were performed with naoh (0.1mol.l-1) as the titration reagent. . for analyses, a 10 ml sample was used; this sample was diluted with 10 ml of distilled water. because of a subsequent formol titration, the sample was not titrated up to the usual ph value of 7.0 but up to the value of 8.1 (the resulting deviation was thereafter considered to be a systematic error). the detection of ph was assured by means of a ph-electrode sentix 21. after the end of titration, the consumption of naoh solution in milliliters was read, with the accuracy of two decimal positions, on the titrator's display. the content of total titratable acidity (in g.l-1) was calculated as follows: the amount of consumed naoh solution was multiplied by the factor of the naoh solution used for the titration; the product of this multiplication was thereafter multiplied by and by the coefficient 0.75. 2.4. estimation of ph the ph value was estimated in an undiluted sample using a ph-meter wtw ph 526 and a ph electrode sentix 21 (both manufactured by the company wtw, germany). estimation of residual sugar and alcohol contents of residual sugar and alcohol were estimated in the apparatus alpha. the alpha apparatus is a compact ftir analyzer that uses the atr sampling technique. this technique helped to process samples before the analysis. before the first measurement, the spectrometer was thoroughly rinsed with deionized water and the background was determined using a blank sample (i.e. of deionized water). for analyses, 1 ml samples were taken with a syringe; of this sample, 0.5 ml was used for rinsing of the system while the remaining volume of 0.5 ml was analyzed three times. depending on the calibration used, the measured values were evaluated automatically using a special software. ital. j. food sci., vol 29, 2017 225 2.5. estimation of contents of aromatic compounds in berries by means of gas chromatography 2.5.1 preparation of samples contents of aromatic compounds present in berries were estimated after their extraction by an organic resolvent. in each sample, altogether 100 g of berries were mixed with 100 μl of 1m k2s2o5 solution (to prevent oxidation) and 10 μl of the gc internal standard. thereafter, the mixture was homogenised in a manual mixer and the juice was separated from the mush using a filter paper. the ph value of the juice was adjusted to 3.0 with 10 m h3po4. the adjusted juice was thereafter poured into a volumetric flask of the volume of 25 ml. the extraction was performed after the sample incubation in the boiling water bath for l hour. it is necessary to mention in this context that each heating results in a disintegration of glycosides and a release of aromatic compounds. the sample heating promote also the terpenic cyclization, but this preparation of sample is easy to perform and for purpose of this experiment is convenient. the product of terpene cyclization is αterpinol, so in each sample we can compare its increasing. the extraction was performed using 1 ml of methyl terc-butyl ether that contained 1 % of cyclohexane. after the separation, the phase of organic matter was dried up with anhydrous magnesium sulphate and used for the gc-ms analysis. 2.5.2 analysis of aromatic compounds by means of gas chromatography concentrations of individual volatile compounds in wine were determined according to until now unpublished method of extraction with methyl-t-butylether. into a 25-ml volumetric flask, 20 ml of wine was pipetted together with 50 µl of 2-nonanol solution in ethanol; this compound was used as an internal standard (in concentration of 400 mg·l-1) and 5 ml of a saturated (nh4)2so4 solution. the flask content was thoroughly stirred and thereafter 0.75 ml of the extraction solvent (mtbe with an addition of 1% cyclohexane) was added. after another thorough stirring and separation of individual phases, the upper organic layer was placed into a micro test tube together with the produced emulsion centrifuged and the clear organic phase was dried up with anhydrous magnesium sulphate. extract samples, adjusted in this way, were thereafter used for the gc-ms analysis. instruments: shimadzu gc-17a, autosampler: aoc – 5000, detector: qp-5050a, software: gcsolution. program: labsolutions, gc ms solution.version 1.20, conditions of separation: column: db-wax 30m x 0.25mm; 0.25μm stationary phase (polyethylene glycol). voltage of the detector 1.5 kv. individual compounds were identified on the base of ms spectrum and retention time using nist 107 library, which contains 107,886 spectra. 2.6. estimation of antioxidant activity by the dpph method 150 µl volume of the reagent (0.095 mm 2,2-diphenyl-1-picrylhydrazyl dpph•) was incubated with 15 µl of wine sample. the absorbance was measured at 505 nm for 10 minutes and the output ratio was calculated as a difference between absorbance values measured at the 10th minute and the 2nd minute of the assay procedure. 3. results and discussions for experiments, aromatic cultivar moravian muscat was used. the weight of 50 berries was 67.8 g at harvest time. the juice was quartered, each of these four parts was macerated ital. j. food sci., vol 29, 2017 226 for a different time interval and thereafter used for wine making. the following qualitative parameters were estimated in each part of experimental juice: weight of berries, sugar content, content of titratable acids, ph, content of yeast assimilable nitrogen and aromatic compounds. contents of aromatic compounds were estimated also in wine made from individual parts of berries. the aim of this experiment was to find out if the length of the maceration period influenced the content of terpenic substances in final wine product. 3.1. estimation of basic analytical parameters values of basic analytical parameters are presented in fig. 1. the lowest content of alcohol was determined in variant 1 (9.79 g.l-1); this value was also correlated with the highest level of residual sugars (8.55 g.l-1). the lowest content of residual sugars and the highest content of alcohol was found out in variant 4 (4.68 and 12.47 g.l-1, respectively). the highest and the lowest contents of total titratable acids (i.e. 12.47 g.l-1and 5.79 g.l-1) were was found out in variants 1 and 4, respectively. it this context it can be concluded that the longer the period of maceration, the lower the amount of total titratable acids in produced wine. this means that their contents decreased with the period of maceration. on the other hand, however, the ph value increased with the period of maceration. the highest ph (3.36) was found out in variant 4. 0 5 12 24 9 10 11 12 13 c on te nt o f al co ho l [ g/ l] 0 5 12 24 2.5 3.0 3.5 4.0 ph 0 5 12 24 5 6 7 8 c on te nt o f to ta l a ci ds [ g/ l] 0 5 12 24 4 6 8 10 c on te nt o f su ga r [g /l ] figure 1. a content of alcohol, b ph, c content of total acids, d content of sugar; in period of maceration: 0, 5, 12 and 24 hours. a b c d ital. j. food sci., vol 29, 2017 227 3.2. monoterpenes linalool is a naturally occurring terpene alcohol that has a characteristic floral scent with spicy and lemon tones. it can be found in the pulp of berries of muscat cultivars. its content does not change too much during the alcoholic fermentation. linalool is changed because its oxidation to α-terpineol that occurs in grapes only in smaller amounts. it can be identified only with difficulties and it does not influence the aroma of wine. the odour of nerol resembles roses and thyme but it is considered to be a little fresher. in the course of fermentation, the content of this monoterpene decreases and it is gradually changed to α-terpineol. it has a scent of roses and citrus fruit. a rose-like scent of this compound participates significantly in the aroma of muscat wines; however, it was not identified in other varieties. during the alcoholic fermentation, its content is slowly decreasing. in the course of wine ageing, geraniol is transformed to α-terpineol (jackson, 2008). in general, the content of monoterpenes increased in dependence on the duration of macerations. the content of linalool doubled within 24 hours of maceration. the content of ho-trienol increased from 78.6 to 155.5 μg.l-1 while that of α-terpineol rose only from 81.7 to 142.4 μg.l-1 and those of nerol and geraniol rose from 5 to 17.1 μg.l-1 and from 20 to 49.8 μg.l-1, respectively. results are presented in table 1. table 1. content of monoterpens. substance time of maceration 0 hours 5 hours 12 hours 24 hours avg. sd avg. sd avg. sd avg. sd linalool [μg.l-1] 362.40 7.49 381.25 5.73 491.63 7.48 724.21 8.48 ho-trienol [μg.l-1] 78.67 1.67 92.52 1.39 117.61 1.80 155.55 3.18 α-terpineol [μg.l-1] 81.73 0.47 89.17 2.03 85.00 2.55 142.41 3.73 ß-citronellol [μg.l-1] 2.96 0.07 4.98 0.03 5.02 0.03 8.05 0.09 nerol [μg.l-1] 5.08 0.10 5.92 0.07 8.03 0.12 17.17 0.29 geraniol [μg.l-1] 20.07 0.70 26.35 0.60 29.60 0.69 49.81 1.06 epoxylinalol 1 [μg.l-1] 129.25 3.03 144.96 4.40 187.47 4.27 346.00 3.46 epoxylinalol 2 [μg.l-1] 78.67 1.67 106.08 2.08 131.77 2.05 206.61 2.40 2,6-dimetyl-3,7-octadiene-2,6-diol [μg.l-1] 607.01 6.01 701.95 6.95 954.45 9.45 1181.55 17.81 3.3. phenols volatile phenolic substance are produced step by step in the course of wine making by means of enzymatic decomposition of phenolic acids present in berries. the principal compounds are the following: 4-vinylphenol, 4-vinylguaiacol, 4-ethylphenol and 4ethylguaiacol. in white wines, vinylphenols (4-vinylphenol and 4-vinylguajacol in concentrations ranging from 10 to 490 and from 70 to 1,150 μg.l-1, respectively) are predominating while in reds the principal compounds are ethylphenols (4-ethylphenol up to 6 000 μg.l-1). in case that the content of ethylphenol is higher than 400 μg.l-1 (sigma 4ethylphenol + 4-ethylguaiacol), the quality of wine may be negatively influenced; this is usually manifested as an unpleasant wine aftertaste resembling horse sweat (rapp and versini, 1996). the content of phenols is presented in table 2. ital. j. food sci., vol 29, 2017 228 the content of 4-vinylguaiacol increased in the course of the whole maceration period. after 24 hours, the initial value of 118.5 μg.l-1 increased to 24 as much as 292.9 μg.l-1. the content of 4-vinylphenol was fluctuating; however, the difference between the initial and final values, i.e. at the beginning and to the end of maceration (0 h and 24 h) was 992 μg.l-1. table 2. content of phenols. substance time of maceration 0 hours 5 hours 12 hours 24 hours avg. sd avg. sd avg. sd avg. sd methionol [μg.l-1] 0.39 0.01 2.58 0.03 1.48 0.03 0.51 0.01 2-methyltetrathio phen-3-on [μg.l-1] 0.68 0.02 0.90 0.04 0.59 0.01 0.51 0.01 4-vinylguajacol [μg.l-1] 118.58 2.42 208.03 4.43 267.96 6.03 292.85 4.41 4-vinylfenol [μg.l-1] 155.43 4.15 1029.88 15.47 653.60 14.90 1147.81 28.79 4-ethylguajacol [μg.l-1] 10.93 0.23 10.07 0.21 9.87 0.15 8.11 0.18 4-ethylfenol [μg.l-1] 16.94 0.26 16.11 0.49 13.13 0.13 12.16 0.18 1,1-diethoxyetan [μg.l-1] 565.00 22.60 211.15 4.94 630.00 12.60 677.49 7.77 3.4. alcohol and ethyls according to dennis (dennis et al., 2012) the content of precursors of acetate esters is dependent on their concentration in the juice and on the technology of processing of grapes. these precursors involve above all alcohols. their concentration increases with the duration of per-fermentation maceration. these precursors are thereafter transformed to aforementioned ethyls and these influence the aromatic profile of wine. the content of ethylacetate increased within the whole period of maceration, from the initial value of 32.6 mg.l-1 to that of 59.7 mg.l-1. contents of ethyl butyrate and ethyl octanoate increased by 150 and 120 μg.l-1, respectively. the content of isoamyl acetate increased by 92 % to the value of 7.6 μg.l-1. as far as 2-phenylethyl acetate was concerned, its content increased 6 times within the first 5 hours and thereafter decreased to the level that corresponded with 2.5 multiple of its initial concentration. the initial content of 1hexyl acetate was 261.7 μg.l-1and decreased to 43.7 μg.l-1 within the first 5 hours; thereafter, its level increased to final 199 μg.l-1 at the end of the 24 hour period of maceration. the content of alcohols is presented in table 3. as far as individual alcohols are concerned, the content of methanol is also important. its level increased within the whole period of maceration. within 24 hours, this value increased from 12 to 42 mg.l-1. within the first 5 hours, the content of (e)-3-hexen-1-ol increased. however, after 12 hours it began to decrease again and reached approximately the initial level. the content of (z)-3-hexen-1-ol increased within the whole 24-hour period of maceration: the initial value of 71 μg.l-1 doubled and was as much as 146 μg.l-1 . the content of alcohols is presented in table 4. ital. j. food sci., vol 29, 2017 229 table 3. content of ethyls. substance time of maceration 0 hours 5 hours 12 hours 24 hours mean sd mean sd mean sd mean sd ethyl acetate [mg.l-1] 32.63 0.74 36.59 0.87 47.32 0.54 59.70 1.20 ethyl propionate [μg.l-1] 109.27 1.27 109.08 1.87 107.71 1.24 85.55 1.81 ethyl isobutyrate [μg.l-1] 28.90 0.17 42.14 1.06 46.92 0.92 46.69 0.54 ethyl butyrate [μg.l-1] 402.18 8.51 470.00 18.80 505.00 8.66 550.80 9.35 ethyl hexanoate [μg.l-1] 518.22 8.05 228.47 4.79 536.90 8.34 573.68 9.84 ethyl octanoate [μg.l-1] 489.36 2.83 512.00 10.24 624.24 12.24 608.91 12.76 ethyl decanoate [μg.l-1] 54.45 0.95 62.16 0.96 1.00 0.02 125.87 2.56 ethyl lactate [mg.l-1] 5.56 0.12 2.71 0.01 2.72 0.03 2.61 0.01 diethyl uccinate [mg.l-1] 0.60 0.00 1.01 0.02 1.71 0.01 1.99 0.06 diethylmalate [mg.l-1] 3.14 0.07 3.50 0.04 3.09 0.08 2.47 0.05 monoethyl succinate [mg.l-1] 2.62 0.04 5.41 0.19 4.63 0.05 3.32 0.10 gama-butyrolactone [mg.l-1] 7.70 0.09 21.36 0.25 20.35 0.43 19.70 0.53 isoamyl acetate [mg.l-1] 3.94 0.10 5.49 0.10 5.82 0.07 7.60 0.08 2-phenylethyl acetate [μg.l-1] 151.50 2.31 932.45 22.12 552.35 12.42 384.28 13.45 1-propyl acetate [μg.l-1] 66.33 0.67 39.78 0.78 43.14 1.51 90.70 3.20 isobutyl acetate [μg.l-1] 116.61 2.94 198.53 4.04 249.00 9.96 283.05 4.34 1-hexyl acetate [μg.l-1] 261.73 7.94 43.71 0.25 93.69 2.37 199.00 0.00 (z)3-hexen-1]-yl acetate [μg.l-1] 6.04 0.12 14.19 0.21 14.28 0.24 14.19 0.16 table 4. content of alcohol. substance time of maceration 0 hours 5 hours 12 hours 24 hours mean sd mean sd mean sd mean sd methanol [mg.l-1] 12.34 0.31 13.91 0.29 32.57 0.57 42.11 1.29 isoamylalcohol [mg.l-1] 56.19 0.32 136.65 4.78 134.64 4.71 109.92 3.33 isobutylalcohol [mg.l-1 6.33 0.07 28.08 0.48 25.00 0.25 22.17 0.78 2-phenylethanol [mg.l-1 ] 9.73 0.20 68.21 1.21 52.29 0.81 19.80 0.00 1-propanol [mg.l-1] 19.69 0.41 9.96 0.24 12.63 0.15 35.14 0.36 1-butanol [μg.l-1] 1240.67 14.42 1247.15 29.19 1967.58 38.58 7130.90 146.01 1-hexanol [μg.l-1] 1508.00 45.24 1292.80 12.80 915.85 19.19 888.00 20.78 (e)-3-hexen-1-ol [μg.l-1] 84.15 1.47 114.00 3.42 101.00 1.00 88.16 2.01 (z)-3-hexen-1-ol [μg.l-1] 71.76 1.10 126.00 5.04 147.46 2.53 146.03 1.71 3-methyl-1]-pentanol [μg.l-1] 6.02 0.03 18.68 0.40 13.95 0.49 11.11 0.19 benzylalkohol [μg.l-1] 111.72 2.28 123.22 3.23 305.00 0.00 410.63 2.38 2,3-butandiol [mg.l-1] 346.61 5.37 552.57 6.29 1309.57 26.81 1497.02 14.82 propandiol [mg.l-1] 6.60 0.07 8.94 0.19 23.64 0.50 19.05 0.29 ital. j. food sci., vol 29, 2017 230 3.5. antioxidation activity as compared with red wines, a lower antioxidant capacity of white ones is caused by a lower content of phenolic compounds (vinson and hontz, 1995). a higher content of phenolic in red wine results is caused by the period of maceration during which phenolic compounds are released from skins, seeds, stalks and pulp of berries (fuhrman et al., 2001). because in white wines the maceration usually does not take place, their content of phenolic substances is limited and also their antioxidant activity is reduced (lamuelaraventos and de la torre-boronat). for that reason the maceration represents an interesting (and natural) step when producing white wines because it enables extraction of phenolic compounds and, thus, production of wine with strong antioxidant properties. in the course of the first 12 hours of maceration, the antioxidant activity (as measured by the dpph assay, increased and thereafter remained without changes (i.e. constant) during 24 hours of measuring. after 12 hours, it was even more than three times higher than at the beginning. dependence of antioxidant activity on length of maceration is depicted in fig. 2. figure 2. antioxidation activity. 3.6. time of maceration correlations existing between contents of individual aromatic substances and the time of maceration are presented in table 5. they are expressed as the pearson's correlation coefficient and characterize the tightness of individual relationships. values between 0.1 and 0.3 indicate a weak correlation while those between 0.4 and 0.6 and between 0.7 – 0.8 indicate medium and strong correlations, respectively. values above 0.9 mean that the correlation is very strong. d pp h (m g g a e/ l) ital. j. food sci., vol 29, 2017 231 table 5. correlations existing between contents of individual substances and the period of maceration. significant correlations (p < .05000) are in red. linalool 0.982 ethyl butyrate 0.939 1-propanol 0.712 ho-trienol 0.998 ethyl hexanoate 0.467 1-butanol 0.926 α-terpineol 0.891 ethyl octanoate 0.823 1-hexanol -0.894 ß-citronellol 0.961 ethyl decanoate 0.531 (e)-3-hexen-1-ol -0.169 nerol 0.963 ethyl lactate -0.679 (z)-3-hexen-1-ol 0.784 geraniol 0.977 diethyl succinate 0.951 3-methyl-1-pentanol 0.105 epoxylinalol 1 0.969 diethylmalate -0.834 benzylalcohol 0.967 epoxylinalol 2 0.994 monoethyl succinate -0.039 2,3-butandiol 0.935 2,6-dimetyl-3,7-octadiene-2,6diol 0.990 gama-butyrolactone 0.575 propandiol 0.745 methionol -0.250 isoamyl acetate 0.969 acetic acid 0.884 2-methyltetrathiophen-3-on -0.698 2-phenylethyl acetate -0.019 propionic acid 0.569 4-vinylguaiacol 0.903 1-propyl acetate 0.587 butyric acid 0.953 4-vinylfenol 0.711 isobutyl acetate 0.923 isobutyrřic acid 0.967 4-ethylguaiacol -0.966 1-hexyl acetate -0.004 isovaleric acid -0.315 4-ethylphenol -0.941 (z)3-hexen-1-yl acetate 0.655 2-methylbutanoic acid -0.003 1,1-diethoxyetane 0.532 methanol 0.962 hexanic acid 0.347 ethyl acetate 0.994 isoamylalcohol 0.386 octanoic acid 0.144 ethyl propionate -0.899 isobutylalcohol 0.451 decanoic acid -0.111 ethyl isobutyrate 0.779 2-phenyletcanol -0.110 dpph ga 0.880 4. conclusions in these experiments, interesting volatile compounds characterizing the cultivar moravian muscat. wine production from aromatic cultivars such a moravian muscat is a complex process depended on the health condition of grapes, temperature and length of the maceration period. results of this study indicate that the content of individual compounds is changing in the course of maceration. for that reason it is important to pay attention to its length and create favourable conditions enabling the development of wine character. the content of terpenic compounds (especially of monoterpenes) is increasing above all with the increasing time of maceration. this increases not only the antioxidant activity of wine but also the content of ethyls that can show a negative effect on the aromatic profile of produced wine. however, the content of total acids decreases. the only exception represents the content of acetic acid that markedly increases with the length of maceration so that the quality of produced wine is deteriorated. references carballeira lois l., cortés diéguez s., gil de la peña m.l. and fernández gómez e. 2001. spe-gc determination of aromatic compounds in two varieties of white grape during ripening. chromatographia 53(1):s350-s355. dennis e.g., keyzers r.a., kalua c.m., maffei s.m., nicholson e.l. and boss p.k. 2012. grape contribution to wine aroma: production of hexyl acetate, octyl acetate, and benzyl acetate during yeast fermentation is dependent upon precursors in the must. j agric food chem. 60(10):2638-2646. ital. j. food sci., vol 29, 2017 232 dziadas m. and jeleń h.h. 2010. analysis of terpenes in white wines using spe–spme–gc/ms approach. analytica chimica acta 677(1):43-49. fuhrman b., volkova n., suraski a. and aviram m. 2001. white wine with red wine-like properties:  increased extraction of grape skin polyphenols improves the antioxidant capacity of the derived white wine. j agric food chem. 49(7):3164-3168. herjavec s., tupajic p. and majdak a. 2001. influence of malolactic fermentation on the quality of riesling wine. agriculturae conspectus scientificus 66(1):59-64. jackson r.s. 2008. wine science: principles and applications. elsevier science. lamuela-raventos r.m. and de la torre-boronat m.c. beneficial effects of white wines. (0378-6501 (print)). peinado r.a., moreno j., bueno j.e., moreno j.a. and mauricio j.c. 2004. comparative study of aromatic compounds in two young white wines subjected to pre-fermentative cryomaceration. food chemistry 84(4):585-590. rapp a. and versini g. 1996. volatile phenolic compounds of wine. dtsch lebensm-rundsch 92(2):42-48. ribéreau-gayon p., dubourdieu d., don?che b. and lonvaud a. 2006. handbook of enology, the microbiology of wine and vinifications. wiley. santiago a.v.c., munoz r. and garcia r.g. 2011. molecular wine microbiology. elsevier science. tao y., li h., wang h. and zhang l. 2008. volatile compounds of young cabernet sauvignon red wine from changli county (china). journal of food composition and analysis 21(8):689-694. vinson j.a. and hontz b.a. 1995. phenol antioxidant index: comparative antioxidant effectiveness of red and white wines. j. agric. food. chem. 43(2):401-403. paper received july 3, 2016 accepted october 28, 2016 ijfs#324_tripodi_bozza   ital. j. food sci., vol 28, 2016 497 paper quality assessment of mediterranean shrimps during frozen storage c. condurso1, g. tripodi1*, f. cincotta1, c. m. lanza2, a. mazzaglia2 and a. verzera1 1 department of chemical sciences, university of messina, viale f. d’alcontres, 31 98166 messina, italy 2 department of agriculture, food and environment, university of catania, via s. sofia 98123 catania, italy *corresponding author. tel.: +39 +0906765440; fax: +39 0906765186 e-mail address: gitripodi@unime.it abstract aim of the research was to evaluate the effects of frozen storage on the quality of two mediterranean wild shrimps, namely parapenaeus longirostris (deepwater pink shrimp) and parapandalus narval (narwal shrimp) in order to promote the marketing of these littleknown shrimp species as frozen products, strengthening and enhancing their economic value. quality changes were determined by sensory evaluation combined with chemical and chemical/physical analyses, including determination of volatile aroma constituents. in particular, raw and cooked shrimp samples were evaluated at various frozen storage intervals up to sixteen months. the variation observed for the chemical and chemicalphysical indices did not diminish the sensory quality of both shrimp species. the results confirmed that freezing allows maintaining a good sensory quality of the considered shrimp species. keywords: deepwater pink shrimp, frozen storage, narwal shrimp, sensory evaluation, volatile aroma compounds   ital. j. food sci., vol 28, 2016 498 1. introduction the mediterranean shrimp species include, among others, the deepwater pink shrimp (parapenaeus longirostris, lucas 1846) and the narwal shrimp (parapandalus narval, fabricius 1787). both species live in deep waters on muddy or muddy-sandy bottoms. parapenaeus longirostris has a wide geographical distribution, being found both in the eastern and western atlantic (olaso, 1990), as well as in the mediterranean and its adjacent seas (massutti, 1963); italy is the country with the largest catches (holthuis, 1980) especially in the channel of sicily and in the ionian sea. parapandalus narval has an eastern atlantic-mediterranean distribution (thessalou-legaki, 1992) and is very common in the sea of ustica island. shrimps presumably represent the most important market for seafoods. in 2014 the global shrimp production was around 7×106 metric tons, of which almost 4×106 from aquaculture (larkin et al., 2015). the international trade is dominated by usa, japan and european countries as importers, whereas developing nations, especially south east asian countries, act as the main shrimp suppliers of the world. italy imports most of its demand mainly as farmed shrimp whereas the two mediterranean wild shrimps, namely parapenaeus longirostris and parapandalus narval, are of little commercial importance, sold only as fresh locally and close to the fishing grounds since their limited shelf life. although freezing is an effective method for preserving foods, some deterioration in frozen food quality can occur during storage, such as colour fading (chandrasekaran, 1994; okpala and bono, 2016), lipid oxidation (riaz and qadri, 1990), denaturation of protein (bhobe and pai, 1986), sublimation and recrystallization of ice (londahl, 1997). these can result in off-flavours, rancidity, dehydration, loss of juiciness, textural changes (bhobe and pai 1986; yamagata and low 1995; londahl, 1997) and increase in volatile basic nitrogen (riaz and qadri 1990; yamagata and low, 1995). some papers are present in literature on the quality changes of frozen shrimps during storage (yamagata and low 1995; bak et al., 1999; boonsumrej et al., 2007; tsironi et al., 2009; bono et al., 2016). only few of these evaluated the sensory quality or related the sensory evaluation to the volatile aroma compounds (rochat et al., 2009; alam and solberg, 2009), but according to our knowledge, no informations are reported on parapenaeus longirostris and parapandalus narval. in view of the fact that frozen shrimp is a product of high commercial value and increasing demand due to its competitive price and extended shelf life (tsironi et al., 2009), the aim of the research was to evaluate the effects of frozen storage time on the quality of the deepwater pink and narwal shrimps in order to promote the marketing of these little-known shrimp species as frozen products, strengthening and enhancing their economic value. since the consumer is the ultimate judge of quality, chemical and instrumental methods were matched with the sensory evaluation: chemical and chemicalphysical indices, volatile aroma constituents and sensory properties were determined at different times during frozen storage both on raw and cooked samples. 2. materials and methods 2.1. sampling shrimp specimens of parapenaeus longirostris, lucas 1846 (fao name: deepwater pink shrimp) and parapandalus narval, fabricius 1787 (fao name: narwal shrimp) were caught   ital. j. food sci., vol 28, 2016 499 off the southeast coast of sicily (porto palo, siracusa, italy fao 37: mediterranean, black sea; subarea 37.2: central mediterranean; division 37.2.2: ionian) in march 2013. samples of frozen shrimps (size: parapenaeus longirostris, 10-15 cm; parapandalus narval, 7 cm) were provided by a sicilian company that owns and operates both the fishing boats and the packing and storage facilities. after catch, shrimps were put into ice, then quick-frozen at 40°c, stored at 18°c under vacuum (-0.8 bar) and transported frozen (at constant 18°c) to the laboratory. all shrimp samples came from the same frozen batch; for each shrimp species eight packages in total were purchased and stored at constant 18°c for sixteen months. the chemical and sensory analyses were carried out immediately after arriving at the lab and at specific intervals during storage. at fixed time, one package was thawed at room temperature and sufficient quantities were used for analyses. unpeeled raw and cooked shrimp samples were analysed immediately after thawing. cooked shrimps were obtained steaming unpeeled specimens for 15 min. all determinations were made in triplicate. 2.2. ph measurement the ph values of the raw samples were determined on homogenates of samples in distilled water (1:2 w⁄w) by using a phmeter mp220 (mettler toledo, milan, italy) at 25°c. 2.3. determination of the total volatile basic nitrogen (tvb-n) for the determination of the total volatile basic nitrogen (tvb-n) of the raw samples, steam distillation of an extract deproteinised by trichloroacetic acid extraction was used according to official method (eu, 1995). results were expressed as mg tvb-n/100 g of wet sample. 2.4. colour measurement quantification of the colour change was based on measurement of cielab values (l*value: lightness; a*-value: redness and greenness; b*-value: yellowness and blueness), using a nr-3000 colourimeter (nippon denshoku ind. co. ltd, tokyo, japan). the instrument was standardized under ‘‘c’’ illuminant condition according to the cie (commission international de l’eclairage) using a standard white reference tile. at predetermined times of storage, according to the design, measurements were conducted for raw and cooked shrimp at five points. all measurements were carried out on three different shrimp samples. the average values were reported and values of ∆e were determined: ∆e = √(l*-l0*)2 + (a*-a0*)2 + (b*-b0*)2 where l0*, a0*, and b0* are the values of l*, a* and b* colour parameters at storage time zero. 2.5. hs-spme sampling the method of headspace solid phase microextraction (hs-spme/gc-ms) was used for the isolation and concentration of volatiles. the analyses were conducted on peeled raw and cooked shrimp samples. to 5 g of each shrimp sample, placed in a 40 ml vial, 14 ml of a nacl saturated aqueous solution were added. extraction was performed in the headspace vial kept at 35°c using a dvb/car/pdms fibre of 50/30 μm film thickness (supelco, bellefonte, pa, usa) housed in its manual holder (supelco, bellefonte, pa,   ital. j. food sci., vol 28, 2016 500 usa). the sample was equilibrated for 30 min and then extracted for 30 min. during the extraction, the sample was continuously stirred. after the sampling, the spme fibre was introduced onto the splitless injector of the gc/ms maintained at 260°c for 3 min for the thermal desorption of the analytes. no artefacts were observed after a spme analysis of the saturated saline solution performed as blank analysis. 2.6. gc-ms analysis a varian 3800 gas chromatograph directly interfaced with a varian 2000 ion trap mass spectrometer (varian spa, turin, italy) was used. the conditions were as follows: injector temperature, 260 °c; injection mode, splitless; capillary column, cp-wax 52 cb, 60 m, 0.25 mm i.d., 0.25 μm film thickness (chrompack italy s.r.l., turin, italy); oven temperature, 45°c held for 5 min, then increased to 200°c at a rate of 5°c/min and to 240°c at 3°c/min; 240°c held for 20 min; carrier gas, helium at a constant pressure of 10 psi; transfer line temperature, 250°c; acquisition range, 40–250 m/z; scan rate, 1 scan/s. each volatile component was identified using mass spectral data, nist 11 library (nist/epa/nih mass spectra library, version 2.0 g, usa), linear retention indices, literature data and injection of standards where available. the linear retention indices (lri) were calculated according to van den dool and kratz (1963) equation. 2.7. sensory analysis the sensory profiles of the shrimp samples were evaluated following the uni 10957, 2003 method. twenty-five judges were submitted to preliminary tests to determine their sensory performance on basic tastes and the aromas associated with shrimps. the sensory profile (uni 10957, 2003) was defined by using a selected panel of twelve judges trained over four sessions. panelists were asked to score appearance and odour of raw peeled shrimp and appearance, odour, texture, and taste of cooked shrimp. a list of descriptors was selected based on the frequency (60 %) of the terms used by the judges in several sessions. reference standards were available to define descriptors. the descriptors were quantified using a nine-point intensity scale, where 1 = “not perceptible” and 9 = “strongly perceptible”. the shrimp samples were tested in triplicate. each judge evaluated the shrimp samples in two sessions. all evaluations were conducted from 10.00 to 12.00 am in individual booths (iso 8589, 2007) illuminated with white light. the order of presentation was randomized among judges and sessions. water was provided for rinsing between shrimp samples. all data were acquired by a direct computerized registration system (fizz biosystemes. ver. 2.00 m, couternon, france). 2.8. statistical analysis chemical and sensory data were subjected to analysis of variance (anova), using statgraphics plus software (ver. 5.1). duncan’s multiple range test was applied to the data to identify any significant differences between the analysed samples. the model was statistically significant with p<0.05. 3. results and discussions figure 1 shows the variation of the ph values during frozen storage in raw deepwater pink and narwal shrimps. for both shrimp species the ph values ranged between 7.3-7.8 in agreement with cadun et al. (2005) who reported ph values of 7.64 for frozen   ital. j. food sci., vol 28, 2016 501 deepwater pink shrimp; no statistically significant (p>0.05) increase resulted till four month. the ph determination is one of the most frequently used physical methods for the quality control of seafood products since they are affected by the changes in the concentrations of free hydrogen and hydroxyl ions because of the shifts in the oxidation– reduction balance of the food by the activity of micro-organisms or enzymes (varlik et al., 2000). generally, the ph value of crustaceans is higher than that of fish and mammal species because of their higher content of nonprotein nitrogenous compounds (shahidi, 1994). from our results, the ph values of analysed shrimp samples during storage resulted always below 8, the critical acceptability limit for most shellfish products (schormuller, 1968). figure 1: ph variations of raw shrimp samples during frozen storage (error bars indicate standard error of measurements). for each species different letters indicate statistically significant differences among mean values at p ≤ 0.05 by duncan’s multiple range test. figure 2 shows the total volatile basic nitrogen (tvb-n) values in raw deepwater pink and narwal shrimps during storage. the tvb-n content resulted around 29 mg/100 g of wet weight in the shrimp samples of the two species at the beginning of storage, and increased reaching the values of 35.09 mg/100 g and 35.85 mg/100 g at the end of storage for deepwater pink and narwal shrimps, respectively. statistically significant increases were observed after four month of storage for both shrimp species. the increase in tvb-n values is consistent with the results of the ph values: the tvb-n increase during the storage turns the medium alkaline and as a result ph increases. the level of tvb-n is considered a useful index of microbial spoilage in different fresh and lightly preserved seafood (ozoğul and ozoğul, 2000). the limit of tvb-n values for seafood products of good sensory quality has been reported to be 30 mg/100 g. however, this limit may be questionable for shrimp species since the average tvb-n values for fresh crustaceans are often higher (oehlenschläger, 1997); in a study by cobb et al. (1973) the initial tvbn content of fresh shrimp tails from different batches of shrimp stored on ice, ranged from 13.5 to 38.2 mg n/100 g; however a high sensory quality of shrimp samples was   ital. j. food sci., vol 28, 2016 502 perceived. as a consequence, tvb-n content cannot be considered as an indicator of freshness by itself but it must be always enhanced by the sensory tests (chen et al., 1995). table 1 illustrates the changes of l*, a* and b* in raw and cooked deepwater pink and narwal shrimps during frozen storage. l* values increased whereas a* and b* values decreased when storage time increased; the colour of shrimp changed from red and yellow to dull lighter colour. in table 1 ∆e values for raw and cooked shrimps are also reported. ∆e values, indicating the total colour changes, increased with an increase in storage time. for raw deepwater pink shrimp statistically significant variations resulted at the beginning and at the end of storage, whereas for cooked samples a significant increase was observed only after thirteen months. regarding the narwal shrimps, ∆e values significantly increased until ten months both in raw and cooked samples. the rate of increase was higher for narwal shrimp samples, both raw and cooked, than for deepwater pink shrimps probably due to the deeper colour of the former as indicated by the higher scores of the colour sensory descriptor (table 3). shrimp colour is linked to the content of astaxanthin and its esters, that are the major pigments in shrimps; drying and storage conditions affect shrimp colour due to the astaxanthin oxidation and isomerisation reactions that lead to colourless compounds and, thus, to the loss of the typical redness and yellowness (chen et al., 1995; niamnuy et al., 2008). table 2 shows the amount of the volatile compounds detected in raw and cooked deepwater and narwal shrimp samples at different storage time. a total of thirty compounds have been observed, such as aldehydes, ketones, alcohols, esters, acids, nitrogenand sulfurcontaining compounds. figure 2: total volatile basic nitrogen values (mg n/100 g shrimp flesh) of raw shrimp samples during frozen storage (error bars indicate standard error of measurements). figure legend: for each species different letters indicate statistically significant differences among mean values at p ≤ 0.05 by duncan’s multiple range test. slight differences in the qualitative and quantitative volatile composition of the two shrimp species and during the storage period, as well as between raw and cooked samples, resulted. in raw shrimps, hexanal, 2-ethyl-1-hexanol and dimethylsulphide were   ital. j. food sci., vol 28, 2016 503 the most abundant compounds, followed by 6-methyl-5-hepten-2-one and nonanal; 1penten-3-ol, octanal (only in deepwater pink shrimp samples), 1-octen-3-one and γ-butyrolactone were also detected in a good amount. some of these volatiles, namely dimethyl sulphide, 1-penten-3-ol, 1-octen-3-one, 2-ethyl-1-hexanol and γ-butyrolactone, decreased during the storage period but they remained among the most abundant components of volatile fraction up to sixteen months. aldheydes, ketones and alcohols containing 6, 8 and 9 carbon atoms are derived from long-chain polyunsaturated fatty acid via 12or 15-lipoxygenases and hydroperoxide lyase (baek and cadwallader, 1997) and are responsible of the pleasant planty, green and melony aromas and flavours of fresh seafoods. in particular hexanal contributes to the distinct green plant-like, grassy and apple-like aromas of fresh shrimp, whereas 2-ethyl-1-hexanol, together with short-chain alcohols, contributes to the typical sweet aroma of shrimps (alasalvar et al., 1997). also dimethyl sulphide provides a pleasant seashore-like smell in fresh seafoods (iida, 1988); in fact, although sulphur compounds are usually associated with deteriorated seafoods, there is evidence that they can be present even in fresh ones and are considered important volatile aroma components in marine crustaceans (alasalvar et al., 1997). γ-butyrolactone has faintly sweet odour reminiscent of rancid butter; lactones have been identified in roasted shrimp and boiled scallops; they derived from aliphatic saturated and unsaturated γ-hydroxycarboxylic and δ-hydroxycarboxylic acids (pan and kuo, 1994). dimethylamine, trimethylamine, acetic acid, and phenol were detected in low amount in fresh samples and slightly increased during frozen storage, except for trimethylamine, whose variations during storage were not statistically significant both in narwal and in deepwater shrimp samples. the formation of trimethylamine in frozen seafoods is prevented by the inhibition of microbial growth, yet dimethylamine is produced enzymatically: castell et al. (1970) reported the increase of dimethylamine content in frozen fish muscle due to enzymatic breakdown of trimethylamine oxide. also acetic acid could be formed by enzymatic decomposition of either lipid autoxidation or secondary hydroperoxides of fatty acids (alasalvar et al., 1997), whereas phenol formation occurs via decarboxylation of phenolic carboxylic acids (spurvey, 1998). in all the samples, 6-methyl-5-hepten-3-one and geranylacetone (spicy and flowery notes) were present; these compounds, deriving from carotenoid degradation, showed a statistically significant increase during storage. their amount and the rate of increase during storage are higher in narwal than in deepwater pink shrimps. 2,4-decadienal, (e,z)-2,4-heptadienal, (e,z)-3,5-octadien-2one and (z)-4-heptenal, deriving from lipid autoxidation during storage, were not detected. the absence of these compounds, responsible of stale and oxidized aromas and of fish cold-stored off-flavours (alasalvar et al., 1997), was probably due to the very low fat content of deepwater pink and narwal shrimps. as expected, the cooked shrimps showed some differences in volatile aroma profile compared to the raw ones. after cooking, hexanal and dimethylsulphide remained the main constituents of the volatile fraction but their amount decreased when freezing time increased. 2-methylbutanal, 3-methylbutanal, 2,6-dimethylpyrazine, dimethylsulfoxide, 1dodecanol were detected only in cooked shrimps samples and their amount did not vary among the cooked samples. among these compounds, 1-dodecanol, with a flower-like odour, has been already recognized as an important volatile of cooked shrimps (mandeville et al., 1992). 2-methylbutanal and 3-methylbutanal are well known as amino acid degradation products and may be generated either thermally (strecker degradation) or enzymatically from isoleucin and leucin.   ital. j. food sci., vol 28, 2016 504 table 1: ∆e, l*, a* and b* values of shrimp samples during frozen storage. months of storage deepwater pink shrimp narwal shrimp raw cooked raw cooked δe l* a* b* δe l* a* b* δe l* a* b* δe l* a* b* 0 31.31a 10.87d -2.03d 45.47a 3.07d -1.46d 43.99a 17.66c 10.84b 23.06a 12.73c 14.63c 2 0.54a(a) 33.86a 9.93c -4.12c 1.13a 58.76b 1.46c -2.59c 0.92a 44.86a 11.73c 9.92b 0.18a 28.88a 12.17c 13.52c 4 0.93b 38.22b 6.41b -4.51c 1.15a 58.69b -1.44b -3.12b 1.18b 45.47a 11.56c 9.04b 0.59b 36.75b 11.38b 11.34b 7 0.99b 39.16b 6.29b -5.41b 1.16a 58.31b -3.22a -3.78b 1.30c 48.72a 8.07b 2.53a 0.78c 38.33b 11.33b 11.13b 10 1.04b 39.79b 4.72a -5.67b 1.17a 58.39b -3.92a -4.12b 1.40d 53.57b 6.82a 1.37a 1.16d 43.52c 10.89b 8.34a 13 1.13c 42.47c 4.41a -6.32b 1.22b 60.13c -4.07a -4.88b 1.41d 58.84b 6.06a 1.11a 1.25d 43.53c 6.61a 6.73a 16 1.19c 48.43c 3.40a -8.29a 1.28b 62.11c -4.97a -6.73a 1.45d 59.98b 5.12a 1.02a 1.29d 46.07c 4.83a 6.68a (a) different letters in the same column indicate statistically significant differences among mean values at p ≤ 0.05 by duncan’s multiple range test.   ital. j. food sci., vol 28, 2016 505 table 2: volatile fraction composition(a) of shrimp samples during frozen storage. months of storage deepwater pink narwal raw cooked raw cooked 0 4 10 16 0 4 10 16 0 4 10 16 0 4 10 16 volatiles(b) dimethylamine 84b(d) 90b 114a 149a 60b 68b 88a 99a 50b 67b 107a 113a 90b 92b 109a 111a trimethylamine 48 46 40 43 52 56 57 54 56 51 51 54 64 67 62 65 dimethyl sulphide 920a 875a 585b 332c 2246a 2352a 1391b 596b 704a 743a 304b 298b 2656a 2021a 1231b 698c 3-hydroxy-2-butanone 25 28 38 43 2-butanone 65 60 53 48 2-methyl butanal -(c) 68 71 75 69 82 79 86 89 3-methyl butanal 52 63 58 57 60 71 68 75 hexanal 1120 1155 1051 1197 1855a 1693a 1076b 1038b 1157 1136 1156 1098 1931a 1832a 1123b 1311b 1-penten-3-ol 232a 160b 115c 48d 96a 41b 38b 16c 104a 88b 41c 48c 29a 17b 10b 9b heptanal 32 42 32 28 23 22 28 11 2-methyl-1-butanol 33 24 35 29 78 59 60 65 29 25 11 12 1-pentanol 39 31 25 17 31 28 14 21 octanal 100 116 115 126 127 143 143 121 21 36 24 43 59 60 89 34 6-methyl-5-hepten-2-one 321b 358b 397b 498a 119b 130b 168b 262a 413b 452b 479b 681a 121b 164b 279a 325a 2,6-dimethylpyrazine 43 37 38 46 46 56 53 47 nonanal 260 287 242 225 350 327 321 312 330 341 294 288 380 372 222 287 ethyl octanoate 85 93 1-octen-3-one 186a 148a 96b 82b 41a 49a 18b 11b 216a 228a 182a 106b acetic acid 99b 108b 120b 160a 236b 246b 254b 294a 89b 99b 111b 142a 47b 54b 116a 135a 2-ethyl-1-hexanol 913a 605b 465c 428c 1589a 1295b 861c 973c dimethyl sulfoxide 59b 68b 125a 130a 19b 31b 49a 66a 1-nonanol 43 39 53 32 36 42 59 48 45 40 28 32 7 7 5 3 γ-butyrolactone 191a 70b 68b 74b 143a 89b 66b 64b 122a 56b 21c 25c 111a 53b 22c 20c 4-butoxy-1-butanol 48 34 39 30 30 25 26 28 58 48 51 63 50 49 47 53 dodecanal 58 63 83 64 91 86 93 89 methyl dodecanoate 8 4 5 5 10 8 15 13 hexanoic acid 23 28 32 35 31 28 36 38 8 7 10 6 31 28 34 36 1-dodecanol 158 174 164 169 201 198 231 243 geranylacetone 11b 15b 42a 50a 166b 164b 236a 242a 62b 45b 125a 175a 185a 179a 265b 271b phenol 54b 46b 71a 86a 26b 23b 42a 53a (a) expressed as peak areas, arbitrary scale. (b) volatile compounds have been reported according to the order of elution on the column cp-wax 52 cb column. (c) not detected. (d) different letters in the same row indicate statistically significant differences among mean values at p < 0.05 by duncan’s multiple range test.   ital. j. food sci., vol 28, 2016 506 table 3: sensory scores of shrimp samples during frozen storage. deepwater pink narwal raw cooked raw cooked months of storage 0 4 10 16 0 4 10 16 0 4 10 16 0 4 10 16 volatiles flesh colour 3.5b(a) 2.9a 2.8a 2.1a 3.5b 3.7b 2.3a 2.6a 7.5c 6.2b 2.7a 2.8a 7.4d 6.8c 6.0b 4.8a sheen 4.5b 4.4b 4.1a 3.9a 6.5c 5.8b 6.0b 4.0a compactness 6.5 6.2 6.0 6.2 6.5 5.9 6.3 6.4 sea aroma 5.1 4.8 4.7 5.0 4.2 4.7 4.2 4.3 5.3 4.8 4.8 5.1 5.8 4.4 4.5 4.1 algae aroma 4.3 4.9 4.5 4.7 4.9 4.9 4.7 4.3 shrimp aroma 5.0 5.9 5.4 5.4 6.9 6.3 6.6 6.3 6.2 5.9 5.4 6.4 7.1 6.3 6.5 6.2 off-odour 2.5 2.7 3.3 3.3 2.2 2.9 3.3 3.4 2.6 2.6 3.7 3.8 2.0 2.8 3.4 3.8 flesh firmness 5.3 4.2 4.5 4.8 4.6 3.3 3.8 3.8 5.1 4.2 5.0 5.5 4.8 3.6 3.3 3.6 bitter 2.5 3.3 2.6 3.0 3.0 3.1 2.8 2.8 salty 3.4 3.0 3.1 2.8 3.8 3.7 4.5 3.7 sour 2.3 2.1 2.0 1.9 2.3 2.1 2.5 1.7 sweet 4.6 3.8 4.0 3.9 5.7 4.6 4.0 4.5 juicy 5.1 4.8 4.1 4.2 5.5 4.4 4.5 4.7 chewy 3.9 4.0 4.2 4.5 3.4 3.6 4.0 4.0 sea flavour 4.8 3.8 3.7 4.6 4.4 4.0 3.9 4.0 shrimp flavour 6.8 5.9 5.9 6.8 7.0 6.2 6.6 6.4 off-flavour 2.2 2.8 3.0 3.0 2.3 3.3 3.5 3.0 overall 7.4 7.1 7.4 7.2 7.6 7.6 7.4 7.5 8.0 8.1 7.8 7.9 8.3 8.2 8.2 8.1 (a) different letters in the same row indicate statistically significant differences among mean values at p < 0.05 by duncan’s multiple range test.   ital. j. food sci., vol 28, 2016 507 since these branched aldehydes were not present in uncooked samples, their thermal generation could be presumed. also 2,6-dimethylpyrazine has a thermal origin: in fact alkylpyrazines may be formed by the involvement of lipid oxidation products in maillard reaction (huang et al., 1987) by heating of food at or above 100 °c. alkylpyrazines, including methylpyrazine, 2,5and 2,6-dimethylpyrazine have been detected in several cooked crustaceous, identified as having a roasted, nutty/meaty aromas in boiled crayfish and considered to contribute more to boiled rather than roasted odours in proteinaceous food (spurvey, 1998). other volatiles such as the 2-ethyl-1-hexanol were not identified in the cooked shrimps. otherwise, as below reported 2-ethyl-1-hexanol, together with short-chain alcohols, contributes to the typical sweet aroma of raw shrimps (alasalvar et al., 1997). as happened for the raw shrimps, both deepwater pink and narwal, the volatile profile of the cooked shrimps considered as a whole, remained almost stable at least until ten months of freezing storage. table 3 reports the results of sensory evaluation of raw and cooked deepwater pink and narwal shrimps. in particular, for the raw samples the descriptors were three for the appearance (colour of flesh, sheen, compactness) four for the aroma (shrimp, sea, algae, off odour) and one for the rheological properties (flesh firmness). regarding the cooked shrimps four descriptors for the aroma (shrimp, sea, algae odour, off odour), three for the flavour (shrimp, sea, off flavour), one for the rheological properties (flesh firmness), four for the taste (bitter, salty, sour, sweet), one for the oral perception (juicy) and finally, one for the texture (chewy) were selected. the analysis of variance applied to the sensory data showed statistically significant differences only for sensory scores linked to colour and sheen descriptors both for raw and cooked deepwater pink and narwal shrimps. this was in accordance with the colour analysis that showed colour changes during storage and with the volatile analysis that evidenced an increase in the amount of carotenoid degradation products. no statistically significant variation was observed for the “overall” descriptor up to sixteen month of frozen storage. the differences observed in chemical indices and volatile aroma constituents little influenced the sensory attributes of raw shrimp samples during the freezing storage. the same occurred for the sensory attributes of the samples cooked after different periods of freezing. 4. conclusions the main goal of this research was to evaluate the effect of freezing storage on the quality of deepwater pink shrimps and narwal shrimps. to this end, ph, tvb-n, colour analysis, volatile fraction analysis and sensory evaluation were carried out. the assessment of shrimp quality mainly considered the impact of the preservation method on the sensorial characteristics since they are major concerns of consumers. the chemical and chemicalphysical data and the volatile profile, considered the most important parameter for shellfish flavour quality, evidenced slight variations during the freezing storage but were unable to affect the sensory quality as confirmed by the maintenance of high scores for the “overall quality” descriptor throughout the entire storage period. our results demonstrate the maintenance of a good sensory quality during the considered period, therefore these two shrimp species could be of great economic interest if marketing as frozen products.   ital. j. food sci., vol 28, 2016 508 references alam a. and solberg c. 2009. physical and sensory quality changes during freeze storage of black tiger shrimp (penaeus monodon). int. j. sustain. agr. 5: 24. alasalvar c., quantick p.c. and grigor j.m. 1997. aroma compounds of fresh and stored mackerel. in shahidi f, cadwallader kr (eds) flavour and lipid chemistry of seafoods, acs symposium series 674, american chemical society, washington dc, pp 39. baek h.h. and cadwallader k.r. 1997. character-impact aroma compounds of crustaceans. in shahidi f, cadwallader kr (eds) flavour and lipid chemistry of seafoods, acs symposium series 674, american chemical society, washington dc, pp 85. bak l.s., jacobsen l. and jørgensen s.s. 1999. characterization of qualitative changes in frozen, unpeeled cold-water shrimp (pandalus borealis) by static headspace gas chromatography and multivariate data analysis. z. lebensm. 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(eds) seafood: chemistry, processing technology and quality, blackie academic & professional, london, pp 85. riaz m. and qadri r.b. 1990. time-temperature tolerance of frozen shrimp 2. biochemical and microbiological changes during storage of frozen glazed shrimps. trop. sci. 30: 343. rochat s., egger j. and chaintreau a. 2009. strategy for the identification of key odourants: application to shrimp aroma. j. chromatogr. a 36: 6424. shahidi f. 1994. seafood proteins and preparation of protein concentrates. in: shahidi f., botta j.r. 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ital. j. food sci., vol 28, 2016 669 paper convective dehydration kinetics and quality evaluation of osmo-convective dried beetroot candy b. singh*1and b. singh hathan2 1cdl state institute of engineering and technology, panniwala mota, sirsa, haryana, india 2sant longowal institute of engineering and technology, longowal, sangrur, punjab, india *corresponding author. bhupimander@rediffmail.com abstract the beetroot cubes were osmotically pretreated in 60°bx of sucrose solution at 55°c osmotic solution temperature for 180 min with fruit to solution ratio 1:4 (w/w). the osmotically dehydrated beetroot cubes were further dehydrated convectively at different drying air temperatures of 55, 65 and 75°c up to final moisture content of 9±1% (w.b). among the models investigated, the page model fitted the experimental data for convective drying of natural and osmosed beetrooot cubes. during convective dehydration, the effective moisture diffusivity of natural samples and osmosed samples at drying air temperatures ranged from 55 to 75°c varied between 8.09917×10-9 to 1.45785 × 10-8 m2s-1 and 1.13388 × 10-8 to 1.61983 × 10-8 m2s-1 respectively. the activation energy of convective dried natural beetroot cubes was 27.92 kj mol-1 as compared to 16.98 kj mol-1 for osmosed samples. finally osmo-convectively dried beetroot cubes were coated with sucrose for candy preparation. keywords: activation energy, beetroot, convective dehydration, effective diffusivity ! ital. j. food sci., vol 28, 2016 670 1. introduction beetroot (beta vulgaris) is considered a very important vegetable from the nutritional point of view, which is rich in valuable, active compounds such as carotenoids (dias et al., 2009), glycine betaine (de zwart et al., 2003), saponins (ata-manova et al., 1975), betacyanines (patkai et al., 1997), betanin, polyphenols and flavonoids (vali et al., 2007). therefore, beetroot ingestion can be considered a factor in cancer prevention (kapadia et al., 1996). the moisture content of fresh fruits and vegetables is more than 80%, they are classified as highly perishable commodities (orsat et al., 2006). the moisture content of fruits and vegetables is removed by drying with an aim to preserve and store them for a long period of time by ensuring microbiological safety. drying is a complex process where heat and mass transfer occurs simultaneously in transient conditions. india is one of the leading producers of fruits and over 20% of the world perishable crops are dried to increase shelf-life and food security (ertekin and yaldiz, 2004). the preservation of fruits and vegetables by dehydration should be accomplished in such a manner that the quality effect on the structural configuration of these products is minimized. among the different methods of food preservation, convective dehydration is the most popular and efficient way to reduce the moisture content and preserve foods. the modes involved in convective drying of foods are heat and mass transfer. transport of the heat energy flow throughout the medium designated as heat transfer that causes the movement of internal moisture within the foods followed by the movement of water vapor from the food surface is called mass transfer that depends upon external conditions of temperature, air humidity, air flow and area of exposed surface in convective drying. a lot of studies have reported that convective drying is the most popular method applied to reduce the moisture content of fruits and vegetables including beetroot (henderson et al., 1961; lewiciki, 2006; shynkaryk et al., 2008). however, this method takes long time and high temperature for drying that affects the nutritive value of products (marfil et al., 2008). product quality notably depends on texture, colour and flavour and they deteriorate with convective dehydration (lenart, 1996). a well-known process to achieve good quality product is freeze drying, but this is an expensive method of food preservation. therefore, there is a need for simple economic and technically feasible alternative drying processes, which have low capital cost and offer a method that save highly perishable products, making them available to the regions away from production zones. furthermore, osmotic dehydration is one of such methods that involves the partial removal of water from food, such as fruit or vegetables, by immersion in a hypertonic solution leaving a material that will need shorter drying time than the original food material, thereby, making this process more economical (lenart, 1996; shi and lemaguer, 2002; fasogbon et al., 2013). osmotic pretreatment can also minimise drying colour losses (raoult-wack et al., 1991), as well as reducing nutrient losses (shi et al., 1999). this paper assesses the improvement of the quality of the final product to prevent oxidative browning and the loss of volatile flavoring constituents, reducing the fruit acidity and structural collapse during subsequent air-drying (dias et al., 2009). it has been reported that when convective drying is conducted between temperatures ranging from 40 to 60°c the moisture curves follow sigmoid shape characteristics of the drying process and shows reduction in drying time with increase in temperature (barroca, 2012). hence, osmo-convective dehydration of beetroot is an interesting alternative for the development of confectionery based functional food with extended shelf life. one of the most important aspects of drying technology is the modeling of the drying process. the basic principle in modeling is based on a set of mathematical equations that adequately explore the system. the solution of these equations must allow calculation of ! ital. j. food sci., vol 28, 2016 671 the process parameters as a function of time at any point in the dryer based only on the primary condition (premi et al., 2010). hence, the use of a simulation model is an important tool for prediction of performance of drying systems. the best model describing the drying characteristics of samples should be chosen as the one with the highest coefficient of determination (r2), reduced chi-square (χ2) and rmse (madamba et al., 1996). (shi and lemaguer, 2002) calculated effective diffusivity using the slope method but this method gives only a single value of diffusivity for the entire process and therefore does not predict the kinetics of the entire osmotic dehydration process, because the value of diffusivity changes with time and with moisture content of the commodity. some researchers calculated effective diffusivity by using only first term of the analytical solution of the fickian model assuming that the effect of terms other than first on the value of diffusivity was non-significant (rastogi et al., 1999; sharma et al., 2003). thermodynamically, the activation energy is the ease with which the water molecules pass the energy barrier when migrating within the product (lopez et al., 2009). in this context, the objective of this work aim to: (i) study the experimental investigation to know the effect of osmotic pre-treatment and drying air temperature on the convective dehydration kinetics and determination of effective moisture diffusivity and activation energy. (ii) develop the preparation of beetroot candy from osmo-convective dried beetroot cubes. 2. materials and methods 2.1. sample preparation fresh, well graded, beetroot was procured from the local market of sirsa, haryana (india). beetroot was washed properly and cut into 1cm×1cm×1cm size with the help of cutter equipped with a knife moving perpendicularly to a horizontal base. two types of samples like natural beetroot and osmotic dehydrated beetroot were used for the study of the osmo-convective dehydration kinetics .the initial moisture content of natural beetroot cubes was found to be between 85.71 to 86.29% (w.b.). 2.2. osmotic dehydration a known amount (20-25 g approximately) of beetroot cubes were transformed in stainless steel containers containing calculated volume of an osmotic solution of different concentrations at pre-set desired temperature in hot water bath. temperature of the osmotic solution was maintained by hot water bath agitating at the rate of 75 oscillations per min to reduce the mass transfer resistance at the surface of beetroot and for good mixing (gupta et al., 2012). optimization of osmotic dehydration process was carried out with the purpose of maximizing water loss, solute gain and quality of the product. the optimum conditions were 60°bx osmotic solution concentration, 55°c osmotic solution temperature and 180 min process duration at fruit to solution ratio 1:4 (w/w). following osmotic pre-treatment at optimum conditions, the moisture content of the beetroot cubes reduced to 74.86% (w.b.) and the solid content increased up to 8% designated as osmotic dehydrated beetroot. ! ital. j. food sci., vol 28, 2016 672 2.3. convective dehydration the natural and osmosed samples were taken separately in plastic containers before drying and then divided into 175 g portions each. to prepare a shelf stable product, the beetroot cubes was dehydrated up to final moisture content 9±1% (w.b.) at an air temperature of 55, 65 and 75°c and air velocity of 1.6 m s-1 (reppa et al., 1999). the dried samples were packed in high-density polyethylene bags after cooling in desiccators and placed at ambient temperature for further analysis. initial moisture content was determined by the oven-drying method, for the natural and osmosed beetroot samples at a temperature of 135°c for 2 h until constant weight was reached with repetition in order to assure moisture content average values (aoac, 2000). 3. engineering analysis of drying data 3.1. determination of moisture content the moisture content of the samples during the drying process was calculated according to the following formula. d dt t w ww m − = (1) where, mt = moisture content at time t (g water/ g dm) wt = dry matter at any time t(g). wd = dry matter (g). 3.2. determination of moisture ratio however, moisture data are used in non-dimensional form so moisture ratio is defined by the following equation: eo et mm mm mr − − = (2) where, mr= moisture ratio, mo and me initial moisture content and equilibrium moisture , g water/ g dry matter content, respectively .mt, moisture content at any time t on dry basis (g water/ g dry matter). 3.3. determination of drying rate to study the drying behavior at different drying air temperature, moisture content (d.b.) and drying rates were calculated. the drying curves were plotted to observe the effect of process variables. corresponding to drying curves, the drying rate curves were also plotted (kar and gupta, 2003). the instantaneous drying rate (dr) was calculated from the drying data by estimating the change in moisture content, which occur in each ! ital. j. food sci., vol 28, 2016 673 consecutive time interval (dt) and was expressed as a gram of water/gram of dry matter per minutes. dr= (mt + dt -mt) /dt (3) where, mt = moisture content at time t (g water/g dm) mt+dt = moisture content at time t+ dt (g water/g dm min). 3.4. selection of models the experimental data were fitted using the four models listed in table 1. in order to find the best suitable model for describing the drying behavior of natural and osmosed beetroot, non-linear regression analysis was used for determination of the constant of each model. the effectiveness of each model used was evaluated critically analyzing coefficient of determination (r2), reduced chi square (χ2) and root mean square error (rmse). the best model describing the convective drying characteristics was chosen based on the higher r2 value and lower χ2 and rmse. table 1: selected convective dehydration models. model name with reference model newton (singh et al., 2007) mr= exp(-kt) henderson and pebis (grabowski et al., 2003) mr= a exp(-kt) page (page, 1949) mr = exp(-ktn) wang and singh (wang and singh, 1978) mr= 1+at+bt2 3.5. effective moisture diffusivity an analytical solution of fick’s model of mass diffusion equation for drying biological products in falling period was explained by (crank, 1975). when the plot of logarithm of moisture ratio (ln mr) versus drying time is linear, the moisture diffusivity assumes an independent function of moisture content. in this case, the change of moisture content can be described by the following equation (lopez et al., 2009) ( ) ! ! " # $ $ % & + − + == − − = ∑ ∞ = 2 22 0 2 4 12 exp )12( 18 l tdn nm m mm mm mr eff no t eo et π π (4) where, deff is the effective moisture diffusivity (m2/sec) and mr is the moisture ratio. since the top surface of slices was only exposed to hot air, the length, l, in eq (4) was the thickness of the slabs. for long drying times; n = 0, then eq. (4) can be written as; ! ital. j. food sci., vol 28, 2016 674 ! ! " # $ $ % & −! " # $ % & = t l d mr eff . 4 .8 ln)ln( 2 2 2 π π (5) the effective moisture diffusivity was calculated using the method of slopes. when logarithm of mr values v/s drying time were plotted in accordance with eq. (5), straight lines were obtained at all temperatures and sample thickness was investigated. with the help of linear regression analysis, numerical values of diffusion coefficients were obtained for different drying conditions from the slope of the straight lines. 3.6. determination of activation energy effective diffusivity dependence on drying air temperature was obtained from arrhenius relationship to calculate the activation energy (lopez et al., 2009). ! ! " # $ $ % & + − = )273( exp tr e dd g a oeff (6) where, t is temperature (°c), rg is gas constant having a constant value of 8.314 kj/mol k, do is the pre-exponential factor of arrhenius equation, m2s-1. ea is the activation energy kj mol-1. the above exponential form of arrhenius equation can be expressed as; ( )273 lnln + −= tr e dd g a oeff (7) a plot of ln deff versus 1/(t+273) gives a straight line of slope ea/rg slope and consequently activation energy. 3.7. preparation of beetroot candy osmo-convectively dried beetroot cubes at different temperatures were immersed in sucrose syrup (70°bx) for candy preparation. sugar in powder form was crystallized on the surface of beetroot cubes during the cooling process (gupta et al., 2012). the resulting beetroot candy was packed in low-density polyethylene bags and kept at room temperature for sensory analysis. 3.8. sensory evaluation organoleptic attributes like color, flavour, taste, texture, and overall acceptability of beetroot candy were determined using a 9-point hedonic scale with the help of a 10member consumer panel (wang et al., 2009). the average scores of all 10 panelists were computed for different characteristics. ! ital. j. food sci., vol 28, 2016 675 3.9. statistical analysis all the data obtained from convective drying of osmosed and natural beetroot were analysed to find out statistical parameters like r2, χ2 , rmse and drying constants for different models using statistical package for social sciences (spss). intra-pair significant differences especially for sensory quality attributes were determined using duncan’s tests at 5% level of significance. 4. results and discussion 4.1. effect of osmotic pre-treatment on convective drying kinetics table 2 indicates that, the total convective dehydration time at 55°c of natural beetroot was 690 min, but was 570 min for samples given osmotic pre-treatment with the sucrose solution. therefore, beetroot samples given osmotic pre-treatment in sucrose solution consequently reduced convective drying time approximately by 120 min when compared with the convective drying of natural beetroot samples (not treated with the sucrose solution) at 55°c air temperature. similar behaviors have also been observed at 65°c and 75°c drying air temperatures where the convective drying time reduced for the beetroot samples given osmotic pretreatment in sucrose solution by approximately 90 min and 140 min respectively. this might have happened due to leaching of some volatile components of the cellular structure during soaking in osmotic solution. this reduces cell wall resistance and increases drying of beetroot, the results obtained were consistent with data reported by other authors for apricot cubes and melons (riva et al., 2004; rodrigues and fernandes, 2007). table 2: total convective drying time for natural and osmosed dried beetroot. temperature (°c) average drying time (min) natural osmosed 55 690 570 65 570 480 75 460 320 4.2. effect of drying air temperature on drying kinetics the drying curves for all the drying experiments performed are reported in figs. 1 and 2. fig 1 shows the variation of moisture content of osmosed and natural beetroot samples with time for different temperatures. to dry natural and osmosed beetroot sample to final moisture content of 9±1% (w.b.), the drying time at 75 0c was lower as compared to drying time at 65°c and 55°c. temperature increase cause diffusion coefficient to get higher values, and then drying rate increases. similar results have been obtained in case of carrot (dias i., 2009), garlic (madamba et al., 1996) and eggplant (doymaz and ismail, 2011). it can be seen (fig. 1) that, the osmosed and natural beetroot did not have any constant rate drying period and complete drying took place during the falling rate period. the absence of a constant rate period was because the product could not provide a constant supply of water for an appreciable period of time for rapid thin-layer drying of ! ital. j. food sci., vol 28, 2016 676 the product for initial stages of drying (lahsansi et al., 2004; prakash et al., 2004). drying in the falling rate period showed that internal mass transfer occurred by diffusion. similar results have been obtained by different authors for drying of vegetables and fruits (dias et al., 2009; madamba et al., 1996). figure 1: effect of air temperature on drying behavior of osmosed and natural beetroot cubes at different drying air temperature. effect of drying temperatures on a variation of the drying rate with moisture content for osmosed and natural beetroot samples is shown in fig. 2. increasing the drying temperature results in an increase of the drying rate and a decrease of the total time of drying. drying rate later decreased with decreasing moisture for natural and osmotically pre-treated samples under all the conditions of convective dehydration. ! ital. j. food sci., vol 28, 2016 677 figure 2: effect of air temperature on drying behavior of natural and osmosed beetroot cubes at different drying temperature. the reason for the reduction of drying rate might be due to the reduction in porosity of the material and also due to shrinkage as the drying process advances. there was a decline in drying rate for both osmosed and natural beetroot. at the end, when moisture content of beetroot cubes neared 1 g [water] g-1 [dm], drying rate curves showed very low drying rate. therefore, a considerably long drying period would be necessary to achieve final moisture content lower than 1 g [water] g-1 [dm]. as indicated by drying rate curves in fig. 2, the migration of moisture to the surface and evaporation (drying) rate from the surface decreased with decreasing moisture in the product. however, closer examination of drying rate for experimental data for values below moisture content of 1 g [water] g-1 [dm] ! ital. j. food sci., vol 28, 2016 678 for natural beetroot having higher drying rate compared to osmosed beetroot. this might be due to the resistance offered by solute gain during osmotic pre-treatment. 4.3. validity of empirical models for convective dehydration the statistical results with respect to terms of r2, χ2, rmse and drying constants k for newton, a and k for henderson-pabis and k, n for page and a, b for wang & singh models are summarized in tables 3 and 4, where t is the drying temperature. table 3: statistical results obtained for different convective drying conditions for natural beetroot cubes. t (oc) model k drying coefficients r 2 rmse χ2 55 newton 0.002 ----- 0.969 0.06848 0.00498 hendersonpabis 0.002 a=1.106 0.972 0.08645 0.00781 page 0.001 n=1.471 0.997 0.05524 0.00293 wang and singh ---- a=-0.002 b=3.97e-07 0.987 0.16862 0.02972 65 newton 0.003 ------ 0.954 0.06888 0.00496 hendersonpabis 0.003 a=1.113 0.973 0.05375 0.00303 page 0.001 n=1.467 0.997 0.04860 0.00243 wang and singh ----- a=0.002 b=0.00001 0.986 0.04919 0.00253 75 newton 0.004 ------- 0.983 0.07998 0.00682 hendersonpabis 0.004 a=1.111 0.958 0.06528 0.00453 page 0.002 n=1.587 0.997 0.04913 0.00298 wang and singh ----- a=-0.003 b=0.000000115 0.985 0.11529 0.01418 r2 values greater than 0.95 indicate a good fit, the appropriateness of the selected model is also confirmed by lower value of rmse and χ2 (dias et al., 2009). among the models selected for convective drying, the page model implies an excellent consistency in all the ranges of drying temperature (bold numbers in tables 3 and 4) and this model may be assumed to represent the drying behavior of beetroot cubes in a convective dryer within examined range. 4.4. effective diffusivity for convective dehydration during convective dehydration, the effective moisture diffusivity of natural samples and osmosed samples at drying air temperatures ranging from 55 to 75°c varied between 8.09917 × 10-9 to 1.45785 × 10-8 m2s-1, between 1.13388 × 10-8 to 1.61983 × 10-8 m2s-1, respectively. the results obtained are in good agreement with that reported in the literature (zogzas et al., 1996). ! ital. j. food sci., vol 28, 2016 679 table 4: statistical results obtained for different convective drying conditions for osmosed beetroot cubes. t (oc) model k drying coefficients r 2 rmse χ2 55 newton 0.003 -------0.957 0.07316 0.00563 hendersonpabis 0.004 a=1.086 0.972 0.05969 0.00375 page 0.002 n=1.415 0.987 0.05543 0.00324 wang & singh ------a=-0.002 b=0.0000013 0.979 0.13709 0.01978 65 newton 0.004 0.961 0.06868 0.00498 hendersonpabis 0.005 a=1.097 0.977 0.05568 0.00327 page 0.001 n=1.373 0.986 0.04717 0.00228 wang & singh ------a=-0.003 b=0.0000029 0.976 0.09645 0.00982 75 newton 0.006 0.966 0.06306 0.00428 hendersonpabis 0.007 a=1.062 0.974 0.05904 0.00375 page 0.001 n=1.451 0.996 0.05869 0.00347 wang & singh ----a=-0.004 b=0.0000047 0.992 0.09136 0.00899 4.5. activation energy for convective dehydration the activation energy of convective dried natural beetroot cubes were 27.92 kj mol-1 compared to 16.98 kj mol-1 for osmosed samples. activation energy was higher for natural beetroot cubes compared to osmosed beetroot cubes, it may be due to the presence of high initial moisture content of 85% (w.b.) for natural beetroot samples compared to 74% (w.b.) for pre-osmosed samples. therefore, more thermal energy would be required to remove greater amounts of water from natural beetroot cubes. effect of convective drying temperature and time of activation energy were also reported for osmosed and natural samples (reppa et al., 1999). similar reports were also found for pears where activation energy was 26.46-31.21 kj mol-1 for pears without osmotic dehydration and 24.34-28.20 kj mol-1 for pears with osmotic dehydration (park et al., 2002). 4.6. sensory analysis for osmo-convective dried beetroot candy the average score of colour of osmo-convective dried beetroot candy at temperature 65°c was 8.73 compared to temperature at 55°c and 75°c which obtained the score of 8.50 and 8.26 (table 5) respectively. table 5: effect of convective drying temperature on sensory quality of beetroot candy. temperature (oc) colour flavour taste texture oa 55 8.50±0.10b 8.30±0.17a 8.26±0.20b 8.3±0.17a 8.45±0.15b 65 8.73±0.25a 8.53±0.40a 8.4±0.15a 8.56±0.42a 8.59±0.16a 75 8.26±0.15a 8.23±0.05a 8.0±0.10a 8.0±0.12a 8.20±0.05a means in the same column with different letters as superscripts are significantly different (p<0.05). ! ital. j. food sci., vol 28, 2016 680 in case of flavor of osmo-convective dried beetroot candy, the average value at temperature 65°c was higher compared to temperature at 55 and 75°c. highest score values of 8.4 and 8.56 were awarded to osmo-convective dried beetroot candy at temperature 65°c for its taste and texture respectively. so the overall acceptability score of beetroot candy dried at temperature 65°c was highest over other two temperature treatments. the above results indicated that osmo-convective dried beetroot candy at temp 65°c was the most-preferred candy. 5. conclusions in the present study for preparing a shelf-stable product having final moisture content of 9 ±1% (w.b.), osmotic pre-treatment before convective dehydration of beetroot cubes results in a decrease of the total convective dehydration time. osmotic pre-treatment also results in increases in drying rate and effective moisture diffusivity and decreasing activation energy during convective dehydration. among the empirical models applied to the data, the page model best describes the convective drying characteristics of natural and osmosed cubes. among osmo-conevctively dried beetroot candies at different temperatures, most preferred candy was obtained at 65°c. nomenclature deff effective moisture diffusivity (m 2s-1) mt moisture content at any time t on dry basis (g water/ g dry matter) ea activation energy mr moisture ratio (dimensionless) r2 coefficient of determination mo initial moisture content t time me equilibrium moisture content t temperature (oc) χ2 reduced chi-square rg gas constant (8.3143kj mol -1k-1) rmse root mean square error do pre-exponential factor of arrhenius equation, m 2s-1 references aoac. 2000. “official method of analysis” 17th ed. association of official analytical chemists, washington, dc. atamanova a., brezhneva t.a., slivkin a.i., nikolaevskii v.a., selemenev v.f. and crank j. 1975. “mathematics of diffusion” 2nd ed. oxford university press, london. barroca m.j. 2012. study of drying kinetic of quince. in proceedings of international conference of agricultural engineering cigr ag, valenica, spain , july 8-12. crank j. 1975. “mathematics of diffusion”, 2nd ed . oxford university press, london. dezwart f.j., slow s., payne r.j., lever m., george p.m., gerard j.a. and chambers s.t. 2003. glycine betaine and glycine betaine analogues in common foods. food chem. 83:197-204. dias m.g., camoes m.f.g.f.c. and oliveira l. 2009. carotenoids in traditional portuguese fruits and vegetables. food chem. 113:808-815. doymaz i. and ismail. 2011. drying characteristics of sweet cherry. food and by products process. 89:31-38. ertekin c. and yaldiz o. 2004. drying of eggplant and selection of a suitable thin layer drying model. j. food eng. 63:349-359. ! ital. j. food sci., vol 28, 2016 681 fasogbon b.m., gbadamosi s.o. and taiwo k.a. 2013. studies on the osmotic dehydration and rehydration characteristics of pineapple slices. food process. technol. 4:4-11. grabowski s., marcotte, m. and ramaswamy h.s. 2003. drying of fruits, vegetables, and spices. ch. 23. in “handbook of postharvest technology:cereals, fruits, vegetables, tea, and spices”. chakraverty a, mujumdar as, raghavan gsv, rawaswamy hs (ed.), p 653-695. marcel dekker publishing co., new york. gupta r., singh b. and shivare u.s. 2012. optimization of osmo-convective dehydration process for the development of honey-ginger candy using response surface methodology. drying tech. 30:750-759. henderson s. m. and pabis s. 1961. grain drying theory. ii:temperature effects on drying coefficients. j. agri. eng. res. 6:169-174. kapadia g.j., tokuda h., konoshima t. and nishino h. 1996. chemoprevention of lung and skin cancer by beta vulgaris (beet) root extract. cancer letters. 100:211-214. kar a. and gupta, d.k. 2003. air drying of osmosed button mushrooms. j. food sci. technol. 40:23-27. lahsasni s., kouhila m., mahrouz m. and jaouhari j. t. 2004. drying kinetics of prickly pear fruit (opuntia ficus indica). j. food eng. 61:173-179. lenart a. 1996. osmotic-convective drying of fruits and vegetables:technology and application. drying technol. 18:951966. lewicki p.p. 2006. design of hot air drying for better foods. trends in food sci. and technol. 17:153-163. lopez r., delta a., and vaca m. 2009. drying of pricky pear cactus cladodes (opuntia ficus indica) in a forced convection tunnel. energy converse. manage. 50:2119-26. madamba p. s., driscoll, r. h. and buckle k. a. 1996. the thin layer drying characteristics of garlic slices. j. food eng. 29:75-97. marfil p.h.m., santos e.m. and telis v.r.n. 2008. ascorbic acid degradation kinetics in tomatoes at different drying conditions. lwt-food sci. technol. 41:1642-1647. orsat v., changrue v. and raghavan g.v. 2006. microwave drying of fruits and vegetables. stewart post-harvest rev. 6:4-9. page g. 1949. factors influencing the maximum rates of air drying shelled corn in thin layer. msc thesis, purade university. lafayette, in, usa. park k. j., bin a. and brod f. p. r. 2002. drying of pear d’anjou with and without osmotic dehdration. j. food eng. 56:97-103. patkai g., barta j. and varsanyi i. 1997. decomposition of anticarcinogen factors of the beetroot during juice and nectar production. cancer letters. 114:105-106. prakash s., jha s. k. and datta n. 2004. performance evaluation of blanched carrots dried by three different driers. j. food eng. 62:305-313. raoult-wack a. l., guilbert s., le maguer m. and andrios g. 1991. simultaneous water and solute transport in shrinking media:application to dewatering and impregnation soaking process analysis (osmotic dehydration). drying technol. 9:589-612. rastogi n.k., eshtiaghi m.n. and knoor d. 1999. accelerated mass transfer during osmotic dehydration of high intensity electrical field pulse pretreated carrots. j. food sci. 64:1020-1023. reppa a., mandela j., kostaropoulos, and saravacos g. d. 1999. influence of solute temperature and concentration on the combined osmotic and drying. drying technol. 17:1449-1458. riva m., campolongo s., leva a. a., maestrelli a. and torreggiani d .2004. structureproperty relationship in osmo-airdehydrated apricot cubes. food res. intern. 38:533-542. rodrigues s. and fernandes f. a. n. 2007. dehydration of melons in a ternary system followed by air drying. j. food eng. 80:678-687. sharma g.p., prasad s. and datta a.k. 2003. drawing kinetics of garlic cloves under convective drawing conditions. j. food sci. technol. 40:45-51. ! ital. j. food sci., vol 28, 2016 682 shi j, le maguer m. 2002. osmotic dehydration of foods:mass transfer modeling aspects. food rev. intern. 18:305–35. shi j., le maguer m., kakuda y., liptay a. and niekamp f. 1999. lycopene degradation and isomerization in tomato dehydration. food res. int. 32:15-21. shynkaryk m.v., lebovka n.i. and vorobiev e. 2008. pulsed electric fields and temperature effects on drying and rehydration of red beetroot. drying technol. 26:695-704. singh b., kumar a. and gupta a. k. 2007. study of mass transfer kinetics and effective diffusivity during osmotic dehydration of carrot cubes. j. food eng. 79:471-480. singh d.b., kingly a.r.p. and jain r.k. 2007. studies on separation techniques of pomegranate arils and their effect on quality of anardana. j. food eng. 79:671-674. vali l., stefanovits-banyai e., szentmihalyi k., febel h., sardi e., lugasi a., kocsis i. and blazovics a. 2007. liverprotecting effects of table beet (beta vulgaris var. rubra) during ischemia-reperfusion. nutrition. 23:172-178. wang c. y. and singh r. p. 1978. a single layer drying equation for rough rice. st. joseph, mi:asae. asae paper no:783001. wang r., zhang m., mujumdar a.s. and sun jin-cai. 2009. microwave freeze–drying characteristics and sensory quality of instant vegetable soup. drying technol. 2009. 27(9):962-968. zogzas n.p., maroulis, z.b. and marinos-kouris d. 1996. moisture diffusivity data compilation in food stuffs. drying technol. 14:2225-53. paper received february 13, 2015 accepted june 5, 2015 #420_caponio_bozza ! ital. j. food sci., vol 28, 2016 705 paper talc effect on the volatiles of virgin olive oil during storage f. caponio*, g. squeo, c. summo, v.m. paradiso and a. pasqualone university of bari aldo moro, department of soil, plant and food sciences, food science and technology unit, via amendola 165/a, 70126 bari, italy *corresponding author. fax: +39 0805443467 e-mail address: francesco.caponio@uniba.it abstract the aim of the study was to assess the influence of talc on the extra virgin olive oil volatile profile during production and storage. the obtained results showed that talc used at 1% level did not cause significant differences, whereas at 2% level showed only slight differences were revealed compared to control. during storage a significant increase of the volatiles deriving from the oxidative process was observed and a concomitant significant decrease of the c6-lox aldehydes. the evolution of the volatile compounds of extra virgin olive oils during storage was not significantly influenced by talc addition, with the exception of the sum of c6-lox aldehydes, which showed a significant higher decrease. keywords: extra virgin olive oil, storage, talc, volatile compounds ! ital. j. food sci., vol 28, 2016 706 1. introduction volatile compounds, together with phenolics, are responsible for the sensory attributes of extra virgin olive oil (morales et al., 1995; aparicio et al., 1996) and play a key role in the marketing process, by influencing the choice of the consumer. volatiles arise during olive (olea europaea l.) ripening (angerosa and basti, 2001; aparicio and morales, 1998) and are influenced by oil extraction processes (angerosa et al., 2000; kiritsakis, 1998). moreover, they may be altered during storage, especially if the main factors promoting product oxidation such as heat, light, and air are not well-managed (frankel, 1985; morales and tsimidou, 2000; brkić bubola et al., 2014). it is well known that in fresh extra virgin olive oils the volatile compounds derive from the degradation of polyunsaturated fatty acids, through the lipoxygenase (lox) pathway (angerosa et al., 2000; kalua et al., 2007), which produce c5 and c6 volatiles, mainly responsible for the “green” odor notes (angerosa et al., 2004). among these volatiles, c6 unsaturated and saturated aldehydes, whose amount depends on the activity of the enzymes involved in lox pathway (angerosa et al., 2001), are the most abundant compounds in high quality virgin olive oils (angerosa et al., 2004). in recent years the use of coadjuvants in the oil mills has increased considerably, bringing increments of extraction efficiency during oil production and decreasing pomace moisture. it is common knowledge that the extraction systems can only recover about 80-90% of the oil contained in the starting olives, with the remainder being trapped in microgels or emulsified with the water phase (aguilera et al., 2010). the extraction yield is further lowered in case of the so-called “difficult pastes” (caponio et al., 2014a), derived by batches of olives having a heterogeneous ripening index. to increase extraction effectiveness, malaxation is the key phase of virgin olive oil production process and, generally, increasing the temperature determines an increase in oil extraction yield, due to a reduction of viscosity in the oily phase; however, the collateral effects of increasing temperature and time at this step are well known (angerosa et al., 2001). more recently, new technologies were also proposed for oil industries, among which ultrasounds, microwaves, and mechanical vibrations (bejaoui et al., 2016; tamborrino et al., 2014; toschi et al., 2013). on the other hand, innovation in the field of olive oil extraction lead to the spread of a new generation of two-way decanters, which do not require the addition of water to the olive paste and result in obtaining high quality oils, without losing the minor compounds responsible for sensory, nutritional, and healthy features of olive oil. however, these decanters generate pomaces with a moisture content of higher than 55% (caponio et al., 2014a; tamborrino et al., 2015), which is an excessively high value for pomace refineries, necessitating to expensive preliminary drying. talc is a commonly used coadjuvant, not excluded by the ec regulation no. 1513/2001 in the production of extra virgin olive oil, due to its exclusively physical action (cert et al., 1996). it is added during malaxation and has a positive effect on oil yield, especially in the case of difficult olive pastes (caponio et al., 2016). all investigations highlighted an effect of talc on the chemical parameters of extra virgin olive oil. nevertheless, few studies are available regarding the influence of talc on the volatile compounds of extra virgin olive oil. in particular, caponio et al. (2015) highlighted that talc addition did not significantly influence the sums of aldehydes, alcohols, and ketones; on the contrary, the sums of acids and esters significantly increased with talc addition. koprivnjak et al. (2016) did not find significant changes of c6 aldehydes and c5 compounds, whereas with 3% of talc a significantly higher value of c6 alcohols was observed. the authors concluded that correlations between the degree of talc addition and levels of volatile compounds are not clearly evident. moreover, no information is available about the evolution of the volatile ! ital. j. food sci., vol 28, 2016 707 compounds of extra virgin olive oil obtained with talc addition to olive paste malaxation during storage. in this framework, the aims of the present work were: i) to further investigate the influence of talc on the volatile compounds of extra virgin olive oils; ii) to evaluate whether the addition of talc to olive paste during malaxation influenced the evolution of the volatile fraction of extra virgin olive oils during storage. olive fruits of coratina, a cultivar that often leads to difficult olive pastes, were processed with and without talc addition at two different levels, and the corresponding oils were analyzed over a period of 6 months. 2. materials and methods 2.1. material olive fruits (olea europaea l.) from coratina cultivar, mechanically harvested in december 2013 were transported, immediately after harvesting, to a local plant (andria, bari, italy) where, after leaf-removal, were milled within 24 h. talc (hydrated magnesium silicate) was kindly furnished by imerys talc (luzenac, france). 2.2. oil extraction process three lots of about 2,000 kg of olives were considered in the trials. each lot was divided into three homogeneous batches: one batch was processed without micronized natural talc addition (co, control) and the remaining two batches were processed by adding 1% and 2% of talc (t1 and t2), respectively. talc was added at the beginning of the malaxation phase. for each batch, the olive paste after crushing was transferred into the malaxer, where it was mixed with talc only for the trials that provided its addition. after malaxation (50 min at 22±1°c) the paste was pumped into a two-phase decanter, operating at 2,800 rpm, with a processing capacity of 3,000 kg h-1. finally, the oily phase was separated from any aqueous residue by centrifugation at 6,400 rpm. then, the obtained oil was poured into 1-l clear glass bottles and hermetically sealed with headspace of about 3 ml. three bottles for each batch were sampled. one bottle for each batch was immediately analyzed (time 0), whereas the others were placed in a carton box and stored in the dark to be analyzed after three (time 3) and six (time 6) months of storage. the samples were stored at room temperature. 2.3. volatile compounds determination for the determination of the volatile compounds, the oil samples (0.5 ± 0.005 g) were weighed into 20 ml vials, sealed with a screw aluminium cap and silicone/ptfe septa, and submitted to the spme/gc-ms in the conditions reported in a previous paper (caponio et al., 2014b). in particular, the extraction was performed by exposing a 75 µm carboxen/polydimethylsiloxane (car/pdms) fiber (supelco, bellefonte, pa, usa) in the headspace of the sample at 40°c for 20 min. when the extraction process was completed, the fiber was inserted into the injector port (set at 230°c) of the gas chromatograph for thermal desorption of volatiles. the gc/ms instrumentation included an agilent model 6850 gas chromatograph coupled to a mass spectrometer agilent 5975. the volatile compounds were separated on a hp-innowax (60 m × 0.25 mm, 0.25 #m film thickness) polar capillary column (agilent) under the following conditions: flow 1.5 ml min-1; injector ! ital. j. food sci., vol 28, 2016 708 temperature, 250°c; pressure of the carrier (helium), 30 kpa. the oven temperature was held for 5 min at 35°c, then increased by 5°c min-1 to 50°c and held constant for 5 min, then raised to 230°c at 5.5°c min-1 and finally held at 230°c for 5 min. the mass spectrometer was operated in the electron impact mode (electron energy = 70 ev), and the ion source temperature was 250°c. a continuous scan mode was employed with a scan time of 7.7 scans/s over a mass range of 33-200 amu. the volatile compounds were identified both by comparison of their mass spectra and retention times with those of authentic reference compounds and by their lri and by comparison with the mass spectra present in the nist and wiley libraries. the volatile compounds were expressed as integrated area and its relative percentage. 2.4. statistical analysis analysis of variance (one-way and two-way anova) was carried out on the experimental data using the minitab software (minitab inc., state college, usa). two-way anova was performed considering the amount of talc added (talc) and oil storage time (time), as well as the first order interaction (talc*time), as independent variables; tukey’s hsd test was applied for multiple comparisons. 3. results and discussion all the samples showed values of free fatty acids, peroxides, k232, k270, and dk which fulfilled the extra virgin olive oil marketing requirements according to current rules (official journal of the european communities, 2011) (data not shown). table 1 reports the volatile compounds detected in the oil samples, obtained with and without the addition of talc during processing, immediately after their production (fresh oils). the volatiles were grouped on the basis of their most probable origin. the overall volatile profile agreed with the findings of other authors for italian extra virgin olive oils (angerosa et al., 2004; kalua et al., 2007), which were found to be richer in c6 volatile compounds and poorer in esters than spanish and moroccan extra virgin olive oils (reiners and grosch, 1998). the low amounts of volatiles generated by autooxidation and/or sugar fermentation evidenced the high quality level of the starting olives. the most represented volatile compound was trans-2-hexenal, accounting for more than 80% headspace. it derives from the lox activity involving polyunsaturated fatty acids and is responsible for bitter, almond, and green notes (morales et al., 1997; bendini et al., 2009). cavalli at el. (2004) analyzing seven french cailletier olive oils, found a content of trans-2-hexenal ranging from 37-64%. the volatile profile of the oils obtained with talc addition generally corresponded to data reported in literature (brkić bubola et al., 2014; caponio et al., 2015; cavalli et al., 2004; koprivnjak et al., 2016). in particular, talc used at 1% level did not cause significant differences, whereas at 2% level a significant increase of ethanol (responsible for alcoholic, ripe apple, and floral notes) (reiners and grosch, 1998; servili et al., 2001) and a decrease of 1-penten-3-one (associated with the green and pungent notes), trans-2-penten-1-ol (responsible for green notes), and ethyl acetate (responsible for sweet and aromatic notes) (angerosa et al., 2004; bendini et al., 2009) were observed. ! ital. j. food sci., vol 28, 2016 709 table 1: integrated area mean value and results of one-way anova (p < 0.05) of the volatile compounds detected in oil samples obtained without (co) and with addition of 1% and 2% of talc during processing (t1 and t2, respectively), analyzed immediately after production (fresh oils). volatile compounds co t1 t2 area % area % area % c6 lox pathway hexanal 6.84e+07 a 3.26 6.96e+07 a 3.38 6.59e+07 a 3.24 trans-2-hexenal 1.72e+09 a 81.90 1.67e+09 a 81.31 1.66e+09 a 81.59 hexan-1-ol 2.61e+07 a 1.25 2.45e+07 a 1.19 2.59e+07 a 1.28 cis-3-hexenal 1.12e+07 a 0.53 9.06e+06 a 0.44 9.02e+06 a 0.44 trans-3-hexen-1-ol 1.01e+06 a 0.05 1.07e+06 a 0.05 1.03e+06 a 0.05 cis-3-hexen-1-ol 4.15e+07 a 1.98 4.42e+07 a 2.15 4.42e+07 a 2.18 cis-2-hexen-1-ol 4.40e+07 a 2.10 4.52e+07 a 2.20 4.63e+07 a 2.28 c5 lox pathway 1-penten-3-one 4.54e+07 a 2.17 4.48e+07 a 2.18 3.92e+07 b 1.93 trans-2-pentenal 6.26e+06 a 0.30 6.67e+06 a 0.32 5.99e+06 a 0.30 1-penten-3-ol 9.99e+06 a 0.48 8.72e+06 a 0.42 9.08e+06 a 0.45 trans-2-penten-1-ol 2.50e+06 a 0.12 2.30e+06 a 0.11 1.85e+06 b 0.09 cis-2-penten-1-ol 2.47e+07 a 1.18 2.69e+07 a 1.31 2.47e+07 a 1.22 carbohydrate fermentation ethyl acetate 1.46e+07 a 0.70 1.26e+07 a 0.61 7.63e+06 b 0.38 ethanol 3.22e+07 a 1.54 4.06e+07 ab 1.97 4.49e+07 b 2.21 acetic acid 1.74e+07 a 0.83 1.44e+07 a 0.70 1.55e+07 a 0.76 other origins methyl acetate 3.34e+06 a 0.16 3.06e+06 a 0.15 3.11e+06 a 0.15 pentan-3-one 1.02e+07 a 0.49 8.53e+06 a 0.41 8.58e+06 a 0.42 cis,cis,cis-1,3,6-octatriene, 3,7dimethyl 3.14e+06 a 0.15 3.17e+06 a 0.15 3.00e+06 a 0.15 nonanal 3.75e+06 a 0.18 4.20e+06 a 0.20 3.95e+06 a 0.19 trans,trans-2,4-hexadienal 5.36e+06 a 0.26 5.79e+06 a 0.28 5.78e+06 a 0.28 trans,trans-2,4-heptadienal 8.28e+05 a 0.04 1.01e+06 a 0.05 7.34e+05 a 0.04 benzaldehyde 1.85e+06 a 0.09 2.29e+06 a 0.11 1.90e+06 a 0.09 propanoic acid 6.20e+05 a 0.03 5.87e+05 a 0.03 7.29e+05 a 0.04 hexanoic acid 1.44e+06 a 0.07 1.32e+06 a 0.06 1.53e+06 a 0.08 phenylethyl alcohol 3.56e+06 a 0.17 4.02e+06 a 0.20 3.45e+06 a 0.17 with the aim of evaluating the effect of talc on the volatiles, the variations of groups of compounds having the same origin, and belonging to the same chemical class, have been monitored during storage in the oils with or without talc addition. moreover, some specific compounds considered to be effective indices of oil oxidations have been focused. ! ital. j. food sci., vol 28, 2016 710 figures 1 and 2 show the evolution of c6 and c5 volatile compounds, respectively, both derived from the lox pathway. figure 1: percent mean value and results of two-way anova (p < 0.05) of c6 volatile compounds deriving from the lipoxygenase (lox) pathway of the oils obtained without (co) and with addition of 1% and 2% of talc (t1 and t2, respectively) during storage: total (a), aldehydes (b), and alcohol (c). 0, fresh oils; 3, oil stored for three months; 6, oil stored for six months. time trial 630 t2t1cot2t1cot2t1co 90 80 70 60 50 40 30 20 10 0 c 6l o x ( % ) a a a ab b c d e e a time trial 630 t2t1cot2t1cot2t1co 90 80 70 60 50 40 30 20 10 0 c 6l o x a ld he yd es ( % ) b a a a b b c d e e time trial 630 t2t1cot2t1cot2t1co 12 10 8 6 4 2 0 c 6l o x a lc oh ol s (% ) c d d d bc c bc ab ab a ! ital. j. food sci., vol 28, 2016 711 figure 2: percent mean value and results of two-way anova (p < 0.05) of c5 volatile compounds deriving from the lipoxygenase (lox) pathway of the oils obtained without (co) and with addition of 1% and 2% of talc (t1 and t2, respectively) during storage. 0, fresh oils; 3, oil stored for three months; 6, oil stored for six months. the c6-lox compounds (fig. 1a), which represent about 90% of the volatile compounds, showed a significant decrease during storage, as observed also by other authors (bendini et al., 2009), more evident in the trials involving the use of talc. the observed trend was mainly attributable to the evolution of the c6-lox aldehydes (fig. 1b), which significantly decrease during storage, while c6-lox alcohols (fig. 1c), that representing about 5% of the volatile fraction, did not show a significant variation among trials. the significant increase of the latter during time, as reported by cavalli et al. (2004), could be attributed to the formation of alcohols by decomposition of methyl linolenate hydroperoxides (frankel, 1980). the c5-lox compounds (fig. 2) showed a significant increase during storage, more evident after 3 months. the increase of c5-lox compounds was mainly due to the increase of trans-2-pentenal (fig. 4b) and of 2-penten-1-ol (data not shown), that could arise from the decomposition of methyl linolenate hydroperoxides (frankel, 1980). in fact, bendini et al. (2009) reported that trans-2-pentenal increases significantly during the oxidation of virgin olive oils in presence of metals. ketones (fig. 3) did not show significant variation up to 3 months of storage whereas a significant increase after six months of storage was observed, in agreement with the findings of cavalli et al. (2004). talc addition did not show to significantly influence the variations of ketones (fig. 3) and of c5-lox compounds (fig. 2). several authors report that the hexanal/nonanal ratio is an appropriate index to detect the beginning of olive oil oxidation and to follow its evolution (morales et al., 1997; kiritsakis, 1998; angerosa et al., 2004; kanavouras et al., 2004; bendini et al., 2009). moreover, also trans-2-pentenal, an unsaturated aldehyde originated by secondary reactions of the primary auto-oxidation products (13-lnooh, 9-looh and 9-oooh, respectively), could effectively monitor the oxidative process evolution of extra virgin olive oil (luna et al., 2006). finally, propanoic and hexanoic acids, the latter due to the oxidation of hexanal, and 2,4-decadienal, derived from fatty acid oxidation (reiners and grosch, 1998), have also been detected during the oxidation (gutierrez et al., 2002; vichi et al., 2003). fig. 4 shows the variations of the above reported indices during storage. time trial 630 t2t1cot2t1cot2t1co 6 5 4 3 2 1 0 c 5 l o x ( % ) de de e b ab a bc ab cd ! ital. j. food sci., vol 28, 2016 712 figure 3: percent mean value and results of two-way anova (p < 0.05) of sum of ketone volatile compounds of the oils obtained without (co) and with addition of 1% and 2% of talc (t1 and t2, respectively) during storage. 0, fresh oils; 3, oil stored for three months; 6, oil stored for six months. time trial 630 t2t1cot2t1cot2t1co 6 5 4 3 2 1 0 k et on es ( % ) b b b b b b a a a time trial 630 t2t1cot2t1cot2t1co 20 15 10 5 0 h ex an al /n on an al a b b c c c d d d a time trial 630 t2t1cot2t1cot2t1co 0.5 0.4 0.3 0.2 0.1 0.0 tr an s2pe nt en al ( % ) b bc b bc bc bc c a a a ! ital. j. food sci., vol 28, 2016 713 figure 4: percent mean value and results of two-way anova (p < 0.05) of hexanal/nonanal ratio (a), trans2-pentenal (b), and sum of propanoic and hexanoic acids (c) of the oils obtained without (co) and with addition of 1% and 2% of talc (t1 and t2, respectively) during storage. 0, fresh oils; 3, oil stored for three months; 6, oil stored for six months. an increase of auto-oxidation phenomena occurred during 6 months, irrespective of the addition of talc, with the only exception of the hexanal/nonanal ratio of fresh oil that indicated a stronger oxidation of talc-added oils than in the control. 4. conclusions the obtained results confirm that talc addition during olive processing determines only slight differences in the volatile compounds profile of the corresponding extra virgin olive oil compared to control obtained without talc addition. in the majority of cases, these differences were devoid of statistical significance. during storage a significant increase of the volatiles deriving from the oxidative process was observed, as shown by the hexanal/nonanal ratio and the sum of propanoic and hexanoic acids, and a concomitant significant decrease of the c6-lox aldehydes responsible for bitter, almond, and green notes of extra virgin olive oil. the evolution of the volatile compounds of extra virgin olive oils during storage was not significantly influenced by talc addition, with the exception of the sum of c6-lox aldehydes which showed a significant higher decrease. references aguilera m.p., beltran g., sanchez-villasclaras s., uceda m. and jimenez a. 2010. kneading olive paste from unripe ‘picual’ fruits: i. effect on oil process yield. j. food eng. 97:533. angerosa f. and basti c. 2001. olive oil volatile compounds from the lipoxigenase pathway in relation to fruit ripeness. ital. j. food sci. 13: 421. angerosa f., mostallino r., basti c. and vito r. 2000. virgin olive oil odour notes: their relationship with volatile compounds from lipoxygenase pathway and secoiridoid compounds. food chem. 68:283. angerosa f., mostallino r., basti c. and vito r. 2001. influence of malaxation temperature and time on the quality of virgin olive oils. food chem. 72:19. time trial 630 t2t1cot2t1cot2t1co 0.30 0.25 0.20 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buxaderas s. and lopez-tamames e. 2003. solid-phase microextraction in the analysis of virgin olive oil volatile fraction: modifications induced by oxidation and suitable markers of oxidative status. j. agric. food chem. 51:6564. paper received february 5, 2016 accepted june 14, 2016 ijfs#670_bozza ital. j. food sci., vol. 29, 2017 591 paper the contamination rate of aflatoxins in ground red peppers, dried figs, walnuts without shell and seedless black raisins commercialized in sakarya city center, turkey s. öztürk yilmaz sakarya university, faculty of engineering, department of food engineering, tr-54187 sakarya, turkey tel.: +90 264 2955832 e-mail address: suzanyilmaz@sakarya.edu.tr abstract the investigation of this study is concerned with the occurrence of aflatoxin total (aft) and aflatoxin b1 (afb1) in 120 specimens randomly bought from retail stores, bazaars, supermarkets and regional stores in sakarya city center. immune affinity (iac) clean-up with high-performance liquid chromatography (hplc) and fluorescence detection (fid) methods were used to investigate the specimens to determine the incidence of aflatoxin (b1, b2, g1, g2) contamination. the findings indicate that the percentages of ground red peppers, dried figs, walnuts without shell and seedless black raisins contaminated with aft are about 72%, 51%, 64% and 64%, respectively. one ground red peppers (18.68-10.49 µg/kg) and one specimen of walnuts without shell (10.26-5.06 µg/kg) exhibit the maximum contamination levels of aft and afb1, respectively. of all the contaminated specimens, two ground red peppers and one dried fig specimens (2.5%) exceed the recommended afb1 (5 µg/kg) limit defined by the turkish food codex (tfc) regulations. this study presents the details of the first inspection regarding the presence of aflatoxins in seedless black raisin specimens in sakarya city center of turkey. keywords: aflatoxin, hplc-fld, pepper, fig, walnut, raisins ital. j. food sci., vol. 29, 2017 592 1. introduction as secondary metabolites, mycotoxins are generated by micro fungi which result in vertebrates contracting illnesses ((luttfullah and hussain, 2011). aflatoxins (afs) from mycotoxin family are immensely deleterious secondary metabolic compounds of aspergillus flavus, a. parasiticus and a. nomius (set and erkmen, 2010). they can cause cancer, mutations and physiological abnormalities in animals and humans (erkmen and bozoglu, 2008). afs are impossible to avoid because they are found in nature as contaminants (iarc 1993). external circumstances such as climate, moisture and the span of precipitation during tilling and crop yield seasons affect the contamination (di̇ni̇ et al., 2013). such products as cereals, oil seeds, nuts, beans and spices are affected the most (reddy et al., 2011). as a toxic chemical, afb1 is one of the most potent substance which causes cancer and is categorized group i by international agency for research on cancer (iarc 1993). the scale of toxicity, afb1>afg1>afb2>afg2, indicates that the fatal furan moiety of afb1 is the significant point to detect the stage of bioactivity of this group of mycotoxins (colak et al., 2006). numerous researches demonstrate that gastrointestinal diseases, hepatic neoplasms and hepatocellular carcinoma observed in humans in africa, philippines and china are related to aflatoxins (luttfullah and hussain, 2011). additionally, it has been manifested that feeding on products polluted by aflatoxin has led to some aflatoxicosis-related epidemics (reddy and raghavender, 2007). aflatoxicosis-related epidemics were observed to affect a large geographical area and result in the demise of 123 people in kenya (cdc, 2004). it is of great importance to safety not only the well-being of people but also the interests of producers by applying an array of action to decrease the aflatoxin contamination to the minimum. if this is not achieved, the situation can have debilitating repercussions for producers in terms of their economic perpetuity and can also debar people from a substantial food source (di̇ni̇ et al., 2013). some studies and investigative research have been conducted in many countries to acquire a prevalent paradigm as regards foods contaminated with aflatoxin (reddy et al., 2011). owing to the fact that afs are toxic and observed quite often, many countries, including turkey, have outlined protocols and tolerance margins for aflatoxins. the turkish food codex (tfc) states that the maximum levels for afb1 and aft in dried fruits are 8 and 10 μg/kg, in nuts or peppers 5 and 10 μg/kg, respectively (tfc, 2011). owing to the considerable risks of health complications caused by aflatoxins in foods, it is of utter importance to collect data regarding the prevalence of these toxic substances in foods in sakarya (turkey). despite a lot of studies on aflatoxin rates in a variety of food which are present in the diet of people in turkey, the research on aflatoxins in ground red peppers, figs and walnuts in sakarya is scarce. therefore, the aim of this study is to specify the aft (afb1, afb2, afg1, and afg2) and afb1 contamination rates in ground red peppers, walnuts without shell, dried figs and seedless black raisins, to show how contamination is a risk to the public health and to compare the findings to the highest aflatoxin tolerance margins defined by the tfc. this is also first survey to determine the incidence of afs contamination in seedless black raisins in sakarya city. 2. materials and methods 2.1. specimen preparation 120 food specimens were purchased in 2014 and 2015 from different retail shops, bazaars, supermarkets and local markets in sakarya city center, turkey. while a few of the ital. j. food sci., vol. 29, 2017 593 specimens were foreign origins, the majority of the specimens were from turkey. they have been produced traditionally and locally consumed in generally. most of the specimens sold in bulk were purchased unpackaged except for a few. the selected commodity groups are 25 ground red peppers and dried fruits including 45 dried figs, 25 walnuts without shell, 25 seedless black raisins (each about 500g). a subsample divider was used to divide the specimens. a 200 g subsample was ground and placed in plastic bags and kept at -20ºc until they were analyzed and put away to avoid sunlight. two specimens were selected and two separate analyses were carried out for each specimen (set and erkmen, 2010). mean results of the four analyses were presented. all the testing and fluxing agents were of lc grade delivered from merck (darmstadt, germany) and sigma (st. louis, usa). the aflatoxin b1, aflatoxin b2, aflatoxin g1, aflatoxin g2 (afb1, afb2, afg1, afg2) mix standard were obtained from supelco (sincer, turkey). standard stock solutions (2600 ng/ml) of mixed aflatoxin were produced using methanol, covered with aluminium foil so that aflatoxins would steadily dissolve under ultraviolet light and be kept for up to 3 months. the stock solutions were thinned to the rates of 0.3 and 1.0 µg/ml (standard solutions) using methanol and kept at -20°c (set and erkmen, 2010). 2.2. aflatoxin analyses in specimens by hplc this study was carried out making a few adjustments using hplc method as described by aoac (2000) and stroka et al. (2000). briefly, 5 g of nacl was put in 50 g of walnuts without shell and afterwards mixed with 200 ml of methanol/water (80:20) and 200 ml of cyclohexane for 3 min. following the parting of the two phases, cyclohexane was done away with. for dried figs, ground red peppers and raisins, specimens were removed only with 200 ml of methanol / water (80:20). extracts were filtrated using a whatman filter paper no. 4 with a pore size of 30 um (luttfullah and hussain, 2011). 10 ml filtrate was thinned using 40 ml phosphate buffer saline (pbs) from sigma (st. louis, mo, usa). 10 ml of filtrate was sent through the immune-affinity column (aflaprep, r-biopharm rhone, scotland) at a speed of 2-3 ml/min. the column was cleaned using 20 ml distilled water. finally, bounded aflatoxins were eluted slowly using 1 ml methanol and air was pushed through the column to collect the last drops of eluate and finally thinned using 1 ml water (özkan et al., 2015). the extract was moved to a 1.8 ml vial for the injection (set and erkmen, 2010). the mobile phase was water-methanol-acetonitrile (5:2:3, v/v/v). the final concentration of the mobile phase was set to be 88.23 mg/l and 120 mg/l by nitric acid (lc grade, sigma-aldrich, germany) and potassium bromide (lc grade, merck, germany) respectively. the mobile phase was filtered through a disposable filter unit (0.45µm). the presence of aflatoxins was observed by hplc (shimadzu, tokyo, japan) using a postcolumn derivatization electrochemically generated bromine (kobra cell) and a fluorescence detector (rf 20a) at 362 nm (excitation) and 450 nm (emission) (set and erkmen, 2010). the intersil ods-3 (25 cm 4.6 mm id, 5 µm, tokyo, japan) column was connected as hplc column and the flow rate was 1 ml/min. 100 µl specimen was injected to hplc automatically. the peaks were then compared with the actual specimen peaks obtained with that of aflatoxin standards. the recovery studies were carried out by spiking to uncontaminated spicemens with two concentration levels of each toxin (aft and afb1) at least 1 hour prior to analysis. the recovery rate of aflatoxins was determined at a rate of 5.2 µg/kg and 2.6 µg/kg in all specimens in triplicate. the fortified spicemens were extracted and analyzed. the limits of detection (lod) and the limits of quantification (loq) were defined according to signal to noise ratio; s/n=3/1 and s/n=10/1, respectively. ital. j. food sci., vol. 29, 2017 594 3. results this study examined aft and afb1 contamination in 25 ground red peppers, 25 walnuts without shell, 25 seedless black raisins and 40 dried fig specimens. the findings were assessed in line with the legal limits for aft and afb1 specified by the turkish food codex (tfc 2011). the following recovery rates were obtained as µg/kg in ground red peppers and dried figs respectively: afb1, 99.8%-106.3%; afb2, 100.6%-100.1%; afg1, 99.0%-98.2% and afg2, 92.3%-102.2%. the limits of detection were as follows: afb1, 0.05 µg/kg; afb2, 0.02 µg/kg; afg1, 0.04 µg/kg and afg2, 0.05 µg/kg. the following recovery rates were calculated as µg/kg in walnuts without shell and seedless black raisins respectively: afb1, 77.4%-84.0%; afb2, 85.1%-107.0%; afg1, 99.0-77.7% and afg2, 75.9%-108.2% (table 1). table 1. recoveries of aflatoxins in the fortified specimens (%). commodities mean recoverya ± rsdb (%) afb1 afg1 afb2 afg2 ground red peppers 99.8±1.5 99.0±4.2 100.6±2.1 92.3±3.4 walnuts without shell 77.4±3.7 99.0±4.5 85.1±2.0 75.9±2.7 seedless black raisins 84.0±1.8 77.7±5.3 107±3.7 108.2±1.8 dried figs 106.3±2.1 98.2±5.3 100.1±3.4 102.2±4.1 anumber of replicates: n= 3; brelative standard deviation. table 2 presents the summary of the results of 120 specimens for aft. the results clearly show that 18 ground red peppers, 23 dried fig specimens, 16 walnuts without shell and 16 seedless black raisins are contaminated with aflatoxins. of all the specimens, ground red peppers have the highest contamination rates. the mean values of ground red peppers, walnuts without shell, dried figs and seedless black raisins are as 2.30 µg/kg, 1.68 µg/kg, 0.40 µg/kg, 1.78 µg/kg, respectively. aft was observed in 73 specimens (68%) and 69 specimens had contamination below rates varying between 0.02 and 3.47 µg/kg; while 3 specimens had contamination varying between 5.30 and 18.68 µg/kg, exceeding the recommended limit for aft. table 2. total aflatoxins in the specimens. commodities contaminated specimens with aft (µg/kg) mean valuea (µg/kg) number of specimens analyzed /positive frequency (%) below limit (<10 µg/kg) above limit (>10 µg/kg) ground red peppers 25/18 72 23 (0.04-3.47)b 2(12.09-18.68) 2.30 walnuts without shell 25/16 64 15(0.66-2.62) 1(10.26) 1.68 seedless black raisins 25/16 64 16(0.02-2.07) 0.40 dried figs 45/23 51 22 (0.16-5.20) 1.78 total 120/73 68 69(0.02-3.47) 3(5.30-18.68) aaverage contamination on positive samples that higher than the lod; btotal aflatoxin range. ital. j. food sci., vol. 29, 2017 595 table 3 demonstrates the results of the analysis of afb1 where there are 34 contaminated specimens at a rate <5 µg/kg, 3 specimens varying between 5 and 10.49 µg/kg. as one can see in table 3, the mean values of ground red peppers, walnuts without shell, dried figs and seedless black raisins are 1.38 µg/kg, 0.86 µg/kg, 0.11 µg/kg, 1.08 µg/kg, respectively. afb1 was observed only in 37 specimens (38%), in addition to 34 not exceeding the established limit and 3 specimens above the established limit. table 3. aflatoxin b1 in the specimens. commodities contaminated specimens with afb1 (µg/kg) mean valuea (µg/kg) number of specimens analyzed /positive frequency (%) below limit (<5-8*µg/kg) above limit (>5-8*µg/kg) ground red peppers 25/8 32 6(0.08-1.96)b 2(7.41-10.49) 1.38 walnuts without shell 25/5 20 5(0.66-2.62) 1(5.06) 0.86 seedless black raisins 25/16 64 16(0.02-0.26) 0.11 dried figs 40/8 20 7(0.29-3.60) 1.08 total 120/37 38 34(0.02-3.60) 3(5.06-10.49) aaverage contamination on positive samples that higher than the lod; baflatoxin b1 range. *aflatoxin b1 maximum limits for dried fruits in turkey two ground red pepper specimens (12.09-18.68 µg/kg) exceed the established limit (10 µg/kg) specified by the turkish regulations for aft (tfc 2011), whereas 18 specimens vary between 0.085 and 3.47 µg/kg with aflatoxins below the established limit. the presence of afs in ground red peppers has been documented by numerous researchers from various countries (juan et al., 2008). in turkey, ground red peppers contaminated with afb1 are 5 to 25 µg/kg in bursa and 0.025 to 40.9 µg/kg in istanbul, respectively (dokuzlu et al., 2001). red pepper specimens contaminated with afb1 are 1.48 to 70.05 µg/kg in kayseri (reddy et al., 2001). 18.2% of ground red peppers contaminated with total aflatoxins (aft) varies between 1.1 and 97.5 µg/kg in şanlıurfa (erdogan, 2004). set and erkmen (2010) report the contamination rates of aft and afb1 ranging from 0.13 to 57.3 µg/kg and from 0.07 to 55.90 µg/kg, respectively, in unpacked ground red peppers and from 0.08 to 2.06 µg/kg and from 0.08 to 1.95 µg/kg, respectively, in packed specimens. özkan et al., (2015) state the content of afb1 exceeds the legal limit in 49 of 180 red chilli pepper specimens and afs exceeds the legal limit in 37 specimens. fazekas et al., (2005) point to the presence of afb1 in 25.7% (18/70) of ground red peppers in hungary. abdulkadar et al., (2000) also suggest that 66.7% (4/6) chilli pepper powder contains aft from 5.60 to 69.28 µg/kg in qatar. romagnoli et al., (2007) observe the aft contamination in 45.5% red pepper specimens varying between 0.57 and 30.7 µg/kg in italy. reddy (2001) also reports afb1 contamination of 39.5% of pepper specimens in india ranging from 10 to 99 µg/kg. the present data indicate that the quality of peppers is better in comparison with the previous studies. among the 25 walnuts without shell, only one specimen is contaminated with aflatoxins rate of 10.26 µg/kg aft and 5.06 µg/kg afb1, which is above the legal limit. various investigators also have reported similar results. for example lutfullah and hussain, (2011) report that three in walnuts without shell contain concentrations varying between 6.0 and 10.8 µg/kg, which exceeds the recommended limit for aft. gürses, (2006) detected that 6 of 24 walnuts are positive within the contamination range 3-28 µg/kg. in a ital. j. food sci., vol. 29, 2017 596 study mekkah determined quantitative afb1 and aft in walnuts 0-17.4 µg/kg and 0.936.6 µg/kg, respectively (el tawila et al., 2013). asghar et al., (2017) state about 37% walnut specimens showing afs contamination, ranged from 0.68−6.66 μg/kg. in another study the contamination levels in walnut specimens vary between 0.56 and 2500 µg/kg for afb1 and between 1.24 and 4320 µg/kg for aft (juan et al., 2008). these values exceed the results presented in this paper. the aft rate in the contaminated specimens of dried figs varies between 0.16 and 5.20 µg/kg, which is below the recommended legal limit. the findings indicate that the afs contamination of the specimens of the dried figs is low according to many studies as follows. of late, the aflatoxin contamination level of the dried figs in turkey has varied between 117.9 to 471.9 µg/kg (karaca and nas, 2006). kaya and tosun, (2013) state that the aflatoxin rates in dried figs varied between 0 and 10.47 µg/kg in 2013. bircan (2009) reports that 34% of the specimens has noticeable levels of aft (0.20-208.75 µg/kg) in 7326 dried figs in aydin province. lutfullah and hussain, (2011) also point out that the contamination level of aft in dried fig specimens varies between 0.8 and 12.5 µg/kg. the values in this study are below these findings. on the other hand, asghar et al., (2017) indicate about 30% samples were contaminated with afs, ranged from 0.69−3.44 μg/kg. the results from current study are similar with these previous findings. in seedless black raisins, all contaminated specimens have lower limits than the maximum rate set by the turkish regulations (tfc 2011). afb1 rates detected in all dried figs and the seedless black raisins do not exceed the turkish acceptable maximum rates (<8 µg/kg). despite the low rates of afb1 observed in most specimens, it still brings with it detrimental risks of health conditions to people who consume those contaminated foods (reddy et al., 2011). lutfullah and hussain (2011) report that one of the two contaminated specimens exceeds the recommended rate of aflatoxin while the other is below the recommended rate. 16% of the dried grapes in brazil was found to contain afb1 and afb2 (iamanaka et al., 2007). several studies have been stated that dried raisins do not exhibit the satisfactory surface or environment for aflatoxins production. however, asghar et al., (2017) determined that 12 (13%) samples out of 90 tested specimens were determined positive with afs. the concentrations ranged from 0.24-4.86 μg/kg. alghalibi et al., (2008) also report that the aflatoxin contamination range of 3 out of 7 dried grape specimens in sana'a city, republic of yemen is between 2678.66 and 11556.88 ng/kg. whereas in the present study, small quantity of aft and afb1 was described in dried seedless black raisins. the concentrations ranged from 0.02-2.07 μg/kg and 0.02-0.26 μg/kg respectively. the results from current study indicate that the afb1 and aft rates of seedless black raisins from sakarya city does not exceed the recommended limit by tfc (tfc 2011). in present research the lowest afb1 and aft mean level detected in seedless black raisins was 0.110.40 μg/kg respectively. in contrast, ground red peppers exhibited equivalent to 1.30-2.30 μg/kg for afb1 and aft, respectively. due to serious toxicity concerned with afaltoxins, various countries established guidelines for the acceptance level of afs in dry fruits and nuts. for example, the european commission has set the maximum legal limits 4-10 μg/kg for aft in dry fruits and nuts, respectively. european maximum limits are 5 and 2μg/kg for afb1 in hazelnuts and dried figs for afb1, respectively. (ec 2010). additionally turkish regulations for aflatoxins consider different limits for unprocessed products and product intended for direct human consumption, as well as for different commodities. for instance tfc has set the maximum legal limits 5-10 μg/kg in nuts and spices; 8-10 μg/kg in dried fruits; 2-4 μg/kg in cereals for afb1 and aft, respectively (tfc 2011). in both turkey (tfc 2011) and the european commission (ec 2010), the maximum legal limits for ground red pepper are 5 μg/kg for afb1 and 10 μg/kg for aft. as a result, the determination of afs showed that there is a ital. j. food sci., vol. 29, 2017 597 powerful need for further research, routine analysis and to establish a monitoring program or project as per food quality control standard and procedures. furthermore to improve and implement some food quality control procedures and standards such as good manufacturing practices (gmp) and the hazard analysis and critical control point (haccp) system to minimize the risk and finally prevent the formation of afs. 4. conclusions the goals of this study are to specify the aft and afb1 contamination rates in ground red peppers, dried figs, walnuts without shell and seedless black raisins, and to draw attention to the effects of contamination in these foods on public health. the results of this study provide insight regarding the danger of afs contained various types of foods. the analysis of all the findings indicates that 68% and 38% of the specimens are contaminated by aft and afb1, respectively. of all the contaminated specimens, 2.5% of specimens exceed the recommended afb1 (5 µg/kg) limit defined by the turkish food codex (tfc) regulations. consequently, aflatoxins entail a health risk for those who consume contaminated foods. cases of afs contamination show that constant surveillance and a better food safety system should be implemented so that the content of afs in foods can be kept at the minimum level (leong et al., 2010). on the other hand, more comprehensive studies should be carried out on a wide range of foods consumed in turkey in order to design appropriate executive projects and to bring forth regulations on afs encompassing all sustenance consumed. this research will pave the way for new studies on afs in order to establish a comprehensive food safety system, which will protect humans’ health. this study is the primary documentation, which addresses the occurrence of commercial seedless black raisins contaminated with aflatoxin in sakarya. acknowledgements this work was supported by research fund of the sakarya university (project number: 201-01-16-011). references abdulkadar a.h.w., al-ali a. and al-jedah j. 2000. aflatoxin contamination in edible nuts imported in qatar. food control 11:157-160. asghar m.a., ahmed a., zahir e., asgher m. a., iqbal j. and walker g. 2017. incidence of aflatoxins contamination in dry fruits and edible nuts collected from pakistan, food control, doi: 10.1016/j.foodcont.2017.02.058 aoac (association of official analytical chemists). 2000. natural toxins, 17th ed. chapter: 9 gaithersburg, maryland. bircan c. 2009. comparison of homogenization techniques and incidence of aflatoxin contamination in dried figs for export. food additives contaminants part b-surveillance 2:171-177. cdc (centres for disease control and prevention). 2004. outbreak of aflatoxin poisoning-eastern and central provinces, kenya, january-july 2004. morbidity and mortality weekly report 3:790-793. colak h., bingöl e.b., hampikyan h., and nazlı b. 2006. determination of aflatoxin contamination in red-scaled, red and black pepper by elisa and hplc. j. of food and drug analysis 14(3):292-296. dini a., khazaeli p., roohbakhsh a. and madadlou a. 2013. aflatoxin contamination level iran’s pistachio nuts during years 2009-2011. food control 30:540-544. dokuzlu c. 2001. aflatoxin in red pepper. uludag university veterinary faculty journal 20:1-2. ital. j. food sci., vol. 29, 2017 598 doster m.a. and michailides t.j. 1994. aspergillus molds and aflatoxin in pistachio nuts in california. phytopathology 84:583-590. ec. european commission, 2010. regulation no: 165/2010 of 26 february 2010 setting maximum levels for certain contaminants in foodstuffs. official journal of the european union 2010, l 50/8-12. el tawila m.m, neamatallah a. and serdar s.a. 2013. incidence of aflatoxin in commercial nuts in the holy city mekkah. food control 29:121-124. erdogan a. 2004. the aflatoxin contamination of some pepper types sold in turkey. chemosphere 56:321-325. erkmen o. and bozoglu t.f. 2008. food microbiology 1: microorganisms in foods, microbial growth, foodborne diseases and detection of microorganisms and their toxins. ilke publishing company, ankara. fazekas b., tar a. and kovacs m. 2005. aflatoxin and ochratoxin a content of spices in hungary. food additives and contaminants 22:856-863. gürses m. 2006. mycoflora and aflatoxin content of hazelnuts, walnuts, peanuts, almonds and roasted 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menna v., gruppioni n. and bergamini c. 2007. aflatoxins in spices, aromatic herbs, herb-teas and medicinal plants marketed in italy. food control 18:697-701. set e. and erkmen o. 2010. the aflatoxin contamination of ground red pepper and pistachio nuts sold in turkey. food and chemical toxicology 48:2532-2537. stroka j., anklam e., jorissen u. and gilbert j. 2000. immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxins in peanut butter, pistachio paste, fig paste, and paprika powder: collaborative study. journal of aoac international 83:320-340. tfc. turkish food codex, 2011. regulation no. 2011/28157, the maximum allowed level of food contaminants. official gazette of publication: 29.12.2011-28157, ankara. paper received november 5, 2016 accepted june 12, 2017 paper ital. j. food sci., vol. 27 2015 1 keywords: microwave, air drying, antioxidant activity, total phenolic content, response surface methodology optimization of microwave and air drying conditions of quince (cydonia oblonga, miller) using response surface methodology cem baltacioğlu*, nurhan uslu and mehmet musa özcan food engineering department, faculty of agriculture, selcuk university, konya, turkey *corresponding author: cembaltacioglu@gmail.com abstract effects of slice thickness of quince (cydonia oblonga miller) , microwave incident power and air drying temperature on antioxidant activity and total phenolic content of quince were investigated during drying in microwave and air drying. optimum conditions were found to be: i) for microwave drying, 285 w and 4.14 mm thick (maximum antioxidant activity) and 285 w and 6.85 mm thick (maximum total phenolic content), and ii) for air drying, 75 ºc and 1.2 mm thick (both maximum antioxidant activity and total phenolic content). drying conditions were optimized by using the response surface methodology. 13 experiments were carried out considering incident microwave powers from 285 to 795 w, air temperature from 46 to 74 ºc and slice thickness from 1.2 to 6.8 mm. 2 ital. j. food sci., vol. 27 2015 1. introduction edible fruits are sources of nutrients such as carbohydrates, vitamins, and minerals as well as non-nutrient compounds such as polyphenols. nowadays, it is commonly admitted that there is a positive relationship between a diet rich in vegetables and fruits and a reduced incidence of degenerative diseases such as cancer and cardiovascular events (gibney et al., 2009). health beneficial properties of quince fruit (cydonia oblonga miller) have known from ancient times. quince is the only species in the genus cydonia, which falls into pomoideae subfamily of the rosaceae along with apple and pear (pacifico et al., 2012). quince is used extensively in europe as a dwarfing rootstock for pear. total world production of fresh quince was 540.337 tons in 2010 and about 25% of this was produced in turkey (tsi, 2012). drying process is one of the most important preserving operations that causes time and energy consumption in the food industry. that is why new methods are aimed to decrease drying time and energy consumption. new methods combined different systems such as using microwave drying together with traditional drying methods to reduce drying time (secmeler, 2003). over the past two decades, there has been an increasing attraction in microwave drying to reduce drying time and increase the removal of water from agricultural products. microwave drying has several advantages such as short drying time, higher drying rate, better quality of the dried products and decrease energy consumption (sanga et al., 2000). response surface methodology (rsm) is one of the most commonly used optimization technique in food science. this method is preferred because of the simplicity and high efficiency. rsm covers a group of techniques used to study the relationship between one or more measured responses and input variables. (artege et al., 1994). it has been successfully applied to optimize food processing operations by many researchers (frank, 2001; lee et al., 2006; luciane et al., 2001; mirhosseini et al., 2008; pietrasik and li-chan, 2002). 2. material and methods 2.1. material fresh quinces were obtained from a local market in konya. these samples were transferred to laboratory in cool bags and they stored in refrigeration temperature (4 °c) until the assay, initial moisture content of fruits was detected as 80% in average. prior to drying, round shaped samples (2 cm in diameter) were obtained from fruit slices. thickness varied from 1.2 to 6.8 mm according to experimental design. 2.2. drying quinces were dried until the moisture content decreased to 40% of the initial moisture content, since burning was observed on the quince slices in microwave drying below this moisture content level. microwave drying experiments were performed using a domestic microwave oven (arcelik armd 580, turkey). the dimensions of the microwave cavity were 345 mm x 340 mm x 225 mm. three power levels were selected as high (720 w), medium (540 w) and low (360 w) for drying experiments. one dish containing 1 slice of sample to make effective drying was placed on the centre of a turntable fitted inside the microwave oven. quinces were placed uniformly as a thin layer onto the stainless steel trays (0.3 m x 0.2 m) and dried using air oven (nüve fn055 ankara, turkey, 55 l volume) at three different temperatures ( 50, 60 and 70 °c). quince slices were placed uniformly as a thin layer onto the stainless steel trays (0.3 m x 0.2 m) and dried under direct sunlight in april in konya, turkey (balladin & headley, 1999). 2.3 extraction the phenolic compounds were isolated from samples using a modified version of the method described by shahidi et al. (2001). one gram of sample was extracted 3 times using 10 ml of 70% (v/v) aqueous methanol (merck, germany) at room temperature by a homogenizer (ika ultra turrax tube disperser) for 1 min. the slurry was centrifuged at 4000 rpm for 15 min. supernatants were collected and combined in a rotary flask and then evaporated at 45 °c under vacuum by a rota vapor r-3000 rotary evaporator (laborato 4001, heidolph). the extracted phenolics were dissolved in 25 ml methanol and then filtered using filter paper. methanolic solutions of phenolic were stored -25 °c until analysis. 2.4 determination of total phenolic content and antioxidant activity total phenolic compounds were prepared by using folin-ciocalteu reagent (yoo et al., 2004). the free radical scavenging activity of the extract was determined using 1, 1-diphenyl-2-picrylhydrazyl (dpph) in order to determine antioxidant activity (lee et al., 1998). total phenolic contents were calculated by a standard calibration curve prepared using gallic acid. total phenolic content results were given as gallic acid equivalents in milligrams per 1000 g fruit. antioxidant activity results were expressed as percentage activity (%). ital. j. food sci., vol. 27 2015 3 2.5 experimental design response surface methodology (rsm) was used to optimize drying conditions, based on better preserve the antioxidant activity and total phenolic content of quince fruit. box– behnken design was selected for rsm analysis. box–behnken designs require only three levels, coded as −1, 0 and +1. the effects of the two independent processing parameters: slice thickness (x 1 , mm), incident microwave power (x 2 , watt) in microwave operation and additionally slice thickness (x 1 , mm), processing temperature level (x 2 , °c) in air drying on two dependent variables (antioxidant activity and total phenolic content) were investigated using rsm. the total number of experiments in this study was 13 based on two levels and a two factor experimental design, with five replicates at the centre of the design for estimation of a pure error sum of squares. minitab 16 (minitab inc. state college, pa) was used for the experimental design, data analysis and regression modeling. the independent variables were; x 1 (2–4 mm), x 2 (360-720 w) in microwave process and x 1 (2 – 4 mm), (x 2 50 70 °c) in air drying. experimental data from the box– behnken design was fitted into a second-order polynomial model. y= b o + b 1 x 1 + b 2 x 2 + b 1 2 x 1 2+ b 2 2 x 2 2 + b 1 b 2 x 1 x 2 (1) where y is the predicted response, x 1 and x 2 are independent variables, b 0 is a constant; b 1 , b 2 , b 1 2, b 2 2, b 1 b 2 are linear, quadratic and interaction coefficients, respectively. 3. results and discussion 3.1 microwave drying independent variables [thickness of slice (x 1 ) and microwave power level (x 2 )] observed and predicted values of antioxidant activity and total phenolic content for microwave drying were given in table 1. during drying in microwave oven antioxidant activity decreased in the studied variables. the influences of interaction between thickness-thickness were determined as statistically important (p≤0.05) whereas the other parameters were found insignificant (p>0.05) (table 2a). statistical analysis indicated that the fitted model (eq.1) to experimental results displayed high performance to predict the antioxidant activity of quince samples within the studied ranges of variables. regression coefficient (r2) and adjusted regression coefficient (r2 adj ) were calculated as 0.994 and 0.990, respectively (table 2a). 3d surface plots for the significant terms were shown in figs. 1 and 2 (minitab 16). surface plot given in fig. 1a indicated that antioxidant activity was not affected by thickness and power level. thickness-thickness square parameter was the only parameter which affected on antioxidant activity (p≤0.05) (table 2a). antioxidant activity of quince increased up to 4 mm of slice thickness and then decreased (fig. 1a). the interaction between thickness-thickness, thickness-power, power-power, power and thickness alone showed significant effects (p≤0.05) (table 2b) on total phenolic content of quince dried in microwave oven. total phenolic content of quince dried in microwave oven was fitted in eq.2. re4 ital. j. food sci., vol. 27 2015 gression coefficient (r2) and adjusted regression coefficient (r2 adj ) were calculated as 0.880 and 0.795, respectively (table 2b). the linear coefficient for thickness was over seventy times greater than that of power level. there was interaction of significance between thickness and power level. y 1 = 80.13 + 5.58 x 1 – 0.66 x 1 2 (1) y 2 = 2088.05 + 290.44 x 1 – 4.87 x 2 + -7.86 x 1 2 -0.39 x 1 x 2 (2) antioxidant activity and total phenolic content values were found higher in microwave drying than that of air drying. drying time decreased 60-120 times in microwave drying. berteli & marsaioli (2005) have studied the influence of drying methods on moisture content of product. very short heating–cooling cycles take place in microwave drying. the use of microwave for drying has become common because it enhances the product quality and processing speed (diaz et al., 2003). fresh fruits are well known for their antioxidant activity which is usually attributed to the polyphenol content (wang et al., 1996). total phenolic content of 2020 mg gae/kg sample and 78% inhibition for antioxidant activity were reported in fresh quince samples before the drying experiment (hamazu et al., 2005). karadeniz et al. (2005) reported that antioxidant activity of some quince varieties in turkey found between 51-68%. ital. j. food sci., vol. 27 2015 5 fig. 1 effect of thickness and power on antioxidant activity during microwave drying (a) and on total phenolic content during microwave drying (b). fig. 2 effects of thickness and temperature on antioxidant activity oven drying (a) and on total phenolic content oven drying (b). 6 ital. j. food sci., vol. 27 2015 3.2 air drying independent variables [thickness of slice (x 1 ) and temperature of air (x 2 )], observed and predicted values of antioxidant activity and total phenolic content for air drying were given in table 3. antioxidant activity of quince samples decreased after air drying. thickness of samples and process temperature had considerable effect on antioxidant activity. additionally, the influences of interaction between thickness-thickness and temperature-temperature were statistically significant (p≤0.05) whereas the other parameters were insignificant (p>0.05) (table 4a). effect of thickness and temperature on the antioxidant activity in air drying was given in fig. 2a. increase in thickness from 2 to 4 mm resulted in lower antioxidant activity at different drying temperatures. the fitted model to antioxidant activity results of quince samples after air drying was given in eq. 3. model parameters for eq. 3 (r2 and r2 adj ) were calculated as 0.953 and 0.920, respectively (table 4a). the interaction between thickness-thickness, temperature-temperature, thickness-temperature and temperature of process showed significant effects (p≤0.05) on total phenolic content after air drying (table 4b). changes in total phenolic content was given in fig. 2b and increase in thickness and power level caused firstly decrease and then increase in total pheital. j. food sci., vol. 27 2015 7 nolic content similar to the antioxidant activity. total phenolic content of quince by air drying was fitted in eq. 4. model parameters for eq 4 (r2 and r2 adj ) were calculated as 0.977 and 0.960, respectively (table 4b). the high values of regression coefficient indicate a high degree of correlation between the experimental and fitted values. y 3 = 449.60 – 20.10 x 1 – 12.54 x 2 + + 2.59 x 1 2+ 0.10 x 2 2 (3) y 4 = 4925.38 – 6.43 x 1 – 161.10 x 2 + + 42.22 x 1 2+ 1.63 x 2 26.27 x 1 x 2 (4) 3.3 sun drying sun drying was chosen as traditional drying method and this process maintained about 24 h. the use of microwave oven seems to be more advantageous considering the time factor. in sun drying, total phenolic content and antioxidant activity were determined as 1544 mg gae/ 1000 g and 74%, respectively. these values were close to the results observed after microwave drying and higher than that of air drying. in order to determine the optimal residual activity, response optimizer tool in minitab 16 (minitab inc. state college, pa) was used. the optimum conditions were found as 4.14 mm thickness and 285 w power level for maximum antioxidant activity, 6.85 mm thickness and 285 w power level for maximum total phenolic content in microwave drying. when air drying was analyzed, 1.2 mm thickness and 75 °c temperature were selected for maximum antioxidant activity and total phenolic content. 4. conclusions the effect of power level and slice thickness on the antioxidant activity and total phenolic content were investigated for quince samples after drying. rsm was used to optimize the factors in order to obtain maximum level of antioxidant activity and total phenolic content of quince samples. all independent variables including thickness of slice (mm), power level (w) and processing temperature (°c) had significant effects on the response values. furthermore square and interaction parameters showed significant effects (p≤0.05). a desirable quadratic mathematical model was built by using box–behnken design. the antioxidant activity and total phenolic content decreased after drying. nevertheless optimum drying conditions were obtained for both microwave and air drying. additionally, the use of microwave provides time saving compared to the other drying methods. references arteaga g.e., li-chan e., vazquez-arteaga m.c. and nakai s. 1994. systematic experimental designs for product formula optimization. trends in food science and technology. 5: 243. balladin d.a. and headley o. 1999. evaluation of solar dried thyme (thymus vulgaris l.) herbs. renewable energy. 17: 523. berteli m.n. and marsaioli a. jr. 2005. evaluation of short cut pasta air dehydration assisted by microwaves as compared to the conventional drying process. journal of food engineering. 68:175. dı´az g.r., martìnez-monzò j., fito p. and chiralt a. 2003. modelling of dehydration–rehydration of orange slices in combined microwave/air drying. innovative food science & emerging technologies. 4: 203. frank r. 2001. blending response surface methodology and principal components analysis to match a target product. food quality and preference. 12: 457. gibney m.j., lanham-new s.a., cassidy a. and vorster h.h. 2009. introduction to human nutrition. 2nd ed. wiley-blackwell. hamazu y., yasui h., inno t., kume c. and omanyuda m. 2005. activity of chinese quince (pseudocydonia sinensis schneid.), quince (cydonia oblonga mill.), and apple (malus domestica mill.) fruits. journal of agricultural and food chemsitry. 53: 928. karadeniz f., burdurlu h.s., koca n. and soyer y. 2005. antioxidant activity of selected fruits and vegetables grown in turkey. turk. j. agric. for. 29: 297. lee w.c., yusof s., sheikh abdul hamid n. and baharin b.s. 2006. optimizing conditions of enzymatic clarification of banana juice using response surface methodology (rsm). journal of food engineering. 73: 55. lee s.k.. mbwambo z.h.. chung h.s.. luyengi l.. games e.j.c. and mehta r.g. 1998. evaluation of the antioxidant potential of natural products. combinational chemistry and high throughput screening. 1: 35. luciane c.m., hilary c.m., aparecida m. and de silva a.p. 2001. optimization of the roasting of robusta coffee (coffea canephora, conillon) using acceptability tests and rsm. food quality and preference. 12: 153. mirhosseini h., tan c.p., sheikh abdul hamid n. and yusof s. 2008. optimization the contents of arabic gum, xanthan and orange oil affecting on turbidity, cloudiness, average particle size, polydispersity index and density in orange beverage emulsion. food hydrocolloids. 22: 1212. pcifico s., gallicchio m., fiorentino a., fischer a., meyer u. and stintzing c. 2012. antioxidant properties and cytotoxic effects on human cancer cell lines of aqueous fermented and lipophilic quince (cydonia oblonga mill.) preparations. food and chemical toxicology. 50: 4130. pietrasik z. and li-chan e.c.y. 2002. response surface methodology study on the effects of salt, microbial transglutaminase and heating temperature on pork batter gel properties. food research international. 35: 387. sanga e., mujumdar a.s. and raghavan g.s.v. 2000. principles and application of microwave drying. in: mujumdar ed. drying technology in agriculture and food sciences. science publication, enfield, usa. secmeler o. 2003. comparison of microwave drying and microwave mixed-bed drying of red peppers. m.sc. thesis, department of food engineering, middle east technical university, ankara, turkey. turkish standardization instutue bulletin. 2012. number :10950. yoo k.m., lee k.w., park j.b., lee h.j. and hwang i.k. 2004. variation in major antioxidants and total antioxidant activity of yuzu (citrus junos sieb ex tanaka) during maturation and between cultivar. journal of agricultural and food chemistry. 52: 5907. wang h., cao g.h. and prior r.l. 1996. total antioxidant capacity of fruits. journal of agricultural and food chemistry. 44: 701. paper received december 11, 2013 accepted may 8, 2014 #747_caponio_bozza ital. j. food sci., vol 29, 2017 370 short communication fatty acids methyl and ethyl esters behaviour during olives processing by means of technological coadjuvants g. squeo, r. silletti, c. summo, v.m. paradiso, a. pasqualone and f. caponio* university of bari aldo moro, department of soil, plant and food sciences, food science and technology unit, via amendola 165/a, i-70126, bari, italy *corresponding author. fax: +39 0805443467 e-mail address: francesco.caponio@uniba.it abstract recently, the quantification of fatty acids alkyl esters has become mandatory for the extra virgin olive oil classification. however, the behaviour of such metabolites, during olives processing, is not yet well understood. thus, the present paper aims to point out the influence of the use of calcium carbonate on the fatty acids alkyl esters content of the oils. the results showed that the content of fatty acids alkyl esters was significantly influenced by the technological coadjuvant. the use of calcium carbonate led to a general reduction of fatty acids alkyl esters compared to the untreated samples. methyl esters of fatty acids were more susceptible to the use of processing aid than the ethyl esters. keywords: alkyl esters, extra virgin olive oil, olives processing ital. j. food sci., vol 29, 2017 371 1. introduction according to the ec regulation (official journal of the european communities, 2001), virgin olive oils are “oils obtained from the fruit of the olive tree solely by mechanical or other physical means under conditions that do not lead to alteration in the oil, which have not undergone any treatment other than washing, decantation, centrifugation or filtration, to the exclusion of oils obtained using solvents or using coadjuvants having a chemical or biochemical action, or by re-esterification process and any mixture with oils of other kinds”. such statement does not take into account the quality of the raw material that, instead, represents one of the main factors, together with the extraction process, affecting the final product quality. to classify virgin olive oils, several parameters must be checked, some of which have very good correlation with the raw material features (salvador et al., 2001; koprivnjak et al., 2010). the eu regulation 61/2011 (official journal of the european union, 2011) added the determination of methyl and ethyl esters of fatty acids, generally recognised as fatty acids alkyl esters (faae), to the list of parameters to be checked for classifying the quality levels of virgin olive oils. later, the eu regulation 1348/2013 (official journal of the european union, 2013) stressed attention only on the fatty acids ethyl esters (faee). alkyl esters originate from the esterification of fatty acids and low molecular weight alcohols, methanol and ethanol, respectively arisen from the progressive pectin degradation during the olive ripening and/or by the bad and/or prolonged storage of drupes (biedermann et al., 2008; pérez-camino et al., 2008). thus, the presence of faae in oils became an established marker of the quality of the raw material employed. the knowledge about the faae is continuously growing. indeed, several researches have been carried out, focused on the correlation between faae and the raw material quality (cert, 2006; mariani and bellan, 2008); the virgin oil sensory characteristics (biedermann et al., 2008; gómez-coca et al., 2012); the olive pomace oil storagerelated changes (ruiz-méndez and ramos-hinojosa, 2003); the olive storage conditions (jabeur et al., 2015) and, recently, on the oil storage temperature and substrate availability (gómez-coca et al., 2016) and on the influence of the washing process (alcalá et al., 2016). virgin olive oil producers try to reach the maximum yield during the extraction process while saving, at the same time, the nutritional, functional and organoleptic features that distinguish virgin olive oil from other oils. several attempts have been done aiming to the improvement of the extraction yield, mostly on those cultivars, which give the so-called “difficult pastes” (di giovacchino and mascolo, 1988). the most common strategies adopted regard: i) malaxation time and temperature increase (stefanoudaki et al., 2011); ii) use of technological coadjuvants able to break down the water-oil emulsions (caponio et al., 2016a; caponio et al., 2016b; squeo et al., 2016). as far as we know, no information are available about the evolution of the faae as a consequence of the use of processing aids, such as calcium carbonate, during the extraction process. considering that the “extra” quality virgin olive oils have the highest price on the market and thus even the profit of the producers and farmers are linked to the compliance with the current legal limits, it is clear that such information could be very useful for both the operators as well as the lawmakers. hence, the aim of this work was the assessment of the ethyl esters and methyl esters of fatty acids in extra virgin olive oils in regard to the use of calcium carbonate during the malaxation step. ital. j. food sci., vol 29, 2017 372 2. materials and methods 2.1. sampling and experimental plans all the experimental trials were carried out on olives (coratina cultivar) milled within 24 hours after the harvest in industrial olive mills. for studying the effect of the calcium carbonate, two olive lots, having 0.51 (lot a) and 1.40 (lot b) pigmentation index (pi), calculated as reported in squeo et al. (2016), were divided in 14 homogeneous batches of about 300 kg. two batches were processed without any treatment (controls) while the others (two batches per each treatment) were processed by using two different types of calcium carbonate (average particle size of 2.7 µm, ca2, and 5.7 µm, ca5, respectively) at three percentages of addition respect to the olives paste weight (1-2-4%). the full experimental plan was reported in table 1. calcium carbonate was kindly furnished by omya spa (milan, italy). after being weighted, the coadjuvant was directly and gradually added into the malaxer at the begin of the malaxation stage without stopping the machine. table 1. experimental plan for olive lots a and b. coadjuvant typology level of addition (%) trial name* none none c calcipur®2 1 ca2-1% calcipur®2 2 ca2-2% calcipur®2 4 ca2-4% calcipur®5 1 ca5-1% calcipur®5 2 ca5-2% calcipur®5 4 ca5-4% *each trial was repeated twice. 2.2. alkyl esters analysis the analyses of the methyl and ethyl esters of fatty acids were carried out according to the official method (official journal of the european union, 2011). the gas chromatographic system was made up of a 7890b agilent technologies (santa clara, ca, usa) chromatograph equipped with a flame ionization detector (fid). the column used was a capillary fused silica db-5ht (length 15 m, i.d. 0.32 mm, film thickness 0.10 µm). the operating conditions were as follows: oven temperature, 80 °c for 1 min and then increased at 20 °c min-1 to 140 °c, then increased at 5 °c min-1 to 335 °c and maintained for 20 min. the detector temperature was 350 °c. helium was used as the carrier gas, with a flow through the column of 2 ml min-1 in splitless mode. 2.3. statistical analysis anova and tukey post-hoc test for multiple comparisons were carried out on the experimental data by means of minitab 17 software (minitab inc., state college, pa, usa). ital. j. food sci., vol 29, 2017 373 3. results and discussions all the virgin olive oils obtained in the industrial trials were classified as extra virgin olive oils according to the eu regulation 1348/2013 (official journal of the european union, 2013) (data not shown). fig. 1 reports the average amounts of alkyl esters in the samples under study. the contents found were in accordance with those reported in literature (biedermann et al., 2008; mariani and bellan, 2008; pérez-camino et al., 2008) and lower than law limits. overall, a gradual decrease of faae than controls (c) was observed as the percentage of calcium carbonate increased. a good discrimination was observed in particular between the samples of the 4% addition trial and those without (c) or with the lowest additions (1%). moreover, the faee content was generally higher than the amount of fatty acids methyl esters (fame). figure 1. fatty acids methyl esters (fame) versus fatty acids ethyl esters (faee) (mg kg-1) detected in the samples. olive processed without calcium carbonate addition (c, control) and with the addition of 1%, 2%, and 4% of calcium carbonate. ca2, calcipur®2 with particle size of 2.7 µm; ca5, calcipur®5 with particle size of 5.7 µm (squeo et al.). the differences observed are clearly shown by fig. 2, in which the differences of means, and the respective 95% confidence intervals obtained by the tukey post-hoc test, are reported in regard to the variables percentage of addition and type of coadjuvant. overall, the differences of means between the treatments and the control shown that the treated oils were poorer in faae (fig. 2 a-d). considering the percentages of addition (fig. 2 a-b), the differences between treatments and control were significant in all cases for the fame, while only 4% of addition led to significantly lower amounts of faee compared to control. among the treatments, significantly lower amounts of fame were found using 2% and 4% of the coadjuvant respect to 1%. no significant differences have been highlighted between 2% and 4% addition (fig. 2a). as regards the faee, a significant difference was reported for the 4%, as compared to 1% of addition (fig. 2b). the coadjuvant granulometry (fig. 2 c-d) induced significant differences only for the fame, with a more noticeable effect of the ca5 than the ca2 respect to the control. no 87654 7 6 5 4 3 faee fa m e 0% 1% 2% 4% ca5ca5 ca5 ca5 ca5 ca5 ca2 ca2 ca2ca2 ca2 ca2 c c ca5ca5 ca5 ca5 ca5 ca5 ca2 ca2 ca2 ca2 ca2 ca2 c c ital. j. food sci., vol 29, 2017 374 statistical difference was highlighted between the different types of calcium carbonate adopted for both the fame and the faee. overall, our findings underline that the use of calcium carbonate in the olive oil mill causes a reduction of the amounts of faae, that seems to be proportional to the amount of processing aid employed, whereas the type of coadjuvant did not show any significant effect. figure 2. tukey 95% confidence intervals for the differences of means for the calcium carbonate trials for both the olives lots. a) means differences in fame as a function of the percentages of addition; b) means differences in faee as a function of the percentages of addition; c) means differences in fame as a function of the type of coadjuvant; d) means differences in faee as a function of the type of coadjuvant (squeo et al.). it is known that the synthesis of the alkyl esters occurs in an acid environment and is catalysed by enzymes and temperature (pérez-camino et al., 2003). in such conditions, methanol and ethanol react with fatty acids (mainly oleic acid) giving rise to the faae. the lowering in the content of these metabolites, observed in the treated oils, might be due to the interference on the enzymatic activity exerted by processing aids during the malaxation step or, more simply, by an absorption of the low weight alcohols, more evident for the methanol. 4. conclusions our findings showed that the content of the fatty acids alkyl esters could be affected by the technological strategies adopted during olives processing into oil. in particular, the use of technological coadjuvants, aimed to increase the extraction yield, influences the amount of such compounds, bringing to a reduction. anyway, this is more evident for the methyl esters, while a weaker influence has been observed on the ethyl ester amount. further ital. j. food sci., vol 29, 2017 375 studies should be carried out in order to confirm these preliminary results and understanding deeply the faae behaviour in the olive oil matrix. as regards the economic point of view, the use of such processing aid will be easily faced by the producers considering a cost increase of about 0.40 € kg-1 of oil in the face of an extraction efficiency increase of about 4%. acknowledgements this research was supported by regione puglia, project qu.al.e.olio. references alcalá s., ocaña m.t., cárdenas j.r., miquel m.a., vilar j., espínola f. and moya m. 2016. alkyl esters content and other quality parameters in oil mill: a response surface methodology study. eur. j. lipid sci. technol. 118:doi: 10.1002/ejlt.201600026. biedermann m., bongartz a., mariani c. and grob k. 2008. fatty acid methyl and ethyl esters as well as wax esters for evaluating the quality of olive oils. eur. food res. technol. 228:65. caponio f., squeo g., difonzo g., pasqualone a., summo c. and paradiso v.m. 2016a. has the use of talc an effect on yield and extra virgin olive oil quality? j. sci. food agric. 96:3292. caponio f., squeo g., summo c., paradiso v.m. and pasqualone a. 2016b. talc effect on the volatiles of virgin olive oil during storage. ital. j. food sci. 28:705. cert a. 2006. meeting of chemists to study methods of analysis olive oils and olive pomace oils. international olive oil council, madrid, t20/doc. no. 53-3. di giovacchino l. and mascolo a. 1988. incidenza delle tecniche operative nell’estrazione dell’olio dalle olive con il sistema continuo. nota ii. riv. ital. sostanze grasse 65:283. gómez-coca r.b., fernandes g.d., pérez-camino m.c. and moreda w. 2016. fatty acid ethyl esters (faee) in extra virgin olive oil: a case study of a quality parameter. lwt-food sci. technol. 66:378. gómez-coca r.b., moreda w. and pérez-camino m.c. 2012. fatty acid alkyl esters presence in olive oil vs. organoleptic assessment. food chem. 135:1205. jabeur h., zribi a., abdelhedi r. and bouaziz m. 2015. effect of olive storage conditions on chemlali olive oil quality and the effective role of fatty acids alkyl esters in checking olive oils authenticity. food chem. 169:289. koprivnjak o., dminić i., kosić u., majetić v., godena s. and valenćić v. 2010. dynamics of oil quality parameters changes related to olive fruit fly attack. eur. j. lipid sci. technol. 112:1033. mariani c. and bellan g. 2008. detection of low quality oils in extra virgin olive oils. riv. ital. sostanze grasse 85:3. official journal of the european communities. 2001. council regulation no. 1513/2001, n. l. 201 of july 26th, publications office of the european union, bruxelles. official journal of the european union. 2011. commission regulation no. 61/2011, n. l. 23 of january 1st, publications office of the european union, bruxelles. official journal of the european union. 2013. european community regulation no. 1348/2013, n. l. 338 of december 17th, publications office of the european union, bruxelles. pérez-camino m.c., cert a., romero-segura a., cert-trujillo r. and moreda w. 2008. alkyl esters of fatty acids a useful tool to detect soft deodorized olive oils. j. agric. food chem. 56:6740. pérez-camino m.c., moreda w., mateos r. and cert a. 2003. simultaneous determination of long-chain aliphatic aldehydes and waxes in olive oils. j. chromatogr. a 983:283. ruiz-méndez m.v. and ramos-hinojosa a.e. 2003. fatty acid esters with short-chain alcohols in two-phase olive pomace oils. eur. j. lipid sci. technol. 105:346. ital. j. food sci., vol 29, 2017 376 salvador m.d., aranda f. and fregapane g. 2001. influence of fruit ripening on cornicabra virgin olive oil quality. a study of four successive crop seasons. food chem. 73:45. squeo g., silletti r., summo c., paradiso v.m. pasqualone a. and caponio f. 2016. influence of calcium carbonate on extraction yield and quality of extra virgin oil from olive (olea europaea l. cv. coratina). food chem. 209:65. stefanoudaki e., koutsaftakis a. and harwood j.l. 2011. influence of malaxation conditions on characteristic qualities of olive oil. food chem. 127:1481. paper received january 9, 2016 accepted february 6, 2017 p u b l i c a t i o n s codon italian journal of food science, 2023; 35 (2): 1–12 issn 1120-1770 online, doi 10.15586/ijfs.v35i2.2304 1 p u b l i c a t i o n s codon effects of acidified apple juice before fermentation on ethyl carbamate and volatile components of apple distillate zhicong su, yingying han, jinhua du* college of food science and engineering, shandong agricultural university, tai’an, shandong, china *corresponding author: jinhua du, college of food science and engineering, shandong agricultural university, tai’an, shandong 271018, china. email: djh@sdau.edu.cn received: 18 november 2022; accepted: 13 march 2023; published: 11 april 2023 © 2023 codon publications open access opinion paper abstract in order to eliminate ethyl carbamate (ec) content in apple distillate, fuji apple juice was acidified to ph 3.0 by sulfuric acid (st), malic acid (mt), lactic acid (lt), or citric acid (ct). the acidified juice was inoculated with yeast, fermented at room temperature, and distilled by double distillation. acid treatment by st (3.23 μg/l), mt (3.20 μg/l), lt (2.93 μg/l), and ct (3.57 μg/l) significantly eliminated ec from apple distillate. combined with the ec content and sensory evaluation, it was suggested that the high-quality apple distillate could be obtained with lower ec if apple juice was treated with st or mt before fermentation. keywords: apple distillate, ethyl carbamate, sulfuric acid, malic acid, lactic acid, citric acid introduction apple is one of the main fruits in china. its annual output is on the top in the world. however, some low quality apples are not able to meet the market demand of fresh sales, resulting in a serious waste of resources. consequently, the preparation of apple distillate not only reduces apple waste but also increases new apple products and greatly improves the economic and social benefits. however, some harmful components, such as ethyl carbamate (ec) and cyanide, are formed during alcoholic fermentation and distillation. ec, a potentially genotoxic and carcinogenic substance (tu et al., 2018), has been recognized as a group 2a carcinogen by the world health organization’s (who) international agency for research on cancer. in france, the maximum allowable level of ec is set as 150 μg/l for distilled spirits. the upper limit for ec in canada is 400 μg/l in fruit spirits, 30 μg/l in wines, and 150 μg/l in wine spirits, brandies, and whiskies (jia et al., 2022). some studies have shown high levels of ec in distillates, for example, plum brandy contains up to 4750 μg/l of ec in tail during distillation (balcerek et al., 2017), and the ec content in some sugarcane spirits is between 42 μg/l and 5589 μg/l (alcarde et al., 2011). it is essential to reduce the content of ec in apple distillate; however, no research has been conducted on the content of ec in apple distillate. ethyl carbamate is found in many fermented food products and alcoholic beverages, such as wine, sake (rice beer), whisky, brandy, etc. (european food safety authority, 2007). ec has the effects of oral toxicity, immunosuppression, and heart rate inhibition (jiao et  al., 2014). thus, the presence of ec in apple distillate is objectionable, and the ec content is expected to be as low as possible. in the process of making wine, five pathways of ec derived from urea, citrulline, cyanide, and 3a,6a dimethylglycoluril react with ethanol (wang et al., 2014b; mailto:djh%40sdau.edu.cn?subject= 2 italian journal of food science, 2023; 35 (2) su z et al. (beijing, china). all the reagents used were of analytical grade. apple distillate making apple juice preparation well-matured apple fruits were selected, washed, crushed, and squeezed to obtain apple juice. the resulting juice was collected, divided into five samples (control, st, mt, lt, and ct). each juice sample (35 l) was transferred into sterile fermenters (40 l) and treated as follows. nothing was added to control. other four juices were prepared by adding 10% st, mt, lt, and ct to adjust their ph to 3.0. all the above-mentioned processes were carried out in triplicate (figure 1). fermentation wine yeast cy 3079, 0.20 g/kg of juice, activated by apple juice was added into each of the above-prepared apple juices. subsequently, the inoculated juice samples were  fermented at room temperature. the fermented apple juice was obtained after the completion of fermentation, that is, the residual sugar in the juice did not decrease over 3 consecutive days. the fermented juice was sampled daily for further analysis. distillation the fermented apple juice was distilled by a double distillation method. the first distillation was carried out in a 35-l dibosk distiller comprising a stainless steel pot and condensing unit. the pot was heated on an induction stove. the first distillate was obtained with 30% (v/v) alcohol. the second distillation was carried out in a 5-l glass conical flask heated on an electric furnace (satora and tuszynski, 2010). during the second distillation, the zimmerli and schlatter, 1991). concerning distillation, urea is decomposed at a high temperature to cyanic acid, which reacts with ethanol to form ec (schaber et al., 2004; taki et al., 1992). many studies have shown that ec production can be affected by controlling ph in wine (uthurry et al., 2006). lower ph affects citrulline metabolism (arena and nadra, 2005) and thus reduces ec production during fermentation. meanwhile, amygdalin in apple is hydrolyzed to hydrocyanic acid by β-glucosidase, which is then oxidized to isocyanic acid, and isocyanic acid reacts with ethanol to form ec, and low ph inhibits β-glucosidase activity. as far as we know, no study has focused on reducing ec content in fruit distillates by adjusting juice’s ph to 3.0 prior to fermentation. sulfuric acid (st), an inorganic acid is widely used in the preparation of ethanol (sun et al., 2011). it is a listed food additive, as a flocculant agent, which can be used in fermentation processes in china (gb/t 2760) (national health and family planning committee of china, 2014). malic acid (mt), lactic acid (lt), and citric acid (ct), as acidity regulators (gb/t 2760) (national health and family planning committee of china, 2014), are widely used in the food industry (marques et al., 2020; won et al., 2015). hence, it is meaningful to apply st, mt, lt, and ct to adjust the ph of apple juice before fermentation. the objective of this study was to investigate the effect of st, mt, lt, and ct on cyanide and ec in apple distillate. apples were washed and juiced. then st, mt, lt, or ct was added to apple juice to adjust its ph to 3.0. the treated juice was fermented at room temperature to get fermented juice. the juice was distillated by a double distillation method to obtain apple distillate. ec, cyanide, and volatile components present in the distillate were investigated. materials and methods materials fuji apple (malus domestica borkh. cv. “red fuji”) fruits were purchased from a local fruit market (tai’an, china). commercial wine yeast lalvin cy 3079 was purchased from shanghai jatou industry and commerce co. ltd. (shanghai, china). st was purchased from laiyang kant chemical co. ltd. (laiyang, china). dl-malic acid (anhui xuelang biotechnology co. ltd, china) was ordered from a local food additives store. ct was purchased from the local food additive store, and lt was purchased from henan jindan lactic acid technology co. ltd. (henan china). volatile standards (chromatographic grade) were obtained from china national research institute of food and fermentation industries apple apples juice control treated juice yeast cy3079 fermented juice distillation apple distillates fermentation malic acid lactic acid citric acid sulfuric acid figure 1. process of making apple distillates. italian journal of food science, 2023; 35 (2) 3 acid treatment removes ethyl carbamate and affects volatile components from apple distillates occurring in the collision cell of triple quadrupoles, with an argon collision gas pressure of approximately 2.0 m torr and an offset voltage of 20 ev. for quantitative analysis, the chosen fragments were monitored in multiple reaction monitoring (mrm) modes: 74, 44, 62, and 89 m/z for ec, and 64 and 76 m/z for ec-d5. then, selective ion monitoring (sim) of 62 m/z (ec) and 64 m/z (ec-d5) was used for the purpose of quantification. for quantification, peak area ratios of ec to ec-d5 were calculated as a function of the concentration of substances. determination of cyanide the determination of cyanide is according to the chinese national standard (national health and family planning committee of china. 2016; gb/t 5009.36). in a 50-ml beaker, 1 ml sample was taken; 5-ml of 2 g/l sodium hydroxide (naoh) solution was added to the sample taken in beaker and and allowed to remain for 10 min. then the beaker was heated on electric heating plate at 120°c till the solution was reduced to about 1 ml. it was transferred into 10-ml stopper colorimetric tube and the volume was adjusted to 5 ml by adding 2 g/l naoh solution. two drops of phenolphthalein indicator were added to the sample and the standard tube separately. acetic acid was added to make the red color of the solution to fade; 2 g/l naoh solution was used to adjust the color to near red. phosphate buffer solution, 2 ml, and chloramine t solution, 0.2 ml, were added in turn with continuous shaking for 3 min. then, 2 ml of isonicotinic-pyrazolone solution was added to the sample and diluted to 10 ml with water. incubation was performed for 40 min in a water bath at 37°c constant temperature. following incubation, the sample was taken out and the zero point was adjusted with a 1-cm colorimetric cup with a blank tube to measure absorbance at 638 nm. after the absorbance of 0-, 0.4-, 0.8-, 1.2-, 1.6-, and 2.0-ml cyanide standard intermediate solution into 10-ml stopper colorimetric tube, colorimetry was conducted according to sample determination to draw a standard curve. the cyanide content of the sample was measured by comparing to the standard curve of cyanide ion standard intermediate. determination of methanol methanol content was determined by gas chromatography with internal standard added according to the official reference method of association of official analytical chemists (aoac, 1994; 940.06). the 100-ml sample was added to 50-ml deionized water and distilled to 100  ml. internal standard (tert-amyl alcohol of 162 mg/l), 1 ml, was added to 10 ml of distillate; 1.0 μl of following three fractions were collected: the head (1% ethanol), the heart (83% ethanol), and the tail (16% ethanol). a final alcoholic concentration of 68%–72% (v/v) was reached in the second distillate (sd). in order to avoid loss of volatiles, all samples were sealed and kept at 4°c until analysis. before analysis, the heart was standardized and the alcohol was diluted to about 40% (v/v). all analyses were performed on 40% (v/v) samples. physicochemical analysis physicochemical analysis was carried out according to the national standards of the people’s republic of china, gb/t 15038-2006. alcohol content (% by volume) and dry extract (g/l) were measured based on the pycnometer method. titrable acidity (g/l, tartaric acid equivalent) was determined by potentiometric titration. ph values were directly measured using a laboratory recording ph meter (fe20, mettler toledo, zurich, switzerland). sugar content was titrated with fehling’s reagent. determination of ethyl carbamate ethyl carbamate was analyzed by gas chromatography– mass spectrometry (gc-ms) method according to our previous study (han et al., 2021) with some modifications. apple distillate samples, 2 ml, were mixed with 100 μl of ec-d5 (2 μg/ml in methanol solution), followed by addition of 0.30-g sodium chloride (nacl). after ultrasonic dissolution for 10 min, the mixture was directly applied to solid phase extraction (spe) cartridge (sbeq-ca3999, cnw technology, germany), and allowed to remain for 10 min for adequate absorption. the column was then washed with 10 ml n-hexane. next, the analytes were extracted using 10-ml 5% ethyl acetate and diethyl ether solution. the eluate was mixed in a test tube and reduced to approximately 0.5 ml by a gentle stream of nitrogen. subsequently, the residual eluents were adjusted to 1 ml with methanol and directly injected into a gc-ms system (gcms-tq8030, shimadzu, tokyo, japan). substances were separated on a fused-silica capillary column (vf-was, 60 m × 0.25 mm × 0.5 μm; agilent, usa). helium was used as a carrier gas at a constant flow rate of 1 ml/min. the injector port was kept at 220°c in splitless mode. the starting temperature was held at 50°c for 1 min, then increased to 180°c at a rate of 8°c/min and held for 5 min. finally, the temperature was increased to 240°c at a rate of 20°c/min and held for 5 min. the ms detector port and ion source temperature were set at 250°c and 230°c, respectively. gc-ms experiments were based on in-source collision-induced dissociation (cid) 4 italian journal of food science, 2023; 35 (2) su z et al. was performed according to the chinese national standard (general administration of quality supervision, inspection and quarantine of the people’s republic of china. 2008; gb/t 11856) with some modifications. the evaluators were provided with 45-ml apple distillate in standard glass cups, coded with random numbers. apple distillates were sniffed and tasted. three aspects, including olfactory, gustatory, and typicality, were used to measure the quality of apple distillates. the descriptors of olfactory were fruity and vinous; for gustatory evaluations, considered descriptors were alcoholic and balance. during each session, expert judges first assessed the smell, and then they evaluated gustatory attributes after a short break. according to the characteristics of the samples, the experts scored each descriptor with the highest score of 20 points. after descriptive analysis, the panelists assessed all samples for typicality according to their olfactory and gustatory tests and rated them with a maximum score of 20 points. finally, the experts wrote their comments on the samples after all the scores were completed. the total score of the samples were the sum of all the scores. all samples were diluted with distilled water to an alcohol concentration of 40% (v/v). mouthwash was used by tasters between analyses of two samples. statistical analysis all the data were processed using spss statistics 22. all pictures were drawn using origin 2022. in figure 3, red indicates positive correlation and blue shows negative correlation; the deeper the color, the greater the absolute value of correlation and stronger the correlation between them. mean differences at p < 0.05 were considered as significant using tukey’s test. all data were the average values of three replicates, analyzed in triplicate for each condition, and presented as mean values and standard deviations. results and discussion changes of titrable acidity and ph during fermentation all apple juice samples were fermented for 7 days, and the total sugar content was reduced to less than 1.4 g/l. in order to explore the influence of different acid treatments on fermented apple juice, titrable acidity and ph were tracked during fermentation. the ph of control was increased to 4.25 on the second day of fermentation and then decreased to 4.06 (figure 2a). on the contrary, the titrable acidity content decreased on the second day of fermentation and then increased (figure 2b). however, the ph of apple juice samples treated with different organic acids increased, but the corresponding titratable acidity content decreased during fermentation, which sample was directly injected into gas chromatography system equipped with a capillary column peg-20 m (30 m × 0.5  mm × 0.25 μm; dalian zhonghuida scientific instrument, china) and a flame ionization detector. the temperature of injector and detector was 220oc. the oven temperature procedure was as follows: the initial temperature was maintained at 40oc for 4 min; it was then increased to 200oc at a rate of 3.5oc/min and held at 200oc for 10 min. the carrier gas was nitrogen with a flow rate of 1.0 ml/min. the split ratio was 50:1. methanol quantification was determined by external standard method, but internal standard method was also used to improve the accuracy of results. volatile compounds analysis the volatile compounds were analyzed by gas chromatography (lópez-vázquez et al., 2010) with some modifications. internal standard (10 μl), including tert-amyl alcohol and n-butyl acetate 162 mg/l, as well as 2-ethyl butyrate 186.6 mg/l, was added into 1 ml of sample. then, 1 μl of sample was directly injected into a shimadzu 2010 chromatograph system with flame ionization detector. a capillary column cp-wax 57cb (50 m × 0.25 mm × 0.2 μm; agilent, usa) was used for this analysis. the temperatures of detector and injector were 260oc and 240oc, respectively. the oven temperature program was maintained at 35oc for 4 min, increased to 60oc at a rate of 2oc/min, continued to rise to 130oc at a rate of 10oc/min, and finally increased to and maintained at 205oc at a rate of 15oc/min. the carrier gas was nitrogen with a flow rate of 1.35 ml/min, and the split ratio was 40:1. the qualitative analyses of volatile compounds were based on the comparison of retention time read from the chromatograms of both samples and standards. the quantitative analysis was performed according to the internal standard method. all detected carbonyl compounds, esters, higher alcohols, and acids were analyzed quantitatively. sensory analysis the panel included three female and eight male analysts, with experience in the evaluation of fruit distillates and were trained to describe and recognize the evaluated odor qualities. the panelists were trained according to the iso 8586 standard (international organization for standardization 2012). prior to and during this study, monthly training was conducted to evaluate multiple flavor standards (acetoin, isoamyl acetate, ethyl acetate, ethyl lactate, acetaldehyde, 1-propanol, 1-hexanol, acetic acid, and head and tail distillation fractions) and different spirits distilled from cider, hawthorn wine, and persimmon wine. the sensory analysis of the samples italian journal of food science, 2023; 35 (2) 5 acid treatment removes ethyl carbamate and affects volatile components from apple distillates control 4.4 (a) (b) 4.2 4.0 3.8 3.2 3.0 0 1 2 3 fermentation time (d) ph 4 5 6 7 st mt lt ct control 10 8 6 4 2 0 0 1 2 3 fermentation time (d) ti tr ab le a ci di ty ( g/ l) 4 5 6 7 st mt lt ct figure 2. changes in ph (a) and titrable acidity (b) during fermentation. control: original apple juice; st: sulfuric acid-treated juice; mt: malic acid-treated juice; lt: lactic acid-treated juice; ct: citric acid-treated juice. might be caused by the degradation of organic acids by yeast and lt bacteria (lerena et al., 2016; zhong et al., 2020). in all organic acid-treated samples, maximum change in titrable acidity was observed in mt sample, which decreased from 9.53 g/l to 7.92 g/l during the fermentation. the titrable acidity of ltand ct-treated samples was reduced by 0.74 g/l and 1.16 g/l, respectively. conversely, the ph of mt increased from 3.00 to 3.21. in summary, the titrable acidity of mt-, lt-, and ct-treated samples showed an overall decreasing trend during the fermentation process, while the titrable acidity of the control and st-treated sample showed an insignificant decreasing trend. physicochemical indices of fermented apple juices the basic physical and chemical indexes of fermented apple juices are shown in table 1. all the fermented apple juice samples achieved complete fermentation, with the total sugar content ranging from 0.9 to 1.3 g/l and the alcohol content from 7.66 to 7.73% (v/v). this proposed that different treatments of apple juice samples before fermentation did not affect the fermentation capacity of yeast. the methanol content of all fermented apple juice samples was between 5.28 mg/l and 6.42 mg/l. the methanol content of st, mt, lt, and ct was significantly lower than the control (p < 0.05), indicating that the methanol content could be significantly reduced by different acid treatments. in addition, ec was not detected in all the fermented apple juices. volatile component of fermented apple juice samples in the fermented apple juice samples, 20 volatile compounds, such as carbonyl compounds, esters, higher alcohols, and acids, were detected by gas chromatography (table 2). three carbonyl compounds were identified in all samples. these could be produced by oxidation of alcohols or decarboxylation of acids (xiao et al., 2015). acetaldehyde contributed to the flavors of fermented apple juice samples with fruity, nut, and dried fruits aroma. however, acetaldehyde could impart pungent aroma if higher quantity is added. it was determined in all fermented apple juices; however, the maximum amount was determined in ct (123.26 mg/l). the lowest acetaldehyde content was observed in st, which was 26.69 mg/l. acetoin was the only ketone detected and it provided a buttery and cream aroma to the fermented apple juice samples (welke et al., 2014). in this study, the content of acetoin in st, mt, lt, and ct were significantly reduced, compared to the control (p < 0.05). acetoin reached the lowest level of 1.61 mg/l in st. although the amount of acetoin in fermented apple juices varies significantly, it is difficult for them to have a significant impact on the flavor of fermented apple juice samples because of its high sensory threshold. esters are one of the most important volatile constituents of fermented apple juices, and were formed by the reaction of alcohol and free organic acids during fermentation (villière et al., 2015). in this study, the contents of ethyl acetate and isoamyl acetate were above the threshold, 6 italian journal of food science, 2023; 35 (2) su z et al. table 1. physicochemical indices of fermented apple juice samples. control st mt lt ct alcohol (%, v/v) 7.71 ± 0.04a 7.66 ± 0.06a 7.70 ± 0.03a 7.73 ± 0.04a 7.66 ± 0.01a total sugar (g/l) 1.3 ± 0.1a 0.9 ± 0.0d 0.9 ± 0.0d 1.2 ± 0.0b 1.1 ± 0.0c titrable acidity (g/l) 2.2 ± 0.0e 4.3 ± 0.0d 7.9 ± 0.0c 8.3 ± 0.0b 9.5 ± 0.0a ph 4.03 ± 0.01a 3.08 ± 0.00e 3.24 ± 0.00b 3.17 ± 0.00c 3.15 ± 0.00d dry extract (g/l) 16.7 ± 0.0d 19.9 ± 0.2c 25.4 ± 0.0a 24.9 ± 0.0b 24.7 ± 0.0b methanol (mg/l) 6.42 ± 0.08a 5.41 ± 0.00b 5.28 ± 0.14b 5.48 ± 0.69b 5.37 ± 0.18b ethyl carbamate (μg/l) – – – – – all values are expressed as mean values ± standard deviations (n = 3); different superscripted lowercase letters in the same row indicate significant difference (p < 0.05). control: fermented apple juice from original apple juice; st: fermented apple juice from sulfuric acid-treated juice; mt: fermented apple juice from malic acid-treated juice; lt: fermented apple juice from lactic acid-treated juice; ct: fermented apple juice from citric acid-treated juice. – indicates not detected. table 2. volatile component in fermented apple juice samples (mg/l). compound threshold control st mt lt ct carbonyl compounds acetaldehyde 0.5(1) 48.76 ± 5.72b 26.69 ± 0.25c 46.48 ± 7.08b 52.56 ± 4.87b 123.26 ± 6.23a acetal 0.05(1) nd nd 0.85 ± 0.14b 0.89 ± 0.09b 1.49 ± 0.07a acetoin 150(2) 31.85 ± 0.98a 1.61 ± 0.11c 12.72 ± 2.71b 12.21 ± 0.22b 14.08 ± 0.68b esters ethyl acetate 7.5 (1) 77.03 ± 4.08a 22.41 ± 2.32b 79.50 ± 3.49a 76.95 ± 0.73a 15.95 ± 0.05c isoamyl acetate 0.03(3) 2.36 ± 0.17a 1.04 ± 0.04c 1.62 ± 0.07b 1.43 ± 0.02b 0.49 ± 0.13d ethyl lactate 150(2) 7.18 ± 0.07c 4.13 ± 0.51c 31.77 ± 7.65b 73.39 ± 2.42a 13.19 ± 0.46c ethyl decanoate 0.5(4) 0.07 ± 0.00a 0.04 ± 0.01b 0.07 ± 0.02a 0.05 ± 0.00a,b 0.04 ± 0.00b higher alcohols 1-propanol 50(3) 62.78 ± 0.97a 19.87 ± 0.16e 39.75 ± 0.34c 41.58 ± 0.52b 38.16 ± 0.21d 2-methyl-1-propanol 40(1) 16.5 ± 0.06d 21.69 ± 0.20b 18.43 ± 0.11c 22.87 ± 0.09a 18.41 ± 0.40c 1-butanol 150(3) 2.02 ± 0.04a 1.96 ± 0.09a 2.16 ± 0.17a 1.99 ± 0.06a 2.05 ± 0.02a 2-methyl-1-butanol 0.32(4) 14.85 ± 0.12c 17.51 ± 0.19a 13.39 ± 0.42d 17.54 ± 0.02a 16.24 ± 0.04b 3-methyl-1-butanol 30(4) 65.38 ± 0.39e 78.98 ± 1.07a 68.36 ± 0.88d 75.33 ± 0.32b 71.68 ± 0.79c 1-hexanol 8(1) 2.39 ± 0.02a 2.16 ± 0.02a 2.1 ± 0.03a 2.55 ± 0.53a 1.95 ± 0.01a 2,3-butanediol – 4.59 ± 0.00c 4.18 ± 0.02d 5.67 ± 0.08b 6.30 ± 0.05a 2.94 ± 0.12e 2-phenylethanol 10(1) 0.53 ± 0.14b 0.8 ± 0.07b 0.82 ± 0.09b 0.76 ± 0.20b 1.99 ± 0.08a acids acetic acid 200(1) 244.87 ± 4.79a 108.3 ± 1.33c 254.22 ± 7.12a 230.22 ± 7.29b 46.68 ± 0.44d propanoic acid 8.1(3) 2.26 ± 0.30a 1.33 ± 0.15b 2.57 ± 0.35a 2.81 ± 0.50a 1.99 ± 0.53a,b butyric acid 10(1) 1.31 ± 0.23b 1.97 ± 0.02a 1.68 ± 0.06a 1.92 ± 0.08a 1.87 ± 0.11a hexanoic acid 3(1) 5.73 ± 0.4a 4.47 ± 0.06c 4.56 ± 0.12c 4.82 ± 0.10b,c 5.36 ± 0.45a,b octanoic acid 0.2(3) 14.92 ± 2.08a,b 10.89 ± 0.68c 13.34 ± 0.18a,b,c 12.06 ± 1.22b,c 15.18 ± 0.60a all values are expressed as mean values ± standard deviations (n = 3); different superscripted lowercase letters in the same row indicate significant difference (p < 0.05). control: fermented apple juice from original apple juice; st: fermented apple juice from sulfuric acid-treated juice; mt: fermented apple juice from malic acid-treated juice; lt: fermented apple juice from lactic acid-treated juice; ct: fermented apple juice from citric acid-treated juice; nd: not detected (guth, 1997;(1) peinado et al., 2004;(2) wang et al., 2017;(3) wei et al., 2020(4)). italian journal of food science, 2023; 35 (2) 7 acid treatment removes ethyl carbamate and affects volatile components from apple distillates fermented apple juices. they were described with cheese, rancid, and fatty notes, and were important for the balance of complexity and fruity aromas of fermented apple juices (sun et al., 2013). physicochemical indices of apple distillates cyanide and ec were not detected in the fermented apple juice or first distillates. as shown in table 3, the highest concentration of cyanide was detected in the control (0.035 mg/l). the cyanide content of other treatment samples was significantly lower than that of the control. in this study, the content of ec in different treatments was in the range of 9.12–2.93 μg/l. compared to the control, the content of ec in st, mt, lt, and ct was significantly reduced (p < 0.05). the ec content of the control was 9.12 μg/l, which was 2.12–3.11 times that of other treatment samples. lt had the best effect on reducing ec by 67.87% compared to the control. the ec reduction effects of st, mt, and ct were 64.58%, 64.91%, and 60.86%, respectively. generally, the concentration of cyanide and ec in apple distillates could be effectively reduced by treating apple juice with different acids before fermentation. volatile components of apple distillates as shown in table 4, 24 volatile compounds were detected in the apple distillates by gas chromatography, including carbonyl compounds, esters, alcohols, and acids. among carbonyl compounds, a total of four aldehydes and one ketone were detected. only the contents of acetaldehyde and acetal exceeded the threshold in all apple distillates. acetaldehyde was the most important aldehyde; its concentration in different apple distillates ranged from 47.64 to 468.48 mg/l, and its content was significantly different (p < 0.05) in all distillates. the highest acetaldehyde content was detected in ct, and the lowest was observed in st. in the control, the contents of acetaldehyde and acetal were 126.60 mg/l and 74.60 mg/l, respectively. it is worth mentioning that except for st, the contents of and had a great influence on the aroma of apple juices. however, compared to the control, the content of ethyl acetate in st and ct was significantly reduced to 54.62 mg/l and 61.08 mg/l, respectively, whereas the content of isoamyl acetate in other treatment samples was significantly decreased (p < 0.05). significantly, although the content of ethyl lactate did not exceed the threshold, the mtand lt-treated apple juice before fermentation could obviously increase the content of ethyl lactate in fermented apple juice samples. this result might be caused by the addition of mt and lt before fermentation, which increased lt content during fermentation, thus increasing the content of ethyl lactate. higher contents of alcohols in cider was synthesized by yeast through the glucose synthesis pathway or the corresponding amino acid catabolism pathway, which was characterized by strong and pungent smell and had an important role in the aroma of fermented apple juices (qin et al., 2018). eight higher alcohols were detected in all treatment samples, of which 3-methyl-1-butanol was the most predominant alcohol in all fermented apple juices and its concentration was in the range of 65.38– 78.98 mg/l. furthermore, both 3-methyl-1-butanol and 2-methyl-1-butanol were above the threshold in all fermented apple juices and provided the smell of alcohol, nail polish, and whiskey to fermented samples (arcari et al., 2017). compared to the control, the contents of both 3-methyl-1-butanol and 2-methyl-1-butanol in st, lt, and ct were significantly increased (p < 0.05). as for 1-propanol, only the control exceeded the threshold of 1-propanol content, which was 62.78 mg/l, indicating that different acid treatments could significantly affect the production of 1-propanol. five different volatile fatty acids were identified across fermented apple juices. among these, acetic acid was the predominant volatile acid in all fermented apple juices. however, the content of acetic acid in st and ct was significantly lower than that in the control (p < 0.05) and did not exceed the threshold, especially its content was only 46.68 mg/l in ct. furthermore, the contents of hexanoic acid and octanoic acid exceeded the threshold in all table 3. physicochemical indices of apple distillates. control st mt lt ct alcohol (%, v/v) 40.2 ± 0.0a 40.2 ± 0.0a 40.2 ± 0.0a 40.1 ± 0.1a 40.2 ± 0.0a cyanide (mg/l anhydrous ethanol) 0.035 ± 0.004a nd 0.015 ± 0.000c 0.010 ± 0.000c 0.028 ± 0.004b ec (μg/l) 9.12 ± 0.40a 3.23 ± 0.16b 3.20 ± 0.27b 2.93 ± 0.09b 3.57 ± 0.22b all values are expressed as mean values ± standard deviations (n = 3); different superscripted lowercase letters in the same row indicate significant difference (p < 0.05). control: apple distillate from original apple juice; st: apple distillate from sulfuric acid-treated juice; mt: apple distillate from malic acid-treated juice; lt: apple distillate from lactic acid-treated juice; ct: apple distillate from citric acid-treated juice; nd: not detected. the concentrations of cyanide and ec were recalculated on the basis of 100% (v/v) ethanol. 8 italian journal of food science, 2023; 35 (2) su z et al. table 4. volatile components of apple distillates (mg/l). threshold control st mt lt ct carbonyl compounds acetaldehyde 19.2(1) 126.60 ± 0.64d 47.64 ± 0.18e 191.39 ± 1.20c 207.92 ± 2.42b 468.48 ± 8.21a 2-methylpropanal 1.3(4) 0.52 ± 0.03e 1.97 ± 0.04b 1.82 ± 0.03c 1.84 ± 0.03c 2.07 ± 0.05a acetal 0.719(1) 74.60 ± 0.45d 59.54 ± 0.46e 92.55 ± 0.03c 111.95 ± 0.60b 148.33 ± 0.92a acetoin – 6.70 ± 0.21b 0.45 ± 0.04e 4.98 ± 0.31c 16.03 ± 0.19a 3.77 ± 0.13d furfural 44(4) 1.77 ± 0.12b,c 1.59 ± 0.06c 2.21 ± 0.02b 3.33 ± 0.39a 1.91 ± 0.04b,c esters ethyl formate – 1.46 ± 0.10d 3.01 ± 0.00c 3.78 ± 0.37b 2.84 ± 0.12c 5.31 ± 0.09a ethyl acetate 32.6(3) 274.22 ± 3.08a 101.63 ± 0.64d 205.82 ± 1.17c 219.37 ± 0.60b 79.55 ± 1.07e isoamyl acetate 0.245(3) 19.95 ± 2.29b 17.59 ± 0.74b,c 14.73 ± 0.39c,d 13.09 ± 0.10d 26.08 ± 1.56a ethyl lactate 128(2) 8.21 ± 0.17d 5.59 ± 0.16e 21.65 ± 0.04b 74.74 ± 0.30a 18.76 ± 0.20c ethyl octanoate 0.147(3) 1.45 ± 0.02a 0.91 ± 0.03c 0.80 ± 0.05d 1.00 ± 0.01b 0.88 ± 0.01c ethyl decanoate 1.120(2) 0.02 ± 0.00b 0.02 ± 0.00a 0.01 ± 0.00c 0.02 ± 0.00b 0.01 ± 0.00c alcohols 1-propanol 54(3) 394.49 ± 3.29a 124.89 ± 0.22e 252.3 ± 0.95b 237.39 ± 0.76c 228.41 ± 0.41d 2-methyl-1-propanol 28.3(3) 107.88 ± 0.76d 152.56 ± 2.72a 125.52 ± 0.07c 144.24 ± 0.40b 122.75 ± 0.32c 1-butanol 2.73(2) 12.60 ± 0.46a 12.87 ± 0.29a 12.94 ± 0.04a 12.74 ± 0.08a 13.18 ± 0.17a 2-methyl-1-butanol 45(1) 126.30 ± 4.58a,b 139.34 ± 1.78a 115.09 ± 4.00b 132.14 ± 7.11a 123.45 ± 10.48a,b 3-methyl-1-butanol 179(3) 470.29 ± 1.84c 614.23 ± 8.07a 513.65 ± 4.54b 524.47 ± 4.88b 515.86 ± 0.81b 1-hexanol 8(3) 21.04 ± 0.56a 20.77 ± 0.01a 19.43 ± 0.13b 20.73 ± 0.28a 19.37 ± 0.17b 2,3-butanediol – 1.47 ± 0.03b 0.95 ± 0.18c 1.35 ± 0.09b 2.87 ± 0.00a 0.24 ± 0.02d 2-phenylethanol 2.6(3) 1.74 ± 0.07b 1.76 ± 0.02b 1.45 ± 0.09c 1.95 ± 0.04a 1.14 ± 0.02d methanol 25.54 ± 0.14a 22.58 ± 0.52c 23.86 ± 0.44b 23.47 ± 0.28b,c 22.45 ± 0.56c acids acetic acid 75.521(3) 4.60 ± 0.07a 2.27 ± 0.60c 1.11 ± 0.04d 3.66 ± 0.40b nd butanoic acid 1.2(3) 1.85 ± 0.24a 0.92 ± 0.14b,c 1.21 ± 0.15b 0.37 ± 0.03d 0.68 ± 0.18c,d hexanoic acid 2.52(3) 8.47 ± 0.35a 5.80 ± 0.31b 5.42 ± 0.25b 5.66 ± 0.29b 4.46 ± 0.33c octanoic acid 2.7(3) 46.87 ± 0.77a 17.96 ± 3.48c,d 24.96 ± 0.28b 21.1 ± 0.30b,c 13.83 ± 2.53d all values are expressed as mean values ± standard deviations (n = 3); different superscripted lowercase letters in the same row indicate significant difference (p < 0.05). control: apple distillate from original apple juice; st: apple distillate from sulfuric acid-treated juice; mt: apple distillate from malic acid-treated juice; lt: apple distillate from lactic acid-treated juice; ct: apple distillate from citric acid-treated juice; nd: not detected. – indicates not found. the concentrations of volatile compounds were recalculated on the basis of ethanol 40% (v/v) (gao et al., 2014;(2) wang et al., 2014a;(4) willner et al., 2013;(1) xiang et al., 2020(3)). acetaldehyde and acetal were significantly increased in other samples (p < 0.05). compared to fermented juices, 2-methylpropanal and furfural were newly observed natural products observed in apple distillates; these were formed due to the chemical reactions caused by high temperature during distillation (awad et al., 2017). six esters were also discovered in apple distillates (table 4), among which the contents of ethyl acetate, isoamyl acetate, and ethyl octanoate were above the threshold in all samples. ethyl acetate was the most important ester, and its presence was consistent with the results in other apple distillates (ledauphin et al., 2010; versini et  al.,  2009). the lowest content of 79.55 mg/l was discovered in ct. the content of ethyl acetate in st, mt, and lt was 101.63, 205.82, and 219.37 mg/l, respectively. its concentration in apple distillates with different treatments was significantly different (p < 0.05). the content of ethyl acetate in apple distillates was significantly reduced by acid treatments. alcohols were the most important volatiles observed in distillates, and nine compounds were discovered in this study. the contents of 1-propanol, 2-methyl-1propanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, and 1-hexanol in all samples exceeded the threshold, italian journal of food science, 2023; 35 (2) 9 acid treatment removes ethyl carbamate and affects volatile components from apple distillates sensory evaluation of apple distillates according to the sensory descriptors mentioned in table 5, we observed that mt had the highest fruity score, which was also consistent with the expert’s description of mt. st had the lowest scores for fruity and typicality, but both vinous and alcoholic had high scores. the results indicated that although st could affect the typicality of apple distillate, but st would not have a seriously adverse effect on the quality of distillate. concerning lt and ct, most of their scores of sensory descriptors were significantly lower than the control hence, lt and ct could have bad effects on the quality of distillate. in sensory evaluation, the control had the highest score of 92.27, but the ct score was lowest (85.73). the sensory evaluation scores from low to high were: lt (86.64), st (88.64), and mt (91.09). the scores of control and mt were close to each other, which could be due to the close content values of esters and alcohols. st had the highest 3-methyl-1 butanol content of 614.23 mg/l (table 4), which could be responsible for its strong alcoholic taste. although ct had the highest acetaldehyde content (468.48 mg/l), it had the lowest content of ethyl acetate (79.55 mg/l), which could be the reason for its flavor defect and bitter taste. in summary, although the treatment of st, mt, lt, and ct on apple juice before fermentation could reduce the ec but 3-methyl-1-butanol had the highest concentration as observed previously for apple distillates (ledauphin et al., 2003). compared to the control, content of 1-propanol in the acid-treated sample was decreased significantly (p < 0.05), and the lowest content found in st was only 124.89 mg/l. however, the concentrations of 2mthyl-1-propanol and 3-methyl-1-butanol in different acid-treated samples were higher than that in the control (p < 0.05), and the highest contents of these two substances in st-treated sample were 152.56 mg/l and 614.23 mg/l, respectively. however, the content of methanol in different treatment samples was decreased significantly (p < 0.05). four different volatile acids were identified in all samples. the content of acetic acid in apple distillates was significantly reduced compared to that in fermented apple juice; this could be due to the removal of tail during distillation. the contents of hexanoic acid and octanoic acid were above the threshold in all samples. in addition, the highest contents of hexanoic acid and octanoic acid in the control were 8.47 mg/l and 46.87 mg/l, respectively. furthermore, the concentrations of these substances in other treatment samples were significantly decreased, and the lowest concentrations of hexanoic acid and octanoic acid were detected in ct sample, which were 4.46 mg/l and 13.83 mg/l, respectively. table 5. effects of different treatments on sensory scores of apple distillates. sample sensory descriptor olfactory gustatory typicality fruity vinous alcoholic balance control 17.91 ± 0.54b 17.55 ± 0.52a 18.09 ± 0.54b 19.27 ± 0.47a 19.45 ± 0.52a st 17.09 ± 0.83c 17.55 ± 0.69a 19.18 ± 0.40a 18.27 ± 0.47b 16.55 ± 0.52c mt 18.82 ± 0.40a 17.82 ± 0.60a 16.91 ± 0.54c 18.27 ± 0.47b 19.27 ± 0.47a lt 17.73 ± 0.47b 16.64 ± 0.50b 16.27 ± 0.47d 17.82 ± 0.60c 18.18 ± 0.40b ct 16.91 ± 0.54c 17.45 ± 0.52a 16.73 ± 0.47c 16.82 ± 0.40d 17.82 ± 0.40b sensory evaluation score control the distillate had a pleasant aroma, a full-bodied palate, and no miscellaneous flavors, and had the typical characteristics of apple distillate. 92.27 ± 0.90a st the aroma of apple was weaker than that of the control and had a plastic odor. strong alcoholic taste in the mouth. the overall coordination was good. 88.64 ± 1.12c mt it had the best apple aroma, no odor, the mouth was clean and refreshing, but the overall coordination was lower than the control. it also had the typical characteristics of apple distillates. 91.09 ± 0.94b lt it had a strong aroma of apple, but a weaker vinous. it also had a faint alcoholic taste in the mouth, which was unpleasant. 86.64 ± 1.21d ct the aroma of the wine was flawed compared to that of the control, with a lighter apple aroma, a bitter taste, and the heaviest wine. the overall coordination was not good. 85.73 ± 0.65e all values are expressed as mean values ± standard deviations (n = 11); different superscripted lowercase letters in the same column indicate significant difference (p < 0.05). control: apple distillate from original apple juice; st: apple distillate from sulfuric acid-treated juice; mt: apple distillate from malic acid-treated juice; lt: apple distillate from lactic acid-treated juice; ct: apple distillate from citric acid-treated juice. 10 italian journal of food science, 2023; 35 (2) su z et al. in distillates had a significantly positive correlation (0.72*). it has been reported that as a precursor substance of ec, cyanide content is significantly correlated with ec content (wang et al., 2021). therefore, the ec content of apple distillate can be decreased by reducing the cyanide content. furthermore, ph affected the production of ethyl acetate (0.73*), ethyl octanoate (0.93***), 1-propanol (0.91***), acetic acid (0.65*), butanoic acid (0.83**), hexanoic acid (0.91***), and octanoic acid (0.96***). in the meantime, ph was negatively correlated with 2-methylpropanal (-0.99***), ethyl formate (-0.69*), 2-methpropanol (-0.77**), and 3-methyl-1-butanol (-0.70*). overall, the ph of fermented apple juice plays an important role in the formation of volatiles in apple distillates. conclusion acid treatment by st, mt, lt, and ct significantly removed ec from apple distillates produced by fuji acidified apple juice. the lower the ph of fermented apple content in the final distillates, it also impacted the aroma, flavor, and taste. therefore, the stored fermented apple juice would be treated with the acids to find their influence on ec and volatiles on the final distillate in of the following research. correlation analysis of volatile constituents in apple distillates and basic physicochemical indexes of fermented apple juice in order to further analyze the effects of different treatments on cyanide, ec, and volatiles found in apple distillates, the correlation analysis chart was used to analyze the relationship of total sugar, titrable acidity, ph, dry extract, and alcohol in fermented apple juice, and that of cyanide, ec and volatiles in distillates. as shown in figure 3, a significantly positive correlation is observed between the ec content in apple distillates and the ph of fermented apple juice (0.98***). it indicates that lowering the ph of fermented apple juice significantly reduces the ec content of apple distillates. in addition, the ec and cyanide contents figure 3. correlation analysis of volatile constituents in apple distillates and basic physicochemical indexes of fermented apple juice. red indicates positive correlation and blue shows negative correlation. the deeper the color, the greater the absolute value of correlation and the stronger the correlation between them. ad: apple distillates. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.0001. -0.29 0.74 -0.39 0.34 0.66 0.72 0.11 -0.73 0.15 0.59 0.27 -0.51 0.63 0.14 0.28 0.83 0.80 -0.55 -0.50 -0.73 0.44 0.39 0.31 0.65 0.26 0.64 0.63 0.019 -0.021 -0.71 0.97 -0.088 -0.13 -0.75 0.75 0.77 0.83 0.27 0.50 0.83 -0.42 0.053 0.53 -0.76 -0.69 0.19 0.53 -0.33 -0.73 -0.016 -0.49 -0.69 -0.74 -0.87 -0.74 -0.010 -0.36 -0.73 0.31 0.73 0.98 -0.25 -0.99 -0.32 0.082 -0.25 -0.69 0.73 0.13 -0.30 0.93 0.073 0.91 -0.49 -0.16 -0.70 0.091 0.20 0.65 0.83 0.91 0.96 0.44 -0.77 -0.0098 -0.26 -0.81 0.58 0.77 0.68 0.30 0.56 0.74 -0.31 0.17 0.56 -0.81 -0.66 -0.39 0.27 -0.36 0.035 -0.71 -0.38 -0.64 -0.70 -0.84 -0.71 0.11 0.45 0.13 0.20 -0.22 -0.35 -0.10 0.60 0.52 -0.41 0.66 -0.52 0.45 0.29 0.031 0.42 -0.10 -0.11 -0.36 0.41 0.39 0.58 0.095 0.32 0.44 0.62 -0.016 0.72 0.47 -0.64 0.36 0.034 -0.22 -0.045 0.34 0.58 -0.25 0.58 -0.49 0.84 -0.94 -0.52 -0.86 -0.10 -0.42 -0.087 0.53 0.39 0.57 -0.26 0.037 -0.23 -0.97 -0.33 -0.030 -0.36 -0.66 0.61 0.27 -0.40 0.94 0.14 0.85 -0.74 -0.080 -0.61 0.45 0.17 0.62 0.81 0.90 0.92 -0.032 -0.46 0.36 0.97 0.072 0.13 0.79 -0.41 0.63 0.16 -0.32 -0.78 0.043 -0.32 -0.41 -0.36 -0.69 -0.76 -0.64 -0.39 -0.58 -0.43 -0.38 0.54 0.42 -0.12 0.21 0.78 -0.77 -0.031 0.26 -0.94 -0.18 -0.87 0.68 0.092 0.63 -0.55 -0.33 -0.74 -0.82 -0.94 -0.98 -0.18 0.54 0.29 0.35 0.75 -0.34 0.45 0.39 -0.35 -0.75 0.011 -0.20 -0.34 -0.35 -0.62 -0.61 -0.55 -0.53 -0.62 -0.48 -0.16 0.47 0.92 -0.28 0.57 -0.50 0.92 0.20 -0.12 0.32 0.052 0.014 -0.41 0.27 0.52 0.49 -0.36 0.098 0.13 0.88 -0.33 -0.032 0.35 -0.57 0.97 -0.15 -0.19 0.023 0.28 0.040 -0.19 0.073 0.41 0.24 -0.57 -0.22 -0.17 0.82 -0.17 -0.77 0.45 -0.036 -0.78 -0.61 -0.49 0.11 -0.27 0.17 -0.83 -0.82 -0.94 -0.55 -0.90 -0.80 -0.55 0.72 -0.50 0.26 0.66 0.0059 0.79 -0.45 -0.23 -0.66 0.44 0.55 0.76 0.52 0.75 0.83 0.67 -0.62 -0.53 0.11 -0.29 0.11 -0.45 -0.12 -0.16 -0.35 -0.70 -0.43 0.089 -0.11 -0.078 -0.81 0.35 -0.16 -0.14 -0.045 0.35 0.074 -0.13 0.092 -0.42 0.23 -0.66 -0.25 -0.24 0.81 -0.13 0.33 0.78 -0.55 0.14 -0.53 0.67 0.41 0.79 0.64 0.91 0.88 0.22 -0.59 -0.29 0.54 0.80 0.60 0.83 0.71 0.58 0.085 0.42 0.15 0.21 -0.35 -0.88 -0.41 -0.93 0.19 0.051 0.51 0.66 0.72 0.86 0.19 -0.39 0.59 0.88 0.19 0.41 -0.071 -0.69 -0.46 -0.66 0.26 -0.35 0.21 -0.45 -0.66 0.65 -0.30 -0.66 -0.53 -0.52 0.59 -0.58 0.51 0.36 -0.25 0.11 -0.18 0.17 0.12 0.18 -0.23 -0.43 -0.41 -0.62 -0.17 0.81 0.88 0.25 0.66 0.50 0.80 0.67 -0.21 0.26 0.23 0.84 0.0036 0.53 0.35 0.36 0.83 0.74 0.76 0.82 0.94 0.50 0.10 to ta l s ug ar ti tr ab le a ci di ty ph d ry e xt ra ct a lc ho l a d c ya ni de a d e th yl c ar ba m at e a d a ce ta ld eh yd e a d 2 -m et hy lp ro pa na l a d a ce ta l a d a ce tio n a d fu rf ur al a d e th yl fo rm at e a d e th yl a ce ta te a d is oa m yl a ce ta te a d e th yl la ct at e a d e th yl o ct on oa te a d e th yl d ec an oa te a d 1 -p ro pa no l a d 2 -m et hy l-1 -p ro pa no l a d 2 -m et hy l-1 -b ut an ol a d 3 -m et hy l-1 -b ut an ol a d 1 -h ex an ol a d 2 ,3 -b ut an ed io l a d 2 -p he ny le th an ol a d a ce tic a ci d a d b ut an oi c ac id a d h ex an oi c ac id a d o ct an oi ca ci d a d 1 -b ut an ol total sugar 1 0.8 0.6 0.4 0.2 0 –0.2 –0.4 –0.6 –0.8 –1 titrable acidity ph dry extract alchol ad cyanide ad ethyl carbamate ad acetaldehyde ad 2-methylpropanal ad acetal ad acetion ad furfural ad ethyl formate ad ethyl acetate ad isoamyl acetate ad ethyl lactate ad ethyl octonoate ad ethyl decanoate ad 1-propanol ad 2-methyl-1-propanol ad 2-methyl-1-butanol ad 3-methyl-1-butanol ad 1-hexanol ad 2,3-butanediol ad 2-phenylethanol ad acetic acid ad butanoic acid ad hexanoic acid ad octanoicacid ad 1-butanol italian journal of food science, 2023; 35 (2) 11 acid treatment removes ethyl carbamate and affects volatile components from apple distillates and alcohol content in ‘heart’ fractions on the concentration of aroma volatiles and undesirable compoundsin plum brandies. j inst brewing. 123: 452–463. https://doi.org/10.1002/jib.441 european food safety authority. 2007. ethyl carbamate and hydrocyanic acid in 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https://doi.org/10.1002/jib.197 https://doi.org/10.1007/s00217-014-2275-z� http://doi.org/10.1080/19440049.2013.878869 http://doi.org/10.1016/j.foodchem.2017.01.007 https://doi.org/10.1007/s00253-021-11348-1� https://doi.org/10.1007/s00253-021-11348-1� https://doi.org/10.1016/j.ijfoodmicro.2019.108471� http://doi.org/10.1016/j.foodres.2014.02.002 https://doi.org/10.1021/jf403024t� https://doi.org/10.3746/pnf.2015.20.4.292� https://doi.org/10.3746/pnf.2015.20.4.292� https://doi.org/10.1016/j.foodres.2020.109388� http://doi.org/10.1016/j.jchromb.2014.12.006 https://doi.org/10.3390/fermentation6010025� https://doi.org/10.1016/0165-1218(91)90126-7� https://doi.org/10.1016/0165-1218(91)90126-7� https://doi.org/10.1016/j.arabjc.2020.10.036� https://doi.org/10.1016/j.arabjc.2020.10.036� http://down.afoodmate.net/standard/sort/3/42543.html� http://down.afoodmate.net/standard/sort/3/42543.html� http://down.foodmate.net/standard/sort/3/50421.html� http://down.foodmate.net/standard/sort/3/50421.html� https://doi.org/10.1016/s0308-8146(03)00282-6� https://doi.org/10.1016/j.foodres.2017.12.003� https://doi.org/10.1016/j.fm.2009.12.005� http://doi.org/10.1016/j.tca.2004.05.018� http://doi.org/10.1016/j.tca.2004.05.018� http://doi.org/10.1016/j.foodchem.2012.12.032 http://doi.org/10.1016/j.foodchem.2012.12.032 https://doi.org/10.1016/j.biortech.2011.09.071� https://doi.org/10.1248/jhs1956.38.498� https://doi.org/10.1016/j.aca.2017.11.022� https://doi.org/10.1248/jhs1956.38.498� https://doi.org/10.1248/jhs1956.38.498� https://doi.org/10.1016/j.foodchem.2008.08.003� https://doi.org/10.1002/jib.197� ole_link17 ole_link19 ole_link94 ole_link95 ole_link22 ole_link21 ole_link18 ole_link20 ole_link84 ole_link85 ole_link1 ole_link2 _hlk124520299 _hlk124520280 ole_link3 ijfs#42_pietrzak-fiećko_bozza   ital. j. food sci., vol 28, 2016 402 paper fatty acid composition in wild boletus edulis from poland r. pietrzak-fiećko1*, m. gałgowska1 and s. bakuła2 1department of commodities and food analysis, university of warmia and mazury in olsztyn, pl. cieszyński 1, 10-719 olsztyn, poland 2 institute of food technology and gastronomy, state college of computer science and business administration in łomża, akademicka 14 str., 18-400 łomża, poland *corresponding author: tel.: +48 8952335 70 e-mail address: renap@uwm.edu.pl abstract the aim of this study was to determine the content of fat and fatty acids profile in wild boletus edulis. the research material consisted of 33 samples of wild boletus edulis in the form of caps and stems, collected from selected regions of poland. methyl esters of fatty acids were prepared by peisker’s method. separation of the examined compounds was performed by gas chromatography (fid). the dominant fatty acids in all samples under study were: c18:2, c18:1 and c16:0. the profile of fatty acids in boletus edulis varied between the regions where the mushrooms were collected as well as the morphological parts of the fruiting body. keywords: boletus edulis, edible mushrooms, fatty acids, human nutrition, poland   ital. j. food sci., vol 28, 2016 403 1. introduction edible wild mushrooms are a raw material consumed in many countries of the world as a delicacy (ribeiro et al., 2009). they are most appreciated by gourmets as well as enthusiasts of mushroom picking which, apart from being a piece of cultural heritage, has recently become a highly valued recreational activity (kalač, 2009; 2013). widespread consumption of mushrooms is related primarily to their taste and smell properties, which give a sophisticated flavor to dishes. in our previous studies we examined the toxicological aspects of edible mushrooms (chlorinated hydrocarbons residues) due to the fact they are considered to be bioindicators of the level of environment contamination (gałgowska et al., 2012). nevertheless, in recent years, researchers have begun to focus on to their significant role in human nutrition. the growing awareness of consumers of food quality has made mushrooms a subject of scientific interest. the attention of researchers has been focused on their chemical composition and content of necessary nutrients essential for basic human diet supplementation. since the last decade, a synoptic knowledge of the composition and nutritional value of the most important species of edible mushrooms has been available. bano suggested that the food value of mushrooms lies between vegetables and meat. mushrooms are source of beneficial bioactive compounds (bano, 1976). they are quite rich in protein, providing all the essential amino acids and contain relatively high amounts of carbohydrates and fiber (kalač, 2009). due to the fact that mushrooms have a low fat content, they are considered low-energy functional foods, which could significantly contribute to the design of healthy dietary patterns (alobo, 2003; barros et al., 2007; kavishree et al., 2008; lee et al., 2011). they contain significant amounts of vitamins and vitamin precursors, minerals and trace elements (kalač, 2009; 2013). mushrooms also include sterols, with the predominance of ergosterol, the precursor of vitamin d (kalogeropoulos et al., 2013). apart from rich composition, mushrooms have therapeutic properties, including prevention of such health problems as: atherosclerosis, diabetes mellitus, chronic inflammation, cancer and aging (yilmaz et al., 2006). physical and psychological development and health maintenance involves supplying the human body with proper nutrients, including animal and vegetable fat (barros et al. 2008, moioli et al., 2007). lipids are a basic component of a diet and play many varied roles in the organism, primarily as the richest and the most concentrated source of energy (gawęcki and hryniewiecki, 2000). lipid consumption provides the body with the proper amounts of fatty acids, including saturated (sfa), monounsaturated (mufa) and polyunsaturated fatty acids (pufa). although each member of the group of fatty acids is important in the human diet, it is also necessary to preserve the relative proportions of their consumption. interest in mufas is related to their function in preventing cardiovascular diseases such as atherosclerosis (hunter et al., 2010; kanu et al., 2007; motard-belanger et al., 2008; von schacky and harris, 2007). the dietetic value of fat is to the highest extent, determined by the presence of pufas in its content, particularly of linoleic (c18:2 n-6) and α-linoleic acid (c18:3 n-3), which form a family of so-called “essential” fatty acids (innis, 2005; przysławski and bolesławska, 2006). they are converted to tissue hormones, which affect the functions of numerous tissues and organs and reinforce or weaken the regulatory effect of the hormonal and nervous systems. efas (essential fatty acids) prevent blood clotting and hypertension. additionally, they increase the blood supply to the heart and contribute to the proper distribution of cholesterol in the body (kris-etherton and etherton, 2003; williams, 2000). polish literature contains only few references concerning the profile of fatty acids in edible mushrooms. based on international reports, mushrooms contain significant amounts of   ital. j. food sci., vol 28, 2016 404 unsaturated acids and low amounts of saturated acids (ribeiro et al., 2009; kalač, 2009; 2013; barros et al., 2007; kavishree et al., 2008; yilmaz et al., 2006; barros et al., 2008; pedneault et al., 2006). this is an important reason to recommend the raw material in various diets. the growing interest in the consumption of edible mushrooms provides an incentive to carry out a broad scope of analytical research concerning the composition of fatty acids in these raw materials. in poland, the picking and consumption of mushrooms is very popular due to the occurrence of large areas of forests. of the many species of mushrooms, boletus edulis, (a member of the boletaceae family), is highly valued by consumers due to its unique flavor characteristics. taking the above into account, the aim of this study was to determine the content of the fat and fatty acid profile in wild boletus edulis, indicate potential differences in the composition of fatty acids between the various morphological parts and examine the impact of the mushrooms’ vegetative sites on the fatty acid profile. 2. materials and methods the research material consisted of 33 samples of whole fruiting bodies of wild boletus edulis from four selected regions of poland (fig. 1) and 29 averaged samples in the form of caps and stems, collected in 2010 during the period from july to september. the samples were prepared (selected, cleaned, dried in a fruit and vegetable drier) according to pn68/a-78508-1968 (pn-68/a-78508-1968). the dried and finely cut-up material was subjected to extraction in a soxhlet’s apparatus in order to obtain lipid substances pn-a78509:2007 (pn-a-78509:2007). methyl esters of fatty acids were prepared by peisker’s method, using a mixture of methyl alcohol, sulphurous acid and chloroform (peisker, 1964). figure 1: location of sampling of boletus edulis.   ital. j. food sci., vol 28, 2016 405 separation of the examined compounds was performed by gas chromatography. the conditions of chromatographic separation involved a gas chromatographer (7890a agilent technologies) and a flame ionization detector (fid); capillary column supelcowax 10: length 30 m, inside diameter 0.32 mm, liquid phase – supelcowax 10, film thickness 0.25 μm; temperature: detector – 250°c, dispenser – 230°c and column – 195°c; carrier gas helium, flow rate 1.5 ml/min (51 cm/s); split 50:1. the identification of fatty acids was carried out on the basis of their retention time in relation to the standard retention time of fatty acid methyl esters. for this purpose, a mixture of 37 standards of supelco 37 component fame mix (10 mg/ml in methylene chloride (varied)) was applied. for the calculation of the percentage share of fatty acids, a chemostation computer program was used. in the experiment, there were 17 fatty acids identified from c12:0 to c24:1, which were divided into three groups: saturated (sfa), monounsaturated (mufa) and polyunsaturated fatty acids (pufa). statistical analyses were conducted with microsoft excel software, which included calculation of the mean values as well as the standard deviations. the significance of difference of the mean values between the samples was determined using statistica 10 software (duncan’s test, analysis at the level of significance of p=0.05). 3. results and discussions the fatty acid profiles of boletus edulis are shown in tables 1 and 2 and in figs. 3 and 4. statistical differences were found in the fat content, depending on the morphological parts of a mushroom. the average fat content in caps and stems of boletus edulis was 4.38% and 1.75%, respectively (fig. 2). in boletus edulis there 17 different fatty acids from c 12:0 to c24:1 were determined. table 1 presents the fatty acids composition in whole fruiting bodies of wild boletus edulis depending on research regions. figure 2: the average content of fat in boletus edulis depending on the morphological parts of fruiting body.   ital. j. food sci., vol 28, 2016 406 the content of every fatty acid was varied depending on the region. analyzing the dominant acids (c16, c18: 1 and 18: 2), the highest percentage of palmitic acid was found in the region b, while the lowest statistically different from the rest in the region a. however, for the region a, was the highest percentage share of c18:1 acid reported. the lowest contribution of this acid was observed for the region b. in the case of c18:2 acid no statistically significant differences were found between regions a and b. the highest content of this acid was indicated for the region d, and the lowest for region c. determinated differences between regions can be related with different composition of the soil, weather conditions, et al. table 1: the fatty acids profile in boletus edulis originating from different regions of poland (n= 33). region fatty acids a b c d (n = 4) (n = 9) (n = 10) (n = 10) c12:0 0.10±0.00a 0.41±0.06c 0.22±0.04b 0.77±0.09d c14:0 0.13±0.01a 1.11±0.18c 0.65±0.11b 0.76±0.13b c15:0 0.10±0.00a 0.89±0.14c 1.00±0.13c 0.50±0.09b c16:0 8.39±0.42a 28.84±3.35c 25.10±1.03c 16.82±1.21b c16:1 0.75±0.06a 1.69±0.14c 1.50±0.18c 1.11±0.19b c17:0 0.10±0.00a 0.24±0.04c 0.32±0.07c 0.19±0.03b c18:0 3.12±0.04b 2.87±0.27ab 2.78±0.38ab 2.49±0.30a c18:1 44.75±0.70d 19.56±3.11a 31.71±2.56c 24.33±0.95b c18:2 40.71±0.51b 40.07±4.02b 32.78±2.60a 50.62±2.00c c18:3 0.31±0.02a 1.30±0.22d 0.60±0.12c 0.42±0.06b c20:0 0.40±0.01b 0.23±0.02a 0.27±0.06ac 0.40±0.07bc c20:1 0.74±0.05b 0.35±0.06a 0.73±0.11b 0.35±0.05a c20:2 0.20±0.01a 0.92±0.16c 0.29±0.04b 0.18±0.02a c22:0 0.26±0.08a 0.68±0.09d 0.54±0.06cd 0.44±0.06bc c22:1 0.16±0.04a 0.23±0.04a 0.53±0.08b 0.43±0.07b c24:0 0.10±0.00a 0.51±0.10b 0.77±0.11c 0.10±0.02a c24:1 0.11±0.01a 0.10±0.00a 0.22±0.05b 0.10±0.01a a–d the significance of difference of the mean values between the samples; p = 0.05. a similar relation in the case of content of predominant fatty acids (linoleic acid (c18:2), oleic (c18:1) and palmitic (c16:0)) in this species was also observed by other authors (kavishree et al., 2008, barros et al. 2008). in presented studies, linoleic acid content ranged from 32.78-50.62%. a similar amount of c18:2 was found by the following authors: barros et al. (2008) 44.32% (portugal), kavishree et al. (2008) 33.80% (india), pedneault et al. (2006) 42.20% (canada), yilmaz et al. (2006) 33.60% (turkey). the content of c18:1 acid in the boletus edulis under study ranged from 19.56 to 44.75%. in their research barros et al. (2008) found 39.72%, pedneault et al. (2006) 36.01%, kavishree et al. (2008) 31.10%, yilmaz et al. (2006) 30.20%. palmitic acid (c16:0) was   ital. j. food sci., vol 28, 2016 407 found in samples in the range of 8.39 28.84%. the content of the acid in the studies of pedneault et al. (2006) was that of 9.80%, of kavishree et al. (2008) 21.60% and of barros et al. (2008) 10.03%. in none of the samples were short-chained fatty acids detected, while barros et al. (2008) and pedneault et al. (2006) observed the presence of trace amounts of c6:0, c8:0 and c10:0 in boletus edulis. the presence of fatty acids in the trans configuration was also not stated. the obtained results varied depending on the morphological parts of the mushroom, what is presented in table 2. table 2: the fatty acids profile in boletus edulis depending on morphological parts of the mushroom. fatty acids caps stems (n = 15) (n = 14) c12:0 0.57±0.16b 0.30±0.07a c14:0 0.69±0.03a 0.61±0.07a c15:0 0.46±0.11a 1.45±0.20b c16:0 15.96±4.77a 35.95±0.95b c16:1 1.11±0.07a 1.94±0.11b c17:0 0.16±0.02a 0.26±0.06b c18:0 2.69±0.48a 2.40±0.14a c18:1 34.51±0.80b 20.69±1.12a c18:2 40.71±1.41b 32.68±1.18a c18:3 0.90±0.20b 0.48±0.04a c20:0 0.36±0.08a 0.33±0.02a c20:1 0.61±0.09b 0.35±0.05a c20:2 0.26±0.06a 0.18±0.03a c22:0 0.36±0.03a 0.85±0.11b c22:1 0.31±0.08a 0.55±0.07b c24:0 0.23±0.02a 0.86±0.12b c24:1 0.10±0.01a 0.11±0.02a a–d the significance of difference of the mean values between the samples; p = 0.05. the predominant fatty acids in caps were c18:2, c18:1 and c16:0 (40.71%, 34.51%, 15.96%, respectively). in the case of three predominant fatty acids in stems, the biggest share had c16:0 (35.95 %), next – c18:2 and c18:1 (32.68%, 20.69%, respectively). in the examined morphological parts of the mushrooms statistically significant differences in the content of c18:2 acid were found, where in the caps 40.71% was determined and 32.68% in the stems. the stems of the same species of mushroom from turkey had 28.40% c18:2 acid content (yilmaz et al., 2006). a statistically significant difference in the content of oleic acid (c18:1) in the caps (34.51%) and stems (20.69%) was found. the content of this acid in the stems at level of 8.30% was determined by yilmaz et al. (2006). the stems of   ital. j. food sci., vol 28, 2016 408 boletus edulis (35.95%) were characterized by a significantly higher content of c16:0 acid compared to caps (15.96%). the content of other fatty acids ranged from 0.10 2.69% and was dependent on the morphological parts of the mushroom. it was found that the percentage share of each group of fatty acids (sfa, mufa pufa) in the studied morphological parts of boletus edulis was statistically different (fig. 3). figure 3: percentage contribution of sfa, mufa, pufa in boletus edulis depending on morphological parts of the mushroom. in the case of saturated fatty acids the share was about twice as high in caps (43.01%) than in the stems (21.48%). the caps were characterized by a higher contribution of monounsaturated (36.64%) and polyunsaturated fatty acids (41.87%) compared to the stems (23.64% and 33.34%, respectively). the main groups of fatty acids present in the mushrooms under study were unsaturated fatty acids (from 64.22 to 87.73 %). pedneault et al. (2006) also obtained similar amounts 84.50%. there were significant statistical differences found in the contents of particular groups of fatty acids (sfa, mufa, pufa) in boletus edulis from different regions (fig. 4). the analyzed samples had the largest share of polyunsaturated (33.67 51.21%) and the lowest contribution of saturated fatty acids 12.70 35.78%. the biggest share of unsaturated fatty acids (41.22% of pufa and 46.51% of mufa) was observed in boletus edulis from region a (87.73%). in the regions b and c there were similar contributions of unsaturated fatty acids (64.22% and 68.35%, respectively) determined. nevertheless the region b characterized the higher content of pufa (42.29%) and only 21.92% of mufa, while in the region c there was indicated 33.67% of pufa and 34.68% of mufa. the mushrooms from the region d were the richest source of pufa (51.21%) (fig. 4).   ital. j. food sci., vol 28, 2016 409 figure 4: percentage contribution of sfa, mufa, pufa in boletus edulis originating from different regions of poland. 4. conclusions the profile of fatty acids in boletus edulis varied between the regions where the mushrooms were collected as well as the morphological parts of the fruiting body. this confirms view of sanmee et al. (2003) and diez and alvarez (2001), that a number of factors usually influence the nutritional composition of mushrooms. these factors include growing site, type of substrates, mushroom type, developmental stages and part of the fungal samples analyzed (diez and alvarez, 2001; sanmee et al., 2003). the observed differences in the fatty acids content in mushrooms under study may result, among others, from the different chemical composition of the substrate in particular regions. however, this thesis requires confirmation in further studies. in terms of human nutrition, caps of boletus edulis are a more valuable raw material than stems due to the significantly higher content of mufa and pufa and lower sfa. among polyunsaturated fatty acids, the most noteworthy is linoleic acid c18:2 (ω-6), which is a precursor of 1-acetic-3-ol the major component of mushrooms giving them a specific aroma (ribeiro et al., 2009; barros et al., 2007). the high content of this acid in polish boletus edulis provides that the mushroom may be recommended in different types of diets for people with high blood cholesterol (kavishree et al., 2008; vaz et al., 2011). references alobo a.p. 2003. proximate composition and functional properties of pleurotus tuberregium sclerotia flour and protein concentrate. plant foods hum. nutr. 58:1-9. bano z. 1976. nutritive value of indian mushroom. ind. mushroom sc. 1, 2:473-484. barros l., baptista p., correia d.m., casal s., oliveira b. and ferreira i.c.f.r. 2007. fatty acid and sugar composition, and nutritional value of five wild edible mushrooms from northest portugal. food chem. 105:140-145.   ital. j. food sci., vol 28, 2016 410 barros l., cruz t., babtista p., estevinho l.m. and ferreira c.f.r. 2008. wild and commercial mushrooms as source of nutritient and nutraceuticals. food chem. toxicol. 46:2742-2747. diez v.a. and alvarez a. 2001. compositional and nutritional studies on two wild edible mushrooms from north west spain. food chem. 75:417-422. 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p.1-24 (in polish). przysławski j. and bolesławska i. 2006. food lipidstherapeutic or pathogenic factor. tłuszcze jadalne 41:179-192 (in polish). ribeiro b., guedes de pinho p., andrade p.b., baptista p. and valentão p. 2009. fatty acid composition of wild edible mushrooms species: a comparative study. microchem. j. 93:29-35. sanmee r., dell b., lumyong p., izumori k. and lumyong s. 2003. nutritive value of popular wild edible mushroom from northern thailand. food chem. 82:527-532.   ital. j. food sci., vol 28, 2016 411 vaz j.a., barros l., martins a., santos-buelga c., vasconcelos m.h. and ferreira i.c.f.r. 2011. chemical composition of wild edible mushrooms and antioxidant properties of their water soluble polysaccharidic and ethanolic fractions. food chem. 126:610-616. von schacky c. and harris w.s. 2007. cardiovascular benefits of omega-3 fatty acids. cardiovasc. res. 73:310-315. williams c.m. 2000. dietary fatty acids and human health. ann. zootech. 49:165-180. yilmaz n., solmaz m., türkekul i. and elmastas m. 2006. fatty acid composition in some wild edible mushrooms growing in the middle black sea region of turkey. food chem. 99:168-174. paper received january 20, 2015 accepted december 12, 2015 paper 64 ital. j. food sci., vol. 28 2016 keywords: d value, inactivation, ultrasound, zygosaccharomyces rouxii inactivation of zygosaccharomyces rouxii using power ultrasound at different temperatures, ph and water activity conditions s. kirimli1 and b. kunduhoglu2* 1institute of science, university of eskisehir osmangazi, 26480 eskisehir, turkey 2department of biology, science and arts faculty, university of eskisehir osmangazi, 26480 eskisehir, turkey *corresponding author: tel. +90 222 2393750 ext. 2845, fax +90 222 2393578, email: bkunduh@gmail.com abstract in this study, the effect of ultrasound treatments (20 khz) combined with mild temperatures (thermo-sonication) on the inactivation of z. rouxii was examined. additionally, the effect of ph (4 and 7) and water activity (aw 0.99 and 0.94) of the sonication medium on yeast inactivation was determined. the d (40-55) values at a thermo-sonication amplitude of 80% were shorter than that obtained at 40%. using thermo-sonication, particularly at a low aw, was associated with a significant synergistic effect for z. rouxii inactivation (p<0.05). in most thermo-sonications at 50° and 55°c, the fda requirement of a 5-log cycle reduction could be achieved (>5.7-log reductions in <0.2-0.2 min). our findings show that sonication offers advantages in terms of reduced duration and temperature of pasteurization, without a reduction in structural and sensory quality particularly for fruit juices. mailto:bkunduh%40gmail.com?subject= ital. j. food sci., vol. 28 2016 65 introduction the yeast zygosaccharomyces rouxii represents a major cause of spoilage of foods and drinks that are packaged according to good manufacturing practices (gmp), including fruit juices, sauces, carbonated drinks, wine, salad dressings, and ketchups (james and strat ford, 2003; pitt and hocking, 1985; loureiro and malfeito-ferreira, 2003; fugelsang and edwards, 2007; deák, 2008). typical physiological characteristics of z. rouxii include tolerance to low-acidity preservatives, extreme osmotolerance, and the ability to adapt to high glucose concentrations, low water activity (aw) and thermal treatment (emmerich and radler, 1983; james and stratford, 2003; martorell et al., 2007). thus, z. rouxii is important to consider in examining spoilage during the processing of foods with low-acidity and high-sugar content. the food industry most frequently uses traditional pasteurization methods such as low temperature long time (ltlt) and high temperature short time (htst) to achieve shelf-life stability for fruit juices and drinks due to these methods’ effectiveness and low cost. however, these procedures are associated with the loss of vitamins and volatile aromatic substances (körmendy, 2007; vasantha rupasinghe and li juan yu, 2012). in addition to thermal pasteurization, other methods that are commonly utilized to prolong shelf-life include chemical preservatives such as potassium sorbate, sodium benzoate (vasantha rupasinghe and li juan yu, 2012), citric acid and sulfur dioxide (wiley, 1994; bates et al., 2001). chemical preservatives used to prolong shelf-life may be associated with adverse health consequences in humans, depending on the characteristics of the consumer population and the frequency of consumption (isman, 2000). although thermal treatment is the most common technique to inactivate microorganisms in food, there is an increased interest in the use of alternative food preservation methods as a response to consumer demand for food with conserved innate characteristics and no artificial preservatives (corbo et al., 2009; vasantha rupasinghe and li juan yu, 2012; alzamora et al., 2003). some of the non-thermal food preservation methods that may represent an alternative to thermal treatment include electric or magnetic fields, microwave radiation, ionizing radiation, high-intensity light pulses and high-hydrostatic pressure (corbo et al., 2009; di benedetto et al., 2010). additionally, power ultrasound (us) is a promising novel technology that minimizes the need for treatment, increases food quality, and conserves the characteristics and sensory qualities of the food. power us is defined as the use of pressure waves between 20 and 100 khz. the lethal effect of ultrasonic processing on microorganisms is achieved through the conversion of electrical energy to ultrasonic sound waves via the ultrasonic transducer and through the formation and collapse of vast numbers of small bubbles in each second during the propagation of ultrasonic waves within liquids. the quick formation and collapse of these bubbles (cavitation) creates very high local temperatures (5500°c) and pressures (50 mpa), which cause disruption of the cell wall and damage to the cell membrane and dna (jiranek et al., 2008; manvell, 1997; knorr et al., 2004; o’donnell et al., 2010; cárcel et al., 2012; leighton, 1998; soria and villamiel, 2010). the duration and temperature of the procedure, the composition and volume of the liquid, and the form and dimensions of the microorganism are among the determinants of the antimicrobial efficiency of ultrasonic processes (bevilacqua et al., 2013). the possible areas of use for microbial inactivation by power us have been relatively well studied in the food industry. it has been reported that to achieve the fda-required 5-log reduction in microorganisms, sonication should be used in combination with mild heat treatment and/or pressure (fda, 2001; walkling-ribeiro et al., 2009; baumann et al., 2005; d’amico et al., 2006; ugarte-romero et al., 2006; salleh-mack and roberts, 2007; tiwari et al., 2009). many studies have reported the synergistic effect of the combination of non-thermal technologies and heat treatment on microbial inactivation (guyot et al., 2007; lee et al., 2009; leistner and gorris, 1995; raso et al., 1998; reddy et al., 2006; ross et al., 2003). however, to our knowledge, there are no studies examining the combined effect of heat, ph and aw on z. rouxii inactivation using us. therefore, the aim of this research was to evaluate the effect of us with heat (thermo-sonication) on the inactivation of z. rouxii at different ph and aw conditions. for this purpose, citrate buffer was chosen as the model medium, and the effect of thermo-sonication on z. rouxii was tested under different ph and aw conditions. thus, the optimum procedural parameters defined for z. rouxii inactivation may be utilized as a model for the us-assisted pasteurization of real fruit juices and other drinks at mild temperature conditions. materials and methods maintenance of test strain zygosaccharomyces rouxii (nrrl y-229) was obtained as a lyophilized culture from the ars culture collection (northern regional research laboratory, united states department of agriculture, midwest area-national center for agricultural utilization research microbial genom66 ital. j. food sci., vol. 28 2016 ics & bioprocessing research unit 1815 n university street, peoria, il 61604). the culture tube was opened aseptically, the contents were transferred to a 2% sabouraud dextrose broth (sdb, merck, germany), and the mixture was incubated for 48-72 h at 30°c. the stock cultures were then grown on sabouraud dextrose agar (sda, merck, germany) slants and stored at 4°c until use. preparation of yeast culture for inactivation studies z. rouxii subcultures were prepared by inoculating a test tube that contained 5 ml of sterile sdb with one single colony from a culture plate. the tubes were then incubated at 30°c for 48 h. erlenmeyer flasks (250 ml) containing 50 ml of sdb were inoculated with this subculture. the flasks were incubated under agitation (130 rpm). the broth cultures were transferred to sterile centrifuge tubes, and pellets were obtained at 5500 rpm for 10 min. the pellets were then washed with saline water (0.85% nacl) and resuspended in the same medium. z. rouxii suspensions prepared in this way were used to inoculate sonication vessels at a final concentration of 108 cfu/ml. preparation of citrate buffer all sonication and control group treatments in this study were applied in citrate buffer medium. citrate buffer was prepared as two stock solutions (stock solution a: 0.1 m citric acid, c 6 h 8 o 7 .h 2 o reagent, carlo erba, italy; and stock solution b: 0.2 m di basic sodium phosphate, na 2 hpo 4 .2h 2 o, merck, germany). the final ph of the citrate buffer was measured using a ph meter (wtw inolab 730, germany). water activity (a w ) of the citrate buffer was adjusted to a w 0.94 with glycerol (merck, germany). the aw values of the citrate buffer medium were measured at room temperature (23-25°c) with an aqualab water activity meter (decagon devices, inc., usa). combined treatments (thermo-sonication treatments; ts-t) sonication was performed with a vc-750 watt us generator and a vibracell® wcx 750 (sonics and materials, ct, usa) model ultrasonic processor at a frequency of 20 khz (maximum 124 μm amplitude). a solid sonication probe (13 mm in diameter) was used in all treatments. levels of 40% (49.6 μm amplitude) and 80% ultrasonic power (99.2 μm amplitude) were applied in each case. most of the sonication treatments were applied for 20 min. a 100 ml sterile water-jacketed vessel (part no. 830-00010, sonics and materials, ct, usa) was used to hold the citrate buffer. the temperature of the citrate buffer in the vessel was controlled by a refrigerated circulating water bath (polyscience-9102, il, usa). the temperature of the medium in the vessel was monitored during the sonication process using the digital thermometer (sonics and materials, ct, usa) of the ultrasonic processor. the vessels and probes were sterilized at 121°c for 15 min before and after each experiment. the preparation of the sonication vessels and the sonication process are described below. additionally, the experimental design of the combined treatments (ts-t at different medium conditions) and thermal treatments alone (t-t: control group treatments, at the same medium conditions) are summarized in table 1. (1) a total of 99 ml of citrate buffer was placed in a water-jacketed vessel. (2) a sonication probe was immersed in the center of the vessel. (3) the sonication procedure produces heat in a liquid medium; thus, to fix the temperature of the citrate buffer in the vessel at the target treatment temperature (40, 45, 50 or 55°c) during the sonication process, the temperature of the circulating water bath was adjusted to 7-10°c less than the target temperature. then sonication was started. (4) immediately after reaching the target temperature, 1 ml of yeast suspension was added to produce a final concentration of 108 cfu/ ml in the citrate buffer in the sonication vessel. (5) at the beginning and during the treatment, 1 ml samples of citrate buffer samples were collected from the vessel and serially diluted in sterile saline water (1:10). if necessary, the sampling intervals and treatment times were adjusted (e.g., in the case of high temperature table 1 summary of the experimental design with thermo-sonication (ts-t) and thermal treatments (t-t) at different ph and aw levels. variables treatment sonication ph aw treatments temperatures levels ts-t t-t 4 0,99 + 0,94 + 7 0,99 + 0,94 + 40% 4 0,99 + 0,94 + 7 0,99 + 0,94 + 80% 4 0,99 + 0,94 + 7 0,99 + 0,94 + -: no sonication. 40°c, 45°c, 50°c and 55°c ital. j. food sci., vol. 28 2016 67 levels). survival was determined using the dropplate and spread-plate techniques. aliquots of 0.02 ml (for drop-plate technique) or 0.1 ml (for spread-plate technique) were taken from the dilutions and plated on sda. the plates were incubated at 30°c for 48 h, and counts of survivors in treated samples were conducted. all experiments were repeated at least two times. thermal treatments (t-t) alone the survival and growth of z. rouxii was also determined in citrate buffer at different temperatures (40, 45, 50 or 55°c) and under different medium conditions (ph 4 and 7 and aw 0.99 and 0.94) without sonication. treatments were performed in a shaking water bath (memmert, germany). the t-t process is described below and given in table 1. (i) a total of 99 ml of citrate buffer was placed in a flask. (ii) to reach the target temperatures (40°, 45°, 50° and, 55°c), 99 ml of citrate buffer in flasks was pre-heated in a shaking water bath. the temperature of the citrate buffer in the flasks was monitored using a digital thermometer. (iii) the citrate buffer reached the target temperature level. (iv) one milliliter of yeast suspension was added to achieve a final concentration of 108 cfu/ ml in the citrate buffer. this step corresponded to the beginning of the treatment time. (v) during the treatment, 1 ml samples of the citrate buffer were collected from the flasks and serially diluted in saline water (1:10). the sampling intervals were 0, 1, 2, 4, 8, 12, 24 and 48 h. viability counts were conducted as described above. as shown in table 1, 48 different (32 ts-t + 16 t-t) treatment conditions were studied to determine yeast inactivation, and each treatment was repeated in parallel at least two times. determination of d values in this study, the inactivation of z. rouxii was described using the first-order inactivation kinetic model. the d values were directly calculated from the k values (the slope of the inactivation curve) and the r2 values. first-order kinetic model: where; no= initial cell number (cfu/ml), t = treatment time (min), n = number of the surviving cells (cfu/ml) after t minutes of treatment, k = slope of inactivation curve (min-1), k´ = log of slope of inactivation curve (min-1), and d = decimal reduction time, or the time required for a 1-log cycle reduction in the microbial population. data were fitted to this model with a linear regression using the microsoft excel program. additionally, log reductions (log cfu/ml) for each process were calculated using data of the initial and final yeast numbers in the vessel. viability of yeast cells in treated samples during storage in this step of the study, we determined the growth of the survivors during storage at different temperatures (4° and 25°c). two samples (10 ml) were taken from each treatment, with 5-log cycle reductions achieved; they were aseptically transferred into 10 ml double-strength glass bottles containing sdb and stored at 4° and 25°c for 60 d in the dark. during storage, 1 ml aliquots were taken predetermined intervals from each bottle and were then transferred into sdb; the tubes were incubated at 30°c for 3-5 days, and yeast growth was checked. the sampling intervals were 1, 7, 15, 30, 45 and 60 d. statistical analysis variance analysis was used to determine the effect of inactivation factors on d values. the plate-count data were logarithmically transformed for statistical analysis. the results (log 10 cfu/ml) were subjected to an analysis of variance (spss ver. 11.5, chicago, il, usa). for all experiments, a p value ≤0.05 was considered to indicate statistical significance. results and discussion in the present study, the inactivating effect of ultrasound waves (20 khz) on z. rouxii was investigated in a model medium (citrate buffer). a total of 48 different experiments were performed to determine the effect of heat (40, 45, 50 and 55°c), ph (4 and 7), and aw (0.99 and 0.94) on ultrasonic inactivation (40 and 80% amplitude) of z. rouxii. during the sonication procedure, periodical sampling from the sonication chamber was conducted to determine the number of viable cells of z. rouxii (cfu/ml). a first-degree kinetics reaction was used to establish inactivation plots for z. rouxii that were subsequently utilized to estimate the “d values” based on slope and r2. additionally, yeast reduction was determined based on a comparison of the cell numbers before and after the procedure. the difference in d values, as defined by the ts-t and t-t processes, were assessed using variance analysis. 68 ital. j. food sci., vol. 28 2016 furthermore, the growth pattern of sublethally injured yeasts following ts-t and t-t processes were evaluated under different storage conditions (at 4° and 25°c for 60 d). inactivation of z. rouxii at 40°c an overall assessment of the results of all combined procedures at 40°c showed a smaller d value at 80% amplitude (0.94 aw and 0.99 aw; ph = 4 and ph = 7) than at 40% amplitude (p<0.05) (fig. 1a). a generally reduced microbiological resistance to heat occurs in an acidic environment. however, in our study, the d 40 values in the combined and thermal procedures at ph= 4 were statistically significantly higher than those at ph=7 (p=0.012). the aw of the medium also had an impact on z. rouxii inactivation. the d 40 values estimated at 0.94 aw in all combined and thermal procedures were higher than those estimated at 0.99 w (p<0.05). thus, a low aw was considered to give z. rouxii a higher resistance to heat and sonication. similar to this study, alvarez et al. (2003) observed a 30-fold increase in the thermal decimal reduction time for salmonella enteriditis by decreasing aw from 1 to 0.96, whereas only a two-fold increase was observed with mano-sonication, and a synergistic lethal effect with the combined use of heat and ultrasound was observed. in our combined treatment procedures at 40°c, the reduction in z. rouxii for the 40% and 80% amplitude levels was 0.4-1.6 log cfu/ml and 0.8-3.6 log cfu/ml, respectively (fig. 1b). in a study by bevilacqua et al. (2013), ultrasound was used to determine the reduction in several spoiling yeasts, including z. rouxii, in fruit juices; similar to our observations, there was a maximum reduction of 1.7 log cfu/ml z. rouxii in orange juice after sonication (40°c, 20 khz, amplitude 60%, time 4 min, pulse 2 s). according to the hurdle concept, if the effect obtained via the combined use of two different inactivation factors is greater than the sum of the separate use of these methods, then a synergistic interaction is said to occur (leistner and gorris, 1995). in the present study, treatment with a ph= 4 or 7 at 0.94 aw, with the combined use of ultrasound (40% and 80%) and heat, resulted in a significant synergistic interaction, although the d 40 value was higher than that observed with an aw of 0.99. in control treatments performed at the same temperature, sonication at 0.94 (ph 4 and 7) and 0.99 aw (ph 4 and 7), the reduction in d 40 values was, respectively 1/8-1/16 and 1/32-1/128 (fig. 1a). inactivation of z. rouxii at 45°c the d 45 values estimated for combined treatments at 0.94 aw were greater than those observed with 0.99 aw; however, the d 45 values were lower than those obtained at 40°c (fig. 2a). overall, our results suggest that increased treatment temperatures resulted in increased yeast inactivation. additionally, all sonications at 80% amplitude (0.94 aw and 0.99 aw; ph 4 and 7) had d values smaller than those found at 40% (p<0.05). the reduction in z. rouxii for the 40% and 80% amplitude levels was 0.5-2.0 log cfu/ml and 1.1-3.9 log cfu/ ml, respectively (fig. 2b). in treatments at 0.99 aw (ph 4 and 7), a synergistic interaction for z. rouxii inactivation was observed with the combined use of heat and ultrasound (40% and 80% amplitude). while synergy was present at 0.94 aw and ph values of 4 and 7 (40% and 80%), the d 45 value was greater than that observed at 0.99 aw. compared with control treatments at the same temperature and ph, the reductions in d 45 obtained with the combined treatments at 0.94 and 0.99 aw were from 1/8-1/32 and 1/641/128, respectively (fig. 2a). lopez-malo et al. (2005) assessed the sonication inactivation (20 khz, 90 µm) of z. bailii in 2% sabouraud glucose broth with a ph of 3.5 and at three different aw (0.99, 0.97 and 0.95) and temperatures (45, 50 and 55°c) levels. consistent with our findings, the d value at 45°c obtained with thermal treatment (tt) was significantly greater than that obtained with thermoultrasonication (tut) (p<0.05). these authors found that at 45°c and at 0.99, 0.97, and 0.95 aw, the d value was reduced from 15.4 to 7.4, 26.8 to 8.6 and 43.5 to 12.9, respectively, with tt and tut. additionally, along with the reduction in aw, an increase in the d values was observed. furthermore, a lower aw was associated with a greater synergistic effect in tut. in the present study, the average d 45 values of z. rouxii for 0.99 and 0.94 aw at 45°c t -t (ph=4) were 98.66 and 140.1 min, respectively. in contrast, treatment ts-t under the same conditions (80% amplitude: 99.2 µm) resulted in d 45 values of 0.58 and 4.33 min at 0.99 and 0.94 aw, respectively. inactivation of z. rouxii at 50°c in our sonication treatments, the minimum possible sampling interval from the sonication vessel was 20 s. therefore, in some combined treatments, especially those conducted with high temperatures and high aw values (i.e., aw=0.99; 50 and 55°c), samples were taken after the first 20 s, and there were typically no viable yeast cells (for this reason, some d values in figs. 3a and 4a are shown as <0.2 min). additionally, yeast reductions are shown as >5.7log cfu/ml because the maximum yeast reduction was determined as 5.7-log cfu/ml in this study (figs. 3b and 4b). similar to our results obtained at 45°c, the estimated d 50 for z. rouxii at 50°c and 0.94 ital. j. food sci., vol. 28 2016 69 fig. 1 d 40 values of z. rouxii obtained from the ts-t and t-t (a) and reductions of z. rouxii after ts-t and t-t at 40°c (b). ba b a fig. 2 d 45 values of z. rouxii obtained from the ts-t and t-t (a) and reductions of z. rouxii after ts-t and t-t at 45°c (b). fig. 3 d 50 values of z. rouxii obtained from the ts-t and t-t (a) and reductions of z. rouxii after ts-t and t-t at 50°c (b). ba 70 ital. j. food sci., vol. 28 2016 fig. 4 d 55 values of z. rouxii obtained from the ts-t and t-t (a) and reductions of z. rouxii after ts-t and t-t at 55°c (b). ba aw was greater than that observed at 0.99 aw (fig. 3a). z. rouxii showed a greater resistance to combined treatments at a ph of 4 than at a ph of 7, most likely because z. rouxii is a yeast with good adaptation to lower ph values. the maximum d 50 at 0.99 aw and ph 4 was 0.8 minutes, whereas the d 50 value at ph 7 was <0.2 minutes. in treatments at 0.99 aw and ph 4, the combined use of ultrasound (40% and 80%) provided a significant synergistic interaction for z. rouxii inactivation. however, no such synergy could be observed at 0.99 aw and ph 7 for the combined treatment. a synergistic effect could be observed at 0.94 aw, with ph values of 4 and 7, with sonication (40% and 80%), and with d 50 values greater than that observed with 0.99 aw. compared with controls under the same temperature conditions, the reduction in d 50 in sonications of 0.94 aw (ph=4 and 7) and 0.99 aw (ph = 4 and 7) was 1/4-1/16 and 1/31/6, respectively. and the reduction in z. rouxii for the 40% and 80% amplitude levels was 1.1>5.7 log cfu/ml and 3.6->5.7 log cfu/ml, respectively (fig. 3b). inactivation of z. rouxii at 55°c compared with control treatments, the d 55 values obtained with the combined treatments at 55°c and 0.99 aw suggested that the use of ultrasound did not result in a significant differences (p>0.05) in yeast inactivation and that heat was the primary determinant of inactivation. in all combined treatments (at 0.94 aw), the d 55 values for z. rouxii were determined to be 0.2 minutes; at 0.99 aw, the d 55 values were determined to be <0.2 minutes (fig. 4a). compared with controls at the same temperature levels, the reduction in d 55 at sonications at 0.94 aw and ph levels of 4 and 7 was 1/8. in a similar study by guerrero et al. (2001), the inactivation of s. cerevisiae was examined at different amplitude levels (20 khz, 71.4 and 107.10 µm), ph values (3 or 5.6), and temperatures (35°, 45°, and 55°c) in sabouraud broth. in line with our findings, the d value at 55°c was lower that obtained at other temperature levels (i.e., 35° and 45°c) (p<0.05), whereas sonication, amplitude, and medium ph were not associated with a change in that reduction. however, in our study, the combined treatment with 0.94 aw resulted in an increased yeast inactivation (p<0.05), regardless of the ph and amplitude, and was associated with a synergistic effect. the d 55 values obtained for all sonication procedures at 80% were lower than those obtained at 40%, although the differences were not statistically significant (p>0.05). the reduction in z. rouxii for the 40% and 80% amplitude levels was 4.7->5.7 log cfu/ml and 5.6->5.7 log cfu/ml, respectively (fig. 4b). viability of yeast cells in treated samples during storage the growth during storage of sublethally injured yeast after combined treatments was tested. samples were taken from treatments in which the 5-log cycle yeast reductions had been achieved (figs. 3b and 4b) and stored for 60 d under different storage temperatures (4° and 25°c). as a result, none of the samples exhibited yeast growth during storage. this findings suggests that thermo-sonication is associated with irreversible cell damage. in a study by marx et al. (2011) examining the effect of continuous and pulsed thermo-sonication (20 khz frequency, at 60°c, 100% amplitude, for 30 min) on s. cerevisiae inactivation, the structural damage occurring in yeast cells after treatment was examined using scanning electron microscopy. they observed more broken cells using continuous rather than pulsed thermo-sonication treatments; however, they did not find any viable cells in their samples. ital. j. food sci., vol. 28 2016 71 conclusions compared with controls, all thermo-sonication procedures at 40, 45, 50 and 55°c resulted in a significant decrease in the d values (p=0.00) for z. rouxii in our study. this finding shows a decreased resistance of z. rouxii cells to heat together with the use of us. the amplitude of the ultrasound waves was effective in the reduction of yeast cells, with lower d values obtained at the 80% amplitude than at 40%. the use of us, particularly in medium with a low aw, resulted in significant synergistic effects for z. rouxii inactivation. however, thermo-sonications performed at low aw (0.94) were associated with a more prolonged d value and a less marked reduction. additionally, low aw was associated with the relative protection of yeast cells against thermo-sonication, particularly at lower temperatures. furthermore, as the sonication temperatures increased, the effects of amplitude, medium ph and aw on yeast reduction tended to weaken. increased sonication temperatures (50° and 55°c) resulted in significant yeast inactivation (>5.7-log reductions). in most of the combined treatments at 50° and 55°c, the fda requirement of a minimum of 5-log cycle reduction (within <0.2-0.2 min) could be met. however, although heat was the primary determinant of the yeast inactivation in combined treatments with high aw (55°c), the synergistic effect of us was more prominent than at 0.94 aw. the absence of yeast growth at 60 d that was observed in the samples obtained from the sonication chamber after combined treatments indicates that thermosonication was associated with irreversible yeast damage. the findings of this study indicate that us combined with mild heat treatments (50° and 55°c) has the potential to inactivate z. rouxii in fruit juices and beverages as an alternative to traditional pasteurization methods. particularly for pasteurizing fruit juices to retain their structural and sensory qualities at higher temperatures, the use of us may offer certain advantages with respect to reducing the duration and temperature of the treatment. acknowledgements this research was supported by the scientific research projects commission of eskişehir osmangazi university (project number: 200419023). we would like to express our gratitude to nafi çoksöyler for his assistance in the kinetic analyses. references alvarez i., manas p., sala f.j. and condon s. 2003. inactivation of salmonella enteriditis by ultrasonic waves under pressure at different water activities. appl. environ. microb. 69: 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process. 82: 102-107. wiley c.w. 1994. “minimally processed refrigerated fruits and vegetables”, 368 p., chapman and hall, new york. paper received august 21, 2014 accepted april 15, 2015 http://www.sciencedirect.com/science/journal/09242244/21/7 http://www.sciencedirect.com/science/journal/13504177/14/3 http://www.sciencedirect.com/science/journal/09242244 http://www.sciencedirect.com/science/journal/09242244 http://www.sciencedirect.com/science/journal/09242244/20/3 http://www.sciencedirect.com/science/journal/09242244/20/3 http://cdn.intechopen.com/pdfs-wm/28909.pdf http://cdn.intechopen.com/pdfs-wm/28909.pdf #224_bernacchia_bozza ! ital. j. food sci., vol 28, 2016 565 review organic and conventional foods: differences in nutrients r. bernacchia*, r. preti and g. vinci laboratory of commodity sciences, department of management, sapienza university of rome, via del castro laurenziano 9, 00161 rome, italy *corresponding author. tel.: +390649766514 e-mail address: roberta.bernacchia@uniroma1.it abstract organic agriculture represents a sustainable crop system focused on producing food without environmental degradation. consumer confidence in organic food is based both in lowering environmental impacts and health diseases. the aim of the present review is to compare nutritional properties of agricultural products cultivated following organic and conventional procedures. the heterogeneity of published results does not permit to conclude definitely the higher presence of nutrients in organic foods; even there are clear evidences for some antioxidants. comparative studies are challenging for the several factors influencing plant quality such as clime, soil type, cultivars and time of storage that should be considered. holistic approaches whit nutritional, technological parameters and sensorial quality of food seem to be the best way for future comparative studies. keywords: food quality, nutrients, organic food, sustainable agriculture ! ital. j. food sci., vol 28, 2016 566 1. introduction organic agriculture consists of many practices that emphasize farming based on ecosystem management, integrated cropping and livestock systems, diversity of products, reliance on natural pest and disease control without the use of chemical inputs. the principal objectives of organic agriculture are to produce healthy and sustainable food only using biological and ecological processes (azadi et al., 2011). the increase in production and consumption of organic foods is one of the major market trends of last years. in fact u.s. sales of organic products were an estimated $28.4 billion in 2012 over 4%of total food sales and will reach an estimated $35 billion at the end of 2014 (usda, 2014). in europe, while sales in some countries were rather stagnating in 2012, others displayed a growth of more than 10% (finland, norway and the netherlands) (fibl, 2014). fresh fruits and vegetables have been the top selling category since the organic food industry started retailing products over 3 decades ago. produce accounted for 43% of u.s. organic food sales in 2012, followed by dairy, packaged/prepared foods, beverages, bread/grains, snack foods, meat/fish/poultry and condiments. the growth of organic agriculture, its production and trade needed of an increase in national and international legislation, in order to set the specific requirements and create the institutional framework for certification. organic farming and production has been regulated in the context of eu farm policy reform since 1991, when the european council of agricultural ministers adopted regulation (eec) no 2092/91 on organic farming and labelling of organic farm produce and foods. evolutions in the regulations are set by council regulation (ec) no 834/2007 defining the official eu aims, objectives and principles of organic farming and production, and by two implementing regulations (no 889/2008 and no 1235/2008) detailing the organic production, labelling and import rules. the principal topics regard sustainable management system for the respect of nature’s cycle based on risk assessment and precautionary measures (exclusion of chemical fertilizers and mechanical treatments, respect of biodiversity, use of natural sources and respect of animal welfare standards). in recent years, several studies confirm people belief in healthier properties of food from organic agriculture as consequence of the environmental-friendly management (harper and makatouni, 2002; yiridoea et al., 2005). but until now, there is a lack of strong scientific evidences that organic food is significantly different from the conventional regarding nutritional properties and health impact. this is due to several important aspects that need to be considered in the experimental design of comparative studies and, consequently, in their interpretation. in fact, many factors impact the nutrient density of crops, whether they are grown organically or conventionally. some factors impact both production systems equally, while a few factors tend to have a larger impact on one production system over the other. in order to carry out a valid comparison between organic and conventional agriculture food products at first, plants have to be of the same cultivar, and have to be cultivated in near farms, with similar soils, under similar climatic conditions (gastol and domagala-swiatkiewicz, 2013). furthermore, products must be sampled at the same time and pre-treated similarly, analyzed by accredited laboratories employing validated methods and results statistically treated. generally, studies are divided into three classes: market-oriented studies, surveys and cultivation tests. in the first type of works, products are taken from organic and conventional shops; no information about origin, ripeness, variety, clime and condition of production is considered during comparisons. in surveys, instead, products derived from selected organic and conventional farms (siderer et al., 2005). environmental factors and condition of production can be used in studies comparison selecting neighbouring farms, ! ital. j. food sci., vol 28, 2016 567 but the precision of these parameters cannot be verified. the last category of works is represented by cultivation tests that permit to assess the difference in quality between organic and conventional products; unfortunately results can be applied only in the specific farm situation considered (rigby and càceres, 2001). organic foods differ from the conventional ones predominantly because the absence of pesticides, fertilizers and heavy metals residues as application of regulated production rules; the majority of literature studies dealing with organic food quantify these compounds to verify the limits. the absence of pesticides use and nitrogen fertilization influences the production of bioactive compounds and plant metabolites; an example is given by those involved in the defensive mechanisms of plants. as consequence, a natural functional food is expected to have higher contents of health promoting substances if cultivated under organic agricultural system. the present review focuses attention on such bioactive compounds, noted to improve human health, to evaluate if their content in foods can be use to discriminate between the two type of agriculture systems. for this scope, in this review we have considered the most relevant scientific papers published in the period 2000-2015, searched in three databases (scopus, pubmed, web of science), written in english and comparing chemical composition of organic and conventional foods. the exposure terms searched were: “organic” and “conventional” combined with “food”, “agricultural crops”, “livestock”, “agriculture” and terms for nutritionally relevant substances. results are organized on the base of polyunsaturated fatty acids, essential amino acids, vitamins, minerals and polyphenols contents. 2. organic vs conventional comparative studies the debate about the differences in nutritional properties between organic and conventional food interested largely researchers, as shown by the consistent number of papers and reviews published in few years. all the reviews existing about this topic reported different results. some of these, concluded that organic foods have higher content of such constituent instead others underlined the absence of differences in nutritional values between the two alternatives (woese et al., 1997; bourn and prescott, 2002; hunter et al., 2011; gastol and domagala-swiatkiewicz 2013; jensen et al., 2013). the opposite outcomes were principally ascribed to the lack of coherence in study design and implementation. in fact, frequently, inaccurate comparisons led to assert the superior quality of organic respect to conventional foods. at first, nutrient content is strictly affected by varieties; furthermore, other factors such as geographic locations of crops, characteristics of soil and clime, maturity from harvest to storage and testing must not be neglected in the way of comparison between organic and conventional agricultural methods. in the last years, systematic reviews were realized to compare the content of chemical compounds in different foods, checking at the same time for differences in study methodologies and implementation. in this way, the available scientific literature on the subject of interest is screened and the outcomes of all articles meeting predefined quality criteria, analysed by a systematic approach. (benbrook et al., 2008; dangour et al., 2009; brandt et al., 2011; smith-spangler et al., 2012; barański et al., 2014). in 2008, the meta-analysis by benbrook et al., analyzed differences in nutrient content between organic and conventional food samples within 236 matched pairs. nutrients considered were vitamin c, beta-carotene, vitamin e, potassium and phosphorous, nitrates, total proteins, total phenolics, total antioxidant capacity, and the polyphenols ! ital. j. food sci., vol 28, 2016 568 quercetin and kaempferol. this review found that total phenolics, vitamin e, vitamin c, quercetin, and total antioxidant capacity of organics exceeded that of conventionally grown produce in the case of total antioxidant capacity, by 80%. conventional products had higher levels of potassium, phosphorous, and total protein, all basic constituents of conventional fertilizers. dangour et al. (2009) systematic review, based only on studies of satisfactory quality including field trials, farm surveys and basket studies, underlined the absence of differences in nutrient parameters between organically and conventionally produced foodstuffs. the small differences in nutrient content detected were ascribed as biologically plausible and generally related to differences in cultivation methods. hunter et al. (2011) evaluated the micronutrient composition of organic and conventional plant foods with a systematic analysis. organic plant foods (vegetables, legumes and fruit) were found to have a 5.7% higher content of vitamins and minerals than their conventionally grown counterparts. irrespective of cultivar, soil type, harvest conditions, and chemical analysis, organic plant foods contained significantly higher amounts of minerals, including phosphorus, compared to conventional foods. these results were explained by the hypothesis of accelerated growth, as a result of conventional agricultural methods, that down-regulates the synthesis of carbon-containing metabolites, such as ascorbic acid. furthermore, it has been proposed that organically produced plants synthesize higher levels of ascorbic acid than conventionally-grown plants, in response to biological and ecological stresses, and the absence of protection conferred by synthetic pesticides. brandt et al. (2011) meta-analysis, focused on secondary metabolites and vitamins content in fruits and vegetables, collecting data from different studies had the scope to detect effects of systematic factors, separately from factors that occur randomly. the results obtained show that organic plant material had higher levels of all analyzed secondary metabolites and of vitamins c than conventional vegetables and fruits, together with higher content of phenolic acids and total phenolics. instead, for the content of flavones and flavonols (non-defence-related compounds), differences between two cultivations systems were heterogeneous, while for the content of carotenes no significant differences were found. the systematic analysis by smith-spangler et al. (2012) based on 223 literature study on nutrient and contaminant levels underlined that heterogeneity in results were too high to estimate significantly higher nutritional properties of organic food. about pesticides residues, lower levels were found in organic than conventional product, but differences in risk for exceeding maximum allowed limits were small. of the entire nutrient evaluated, only phosphorus content was homogeneous and significantly higher in organic food, but this difference was not statistically significant. gastol and domagala-swiatkiewicz (2013) made an evaluation on polish organic and conventional fruit and vegetables juices. the results of this comparative study that covered 33 neighbouring pairs of organic/conventional fields with six evaluated species, confirmed the absence of differences in nutritional properties between the organic and conventional food analyzed. only in few species, parameters such as polyphenolic content, antioxidant activity and dry matter significantly discriminate the two types of cultivation products. the systematic review by jensen et al. (2013) dealt with the comparison between organic and conventional agriculture in terms of nutrients content, bioavailability of nutrients and potential effect on human health. about nutritional differences between the two type of agricultural products, results highlighted the difficulty of make direct comparison because of the variability of influencing factors and study designs. the approach of using a systematic review permitted to conclude that organic food contained higher levels of ! ital. j. food sci., vol 28, 2016 569 vitamin e, vitamin c, phosphorus and lower content of pesticides than organic produce; no clear effects were established on health-related biomarkers. the most recent systematic review by barański et al. (2014), based on an extensive data set of 343 peer-reviewed publications, indicated that organic crops and foods have a higher antioxidant activity and contain higher concentrations of a wide range of nutritionally desirable antioxidants/polyphenolics, but lower concentrations of cd metal. this study, for plant secondary metabolites is in accordance with results carried out by brandt et al. (2011), but it contradicts the results of the most systematic reviews/metaanalyses published previously, which indicated that there are no significant composition differences between organic and conventional crops. the main reason for the inability of previous studies to detect composition differences was addicted, by the authors, probably to the highly limited number of studies or data sets available or included in the analyses, which could decrease the statistical power of the meta-analyses. in addition, authors used a weighted meta-analysis based on smd (standardised mean difference), not used in most of the previous studies that is recommended when combining data from studies that measure the same parameter (e.g. the major phenolic compounds found in different crops), but use different scales. results of relevant scientific works considered in this review, from cultivation tests and surveys, are described below. differences are underlined considering the content of functional compounds (polyunsaturated fatty acids, essential amino acids, vitamins and minerals, polyphenols) in organic and conventional foods. 2.1. polyunsaturated fatty acids the majority of studies dealing with pufas in conventional and organic products are focused on olive oil, for high contents in this matrix (table1). garcía-gonzález et al. (2014) compared certified organic and conventional olive oil samples from different spanish cultivars (arbequina, cornicabra, hojiblanca and picual). by application of multivariate algorithms (principal component analysis and multidimensional scaling), authors concluded that fatty acids and sterols profile and content were only able to discriminate olive oil samples according to fruit cultivar. differences in total pufas content were not statistically significant between organic (0.28±0.06 mgkg-1) and conventional (0.28±0.07 mgkg-1) oils. also samman et al. (2008), comparing organic and conventional edible oils (coconut oil, olive oil, canola oil, mustard oil seed and sesame oil) found insignificant differences in the content of pufas. the influence of the cultivation method on the quality indices of virgin olive oils was also investigated by anastasopoulos et al. (2013). olive oils (koroneiki cv.) produced in different geographical origins and seasons, years 2000 and 2004, showed differences in pufas content. those from organic production system and season 2004 had a pufas level (expressed as mean±sd% on total content) of 79.25±0.18 that resulted significantly higher of 79.05±0.18, content of conventional products. rouphael et al. (2015) investigated the presence of pufas in the seeds of perilla, an annual plant of the mint family lamiaceae. irrespective of the farming systems, the linolenic acid was the predominant fatty acid in seeds, representing 62% of the total fatty acids in the lipid fraction. no differences were recorded among treatments for the content of palmitic c16:0 (avg. 6.0%), stearic c18:0 (avg. 1.0%), and oleic c18:1 (avg. 14.0%), and linoleic acid (avg. 15.0%). ! ital. j. food sci., vol 28, 2016 570 table 1: comparison of polyunsaturated fatty acids contents in organic and conventional foods. 2.2. essential amino acids few literature studies investigated for differences in amino acids content in organic and conventional foods (table 2). furthermore, essential and non-essential amino acids were generally analyzed together in the same food. röhling and engel (2010) analyzed the influence of the input system on several metabolites of three maize cultivars (amadeo, lukas and flavi). in the text numeric data results were not shown. authors only reported results of statistical tools used: principal component analysis (pca) and analysis of variance (anova). amino acids levels, as well other compounds investigated (polar compounds, organic acids, sugar and sugar alcohols) showed the absence of a direct relationship between metabolites content and agricultural practice. authors suggested that only genotype and environment contributed to differentiations in metabolite profiles of maize. an absence of differences among organic and conventional food was also found by mader et al. (2007) analyzing protein and amino acids content in wheat (triticum aestivum l.). maggio et al. (2008), investigating in tubers (agria and merit cv), found that the organically grown ones contained only significant higher levels of threonine (24.5 mg 100g-1 of fresh weight) respect to conventional tubers (24.0 mg 100g-1 of fresh weight). references study design compounds analyzed compounds contents units statistical comparison organic conventional garcía gonzález et al., 2014 16 samples of extra virgin olive oil of 4 spanish cultivars taken from cooperative societies. palmitic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid. 0.28±0.06 0.28±0.07 means ± sd of pufas in mg kg-1 no differences in pufa content samman et al., 2008 59 certified organic and 53 conventional oils purchased from markets in sydney. edible oils considered: coconut oil (1), olive oil (2), canola oil (3), mustard seel oil (4), sesame oil (5). palmitic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid. (1) 2.63±0.86 3.86±0.46 means ±sd % of pufa on total fatty acids content no differences in pufa content (2)10.59±3.26 12.09±9.6 (3)25.73±9.08 29.51±0.53 (4)46.07±0.45 28.21±13.79 (5)48.2±1.09 44.18±3.8 anastasopo ulos et al., 2013 virgin olive oil (koroneiki variety) produced in messinia, peloponnesus, greece from different harvesting periods: season 2000(1) and 2004(2). palmitic acid, palmitoleic acid,oleic acid, linoleic acid,linolenic acid,arachidic acid. (1)77.43±1.95 77.90±1.40 means±sd% of pufas on total content higher pufa content in organic olive oil (2)79.25±0.18 79.05±0.18 rouphael et al., 2015 perilla plants grown under conventional and organic farming in a typical mediterranean area such as southern italy,season 2005. palmitic acid 6.3 6.3 means% of pufas on total content no differences in pufa content oleic acid 13.5 13.9 linoleic acid 14.7 14.6 linolenic acid stearic acid 61.9 1.9 61.6 1.9 ! ital. j. food sci., vol 28, 2016 571 table 2: comparison of essential aminoacids contents in organic and conventional foods. conventional potatoes were grown with varying levels of nitrogen fertilization and as result, a significant decrease in some amino acids content (including alanine, glutamate and histidine) was correlated to a highest nitrogen levels used. other amino acids levels varied principally with cultivar variety. pieper and barret (2009) analyzed the effect of production system on quality and nutrient content of tomatoes (lycopersicon esculentum var. ab2) from two consequential years of cultivation, 2006 and 2007. results indicated that differences in nutrient content were not statistically significant between production systems. furthermore, non-essential amino acids such as glutamate, glutamine and tyrosine, were significantly higher in conventional tomatoes. authors justified results obtained depending by the amount of available nitrogen, generally greater in conventional crops. 2.3. vitamins and minerals many studies investigated micronutrient levels in organic and conventional products, due to their vital importance in human diet (table 3). reference study design compounds analyzed compounds contents units statistical comparison organic conventional rohling and engel, 2010 three maize cultivars grown in the season 2004 at two locations. the same procedure was repeated in the season 2005. histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine numeric data not reported. numeric data not reported. no differences in essential amino acids content mader et al., 2007 wheat (triticum aestivum l.) grown in a 21 year agrosystem comparison in central europe histidine isoleucine leucine lysine methionine phenylalanine threonine tryptophan valine 47.2 23.1 34.6 67.1 26.3 13.5 45.9 31.0 11.4 42.8 46.6 22.8 35.4 67.0 25.4 13.3 46.4 30.5 10.8 42.8 means g kg−1 total protein no differences in essential amino acids content maggio et al., 2008 tubers harvested on july, 2003. six potatoes, collected from each plot, treated and analyzed histidine isoleucine leucine lysine methionine phenylalanine threonine tryptophan valine 104.9 14.8 11.8 36.6 16.7 16.0 24.5 10.6 34.1 98.5 17.1 15.1 38.4 16.7 19.2 24.0 11.2 37.3 mg 100g-1 fresh weight only threonine is in significant higher levels in organic tubers pieper and barret, 2009 processing tomatoes of the same cultivar (lycopersicon esculentum var. ab2) grown and harvested in season 2006 (1)and 2007(2) from three commercial growers in california. phenylalanine histidine methionine lysine threonine (2) 1.41±0.27 0.56±0.09 0.10±0.02 0.68±0.10 0.76±0.15 (1) 1.81±0.28 0.78±0.14 0.15±0.03 0.89±0.17 1.15±0.28 g kg−1 fresh weight no differences in essential amino acids content ! ital. j. food sci., vol 28, 2016 572 table 3: comparison in minerals and vitamins content in organic and conventional foods. references study design compounds analyzed compounds content units statistical comparison organic conventional gastol et al., 2013 66 fruit and vegetable fields (36 farms) producing organic and conventional crops cu, b, fe, mn, zn, ni, pb, cd, ca, p, mg, s, na, numeric data not shown numeric data not shown higher amounts in organic product colla et al., 2002 10 years of organic and conventional management practices on soil chemical properties, processing tomato yields and fruit mineral composition n,p,k,ca,mg, na numeric data not shown numeric data not shown organic fruit contain higher amounts of ca and p laursen et al., 2011 samples of winter wheat,spring barley, faba bean, and potato(1), obtained from field trials undertaken in 2007 and 2008 at three different danish geographical locations. k mg p s ca fe mn b zn cu mb sr na (1) 2.28±0.24 0.11±0.01 0.24±0.03 0.16±0.01 180±67.4 21.0±7.66 6.16±1.01 4.46±0.46 11.6±0.77 5.30±0.99 0.35±0.07 0.75±0.20 69.3±16.0 2.08±0.16 0.11±0.01 0.21±0.04 0.15±0.01 206±78.4 20.0±7.57 6.19±0.90 4.57±0.51 10.1±0.51 4.31±0.78 0.21±0.02 0.91±0.26 48.7±15.4 mg kg-1 no differences in mineral levels gorenjak et al.,2012 52 samples of lettuce, conventional and organic, from 15 different areas of northeast slovenia. nitrate/nitrite concentration 1258±1018.3 1359±960.6 mg kg-1 fresh weight basis nitrate levels higher in conventional foods ismail and fun, 2003 5 types of green vegetables grown organically and conventionally selected based on popular consumption among malaysian market. vitamin c 124.80 114.70 mg 100 g-1 no differences in vitamins content weibel et al., 2000 apples (golden delicious cv) harvested of 5 pair of organic/conventional fruit farms with similar micro climate, soil condition and planting system. p vitamin c numeric data not shown numeric data not shown numeric data not shown numeric data not shown no differences in vitamin c content the most investigated essential elements were phosphorus (p), potassium (k), magnesium (mg), iron (fe), copper (cu), sulphur (s), calcium (ca), zinc (zn) and sodium (na). differently by vitamins, minerals are present in the soil and are bio-available for plant acquisition. in conventional agricultural management many minerals (p, k and n), are commonly used in the form of soluble chemical fertilizers, so it could be expected a highest quantity of such minerals in conventional products than in the organic alternatives. but different results were obtained considering levels of minerals in organic and conventional fruit and vegetables (brandt et al., 2011). many scientific studies showed how different agricultural systems have a strong influence on mineral content in products; in particular organic food were higher in such compounds content (table 3). colla et al. (2002), investigating tomatoes elemental composition, reported only ! ital. j. food sci., vol 28, 2016 573 statistical results from which, organically grown fruits had highest amounts of p and ca instead of conventionally grown tomatoes richest in n and na. gastol and domagala-swiatkiewicz (2012), found none relationships between agricultural processes and minerals content in some fruits because of heterogeneity in results. only in blackcurrant juice, results shown with the use of histograms, authors found significant higher amounts of minerals in the organic fruit. further, a multi-element fingerprinting of potatoes and cereals, stated the absence of systematic differences between levels of minerals in products and different factors (crop management, location and year of cultivation) (laursen et al., 2011). in table 3 levels of minerals are indicated only for potatoes. also nitrate content was evaluated in organic food for the risks on human health. gorenjak et al. (2012), estimated nitrate content in lettuce (lactuca sativa) of different geographical origins. the mean of nitrate content, expressed as mg kg-1 of fresh weight basis, was significantly lower in organically cultivated lettuce (1258±1018.3) than in conventional products (1359±960.6). on the contrary of minerals, plant itself is responsible for the production of vitamins depending manly by variety, ripeness of fruits and crop size. for such these factors, heterogeneous results came from research studies about the content of vitamins in organic and conventional plant foods. some studies underlined a significant relation between vitamin c content in fruit and vegetables and organic procedure; others didn’t found a consistent trend (weibel et al., 2000; rembialkowska, 2007). few studies focused on the presence of other vitamins and their precursor. ismail and fun (2003) determined vitamin c, β-carotene and riboflavin contents in five green vegetables from organic and conventional systems. only organic swamp cabbage was highest in all vitamins content; for this vegetable only statistical results are shown. organic chinese mustard resulted in significant higher levels of β-carotene and in lower content of riboflavin. no significant differences were found in β-carotene content for chinese kale, lettuce and spinach grown using the two different agricultural techniques while riboflavin content in conventionally grown chinese kale and spinach was not detected compared to the organically grown vegetables. to understand evidences of higher content of some vitamins in organic fruit and vegetables, influence on nitrogen fertilization was studied in deep. nitrogen fertilization, belonged to conventional agriculture, was found to decrease vitamin c content in different fruits and vegetables (potatoes, tomatoes and citrus fruit) as well as increase beta-carotene content (mozafar, 1993; lee and kader, 2000). 2.4. polyphenols higher contents of polyphenolic compounds in organic fruits and vegetables were demonstrated in different reports (table 4). organic apples (cv. golden delicious) originated from ten neighbouring organic and conventional fruit farms in switzerland were examined by weibel et al. (2000). the content of phenolic compounds (in particular flavanols) was 19% higher in organic apple. the effect of cultivation methods on the antioxidant capacity of bluesberry (cv. vaccinum corymbosum l.) was evaluated in random samples of commercial late harvest fileds in new jersey by wang et al. (2008). results showed that blueberry fruit grown from organic culture contained significantly higher total phenolics (67.8%), total anthocyanins (59.2%), and antioxidant activity (49.8%) than fruit from the conventional culture. higher levels of some polyphenolic compounds were also found in organic peach (cv. regina bianca) and pear(cv. williams) respect to the corresponding conventional samples (carbonaro et al., 2002). ! ital. j. food sci., vol 28, 2016 574 table 4: comparison of polyphenols contents in organic and conventional foods. reference study design compounds analyzed compounds content units statistical comparison organic conventional weibel et al., 2000 apples harvested of 5 pair of organicconventional fruit farms with similar micro climate, soil condition and planting system. flavanols,cinnamon acids, phloretinglycosides, quercetinglycosides only statistical result reported only statistical result reported higher levels of phenolics in organic food wang et al., 2008 organic and conventional samples of blueberry collected from 5 certified organic farms in new jersey. anthocyanins: delphinidin 3gal. delphinidin 3-glu. cyanidin 3-gal. delphinidin 3-ara. petunidin 3-gal. petunidin 3-glu. petunidin 3-ara. malvidin 3-gal. malvidin 3-gluc. malvidin 3-ara. 171.59 69.77 29.22 93.53 184.86 127.13 95.70 303.03 303.35 227.12 41.74 24.64 14.24 37.00 75.26 79.00 66.70 289.38 184.92 199.62 µg g-1 higher levels of anthocyanins in organic food carbonaro et al., 2002 peaches (1) and pears (2), either grown on tilled soil (of the same age, 5 years), obtained from the istituto sperimentale per la frutticoltura (ciampino, rome). caffeic acid chlorogenic acid catechol α-tocopherol ɤ-tocopherol tocopherolquinone caffeic acid chlorogenic acid catechol α-tocopherol ɤ-tocopherol tocopherolquinon (1) 2174.50±198.20 2655.30±171.20 0.57±0.01 0.37±0.01 1.34±0.03 (2) 865.1±43.8 3020.7±235.4 401.4±110.3 0.71±0.05* 0.68±0.15 2.0±0.10 2451.9±126.4 2053.2±145.0 0.65±0.02 0.46±0.01 1.80±0.27 674.2±50.5 959.1±100.9 557.1±143.2 0.58±0.03 0.68±0.09 1.43±0.30 ppo activity (unit min1/100 g f.w.) µg 100 g-1 fresh weight ppo activity (unit min1/100 g f.w.) µg 100 g-1 fresh weight higher levels of polyphenolics in organic peach and pear lombardiboccia et al., 2004 yellow plums,conventio nally or organically grown in the same farm (fruit farminginstitute, rome, italy). caffeic acid trans-p-cumarico acid ferulic acid chlorogenic acid neo-chlorogenic acid myricetin quercetin kaempferol 22.6±1.05 8.9±0.32 9.3±0.42 37.5±2.94 46.0±6.9 1.1±0.1 30.2±0.8 0.6±0.2 20.6±1.23 8.5±0.34 8.0±0.63 25.2±1.25 52.0±2.76 0.9±0.2 19.6±1.2 1.7±0.3 mg kg-1 fresh weight higher polyphenols content in conventional plums gastol et al., 2013 66 fruit and vegetable fields (36 farms) producing organic and conventional crops. total polyphenols data not shown data not shown no differences in polyphenols content valverde et al., 2015 2 varieties of broccoli grown over 2 years (1),(2) in a splitplot factorial system comparison trial. total phenolics total flavonoids (1) 345.70±51.30 16.60±6.90 290.80±3.90 10.20±1.80 mg 100 g−1 fresh weight no differences in polyphenols content granato et al., 2015 purple grape juices (n = 31) produced in europe total phenolics 826.60±382.99 714.42±244.63 mg of chlorogenic acid equivalents per liter of juice no differences in polyphenols content ! ital. j. food sci., vol 28, 2016 575 in particular organic peach had statistically higher levels of chlorogenic acid (29.3%) and of total polyphenols (36.1%); organic pear had higher levels of total polyphenols (10.4%). and of caffeic acid (28.3%). lombardi-boccia et al. (2004) found conventional plums (cv. shiro) richest in total polyphenols content. quercetin was higher in conventional plums (54.1%), but myrecitin (22.2%) and kaempferol (183.3%) were higher in organic plums; both in organic and conventional fruits caffeic acid, chlorogenic acid and quercetin were the predominant compounds. in opposite to these results, in vegetable juices from celery, carrot and red beet, gastol et al. (2013), did not found differences in polyphenolic content between organic and conventional products. in broccoli (brassica oleracea var. italica), grown over two years of a split-plot factorial system trial, valverde et al. (2015) didn’t find differences in total phenols and flavonoids levels between the two agricultural systems. no differences in total phenolics contents was also found by granato et al. (2015) analyzing organic and conventional purple grape juices. polyphenols, as phytonutrient compounds, are involved in the defensive mechanism of plants after attack by pests or diseases (faller and fialho, 2010). many hypotheses were formulated to justify higher concentration of such compounds in organic crops and foods. at first, plant subjected to stress, tend to accumulate higher content of secondary compounds. when pesticides are avoided, these compounds are also accumulating by natural protection system of plants. furthermore, polyphenols are mainly synthesized during ripening of plant products. conventional crop management consisting in higher amounts of nitrogen fertilizers than organic farming, generally accelerates plant growth with the consequent decreasing of plant metabolites production. 4. conclusions food quality aspects, human health and environmental concerns influence organic food consumer preferences. the rapid growth of organic market, in recent years, is due principally to people belief of more nutritive properties of foods from organic agriculture. in this regard, literature studies considered in this review, dealing with the comparison of nutritive properties between organic and conventional fruits and vegetables, showed a high variability in results. small differences in nutritive contents exist in foodstuffs belonged to the different cultivation methods. but in almost every study claiming large nutritional and sensory quality differences between organically and conventionally grown produce, the experimenters failed to control or to “pair-up” similar environmental and cultivar inputs that affect plant and fruit development, yield and quality. when this lack of methodological rigor was overcome by the application of a systematic review and metaanalysis approach, significant higher levels of antioxidant compounds and lower cadmium levels in organic food products were demonstrated. a way to improve comparative researches on nutritional value of organic food is to unify the methodologies applied and to better consider the various factors influencing nutrients content of agricultural food. there is also the need to include analysis of food during every step of the production chain and to consider further the effect of processing on nutritional parameters content. in accordance to this aspect, some authors suggest for future studies, the holistic approach in which nutritional and technological parameters together with sensor quality are needed to evaluate in total quality of foods (kahl et al., 2010). ! ital. j. food sci., vol 28, 2016 576 references anastasopoulos e., kalogeropoulos n., kaliora a.c., kountouri a.m. and andrikopoulos n.k. 2011. the influence of ripening and crop year on quality indices, polyphenols, terpenic acids, squalene, fatty acid profile, and sterols in virgin olive oil (koroneiki cv.) produced by organic versus non-organic cultivation method. international j. food sci. & tech. 46:170. azadi h., 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consumer perceptions and preference toward organic versus conventionally produced foods: a review and update of the literature. renew. agr. food syst. 20:193. paper received july 27, 2015 accepted april 14, 2016 #449_caponio_bozza ital. j. food sci., vol 29, 2017 302 paper new formulations of olive-based pâté: development and quality l. cosmai, f. caponio*, c. summo, v.m. paradiso, a. cassone and a. pasqualone *department of soil, plant and food science (disspa), university of bari aldo moro, via amendola, 165/a, i-70126 bari, italy *corresponding author. fax: +39 0805443467 e-mail address: francesco.caponio@uniba.it abstract the aim of this research was the chemical, microbiological, and sensory characterization of new ingredient formulations of olive-based pâtés, since few studies are present in the literature regarding table olive-based processed products. three ingredient formulations were selected by a consumer test on the basis of the scores of odour and taste descriptors, higher than 6 (linear scale 0-10 cm). the chemical and sensory analyses allowed to clearly differentiate all ingredient formulations on the basis of both composition and volatile profile. microbiological data corresponded to levels usually detected for similar products and excluded the presence of pathogens. finally, no off-flavour was perceveid in the tested formulations. keywords: characterization olive-based pâté, sensory analysis, table olives, volatile profile ital. j. food sci., vol 29, 2017 303 1. introduction table olives (olea europaea l.) are one of the most important traditional fermented vegetables in the western world (bleve et al., 2015) and, similarly to olive oil, one of the most characteristic components of the diet in mediterranean countries. the world production of table olives has shown a steady increase for the last 20 years and is mainly concentrated in spain, turkey, egypt, syria, morocco, italy, and greece. the international olive council (ioc, 2014) estimated that table olive production (2011/2012 season) was above 2.4 milliontons. according to the trade standard applying to table olives (ioc, 2004), table olives are prepared from sound olive fruits whose volume, shape, flesh-to-stone ratio, firmness, and taste make them particularly suitable for processing. moreover, table olives have to be treated to remove bitterness by natural fermentation or by heat treatment, with or without the addition of preservatives. table olives have been attracting increasing interest, due to their postulated health benefits, that seem to be intrinsically linked to the high monounsaturated fatty acid content, as well as to their important antioxidant capacity, antimicrobial activity, and protection against mycotoxin effects due to minor constituents like tocopherols and phenolic compounds (malheiro et al., 2011). table olives consumption is greatly varied: the fruits, conveniently processed, can be served as an appetizer or as a complement to salads, pasta, pizza, fish, and meat. even bread can be prepared by adding green/black olives to the dough (sabatini et al., 2009; lanza, 2012). as reported by ioc (2004), table olives consumption showed an increase primarly attributable to the marketing efforts by manufacturers essentially aimed at the introduction of new products, to satisfy the growing consumers’ awareness of their health benefits. in this context, the table olives characterized by defects in terms of size and/or shape, unsuitable to be marketed as such, could represent the base for the preparation of new commercial products among which olive-based pâtés. the term “pâté” indicates a processed product that has important gastronomic tradition and good sensory properties with a coarse texture (ünlüsayin et al., 2007) in which the main ingredients are more or less finely ground and mixed with various ingredients, considered essential for their binding capacity. with respect to other types of vegetable pâtés – for example tomato-based pâtés, characterized by a complex ingredient formulation (the starting vegetable is usually associated with other kinds of ingredients, such as mushrooms, eggplants, peppers, oil, spices, and herbs) – usually commercial olive-based pâtés contain only table olives and olive oil. table olives have been studied from microbiological (pereira et al., 2008; tassou et al., 2002; hurtado et al., 2008; arroyo-lópez et al., 2008; panagoua et al., 2008; campaniello et al., 2005), chemical (montaňo et al., 2003; aponte et al., 2010; romeo et al., 2010; pasqualone et al., 2014), and sensory points of view (sabatini and marsilio, 2008; pérez et al., 2007). however, no studies are present in the literature regarding table olive-based processed products, except for a study carried out by alvarenga et al. (2012). on the basis of these considerations, the aim of this study was the development of new formulations of olive-based pâtès to satisfy more dynamic, complex and differentiated consumer demands for traditional and functional foods. it is crucial to consider food safety as well as nutritional, sensory and commercial qualities. in this context sensory, microbiological, and chemical properties of olive-based pâtès were reported. ital. j. food sci., vol 29, 2017 304 2. materials and methods 2.1. sampling the trials were carried out utilizing cv. bella di cerignola table olives, debittered by the spanish method (pasqualone et al., 2014), purchased at local retailers without defects. the experimental plan required that the olives represent at least 50% of the total ingredient formulation, while the choice of the other ingredients was made through preliminary tasting sessions that were designed to assess which combinations were judged the best among different ingredients. extra virgin olive oil (evoo) of coratina cultivar was used in the experimental trials. a total of six formulations (f1, f2, f3, f4, f5, f6) were evaluated: • f1, table olives (500 g kg-1), evoo (170 g kg-1), sweet shelled almonds (165 g kg-1), water (165 g kg-1); • f2, table olives (640 g kg-1), evoo (100 g kg-1), olive-oil tuna (130 g kg-1), dried tomatoes (130 g kg-1); • f3, table olives (770 g kg-1), evoo (155 g kg-1), zucchini (75 g kg-1); • f4, table olives (750 g kg-1), evoo (50 g kg-1), dried tomatoes (50 g kg-1), red peppers (50 g kg-1) eggplants (50 g kg-1), mushrooms (pleurotus eryngii) (50 g kg-1), capers (1 no.); • f5, table olives (770 g kg-1), evoo (150 g kg-1), salted anchovies (40 g kg-1), red onion (40 g kg-1), leaves of arugula (2-3 no.), drop balsamic vinegar (5 no.); • f6, table olives (750 g kg-1), evoo (130 g kg-1), salted anchovies (60 g kg-1), red onion (60 g kg-1). before the olive-based pâtés production, some raw materials were subjected to preliminary treatments: table olives were washed and pitted; dried tomatoes and mushrooms were blanched in boiling water or water/vinegar (1:1 v/v), respectively, for two minutes; zucchini, red peppers, and eggplants were roasted. all ingredients were mixed by means of a homogenizer (waring lb 20 es, rome, italy) until obtaining a homogeneous creamy paste (about 5 minutes at 12,000 rpm). after mixing, the product was transferred and packed in plastic boxes having the capacity of 100 g. 2.2. consumer test a consumer test was conducted for the six different formulations. a total of 90 people (mean age 27 years, range 17-60 years, 45 males and 45 females) participated in the consumer test, which was performed within 4 hours from pâté production. the participants were selected among people who regularly consumed vegetable pâtés (i.e. 1-4 times a month). an alpha-numeric code was assigned to each pâté, served in white plastic cups (about 10 g). after trying, several different palate cleansers to avoid carry-over effects and adaptation to sensory stimuli, mineral water and a 1-minute break between samples were chosen. before starting the evaluation of products, the investigator read aloud the instructions to the participants. each subject was asked to fill a sensory evaluation sheet composed of a first part regarding personal information (age and sex), and a second part that included a continuous 10 cm-line on which consumers expressed an acceptability judgment for odour pleasant and taste pleasant descriptors, respectively. testing was performed at room temperature (20 °c) with artificial lighting, simulating daylight. ital. j. food sci., vol 29, 2017 305 2.3. sensory analysis on the basis of the results of consumer test, the three most appreciated pâté were submitted to a descriptive sensory analysis by a trained panel of assessors. the first step has provided the identification of the sensory descriptors, in order to develop a common vocabulary for the description of the sensory attributes and to familiarize with scales and procedures. each attribute term was extensively described and explained to avoid any doubt about the relevant meaning. on the basis of the citation frequency (>60%), eleven descriptors were selected and related to the rheological characteristics (consistency and phase separation), visual characteristics (brightness and colour homogeneity), taste (bitter, acid, sweet, salt), and flavour (olive flavour and off-flavour). each descriptor was scored on a non-structured line scale of 10 cm. a panel of eight assessors, aged between 25 and 50 years, was trained through preliminary sessions to ensure that each panelist interpret the same descriptor in the same way. the same product was also subjected several times, without their knowledge, to repeatedly tasting to assess the ability of panelists to detect the intensity of each descriptor always in the same way. the panel consisted of teachers and students, and all sensory tests were performed in the sensory laboratory of food science and technology section of bari university, which fulfills the requirements of the international standards (iso, 1988). during the evaluation phase, the panelists were seated in private booths equipped with air conditioning and under incandescent/fluorescent light. the sample presentation order was randomized for each panelist. the samples, identified by an alpha-numeric code, were served to each panelist in plastic cups at room temperature, and tap water was provided between samples to cleanse the palate. samples were analyzed in duplicate. 2.4. microbiological analysis microbiological analysis included the determination of contaminating micro-organisms and pathogens (coliforms, salmonella spp., listeria monocytogenes and escherichia coli). all cultural media, supplements, and diagnostic kits were provided by oxoid (basingstoke, uk). coliforms were determined by count in plates of selective medium violet red bile glucose agar (38.5 g/l in distilled water boiled for 2 min), seeding 1 ml of decimal dilutions for inclusion and incubating the plates at 30 °c for 24 h (anon, 1996). the counting was done considering only the characteristic colonies (dark red with diameter > 0.5 mm, with or without the surrounding precipitate), while the others were submitted to the confirmation. this was made inoculating 3-5 colonies with a loop in tubes of sterile broth of brilliant green lactose then incubated at 30 °c for 24 h. all colonies developed in the culture broth with gas production were included in the count of coliform. the monitoring of l. monocytogenes (anon, 1996) consists of several stages: i) the primary selective enrichment: 25 g of sample were spiked with 225 ml of half fraser and the incubated at 30 °c for 24-48 h; ii) secondary selective enrichment: 0.1 ml of the primary enrichment culture were incubated in 10 ml of medium fraser broth at 37 °c for 24-48 h; iii) isolation: a loopful of enrichment culture was streaked on distinct plates of oxford agar and palcam agar, incubated at 37 °c for 24-48 h (in microarofilia conditions with 512% co2, 5-15% o2, 75% n2 only for palcam agar plates); iv) confirmation and identification: by each isolation medium were taken at least 5 characteristic colonies to perform subculture by streaking on the tryptone soy yeast extract agar, incubated at 37 °c for 24-48 h. the identification was performed with biochemical tests of catalase, gram ital. j. food sci., vol 29, 2017 306 stain and hemolysis on plates of blood agar bases supplemented with defibrinated sheep blood after 24-48 h of incubation at 37 °c. for the determination of e. coli, 25 g of each pâté were homogenized in 225 ml of the solution of tryptone soy broth and incubated at 37 °c for 18 h under stirring (150 rpm). from each crop, inocula of 0.1 ml were made through smear on macconkey sorbitol agar enriched with cefixime-tellurite supplement. the plates, produced in triplicates, were incubated at 35 °c for 20-22 h before counting the probable colonies of e. coli 0157:h7; identification was made with the use of e. coli o157 test kit (abdul-raouf et al., 1993). the results were expressed as cfu/g of sample. 2.5. chemical analysis the measurements of ph were conducted by using a basic 20 ph meter (crison instruments s.a., barcelona, spain) provided with a conductivity penetration probe (codito 50 10t, crison instruments s.a., barcelona, spain) and a temperature compensator. moisture content was estimated by drying the samples at 105±5 °c, and ash content by incineration, both until constant weight (aoac, 2000). fat content was determined by soxhlet extraction with 40-60 °c diethyl ether for 6 hours, followed by evaporation of the solvent (iupac, 1979). protein content was determined by kjeldahl method (n × 6.25) (aoac, 2000). carbohydrate content was estimated by subtracting the weight of the other components to the total weight. 2.6. headspace analyses volatile compounds were extracted by solid-phase micro-extraction (spme) and analyzed by a gas-chromatographic system equipped with mass spectrometer (gc-ms). in particular, an aliquot of sample (1 g ± 0.05) was placed inside 12-ml glass vials, closed by silicone/ptfe septa and an aluminum seal. the pâté sample was homogenized for 2 min using a laboratory vortex shaker. before extraction, stabilization of the headspace in the vial was achieved by equilibration for 5 min at 40 °c. the extraction was performed by exposing a 75-μm polydimethylsiloxane/divinylbenzene/carboxen (pdms/dvb/car) fiber (supelco, bellefonte, pa., usa) in the headspace of the sample at 40 °c for 15 min. when the extraction process was completed, the fiber was removed from the vial and desorbed in the injection port of the gc in a splitless mode. the gc-ms instrumentation included an agilent 6850 gas-chromatograph (milan, italy) equipped with an agilent 5975 single quadruple mass-spectrometer. compounds were resolved on a hp-innowax (20 m × 0.18 mm, 0.18 µm film thickness) polar capillary column (agilent, milan, italy) under the following conditions: injector temperature, 220 °c; helium as the carrier gas at a flow rate of 15 ml/min for 7 min; oven temperature was at 40 °c/0.70 ml/min (linear speed of 36 cm/sec), then increased at 18 °c/min up to 180 °c, then increased at 20 °c/min up to 220 °c. the mass spectrometer was operated in the electron impact mode (electron energy = 70 ev) and the ion source temperature was 250 °c. the mass range was m/z 20-250. the volatile compounds were identified by comparison with the mass spectra present in the nist and wiley libraries, quantified and expressed in terms of integrated area. 2.7. statistical analysis the results were expressed as mean and standard deviation of three different trials and all the analytical determinations were carried out in triplicate. analysis of variance (one-way anova) was carried out on the chemical and microbiological analyses, whereas two-way ital. j. food sci., vol 29, 2017 307 anova was used on sensory analysis, considering formulation and panelist as independent variables. significant differences among the values of all parameters were determined at p≤ 0.05. all data were processed by the xlstat software (addinsoft sarl, new york, ny, usa). 3. results and discussion table 1 shows the mean values and the results of a statistical analysis (one-way anova) of the scores attributed to odour and taste during the consumer test performed on six different types of olive-based pâtés. the acceptability of the formulations was evaluated in order to identify the most appreciated, by considering only formulations scored more than 6 on a 0-10 scale. table 1. means values and standard deviation of the results of consumer test performed for the six different ingredient formulations of olive-based pâtés. pâtés pleasant odour pleasant taste f1 4.55±2.22 d 5.19±2.15 c f2 5.16±2.49 c 6.42±2.74 b f3 6.01±2.60 b 6.62±2.45 b f4 6.50±2.12 ab 7.22±2.26 a f5 7.00±2.03 a 6.64±2.00 b f6 5.80±2.31 bc 6.37±2.21 b a-d, different letters indicate a significant difference at p≤0.05. the data showed significant differences among all the considered samples. relatively to odour descriptor, scores higher 6, on linear scale 0-10 cm, were observed in f3, f4, and f5 pâtés. the f4 sample showed the highest taste score, even if all the other formulations (except f1) were scored more than 6, with no significant differences among them. the most appreciated formulations in terms of odour were characterized by completely different ingredients among them, demonstrating that it is possible to set up several pleasant combinations, assuming that the components are quantitatively well-balanced. in particular, f3 and f4 were characterized by the presence of different vegetables that contributed to make a specific odour more intense which would have otherwise been flat if only table olives had been used, as evidenced for f1. moreover, in the f1 sample probably the use of almonds flattened both the odour and the taste. f5 had a higher score than f6, although containing similar ingredients. this result could be attributed to a positive effect of additional ingredients, such as balsamic vinegar and arugula, or salted anchovies. on the basis of the scores obtained for both odour and taste, three ingredient formulations were selected: f3, f4, and f5. they reached a mean odour score of 6.01, 6.50, and 7.00, respectively, and a mean taste score of 6.62, 7.22, and 6.64, respectively. these results evidenced a real possibility to market these products. table 2 shows the chemical composition of the three selected pâté formulations and the results of one-way anova. the ph level ranged between 4.95 and 6.76, with significantly lower values in f4, due to the use of dried tomatoes and mushrooms (which needed to be blanched in water/vinegar, 1:1 v/v). higher values of ph observed in the other formulations were due to the use of olives, which underwent the spanish debittering ital. j. food sci., vol 29, 2017 308 method, involving the use of naoh. moisture content was higher in f4, due to the use of a greater amount of vegetables, than in f3 and f5. fat content, on the contrary, was higher in f3 and f5 than in f4 due to the higher amount of olive and oil used in their recipe. protein and ash content were significantly higher in f5, probably due to the presence of salted anchovies, whereas carbohydrate content was higher in f4. microbiological analyses (table 3) performed on the selected pâté formulations shown as coliforms and e. coli cell density were below the limit indicated in the ec regulation no 2073/2005 on microbiological criteria for foodstuffs. significantly higher values were determined in f5, probably due to the use of salted anchovies (patir et al., 2006). biochemical and serological tests on salmonella spp. and l. monocytogenes allow to exclude the presence of pathogens, in agreement with the requirements of the above ec regulation no 2073/2005. table 2. mean values and standard deviation of percent composition of three selected olive-based pâtés. pâtès ph moisture fat proteins carbohydrates ashes f3 6.76±0.03a 59.65±0.98b 31.00±0.20a 1.13±0.02c 6.86±1.06b 1.36±0.11c f4 4.95±0.04b 67.72±0.84a 21.27±0.44b 1.63±0.02b 7.37±0.78a 2.01±0.15b f5 6.13±0.01a 58.94±0.32b 31.85±0.40a 1.82±0.02a 4.94±0.18c 2.45±0.19a a-c, different letters indicate a significant difference at p≤0.05. table 3. mean values (cfu/g) and standard deviation of the results of microbiological analyses of three selected olive-based pâtés. samples coliforms salmonella spp. listeria monocytogenes escherichia coli f3 <1000 b not found in 25 g not found in 25 g <100 (not encountered pathogenic strains) f4 <1000 b not found in 25 g not found in 25 g <100 (not encountered pathogenic strains) f5 (2.0±0.2) × 103 a not found in 25 g not found in 25 g <100 (not encountered pathogenic strains) a-b, different letters indicate a significant difference at p≤0.05. cfu, colony-forming units. fig. 1 shows the gc-ms chromatograms related to headspace analysis of f3, f4, and f5 pâtés. it is possible evidence the good peaks separation and the more complex volatile compounds pattern in f4, in which the main volatile compounds were evidenced. in particular, a total of 77 volatile compounds were identified and grouped in relation to the chemical class they belonged to (table 4). generally, the volatile compounds of the examined pâtés were affected by the different ingredients used, but the exact contribution of each ingredient to the volatile fraction of pâtés was difficult to point out: in fact, the major volatile compounds identified were shared by different raw materials, as evidenced by their preliminary headspace analysis (data not showed). in particular, the most abundant volatile compound was acetic acid, significantly more represented in f4 than in f3 and f5. the analysis of raw materials evidenced that the abundance of this compound was mainly attributable to the presence of eggplants, capers, dried tomatoes, and mushrooms, the latter blanched in water/vinegar. the use of capers could justify also the preponderance of other carboxylic acids in f4, with respect to f3 and f5, such as hexanoic and heptanoic acids (romeo et al., 2007). ital. j. food sci., vol 29, 2017 309 figure 1. chromatograms of the headspace of f3, f4, f5 formulations. number peaks identify the main volatile compounds. 1 = ethanol; 2 = hexanal; 3 = eucalyptol; 4 = 2-hexenal; 5 = 6-methyl-5-hepten-2-one; 6 = 1-hexanol; 7 = (z)-3-hexen-1-ol; 8 = 2-hexen-1-ol; 9 = acetic acid; 10 = hexanoic acid; 11 = heptanoic acid (cosmai et al.). the presence of vegetables (eggplants, red peppers, mushrooms, and dried tomatoes) made f4 richer in terpenic compounds more than f3 and f5, such as eucalyptol (contributed by olives and eggplants), d-limonene (from olives, mushrooms, and dried tomatoes), terpinen-4-ol (from olives, dried tomatoes, and eggplants) and linalool (from olives and dried tomatoes). the latter was very abundant also in f5, where it was contributed by olives and arugula. the f4 pâté was also characterized by higher levels of 6-methyl-5-hepten-2-one, related to pungent and green sensory notes. this volatile ital. j. food sci., vol 29, 2017 310 derived from the dried tomatoes, being considered as a marker of the lycopene degradation (cremer and eichner, 2000). table 4. mean values of integrated total area of volatile compounds of three selected olive-based pâtés. volatile compounds f3 f4 f5 p-value terpenes, phenols, lactones and thiols α-pinene nd 3.39a 2.00b <0.001 β-pinene 3.59a 4.47a 2.30b 0.010 α-phellandrene 2.56 nd nd <0.001 (+)-4-carene 1.31 nd nd 0.085 d-limonene 17.91ab 18.75a 12.10b 0.095 eucalyptol 114.19b 159.50a 127.60b <0.001 2,4-quinolinediol nd 4.23ns 5.07ns <0.001 acetophenone nd 6.94 nd <0.001 butyrolactone 3.03ns 3.03ns nd 0.005 benzothiazole nd 4.67 nd <0.001 α-farnesene 2.75ns nd 3.20ns <0.001 2-ethylphenol 3.30 nd nd <0.001 linalool 8.12b 14.34a 12.86a <0.001 terpinen-4-ol 4.53c 14.33a 10.73b <0.001 esters methyl acetate 2.34b 3.09b 4.59a 0.006 ethyl acetate 12.91b 13.84b 24.23a <0.001 ethyl butanoate 3.10a 1.14b 3.04a <0.001 ethyl 2-methylbutanoate 3.77b 2.38c 4.84a <0.001 ethyl 3-methylbutanoate 3.78c 6.75a 5.11b <0.001 hexyl acetate nd nd 1.04 <0.001 (z)-3-hexen-1-ol acetate 3.51c 9.76a 4.61b <0.001 methyl benzoate 0.88b 3.76a nd <0.001 alcohols ethanol 83.65ns 80.46ns 91.49ns 0.363 1-butanol nd 51.34ns 36.14ns 0.007 1-penten-3-ol nd nd 8.80 <0.001 2-methyl-1-butanol 2.68ns 2.26ns 2.49ns 0.514 3-methyl-1-butanol 4.59ns 4.72ns 5.11ns 0.376 1-pentanol 2.85b 4.37a 3.24b 0.001 (z)-2-penten-1-ol 4.68c 8.00b 11.17a <0.001 1-hexanol 68.98a 71.78a 54.50b 0.001 (z)-3-hexen-1-ol 93.94a 43.14b 95.96a <0.001 (z)-2-hexen-1-ol 82.38a 36.06b 28.96c <0.001 1-octen-3-ol 1.66b 4.85a 4.75a 0.017 1-heptanol 0.89b 4.69a 3.58a 0.009 1-octanol 4.76b 7.51a 7.65a 0.082 aldehydes propanal nd nd 9.38 <0.001 2-methylpropanal nd nd 2.68 <0.001 2,2-dimethylpropanal-3-pentanone 12.41 nd nd <0.001 ital. j. food sci., vol 29, 2017 311 2-methylbutanal nd 5.57b 10.54a <0.001 3-methylbutanal nd 9.41b 26.29a <0.001 pentanal nd 24.62 nd <0.001 hexanal 6.20c 74.17a 26.94b <0.001 2-hexenal 2.47c 70.11a 28.47b <0.001 (e)-2-heptenal 3.44b 8.34a 10.96a 0.010 nonanal nd 4.83b 12.64a <0.001 (e,e)-2,4-heptadienal nd nd 5.36 <0.001 furfural nd 9.34a 2.45b <0.001 benzaldehyde nd 21.74a 6.91b <0.001 2-decenal nd 2.53b 3.13a <0.001 3,5-dimethylbenzaldehyde 1.36b 2.92a nd 0.035 ketones acetone 1.34b 3.35a 2.73a 0.009 2-butanone 2.79b 4.68a 2.90b 0.012 3-hexanone nd nd 1.94 <0.001 6-methyl-5-hepten-2-one 4.52b 47.14a 6.07b <0.001 sulfur compounds dimethyl sulfide 1.11ns 2.16ns 2.05ns 0.112 dipropyl disulfide nd nd 12.55 <0.001 furans 2-pentylfuran nd 2.05 nd <0.001 acids acetic acid 43.21c 517.62a 218.83b <0.001 propanoic acid nd 4.22 nd <0.001 pentanoic acid nd 3.63 nd 0.001 hexanoic acid nd 21.94a 2.33b <0.001 heptanoic acid nd 10.71a 2.94b <0.001 2-methyl-2-propenoic acid nd 2.94 nd <0.001 other compounds octane 19.54ns 20.45ns 26.40ns 0.132 1-octene 0.73ns nd 0.75ns 0.089 (z)-2-octene nd nd 0.79 <0.001 decane nd 2.94ns 2.53ns 0.003 3-ethyl-1,5-octadiene nd nd 4.82 <0.001 4,8-dimethyl-1,7-nonadiene nd nd 4.26 <0.001 ethylbenzene 1.39b 2.98a nd 0.037 p-xylene 2.14b 2.80b 4.62a <0.001 (e)-5-octadecene nd 2.56b 9.43a <0.001 styrene 1.43ns 2.74ns 1.36ns 0.216 3-cyclohexene-1-methanol α-α, 4-trimethyl 8.44a 8.50a 6.32b 0.039 α-α, dimethyl benzyl alcohol nd 2.49 nd <0.001 1,2-dimethoxy-4-(2-propenyl)benzene 2.59b 4.90a 2.18b <0.001 methoxy-phenyl-oxime nd 8.20 nd <0.001 a-c, different letters indicate a significant difference at p≤0.05. least squares means expressed as total area counts x 10-5. nd, not detected. ns, not significant. ital. j. food sci., vol 29, 2017 312 the headspace composition of the examined pâtés also was characterized by the presence of high amounts of alcohols, above all ethanol, 1-butanol, 1-hexanol, (e)-3-hexen-1-ol, and (z)-2-hexen-1-ol, as well as by aldehydes, such as hexanal and 2-hexenal (significantly higher in f4 and attributed to the presence of mushrooms and dried tomatoes). the majority of alcohols are by-products of some pathways involving the aldehydes. once formed, the aldehydes undergo a series of enzymatic transformations mediated by isomerases and alcohol dehydrogenases that generate c6 alcohols (cavalli et al., 2004). c6 volatile alcohols are also important components of the flavour of fruits, vegetables, and leaves (schwab et al., 2008). regarding the most abundant alcohols, no significant differences were observed for ethanol in the three selected formulations, and for 1-butanol between f4 and f5 (attributable to the use of capers and salted anchovies, respectively). high levels of 1hexanol were observed for f3 and f4 pâtés, as well as of (z)-3-hexen-1-ol for f3 and f5, and (z)-2-hexen-1-ol for f3. the presence of these volatiles is principally linked to the use of table olives and extra-virgin olive oil; a further contribution of 1-hexanol could be attributed to mushrooms and, above all, to dried tomatoes where it is considered one of the most important volatile compounds (christensen et al., 2007). on the other hand, (z)-3-hexen-1-ol could be related to arugula, whose headspace analysis showed this compound as the major contributor of aroma (data not shown). significantly higher values of benzaldheyde were found in f4 more than in the other two formulations. this compound, present in all the examined raw materials, but at particularly high levels in roasted vegetables, is a powerful volatile having an aroma of bitter almond. it can be thermally generated from phenylalanine (chu and yaylayan, 2008), via the strecker degradation. the presence of salted anchovies in f5 could justify the significantly higher content of 2methylbutanal, 3-methylbutanal, (e)-2-heptenal, (e,e)-2,4-heptadienal, and nonanal. the first two volatile compounds derived from catabolism of specific amino acids such as isoleucine and leucine, respectively, through the non-enzymatic strecker reaction (estévez et al., 2011) while the other volatile compounds were oxidation-derived compounds, dueto the effect of salting that caused a marked increase of these compounds with respect to fresh sample (soliman et al., 1983). in particular, (e)-2-heptenal is associated to oxidized, tallow, pungent aroma; (e,e)-2,4-heptadienal to boiled-potato like odour (rustad, 2009) and fatty, rancid aroma; nonanal to fatty, waxy, and pungent aroma. the f5 pâté was also rich in esters, especially ethyl acetate, related to the presence of balsamic vinegar (del signore, 2001) and anchovies (humaid and jamal, 2014) among the ingredients. moreover, the presence of dipropyl disulfide in f5 is due to the addition of onions to its formulation (carson, 1987). fig. 2 shows the results of quantitative descriptive sensory analysis and the results of twoway anova, performed on the three selected formulations. from the textural point of view, the three formulations did not show a phase separation, indicating a good homogenization and consequently an appropriate ratio among the ingredients. no significant differences were found among the tested formulations. the consistency (fig. 3), instead, was significantly higher in f3 than f4 and f5, probably due to the different water content of the ingredients used. in terms of colour, all the examined samples showed generally low and high values for brightness and colour homogeneity, respectively. ital. j. food sci., vol 29, 2017 313 figure 2. sensory profile of the three selected olive-based pâtés. a-b, different letters indicate a significant difference at p≤0.05 (cosmai et al.). 0 2 4 6 8 brightness colour homogeneity phase separa9on bi:er acid sweet salt olive flavour off-flavour f3 a b ab b 0 2 4 6 8 brightness colour homogeneity phase separation bitter acid sweet salt olive flavour off-clavour f4 a b b a 0 2 4 6 8 brightness colour homogeneity phase separation bitter acid sweet salt olive flavour off-clavour f5 b a a b ital. j. food sci., vol 29, 2017 314 about taste descriptors, the intensity of their perception was low, except in f5 for bitter where was attributable to the use of onion and arugula and for salty sensations, due to the use of salted anchovies. moreover, the presence of anchovies could explain the significant lower olive flavour assessed in f5. no off-flavour was perceveid in the tested formulations. figure 3. consistency values of the three selected olive-based pâtés. a-b, different letters indicate a significant difference at p≤0.05 (cosmai et al.). 4. conclusions the obtained results showed a good overall acceptability of the examined pâtés. the ingredients used in the recipe allowed their clear differentiation and identification. the microbiological analyses ensured the safeness of the product. moreover, the obtained results evidenced that it is possible to produce innovative formulations of olive-based pâtés by adding new ingredients to their recipes, and representing also a viable alternative use to unsuitable table olives not to be marketed as such. acknowledgements this work was funded by ministero dell’istruzione, dell’università e della ricerca, ministero dello sviluppo economico and fondo europeo di sviluppo regionale (pon02_00186_3417037, project proinno_bit). references abdul-raouf u.m., beuchat l.r. and ammar m.s. 1993. survival and growth of escherichia coli o157:h7 on salad vegetables. appl. environ. microbiol. 59:1999. alvarenga n.b., lidon f.j.c., silva a., martins g., cruz t., palma v. and canada j. 2012. production and characterization of green and black olive paste using cream of animal and vegetable origins. emir. j. food agric. 24:12. anon. 1996. rapporti istisan issn 1123–3117, 96/35. aponte m., ventorino v., blaiotta g., volpe g., farina v., avellone g., lanza c.m. and moschetti g. 2010. study of green sicilian 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institute of applied scientific higher education of jahad-e agriculture, imam khomeini agricultural higher education center, karaj, iran *corresponding author: tel. 09352394098, email: fariba2022@yahoo.com abstract this study was conducted to evaluate the impacts of nutrient elements on phytochemistry characters and qualities of strawberry in soilless culture system. the experiment was carried out in a factorial experiment based on randomized complete design with three replications. treatments consisted of 6 groups of strawberry growing on soilless medium made of perlite and coco peat that were treated with different ratio of nutrient solutions. according to the results modified nutrient improved fruit nutritional characters but it was not unique. tss s , ta, vitamin c and ph often were increased by increasing nutritional elements but anthocyanin was decreased by increasing some nutritional elements. 2 ital. j. food sci., vol. 27 2015 introduction strawberry (fragaria × ananassa duch.) is one of the most commonly consumed berries, both in fresh and processed forms such as jams, yoghurts, desserts or juices. the relevant nutritional value of strawberry fruits has been remarkably correlated (hannum 2004) with the high level of micronutrients such as minerals, vitamin c and folate which are essential for health, and, more recently, to the high levels and different phytochemical constituents (tulipani et al., 2009). since strawberry is an adaptable plant, and its fruit can be obtained almost in all seasons, its growing areas are being widely expanded in the world. on the other hand since the fruits can be obtained early in the season, when there are no fresh fruits in the markets, its marketability is high. another important aspect is that it can bring back the investment in a short period; therefore it is suitable for family hobby (ilgm, 2006). because of increased demand for more products with high quality and offseason, greenhouse production is increasing. soilless media are popularly used in greenhouse crop production because they are relatively lightweight, free from diseases, readily available, more uniform and more suitable for growing in containers than soil (yuan et al., 1996). the choice of the medium should be based on physical characteristics as well as availability and cost (lieten et al., 2004; tabatabaei and mohammadrezaei, 2006). hydroponics is a method of growing plants using mineral nutrient solutions without soil. in this method, growing substrate may be an organic material (peat moss, shredded bark, foam or other organic materials) or an inor ganic material such as sand, perlite, vermiculite and rock wool. to support and anchor the root system; plant nutrition is provided through a nutrient solution circulating in the substrate. one of the advantages of plant nutrition in soilless culture derives from the possibility of precise control of nutrient elements (johnston et al., 2010) which is not possible with soil substrate (arzani, 2007). coco peat is the best med i u m for growing summer crops, flowers and strawberry because i t has high porosity, and it has a good capacity of holding water and nutrients. porosity in perlite provides good air exchanges and soil watering and it improves soil aeration promoting the growth of the root system (noguera et al., 2003). perlite has rich inorganic materials such as iron, sodium, calcium and rare organic materials, since it is based an organic feature (djedidi, 1999; ebrahimi et al., 2012). strawberry requires high amounts of potassium because this element is a major component of the fruit and has a positive correlation with fruits size, color and acidity (behnamiyan and masiha, 2002). the research was designed and performed to evaluate the effect of different concentrations of nutrients elements via nutrient solution in soilless system composed of 50% perlite and 50% coco peat on phytochemical and quality characters of greenhouse grown strawberries cultivar gaviata (ameri et al., 2012). material and method plant material and growth conditions the study was conducted from march to august, 2013, in an experimental greenhouse of the plant production department, imam khomeini higher educational center karaj, iran. strawberry plants cv gavieta were grown in 2 liters pots on a soilless medium made of 50% perlite and 50% coco peat (v:v) with three plants per pot. day/ night temperatures were kept at 22/17°c. and were treated with different ratios of a nutrient solution. the full nutrient solution formula was made up with the following stock solutions of the different nutrients: 2.6 kh 2 po 4 , 1.9 kno 3 , 2.4 ca(no 3 ) 2 4h 2 o, 0.65 mgso 4 7h 2 o and 0.46 k 2 so 4 . microelements for the full nutrition solution were provided in the following amounts: 0.16 h3bo3, 0.09 mnso4, 0.07 znso4, 0.01 cuso 4 and 0.002 h 2 moo 4. to provide iron, a stock solution containing 0.1 fe-edta was prepared (arzani, 2007). the following treatments were applied: full nutrient solution (g1), a modified nutrient solution with either 10% less (g2) or more (g3) amount of fe, znso 4 , b 2 so 4 , mgso 4 , kno 3, a modified nutrient solution with either 10% more (g4) or less (g5) amount of fe, ca(no 3 )2, khpo4, mnso 4 , cuso 4 , moso 4 and a modified nutrient solution consisting of +10% ca (no 3 )2, khpo4, mnso4, cuso 4 , moso 4 (g6). the hydroponic system was open. nutrient solution formula for the group containing chemical treatment was prepared according to kerej et al. (1999) instruction (table 1). the ph and ec of nutrient solution were adjusted to 5.7 and from 0.9 to 1.4 ds m-1, respectively. the pots were arranged in the glasshouse according to a randomized complete design with three replications per treatment. determination of total anthocyanin content (acy) the acy of the hydroalcoholic extract of fruits was determined using the ph differential method previously described by giusti (2001). acy concentration was calculated from the calibraital. j. food sci., vol. 27 2015 3 tion curve using pelargonidin3-glucoside (pg3-gluc) as a standard. results are expressed as mg of pg-3-gluc equivalents per 100 g of fresh weight (fw) of strawberry. data are reported as a mean value (sd for six measurements) (tulipani et al., 2008). total soluble solids (tsss), total titratable acidity (ta), and ph determinations twenty fruits from each replicate were wrapped in cheesecloth and squeezed with a hand press, and the juice was analyzed. tsss, was determined at 20°c by an atago dbx-55 refractometer (atago co. ltd, tokyo, japan). ph was measured with a ph meter. ta was determined by titrating to ph 8.2 using 0.1 mol/l naoh after appropriate dilution (aoac, 2000). determination of vitamin c ascorbic acid was measured by hplc as described by helsper et al. (2003). briefly, vitamin c was extracted by sonication of 0.5 g of wet frozen powder in 2 ml of ice cold water with 5% metaphosphoric acid and 1 mm dtpa, followed by centrifugation at 2500 rpm for 10 min, filtering, and immediate analysis on an hplc system. quantification was made through a standard calibration curve prepared by running standard concentrations of vitamin c prepared similarly and measured in duplicate at the beginning and end of the analysis. results are expressed as mg of vitamin c per g of fw (tulipani et al., 2008). statistical analyses data were analyzed by the general linear model anova by minitab® release 13.2 (minitab inc.). following anova, treatment means were compared using the lsd test at p = 0.05. statistical procedures were performed using the pc sas software package. results the results showed under g 1 nutrient solution (first group) the fruit regarded indexes that affect its aroma and taste are in acceptable level, however when we used modified nutritional solution for the other groups (g 2 -g 6 ), increasing levels of some of the characters was detectable. the highest level of tss and vitamin c observed in group four that had higher percentage of fe, n, p, s, mn, cu and k against the group that treated with g 1 nutrient solution (first group). fruits produced under treatment g 3 (group3) that received higher amount of fe, s, mg, zn, n and k than the other groups exhibited the highest level of ta, whereas levels of tss and vitamin c were almost similar to group g 4 . fruits treated with modified nutrient solution number 5 that contained lower level of fe, n, p, s, mn, cu and k against normal nutrient solution, showed the highest level of anthocyanin and the highest amount of ph was detectable in fruits treated with modified nutrient solutions g 6 and g 2 that received higher level of n,ca,p,k,s,mn,cu and mo and fe, zn, b,s,n,k,mg respectively (table 2). table 1 analysis of variance for strawberry phytochemical characters under different nutrient solutions. treatment df phythochemical characters tss ta vitamin total anthocyanin ph (brix) (mg/100 g f.w.) c(mg/g f.w.) (mg/100 g f.w.) nutrient solution 5 2.957* 0.0024* 0.0306* 7527570.5** 0.045** error 15 0.944 0.0008 0.0084 435379.3 0.006 cv 10.1 10.9 8.3 26.6 1.9 **,*, ns and cv, significant at 1, 5% level of probability, non-significant and coefficient of variation, respectively. table 2 effect of different concentration of nutrient solution on strawberry phytochemical characters. treatment phythochemical characters ph tss ta vitamin c total anthocyanin (brix) (mg/100 gf.w.) (mg/g f.w.) (mg/100 g f.w.) g1 10.33ab 0.28b 1.03ab 3997.3ab 3.77b g2 9.00b 0.26b 1.10ab 3744.3b 4.02a g3 9.67ab 0.32a 1.10ab 2343.0c 3.85b g4 11.00a 0.24b 1.26a 2067.3c 3.81b g5 9.17b 0.25b 1.13ab 4996.8a 3.85b g6 9.00b 0.26b 1.20ab 1378.1c 4.06a 4 ital. j. food sci., vol. 27 2015 analysis of variance showed that interaction between nutritional elements of nutrient solution supplied with nutritional characters of the fruits was significant; tss, ta and vitamin c in 5% level and anthocyanin and ph in 1% level of probability (table 1). nutrient solution treatment with different ratio chemical nutritional elements showed significantly influence on total soluble solids (tss), total acidity (ta), vitamin c (p>0.05), anthocyanin and ph (p<0.001). discussion considering the results of the present study using a modified nutrient improved fruit nutritional characters but it was not unique; different nutritional formula showed different effect on each index. however often characters increased following enhanced nutritional elements (micro and macro), but anthocyanin decreased with increasing some nutritional elements (micro and macro) (table 2). seyyedi (2005) studied the effect of four kinds of nutrient solution in hydroculture system on the quantitative and qualitative traits of silva strawberry. he showed that by increasing potassium up to 3meq/l in nutrient solution, the soluble solid material increases. farzaneh et al. (2009) studied the effect of different nitrogen and potassium levels on yield and density of nitrogen and potassium of tomato leaf in perlite environment and reported that the most yield of fruit was gained with 200 mg/l nitrogen consumption, and higher levels of nitrogen reduced the yield and different levels of potassium did not have any significant effect on the yield. hartz et al. (1999) studied different levels of potassium on the quality of muskmelon. they found that 240 mg/l potassium level caused a significant increase in total sugar, tss, glutamic acid, aspartic acid and acetate volatile components in fruit flesh, which have an effect on its taste and flavor. mashhadi-jafarloo et al. (2009) showed that the most strawberry yield was obtained in 100% coconut medium and cocopeat + perlite (50% + 50%) placed in the next stages (ebrahimi et al., 2012; aniel et al., 2007). according previous studies the result showed that increasing nutritional element until a definite level has positive effect on the fruit nutritional characters. whereas fruits treated with nutrient solution formula exhibited significant effect on nutritional characters in fruit quality, were not the highest level but almost all of them were in limited amount. however with using of modified nutrient solution, some of the indexes decreased though some of them increased, so it seems that under a usual condition and using g 1 nutrient solution we have strawberries with satisfying aroma and taste. when we used a growing medium containing 50% perlit and 50% coco peat treated with g 1 nutrient solution formula, produced strawberries with more acceptable nutritional characters comparing the fruits that produced under other nutrient solution. however in these conditions some nutritional characters exhibited to increase but the others decreased. therefore based on the experiment results fruits produced under g 1 conditions are the best. references ameri a., tehranifar a., shoor m. and davarynejad g.h. 2012. effect of substrate and cultivar on growth characteristic of strawberry in soilless culture system, afri. j. biotechnol. 11(56): 11960-11966. aniel j., cantliffe j., castellanos z. and paranjpe, a.v. 2007. yield and quality of greenhouse-grown strawberries as affected by nitrogen level in coco coir and pine bark media. proc. fla. state hort. soc. 120:157-161. aoac. 2000. “official method of analysis” 17th ed. gaithersburg, md, usa: association of official analytical chemists, no. 967.21. arzani m. 2007. “cultivation without soil (hydroponic), commercial and house-made”1st ed. isfahan industrial press, isfahan behnamiyan m. and masiha s. 2002. “strawberry” 1 st ed. sotoudeh press, iran djedidi m., gerasopoulos d. and maloupa, e. 1999. the effect of different substrates on the quality of f. carmello tomatoes (lycopersicom esculentum mill.) grown under protection in a hydroponic system. cahier option mediterranneenes, 31: 379-383. ebrahimi r., souri m.k., ebrahimi f. and ahmadizadeh m. 2012. effect of different substrates on herbaceous pigments and chlorophyll amount of strawberry in hydroponic cultivation system american-eurasian, j. agric. & environ. sci., 12 (2): 154-158. farzaneh n., golchin a. and hashemi m. 2009. the effect of different levels of nitrogen and complement potassium of nutrient solutions on performance and the concentration of nitrogen potassium of tomato leaves. presented at 1st national congress of hydroponics and greenhouse production. isfahan university of echology, iran. giust, m.m. and wrolstad r.e. characterization and measurement of anthocyanins by uv-visible spectroscopy. curr.protocols food anal. chem. 2001, doi 10.1002/ 047112913. faf0102s00 hannum s.m. 2004 potential impact of strawberries on human health: a review of the science. crit rev food sci nutr. 44: 1-17. hartz h.k., miyao g. and mullen r.j. 1999. potassium requirements for maximum yield and fruit quality of processing tomato. j. am. soc. hort. sci., 124(2): 199-204. helsper j.p.f.g., de vos c.h.r., maas f.m., jonker h.h., van den broeck, h.c., jordi w., pot c.s., keizer l.c.p. and schapendonk a.h.c.m. 2003. response of selected antioxidants and pigments in tissues of rosa hybrida and fuchsia hybrida to supplemental uv-a exposure. physiol. planta, 117: 171-187. kerej c., voogt w. and bass r. 1999.nutrition solution and water quality for soilless cultures. brochure of research station for floriculture and glasshouse vegetables.netherland. ilgin m., colak a. and kaska n. 2006. effects of the different growing media on the yield and quality of some strawberry (fragaria x ananassa) cultivars. j. biologi. sci. 6 (3): 501-506. johnson j.r., hochmuth g.j. and maynard d.n. 2010. soilless culture of greenhouse vegetables. institute of food and agricultural sciences. university of florida, 218: 19-22. lieten f., longuesserre j., baruzzi g., lopezmedina j., navatel j.c., krueger e., matala v. and paroussi g. 2004. ital. j. food sci., vol. 27 2015 5 paper received february 27, 2014 accepted june 9, 2014 recent situation of strawberry substrate culture in europe. acta hortic. 649: 193-196. mashhadijafarloo a., naseri l., samadi a. and hanare m. 2009. determination of circulation and the suitable cultivation bed in hydroponic cultivation system of selva strawberry. presented at 1 st national congress of hydroponics and greenhouse production, isfahan university of technology, iran. noguera p., abad m., puchades r., maquieira a. and noguera v. 2003. influence of particle size on physical and chemical properties of coconut coir dust as container medium. soil sci. plant anal. 34:593-605. seyyedi a., 2005. the effect of nutrient solution’s potassium and density of cultivation on the quality and quantity of selva strawberry in hydroponic cultivation system. m.sc. theses on plant protection, tehran university, iran. tabatabaei j. and mohammadrezaei r. 2006. the effect of different substrate cultivation on the growth and performance of greenhouse cucumber in watery cultivation system (hydroponic). j. agric. sci. 16(2): 35-34. tulipani,s., mezzetti b., capocasa f., bompadre s. and beekwilder j. 2008.antioxidants, phenolic compounds, and nutritional quality of different strawberry genotypes. j. agric. food chem. 56: 696-704. tulipani s., mezzetti b. and battino m. 2009. impact of strawberries on human health: insight into marginally discussed bioactive compounds for the mediterranean diet, public health nutrition, 12(9a): 1656-1662 yuan l.p., jaj e.h. and jonathan p.l. 1996. marigold growth and phosphorus leaching in a soilless medium amended with phosphorus charged alumina. j. hortic. sci., 31: 94-98. paper ital. j. food sci., vol. 27 2015 459 keywords: eggplant, air drying, infrared determination of drying characteristics and quality properties of eggplant in different drying conditions gözde bayraktaroğlu urun1*, ünal riza yaman2 and ergun köse1 1department of food engineering, faculty of engineering, celal bayar university, muradiye, manisa, turkey 2department of fruit-vegetable processing and engineering tire-kutsan vocational training school, ege university, tire, i̇zmir, turkey *corresponding author: gozdebayraktarurun@yahoo.com abstract drying is the most traditional process used for preserving eggplant a long time. the aim of this study was to determining drying characteristics and quality properties of eggplant dried by sun drying, hot air convective drying and infrared assisted convective drying. convective drying and infrared assisted convective were carried out in a convective dryer at three different temperatures (40°, 50°, 60°c) and air velocity at 5 m/s. the increasing of temperatures during the drying of eggplant led to a significant reduction of the drying time. however loss of nutrition was observed in eggplant samples dried at higher temperature. the biggest change in colour parameters was observed in samples dried with sun drying. so it was thought that sun drying had a negative effect on quality properties of eggplant samples. mailto:gozdebayraktarurun%40yahoo.com?subject= 460 ital. j. food sci., vol. 27 2015 introduction eggplant (solanum melongena l.) is a common annual vegetable crop grown in the subtropics and tropics (concellon, 2012). eggplant is an important market vegetable of asian and mediterranean countries and has a very limited shelf life for freshness (wu et al., 2007; boulekbache-makhlouf et al., 2013). its shelf-life at temperature of 10–15°c is about 10 days (hu et al. 2010). the limited shelf-life constitutes a heavy drawback for commercial purpose (brasiello et al., 2013). drying which is the process of removal of most of the moisture present in the food is the oldest preservation method applied since ancient times (ayhan and ali̇baş, 2005; er and akbulut, 2011; ali̇baş, 2012). the removal of moisture from the food materials prevents the growth and reproduction of spoilage microorganisms, slows down the action of enzymes and minimizes many of the physical and chemical reactions (ceylan et al., 2006; wu et al., 2007; guine et al., 2012a). nowadays drying process of product is carried out by various methods such as sun drying, contact, convective, radiation, dielectric, vacuum, freeze drying and osmotic drying (karabayir, 2006). natural sun drying is practiced widely in the world and also turkey, but has some problems related to the contamination by dirt and dust and infestation by insects, rodents and other animals (kocabiyik and demirtürk, 2008). therefore, the convective drying process carried out in closed equipments is preferred (ertekin and yaldiz, 2004). convective drying is the most traditional dehydration method used to preserve foods; it mainly consists of forcing air through the product to be dried. the surface area of the product to be dried, the drying time, drying temperature, air velocity, moisture content of air and atmospheric pressure determine drying efficiency (cemeroğlu, 2004). convective drying processing effectively extends the shelf life of agricultural products, however this drying process involves chemical, physical, structur al and nutritional changes, linked to the water loss and the high temperatures applied, which affect the product quality (garcia-perez et al., 2012). loss of sensory and nutritive qualities is considered inevitable during traditional drying process due to the undesirable textural and biochemical changes (wu et al., 2007). the expansion of dehydrated food market demands high quality products that maintain at a very high level the nutritional and sensorial properties of the initial fresh product (russo et al., 2013). infrared radiation has significant advantages over conventional drying. these advantages are higher drying rate, energy saving, and uniform temperature distribution giving a better quality product. at present, many driers use infrared radiator to improve drying efficiency, save space and provide clean working environment, etc. therefore infrared drying can become popular as an energy saving drying method (wang and sheng, 2006). drying times of carrot pomace dried at the infrared power levels of 83, 125, 167 and 209 w were studied. according to the results, it was determined that drying rate increased and drying time decreased with increasing infrared power level (doymaz, 2013). the effect of harvest time and drying techniques on the quality characteristics, which are specifically important for maize crop were investigated. energy expenses of the drying techniques were calculated for all harvest periods and it was found out that the expenses to reduce the moisture level from 15 to 13% with hot air drying are higher than the expenses to reduce moisture level from 29 to 13% with infrared-hot air drying combination (yilmaz and tunçel, 2008). abdelmotaleb et al. (2009) investigated thin layer drying of garlic slices under convection and combined infrared–convection heating modes and observed increases in drying rate, thermal efficiency, rehydration ratio, flavor strength and colour difference and decreases in drying time and specific energy consumption for the combined (infrared–convection) heating mode in comparison with convection only. effects of infrared power, air temperature and air velocity on drying rate and quality of onion slices dried in infrared-convective dryer were studied. it was found that drying time of onion slices increased with increasing air velocity, and decreased with increasing temperature and infrared power (sharma et al., 2005). the objectives of this study were to investigate the drying characteristics of the eggplant samples, to examine the effect of drying conditions on the drying process, and to choose optimum drying method for quality of dried eggplant samples. materials and methods sample preparation fresh eggplants were obtained from öcal agricultural product limited company (turgutlu, manisa, turkey). vegetables were washed and sliced (30 mm diameter and 6 mm thickness). eggplant slices were placed over a metal grating in a convective oven operating at constant temperature (russo et al., 2013). drying experiments eggplant slices were subjected to drying with three methods. these drying methods were: ital. j. food sci., vol. 27 2015 461 sun drying, hot air convective drying and infrared assisted convective drying. convective drying and infrared assisted convective drying of eggplants were carried out in a drying system consisting of solar collector, carbon fiber infrared heaters, drying chamber, condenser, heat exchanger, hot water tank, and plc (programmable logic controller) panel at three different temperatures (40°, 50°, 60°c) and air velocity of 5 m/s. drying kinetics in our study drying time was defined as the time passing from initial moisture content of the samples until final moisture content of samples. drying rate was described as the amount of water removed from the sample per unit of time. effect of temperature on drying time and drying rate of eggplant samples was determined (nasiroğlu and kocabiyik, 2007). specific energy consumption specific energy consumption is amount of energy required for removing unit amount of water from samples during drying of samples. specific energy consumption of eggplant samples dried in different conditions was calculated as follows eq. (1): e s = e t /w r (1) e s : specific energy consumption (mj/kg), e t : total energy (mj), w r : amount of water removed during drying (kg) (sharma and prasad, 2006). shrinkage shrinkage, which occurred during drying as a result of water evaporation, was evaluated by determination of the relative volume of dried material. the relative volume was the ratio of eggplant slices volume after drying to that before drying as follows eq. (2): v s = v/v 0 (2) v s = shrinkage v = volume of dried samples v 0 = volume of fresh samples (figiel, 2010). rehydration rehydration kinetics study was carried out for dried eggplant slices. the samples were placed in water at 45°c and waited for 5 h. the rehydrated samples were spread on absorbent paper for the removal of free water on the surface of vegetable. the change in weight was recorded after a regular interval of time. the rehydration capacity was calculated from the ratio of sample weight after and before the rehydration as follows eq. (3): r r = m r /m d (3) r r = rehydration ratio m r = weight of rehydrated samples m d = weight of dried samples (russo et al., 2013). colour parameters colour of dried and fresh samples was evaluated by means of a minolta chroma meter cr-300 (minolta co. ltd., osaka, japan). instrumental colour data were expressed as cie l*, a*, b* coordinates, which define the colour in a threedimensional space: l* (dark–light), a* (redness– green) and b* (yellowness–blueness). total colour difference (∆e), chroma (c), hue angle (h) and r (a/b) values were calculated by using l*, a*, b* values in eqs. (4)-(7). eggplant slices were placed in container without space. colour measurements were performed twice (demir and akbulut 2010; nasiroğlu and kocabiyik, 2007). (4) (5) (6) (7) textural properties for determining the textural properties of fresh and rehydrated eggplant slices, texture profile analysis (tpa) was performed using a texture analyser (model ta.xt.plus). the texture profile analysis was carried out by two compression cycles between parallel plates performed on cylindrical samples (diameter 10 mm, height 3 mm) using a flat 35 mm diameter plunger, with a 5 s of time between cycles. the parameters that have been used were the following: 50 kg force load cell and 0.5 mm s−1 test speed (nayak et al., 2007; gui̇ne and barroca, 2012b; russo et al., 2013). the textural properties: hardness, springiness, cohesiveness, gumminess and chewiness were calculated after eqs. (8)-(12): hardness, h=f1 (8) springiness, s=∆t2/∆t1 (9) cohesiveness, c=a2/a1 (10) gumminess, g=h x c (11) chewiness=h x s x c (12) 462 ital. j. food sci., vol. 27 2015 total dry matter dry matter of dried and fresh samples was determined by drying the samples cut into small pieces at 105°c to constant weight. total dry matter content of the samples was calculated from the difference in mass before and after the drying process (cemeroğlu, 2007; öztürk and çapur, 2010). water activity water activity measurement set was used for determination of water activity values of all the samples. in this system, the product to measured water activity was cut into small pieces, placed in a hermetic steel chamber. when humidity of air inside the container reached equilibrium with the product, equilibrium moisture content of samples was measured by probe in the container (hastürk-şahi̇n and ülger, 2010). ascorbic acid (vitamin c) a spectrophotometric method was used to determine the total amount of vitamin c in the eggplant slices. the absorbance value of samples was measured by means of a uv-visible spectrophotometer (shimadzu corp., kyoto, japan) with wavelength at 518 nm. ascorbic acid of samples were calculated from standard curve showed absorbance values to concentrations of ascorbic acid and expressed as microgram of ascorbic acid per 100 gram of sample (hişil, 2010). statistical analysis in our study drying methods in different conditions were designed as applications and a completely randomized design was used for statistical analysis. these applications were sun drying, hot air convective drying (40°, 50°, 60°c) and infrared assisted convective drying (40, 50, 60°c). effect of different drying methods and drying conditions on drying characteristics, chemical, physical and textural properties of eggplant samples was determined. number of replication was two. in order to determine the differences between applications, analysis of variance (anova) was carried out using statistical analysis software (sas, 2001). data found important in result of anova were evaluated with proc mixed procedure. for every data lsmeans values were determined and least significant differences (lsd) between data were calculated. results and discussion drying kinetics of eggplant samples in our project it was observed that drying method affected drying time of eggplant slices. drying time of infrared assisted convective drying and convective drying was shorter than drying time of sun drying. infrared application decreased drying time of eggplant samples during convective drying at air temperature of 50° and 60°c. also umesh-hebbar et al. (2004) reported that the combined infrared and hot air dryer reduced the processing time dramatically (48%), in addition to consuming less energy (63%) for water evaporation compared to hot air drying. in convective drying, drying time of the eggplant samples showed reduction with increasing temperature. however drying rate of eggplant samples increased at higher temperature (fig. 1). similarly the effect of temperature on the drying kinetics and quality attributes of apple (var. granny smith) slices during drying was investigated and the experimental results of study showed that dehydration were faster fig. 1 drying curves of eggplant samples dried with convective drying at three different temperatures. ital. j. food sci., vol. 27 2015 463 when air temperature increased (vega-galvez et al., 2012). effect of air temperature on drying time of cornelian cherry fruits dried in convective dryer was investigated, it was observed that increasing air temperature reduced drying time by 34% (kaya and aydin, 2008). similar result were described by ertekin and yaldiz (2004) working about drying characteristics of eggplants dried using heated ambient at air temperatures from 30° to 70°c and it was stated that drying time decreased with increasing drying air temperature. during the infrared assisted convective drying, drying time of the eggplant samples showed reduction with increasing temperature. however drying rate of eggplant samples increased at higher temperature (fig. 2). toğrul et al. (2005) studied drying characteristics of banana slices dried in infrared dryer at drying temperature ranging from 50° to 80°c and it was found that drying rate increased with increasing drying temperature. when eggplant samples were dried with sun drying, it was determined that sun drying took a longer time than convective drying and infrared assisted convective drying (fig. 3). fig. 2 drying curves of eggplant samples dried with infrared-convective drying at three different temperatures. fig. 3 drying curves of eggplant samples dried with sun drying. 464 ital. j. food sci., vol. 27 2015 table 1 specific energy consumption values of dried eggplant samples (kj/kg). drying methods eggplant cd (40°c) 0,0023±0,001 cd (50°c) 0,0040±0,004 cd (60°c) 0,0033±0,000 icd (40°c) 0,0018±0,001 icd (50°c) 0,0036±0,001 icd (60°c) 0,0038±0,0001 sd 0±0,000 p=0.3071 cd: convective drying, icd: infrared assisted convective drying, sd: sun drying. table 2 rehydration ratios of dried eggplant samples. drying methods eggplant cd (40°c) 5,27±0,23c cd (50°c) 5,71±0,26cb cd (60°c) 5,81±0,35ab icd (40°c) 5,42±0,07cb icd (50°c) 5,92±0,10ab icd (60°c) 6,33±0,08a sd 5,50±0,31cb p=0.0283 lsd=0.539 fig. 4 change of rehydration for eggplant samples dried at different conditions. specific energy consumption of dried eggplant samples in our study amount of energy required for drying of samples was calculated. specific energy consumptions of dried eggplant samples are showed in table 1. as a result of statistical analysis, it was determined that there were not important differences between specific energy consumption values of dried eggplant samples at different drying methods (p>0.05). the lowest specific energy consumption of eggplant slices was measured during infrared assisted convective drying at air temperature of 40°c. according to results obtained from previous study, specific energy for drying of mushroom slices in a hot air flow-infrared combination dryer increased with increasing temperature, while the specific energy for mushroom drying in the convection dryer decreased with increasing temperature (mi̇naei̇ et al., 2011). rehydration ratio of dried eggplant samples in our project rehydration ratio values of eggplant samples dried with different drying methods were determined. data obtained as a result of analysis were given in table 2. increasing air temperature increased rehydration ratio values of eggplant samples. similarly russo et al. (2013) did scientific study about dried and rehydrated eggplant and state that samples dried at higher temperature showed faster water uptake during rehydration because of wrinkled structure. it was determined that drying methods had a significant effect on rehydration ratio values of dried eggplant samples (p<0.05). especially it was observed that there was important difference between the rehydration ratio values of eggplant samples dried with convective drying at 40°c and the rehydration ratio values of eggplant samples dried with infrared assisted convective drying at 60°c. change in rehydration ratio of dried eggplant samples was given in fig. 4. rehydration ratio of dried eggplant samples reached to maximum value in five hour. drying method did not affect rehydration time of dried eggplant samples. ital. j. food sci., vol. 27 2015 465 table 3 shrinkage of dried eggplant samples. drying methods eggplant cd (40°c) 0,225±0,05 cd (50°c) 0,239±0,02 cd (60°c) 0,278±0,024 icd (40°c) 0,217±0,032 icd (50°c) 0,254±0,076 icd (60°c) 0,271±0,012 sd 0,230±0,034 p=0.6868 shrinkage of dried eggplant samples shrinkage values of dried eggplant were determined with analysis and showed in table 3. increasing of air temperature in convective dryer caused increasing of shrinkage values of eggplant slice. as a result of statistical analysis it was found that drying conditions did not have an important effect on shrinkage values of dried eggplant samples (p>0.05). however lewicki and jakubczyk (2004) investigated mechanical properties of apples dried at drying temperature ranging from 50° to 80°c in a laboratory convection dryer and found that the increasing drying temperature caused the gradual decrease of shrinkage values. chemical properties of eggplant samples in our project total dry matter, water activity, ascorbic acid and loss of ascorbic acid of eggplant samples were detected. these data were showed in table 4. eggplant samples were dried to 90% dry matter content. the highest ascorbic acid loss in dried samples was determined in eggplant samples dried with infrared assisted convective drying at 60°c. the lowest ascorbic acid loss was observed in samples dried with sun drying. effect of different drying methods on chemical properties of eggplant samples was examined as statistical, it was determined that drying methods had a significant effect on total dry matter, water activity, ascorbic acid and loss of ascorbic acid of eggplant samples (p<0.05). textural properties of eggplant samples in our thesis project hardness, springiness, cohesiveness, gumminess and chewiness values of fresh and rehydrated samples were measured for determining textural properties of eggplant samples. textural properties of eggplant samples were given in table 5. as a result of statistical examination of textural properties of rehydrated samples, rehydration process was found important in terms of hardtable 4 chemical properties of eggplant samples. samples total dry matter water activity ascorbic acid loss of ascorbic (mg/100g) acid (%) fresh 8,81±0,516a 0,991±0,010a 16,22±0,08a cd (40°c) 90,73±2,520b 0,594±0,013b 12,36±0,625bc 23,76±4,20cd cd (50°c) 92,12±0,177b 0,501±0,026d 11,44±0,577c 29,46±3,24bc cd (60°c) 90,25±1,582b 0,555±0,033bcd 9,74±0,481d 39,92±3,25ab icd (40°c) 90,27±1,117b 0,568±0,002bc 12,53±0,577bc 22,73±3,21cd icd (50°c) 91,43±4,582b 0,584±0,040bc 11,37±0,866c 29,86±5,03bc icd (60°c) 89,21±4,509b 0,591±0,050b 9,54±0,962d 41,18±5,65a sd 87,75±0,080b 0,522±0,005cd 13,76±0,962b 15,18±5,57d p<0.0001 p<0.0001 p=0.0002 p=0.0053 lsd=5.864 lsd=0.064 lsd=1.6135 lsd=10.465 table 5 textural properties of fresh and rehydrated eggplant samples. samples hardness (n) springiness cohesiveness gumminess (n) chewiness (n) fresh 148,8±2,73a 0,670±0,026 0,577±0,005e 85,90±2,28a 57,17±0,160a rcd (40°c) 50,78±8,23b 1,877±1,428 0,708±0,023dc 36,00±4,29b 32,12±2,56b rcd (50°c) 47,27±3,88b 0,816±0,003 0,693±0,019d 32,81±3,47b 27,49±3,56b rcd (60°c) 44,65±7,71b 1,895±1,465 0,705±0,017dc 31,48±6,02b 27,19±4,28b ricd (40°c) 52,59±3,67b 1,331±0,792 0,754±0,043ab 39,22±0,814b 32,39±2,13b ricd (50°c) 47,77±9,51b 0,878±0,084 0,686±0,010d 32,55±5,77b 28,77±7,84b ricd (60°c) 42,08±8,68b 0,889±0,013 0,747±0,016abc 31,21±7,27b 27,69±5,71b rsd 41,63±7,04b 0,881±0,033 0,774±0,009a 32,26±5,10b 28,48±3,56b p<0.0001 p=0.6138 p=0.0003 p<0.0001 p=0.0015 lsd=15.879 lsd=0.048 lsd=11.07 lsd=9.9053 rcd: rehydrated convective drying. 466 ital. j. food sci., vol. 27 2015 ness, cohesiveness, gumminess and chewiness values of samples (p<0.05). however it was determined that there was not an important difference between the springiness values of eggplant samples (p>0.05). results obtained from research indicated that hardness, gumminess and chewiness values of rehydrated samples were smaller than those of fresh samples. vega-galvez et al. (2008) studied with red pepper samples (capsicum annuum l.) dried at four air inlet temperatures from 50° to 80°c and rehydrated in water at 30°c and found that firmness was significantly affected by the temperature used during drying. colour parameters of eggplant samples in our project l*, a*, b* values of fresh and dried eggplant samples were measured during determining colour parameters of samples. chroma, hue angle, r(a/b) and total colour difference (∆e) values were calculated by using l*, a*, b* values of samples. colour parameters of samples were showed in table 6. as a result of statistical examination, it found that drying method did not affect to a* and ∆e values of eggplant samples (p>0.05), however it was determined that there were significant difference between l*, b*, chroma, hue angle, r(a/b) values of samples (p<0.05). so it was concluded that drying process had a significant effect on colour parameters of fresh samples. ertekin and yaldiz (2004) also investigated the effect of drying air temperature on colour parameters of the eggplant samples and suggested that increasing drying air temperature decreased the colour lightness and raised the saturation. from the results of the present work it was concluded that drying process increased a*, r(a/b) values of eggplant samples, while decreased l*, b*, chroma and hue angle values of eggplant samples. the biggest change in colour parameters was observed in samples dried with sun drying during the examination of colour parameters of fresh and dried eggplant samples. therefore it was possible to determined that sun drying had a negative effect on quality properties of eggplant samples. table 6 colour parameters of eggplant samples. samples l* a* b* chroma hue angle r (a/b) δe fresh 61,64±1,23a 3,74±0,02 19,55±0,648a 19,91±0,636a 79,27±0,197a 0,190±0,004b cd (40°c) 50,10±0,108b 6,97±1,72 17,96±0,616ab 19,91±0,308a 69,69±4,98b 0,378±0,107a 12,53±2,12 cd (50°c) 47,52±2,52bc 7,20±0,101 16,98±0,062bc 18,45±0,015ab 66,95±0,372b 0,426±0,008a 14,84±1,37 cd (60°c) 47,85±2,28bc 6,09±0,318 15,45±0,847c 16,61±0,670c 68,48±2,16b 0,395±0,044a 14,72±1,34 icd (40°c) 47,81±0,986bc 5,67±1,02 15,31±0,554c 16,34±0,872c 69,77±2,62b 0,369±0,052a 14,26±0,522 icd (50°c) 47,91±1,64bc 6,48±0,621 17,87±1,37ab 19,01±1,50ab 70,05±0,325b 0,363±0,006a 13,93±2,40 icd (60°c) 50,17±1,00b 5,30±0,477 17,04±1,25bc 17,86±1,05bc 72,58±2,66b 0,315±0,051a 12,10±0,055 sd 44,77±4,01c 5,85±1,03 16,79±0,268bc 17,80±0,082bc 70,79±3,41b 0,349±0,067a 17,27±5,16 p=0.001 p=0.0591 p=0.0126 p=0.0121 p=0.0343 p=0.0491 p=0.496 lsd=4.7391 lsd=1.8826 lsd=1.832 lsd=6.0647 lsd=0.1246 conclusions in our thesis project, the drying characteristics of the eggplant slices dried by sun drying, hot air convective drying (40°, 50°, 60°c) and infrared assisted convective drying (40°, 50°, 60°c) were studied. air temperature in a convective dryer affected drying time of eggplant slices. increasing drying air temperature decreased drying time and increased drying rate. ascorbic acid value of eggplant samples dried by sun drying was quite high. however quality losses were observed in eggplant samples dried by sun drying. convective drying at low temperature should be applied to maintain ascorbic acid content and quality of eggplant slices. references abdelmotaleb a., el-kholy m.m., abou-el-hana n.h. and younis m.a. 2009. thin layer drying of garlic slices using convection and combined (convection infrared) heating modes. misr j. ag. eng. 26(1): 251-281. alibaş i̇. 2012. microwave drying of grapevine (vitis vinifera l.) leaves and determination of some quality parameters. tarım bilimleri dergisi-journal of agricultural sciences 18: 43-53. ayhan a. and alibaş k. 2005. determination of dehydration parameters of some agricultural products dehydrated by vacuum. master’s thesis uludağ üniversitesi, bursa. boulekbache-makhlouf l., medouni l., medouni-adrar s., arkoub l. and madani k. 2013. effect of solvents extraction on phenolic 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microstructural changes in eggplant (solanum melongena l.) during conventional and ultrasonically assisted convective drying. food and bioproducts processing 90: 624-632. russo p., adiletta g. and di matteo m. 2013. the influence of drying air temperature on the physical properties of dried and rehydrated eggplant. food and bioproduct processing 91: 249-256. sas 2001. sas user’s guide cary nc : sas institute, inc. sharma g.p., verma r.c. and pathare p.b. 2005. thin-layer infrared radiation drying of onion slices. journal of food engineering 67: 361-366. sharma g.p. and prasad s. 2006. specific energy consumption in microwave drying of garlic cloves. enerji 31: 1921-1926. toğrul h., toğrul i̇. and i̇spir a. 2005. examination of drying kinetics of banana in infrared dryer. iii.tarımsal ürünleri kurutma çalıştayı, antalya. umesh-hebbar h., vishwanathan k.h. and ramesh m.n. 2004. development of combined infrared and hot air dryer for vegetables. journal of food engineering 65: 557–563. 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january 27, 2015 #623_rolle_bozza ital. j. food sci., vol 29, 2017 243 paper changes in texture analysis parameters of wine grape berries at two ripeness stages: a study on varietal effect l. brillantea,b, f. gaiottia, l. lovata, s. giacosac, s. río segadec, s. vincenzid, f. torchioe, l. rollec* and d. tomasia acrea-vit council for agricultural research and economics, viticulture research center, conegliano, tv, italy buniversity of california davis, department of viticulture and enology, davis, ca, usa cuniversity of turin, dipartimento di scienze agrarie, forestali e alimentari, grugliasco, to, italy duniversity of padova, centro interdipartimentale per la ricerca in viticoltura ed enologia, legnaro, pd, italy euniversità cattolica del sacro cuore, istituto di enologia e ingegneria agro-alimentare, piacenza, italy *corresponding author. tel.: +39 0116708558; fax: +390116708749 e-mail address: luca.rolle@unito.it abstract ripening of grapes is associated with great modifications at both the chemical and physical level. the aim of this work was to describe the changes in physical-mechanical parameters associated to ripening of wine grape berries, as evaluated by texture analysis, in order to understand if these modifications are stable across cultivars, or they are cultivar-specific. berries from 21 different cultivars were sorted by flotation in different saline solutions, separated in two ripening stages differentiated by the amount of sugars (183 and 217 g l-1) and then analysed. multivariate and univariate variations in texture analysis parameters were found, which were not constant across the studied grapevine varieties. however, a general behaviour was observed for skin weight, which had the largest variation between the two ripening stages. other parameters showed significant differences between the ripening stages: skin thickness, berry gumminess, chewiness, and springiness, but the variation was not common to all cultivars. the work therefore evidenced the existence of cultivar-specific differences in the behaviour of physicalmechanical parameters between ripening stages. keywords: berry ripening, physical-mechanical properties, texture analysis, skin weight, wine grapes, vitis vinifera l. ital. j. food sci., vol 29, 2017 244 1. introduction ripening of grape berries is a complex process, which happens according to a double sigmoid growth curve composed of three different phases (coombe, 1992). during the first period of growth, cell number per berry increases because of mitosis divisions, while cell expansion is limited. generally, at the end of this period the grapevine reaches the phenological stage of bunch closure. this first growth period is followed by a lag phase during which enlargement slows and the seed develops. the phenological stage at the transition between this second and the third final phase is called véraison, which corresponds to the onset of ripening, when berries start to soften and change in colours because of anthocyanin synthesis (in red/black grapes). in the third stage, cells enlarge as a result of solutes (principally glucose and fructose) and water accumulation, and berries approximately double in size (conde et al., 2007). this last step is crucial because important changes in secondary metabolites occur. these compounds are responsible for flavour, aroma, colour and mouth feel of grapes as well as wines. modifications of pectins during this stage cause the progressive loss of firmness in ripe berries (nunan, 1998; nunan et al., 2001). such modifications are principally due to an increase in the enzymatic activity of pectin methylesterase, α-galactosidase and β-galactosidase, which has been registered after véraison (nunan et al., 2001; deytieux-bellau et al., 2008; ortega-regules et al., 2008). however, only in recent years, scientific studies have begun to instrumentally measure these visual and tactile changes, as summarized by rolle et al. (2012). the structure and composition of skin cell walls directly impact textural characteristics and have been linked to phenol extractability (ortega-regules et al., 2006; bindon et al., 2012; hernández-hierro et al., 2014). several studies have shown that the mechanical properties of whole berry and berry skin are significantly related to anthocyanin and flavanol extractability (rolle et al., 2008; río segade et al., 2011a, río segade et al., 2011b). these studies are based on the use of texture analysis (ta) test, which is an effective instrumental texture analysis test for a quantitative evaluation of physical-mechanical characteristics of grape berries (letaief et al., 2008). the technique is rapid and cost-effective since it does not require long times for sample preparation and analysis. however, literature describing the changes in physical-mechanical parameters according to different berry ripening stages is yet scarce and focuses on a very limited number of winegrape varieties (maury et al., 2009; zouid et al., 2010; río segade et al., 2011c; rolle et al., 2011a). this study evaluates, on a heterogeneous dataset from 21 different grapevine cultivars, which texture analysis (ta) parameters change between two different sugar contents (i.e. stages of ripening). in particular, the aims of the work were i) to study if physicalmechanical parameters, as assessed by ta, can significantly vary between the two sampled ripening groups, ii) to evaluate if ta measurements allow to discriminate and classify the two ripening classes, therefore assessing the validity of the methods to describe variations in physical-chemical properties with ripening, and iii) to evaluate differences in physical-mechanical modifications with ripening across cultivars. 2. materials and methods 2.1. plant material and grape sampling vitis vinifera l. grapes from 21 red grapevine varieties were sampled in the crea-vit experimental collection (1.2 ha) located in susegana (tv, veneto, north-east italy), in ital. j. food sci., vol 29, 2017 245 2011. sampled cultivars were ancellotta, barbera, bonarda, cabernet-franc, cannonau, corvina, croatina, franconia, gamay, malbech, malvasia nera di lecce, marzemino, merlot, montepulciano, negramaro, pinot noir, primitivo, raboso, refosco, schiava gentile, and teroldego. vines were 15 years old, grafted on so4 rootstock (interspecific cross between vitis riparia michx. and vitis berlandieri planch.), and planted at 3.0 m between rows and 1.5 m between vines. they were sylvoz pruned and trained with a vertical shoot position system. for each cultivar, samples were composed of about 3 kg of grape berries, which were picked up randomly from ten vines. the berries were sampled at two ripening stages (time lag of two weeks) of difference, and sorted using a densimetric method by berry flotation in different saline solutions (rolle et al., 2011a). the two selected groups, called a (early harvest) and b (full ripeness), had respectively 183±8 and 217±8 g l-1 of sugars corresponding about to 11.0 ± 0.5% and 13.0 ± 0.5% potential alcohol content by volume, respectively. the sorted berries were visually inspected before analysis; those with damaged skins were discarded. for each variety studied, a sub-sample of 36 sorted berries (therefore a total of 756 berries for all cultivars together) was randomly selected for the determination of the physical-mechanical properties. 2.2. physical-mechanical measurements grape berries were singularly weighed (g, bw parameter), with an analytical laboratory balance (radwag as 220/x, radwag, radom, poland), and a texture profile analysis (tpa) non-destructive mechanical test was then performed for each of them as described by letaief et al. (2008). analyses were made using a universal testing machine (utm) taxt2i texture analyzer (stable micro systems, godalming, surrey, uk) equipped with a 5 kg load cell and a hdp/90 platform. a sms p/35 flat cylindrical probe was used, and the test was carried out on each berry in the equatorial position under 25% deformation, with a waiting period of 2 s between the two compressions and a test speed of 1 mm s-1. all force-deformation curves were acquired at 400 hz and evaluated using the texture expert exceed software package (stable micro systems). tpa parameters calculated were berry hardness (n, as h), cohesiveness (adimensional, as co), gumminess (n, as g), springiness (mm, as s), chewiness (mj, as ch) and resilience (adimensional, as r). after the tpa test, each berry was manually peeled with a razor blade, and skin weight (g, as sw) and skin thickness (μm, as spsk) were singularly measured. for the latter test, the same utm texture analyzer was used, equipped with a sms p/2 flat cylindrical probe, and setting a compression test at 0.2 mm s-1 test speed (battista et al., 2015). 2.3. statistical analysis analysis of covariance (ancova) was performed to evaluate the univariate differences in each physical-mechanical parameter between the two ripening classes, treating bw as a nuisance factor. the ta data were also normalised by bw as described in santini et al. (2011), and the resulting data were analysed by one-way anova. robust multivariate analysis of variance (robust manova) was used to investigate multivariate differences for physical-mechanical parameters between the ripening groups. multiple logistic regression was used to understand if texture analysis (ta, including tpa and spsk) measurements can differentiate the two ripening classes, and results were interpreted to assess if relationships between mechanical parameters and ripening classification are stable across cultivars. the choice of the ta parameters in the model (independent variables) was based on an exhaustive search in order to minimise the bayesian information criterion (bic) and the akaike information criterion (aic). finally, ital. j. food sci., vol 29, 2017 246 a cross-validation procedure was used to choose between the minimal aic and bic proposed models. the statistical analysis presented in this work was performed in r v.3.2.0 (r core team, 2015). robust multivariate analysis of variance was performed using the rrcov package (todorov and filzmoser, 2009), and best subset logistic regression with the ‘bestglm’ package (mcleod and xu, 2014). 3. results 3.1. differences in physical-mechanical parameters between ripeness levels to observe differences in physical-mechanical parameters with ripening, the first step was to exclude possible differences in bw between classes because this parameter is correlated to other berry physical-mechanical parameters. as an example, lighter berries have lower s and ch values than heavier ones (r = 0.88 between bw and s, r = 0.44 between bw and ch), but also lower skin weight (sw) (r = 0.76). it would be logical to assume that the ripe a group (early harvest) could have different bw than the ripe b group (full ripeness), for some reason such as sugar accumulation, water accumulation or loss, etc. this hypothesis is rejected by an anova test, which excludes a significant difference in bw means between the two groups (p-value = 0.19). the same results were obtained when performing the anova test individually by cultivars: the difference in bw was not significant (p-value > 0.05) between ripe a and b groups for each cultivar sample in the experiment. analysis was performed by taking into account the multiplicity problem by using the bonferroni correction. however, in order to increase the robustness of the statistical analysis, bw was treated as a nuisance parameter and then an ancova test was performed to adjust, for any treatment, group differences in bw before assessing the impact of the ripening stage. the inclusion of bw in the model permits to exclude the effect of berry weight on the observed differences in physical-mechanical parameters. the test was first performed including the interaction between bw and ripening class in order to assess if the assumption of slope equality is respected, which is necessary to the analysis. because the interaction term was never significant, the assumption was respected and the interaction term excluded. this also means that the relation between bw and all tested mechanical parameters did not depend on the level of ripening. in all cultivars pooled, the only significant difference between ripe a and b berries for all studied physical-mechanical parameters were found in sw (p-value < 5-04). the difference remained significant even after a bonferroni correction accounting for the increase in risk because of the multiple comparisons (p-value < 0.01 after correction). while bw was not different between the two ripening stages considered, a difference in sw exists and berries in the b group had skins 20 mg (approx. 10% over the mean) heavier than those in the a group. the ancova analysis for sw is presented in fig. 1. the analysis was also performed by normalising each ta parameter by bw, as already made in santini et al. (2011). however, even when normalising data by bw, these results were confirmed. when ta parameters were analysed singularly in a univariate way, differences between the classes were too little to be significantly noticed, with the exception of sw. nevertheless, considering the variations in all ta parameters as a whole in a multivariate way (robust manova), significant differences were found in the texture parameters between the two ripening groups, even when sw was removed from the dataset (wilks' λ = 0.82, p-value < 1-05). ital. j. food sci., vol 29, 2017 247 figure 1. results of the ancova with equation sw = bw + mat. the analysis shows differences in sw between the two ripening stage, while controlling for the nuisance effect of bw. 3.2. classifying berry ripening classes using ta parameters because the two ripening classes were significantly different in the berry texture characteristics, the analysis was extended to evaluate if ta parameters were able to discriminate ripe a and b berries. the reason of this analysis was to evaluate the performances of the classification across cultivars, and therefore to know if, at this late stage of ripening, different cultivars show similar changes in ta parameters, or not. if cultivars show similar changes, the performance of the classification should be equal for all cultivars; conversely, if changes in ta parameters are cultivar-specific, the performance should vary across cultivars, with cultivars having greater changes being easier to classify than cultivars with little changes. this was evaluated, in a straightforward and single step approach, using a multiple logistic regression, where the response was the ripening class (which was binary) and the predictors were the physical-mechanical parameters. because the number of ta parameters was large, feature selection was performed to identify the most informative independent variables. two models were selected, the first minimised the bic and just included sw and s as independent variables. both sw and s parameters were significant in the model (p-value < 0.01). this regression model correctly classified 58% of the observations. the second model minimised the aic, and contained as variables the sw, g, ch, and spsk (p-value < 0.01 for g, ch and sw, p-value < 0.1 for spsk); the model correctly classified 62% of the observations. it is worth noting that s is correlated to g and ch (r = 0.61 and 0.38, respectively, p-value < 0.05). this can be a reason why s is excluded in this second model when g and ch are included. the two models were then compared using cross-validation, which suggested the selection of the second model (aic minimised). however, the significant effect of s in the model can also be taken into account for a future discussion. the final logistic regression predicting the probability that an observed sample x belongs to the b group has equation 1: p̂ x( )= e−2.448−0.008spsk+13.205sw−4.243g−1.606ch 1+e−2.448−0.008spsk+13.205sw−4.243g−1.606ch (1) ital. j. food sci., vol 29, 2017 248 where p(x) is the probability of x, e is the natural logarithm base, spsk is skin thickness, sw is skin weight, g is gumminess, ch is chewiness. table 1 shows coefficient estimate, standard error and p-value for the regressions minimising either the aic or the bic. errors do not appear equally spread across all cultivars in the experiment. table 1. coefficients of the logistic regression classifying berries in two ripening classes according to their physical-mechanical characteristics. logistic regression minimising aic (equation 1) estimate std. error p-value intercept -2.448 0.948 9.8e-03 spsk -0.008 0.004 5.5e-02 sw 13.205 3.293 6.1e-05 g -4.243 1.031 3.9e-05 ch -1.606 0.375 1.9e-05 logistic regression minimising bic intercept 3.113 1.277 1.5e-02 s 7.820 2.405 1.1e-03 sw -2.135 0.652 1.1e-03 spsk: skin thickness; sw: skin weight; g: gumminess; ch: chewiness; s: springiness; sw: skin weight. cultivar-specific errors in the classification are presented in table 2 (12 samples of three berries for cultivar). in eight of the 21 cultivars (cannonau-grenache, croatina, franconia, malbech, malvasia nera di lecce, marzemino, merlot, and raboso), the classification is not higher than that attended by chance. table 2. percentage of correctly classified samples for the logistic regression in equation 1 across cultivars. cultivars correctly classified samples (%) cultivars correctly classified samples (%) merlot 50 cabernet-franc 67 raboso 50 corvina 67 cannonau 50 gamay 67 malvasia nera di lecce 50 montepulciano 67 croatina 50 pinot noir 67 franconia 50 ancellotta 75 marzemino 50 refosco 75 malbech 50 negramaro 75 bonarda 58 barbera 83 schiava gentile 58 primitivo 92 teroldego 58 ital. j. food sci., vol 29, 2017 249 therefore, in these cultivars at this stage of ripening, the changes in physical-mechanical parameters do not consistently vary and do not allow to differentiate between the two classes. in all other cultivars, the results of the classification are sensibly better than chance. very good results were reached for primitivo, barbera, ancellotta, negramaro, and refosco (correct classification equal to 92%, 83%, and 75% for the last three cultivars, respectively). in this last group of cultivars, changes in physical-mechanical properties continue even in a late period of ripening. differences in the variation percentage between the two ripening stages are summarised in fig. 2. the well classified cultivars show higher variation between the two stages than the bad classified cultivars; for bad classified cultivars, variation is close to zero excepting for sw. this suggests that the changes of physical-mechanical properties in the last ripening stages are cultivar dependent. figure 2. variation percentage between the two ripening stages for ta parameters and sw. 4. discussion the production of high-quality red wines requires the assessment of grape phenolic ripening indexes through the determination of the content of phenolic compounds and of their extractability during winemaking (río segade et al., 2008). texture analysis has been already used to develop rapid methods for the evaluation of total phenolic content and phenol extractability in grape seeds (rolle et al., 2012), and of anthocyanin extractability in grape skins with a good accuracy (rolle et al., 2008; río segade et al., 2011a; río segade et al., 2011b). the scientific literature is scarce on the description of changes in physical-mechanical parameters, instrumentally measured by ta, in the late stages of ripening, when variation in sugar content is not huge. in fact, several studies suggested that a steady value is achieved close to ripeness for some mechanical parameters, which could limit their choice as ripeness indicators in grape berries (maury et al., 2009). in the present work, this observation is confirmed for a group of the cultivars studied: cannonau-grenache, croatina, franconia, malbech, malvasia nera di lecce, ital. j. food sci., vol 29, 2017 250 marzemino, merlot, and raboso. nevertheless, the observation is not confirmed for other cultivars in this study (primitivo, barbera, ancellotta, negramaro, and refosco), which can be easily classified because physical-mechanical properties still change in the late ripening. the reason of this variation can have a genetic origin, and it deserves to be further investigated in future studies. a variety effect was already found in the relationship between flavonoid content and ta parameters (brillante et al., 2015a; brillante et al., 2015b). variability in physical-mechanical parameters across cultivars can also be related to the climatic conditions of the ripening period, as shown in rolle et al., 2011b. that study showed that while the differences between cultivars for some texture parameters are, at least qualitatively, stable across vintages (an example is fsk), others can also be affected by the climate (an example is wsk). this work carefully treats the bw effect on physical-mechanical parameters. when the bw effect is excluded from the analysis, results for other physical-mechanical characteristics are more reliable. in details, the study evidences an increase in sw with ripening. although some authors showed that the percentage of skin cell wall material decreases during ripening (hernández-hierro et al., 2014) probably due to the cell walls become thinner (ortega-regules et al., 2006), others reported that cell wall material slightly but continuously increases as ripening progresses before decreasing (vicens et al., 2009). this increase in sw, even if modest in absolute terms, can become significant when considered as a ratio of the sw with bw, and is equal to 10%. this is a huge variation, especially if we consider that the accumulation of sugars in berries between the two ripening classes (approx. 34 g) accounts for just the 3% in average of bw. among the mechanical parameters measured by ta, and therefore excluding sw, whole berry characteristics, such as s, g, and ch, were better related to berry ripeness than skin properties. among all tested skin-related properties, spsk was the only one showing a little effect. since the texture properties of the whole berry depend on different characteristics, such as cell wall composition, cell structure and pulp turgescence (goulao and oliveira, 2008), and fruit softening occurring during ripening (nunan 1998), it is not surprising to find larger evidence in the modification of the mechanical properties of the whole berry, as also reported in zouid et al. (2013). in future, it could be interesting to couple ta analysis to the determination of pectins in grape berries. this will probably allow to better understand the direct relations between the physiological activity in grape berries during ripening, molecular structure and the macroscopic modifications of texture. 5. conclusions this work highlights that changes in physical-mechanical properties of grape berries are cultivar-specific in the final ripening stages. however, among all tested physicalmechanical parameters, a general behaviour was shown by skin weight. this parameter showed larger variation with ripening than the others considered. the observed increase in sw is particularly evident once considered its ratio over berry weight, which is equal to 10%. proportionally, between early harvested and full ripe berries, berry weight changed more because of increased skin weight than because of sugar accumulation. differences in sugar content between the two ripening classes accounted for just 3% in average of bw. references battista f., tomasi d., porro d., caicci f., giacosa s. and rolle l. 2015. winegrape berry skin thickness determination: comparison between histological observation and texture analysis determination. ital. j. food sci. 27:136-141. ital. j. food sci., vol 29, 2017 251 bindon k.a., bacic a. and kennedy j.a. 2012. tissue-specific and developmental modifications of grape cell walls influence the adsorption of proanthocyanidins. j. agric. food chem. 60:9249-9260. brillante l., gaiotti f., lovat l., vincenzi s., giacosa s., torchio f., río segade s., rolle l. and tomasi d. 2015a. investigating the use of gradient boosting machine, random forest and their ensemble to predict skin flavonoid content from berry physical-mechanical characteristics in wine grapes. comput. electron. agr. 117:186-193. brillante l.; 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giacosa s. and rolle l. 2011c. instrumental texture analysis parameters as winegrapes varietal markers and ripeness predictors. int. j. food prop. 14:1318-1329. rolle l., torchio f., zeppa g. and gerbi v. 2008. anthocyanin extractability assessment of grape skins by texture analysis. j. int. sci. vigne vin, 42(3):157-162. ital. j. food sci., vol 29, 2017 252 rolle l., río segade s., torchio f., giacosa s., cagnasso e., marengo f. and gerbi v. 2011a. influence of grape density and harvest date on changes in phenolic composition, phenol extractability indices, and instrumental texture properties during ripening. j. agric. food chem. 59:8796-8805. rolle l., gerbi v., schneider a., spanna f., and río segade s. 2011b. varietal relationship between instrumental skin hardness and climate for grapevines (vitis vinifera l.). j. agric. food chem. 2011, 59:10624-10634 rolle l., siret r., río segade s., maury c., gerbi v. and jourjon f. 2012. instrumental texture analysis parameters as markers of table-grapes and winegrape quality: a review. am. j. enol. vitic. 63:11-28. santini d., rolle l., cascio p. and mannini f. 2011. modifications in chemical, physical and mechanical properties of nebbiolo (vitis vinifera l.) grape berries induced by mixed virus infection. s. afr. j. enol. vitic. 32:183-189. todorov v. and filzmoser p. 2009. an object-oriented framework for robust multivariate analysis. j. stat. softw. 32(3):147. vicens a., fournand d., williams p., sidhoum l., moutounet m. and doco t. 2009. changes in polysaccharide and protein composition of cell walls in grape berry skin (cv. shiraz) during ripening and over-ripening. j. agric. food chem. 57:2955-2960. zouid i., siret r., mehinagic e., maury c., chevalier m. and jourjon f. 2010. evolution of grape berries during ripening: investigations into the links between their mechanical properties and the extractability of their skin anthocyanins. j. int. sci. vigne vin. 44:87-99. zouid i., siret r., jourjon f., mehinagic e. and rolle l. 2013. impact of grapes heterogeneity according to sugar level on both physical and mechanical berries properties and their anthocyanins extractability at harvest. j. texture stud. 44:95103. paper received september 25, 2016 accepted november 3, 2016 ijfs#838_bozza ital. j. food sci., vol. 29, 2017 766 short communication phenolic acids, flavonoids, ascorbic acid, β-glucans and antioxidant activity in mexican wild edible mushrooms e. lópez-vázqueza, f. prieto-garcía*a, m. gayosso-canalesb, e.m. otazo sáncheza and j.r. villagómez ibarraa ainstituto de ciencias básicas e ingeniería, universidad autónoma del estado de hidalgo carretera pachucatulancingo km. 4.5, c.p. 42076, pachuca, hidalgo, méxico binstituto de ciencias agropecuarias, universidad autónoma del estado de hidalgo, rancho universitario av. universidad km. 1, ex–hda. de aquetzalpa c.p. 43600, tulancingo, hidalgo, méxico *corresponding author. tel.: +52 7712211289; e-mail address: prietog@uaeh.edu.mx abstract five wild edible mushrooms (amanita caesarea, boletus edulis, cantharellus cibarius, lactarius indigo and ramaria sp.) from mexico were studied for their total phenolic content, antioxidant activity, flavonoids, ascorbic acid, gallic acid, and cinnamic and chlorogenic acids. b. edulis showed the highest contents, expressed in dry weight, of flavonoids (0.92 mg of quercetin equivalents/g), total phenolic acids (8.66 mg of gallic acid equivalents/g), chlorogenic acid (1001.67 μg/g) and cinnamic acid (10.68 μg/g), and it also presented the greatest antioxidant activity (48.6% dpph scavenging activity and 59.48% reductive power) among the species analyzed. overall, the mexican wild edible mushrooms had high concentrations of chlorogenic and ascorbic acids and flavonoids. this study constitutes the first report in mexico about nutraceuticals from wild edible mushrooms. keywords: wild mushrooms, antioxidants, flavonoids, phenolics, β-glucans ital. j. food sci., vol. 29, 2017 767 1. introduction some species of mushrooms are appreciated as food not only for their color, texture, flavor and odor but also as valuable sources of nutrients and nutraceuticals (taofiq et al., 2015; wang et al., 2014). several studies of mushrooms from portugal (heleno et al., 2015a), italy (manzi et al., 2004), poland (nowacka et al., 2014), china (siu et al., 2014), ethiopia (woldegiorgis et al., 2014) and turkey (sarikurcku et al., 2015) have described mushrooms as naturally rich sources of phenolics and flavonoids. these compounds exhibit antioxidant, antibacterial, antiviral, anticarcinogenic and antiinflammatory activities (heleno et al., 2015b; reis et al., 2011; wang et al., 2014). in addition, mushrooms are recognized for having other bioactive compounds that are beneficial for human health such as β-glucans and ascorbic acid (kagimura et al., 2015; ruthes et al., 2015). β-glucans are polysaccharidic compounds that display anticoagulant, antithrombotic, antioxidant, and anti-inflammatory activities. therefore, these compounds play an effective role in the prevention of cardiovascular problems, the reduction of blood cholesterol levels and treatment of illnesses such as various cancers and diabetes (zhu et al., 2015). ascorbic acid is an antioxidant vitamin common in mushrooms (vaz et al., 2011). in spite of the fact that since precolonial times in mexico, the fruiting body of mushrooms has been known as nanacatl (mushroom) from nahuatl, and that a great edible mushroom diversity of approximately 300 species is known in the country (moreno, 2014), there are no reports with respect to the content of phenolic compounds and phenolics acids in mushrooms. therefore, thorough studies of the chemical composition of the wild edible mushrooms are needed to identify and quantify the bioactive compounds. the selection of the mushroom species was based on their appreciation as food in mexico: a. caesarea, b. edulis, c. cibarius, l. indigo and ramaria sp. (moreno, 2014). the aim of this study was to determine the chemical composition of five mexican wild edible mushrooms in terms of flavonoids, phenolic acids, ascorbic acid, β-glucans and antioxidant activity. 2. materials and methods 2.1. samples, standards and reagents the mushroom samples were collected in different regions of hidalgo state in mexico. the regions were los reyes, acaxochitlán (latitude 20.152° and longitude -98.185°, 2199 m a.s.l.), el susto (latitude 20.074° and longitude -98.509°, 2620 m a.s.l.) and paxtepec (latitude 20.033° and longitude -98.426°, 2381 m a.s.l.). the sampling was conducted during the rainy season (august-october) of 2014. the taxonomic identification of the sporocarps was made according to mycology keys (læssøe, 2013). the standards of chlorogenic, cinnamic, gallic, and (l)-ascorbic acid and schizophyllan, the reagents folin-ciocalteu (fc) reagent, 2,2-diphenyl-1-picrilhydrazyl radical (dpph) and 2,6-dichloroindophenol, and the lc solvents methanol 99.8% and acetonitrile 99.9% were all purchased from sigma-aldrich fine chemicals (st. louis, mo, usa). the methanol, hydrochloric acid, phosphoric acid and red congo dye were purchased from j. t. baker (philipsburg, usa). all other chemicals of analytical grade, including sodium hydroxide, metaphosphoric acid, potassium hydroxide, sodium carbonate, citric acid, sodium nitrite, aluminum chloride, ferric chloride, trichloroacetic acid, potassium ferricyanide, sodium phosphate mono and dibasic, were purchased from meyer co (mexico). ital. j. food sci., vol. 29, 2017 768 the fruiting bodies were cleaned with a brush, cut into slices and dried in a drying oven in a range between 40-60°c until a constant weight was achieved. the dried samples were reduced to fine powder (20 mesh) using an electric mill (ika a11 basic) and stored in amber plastic bottles until analysis. 2.2. analysis of antioxidant activity preparation of the extracts. the preparation of the extracts was made using the methodologies proposed by pan et al. (2003) and özyurek et al. (2014) with some modifications. each sample (∼200 mg) was extracted with methanol:water (80:20, v/v; 25 ml), using a conventional microwave oven (900 w, 10% power). the mixtures were irradiated as follows: 7 s power on (heating without superboiling of the solution) and 30 s power off (cooling in an ice bath and mixed on a vortex) then 7 s power on and 30 s power off and so on to reach 5 min of heating time. the obtained extracts were filtered through a filter paper (whatman no. 4), then the filtered volume was adjusted with methanol:water (80:20 v/v) to 25 ml and kept at 4°c in amber bottles until analysis. dpph radical scavenging activity assay. aliquots of 60 µl of extract were mixed with 540 µl of a methanolic dpph radical solution (0.06 mm). the mixture was left to stand for 60 min in the dark. the reduction of the dpph radical was determined by measuring the absorption at 515 nm (genesys 10s vis, thermo scientific, usa). l-ascorbic acid (8 mg/ml) was used as a standard. the radical scavenging activity (rsa) was calculated as a percentage of the dpph pink discoloration using the following equation: % rsa = [(adpph–as)/adpph] x 100 where as is the absorbance of the solution containing the sample, and adpph is the absorbance of the dpph solution (vaz et al., 2011). reducing power. the reducing power was determined according to barros et al. (2011). the methanolic extract (500 µl) was mixed with a sodium phosphate buffer (500 µl, 200 mm, ph 6.6) and potassium ferricyanide (500 µl, 1% w/v). the mixture was incubated at 50-60°c for 20 min, then trichloroacetic acid (500 µl, 10% w/v) was added. an aliquot of the reaction mixture (800 µl) was transferred to vials containing distilled water (800 µl) and ferric chloride (160 µl, 1% w/v). after 90 min, the absorbance was measured at 690 nm (genesys 10s vis spectrophotometer, thermo scientific). the reducing power was obtained as a percentage of the conversion of an orange fe3+ ferricyanide complex to the prussian blue fe2+ferrocyanide form in the presence of reductants (antioxidants from the mushroom extracts). bht (1 mg/ml) was used as a control by measuring its absorbance in the assay and assigning it 100% of the reductive power. total phenolics. the total phenolic content was determined spectrophotometrically with the folin-ciocalteu (fc) test, which is an assay based on the capability of the phenolic compounds in an alkaline solution to reduce a colorimetric reagent composed of a mixture of phosphotungstic and phosphomolibdic acids from mo(vi) to mo(v) to produce a blue color (huang et al., 2002). according to heleno et al. (2015a) 100 µl of methanolic extract were mixed with the fc reagent (750 µl, 10% v/v) for 1 min. after 5 min, a solution of na2co3 (750 µl, 10% w/v) was added to the mixture. the reaction was kept in the dark for 90 min, after which the absorbance was read at 725 nm (genesys 10s vis spectrophotometer, thermo scientific, usa). gallic acid was used to obtain the standard curve (20 600 µg/ml). the total phenolic content is expressed as mg of gallic acid equivalents (gae) per g of dry mushroom. ital. j. food sci., vol. 29, 2017 769 2.3. determination of phenolic compounds by hplc the methanolic extracts were filtered through a 0.45 µm disposable lc filter disk for the hplc analysis. the phenolic acids determination was performed using an agilent 1260 ca, usa series liquid chromatograph equipped with a diode array detector. the separation was carried out using 5 µl of extract on a poroshell 120 ec-c18 agilent ca, usa, column (4.6 x 50 mm, 2.7 µm) thermostated at 25°c. the mobile phase was (a) 0.1% phosphoric acid in water, (b) hplc-grade acetonitrile and (c) hplc-grade methanol. the gradient used was 95% a, 5% b, and a flow rate of 1.0 ml/min for 2 min, 50% a, 25% b, 25% c, and a flow rate to 0.5 ml/min for 8 min. the phenolic acids were quantified by comparison of the area of their peaks recorded at 280 nm with the calibration curves obtained from the commercial standards of each acid. the calibration curves were obtained from a concentration range between 20-1000 µg/ml and 2-100 µg/ml for chlorogenic and cynamic acid respectively. the results are expressed in μg per g of dry mushroom. 2.4. extraction and quantification of β-glucans mushroom β-glucans were isolated and quantified according to nitschke et al. (2011): 750 mg of dry mushroom powder was heated with 60 ml 1 m koh during 20 min at 60°c under constant stirring. then, the suspension was filtered, the filter cake was washed with distilled water and the filtrate was collected and neutralized with 6 m hcl. the neutralized filtrate volume was adjusted to 100 ml with distilled water in a volumetric flask. this fraction was called koh-fraction. the filter cake was resuspended in 65 ml of 0.58 m hcl and heated in an oil bath at 100°c for 1 h. the suspension was again filtered and the filter cake was washed with distilled water. the collected filtrate was neutralized with 6 m naoh, transferred to a 100 ml volumetric flask and the volume adjusted with distilled water. this fraction was named hcl-fraction. the filter cake was again resuspended with 60 ml of 1 m naoh and heated at 60°c for 20 min. the suspension was filtered and the filter cake was washed with distilled water, then the filtrate was neutralized with 6 m hcl. the filtrate was transferred to a 100 ml volumetric flask and the volume was augmented with distilled water. this fraction was called naoh-fraction. the three fractions were used for the β-glucans determination. for quantification of the β-glucans, 350 µl of each fraction were mixed with 300 µl of 0.2 m citric acid/sodium hydroxide buffer ph 7 and 50 µl of dye solution (8 mg of congo red diluted in 10 ml of buffer) was added. the mixture absorbances were read at 523 nm against 350 µl of distilled water, 300 µl of buffer and 50 µl of dye solution as a blank. because of the light brownish color of some of the fractions, a measurement of the background absorption at 523 nm was necessary. therefore, 350 µl of the sample was mixed with 350 µl of the buffer, and the absorption was measured at 523 nm. the calibration curve was obtained with stock schizophyllan solutions in the range of 225-600 µg/ml. all analyses were performed in triplicate. the total content of the β-glucan is expressed as mg of β-glucan per g of dry mushroom. 2.5. ascorbic acid for the ascorbic acid determination, the mushroom powder (150 mg) was extracted with 10 of 1% (w/v) metaphosphoric acid for 45 min at room temperature under constant stirring and filtered through a whatman n° 4 filter paper. the filtrate (0.1 ml) was mixed with 0.9 ml of 2,6-dichloroindophenol (0.00125% w/v) and was left to stand 30 min, then the absorbance was measured at 515 nm against a blank (vaz et al., 2011). the ascorbic ital. j. food sci., vol. 29, 2017 770 acid content was calculated based on the calibration curve of the l-ascorbic acid (0.5-2.01 µg/ml), and the results are expressed as mg of ascorbic acid per g of dry mushroom. 2.6. flavonoids the flavonoid quantification was carried out according to pereira et al. (2012). a total of 500 µl of methanolic extract was mixed with 150 µl of a 5% sodium nitrite solution and 2 ml of distilled water. after 5 min, 150 µl of a 10% aluminum chloride solution was added, the reaction was kept for 6 min, then 2 ml of 4% sodium hydroxide solution and 200 µl of distilled water were added to the mixture. the reaction was mixed, and after 15 min the absorbance was measured at 510 nm. quercetin was used to calculate the calibration curve (1 20 µg/ml) and the results are expressed as mg of quercetin equivalents (qe) per g of dry mushroom. 2.7. statistical analysis all assays were carried out in triplicate. the results are expressed as the mean values and standard deviation (sd). the results were analyzed using a one-way analysis of variance (anova) followed by tukey’s hsd test with α = 0.05. these analyses were carried out using the spss v. 22.0 program (ibm corp., usa). 3. results and conclusions 3.1. antioxidant activity and total phenolics the antioxidant activity of extracts was evaluated through the scavenging activity on the dpph radicals and the reducing power. the antioxidant activity showed highly significant differences (p< 0.001) between the different mushrooms in both assays. the b. edulis extracts had the highest dpph radical scavenging activity (48.06%) and reducing power (59.48%). these results are consistent with its higher content in phenolic compounds (8.66 gae mg/g of dry mushroom) compared to the other mushrooms (table 1). table 1. total phenolics and antioxidant activity of methanolic extracts of wild edible mushrooms from mexico. mushroom phenolics (mg of gae/g) % dpph inhibition % reducing power a. caesarea 2.90±0.16b 6.15±1.76d 36.69±2.37b b. edulis 8.66±0.69a 48.06±6.60a 59.48±0.99a c. cibarius 1.47±0.10c 30.48±3.94b 23.30±1.97c l. indigo 1.91±0.23c 31.28±2.42b 18.63±0.55c ramaria sp 2.07±0.03c 20.88±3.91c 14.73±5.11c in each column, the letters imply significant differences (p< 0.001). mean±sd; n = 3. the l. indigo, c. cibarius and ramaria sp. presented similar levels of the dpph scavenging activity, reducing power and total phenolics. despite the a. caesarea extracts showing the lowest (6.15%) dpph scavenging activity, these extracts registered a high reducing power ital. j. food sci., vol. 29, 2017 771 (36.69%) and a low phenolic compound content (2.90 gae mg/g of dry mushroom). the scavenging effect of the l-ascorbic acid was higher with 94.4% inhibition at 8 mg/ml. palacios et al. (2011) reported the phenolics content for the b. edulis and c. cibarius of 5.5 and 2.5 mg of gae/g of dry mushroom respectively, while we found 8.66 and 1.47. the differences observed may be due to the nutrimental and environmental conditions that affect the production of these metabolites (heleno et al., 2015a). the concentrations of chlorogenic acid range from 108.22 to 1001.67 µg/g of dry mushroom (table 2), thus this acid constitutes 7.36 to 24.64% of the total phenolics present in the wild edible mushrooms studied. b. edulis showed the highest concentration of chlorogenic acid, while c. cibarius registered the lowest concentration. other studies report chlorogenic acid concentrations of 4.55 to 63.73 µg/g (kim et al., 2008, palacios et al., 2011; woldegiorgis et al., 2014) in mushrooms. compared to those reports, the mushrooms evaluated in this study had very high amounts of chlorogenic acid. our results suggest that chlorogenic acid could make a significant contribution to the dpph radical scavenging activity of all mushrooms evaluated, except a. caesarea. cinnamic acid was only detected in b. edulis (10.68 µg/g of dry mushroom) and in a. caesarea (4.93 µg/g of dry mushroom). while heleno et al. (2015a) report 3.1 µg/g of cinnamic acid in b. edulis, taofiq et al., (2015) found 14.2 µg/g. the content of this acid in a. caesarea reported by fernandes et al. (2015) was 24.8 µg/g, and reis et al. (2011) found 0.3 µg/g. thus, the results obtained in this study are somewhat comparable with those studies. table 2. content of chlorogenic and cinnamic acids (µg/g of dry weight) in wild edible mushrooms from mexico. mushroom species chlorogenic acid cinnamic acid a. caesarea 564.45±41.99b 4.93±0.61 b. edulis 1001.67±2.74a 10.68±2.15 c. cibarius 108.22±2.61d nd l. indigo 222.42±1.55c nd ramaria sp 510.11±8.13b nd in each row, different letters imply significant differences (p< 0.05). mean±sd; n = 3. 3.2. ascorbic acid the ascorbic acid concentration ranging from 2.08 to 3.65 mg/g of dry mushroom reported elsewhere for the l. indigo reveals highly significant differences (p< 0.01) as compared to the mushrooms investigated herein (table 3). the l. indigo registered the highest concentration and the a. caesarea registered the lowest. although barros et al. (2007) report amounts of ascorbic acid of 0.13 to 0.35 mg/g in mushrooms, reis et al., (2011) found 1.75 to 8.99 mg/g. hence, our results are in agreement with the latter study. the presence of ascorbic acid in the mushroom is not surprising as this metabolite is required as part of the defense mechanisms against radicals to avoid oxidative stress; therefore, the concentrations of ascorbic acid may be subject to the effects of geographic locations, maturating stage of the fruiting body, genotype and weather conditions (ferreira et al., 2009). ital. j. food sci., vol. 29, 2017 772 3.3. flavonoids the flavonoid content of the mushrooms studied is shown in the table 3. the concentration varies depending the mushroom species, and the differences are highly significant (p < 0.001). the results of the present study are similar to those reported by woldegiorgis et al. (2014) who found concentrations of 0.17 to 1.97 mg catechin equivalent/g. although korzaski et al. (2015) report that c. cibarius is a rich source of flavonoids (42.9 mg of catechin equivalents/g of dry mushroom), this does not agree with our finding. the results suggest that flavonoids may play an important role in the total antioxidant activity, specifically in reducing power. table 3. concentration of ascorbic acid, flavonoids and β-glucans in wild edible mushrooms from mexico. mushroom ascorbic acid (mg/g) flavonoids (mg qe/g) β-glucans (mg/g) a. caesarea 2.08±0.16d 0.39±0.04b 214.92±2.09a b. edulis 2.61±0.05c 0.92±0.02a 39.97±0.91c c. cibarius 2.63±0.09c 0.34±0.02b nd l. indigo 3.65±0.00a 0.25±0.00c 88.34±3.62b ramaria sp 3.14±0.04b nd nd nd = not detected. in each row, different letters imply significant differences (p< 0.001). mean±sd; n = 3. 3.4. β-glucans β-glucans are the most abundant polysaccharide on the fungal cell wall (fesel and zucaro, 2015) and the colorimetric method with congo red detects β-1,3-1,6 glucans from mushrooms with high precision and without extensive clean-up (zhu et al., 2015). thus, the β-glucans content determined by this study showed highly significant differences (p< 0.01) between the mushroom species (table 3). the highest β-glucans content was detected in a. caesarea with 214.92 mg/g of dry mushroom, while ramaria sp. and c. cibarius did not show detectable β-glucans contents. despite the fact that there are several reports on β-glucans in mushrooms in other countries (synytsya and novák, 2013; ruthes et al., 2015), the species described here have been not explored yet, except for b. edulis. manzi et al. (2004) reported that rehydrated dry samples of b. edulis had a βglucans content of 43.3 mg/g, which is similar to our finding for this mushroom (39.97 mg/g). it seems that a. caesarea is a rich source of β-glucans, which will produce health benefits when consumed. the mexican wild edible mushrooms studied in this research shown high contents of chlorogenic and ascorbic acids and flavonoids. b. edulis has the highest antioxidant activity, which correlates with its contents of chlorogenic and cinnamic acids and flavonoids. l. indigo has the highest content of ascorbic acid, and a. caesarea registered the highest concentration of β-glucans. the presence of these biologically active molecules in the mushrooms studied reveals the nutraceutical potential of the mushrooms. ital. j. food sci., vol. 29, 2017 773 references barros l., ferreira j.m., queiroz b. and ferreira i.c.f. r. 2007. total phenols, ascorbic acid, b-carotene and lycopene in portuguese wild edible mushrooms and their 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(2014). antioxidant property of edible mushrooms collected from ethiopia. food chem. 157:30. zhu, f., du, b., bian, z. and xu, b. 2015. beta-glucans from edible and medicinal mushrooms: characteristics, physicochemical and biological activities. j. food comp. anal. 41:165. paper received april 24, 2017 accepted september 8, 2017 paper ital. j. food sci., vol. 28 2016 43 keywords: antinutrients, nutrients, cissus rotundifolia, evaluation nutritional evaluation of wild plant cissus rotundifolia mohamed korish1,2 1arid land agriculture department, faculty of meteorology, environment and arid land agriculture, king abdulaziz university, p.o. box 80208, jeddah 21589, saudi arabia 2department of food and dairy science &technology, faculty of agriculture, damanhour university, damanhour 22516, egypt tel. +00966 6952366, fax +00966 6952364, email: mmkorish@yahoo.com abstract this study aimed to evaluate the nutritional and antinutritional components of cissus rotundifolia leaves. they contain an appreciable amount of protein (12.5% db ), fat (7.45% db ), crude fiber (8.34 % db ) and minerals (16.32% db ). the protein fraction contains a relatively high level of essential amino acids, which accounted for 44.3% of the total amino acids. the fat contains a high concentration of unsaturated fatty acids that comprises 55.1% of total fatty acids. the mineral profile is composed of macroand microelements. the antinutritional factors oxalate, phytate, tannins and cyanogenic glycosides are present at very low concentrations. cissus rotundifolia leaves can be considered a potential source of nutritional components for healthy food purposes. mailto:mmkorish%40yahoo.com?subject= 44 ital. j. food sci., vol. 28 2016 introduction wild edible plants are species of plants that grow freely in the wild habitat without any agricultural treatments and can be consumed as a food (beluhan and ranogajec, 2010). these types of plants are consumed worldwide, from developing and developed nations alike, and provide nutrition and food security for poor rural communities in several regions across the world (sundriyal et al., 2003; afolayan and jimoh, 2009) while serving as a diet supplement in japan, europe and north american (chen and qiu, 2012; burlingame, 2000; redzic, 2006). wild edible plants are rich in minerals, vitamins, dietary fiber, fatty acids and amino acids (barros et al., 2010; luczaj, 2010). the nutritional values of these plant species are comparable to, or even exceed, the corresponding domesticated types of plants (burlingame, 2000; tardio et al., 2006; afolayan and jimoh, 2009). moreover, wild edible plants are considered a good source of phytochemicals for human therapeutics (penny et al., 2002; masuda et al., 2003; vardavas et al., 2006). however, the presence of antinutritional principles in some species of wild plants, such as phytic acid, tannins, saponins, alkaloids and oxalates, can limit their exploitation (guil et al., 1997; gupta et al., 2005; lachumy et al., 2010). previous studies have shown that the corresponding domesticated types of these plants contain similar levels of antinutritional factors (shad et al., 2013). moreover, some of the antinutritional factors have therapeutic potential; for example, phytic acid has been shown to have anticancer and antioxidant activity (jariwalla, 2001; shamsuddin, 2002). thus, the compositional analysis and nutritional evaluation of such wild plants are necessary for understanding their impacts on consumer’s health (guil et al., 1997). cissus rotundifolia (forsk) vahl. is a perennial, evergreen, climber, wild plant and is a species of cissus belonging to the family of vitaceae (grape family). it is known as a common arabian wax cissus, peruvian grape ivy, venezuelan tree bine and locally (in south saudi arabia) as algalaf. this wild plant is commonly used as food thickeners in rural nigeria. moreover, it was found to have many therapeutic effects as hypoglycemic (onyechi et al. 1998), hypolipidemic (bell et al.1993). in addition, its extract exhibits antibacterial activity (alzoreky and nakahara, 2003). cissus rotundifolia grows extensively in the southern region of saudi arabia, and their leaves only are widely consumed after cooking by local people as leafy vegetables. although it is commonly used to prepare various dishes according to traditional dietary culture of locals, its nutritional potential has not been assessed. therefore, this study aimed to evaluate the nutritional and antinutritional components of cissus rotundifolia leaves (crl). these data would increase the awareness about the exploitation of this renewable natural resource as a food. materials and methods sample collection and preparation the leaves of cissus rotundifolia (20 kg) were collected from the abha region in southern saudi arabia. the leaves were washed with distilled water, dried in a hot air oven at 50°c to a constant weight, ground to a fine powder and stored in airtight plastic bags at 4°c until analysis. proximate composition analysis the moisture, ash, crude lipid, crude fiber and crude protein (n×6.25) contents were determined according to the standard methods of (aoac, 2000). amino acids analysis the defatted samples (0.2 g) were hydrolyzed with 6 n hcl (10 ml) in a sealed tube at 100°c for 24 hours. the hydrolyzates were completed to 25 ml with deionized water. five ml of each hydrolyzate were evaporated until free from hcl vapor and dissolved in citrate buffer (csomos and simon-sarkadi, 2002). the identification and determination of amino acids were conducted using the amino acid analyzer aaa-400 (ingos, czech republic) equipped with an (ostion lg anb, ingos) ion-exchange column (200 x 3.7 mm) and a flow photometer detector. the elution was carried out using a different ph gradient of sodium-citrate buffers. chromatographic data processing including calculation of retention times and peak areas of separated amino acids were performed using amik software 3.0 (czech republic). a mixture of standard amino acids (ingos, czech republic) was utilized as external standards. fatty acid analysis the lipids were extracted according to the method outlined by egan et al. (1981) and gressler et al. (2010). briefly, 10 g of the sample was digested with 10 ml of hot concentrated hcl using a boiling water bath and vigorous stirring before the color of the content turned brown. the lipid was extracted by shaking with 30 ml of diethyl ether and was repeated three times. the solvent was evaporated and the total amount of lipid was gravimetrically estimated. the fatty acid was transmethylated into their corresponding methyl esters (radwan, 1978). the lipids (50 mg) were redissolved in 2 ml benzene, aliquots of 2 ml of methanolic sulfuric acid (1%, v/v) were added and the tubes were stoppered with nitrogen and kept in a water bath at 90°c for 90 min. water (8 ml) was added, the methylated fatty acids were extracted with 5 ml petroleum ether and the mixture was evaporated to dryness. two microliters of the ital. j. food sci., vol. 28 2016 45 fatty acid methyl esters solution were injected into a hp (hewlett packard) 6890 gc, coupled with a splitless injector mode, a flameionization detector (fid) and a hp-5 column (5% diphenyl, 95% dimethyl polysiloxane, 30 m, 0.32 mm id, 0.25 μm film thickness). the following operating conditions were used: injector temperature 220oc, oven temperature program: initial temperature 150°c for 2 min, raised to 200°c at a rate of 10 °c /min, then increased to 250°c at a rate of 5°c /min and held at 250°c for 9 min, detector temperature: 250 °c, carrier gas was nitrogen at a flow rate of 1 ml/min. the mixture of fatty acid standards was subjected to the same treatments of the samples and used to identify and quantify the fatty acids in the samples. mineral analysis the samples were digested as described by amin et al. (2013). briefly, leaf powder (0.5 g) was digested with 4 ml of concentrated nitric acid and 1 ml of perchloric acid, cooled and filtered with whatman no.42 filter paper. the supernatant was completed to 50 ml with distilled water. the blanks were carried out using the same procedure. the mineral concentrations of the digested diluents were determined against a multielement standard solution (campro scientific, berlin, germany) using inductively coupled plasma-optical emission spectrophotometry icp-oes (varian 720-es, varian inc, palo alto, ca, usa). determination of antinutrients the content of oxalate was measured using the titrimetric method of sanchez-alonso and lachica (1987). phytic acid in leaves was quantified according to the method of lucas and markakas (1975). the spectrophotometric method described by sarkiyaki and agar (2010) was used to estimate the amount of cyanogenic glycoside in leaves. the tannin content was estimated using spectrophotometric analysis according to the method of polshettiwar et al. (2007). statistical analysis all measurements were achieved in triplicate and the results were expressed as the mean value ± standard deviation of three measurements, using spss 13.0 (spss inc., il, usa). results and discussion proximate compositions the nutritional composition of the leaves (table 1) was compared with those of the most widely consumed foods (wheat, rice and potato) throughout the world. this comparison is justified by the fact that in the countries of origin leaves are used in two forms: fresh and sundried powder. the latter one is consumed as a partial replacer of wheat flour, corn flour and rice, to overcome a deficient of these foods. the determined nutrients of the leaves were superior to those of wheat, rice and potato. this emphasizes their value as a good source of nutrients. a relatively high ash content in the leaves was associated with the amount of mineral elements. amino acid composition and protein quality by the amino acid analysis (table 2) fifteen amino acids were identified in crl protein fraction. among the detected amino acids, eight of essential amino acids (eaas), which amounted to 358.5 mg/g crude protein, was identified. this exceeded the value of eaas that is recommended by fao for adults (2013). the amount of eaas comprised 44.3% of the total individual amino acids, which is a ratio similar to that reported for the domesticated vegetable kale leaves (lisiewska et al., 2011). the present analyses also indicated that the protein in crl contained a considerable level (69.9 mg/g protein) of aromatic amino acids (aaa) (histidine, phenylalanine and tyrosine), which is much higher than the aaa scoring pattern recommended by fao for adults (38 mg/g) (2013). similar to previous studies performed on many domesticated vegetable species (lisiewska et al., 2011; kmiecik et al., 2009), glutamic acid was the major amino acid identified in crl protein. cysteine, methionine and tryptophan were excluded in this study because they were destroyed during acid hydrolysis. all individual eaas in leaf proteins (table 2) compared favorably with the corresponding amino acid reference that is recommended for adults by fao (2013) except for histidine, which had a score slightly below what is recommended. therefore, crl can be considered a good source of balanced protein. table 1 proximate composition (g/ 100g) of crl compared with wheat, rice and potato. constituent (%)a crl wheatb riceb potatoc moisture 93.1±0.2 12.6 13.0 75.7 crude protein (dry basis) 12.5±0.1 11.3 7.70 8.27 crude fat (dry basis) 7.45±0.1 1.80 2.20 1.11 crude fiber (dry basis) 8.34±0.2 13.2 2.20 9.94 ash (dry basis) 16.3±0.2 1.70 1.20 3.98 avalues are expressed as the means ± sd of three separate determinations). source: bkoehler and wieser (2013); cgumul et al. (2011) 46 ital. j. food sci., vol. 28 2016 table 2 amino acid profile of crl protein. amino acids mg/g proteina fao pattern 2013 % of total essential amino acids histidine 16.4±0.2 15 2.03 isoleucine 47.5±0.2 30 5.88 leucine 96.6±0.4 59 11.9 lysine 38.7±0.1 45 4.79 phenylalanine 37.9±0.1 4.69 threonine 23.7±0.2 23 2.94 valine 69.9±0.6 39 8.65 arginine 27.4±0.2 3.39 non-essential amino acids alanine 98.5±0.7 12.1 aspartic acid 64.9 ± 0.1 8.03 glutamic acid 127.3±0.6 15.7 glycine 97.7±0.6 12.0 proline 7.77±0.1 0.96 serine 38.2±0.2 4.72 tyrosine 15.5±0.1 1.92 total eaasb 358.5 total noneaas 450.0 total individual amino acids (mg/g protein) 808.5 total aaac 69.9 % of eaas 44.3 % of noneaas 55.7 avalues are expressed as the means ± sd of three separate determinations on dry weight basis; bessential amino acids; caromatic amino acids (phenylalanine+ histidine +tyrosine). table 3 fatty acid composition of crl. fatty acid fa (µg/g)a % of total caprylic acid (c8:0) 7.56±0.3 0.23 capric acid (c10:0) 12.4±0.2 0.38 lauric acid (c12:0) 35.6±0.2 1.09 tridecylic acid (c13:0) 63.1±0.1 1.93 myristoleic acid (c14:1) 101.2±0.2 3.09 myristic acid (c14:0) 39.8±0.2 1.21 pentadecenoic acid (c15:1) 110.5±0.1 3.38 pentadecanoic acid (c15:0) 92.8±0.2 2.83 palmitic acid (c16:0) 1036.5±0.4 31.7 linoleic acid (c18:2c) 750.2±0.1 22.9 oleic acid (c18:1c) 841.5±0.5 25.7 stearic acid (c18:0) 181±0.7 5.53 total unsaturated fatty acids 1803.4 total saturated fatty acids 1468.8 total individual fatty acids 3272.1 % of total unsaturated fatty acids 55.1 % of total saturated fatty acids 44.9 avalues are expressed as the means ± sd of three separate determinations on dry weight basis. fatty acid profile of crl the data in table 3 show that 12 fatty acids were determined in the leaf lipidic extract, four out of which are unsaturated fatty acids and comprised more than half (55.1%) of the total fatty acid content. this high level of unsaturated fatty acids makes the crl of main health interest. palmitic acid, oleic acid and linoleic acid were the three major components present in the leaves, representing 31.7%, 25.7% and 22.9% of the total individual fatty acids, respectively. palmitic acid is commonly found in both animal and plant foods. who (2003), reported that, dietary intake of palmitic acid increases the risk of cardiovascular diseases. however, in moderation, palmitic acid may not be entirely bad, as it does display mild antioxidant and anti-atherosclerotic properties (cho et al., 2010). the high proportion of both oleic acid (omega-9 fatty acids) and linoleic acid (omega-6 fatty acids) in leaves raises the biological value; therefore, consuming the leaves could be healthy and meet a part of the essential fatty acids requirements. the data also show that the leaf lipids contain odd-numbered fatty acids (tridecylic, pentadecanoic and pentadecenoic acid) in its composition. such fatty acids have been found in many daily consumed foods such as human milk (nishimura et al., 2013; koletzko et al., i988), ruminants milk (brevik et al., 2005), fish (ates̨ et al., 2013), and commonly consumed vegetables (batista et al., 2011). concerning the impact of odd-numbered fatty acids on health, martysiak-zurowska (2008) reported that there is no risk of presence of odd-numbered fatty acids in food as it is found in mother’s milk and ruminant’s milk. ital. j. food sci., vol. 28 2016 47 mineral content of crl the contents of both macroand microelements in leaves are presented in table 4. calcium, which is required for the formation of bone and neurological function (brini et al., 2013), was the predominant element in leaves (15.1 mg/g). a modest consumption of 66.5 g of leaves per day would satisfy the adult daily requirement of calcium (1,000 mg/day), according to the institute of medicine (2011). therefore, crl could be a good source of calcium. sodium was the second abundant element found in crl, followed by potassium. potassium and sodium play an important role in regulating blood pressure and body acid-base balance (clausen et al., 2013; siddhuraju et al., 2001). an appreciable concentration of magnesium was determined in the leaves. magnesium is needed to prevent heart disease and growth retardation (chaturvedi et al., 2004). crl could be considered a rich source of iron and an intake of 47.4 g of leaves could satisfy the recommended adult dietary intake (6 mg/ day) of iron according to the institute of medicine (usa, 2001). zinc, which is a component of many enzymes and a wide array of cellular and biochemical processes (karcioglu, 1982; coleman, 1992), is present in a moderate amount in leaves. significant amounts of both copper and chromium, which are a component of many respiration enzymes and glucose tolerance factor, respectively (sands and smith, 2002; mertz, 1993), were observed in the leaves (failla et al., 2001; kelvay, 2000). antinutritional factors the edibility of any wild plant depends on the content of anti-nutritional factors. analyses were carried out in crl and results are shown in table 5. the oxalate content was equal to 3.05mg/100 g, value lower than that reported (14.9 g/100 g) in common green leafy vegetable spinach (spinacia oleracia) (yadav and sehgal, 2003). the phytate level (0.76 mg/100 g) in leaves was found to be less compared with that reported in domesticated crops of solanum indicum (695.8 mg/100 g, aberoumand, 2012), lima beans (234 mg/100 g, egbe and akinyele, 1990) and underutilized green leafy vegetables (0.92–13.06 mg/100 g, gupta et al., 2005), indicating that the lower phytic acid content in crl will provide a better bioavailability of minerals. the estimated tannin value in leaves is considerably lower compared with those (0.59 mg/100 g) reported in lima beans (phaseolus lunatus) by egbe and akinyele (1990). the detected level of cyanogenic glycosides (0.023 mg/100 g) can be consider inappreciable compared with those of lima beans (colored) (3120 mg hcn/kg) (speijers, 1993) and is much lower than the reported lethal dose (3.70 hcn mg/ kg bw) for mouse (conn, 1979). these results reveal that antinutritional factors exist in crl, but at lower levels compared with many dailyconsumed foods. conclusions the present study serves as a basis to encourage the local communities to exploit the nutritive potentials of the wild plant cissus rotundifolia. results of analyses demonstrated good nutritional qualities and crl could, thus, contribute to overcome the nutritional deficiency especially in arid climates. therefore, it is now imperative that a nutritional database of this wild plant is set up to retain the information for a better management and conservation of this natural resource and habitats related to it. acknowledgements this article was funded by the deanship of scientific research (dsr), at king abdulaziz university, jeddah. the author, therefore, acknowledges with thanks dsr for technical and financial support. the author thanks dr. abdullah al-shehry, faculty of meteorology, environment and arid land agriculture, at king abdulaziz university, for providing the plant samples. table 4 mineral composition of crl. mineral concentrationa macroelements mg/g calcium (ca) 15.1±0.2 magnesium (mg) 3.55±0.1 sodium (na) 11.2±0.2 potassium (k) 8.09±0.3 microelements µg/g iron (fe) 126.6±3 zinc (zn) 51.6±0.3 manganese (mn) 31.3±0.6 copper (cu) 3.21±0.3 chromium (cr) 2.38±0.2 avalues are expressed as the means ± sd of three separate determinations on dry weight basis. table 5 antinutrients contents in crl. compound content (mg/100g) a oxalate 3.05±0.1 phytate 0.76±0.1 tannins 0.26±0.1 cyanogenic glycosides 0.023±0.0 avalues are expressed as the means ± sd of three separate determinations on dry weight basis. 48 ital. j. food sci., vol. 28 2016 references aberoumand a. 2012. screening of phytochemical compounds and toxic proteinaceous protease inhibitor in some lesser-known food based plants and their effects and potential applications in food. int. j. 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(1993) showed that the consumption of 110 g of apples a day reduces the risk of heart attack by 49% when compared with the daily consumption of only 18 g of apples. dehydration by hot-air is probably the most common and effective preservation method, used to imbue a food product with long shelf-life. drying adds new values to food by limiting the spoilage and reducing the mass of the product (mulet et al., 2003; chong et al., 2008; gamboa-santos et al., 2013; kek et al., 2013, witrowa-rajchert et al., 2014). however, the convective drying conditions such as temperature and air velocity may negatively affect the quality of a final product, causing changes in the microstructure, physical properties and nutritional value of food products (gamboa-santos et al., 2013; chong et al., 2014). the texture modification, the degradation of vitamin, the loss of essential amino acids and changes in colour and flavour occur during hot-air drying (azoubel et al., 2010; ozuna et al., 2014). in order to determine the changes that appear during treatment and after drying food is tested on its reconstitution properties such as rehydration and hygroscopic properties (rząca and witrowa-rajchert, 2007). although convective drying is widely used, the method is related to high energy consumption, which results in the high cost of this technique (gamboa-santos et al., 2014). because of the growing need for the production of higher quality dried food products at lower processing cost, the traditional convective drying is combined with non-thermal techniques of pre-treatment such as high hydrostatic pressure, pulsed electric field and power ultrasound (witrowa-rajchert et al., 2014). due to the low heating effect, the ultrasound treatment is a very promising method in the food industry (ozuna et al., 2014). ultrasound waves indicate compression and expansion cycles in the material, which leads to micro-channels formation (fernandes et al., 2008a; nowacka et al., 2014; nowacka and wedzik, 2016). this phenomenon improves the rate of mass transfer and accelerates diffusion during dehydration (gamboa-santos et al., 2013; rodríguez et al., 2014). moreover, the sonication generates cavitation, which can cause the removal of strongly attached moisture (mulet et al., 2003; gamboa-santos et al., 2014). for example, the ultrasound pre-treatment reduced the drying time by 5-40% in the case of dried banana (azoubel et al., 2010), by 31-40% in the case of dried apples (nowacka et al., 2012) and in the case of pineapple by over 30% (fernandes et al., 2008c). the ultrasound application reduces the drying time, and the process can be carried out at a lower temperature (nowacka et al., 2012), which is relevant for food containing thermo-labile compounds. the ultrasonic effects during dehydration depend on the kind of the product and the ultrasound power. the visual appearance such as colour and texture are important sensory criteria for both the manufacturer and the customer (cybulska et al., 2011; pingret et al., 2013). the drying process linked to the ultrasound pre-treatment can improve the quality of dried fruits and modify food properties (ozuna et al., 2014). the purpose of the research was to investigate the effects of ultrasound pre-treatment at different treatment times on the quality factors of dried products, which affect the consumers’ choice such as colour, mechanical, rehydration and hygroscopic properties. moreover, the influence of sonication on the kinetics of apple drying was analysed. ital. j. food sci., vol 29, 2017 345 2. materials and methods 2.1. sample preparation apples (var. idared) from the experimental fields (orchards) located in the district of wilanow in warsaw served as the experimental material. the production of apples is carried out by employees of the faculty horticulture, biotechnology and landscape architecture (warsaw university of life sciences wuls-sggw). apples were harvested and transported to the faculty of food sciences in wuls-sggw. while the experiments were performed, the apples were stored at 90% air humidity and a temperature of 4-8 °c for 1 month. the material was cut into slices of a thickness of 0.005±0.001 m and a diameter of 0.030±0.001 m, excluding peel and core. in order to prevent enzymatic browning reactions, apple slices were immersed in the 0.1% citric acid solution, then blotted with filter paper and pre-treated with ultrasound. 2.2. ultrasound pre-treatment the material was submitted to ultrasound treatment for 10, 20 and 30 minutes in an ultrasonic bath with a frequency of 35 khz (intersonic, olsztyn, poland, is-3 model, internal dimensions: 240×135×100 mm), while the ultrasound intensity equalled 4 w/cm2. pre-treatment was carried out in distilled water at room temperature (22±1 °c) and, in order to prevent flowing out of the samples, the slices were covered with a metal net. the ratio of raw material to water was 1:4, as recommended by nowacka et al. (2014). afterwards, the samples were blotted with filter paper and placed in a dryer. the material mass, the content of dry matter and a temperature of medium were measured before and after pre-treatment. the experiments were performed in duplicate. 2.3. convective drying the apple slices were dried in a laboratory convective dryer (warsaw, poland) at a temperature of 70 °c with parallel air-flow at an air velocity of 2 m/s. the scheme of the dryer is presented in fig. 1. the main part of the dryer is the chamber within the shelf, where the dried product is placed. the shelf is connected with the balance. the product is dried in the hot air flow, which is heated by the heater system placed on the air inlet. the temperature of the air flow is measured by the thermocouples. the thermal sensors and the balance are connected to the computer, which records the data during the drying process. the heaters and thermal sensors are connected to the control panel, which is used to set up temperature and air velocity. firstly, the dryer was preheated to a set-point temperature and then loaded with 0.25 kg (1,92 kg/m2) of apple slices spread over the two nets in a single layer. drying was carried out until constant weight was achieved. the experiments were conducted in duplicate. the relative moisture ratio and the effective water diffusivities (deff) were calculated using the equations (1) and (2), respectively (sledz et al., 2013): 𝑀𝑅 = ! !! (1) where: u – water content during drying (kg moisture/kg d.m.), uo – initial water content (kg moisture/kg d.m.) ital. j. food sci., vol 29, 2017 346 ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⋅ ⋅⋅− ⋅= 2 2 2 4 exp 8 l d mr eff τπ π (2) where: deff – diffusion coefficient (m2/s), τ drying time (s), l – half-thickness of the sample (m). figure 1. the scheme of a laboratory convective dryer (figure made by www.ct.waw.pl) 2.4. physical properties the dry matter in the raw, pre-treated, dried and rehydrated material was measured according to the polish standard pn-90/a-75101/03 by drying at a temperature of 105 °c to the constant weight. the water activity of the untreated, ultrasound treated and dried samples were examined using hygrometer aqua lab cx-2 (dekagon device inc., usa) with the accuracy of ±0.001. the equipment was switched on around 30 minutes before measuring and checked with the water. the raw apple slices were placed in a plastic vessel, covering the bottom of the vessel. after ultrasound pre-treatment the samples were blotted with filter paper and placed in the vessel. the apple slices after drying were stored in a sealed bag for three days to compensate the humidity of the sample after drying. the measurements were made thrice for each sample at a temperature of 25 °c. based on the measurement of the mass and volume of the dried sample, density and porosity were calculated (andrés et al., 2004). 2.5. colour measurement a hand-held minolta cr-300 chromameter (minolta, japan) was used to measure the colour of the dried apple slices (diffuse illumination, 0º viewing geometry). colour was recorded with the cie l*a*b* system. in this colour space, the l* parameter defines ital. j. food sci., vol 29, 2017 347 lightness and ranges from 0 to 100, the a* parameter denotes chromaticity on a green (-) to red (+) axis and the b* parameter represents chromaticity on a blue (-) to yellow (+) axis. the l*, a* and b* coefficient were used to calculate the chroma (c*) which indicates the purity and saturation of colour and the value of the colour difference (∆e) (gonçalves et al., 2007; fijalkowska et al., 2016) according to equations (3) and (4), respectively. 𝐶∗ = (𝑎∗)! + (𝑏∗)! (3) ∆𝐸 = (∆𝐿∗)! + (∆𝑎∗)! + (∆𝑏∗)! (4) where: ∆l*, ∆a*, ∆b* the change of lightness, a* and b* parameter value between pretreated and untreated dried apple. the experiments were performed in 15 repetitions. 2.6. structure the internal structure of dried tissue was examined using a scanning electron microscope hitachi tm 3000 tabletop microscope (tokio, japan). the internal part of the slices was cut in half with a scalpel and put into the vacuum chamber of the microscope. the images were examined under the magnification of 100. 2.7. mechanical properties mechanical properties were analysed using the cutting test in a texture analyser (ta-tx2i model, stable micro systems, godalming, england). in order to conduct this test, a knife of 0.062 m in length, 0.024 m in width and 0,0005 m in thickness was slid inside a metal table with a slot. the load cell was calibrated to 250 n and the cutting was applied at a velocity of 0.001 m/s. the cutting began when the sample resisted and carried out to complete intersection. both the maximum cutting force and the cutting energy were recorded using program “texture” and then calculated. the mean value for ten replicates of apple slice was averaged for each ultrasound treatment. 2.8. rehydration properties in order to analyse rehydration kinetics, two slices of dried apples were weighed with an accuracy of ±0.1·10-6 kg and placed in a glass with 10-4 m3 of distilled water. the process was carried out at a temperature of 20 °c and the samples were immersed for 30, 60 and 180 minutes. after a given rehydration time, the slices were drained on a sieve and then on a blotting paper. the experiments were repeated twice for each kind of the dried material. the rehydration properties were calculated according to the following equation: 𝑋 = !! !! (5) where: xτ – moisture of a rehydrated sample at time τ (kg water/ kg d.m.), x0 – initial moisture of a fresh sample (kg water/kg d.m.). ital. j. food sci., vol 29, 2017 348 the soluble solid loss during rehydration was calculated using the following equation: 𝑆𝑆𝐿 = !!∙!"! !!∙!"! (6) where: mτ – sample weight after rehydration at time τ (kg), m0 – sample weight before rehydration (kg), dmτ – dry matter content of a sample after rehydration at time τ (%), dm0 – dry matter content of a sample before rehydration (%). 2.9. hygroscopic properties in order to determine the hygroscopic properties of the apple tissue after drying the samples were weighed with an accuracy of ±0.1·10-6 kg and placed in a desiccator over nacl solution of a water activity aw = 0.75. the kinetics of adsorption was determined for 72 hours at 25 °c. after a specific time of 0.5, 1, 3, 5, 8, 10, 24, 48 and 72 h the samples were weighed again. the experiments were performed in triplicate for each kind of the dried material 2.10. statistical analysis the significance of the ultrasound treatment and the drying process were examined by the analysis of variance (anova) using statgraphics plus 5.0 programme. the homogeneity of variance was verified using levene’s test. duncan’s multiple range tests with a probability of 0.05 were used to determine homogeneous groups. 3. results 3.1. ultrasound treatment during sonication, the distilled water temperature increased by 4.3, 7.3 and 10.8 °c for 10, 20 and 30 minutes of treatment, respectively (table 1). due to a series of rapid compressions and expansions of plant tissue generated by ultrasound waves, water included in the raw material could flow out to the surroundings. after 10 and 20 minutes of pre-treatment, the apples lost 0.89±0.02 and 0.59±0.03% of weight, respectively (table 1), whereas 30 minutes of ultrasound application increased the weight of the samples (1.47±0.10%). the phenomenon that occurred during a longer time of immersion, can be caused by water penetration into the tissue as a result of osmotic concentration differences. statistical analysis showed significant differences between mass changes for all pre-treatment time. the similar relation was observed in nowacka et al. (2012) research where the smallest weight loss of apple tissue (0.8±0.4%) was obtained for samples pre-treated for 30 minutes with an ultrasound frequency of 35 khz. for 10 and 20 minutes of sonication, the weight loss was equal to 2.3±0.1 and 3.0±0.2%, respectively. the opposite tendency was observed by fernandes et al. (2008b), who subjected papaya tissue to ultrasound waves with a frequency of 25 khz. the enhancement of water loss was obtained with increasing time of treatment in the range of 10 to 30 minutes. raw apple tissue contains 15.1±0.8% of dry matter. the samples submitted to ultrasonic treatment lost from 26.5 to 28.4% of soluble solids because of their flowing out to a liquid medium. the decrease of this parameter was significant in the case of each time of pretreatment in comparison with the untreated apple slices (table 1). the length of ital. j. food sci., vol 29, 2017 349 ultrasound application did not have statistically meaningful influence on the dry matter content of the material. table 1. changes of mass, dry matter content and medium temperature after ultrasound pre-treatment. type of treatment weight gain (+)/loss(-) [%] dry matter [%] medium temperature increase [ºc] untreated 15.1±0.8a us 10 min -0.89±0.02a 10.8±1.4b 4.3±0.8a us 20 min -0.59±0.03b 11.0±0.5b 7.3±0.3b us 30 min 1.47±0.10c 11.1±0.5b 10.8±0.8c a, b, c: the same letters indicate homogeneous groups. 3.2. drying characteristics the application of ultrasound resulted in a reduction of the convective drying time by 513% in relation to the untreated material. the drying time decreased significantly with the increasing time of ultrasound pre-treatment (table 2). similarly, the shorter time of drying process after ultrasound treatment was noticed in the literature where a time reduction of 4.5% was observed for banana (azoubel et al., 2010), 3-22% for papaya (fernandes et al., 2008b), 7-39% for pineapple (fernandes et al., 2008c), 27% for apple cubes, 18-23% for red bell pepper (schӧssler et al., 2012) and 50-75% for eggplant (puig et al., 2012). these examples confirm the assumptions of feunte-blanco et al. (2006) who noticed in the influence of ultrasound on the fruit tissue facilitating water diffusion during airdrying. this phenomenon can be caused by micro-channels formation during sonication, which can enable easier water diffusion from interior material to the surface (fernandes et al., 2008a). table 2. drying time of the apple slices to obtain 0.09 kg moisture/kg d.m., the value of the effective moisture diffusion coefficient (deff), dry matter content and water activity of the dried apples. type of treatment drying time [min] deff·109 [m2/s] dry matter [%] density [kg/m3] porosity [%] water activity [%] untreated 133±3c 1.037 92.3±0.9a 524± 100bc 65.6±5.7 a 0.264±0.012b us 10 min 126±1b 1.058 92.1±0.2a 464±30ab 69.5±1.8ab 0.286±0.010c us 20 min 123±1b 1.064 93.2±0.4a 545±60bc 64.2±3.5 a 0.246±0.011a us 30 min 116±2a 1.102 93.1±1.0a 388±40a 74.5±2.2 b 0.290±0.009c a, b, c: the same letters indicate homogeneous groups. furthermore, the values of the effective water diffusivity (deff) for the ultrasound treated samples were growing with the increase of treatment time, and the highest value of this parameter was noted for the apples subjected to 30 minutes of sonication. in this case the effective moisture diffusion coefficient increased by 6% in comparison with the untreated material. the values of this coefficient were related to drying time (nowacka et al., 2012), which was proved by the shortest time of drying in the case of the apples treated by ultrasound for 30 minutes (table 2). the apple slices were dried to obtain 0.09 kg moisture/kg d.m., where the dry matter content of untreated tissue equalled 92.3±0.9% and there was no significant influence of ital. j. food sci., vol 29, 2017 350 ultrasound treatment on this parameter value (table 2). however, statistical analysis confirmed that sonication caused significant changes in the density and porosity of the dried tissue (table 2). the density of the material is closely related to its porosity. a low density product must possess high porosity. the tissue exposed to ultrasound waves for 30 minutes after drying exhibited the lowest density of 388±40 kg/m3 and the highest porosity (74.5±2.2%). the difference between the dried treated samples and the untreated ones was statistically significant. similar results were reported by nowacka et al. (2012) for dried cubes of apples. moreover, the statistical changes were found in the water activity of the ultrasound treated samples. the samples that were subjected to ultrasound for 10 and 30 minutes obtained higher values of water activity in comparison to the untreated material, whereas the tissue treated for 20 minutes was characterized by a lower value of this parameter. however, all dried samples revealed low water activity, which proves the microbiological safety of food. 3.3. optical properties colour is one of the most important factors of raw and dried fruits’ quality because the external appearance influences the consumer acceptability (singh and reddy, 2006; nuncio-jáuregui et al., 2014). the colour parameters l*, a*, b*, c* and ∆e of the untreated and pre-treated dried apple slices are presented in table 3. in the case on lightness, the l* value of dried apples significantly increased after 30 minutes of sonication in comparison with the untreated sample. however, a shorter time of ultrasound treatment did not have any significant impact on this colour parameter. the higher value of a* parameter denotes the increase of red colour saturation, which can be associated with the enzymatic browning reaction occurring during pre-treatment and the drying process (vadivambal and jayas, 2007). this trend can be observed for the samples treated with ultrasound waves for 20 minutes, where parameter a* equalled 1.45±0.75, but statistical analysis did not show any significant differences in relation to the untreated dried apple tissue (table 3). however, the lowest values of this parameter were obtained by the samples subjected to ultrasound for 10 and 30 minutes. as compared to the untreated material the statistically significant effect occurred only after 30 minutes of pre-treatment, where a* value was equal to -0.53±0.83. table 3. l*, a*, b*, chroma c* values of the dried apple slices and total colour differences (∆e) in comparison to the dried untreated apples. type of treatment l* a* b* c* δe untreated 82.07±1.87a 0.91±0,98 bc 21.42±3.23 a 21.45±2.93 a us 10 min 84.27±1.33ab 0.02±0.75 ab 19.81±0.92 a 19.82±0.81 a 2.93±1.42 us 20 min 82.01±1.22a 1.45±0.75 c 22.01±1.27 a 22.07±1.15 a 1.36±1.24 us 30 min 84.57±0.73b -0.53±0.83a 20.77±2.21a 20.79±1.82a 3.57±0.33 a, b, c: the same letters indicate homogeneous groups. in the case of b* parameter, the lowest value was observed after 10 minutes of ultrasound application (19.81±0.92) (table 3), whereas the highest level of this parameter was noted for the samples subjected to ultrasound for 20 minutes (22.01±1.27). the statistical analysis did not show any significant differences between the untreated and ultrasound pre-treated ital. j. food sci., vol 29, 2017 351 samples. the same tendency was noticed for chromaticity c* (table 4) which determines the purity or saturation of the colour (gonçalves et al., 2007). the l*,a* and b* colour parameters were used to calculate the total colour differences (∆e). choi et al. (2002) revealed that ∆e value higher than 2 confirms the visible difference. after 10 and 30 minutes of pre-treatment, the obtained results showed higher ∆e value than 2, which means that the colour was changed noticeably in these cases (table 3). however, the invisible colour changes were observed for the apple tissue treated with ultrasound for 20 minutes, hence ∆e value was lower than 2. 3.4. structure of the ultrasound treated apple tissue fig. 2 shows a photo of the dried apple tissue with and without ultrasound treatment. dried untreated apple tissue is characterized by high density and small pores with elongated cell shape. in the case of the dried tissue subjected to ultrasound treatment, the changes in the structure were observed (fig. 1). the results confirm that the ultrasound treated material exhibited a lower density and more porous form (table 2). however, it was impossible to clearly determine the effect of ultrasound treatment time on microstructure changes, as it was done by other researchers (fernandes et al., 2008a; fenandes and rodrigues, 2009; nowacka et al., 2012; nowacka and wedzik, 2016). moreover, it was not noticed that ultrasound treatment before drying caused the formation of a microscopic channel in dried tissue as reported by nowacka and wedzik (2016) in carrot or fernandes et al. (2008a) in pineapple. figure 2. photos of dried apples taken by using scanning electronic microscopy at a magnification of 100: untreated samples (a), 10 min (b), 20 min (c), 30 min (d) ultrasound treated samples. 3.5. mechanical properties texture evaluation of the product is an important feature to determine the quality of dried fruits (chong et al., 2008; kek et al., 2013). fig. 3 shows texture changes of the ultrasound treated dried apple slices, where results of the maximum cutting force and the ital. j. food sci., vol 29, 2017 352 cutting energy of dried samples were presented. the ultrasound treatment caused alteration of apple tissue, which resulted in changes of textural properties of the dried material. moreover, it was observed that the maximum cutting force and cutting energy decreased with increasing ultrasound treatment time. taking into account the maximum cutting force and cutting energy, the apple slices subjected to ultrasound for 10 minutes were the most similar to the untreated dried material. the dried material was characterized by the highest hardness; however, the differences were not statistically significant. the lowest hardness of dried apple slices was observed for the material treated with ultrasound for 30 minutes. these samples had significantly lower values of maximum cutting force (196.0±37.5 n) and cutting energy (233.4±47.2 mj) in comparison with the untreated dried tissue, which was probably related to lower density and porosity. figure 3. the maximum cutting force and the cutting energy of dried apple; a, b: the same letters indicate homogeneous groups for maximum cutting force; a, b, c: the same letters indicate homogeneous groups for cutting energy. 3.6. rehydration properties a rehydration is a significant quality criterion of dried food. high temperature during drying causes irreversible structure changes of plant tissue. in order to determine these changes, the reconstitution characteristics are investigated (ciurzynska et al., 2011). the gain of weight and volume occur during the rehydration process due to water uptake, while the water soluble compounds are removed from the interior of tissue to the surrounding. the rate of the rehydration process is determined by the level of tissue destruction (nowacka et al., 2012). the changes of rehydration properties are presented in table 4. the obtained results showed that the prolongation of rehydration time resulted in the increase of moisture uptake. the same tendency was observed during rehydration of apple cylinders var. fuji (deng and zhao, 2008) and apple cubes var. idared (nowacka et al., 2012). however, the use of different ultrasound frequencies (fijalkowska et al., 2016) and different treatment times (nowacka et al., 2012) did not significantly influence rehydration b b ab a bc c b a 0 50 100 150 200 250 300 350 400 untreated us 10 min us 20 min us 30 min maximum cutting force [n] cutting energy [mj] ital. j. food sci., vol 29, 2017 353 properties. the amount of water content in the untreated apple slices after 3 hours of rehydration was equal to 12.0±2.2 kg moisture/kg d.m., while the ultrasound pre-treated samples were characterized by higher levels of moisture content, but the statistical analysis showed that these differences were not significant. the results calculated to the initial moisture of the fresh sample confirmed that after 30 minutes of rehydration the dried apples did not obtain the initial water content. after 60 minutes of rehydration the water content of the samples subjected to ultrasound for 10 and 20 minutes were similar to the fresh apple, while the untreated samples and the samples treated with ultrasound for 30 minutes and after 180 minutes of rehydration were characterized by a higher moisture content than the fresh raw material (table 4). the rehydration process is also related to the loss of water soluble components of dry matter the loss is growing with the increasing time of rehydration. after 30 minutes of dried apple rehydration, the amount of their dry matter content was higher than for the fresh sample. the rehydration for 60 minutes of the samples treated with ultrasound for 10 and 20 minutes resulted in the amount of dry matter content similar to the intact sample. however, the untreated apples and the ones subjected to ultrasound for 30 minutes and rehydrated for 60 minutes and all the samples rehydrated for longer time (180 min) were characterized by lower dry matter content in comparison to the raw material, which was associated with high loss of water soluble solids from the dried tissue (table 4). table 4. water content and loss of water soluble solids after different time of rehydration expressed in kg moisture/kg d.m. and calculated percentage of the initial moisture recovery compared to the fresh fruit. type of treatment rehydration time [min] 30 60 180 30 60 180 water content [kg moisture/kg d.m.] loss of water soluble solids [kg d.m./kg d.m.] untreated 4.0±0.6a 6.3±1.0a 12.0±2.4a 0.71±0.03ab 0.58±0.06a 0.39±0.06a us 10 min 4.5±0.4a 5.5±0.6a 13.3±1.7a 0.67±0.02 b 0.65±0.06a 0.37±0.02a us 20 min 4.2±0.4a 5.5±0.6a 15.3±1.7a 0.73±0.03 a 0.64±0.03a 0.32±0.03a us 30 min 4.7±0.7a 6.5±0.6a 13.7±2.8a 0.67±0.05ab 0.56±0.04a 0.36±0.05a water content [%] loss of water soluble solids [%] untreated -29±11a 12±18a 113±43a 34±16ab -8±13a -48±9a us 10 min -20±8a -3±11a 136±30a 22±10 b 3± 9a -53±6a us 20 min -26±8a -2±11a 172±30a 29±11 a 2±10a -59±5a us 30 min -17±12a 16±11a 144±49a 19±16ab -11±8a -54±8a a,b: the same letters indicate homogeneous groups in column. 3.7. hygroscopic properties hygroscopic properties of plant materials can specify the changes induced by the drying process and other treatment. the course of adsorption kinetics is mainly influenced by the method and drying parameters, as well as by the structure, shrinkage and porosity of a dried product. the smaller shrinkage and the higher porosity result in faster absorption of water vapour (rząca and witrowa-rajchert, 2007; acevedo et al., 2008). according to the creation of microchannels by ultrasonic waves (fernandes et al., 2008a; nowacka et al., 2014; nowacka and wedzik, 2016) the hygroscopic properties may provide information about changes in the structure of the tissue. ital. j. food sci., vol 29, 2017 354 after 72 hours of water vapour absorption from the nacl solution with a water activity of 0.75, the dried apple rings absorbed from 0.36 to 0.38 g h2o/g d.m. (fig. 4). analyzing the water sorption curves, it was observed that the process was most intense in the initial phase for 24 hours. at a later stage, there was a decrease in the rate of moisture adsorption. the untreated samples displayed the greatest ability to absorb water. in most cases, the amount of absorbed water did not depend on the type of pre-treatment. however, the apple tissue treated with ultrasound waves for 30 minutes before drying demonstrated lower hygroscopic properties. this dried tissue was characterized by a higher porosity and a lower density (table 2), which may affect the water binding capacity reduction. at the same time, the lower ability of water absorption could be related to the fact that longer ultrasound treatment resulted in greater damage to the structure of the raw material (fig. 1), which has been proved by other researchers (fernandes et al., 2008a; nowacka et al., 2014; nowacka and wedzik, 2016). figure 4. the hygroscopic properties of the ultrasound treated dried apples a, b: the same letters indicate homogeneous groups after 72 h of water vapour absorption. 4. conclusions ultrasound waves used as a method of pre-treatment enhanced the drying of apple slices. the application of ultrasound induced a reduction of the convective drying time by 5-13% in relation to the untreated material. moreover, the effective water diffusivity was the highest for the apples treated with ultrasound for 30 minutes and it increased by 6% in comparison with the untreated material. furthermore, ultrasound treatment impacted on physical, optical and reconstitution properties of the dried apple tissue; however, the effect of the overall quality of the product was not obviously stated. the ultrasound treatment resulted in the decrease of dry matter content, which is not a very favourable effect due to the loss of the tissue watersoluble components. moreover, after 30 minutes of treatment the significant decrease of density and increase of porosity was observed, which was confirmed by micro-structure assessment. the changes in the structure had an impact on the acceleration of the drying process. the conducted research indicated that all dried samples demonstrated low water activity, which provides microbiological safety of food. additionally, the changes of total colour ital. j. food sci., vol 29, 2017 355 differences (∆e) were unnoticeable for the samples sonicated for 20 minutes, or slightly changed for the tissues ultrasound treated for 10 and 30 minutes. on the other hand, longer time of ultrasound pre-treatment resulted in a significant decrease of dried slices hardness, which could be considered as a negative effect of ultrasonic treatment because a product that should be crispy like apple chips is softened. furthermore, it was observed that with increasing ultrasound treatment time the maximum cutting force and the cutting energy of the dried samples decreased. furthermore, the apples subjected to ultrasonic 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effect of continuous and intermittent ultrasound on drying time and effective diffusivity during convective drying of apple and red bell pepper. j. food eng. 108(1):103-110. singh k.k. and reddy b.s. 2006. post-harvest physic-mechanical properties of orange peel and fruit. j. food eng. 73:112120. sledz m., nowacka m., wiktor a. and witrowa-rajchert d. 2013. selected chemical and physico-chemical properties of microwave-convective dried herbs. food and bioprod. process. 91(4):421-428. vadivambal r. and jayas d.s. 2007. changes in quality of microwave-treated agricultural products-a review. biosystems eng. 98(1):1-16. witrowa-rajchert d., wiktor a., sledz m. and nowacka m. 2014. selected emerging technologies to enhance the drying process. a review. dry. technol. 32(11):1386-1396. paper received october 18, 2016 accepted january 18, 2017 ijfs#697_bozza ital. j. food sci., vol 29, 2017 454 paper effect of storage on the content of selected antioxidants and quality attributes in convection and freeze-dried pears (pyrus communis l.) p. gębczyński*, r. skoczeń-słupska and k. kur university of agriculture in krakow, faculty of food technology, department of fruit, vegetable and mushroom processing, balicka 122, 30-149 krakow, poland *corresponding author. p.gebczynski@ur.krakow.pl abstract fresh, convection dried and freeze-dried pears were examined for selected quality parameters vitamin c and e, total polyphenols, antioxidant activity, rehydration, and colour. both products were analyzed immediately after drying and after long-term (12 months) storage at 2±1 ºc and 20±2 ºc. retention in freeze-dried pears was superior to that in convection-dried products for vitamins and was similar for polyphenols and antioxidant activity. there were no significant differences in lightness between convection and freeze-dried products, either immediately after drying or throughout the storage period. 12-month storage led to a significant increase in the proportion of yellow color in both types of dried product compared to the raw material, and compared with the product after drying. the differences were significant in most cases except for the convection dried pear kept in cold store. keywords: fruit, drying, storing, antioxidants, rehydration, colour ital. j. food sci., vol 29, 2017 455 1. introduction fruits are recognized as a good or very good source of antioxidants in the human diet. these substances form a large group, which comprises polyphenols, vitamins, carotenoids and many others. medical studies have shown a correlation between the consumption of antioxidants and decreased risk of cardiovascular disease and some cancer types (lila, 2004; john et al., 1996; ollson et al., 2004). in view of the seasonal availability most of the fresh fruits, there is a need to find relatively inexpensive methods of preservation that will give products with a similar nutritive value to that of the raw material. although dried fruits have long been a part of the human diet, there is little in the literature on the levels of antioxidant compounds they contain, not excluding even the popular fruit. one such species known is pear pyrus communis (sansavini, 2002). pears are a good source of many valuable nutrients (chen et al., 2007; komes et al., 2013). it is a typical fruit of temperate zones. due to its nutritive values and organoleptic properties, the pear is popular fruit among consumers. it is consumed as fresh fruit but also is popular as processed products, and it is used in juices, nectars, marmalades and purees, dried product, milk products (park et al., 2003). drying fruits allows their preservation by removing most of the free water content, and thus inhibiting microbial and fruit own enzymes activity. dehydration also reduces the weight and volume of the raw material. this method gives the benefit due to the cost of packaging, transport and storage (brennan and lancaster, 1994; guiné and castro, 2003). convection drying (using air circulation) is more widely used in industrial processing than freeze-drying due the high costs of the latter, both in terms of equipment and the process itself. although convection drying is a cheaper process, the resulting product is less abundant in nutritive compounds and more difficult to rehydrate owing to the higher drying temperature and intensive aeration of the material among other factors (michalczyk et al., 2008). apart from the drying method applied, the quality of the dried product may also be affected by the conditions and length of storage, two factors which have received little attention in the literature. the aim of this paper was, therefore, to compare convection dried and freeze-dried pears in terms of the selected quality parameters, antioxidants, rehydration and colour, in each product and the extent to which quality is affected by the conditions and length of storage. 2. material and methods 2.1. material the experimental material consisted of whole and sound pears of the conference cultivar, of uniform size gathered at consumption maturity. fruits were obtained from the orchard experimental station of the university of agriculture in cracow (garlica murowana, cracow district, 50°08’23.3n, 19°55’45.6e). healthy and shaped fruit with a weight of 150.0-180.0 g were washed, peeled, removed the seeds, and sliced into eighths. peeled and sliced pears were blanched in water containing 0.1 % sodium metabisulfite and 0.5% citric acid. blanching time required to inactivate the peroxidase was 60 seconds at a temperature of 96-98 °c. after blanching the material is cooled by spraying cold water and allowed sieves for 30 minutes to drain any residual water and dried in a stream of air. representative samples were then taken to determine the level of the selected indicators in the raw material. the remaining fruits were divided into two batches, one each for convection (cd) and freeze-drying (fd). ital. j. food sci., vol 29, 2017 456 for convection drying, electric dryers designed for drying fruits, vegetables and mushrooms (zorpot zalmet. poland) were used. the process was carried out at 60 ºc for 10 hours to a moisture content of about 10%. for freeze-drying, pears were first frozen at 40 °c in a feutron 3626-51 (ilka feutron, germany) fast freezing chamber (korus, 2012). next, sublimation was performed using a gamma 1-16 lsc freeze dryer (christ, germany). the process was conducted under the following parameters: initial temperature of the frozen raw material: -30 °c; condenser temperature: -52 °c, shelf temperature: +20 °c; duration of secondary drying: 6 hours; shelf temperature: +30 °c. the overall time required to achieve a water content of less than 3% using this method was 20 hours. immediately after drying, the pears in each separate type of dried product (convection and freeze-dried) were thoroughly mixed, placed in airtight plastic containers, left for 7 days to allow for any equilibration of humidity, and mixed once more. next, the containers were opened in conditions of low humidity (< 40%) in order to collect samples for analysis of indicators of chemical composition and to determine rehydration ability at the stage described in this work as “immediately after drying 0 months storage”. the remaining dried product was then packed in a twist off jars, divided into two groups and stored without exposure to light. one group was placed in chilled storage (2±1 °c) and the other stored at room temperature (20±2 °c). 2.1. chemical analysis and colour evaluation the content of vitamin c, e, total polyphenols and antioxidant activity were determined in the raw material, and in products immediately after drying and after 4, 8 and 12 months of storage. additionally, rehydration ability and colour were determined immediately after drying and again after 12-month storage. water content was established by the oven method (aoac, 1984), vitamin c and e content using high-performance liquid chromatography (hplc) (pn-en, 2003; pn-en, 2002). total polyphenols were determined by the folin-ciocalteu spectrophotometric method (singleton et al., 1999) while total antioxidant activity was measured by means of the dpph (2.2-diphenyl-1picryhydrazyl) (pekkarinen et al. 1999). immediately after production and after 12month storage, dried products were also examined for water absorption ability (pn, 1990) as well as for colour by an instrumental method with a minolta cm-3500d spectroscope setting l*a*b* parameters. analyses were made in four replications. the results were statistically evaluated using single-factor analysis of variance and lsd test (statistica v. 12, statsoft, inc.). the standard deviation was calculated for the results obtained. 3. results and discussions antioxidant levels in fresh fruits, including pears, have been discussed in the literature (prior et al., 1998; oms-oliu et al., 2008; markowski et al., 2012). however, there are few works concerned exclusively with preserved products, including dried fruits (chong et al., 2013; vega-gálvez et al., 2012). vitamin c, regarded as a fundamental antioxidant in fruits (santos and silva, 2008), is susceptible to degradation by high ph, increased temperatures, exposure to light and the presence of oxygen, enzymes and such metals as iron and copper (moser and bendich, 1991). it has been observed that good l-ascorbic acid retention during technological treatment is accompanied by similar retention of other nutritive compounds (santos and silva, 2008). the level of vitamin c may, therefore, be an indicator of the degradation of other biologically active substances. ital. j. food sci., vol 29, 2017 457 fresh pears contained 41.7 mg vitamin c/100 g dry matter (6.7 mg/100 g fresh matter) (table 1). similar values, less than 10 mg/100 g fm, gives silva et al. (2010) and tavarini et al. (2010), but ozturka et al. (2015) found in different cultivars of pears 930 mg/100 g fm. table 1. effect of drying methods and storage temperature on the nutrient content in the dried pears. object vitamin c [mg/100 g dry matter] vitamin e [mg/100 g dry matter] total polyphenol [mg/100 g dry matter] antioxidant activity [μm trolox /1g dry matter] raw material 41.7±1.9 0.94±0.03 597±27 100±3 dried fruits, time and temperature of storage [months] [ºc] cd fd cd fd cd fd cd fd 0 12.9±0.7 18.5±0.9 0.50±0.03 0.79±0.05 528±20 555±21 71±3 91±4 4 2±1 11.6±0.5 17.6±0.8 0.31±0.02 0.51±0.01 505±18 527±22 61±3 61±4 20±2 10.7±0.5 16.2±0.6 0.25±0.02 0.48±0.01 486±19 501±21 54±3 56±4 8 2±1 10.7±0.5 16.6±0.6 0.44±0.01 0.42±0.03 485±24 494±18 57±4 61±3 20±2 9.9±0.5 14.5±0.7 0.20±0.03 0.34±0.01 472±21 453±22 50±3 49±2 12 2±1 10.3±0.4 15.6±0.4 0.22±0.01 0.36±0.02 463±16 473±17 54±3 56±3 20±2 9.6±0.6 12.5±0.7 0.14±0.01 0.27±0.01 439±16 427±18 45±2 45±3 lsd (α = 0.05) 1.10 0.031 28.8 4.5 cd convention drying, fd freeze-drying. drying caused significant vitamin c loss in both convection and freeze dried pears: 69% and 56% respectively. this confirms the earlier findings for strawberry and american cultivars of blackberry, in which freeze-drying resulted in better l-ascorbic acid retention than other drying methods. this being attributed to lack of oxygen and lower temperature of the process (asami et al., 2003). reduction of vitamin c losses can be achieved by using neutral gas instead of air in the convection drying (ramesh et al., 1999). vitamin c content fell steadily throughout the 12 month period of storage at both storage temperatures. although at every stage of evaluation. the freeze-dried product contained significantly more vitamin c than convection dried. in addition, vitamin c levels were higher in products stored at the lower temperature. after 12 months of storage vitamin c retention, compared with the raw material, was 23-25% in the convection dried product and 30-37% in the freeze-dried product; and 74-79% and 68-84% respectively compared with the product immediately after drying (the two values refer to the higher and lower storage temperature respectively). vitamin e, which comprises a number of tocopheroland tocotrienol-derived compounds, is subject to degradation from exposure to oxygen and uv radiation and the presence of iron (lin et al., 2006). vitamin e content in fresh pears was 0.94 mg/100g dry matter (0.157 mg/100 g fm) (table 1). according to lin et al. (2006), the edible part of the pears had about 0.2 mg vitamin e per 100 g fm. the drying process caused significant though but moderate losses in vitamin e content compared with the raw material: 47% in the convection dried product and 26% in the freeze-dried product. examination of vitamin content in apricots after microwave and radiation drying showed that the shorter exposure to high temperature in microwave drying resulted in better vitamin e retention (karatas and kamişli, 2007). daood et al. (1996) comparing natural drying of paprika under ambient conditions with forced-air ital. j. food sci., vol 29, 2017 458 dehydration, showed that the former method led to higher losses of α-tocopherol. vitamin e loss after 12 months’ storage was significant; their levels in convection and freeze-dried products were 15-22% and 28-38% respectively of those found in the raw material and 2844% and 34-46% of those in the product immediately after drying (the two values refer to higher and lower storage temperature respectively). industrially dried peaches, pears, and plums contained respectively 75, 76, and 48% of the vitamin e levels in fresh fruits, although there is no information concerning the conditions and length of storage (chun et al., 2007). in the convection dried and comminuted paprika observed falls in αtocopherol content were 70%, 90% and 100% in products stored for 30, 60 and 90 days respectively (daood et al., 1996). polyphenols form one of the principal groups of plant secondary metabolites. pear fruits are characterized by moderate polyphenol content (naczk and shahidi, 2006). in fresh pears total polyphenols amounted to 597 mg/100 g dry matter (96 mg/100 g fm). the content of this substances can vary over a wide range, for example, catechin can range from 40-544 mg/kg fm although considerably lower levels of 525 mg/100 g and 429 mg/100 g fm (ozturka et al., 2015). total phenols can vary from 30 mg in italian coscia cultivar (tavarini et al., 2010) up to 232 mg/100 g fm in unidentified thai cultivar of pyrus pyrifolia (chong et al., 2013). convection and freeze-drying caused moderate though still significant reductions in polyphenol content of 47 and 26% respectively compared with the raw material. chong et al. (2013) reported losses in dried pears of 13-66%, depends on the used drying methods. further slight losses in total polyphenols were observed throughout the 12-month storage period, becoming significant after 8 months. the effect of both the drying method and lower temperature was not always proved statistically. after 12 months’ storage polyphenol retention in convection and freeze-dried products was 74-78% and 72-79% respectively compared with the raw material, and 82-88% and 77-85% compared with the product immediately after drying (the two values refer to storage at 20±2 ºc and 2±1 ºc respectively). the level of antioxidant activity depends on the fruit species, cultivation conditions, the length of storage and method of measurement (connor et al., 2002; kalt et al., 1999). antioxidant activity in fresh pears was 100 μm trolox eq/1 g dry matter (16.1 μm trolox eq/1 g fm). chong et al. (2013) using an identical method, reported a value of 16.6 µm trolox eq/g, while kevers et al. (2011) who applied the oxygen radical absorbance capacity (orac) method, recorded 27.5 µm trolox eq/g in an extract of conference pear, and 14.6-42.5 µm trolox eq/g fm for five other cultivars. convection drying and freezedrying caused 29 and 9% reductions in antioxidant activity. storage of products, however, led to larger losses, becoming significant after 4 and 12 months in air-dried product and after first 4 and 8 months in freeze-dried ones. antioxidant activity was not significantly higher in freeze-dried than in convection-dried products at all stages of storage experiment. the lower storage temperature was found to have a beneficial effect. after 12 months of storage, antioxidant activity in convection and freeze-dried products was lower by 46-55 %, and by 44-55% compared to the raw material, and by 63-76% and by 49-62% compared to the product immediately after drying (the two values refer to storage at 20±2 ºc and 2±1 ºc respectively). the content of vitamin c and polyphenols in fresh berry fruits was positively correlated with the level of antioxidant activity (connor et al., 2002; kalt et al., 1999; kevers et al., 2007). in comparison with other fruit species, extracts of pears had moderate amounts of polyphenols and lower amounts of vitamin c (garcia-alonso et al., 2004). hence, wang et al. (1996) reported that vitamin c did not account for more than 15% of total antioxidant activity. our results showed that for dried pear products stored for 12 months the correlation coefficients calculated between antioxidant activity and polyphenols, and ital. j. food sci., vol 29, 2017 459 vitamin c were 0.89 and 0.80 respectively, regardless of the drying method or storage temperature. the water content in the raw material affects the yield of the dried product, its quality and tendency to go mouldy. fresh pears contained 83.97 g water per 100 g. convection drying removed water to the level of 9.13 g/100 g immediately after production. in freeze-drying, the respective value was 2.89 g/100 g. therefore, both products conformed to the methodical assumptions of this research with water contents after 12-month storage of 9.58-9.66 g in the convection dried product and 2.95-2.97 g/100 g in the freeze-dried product. good rehydration properties are an essential characteristic of quality in dried products (ratti, 2001). when apples, bananas, carrots, and potatoes were dried using five different methods, freeze-drying resulted in the highest porosity and natural drying in the lowest [krokida and maroulis, 1997]. this statement agrees with our observations because the higher water absorption ability of fd pears could be explained, above all, by higher porosity. immediately after drying, 100 g of convection dried pears absorbed 360 cm3 of water, while freeze-dried ones absorbed 19% more (table 2). this tendency remained unchanged after 12 months of storage. although absorption power decreased by 12-14% and 9-11% in convection and freeze-dried products respectively (the two values refer to storage at 20±2 ºc and 2±1 ºc respectively). table 2. the ability of water absorption by dried pears immediately after drying and after 12 months of storage, ml/100 g dried fruits. colour is a crucial factor determining the sensory attractiveness of fruits. changes in colour may indicate deterioration in the quality of a product due to processing and storage. colour is determined by the presence of natural pigments and the degree of their decomposition as well as the interactions and degradation of other components in fruit, which occur, for example, during the process of enzymatic and non-enzymatic browning (chong et al., 2013; pasławska, 2005). in the present work, the colour of the raw material and dried products was determined according to the cie (l*a*b*) system (table 3). the drying process caused significant increase of lightness (the l* value increased by 15-17% in convection and freeze-dried pears). only freeze-drying resulted in a significant change in a* value, i.e., the decrease in the red colour, as compared to the raw material. this occurrence can be explained by the use of blanching before drying. this operation in aqueous solution could cause rinsing of the ingredients responsible for a* parameter in fresh fruits, e.g., water-soluble polyphenols. then, low fd drying temperatures and lack of oxidation enzymes in the blanched material caused that the darkening no took place. in turn, the cd pears were dehydrated at a temperature that could induce maillard reactions (vega-galvez et al., 2012) and thus increase a*. the above explanation also seems to confirm minor changes in b*. positive values of b* parameter (b* > 0) correspond to the yellow colour formed mainly by water-insoluble carotenoids (guiné and barroca, 2012). however, it should be noted that the values of parameters a* and b* were small, with a* close to zero and parameter l* was over 80, which translated into the colour of dried material after storage time [months] storage temperature [ºc] convention drying freeze-drying 0 360±12 428±10 12 2±1 318±13 398±8 20±2 308±10 380±8 lsd (α = 0.05) 12.3 ital. j. food sci., vol 29, 2017 460 fresh and dried pears similar to white or cream-white. conversely, the b* value determined in the convection dried product was 14% higher than in the raw material, while in the freeze-dried product this difference was insignificant. table 3. effect of drying method and storage temperature on changes of colour parameters l*a*b* in the dried pears. cd convention drying,. fd freeze-drying. 4. conclusions retention rates in dried products stored for 12 months were similar for vitamin c and vitamin e, 23-37% and 15-38% respectively, and over 70% total polyphenols. retention rates for antioxidant activity against the dpph radical were between these values, 45-56%. retention in freeze-dried products was superior to that in convection-dried products for both vitamins and was similar for total polyphenols and total antioxidant activity. in addition, retention rates were almost ever significantly higher at the lower storage temperature. average losses of vitamin c and total polyphenols were higher during drying than over the 12-month storage period, while for vitamin e and antioxidant activity the losses were lower, slightly in the case of the former, and distinctly so for the latter. there were no significant differences in l* value between convection and freeze-dried products, either immediately after drying or throughout the storage period. 12-month storage led to a significant increase in the proportion of yellow colour in both types of dried product compared to the raw material; however, compared with the product immediately after drying, the differences found were significant in most cases except for the convection dried product kept in chilled storage. acknowledgements this research was supported by ministry of science and higher education project nn312 441837 (2009-2012). references aoac. 1984. 32.064 official methods of analysis. 14th ed. association of official analytical chemists, arlington va, usa. asami d.k., hong y.j., barrett d.m. and mitchell a.e. 2003. comparison of the total phenolic and ascorbic acid content of freeze-dried and air-dried marionberry, strawberry, and corn grown using conventional. 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reagent. methods in enzymol. 299:152-178. tavarini s., trinci l., degl’innocenti e. and guidi l. 2010. different sensitivity to browning in fresh-cut pineapple, apple and pear: the role of endogenous vitamin c. ital. j. food sci. 22:171-179. vega-gálvez a., ah-hen k., chacana m., vergara j., martínez-monzó j., garcía-segovia p., lemus-mondaca r. and di scala k. 2012. effect of temperature and air velocity on drying kinetics, antioxidant capacity, total phenolic content, colour, texture and microstructure of apple (var. granny smith) slices. food chem. 132: 51-59. http://dx.doi.org/10.1016/j.foodchem.2011.10.029 wang h., cao g. and prior r.l. 1996. total antioxidant capacity of fruits. j. agric. food chem. 44:701-705. http://dx.doi.org/10.1021/jf950579y paper received november 23, 2016 accepted march 22, 2017 paper ital. j. food sci., vol. 28 2016 25 keywords: polygonum cuspidatum, antioxidant, polyphenol, neochlorogenic acid identification of neochlorogenic acid as the predominant antioxidant in polygonum cuspidatum leaves serika kurita, takehiro kashiwagi*, tomoyo ebisu, tomoko shimamura and hiroyuki ukeda faculty of agriculture, kochi university, b 200 monobe, nankoku city, kochi prefecture, japan *corresponding author: tel. +81 88 864 5184, fax +81 88 8645189, email: tkashi@kochi-u.ac.jp abstract to identify the predominant antioxidant compound in polygonum cuspidatum leaves, the methanol extract of fresh samples were separated by liquid–liquid partitioning, octadecylsilyl sep-pak® cartridge and high-performance liquid chromatography. the main active compound was identified as (1r,3r,4s,5r)-3-{[(2e)-3-(3,4-dihydroxyphenyl)-2-propenoyl]oxy}-1,4,5-trihydroxycyclohexanecarboxylic acid (neochlorogenic acid) by nuclear magnetic resonance and liquid chromatographymass spectroscopic analysis. its content was found to be 2.31 mg/g of fresh leaves. as shown by 1,1-diphenyl-2-picrylhydrazyl (dpph) radical and superoxide anion scavenging assays, the contributions of neochlorogenic acid as an antioxidant were 16.5% and 36.5%, respectively, suggesting that neochlorogenic acid is the predominant antioxidant in p. cuspidatum leaves. mailto:tkashi%40kochi-u.ac.jp?subject= 26 ital. j. food sci., vol. 28 2016 introduction polygonum cuspidatum, commonly known as japanese knotweed, originated in east asia and has spread widely to european and american countries where it has been listed as one of the most invasive plants. in some invaded areas it has become a severe environmental problem and governmental actions have been taken to thwart its spread (grevstad et al., 2013). however, the chemical and mechanical methods that have been used have not been successful in eliminating this plant, owing to its viability. contrastingly, in other areas, p. cuspidatum has been used as medicine and consumed as a food. for example, in china its dried rhizomes are used in traditional chinese medicine to treat inflammatory diseases, hepatitis, tumors, and diarrhea (chen et al., 2013). it is also reported that the young stems of p. cuspidatum were consumed by native people of north america (chen et al., 2013). in some areas of japan, such as kochi prefecture, the edible portions of young stems are pickled and cooked to be served as traditional dishes even today. the young leaves have also been recognized as edible (hashimoto, 2003). over the past few decades the health-promoting effects of p. cuspidatum have attracted the attention of researchers and several bioactive compounds, particularly those with antioxidant activity, have been identified. resveratrol, or trans-3,5,4′-trihydroxystilbene, also found in grape skins and wine, is abundant in the rhizomes of p. cuspidatum. numerous health-promoting effects of resveratrol, including anticancer, anti-inflammatory, antiviral, and antifungal activities have been described (peng et al., 2013). polydatin, a glycoside precursor to resveratrol, is also found in abundance in the rhizomes of p. cuspidatum. polydatin has been linked with beneficial lipid-regulating, melanogenesis-inhibitory and hepatoprotective effects (peng et al., 2013; chu et al., 2005). besides stilbene compounds, other antioxidants including anthraquinones, such as emodin and physcion, and flavonoids, such as catechin and quercetin, that possess health-promoting properties have also been found in the rhizomes of p. cuspidatum (chen et al. 2013; peng et al., 2003; chu et al., 2005). less research has been performed on the different parts of the plant. the rhizomes have been the most studied however the health-promoting effects of other parts of p. cuspidatum have not been studied. although the stems and leaves are not as commonly used as the rhizomes, in a previous study we observed the antioxidant effect of the leaves was comparable to that of the rhizomes (kurita et al., 2014). despite their high antioxidant capacity, only a few studies have been performed to identify antioxidant compounds in the leaves. in this study, we isolated and identified the predominant antioxidant compounds in the leaves of p. cuspidatum. materials and methods instruments to determine 1,1-diphenyl-2-picrylhydrazyl (dpph) radical scavenging activity, a tecan cts-r r-10 microplate reader (tecan, mannedorf, switzerland) was used. high-performance liquid chromatography (hplc) was performed with an lc-7100 pump, l-2300 column oven, and l-2420 uv vis detector (hitachi, tokyo, japan). liquid chromatography-mass spectroscopy (lc-ms) was performed with a waters acquity uplc system (waters, milford, usa) with a cosmosil® 5c 18 ar-ii column (150 × 4.6 mm i.d., particle size 5 µm, pore size 12 nm), (nacalai tesque inc., kyoto, japan). the mobile phase of lc-ms included 20% meoh, 1% acetic acid and 79% h 2 o at a flow rate of 0.5 ml. positive ion esi with the capillary voltage at 3 kv was used. the source and desolvation temperatures were 150°c and 400°c, respectively, and the eluted compounds were detected at 254 nm. 1hand 13c-nmr data for compound 1 were measured using a jeol jnm-ecx500 (jeol resonance inc., tokyo, japan) at 500 mhz. the letters (br.) s, d, t, q and m represent (broad)singlet, doublet, triplet, quartet, and multiplet, respectively, and coupling constants are expressed in hz. specific rotation was determined by horiba sepa-500 (horiba ltd., kyoto, japan), and the uv spectrum was measured with a pharmacia biotech ultraspec 3000 uv/visible spectrophotometer (ge healthcare uk ltd., buckinghamshire, uk). for the folin–ciocalteu method, uvmini-1240 uv-vis spectrophotometer (shimadzu, kyoto, japan) was used for measurement. chemicals and reagents all reagents used were of analytical grade or better. dpph and hplc-grade methanol were purchased from wako pure chemical industries (osaka, japan). neochlorogenic acid was obtained from sigma chemical co. (st. louis, usa), and chlorogenic acid was from mp biomedicals, lcc (santa ana, usa). phenol reagent solution for folin–ciocalteu assay was purchased from nacalai tesque inc. (kyoto, japan). superoxide dismutase (sod) assay kitwst was purchased from dojindo laboratories (kumamoto, japan). isolation of antioxidants from p. cuspidatum sample materials were collected in murotoshi, kochi prefecture, japan, in may 2013. the roots, stems, and leaves of p. cuspidatum were separated and extracted in an aqueous solution containing 80% methanol (meoh) for 24 h, and the extraction was repeated twice. the extract was filtered using minisart® rc 15 syringe filters made from regenerated cellulose ital. j. food sci., vol. 28 2016 27 with a pore size of 0.45 µm (sartorius stedium, göttingen, germany). twenty grams equivalents of fresh leaf weight (f.w.) were evaporated until dry under reduced pressure (1110 mg) and subjected to liquid–liquid partitioning. the residue of the meoh extract was dissolved in 27.7 ml of water, and the solution was partitioned between hexane (19.6 ml × 3) and water and then between ethyl acetate (19.6 ml × 3) and water. the hexane (53.3 mg), ethyl acetate (93.2 mg), and water (960 mg) layers were collected. the water layer (1 g f.w. equivalent) was applied to a sep-pak® plus c18 cartridge (waters, milford, usa), containing 360 mg of octadecylsilyl (ods), and eluted with increasing concentrations of meoh to obtain four fractions: 0% meoh (25.6 mg), 20% meoh (8 mg), 40% meoh (3.6 mg), and 100% meoh (trace amount) fractions. the ods 20% meoh fraction was further separated into six fractions by reverse-phase semipreparative hplc (cosmosil® 5c 18 ar-ii column, 250 × 10 mm i.d., particle size 5 µm, pore size 12 nm, nacalai tesque inc.) and eluting with 20% meoh containing 1% acetic acid at a flow rate of 3 ml/min and detected at 254 nm. compound 1 was isolated from fraction 2, and its structure is (1r,3r,4s,5r)-3-{[(2e)-3-(3,4dihydroxyphenyl)-2-propenoyl]oxy}-1,4,5-trihydroxycyclohexanecarboxylic acid, neochlorogenic acid. [α] d 20 + 12.00° (c = 0.02, meoh). uv λ max (meoh) nm (ε): 238.5 (5383), 324.2 (8729). positive-ion esi-ms: m/z 355 [m+h]+, 163 [m-quinic acid]+. nmr spectral data were as follows. 1hnmr (500 mhz, dmso-d 6 ) δ: 7.44 (d, 1h, j = 16.0 hz, h c -3), 7.00 (d, 1h, j = 2.5 hz, h c -2′), 6.93 (dd, 1h, j = 8.0, 2.5, hz, h c -6′), 6.75 (d, 1h, j = 8.0 hz, h c -5′), 6.21 (d, 1h, j = 16.0 hz, h c 2), 5.16 (dt, 1h, j = 3.5, 8.5 hz, h q -3), 3.84 (dt, 1h, j = 7.5, 4.0 hz, h q -5), 3.15 (m, 1h, h q -4), 2.00 (dd, 1h, j = 15.0, 4.0 hz, h q -2′), 1.89 (dd, 1h, j = 15.0, 7.5hz, h q -2), 1.83 (m, 2h, h q -6). 13c-nmr (125 mhz, dmso-d 6 ) δ: 176.1 (c q -7, s), 166.2 (c c -1, s), 148.2 (c c -4’, s), 145.6 (c c -3’, s), 144.5 (c c -3, d), 125.9 (c c -1’, s), 121.2 (c c -6’, d), 115.9 (c c -5’, d), 115.2 (c c -2, d), 114.6 (c c 2’, d), 73.1 (c q -1, s), 71.6 (c q -5, d), 71.1 (c q -3, d), 67.2 (c q -4, d), 39.5 (c q -2, t), δ 35.2 (c q -6, t). structural determination of compound 1 the structure of compound 1 was established by independent injection and co-injection of fraction 2 with an authentic preparation in hplc to confirm the retention times. the following conditions were used to identify the compound found in fraction 2: a cosmosil® 5c 18 ar-ii column (150 × 4.6 mm i.d., particle size 5 µm, pore size 12 nm, nacalai tesque inc.) was used with a mobile phase of 20% meoh containing 1% acetic acid at a flow rate of 0.5 ml/min, and uv detection was set at 254 nm. determination of total phenolic content the polyphenol content of p. cuspidatum leaves was determined by the folin-ciocalteu method as described by singleton et al. with some modifications (singleton et al., 1999). in a test tube, 0.25 ml of sample solution, 0.1 ml of phenol reagent (1.8 n), and 0.25 ml of saturated sodium carbonate were added within 15 s and mixed. then, 2.15 ml of water was added and mixed, followed by 1 h of incubation at room temperature. after incubation, the sample was measured at 725 nm. the measured value for the crude extract was expressed as gallic acid equivalent (gae) per gram of the sample material. dpph radical scavenging activity assay antioxidant activity was measured using the dpph method as described in our previous study (kurita et al., 2014). in a 96-well plate, 20 µl of sample solution, 80 µl of 0.1 m tris-hcl buffer (ph 7.4), and 0.2 mm dpph in ethanol solution were added and mixed. the mixture was incubated in the dark at room temperature for exactly 30 min. the radical scavenging rates of each sample and a control solution were measured at 517 nm. all experiments were performed in triplicate. the radical scavenging rate was calculated using following equation: scavenging rate (%) = = (a control – a sample ) / a control × 100 where a control is the absorbance of the control and a sample is that of the sample. sc 50 , which is the sample concentration at 50% of the scavenging ratio, was used to express the antioxidant capacity of each sample. to determine the contribution rate, sc 50 was then converted to 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) equivalent (te) antioxidant capacity, teac, using the following equation (shimamura et al., 2014): teac (mg te/mg) = trolox sc 50 (mg/ml)/ /sample sc 50 (mg /ml) the contribution rate of the active compound was calculated using the following equation: contribution rate (%) = (teac of active compound × concentration of active compound in p. cuspidatum) / (teac of crude extract) × 100 superoxide anion scavenging assay a sod assay kit-wst was used to determine the superoxide scavenging activity (sosa) of each sample. the assay was performed according to the manufacturer’s procedure. the resulting 50% inhibitory concentration (ic 50 ) was used to determine the sosa, which was further used to evaluate the contribution of compound 1 to 28 ital. j. food sci., vol. 28 2016 the total antioxidative capacity. sosa was defined using following equation: sosa (unit/g) = [1/ic 50 (mg/ml)] × 0.02 ml × × 1000 mg/g the contribution rate from sosa was calculated using the following equation: contribution rate (%) = (sosa of active compound × concentration of active compound in p. cuspidatum) / (sosa of crude extract) × 100 results antioxidant capacities of different parts of p. cuspidatum all results of dpph radical scavenging activity assays had relative standard deviations (rsd) of < 5%. among the meoh extracts of the different parts of p. cuspidatum, the strongest activity was observed in the leaves (sc 50 : 1.24 mg f.w./ml), followed by the rhizomes (sc 50 : 1.63 mg f.w./ml) and stems (sc 50 : 14.1 mg f.w./ml). this is consistent with our previous study which also found that the leaves and rhizomes showed almost equivalent antioxidant capacities (kurita et al. 2014). fractionation and antioxidant activity of the leaf extract the fractionated leaf extracts and antioxidant activities are shown in fig. 1. among all the layers, the water layer showed the highest activity (sc 50 : 1.90 mg f.w./ml), followed by the ethyl acetate layer (sc 50 : 13.2 mg f.w./ml). the separated hexane, ethyl acetate, and water layers were further combined for measurement. the combined sample yielded an sc 50 of 1.8 mg f.w./ml, although some activity had been lost in the separation process. the combinations of the hexane and water layers (sc 50 : 1.93 mg f.w./ml), and the ethyl acetate and water layers (sc 50 : 1.69 mg f.w./ml) were measured and compared with the water layer only. the results suggest that the antioxidants were present mainly in the water layer because the activities of these combinations were close to that of the water layer only. antioxidant activities were observed in the ods water and in the 20% and 40% meoh fractions (fig. 1). the ods 20% meoh fraction showed the highest activity, yielding an sc 50 of 4.3 mg f.w./ ml. all the fractions combined had an sc 50 of 1.55 mg f.w./ml. when the ods 20% meoh fraction was combined with the second highest fraction, the ods water fraction (sc 50 : 5.56 mg f.w./ml), the sc 50 of the combined sample was 1.68 mg f.w./ml. this suggests that the ods water and the 20% meoh fractions account for the majority of the antioxidant capacity of the water layer. the ods 20% meoh fraction was further fractionated by reversed phase semipreparative hplc, and the chromatogram is shown in fig. 2. the highest antioxidant capacity was seen in fraction 6 (sc 50 : 22.4 mg f.w./ml), followed by fraction 1 (sc 50 : 32.4 mg f.w./ml) and fraction 2 (sc 50 : 36.1 mg f.w./ml). a further hplc analysis with multiple-wavelength detection using a spd-m10a photodiode array detector (shimadzu, kyoto, japan) detected no other distinct peaks in fraction 1 or 6. fractions 1 and 6 were further separated to isolate and identify the compound; however, the antioxidant activity was dispersed during the process. in contrast fraction 2, which exhibited relatively high antioxidant activity, contained a major single peak at the retention time of 10.07 min. this major peak was assigned as compound 1, which was further purified. compound fig. 1 the separation process of the leaf extract and the antioxidant capacity of each fraction. ital. j. food sci., vol. 28 2016 29 1 was present not only in the ods 20% meoh fraction but also in the ods water fraction, which showed the second highest antioxidative activity among the ods fractions. in the ods water fraction, compound 1 was also found abundantly and accordingly was inferred to be the major compound in the water layer of the leaf extract. identification of compound 1 compound 1 was found to have sixteen carbon atoms consisting of two methylene, eight methine, and six quaternary carbon atoms including two carbonyl groups (c q -7, δ 176.1 and c c -1, δ 166.2) as a result of 13c-nmr. this result was consistent with 1h-nmr, which showed the presence of twelve hydrogen atoms in the spectrum. this compound contains a trans-form double bond (c c -2, δ 115.2 and c c -3, δ 144.5) signified by two hydrogen signals (δ 6.21 and δ 7.44) corresponding to a double bond where doublet with 16 hz coupling constant. this double bond and a carbonyl group (c c -1, δ176.1) were observed to be conjugated, consistent with these chemical shifts and the result from heteronuclear multiple bond correlation (hmbc). because the observed six aromatic carbons in the 13c-nmr spectrum corresponded to an abx system at δ 6.75 (hc-5′, d, j = 8 hz), δ 6.93 (hc-6′, dd, j = 2, 8 hz), and δ 7.00 (hc-2′, d, j = 2 hz) in 1h-nmr, compound 1 was found to contain a 1,2,4-trisubstituted benzene ring. for the abovementioned reasons, compound 1 was inferred to contain a caffeic acid moiety. in the rest of the structure, three methine carbon atoms with oxygen atoms, one quaternary carbon, two methylene carbon atoms, and one carbonyl carbon were found. two-dimensional nmr spectral data imply a six-membered ring substituted with four oxygen atoms. the methylene proton at δ 1.85 and the carbonyl carbon (c q -7, δ 176.1) were interrelated in hmbc spectroscopy. the other moiety was thus determined to be a quinic acid derivative. the proton corresponding to the carbon of quinic acid (c q -3, δ 71.1) showed a downfield shift at 5.16 ppm, suggesting that this compound formed a caffeate ester. the molecular formula of a caffeoylquinic acid is c 16 h 18 o 9 and its molecular weight is calculated to be 354. based on the esi mass data (m/z 355 [m+h]+), the molecular weight of compound 1 was found to be 354; therefore, compound 1 was assigned the molecular formula c 16 h 18 o 9. the data in the literature from 13c-nmr and 1h-nmr studies on chlorogenic acid (5-caffeoylquinic acid), cryptochlorogenic acid (4-caffeoylquinic acid) and neochlorogenic acid (3-caffeoylquinic acid) were compared with our observed data and most of the values for compound 1 matched with those of neochlorogenic acid (fig. 3) (qin et al., 2006; hyun et al., 2010). the specific rotation value of compound 1 was also consistent with that of a neochlorogenic acid standard. to determine the structure, fraction 2 was further analyzed using hplc under the conditions described in determination of structure of compound 1, and the result is shown in fig. 4. the peak of compound 1 was observed at 8.1 min (fig. 4a). the retention time of neochlorogenic acid was clearly different from that of chlorogenic acid; the neochlorogenic acid peak appeared at 8.04 min, whereas the peak of chlorogenic acid appeared at 16.67 min (fig. 4b and 4c). co-injection analysis showed that the peak of compound 1 was identical to that of neochlorogenic acid. accordingly, compound 1 was assigned as neochlorogenic acid. quantification of neochlorogenic acid and its contribution to the whole leaf extract the leaves of p. cuspidatum were freshly collected in otoyo-cho in may 2014 to determine neochlorogenic acid content. one gram of fresh p. cusfig. 2 the chromatogram of ods 20% meoh fraction of the leaf extract. the sc 50 of fr. 1, 2, 3, and 6 were 32.4, 36.1, 62.4 and 22.4 mg f.w./ml, respectively. fr. 4 and 5 was not determined since their sc 50 were over 200 mg f.w./ml. 30 ital. j. food sci., vol. 28 2016 pidatum leaves contained 2.31 mg of neochlorogenic acid. by the folin–ciocalteu method, 17.9 mg gae of phenolic compounds were found to be present in the fresh leaves; thus, neochlorogenic acid comprises 12.8% of the total polyphenol content. to evaluate the antioxidant capacity of neochlorogenic acid in p. cuspidatum two different assays, each measuring the sample’s ability to quench reactive oxygen species in a different way, were performed. antioxidant capacity cannot be evaluated by a single method because reactive oxygen species in the body do not always operate through the same mechanisms. the assays we used in this study were the dpph radical scavenging (teac) and superoxide anion scavenging assays (sosa). the teac values of the crude extract and neochlorogenic acid were 59.7 mg te/g f.w. and 4.25 mg te/mg, respectively, indicating the neochlorogenic acid contribution is 16.5%. fig. 3 the structures of neochlorogenic acid and chlorogenic acid. however, by the superoxide anion scavenging assay, the sosa values of the crude extract and neochlorogenic acid were 22.7 unit/g f.w. and 3.57 unit/mg, respectively, suggesting 36.5% of the antioxidant activity is by neochlorogenic acid. the disparate results may be explained by the different mechanisms of the two antioxidant activities (shimamura et al., 2007). in the dpph method, free radical scavenging activity is achieved by single electron transfer, and the assay simply measures the rate of free radical quenching. the superoxide anion scavenging assay, however, measures the sample’s ability to scavenge superoxide anions produced by xanthine oxidase, thus evaluating the sod-like activity of the sample. the superoxide anion further reduces 2-(4-iodophenyl)3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2h-tetrazolium (wst-1) to produce formazan, which is detectable at a wavelength of 450 nm. thus, the superoxide anion scavenging assay involves competition by the sample antioxidants with wst-1 in addition to the enzymatic reactions of xanthine oxidase. taken together our results indicate that neochlorogenic acid in p. cuspidatum contributes a large part of its antioxidant activity, particularly as a superoxide anion scavenger. in a study by kirino et al. (2012) chlorogenic acid was reported as one of the major polyphenols in the leaves of p. cuspidatum (kirino et al., 2012). the amount of chlorogenic acid was reported to be 0.36 mg/g of fresh leaves, which is only 1/6th of the neochlorogenic acid content observed in this study. in the chromatogram in fig. 4, chlorogenic acid appeared to be a small peak in the water layer of the leaf extract. however, according to the data from kirino et al., the peak of chlorogenic acid was much more distinct than in our study. the contents of such antioxidants in p. cuspidatum may differ depending on its origin and harvest season, as we mentioned in a previous report (kurita et al., 2014). stress factors such as sunlight and insects can influence antioxidant production levels as well. comparison of neochlorogenic acid contents in other food sources and its possible effects on human health to the best of our knowledge, this is the first study to report the presence of neochlorogenic acid in p. cuspidatum leaves. neochlorogenic acid is also found in rosaceae fruits such as plums, cherries, and apples and brassica vegetables such as broccoli and kale (ballistreri et al., 2013; kim et al., 2003; kaulmann et al., 2014). among different kinds of sweet cherries, its content varied between 6.27–71.5 mg/100 g f.w. (ballistreri et al., 2013). plums contain even higher amounts of up to 179 mg/100 g f.w., unsurprisingly neochlorogenic acid has been recognized as the predominant polyphenol in plums (kim et al., 2003). brassica vegetables are also rich in the compound. green vegetables such fig. 4 the chromatogram of the water layer of the leaf extract, neochlorogenic acid and chlorogenic acid. in the water layer of leaf extract (a), compound 1 was observed at 8.11 min. neochlorogenic acid (b) was found at 8.04 min whereas chlorogenic acid (c) was at 16.67 min. ital. j. food sci., vol. 28 2016 31 as kale, broccoli, and brussels sprouts contain 7.06, 5.61, and 4.59 mg/100 g f.w., respectively, of neochlorogenic acid (kaulmann et al., 2014). in comparison with these neochlorogenic-rich fruits and vegetables, the content was much higher in the leaves of p. cuspidatum, which yielded 231 mg of neochlorogenic acid per 100 g of fresh material. our study suggests that the leaves of p. cuspidatum are a rich source of neochlorogenic acid. besides its antioxidant activity, neochlorogenic acid has been shown to exert health-promoting effects. as an antitumor agent, neochlorogenic acid has been found to suppress the growth of estrogen-independent mda-mb-435 breast cancer cells (noratto et al., 2009). this suppressive effect is selective for cancer cells and is more pronounced than that of chlorogenic acid. the compound has also been investigated in a weight-control study (shimoda et al., 2006). in the study performed by shimoda et al. (2006), experimental mice were fed a diet containing neochlorogenic acid (0.028% and 0.055%, respectively) extracted from green coffee beans for 6 days. the hepatic carnitine palmitoyltransferase activity of the experimental mice increased, indicating they had improved fat metabolism. these studies suggest that neochlorogenic acid could play a role in preventing chronic diseases and preserving healthy body weight when consumed in the diet. as a natural source of neochlorogenic acid, the leaves of p. cuspidatum may be used to improve human health in modern society. conclusions for their medicinal effects the antioxidants in p. cuspidatum have been of interest to researchers, but other than the rhizomes the plant has not been extensively studied. the leaves possess high antioxidant activity and can be consumed in the diet as they currently are in japan. given the reports of health-promoting effects of neochlorogenic acid, our result that neochlorogenic acid is a main antioxidant in the leaves of p. cuspidatum may increase the utility of this hardy and prolific plant. acknowledgements we thank soichiro ueta from npo sakihama genki project for providing the samples for our study. references ballistreri g., continella a., gentile a., amenta m., fabroni s. and rapisarda p. 2013. fruit quality and bioactive compounds relevant to human health of sweet cherry (prunus avium l.) cultivars grown in italy. food chem. 140(4): 630-8. chen h., tuck t., ji x., zhou x., kelly g., cuerrier a. and zhang j. 2013. quality assessment of japanese knotweed (fallopia japonica) grown on prince edward island as a source of resveratrol. j. agric. food chem. 61(26): 6383-92. chu x., sun a. and liu r. 2005. preparative isolation and purification of five compounds from the chinese medicinal herb polygonum cuspidatum sieb. et zucc by highspeed counter-current chromatography. j. chromatogr. a. 1097(1-2): 33-9. grevstad f., shaw r., bourchier r., sanguankeo p., cortat g. and reardon r.c. 2013. 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enzymol. 299: 152-178. paper received november 5, 2014 accepted february 19, 2015 #686_ulloa_bozza ital. j. food sci., vol 29, 2017 288 paper modelling of hydration of bean (phaseolus vulgaris l.): effect of the low-frequency ultrasound l.r. lópez lópeza, j.a. ulloa*a,b, p. rosas ulloab, j.c. ramírez ramírezc, y. silva carrillod and a. quintero ramose aposgrado en ciencias biológico agropecuarias. unidad académica de agricultura, universidad autónoma de nayarit, carretera tepic-compostela km 9, cp 63780 xalisco, nayarit, méxico bcentro de tecnología de alimentos, universidad autónoma de nayarit, ciudad de la cultura amado nervo, c.p. 63155 tepic, nayarit, méxico cunidad académica de medicina veterinaria y zootecnia, universidad autónoma de nayarit, carretera a chapalilla km 3.5, cp 63700 compostela, nayarit, méxico dunidad académica de agricultura, universidad autónoma de nayarit, carretera tepic-compostela km 9, cp 63780 xalisco, nayarit, méxico edepartamento de investigación y posgrado, facultad de ciencias químicas, universidad autónoma de chihuahua, circuito universitario s/n, campus universitario no. 2, cp 31240 chihuahua, chihuahua, méxico *corresponding author. tel. +52 3112118851; fax +52 3112118861 e-mail address: arulloa5@gmail.com abstract beans of six varieties were soaked in distilled water at 30 °c and exposed to ultrasound at powers of 5, 12 or 19 w, in addition to treatment control without ultrasound to attain the equilibrium moisture. from four model studied, the weibull model presented the best fit (r2 0.986-0.999) for the experimental data of the hydration kinetics. soaking time was reduced from 52.6 % to 77.2 %, while the effective diffusivity was increased from 2.25 times to 3.50 times at 19 w, depending on the bean variety. the ultrasound power improved the hydration capability being suitable for possible use in industrial applications. keywords: beans, hydration, mathematical modelling, ultrasound, water diffusivity, weibull´s model ital. j. food sci., vol 29, 2017 289 1. introduction common beans (phaseolus vulgaris l.) are a grain legumes that belong to the family of fabaceae. they provide an affordable source of protein (16-33%), almost two to three times that of cereals), and also are a rich source of dietary fibre, starch, minerals and vitamins (mkanda et al., 2007). bioactive compounds present in common bean have been associated with the prevention and/or regulation of chronic degenerative diseases such as obesity, diabetes, coronary heart disease and cancer (campos-vega et al., 2010; plans et al., 2013). on the other hand, the world health organization has issued several recommendations for reducing overweight, obesity and cardiovascular diseases that also are likely to reduce the risk of diet-related diseases, such as type 2 diabetes and obesity. these recommendations include the achievement of adequate intakes of non-starch polysaccharides through regular consumption of wholegrain cereals, legumes, fruits and vegetables (who/fao, 2003). of the leguminous plants consumed by humans, one of those of greatest importance on a worldwide level is the common bean (olmedillaalonso et al., 2013). cooked dry beans along with whole grains have been emphasized as the primary nutritional shifts in food intake patterns to a plant-based diet by the 2010 dietary guidelines advisory committee (campos-vega et al., 2012). before the cooking step, beans are hydrated in water until maximum weight is reached (ulloa et al., 2016). hydration capacity is dependent on the ease of water absorption through the seed coat to the cotyledons (mkanda et al., 2007). however, the soaking process is a time-consuming step, requiring approximately 12 h at room temperature (piergiovanni, 2011), and many attempts have been directed towards shortening it (abu-ghannam and mckenna, 1997). ultrasound power is a novel technology in the food industry, and research on its application is a rapidly growing field. ultrasonic waves can cause a rapid series of alternative compressions and expansions similar to a sponge when it is squeezed and released repeatedly, a phenomenon known as cavitation (chemat et al., 2011). ultrasound cavitation results in the occurrence of microstreaming, which enhances heat and mass transfer (cárcel et al., 2012). ultrasound applications were reported to enhances the hydration of chickpeas (yildirim et al., 2011), sorghum grains (patero and augusto, 2015), and navy beans (ghafoor et al., 2014). however, as soaking conditions vary depending on the particular legume or food material, it is necessary for practical applications to characterise and optimise these conditions. therefore, the objective of the present study was to evaluate the effect of low-frequency ultrasound on the kinetics and modeling of the hydration of the six varieties of the most consumed beans in mexico. 2. materials and methods 2.1. material common bean seeds from six varieties, which are highly consumed in mexico (rodríguez-licea et al., 2010) and classified as most preferred (azufrado, mayacoba, pinto, peruano bola, flor de mayo and negro jamapa), were used for this study and were obtained from the mercado de abastos, located in tepic, nayarit, mexico. these food legumes were separated from broken, small and split. seeds were cleaned and size-graded ital. j. food sci., vol 29, 2017 290 manually. samples were stored in hermetically sealed bags inside closed plastic jars at room temperature (25 °c) in a dark room. 2.2. chemical and morphological characterization of the bean seeds the chemical composition (moisture, protein, fat, ash, and carbohydrates) of the beans was determined following the official methods of the aoac (2002). for the morphological characterization, 100 beans of each variety were selected, and the weight (we), length (l), width (w) and depth (d) were measured to determine the geometric mean diameter (gm), arithmetic mean diameter (am), square mean diameter (sm) and the radius of an equivalent sphere (r) according to the methods reported by gafhoor et al. (2014). 2.3. seed coat content to determine the percentage of the coat of the seeds, the tizazu and emire (2010) method with some modifications was used. thirty bean seeds were soaked in distilled water for 8 h, and the coat was removed manually, separating it from the cotyledon. the seed coats collected were dried in an oven at 60° c for 24 h, followed cooling in a desiccator. it was then weighed and the percentage of seed coat was calculated. 2.4. ultrasound soaking treatments a sample of 5 g of beans was used in each one of the three replicates. the bean samples were soaked in a 100-ml beaker with 50 ml of distilled water and exposed to ultrasound at powers of 5, 12 or 19 w (20 khz, 30 °c), using a ge-130 ultrasonic processor (cole parmer, vernon hills, connecticut, illinois) provided of a titanium probe of 6 mm. at specific time intervals (10 min for ultrasound treatments and 30 min for control, until stabilization), the seeds were removed from the water, drained, superficially blotted with absorbent paper, weighed and returned to the water. 2.5. modeling the kinetics of hydration for fitting the moisture uptake of soaked bean, four models were used to estimate the parameters associated with each model. the list of the models, and the respective equations used in this study, is presented in table 1. the best fitted model was determined by the highest coefficient of determination (r2) and the lowest values of the root mean square error (rmse) and chi-square (χ2) amongst the predicted and experimental results (cox et al., 2012). 2.6. effective diffusivity (deff) deff was determined according to the method reported by kaptso et al. (2008) with the following equation: 𝑐 = ! !!!"" !! eq. 1 where deff (m2/s) is the effective diffusivity, c (s-1) is the rate constant of the water absorption of the mathematical model with the best fit and r (m) is the value of radius of the equivalent sphere. ital. j. food sci., vol 29, 2017 291 table 1. models used to describe moisture content and rate of moisture uptake by effect of ultrasound. model and reference equation peleg (peleg, 1988) m! = m! + ! !!!!!! (2) sigmoid (leal-oliveira et al., 2013) m! = !! !!!"# !!(!!!) (3) first order (ghafoor et al., 2014) mr = 1 − exp (−k!t) (4) weibull (zura et al., 2013) mr = 1 − exp − ! ! ! (5) mr is the rate of moisture uptake, and is given by the equation: 𝑀𝑅 = !!!!! !!!!! where m0 is the initial moisture content of the bean, mt is the moisture content of bean at time t, and me is the final moisture content at equilibrium. t is the hydration duration (in min), and the variables c1, c2, k1, k, τ, α and β are the coefficients used in nonlinear regression analysis with the various models. 2.7. statistical analysis one-way analysis of variance and tukey tests were performed to determine the difference between the values of seed morphological traits (we, l, w, d, gm, am, sm and r), chemical composition (moisture, proteins, fats, ashes and total carbohydrates), %cs and the kinetic parameters of the soaking treatments (me and deff) of the different varieties of beans, using statgraphics plus 5.0 statistical package (statistical graphics corp., md, usa). significance level were tested at p < 0.05. 3. results and discussion 3.1. chemical and morphological characterization of the bean seeds table 2 shows the results of we, l, w, d, gm, am, sm and r of the six bean varieties studied. according to the obtained results, the azufrado bean variety presented the lowest values in the majority of the morphological characteristics studied, and the mayocoba and flor de mayo varieties presented the highest values. although flor de mayo and azufrado beans had the highest r (0.41 cm) and the lowest r (0.30 cm), respectively, the majority of the varieties of bean used in this study presented a higher r value in comparison with the navy bean (ghafoor et al., 2014). table 3 shows the chemical composition of the six varieties of beans studied. the values of the proximal composition of the six bean varieties of this study are similar to those reported by wani et al. (2015) for other bean varieties, with the exception of the moisture content for the mayocoba variety, which was the highest, and the fat content for the pinto variety, which was the lowest. 3.2. kinetics of water absorption the water absorption kinetics of the different varieties of bean studied are shown in fig. 1. the time to attain the me ranged from 330 min to 660 min depending of the bean variety, being the shortest time for mayacoba and longest time for negro jamapa. according to this finding, exposure to ultrasound reduced the soaking time of the bean; however, at a higher power of ultrasound, the time lapse was even lower. from the six different varieties of bean studied, after of the ultrasound treatments at 5w, 10 w and 19 w, the shortest and longest soaking times were 173 min and 280 min, 108 and 360 min, and 123 min and 313 ital. j. food sci., vol 29, 2017 292 min, respectively, corresponding to mayacoba and peruano bola, mayacoba and negro jamapa, and pinto and negro jamapa. table 2. characteristics of the six varieties of common bean (phaseolus vulgaris) seeds. parameters bean varieties mayocoba azufrado pinto peruano bola flor de mayo negro jamapa we (g) 0.39±0.03a 0.25±0.02d 0.36±0.05b 0.32±0.04c 0.35±0.06b 0.24±0.04d l (cm) 1.29±0.06a 1.08±0.06c 1.25±0.08b 1.03±0.06d 1.25±0.01b 0.96±0.07e w (cm) 0.70±0.04cd 0.66±0.03e 0.73±0.05b 0.72±0.03bc 0.76±0.05a 0.69±0.08d d (cm) 0.59±0.05a 0.47±0.04d 0.56±0.05b 0.61±0.03a 0.52±0.04c 0.53±0.04c gm (cm) 0.82±0.04ª 0.70±0.03 d 0.79±.0.04ab 0.76±0.03c 0.79±0.06b 0.71±0.04d am (cm) 0.86±0.04ª 0.74±0.03 d 0.85±0.05ab 0.78±0.03c 0.84±0.06b 0.73±0.04b sm (cm) 0.41±0.01 d 0.37±0.01e 0.41±0.01d 0.84±0.02b 0.87±0.04a 0.80±0.03c r (cm) 0.35±0.01d 0.30±0.00e 0.34±0.02d 0.39±0.01b 0.41±0.02a 0.37±0.02c values are given as means±standard deviation (n = 100). different superscript letters in the same row indicate significant differences (p < 0.05). we: weight; l: length; w: width; d: depth; gm: geometric mean diameter; am: arithmetic mean diameter; sm: square mean diameter; r: radius of an equivalent sphere. table 3. seed proximate composition of the six varieties of common bean (phaseolus vulgaris). component bean varieties mayocoba azufrado pinto peruano bola flor de mayo negro jamapa moisture (%) 12.8 ±0.1a 11.2±0.1bc 11.0±0.4bcd 11.4±0.3b 9.5±0.1e 8.9±0.1f fat (%) 1.6±0.2bc 2.1±0.2b 0.6±0.1d 4.7±0.5a 1.0±0.1cd 1.5± 0.1bcd ash (%) 4.5±0.1a 4.8±0.2a 4.1±0.1a 4.5±0.1a 4.0±0.2a 4.6±0.1a protein (%, n x 6.25) 23.5± 0.5a 23.6±0.1a 23.9±0.5a 23.8±0.4a 23.2±0.2a 23.9±0.5a total carbohydrates (%) 57.6±0.5 58.3±0.2 60.4±0.5 55.6±0.5 62.3±0.2 61.1±0.5 values are given as means±standard deviation (n = 3). different superscript letters in the same row indicate significant differences (p < 0.05). on the other hand, the azufrado and peruano bola bean varieties presented an initial lag phase or lateness (period with a low water absorption rate) for the 5 w ultrasound treatment and the control treatment, whereas the pinto, flor de mayo and negro jamapa bean varieties presented initial lag phase for the ultrasound treatments of 5 w and 12 w, as well as the control treatment. the behavior of the hydration kinetics of this study has been observed in the conventional soaking (25-55° c) of common bean by piergiovanni (2011), who classified the bean varieties into three groups as a function of the rate of hydration (fast, intermediate and slow); the fast and intermediate bean varieties did not present an initial lag phase, but the slow hydration bean varieties did have an initial lag phase. according to this classification, the mayocoba bean variety can be considered to have a rapid hydration rate, in contrast to the rest of the bean varieties studied, even though with the application of the ultrasound power, especially at 19 w, the initial lag phase of the bean hydration of the varieties of ital. j. food sci., vol 29, 2017 293 azufrado, pinto, peruano bola and flor de mayo was reduced. kaptso et al. (2008) reported that the application of ultrasound for soaking has an effect similar to increasing the soaking temperature, thus overcoming the defect of low water absorption observed in the seeds. figure 1. water absorption kinetic of (a) mayocoba, (b) azufrado, (c) pinto, (d) peruano bola, (e) flor de mayo and (f) negro jamapa bean varieties during soaking at different ultrasound powers. solid lines represent the corresponding weibull model fitted to the experimental data. the statistical parameters generated from the application of the different mathematical models (weibull, peleg, first order, and sigmoid) describing the kinetics of water absorption for the bean varieties are presented in table 4. in general, the sigmoid model ital. j. food sci., vol 29, 2017 294 presented higher values of r2 (0.998) and lower χ2 (0.000) and rsme (0.019) than the peleg and first order models (table 4); in addition, sigmoid model can describe the initial lag phase followed by a high rate absorption phase and, finally, a stationary phase (lealoliveira et al., 2013). however, the weibull model presented the best fit, giving the highest values of r2 (0.986-0.999) and the lowest of χ2 (0.000-0.002) and rsme (0.013-0.044), in accordance with marabi et al. (2003), who found that such model is one of the best for describing the kinetics of food hydration. ital. j. food sci., vol 29, 2017 295 table 4. statistical parameters of the mathematical models fitted to the kinetics of hydration of six bean varieties. model ultrasound power (w) mayocoba azufrado pinto peruano bola flor de mayo negro jamapa r2 χ2 rsme r2 χ2 rsme r2 χ2 rsme r2 χ2 rsme r2 χ2 rsme r2 χ2 rsme sigmoidal control 0.961 0.003 0.050 0.997 0.001 0.023 0.997 0.001 0.027 0.989 0.002 0.035 0.991 0.002 0.036 0.993 0.001 0.016 5 0.970 0.002 0.046 0.998 0.000 0.019 0.996 0.001 0.024 0.996 0.001 0.022 0.987 0.002 0.039 0.996 0.001 0.020 12 0.983 0.002 0.040 0.996 0.001 0.024 0.998 0.000 0.019 0.994 0.001 0.029 0.997 0.001 0.020 0.995 0.001 0.025 19 0.985 0.002 0.036 0.996 0.001 0.024 0.997 0.001 0.023 0.985 0.002 0.040 0.992 0.001 0.034 0.995 0.001 0.028 first order control 0.989 0.001 0.026 0.971 0.006 0.070 0.991 0.002 0.046 0.961 0.006 0.070 0.959 0.006 0.076 0.906 0.016 0.123 5 0.990 0.001 0.026 0.921 0.013 0.110 0.940 0.009 0.091 0.949 0.007 0.084 0.981 0.002 0.046 0.933 0.010 0.095 12 0.966 0.004 0.056 0.929 0.013 0.108 0.922 0.014 0.112 0.943 0.009 0.092 0.966 0.005 0.069 0.911 0.014 0.113 19 0.991 0.001 0.028 0.949 0.009 0.090 0.951 0.009 0.087 0.956 0.007 0.080 0.977 0.004 0.059 0.942 0.008 0.087 peleg control 0.994 0.000 0.020 0.977 0.004 0.062 0.994 0.002 0.038 0.973 0.004 0.059 0.962 0.006 0.075 0.959 0.007 0.081 5 0.979 0.002 0.038 0.961 0.007 0.077 0.979 0.003 0.053 0.972 0.004 0.062 0.986 0.002 0.039 0.988 0.002 0.039 12 0.948 0.006 0.070 0.975 0.005 0.064 0.971 0.005 0.067 0.959 0.007 0.078 0.986 0.002 0.0433 0.987 0.002 0.041 19 0.979 0.002 0.044 0.980 0.004 0.056 0.979 0.004 0.057 0.970 0.005 0.059 0.993 0.001 0.031 0.993 0.001 0.031 weibull control 0.995 0.000 0.017 0.996 0.001 0.022 0.999 0.000 0.013 0.997 0.000 0.0171 0.995 0.001 0.025 0.998 0.001 0.018 5 0.991 0.001 0.025 0.997 0.001 0.022 0.998 0.000 0.016 0.999 0.001 0.010 0.997 0.001 0.018 0.991 0.001 0.033 12 0.986 0.002 0.036 0.998 0.000 0.019 0.999 0.000 0.014 0.996 0.001 0.0213 0.996 0.001 0.021 0.991 0.002 0.035 19 0.993 0.001 0.025 0.999 0.000 0.015 0.999 0.000 0.013 0.997 0.000 0.0163 0.993 0.001 0.033 0.984 0.002 0.044 ital. j. food sci., vol 29, 2017 296 figure 1 also shows the kinetics of bean hydration fitted to the weibull model where α is a scale parameter and β a shape parameter. the scale parameter defines the rate of moisture uptake process (α is the reciprocal of the process rate constant) and represents the time needed to accomplish the approximately 63% of the moisture uptake process, while the shape parameter is a behavior index, which depends on the process mechanism. the kinetic parameters generated from the application of the different mathematical models for evaluating the hydration kinetics of beans by effect of the ultrasound treatment are shown in table 5. according to the results obtained, the constants of hydration rate, k and k1, of the sigmoidal and first order models, respectively, were increased for effect of ultrasound power from 1.49 to 4.04 times and from 1.93 to 3.56 times in comparison with the control treatment, depending of the bean variety. regarding peleg’s model, the first constant, c1, which has been shown to be linked to the hydration was reduced for all bean varieties by effect of ultrasound in comparison with the control treatment. therefore, the application of ultrasound and its increasing of power level improved the hydration properties such as reflected in the rate constants of the sigmoidal, first order and peleg models. in relation to the α parameter of weibull model, its value decreased with increasing ultrasound power for all the bean varieties (table 5), in agreement with the reported results by ghafoor et al. (2014) for navy bean. on the other hand, the values of kinetic parameter of hydration β of weibull model (table 5) for the soaking treatments of the different varieties of beans were high (0.834-1.995). according to machado et al. (1999), the weibull model predicts the initial lag phase of the hydration kinetics when β is greater than 1, as it was observed for the different treatments and bean varieties in this study, except for the control treatment in the mayocoba variety. 3.3. seed coat content the flor de mayo and mayacoba bean varieties presented the highest %cs (9.85±0.62 and 8.82±0.62, respectively), followed by the negro jamapa (8.65±0.02), peruano bola (7.92±0.36), azufrado (7.56±0.19) and pinto (7.48±0.50) varieties bean. in this study, the varieties of bean with the first and third highest %cs (flor de mayo and negro jamapa) required a higher soaking lapse to attain me in comparison with the fourth and fifth highest %cs (peruano bola and azufrado). however, the time to attain me of the pinto variety with a %cs lower than the mayacoba variety was longer (fig. 1). on the other hand, the varieties of bean with a higher %cs presented a higher coat rupture by effect of the ultrasound (table 6). in the varieties of azufrado, pinto, peruano bola, flor de mayo and negro jamapa, the percentage of seeds with a higher percentage of coat rupture increased as the ultrasound power was higher (5, 12 and 19 w). the negro jamapa and flor de mayo varieties presented coat rupture in all soaking treatments, including the control. the results suggest that the percentage of the coat rupture depended on bean variety and the ultrasound power used in the soaking process, agreeing with the report of pan et al. (2010). ital. j. food sci., vol 29, 2017 297 table 5. kinetic parameters for the models fitted to the hydration kinetics data of six bean varieties. model soaking treatment bean varieties mayocoba azufrado pinto peruano bola flor de mayo negro jamapa sigmoidal control k = 5.351x10 -2 τ = 26.906 k = 1.740x10-2 τ = 104.356 k = 1.261x10-2 τ = 131.756 k = 2.034x10-2 τ = 87.458 k = 1.803x10-2 τ = 114.714 k = 1.111x10-2 τ = 225.728 5 w k = 5.433x10 -2 τ = 24.634 k = 2.666x10-2 τ = 84.062 k = 1.900x10-2 τ = 104.513 k = 2.571x10-2 τ = 76.631 k = 2.267x10-2 τ = 71.294 k = 1.436x10-2 τ = 139.061 12 w k = 11.354x10-2 τ = 16.368 k = 3.920x10-2 τ = 54.383 k = 2.681x10-2 τ = 82.217 k = 3.739x10-2 τ = 56.192 k = 2.754x10-2 τ = 65.193 k = 1.595x10-2 τ = 143.056 19 w k = 15.757x10 -2 τ = 10.395 k = 5.020x10-2 τ = 39.387 k = 5.100x10-2 τ = 38.667 k = 4.524x10-2 τ = 43.394 k = 3.540x10-2 τ = 46.346 k = 1.659x10-2 τ = 110.453 first order control k1 = 2.458x10 -2 k1 = 0.750x10 -2 k1 = 0.576x10 -2 k1 =0.8445x10 -2 k1 = 0.694x10 -2 k1 = 3.678x10 -3 5 w k1 = 2.711x10 -2 k1 = 0.945x10 -2 k1 = 0.742x10 -2 k1 = 1.051x10 -2 k1 = 1.060x10 -2 k1 = 5.729x10 -3 12 w k1 = 4.425x10 -2 k1 = 1.460x10 -2 k1 = 0.965x10 -2 k1 = 1.422x10 -2 k1 = 1.219x10 -2 k1 = 5.575x10 -3 19 w k1 = 6.848x10 -2 k1 = 2.000x10 -2 k1 = 2.050x10 -2 k1 = 1.808x10 -2 k1 = 1.703x10 -2 k1 = 7.121x10 -3 peleg control c1 = 23.619 c2 = 0.959 c1 = 123.560 c2 = 0.568 c1 = 160.676 c2 = 0.569 c1 = 100.512 c2 = 0.626 c1 = 123.575 c2 =0.558 c1 = 329.984 c2 = 0.321 5 w c1 = 30.503 c2 = 0.884 c1 = 123.423 c2 = 0.374 c1 = 156.812 c2 = 0.3657 c1 = 102.118 c2 = 0.471 c1 = 81.568 c2 = 0.564 c1 = 223.034 c2 = 0.274 12 w c1 = 18.995 c2 = 0.818 c1 = 79.673 c2 = 0.328 c1 = 123.713 c2 = 0.3115 c1 = 69.255 c2 = 0.505 c1 = 79.689 c2 = 0.468 c1 = 234.070 c2 = 0.159 19 w c1 = 10.078 c2 = 0.868 c1 = 54.001 c2 = 0.402 c1 = 51.482 c2 = 0.4301 c1 = 54.229 c2 = 0.506 c1 = 52.746 c2 = 0.475 c1 = 163.072 c2 = 0.297 weibull control α = 42.481 β = 0.834 α = 141.833 β = 1.493 α = 181.544 β = 1.336 α =124.212 β = 1.372 α = 150.984 β = 1.534 α = 286.092 β = 1.995 5 w α = 37.404 β = 1.076 α = 110.515 β = 1.838 α = 139.957 β = 1.607 α = 102.955 β = 1.593 α = 96.963 β = 1.259 α = 182.245 β = 1.6121 12 w α = 23.207 β = 1.514 α = 71.718 β = 1.732 α = 107.840 β = 1.788 α = 74.790 β = 1.660 α = 86.939 β = 1.425 α = 183.437 β = 1.880 19 w α = 15.019 β = 1.159 α = 52.644 β = 1.599 α = 51.570 β = 1.588 α = 58.377 β = 1.545 α = 62.154 β = 1.287 α = 146.326 β = 1.483 ital. j. food sci., vol 29, 2017 298 3.4. equilibrium moisture content (me) the varieties of beans studied presented a me that oscillated from 107.18 to 133.99 % d.b. (table 6). the me in the soaking control treatments increased for all the bean varieties, except for peruano bola (table 6). the effect caused by the ultrasound power in the bean hydration is similar to the effect caused by the increase of temperature for the conventional soaking of bean (shafaei et al., 2014), sesame seeds (khazaei and mohammadi, 2009), rice (cheevitsopon and noomhorm, 2011), and botswana bambara (jideani and mpotokwana, 2009). table 6. effect of soaking treatment on the hydration properties of beans at 30 °c. variety/property soaking treatments control 5 w 12 w 19 w mayocoba me (% g/g d.b.) 110.08b 107.18c 111.46ab 113.98a rupture of coat (%) 0.0a 0.0a 0.0a 0.0a deff (x 10 -10 m2/s) 4.845c 5.501c 8.852b 13.717a azufrado me (% g/g d.b.) 124.47b 124.32b 128.60a 128.38a rupture of coat (%) 0.0b 0.0b 0.0b 5.0a deff (x 10 -10 m2/s) 0.380d 0.488c 0.752b 1.026a pinto me (% g/g d.b.) 123.72b 124.46b 127.24a 127.69a rupture of coat (%) 0.0b 0.0b 0.0b 16.6a deff (x 10 -10 m2/s) 1.097d 1.413c 1.839b 3.847a peruano bola me (% g/g d.b.) 124.47ª 125.08a 127.18a 126.96a rupture of coat (%) 0.0b 0.0b 0.0b 1.5a deff (x 10 -10 m2/s) 2.135b 2.616b 4.168a 4.633a flor de mayo me (% g/g d.b.) 125.96b 125.59b 128.39ab 133.99a rupture of coat (%) 15.5c 29.0b 40.0a 49.0a deff (x 10 -10 m2/s) 1.967c 3.056b 3.414b 5.025a negro jamapa me (% g/g d.b.) 123.07c 124.68bc 126.22ab 128.05a rupture of coat (%) 1.6b 10.0a 13.3a 21.6a deff (x 10 -10 m2/s) 0.711c 1.287b 1.275b 1.60a values are given as means±standard deviation (n = 3). different superscript letters in the same row indicate significant differences (p < 0.05). 3.5. effect of ultrasound on the effective diffusivity (deff) to calculate deff (eq. 6), the inverse of α was used (1/α) as the rate constant of water absorption. table 6 shows the effects of the ultrasound treatments during the bean soaking process on deff for the distinct studied varieties. ital. j. food sci., vol 29, 2017 299 the soaking treatment with ultrasound at 19 w obtained the highest values of deff for all bean varieties. the increase of deff observed in this study as the ultrasound power was increased is congruent with a study on chickpeas, where the value of deff of the soaking control treatment at 30 °c was 1.87 x 1010 m2/s and it was increased with the application of ultrasound to values of 2.10 x 1010 and 2.62 x 1010 (m2/s) for the soaking treatments at 25 khz and 100 w and 25 khz and 300 w, respectively (yildirim et al., 2011). the increase of the absorption of water during the assisted soaking process with ultrasound is due to the formation of microscopic channels in the grains, which reduce the internal resistance to mass transference (fuente-blanco et al., 2006). in this study we observed that increasing the ultrasound power in the soaking treatments of the beans generated a proportional increase on the values of deff (fig. 2), in similarity to the proportional increasing of the water absorption during soaking of bean by effect of increase of temperature (shafaei et al., 2014). figure 2. effect of low frequency ultrasound power (20 khz) on the deff in the soaking of the mayocoba (a), azufrado (b), pinto (c), peruano bola (d), flor de mayo (e) and negro jamapa (f) bean varieties. ital. j. food sci., vol 29, 2017 300 4. conclusions the application of ultrasound reduced the lapse of soaking in the six varieties of beans studied, although the reduction depended of the bean variety and the ultrasound power. the soaking treatment at 19 w presented the highest values of deff, me and the least time to attain me. however, this ultrasound treatment also presented coat rupture of the beans, in different proportion, except for the mayocoba variety. of the models used, the weibull model presented the best fit for the experimental data of the hydration kinetics, in the different soaking treatments and for the majority of the bean varieties. acknowledgements during the devolvement of this study, financial support was obtained from the consejo nacional de ciencia y tecnología (conacyt) with scholarship number 550780. references abu-ghannam, n. and mckenna, b. 1997. hydration kinetics of red kidney beans (phaseolus vulgaris l.). j. food sci. 62, 520-523. aoac. 2002. “official methods of analysis of aoac international” association of official analytical chemists, gaithersburg, md. campos-vega r., loarca-piña g. and oomah b.d. 2010. minor components of pulses and their potential impact on human health. food res int. 43:461-482 campos-vega r., vergara-castañeda h.a. and oomah b.d. 2012. in: “beans: nutrition, consumption and health: functional food sources: beans in sight”. e. popoescu, i. golubev (ed.), p 1-56. nova science publishers, new york. cárcel j.a., garcía-pérez j.v., benedito j. and mulet a. 2012. food process innovation through new technologies: use of ultrasound. j. food eng. 110: 200-207. cheevitsopon e. and noomhorm a. 2011. kinetics of hydration and dimensional changes of 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(lupinus albus) seeds grown in ethiopia. afr. j. food agric. nutr. dev. 10: 3029-3046. wani i.a., sogi d.s., wani a.a. and gill b.s. 2015. physical and cooking characteristics of some indian kidney bean (phaseolus vulgaris l.) cultivars. journal of the saudi society of agricultural sciences doi: 10.1016/j.jssas.2014.12.002. who/fao. 2003. diet, nutrition and the prevention of chronic diseases. who technical report series 916, geneva. yildirim a., öner m.d. and bayram m. 2011. fitting fick’s model to analyze water diffusion into chickpeas during soaking with ultrasound treatment. j. food eng. 104:134-142. zura l., uribe e., lemus-mondaca r., saavedra-torrico, j., vega-gálvez, a. and di scala k. 2013. rehydration capacity of chilean papaya (vasconcellea pubescens): effect of process temperature on kinetic parameters and functional properties food bioprocess technol. 6:844-850. paper received november 17, 2016 accepted december 31, 2016 paper ital. j. food sci., vol. 27 2015 1 keywords: mycotoxins, seed-borne, coffea arabica l., high-performance liquid chromatography, metabolites coffee bean myco-contaminants and oxalic acid producing aspergillus niger mohamed a. yassin1,2*, abd el-rahim m.a. el-samawaty1,2, mohamed a. moslem2 and abdullah a. al-arfaj2 1botany and microbiology department, faculty of science, king saud university, riyadh, saudi arabia 2agricultural research center, plant pathology research institute, giza, egypt *corresponding author: mohamdyassin@gmail.com, myassin@ksu.edu.sa abstract coffee bean-contaminating fungi were determined in random samples collected in riyadh, kingdom of saudi arabia, using the direct plating technique. forty-five samples were examined and 12 fungal species belonging to 5 genera were isolated. aspergillus niger was the most widely distributed and most frequently isolated fungus (86.67%). the ability of the predominant fungus, a. niger, to produce oxalic acid was evaluated using high-performance liquid chromatography. about 50% of the tested a. niger isolates produced oxalic acid; the amount produced was in the range of 90–550 ppm of oxalic acid. because a. niger was the predominant and most widely distributed toxigenic fungus in the examined samples, more efforts should be directed to minimize the risk of oxalic acid contamination of commoditized coffee beans in the kingdom of saudi arabia. 2 ital. j. food sci., vol. 27 2015 introduction coffea arabica l. is considered to have the best flavour and quality, and coffee is one of the most popular beverages consumed in countries of the arabian peninsula. however, coffee beans may become poisonous because of contamination with mycotoxigenic fungi that may occur throughout all pre and/ or post-harvest stages (batista et al., 2003; noonim et al., 2008). several fungal genera were found to contaminate coffee beans at each stage, from the farmer to the consumer (vega et al., 2008; batista et al., 2009; vilela et al., 2010). the main mycotoxigenic fungal genera found to be associated with commoditized coffee beans belong to the genera aspergillus, penicillium, and fusarium (pardo et al., 2004; bokhari, 2007; leong et al., 2007). these fungi not only affect the quality of coffee beans but also produce toxic secondary metabolites that are harmful to the consumers (bernnett and klich, 2003, vilela et al., 2010). one of the most important mycotoxigenic and organotoxic metabolite-producing contaminant is aspergillus niger, which is considered a class 1 containment agent (u.s. npa, 1977; schuster et al., 2002; ilic et al., 2007). this fungus is responsible for the in vitro and/or in vivo secretion of the nephrotoxic compound oxalic acid (mandal et al., 2005; magnoli et al., 2008). the toxicity of oxalic acid is due to the deposition of calcium-oxalate complexes in renal tubules causing renal failure; in humans, the minimum lethal dose of orally ingested oxalic acid is 600 mg/kg (safety officer in physical chemistry, 2005; botha et al., 2009). the purpose of this study was to examine coffee beans commoditized in saudi arabian markets for the presence of toxigenic fungi. furthermore, we evaluated the production of oxalic acid by the isolated a. niger strains. materials and methods mycological analysis mycotoxin-producing fungi were determined in 45 random samples of coffee beans collected in riyadh city, kingdom of saudi arabia. for the isolation of fungi associated with coffee beans, the direct plating technique was applied using potato dextrose agar (pda) medium. beans were plated directly onto pda medium after surface disinfection by 5% sodium hypochlorite solution. the plates were incubated for 5–7 days at 25°c and the growing fungal colonies were purified. the obtained fungal isolates were then identified to the species levels at the mycological centre, assiut university, egypt. mycotoxigenicity the oxalic acid production of the tested a. niger isolates was determined using high-performance liquid chromatography. briefly, 50 ml of czapek-dox broth medium was placed in 250-ml erlenmeyer flasks and inoculated with a. niger. inoculated media were incubated in triplicates at 30°c on an orbital shaker maintained at 215 rpm for 7 days. the culture supernatants were then analysed for their oxalic acid content. separation of oxalic acid was carried out using a clc-c825 cm cation exchange column. the mobile phase was 90% h 2 o and 10% ch 3 oh. the flow rate was 1 ml/ min and the temperature was 35°c (ghorbani et al., 2007). statistical analysis the spss-16 statistical package was used for the analysis of variance and correlation and cluster analyses. cluster analysis was per formed by the unweighted pair -group method with arithmetic averages. data were processed by root square transformation of % frequencies + 0.5 to normalize and stabilize the variance before subsequent analyses were carried out. means were statistically compared using the least significant difference test. table 1 distribution of isolated fungi in coffee bean samples. fungi distribution % 1. a. alternata 8.89 2. a. f.columnaris 17.78 3. a. niger 86.67 4. a. ochraceus 4.44 5. a. terreus 2.22 6. nigrospora sp. 2.22 7. p. brevicompactum 8.89 8. p. corylophilum 6.67 9. p. variabile 2.22 10. p. suchlasporia 2.22 11. r. stolonifer 44.44 12.t. flavus 2.22 table 2 anova of the isolation frequencies of mycotoxigenic fungi in coffee beans. source of variance df m s f sig. r.c.* samples 44 2.070 1.558 0.012 0.35 fungi 11 575.637 433.265 0.000 98.60 samples x fungi 484 6.086 4.580 0.000 1.04 error 1620 1.329 *relative contribution. 3 2 ta bl e 3 c om pa ris on o f t he is ol at io n fr eq ue nc ie s o f f un gi is ol at ed fr om c of fe e be an s. s. f1 f2 f3 f4 f5 f6 f7 f8 f9 f1 0 f1 1 f1 2 n o fr eq . tr an s. fr eq . tr an s. fr eq . tr an s. fr eq . tr an s. fr eq . tr an s. fr eq . tr an s. fr eq . tr an s. fr eq . tr an s. fr eq . tr an s. fr eq . tr an s. fr eq . tr an s. fr eq . tr an s. 1 0. 00 0. 71 25 .0 0 3. 04 50 .0 0 5. 37 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 2 0. 00 0. 71 25 .0 0 3. 04 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 3 7. 30 2. 29 0. 00 0. 71 36 .4 6 4. 63 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 1. 79 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 4 0. 00 0. 71 0. 00 0. 71 62 .5 0 7. 03 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 6. 25 1. 79 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 6. 25 1. 79 0. 00 0. 71 5 23 .7 5 4. 35 23 .7 5 4. 35 46 .2 5 6. 76 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 6. 25 1. 79 0. 00 0. 71 6 0. 00 0. 71 0. 00 0. 71 10 0. 00 10 .0 3 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 7 50 .0 0 5. 37 25 .0 0 3. 04 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 8 0. 00 0. 71 5. 00 1. 66 95 .0 0 9. 76 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 9 0. 00 0. 71 3. 57 1. 49 42 .8 6 4. 98 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 3. 57 1. 49 0. 00 0. 71 10 0. 00 0. 71 0. 00 0. 71 20 .0 9 3. 54 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 6. 70 2. 22 23 .2 1 3. 78 11 0. 00 0. 71 0. 00 0. 71 70 .8 3 7. 48 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 4. 18 1. 57 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 12 0. 00 0. 71 0. 00 0. 71 64 .2 8 8. 01 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 25 .0 0 4. 38 10 .7 2 2. 66 0. 00 0. 71 3. 57 1. 49 0. 00 0. 71 13 0. 00 0. 71 0. 00 0. 71 10 0. 00 10 .0 3 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 14 0. 00 0. 71 0. 00 0. 71 10 .0 0 2. 12 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 15 .0 0 2. 48 0. 00 0. 71 15 0. 00 0. 71 0. 00 0. 71 83 .3 3 9. 16 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 16 .7 0 4. 15 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 16 0. 00 0. 71 13 .4 4 2. 97 48 .2 1 6. 19 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 13 .3 9 2. 96 0. 00 0. 71 17 0. 00 0. 71 0. 00 0. 71 37 .5 0 4. 64 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 37 .5 0 4. 64 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 18 0. 00 0. 71 3. 57 1. 49 88 .6 9 9. 42 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 7. 75 2. 35 0. 00 0. 71 19 0. 00 0. 71 0. 00 0. 71 10 0. 00 10 .0 3 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 20 0. 00 0. 71 0. 00 0. 71 91 .6 7 9. 59 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 8. 35 2. 43 0. 00 0. 71 21 0. 00 0. 71 0. 00 0. 71 95 .8 3 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0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 35 0. 00 0. 71 0. 00 0. 71 95 .8 3 9. 81 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 11 .3 2 2. 74 0. 00 0. 71 36 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 50 .0 0 5. 37 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 37 0. 00 0. 71 0. 00 0. 71 38 .6 9 4. 76 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 11 .3 2 2. 74 0. 00 0. 71 38 75 .0 0 7. 70 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 39 0. 00 0. 71 0. 00 0. 71 10 0. 00 10 .0 3 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 40 0. 00 0. 71 0. 00 0. 71 95 .8 3 9. 81 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 4. 18 1. 57 0. 00 0. 71 41 0. 00 0. 71 0. 00 0. 71 45 .0 0 5. 10 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 5. 00 1. 66 0. 00 0. 71 42 0. 00 0. 71 0. 00 0. 71 41 .6 7 4. 93 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 8. 35 2. 43 0. 00 0. 71 43 0. 00 0. 71 0. 00 0. 71 10 0. 00 10 .0 3 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 44 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 75 .0 0 7. 70 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 45 0. 00 0. 71 0. 00 0. 71 75 .0 0 8. 57 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 0. 00 0. 71 25 .0 0 3. 91 0. 00 0. 71 f1 -f 12 = a. a lte rn at e, a . f . c ol um na ri s, a. n ig er , a . o ch ra ce us , a . t er re us , n ig ro sp or a sp ., p. b re vi co m pa ct um , p . c or yl op hi lu m , p . v ar ia bi le , p . s uc hl as po ri a, r . st ol on ife r a nd t . f la vu s r es pe ct iv el y. fr eq . = f re qu en cy o f f un ga l i so la tio n. tr an s. = tra ns fo rm ed d at a in to ro ot sq ua re o f % v al ue s + 0 .5 . f1 -f 12 = a . a lte rn at e, a . f . c ol um na ris , a . n ig er , a . o ch ra ce us , a . t er re us , n ig ro sp or a sp ., p. b re vi co m pa ct um , p . c or yl op hi lu m , p . v ar ia bi le , p . s uc hl as po ria , r . s to lo ni fe r a nd t . fl av us re sp ec tiv el y. fr eq . = f re qu en cy o f f un ga l i so la tio n. tr an s. = tr an sf or m ed d at a in to ro ot s qu ar e of % v al ue s + 0. 5. t a b le 3 c o m p a ri so n o f th e is o la ti o n f re q u en ci es o f fu n g i is o la te d f ro m c o ff ee b ea n s. 4 ital. j. food sci., vol. 27 2015 table 4 correlation among frequencies of fungi isolated from coffee beans. fungi 1 2 3 4 5 6 7 8 9 10 11 12 1. a. alternata 1 0.365* -0.427** -0.058 -0.042 -0.042 -0.048 -0.075 -0.042 -0.042 -0.133 -0.042 2. a. f.columnaris 1 -0.293 -0.084 -0.062 -0.062 -0.107 -0.109 -0.062 -0.062 -0.003 -0.062 3. a. niger 1 -0.306* -0.060 0.078 -0.310* 0.007 0.045 0.027 -0.005 -0.172 4. a. ochraceus 1 0.439** -0.031 -0.053 -0.055 -0.031 -0.031 -0.144 -0.031 5. a. terreus 1 -0.023 -0.039 -0.040 -0.023 -0.023 -0.106 -0.023 6. nigrospora sp. 1 -0.039 -0.040 -0.023 -0.023 -0.106 -0.023 7. p. brevicompactum 1 -0.069 -0.039 -0.039 -0.108 -0.039 8. p. corylophilum 1 0.562** -0.040 -0.119 -0.040 9. p. variabile 1 -0.023 0.010 -0.023 10. p. suchlasporia 1 0.130 -0.023 11. r. stolonifer 1 0.116 12. t.fl avus 1 fig. 1 phenogram based on average linkage cluster analysis of frequencies of fungi recovered from coffee beans. results and discussion the mycological examination of the coffee bean samples (table 1) revealed the presence of 12 fungal species that belonged to 5 genera (silva et al., 2008; batista et al., 2009). the predominance of a. niger in the tested coffee bean samples was in agreement with previously reported data (urbano et al., 2001; nehad et al., 2007; noonim et al. 2008). the main mycotoxigenic fungal genera, i.e. aspergillus and penicillium, have frequently been associated with coffee beans (joosten et al., 2001; pardo et al., 2004; bokhari, 2007; leong et al., 2007). anova indicated that the effects of fungus, sample, and fungus × sample interaction were highly signifi cant sources of variation in the fungal isolation frequencies. compared to the other sources, fungus was the most important source of variation (table 2). the isolation frequencies varied according to the tested samples. for example, alternaria alternata and a. fl avus var. columnaris showed equal isolation frequencies in sample no. 5, but the former was signifi cantly more frequently isolated from sample no. 7 than the latter. although a. fl avus var. columnaris was isolated from samples no. 9 and 18 at equal frequencies, the frequency at which a. niger was isolated from these 2 samples varied signifi cantly. whereas penicillium brevicompactum and rhizopus stolonifer were isolated from sample no. 4 at equal frequencies, p. brevicompactum was signifi cantly more frequently isolated than r. stolonifer (table 3). however, the contamination of coffee beans with mycotoxigenic fungal genera (noonim et al., 2008; vilela et al., 2010) may start in the fi eld and subsequently extend to storages and markets, in particular under conducive conditions (jestoi et al., 2004; juan et al., 2008; paterson and lima, 2010). correlation analysis showed that the fungal isolation frequencies were positively and/or negatively correlated with each other (table 4). some correlations between isolated fungal sp ecies and their isolation frequencies were significant or highly signifi cant. highly signifi cant positive correlations were noted among asperital. j. food sci., vol. 27 2015 5 gillus ochraceus and aspergillus terreus as well as among penicillium corylophilum and penicillium variabile. furthermore, a highly significant negative correlation was found between a. alternata and a. niger. the highly significant positive correlations between some isolated fungi found in this study imply that similar colonizing conditions are provided by the coffee beans for those fungi; the opposite conclusion can be drawn for negatively correlated fungi (yassin et al., 2013). the phenogram (fig. 1) illustrates the cluster analysis of fungal isolation frequencies (%) on the basis of their distribution patterns using all samples. three distinct groups of isolated fungi are shown; each of them is divided into subgroups. strongly and positively associated fungi were grouped in the same cluster. the grouping pattern of the isolated fungi in the cluster analysis suggests the potential existence of sample(environment-) related fungal groups (yassin et al., 2011). with regard to the mycotoxigenicity, 50% of the tested a. niger isolates were able to produce oxalic acid. other tested isolates failed to produce any detectable amounts of oxalic acid (table 5). aspergillus species are well known to be responsible for the secretion of different toxic metabolites (al-abdalall, 2009; yassin et al., 2010; el-samawaty et al., 2011). moreover, the organotoxic metabolite oxalic acid has also been shown to be produced by a. niger isolated from commoditized agricultural products (bahkali et al., 2013; yassin et al., 2013). accumulation of such compounds produced by mycotoxigenic fungi could affect the quality of coffee (arrusa et al., 2005; kumar et al., 2008) and harm human consumers (palanee et al., 2001; david et al., 2005). conclusions the coffee beans that were examined in this study were found to be contaminated with many fungal genera that might affect the quality of coffee. a. niger was the most widely distributed and most frequently isolated fungus (86.67%). furthermore, 50% of the tested a. niger isolates were mycotoxigenic. they were able to produce oxalic acid in the range of 90–550 ppm. because a. niger was the predominant and most widely distributed fungus in the examined coffee bean samples, more efforts should be directed to minimize the risk of oxalic acid contamination of commoditized coffee beans in ksa. acknowledgements the authors would like to extend their sincere appreciation to the deanship of scientific research at king saud university for its funding of this research through the research group project no. rgp-vpp-298. references arrusa k., blanka g., abramsonb d., clearc r. and holleya r.a. 2005. aflatoxin production by aspergillus flavus in brazil nuts. j. stored prod. res. 41:513-527. al-abdalall a.h.a. 2009. production of aflatoxins by aspergillus flavus and aspergillus niger strains isolated from seeds of pulses. j. food, agri. environ. 7 (2): 33 39. schuster e., dunn-coleman n., frisvad j.c. and van dijck p.w.m. 2002. on the safety of aspergillus niger – a review. appl. microbiol. biotechnol. 59: 426-435. bahkali a.h., el-samawaty a.m.a. and yassin m.a. 2013. toxigenic fungal biota associated with walnut in saudi arabia. j. pure appl. microbiol. 7(2), p. 1079-1086. batista l.r., chalfoun s.m., prado g., schwan r.f. and whealsa e. 2003. toxigenic fungi associated with processed (green) coffee beans (coffeaar abica l.). int. j. food microbiol. 85:293-300. batista l.r., chalfoun s.m., silva c.f., cirillo m., varga e.a. and schwan r.f. 2009. ochratoxin a in coffee beans (coffea arabica l.) processed by dry and wet methods. food cont. 20:784-790. bernnett j.w. and klich m. 2003. mycotoxins. clin microbiol rev 16:497-516. bokhari f.m. 2007. mycotoxins and toxigenic fungi in arabic coffee beans in saudi arabia. adv. biol. res. 1:56-66. botha c.j., truter m., bredell t., lange l. and mülders m.s.g. 2009. putative aspergillus niger-induced oxalate nephrosis in sheep. journal of the south african veterinary association. 80(1):50–53. david m.s., manfred m. and leane l. 2005. mutagenicity of the mycotoxin patulin in cultured chinese hamster v79 cells, and its modulation by intracellular glutathione. arch. toxicol. 79:110-121. ghorbani y., oliazadeh m., shahvedi a., roohi r., and pirayehgar a. 2007. use of some isolated fungi in biological leaching of aluminum from low grade bauxite. afri. j. biotechnol. 6(11): 1284-1288. el-samawaty a.m.a., yassin m.a., bahkali a., moslem m.a. and abd-elsalam k.a. 2011. biofungal activity of aloe vera sap against mycotoxigenic seed-borne fungi. fresenius environ. bull. 20(6):1352-1359. ilic z., bui t., tran-dinh n., dang m.h.v., kennedy i. and carter d. 2007. survey of vietnamese coffee beans for the presence of ochratoxigenic aspergilli mycopathol. 163:177–182. jestoi m, somma m.v., kouva m., veijalainen p., rizzo a., ritieni a., peltonen k. 2004. levels of mycotoxins and sample cytotoxicity of selected organic and conventional grainbased products purchased from finnish and italian markets. mol. nutr. food res. 48:229-307. joosten h.m.l.j., goetz j., pittet a., schellenberg m. and bucheli p. 2001. production of ochatoxin a by aspergillus carbonarius on coffee cherries. int. j. food microbiol. 65:39-44. table 5 production of oxalic acid by a. niger isolates. a. niger isolates oxalic acid (ppm) a.n.1 00.00 a.n.2 200.00 a.n.3 150.00 a.n.4 00.00 a.n.5 00.00 a.n.6 00.00 a.n.7 00.00 a.n.8 550.00 a.n.9 100.00 a.n.10 90.00 6 ital. j. food sci., vol. 27 2015 juan c., moltó j.c., lino c.m. and mañes j. 2008. determinaton of ochratoxin a in organic and non-organic cereals and cereal products from spain and portugal. food chem. 107:525-530. kumar v., basu m.s. and rajendran t.p. 2008. mycotoxin research and mycoflora in some commercially important agricultural commodities. crop protec. 27: 891-905. leong s.l., hien l.t., an t.v., trang n.t., hocking a.d. and scott e.s. 2007 ochratoxin a-producing aspergillii in vietnamese green coffee beans. lett. appl. microbiol. 45:301-306. magnoli c.e., astoreca a.l., ponsone m.l., barberis c.l., fernández-juri m.g. and dalcero a.m. 2008. ochratoxinand aflatoxin-producing fungi associated with green and roasted coffee samples consumed in argentina world myco. j. 1(4): 419-427. mandal s. k. and banerjee p.c. 2005. submerged production of oxalic acid from glucose by immobilized aspergillus niger. process biochem. 40: 1605–10 nehad e.a., m.m. farag, m.s. kawther, a.k.m. abdel-samad and khayria naguib 2007. an exposure and intake assessment of ochratoxin a from imported coffee beans in egypt. world j. agric. sci. 3(3):285-294. noonim p., mahakarnchanakul w., nielsen k.f., frisvad j.c. and samson r.a. 2008. isolation, identification and toxigenic potential of ochratoxin aproducing aspergillus species from coffee beans grown in two regions of thailand. int. j. food microbiol. 128:197-202. palanee t., dutton m.f. and chuturgoon a. 2001. cytotoxicity of aflatoxin b1 and its chemically synthesized epoxide derivative on the a459 human epithelioid lung cell line. mycopathol. 151: 155-159. pardo e., marín s., ramos a.j. and sanchis v. 2004. occurrence of ochratoxigenic fungi and ochratoxin a in green coffee from different origins. food sci. technol. int. 10:45-50. paterson r.r.m. and lima n. 2010. how will climate change affect mycotoxins in food? food. res. int. 43:1902-1914. safety officer in physical chemistry. 2005. “safety (msds) data for oxalic acid dihydrate”. oxford university. retrieved december 30, 2009. silva c.f., batista l.b. and schwan r.f. 2008. incidence and distribution of filamentous fungi during fermentation, drying and storage of coffee (coffea arabica l.) beans. braz. j. microbiol. 39:521-526. u.s. environmental agency 1977. aspergillus niger final risk assessment. biotechnology program under the toxic substances control act (tsca) http://www.epa.gov/ biotech_rule/pubs/fra/fra006.htm. urbano g.r., taniwaki, m.h., leitao, m.f.d.f. and vicentini, m.c. 2001. occurrence of ochratoxin aproducingfung i in raw brazilian coffee. j. food protec. 68, 1226–1230. vega f.e, posada f, aime mc, peterson sw, rehner sa, f, (2008). fungal endophytes in green coffee seeds. mycosystema. 27(1): 75-84. vilela d.m., pereira g.v., silva c.f., batista r., schwan r.f. 2010. molecular ecology and polyphasic characterization of the microbiota associated with semi-dry processed coffee (coffeaarabical.). food microbiol. 27:1128-1135. yassin m.a., el-samawaty a.m.a., bahkali a., moslem m., abd-elsalam k.a. and hyde k.d. 2010. mycotoxin-producing fungi occurring in sorghum grains from saudi arabia. fungal diver. 44:45-52. yassin m.a., el-samawaty, a.m.a., bahkali a., and abdelsalam k. 2011. fungal biota and occurrence of aflatoxigenic aspergillus associated with postharvest corn grains. fresenius environ. bull. 20(4):903-909. yassin m.a., el-samawaty a.m.a., moslem m.a., and el-naggar m.a. 2013. mycobiota of almond seeds and the toxigenicity of some involved genera. life sci. j. 10(4):10881093. paper received february 10, 2014 accepted august 23, 2014 ijfs#611_bozza ital. j. food sci., vol 29, 2017 487 paper impacts of plasticizer and pre-heating conditions on properties of bovine and fish gelatin films fabricated by thermo-compression molding technique k. chuaynukul1, m. nagarajan1, t. prodpran*1, s. benjakul2 and s. prasarpran1 1department of material product technology, faculty of agro-industry, prince of songkla university, 15 kanchanawanich road, hat yai, songkhla 90112, thailand 2department of food technology, faculty of agro-industry, prince of songkla university, 15 kanchanawanich road, hat yai, songkhla 90112, thailand *corresponding author. tel.: +66 74286357; fax: +66 74558866 e-mail address: thummanoon.p@psu.ac.th abstract bovine and fish gelatin films were prepared by thermo-compression molding technique. this study investigated the effects of glycerol levels (15-35%) and resin pre-heating conditions including pre-heating temperatures (120, 140 and 160°c) and times (5 and 10 min) on film properties. tensile strength (ts), elastic modulus (em) and yellowness of films decreased, but elongation at break (eab), water-vapor permeability (wvp) and transparency increased with increasing glycerol level. the gelatin films generally had decreased ts, em, wvp and transparency, but increased yellowness as the pre-heating temperature and time increased. therefore, the glycerol level and condition used for resin pre-heating directly influenced the film properties. keywords: compression molding, film, gelatin, glycerol level, pre-heating conditions, wvp ital. j. food sci., vol 29, 2017 488 1. introduction proteins are natural polymers frequently used for preparing bio-degradable/edible films. these films are alternatives to synthetic plastic films, which serve as one of the ways of reducing environmental pollution (gomez-guillen et al., 2009). in general, protein films exhibit excellent barrier properties to oxygen, lipids and aromas, and have been reported to possess moderate mechanical properties, as well as a high nutritional value, but with low water vapor barrier property due to the hydrophilic character of these macromolecules (gennadios, 2002; ou et al., 2004). protein films can be produced from several protein sources including vegetable proteins (corn zein, wheat gluten, soy protein, peanuts and cottonseed protein) and animal proteins (milk proteins, collagen, gelatin, keratin, egg albumin and myofibrillar protein) (cuq et al., 1998). gelatin is an animal protein derived from the partial hydrolysis of native collagens, which are the most abundant structural proteins found in skins, bones and connective tissues (karim and bhat, 2009). it possesses a good film-forming property and is one of the first materials applied as edible coatings and films (klose et al., 1952; gennadios et al., 1994). skin and bone from bovine and porcine sources are utilized commercially in gelatin production (johnston-banks, 1990; venien and levieux, 2005). as a result of religious objections to the consumption of bovine materials and health concerns about the spread of diseases (such as bovine spongiform encephalopathy) to humans, fish gelatin is increasingly gaining attention as an alternative to replace mammalian gelatin (gomezguillen et al., 2009; karim and bhat, 2009). among all proteins, gelatin has received considerable attention in the development of edible films owing to its abundance and biodegradability (bigi et al., 2002; jongjareonrak et al., 2006). in addition, it is unique among hydrocolloids in forming thermo-reversible products with a melting point close to body temperature, which is particularly significant in edible and pharmaceutical applications (norland, 1990; gomez-guillen et al., 2009). with regards to film with many outstanding properties, such as transparency, bio-degradability and barrier properties (gases and aroma), gelatin is suitable for application in bio-degradable packaging (martucci and ruseckaite, 2009; nagarajan et al., 2015). however, the properties of gelatin films depend on the characteristics of the raw materials and manufacturing processes. in general, there are two main methods used for preparing protein films: 1) the dry and 2) the wet processes. the wet process or solution casting is the most widely used filmforming method (zhang et al., 2007; wang et al., 2009; limpisophon et al., 2010). the dry process (or thermal possessing method), such as compression molding and extrusion, is based on the thermoplastic properties of proteins when plasticized and heated above their glass transition temperature (tg) under low water content. heating above tg produces soft and rubbery material and may permit their incorporation into specific products. cooling to room temperature can reconvert rubbery material to glassy materials, giving more or less rigid forms with the desired structure (cuq et al., 1997). this thermal process may affect film properties differently as compared with the casting method, but it enhances the commercial potential for large-scale production of bio-degradable/edible films. moreover, this method allows a much shorter period of time for film preparation and the use of conventional techniques which are more convenient for industrial applications than casting. among various thermal processing techniques, thermocompression molding can be applied for film preparation from different polymers. although this technique is feasible at the laboratory level, it is not a potential technique for realistic packaging applications. this technique is still widely used especially in preliminary studies to ascertain the feasibility of using thermal processing technique and ital. j. food sci., vol 29, 2017 489 to standardize the processing conditions prior to developing such biodegradable film via the continuous thermal processing techniques. however, there is limited information regarding the preparation and properties of gelatin films fabricated by the thermal method. among thermal processing methods, compression molding is the most common and simple method of molding, and is typically used to investigate the feasibility of converting any polymer to the desired product via thermoplastic processing. one of the requirements of thermal processing in film making from biopolymers including gelatin, is the possession of a sufficient melt flow under the processing conditions. gelatin normally involves high molecular interaction, which results in high tg and melt viscosity, but low melt flow-ability. as a consequence, the incorporation of a proper plasticizer to gelatin at suitable levels will allow a sufficient flow of the melt, thereby producing the gelatin film by thermal processing. moreover, the processing conditions of the thermal technique used are also crucial for obtaining a gelatin film with good properties, which may vary based on gelatin type. the compression molding technique generally involves several fundamental steps including pre-heating (without applied pressure), degassing, heating and pressurizing (compressing), as well as cooling. the conditions used in each step have to be properly manipulated. therefore, this study was carried out to investigate the feasibility of gelatin film production by thermo-compression molding technique. in particular, the effects of plasticizer (glycerol) level, pre-heating temperature and time of compression molding on film-forming ability, as well as the properties of films from bovine-hide and fish-skin gelatins were studied. 2. materials and methods 2.1. materials and chemicals this experiment utilized commercial bovine-hide gelatin (~240 bloom) and fish-skin gelatin (~240 bloom), purchased from halamic company (bangkok, thailand) and lapi gelatine s.p.a. (empoli, italy), respectively. the glycerol (food grade), used as plasticizer being approved as safe by the food and drug administration (fda), was purchased from wako pure chemical industry, ltd., tokyo, japan. 2.2. preparation of molding compound resin prior to molding, compound resin based on gelatin and plasticizer, which is used as raw material for compression molding, was prepared. from the preliminary investigations of this study, the compound resin in the pellet form could not be successfully prepared by dry blending or melt compounding using twin-screw extrusion technique. for dry blending, the glycerol could not be well dispersed with gelatin powder and as such, the resin could not flow properly upon heat compression and became easily degraded. for melt blending, the gelatin melt was too viscous and tended to stick to the screw surface and became degraded upon mixing, mainly due to the evaporation of water acting as a plasticizer at high temperature. thus, the plasticized-gelatin molding compound resin was prepared by solution blending. molding compound resin (a mixture of gelatin and plasticizer) was prepared according to the method of park et al. (2008) with some modifications. bovine or fish gelatin powders were first dissolved in hot (65 °c) deionized water to obtain a protein concentration (aoac, 2000) of 20% (w/v). glycerol was added to gelatin solution at different concentrations (15, 25 and 35% of protein, w/w). the filmforming solutions (ffs) were then heated at 90 °c for 2 h in a water bath. upon heat ital. j. food sci., vol 29, 2017 490 pretreatment, the ffss were gently stirred. thereafter, the ffss were poured onto a stainless tray and partially dried for 12 h at ambient temperature. these semi-dried resins were cut into small pellets (~0.5×0.3×0.3 cm3) and further dried in a vacuum oven at 35 °c for 48 h. the obtained resins (fig. 1) were conditioned at 25 °c and 60% rh in an environmental chamber (tk120, nuve, belgium) for 48 h, before being used for the preparation of films via thermo-compression molding. the conditioned resins obtained had a moisture content of 15±3%. 2.3. effects of glycerol level on film formation and film properties 2.3.1. film fabrication by thermo-compression molding the conditioned bovine and fish gelatin resins containing different glycerol levels (about 3 g) were placed between two stainless steel plates (10x10 inch2) covered with mylar sheets. a spacer with a thickness of 0.1 mm was inserted between the plates. the set was inserted between heating platens of the compression molder previously heated to 120 °c. to melt the resin, it was pre-heated without applying pressure at the aforementioned temperature for 10 min. the molten resin was subsequently pressed to form a film in the compression molder at that temperature. a pressure of 20 mpa was applied for 2 min followed by the removal of the set from the compression molder. the samples were cooled down to room temperature. the gelatin film was then removed from the plates and subjected to analyses. figure 1. photographs of molding compound resins from bovine and fish gelatins. 2.3.2. analyses of thermo-compression molded gelatin films prior to testing, film samples were conditioned in an environmental chamber for 48 h at 25±0.5 °c and 50±5% rh. 2.3.2.1 film thickness the film thickness was measured using a digital micrometer (gotech, model gt-313-a, gotech testing machines inc., tawain) (chuaynukul et al., 2015). five random thickness measurements were recorded and the average was taken as the result. ital. j. food sci., vol 29, 2017 491 2.3.2.2 mechanical properties the tensile strength (ts), elastic modulus (em) and elongation at break (eab) of the films were measured according to the astm-d882-01 (2002a) method, as described by iwata et al. (2000), using a universal testing machine (lloyd instruments, hampshire, uk). the specimen strip (50 x 20 mm2) was clamped between the grips with initial separation of 30 mm and then pulled apart at a cross-head speed of 30 mm/min until it was broken. the ts was calculated by dividing the maximum force at break by the cross-sectional area of the film. the eab was calculated by dividing the length extended (∆l) by the original length (l0) of the film. em was derived from the initial slope of the linear portion of the stress-strain curve. ten specimens were tested for each treatment. 2.3.2.3 water vapor permeability (wvp) wvp was determined using a modified astm e-96-01 (2002b) method as described by shiku et al. (2004). the pre-conditioned film was sealed onto the opening of an aluminum permeation cup (30 mm internal diameter) containing dried silica gel (0% rh) with silicone vacuum grease and rubber gasket. the cup was kept in a controlled chamber at 30±0.5 °c and 65±5% rh. the cup was weighed every 1 h until 8 h, under this controlled environment. the wvp of the gelatin film was calculated using the following equation: wpv = q ⋅l t ⋅ a ⋅δp where l is the average thickness of the film sample (mm); a is the exposed area of the film (m2); δp is the difference of the partial vapor pressure (pa) across the film and the term q/t was calculated by linear regression from the plot of weight gain and time, in the constant rate period. the wvp value was expressed in g.m/m2.s.pa. 2.3.2.4 color the color of the film was determined using a cie colorimeter (hunter associates laboratory, inc., va, usa). also, d65 (day light) and a measure cell with an opening of 30 mm were used. dried film samples were directly placed on the sample compartment and covered with a white standard plate. the color of the film was expressed as l*(lightness/brightness), a*-(redness/greenness) and b*-(yellowness/blueness) values. the total difference in color (∆e*) was calculated according to the equation of gennadios et al. (1996a) as follows: where, ∆l*, ∆a* and ∆b* are the differences between the color parameter of corresponding film samples and that of the white standard (l*= 92.82, a*= -1.29 and b*= 0.51). 2.3.2.5 light transmittance and transparency value the transmission of visible light of gelatin films was measured at a wavelength of 600 nm, using uv-vis spectrophotometer (model no. 1601, shimadzu, kyoto, japan) as described ∆𝐸∗ = %(∆𝐿∗)2 + (∆𝑎∗)2 + (∆𝑏∗)2 ital. j. food sci., vol 29, 2017 492 by han and floros (1997). the transparency value of the films was then calculated using the following equation: x t valuecytransparen 600 log − = where t600 is the fractional value of transmittance at 600 nm and x is the film thickness (mm). according to this equation, a higher transparency value indicates a lower degree of film transparency. 2.4. effects of pre-heating conditions on film formation and film properties the conditioned bovine and fish gelatin resins containing glycerol at 25% of protein were used to prepare films via compression molding using the same procedure as described above. in this study, different conditions in the pre-heating step prior to pressing were applied, including pre-heating temperatures (120, 140 and 160 °c) and times (5 and 10 min). thereafter, the pre-heated resins were compressed at the aforementioned temperatures for 2 min with pressure of 20 mpa, followed by cooling as described above. the obtained films from bovine and fish gelatins were then subjected to analyses as mentioned earlier. 2.5. statistical analysis all experiments were performed in triplicates (n=3) and a completely randomized design (crd) was used. analysis of variance (anova) was performed and the mean comparisons were done by duncan’s multiple range tests (steel and torrie, 1980). data are presented as mean±standard deviation and the probability value of p<0.05 was considered as significant. statistical analysis was performed using the statistical package for social sciences (spss 17.0 for windows, spss inc., chicago, il, usa). 3. results and discussion 3.1. effect of glycerol levels on the properties of thermo-compression molded gelatin films 3.1.1. film forming ability and visualized appearance of films glycerol, as a plasticizer, was incorporated into gelatin at different levels (15, 25 and 35% of protein, w/w). the addition of glycerol was expected to influence not only the properties of obtained films, but also the flow-ability and fusion behavior of molding compound resin during thermo-compression. the later effects were important for the feasibility of film-formation via thermal technique. it was found that all levels of glycerol used were able to promote sufficient flow-ability and fusion of the molten resins under hot-pressing (at 120 °c and 20 mpa). thus, continuous, flexible films with average thickness in the range of 80.2-127.3 µm were formed, regardless of gelatin type. as the glycerol level increased, the average thickness of the films decreased (table 1). this can be attributed to the fact that an increase in glycerol level resulted in increased flow-ability of the molten gelatin (at 120 °c), probably due to a decrease in its melt viscosity. theoretically, the addition of a plasticizer to the polymer can also act as a processing aid ital. j. food sci., vol 29, 2017 493 which can decrease the melt viscosity of the polymer, thereby enhancing the flow-ability of the polymer melt (park et al., 2008; martucci and ruseckaite, 2009). park et al. (2008) prepared gelatin resins plasticized with 20% glycerol, sorbitol or a mixture of glycerol and sorbitol, and the resins were extruded to produce stretchable films. the results suggested that the film prepared only with sorbitol, as the plasticizer was not suitable for the extrusion film process due to its low fluidity. however, the films can be successfully extruded from resins plasticized with 20% glycerol and the glycerol-sorbitol mixture. therefore, the glycerol level had an impact on the flow-ability and thus, the filmforming ability via compression molding of gelatin. table 1. thickness and mechanical properties of thermo-compression molded gelatin films from resins containing different glycerol levels. gelatin glycerol (%) thickness (µm) em# (x102mpa) ts# (mpa) eab# (%) bovine 15 127.3±7.1c 5.22±0.20e 26.68±0.79e 6.86±1.57a¥ 25 98.5±8.2b 3.84±0.24c 21.65±0.55d 36.29±14.67c 35 83.2±7.4a 2.05±0.16b 10.24±0.85b 125.18±11.83d fish 15 122.7±8.3c 4.38±0.24d 25.45±1.69e 4.98±0.87a 25 94.2±9.4b 3.46±0.30c 19.74±1.00c 24.10±8.73b 35 80.2±8.4a 1.30±0.22a 7.83±0.68a 124.21±6.79d #mean ± sd (n=3). ¥different superscripts in the same column indicate significant differences (p<0.05). 3.1.2. mechanical properties the mechanical properties of thermo-compression molded (120 °c, 20 mpa) bovine and fish gelatin films from resins incorporated with glycerol at various levels are shown in table 1. as shown in the table, gelatin films had decreased ts and em, but increased eab with increasing glycerol levels, regardless of gelatin type (p<0.05). film containing 35% glycerol had the highest eab (125.18 and 124.21% for bovine and fish gelatin films, respectively). however, it exhibited a rather sticky surface, which could be attributed to the excessive addition of glycerol. these results indicated the plasticizing effect caused by glycerol, as well as some adsorbed water in the film matrix. the addition of a miscible plasticizer can reduce inter-chain interactions to overcome the brittleness of films and improve film flexibility (limpisophon et al., 2010). when glycerol is used, its hydroxyl groups could interact with the amino acid groups in the protein, thereby decreasing inter and intra-molecular interactions between protein chains, such as hydrogen bonds, and improving the motion ability of protein macromolecules, thereby resulting in increased flexibility (i.e. reducing the em) of the films. as a result, the behavior of gelatin films changed from brittle to flexible. moreover, glycerol, a small molecule with highly hygroscopic characteristic, is easily inserted between protein chains and attracts more water to the structure of the film, thereby becoming more flexible. similar results have been reported for protein films prepared using thermal techniques (cunningham et al., 2000; zhang et al., 2001; sothornvit et al., 2007). however, the required amount of plasticizer, which is appropriate for the film-forming ability and better properties of film prepared from thermal processing, is dependent on protein type, technique and condition used (sothornvit et al., 2007; park et al., 2008). park et al. (2008) produced a gelatin film from resin that was plasticized with 20% ital. j. food sci., vol 29, 2017 494 glycerol by extrusion. thus, it was found that the extruded film could not be produced from resin plasticized with sorbitol, since it had low fluidity during processing. sothornvit et al. (2007) prepared a protein sheet from mixtures of whey protein isolates (wpi) and glycerol (30, 40 and 50%) using the compression molding method. they revealed that the increasing glycerol content not only decreased ts and em, but also increased the eab of the wpi-sheet. they suggested that by using the compression molding method, a minimum of 30% glycerol should be added in the production of wpisheets, since lower glycerol levels produce sheets which are too brittle to handle and test. zhang et al. (2001) prepared soy protein sheets with various glycerol levels (10, 20, 30, 40 and 50%) by extrusion. they reported that films with 10% glycerol were very difficult to produce and very brittle after losing moisture. when glycerol of 20 to 30% was added, the strength of the sheet decreased a little, but it was good in terms of greater eab. an increase in glycerol level (above 30%) resulted in a large decrease in yield stress and continuous increase in eab. from the results, it was found that the effect of the added glycerol on the compression-molded gelatin film showed a similar trend to the gelatin film developed using the casting technique (vanin et al., 2005; jongjareonrak et al., 2006; hoque et al., 2011). for the same level of glycerol used, bovine gelatin films showed greater mechanical resistance than fish gelatin films as indicated by the higher ts, em and eab of the films. this can be attributed to the higher imino acid content in bovine gelatin (arvanitoyannis et al., 2002; avena-bustillos et al., 2006). the proline and hydroxyproline contents are approximately 30% for mammalian gelatins, 22-25% for warm-water fish gelatins (tilapia and nile perch), and 17% for cold-water fish gelatins (cod) (avena-bustillos et al., 2006; chiou et al., 2008). mammalian gelatins commonly have better physical properties and thermal stability than most fish gelatins (arvanitoyannis et al., 2002; muyonga et al., 2004; avena-bustillos et al., 2006). nevertheless, the incorporation of glycerol at 25% protein, produced continuous gelatin films with sufficient flexibility to be handled, irrespective of gelatin type. thus, the glycerol levels used had a significant influence on the film-forming ability and properties of the resulting compression-molded gelatin films. 3.1.3. water vapor permeability (wvp) the wvp of compression-molded bovine and fish gelatin films from resins incorporated with different glycerol levels is shown in fig. 2. generally, the wvp of all gelatin films increased with increasing glycerol levels (p<0.05), regardless of gelatin type. glycerol, a highly hygroscopic substance, which consists of 3 hydroxyl groups, was able to attract water to the plasticized protein system (sothornvit et al., 2001). plasticizers not only improve the mechanical properties of protein films, but also increase the film permeability (cuq et al., 1997; sothornvit et al., 2001). besides the contribution from solubility, polymer permeability is also dependent on its diffusion coefficient. the diffusion coefficient and thus, the permeability increased with an increase in free volume, as well as the interstitial space between the polymer molecules. from the result, the increase in wvp of both gelatin films with increasing glycerol level could also be explained by an increase in free volume of the film matrix. the insertion of plasticizers between protein molecules increased the free volume of the system and favored the mobility of polymeric chains. consequently, the network structure of the film became less dense and more permeable (jongjareonrak et al., 2006). orliac et al. (2003) reported that an increase in hydrophilic plasticizer levels enhanced the absorption of more water to the film network and increased water vapor transfer. ital. j. food sci., vol 29, 2017 495 the results of this study are in agreement with the reports of various protein films prepared using solution casting such as gelatin films from cuttlefish (sepia pharaonis) skin (hoque et al., 2011), brown stripe red snapper and big-eye snapper skin (jongjareonrak et al., 2006), pig skin (vanin et al., 2005), muscle protein of nile tilapia (paschoalick et al., 2003), wheat gluten (gontard et al., 1993) and protein films prepared using thermal method such as sun flower protein film (orliac et al., 2003), soy protein sheet (zhang et al., 2001) and soy protein isolate film (cunningham et al., 2000). glycerol level w v p (x 10 -1 0 g .m /m 2 . s. p a) 2 3 4 5 15% 25% 35% 15% 25% 35% b e bovine fish a c d f figure 2. the wvp of thermo-compression molded gelatin films from resins containing different glycerol levels. each bar represents the standard deviation (n=3). different letters indicate significant differences (p<0.05). at the same glycerol level, fish gelatin films showed lower wvp than bovine gelatin films (p<0.05). this may be attributed to the lower concentrations of proline and hydroxyproline in fish gelatins as compared with mammalian gelatins (norland, 1990; gomezguillen et al., 2009). moreover, it has been reported that the lower wvp of fish gelatin films could be useful, particularly for applications related to reducing water loss from encapsulated drugs and refrigerated or frozen food systems (norland, 1990). therefore, the amount of glycerol added influenced not only the flow-ability, film-forming ability and mechanical properties but also the barrier properties of the compressionmolded gelatin films. 3.1.4. color and transparency value the color of the thermo-compression molded gelatin films from resins containing different glycerol levels is shown in table 2. in general, as the glycerol concentration increased from 15 to 25%, both types of gelatin films exhibited almost similar l*and a*-values (p>0.05). when the glycerol content was further increased to 35%, the fish gelatin film showed higher l*and a*-values (p<0.05). however, the b*and ∆e*-values of films from both gelatins tended to decrease with increasing glycerol level. the behavior of total color difference (∆e*) was mainly as a result of the variation in b*-value. overall, the results indicated that when the concentration of glycerol increased, the film showed higher ital. j. food sci., vol 29, 2017 496 lightness and less color, especially yellowness. this can be attributed to the effect of the dilution of proteins by the addition of glycerol, which is a colorless plasticizer (paschoalick et al., 2003; sobral et al., 2005). however, vanin et al. (2005) did not observe the effect of plasticizer type (glycerol, polypropylene glycol, ethylene glycol and di-ethylene glycol) and plasticizer concentration (10, 15, 20, 25 and 30%) on the color difference and opacity of gelatin-based films. the different observations might be due to differences in amino acid composition and molecular weight of gelatin, as well as the alignment of gelatin molecules in the film network. at the same glycerol levels, bovine gelatin films were darker in color and had much higher yellowness and slightly lower lightness as evidenced by a greater b*-value and lesser l*-value, respectively, when compared with fish gelatin films (p<0.05). this was mainly due to the differences in color of both gelatin powders used as raw materials. table 2. color of thermo-compression molded gelatin films from resins containing different glycerol levels. gelatin glycerol (%) l*# a*# b*# ∆e*# bovine 15 88.98±0.02a -1.58±0.02a 6.70±0.16e 7.95±0.14e¥ 25 89.56±0.04ab -1.41±0.02b 4.88±0.22d 6.17±0.19d 35 89.85±0.08bc -1.33±0.04b 3.99±0.18c 5.31±0.18c fish 15 90.53±0.08c -1.21±0.04c 2.46±0.05b 3.81±0.05b 25 90.46±0.08c -1.17±0.07c 2.27±0.09b 3.76±0.08b 35 92.01±0.88d -0.44±0.10d 0.17±0.08a 1.60±0.20a #mean ± sd (n=3). ¥different superscripts in the same column indicate significant differences (p<0.05). the transparency value of compression-molded gelatin films, plasticized with glycerol at different levels, is shown in fig. 3. glycerol level tr an sp ar en cy v al ue 0.50 0.55 0.60 0.65 0.70 0.75 15% 25% 35% 15% 25% 35% d cd ab cd abc a bovine fish figure 3. transparency value of thermo-compression molded gelatin films from resins containing different glycerol levels. each bar represents the standard deviation (n=3). different letters indicate significant differences (p<0.05). ital. j. food sci., vol 29, 2017 497 generally, the glycerol content used had a slight impact on the transparency of the compression-molded films of both gelatins. the transparency value of the film tended to decrease with increasing glycerol levels. for both gelatins, films plasticized with 35% glycerol showed a lower transparency value than those with 15% glycerol (p<0.05). based on the equation used for the calculation of the transparency value, the film sample with lower transparency value was more transparent. from the results, films were more transparent when a high level of glycerol was incorporated. this could be attributed to the fact that the added glycerol decreased protein-protein interactions with concomitant increase in free volume in the film matrix; hence, light could pass through such a network more easily. this result is in accordance with other reports in which films became more transparent with increasing plasticizer level (paschoalick et al., 2003; sobral et al., 2005). 3.2. effect of pre-heating conditions on properties of thermo-compression molded gelatin films the result of this study showed that films from bovine and fish gelatins to which 25% glycerol was added had satisfactory mechanical and water vapor barrier properties. furthermore, the film obtained was not sticky and easy to handle. therefore, to study the effect of pre-heating conditions on gelatin resins, 25% glycerol level was used. 3.2.1. film forming ability and visualized appearance of films to investigate the film forming ability of plasticized gelatins in the melt state via thermocompression molding, pre-heating of resin prior to pressing was performed at various temperatures (120, 140 and 160 °c) and duration (5 and 10 min). table 3 shows the overall visualized appearance of films obtained under different pre-heating conditions. table 3. appearance of thermo-compression molded gelatin films prepared under different pre-heating conditions. gelatin preheating conditions visual appearance temp.(oc) time (min) bovine 120 5 film with many voids or pinholes 10 140 5 continuous, flexible, transparent and yellow film 10 160 5 opaque and very dark yellow brittle film 10 low fluidity of the melt; too brittle film fish 120 5 10 continuous, flexible, transparent and less yellow film. 140 5 10 160 5 rather opaque and slightly yellow brittle film 10 low fluidity of the melt; too brittle film ital. j. food sci., vol 29, 2017 498 the thickest films with a lot of visualized voids were obtained for both gelatin types when the processing temperatures used were lower than 120 °c. this might be due to the fact that the molten resins could not flow properly because of its high melt viscosity. moreover, processing temperatures higher than 160 °c resulted in dramatic degradation. it was found that pre-heating of bovine gelatin resin at 120°c for 5 min rendered the film with numerous visualized voids or pin-holes. under this pre-heating condition, the plasticized-bovine gelatin melt might still possess high viscosity. as a result, the air bubbles could not be evacuated from the melt before compressing and thus, were entrapped in the film after cooling. however, it was not noticed for fish gelatin resin preheated under the same condition, possibly due to the lower melt viscosity of fish gelatin as compared with bovine gelatin. continuous and flexible films without visualized pin-holes were formed when the pre-heating temperature and time were increased up to 160 °c and 5 min, regardless of gelatin type. the melt viscosity of both gelatin resins might decrease sufficiently and allowed the gelatin melt to flow easily. as a result, the gelatin molecules could form a continuous network structure of film. generally, the melt viscosity of the polymer decreased with increasing processing temperature, which is a significant factor in determining the flowability and processibility of the polymer via molding processes (cunningham et al., 2000; zhang et al., 2001; park et al., 2008). however, very brittle films with obvious degradation were obtained for both gelatins when the pre-heating of resins at 160 °c for 10 min was implemented. the application of a higher temperature for a longer period of time resulted in a dramatic degradation of gelatin, as well as the evaporation of bounded water. chuaynukul (2011) reported that partial degradation was noticed in the gelatin film compression molded at 120 °c, as evidenced by the increased free-amino group content. therefore, the condition used in the pre-heating step was crucial for determining the gelatin film processibility via compression molding. the studied pre-heating conditions also affected the properties of bovine and fish gelatin films differently. 3.2.2. mechanical properties the mechanical properties of gelatin films prepared by different resin pre-heating conditions are shown in table 4. the ts and em of the films decreased, whilst the eab increased with increasing pre-heating temperature and time (p<0.05), regardless of gelatin type. gelatin films exhibited the highest values of ts and em at pre-heating temperature of 120 °c (20.82 mpa, 6.17 x102 mpa and 18.90 mpa, 4.76.x102 mpa for bovine and fish gelatin films, respectively). the ts and em decreased when the pre-heating temperature was increased. this was probably due to the partial degradation of gelatin, which took place at higher temperature and extended time. the shorter gelatin molecules formed the lower intermolecular interactions, resulting in films with lower strength and stiffness (that is lower ts and em, respectively). chuaynukul (2011) observed that the partial degradation of gelatins took place in resins and the resulting films when the resins were subjected to higher temperature prior to molding, as reconfirmed by the increased freeamino group content of the gelatin. the impact of heating on the degradation of gelatin has been investigated and reported by hoque et al. (2010). it was pointed out that the partial degradation of gelatin occurred when the gelatin solution was heated at a temperature higher than 70°c. this caused a decrease in the ts of the resulting films, because the shorter chains of gelatin molecules could not form the strong film network. the presence of degradation of protein molecules upon being processed at higher temperature was similarly observed in various protein-based films fabricated using ital. j. food sci., vol 29, 2017 499 thermal processing techniques (sothornvit et al., 2007; katoh et al., 2004; jansens et al., 2013). table 4. mechanical properties and water vapor permeability (wvp) of thermo-compression molded gelatin films prepared under different pre-heating conditions. g el at in pre-heating conditions em# (x102mpa) ts# (mpa) eab# (%) wvp# (x10-10g.m/m2.s.pa) temp. (°c) time (min) b ov in e 120 5 nd nd nd nd 10 6.17±1.37f 20.82±2.32f 33.61±6.75a 3.29±0.26f ¥ 140 5 4.33±0.87de 17.15±0.43de 57.87±12.96b 3.07±0.30ef 10 3.48±0.71c 13.85±1.33c 126.29±11.49e 2.80±0.04cd 160 5 2.07±0.73c 11.56±1.40a 90.12±9.63d 2.47±0.11ab 10 nd nd nd nd fi sh 120 5 4.76±0.52e 18.90±1.41e 24.68±5.94a 3.12±0.05ef 10 3.63±0.07cd 16.67±1.63d 31.50±5.79a 2.97±0.26de 140 5 2.79±0.25bc 13.52±0.92bc 79.90±7.18cd 2.61±0.08bc 10 1.91±0.57ab 11.81±1.72ab 105.52±15.1e 2.50±0.07ab 160 5 1.60±0.36a 11.42±0.56a 82.24±12.27c 2.25±0.06a 10 nd nd nd nd #mean ± sd (n=3). ¥different superscripts in the same column indicate significant differences (p<0.05). nd = not determined. for the eab value, pre-heating at higher temperature resulted in increased eab of the films in both gelatins (p<0.05). at the same pre-heating temperature, films pre-heated for 10 min had higher eab than those pre-heated for 5 min (p<0.05), regardless of gelatin type. this suggested an increase in film extensibility or stretch-ability. from the result, films prepared from the same type of gelatin had the highest eab when pre-heating at 140 °c for 10 min was implemented. an increase in eab was observed with a concomitant decrease in ts (gennadios et al., 1996b; jongjareonrak et al., 2006). additionally, with increasing temperature from 120 to 140°c, gelatin might undergo partial degradation, leading to the formation of shorter chains. this may result in lower and/or weaker interaction between gelatin molecules (resulted in decreased ts), which could allow the molecules to slip around each other with more ease and a larger degree upon tensile deformation before breaking of the film specimen (leading to increased eab) (hoque et al., 2010). muyonga et al. (2004) observed that the gelatin extracted from nile perch bones, which consisted of a higher proportion of low-molecular weight fractions, had considerably lower tensile strength, but higher percentage of elongation. however, when the pre-heating temperature was further increased to 160 °c, both gelatin films exhibited decreased eab. at this temperature, a higher degradation of gelatin might take place resulting in shorter chains. these shorter gelatin chains may be unable to form a strong network, leading to a decrease in both ts and eab. when the films were compared based on different gelatin sources, fish gelatin films had lower ts, em and eab as compared with bovine gelatin films fabricated under the same pre-heating condition. this might be due to the presence of a higher number of imino acid residues in the α-chain of bovine gelatin, which contributed to the high number of ital. j. food sci., vol 29, 2017 500 hydrogen bonds (norland, 1990). chiou et al. (2008) prepared fish gelatin films from alaska pollock (theragra chalcogramma) and alaska pink salmon (oncorhynchus gorbuscha) and compared their mechanical properties to bovine and porcine gelatin films. they reported that pollock and salmon gelatin films had lower ts and eab than mammalian gelatin films. therefore, the pre-heating temperature and time used in thermocompression molding are essential to controlling the mechanical properties of the resulting films. 3.2.3. water vapor permeability (wvp) the wvp of the thermo-compression molded gelatin films prepared from different preheating conditions is shown in table 4. the wvp of the films seemed to decrease with an increase in pre-heating temperature and time used, irrespective of gelatin type (p<0.05). at the same pre-heating temperature, films prepared with pre-heating time of 10 min showed lower wvp than those prepared with pre-heating time of 5 min (p<0.05). films obtained from higher pre-heating temperature also exhibited lower wvp (p<0.05). these results indicated that pre-heating at higher temperature led to increase in the water vapor barrier of the compression-molded gelatin films. this may be attributed to the exposure of the hydrophobic domains of gelatin chains upon heating, resulting in increased hydrophobic interaction and hydrophobicity of the films; thus, lowering the tendency of water absorbance by the film matrix (kim et al., 2002). furthermore, the regular arrangement of shorter chain gelatin obtained after heating at higher temperatures, plausibly led to a compact matrix of gelatin during film formation (kim et al., 2002). as a result, the diffusion of water molecules could be retarded, which resulted in decreased wvp. katoh et al. (2004) reported that molding at higher temperature caused the formation of a more compact structure of keratin film, which prevented the film from absorbing water. moreover, the high temperature treatment of proteins might provoke additional interaction and crosslinking of protein molecules. jansens et al. (2013) recorded an increase in water absorption of the compression-molded gluten protein film, as higher processing temperatures and longer times were used. this is mostly due to the presence of a higher degree of protein cross-linking during compression molding. a similar result was also reported by kim et al. (2002) for heated soy protein films, in which the wvp of the film decreased during heating, and this was probably caused by the formation of crosslinks. under the same pre-heating condition, fish gelatin film had lower wvp as compared with bovine gelatin film (p<0.05). this can be attributed to the higher hydrophobicity of fish gelatin, which had lower concentrations of proline and hydroxyproline as compared with mammalian gelatins (norland, 1990; karim and bhat, 2009). avena-bustillos et al. (2006) reported the wvp of fish-gelatin films to be significantly lower than that of films made from mammalian gelatin. they explained the tendency of fish-gelatin films to exhibit lower wvp values than land animal-gelatin films in terms of amino acid composition. fish gelatins, especially cold-water fish gelatins, are known to contain higher amounts of hydrophobic amino acids and lower amounts of hydroxyproline (avenabustillos et al., 2006). 3.2.4. color and transparency value of films the color and transparency value of compression-molded gelatin films prepared under different pre-heating conditions are shown in table 5. as the pre-heating temperature and time increased, the films had increased yellowness (b*-value) and total color difference (δe*-value), as well as slightly decreased film transparency (as indicated by an increase in ital. j. food sci., vol 29, 2017 501 the transparency value) (p<0.05). the increased yellowness of the films pre-heated at higher temperature and longer time was plausibly due to maillard reaction, which is associated with the heat-induced process (hoque et al., 2010). as the pre-heating temperature and time increased, the lightness (l*) of the bovine gelatin films showed a slight decreasing trend, whereas fish gelatin films showed no difference in l*-value (90.12-90.62). paschoalick et al. (2003) suggested that the increase in temperature caused a slight increase in the color of films, possibly due to the occurrence of a reaction among the glycerol molecules and the reactive group of lysine. for films fabricated under the same pre-heating condition, the fish gelatin films exhibited a lighter color but they were slightly more transparent than the bovine gelatin films. the differences in color and lightness of both films observed from the results were most likely influenced by the different gelatin types and manufacturing processes. the slight difference in transparency value of bovine and fish gelatin films might be due to differences in the intrinsic characteristics of each gelatin type, such as composition, density, aggregation or alignment of gelatin molecules in the films, as well as type of extraction treatments used to prepare the gelatin (bailey et al., 1989). the purification procedure might increase the lightness of gelatin powder (norland, 1990). table 5. color and transparency values of thermo-compression molded gelatin films prepared under different pre-heating conditions. g el at in pre-heating conditions l*# a*# b*# ∆e*# transparency value# temp. (°c) time (min) b ov in e 120 5 nd nd nd nd nd 10 89.78±0.07cd -1.55±0.06de 5.09±0.14d 6.05±0.12c 0.64±0.03ab¥ 140 5 89.10±0.05bc -1.82±0.03c 7.49±0.11e 8.42±0.09d 0.70±0.03c 10 88.65±0.13b -1.97±0.16b 8.36±0.71f 9.4±0.65e 0.71±0.03c 160 5 87.16±1.41a -3.06±0.12a 12.04±0.62g 13.43±1.22f 0.81±0.02d 10 nd nd nd nd nd fi sh 120 5 90.62±0.03d -1.27±0.02f 1.73±0.08a 3.53±0.06a 0.58±0.04a 10 90.59±003d -1.30±0.01f 1.90±0.06a 3.60±0.05a 0.60±0.03a 140 5 90.50±0.06d -1.43±0.01e 3.32±0.18b 4.62±0.20b 0.63±0.02ab 10 90.47±0.05d -1.46±0.03e 3.49±0.14b 4.69±0.05b 0.63±0.03ab 160 5 90.12±0.07d -1.64±0.05d 4.17±0.07c 5.46±0.07bc 0.68±0.02bc 10 nd nd nd nd nd #mean ± sd (n=3). ¥different superscripts in the same column indicate significant differences (p<0.05). nd = not determined. 4. conclusions films from bovine and fish gelatins were successfully prepared using the thermocompression molding technique. increasing the glycerol level resulted in decreased ts, em and yellowness, but increased eab, wvp and transparency of the resulting films. gelatin films had generally decreased ts, em, wvp and transparency, whilst increased yellowness occurred as pre-heating temperature and time increased. the compressionmolded gelatin films incorporated with 25% glycerol (based on protein) had sufficient ital. j. food sci., vol 29, 2017 502 flexibility (that is, eab). film processing conditions had significant impact on the processibility and properties of obtained compression-molded gelatin films. among the major steps involved in the compression-molding of gelatin films, the pre-heating of resin prior to molding seemed to be the most crucial step in determining the processibility and properties of the obtained films. therefore, the plasticizer level, pre-heating temperature and time have to be controlled for optimal flow-ability and fusion of the molten gelatin before compression/molding into a film. acknowledgements the authors would like to thank the prince of songkla university, thailand for the financial support. the trf distinguished research professor grant is also acknowledged. psu – postdoctoral fellowship from prince of songkla university for muralidharan nagarajan is acknowledged. references aoac. 2000. “official methods of analysis” 17th ed. association of official analytical chemists, gaithersberg. arvanitoyannis i.s. 2002. formation and properties of collagen and gelatin films and coatings. in “protein-based films and coatings” a. gennadios (ed.) pp. 275-304. crc press, boca raton, florida. astm. 2002a. standard test method for tensile properties of thin plastic sheeting (d882-01). in: “annual book of astm standards” (ed.). pp. 162-170. astm (american society for testing and materials), pennsylvania. astm. 2002b. standard test method for water vapor transmission of materials (e96-01). in: “annual book of astm standards” (ed.). pp. 162-170. astm (american society for testing and materials), pennsylvania. avena-bustillos r.j., olsen c.w., chiou b., yee e., bechtel p.j. and mchugh t.h. 2006. water vapor permeability of mammalian and fish gelatin films. j. food sci. 71:202-207. bailey a.j. and light n.d. 1989. connective tissue in meat and meat products. elsevier applied science, london. bigi a., cojazzi g., panzavolta s., roveri n. and rubini k. 2002. stabilization of gelatin films by cross-linking with genipin. biomaterials. 23:4827-4832. chiou b.s., avena-bustillos r.j., bechtel p.j., jafri h., narayan r., imama s.h. et al. 2008. cold water fish gelatine films: effects of cross-link on thermal, mechanical, barrier and biodegradation properties. eur. polym. j. 44:3748-3753. chuaynukul k., prodpran t. and benjakul s. 2015. properties of thermo-compression molded bovine and fish gelatin films as influenced by resin preparation condition. int. food res. j. 22:1095-1102. chuaynukul k. 2011. properties of gelatin film prepared by thermo-compression molding technique as affected by forming conditions. master of science thesis, prince of songkla university, thailand. cunningham p., ogale a.a., dawson p.l. and acton j.c. 2000. tensile properties of soy protein isolate films produced by a thermal compaction technique. j. food sci. 65:668-671. cuq b., gontard n. and guilbert n. 1998. proteins as agricultural polymers for packaging production. j. cereal chem. 75:1-9. cuq b., gontard n. and guilbert s. 1997. thermal properties of fish myofibrillar protein-based films as affected by moisture content. polymer. 38:2399-2405. gennadios a. 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325-333. technomic publishing co. inc., lancaster, pennsylvania. orliac o., rouilly a., silvestre f. and rigal l. 2003. effect of various plasticizers on the mechanical properties, water resistance and aging of thermo-moulded films made from sunflower proteins. ind. crop. prod. 18:91-100. ou s.y., kwok k.c. and kang y.j. 2004. changes in vitro digestibility and available lysine of soy protein isolate after formation of film. j. food eng. 64:301-305. park j.w., whiteside w.s. and cho s.y. 2008. mechanical and water vapor barrier properties of extruded and heatpressed gelatin films. lwt-food sci. technol. 41:692-700. ital. j. food sci., vol 29, 2017 504 paschoalick t.m., garcia f.t., sobral p.j.a. and habitante a.m.q.b. 2003. characterization of some functional properties of edible films based on muscle proteins of nile tilapia. food hydrocolloid. 17:419-427. shiku y., hamaguchi p.y., benjakul s., visessanguan w. and tanaka m. 2004. effect of surimi quality on properties of edible films 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(ed.). 1980. principles and procedures of statistics: a biometrical approach. mcgraw-hill, new york. vanin f.m., sobral p.j.a., menegalli f.c., carvalho r.a. and habitante a.m.q.b. 2005. effects of plasticizers and their concentrations on thermal and functional properties of gelatin-based films. food hydrocolloid. 19:899-907. venien a. and levieux d. 2005. differentiation of bovine from porcine gelatins using polyclonal anti-peptide antibodies in indirect and competitive indirect elisa. j. pharmaceut. biomed. 39:418-424. wang l., auty m.a.e., rau a., kerry j.f. and kerry j.p. 2009. effect of ph and addition of corn oil on the properties of gelatin-based biopolymer films. j. food eng. 90:11-19. zhang s., wang y., herring j.l. and oh j.h. 2007. characterization of edible film fabricated with channel catfish (ictaluruspunctatus) gelatin extract using selected pretreatment methods. j. food sci. 72:498-503. zhang j., mungara p. and jane j. 2001. mechanical and thermal properties of extruded soy protein sheets. polymer. 42:2569-2578. paper received september 3, 2016 accepted december 30, 2016 #581_hac-szymanczuk_bozza ital. j. food sci., vol 29, 2017 329 paper application of rosemary for the prolongation of microbial and oxidative stability in mechanically deboned poultry meat from chickens e. hać-szymańczuk*1, a. cegiełka2, e. lipińska1 and k. piwowarek1 1department of biotechnology, microbiology and food evaluation, faculty of food sciences, warsaw university of life sciences sggw (wuls sggw), 159c nowoursynowska street, 02-787 warsaw, poland 2department of food technology, faculty of food sciences, warsaw university of life sciences sggw (wuls sggw), 159c nowoursynowska street, 02-787 warsaw, poland *corresponding author. elzbieta_hac_szymanczuk@sggw.pl abstract in this study, we aimed to determine the effect of rosemary (rosmarinus officinalis l.) preparations on microbial quality and oxidative stability in vacuum-packed mechanically deboned poultry meat (mdpm) from chickens stored at -18 °c for 4 months. we used mdpm originating from four production batches in which rosemary was added to in the form of dried spice (2.0%), extracts (2.0%) such as aqueous and ethanol (40 and 70% (v/v)), and essential oil (0.2%). mdpm control sample did not contain added rosemary. according to the results, the microbial quality of mdpm depended on the type of rosemary preparation used. compared to the control sample, total bacterial count was considerably lower in samples with added essential oil and ethanol extract (70%, v/v). essential oil was found to be the most effective in inhibiting psychrotrophic bacteria growth in vacuum-packed mdpm during storage. during the entire storage period, the use of rosemary preparations did not have a significant effect on the count of enterobacteriaceae, but it significantly limited the growth of the coliform bacteria. based on the index value of thiobarbituric acid reactive substances, rosemary preparations also showed, except for aqueous extract, a decrease in lipid oxidation in vacuum-packed mdpm from chickens stored at -18 °c for 4 months. keywords: antimicrobial effect, antioxidant effect, mechanically deboned poultry meat, rosemary, storage ital. j. food sci., vol 29, 2017 330 1. introduction mechanically deboned poultry meat (mdpm) obtained from chickens constitutes raw material commonly used in the meat industry, particularly in the production of homogenized products. the use of mdpm is justified for economic reasons as well as for the pursuit of rational usage of the elements of carcasses, which would be difficult to use otherwise (pietrzak et al., 2011). the basic raw material to obtain mdpm from chickens is bones that remains from the deboning of the largest muscles (breast and thigh) and carcasses of chicken of lower quality (stangierski et al., 2011). the mdpm production, storage, and processing conditions have been established in regulation (2004). despite the continuous improvements in methods and machines used to obtain mdpm, polish and european poultry industries most commonly use highpressure methods for the production of mdpm, which is destructive for the bone structure (nagy et al., 2007; botka-petrak et al., 2011; bełkot et al., 2013). this leads to the lower stability of the raw material upon storage than hand-trimmed or machine-trimmed chicken meat. this poor stability is primarily due to the high level of fragmentation and aeration during production, which contributes to a higher susceptibility toward oxidation processes and an increase in the growth of microflora (grabowski and kijowski, 2004; michalski and pomykała, 2008). in a situation of inability for immediate use of mdpm in processing, the raw material is stored in a frozen state (grabowski and kijowski, 2004). addition of natural substances from plant origin, exhibiting antimicrobial and antioxidant effect, constitutes an additional factor in prolonging the stability of meat and meat products during storage. however, the latest literature search (shah et al., 2014) reveals that the majority of the studies of the effectiveness of plant preparations on the stability of meat products during storage involves mammal meat and its products. study results demonstrate that inter alia rosemary preparations may be used to minimize the oxidative changes in meat and meat products such as aged beef (colle et al., 2016), raw pork batters (hernándezhernández et al., 2009), fresh (georgantelis et. al., 2007) and thermally processed pork sausage (sebranek et al., 2005), wiener (coronado et al., 2002) and bologna sausages (viuda-martos et al., 2010), frankfurters (estévez and cava, 2006), and reduced nitrite liver pâtés (doolaege et al., 2012). due to the application of rosemary preparations, microbial quality of different meat products can be improved, inter alia in modified atmosphere-packaged fresh pork and vacuum-packed ham slices (zhang et al., 2009), and in the african fresh sausage (mathenjwa et al., 2012). however, there is little information on the application possibilities of plant preparations for the prolongation of storage stability of mdpm (hassan and lam swet fan, 2005; hać-szymańczuk et al., 2014) and its products (mohamed and mansour, 2012; jiridi et al., 2015). rosemary (rosmarinus officinalis l.), from the lamiaceae family, is a plant with both strong antioxidant and antimicrobial activities. it is used in food in the form of fresh or dried leaves, essential oil, and aqueous and alcoholic extracts from leaves (erkan et al., 2008; hać-szymańczuk et al., 2010). various studies have demonstrated that the complex biologically active substances of rosemary have an inhibitory effect on a wide spectrum of bacteria, including enterococcus faecalis, staphylococcus aureus, staphylococcus epidermidis, bacillus subtilis, and klebsiella pneumoniae (dimitrijevic et al., 2007; zhang et al., 2009; hać-szymańczuk et al., 2010). as an alternative to synthetic antioxidants, rosemary preparations have been used in food processing (balentine et al., 2006; velasco and williams, 2011). in this study, we aimed to determine the effect of rosemary (r. officinalis l.) on lipid oxidation and microbial quality of mdpm from chickens stored at -18 °c for 4 months. ital. j. food sci., vol 29, 2017 331 the results of this study may contribute to the understanding of the innovative methods of mdpm preservation. 2. materials and methods we used mdpm that was obtained from a production plant in northeast poland. mdpm was prepared using high-pressure separation method on breast muscle scraps of broilers. the chilled mdpm (6 kg) was collected from the wholesalers in warsaw and transported to the division of food biotechnology and microbiology of the faculty of food sciences under refrigerated conditions. rosemary (r. officinalis l.) was added to the mdpm in dried form (“kamis,” mccormic, stefanowo, poland) and as an aqueous extract, ethanol extracts, and essential oil (own production) under laboratory conditions. in the following parts of the paper, they will be called rosemary preparations. each of the batches of mdpm from chickens was analyzed for fat, protein, and water content. the determination of these chemical components in mdpm was performed in accordance with the requirements of the association of official analytical chemists (aoac, 2007). we used a foodscan™lab near-infrared spectrometer (foss analytical a/s, hillerød, denmark) working in the spectral range of 850-1050 nm and using a calibration based on the artificial neural network model. the aqueous extract and ethanol extracts from dried rosemary (“kamis,” mccormic, stefanowo, poland) were obtained via continuous extraction in a soxhlet apparatus (a universal extraction system b-811, büchi labortechnik ag, flawil, switzerland). the extraction process parameters were established in the preliminary study (results unpublished). for the preparation of each extract, 40 g of dried rosemary was distributed onto 8 extraction thimbles (5 g per thimble). distilled water and ethyl alcohol with 40 and 70% (v/v) concentration were, respectively, used as solvents. the raw material in each thimble was extracted with 150 ml of the appropriate solvent for 15 cycles, maintaining the boiling point of a solvent. the portions obtained from each extract were combined, resulting in approximately 550 ml of raw extracts. the raw extracts were filtered using 180-µm thick filter paper (whatman ge, laboplus sp. z o.o., warsaw, poland). subsequently, each extract was concentrated in a rotary evaporator (rotovaporator r-205; büchi labortechnik ag) until approximately 40 g of the extract was left, corresponding to the weight of dried rosemary used to obtain the extract. to obtain essential oil from rosemary, the method of białecka-floriańczyk and włostowska (2007) was followed. around 30 g of fresh rosemary leaves were crumbled and covered with 400 ml of water. this was subjected to distillation in a deryng apparatus by simax until essential oils were obtained. the chilled distillate was four times extracted using dichloromethane in a separatory funnel. then, water was removed by adding anhydrous magnesium sulfate. the obtained extract was concentrated in a rotary evaporator (rotovaporator r-205). the solvent was evaporated at a temperature of 30 °c and at a pressure of 540-560 hpa. the chemical composition of the aqueous extract, ethanol extracts, and essential oil from rosemary was analyzed for the identification and determination of chemical compounds. the determination of volatile compounds in essential oil was performed by gas chromatography (gc) equipped with flame ionization detector (fid) (perkin elmer, autosystem xl) based on the literature (burt, 2004; djeddi et al., 2007). the following parameters were used for separation: hp-5 column (30 m × 0.32 mm × 0.25 µm), helium as a carrier gas (3 cm3/min), split mode (1:100) for sample injection, injection temperature at 270 °c, and fid temperature at 300 °c. the following program of oven temperature was used: initial temperature 35 °c/5 min, 30 °c/min temperature increase up to 60 °c ital. j. food sci., vol 29, 2017 332 followed by 6 °c/min to 200 °c and 30 °c/min until a temperature of 280 °c was achieved. the identification and determination of the amount of selected chemical compounds in rosemary extracts was performed based on the literature (longaray delamare et al., 2007, tawaha et al., 2007). in this study, we performed high performance liquid chromatography (hplc) using agilent 1200 liquid chromatography coupled with diode array detector (dad). zorba eclipse xdb c18 (4.6 × 150 mm) column was used with the following parameters: 5 μl injection volume, 0.8 cm3/min flow rate, and uv detection at a wavelength of 210 and 325 nm. separation was performed in gradient elution with two eluents: a-acetonitrile and b-0.05% trifluoroacetic acid. data were analyzed using ez elite chrome program. in each experimental series, six samples of mdpm were prepared (each weighing 1 kg), differing in the type of rosemary preparation added: control-sample without addition of rosemary, d-2.0% addition of dried rosemary, we-2.0% addition of aqueous extract from rosemary, e40-2.0% addition of 40% (v/v) ethanol extract from rosemary, e70-2.0% addition of 70% (v/v) ethanol extract from rosemary, and eos-0.2% addition of essential oil from rosemary. the amount of rosemary preparations added to mdpm was established based on the recommendations of the producer or based on the literature (georgantelis et al., 2007). after mdpm samples were thoroughly mixed with rosemary preparations, each sample was divided into four portions (250 g each) and vacuum-packed in plastic bags (pe/pa, thickness 75 µm) using a vacuum machine c200 (multivac sepp haggenmüller gmbh & co. k.g., wolfertschwenden, germany). these samples were stored at a temperature of -18° c for 4 months. after each month, microbial analyzes were performed and tbars index values were determined for all mdpm samples including the control sample. before the analyzes, each sample was defrosted (+4 °c, 4 h) without opening the packaging. moreover, directly after the delivery of the raw material to the laboratory, the same determinations were carried out only on the mdpm control sample. the microbial analyzes were conducted following polish standard (pn-en iso, 2005). they include the determination of the total bacteria count (tbc) (pn-en iso, 2013), number of psychrotrophic bacteria (pn-iso, 2004), enterobacteriaceae (pn-iso, 2005), coliform bacteria (pn-iso, 2007), and salmonella spp. (pn-en iso, 2003). the number of bacteria was expressed as log10 colony forming units per gram (log cfu/g). tbars was determined using the extraction method of pikul et al. (1989). tbars index value was expressed in milligram of malondialdehyde per kilogram of sample (mg mad/kg). the experiment was repeated four times, preparing mdpm samples from different production batches. the statistical analysis of the results was performed using statistica version 10.0 program (2011). the significance was tested using one-way analysis of variance (anova) and tukey’s honest significant difference (hsd) test at a significance level of α=0.05. 3. results in this study, the analysis of chemical composition of rosemary preparations revealed that they differed in the chemical profile. each of them consisted of a complex mixture of different substances. the results of an earlier work (hać-szymańczuk et al., 2015) demonstrated that the dominating compounds of the aqueous extract of rosemary were rosmarinic, ferulic, and chlorogenic acids. in the ethanol extracts of rosemary, the major compounds present were rosmarinic acid, carnosol, and ferulic acid (table 1), whereas in ital. j. food sci., vol 29, 2017 333 the essential oil, the major compounds present were camphor, borneol, and r(+)limonene (table 2). table 1. chemical composition of extracts from rosemary. chemical compound retention time (min) ethanol extract (40%, v/v) ethanol extract (70%, v/v) concentration (mg/cm3) chlorogenic acid 2.83 0.068 0.108 epicatechin 3.80 nd 0.002 caffeic acid 4.33 0.012 0.026 rutoside 5.83 0.060 0.104 p-coumaric acid 7.04 0.006 0.020 ferrulic acid 8.08 0.112 0.166 benzoic acid 11.87 0.012 0.018 rosemarinic acid 12.70 4.006 5.756 myricetin 12.80 nd 0.002 resveratrol 15.51 nd 0.002 quercetyn 18.69 0.006 0.012 carnosol 25.73 0.468 0.178 curcumin 26.03 nd 0.026 nd – not detected table 2. chemical composition of essential oil from rosemary. chemical compound retention time (min) concentration (mg/cm3) α-pinene 7.88 0.20 β-pinene 8.79 0.10 myrcene 9.21 0.22 1,4-cineole 9.72 0.10 p-cymene 9.92 0.53 r(+) limonene 10.13 22.47 γ-terpinene 10.71 0.75 linalol 11.70 1.33 camphor 12.26 51.87 borneol 13.15 27.90 carvone 14.93 0.44 bergamol 15.23 0.18 thymol 15.99 0.11 carwacrol 16.20 6.70 eugenol 17.42 0.92 β-caryophyllene 18.73 0.62 based on the results of microbial analysis of mdpm during storage (figs. 1-4), it was found that the tested rosemary preparations showed different antimicrobial activity. during the storage period, tbc was found to be highest in the control sample (fig. 1). in the eos, e70, and e40 samples, a reduction in tbc was observed from 2 months of ital. j. food sci., vol 29, 2017 334 storage. after 4 months of storage, the eos and e70 samples were characterized by significantly (p≤0.05) lower tbc than control sample. of all the tested rosemary preparations, essential oil was found to be the most efficient in inhibiting the growth of psychrotrophic microorganisms (fig. 2). eos significantly lowered the number of microorganisms compared to control, d, and we after 4 months of storage. in each of the examined mdpm samples, a significantly (p≤0.05) higher enterobacteriaceae bacterial count was found after 2 months of storage (fig. 3). the use of rosemary preparations did not significantly (p>0.05) influence the count of enterobacteriaceae in the mdpm samples during the entire storage period. in each of the examined mdpm samples, coliform bacteria was also detected (fig. 4). however, in comparison to the control sample, addition of rosemary preparations to mdpm significantly (p≤0.05) restricted the growth of coliform bacteria during the entire storage period. salmonella spp. was not determined in any of the examined mdpm samples. the fat, protein, and water content in mdpm from chickens was on average 15.93, 15.72, and 66.31%, respectively. based on tbars index values (fig. 5.), the addition of rosemary preparations to mdpm had an influence on the course of oxidative changes in lipids. among the tested preparations, the weakest antioxidant activity was exhibited by the aqueous rosemary extract. however, other rosemary preparations significantly (p≤0.05) slowed down the processes of lipid oxidation in the mdpm samples during the storage period. our results also demonstrated that for eos and e70 samples, the tbars index value after 4 months of storage was significantly (p≤0.05) lower than the tbars index value after 1 month of storage. figure 1. effect of addition of rosemary preparations on the total bacteria count in mechanically deboned poultry meat (mdpm) from chickens stored at -18 °c. notes: control-control sample, without addition of rosemary; d-2.0% addition of dried rosemary; we-2.0% addition of aqueous extract from rosemary; e402.0% addition of 40% (v/v) ethanol extract from rosemary; e70-2.0% addition of 70% (v/v) ethanol extract from rosemary; eos-0.2% addition of essential oil from rosemary. ital. j. food sci., vol 29, 2017 335 figure 2. effect of addition of rosemary preparations on the number of psychrotrophic bacteria in mechanically deboned poultry meat (mdpm) from chickens stored at -18 °c. notes: control-control sample, without addition of rosemary; d-2.0% addition of dried rosemary; we-2.0% addition of aqueous extract from rosemary; e40-2.0% addition of 40% (v/v) ethanol extract from rosemary; e70-2.0% addition of 70% (v/v) ethanol extract from rosemary; eos-0.2% addition of essential oil from rosemary. figure 3. effect of addition of rosemary preparations on the number of enterobacteriaceae bacteria in mechanically deboned poultry meat (mdpm) from chickens stored at -18 °c. notes: control-control sample, without addition of rosemary; d-2.0% addition of dried rosemary; we-2.0% addition of aqueous extract from rosemary; e40-2.0% addition of 40% (v/v) ethanol extract from rosemary; e70-2.0% addition of 70% (v/v) ethanol extract from rosemary; eos-0.2% addition of essential oil from rosemary. ital. j. food sci., vol 29, 2017 336 figure 4. effect of addition of rosemary preparations on the number of coliform bacteria in mechanically deboned poultry meat (mdpm) from chickens stored at -18 °c. notes: control-control sample, without addition of rosemary; d-2.0% addition of dried rosemary; we-2.0% addition of aqueous extract from rosemary; e40-2.0% addition of 40% (v/v) ethanol extract from rosemary; e70-2.0% addition of 70% (v/v) ethanol extract from rosemary; eos-0.2% addition of essential oil from rosemary. figure 5. effect of addition of rosemary preparations on thiobarbituric acid reactive substances (tbars) index value in mechanically deboned poultry meat (mdpm) from chickens stored at -18 °c. notes: controlcontrol sample, without addition of rosemary; d-2.0% addition of dried rosemary; we-2.0% addition of aqueous extract from rosemary; e40-2.0% addition of 40% (v/v) ethanol extract from rosemary; e70-2.0% addition of 70% (v/v) ethanol extract from rosemary; eos-0.2% addition of essential oil from rosemary. 4. discussion the results of this study showed that mdpm from chickens contained a high amount of fat (15.93%). however, it was in compliance with the requirements of non-compulsory polish standard (pn, 1992), which states that mdpm from burring poultry should not contain fat more than 20%, protein less than 12%, and water more than 75%. the fat present in mdpm is susceptible to oxidation, which might be due to the presence of the unsaturated fatty acids and phospholipids along with the catalytic effects of heme iron ital. j. food sci., vol 29, 2017 337 (grabowski and kijowski, 2004; pietrzak et al., 2011; bełkot et al., 2013). since lipid oxidation is the major cause of quality loss in mdpm, in our opinion the application of rosemary preparations as sources of natural antioxidants seems to be interesting option for preserving the shelf life of this raw material. the raw material used in this study met the food safety criteria with respect to salmonella and aerobic bacteria and e. coli count as specified in commission regulation (2005). more discussion in this area is difficult because the available literature lacks the information on antimicrobial effect of rosemary extracts in mdpm. hać-szymańczuk et al. (2009) and okoh et al. (2010) have found that the antimicrobial efficiency of rosemary extracts varies, which could be attributed to the type and method of its preparation (e.g., distillation and extraction). this could be due to the different chemical profiles of these preparations. in their study, okoh et al. (2010) found that rosemary oil obtained by solvent free microwave extraction exhibited stronger inhibitory effect on the examined bacteria (staphylococcus aureus, e. coli, bacillus subtilis, and klebsiella pneumoniae) in comparison to the oil obtained through hydro-distillation. however, hać-szymańczuk et al. (2009) found that rosemary oil as well as aqueous extract did not inhibit the growth of s. aureus and k. pneumoniae on mueller-hinton agar medium. romano et al. (2009) found that the addition of rosemary leaf extract limited the growth of e. coli. according to the authors the minimal inhibitory concentration (mic) of this extract was 105 μg/ml. they found a stronger antimicrobial activity than benzoic acid and butylated hydroxytoluene (bht), whose concentration was 250 μg/ml. however, based on the comparative study of antimicrobial properties of essential oils from lamiaceae plants, žižovic et al. (2009) found that the minimum inhibitory concentration (mic) for escherichia and salmonella bacteria was above 1250 µg/ml. similar studies on the use of rosemary preparations, solely or mixed with other components of plant origin, have demonstrated efficiency in inhibiting the growth of microflora in meat and meat products. according to abdel-hamied et al. (2009), a significant inhibition of psychrotrophic microorganisms in minced meat stored at 4 °c and -18 °c was obtained by using a mixed addition of rosemary and salvia extracts. according to them, the addition of 0.05% of extracts to the meat stored at 4 °c for 10 days reduced the number of psychrotrophic microbes from 31.64 log cfu/g in the control to 14.12 log cfu/g in the extract sample. for meat stored at -18 °c for 100 days, the bacterial count was 7.16 and 20.31 log cfu/g, respectively, for the extracts and control samples. zhang et al. (2009) studied antimicrobial activity of 14 different extracts toward pathogenic bacteria causing pork meat spoilage such as listeria monocytogenes, e. coli, pseudomonas fluorescens, and lactobacillus sake. according to their results, the modifiedatmosphere-packed meat stored at 4 °c for 28 days demonstrated the effectiveness of combination of rosemary and liquorice extracts as a natural preservative, which significantly inhibited the growth of the studied microorganisms. according to pham et al. (2013), a mixture of rosemary extract (addition level: 2000 ppm) and green tea extract (100–300 ppm) can be used to limit the growth of psychrotrophic bacteria in raw pork sausage stored at -20 °c for 6 months. according to mathenjwa et al. (2012), the use of plant extracts and chitosan in the production of traditional south african pork and beef sausage can lower or eliminate the addition of sulfur dioxide (so2) as a preservative. they found that the combination of rosemary extract (addition level: 260 mg/kg), chitosan (10 mg/kg), and so2 (100 mg/kg) or rosemary extract with chitosan had an equally efficient antimicrobial effect in sausages as so2 (250 mg/kg). the results of microbiological quality evaluation of mdpm obtained in this study confirm the bacteriostatic properties of rosemary formulations. literature data indicate, however, ital. j. food sci., vol 29, 2017 338 that some of the active substances present in these preparations may exhibit bactericidal effect. thymol and carvacrol are chemical compounds whose mechanism of action on bacterial cells has been most comprehensively evaluated so far. the presence of these compounds has been confirmed in rosemary oil used in this study. their mechanism of action on gram-negative bacteria is based on the disintegration of the cell membrane, by releasing lipopolysaccharides (lps) and increasing the permeability of the plasma membrane for adenosine triphosphate (atp), the loss of which ultimately leads to cell death (helander et al., 1998). in case of gram-positive bacteria, carvacrol interacts with the cell membrane, changing its permeability toward h+ and k+ cations. change in the gradient of these cations causes disruption of the basic processes in cell and ultimately leads to cell death. in grampositive bacteria, increase in membrane permeability toward atp is not observed as for gram-negative bacteria (ultee et al., 2002). the present literature does not provide information on the antioxidant activity of rosemary extracts in mdpm, and the majority of studies concern the storage stability of slaughtered mammal meat and its products (hernández-hernández et al., 2009; wójciak et al., 2011; pham et al., 2013; armenteros et al., 2016). wójciak et al. (2011) compared the antioxidant activity of aqueous extracts from different plants added to pork meat and found that after 30 days of storage under refrigeration conditions, the highest efficiency was observed in rosemary extract. however, hernández-hernández et al. (2009) recommend the addition of alcoholic extract of rosemary based on the study performed on model pork batters to slow down the lipid oxidation processes. according to them, the strong antioxidant property of the extract might be due to high concentration of carnosic acid and carnosol and the presence of numerous other active components. the antioxidant activity of rosemary extracts was also confirmed by pham et al. (2013) on raw pork sausage and by mathenjwa et al. (2012) on pork and beef sausage, which were stored in a frozen state for 180 and 100 days, respectively. according to mielnik et al. (2003), the use of commercial rosemary preparations may also constitute an alternative method for the improvement of oxidative stability and prolongation of mdpm from turkey upon storage. to obtain a satisfactory quality of vacuum-packed raw material stored at -25 °c for 7 months, an individual selection of the type and amount of rosemary preparation is necessary, which complies with our results. in contrast to the above-cited literature, colle et al. (2016) reported that the use of rosemary extract together with ascorbic acid did not significantly limit the processes of lipid oxidation in beef steaks in comparison to the control product. szczepanik (2007) conducted a comparative study of antioxidant activity of extracts from dill, coltsfoot, rosemary, horsetail, salvia, and thyme in the breast muscle of chickens and turkeys during a 6-month frozen storage (-25 °c). the author also found that none of the used extracts significantly slowed down the oxidation process of lipids contained in the chicken muscles. although we did not evaluate it in this work, the use of natural preservatives of plant origin could be helpful in controlling the oxidation of other ingredients that exhibit nutritional value in meat products. nieto et al. (2013) explored the mechanisms behind the protection of protein against oxidation by natural plant antioxidants. the oxidative stability of the meat proteins in pork patties was evaluated as loss of thiols and as formation of myosin cross-links. essential oil of rosemary was found to have an antioxidative effect on protein thiol loss. furthermore, protein disulfide cross-link formation was inhibited in pork patties with added essential oil of rosemary. these and other properties of rosemary preparations are due to a large range of chemical compounds. ital. j. food sci., vol 29, 2017 339 both the results of the analysis of the chemical composition of rosemary preparations obtained by the authors of this study and those presented by other researchers (burt, 2004; djeddi et al., 2007) demonstrate that these preparations are mixtures of many different compounds. according to djeddi et al. (2007) the chemical profile of preparations from rosemary depends not only on the methods of obtaining them but also on the habitat of plants. when reviewing the literature, the authors concluded that the climatic differences between south europe and north africa mediterranean areas might have a significant impact on the content of ingredients such as 1,8-cineole, α-pinene, and camphor in rosemary essential oil. kasparavičiene et al. (2013) reported that ethanol extracts of rosemary contain primarily three groups of compounds: phenolic diterpenes, flavonoids, and phenolic acids. abramovič et al. (2012), among the dominant phenolic diterpenes of these extracts, mentioned-after couvelier et al. (1996) carnosol, carnosic acid, methyl carnosate, and phenolic acids from caffeinic and rosmarinic acid. 5. conclusions our results indicate that the addition of rosemary preparations constitute an auxiliary factor in the preservation of mdpm from chickens stored in a frozen state for 4 months. the tested preparations differed in their chemical composition and antimicrobial and antioxidant activities. the addition of 0.2% essential oil and 2.0% of 70% (v/v) ethanol extract was the most efficient in restricting the growth of microflora and inhibiting lipid oxidation in mdpm from chickens. acknowledgements this work is a part of the project titled “studies on the antimicrobial and antioxidant effects of extracts, essential oils and dried spices in poultry meat,” which was financially supported by the polish ministry of science and higher education in 2011–2015 (grant no. n n312 257040). references abdel-hamied a.a, nassar a.g. and el-badry n. 2009. investigations on antioxidant and antibacterial activities of some natural extracts. world j. dairy food sci. 4:1-7. abramovič h., terpinc p., generalia i., skroza d., klančnik a., katalinic v. and možina s.s. 2012. antioxidant and antimicrobial activity of extracts obtained from rosemary (rosmarinus officinalis) and vine (vitis vinifera) leaves. croat. j. food sci. technol. 4(1):1-8. aoac. 2007. “official methods of analysis” 18th ed. association of official analytical chemists, gaithesburg, md, usa. armenteros m., morcuende d., ventanas j. and estévez m. 2016. the application of natural antioxidants via brine injection protects iberian cooked hams against lipid and protein oxidation. meat sci. 116:253-259. balentine c., crandall p., o’bryan c., duong d. and pohlman f. 2006. the preand post-grinding application of rosemary and its effects on lipid oxidation and color during storage of ground beef. meat sci. 73:413-421. bełkot z., ziomek m. and gondek m. 2013. nutritional value of mechanically recovered goose and chicken meat. med. weter. 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nuernberg et al., 2008). beef and mutton are regarded as having a higher saturated fatty acid content and cholester ol level than other red meat and poultry (karaca and kor, 2007). however, conjugate linoleic acid, a derivative of linoleic acid of unsaturated fatty acids, has anti-carcinogenic and beneficial effects on human health, such as decreasing body fatty acids and improving immunity. previous research has revealed that lamb has higher rates of this fatty acid than other meat sources (inanç, 2006; kurban and mehmetoğlu, 2006). along with ever developing and changing consumer demand, there is a need for studies about fatty acids and the cholesterol contents of muttons of local sheep breeds and such studies will unquestionably provide a great contribution to the preservation of local breeds and gene source. in lambs, meat quality is significantly affected by genotypes (esenbuğa et al., 2001; purchas et al., 2002; mar tínez-cerezo et al., 2005), slaughter weights (sws) (jeremiah et al., 1998; purchas et al., 2002; mar tínez-cerezo et al., 2005), gender (dransfield et al., 1990), pre-slaughter stress (teixeira et al., 2005), carcass cooling ratio (teixeira et al., 2005), raising system (velasco et al., 2004; carrasco et al., 2009) and maturation duration (teixeira et al., 2005). karayaka sheep have low fertility (52-103%) (akçapinar et al., 2002; aksoy, 2008), milk production (40-45 kg) and live weight (35-50 kg) (sönmez et al., 2009), while the quality of meat traits is better than that of other local breeds such as red karaman, anatolian merino and awassi. karayaka sheep constitute about 4-5% of the total turkish sheep population and are extensively reared in the black sea region of turkey (ulutas et al., 2008). the present study was conducted to determine the meat quality traits of karayaka lambs with different sws. material and methods the present research was conducted in the sheep barns of the agricultural research farm of gaziosmanpaşa university (2011-hadyek-046 numbered local ethics committee approval). singleton-born karayaka male lambs (n = 30) with an average live-weight of 20 kg and weaned at 2.5-3 months of age were considered for the study. the sws and age of lambs at slaughter were 30 kg and 104.8±4.83 days; 35 kg and 119.2±4.29 days; 40 kg and 135.8±1.87 days; 45 kg and 154.6±1.99 days; 50 kg and 163.6±3.26 days, respectively. the animals with pre-specified sws were divided into sw groups in a fully randomized design. lambs housed together in 5 × 8 meter pens. before the initiation of fattening, the lambs were disinfected against internal and external parasites. following an initial one-week feeding adaptation period, the actual fattening was commenced and lambs were fed until they reach sws of 30, 35, 40, 45 and 50 kg. six lambs were slaughtered from each weight group. lamb fattening feed (concentrated feed) and lentil straw (coarse fodder) were used as the feed material. during the fattening period, lamb-fattening feed was supplied ad libitum and coarse fodder was supplied at a ratio of 100 g/head/day. the nutrient contents of the concentrated feed and coarse fodder are provided in table 1. fresh water and licking stones were continuously supplied to animals during the experiments. the lambs with the desired sws were taken into private pens. the animals were not fed for 12 hours prior to slaughter; they were then were transported for 10 minutes to a local licensed abattoir. after holding them in the paddock of the slaughterhouse for two hours, they were slaughtered following the standard commercial slaughter procedures (tsi, 1987). the lambs were brought to slaughter within ±1 kg of the expected sws. after slaughter, the carcasses were kept at +4°c for 24 h and then the m. longissimus dorsi et thoracis (ld) muscles were isolated for meat quality analyses. sufficient samples taken from these muscles were vacuumed and stored at +4 °c for analysis, at -20 °c for moistable 1 the chemical composition of concentrated feed and coarse fodder. nutrient content concentrated feed lentil straw dry matter (%) 92.00 91.30 crude protein (%) 20.63 5.78 adf (%) 26.39 55.59 ndf (%) 37.96 56.29 crude fat (%) 2.60 1.49 crude ash (%) 10.40 9.60 metabolic energy (kcal/kg) 2658 2012 adf:acid detergent fiber ndf:neutral detergent fiber ital. j. food sci., vol. 28 2016 133 ture (m), crude ash (ca), crude protein (cp), intramuscular fat (if) and at -80 °c for defrosting and cooking loss (cl), texture, fatty acid composition and cholesterol analyses. the ph of the ld muscle samples was measured at the 45th minute and 24th hour after slaughter with a meat ph meter (testo 205, germany). measurements were taken from three different locations of the samples and an average of those three measurements was taken as the ph value of that sample (ramírez and cava, 2007). meat color measurements were performed on the ld at the level of the 12th and 13th ribs, one and 24 hours after slaughter with a konica minolta cr-400 (japan) spectro-colorimeter. commission international de i’eclairage (cie) (1976) standards were used for the measurements (cie, 1986). the color parameters (l*-lightness, a*-redness, b*-yellowness) were measured from five different sections of each sample. a data set was created by taking the average of measurements for each of the three parameters (önenç et al., 1999a,b). then c (chroma = (a*2+b*2)1/2) and h° (hue = tan-1(b*/a*) values were calculated (önenç, 2003). water holding capacities (whcs) were measured in accordance with the press method developed by grau and hamm (1956). a 25 g meat sample was taken from each main sample and ground in an aura type 103 (turkey) brand mini chopper. then, 1 g of chopped sample was placed in between two filter papers (whatman 1 qualitative circles 125mm ø cat no: 1001 125); glass plates were placed above and below the filter papers and a 2.250 kg weight was placed on them. after five minutes, samples were taken out the filter papers and re-weighed (barton-gade et al., 1993). then, whc was calculated, using the equation of “whc (%) = ((initial sample weight – pressed sample weight) / initial sample weight) x 100”. to determine drip loss (dl), 20-25 g samples were taken from ld muscle and vacuumed into plastic bags. the vacuumed samples were stored at 4°c. the samples were then taken out of the vacuum bags three and seven days later, dried without any pressure, and reweighed. the ratio of the difference between the initial and final weights was calculated to find dl% after three and seven days (bond and warner, 2007). to determine the cl, 40-50 g samples were taken from the ld muscle, placed into vacuum bags and cooked in a water bath (70°c) for 40 min. the samples were then placed under a running tap for 30 minutes to lower the sample temperature to 25°c (mitchaothai et al., 2006). then the samples were taken out of the bags, blotted without any added pressure and reweighed. the cl was calculated using the equation of “cl (%) = ((initial sample weight – cooked sample weight) / initial sample weight) x 100. textural characteristics were determined at room temperature, using the p36/r probe of a texture analyzer (ta.xp plus stable micro systems, godalming, uk) (marti̇nez et al., 2004). sample dimensions were arranged into 1x1x1 cm (cubic) cubes and before, during and after, probe speeds were respectively set as 1, 5 and 5 mm/s. the m, cp and ca contents of the ld muscle samples were determined in accordance with aoac (1990). the if contents were determined, according to the heat extraction method with an ankom (xt10, spain) extractor device (okeudo et al., 2007). the extraction of lipids for fatty acid analysis was performed with chloroform/methanol (2:1), as described by folch et al. (1957). triglycerides in the cold-extracted lipids were converted into fatty acid methyl esters, in accordance with aocs (1993). the fatty acid composition of the samples were determined using a perkin elmer clarus 500 (usa) gas chromatography device, equipped with a fid (flame ionization detector) detector and a thermo scientific tr 70 capillary column (30 m x 0,25 mm and 0,25 μ film thickness). helium (1 ml/min) was used as a carrier gas. split ratio was set as 1/50, operational temperature for injection block as 250°c and for detector as 260°c. the temperature increase rate was 1°c/min, to increase the column temperature from 140°c to 180°c and 2°c from 180°c to 200°c. samples were kept at a final temperature of 200°c for eight minutes. a supelco 37 fame mix (c4-c24) (bellefonte, pa, usa) was used as the standard by which to define the fatty acids. the results were expressed in % methyl esters. about 0.3-0.5 g of lipid samples was taken from the lipid, cold-extracted from the ld muscle, and the samples were placed into closed glass tubes. then, 0.3 ml 33% koh and 3 ml 95% ethyl alcohol solution was added, and the mixture roughly mixed and saponificated in a water bath at 60°c for 15 min. the tubes were cooled down, 10 ml hexane and 3 ml of distilled water was added and the roughly mixed samples were then kept for 10 minutes for phase separation. to determine cholesterol content, a 1 ml sample was removed from the hexane fraction into a test tube. the hexane was removed using nitrogen gas. a fecl 3 stock solution was prepared with 840 mg fecl 3 and 10 ml concentrated glacial acetic acid, and 1 ml of this stock solution was increased to 100 ml with a concentrated glacial acetic acid, to prepare the fecl 3 working solution. later on, the 1.5 ml fecl 3 working solution was added to test tube and the resulting solution was roughly mixed. after 15 minutes, 1 ml of concentrated sulphuric acid was added and the samples were mixed in a tube mixer for 1 min. the tubes were placed in the dark for 45 min. the absorbance values of the resulting purple color were read at 560 nm wavelength of a unicam uv/vis model spectrophotometer. cholesterol standard curves were crehttp://www.sciencedirect.com/science/article/pii/s0956713503001300 134 ital. j. food sci., vol. 28 2016 table 2 meat quality characteristics of m. longissimus dorsi et thoracis (ld). traits slaughter weight (kg) mse p 30 35 40 45 50 ph 45m 6.15c 6.10c 6.31b 6.14c 6.46a 0.01 *** ph 24h 5.55c 5.60c 5.75ab 5.70b 5.80a 0.01 *** color 60m l* 33.99a 33.90ab 33.23b 33.59ab 32.10c 0.10 *** a* 12.55a 12.25a 10.47b 10.27b 10.49b 0.08 *** b* 3.15a 3.04a 1.30b 1.07b 0.94b 0.07 *** c* 12.94a 12.64a 10.57b 10.33b 10.53b 0.09 *** h° 14.38a 13.41a 6.50b 5.90b 5.34b 0.29 *** color 24h l* 41.04a 39.70ab 39.68ab 39.58ab 38.60b 0.22 * a* 13.27d 14.35ab 14.12bc 13.75c 14.61a 0.06 *** b* 5.03a 5.35a 4.08b 4.18b 5.02a 0.06 *** c* 14.21b 15.36a 14.64b 14.39b 15.37a 0.07 *** h° 20.83a 20.21a 16.25c 16.55c 18.79b 0.20 *** drip loss (%) 3rd day 8.10a 8.71a 7.15b 9.67a 9.94a 0.20 *** 7th day 12.22ab 11.73ab 9.35c 13.20a 10.94b 0.24 *** cooking loss (%) 28.25a 27.23a 26.11ab 25.03b 24.73b 0.29 ** whc (%) 34.37d 36.20c 36.28c 37.74b 39.15a 0.21 *** texture (kg/cm2) 4.51 4.91 5.18 5.96 7.29 0.35 whc: water holding capacity; mse: mean standard error -: non-significant, *: p<0.05, **: p<0.01, ***: p<0.001 means within a row with different letters differ significantly (p<0.05) ated and the cholesterol content of the samples was expressed as mg cholesterol/100 g sample (rudel and morris, 1973). statistical analyses were performed using spss (1999) software. the duncan’s test was used to determine differences among the means (düzgüneş et al., 1987). results and discussion mean values, for the meat quality traits of the ld muscles of karayaka lambs with different sws, are shown in table 2, the compositional nutrient content in table 3 and, fatty acid composition and cholesterol contents in table 4. table 3 compositional properties of m. longissimus dorsi et thoracis (ld) (%). traits slaughter weight (kg) mse p 30 35 40 45 50 moisture 75.92ab 75.08cd 76.18a 74.46d 75.33bc 0.12 *** protein 20.14ab 20.82a 20.13ab 20.68a 19.85b 0.11 * if 2.59b 2.67b 2.41b 3.44a 2.98ab 0.08 ** ash 1.08 1.06 1.07 1.08 1.06 0.01 if: intramuscular fat; mse: mean standard error -: non-significant, *: p<0.05, **: p<0.01, ***: p<0.001 means within a row with different letters differ significantly (p<0.05) meat ph values have distinctive impacts on meat quality traits, such as color, whc and texture. therefore, the ph plays a significant role in the quality assessment of meat (karaca, 2010). in the present study, ph measurements were performed 45 minutes (ph 45m ) and 24 hours (ph 24h ) after the slaughter. in both measurement times, the differences in muscle ph values of the slaughter groups were found to be significant (p<0.001; table 2). similar to the current findings, the significant effects of sws on final ph values were reported in previous studies (beriain et al., 2000; yakan and ünal, 2010); however, others reported insignificant effects (martínez-cerezo et al., 2005). increasing ph 24h values were observed in this study with increasital. j. food sci., vol. 28 2016 135 ing sws and the relevant values varied between 5.55 – 5.80. based on the assumption that a final ph value above 5.8 is considered undesirable, it can be said that the final ph ranges were both appropriate and inside normal range (yakan and ünal, 2010). whc is closely related to ph and therefore it is considered as a significant parameter for meat quality assessments (yakan, 2008). the differences between whcs of the slaughter groups were also found to be significant (p<0.001; table 2). increasing whc values were observed with increasing sws. lawrie and ledward (2006) reported increasing whcs with increasing ph values. however, current findings were contrary to those reports. cold-induced contraction might have such effects on whc. such contractions have higher impacts on carcasses with high ph levels. cold carcass contractions result in decreasing intra-myofibril spaces and water release from the meat (karaca, 2010). the whcs of lambs fed with concentrated feed were reported as between 9.76 28.27 (beriain et al., 2000; ekiz et al., 2009; yakan and ünal, 2010). consumers commonly assess the meat they buy based on fattiness, general appearance and color; regarding light colored meat as that of young animals, which they prefer to buy (sañudo et al., 2007). in the present study, the color parameters l*, a* and b* were measured over hot carcasses (60 minutes after slaughter) and cold carcasses (24 hours after slaughter) of ld muscle samples and significant differences were observed between slaughter groups with regard to l*, a*, b*, c* and h° values, in both measurement periods (p<0.05; table 2). similar to the current findings, beriain et al. (2000) and martinez-cerezo et al. (2005) reported significant effects of sws on the color parameters. the decreasing l* values observed in this study were concomitant with increasing sws. the a* values recorded at 24h in 50 kg group was higher than those recorded in 40 and 45 kg groups and similar to those recorded in 35 kg group. beriain et al. (2000) carried out a study on lacha and rasa aragonesa lambs with different sws (12, 24 and 36 kg) and reported decreasing l* values and increasing a* values with increasing sws. in other studies carried out with local lamb breeds, fed by concentrated feeds, l* values (24 hours after slaughter) were reported as between 37.91 – 42.72; a* values as between table 4 cholesterol content (mg/100 g meat) and fatty acid composition (%) of lipids of m. longissimus dorsi et thoracis (ld). traits slaughter weight (kg) mse p 30 35 40 45 50 c8:0 0.210 0.170 0.172 0.156 0.154 0.01 c10:0 0.310 0.258 0.220 0.222 0.242 0.01 c11:0 7.188 6.156 6.123 5.664 5.530 0.14 c12:0 0.415 0.224 0.172 0.138 0.142 0.03 c14:0 2.927a 2.940a 2.725ab 2.530ab 2.380b 0.07 * c14:1 0.215 0.134 0.090 0.110 0.122 0.01 c15:0 0.300 0.204 0.165 0.104 0.200 0.02 c16:0 23.135 23.206 23.493 23.914 24.326 0.21 c16:1 1.055 1.278 0.928 1.234 0.808 0.11 c17:0 0.750 0.968 0.717 0.818 0.808 0.07 c17:1 0.550 0.695 0.537 0.612 0.584 0.05 c18:0 13.792b 14.374b 14.350b 13.964b 16.306a 0.25 ** c18:1 37.867 39.622 39.980 43.132 40.294 0.59 c18:2 (n-6) 7.572a 6.790ab 7.380a 5.334b 5.878ab 0.28 * c18:3 (n-6) 0.028 0.098 0.012 0.008 0.014 0.01 c18:3 (n-3) 0.023 0.030 0.005 0.012 0.010 0.01 c20:0 0.140 0.060 0.065 0.042 0.036 0.01 c20:1 0.247 0.244 0.213 0.138 0.178 0.03 c20:3 (n-3) 0.180 0.102 0.060 0.058 0.102 0.01 c21:0 0.188 0.026 0.015 0.034 0.016 0.02 c22:1 2.725a 2.442a 2.527a 1.722b 1.832b 0.11 ** σsfa 48.947ab 48.586b 48.217b 47.586b 50.140a 0.21 ** σmufa 42.660b 44.414b 44.275b 46.948a 43.818b 0.38 * σpufa 7.779a 6.980ab 7.449a 5.362b 5.979ab 0.27 * (σmufa+σpufa)/σsfa 1.033bc 1.059abc 1.073ab 1.102a 0.995c 0.09 * σpufa/σsfa 0.159a 0.144ab 0.154abc 0.113c 0.119bc 0.05 * total cholesterol 199.799b 194.143bc 162.044d 224.326a 190.381c 3.03 *** mse: mean standard error -: non-significant, *: p<0.05, **: p<0.01, ***: p<0.001; means within a row with different letters differ significantly (p<0.05); sfa: saturated fatty acid; mufa: mono-unsaturated fatty acid; pufa: poly-unsaturated fatty acid 136 ital. j. food sci., vol. 28 2016 16.08 – 21.26 and b* values as between 5.10 – 8.45 (ekiz et. al., 2009; esenbuğa et. al., 2009; karaca, 2010; yakan and ünal, 2010). various researchers have shown carcass weight as the most significant factor indicating lamb carcass and meat quality (di̇az et al., 2002; vergara et al., 1999). peña et al. (2005) reported darkened meat color with increasing lamb carcass weights. similar to the current findings, sañudo et al. (2000) reported decreasing a* values and increasing l* values with decreasing carcass fat ratios. texture is another factor affecting meat quality. consumers specify meat hardness as a significant quality indicator (karaca, 2010). shackelford et al. (1991) reported that consumers and taste panelists indicated meats with a hardness value over 5.5 kg/cm2 as hard meats. for karayaka lambs in the present study, except for the sw groups of 45 kg (5.96 kg/cm2) and 50 kg (7.29 kg/ cm2), the hardness values were within the limits specified by shackelford et al. (1991). although not significant (p>0.05), increasing hardness values were observed in this study with increasing sws (table 2). the hardness value of entire sw groups of karayaka lambs were lower than the values reported by esenbuğa et al. (2001) for awassi and red karaman. the hardness value of 40 kg sw group of the present study were higher than those reported by ekiz et al. (2009) for merino, ramlıç, kivircik lambs (40-41 kg sw); and by perlo et al.. (2008), for corriedale lambs (41 kg sw). some other researchers reported the hardness values of lambs fed with concentrated feeds (24-30 sw) as between 3.35-4.01 kg/cm2 (santos-silva et al., 2002a; ekiz et al., 2009; yakan and ünal, 2010). with regard to dl on 3rd and 7th days, the differences between the slaughter groups were found to be significant (p<0.001; table 2). increasing sws resulted in increasing dls on the third day. the highest dl on 3rd and 7th days was observed in the 50 kg (9.94%) and 45 kg (13.20%) weight groups. the cl values decreased with increasing sws (p<0.01; table 2). although gökalp et al. (1993) indicated lower cl values for high whc meats, contrary results were observed in this study. ekiz et al. (2009) slaughtered merino, ramlıc and kivircik lambs fed with concentrated feeds at 40-41 kg weights and observed the cl, respectively as 27.14, 25.57 and 29.54%. the cl for 40 kg sw of the present study (26.11%) was higher than the value determined by ekiz et al. (2009) for the same-weight ramlıc lambs, and lower than merino and kivircik lambs. the cl determined for 30 kg sw groups of karayaka lambs was similar to that reported by the same researchers for 26 kg imroz lambs (28.91%) and higher than the value reported by chios lambs (27.81%). while the differences between sw groups were found to be significant with regard to cp and m contents (p<0.05), the differences in ca contents of the groups were insignificant (p>0.05; table 3). if in 50 kg was similar to if observed in all the other sw groups. the highest value was observed in the 45 kg (3.44%) groups and the lowest value was seen in the 40 kg (2.41%) sw groups. yakan (2008) reported a decreasing if content in bafra lambs with increasing sws, with the highest value for 30 kg (4.20%) and the lowest value for 40 kg (2.80%) weight groups. the cp ratios for the karayaka lambs in the present study were similar to the values determined by previous researchers for local, crossbred and heritage breeds of lamb (beriain et al., 2000; macit et al., 2003; perlo et al., 2008; esenbuğa et al., 2009). the ld muscle m contents of karayaka lambs of the present study (74-76%) were similar to values reported by the other researchers for the same muscle (73-76%) (beriain et al., 2000; perlo et al., 2008; esenbuğa et al., 2009). in ruminants, almost all of the fats are localized as triglycerides in adipose, and fatty acids are localized as c16 and c18. in general, more than 80% of the fatty acids are composed of c14:0 (myristic acid); c16:0 (palmitic acid), c18:0 (stearic acid) and c18:1 (oleic acid) (karaca, 2010). the order of those primary fatty acids in the present study was observed as c18:1, c16:0 and c18:0 in all sw groups and only the differences in the c14:0 and c18:0 fatty acids were found to be significant (p<0.05; table 4). with regard to unsaturated fatty acids, the differences in c18:2 (n-6) (linoleic acid) and c22:1 (erucic acid) fatty acids of the weight groups were found to be significant (p<0.05). on the other hand, differences in the monounsaturated fatty acid contents of the groups were insignificant (p>0.05). the differences between the sw groups were also found to be significant, with regard to total monounsaturated fatty acids, total polyunsaturated fatty acids, total unsaturated fatty acid/total saturated fatty acid ratios and total polyunsaturated fatty acid/total saturated fatty acid ratios (p<0.05). the highest total saturated fatty acid content was observed in the 50 kg (50.14%) and the lowest in the 45 kg (47.58%) sw group. in general, a decreasing total of saturated fatty acid contents were observed with increasing sws. while such decreases comply with the findings of some previous research (díaz et al., 2005; oriani et al., 2005; yakan and ünal, 2010), they differed from an other study (santos-silva et al., 2002b). the total unsaturated fatty acid / total saturated fatty acid ratios of karayaka lambs of the present study varied between 0.99-1.10. such values were reported in previous studies as between 0.09 – 0.95 for the lambs fed with concentrated feeds (rowe et al., 1999; díaz et al., 2002; karabacak, 2007). the total unsaturated fatty acid / total saturated fatty acid ratios of the karayaka lambs in the present study were higher than the other studies. such differences were mainly due to differences in genotype and the age of slaughter, since http://www.sciencedirect.com/science/article/pii/s0921448802000160 http://www.sciencedirect.com/science/article/pii/s0921448802000160 ital. j. food sci., vol. 28 2016 137 genotype, age of slaughter, gender and type of fat stores, and the anatomic location of muscles and fats are major factors affecting the fatty acid composition of meat. the differences in cholesterol levels of the sw groups were found to be significant (p<0.001; table 4). the highest total cholesterol level was observed in the 45 kg (224.32 mg/100 g meat), the lowest value in the 40 kg (162.04 mg/100 g meat) weight groups. yakan and unal (2010) carried out a study on bafra lambs and reported the highest total cholesterol levels for the 45 kg (63.00 mg/100 g meat) and the lowest levels for the 35 kg (53.80 mg/100 g meat) sw groups. bunch et al. (2004) reported the total cholesterol level of wool lambs with 46-54 kg sws and fed with concentrated feed, as 117 mg/100 g meat; as 73 mg/100 g meat for callpyge wool x st. croix lambs; 50 mg/100 g meat for callpyge wool x wool lambs; 149 mg/100 g meat for dorper x wool lambs and 131mg/100 g meat for dorper x st. croix lambs. similarly, salvatori et al. (2004) reported the total cholesterol level of extensively fed ile de france x parliarola and gentile di puglia sopravissana lambs respectively as 63.0 and 60.3 mg/100 g meat. in another study carried out on corriedale and corriedale crossbreds, the total cholesterol level was reported as 62.03 for the lambs fed with concentrated feeds and as 57.76 mg/100 g meat for range-fed lambs (rowe et al., 1999). the total cholesterol values of the ld muscle of the karayaka lambs in the present study were higher than thoses values reported by rowe et al. (1999), salvatori et al. (2004) and bunch et al. (2004). conclusions in conclusion, with regard to meat quality parameters, except for ca and hardness, the differences in entire traits of ld muscle of the different sw groups of karayaka lambs of the present study were found to be significant. increasing sws resulted in increasing whc and hardness values, and decreasing cl values, but the differences between the hardness values of the samples were not found to be significant. among the fatty acids, except for c14:0, c18:0, c18:2 (n-6) and c22:1, differences in the entire fatty acid contents of sw groups were found to be insignificant. acknowledgements the present research was derived from the ph.d. thesis entitled: the determination of carcass and meat quality characteristics of karayaka lambs with different slaughter weights. the authors wish to thank tubi̇tak (project no: tovag-111o848) and general directorate of agricultural research and policy (public small-head animal breeding “karayaka sheep breeding ii – university nucleus herd 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http://www.tuik.gov.tr/veribilgi.do%3falt_id ijfs#703_bozza ital. j. food sci., vol. 30, 2018 13 paper effect of hydrogenated fat replacement with white sesame seed oil on physical, chemical and nutritional properties of cookies f. aslam1, s. iqbal*1, m. nasir 1, a.a. anjum2, p. swan3 and k. sweazea3 1department of food science and human nutrition, faculty of biosciences, university of veterinary and animal sciences lahore, syed abdul qadir jillani (out fall) road, lahore, pakistan 2department of microbiology, faculty of veterinary sciences, university of veterinary and animal sciences lahore, syed abdul qadir jillani (out fall) road, lahore, pakistan 3school of nutrition and health promotion, arizona state university, phoenix, az, usa *corresponding author. tel.: +92 4299211449 ext. 284 e-mail address: sanaullah.iqbal@uvas.edu.pk abstract sesame seed oil has known antioxidant properties that may improve both nutritional importance and shelf-life of the product. three aims of the study were to: a) examine nutritional value and physicochemical properties of white sesame seed oil (wsso) and hydrogenated vegetable fat (hvf) cookies, b) compare the antioxidant potential of the cookies and c) determine the effects of storage and treatment conditions on palatability of the cookies. results showed that energy and fat% were significantly higher (p < 0.05) in wsso than hvf cookies. at 60th day, mean moisture, peroxide value, and acidity were higher (p < 0.05) in hvf cookies. over time, protein and fiber% decreased significantly (p < 0.05) in both cookies but remained higher (p < 0.05) in wsso at 60 days. wsso cookies had longer shelf life, greater palatability, improved physical properties and greater antioxidant potential. keywords: antioxidant potential, food product development, palatability, physicochemical properties, sesame oil ital. j. food sci., vol. 30, 2018 14 1. introduction food industries are interested in developing plant products that provide both functional as well as nutritional value (jisha et al., 2009; oomah and mazza, 1998; tripathy et al., 2003). in addition, the increasing trend toward consuming “pre-packaged” “ready-toeat” products have increased the need for improving the nutritional quality, palatability and shelf life of these food products (nanditha and prabhasankar, 2009). along with proteins and carbohydrates, fats are a critical component of a healthful diet. fats are important for improving both the taste and texture of food as well as stimulating neurological sensory signals of “fullness” after consumption (rolls, 1995). some oil containing foods are rich in antioxidants, which may be able to decrease harmful inflammatory conditions resulting from oxidative stress. these same antioxidative properties may also be able to increase the shelf life of food products by reducing the undesirable progression of oxidation that causes rancidity (sims and fioriti, 1977). furthermore, increasing shelf life also influences the economic cost of the product by reducing the waste of discarding unused “out of date” products (reddy et al., 2005). finally, there is an increasing need to replace partially hydrogenated fats that are now known to be unhealthy, with healthy replacements without affecting the physical and sensory properties of the end products (wang et al., 2016). white sesame seed oil (wsso) (sesamum indicum l.) specifically has been recognized for its potential role in a healthful diet. the oil in sesame seeds contains appreciable amounts of bioactive components with powerful antioxidant properties identifying it as a promising nutraceutical in the treatment of chronic inflammatory conditions such as cardiovascular disease and diabetes (bhuvaneswari and krishnakumari, 2011; hemalatha and ghafoorunissa, 2004; latif and anwar, 2011). wsso is readily cultivated in tropical and sub-tropical regions of the world and has been used in food preparation and baking and for centuries (reshma et al., 2010). sesame oil (so) is considered as an extremely stable oil against oxidation because of the high proportion of natural antioxidants or lignans, such as sesamin and sesamolin (anilkumar et al., 2010). the presence of these antioxidants may also improve the oxidative stability of so (el-adawy, 1997; latif and anwar, 2011; reshma et al., 2010). it is plausible that exchanging the fat source in cookies from standard hydrogenated vegetable fat with so, would improve the antioxidant potential, shelf life and palatability of cookies. sowmya et al., (2009) described the effect of fat replacement with so, hydrocolloids and emulsifiers on changes in the fatty acid profile and microstructural qualities of cakes. they determined that a combination of 50% so combined with hydroxypropylmethylcellulose and emulsifiers greatly improved the palatability of the product and provided better results than the control (vegetable fat) cake in all aspects. in fact, replacement of fat with so also decreased the saturated fatty acid content of the cake (sowmya et al., 2009). lim and lee (2015) described that that by incorporating black sesame powder into cookies, the functional properties of the cookies was improved without affecting the consumer acceptability. in addition, cookies in which butter was replaced with an oil emulsion, showed better fracture properties and higher consumer acceptability and potentially overall healthier properties (giarnetti et al., 2015). finally (rangrej et al., 2015) noted that the replacement of hydrogenated fat with seed oil improved the physical, textural and sensory properties of cookies. to our knowledge, no previous studies have examined the effect of replacing the fat source in cookies with so. it is currently unknown whether replacing the fat source in cookies would improve the nutritional, antioxidative or perhaps more importantly, the taste and palatability of the cookies. the purpose of this investigation was to study the differences in nutritional ital. j. food sci., vol. 30, 2018 15 properties as well as the palatability of cookies made with so compared to cookies made with hydrogenated vegetable fat (hvf). the specific aims of this study were three-fold: 1) to examine differences in nutritional value and physicochemical properties of cookies made with so compared to hvf, 2) to compare the antioxidant potential of the cookies and 3) to determine the effects of time and oil type on palatability of the cookies. 2. material and methods 2.1. materials two types of cookies were prepared, one with 100% hydrogenated vegetable fat (hvf) and other with 100% white sesame seed oil (wsso). vegetable fat (vf) was the hydrogenated fat and it was the blend of soybean, palm and canola vegetable oil. cookies were prepared according to the method given by the american association of cereal chemists (aacc, 2000) with modifications to fat replacement and baking time & temperature. baking ingredients were procured from the local market. all ingredients were weighed as per their percentage in the recipe that included: fine (cake) flour (45%), whole wheat flour (10%), white sesame seed oil (33%) or vegetable fat (hydrogenated fat composed of soybean, palm and canola vegetable oil), salt (0.30%), egg (5%), stevia (0.15%), baking powder (5%), sugar (1.5%), and vanilla extract (0.05%). cookies were prepared in a commercial bakery unit. the white sesame seed oil was extracted from seeds (pb-till 90) that were procured from the ayub agriculture research institute faisalabad, pakistan. sesame seed oil was extracted from seeds through solvent extraction method (latif and anwar, 2011) then this oil was used for the preparation of cookies. dry ingredients (flour, whole wheat flour, salt, sugar, stevia and baking powder) were placed in a mixer at low speed for 2-3 minutes to ensure thorough mixing. next, eggs were added to the dry mixed ingredients during mixing. the fat source (either so or hvf) was then added in small amount to facilitate thorough blending of fat with the ingredients and the mixer speed was increased up to 50-60 rpm (medium) and the ingredients were mixed for additional 4-5 minutes. then vanilla extract was added. the whole mixing process was completed in approximately 10-15 minutes. two batches of 15 kg each, one from so and the other from hvf were made. cost of recipe for both type of cookies was almost same and these was not much difference. the mixture was dispersed onto a cookie sheet in 12-15 gm aliquots to produce a total of 950-970 cookies. cookies were baked in a preheated commercial oven at 175°c for approximately 15-20 minutes until a golden color was achieved. baked cookies were then allowed to cool on a rack and the weight of each cookie was noted. cookies were then divided into 12 air-tight glass containers (6 with so and 6 with hvf) holding 160-170 cookies each and placed in a cabinet in the laboratory at an ambient room temperature (25 ± 5°c) away from sunlight for a period of 60 days. one container of each type of cookie (so and hvf); were opened and the cookies were analyzed at each data collection period (baseline, 30th and 60th day). 2.2. methods 2.2.0. proximate analysis of cookies proximate composition analyses followed the specific methodology as described by aacc, (2000). these analyses included, moisture (method no. 44-15a), protein (method ital. j. food sci., vol. 30, 2018 16 no. 46-30), fat (method no. 30-25), fiber (method 32-10), ash (method no. 08-01). energy (kcal) was estimated by calculating kcal per gram of the individual macronutrients. 2.2.1 moisture moisture was determined by following the method (method no. 44-15a) aacc (2000). air dried oven (blodgett; ctb/ctbr, usa) was used to determine the moisture. the percentage of moisture was calculated according to the expression given below. w1 x 100 moisture (%) = ---------------- w where, w1 = loss in gm of the material on drying w = weight in gm of the material taken for test 2.2.2 protein protein content in each sample was estimated according to the kjeldahl’s method (method no. 46-30) as described in aacc, (2000). 2.2.3 fat fat (%) content in each sample was determined by taking 5 gm dried sample and running through soxhlet apparatus for 04 hours using n-hexane as a solvent by following the procedure described in aacc, (2000) method no. 30-25. the percentage of fat was calculated according to the expression given below. wt. of fat fat (%) = ----------------------x 100 wt. of sample 2.2.4 fiber the crude fiber was estimated according to the procedure as outlined in aacc, (2000) method 32-10. muffle furnace (thermo scientific thermolyne f48010-33, usa) was used to determine the fiber. the percentage of fiber was calculated after igniting the samples according to the expression given below. weight loss fiber (%) = -------------------------------x 100 weight of sample 2.2.5 ash ash was estimated according to the procedure as outlined in aacc, (2000) method no. 0801. ash content was determined by high temperature incineration in an electric muffle furnace (thermo scientific thermolyne f48010-33, usa). ital. j. food sci., vol. 30, 2018 17 a-b ash (%) = --------------x 100 c where: a = weight of crucible with sample (gm)� b = weight of crucible with ash (gm) �c = weight of sample (gm) 2.3. antioxidant potential antioxidant potential was determined by measuring peroxide value, total acidity, energy, nitrogen free extract, and thiobarbituric acid value according to their respective methods described in aacc, (2000). furthermore, the lignan content (sesamin, and sesamol) of the cookies were assessed following the method of schwertner and rios, (2010). 2.3.1 peroxide value ash was estimated according to the procedure as outlined in aacc, (2000) method. sample was melted and filtered through the filter paper to remove any impurities. a blank reading was taken under the similar conditions at the same time. the peroxide value was calculated by using the relationship (b-a) × n × 1000 peroxide value = ---------------------- w b = vol. of na2s2o3 used for blank a = vol. of na2s2o3 used for sample n = normality of na2s2o3 w = weight of the oil taken. 2.3.2 total acidity total was estimated according to the procedure as outlined in aacc, (2000) method. total acidity was calculated according to the expression given below. calculation: acid value = 56.1vn, where: v = volume in ml of standard koh or naoh used n = normality of the koh solution or naoh solution; and w = weight in gm of the sample 2.3.3 nitrogen free extract (nfe) the nfe was calculated by the following expression. nfe% = 100 – (moisture% + ash%+ fat%+ fibre% + protein%) ital. j. food sci., vol. 30, 2018 18 2.3.4 thiobarbituric acid value tba reagent (0.2883g/100ml of 90% glacial acetic acid), heated in water bath for 35 min with a blank sample. the tubes were cooled in water for 10 min and absorbance (d) against blank sample was taken by adjusting spectrophotometer (cecil ce-7200, uk) on 538nm wavelength (aacc, 2000). tba no. was calculated by using the following expression: tba no. (mg malenaldehyde per kg sample) = 7.8 x d 2.3.5 lignans lignans (sesamin and sesamol) were analyzed by adopting the method of (schwertner and rios, (2010); schwingshackl and hoffmann, (2012))with little modifications in it. a 2ml of sesame seed oil sample was taken in glass tubes and poured in it 20ml of methanol and vortex it for 30 minutes. the sample was then centrifuged at 2500 rpm for 30 minutes. after centrifugation, the upper layer was separated and again extraction was done by adding methanol again in it. the two extractions were combined and then evaporated under nitrogen. then 2 ml of methanol was added to reconstitute it and vortex it. 20 μl of sample was injected into hplc (model: perkin elmer series 200 usa) equipped with c18 (4.6mm x 150 mm). the mobile phase was a mixture of methanol and water (70:30, v/v) and the flow rate was 1 ml/min. the uv detector was set at 288 nm. sesamin, sesamolin and sesamol were quantified by comparing with standards. 2.4. physical properties and palatability to evaluate the palatability of cookies, taste tests were conducted using a sensory evaluation assessment tool described by meilgaard et al., (2007). briefly, 100 men and women were recruited from university student, faculty, and staff members, and the local community to participate in a series of consumer taste tests. the same individuals were asked to evaluate the cookies at each time period. among these volunteers, 75 people completed the testing at all three-time points. cookies were placed on a table in small cups coded by type. both researchers and panelists were blinded to the coding scheme. panelists were provided water to neutralize the taste after chewing and between tasting each cookie. each panelist was asked to taste 3 cookies of each type and asked to evaluate the cookies for colour, flavor, taste, texture, crispness and overall acceptability using a 9point (1= extremely poor to 9 = excellent), hedonic scale (meilgaard et al., 2007). the physical properties such as cookie size and diameter, thickness, and spread factor were determined by investigators on three cookies from each group at the three time points (aacc, 2000). 2.5. statistical analyses data was expressed as mean ± se. for composition analyses and physical properties, a total of 3 cookies from each group (hvf or so) were evaluated at each time point. for palatability studies, evaluation data from 75 subjects was compared for each variable at each time point. all data were analyzed by repeated measures anova. where significant effects occurred, tukey post-hoc analyses were performed. a p-value of ≤ 0.05 was considered statistically significant. ital. j. food sci., vol. 30, 2018 19 3. results and discussions 3.1. proximate analysis and antioxidant potential of cookies as previously described, two types of cookies were prepared, one with 100% so and other with 100% hvf and both were stored for up to 60 days in an air tight container. a description of the proximate composition of each of the cookies type for each time period is shown in table 1. at baseline, both so and hvf cookies had similar properties of moisture, fiber and ash. compared to hvf, so had significantly higher initial percentages of protein (6%), fat (8.5%), and energy kcal/100gm (15.4%). by the end of the 60 days of storage time, moisture content in so cookies increased approximately 34% (p < 0.05), while other components decreased significantly (p < 0.05) over time; (protein: -0.2%, fat: -3%, fiber: -5.5%, and ash: -7.9%). in hvf cookies, a similar trend was observed. in hvf cookies, moisture increased by about 52% (p < 0.05), while other components decreased (p <0.05); (protein: -2.5 %, fat: -3.4 %, fiber: -6.9 %, and ash: 16.4 %) from baseline to 60 days of storage. energy (kcal/100gm) did not change over time in either cookie group. table 1. cookies proximate analysis. component day 0 day 30 day 60 moisture (%) sso 2.49 ± 0.04 (2.63) 3.19 ± 0.05 (2.87)a 3.35 ± 0.05 (2.60)a,b,c hvf 2.41±0.02 (1.66) 3.08±0.03 (1.49)a 3.66±0.06 (2.96)a, protein (%) sso 9.11±0.02 (0.33)c 9.05±0.04 (0.83)c 9.00±0.03 (0.62)a,c hvf 8.57±0.02 (0.40) 8.43±0.03 (0.54)a 8.36±0.02 (0.43)a fat (%) sso 39.62±0.59 (2.57)c 38.92±0.04 (0.18)c 38.41±0.31 (1.40)c hvf 36.51±0.39 (1.83) 35.82±0.07 (0.33) 35.31±0.05 (0.22) fiber (%) sso 2.09±0.02 (1.27) 2.01±0.02 (1.32)a,c 1.98±0.03 (2.31) a,c hvf 2.01±0.02 (1.99) 1.92±0.03 (2.90)a 1.88±0.04 (3.72)a ash (%) sso 0.95±0.01 (2.11) 0.93±0.02 (2.84)c 0.88±0.02 (4.10)a,c hvf 0.92±0.02 (3.92) 0.83±0.03 (5.52) 0.79±0.03 (6.70) energy (kcal/100gm) sso 535.67±2.97 (0.96)c 535.58±0.24 (0.08)c 534.42±2.59 (0.84)c hvf 464.33±2.96 (1.11) 463.89±2.77 (1.03) 463.12±2.82 (1.06) mean±se (%coefficient of variance); the values are replicate of at least three. sig. ap<0.05 from day 0; sig. bp<0.05 from day 30; sig. cp<0.05 sesame seed oil cookies vs vegetable fat cookies. at 60 days there were significant (p < 0.05) differences between groups. moisture was significantly higher in hvf verses so, whereas all other components were significantly (p < 0.05) lower in hvf group compared to so group; (protein: -7.6%, fat: -9%, fiber: -5% and ash: -11 %). ital. j. food sci., vol. 30, 2018 20 table 2 shows antioxidant potential of both so and hvf cookies. at baseline, both so and hvf had similar properties of nitrogen free extract and thiobarbituric acid value. compared to so, hvf had significantly higher initial percentages of peroxide value (37.5%) and acidity (20%). lignans with antioxidant potential were detected in so but were absent in hvf. over time, from baseline to 60 days, peroxide value increased approximately 252% in so cookies. additionally, in so, acidity, nitrogen free extract, and thiobarbituric acid values increased (35%, 3%, 54% respectively), while bioactive components, sesamin and sesamol, decreased significantly (p <0.05) over time (i.e., -0.22% and -1.2% respectively). a similar trend was observed in hvf cookies. in hvf cookies significant (p < 0.05) increases were observed in peroxide (+182.5%), acidity (+24%), nitrogen free extract (+5%) and thiobarbituric acid (+ 53%) from baseline. there were no bioactive components detected in hvf cookies. table 2. antioxidant potential of cookies. component day 0 day 30 day 60 peroxide value (meq/kg) sso 0.133±0.01 (17.3)c 0.341±0.00 (1.83)a,c 0.469±0.00 (0.93)a,b,c hvf 0.183±0.01 (8.67) 0.428±0.00 (0.62)a 0.517±0.00 (1.02)a,b acidity (%) sso 0.142±0.01 (11.3)c 0.188±0.01 (4.88)a 0.192±0.00 (4.26)a,c hvf 0.171±0.00 (0.58) 0.198±0.00 (1.34)a,b 0.212±0.00 (1.70)a nitrogen free extract (%) sso 41.27±1.01 (4.23) 42.21±1.24 (5.10) 42.57±1.21 (4.93) hvf 41.87±0.51 (2.11) 42.45±0.70 (2.84) 43.94±0.58 (2.29) thiobarbituric acid value (mg malonaldehyde/kg-oil) sso 0.046±0.00 (8.70) 0.068±0.01 (14.0)a 0.071±0.01 (18.3)a hvf 0.055±0.00 (6.56) 0.062±0.00 (8.98) 0.084±0.00 (3.15)a,b sesamin (mg/kg) sso 8.093±0.00 (0.01) 8.077±0.00 (0.09)a 8.075±0.00 (0.09)a hvf na na na sesamol (mg/kg) sso 18.64±0.05 (0.46) 18.48±0.02 (0.20)a 18.42±0.02 (0.20)a hvf na na na mean±se (%cv) the values are replicate of at least three sig. ap<0.05 vs day 0; bp<0.05 vs day 30; cp<0.05 vs hvf the moisture content of both the cookies increased over the 60 days of storage period in both cookies. however, it increased significantly more in the hvf cookies. during storage, the rise in moisture content in cookies and cakes has been well documented by studies conducted by (leelavathi and rao, 1993; nagi et al., 2012; robertson, 1993). the hygroscopic nature of the dry ingredients of cookies is known to influence moisture content during storage. additionally, this rise in moisture can influence the shelf life of the products by increasing peroxide value, acidity, nitrogen free extract, and thiobarbituric ital. j. food sci., vol. 30, 2018 21 acid value; while decreasing protein, fat, fiber and ash content. ash helps with the overall absorption of moisture. thus a reduction of ash results in a corresponding increase in moisture. this storage phenomenon whereby a decrease in ash results in increasing moisture, has been well documented (pasha et al., 2002; reddy et al., 2005; sharif et al., 2003; waheed et al., 2010). when compared with other vegetable oil; sesame seed oil has high degree of monounsaturated and polyunsaturated fatty acids i.e., is approximately 39% and 46% respectively that collectively makes almost 85% of unsaturated oil and low saturated fatty acids i.e., is approximately 14% (schwingshackl and hoffmann, 2012). replacement of normal shortening in cookies with vegetable oils showed significant effect on moisture, fat content and nfe during storage, while change in fiber and ash content was not significant (sharif et al., 2005). bioactive components (i.e., sesamin, sesamolin, and sesamol) in the so, were also affected during the storage period. high temperature (160-250ᵒc) does not affect the bioactive components of sesame seed oil especially lignans and their concentration almost remains the same that is the reason behind the strong antioxidant potential of sesame seed oil even at high temperature it sustain its properties (yoshida and takagi, 1997). nutritional improvement is not in a sense that it increases anything, but here it’s in a context that it increases the stability of wsso cookies against oxidation during study period due to antioxidant potential of white sesame seed oil. antioxidant potential is evident from the results. storage in polythene bags showed a significant change in moisture, peroxide value and overall acceptability of cookies (rangrej et al., 2015). as the moisture increased with storage time, the antioxidant properties decreased. however, although moisture increased in both groups of cookies, because so cookies had greater antioxidant potential to begin with, the so cookies showed more stability than hvf cookies. nanditha and prabhasankar, (2009) presented in their studies that natural antioxidants are very effective in enhancing the shelf life of bakery products. sharif et al., (2003) made cookies from oil extracted from natural source that has functional properties and natural antioxidants, which increased the shelf life of cookies by enhancing their antioxidant potential. similar to our findings various studies reported that so cookies are not only more stable in their proximate composition analysis but also in the antioxidants available by day 60 (quilez et al., 2006; reddy et al., 2005). 3.3. sensory evaluation a total of 75 people completed the study at all three time periods. before sensory evaluation, training and instructions were given to the all participants so that they can evaluate the products for sensory evaluation in the right way. the mean rating scores of the 9 points (low to high) sensory evaluation scale of colour, flavour, taste, crispness and overall acceptability are shown in table 3. table 3 indicates that at baseline, so cookies had significantly (p < 0.05) higher evaluation for colour (16%), flavour (10%), taste (5%), texture (9%), crispness (9%) and overall acceptability (12%) compared to hvf cookies. over the period from baseline to 60 days, the mean rating on each attribute decreased significantly (p < 0.05) for each cookie type. for so cookies, colour decreased by about 5.5%, flavour -8%, taste -16%, texture -11.6%, crispness -8% and overall acceptability by 14%. a similar trend was observed in hvf cookies. in hvf cookies, the mean rating for colour decreased -9%, flavour decreased by -11%, taste decreased by -11%, texture decreased by -12%, crispness decreased by -7% and overall acceptability decreased by 5.5%. by day 60, there were significant (p < 0.05) differences in the sensory rating between groups. compared to the hvf group, colour, flavor, texture and crispness were rated ital. j. food sci., vol. 30, 2018 22 higher in the so group (range 8-20%). taste and overall acceptability were lower than baseline but were not significantly different between groups by day 60. table 3. palatability of cookies. characteristic day 0 day 30 day 60 color sso 8.51±0.07 (6.80)c 8.21±0.05 (5.02)a,c 8.05±0.03 (2.81)a,c vegetable oil 7.35±0.10 (12.2) 6.91±0.09 (10.7)a 6.71±0.09 (11.4)a flavor sso 7.51±0.06 (6.71)c 7.11±0.09 (11.0)a,c 6.93±0.08 (9.58)a,c vegetable oil 6.80±0.09 (11.1) 6.43±0.08 (10.6)a 6.12±0.05 (7.09)a,b taste sso 8.00±0.09 (9.86)c 7.15±0.09 (11.2)a 6.87±0.07 (8.74)a,b vegetable oil 7.65±0.09 (10.2) 7.15±0.08 (10.2)a 6.92±0.09 (11.6)a texture sso 7.49±0.09 (9.90)c 6.95±0.10 (12.5)a,c 6.72±0.08 (9.96)a,b,c vegetable oil 6.89±0.09 (11.1) 6.25±0.05 (7.48)a 6.17±0.05 (6.72)a crispness sso 7.40±0.10 (11.5)c 7.05±0.09 (11.4)a,c 6.84±0.07 (9.00)a,b,c vegetable oil 6.79±0.08 (9.78) 6.45±0.06 (8.17)a 6.33±0.06 (7.93)a overall acceptability sso 8.00±0.06 (6.82)c 7.40±0.09 (10.4)a,c 7.03±0.07 (9.05)a,b,c vegetable oil 7.12±0.08 (9.51) 6.97±0.08 (10.5) 6.75±0.06 (7.35)a scale 1= low; 9 = high mean±se (%cv) the values are replicate of at least three ap<0.05 vs day 0; bp<0.05 vs day 30; cp<0.05 vs. vegetable oil cookies at each time period, both so and hvf cookies had similar physical properties as shown in table 4. by 60 days, the diameter in hvf cookies increased significantly (p < 0.05) by about 3% from baseline. in both groups, the rating for colour and flavor decreased over time. during storage, a common oxidation process that is stimulated by an increase in moisture known as maillard reactions, stimulates increased oxidation of the fat and increases in free fatty acids which could affect colour and flavor of the cookie. tbars is an important indicator for the quality of stored food (butt et al., 2007; wada, 1998). slow increase in moisture of wsso cookies caused the sustainability of total acidity and peroxide value that is directly related to antioxidant potential of wsso cookies. as noted previously, the increase in moisture with storage resulted in increased peroxide and acidity which will negatively affect most of the sensory attributes. in fact, several authors (bender, 1996; sharif et al., 2003; waheed et al., 2010), have reported a similar trend between increasing moisture with decreasing palatability with storage. previous research has also indicated that the addition of sesame flour improved the aroma, taste and overall acceptability of cookies and there were no significant differences from the control cookies (olagunju and ifesan, 2013). also, lim and lee reported that the addition of sesame ital. j. food sci., vol. 30, 2018 23 powder did not affect the overall acceptance of cookies (lim and lee, 2015). further, when bioactive components like phytosterol, α tocopherol and β phytosterol were added, there were no changes in the sensory and chemical properties of cookies (quilez et al., 2006). lastly, extra moisture is known to change the diameter, thickness, and spread factor of the cookie. during storage, the amount of moisture absorbed will increase the diameter of cookies and decrease their thickness. physical properties are also directly related to composition of cookies. as in composition of both cookies the main difference was the wsso and hydrogenated vegetable fat that creates the difference during storage. table 4. physical properties of cookies. characteristic day 0 day 30 day 60 diameter sso 63.25±0.26 (0.70)c 63.02±0.04 (0.11)c 62.96±0.06 (0.16)c hvf 62.08±0.14 (0.38) 62.01±0.10 (0.27) 63.93±0.03 (0.08)a,b thickness sso 9.80±0.03 (0.51) 9.76±0.01 (0.20) 9.71±0.03 (0.47) hvf 9.75±0.01 (0.21) 9.72±0.03 (0.45) 9.69±0.06 (1.00) spread factor sso 64.54±0.42 (1.13)c 64.56±0.05 (0.12) 64.84±0.08 (0.20)c hvf 63.67±0.08 (0.21) 63.79±0.23 (0.64) 63.91±0.40 (1.08) mean±se (%cv) the values are replicate of at least three ap<0.05 vs day 0; bp<0.05 vs day 60; cp<0.05 vs. hvf cookies 5. conclusions the results of this study indicate that exchanging the fat source used in a cookie significantly affects its physiochemical properties, antioxidant potential, palatability, and physical properties. although, following 60 days of storage, the overall properties of both types of cookies decreased, the cookies made with white sesame seed oil had better organoleptic properties and were found to be more palatable than the hvf cookies. furthermore, since so cookies have greater antioxidant potential than cookies made with hvf, they may be a healthier cookie choice. the results of this study indicate that wsso improved the overall functional importance of so cookies in terms of their physicochemical properties and bioactive components. the enhanced stability of wsso cookies against oxidation and their improved antioxidant potential may be particularly important for food industries that are interested in developing cookies with functional value. by replacing the fat source with sesame oil, cookie manufacturers may be able to meet high standards for nutritional potential without sacrificing palatability or shelf 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a.g. 2016. novel trans fat replacement strategies. current opinion in food science 7:27. yoshida h and takagi s. 1997. effects of seed roasting temperature and time on the quality characteristics of sesame (sesamum indicum) oil. j. sci. food agric. 75(1):19. paper received december 1, 2016 accepted august 6, 2017 survey ital. j. food sci., vol. 27 2015 1 keywords: agri-food, consumer, market, marketing analysis of a direct selling network for agrifood products p. rapisarda*1, m. rizzo1 and a. scuderi2 1department of economics and business, university of catania, italy 2departement of agrifood and environmental systems and management, university of catania, italy *corresponding author: placido.rapisarda@unict.it abstract sicily has become a food and wine area of great interest. however, conflicts within the supply chains have caused the selling process to become long and complex to the disadvantage of farmers, thereby leading to an information asymmetry between producers and consumers. in order to meet the new needs of the agrifood sector, we developed a theoretical model of organized direct selling that goes beyond regional boundaries, which is an alternative model to farmers’ markets and that helps to promote the creation of a network among the operators of sicilian agrifood supply chains. the aims of this study was to verify the potential of the proposed theoretical model based on a swot analysis, which was achieved by collecting data from interviews with the producers involved in the sicilian agrifood supply chains, and with the main stakeholders involved. 2 ital. j. food sci., vol. 27 2015 introduction farmers have always tried to shorten the supply chain between producers and consumers. they started by setting up consumer cooperatives and farmers’ points of sale1. recently, farmers have incorporated e-commerce within their selling activities, as well as farmers’ markets, box schemes, pick-your-own initiatives, and community-supported purchasing groups (abel et al., 1999; aguglia, 2009; brunori et al., 2009; la trobe, 2001). national and international studies make numerous references to these practices, which have focused on producers, consumers, those outside the established sources of supply, which have been compare with consolidated distribution systems (murdoch et al., 2000) with respect to environmental or legislative issues. in particular, these studies focus on short supply chains, direct selling, alternative food networks (holloway and kneafsey, 2004), short food supply chains (renting et al., 2003), communitysupported agriculture (raffaelli et al., 2009), and food community networks (lombardi et al., 2012; pascucci, 2010). one of the main features that are debated frequently concerns the effective advantages of short supply chains for producers, consumers, and the community as a whole. producers may have higher economic margins compared with those in the traditional agrifood supply chains, where different mediators take away part of the producers’ margins (van der ploeg, 2006). however, a feature that is often ignored is the fundamental difference between the roles, tasks, attitudes, and capacities of farmers and market experts, who have specific skills to address the market in an effective manner. it is inconceivable in terms of education, culture, or tradition for farmers to occupy specific commercial roles or to confront the “unfair” challenge of the largescale retail trade, which has been present in cities for decades. instead, it is possible to suggest the organization of an innovative market system, which can use its available efficiencies to move agriculture closer to urban consumers in different but stable ways, because consumers are increasingly keen to retain the precious nutritional value of food in general and of the “mediterranean diet”2 in particular, where unesco has recognized the latter as an intangible heritage of humanity (grosso et al., 2013). at present, the food industry and large-scale retailers are trying to exploit the information asymmetry that exists to direct consumption toward their needs and targets. this can only be overcome by farmers, particularly the producers of high quality food with good organoleptic characteristics (abel et al., 1999; hunt, 2007). the real advantages for consumers of short supply chains are lower prices but also better information about the nutritional value of raw materials, the characteristics of the production process, agri-industrial processing techniques, and food preservation techniques (hinrichs, 2000; la trobe, 2001). accordingly, our goal was to design an experimental model of direct selling that may represent an innovative method for agrifood products and to promote the products from specific areas, thereby implementing an alternative networked commercial system that can communicate effectively and reliably with consumers about the value of the food produced. analysis and reference context italian legislation has supported the direct commercialization of agrifood products since the 1960s (law no. 59 of 1963) and legislative decree no. 228 of 20013 simplified the relative procedures for farmers who practice direct selling4 (alabrese, 2008). subsequently, a decree has been applied to farmers’ markets only (belletti et al., 2010). the growth of these markets was supported further by the ministry of agricultural, food, and forestry policies on november 20, 20075 but the current economic recession is limiting food habits and life style, and reducing the purchas1 for example, citrus fruit producers’ organizations in the province of catania promote a network of points of sale in north-eastern italy, which are run directly by the members (rizzo and mazzamuto, 2009). 2 hedonistic reasons accompany healthy ones in defining the value of food products. the increasing number of press columns and television formats dedicated to food confirm the widespread research into the pleasure of good food and conviviality, as well as best sellers and editorials about regional recipes and the increasing number of concept stores where furniture, atmosphere, and menus make consumers feel relaxed and comfortable while enjoying simple, traditional, but creative meals based on the mediterranean diet. 3 article 4 of l.d. no. 228 dated 18 may 2001, “orientation and modernization of the agrifood sector, according to art. 7 of law, no. 57 dated 5 march 2001”. 4 the new decree also allowed direct selling for products not produced directly by the farm itself. the previous law, no. 59/63, “rules for farmers to directly sell agrifood products,” limited selling by farmers to the products obtained exclusively from their farms. they were identified as “owners of the land where they grow, their cooperatives or associations.” 5 this is an unprescribed decree since the legislative competence concerning commerce and agriculture is limited exclusively to regional governments according to article 117 of the constitution. only regional governments can issue rules and regulations about this matter. thus, it is a decree aimed at guidance that is not mandatory (alabrese, 2008). ital. j. food sci., vol. 27 2015 3 ing power of families (fantuzzi and brugnoli, 2010). producers use direct selling as a way to sell their products in nearby markets. there are two reasons for this: first, the greater the distance from the origin of the food, the greater the problems of information asymmetry between producers and consumers become, while the information and communication costs related to products and production techniques are also higher (briamonte, 2010; gardini et al., 2007; guidi, 2008); second, producers are hampered by the lack of organization, logistics, and the availability of finance when operating in distant markets. this makes it difficult for small-medium enterprises to place their products and operate at national and international levels (chiffoleau, 2009). several regional governments have issued specific regulations and financially supported market development6, including numerous measures in the rural development plan (rdp). in sicily, measure 321/a1 of the 2007/2013 rdp, supports the development of equipped public areas for farmers’ markets of typical products and handicrafts. fig. 1 shows the intervention areas, which include 85 projects and a total investment of 9 million euros to develop farmers’ markets, 79 of which will be activated in local action groups (lags) areas and the remaining six in other areas. however, direct selling within farmers’ markets has limits and critical points (verhaegen and van huylenbroeck, 2001; chiffolaeu, 2009). thus, previous studies have noted that farmers’ markets provide a direct relationship between producers and consumers, which may guarantee fresh products, because of their excellent locations and temporal discontinuity, but they do not provide an effective organization for selling, they do not obtain appropriate sales volumes, and they do not meet demand in full (brunori et al., 2009). farmers’ markets are often combined with village fairs and with folkloristic and cultural characteristics, but they are rarely oriented towards a modern organization7. the exclusive selling of products grown and consumed in the same area cannot allow for temporal continuity or completeness in terms of product diversification and the quality level, which is the basis of the modern distribution fig. 1 farmers’ market in sicily (source: data elaboration 2007-2013 rdp sicily). 6 the sicilian regional government intervened on this issue by article 83 of l.r. 11/2010, by allowing direct selling, particularly by certified farmers who carry out their activities within the sicilian territory. 7 the regulation gives municipalities central powers to organize, authorize, and finance such markets. despite the marginal role assigned to regional governments by national legislation, the majority have regulated and financially sustained farmers’ markets. 4 ital. j. food sci., vol. 27 2015 system thanks to the evolution of preservation techniques, transportation systems, and logistics (hinrichs, 2000). several studies (rizzo and vecchio, 2008) have shown that this approach is not a sustainable alternative because the volumes are too small. these low volumes are due to the lack of temporal continuity of production and the incapacity of the local agrifood system to offer an articulated range of products. if direct selling of agrifood products is to gain a higher economic weight, it cannot be confined to the farm location itself or to the neighboring area. italian legislation has not limited direct selling to local products alone, which has allowed it to spread throughout the territory of the republic after communication with the municipality to which the farm belongs8 (tudisca et al., 2014). recently, the sicilian regional government issued a regulation (article 10 of law 25/20119) that goes beyond regional boundaries to support and promote “the direct and market selling activities” of sicilian agrifood products via networked regional structures (section 1), that may interact in synergy with analogous networked structures at the national level (section 5) and at community level (section 6). prerequisites of the network model the european agrifood sector, particularly fruits and vegetables, has not experienced consumption increases for many years. by contrast, demand has diversified greatly where the dynamics have affected the structure of the offer (cso 2012 data). consumers are increasingly keen to look for products that better meet their needs and their respect for a renewed linkage between purchasing processes. the points of sale within the framework of the complex income dynamics suggest new market segmentation models, which are function of a different “perception” of the quality-price relationship. indeed, quality now includes aspects that go beyond its traditional concept, which was bound only to the organoleptic characteristics of products (brunori et al, 2009; chinnici et al., 2013). an orientation toward “responsible” purchasing has been added to the reasons to buy, especially from the particular segment of consumers who have a mature awareness of high value products because of their organoleptic, nutritional, healthy, evocative, ethical, and solidaritybased characteristics compared with commodities (di vita et al., 2013). this value is derived from, either jointly or singly, the fact that products come from specific territorial contexts (abel et al., 1999), where they are grown with traditional and/or organic production techniques, thus the offer is organized directly by producers who bet their reputation on their products and they only receive a premium price. accordingly, sicily’s pedoclimatic characteristics may allow it to produce a wide range of agrifood and zootechnical products, which may satisfy all the nutritional needs of the regional market, but also the national market. sicily is a food and wine “continent” because of the wide range of high quality agrifood products it offers and its millennial culinary tradition. based on its range of high quality agrifood products, sicily (graphic 1) is the third highest ranked region in terms of the number of registered products (28), especially for fruits, vegetables, oils, and cheeses. despite the acknowledged excellence of several quality products, the tendency for territorial specialization and exploitation has not diminished, especially in those territories where the community politics for years have favored monocultures destined for “global” markets. in sicily, this tendency has caused (fig. 2) the sellable gross production (sgp) to rely on a few typical products, i.e., citrus fruits, grapes, oil, wine, vegetables, and a wide range of zootechnical products, which together comprise 70% of the sgp of sicilian agriculture, while 30% of the sgp includes other fruits, vegetables, and livestock, which have lower value despite their high quality. although sicily may rely on a good range of agrifood products, its supply chains are inadequate due to a lack of organization and production volumes. thus, it cannot account for a significant market share. in many cases, single companies cannot face the problems related to the planning and management of the necessary promotion and communication activities. in addition, they cannot easily obtain the necessary information about the market situation and consumers’ preferences in order to tailor their offer according to consumer needs. for example, it is difficult for them to standardize their quality, arrange for suitable packaging, ensure the constant presence of their product, or adopt an advanced traceability system (hausmann and de amicis, 2007; rapisarda and rizzo, 2010). all of this would require 8 the first national organization to undertake direct selling and overcome these critical points was sponsored by coldiretti, an organization that represents italian farmers. coldiretti, via its initiative called “campagna amica,” is promoting points of sale throughout the national territory. these points of sale are gathered in a single commercial network that offers consumers the products of their members regardless of the geographical location of the growing area within italy. 9 regional law dated november 24, 2011, no. 25, issued on gurs no. 50 of december 2, 2011, entitled: “interventions to support agriculture and fishing. regulations for handicrafts, cooperation, and commerce.” ital. j. food sci., vol. 27 2015 5 resources, competencies, and a critical mass of products that smes often lack. the only way to overcome these problems is via group initiatives (galisai et al., 2009; lombardi et al., 2012) that combine production as well as some steps of the distribution processes and logistic arrangements (belletti and marescotti, 2012). based on these requirements and legislative interventions of the sicilian regional government, we aimed to develop an organized direct selling (ods) theoretical model, which goes beyond regional boundaries and provides an alternative to the farmers’ markets, thereby promoting the creation of networks among sicilian agrifood supply chain operators. the model is structured and includes the following subject typologies. subjects in the supply chains: farmers’ associations: these subjects have to organize the offer and services for each farmer’s direct shop10 (from product preservation to shipment). subjects for farmer’s direct shop management: specific or pre-existing companies will organize the farmer’s direct shops at national and international levels, such as the organization of promotional events and the management of market activities. they will have the functional prerequisite of collaborating with the subjects of the supply chains to guarantee the direct selling of products and to form a network that agrees to perform all of the other related activities. network junction: this is the organizational structure required to deal with the relationship between the subjects of the supply chains and graphic 1 pdo, pgi and tsg registered products in italy (source: qualivita data direct elaboration). 10 direct selling shop, a “closed area,” which is independent and included in an articulated structure with dedicated corner for the subjects of the supply chains to carry out their activities. graphic 2 sicilian gross sellable productions (2012) (source: inea data direct elaboration). 6 ital. j. food sci., vol. 27 2015 the subjects of the farmer’s direct shop management to coordinate and organize common activities, and to ensure that regulations are observed (rizzo and giudice, 2013). this ods model allows the possibility of overcoming the limits of the most common short supply chains in the italian territory, by joining producers as subjects in the supply chains, which are separated based on products, and by proposing the management of the direct selling commercial activities via a direct selling shop manager, who is a third subject. this theoretical management model is based on the network junction, the function of which is to coordinate the bidirectional inputs from the subjects of the supply chains and the subjects of the farmer’s direct shop management. the results of this information exchange will generate the product typology, the packaging typology, and the selling price, as well as linking producers directly to the subjects of the farmer’s direct shop management, who have direct daily contact with consumers. all of the subjects of the supply chains will be represented inside farmer’s direct shops with promotional and tasting initiatives for their products. in order to make the model stronger and more significant, side activities are included within the agrifood product direct selling scope, such as the following: tasting and distribution of quality regional agrifood products; organization of “satellite market spots” within the commercial area of reference of each farmer’s direct shop; promotion of regional quality production within the hotel, restaurant, and café (ho. re.ca.), and ethical purchasing groups (gas) commercial scopes of reference; acting as a structure that manages the organization of promotional activities at a regional level, including territorial marketing and customer retention; acting as an intermediate logistics centre to carry out e-commerce activities. this network includes a union point where producers and consumers meet to increase knowledge of the organoleptic and nutritional qualities of products based on tasting as well as on information that, thanks to modern it tools, has become widely available and is articulated and updated in real time. in order to comply with the aim of this research and combine tradition, culture, gastronomy, and diet inside farmers’ direct shops, traditional promotional activities will be developed, such as tasting, cultural, educational, and gastronomic activities involving the products and the territory. the truly innovative element is the promotional function of farmer’s direct shops, which will be integrated within the market and promotional activities, thereby producing a synergy between promotion and selling to help overcome the limits of the promotional activities carried out by public and/or territorial bodies, which often develop out of the commercial logic of private operators. integration, in addition to the physical level, is a common operative project between the public promotional activity and the private commercial activity, which is a highly innovative element of the model suggested to the regional government. methodology we decided to verify the potential of the proposed theoretical model by carrying out a swot analysis using data collected from interviews with producers and the main stakeholders involved with sicilian agrifood supply chains. swot analysis is a strategic planning tool used to evaluate the strengths, weaknesses, opportunities, and threats related to the ods model in order to fulfill its goals. the swot analysis includes: strengths: the aspects of the model that help to fulfill its goals; weaknesses: the aspects of the model that hamper the fulfillment of its goals; opportunities: useful conditions outside the model that help to fulfill its goals; threats: external conditions that may damage the performance of the model. the analysis used aimed to meet the goals of our research. in fact, it links the knowledge of the context where producers operate to the politics of the economic development and promotion of agrifood products. the analysis group collected information concerning the difficulties of sicilian farmers, commercial solutions, market dynamics, the specific needs of producers who adopt short supply chains at organizational and management levels, and objective and official data related to the agrifood system, which was obtained from the main research institutes of ismea, inea, istat, the osservatorio sulla vendita diretta, and nomisma. this study helped to identify the strengths and weaknesses of the most widespread forms of short supply chains, specific data concerning sicilian agrifood productions, the importance of this phenomenon, legislative aspects, and the fiscal and administrative supports of direct selling. direct interviews were conducted during 2013 in collaboration with the technical assistance operational sections (soats) of the sicilian regional government, which allowed the nine provinces of reference to select farms that were interested in the proposed direct selling model, where 126 operators in the sicilian agrifood supply chains were interviewed either jointly or singly. the number of interviews was quite significant compared with the number of operators involved. the answers to the questionnaire and swot analysis entries were selected based on ital. j. food sci., vol. 27 2015 7 the number of times they were iterated. answers were given according to the personal experiences of the interviewees. the originality of this research concerns the definition of a functional model of direct selling that includes producers, market managers, and consumers, as well as the creation of farmer’s direct shops with the primary function of selling agrifood products, but also with a wider and complex economic meaning, which combines articulated functions, such as “farmer’s direct shops”, promotion, marketing, and tasting that are linked to the production territory. results the results of this study show that “local food” has emerged as an increasing interest due to the economic weight it is gaining in terms of “proximity,” i.e., the physical distance between producers and consumers, but also because of the growing importance consumers allocate to the quality of products that come from a specific territory. numerous typical sicilian products possess the necessary characteristics to develop their own local market and to find places in the market that differ from their original roles, especially if they are characterized by clear traceability, sufficient critical mass, and the will to create a network of all supply chains and services for farms. the swot analysis highlighted the main strengths, weaknesses, and threats, but also the opportunities that the proposed model offers to support sicilian agrifood productions and to strengthen the role of producers in the supply chains (table 1). the swot analysis suggest that the ods model has a strategic meaning and it may achieve the following. promote a base of knowledge and excellence beyond the regional scope, thereby spreading information and stimulating consumption. improve the competitiveness of producers who cannot easily find commercialization channels table 1 swot analysis of the organized direct selling experimental model. source: elaborations of direct surveys and nomisma dat table 1 swot analysis of the organized direct selling experimental model. source: elaborations of direct surveys and nomisma data strengths weaknesses ▪ wide product range ▪ creation of a network between producers and consumers ▪ selling far from the place of origin guaranteeing the origin of products, quality, freshness, product seasonality ▪ valorization and promotion of products within the selling stage ▪ higher added value for producers along the value chain ▪ daily selling activity ▪ logistics organization ▪ remote management of points of sale ▪ logistics and transportation costs opportunities threats ▪ creation of alternative selling channels ▪ promotion of the territory of origin ▪ possible interaction with local bodies and associations to develop community initiatives to the advantage of the territory ▪ diversification towards non-agriculturerelated activities (ho.re.ca; catering) ▪ advertising of the mediterranean diet and of the “born in sicily” label ▪ management cost of points of sale ▪ purchase frequency ▪ management of returns author 30/9/y 14.59 formattato: tipo di carattere:times 8 ital. j. food sci., vol. 27 2015 beyond the regional scope, thereby motivating production differentiation in sicilian agriculture and making traditional producers economically sustainable. indeed, the latter remains an expression of the biodiversity of specific territorial contexts. exploit the well known advantages of short supply chains to allow the agrifood world to become closer, both significantly and stably, to the growing segment of consumers who look for and buy quality agrifood products. stimulating farmers’ associations to concentrate, organize, and commercialize their offer, thereby improving the performance and competitiveness of members. in addition, several weaknesses of direct selling in farmers’ markets may be overcome by creating an organized network that strengthens the role of producers within the supply chains. however, the weaknesses and threats show that there is a need to strengthen the concept of direct selling within farmer’s direct shops. by contrast, the model may become distorted given the difficulties of maintaining the producer-consumer relationship directly from a legislative-fiscal point of view. overall, this model is an example of organized supply chains with defined roles and a vertical distribution strategy directly from producers to consumers, thereby providing the opportunity to design intervention proposals and strategies to define the offer based on the specific characteristics of the demand. conclusions the research results allowed us to evaluating our experimental model that aims to promote a network of direct selling operating promotional farmer’s direct shops and points of sale throughout the national territory, thereby promoting sicilian agrifood products. this is a step forward compared with today’s “country markets” because it shortens the physical distance from the field to the table and optimizes the organizational and economic structure of this sector. the proposed short supply chain management model provides tools that are more flexible for producers, by overcoming the current difficulty of being present in different places at the same time to meet consumers. a direct relationship with the farmer makes product commercialization easier but not all farmers are ready to assume this role. indeed, many would prefer to continue playing their existing role, which is to dedicate themselves to their production activities because they lack sufficient time, resources, or the correct attitude to participate in selling activities. sicilian farmers may not face price competition, but the hypothetical shortening of the distribution chain by proposing direct contacts between the consumer (national, according to the whole range of the legislative intervention under study) and the producer (sicilian) cannot be separated by the segmentation of a specific target group of consumers. thus, the proposal of a “pact” as a sign of a philosophy that favors some qualitative aspects but does not aim to 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2001. cost and benefits for farmers participating in innovative marketing channels for quality food products. j. rural stud. 17 (4). paper 90 ital. j. food sci., vol. 28 2016 keywords: chlorophyll, kinetics, thermal degradation, virgin olive oil differential method to determine thermal degradation kinetics of chlorophyll in virgin olive oil ahmet levent inanc department of food engineering, engineering and architecture faculty, kahramanmaras sutcu imam university, 46100 kahramanmaras, turkey email: linanc@ksu.edu.tr abstract differential method is presented to study thermal degradation kinetics of chlorophyll in virgin olive oil. the oil samples, naturally containing 20.0 mg/kg chlorophyll were stored at 150°, 160°, 170°, 180°, 190° and 200°c until the time at which chlorophyll contents had reduced to the certain amounts. the concentration gradually decreased as heating time increased. a half order equation was found as the best model for the present experimental data. differential method with graphic and substitution methods was compared for the determination of the rate constant and the half-time. the rate constants and half life at 150°c were determined in the range of 0.20-0.22 and 12.14-13.12 for the thermal process of chlorophyll in virgin olive oil, respectively. the reaction rates increased approximately 1.1 times with increment of every 10°c from temperature of 150°c. conversely, the half lifes decreased 0.9 times for increment of every 10°c. the activation energies were approximately 24 j/kg for differential method, and 22 j/kg for graphic and substitution methods. mailto:linanc%40ksu.edu.tr?subject= ital. j. food sci., vol. 28 2016 91 introduction chlorophylls are responsible for the green color of all vegetables and fruits. animal tissues can’t synthesis chlorophylls, though animal cells can chemically modify them for assimilation. these compounds should be supplied from food (giuffrida et al., 2007). chlorophyll and its various derivatives have been used in traditional medicine and for therapeutic purposes for many years and perhaps have the potential role of these pigments in the prevention of human cancers that has drawn more recent attention (ferruzzi and blakeslee, 2007). the color of olive oil is principally related to its perceived quality, and therefore to its acceptability. the economic importance of the appearance of the oils is unquestionable. the color of virgin olive oil is due to the natural pigments chlorophylls, and carotenoids (minguez mosquera et al., 1994). olive oil contains originally the chlorophylls a and b. chlorophyll a, pheophytin a, is typically found in higher amounts than chlorophyll b. the distribution and content of chlorophyll in olive oil are dependent on a number of factors including species, agroclimactic conditions, pre and postharvest treatment, and type and degree of food processing (minguez-mosquera et al., 1990, gandul-rojas et al., 1996, giuffrida et al., 2007, criado et al., 2008, cerretani et al., 2008, giuffrida et al., 2011). the grades of oil extracted from the olive fruit are classified as virgin, lampante, refined and olive pomace oil. virgin oil is produced by the use of mechanical means only, with no chemical treatment or heat. virgin oil includes both virgin olive oil (voo) and extra-virgin olive oil (evoo) products, depending on quality. therefore, virgin olive oil should be preferably added as the final seasoning in fresh salads, soups, or more elaborated dishes (carla et al., 2013) but olive oil like other vegetable oils is used in several cooking processes such as deep-frying, pan-frying, roasting, microwave cooking, etc. (waterman and lockwood, 2007; boskou, 2009). each thermal processing type has particular characteristics as depending on process temperature and time. carla et al. (2013) summarized several works related to olive oil that used as the cooking base, grouped the works by real and simulated cooking method and showed the analytical parameters chosen by the authors to evaluate olive oil performance. for example, in frying process the both methods were tested with several olive oil commercial grades, at temperatures ranging from 170°c to 180°c in real frying, and from 160°c to 190°c in simulated frying, i.e. being the olive oil heated without any food. some authors also compared the effects of adding fresh oil between frying sessions in the oil performance. in the previous studies it was made on thermal stability of olive oil. the studies on thermal stability of olive include the thermal decomposition of commercial vegetable oils of some of their thermal properties (dweck et al., 2004), the thermal degradation study of four unsaturated or saturated esterified c18 fatty acids with glycerol (vecchio et al., 2008), stability of olive oil during heating (berasategi et al., 2012), the heat-oxidation stability of binary blends made with palm oil and several extra virgin olive oils (de leonardis and macciola, 2012) and effects of the main virgin olive oil antioxidants under mild temperature conditions (mancebo-campos et al., 2014) but, virgin olive oil contains minor constitutes together with triglycerides, the thermal effect on chlorophyll stability and degradation in olive oil has not been studied extensively. kinetic modelling recently gaining increasing interest in food science gives the possibility of controlling changes in foods such as to control food quality during processing and shelf life (niamnuy et al., 2012; goula, 2013; grauwet et al., 2014; remini et al., 2015). microbiological changes which are called as predictive microbiology have been worked up to recent years but it can also be applied for biological, chemical and physical changes. the rate of a reaction and its temperature dependence, the occurrence of such a reaction can be predicted and controlled under specified conditions. the difficulties in kinetic modelling are choosing the right model for a reaction. for example; one of the difficulties is that too few data points are available to decide for the correct order. in general, researchers in food science have limited themselves often to simple reaction kinetics. i.e. it is trying to fit a zero-, firstor second order model to their data (van boekel, 1996; van boekel, 1999). the present study focused on the determination of rate order and characterization of the arrhenius parameters governing the thermal degradation reactions of chlorophyll in virgin olive oil by using the differential method and was to compare it with other two rate order determination methods. 2. material and methods 2.1. materials olive oil from olive fruits harvested in 20012 2013 season were obtained from a local olive oil plant (demirkol ltd., kahramanmaras, turkey). working principle of the plant is that olives are stored in the hopper of olive elevator and transported to washing machine. first leaves of olives are removed by leaf remover. then olives are washed without giving harms to its pulp in the olive washing unit. olives are transported to crusher by crusher elevator. olives are crushed and become semi paste in crusher. semi paste olives are mixed to obtain oil in malaxers. crushed olive is fed into the decanter without water through a 92 ital. j. food sci., vol. 28 2016 pulp pump. input product comes out of decanter as oil and pomace with black water. the characteristic of the olive oil are as follows: free acidity, 0.49 % oleic acid; peroxide value, 5.22 meq o 2 /kg; k 232 and k 270 extinction coefficients, 1.89 and 0.15; respectively, according to the analytical methods described in european regulation eec 2568/91 (eec, 1991) and chlorophyll content, 20.0 mg/kg (pokorny et al., 1998). the oil samples (25  ml each) were transferred into 50 ml glass bottles. the bottles were sealed with teflon-coated rubber seals and aluminum caps and stored at 150°, 160°, 170°, 180°, 190° and 200°c under dark condition in a forced air oven. chlorophyll content was measured with 2-h intervals from initial time until the time at which chlorophyll contents had reduced to 1 mg/kg all samples were prepared in duplicate. 2.2. determination of chlorophyll content in olive oil the chlorophyll content of olive oil was analyzed using the method described by pokorny et al. (1998). the sample was measured at 630 nm, 670 nm and 710 nm in a 10 mm spectrophotometer cell against air, instead of a reference cell. the method is suitable for the determination of quantities of chlorophyll pigments higher than 1 mg/kg. the following equation was used for determining the chlorophyll content where: [c] = content of chlorophyll pigments in mg of pheophytin a in 1 kg of oil, a = absorbance at the respective wavelength (nm), l = thickness of the spectrophotometer cell (mm). 2.3. kinetic theory differential method was used for determination of the degradation rate order and the rate constant of chlorophylls in olive oil. it was expressed the concentration at any temperature as a function of time in a power series, with constants a, b, c by deriving from the experimental concentration-time data [c] = at2 + bt + c where concentration ([c]) and time (t) were expressed in mg/kg and in hour. rate of reaction in mg/kgh (v) was estimated from the following equation; the most simple general rate equation was used for a single reactant at concentration [c]: where n = rate order, k n = rate constant at order n. by taking the logarithm of the above equation to base e it follows that: ln v = nln [c] + ln k n rate order and rate constants at different temperatures were determined by plotting graph ln v versus ln [c] the half-life value (t 1/2 ) of chlorophyll degradation was calculated using the equation given below after founding rate order and rate constants: lnk 1/2 was plotted versus 1/t to determine arrhenius parameters (a and e a ) by taking the logarithm of arrhenius equation; k 1/2 = aexp(e a /rt) to base e; where e a is the activation energy (j/kg), a is the pre-exponential factor or arrhenius constant, r* is the specific gas constant for pheophytin a (9.543 j/kg k), and t is the absolute temperature (k). differential method using for determination of the rate constant was compared with substitution and graphic methods. in substitution method the k value at a temperature was calculated by substituting initial concentration, concentration at any time and time values into the following half order rate equation: k 1/2 = 2/t×([c 0 ]1/2 [c]1/2) for n = ½ in graphic method the above equation was rearranged as [c]1/2 = [c 0 ]1/2 (k 1/2 /2)×t and [c]1/2was plotted versus t to determine k 1/2 value (the plot not shown). 3. results and discussion virgin olive oil is a food matrix contains triglyceride having a high percentage of monounsaturated fatty acids and also other minor constituents such as the phenols, chlorophyll and carotenoids fundamental in contributing to specific characteristics of virgin olive oil. therefore the kinetic study and characterization of the arrhenius parameters related with the thermal degradation reactions of chlorophyll in voo were performed in an oil matrix system to establish mathematical models enabling the prediction of the degradation of this pigment during voo thermal processing. changes with respect to the time in chlorophyll concentration in oil matrix during thermal ital. j. food sci., vol. 28 2016 93 processing, expressed in mg/kg, were shown in fig. 1. the chlorophyll concentrations gradually decreased while heating times increased. the experimental data was transferred to sigmaplot (version 12.0) program and trial and error method was applied to find the best fit curve equation on the data. the chlorophyll concentration at any temperature was expressed as a function of time. the best fit mathematical equations for the changes in the experimental data with the reaction time were selected to verify the rates of reaction at any temperature. the equations and their constants are shown in table 1. the initial concentration of chlorophyll was arbitrarily set at 20.0 units. the reaction mechanism for chlorophyll degradation kinetics was assumed as a simple reaction type; pheophytin a → colorless products where k n = rate constant for n order the rates of reaction were obtained by taking derivatives of the concentrations with respect to time. so lnv versus ln[c] was plotted to estimate the rate order and rate constants at different temperatures (fig. 2). table 2 shows the best fit equations for lnk ln[c] data. after estimating rate order as half order reaction it was calculated coefficients of the best equations for it (table 3). an assumption had been made for order of reaction of thermal chlorophyll degradation in a lot of previous studies on the processes of different food matrices such as fermentation of pickles coleslaw and olives (minguez-mosquera et al., 1992, minguez-mosquera et al., 1994; heaton et al., 1996) or thermal processing of spinach (canjura et al., 1991; yongxi et al., 2000) and also such as the visual green color a degradation (steet and tong, 1996; weemeas et al., 1999; ahmed et al., 2002; thron et al., 2001; ahmed et al., 2004; aparicio-ruiz et al., 2011, ahmed et al., 2013; mercali et al., 2014; dong et al., 2014), and kinetics studies had been gone on assuming an order of 1. but van boekel (2009) reported that the best model for the decomposition of chlorophyll is not only first-order equafig 1 changes with respect to the time in chlorophyll concentration in oil matrix during thermal processing. table 1 best fit equations for the concentration-time data. t (oc) [c]=at2-bt+c r² a b c 150 0.012 0.970 20.0 0.999 160 0.014 1.066 0.998 170 0.018 1.183 0.993 180 0.028 1.489 0.993 190 0.030 1.558 0.991 200 0.035 1.678 0.987 [c]: chlorophyll concentration; t: time; a, b and c: function coefficients. fig 2 lnv ln[c] plot to estimate the rate order and rate constants at different temperatures. table 2 best fit equations for lnk ln[c] data. t (oc) lnv=n×ln[c]+lnkn r² n lnkn 150 0.53 -1.59 0.998 160 0.51 -1.45 0.999 170 0.49 -1.32 0.999 180 0.47 -1.04 0.997 190 0.53 -1.11 0.998 200 0.50 -0.97 0.999 [c]: chlorophyll concentration; v: reaction rate; k: reaction constant; n: reaction order. table 3 best fit equations for half-order rate. t (oc) [c]1/2=[c0]1/2 k1/2/2×t r² k1/2 [c0]1/2 150 0.21 4.47 0.998 160 0.24 170 0.26 180 0.33 190 0.34 200 0.38 94 ital. j. food sci., vol. 28 2016 tion, but also could be half-order equation; for example, applying nonlinear regression to the data of schwartz and von elbe (1983), the best order n is 0.5 ± 0.5 for chlorophyll a and 0.6 ± 0.4 for chlorophyll b (± 95% confidence interval). thus, the present data similar to the data of van boekel (2009). it was compared the differential method with the other two methods; substitution and graphic method. the results obtained from the three different methods are shown in table 4. it was found that the rate constants and half-lifes at each temperature determined by three methods were close together. the reaction rates increased approximately 1.1 times with increment of every 10°c from temperature of 150°c. but, in general the reaction rate doubles for each 10°c increase in temperature (aparicio-ruiz et al., 2010). however, clark (2009) reports that this approximation (about the rate of a reaction doubling for a 10 degree rise in temperature) only works for reactions with activation energies of about 50 kj/mol fairly close to room temperature, and the rate constant goes on increasing as the temperature rise up, but the rate of increase falls off quite rapidly at higher temperatures. the half-life of a reaction is defined as the time at which the concentration of component a is at half its initial value. it provides a highly detailed description of how fast a reaction is occurring. in the present work, the half-life decreased 0.9 times for each 10 °c increase in temperature. the activation parameters were determined for the thermal process of chlorophyll in virgin olive oil in the range between 150°c and fig 3 lnk versus 1/t plot for the thermal process of chlorophyll in virgin olive oil. table 4 comparison of methods used for determination of the rate constant and the half-life. t (oc) rate constant (k1/2) half-life (t1/2) d g s d g s 150 0.20 0.21 0.22 13.12 12.44 12.14 160 0.24 0.24 0.24 11.36 11.22 11.12 170 0.27 0.26 0.26 9.96 10.09 10.27 180 0.35 0.33 0.33 7.55 8.12 8.09 190 0.33 0.34 0.35 8.12 7.79 7.63 200 0.38 0.38 0.38 7.04 7.11 7.03 d: differential method. g: graphic method. s: substitution method. table 5 arrhenius constant, and activation energy for chlorophyll. method k=aexp(-ea/r*t) best fit equations for lnk1/2 vs 1/t data a ea (j/kg) r*(j/kg.k) r² d 79.84 24.05 9.543 lnk1/2= -2.52x103(1/t) + 4.38 0.93 g 55.70 22.43 lnk1/2= -2.35x103(1/t) + 4.02 0.97 s 55.15 22.43 lnk1/2= -2.35x103(1/t) + 4.01 0.97 d: differential method. g: graphic method. s: substitution method. 200°c. the resulting logarithmic plot is shown in fig. 3. the estimated values used in the arrhenius equation for chlorophyll degradation reaction during heating by using three methods is shown in table 5. the ea determined by graphic method (22.43 j/kg) was the same value found in substitution method whilst the value in differential method was 24.05 j/kg. average activation energies for chlorophyll with respect to first order reaction were reported to be in range of 14.8 and 15.3 kcal/mol in the different temperatures and ph range (ryan-stoneham and tong, 2000; 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april 18, 2015 paper ital. j. food sci., vol. 27 2015 505 keywords: antioxidant activity, abts, dpph, frap, wine, sulfites effect of sulfites on the in vitro antioxidant activity of wines c. d. di mattia, a. piva, m. martuscelli, d. mastrocola and g. sacchetti* faculty of bioscience and technology for agriculture, food and environment, university of teramo, via c.r. lerici 1, mosciano s. angelo, 64023 teramo, italy *corresponding author: tel. +39 0861 266913, fax +39 0861 266915, email: gsacchetti@unite.it abstract the objective of this study was to assess the contribution of so 2 to the overall antioxidant activity of wines. in this study, white, red, and model wines, with increasing sulfite content, were used. the radical scavenging activity of the wines was evaluated by abts and dpph assays, while the reducing capacity of the wines was assessed by the frap assay. so 2 positively affected the antioxidant properties of the wines and, in some cases, its contribution to the overall antioxidant activity of wines was higher than that of naturally occurring antioxidants. depending on the assay, so 2 showed both synergistic and antagonistic effects with the antioxidants naturally present in wines. 506 ital. j. food sci., vol. 27 2015 introduction wine is one of the most important dietary sources of antioxidants with both in vitro and in vivo antioxidant activity (manzocco et al., 1998; frankel et al., 1995; vinson and hontz, 1995; serafini et al., 1998; tsang et al., 2005). the in vitro antioxidant activity of wine, which has been studied in depth for decades, is highly correlated to its phenolic content (simonetti et al., 1997; burns et al., 2000; alonso et al., 2002; fernández-pachón et al., 2004; yildirim et al., 2005). in vivo studies have shown that the consumption of wine modulates the serum non-enzymatic antioxidant capacity in humans. however, the direct antioxidant effect of polyphenols in vivo is still under debate (serafini et al., 2011; hollman et al., 2011). even though the antioxidant activity of wines has been mostly attributed to the presence of phenolic compounds (manzocco et al., 1998; yildirim et al., 2005; burns et al., 2001; villaño et al., 2006), exogenous antioxidants, such as sulfites, are added during the wine-making process. sulfur dioxide is one of the most commonly used additives in the food and beverage industries (who, 1998) due to its antioxidant, antiseptic, and preservative properties (branen et al., 2002). in wines, sulfur dioxide has positive effects by inhibiting oxidation and microbial growth, increasing pigment extraction, and reducing color loss and phenolic polymerization (ribéreau-gayon et al., 2000). however, the use of sulfites in certain food products has either been banned (fda, 1986) or strictly limited (eec, 1995) and is currently under regulation due to its allergenic effects in hypersensitive individuals (efsa, 2004). even though sulfites have reducing and antioxidant properties, there are contradictory findings on the contribution of sufur dioxide to the overall antioxidant capacity of wines. some studies have reported that sulfur dioxide reacts with dpph radicals and improves the radical scavenging activity of wines (abramovic et al. 2015). other studies have found that sulfur dioxide plays a minor role in the antioxidant capacity of wines (manzocco et al., 1998; cimino et al., 2007). additionally, as reported by kilmartin et al. (2001), the contribution of sulfur-containing antioxidants is lost when their reducing properties are determined by cyclic voltammetry methods equipped with glassy carbon electrodes. on the other hand, authors have reported that sulfur dioxide might play a significant role in the antioxidant activity of beverages and sauces (long et al., 2000; lachman et al., 2009; mitsuhashi et al., 2001), especially of white wines, which contain high sulfite levels and low natural antioxidant levels. additionally, the in vivo effect of sulfites on the antioxidant activity of foods is still unknown (campanella et al., 2004; laggner et al., 2005). the objective of this study was to evaluate the contribution of sulfur dioxide to the overall in vitro antioxidant capacity of wines using the abts, dpph, and ferric reducing antioxidant power (frap) assays. these assays differ in several properties including mechanism of action (radical or redox reaction) and environmental conditions (solvent polarity and ph). in this study, three wine types (white wine, red wine, and a model wine) were used. this experimental approach was used to assess possible matrix effects which, to the best of the authors’ knowledge, have not been evaluated. materials and methods materials three types of wines were analyzed: white wine (trebbiano d’abruzzo pietrosa, 2003 vintage, winery dora sarchese), red wine (montepulciano d’abruzzo, 2004 vintage, miglianico social winery), and a model wine made from distilled water, ethanol (12% v/v), and tartaric acid (0.033 m, ph 3.6). the content of alcohol, total polyphenols, and sufur dioxide, and the ph value of the three wines are shown in table 1. different levels of sodium metabisulphite (k 2 s 2 o 5 ) were added to the wines. all reagents used in this study were of analytical grade. chemical and chemico-physical analyses alcohol content, total and free sulfur dioxide content, and ph values were determined by official eu methods (eec, 1990). total polyphetable 1 total and free sulphur dioxide content, ph, alcohol amount, total polyphenol index and total dry extracts of the white and red wines under investigations. samples so 2 tot so 2 free ph alcohol tpi total dry extract (mg l-1) (mg l-1) (%v/v) (mg gae l-1) (g l-1) white wine 77 65 3.24 12.90 1052 22.80 red wine 70 54 3.27 13.05 1837 23.55 data coefficient of variation <2%. ital. j. food sci., vol. 27 2015 507 nol content was determined by the method reported by singleton and rossi (1965). abts assay the radical-scavenging activity of the samples was determined by the abts (2,2’-azino-bis(3ethylbenzothiazoline-6-sulphonic acid) radical decolorization assay (re et al., 1999). the bleaching rate of the abts radical in the presence of sample was monitored at 734 nm. abts radical solution (2.97 ml; abs = 0.70±0.02) was mixed with 30 µl of diluted wine samples (1:2, 1:5, 1:10, and 1:20) using the model wine as diluent. abts radical bleaching was monitored at 25°c for 60 min; the decoloration degree after 5 min was used as an indicator of antioxidant activity. in the dilution range considered, the abts radical bleaching was proportional to the concentration of sample added to the medium; a dose-response curve was fitted to a linear model. antioxidant activity, which was calculated as the ratio between the regression coefficient of the dose-response curve of the sample and the regression coefficient of the dose-response curve of trolox (hydrophilic homologue of tocopherol), was expressed as µmoles of trolox equivalents per ml of sample (teac abts : trolox equivalent antioxidant capacity). frap assay the reducing activity of the samples was determined according to the method described by benzie and strain (1996), with slight modifications. sample (0.1 ml) was mixed with frap reagent (2.9 ml) obtained by mixing 300 mm acetate buffer (ph 3.6), 10 mm tptz (2,4,6-tripyridyl-s-triazine) solubilized in 40 mm hcl, and 20 mm fecl 3 in a 10:1:1 ratio. absorbance was measured at 593 nm for 6 min. a calibration plot was generated based on feso 4 7h 2 o; the results were expressed as mm fe2+. dpph• assay the antiradical activity of the samples was measured by the dpph (2,2-diphenyl-1-picrylhydrazyl) decolorization assay as reported by brand-williams et al. (1995), with slight modifications in data computation. a dose-response curve was generated by adding 0.1 ml of sample at different dilutions (1:2, 1:5, 1:10, and 1:20) to 2.9 ml of a 6.1⋅10-5 m dpph-methanol solution. radical bleaching was monitored at 25°c for 60 min. dilutions were performed with the model wine as diluent. the teac dpph value was calculated as the ratio between the regression coefficient of the dose-response curve of the sample and the regression coefficient of the dose-response curve of trolox and expressed as µmoles of trolox equivalents per ml of sample. statistical analyses three aliquots were sampled from each wine; each aliquot had different levels of sodium metabisulfite. all analytical determinations were carried out in triplicate. data were reported as mean ± standard deviations. linear regression was applied to assess the relationship between sulfite content and antioxidant activity; the goodness of fit was evaluated by the coefficient of determination (r2). the antioxidant activity of wines in the absence of sulfites was obtained by extrapolation of the intercept value; the accuracy of the predicted values was assessed from the standard deviation. all statistical analyses were performed with statistica for windows (statsoft, tulsa, ok). results and discussion the proximate composition and sulfite content of the wines are shown in table 1. the polyphenol content of the white wine was quite high because the wine was processed by cryo-maceration, while that of red wine was relatively low because it was a ‘cerasuolo-type’ red wine. these two types of wine were selected for this study because they had similar alcohol and total dry extract contents. the sulfite content of the three wines increased with increasing sodium metabisulfite addition. the total sulfur dioxide content, which was assessed by titration, was 50, 100, 150, and 200 mg l-1 in the model wine; 77, 113, 125, 153, 185, and 209 mg l-1 in the white wine; and 70, 100, 125, 150, 175, and 200 mg l-1 in the red wine. the amount of sodium metabisulfite added to the wines was calculated using data in table 1. the free sulfur dioxide content was measured immediately and 2 h after sodium metabisulfite addition; no significant changes in bound sulfilte levels were obtained between these two time points. this time lapse is usually required for antioxidant activity determinations. antioxidant activity was determined by abts (teac method), dpph, and frap assays. the abts and dpph assays have similar mechanism of action towards ar-oh, because they can be neutralized either by direct reduction via electron transfer or by radical quenching via h atom transfer (prior et al, 2005), even though in the case of dpph radical, the hydrogen atom removal from ar-oh could be considered as a marginal reaction because it occurs very slowly in strong hydrogen bond-accepting solvents such as methanol (huang et al., 2005). the environmental conditions of the two radical scavenging assays are quite different because the abts assay is performed in aqueous media versus pure methanol in the dpph assay. 508 ital. j. food sci., vol. 27 2015 radical scavenging activity as determined by the abts radical decolorization assay the antiradical activity of the wines, as determined by the abts decolorization assay, is shown in fig. 1. antiradical activity improved with increasing sulfur dioxide concentration. the model wine containing 50 ppm so 2 , an amount that is likely to occur in real wines, was characterized by a teac abts value of 0.85 µmoles trolox equivalents per ml of sample, while the 200 ppm model wine had a teac abts value of 3.48 µmoles trolox equivalents per ml of sample. taking into account the fact that the antioxidant activity of the white wine measured by the abts assay may vary between 0.8 and 4.24 µmoles trolox equivalents per ml (alonso et al., 2002; de beer et al., 2003; villaño et al., 2004), these results suggest that sulfites may play a more significant role in the antiradical properties of wines than polyphenols. however, all wine samples had so 2 added in its free form; commercial wines are likely to have so 2 bound to different compounds. to evaluate the effect of sulfur dioxide on the antioxidant capacity in a real wine, the antiradical activity determinations were carried out in white wine with different contents of total sulfur dioxide. the results revealed that the white wine, with a total so 2 content of 77 mg l-1, was characterized by a teac abts value of 1.16 µmoles of trolox equivalents per ml. taking into account that 77 mg l-1 of sulfur dioxide in the model wine exerted a teac value of 1.30, it can be hypothesized that most of the antioxidant capacity of white wine is attributed to its sulfur dioxide content. by extrapolating the antioxidant activity of white wine without sulfites from the regression curve (fig. 1), the wine had a teac abts value of 0.40 µmoles of trolox equivalents per ml of sample. therefore, sulfur dioxide contributed to the antioxidant activity of white wine to such an extent that an amount of 50 mg l-1 sulfur dioxide can double the teac abts value. these results were in agreement with those obtained by long et al. (2000), who reported that small quantities of sulfites can affect the total antioxidant activity of the product. in white wine, an increase in sulfur dioxide concentration from 77 to 200 mg l-1 doubled its antioxidant activity. this result is quite significant because most wine research studies have not evaluated sulfite interference or sulfite contribution to the overall wine antioxidant activity (villaño et al., 2006; lachman et al., 2009; 24, villaño et al., 2004; arnao, 2000), even when the fractionation of polyphenolic compounds could not explain the overall antioxidant activity of the samples (fernández-pachón et al., 2004). the regression coefficient of the dose-response curve of the white wine was lower than that of the model wine (fig. 1), which could be attributed to matrix effects. to further investigate the matrix effect on the antioxidant capacity of sulfur dioxide, the antiradical activity was also determined in red wine. fig. 1 shows that the red wine, with a sulfur dioxide content of 70 mg l-1, had a teac abts value of 1.70 µmoles of trolox equivalents per ml. considering that the model wine with similar sulfur dioxide content had a teac abts value of 1.18, it could be hypothesized that a considerable percentage of the antiradical activity of red wine is attributed to its sulfur dioxide content. however, when the antioxidant activity of the red wine with no sulfites was extrapolated in the regression curve (fig. 1), the teac abts value was 1.52. taking into account the regression equafig. 1 antiradical activity as evaluated by the abts radical decolorization assay of the model wine solutions and of the white and red wines as a function of free so 2 concentration. ital. j. food sci., vol. 27 2015 509 tion, the sulfur dioxide contribution to the overall antiradical capacity was 10–38% in the tested concentration range, which was lower than that of white wine. in decreasing order of regression coefficient magnitude, the wines were model wine > white wine > red wine. this result confirmed the presence of a matrix effect on the determination of antioxidant activity; this matrix effect was higher in the red wine than in the white wine. it has been extensively reported that sulfites in wine can bind to several compounds such as acetaldehyde and polyphenols. polyphenol content is usually much higher in red wines than in white wines (alonso et al., 2002; de beer et al., 2003). additionally, red wines contain a high amount of anthocyanins, which bind to sulfur dioxide (antonelli and arfelli, 1993; timberlake and bridle, 1967). however, in this study, the free sulfite content was taken into consideration (fig. 1); therefore, in our experimental conditions it could be assumed that natural antioxidants and sulfites interfered with the antioxidant activity assays. in fact, both synergistic and antagonistic effects among antioxidants were observed in different in vitro antioxidant activity assays. reducing activity as determined by the frap method the antioxidant properties of the wines were evaluated with the frap assay (benzie and strain, 1996). in contrast with the previously described methods, this method is based on the reducing capacity of a compound rather than its antiradical activity. the frap values of the model, white, and red wines are shown in fig. 2. the model wine had low reducing activity; however, the addition of sulfites (70–200 ppm) resulted in a threefold increase relative to the initial value. the addition of sodium metabisulfite to the white and red wines increased their reducing power. the higher the sulfite content, the higher the reducing properties, likely due to the protective role of sulfur dioxide against polyphenol oxidation. an increase in sulfur dioxide from 71 to 200 ppm contributed to a 73% and 158% increase in the reducing capacity of the red and white wines, respectively. in the red and model wines, it was possible to extrapolate the frap value without sulfite addition. based on the results, sulfur dioxide is responsible for most of the reducing power of the wine samples. however, with respect to the white wine without sulfite addition, the experimental data did not allow an accurate estimation of the frap value because of a non-linear response (fig. 2). this result could be attributed to synergistic effects between natural antioxidants and sulfur dioxide. the synergistic effects between natural antioxidants and sulfur dioxide could account for a non-linear response between the frap assay and the so 2 dose, which was evident in the red and white wines (fig. 2). if the individual effect of an antioxidant on frap is linear within a certain concentration range, the synergistic effect of two antioxidants could show an increase or decrease in the response due to variations in their molar ratios (hidalgo et al., 2010). in order of decreasing regression coefficient magnitude, the wines were red wine >white wine > model wine. there were no negative matrix effects in the red and white wines. contrary to the results obtained from the abts assay, sulfites fig. 2 reducing capacity as evaluated by the frap method of the model wine solutions and of the white and red wines as a function of free so 2 concentration. 510 ital. j. food sci., vol. 27 2015 and naturally occurring antioxidants (i.e., polyphenols) had a synergistic effect on the reducing power of wines (fig. 3). this result may be attributed to several factors: (i) in the experimental conditions of the frap assay (ph= 3), there is a lower amount of bound so 2 (ribéreau-gayon et al., 2000) than in the abts assay (ph= 7); (ii) free so 2 could scavenge hydrogen peroxides produced via the fenton reaction from catechols; and (iii) polyphenols could prevent the prooxidant action of peroxomonosulfate radicals resulting from fe(iii)-initiated bisulfite oxidation (danilewicz, 2007; danilewicz et al., 2008). the synergistic effect between sulfites and polyphenols support the facts that so 2 and catechols are not individual antioxidants, and that the antioxidant activity of wine is a result of multiple antioxidants (danilewicz et al., 2008). fig. 4 antiradical activity as evaluated by the dpph radical decolorization assay of the model wine solutions (secondary y axe) and of the white and red wines (primary y axe) as a function of free so 2 concentration. fig. 3 reducing capacity of the samples with no sulfites (*obtained by extrapolation of data linear regression) and in the presence of 150 and 200 mg l-1 total sulphur dioxide content. radical scavenging activity as determined by the dpph• radical cation decolorization assay the antiradical activity of sulfur dioxide was evaluated by the dpph decolorization assay (brand-williams et al., 1995), which relies on a methanol-soluble stable radical in an amphiphilic environment. dose response curves were generated with different dilutions of hydroalcoholic solutions containing increasing amounts of total sulfur dioxide. fig. 4 shows the antioxidant activities of the model wine, expressed as teac dpph , plotted against the total sulfur dioxide content (25–200 mg l-1). the model wine containing sulfites had limited antiradical activity in the amphiphilic environment (manzocco et al., 1998). water-methaital. j. food sci., vol. 27 2015 511 nol mixtures are not ideal solutions; in such mixtures, small volumes of water in methanol result in the stabilization of methanol clusters as a result of hydrophobic and hydrogen bond interactions (takamuku et al., 2000; wakisaka et al., 1998; okasaki et al., 1984), resulting in a phase separation that could limit the contact between the water soluble antioxidant (sulfites) and the methanol-soluble radicals with negative effects on the estimation of the antioxidant capacity. fig. 4 shows the dose response curves of the white and red wines. in the white wine, increasing sulfur dioxide content from 77 to 200 mg l-1 caused a slight increase in the antiradical activity (+13%). in the red wine, the increase in sulfur dioxide contributed to increased but fluctuating antiradical activity values. a reduction in antioxidant activity due to sulfite addition (>150 mg l-1) was observed in the model wine (fig. 4). based on the results obtained from the dpph• assay, there were no negative matrix effects on the antiradical activity of sulfur dioxide. white and red wines, as opposed to the model wine of this study, contain phenolic compounds, which exhibit a surface activity that affects their radical scavenging efficiency in multiphasic systems (di mattia et al., 2009; 2010). a possible explanation for this result is that amphiphilic compounds like polyphenols could have acted as surfactants allowing sulfur dioxide to exert its antiradical activity with positive effects on the teac dpph value. in this case, the interfacial effect of polyphenols may justify the positive combined effects between the two antioxidants. another possible explanation is the synergistic effects between polyphenols and so 2 on reducing activity. the synergistic effects between natural antioxidants and sulfur dioxide could account for a non-linear response between the dpph assay results and the so 2 dose, which was evident in the red wine (fig. 4). in the case of the frap assay, a non-linear response was observed in the white wine, which contains less polyphenols (fig. 2), while in the case of the dpph• assay, a non-linear response was evident in the red wine, which contains more polyphenols (fig. 4). this result could be due to the different solvents used in the two assays: hydrophilic solvents in the frap assay and amphiphilic solvents in the dpph• assay. additionally, the different activities of phenolic antioxidants in the two assays is ph dependent (jovanovich et al., 1994). conclusions sulfur dioxide had antioxidant activity in the white, red, and model wines. in some cases, the contribution of sulfur dioxide to the overall antioxidant capacity of the wines was higher than that of naturally occurring antioxidants. moreover, in wines, sulfur dioxide had both antagonistic and synergistic effects with naturally occurring antioxidants on the total antioxidant activity of wines. the role of sulfur dioxide on the total antioxidant capacity of wines is of utmost importance to assess the technological and potentially healthpromoting properties of different products. future studies should evaluate the in vivo antioxidant effects of sulfur dioxide in wines. references abramovic h., kosmerl t., poklar ulrich n. and cigic b. 2015. contribution of so 2 to antioxidant potential of white wine. food chem. 174: 147. alonso a.m., dominguez c., guillen d.a. and barroso c.g. 2002. determination of antioxidant power of red and white wines by a new electrochemical method and its correlation with polyphenolic content. j. agric. food chem. 50: 3112. antonelli a. and arfelli, g. 1993. l’anidride solforosa. vignevini 20: 39. arnao m.b. 2000. some methodological problems in the determination of antioxidant activity using chromogen radicals: a practical case. trends food sci. tech. 11: 419. benzie i.f.f. and strain j.j.1996. the ferric reducing ability of plasma (frap) as a measure of “antioxidant power”: the frap assay. anal. biochem. 239: 70. brand-williams w., cuvelier m.e. and berset c. 1995. use of a free radical method to evaluate antioxidant activity. lebensm-wiss u-technol. 28: 25. branen a.l., davidson p.m., salminen s. and thorngate j.h. 2002. food additives. 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fax: +39 0116708549 e-mail address: giuseppe.zeppa@unito.it abstract ultrasound-assisted extraction (uae) and microwave-assisted extraction (mae) of caffeoylquinic acids and caffeine from coffee silverskin (cs) at two particle size were investigated using response surface methodology and compared to a conventional solvent extraction (cse). the impact of time and temperature on the extraction process was evaluated and extraction efficiency was optimised by measuring total phenolic content (tpc), radical scavenging capacity (rsc), caffeoylquinic acids (totcqas) and caffeine content. uae allowed to obtain extracts with values of tpc, rsc and totcqas higher or similar to cse; moreover uae produced the highest caffeine content (14.24 g kg-1 dw) with a significant reduction of extraction time. keywords: caffeine, coffee silverskin, microwave-assisted extraction (mae), polyphenolic compounds; ultrasound-assisted extraction (uae), response surface methodology (rsm) ital. j. food sci., vol 29, 2017 410 1. introduction coffee is the second largest traded commodity in the world after petroleum with a production of 8.8 million tons in 2015 (international coffee organization, 2015). since approximately 90% of the coffee cherry is discarded during the conversion of the cherry into coffee brew (del castillo et al., 2016), million tons of by-products are generated. coffee silverskin (cs) is a thin tegument of the outer layer of the two beans forming the green coffee seed obtained as a by-product of the roasting process. since cs represents about 4.2 % (w/w) of coffee beans (del castillo et al., 2016), hundreds of thousands tons of waste are produced generating a disposal cost for industry. in the last years cs has attracted great attention as an abundant source of bioactive compounds such as caffeoylquinic acids and caffeine (bresciani et al., 2014). in particular caffeoylquinic acids are phenolic compounds that belong to the family of chlorogenic acids; different studies have evaluated antifungal, antibacterial, anti-inflammatory, antioxidant, antiglycative, anti-carcinogenic and neuroprotective properties of these compounds (del castillo et al., 2016). considering caffeine, a moderate amount of this alkaloid increases energy availability, cognitive performance and neuromuscular coordination (glade, 2010). in order to optimise the extraction of bioactive compounds from cs, in the last years several extraction methods with different yield, complexity and cost, such as soxhlet extraction, solid-state fermentation (ssf), subcritical water and solid-liquid extraction were applied (murthy and naidu, 2010; machado et al., 2012; narita and inouye, 2012; ballesteros et al., 2014; costa et al., 2014). among the extraction techniques, ultrasound-assisted extraction (uae) and microwaveassisted extraction (mae) are considered efficient for extracting analytes reducing extraction time and energy consumption. regarding uae, the responsive of ultrasonic effect are the cavitation bubbles that grow during rarefaction phases and decrease in size during compression cycles; when the size of the bubbles reaches a critical point, they collapse during a compression cycle and release large amounts of energy that destroys the cell walls of the plant matrix producing the discharge of cell content into the medium (chemat et al., 2011). uae applied on spent coffee ground (scg), the solid waste obtained after coffee brewing and having similar composition to cs, has been reported to be an efficient method to improve the extraction of antioxidant compounds (severini et al., 2016). mae is an efficient method that has garnered increasing interest in various fields mainly due to its particular heating mechanism and its moderate capital cost (chan et al., 2011); microwaves can penetrate into certain materials and interact with the polar components of the matrix to generate heat. mae has been found to be an optimal extraction technique to recover caffeine from a plant matrix such as tea (wang et al., 2011). an important role in the extraction of caffeoylquinic acids and caffeine from a plant matrix is represented by the particle size of the sample (pinelo et al., 2007; astill et al., 2001). in literature no studies are reported on the use of ultrasounds and microwaves for the extraction of bioactive compounds from cs and on the influence of cs particle size on the extraction process. response surface methodology (rsm) is a multivariate statistic technique used to optimise extraction processes of bioactive compounds from plant matrices; in particular different studies reported the application of rsm to optimise the extraction of caffeine and chlorogenic acid from a plant matrix (d’archivio et al., 2016; bae et al., 2015). this study aimed to optimise uae and mae of the three most abundant caffeoylquinic acids and caffeine from cs using rsm; this statistical approach was used to evaluate the impact of time and temperature on total phenolic content, radical scavenging capacity, caffeoylquinic acids and caffeine content of the extracts. coffee silverskin was used at 80 ital. j. food sci., vol 29, 2017 411 and 250 µm particle size in order to test a possible effect of the grain size on the extraction process. finally extraction efficiency of uae and mae methods was compared to a conventional solvent extraction (cse). 2.materials and methods 2.1. plant material and chemical reagents a cs blend produced by roasting arabica (coffea arabica) and robusta (coffea canephora) coffee beans was provided by torrefazione della piazza (sant’antonino di susa, turin, italy). caffeine, 3-o-caffeoylquinic acid, 4-o-caffeoylquinic acid, 5-o-caffeoylquinic acid, ethanol, methanol, formic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), 2,2-diphenyl-1-picrylhydrazyl (dpph), gallic acid, folin-ciocalteau reagent and sodium carbonate were purchased from sigma-aldrich (milano, italy). ultrapure water was produced using a milli-q system (millipore, milan, italy). 2.2. sample preparation cs was ground to obtain a powder with 80and 250-µm particle size using an ultracentrifugal mill retsch zm 200 (retsch gmbh, haan, germany) and stored in vacuum bags at +4ºc until use. the moisture content of cs (6.9±0.3%) was determined using an electronic moisture balance (eurotherm, gibertini elettronica, milan, italy) with 5 g of sample in order to express the results on dry weight basis (dw). 2.3. extraction procedure according to the optimization study performed on cs by ballesteros et al. (2014), 60% (v/v) ethanol and solvent/solid ratio of 35 ml g-1 were used as fixed parameters for uae, mae and cse. after the extractions, extracts were cooled in an ice bath to stop the extraction process and centrifuged at 16,800 g for 10 min; supernatants were then filtered (0.45 µm) and immediately analysed. 2.3.1. ultrasound-assisted extraction (uae) uae was carried out using an ultrasonic bath (sonica® 3300ep s3; soltec, milan, italy) at a 40 khz frequency with 300 w of power. the flasks containing 1 g of cs and 35 ml of 60% (v/v) ethanol were immersed into the ultrasonic bath, where the water level was fixed at 2 cm above the liquid surface in the flask. for each experimental condition of the central composite design (ccd) reported in table 1, water bath temperature was set and maintained constant (± 1 ºc) using an external chiller. 2.3.2. microwave-assisted extraction (mae) mae was carried out using a start d microwave digestion system (milestone, bergamo, italy) with an sk rotor equipped with digestion vessels composed of high-purity ptfe. cs (1 g) and 35 ml of 60% (v/v) ethanol were put into the digestion vessels and a power of 280 w was then applied. for each experimental condition of ccd (table 1), temperature was measured using the internal temperature probe of the apparatus. ital. j. food sci., vol 29, 2017 412 table 1. ccd experimental matrix. run extraction time (coded value) (x1) extraction temperature (coded value) (x2) extraction time (real value) (x1, min) extraction temperature (real value) (x2, °c) 1 -1 -1 19 37 2 1 -1 41 37 3 1 1 41 73 4 -1 1 19 73 5 -1.4141 0 15 55 6 1.4141 0 45 55 7 0 -1.4141 30 30 8 0 1.4141 30 80 9-13 0 0 30 55 2.3.3. conventional solvent extraction (cse) cse was carried out mixing 1 g of cs and 35 ml of 60% (v/v) ethanol into a 40 ml amber vial inserted into a digital pulse mixer (glas-col, u.s.a.); stirring was carried out at 200 rpm, 70% duty cycle and 70 pulses per minute. 2.4. total phenolic content (tpc) total phenolic content (tpc) was measured following folin-ciocalteu's method (singleton and rossi, 1965) with modifications as described by zhou and yu (2006). briefly, 0.05 ml of phenolic extract was mixed with 0.250 ml of folin-ciocalteu's reagent and 3 ml of ultrapure water. the mixture was incubated at room temperature for 3 minutes; then 0.75 ml of 20% (w/v) sodium carbonate was added and the obtained mixture was incubated in the dark at room temperature for 2 h. a mixture of the solvent and reagents was used as blank. the specific absorbance at 765 nm was measured using a uv-visible spectrophotometer (uv-1800 pharmaspec, shimadzu, milan, italy). gallic acid was used to construct the calibration curve (linearity range 0-500 mg/l, r2=0.998). tpc was expressed as g gallic acid equivalents (gae) kg-1 of cs on dry weight basis (dw). 2.5. radical scavenging capacity (rsc) radical scavenging capacity (rsc) of the extracts was determined according to gadow et al. (1997). phenolic extract (75 μl) was mixed with 3 ml of 6.1×10-5 m dpph• solution in methanol and incubated for 1 h at room temperature in the dark (sharma and bhat, 2009). discolouration of the purple dpph• solution was measured at 515 nm. methanol was used as a control, and a methanol solution of dpph was used as a blank. the inhibition percentage (ip) of the dpph• by the phenolic extract was calculated using the following equation: 2.6. hplc–pda analysis the three caffeoylquinic acids considered (3-o-caffeoylquinic acid, 3-cqa; 4-ocaffeoylquinic acid, 4-cqa; 5-o-caffeoylquinic acid, 5-cqa) and caffeine were quantified using an hplc-pda thermo-finnigan spectra system (thermo-finnigan, waltham, usa) equipped with a finnigan surveyor pda plus detector. chromquest software (version ital. j. food sci., vol 29, 2017 413 5.0) was used for instrument control and data processing. compounds were separated using a kinetex phenyl-hexyl c18 column 5 µm, 150 × 4.6 mm (phenomenex, castel maggiore, italy). flow rate was set at 1.0 ml/min, temperature at 35°c and injection volume at 10 μl. a gradient elution with 0.1% formic acid (a) and methanol (b) as the solvents was applied as follows: 0.0–3.0 min, 10–30% of b; 3.0– 8.0 min, 30–35% of b; 8.0– 11.0 min, 35–40% of b; 11.0–30.0 min, 40–80% of b; 30.0–32.0 min, 80-10% of b. pda spectra were recorded using a full scan modality over the wavelength (λ) range 200 to 400 nm, and data were quantified using the external standard method with six-point calibration curves. in particular, the caffeoylquinic acids were quantified at 325 nm while caffeine at 273 nm with the respective standards (r2 = 0.9987, lod = 0.20 µg/ml, loq = 0.62 µg/ml for 3-o-caffeoylquinic acid, r2 = 0.9893, lod = 0.25 µg/ml, loq = 0.66 µg/ml for 4-o-caffeoylquinic acid, r2 = 0.9993 for 5-o-caffeoylquinic acid, lod = 0.16 µg/ml, loq = 0.50 µg/ml and r2 = 0.9837, lod = 0.014 µg/ml, loq = 0.046 µg/ml for caffeine). data were expressed as g kg-1 of cs on dw. 2.7. experimental design and statistical analysis a two factorial 22 central composite design (ccd) was employed to evaluate the effects of time (x1) and temperature (x2) on tpc, rsc, caffeine and totcqas (as the sum of 3-cqa, 4-cqa and 5-cqa) values of the extracts obtained using uae, mae and cse methods. extraction experiments were performed in triplicate for each experimental condition of the central composite design (table 1). as reported in table 1, variables were codified such that their values ranged between ±1.414 and the central point was repeated five times. the values of the independent variables were coded using following equation: xi = (xi xm)/ ∆x where xi is the coded value of an independent variable, xi is the real value of an independent variable, xm is the mean of the real values of an independent variable at the central point, and ∆x is the step change value. according to pavlić et al. (2016), response surface regressions were used to analyse tpc, rsc, totcqas and caffeine of obtained extracts and were fitted to the following secondorder polynomial model: y = b0 + b1x1 + b2x2 + b11x12 + b22x22 + b12x1x2 where y are the predicted responses (tpc, rsc, totcqas and caffeine), x1 and x2 correspond to independent variables time and temperature, b0 is the costant coefficient, b1 and b2 represent the linear coefficients, b11 and b22 the quadratic effects and b12 the crossproduct coefficient. the 24 second-degree polynomial equations, describing the four predicted responses obtained for each of the extraction method applied on the two cs particle sizes, were calculated using statistica software (version 7.0, statsoft inc., tulsa, ok, usa). also the respective surface plots were developed using statistica software. the values of tpc, rsc, totcqas and caffeine registered in the 13 experiments of ccd and observed at optimal conditions were compared among the extraction methods by analysis of variance (anova) in order to evaluate the extraction efficiency of uae and mae compared to cse; the means of the triplicates were separated at a 95% confidence interval using duncan’s test. ital. j. food sci., vol 29, 2017 414 3. results and discussions 3.1. model fitting second order polynomial model is the empirical model most commonly used for optimization methodology. least-squares regression analysis of variables was used to determine the corresponding coefficients within the quadratic models and their ability to predict the responses (table 2). the quality of the generated models was evaluated by analysis of variance (anova) and r square of the models; as shown in table 2, anova results showed that all the models had very low p values (≤ 0.0001). in addition, high r square values suggest that the proposed models were generally adequate to explain most of the variability (table 2). 3.2. effects of the process variables on total phenolic content (tpc) experimental data of tpc obtained using uae, cse and mae on cs at 80and 250 µm particle size (p.s.) are reported in table 3. tpc value ranged from 5.23 gae kg-1 dw (mae, 30 min, 80ºc, 250 µm p.s.) to 10.58 gae kg-1 dw (cse, 30 min, 80ºc, 80 µm p.s.). as shown in the anova test reported in table 2, tpc value was affected by the linear and quadratic terms of time for uae applied on cs at 80and 250 µm and for mae applied on cs at 80 µm; the linear and quadratic terms of temperature affected tpc value for all the extraction methods applied on cs at 80 µm and for cse and mae applied on cs at 250 µm. according to ballesteros et al. (2014), 30 min may be considered an enough time to be used in the three extraction processes to obtain a high phenolic content. temperature exerted a higher effect on tpc than extraction time, especially for cse and mae; an increase in temperature favors the extraction of phenolics by enhancing the diffusion coefficient of solvent, solubility of solutes, diffusion rate of analytes, and reducing solvent viscosity and surface tension (ju and howard, 2003). response surface plots representing the effect of time and temperature on total phenolic content (tpc) of the coffee silverskin at 80 µm particle size extracts obtained using conventional solvent extraction (cse), ultrasound-assisted extraction (uae) and microwave-assisted extraction (mae) have been reported in figs. 1, 2 and 3 respectively. since no significant differences were observed in the extraction efficiency using the two cs particle sizes, data related to 250 µm particle size have been reported in the text without graphical representations. considering mae, above 51.5ºc temperature exerted a negative effect on tpc value (fig. 3). other studies have shown that chlorogenic acids, phenolic compounds to whom the selected caffeoylquinic acids belong, decreased at temperatures higher than 50ºc using mae (upadhyay et al., 2012). phenolic compounds with several hydroxyl groups in their aromatic rings such as caffeoylquinic acids are unstable in a solvent and can easily degrade under microwave radiation combined with high temperatures (ince et al., 2014). finally the increase of tpc value at high temperatures using cse and uae could confirm this hypothesis, since temperature without microwaves did not produce a degradation of the phenolic compounds (figs. 1 and 2). ital. j. food sci., vol 29, 2017 415 table 2. regression coefficients of the predicted quadratic polynomial models of tpc, rsc, totcqas and caffeine obtained using cse, uae, and mae extraction methods on coffee silverskin at 80and 250 µm particle sizes. coffee silverskin (80-µm particle size) coffee silverskin (250-µm particle size) csea tpc rsc totcqas caffeine csea tpc rsc totcqas caffeine b0 7.9106 *** 33.9350*** 180.0969*** 35.97ns b0 5.6448 *** 22.0388*** 156.2306*** 192.9056** b1, time 0.0239 ns 0.0358* 2.0294*** 28.13*** b1, time 0.0441 * 0.1896*** 3.3476*** 21.4610*** b2, temperature -0.0064 ns 0.1387*** 0.5358ns 21.63*** b2, temperature 0.0597 *** 0.4139*** 1.2105*** 19.2199*** b11 -0.0003 ns -0.0001ns -0.0039ns -0.24*** b11 -0.0006 ns -0.0015** -0.0427*** -0.1459*** b22 0.0003 *** -0.0009** 0.0069** -0.11*** b22 -0.0003 ** -0.0027*** -0.0080*** -0.1030*** b12 0.0002 ns -0.0004*** -0.0230*** -0.19*** b12 0.0002 ns -0.0014*** -0.0036ns -0.1533*** r2 0.96 0.96 0.91 0.92 r2 0.92 0.97 0.90 0.94 p value ˂ 0.0001 ˂ 0.0001 ˂ 0.0001 ˂ 0.0001 p value ˂ 0.0001 ˂ 0.0001 ˂ 0.0001 ˂ 0.0001 uaeb tpc rsc totcqas caffeine uaeb tpc rsc totcqas caffeine b0 4.1174 *** -5.0522* -30.0050ns 573.9909*** b0 4.6483 ** 16.8912*** -62.2672* 691.4779*** b1, time 0.1676 *** 0.7384*** 4.9254*** -2.6802ns b1, time 0.2595 *** 0.8034*** 7.8861*** 7.7372** b2, temperature 0.0714 *** 1.0476*** 6.0426*** 27.6387*** b2, temperature -0.0321 ns 0.2369*** 5.9860*** 16.4356*** b11 -0.0018 *** -0.0022ns -0.0201ns 0.0956* b11 -0.0029 *** -0.0075*** -0.0648*** -0.0045ns b22 -0.0002 * -0.0057*** -0.0277*** -0.2021*** b22 0.0009 ** -0.0005ns -0.0279*** -0.0866*** b12 -0.0011 *** -0.0098*** -0.0703*** -0.0704* b12 -0.0011 * -0.0042*** -0.0679*** -0.1212*** r2 0.92 0.94 0.86 0.94 r2 0.74 0.90 0.86 0.89 p value ˂ 0.0001 ˂ 0.0001 ˂ 0.0001 ˂ 0.0001 p value ˂ 0.0001 ˂ 0.0001 ˂ 0.0001 ˂ 0.0001 maec tpc rsc totcqas caffeine maec tpc rsc totcqas caffeine b0 1.9917 *** 26.2296*** 125.0379*** 108.9807ns b0 3.6921 *** 25.2296*** 110.1193*** 119.3925ns b1, time 0.1011 *** -0.0880** -1.0855ns 33.1830*** b1, time 0.0543 ns -0.0880** -0.1901ns 39.3521*** b2, temperature 0.1680 *** 0.2242*** 4.5193*** 18.0169*** b2, temperature 0.1100 *** 0.2242*** 4.6963*** 14.0377*** b11 -0.0015 *** 0.0011** -0.0009ns -0.3733*** b11 -0.0015 ** 0.0011** -0.0136ns -0.3500*** b22 -0.0016 *** -0.0024*** -0.0627*** -0.0791*** b22 -0.0015 *** -0.0024*** -0.0630*** -0.0254ns b12 -0.0001 ns 0.0005ns 0.0281* -0.1912*** b12 0.0008 * 0.0005ns 0.0241* -0.2891*** r2 0.92 0.94 0.92 0.84 r2 0.86 0.94 0.93 0.89 p value ˂ 0.0001 ˂ 0.0001 ˂ 0.0001 ˂ 0.0001 p value ˂ 0.0001 ˂ 0.0001 ˂ 0.0001 ˂ 0.0001 a conventional solvent extraction; bultrasound-assisted extraction, cmicrowave-assisted extraction. p value, probability of f value for the model. * p < 0.05, ** p < 0.01, *** p < 0.001, ns= not significant. ital. j. food sci., vol 29, 2017 416 table 3. central composite design with the observed tpc, rsc, totcqas and caffeine values of coffee silverskin (80and 250 µm particle sizes) extracts obtained using cse, uae, and mae. coffe silverskin at 80-µm particle size run tpc (g gae kg -1 dw) rsc (μmol te g-1 dw) csea uaeb maec p csea uaeb maec p 1 8.73±0.02a 8.35±0.10b 7.51±0.02c *** 38.28±0.21a 32.05±0.72b 30.47±0.02c *** 2 8.94±0.06a 8.58±0.01b 7.55±0.02c *** 38.63±0.04a 38.41±0.12b 30.01±0.04c *** 3 10.15±0.06a 8.76±0.02b 7.14±0.04c *** 39.40±0.04a 38.47±0.16b 29.45±0.04c *** 4 9.77±0.04a 9.35±0.05b 7.17±0.03c *** 39.38±0.02b 39.47±0.06a 29.55±0.04c *** 5 8.98±0.08a 8.60±0.02b 7.55±0.10c *** 39.06±0.08a 38.67±0.27b 30.57±0.09c *** 6 9.57±0.14a 8.67±0.02b 7.89±0.01c *** 39.16±0.08a 39.02±0.33a 31.09±0.10b *** 7 8.53±0.06a 8.46±0.04a 7.15±0.04b *** 37.80±0.11a 32.27±0.28b 29.62±0.06c *** 8 10.58±0.10a 9.41±0.06b 7.01±0.07c *** 39.37±0.04a 39.22±0.38a 28.62±0.08b *** 9 9.31±0.07a 8.92±0.05b 7.90±0.08c *** 39.30±0.05a 39.16±0.22a 30.75±0.03b *** 10 9.33±0.13a 9.19±0.08a 8.01±0.06b *** 39.10±0.02b 39.29±0.12a 30.63±0.07c *** 11 9.43±0.02a 9.03±0.11b 7.90±0.04c *** 39.17±0.10b 39.34±0.05a 30.44±0.06c *** 12 9.26±0.09a 8.93±0.03b 8.16±0.11c *** 39.16±0.07a 38.97±0.35a 30.44±0.02b *** 13 9.38±0.06a 9.13±0.03b 8.17±0.06c *** 39.12±0.09b 39.41±0.10a 30.44±0.16c *** run totcqas (g kg-1 dw) caffeine (g kg-1 dw) csea uaeb maec p csea uaeb maec p 1 2.34±0.00a 2.04±0.01b 2.00±0.07b *** 9.81.00±0.03c 12.86±0.05a 9.93±0.04b *** 2 2.51±0.01a 2.20±0.09b 2.00±0.03c *** 11.31±0.18b 12.89±0.12a 11.01±0.10c *** 3 2.63±0.03a 2.18±0.06b 1.71±0.01c *** 12.03±0.02b 13.49±0.06a 11.93±0.06b *** 4 2.63±0.02a 2.54±0.02b 1.50±0.03c *** 11.91±0.04c 13.98±0.03a 12.29±0.07b *** 5 2.38±0.01a 2.21±0.02b 1.89±0.06c *** 11.10±0.09c 13.91±0.17a 11.47±0.06b *** 6 2.58±0.03a 2.26±0.01b 1.99±0.01c *** 11.99±0.09b 13.75±0.15a 11.12±0.08c *** 7 2.37±0.02a 1.84±0.01c 2.04±0.02b *** 10.63±0.07c 11.31±0.05a 11.01±0.04b *** 8 2.68±0.02a 2.38±0.07b 1.06±0.03c *** 12.12±0.15b 13.39±0.13a 12.27±0.08b *** 9 2.53±0.01a 2.31±0.02b 1.98±0.07c *** 11.81±0.17b 13.70±0.10a 11.70±0.35b *** 10 2.49±0.01a 2.31±0.04b 1.96±0.02c *** 11.86±0.21b 13.74±0.05a 11.94±0.33b *** 11 2.53±0.04a 2.37±0.02b 1.87±0.03c *** 12.05±0.19b 13.75±0.09a 12.08±0.08b *** 12 2.49±0.05a 2.32±0.06b 2.02±0.07c *** 12.08±0.38b 13.73±0.13a 12.28±0.11b *** 13 2.46±0.03a 2.28±0.07b 2.03±0.04c *** 12.17±0.28b 13.69±0.07a 12.23±0.22b *** coffe silverskin at 250-µm particle size run tpc (g gae kg -1 dw) rsc (μmol te g-1 dw) csea uaeb maec p csea uaeb maec p 1 8.31±0.05a 8.14 ±0.02a 6.67±0.25b *** 36.13±0.09a 34.42±0.31b 29.47±0.02c *** 2 8.75±0.02a 8.59 ±0.09b 6.61±0.05c *** 37.13±0.17b 38.41±0.25a 29.01±0.04c *** 3 9.87±0.04a 8.87 ±0.01b 5.88±0.29c *** 39.00±0.09b 39.38±0.07a 28.45±0.04c *** 4 9.25±0.18a 9.28 ±0.01a 5.35±0.16b *** 39.05±0.14a 38.56±0.67a 28.55±0.04b *** 5 8.59±0.02a 7.56 ±0.13b 6.52±0.35c *** 38.07±0.31a 35.18±0.85b 29.57±0.09c *** 6 9.24±0.12a 9.00 ±0.08a 6.63±0.18b *** 38.62±0.07b 39.20±0.07a 30.09±0.10c *** 7 8.05±0.21a 8.49 ±0.34a 6.73±0.14b *** 34.74±0.19b 37.68±0.39a 28.62±0.06c *** 8 9.63±0.04b 10.46±0.07a 5.23±0.13c *** 39.31±0.06a 39.40±0.50a 27.62±0.08b *** 9 9.29±0.05a 8.88±0.51a 6.95±0.16b *** 38.84±0.13a 38.53±0.19b 29.75±0.03c *** 10 9.25±0.09a 9.35±0.05a 6.92±0.21b *** 38.83±0.19b 39.13±0.04a 29.63±0.07c *** 11 8.98±0.16a 8.53±0.08a 6.80±0.37b *** 38.65±0.21a 38.81±0.31a 29.44±0.06b *** 12 9.06±0.20a 8.73±0.30a 6.53±0.25b *** 38.66±0.20a 38.61±0.52a 29.44±0.02b *** 13 9.08±0.09a 8.74±0.27a 6.97±0.10b *** 38.81±0.19a 38.80±0.83a 29.44±0.16b *** run totcqas (g kg-1 dw) caffeine (g kg-1 dw) csea uaeb maec p csea uaeb maec p 1 2.38±0.01a 2.09±0.01a 2.03±0.05b *** 9.88±0.07b 12.64±0.04a 9.93±0.03b *** 2 2.52±0.02a 2.38±0.02b 1.99±0.05c *** 11.69±0.09c 13.41±0.07a 11.85±0.05b *** 3 2.58±0.02a 2.43±0.01a 1.60±0.15b *** 12.40±0.02b 13.80±0.08a 12.02±0.02c *** 4 2.47±0.03b 2.64±0.02a 1.47±0.08c *** 11.73±0.05c 13.93±0.03a 12.27±0.08b *** 5 2.35±0.01a 2.23±0.03b 1.91±0.07c *** 11.31±0.18b 13.48±0.10a 11.14±0.03b *** 6 2.52±0.004a 2.34±0.03b 2.04±0.01c *** 12.12±0.04b 13.51±0.02a 11.43±0.13c *** 7 2.42±0.02a 2.03±0.03b 2.03±0.03b *** 10.66±0.06c 11.92±0.06a 11.52±0.03b *** ital. j. food sci., vol 29, 2017 417 8 2.54±0.02a 2.48±0.04a 1.19±0.05b *** 12.14±0.07c 14.01±0.03a 12.31±0.08b *** 9 2.57±0.02a 2.55±0.03a 2.02±0.04b *** 11.97±0.09b 13.67±0.15a 11.99±0.07b *** 10 2.55±0.03a 2.53±0.03a 2.02±0.01b *** 11.94±0.03c 13.54±0.12a 12.10±0.05b *** 11 2.54±0.04a 2.53±0.05a 1.94±0.07b *** 11.99±0.03b 13.54±0.15a 11.94±0.17b *** 12 2.55±0.02a 2.43±0.06a 1.93±0.09b *** 12.06±0.13b 13.61±0.09a 12.02±0.24b *** 13 2.52±0.02a 2.39±0.05b 2.06±0.04c *** 11.93±0.12b 13.70±0.06a 12.11±0.09b *** data are expressed as the means±standard deviation (n = 3). values in each row having different capitals letters indicate significant differences at p < 0.05 (duncan’s test). * p < 0.05, ** p < 0.01, *** p < 0.001, ns= not significant dw: dry weight; totcqas: total caffeoylquinic acids. a conventional solvent extraction; b ultrasound-assisted extraction, c microwave-assisted extraction. figure 1. response surface plots representing the effect of time and temperature on total phenolic content (tpc), radical scavenging capacity (rsc), total caffeoylquinic acids (totcqas) and caffeine content of the coffee silverskin (80 µm particle size) extracts obtained using conventional solvent extraction (cse). figure 2. response surface plots representing the effect of time and temperature on total phenolic content (tpc), radical scavenging capacity (rsc), total caffeoylquinic acids (totcqas) and caffeine content of the coffee silverskin (80 µm particle size) extracts obtained using ultrasound-assisted extraction (uae). ital. j. food sci., vol 29, 2017 418 figure 3. response surface plots representing the effect of time and temperature on total phenolic content (tpc), radical scavenging capacity (rsc), total caffeoylquinic acids (totcqas) and caffeine content of the coffee silverskin (80 µm particle size) extracts obtained using microwave-assisted extraction (mae). 3.3. effects of the process variables on the dpph radical scavenging capacity (rsc) as shown in table 3, rsc values ranged from 27.62 μmol te/g dw (mae, 30 min, 80ºc, 250 µm p.s.) to 39.47 μmol te/g dw (uae, 19 min, 73ºc, 80 µm p.s.). table 2 shows how rsc value was affected by linear and quadratic terms of temperature for all the extraction methods applied on cs at 80 µm and for cse and mae applied on cs at 250 µm particle size. moreover rsc value was affected by both linear and quadratic terms of extraction time for mae applied on cs at 80 µm. as seen for tpc, also rsc was generally more affected by temperature than time, especially for cse and mae methods. considering the surface plots reported in figs. 1, 2 and 3, a positive effect of extraction time was observed for uae while a temperature increase caused a rapid increase of rsc value for all the extraction methods, except mae applied at temperatures higher than 50 °c, as seen for tpc. according to narita and inouye (2012), a correlation between tpc and rsc trends has been observed meaning the high contribute of phenolic compounds in antioxidant activity of cs extracts. 3.4. effects of the process variables on total caffeoylquinic acids (totcqas) table 3 shows how totcqas ranged from 1.06 g kg-1 dw (mae, 30 min, 80ºc, 80 µm p.s.) to 2.68 g kg-1 (cse, 30 min, 80ºc, 80 µm p.s.). considering the anova test on cs extracts at 80and 250 µm particle sizes (table 2), totcqas value was affected by linear terms of extraction time and temperature for uae and by linear and quadratic terms of temperature for mae. as seen for tpc and rsc, totcqas was more affected by temperature than time for all the extraction methods. also surface plots of totcqas were generally comparable, for each extraction method, to that observed for tpc and rsc (figs. 1, 2 and 3); these similar trends could confirm that 3-cqa, 4-cqa and 5-cqa are the main compounds in phenolic content of cs and the main responsible of cs antioxidant activity, as reported by bresciani et al. (2014). according to upadhyay et al. (2012), caffeoylquinic acids content decreased at temperatures above 50ºc using mae, confirming a possible degradation of the selected compounds caused by the combination of microwaves and high temperatures (fig. 3). ital. j. food sci., vol 29, 2017 419 3.5. effects of the process variables on caffeine content as reported in table 3, caffeine content ranged from 9.81 g kg-1 dw (cse, 19 min, 37ºc, 80 µm p.s.) to 14.01 g kg-1 dw (uae, 30 min, 80ºc, 250 µm p.s). table 2 shows how the caffeine content was affected by linear and quadratic terms of time and temperature for cse and mae applied on cs at the two particle sizes while uae was especially affected by extraction temperature. as seen for tpc, rsc and totcqas, also in this case temperature was the variable that produced the most relevant effects on the extraction processes. in contrast with totcqas values, caffeine content obtained by mae was rapidly increased also at high temperatures (fig. 3); caffeine stability in these conditions has been reported by liu et al. (2012) that optimised microwave-assisted extraction of caffeine from coffee at 120ºc. 3.6. optimization and verification of the models optimization of cse, uae and mae methods was carried out to obtain extracts with the highest predicted tpc, rsc, totcqas and caffeine values (table 4). following the model proposed by zielinski et al. (2015), the errors in relation to the predicted models of the optimization variables were verified. an external validation was performed using the optimal conditions of time and temperature obtained by the predict models; all the observed values were within the predicted interval to a level of 95 % (table 4), meaning that experimental data matched well the predicted values and interval ranges generated by rsm. comparing the observed values at optimal conditions with the ones reported in literature, tpc values of 10.01 g gae kg-1 dw (cse, 45 min, 80ºc, 80 µm p.s.) and 9.91 g gae kg-1 dw (uae, 29.5 min, 80ºc, 250 µm p.s.) were higher than the one obtained during cs extraction with water at 80ºc during 60 min (7.00 g gae kg-1 dw) performed by narita and inouye (2012); moreover uae allowed to split in half the extraction time. observed totcqas values of 2.61 g kg-1 dw (cse, 24.5 min, 80ºc, 80 µm p.s.) and 2.57 g kg-1 dw (uae, 15 min, 80ºc, 80 µm p.s.) can be compared with the sum of 3cqa, 4-cqa and 5-cqa (4.31 g kg-1 dw) obtained by bresciani et al. (2014) using two times a sonic bath for 30 min and then a dubnoff bath for 1 h at 70°c; cse and uae optimized in our study allowed to obtain a slightly lower totcqas value balanced by a significant time reduction (86.4 % for cse and 91.7 % for uae). finally the highest caffeine content of 14.24 g kg-1 dw (uae, 15 min, 66ºc, 80 µm p.s.) obtained in our study was higher than the one reported by narita and inouye (2012) using subcritical water at 210ºc (4.1 g kg-1 dw). considering uae, highest predicted and observed values were generally obtained at high temperatures according to other studies that reported a maximum uae extraction of several bioactive compounds at 80ºc (tomšik et al., 2016; zhang et al., 2011; zhu et al., 2017). 3.7. comparison among the combinations of extraction method and cs particle size the observed values of tpc, rsc, totcqas and caffeine obtained at optimal time and temperature conditions by each combination of extraction method and cs particle size were compared in order to evaluate the most efficient combination for each response (fig. 4). ital. j. food sci., vol 29, 2017 420 table 4. predicted and observed values of tpc, rsc, totcqas and caffeine at optimal time and temperature conditions obtained using cse, uae, and mae extraction methods on cs at 80and 250-µm particle sizes. cs 80 µm cse response variables optimal conditions observed value predicted value -95 % pred +95 % pred t (min) t (°c) tpc (g gae kg-1 dw) 45 80 10.01 ± 0.21 10.51 9.97 10.75 rsc (μmol te g-1 dw) 45 67 39.01 ± 0.46 39.41 38.96 39.86 totcqas (g kg-1 dw) 24.5 80 2.61 ± 0.05 2.69 2.61 2.78 caffeine (g kg-1 dw) 31.5 69 12.11 ± 0.12 12.26 11.80 12.72 cs 250 µm cse response variables optimal conditions observed value predicted value -95 % pred +95 % pred t (min) t (°c) tpc (g gae kg-1 dw) 45 80 9.83 ± 0.15 9.99 9.59 10.39 rsc (μmol te g-1 dw) 31 68.5 39.01 ± 0.50 39.19 38.66 39.72 totcqas (g kg-1 dw) 36.5 67.5 2.54 ± 0.05 2.58 2.53 2.63 caffeine (g kg-1 dw) 40 63.5 12.13 ± 0.11 12.34 11.94 12.73 cs 80 µm uae response variables optimal conditions observed value predicted value -95 % pred +95 % pred t (min) t (°c) tpc (g gae kg-1 dw) 22 80 9.25 ± 0.16 9.43 9.25 9.61 rsc (μmol te g-1 dw) 15 79 40.81 ± 0.76 41.1 39.39 42.81 totcqas (g kg-1 dw) 15 80 2.57 ± 0.05 2.61 2.42 2.80 caffeine (g kg-1 dw) 15 66 14.24 ± 0.15 14.29 13.86 14.72 cs 250 µm uae response variables optimal conditions observed value predicted value -95 % pred +95 % pred t (min) t (°c) tpc (g gae kg-1 dw) 29.5 80 9.91 ± 0.24 10.38 9.54 11.22 rsc (μmol te g-1 dw) 31 80 39.21 ± 1.80 39.93 38.73 41.13 totcqas (g kg-1 dw) 19 80 2.54 ± 0.05 2.61 2.44 2.79 caffeine (g kg-1 dw) 15 80 14.02 ± 0.21 14.22 13.69 14.74 cs 80 µm mae response variables optimal conditions observed value predicted value -95 % pred +95 % pred t (min) t (°c) tpc (g gae kg-1 dw) 32 51.5 7.34 ± 0.23 7.78 7.02 8.55 rsc (μmol te g-1 dw) 45 51.5 26.44 ± 0.72 26.61 26.20 27.01 totcqas (g kg-1 dw) 45 46 1.98 ± 0.06 2.02 1.82 2.22 caffeine (g kg-1 dw) 24 80 11.58 ± 0.21 11.96 11.28 12.64 cs 250 µm mae response variables optimal conditions observed value predicted value -95 % pred +95 % pred t (min) t (°c) tpc (g gae kg-1 dw) 30 44.5 6.76 ± 0.20 6.85 6.35 7.35 rsc (μmol te g-1 dw) 45 51.5 28.44 ± 0.26 28.68 28.28 29.09 totcqas (g kg-1 dw) 31.5 43.5 2.08 ± 0.02 2.08 1.93 2.24 caffeine (g kg-1 dw) 23 80 7.18 ± 0.02 7.86 7.16 8.57 the data are expressed as the means±standard deviation (n = 3). a conventional solvent extraction; b ultrasound-assisted extraction, c microwave-assisted extraction. ital. j. food sci., vol 29, 2017 421 generally lower values were obtained using mae than cse or uae, regardless of cs particle size. other studies showed a better capacity of uae compared to mae to extract chlorogenic acids (routray and orsat, 2014); as shown in fig. 4, uae and cse exhibited comparable maximum observed values of tpc and totcqas, while rsc was higher using uae than cse, regardless of the particle size; considering tpc, rsc and totcqas values, uae allowed to obtain higher or similar values to cse with a significant reduction of extraction time, especially at 80 µm particle size (table 4). according to choung et al. (2014), caffeine content was significantly (p ≤ 0.05) higher using uae than cse or mae (fig. 4). generally maximum observed values were significantly affected by extraction method while no significant differences were observed between the two particle sizes of cs, with the only exception of caffeine content for mae (fig. 4). nevertheless a lower cs particle size allowed to reduce extraction time up to 32.9% for totcqas obtained with cse and up to 51.6% for rsc obtained with uae (table 4). figure 4. comparison of the observed values (mean±standard deviation) of total phenolic content (tpc), radical scavenging capacity (rsc), total caffeoylquinic acids (totcqas) and caffeine content obtained at optimal time and temperature conditions by each combination of extraction method and cs particle size. the standard deviation bars with different letters are significant different (p ≤ 0.05). 4. conclusions cse, uae and mae of phenolic compounds and caffeine from coffee silverskin at 80and 250 µm particle sizes were optimised using rsm approach. all the quadratic polynomial models were able to predict and optimise cse, uae and mae processes. anova showed that temperature was the process variable that most affected the extraction processes. in particular a positive correlation was observed between an increase of temperature and tpc, rsc, totcqas and caffeine values for cse and uae; using mae above 50°c, a negative effect on tpc, rsc and totcqas was observed meaning a possible degradation of the selected caffeoylquinic acids due to a combination of high temperatures ital. j. food sci., vol 29, 2017 422 and microwaves. comparing extraction methods, lowest values of tpc, rsc, totcqas and caffeine were obtained using mae; uae allowed to obtain extracts with values of tpc, rsc and totcqas higher or similar to cse with a significant reduction of extraction time, especially at 80 µm particle size. moreover, uae produced a significant higher content of caffeine compared to cse in the 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technology in bydgoszcz, prof. s. kaliskiego 7, 85796 bydgoszcz, poland 3department of agronomy, west pomeranian university of technology in szczecin, papieża pawła vi 3, 71434 szczecin, poland 4institute of plant production, university of agriculture in cracow, mickiewicza 21, 31120 krakow, poland 5department of human nutrition, university of agriculture in cracow, balicka 122, 30149 krakow, poland 6department of plant physiology and biochemistry, west pomeranian university of technology in szczecin, słowackiego 17, 71434 szczecin, poland *corresponding author. tel.: +48914996292; e-mail address: anna.jaroszewska@zut.edu.pl abstract the aim was to assess the entire plant of three species of amaranthus l. with regard to their chemical content, determination of the most valuable genotype, and the optimum harvest time in relation to the species' nutritional value. the amaranth harvested later was found to contain less protein and ash, but clearly more fibre, and nitrogen-free extracts. the highest content of mineral compounds was observed at the beginning of blossoming. the genotypes were characterized by very high levels of radical scavenging activity, which was dependent on harvest timing. betanine and amaranthine concentration decreased with the delay in harvesting. keywords: amaranth, antioxidant activity, betacyanins, chemical composition, genotype, harvest time ital. j. food sci., vol. 29, 2017 729 1. introduction the effects of globalization and the observed unification of food production around the world have limited the number of cultivated plant species. to counteract this trend, new plants previously not used on a massive scale, but with a proven nutritional value have been introduced in commercial agriculture. this group includes one of the oldest cultivated plants, belonging to the genus amaranthus. the value of amaranth depends mainly on the unique chemical composition of its seed (mota et al., 2016). the chemical composition of the foliage is also very interesting and presents significant nutritional value. the content of high quality protein in the leaves amounts to 19,4%-30% of dry matter (andini et al., 2013; ngugi et al., 2017). the entire amaranth plant contains biologically active components such as anthocyanins, flavonoids, and phenyl acids, which are responsible for the plants’ pharmacological properties (li et al., 2015). apart from the aforementioned nutritional value of the whole amaranth plant, there are other reasons justifying efforts to increase its cultivation volume. two such economic factors are the relatively undemanding cultivation and the high yield. in order for the plants to produce valuable flowers and seeds, a relatively low crop density is recommended (from 10 to 30 plants per m2) (yarnia, 2010). in the case of forage crop cultivation the plants should produce delicate and thin shoots (o’brien and price, 2008). an increase in crop density to 140 plants per m2 results in better parameters for green foliage (required ratio of leaf mass to shoot mass). a lower crop density results in higher shoot mass and higher fibre content. in the case of high crop density the produced shoots are narrower, and tend to drop older leaves that receive less light. the choice of the best time for forage crop harvesting is related to the size of the harvest and its nutritional value, which could be determined in two ways: by choosing a specific day after sowing (abbasi et al., 2012) or by following the plant’s development phase (pospišil and pospišil, 2008). regardless of the harvest method, all the mentioned papers report that the most beneficial period for harvest in terms of the nutritional value and digestibility of the plant is the end of its vegetative stage, and if the size of the collected yield is concerned – full bloom stage. a delay in harvest quite negatively influences the quality of the collected mass. in this study, we attempted to assess whether the species of the genus amaranthus l. differentiates chemical composition of the whole plants, and whether the nutritional value whole plants of amaranthus l. is dependent on the harvest time. therefore, the aim of study was to assess the chemical composition of the whole plant of three species of the genus amaranthus l. and to determine the most valuable genotype, as well as the optimum harvest time with regard to the plants’ nutritional value. 2. materials and methods 2.1. study sites the experiment was conducted in 2014 at the mochełek experimental station near bydgoszcz, poland (53°12′24″n, 17°51′40″e). the experiment was conducted in a totally random system and was repeated three times. the soil in which the crops were grown is typical haplic luvisols, created from fluvioglacial sands on sandy loam, class iva. the soil ph was slightly acidic (ph 5.6 in kcl). the content of silt and clay in the arable layer stood at 15%. analysis of soil mineral content showed high levels of phosphorus (20.9 mg/100g), very low levels of magnesium (2.2 mg/100g), and low levels of potassium (8.0 mg/100g). sowing was carried out on 15th may with a spacing of 0.45 m, 5 kg per ha, at 1-2 cm depth ital. j. food sci., vol. 29, 2017 730 by spacing seeder. the area of the harvest plot was 10 m2. no mineral fertilization was used. the plant maintenance included mechanical weeding of the interrows. the fore crop for amaranth was spring barley. 2.2. plant material the first experimental factor (a) was genotype: (a1) amaranthus cruentus l. ‘rawa’ cultivar, (a2) amaranthus hypochondriacus l. ‘aztek’ cultivar, (a3) amaranthus caudatus l. ‘oscar blanco’, and ‘phule kartiki’ cultivar. the second factor (b) was harvest time – the number of days after sowing: (b1) the beginning of blossoming, (b2) full bloom, and (b3) full seed development. the choice of experimental factor (harvest time) was made based on observation of the developmental stage of the majority of plants in the plot field. the term of specific agrophenophases was established a priori and not using the calendar. on the day prior to harvest, random plant samples were collected for chemical analysis (20 samples of each cultivar). 2.3. climatic conditions the atmospheric conditions for the year of the study are presented in table 1. in comparison with the mean values of multi-year meteorological parameters (1996-2013) the humidity during sowing and germination periods was higher (the sum of precipitation was higher by 45%), but in the following months it was relatively lower. in addition, very high temperatures were registered over the entire vegetative phase. as a result, the values of seljaninov hydrothermal indicator in june, july, and august were lower than the mean, which confirms the occurrence of drought. despite this, based on long-term research on amaranth cultivation conducted at the same location, it could be assumed that the weather during the year 2014 was particularly beneficial for the growth and development of all the chosen amaranth species. table 1. temperature, precipitation and hydrothermal index during the experiment as compared with multiyear average [1996-2013]. years the meteorological parameter decade of the month month april may june july august 2014 the average air temperature (oc) i 7.3 10.4 17.6 21.1 21.8 ii 8.7 12.8 15.8 20.6 16.1 ii 13.7 16.3 14.5 22.8 14.1 average monthly temperature 9.9 13.3 16.0 21.5 17.2 precipitation (mm) i 17.6 32.1 10.2 14.4 18.9 ii 18.6 10.6 12.0 7.7 16.2 ii 4.5 23.0 22.7 33.3 22.2 monthly amounts of precipitation [mm] 40.7 65.7 44.9 55.4 57.3 hydrothermal index 1.37 1.59 0.94 0.83 1.07 19962013 the average air temperature (oc) 8.0 13.2 16.3 18.5 17.8 rainfall (mm) 28.0 60.9 53.5 88.8 67.0 hydrothermal index 1.17 1.49 1.09 1.55 1.21 ital. j. food sci., vol. 29, 2017 731 2.4. chemical analyses the chemical composition of samples were determined according to procedures of the association of official analytical chemists (aoac, 2012): dry matter was determined by drying at 105ºc to a constant weight, crude fat by soxhlet extraction with diethyl ether, crude ash by incineration in a muffle furnace at 580ºc for 8 h. crude protein (n × 6.25) by kjeldahl method using a büchi distillation unit b 324 (büchi labortechnik ag, switzerland). crude fibre was determined in an ankom 220 fibre analyzer (ankom technology, usa). nitrogen-free extract (nfes) was calculated as: nfes = 100 − (moisture + crude protein + crude fat + crude ash + crude fibre). the fibre components were determined using the detergent method according to van soest et al. (1991) performed with the ankom 220 fibre analyzer. determination of neutral detergent fibre (ndf) was conducted on an ash-free basis and included sodium dodecyl sulphate (merc 822050). determination of acid detergent fibre (adf) included hexadecyl-trimethyl-ammonium bromide (merc 102342), while acid detergent lignin (adl) was determined by hydrolysis of adf samples in 72% sulphuric acid. hemicellulose content was calculated as the difference between ndf and adf, while cellulose content as the difference between adf and adl. the material for analyses of the major dietary element concentrations was subjected to mineralization in concentrated h2so4 and hclo4 acids, whilst the material for analyses of the micro-compound concentrations was subjected to mineralization in a mixture of hno3 and hclo4. the concentration of phosphorus (p) were determined by colorimetric method, with ammonium molybdate, at wavelength 660 nm, using a specol 221 apparatus. an atomic absorption spectrometer apparatus (ice 3000 series, thermo fisher scientific) was used to determine potassium (k), sodium (na) and calcium (ca) by means of emulsion flame spectroscopy, whilst magnesium (mg), zinc (zn), iron (fe), manganese (mn) and copper (cu) by means of absorption flame spectroscopy. assessment of betacyanins was carried out using a spectrophotometer; uv-1800r. exactly 2g of ground plant material was placed in a mortar before homogenization with 10cc demineralized water to ease extraction. the ph of the resulting solution was adjusted to 5.4 (stintzing et al., 2004). samples were diluted in a 0.05 m phosphate buffer (ph 6.5) as described by stintzing et al. (2003) using the extinction coefficients of betanin (ε = 60000 dm3/mol/cm; λ = 538 nm; molecular weight = 550) (wyler and meuer, 1979) and of amaranthine (ε = 56600 dm3/mol/cm; λ = 538 nm; molecular weight = 726) (piattelli et al., 1969). the level of polyphenols was determined by the poli-swain and hillis (1959) method, using folin-ciocalteu reagent. the extract used for determination of polyphenols was obtained by grinding dried samples of amaranth green forage in the lab sample mill (og 109), followed by extraction with 40 ml 0.08 m hcl in 80% methanol, at a temperature of 18-22ºc for two hours. the extract from 5g of green forage was centrifuged at 1500 g for 15 minutes and the remains were re-extracted twice with 40 ml 70% acetone for two hours. the content of polyphenols was expressed in mg of chlorogenic acid in 100g of the product. the extract’s capacity to eliminate free radicals was assessed using the method described by re et al. (1999) using abts•+. abts•+ free radical diluted in a solution of potassium persulfate adjusted to provide an absorbance of 0.74-075 at 734 nm. the methanol-acetone extract (0.8 ml) was topped up to 1 ml with a 1:1 mixture of acetone and methanol. subsequently, 2 ml of abts•+ free radicals were added to the mixture. the extract was incubated at 3ºc for 6 minutes and the absorption measurement was subsequently carried out at 734 nm. ital. j. food sci., vol. 29, 2017 732 the capacity to eliminate free rsa (radical scavenging activity) was derived from the following equation: rsa = (e1–e2) /e1 where: e1 – sample absorbance before incubation, e2 – sample absorbance after incubation. 2.5. statistical analysis the data were subjected to statistical analysis using anova in a randomized block design. the separate factors in the analysis were genotype and harvest time. the sum of the errors and the interaction of genotype × harvest date were used to test the significance of the main effects. the significance of differences between means shown in the form of homogeneous groups was assessed using a duncan test at p = 0.05. results are presented as mean±sd (standard deviation). all chemical analyses were performed in three replications. 3. results and discussions 3.1. chemical composition traditional vegetables provide low-cost quality nutrition for large parts of the population in both rural and urban areas. one such example is amaranth, containing more nutrients than typical leafy vegetables (venskutonis and kraujalis, 2013) in this study, we found statistically significant differences were found in the dry matter content, crude ash, crude protein, crude fibre, and nitrogen-free extract (nfe) in the whole plant between the studied genotypes (table 2). the harvest time influenced amaranth chemical composition. similar to amaducci et al. (2000) and fraser et al. (2001), who showed that the delay in harvesting of plants caused changes in their chemical content, in our study a later harvesting time caused an increase in dry matter and crude fibre content (table 3), but a decrease in crude protein content. green leafy vegetables have long been recognized as the cheapest and most abundant potential source of protein because of their ability to synthesize amino acids from a wide range of virtually unlimited and readily available primary materials such as water, co2, and atmospheric nitrogen (aletor et al., 2002). our results concerning crude protein in amaranth leaves do not provide a clear indication of statistical difference between the studied species, with protein content ranging between 95.9 and 101 g per kg of dry matter, similar to the results of other authors (akubugwo et al., 2007; modil, 2007). however, the results of our analysis permit the claim that amaranthus hypochondriacus l. (‘aztek’) tends to have a slightly higher protein level than the other studied genotypes. the highest amounts of protein were found in plants harvested at the beginning of blossoming (128 g/kg dm), being 37% and 32% higher than the samples from the two consecutive terms, respectively. even higher crude protein levels in amaranth biomass (152 to 216·kg dm) were reported by pospišil et al. (2009), which depended on the cultivar and the year. crude fat content in the whole plant differed between the studied amaranth cultivars, with the highest amount found in the ‘rawa’ cultivar. levels depended not only on the genotype but also on the harvest time. ital. j. food sci., vol. 29, 2017 733 table 2. the tested the whole plant of amaranth composition [g/kg dm]. specification dry matter crude ash crude protein crude fat crude fibre nfes cultivar (a)* (a1) ‘rawa’ 939±0.26 146±1.15 95.9±0.79 22.6a±0.12 235±0.18 499±0.65 (a2) ’aztek’ 936±0.40 144±0.20 101.0±0.74 17.6ab±0.17 240±1.27 495±0.16 (a3) ‘phule kartiki’ 941±0.75 163±1.37 98.0±0.81 11.3b±0.31 219±0.34 508±2.15 (a4) ‘oscar blanco’ 938±0.38 154±0.58 97.9±0.02 12.2b±0.45 249±1.00 486±0.05 term of the mowing no. of days after sowing date (b)** (b1) 60 934±0.12 183a±0.83 128.0a±0.66 15.4b±0.31 203b±0.90 470c±0.76 (b2) 90 942±0.25 145b±0.26 80.3b±0.53 11.5b±0.35 268a±0.86 494b±0.78 (b3) 120 939±0.41 128b±0.67 86.9b±0.57 20.8a±0.27 236ab±1.36 527a±18.9 *the cultivar's means denoted by different letters differ statistically at (for all columns separately). **the term of mowing's means denoted by different letters differ statistically at (for all columns separately). 3.2. fibre fractions recently, increasing attention has been paid to the components that are difficult to digest in the gastrointestinal tract of humans, belonging to the dietary fibre group. the source of the dietary fibre and the ratios of its fractions determine its properties and applications; the chemical and physical properties of its many structures have various effects on the physiology of the human body (mann et al., 2009). in our study, the content of crude fibre in the selected species was affected only by harvest time; a delay in harvest increased the content of crude fibre in the whole plant. the levels of specific fibre fractions were also determined (table 3) and indicated that a later harvest time caused a increase in ndf, adf, adl and cellulose content. hcel are best at binding ions of heavy metals (hu et al., 2010). celluloses and lignin also have these properties, but to a lesser degree and are dependent on the fraction’s origin. cellulose does not have good ion exchange properties, nor does it bind bile acids or salts (kahlon et al., 2007). cellulose fibres are virtually undigested in the gastrointestinal tract; however, they aid intestine peristalsis. table 3. the tested the whole plant of amaranth dietary fibre [g/kg dm]. specification ndf*** adf adl hcel cel cultivar (a)* (a1) ‘rawa’ 440±3.02 304±1.06 51.2b±1.99 136±4.08 253±0.93 (a2) ‘aztek’ 440±2.57 332±0.16 63.1a±2.42 109±2.73 269±2.57 (a3) ‘phule kartiki’ 415±0.29 291±3.91 54.5ab±0.88 126±4.20 236±3.03 (a4) ‘oscar blanco’ 453±3.39 329±2.61 55.1ab±0.16 124±5.99 274±2.76 term of the mowing no. of days after sowing date (b)** (b1) 60 408b±1.83 288b±2.20 51.8b±0.30 121±0.38 236b±2.50 (b2) 90 460a±0.83 347a±0.01 54.1b±1.05 113±0.83 293a±1.05 (b3) 120 444a±0.01 307ab±1.90 62.1a±1.18 137±1.90 245b±0.72 *the cultivar's means denoted by different letters differ statistically at (for all columns separately). **the term of mowing's means denoted by different letters differ statistically at (for all columns separately). ***ndf, neutral detergent fibre, adf, acid detergent fibre, adl, acid detergent lignin, cel, cellulose, hcel, hemicelluloses. ital. j. food sci., vol. 29, 2017 734 in our study, the highest relative amount of cellulose was found at the second harvest time (almost 20% more in comparison to the first harvest term), which is the result of an increase in content in tissues during the development and aging of the plant. lignin accumulates in the cell wall at the end of cell growth, after the formation of polysaccharide scaffolding of the wall is completed. the highest level of adl fraction was present in the dry matter from the last harvest term. this fraction does not present a high capacity for binding heavy metals. however, similar to cellulose, it also aids in intestine peristalsis. ndf had a highest share in dietary fibre, followed by adf, which includes lignin and cellulose. a later harvest time increased the amount of these fractions in the whole amaranth plant, which was probably caused by the production of cell wall components, as well as more polyphenols as a result of temperature stress. the delay in harvest promoted lignification as well as an increase of cell wall participation in cells of plant tissue (amaducci et al., 2000). 3.3. macronutrients a special role is assigned to mineral components due to their participation in numerous anti-oxidation processes. the enzyme peroxide dismutase is activated by copper, zinc, and manganese. deficiency of the mentioned elements, or magnesium, calcium and potassium, decreases the efficiency of internal anti-oxidant mechanisms causing an increased risk of degenerative diseases (ames, 2010; prashanth et al., 2015). in our study, we assessed the level of selected elements in the amaranth samples and found that they were influenced by the experimental factors (tables 4 and 5). the content of the tested mineral components in the studied cultivars of amaranth differed, which indicates various capacities to absorb and accumulate the mentioned components in the biomass by the different cultivars growing in similar conditions. the highest content of calcium and magnesium were found in the ‘aztek’ cv., while the lowest calcium was detected in ‘rawa’ cv., and the lowest magnesium in ‘phule kartiki’ cv. (table 4). ‘rawa’, ‘phule kartiki’ and ’oscar blanco’ cultivars had higher phosphorus concentrations than ‘aztek’ cv. (6.8 g/kg dm). the content of potassium in ‘oscar blanco’ cv. (63.7 g/kg dm) was the highest and differed from other cultivars. present research confirmed that amaranth was a rich source of potassium. the richest in potassium was ‘oscar blanco’ cv., containing 10 g/kg more of this element than spinach (matraszek et al., 2002), which belongs to the superfood group and is considered a rich source of potassium. ‘aztek’ cultivar contained the highest level of magnesium (4.4 g/kg dm). the highest concentration of sodium was recorded for ‘rawa’ cv. (111 g/kg dm) and ‘oscar blanco’ cv. (95.8 g/kg dm). phosphorus was the only element not influenced by the time of harvest, and its mean concentration in the studied genotypes of amaranth amounted to 8.9 g/kg dm. differences in ca and na content were recorded between the two harvest times. a tendency was found for less of the elements in the green mass of amaranth in the consecutive harvest terms (table 4). the amaranth is a valuable product and could be an important source of necessary nutrients in the human and the animal diet (akubugwo et al., 2007; onwordi et al., 2009; mlyneková et al., 2014). the application of food minerals in an organism largely depends on the ratios. the requirements regarding particular mineral components and their ratios in the animal diet depend, inter alia, on the species of animal, age, and physiological stage. the potassium to sodium ratio (k:na) in humans plays an important role in the regulation of blood pressure; it should be less than one to avoid adverse effects (yusuf et al., 2007). as shosphorus metabolism is linked to calcium metabolism in the system, calcium-phosphate homeostasis crucially depends on an appropriate ca:p ratio in food (close to 1). an inappropriate ca:p ratio decreases calcium absorption, which negatively affects the skeletal system and physiological ital. j. food sci., vol. 29, 2017 735 processes (driver et al., 2006; ye et al., 2006). a ratio lower than 1:2 impairs calcium absorption and vitamin d synthesis. this results in higher levels of parathyroid hormone and accelerates bone resorption processes (kemi et al., 2010). ‘rawa’, ‘phule kartiki’, and ‘oscar blanco’ cultivars all fulfilled the aforementioned requirements, which confirms the results of akubugwo et al. (2007). in addition, the ‘phule kartiki’ and ‘aztek’ cultivars had a k:na ratio close to 1, which qualifies their use in poultry feeds. table 4. the tested the whole plant of amaranth macronutrients [g/kg dm]. specification ca p k mg na ca:p k:na cultivar (a)* (a1) ‘rawa’ 10.4b±0.13 9.6a±0.01 55.6b±0.33 3.8b±0.01 111.8a±0.56 1.1b±0.01 0.5c±0.01 (a2)’ aztek’ 13.7a±0.07 6.8b±0.05 54.3b±0.41 4.4a±0.01 65.4b±1.83 2.0a±0.01 0.8a±0.02 (a3) ‘phule kartiki’ 10.7ab±0.10 9.1ab±0.09 54.6b±0.14 3.6b±0.01 60.9b±1.17 1.2b±0.02 0.9a±0.01 (a4) ‘oscar blanco’ 10.8ab±0.13 10.1a±0.06 63.7a±0.51 3.7b±0.01 95.8a±0.69 1.0b±0.02 0.7b±0.01 term of the mowing no. of days after sowing date (b)** (b1) 60 14.6a±0.16 10.3±0.01 65.6a±0.26 4.7a±0.02 90.4a±0.25 1.5a±0.01 0.8a±0.01 (b2) 90 9.7b±0.08 8.2±0.04 62.1b±0.51 3.1c±0.01 70.1b±0.46 1.3ab±0.01 0.9a±0.01 (b3) 120 9.9b±0.07 8.3±0.13 43.5c±0.27 3.8b±0.01 89.5a±0.79 1.2b±0.02 0.5b±0.01 *the cultivar's means denoted by different letters differ statistically at (for all columns separately). **the term of mowing's means denoted by different letters differ statistically at (for all columns separately). 3.4. micronutrients iron and zinc are both responsible for the proper functioning of specific and non-specific immune responses. the studied amaranth genotypes turned out to be a rich source of iron. the harvest time did not affect the content of this component (table 5). the whole plant of amaranth was found to contain on average 236.4 mg/kg of iron, which confirms the results of kamga et al. (2013). by comparison, matraszek et al. (2002) recorded 30% and 7% less iron in the respective dry matter of lettuce (lactuca sativa l.) and spinach (spinacia oleracea l.). as confirmed in the samples in the presented research, funke (2011) found 40% more iron in amaranth leaves cultivated in nigeria than in lettuce. the daily diet should also provide proper amount of zinc, since it is not accumulated in tissues, but excreted from the system. zinc has a beneficial effect on the production, maturation and activity of leukocytes. however, excessive consumption may limit iron and copper absorption, which can lead to anaemia. in our study, both the cultivar and the harvest time had an effect on the content of zn. ‘phule kartiki’ cv. biomass had the highest content of zn. the later the harvest, the lower the amount of zn in the selected amaranth cultivars. iron metabolism also involves copper, and the highest content of copper was found in ‘aztek’ cv. the highest levels of copper were also found in the amaranth at the beginning of blossoming (table 5). manganese is an essential trace element, necessary for development and growth of the organism. it is a component of metalloenzymes such as superoxide dismutase, arginase, and pyruvate carboxylase, and is involved in amino acid, lipid and carbohydrate metabolism. disturbances in manganese absorption and retention may play a role in the etiopathogenesis of several diseases and disorders. there has been no specific manganese deficiency syndrome described in humans (zabłockasłowińska and grajeta, 2012; panel on dietetic products, nutrition and allergies, 2013). manganese content in the studied plants was significantly ital. j. food sci., vol. 29, 2017 736 correlated to both cultivar type and harvest time. the highest content of manganese was reported in the ‘phule kartiki’ cv. (177.5 mg/kg dm). the mean content of manganese in the studied cultivars of amaranth stood at 150.8 mg/kg dm. the highest manganese content was reported in amaranth collected during the full bloom period. manganese concentration in vegetable leaves, including amaranth, varies between 2.54 mg/kg dm in indian spinach (basella alba l.), 5.46 mg/kg dm in bush buck (gongronema latifolium l.), 6.14 mg/kg dm in roselle plant (hibiscus sabdariffa l.) and 10.6 mg/kg dm in smooth amaranth (amaranthus hybridus l.) (asaolu et al., 2012). table 5. the tested the whole plant of amaranth micronutrients [g/kg dm]. specification zn fe mn cu cultivar (a)* (a1)’ rawa’ 37.8b±0.36 231.1ab±1.23 132.6b±0.01 2.8c±0.03 (a2) ‘aztek’ 44.4b±0.04 237.4ab±0.17 140.4b±0.20 3.7a±0.30 (a3) ‘phule kartiki’ 82.4a±0.06 272.3a±0.14 177.5a±0.10 3.5ab±0.10 (a4) ‘oscar blanco’ 41.0b±0.09 205.0c±1.03 152.7ab±0.18 3.0bc±0.02 term of the mowing no. of days after sowing date (b)** (b1) 60 60.0a±0.25 248.4±1.11 147.9ab±0.02 4.3a±0.20 (b2) 90 48.6b±0.04 219.7±0.47 171.9a±0.22 2.5b±0.01 (b3) 120 45.6b±0.04 241.1±1.07 132.7b±0.12 2.8b±0.09 *the cultivar's means denoted by different letters differ statistically at (for all columns separately). **the term of mowing's means denoted by different letters differ statistically at (for all columns separately). 3.5. polyphenol, pigments and scavenging ability betanins are water-soluble nitric herbal dyes and can be found in the cell fluids. their stability within a wide ph range from 3.5 to 7.0 contributes to the fact that they are excellent food dyes and good substitutes for anthocyanins (moreno et al., 2008). in a collective summary for the amaranthaceae family, khan and giridhar (2015) found the betanin content between 7.6 and 117.0 mg/kg dm (in celosia spp., achyrranthes spp., aerva sanguinolenta spp., alternanthera spp., iresine herbstii spp., gomphrrena globose spp.). in the studied whole plant of amaranth, betanin levels of 11.4-17.3 mg/kg dm and amaranthine of 106.5-161.1 mg/kg in air dried mass of different cultivars of amaranth were found. in amaranth shoots, venskutonis and kraujalis (2013) recorded 1.77 mg/100g of betanin. they also showed that amaranthine and isoamaranthine are the dyes found in the highest quantities, at 15.3 and 5.87 mg/100g, respectively. the presence of amaranthine and the absence of isoamaranthine were reported in celosia argentea l. (schliemann et al., 2001). in our study, a content of betanin in the whole plant of amaranth ranged between 1.19 and 1.54 mg/100g and depended on genotype (table 6). the highest content was reported in the leaves of the ‘aztek’ and ‘oscar blanco’ cultivars. analysis of the effect of harvest time on betanin content showed that a delay in harvest decreased the content of this compound in the whole plant. the highest content of amaranthine was reported in dry matter of plants from the first harvest, as well as in the dry matter of the ‘aztek‘ and ’oscar blanco’ cultivars. betacyanins also display a free radical scavenging capacity, and betanin and its metabolites maintain their properties in acidic conditions (taira et al., 2015). the free radical scavenging activity (rsa) of bethanidine is comparable to that of vitamin e (tesoriere et al., 2009). the concentrations found in the ital. j. food sci., vol. 29, 2017 737 various cultivars fell within the range 2.9 mg/g to 35 mg/g dm. free radial scavenging activity is also correlated with the content of polyphenols in raw material. the statistical analysis of the genotypes allowed their classification into two homogenous groups. the group with the highest content of polyphenols was the ‘phule kartiki‘ cv. (2792.5 mg/100g dm). such a high content of total polyphenols in the whole plant of this cultivar did not result in statistically significantly higher antioxidant activity compared to other cultivars. the high capacity to remove free radicals in the studied cultivars oscillated within a very narrow range (97.5% to 98.4%), which can be ascribed to harvest time, with rsa slightly higher in the second and third terms. although the highest levels of polyphenols were found in the second harvest term (2884.2 mg/kg dm), and the lowest in the third term (1482.3 mg/kg dm), free radical scavenger properties were statistically unchanged. differences in phenol compounds content between leafy vegetables, including amaranthus cruentus l. and amaranthus hybridus l. were shown by ademoyegun et al. (2013). of these two species, it was a. hybridus that had higher phenol content and higher anti-radical activity. due to the fact that these compounds play an important role both for these plants’ ontogenesis, and their health promoting and sensory properties, it seems they should be considered as potential food supplements. in this regard, amaranth is more beneficial than sprouts and leaves of oat and buckwheat, commonly known for their antioxidant potential; no less important is the presence of betacyanines in amaranth (piątkowska et al., 2015; witkowicz et al., 2015). table 6. polyphenol content [mg/100g dm], pigments content [mg/kg dm] and scavenging ability rsa [%] in the tested the whole plant of amaranth. specification polyphenol rsa betanine amaranthine cultivar (a)* (a1) ‘rawa’ 1669.7b±49.3 98.0±0.22 11.9b±0.31 111.3b±2.96 (a2) ‘aztek’ 1846.3b±53.3 98.1±0.22 14.4a±0.76 134.5a±7.07 (a3)’ phule kartiki’ 2792.5a±182.8 97.8±0.31 12.1b±0.35 113.3b±3.33 (a4) ‘oscar blanco’ 1768.3b±61.73 98.0±0.22 15.4a±0.02 143.7a±0.18 term of the mowing no. of days after sowing date (b)** (b1) 60 1690.9b±55.6 97.5b±0.09 17.3a±0.09 161.1a±0.82 (b2) 90 2884.2a±172.2 98.1a±0.09 11.7b±0.13 109.5b±1.30 (b3) 120 1482.3b±32.6 98.4a±0.21 11.4b±0.10 106.5b±0.93 *the cultivar's means denoted by different letters differ statistically at (for all columns separately). **the term of mowing's means denoted by different letters differ statistically at (for all columns separately). 4. conclusions the weather in the vegetative season in 2014 was particularly beneficial for the growth and development of the cultivated amaranth species. the factor that determined the nutritional value of the studied plants was the harvest time. a delay in harvest time decreased the amount of crude protein and mineral compounds (determined in ash). the amaranth from later harvests (full bloom and full seed development) had increased concentration of crude fibre and total carbohydrates. the content of mineral compounds was different, which indicated differences in the abilities to absorb and accumulate particular elements by the studied cultivars. with a delay in harvest the concentrations of the macro and micro-compounds decreased. the studied amaranth demonstrated very ital. j. food sci., vol. 29, 2017 738 high free radical scavenging activity, with the highest rsa values recorded in the two later harvest terms. the content of pigments in amaranth was correlated to both the genotype and the harvest term. the highest concentrations of pigments were observed in ‘oscar blanco’ cv. and ‘aztek’ cv. this content then decreased with the delay in harvest. the amaranth is a valuable product and may be an excellent source of essential nutritional components in the human and animal diet. however, based on the presented results, it is difficult to unequivocally indicate the most valuable genotype and optimal harvest term with reference to the nutritional value. references abbasi d., rouzbehan 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zuccarato giallo and prunu niuru presented tss higher than the commercial cultivars (24.9 and 21.6 °brix respectively) and interesting data obtained on the nutraceutical compounds values suggested these local cultivars as sources of polyphenols (zuccarato giallo with 663 mg ga/100 gfw) and natural antioxidants (pruno regina with 47.46 fe2+/100 gfw). the characterization of these plums could represent also an important resource for the international activity in the genetic improving and the collection of the more interesting quality traits could be useful for improving the prunus database actually in use. ital. j. food sci., vol. 27 2015 321 1. introduction the varietal diversity is one of the agricultural biodiversity (heywood, 1999) and the management by farmers at the local community level is one of the factors involved to preserve it. the international treaty on plant genetic resources for food and agriculture approved in 2001 has assigned to all joined nations the duty to take specific local actions in terms of preservation of genetic resources, particularly those that are directly related with the food and agriculture, ie. agrobiodiversity. since that, many national initiatives have been set up aiming at the recovery and characterization of the genetic autochthonous resources as well as the enhancement in both nutritional and nutraceuticals traits. the need to maintain, to protect and to manage agobiodiversity is increasing; the identification of the composition of locally cultivated food as sources of nutraceutical compounds is essential to promote a more food-base approach to nutrition and health (scoones, 1992). plums are the most taxonomically diverse of stone fruits (das et al., 2011) and the varietal diversity is strongly related to the high percentage of selfincompatibility that led over the centuries to various cross-pollinations with recombinations of characters (sottile et al., 2010a). the management in situ is one of the commonly recommended germplasm conservation approaches (maxted et al., 2010) and in sicily plum fruits were cultivated since the sixteenth and seveteenth centuries as reported in literature (cupani, 1696; nicosia, 1735) due the propitious pedoclimatic conditions of the territory for their development and genetic diversification (impallari et al., 2010). the most representative areas for the diffusion of plum trees are described by these authors as the palermo and trapani province which today have an important rule to improve and to diffuse the cultivation of the specie by using its natural and favorable climatic conditions. the characterization and identification of plum varieties usually is performed on morphological data (sottile et al., 2010b), phenototic traits (horvath et al., 2011) and more recently on some molecular markers (gregor et al., 1994; ortiz et al., 1997; gharb et al., 2014), but the study of the nutraceutical compounds (sottile et al., 2010b), represent today an important tool to improve the collected data and to describe better the varietal diversity (vasantha rupasinghe et al., 2006; díaz-mula et al., 2009). the replacement of local cultivars with the new one introduced by genetic improvement programs, due to a higher productivity, as well as resistance or tolerance to pests and diseases, or to abiotic stress, has caused a strong genetic erosion of the indigenous fruit tree species germplasm (impallari et al., 2010). as a consequence of the globalization process, the homologation involved the fruit consumption and deeply contributed in the loss of the unique taste of these fruits. many studies have showed that the locally available cultivars, varieties and wild underutilized ecotypes; jablonska-rys et al., 2009; petruccelli et al., 2013;) are in many cases more rich in nutrients than similar commercially foods, confirming the old ecotypes as genetic resources of fruit nutritional traits. plums have the potential to contribute greatly to human nutrition because of their richness in fiber and antioxidants (stacewicz-sapuntzakis et al., 2001; sottile et al., 2010b). in many cases the genetic resources with a higher relevance in terms of nutraceutical facts are related to old varieties and reported with a high risk of erosion. to limit the loss of biodiversity and to adopt collaborative conservation strategies it is necessary to improve the knowledge of the genetic resources and their horticultural aspects. the reduction in the genetic variability is increasing from the past century so as established by international approaches to biodiversity preservation protocols, each country is responsible for its own genetic resources (das et al., 2011). no detailed study concerning physical and nutritional properties of old sicilian plum ecotypes have been performed up to now, so the aim of this study was to determine some qualitative and nutritional traits of plum fruits belonging to some local cultivars identified in the sicily island. 2. materials and methods 2.1 plant material and collection of data one hundred forty-three georeferenced plum cultivars and accessions were identified in the sicilian island from existing bibliography and a territorial investigation but in this preliminary study only thirty-seven varieties are considered. in the table 1 the total 37 local cultivars and accessions of plums used for the qualitative and nutraceutical analysis on fruits are reported. these included 34 plum trees from different locations in the sicily region and 3 commercial varieties respectively of prunus domestica l. (cv. stanley) and prunus salicina lindl. (cv. shiro and cv. angeleno), as references. the investigated area is located both in the west and in the eastern part of sicily. for each local cultivars and accession 30 fruits randomly collected from the entire production were used. the plants of the sicilian germplasm were maintained on site in their natural habitat where they are routinely grown by local farmers. in all cases minimal cultural techniques have been applied, without any fruit thinning. all cultivars produce primarily on spurs and pruning is not commonly carried out. an integrated pest management approach is ordinary adopted by 322 ital. j. food sci., vol. 27 2015 growers by following the regional governmental rules for plum. the fruits were picked by hand at the ripe stage (table 2). the fruits damaged were removed, were graded for color and size uniformity and they were immediately transported to the pomological laboratory for analysis. 2.2 fruit quality traits the weight was obtained measuring individually 30 fruits per each local cultivars and accessions. the data were expressed as the mean ± se. fruit weight (g) was performed using an electronic balance (se622, wvr, usa) with an accuracy of 0.01 g. the fresh fruit firmness (fff) was measured using an effegi hand-held penetrometer (turoni, italy) with a 5-mm-diameter plunger in accordance with standard industry practice. the skin of the fruits was partially removed before measuring. two measurements (30 fruits) were made on opposite sides of the central zone of the fruits and then averaged to yield a mean value for the fruit. the measurements were reported in kg force (kgf) cm−2. after the firmness measurements for the total soluble solids (tss) and the titratable acidity (ta) determination, the same fruits were completely hand-peeled and skin and pulp were cut in small pieces to obtain homogeneous samples. for tss and ta determination 10 g of pulp samples were squeezed using a commercial blender and the extracted juice was later sieved and centrifuged at 8,000 × g for 20 min (sigma 3-18 k, osterode and harz, germany). an aliquot of this supernatant was used to determine tss with a digital pocket refractometer atago pal1 (atago co. ltd., japan) calibrated at 20°c to 0% with distilled water, and expressed as pertable 1 list of cultivars and accessions plums collected from the local germplasm of sicily and sampling locations. authoctonous cultivars location* and accession name 1 69sus005p messina (me) 2 lazzarino palermo (pa) 3 sanacore tardivo palermo (pa) 4 zuccarino rosa messina (me) 5 prunu nucidda messina (me) 6 cuore di bue catania (ct) 7 pruno regina catania (ct) 8 107sus009e trapani (tp) 9 107sus008b trapani (tp) 10 107sus007b trapani (tp) 11 rapparinu russu trapani (tp) 12 sanacore palermo (pa) 13 prunu niuru catania (ct) 14 ariddo di core palermo (pa) 15 occhio di bue catania (ct) 16 ranco’ nero catania (ct) 17 don ciccino catania (ct) 18 cugghiuni di mulu catania (ct) 19 papale catania (ct) 20 71sus028b catania (ct) 21 president catania (ct) 22 pruna di s. antonio messina (me) 23 susine nere messina (me) 24 nivuru purmintia messina (me) 25 nivuru messina (me) 26 pruna i sceccu messina (me) 27 santu vitu messina (me) 28 66sus052p messina (me) 29 prunu ciraseddu catania (ct) 30 prugnolo rosso messina (me) 31 pruno rosa palermo (pa) 32 primintio palermo (pa) 33 prunu nivuru codulusu trapani (tp) 34 zuccarato giallo messina (me) 35 shiro palermo (pa) 36 stanley palermo (pa) 37 angeleno palermo (pa) *data on geographical position (latitude and longitude) are available for all plums. fig. 1 total anthocyanins of local sicilian plums ital. j. food sci., vol. 27 2015 323 t a b le 2 h a rv es ti n g d a te s fo r th e a u th o ch to n o u s s ic il ia n p lu m s. p lu m s j u n e j u ly a u g u s t s e p te m b e r 5 10 15 2 0 2 5 3 0 5 10 15 2 0 2 5 3 0 5 10 15 2 0 2 5 3 0 5 10 15 2 0 2 5 3 0 6 9 s u s 0 0 5 p l a zz a ri n o s a n a c o re t a rd iv o z u c c a ri n o r o s a p ru n u n u c id d a c u o re d i b u e p ru n o r e g in a 10 7 s u s 0 0 9 e 10 7 s u s 0 0 8 b 10 7 s u s 0 0 7 b r a p p a ri n u r u s s u s a n a c o re p ru n u n iu ru a ri d d o d i c o re o c c h io d i b u e r a n c o ’ n e ro d o n c ic c in o c u g g h iu n i d i m u lu p a p a le 7 1 s u s 0 2 8 b p re s id e n t p ru n a d i s . a n to n io s u s in e n e re n iv u ru p u rm in ti a n iv u ru p ru n a i s c e c c u s a n tu v it u 6 6 s u s 0 5 2 p p ru n u c ir a s e d d u p ru g n o lo r o s s o p ru n o r o s a p ri m in ti o p ru n u n iv u ru c o d u lu s u z u c c a ra to g ia ll o s h ir o s ta n le y a n g e le n o 324 ital. j. food sci., vol. 27 2015 table 3 fruit quality traits of 37 authochtonous sicilian plums. fruit weight fff tss ta mi (g) kgf cm-2 °brix g malic acid l-1 plums 69sus005p 34.20±4.51 g-i 0.88±0.21 d-i 19.6±0.21 b-e 7.76±1.74 g-o 2.5 lazzarino 16.77±2.90 o-s 0.58±0.26 l-r 16.5±1.47 f-i 6.28±0.41 i-q 2.6 sanacore tardivo 28.98±7.13 l-m 0.80±0.22 g-n 15.3±0.06 h-n 7.72±0.26 g-p 2.0 zuccarino rosa 13.67±1.99 r-u 0.31±0.12 s-u 15.1±0.70 h-n 9.02±0.14 f-l 1.7 pruno nucidda 12.06±4.32 s-u 1.14±0.36 d 21.2±0.28 b-c 9.00±0.13 f-l 2.4 cuore di bue 83.79±9.02 a 0.76±0.21 h-o 13.3±0.21 l-p 12.21±1.22 c-e 1.1 pruno regina 33.62±3.58 h-i 0.76±0.22 h-o 18.4±0.21 c-g 7.95±0.09 g-n 2.3 107sus009e 21.07±7.06 n-o 0.63±0.20 i-q 11.5±0.21 o-q 16.56±0.57 b 0.7 107sus008b 14.26±2.44 q-u 0.40±0.12 q-t 12.8±0.00 m-p 11.97±1.02 c-e 1.1 107sus007b 14.93±3.43 p-u 0.40±0.11 q-t 15.5±0.42 g-n 10.56±0.15 e-h 1.5 rapparinu rosso 13.75±2.25 q-u 0.58±0.21 l-r 15.7±1.91 g-n 4.27±0.75 m-q 3.7 sanacore 19.26±5.86 o-q 0.93±0.30 d-h 16.0±1.06 f-l 7.98±0.43 g-m 2.0 prunu niuru 26.36±4.44 l-n 0.69±0.15 g-p 21.6±1.49 b 4.93±0.25 m-q 4.4 ariddo di core 16.75±3.19 o-s 1.07±0.35 d-f 20.1±2.03 b-d 6.13±0.76 i-q 3.3 occhio di bue 41.76±8.28 e-f 0.55±0.10 n-s 15.6±0.28 g-n 4.86±0.08 m-q 3.2 ranco’ nero 44.13±5.39 e 0.60±0.10 l-r 16.6±0.07 e-i 7.29±0.09 h-p 2.3 don ciccino 37.22±5.92 f-h 0.47±0.06 p-s 19.1±0.28 b-f 10.12±0.21 e-i 1.9 cugghiuni di mulu 61.53±8.92 c 0.56±0.13 m-s 14.6±0.06 i-o 8.24±0.39 g-m 1.8 papale 45.19±6.46 d-e 0.57±0.17 m-r 15.4±0.10 g-n 9.60±1.17 e-i 1.6 71sus028b 49.99±8.77 d 0.55±0.07 n-s 16.7±0.15 e-i 7.67±0.24 g-p 2.2 71sus024c 66.52±12.77 b-c 0.87±0.27 e-i 13.7±0.06 i-p 12.78±0.33 c-e 1.1 66sus006s 16.16±4.37 o-t 0.65±0.17 i-q 14.8±0.07 h-n 9.43±0.91 e-i 1.6 susine nere 9.48±1.69 u 0.52±0.05 o-s 14.2±0.07 i-o 11.83±0.99 c-e 1.2 nivuru primintia 40.70±7.23 e-f 0.82±0.17 f-m 14.5±0.96 i-o 10.84±0.26 e-g 1.3 nivuru 15.21±2.91 p-t 0.69±0.26 h-p 14.9±0.07 h-n 9.65±1.26 e-i 1.5 pruna i sceccu 39.79±7.30 e-g 0.83±0.31 f-l 11.0±0.28 p-q 22.62±0.59 a 0.5 santu vitu 20.41±2.48 o-p 0.52±0.25 o-s 14.0±3.02 i-p 4.45±0.45 p-q 3.1 66sus052p 65.64±10.75 b-c 1.04±0.64 d-g 12.8±0.35 n-q 14.56±0.47 b-c 0.9 prunu ciraseddu 14.21±2.05 q-u 0.35±0.04 r-u 10.1±0.14 q 3.60±0.18 q 2.8 prugnolo rosso 18.45±2.07 o-r 1.10±0.27 d-e 17.7±0.78 d-h 6.76±0.42 i-p 2.6 pruno rosa 17.90±3.32 o-r 0.55±0.26 n-s 16.2±0.74 f-l 8.66±1.07 g-l 1.9 primintio 21.09±5.33 n-o 0.46±0.17 p-s 15.7±1.44 g-n 10.94±2.15 d-g 1.4 prunu nivuru codulusu 10.78±1.68 tu 0.12±0.04 u 16.7±0.07 e-i 14.23±0.40 b-d 1.2 zuccarato giallo 13.57±2.89 r-u 0.16±0.05 t-u 25.0±0.35 a 5.86±0.27 l-q 4.3 shiro 36.62±5.16 f-h 1.57±0.09 c 15.0±0.40 g-m 10.63±0.81 e-h 1.4 stanley 40.10±3.35 e-f 2.46±0.10 b 15.9±0.36 g-m 5.23±0.20 l-q 3.0 angeleno 67.70±9.80 b 4.33±0.39 a 21.2±1.28 b-c 4.64±0.61 p-q 4.6 data are means ± sd. values with the same letter at the column level are not statistically different with the tukey’s test (0.05). centage (°brix). ta was determined in 1 ml of the above supernatant diluted in 25 ml of distilled water by titration with 0.1 n naoh up to ph 8.1, using an automatic titration device (484 titrino plus, metrohm, switzerland) and results expressed as grams of malic acid l−1. three replicates per measurement were used and the data reported are the mean ± se. the tss:ta ratio was calculated for individual fruit from the tss and ta results and it expressed the maturity index (mi). 2.3 total anthocyanins, phenolic content and antioxidant activity to determine the total anthocyanin content, the total phenolic content and the total antioxidant capacity, fruit extract was obtained using 10 g of fruit added to 25 ml of extraction buffer (500 ml methanol, 23.8 ml deionized water and 1.4 ml hydrochloric acid 37%). after 1 h in the dark at room temperature, the samples were thoroughly homogenized for a few minutes with an ultra turrax (ika, staufen, germany) and centrifuged for 15 min at 3,000 rpm. the clear supernatant fluid was collected and stored at -20 °c until analysis. the total anthocyanin content was quantified according to the ph differential method of cheng and breen (1991). anthocyanins were estimated by their difference of absorbance at 510 and 700 nm in a buffer at ph 1.0 and ph 4.5, where a tot = (a 515 a 700 ) ph 1.0 (a 515 a 700 ) ph 4.5. the results are expressed as milligrams of cyanidin-3-glucoside (c3g) equivalent per 100 g of fresh weight (fw). the total phenolics content was measured using a folin-ciocalteu reagent with gallic acid ital. j. food sci., vol. 27 2015 325 as a standard at 765 nm following the method of slinkard and singleton (1977). the results are expressed as milligrams of gallic acid equivalents (gae) per 100 g of fresh weight (fw). the antioxidant activity was determined using the frap (ferric reducing antioxidant power) assay, according to benzie and strain (1996) method modified by pellegrini et al. (2003). the antioxidant capacity of the dilute fruits extract was determined by its ability to reduce ferric iron to ferrous iron in a solution of 2,4,6-tris(2pyridyl)-s-tri-azine (tptz) prepared in sodium acetate at ph 3.6. the reduction of iron in the tptz–ferric chloride solution (frap reagent) results in the formation of a blue-coloured product (ferrous tripyridyltriazine complex), the absorbance of which was read spectrophotometrically at 595 nm 4 min after the addition of appropriately diluted fruits extracts or antioxidant standards to the frap reagent. the results were expressed as mmol fe2+ equivalents per kilogram of fresh fruits. all of these analyses were performed using a uv-vis spectrophotometer 1600-vwr. three replicates per measurement were used. 2.4 statistical analysis the data obtained were treated with one-way analysis of variance (anova) using spss for windows version 20.0 and the means were separated using the tuckey test (p ≤ 0.05). 3. results 3.1. quality parameters in the table 3 the qualitative traits studied for the different plums are reported. as already reported, fruit size is an important quality parameter to evaluate the economic value for the consumption of fresh fruits (petruccelli et al., 2013) and to determine the category of the ranges of marketability of many fruits. it is affected by a number of variables, including source–sink relationship (snelgar et al., 1998), water availability (intrigliolo and castel, 2006) as well as temperature and growing conditions in general. in our study a great variability on fruit weight has been reported among the different plums suggesting a different fruit surface/volume (eifert et al., 2006), a fruit size distribution into different classes and a different correlation to physical-chemical parameters, such tss and ta. fruits with greater weight would have a greater proportion of edible flesh (franco-mora et al., 2009). the fresh weight of all plums analysed revealed a mean value of 30.91 g ranging from 83.79 to 9.48 g; the maximum value was observed for the cuore di bue cultivar while the lowest was observed for the susine nere plums. less than half of the plums (sixteen) showed higher values than the mean value. the don ciccino cultivar was the only one to show the weight (37.22 g) similar to the values of the commercial cv. shiro (36.62 g) while nivuru primintia and occhio di bue with respectively 40.70 and 41.76 g showed similar weight to the commercial cv. stanley (40.10 g). all plums showed statistically differences from the commercial cv. angeleno which weight was of 67.70 g. flesh firmness is a key quality parameter, since it is directly related to fruit ripeness, and is often a good indicator of shelf-life potential (valero et al., 2007). the highest pulp firmness was observed for all the three commercial cultivars, the cv. angeleno with 4.33 (kgf) cm−2 was the higher value followed by the cv. stanley with 2.46 (kgf) cm−2 and the cv. shiro with 1.57 (kgf) cm−2. all autochthonous plums (34) showed statistically differences from these last and only 11% of them showed fff value major than 1(kgf) cm−2. the mean value was of 0.82 (kgf) cm−2, the lowest value was reached by the prunu nivuru codulusu probably due to the low weight (10.78 g), while the highest was observed for the pruno nucidda (1.14) (kgf) cm−2. the physical-chemical parameters, such tss and ta, strongly influence the consumer preference for stone fruit quality and the aromatic profile for the plums consumption (crisosto et al., 2007). the zuccarato giallo and the prunu niuru showed the highest tss content (25.0 and 21.6 °brix, respectively) while the lowest tss value (10.1 °brix) was scored by the prunu ciraseddu cultivar. observing the table 2 there is a tendency for late season plums to have higher tss than early season plums. less than half of the plums (43%) showed the tss content greater than the mean value (16.6°brix). the 69sus005p, pruno nucidda, pruno regina, prunu niuru, ariddo di core and don ciccino authoctonous plums showed similar values to the commercial cv. angeleno (21.2 °brix) and no statistically significant differences were observed between these fruits. the 66% of the plums showed value not statistically different from the commercial cv. shiro and stanley which scored 15.0 and 16.0 °brix respectively. the tss values measured in the local cultivars and accession are quite high when compared to the value find in the literature; in fact tss between 14% and 16% (westwood, 1978) or 10 and 15% (díaz-mula et al., 2009) could suggest edible fruit ready for consumption. the ta mean value was of 9.10 g malic acid l-1 and the 58% of the local plums showed ta values inferior to the average. the lowest acidity was for the prunu ciraseddu (3.60 g malic acid l-1) while the highest value was for the pruna i sceccu (22.62 g malic acid l-1). the variations observed in tss and ta affected the values of the maturity index (mi). great 326 ital. j. food sci., vol. 27 2015 differences were observed among values which ranged from a minimum of 0.5 in the case of the pruna i sceccu to a maximum of 4.6 for the commercial cv. angeleno. the prunu niuru and the zuccarato giallo with the mi of 4.4 and 4.3 showed tss/ta ratio similar to the cv. angeleno while primintio (1.4) and santu vitu (3.0) were similar to the cv. shiro and the cv. stanley respectively. 3.2 total anthocyanins, phenolic content, and antioxidant activity the evaluation of the total anthocyanins content (fig. 1), in general, has shown that this component does not assume, in percentage terms, a high importance in the context of polyphenolic compounds (fig. 4). the primintio accession had significantly more total anthocyanins content (65.22 mg of cyanidin-3-glucoside /100 g fw) than other fruits, although it is only 34.5% of the total polyphenols (fig. 4). the lowest value was for ariddo di core (1.22 mg of cyanidin-3-glucoside/100 g fw) which content was similar to the commercial cv. shiro and the fraction measured on the total content of the total polyphenols was of 0.3 %. although the anthocyanin fraction mean value was of the 5.8% of the total polyphenol content it was observed that the absolute values detected in some local cultivars and accessions such as primintio, 107sus007b and pruna di s. antonio were higher than values previously reported for other varieties (tomásbarberán et al., 2001; cevallos-casals et al., 2006; usenik et al., 2009). polyphenols represent the largest group of water-soluble phytochemicals. they have been known to be chemotaxonomic markers for classification purposes in plum fruits (treutter et al., 2012) and their content could contribute strongly to the antioxidant activity in fresh fruits. generally the polyphenol composition is related to the cultural practices and abiotic factors such as the outside air temperature and the rainfall rate (salgado et al., 2008; miletic et al., 2012). strong variations in the total polyphenol content were observed among the plums of the study (fig. 2) whose mean value was of 301.67 mg ga/100 g fw. the minimum value of 104.87 mg ga/100 g fw was observed for the sanacore tardivo while the maximum value of 663.99 mg ga/100 g fw was observed for the zuccarato giallo. the total phenolic content in the commercial cultivars used as references was lower than that reported by cevallos-cavals et al., 2006 (298 to 563 mg/100 g fw) for prunus salicina cv. shiro (191.17 mg ga/100 g fw) and cv. angeleno (242.01 mg ga/100 g fw) while according to los et al., (2000) it was in the range for prunus domestica (160-300 mg/100 g) (cv. stanley 211.23 mg ga/100 g fw). the 58% of the local plums included a total polyphenol content major than cv. angeleno. previous studies showed as the averages of the total phenolic content of plums were significantly higher than the content in other fruits such as apples (lee and smith, 2000; proteggente et al., 2002) and our data confirmed that. the total antioxidant capacity of fresh fruits (fig. 3), expressed as mmol fe2+ per kg of fresh fruits ranged from a maximum value of 47.46 measured for pruno regina and a minimum value of 4.14 for the rapparinu russu accessions. no statistically significantly differences were obfig. 2 total phenolics content of local sicilian plums ital. j. food sci., vol. 27 2015 327 served among the cv. shiro, stanley and angeleno which values ranged between 4.86 and 9.59 mmol fe2+/100 g fw and the 20% of the fruits showed values major than the average (13.45 mmol fe2+/100 g fw). as described by frankel and mayer (2000), the measure of fruit antioxidant capacity is influenced by the analytical method used and this could represent a limit for the evaluation. according to miletic et al., (2012), fruits containing the highest total phenols do not necessarily exhibit the highest antioxidant capacity. in fact the highest value observed fig. 3 total antioxidant activity of local sicilian plums for the pruno regina accession is related to the relative high phenolic content but the same correlation wasn’t observed for the zuccarato giallo which corresponded the highest phenolic content (663.99 mg ga/100 g fw) but the low total antioxidant activity of 11.86 mmol fe2+/100 g fw. comparing the total antioxidant activity of the studied plums to the total frap of other fruits reported in previous work (guo et al., 2003) it is interesting to observe the high values find that could suggest interesting uptake of plums for the human diet. fig. 4 total anthocyanins and phenolics distribution (%) in authochtonous sicilian plums. 328 ital. j. food sci., vol. 27 2015 4. conclusions this study provides important data for qualitative and nutraceutical properties of the fruits. the results obtained by this preliminary study reveals that the authocthonous sicilan plums contains important amounts of anthocyanins and phenols; often their concentrations is higher than found not only in the commercial cultivars but also in fruits that are reported in literature to have high content of nutraceutical compounds. considerable and significantly different among the plums were observed and the environmental conditions and the activity of propagation and exchange of genetic material results of the local farmers could be both responsible for the differentiation of the germplasm collected in the investigated areas of the sicilian territory. the maintaining of local genetic materials is important for the biodiversity and the action of localization and characterization of old fruits cultivar and accessions is fundamental to improve the management of the european prunus database for plum (epdp) making them available for research and genetic improvement. the knowledge of the qualitative traits of fruits represent a good opportunity not only for the health advantages but also for the general consumption; in fact the enhancement and the safeguarding of these fruits can be thought as an opportunity of new marketing channel but more studies need to be undertaken about the evolution of the qualitative and nutraceutical compounds during storage to answer the consumer’s demands and expectations. acknowledgements this work is part of the activity funded by the sicilian regional government with the project “risorse genetiche vegetali sicilia’ – scientific coordinator francesco sottile, university of palermo. references benzie i.f.f. and strain j.j. 1996. the ferric reducing ability of plasma (frap) as a measure of ‘antioxidant power’: the frap assay. anal. biochem. 239: 70. cevallos-casals b.a., byrne d., okie w. r. and cisneroszevallos l. 2006. selecting new peach and plum genotypes rich in phenolic compounds and enhanced functional properties. food chem. 96: 273. cheng g.w. and breen p.j. 1991. activity of phenylalanine ammonia-lyase (pal) and concentrations of anthocyanins and phenolics in developing strawberry fruit. j. am. soc. hortic. sci. 116: 865. crisosto g.m., crisosto c.h., echeverria g. and puy j. 2007. segregation of plum and pluot cultivars according to their organoleptic characteristics. postharvest biol. technol. 44: 271. cupani f., 1696. hortus catholicus, 2nd ed. benzi, napoli. das b., ahmed n. and singh p. 2011. prunus diversity. eearly and present development: a review. intern. j. of biodiversity and conservation 3: 721. díaz-mula h.m., zapata p.j., guillen f., martinez-romero d., castillo s., serrano m. and valero d. 2009. changes in hydrophilic and lipophilic antioxidant activity and related bio-active compounds during postharvest storage of yellow and purple plum cultivars. postharvest biol. technol. 51: 354. eifert j. d., sanglay g. c., lee d.j., sumner s. s. and pierson m. d. 2006. prediction of raw produce surface area from weight measurement. j. food eng. 74: 552. franco-mora v.h., franco-mora o., lopez-sandoval j.a., de jesus perez-lopez d. and balbuena-melgarejo a. 2009. characterization of wild plum (ximenia americana l. var. americana; 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among european plum genotypes. sci. hortic. 108: 243. westwood m.n. 1978. temperate-zone-pomology (postharvest, storage and nutritional value), new york: w.n. freeman and company, p. 428. ijfs#920_bozza ital. j. food sci., vol. 30, 2018 116 paper human health risk assessment of organochlorine compounds associated with raw milk consumption in a romanian industrial area m. micleana,b, o. cadar*a, e.a. leveia and d.a. todeac aincdo-inoe 2000, research institute for analytical instrumentation, 67 donath street, 400293 clujnapoca, romania bsomes-tisa water basin administration, 17 vanatorului street, 400213 cluj napoca, romania cuniversity of medicine and pharmacy "iuliu hatieganu", 6 bp hasdeu street, 400371 cluj-napoca, romania *corresponding author. tel.: +40 264420590; fax: +40 264420667 *e-mail address: oana.cadar@icia.ro abstract dietary exposure to organochlorine pesticides (ocps) and polychlorinated biphenyls (pcbs), generically named organochlorine compounds (occs), now represents a significant health risk for humans due to their endocrine-disrupting and carcinogenic effects. to assess the potential health risk associated with raw milk consumption, 10 milk samples were collected from a local market in the baia-mare industrial area, romania. the concentrations of ocps and pcb congeners in these samples were determined by capillary gas chromatography with electron-capture detection, after liquid–liquid extraction. in all samples, the occs were below the maximum admitted concentrations set by european legislation. the predominant compounds were 4,4’-dde (11.5 ng/g lipid wt.), β-hch (10.1 ng/g lipid wt.) and pcb180 (5.08 ng/g lipid wt.). exposure assessment through milk consumption was performed for male, female and children by calculating the estimated daily intakes (edis) and the hazard indices (his) for non-carcinogenic effects. the obtained edis were lower than the acceptable daily intake values, and his were far below 1, indicating no potential health risk for the investigated population. keywords: health risk assessment, ocps, pcbs, raw milk ital. j. food sci., vol. 30, 2018 117 1. introduction foods of animal origin play an essential role in human nutrition. milk and dairy products are a source of microand macro-elements and active compounds that play an important role in nutrition and health (cadar et al., 2015). the microbial and chemical contamination of raw milk during animal feed production, dairy processing or packaging is of great concern for public health, especially for products purchased from local producers. the primary chemical contaminants in milk and dairy products are antibiotics, anthelmintic drugs, hormones, pesticides, heavy metals, mycotoxins, nitrites, etc. (khaniki, 2007; selim et al., 2015). organochlorine pesticides (ocps) and polychlorinated biphenyls (pcbs) are persistent in the environment and have the potential to bioaccumulate along the food chain, and this may cause adverse health effects (tsakiris et al., 2015; yu et al., 2011). most ocps have endocrine-disrupting effects and can cause hepatotoxicity, immunotoxicity and developmental abnormalities as well as have neurobehavioral effects (martins et al., 2013). despite their international ban starting from the 1980s, they are still detected in the environment, food, biota and humans (henríquez-hernández et al., 2017). the international agency for research on cancer (iarc) has classified most of the ocps as possibly carcinogenic to humans (group 2b) and “dioxin-like” pcb congeners as carcinogenic to humans (group 1) (klincic et al., 2016). bioaccumulation of occs through the food chain makes foods of animal origin the main source of human exposure to these contaminants (caspersen et al., 2016). the exposure of human organisms to environmental contaminants, through food, cannot be avoided, and this can lead to acute (short-term exposure) or chronic (long-term exposure) effects. the human risk assessment can be based on deterministic approaches by comparing it to a threshold toxicity value (cerna et al., 2016). following the banning of occs, the concentrations of occs in foodstuff decreased significantly (li, 1999). there are several papers presenting the level of pesticides in milk and dairy products (santos et al., 2015; santos et al., 2006; luzardo et al., 2012; avancini et al., 2013; deti et al., 2014) all over the world. also, there are papers on the human health risks associated with various pesticides in foodstuff (zhang et al., 2017; cui et al., 2015; lei et al., 2015; zhao et al., 2014). however, there is only limited information on health risk from pesticides in milk and dairy products (bedi et al., 2015; witzak et al., 2016). the aim of this study was to establish the level of 19 ocps and 7 pcbs in raw milk collected from a local market in baia mare city, north western romania, and to assess the risk associated with raw milk consumption for the local children and adults according to gender (male, female). 2.materials and methods 2.1. sampling a number of 10 raw cow milk samples were purchased from a local market in baia mare city, nw romania, during 2016. the cow milk is widely consumed by the local inhabitants. samples were collected in chemical-free glass bottles with teflon seals and frozen at -20°c until chemical analysis according to the procedure described by heck et al. (2007). ital. j. food sci., vol. 30, 2018 118 2.2. reagents, standard solution and crms the used solvents (acetonitrile, dichloromethane, ethanol and n-hexane) were of gas chromatography grade (merck, germany). anhydrous sodium sulphate, and florisil were used after heating overnight at 120°c. mix standard solution (en iso 6468 certan, ne7550) for ocps and pcbs was purchased from lgc standards (germany). the working standard solutions were prepared by diluting accurate volumes of mix standard solution in dichloromethane. milk powder certified reference materials (crms) purchased from lgc standards (germany) bcr-188 and bcr-450 were used for the quality control of the results. 2.3. sample preparation the extraction and clean-up of milk samples were carried out according to the method described by ennaceur et al. (2007). the milk samples were thawed at room temperature and homogenized. 20 ml sample was extracted 3 times with a 20/5/1 mixture (v/v/v) of n-hexane/acetonitrile/ethanol. the hexane layers were filtered over anhydrous sodium sulphate, and evaporated to 5 ml. afterwards, 1 ml was pipetted in a pre-weighed flask and evaporated to dryness. the difference between the final and the initial weight of the empty flask was used to calculate the lipid content of the sample. 4 ml of extract was purified with florisil and anhydrous sodium sulphate in a chromatographic mini-column. the extract eluted with a 27/3 elution mixture (v/v) of dichloromethane/n-hexane and evaporated to 1 ml, using a eva-ec1-s sample concentrator (vlm, germany) 2.4. sample analysis in this study the following compounds were determined: α-, β-, γ-, δ-, ε-isomers of hexachlorocyclohexane (expressed as hchs), 1,1,1-trichloro-2,2-bis-(chlorophenyl)ethane (ddt), 1,1-dichloro-2,2,-bis-(chlorophenyl)ethane (ddd), and dichlorodiphenylchloroethylene (dde), each with their isomers 4,4′and 2,4′-, expressed as ddts; aldrin, dieldrin, heptachlor, heptachlor epoxide isomer a, heptachlor epoxide isomer b, αendosulfan, β-endosulfan, hexachlorobenzene (hcb) and pcb congeners: tri (28), tetra (52), penta (101), hexa (138, 153), hepta (180) and octa (194). in order to separate, detect and quantify the occs, an agilent technologies 6890n gas chromatography equipped with a 63ni µ-electron-capture detector (gc-ecd) and an agilent j&w, db-1 capillary column (30 m l × 0.32 mm i.d. with film thickness 3.0 μm) were used. subsequent to extraction and evaporation, 1 μl of purified extract was injected in splitless mode, at 280°c. the gc oven temperature program consists of 4 stages: from 80°c to 196°c (rate 4°c/min, 2 min), from 196°c to 224°c (rate 4°c/min, 2 min), from 224°c to 240°c (rate 4°c/min, 2 min) and from 240°c to 275°c (rate 4°c/min, 2 min). occs were identified by comparison of each relative retention time with the calibration standards. confirmation of compounds was performed using an agilent technologies 6890n coupled with 5975b agilent technologies quadrupole gc-ms system. for quantification, multi-level calibration curves were created using standard solutions, and good correlations (r2=0.995) were achieved. 2.5. human health risk assessment for estimating long-term exposure through food intake, the average consumption over a period of time and the ratio between the average consumption and the reference values of ital. j. food sci., vol. 30, 2018 119 the contaminant amount (µg/kg body weight) that can be consumed daily over a lifetime without appreciable health risks were considered (lemos et al., 2016). the exposure to occs through food ingestion was evaluated by estimating the dietary exposure, by calculating the estimated daily intake (edi) from the amount of analyte found in milk and the daily milk consumption by population. edi is expressed in µg contaminant/person/day. the risk assessment was evaluated by calculation of hazard indices (his), expressed as the ratio between exposure and reference dose. if hi<1, the daily exposure does not have potentially adverse effects on the consumers’ health over the lifetime (lemos et al., 2016; li et al., 2015). 3. results and discussions 3.1. organochlorine pesticides in milk samples the obtained results for both crms were in agreement with the certified values. ocps, 4,4’-dde and 4,4’-ddt metabolites were found in all investigated milk samples. the most frequently found compounds were 4,4’-dde (11.5 ng/g lipid wt.), β-hch (10.1 ng/g lipid wt.) and pcb180 (5.08 ng/g lipid wt.). the concentration range, average and standard deviation along the incidence of ocps in the investigated milk samples are shown in table 1. for statistical purposes, the concentrations below the quantification limit (lq<0.05 ng/g lipid wt.) were considered equal to 0.5lq (le faouder et al., 2007). to compare the found ocp concentrations with the legislative threshold values, the maximum admitted concentrations (macs) expressed in µg/kg (order 23, 2007) were converted to ng/g lipid wt., considering a mean lipid wt. of 4% in milk (gebremichael et al., 2013). both the individual and the sum of ocp concentrations were below macs, according to romanian legislation (order 23, 2007). the graphical representation of the sum of hch, sum of cyclodienes, sum of endosulfans and sum of chloro-diphenyl aliphatic compounds concentrations is shown in fig. 1. hcb was detected in 80% of samples, with a maximum value of 4.32 ng/g lipid wt., significantly lower than mac. endosulfan (α and/or β isomer) was detected in 90% of investigated samples, in relatively low concentrations. the average concentration of αendosulfan was lower than β-endosulfan β isomer, in accordance with the higher predilection of β isomer compared to α isomer (tsiplakou et al., 2010). hch compounds were detected in all the analysed samples. the average concentrations of hch isomers varied in the following order: β-hch>γ-hch>α-hch>δ-hch>ε-hch. the total concentrations of hchs varied between 3.64 and 37.8 ng/g lipid wt., with average and standard deviation values of 6.53 and 3.55 ng/g lipid wt., respectively. compounds from cyclodiene group were detected in all milk samples, except one, but the obtained values were low. the average values ranged in the following order: dieldrin>heptachlor>heptachlor epoxide β>aldrin>heptachlor epoxide α. the concentrations of aldrin and dieldrin were much lower than their corresponding macs. chloro-diphenyl aliphatic compounds were determined in all analysed samples, the highest contents of total ddts were recorded in samples 8 (29.3 ng/g lipid wt.), 9 (18.4 ng/g lipid wt.) and 1 (17.8 ng/g lipid wt.), and the predominant component was 4,4’dde. the lowest value was reported in sample 5 (3.26 ng/g lipid wt.). ital. j. food sci., vol. 30, 2018 120 table 1. range, average and standard deviation values of ocps in milk samples. compound range (ng/g lipid wt.) average (ng/g lipid wt.) standard deviation (ng/g lipid wt.) mac (µg/kg / ng/g lipid wt.) samples > mac (%) incidence (%) hcb <0.05-4.32 2.18 1.61 10*/250 0 80 hexachlorcyclohexanes (hch) α-hch <0.05-6.49 1.65 2.01 4*/100 0 90 β-hch <0.05-17.4 10.14 5.21 3*/75 0 90 γ-hch (lindane) <0.05-7.16 2.92 2.81 8*/200 0 90 δ-hch 0.25-4.34 1.70 1.37 90 ε-hch <0.05-2.41 0.68 0.78 80 σhchs 3.64-37.8 17.1 10.3 cyclodienes aldrin <0.05-1.71 0.63 0.62 6*/150 0 70 dieldrin <0.05-6.49 2.45 2.04 6*/150 0 80 heptachlor <0.05-6.25 2.30 1.93 90 heptachlor epoxide β <0.05-2.03 0.94 0.66 90 heptachlor epoxide α <0.05-0.69 0.22 0.23 80 endosulfans β-endosulfan <0.05-1.82 0.57 0.44 80 α-endosulfan <0.05-1.27 0.42 0.47 70 σendosulfan <0.10-2.63 0.98 0.69 4*/100** 0 chloro-diphenyl aliphatic compounds 2,4’-dde <0.05-0.13 0.07 0.04 70 4,4’-dde 2.93-28.4 11.52 8.15 100 2,4’-ddd <0.05-0.48 0.12 0.15 70 4,4’-ddd <0.05-0.44 0.13 0.14 70 2,4’-ddt <0.05-0.24 0.12 0.08 80 4,4’-ddt 0.05-3.67 0.58 1.17 100 σddts*** 3.21-28.9 12.3 7.96 40*/1000 0 σocps 12.7-68.1 37.1 16.6 *according to romanian legislation (order 23, 2007). **according to codex alimentarius (fao/who, 2006). ***σ(4,4’-dde+4,4’-ddd+2,4’-ddt+4,4’-ddt). ital. j. food sci., vol. 30, 2018 121 figure 1. concentrations of total hchs, cyclodienes, endosulfans and total ddt (ng/g lipid wt.) in milk samples. the obtained values for ocps in milk samples were comparable with those reported by heck et al. (2007) in milk samples consumed in rio grande do sul, brazil, for hcb, αhch, 4,4’-dde and lower for lindan, aldrin, 2,4’-ddd and 2,4’-ddt. also, the average values obtained for σddt were comparable with the average values reported in yugoslavia and canada and lower than in egypt, mexico, ethiopia, ghana, india, tunisia and iran (gebremichael et al., 2013). the obtained average values were comparable with those reported by miclean et al. (2011) in milk samples collected in cluj-napoca area (romania), except for β-hch and β-endosulfan, which were lower, while dieldrin, heptachlor, 4,4’-ddd and 4,4’-ddt were higher. 3.2. polychlorinated biphenyls in milk samples pcbs were detected in all samples. the range, average and standard deviation values of pcb concentrations in milk samples are shown in table 2. the highest contribution to the total pcb content is represented by congener pcb180. the total concentrations of pcbs (expressed as the sum of the seven congeners: pcb 28, 52, 101, 138, 153, 180 and 194) varied between <0.60 ng/g lipid wt. (sample 7) and 15.3 ng/g lipid wt. (sample 4), with average and standard deviation of 9.12 ng/g lipid wt. and 4.99 ng/g lipid wt., respectively. in the case of non-dioxin like pcbs, the european commission set the mac to 100 ng/g lipid wt. from milk for the sum of the seven pcb congeners (pcb 28, 52, 101, 138, 153, 180, 194) (efsa, 2005; ec, 2006a,b) and recently set the mac as 40 ng/g lipid wt. from milk for the sum of six congeners (pcb 28, 52, 101, 138, 153 and 180) (ec, 2011) (pérez et al., 2012). in the analysed milk samples, the mac was not exceeded. the contribution of the pcb congeners to the total pcb content in analysed milk samples is shown in fig. 2. 0 5 10 15 20 25 30 35 40 1 2 3 4 5 6 7 8 9 10 concentration (ng/g fat) sa m pl in g po in t total hchs 0 2 4 6 8 10 12 1 2 3 4 5 6 7 8 9 10 concentration (ng/g fat) sa m pl in g po in t total cyclodienes 0 0,5 1 1,5 2 2,5 3 1 2 3 4 5 6 7 8 9 10 concentration (ng/g fat) sa m pl in g po in t total endosulfans 0 5 10 15 20 25 30 1 2 3 4 5 6 7 8 9 10 concentration (ng/g fat) sa m pl in g po in t total ddts ital. j. food sci., vol. 30, 2018 122 table 2. range, average and standard deviation values of pcbs in milk samples compound range (ng/g lipid wt.) average (ng/g lipid wt.) standard deviation (ng/g lipid wt.) incidence (%) pcb28 <0.05-0.44 0.18 0.17 70 pcb52 <0.05-0.23 0.09 0.07 80 pcb101 <0.05-3.71 0.89 1.42 50 pcb138 <0.05-2.26 0.54 0.75 80 pcb153 <0.05-5.37 1.09 1.88 40 pcb180 <0.05-10.4 5.08 3.30 80 pcb194 <0.05-5.32 1.25 2.11 30 σpcb* <0.60-15.3 9.12 4.99 *σpcb – sum of pcb congeners (pcb 28, 52, 101, 138, 153, 180, 194). figure 2. pcb congeners contribution of total pcb content in milk samples. 3.3. estimated daily intake in this study, the long-term exposure was assessed by determination of the average intake of ocps and pcbs through milk consumption for the three population groups male, female and children and their comparison with the reference values, namely acceptable daily intake (adi), set by the joint fao/who expert committee on food additives (fao/who, 2006). the daily intake of milk for the three investigated population groups was determined based on milk consumption frequency questionnaires. these questionnaires were filled out by 75 individuals 55 adults (18-75 years old), among them 28 male, 27 female and 20 children (6-10 years old) and indicated an average milk daily intake of 300 g milk/person/day for male, 200 g milk/person/day for female and 500 g milk/person/day for children and the average body weight 75 kg for male, 65 kg for female and 30 kg for children. the values obtained for the estimated daily intake (edi) associated with milk consumption are shown in table 3. in calculations, the pesticides concentrations lower than lq were considered zero (luzardo et al., 2012). 0% 20% 40% 60% 80% 100% 1 2 3 4 5 6 7 8 9 10 concentration (ng/g fat) sa m pl in g po in t pcb2 8 pcb5 2 pcb1 01 pcb1 38 ital. j. food sci., vol. 30, 2018 123 table 3. average estimated daily intake (edi) and acceptable daily intake (adi) of organochlorine compounds (µg/kg bw*/day). compound edi, µg/kg bw/day adi, µg/kg bw/day*** female male children hexachlorbenzene 0.00033 0.00043 0.00181 hexachlorcyclohexanes (hch) α-hch 0.00034 0.00044 0.00185 β-hch 0.00138 0.00180 0.00748 γ-hch (lindane) 0.00039 0.00051 0.00213 5 δ-hch 0.00023 0.00030 0.00124 ε-hch 0.00010 0.00013 0.00056 σhch 0.00223 0.00255 0.01207 0.3 cyclodienes aldrin 0.00011 0.00014 0.00060 0.1 dieldrin 0.00037 0.00048 0.00199 0.1 heptachlor 0.00032 0.00041 0.00171 heptachlor epoxide β 0.00013 0.00017 0.00071 heptachlor epoxide α 0.00004 0.00005 0.00020 σheptachlor 0.00043 0.00056 0.00232 0.1 endosulfans α-endosulfan 0.00009 0.00011 0.00046 β-endosulfan 0.00007 0.00009 0.00038 σendosulfans 0.00016 0.00020 0.00084 6 chloro-diphenyl aliphatic compounds 2,4’-dde 0.00001 0.00001 0.00006 4,4’-dde 0.00142 0.00184 0.00768 2,4’-ddd 0.00002 0.00003 0.00011 4,4’-ddd 0.00002 0.00003 0.00011 2,4’-ddt 0.00002 0.00002 0.00009 4,4’-ddt 0.00008 0.00009 0.00039 σddt** 0.00154 0.00200 0.00834 10 σocps 0.00495 0.00644 0.02682 pcb2013 pcb28 0.00003 0.00004 0.00017 pcb52 0.00001 0.00002 0.00008 pcb101 0.00022 0.00028 0.00118 pcb138 0.00008 0.00010 0.00043 pcb153 0.00035 0.00045 0.00187 pcb180 0.00077 0.00100 0.00417 pcb194 0.00053 0.00069 0.00286 σoccs 0.00607 0.00789 0.03287 *bw=body weight. ** 2,4’-dde + 4,4’-dde + 2,4’-ddd + 4,4’-ddd + 2,4’-ddt + 4,4’-ddt. *** efsa, 2013. ital. j. food sci., vol. 30, 2018 124 the obtained values for the estimated daily intake of investigated organochlorine compounds, through milk consumption, for adults (male and female) and children, in baia mare area were far below adi values set by the european food safety authority (efsa), according to table 3 (efsa, 2013). for each investigated analyte, the average estimated daily intake varied in the following order: edichildren>edimale>edifemale, according to the daily milk consumption and body weight. for the investigated children, the edi value calculated for σheptachlor provides 2.32% from adi value, set for the sum of these cyclodienes. the values obtained for edi of total ddt were below the efsa recommended value (10 µg/kg bw) and were much lower (4 orders of magnitude). the obtained edi values for total ddt were comparable (the same order of magnitude) with the values reported in spain, poland and china and were lower (with an order of magnitude) than those obtained in ethiopia, mexico, egypt and iran, through cow milk consumption (gebremichael et al., 2013). the edis obtained in this study were comparable with those determined by luzardo et al. (2012) for adult and child consumers of milk in spain for hexachlorobenzene, dieldrin, heptachlor and higher (but the same order of magnitude in both studies) for α-, β, γ-, δhch, aldrin, endosulfan, 4,4’-ddt. the average edi values for total ocps were comparable in both studies for adults and children. 3.4. risk assessment in order to assess the non-carcinogenic risk from exposure to milk through consumption of the three investigated population groups, the his were calculated by dividing the edi values to the us epa reference doses (rfds) (tsakiris et al., 2015). us epa provides rfd values only for γ-hch (0.3 μg/kg bw/day) and 4,4’-ddt (0.5 μg/kg bw/day) (usepa, 2006). the obtained values for his are shown in table 4. table 4. hazard indices for lindane and 4,4’-ddt. compound hazard index female male children γ-hch (lindane) 0.0013 0.0017 0.0071 4,4’-ddt 0.00016 0.00018 0.00078 the values of his for lindane and 4,4’-ddt were far below 1, indicating no health risk for the investigated population groups through raw milk consumption, in the studied area. 4. conclusions the tendency of organochlorine compounds to accumulate in fatty tissues, their long persistence and high acute health risk raise concerns about their impact on health due to chronic dietary exposure to low concentrations. this study revealed low dietary intake of organochlorine compounds from milk consumption, in females, males and children. the health risk assessment was calculated based on specific values for the investigated area and the intake of organochlorine pesticides. the specific exposure factors were obtained using questionnaires regarding the structure of the diet for three consecutive days. for each investigated analyte, the average estimated dietary exposure varied in order edichildren>edimen>edifemale according to the daily consumption of milk. the average daily ital. j. food sci., vol. 30, 2018 125 intake of organochlorine compounds through milk for the investigated residents exposed to 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ddts and hchs through dietary exposure in nanjing, china, chemosphere 177:211-216. zhao z., zhang l., cai y. and chen y. 2014. distribution of polycyclic aromatic hydrocarbon (pah) residues in several tissues of edible fishes from the largest freshwater lake in china, poyang lake, and associated human health risk assessment, ecotox. environ. safe. 104:323-331. paper received june 2, 2017 accepted november 9, 2017 paper 148 ital. j. food sci., vol. 28 2016 keywords: removal, peracetic acid, cutting boards, escherichia coli effectiveness of sanitizing agents in inactivating escherichia coli (atcc 25922) in food cutting board surfaces. removal e. coli using different sanitizers cezar augusto beltrame, eduarda boff martelo, raíza de almeida mesquita, ieda rottava, juliana barbosa, clarice steffens*, geciane toniazzo, eunice valduga and rogério luis cansian department of food engineering, uri, erechim, av. 7 de setembro, 1621, cep 99700-000, erechim, rs, brazil, *corresponding author: tel. 550xx543520-9000, email: claristeffens@yahoo.com.br abstract the objective of this study was to investigate escherichia coli adhesion on new and used polyethylene cutting board surface and evaluate it’s removal using different sanitizer (peracetic acid, chlorhexidine, sodium hypochlorite and organic acids). results indicated that the number of adherent cells increased with time in both surfaces evaluated. evaluating the sanitizer action, 0.5% peracetic acid was more effective in removal e. coli than chlorhexidine and organic acids at same concentration in both surfaces. peracetic acid and sodium hypochlorite also showed effectiveness at concentrations of 0.2% and 0.5% on new surfaces, respectively. 0.8% of chlorhexidine and 2.0% of organic acids showed similar effectiveness in the removal e. coli on new and used surfaces, respectively. these results suggest that peracetic acid is considerable promise sanitizer for application in surfaces of the food processing industry. mailto:claristeffens%40yahoo.com.br?subject= ital. j. food sci., vol. 28 2016 149 introduction e. coli is a gram-negative bacteria that present surface layer organizations of the type fimbriae, exopolysaccharides (eps) or flagella, that favor the adherence to materials or host cell surfaces motility and pathogenicity. food can become contaminated with e. coli when animals are slaughtered or processed, even if precautions are taken and also when it is handled by a person infected with e. coli, or from cross-contamination (beumer and kusumaningrum, 2003). food residues left on food processing or handling equipment may provide a niche of microor ganisms that can rapidly grow. the growth of pathogenic bacteria can result in crosscontamination from food processing surfaces such as cutting boards to food products (montville et al., 2012). in the food industry, good manufacturing, hygienic production and regular cleaning and disinfection procedures are very important, since food safety and quality are determined by the efficacy of sanitizer agents (krolasik et al., 2010). bacteria have the ability to adhere to any surface including, but not limited to, glass, stainless steel, polypropylene, rubber and wood (coquet et al., 2002; teixeira et al., 2008). to prevent bacterial attachment on surfaces the choosing an appropriate sanitizer is very important for achieving a satisfactory end result in microbiological indexes. many sanitizers have been broadly used across many industries to reduce pathogenic bacterial contamination in food products or on kitchen utensils, because these compounds have been shown to effectively inactivate foodborne pathogenic bacteria (cabeça et al., 2012; frank, 2003; rossoni and gaylarde, 2000). therefore, more studies into the bactericidal properties of sanitizers at different concentrations and contact times are required to define the correct application. many researchers have examined materials employed in manufacturing of foods contact surfaces such as stainless steel (cabeça et al., 2012; frank, 2003; krolasik et al., 2010; rossoni and gaylarde, 2000; ryu and beuchat, 2005), but few reports bacterial removal on commercial polyethylene cutting boards used in industrial food preparation have been published to date. the objective of this study was to evaluate the e. coli adhesion on new and used cutting board surfaces and removal with different sanitizers used in food industry (peracetic acid, chlorhexidine, sodium hypochlorite and organic acids). for each sanitizer tested, different concentrations were evaluated over 72 h, determining the sanitizer’s effectiveness on new and used polyethylene cutting boards. material and methods surface material the food processing surfaces evaluated in this study was new and used polyethylene cutting board, white high-density polyethylene (hdpe plastic). the boards were obtained from cutting room of a slaughter unit, where the used surfaces had around of 45 days of handle. surfaces materials with 1.0 cm x 1.0 cm plates were cut, cleaned by brushing employing liquid detergent and water, and rinsed with distilled water. they were immersed in 70% ethanol, for 1 h, to fat removal, and again rinsed with distilled water and air dried. the surfaces were exposed to ultraviolet light 254 nm for 1 h to sanitize them, as described by parizzi (1999), before deposition of any bacterial cultures. adhesion of escherichia coli on food processing surfaces bacterial strains were obtained from seattle, usa, 1946 (american type culture collection; rockville, md, usa). for the study of adherence was used an e. coli (atcc 25922) strain, grown previously in luria bertani broth lb (tryptone 10.0 g l-1, yeast extract 5.0 g l-1, nacl 5.0 g l-1) and incubated at 35ºc (±2) for 24 h. e. coli was chosen as indicator organism, commonly present in industrial food plants. the cleaned surfaces were immersed, at 25ºc, in erlenmeyer containing 100 ml of lb supplemented with a suspension of bacterial cells in order to obtain a count of 103 cfu ml-1. the sterilized surface, for each time, was immersed in these erlenmeyer with sterilized forceps and incubated at 35°c in lb broth. the quantities of adhered cells per square centimeter were evaluated for 72 h of contact time (0.1, 1, 3, 6, 12, 24, 48, and 72 h) on new and used surface. the initial time (0 h) corresponds to the analysis performed immediately after the immersion of the surfaces in the erlenmeyer containing the medium culture and the bacterial suspension. triplicates were performed for each treatment. after the incubation, the surfaces were withdrawn from the bacterial suspension e. coli and transferred to tubes, containing 10 ml of peptone water 0.1% (p/v) for 1 min, to remove planktonic cells. subsequently, immersed in tubes containing 5 ml of the same diluent solution and vortex for 1 min, to remove sessile cells (parizzi, 1999). the contact areas were swabbed and the adhered microorganisms in the swabs were transferred to tubes, containing 10 ml of peptone water 0.1% (p/v) sterilized at 121°c, for 15 min. the tube was stirred using a vortex for 10 s to release the bacteria from the swab. next, 1 ml of solution was carefully plated on lb agar, incubated at 35-37°c for 24 h, to colony counting. 150 ital. j. food sci., vol. 28 2016 efficiency of different sanitizers against escherichia coli on food processing surfaces the sanitizers used in this study were chosen to represent those used in the food industry. the following sanitizers were used: peracetic acid 15% (johnson diversey, são paulo-sp, brazil), chlorohexidine 20% (ad foods industry ltda, laguna-sc, brazil), sodium hypochlorite 10% (csm chemical products ltda, chapecósc, brazil) and organic acids (formulated with lactic acid-30%, citric acid-3%, ascorbic acid3%, and salts of fatty acids-7% in water). for each sanitizer, different concentrations (0, 0.2, 0.5, 0.8 and 2.0%) were investigated for 10 min of exposure, to evaluate their efficiency in removal the adhered cells. these agents were diluted in sterilized distilled water according to the supplier’s instructions. after this treatment, the surfaces were immersed (separately) in 10 ml of sterilized water, for 1 min and repeated twice to removal the excess of sanitizer. the counts of bacterial adhesion and inactivation by sanitizers were carried out using swab on cutting boards, evaluated through the standard plate count method. then, plated on lb agar, incubated at 35-37°c for 24 h to colony counting. all determinations were performed in triplicate and the results expressed in terms of mean values (parizzi et al., 2004). statistical analysis descriptive analyses, including the mean value and variability (standard deviation) and graphic displays were performed. results obtained in experimental design described previously were performed considering a 95% confidence level (p<0.05) by the tukey’s test, using the software statistica 8.0 (statsoft inc®, usa). results e. coli adhesion in food processing surfaces fig. 1 show the number of e. coli adhered on new and used cutting board surfaces with different contact times. numbers of e. coli were estimated and expressed as log 10 colony forming units per cm2 (log cfu cm-2). a fast adhesion of e. coli on both surfaces studied were observed for up to 12h, becoming constant after 24h on used surfaces, when the maximum population reached (6.92 log cfu cm-2). a significant difference (p<0.05) was observed in the intensity of adhesion between the surfaces until 24h. effect of different sanitizers for inactivating escherichia coli figs. 2, 3, 4 and 5 show the data’s of inactivation e. coli on new and used cutting boards fig. 1 counts of e. coli on new and used cutting board surfaces without the presence of sanitizers, over 72 h of contact time. bars represent the standard errors of the mean from triplicate experiments and * simbolize significant differece (p<0.05). fig. 2 the efficacy of different concentrations of peracetic acid (0, 0.2, 0.5, 0.8 and 2.0%) on the reduction of e. coli on: (a) new and (b) used cutting board surfaces, over 72 h. bars represent the standard errors of the mean from triplicate experiments. a b ital. j. food sci., vol. 28 2016 151 sanitizer (2.0%) was effective until 1h of contact (fig. 3 b), and reduce around 2.5 log cfu cm-2 of cells after 72 h. in this way, chlorhexidine sanitization had a better effect on removal attached cell on new surfaces. for 0.8% chlorhexidine was observed completely e. coli removed on new boards with 1h of contact, but not was effective on the used boards. consequently, the lower concentrations investigated (0.2 and 0.5%) not show complete inactivation. on used cutting board, all concentrations of sanitizer studied not inactive bacteria after 1 h of contact. according to the suppliers, also organic acids are suggested in a concentration of 0.5%. in this way, this concentration showed efficiency only for 10 min, on both surfaces evaluated. higher concentrations, 0.8 and 2.0%, were effective for removing e. coli up to 1 and 3 h of contact on new surfaces, respectively (fig. 4a). the results also indicated that the amount of adherent cells reduced 2.4 log with 2.0% organic acid and was efficient for 1h on used surfaces (fig. 4b). this fig. 3 the efficacy of different concentrations of chlorhexidine (0, 0.2, 0.5, 0.8 and 2.0%) on the reduction of e. coli on: (a) new and (b) used cutting board surfaces, over 72 h. bars represent the standard errors of the mean from triplicate experiments. fig. 4 the efficacy of different concentrations of organic acid (0, 0.2, 0.5, 0.8 and 2.0%) on the reduction of e. coli on: (a) new and (b) used cutting board surfaces, over 72 h. bars represent the standard errors of the mean from triplicate experiments. with different concentrations (0, 0.2, 0.5, 0.8 and 2.0%) of peracetic acid, chlorhexidine, organic acid and sodium hypochlorite sanitizers, respectively, over 72 h of contact time. fig. 2 a and b demonstrates that the bacteria exhibited a significant decrease in the survival rate of viable cells after treatment with peracetic acid. the concentration of 0.5% peracetic acid indicated by the supplier was completely effective for inactivating e. coli at all times investigated on new surfaces, while 0.2% peracetic acid was effective for up to 6 h, and able to reduce the number of adhered cells of 4.4 and 5.0 log for 48 and 72 h, respectively (fig. 2a). in fig. 3 a is possible to observe that only the highest concentration of chlorhexidine (2.0%) was completely effective for inactivating e. coli on new surfaces, for 72 h. in used surfaces this a b a b 152 ital. j. food sci., vol. 28 2016 low efficiency of organic acids can be explained by the fact that the compounds are in a dissociated form at the product application moment and dilute the sanitizer, so a higher dissociation leads to lower efficiency (beltrame et al., 2012). fig. 5 demonstrates the efficiency of sodium hypochlorite against e. coli. the concentration (0.5%) indicated by the supplier was able to remove bacteria cells, at all exposure times, on new surface (fig 5a). on the other hand, to obtain the same effect, on the used surface, a concentration of 2.0% was required (fig 5b). effectiveness correlation between different sanitizers the sanitation in food surfaces, including cutting boards is critical for the control of microbial contamination of foods and is a significant concern of food preparation and processing industries and public health agencies. in this way, to compare the efficacy of sanitizers (peracetic acid, chlorhexidine, sodium hypochlorite and organic acids) used in the food industries was evaluated a concentration of 0.5%, after 3 h of contact, on new and used surfaces for e. coli removal (fig. 6). the concentration of 0.5% correspond the minimum recommended by the supplier and 3 h of contact is the maximum time (practiced by the food industry) for disinfecting surfaces used. comparing the sanitizers, the peracetic acid was completely effective in removing e. coli on new and used surfaces (p<0.05), as well as for sodium hypochlorite only new surfaces. it was found that chlorhexidine and organic acids exhibit reductions on new and used cutting boards (fig. 6), without significant difference between the sanitizers (p>0.05), but less effectively than other sanitizers evaluated in this work (p<0.05). discussion the differences of adhesion on cutting boards could be due microbiological, physical and chemical parameters related to the polyethylene. particularly, in this study can be verify that the used surfaces have higher counts until 24 h (fig. 1), possibly due to the surface characteristics, which visually present more cracks and wear by 45 days of use in the slaughter unit. the surface topography has been widely studied, since microorganisms adhere more easily in fissures or cracks, and can resist cleaning and disinfecting procedures (hilbert et al., 2003; parizzi et al., 2004). thus, macroscopic and microscopic characteristics are crucial for microbial adhesion, reflected in the food contamination by spoilage or pathogenic microorganisms (vadillo-rodríguez et al., 2004). after 48 h the number of adherent cells remained constant over time in both surfaces. this was fig. 5 the efficacy of different concentrations of sodium hypochlorite (0, 0.2, 0.5, 0.8 and 2.0%) on the reduction of e. coli on: (a) new and (b) used cutting board surfaces, over 72 h. bars represent the standard errors of the mean from triplicate experiments. fig. 6 the efficacy of different sanitizers (concentration of 0.5%), over 3 h, on the reduction e. coli from new and used cutting board surfaces. means (± standard deviations) followed by the same letters represents no significant difference at 5% level (tukey’s test) between the sanitizers and surfaces. a b ital. j. food sci., vol. 28 2016 153 also observed in surface reaches saturation level with greater numbers of planktonic cells and not result in greater number of adherent cells (hood and zottola, 1997). the results of bacteria removal demonstrate that from 48 h of contact (in used surface), even with concentrations 4 times superior than recommended by suppliers, peracetic acid was not effective. this suggests that the attachment increase during the contact time. similar results was found by other researcher (adetunji and isola, 2011). miller et al. (1996) evaluated the potential of water for removal e. coli 0157:h7 from polyethylene cutting boards, and the microorganism was incubated for 0 to 30 h, at 37°c, to determine their inhibitory potential. the authors observed an increase in bacteria cells on the boards during the first 30 min of contact, and the water removed 2.3 log cfu cm-2 from the surface. cabeça et al. (2012) carried out a study of disinfection on stainless steel surfaces, using biguanide and peracetic acid, and verified that they were able to reduce e. coli cells adhered of 2.2 and 2.1 log cfu cm-2 for 10 min, respectively, with a concentration of 0.5% (w/v). in the present work was possible reduce 3.5 log cfu cm-2 after for 3 h, at the same concentration of peracetic acid on new and used polyethylene cutting boards. peracetic acid disinfectant activity is based on the release of active oxygen. it disrupts the chemiosmotic function of the lipoprotein cytoplasmic membrane and transports through dislocation or rupture of cell walls. it may also be effective on outer membrane lipoproteins, facilitating action against gram-negative bacteria. intracellular peracetic acid can also oxidize essential enzymes. thus, vital biochemical pathways, transported through the membrane and intracellular solute levels of are damaged, and alterations in the dna molecule (kitis, 2004). in this study, all concentrations of chlorhexidine not were effective for the removal of bacteria after 1 h. this low activity may be due mechanism action, rapid absorption of bacterial cells, resulting in several cytological modifications that affect permeability and optical properties. studies have shown that chlorhexidine reacts with the cell from lipophobic groups, causing a disorientation of the lipoprotein membrane and generating a change in osmotic barrier function (kudavidanage et al., 2009). chlorhexidine is a cationic molecule with a wide antimicrobial spectrum against both gram-positive and gram-negative bacteria (mohammadi and abbott, 2009). this group of biguanides differs from other cationic biocides that interact only superficially with the lipid bilayer altering fluidity through cation displacement and head group bridging (gilbert and moore, 2005). in a study performed by houari and di martino (2007) the authors verified that chlorhexidine diacetate (fluka) was able to inhibit the biofilm formation of different bacteria such as e. coli, klebsiella pneumoniae, pseudomonas aeruginosa and staphylococcus epidermidis at conventional in-use concentrations. second patel (2005), the bacteria resistance to antimicrobial agents begins at the attachment phase and increase with the biofilm age. although, bacteria in biofilms are surrounded by an extracellular matrix that might physically restrict the diffusion of antimicrobial agents, this does not seem to be a predominant mechanism of biofilm-associated antimicrobial resistance. another indication of high counts are the surface roughness and hydrophobicity that can significantly affect the attachment, formation places for microorganism’s accommodation and permanent adhesion. movassagh et al. (2010), showed counts of 7.69 log ufc cm-2 for e. coli o 111 on polyethylene surfaces. second the authors, bacteria encountered in food processing environments can be very hardy and difficult to remove. bacterial attachment and subsequent survival involved interactions between a bacterial cell, surface and surrounding microenvironment. the removal bacteria by sodium hypochlorite can be associated with water forms hypochlorous acid, which contains active chlorine (a strong oxidizing agent). chlorine exerts its antibacterial action by irreversible oxidation of a sulfhydryl group of essential enzymes to microorganisms, disabling metabolic functions of the bacterial cell (poggio et al., 2012). sodium hypochlorite may also have a deleterious effect on the bacterial dna, involving the formation of chlorinated derivatives of nucleotide bases. furthermore, it has been reported that sodium hypochlorite can induce disruption of the bacterial membrane (mc donnel and russel, 1999). organic acids have an inhibitory action in the undissociated form, from 100 to 600 times greater than the dissociated form. undissociated organic acid can permeate the cell membrane by diffusion and release protons in the cytoplasm of the cell. the influx of protons induces acidification of the cytoplasm and dissipates the membrane proton potential (kitko et al., 2009). this inhibits the transport mechanism for the substrate, energy generation and synthesis of macromolecules (stopforth et al., 2003). conclusions in both surfaces studied it was observed a fast adhesion of e. coli and present lower counts in new surface when compared with used. the biofilm formed on used polyethylene cutting boards reduces significantly the action of sanitizers. among the sanitizers evaluated, peracetic acid was the most efficient for reducing e. coli counts. on the new cutting boards concentration of 0.5% peracetic acid was effective in eliminating e. coli adhesion during 72 h evaluated and un154 ital. j. food sci., vol. 28 2016 til 1 h in used surface. hypochlorite, chlorhexidine and organic acids demonstrated similar effects until 1h, reducing the total adhesion with 0.8 and 2.0% on new and used cutting boards, respectively, although 2.0% sodium hypochlorite has been effective for total removal until 72 h. the order of efficacy in removing e. coli was as follows: peracetic acid, sodium hypochlorite, chlorhexidine and organic acids. the results of the study showed the importance of hygiene procedures on surfaces that come into contact with food. it was found that biofilm formation can occur over a short time, which emphasizes the need for good cleaning procedures during food processing.  acknowledgements the authors thank cnpq, capes, fapergs, science and technology secretary rs and uri erechim for the financial support for this research. references adetunji v.o. and isola t.o. 2011. adhesion of e. coli and e. coli o157: h7 isolates from a typical tropical abattoir on wood, steel and glass 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(reading, england). 150:1015-1022. paper received october 7, 2014 accepted july 10, 2015 90 issn 1120-1770 online, doi 10.15586/ijfs.v35i3.2354 p u b l i c a t i o n s codon italian journal of food science, 2023; 35 (3): 90–98 ganoderma lucidum extract reverses hepatocellular carcinoma multidrug resistance via inhibiting the function of p-glycoprotein in vitro and in vivo jing li1, lin cao1, cheng yuan1, zhaojian jiang1, hongfei cai1, wendong xu1, yaming han1, liang chen1, qin zhang1, renwang jiang2*, juyan liu1,3* 1guangzhou hanfang pharmaceutical company limited, national engineering research center of pharmaceutical processing technology of traditional chinese medicine and drug innovation, guangdong provincial key laboratory of medicinal lipid, guangzhou, china; 2jinan university, guangzhou, china; 3guangzhou pharmaceutical holdings limited, guangzhou, china *corresponding authors: renwang jiang, jinan university, guangzhou, china. email: rwjiang2008@126.com; juyan liu, guangzhou pharmaceutical holdings limited, guangzhou, china email: ljyan65@163.com received: 10 april 2023; accepted: 12 july 2023; published: 12 august 2023 © 2023 codon publications open access paper abstract cancer is a leading cause of death globally. chemotherapy still plays an indispensable role in the clinical treatment of cancer. however, the emergence of multidrug resistance (mdr) has greatly obstructed the further application of chemotherapy agents. ganoderma lucidum (g. lucidum) is a traditional chinese medicine as well as an edible mushroom. in this study, we first explored the effect of six extract samples derived from g. lucidum on the cell viability of adriamycin-resistant human hepatocellular carcinoma multidrug-resistant cell subline (hepg-2/ adm). all these samples showed no obvious toxicity to cells; however, only g. lucidum ethanol extract could reverse resistance to doxorubicin and paclitaxel. then the p-glycoprotein (p-gp) function in hepatocellular carcinoma g2 (hepg-2) and hepg-2/adm cells was determined after incubated with these samples, and we found that g. lucidum ethanol extract could inhibit p-gp function in vitro. furthermore, g. lucidum ethanol extract could reverse resistance to paclitaxel in hepg-2/adm tumor-bearing mice in vivo, while the protein expression level of p-gp was unchanged. taken together, these results indicated the potential role of g. lucidum ethanol extract in reversing mdr in the clinical treatment of hepatocellular carcinoma. keywords: ganoderma lucidum, hepatocellular carcinoma, multidrug resistance, paclitaxel, p-glycoprotein introduction cancer has become a leading cause of death globally, and has severely threatened the life expectancy of humans (bray et  al., 2021). it is estimated that about 19.3 million new cases and 10 million cancer deaths occurred worldwide in 2020 (sung et  al., 2021). although different approach to treating cancer have developed successfully, the incidence and mortality are still increasing rapidly. the main cause of failure of anti-cancer therapy is the emergence of acquired resistance, among which multidrug resistance (mdr) has played a major role (gottesman et al., 2002). mdr is a phenomenon in which cancer cells develop resistance to multiple chemotherapy agents with distinct structures or mechanisms (kumar and jaitak, 2019). p-glycoprotein (p-gp), encoded by adenosine triphosphate (atp)-binding cassette (abc) subfamily b member 1 (abcb1) is the extensively studied factor in the development of mdr. abcb1 could utilize the energy of atp to actively transport drugs out of cytoplasm, resulting in the survival of cancer cells (kumar and jaitak, 2019). p-gp has been confirmed to be involved in the resistance to several anti-cancer agents, such as paclitaxel, doxorubicin, cisplatin, etoposide, etc. (gottesman et  al., 2002; szakacs et  al., 2006; zeino et  al., 2015). in spite of great efforts in exploring p-gp inhibitors, most of italian journal of food science, 2023; 35 (3) 91 ganoderma lucidum extract reverses multidrug resistant hcc cell lines and culture human hepatocellular carcinoma (hcc) cell line hepg-2 and the multidrug-resistant p-gp overexpressing hcc cell line hepg-2/adm were provided by cancer institute & hospital, chinese academy of medical sciences (beijing, china). all cell lines were cultured in dulbecco’s modified eagle’s medium (dmem; corning, new york, us) containing 10% fetal bovine serum (fbs; life technologies, new york, us) and 1% penicillin– streptomycin solution (life technologies). cell lines were maintained at 37°c in a humidified atmosphere with 95% air and 5% co2. cell viability assay cells were seeded in 96-well plates at a density of 4×103 cells per well and cultured overnight. the cells were treated with various concentrations of samples 1–6 or compounds in the presence or absence of verapamil for 48 h. mtt, 20 μl, was added into each well and incubated for an additional 4 h. finally, purple formazan crystals were dissolved in 150-μl dimethyl sulfoxide (dmso) and the absorbance was observed at 490 nm by multimode microplate reader (bio-rad laboratories, ca, us). rhodamine-123 intracellular accumulation assay cells were seeded in six-well plates at a density of 1×105 cells per well and cultured overnight. the cells were treated with 500 μg/ml of samples 1–6 for 48 h and incubated with 10-μm rh-123 for an additional 4 h in dark at 37°c. then the cells were collected, washed with phosphate-buffered saline (pbs) thrice, and analyzed by flow cytometer (bd bioscience, san jose, ca, us). the data were analyzed using the flowjo 7.6.1 software. xenograft mouse model experiments balb/c nude mice were purchased from guangdong province medical animal center and monitored under specific pathogen-free conditions. all animal experiments complied with the national institute of health guide for the care and use of laboratory animals, 8th edition (national institutes of health, 2011). hepg-2/ adm cells (1×107 in 200 μl) were collected and injected subcutaneously into the right flank of mice. treatments were initiated when tumors reached a mean volume of 100 mm3. mice were randomly divided into the following four groups (n = 5): (1) control group (mice were given normal saline [ns]); (2) paclitaxel group (mice were given 18-mg/kg paclitaxel); (3) sample 6 group (mice were given 400-mg/kg sample 6); and (4) sample 6 + paclitaxel them failed to show ideal efficacy while possessing major toxicities (chen et al., 2016). therefore, discovering novel p-gp inhibitors with potent efficacy and little toxicity is required urgently. ganoderma lucidum (g. lucidum) is a traditional chinese medicine widely used in china (named ling zhi) for hundreds of years (ahmad, 2018). as a medicinal and edible mushroom, g. lucidum has proved to possess diverse pharmacological activities, including antioxidant (zhang et  al., 2021), antidiabetic (ma et  al., 2015), antimicrobial (cor et al., 2018), anti-inflammatory (cai et al., 2016), immunomodulatory (li et al., 2020), and anticancer effects (jin et al., 2016). researchers have found that g. lucidum could exert its anticancer effects by directly killing cancer cells, inhibiting angiogenesis, inducing cell differentiation, and activating immune response of the host (ahmad, 2018). however, whether constituents of g. lucidum could reverse p-gp-related mdr in vivo has not been studied amply. in this study, we explored the effect of six extract samples derived from g. lucidum on the cell viability and mdr of adriamycin-resistant human hepatocellular carcinoma multidrug-resistant cell subline (hepg-2/ adm). then the difference in p-gp function between hepatocellular carcinoma g2 (hepg-2) and hepg-2/ adm cells was compared, and we further investigated the effect of extract samples on p-gp function. finally, the effect of g. lucidum ethanol extract alone and in combination with paclitaxel on tumor growth and expression level of p-gp was measured in hepg-2/adm tumor-bearing mice in vivo. materials and methods materials and chemicals samples 1–6 were obtained from guangzhou hanfang pharmaceutical co. ltd. (guangzhou, china). sample 1 was the major product of g. lucidum spore oil using supercritical fluid co2. sample 2 was the product of g. lucidum spore oil using supercritical fluid co2 with modifier ethanol. sample 3 was ethanol extract of the remains after using supercritical fluid co2. sample 4 was water extract of the remains after using supercritical fluid co2. sample 5 was the water extract of g. lucidum. sample 6 was the ethanol extract of g.  lucidum. verapamil, paclitaxel, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-2h-tetrazolium bromide (mtt), and rhodamine-123 (rh-123) were purchased from sigmaaldrich (deisenhofer, germany). doxorubicin (dox) was obtained from zhejiang hisun pharmaceuticals co. ltd. (zhejiang, china). 92 italian journal of food science, 2023; 35 (3) li j et al. one-way anova. two-tailed student's t-test was used to compare difference between two groups. statistical analysis was performed using the spss 22.0 software; p < 0.05 were considered statistically significant. results g. lucidum samples showed no obvious toxicity to hepg-2/adm cells we measured the viability of hepg-2/adm cells after incubation with various concentrations of samples 1–6 using mtt assay to evaluate whether the six samples derived from g. lucidum possessed toxicity to multidrugresistant hcc cells. as shown in figure 1, all samples showed little toxicity for hepg-2/adm cells. over 90% cells were alive even when the concentration reached 1,000 μg/ml. the results indicated that these samples derived from g. lucidum had no obvious toxicity to multidrug-resistant hcc cells, which could be candidates for investigating the effect of g. lucidum extract on mdr. g. lucidum ethanol extract reversed multidrug resistance in hepg-2/adm cells in order to investigate the effect of g. lucidum extract on the mdr of hepg-2/adm cells, we measured cell viability after incubation with doxorubicin or paclitaxel in combination with samples 1–6 or a classic inhibitor of p-gp (verapamil). as shown in table 1, verapamil could potently suppress the function of p-gp and diminish cell viability at the 50% inhibitory concentration (ic50) of doxorubicin and paclitaxel, which proved the mdr characteristic of hepg-2/adm cells. to our surprise, we also observed that only sample 6 could effectively reverse resistance to doxorubicin and paclitaxel at ic50 and 20% inhibitory concentration (ic20) and further reduced the viability of hepg-2/adm cells. the fold reversal of resistance to doxorubicin and paclitaxel was almost three times at ic50, while that was nearly five to seven times at ic20. these data indicated that g. lucidum ethanol extract could reverse mdr in hcc cells. g. lucidum ethanol extract inhibited the function of p-gp in hepg-2/adm cells rh-123 is a specific fluorescent substrate of p-gp used to monitor the efflux function of p-gp. therefore, we first treated hepg-2 cells and hepg-2/adm cells with rh-123 to validate their difference in p-gp function. as shown in figure 2a, the fluorescent intensity of rh-123 was significantly higher in hepg-2 cells than that in group (mice were given 400-mg/kg sample 6 + 18-mg/kg paclitaxel). mice were daily administrated sample 6 orally. mice were injected with paclitaxel intraperitoneally every 2 days. body weight and tumor volume were determined every 2 days, and tumor volume was calculated using the following formula: v = π (length × width2)/6. animals were euthanized with co2 when the average tumor volume in the control group reached 1,300 mm3, and tumors were collected and weighed for the following experiments. western blot analysis tumor tissues were homogenized and total protein was quantified by bradford assay. protein extract was separated in 4–12% sodium dodecyl sulfate– polyacrylamide gel electrophoresis (sds-page) and transferred on polyvinylidene fluoride (pvdf) membranes. after blocking with 5% skimmed milk for 2 h, the membranes were incubated overnight at 4°c with primary antibodies against p-gp/abcb1 and glyceraldehyde 3-phosphate dehydrogenase (gapdh) (cell signaling technology, beverly, ma, us). next, the membranes were incubated with a rabbit secondary horseradish peroxidase conjugated antibody (cst) for 1 h at 37°c. protein–antibody complexes were discovered using electrochemiluminescence (ecl) kit (thermo fisher, il, us), and the intensity of protein bands was quantitated by the imagej software. hematoxylin and eosin (h&e) staining tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned with a thickness of 5 μm. the sections were stained with h&e according to a standard protocol, and observed using leica dm750 microscope (leica, heidelberg, germany). assay for alanine aminotransferase (alt) and aspartate aminotransferase (ast) the blood samples were collected and centrifuged at 3,500 rpm for 5 min at 4°c. the supernatant was collected and the levels of serum alt and ast were analyzed using drew trilogy analyzer (diamond diagnostics, holliston, ma, us). statistical analysis data were expressed as mean ± sd. comparison of differences between multiple groups was performed using italian journal of food science, 2023; 35 (3) 93 ganoderma lucidum extract reverses multidrug resistant hcc g. lucidum ethanol extract reversed resistance to paclitaxel in hepg-2/adm tumor-bearing mice without additional toxicity we established hepg-2/adm tumor-bearing mice models and administrated with paclitaxel and sample 6. as shown in figures 3a–3c, paclitaxel treatment alone failed to inhibit the growth of tumor, indicating that resistance to paclitaxel was maintained in the xenografted tumor model. furthermore, although treatment with sample 6 alone did not inhibit the growth of hepg-2/ adm tumors in vivo, it was apparent that sample 6 could hepg-2/adm cells, indicating that there was less rh-123 accumulation in multidrug-resistant hcc cells. we further treated hepg-2/adm cells with 500 μg/ml of samples 1–6 to investigate their effect on p-gp function. as shown in figure 2b, samples 1–5 failed to change the accumulation of rh-123 in hepg-2/adm cells, while only sample 6 demonstrated a significant inhibitory effect on p-gp function and elevated fluorescent intensity, which was in consistent with the reversal effect assay. the results indicated that g. lucidum ethanol extract could inhibit the function of p-gp in multidrug-resistant hcc cells. figure 1. samples derived from g. lucidum showed no obvious toxicity to hepg-2/adm cells. hepg-2/adm cells were treated with samples 1–6, and the cell viability was measured by mtt assay (n = 5). data are presented as mean ± sd. 150 100 50 0 0 31 .2 5 62 .5 12 5 25 0 50 0 10 00 concentration (µg/ml) sample 1 c e ll vi a b ili ty ( % c o n tr o l) 150 100 50 0 0 31 .2 5 62 .5 12 5 25 0 50 0 10 00 concentration (µg/ml) sample 2 c e ll vi a b ili ty ( % c o n tr o l) 150 100 50 0 0 31 .2 5 62 .5 12 5 25 0 50 0 10 00 concentration (µg/ml) sample 3 c e ll vi a b ili ty ( % c o n tr o l) 150 100 50 0 0 31 .2 5 62 .5 12 5 25 0 50 0 10 00 concentration (µg/ml) sample 4 c e ll vi a b ili ty ( % c o n tr o l) 150 100 50 0 0 31 .2 5 62 .5 12 5 25 0 50 0 10 00 concentration (µg/ml) sample 5 c e ll vi a b ili ty ( % c o n tr o l) 150 100 50 0 0 31 .2 5 62 .5 12 5 25 0 50 0 10 00 concentration (µg/ml) sample 6 c e ll vi a b ili ty ( % c o n tr o l) 94 italian journal of food science, 2023; 35 (3) li j et al. figure 2. g. lucidum ethanol extract inhibited the function of p-gp in hepg-2/adm cells. (a) hepg-2 and hepg-2/adm cells were incubated with rh-123, and the fluorescent intensity was measured by flow cytometry. (b) hepg-2/adm cells were treated with samples 1–6 and incubated with rh-123, and the fluorescent intensity was measured by flow cytometry. table 1. the reversal effect of g. lucidum ethanol extract on the mdr of hepg-2/adm cells (n = 5). cell viability ± sd (%) (fold-reversal) doxorubicin (ic 50 ) 57.393 ± 4.889 +sample 6 (500 μg/ml) 16.118 ± 4.093*** (3.561) +sample 6 (250 μg/ml) 21.346 ± 8.690*** (2.689) +verapamil (5 μmol) 4.646 ± 0.049*** (12.353) doxorubicin (ic 20 ) 84.807 ± 3.378 +sample 6 (500 μg/ml) 11.054 ± 3.366*** (7.672) +sample 6 (250 μg/ml) 13.922 ± 11.810*** (6.092) paclitaxel (ic 50 ) 47.507 ± 10.849 +sample 6 (500 μg/ml) 15.402 ± 3.538### (3.085) +sample 6 (250 μg/ml) 16.279 ± 4.660### (2.918) +verapamil (5 μmol) 0.942 ± 0.121### (50.43) paclitaxel (ic 20 ) 83.588 ± 11.879 +sample 6 (500 μg/ml) 14.311 ± 2.058### (5.841) +sample 6 (250 μg/ml) 16.989 ± 4.604### (4.920) data are presented as mean ± sd, and significant differences are indicated as ***p < 0.001 vs the doxorubicin group, ###p < 0.001 vs the paclitaxel group. reverse resistance to paclitaxel and decrease tumor volume and weight combined with paclitaxel. h&e staining of tumor sections showed that after treatment with paclitaxel + sample 6, tumor tissues demonstrated large areas of nuclear consolidation, cell vacuolization, and necrosis, with lymphocyte-infiltrated hyperemia in figure 3e. we also measured the body weight and serum levels of alt and ast in mice, and the results showed that neither sample 6 nor paclitaxel was toxic to mice as shown in figures 3d and 3f. the results showed that g. lucidum ethanol extract could reverse resistance to paclitaxel in hepg-2/adm tumor-bearing mice without additional toxicity. g. lucidum ethanol extract showed no effect on the protein expression level of p-gp in order to investigate the underlying mechanism of the reversal effect of sample 6 on mdr of hcc tumor, we extracted the total protein of tumor tissues and carried out western blot assay. as shown in figure 4, administration of paclitaxel could slightly increase the expression level of p-gp; however, sample 6 showed no additional effect on p-gp expression level. the results indicated that the reversal effect of g. lucidum ethanol extract on mdr (a) (b) hepg-2 blank control rh-123 accumulation in hepg-2/adm rh-123 accumulation in hepg-2 c o u n t c o u n t 0 0 100 101 102 103 104 100 101 102 103 104 sample 6 500 �g/ml sample 5 500 �g/ml sample 4 500 �g/ml sample 3 500 �g/ml sample 2 500 �g/ml sample 1 500 �g/ml hepg-2/adm control hepg-2/adm blank control italian journal of food science, 2023; 35 (3) 95 ganoderma lucidum extract reverses multidrug resistant hcc control paclitaxel sample 6 before after sample 6 + paclitaxel control paclitaxel sample 6 sample 6 + paclitaxel hepg-2/adm xenograft co nt ro l pa cl ita xe l sa m pl e 6 sa m pl e 6 + pa cl ita xe l co nt ro l pa cl ita xe l sa m pl e 6 sa m pl e 6 + pa cl ita xe l alt ast u /l co nt ro l pa cl ita xe l sa m pl e 6 sa m pl e 6 + pa cl ita xe l b o d y w e ig h t (g ) tu m o r w e ig h t (g ) 0 2 4 6 8 10 treatment period (days) tu m o r v o lu m e (m m 3 ) 12 14 16 control saline 18 mg/kg 400 mg/kg paclitaxel sample 6 sample 6 + paclitaxel 1500 1200 900 600 300 0 30 25 20 15 10 5 0 1.0 0.8 0.6 0.4 0.2 0.0 60 45 30 15 0 figure 3. g. lucidum ethanol extract reversed resistance to paclitaxel in hepg-2/adm tumor-bearing mice without additional toxicity. (a) tumors from nude mice in each treatment group. (b) tumor volume and (c) tumor weight in each treatment group (n = 5). (d) body weight of mice in each treatment group (n = 5). (e) h&e staining of paraffin-embedded tumor tissue sections in each treatment group (n = 5). (f) serum levels of alt and ast in each treatment group (n = 5). data are presented as mean ± sd., and significant differences are indicated as **p < 0.01 vs the control group, ##p < 0.01 vs the sample 6 + paclitaxel group. could be related to the direct inhibition of p-gp function, rather than down-regulation of protein expression level. discussion in the current study, the effect of six extract samples derived from g. lucidum on the viability of hepg-2/adm human hepatocellular carcinoma multidrug-resistant cells was determined, and they exhibited no obvious toxicity. however, g. lucidum ethanol extract could reverse resistance to doxorubicin and paclitaxel. then the p-gp function in hepg-2 and hepg-2/adm cells was determined after incubated with these samples, and it was found that g. lucidum ethanol extract could inhibit p-gp function in vitro. furthermore, g. lucidum ethanol extract could reverse resistance to paclitaxel in hepg-2/adm tumor-bearing mice in vivo, without (a) (c) (e) (b) (d) (f) 96 italian journal of food science, 2023; 35 (3) li j et al. p-gp, a member of the abc transporter family, has been identified as a major factor that leads to mdr via utilizing the energy of two atp molecules. p-gp could fulfill one catalytic cycle and the following conformational change, and pump the substrate out of the cytoplasm, resulting in resistance to several cytotoxic chemotherapy agents (zeino et  al., 2015). the overexpression of p-gp has been confirmed in different malignancies, such as hepatocellular carcinoma (komori et  al., 2014), breast carcinoma (ding et  al., 2021), colorectal carcinoma (hu et  al., 2014), and chronic myeloid leukemia (ammar et  al., 2020). although great efforts have been invested in the research of p-gp inhibitors, most candidates failed because of inevitable toxicity and frustrating efficacy. nowadays, natural products have demonstrated promising capabilities in regulating the function of p-gp while providing safer outcomes (kumar and jaitak, 2019). in this study, we first applied a classic p-gp inhibitor, verapamil, and found that it could dramatically reverse the resistance of hepg-2/adm cells to doxorubicin or paclitaxel, implying the role of p-gp in the mdr of hepg-2/ adm cells. interestingly, we further measured the reversal effect of g. lucidum extract samples and discovered that only the ethanol extract sample 6 could significantly reverse resistance to doxorubicin and paclitaxel. therefore, in order to investigate the effect of g. lucidum components on p-gp function, we applied rh-123, a specific fluorescent substrate of p-gp, and performed flow cytometry experiments. our results showed that, compared to hepg-2 cells, the multidrug-resistant hepg-2/ affecting the protein expression level of p-gp, indicating that g. lucidum ethanol extract might directly inhibit the function of p-gp. in the past decades, cancer has become one of the major threats to human health worldwide, with increased incidence and mortality (sung et al., 2021). currently, in the clinical treatment of cancer, chemotherapy still plays an indispensable role because of good response and wide utility. unfortunately, the development of mdr has severely limited the use of chemotherapeutic drugs, such as paclitaxel, doxorubicin, etoposide, and cisplatin (robey et al., 2018). g. lucidum, a basidiomycete, has been recognized as a traditional chinese medicine with a history of hundreds of years in ancient china. it was believed that g. lucidum could promote immunity and extend life expectancy (chan et al., 2021). recently, researchers have discovered that g. lucidum exhibits multiple biological activities, including antioxidant, anticancer, anti-inflammatory, and immunomodulatory effects (cor et al., 2018). in this study, to further investigate the anticancer characteristics of g. lucidum, we prepared six samples with different isolation methods, and measured their effect on the viability of hepg-2/adm cancer cells. the results revealed that, within a range of experimental dose, none of the six samples showed any inhibitory effects on cell growth, indicating that they posed no direct toxicity to hepg-2/adm cancer cells. figure 4. g. lucidum ethanol extract showed no effect on the protein expression level of p-gp in vivo. the protein expression level of p-gp was assessed in hepg-2/adm xenografts (n = 3). data are presented as mean ± sd. co nt ro l p-gp gapdh co nt ro l pa cl ita xe l sa m pl e 6 sa m pl e 6 + pa cl ita xe l sa m pl e 6 + pa cl ita xe l co nt ro l sa m pl e 6 sa m pl e 6 + pa cl ita xe l co nt ro l r e la ti ve p -g p e xp re ss io n le ve ls r e la ti ve p -g p e xp re ss io n le ve ls pa cl ita xe l sa m pl e 6 + pa cl ita xe l 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 italian journal of food science, 2023; 35 (3) 97 ganoderma lucidum extract reverses multidrug resistant hcc in guangdong province (no. 2020b1212070024), the guangdong province key areas r&d program project (no. 2020b1111120002), and the national key r&d program of china (no. 2022yfc3500302). authors of this research are in deep gratitude toward professor renwang jiang of jinan university for his guidance and support to this work. conflict of interest the authors stated that they had no conflict of interest to declare. author contributions renwang jiang, juyan liu, and jing li designed the study. lin cao wrote the manuscript. jing li, cheng yuan, wendong xu, yaming han, and juyan liu revised the manuscript. jing li, hongfei cai, liang chen, and qin zhang carried out the experiments. lin cao, zhaojian jiang, and hongfei cai analyzed the data. all authors approved the final version of the paper. references ahmad, m.f., 2018. ganoderma lucidum: persuasive biologically active constituents and their health endorsement. biomed pharmacother. 107: 507–519. https://doi.org/10.1016/j. biopha.2018.08.036 ammar, m., louati, n., frikha, i., medhaffar, m., ghozzi, h., elloumi, m., menif, h., 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zhang, l., zhu, p., deng, m., huang, c., hu, t., jiang, l. and li, j., 2016. mammalian drug efflux transporters of the atp binding cassette (abc) family in multidrug adm cells accumulated less rh-123 in cytoplasm, indicating that the elevated function of p-gp contributed to mdr in hepg-2/adm cells. moreover, g. lucidum ethanol extract sample 6 could inhibit p-gp and elevate the level of rh-123 in hepg-2/adm cells, which further validated our previous results in the assay of reversal effect. based on the above results, xenograft hepg-2/adm tumor-bearing mouse models were created and administered with paclitaxel and sample 6 to investigate whether g. lucidum ethanol extract could reverse mdr in hepg-2/adm cells in vivo. the results demonstrated that paclitaxel had little effect on the growth of hepg-2/ adm tumors, confirming the mdr characteristic of xenografted tumor model. further, g. lucidum ethanol extract sample 6 could reverse the mdr of xenografted tumors in combination with paclitaxel, resulting in the suppression of tumor volume and weight, accompanied with deteriorated tumor pathologic conditions. the results indicated the reversal effect of g. lucidum ethanol extract on mdr in vivo. meanwhile, we also measured the body weight and serum levels of alt and ast, and the data showed that the administration of sample 6 did not alter body weight and liver functions of mice, implying that g. lucidum ethanol extract had no obvious toxicity in vivo. further, we extracted the total protein of tumor tissues and determined the expression level of p-gp in each treatment group. however, the results showed that sample 6 did not change the protein expression level of p-gp, while paclitaxel could slightly increase the expression of p-gp in vivo. based on these results, we deduced that sample 6 could exert the reversal effect of mdr by directly inhibiting the function of p-gp, rather than affecting protein expression level. therefore, our results indicated that g. 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honda, m., hayashi, k., ishigami, m., katano, y., goto, h., ueyama, j., ishikawa, t. and wakusawa, s., 2014. ursodeoxycholic acid inhibits overexpression of p-glycoprotein induced by doxorubicin in hepg2 cells. eur j pharmacol. 724: 161–167. https://doi.org/10.1016/j.ejphar.2013.12.023 ijfs#736_bozza ital. j. food sci., vol. 29, 2017 954 paper nutritional value of raw and processed fillets of bolsena lake whitefish (coregonus lavaretus l.) s. mattioli1*, a. dal bosco1, e. zingone1, d. ranucci2, c. castellini1 and r. branciari2 1department of agricultural, food and environmental science, university of perugia, borgo xx giugno 74, 06121 perugia, italy 2department of veterinary science, university of perugia, via s. costanzo 1, 06124 perugia, italy *e-mail address: simona.mattioli@hotmail.it abstract the aim of the study was to investigate and compare the nutritional value of raw and processed fillets (marinated and smoked) of whitefish from bolsena lake (italy). the study was carried out in collaboration with the “lago vivo” fisherman cooperative using 40 whitefish caught by net. the chemical composition showed increased nutrient (protein, lipid and carbohydrate) concentration with processing due to dehydration. regarding the fatty acid profile of fillets, marinating was associated with higher levels of n-6 pufas, whereas the smoked and raw fillets showed higher concentrations of α-linolenic, eicosapentaenoic (epa) and docosahexaenoic (dha) acids. whitefish fillets were characterized by high nutritional value and good oxidative stability, mainly as smoked products. keywords: fillet, nutritional quality, processed, whitefish, fatty acids, food value ital. j. food sci., vol. 29, 2017 955 1. introduction bolsena lake is the largest volcanic lake in europe and the fifth largest in italy. it is located in the province of viterbo in northern lazio on the border with umbria and tuscany. it has a drainage basin of 243 km2, nearly half of which is occupied by the lake itself. unlike other italian lakes, bolsena lake is populated by a large number of fish species, including native herbivorous or insectivorous species (i.e. tench, chub, carp, rudd and sand smelt) and some predatory species such as pike, perch and eel. these are supported by other fish species added for specific human interest, such as whitefish and gambusia, or placed unconsciously as in the cases of bluegill, a voracious predator of young fish and eggs. the european whitefish (coregonus lavaretus), widely distributed in the freshwaters of northern europe (goebel et al., 2016) and introduced into the major lakes of northern italy, is present in the lakes of central italy mainly as result of stock enhancement practices. however, it is an uncommon species in our country and rarely present on consumer tables. in addition, the reduced surface area of the bolsena lake basins and the strong national tradition linked mainly to marine species of our country are limiting marketing and thus the spread of freshwater fish species in the domestic markets. as result, there is very little knowledge of whitefish nutritive characteristics among consumers. the high value of meat and the economic interest in the bolsena lake whitefish has placed a spotlight on the return of the product under the collective brand “tuscia-viterbese”, which is also very popular. a license for this kind of product can be issued by fishing companies and/or companies that process and/or market the species covered by this brand; for this reason, it is considered a local product. furthermore, considering the extreme perishability of foodstuffs, conservation techniques for whitefish products are needed (yeannes and casales, 2007), some of which have already come up from fishermen 2000 years ago. nowadays, the various storage systems are also largely used to distinguish products and to attract consumers (sogn-grundvag et al., 2014). accordingly, a potential contribution to the economy of the fishermen's cooperative is certainly represented by the possibility of placing on the market differentiated products, such as smoked or marinated fillets, and to define their nutritional characteristics. indeed, in accordance with ec regulation 1169/2011, nutrition labelling on food is mandatory to achieve a high level of health protection for consumers and to guarantee access to accurate information. in a previous work, orban et al. (2006) studied the nutritional quality and safety of whitefish caught in three different italian lakes, underlining their good protein and mineral contents and low lipid levels in the fillet of such species; beginning with this study, we wanted to deepen the existing knowledge on nutritional aspects of whitefish living in bolsena lake, both raw and processed (marinated and smoked), in order to add value to local products on the market. 2. materials and methods the study was carried out in collaboration with the “lago vivo” fisherman’s cooperative of bolsena (viterbo, italy). forty whitefish were caught by net in april 2016. all fish were immediately dipped in a mixture of water and ice and successively transferred to polystyrene boxes containing ice and transported under refrigerated conditions to the laboratory of the cooperative for processing as described below. ital. j. food sci., vol. 29, 2017 956 2.1. fillets and processed preparation whitefish with an average live weight of 250 g were eviscerated after washing with running water, the heads and tails were removed, dorsal and ventral fillets were dissected and 20 of them were taken for analysis. the preparation procedure of the threads and the process (smoking and marinating) adopted provided for manual filleting with subsequent removal of the skin and scraps. at this point, the process followed two different paths: firstly, for the smoked product, the obtained fillets (approximately 100 g per fish) were salted and then smoked at 60 ± 5°c over beech wood for 4 hours; the next step was vacuum packaging and storage in a special cold storage (0-4°c) awaiting shipment to the various sales outlets. concerning the production of processed fillets, these were marinated for almost 12 hours in vinegar, lemon juice and spices with addition of olive oil, onion, celery, pepper and salt (approximately 30 g/kg of fillet), then the whole fillet was packed in packages of various weights (from 100 to 1000 g) in a modified atmosphere and stored in special refrigerated cells (0-4°c) awaiting shipment to different outlets. all fillet samples, raw and processed (n=20 per type), were stored at -30°c at the laboratories of the department of agricultural, food and environmental science and department of veterinary medicine of the university of perugia until analysis (2 weeks later). 2.2. proximate analyses and fatty acid composition all samples were analysed in duplicate to determine the proximate composition. in detail, moisture, ash and total nitrogen were assessed using the aoac methods (2000 n. 950.46, 923.03 and 991.15, respectively). total protein nitrogen was calculated by the kjeldahl method using 6.25 as the conversion factor. total lipids were extracted in duplicate from 10 g of each homogenized sample and calculated gravimetrically (franceschini et al., 2015). the energy value and caloric value of the raw and processed fillets expressed in kj/g and kcal/g, respectively, was calculated following the criteria of eu regulation 1169/2011. fatty acids (fa) were determined by gas chromatography after lipid extraction according to the method proposed by franceschini et al. (2015). approximately 10 g of fish were homogenized with 5 ml of 0.5 m sodium acetate-water solution using an ultraturrax (t25 basic, ika, labortechnik, germany). then, another 8 ml of sodium acetate solution, 8 ml of methanol and 4 ml of chloroform were added to 4 g of this mixture and mechanically shaken for 3 minutes. after addition of 4 ml of chloroform, the mixture was shaken again for 2 minutes, and 8 ml of water was added in order to separate the chloroform and methanol phases. after centrifugation, the lower phase was collected in a flask. fatty acid methyl esters (fame) were obtained as described by branciari et al. (2017). fifty milligrams of the lipid fraction were dissolved in 2 ml of hexane, then 1 ml of hexane containing internal standard (methyl nonadecanoate 0.4 mg/ml; sigma-aldrich, bellefonte, pa, usa) and 2 n koh in methanol (0.5 ml) were added. the mixture was then shaken vigorously for 3 min, water (3 ml) was added and the upper organic phase was dried over anhydrous sodium sulphate, cooled in an ice bath and immediately injected into a high-resolution gas chromatograph. separation of fame was carried out on a capillary column cp-select cb (100 m x 0.25 mm i.d., 0.39 µm, j&w, agilent technologies, palo alto, ca, us). we used helium as a carrier gas at a flow rate of 1.6 ml/min. the injector temperature was set at 270°c and the detector at 300°c. the oven temperature program was the following: starting from 60°c (maintained for 1 minute), the temperature was increased to 30°c/min up to 150°c; after 3 min, with an increase of 0.5°c/min, then, after 1 minute, it was increased to 220°c in increments of 1.5°c/min and ital. j. food sci., vol. 29, 2017 957 then maintained for 15 min. one μl of sample was injected in a split/splitless system (split ratio 1:5). individual fame were identified by comparison with a standard mixture (pufa no. 1, marine source, 37 fames by sigma, methyl cis-7,10,13,16,19docosapentaenoate, trans-11-vaccenic methyl ester, cis-11-vaccenic methyl ester, supelco, bellefonte pa, usa). the percentage of each fa was calculated by using the peak area of the samples corrected with the respective correction factors (aoac, 2012). to assess the actual nutritional quality of fish fillets, fatty acids were quantified and expressed in mg/100 g tissue using the internal standard method outlined by joseph and ackman (1992). the following equation was applied: fatty acids (mg/100 g food) = [(ax × wis × crfx × cnfx)/(ais × ws)] × 1000 × wl, where ax is the eicosapentaenoic acid (epa) or docosahexaenoic acid (dha) area, ais is the internal standard area, crfx is the theoretical correction factor for epa and dha, cnfx is the conversion factor from fame to the corresponding fatty acid, wis is the weight of the internal standard added to the lipids, ws is the weight of the derivatized lipids and wl is the percentage of sample lipid. based on current knowledge regarding the effect of specific fatty acids on cholesterol metabolism, the ratio between hypocholesterolaemic and hypercholesterolemic fatty acids (hh) was calculated using the following mathematical equation (santos-silva et al., 2002): hh = (c18:1n-9 + c18:2n-6 + c20:4n-6 + c18:3n-3 + c20:5n-3 + c22:5n-3 + +c22:6n-3)/(c14:0 + c16:0) the mean value of each fatty acid was used to calculate the sum of the saturated (sfa), monounsaturated (mufa) and polyunsaturated (pufa) fatty acids and to calculate the peroxidability index (pi) according to the equation proposed by arakawa and sagai (1986): pi = (% monoenoic x 0.025) + (% dienoic x 1) + (% trienoic x 2) + (% tetraenoic x 4) + +(% pentaenoic x 6) + (% hexaenoic x 8). the amount of each fatty acid was also used to calculate the atherogenicity (ai) and thrombogenicity (ti) indexes as proposed by ulbricht and southgate (1991): ai = (c12:0 + 4xc14:0 + c16:0)/[(∑mufa + ∑ (n-6) + ∑ (n-3) ] ti = (c14:0 + c16:0 + c18:0)/[ (0.5 x ∑mufa + 0.5 x (n-6) + 3x (n-3) + (n-3)/(n-6)] the index of nutritional quality (inq) was calculated on the basis of the epa + dha acid level using the formula suggested by godbe (1994). 2.3. assessment of oxidative stability the extent of lipid oxidation was quantified by spectrophotometry (shimadzu 2025, kyoto, japan) as thiobarbituric acid reactive substances (tbars) according to the method reported by ke et al. (1977) and using a molar extinction coefficient of 156 x 103 m/cm at λ = 532 nm. results are expressed as malondialdehyde (mda) equivalents per kg of tissue (mg mda/kg). ital. j. food sci., vol. 29, 2017 958 2.4. reagents unless otherwise noted, all chemicals were analytical grade and were purchased from sigma chemical co (st louis, mo, usa). 2.5. statistical evaluation the data were analysed with a one-way linear model (statacorp®, 2015) evaluating the effect of technological treatment. differences among whitefish products were evaluated by multiple samples t-test, reporting the mean and standard error of the mean (sem). results were considered significant at p < 0.05. 3. results and discussions 3.1. food labels of raw and processed whitefish fillets the chemical characteristics of analysed whitefish fillets are reported in table 1. the processing of whitefish, i.e. smoking and marinating, significantly affected the chemical parameters of fillets. in particular, smoking reduced the water content of muscle fibres with respect to raw filets (64.82% vs. 76.53%) due to surface drying of the fillet and consequently, the fillets showed nutrient concentration. even in marinated fillets, an increase in nutrient content due to dehydration was found (65.26%). table 1. raw and processed whitefish fillets food label. fillets raw smoked marinated sem moisture 76.53b 64.82a 65.26a 4.52 lipids 1.97a 2.86a 9.77b 0.59 of which saturates 30.82b 30.21b 14.50a 1.18 monounsaturates 33.60 33.54 36.35 2.11 polyunsaturates 35.57a 36.23a 49.14b 2.56 carbohydrates 0.97a 1.05a 2.63b 0.29 of which sugar n.d n.d. 0.41 0.18 protein 19.23a 28.53b 20.44a 2.01 ash 1.30a 2.74b 1.90ab 0.35 energy* 98a 144b 180c 20.15 416a 602b 754c 41.18 n=20 per group. a..c values within a row with different superscripts indicate a significant difference at p < 0.05. proximate composition was expressed as %; energy was expressed as kcal/100 g on the first line values and kj/100 g on the second line values. * eu regulation 1169/2011. ital. j. food sci., vol. 29, 2017 959 the percent protein content of smoked fillets was significantly higher than that of raw and marinated ones (28.53% vs. 19.23% and 20.44%, respectively). however, the smoking process can cause a reduction of protein digestibility attributable to the formation of oxidized forms of sulphur amino acids and carbohydrate-protein complexes called maillard compounds (mensa-wilmot et al., 2001), as demonstrated by the low percentage of carbohydrates relative to that in the marinated fillets (1.05% vs. 2.63%). instead, the higher proportion of carbohydrates in marinated samples was the result of the addition of onion and celery as ingredients, which contain approximately 5.7% and 2.4% carbohydrates, respectively (marletta and carnovale, 2000). a prominent difference was found in the lipid content, which was higher in the marinated fillet (9.77%), probably because of the addition of olive oil; indeed, the main fatty acids were represented by pufa and mufa. the total mineral content was significantly different only between raw and smoked fillets, with lower values in the former (1.30% vs. 2.74%), possibly attributed to the concentration process previously cited. fuentes et al. (2010) reported a strong correlation of the smoking process with several physico-chemical parameters (moisture, water activity, ph and colour) in commercial smoked products (anchovy and salmon). they suggested that the ability of the smoking process to preserve fish is due to the synergistic action of salt incorporation, smoke compounds and dehydration during the smoking process. similarly, marinated fish are products consisting of raw, frozen or salted fish or portions of fish processed by treatment with edible organic acids, usually acetic acid, and salt and added to sauces, creams or oil (meyer, 1965). they represent semi-preserved fish products, ready-to-eat with no heat treatment (gram and huss, 1996), and they are considered as a high-value delicacy, as are cold-smoked fish. in the present study, marinated fish were prepared with lemon juice and vinegar, then with natural citric and acetic acids. hamm (1960) outlined that, in the presence of acid alone, the ph of the muscle was on the acidic side of the isoelectric point, and the electrostatic repulsion allowed for an increase in water holding capacity and a decrease in firmness. however, when salt was added (as in our case), the repulsion decreased and the structure became firmer. the differing proportions of nutrients in the raw and processed fillets also affected the energy content of the studied products, with higher values found in marinated fillets, followed by smoked and non-processed whitefish (180, 144 and 95 kcal/100 g and 754, 602 and 416 kj/100 g, respectively). 3.2. fatty acid profiles of raw and processed whitefish fillets the fatty acid composition (mg/100 g) of the samples is shown in table 2. a total of 33 fatty acids were identified in the present study. the fatty acid composition of raw and smoked fillets was nearly identical. the slight differences in concentration were due to the lower moisture content of the smoked fillets, as demonstrated by chemical composition analysis. in raw and smoked fillets, the most represented fatty acid was oleic acid (c18:1n9cis); this mufa was present in an amount equal to 327.26 mg/100 g in raw fillets, whereas in smoked ones, a concentration of 483.85 mg/100 g was recorded, followed by palmitic acid (c16:0). conversely, orban et al. (2006) found a higher proportion of the latter fatty acid, followed by oleic (c18:1n-9) and palmitoleic (c16:1) acids in raw whitefish fillets. in the marinated fillet, the most represented fatty acid was linoleic acid (c18:2n-6, la; 4005.84 mg/100 g). this higher value, compared with that recorded in raw and smoked fillets, was justified by the marinating preparation method, which included the addition of olive oil with an average linoleic acid value of 12%. the same was also true for oleic acid ital. j. food sci., vol. 29, 2017 960 (2999.25 mg/100 g), which is present at almost 83% in olive oil, and for palmitic and stearic acids (786.81, 296 and 17 mg/100 g, respectively), which are present from 5% to 20% (boskou, 2015). table 2. fatty acid composition (mg/100 g tissue) of raw and processed whitefish fillets. n=20 per group. a..c values within a row with different superscripts indicate a significant difference at p < 0.05. oleic acid plays an important role in physical well-being and is responsible for the reduction of plasma cholesterol levels and the improvement of the low density/high fillets raw smoked marinated sem c12:0 1.18a 1.68a 8.73b 0.98 c13:0 0.57b 0.86b 0.00a 0.12 c14:0 119.08b 174.69c 63.93a 15.21 c14:1 22.62b 33.77c 12.74a 1.25 c15:0 15.19b 22.21c 9.70a 1.05 c15:1 0.80b 1.07b 0.00a 0.08 c16:0 319.22a 444.88a 786.81b 50.16 c16:1 214.52b 303.12c 147.19a 17.51 c17:0 9.08a 13.24b 8.11a 0.89 c17:1 0.54b 0.73b 0.00a 0.10 c18:0 50.42a 77.03a 296.17b 20.31 c18:1n-9t 4.52b 7.25b 0.00a 0.32 c18:1n-9c 327.26a 483.85a 2999.25b 124.21 c18:1n-7c 67.31a 93.46b 124.66c 24.15 c18:2n-6t 0.62b 0.97b 0.00a 0.12 c18:2n-6c 93.52a 136.69a 4005.84b 66.58 c18:3n-6 8.93a 13.54b 6.00a 1.24 c18:3n-3 138.30b 198.28c 82.65a 26.21 c20:0 3.22a 4.45a 24.20b 2.14 c18:4n-3 86.75b 128.08c 38.69a 15.48 c20:1n-9 8.98a 12.80a 20.39b 2.36 c21:0 5.30a 7.35b 7.32b 0.87 c20:3n-6 3.46a 5.20b 3.13a 0.69 c20:4n-6 41.20b 59.20c 28.71a 3.25 c20:3n-3 8.96b 12.12c 4.58a 1.05 c22:0 5.49a 8.15a 48.87b 2.36 c22:1n-9 1.37b 1.93b 0.00a 0.14 c20:5n-3 128.64b 186.87c 70.58a 3.36 c22:2 0.94a 1.32b 2.73c 0.25 c24:0 0.98a 1.40a 17.46b 1.28 c24:1 1.47a 2.09a 7.44b 0.86 c22:5n-3 38.10b 59.11c 27.32a 3.72 c22:6n-3 61.96b 105.25c 37.68a 3.14 ital. j. food sci., vol. 29, 2017 961 density lipoprotein ratio (ldl/hdl, secchiari, 2008), both of which are well-known, important risk factors for cardiovascular disease (althaus et al., 1988). furthermore, considering the cultural value of olive oil in our country (de leonardis, 2014), its use in fishery products could represent an important value added. concerning pufa, the fatty acid with the highest concentration was α-linolenic acid (ala, c18:3n-3), followed by epa (c20:5n-3) in all studied samples. even the ala content, both in raw and smoked fillets was higher than that in marinated ones (138.30 and 198.28 vs 82.65 mg/100 g, respectively). epa and dha are long-chain n-3 fatty acids, synthesized by the human body only in small amount (de filippis and sperling, 2006); therefore, their dietary consumption is required. in particular, epa and dha intake has demonstrated physiological benefits in terms of blood pressure, heart rate, triglycerides and inflammation; moreover, a reduced risk of foetal coronary heart disease (chd) and sudden cardiac death has been associated with the consumption of ~250 mg/day of epa plus dha (gissi-hf, 2008; mozaffarian and rimm, 2006). accordingly, whitefish fillet is an excellent source of long-chain n-3 pufa, as 100 g of raw fillet provides approximately 190 mg of epa + dha, and smoked filet exceeds the recommended requirement (292.18 mg/100 g). 3.3. nutritional indexes and oxidative stability of raw and processed whitefish fillets the total amounts of different fatty acid series, nutritional indexes and oxidative stability of whitefish fillets are reported in table 3. when the three processing methods were compared, raw and smoked fillets had different amounts of sfa (529.73 vs 755.94 mg/100 g, respectively), due, as previously mentioned, to the concentration of nutrients during the smoking process. the same trend was observed in the mufa and pufa levels. table 3. total saturated, monounsaturated and polyunsaturated fatty acids (mg/100 g), nutritional indexes and oxidative status of raw and processed whitefish fillets. n=20 per group. a..c values within a row with different superscripts indicate a significant difference at p < 0.05. sfa, mufa, pufa, σ n-3 and σ n-6 are expressed as mg/100 g tissue; tbars are expressed as mg mda/kg tissue. fillets raw smoked marinated sem sfa 529.73a 755.94b 1271.28c 50.26 mufa 577.57c 839.37b 3187.01c 62.52 pufa 611.39a 906.63b 4307.91c 59.85 σ n-3 462.71b 689.70c 261.50a 21.36 σ n-6 147.74a 215.61b 4043.68c 78.15 n-6/n-3 0.32a 0.31a 15.46b 2.26 inq 5.95b 6.24b 1.85a 1.85 hh 1.16a 1.21a 5.00b 5.00 peroxidability index 112.60 b 117.83b 91.14a 21.14 atherogenic index 0.67 b 0.66b 0.14a 0.14 trombogenic index 0.28 0.27 0.26 0.26 tbars 0.22 a 0.11a 3.60b 0.60 ital. j. food sci., vol. 29, 2017 962 several studies have shown a direct relationship between the consumption of sfa in the diet and the risk of cardiovascular disease (dayton et al., 1968). the negative effect of dietary sfa is mainly due to the increase in blood ldl cholesterol (mensink et al., 1992). however, the heterogeneity of the saturated fatty acids and their effect as risk factors are worthy of note. for example, stearic acid, little represented in the raw fillet, is not a hypercholesterolemic agent (hunter et al., 2010), whereas the myristic acid, which was present in a higher amount than stearic acid, is dangerous to human health because it increases serum cholesterol by four times more than palmitic acid (secchiari, 2008). regarding the marinated fillet, the levels of sfa, mufa and pufa were significantly higher respect to control or smoked one. the sfa and mufa amounts were 1271.28 mg/100 g and 3187.01 mg/100 g, respectively, mainly due to the addition of olive oil to the product (table 3). this ingredient affected also the pufa levels (4307.91 mg/100 g), which were greatly elevated (boskou, 2015). the prevalence of pufa over mufa and sfa may reflect a high inherent capability of freshwater fish to desaturate and elongate enzymatically dietary precursors into long-chain highly unsaturated fatty acids (henderson and tocher, 1987). the smoked fillet had 689.70 mg/100 g of n-3 fatty acids, whereas during the marinating process, the n-3 fatty acid concentration decreased to 261.50 mg/100 g, of which 70.58 mg was epa, and 37.68 mg was dha. these results were not due to the oxidation of the product resulting from manipulation (frankel et al., 2014) but rather to the high quantity of pufa found. in fact, the lipid oxidative status, evaluated with the tbars test, was worse in marinated than in raw and smoked fillets (3.60 in marinated vs. 0.22 and 0.11 mg mda/kg in raw and smoked samples, respectively), whereas the ip index, which measures the susceptibility to oxidation on the basis of the fatty acid composition, was lower in marinated than in raw and smoked fillets (91.14 in marinated vs. 112.60 and 117.83, in raw and smoked samples, respectively). we observed a correlation between the amount of long chain n-3 fatty acids and oxidative status. as already mentioned, the raw and smoked fillets had a higher content of n-3 fatty acids than the marinated fillet. for fish products subjected to processing after slaughter, it is necessary to take precautions to avoid over-oxygenation of the product, which would make it more susceptible to oxidation. the higher tbars value was related to the worse oxidative status found in the marinated samples, and to the content of unsaturated fatty acids, which are the primary targets of the free radicals (dal bosco et al., 2010). the trend for n-6 fatty acids was different to that of n-3 fatty acids. raw and smoked fillets showed a lower content of total n-6 fatty acids (147.74 and 215.61 mg/100 g, respectively), while in the marinated fillet, the concentration is almost 20-times higher, as also shown by the n-6/n-3 ratio (table 3). nowadays, fish products are the main source of n-3 pufa in the human diet and, in consideration of the high intake of n-6 pufa in industrialized countries, an increment of their consumption is recommended by dietary guidelines in order to re-establish a healthy balance between n-3 and n-6 pufa (simopoulos, 2003). several studies have demonstrated the role of n-3 fatty acids in the prevention of human diseases. in particular, these compounds also have a beneficial effect on the control and prevention of cardiovascular diseases and play crucial roles in brain development and visual activity (rizos et al., 2012). all the studied indexes reflect the composition of single fatty acids of fillets (table 3). the hh index measures the ratio of hypocholesterolemic and hypercolosterolemic fatty acids. the marinated fillet contained a higher concentration of hypocholesterolemic fatty acids (mufa + pufa), with a hh value of 5.00, than raw and smoked fillets. this result is noteworthy considering that the main factor in the onset of cardiovascular diseases is the oxidation of low-density lipoproteins (ldl cholesterol; hu and willett, 2002); ital. j. food sci., vol. 29, 2017 963 therefore, fish products have an important role in the prevention of such pathologies (valfrè et al., 2003). the inq index describes the nutritional quality of a food, based on the satisfaction of the daily requirements of epa + dha. in the present study, the raw and smoked fillet had higher values than the marinated ones (5.95 and 6.24 vs. 1.85, respectively), in agreement with the epa + dha contents previously described. finally, the atherogenic (ai) and thrombogenicity indexes (ti), which measure the quality of the lipids in the fillets, are inversely correlated with the ability of fatty acids to reduce the lipid content in the blood and to reduce platelet activity, respectively (ulbricht and southgate, 1991). the results obtained differed significantly between all three sample treatments, and they were better in the marinated fillet. in raw and smoked fillets, ai values of 0.67 and 0.66, respectively, were recorded, whereas for the marinated fillet, this value was 0.14. however, the ti value remained constant in all the samples (0.28, 0.27 and 0.26 in raw, smoked and marinated, respectively). these similar results could be explained by n-3 and n-6 pufa values considered in the equations suggested by ulbricht and southgate (1991). indeed, they were equally weighted for the ai index, but not for the ti, where n-3 pufa obtained a higher weight and then, equalizing the ratio between n-6 and n-3, was not significantly different between groups. 4. conclusions the data from the present study suggest that whitefish from bolsena lake is characterized by high nutritional quality (mainly due to epa and dha content) and a good oxidative stability of raw fillets. however, nutritional characteristics of fillets differed between processing methods: the smoked fillet showed a higher inq and a lower n-6/n-3 ratio, hh index and tbars value compared with the marinated one. in contrast, the marinated fillet offered an added value given by olive oil presence (lowest pi and highest pufa content). to conclude, the nutritional characterization of local products represents a good strategy to increase their economic value. further studies focusing on the environment in which the fish lives and the one from which it originates, or on its history (as it is caught and processed on board the grounded boats and transformed yourself), are needed to evaluate such umbrian fishery products (raw or processed). acknowledgements authors are grateful to “lago vivo” fisherman cooperative of bolsena (viterbo, italy). references althaus b.u., staub j.j., leche a.r.d.e., oberhänsli a, stähelin h.b. 1988. ldl/hdl changes in subclinical 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science and technology 28(4):798-803. paper received january 1, 2017 accepted july 20, 2017 #551_vincenzi_bozza ital. j. food sci., vol 29, 2017 50 paper characterization of chitinase isoforms from grape juice d. gazzola1, g. pasini1, s. tolin2, a. curioni1 and s. vincenzi*1 1department of agronomy, food, natural resources, animals and environment (dafnae), university of padova, viale dell' università 16, 35020 legnaro (pd), italy; centro interdipartimentale per la ricerca in viticoltura ed enologia (cirve), university of padova, viale xxviii aprile 14, 31015 conegliano (tv), italy 2proteomics center, university of padova, via g. orus 2b, 35129 padova, italy *corresponding author. tel.: +39 0438453052; fax: +39 0438453736 e-mail address: simone.vincenzi@unipd.it abstract grape chitinases are recognized as being mainly responsible for protein haze formation in white wines. vitis vinifera l. cv. manzoni bianco grape juice proteins were fractionated using anion exchange and hydrophobic interaction chromatographies. according to sdspage and zymography, six protein bands with chitinolytic activity were subjected to mass spectrometry (maldi-tof/tof ms), which assigned all the bands to vitis vinifera class iv chitinases. these grape chitinase isoforms showing different electrophoretic and chromatographic behaviours are likely to be also distinct in their functionality in wine. this could be relevant to understand the involvement of single chitinase components in wine hazing and to develop specific winemaking techniques for their removal from wine. keywords: chitinase, electrophoresis, glycol chitin, grape juice, isoform, mass spectrometry ital. j. food sci., vol 29, 2017 51 1. introduction the problem of protein haze formation in white wines is still unresolved, despite wine hazing being a serious quality defect because consumers perceive hazy wines as faulty products. protein haze is caused by the presence of relatively low concentrations (from 15 to 700 mg/l) of pathogenesis-related (pr) proteins, namely thaumatin-like proteins (tlps) and chitinases (ferreira et al., 2001; waters et al., 2005; vincenzi et al., 2005; van sluyter et al., 2015). chitinases are the most active protein components in causing wine turbidity (falconer et al., 2010; marangon et al., 2011). these proteins derive from grapes, are present in different isoforms (marangon et al., 2011; gazzola et al., 2012), are tolerant to low ph in juice and wine and are resistant to proteolytic enzymes, as most of the pr proteins (ferreira et al., 2001; waters et al., 2005; van sluyter et al., 2015). the first step of the mechanism leading to haze formation in wines should involve protein denaturation (van sluyter et al., 2015). grape chitinases can denature within minutes at temperatures >40°c, compared to weeks for tlps under the same conditions, with a predicted half-lives of only 14 hours at a realistic temperature of 35°c (falconer et al., 2010). moreover, these grape enzymes seem to maintain their activity in wine at least for some months after alcoholic fermentation (manteau et al., 2003) and the consequences of this activity on wine quality are unknown. chitinases have antifungal properties resulting from their activity toward chitin, a major structural component of many fungal cell walls (graham and sticklen, 1994). however, a chitinase purified from vitis vinifera l. cv. manzoni bianco grape juice, although showing both endoand exo-chitinase activities, was not able to inhibit wine yeast growth (vincenzi et al., 2014). chitinases have been successfully purified by others (marangon et al., 2009; van sluyter et al., 2009; dufrechou et al., 2013), despite their low concentration and strong interaction with endogenous polyphenols and other non-protein compounds (ferreira et al., 2001; gazzola et al., 2012). in spite the interest of these type of wine components, in-depth knowledge surrounding them is still incomplete. therefore it is important to develop robust systems for a better characterisation of these components and in particular to establish the role of each chitinase isoform found in grape in wine haze formation and development. in this paper, the purification, the electrophoretic characterisation and the mass spectrometry identification of some chitinase isoforms from manzoni bianco grape juice is described. 2. materials and methods 2.1. protein extraction from grape juice according to vincenzi et al. (2014) with minor modifications, fifteen kg of vitis vinifera l. cv. manzoni bianco berries were manually crushed and treated with 7.5 g/kg polyvinylpolypyrrolidone (pvpp) (sigma-aldrich, st. louis, mo), 0.15 g/kg ascorbic acid (baker, deventer, holland) and 0.375 g/kg potassium metabisulfite (carlo erba, milano, italy). the grape juice (10 l) was treated overnight at 4°c with 3 g/l of pectolytic enzymes (pectazina dc, dal cin spa, milano, italy), and centrifuged (5000 g, 20 min, 4°c). the free run juice was dialysed (3500 da cut-off) against distilled water, concentrated by ultrafiltration (3000 da cut-off) and freeze-dried. ital. j. food sci., vol 29, 2017 52 2.2. protein separation by chromatography a two-step chromatographic separation was performed using an äkta purifier fplc (ge-healthcare, uppsala, sweden) equipped with an uv detector. data were processed by the unicorn 5.11 software (ge-healthcare). each solution used and samples to load were previously filtered with 0.20 µm cellulose acetate filters (millipore, vimodrone, italy). the first chromatographic step was anion exchange chromatography (aec). ≈ 50 mg of freeze-dried extract were dissolved in 20 mm tris-hcl ph 9.0 (buffer a) and loaded onto a tricorn monoq 5/50 column (ge-healthcare) equilibrated with buffer a at a flow rate of 1 ml/min. bound proteins were eluted at 1 ml/min with a gradient of buffer b (20 mm tris-hcl, 1 m nacl, ph 9.0) as follows: 0 to 14% b in 70 min and 14 to 100% b in 3 min (vincenzi et al., 2011 with minor modifications). aec fractions were pooled on the basis of 280 nm elution profiles and analysed by sds-page after being concentrated and dialysed against water (vivaspin 50, 3000 da cutoff, sartorius, göttingen, germany). the second purification step was performed by hydrophobic interaction chromatography (hic) according to vincenzi et al. (2014). the pooled and selected aec fractions were fractionated at 0.5 ml/min on a hic biosuite phenyl 10 µm hic 7.5 x 7.5 mm column (waters, milford, ma) with a 60 min linear gradient to 100% buffer b (20 mm tartaric acid ph 3.5) in buffer a (20 mm tartaric acid ph 3.5 containig 1.25 m ammonium sulfate). 2.3. sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) sds-page analyses were performed according to laemmli (1970) in a mini-protean iii apparatus (bio-rad laboratories, hercules, ca, usa). samples were prepared by precipitating proteins from 5 to 50 µl (depending on protein concentration on samples) of pooled chromatographic fractions by the kds method (vincenzi et al., 2005; gazzola et al., 2015). precipitated proteins were solubilized in 20 µl of 0.5 m tris-hcl buffer, ph 6.8, containing 15% (w/v) glycerol and 1.5% (w/v) sds (bio-rad laboratories) and heated at 100°c for 5 minutes before loading. in order to detect the presence of disulphide linked protein aggregates, sds-page was performed also in reducing conditions. this was done by adding 4% (v/v) 2-mercaptoethanol to the loading buffer. electrophoresis was carried out at 25 ma constant current until the tracking dye bromophenol blue ran off the gel. the molecular weight standard proteins were the broad range molecular weight markers (bio-rad laboratories). 1.5 mm thick gels were prepared with t = 12% (sds-page in fig. 5) or 14% (acrylamide-n, n’ metilenbisacrylamide 29:1; sigma-aldrich) and stained with colloidal coomassie brilliant blue g-250 (sigma-aldrich) or with the pas (periodic acid-schiff) method for glycocompounds detection (segrest and jackson, 1972). 2.4. chitinolytic activity detection on sds-page gels chitinolytic activity detection was assayed according to vincenzi and curioni (2005). samples were prepared with the same reagents used for sds-page and loaded into a gel (t = 14%) containing glycol-chitin (0.01% or 0.05% w/v). after protein separation, the gels were incubated overnight at room temperature in a 50 mm sodium acetate buffer ph 5.5 containing 1% (w/v) triton x-100 (sigma-aldrich). afterwards, gels were incubated for 20 minutes in 0.5 m tris-hcl buffer ph 8.9, containing 0.01 % (w/v) calcofluor white mr2 (sigma-aldrich), followed by a wash in distilled water (for at least 1 h). protein bands with chitinolytic activity were digitized with an edas290 image capturing system (kodak, rochester, ny). ital. j. food sci., vol 29, 2017 53 2.5. protein identification by maldi-tof/tof ms the selected bands were excised from sds-page gels, dehydrated with acetonitrile for 10 min and dried in speed vac concentrator. disulphide bridges were reduced with 10 mm dithiotreitol (1 h, 56°c, in the dark) and cysteines were alkylated with 55 mm iodoacetamide (1 h, room temperature, in the dark). gel bands were repeatedly washed with 50 mm nh4hco3 and acetonitrile, and dried under vacuum. in gel protein digestion was performed using sequencing grade modified trypsin (promega, madison, wi). 10 μl of trypsin (12.5 ng/μl in 50 mm nh4hco3) were added to each band, and digestion was carried out at 37°c overnight. peptides were extracted with 50 μl of 50% acetonitrile and 1% formic acid (3 times), dried under vacuum and dissolved in 10 μl of 0.1% formic acid. the digested sample was mixed with an equal volume of matrix solution (a-cyano-4hydroxycinnamic acid, 5 mg/ml in 70% acetonitrile, 0.1% trifluoroacetic acid) and 1 µl was spotted on a 384-well ab optitof maldi stainless steel target plate (shevchenko et al., 2006). samples were analysed using a maldi-tof/tof 4800 analyzer (applied biosystems, toronto, canada) with 4000 series explorer v3.5.3 software. ms data were acquired automatically over a mass range of 900–3500 da in the positive-ion reflector mode. in the ms spectrum, the 10 most abundant ms peaks were selected for ms/ms. ms/ms data were searched using the mascot search engine (matrix science, london, uk) against the msdb database (3239079 sequences; 1079594700 residues; taxonomy: viridiplantae, 247880 sequences). enzyme specificity was set to trypsin with one missed cleavage using a mass tolerance window of 50 ppm for the precursor ion and 0.3 da for the fragment ions and carbamidomethylcysteine as fixed modification. 3. results and discussions 3.1. sds-page analysis of the grape juice proteins vitis vinifera l. cv. manzoni bianco (riesling renano x pinot bianco) was used for the grape protein extraction, as this variety shows a high protein content and gives wines generally requiring fining treatments with significant amounts of bentonite for protein stabilization (vincenzi et al., 2011). from the free run juice, 2.6 g of grape juice macromolecular powder (crude extract, ce) was obtained. an aliquot of ce was analyzed by sds-page, showing main protein bands in the region between 20 and 30 kda (fig. 1a). these bands have been previously identified as grape pathogenesis-related (pr) proteins including thaumatin-like proteins (tlps) and chitinases (waters et al., 1996; monteiro et al., 2007; van sluyter et al., 2015). high molecular weight protein bands were also evident in the 45-80 kda range. the protein with relative molecular mass (mr) of 65 kda is likely to be the grape vacuolar invertase which is known to be one of the most abundant proteins in grape juice and wine, reaching 14% of chardonnay wine proteins (dambrouck et al., 2005). the minor bands with mr ranging from 45 to 60 kda have also been identified by proteomic analysis in a semillon grape juice as (vitis vinifera) “unnamed protein product” and class iv chitinase (marangon et al., 2009). finally, the band of 12 kda is likely to correspond to the lipid transfer protein (ltp), whose presence has already been reported in grapes where was indicated as one of the major allergens (pastorello et al., 2003). staining the gel for sugar residues confirmed the presence of glycocompounds, probably polysaccharides, which barely entered the gel (fig. 1b) (vincenzi et al., 2012). ital. j. food sci., vol 29, 2017 54 figure 1 non-reducing sds-page of the grape berries crude extract (ce), stained for proteins (a) and glycocompounds (b). molecular weight standard proteins are on the left. 3.2. protein fractionation and characterization starting from the ce, the grape juice macromolecules (>3.5 kda) were initially fractionated by anion exchange chromatography (aec). since the grape proteins have very similar mws and different pi (monteiro et al., 2001), this chromatographic technique proved to be very effective at this stage. aec has already been applied previously to those compounds (waters et al., 1992; dorrestein et al., 1995; pastorello et al., 2003), allowing to obtain a good resolution of protein peaks. a representative aec chromatogram for grape juice macromolecules (≈ 50 mg) is shown in fig. 2. the material not retained by the column (flow through) was very little or at least had a low uv absorption. almost all peaks were eluted at relatively low nacl concentrations (0.08-0.12 m), while a last peak was obtained with a high nacl concentration (1 m), indicating that at ph 9 the eluting fractions have different charge properties. six separated fractions were collected (fig. 2) and analysed by sds-page in non-reducing conditions (fig. 3). as expected, the flow through did not contain any proteins. the first two peaks (1a and 1b) both displayed a band at ≈ 20 kda. fraction 1b contained also some minor bands around 25 kda. all these bands probably correspond to tlps according to literature data (waters et al., 1996; marangon et al., 2011; marangon et al., 2014). moreover, peak 1b showed a 40 kda band which could correspond to a β-glucanase (esteruelas et al., 2009; sauvage et al., 2010). peak 2 contained only one band that showed a mr similar to that of tlps (waters et al., 1996; marangon et al., 2011; marangon et al., 2014). peaks 3a and 3b showed several bands with mrs similar to that of tlps and a protein of ≈ 66 kda, probably corresponding to invertase (marchal et al., 1996). ital. j. food sci., vol 29, 2017 55 figure 2. anion exchange chromatogram for manzoni bianco crude extract (50 mg). collected fractions are indicated by numbered boxes. the dotted line indicates the salt gradient. figure 3. non-reducing sds-page of the fractions collected from anion exchange chromatography. molecular weight standard proteins are on the left. the presence of faint bands with mws of ≈ 31 and ≈ 32 kda is noteworthy, which could correspond to grape chitinases (waters et al., 1996; marangon et al., 2009). peak 3b also contained a ≈ 52 kda band, whose identity was investigated in this work. finally, peak 4 showed another band with the same mobility of tlps and a low mw band that could correspond to the grape ltp (pastorello et al., 2003) (fig. 3). according to this chromatographic behaviour it is clear that different protein isoforms assigned to both tlps and chitinases on the basis of their sds-page mobility show different charge properties at ph 9 being eluted from the aec column at different nacl concentrations (from ≈ 0.08 to ≈ 0.12 m). this is obviously related to a heterogeneity of the grape juice proteins at the amino acid level (monteiro et al., 2001) which is likely to affect their net ital. j. food sci., vol 29, 2017 56 charge also at the low ph of the wine. since charge is one of the main factors involved in protein functionality in terms of colloidal behavior (vincenzi et al., 2011), it is likely that different forms of the same protein detected by sds-page in the different aec fractions play specific roles in the phenomena leading to haze formation in wine. indeed the phdependent variation of protein charges has been indicated as one of the factors that strongly affect protein aggregation in wine (dufrechou et al., 2012). the chitinasecontaining peaks (3a and 3b in figures 2 and 3), from 15 chromatographic separations of 50 mg of protein each, all giving the same results (not shown), were combined and freeze dried. since this sample (from now on named “peak 3”) was contaminated by other proteins (fig. 3, lanes 3a and 3b), a further purification step involving hydrophobic interaction chromatography (hic) was used, resulting in the separation of protein fractions differing in surface hydrophobicity (van sluyter et al., 2009). the protein peak 3 from aec gave six peaks after hic (fig. 4). sds-page analysis of the proteins of hic peaks under reducing and non-reducing conditions, showed several protein bands, differing in both mr and staining intensity (fig. 5). also in this case, proteins with the same sds-page mobility were detected in more than one peak, indicating differences in surface hydrophobicity of components showing very similar charges (those of fractions 3a and 3b of the aec) and apparent molecular weight (by sds-page). hydrophobicity is also an important property affecting the interaction of a protein with other components (siebert et al., 1996). therefore the propensity to form haze in wine can be different for wine protein isoforms with different hydrophobicity, as demonstrated, for example, by studying the reactivity of wine protein fractions differing for this parameter with tannins (marangon et al., 2010). figure 4. hydrophobic interaction chromatogram of aec fraction 3 (pooled fractions 3a and 3b). collected fractions are indicated by numbered boxes. the dotted line indicates the linear gradient. the bands of hic fractions 3 and 4 were found to be almost pure when analysed by sdspage in reducing conditions (fig. 5a). however, when the same samples were analysed in non-reducing conditions a variation of the electrophoretic patterns was noted. in addition to a shift of the main bands to a slightly higher apparent molecular weight, a minor low mobility band of ≈ 52 kda was detected when the gel was run under nonreducing conditions (fig. 5b). similar bands of 50-52 kda found by analysing the proteins present in the natural wine haze, and not directly in the grape juice as done here, where ital. j. food sci., vol 29, 2017 57 identified by nanolc-ms/ms as vitis vinifera class iv chitinase (marangon et al., 2011). however, in that study it was not clear the origin of these high mw chitinase bands, which could be artefacts produced during the extraction procedure (marangon et al., 2011), but also the result of the protein aggregation leading to haze. in contrast, the 52 kda bands here detected, which were obtained directly from the grape juice, are clearly due to the presence of disulphide-linked proteins, being present only when the fractions were not treated with a reducing agent (fig. 5b). figure 5. reducing (a) and non-reducing (b) sds-page of the fractions (1 to 6) from hydrophobic interaction chromatography. molecular weight standard proteins are on the left. when tested for chitinolytic activity on gel (vincenzi and curioni, 2005), peaks 3 and 4 confirmed to contain active chitinases corresponding to the bands with the mw expected for grape chitinases (chi 1, chi 2, chi 3 and chi not delayed) (in the range 30-32 kda) but also to that of the disulphide linked components of ≈ 52 kda (chi dimer i and ii) (fig. 6). in contrast the other chromatographic fraction did not show bands with well marked chitinolytic activity (not shown). confirming what was previously reported (vincenzi and curioni, 2005), the presence of glycol chitin in the non-reducing sds-page gel caused a mr decrease of the chitinase bands chi 1, chi 2, chi 3 and this shift was proportional to the quantity of glycol chitin incorporated (fig. 6). this result indicates that grape chitinases interact with the substrate during the electrophoretic migration if not reduced, likely due to the presence of the chitin-binding domain typical of the type iv chitinases (collinge et al., 1993). however, one minor chitinase isoform (chi not delayed) did not show the same behaviour (fig. 6), suggesting that the chitin-binding domain involved in the interaction with chitin was lacking in this component. it is also interesting to note that the ≈ 52 kda band was retarded in these conditions, showing the same behaviour of the main bands with higher mr. this result and the disappearance of the ≈ 52 kda band in reducing conditions suggest that this protein could be a dimer of chitinases linked by s-s bonds. as a matter of fact, all these bands, including that at ≈ 52 kda, showed chitinolytic activity after staining the gels for its detection (fig. 6). ital. j. food sci., vol 29, 2017 58 figure 6. sds-page analysis of hic fractions 3 and 4 under reducing (r) and non-reducing (nr) conditions. gels contained 0 (a), 0.01 (b, d), and 0.05 (c, e) % glycol chitin (gc). panels a-c: staining for proteins. panel de: staining for chitinolytic activity. the arrowheads indicate bands retarded in the presence of glycol chitin. the bands selected for maldi-tof/tof ms analysis are indicated in panels a and b. molecular weight standard proteins are on the left. 3.3. ms protein identification the six bands showing chitinolytic activity (chi 1, 2 and 3, chi dimers i and ii, and chi not delayed, fig. 6) were excised from the sds-page gels and analysed by malditof/tof ms. according to database searching using mascot, all bands were found to belong to vitis vinifera class iv chitinases, including that named “chi not delayed”. as mentioned above, these chitinases are characterised by the presence of a chitin binding domain of the hevein type in the n-terminal region (collinge et al., 1993), and this would be the reason for being retarded in glycol chitin containing gel (vincenzi and curioni, 2005). therefore, since the electrophoretic migration of the minor “chi not delayed” band was unaffected by the presence of glycol chitin in the gel, it is likely that this grape chitinase has in common with the typical class iv chitinases only one part of its structure, but not the chitin binding domain. in most of the cases the analysed proteins were assigned to two isoforms of class iv chitinases (accessions>gi|2306811|gb|aab65776.1| and >gi|33329392|gb|aaq10093.1|). only for the band named ‘chi dimer ii’ there was only one sequence matched (>gi|2306811|gb|aab65776.1|), and three in the case of ‘chi 3’ (table 1). ital. j. food sci., vol 29, 2017 59 table 1. selected proteins identified by maldi-tof/tof ms. sample protein identification name ncbi accession number sequence coverage (%) number of peptides matched chi 1 class iv endochitinase [vitis vinifera] class iv chitinase [vitis vinifera] >gi|2306811|gb|aab65776.1| >gi|33329392|gb|aaq10093.1| 17% 17% 5 5 chi 2 class iv endochitinase [vitis vinifera] class iv chitinase [vitis vinifera] >gi|2306811|gb|aab65776.1| >gi|33329392|gb|aaq10093.1| 20% 20% 6 6 chi 3 class iv endochitinase [vitis vinifera] class iv endochitinase [vitis vinifera] class iv chitinase [vitis vinifera] >gi|2306811|gb|aab65776.1| >gi|2306813|gb|aab65777| >gi|33329392|gb|aaq10093.1| 15% 15% 15% 5 5 5 chi not delayed class iv chitinase [vitis vinifera] class iv endochitinase [vitis vinifera] >gi|33329392|gb|aaq10093.1| >gi|2306811|gb|aab65776.1| 20% 20% 6 6 chi dimer i class iv chitinase [vitis vinifera] class iv endochitinase [vitis vinifera] >gi|33329392|gb|aaq10093.1| >gi|2306811|gb|aab65776.1| 17% 17% 5 5 chi dimer ii class iv endochitinase [vitis vinifera] >gi|2306811|gb|aab65776.1| 18% 5 ital. j. food sci., vol 29, 2017 60 two reasonable hypotheses can be given to explain why bands with different electrophoretic and chromatographic behaviour are recognised as chitinases corresponding to the same isoforms: i) the ms data do not provide complete coverage of any sequence and therefore a precise identification of the proteins is not possible. in addition, because of the partial lack of available grape protein sequences, there is a chance that the selected peptides do not exactly match corresponding database entries; ii) the proteins could be modified forms of the same original chitinase isoforms, affecting only the chromatographic and electrophoretic behaviour but not the catalytic activity or the capacity to bind chitin. indeed, it has been shown that some partial modification of the chitinases could occur during juice preparation (waters et al., 1998; manteau et al., 2003; dahiya et al., 2006). overall, the results of the ms analysis are the same of those reported for the proteins found in natural wine haze (marangon et al. 2011), confirming that grape protein components related to type iv chitinases are actually those mainly involved in haze formation in wines. 4. conclusions grape chitinases are considered as one of the main protein components involved in protein haze formation in wines (falconer et al., 2010; marangon et al., 2011). here we have confirmed that these proteins are present in the grape juice as different isoforms, which, although sharing common amino acid sequences related to type iv chitinases, can be distinguished on the basis of their electrophoretic and chromatographic behaviours. moreover, chitinases are present in the grape juice also in the form of s-s-linked dimers, and also in a form apparently lacking the chitin-binding domain. since all these characteristics can be related to differences in the functional properties of the single components, it is likely that the different chitinase isoforms found in grape juice have different impacts on their hazing potential in wines, as it has been demonstrated for tlps (gazzola et al., 2012; marangon et al., 2014). in particular, differences in charge, hydrophobicity and also molecular weight can affect the interactions of the single chitinase components when they are present in a complex colloidal system as wine, leading to different tendencies to form haze. this can be an important point to be clarified, not only to better understand the mechanisms of wine hazing, but also for practical pourposes. for example, the identification of the most unstable protein components will help to develop protein instability tests much more specific than those currently in use, thus allowing the winemaker to be more precise in applying the wine stabilisation treatments. moreover, also these treatments can be improved by a deep knowledge of the molecular characteristics and functionality of the single wine protein components, which will allow to design stabilisation treatments tailored to specifically remove the desired proteins and not the others. for example, the discovery that wine chitinases are able to bind chitin was the rational basis for the application of chitin as a specific adsorbent to remove these unstable proteins from wine (vincenzi et al., 2005). in conclusion, the biochemical and molecular characterisation of the different protein components of grape, as done here, can be of great help to develop “precision” winemaking techniques aimed to improve wine quality. acknowledgements this work was 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williams p.j. 1998. sequence analysis of grape (vitis vinifera) berry chitinases that cause haze formation in wines. j. agric. food chem. 46:4950. waters e.j., shirley n.j. and williams p.j. 1996. nuisance proteins of wine are grape pathogenesis-related proteins. j. agric. food chem. 44:3. waters e.j., wallace w. and williams p.j. 1992. identification of heat-unstable wine proteins and their resistance to peptidases. j. agric. food chem. 40:1514. paper received july 7, 2016 accepted august 7, 2016 p u b l i c a t i o n s codon italian journal of food science, 2023; 35 (1): 91–105 issn 1120-1770 online, doi 10.15586/ijfs.v35i1.2279 91 p u b l i c a t i o n s codon fermented nondairy functional foods based on probiotics pınar şanlıbaba department of food engineering, faculty of engineering, ankara university, ankara, turkey *corresponding author: pınar şanlıbaba, department of food engineering, faculty of engineering, ankara university, ankara, turkey. email: sanlibab@ankara.edu.tr received: 9 september 2022; accepted: 20 february 2023; published: 10 march 2023 © 2023 codon publications open access paper abstract functional foods containing probiotic bacteria are consumed worldwide. according to the food and drug administration (fda), probiotics are living microorganisms that contribute to the host’s overall health when provided in adequate amounts. fermented dairy products, especially yogurt and other dairy-based products, are good substrates for probiotic delivery. however, recently, consumers have begun to seek alternatives due to lactose intolerance and high fat and cholesterol contents. moreover, the growing vegetarianism has increased the demand for nondairy probiotic foods. thus worldwide, researchers are studying probiotic bacteria feasibility in nondairy products, including fruits, vegetables, and cereals. this study aims to give an overview of various nondairy based products that contain probiotic bacterial strains available worldwide based on fruits, vegetables, cereals, chocolate-based products, and meat products. moreover, the latest globally available commercial products are also summarized. keywords: functional foods, nondairy-based products, probiotic introduction functional food was introduced in the mid 1980s by the japanese government (min et al., 2019; zamfir et al., 2022). in 1991, the japanese ministry of health, welfare, and labor established a systematic regulatory system for functional foods, and “food for specified health uses” (foshu) became the legal term (iwatani and yamamoto, 2019; gomes et al., 2021). however, the interest in functional foods in europe started in the 1990s. later, the european commission established a commission called functional food science in europe (fufose) to explore functional foods (perricone et al., 2015). functional foods are used to maintain or regulate specific health conditions, such as decreased cancer risk, improve heart health, enhancement of the immune system, to reduce menopause symptoms, improvement of gastrointestinal health, preservation of urinary tract health, anti-inflammatory influences, decrease in blood pressure and cholesterol levels, reduced osteoporosis, and anti-obesity influences (al-sheraji et al., 2013; aspri et al., 2020). functional food is classified into four different categories by the american dietetic association (ada): (1) conventional foods, (2) modified foods, (3) medical foods, and (4) foods for special dietary use. of these functional foods, probiotic foods have currently received maximum attention as health promoters, accounting for approximately 60–70% of the total functional food market (aspri et al., 2020; misra et al., 2019; küçükgöz and trzaskowska, 2022). generally, three main ingredients designed for gut health are added to functional foods. they are living microorganisms (probiotics), nondigestible carbohydrates (prebiotics), and secondary plant metabolites (polyphenol compounds) (panghal et al., 2018). elie metchnikoff, a russian-born nobel prize winner and professor at the pasteur institute in paris, first expressed the probiotic concept. he suggested that some selected bacteria may have positive effects on the human gastrointestinal tract. he observed that many bulgarian 92 italian journal of food science, 2023; 35 (1) şanlıbaba p bifidobacterium spp. in addition, bacillus, pediococcus, clostridium, and some yeasts such as saccharomyces (e.g., saccharomyces cerevisiae and saccharomyces bou­ lardii) have also been used as probiotic candidates (kumar et al., 2022; soccol et al., 2010). most probiotic cultures are gram-positive, usually catalase-negative, non-motile, non-spore-forming, and non-flagellated. most probiotics grow optimum at 37°c and ph of 6.5–7.5, but some prefer 30°c, for example, lactobacillus casei (song et al., 2012). who, fao, and efsa (the european food safety authority) suggested that before using them as a probiotic in food application, safety, technological, and functional characteristics of probiotic organisms must be considered (markowiak and slizewska, 2017). hence, some criteria need to be fulfilled. some of the safety properties of probiotics are summarized as; (1) must be isolated from healthy human or animal gastrointestinal tract, (2) have a history of safe use, (3) must have diagnostic identification traits (phenotype and genotype traits), (4) no adverse effects, (5) absence of genes responsible for antibiotic resistance, (6) nonpathogenic and nontoxic, ability to interact or send signals to immune modulator activity, and (7) survive in the presence of administered drugs and other antimicrobial compounds. functional criteria are defined as (1) resistance to low ph for surviving and processing in the gut condition, (2) the ability to resist gastric juices, enzymes and the exposure to bile acid, which is crucial for oral administration, (3) ability to pass through gastrointestinal tract at low ph and in contact with bile salts, (4) antimicrobial activity against pathogenic bacteria such as salmonella spp., listeria monocytogenes, clostridium spp., (5) ability to influence local metabolic activity, (6) health effects on human bodies such as anti-carcinogenicity and anti-mutagenic activity, and cholesterol-lowering effect, (7) to create a beneficial effect on the host by increasing disease resistance, and (8) to have the power of restore and replace the intestinal microflora (kumar et al., 2022; pimentel et al., 2021). finally, technological aspects are detailed: (1) to have excessive cell viability, (2) to have genetic stability, (3) must be stable, safe, effective, and equipped for staying viable under storage conditions, (4) ability to be viable and stable during the food processes and (5) ability to have large scale production (abatenh et al., 2018; kumar et al., 2022; markowiak and slizewska, 2017). table 1 shows the frequently used probiotic cultures applied in food systems, some of which are produced commercially. as an example, lactobacillus acidophilus la–1, lactobacillus acidophilus la–5, lactobacillus paracasei crl 431, bifidobacterium animalis bb–12, bifidobacterium bifidum bb-11, lacto bacillus paracasei crl 431, and bifidobacterium lactis bb–12 from chr. hansen (horsholm, denmark); lactobacillus casei shirota and bifidobacterium breve strain yakult from yakult (tokyo, japan); lactobacillus acidophilus r0011 peasants had long and healthy lives as they consumed large quantities of fermented dairy products, including beneficial organisms. therefore, he suggested the possibility to direct the microflora in human bodies to replace harmful microorganisms with beneficial ones (shorkryazdan et al., 2017). research on this concept has been developed further to date. this study summarizes some characteristics of probiotics as a functional ingredient in food and nondairy-based probiotic foods. probiotics, prebiotics, and synbiotics probiotics, prebiotics, and synbiotics have attracted attention for developing balance in gastrointestinal microflora (küçükgöz and trzaskowska, 2022). the term “probiotic” comes from the greek meaning “for life”. in 1954, ferdinand vergin first used this term in his article entitled “anti-und probiotika”, comparing the beneficial effects of antibiotics and other antibacterial agents of some useful bacteria. later, lilly and stillwell, in 1965, determined that probiotics were substances produced by microorganisms that support the growth of other organisms. subsequently, the definition of probiotics was modified several times and changed by many scientists till today. in 1989, fuller emphasized that these organisms must be viable and have some beneficial health effects on humans. the current definition of 2002, by fao (food and agriculture organization of the united nations) and who (world health organization), stated that “probiotics are living strains of strictly selected microorganisms that, when administered in sufficient quantities, provide a health benefit to the host” (markowiak and slizewska, 2017). the last updated definition was made in 2013 by a panel of expert teams from the international scientific association for probiotics and prebiotics, and fao/ who’s definition was reinforced with some minor grammatical corrections. the final definition is “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.” three main key aspects, such as viable, microbial, and beneficial to health, were included in this definition (kumar et al., 2022; shorkryazdan et al., 2017). generally, the level of viable probiotic organisms should be at least a minimum of 106 cfu (colony forming units)/g of viable cells during the food shelf life to show the minimum therapeutic effect on the human body. it was also suggested that a daily intake of 108–109 cfu/g probiotic microorganisms could pass the upper ingestion to show their beneficial physiological effects on the human body. a daily intake of approximately 100 g of probiotic food products should be consumed to reach these counts of viable probiotic cells (küçükgöz and trzaskowska, 2022; terpou et al., 2019). the most important probiotic microorganisms are lactic acid bacteria (lab) that include lactobacillus spp. and italian journal of food science, 2023; 35 (1) 93 nondairy functional probiotic foods in pharmaceutical and food systems, the selection of probiotic cells and their differentiation are crucial. foodbased probiotic products are classified into two distinct groups: dairy products and nondairy products (dahiya and singh-nee nigam, 2022; terpou et al., 2019). the purpose of this study was to provide information about nondairy probiotic foods. two terms entitled “prebiotics” and “synbiotics” are also significant. prebiotics are short–chain carbohydrates (sccs), mostly nondigestible food ingredients, which positively affect the host’s health by selectively stimulating the growth and/or activity of specific microorganisms in the colon such as lactobacillus spp. and bifido­ bacterium spp. (aladeboyeje and sanli 2021; al–sheraji et al., 2013). when prebiotics passes from the small intestine to the lower gut, health-promoting bacteria such as probiotics selectively ferment and use these fibers as an energy source by breaking them down. in addition, they also boost the human immune response by increasing the gut microbial activity and the production of short-chain fatty acids (scfa), particularly acetic acid, propionic acid, and butyric acid (priya, 2020). among the beneficial effects of prebiotics, scfa production stimulation is essential (nagpal et al., 2012). these products have some positive impact on the human body. they participate in different host signaling mechanisms, affect t-helper 2 cells in the airways and macrophages, decrease the colon ph, stimulate gut hormone release, shape the gut environment, and influence the colon physiology. they are also used as energy sources by colonic epithelial cells and the intestinal microflora (lockyer and stanner, 2019). the term “prebiotic” was first defined by glenn and lactobacillus rhamnosus r0052 from institut rosell (montreal, canada); lactobacillus johnsonii la1 (same as lj1) from nestle’ (lausanne, switzerland); lactobacillus plantarum 299v, lacto bacillus plantarum lp01 and lactobacillus rhamnosus 271 from probi ab (lund, sweden); lactobacillus rhamnosus gg from valio dairy (helsinki, finland); saccharomyces boulardii from biocodex inc. (seattle, wa); bifidobacterium longum bb536 from morinaga milk industry co., ltd (zama– city, japan); lactobacillus casei dn014001 and bifido­ bacterium essencis from danone le plessis robinson (paris, france); lactobacillus salivarius from ucc118 university college (cork, ireland); bacillus lactis dr10 from danisco (copenhagen, denmark) (nagpal et al., 2012; pimentel et al., 2021; soccol et al., 2010). the health benefits of probiotics depend on the specific strain and dosage. one strain cannot show all positive health effects. foods containing probiotic strains from different species rather than a single strain show better success in providing human health benefits and broader efficacy (žuntar et al., 2020). probiotic cultures differ in health effects on humans. the mechanism of probiotics on human health is still unclear (küçükgöz and trzaskowska, 2022). however, some of the potential effects of probiotic microorganisms are summarized in table 2. nowadays, three different types of food products supplemented with probiotic cells are available for direct or indirect human consumption. these are (1) fermented or nonfermented form, (2) dried or deep–frozen for industrial or home uses, and (3) drugs in powder, capsule, or tablet meaning pharmaceutical form (misra et al., 2019). table 1. commonly used probiotic organisms in food application, both dairy and non-dairy foods. (*) lactobacillus spp. lactobacillus acidophilus, lactobacillus amylovorus, lactobacillus brevis, lactobacillus casei, lactobacillus rhamnosus, lactobacillus crispatus, lactobacillus delbrueckii subsp. bulgaricus, lactobacillus fermentum, lactobacillus gasseri, lactobacillus helveticus, lactobacillus johnsonii, lactobacillus lactis, lactobacillus paracasei, lactobacillus plantarum, lactobacillus reuteri, lactobacillus salivarius, lactobacillus gallinarum, lactobacillus cellobiosus, lactobacillus curvatus, lactobacillus gasseri bifidobacterium spp. bifidobacterium adolescentis, bifidobacterium animalis, bifidobacterium breve, bifidobacterium bifidum, bifidobacterium infantis, bifidobacterium lactis, bifidobacterium longum, bifidobacterium thermophilum bacillus spp. bacillus coagulans, bacillus cereus, bacillus subtilis, bacillus lentus, bacillus licheniformis, bacillus cereus var. toyoi, bacillus clausii, bacillus laterosporus, bacillus pumilus, bacillus racemilacticus pediococcus spp. pediococcus acidilactici, pediococcus cerevisiae, pediococcus pentosaceus streptococcus spp. streptococcus salivarius subsp. thermophilus, streptococcus intermedius bacteriodes spp. bacteriodes capillus, bacteriodes suis, bacteriodes ruminicola, bacteriodes amylophilus propionibacterium spp. propionibacterium freudenreichii, propionibacterium shermanii leuconostoc spp. leuconostoc mesenteroides subsp. dextranicum lactococcus spp. lactococcus lactis subsp. cremoris, lactococcus lactis subsp. lactis enterococcus spp. enterococcus faecalis, enterococcus faecium, enterococcus durans other bacteria clostridium butyricum, escherichia coli nissle 1917 yeast and mold aspergillus niger, aspergillus oryzae, saccharomyces boulardii, candida torulopsis, candida pintolopesii adapted from abatenh et al., 2018; žuntar et al., 2020. 94 italian journal of food science, 2023; 35 (1) şanlıbaba p table 2. potential health effect of probiotics and prebiotics.(*) probiotics prebiotics 1. reduction of food allergy symptoms such as atopic dermatitis 1. helping increase the count of useful organisms in intestines, hence possibly help in preventing gastroenteritis 2. lowering cholesterol level 2. decreasing inflammatory bowel disease related to the intestinal microbiota pathogenesis 3. elimination of pathogens or decrease in pathogenic adherence by lactic acid and bacteriocin 3. reduction of colorectal cancer risk (decreasing the activity of genotoxic enzyme via the administration of prebiotics) 4. pediatric gastroprotection 4. prevention and treatment of allergy 5. increasing the body’s immunity (immune stimulation and immunomodulatory activity) 5. effective in bone mineralization (prebiotics play a crucial role in adult bone mass depending on the bioavailability of calcium) 6. elimination of helicobacter pylori, inflammatory bowel disease (crohn’s disease, pouchitis, and ulcerative colitis) 6. plays an active role in preventing atherosclerosis and cardiovascular disease 7. reduction of irritable bowel syndrome 7. selectively stimulating the growth and/or activity of certain types of organisms in the colon 8. treatment of diseases such as obesity, insulin-resistant syndrome, and type-2 diabetes 8. improving immune response by increasing the gut microbial activity 9. having anti-carcinogenic effects such as colorectal cancer and anti-mutagenic activities 9. treatment of hepatic encephalopathy 10. decreasing incidence and duration of antibiotic-induced diarrhea 10. lowering serum lipid concentration 11. decreasing blood pressure 11. insulin sensitivity improvement 12. improving child growth 12. laxative agent 13. formation of useful compounds, such as vitamins, short-chain fatty acids, and conjugated linoleic acid 13. lowering the glycemic index and body weight 14. showing antioxidant activity 15. reducing symptoms related to lactose intolerance 16. reduction of toxin-binding and detoxification activity 17. effective in nutrient absorption 18. pathogen interference, exclusion, and antagonism 19. having neuropsychiatric disorders via the enteric and central nervous system 20. maintenance of mucosal integrity adapted from markowiak and slizewska, 2017; priya, 2020; shorkryazdan et al., 2017; younis et al., 2015. gibson and marcel roberfroin in 1995. a compound should have several requirements to be prebiotic. these are summarized as: (1) not be hydrolyzed by mammalian enzyme, (2) not be absorbed in the upper part of the gastrointestinal tract, (3) fermented selectively by intestinal flora, (4) promote the growth of selected beneficial bacteria, (5) resistant to gastric acidity, and (6) not confer negative consequences to the host, such as excess gas production or the growth of pathogenic organisms (lockyer and stanner, 2019; younis et al., 2015). the ways of isolating the most commonly known prebiotics and its candidates are summarized as follows: (1) extraction from plants (such as chicory root, onions, whole grains, bananas, and garlic), (2) enzymatic hydrolysis (like oligofructose from inulin), (3) synthesis from monoor disaccharides (such as fructo-oligosaccharides [fos] from sucrose, or galactooligosaccharides [gos] and transgalactosylated oligosaccharides from lactose), or (4) microbiological production. among prebiotics, inulin and oligosaccharides are most recognized as dietary fibers in the world. in addition, fos, gos, inulin, isomalto oligosaccharide (imo), and beta-glucan are also commonly used as prebiotics in food applications (singla and chakkaravarthi, 2017; younis et al., 2015). some health effects of prebiotics are summarized in table 2. enhancing organoleptic characteristics and improving both taste and mouthfeel are among the positive effects of prebiotics in food applications. prebiotics must also be chemically stable in food processing, such as high temperature, low ph, and maillard reaction conditions. when prebiotics are broken down into their components, such as monoand disaccharides, or chemically altered, they no longer provide selective stimulation of beneficial microorganisms called as probiotics (gomes et al., 2021; younis et al., 2015). in 2015, the united kingdom scientific advisory committee on nutrition’s (sacn) recommended an average intake of approximately 30 g/day for adults, 15 g/day for children aged italian journal of food science, 2023; 35 (1) 95 nondairy functional probiotic foods gas production in the gastrointestinal tract, and diarrhea are common symptoms of lactose intolerance (gomes et al., 2021; panghal et al., 2018). moreover, vegetarianism is increasing in both developed and developing countries, thus demanding plant-based probiotic products. high cholesterol level in human is another important health problem. milk contains 4–5% fat. consuming high-fat dairy products causes an increase in total cholesterol and low-density lipoprotein (ldl) cholesterol contents in the blood, thereby leading to coronary heart disease. this can be eliminated by lowering the consumption of high ldl-cholesterol foods (hassan et al., 2010; kumar et al., 2022). these reasons make investigating the potential of nondairy foods in supporting probiotic cultures of immense importance. therefore, in recent years, probiotic cultures have been successfully applied to other types of food matrices, including meats, beverages, cereals, vegetables, and fruit (aspri et al., 2020). the first nondairy probiotic food, named proviva, was formulated and manufactured by a swedish company, skane dairy, in 1994. the main substrate in this product is oatmeal gruel fermented by lactobacillus plantarum 299v. then, malted barley was added to improve the liquefaction of this product. finally, fermented food was mixed at a concentration of 5% with different fruit juices such as strawberry, blueberry, rosehip, or any tropical fruit. proviva contains approximately 5 × 1010 cfu/l of viable l.actobacillus plantarum 299v. like proviva, goodbelly prepared from oatmeal and fermented with lactobacillus plantarum 299v was the first nondairy probiotic drink in the us market in 2006. today, many nondairy based probiotic products are commercially available (table 3). cereal-based probiotic products recently interest in cereal-based probiotic foods has been increasing. cereals are the most important sources of nutrients and bioactive compounds in people’s diets. moreover, they are an excellent resource for nondigestible carbohydrates such as fiber and oligosaccharides, which stimulate probiotic culture growth (kockova and valik, 2014; küçükgöz and trzaskowska, 2022). dietary fibers are divided into two groups according to their solubility in water, namely insoluble and soluble fibers. cereals usually have insoluble fiber, including cellulose, hemicellulose, and lignin (fernandes et al., 2018). however, some cereals also contain water-soluble fiber, such as β-glucan and arabinoxylan (vasudha and mishra, 2013). consumption of whole grains also has some positive effects on human health. these effects are reducing the risk of type-2 diabetes, cardiovascular disease, obesity, and certain types of cancer (lamsal and faubion, 2009). rice, millets, rye, barley, sorghum, maize, and wheat are the most consumed cereals, while buckwheat, 2–5 years, 20 g/day for children aged 5–11 years, 25 g/day for children aged 11–16 years, and 30 g/day for adolescents aged 16–18 years of fiber from natural dietary sources (lockyer and stanner, 2019). formulating a probiotic product with prebiotics gives synbiotics properties to the food item. in 1995, gibson and roberfroid introduced this term to describe a combination of probiotics and prebiotics. synbiotics means synergistically acting probiotics and prebiotics. for example, lactobacillus rhamnosus, bifidobacterium spp., lacto­ bacillus acidophilus, and lactobacillus casei, when combined with prebiotics such as raftiloses p95, improve their viabilities at 4°c on 4 weeks of storage. pear and apple fibers combined with raffinose also increased the activity and viability of probiotic bacteria. it is recommended that 10 g/day of gos is sufficient to improve fecal bifidobacteria levels. moreover, xylo-oligosaccharides 2 g/day are necessary for the bifidogenic effect. similarly, 2–10 g/day fos intake is essential as a bifidogenic stimulus (al-sheraji et al., 2013). european milk and yogurt producers such as aktifit (emmi, switzerland), proghurt (ja naturlich naturprodukte, austria), vifit (belgium, uk), and fysiq (netherlands) are the widely used synbiotics concept to produce probiotic foods (dahiya and singh-nee nigam, 2022; nagpal et al., 2012). why nondairy probiotics? traditionally for about a century, probiotic foods have been commonly recognized as dairy-based derived products due to more demand and easy availability. besides, yogurt has always been the top probiotic food preference. some other examples of dairy-based products are fermented milk, milk powder, sour cream, cheese, fermented dairy beverage, buttermilk, dairy desserts, flavored liquid milk, baby foods, and ice cream (misra et al., 2019). consumer vegetarianism, dyslipidemia, lactose intolerance, milk protein allergies (casein, cholesterol phobia), the requirement of cold storage, and economic reasons associated with dairy-based probiotic products are the main causes restricting their consumption (chaudhary, 2019; zamfir et al., 2022). lactose intolerance is the most important health problem, which accounts for 75% of the world’s population, according to the national institute of diabetes and digestive and kidney diseases (niddk). the absence of lactase enzyme activity, which hydrolyses lactose into glucose and galactose, is the leading cause of this disease. lactose, known as milk sugar, is a crucial carbohydrate for the health of a newborn. during the postnatal period, this intestinal enzyme is at the highest in infants. however, two distinct groups, entitled lactose nonpersistence, and lactose persistence occurred among 2–12-year-old children. bloating gastric pain and cramps, 96 italian journal of food science, 2023; 35 (1) şanlıbaba p ta bl e 3. c om m er ci al ly a va ila bl e no nd ai ry p ro bi ot ic fo od s. * p ro du ct n am e m an uf ac tu re r p ro bi ot ic s tr ai ns m aj or c ha ra ct er is tic s fr ui tba se d b io la r ti n e b a , n or w ay la ct ob ac ill us rh am no su s g g m ix tu re o f ap pl epe ar a nd o ra ng em an go ju ic e, 9 5% fr ui t, no s ug ar . b ra vo f ris cu s p ro bi a b, s w ed en la ct ob ac ill us p la nt ar um h e a l+ a nd l b. p ar ac as ei 87 00 :2 o ra ng e ap pl e an d tro pi ca l f ru it ju ic es k e v it a k e v it a , u s a la ct ob ac ill us rh am no su s, l ac to ba ci llu s pl an ta ru m , la ct ob ac ill us p ar ac as ei , b ac ill us c oa gu la ns g b i-3 0 60 86 va rio us fr ui t m ix tu re (s tra w be rr y, a nd c oc on ut , l im e, m in t a nd c oc on ut , pi ne ap pl e an d co co nu t) m al ee p ro bi ot ic s m al ee e nt er pr is e c om pa ny , t ha ila nd la ct ob ac ill us p ar ac as ei p ru ne , g ra pe a nd o ra ng e ju ic e p ro bi ot ic m ac hi ne t ro pi ca l m an go pe ps i c om pa ny (p ep si c o) b ifi do ba ct er iu m s pp . m ix tu re o f ap pl e, o ra ng e an d pi ne ap pl e ju ic e, m an go p ur ee , b an an a pu re e, fo s a nd n at ur al fl av or s tr op ic an a p ro bi ot ic s tr op ic an a, u s a b ifi do ba ct er iu m la ct is fr ui t j ui ce m ix tu re s uc h as s tra w be rr y, b an an a, p in ea pp le , m an go a nd p ea ch pa ss io n fr ui t p ro v iv a e m e a p ro bi a b, s w ed en la ct ob ac ill us p la nt ar um 2 99 v fr ui t j ui ce (o ra ng e, s tra w be rr y, o r b la ck cu rr an t) p e r k ii p ro bi ot ic w at er p e r k ii, a us tra lia la ct ob ac ill us c as ei l c4 31 fr ui t j ui ce m ix tu re (s uc h as m an go , s tra w be rr y, li m e, c oc on ut , w at er m el on , ra sp be rr y, p om eg ra na te , p as si on fr ui t) b io -l iv e g ol d b io -l iv e/ m ic ob z lt d. , u k m ix tu re o f 13 s tra in s, in cl ud in g la ct ob ac ill us ac id op hi lu s, l ac to ba ci llu s bu lg ar ic us , l ac to ba ci llu s ca se i, la ct ob ac ill us fe rm en tu m , l ac to ba ci llu s pl an ta ru m , l ac to co cc us la ct is , b ac ill us s ub til is , b ifi do ba ct er iu m b ifi du m , b ifi do ba ct er iu m in fa nt is , b ifi do ba ct er iu m lo ng um , s tr ep to co cc us th er m op hi lu s, s ac ch ar om yc es c er ev is ia e m ix tu re o f fr ui t j ui ce s su ch a s ac ai , b er ry , c he rr y, g oj i, no ni , l em on , a nd va rio us h er bs . m al ee p ro bi ot ic s m al ee e nt er pr is e c om pa ny l td ., th ai la nd la ct ob ac ill us p ar ac as ei fr ui t j ui ce s su ch a s pr un e, g ra pe , a nd o ra ng e c er ea l-b as ed b us he ra b as ill ia f oo ds , c en tu rio n, g au te ng , s ou th a fri ca la ct ob ac ill us b re vi s, l ac to ba ci llu s de lb ru ec ki i, la ct ob ac ill us p ar ac as ei , l ac to ba ci llu s pl an ta ru m s or gh um , m ill et fl ou r m ah ew u b as ill ia f oo ds , c en tu rio n, g au te ng , s ou th a fri ca la ct oc oc cu s la ct is s ub sp . l ac tic m ai ze , s or gh um , m ill et m al t, w he at av en ly v el le av en ly o y lt d. , f in la nd la ct ob ac ill us a nd b ifi do ba ct er iu m o at b as ed d rin k g ra in fie ld s w ho le gr ai n liq ui d ag m f oo ds p vt . l td ., a us tra lia la ct ob ac ill us a ci do ph ilu s, l ac to ba ci llu sd el br ue ck ii, s ac ch ar om yc es b ou la rd ii, s ac ch ar om yc es c er ev is ia e g ra in s, b ea ns a nd s ee ds italian journal of food science, 2023; 35 (1) 97 nondairy functional probiotic foods k efi r s oy li fe w ay , g re ek k lu yv er om yc es m ar xi an us , l ac to ba ci llu s ke fir , k lu yv er om yc es la ct is , l ac to ba ci llu s br ev is , le uc on os to c m es en te ro id es , l ac to ba ci llu s he lv et ic us s oy a be an s o gi w es t a fri ca la ct ob ac ill us a ci do ph ilu s, l ac to ba ci llu s ag ili s, la ct ob ac ill us c el lo bi os us , l ac to ba ci llu s co nf us us , la ct ob ac ill us m ur in us , l ac to ba ci llu s pl an ta ru m g ru el m uc ilo n n es tle b ifi du s b l o at a nd r ic e b oz a ve fa , t ur ke y la ct ob ac ill us p la nt ar um , l ac to ba ci llu s ac id op hi lu s, le uc on os to c ra ffi no la ct is , l ac to ba ci llu s br ev is , la ct ob ac ill us fe rm en tu m , l eu co no st oc m es en te ro id es r ye , m ill et , w he at , c er ea l, co rn ve ge ta bl eba se d ve ge ta bl e ju ic es a nd p ic kl e h ea lth y lif e p ro bi ot ic s, g ol de n c irc le , a us tra lia ; b io pr ofi t, g efi lu s, v al io l td , f in la nd la ct ob ac ill us p ar ac as ei 8 70 0: 2, l ac to ba ci llu s pl an ta ru m h ea l9 , l ac to ba ci llu s rh am no su s g g , p ro pi on ib ac te riu m fr eu de nr ei ch ii ss p. s he rm an ii js , la ct ob ac ill us a ci do ph ilu s, l ac to ba ci llu s pl an ta ru m , la ct ob ac ill us c as ei , b ifi do ba ct er iu m lo ng um , la ct ob ac ill us d el br ue ck ii c ar ro ts , b ee ts , g in ge r, to m at oe s, c ab ba ge , o ni on s, p ea nu ts k im ch i k or ea g in se ng c or po ra tio n, k or ea s tr ep to co cc us , p ed io co cc us , l eu co no st oc s pp ., e nt er oc oc cu s, l ac to co cc us c ab ba ge , g ar lic , r ed p ep pe r, on io n, g in ge r, ra di sh k ev ita h -e -b , u s a b ac ill us c oa gu la ns g b i-3 06 08 6, l ac to ba ci llu s pa ra ca se i 8 70 0: 2, l ac to ba ci llu s pl an ta ru m h e a l 9 s pa rk lin g le m on a nd g in ge r d rin k *a da pt ed fr om a sp ri et a l., 2 02 0; c ha ud ha ry , 2 01 9; r an ad he er a et a l., 2 01 7. 98 italian journal of food science, 2023; 35 (1) şanlıbaba p quinoa, and amaranth are pseudo-cereals connected to the diet (fernandes et al., 2018). nowadays, several nondairy cereal-based probiotic products exist. these products have the potential to harbor probiotic cultures, such as boza, bushera, mahewu, pozol, kvass, ben-saalga, degue, kenkey, koko, kanun-zaki, mawe, munkoyo, thobwa, ting, uji, and togwa (dahiya and singh-nee nigam, 2022; misra et al., 2019). however, this chapter is restricted to cereal-based probiotic products sold in different parts of the world. boza is popular in turkey, kazakhstan, kyrgyzstan, albania, bulgaria, macedonia, montenegro, bosnia and herzegovina, and parts of romania and serbia. boza, the turkish name, comes from the persian word, buze, which means millet. boza is made from various cereals such as wheat, rye, millet, maize, but the variety with the best quality and taste is made of millet flour. boza production steps are as follows: (1) preparation of the raw materials, (2) boiling, (3) cooling, (4) sugar addition, and (5) fermentation (aladeboyeje and sanli, 2021). boza is a viscous, low-alcohol beverage that ranges from creamy white and beige to light brownish colors, with a pleasant sweet taste and slightly acidic taste. two types of fermentation co-occur during boza fermentation. alcohol fermentation produces carbon dioxide bubbles and increases the volume, while lactic acid fermentation produces lactic acid and provides its acidic character. the microbiota responsible for its fermentation shows a diversity of both homoor hetero-fermentative lab and yeasts. lab strains are lactobacillus, lactococcus, leuconostoc, pediococcus, enterococcus, oenococcus, and weissella. these include lactobacillus paracasei subsp. paracasei, lactobacillus pentosus, lactobacillus planta­ rum, lactobacillus brevis, lactobacillus rhamnosus, lactobacillus fermentum, lactobacillus acidophilus, lacto­ bacillus coprophilus, lactobacillus coryniformis, lactobacillus sanfranciscensis, leuconostoc lactis, leuconostoc mesenteroides, leuconostoc mesenteroides subsp. dextranicum, leuconostoc raffinolactis, weissella kimchi, and weissella paramesenteroides. however, lab cocci, including enterococcus faecium, oenococcus oeni, lactococcus lactis subsp. lactis and pediococcus pentosa­ ceus are rare. yeast isolates in boza are candida diversa, candida inconspicua, candida pararugosa, issatchenkia orientalis, pichia fermentans, pichia guilliermondii, pichia norvegensis, rhodotorula mucilaginosa, and torulaspora delbrueckii (petrova and petrov, 2017). bushera is an other traditional beverage most commonly consumed by children and adults in uganda’s urban and rural areas. it is produced by mixing sorghum or millet flour with boiling water and then fermenting at room temperature. the dominant flora consists of five lab genera (lactobacillus spp., mainly lactobacillus brevis, lactococcus spp., leuconostoc spp., enterococcus spp., and streptococcus spp.). although studies on the probiotic potential of bushera are still limited, there is evidence of its antidiarrheal effects (panghal et al., 2018). a fermented corn product, ogi is a popular breakfast cereal in sub-saharan african communities, primarily nigeria. traditional production begins with the fermentation of corn grains by soaking them in the water for 2–4 days at room temperature, then resting the milled and sieved slurry for 2–3 days at room temperature (enujiugha and badejo, 2017). the main microorganisms isolated were lab, mainly lactobacillus (lactobacillus acidophilus, lactobacillus plantarum, lactobacillus brevis, and lactobacillus fermentum) and yeast (saccharomyces cerevisiae, rhodotorula graminis, candida krusei, candida tropicalis, geotrichum can­ didum, and geotrichum fermentum) (omemu, 2011). in many parts of nigeria, it is a tradition for mothers to give their babies ogi liqueur (water prepared with fermented grain paste) to treat diseases such as diarrhea and abdominal pain (adebolu et al., 2007). ogi is commonly consumed as semisolid oatmeal called “akamu” or steamed pudding called “agidi.” therefore, heat treatment applied in both forms of ogi eliminates the existing lab and, thereby, the ogi probiotic effect (enujiugha and badejo, 2017). mahewu is also a nondairy cereal-based probiotic product preferred by consumers of all age groups in south africa. mahewu is an alcohol-free beverage made by a multigrain mix, including maize, sorghum, millet, malt, and wheat flour. in the traditional production process, a multigrain mix is kept at room temperature for approximately 24 h for spontaneous fermentation. after fermentation, various fruit flavors may be added to the mixture to enhance the flavor. the major microorganisms responsible for mahewu fermentation is lactococcus lactis subsp. lactis (bansal et al., 2016; enujiugha and badejo, 2017). moreover, togwa, a traditional fermented beverage in tanzania, is produced by fermenting multigrains such as maize, sorghum, finger millet, rice, cassava, and cornflour. due to the unhygienic preparation and variable end product characteristics, togwa is consumed by low income people (panghal et al., 2018). many lactobacillus species, predominantly lactobacillus plantarum, weissella confuse, and pediococcus pentosaceus and yeast (issatchenkia orientalis, saccharomyces cerevisiae, candida tropicalis, and candida pelliculosa), are the main microflora of togwa (aspri et al., 2020; mugula et al., 2003). in addition, pozol, a nonalcoholic beverage, is mostly consumed in southeast mexico. pozol is produced by cooking italian journal of food science, 2023; 35 (1) 99 nondairy functional probiotic foods soymilk with probiotic cultures has some advantages, for example, decreasing the problems of beany flavor and flatulence connected to the oligosaccharide constituents (hassanzadeh-rostami et al., 2015). božanić et al. (2011) studied soy milk fermentation by the probiotic culture abt5 (a mixture of lactobacillus acidophilus, bifido­ bacterium spp., and streptococcus thermophilus) at two different temperatures (37°c and 42°c). soymilk fermentation time was 7 h at 42°c and 8 h at 37°c. however, lactobacillus acidophilus grew poorly in both fermentation procedures. the viable cell count of bifidobacteria spp. was approximately 107 cfu/ml during 28 days of cold storage, better than lactobacillus acidophilus. the authors emphasized that soymilk is a good substrate for bifidobacterium spp. fruitand vegetable-based probiotic products fruits and vegetables are rich in several nutrients such as phytochemicals, minerals, vitamins, soluble dietary fibers, and antioxidants. moreover, they do not contain allergens and cholesterol. thus, several food industries have produced healthy fruitand vegetable-based probiotic products. vegetable-based drinks, fermented or nonfermented fruit-based drinks, fermented banana pulp, beets-based and tomato-based drinks, peanut milk, dried fruits, green coconut water, and ginger juice are examples of some fruitand vegetable-based probiotic products (kumar et al., 2022; misra et al., 2019; song et al., 2012). these fruit and vegetable juices are a novel and an appropriate probiotic vehicle that are investigated lately (coşkun, 2017a, 2017b; fazali et al., 2007; fu et al., 2014; garcia et al. 2020; khatoon and gupta, 2015; lu et al., 2018; nguyen et al., 2019; pakbin et al., 2014; panghal et al., 2018; pereira et al., 2013; swain et al., 2014; thakur and joshi, 2017; yoon et al., 2006). several factors limit the viability and survival of probiotic cultures in fruit and vegetable juices. the major parameters are (1) food matrix such as titratable acidity, water activity, ph, molecular oxygen, presence of sugar, artificial flavoring and coloring agents, (2) processing parameters such as heat treatment, cooling rate, packaging materials, and storage techniques, and (3) microbiological factors including probiotic strains, inoculum proportion, and rate of culture growth (kumar et al., 2022; patel, 2017). there are two applications in obtaining probiotic fruits and vegetables: fermented and nonfermented probiotic products (chaudhary, 2019). many strains of lactobacillus plantarum, lactobacillus acidophilus, lactobacillus bifidus, and lactobacillus casei can grow on fruit and vegetable matrices due to their tolerance to acidic environments (perricone et al., 2015). lactic fermentation occurs in fruits and vegetables in three ways: (1) spontaneous fermentation by natural microflora, corn in about 1% (w/v) lime solution, washing with water, grinding to make the dough known as nixtamal. they form into balls, wrapped in banana leaves, and fermented for half a day to 4 days at ambient temperature. prior to consumption, the fermented dough is suspended in water. pozol dominant microflora is amylolytic lab strains, clodosporium guilliermondii var. guillier–mondii, cladosporium cladosporioides, and geotrichum can­ didum (bansal et al., 2016; panghal et al., 2018). finally, soybean is the most important cereal because of its high nutritional value (soccol et al., 2010). functional soybean products are fermented commonly by the lab such as lactobacillus paracasei, lactobacillus casei, lacto bacillus mali, and bifidobacterium breve. traditionally in the past, soy products were made from china. today, they are known in other parts of asia and europe. soy products can be classified into two forms, including fermented soy products such as tempeh, natto, shoyu, miso, and sufu, and unfermented soy products include soy milk and tofu. soy cheese is often called tofu, a high-protein unfermented product (zielińska et al., 2015). zielińska et al. (2015) aimed to produce tofu with probiotic bacteria such as bifidobacterium animalis ssp. lactis bb–12 and lactobacillus casei łock 0900. it was concluded that the viable count of lactobacillus casei łock 0900 was 109–1010 cfu/g, but bifidobacterium animalis ssp. lactis bb–12 did not exceed 103 cfu/g after 15 days of storage of tofu at 4°c. tempeh is a fermented soybean product that originated from indonesia. tempeh is rich in soy protein and genistein, with beneficial effects on high blood sugar regulation and prevents diabetes. tempeh is fermented with rhizopus microspo­ rus var. oligosporus. moreover, acetobacter indonesiensis, klebsiella pneumoniae, bacillus subtilis, flavobacterium spp., brevundimonas spp., pseudomonas putida also play an important role in producing indonesian tempeh. lactobacillus families, such as lactobacillus agilis, lactobacillus fermentum, and enterobacteria cecorum are commonly isolated from tempeh (subandi et al., 2019). miso is a traditional japanese fermented soy paste. there are three main types, including bean miso, rice miso, and barley miso, based on the molt material. miso is commonly produced by fermentation of soybeans with aspergillus oryzae together with some strains of lab, including tetragenococcus halophilus and yeast such as saccharomyces cerevisiae (kumazawa et al., 2018). natto is a japanese cheese-like product made from soybeans by fermentation with bacillus subtilis var. natto (former name, bacillus natto). bacillus natto, as a probiotic strain, could be used as a functional ingredient in natto production (fujiwara et al., 2008). soymilk is a lactose-free product and contains gos considered prebiotics as a source of energy due to the β-galactosidases in soybean. soymilk can be obtained from the water extract of soybean and supports probiotic bacteria growth in probiotic foods. 100 italian journal of food science, 2023; 35 (1) şanlıbaba p are: (1) high antioxidant effect, (2) prevents oxidative stress to inhibit cancer cells formation, and (3) decreases plasma lipid peroxidation parameters and serum homocysteine concentration (coşkun, 2017a). apple juice was fermented for 72 h by lactobacillus plantarum by thakur and joshi (2017). after storage for four weeks at 4°c, the initial probiotic cell count decreased from 107 cfu/ml to 4.76–6.00 × 106 cfu/ml. the authors concluded that lactobacillus plantarum is suitable for apple juices as a probiotic culture. tempoyak produced from durian fruit widely grown in southeast asian countries such as malaysia, thailand, and the philippines is a traditional fermented beverage. durian pulp is fermented by spontaneous lactic acid fermentation. lactobacillus species like lactobacillus mali, lactobacillus brevis, lactobacillus durianis, lactobacillus fermentum, and leuconostoc mes­ enteroides are used to ferment tempoyak (lu et al., 2018; swain et al., 2014). in a similar study by nguyen et al. (2019), pineapple juice fermented with bifidobacterium lactis bb–12, lactobacillus plantarum 299v, and lactobacillus acidophilus la5 was investigated. the viable cell counts of lactobacillus strains were approximately 109 and 1010 cfu/ml, while the bifidobacterium strain was in the range of 108 and 109 cfu/ml in the first month of storage at 4°c. the authors suggested that probiotic lactobacillus. plantarum 299v was more suitable for developing probiotic pineapple juice drinks. cabbage, sauerkraut, fermented cucumbers, carrot root, potato, tomato, beet, onion, ginger, peanuts, and kimchi are the most studied vegetable-based probiotic products (garcia et al., 2020). some examples of these products are summarized below. firstly, shalgam is a red and sour soft traditional turkish beverage made from black or purple carrot or their mixture. shalgam, also called turnip juice, shalgam juice, or shalgam water, is produced by two methods with different formulations. its microbiota mainly include yeasts such as saccharomyces cerevisiae and lactobacillus spp. (lactobacillus plantarum, lacto­ bacillus arabinosus, lactobacillus fermentum, lacto bacillus buchneri, lactobacillus pentosus, lactobacillus brevis, and lactobacillus paracasei subsp. paracasei) (89.63%), leuconostoc spp. (leuconostoc mesenteroides subsp. mes­ enteroides) (9.63%) and pediococcus spp. (pediococcus pentosaceaceus) (0.74%). generally, shalgam fermentation occurs in about 7 days at 25°c with a 3–4 months shelf life stored at 4 °c in a closed container. its shelf life increases to about 6 months at 4–20°c if sterile filtration is used (coşkun, 2017b; garcia et al., 2020). the other product is cabbage juice. yoon et al. (2006) determined cabbage availability for probiotic cabbage juice production using lactobacillus l. plantarum c3, lactobacillus casei a4, and lactobacillus delbrueckii d7 as probiotic cultures. during the fermentation at 30°c, lactobacillus casei, lactobacillus delbrueckii, and lactobacillus plantarum (2) fermentation by starter cultures added to raw material, and (3) fermentation by heat treatment materials by starter cultures (chaudhary, 2019). the stability and viability of probiotic culture can be improved in juices by adding some prebiotics (dietary fiber, cellulose) or some ingredients that protect their effect (perricone et al., 2015). microencapsulation techniques have also been investigated to preserve probiotic viability in these juices (white and hekmat, 2018). probiotic fruit juices such as pineapple, orange, apple, mango, grapes, sweet lime, watermelon juice, cashew apple juice, blackcurrant juice, and cranberry are the most popular products in this category (panghal et al., 2018). watermelon juice was produced using four lactobacillus strains (lactobacillus casei, lactobacillus. acidophilus, lactobacillus fermentum, and lactobacillus plantarum) by fazali et al. (2007). watermelon juice was first pasteurized at 63ºc for 30 min and then inoculated with a 24 h–old culture of individual probiotic cells. after incubation at 37ºc, the lactobacillus strains grew in watermelon juice and reached a viable cell count of 108 cfu/ml after 48 h. the antibacterial activity of probiotic watermelon juice against salmonella typhimurium was also studied. all the lactobacillus strains could completely inhibit the growth of salmonella typhimurium after 2–6 h. moreover, pereira et al. (2013) optimized cashew apple juice fermentation by lactobacillus casei nrrl b-442. the viable probiotic cell counts were observed to be higher than 8.0 log cfu/ml at 4°c for 42 days. pakbin et al. (2014) also reported that lactobacillus delbrueckii grew well in peach juice, and viable cell count reached nearly 10 × 109 cfu/ml after 48 h of fermentation at 30°c. after 4 weeks of cold storage at 4°c, the viable cell count of lactobacillus delbrueckii was 1.72 × 107 cfu/ml. thus, this juice can be consumed as a healthy probiotic beverage by vegetarians and lactose-allergic consumers. khatoon and gupta (2015) found that the viable cell count of lactobacillus acidophilus cultures in sugarcane juice and sweet lime juices reached 108 cfu/ ml after 24 h of fermentation at 37ºc. after three weeks of storage at 4ºc, the viable cell count of lactobacillus acidophilus in the sugarcane juice was 4.0 × 108 cfu/ml, while culture viability was lost in sweet lime juices after the second week of storage due to higher acidic conditions. the authors implied that fermented sweet lime and sugarcane juice could be further developed as a functional probiotic beverage. grape juice, also called hardaliye, is a traditional fermented nonalcoholic beverage produced in thrace, the european part of turkey. lactobacillus sanfranciscensis, lactobacillus acetotoler­ ans, lactobacillus pontis, lactobacillus paracasei ssp. paracasei, lactobacillus brevis, and lactobacillus vacci­ nostercus are used as the probiotic bacteria in the fermentation of hardaliye. the health benefits of hardaliye italian journal of food science, 2023; 35 (1) 101 nondairy functional probiotic foods garlic, green onion, ginger, red pepper, mustard, parsley, jeotgal (fermented seafood), carrot, and salt are used for producing kimchi. the main microflora of kimchi were various lab strains, with lactobacillus kimchii being the typical fermenting species, and also fermented kimchi contained high levels of lab (about 107–109 cfu/g). in addition, many leuconostoc spp. and lactobacillus spp., including leuconostoc citreum, leuconostoc gasicomi­ tatum, leuconostoc gelidum, lactobacillus sakei, and lactobacillus brevis are the predominant species. many active compounds in kimchi showed anticancer, antiobesity, and antiatherosclerotic functions (park et al., 2014). gundruk is another pickled product from nepal. gundruk is a nonsalted, fermented, acidic vegetable product obtained by fermenting leafy vegetables (rayosag, mustard leaves, cauliflower leaves, and cabbages). its fermentation is usually dominated by pediococcus spp. (pediococcus pentosaceus) and lactobacillus spp., (lactobacillus fermentum, lacto bacillus casei, lacto­ bacillus casei subsp. pseudoplantarum, lactobacillus cellobiosus, and lactobacillus plantarum) the initiators of fermentation. fermentation time is reported to be about 15–22 days (swain et al., 2014). khalpi, also called cucumber, is a pickled cucumber from nepal. lacto­ bacillus plantarum, lactobacillus brevis, and leuconostoc fallax are commonly involved in khalpi fermentation. moreover, the cucumber can be fermented using a pure or mixed culture of lactobacillus plantarum and saccharomyces cerevisiae. khalpi fermentation occurs in 4–7 days at room temperature (behera et al., 2020). turşu, a traditional fermented turkish pickle, is made of different vegetables such as cabbage, cucumber, carrot, beet, pepper, turnip, eggplant, and beans. lactobacillus plantarum, lactobacillus brevis, leuconostoc mesenteroi­ des, and pediococcus pentosaceus are the dominant microorganisms during fermentation (irkın and songun, 2012). al-shawi et al. (2019) investigated the effect of adding probiotic bacteria (lactobacillus acidophilus) and synbiotic (lactobacillus acidophilus + inulin) in tursu. the cell count of lactobacillus acidophilus was higher in the synbiotic (log 9.68 cfu/ml) than the probiotic (log 9.54 cfu/ml) and control (log 3.97 cfu/ml) at the end of the storage period. after 30 days, the total cell count was higher in the control sample (log 6.99 cfu/ml) than probiotic (log 6.90 cfu/ml) and synbiotic samples (log 6.52 cfu/ml), respectively. the authors implied that synbiotic pickle had more desirable characteristics compared with probiotic and control pickle. sunki is a traditional japanese nonsalted pickle made from the fermentation of the leaves of red turnips. lactobacillus kisonensis, lactobacillus otakiensis, lacto­ bacillus rapi, and lactobacillus sunkii are the predominant species in sunki. however, lactobacillus delbrueckii identified as the subdominant lab strain (kudo et al., 2012). reached nearly 10 × 108 cfu/ml. after 4 weeks at 4°c, the viable cell counts of lactobacillus plantarum and lactobacillus delbrueckii was 4.1 × 107 cfu/ml and 4.5 × 105 cfu/ml, respectively, but lactobacillus casei did not survive after 2 weeks because of low acidity. the authors concluded that lactobacillus plantarum and lactobacillus delbrueckii could be used as probiotic cultures to produce cabbage beverage. do and fan (2019) studied the suitability of a mixture of juices from jicama, winter melon, and carrot to produce a probiotic juice using lactobacillus plantarum cicc22696 and lactobacillus acidophilus cicc20710 strains. both strains grew well in the vegetable juice mixtures after 24 h of fermentation at 37°c and reached nearly 9 and 8 log cfu/ml when inoculated with lactobacillus plantarum and lactobacillus acidophilus, respectively. at the end of the storage, the viability of lactobacillus plantarum was approximately 8 log cfu/ml, whereas lactobacillus acidophilus was 4.57 log cfu/ml. kombucha is another vegetable-based probiotic drink consumed worldwide as a medicinal health promoting drink (kozyrovska et al., 2012). kombucha beverage is produced by fermenting sugared tea with a symbiosis of yeast spp., fungi, and acetic acid bacteria at ambient temperature for about 7–14 days. besides, this beverage is composed of some probiotic lactic acid bacteria. acetic acid bacteria species such as acetobacter xylinoides, acetobacter pasteurianus, acetobacter xyli­ num, acetobacter aceti, and bacterium gluconicum are the main microflora in kombucha. moreover, yeasts species, including brettanomyces bruxellensis, brettanomyces lambicus, brettanomyces custersii, kloeckera imchite, saccharomycodes ludwigii, schizosaccharomyces pombe, saccharomyces cerevisiae, zygosaccharomyces bailii, candida, and pichia were also isolated from kombucha (fu et al., 2014). tomato juice is also another product based on vegetable probiotic juice. yoon et al. (2004) determined the suitability of tomato juice as a raw material for probiotic juice production using lactobacillus aci­ dophilus la39, lactobacillus plantarum c3, lactobacillus casei a4, and lactobacillus delbrueckii d7. tomato juice was inoculated with 24 h–old probiotic cultures and incubated at 30°c. the viable cell count reached nearly from 1.0 to 9.0 × 109 cfu/ml after 72 h. moreover, the four lab strains’ viable cell count ranged from 106 to 108 cfu/ ml after 4 weeks at 4°c. traditionally, vegetable-based fermented pickles have a long history in different cultures worldwide (fan et al., 2017). it can be produced by lactic fermentation (vegetables and fruits), alcoholic fermentation (cassava and rice), high salt fermentation (fish and soy sauce), and mold fermentation (peanut press cake and soybeans) (behera et al., 2020). there are several pickles produced under different names in the world. kimchi is a traditional pickled vegetable from korean culture. cabbages, radish, 102 italian journal of food science, 2023; 35 (1) şanlıbaba p lacto coccus lactis subsp. lactis, lactobacillus plantarum, lacto bacillus reuteri, lactobacillus fermentum, bifido­ bacterium animalis, bifidobacterium lactis, and pedio­ coccus pentosaceus have also been used in fermentation and ripening of these meat products as bioprotectors against pathogenic strains (cavalheiro et al., 2015). thus the selection of suitable probiotic starter culture is vital in the production of dry-fermented sausage products. the most important probiotic selection criteria are that probiotic strains can compete with the bacteria in meat, survive the production processes such as fermentation and drying, refrigeration, and storage. moreover, they should have sufficient numbers in the final products for expected health effects (khan et al., 2011; kumar et al., 2022). lab plays the central role in sausage production by producing organic acids (mainly lactic acid) that inhibit undesirable microorganism growth. at the beginning of the sausage fermentation, the lab count ranged between 3.2 and 5.3 log cfu/g. however, lab count reached 7–9 log cfu/g in the first days and remained at this level during ripening (maksimović et al., 2015). jofré et al. (2015) tested the efficiency of lactobacillus casei/paracasei ctc1677, lactobacillus casei/paracasei ctc1678, lactobacillus rhamnosus ctc1679, lacto­ bacillus gasseri ctc1700, lactobacillus gasseri ctc1704, and lactobacillus fermentum ctc1693 as potential probiotic strains in model sausage systems. only ctc1677, ctc1678, and ctc1679 were able to grow and dominate at 15ºc after 9 days (108 cfu/g) during ripening. probiotic strain lactobacillus casei crl431 was added in traditional turkish dry–fermented sausage, namely sucuk (bağdatli and kundakci, 2016). at the end of the fermentation and ripening period, the number of lactobacillus casei crl–431 reached a sufficient microbial count (approximately 106 cfu/g). thus, the authors implied that lactobacillus casei crl 431 could be used as a probiotic strain in turkish sausage production. nedelcheva et al. (2014) prepared the rawdried meat sausages using a probiotic strain lactobacillus plantarum nbimcc 2415. the viable cell count of lactobacillus plantarum nbimcc 2415 was approximately 1012 cfu/cm3 on day 6 at 15–18oс. this probiotic strain completely inhibited the growth of pathogenic bacteria such as escherichia coli atcc 25922, proteus vulgaris g, salmonella abony ntcc 6017, staphylococcus aureus atcc 25093, and listeria monocytogenes i at 15–18°c during the meat production. conclusion probiotic foods comprise 60–70% of the total functional food market. dairy-based probiotic products are the primary vehicle of probiotic cultures to humans and meat-based probiotic products meat and meat products are essential components of human nutrition. raw-cured and ripened meat products are traditionally produced by fermentation worldwide. moreover, drying is the oldest food preserving methods in the world. meat-based probiotic products are relatively new and recently adopted by the meat industry (kołozynkrajewska and dolatowski, 2012; kumar et al., 2022). lab (lactobacillus casei, lactobacillus curvatus, lacto­ bacillus pentosus, lactobacillus plantarum, lactobacillus sakei, pediococcus acidilactici, and pediococcus pentosa­ ceus), staphylococci (staphylococcus xylosus, staphylo­ coccus saprophyticus or staphylococcus carnosus), and micrococci (micrococcus varians) are frequently used as a starter culture in the meat fermentation process (song et al., 2012). in general, a single lab species or together with other bacteria are used as a starter culture. due to its high moisture content (70–80%), raw meat is easily contaminated with pathogenic microorganisms transmitted from animal skin to raw meat during slaughter (holck et al., 2017; kumar et al., 2022). lab strains used in meat fermentation strongly inhibit pathogens. therefore, lab is widely used to preserve meat products for consumer quality purposes (fraqueza et al., 2020). various factors influence the viability of probiotic cultures in fermented meat products. these are low ph, low water activity, acidity, organic acid concentration, microorganisms (native microflora of meat), processing and storage temperature, oxygen content, salt, low content of natural sugars, and additives (nitrite and nitrate) (aspri et al., 2020). dry-fermented sausages are the only example of a probiotic application in fermented meats (song et al., 2012). dry-fermented meat products are popular in the world. depending on the geographic region, there are many types of dry-fermented sausages. for example, in portugal, chouriço, paio, catalão, salsichao, linguiça, palaio, salpicao, butelo, cacholeira, alheira, farinheira, and morcela are produced as a dry-fermented sausage (rocha and elias, 2016). salami in italy, sucuk in turkey, and salchichon and chorizo-like mediterranean-type sausages in spain are the other examples of dry-fermented sausage types (holck et al., 2017). however, producing these food products has a high contamination risk, especially during drying, slicing, and packaging steps (song et al., 2012). thermal processes during the production of dry-fermented meat are usually not used or only heated mildly. this temperature is not harmful to probiotic strains, so the transfer of probiotic microorganisms into the human gastrointestinal tract is provided easily (rocha and elias, 2016). some probiotic microorganisms such as lactobacillus acidophilus, lactobacillus casei, lactobacillus paracasei, lactobacillus rhamnosus, italian journal of food science, 2023; 35 (1) 103 nondairy functional probiotic foods behera, s.s., el sheikha, a.f., hammami, r. and kumar, a., 2020. traditionally fermented pickles: how the microbial diversity associated with their nutritional and health benefits?. journal of functional foods. 70: 103971. https://doi.org/10.1016/j.jff.2020.103971 božanić, r., lovković, s. and jeličić, i., 2011. optimising fermentation of soymilk with probiotic bacteria. czech journal of food 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2017. probiotics: from isolation to application. journal of the american college of nutrition. 36(8): 666–676. https://doi.org/ 10.1080/07315724.2017.1337529 survey ital. j. food sci., vol. 27 2015 1 keywords: direct sales, entrepreneurial strategies, short supply chain, small and medium enterprises (sme) socio-economic assessment of direct sales in sicilian farms s. tudisca, a.m. di trapani, f. sgroi* and r. testa department of agricultural and forestry sciences, university of palermo, viale delle scienze, edificio 4, 90128 palermo, italy *corresponding author: tel. +39 091 23896615, fax +39 091 484035, email: filippo.sgroi@unipa.it abstract many farmers today adopt direct sales as an entrepreneurial strategy in order to achieve a competitive advantage. the aim of this study has been to analyze the role that direct sales play in sicilian farms and how the short food supply chain is able to valorize the endogenous resources of rural areas and increase the net income of farmer. our results showed that direct sales, in conjunction with conventional sales, can represent a growing opportunity for farmers and lead to an improvement in the economic performances of agricultural businesses, an increase in farm investments and the creation of new job opportunities. 2 ital. j. food sci., vol. 27 2015 1. introduction competitive advantage represents the result of a strategy that leads an enterprise to occupy and maintain a favorable position in the market in which it operates, thereby obtaining a higher profitability than its competitors (tudisca et al., 2013a). in order to compete in the global market, farmers have to be able to change their entrepreneurial strategies and improve their economic performance, thus incorporating ‘added value’ (chinnici et al., 2013a; sturiale and scuderi, 2013; veidal and korneliussen, 2013). the higher profit margin obtained allows a higher level of self-financing and a greater return on the invested risk capital (santeramo et al., 2012). considering the difficulties that farms have to be competitive (high production costs, low sale prices of agricultural products, shortage of labour), direct sales could represent a way to achieve a competitive advantage and improve profit margins. direct sales, through the reduction of intermediaries along the supply chain, can affect the annual budget of a farm by allowing the farmer’s family to obtain a dignified remuneration for the use of their productive factors (rizzo and mazzamuto, 2009; polidori et al., 2008). with direct sales, the farmers are not subject to the price offered to them; they may decide to become price-makers (saccomandi, 1999) and apply a different price, one that is higher than the one that is determined in the case of sales to fruit and vegetable wholesale markets or contracts with the large organized distribution (lod). this type of sale fully utilizes the work of the farmer’s family and produces positive effects on the farm’s economic performance because it increases the available liquid assets in the business and lowers the capital required for the coverage of the short-term debts that are present during the management activity of the farm (di trapani et al., 2013). this is a worthy outcome, especially in rural territories where agriculture is the main economic activity and where the pricing of farm production represents the strategic variable for the success needed to relaunch farm competitiveness and boost the local economy, thereby avoiding the phenomena of rural exodus (tudisca et al., 2014a; rizzo and giudice, 2013; tudisca et al., 2011; bulin, 2011; brunori et al., 2002). all of this is fostered by a general increase in the public interest in issues such as ecology and the health and welfare of animals, as well as a growing distrust in the quality of food products derived from conventional agriculture (tudisca et al., 2014b; chinnici et al., 2013b; brunori et al., 2012; briamonte and giuca, 2010; harvey et al., 2004). direct sales allow consumers to obtain more reasonably priced fresh, healthy food and foster ecological sustainability, as represented by the reduced food miles and carbon emissions flowing from sustainable farming (little et al., 2009; feagan, 2008). the decreased amount of food miles can result in a lower level of environmental pressure due to a reduction in key factors such as air pollution, soil pollution, loss of biodiversity and noise pollution and can also reduce social pressures that can contribute to problems arising from road accidents and animal welfare issues (van passel, 2013). finally, direct sales can reconfigure relations between producers and consumers, assuming a social justice characteristic (feagan, 2007), and can encourage more harmonious community relations (winter, 2003) and more democratic participation of participants in the food supply chain (hinrichs, 2003). the aim of this paper, as well as in other studies (peter et al., 2010; holloway, 2008; sonnino and marsden, 2006), has been to analyze the role of direct sales in sicilian farms and to determine how the short food supply chain is able to valorize the endogenous resources of rural areas and, consequently, the economic profitability of farmers, who seek to enhance their value along the food supply chain. in particular, it has been carried out an empirical analysis on a sample of farms that adopted the direct sales in order to analyze their structural characteristics, the motivations that led farmers to undertake this selling strategy and its benefits for farms. 2. materials and methods in order to analyse how direct sales can contribute to obtaining a competitive advantage for sicilian farms, we carried out an empirical analysis on 30 small and medium enterprises (smes) that adopted this entrepreneurial strategy. the survey was conducted in 2013 by means of faceto-face interviews with farmers, using a specific questionnaire (tudisca et al., 2014c; raffaelli et al., 2009; marbach, 2000) divided into three parts. in particular, in the first section we collected the information related to the structural characteristics of farms, their agri food products (product portfolios), the socio-demographic characteristics of farmers, the quota of farm production destined to direct sales and its sales modality (farm outlets and/or farmers’ markets). in the second one we asked to farmers the reasons that led them to adopt a short supply chain strategy and the benefits reflected in the business performance by its adoption. in this case interviewees had to assign a score to a 1-5 scale to each possible predefined question (tudisca et al., 2013c; trabalzi and de rosa, 2012). this scale, better know as likert scale (likert, 1932), is a psychometric scale commonly involved in research that employs questionnaires. it is the most widely used approach to scaling responses in survey research. when responding to a likert questionnaire item, respondents specify their level of agreement or disagreement (strongly agree, agree, uncertain, disagree, strongly disagree) on a symmetric agreedisagree scale for a series of statements by attribital. j. food sci., vol. 27 2015 3 uting a score for each one (5, 4, 3, 2, 1). thus, the range captures the intensity of their feelings for a given item (norman, 2010; burns and burns, 2008; allen and seaman, 2007). in the third section, we collected information in order to determine the net income of farmers, by means of the following formula: ni = gpv – σci (1) where: ni = net income of farmer; gpv = gross production value of agri food products; c i = costs of productive factors that have not been conferred by entrepreneur. finally, in order to better quantify how direct sales allowed farmers to remain competitive in the market, it has been compared this value to the net income that farmer would obtain by conferring all farm production exclusively on traditional sales channels. the empirical survey responses showed that the surveyed farms had an average area of 8.39 ha and ranged from a minimum of 4.50 ha to a maximum of 13.56 ha (table 1). the majority of enterprises produced fruit and vegetables (8), followed by milk, cheese and dairy products (6), olive oil and fruit (5), vegetables (4), grapes, wine and fruit (4), grapes and wine (2) and olive oil (1). all of the surveyed farms were worked directly by the farmer’s family. the majority of our sample (18 farmers) had used direct sales for more than five years, while the remaining farmers had applied this entrepreneurial strategy in the past three years. males accounted for 63.3% cent of the entrepreneurs and 36.7% were females. the majority of the entrepreneurs (60.0%) were aged between 31 and 40 years; only 13.3% were over 60 years. these responses highlighted how direct sales strategies have been used mainly by young entrepreneurs, unlike the sicilian primary sector, where farmers are generally of an advanced age (massoli table 1 surveyed farms and socio-demographic characteristics of farmers. 4 ital. j. food sci., vol. 27 2015 and de gaetano, 2004). young farmers are able to respond significantly better to new opportunities and market changes than older entrepreneurs (parker, 2006) and are able to change their entrepreneurial strategies and skills in order to remain competitive in an increasingly competitive market (prashantham and young, 2013). our surveyed entrepreneurs had a medium-high level of school education. in particular, 46.6% of farmers had higher school qualifications and 33.4% had a degree, indicating that the adoption of a new entrepreneurial strategy is correlated positively with the level of education (najjar et al., 2013; mancini et al., 2008). 3. results and discussion the results showed that direct sales of agrifood products were in all cases a portion of the agricultural production (ranging from 18 to 35%) of the farms we surveyed; the remaining production was marketed through traditional channels (fruit and vegetable wholesale markets, ldo, packing centres). the direct sale was conducted by the entrepreneur or his family (especially women and young people) and was crucial to achieving increases in the remuneration of the enterprise (henke and salvioni, 2010). our empirical survey showed that the majority of farmers (20) sold their products only in farm outlets. this was because it is relatively easy for a farmer to sell his products directly at the place of production; it requires only a simple organization and helps to improve the farm’s image (uematsu and mishra, 2011). eight of these farmers also conducted guided tours of their farms, with positive outcomes for the visitors such as nutrition education, the diffusion of rural culture and the valorization of territory and local products (mettepenningen et al., 2012). the influx of customers was distributed throughout the year, thanks to the sicilian favourable climatic conditions (grillone et al., 2014; d’asaro and grillone, 2012; agnese et al., 2008). six of the farmers sold their products exclusively in farmers’ markets and four adopted both sales channels. participation in farmers’ markets allowed the entrepreneurs to also sell their agrifood products in urban and periurban areas, thereby promoting their farm and increasing customers and annual revenues (psarikidou and szerszynski, 2012; brunori et al., 2009; brown and miller, 2008). entrepreneurs adopted direct sales mainly because of the low sale prices of agrifood products, by assigning them the highest score (140) (fig. 1). this was essentially attributable to the absence of intermediaries, so that despite the lower gross sales prices for agrifood products compared with conventional markets, the farmers were able to obtain a higher net level of remuneration for their productive factors, by obtaining a competitive advantage (holloway et al., 2006; renting et al., 2003). they were essentially appropriating a portion of the value that is usually dispersed in the various stages of the long supply chain (bandarra, 2011). the second motivation, in order of importance, was customer loyalty with a score equal to 126. this is due to the fact that, compared to the traditional supply chain, direct sales can create a relationship between the consumer and producer that allows the farmer to valorise his production and to transmit his knowledge and links with the territory (guarino and doneddu, 2011; renko et al., 2010). consumers have the opportunity to purchase agrifood products at lower prices compared to traditional sales channels (seyfang, 2008; taylor et al., 2005; knickel and renting, 2000) and the growth of face-to-face transactions has stimulated the development of markets in the region, which are considered for their status as oppositional sites to the mainstream food industry (sage, 2003). the third motivation, in order of importance, was environmental sustainability (112). this denotes a new multifunctional vision of farming that meets the eu guidelines and the new consumer’s needs, respecting and recovering territorial, environmental and ecological values (tudisca et al., 2013b; renting et al., 2009; renting et al., 2008; thilmany et al., 2008). direct sales represent a sustainable alternative as the food miles (the distance between the place of production and consumption) are minimized in passing ‘from farm to fork’ marketing strategy diversification 2 farm geographical location 2 environmental sustainability 3 customer’s loyalty 4 low sales prices 5 fig. 1 reasons that led entrepreneurs to adopt direct sales. ital. j. food sci., vol. 27 2015 5 and the area of distribution is limited. this results in a significant reduction of the negative externalities associated with transport over long distances, such as co 2 emissions, traffic, road accidents and noise pollution (lanfranchi and giannetto, 2013). in contrast to the results of other studies (harris, 2010), farmers assigned to the geographical location of the farm and the diversification of the business marketing strategy the lowest score (80). our empirical analysis showed that the farmers felt that the increase in farm profitability was the main benefit to their business performance through the adoption of direct sales. in common with other authors (traversac et al., 2011), they assigned it the highest importance by attributing a score equal to 145 (fig. 2). another benefit considered to be of fundamental importance was the increase in liquid assets (134), because timely availability of capital can lead to the adoption of modern technologies, which increase farm production and ultimately the growth rate (riaz et al., 2012). the third highest-ranking benefit from adopting direct sales was the increase in investments (98). this was correlated to the increase in farm profitability that allowed for an increase in self-financing and thereby enabled farmers to realize investments (crnčan et al., 2011). finally, the farmers also identified direct sales as an opportunity for human resources optimization, by attributing it a score equal to 77. however, this presupposes that in the farm family there is a state of under-employment, because the possible economic advantage created would otherwise be absorbed by the need to employ external sales staff (tudisca et al., 2014d). our results showed that farmers who adopted direct sales were able to update their skills and modify the market orientation of their enterprises in order to compete effectively in the current competitive system (frank et al., 2012). this highlights the importance of human capital in an enterprise (gurǎu et al., 2010). economic importance of direct sales adoption is showed by table 2. results denoted that in surveyed farms the net income deriving from the adoption of direct sales ranged from a minimum human resources optimisation 2 increase of investments 3 increase of liquid assets 4 increase of farm profitability 5 table 2 net incomes in surveyed farms fig. 2 obtained benefits on the business performance by adopting direct sales. 6 ital. j. food sci., vol. 27 2015 of 3,052.00 to a maximum of 14,630.00 euro. in all farms, as well as in other studies (hardesty and leff, 2010), it has been highlighted an increase of net income ranged from 5.5 to 60.9% respect to the one deriving from the selling of farm production exclusively through traditional channels. the higher increases were in farms that sold their products both in farm outlets and farmers’ markets as they were able to reach a greater number of consumers. 4. conclusions our results showed that smes employing a direct sales strategy are part of an entrepreneurial network characterized by entrepreneurs who have been able to reorient their business strategy in order to remain competitive in the market. the most important reason that has driven entrepreneurs to adopt direct sales was the need to overcome the consistently low sales prices of agrifood products and thereby obtain higher profit margins and create a competitive advantage. the increase in farm profitability resulted in higher liquid assets and an increase in farm investments and enabled the human resources in the farmer’s family to be optimized. this highlights the fact that the use of direct sales in agriculture nowadays can have a positive impact on the many components of the territorial system in which it operates. nevertheless, direct sales cannot be the only marketing strategy for a farm because the produced quantities cannot be absorbed exclusively by local demand. however, direct sales can represent a winning strategy for a farm if it is inserted within a wider business marketing strategy or if this strategy is used in conjunction with traditional sales methods, such as fruit and vegetable wholesale markets and contracts with lod. acknowledgements this paper is a result of the full collaboration of all the authors. however, s. tudisca wrote conclusions, a.m. di trapani wrote materials and methods, f. sgroi wrote 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uematsu h. and mishra a.k. 2011. use of direct marketing strategies by farmers and their impact on farm business income. agricultural and resource economics review 40 (1): 1-19. van passel s. 2013. food miles to assess sustainability: a revision. sustainable development 21 (1): 1-17. veidal a. and korneliussen t. 2013. entrepreneurial orientation and market orientation as antecedents of organisational innovation and performance. international journal of entrepreneurship and small business 19 (2): 234-250. winter m. 2003. embeddedness, the new food economy and defensive localism. journal of rural studies 19: 23-32. paper received january 15, 2014 accepted april 8, 2014 ijfs#671_bozza ital. j. food sci., vol 29, 2017 559 short communication rapid screening method to assess tannin antioxidant activity in food-grade botanical extract a. schwertner palmaa,b, a. riccia, g.p. parpinello*a and a. versaria adepartment of agricultural and food sciences, university of bologna, piazza goidanich 60, 47521 cesena (fc), italy bcapes foundation, ministry of education of brazil, 70040-020 brasilia (df), brazil *corresponding author. giusi.parpinello@unibo.it abstract fourier transform infrared (ftir) spectroscopy measurements were used for the prediction of commercial tannins antioxidant capacity, i.e. dpph, through partial-least squares (pls) regression. plot of the leave-one-out full cross-validated pls predicted scavenging activity values (dpph %) with a good correlation (r= 0.82), proving ftir succeeded to rapidly provide information on commercial tannins antioxidant capacity. keywords: chemometric, dpph, ftir, pls, tannins ital. j. food sci., vol 29, 2017 560 1. introduction tannins are naturally occurring polyphenols produced by plants via secondary metabolic processes. their ability to bind proteins, pigments, and complex metallic ions, together with their flavouring effect are the basis for their extensive use as additives in the food industry. tannins are commercially available in water suspension liquids, which can be stabilized by the addition of (poly)saccharides, or as lyophilized powders, which can be either a single extract or a blend of two or more tannins, which can have one or more degree of purity (versari et al. 2013). taking into account their nutritional and technological potentials, the main challenge is their analytical characterization, since suppliers seldom label their composition and the degree of purity. manufacturers provide general information only regarding the source of the product and its use, whereas additional details such as their antioxidant activity – which makes tannins of great interest in the wine industry, due to a diminished addition of sulphur dioxide in this beverage – are often needed. in this view, there is an ongoing interest to improve the measurement of the tannin antioxidant activity. it is well known that tannins bind proteins therefore the assays based on reaction catalysed by enzymes for generating radicals are inadequate because they use proteins sensible to oxidation to measure the antioxidant activity. these leads to the disability of tannins to be measured with, since there is the possibility that tannins can interact with the radical generator per se, or sensor, or even to scavenging the radicals themselves. it is important to notice that these both ways of measurements can be seen as antioxidant activity, but it may not be clear whether the tannins are acting or not (hagerman et al., 1998; riedl et al., 2002; miller et al., 1993; cao et al., 1993). currently there are several antioxidant assays available: abts (2,2’-azino-bis (3ethylbenzthiazoline-6-sulphonic acid) cation radical (abts•+) scavenging ability), dpph (2,2-diphenyl-1-picrylhydrazyl radical, dpph• scavenging capacity), frap (ferric reducing antioxidant power), electrochemistry (hagerman et al., 1998; bouchet et al., 1998; muccilli et al., 2017; oszmianski et al., 2007), each of them presenting both advantages and limitations. for example, the abts assay needs the time of incubation to be optimized depending on each and every substrate (walker and everette, 2009). concerning electrochemical analysis, it is necessary to have trained professionals as an expensive apparatus. conversely, the application of fourier transforms infrared spectroscopy (ftir) analysis in oenology has had a considerable boost in recent times, due to its fastness, reliability and versatility of use. analytical devices based on the vibrational spectroscopy method are widespread in analytical laboratories of oenological companies, providing the most important quality parameters and supporting oenologists in different process stages. vibrational spectroscopy enables to disclose the molecular composition of unknown samples, also including complex matrices, and to highlight interactions involved in the molecular arrangements; this latter is especially useful to determine the occurrence of intermolecular interactions. therefore, this study aimed to develop a fast analytical approach suitable for screening antioxidant activity of tannins, which can provide reliable information in the wine and food industry to tailor at best the use of commercial tannins. ital. j. food sci., vol 29, 2017 561 2. materials and methods 2.1. samples thirty commercial tannins were provided by suppliers as powder and dissolved in model wine (ph 3.6, 12% etoh) in a concentration of 1 g l-1. 2.2. dpph scavenging activity the amount of ‘bioactive tannins’ was calculated with the method of scavenging the dpph radical (brand-williams, 1995), which is based on ability of the sample to act as antioxidant agent within the methanolic solution of the free radical 2, 2 diphenyl-1 picrylhydrazyl (dpph), which shows a maximum of absorption at 517 nm. once the antioxidant is added to the solution, absorption diminishes. the chemical reactions that take place are as follows: dpph• +ah →dpph-h +a dpph• +r• → dpph-r dpph radical reagent was prepared in methanol (25 mg l-1). tannin solutions (100 μl) were mixed with 2.9 ml dpph radical solution (nixdorf and hermosíngutiérrez, 2010) and after 60 min, absorbance were measured at 517 nm against methanol. the results are given considering the solution of 2.9 ml of radical and 100 μl as control (100%), and to tannins sample it was considered their ability to neutralize the dpph radical, in relation to control, according to the following equation: % inhibition= [(absdpph – abstannin) / absdpph)] x 100 2.3. ftir analysis spectra acquisition fourier-transform mid-infrared (ft-mir) spectral analysis was performed using a tensor 27 spectrometer (bruker optics) equipped with a horizontal attenuated total reflectance (atr) zinc selenide (znse) crystal (hatr, pike technologies, madison, us). a remotecontrol thermosetting device was used to keep samples (1 ml) at 40 ±1°c for the whole duration of measurements. spectra, with a spectral resolution of 4 cm-1 and 64 scans averaged for each spectrum, were recorded in duplicates from 4000 to 700 cm-1 for each tannin. the same number of scans was used for background subtraction. prior to data analysis each spectrum was corrected for the variation in effective path-length using the atr correction option available in the spectrum one 5.3.1 software (perkin-elmer, waltham, ma). the spectra were exported in ascii format to the statistical software for statistical analysis. 2.4. chemometrics and data analysis partial least square regression (pls) (unscrambler 9.7, camo, oslo, norway) was used to model the relationships between the amount of bioactive tannins in commercial samples and the selected wavelengths of ftir spectra. multivariate analyses were performed using full cross-validation, i.e. the leave-one-out model, and with x-variables pre-processed as ‘standard normal variate’ (snv). ital. j. food sci., vol 29, 2017 562 3. results and discussion the tannins were coded by manufacturers (same letter), with info on their chemical classification, if available (table 1). table 1. code, chemical classification and dpph scavenging activity (%) of commercial tannins. dpph values ranged between 18.7-49.6%, which can be considered a representative interval suitable for pls modelling of ftir spectra. although the entire ir spectra was acquired, after a preliminary attempt using the whole ir signal (4000 to 700 cm-1), a specific region 1750-900 cm-1 – which is well known as ‘fingerprint’ for polyphenolic compounds – was considered for the pls modelling, using 6 latent variables explaining up to 77% total x-variance, and 61% total y-variance. the selection of spectral regions was consistent with code chemical classification dpph scavenging activity (%) (mean±sd) a1 not available 25.7±0.2 a2 condensed 44.3±0.7 a3 hydrolysable / condensed 29.1±0.6 a4 condensed 18.7±0.3 a5 hydrolysable / condensed 26.4±0.01 a6 hydrolysable / condensed 49.6±2.0 a7 hydrolysable 19.3±0.2 a8 hydrolysable 25.0±0.3 a9 hydrolysable / condensed 40.8±0.9 a10 not available 66.9±2.0 a11 hydrolysable 34.7±1.6 a12 hydrolysable stabilized with natural polysaccharides 27.8±0.7 a13 hydrolysable / condensed 41.9±0.2 a14 hydrolysable 36.0±0.4 b1 hydrolysable 42.8±0.4 b2 hydrolysable 59.4±0.5 b3 condensed 30.7±0.6 b4 hydrolysable 38.3±0.8 b5 hydrolysable 37.9±0.01 b6 hydrolysable 29.6±0.7 c1 hydrolysable 28.8±0.7 c2 hydrolysable / condensed 29.8±0.4 c3 hydrolysable 34.6±0.1 c4 condensed 24.2±1.2 c5 condensed 25.4±0.4 c6 condensed 32.5±0.3 c7 condensed 22.0±0.8 d1 hydrolysable 21.3±2.7 d2 hydrolysable 36.9±0.2 d3 hydrolysable 29.3±2.6 d4 hydrolysable 26.2±0.4 ital. j. food sci., vol 29, 2017 563 info from the literature (jensen et al. 2008; ricci et al. 2015), which identified two ir regions, 1485-1425 and 1060-995 cm-1, as mostly important for tannin quantification. the fast-molecular screening provided by mir spectroscopy showed satisfactory correlation with the effective dpph scavenging activity of commercial tannins (r=0.817; slope 0.82) with the root mean square error of full cross-validation (rmsecv) of 6.6 (fig. 1). this result is consistent with previous finding (versari et al., 2010), therefore confirmed the reliability of ftir analysis combined with pls regression as a fast screening method for antioxidant prediction in foodstuffs. moreover, it is also known that ftir associated with chemometric can be useful for the classification of tannin, and fraud disclosure (grasel et al., 2016a; grasel and ferrao, 2016b). figure 1. prediction of antioxidant activity (dpph) of commercial tannins using ftir and full cross pls validation. 4. conclusions the preliminary findings of this short communication suggest that ftir spectroscopy is suitable as a rapid screening tool to provide information on antioxidant activity of commercial tannins. further ftir analysis on a larger number of samples is needed to improve the prediction model at best using an independent set of samples. acknowledgements author 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parpinello g.p. 2013. oenological tannins:a review. aust. j. grape wine res. 19:1. walker r.b. and everette j.d.j. 2009. comparative reaction rates of various antioxidants with abts radical cation. j. agric. food chem. 57:1156. paper received november 8, 2016 accepted february 26, 2017 ijfs#756_bozza ital. j. food sci., vol. 30, 2018 336 paper nutraceutical value of edible flowers upon cold storage m. landi*a, b. ruffonib, l. combournacc and l. guidia,d adepartment of agriculture, food and environment (dafe), university of pisa, via del borghetto 80, 56124 pisa, italy bcrea research centre for vegetable and ornamental crops, corso inglesi 508, 18038 sanremo, italy ccreat-chambre d'agriculture des alpes-maritimes, min fleurs 17 box 85, nice cedex, france dinterdepartmental research center “nutraceuticals and food for health”, university of pisa, via del borghetto 80, 56124 pisa, italy *corresponding author: tel. +39 0502216620 e-mail address: marco.landi@for.unipi.it abstract the attraction and quality of edible flowers correlates with their high perishability. few studies have evaluated whether edible flowers decay faster than they lose their nutraceutical value. in this experiment, ascorbic acid was negatively affected by cold storage in all the edible flowers investigated, whereas phenolic, flavonoid, and anthocyanin content were affected only in some cases. no decrease in total antioxidant activity was detected in any of the edible flowers at the end of their shelf life. our dataset highlights that (i) the selection of edible flowers with low moisture content is key in ensuring a longer shelf life, and (ii) more effort should focus on preventing water loss in edible flowers. keywords: ascorbic acid, anthocyanins, brightness, edible flowers, nutraceutical value, phenolics ital. j. food sci., vol. 30, 2018 337 1. introduction the demand for more attractive and high quality foodstuffs is increasing in the west. the appeal of food dishes can be enhanced by edible flowers, which is why they are becoming more and more popular (aquino-bolaños et al., 2013). edible flowers are mainly purchased by consumers for use in dishes as a garnish or ingredient although more often they are referred to in the literature in terms of their biologically active compounds. some papers have extensively investigated the chemical composition of many edible flowers (li et al., 2007; garzon et al., 2009; kaisoon et al., 2011; garzon et al., 2015; loizzo et al., 2015), highlighting that they are a substantial source of chemical compounds with a high antioxidant activity (fu and mao, 2008; garzon et al., 2015). phenolic acids, flavonoids, including anthocyanins, have been recognized as the most representative biologically-active compounds found in the petals of fresh edible flowers (mlcek and rop, 2011; navarro-gonzalez et al., 2015). unlike freshly-marketed edible flowers, in which the profile of bioactive compounds has been extensively investigated (for a review see mlcek and rop, 2011), only a few papers have evaluated the stability of edible flower phytochemicals during storage (das et al., 2010; kazaz et al., 2010; aquino-bolaños et al., 2013; landi et al., 2015a). results are sometimes conflicting. in some cases cold storage has been found to have a negative impact on the nutraceutical value of edible flowers (das et al., 2010; aquinobolaños et al., 2013), but not in other cases (friedman et al., 2007). we investigated the effect of cold storage on various biologically-active compounds namely phenolics, flavonoids, anthocyanins and ascorbic acid in seven edible flowers belonging to five species (acmella oleracea l., begonia semperflorens l. with white, pink, and dark-pink, salvia discolor kunth, tulbaghia cominsii vosa, tropaeolum majus l.) with different sizes, shapes and colors (the features of each edible flower are summarized in table 1). given that consumers are influenced by the visual appeal of edible flowers, and only high-quality produce encourages repeat purchases, we attempted to establish whether the loss of the visual appeal of edible flowers proceeds faster than the loss of their nutritional value. the overall aim was to address future research aimed at extending the shelf life of edible flowers. 2. materials and methods 2.1. chemicals methanol (lc/ms grade; > 99.95 % solvent purity), and hcl (acs reagent, 37%) were purchased from carlo erba reagents s.r.l., milan, italy. all the other reagents were purchased from sigma-aldrich s.r.l., milan, italy. 2.2. flower harvest and processing flowers of a. oleracea (ao), b. semperflorens (with white; bsw, pink; bsp, and dark-pink petals; bsdp), s. discolor (sd), t. cominsii (tc), t. majus (tm) were kindly provided by creat-chambre d'agriculture des alpes-maritimes (nice, france) (table 1). ital. j. food sci., vol. 30, 2018 338 table 1. features of the selected edible flowers studied in this work. species family abbreviation color flower size acmella oleracea l. asteraceae ao yellow small begonia semperflorens l. begoniaceae bsdp dark pink medium begonia semperflorens l. begoniaceae bsp pink medium begonia semperflorens l. begoniaceae bsw white medium salvia discolor kunth lamiaceae sd violet small/ medium tulbaghia cominsii vosa amaryllidaceae tc light pink small tropaeolum majus l. tropaeolaceae tm orange big fresh flowers at maturity stage (june, 2014) were harvested early in the morning, transported in refrigerated containers (4 °c) and processed within a few hours in an aseptic laboratory in accordance with kelley et al. (2003). for each species, some flowers (about 2 g) were finely ground with liquid nitrogen and stored at -80 °c until analysis. these samples represented the first day of storage (t0). the flowers were then randomized in air-tight hinged boxes (500 cm3, comital cofresco, italy) made from polyethylene terephthalate and stored at 4 °c under light to simulate commercial shelf conditions. each box contained about 20 g of fresh flowers. flowers were inspected to ensure there was no visually detectable damage prior to being placed in each container. samples were collected following the same procedure after 2, 5 and 8 d of storage for biochemical analysis. for the 8-day storage, samples were collected only for edible flowers that would still have been marketable at that time. before being ground, some of the sample flowers were used for color determination. 2.3. determination of moisture content initial (t0) fresh weight (fw) was evaluated immediately after the preparation of the flower containers. to determine flower dry weight (dw), florets were desiccated in a ventilated oven at 80 °c until constant weight. at each sampling data (2, 5, 8 d), moisture content was evaluated as the difference between fw and dw of the flowers contained in each box, and expressed as percentage moisture content. 2.4. color determination for each species, color measurements were performed on five randomly selected flowers (n=5) at different storage times (0, 2, 5, 8 d). the value of each replicate was the mean of three independent spot measurements on each flower’s surface evaluated by standard cie l*a*b* color space coordinates determined by an ocean optic hr2000-uv-vis-nir spectrometer coupled with a tungsten halogen dh2000 light source (ocean optics, usa) as reported in landi et al. (2015b). among all colorimetric parameters, l* represents the lightness of colors (lightness index scale; 0 for black to 100 for white) and is a good parameter for monitoring the development of tissue darkening. 2.5. postharvest visual quality rating the visual appeal of flowers was scored on a 9-point scale based on visual observation of the degree of decay, as described by aquino-bolaños et al. (2013). for the sake of ital. j. food sci., vol. 30, 2018 339 simplicity, score points were grouped as follows: 9 to 7 = fresh appearance (flower with no defects or the slight beginning of decay; classified with a green triangle in fig. 1), 6 to 4 = limit of marketability (moderately deteriorating flower with between a quarter or and half the surface area decayed; yellow triangle in fig. 1), 3 = not suitable for sail (water-soaked, dark and wilted flower with more than half the surface area decayed; red triangle in fig. 1). the visual quality was assessed by a panel of five people with expertise in browning phenomena and post-harvest loss of quality. 2.6. total phenolics, flavonoids, and anthocyanins extraction of total phenolics and total flavonoids was based on a slight modification of the method reported by du et al. (2009). an aliquot of 100 mg of flower sample was homogenized in 1 ml of ethanol:acetone (7:3, v/v) and shaken overnight at 4 °c. the extract was centrifuged at 1,000 g for 15 min at 4 °c and the supernatant was filtered using minisart filters (pore size 0.45 µm). the filtrate was collected and stored at −20 °c until analysis. total phenolic content was determined using the folin-ciocalteau assay, according to dewanto et al. (2002), using 10 µl of extract. the absorbance was read at 760 nm and the total phenolic concentration was expressed as gallic acid equivalents (mg gae g-1 dw) using a calibration curve (50-600 µg ml-1). content of total flavonoids was determined according to du et al. (2009) with a few modifications. in a 2 ml eppendorf tube, 100 µl of flower extract were added in 1 ml ethanol 30% (v/v), 45 µl of 50 mm nano2, 45 µl of alcl3 x 6h2o 0.3 m. after 5 min at room temperature, 300 µl of 1 m naoh were added and the mixture absorbance was measured at 506 nm. the content of total flavonoids was expressed as rutin equivalents (mg re 100 g-1 dw) using a calibration curve as a standard (6.25-1000 µg ml-1). total anthocyanins were extracted as reported by landi et al. (2014). briefly, 100 mg of ground samples were mixed with 1 ml of acidified methanol (1.5% hcl v/v) and shaken overnight at room temperature. the supernatant was filtered using minisart filters (pore size 0.45 µm); anthocyanin-containing flower extract (50 μl) was added to 950 µl of acidified methanol (1.5% hcl v/v) and the absorbance was read from 408 to 560 nm against a blank. total anthocyanin content was expressed as the mean value of abs in the range 408-560 nm per 100 mg-1 dw. an ultrospec 2100 pro spectrophotometer (ge healthcare ltd, little chalfont, england) was used for the analyses of total phenolics, flavonoids and anthocyanins, together with all the other spectrophotometric determinations. 2.7. ascorbic acid determination total ascorbate (asatot), reduced ascorbate (asa), and dehydroascorbate (dha) were spectrophotometrically determined as described by kampfenkel et al. (1995). the assay is based on the reduction of fe3+ to fe2+ by asa and the spectrophotometric detection of fe2+ complexed with 2,2’-dipyridyl. dha is calculated as the difference between asatot and asa, and data were expressed as µg g-1 dw. 2.8. dpph scavenging activity the antioxidant activity of each sample was determined using a modified version of the 2,2-diphenyl-1-picrylhydrazyl radical (dpph) free radical scavenging assay, as described by kim et al. (2005). the methanolic flower extract (20 μl) was diluted to 100 μl with 80% aqueous methanol. it was then added to 0.4 ml of 0.1 m tris-hcl buffer and 0.5 ml of 0.3 mm dpph in methanol. the solution was mixed thoroughly and incubated in the dark for ital. j. food sci., vol. 30, 2018 340 20 min at room temperature. the absorbance of the sample mixture (asample = asample t20’ asample t0) was monitored at 517 nm. the absorbance of a control sample (acontrol = acontrol t20’ acontrol t0) containing only methanol/tris-hcl, and dpph versus a methanol/tris-hcl blank was also analyzed. the percentage dpph free radical scavenging activity was calculated according to the following equation: % dpph free radical scavenging = [1-(asample/acontrol)] x 100 the antioxidant activity was determined by comparing the percentage dpph free radical scavenging of each sample to a calibration curve prepared with trolox. antioxidant activity was expressed as trolox equivalents (te; mmol of te g-1 fw) for direct comparison of the free radical scavenging capabilities between all the samples. 2.9. statistical analysis visual quality was assessed by each expert in five randomly selected flowers per species at each sampling time. reported data for flower moisture and phytochemical contents are the means (±sd) of five independent replicates (n=5), where each box was considered as a replicate. means were compared by one-way anova, following bartlett’s test to assess the homogeneity of variance among samples, considering storage as the variability factor. percentage values were arcsine transformed prior to the analyses. means with different letters within species are significantly different after fisher’s least-significant difference test (lsd) for p=0.05. for some comparisons among species (when discussed), one-way anova was applied with species as the variability factor. all statistical analyses were performed using costat (cohorttm software, berkeley, ca). 3. results and discussions 3.1. physiological weight loss and visual quality values of l* are indicative of tissue darkening as browning is commonly associated with the oxidation of phenolics and their polymerization into dark brown pigments (martindiana et al., 2015; landi et al., 2015c, remorini et al., 2015). as expected, in this experiment l* values decreased in all the efs under investigation, at least at the end of the storage period (8d, fig. 1). flowers of a. oleracea and t. cominsii were considered to be still marketable after 8 d of storage at 4 °c, whereas all the other species were classified as not suitable for sale at this storage time (fig. 1). differences in the shelf life of edible flowers subjected to cold storage have already been reported (kuo et al., 2012; kelley et al., 2003). unlike reported by kelley et al. (2003), five out of the seven edible flowers that we evaluated (including nasturtium, which was one of the edible flowers considered by kelley et al) had a shorter shelf life at 5 °c (5 d). these differences can be attributable to the different packaging: kelley et al. (2003) used polyethylene bags, whereas we used polyethylene terephthalate boxes in order to preserve the delicate flowers. despite the evident differences in terms of shelf life, the main determinants that contribute are less clear from the literature. ital. j. food sci., vol. 30, 2018 341 days of storage 0 2 5 8 81.2±3.0 a 80.2±4.4 a 74.7±8.7 b 72.5±1.7 b 39.1±2.2 a 39.9±10.9 a 39.8±2.9 a 28.3±7.9 b 67.5±5.5 a 69.5±3.1 a 60.3±5.8 b 54.8±3.2 b 84.3±3.0 a 85.2±7.9 a 77.6±4.2 b 70.7±2.5 c 16.6±0.5 a 17.0±0.6 a 9.1±2.7 b 4.4±1.8 c 78.4±5.2 a 80.0±1.1 a 74.9±6.2 b 66.8±3.4 b 41.0±3.9 a 37.7±2.8 b 34.1±1.9 c 29.5±2.3 d : excellent : limit of marketability : not fit for sale figure 1. visual appearance, marketability and lightness (l*) of edible flowers. reported results for l* values are the mean of five replicates (n=5;±sd). means flanked by different letters are significantly different within each flower species upon storage for p = 0.05 after one-way anova followed by lsd test. colored triangles at the top right of each figure represent excellent product (green), limit of marketability (yellow), or unsaleable product (red). bars = 1 cm. begonia semperflorens acmella oleracea tropaeolum majus tulbaghia cominsii dark pink pink begonia semperflorens 1 cm white begonia semperflorens salvia discolor ital. j. food sci., vol. 30, 2018 342 our data suggest that the constitutive moisture content of edible flowers has the most impact on shelf life. at t0, flowers of b. semperflorens with dark-pink, pink and white petals and t. majus had a significantly higher (p<0.001) moisture % (which averaged 97.34% in b. semperflorens with dark-pink, pink and white petals and t. majus) than a. oleracea and t. cominsiii (87.82 and 88.19%, respectively), and had a 5-d versus a 8-d shelf life for a. oleracea and t. cominsiii (table 2 and fig. 1). the hypothesis of reduced dehydration related to longer flower shelf life is also in agreement with kou et al. (2012). this hypothesis does not seem applicable to s. discolor flowers which had a simultaneously lower moisture content than b. semperflorens with dark-pink, pink and white petals and t. majus, but only 5 d of marketability. in this species, the reduced marketability seems mainly related to the loss of anthocyanin content (table 3) as s. discolor was the only edible flower in which anthocyanin decreased during storage. although not measured in our experiments, the high perishability of edible flowers might be connected to their respiration rate and the production of ethylene, as is the case with other horticultural commodities (kader and saltveit, 2003). however, kou et al. (2012) demonstrated that after 7 d of storage the decay index of carnation increased to a similar extent and irrespectively of the use of modified atmosphere packaging and/or 1methylcyclopropene, which is a commonly used ethylene inhibitor. friedman et al. (2007) also found that t. majus flower quality was not related to co2 or to ethylene levels inside the packaging in a short-term storage period. villalta et al. (2004) found that the respiratory rate of yellow summer blossom remained relative constant and low during the 8-d storage period at 5 °c. table 2. moisture content of edible flowers of acmella oleracea l. (ao), begonia semperflorens l. (with white; bsw, pink; bsp, and dark-pink petals; bsdp), salvia discolor kunth (sd), tulbaghia cominsii vosa (tc), tropaeolum majus l. (tm) during storage. storage (d) species 0 2 5 8 moisture content (%) ao 87.82±1.74 a 87.08±2.03 a 79.15±5.39 b 61.44±3.41 c bsdp 97.33±0.39 a 83.63±1.95 b 69.15±3.22 c 47.23±4.66 d bsp 97.41±0.03 a 92.89±1.08 b 82.87±1.80 c 61.67±3.18 d bsw 97.38±0.35 a 89.21±1.26 b 74.13±3.95 c 47.85±0.12 d sd 83.09±0.57 a 80.11±1.48 b 73.41±2.60 c 57.25±1.65 d tc 88.19±0.08 a 86.43±1.55 a 83.22±3.40 b 77.56±3.67 c tm 97.27±0.54 a 91.66±4.10 b 80.31±6.45 c 45.03±7.24 b data represent the mean±sd (n=5). means flanked by different letters are significantly different within each flower species upon storage for p = 0.05 after one-way anova followed by lsd test. this evidence weakens the hypothesis that the flower’s respiration rate is the key factor in extending an edible flower’s shelf life, at least for short-term storage. on the other hand, controlling the respiration rate seems to be more important for longer storage periods (at least two weeks; kuo et al., 2012). 3.2. bioactive compounds and antioxidant activity phenols (including phenolic acid, flavonoids and anthocyanins) are currently the target of numerous studies since their intake has been associated with the decreased risk of cancer, cardiovascular diseases and neurodegenerative disorders (salem et al., 2011). in our ital. j. food sci., vol. 30, 2018 343 study, constitutive levels of total phenolics ranged from 10.02 mg gae g-1 dw in a. oleracea to 194.33 in t. majus (table 3). table 3. phytochemical content of edible flowers of acmella oleracea l. (ao), begonia semperflorens l. (with white; bsw, pink; bsp, and dark-pink petals; bsdp), salvia discolor kunth (sd), tulbaghia cominsii vosa (tc), tropaeolum majus l. (tm) during storage. species storage (d) total phenols total flavonoids total anthocyanins dpph (mg gae g-1 dw) (mg re g-1 dw) (absb 100 mg-1 dw) (mmol te g-1 dw) ao 0 10.02±2.93 c 4.85±1.05 a nd 26.25±2.58 b 2 15.00±0.35 a 6.17±1.39 a nd 32.04±1.39 a 5 12.62±1.59 b 4.94±1.06 a nd 30.77±4.16 a 8 11.83±0.18 bc 4.66±0.09 a nd 30.73±1.27 a bsdp 0 64.21±1.56 b 37.81±3.63 a 2.20±0.37 a 96.21±8.80 a 2 77.78±4.70 a 32.77±2.23 b 1.92±0.64 a 90.42±12.03 a 5 63.85±3.86 b 29.11±0.43 c 1.94±0.47 a 89.37±8.14 a 8 bsp 0 51.72±4.30 c 41.79±13.87 a 0.84±0.16 a 74.71±4.87 c 2 94.21±8.80 a 36.50±3.10 ab 0.76±0.21 a 94.53±0.52 a 5 84.54±11.3 b 27.43±0.97 b 0.65±0.15 a 83.80±5.81 b 8 bsw 0 77.77±9.50 b 32.01±6.50 a nd 67.94±17.34 b 2 95.27±1.02 a 36.09±1.53 a nd 93.10±8.47 a 5 69.29±6.01 b 28.37±4.60 a nd 63.40±9.25 b 8 nd sd 0 26.76±0.92 a 11.35±1.59 a 0.27±0.03 a 32.62±0.41 a 2 19.29±1.53 b 9.60±1.45 a 0.23±0.03 ab 31.37±3.47 a 5 19.20±3.05 b 9.76±1.22 a 0.19±0.06 b 29.17±0.15 a 8 tc 0 30.51±2.16 a 3.02±0.24 a 0.09±0.03 a 44.85±0.36 a 2 28.55±0.47 b 2.74±1.01 a 0.10±0.04 a 47.59±1.53 a 5 28.13±1.28 b 3.11±0.58 a 0.13±0.01 a 46.79±2.10 a 8 25.1±1.02 c 3.51±0.78 a 0.13±0.01 a 47.40±0.78 a tm 0 194.33±16.50 a 28.34±3.70 a 10.10±3.34 a 142.13±22.16 c 2 140.51±9.98 c 28.91±0.50 a 9.81±1.70 a 156.33±4.00 b 5 163.50±2.33 b 31.55±1.41 a 11.22±1.27 a 181.02±1.40 a 8 data represent the mean±sd (n=5). means flanked by different letters are significantly different within each flower species upon storage for p = 0.05 after one way anova followed by lsd test. abs, absorbance; dpph, total antioxidant activity evaluated by 2,2-diphenyl-1-picrylhydrazyl radical; d, days; dw, dry weight; gae, gallic acid equivalents; nd, not detectable; re, rutin equivalent; te, trolox equivalents. b, mean value of absorbance in the range 408-560 nm. ital. j. food sci., vol. 30, 2018 344 these values are common in flower species, as also testified by other studies (rop et al., 2012; li et al., 2014). in many cases, the total phenol values in all the edible flowers studied here were abundantly higher than those reported for other vegetables and fruits, which are usually considered as good sources of phenols (kähkönen et al., 1999). edible flower species could thus represent an interesting source of phenolic compounds, despite the small amount usually consumed compared to other fruits and vegetables. interestingly, the levels of total phenols found in t. majus flowers are two and a half times higher than those reported for blueberry genotypes, which are usually classified as some of the richest sources of phenols, in particular due to their high level of anthocyanins (castrejón et al. 2008). overall, we found that 8 d of cold storage affected the concentration of all the phenolic bioactive compounds evaluated here only in some cases (table 3). a reduction in total phenols was recorded only in s. discolor, t. cominsii, and t. majus flowers. total flavonoids decreased only in pigmented b. semperflorens, whereas the loss of anthocyanins was observed only in s. discolor. results regarding the effect of storage on phenolic compounds are scarce and conflicting. for example, aquino-bolaños et al. (2013) found a reduction in total phenols in yellow summer squash flowers during postharvest storage. friedman et al. (2007) found no difference in anthocyanin content in b. semperflorens flowers and landi et al. (2015a) found no significant changes in total phenols in sage flowers during storage. we are not aware of any other work focused on the variation in phenolic content and profile in edible flowers upon storage. ascorbic acid is a well-known key antioxidant in plants and is an essential vitamin for humans. unlike the polyphenols mentioned above, the concentration of asatot decreased significantly during storage (at least at the end of the marketability stage) in almost all the edible flowers under investigation (a. oleracea, b. semperflorens with dark-pink and white petals, t. cominsii, and t. majus) (table 4). notably, in b. semperflorens with pink petals and s. discolor, whose level of asatot was unchanged, we found an increased level of the oxidized form of ascorbate (decremented ratio asa/asatot). thus, storage may have negatively affected the level of asa (which is the biological active form of ascorbic acid) in all the edible flowers under investigation. a reduction in ascorbate levels in edible flowers has also been reported by das et al. (2010) and by aquino-bolaños et al. (2013) under cold storage. aquino-bolaños et al. (2013) attributed the loss of ascorbate to the loss of cell integrity and compartmentalization, which expose ascorbic acid to oxygen, which, in turn, decreases the reducing power of this key antioxidant. the biological activities of phenolic compounds and ascorbate seem to be related to their strong antioxidant capacity in vitro, as also reported for many edible flower-derived compounds (kaisoon et al., 2011; salem et al., 2011; li et al., 2014; garzon et al., 2015; loizzo et al., 2015). interestingly, the total antioxidant activity of some edible flowers, including some of the flowers tested in our investigation (i.e., s. discolor and t. cominsii), is even higher than that of many blueberries varieties (v. corymbosum l.), which is one of the richest reported antioxidants (giovannelli and buratti, 2009). the total antioxidant activity of edible flower extracts did not decrease in any of the edible flowers upon storage (table 3). conversely, in others (i.e., a. oleracea, b. semperflorens with pink petals, and t. majus) the total antioxidant activity was also found to increase during the storage. our findings are in agreement with friedman et al. (2007), who reported that 7-8 d of cold storage (2-5 °c) did not reduce the antioxidant activity of b. semperflorens flowers. the stability of the antioxidant capacity of edible flowers seems principally related to the relative stability of the total phenol content given that many researchers have found a strong linear relation between total phenol content and the antioxidant activity of edible flowers (r2 > 0.93) (li et al., 2014; navarro-gonzalez et al., 2015). ital. j. food sci., vol. 30, 2018 345 table 4. ascorbic acid content of edible flowers of acmella oleracea l. (ao), begonia semperflorens l. (with white; bsw, pink; bsp, and dark-pink petals; bsdp), salvia discolor kunth (sd), tulbaghia cominsii vosa (tc), tropaeolum majus l. (tm) during storage. storage (d) species 0 2 5 8 asatot (mg g -1 dw) ao 2.51±0.12 b 3.05±0.12 a 2.40±0.53 b 1.04±0.09 c bsdp 16.87±3.63 a 16.06±3.12 a 11.53±2.14 b bsp 16.90±3.63 a 17.52±2.06 a 16.38±1.93 a bsw 5.13±0.59 a 5.76±0.51 a 2.31±0.46 b sd 8.13±2.00 a 8.18±2.66 a 6.75±1.45 a tc 12.84±0.12 a 8.07±1.29 b 7.43±2.67 c 8.80±1.12 b tm 89.25±15.88 a 68.75±14.10 b 47.01±1.33 c asa/asatot ao 0.45±0.01 a 0.41±0.05 a 0.47±0.04 a 0.50±0.03 a bsdp 0.74±0.01 a 0.57±0.01 b 0.77±0.06 a bsp 0.46±0.02 a 0.39±0.04 a 0.39±0.02 b bsw 0.53±0.08 a 0.46±0.15 a 0.58±0.13 a sd 0.73±0.10 a 0.54±0.12 b 0.58±0.13 b tc 0.86±0.03 a 0.89±0.01 a 0.87±0.01 a 0.75±0.12 b tm 0.62±0.10 a 0.58±0.18 a 0.64±0.02 a data represent the mean±sd (n=5). means flanked by different letters are significantly different within each flower species upon storage for p = 0.05 after one-way anova followed by lsd test. asa, ascorbic acid (reduced form); asatot total ascorbate (sum of oxidized and reduced form); dw, dry weight. our data also highlight a good correlation between total phenol content and total antioxidant activity, although we found a lower r2 than that mentioned above (r2 = 0.655). this determination coefficient is however in agreement with that reported by li et al. (2009) (r2 = 0.664) and tai et al. (2011) (r2 = 0.652) in flowers of peony and sophora viciifolia, respectively. it suggests that, despite the main role of phenolics as antioxidants, other antioxidant compounds contribute significantly to the flower antioxidant activity. this would also justify the reduction in total phenolic content found in s. discolor, t. cominsii, and t. majus associated with unchanged (though increased in t. majus) levels of their total antioxidant activity. in our experiments, the contribution of ascorbic acid to the total antioxidant activity seems less significant than total phenolics, given that ascorbic acid levels were negatively affected by the storage, while the total antioxidant activity was not. 4. conclusions our data suggest that the loss of visual appeal of most edible flowers proceeds faster than the loss of their bioactive compound content. only ascorbic acid was found to be highly susceptible to the storage process. the content of its reduced form (asa) decreased upon storage in all the edible flowers under examination before the end of their shelf life. on the other hand, phenolic moieties were less affected by the storage. the total antioxidant activity of all the edible flowers evaluated here was stable under cold storage up to the end of their shelf life. this suggests that edible flower decay is directly related to their constitutive water content, thus (i) the selection of edible flowers with low moisture is a key factor in ensuring their longer marketability; (ii) more efforts should focus on the processes and technologies aimed at preserving (or delaying) edible flowers from water ital. j. food sci., vol. 30, 2018 346 loss, such as the use of boxes or bags made from appropriate plastic material and/or appropriately modified atmospheres, in order to extend the shelf life of edible flowers. acknowledgements this study was funded by the italian project interreg – alcotra 2007-2012 “aroma” (n°.68) “le piante aromatiche tra ambiente e attività produttive”. 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o., kangsadalampai k. and tongyonk l. 2010. antimutagenicity of some flowers grown in thailand. food chem. toxicol. 48:1045. paper received january 20, 2017 accepted may 9, 2017 ijfs#939_bozza ital. j. food sci., vol. 30, 2018 256 paper apple slices enriched with aloe vera by vacuum impregnation a. derossi, i. ricci, a.g. fiore and c. severini* department of science of agriculture, food and environment, university of foggia, via napoli 25, 71122 foggia, italy *e-mail address: carla.severini@unifg.it abstract recently, the interest in aloe vera has been increased accordingly with its content in polymannas, which show many healthy effects. the vacuum impregnation (vi) was used to enrich apple slices with aloe vera gel. the effects of vacuum level, vacuum and relaxation times on the main chemical and physical attributes were described. results showed as, applying the best operating conditions, vi allowed to introduce aloe vera gel into the pores of apple tissue reaching a content of polymannan between 1 and 8 mg/100 g of fresh apples. keywords: fresh cut apples, polymannans, enriched fruit, aloe vera gel ital. j. food sci., vol. 30, 2018 257 1. introduction aloe vera (aloe barbadensis mill.) is a perennial xerophyte belongs to the liliaceae family, which consists in about 360 species. the internal fraction of the leaves is a large thinwalled parenchyma cells in which the water is held as a gel (newton, 2004). the aloe vera’s gel consists of water (99.5%) and solids (0.5%) such as polysaccharides, vitamins, minerals, enzymes, phenolic compounds and organic acids (eshun and he, 2004; boudreau and beland, 2006). however, a mass fraction of 60% of the total solids is constituted by polysaccharides (mcanalley, 1993). recently, the interest on aloe vera gel has increased on the basis of its healthy properties. back in the past, peoples used the aloe vera for its curative and therapeutic properties. recently the intake of the gel has been proved to have positive effects in the treatment of gastrointestinal, kidney and cardiovascular deseases. also, the gel has used to reduce the cholesterol and triglyceride levels in human blood (lim et al., 2003; geremias et al., 2006). furthermore, antiinflammatory and antibiotic properties, as well as positive effects against several diseases, have been reported (reynolds and dweck, 1999; eshun and he, 2004). several scientific papers (hamman, 2008; rodriguez et al., 2010) proved the most of the health benefits may be attributed to polysaccharides (dagne et al., 2000; ni et al., 2004; habeeb et al., 2007) such as cellulose, hemicellulose, glucomannans, mannose derivatives and acetylated mannans (robert and travis, 1995; femenia et al., 1999; lee et al., 2001). given these considerations, food industries have been attracted from the use of aloe vera as an ingredient to improve the functional properties of food products. moreover, the fda approved the use of aloe vera gel extracted as a “dietary supplement” (ramachandra and srinivasa rao, 2008). thus, a wide number of enriched foods with aloe vera such as yogurt, juice, pasta are available on the market. however, food processing could induce irreversible modifications of the polysaccharides, reducing or inactivating the health effects (femenia et al., 2003; eshun and he, 2004; chang et al., 2006; vega-gálvez et al., 2011). the vacuum impregnation (vi) is a very interesting technique that allows to introduce, dissolved or suspended substances in the void fraction (i.e. the pores) of food in a controlled manner (gras et al., 2002). vi occurs at room temperature, avoiding the thermal degradation of nutritional and functional compounds. in the last years, several authors studied the application of vi to obtain foods enriched with structural compounds (martinez-monzo et al., 1998), probiotic microorganisms (puente et al., 2009), fruit juices (betoret et al., 2012; castagnini et al., 2015), phenols (schulze et al., 2014), folic acid (moreno et al., 2016), sugars (neri et al., 2016), etc. sanzana et al. (2011) evaluated the effects of vi with aloe vera on the respiration rate of some vegetables (endive, cauliflower, broccoli and carrots). in some previous papers we applied the vacuum impregnation in order to accelerate the acidification of some vegetables (derossi et al., 2013a,b) as well as to improve the quality of thawed truffles by introducing antifreezing proteins (derossi et al., 2015a). given these considerations, the main aim of this paper was to study the application of vacuum imprengation to improve the functional properties of apple slices by using an extract of aloe vera gel. specifically, the effects of the main process variables of vi on the level of enrichment as well as on the main physical properties of apples slices were studied using the response surface methodology. ital. j. food sci., vol. 30, 2018 258 2. materials and methods 2.1. fresh apple and aloe vera plant fresh apples (cv. golden delicious) were purchased to the local market in september 2015 and stored for a maximum of 3 days at 4°c. before treatments, the apples were equilibrated at room temperature. aloe vera plant (aloe barbadensis mill.) of three years old was supplied by ricciotti gardens (foggia, italy). the gel was prepared by cutting the fresh leaves and separating the outer green rind from the inner parenchyma. 2.2. vacuum impregnation treatments after washing and peeling, the apples were manually cut in slices with a thickness of 0.5 cm. the aloe vera extract was prepared by dissolving 25 g of the aloe vera gel in 125 ml of distilled water at room temperature under agitation at 300 g-1 for 90 min. a product/solution mass ratio of 1:5 (w/w) was used for each experiment. 2.3. experimental design a factorial design was used to study the effects of three variables (pressure, p, vacuum time, t1, and relaxation time, t2) on the main quality attributes of apples (box and behnken, 1960). the pressure was modified between 50 and 450 mbar while vacuum times and relaxation times were studied in the ranges of 1-5 min and 5-15 min, respectively. these values were chosen on the basis of preliminary experiments performed in order to define wide ranges able to significantly increase the weight of apple slices that is a rough index of the filling of pores. more specifically, a 3(k-p) fractional factorial design was implemented with k = 3 and p = 1 obtaining a total of 9 experimental conditions. each vi test was repeated in triplicate by using the experimental conditions reported in table 1. table 1. experimental conditions of vacuum impregnation experiments. experiments pressure vacuum time relaxation time (mbar) (t2, min) (t1, min) 1 450 5 15 2 450 1 10 3 450 3 5 4 250 1 15 5 250 5 5 6 250 3 10 7 50 1 5 8 50 3 15 9 50 5 10 2.4. chemical and physical analysis moisture (xw) and solid (xs) content of samples were gravimetrically measured by drying 5 g of vegetable tissue at 65°c until a constant weight (cabezas-serrano et al., 2009). ital. j. food sci., vol. 30, 2018 259 2.5. changes in porosity porosity values of fresh and vi samples were obtained comparing apparent (ρa) and real solid-liquid (ρr) density values. all porosity values were expressed as kg/m3. pycnometer method was used to determine the apparent density (ρa) using an isotonic sucrose solution as a reference. real (ρr) density and porosity fraction (ε) were estimated as reported by gras et al. (2002). the weight increase (de) was expressed as relative differences on the basis of fresh weight. 2.6. determination of total polymannans content the total polymannans content was determinated by using the colorimetric assay proposed from eberendu et al. (2005) with some minor changes. a volume of 400 ml of aqueous extract, 500 ml of naoh (0.1 m) and 1 ml of congo red (sigma aldrich) dye (diluted by a factor of 500) were mixed and agitated for 2 h, then the changes in colour were analysed at 540 nm with a spectrophotometer (perkin elmer lambda 25 uv/vis). the calibration curve was performed by using a solution of β-glucan (sigma aldrich inc.) in the range between 3.2 and 100 mg/l as reported by pellizzoni et al. (2012). 2.7. statistical analysis the effect of each independent variable on the quality indexes of apple samples was evaluated by anova test with a significant level of 0.05. also, the results were described by pareto’s charts. moreover, 3d plots describing the changes of each dependent variable as a function of experimental conditions were obtained by fitting the experimental data with a polynomial model as reported by derossi et al., (2015b). all statistical analyses were performed using statistica ver. 10.0 (statsoft tulsa, usa). 3. results and discussion table 2 shows the main physico-chemical attributes of fresh apples. average values of moisture and solids content of golden delicious were of 0.88±0.01 g h2o /g f.w. and 0.12±0.01 g h2o /g f.w., respectively. these values were in good agreement with other authors (mujica-paz et al., 2003; paes et al., 2007; wu et al., 2007). table 2. physico-chemical properties of fresh apples. parameter value ± se moisture content (xw) (g h2o/g f.w.) 0.88±0.01 solids content (xs) (g/g f.w.) 0.12±0.01 apparent density (ρa) (kg/m 3) 822±0.04 real density (ρr) (kg/m 3) 1047±0.04 porosity fraction (ε) (%) 21.5±0.04 moreover, a porosity fraction of 21.4±3.69 % indicates slight variability in porosity as a consequence of their biological variance, which includes the ripening degree, the environmental conditions, varieties, etc. in general, literature has reported a significant variability in porosity of apples with values between ~18 % and ~27 % (salvatori et al., ital. j. food sci., vol. 30, 2018 260 1998; martinez-monzò et al., 2000; mujica-paz et al., 2003; paes et al., 2007) which are in agreement with our data. as expected, polymannans were not revealed in fresh apples. with the aim to quantify the content in polymannans in aqueous extracts of aloe vera the distribution function of about 40 measurements performed on extracts prepared from different part of aloe vera plant, is reported in fig. 1. a normal distribution was proved by the shapiro-wilk’s test that exhibited a value of 0.98. the mean value was of 0.355 ± 0.078 mg/g enables to state that the most of the observation fell in the range of 0.3 and 0.4 mg/g. also, minimum and maximum values of 0.188 mg/g and 0.546 mg/g were also observed. figure 1. the normal probability function of polymannans content of the aloe vera gel extracts. the results of statistical analysis showed that the pressure value and the relaxation time linearly affected the changes in porosity of apples showing standardized effects of 6.97 and -3.53, respectively (fig. 2a). this means that as the pressure decreased as the porosity decreased too, while the relaxation time showed an inverse relationship with the void fraction of apple tissue. since that the driving force of vi is the difference between internal (i.e. inside the pores) and external pressures, the higher was the vacuum, the greater was the impregnation. also, the longer was the relaxation times, the higher was the reduction in porosity, because there was more time for impregnation and relaxation phenomenon (gras et al., 2002; mujica-paz et al., 2003; neri et al., 2016). figure 2b shows the 3d plot describing the effects of relaxation time and pressure on the porosity fraction of apples. at first, taking into account the treatment performed at the lower vacuum of 450 mbar and the minimum relaxation time of 5 min, a significant reduction in porosity from fresh apple (ε = 21.4±3.69%) to 14% was observed. moreover, according to pareto chart of fig. 2a the pressure had the highest effect in the reduction of porosity fraction of the samples. according to this results porosity fraction reduced from 0.14 to ∼0.08% when the pressures of 450 and 50 mbar were applied, respectively, with a minimum t2 of 5 min (fig. 2b). ital. j. food sci., vol. 30, 2018 261 figure 2. a) estimated effects of the independent variables on the porosity fraction of apple slices submitted to vi treatments with aloe vera gel extract. l and q refer to the linear and no linear (quadratic) effect, respectively. b) 3d plot describing the effects of relaxation time and pressure on the fraction porosity of apple slices submitted to vi treatments with aloe vera gel extract. on the other hand, a negligible reduction in porosity was observed by applying relaxation time from 5 to 15 min for some pressure applied. for instance, appling a pressure of 50 mbar, values of 0.08 and 0.03 were measured progressively increasing t2. moreover, the results stated the very high reduction in porosity fraction of apples tissue independently from the length of vacuum time; in fact, by reducing t1 a change in porosity only of 0.4% was achieved (data not shown). however, experimental data show a high variability, which cannot be underestimated. this could be the result of the variance in microstructure properties such as the porosity of fresh apples, the presence of closed pores, the size and dimension of capillaries, their tortuosity, etc. however, the decrease of the porosity fraction after vi treatment cannot assure that the impregnation by external solution ital. j. food sci., vol. 30, 2018 262 occurred. as well known, during relaxation time the impregnation and the compression of capillaries are involved, both allowing to reduce the porosity fraction of fruit. the equilibrium between the impregnation and compression level is controlled by several variables such as the viscosity of the solution, the rigidity of vegetable tissue, etc., most of which cannot be manually controlled. the pareto chart (fig. 3a) shows the effect of the independent variables on the weight increase of apples. the pressure linearly affected the weight increase of apple samples exhibiting a standardized effect of -3.58, while the other variables did not show any effect. the variation in weight as a function of vacuum time and pressure are shown in fig. 3b. figure 3. a) estimated effects of independent variables on the weight increase of apples slice submitted to vi treatment with aloe vera gel extracts. l and q refer to the linear and no linear (quadratic) effect, respectively. b) 3d plot describing the effect of vacuum time and pressure on the weight increase of apple slices. ital. j. food sci., vol. 30, 2018 263 according to pareto chart, only the pressure exhibited a significant effect. even considering the lower t1 = 1 min the weight of apple slices increased from ∼0.18 to ∼0.26 g/g by reducing the pressure from 450 to 50 mbar. that means that a significant amount of aloe vera extract was introduced in the void phase of apple. on the other hand, any differences were not observed increasing the vacuum time until 5 min. also in this case, the experimental data showed a not negligible variability that could be the result of differences in microstructure (porosity, connectivity, pore size distribution, etc.). fig. 4a reports the standardized effects of the independent variables on polymannans content of apple slices. the pressure and the relaxation time were the most important variables affecting the enrichment of apple slices. the standardized effects of -3.15 and 2.55 showed as the pressure exibhited the greater effect on enrichment of apple samples. fig. 4b shows the effect of the vacuum level on the polymannans content of apples. figure 4. a) standardized effect of the independent variables on the polymannan content of apple slice submitted to vacuum impregnation with aloe vera gel extract. l and q refer to the linear and no linear (quadratic) effect, respectively b) changes in polymannas content of apple slices submitted to vacuum impregnation as a function of pressure. ital. j. food sci., vol. 30, 2018 264 more specifically, the figure was obtained by grouping all experimental data for the pressure values used during experiments while the other two variables, t1 and t2, were free to change. the positive effect of the vacuum impregnation is clearly observed. by reducing the pressure from 450 to 50 mbar, the average content in polymannans increased from 2 to 5 mg/100 g f.w. this means that vi treatments may be considered a useful method to enrich apples with the bioactive compounds of aloe vera. of course, the high variability of the data (i.e. error bars) was caused by the different relaxation times as well as by the effects of the microstructure variability of fresh apple. fig. 5 shows the 3d plot describing the effects of pressure and relaxation time on the polymannans content of apple slices. the enrichment in polymannans was obtained for any experimental condition with values ranged between 1 and 8 mg/100 g f.w.. for any relaxation time, by increasing the vacuum level, it was possible to enrich apple slices with polymannans. an average increase of 7 mg/100 g f.w. was obtained decreasing the pressure, progressively, with a fixed relation time of 5 or 15 min. for a t2 of 10 min a peak of polymannans content was reached according to the non-linear effect reported in figure 4a. this means that a negative effect was observed for relaxation time longer than 10 min, probably because a prolonged compression damaged apple tissue favoring the release of aloe vera extracts from the capillaries. on these bases, further experiments, focused on the changes in void and solid matrix phases as well as in terms of microstructure, would be necessary to improve the understanding of the behavior of apple tissues under vi treatment and to optimize the enrichment in polymannans. figure 5. 3d plot describing the effect of relaxation time and pressure on the changes in polymannas content of apples slice submitted to vacuum impregnation treatments. 4. conclusions by vacuum impregnation is possible to obtain fresh-cut apple slices with improved healthy properties by filling their pores with an aloe vera gel extract. pressure and relaxation time significantly affected the porosity fraction and the polymannans content of apples, while any effect was not 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ital. j. food sci., vol 28, 2016 598 paper potential technological interest of indigenous lactic acid bacteria from algerian camel milk k. belkheir1, j.a. centeno2, h. zadi-karam1, n.-e. karam1 and j. carballo*2 1laboratoire de biologie des microorganismes et biotechnologie (lbmb), université ahmed ben bella d’oran 1, bp 1524, el m'naouer, 31000 oran, algérie 2food technology area, faculty of sciences, university of vigo, 32004 ourense, spain *corresponding author. tel.: +34 988387052; fax: +34 988387001 e-mail address: carbatec@uvigo.es abstract nine isolates of lactic acid bacteria (lab) obtained from the predominant microbiota of different camel milk samples collected in south-west algeria, were selected in accordance with their growth ability in (cow) milk. the isolates were phenotypically and genotypically assigned to the following species: 4 leuconostoc mesenteroides subsp. dextranicum; 2 lactobacillus brevis; 2 lb. plantarum; and 1 lactococcus lactis subsp. lactis. one isolate from each of the leuconostoc and lactobacillus species were selected on the basis of their highest proteolytic and aminopeptidase activities. the selected isolates were used in combination with a commercial mesophilic o-type culture to make fermented milks. sulfury flavor was detected as predominant in the sensory analysis of the milks made with only the ln. mesenteroides and lb. brevis adjuncts, which were characterized by the highest abundances of sulfur volatile compounds. butter flavor was perceived in the milks made with the lb. plantarum(cit+) adjunct, and was related to the presence of acetoin. finally, cheese flavor prevailed in the milks made with both the lb. brevis and the lb. plantarum adjuncts, characterized by their high contents of short-chain free fatty acids. the results suggest the potential interest of these microorganisms in the manufacture of dairy products, particularly the combination of the lb. brevis and lb. plantarum isolates for cheese making. keywords: camel milk, lactic acid bacteria, lactobacillus brevis, lactobacillus plantarum, sensory analysis, volatile compounds ! ital. j. food sci., vol 28, 2016 599 1. introduction one of the main challenges of modern dairy industry is the development of a range of varied products to meet the needs and tastes of the various sectors of an increasingly numerous and demanding population. the request for novel cultured products development having different and improved sensorial attributes requires the use of microbial strains with interesting properties for application in dairy fermentations (wouters et al., 2002). the strict hygiene measures and the high standardization of the production systems make the milk and dairy cultured products manufactured in the developed countries a poor source of novel and distinctive lab strains. so, novel strains should be searched in little standardized raw materials and products, and in ecological niches with particular and unusual environmental conditions. camel (camelus dromedarius) milk, besides being produced in traditional low-tech systems, has compositional characteristics that make it very special milk with clearly different environmental conditions (farah, 1993; konuspayeva et al., 2009) that may influence the metabolic abilities of the microbiota that settles it. the majority of the studies carried out on camel milk in the world, and particularly in algeria focuses on the problem of its low clotting ability, which has been widely investigated (farah and bachman, 1987; boudjenah-haroun et al., 2012). however, few works have been conducted regarding the microbiota of camelus dromedarius milk or the biochemical and technological properties of lab isolated from this source (ashmaig et al., 2009; drici et al., 2010; bendimerad et al., 2012; akhmetsadykova et al., 2015). in this context, the aims of the present study were: (i) to identify lab isolates with potential technological interest belonging to the predominant microbiota of algerian camel milk, and (ii) to characterize and to assay the isolates with a view to their use as adjunct cultures in the manufacture of fermented milks and cheeses with differentiated sensory characteristics. 2. materials and methods 2.1. bacterial cultures, media, and growth conditions nine lab isolates obtained on ph 5.5 mrs medium (oxoid ltd., basingstoke, uk) from six different camel milk samples collected in bechar and tindouf cities (southwest algeria) were selected for this study. the selected isolates were able to clot cow milk after 12-24 h incubation at 30ºc. the bacterial cultures were maintained at –30ºc in mrs broth containing 20% glycerol (v/v). working cultures were prepared by two consecutive transfers in mrs broth at 30 ºc. lyophilized commercial mesophilic o-type culture fddvs r-704 (chr. hansen, denmark), containing lactococcus lactis subsp. lactis and lc. lactis subsp. cremoris (phage-resistant) strains was used alone or in combination with selected indigenous lab cultures for the preparation of fermented milks. this culture was stored at –20ºc and was directly inoculated in milk following the manufacturer’s instructions. 2.2. phenotypic identification of lab isolates isolates were phenotypically assigned to the genus level as described by garabal et al. (2008). utilization of citrate was also determined on citrate calcium agar (kca) (nickels and leesment, 1964), after 72 h incubation at 30ºc. criteria followed for phenotypic identification were those compiled by wood and holzapfel (1995). assignation to species level was made by means of the api 50 chl carbohydrate fermentation strips ! ital. j. food sci., vol 28, 2016 600 (biomérieux, marcy l’etoile, france), following the manufacturer’s instructions. the results at 48 h were analyzed using the api ch lab software package (biomérieux). 2.3. genotypic identification of lab isolates for molecular identification of (presumptive) leuconostocs and lactobacilli isolates, chromosomal dna from bacterial isolates was extracted from single-colony by using the speedtools dna extraction kit (biotools b&m labs, s.a., madrid, spain) following the manufacturer’s instructions. from the obtained dna, 20 !l aliquots were made and stored at – 20ºc until needed. all dna samples were tested using universal primers amplifying a 1000-bp region of the 16s rrna gene 616v (forward): 5’-agagtttgatymtggctc ag-3’ and 699r (reverse): 5’-rgggttgcgctcgtt-3’ (arahal et al., 2008). the primers were synthesized by invitrogen co. (invitrogen, carlsbad, ca, usa), diluted at a final concentration of 1 !g/!l with sterilized deionized water upon reception and stored at –20ºc. pcrs were performed in a reaction volume of 50 !l, containing 20 pm each of forward and reverse primers and 1 ml dna template and prepared by using the dreamtaq dna polymerase kit (thermo scientific, waltham, ma, usa). pcr amplifications were monitored in a gene amp thermal pcr system 2700 thermal cycler (applied biosystems, foster city, ca, usa) under the following conditions: an initial cycle of 95ºc for 15 min; then 35 cycles of 95ºc for 30 s, 55ºc for 30 s, 72ºc for 1 min; and a final elongation step of 72ºc for 10 min. the pcr products were purified using the nucleospin® gel and pcr clean-up kit (macherey-nagel gmbh & co. kg, düren, germany). dna sequencing was carried out with the v3.1 bigdye terminator cycle sequencing kit (applied biosystems) on the abi 3130xl capillary automate sequencer (applied biosystems) following the manufacturer’s instructions. the obtained sequences were aligned to 16s rrna gene sequences in the gen bank ncbi data base (national center for biotechnology information, http://www.ncbi.nlm.nih.gov) using the blast program (http://www.ncbi.nlm.nih.gov/blast) database. 2.4. technological characterization of the selected lab isolates leuconostocs and lactobacilli isolates were characterized for the following abilities: (a) carbohydrate fermentation, using the miniaturized api 50chl system (biomérieux), after 48 h incubation at 30ºc; (b) acidifying activity in sterile (110ºc, 15 min) reconstituted (10%, w/v) skim milk (ph 6.70) (oxoid) after 6 h incubation at 30ºc (idf, 1995); (c) proteolytic activity in sterile reconstituted skim milk (oxoid) after 24 h incubation at 30ºc, as evaluated by the opa method (church et al. 1983); (d) aminopeptidase (ap) activity in sterile reconstituted skim milk (oxoid) using leucineand lysine-p-nitroanilide (both reagents from sigma-aldrich corp., st. louis, mo, usa) as substrates (el soda and desmazeaud, 1982); (e) diacetyl-acetoin production in sterile reconstituted skim milk (oxoid) after 48 h incubation at 30ºc (idf 1997); (f) extracellular proteolytic activity on calcium caseinate agar (merck gmbh, darmstadt, germany) (rodríguez-alonso et al., 2008); (g) extracellular lipolytic activity on 1% (w/v) tributyrin agar (merck), containing 1% arabic gum (panreac) (rodríguez-alonso et al., 2008); and (h) antimicrobial activity of the nine isolates against each other and against the commercial culture r-704 (chr. hansen, hørsholm, denmark), determined by the agar well diffusion assay as described by centeno et al. (2002). all assays for testing technological abilities were performed in triplicate, and numerical results were expressed as the mean values obtained for each isolate. ! ital. j. food sci., vol 28, 2016 601 2.5. preparation of fermented milks eight fermented or acidified milks were prepared per assay. one of the milks was acidified with the mesophilic commercial culture r-704 alone, and the other seven milks were fermented with the commercial culture plus one among three selected isolates (leuconostoc mesenteroides subsp. mesenteroides c8m, lactobacillus brevis c21b, lactobacillus plantarum c22p), or their combinations (c8m+c21b, c8m+c22p, c21b+c22p, and c8m+c21b+c22p). fermented milks were prepared in 1 l of retail pasteurized (80ºc, 20 s) homogenized whole (3.6 g fat 100 ml-1) milk (leyma, a coruña, spain) contained in polyethylene bottles. the milks used for each assay corresponded to the same industrial batch and had been pasteurized the day before purchasing. the bottles were inoculated with 0.1 units of the dvs r-704 commercial culture. the dvs culture was previously rehydrated and strongly stirred in sterile (reconstituted) skim milk (oxoid) at a ratio of 10 units per liter of milk, and then inoculated at 1% (v/v) in the pasteurized milk. the lab isolates were cultured in sterile skim milk (oxoid) at 30ºc for 16 h, and then inoculated at 1% (v/v) in the pasteurized milk. the inoculated milks were incubated at 30ºc for 24 h. assays were made in triplicate. 2.6. sensory analysis and volatile compounds produced in the fermented milks for sensory analysis, the (clotted) fermented milks were intensely agitated and then distributed in 50 ml amounts in sterile polyethylene containers. flavors of the fermented milks were perceived by smelling and tasting, as evaluated by a panel of ten regular consumers of fermented milks who had been trained as previously described (rodríguez-alonso et al., 2008). the judges were asked to agitate the milks before smelling and tasting. odor and taste preference as well as overall acceptance were scored on a scale from 1 to 7. the acceptability indexes (ai) of the fermented milks were calculated by the formula: overall acceptance × 100/7 (dutcosky, 1996). volatile compounds were determined in 20 ml samples taken after agitation of the clotted milks, and stored at –80 ºc until analysis. volatile compounds were extracted by using the solid phase microextraction (spme) technique and detected by gas chromatography coupled to mass spectrometry (gc–ms). for samples equilibration, 10 ml headspace vials containing 1 g of fermented milk were sealed with a ptfe-faced silicone septum (supelco, bellefonte, pa, usa) and maintained at 35ºc in a thermo block (memmert model 100-800, schwabach, germany) during 15 min. then, a 75 µm film thickness carboxen/polydimethylsiloxane (car/pdms) fibre (supelco) was exposed to the headspace while maintaining the sample at 35°c during 30 min. the spme adsorbed compounds were injected to the chromatograph with a splitless mode injection at 260ºc for 8 min. the separation of volatiles was performed on a hewlett-packard 6890n (agilent technologies, santa clara, ca, usa) gas chromatograph equipped with a db-624 capillary column (30m × 0.25 mm id, 1.4 µm film thickness; j&w scientific, folsom, ca, usa), following the method described by lorenzo and fonseca (2014). compounds were identified by comparing their mass spectra with those contained in the nist05 (national institute of standards and technology, gaithersburg, md, usa) library, and by comparing their mass spectra and retention time with authentic standards (supelco). abundances of volatile compounds are provided as peak area units/106 values. 2.7 statistical analysis numerical data corresponding to technological characteristics, sensory analysis and volatile compounds were subjected to analysis of variance (anova) and where statistical ! ital. j. food sci., vol 28, 2016 602 differences were noted, differences among the distinct groups (isolates or fermented milks) were determined by the duncan’s test at a significance level of p < 0.05. all statistical procedures were performed with the spss software, version 20.0 (spss inc., chicago, il, usa). 3. results and discussions 3.1. isolation and identification of lab isolates low quantities (mean value of 2.50 log cfu ml-1) of lab were found in the six samples of camel milk used for lab isolation (data not shown). this fact may be attributed to the high content of lysozyme and ascorbic acid in camel milk (farah, 1993). among the 45 lab isolates initially obtained, 27 (60%) were phenotypically assigned to leuconostoc/weissella genus (data not shown). similarly to the results of this study leuconostocs have often been found as the major lab in camel milk (ashmaig et al., 2009; akhmetsadykova et al., 2015), this may be attributed to the higher resistance to lysozime of this microbial group as compared to other lab (limonet et al., 2004). as regards the remaining 18 isolates, eight were identified as mesophilic lactobacilli, one was assigned to lactococcus genus, and nine lab isolates (unable to clot cow milk) could not be assigned to genus level (data not shown). the selected nine isolates able to clot (cow) milk after 12-24 h incubation at 30ºc were identified by means of the api 50 chl system as follows: four leuconostoc mesenteroides subsp. dextranicum (similarity level of 95.2-99.8%); two lactobacillus brevis (99.7% similarity); two lactobacillus plantarum (77.8-79.3% similarity); and one lactococcus lactis subsp. lactis (72.3% similarity) (data not shown). both the lb. plantarum and the lc. lactis subsp. lactis isolates were able to metabolize citrate on kca medium (data not shown). the low proportion of leuconostoc isolates showing the ability to coagulate milk may be explained by the fact that these microorganisms are adapted to growth on vegetables and roots and therefore lack sufficient proteolytic ability to grow in milk (vedamuthu, 1994). the lactococcal isolate was not further considered in this study because of the known susceptibility of lactococcal cells to bacteriophage infection, as lytic phage infection is at present a major cause of fermentation failure (garneau and moineau, 2011). phenotypic identification of the selected (presumptive) leuconostocs and lactobacilli isolates was confirmed by sequencing of the fragments of the 16s rdna gen amplified by pcr (data not shown). sequences obtained from the four presumptive ln. mesenteroides isolates produced significant (97-99%) alignments with the complete genome of ln. mesenteroides subsp. dextranicum dsm 20484. sequences from the two presumptive lb. brevis isolates aligned (97%) with that of lb. brevis atcc 367. lower percentages of similarities (91-94%) were found between the presumptive lb. plantarum isolates and the lb. plantarum jdm1 strain (genomes of all the type strains included in the ncbi data base). similarly to the results of this study, the species lb. plantarum and lb. brevis have been isolated from sudanese fermented camel milk (ashmaig et al., 2009), and lc. lactis subsp. lactiscit+ has been found in algerian fermented camel milk (drici et al., 2010; bendimerad et al., 2012). 3.2. technological properties of the selected leuconostocs and lactobacilli isolates the most relevant carbohydrate fermentation abilities exhibited by the selected lab strains are shown in table 1. despite of their ability to hydrolyze lactose in the api 50 chl test tube, all the selected isolates (ln. mesenteroides subsp. dextranicum c2m, c5m, ! ital. j. food sci., vol 28, 2016 603 c8m and c14m; lb. brevis c21b and c27b; and lb. plantarum c14p and c22p) showed low acidifying activities (mean ph values of 6.42-6.65, and mean titratable acidities of 0.06-0.21 g lactic acid 100 ml-1) after 6 h incubation at 30ºc (table 1). the ph values of the milk cultures of the lactobacilli isolates were significantly (p < 0.05) lower than those of the leuconostocs isolates. the lowest mean ph values corresponded to the two isolates of lb. brevis (table 1). on the basis of their low acidifying activities, lb. brevis, lb. plantarum and ln. mesenteroides have been generally classified as adjunct lab to be used together with a lactococcal starter in order to enhance the acidification of milk (settanni and moschetti, 2010). the proteolytic activities evaluated by means of the opa test ranged between 0.124 lysine mm for one lb. brevis isolate and 0.230 lysine mm for one ln. mesenteroides isolate (table 1). these values are much lower than those reported by herreros et al. (2003) for 17 isolates of leuconostoc and mesophilic lactobacilli obtained from raw goat’s milk cheeses (mean value equivalent to 0.296 lysine mm) and by garabal et al. (2008) for 42 isolates of mesophilic lactobacilli from raw cow’s milk cheeses (mean value equivalent to 0.298 lysine mm). nevertheless, this low proteolytic activity could be a suitable trait if these bacteria are used as adjunct cultures, in order to prevent development of bitterness in the final product (nandan et al., 2010). all the leuconostocs and lactobacilli isolates exhibited both leuand lys-aminopeptidase activities, with higher lys-ap than leu-ap values (table 1). differences among species were pronounced, with the two lb. brevis isolates showing significant (p < 0.05) higher values for both activities (highest mean values of 200 u for leu-ap and 530 u for lys-ap) than the lb. plantarum and ln. mesenteroides isolates. values obtained for the lb. plantarum isolates were also significantly (p < 0.05) higher than those of the ln. mesenteroides isolates (table 1). high leuand lys-aminopeptidase activities have been detected for cell-free extracts of some strains of lb. brevis, lb. plantarum and ln. mesenteroides isolated from raw ewe’s and goat’s milk cheeses (macedo et al., 2000; herreros et al., 2003). similarly to the results of the present study, nieto-arribas et al. (2009) found low leuand lys-ap activities for intact cells of 19 ln. mesenteroides subsp. dextranicum isolates obtained from artisanal manchego ewe’s cheese, with lys-ap activity being higher than leu-ap activity for all the isolates. the same authors (nieto-arribas et al., 2010) reported higher values of both leu-ap and lys-ap activities for most of 10 lb. plantarum isolates than those of ln. mesenteroides isolates from the same cheese variety. aminopeptidases play a key role in the degradation of bitter peptides and flavor formation during cheese ripening (urbach et al., 1995; nandan et al., 2010). only the two lb. plantarum isolates able to metabolize citrate on kca produced diacetylacetoin in skim milk (table 1). the production of diacetyl-acetoin by lb. plantarum c22p (120 mg diacetyl l-1) was significantly (p < 0.05) higher than those observed for lb. plantarum c14p (45 mg l-1) and for the initially selected lc. lactis subsp. lactis(cit+) isolate (35 mg l-1, data not shown). garabal et al. (2008) reported a mean production equivalent to 84 mg diacetyl l-1 for 33 isolates of mesophilic facultatively heterofermentative lactobacilli obtained from galician (northwest spain) raw cow’s milk cheeses. production of diacetyl by lab could be considered an interesting technological property since this compound is related to positive (butter) flavor and antimicrobial effect in dairy products (drici et al., 2010). none of the isolates exhibited extracellular proteolytic or lipolytic activities in spite of their growth on calcium caseinate agar and on tributyrin agar media (data not shown). ! ital. j. food sci., vol 28, 2016 604 table 1: technological characterization of the selected lab isolates obtained from camel milk (quantitative results are means of three replicates). leuconostoc mesenteroides lactobacillus brevis lb. plantarum s.e.m. 1 p value fermentation of: c2m c5m c8m c14m c21b c27b c22p c14p larabinose – – – – + + + + d-cellobiose – – – – – – + + dfructose + + + + + + + + d-galactose + + – – + + + + d-glucose + + + + + + + + d-lactose + + + w2 + + + + d-maltose + + + + + + + + d-mannose + + + + – – + + d-melibiose – – – – + + + + d-raffinose – – – – – – – – l-rhamnose – – – – – – – – d-ribose w – w w + + + + d-saccharose + + + + – w + + l-sorbose – – – – – – – – d-trehalose + + + + – – + + d-xylose w w w w + + – – acidifying activity (skim milk; 30 ºc, 6 h) ph 6.60b 6.65a 6.63a 6.65a 6.42d 6.45cd 6.48c 6.50c 0.003 0.000 titratable acidity3 0.10 bc 0.06d 0.08cd 0.09c 0.21a 0.21a 0.14b 0.12b 0.0002 0.000 proteolytic activity (opa)4 0.210 ab 0.212ab 0.230a 0.189b 0.126d 0.124d 0.180bc 0.178c 0.009 0.012 aminopeptidase activity5 leu-ap 17.2d 14.6d 24.0d 19.4d 200a 197a 109b 51c 66 0.000 lys-ap 73c 68c 73c 71c 530a 528a 129b 134b 161 0.000 producton of diacetylacetoin6 0.0c 0.0c 0.0c 0.0c 0.0c 0.0c 120a 45b 6.9 0.000 a-dmean values within a row with different superscripts are significantly different (p < 0.05; duncan’s test) 1s.e.m.: standard error of the mean 2w: weak reaction 3expressed as g lactic acid 100 ml-1 4expressed as mm lysine 5expressed as enzymatic units (1 enzymatic unit = amount of enzyme giving an absorbance of 0.001 units at 410 nm min-1) 6expressed as mg of diacetyl per liter of milk ! ital. j. food sci., vol 28, 2016 605 the absence of extracellular enzymatic activities in lab other than enterococci has been generally reported (herrero et al., 1996; buffa et al., 2004; nieto-arribas et al., 2009, 2010). according to herrero et al. (1996), lab used as starter cultures should ideally present low lipolytic activity, as the degradation of milk fat must be slight in order to induce aroma production without giving rancid flavors. one isolate from each one of the identified species (ln. mesenteroides subsp. dextranicum c8m; lb. brevis c21b; and lb. plantarum c22p) were selected in accordance with their expected better technological performance (highest proteolytic and ap activities, and production of diacetyl for lb. plantarum isolates). it should be pointed out that the similarity in the results of biochemical and genotypic (data not shown), and technological assays for lb. brevis c21b and c27b could indicate that these two isolates probably belong to the same strain, although they were obtained from different camel milk samples (data not shown). the selected isolates were used to make fermented milks in combination with the commercial culture r-704 (chr. hansen) after confirming the absence of antibacterial activity against each other and against the commercial culture (data not shown). 3.3. sensory analysis and volatile compounds produced in the fermented milks the milks fermented with the commercial culture alone (control) and those cultured with the commercial starter in combination with both ln. mesenteroides c8m and lb. plantarum c22p, and in combination with the three selected isolates received the highest scores for sensory attributes (table 2). the scores reached for the odor attribute were significantly (p < 0.05) higher for these last two fermented milks than for the other milks, and the scores for the taste attribute were significantly (p < 0.05) higher for the milk fermented with both the c8m and c22p adjuncts than for the other cultured milks. the highest acceptability index (86.1%) corresponded to the milk cultured with both the c8m and c22p isolates, followed by the milk fermented with the three selected isolates (76.7%) and by the control milk (74.5%) (table 2). table 2: mean sensory scores and main flavor descriptors of the fermented milks made with the commercial fd-dvs r-704 o-type starter alone (control) and in combination with cultures of the selected isolates obtained from camel milk. control +c8m +c21b +c22p +c8m +c21b +c8m +c22p +c21b +c22p +c8m +c21b +c22p s.e.m.1 p value odor 5.0b 4.8bc 4.7bc 4.9bc 4.4c 5.8a 4.8bc 5.7a 0.060 0.039 taste 5.3b 3.9d 3.8d 4.8bc 5.3b 6.2a 5.0bc 5.0bc 0.087 0.000 main flavor descriptors acid yoghurt sulfury metallic sulfury garlic butter vanilla sulfury sour butter yoghurt cheese butter cheese butter ai (%)2 74.5bc 62.8e 61.5e 70.1d 70.2d 86.1a 71.7cd 76.7b 1.9 0.000 a-emean values within a row with different superscripts are significantly different (p < 0.05; duncan’s test) 1s.e.m.: standard error of the mean 2ai (%): acceptability index of the fermented milk calculated by the formula: overall acceptance × 100/7 (in accordance with the used 7-point scale) the flavor descriptors used by the majority of the judges include: acid and yoghurt for the control milk; butter and yoghurt for the milk fermented with both the c8m and c22p adjuncts; and butter and cheese for the milk cultured with the three selected isolates (table ! ital. j. food sci., vol 28, 2016 606 2). the butter and vanilla notes described for the milks made with the lb. plantarum c22p adjunct may be related to the production of diacetyl and acetoin by this culture. these compounds are responsible for butter and nuts flavors in cheese (kondyli et al., 2003; kaminarides et al., 2007). sulfury and garlic nuances were detected by most of the judges in the milk fermented with the sole lb. brevis c21b adjunct, which showed the lowest ai (61.5%) (table 2). the sulfur flavors are related to the production of sulfur compounds principally from l-methionine (met). it has been reported that sulfur compounds provide particular flavor notes in cheese such as garlic taste (kaminarides et al., 2007). the milks fermented with the c21b adjunct in combination with either the c8m or the c22p isolates received significant (p < 0.05) higher scores for the taste attribute than the milk made with the c21b isolate as the only adjunct culture (table 2). this suggests that the “sulfury defect” was moderated due to the presence of other flavor compounds. only ten volatile compounds were definitely identified in the fermented milks with the methodology used in the present study. these volatiles include: the alcohol 2-heptanol; the ketones 3-hydroxy 2-butanone (acetoin), 2-heptanone and 2-nonanone; the fatty acids acetic acid, butanoic acid, hexanoic acid and octanoic acid; and the sulfur compounds dimethyl disulfide (dmds) and dimethyl trisulfide (dmts) (table 3). table 3: mean abundances (expressed as peak area units / 106) of each of the volatile compounds determined in the fermented milks made with the commercial fd-dvs r-704 o-type starter alone (control) and in combination with cultures of the selected isolates obtained from camel milk. control +c8m +c21b +c22p +c8m +c21b +c8m +c22p +c21b +c22p +c8m +c21b +c22p s.e.m.1 p value alcohols 2-heptanol ndd2 4.1c 5.2c 14.0a 8.6b 14.2a 10.8ab 9.8ab 0.67 0.003 ketones 3-hydroxy 2butanone nd d ndd ndd 93a ndd 20.1b ndd 5.4c 21.8 0.031 2-heptanone 1.1b 2.8b 1.5b 29.3a 0.8b 28.2a 4.8b 4.7b 3.06 0.001 2-nonanone ndd 1.3b 0.4c 3.1a ndd 2.8a ndd ndd 0.39 0.043 fatty acids acetic acid 128e 239c 149de 329a 160d 265bc 131e 281b 18.9 0.005 butanoic acid 3.8e 5.8d 9.7bc 9.2bc 8.1c 7.4c 15.4a 10.7b 0.66 0.006 hexanoic acid ndf 2.7e 7.3bc 6.5c 5.6d 6.7c 12.7a 7.8b 0.56 0.004 octanoic acid ndc ndc 0.8ab 0.5b 0.5b 0.5b 1.2a 1.1a 0.073 0.019 sulfur compounds dimethyl disulfide 0.7cd 2.0a 2.3a 1.3b 2.1a 1.0bc 0.5d 0.6d 0.21 0.011 dimethyl trisulfide ndb ndb 0.3a ndb 0.2a ndb ndb ndb 0.01 0.001 a-fmean values within a row with different superscripts are significantly different (p < 0.05; duncan’s test) 1s.e.m.: standard error of the mean 2nd: compounds not detected (considered as 0.0 values for statistical analysis) ! ital. j. food sci., vol 28, 2016 607 the poor volatile profiles of the fermented milks, with the absence of aldehydes and the practical absence of alcohols, may be partially explained on the basis of the spme fiber performance. it has been reported that the porous car/pdms fiber coating shows a higher affinitiy for low-molecular-weight compounds, including a high proportion of ketones (lorenzo, 2014). in addition, absorption for the most compounds may reach equilibrium with a longer time of exposure than that assayed in this work (marco et al., 2004), although longer exposure times could result inappropriate due to oxidation phenomena that can produce competitive effects between compounds (lorenzo, 2014). the secondary alcohol 2-heptanol, and its probable precursor 2-heptanone were present in all the fermented milks made with the adjunct cultures, but not in the control milk. the highest abundances of (2-heptanone and) 2-heptanol were found in the milk fermented with the lb. plantarum c22p adjunct. the abundances of 2-heptanol in the milks made with the sole c22p adjunct and with the combination of the c8m and c22p adjuncts were significantly (p < 0.05) higher than those of the milks made without the c22p adjunct culture. this compound, associated to herbaceous and fruity aromas has been considered as a key odorant in many cheese varieties (delgado et al., 2011). regarding the ketones group, 3-hydroxy 2-butanone (acetoin) was only detected in the milks cultured with the lb. plantarum c22p adjunct (table 3), as it could be expected from the results of the technological assays. acetoin is produced by the reduction of diacetyl originated from citrate metabolism, or by decarboxylation of α-acetolactate (mcsweeney et al., 2000). diacetyl, and to a lesser extent acetoin are generally appreciated for their buttery and nut-like notes (curioni et al., 2002), and may be responsible to some extent for the butter flavors described in the sensory analysis. the abundances of the methyl ketones 2-heptanone and 2-nonanone were significantly (p < 0.05) higher in the milks made with the sole lb. plantarum c22p adjunct culture and with the combination of the c8m and c22p adjuncts than in the other fermented milks (table 3). the methyl ketones 2heptanone and 2-nonanone are associated with cheesy odors, particularly with blue cheese notes (curioni et al., 2002). methyl ketones containing odd numbers of c atoms are produced from β-oxidation of fatty acids (mcsweeney et al., 2000), which may have been released through the esterase and/or lipase activities of the lactobacilli. the four fatty acids identified were detected in all the fermented milks made with the adjunct cultures, with the exception of octanoic acid in the milk fermented with the sole c8m leuconostoc adjunct. the abundances of acetic acid were significantly (p < 0.05) higher in the milks made with the sole lb. plantarum c22p adjunct culture than in the other fermented milks (table 3). citrate-fermenting microorganisms convert this compound to pyruvate, carbon dioxide, and acetic acid, together with various carbonyl compounds such as diacetyl and acetoin. facultatively heterofermentative lactobacilli such as lb. plantarum may also produce acetate from lactose or from amino acids (buffa et al., 2004). acetate is primarily formed from citrate and this may be the main reason why this compound is more abundant in the milks fermented with the lb. plantarum c22p adjunct. acetate also comes from pyruvate originated in the glycolysis from carbohydrates, and this would explain its presence in milks fermented with adjuncts unable to metabolize citrate. acetic acid is responsible for sour flavor in dairy products (kaminarides et al., 2007). the abundances of butanoic and hexanoic acids were significantly (p < 0.05) higher in the milks made with the combination of lb. brevis c21b and lb. plantarum c22p adjunct cultures than in the other fermented milks (table 3). menéndez et al. (2000) found higher volatile free fatty acids contents (3.05 vs. 1.33 meq/100 g) in 1-day samples of cow’s cheeses made with a lb. plantarum adjunct culture than in control samples. butanoic acid may be derived from lipolysis of milk fat or produced through the fermentation of lactose and lactic acid (kaminarides et al., 2007). however, and despite the fact that the strains tested in the present study did not exhibit (exocellular) lipolytic activity when growing on ! ital. j. food sci., vol 28, 2016 608 tributyrin agar, hexanoic and octanoic acids are probably released from triglycerides through the action of non-specific bacterial esterases and lipases as reported by gonzález de llano et al. (1996) and buffa et al. (2004). short-chain fatty acids are related to rancid and pungent flavors (mcsweeney et al., 2000; delgado et al., 2011), and butanoic acid has been found to be a potent odorant in cheese (kaminarides et al., 2007). the high contents of butanoic and hexanoic acids may relate to the cheese flavors detected in the milks made with both the c21b and c22p adjuncts. as regards sulfur compounds, the abundances of dmds were significantly (p < 0.05) higher in the milks made with the ln. mesenteroides c8m and the lb. brevis c21b adjuncts, both alone or in combination, than in the other fermented milks (table 3). dmts was only detected in the milks made with the sole c21b adjunct and with the combination c8m+c21b. the thioesters dmds and dmts are expected to be formed mainly from the amino acid met by the metabolism of lab and secondary microbiota (urbach, 1995; engels et al., 1997). it has been reported that a number of lactobacilli strains, including lb. brevis are able to degrade met (engels et al., 1997; sreekumar et al., 2009) which agrees with our findings regarding the lb. brevis c21b adjunct assayed in the present study. the sulfury flavors detected in the sensory analysis of milks made with the ln. mesenteroides c8m and the lb. brevis c21b adjuncts, and the garlic nuance described in the milk made with the sole c21b adjunct culture may be attributed to the high contents of sulfur compounds. 4. conclusions the selected leuconostocs and lactobacilli isolates from camel milk used in the present study as adjunct cultures to manufacture fermented milks in combination with an acidifying starter, conferred different flavor nuances and were responsible for different volatile profiles of the products. sulfury flavor was detected as a main flavor in the milks made with only the ln. mesenteroides and lb. brevis adjuncts, characterized by the highest abundances of volatile sulfur compounds. butter flavor, related to the presence of the volatile 3-hydroxy 2-butanone (acetoin) was perceived in the milks made with the lb. plantarum(cit+) adjunct, and cheese flavor, associated to high contents of volatile free fatty acids prevailed in the milks made with both the lb. brevis and the lb. plantarum adjuncts. the results suggest the potential interest of these lab isolates in the manufacture of dairy products, in particular the combination of the lb. brevis and lb. plantarum isolates for cheese making. acknowledgements authors are grateful to dr. josé manuel lorenzo (food technology center of galicia, ourense, spain) for assistance with spme–gc–ms methodology. this study was financially supported by the government of algeria, within the program national exceptional (pne). k. belkheir acknowledge receive a research fellowship from the algerian ministry of high studies and scientific research, and the university of relizane during these studies. this work was also partially supported by the xunta de galicia (the spanish regional government) under the consolidation and 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use of electronic nose to discriminate meats from bulls fed diet with or without flaxseed inclusion and subjected to different aging periods m. moschini*a, s. sigoloa, a. brugiapagliab, m. rennab, c. lussianab and a. prandinia adepartment of animal science, food and nutrition (diana), università cattolica del sacro cuore, via emilia parmense 84, 29122 piacenza, italy bdepartment of agricultural, forest and food sciences (disafa), università di torino, largo paolo braccini 2, 10095 grugliasco, torino, italy *corresponding author: tel.: +39 0523599192; fax: +39 0523599259 e-mail address: maurizio.moschini@unicatt.it abstract a metal-oxide sensors array electronic nose (e-nose) was used to discriminate beef loins (longissimus thoracis) obtained from piemontese bulls fed without or with flaxseed and subjected to 3 different aging periods (2, 7, 10 days) at 4°c. at 7 days of aging, samples were also assessed for flavor intensity by panelists. a comparison between e-nose and panel assessments was performed subjecting a 7 days e-nose reading on cooked meat to partial least square regression for flavor prediction. the e-nose could not discriminate populations in meat samples, however it could represent a valuable tool in supporting flavor scoring from sensory evaluation. keywords: aging, beef meat, electronic nose, flaxseed, sensory evaluation ital. j. food sci., vol. 30, 2018 441 1. introduction red meat has been addressed has having high content of saturated fatty acids (sfa). several studies have shown a positive relationship between dietary sfa and the onset and development of several widespread human pathologies, such as cardiovascular diseases and various forms of cancer (boada et al., 2016). in response to consumers demand, in recent years different feeding strategies aiming at reducing sfa and contemporarily at increasing polyunsaturated fatty acids (pufa), particularly omega-3 fatty acids, in ruminant-derived food products have been developed (shingfield et al., 2013). omega3 pufa bring numerous beneficial effects on human health as they favor normal embryogenesis and brain development, and protect against cancer, cardiovascular and neurodegenerative diseases (calder, 2013). flaxseed, one of the richest natural sources of α-linolenic acid (c18:3 n-3), has been shown to be an effective feed ingredient in increasing the content of omega-3 pufa in beef (juárez et al., 2011). however, an increase of highly unsaturated fats may pose to alterations of meat flavor, mainly because of the derived greater susceptibility to oxidative breakdown (juárez et al., 2012). moreover, when the proportion of c18:3 n-3 approaches 3% of muscle fatty acids, flavor liking scores assessed by human panelists can be significantly altered, even in case of only slight decreases of lipid oxidative stability (wood et al., 2008). therefore, when applying feeding strategies to increase the omega-3 pufa content of meat, the associated investigation of meat flavor is determinant. flavor is a very complex attribute of meat palatability; it chemically acts on taste and smell receptors, and plays a key role in acceptability by consumers (khan et al., 2015). meat flavor has traditionally been evaluated either by trained assessors or by head-space gas chromatography or mass spectrometry, these methods being time-consuming, labor-intensive and costly, particularly for routine quality control application. the development of objective automated non-destructive techniques that can easily and rapidly characterize meat flavor is an impelling need for the meat industry (narsaiah and jha, 2012). chemical sensor systems (i.e., electronic noses) are technologies for the ator on-line discrimination of populations according to volatile compounds. these systems involve various types of electronic chemical gas sensors and with partial specificity which, combined to suitable statistical methods, allow for pattern recognition of simple or complex families of volatile chemical compounds (ghasemi-varnamkhasti et al., 2009). over the last twenty years, several studies have been carried out using the electronic nose (e-nose), as a rapid and non-destructive method, to assess meat quality (ghasemi-varnamkhasti et al., 2009; hong et al., 2012; loutfi et al., 2015). some studies also showed the potential of enose to aid or replace olfactory sensory analysis of meat performed by trained panelists (mildner-szkudlarz et al., 2007), but limited literature is currently available correlating e-nose response to flavor intensity assessed by sensory panels (loutfi et al., 2015). the aims of this study were: (i) to evaluate whether the e-nose could be used to discriminate meat beef loins (longissimus thoracis muscle; lm) samples obtained from bulls fed diets without or with flaxseed and aged for 2, 7 or 10 days, and (ii) to compare, at 7 days of aging, e-nose reading and sensory panel evaluation in the assessment of meat flavor intensity. ital. j. food sci., vol. 30, 2018 442 2. materials and methods 2.1. animals, dietary treatments and sampling procedures animal care and experimental procedures were carried out in compliance with european union legislation on the protection of animals used for scientific purposes (european parliament and the council of the european union, 2010). eighteen male calves of the piemontese breed (4.5 ± 0.59 months old; mean ± sd) were purchased from a local dealer and randomly allotted into two pens (9 animals/pen). animals had free access to fresh water and were fed for 172 days the same base diet (adaptation period) consisting of a commercial concentrate for fattening cattle, ryegrass hay, corn meal, distillers dried grains, dried beet pulp and soybean meal. the adaptation period was followed by a treatment period of 135 days during which pens were randomly assigned to two treatment diets: control or flaxseed diet. the amount of ground flaxseed (dry matter (dm): 917 g/kg; ether extract: 360 g/kg dm; α-linolenic acid: 200 g/kg dm) in the flaxseed diet was set at 100 g/kg dm. all diets (table 1) were formulated according to nrc (nrc, 2000) to fulfill the nutritional requirements of piemontese young bulls. at the end of the treatment period animals were slaughtered. a portion of the lm between the 8th and the 10th thoracic vertebra from the right side of the carcass was taken 24 h after slaughter and transferred under refrigerated conditions to the lab. then, the lm was cut into three 2 cm thick steaks. four equal subsamples were obtained from the first steak; each subsample was sealed in a commercial food grade polymer bag and kept for 2, 7 (two subsamples) or 10 days (d) in a controlled environment at 4°c, away from direct light, until e-nose headspace analysis. one subsample was aged for 7d at 4°c, then vacuum-packed and stored at -80°c until sensory evaluation by panelists. 2.2. analysis of feed aoac international (2000; 2003) procedures were used to determine dm, ash, crude protein (cp), ether extract (ee) in flaxseed and diets. feed chemical composition was expressed as g/kg dm. feed fatty acid (fa) composition was assessed as described by renna et al. (2014). feed fa results are reported as g/kg of total detected fa. the proximate and the main fa compositions of the diets are reported in table 1. 2.3. e-nose procedure a pen 3 portable electronic nose (airsense analytics gmbh, schwerin, germany) equipped with an array of 10 metal-oxide sensors (table 2) and a pattern recognition software for data recording and processing (winmuster, v. 1.6.2.13) was used. the meat subsamples were subjected to e-nose reading either as raw (2, 7 and 10d) or cooked (7d). the meat was cooked in a flask in a water-bath at 70°c for 30 minutes. before starting the e-nose assay a calibration procedure was carried out to account for variations in relative humidity of the air, temperature and possible drift of sensors over time. the air filtered through an active carbon filter was used as zero gas. at the end of the calibration procedure the sensors responses were recorded (g0). then, about 50 grams of raw or cooked meat were cut into 2 cm3 particles and put into a 250 ml flask equipped with teflon/silicon septum cup and let it stand for 30 minutes at 25°c to allow for a uniform distribution of gasses in the flask headspace before the e-nose analysis. upon analysis, a needle connected to the e-nose was used to perforate the septum of the flask containing the meat sample and air of the headspace was absorbed into the air detection chamber ital. j. food sci., vol. 30, 2018 443 with a flow rate of 400 ml/min. before each sample reading, the detection chamber was flushed for 330 s with reference air (air filtered through an active carbon filter) for sensors recovery. then, upon flowing of headspace sample air the sensors’ responses (g) were recorded once per second and for 60 s. the 60 s measurement interval was selected to allow sensors to reach a stable signal value. the sensor response to the substances in the headspace was defined by the conductance ratio g/g0. a g/g0 threshold value of 6 was set for the sensor number 2 in the array (w5s) through an automatic dilution system to protect the sensor array from overloading. table 1. ingredients, proximate composition and main fatty acid profile of the experimental diets fed in the treatment period. control flaxseed 333 258 254 294 160 102 121 151 78 61 0 100 35 14 ingredients (g kg-1 dm) concentrate a corn meal dried sugar beet pulp ryegrass hay barley meal ground flaxseed soybean meal concentrate b 22 23 86 87 173 170 38 75 66 60 proximate composition (g kg-1 dm) dm (%) cp ee ash net energy (mj kg-1dm) 7.92 8.19 173.20 109.20 22.60 24.90 260.65 240.95 492.50 319.15 main fatty acid composition (g kg-1 of tfa) c16:0 c18:0 c18:1 n-9 c18:2 n-6 c18:3 n-3 31.15 292.80 abbreviations: dm, dry matter; cp, crude protein; ee, ether extract; tfa, total fatty acids. concentrate a: corn, wheat middlings, sunflower meal, roasted dehulled soybean meal, wheat bran, roasted soybean meal, vitamins and minerals; concentrate b: corn germ meal, wheat middlings, wheat bran, corn, vitamins and minerals. ital. j. food sci., vol. 30, 2018 444 table 2. sensitivity and selectivity of the sensors in the portable electronic nose device (pen 3 portable electronic nose, airsense analytics gmbh, schwerin, germany). number in array sensor general description reference 1 w1c aromatic aromatic compounds toluene, 10 ppm 2 w5s broad range broad range sensitivity react on nitrogen oxides and ozone, very sensitive with negative signal no2, 1 ppm 3 w3c aromatic ammonia, used as sensor for aromatic compounds benzene, 10 ppm 4 w6s hydrogen mainly hydrogen, selectively (breath gases) h2, 100 ppb 5 w5c aromatic-aliphatic alkanes, aromatic compounds, less polar compounds propane, 1 ppm 6 w1s broad methane sensitive to methane (environment) ca. 10ppm, broad range, similar to w2s ch4, 100 ppm 7 w1w sulphur organic reacts on sulphur compounds (h2s 0,1ppm) otherwise sensitive to many terpenes and sulphur organic compounds, which are important for smell (limonene, pyrazine) h2s, 1 ppm 8 w2s broad alcohol detects alcohol’s, partially aromatic compounds, broad range co, 100 ppm 9 w2w sulphur-chlorine aromatic compounds, sulfur organic compounds h2s, 1 ppm 10 w3s methane-aliphatic reacts on high concentrations >100ppm sometimes very selective (methane) ch4, 100 ppm ital. j. food sci., vol. 30, 2018 445 2.4. sensory evaluation the steaks were placed in a refrigerator to thaw for 24 h at 4°c, then cooked without salt or spice addition in a double plate grill, preheated at 250°c, until the final internal temperature reached 70°c, which was monitored by individual thermocouples inserted into the geometric center of each steak (american meat science association (amsa), 1995). upon reaching 70°c, the steaks were trimmed of external connective tissue, cut into 1.3 x 1.3 x 2 cm samples, wrapped in a foil pouch and labeled with three-digit random numbers. a sensory quantitative affective test based on intensity scale (meilgaard et al., 2006) was performed by 39 males and 24 females consumers, ranging in age from 21 to 60 years old. panelists recruited for testing the samples, previously involved in surveys on beef preference/acceptance tests, were regular consumers of beef and had not diet restriction or allergies. samples from each treatment were randomly served one at a time to each panelist. five sessions with approximately 12 panelists per session were carried out in individual booths in a sensory testing laboratory under artificial white lighting. four samples (two per dietary treatment), each served 5 minutes apart, were offered to each consumer per session, for a total of 252 assessments over the five sessions. panelists evaluated beef flavor intensity using an unstructured scale, consisting of a 15 cm long horizontal line, with anchor points labeled with the expression "extremely bland" (0 cm) and "extremely intense" (15 cm) (meilgaard et al., 2006). the panelists expressed each evaluation by making a vertical line across the horizontal line at the point best reflecting their perception of the magnitude of flavor. the panelists were asked to rinse their mouth with still water served at room temperature during the one minute break imposed between consecutive samples. 2.5. statistical analysis all analyses were performed using sas (statistical analytical system (sas), 2003). significance was declared at p≤0.05. the e-nose measurement produced 60 readings for each sensor for a total of 600 readings for each sample. however, multiple readings from a sensor are correlated each other. therefore, among the sixty available readings from each sensor only one (59th) and in a plateau condition was considered for subsequent analysis, for a total of 10 values/sample. the mahalanobis distance (md) was used to calculate similarity between samples within classes (i.e. aging and diet). the clustering of samples was investigated based on the sensors activation patterns. at first, variables (w1c, w5s, w3c, w6s, w5c, w1s, w1w, w2s, w2w, w3s) were subjected to the stepdisc procedure and all of them were suited for entering the discriminant analysis. then, the dataset was subjected to the factor procedure for principal component analysis (pca) and the candisc procedure for canonical discriminant analysis (cda). sensory evaluation data were subjected to the glm procedure. the model included the diet as fixed effect whereas the panelist and diet x panelist interaction entered the model as random effects (naes et al., 2011). the relationship between e-nose data, either on raw or cooked samples, and sensory scores assessed by panelists was analyzed by partial least square regression (pls) for flavor prediction. ital. j. food sci., vol. 30, 2018 446 3. results and discussion 3.1. e-nose analysis when exposed to the headspace gas the sensor array produces a particular pattern in which each curve represents a different transient sensor response (fig. 1). the x-axis represents the time of reading and the y-axis the sensors’ ratio of conductance. for some sensors, the conductivity grows rapidly and then decreases to a stable condition whereas for some others the change in resistance, and therefore the g/g0 ratio is minimal or below the unity. only one point toward the end of sample measurement was considered for each sensor and their mean responses within classification groups (i.e. diet and aging) are shown in fig. 2. the polar plot suggests some sensors are more relevant than others in terms of signal response between control and flaxseed within the day of aging. sensors w1c, w3c, w5c, w1w, w2w and w3s differentiate between diets at 7d of aging (p<0.05). detailed information about sensors characteristics are not available in literature (smyth and cozzolino, 2013), as well as there are no reports that interrelate the response of a sensor to a particular chemical component. nevertheless, association between sensors and groups or families of substances are outlined (table 2) and from this we can address strongest reactive sensors at 7d (except broad range sensors: w1s, w5s) into three groups: sensors reactive to aromatic compounds (w1c, w3c and w5c; in which samples from flaxseed fed animals were in average 12% lower than control), sensors reactive to sulfur compounds (w1w, w2w; in which samples from flaxseed fed animals were in average 37% lower than control) and sensor w3s reactive to high concentration of methane in which samples from flaxseed fed animals were in average 10% higher than control. figure 1. example of electronic nose reading. the sensor gas response is expressed as g/g0 or g0/g (for sensors showing a negative behavior in presence of chemical compounds; w1c, w3c, w5c), where g and g0 represent the resistance of the sensor in sample gas and in zero gas air, respectively. ital. j. food sci., vol. 30, 2018 447 figure 2. polar plot of the average responses of sensors when exposed to raw meat samples at different aging (2, 7 or 10 days) and from animals fed control or flaxseed diet (n = 9). the gas response is expressed as the g/g0 ratio, where g and g0 represent the resistance of the sensor in sample gas and in zero gas air, respectively. values with superscript (*) and within the 7 days aging differentiate for p<0.05 the md classifies the observation into the nearest population by calculating the distance between the unit vector and the centroid for population. the md takes into account the correlation of the data within the cluster, it is unit less and it measures how many deviations is the value from the cluster centroid. the md enlarged with the increase of aging (table 3). table 3. mahalanobis distance of sensors (between class means). control flaxseed day 2 7 10 2 7 10 2 0 9.176 14.909 0 4.914 12.117 7 9.176 0 7.344 4.914 0 8.714 10 14.909 7.344 0 12.117 8.714 0 as previously reported (hong et al., 2012) also in the present work the e-nose seems capable of suggesting divergences between samples kept for different length of time at 4°c. the increments of the distances were different among samples from animals being fed different diets. the distances in flaxseed were 54% and 81% compared with control, respectively at 7d and 10d. even though less pronounced (hong et al., 2012), we could speculate that in our condition the large md in the control group might suggest an early ital. j. food sci., vol. 30, 2018 448 start of the aging related modification leading to different responses of the e-nose sensor array. nevertheless, after an initial latency time, at 10d the md of flaxseed seemed similar to the control. correlation patterns with sensors were obtained with pca to help in the discrimination process among meat samples. the stepdisc procedure suggested all sensors could be included in the discriminant analysis (sas, 2003) and a 0.66 value for the kaiser measure of sampling adequacy exceeded the threshold value of 0.60 (stevens, 2009), therefore supporting the data set as suitable for the pca analysis (cerny and kaiser, 1977). the pca is a variable reduction method yielding linear combination of original variables (principal components, pc). the maximum number of pc equals the number of considered variable (i.e. number of sensors). the latent constructs were obtained with the prin method of the proc factor procedure, with varimax rotation, and retained in accordance to the eigenvalue-one criterion (stevens, 2009). then, variables loading vectors and pc scores were obtained. following a varimax orthogonal rotation, three factors were extracted explaining 95.2% of the total variability of data (table 4). by giving a magnitude of at least 0.4 as indicator of a salient variable-factor relationship, the sensors w5s, w1w, w2w and w3s loaded on pc1 (32.89%), sensors w1c, w3c and w5c loaded on pc2 (31.39%) whereas sensors w6s, w1s and w2s loaded on pc3 (30.95%). the orthogonal factor rotation simplifies the interpretation of extracted factors and from that we could suggest pc1 as related mainly to the proteolysis activity, pc2 mainly addressing processes leading to aromatic compounds formation, whereas pc3 included the broad range sensors and a sensor reactive to hydrogen. to identify pattern of correlation among sensors responses, score coefficients for each variable were obtained and principal component scores for each sample were calculated. fig. 3 shows a two-dimensional plot of the analysis score of meat samples with pc1 and pc2. the control at 7d and 10d and the flaxseed at 10d tended to cluster in the positive quarter for the considered pcs. table 4. loading vectors of sensors on varimax rotated extracted pc and proportion of explained variance. pc1 pc2 pc3 w1c 0.02 0.96* 0.03 w5s 0.90* -0.28 0.08 w3c 0.11 0.99* -0.04 w6s 0.32 0.02 0.94* w5c 0.17 0.97* -0.08 w1s 0.16 -0.07 0.98* w1w 0.84* 0.31 0.36 w2s 0.22 -0.06 0.97* w2w 0.87* 0.29 0.36 w3s -0.90* -0.19 -0.20 proportion 32.89 31.39 30.95 pc = principal component, *variables loaded on extracted components (i.e. loading vectors higher than 0.40). ital. j. food sci., vol. 30, 2018 449 figure 3. score plot of principal component analysis (pc) of raw meat samples at different aging (2, 7 or 10 days). as in pca, also the cda performs a dimension-reduction through linear combination of quantitative variables and helps to discriminate differences among classes. when performing the cda the r squares indicated all sensors showed a significant (p<0.05) difference between the classification groups for all canonical variables (can). the raw canonical coefficients of the first canonical variable showed that classes differ on the linear combination of the sensors selective for aromatic compounds (table 5). the plotting of the first two canonical variables (fig. 4) revealed can1 has more discriminatory power and it is capable of discriminating meat samples into four groups: flaxseed at 2d, control at 2d and flaxseed at 7d, control at 7d and flaxseed at 10d, control at 10d. however, the better result in samples discrimination compared to pca was inherent to the algorithm used for group separation since the cda is a supervised learning method relying on group labels. 3.2. sensory analysis sensory analysis was performed after 7d of aging when usually beef is offered for sale as retail cuts. the amount of fat in meat was similar between samples coming from differently fed animals (5.2 vs. 4.6 g kg-1, respectively for control or flaxseed; data not shown). the flavor intensity was higher (p<0.05) in meat samples from flaxseed-fed animal compared with meat samples from control-fed animals (7.84 and 6.74, respectively). feeding flaxseed doubled the intramuscular content of total n-3 pufa (from 21.1 to 46.7 g/kg of total detected fa – data not shown) and the proportion of c18:3 n-3 in lm from flaxseed-fed bulls reached 3.0% while in control-fed bulls remained lower than 0.9% (data not shown); such modifications of the fatty acid profile of meat might explain the higher flavor intensity scored by panelists. ital. j. food sci., vol. 30, 2018 450 table 5. raw canonical coefficients for canonical variables. can1 can2 can3 can4 can5 w1c 64.874 75.721 24.026 -118.107 1.820 w5s 0.798 -0.783 1.410 -0.636 -1.294 w3c -71.757 -81.159 -137.678 96.442 -125.821 w6s -5.924 -0.211 2.979 5.177 -5.727 w5c 57.716 20.960 150.921 -7.927 115.196 w1s 3.565 -3.336 -7.029 -6.538 6.833 w1w -4.616 -5.562 13.041 3.326 0.050 w2s 3.976 6.841 7.977 4.485 -3.202 w2w 7.278 6.173 -15.159 -4.777 -0.785 w3s -21.962 13.150 7.314 2.614 -25.212 proportion, % 85.65 6.04 4.49 2.32 1.50 r2 0.98 0.75 0.69 0.53 0.42 can = canonical variable. figure 4. score plot of canonical discriminant analysis (can) of raw meat samples at different aging (2, 7 or 10 days). several studies have shown that animal diet can strongly influence the fatty acid composition of meat (ponnampalam et al., 2001; wood et al., 2003; bas et al., 2007; vahmani et al., 2015). the variation of fatty acid compositions has profound effects on meat quality, because fatty acid composition determines the firmness/oiliness of adipose ital. j. food sci., vol. 30, 2018 451 tissue and the oxidative stability of muscle, which in turn affects flavor and muscle color. high pufa levels may produce alterations in meat flavor due to their susceptibility to oxidation and the production of unpleasant volatile components during cooking (wood et al., 1999). even if increases in overall liking scores were reported (vatansever et al., 2000), most studies have shown decreases in panelist preferences for meat from animals fed diets high in unsaturated fatty acids (campo et al., 2006), sometimes due to the related increase of oxidation products (yang et al., 2002). 3.3. relationship between e-nose data and sensory scores the 7d e-nose data on cooked meat were analyzed by pls to investigate the relationship between sensors readings and flavor scores. meat samples used in the two assays were cocked with different methods, water bath for e-nose and in a double plate grill for sensory evaluation. while the cocking methods could lead to different textural attributes of meat, the flavor is however not affected (choi et al., 2016). the water-bath method selected for the e-nose assay was to minimize maillard products and their reaction with volatile compounds (aaslyng and meinert, 2017) and effects of high grilling temperatures on variability of volatile compounds and therefore on pattern observed during the e-nose assay. the e-nose sensor responses (predictor variable) were used to predict the flavor score (dependent variable) from sensory evaluation. since the restricted number of samples, an independent data set for validation was not possible, therefore a one at a time cross-validation method was used to choose the number of extracted factors minimizing the predicted residual sum of squares (press) and the van der voet’s test (van der voet, 1994) was used to select the fewest number of factors (i.e. with residual press not statistically different than the minimum press) (table 6). the contribution of each sensor in fitting the pls model was based on the variable importance for projection (vip) statistic of wold (wold, 1994) with a minimum threshold value of 0.8. the vip shows the contribution of sensors in fitting the pls model for both sensors and flavor (table 7). a small (in absolute value) coefficient of center and scaled parameter and a small vip (i.e. <0.8) suggest low importance of the predictor in the pls model. the parameter estimate in original scale represents the coefficients of each predictor in the pls model. the predicted results by pls vs observed results from sensory evaluation are shown in fig. 5. the retained factors in pls explained 99.6% and 82.5% of the variance of independent (sensors) and dependent (flavor) variables, suggesting in our condition the enose could represent a tool supporting the sensory evaluation by panelists. 4. conclusions the approach used in data evaluation could not clearly indicate the e-nose as capable of discriminating populations in meat samples from differently fed animals and with different days of aging. within the sensor array used, sensors having major importance in discriminating power were the ones reacting to aromatic compounds, followed by sensors that could be related to proteolysis reactions. differences among samples were observed at the 7d of aging. in our condition, when performing pls regression the e-nose proved to be a valuable tool supporting the sensory evaluation. additional efforts are needed to better understand the relationship between sensor activation and flavor intensity toward the identification of the substances acting in flavor intensity. ital. j. food sci., vol. 30, 2018 452 table 6. steps of the partial least square (pls) method with cross-validation for the 7d cooked meat samples. pls factors cross-validation root mean press comparison significance 0 1.059 0.034 1 1.035 0.047 2 0.997 0.072 3 0.869 0.095 4 0.912 0.079 5 0.911 0.04 6 0.956 0.036 7 0.792 0.101 8 0.931 0.011 9 0.659 0.064 10 0.613 1 minimum root mean press 0.613 minimizing number of factors 10 smallest number of factors with p>0.1 7 retained factors percent variation accounted for independent variable dependent variables current total current total 1 32.338 32.338 48.745 48.745 2 35.152 67.490 7.229 55.974 3 26.017 93.507 2.970 58.944 4 3.608 97.115 11.002 69.946 5 1.896 99.011 2.042 71.987 6 0.514 99.525 4.166 76.153 7 0.066 99.591 6.364 82.519 table 7. variable importance for projection (vip) and regression coefficients values for sensors in the prediction of flavor from e-nose reading on 7d cooked meat. variable vip centered and scale parameter estimates parameter estimates in original scale intercept 0 31.891 w1c 0.748 -0.247 -8.208 w5s 0.878 0.620 0.305 w3c 0.722 2.223 48.322 w6s 1.411 3.075 5.115 w5c 0.878 -3.922 -76.491 w1s 1.404 -1.693 -2.259 w1w 0.660 0.056 0.138 w2s 1.337 -1.624 -4.393 w2w 0.645 0.104 0.246 w3s 0.881 0.488 3.451 ital. j. food 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whittington f.m. 2008. fat deposition, fatty acid composition and meat quality: a review. meat sci. 78:343-358. yang a., brewster m.j., beilken s.l., lanari m.c., taylor d.g. and tume r. k. 2002. warmed-over flavor and lipid stability of beef: effects of prior nutrition. j. food sci. 67(9):3309-3313. paper received june 24, 2017 accepted february 12, 2018 ijfs#604_bozza ital. j. food sci., vol 29, 2017 463 paper quality and sensory profile of ultrasound-treated beef e.m. peña-gonzález1, a.d. alarcón-rojo*1, a. rentería1, i. garcía1, e. santellano1, a. quintero2 and l. luna3 1facultad de zootecnia y ecología, universidad autónoma de chihuahua, periférico francisco r. almada km 1, chihuahua chih., c.p. 31453 2facultad de ciencias químicas universidad autónoma de chihuahua, campus universitario no. 2, circuito universitario chihuahua, chih. méxico, c.p. 31125 3catedrático conacyt facultad de zootecnia y ecología, universidad autónoma de chihuahua, periférico francisco, r. almada km 1, chihuahua chih., c.p. 31453. *corresponding author. aalarcon@uach.mx abstract the effects of high-intensity ultrasound treatment on beef (m. longissimus dorsi) quality and sensory attributes were evaluated. ultrasound treatment (40 khz, 11 wcm-2) was applied for 60 min. control and ultrasound-treated samples were stored at 4°c and evaluated at 0, 7, and 14 days. after 14 days of storage, lipid oxidation of the ultrasoundtreated samples increased (p < 0.0089), shear force decreased (p < 0.0001), and the treated meat was perceived as more tender and juicy. the application of ultrasound increased perception of tenderness without changing other sensory attributes. keywords: lipid oxidation, meat aging, meat tenderness, sensory attributes, ultrasonic treatment ital. j. food sci., vol 29, 2017 464 1. introduction various technological alternatives have been explored to enable minimally processed meat preservation, including novel thermal and nonthermal processing tools that have been successfully applied throughout the food supply chain (demirdöven and baysal, 2009) without affecting the functional or sensory properties of fresh meat and meat products. sensory attributes are important quality factors in the meat industry and are responsible for consumers’ meat choices (mandour et al., 2014). for this reason, methods are needed to ensure the safety, nutritional, and sensory qualities of meat. the use of ultrasound technology in meat processing is emerging (gallego-juárez, 2010; chemat et al., 2011). ultrasound is an acoustic energy, and is considered mechanical, nonionizing, and nonpolluting (ünver, 2016) with great potential for use in high-quality food production processes. ultrasound changes the physical, chemical, and functional properties (terefe et al., 2016) of food products; can therefore, influence the quality of various food systems (kentish and feng, 2014). low intensity ultrasound has been used to evaluate the composition of meat, fish, and poultry products through food quality analysis (knorr et al., 2004) but also it has been reported as successful in the processes of mass transfer (cárcel et al., 2007), marination, softening, and inactivation of microorganisms (ünver, 2016). ultrasound is an alternative to traditional meat aging methods for the tenderization and improvement of meat quality. exposure to highintensity ultrasound can induce tenderness due to the cavitation effects that weaken the cell structure, release lysosomes and proteases, and cause protein denaturation (siró et al., 2009). the muscle tissue can be weakened to increase meat tenderness (stadnik and dolatowski, 2011; hai-jun et al., 2012). therefore, the aging period can also be reduced while preserving the quality parameters of meat (dolatowski et al., 2007) without compromising the oxidative stability of meat (stadnik et al., 2008). however, this method must be developed further before it can be considered for industry-wide use. to date, no study has examined changes in the sensory properties of fresh or aged meat caused by high-intensity ultrasound. thus, the aim of this study was to evaluate the effects of high-intensity ultrasound treatment on sensory quality, texture, and lipid oxidation (lo) of beef stored at 4ºc. 2. materials and methods 2.1. meat and sample preparation the samples for all experiments were beef from m. longissimus dorsi (hereford), obtained from a local supplier 2 days post mortem and then vacuum-packed. muscles were stored at 4°c for 24 h prior to treatment. the ph of the meat was between 5.6-5.9. visible fat was manually removed from each muscle prior to treatment. samples were sliced similarly in terms of weight and size (130 × 90 × 25 mm, length × width × height). the location of the sample was randomly assigned to each treatment and a new muscle was used for each experimental replication. a total of 12 replicates were used. 2.2. treatments samples were designated as; control (c) and ultrasound-treated (u). based on the storage length (0, 7, or 14 days at 4°c), samples were further identified as; c0, c7, and c14 and u0, u7, and u14, respectively. ultrasound treatment (40 khz, 11 wcm-2) was applied to the u samples at the end of each storage period. the samples were treated for 60 min (30 ital. j. food sci., vol 29, 2017 465 min/side) in a modified-intensity ultrasonic bath (branson® 1510 model 1510r-mth; branson ultrasonics corporation, danbury, ct, usa) using distilled water as the diffusion medium. the effective power of the ultrasound system was determined using a calorimetric technique previously described (margulis and margulis, 2003). temperature was kept constant at 4ºc and the intensity was modified to obtain 11 wcm-2. after sonication (or no treatment), meat was vacuum packed and prepared for analysis. 2.3. shear force measurements shear force (sf) was measured using the method outlined by maher et al. (2004); the samples were placed in airtight plastic bags and cooked in a water bath (isotemp 215: fisher scientific, pittsburgh, pa, usa) until the temperature at the geometrical center of the sample reached 72°c. cooked samples were tempered at room temperature and cooled at 4°c overnight, then drained and stored at 4°c for 24 h. after this period, 1 cm diameter cylinders were cut in the muscle parallel to the fibers using a punch. cored samples were sheared using a ta-xt2i (stable micro systems, surrey, uk) with a vshaped blade (warner-bratzler meat shear-compression) attached to a 100 n load cell and a crosshead speed of 200 mm min-1. average values of 8 replicates for each sample were performed and the sf values were reported as newtons. 2.4. lipid oxidation measurement the degree of lipid oxidation (lo) was determined by measuring thiobarbituric acid (tba)-reactive substances (tbars) according to the technique described by piccini et al. (1986). ten grams of muscle were homogenized (esge bio homogenizer model m133/1281-0; bio spec products inc., bartlesville, ok, usa) with a 10% solution of 6 n hcl for 40 s, the resulting suspension was subjected to distillation and 50 ml aliquots were collected. afterwards a 2.5 ml of distillate was taken and mixed with 2.5 ml tba at 0.02 m tba. the mixture was incubated in a boiling water bath (100°c) for 40 min. a sample containing 2.5 ml of distilled water and 2.5 ml of tba was used as a blank. both were cooled for 10 min in running tap water and absorbance was measured at 535 nm on a spectrophotometer (genesys 20, model 4001/4; thermo spectronic, waltham, ma, usa). the results were plotted against a standard curve prepared with known concentrations of tetraethoxypropane. this determination was performed in triplicate and the results expressed as mg of malondialdehyde (mda) per kg of meat (mg mda/kg meat). 2.5. sensory evaluation 2.5.1 selection and training of panellists twelve panellists were recruited and trained using the quantitative descriptive analysis technique described by stone et al. (2004). the panellists were selected by the basic taste test. in the second stage of selection, the farnsworth-munsell 100 hue test for color sensitivity and test was used for taste, using the triangular test (international organization for standardization, iso, 8586-1, 1993). total training duration was 80 h, training included familiarization with relevant descriptive terms and ways of perceiving the selection and quantification of the sensory characteristics of cooked meat as well as the use of intensity scales iso 4121 (2003). representative samples were offered to the panel to determine relevant attributes. for the evaluation of appearance (i.e., color), a modified version of the amsa (2012) protocol was used. meat color was evaluated using reference scales. the panellists then generated individual lists of descriptors for each sensory characteristic (i.e., ital. j. food sci., vol 29, 2017 466 odor, appearance, flavor and texture) to characterize the meat samples (iso 8586-2, 1994). next, consensus lists of five descriptors per sensory modality were created and these terms were used for preparation of a lexicon that was used for further panellist training. the selected attributes were: whitish, pink, grayish, light-brown, and pale appearance; raw meat, grilled meat, fresh-cooked meat, boiled meat, and metallic odors; fresh bovine cooked meat, greasy, dry meat, bovine meat, and metallic flavors; soft, juicy, fibrous, tough, and elastic textures. the descriptor intensities per attribute were evaluated on a 10 cm linear scale with two anchor points. the final lexicon terms (descriptors) and definitions used to train the panellists are shown in table 1. table 1. lexicon developed (descriptors that characterize a beef sample) by panellists and used in the evaluated quantitative descriptive analysis. attribute descriptor definition appearance whitish perception of greater amount of white light on the surface of the meat. pink pale shade of red. grayish meat with less intense hue and brown tone. light-brown brown hue reflecting more light. pale meat color is observed to be less saturated. odor raw meat amount of beef odor in the sample; beef identity. grilled meat full aromatic generally associated with beef suet that has been grilled. fresh-cooked meat odor or note of aromatic fresh-cooked beef. boiled meat aromatic notes associated with boiled meat or soup stock. metallic aromatics associated with impression of slightly oxidized metal. flavor metallic taste associated with undercooked meat (bloody taste). fresh bovine cooked meat taste characteristic of all meat, the aromatics associated commonly in partially cooked meat. greasy flavor associated with fat heated to a high temperature. dry meat flavor associated with meat that is overcooked and charred on the outside. bovine meat the aromatics commonly associated with matured cooked beef muscle products (boiled beef broth). texture soft describes beef meat that is easy to bite between the teeth (low hardness). juicy perception of the amount of water released by the product during the first bites. fibrous indicate that the orientation of particles in meat beef is similar to that perceived in celery. tough the number of chews required to masticate beef meat into a state ready for swallowing is similar to that necessary for old cow meat. elastic describes the rapidity of recovery from a deforming force. sources: amsa, 1995; byrne et al., 2001; nollet and toldrá, 2011. panellists’ performance was evaluated by applying a test of homogeneity of variances using the proc glm procedure in the sas statistical package (sas institute, cary, nc, usa). consistency among the panel on sensory modalities was statistically significant for appearance (color) (p < 0.0001), odor (p < 0.05), flavor (p < 0.0001), and texture (p < 0.0001). the coefficient of the descriptors was estimated using xlstat-sensory software (version 2015.6.01.25740; addinsoft, paris, france). ital. j. food sci., vol 29, 2017 467 2.5.2 sensory test samples were cooked in an oblong electric skillet (the west bend company, west bend, wi, usa) to an internal temperature of 72 ºc, following amsa (1995)-established methods. samples were cut into six equal pieces and maintained at 35ºc until sensory analysis (≤30 min). the test was conducted under white light. panelists were instructed to cleanse their palates with water between samples. the sensory tests were conducted for the sonicated (u) and untreated (c) samples in three sessions. in each session, panelists received randomly a (30 g) sample from each treatment, identified by a three-digit code. the panellists evaluated the samples using an unstructured 10-cm linear scale (0 = none, 10 = very). data were recorded (values in cm) as intensity points for each descriptor. 2.6. statistical analyses the test variables (sf, lo, and sensory attribute intensity) were analyzed using the generalized linear model procedure (sas software, sas institute) and the statistical model yijk = µ + ai + bj + (ab)ij + eijk;, where yijk = response variables, µ = general average, ai = effect of ultrasound treatment, bj = effect of storage time, (ab)ij = effect of interaction between ultrasound treatment and storage time, and eijk = random error. when the effect of a factor or interaction on one or more variables was significant (p ≤ 0.05), tukey’s statistical test was performed to compare the averages. analysis of variance was also performed to determine the discriminant power of the descriptors and their estimated coefficients, using the xlstat-sensory software package (version 2015.6.01.25740; addinsoft). 3. results and discussions 3.1. shear force sf differed significantly between treatments and storage periods (p < 0.0001; fig. 1). sf values were higher on day 0 of storage and declined significantly by day 14 for both u and c samples. u samples had significantly reduced sf (p < 0.0001) compared to the c samples, which showed higher sf at all storage times. these results corroborate those reported previously (jayasooriya et al., 2007; zhou et al., 2010). stadnik and dolatowski (2011) highlighted the potential of using low-frequency and low-intensity postmortem. they found reduced meat toughness at 48 and 72 h postmortem. the effect of high-intensity ultrasound on sf reduction has also been reported for the following parameters: 24 khz and 12 wcm-2 for 4 min in bovine meat (jayasooriya et al., 2007), 24 khz and 12 wcm-2 for 4 min in poultry after 7 days of storage (xiong et al., 2012), and 2.53 wcm-2 for 180 min in pork (siró et al., 2009). sikes et al. (2014) also observed a reduction in sf with aging at 4°c for 7 days (p < 0.001), but no interaction between ultrasound treatment and storage. other results have differed; as no effect was observed on sf with 62 wcm-2 (lyng et al., 1998), 22 wcm-2 (pohlman et al., 1997a), or 4-19 wcm-2 (mcdonnell et al., 2014), although ultrasound treatment decreased gumminess and cohesiveness of salted pork in the latter study. ital. j. food sci., vol 29, 2017 468 figure 1. effects of treatment and storage time on instrumental texture, measured as shear force (n) for bovine m. longissimus dorsi treated and not treated with ultrasound (40 khz, 11 wcm-2) and stored at 4°c for 0, 7, or 14 days (mean ± standard error bars). c= control (no ultrasound); u= ultrasound-treated. a, b different letters indicate significant differences with ultrasound (p < 0.0001). x, y, z different letters indicate significant differences between storage time (p < 0.0001). ultrasound treatment affects meat tenderization via acoustic cavitation, as bubble formation, growth, and eventual collapse have thermal, chemical, and mechanical effects (yusaf and al-juboori, 2014). asymmetric collapse causes an eruption of fluid, producing a microburst affecting the integrity of muscle structure (bhaskaracharya et al., 2009). this process is associated with postmortem hydrolysis of myofibrillar proteins in the aging stage, which leads to greater meat tenderization (geesink et al., 2001) and explains the sf reduction observed in the present study. likewise, depending on ultrasound frequency, alternating positive and negative pressures are produced, causing expansion or compression and resulting in cell rupture. this process also causes water hydrolysis (awad et al., 2012), leading to the formation of chemically active free radicals (h+ and oh-), which intervene in the structural stability and catalytic functions of proteins. thus, ultrasound treatment may modify the availability of adenosine triphosphate in the pre-rigor muscle (sikes et al., 2014), which also accelerates the start of rigor mortis (dolatowski et al., 2004; stadnik and dolatowski, 2011) and therefore increases the aging rate of meat (chandrapala, 2015). 3.2. lipid oxidation the degree of lo in the samples differed significantly according to the interaction of treatment and storage factors (p < 0.01; fig. 2). both ultrasound and control meat presented lower lipid oxidation at day 0 of storage and these values increased significantly after 14 days of storage in treated samples (p < 0.01). the degree of lo in all samples fell below the rancidity threshold of 1-2 mg mda/kg (vieira et al., 2009) and was also lower than the oxidation odor detection threshold (0.5-1 mg mda/kg) (tarladgis et al., 1960). these results agree with those reported by stadnik (2009), who obtained tbars values that indicated no compromise to the oxidative stability of ultrasound-treated (45 khz, 2 wcm-2 for 120 s) meat samples stored under refrigeration. ultrasound breaks down cell membranes, fragments collagen, denatures proteins by bubble pulsation and cavitation, and promotes the formation of free radicals (kuijpers et al., 2002). consequently, it intensifies meat oxidation by increasing the speed of chemical reactions (awad et al., ital. j. food sci., vol 29, 2017 469 2012). furthermore, aging represents a change in both structures and chemical composition of beef. for example, free radicals are produced during aging, mainly from metal release. furthermore, fat and fat-like membrane molecules are degraded to fatty acids during aging (dashdorj et al., 2016). these two factors together may explain why ultrasonicated meat is slightly more oxidized after 14 days of storage. possibly, polyunsaturated fatty acids (pufas) released from phospholipids (membranes) during aging become more exposed to released free radicals during sonication (i.e., iron), interacting more rapidly during the same processes. since lipid peroxidation is more strongly influenced by oxidation of membrane components such as pufas (faustman et al., 2010), the exposition of these fatty acids could be responsible for the slight increase in sonicated meat. however, the values obtained from treated samples in our study indicated minimal changes in lo during storage. figure 2. effects of treatment and storage times on the lipid oxidation index (mg mda/kg of meat) for bovine m. longissimus dorsi treated and not treated with ultrasound (40 khz, 11 wcm-2) and stored at 4°c for 0, 7 or 14 days (mean ± standard error bars). c= control (no ultrasound); u= ultrasound-treated. a, b, c different letters indicate significant differences by interaction treatment of ultrasound and storage time (p < 0.0089). 3.3. sensory properties the effects of ultrasound treatment and storage on odor and flavor characteristics differed significantly with an interaction between these factors (p < 0.01 and p < 0.0001, respectively; fig. 3a and 3c). after 7 and 14 days of storage, untreated samples had a more intense odor and flavor (raw meat odor, p < 0.0001; fresh-cooked meat odor p < 0.0006; and fresh bovine cooked meat flavors, p < 0.0001) than meat without storage, but also a more intense pleasant boiled meat odor (p < 0.0001) compared to samples treated with ultrasound. ultrasound treatment also increased the perception of unpleasant greasy flavor (p < 0.0034), which was more noticeable after storage for 14 days (p < 0.0001). the untreated samples were perceived as less greasy on day 0 (p < 0.0001) maybe because of the structural damage or the liberation of cooked-meat flavor precursor lipids. metallic flavor, dry meat flavor, and metallic odor showed no significant difference according to storage period and treatment. the intensity of fresh-cooked meat odor was lower after storage for 7 days in ultrasound-treated samples (p < 0.0001), which may be due to the concentration of volatile compounds (aromatic molecules) that may be lower during this ital. j. food sci., vol 29, 2017 470 period. stetzer et al. (2007, 2008) reported that positive flavor compounds decrease with aging (between 7 and 14 days of storage) and negative compounds increase. pentanal and 3-hydroxy-2-butanone decrease with aging while nonanal, butanoic acid and 1-octene-3-ol increase. both sonicated and untreated meat showed more whitish and pink colors at 14 days of storage (p < 0.0001; fig. 3b) compared to treated and untreated samples in other periods of storage. figure 3. quality descriptors for bovine m. longissimus dorsi with and without ultrasound treatment (40 khz, 11 wcm-2) after storage at 4 °c for 0, 7 and 14 days. a) odor descriptors, b) color descriptors, c) flavor descriptors, d) texture descriptors. c0= control (not ultrasound, yellow); 0 days of storage; c7= control (not ultrasound, red); 7 days of storage; c14= control (not ultrasound, green); 14 days of storage; u0= ultrasound, 0 day of storage, (purple); u7= ultrasound, 7 days of storage, (blue); u14= ultrasound, 14 days of storage, (orange). untreated meat tended to have a grayish color, with significant interaction observed for 0 and 7 days of storage (p < 0.0001). on day 0, untreated meat had a more intense lightbrown color (p < 0.0002) than sonicated meat; contrarily, the lowest intensity of this attribute was observed in ultrasound-treated samples after 7 days of storage. the palest color was registered for ultrasound-treated meat at 14 days of storage (p < 0.0001). this may be related to the results obtained by jayasooriya et al. (2007) and hai-hun et al. (2012), who indicated that ultrasound application generates an increase in muscle temperature. therefore, the thermal denaturation and oxidation of the meat pigments could affect the color of the meat, making it paler and less red. ital. j. food sci., vol 29, 2017 471 the ultrasound-treated meat stored for 14 days was the softest and juiciest of all samples (p < 0.0111 and p < 0.004, respectively; fig. 3c), but it had a more fibrous texture, associated with lower sf values. meat not treated with ultrasound and stored for 7 days had the most elasticity (p < 0.0058). untreated meat on day 0 of storage had the toughest perceived texture, in agreement with the instrumental texture results (greater sf value). these results coincide with lyng et al. (1998) who indicated that lamb treated with ultrasound and storage for 7 days was perceived as softer probably associated with the process of proteolysis during storage and the cavitation effect of ultrasound. in contrast, pohlman et al. (1997b) reported no effect of treatment with ultrasound and storage time on bovine m. pectoralis because of the greater presence of connective tissue. it has been asserted (dolatowski et al., 2007; stadnik and dolatowski, 2011) that a softer meat texture after ultrasound can be explained by the physical weakening of the muscular structure, affecting the cellular membranes by accelerating proteolysis and releasing cathepsins from the lysosomes and/or calcium ions of the intracellular storage. the descriptors with the strongest discriminating factors for sample characterization were texture attributes, with the exception of untreated meat after 14 days of storage and ultrasound-treated meat after 7 days of storage (fig 4). human perception is conditioned by the sensory interaction of physical processes, such as chewing; thus, sensory properties are linked to physical characteristics (caine et al., 2003) and ultrasound wave propagation in meat depends on meat properties (damez and clerjon, 2008). the results of the present study show that exposure to high-intensity ultrasound increases meat tenderness, as perceived by trained panellists who characterized the ultrasound-treated sample that had been stored for 14 days as the most tender. sensory attributes resulting from proteolysis, such as odor, flavor, tenderness, and juiciness became evident due to the aging process, as the storage period increased. ultrasound treatment resulted in additional softness and juiciness effects over the storage period. these panel results and sf values are similar to those obtained in other studies. pohlman et al. (1997a) conducted a sensory analysis of beef samples (m. pectoralis and m. longissimus thoracis) subjected to ultrasound aging (20 khz, 1000 wcm-2) or cooked by convection, and found increased myofibrillar tenderness (p < 0.05) and reduced flavor intensity in ultrasound-treated samples. muscle treated with ultrasound had greater postcooking moisture, but no difference in juiciness was observed; the quantity of connective tissue and tenderness in general were unaffected by the aging method. lyng et al. (1998) reported that sensory evaluation of bovine m. longissimus thoracis, m. lumborum, and m. semimembranosus treated with ultrasound (20 khz and 62 wcm-2 for 15 s) showed no difference in tenderness, general texture, or global acceptance after 0, 3 or 14 days of storage. however, they found that storage time significantly improved chewability. in spite of the difficulties with comparing different experiments due to differences in frequency/intensity/time combinations of the ultrasound applied to meat, the majority of studies describe the favorable effects of ultrasound on meat texture (alarcon-rojo et al., 2015) and that effect has been corroborated herein. ital. j. food sci., vol 29, 2017 472 figure 4. estimated coefficient of descriptors for bovine m. longissimus dorsi with ultrasound treatment after storage at 4 ºc for 0, 7, or14 days. (confidence interval 95 % model y= p+j). c0 = control, 0 days of storage; c7 = control, 7 days of storage; c14 = control, 14 days of storage; u0 = ultrasound, 0 days of storage; u7 = ultrasound, 7 days of storage; u14 = ultrasound, 14 days of storage. rm= raw meat; gb= grilled beef; fcm= fresh-cooked meat; bm= boiled meat; mo= metallic odors; w= whitish; p= pink; g= grayish; lb= lightbrown; pc= pale color; fbcm= fresh bovine cooked meat; gf= greasy flavor; dm= dry meat; bmf= bovine meat flavor; mf= metallic flavor; s= soft; j= juicy; f= fibrous; t= tough; et= elastic texture. ital. j. food sci., vol 29, 2017 473 4. conclusions high-intensity ultrasound reduces the warner-bratzler sf of beef, and although meat lo increases, it does not negatively affect the quality. thus, ultrasound application may be a feasible way to preserve the sensory properties of meat while significantly reducing aging time. ultrasound technology can be applied to improve meat texture, as confirmed by our finding that high-intensity ultrasound increased meat tenderness. in this context, factors related to muscle (species, gender, age, diet or muscle type) and those related to ultrasound (frequency, intensity, time or ultrasound system) should be considered. results of sensory analyses indicate that ultrasound does not change panellists’ perception of beef quality. these findings should be complemented by consumer evaluation to rule out any detriment to meat quality. acknowledgements this work was supported by the national council for science and technology (conacyt) of mexico, project catedras 100. references amsa. 1995. american meat science association. research guidelines for cooker, sensory evaluation and instrumental tenderness measurements of fresh meat. national livestock and meat board chicago, iii. http://www.meatscience.org/docs/default-source/publications-resources/amsa-sensory-and-tenderness-evaluationguidelines/research-guide/2015-amsa-sensory-guidelines-1-0.pdf?sfvrsn=6. amsa. 2012. american meat science association meat colour measurement guidelines. champaign, illinois usa. http://www.meatscience.org/docs/default-source/publications-resources/hottopics/2012_12_meat_clr_guide.pdf?sfvrsn=0 alarcon-rojo a.d., janacua h., rodríguez j.c., paniwnyk l. and mason t.j. 2015. power ultrasound in meat processing. meat sci. 107:86. https://curve.coventry.ac.uk/open/file/207a92c3-ea93-4d1a-a6af-17afcc03fcca/1/1-s2.0s0309174015001126-main.pdf awad t.s., moharram h.a., shaltout o.e., asker d. and youssef m.m. 2012. applications of ultrasound in analysis, processing and quality control of food: a review. food res. int. 48: 420. http://www.pfigueiredo.org/mai8.pdf. bhaskaracharya r.k., kentish s. and ashokkumar m. 2009. selected applications of ultrasonics in food processing. food eng. rev. 1: 31. http://download.springer.com/static/pdf/699/art%253a10.1007%252fs12393-009-90037.pdf?originurl=http%3a%2f%2flink.springer.com%2farticle%2f10.1007%2fs12393-009-90037&token2=exp=1472585421~acl=%2fstatic%2fpdf%2f699%2fart%25253a10.1007%25252fs12393-009-90037.pdf%3foriginurl%3dhttp%253a%252f%252flink.springer.com%252farticle%252f10.1007%252fs12393-009-90037*~hmac=c1b821f61286b3d43229e65560870b408f557e14665d4968a0154b9586456001 byrne d.v., o´sullivan m.g., dijksterhuis g.b., bredie w.l.p. and martens m. 2001. sensory panel consistency during development of a vocabulary for warmed-over flavour. food qual. prefer. 12:171. http://www.sciencedirect.com/science/article/pii/s0950329300000434 caine w.r., aalhus j.l., best d.r., dugan m.e.r. and jeremiah l.e. 2003. relationship of texture profile analysis and warner-bratzler shear force with sensory characteristics of beef rib steaks. meat sci. 64:333. http://www.sciencedirect.com/science/article/pii/s0309174002001109 cárcel j.a., benedito j., bon j. and mulet a. 2007. high intensity ultrasound effects on meat brining. meat sci. 76:611. http://www.sciencedirect.com/science/article/pii/s0309174007000381 chandrapala j. 2015. low intensity ultrasound applications on food systems. int. food res. j. 22(3):888. http://www.ifrj.upm.edu.my/22%20(03)%202015/(2).pdf. chemat f., zill-e-huma and khan m.k. 2011. applications of ultrasound in food technology: processing, preservation and extraction. ultrason. sonochem. 18:813. http://www.sciencedirect.com/science/article/pii/s1350417710002385 ital. j. food sci., vol 29, 2017 474 damez j.l. and clerjon s. 2008. meat quality assessment using biophysical methods related to meat structure. meat sci. 80:132. http://www.sciencedirect.com/science/article/pii/s0309174008001757 dashdorj d., tripathi v.k., cho s., younghoon kim. and hwang i. 2016. erratum to: dry aging of beef; 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maria.paciulli@nemo.unipr.it abstract the current study assessed the effect of different soils (clay, evoosc, stony, evooss, brown, evoosb, limestone and gypsum, evooslg) on the chemico-physical (free acidity, peroxide value, oxidative stability, fatty acids, pigments, colour, viscosity, heat capacity) and thermal (upon dsc) quality of four 'chemlali' extra virgin olive oils. evoosc showed the lowest peroxide value, the highest amount of monounsaturated fatty acids and the narrowest crystallization range. evooslg had the highest content of saturated and polyunsaturated fatty acids as well as the lowest stability to oxidation and melting enthalpy. evooss and evoosb revealed the highest viscosity, while the heat capacity measurement didn’t show any difference among the oils. evoosb exhibited the highest b* colour parameter in relation to the highest carotenoids content. these preliminary findings have showed that the nature of the soil has an effect on the final quality of evoo, as also revealed by the pca analysis. keywords: composition, dsc, extra virgin olive oil, physical properties, soil ital. j. food sci., vol 29, 2017 75 1. introduction evoo is a traditional product of the mediterranean countries. among these the olive culture represents one of the main economic and agricultural strategic sectors in tunisia. about 60 million olive trees are distributed and spread on 1.6 million hectares extended from the northern to the southern regions of the country. tunisia contributes to more than 4% of the world olive oil production the 'chemlali' being the most abundant olive variety (issaoui et al., 2010). the chemical characterization of 'chemlali' olive oils, as influenced by different either natural or human factors, was the object of several scientific studies (ben hassine et al., 2014; hannachi et al., 2007; ben hassine et al., 2015). significant variability on fatty acids (fa) composition other than minor components such as polyphenols and/or pigments and oxidative stability was found. closely related to chemical composition, but less debated in literature, is the physical and thermal characterization of the oils, which could be considered of great interest for consumers and industries. the use of differential scanning calorimetry (dsc) has been mainly suggested as an alternative interesting approach for the characterization of the oils from different vegetable sources (tan and che man, 2002) or for the evaluation of different oxidative stability (chiavaro et al., 2009; chiavaro et al., 2011; chiavaro et al., 2012) by means of the phase transition behaviour. recently, dsc has also been applied for the characterization of tunisian olive oils on the basis of the cultivar– environment interaction showing good correlations between thermal and chemical properties (kotti et al., 2009). dsc can be also used for the determination of the heat capacity (cp). santos et al. (2005) reported an increase in the cp of vegetable oils, measured by dsc, as a function of the fatty acids saturation. the measurement of viscosity along with the heat capacity provides useful information to optimize the equipment design (settling and storage tanks, centrifuges, pumps, etc.) thanks to the determination of their behaviour during different technological processes. the correlation between viscosity and chemical composition of vegetable oils was slightly mentioned only when, after prolonged heating, an increasing of the viscosity was observed in relation with the increased saturation of the oil constituents and the generation of different polymer compound classes (kalogianni et al., 2011). the colour is a basic criterion affecting the consumer preference although the european union regulations do not require its measurement for an assessment of the virgin olive oil quality. olive fruits contain two main classes of pigments that are transferred to the virgin olive oil during the extraction process: the green chlorophylls and the yellow and orange carotenoids. they can be used as indicator of the olives genetic make-up other than on the habitat where the olive trees are grown (cerretani et al., 2008). it is well-known that the unique nutritional and organoleptic properties of the olive oil are closely related to olives genotype other than agronomic, environmental and technological factors (garcía-gonzález et al., 2010). among these factors, the effect of the soil nature on the chemical composition of the oils is barely debated and often just regarded to as a combined effect with other factors (bedbabis et al., 2015; papadia et al., 2011; romero et al., 2015). moreover, to the authors’ best knowledge, no study on the impact of the soil nature on physical and thermal properties of olive oils has been reported in literature so far. this literature gap has led us to perform a complete characterization, in terms of composition (fatty acids, quality parameters, main pigments), oxidative stability, physical (viscosity, colour, heat capacity) and thermal properties, of four monovarietal 'chemlali' evoos, differing only in the nature of the cultivation soils being cultivated in the same area under the same agricultural conditions and harvested in the same season at the same ripening index. ital. j. food sci., vol 29, 2017 76 2. materials and methods 2.1. oil samples preparations evoo samples were obtained from fruits of the main tunisian olive cultivar, 'chemlali', which were picked by hand at the same stage of maturity, according to the iooc (2004) classification, from three trees, during the crop season 2012/ 2013 (october), placed in 4 locations with different soil nature: clay (evoosc), stony (evooss), brown (evoosb), limestone and gypsum (evooslg), located in sousse center of tunisia (35°.49’ n, 10°.30’ e). olive trees were subjected to identical fertilisation regime and to all common olive cultivation practices. the same laboratory mill was used to prepare the olive oil samples. only healthy fruits without any kind of infection or physical damage were processed. after harvesting, fresh olives (1.5-2.0 kg) were washed and defoliated, smashed with a hammer crusher and then paste mixed at 25°c for 30 min, centrifuged without addition of warm water (oil produced from each extraction was 200-250 ml/kg) and then transferred into dark glass bottles and stored in the dark at 4°c until analysis. three samples of each oil were analysed and triplicate analyses were carried out for each sample. 2.2. chemical analysis free acidity, expressed in oleic acid (18:1) percentage, and peroxide value (pov), given as milli-equivalents of active oxygen per kilogram of oil (meqo2/kg) were determined according to the analytical methods described in the european union commission regulations (eec/2568/91; eec/1429/92). oxidative stability (osi) was evaluated by the rancimat method (gutiérrez rosales et al., 1989). stability was expressed as the oxidation induction time (h), measured with the rancimat 743 apparatus (metrohm, herisau switzerland), using an oil sample of 3.6 g. the oil temperature was 101.6°c and the air flow was 10 l/h. chlorophyll and carotenoid contents were determined colorimetrically, as previously described (minguez-mosquera et al., 1991). briefly, the method consists in quantitatively assessing the chlorophyll fraction by measuring the absorbance of the olive oils at 670 nm and the carotenoid fraction at 470 nm through the use of appropriate molar absorption coefficients. the results were expressed in g/kg. the fatty acids (fa) were converted to fatty acid methyl esters before analysis by shaking a solution of 0.2 g oil and 3 ml of hexane with 0.4 ml of 2-n methanolic potassium hydroxide, and analyzed using a hewlett-packard (hp 4890d; hewlett-packar company, wilmington, de) chromatograph equipped with a capillary column (supelcowax: 30 m × 0.53 mm; 0.25 mm), a split/splitless injector and a flame ionization detection (fid) detector. the carrier gas was nitrogen at a flow rate of 1 ml/min. the temperatures of the injector, the detector and the oven were held at 220, 250 and 210°c, respectively. the injection volume was 1 μl. the results are expressed as relative area percentage of total fas. 2.3. thermal analysis evoo samples (8-10 mg) were weighed into aluminium pans, covers were sealed into place and the whole analyzed with a dsc q100 (ta instruments, new castle, de). indium (melting temperature 156.6°c, ∆hf= 28.45 j/g) and n-dodecane (melting temperature -9.65°c, ∆hf= 216.73 j/g) were used to calibrate the instrument and an empty pan was used as reference. oil samples were equilibrated at 30°c for 8 min and then cooled at -80°c at the rate of 2°c/min, equilibrated at -80°c for 8 min and then heated ital. j. food sci., vol 29, 2017 77 from -80 to 30°c at 2°c/min. dry nitrogen was purged in the dsc cell at 50 cm3/min. dsc curves were analysed with universal analysis software (version 3.9a, ta instruments) to obtain enthalpy change for transition (∆h, j/g), onset temperature of transition (ton,°c), offset temperature of transition (toff,°c) and peak temperature at the maximum (tp) for the two main events of cooling and heating transitions (tp1 and tp2,°c). range of transition was calculated as the temperature difference between ton and toff. heat flow (w/g) of the main peak (p1) of both cooling and heating was also calculated. heat capacity (cp, j/g°c) measurements were taken with a dsc q100 (ta instruments, new castle, de). each determination was performed in triplicate according to the combination of two methods (astm e1269-05; heidenreich et al., 2007). according to the method (minguez-mosquera et al., 1991) three individual dsc experiments referred to as: baseline, reference, and sample were performed using the same procedure: equilibration at 5°c; isothermal for 15 min; ramp at 5°c/min up to 300°c; isothermal for 20 min. for determination of the heat capacity calibration constant e(t), the heat capacity of the sapphire (cp,s), the mass of the sapphire (ms) and the temperature-dependent heat flow of sapphire (qs) and of the empty pan (qe) were considered in eq.1. e(t) = !!,!!!! !" !! ! !!! ! eq. 1 the constant e(t) was then used to calculate the heat capacity of the sample by using the appropriate heat flow and mass (qc and mc, respectively). 𝑐!= !"!(!) !! ! !!! (!) !!! eq. 2 2.4. viscosity measurements measurements were made by means of a concentric cylinder brookfield® dv-i prime rotational viscosimeter (brookfield, middleboro, massachusetts, usa). data capture was obtained connecting the viscosimeter to a computer, monitoring rotation per minutes (rpm) and the apparent viscosity (centipoise) at 1s intervals. control of temperature was obtained connecting the jacket of the measuring cell to a water bath whose temperature was checked at 25°c±0.5. two cylindrical spindles were used for the rheological analyses. the ula spindle (brookfield, middleboro, ma, usa) (viscosity range from 0.06 to 2000 mpa s) and the sc4-18/13r spindle (brookfield, middleboro, ma, usa)(viscosity range from 0.3 to 9998 mpa s). rheological behaviour was described in terms of viscosity (mpa s) at various shear rates (dv/dy). since the apparatus measures the viscosity as a function of spindle rpm, shear rate values (1/s) were obtained multiplying rpm by a specific constant for every spindle (e.g.: 1.224 for ultra low adapter, and 1.32 for the spindle sc418 of the small sample adapter). the viscosity (µ) value was obtained from the newton’s law σ=µ𝛾 eq. 3 where σ is shear stress (mpa),𝛾 is the shear rate (1/s) and µ is viscosity (mpa s). 2.5. instrumental colour the software imagej, v.1.38x, fitted with the plugin color inspector 3d v. 2.3, was used to assess the oil colour applying the cielab colorimetric system. each time 20 ml of samples ital. j. food sci., vol 29, 2017 78 were put into a glass petri dish. the images of each petri dish were acquired with a scanner (hewlett packard, palo alto, ca, usa) at 600 dots per inch (dpi). the colour brightness coordinate l* measures the whiteness value of a colour and ranges from black at 0 to white at 100. the chromaticity coordinate a* measures red when positive and green when negative, and chromaticity coordinate b* measures yellow when positive and blue when negative (moyano et al., 2008). 2.6. statistical analysis means and standard deviations were calculated with spss (version 22.0 spss inc., chicago, il, usa) statistical software. spss was used to perform one-way analysis of variance (anova) and tukey’s honest significant difference test (hsd) at a 95% confidence level (p<0.05) to identify differences among samples. pearson correlation coefficients were calculated among the variables at a 95% and 99% confidence levels (p<0.05 and p<0.01). principal component analysis (pca) was also performed by means of statistica software (version 8.0, stat‐soft, tulsa, ok, usa). pca has been used as descriptive statistical technique plotting the selected vectors (independent variables) versus all cases (samples) with the aim to find relationships among the variables, able to describe differences among the four cases. 3. results and discussion 3.1. chemical analysis 3.1.1 quality parameters, oxidative stability and main pigment content chemical quality parameters and oxidative stability of the four samples are shown in table 1. table 1. chemical quality parameters of the evoo samples. evoosc evooss evoosb evooslg free acidity (%) 0.70±1.00a 0.75±0.05a 0.55± 0.15a 0.70±0.01a pov (meqo2/kg) 2.5±0.50 c 4.5±0.51b 4.5±0.50b 10.5±0.49a osi (h) 15.58±0.41b 14.84±0.15c 18.01±0.16a 3.34±0.27d chlorophylls (mg/kg) 3.48±0.09b 3.01±0.22b 4.32±0.10a 4.68±0.44a carotenoids (mg/kg) 1.27±0.00b 1.20±0.11b 1.83±0.02a 1.60±0.12a data are expressed as mean±standard deviation of three determinations (n = 3, p<0.05). different letters in the same row are statistically different (p<0.05). evoosc, extra virgin olive oil from soil clay, evooss extra virgin olive oil from soil stony, evoosb, extra virgin olive oil from soil brown, evooslg extra virgin olive oil from soil limestone and gypsum. the free acidity value (5.5-7.5 g/kg) was lower than the legal limit for the commercial category of extra virgin olive oil in all the samples (ecc/61/2011) and it does not seem to be affected by the nature of soil. similarly other authors didn’t find any difference in the free acidity on 'chemlali' olive oils obtained from olives cultivated on soils irrigated with ital. j. food sci., vol 29, 2017 79 different quality waters (bedbabis et al., 2015) or from different geographical origins (ben hassine et al., 2014). the peroxide value (pov), on the other hand, showed (table 1) significant differences among the four 'chemlali' evoo according to the nature of the soil. among all the samples, evoosc showed the lowest pov value (2.5 meqo2/kg), while evooslg had the highest one (10.5 meqo2/kg) (table 1); however they were all below the legal limit of 20 meqo2/kg of oil for extra virgin olive oil (eec/2568/91). contradictory results were previously reported in literature according to this quality parameter in evoo. bedabis et al. (2015) didn’t find any difference among 'chemlali' evoos obtained from olives irrigated with different types of water. other authors (issaoui et al., 2010; ben hassine et al., 2014) found differences comparing 'chemlali' olive oils belonging to different geographical sites in tunisia. the oxidative stability (osi) of the 'chemlali' samples turned out to be influenced by pedologic conditions as well significant differences among the four oils (table 1) have been observed and evoosb being the most stable (18 h). in accordance with the highest pov values evooslg reported the lowest oxidative stability (3h). in general, the oxidative status and the stability to the oxidation of an olive oil depend on the oil composition, both in terms of lipids profile and micro-components (ben mansour et al., 2015). the oil composition is demonstrated to be itself affected by several environmental factors (issaoui et al., 2010; ben hassine et al., 2014; hannachi et al., 2007), including the soil nature (papadia et al., 2011). as shown in table 1, the oils pigments content seems to be influenced by the nature of the soils, showing significant differences among the samples. the chlorophyll content was in the range 3.0-4.7 mg/kg with evooslg and evooss as the richest and the poorest samples, respectively. the same trend was observed for the carotenoids, being however in the concentration range of 1.2-1.8 mg/kg (table 1). several authors reported different pigments concentration in 'chemlali' olive oils as affected by the growing area in tunisia (issaoui et al., 2010; ben hassine et al., 2014; ben mansour et al., 2015) being however in the same order of magnitude of this study. bedbabis et al. (2015) reported different pigments contents in 'chemlali' evoos obtained from olives irrigated with different types of water. these authors outlined that the pigments content of the olive oils is influenced by the olives ripening, being the latter itself influenced by the salinity of the soil. 3.1.2 fatty acid composition the fatty acids (fa) composition of the four 'chemlali' evoo is shown in table 2. oleic, linoleic and palmitic acids were the main fa present in the samples being oleic the main abundant compound. the fa distribution was within the range expected for high quality olive oils ecc/61/2011. the fa composition found in this study traced the ones found for extravirgin 'chemlali' oils from other authors (issaoui et al., 2010; ben hassine et al., 2015; ben mansour et al., 2015), with differences attributable to the environmental factors. as summarized in table 2, evooslg exhibited the lowest c18:1 content (471.6 g/kg) and the highest amount of c16:0 and c18:2 (206.6 and 256.4 g/kg, respectively). on the contrary, evoosc showed significantly highest content of c18:1(610.5 g/kg) and lowest of c16:0 and c18:2 (180.6, 158 g/kg, respectively). stearic acid content did not show a significant difference among samples, showing concentration around 20 g/kg. evooss and evoosb exhibited a very similar composition and intermediate between the ones of the other two oils. while evooslg exhibited the highest amount of sfa and pufa other than the lowest amount of mufa, evoosc had the highest content of this fatty acid category, among all. in particular, mufa correlated positively with osi (p≤ 0.01, r=0.738) and negatively with pov (p≤ 0.01, r= -0.922), ital. j. food sci., vol 29, 2017 80 inversely pufa correlated positively with pov (p≤ 0.01, r=0.948) and negatively with osi (p≤ 0.01, r= -0.780). similarly, ben hassine et al. (2015) associated the oxidative stability of an olive oil to the value of the oleic/ linoleic acids ratio. interestingly, papadia et al., (2011) found significant correlations between the amount of oleic and linoleic acids in mono-varietal extra virgin olive oils and the concentration of b, cr, mn and zn in the cultivation soil. table 2. main fatty acid composition (%) of the evoo samples. evoosc evooss evoosb evooslg c16:0 18.06±0.52c 20.40±0.30ab 19.77±0.02b 21.07±0.43a c16:1 2.27±0.07c 3.13±0.11ab 2.88±0.01b 3.40±0.16a c18:0 1.99±0.06a 2.00±0.07a 1.99±0.02a 1.91±0.05a c18:1 61.05±0.53a 53.66±0.30b 53.65±0.08b 47.16±0.75c c18:2 15.80±0.05d 20.14±0.07c 20.66±0.08b 25.64±0.20a c18:3 0.82±0.02a 0.67±0.02b 0.85±0.03a 0.82±0.01a sfa 20.05±0.52c 22.40±0.24ab 21.95±0.02b 22.98±0.38a mufa 63.32±0.46a 56.79±0.19b 56.53±0.08b 50.57±0.59c pufa 16.63±0.07d 20.80±0.08c 21.51±0.11b 26.45±0.21a data are expressed as mean ±standard deviation of three determinations (n = 3, p<0.05). evoosc, extra virgin olive oil from soil clay, evooss extra virgin olive oil from soil stony, evoosb, extra virgin olive oil from soil brown, evooslg extra virgin olive oil from soil limestone and gypsum. fa, fatty acids; sfa, saturated fatty acids; mufa, monounsaturated fatty acids; pufa, polyunsaturatedfatty acids. (c16:0) palmitic, (c16:1) palmitoleic, (c18:0) stearic, (c18:1) oleic, (c18:2) linoleic, (c18:3) linolenic. 3.2. thermal analysis 3.2.1 cooling curves the dsc cooling curves of the four 'chemlali' evoo (fig. 1a) were similar to that previously reported for extra virgin olive oils (chiavaro et al., 2010) and those of 'chemlali' samples in particular (kotti et al., 2009). the dsc cooling curves of the olive oils are well known to be simply influenced by the chemical composition of the oils (tan and che man, 2000). all the cooling profiles (fig. 1a) showed two well distinguishable exothermic events. in all the samples, the major event (peak 1 of fig. 1a), at lower temperature (~ -45°c), showed a symmetrical line shape, indicating a highly cooperative and more ordered transition due to the homogeneity of the crystallizing molecules; the minor exothermic transition (peak 2 of fig. 1a), (~ -10°c) showed an asymmetrical line shape, suggesting a more heterogeneous crystallization process (tan and che man, 2000). the major exothermic event was previously attributed to the crystallization of tag rich in oleic acid, while the other was attributed to the crystallization of the more saturated tag (tan and che man, 2000; tan and che man, 2002; chiavaro et al., 2010). besides, a less defined exothermic event was also quite evident appearing as a shoulder of the major peak. ital. j. food sci., vol 29, 2017 81 the thermal parameters extrapolated from the cooling thermograms (table 3) exhibited differences among the four studied evoos, probably attributable to the nature of the soil on which they were grown. figure 1. cooling a) and heating b) curves of the evoo samples. the main transitions are indicated with number 1 and 2. evoosc, extra virgin olive oil from soil clay; evooss extra virgin olive oil from soil stony; evoosb, extra virgin olive oil from soil brown; evooslg, extra virgin olive oil from soil limestone and gypsum. very different values were found for evoosc and evooslg, while similar parameters were registered for evoosb and evooss, intermediate from the other two. for evoosc, the major exothermal peak (fig. 1a-peak 1) resulted as the tallest among all the samples, as visible from the significant highest heat flow (table 3). this observation could be related to the highest content of mufa (chiavaro et al., 2010), in particular oleic acid, of this olive oil (table 2). moreover, it is reported that olive oils rich in oleic acid and mufa, crystallized at lower temperature than samples rich in palmitic acid and sfa (chiavaro et al., 2010), as confirmed from our results (tables 2-3). the minor exothermal event (fig. 1a-peak 2) of evooslg was peaked at higher temperatures (table 3) than the other oils. this may be related to the significantly higher degree of lipid saturation (sfa content) of evooslg (table 2), as already reported for olive oil (chiavaro et al., 2010; kotti et al., 2009). evooslg and evoosc exhibited respectively the broadest and the narrowest range of transition (table3). narrow range of transition has been associated to high oleic virgin olive oil (jimenez marquez et al., 2003). about this, a high statistical correlation was found between the oleic acid content and the range of crystallization (p≤0.01; r= 0.964). moreover high statistical correlations were also found between the range of crystallization and oxidative status of the oils by means of peroxide values (p≤0.01; r= 0.912) and oxidative stability (p≤0.01; r= 0.797). vittadini et al. (2003) reported that the ital. j. food sci., vol 29, 2017 82 broadening of the crystallization phenomena is one of the changes related to the formation of lipid oxidation products that could form mixed and disordered triglyceride crystals. these crystals require lower energy to crystallize, and retard the occurrence of phase transition. the crystallization enthalpy on the other hand was not able to discriminate among the four evoo according to the nature of the soil (tab.3), as already observed for different 'chemlali' evoo from different geographical origin (kotti et al., 2009). table 3. dsc data obtained from the cooling and heating thermograms of the different samples. enthalpy (j/g) ton (c°) toff (c°) range (c°) heat flow (w/g) tp1 (°c) tp2 (°c) cooling evoosc 73.46±0.34 a -8.83±0.32 c -52.95±0.67 a 44.12±0.83 c 0.37±0.00 a -43.84±0.45 a -11.36±0.07 c evooss 74.72±2.38 a -6.79±0.14 b -56.79±0.58 b 50.00±0.67 b 0.32±0.02 bc -46.38±0.45 b -9.60±0.51 b evoosb 77.52±1.81 a -7.21±0.00 b -55.70±0.29 b 48.49±0.29 b 0.36±0.02 ab -45.51±0.34 b -10.12±0.16 b evooslg 75.30±4.30 a -5.88±0.08 a -60.77±1.00 c 54.89±1.00 a 0.28±0.01 c -49.67±0.29 c -8.79±0.09 a heating evoosc 83.78±1.56 a -24.75±0.22 a 13.31±0.08 a 38.06±0.17 c 0.30±0.01 c -7.11±0.63 a 9.75±0.07 a evooss 76.20±3.82 ab -27.31±0.22 b 13.07±0.16 a 40.38±0.25 b 0.25±0.01 ab -7.32±0.79 ab 9.81±0.09 a evoosb 79.54±2.08 a -27.26±0.14 b 12.74±0.08 b 40.00±0.08 b 0.28±0.01 bc -8.49±0.14 b 9.90±0.08 a evooslg 70.76±4.23 c -30.25±0.14 c 11.79±0.16 c 42.04±0.21 a 0.24±0.01 a -7.11±0.25 a 8.52±0.08 b data are expressed as mean±standard deviation of three determinations (n = 3, p<0.05). same letters within each column do not significantly differ. range(c°): temperature difference between ton and toff; heat flow (w/g): value measured on the peak 1. evoosc, extra virgin olive oil from soil clay, evooss extra virgin olive oil from soil stony, evoosb, extra virgin olive oil from soil brown, evooslg extra virgin olive oil from soil limestone and gypsum. 3.2.2 heating curves the heating curves of the four evoo (fig. 1b) showed shapes similar to those reported in literature for 'chemlali' samples (kotti et al., 2009). the thermal properties during heating were previously found to be largely influenced by difference in macrocomponents (fa) (chiavaro et al., 2009), despite those curves appeared to be more complex than cooling ones, due to the polymorphism of oils and fats (tan and che man, 2000). multiple exo-endothermic events were observed for all the studied samples (fig. 1b). a first exothermic peak, visible around -25°c could be attributed to the crystallization of the minor components that did not solidify under the cooling regime used in this study and/or to a solid–solid polymorphic transformation resulting from the rearrangement of a portion of the crystals formed during cooling (barba et al., 2013). the following increase of temperature caused two major endothermic events: the melting of the unsaturated components (pufa, mufa) (fig. 1b-peak1), at lower temperatures (~ 7.5°c), followed by that of the sfa ones (fig. 1b-peak2), at higher temperatures (~ 9.5°c), as previously reported (jimenez marquez et al., 2013). peak 1 exhibited a quite different profile between the samples (fig. 1b). it appeared more symmetric for evoosc, ital. j. food sci., vol 29, 2017 83 probably in relation to the highest content of oleic acid and thus, to a more cooperative transition. the presence of a small endothermic event, as a shoulder of the major endothermic peak, was also observed in all the samples (∼ -20°c), and with a lower intensity for evoosc. it is generically attributed to the melting of lowest stables polymorphic forms of tag (e.g. α) (barba et al., 2013). as shown in table 3, the thermal parameters of heating thermograms showed significant difference among the studied 'chemlali' evoo, according to the different nature of the soils. heating enthalpy values were significantly different among samples; evoosc exhibited the highest value and evooslg showed the lowest one. positive correlations between heating enthalpy and oleic acid were found (p≤0.01; r= 0.829) in this study, as also previously observed on evoo (ilyasoglu et al., 2011). evoosc exhibited the highest and evooslg the lowest ton of heating. evooslg moreover had a significant lowest toff than the other oils, in relation to the high instauration of its fa profile, as previously suggested (kotti et al., 2009). finally, evoosb and evooss presented similar values for mostly of the heating thermal properties, probably in relation with the similarity shown in their chemical composition. 3.2.3 heat capacity the variation of the heat capacity as a function of temperature, in the range 26.85 – 296.85°c, is reported in fig. 2 for all the samples. figure 2. heat capacity as a function of temperature (16.85 – 296.85°c) for the four evoo samples. evoosc, extra virgin olive oil from soil clay; evooss extra virgin olive oil from soil stony; evoosb, extra virgin olive oil from soil brown; evooslg, extra virgin olive oil from soil limestone and gypsum. the values obtained at 50°c were in accordance with those reported in literature for olive oil and calculated with the same method (1.99 ± 0.02) (heidenreich et al., 2007). a linear increase of the heat capacity was observed with temperature, showing coefficient of linearity (r2) of 0.9943, 0.9876, 0.9894 and 0.9905 for evoosc, evooss, evoosb and evooslg respectively. the values obtained, by means of interpolation, at room temperature (25°c) are summarized in table 4 ranging from 2.1 to 1.8 (j/gc°) for evooss and evoosc 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 0 50 100 150 200 250 300 350 evoosc evooss evoosb evooslg heat capacity (j/g°c) temperature (°c) ital. j. food sci., vol 29, 2017 84 respectively, without any significant differences. thus, the measurement of the heat capacity seems to not discriminate among the evoo according to the nature of the soil. 3.3. physical analysis 3.3.1 viscosity edible oils are almost all newtonian liquids (kalogianni et al., 2011). the linear relationship of shear stress to shear rate found in this study (data not shown) indicates that all the vegetable oil samples in the tested shear rate conditions exhibited a newtonian behaviour. the viscosity value at room temperature (25°c) was therefore obtained from the fitting of the slope of experimental shear stress-shear rate data to the newton’s law of viscosity equation (eq. 1) (fasina et al., 2008) and reported in table 4. table 4. physical quality parameters of evoo samples. evoosc evooss evoosb evooslg viscosity (mpa s) 59.21±0.12b 61.58±0.24a 61.79±0.19a 59.24±0.12b heat capacity (j/gc°) 1.81±0.07a 2.06±0.18a 1.89±0.07a 1.88±0.09a colour l* 57.67±1.15a 57.67±0.58a 56.00±0.00a 57.67±0.58 a a* -3.67±0.58a -5.00±0.00b -5.33±0.61b -3.33±0.59a b* 14.33±1.53c 21.00±0.00b 41.0±2.65a 14.0±1.73c data are expressed as mean standard deviation of three determinations. different letters in the same row are statistically different (p<0.05). evoosc, extra virgin olive oil from soil clay, evooss extra virgin olive oil from soil stony, evoosb, extra virgin olive oil from soil brown, evooslg extra virgin olive oil from soil limestone and gypsum. the values of viscosity found in this study are in accordance to that reported from other authors (gila et al., 2015; bonnet et al., 2011) on virgin olive oils at room temperature. evoosb and evooss showed values of viscosity (61.6 and 61.8 mpas respectively) significantly higher than that of evoosc and evooslg (59.2 mpas for both). some authors reported slight but significant correlation between viscosity and olive oil composition (gila et al. 2015). gila et al. (2015) observed an increase of viscosity increasing the oleic acid content of virgin olive oils from different varieties. bonnet et al. (2011) affirmed however that the intravarietal variance of the fatty acid and tag compositions is much smaller than the intervarietal variance, therefore viscosity differences should not be visible. thus, the differences found in this study may be ascribable to other factors influencing the final composition, such as the oxidative status, as effect of the soil nature. in support of this hypothesis, a correlation was found in this study between viscosity measurement and the oxidative stability of the oils (p≤0.05 r=0.617). it is reported that lipid oxidation products may weak and/or hinder intermolecular bonding between tag molecules leading to the increase of the oil viscosity (vittadini et al., 2003). ital. j. food sci., vol 29, 2017 85 3.3.2 instrumental colour the colour of the oils showed significant differences related to the nature of the soil, except for l* (table 4), which was found to be not statistically different among the samples, even if the lowest value was registered for the evoosb, the sample richest in carotenoids. in general, l* is reported to increase with the reduction of the pigment content in the oils, as pigments would capture part of the light, instead of transmitting it (cerretani et al., 2008). the values of a*, ranging from -3.3 to -5.3, described a green colour for all the samples. it resulted significantly higher for evoosc and evooslg than for the other two oils. the values of b*, representing the yellow colour, ranged from 41 to 14. evoosb resulted the most yellow among all the samples in relation with the highest content of carotenoids, as already reported (moyano et al., 2008). two pearson’s correlations were also found between the chlorophylls/carotenoids ratio and the chromatic coordinates a* (p<0.01, r=0.841) and b* (p<0.01, r= -0.828), confirming the function of the pigments on the color of the studied olive oils, as affected by the soil nature. 3.4. pca analysis based on previous works (caponio et al., 2013; chiavaro et al., 2013), in which pca analysis was successfully performed to discriminate among olive oils according to different refined steps or oxidative status by means of the correlation among thermal and chemical properties, it was again proposed in this study to tentatively discriminate among the four evoo according to the nature of the soils. twenty one variables were selected after factor extraction using as selection criteria loading values higher than 0.7, with pc1 and pc2 being the first two principal components that explained about 85% of the total variance. in fig. 3a, the projection of the variables on the factor plane is reported. all the dsc thermal properties, except the cooling enthalpy, were represented on the plane and better described by the pc1. most of the dsc thermal properties showed positive factor loadings on pc1, being instead negative only for onset temperature of cooling (ton cooling) and peak 2 cooling temperature at the maximum (tp2 cooling). as far as the chemical properties are concerned, positive factor loadings on pc1 were observed for mufa and osi, being instead negative for sfa, pufa and pov. few variables were represented on pc2, having this component a low contribution on the total results, as revealed from the high distance of the vectors from the boarders if compared to the ones on pc1. positive factor loading on pc2 were showed by viscosity, carotenoids and colour parameter b*, being instead negatives for l*, a* and peak 1 heating temperature at the maximum (tp1 heating). the heat capacity was not able to discriminate among the four evoos, thus it was excluded from the factor analysis. the score plot obtained for the samples was shown in fig. 3b. pc1 clearly divided evooslg and evoosc in different clusters, having the first negative scores and the second positive ones. evooss showed an intermediate behavior among all the samples, being instead evoosb better described by pc2 with positive scores. by comparing the score with loading plots it is evident how evooslg and evoosc were well discriminate by the thermal parameters, being themselves influenced by the fatty acids composition and the oxidative stability. in particular, evoosc, the evoo richest in mufa, resulted well discriminated by the thermal properties of the cooling and heating major peak, confirming previous correlation found among this peaks with this class of fatty acids (chiavaro et al., 2007). ital. j. food sci., vol 29, 2017 86 figure 3. principal component analysis (pca) results obtained for the two principal components, showing a) the projection of the cases on the factor plane, and b) the projection of the variables on the factor plane. sfa, saturated fatty acids; mufa, monounsaturated fatty acids; pufa, polyunsaturated fatty acids; osi, oxidative stability; pov, peroxide value; ton, onset temperature of transition; toff, offset temperature of transition; hfp, peak heat flow at the maximum; tp, peak temperature at the maximum; ∆h, enthalpy change of transition; evoosc, extra virgin olive oil from soil clay; evooss extra virgin olive oil from soil stony; evoosb, extra virgin olive oil from soil brown; evooslg extra virgin olive oil from soil limestone and gypsum. ital. j. food sci., vol 29, 2017 87 evooslg on the other hand, with its high content of sfa and pufa and the related pov value, was well described by the cooling parameters of the thermal event peaking at higher temperatures: tp2 and ton of cooling, having also an inverse relation with the heating enthalpy. evooss and evoosb had an intermediate composition in comparison to the other two oils, thus they showed intermediate values on pc1. evoosb was better discriminated on pc2 particularly from the viscosity value and from the b* colour parameter, being the latter related to the carotenoid content, as observed in this study and already reported in literature (moyano et al., 2008). 4. conclusions the current study has investigated for the first time how the soil may impact on different quality parameters of four 'chemlali' extra virgin olive oils considering physical and thermal variables other than the traditional chemical parameters. the pca analysis helped in identifying the variables able to discriminate among the four studied evoos. evoosc and evooslg resulted better discriminated by their chemical composition and thus by the related thermal properties. in particular, evoosc resulted as the sample with the highest mufa content, exhibiting the highest heat flow of the first cooling peak and the narrowest cooling range in relation with lowest pov. evooslg was the richest in sfa and pufa showing the lowest heating enthalpy and the highest cooling and heating ranges together with the lowest osi value. evoosb was distinguished on the base of its colour, being more yellow than the others, as revealed from the b* parameter in relation to the highest carotenoid content, and having also the highest osi value. evooss showed intermediate properties to the other samples. these first encouraging results have revealed the effective influence of the soil on chemicophysical and thermal properties of 'chemlali' evoos which need further confirmation through the analysis of a larger set of samples to sort out the best pedologic conditions for the olive cultivation. their relation with the soil composition should be also considered in the future. references astm e1269-05 “test method for determining specific heat capacity by differential scanning calorimetry,” astm international, west conshohocken, pa. barba l., arrighetti g. and calligaris l. 2013. crystallization and melting properties of extra virgin olive oil studied by synchrotron xrd and dsc. eur. j. lipid sci. technol. 115(3):322. bedbabis s., trigui d., ahmed c.b., clodoveo m.l., camposeo s., vivaldi g.a. and rouina b.b. 2015. long-terms effects of irrigation with treated municipal wastewater on soil, yield and olive oil quality. agric. water manage. 160:14. ben hassine k., el riachy m., taamalli a., malouche d., ayadi m., talmoudi k., aouini m., jlassi y., benincasa c., romano e., perri e., kiristakis a., hamdi m., grati‐kammoun n. and hammami m. 2014. consumer discrimination of chemlali and arbequina olive oil cultivars according to their cultivar, geographical origin, and processing system. eur. j. lipid sci. technol. 116(7):812. ben hassine k., taamalli a., slama m.b., khouloud t., kiristakis a., benincasa c., perri e., malouche d., hammami m., bornaz s. and grati-kammoun n. 2015. characterization and preference mapping of autochthonous and introduced olive oil cultivars in tunisia. eur. j. lipid sci. technol. 117(1):112. ben mansour a., gargouri b., flamini g. and bouaziz m. 2015. effect of agricultural sites on differentiation between chemlali and neb jmel olive oils. j. oleo sci. 64(4):381. bonnet j.p., devesvre l., artaud j. and moulin p. 2011. dynamic viscosity of olive oil as a function of composition and temperature: a first approach. eur. j. lipid sci. technol. 113(8):1019. ital. j. food sci., vol 29, 2017 88 caponio f., chiavaro e., paradiso v.m., paciulli m., summo c., cerretani l. and gomes t. 2013. chemical and thermal evaluation of olive oil refining at different oxidative levels. eur. j. lipid sci. tech. 115(10):1146. cerretani l., motilva m.j., romero m.p., bendini a. and lercker g. 2008. pigment profile and chromatic parameters of monovarietal virgin olive oils from different italian cultivars. eur. food res. technol. 226(6):1251. chiavaro e., barnaba c., vittadini e., rodriguez-estrada m.t., cerretani l. and bendini a. 2009. microwave heating of different commercial categories of olive oil: part ii. effect on thermal properties. food chem. 115(4):1393. chiavaro e., cerretani l., paciulli m., vecchio s. 2012. kinetic evaluation of non-isothermal crystallization of oxidized extra virgin olive oil. j. therm. anal. calorim. 108(2):799. chiavaro e., cerretani l., paradiso v.m., summo c., paciulli m., toschi t.g. and caponio f. 2013. thermal and chemical evaluation of naturally auto-oxidised virgin olive oils: a correlation study. j. sci. food agric. 93(12):2909. chiavaro e., mahesar s.a., bendini a., foroni e., valli e. and cerretani l. 2011. dsc evaluation of olive oil during accelerated oxidation. ital. j. food sci. 23(2):164. chiavaro e., rodriguez-estrada m.t., bendini a. and cerretani l. 2010. correlation between thermal properties and chemical composition of italian virgin olive oils. eur. j. lipid sci. tech. 112(5):580. commission regulation 2568/91. official journal of the european communities. 1991. l248, 1-82. commission regulation 1429/92. official journal of the european communities 1992. l150, 17-20. european community. 2011. commission regulation no. 61/2011 of 24 january 2011 amending regulations no. 2568/91eec on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis. off. j. eur. commun. l23, 1-14. fasina o.o. and colley z. 2008. viscosity and specific heat of vegetable oils as a function of temperature: 35°c to 180°c. int. j. food prop. 11(4):738. garcía-gonzález d.l and aparicio r. 2010. research in olive oil: challenges for the near future. j. agric. food chem. 58(24):12569. gila a., jiménez a., beltrán g. and romero a. 2015. correlation of fatty acid composition of virgin olive oil with thermal and physical properties. eur. j. lipid sci. technol. 117(3):366. gutiérrez rosales f. 1989. determinación de la estabilidad oxidativa de aceites de oliva vírgens. comparación entre el método del oxígeno activo (aom) y el método rancimat (determinación of the oxidative stability of virgin olive oils. comparison between aom and rancimat methods). grasas y aceites 40:1. hannachi h., msallem m., ben elhadj s. and el gazzah m. 2007. influence du site géographique sur les potentialités agronomiques et technologiques de l’olivier (olea europaea l.) en tunisie. c r biol. 330(2):135. heidenreich s., langner t. and rohm h. 2007. heat capacity of cheese. determination or calculation? j. therm. anal. calorim. 89(3):815. ilyasoglu h. and ozcelik b. 2011. determination of seasonal changes in olive oil by using differential scanning calorimetry heating thermograms. j. am. oil chem. soc. 88(7):907. iooc 2004. olive oil exportations. international olive oil council. 2011. http://www.internationaloliveoil.org. issaoui m, flamini g, brahmi f, dabboua s, ben hassine k, taamali a, chehab h, ellouz m, zarrouk m and hammami m. 2010. effect of the growing area conditions on differentiation between chemlali and chétoui olive oils. food chem. 119(1):220. jimenez marquez a. 2003. preliminary results of the characterization of mixtures of olive oil by differential scanning calorimetry. cienc. technol. aliment. 4:47. kalogianni e.p., karapantsios t.d. and miller r. 2011. effect of repeated frying on the viscosity, density and dynamic interfacial tension of palm and olive oil. j. food eng. 105(1):169. kotti f., chiavaro e., cerretani l., barnaba c., gargouri m. and bendini a. 2009. chemical and thermal characterization of tunisian extra virgin olive oil from chetoui and chemlali cultivars and different geographical origin. eur. food res. technol. 228(5):735. ital. j. food sci., vol 29, 2017 89 minguez-mosquera m.i., rejano-navarro l., gandulrojas b., sanchez gomez a.h. and garrido-fernandez j. 1991. colorpigment correlation in virgin olive oil. j. am. oil chem. soc. 86(5):332. moyano m.j., melendez martinez a.j., alba j. and heredia f.j. 2008. a comprehensive study on the colour of virgin olive oils and its relationship with their chlorophylls and carotenoids indexes (i): ciexyz non-uniform colour space. food res. inter. 41(5):505. papadia p., del coco l., muzzalupo i., rizzi m., perri e., cesari g., simeone v., mondelli d., schena f.p. and fanizzi f.p. 2011. multivariate analysis of 1h-nmr spectra of genetically characterized extra virgin olive oils and growth soil correlations. j. am. oil chem. soc. 88(10):1463. romero n., saavedra j., tapia f., sepúlvedaa b. and aparicio r. 2016. influence of agroclimatic parameters on phenolic and volatile compounds of chilean virgin olive oils and characterization based on geographical origin, cultivar and ripening stage. j. sci. food agric. 96(2):583. santos j.c.o., santos m.g.o., dantas j.p., conceição m.m., athaide-filho p.f. and souza a.g. 2005. comparative study of specific heat capacities of some vegetable oils obtained by dsc and microwave oven. j. therm. anal. calorim. 79(2):283. tan c.p. and che man y.b. 2000. differential scanning calorimetric analysis of edible oils: comparison of thermal properties and chemical composition. j. am. oil chem. soc. 77(2):142. tan c.p. and che man y.b. 2002. differential scanning calorimetric analysis of palm oil, palm oil based products and coconut oil: effect of scanning rate variation. food chem. 76(1):89. vittadini e., lee j.h., frega n.g., min d.b. and vodovotz y. 2003. dsc determination of thermally oxidized olive oil. j. am. oil. chem. soc. 80(6):533. paper received march 23, 2016 accepted july 22, 2016 ijfs#619_bozza ital. j. food sci., vol 29, 2017 434 paper grey relation analysis of solar drying process parameter on copra g. padmanaban*1, p.k. palani2 and v.m.m. thilak1 1 department of mechanical engineering, rathinam technical campus, coimbatore, tamilnadu, india 2 department of mechanical engineering, govt. college of technology, coimbatore, tamilnadu, india *corresponding author. agpn1977@gmail.com abstract the methodology for the optimization of the drying parameters on solar drying of copra was investigated and studied in this paper. this paper investigates the influence of the process parameters like initial mass, inclination angle and time period on the output parameters such as weight reduction rate and moisture content. based on the analysis, optimal levels of parameters were determined and the same was validated through the confirmation test. the confirmation results reveal that, there is considerable improvement in the weight reduction rate, moisture content and grey relational grade and they improved by 37.36%, 32.28% and 32.94 % respectively. it is observed that the drying performance can be effectively improved with respect to the initial parametric setting. keywords: weight reduction rate (wrr), moisture content, taguchi, grey relational analysis & design of experiment ital. j. food sci., vol 29, 2017 435 1. introduction the use of solar dryers in the drying of agricultural products can significantly reduce or eliminate product wastage, food poisoning and so on, thereby, enhancing productivity of the farmers in obtaining higher revenue. drying using solar radiation, that is, drying under direct sunlight, is one of the oldest techniques used by man to preserve agriculture based food and non-food products (chandrakumar and jiwanlal, 2013). this form of energy is free, renewable and abundant in any part of the world, especially for countries situated in the tropics. however, in order to maximize its advantage and optimize the efficiency of drying using solar radiation, appropriate measures in terms of technology need to be taken in order to make this technique a sustainable one. such technology is known as solar drying and it is fast becoming a popular option to replace mechanical thermal dryers, owing to the high cost of fossil fuels which is growing in demand, but dwindling in supply. srinivasan and balusamy (2015) have studied the performance of forced convection solar dryer, integrated with heat storage materials for processing copra. pardhi and bhagoria (2013) have carried out preliminary investigations and under controlled condition of drying experiments constructed a mixed-mode solar dryer with forced convection, using smooth and rough plate solar collector. dharmalingam et al. (2015) have used l27 orthogonal array to determine the signal-to-noise ratio (s/n ratio) and analysis of variance (anova) was conducted. based on the experiments, they concluded that pulse on-time and electrolyte concentration are the most significant parameters for material removal rate (mrr). gap current and electrolyte concentration which are the influencing parameters for lesser overcut were experimentally investigated. dharmalingam et al. (2014a) have found that the influence of the process parameters is through the response surface methodology and grey relational analysis. dhanushkodi et al. (2014) reported that a directly forced convection solar drier, integrated with recirculation of air has been developed and its performance is tested for drying grapes under the meteorological conditions of coimbatore, india. the specific moisture extraction rate was estimated to be 0.87 kg/kwh3. dharmalingam et al. (2015) also stated that anova was used for identifying the significant parameters affecting the responses. 2. materials and methods 2.1 description of the solar dryer the developed solar dryer (fig. 2.1) consists of a glass sheet-covered flat plate solar collector, used to simplify construction and reduce costs. the solar collector is connected directly to the drying chamber without any additional air ducts. the top surface of the insulator in the collector is painted black to absorb solar radiation. the collector is covered with a transparent ultra violet (u.v)-stabilized glass sheet that is fixed to the collector frame using reinforced plastic clamps in the drying chamber, and a wire mesh is placed on top of the insulators. a glass is placed on top of the black v groove sheet, on which the product to be dried are spread. this arrangement allows drying air to flow around the whole surface of the product being dried. one side of this sheet is fixed to the drying chamber frame and the other side is fixed to a metal tube, allowing the sheet to be rolled up and down for loading and unloading the dryer. this fixing method is designed to facilitate the replacement of the sheets. in general, the transparent sheet can be used for 1-2 years and the air could last for 3-5 years. the glass is installed at the back of the collector ital. j. food sci., vol 29, 2017 436 to suck ambient air into the collector. the blowers are intentionally installed below to constantly reduce its temperature, thus, maintaining its efficiency. both the collector and the drying chamber are installed on mild steel block substructures. all parts of the dryer, including the back insulator and metal frames were designed using a modular concept, which facilitates the transport and installation of the dryer. this solar dryer uses solar energy both in the thermal form of the drying process and in the electrical form for driving the blower, by means of the solar collector and solar module, respectively. therefore, the dryer could be used in rural areas where there is no supply of electricity. the dryer is a passive system in the sense that it has no moving parts. the sun rays entering through the collector glazing energizes it. the absorption of the rays is enhanced by the inside surface of the collector painted black and the absorbed energy heats the air inside the collector. the greenhouse effect achieved within the collector drives the air current through the drying chamber. if the vents are open, the hot air rises and escapes through the upper vent in the drying chamber, while cooler air at ambient temperature enters through the lower vent in the collector. the fig. 2.2 shows the copra drying process in the solar dryer. figure 2.1. arrangement of solar dryer. figure 2.2. copra in solar dryer. ital. j. food sci., vol 29, 2017 437 2.2. optimization methodology the optimization of process parameters is the key step in the taguchi method (dharmalingam et al. 2014b). twenty seven experimental runs (l27) based on the orthogonal array (oa) of taguchi methods have been carried out (dharmalingam et al. 2014c). the multi-response optimization (grey relational analysis) of the process parameters has been performed for drying, using weight reduction rate and moisture content. the drying time, weight reduction rate and moisture content are noted twice for every trial. 3. results and conclusions 3.1. major results and inferences the assignment of factors with their levels identified inthis investigation are given in table 3.1. table 3.1. drying process parameters and their corresponding levels. symbol factors level 1 level 2 level 3 a initial mass mi (g) 1000 750 500 b inclination angle(°) 30 45 60 c time period (hr) 11am -12 noon 1-2 pm 3-4 pm based on the taguchi’s l27 oa, drying experiments were conducted on solar dryer for copra. the experimental results were gathered for each trial. the s/n ratios were calculated for all the responses since the objective of this work was to maximize the weight reduction rate and the minimization of moisture content. therefore, for weight reduction rate (wrr), the larger-is-better type was considered and for moisture content, smaller-isbetter type was considered for the analysis. the s/n ratio was computed for each of the twenty-seven trial conditions for the weight reduction rate and moisture content and is shown in table 3.2 below. the weight reduction rate (%) in the table 3.2 was calculated by measuring the weight of copra after drying between different time intervals. the optimal setting levels for each factor resulting from the s/n ratios are shown in table 3.3. ital. j. food sci., vol 29, 2017 438 table 3.2. experimental results for l27 oa of copra. trial no. initial mass (mi) g inclination angle (º) time period weight reduction rate (%) moisture content s/n ratio for wrr s/n ratio for moisture content grey relational weight reduction rate (%) grey relational moisture content grey grade 1 1000 30 11-12 23.25 69.38 27.328 -36.825 0.5052 0.3358 0.4205 2 1000 30 1-2 36.37 42.18 31.215 -32.502 0.8871 0.4159 0.6515 3 1000 30 3-4 15.12 21.08 23.591 -26.477 0.3940 0.6233 0.5086 4 1000 45 11-12 27.12 33.09 28.666 -30.394 0.5878 0.4707 0.5293 5 1000 45 1-2 34.32 45.60 30.711 -33.179 0.8144 0.4010 0.6077 6 1000 45 3-4 14.46 22.36 23.203 -26.989 0.3831 0.5980 0.4905 7 1000 60 11-12 26.32 35.12 28.406 -30.911 0.6160 0.4560 0.5360 8 1000 60 1-2 33.35 44.06 30.462 -32.881 0.7827 0.4074 0.5951 9 1000 60 3-4 12.21 24.06 21.734 -27.626 0.3468 0.5692 0.4580 10 750 30 11-12 25.32 37.06 28.069 -31.378 0.5479 0.4435 0.4957 11 750 30 1-2 37.35 51.56 31.446 -34.246 0.9249 0.3794 0.6522 12 750 30 3-4 13.35 14.86 22.510 -23.440 0.3333 0.8325 0.5829 13 750 45 11-12 24.35 35.06 27.730 -30.896 0.5704 0.4564 0.5134 14 750 45 1-2 37.31 50.46 31.437 -34.059 0.9233 0.3830 0.6532 15 750 45 3-4 13.35 18.48 22.510 -25.334 0.3650 0.6884 0.5267 16 750 60 11-12 28.52 32.18 29.103 -30.152 0.6716 0.4780 0.5748 17 750 60 1-2 35.51 70.72 31.007 -36.991 0.8556 0.3333 0.5945 18 750 60 3-4 14.14 12.48 23.009 -21.924 0.3457 1.0000 0.6729 19 500 30 11-12 29.35 35.42 29.352 -30.985 0.6414 0.4540 0.5477 20 500 30 1-2 38.36 46.98 31.678 -33.438 0.9663 0.3955 0.6809 21 500 30 3-4 15.12 15.90 23.591 -24.028 0.3940 0.7817 0.5879 22 500 45 11-12 28.20 28.08 29.005 -28.968 0.6631 0.5168 0.5900 23 500 45 1-2 38.35 47.42 31.675 -33.519 0.9659 0.3938 0.6798 24 500 45 3-4 16.98 21.90 24.599 -26.809 0.4258 0.6066 0.5162 25 500 60 11-12 26.12 38.48 28.339 -31.705 0.6112 0.4351 0.5232 26 500 60 1-2 39.14 47.00 31.852 -33.442 1.0000 0.3954 0.6977 27 500 60 3-4 13.35 23.20 22.510 -27.310 0.3650 0.5831 0.4741 footnote: the values showed in bold are optimized values. ital. j. food sci., vol 29, 2017 439 fig. 3.1 shows the residual plots for grey grade. table 3.4 shows the anova table, which indicates the significance of process parameters on the weight reduction rate and moisture content. in the table 3.4, the f-test is a matter of including the correct variances in the ratio. the f-statistic is this ratio of variation between sample means to the variation within the samples. table 3.3. response table for the grey relational grade. table 3.4. anova for grey relation grade. factors design of factor sum of squares mean square=sumof squares /2 f value f 0.05 %of contribution=sum of squares/total sum of squares initial mass 2 0.002141 0.00107 13.55 0 1.56 significant inclination angle 2 0.015905 0.007952 100.69 0 11.62 significant time period 2 0.117244 0.056822 742.26 0 85.66 significant error 20 0.00158 0.000079 1.15 total 26 0.13687 100% s = 0.00888699; r-sq = 98.85%; r-sq (adj) = 98.50%. footnote: the values showed in bold are optimized values. figure 3.1. residual plots for grey grade. process parameters level 1 level 2 level 3 initial mass mi (g) 0.5330 0.5851 0.5886 inclination angle(0c) 0.5698 0.5674 0.5696 time period 0.5256 0.6458 0.5353 *optimum levels mean grey grade = 0.5466 ital. j. food sci., vol 29, 2017 440 3.2. analysis for weight reduction rate and moisture content of copra the optimal values for the maximum weight reduction rate has an initial mass of 500 g, inclination angle is 300 and time period is between 1-2 an (after noon). the interaction has been plotted to pictorially depict the interaction process parameters on weight reduction rate and moisture content. in the full interaction plot, two panels per pair of process parameters have been shown in figs. 3.2 and 3.3. figure 3.2. interaction plot for wrr. figure 3.3. interaction plot for mc. ital. j. food sci., vol 29, 2017 441 3.3 confirmation test after identifying the most influential parameters, the final phase is to verify the predicted results (wrr and moisture content) by conducting the confirmation test. the a3b1c2 through the gra is an optimal parameter combination of the drying process. therefore, the combination a3b1c2 was seen as a confirmatory test. the predicted grey relational grade can be calculated using the optimum parameters as where, αo = average grey relational grade of the optimal level, αm = overallmean grey relational grade. α predicted= 0.57045 +(0.58534 0.57045)+(0.645329-0.57045)+(0.58534 0.57045) = 0.6722 table 3.4 shows the confirmatory test for weight reduction and moisture content of copra. based on the confirmatory test, the weight reduction and moisture content were improved by 37.36 % and 32.28 % respectively, with respect to the initial parametric setting. the optimized parameter combination suggested for the higher wrr and lower moisture content has an intial mass of 500 g, the inclination angle of 30o and time period of 1-2 hours. the percentage contribution of factors on grey relational grade within a period of time is 86% and inclination angle is 12%. the error and initial mass percentage of contribution of factors are 1% respectively. table 3.4. confirmatory test table. the following data were observed from the present investigationwhich focuses on optimization and analysis of the solar drying process parameters of copra using grey relational analysis and anova. a. based on the confirmatory test, improvement in weight reduction rateand moisture content is 37.36 % and 32.28 % respectively. b. the grey relational grade is improved by 32.94 %. c. the parameter combination suggested for the higher wrr and lesser moisture content have an initial mass of 500 g, inclination angle of 300 and time period 1-2 an. d. the results of anova, the time period and inclinational angle are the significant drying parameters which affect the weight reduction rate and moisture content. the modified design of the direct air dryer modelled during this research will maximize the efficiency of drying using solar radiation. this modified design can be used to replace initial levels of drying parameters optimal combination levels of drying parameters prediction experiment improvement level a1b1c1 a3b1c2 a3b1c2 wrr (gr) 23.25 38.36 38.36 37.36% moisture content 69.38 46.98 46.98 32.28% grey grade 0.5123 0.6722 0.6811 32.94% ital. j. food sci., vol 29, 2017 442 mechanicaland thermal dryers which are dependent on high cost fuel. this modified design is brought into existence by keeping the copra as the food component. the researches on carrying out direct drying of other food components using this modified design can be carried out as future research work. references chandrakumar b.p and jiwanlal l.b. 2013. development and performance evaluation of mixed-mode solar dryer with forced convection. int. j. energy environ. 4(1):23. dhanushkodi s., wilson v.h., sudhakar k. 2014. thermal performance evaluation of indirect forced cabinet solar dryer for cashew drying. american-eurasian j. agric. & environ. sci. 14(11):1248. dharmalingam s,. marimuthu p., raja k., nithyapathi c., babu b. and siva m. 2014a. experimental investigation on electrochemical micro machining of al-10%wt sicp based on taguchi design of experiments, int. j. r. mec. eng. 8(1):80. dharmalingam s., marimuthu p. and raja k. 2014b. machinability study on al10% tic composites and optimum setting of drilling parameters in electrochemical micro machining using grey relational analysis. int. j. lat. amer. appd res. 44(4):331. dharmalingam s., marimuthu p., raja k., pandyrajan. r. and surendar s. 2014c. optimization of process parameters on mrr and overcut in electrochemical micro machining on metal matrix composites using grey relational analysis. int. j. eng. tech. 6(2):519. pardhi c.b. and j.l bhagoria. 2013. development and performance evaluation of mixed-mode solar dryer with forced convection, int. j. energy environ. 4:23. srinivasan r and balusamy t. 2015. comparative studies on open sun drying and forced type (mixed mode) solar drying of bitter gourd. j. chem. pharm. sci. 974:2115. paper received september 10, 2016 accepted march 11, 2017 ijfs#767_bozza ital. j. food sci., vol 29, 2017 518 paper saccharomyces cerevisiae biodiversity in monferrato, north west italy, and selection of indigenous starter cultures for barbera wine production k. rantsiou*a, f. marengoa, v. englezosa, f. torchiob, s. giacosaa, l. rollea, v. gerbia and l. cocolina a university of turin, department of agriculture, forest and food sciences, largo paolo braccini 2, 10095 grugliasco, torino, italy b università cattolica del sacro cuore, istituto di enologia e ingegneria agro-alimentare, piacenza, italy *corresponding author. tel.: +39 011 670 8870; fax: +39 011 670 8549 e-mail address: kalliopi.rantsiou@unito.it abstract the aim of this study was to examine the biodiversity of saccharomyces cerevisiae isolates from barbera grapes and musts, from the monferrato area, in the piedmont region – north west italy. an interdelta element pcr analysis was used to identify and discriminate 636 s. cerevisiae isolates at a strain level. ninety-six s. cerevisiae that showed different molecular fingerprints were characterized through physiological tests and laboratory scale fermentations. a chemical analysis of experimental wines obtained from inoculated fermentations showed significant differences between the wines. the main variables considered in the strain differentiation were the residual sugars and the production of acetic acid, which ranged from 148.64 to 3.44 g/l and from 0.20 to 0.60 g/l, respectively. as a consequence, strain variability should be considered as a relevant resource to select suitable starter cultures in order to improve or characterize wines with a close bond to the geographic region. keywords: saccharomyces cerevisiae, yeast biodiversity, indigenous starter, interdelta pcr, selection ital. j. food sci., vol 29, 2017 519 1. introduction wine production is an ancient tradition that has been carried out for centuries through the spontaneous fermentation of grape juice, which takes place due to the presence of indigenous yeasts from different genera and species (fleet, 2003; pretorius, 2000; romano et al., 2003). the number of species and their presence during fermentation depends on several factors (pretorius et al., 1999), which lead to subsequent wine quality variations from region to region, but also from one year to another, and all this makes the outcome of spontaneous fermentation difficult to predict (pretorius, 2000). in an attempt to address this issue, many winemakers have used pure commercial saccharomyces cerevisiae cultures inoculated into the must (pretorius, 2000). however, it has been suggested that native s. cerevisiae strains are better suited to the micro-area climatic conditions of the wine production region (lopes et al., 2002) and can therefore more easily dominate the natural biota (la jeune et al., 2006). s. cerevisiae, the most relevant species in winemaking, is usually chosen as the wine yeast, and the particular strain is chosen according to a set of physiological features that are indicative of their potential usefulness for industrial wine production. in addition to the primary end products of the glycolytic fermentation of glucose and fructose, certain oenological criteria must be considered in order to select yeast strains that show desirable characteristics, including: tolerance and high ethanol production, exhaustion of the sugar in must and high fermentation activity, growth at high sugar concentrations, high glycerol production, resistance and low sulphur dioxide production, good enzymatic profile (high β-glucosidase and proteolytic activities) and low acetic acid formation (estevezarzoso et al., 2000). at present, there is increasing interest, in the wine community, in the use of indigenous s. cerevisiae strains that may contribute to the overall sensorial quality of wine and reflect the characteristics of a given region, even in guided fermentations using selected s. cerevisiae starter cultures (capece et al., 2010; suzzi et al., 2012). recently, in an attempt to respond to these aspects coupled with the current emphasis on the preservation of all forms of genetic biodiversity, some research groups have focused on the selection of yeasts from restricted areas (settanni et al., 2012; francesca et al., 2009; orlic et al., 2007; lopes et al., 2007). we have previously extensively studied the indigenous mycobiota originating from the barbera grapes from the monferrato area, piedmont region, north-west italy (alessandria et al., 2015). barbera grapes produce a ruby-red coloured wine with berry, cherry, plum and spicy flavours, depending on the clone, as well as the location and the age of the plant (bosso et al., 2011). in this study, s. cerevisiae isolates were characterized to establish their genetic and technological variability in order to contribute to the preservation of the s. cerevisiae genetic resources of the barbera of monferrato terroir. interdelta-pcr was used to help establish the genetic diversity of the isolates. physiological tests, which focused on the production of extracellular hydrolytic enzymes and on their growth in different ethanol and total so2 concentrations, were then conducted. finally, selected genotypes were used to ferment barbera must, during micro-fermentation trials, in order to evaluate their fermentation potential. 2. materials and methods 2.1. fermentation set up and yeast isolation spontaneous fermentations were conducted using vitis vinifera l. barbera grapes obtained from fifteen different vineyards (fig. 1), located in five areas in the asti and alessandria ital. j. food sci., vol 29, 2017 520 districts of the piedmont region (alessandria et al., 2015). the vineyards, which were identified on the basis of their geographical locations, were: 1 (murisengo), 2 (san martino alfieri), 3 (costigliole d'asti), 4 (isola d'asti), 5 (montegrosso d'asti), 6 (agliano terme), 7 (vinchio), 8 (nizza monferrato), 9 (incisa scapaccino), 10 (loazzolo), 11 (ricaldone), 12 (alice bel colle), 13 (acqui -terme crocera south west zone), 14 (acqui terme crocera south est zone) and 15 (acqui terme dannona zone). approximately 25 kg of healthy grapes, without signs of bird damage or botryotinia fuckeliana infection, were harvested. the grapes were crushed in the laboratory and the obtained juice (about 15 l volume) was transferred to sterile jugs where it underwent spontaneous fermentation at room temperature (between 22 and 25°c). yeasts were isolated from each container at the beginning of the fermentation (after 1 day and 3 days), in the middle (after 7 days) and when alcoholic fermentation was completed. the alcoholic fermentation was monitored with a densitometer. aliquots (0.1 ml each) of several decimal dilutions, in a 0.1% ringer solution (oxoid, milan, italy), were plated on wallerstein laboratory nutrient (wln) medium (oxoid) (pallmann et al., 2001). the plates were incubated at 28°c for 5 days. wln allows the presumptive identification of the yeast species according to the colony morphology and colour (urso et al., 2008). at least 10 colonies were selected from each sample and at each fermentation stage and were isolated on wln; priority was given to putative colonies of saccharomyces spp. the isolates were stored at −80 °c in ypd broth (1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose, all obtained from oxoid) after the addition of glycerol (30%, v/v) (sigmaaldrich, milan, italy). 2.2. yeast identification the putative saccharomyces spp. isolates were subsequently identified and simultaneously differentiated at a strain level on the basis of a pcr interdelta element analysis (δ-pcr). in order to conduct the δ-pcr analysis, the total dna was extracted from 1 millilitre of an overnight culture in ypd broth, according to cocolin et al. (2000), quantified using a nanodrop nd-1000 spectrophotometer (celbio, milan, italy) and standardized at 100 ng/µl. the delta12 (5′-tcaacaatggaatcccaac-3′) and delta21 (5′catcttaacaccgtatatga-3′) oligonucleotide primers were used to amplify regions between the repeated interspersed delta sequences (legras and karst, 2003). amplification reactions were performed with a ptc-200 dna engine mj research thermal cycler (biorad, milan, italy) using the following programme: initial denaturation at 95°c (5 min), 35 cycles of denaturing at 94°c (1 min), annealing at 50°c (1 min), extension at 72°c (1 min) and a final extension at 72°c (10 min). the pcr products were separated in 1.5% agarose gels and stained with ethidium bromide. the resulting fingerprints were analyzed by means of the bionumerics v4.0 software package (applied-maths, sint-martenslatem, belgium). similarity among the digitized profiles was calculated using the pearson correlation, and an average linkage (upgma) dendrogram was derived from the profiles. a coefficient of correlation of 85% was arbitrarily selected to distinguish the clusters. the yeasts that were not amplified with δ-pcr, were subsequently identified by using the rflp of the ribosomal region method as described in alessandria et al. (2015). 2.3. physiological characterization 2.3.1 hydrogen sulphite production the ability to produce hydrogen sulphite was determined by streaking single colonies onto biggy agar (oxoid) and incubating them at 25°c for 48-72 h. colony colour was observed and scored as being white, pale hazel, hazel, dark hazel or black. ital. j. food sci., vol 29, 2017 521 2.3.2 enzymatic activities the esterase, protease and β-glucosidase activities of the isolates were screened as described by englezos et al. (2015). 2.3.3. ethanol and so2 tolerance assays ethanol tolerance and so2 tolerance were determined in microplates, according to the method proposed by arroyo-lopez et al. (2010) and tofalo et al. (2012), with some modifications. yeast nitrogen base with amino acids (ynb, 6.7 g/l, [remel, lenexa, ks, usa]) and ph 5.5 was supplemented with 20 g/l of glucose and sterilized by filtration using a 0.2 μm membrane filter (vwr, milan, italy). the medium was supplemented with different concentrations of ethanol (sigma) (final concentrations of 0, 12, 14 and 16% v/v) in order to test for ethanol tolerance, while, in order to establish so2 resistance, different amounts of total so2 were added (after adjustment to ph 3.0) until final concentrations of 0, 50, 100 and 150 mg/l. cells for the inoculation were prepared from an overnight culture in 1 ml of ypd medium, centrifuged at 9000 rpm for 10 min. the obtained pellet was washed twice in a sterile salt solution (8 g/l nacl) and then re-suspended in the same solution to obtain a concentration of about 106 cells/ml. the diluted cells (20 μl) were mixed with 180 μl of ynb, prepared as above. the microplates were incubated at 25 °c and the optical density (od) was measured at 600 nm using a microtiter plate reader (savatec instruments, torino, italy) at 24 and 48 hours after an orbital shaking of 30 s, in order to re-suspend the cells in the medium before the measurement. ynb without ethanol or so2 was used as the control. cell growth was determined on the basis of the ratio (%) of od values obtained in medium with and without ethanol or so2 for the specific incubation times. tests were carried out in triplicate. isolates with a percentage of growth < 10% were considered sensitive. 2.4. microfermentations the fermentation potential of the different genotypes was evaluated in microfermentation trials. these were carried out in duplicate for 14 days, in 50 ml tubes containing 25 ml of barbera grape must (120 g/l glucose, 124 g/l fructose, 5.25 titratable acidity as g/l of tartaric acid, ph 3.20 and 184 mg/l yeast available nitrogen (yan)). before the inoculation, the must was thermally treated at 60 °c for 50 min, and the absence of viable populations was evaluated by plating 100 μl of the must after the treatment on the wln medium, followed by incubation at 28 °c for 5 days. pre-cultures were prepared in must at 25 °c for 24 h, and then used to inoculate each fermentation with a cell concentration of 106 per ml, which was determined through a microscopical cell count. the fermentations were carried out under static conditions at 25 °c. 2.5. chemical analysis after 14 days of fermentation, the sugar consumption (glucose and fructose) and the ethanol, glycerol and acetic acid production were evaluated directly by means of hplc, according to the method proposed by rolle et al. (2012). yan was measured following the protocols reported in englezos et al., 2016). ital. j. food sci., vol 29, 2017 522 2.6. data analysis the results of the chemical composition of the wines obtained from the microfermentation trials were subjected to a principal component analysis (pca), in order to evaluate the intraspecific biodiversity of s. cerevisiae isolates. statistical analyses were performed using the ibm spss statistics software package (version 19.0, ibm corp., armonk, ny, usa). 3. results and discussions the possible association between territory and yeasts is being actively investigated in recent years and is believed to have a positive impact and influence purchase decisionmaking by the consumer. the use of indigenous selected yeasts could represent a useful alternative to spontaneous fermentation in order to optimize the typical attributes of the grape variety (clemente jimenez et al., 2004; rementeria et al., 2003; romano et al., 2008). the goal of this study was to isolate and characterize indigenous s. cerevisiae yeasts present on barbera grapes in the vineyards of the monferrato area. fifteen vineyards, located in the piedmont region (fig. 1) and cultivated with the barbera grape variety, were studied during the 2012 harvest season and the collected grapes were crushed to obtain 15 spontaneous alcoholic fermentations. figure 1. geographic location of the fifteen vineyards in the monferrato region (asti and alessandria) with indication of the 5 areas considered. the distribution of the vineyards in the five areas was as follows: a (murisengo), b (montegrosso d'asti, costigliole d'asti, san martino alfieri, agliano terme, isola d'asti), c (vinchio, nizza monferrato, incisa scapaccino), d (loazzolo) and e (ricaldone, aqui terme, alice bel colle). overall, 943 yeast colonies were isolated during the fermentations, and after molecular identification, most of them (636 isolates) were identified, through the use of δ-pcr, as s. cerevisiae. other species, namely hanseniaspora uvarum (248 isolates), starmerella bacillaris (synonym candida zemplinina) (11 isolates), pichia anomala (7 isolates) and torulaspora delbruecki (7 isolates) were also isolated, mainly at the beginning and middle of the fermentations. ital. j. food sci., vol 29, 2017 523 the pcr amplification of the δ interspersed sequences was also used to identify the genetic differences among the s. cerevisiae isolated during the fermentations. the molecular fingerprinting analysis, using a coefficient of similarity of 85%, allowed numerous strains among the isolates to be distinguished. the dendrogram resulting from the analysis of 636 s. cerevisiae isolates highlighted the presence of 62 clusters and 37 strains, which were unique and did not cluster with any other isolate. the most numerous clusters were: xvii and xli with 60 and 44 isolates, respectively (table 1). it is interesting to notice the different level of heterogeneity of s. cerevisiae isolated from the different vineyards. for example, most of the isolates from vineyard 1 grouped in one single cluster (xli), while in other cases (vineyards 12, 13 and 14) a high level of diversity was observed. only 37 s. cerevisiae δ-pcr patterns were unique, demonstrating a feeble biodiversity of indigenous s. cerevisiae strains in monferrato area. considering the ratio between the number of s. cerevisiae isolated and the number of observed patterns, as an approximate biodiversity estimation, our results showed similar values to those found in portugal (schuller et al., 2005) and in france (valero et al., 2007). in order to investigate further the s. cerevisiae diversity, 96 strains were selected on the basis of the cluster analysis, and screened for desirable oenological characteristics (esteve-zarzoso et al., 2000) such as: a low production of hydrogen sulphide and tolerance to a final concentration of 150 mg/l total sulphur dioxide and 16% (v/v) of ethanol (table 2). all the strains exhibited a medium hydrogen sulphide production level; 5% of them appeared to be pale hazel on biggy agar, while the others were hazel. concerning the results of the tolerance to so2, the selected strains were able to grow in the presence of 50 and 100 mg/l of so2 (83% and 60% of the isolates, respectively), while only a few isolates (32%) grew at 150 mg/l of so2 within 24 h. extending the incubation time to 48 h, the number of the isolates that were able to grow at 150 mg/l of so2 increased to 63%. only one strain (scba20) was totally inhibited by so2. as far as ethanol tolerance is concerned, 60% of the strains grew at 14% v/v within 24 h. ethanol mainly affected yeast growth by increasing the lag phase, and this evidence explains why after increasing the incubation time to 48 h, 95% of the strains were able to grow in all the ethanol concentrations (table 2). β-glucosidase activity was found in only 2.7% of the strains, thus indicating possible production and activity during the fermentation. protease activity was detected in 37.8% of the tested s. cerevisiae, while 21.6% were able to hydrolyse esters (data not shown). in order to extend the information on the 96 s. cerevisiae strains of the monferrato area, alcoholic fermentations were carried out in barbera grape must. the experimental wines obtained were analyzed to establish the content of some by-products correlated to the organoleptic quality of the wine and the obtained results are reported in table 3. the values of the residual sugars ranged from 3.44 to 148.64 g/l. only seven isolates (scba4, scba5, scba13, scba26, scba44, scba60 and scba63) were able to leave less than 5 g/l of residual sugars after 14 days of fermentation. all the strains, except scba12, scba47, scba48 and scba57, were capable of consuming almost all the glucose of the must, confirming the glucophylic character of this species. all the yeast strains, that completed the fermentation, formed low amounts of acetic acid in the wines (less than 0.6 g/l). glycerol production was relatively low, ranging from 5.20 to 7.86 g/l. the fermentation purity was high; most strains had a low ratio between the produced acetic acid and ethanol (range 0.01-0.09) and only three strains showed values above 0.04. as regards ethanol production, 52% of the strains produced more than 13%, and 6% of them managed to develop more than 14%. ital. j. food sci., vol 29, 2017 524 table 1. clusters obtained from a comparison of the different fingerprinting profiles of the s. cerevisiae isolates examined in this study by means of the molecular technique. the arbitrarily selected coefficient of similarity was 85%. the table shows their composition according to the geographical locations (vineyard) from which the isolates were obtained. monferrato's vineyards cluster number of strains in the cluster 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 i 5 / / / / / / / / / / 5 / / / / ii 15 / / / / 11 / 4 / / / / / / / / iii 14 / / 6 / / / 4 / / / 2 / 2 / / iv 6 / / 6 / / / / / / / / / / / / v 3 / 3 / / / / / / / / / / / / / vi 6 / / / / / / / / / 2 / / / 3 1 vii 2 / / / / / / / / / / / / / / 2 viii 24 / 14 5 / / / / 2 / / / / 1 / 2 ix 4 / 4 / / / / / / / / / / / / / x 7 / / / / 3 / / / / / 4 / / / / xi 10 / / / / / / / / 3 / / / 7 / / xii 8 / / / / / / / / 4 / / / 4 / / xiii 7 / 7 / / / / / / / / / / / / / xiv 18 / 4 / / 10 / / / / / 1 / 2 1 / xv 4 / / / / / 2 / / / / / / 2 / / xvi 2 / / / / / / / / / 1 / / / 1 / xvii 60 6 4 3 2 1 / 3 8 / 2 / 2 5 9 15 xviii 6 / / / / / / 2 2 / / / / 2 / xix 10 / / 4 / / / / / / / / / 4 / 2 xx 15 1 / / 9 2 / / / / / / / / 2 1 xxi 13 / / / 2 2 / / 2 / 2 / / 2 2 1 xxii 28 2 / 1 / / / / 1 / / / 1 1 4 18 xxiii 2 / / 1 / / / / / / / / / 1 / / xxiv 3 / / / / / / / / / 3 / / / / / xxv 3 / / / / / / / / / 3 / / / / / xxvi 4 / / / / / / / / / / / / / / 4 xxvii 5 / / 3 / / / / / / / / / / 1 1 xviii 2 / / / / / / / / / / / / 2 / / xxix 10 / / / / / / / / / / / / 10 / / xxx 6 / / / / / / / / / / / / 3 3 / xxxi 7 / / / / / / / / / 3 / / 3 / 1 xxxii 3 1 / / / / / / / / 2 / / / / / xxxiii 6 / / / / / / / / / / / 5 1 / / xxxiv 5 / / / / / / / / / / / 5 / / / xxxv 3 / / / 1 / / / / / / / / 2 / / xxxvi 5 / / / / / 5 / / / / / / / / / xxxvii 9 / / / 2 / / / 2 / 4 / / / / 1 xxxviii 4 / / / / / / / / / / / / / / 4 ital. j. food sci., vol 29, 2017 525 table 1. continues. monferrato's vineyards cluster number of strains in the cluster 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 xxxix 2 / / / / / / / 2 / / / / / / / xl 7 / / / / / / / / / / / / 7 / / xli 44 38 / / / / / / / / / / 3 3 / / xlii 7 / / / / / 2 / 1 4 / / / / / / xliii 9 / / 5 / / / / / 1 / / 2 / 1 / xliv 7 / / / / / / / 3 3 1 / / / / / xlv 6 / / / / / / / / / / / / 6 / / xlvi 5 / / / / / / 5 / / / / / / / / xlvii 10 / / / 9 / / / / / / / / 1 / / xlviii 15 / / / / / / / / / / 13 2 / / / xlix 6 / / / / 2 4 / / / / / / / / / l 10 / 3 / / / / / 6 / 1 / / / / / li 4 / / / / / / / / 3 / 1 / / / / lii 30 / / / / 2 16 10 / / / / / 2 / / liii 12 / / / / 2 / 7 / / / / 1 / 2 / liv 2 / / / / / / / / / / / / / 2 / lv 10 / / / / / / / / / / / / / / 10 lvi 5 / / / / 3 2 / / / / / / / / / lvii 19 2 / / / 2 / / 1 / / / / 6 8 / lviii 15 / / / / 3 / / / / 2 / 10 / / / lix 25 / / / / / 5 7 / / / / 1 / 12 / lx 22 / / / / / / / 1 / 7 / / 12 1 1 lxi 3 / / / / / / / / / 1 / / 2 / / lxii 17 / 3 / / / / 1 / / / 4 8 / 1 / ital. j. food sci., vol 29, 2017 526 table 2. results of the resistance to ethanol and so2 of the tested strains. the values presented are the ratio between the od of the isolates in broth with and without ethanol or so2 times 100 at the specific incubation times. the values are the means of triplicate experiments. strains so2 growth (mg/l) ethanol growth (% vol.) 24 hours of incubation 48 hours of incubation 24 hours of incubation 48 hours of incubation 50 100 150 50 100 150 12 14 16 12 14 16 scba1 36 6 14 81 74 81 0 0 0 64 0 0 scba2 82 87 71 93 99 89 100 93 100 100 100 100 scba3 81 46 36 100 91 100 36 6 7 97 47 0 scba4 34 3 4 100 72 36 2 1 1 17 0 0 scba5 81 46 36 98 99 96 36 6 7 98 73 4 scba6 44 46 16 87 67 52 11 1 1 13 1 1 scba7 58 48 42 95 88 61 9 4 4 90 6 5 scba8 76 49 42 98 94 93 8 4 5 85 7 5 scba9 70 42 43 96 92 94 5 4 5 72 4 5 scba10 26 15 9 70 69 60 19 1 0 70 0 0 scba11 22 6 6 87 83 10 2 1 1 5 0 0 scba12 9 6 2 44 7 7 4 2 2 3 1 1 scba13 60 32 32 99 93 83 9 3 3 97 3 3 scba14 38 2 1 85 55 16 51 1 0 61 0 0 scba15 26 3 3 72 30 14 49 0 0 58 0 0 scba16 2 4 23 6 40 77 51 0 0 61 0 0 scba17 12 0 0 91 54 17 37 0 0 76 0 1 scba18 47 0 0 55 9 0 1 1 0 38 0 0 scba19 87 58 52 85 84 75 40 0 0 77 0 0 scba20 63 0 0 64 2 0 0 0 0 13 0 0 scba21 52 31 46 60 59 50 55 0 0 83 0 0 scba22 96 91 83 100 97 96 100 95 97 98 93 99 scba23 67 63 54 96 66 40 96 70 71 100 92 100 scba24 28 16 8 85 87 78 100 69 75 100 71 84 scba25 76 76 75 95 82 58 73 71 52 81 67 57 scba26 39 31 21 74 40 22 79 17 8 69 65 66 scba27 14 11 10 74 40 22 67 59 59 69 65 66 scba28 83 65 62 88 86 76 92 83 71 100 94 90 scba29 95 81 65 100 98 89 89 77 58 100 92 83 scba30 44 33 7 88 91 38 69 55 64 71 63 68 scba31 100 90 86 100 98 99 80 52 1 90 78 0 scba32 100 84 83 100 97 98 99 90 5 100 98 7 scba33 100 81 75 100 98 97 98 90 5 99 97 1 ital. j. food sci., vol 29, 2017 527 scba34 45 37 10 97 87 67 77 50 1 79 63 0 scba35 36 22 28 50 39 30 44 32 27 87 83 67 scba36 65 62 33 100 95 84 96 83 86 88 84 87 scba37 66 32 23 100 68 65 100 95 97 100 100 100 scba38 70 43 29 100 70 66 100 98 100 100 98 100 scba39 60 34 28 100 96 98 97 86 73 82 67 44 scba40 27 9 14 100 99 98 92 87 78 100 96 98 scba41 25 8 7 72 30 14 51 28 0 87 44 1 scba42 24 14 20 100 97 97 96 91 74 99 96 90 scba43 43 18 21 100 98 86 80 62 13 99 92 75 scba44 66 55 38 89 86 83 100 96 99 100 98 100 scba45 52 35 25 88 89 77 99 91 85 100 93 91 scba46 40 26 22 98 90 76 76 77 86 93 88 94 scba47 29 28 9 100 64 70 83 0 0 100 100 100 scba48 14 4 6 69 63 46 90 27 4 100 100 100 scba49 59 53 52 90 85 76 98 89 85 97 88 86 scba50 57 50 44 77 86 88 95 94 89 95 96 95 scba51 58 51 55 80 83 70 100 91 85 100 89 87 scba52 100 96 100 99 96 99 100 99 100 100 98 100 scba53 91 91 87 90 91 87 91 88 91 92 93 93 scba54 100 98 100 100 97 100 100 100 100 100 100 100 scba55 100 98 99 100 99 99 100 100 100 100 100 100 scba56 98 73 90 100 95 99 100 100 99 100 100 99 scba57 20 12 13 70 16 11 100 93 63 100 100 100 scba58 72 19 12 100 88 71 100 98 92 100 100 96 scba59 80 45 27 100 98 99 100 98 96 100 98 96 scba60 70 76 53 100 100 96 97 77 92 100 100 100 scba61 86 87 62 99 97 98 100 95 81 100 100 91 scba62 88 69 73 100 98 100 96 94 90 100 94 87 scba63 80 54 49 100 100 97 98 98 92 100 99 93 scba64 86 49 31 100 100 98 96 96 91 98 96 90 scba65 100 93 94 100 100 100 100 95 100 100 96 99 scba66 35 12 15 67 50 61 95 92 72 95 95 88 scba67 30 7 15 70 70 66 95 83 61 100 97 92 scba68 36 6 8 72 68 65 99 99 97 100 100 100 scba69 25 7 8 79 70 73 100 100 100 100 100 100 scba70 65 79 42 79 82 61 100 97 83 100 99 85 scba71 18 35 18 70 74 71 97 81 78 100 95 88 scba72 80 77 42 100 93 78 100 96 96 100 100 99 ital. j. food sci., vol 29, 2017 528 scba73 54 59 32 83 81 73 97 96 85 100 100 100 scba74 93 86 68 100 98 91 100 95 84 100 97 100 scba75 57 59 59 89 55 56 66 66 51 66 67 53 scba76 88 85 86 86 83 84 97 97 74 97 99 79 scba77 87 85 85 84 82 86 95 88 71 100 92 76 scba78 100 99 95 99 97 97 100 99 99 100 100 100 scba79 100 100 99 100 100 100 100 100 100 100 100 100 scba80 91 92 88 90 91 88 83 70 65 88 71 66 scba81 81 72 43 100 97 98 100 96 93 100 100 100 scba82 74 79 63 100 97 100 100 92 98 100 97 100 scba83 100 99 99 100 99 99 100 100 100 100 100 100 scba84 84 84 86 84 84 86 87 69 62 93 72 63 scba85 92 90 91 93 91 92 99 86 75 100 97 93 scba86 91 93 93 91 92 93 100 100 100 100 100 100 scba87 92 91 92 89 87 88 86 71 64 91 70 62 scba88 91 86 90 91 86 90 99 92 83 100 96 89 scba89 93 83 89 100 96 88 100 97 97 100 100 100 scba90 97 95 99 99 96 100 100 96 90 100 100 92 scba91 80 84 63 93 95 86 100 100 92 100 100 100 scba92 99 90 100 99 96 100 100 99 100 100 100 100 scba93 100 79 72 100 75 68 100 98 99 100 100 100 scba94 100 100 100 100 100 99 100 100 100 100 100 99 scba95 100 98 94 100 99 95 100 85 86 100 86 88 scba96 100 90 65 100 90 66 100 97 97 100 98 99 ital. j. food sci., vol 29, 2017 529 table 3. chemical analysis of the wines obtained from the fermentation of the pure indigenous s. cerevisiae cultures. the data are means±standard deviations. with * were reported strains which were unique and did not cluster with any other isolate. strains vineyard cluster residual glucose (g/l) residual fructose (g/l) glycerol (g/l) acetic acid (g/l) ethanol (%v/v) fermentatio n puritya ethanol yieldb glycerol yieldc acetic acid yieldd scba1 11 i 2.18±0.54 24.23±2.14 7.00±0.15 0.45±0.00 12.57±0.19 0.036±0.001 0.058±0.001 0.032±0.001 0.0359±0.0008 scba2 13 * 0.88±0.07 8.68±0.80 6.86±0.05 0.51±0.02 13.77±0.05 0.037±0.001 0.059±0.001 0.029±0.001 0.0367±0.0012 scba3 5 ii 0.65±0.08 4.43±1.04 7.01±0.01 0.56±0.01 14.11±0.11 0.039±0.001 0.059±0.001 0.029±0.001 0.0395±0.0005 scba4 7 * 0.68±0.01 2.77±1.47 7.42±0.82 0.29±0.03 14.14±0.11 0.021±0.002 0.059±0.001 0.031±0.003 0.0208±0.0023 scba5 7 iii 0.38±0.27 3.08±1.84 6.89±0.10 0.30±0.05 14.27±0.01 0.021±0.004 0.059±0.001 0.029±0.001 0.0207±0.0037 scba6 7 * 1.14±0.37 6.57±2.71 7.08±0.03 0.31±0.07 13.89±0.35 0.022±0.006 0.059±0.001 0.026±0.001 0.0224±0.0056 scba7 3 iv 1.58±0.73 12.15±1.93 6.92±0.16 0.33±0.00 13.82±0.03 0.024±0.001 0.060±0.001 0.030±0.001 0.0240±0.001 scba8 2 v 2.98±3.16 16.78±18.98 6.54±0.43 0.47±0.34 12.60±0.90 0.037±0.025 0.056±0.002 0.029±0.005 0.0367±0.0247 scba9 5 * 0.69±0.25 5.68±4.83 7.07±0.01 0.29±0.02 13.73±0.20 0.021±0.001 0.058±0.002 0.030±0.001 0.0215±0.0014 scba10 10 vi 1.65±0.49 15.33±2.45 6.81±0.14 0.41±0.04 13.46±0.13 0.031±0.002 0.059±0.001 0.030±0.001 0.0307±0.0025 scba11 15 vii 1.11±0.96 11.44±10.36 7.44±0.08 0.35±0.01 13.46±0.58 0.026±0.001 0.058±0.001 0.032±0.001 0.026±0.0007 scba12 2 viii 78.13±0.19 70.52±0.15 5.20±0.10 0.43±0.01 4.90±0.09 0.088±0.001 0.051±0.001 0.054±0.001 0.0879±0.0001 scba13 2 ix 0.82±0.25 2.80±0.68 6.63±0.08 0.20±0.02 13.87±0.28 0.014±0.002 0.058±0.001 0.028±0.001 0.0144±0.0015 scba14 11 x 1.14±0.72 13.37±5.12 6.99±0.07 0.29±0.03 13.59±0.45 0.021±0.003 0.059±0.001 0.030±0.001 0.0213±0.0033 scba15 14 xi 2.88±0.61 28.44±1.79 7.10±0.03 0.44±0.01 12.38±0.05 0.035±0.001 0.058±0.001 0.033±0.001 0.0352±0.0008 scba16 14 xii 3.71±0.58 28.71±1.54 6.82±0.22 0.38±0.04 12.04±0.14 0.032±0.003 0.057±0.001 0.032±0.001 0.0319±0.0028 scba17 2 xiii 1.09±0.07 6.93±0.34 7.37±0.01 0.39±0.01 14.03±0.03 0.028±0.001 0.059±0.001 0.031±0.001 0.0275±0.0004 scba18 2 * 0.89±0.37 11.64±5.35 6.46±0.65 0.41±0.18 13.00±0.32 0.031±0.013 0.056±0.001 0.028±0.004 0.0314±0.0129 scba19 5 xiv 0.83±0.23 8.09±5.27 6.85±0.09 0.36±0.05 13.34±0.71 0.028±0.006 0.056±0.004 0.029±0.001 0.0275±0.0055 scba20 14 xv 0.63±0.24 7.80±2.79 7.26±0.02 0.41±0.00 13.89±0.07 0.029±0.001 0.060±0.001 0.031±0.001 0.0292±0.0004 scba21 10 xvi 1.58±0.21 10.27±2.32 6.72±0.04 0.44±0.06 13.40±0.15 0.030±0.001 0.0059 ±0.001 0.030 ±0.001 0.0234±0.003 scba22 15 xvii 1.54±0.62 17.88±4.13 7.88±0.12 0.38±0.04 12.88±0.14 0.030±0.004 0.057±0.001 0.035±0.001 0.0297±0.0037 scba23 13 * 1.04±0.65 13.68±5.92 7.35±0.04 0.34±0.01 13.21±0.26 0.026±0.001 0.058±0.001 0.032±0.001 0.026±0.0012 scba24 14 * 1.18±0.04 15.75±0.21 7.13±0.20 0.34±0.01 13.14±0.19 0.026±0.001 0.058±0.001 0.031±0.001 0.0261±0.0007 scba25 7 * 3.86±1.76 22.9±5.06 6.81±0.17 0.26±0.01 12.82±0.71 0.021±0.001 0.059±0.001 0.031±0.001 0.0206±0.0006 ital. j. food sci., vol 29, 2017 530 scba26 7 xviii 0.69±0.08 4.20±1.12 7.13±0.21 0.25±0.04 13.96±0.22 0.018±0.003 0.058±0.001 0.030±0.001 0.0181±0.0035 scba27 14 xix 0.73±0.48 9.18±6.55 6.88±0.13 0.24±0.04 13.72±0.64 0.017±0.004 0.059±0.001 0.029±0.001 0.0174±0.0037 scba28 4 xx 0.75±0.10 7.31±2.50 6.65±0.12 0.32±0.07 13.68±0.30 0.024±0.006 0.058±0.001 0.028±0.001 0.0237±0.0058 scba29 5 xxi 1.84±0.13 16.38±0.98 6.80±0.11 0.41±0.05 13.32±0.20 0.031±0.004 0.059±0.001 0.030±0.001 0.0307±0.0043 scba30 15 xxii 5.12±2.03 31.28±5.30 6.65±0.03 0.44±0.01 11.8±0.54 0.037±0.001 0.057±0.001 0.032±0.001 0.037±0.0008 scba31 2 xxiii 1.15±0.44 11.07±4.09 6.59±0.01 0.40±0.06 13.56±0.41 0.029±0.005 0.058±0.001 0.028±0.001 0.0293±0.0051 scba32 10 xxiv 0.96±0.47 6.09±0.38 7.03±0.84 0.37±0.02 13.67±0.17 0.027±0.002 0.058±0.001 0.03±0.004 0.0269±0.0015 scba33 10 xxv 0.65±0.17 5.69±1.43 6.42±0.17 0.44±0.03 13.97±0.27 0.031±0.003 0.059±0.002 0.027±0.001 0.0313±0.003 scba34 13 * 1.13±0.54 18.34±1.89 7.78±0.05 0.32±0.00 12.82±0.01 0.025±0.001 0.057±0.001 0.035±0.001 0.0249±0.0001 scba35 7 * 0.62±0.20 5.13±1.56 7.44±0.03 0.52±0.02 13.93±0.20 0.037±0.002 0.058±0.001 0.031±0.001 0.0372±0.0019 scba36 15 xxvi 1.14±0.42 15.81±2.76 6.69±0.08 0.40±0.06 12.99±0.12 0.031±0.004 0.057±0.001 0.029±0.001 0.0306±0.004 scba37 3 xxvii 0.57±0.13 5.55±2.26 6.59±0.07 0.33±0.01 13.87±0.12 0.024±0.001 0.058±0.001 0.028±0.001 0.0239±0.0012 scba38 10 * 0.79±0.02 7.81±1.69 6.99±0.32 0.41±0.02 13.23±0.50 0.031±0.002 0.056±0.003 0.030±0.001 0.0310±0.0002 scba39 14 xxviii 1.58±0.35 17.36±1.76 7.86±0.10 0.31±0.01 12.99±0.23 0.024±0.001 0.058±0.001 0.035±0.001 0.0240±0.0010 scba40 14 xxix 2.68±0.47 24.44±0.38 7.49±0.68 0.42±0.05 12.24±0.90 0.036±0.007 0.054±0.004 0.034±0.003 0.0356±0.0068 scba41 12 * 1.40±0.86 19.33±4.79 7.12±0.16 0.39±0.02 13.01±0.28 0.03±0.002 0.058±0.001 0.032±0.001 0.0297±0.0025 scba42 13 xxx 2.52±0.62 25.2±1.99 6.98±0.23 0.36±0.00 12.6±0.17 0.029±0.001 0.058±0.001 0.032±0.001 0.0287±0.0003 scba43 15 xxxi 3.07±1.42 26.09±4.50 6.91±0.20 0.36±0.02 12.50±0.68 0.029±0.001 0.058±0.002 0.032±0.001 0.0289±0.0001 scba44 1 xxxii 0.62±0.01 3.63±0.59 6.99±0.08 0.27±0.01 14.05±0.04 0.019±0.001 0.058±0.001 0.029±0.001 0.0189±0.0003 scba45 12 xxxiii 3.88±0.02 30.01±0.48 6.86±0.11 0.41±0.03 12.11±0.02 0.034±0.002 0.058±0.001 0.033±0.001 0.0338±0.0020 scba46 12 xxxiv 3.32±0.14 24.06±0.06 7.69±0.27 0.39±0.00 12.43±0.22 0.031±0.001 0.057±0.001 0.035±0.001 0.0313±0.0003 scba47 4 xxxv 61.26±8.62 48.21±7.22 5.65±0.44 0.52±0.02 7.42±0.89 0.071±0.006 0.055±0.001 0.042±0.002 0.0708±0.0058 scba48 6 xxxvi 68.47±0.49 53.25±1.42 5.51±0.29 0.54±0.02 6.86±0.28 0.079±0.001 0.056±0.002 0.045±0.002 0.0786±0.0003 scba49 15 xxxvii 1.39±0.90 17.01±3.35 6.94±0.05 0.37±0.00 12.88±0.40 0.029±0.001 0.057±0.001 0.031±0.001 0.0288±0.0012 scba50 9 * 2.78±0.19 25.25±0.23 6.75±0.15 0.39±0.01 12.6±0.08 0.031±0.001 0.058±0.001 0.031±0.001 0.0311±0.0003 scba51 15 xxxviii 2.48±0.03 24.83±0.26 6.81±0.18 0.36±0.01 12.49±0.22 0.029±0.001 0.058±0.001 0.031±0.001 0.0291±0.0001 scba52 8 xxxix 0.71±0.09 8.32±0.62 6.67±0.11 0.43±0.02 13.73±0.08 0.031±0.001 0.058±0.001 0.028±0.001 0.0312±0.0010 scba53 11 * 1.78±1.01 21.99±5.78 6.80±0.04 0.42±0.03 12.60±0.14 0.033±0.002 0.057±0.001 0.031±0.001 0.0333±0.0019 scba54 14 xl 1.72±1.39 15.94±7.53 7.06±0.01 0.31±0.00 13.24±0.42 0.023±0.001 0.058±0.001 0.031±0.001 0.0233±0.0011 scba55 1 xli 2.11±1.08 17.06±5.14 6.70±0.06 0.24±0.01 13.18±0.48 0.018±0.001 0.059±0.001 0.030±0.001 0.0181±0.0015 scba56 6 xlii 1.85±0.39 15.86±1.64 6.78±0.01 0.32±0.01 13.35±0.16 0.024±0.001 0.059±0.001 0.030±0.001 0.0238±0.0007 ital. j. food sci., vol 29, 2017 531 scba57 3 xliii 68.19±0.5 45.76±2.71 5.21±0.13 0.60±0.00 7.39±0.17 0.081±0.002 0.057±0.001 0.040±0.002 0.0811±0.0024 scba58 8 xliv 4.84±3.73 30.43±10.18 6.53±0.20 0.43±0.01 11.73±1.22 0.038±0.004 0.055±0.002 0.031±0.003 0.0375±0.0045 scba59 1 * 3.60±1.45 28.12±4.22 6.84±0.01 0.42±0.00 12.20±0.46 0.034±0.001 0.057±0.001 0.032±0.001 0.0341±0.0011 scba60 10 xliv 0.69±0.20 4.12±2.80 7.81±0.20 0.30±0.02 14.08±0.03 0.022±0.001 0.059±0.001 0.033±0.001 0.0216±0.0013 scba61 14 xlv 0.56±0.05 7.10±0.59 6.96±0.19 0.42±0.00 13.79±0.18 0.030±0.001 0.058±0.001 0.029±0.001 0.0305±0.0007 scba62 13 * 0.98±0.08 13.73±0.37 6.43±0.17 0.32±0.01 13.21±0.28 0.024±0.001 0.058±0.001 0.028±0.001 0.0239±0.0001 scba63 14 * 0.39±0.15 3.85±1.71 7.20±0.07 0.50±0.14 13.58±0.48 0.038±0.012 0.056±0.002 0.030±0.001 0.0377±0.0116 scba64 14 * 0.98±0.54 7.81±1.64 6.87±0.14 0.32±0.09 13.33±0.61 0.025±0.008 0.056±0.002 0.029±0.001 0.0248±0.0079 scba65 7 xlvi 1.07±0.51 9.91±4.72 6.46±0.09 0.23±0.01 13.42±0.50 0.017±0.001 0.057±0.001 0.028±0.001 0.0173±0.0013 scba66 4 xlvii 3.00±0.12 25.73±0.44 6.76±0.01 0.39±0.01 12.15±0.07 0.032±0.001 0.056±0.001 0.031±0.001 0.0319±0.0005 scba67 4 * 3.17±1.05 25.26±2.74 7.72±1.37 0.37±0.01 12.71±0.56 0.029±0.001 0.059±0.004 0.036±0.007 0.0292±0.0002 scba68 4 * 0.89±0.22 9.31±2.35 6.60±0.22 0.22±0.03 13.46±0.31 0.016±0.002 0.057±0.001 0.028±0.001 0.0162±0.0017 scba69 4 * 0.85±0.42 8.44±4.20 6.54±0.03 0.24±0.02 13.41±0.27 0.018±0.002 0.057±0.001 0.028±0.001 0.0182±0.0021 scba70 12 xlviii 4.27±1.33 31.23±3.89 6.88±0.02 0.40±0.03 12.27±0.27 0.033±0.001 0.059±0.003 0.033±0.001 0.0330±0.0014 scba71 11 * 0.72±0.33 4.54±2.92 6.72±0.09 0.30±0.01 13.88±0.39 0.021±0.001 0.058±0.001 0.028±0.001 0.0214±0.0001 scba72 6 xlix 0.66±0.09 7.05±0.76 6.84±0.01 0.40±0.01 13.78±0.08 0.029±0.001 0.058±0.001 0.029±0.001 0.0291±0.0009 scba73 14 * 0.78±0.04 7.81±0.64 7.10±0.04 0.42±0.00 13.82±0.02 0.031±0.001 0.059±0.001 0.030±0.001 0.0307±0.0002 scba74 8 l 0.93±0.28 9.96±2.72 6.64±0.01 0.40±0.01 13.49±0.11 0.030±0.001 0.058±0.001 0.028±0.001 0.0300±0.0007 scba75 9 li 3.57±2.41 27.49±8.01 6.79±0.13 0.38±0.01 12.35±0.66 0.031±0.002 0.058±0.001 0.032±0.001 0.0309±0.0022 scba76 9 * 3.63±1.97 30.74±4.86 6.58±0.05 0.37±0.00 12.01±0.29 0.031±0.001 0.057±0.001 0.031±0.001 0.0308±0.0008 scba77 9 * 3.96±1.15 30.12±3.88 6.61±0.04 0.37±0.03 11.93±0.41 0.031±0.001 0.057±0.001 0.031±0.001 0.0314±0.0011 scba78 7 * 1.02±0.22 9.71±2.42 6.15±0.11 0.31±0.04 13.42±0.11 0.023±0.003 0.057±0.001 0.026±0.001 0.0228±0.0033 scba79 11 * 1.23±0.37 12.73±2.91 6.52±0.13 0.23±0.01 13.37±0.06 0.017±0.001 0.058±0.001 0.028±0.001 0.0169±0.0007 scba80 8 * 6.79±2.11 36.88±3.98 6.57±0.00 0.41±0.02 11.22±0.36 0.037±0.003 0.056±0.001 0.033±0.001 0.0366±0.0032 scba81 7 lii 1.96±1.25 15.41±6.55 6.96±0.03 0.40±0.01 13.30±0.38 0.030±0.002 0.059±0.001 0.031±0.001 0.0299±0.0017 scba82 6 liii 1.00±0.06 10.89±0.92 6.65±0.04 0.21±0.00 13.36±0.07 0.016±0.001 0.057±0.001 0.029±0.001 0.0157±0.0001 scba83 5 liv 2.43±1.29 17.35±5.92 6.66±0.15 0.38±0.01 12.82±0.45 0.030±0.002 0.057±0.001 0.030±0.001 0.0298±0.0017 scba84 15 lv 2.14±0.86 23.56±3.33 6.68±0.05 0.40±0.02 12.47±0.29 0.032±0.002 0.057±0.001 0.031±0.001 0.0318±0.0022 scba85 14 * 3.92±1.20 29.25±3.40 6.70±0.02 0.38±0.00 12.10±0.16 0.031±0.001 0.057±0.001 0.032±0.001 0.0314±0.0008 scba86 6 lvi 1.06±0.15 10.25±1.61 6.73±0.13 0.42±0.01 13.60±0.20 0.031±0.001 0.058±0.002 0.029±0.001 0.0307±0.0011 scba87 1 lvii 1.74±0.96 21.21±4.94 6.85±0.07 0.39±0.02 12.54±0.20 0.031±0.002 0.057±0.001 0.031±0.001 0.0311±0.0021 ital. j. food sci., vol 29, 2017 532 scba88 12 lviii 2.85±2.21 25.68±9.50 6.64±0.24 0.37±0.02 12.39±0.02 0.029±0.002 0.058±0.003 0.031±0.003 0.0295±0.0016 scba89 13 * 2.24±0.24 19.4±0.70 7.12±0.07 0.33±0.02 13.07±0.18 0.025±0.001 0.059±0.001 0.032±0.001 0.0253±0.0009 scba90 13 lix 2.55±0.28 24.87±0.73 7.43±0.20 0.32±0.01 12.29±0.13 0.026±0.001 0.057±0.002 0.034±0.001 0.0258±0.0011 scba91 10 lx 2.64±0.41 19.53±1.64 6.36±0.17 0.37±0.01 12.64±0.11 0.029±0.001 0.057±0.001 0.029±0.001 0.0292±0.0007 scba92 14 lxi 3.43±2.51 26.05±8.16 6.49±0.02 0.36±0.01 12.06±0.33 0.030±0.001 0.056±0.001 0.030±0.002 0.0301±0.0001 scba93 6 lxii 0.67±0.31 6.69±5.00 6.46±0.18 0.21±0.01 13.55±0.34 0.016±0.001 0.057±0.002 0.027±0.001 0.0156±0.0001 scba94 5 xx 0.83±0.05 10.16±0.53 6.59±0.05 0.28±0.00 13.46±0.05 0.021±0.001 0.058±0.001 0.028±0.001 0.0205±0.0003 scba95 15 lxii 2.42±0.73 24.31±2.48 6.55±0.17 0.37±0.01 12.31±0.24 0.030±0.002 0.057±0.002 0.030±0.001 0.0301±0.0017 scba96 12 lix 3.18±1.87 25.13±5.26 6.67±0.06 0.40±0.02 12.12±0.28 0.033±0.001 0.056±0.001 0.031±0.001 0.0329±0.0007 a fermentation purity: acetic acid (g/l)/ethanol % (v/v), b ethanol yield: ethanol % (v/v)/sugar consumption (g/l), c glycerol yield: glycerol (g/l)/sugar consumption (g/l), d acetic acid: acetic acid (g/l)/sugar consumption(g/l). ital. j. food sci., vol 29, 2017 533 the physiological data from the growth tests at 50 mg/l of so2 after 24 h (enological conditions, variable: “ability to grow”), the presence or absence of enzymatic activities (esterase and protease activity), h2s production and the chemical composition (glucose, fructose, malic and acetic acids, glycerol, and ethanol) of the wines obtained after 14 days of fermentation were used to evaluate the technological diversity of the strains. a principal component analysis (pca) was carried out and the outcome is presented in fig. 2, including the loadings plot (fig. 2a) and the scatter plot (fig. 2b). figure 2. principal component analysis of the s. cerevisiae strains from the fifteen vineyards (as reported in figure 1), according to the wine chemical composition: loadings plot (a) and scatter plot (b). the fifteen vineyard considered were: 1 (murisengo), 2 (san martino alfieri), 3 (costigliole d'asti), 4 (isola d'asti), 5 (montegrosso d'asti), 6 (agliano terme), 7 (vinchio), 8 (nizza monferrato),9 (incisa scapaccino), 10 (loazzolo), 11 (ricaldone), 12 (alice bel colle), 13 (acqui terme crocera south west), 14 (acqui terme crocera south est) and 15 (acqui terme dannona). ital. j. food sci., vol 29, 2017 534 pc1 (35.0 % of variance explained) was better correlated with the strains that left high level of residual sugars present in the wine and that produced high amount of acetic acid, while pc2 (14.4 % of variance explained) was mainly correlated to the high production of glycerol and low degradation of malic acid (fig. 2a). two groups may be differentiated according to the scatter plot (pc1 or pc2 with values close or higher than 2, respectively). some strains from vineyards 4, 13, 14, and 15 produced high glycerol amounts and preserved malic acid contents, and two isolates from vineyards 10 and 12 were unsatisfactory due to their sugars degradation. in addition, all the four isolates from vineyard 9 were present either in the first or in the second group (fig. 2b). 4. conclusions the present study investigated the genetic and technological diversity of autochthonous s. cerevisiae in the north-west of italy, in the monferrato area. it was possible to genetically and phenotypically differentiate the strains. in order to investigate the ability of the isolated strains to properly ferment barbera must, at pilot scale level first and in industrial settings after, relevant technological characteristics, such as sugar consumption and acetic acid production, should be taken into consideration. all the data presented here were obtained from pasteurized natural must, and the ability of the selected strains to dominate the natural grape and must mycobiota should therefore be determined throughout the fermentation process. in parallel, the ability to complete the fermentation in the competitive environment of a natural grape should also be confirmed. finally, the production of compounds, such as alcohols, esters, carbonyl compounds and fatty acids that have an impact on the sensory characteristics of wine should also be evaluated. acknowledgements this work has been funded by eu fp7 under grant agreement 315065-wilwine (http://www.wildwine.eu/). the information in this document only reflects the authors’ views and the community is not liable for any use that may be made of the information contained herein. references alessandria v., marengo f., englezos v., gerbi v., rantsiou k. and cocolin l. 2015. mycobiota of barbera grapes from the piedmont region from a single vintage year. am. j. enol. vitic. 66: 244-250. arroyo-lopez f.n., salvadó z., tronchoni j., guillamón j.m., barrio e. and querol a. 2010. susceptibility and resistance to ethanol on saccharomyces strains isolated from wild and fermentative environments. yeast 27: 1005-1015. bosso a., panero l., petrozziello m., follis r., motta s. and guaita m. 2011. influence of submerged-cap vinification on polyphenolic composition and volatile compounds of barbera wines. am. j. 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and the genetics of populations. variability within and among natural populations. univ. chi. press, chi. paper received february 9, 2017 accepted may 19, 2017 paper ital. j. food sci., vol. 27 2015 1 keywords: anticancer activity, antifungal activity, antioxidant activity, flammulina velutipes, se-enriched mycelium se effect on biological activity of flammulina velutipes i. milovanović1, t. stanojković2, m. stajić1*, j. vukojević1 and a. knežević1 1university of belgrade, faculty of biology, takovska 43, 11000 belgrade, serbia 2institute of oncology and radiology of serbia, pasterova 14, 11000 belgrade, serbia *corresponding author: stajicm@bio.bg.ac.rs abstract the goals of the study were evaluation of antioxidant, antifungal and anticancer potential of flammulina velutipes mycelium ethanol extract and examination of se effect on those activities. both se-amended and non-amended mycelium extracts exhibited significant antioxidant and antifungal potential. se-enriched extract was more effective against candida krusei and c. albicans and better dpph• scavengers than non enriched one. carriers of the antioxidant activity were phenol compounds. contrary to antioxidant and antifungal potential, tested extracts were many times weaker cytotoxic agents against hela and ls174 cell lines than cis-ddp. thus, se-enriched mycelium could be supplement with antioxidant and antifungal capacity. 2 ital. j. food sci., vol. 27 2015 introduction a need for new antimicrobial agents exist for a long time due to the emergence of microorganism resistance as a result of the uncontrolled usage of commercial antibiotics and antimycotics. preference is given to natural compounds due to their health-beneficiary and environmentallyfriendly effect. besides diseases caused by microorganisms, nowadays, cancer, diabetes, atherosclerosis, as well as neurodegenerative disorders occur frequently. one of the significant triggers for the mentioned diseases and disorders is oxidative stress (limón-pacheco and gonsebatt, 2009). since the capacity of cellular antioxidant defence is insufficient in some cases and synthetic antioxidants could have toxic and mutagenic effects, and as awareness about healthy lifestyle is permanently raised, search for natural antioxidants presents current trend. nowadays, natural remedies are also favoured for cancer treatment, because of the many side effects of the chemotherapy, such as the same effect on cancer and healthy cells, occurrence of mutations which could pass into future generations, as well as more aggressive metastasis of returned disease. based on traditional experience and scientific data, numerous mushroom species represent important resources of antimicrobial, antioxidant, and anticancer agents. although special attention is given to the species of the genera ganoderma, lentinus, trametes, and pleurotus, other mushrooms also produce active compounds and their biological activities should be tested (zhang et al., 2007; chen et al., 2008; shi et al., 2012). one of them is flammulina velutipes (curt. fr.) karst, known as golden needle mushroom, enoki, enokidake and enokitake. this species is a famous edible and medicinal mushroom due to synthesis of numerous bioactive molecules, such as polysaccharides, proteins, sterols etc. significant immunomodulating, antitumor, antioxidant, and antiviral potential of its extracts and compounds were reported (bao et al., 2010; chang et al., 2010; yang et al., 2012). chang et al. (2010) demonstrated cytotoxic effect of immunomodulatory protein fve against murine hepatocellular carcinoma, and yang et al. (2012) showed anti-proliferation activity of polysaccharide fvp against human gastric and lung cancer cells. however, antibacterial and especially antifungal activities of the extracts were poorly studied (rodrigues de melo et al., 2009; wang et al., 2012). among essential microelements, se takes significant place in animal and human diet, due to its participation in biosynthesis of selenoproteins and selenoenzymes involved in cell protection against free radicals and indirectly in cell prevention from malignant transformation (rayman, 2005; brigelius-flohé, 2006). organic se compounds, depending on form and concentration, also have antimicrobial activity based on catalysis of o 2 production which causes oxidative damage of bacterial cell membrane and cell disfunction (xu et al., 2008). considering that se bioavailability from organic forms is better than from inorganic ones, and that mushrooms have ability to absorb inorganic se and convert it to organic, their usage as food and dietary supplements could contribute the prevention of disease appearance (kalać , 2010). however, there are just a few reports about biological activity of seenriched mushroom mycelia (malinowska et al., 2009; turlo et al., 2010; shi et al., 2010), and there are no available data for f. velutipes. considering everything mentioned, the aims of the study were to evaluate antioxidant, antifungal and anticancer potential of f. velutipes mycelial extract and examine in which way mycelium enrichment with se affects those activities. materials and methods organism and cultivation conditions the culture of flammulina velutipes hai 966 was obtained from institute of evolution, university of haifa, israel (hai), and maintained on malt agar medium in the culture collection of the institute of botany, faculty of biology, university of belgrade. the inoculum preparation involved the following steps: (i) inoculation of 100.0 ml of synthetic medium (glucose, 10.0 g/l; nh 4 no 3 , 2.0 g/l; k 2 hpo 4 , 1.0 g/l; nah 2 po 4 x h 2 o, 0.4 g/l; mgso 4 x 7h 2 o, 0.5 g/l; yeast extract, 2.0 g/l; ph 6.5) with 25 mycelial discs (ø 0.5 cm, from 7-day-old culture from malt agar); (ii) incubation at room temperature (22 ± 2ºc), on a rotary shaker (100 rpm), for 7 days; (iii) washing of obtained biomass (3 times) by sterile distilled water (dh 2 o); (iv) biomass homogenization with 100.0 ml of sterile dh 2 o in a laboratory blender. homogenized inoculum (30.0 ml) was used for inoculation of 400.0 ml modified synthetic medium (with glucose in the amount of 65.0 g/l and peptone in the concentration of 2.0 g/l, previously determined as the optimal carbon and nitrogen sources and concentrations for biomass production) enriched with sodium selenite (na2 seo 3 ) in the initial se concentration of 1.3 mg/l. the medium without se was used as the control. submerged cultivation was carried out at room temperature on rotary shaker for 21 days. the obtained biomass was filtered, washed 3 times with dh 2 o at magnetic stirrer and temperature of 30°c with the aim of removing the remaining se from cell wall, and dried at 50°c to constant weight. preparation of the fungal extracts dry se-amended and non-amended mycelia (3.0 g) were extracted by stirring with 90.0 ml of 96% ethanol at the 30°c for 72 h. the ital. j. food sci., vol. 27 2015 3 obtained extracts were filtered through whatman no. 4 filter paper, concentrated under reduced pressure in a rotary evaporator (büchi r-114, switzerland) at 40°c to dryness, and redissolved in 96% ethanol (for the testing of antioxidant activity) or in 5% dimethylsulphoxide (dmso) (for the analysis of antifungal and anticancer activity). antioxidant activity dpph assay antioxidant activity was defined by measuring bleaching of the purple-coloured methanol solution of stable 1,1-diphenyl-2-picryl-hydrazil radical (dpph•) (blois, 1958). 1800.0 µl of 4% methanol solution of dpph• and 200.0 µl of extract of defined concentration (series of double dilutions from 32.0 mg/ml to 0.5 mg/ml) were mixed and shaken. after 30 min of incubation in the darkness, the absorbances of reactive mixtures were measured at 517 nm against methanol as blank by spectrophotometer (cecil ce2501, u.k.). the negative control contained all the reaction reagents except the extract. scavenging effects was calculated by equation: dpph scavenging effect (%) = = [(a 0 a sample )/a 0 ] x 100, a 0 the absorbance of the negative control; a sample the absorbance of reaction mixture. the ec 50 value (mg extract/ml) is the effective concentration at which the dpph• were scavenged by 50% and was obtained by interpolation from linear regression analysis. commercial antioxidant, butylated hydroxyanisole (bha) was used as a positive control. determination of total phenolic content total soluble phenolic compounds in the ethanolic extracts of se-amended and non-amended mycelium were estimated with folin-ciocalteu reagent according to the method of singleton and rossi (1965), using galic acid as a standard. 1000.0 µl of 10% folin-ciocalteu reagent and 200.0 µl of the extract were reacted in the dark for 6 min before addition of 800.0 µl of 7,5% na 2 co 3 . the reaction mixture was vortexed vigorously and incubated on a rotary shaker (100 rpm) in the dark and at the room temperature for 2 h. the absorbance was measured at 760 nm by spectrophotometer against blank (mixture without extract). the total concentration of phenolic compounds in tested extracts determined as µg of galic acid equivalents (gae) per mg of dry extract, using an equation that was obtained from standard galic acid graph as: absorbance = 0.012 x total phenols + + 0.041 (r2 = 0.999) determination of total flavonoid content total flavonoid content was determined using the methods of park et al. (1997). 1000.0 µl of the mycelium extract was diluted with 4300.0 µl mixture containing 4100.0 µl of 80% ethanol, 100.0 µl of 10% aluminium nitrate and 100.0 µl of 1 m aqueous potassium acetate. the mixture was incubated at room temperature for 40 min, and absorbance was measured spectrophotometrically at 415 nm. the amount of total flavonoids was expressed as µg of quercetine equivalents (qe) per mg of dry extract, using an equation that was obtained from standard quercetin hydrate graph as: absorbance = 0.014 x total flavonoid 0.072 (r2 = 0.989) antifungal activity the tested micromycetes (table 1) are maintained on malt agar at 4°c in the culture collection of the institute of botany, faculty of biology, university of belgrade. tested micromycetes were cultivated on sabouraud dextrose agar (sda) at temperature of 25 ± 2°c for 21 days. spore suspensions were prepared by washing of agar surface with sterile 0.9% saline containing 0.1% tween 80 (v/v). turbidity was determined spectrophotometrically at 530 nm and spore number was adjusted to 106 cfu/ml (nccls, 1998). dmso extracts of se-amended and non-amended mycelia were sterilized by filtration through whatman no. 4 filter paper and 0.2 µm membrane filter. antifungal potential of the tested extracts was studied by microdilution method using 96-well microtiter plate (sarker et al., 2007). series of double extract dilutions (from 32.0 mg/ml to 0.5 mg/ml) was analyzed. each well contained sda, spore suspension, resazurine, and crude ethanol extract of defined concentration. the mixture without extract was used as the negative control, while positive control contained commercial antimycotic, ketoconazole, instead extract. tested ketoconazole concentrations ranged from 0.0313 mg/ ml to 0.0019 mg/ml (series of double dilutions). effect of 5% dmso on the spore germination was also analysed by its addition in the mixture instead sda. microtiter plates were incubated at 25 ± 2°c for 72 h. the lowest extract concentration without visible mycelium growth was defined as minimal inhibitory concentration (mic). minimal fungicidal concentration (mfc) was determined as the lowest extract concentration with no mycelial growth after reinoculation of 2 µl of the mixture on sda. the experiments were repeated three times. cytotoxic activity cell lines human cervix adenocarcinoma hela and human colon carcinoma ls174 cell lines were ob4 ital. j. food sci., vol. 27 2015 tained from the american type culture collection (atcc) (manassas, va, usa). both cancer cell lines were maintained in the recommended roswell park memorial institute (rpmi) 1640 medium supplemented with 100.0 g/l heat-inactivated (56°c) fetal bovine serum (fbs), 3 mm l-glutamine, 100.0 mg/ml streptomycin, 100.0 iu/ml penicillin, and 25 mm 4-(2 hydroxyethyl)-1-piperazineethanesulfonic acid (hepes) and adjusted to ph 7.2 with bicarbonate solution. cells were grown in a humidified atmosphere of 95% air/5% co 2 (v/v) at 37°c. treatment of cell lines stock solutions (100.0 mg/ml) of extracts, made in 50.0 g/l dmso, were dissolved in enriched rpmi 1640 medium to the required working concentrations. neoplastic hela cells (2000 cells per well) and neoplastic ls174 cells (7000 cells per well) were seeded into 96-well microtiter plates. 24 h later, after cell adherence, five different doubly diluted concentrations of the extracts were added to the wells. the final concentrations applied to target cells were 200.0, 100.0, 50.0, 25.0 and 12.5 µg/ml, except in the control wells where only the nutrient medium was added to the cells. the cultures were incubated for 72 h. determination of cell survival (mtt test) the effect of extracts on cancer cell survival was determined by microculture tetrazolium test (mtt test), according to mosmann (1983) with modification by ohno and abe (1991). 20.0 µl of mtt solution [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in phosphate-buffering saline] of concentration of 5.0 mg/ml was added to each well. samples were incubated for 4 h at 37°c in a humidified atmosphere of 95% air/5% co 2 (v/v). then, 100.0 µl of 10% sodium dodecyl sulfate was added to extract to dissolve the insoluble product formazan resulting from the conversion of the mtt dye by viable cells. the number of viable cells in each well was proportional to the intensity of the light absorbance (a), which was read in an enzyme-linked immunosorbent assay (elisa) plate reader at 570 nm 24 h later. the inhibition rate was calculated according to the formula: cell growth inhibition rate (%) = = (a control – a sample )/a control x 100 it was implied that the a of the blank was always subtracted from the a of the corresponding sample with target cells. ic 50 was defined as the concentration of the extracts inhibiting cell survival by 50%, compared with a vehicle-treated control. cis-diamminedichloroplatinum (cis-ddp) was used as a positive control. all experiments were done in triplicate. statistical analysis the results were expressed as the mean ± standard error of data obtained from three parallel measurements. one-way analysis of variance (anova) followed by lsd post-hoc determinations were performed using statistika software, version 5.0 (statsoft, inc) to test any significant differences. p-values less then 0.01 were considered statistically significant. table 1 antifungal activity (mic and mfc) of ethanol extracts of se-non amended and se-amended mycelium of flammulina velutipes and commercial antimycotic. tested organisms flammulina velutipes ketoconazole mic (mg/ml) mfc (mg/ml) mic mfc (mg/ml) (mg/ml) se non-amended se-amended se non-amended se-amended mycelium mycelium mycelium mycelium acremonium strictum w. gams 4.0 4.0 0.0078 0.0156 aspergillus flavus link 8.0 4.0 0.0078 0.0078 aspergillus fumigatus fresen. 32.0 16.0 0.0078 0.0156 aspergillus niger teigh. 32.0 16.0 0.0156 0.0313 aspergillus terreus thom 16.0 8.0 0.0156 0.0313 candida albicans (c.p. robin) berkhout 8.0 16.0 0.0078 0.0156 candida krusei (castell.) berkhout 8.0 2.0 0.0078 0.0156 candida parapsilosis (ashford) langeron & talice 16.0 2.0 0.0078 0.0156 cladosporium sp. 0.0078 0.0039 fusarium verticillioides (sacc.) nirenberg 8.0 4.0 0.0156 0.0156 microsporum gypseum (e.bodin) guiart & grigoraki 0.0019 0.0039 penicillium funiculosum thom 8.0 4.0 0.0039 0.0078 trichoderma viride pers. 8.0 8.0 0.0039 0.0078 trichophyton mentagrophytes (c.p. robin) sabour. 0.0019 0.0039 ital. j. food sci., vol. 27 2015 5 fig. 1 dpph radical scavenging capacity of ethanol extracts of flammulina velutipes mycelium. se non-amended (◻); seamended (◾). (data represent mean value of activities of three different samples. variations are given as standard errors). results antioxidant activity ethanol extracts of both se-amended and nonamended f. velutipes mycelium showed good antioxidant potential that was dependent on the concentration, at higher concentrations extracts were more effective in dpph• scavenging (fig. 1). however, significant difference between the extracts, especially at higher concentrations, was noted (p<0.01). se-enriched mycelium extract had higher scavenging effect than the control, and it exhibited a progressive increase of the activity at concentrations up to 8.0 mg/ml (from 4% to even 15% at concentration of 32.0 mg/ml). these results were confirmed by ec 50 values, which were 30.5 ± 0.3 mg/ml in se-enriched mycelium extract and 43.8 ± 0.6 mg/ml in non-amended one. commercial antioxidant bha was more efficient comparing with f. velutipes mycelium extracts, with ec 50 value of 13.4 µg/ml. total phenol compounds were detected in both tested extracts. their content in the non-amended extract was 9.5 ± 1.8 µg/mg, while the higher concentration of 14.5 ± 1.1 µg/mg was noted in se-amended extract. however, flavonoids were not detected in any of the extracts. direct correlation between phenol content and dpph• scavenging effect existed, and linear relationship with r2 = 0.989 in non-amended mycelium extract and r2 = 0.957 in se-amended one were noted. antifungal activity the antifungal potential of ethanol extracts of se-amended and non-amended mycelium was tested against 14 micromycetes including saprobes as well as plant, animal and human pathogens. in the most cases, mics of se-enriched mycelium extract were lower, except for candida albicans where control extract had twice more effect, and for acremonium strictum and trichoderma viride where mics of se-amended and nonamended extracts were the same (table 1). the most sensitive species, with mic of se-enriched mycelium extract of 2.0 mg/ml, were c. krusei and c. parapsilosis. the most resistant species were aspergillus fumigatus and a. niger, which growth was inhibited only with the maximum extract concentration (32.0 mg/ml for non-amended and 16.0 mg/ml for se-amended one). tested extract concentrations (from 0.5 mg/ml to 32.0 mg/ ml) have no inhibitory effect on cladosporium sp. and both causal agents of dermatomycosis, microsporium gypseum and trichophyton mentagrophytes. the maximum tested concentration of extract (32.0 mg/ml) did not show fungicidal effect for any tested fungal species (table 1). sensitivity of the tested species to commercial antimycotic, ketoconazole, was more higher. thus, the lowest tested concentration of 0.0019 mg/ml was mic for cladosporium sp., m. gypseum, and t. mentagrophytes. the concentration of 0.0039 mg/ml was mic for penicillium funiculosum and t. viride and mfc for cladosporium sp., m. gypseum, and t. mentagrophytes. mycelium growth of a. strictum, a. flavus, a. fumigatus, c, albicans, c. krusei, and c. parapsilosis was inhibited at ketoconazole concentration of 0.0078 mg/ ml, which was also mfc for a. flavus, p. funiculosum and t. viride. concentration of 0.0156 mg/ ml was mic for a. niger, a. terreus, and fusarium verticillioides and mfc for most of the tested species, while the highest tested concentratable 2 cytotoxic activity (ic 50 ) of se-non amended and se-amended flammulina velutipes mycelium extracts and commercial cytostatic against hela and ls 174 cell lines. mycelial extract/cytostatic ic 50 (µg/ml) hela ls 174 se-non se-amended se-non se-amended amended extracts extracts amended extracts extracts f. velutipes 259.69 ± 0.70 331.91 ± 0.49 338.47 ± 0.97 348.46 ± 0.34 cis-ddp 0.72 ± 0.14 2.61 ± 0.11 6 ital. j. food sci., vol. 27 2015 tion (0.0313 mg/ml) was mfc for a. niger and a. terreus (table 1). 5% dmso, used as a negative control, had no inhibitory effect on the tested micromycetes. cytotoxic activity comparing with cis-ddp, which was used as the positive control, the tested extracts showed low cytotoxic activity against both hela and ls174 cell line (table 2). ic 50 of non-amended mycelium extract against hela cells was 360and 130fold, respectively, higher than values obtained for cis-ddp. se-amended mycelium extract was weaker cytotoxic agent than control one, especially for hela cell line. discussion enrichment of cultivation medium with se in the initial concentration of 1.3 mg/l and ability of f. velutipes hai 966 mycelium to absorb and incorporate it in significant amount (10.0 µg/g) (milovanović et al., 2013) led to enhancement of antioxidant and antifungal capacity, while cytotoxic activity was reduced and was not in correlation with antioxidant potential. dpph• scavenging activity of f. velutipes ethanol extracts was reported by bao et al. (2010), who demonstrated higher efficiency of mycelium extract than fruiting body one in almost 4-fold. according to saltarelli et al. (2009), the activity bases on redox properties of phenols, which enable them to act as reducing agents and hydrogen donators. direct dependence of antioxidant capacity on phenol content was confirmed by chen et al. (2008) and bao et al. (2010). lower phenol content in f. velutipes hai 966 mycelium extracts, compared with that in reported data, was responsible for negligible antioxidant potential that was significant only at the highest concentration. lack of flavonoid in the ethanol extracts was in accordance with results of karaman et al. (2009) who also have not found those compounds in mycelium extracts of f. velutipes. however, other compounds from those extracts could also be carriers of antioxidant activity (shi et al., 2012; wang et al., 2012). thus, shi et al. (2012) demonstrated significant scavenging effects of polysaccharides, even 90% at concentration of 2.5 mg/ml, while wang et al. (2012) have noted high efficiency of sesquiterpenoids. enhanced antioxidant potential of se-amended f. velutipes hai 966 mycelium extract is in accordance with results of turlo et al. (2010) who noted higher level of antioxidant activity in lentinus edodes after cultivation in se-enriched medium. the better effect could be explained by the fact that selenomolecules react with free radicals (•oh, •h) and successfully neutralize them (shen et al., 2010). wang et al. (2012) tested antifungal potential of sesquiterpenoids isolated from f. velutipes mycelium against numerous species and reported weak activity against a. fumigatus and absence of any inhibitory effect on c. albicans. similar, negligible activity of ethanol extract of f. velutipes hai 966 mycelium against a. fumigatus was also noted but it was enhanced with se addition. however, tested candida spp. were very sensitive to the extracts, especially in the se presence, which was contrary to the results of wang et al. (2012). results of this study showed that se acts as an antifungal agent which is in accordance with results of shahverdi et al. (2010) who clearly demonstrated antifungal activity of biogenic se against selected clinical micromycetes. mycelium enrichment with this element presents a way for getting biogenic se nanoparticles that could be a potent ingredient for the preparation of antifungal formulations (shahverdi et al. 2010). contrary to se stimulatory effect on antioxidant activity, this trace element caused regression of cytotoxic activity. numerous biologically active compounds, such as diverse types of sesquiterpenes, polysaccharides, glycoproteins, ribosome inactivating proteins, and sterols, isolated from mycelium of f. velutipes, take important place as cytotoxic agents against various cancer cell lines (leung et al., 1997; ng and wang, 2004; wang et al., 2012; yang et al., 2012; yi et al., 2013). leung et al. (1997) showed that soluble homopolysaccharide, composed of glucose and isolated from f. velutipes fruiting bodies, were very efficient in sarcoma-180 cells regression in vivo. the same effect was noted by yang et al. (2012) for alkaline-soluble heteropolysaccharides with a glucan as backbone chain and triple helix structure, which also have strong anti-proliferation activity against lung cancer cells (a549) and human gastric cancer cells (bgc-823) (inhibitory rate was 32.3% and 95%, respectively). the cytotoxic effect of polysaccharides was based on proliferation of b-cell, t-cell or both cells and not on tumor cell kill. activation of immune system and in such a way production of interferon-gamma with antiproliferate effect on tumor cells was also lied in the base of antitumor activity of f. velutipes glycoprotein fve (chang et al., 2010). however, ribosome inactivating proteins (flammulin, velutin, flammin, and velin) stopped cancer cell proliferation by ribosome inactivation and translation inhibition (ng and wang, 2004). efficiency of sesquiterpenoids against human liver carcinoma cell line (hepg2), breast cancer cell line (mcf-7), human gastric cancer cell line (sgc7901) and a549 was modest (ic 50 was in range between 20.0 and 100.0 µm) (wang et al., 2012), while inhibition rate of human glioma cell line (u251) by sterols, at concentration of 20 µg/ml, was even 57% (yi et al., 2013). however, the sterol was not so efficient against hela cell line (ic 50 > 40.0 µg/ml), but more efficient comparing with tested se-amended and non-amended mycelium extract. although tested extracts showed lower biologital. j. food sci., vol. 27 2015 7 ically activities than commercial antimycotic, antioxidant and cytostatic, preference should be given to natural products. therefore, f. velutipes is not only food but also could be considered as a possible base for natural safe remedies for human therapy. acknowledgents this study was carried out with financial support of ministry of education, science, and technological development of republic of serbia, project no. 173032. references bao h.n.d., ochiai y. and ohshima t. 2010. antioxidative activities of hydrophilic extracts prepared from the fruiting body and spent culture medium of flammulina velutipes. bioresource technol. 101: 6248-6255. blois m.s. 1958. antioxidant determination by the use of stabile free radical. nature, 181: 1199-1200. brigelius-flohé r. 2006. glutathione peroxidases and redox-regulated transcription factors. biol. chem. 387: 1329-1335. chang h.h., hsieh k.y., yeh c.h., tu y.p. and sheu f. 2010. oral administration of an enoki mushroom protein fve activates innate and adaptive immunity and induces anti-tumor activity against murine hepatocellular carcinoma. int. immunopharmacol. 10: 239-246. chen y., xie m.y., nie s.p., li c. and wang y.x. 2008. purification, composition analysis and antioxidant activity of a polysaccharide from the fruiting bodies of ganoderma atrum. food chem. 107: 231-241. kalač p. 2010. trace element contents in european species of wild growing edible mushrooms: a review for the period 2000-2009. food chem.122: 2-15. karaman m., mimica-dukič n.m. and matavulj m. 2009. lignicolous 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assay for cellular growth and survival: application to proliferation and cytotoxicity assays. j. immunol. method. 65: 55-63. nccls. 1998. reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi: proposed standard m38-p.wayne, pa, usa. ng t.b. and wang h.x. 2004. flammin and velin: new ribosome inactivating polypeptides from the mushroom flammulina velutipes. peptides. 25: 929-933. ohno m. and abe t. 1991. rapid colorimetric assay for the quantification of leukemia inhibitory factor (lif) and interleukin-6 (il-6). j. immunol. method. 145: 199-203. park y.k., koo m.h., ikegaki m. and contado j.l. 1997. comparison of the flavonoid aglycone contents of apis mellifera propolis from various regions of brazil. braz. arch. biol. technol. 40: 97-106. rayman m.p. 2005. selenium in cancer prevention: a review of the evidence and mechanism of action. proc. nutr. soc. 64: 527-542. rodrigues de melo m., paccola-meirelles l.d., faria t.d.j. and ishikawa n.k. 2009. influence of flammulina velutipes mycelia culture conditions on antimicrobial metabolite production. mycoscience 50:78–81. saltarelli r., ceccaroli p., iotti m., zambonelli a., buffalini m., casadei l., vallorani l. and stocchi v. 2009. biochemical characterisation and antioxidant activity of mycelium of ganoderma lucidum from central italy. food chem. 116: 143-151. sarker s.d., nahar l. and kumarasamy y. 2007. microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. methods 42: 321-324. shahverdi a.r., fakhimi a., mosavat g., jafari-fesharaki p., rezaie s. and rezayat s.m. 2010. antifungal activity of biogenic selenium nanoparticles. world appl. sci. j. 10: 918-922. shen q., zhang b., xu r., wang y., ding x. and li p. 2010. antioxidant activity in vitro of the selenium-contained protein from the se-enriched bifidobacterium animalis 01. anaerobe 16: 380-386. shi w.l., han h, chen g.z., chen x., hong y.k., chen l.k., chen d. and lu z. 2010. extraction, characterization of the polysaccharide extracts from se-enriched g. lucidum (se-glp) and its inhibition against oxidative damage in ischemic reperfusion mice. carbohyd. polym. 80: 774–778. shi m., yang y., guan d., zhang y. and zhang z. 2012. bioactivity of the crude polysaccharides from fermented soybean curd residue by flammulina velutipes. carbohyd. polym. 89: 1268-1276. singleton v.l. and rossi j.a. 1965. colometric of total phenolics with phosphomolybdic-phosphotungstic acid reagents. ajev. 16: 144-158. turlo j., gutkowska b. and herold f. 2010. effect of selenium enrichment on antioxidant activities and chemical composition of lentinula edodes mycelial extracts. food chem. toxicol. 48: 1085-1091. wang y., bao l., liu d., yang x., li s., gao h., yao x., wen h. and liu h. 2012. two new sesquiterpenes and six norsesquiterpenes from the solid culture of the edible mushroom flammulina velutipes. tetrahedron 68: 3012-3018. xu x.j., xue z., xiao q., hou a.x. and liu y. 2008. antibacterial activities of novel diselenide-bridged bis(porphyrin) s on staphylococcus aureus investigated by microcalorimetry. biol. trace elem. res. 125: 185-192. yang w., pei f., shi y., zhao l., fang y. and hu, q. 2012. purification, characterization and anti-proliferation activity of polysaccharides from flammulina velutipes. carbohyd. polym. 88: 474480. yi c., sun c., tong s., cao x., feng y., firempong c.k., jiang x., xu x. and yu j. 2013. cytotoxic effect of novel flammulina velutipes sterols and its oral bioavailability via mixed micellar nanoformulation. int. j. pharm. 448: 44-50. zhang m., cui s.w., cheung p.c.k. and wang q. 2007. antitumor polysaccharides from mushrooms: a review on their isolation process, structural characteristics and antitumor activity. trends food sci. technol. 18: 4-19. paper received february 4, 2014 accepted may 26, 2014 ijfs#870_bozza ital. j. food sci., vol. 30, 2018 348 paper volatile compounds and sensory properties of coleslaw mix packaged in modified atmosphere e. radziejewska-kubzdela*, h. jeleń and r. biegańska-marecik institute of technology of plant origin food, poznan university of life sciences, wojska polskiego 31, 60-624 poznan, poland *e-mail address: elarad@up.poznan.pl abstract the effect of modified atmosphere and film microperforation on the aroma of a coleslaw mix stored for 12 days at 4˚c was detected. samples were packaged in air and modified atmosphere (5/10/85, 70/30/0% o2/co2/n2) and sealed with the film with or without microperforations. the application of microperforated film makes it possible to maintain a desirable aroma for 12 days. use of the unmicroperforated film resulted to increase in allyl isothiocyanate and dimethyl trisulfide concentration. a strong correlation was observed between the following aroma attributes and volatile compounds: sharp allyl isothiocyanate (r2=0.80), dimethyl trisulfide (r2=0.83); and carrot – p-cymene (r2=0.71). keywords: aroma, cabbage, carrot, minimally processed, modified atmosphere ital. j. food sci., vol. 30, 2018 349 1. introduction in minimal processing, the modified atmosphere packaging (map) is frequently used to extend the shelf life of product. in the case of fruits and vegetables, the recommended solution is to expose them to the atmosphere containing 1-5% oxygen and 5-10% carbon dioxide (balanced with nitrogen). a new trend is packaging in superatmospheric oxygen atmospheres (radziejewska-kubzdela and czaczyk, 2016). from literature, it was observed that an increased oxygen content in the atmosphere may inhibit enzymatic discoloration, prevent anaerobic fermentation reaction, influence aerobic and anaerobic microbial growth, reduce decay of the fresh vegetable and also prevent odour losses (day, 1996). the studies of cliffe-byrnes et al. (2003), and radziejewskakubzdela and biegańska-marecik (2009) indicated that aroma is one of the most important factors determining the quality of minimally processed cabbage. available literature lacks data concerning both the profile of aroma compounds in coleslaw mix and the effect of packaging conditions on the aroma of this product. research is limited to minimally processed broccoli, carrot, york cabbage or lettuce (cliffe-byrnes et al., 2007; deza-durand and petersen, 2014; jacobsson et al., 2004; lonchamp et al., 2009). the profile of aroma compounds may be a significant determinant of quality for such products. during storage in modified atmosphere, the respiration rate of tissue may lead to a decreased o2 content and an increase in the co2 level inside the package if the gas composition and the permeability of the film is insufficient. this change may result in the formation of fermentative metabolites (such as ethanol, ethyl, acetate, acetaldehyde, methyl acetate and acetone) occurring during anaerobic respiration. aroma of product from the brassicaceae family is also determined by sulfurous compounds. during the loss of intracellular compartmentalization, enzymes can react with substrates causing strong off-odors. there are mainly products of s-methyl-l-cysteine and s-methylcysteine sulfoxide degradation by the action of cysteine sulfoxide lyase (methanethiol, dimethyl disulfide and dimethyl trisulfide) as well as degradation products of glucosinolates formed as a result of myrosinase activity (akpolat and barringerb, 2015; jang et al., 2015). aroma of coleslaw mixes may also be influenced by terpenes originating both from carrot roots and cabbage (vuorinen et al., 2004; yahyaa et al., 2015). kjeldsen et al. (2003) reported that during cold storage of carrot, an increase in the contents of terpene compounds to moderate amounts (10-30 ppm) was connected with the carrot aroma, whereas at >35-40 ppm, terpenes produced a harsh and burning turpentine-like flavor. correlation between aroma and volatile compounds is dependent on the composition of product. relationship between cabbage and carrot in coleslaw mix was not tested. a better understanding of these relations is needed to facilitate the development of strategies in order to prevent off-odor formation in package. our previous studies indicate that the application of modified atmosphere composed of 70/30/0% o2/co2/n2 in packaging of a coleslaw mix with a microperforated barrier film resulted in a good sensory and microbiological quality during the 12 days of storage (radziejewska-kubzdela and czaczyk, 2016). for this reason, an analysis of the profile of volatiles in the coleslaw mix was conducted using the above-mentioned composition in the atmosphere, while for comparison, samples packaged in air and in the atmosphere of 5/10/85% o2/co2/n2 were also tested. in the case of an atmosphere with a 70% oxygen content, a barrier film was also applied in product packaging, as it is frequently recommended for superatmospheric oxygen atmospheres in packaging products of plant origin. the aim of this study was to determine the effect of modified atmosphere, and film microperforation on the aroma of a coleslaw mix stored for 12 days at 4˚c. correlations ital. j. food sci., vol. 30, 2018 350 between sensory aroma attributes and contents of volatile compounds in the atmosphere composition were also investigated. 2. materials and methods 2.1. materials coleslaw mix was produced by mixing shredded white cabbage cv. galaxy and carrot cv. perfekcja. the raw materials came from a farm located in western poland. prior to technological process, white cabbage and carrot were stored for 1 week at 1°c (this step is not necessary). 2.2. technological process white cabbages and carrots were washed in tap water. thereafter, the outer leaves of white cabbage were removed and the heads were cored using a sharp knife. carrots were hand-peeled. the vegetables were washed in tap water and dried with absorbent paper. after drying, the vegetables were shredded mechanically, a nagema hu-1 device (dresden, germany) was used for cabbage, while a robot coupe cl 50 ultra device (vincennes, france) was used for carrot. shredded cabbage and carrot were mixed at a ratio of 80/20 (w/w). the anti-microbial treatment involved dipping coleslaw for 5 min with agitation in 5 g/l ascorbic acid and 5 g/l citric acid solution. the adherent water after pretreatments was removed using a manual vegetable spinner (zepter, viersen, germany). after that, the shredded material was weighed out (160 g white head cabbage and 40 g carrot) and placed on 205 x 160 x 60 mm polypropylene trays with an oxygen transmission rate of 7-8 cm3/m2/24 h. the selected atmosphere concentrations % of o2/co2/n2: 5/10/85 and 70/30/0, and air atmosphere were introduced into the packages before thermally sealed with a gas packaging device gas mixer (witt-gasetechnik, witten, germany), multivac t 200 packaging machine (wolfertschwenden, germany). the trays were sealed with an opalen hb 55 packaging film with oxygen permeability of 35 cm3/m2/24 h * atm at 23°c and 85% rh (according to data provided by the film manufacturer) (bemis, soignies, belgium). the film was then microperforated. the microperforations had been made by the multivac system (wolfertschwenden, germany) using one cylinder with 10 needles of 70 μm in diameter (10 microopenings in the film, sealing the tray – 333 holes/m2 of the film). in the case of modified atmosphere composed of 70/30/0% o2/co2/n2, the trays were also sealed with the same film but without microperforations. 2.3. gas composition contents of oxygen and carbon dioxide inside the packages were determined using an oxybaby gas analyzer by witt-gasetechnik (witten, germany). the results were reported as means of three experimental determinations for separated sample ((radziejewska-kubzdela and czaczyk, 2016). 2.4. analysis of volatile compounds the volatile compounds were analyzed after 1, 6, 9 and 12 days of storage at 4°c using solid phase microextraction (spme). a spme fiber coated with ital. j. food sci., vol. 30, 2018 351 divinylbenzene/carboxen/polydimethylsiloxane (dvb/car/pdms) was used to collect volatile compounds within each package. to facilitate the headspace-spme analysis in a semi-quantitative way deuterated (d8) naphthalene (sigma aldrich chemie gmbh, taufkirchen, germany) was used as an internal standard (is). as addition of the internal standard to sampled coleslaw would result in non-uniform distribution of standard, is adsorption on the fiber was used before sample extraction option. for this purpose, before each coleslaw sampling, spme fiber was exposed for 5 minutes to is solution (10 µg/ml) in silicon oil. the solute (10 ml) was placed in a 20 ml headspace vial capped with silicon rubber/ptfe membrane. both is sampling and subsequent coleslaw sampling was performed at room temperature (20°c ± 1°c). the high concentration of is in the solution compared to fiber capacity allowed for multiple extraction of is from a single vial. the fiber was exposed to a headspace of the sample for 30 min and after extraction time, the fiber was transferred immediately to an injection port of a gas chromatograph and desorbed for 5 min at 250°c in a splitless mode. compound identification was performed using an agilent 7890a gas chromatograph coupled to a 5975c tad single quadrupole mass spectrometer (agilent technologies, santa clara, ca) with a db-5ms column (25 m ×0.200 mm×0.33 µm, agilent technologies, santa clara, ca). the carrier gas was helium at a flow rate of 0.8 ml min-1, while oven temperature was 40°c for 1 min, followed by an increase of 8°c min-1 to 220°c and 20 °c min-1 to 280°c. mass spectra were recorded in an electron impact mode (70 ev) in a scan range of m/z 33–333. the mass spectra of volatile compounds were identified tentatively by comparison with spectra of the nist 05 mass spectral library. volatile peaks (total ion current) were compared to that of is and the results provided in ng/g of fresh weight of coleslaw. no correction factors were used. 2.5. sensory evaluation of aroma quantitative descriptive analysis was used to characterize the aroma of coleslaw mixes packaged in air and modified atmosphere during 12 days of storage at 4 °c. the panel consisted of 10 members (all employed at the poznan university of life sciences) who were trained in evaluation according to iso 8586-1. sensory attributes were selected from literature and from orientation sessions, in accordance with iso 11035. a total of 8 descriptors were established as the final list. panelists assessed all descriptive attributes on a 10 cm unstructured line. the results from linear scale were converted into numerical values for data analysis. the 0 value indicated the lowest value intensity and 9 – the highest. 2.6. statistical analysis the two-way variance analysis (anova) and fisher’s least significant difference (lsd) were performed and pearson’s correlation coefficients between aroma attributes and contents of volatile compounds were calculated. statistically significant differences were reported at p = 0.05. principal component analysis (pca) and partial least-squares (pls) regression analysis were performed using the statistica version 9.1 computer software (statsoft inc., tulsa, usa). ital. j. food sci., vol. 30, 2018 352 3. results and discussion 3.1. o2 and co2 contents the results concerning changes in o2 and co2 contents in stored samples are presented in table 1. after 9 days to the end of the assumed storage time, the oxygen content in samples packaged with microperforated film was uniform and remained at 11.2% to 11.7% in the case of samples packaged in the atmosphere containing 70/30/0% o2/co2/n2 in the film with no microperforation a superatmospheric oxygen level was maintained throughout storage and after 12 days it was 44.1%. in samples packaged in an atmosphere with a 30% content of co2, sealed with microperforated film, a significantly (p=0.05) higher level of this gas lasted till day 6 of storage. after 9 days of storage, co2 contents was observed (from 11.3% to 12.1%) in all tested salad mixes packaged in microperforated film equalization. in turn, in samples packaged in modified atmosphere (70/30/0% o2/co2/n2) and in film with no microperforation the carbon dioxide content is significantly (p=0.05) increased during storage and after 12 days amounted to 46.8%. table 1. o2 and co2 contents in coleslaw mix stored for 12 days at 4˚c. content of gas (%) storage time (days) air with film microperforation 5/10/85 70/30/0 70/30/0 % o2/co2/ n2 % o2/co2/ n2 % o2/co2/ n2 with film with film without film microperforation microperforation microperforation o2 1 6 9 12 15.3±0.9 12.9±1.0 11.2±0.2 11.4±0.4 e cd b b 8.8±0.9 11.9±0.5 11.7±0.5 11.5±0.6 a bc bc b 43.1±0.2 13.9±0.9 11.2±0.6 11.5±0.8 f d b b 60,1±0.7 51.7±0.8 48.3±1.4 44.1±1.2 i h g f co2 1 6 9 12 5.8±0.5 9.2±0.2 11.5±0.4 11.7±0.8 a b c c 10.5±0.6 11.8±0.6 11.8±1.1 12.1±1.3 bc c c c 20.2±1.2 14.9±2.1 11.3±1.2 11.7±0.8 e d c c 30.2±0.4 33.5±0.7 44.3±1.9 46.8±1.0 f g h i mean ± standard deviation (n=3) for parameter with different letters are significantly different at p = 0.05. contents of oxygen and carbon dioxide in the atmosphere inside the packaging are influenced both by film permeability and intensity of tissue respiration processes (radziejewska-kubzdela and czaczyk, 2015). the composition of the atmosphere inside the packaging at the application of the barrier film with no microperforation is influenced first of all by respiration processes. a study by jacxsens et al. (2001) showed that packaging of grated celeriac, mushroom slices and shredded chicory endive in a modified atmosphere with a 95% oxygen content in barrier film caused a considerable reduction in oxygen content in the atmosphere of the packaging (to as little as 19.9%, 6.8%, 12.0%, respectively) and an increase in carbon dioxide levels (45.5% for grated celeriac and 47.5% for mushroom slices). in the case of the coleslaw mix, the application of superatmospheric oxygen atmosphere in combination with an elevated content of co2 seems to be a factor inhibiting the intensity of respiration processes in finely comminuted tissue. in the tested samples after 12 days of storage the content of co2 was approximately 17% higher than after packaging. the effect of the reduced respiration intensity in ital. j. food sci., vol. 30, 2018 353 butterhead lettuce at the application of a superatmospheric oxygen atmosphere with an elevated (10-20%) content of carbon dioxide was reported by escalona et al. (2006). 3.2. volatile compounds the figs. 1, 2, 3 and 4 presented the content of volatile compounds in stored coleslaw mixes. volatile compounds identified in the coleslaw mix included monoterpenes, sesquiterpene, sulfur compounds and alcohol. the presence of sulfur compounds was recorded only in samples packaged in a modified atmosphere of 70/30/0% o2/co2/n2 in the film with no microperforation. allyl isothiocyanate was detected after 6, 9 and 12 days of storage, while dimethyl trisulfide was detected after 12 days of storage. the content of allyl isothiocyanate significantly (p=0.05) increased during storage. from literature, it was observed that these compounds are responsible for the sharp, sulfury aroma, which in the case of a minimally processed product may be considered undesirable (akpolat and barringerb, 2015). allyl isothiocyanate is formed as a result of enzymatic (myrosinase) degradation of sinigrin. the accumulation of this compound in the above-mentioned samples may result both from the lack of film microperforation and the high concentration of carbon dioxide inside the packaging. studies conducted by radziejewska-kubzdela and czaczyk (2015) showed that a high level of co2 in the atmosphere may damage cell membrane integrity. in turn, the formation of dimethyl trisulfide is most probably connected with the degradation of s-methylcysteine sulfoxide, under the influence of cysteine sulfoxide lyase (jones et al., 2004). monoterpenes found in the coleslaw mix may originate both from carrot and cabbage. a study by vuorinen et al. (2004) showed that such compounds as α-thujene, α-pinene, βpinene, sabinene, limonene and γ-terpinene are released as a result of break in continuity of the cabbage tissue. kjeldsen et al. (2003) and cliffe-byrnes et al. (2007) indicated the presence of these compounds also in the aroma profile of carrot. moreover, in that raw material, they identified camphene, myrcene, α-phellandrene, α-terpinene, p-cymene, βphellandrene, ocimene and terpinollene and indicated the presence of sesquiterpenes, of which only β-caryophyllene was identified in the coleslaw mix. however, the profile of volatile compounds determined by kjeldsen et al. (2003), and yahyaa et al. (2015) in carrot and cabbage was much broader. a derivative of the octadecadienoic pathway, 3-hexen-1-ol, was identified only in salads packaged in the atmosphere with a superatmospheric oxygen content after 1 day of storage (fig. 1). this compound is emitted after mechanical tissue damage and it is labelled as the green leaf aroma. after 1-day storage, salads packaged in air contained camphene, myrcene and βphellandrene. in turn, in all the tested samples α-phellandrene and α-terpinene were detected. in samples sealed with microperforated film α-thujene, sabinene and β-pinene were detected up till day 9 of storage. however, during storage, a significant (p=0.05) decrease in their contents was recorded. in samples packaged in the film with no microperforation the depletion of α-thujene and sabinene was observed after 6 and 9 days of storage, respectively, while β-pinene was present till the end of the assumed storage period. in the case of the latter compound its content was found to increase significantly (p=0.05) during storage. ocimene, γ-terpinene and terpinolene were found in all the samples only after 1 day of storage. ital. j. food sci., vol. 30, 2018 354 figure 1. volatile compounds in coleslaw mix after 1 day storage (means (n=3) and standard deviations, different lowercase letters are significantly different (p=0.05). figure 2. volatile compounds in coleslaw mix after 6 days storage (means (n=3) and standard deviations, different lowercase letters are significantly different (p=0.05). ital. j. food sci., vol. 30, 2018 355 figure 3. volatile compounds in coleslaw mix after 9 days storage (means (n=3) and standard deviations, different lowercase letters are significantly different (p=0.05). figure 4. volatile compounds in coleslaw mix after 12 days storage (means (n=3) and standard deviations, different lowercase letters are significantly different (p=0.05). terpinolene remained present in salads packaged in air and in the modified atmosphere of 70/30/0% o2/co2/n2 up to day 6 of storage. after 9 and 12 days, it was detected only in samples packaged in superatmospheric oxygen atmosphere, while its content was ital. j. food sci., vol. 30, 2018 356 significantly (p=0.05) greater in salad sealed with film and with no microperforation. ocimene and γ-terpinene after 6 days were still found in samples packaged in the atmosphere of 70/30/0% o2/co2/n2, while after 9 and 12 days, they were detected only in samples sealed with film without microperforation. content of ocimene in that sample after 12-day storage was identical to that after 1 day of storage, whereas in the case of γterpinene and terpinolene, it was significantly (p=0.05) lower. throughout the entire storage period α-pinene, p-cymene, limonene and β-caryophyllene were recorded in all the tested samples. their contents were also dominant (figs. 1, 2, 3, 4). in carrot, kjeldsen et al. (2003) among the main monoand sesquiterpenes also indicated the presence of αpinene, limonene, p-cymene and β-caryophyllene, as well as sabinene, β-myrcene, γterpinene and α-humulene, (e)and (z)-γ-bisabolene. during storage in the tested samples, a significant (p=0.05) decrease in the level of α-pinene was observed, except for the sample packaged in the atmosphere of 5/10/85% o2/co2/n2. after 12 days of storage, the highest content of α-pinene was recorded in the sample packaged using film with no microperforation. in the case of limonene, its contents were found to initially decrease, followed by an increase. after 12 days of storage, the content of limonene in samples packaged with microperforated film was significantly (p=0.05) greater than after 1 day, while in the sample sealed using film with no microperforation it was lower. limonene is a possible oxidation product of chlorophyll in leafy green vegetables, thus its greater concentration in samples packaged in microperforated film may be connected with the depletion of chlorophyll in cabbage leaves (lonchamp et al., 2009). in the case of pcymene and β-caryophyllene, their contents were found to increase during storage. after 12 days the content of these compounds in tested samples did not vary significantly (p=0.05) (fig. 1, 2, 3, 4). an increase in contents of monoand sequiterpenes during cold storage of carrot was also observed by kjeldsen et al. (2003). the terpenoids may be synthesized in response to physiological stress. β-caryophyllene may be produced by the mevalonate pathway (lonchamp et al., 2009) and monoterpenes by the methylerythritol phosphate pathway (bouvier et al., 2005). 3.3. sensory analysis table 2 illustrated the sensory attributes for stored coleslaw determined by quantitative descriptive analysis. after 1 day of storage in all the examined samples, aroma attributes defined as carrot and cabbage predominated. after 6 days of storage, a significant (p=0.05) deterioration of aroma was observed in samples packaged in film with no microperforation. it was related with an increased intensity of perceived aroma attributes defined as sour and off-odor and a decrease in the intensity of the cabbage aroma. in all the tested samples, an aroma defined as terpene appeared and the green aroma disappeared. after 9 days, a further significant (p=0.05) deterioration of aroma was observed in the sample sealed with film without microperforation, which could have been caused mainly by the appearance of the sharp aroma and a greater intensity of terpene aroma. the intensity of cabbage aroma attributes significantly (p=0.05) decreased in salads packaged in modified atmosphere using microperforated film. after 12 days of storage, a further significant (p=0.05) increase in the intensity of sharp aroma was recorded in coleslaw mixes sealed with film with no microperforation. in those samples in comparison with other samples a greater intensity of sour, terpene and off-odor aroma was recorded at a significantly (p=0.05) lesser intensity of cabbage aroma. in salads packaged in microperforated film, the aroma defined as earth ital. j. food sci., vol. 30, 2018 357 and off-odor was reported. in the case of samples packaged in modified atmosphere a significantly (p=0.05) greater intensity of aroma defined as sour was also found. table 2. aroma profile of coleslaw mix stored for 12 days at 4˚c. aroma descriptive attributes storage time (days) air with film microperforation 5/10/85 70/30/0 70/30/0 % o2/ co2/ n2 % o2/ co2/ n2 % o2/ co2/ n2 with film with film without microperforation microperforation microperforation sharp 1 6 9 12 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 a a a a 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 a a a a 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 a a a a 0.0±0.0 0.0±0.0 5.3±0.2 8.0±0.4 a a b c carrot 1 6 9 12 5.7±0.3 8.1±0.4 8.3±0.5 7.3±0.5 ab e e d 5.2±0.2 7.2±0.4 8.0±0.4 7.1±0.3 a d e d 6.5±0.4 6.3±0.4 6.9±0.4 6.3±0.4 cd bc cd bc 5.9±0.3 5.9±0.2 6.7±0.2 7.0±0.2 b b cd d sour 1 6 9 12 3.3±0.2 2.2±0.2 0.0±0.0 0.0±0.0 de b a a 3.0±0.3 0.0±0.0 3.2±0.4 3.9±0.4 cd a de e 2.8±0.2 2.3±0.2 3.4±0.2 2.8±0.1 c b d c 4.2±0.3 5.8±0.2 5.9±0.2 5.3±0.2 e g g f green 1 6 9 12 2.2±0.2 0.0±0.0 0.0±0.0 0.0±0.0 b a a a 2.4±0.3 0.0±0.0 0.0±0.0 0.0±0.0 c a a a 2.5±0.3 0.0±0.0 0.0±0.0 0.0±0.0 c a a a 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 a a a a earth 1 6 9 12 0.0±0.0 0.0±0.0 0.0±0.0 1.3±0.1 a a a c 0.0±0.0 0.0±0.0 0.0±0.0 1.0±0.1 a a a b 0.0±0.0 0.0±0.0 0.0±0.0 1.1±0.2 a a a c 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 a a a a terpene 1 6 9 12 0.0±0.0 1.3±0.2 1.3±0.2 0.9±0.2 a c c b 0.0±0.0 1.0±0.1 1.0±0.2 1.3±0.3 a bc bc c 0.0±0.0 1.1±0.3 1.1±0.3 1.8±0.3 a bc bc d 0.0±0.0 1.2±0.2 2.7±0.1 4.2±0.1 a bc d d off-odour 1 6 9 12 0.0±0.0 0.0±0.0 0.0±0.0 2.2±0.2 a a a c 0.0±0.0 0.0±0.0 0.0±0.0 1.2±0.2 a a a b 0.0±0.0 0.0±0.0 1.2±0.2 1.0±0.2 a a b b 0.0±0.0 4.3±0.2 4.2±0.3 4.3±0.3 a d d d cabbage 1 6 9 12 6.2±0.4 5.3±0.3 2.8±0.3 3.2±0.2 e c b b 6.3±0.5 4.9±0.4 2.9±0.2 3.0±0.2 e c b b 5.9±0.3 5.4±0.3 2.8±0.3 3.1±0.3 de cd b b 8.1±0.5 2.9±0.3 3.2±0.3 1.8±0.3 f b b a mean ± standard deviation (n=3) for parameter with different letters are significantly different at p = 0.05. in the tested samples, a strong correlation at p≤0.05 was observed between the following aroma attributes and volatile compounds: sharp allyl isothiocyanate (r2=0.80), sharp dimethyl trisulfide (r2=0.83) and carrot – p-cymene (r2=0.71). applying the partial least squares techniques, we determined the contribution of volatile compounds to each attribute. in these models, the aroma attributes were y variables, while the concentration measured by gc–ms were x variables. for each aroma attribute, a proper pls regression model was established and interpreted using r2 coefficient. the importance of each volatile for the sensory attributes was determined by variable importance for the projection (vip) values (table 3). the r2 value were satisfactory for sharp, carror, terpene, off-odour and cabbage. allyl isothiocyanate and dimethyl trisulfide was the most important contributor to sharp and p-cymene for carrot. this confirms dependencies ital. j. food sci., vol. 30, 2018 358 determined by pearson’s correlation coefficients. additionally, the dependency between terpene, off-odour, cabbage attributes and volatile compounds was determined (table 3). allyl isothiocyanate, dimethyl trisulfide, sabinene and α-phellandrene were the most important contributor to terpene and ocimene for off-odour. sabinene and α-phellandrene, 3-hexen-1-ol, α-terpinene, allyl isothiocyanate and γ-terpinen were the most important contributor to cabbage. allyl isothiocyanate is also determined as pungent in flavor net. kjeldsen et al. (2003) divided terpene compounds present in carrot into carrot (α-pinene, β-pinene, sabinene, α-phellandrene, myrcene, p-cymene), sweet, citrus, fruity (limonene, γterpinene, terpinolene) and terpene-like, spicy, woody aroma compounds (βcaryophyllene). in the tested product only a dependence between p-cymene and carrot aroma was observed. such a correlation may also be indicated by the very low levels of the odor threshold (13 ppb) for p-cymene in comparison with α-pinene (1000 ppb), β-pinene (140 ppb) and sabinene (75 ppb) (kjeldsen et al., 2003). moreover, the concentration of pcymene in the tested samples was significantly (p=0.05) greater than in the case of other above-mentioned monoterpenes, thus indicating their greater odor activity value. in the case of terpene aroma, sabinene, α-phellandrene are determined as turpentine in flavor net. sabinene and γ-terpinene were also detected as a result of break in continuity of the cabbage tissue as reported by vuorinen et al. (2004) table 3. r2 for pls regression model and vips affected the aroma attributes. attributes r2 volatiles vip sharp 0.91 allyl isothiocyanate 2.457 dimethyl trisulfide 2.240 carrot 0.81 p-cymene 2.133 sour 0.54 green 0.47 earth 0.45 terpene 0.84 allyl isothiocyanate 1.870 dimethyl trisulfide 1.643 sabinene 1.352 α-phellandrene 1.237 off-odour 0.77 allyl isothiocyanate 2.207 dimethyl trisulfide 1.396 sabinene 1.356 ocimene 1.289 α-phellandrene 1.093 cabbage 0.77 sabinene 1.480 α-phellandrene 1.462 3-hexen-1-ol 1.316 α-terpinene 1.190 allyl isothiocyanate 1.166 γ-terpinene 1.038 ital. j. food sci., vol. 30, 2018 359 3.4. pca based on the correlation matrix, 2 principal components were identified, explaining 63% total variability for which correlations with input variables are presented in fig. 5. the pc1 was positively correlated with contents of α-thujene, α-pinene, camphene, sabinene, myrcene, 3-hexen-1-ol, α-phellandrene, α-terpinene, limonene, β-phellandrene, γ-terpinene, terpinolene as well as aroma attributes, that is green and cabbage, while it was negatively correlated with β-caryophyllene, p-cymene, carrot and earth aroma. the pc2 was negatively correlated with contents of oxygen and carbon dioxide, allyl isothiocyanate, dimethyl trisulfide, β-pinene, ocimene and aroma attributes, that is sharp, sour, terpene and off-odour (fig. 5). figure 5. pca of volatile compounds, aroma attributes and content o2 and co2. figure 6 presents values of pc1 and pc2 for sample maps. among the tested samples, the lowest pc2 values were found for salads packaged in film with no microperforation after 6, 9 and 12 days of storage. this was connected with the greatest contents of oxygen, carbon dioxide, allyl isothiocyanate, dimethyl trisulfide, ocimene and β-pinene inside the packaging and aroma defined as sharp, sour, terpene and off-odor. after 12-days of storage the other samples had negative pc1 values, which indicates high contents of βcaryophyllene and p-cymene and aroma defined as carrot and to a limited extent earth aroma. ital. j. food sci., vol. 30, 2018 360 figure 6. sample map. 4. conclusions the application of microperforated film in packaging of coleslaw mix makes it possible to maintain the desirable product aroma for 12 days of storage at 4 °c. it also guarantees maintenance of aerobic conditions and does not contribute to the accumulation of co2 inside the packaging. although it may be assumed that in those samples we observed the degradation of chlorophyll found in comminuted cabbage leaves, it may indicate a significantly (p=0.05) greater concentration of limonene than in samples packaged in film with no microperforation. the broadest profile of aroma compounds during storage was found in samples sealed in the atmosphere of 70/30/0% o2/co2/n2. in the case of packaging the coleslaw mix in the superatmospheric oxygen atmosphere using film with no microperforation, the significant (p=0.05) deterioration of aroma in the sensory analysis was the effect of allyl isothiocyanate and dimethyl trisulfide accumulation inside the packaging. partial least squares regression analysis determined the quantitative contributions of volatile compounds to sharp, carrot, terpene, off-odour and cabbage in coleslaw mix aroma. acknowledgements this work was supported by the ministry of science and higher education of poland (grant no. n n312 151134). references akpolat h. and barringer s.a. 2015. the effect of ph and temperature on cabbage volatiles during storage. j. food sci. 80:s1878-s1884. bouvier f., rahier a. and camara b. 2005. biogenesis, molecular regulation and function of plant isoprenoids. prog. lipid res. 44:357-429. ital. j. food sci., vol. 30, 2018 361 cliffe byrnes v., brennan l. and o’beirne d. 2007. the effects of preparatory procedures and storage temperature on the quality of carrot discs packaged in modified atmospheres. int. j. food sci. tech. 42:482-494. cliffe byrnes v., mc laughlin c. p. and o'beirne, d. 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2003. changes in volatile compounds of carrots (daucus carota l.) during refrigerated and frozen storage. j. agr. food chem. 51:5400-5407. lonchamp j., barry-ryan c. and devereux m. 2009. identification of volatile quality markers of ready-to-use lettuce and cabbage. food res. int. 42:1077-1086. radziejewska-kubzdela e. and biegańska-marecik r. 2009. the effect of composition of modified atmosphere and type of packaging film on sensory quality and physico-chemical properties of coleslaw mix. ejpau, 12. radziejewska-kubzdela e. and czaczyk k. 2015. effect of pretreatment and modified atmosphere packaging on quality of dry coleslaw mix. packag. technol. sci. 28:1011-1026. radziejewska-kubzdela e. and czaczyk k. 2017. the effect of organic acid pretreatment and modified atmosphere on shelf life of dry coleslaw mix. j. food process. pres. 41:1-11. vuorinen t., nerg a.m. ibrahim m.a., reddy g.v.p. and holopainen j.k. 2004. emission of plutella xylostella-induced compounds from cabbages grown at elevated co2 and orientation behavior of the natural enemies. plant physiol. 135:1984-1992. yahyaa m., tholl d., cormier g., jensen r., simon p.w. and ibdah m. 2015. identification and characterization of terpene synthases potentially involved in the formation of volatile terpenes in carrot (daucus carota l.) roots. j. agr. food chem. 63:4870-4878. paper received may 5, 2017 accepted december 20, 2017 p u b l i c a t i o n s codon italian journal of food science, 2021; 33 (3): 47–69 issn 1120-1770 online, doi 10.15586/ijfs.v33i3.2123 47 circular economy in the brewing chain alessio cimini, mauro moresi* department for innovation in the biological, agrofood and forestry systems, university of tuscia, via s.c. de lellis, viterbo, italy *corresponding author: mauro moresi, department for innovation in the biological, agrofood and forestry systems, university of tuscia, via s.c. de lellis, 01100 viterbo, italy. email: mmoresi@unitus.it received: 21 september 2021; accepted: 3 december 2021; published: 14 december 2021 © 2021 codon publications open access review abstract the main aim of this review was to check for the applicability of the concept of circular economy to brewing chain. by analyzing the beer brewing process, it was possible to identify the main brewery wastes formed and packaging materials used as well as their range of composition and yields. in order to reduce the contribution of packaging material to the carbon footprint of beer, it would be necessary to replace one-way containers used nowadays with lighter, reusable, or recycled ones. even if the contribution of beer consumption phase was taken into account, there was no definitive solution about the less environmentally impacting beer packaging format. the direct management of polyethylene terephthalate (pet) packaging for liquid foodstuffs could make available 100% recycled pet flakes to be reconverted into food-grade bottles with minimum downcycling to other non-food usage. the countless potential uses of brewery wastes in nutritional and biotechnological fields were tested in laboratory by disregarding any cost–benefit or market analysis. this was mainly because the estimated market price of dried brewer’s spent grain (bsg) resulted to be about 450% higher than that of conventional lignocellulose residues. all the alternative uses hailed in the literature appeared to be more useful for publishing articles than for defining any economically feasible reusing procedure for all brewery wastes. owing to their high moisture content, such wastes are so perishable as to prevent their safe usage in the human food chain. currently, their use as-is in animal feeding is the disposal method not only economically feasible but also able to reduce the greenhouse gas load of beer packed in glass bottles (gb) by about one-third of that associated with packaging materials. not by chance, it is practiced by most industrial and craft breweries. keywords: beer chain; beer packaging formats; brewer’s spent grain; brewer’s spent yeast; carbon footprint; environmental impact; hot trub; post-consumer packaging waste; disposal methods; spent hops introduction beer is a globally consumed alcoholic beverage (about 1.91 billion hectoliter (hl) in 2019; statista, 2021), with its overall market in 2020 amounting to us$623.2 billion (imarc group, 2021). in italy, the overall production of beer in 2020 was about 15.8 million hl, about 71% of which being produced by five major players, such as heineken italia with a share of 33.3%, birra peroni with 18.3%, anheuser-busch inbev with 8.6%, birra castello with 5.8%, and carlsberg italia with 5.3% share (associazione dei birrai e dei maltatori [assobirra], 2020). in 2020, 756 craft breweries (i.e., 624 micro breweries and 132 brewpubs) produced 361,000 hl of beer with an average specific gravity of 14° plato, this being equal to ∼3.1% of the italian beer production (assobirra, 2020). in italy, the per capita consumption of beer in 2019 was about 35.2 l. standard lager is the most popular beer type, representing 84.2% of the overall beer consumption, followed by specialty beers (14.5%) 48 italian journal of food science, 2021; 33 (3) cimini a and moresi m and distribution (e.g., ghg emissions, and disposal of wastewaters and post-consumer packaging wastes) using circular thinking approaches. the author cited either quite exiguous initiatives (e.g., 100% biodegradable, edible six-pack ring pulling on cans prepared from barley and wheat ribbons, adopted by saltwater brewery to replace the conventional plastic ones) or more significant ones (e.g., use of waste bread instead of malted barley by toast ale in belgium, wind turbines and solar panels as sources of nonrenewable energy by heineken in holland and italy). the main aim of this review was to further verify the applicability of circular economy concepts to the brewing chain. to this end, the main steps of the beer brewing process were outlined to point out main brewery wastes in terms of composition and yield factors. then the real and effective reuses of packaging and biotic wastes were critically reviewed based on their techno-economic feasibility. inventory analysis of the brewing process figure 1 shows block diagram of the beer production process from its basic raw material, that is, barley. the average chemical composition of barley is provided in table 1. it differs with barley variety and environmental conditions. the starch, β-glucan, protein, fat, and ash contents vary in the ranges of 65–68%, 4–9%, 10–17%, 2–3%, and 1.5–2.5% (dry basis), respectively (alijošius et al., 2016; gupta et al., 2010). an ideal protein content of barley destined to brewing ranges from 9.5% to 12.8% (dry basis), while higher protein contents are suitable for producing malt for distilling (grains research & development corporation (grdc), 2018; paynter, 1996). malting is the first step of the brewing process. it consists of three different unit operations: steeping, germination, and drying. during steeping, barley seeds are soaked in water until imbibed with sufficient water to start their sprouting process. the germination phase allows a series of amylases, proteases, and other endogenous hydrolytic enzymes to be produced and/or activated. final drying stops further growth of germs, reduces water activity, and thus yields a shelf-stable product with active enzymes (i.e., barley malt). the average malt-to-barley ratio ranges from 0.75 (climate conservancy, 2008) to 0.79 (food and agriculture organization [fao], 2009). the main byproduct of malting (i.e., barley rootlets, also known as malt culms, coombes, or sprouts) represents 3–5% (w/w) of the malt produced. it may contain other wastes, such as malt dust, small and broken barley grains, barley dust, acrospires, and husk fractions. as depicted by neylon et  al. (2020), its range of composition is provided in table 1. and low or nonalcoholic ones (1.3%). owing to decrease in beer consumption  outside  the  home  from 45.5% in 1999 to 36.1% in 2019, and conversely the increase in off-sales, the prevailing beer packaging format is dominated by glass bottles (gb; 80.8%), followed by stainless steel kegs (ssk; 11.7%), and finally aluminum cans (ac; 7.5%) (assobirra, 2020).  most consumers purchase beer in glass bottles (73.0% and 7.8% of which being produced from nonreturnable and returnable gbs, respectively) or aluminum cans, while beer packaged in stainless steel kegs is chiefly for commercial use. owing to predictable increase in the global demand of food, this currently representing from 22% to 37% of the world anthropogenic greenhouse gas (ghg) emissions (rogissart et al., 2019), the economic growth of the food and beverage industry is expected to be greatly hampered by climate-related risks to food security, and water and energy supply (intergovernmental panel on climate change [ipcc], 2014). as reported by the beverage industry environmental roundtable (bier, 2012), the beverage sector has not only started to reduce its impact on the global climate but also to rethink its business models, products, and processes according to the principles  of circular economy (bocconi university et al., 2021). over the last 20 years, several business-tobusiness or business-to-consumer studies (amienyo and azapagic, 2016; bier, 2012; cimini and moresi, 2016; environmental product declaration® [epd], 2011a, 2011b, 2014a, 2014b; hospido et al., 2005; koroneos et al., 2005; muñoz et al., 2012; narayanaswamy et al., 2005; shin and searcy, 2018; talve, 2001; williams and mekonen, 2014) have been conducted to evaluate the environmental impact of beer as packed in different formats, as summarized by cimini and moresi (2018c). glass bottle or aluminum-can manufacturing and barley cultivation represented the main hot spots of beer life cycle (amienyo and azapagic, 2016; cimini and moresi, 2016). only when using reusable steel kegs, barley production was the most impacting step, followed by brewing and distribution (cimini and moresi, 2016). as estimated by mata and costa (2001) and confirmed by amienyo and azapagic (2016), the contribution of returnable glass bottles to the carbon footprint (cf), acidification, photochemical ozone creation, human toxicity, and energy and raw material consumption was smaller than that of nonreturnable bottles after the second reuse, while that to eutrophication, ozone depletion, solid waste, water and auxiliary material consumption was larger even after several reuses. holland (2021) described a few means to address the most environmentally altering effects of beer production italian journal of food science, 2021; 33 (3) 49 circular economy in the brewing chain mashing is the third step. the grit is suspended into hot water to allow starch sugars, proteins, and tannins to be dissolved in the so-called malt extract. in european breweries, malted barley consumption ranges from 15 kg/hl to 18 kg/hl of beer produced (united nations environment program [unep], 1996), while in italian industrial breweries, 1 hl of beer at a specific gravity of 12°plato (equivalent to an ethanol content of about 5% v/v) needs approximately 12-kg malt and 4-kg unmalted cereals, such as corn grits (assobirra, 2020). the specific consumption of malted barley appears to be inversely proportional to the brewery size. it is as high as 28–32 kg hl-1 in the case of craft breweries with an annual capacity of about 1,000 hl of beer (beloborodko et al., 2014; sturm et al., 2012) and as low as 18–20 kg hl-1 in the case of industrial breweries with an overall capacity of more than 1 million hl/yr (cimini and moresi, 2018c). referring to the overall volume of abstracted water during malting, brewing, and clearing steps, the specific water consumption was about 4.2 l per liter of beer produced in italian breweries (assobirra, 2020), and it can range from 3.5 to 10 l per liter of beer (olajire, 2020) or to as high as 19 l (sturm et al., 2013) or 34 l of specific water per liter of beer produced (pauli, 1997) in old micro-breweries. lautering is the fourth step of the brewing process. it is carried out in the lauter tun to fractionate wort from the so-called wet brewer’s spent grains (bsgs), these being the chief byproduct of this process with an average amount of 16 kg hl-1 of beer produced (assobirra, 2020). table 1 provides their range of composition and amount, as described by several authors along with the international finance corporation (ifc, 2007) and unep (1996). wort boiling or hopping is the fifth step, which comprises the addition of different types and amounts of hops to boiling wort according to the style  of  beer to be produced. as boiling proceeds, the malt enzymatic pool is deactivated, and as water is evaporated, malt proteins and hop tannins tend to precipitate at the bottom of the concentrated hopped wort. the average amount of hop pellets used in the italian breweries is around 260 g hl-1 (assobirra, 2020), even if it is quite lower (∼92 g hl-1) in the case of lager (cimini and moresi, 2016). wort clarification is the sixth step. by feeding tangentially the hopped wort in a whirlpool separator, the resulting moderate centrifugal action allows the hot trub and spent hops to be separated from clear wort. table 1 provides the range of composition of such a proteinaceous residue, as described by rachwał et al. (2020). moreover, its overall quantity varies from 0.2 to 0.4 kg hl-1 depending on the amount and type of protein present in the used barley, which, in turn, is dependent on crop location, seasonal factors, and genetics (barchet, 2019). malt milling is the second step. it allows the malt to be separated from its  chaff, coarsely ground, and sifted into  three fractions, namely, the husk, grits, and flour. the smaller the particle size, the greater the extract (and the smaller the filtration rate). husk is the outer layer of barley kernel undergoing grinding and must be kept intact as possible to allow the formation of porous filter beds and thus minimize filtration time. larger particles can be reground, thus yielding other byproducts, such as hull fractions and fine malt powders (crescenzi, 1987; stubits et al., 1986). figure 1. schematic of the beer production process and its main brewery byproducts. 50 italian journal of food science, 2021; 33 (3) cimini a and moresi m maturation is the ninth step of the process. the freshly brewed liquid undergoes the second fermentation, providing beer its characteristic color in as long as 3 weeks to 3 months depending on the type of beer being produced. beer clarification and stabilization is the tenth step. proteins, yeast particles, and resins from the hop left in the beer after the first and second fermentation phases are generally removed by filtration in the presence of filter aids (i.e., kieselguhr slurry or diatomaceous earth [de]). then, to avoid permanent or chill haze (siebert et  al., 1996), haze active polyphenols are removed by using polyvinylpolypyrrolidone (pvpp), while haze active proteins are removed by means of silica hydrogel or tannic acid. in european breweries, the specific consumption of de ranges from 80 to 570 g/hl of beer (ifc, 2007; unep, 1996), while that of non-regenerable pvpp ranges from 20 to 40 g (gopal and rehmanji, 2000) and that of regenerable pvpp by around 0.1 g hl-1 (cimini and moresi, 2015). filling is the final step of the beer brewing process. if the finished beer is to be kept in glass bottles, it is first bottled and then batch-pasteurized to prolong its shelf life. when using cans or kegs, clear beer undergoes flashpasteurization and is packed aseptically. by referring to main packaging formats in use (cimini and moresi, 2016, 2018c), 1 hl of lager (weighing ∼100.5 kg) would require as much as 43.9 or 56.1 kg of 66-cl or 33-cl amber glass bottles, just 4.4 kg of 66-cl polyethylene terephthalate (pet) bottles, 4.9 kg of 33-cl aluminum cans, and as much as 32.0 kg of 30-l stainless steel kegs (table 2). this clearly the great contribution of packaging materials per unit volume of beer delivered, especially in the case of glass bottles and stainless steel kegs. for further details of the brewing process, refer to eßlinger (2009). wort cooling is the seventh step. it allows the clear wort to be cooled to a temperature depending on the yeast used and the style of beer being produced. it usually ranges from 16 to 20°c and from 10 to 13°c in the case of ale and lager production, respectively. if the wort coming out of the whirlpool separator has a higher strength than that required for fermentation, it is diluted with water to obtain its correct specific gravity. moreover, to allow yeast replication in the early stages of fermentation and guarantee adequate fermentation, an appropriate level (7–18 mg/l) of dissolved oxygen in the wort is provided by aerating the wort on hot or cold side of wort heat exchanger. the oxygen consumption was 1.43 g/hl of lager produced (cimini and moresi, 2016). wort fermentation is the eighth step of the brewing process by which fermentable sugars are converted into ethanol, carbon dioxide, and several other metabolic byproducts by brewer’s yeast. these have a significant effect on the taste, aroma, and other characteristic properties of the style of beer under production. on stoichiometric basis, 1-g maltose must be theoretically  converted  into  0.538-g ethanol. the average inoculation rate in italian breweries is 0.8 kg hl-1 of beer (assobirra, 2020). such a phase is usually accomplished in cylindroconical fermenters. their angle at the bottom of tanks allows the yeast to settle in the bottom of conic vessel at the end of primary fermentation before being collected without exposure to air. thus, a rough beer relatively free of yeast could be discharged. after harvesting, the yeast is stored with its own liquid under gentle agitation at 0°c for next fermentation. in this manner, brewer’s yeast could be used sequentially for four to six times (karlović et al., 2020). the range of composition of brewer’s surplus yeast (bsy) is provided in table 1, as described by different studies. its amount ranges from 2 to 4 kg hl-1 of the beer produced (ifc, 2007; unep, 1996); its mean value in italy was 1.6 kg hl-1 by assuming a 10% dry matter (dm) content (assobirra, 2020). table 1. range of chemical composition of barley and its main brewery byproducts, namely, malt barley rootlets (mbr), brewer’s spent grain (bsg), spent hops/hot trub (ht), and brewer’s spent yeast (bsy). component barley mbr bsg ht bsy unit moisture 12.8 8.2–12.9 75–90 80–90 74–86 g/100 g carbohydrates 0.624 0.51–0.60 0.45–0.61 0.20 0.4 g/g dm protein 0.113 0.203–0.387 0.142–0.300 0.40–0.70 0.15–0.49 g/g dm fat 0.019 0.017–0.044 0.06–0.13 0.045 0.04–0.10 g/g dm ash 0.03 0.028–0.087 0.011–0.050 0.06–0.25 0.02–0.085 g/g dm total fiber 0.215 0.43 0.44–0.84 0.23–0.26 0.25–0.53 g/g dm specific amount 0.03–0.05 kg kg–1 malt 14–19 0.2–0.4 2–4 kg/hl beer references alijošius et al., 2016 neylon et al., 2020 jackowski et al., 2020; karlović et al., 2020; rachwał et al., 2020 dm: dry matter; hl: hectoliter. italian journal of food science, 2021; 33 (3) 51 circular economy in the brewing chain must be refurnished to be reused, recycled, submitted to other recovery options, and, as the least preferred option, disposed of via landfilling or incineration with no recovery of energy. such a waste hierarchy was further detailed by garcia-garcia et al. (2015), as shown in figure 3. prevention of wastage of food is the alternative preferred mostly, followed by food redistribution to people in need, and then to animals, unless it is composed of products of animal origin or is a catering waste. if such options are not applied, then food waste can be regarded as a source from which several valuable products (e.g., fats, proteins, polysaccharides, polyphenols, etc.) could be extracted selectively. then it may be submitted for anaerobic digestion, composting, thermal valorization, or spread for  land fertilization or improvement. waste burning with no energy recovery and landfilling are regarded as the least preferable management options to use (garcia-garcia et al., 2015). by referring to the brewing chain, application of the concept of circular economy essentially refers to the following two aspects: 1. the reuse of abiotic materials, such as packaging materials, and spent kieselguhr, their overall weight being mainly made of glass bottles, which are, for instance, used to pack about 81% of the entire beer produced in italy (assobirra, 2020). 2. the reuse of biotic materials, namely, the main byproducts of the beer brewing process. end of life of abiotic materials packaging materials all abiotic materials arising from beer packaging and pallet management at distribution centers (dc) generally undergo separate waste collection to allow recycling of application of the concept of circular economy to the beer brewing process the linear economy model reflects man-made ecosystems for food production, which requires not only a continuous supply of energy and mass from outside, as nutrients are not recycled at the crop cultivation site, but also the treatment of wastes. on the contrary, the so-called circular economy model refers to natural ecosystems, which are capable of self-regeneration. as sketched in figure 2, its priority area is aimed at eliminating waste and pollution, keeping products and materials in use, and regenerating natural systems (bocconi university et al., 2021). according to the waste hierarchy set out in article 4 of the revised waste framework (directive 2008/98/ec; european union [eu], 2008), any waste must be handled in a manner that does not have a negative impact on the environment or human health. first, its formation must be prevented using, for instance, less material in design and manufacture. when the waste has been formed, its entire apparatus or replacement parts figure 2. schematic of the circular economy concept. table 2. mass of the packaging materials used to pack 1 hl of beer in different formats (66-cl and 33-cl amber glass bottles [gb]; 66-cl pet bottles [pb]; 33-cl aluminum cans [ac]; 30-l stainless steel kegs [ssk]), as described by cimini and moresi (2016, 2018c). packaging format packaging materials 66-cl gb 33-cl gb 66-cl pb 33-cl ac 30-l ssk unit glass 43.9 56.1 0.0 0.0 0.0 kg/hl paper & cardboard 3.0 3.4 3.0 1.2 0.0 kg/hl plastic 0.1 0.1 4.4 0.3 0.0 kg/hl steel 0.3 0.6 0.0 0.0 32.0 kg/hl aluminum 0.0 0.0 0.0 4.9 0.0 kg/hl wood 2.8 3.2 3.0 1.9 2.5 kg/hl adhesive materials 0.2 0.2 0.2 0.2 0.0 kg/hl overall 50.3 63.6 10.6 8.5 34.5 kg/hl 52 italian journal of food science, 2021; 33 (3) cimini a and moresi m the contribution of climate change and its related impact categories would represent 50% or 53% of the overall weighted endpoint score when including or excluding the toxicity-related impact categories (sala et  al., 2018). owing to the diverse contribution of packaging materials and transportation, the beer packaging format exerted a diverse influence on the ghgs emitted to produce and distribute industrial beer from the factory gate to the distribution centers, as assessed previously (cimini and moresi, 2016). by disregarding the ghg credits derived from the use of brewery wastes as cattle feed, these being about 12 kg of carbon dioxide equivalent (co2e) per hl of beer (cimini and moresi, 2016), the business-to-business product carbon footprint (cf) of beer was found to be of the order of 69 or 78 kg co2e hl -1, or 81 or 37 kg co2e hl -1 if the beer was packed in 66or 33-cl glass bottles, or 33-cl aluminum cans or 30-l stainless steel kegs, respectively (table 4). since kegs, on average, are glass, plastic, aluminum, steel, paper, or wood for their recycling. the discarded fraction of packaging components varies from 0.4% in the case of glass bottles to 3.5% in the case of the stretch wrap film, as detected in an industrial brewery (cimini and moresi, 2016). the disposal scenarios of all packaging wastes, resulting from beer processing and post-consumption, generally coincide with those of municipal solid waste. for instance, table 3 refers to the overall italian management scenarios in 2019 (mariotta and tuscano, 2020). it is noted that the minimum objective (65%) of recycling, in terms of weight, for all packaging wastes, to be met by 31 december 2025 according to directive 2018/852/eu (eu, 2018), has been achieved nationwide since 2019, although there are differences in some districts of south italy and plastic recycling rate is still lower than the target value of 50% (mariotta and tuscano, 2020). in order to measure the environmental impact of main packaging materials used to pack beer, we referred to the only effect of beer on climate change, since this impact category was found to be highly correlated to the fossil cumulative energy demand, which, in turn, affected several other categories, such as acidification, eutrophication, and photochemical ozone formation (huijbregts et  al., 2006) as well as the nitrogen and phosphorous emissions resulting from synthetically fertilized soils used for agri-food cultivation (huijbregts et al., 2010). under these circumstances, by accounting for the overall weight factors attributed to the effects of such impact categories in the product environmental footprint methodology, table 3. overall italian waste management scenarios for packaging wastes in 2019, as described by mariotta and tuscano (2020). waste management scenarios landfill recycling incineration waste (%) (%) (%) glass 22.7 77.3 0 paper and cardboard 11.6 80.8 7.6 iron 17.8 82.2 0 plastic 10.1 45.5 44.4 aluminum 23.9 70.0 6.1 wood 34.8 63.1 2.1 overall 19.2 70.0 10.8 figure 3. food waste hierarchy used to select different waste management alternatives according to their environmental preference, as reworked by garcia-garcia et al. (2015). italian journal of food science, 2021; 33 (3) 53 circular economy in the brewing chain nevertheless, as expected, the use of such a lighter packaging material was not so effective, since the cradle to-grave ghg burden of beer decreased by as low as ∼1.7–3.3%. this was a direct consequence of different average recycling rates of glass (70.3%) and plastic (37.9%) wastes registered in italy in 2014 (cimini and moresi, 2018c). in fact, the ghg load of brewery and post-consumer packaging waste disposal was negative in the case of glass bottles (–11 kg co2e hl -1) but positive in the case of pet bottles (+4.6 kg co2e hl -1). by referring to the current higher recycling rates of such packaging wastes (table 3), the glass and plastic recycling rates, respectively, increased by circa +10% and +20% with respect to the aforementioned basic values. this involved a reduction in the cradle-to-grave carbon footprint of 0.9–1.3% and 0.6–0.8% with respect to the basic cases, as the brewery capacity increased from 500 to 2 million hl per year, as estimated by using the same life-cycle assessment (lca) model of beer production developed previously (cimini and moresi, 2018c). moreover, the use of steel cans would give rise to lower effect than usage of glass bottles and aluminum cans not only on ghg emissions (bier, 2012) but also on other environmental impact categories, such as eutrophication, creation of photochemical oxidants, and freshwater aquatic ecotoxicity potentials (amienyo and azapagic, 2016). any increase in glass or pet recycled content would improve the environmental sustainability of resulting bottles either for the lower energy needed for their manufacture or the minor quantity of post-consumption packaging waste to be landfilled. in fact, since the bottle emission factor linearly decreases as the recycled material content increases, 10% increase in the recycled glass or pet content reduced the carbon footprint of beer by 2.2–2.5% depending on the size of brewery (cimini used for 72 times, the contribution of packaging materials was just 5% of the overall ghg burden, while that of glass bottles and aluminum cans was from 5 to 6 times higher, respectively. on the contrary, the contribution of transportation increased to 25% in the case of kegs in consequence of their tare (9.6 kg), while it was, respectively, 14% or 10% if glass bottles or aluminum cans are used (table 4). thus, to reduce the contribution of packaging materials to the carbon footprint of beer, it would be necessary to resort to: 1. lighter bottles or kegs, 2. bottles, including greater percentage of recycled materials, 3. containers reusable as many times as possible. in the case of beer packed in glass bottles, 10% decrease in the mass of glass bottles would reduce the carbon footprint of beer by 2.4–2.6% (cimini and moresi, 2016) to a maximum of 5% (amienyo and azapagic, 2016) due to lower impact for their manufacture and transportation. approximately 70% less ghg emissions were estimated when the tuborg® beer was packed in 20-l plastic drums weighing 290 g each (epd, 2011a). further savings are expected by the replacement of glass bottles or aluminum cans with nanoclay-enriched polyethylene terephthalate (pet) bottles, their empty bottle weight being ∼26 g, and their carbon footprint near to one-third of that (∼9 kg co2e kg -1) of 50% recycled aluminum cans (cimini and moresi, 2016, 2018c). as provided in table 5, when beer was packed in pet bottles instead of glass bottles, the ghg contribution of packaging materials reduced by about 29%, while that of transportation by 25–57%, as the brewery capacity was reduced from 2 million to 500 hl per year. table 4. contribution of different life cycle phases to the ghg emitted to produce and distribute 1 hl of lager as packed in containers of different volumes and masses, as described by cimini and moresi (2016). beer primary packaging type gb ac ssk volume (l)/mass (kg) 0.66/0.290 0.33/0.185 0.33/00123 30.00/9.6 life cycle phases ghg emissions (kg co 2e hl-1) raw materials & processing aids 16.88 16.88 16.88 16.88 brewing processing & packaging 8.41 8.4 8.33 8.41 packaging materials 33.33 42.19 47.55 1.86 transportation 9.71 10.67 8.09 9.26 waste disposal 0.58 0.58 0.57 0.61 beer production and distribution 68.91 78.71 81.42 37.02 ghg: greenhouse gas; gb: glass bottles; ac: aluminum cans; ssk: stainless steel kegs. 54 italian journal of food science, 2021; 33 (3) cimini a and moresi m direct management of pet packaging of liquid foodstuffs by the italian ministry of environment (see decree no. 44 of 28 july 2021). thanks to the deposit system adopted in germany, 99% of refillable bottles and 97% of one-way bottles are returned to supermarkets and grocery stores (anon, 2017). thus, such a packaging waste recycling system must theoretically give rise to pet recycling rates near to 100%. to this end, coripet is intended to reach 25% pet recycling by 2025. in germany, 96–98% r-pet recovery in 2015 supported the formation of new pet bottles from about 34% of total recovery, the remainder being directed to non-food uses, such as plastic sheets and films (27%), textile fibers (22.6), and so forth (deutsche welle [dw], n.d.). the german container deposit legislation appears to be an appropriate incentive for transiting toward a circular economy, even if it has so far given rise to a greater aliquot for downcycling (66%) than for effective recycling and reuse, which indeed would strictly require the conversion of empty bottles into new useable bottles for food purposes. strictly speaking, in the case of beverage packaging, a sustainable waste management must make use of refillable bottles only (anon, 2017). since it is useless to reinvent the wheel, it is worth remembering that up to the early 1960s, the italian customers used to pay a deposit on each glass bottle bought, which they reclaimed by returning the empty bottle. obviously, such a system could become valid not only for glass bottles but also for plastic ones, even if the latter are refilled for 20–25 times and the former for up to 50 times (dw, n.d.). of course, the reintroduction of reusable bottles demands not only for new infrastructure, and moresi, 2018c) or by approximately 3% in the case of glass bottles as estimated by amienyo and azapagic (2016). except for water demand, all other environmental impact categories reduced by 0.5% in the case of eutrophication potential, and 2% in the case of abiotic depletion potential (amienyo and azapagic, 2016). thus, the idea of increasing recycling rate has proliferated in several countries. for instance, in france, mineral water in bottles made of 100% recycled pet (r-pet) has been commercialized since 2019. in italy, usage of r-pet for producing bottles and trays for food was approved by the 2021 budget law on 31 december 2020 with the condition that the material derived from other bottles would be used for food purposes only. in germany, thanks to the container deposit legislation operating since 1 january 2003 and despite the opposition of the german bottling industry and retailers, any empty plastic or glass bottle returned to a grocery store receives a credit ranging from €8 to 25 cents to be discounted at the cash desk. to avoid any contamination problem, recycling companies currently submit plastic bottles to the following procedure. first, such items are automatically collected using the barcode system, sorted by type and color, and aggregated in bales. once foreign materials, labels and caps are removed by infrared-ray sorting and pre-washing, the resulting material is shredded into flakes, which are then dried at 150–180°c. the food reuse of such r-pet flakes involves a decontamination process of thermal or chemical type at 280°c or with a caustic detergent. this procedure has been also adopted by coripet (https:// coripet.it/), a voluntary nonprofit consortium of producers, converters, and recyclers of pet bottles. the procedure was recognized as an autonomous system for the table 5. contribution of main beer life cycle phases to cradle-to-grave (c2g) carbon footprint (cf c2g ) of 1-hl beer packed in 66-cl glass or pet bottles by breweries of different annual capacity, as described by cimini and moresi (2018c). brewery capacity (hl/year) 2 × 106 5 × 105 5 × 104 5 × 102 beer primary packaging type gb pb gb pb gb pb gb pb life cycle phases ghg emissions (kg co 2e hl-1) raw materials and processing aids 23.9 27.0 30.6 41.7 brewing and packaging processing 12.1 13.7 16.8 49.5 packaging materials 34.0 24.0 34.3 24.3 33.6 23.7 33.6 23.7 waste and effluent disposal 1.9 2.0 3.1 3.1 0.6 0.6 0.6 0.6 co 2e credits from byproduct use as feed –2.2 –2.2 –2.5 –2.5 –2.9 –2.9 –3.8 –3.8 transportation to dcs 19.8 15.1 20.5 13.5 22.5 12.6 15.6 5.9 transportation from dcs to retailers 9.7 7.1 7.3 5.3 2.4 1.8 2.4 1.8 retailer refrigeration 0.3 0.3 0.3 0.3 consumer phase 18.1 18.1 18.1 18.1 post-consumer waste disposal –11.0 4.6 –11.0 4.6 –11.0 4.6 –11.0 4.6 cf c2g 106.7 104.9 110.8 107.5 111.0 106.1 147.1 142.3 ghg: greenhouse gases; dcs: distribution center; gb: glass bottles; pb: pet bottles. italian journal of food science, 2021; 33 (3) 55 circular economy in the brewing chain spent filtration aids generally, spent filtration aids resulting from rough beer filtration are in the form of de or kieselguhr slurry, which is rich in suspended solids (e.g., diatom frustules, yeast, and hop and malt residues) and thus highly pollutant (olajire, 2020). world health organization has classified such slurry as hazardous waste; moreover, its disposal costs in agriculture are as high as €170 per metric tons (mg) (fillaudeau et al., 2006). nevertheless, owing to their high filtration rate and efficiency, de dead-end filters are still largely used by the majority of breweries. in a large-size brewery, the specific consumption of de is around 112 g hl-1 of beer, giving rise to ∼336 kg hl-1 of spent de sludge (cimini and moresi, 2016). after beer filtration, sludge is not recycled and generally landfilled. however, thanks to its high contents of available phosphorus (0.37–0.42 g kg-1), potassium (0.9–3.3 g kg-1), organic carbon (0.3 kg kg-1), and total nitrogen (0.02 kg kg-1), it is used in agriculture to improve soil fertility with no significant risk to the environment (dessalew et al., 2017). the main potential reuse opportunities of this material include: (1) its recycling as additive to construction masonry materials, such as concrete, cement, and brick (ferraz et al., 2011); and (2) its regeneration via chemical, physical, or biological methods. the latter up to now is unable to replace totally virgin de (olajire, 2020). moreover, the thermal and acid or alkaline agent regeneration methods cannot be regarded as sustainable recycling methods because of their low efficiency and serious secondary pollution to the environment as well as high processing costs (li et al., 2015). on the contrary, the biological methods appeared to be not only almost zero cost-effective but also capable of improving the adsorption capability of brewery-spent diatomite toward dyes and heavy metals from polluted wastewaters (gong et al., 2019). in order to decrease de consumption and thus reduce de sludge formation, it would be possible to resort to a cleaner filtration technology, such as cross flow microfiltration, which has been applied successfully in other food sectors over a long period of time (cheryan, 1998). unfortunately, so far, its application to beer clarification has been penalized by average permeation fluxes (50–100 l m-2 h-1) of about one-fifth of that obtainable (250–500 l m-2 h-1) with conventional kieselguhr filters (buttrick, 2010), which are also dependent on the initial turbidity of rough beer (cimini and moresi, 2014). only by submitting pre-centrifuged rough beer to a specific enzymatic (i.e., brewers clarex®) treatment and then to clarification at 30°c using ceramic 1.4-μm hollow-fiber membrane modules, it was possible to limit the in-bottle chill-haze formation more effectively than with the pvpp treatment, and what is more to enhance the average permeation flux up to 2,000 l m-2 h-1 with no filtration residues financial incentives, and behavioral changes (amienyo and azapagic, 2016) but also for several other factors such as the distance empty bottles travel by road for cleaning and refilling, and the water and detergents used for the cleaning process. such factors were accounted for the carbon footprint of beer in reusable 30-l stainless steel kegs was half of that of beer packed in 66-cl glass bottles (table 4). nevertheless, this cannot be decisive for reducing the environmental impact of beer, since 80.8% of beer sales are in glass bottles and just 11.7% in stainless steel kegs (assobirra, 2020).  the environmental impact of returnable glass bottles was assessed either for beer in portugal (mata and costa, 2001) or for carbonated soft drinks in the united kingdom (amienyo et al., 2013). in both studies, the environmental load depended on the percentage of bottles returned and the number of times each bottle was reused. in the case of 50% reuse and up to six reuse cycles, returnable bottles reduced several impact categories, except eutrophication and ozone layer depletion (mata and costa, 2001). global warming was reduced by ∼40%, as the glass bottles were reused just once, its minimum asymptotic value being achieved for eight reuses (amienyo et al., 2013). before deciding which is really less environmentally impacting among one-way, recycled, and or reusable beer packaging, the contribution of beer consumption phase (embracing not only beer refrigeration, dispensing, and losses but also consumer displacement and treatment of the wastewater formed) should be assessed, as specified by the beer product category rules (epd, 2019; technical secretariat for the beer pilot [tsbp], 2016). normand et al. (2012) and watson (2008) recommended the consumption of beer in kegs directly in pub, within a walking distance to avoid car use, especially if the pub was supplied directly from the neighboring brewery via pipelines. in addition, keg distribution was found to affect negatively the local traffic at historic sites, such as bruges in belgium (afp, 2014), or during beer festivals, such as the october fest in munich, germany (becker, 2014). since 64% of the italian beer consumption is internal (assobirra, 2020), it would probably be useful to attempt reducing the environmental impact of beer consumption by favoring the diffusion of returnable 10to 30-l keykegs, made from 100% r-pet (https://www.keykeg. com), for summer time get-together parties at the expense of present day most popular beer formats available in the market (i.e., glass bottles and cans). in this manner, it could be possible to emulate the current success of 3to 15-l bag-in-boxes for red and white wines available at both physical stores and online shops. 56 italian journal of food science, 2021; 33 (3) cimini a and moresi m the techno-scientific literature is full of proposals about the potential uses of brewery wastes (aliyu and bala, 2011; cook, 2011; huige, 2006; jackowski et al., 2020; karlović et al., 2020; kusch-brandt et al., 2019; mussatto, 2009; neylon et al., 2020; rachwal et al., 2020). despite, they are acclaimed as a panacea for most of the world’s problems, their high moisture content (table 1) makes them perishable very quickly and de facto unreusable, especially in the human food chain. by accounting for the waste hierarchy set out by directive 2008/98/ec (eu, 2008) and garcia-garcia et al. (2015), figure 4 shows a ranking of their potential upgrading proposals. malt barley rootlets malt barley rootlets (mbr) are removed from malted barley, since they impart a bitter aftertaste to beer (karlović et al. 2020). table 6 classifies their potential applications in accordance with the food waste hierarchy illustrated in figure 4. if their mycotoxin content is low, mbrs should be first used as a food ingredient, thanks to their high protein and fiber contents (table 1). some of their applications are summarized in table 6. regardless of representing the second priority choice (figure 4), mbrs are nowadays quite exclusively utilized by the animal feed industry (table 6). as the third priority choice, mbrs could be used as substrate for extracting several valuable products, such as enzymes and antioxidants, or for microbial cultivation and fermentation. the achilles’ heel of their extraction processes is the need for complex and expensive purification steps to fractionate the enzyme of choice from quite numerous other unsought enzymes. in fact, it was found to be more effective and easier to obtain nucleotide extracts from the autolysis of selected highribonucleic acid containing baker’s yeasts than from mbrs (sombutyanuchit et al., 2001). in addition, the commercial interest for using such extracts in food and cosmetics is limited due to high operating costs of their extraction processes (bonnely et al., 2000). concerning their use as an economic alternative to the conventional de mann, rogosa, sharpe growth media (laitila et al., 2004), it must be remarked that such investigations were performed in laboratories only and did not account for the market size of such a growth medium and thus for its real processing costs, as well as the impact of mbr market price on the final product. finally, the so-called optimized lactic acid production from bsg and mbr hydrolysate by radosavljević et al. (2020) did not account for the problematic recovery of lactate from such an exhausted production medium. this phase would be by far more complex than the traditional one (moresi and parente, 1999), which relies on the purer carbon sources (e.g., raw sugar extracted from sugar beet or sugarcane, and corn starch hydrolysates) used by world’s largest to be disposed of (cimini and moresi, 2018a,b, 2020). final polishing of the resulting beer permeates through 0.45-μm cartridge filter, resulting in a brilliant, colloidally stable, and microbiologically safe beer ready to be packed aseptically without any thermal pasteurization (cimini and moresi, 2020). end of life of brewing biotic materials in large-size breweries, wet bsgs, as well as hot trub and bsy, are generally used as feed supplement for both ruminants and nonruminants (cimini and moresi, 2016; kerby and vriesekoop, 2017). how craft breweries generally deal with the disposal of their byproducts is practically unknown, especially in italy. by resorting to the information provided by 90 british craft brewers interviewed by kerby and vriesekoop (2017), bsgs were destined to feed formulation by about 94% of the rural craft breweries, while the remainder was nearly equally directed to composting or landspreading. the urban counterparts exhibited almost the same disposal scenario, although in smaller craft breweries the percentage of bsgs used as feed ingredient reduced to ∼76% at the expense of that converted into compost (20%). it was also noted that a large rural brewery worked in partnership with a local pig farmer to breed pigs with bsg and serve the resulting pork meat in its own tap house. altogether, animal feed was the primary route of bsg disposal, this mirroring the practices of industrial-size breweries. regarding spent hops/hot trub, their residual bitterness prevents them from being used as an animal feed. nevertheless, owing to its minimum amount (0.2–0.4 kg hl-1 beer; table 1), such a byproduct can be appropriately added to bsg to formulate feed that is not rejected by cattle. nevertheless, the uk craft breweries appeared to reuse it as fertilizer (∼40%) or compost (∼40%) or dispose it of in the landfill (7–10%), as reported by kerby and vriesekoop (2017). even if the majority of breweries reuses yeast to inoculate the next batch of wort, 2–4 kg of surplus yeast per hectoliter of beer (table 1) is disposed of as bsy. among the smaller rural and urban craft breweries, the primary disposal method (55–60% of the overall amount) was through municipal sewage system, although such percentage decreased with increase in the size of brewery. altogether, 10% of small urban craft breweries, as well as 20% of mediumand larger-size rural craft breweries, got rid of bsy as animal feed. similar proportions were used for composting and fertilizing purposes. other uses, such as mixed substrate for anaerobic digestion or inoculum for the fermentation step of a distillery, were additionally pointed out by kerby and vriesekoop (2017). italian journal of food science, 2021; 33 (3) 57 circular economy in the brewing chain figure 4. potential uses of main brewery wastes (e.g., barley rootlets, brewer’s spent grain, spent hops/hot trub, brewer’s spent yeast) as described in the literature and ranked according to the food waste hierarchy set out by directive 2008/98/ec (eu, 2008) and garcia-garcia et al. (2015). table 6. main potential uses of malt barley rootlets (mbr) as classified according to the food waste hierarchy shown in figure 3. food waste hierarchy main mbr reuses remarks and references 1 food formulation neylon et al. (2020) listed a series of food products, such as bread, biscuits, and sausages, enriched with different aliquots of mbrs as such (chiş et al., 2020) or fermented with lactobacillus plantarum sp. (waters et al., 2013) to improve their nutritional properties. 2 feed additive mbrs are generally blended with other malting byproducts (e.g., barley dust, malt dust, and small-size barley grains) and compressed to obtain the so-called malt residual pellets with a bulk density and a protein content of about 600 kg m-3 and 18% (w/w), respectively (the maltsters association of great britain [magb], n.d.). owing to the potential high risk of being contaminated by mycotoxins, such pellets must be appropriately dosed before feeding, for instance, weaner piglets, which are as sensitive to zearalenone as humans (magb, n.d.). 3 source of enzymes mbrs are a source of invertase, superoxide dismutase, nucleases, phosphotransferase, phosphomonoesterase, and especially 50-phosphodiesterase (neylon et al., 2020). in particular, the latter is used commercially to make nucleotides (sakaguchi et al., 1963; sakaguchi and kuninaka, 1965) for enhancing the flavor of broths and soups (yamaguchi, 1998). source of multicomponent extracts mbrs are also a source of natural antioxidants, including ascorbic acid and glutathione, potentially useable in food and cosmetics (bonnely et al., 2000). microbial growth substrate mbrs were used as a cheap growth and storage medium for lactic acid bacteria (neylon et al., 2020). it had an estimated 20% lower price with respect to that of the conventional de mann, rogosa, sharpe growth media (laitila et al., 2004). bioproduct substrate hydrolysates of mbrs and brewers’ spent grains were used as substrate for lactic acid production (radosavljević et al., 2020). 5 activated carbon mbrs were converted into biochar upon heating at ∼450°c in a pyrolysis plant (chan et al., 2007). its application at rates more than 50 mg/ha in conjunction with n fertilizer (100 kg n/ha) improved not only the fertilizer effectiveness but also soil quality (chan et al., 2007). the biochar sorbent properties for several water contaminants (e.g., chlorine, chloroform, chromium, mercury, methylene blue, phenanthrene, trimethoprim, and uranium) were reported by grilla et al. (2020) and neylon et al. (2020). untreated mbr biochar was also used as a catalyst in the transesterification step of biodiesel production (tsavatopoulou et al., 2020). 6 composting 9 landfilling lactate manufacturers such as basf, galactic, musashino chemical, and dow (grand view research [gvr], 2021). as the fifth priority choice being reported in table 6, mbrs are converted into biochar, its use for soil amendment (chan et al., 2007), as a sorbent for several water contaminants (grilla et al., 2020; neylon et al., 2020), or as a catalyst in the transesterification step of biodiesel production (tsavatopoulou et al., 2020). finally, as the least preferable waste management options, mbrs might in all probability be used for composting and landfilling (table 6). brewer’s spent grains figure 4 shows how the food waste hierarchy specified by directive 2008/98/ec (eu, 2008) could be applied to manage the disposal of bsg according to circular economy template. in the first place, because of their high protein and fiber contents (table 1), such spent grains could be used in the food industry. the technical literature reports quite an innumerable list of food applications (aliyu and bala, 58 italian journal of food science, 2021; 33 (3) cimini a and moresi m fat (choi et  al., 2014). on the contrary, conventional chicken patties exhibited net improvement in their cooking loss, consistency, color, and sensory properties if they included no more than 3% bsg (kim et al., 2013). bsg was also used to prepare a probiotic drink (tan et al., 2020). however, since experimental trials have been so far performed in erlenmeyer flasks with no sensory tests and no cost– benefit analysis, such alternative use of bsg as a novel nutritional beverage appears to be very premature. even other food applications listed in table 7 have been tested in laboratory only. nevertheless, as anheuser-busch inbev, a leading brewer of the united states, realized that it had to dispose of about 1.4 million mg of bsg annually, it started a new company called evergrain to convert bsg into a low-starch, proteinaceous, and fibrous material at a pilot-scale plant at newark (new jersey, usa). owing to its successful use in the preparation of several foods and beverages (e.g., plant-based milk, bread, pizza crust, pasta, granola bars, meat alternatives, and smoothies), evergrain decided to 2011; cook, 2011; huige, 2006; jackowski et al., 2020; karlović et al., 2020; mussatto, 2009; rachwal et al., 2020). table 7 lists such applications in accordance with the food waste hierarchy shown in figure 4. in particular, the bsg fortification of food products had no effect on the taste, smell, and consistency of the final product of choice, as well as on its appreciation  by the  end  consumer on condition that it was not greater than 10% (w/w) in dry pasta (nocente et al., 2019) or 25–30% (w/w) in bread (stojceska et al., 2008) and snacks (petrovic et al., 2017). of course, such fortified foods had greater fiber content and a lower glycemic index (kirjoranta et al., 2016). when 15 parts of bsg were homogenized with a pre-emulsion made of 5 parts of carboxymethyl cellulose and 80 parts of ice, such addition to chicken meat batters at 20–25% level resulted in reduced-fat chicken sausages with an overall sensory acceptability not statistically different from that of a reference product prepared with 15% pork back table 7. main potential uses of brewer’s spent grain (bsg) as classified according to the food waste hierarchy shown in figure 3. food waste hierarchy main bsg reuses remarks and references 1 partially exhausted raw material it can be recovered from the uppermost layers of bsg discharged after lautering. since it contains undigested starch, it might be integrated with appropriate doses of fresh malt and reused in the subsequent wort batch to produce low-alcohol or alcohol-free beers (zürcher and gruss, 1990). high-protein and high-fiber containing ingredient it was used to: (a) enrich soft wheat flour and formulate: (i) breads (steinmacher et al., 2012), (ii) breadsticks (ktenioudaki et al., 2012), (iii) cookies (kissell et al., 1979; petrovic et al., 2017), and (iv) baked snacks (kirjoranta et al., 2016; ktenioudaki et al., 2013). (b) enrich hard wheat semolina to prepare several dry pastas (cappa and alamprese, 2017; nocente et al., 2019). (c) reduce fat content in some meat products: (i) frankfurters (özvural et al., 2009), (ii) smoked sausages (nagy et al., 2017), (iii) chicken sausages (choi et al., 2014), and (iv) chicken patties (kim et al., 2013). main substrate for probiotic beverages upon suspension of 200 g l-1 of pre-ground bsg in sterile water, and fermentation of the resulting medium with bacillus subtilis wx-17 (i.e., rod-shaped, gram-positive bacteria generally recognized as key health promoter), it was recovered as a liquor rich in viable cells (7.2 × 109 cfu ml-1), several essential amino acids, and citric acid cycle intermediate metabolites, and with a high antioxidant activity (tan et al., 2020). 2 feed additive bsg can be used to feed (i) cattle (cimini and moresi, 2016), (ii) pigs (kerby and vriesekoop, 2017), (iii) aquaculture fish (nazzaro et al., 2021), (iv) poultry (rachwaƚ et al., 2020), and (v) edible insects (mancini et al., 2019). 3 source of proteins the recovery of proteins, as such or hydrolyzed to formulate vegan foods, asks for quite complex extraction and purification processes using alkaline (du et al., 2020) and/or acid solutions (qin et al., 2018), subcritical water at 200°c and 40 bar (du et al., 2020) or 185°c and 50 bar (alonso-riaño et al., 2021), hydrothermal pretreatment at 60°c, ultrasound-assisted enzymatic pretreatment (yu et al., 2020), or steam explosion (rommi et al., 2018). source of polyphenolics recovery of polyphenolics was performed using quite different processes, namely alkaline hydrolysis, enzymatic hydrolysis, acetone–water, or ethanol–water extraction as such or assisted by ultrasound or microwave, or supercritical carbon dioxide extraction (jackowski et al., 2020; karlović et al., 2020; rachwal et al., 2020; stefanello et al., 2018. italian journal of food science, 2021; 33 (3) 59 circular economy in the brewing chain (continued) food waste hierarchy main bsg reuses remarks and references source of arabinoxylan (ax) such polysaccharide consists of two monomers (xylose and arabinose) and may be recovered from bsg using the integrated process as set up by vieira et al. (2014) where increasing concentrations of koh or naoh allowed ∼83% of total proteins and ∼70% of total arabinoxylan to be extracted sequentially. the efficiency of such a process was further improved with the help of ultrasound (reis et al., 2015) or microwaves (coelho et al., 2014). source of multicomponent extracts these were recovered by submitting bsg or other brewery wastes to water leaching under moderate conditions (almendinger et al., 2020). their carbohydrate or amino acid concentration was generally smaller than 10 mg per g dm or 2 mg per g dm, respectively. thus, their biological activity should be significantly enhanced to be properly utilized in cosmetic products (almendinger et al., 2020). source of cellulose nanofibers such nanofibers could be used as emulsion or dispersion agents in food preparations (rachwal et al., 2020). their recovery from dried bsg required quite a complex procedure consisting of the following steps: primary alkaline treatment with 0.1-m naoh at 60°c for 2 h to get rid of proteinaceous matter; bleaching of the lignocellulose residue with 0.7% (w/v) sodium chlorite at a boiling point for 2 h; filtering and residue resuspension in 5% (w/v) sodium bisulfite at room temperature for 1 h; filtering and washing with distilled water; secondary alkaline treatment with 17.5% naoh at room temperature for 8 h; washing and dispersion in water at 1.5% (w/v); and final homogenization at 700–800 bar for 20 cycles (mishra et al., 2017). however, no information about their processing costs is available. microbial growth substrate it was used as a growth substrate for several microorganisms, such as escherichia coli, actinobacteria, bifidobacterium adolescentis, lactobacillus spp., and yeasts in alternative to expensive nitrogen sources, such as yeast extract and peptone (cooray et al., 2017; rachwał et al., 2020). mushroom substrate it was used to cultivate mushrooms, such as pleurotus ostreatus, lentinula edodes, and hericium erinaceus. the trials carried out at the mycoterra farm (westhampton, ma, usa) suggested not only that bsg should be handled with care to avoid cross-contamination of laboratory environment but also that grain savings from bsg substitution were not so significant to support such a use financially, especially in spawn stages (mycoterra farm, 2015). bioproduct substrate bsg was used as substrate for several bioproducts (rachwał et al., 2020), such as succinic acid (cooray et al., 2017), microbial oil (saenge et al., 2011), fatty acids and carotenoids (zalynthios and varzakas, 2016), xylitol (mussatto and roberto, 2008), pullulan (singh and saini, 2012), or citric acid (femi-ola and atere, 2013). microbeimmobilizing carrier it was used to immobilize yeasts (brányik et al., 2001). 4 additive for bio-composites bsg was used as an environment-friendly reinforcement or filler component in: 1. polyurethane foam composites, even if the foam matrix was found to be less compatible than that using ground tire rubber (formela et al., 2017); 2. food packaging trays made of bsg, potato starch, glycerol, and chitosan or glyoxal in replacement of expanded polystyrene, even if their flexural strength (∼3.8 mpa) decreased to 0.4 mpa after contact with water (ferreira et al., 2019); 3. clay bricks as substitute for sawdust at 5–15% of dried bsg in brick making (ferraz et al., 2013); addition of just 3.5% (w/w) of bsg yielded stronger, more porous, and less dense bricks than standard ones in large-scale tests (russ et al., 2005); 4. wood polymer composites by twin-screw extrusion of pre-dried bsg at 120–180°c, this lowering the specific mechanical energy consumption by 20% and improving their thermal stability (hejna et al., 2021). 5 activated carbon bsg, as such or pelletized, was converted into biochar via pyrolysis and micro-gasification under high-temperature (400–500°c) and low-oxygen conditions with an average yield of 18.6% (w/w) (sperandio et al., 2017). activated carbon from bsg exhibited adsorption capacity for metallic ions, phenolic compounds, and color quite similar or even effective than that of their commercial counterparts (mussatto et al., 2010). 6 composting a proper dosage of wet bsg with a lignocellulosic bulking agent (e.g., wheat straw) and sheep or pig manure favored its appropriate composting (assandri et al., 2021). 7 biomass fuel bsg could be used as a: (i) solid biomass having a lower calorific value (lcv) of 13.7 ± 0.7 mj kg-1 at ∼8% (w/w) moisture content, and a positive economic return, its estimated production cost and its market price being €110–140 kg-1 and €230–270 kg-1, respectively (sperandio et al., 2017); (ii) hydrochar, a coal-like product obtained by hydrothermal carbonization in a closed reactor at 180–280°c and 2–6 mpa for 5– 240 min (jackowski et al., 2019); (iii) substrate for production of bioethanol upon acid pretreatment and inoculation of single or mixed microbial cultures, such as pichia stipitis and kluyveromyces marxianus (white et al., 2008), saccharomyces cerevisiae and aspergillus oryzae (wilkinson et al., 2017), and fusarium oxysporum (xiros et al., 2008); (iv) substrate for bsg anaerobic digestion in continuously stirred bioreactors yielding from 0.56 g (wang et al., 2015) to 0.81 g (vitanza et al., 2016) of biomethane per gram of total organic matter, even if both yields and kinetics were implemented by resorting to microwave-assisted alkaline pre-treatment (kan et al., 2018) or by supplementing 5% biochar (dudek et al., 2019) or trace elements (bougrier et al., 2018). 60 italian journal of food science, 2021; 33 (3) cimini a and moresi m pretreatments boosted the extraction yield to ∼95% with the counter effects of greater solubilization of carbohydrates and lignin and a lower purity of proteins—this making by far more difficult the protein separation and purification steps (qin et al., 2018). similar problems affect the recovery of polyphenolics and arabinoxylans from bsg (jackowski et al., 2020; karlović et al., 2020; rachwal et  al., 2020). moreover, the resulting extracts rich in ferulic and p-coumaric acids had to be micro-encapsulated not only to mask their pungent odor and bitter taste conveyed to fortified fish burger but also to prevent their degradation (spinelli et al., 2016). finally, the suggested use of bsg as inexpensive substrate for several bioproducts (rachwał et al., 2020) relies on tests carried out in laboratory with no account for their feasibility and processing costs in pilot and/or industrial plant. the use of bsg as a remunerative substrate per citric acid production was, for instance, just puerile, since it was drawn in the absence of any comparison among the citric acid yield factors and production rates in laboratory and industrial plant. moreover, it is not known which bioproduct recovery and purification steps are to be used when dealing with a multicomponent matrix such as bsg instead of glucose syrups currently utilized (moresi and parente, 1999) by the world’s largest citric acid manufacturers such as anhui, archer daniels midland, cargill, huangshi xinghua biochemical, jungbunzlauer, tate & lyle, etc. (gr-store, 2021). in accordance with the fourth and fifth waste management options (figure 4), bsg could be used to reinforce different biocomposites or produce activated carbon and biochar, as provided in table 7. even if the adsorption capability of biochar might help to improve water and chemical fertilizer retention in agricultural soils, as well as limit their nitrate leaching and n2o and ch4 emissions, no cost–benefit analysis has been carried out so far. concerning the sixth waste management option (figure  4), bsg may be composted on condition that it is mixed with wheat straw and sheep or pig manure to adjust its initial moisture content (60–65% w/w), carbon/ build its first full-scale production facility at anheuserbusch’s historic headquarter in st. louis, missouri (evergrain, 2021). thus, it is highly probable that more amounts of bsg would be utilized in the food sector and no more diverted to the second option  of food waste hierarchy depicted in figure 4. the second waste disposal prospect (figure 4) is used by most of the industrial and craft breweries to dispose of fresh bsg as feed additive in animal and insect nutrition (table 7). to this end, under the eu regulation no. 183/2005 (eu, 2005), food companies (including breweries) willing to sell their byproducts as feed materials are to register with their local authorities and develop a specific hazard analysis and critical control point (haccp)  plan to comply with traceability requirement and keep the risk of biological, chemical, and physical contamination of food wastes as low as practically attainable. additionally, because of high moisture content (table 1), bsg is so perishable that it must be fed within 2 or 3 days of manufacture unless it is stored at 5°c, dried or pickled (jackowski et al., 2020). whereas its drying is hardly practiced, since the high operating costs are not rewarded by the final feed use, pickling is a low-cost operation capable of extending the shelf life of bsg with no counter effects on its quality (jackowski et al., 2020). the third waste disposal prospect (figure 4) involved the use of bsg as substrate for extracting proteins, polyphenolics, arabinoxylan (ax), and cellulose nanofibers, or for microbial growth and fermentation, as summarized in table 7. the recovery of proteins from bsg is not an easy task. for instance, upon suspending about 100-g bsg in 1 l of aqueous naoh (ph > 11) at 40°c for 2 h, and centrifuging, it was possible to recover a protein-rich precipitate with an extraction yield of about 21% and purity of 60% (du et al., 2020). further extraction of such a residue with subcritical water at 200°c and 40 bar for 20 min enhanced protein extraction yield by an extra 7% (du et  al., 2020). subsequent alkaline and dilute acid food waste hierarchy main bsg reuses remarks and references 8 organic fertilizer bsg might be used as: (i) organic fertilizer because of its p, k, protein, cellulose, lignin, and hemicellulose contents; the mixture of bsg (5 mg ha-1) and npk fertilizer (200 kg ha-1) affecting positively the growth of maize and increasing soil aggregation (nsoanya and nweke, 2015); (ii) biofertilizer useful against soil-born insects; once bsg is inoculated with the spores of entomopathogenic fungi beauveria bassiana the, accumulation of 10 metabolic compounds in the fermented biomass is found to be effective against galleria mellonella larvae (qiu et al., 2019). 9 landfilling wet bsg is landfilled by 7–10% of the uk craft breweries (kerby and vriesekoop, 2017). table 7. (continued) italian journal of food science, 2021; 33 (3) 61 circular economy in the brewing chain above-mentioned food waste hierarchy. beyond the methods reviewed by kerby and vriesekoop (2017), it is worthy pointing out the possibility of fractionating about 0.11 g of a series of monoand sesqui-terpenes from 100 g of dry ht by hydrodistillation—these essential oils acting as natural repellents against two coleoptera (i.e., rhyzopertha dominica and sitophilus granaries) that cause big  economic loss  to stored foods (bedini et al., 2015). obviously, even in this case no cost–benefit analysis was carried out to measure the real applicability for such eco-friendly repellents. brewer’s spent yeast figure 4 illustrates the real and potential disposal methods of bsy as classified according to the above mentioned food waste hierarchy. although bsy has long been used to produce a darkbrown food spread named marmite (this being invented by the german scientist, justus  freiherr  von liebig, and nowadays produced by unilever in the united kingdom), none of the 90 craft breweries interviewed by kerby and vriesekoop (2017) supplied bsy to any marmite factory, probably because of the small amounts available. marmite is a rich source of  vitamin b complex, which is spread on bread,  toast, or  crackers and imparts the so-called umami taste, typical of the  amino acid  l-glutamate  and 5’-ribonucleotides. its main analogues are the australian  vegemite, swiss cenovis, brazilian cenovit, and german  vitam-r (wikipedia, 2021). owing to its high protein content (table 1), bsy can be converted into protein concentrates and isolates (karlović et al., 2020). it is directly added to energy bars in the proportion of 10–30% (w/w) to significantly increase their protein and phytic acid contents as well as density (stojceska et al., 2008). bsy is used as a source of food-grade yeast extracts. naclor saponin-induced autolysis of cell yeast gives rise to yeast extracts containing different free amino acid contents as well as peptides of diverse molecular masses. in this manner, these can be used to tailor-make novel functional foods having peculiar taste profiles, or more conventionally to enhance the flavor of food products by dosing appropriately specific components, namely, nucleotides, peptides, and amino acids (mainly glutamic acid; podpora et al., 2016). notwithstanding the fact that it is used to formulate animal feed as the second option  of  the above-mentioned food waste hierarchy (figure 4), wet bsy combined with bsg and hot trub is primarily sold to farmers as a low-cost feed additive, especially by large-size breweries (cimini and moresi, 2016). nitrogen (c/n) ratio (20:30), and ph (5.5–7.5) (assandri et al., 2021). the seventh waste disposal option shown in figure 4 refers to the utilization of dried bsg as a solid biomass— its lower heating value ranging from 76 to 90% of 15–18 mj kg-1 of the majority of solid biomasses with ∼10% moisture content (paládi, 2013). regarding hydrothermal carbonization of wet bsg, the resulting hydrochar could be used as biofuel, feedstock for gasification, soil additive for nutrient enrichment, adsorbent, or precursor of activated carbon  (jackowski et al., 2019). although no information is currently available about the economic practicability of bsg conversion into hydrobiochar or bioethanol, production of biogas from bsg was regarded as economically unviable unless the process included coproduction of other more profitable products (gonzález-garcía et al., 2018). the enzymatic hydrolysis of cellulose, hemicellulose, and lignin would facilitate the release of fermentable sugars and thus improve the conversion yield into bioethanol or biomethane per unit mass of the lignocellulosic material consumed. however, it is still unknown, how the market price of commercial cellulolytic enzymes would affect the biofuel production costs. as given in table 7, bsg might be disposed of as an organic fertilizer per se or pre-fermented to be effective against soil-born insects (qiu et al., 2019). finally, bsg could be landfilled as the least preferable waste management alternative as shown in figure 4. most of the above-mentioned potential uses of bsg are also elucidated by a recent bibliometric analysis carried out by sganzerla et al. (2021), where up to 510 papers over the last 30 years were retrieved from the web of science© database. globally, 65 countries have been involved in studies linked to bsg, and brazil has been the most productive nation with up to 70 papers. it was demonstrated that a great interest existed in the valorization of bsg, even if no feasibility study was identified to sustain any of such research activities on an industrial scale. although a bibliometric study pointed out the possibility of developing a biorefinery using bsg as raw material, it underlined the difficulty of identifying the most appropriate and economically viable chemico-physical or enzymatic processes applicable to upgrade the product yield of choice as well as to lower their environmental effects. spent hops/hot trub figure 4 shows the main disposal methods of spent hops/hot trub, these being ranked according to the 62 italian journal of food science, 2021; 33 (3) cimini a and moresi m is the primary option for small-, medium-, and large-size breweries, since it represents an avoided production of feed and gives rise to quite a significant co2e credits equaling to about one-third of the contribution of packaging materials (table 5). conclusions in the light of the concept of circular economy, the brewing chain is expected to deal with the reuse of abiotic (e.g., packaging materials and spent kieselguhr sludge) and biotic (e.g., brewery wastes) materials to approach the zero-waste objective. the pros and cons of different packaging alternatives (e.g., one-way, lighter, reusable, or recycled containers) were analyzed. even if the contribution of beer consumption phase was taken into account, there was no definitive result about the less environmentimpacting beer packaging format. autonomous system for the direct management of pet packaging for liquid foodstuffs, recently recognized by the italian ministry of the environment, might help to make available 100% r-pet flakes ready to be reconverted into food-grade bottles with minimum downcycling to other non-food uses. concerning numerous studies suggesting alternative utilization of brewery wastes in nutritional as well as biotechnological fields, it was pointed out that the majority of these was just tested in laboratory and included no cost–benefit or market analysis. even when the concept of biorefinery was stressed upon as an interesting strategy to upgrade brewery wastes, none of the final bioproducts obtained seems to be market-justifiable, mainly because the estimated market price of dried bsg was about 450% higher than that of conventional lignocellulose residues. except for the anheuser-busch’s initiative of quickly converting wet brewer’s grains into a low-starch and high-protein and high-fiber containing ingredient for foods and beverages, the high moisture content of all brewery wastes makes them perishable to prevent their safe usage in the human food chain. their prompt use as an animal feed appears to be the only disposable method not only economically feasible but also able to lower by about one-third the ghg load of packaging materials. not by chance, it is currently practiced by both industrial and craft breweries. all other alternative uses, hailed in the literature as a panacea for most of global problems, appear to be more useful for publishing articles than for defining any economically feasible reusing procedure for all the brewery wastes of concern. under these circumstances, to support its transition toward a circular economy, the beer industry must primarily reduce, reuse, recycle, and recover as much as possible the beer packaging materials. owing to its high moisture content and chemical composition (table 1), bsy degrades easily. thus, before being administrated to animals, bsy must be dried or stabilized by adding organic acids to avoid its fermentation in the gastrointestinal tract of animals, especially pigs, which are highly sensitive to such disorders (crawshaw, 2004). wet bsy is mainly used to feed cattle, but its high digestibility has to be checked for other animals, such as fish, horses, turkeys, hens, and swine (crawshaw, 2004). the third waste management option  in figure 4 suggests using bsy as a source of several useful compounds, such as enzymes (ferreira et al., 2010) and especially invertase (de león-gonzález et al., 2016), polyphenolics (vieira et al., 2016), ergocalciferol used in vitamin d deficiency (metzger et al., 2012), saccharides such as β-glucans (thammakiti et al., 2004), and trehalose (mahmud et al., 2010). in particular, β-glucans extracted from bsy are used to replace partially fat in mayonnaise to lower its energy value and improve its storage stability (worrasinchai et al., 2006). bsy are also used as a growth medium for lactobacilli and pediococci (champagne et al., 2003). all the above-mentioned bsy applications are experimented in laboratory without any cost–benefit analysis to assess their real feasibility. finally, bsy can be used as a fertilizer by composting or land spreading, although these waste management options are the least preferred ones to be applied (figure 4). concluding remarks to conclude, this analysis was about the potential utilization of brewery wastes. it is worth summarizing the results of an economic market analysis carried out by buffington (2014) on the assumption of feeding a bio refinery located centrally with respect to two large-size beer manufacturers in the united states with their bsgs shipped “as-is,” that is, with an average moisture content of 70% (w/w). since the current acquisition cost of other alternative agroforestry wastes, such as stalks and straws, at an average moisture content of ∼10% (w/w) is around us$40 mg-1, the effective acquisition cost for bsg would be us$133.30 per dry mg. moreover, accounting for the depreciation costs of bio-refinery, drying and storage processing costs of bsg, logistic costs, and a 5% net profit, the processed bsg market price would 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district, tamil nadu state, india 4national research centre on equines, hisar, haryana 125001, india 5instructional livestock farm complex, veterinary college and research institute, tirunelveli, tamil nadu 627 358, india *corresponding author. palbert1982@gmail.com abstract the objective of the present study was to elaborate papaya jam by substituting 5% and 10% papaya pulp with same amount of paneer whey. physico-chemical, sensory, microbial and texture analysis were performed on 0, 30th and 60th day of storage. upon whey substitution in jams, protein content and texture of final products were significantly improved. slight but not significant increase of acidity and decrease of ph were observed in all the formulations and during shelf life. reducing sugar content in jam increased from16.20 to 37.49 and that of non-reducing sugar decreased from 45.18 to 26.23% during the storage period of 60 days. absence of microbial growth was observed in all the jam formulations throughout the storage period. taken together, whey can be efficiently substituted in papaya jam to improve nutritive value and texture of the product. keywords: jam, papaya pulp, protein, storage, substitution, texture, whey ital. j. food sci., vol 29, 2017 172 1. introduction jam is the most popular and shelf stable product made from fruits at household and commercial level. fruit jams are good source of energy but poor source of protein (naeem et al 2015). ingredients and fruit pulps used for preparing jams are commonly poor in protein content and ultimately results in less protein level in jam (naeem et al 2015). commercially available jams are poor in protein content as reported by several workers i.e. apple jam 0.04%, mango jam – 0.15%, jackfruit jam – 0.19%, papaya jam – 0.26%, blueberry jam – 0.31%, strawberry jam – 0.41%, grape jam – 0.27% and apricot jam – 0.43% pineapple jam – 0.46% and orange marmalade jam 0.79% (ahmmed et al., 2015, teangpook and paosantong 2013, eke-ejiofor and owuno, 2013 naeem et al., 2015). so there is a need to improve the protein content in jam to make it still a better nutritional food. whey is a valuable by-product obtained during manufacture of cheese, channa, casein paneer and shrikand as watery portion of milk after coagulation and removal of curd. whey contains about 50% of milk solids together with 100% of lactose and 20% of protein. lactose makes up about 75% of total whey solids (siso, 1996). whey represents about 80 to 90% of the volume of milk from which it is obtained (khamrui and rajorhia, 1998). whey protein is a complete, high quality source of protein with a rich amino acid profile. whey has high protein efficiency ratio (3.6), biological value (104) and net protein utilization (95) is next only to egg protein in terms of nutritive value (renner, 1990). about 3 million tonnes of whey is produced annually in india containing about 2 lakh tonnes of valuable milk nutrients (naik et al., 2009). about 40% of total global production of whey is disposed as raw whey (reddy et al., 1987) causing serious environmental pollution. the disposal of whey is problematic as the biological oxygen demand (bod) of whey is 38,000 to 46,000 ppm (due to its high organic content) as compared to 200 ppm permissible limit for domestic sewage (mishra, 2008). whey has to be treated appropriately to obtain commercial products (gupte and nair, 2010) or preheated before its discharge in inland water or rivers as per environmental protection act (1986). so it is important to find alternative uses of whey to reduce the economic and environmental impact. whey is used in food industry for its high nutritional value, excellent functional properties and for reducing the cost of production of food products. whey preparations are used in meat and meat products, reduced fat products, yoghurt, ice cream, cheese, bakery products, confectionary and pastry products, infant formula, whey beverage and for encapsulation of sensitive foods and edible coating of foods. the functional properties of whey proteins, mainly used in the production of food products are solubility, gelling, emulsifying, and water binding properties, antioxidant activity, flavour improvement and fat mimetics (królczyk et al., 2016). papaya is considered as power house of nutrients as it is a rich source of carotenoids, vitamin c, vitamin e, niacin, riboflavin, vitamin k, carbohydrate, folate, pantothenic acid and dietary fibre. it is also rich in fe, na, k, ca, mg, p, cu, zn and mn (aravind et al., 2013). papaya is popularly used as dessert or processed into jam, puree or wine (matsuura et al., 2004). in the present study, different percentages of papaya pulp were substituted with paneer whey in formulations for papaya jam. upon substitution, its effects on functional and nutritional properties of jam were studied along with sensory and microbial analysis during storage period of two months. ital. j. food sci., vol 29, 2017 173 2. materials and methods plain condensed whey obtained from paneer (dairy plant, college of veterinary and animal science, thrissur) was utilized for study. papaya used in the experiment was obtained from the local market. pectin, sugar (sucrose), citric acid (food grades) was purchased from local market. 2.1 preparation of papaya jam three type’s papaya jams were prepared by substituting papaya pulp with paneer whey. control jam group (t0) served as control without whey substitution in papaya pulp (100 g papaya pulp + 0 g whey). substitution of papaya pulp with condensed whey both for t1 and t2 are 5%( 95 g papaya pulp + 5 g whey) and 10%( 90 g papaya pulp + 10 g whey) respectively. composition of prepared papaya jam was given in table 1 and method of preparation was given in fig. 1. for substitution in jam, plain condensed whey (semisolid form) was allowed for slow hydration for a period of 30 min by mixing with water at 60°c at the ratio of 1:2. hydration was performed for optimal performance of whey protein during heat processing (zhang and zhong, 2010). the normal range ph range of jam was 2.5 to 3.5 (broomfield, 1996) and this ph range of jam helps in stability of whey protein.βlactoglobulin in whey is heat stable at ph 3 (boye et al., 1996). heat stability of whey protein also further improved by presence of sucrose in jam (kulmyrzaev et al., 2000). according to duranti et al. (1989) heating to 85°c is critical for whey protein denaturation. so concentrated whey was substituted in the jam just 1 or 2 minutes before reaching end point of jam preparation (105.5ºc) to avoid denaturation. prepared jams were stored in sterilized glass jars at room temperature (30ºc). table 1. formulation of jam. composition control jam (t0) t1 t2 papaya pulp (g) 100 95 90 whey (g) 0 5 10 sucrose (g) 75 75 75 pectin (g) 1 1 1 citric acid (g) 0.6 0.6 0.6 2.2. physical-chemical analysis physical-chemical analyses were performed in jam samples on 0th, 30th and 60th days of storage and results were expressed as mean±standard error of mean. following analysis were performed according to regulations and protocols described by association of official analytical chemists (2000): ph using a digital ph meter, total soluble solids (tss,°brix) by using an abbe refractometer, water activity (aw) using aw sprint novasina th-500®, crude protein using micro kjeldahl method,% acidity by titration with naoh (0.1 m), ash by muffle furnace and moisture by drying in kiln. estimation of fat in condensed whey was performed as per protocol described by aoac (2000). sugars in jams (total sugars, reducing sugar and non-reducing sugar) were determined as per lane and eynon method (1923). ital. j. food sci., vol 29, 2017 174 figure 1. flow chart for jam preparation. 2.3. microbial analysis standard total plate count, spore count, coliform count, yeast and mould count of jams were performed as per ranganna (1986) on 0th, 30th and 60th day of storage period for t0, t1 and t2 jams. 2.4. sensory evaluation jam for organoleptic evaluation was prepared aseptically in clean transparent disposable closed containers and served fresh on test day in a perfectly homogeneous way, i.e. identical conditions of preparation, conservation and presentation. sixteen trained members of panel were selected from the university community among postgraduate students for evaluating sensory characteristics (color and appearance, taste, texture and overall acceptability) of the samples using a 9-point hedonic scale as per ranganna (2008). during product test, panel members were allowed to clean their mouth at intervals with water. the sensory evaluation of jams was performed on 0th, 30th and 60th days of storage. ital. j. food sci., vol 29, 2017 175 2.5. texture analysis texture analysis was performed directly in jar containing jam at the ambient temperature with a texture analyzer ta.xtplus® (stable micro system, united kingdom), using back extrusion procedure. it was used to measure the force – time curve for a two cycle compression. a cylindrical probe (1 inch) was used to compress the samples. on the basis of the preliminary work, instrument working parameters were determined with test mode compression, pretest speed at 1.0 mm/s, test speed at 1.0 mm/s, post-test speed at 10.0 mm/s, distance 10.0 mm, trigger force at 10.0 g and data acquisition rate at 200 pp (younis et al., 2015). data were analyzed using texture expert version 1.22® software (stable micro system, united kingdom) to measure hardness, consistency, cohesiveness and index of viscosity in the samples. all measurements were done in triplicates of jam samples. 2.6. statistical analysis anova with tukey's t-test, at the 5% level, was applied to data to establish significance of difference among the samples. statistical analyses were performed using the statistical analysis package statistica 7.0®. the experiment was carried out with 6 replications 3. results and discussions the composition of plain condensed whey obtained from paneer is presented in table 2. the papaya pulp had ph of 4.46, total soluble solid 10±0.56% and acidity 2.05±0.05% (table 3). similar findings were reported by zaman et al. (2006) where ph of papaya fruit pulp ranged from 4.2 to 4.5, total soluble solids varied from 9.0 to 13.0%, the acidity (as citric acid) ranged between 2.00 to 2.30%, total sugar ranged from 6.96 to 10.5%, reducing sugar ranged from 3.42 to 6.92% and non-reducing sugar ranged from 3.17 to 3.58%. aravind et al. (2013) reported that papaya pulp contain 0.61% crude protein. saran and choudhary (2013) reported that ash content of papaya pulp range from 0.31 to 0.66% and moisture from 85.9 to 92.6. table 2. composition of plain condensed whey. total solids% acidity% ash% fat% crude protein% plain condensed whey(mean±sem) 58.96±0.69 0.48±0.05 6.54±0.21 0.68±0.71 7.35±1.61 sem – standard error of mean 3.1. physical-chemical analysis of jam the physical chemical properties of jam are presented in table 4. in this experiment, acidity values of all jams ranged from 0.55% to 0.59% during the storage period of two months). mamede et al., 2013 reported that acidity of jam ranges between 0.5% and 0.8% of citric acid and jam with acidity above 1% shows syneresis as higher acidity value causes exudation of liquid from jam. substitution with whey at 5% (t1) and 10% (t2) level did not affect significantly the acidity of jam; even if a slight but not significant rise in the acidity of all jams was observe during the storage period. teangpook and paosantong ital. j. food sci., vol 29, 2017 176 (2013) reported that acidity of low sucrose lime juice papaya jam increased from 0.63 to 0.70% during the storage period of 6 months. similarly shakir et al. (2008) reported that there was increase in acidity from 0.6 to 0.78% in apple and pear mixed jam during the storage period of 3 months. increase in acidity might be ascribed to rise in concentration of weakly ionized acids and their salts during storage. further, rise in acidity might also be due to formation of acids by degradation of polysaccharides and oxidation of reducing sugars or by breakdown of pectic substances and uronic acid (hussain et al., 2008). table 3. chemical composition of fresh papaya pulp. ph total soluble solids % acidity % total sugar % reducing sugar % nonreducing sugar % crude protein% ash % moisture % papaya pulp (mean± sem) 4.46±0.02 10±0.56 2.05±0.05 7.26±0.1 3.92±0.6 3.34±0.01 0.65±0.25 0.45±0.06 88.16±0.23 sem – standard error of mean in jam, total soluble solids or obrix is a measure of all soluble solids from natural fruit components, added sugar, acid, pectin and other ingredients. according to food safety and standards regulations (2011) total soluble solids of jam should be not less than 65%. for optimum gel formation in jam with good texture and sensory acceptance, the total soluble solids should range between 65 and 68% (macrae et al., 1993; damiani et al., 2008). in current experiment, total soluble solids of all jams were > 65.0% without any significant differences among them. substitution of papaya pulp with whey in jam at 5% (t1) and 10% (t2) level not caused any changes in total soluble solids level (table 4). during storage period, all jams showed slight but not significant rise in tss. tss of t1 increased from 66.25 to 67.06, t2 from 67.52 to 68.01 and jam c from 68.56 to 69.18. similarly ehsan et al. (2002 and2003) reported rise in tss of watermelon lemon jam from 68.6 to 68.9 and grape fruit apple marmalade from 70.0 to 70.8 after 60 days of storage. shakir et al. (2008) reported that there was increase in total soluble solids of apple and pear mixed fruit jam from 68.5 to 70.6 during the storage period of 90 days. increase in tss during storage might be due to acid hydrolysis of polysaccharides especially gums and pectin (luh and woodroof, 1975). setting quality of jam can be improved by adequate ph maintenance. ph and titratable acidity are indicators for quantity of organic acids and their salts contained in a fruit. in our experiment ph value of all jams were ranged between 3.08and 3.36. this ph range was close to the optimal ph suggested by rauch (1965), which ranges from 2.5 (hard jam) to 3.45 (soft jam). similarly, broomfield (1996) also reported that the ph range of 2.5 to 3.5 in jam was suggested for stable pectin-acidsugar gel structure. the formation of gel structure in jam depends on the concentration of hydrogen ions and not that of the acidity. teangpook and paosantong (2013) reported that low sucrose lime juice papaya jam had ph of 3.22. there was slight decrease in ph of t0 from 3.31 to 3.09, t1from 3.36 to 3.15 andt2 from3.27 to 3.08 during storage period of two month without any significant difference). khan et al. (2012) reported that ph value of the strawberry jam decreased from 3.20 to 2.91 during the storage period of 60 days. ital. j. food sci., vol 29, 2017 177 table 4. physico-chemical analysis of jam. parameters control jam (t0) mean±sem t1 mean±sem t2 mean±sem acidity (%) 0th day 0.55±0.017 0.58±0.03 0.58±0.02 30th day 0.57±0.04 0.58±1.25 0.59±1.28 60th day 0.57±0.05 0.59±0.56 0.59±1.58 total soluble solids ( 0brix) 0th day 66.25±0.029 67.52±0.028 68.56±0.025 30th day 66.78±0.09 67.91±0.18 68.92±0.08 60th day 67.06±0.24 68.01±0.87 69.18±0.14 ph 0th day 3.31±0.12 3.36±0.25 3.27±0.71 30th day 3.25±0.53 3.21 ±0.98 3.15±0.59 60th day 3.09±0.78 3.15±0.65 3.08±0.27 ash (%) 0th day 0.323±0.002 0.323±0.002 0.323±0.007 30th day 0.323±0.006 0.324±0.006 0.324±0.006 60th day 0.324±0.009 0.324±0.005 0.324±0.005 moisture (%) 0th day 26.24±0.21aa 28.74±0.21ba 30.32±0.25ca 30th day 25.22±0.36ab 27.42±0.78bb 29.60±0.39cb 60th day 24.36±0.54ac 26.19±0.87bc 28.91±0.17cc crude protein (%) 0th day 0.92±0.028a 3.15± 0.028b 4.23±0.03c 30th day 0.85±0.58a 3.14±0.13b 4.20±0.04c 60th day 0.84±0.24a 3.12±0.54b 4.20±0.03c reducing sugars (%) 0th day 16.20 ±0.06aa 19.54 ±0.02 ba 22.17 ±0.01ca 30th day 18.47±0.03ab 25.63±0.01 bb 29.35 ±0.05cb 60th day 24.30 ±0.18 ac 32.44 ±0.01bc 37.49 ±0.02cc non reducing sugar (%) 0th day 45.18±0.01 ac 43.16 ±0.01bc 40.28 ±0.02cc 30th day 43.19±0.12ab 37.24 ±0.01 bb 32.50 ±0.02cb 60th day 38.54 ±0.29aa 31.55 ±0.08 ba 26.23 ±0.03ca water activity (aw) 0th day 0.80±0.001c 0.78±0.002 b 0.75±0.003a 30th day 0.80±0.001c 0.78±0.002 b 0.75±0.003a 60th day 0.80±0.001c 0.78±0.002 b 0.75±0.003a microbial analysis 0th day nil nil nil 30th day nil nil nil 60th day nil nil nil sem – standard error of mean. abc means on the same line without a common letter are significantly different at p < 0.05. (all samples at the same time). abc means on the same column without a common letter are significantly different at p < 0.05. (single sample during storage period). jam significantly (p < 0.05) decreased with 5% whey substitution in t1 (0.78) and with of 10% whey substitution in t2 (0.75) substitution when compared to that of control jam t0(0.80) teangpook and paosantong (2013) reported that low sucrose lime juice papaya jam had water activity of0.9. santos et al. (2013) showed that water activity of gabiroba jams prepared with sucrose and sucralose were 0.78 and 0.80 respectively. in this study there was no change in water activity of all jams during the storage period of 60 days. ital. j. food sci., vol 29, 2017 178 the decrease in ph of jam during storage may be attributed to formation of free acids by degradation of polysaccharides, oxidation of reducing sugar, ascorbic acid degradation and hydrolysis of pectin (hussain and shakir, 2010). there was no significant rise (p>0.05) in ash content of both t1 and t2due to whey substitution). ash content of all jams ranged from 0.323% to 0.324% without any significant changes during storage. teangpook and paosantong (2013) reported low sucrose lime juice papaya jam had ash content of 0.51%. ahmed et al. (2011) reported ash content of sapota jam as 0.42%. vidhya and narain (2011) reported that there was no change in ash content of wood apple jam during storage period of 90 days. statistical analyses revealed that addition of whey significantly (p<0.05) increased the moisture content of t1 (28.74%) and t2 (30.32%) when compared to that of control jam t0 (26.24%). this may be due to higher water retention property of protein present in whey (vidigal et al., 2012). t2jam has significantly higher (p<0.05 moisture content than that of t1. eke-ejiofor and owuno (2013) reported that moisture content of jack fruit jam and pineapple jam in their experiments were 24.60% and 23.29% respectively. mamede et al. (2013) observed that the moisture content of dietetic jam prepared from umbucaja fruit ranged from 26.7 to 31.98%.the moisture content of jam produced from dehydrated fruits (tamarind guava and kumquarts) ranges normally between 28.6 – 30.1% (winus,2011). it is important to consider that the moisture content is directly related to the conservation of the product during storage. reduction in moisture content of jam may decrease gel strength and thereby causes firmer jam with poor spreading ability (fasogbon, 2013). in this experiment, there was significant reduction (p>0.05) in moisture content of all jams t0 (from 26.24 to 24.36%), t1 (from 28.74 to 26.19%), and t2 (from 30.32 to 28.91%), during storage period of 60 days. similarly, hussain and shakir (2010) observed a decreasing trend in the moisture content of apricot and apple jam (from 16.08 to 4.3%) during the storage period of 60 days. anjum et al. (2000) observed decreased in% moisture from 79% to 77% after 60 days of storage in dried apricot diet jam. the loss of moisture in jam that is stored in sterilized glass container in room temperature is due to the exchange of moisture between the outside and inside of the glass by desorption, trapping the free water during gel formation in jam and due to maillard reaction, which occurs at high temperatures (even at 25°c) in high-sugar products with low ph values by using the freely available water in jam (damiani et al 2012). protein content of most fruit jam is very low due to the low protein content of most of fruit pulp and none of the ingredients used in jam preparation are an abundant source of protein (naeem et al 2015) in this experiment, t2 contain significantly (p < 0.05) highest crude protein content (4.23%) due to 10% whey substitution. t1 with 5% whey substitution contain crude protein content (3.15%) significantly (p < 0.05) higher than control t0 (0.92%). teangpook and paosantong (2013) reported that low sucrose lime juice papaya jam had protein content of only 0.26%. eke-ejiofor and owuno (2013) prepared jackfruit jam using pineapple jam as control and reported that protein content ranged from 0.19% 1.12% with pineapple jam had lowest and jackfruit jam had highest of the products. salvador et al. (2012) reported that proteins were present in small amounts in yacon jams as yacon pulp had low protein content. carvalho et al. (2013) estimated that protein content of diet strawberry jam ranged between 1.31 and 1.35%. gebhardt and thomas (2002) reported that protein content present in one tablespoon (20 gram) of jam was in traces. chaudhary and verma (2011) analysed the physicochemical properties of the processed fruits and vegetable products and found that protein content of jam was 0.48% which is least when compared to the protein content of other processed products (tomato sauce – 0.82%, pickle – 1.98 and orange juice – 1.97%). no significant change was observed in the value of protein content of all the jam samples during the storage period of two months. ital. j. food sci., vol 29, 2017 179 sugars that have high tendency to crystallize like pure dextrose (glucose) were not used in this study, in contrary refined sucrose is known to be a good sugar for addition to jam because of low tendency to recrystallization was used. sucrose is partly inverted to glucose and fructose in the manufacturing process when the ph of the product is low. this fact was important because as it reduces tendency of sugar to form crystals (cancela et al., 2005). statistical analyses revealed that substitution of whey significantly (p<0.05) increased the reducing sugar content in t1 (19.54%) and t2 (22.17%) when compared to control jam t0 (16.20%). this increase in reducing sugar content in jam is due to the presence of lactose in whey and lactose itself is a reducing sugar (jenness and patton, 1959). presence of lactose may lead to formation of maillard reaction products, which are known to have antioxidant properties in food systems (de wit 2001). the rise in reducing sugar by whey substitution has correspondingly significant decrease (p > 0.05) in non-reducing sugar content of jam t1(43.16%) and t2 (40.28%) when compared to t0 (45.18%). during storage period, there was significant (p < 0.05) rise in reducing sugar and decline in non-reducing sugar content of all jams. the results of reduction in nonreducing sugars in jams were in accordance with riaz et al. (1999) who reported reduction in non-reducing sugars from 44.64 to 32.35% in strawberry jam during storage period of 3 months. ehsan et al. (2003) observed drop in non-reducing sugars of grape fruit apple marmalade during storage period of 60 days. rise in reducing sugar may be due to inversion of non-reducing (sucrose) to reducing sugar (glucose + fructose) because of acid and high temperature during storage (muhammad et al., 2008). riaz et al. (1999) observed increasing trend in reducing sugars of strawberry jam during 3 months storage period. anjum et al. (2000) while working on apricot diet jam also observed similar increase in reducing sugars during storage period of 60 days. ehsan et al. (2003) reported increasing trend in reducing sugars of grape fruit apple marmalade where reducing sugars increased from 16.55% to 31.36% after 60 days of storage. water activity (aw) determines the lower limit of available water for microbial growth. water activity for jams must be less than 0.95 in order to prevent the growth of pathogenic bacteria. in general, minimum aw for most moulds were 0.8, yeasts 0.85 and osmophillic yeasts0.6-0.7 (ray and bhunia, 2014). most yeasts, molds and bacteria do not survive at low water activities, which were directly correlated with a long shelf life (beuchat, 1981) in this experiment water activity of papaya jam significantly (p < 0.05) decreased with 5% whey substitution in t1 (0.78) and with of 10% whey substitution in t2 (0.75) substitution when compared to that of control jam t0 (0.80) teangpook and paosantong (2013) reported that low sucrose lime juice papaya jam had water activity of0.9. santos et al. (2013) showed that water activity of gabiroba jams prepared with sucrose and sucralose were 0.78 and 0.80 respectively. in this study there was no change in water activity of all jams during the storage period of 60 days. 3.2. microbial analysis microbial analysis of jam during 0th, 30th and 60th day of storage revealed no bacterial or fungal growth in all three types of jam (t0, t1 and t2). the presence of total soluble solids about 66 to 70% protects jam from microbial deterioration (taufik and karim, 1992). in this experiment the total soluble solids of all jam ranges between 66.25 to 69.18%, which resulted in the absence of microbial growth during the storage period of two months. jam was also prepared with hygienic measures during handling and storage to make it microbiologically safe. the microbial results of this study were in agreement with findings of many authors like parsi ros (1976) who stated that papaya jam stored for 180 days at 29.4οc were found to be microbiologically safe. joy and rani (2013) reported that microbial analysis showed ital. j. food sci., vol 29, 2017 180 no growth of any kind of bacteria or fungi in the plates of aloe vera strawberry jam, papaya jam and pineapple jam till the 60th day of storage. kerdsupa and nakneana (2013) observed no bacteria, fungi and yeast growth in low-sugar mango jam over 6 weeks of storage. 3.3. texture analysis textural properties of papaya jam were significantly (p<0.05) increased by whey substitution (table 5). water retention capacity and gelling properties of whey protein increased texture of jam by avoiding syneresis. antunes et al. (2003) also reported that βlactoglobulin in the whey act as a main gelling agent due to presence of free sulfhydryl group. firmness of t2jam (283.83 n) was significantly higher (p<0.05) than t1 jam (209.92 n) and control jam t0 (147.83 n). firmness of t1 jam was significantly higher (p<0.05) than control jam t0.vidigal et al. (2012) reported that addition of whey protein concentrate improved firmness but not adhesiveness and cohesiveness of ice cream. adhesiveness of t2jam (-1662.8 ns) was significantly higher (p<0.05) than t1 jam (-1543.3ns) and control jam t0 (-755.83 ns). adhesiveness of t1 jam was significantly higher (p<0.05) than control jam t0. herrero and requena (2006) showed that addition of whey protein concentrate enhanced textural characteristics of yoghurt prepared from goat milk by increasing its firmness, hardness and adhesiveness. viscosity of t2jam (10276.0 cp) was significantly higher (p<0.05) than t1 jam (8739.20 cp) and control jam t0 (5072.8 cp). viscosity of t1 jam was significantly higher (p<0.05) than control jam t0. increased viscosity is important in fruit jams as it gives better mouth feel, a greater sense of fruitiness and sweetness in final product (javanmard and endan, 2010). table 5. texture analysis of jam. sem – standard error of mean. abc means on the same line without a common letter are significantly different at p < 0.05. (all samples at the same time). parameters control jam (t0) mean±sem t1mean±sem t2mean±sem firmness (n) 0th day 147.83 ±1.51a 209.92 ±0.75 b 283.83 ±0.87 c 30th day 153.3 ±0.49 a 210.60 ±0.91 b 285.50±1.33 c 60th day 155.5 ±0.56 a 221.1 ±0.47 b 291.5 ±0.67 c consistency (ns) 0th day 1672.2 ±6.01 a 2021.8±3.96 b 2549.0±4.42 c 30th day 1679.0 ±2.85 a 2033.16 ±2.41 b 2561.16 ±2.77 c 60th day 1683.0 ±4.4 a 2048.66 ±2.02 b 2579.33 ±2.11 c adhesiveness (ns) 0th day -755.83±1.30 a -1543.3±2.72 b -1662.8±6.18 c 30th day -764.66±0.88 a -1558.66±2.62 b -1671.33±1.23 c 60th day -782.16±1.85 a -1575.16±1.94 b -1684.33±1.54 c viscosity (cp) 0th day 5072.8±7.74 a 8739.20±9.60 b 10276.0±1.30 c 30th day 5119.33±3.45 a 8763.33±2.24 b 10313.66±1.87 c 60th day 5152.16±2.12 a 8787.16±1.68 b 10344.16±1.74 c cohesiveness (n) 0th day 264.66±1.14 a 371.83±1.10 b 486.33±4.88 c 30th day 272.00±0.96 a 381.50±0.71 b 504.83±1.13 c 60th day 282.33±0.84 a 392.33±1.25 b 519.33±1.11 c ital. j. food sci., vol 29, 2017 181 li and guo (2006) showed that incorporation of polymerized whey protein (prepared by thermal denaturation method) in yoghurt significantly improved viscosity. consistency of t2jam (2549.0 ns) was significantly higher (p<0.05) than t1 jam (2021.8 ns) and control jam t0 (1672.2 ns). consistency of t1 jam was significantly higher (p<0.05) than control jam t0.whey proteins were added to yoghurts to increase total solid content of milk in order to provide better consistency, texture and creaminess to the product (martinez et al., 2002). cohesiveness of t2 jam (486.33 n) was significantly higher (p<0.05) than t1 jam (371.83 n) and control jam t0 (264.66 n). cohesiveness of t1 jam was significantly higher (p<0.05) than control jam t0.raju et al. (2007) substituted refined wheat flour with whey protein up to 30% in biscuit manufacture and observed increase in cohesiveness of biscuit dough which reduced the fracture stress of biscuit. addition of whey protein increased hardness, adhesiveness, gumminess and chewiness of frankfurters prepared with 5% and 12% fat (hughes et al., 1998). the results of textural parameters of jam from both sensory evaluation by sensory panel and instrument (texture analyzer) were in accordance to each other. jam with 10% whey substitution (t2) showed significantly higher value than other jams (t0 and t1) in textural parameters both through textural analyzer and sensory panel. 3.4. organoleptic evaluation sensory scores of jam samples (table 6) was performed based on 9 point hedonic scale to evaluate colour and appearance, taste, flavour, texture and overall acceptability with 9 = like extremely, 5= neither like nor dislike and 1= dislike extremely. appearance and texture revealed that there was no crystallization of sugars in all jams. presence of off flavor was not noticed in jam samples. table 6. sensory eevaluation of jam. sem – standard error of mean abc means on the same line without a common letter are significantly different at p < 0.05 (all samples at the same time). parameters control jam(t0) mean±sem t1 mean±sem t2 mean±sem color and appearance 0th day 8.5± 0.03 a 7.12±0.21b 7.75± 0.11ab 30th day 8.4± 0.16a 7.11± 0.08b 7.64± 0.17ab 60th day 8.3± 0.03a 7.08± 0.27b 7.52± 0.11ab taste 0th day 8.3± 0.08a 7.37± 0.03b 7.37± 0.15b 30th day 8.1± 0.07a 7.14± 0.24b 7.22± 0.19b 60th day 8.0± 0.23a 7.18± 0.31b 7.18± 0.43b flavor 0th day 8.93± 0.05b 7.06± 0.26a 7.93± 0.38ab 30th day 8.89± 0.17b 7.18± 0.27a 7.85± 0.19ab 60th day 8.74±0.05b 7.29 ±0.07a 7.71±0.22ab texture 0th day 7.32±0.06a 8.06±0.28ab 8.75±0.03b 30th day 7.44±0.08a 8.08±0.34ab 8.55±0.11b 60th day 7.62±0.51a 8.18±0.44ab 8.60±0.18b overall acceptability 0th day 8.37±0.08a 7.87±0.01b 7.75± 0.09b 30th day 8.25±0.31a 7.78±0.13b 7.65± 0.21b 60th day 8.19±0.07a 7.71±0.04b 7.64± 0.06b ital. j. food sci., vol 29, 2017 182 the flavour, colour and appearance of control jam were significantly higher (p < 0.05) from that of jam t1. the taste and overall acceptability of control jam was significantly higher (p < 0.05) from that of jam t1 and jam t2. texture of jam t2 was significantly higher (p < 0.05) than that of control jam. all the parameters of sensory evaluation were within liked limit (sensory score between 7.18 and 8.75) for the jam when substituted with whey both at 5% (t1) and 10% (t2). thus sensory property of papaya jam is not affected by the substitution of whey up to 10% of papaya pulp. 4. conclusions papaya jam with 10% whey substitution can be effectively assigned as best formulation as it has better textural property which is necessary for jam product, with normal range of ph, acidity, total solids and ash, with better water activity, reducing sugar and significant amount of protein content. the crude protein level in control jam (t0), t1, and t2 jam are 0.92%, 3.15% and 4.23% respectively. further, it was substantially proved that whey can be efficiently substituted in jam with advantage of improving its nutritive, physico chemical and textural properties without any detrimental effect on sensory properties and also avoiding much denaturation of whey protein. usually, fruit pulps incorporated in jam had less protein level, so whey can be used as economic protein source to improve its nutritive value. this experiment also paves a way for effective utilization of whey, which is produced abundantly as by product in dairy industry. acknowledgements we would like to thank department of dairy science and dairy plant, college of veterinary and animal sciences, thrissur, kerala, for kind support and facilities to conduct research. further, thanks are due to indian council of agricultural research (icar), new delhi, india for financial assistance to research. references 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thutrangvu1981@yahoo.com abstract the optimization of process parameters for the production of aminoreductone (ar), a bioactive product formed in the initial stage of maillard reaction was investigated using response surface methodology (rsm) and box-behnken design technique. the optimum process conditions were determined by analyzing the response surface of three-dimensional surface plot and solving the regression model equation with the design expert software. the optimum conditions include: heating time of 15 min, temperature of 112.85°c, ph of 8.33 and buffer concentration of 0.53 which were used to obtain the maximum ar yield (76.6 mm) in the model solution of lactose (0.3 m) and butylamine (0.3 m). 2 ital. j. food sci., vol. 27 2015 introduction maillard reaction products are responsible for the change of color, taste, flavor and the nutritional value of food products (ramonaityte et al., 2009). therefore, the maillard reaction is the most important influence on food quality and acceptance (jaeger et al., 2010). however, the evaluation of the extent of the maillard reaction is difficult with many parallel and consecutive reactions (morales and jimenez-perez, 1998). several studies have reported detection methods for estimating the extent of maillard reaction by the detection of an intermediate product as hydroxymethylfurfural (shimamura et al., 2004) or the final polymerized products such as melanoidins (boekel, 1998). in the early stage of the maillard reaction, aminoreductone (ar) is formed (boekel, 1998). therefore, the detection of ar would be more effective in the indication of the maillard reaction and heat treatment of food than other methods (shimamura et al., 2004). thus, the role and characteristics of ar is, of great interest to food scientists. a number of functionalities of ar such as an antioxidative activity, have been found (pischetsrieder et al., 1998), a protective ability on photo-degradation of riboflavin in milk (trang et al., 2008), and antimicrobial activities against pathogenic bacteria: helicobacter pylori (trang et al., 2009), pseudomonas aeruginosa (pa), multi-drug resistant pseudomonas aeruginosa (mdrp), escherichia coli (ec), methicillinsusceptible staphylococcus aureus (mssa) and methicillin-resistant s. aureus (mrsa) (trang et al., 2011, 2013). in the field of food technology, food scientists and producers always consider many factors that can contribute to a good and healthy product. as ar presents the potential properties in medical practices (trang et al., 2009, 2011) and contributes to food quality (katsuno et al., 2013), it can be used as a functional additive ingredient in food to improve the quality of food. as a product of the maillard reaction, the formation of ar depends on multiple parameters such as, the heating time, the heating temperature, the ph and the buffer concentration (boekel, 1998). conventionally, the formation of ar might be optimized using a single factor optimization to evaluate the optimum producing condition, which is relatively simple and does not require statistical analysis. however, the single variable optimization strategy is not only tedious, but can also lead to misinterpretation of results, especially since the interaction between different factors are overlooked (mannan et al., 2007). the response surface methodology (rsm) as a combination of mathematical and statistical techniques was employed to overcome this major problem in the optimization study (li et al., 2008). in this method, statistically designed experiments used a small set of carefully planned experiments, to build models, evaluate the effects of factors and find the optimum conditions for desirable responses (li et al., 2002). it can simultaneously study several variables with a small number of observations, less time consumed and cost effects (deepak et al., 2008). for this reason, the purpose of this study is to optimize the technological conditions favoring the production of ar in a model system using a statistical approach: response surface methodology. materials and methods reagents x t t ( 2 , 3 b i s [ 2 m e t h o x y 4 n i t r o 5-sulfophenyl]-2h-tetrezolium-5-carboxanilide) was purchased from sigma chemical co. (st louis, mo, usa). lactose monohydrate was purchased from nacalai tesque, inc. (kyoto, japan). n-butylamine was obtained from wako pure chemical industries (osaka, japan). all other reagents were of the highest commercial grade available. milli-q water was used in all procedures. model solutions the solutions containing lactose and butylamine were used as a model system of by ar production in the maillard reaction (model solution). the solutions were prepared according to the previous report (shimamura et al., 2004; trang et al., 2011). lactose monohydrate (0.3 m) and butylamine (0.3 m) were dissolved in phosphate buffer. one milliliter of the model sotable 1 factors in actual and coded levels for the box-behnken design. no factors symbols coded and actual level -1 0 +1 1 heating temperature (°c) a 90 110 130 2 heating time (min) b 5 15 25 3 ph c 7 8 9 4 buffer concentration (m) d 0.3 0.5 0.7 ital. j. food sci., vol. 27 2015 3 lutions was heated under the indicated condition. immediately after heating, the heated solutions were cooled in ice and used for the determination of ar formation. determination of aminoreductone formation the formations of ar in the heated model solutions were determined using a xtt assay, performed in a 96-well microtiter plate according to the method described by shimamura et al. (2011). each well contained 60 μl of 0.5 mm xtt prepared with 0.2 m potassium phosphate buffer (ph 7.0) saturated with menadione. a sample (40 μl) was added to the well and after mixing in a microplate shaker at a speed of 500 rpm for 15 s, the difference in the absorbance between 492 nm and 600 nm was measured using a microplate reader (mpr a4i, tosoh, tokyo, japan) as the absorbance at 0 min. after 20 min at room temperature, the difference in absorbance was again measured and the increase in the absorbance was recorded as the ability of a sample to reduce xtt (xtt reducibility). the concentration of ar was estimated by the following equation: y = 0.606 x + 0.046, where x and y represent the concentration of ar (mm) and the reducibility of xtt, respectively (trang et al., 2008). experiment design and procedure box-behnken design with three levels (low, medium, and high, coded as -1, 0, and +1) is more efficient and easier to arrange and to interpret when compared with the others, such as the plackett-burman design, the central composite design and the graeco-latin square design (francis et al., 2003). this statistical technique was therefore used in this study. a total of 27 runs was used to optimize the producing parameters namely: ph, buffer concentration, heating temperature and heating time (boekel, 1998). factors in actual and coded levels considered in this study are listed in table 1. the experiments were designed according to the box-behnken design using 24 axial points and three central points as shown in table 2. individual experiments were carried out in random order. the average of two replicated values of each run was taken as dependent variables or responses. design-expert 7.1 (stat-easse, inc., minneapolis, mn, usa) was used for the experimental design, data analysis, quadratic model building, graph (three-dimensional response surface and contour) plotting and to optimize by desirability methodology. table 2 experimental design and results of the box-behnken design. run factor 1 factor 2 factor 3 factor 4 ar concentration a (°c) b (min) c d (m) (mm) 1 90 5 8 0.5 0.032 2 130 5 8 0.5 34.855 3 90 25 8 0.5 26.244 4 130 25 8 0.5 15.270 5 110 15 7 0.3 45.056 6 110 15 9 0.3 64.033 7 110 15 7 0.7 49.940 8 110 15 9 0.7 74.594 9 90 15 8 0.3 8.802 10 130 15 8 0.3 35.402 11 90 15 8 0.7 19.148 12 130 15 8 0.7 36.247 13 110 5 7 0.5 10.947 14 110 25 7 0.5 33.1 15 110 5 9 0.5 25.3 16 110 25 9 0.5 43.9 17 90 15 7 0.5 10.811 18 130 15 7 0.5 34.91 19 90 15 9 0.5 24.954 20 130 15 9 0.5 46.294 21 110 5 8 0.3 20.89 22 110 25 8 0.3 44.396 23 110 5 8 0.7 35.508 24 110 25 8 0.7 44.478 25a 110 15 8 0.5 76.657 26a 110 15 8 0.5 76.657 27a 110 15 8 0.5 69.672 a center points 4 ital. j. food sci., vol. 27 2015 results and discussions effects of individual factors on aminoreductone formation. variables and factor levels of the experimental design to optimize the variables of ar formation, the key factor affecting the ar formation as well as the range of experimental values must be determined. the mechanism of ar formation in the maillard reaction in the model solution of lactose and butylamine has been investigated (trang et al., 2011). based on the results of those experiments, the solution of lactose and butylamine at 1:1 in concentration ratio (0.3:0.3 (m)) was the best model system for the formation of ar and was used in our previous studies (trang et al., 2011). thus, we used it in the production of ar in this study. the effects of individual factors in the formation of ar while keeping other variables constant were shown in fig. 1. in the model solution consisting lactose and butylamine, the extent of the ar formation strongly relied on the heating temperature and heating time (fig. 1a and 1b). this phenomenon was also similar to our previous studies (shimamura et al., 2004; trang et al., 2011). as soon as the maximum amount of ar was obtained, xtt value decreased. these might be referred to as the progress of the advanced reactions of ar that commonly takes place in the complicated sequences of the maillard reaction during heating (trang et al., 2011) or by the competition of isomerisation⁄degradation reaction to lactose at the high heating temperature (more than 100°c) leading to the reduction of lactose for the maillard reaction (boekel, 1998). the maximum value of ar obtained in fig. 1a and 1b might also change depending on the change of heating time and heating temperature, respectively. thus, the heating time (with a range of 5-25 min) and heating temperature (with a range of 90-130°c) were chosen as the main factors and their levels for the experimental design on the response surface methodology. boekel (1998) suggested that changes in ph can have an effect on reaction rates (boekel, 1998). at the same, heating time and temperature, reactivity of sugar and amino group are also influenced by ph (martins et al., 2001). similar results were also found in this study (fig. 1c). in the maillard reaction of lactose and butylamine fig. 1 effects of individual factors in the formation of aminoreductone. the effect of heating temperature (fig. 1a), heating time (fig. 1b), ph (fing. 1c), buffer concentration (fig. 1d) on the ar formation while other factors were controlled (heating time of 15 min; heating temperature of 110°c; buffer concentration of 0.2 m and ph of 7). ital. j. food sci., vol. 27 2015 5 at 110°c, 15 min of heating time, buffer concentration of 0.2m, the formation of ar rapidly reached maximum values from ph 5 to 8 and did not change much at higher ph values. the ph value that was higher, was used. the more complicated equipment design and handling for ar producing was required. thus, the range of ph value from 7 to 9 for ar production was chosen for experimental design. limited data exist on the effects these buffers have on the maillard reaction and the formation of ar. besides, buffer agents were added in diverse foods to control the ph of the system (bell, 1997). to find out the suitable conditions and establish the good model solution for the production of ar, the effect of phosphate buffer concentration in the formation of ar was also investigated. as shown in fig. 1d, the rates of ar formation increased with the increasing phosphate buffer concentration from 0.1 to 0.5 and slightly decreased with higher concentrations. similar observations of the increase in the maillard reaction rate with the increasing buffer concentration was also presented in the model system of glycine and glucose (bell, 1997). the results obtained in our study indicated that phosphate anion should be used as a catalytic compound for the production of ar. the range of buffer concentration from 0.3 to 0.7 which contains an optimized buffer concentration for ar production was chosen for experimental design. the parameters of optimization for ar production were investigated using a box-behnken design under rsm. parameters such as, temperature of 110oc, time of 15 min, ph of 8 and buffer concentration of 0.5 were chosen as center points from the above pre-screening on the effect of individual factors for the formation of ar. evaluation of aminoreductone formation the design matrix of the factors is shown in table 2, along with the experimental response values. using the software design expert, the results of the experiment of ar formation were used to calculate the coefficients of the quadratic polynomial equations, which were used to predict the formation of ar. the statistical model was checked by f-test, and the analysis of variance (anova) for the response surface quadratic model was summarized (table 3). as shown in table 3, the model’s f-value of 10.27 and the p value of 0.0001 (< α = 0.05) implied that the model was highly significant in which a, b, c, ab, a2, b2, c2, d2 (p < 0.05) are significant model terms (table 3). because d2 had a significant effect, the corresponding main effect of d is included in the regression model. after excluding these insignificant effects from the model and rerunning the software design expert, the model for ar production might be expressed by: y = + 74.33 + 9.42*a + 6.65b + 7.86*c + 3.44*d 11.45*a*b33.26*a2 28.01*b2 11.90*c2 10.07*d2 where y (yield) is the yield of ar (mm); a, b, c, d is the coded values of the heating temperature, the heating time, the ph and the buffer concentration, respectively; r2 = 0.9155; adjusted-r2= 0.8708. the goodness of the model can be checked by the determination coefficient r2 and the adjusted-r2. the determination coefficient r2 of table 3 results of the regression analysis of the box-behnken design for aminoreductone production. factor variable regression coefficient f-value p-value (probability) > f model 10.27 0.0001 b o + 74.33 linear a b 1 + 9.42 14.01 0.0028* b b 2 + 6.65 7.00 0.0214* c b 3 + 7.86 9.76 0.0088* d b 4 + 3.44 1.87 0.1960 interaction ab b 12 11.45 6.90 0.0221* ac b 13 0.69 0.025 0.8769 ad b 14 2.38 0.30 0.5957 bc b 23 0.89 0.042 0.8419 bd b 24 3.63 0.70 0.4206 cd b 34 + 1.42 0.11 0.7503 quadric a2 b 11 33.26 77.65 < 0.0001* b2 b 22 28.01 55.09 < 0.0001* c2 b 33 11.90 9.94 0.0083* d2 b 44 10.07 7.11 0.0205* *, insignificant model terms. 6 ital. j. food sci., vol. 27 2015 0.9155 indicated that the model could explain 91.55% of the variance (wang and lu, 2005). the value of adjusted-r2 closed to r2 and 1, showed the good correlation between the experimental and predicted values. thus, this model can be used to predict the formation of ar in the maillard reaction. analysis of response surfaces three-dimensional response surfaces were plotted on the basis of the model equation by the design expert program to investigate the interaction among the variables and to determine the optimum condition of each factor for maximum ar fig. 2 -response surface plot for aminoreductone production. the interaction between (a) heating time and heating temperature, (b) ph and heating temperature, (c) buffer concentration and heating temperature, (d) ph and heating time, (e) buffer concentration and heating time, (f) buffer concentration and ph. ital. j. food sci., vol. 27 2015 7 fig. 3 perturbation graph showing the effect of each independent factors on aminoreductone production while keeping other factors at their respective midpoint levels. (a) heating temperature, (b) heating time, (c) ph, (d) buffer concentration. production in the model solution of lactose and butylamine (fig. 1). by keeping other variables at their center point values, three dimensional plots of two factors versus the ar formation were drawn (fig. 2). perturbation graph showed the effect of each independent factors on ar production while keeping other factors at their respective midpoint levels (fig. 3). from the response surface (fig. 2) and perturbation plot (fig. 3), it is obvious that heating temperature and time had a significant effect on ar production compared with other variables. although, ph was reported to influence the maillard reaction (martins et al., 2001), the results of this study indicated an unimportant effect of ph on the formation of ar. the perturbation graph clearly showed that the two variables (buffer concentration and ph) did play any significant role in the ar production. optimization of conditions for aminoreductone formation based on the analysis of the response surface of the regression equation, the optimum process parameters were found to be 112.85oc for heating temperature 15 min for heating time with ph 8.33 and buffer concentration 0.53 m resulting in the predicted maximum ar formation which was 76.6 mm. validation of the models the trail experiments were conducted under optimized process conditions with temperature of 112.8oc, heating time of 15 min, ph of 8.3 and buffer concentration of 0.5 m. the results of ar formation were founded to be 75.76 ± 0.02 mm, which was very close to the predicted ar formation obtained from the regression equation (76.6 mm). thus, the model could be used to predict the ar content formed in the food during heating; and to find the suitable heating, condition (temperature and time) that favored the formation of aminoreductone in the specific food system which the ph and food components were clarified. the chemistry underlying the maillard reaction is very complex. it encompasses not only one reaction pathway, but a whole network of various reactions with thousand products (martins et al., 2001). by applying the rsm, the optimum process parameters for ar production were found. these were both helpful for studying further the application of ar in the health and medical fields, as well as providing provide useful information for identifying the technological conditions that favors the formation of ar as a functional ingredient in food. 8 ital. j. food sci., vol. 27 2015 conclusions in this study, rsm, box-behnken design was used to model and establish a regression equation between the response (ar formation) and four statistically significant factors: the heating time, the heating temperature, the ph, the buffer concentration. in four variables, the two factors of heating time and heating temperature showed the effect on the ar formation. finally, the optimal solutions were sought on the basis of the influence of the four parameters in the formation of ar in the maillard reaction. predicted values obtained using the model equations were in agreement with the observed values. heating time of 15 min, heating temperature of 112.85oc, ph of 8.33 and buffer concentration of 0.53 has been determined as optimum levels of the process parameters to achieve the maximum amount of ar formed in the maillard reaction of lactose and butylamine. these optimum conditions were used to evaluate the trail experiment and the maximum yield of ar formation was recorded as 75.76 mm. the results indicated that optimization using rsm can be useful to control and predict the production of ar in the maillard reaction. the results obtained from this study would be applied in ar production for therapeutic application as an efficient source of antimicrobial agents, an antioxidant compound in functional food. acknowldgments this research was funded by the vietnam national foundation for science and technology development (nafosted, 106.99-2011.22.). references bell l.n. 1997. maillard reaction as influenced by buffer type and concentration. food chem. 59: 143. boekel, m.a.j.s.v. 1998. effect of heating on maillard reactions in milk. food chem. 62: 403. deepak v., kalishwaralal k., ramkumarpandian s., babu v.s., senthilkumar, s.k. and sangiliyandi, g. 2008. optimization of media composition for nattokinase production by bacillus subtilis using response surface methodology. biores. technol., 99: 8170. design-expert. 2008. design-expert 7.1 user’s guide, release 7.1, minneapolis, mn. francis f., sabu, a. and nampoothiri, k.m. 2003. use of response surface methodology for optimizing process parameters for the production of a-amylase by aspergillus oryzae. biochem. engineer. j. 15: 107. katsuno s., shimamura t., kashiwagi t., izawa n. and ukeda h. 2013. effects of dissolved oxygen on the maillard reaction during heat treatment of milk. int.dairy j. 33: 34. li c., bai j. and cai z. 2002. optimization of a cultural medium for bacteriocin production by lactococcus lactis using response surface methodology. j. biotechnol. 93: 27. li x., xu t., ma x., guo k., kai l., zhao y., jia x. and mad y. 2008. optimization of culture conditions for production of cis-epoxysuccinic acid hydrolase using response surface methodology. biores. technol. 99 (13): 5391. mannan s., fakhru’l-razi’ a. and alam m. 2007. optimization of process parameters for the bioconversion of activated sludge by penicillium corylophilum using response surface methodology. j. environmental sci. 19: 23. martins s.i.s., jongen w.m.f. and boekel m.a.j.s.v. 2001. a review of maillard reaction in food and implications on kinetic modeling. trends food sci. technol. 11: 364. morales f. j. and jimenez-perez s. (1998) study of hydroxymethylfurfural formation from acid degradation of the amadori product in milk-resembling systems. j. agric. food chem. 46: 3885-3890. pischetsrieder m., schoetter c. and severin t. (1998). formation of an aminoreductone during the maillard reaction of lactose with nα-acetyllysine or proteins. j. agric. food chem. 46: 928-931. ramonaityte d. t., kersiene m., adams a. and tehrani k. a. (2009) the interaction of metal ions with maillard reaction products in a lactose-glycine model system. food res. int. 42: 331-336. jaeger h., janositz a. and knorr d. (2010) the maillard reaction and its control during food processing. the potential of emerging technologies. pathologie biologie, 58: 207-213. shimamura t., ukeda h. and sawamura m. 2004. relationship between the xtt reducibility and aminoreductone formed during the maillard reaction of lactose: the detection of aminoreductone by hplc. food sci. technol. res. 10: 6. shimamura t., kurogi y., katsuno s., kashiwagi t. and ukeda h. 2011. demonstration of the presence of aminoreductone formed during the maillard reaction in milk. food chem. 129: 1088. trang v.t., kurogi y., katsuno s., shimamura t. and ukeda h. 2008. protective effect of aminoreductone on photo-degradation of riboflavin. int. dairy j. 18: 344. trang, v. t., takeuchi, h., kudo, h., aoki, a., katsuno, s., shimamura, t., sugiura, t. and ukeda, h. (2009). antimicrobial activity of aminoreductone against helicobacter pylori. j. agric. food chem., 57, 11343-11348. trang v.t., shimamura t., kashiwagi t., ukeda h. and katsuno s. (2011). elucidation of mechanism of aminoreductone formation in the maillard reaction of lactose. int. j. dairy technol. 64 (2): 188. trang v.t., son v. h., thanh l. x., sarter s., shimamura t., ukeda h. and takeuchi h. (2013). functional properties of maillard reaction products in food: antimicrobial activity of amnireductone against pathogenic bacteria. food sci. technol. res. 19 (5): 833. wang y.x. and lu z.x. 2005. optimization of processing parameters for the mycelial growth and extracellular polysaccharide production by boletus spp. accc 50328. process biochem. 40: 104 paper received july 23, 2014 accepted september 19, 2014 survey ital. j. food sci., vol. 27 2015 1 keywords: cow milk, derogations, somatic cell count, total bacterial count end of the derogations to regulation (ec) 853/2004 for cow’s milk in italy g. bolzoni*, a. marcolini and e. buffoli national reference center for bovine milk quality izsler, via bianchi 9, 25100 brescia, italy *corresponding author: tel. 0039 030 2290541, email: giuseppe.bolzoni@izsler.it abstract derogations for somatic cell and total bacterial count limits had allowed non-compliant milk to be used for cheesemaking in italy. commercial and health considerations prompted a decision to implement a program to gradually repeal the derogations. in this study, we report the statistical evaluation of the situation in 2007-2008, the outcomes of the program to close the derogation and observations of its effects during its implementation from 2010-2013 in the lombardy region. the introduction of a progressive decrease of the limit allowed regulators to minimize the negative impact on production levels by focusing on the management of the most non-compliant farms first. 2 ital. j. food sci., vol. 27 2015 introduction after the adoption of european regulations for food safety (reg. ec 178/2002, 852-853854/2004), italian farmers were still able to sell cow’s milk that was non-compliant in somatic cell count (scc) and total bacterial count (tbc), indicators of presence of udder pathogens and insufficient hygiene during production and storage, respectively, because of the derogation of article 10 of the regulation (ec) 853/2004. specifically the derogation allowed for cow’s milk with geometric means exceeding the legal limits (400,000 cells/ml for scc and 100,000 cfu/ml for tbc) to be used for the production of cheese with ripening periods of at least 60 days. this derogation was based on the knowledge that potential risks linked to high scc and tbc can be significantly reduced or even eliminated during the production processes and ripening periods (annex iv of the reg. ec 854/2004). in fact, many stages of processing have antimicrobial effects including: the cooking of curd, the acidification of curd, the salting of cheese and the reduction of free water. however, over the years unfavorable opinions about the use of non-compliant milk have increased in italy. aged dairy products, like grana padano and parmigiano reggiano cheese, represent the uniqueness and tradition of italian raw milk cheeses. given that they are some of the most popular cheeses on the international market, is the use of the “worst” milk appropriate for the “best” cheeses of italy? it became clear that the presence of the derogation did not promote improvement in the quality of italian milk. furthermore, according to the principles of the community regulations, a derogation has to be considered “temporary” and contingent on specific issues. a derogation, if it affects food safety, must always provide a time limit or an exit strategy that will lead to conformity with the other nations. for these reasons italy began a gradual process to repeal the derogation. the project was developed in 2008, after which it was communicated to the european commission (notification number 134/2010) and it was formalized by the “agreement between the government, regions and autonomous provinces of 09 september 2010”. the goals of this study were to conduct a preliminary evaluation of the problem based on data from 2005 to 2008, to create a plan to phase out the derogation and to evaluate the results of its application over 2 years, from january 2011 to june 2013, in the lombardy region (an area responsible for more than 40% of the national milk production). materials and methods the data come from the analysis of the milk quality payment system instituted in the lombardy region. the system requires at least 2 samples per month from each farm. the samples are taken from farms by trained and qualified dairy industry operators. for statistical evaluation and calculation of the geometric mean we selected the farms with continuous production and active participation in the milk quality payment system during the 2-year study period (roughly 4600 farms out of 6,000 total active farms in the region). the majority of farms can be characterized by rearing holstein friesian (85%) or brown swiss (15%) cows in loose housing with cubicles, with milking parlor, fed with mixed ration of corn silage, hay and concentrate. on these farms the average herd size is 70 cows and the average milk yield is 9,400 kg per cow per year. tbc was determined with bactoscan fc and the scc was obtained using fossomatic 5000 (foss, dk). for descriptive statistics (frequency distribution of farms’ geometric means of scc and tbc) the free “r” software environment was used. results and discussion analysis of compliance the distribution of the farms’ yearly scc and tbc geometric means during the year 2008 are represented in figures 1 and 2, respectively (5,200 farms). the “yearly” geometric means were calculated for each farm from 24 or more samples per year. from the figures it is evident that the scc situation was more critical than that of tbc with respect to regulation 853/2004 limits (bertocchi et al., 2012), (bolzoni et al., 2007). in table 1 an evaluation of the farms’ scc rolling geometric means from 2005 to 2007 is presented. the “rolling” geometric means were calculated over periods of 2 or 3 months for tbc and scc, respectively. the data show that 44% of the farms were consistently under the scc limit, while 29% of farms exceeded it one or more times but returned under the limit within the 3-months observation period; the remaining 27% of the farms were still non-compliant after the observation period. further analysis of the last group of non-compliant farms (figure 3) revealed that only a small fraction of these farms returned to compliance shortly after the 3 months of observation. most of the farms spent a long time in non-compliance and even some farms never became compliant. these were the farms that were able to avoid compliance by commercializing their non-compliant milk in a geographic area where most of the milk is used for aged cheese. the end of the derogation: potential effects according to the abovementioned observations, it could be hypothesized that closure of the derogation would cause economic problems ital. j. food sci., vol. 27 2015 3 table 1 somatic cell count compliance among 4,595 farms from 2005 to 2007. rolling geometric mean (cells/ml) farms (n) farms (%) always < 400,000 2,032 44% > 400,000 with recuperation < 90 days 1,313 29% > 400,000 without recuperation < 90 days 1,250 27% fig. 1. fig. 2. fig. 3. for about 30% of the farms based on their scc values and 4% of the farms based on their tbc values (data not shown). to explore these effects, we performed a statistical simulation on the 2008 data to quantify the effect of a gradual repeal of the derogation and its effects on farm production. the results of the simulation for scc are presented in table 2. program to repeal the derogation a regional program was developed with a series of decreasing temporary limits. given that controlling scc levels is known to require both mediumand longterm actions, it was expected that some of the farmers would become proactive with control measures and improvements in order to reach compliance in time for the more restrictive future limits. the plan was approved by the ministry of health and then it was expanded into a national program with the “agreement between the government, regions 4 ital. j. food sci., vol. 27 2015 table 3 temporary limits for compliance during the program to close the derogation (geometric means calculated over periods of 2 or 3 months, respectively, for tbc and scc). period total bacterial count somatic cell count (rolling gm calculated over 2 months) (rolling gm calculated over 3 months) january 2011 june 2011 < 200,000 < 700,000 july 2011 june 2012 < 100,000 < 600,000 july 2012 june 2013 no derogation < 500,000 from july 2013 no derogation < 400,000 abbreviation: gm, geometric mean. and autonomous provinces”. it banned the use of non-compliant milk for human consumption and set temporary limits for tbc and scc as shown in table 3. currently milk with rolling geometric means > 400,000 for scc or > 100,000 for tbc continues to be used in the production of cheeses with over 60 days of ripening. table 2 geometric means of the compliance of 4,669 farms at different somatic cell count limits in 2008. somatic cell limit % of farms always % of farms over the limit % of farms over the limit (cells/ml) under limit with recuperation in 90 days without recuperation in 90 days 700,000 89 8.4 2.7 600,000 83 12.1 4.6 500,000 73 19.6 7.7 400,000 50 32.1 18 fig. 4. program application and results ten-year trends in scc and tbc levels in the lombardy region are shown in figures 4 and 5, respectively. from these graphs it is possible to deduce a preliminary and general trend of decline in the two parameters. in particular, in figital. j. food sci., vol. 27 2015 5 ure 5, the low percentage of samples over 100,000 ufc/ml (colored bars) and the low and decreasing value of the yearly regional mean (blue line), indirectly confirm the very low number of noncompliant farms in tbc during the last year. the number of samples used to generate the yearly fig. 5. means varies from a high of 165,000 in 2003 to a low of about 100,000 in 2013 which reflects the significant reduction in the number active farms in the region during this period. fig. 6 presents a specific assessment of the effect of the progressive application of the profig. 6. 6 ital. j. food sci., vol. 27 2015 gram where the blue line indicates the distribution of the farms’ geometric means in the first year (2011) while the blue bars indicate the situation in second year (2012). the difference between the two years is particularly evident in some areas (e.g. 200,000-250,000 scc). it is interesting to note that there is a decrease in the percentage of farms presenting with 400,000500,000 cells/ml in 2012 even though the limit was still set at 700,000-600,000 cells/ml. this again suggests that some farmers took preventive actions early with an eye to the more restrictive limits of 2013. figure 7 presents the same comparison for the tbc. in this case, it is not possible to appreciate graphically the differences between the two years because of the minimal variations of the data across the board. one further analysis was performed on scc to compare the first semester of 2010 (before the start of the program) and the first semester of 2013 and the data are presented in figure 8; figure 9 shows the same comparison between the first semesters of 2012 and 2013. it is evident that the percentage of geometric means with a lower scc range increased over time while the higher scc values decreased. for example, in figure 8 the percentages of farms in the ranges of 300,000-350,000 and 350,000-400,000 cells/ ml both decreased while the percentage of farms in the ranges of 100,000-150,000 and 150,000200,000 cells/ml both increased almost equally. even though the data come from thousands of different farms, the combination of these representations enabled us to notice a shift to the right in the distribution and through it the posfig. 7. itive effects of the introduction of this progressive program. its effects will be fully appreciated, of course, only in 2014 when the program will have been finished for over 6 months. however we still hypothesize that the impact on compliant productivity will be minimal due to the progressive shift of the majority of the farms to adopting long-term practices that should ensure their continued conformity within scc limits (kelly et al., 2009; norman et al., 1995; shukken et al., 2003). the number of farms with tbc over the limit has already been negligible since 2011, likely because corrective actions against tbc can be effective in a very short time (kelly et al., 2009). conclusions the decision to repeal the derogations for scc and tbc in raw milk was made for several reasons but particularly because of the need to stimulate improvements in the quality of milk and traditional dairy products. statistical evaluation of the data from the previous years (2005-2007) suggested that a sudden closure of the derogations, especially for scc, would create difficulties for farmers with downstream repercussions on the dairy industry as well as the health authority (regional veterinary service in italy). the introduction of a progressive decrease of the limits allowed regulators to minimize these consequences and focus on the most non-compliant producers. at the same time, it allowed farmers to make improvements and preventive measures during the ital. j. food sci., vol. 27 2015 7 24-month program, without excessive conflict or serious effects on farm productivity. the progressive reduction of the scc limit also allowed the regional veterinary service to split the management of the non-compliant farms, giving priority to solving the most serious and fig. 8. significant problems first (starting from the first and highest limit of 700,000 cells/ml). the issue of non-compliance in tbc has been nearly resolved; since 2012 the cases of tbc non-compliance appear only occasionally and are often quickly resolved. fig. 9. 8 ital. j. food sci., vol. 27 2015 references agreement between the government, regions and autonomous provinces of trento and bolzano on the transitional use of raw bovine milk not meeting the criteria laid down in annex iii, sec. ix, of reg. (ec) n. 853/2004 as regards plate count and somatic cell count for the manufacture of cheeses with an ageing or ripening period of at least sixty days – gu general series n. 250 of 10/25/2010 the r project for statistical computing. available at: http:// www.r-project.org/ bertocchi l., zanardi g., bolzoni g. daga s. andvismara f. 2012. relation between dairy cattle welfare and bulk milk somatic cell count. presented at xxvii world buiatrics congress, lisbon, portugal, june 3-8 bolzoni g., varisco g., marcolini a., benicchio s. and ghilardi c. 2007. pagamento del latte in base alla qualità in lombardia: ci sono le premesse per un nuovo sviluppo. il mondo del latte 61 (5): 37-43 kelly p.t., o’sullivan k., berry d.p., more s.j., meaney w.j., o’callaghan e.j. and o’brien b. 2009. farm management factors associated with bulk tank somatic cell count in irish dairy herds. ir vet j. 62(suppl 4): s45-s51 paper received november 19, 2013 accepted may 30, 2014 kelly p.t., o’sullivan k., berry d.p., more s.j., meaney w.j., o’callaghan e.j. and o’brien b. 2009. farm management factors associated with bulk tank total bacterial count in irish dairy herds during 2006/2007. ir vet j. 62(1):36-42. norman h.d., miller h.d., wright j.r. and wiggans g.r. 2000. herd and state means for somatic cell count from dairy herd improvement. jds. 83 (12): 2782-2788 regulation (ec) n. 178/2002 28 january 2002 laying down the general principles and requirements of food law, establishing the european food safety authority and laying down procedures in matters of food safety regulation (ec) n. 852/2004 29 april 2004 on the hygiene of foodstuffs regulation (ec) n. 853/2004 29 april 2004 laying down specific hygiene rules for on the hygiene of foodstuffs regulation (ec) n. 854/2004 29 april 2004 laying down specific rules for the organization of official controls on products of animal origin intended for human consumption schukken y.h., wilson d.j., welcome f., garrison-tikofsky l. and gonzalez r.n. 2003. monitoring udder health and milk quality using somatic cell counts. vet res. 34:579-596. paper ital. j. food sci., vol. 28 2016 1 keywords: squash, thermal treatment, texture properties, convection steam oven changes in the texture of butternut squash following thermal treatment b. ślaska-grzywna1, a. blicharz-kania1, a. sagan1*, r. nadulski2, z. hanusz3, d. andrejko1 , m. szmigielski1 university of life sciences in lublin, poland 1 department of biological bases of food and feed technologies 2 department of food process engineering and machines 3 department of applied mathematics and computer science *corresponding author: agnieszka.sagan@up.lublin.pl abstract samples of butternut squash were heated in a convection steam oven at the temperature of 80°c and 100°c without any/or with addition of steam. the most significant changes of texture properties in the pulp were registered regarding its hardness and chewiness, while the alterations of its springiness and cohesiveness occurred within a smaller range. the decisive influence on changing the hardness and chewiness of butternut pulp was observed for the addition of steam, and, to a lower extent, for the time and temperature of treatment; in case of springiness the vital factor was the temperature of the process. mailto:agnieszka.sagan%40up.lublin.pl?subject= 2 ital. j. food sci., vol. 28 2016 introduction pumpkin belongs to the family of cucurbitaceae. its edible part is the pulp of the fruit at different stages of ripeness, as well as its seeds (giant pumpkin and summer squash). the nutritional values of pumpkin fruit are high. this is determined primarily by a high content of carotenoids (from 2 to 10 mg·100 g-1), which are characterized by antioxidant and anticancerous properties. pumpkin fruit provides a good source of vitamins c, a and b, as well as minerals, such as potassium, phosphorus, calcium, magnesium, iron and selenium. they also contain organic acids (citric, malic and fumaric). studies have revealed that the polysaccharides extracted from pumpkin have hypolipidemic activity. additionally, pumpkin has low content of calories. due to the presence of numerous, easily absorbed nutrients, it can be used as a component of slimming diets (carvalho et al., 2012; nawirska-olszańska et al., 2014; rakacjeva et al., 2011; wojdyla et al., 2007; zhao et al., 2014). pumpkin pulp may be a healthy and valuable component of many dishes and fruit products. it is used to manufacture juices, baby foods and canned foods. a disadvantage of pumpkin, which may contribute to its low consumption, is its bland flavor and specific cucumber-like smell. this problem may be resolved by mixing pumpkin with other materials (e.g. cornel berries or quinces) in order to obtain food products of better sensory properties and chemical composition (nawirska-olszańska et al., 2012). pumpkin seeds are used in bakery, oil manufacturing and in pharmaceutical industry. oil from pumpkin seeds contains valuable bioactive elements: squalene, unsaturated fatty acids, tocopherols (obiedziñska and waszkiewicz-robak, 2012). prior to consumption, pumpkin fruits are subjected to different types of treatment, most often thermal processing, during which their properties undergo changes (mayor et al., 2011; œlaska-grzywna et al., 2013), hence the aim of the study was to determine the changes in texture properties caused by thermal treatment in a convection steam oven with different parameters of the processes. materials and methods raw material research material was provided by butternut squash (cucurbita moschata duch.) originating from portugal and purchased in london chain supermarkets. butternut squash is an annual plant belonging to the gourd family (cucurbitaceae), from latin america. 100 g of butternut squash contains (after cooking): 0.9 g of protein, 7.4 g of carbohydrates, including 3.9 g sugars, 0.1 g of fat, 1.4 g of fiber, trace amounts of salt, 15 mg of (19% of rda) vitamin c. its caloric value is 156 kj/37 kcal in 100 g. in the study we used ripe, healthy fruits, without any mechanical damage. treatment the pumpkin was subjected to preliminary treatment: washing, peeling, removing the seeds. such material was used to cut out samples for analyses. the pumpkin was sliced into 1-centimeter-thick slices. next, cylinders of 2-centimeter diameter were cut out from the central part of the slices with the use of a calibrator. in this way cylinders were obtained of 1-centimeter height (h) and the diameter φ = 2 cm. six representative samples (cylinders) were selected for tests from each measurement series. treatment was conducted in a convection steam oven (hounӧ combislim cpe 2306 model, randers, denmark) at the temperature of 80 °c and 100 °c; 0, 20, 40, 60, 80 and 100% of steam added in relation to the initial humidity in the oven chamber; treatment time: 5, 10, 15, 20, 25 and 30 min. for the temperature of 100 °c only the 0, 20, 40, 60% of steam addition were conducted. in case of steam addition of 80 and 100% resulted in structural changes going too far (overcooking), which made it impossible to carry out strength tests. strength tests immediately after thermal treatment warm samples were subjected to strength tests. compression strength measurement for pumpkin samples was performed in the strength test machine, zwick/roell z.5. (zwick roell polska, łódź, poland). the material was subjected to double compression at the speed of head movement equal to 50 mm·min-1. the process of compression was carried out at a stable deformation of the plates equal to 50% of their height, while the interval between the series was 5 s. the measurements were performed in 6 replications. on the basis of the measurements obtained in the form of texturegrams in the arrangement of two coordinates of strength and time, the following texture parameters were determined: hardness, springiness, chewiness and cohesiveness. after the tests, the results of the measurement were subjected to a statistical analysis. namely, a double variance analysis was performed with the interaction for each of the analyzed properties with six variations for the temperature of 80°c and four variations for the temperature of 100°c. statistical analysis detailed comparisons of the mean values in pairs were performed on the basis of tukey’s multiple confidence intervals. also, a comital. j. food sci., vol. 28 2016 3 bined analysis of the four variations was performed for the temperatures of 80°c and 100°c, with the use of triple cross classification with interactions. calculations were done in the sas enterprise guide 5.1 software, adopting the significance level of 0.05 in all the statistical analyses. results the results regarding changes in the hardness of pumpkin pulp resulting from thermal treatment at the temperature of 80°c and 100°c during the time from 5 to 30 min at different levels of steam addition [%] are presented in fig. 1(a,b). a significant impact of heating time and the amount of steam on the hardness of pumpkin hardness was observed. in case of pumpkin heated at the temperature of 80ºc, the hardness of the pulp was decreasing along with increasing the amount of steam added for all the analyzed heating time spans (fig. 1a). on the other hand, the hardness of pumpkin pulp heated at the temperature of 100ºc was decreasing along with the amount of steam added for all time spans adopted in the research program, except for the shortest period of 5 min (fig. 1b). with this particular heating time and steam addition, an increase in pulp hardness was noted from 40 to 60%. regardless of the adopted heating temperature, the lowest hardness was recorded for pumpkin pulp after heating it for 30 min. to analyze the effect of heating time and the amount of steam added during heating we used double cross classification with interaction. the analysis conducted suggests that heating time, the amount of steam added and the interaction between the heating time and the amount of steam added significantly differentiate pumpkin hardness. the results of a detailed comparative analysis of mean pumpkin hardness, based on tukey›s multiple comparisons are presented in table 1 for butternut squash heated at the temperature of 80ºc, and 100ºc. the data in table 1 suggest that the average hardness of pumpkin pulp heated at the temperature of 80ºc for 5, 10 and 15 min does not differ significantly, and it is significantly higher than the hardness of pumpkin pulp heated for 20, 25 and 30 min. analyzing the amount of steam added, the significantly highest value was recorded when no steam was added, while the significantly lowest value was with a 100% addition of steam. the analysis of table 1 demonstrates that the average hardness of pumpkin pulp heated at the temperature of 100 ºc for 5 min was significantly higher than the hardness of squash heated for a longer time. the significantly lowest hardness of pumpkin pulp was obtained for the heating periods of 20, 25 and 30 min. the significantly highest hardness of squash was noted when no steam was added. no significant differences in the mean hardness of pumpkin pulp were observed with adding 20, 40 and 60% of steam. the results of studies on the springiness of pumpkin pulp heated at the temperature of 80 ºc and 100ºc depending on the amount of steam added [%] and the time of heating [min] are presented in fig. 1(c,d). the springiness of pumpkin pulp heated at the temperature of 80ºc decreased when the amount of steam added increased from 0 to 40%, while a further increase in the amount of steam from 40% to 100% resulted in an increased value of springiness for the majority of heating periods. a different course of changes in springiness of squash heated during the shortest time of 5 min may be observed. the springiness of squash heated at the temperature of 100 ºc increased along with the amount of steam added for the analyzed heating periods, except for the time of 15 min (fig. 1d). for this particular period springiness decreased with increasing the table 1 the results of tukey’s studentized range test for mean hardness values of pumpkin pulp heated at the temperature of 80ºc and 100ºc depending on heating time and the amount of steam added. temperature 80ºc time [min] 5 10 15 20 25 30 mean values 205.92a 187.79a 185.51a 150.30b 129.10b 133.80b sd 27.07 25.19 26.02 24.20 19.01 25.58 steam [%] 0 20 40 60 80 100 mean values 442.17a 230.78b 118.33c 107.69c 68.39d 25.06e sd 12.05 14.06 7.38 8.82 3.82 1.09 100ºc time [min] 5 10 15 20 25 30 mean values 126.96a 86.85b 49.08bc 32.67cd 33.11d 67.10d sd 24.96 20.78 11.60 6.26 6.32 20.99 steam [%] 0 20 40 60 mean values 184.38a 33.74b 21.72b 23.99b sd 17.43 1.58 1.70 5.40 means with the same letter are not significantly different at 0.05 significance level. 4 ital. j. food sci., vol. 28 2016 fig. 1 analyzed properties of pumpkin pulp depending on amount of steam added and heating time: hardness of pumpkin pulp heated at the temperature of 80ºc (a) and 100ºc (b); springiness of pumpkin pulp heated at the temperature of 80ºc (c) and 100ºc (d); chewiness of pumpkin pulp heated at the temperature of 80ºc (e) and 100ºc (f); cohesiveness of pumpkin pulp heated at the temperature of 80ºc (g) and 100ºc (h). ital. j. food sci., vol. 28 2016 5 amount of steam from 40 to 60%. the highest springiness of pulp was recorded for squash heated for the shortest time. on the basis of double cross classification with interaction it can be concluded that the heating time, the amount of steam added and the interaction of heating time and the amount of steam added significantly differentiate the springiness of pumpkin pulp subjected to thermal treatment both at the temperature of 80°c and 100°c. the results of a detailed comparative analysis of mean springiness of pumpkin pulp on the basis of tukey’s studentized range test are presented in table 2. analyzing the results presented in table 2, it should be noted that the average springiness of pumpkin pulp heated at the temperature of 80°c for 5 min was the highest, yet it was not significantly higher than the mean springiness of squash for the heating time of 20, 25 and 30 min. the lowest mean springiness was obtained for the heating time of 10 min. on the other hand, the highest mean springiness of pumpkin pulp was obtained after adding 100% of steam, while the significantly lowest mean values of springiness were recorded in the situation when 20, 40 and 60% of steam was added. table 2 suggests that the mean springiness of squash heated at the temperature of 100°c for the time of 5 min was significantly highest at the level of significance of 0.05. the significantly lowest springiness of squash was obtained for the heating periods of 25 and 30 min. analyzing the amount of steam added, the significantly highest springiness of squash was recorded with adding 60% of steam. no significant differences of mean squash springiness were noted when there was no steam addition or when 20% of steam was added. the results of studies on the chewiness of squash heated at the temperature of 80°c and at 100°c depending on heating time and the amount of steam added are presented in fig. 1(e,f). the chewiness of squash heated at the temperature of 80°c was decreasing when the amount of steam added increased from 0 to 20 and 40%, and when the addition of steam continued increasing no drop in chewiness was observed. the course of curves presented in fig. 1f suggests that the chewiness of squash heated at 100°c for 5 min differed from the chewiness of squash heated for longer periods included in the research program. in case of the periods of 10, 15, 20, 25 and 30 min the chewiness of squash altered only slightly. double cross classification with interaction used to analyze the effect of heating time and the amount of steam added during heating on changing chewiness of butternut squash revealed that the heating time, the amount of steam added and the interaction of the heating time and the amount of steam added do not differentiate significantly the chewiness of squash. the results of a detailed comparative analysis of average chewiness of pumpkin pulp based on tukey’s multiple comparisons are presented in table 3. while analyzing the results presented in table 3, it should be noted that the mean chewiness of squash heated at the temperature of 80ºc for 5, 10 and 15 min was the highest and it was significantly higher than the mean chewiness of squash for the heating time of 20, 25 and 30 min. on the other hand, the significantly highest chewiness of squash was obtained when there was no steam addition. no significant differentiation in the chewiness of squash was observed when 15, 20, 25 and 30% of steam was added. the analysis of table 3 demonstrates that the mean chewiness of squash heated at the temperature of 100ºc for 5 min was significantly highest at the significance level of 0.05. the significantly lowest chewiness of squash was observed for the periods of 15, 20, 25 and 30 min. analyzing the amount of steam, the significantly highest chewiness of squash was obtained when no steam table 2 the results of tukey’s studentized range test for mean springiness values of pumpkin pulp heated at the temperature of 80ºc and 100ºc depending on heating time and the amount of steam added. temperature 80ºc time [min] 5 10 15 20 25 30 mean values 0.390a 0.275c 0.325bc 0.350ab 0.339ab 0.362ab sd 0.030 0.023 0.030 0.027 0.023 0.027 steam [%] 0 20 40 60 80 100 mean values 0.299c 0.239d 0.237d 0.294cd 0.426b 0.545a sd 0.008 0.005 0.027 0.021 0.020 0.028 100ºc time [min] 5 10 15 20 25 30 mean values 0.568a 0.467b 0.297cd 0.338c 0.207de 0.140e sd 0.044 0.041 0.028 0.031 0.020 0.009 steam [%] 0 20 40 60 mean values 0.268c 0.292bc 0.353b 0.431a sd 0.022 0.022 0.028 0.053 means with the same letter are not significantly different at 0.05 significance level. 6 ital. j. food sci., vol. 28 2016 was added. no significant differences of mean values of chewiness were noted when adding 20, 40 and 60% of steam. the results of studies on the cohesiveness of squash heated at the temperature of 80 oc and at 100°c depending on heating time [min] and the amount of steam added [%] are presented in fig. 1(g,h). the cohesiveness of squash heated at the temperature of 80ºc was changing depending on the amount of steam added and the course varied depending on the heating time (fig. 1g). the highest cohesiveness was observed for squash to which no steam was added and when 100% of steam was supplied. in case of treatment at the temperature of 100°c for the heating periods of 10, 15, 20, 25 and 30 min the cohesiveness of squash generally increased along with the amount of steam added and it reached its highest values for 30% of steam (fig. 1h). after heating for 5 min the cohesiveness of squash decreased along with increasing the amount of steam added. the results of double cross classification with table 3 the results of tukey’s studentized range test for mean chewiness values of pumpkin pulp heated at the temperature of 80ºc and 100ºc depending on heating time and the amount of steam added. temperature 80ºc time [min] 5 10 15 20 25 30 mean values 2.499a 2.005a 1.863ab 1.211bc 0.879c 1.249bc sd 0.557 0.492 0.420 0.228 0.114 0.281 steam [%] 0 20 40 60 80 100 mean values 5.7231a 1.5236b 0.7317c 0.5767c 0.6447c 0.5058c sd 0.5429 0.0946 0.1416 0.0233 0.0295 0.0372 100ºc time [min] 5 10 15 20 25 30 mean values 3.349a 1.023b 0.411c 0.356c 0.282c 0.363c sd 0.836 0.172 0.071 0.030 0.051 0.093 steam [%] 0 20 40 60 mean values 2.523a 0.408b 0.356b 0.569b sd 0.591 0.035 0.049 0.134 means with the same letter are not significantly different at 0.05 significance level. interaction used to analyze the effect of heating time and the amount of steam added during heating on changing cohesiveness of butternut squash revealed that the heating time, the amount of steam added and the interaction of the heating time and the amount of steam added significantly differentiate the cohesiveness of squash. a detailed comparative analysis of mean values of squash cohesiveness on the basis of tukey’s multiple comparisons revealed that the average cohesiveness of squash heated at the temperature of 80ºc for 5 and 10 min was significantly higher, as compared with the mean cohesiveness of squash heated for 20 and 25 min (table 4). the significantly highest cohesiveness of pumpkin pulp was recorded after heating with no steam addition and after adding 100% of steam. no significant differentiation of squash cohesiveness was observed after adding 15, 20, 25 and 30% of steam. an analysis of table 4 shows that the mean cohesiveness of squash heated at the temperature of 100ºc for 20, 25 and 30 min was significantly table 4 the results of tukey’s studentized range test for mean cohesiveness values of pumpkin pulp heated at the temperature of 80ºc and 100ºc depending on heating time and the amount of steam added. temperature 80ºc time [min] 5 10 15 20 25 30 mean values 0.0322a 0.0311a 0.0286ab 0.0264b 0.0261b 0.0292ab sd 0.0022 0.0025 0.0020 0.0014 0.0011 0.0016 steam [%] 0 20 40 60 80 100 mean values 0.0408a 0.0278b 0.0222c 0.0211c 0.0244bc 0.0372a sd 0.0029 0.0009 0.0008 0.0005 0.0011 0.0010 100ºc time [min] 5 10 15 20 25 30 mean values 0.0404c 0.0388c 0.0458b 0.0525a 0.0538a 0.0513a sd 0.0034 0.0023 0.0041 0.0040 0.0041 0.0026 steam [%] 0 20 40 60 mean values 0.0361c 0.0419b 0.0453b 0.0650a sd 0.0027 0.0011 0.0015 0.0035 means with the same letter are not significantly different at 0.05 significance level. ital. j. food sci., vol. 28 2016 7 highest at the significance level of 0.05. the significantly lowest mean cohesiveness of butternut squash was observed after 5 and 10 min of heating. analyzing the amount of steam added, the significantly highest mean cohesiveness of squash was recorded after treatment with the addition of 100% of steam, while the significantly lowest values were noted when no steam was used. the results of three-agent variance analyses for the studied properties, namely hardness, springiness, chewiness and cohesiveness provide a basis for claiming that all the agents analyzed in the present work, namely temperature, the amount of steam added, heating time and the interactions occurring between these agents significantly differentiate the studied qualities of butternut squash. the results of tukey’s multiple comparisons in pairs for the analyzed agents and for all the properties are presented in table 5. these results show that there were significant differences concerning the analyzed properties of squash at the temperature of 80ºc and 100ºc. for hardness and chewiness the mean values of the properties at the temperature of 80ºc were significantly higher, as compared with the mean values of the properties at the temperature of 100ºc. in case of springiness and cohesiveness, it may be claimed that the mean value at the temperature of 100ºc is significantly higher than the mean value at 80ºc. analyzing the comparisons of mean values for the properties with the use of different amounts of steam, it may be noted that the significantly highest mean hardness and chewiness were observed when no steam was added. on the other hand, the significantly highest springiness and cohesiveness were observed for the biggest amount of steam added, amounting to 60%. the most varied mean values of the studied properties were observed for the periods of treatment studied in the work. only in case of the mean cohesiveness there were no significant differences for the pairs of mean values compared. the highest values of mean hardness, springiness and chewiness were noted for the shortest treatment time. structural and rheological properties determine behavior of the squash pulp under compression (shirmohammadi et al., 2014). recognizing the mechanical properties of squash enables improvement of processing its pulp (sosińska et al., 2012). the texture of the squash pulp exhibits characteristics of chewiness and springiness, which can be modified by thermal treatment. earlier studies concerning thermal treatment of pumpkin pulp in a convection steam oven revealed significant modifications in its texture properties (ślaska-grzywna et al., 2013). it was noted that the most significant effect on changing hardness, springiness and chewiness of squash resulted from the amount of steam added, and to a lower degree from the time and temperature of treatment. similarly, works by gonçalves et al. (2007) suggest a significant decrease in the firmness of squash during thermal treatment at the temperature of 75-95oc for 50 min. prior to thermal treatment the firmness of squash was ca. 60 n, while after the treatment it did not exceed 10 n. changes in the texture of pumpkin pulp following thermal treatment during its storage were studied by ratnayaake et al. (2004). in their studies with the help of double-compression test they observed the most significant changes in the texture of pumpkin pulp in case of measuring its hardness and chewiness, while the changes were only slight in case of springiness and cohesiveness. the key factor affecting rheological qualities of vegetables is turgor (lin ta-te and pitt, 1986). softening of tissues is related to the loss of turgor cells and their easier separation (greve et al., 1994). plant tissue is built of cells mutually linked by middle lamella. the cell wall is kept rigid due to hydrostatic pressure inside the cell, which normally amounts to 1-8 bar (0.1 0.8 mpa) (aguillera et al. 1998). cellulose present in the cell wall affects the rigiditable 5 the results of tukey’s studentized range test for pairs of mean values for the three agents and analyzed properties. analyzed agent level of agent analyzed property hardness springiness chewiness cohesiveness temperature 80 224.74a 0.2674b 2.139a 0.0280b 100 65.96b 0.3360a 0.964b 0.0471a steam 0 313.27a 0.2833b 4.123a 0.0385b 20 132.26b 0.2657b 0.966b 0.0349c 40 70.03c 0.2953b 0.544b 0.0338c 60 65.84c 0.3625a 0.573b 0.0431a time 5 207.96a 0.4498a 3.3754a 0.035833a 10 167.42b 0.3527b 1.8921b 0.035208a 15 150.48b 0.2642cd 1.4421bc 0.037917a 20 117.53cd 0.2960c 0.9350cd 0.038958a 25 103.19d 0.2356de 0.6769d 0.039167a 30 125.53c 0.2119e 0.9869cd 0.038125a means with the same letter are not significantly different at 0.05 significance level. 8 ital. j. food sci., vol. 28 2016 ty and strength of the plant tissue, while pectins and hemicellulose present in middle lamella are responsible for its plasticity (lewicki and pawlak, 2003). thermal treatment of vegetables results in structure alterations, tissue disintegration, enzyme inactivation, washing out soluble components, loss of firmness and, consequently, their softening (cruz et al. 2011; galindo et al., 2005). according to researchers, both raw and cooked squash pulp provides numerous health benefits and can be used in prevention and treatment of certain diseases (caili et al.; 2006, niewczas et al.; 2005, stirg, 1997). studies suggest a possibility of selecting adequate parameters of thermal treatment helping to maintain the texture most required by consumers. moreover, the research results will be useful for food producers, allowing them to select the optimal parameters of thermal treatment of squash pulp. conclusions thermal treatment in a convection steam oven results in statistically significant changes of all the studied parameters of squash texture, namely its hardness, springiness, chewiness and cohesiveness. the range and dynamics of texture properties of butternut squash depends on the parameters of thermal treatment, such as temperature, amount of steam added and time. the most significant range of modifications concerning texture qualities of squash were registered for its hardness and chewiness, while changes in its springiness and cohesiveness occurred to a smaller extent. the decisive influence on changing the hardness and chewiness of squash was exerted by the addition of steam, while treatment time and temperature were less significant. in case of springiness the key agent was treatment time, while with cohesiveness it was the temperature of treatment. conducted studies will allow food producers to select the optimal parameters of thermal treatment of squash pulp for consumption purposes. references aguilera j. m., cuadros, t. r. and del valle j. m. 1998. differential scanning calorimetry of low-moisture apple products. carbohydrate polymers 37: 79. astorg p. 1997. food carotenoids and cancer prevention: an overview of current research. trends in food science & technology 8(120): 406. caili f., huan s. and quanhong l. 2006. a review on pharmacological activites and utilizacion technologies of pumpkin. plant foods for human nutrition 61: 73. carvalho l.m.j., gomes p.b., oliveira godoy r.l., pacheco s., monte p.h.f., 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biological macromolecules 64: 137. paper received december, 1, 2014 accepted february 5, 2015 #646_tateo_bozza ital. j. food sci., vol 29, 2017 233 paper triglycerides variability in donkey milk m. bononi1*, f. tateo1 and a. tateo2 1department of agricultural and environmental science, university of milan, via celoria 2, milan, italy 2department of veterinary medicine, university “aldo moro” of bari, valenzano, ba, italy *corresponding author. tel.: +39 0250316538 e-mail address: monica.bononi@unimi.it abstract the distribution of triacylglycerols (tags) in donkey milk and of fatty acids in the glycerol backbone affect assimilation and lipolysis, so the utility of analytical methods to characterise fat fractions is proved. in this study an optimised gas chromatography/oncolumn injector method was used to study the tag variability of various milk species. this method is useful for quality control aimed at standardising formulated donkey milk and was used to compare the tag composition of lyophilised donkey milk distributed on the italian market. three tag groups, based on variability degree, were detected and the most characterising tag fraction was identified. keywords: donkey milk, gc-oci technique, quality control, triglycerides variability ital. j. food sci., vol 29, 2017 234 1. introduction the use of donkey milk for human consumption is justified by various nutritional values and its tolerability (barłowska et al., 2011; do nascimento rangel et al., 2015; malissiova et al., 2016 polidori and vincenzetti, 2013). the presence of bioactive and functional components has been largely reviewed (martini et al., 2014; salimei et al., 2004; salimei, 2011; salimei and fantuz, 2012;). moreover, the consumption of donkey milk is an alternative when there is an intolerance to cow’s milk (høst and halken, 2004; lara-villoslada et al., 2005; monti et al., 2007; restani et al., 2009). previous studies (chianese et al., 2010; guo et al., 2007; marconi and panfili, 2002; summer et al., 2004) support the optimisation of infant formula milk and the production of nutraceutical compounds in donkey milk for the following reasons: (1) the lipid content in donkey milk is essentially lower than in milk from humans or cows; (2) the carbohydrate content in donkey milk is comparable with human milk but higher than in cow’s milk; and (3) the protein content in donkey milk is higher than in human milk but lower than in cow’s milk. this research is part of a larger project concerning the industrial formulation of humanised milk and nutraceutical compounds using lyophilised donkey milk. the standardisation of these commercial products is possible only if they are derived from ingredients characterised by standard parameters. groups have analysed the triacylglycerol (tag) composition to identify the fatty acids in the glycerol backbones (gastaldi et al., 2010), but the analytical methods used for this aim are not suitable for routine industrial quality control. some authors also reported comparative results for the fatty acid and tag compositions of various milks species (blasi et al., 2008; breckenridge and kuksis, 1967; chiofalo et al., 2011; cossignani et al., 2011; dugo et al., 2005; gantner et al., 2015gastaldi et al., 2010; jensen, 1999; martemucci and d’alessandro, 2012; zou et al., 2013). there is often not ideal peak resolution in tag analysis using high performance liquid chromatography with an evaporative light scattering detector (hplc/elsd) (zou et al., 2013) and there are difficulties with tag analysis when using hplc/atmospheric pressure chemical ionisation (apci)/mass spectrometry (ms) techniques with correction factors to estimate the proportion of the different tags in each type of milk (gastaldi et al., 2010). various authors reported comparative results concerning the fatty acid composition and tag distribution of the fatty acids in the glycerol backbone of various milk species, but the analytical methods used for tag analysis are not useful for routine quality control (blasi et al., 2008; breckenridge and kuksis, 1967; chiofalo et al., 2011; cossignani et al., 2011; dugo et al., 2005; gantner et al., 2015; gastaldi et al., 2010; jensen, 1999; martemucci and d’alessandro, 2012; zou et al., 2013). this paper discusses the advantages of adopting a rapid gas-chromatography (gc) method based on the identification sequence of peaks, with each one representing the total carbon number (cn) of a single tag, in order of increasing molecular weight. the triglyceride composition of lyophilised donkey milk products available on the italian market was compared and the characterisation of donkey milk by this easy method was useful for the purposes of industrial quality control and to determine the fat variability composition in these lyophilised donkey milk. ital. j. food sci., vol 29, 2017 235 2. materials and methods 2.1. experimental design a method that was able to produce a complete pattern of tags by direct injection was first reported in the commission regulation (ec) no 213/2001 to determine the genuineness of butter. then, the authors modified the operative conditions to produce the first repeatability data (bononi et al., 2001) and adopted the modified method for the identification of synthesised tags used as support in butter flavours (tateo and bononi, 2003). the same method was also applied for quality control of vegetable and animal fats (tateo and bononi, 2002; tateo and bononi, 2004). some authors (lozada et al., 1995; molkentin and precht, 1994; molkentin and precht, 1995) quantitatively determined the tag composition of milk using capillary columns. in the present paper, we used a petrocol capillary column with bonded phase, hydrogen as the carrier gas, optimised operative conditions for an on-column injector (oci), and programmed oven temperatures. the optimisation of the chromatographic conditions was necessary strictly to obtain repeatability data that were useful for various quality control aims. we used the proposed method to analyse a mass sample of donkey milk from a traditional farming area of martina franca in apulia (italy) and the tag data were compared with milk samples from humans, cows, sheep and goats obtained with the same method. the lyophilised donkey milk from the farming area of martina franca was compared with other lyophilised donkey milk products available from the specialised italian market. 2.2. milk samples from different species first, five samples of milk from different species were compared to highlight the significant differences in the tag composition of the fat fractions using the proposed method (described below). these samples were: a) donkey milk from an autochthonous breed from the apulia region (martina franca), produced by donkeys bred using a semi-extensive method at the farm “masseria lamacarvotta” in laterza (italy); b) cow, sheep, and goat milk selected from italian farms and collected at the mid lactation stage; c) human milk kindly provided by the neonatology unit (milkbank) at the santa chiara hospital in trento (italy). all five samples were stored at 4°c strictly during the time to the laboratory and then were immediately frozen at -20°c before lyophilisation. subsequently, the samples of milk were lyophilised using a scanvac coolsafe 110-4 pro freeze dryer (labogene) at -105°c. 2.3. lyophilised, commercially available donkey milk four samples of lyophilised donkey milk, commercially available in italy, were compared with the donkey milk produced from the martina franca breed and lyophilised in our pilot equipment, and also with a donkey milk available in italy that was lyophilised in our laboratory using the same conditions adopted for the liquid samples presented in table 1. this liquid donkey sample was sterilized by uperisation. ital. j. food sci., vol 29, 2017 236 2.4. gc analysis of triglycerides for the analysis of the tag profiles in donkey, human, cow, sheep, and goat milk fat, ~5 ml of water was added to ~1-2 g of the lyophilised sample. the sample was vortexed for 1 min, and then sonicated in an ultrasonic bath for 5 min with 2 ml of isooctane (sigma aldrich). the mixture was centrifuged at 4000 rpm for 5 min. the isooctane phase (2 ml) was introduced manually in the on-column injector (oci) at 40°c. the tag analyses were performed on a hrgc 5160 mega series (carlo erba instruments) equipped with a bonded phase poly (dimethyl siloxane) petrocol ex 2887 capillary column (supelco) with dimensions of: 5 m x 0.53 mm i.d. and 0.1 μm film thickness. the oven temperature program was 150°c, increased to 200°c at a rate of 10°c min-1 and then increased to 340°c at a rate of 5°c min-1 (held for 30 min). the detector temperature (fid) was 350°c and the carrier gas was h2 at 20 kpa pressure. an anhydrous butter fat standard certified by the community bureau of reference (crm 519) was used for peak identification. table 1. triacylglycerol composition (tag expressed as % of peaks identified with total carbon number) of donkey milk produced in farm “masseria lamacarvotta” (laterza, italy) compared with human, cow, sheep and goat milk samples. tga donkey human cow sheep goat c22 0.01 n.d. 0.07 0.06 n.d. c24 0.49 0.34 0.56 0.65 0.36 c26 0.21 n.d. 0.46 0.85 n.d. c28 0.65 n.d. 0.86 1.80 0.38 c30 1.65 n.d. 1.27 2.93 0.89 c32 2.72 n.d. 2.33 4.35 1.72 c34 3.44 0.20 5.30 6.05 3.59 c36 4.57 0.56 10.04 8.69 5.80 c38 6.13 1.11 12.44 13.15 8.21 c40 7.82 2.24 9.67 13.62 9.22 c42 10.15 4.26 6.17 8.77 9.75 c44 12.49 7.39 5.75 7.25 10.16 c46 9.83 11.17 6.55 6.40 10.02 c48 6.90 12.68 8.59 5.57 9.60 c50 8.76 17.79 12.50 6.81 11.75 c52 16.22 32.71 12.08 8.46 15.23 c54 7.59 9.12 5.35 4.60 3.33 c56 0.37 0.42 n.d. n.d. n.d. 3. results the mean tag compositions (table 1) were derived from three replicate gc-oci analyses of samples of donkey milk from the martina franca farm and of samples from other species. examples of the gc-oci traces of milk from donkey, human, cow, sheep and goat are shown in fig. 1. ital. j. food sci., vol 29, 2017 237 figure 1. examples of gc-oci traces produced for fat fraction of milk of different species and expressed as total carbon number (cn). the nmkl procedure no. 5 (nordic committee on food analysis, 1997) was used to evaluate the relative repeatability standard deviation (rsdr) using three samples for each milk species, and the following results were obtained: 0.022 (donkey milk), 0.032 (human milk), 0.018 (cow milk), 0.034 (sheep milk), and 0.011 (goat milk). the tag composition (table 2) of four lyophilised samples present on the italian market were compared with two lyophilised samples produced from the reference liquid donkey milk of martina franca and from the liquid donkey milk distributed in italy. from these data, it is possible to deduce average data and to calculate dmax values for each tag class: ital. j. food sci., vol 29, 2017 238 the dmax values are simply derived from the minimum and maximum values identified in table 2. to estimate the variability from the mean values, we defined three ranges (<0.3, 0.3-0.6, and >0.6) deduced from the ratio “dmax/mean” that identify tag classes with low, medium, and high variability. table 2. tag composition of the fat fraction of four lyophilised donkey milk products distributed in italy (a-d) compared with the martina franca donkey milk (e) and a donkey milk distributed in italy (f) lyophilised in laboratory pilot equipment. a b c d e f tga average dmax dmax/average c22 0.15 0.03 0.02 0.04 0.01 0.68 0.16 0.67 4.32 *** c24 0.74 0.56 0.70 0.47 0.49 1.19 0.69 0.72 1.04 *** c26 0.13 0.25 0.24 0.32 0.21 0.20 0.23 0.19 0.84 *** c28 0.54 0.86 0.98 0.66 0.65 0.80 0.75 0.44 0.59 ** c30 1.67 2.31 2.54 1.72 1.65 1.87 1.96 0.89 0.45 ** c32 2.80 3.66 4.22 2.82 2.72 4.30 3.42 1.58 0.46 ** c34 3.79 4.72 5.16 3.65 3.44 4.07 4.14 1.72 0.42 ** c36 5.16 6.32 6.58 4.89 4.57 5.19 5.45 2.01 0.37 ** c38 6.75 8.21 8.23 6.40 6.13 6.89 7.10 2.10 0.30 * c40 8.54 10.54 9.83 8.19 7.82 8.82 8.96 2.72 0.30 * c42 11.39 13.61 11.69 10.78 10.15 11.55 11.53 3.46 0.30 * c44 12.61 14.96 12.71 12.41 12.49 12.94 13.02 2.55 0.20 * c46 9.06 9.52 9.13 9.13 9.83 9.43 9.35 0.77 0.08 * c48 6.93 7.14 6.10 6.79 6.90 6.75 6.77 1.04 0.15 * c50 10.41 6.02 7.10 9.80 8.76 8.92 8.50 4.39 0.52 ** c52 14.19 9.80 10.28 15.20 16.22 12.17 12.98 6.42 0.49 ** c54 4.99 1.45 4.36 6.50 7.59 4.23 4.85 6.14 1.27 *** c56 0.15 0.04 0.13 0.23 0.37 n.d. 0.18 0.33 1.79 *** the variability classes for tag are expressed as the total carbon number (cn). data for all samples were derived from five replicates. dmax/average * < 0.3 little variability ** from 0.3 to 0.6 medium variability *** > 0.6 high variability the tags characterised by the lower variability are included in six tag classes from c38 to c48. in each donkey milk sample, the classes with lower variability represented more than 50% of the total fat fraction. in particular, for five samples, the tags with lower variability represented 53-58% of the total tag content, and for sample b they represented 64% of the total fat. the tags characterised by the higher variability are included in four tag classes (c24, c26, c54, and c56) that represented 5-9% of the total fat fraction for five samples and 2% of sample b, while the tags with medium variability (c28, c30, c32, c34, c36, c50, and c52) represented 34-39% of the total fat fraction. ital. j. food sci., vol 29, 2017 239 considering the identification of donkey milk fatty acids on glycerol backbone (gastaldi et al., 2010) and data in table 2, we deduced table 3 that shows the tags composition of three variability classes. table 4 reports fatty acid symbols and names used in table 3. table 3. tga % composition with lower, medium and higher variability classes in six donkey milk samples of italian market. lower variability tga a b c d e f c38 6.75 8.21 8.23 6.40 6.13 6.89 bu-p-ln, cy-c-me, c-la-po, co-p-p, c-la-p, la-la-m c40 8.54 10.54 9.83 8.19 7.82 8.82 c-la-ln, co-p-ln, cy-m-ln, c-la-o, c-m-p, cy-p-p c42 11.39 13.61 11.69 10.78 10.15 11.55 cy-p-ln, c-m-ln, la-ln-ln, cy-p-o, c-po-p, c-p-p c44 12.61 14.96 12.71 12.41 12.49 12.94 cy-ln-l, cy-ln-o, c-p-ln, cy-o-o, c-p-o, la-p-p c46 9.06 9.52 9.13 9.13 9.83 9.43 c-ln-ln, c-ln-l, c-ln-o, c-l-o, la-p-ln, c-o-o, la-p-pl, la-p-o-, m-p-p c48 6.93 7.14 6.10 6.79 6.90 6.75 la-ln-o, la-l-o, mo-p-l, m-p-l, m-po-o, m-p-o, m-p-s, p-p-p σ 55.28 63.98 57.69 53.70 53.32 56.38 medium variability tga a b c d e f c28 0.54 0.86 0.98 0.66 0.65 0.80 cy-cy-la c30 1.67 2.31 2.54 1.72 1.65 1.87 cy-c-la c32 2.80 3.66 4.22 2.82 2.72 4.30 c-c-la c34 3.79 4.72 5.16 3.65 3.44 4.07 cy-cy-ln, c-c-m, cy-c-p, c-la-la c36 5.16 6.32 6.58 4.89 4.52 5.19 cy-cl-ln, cy-c-ln, cy-c-o, cy-la-p c50 10.41 6.02 7.10 9.80 8.76 8.92 m-ln-ln, po-p-ln, p-p-ln, p-p-l, po-p-o, p-p-o, p-p-s c52 14.19 9.80 10.28 15.20 16.22 12.17 p-ln-ln, p-ln-l, po-ln-o, p-l-l, p-ln-o, po-l-o, p-l-o, po-o-o, p-o-o-, p-o-s σ 38.56 33.69 36.86 38.74 38.01 37.32 higher variability tga a b c d e f c22 tr. tr. tr. tr. tr. tr. c24 0.74 0.56 0.70 0.47 0.49 1.19 bu-bu-p c26 0.13 0.25 0.24 0.32 0.21 0.20 cy-cy-c c54 4.99 1.45 4.36 6.50 7.59 4.23 ln-ln-l, ln-l-l, ln-ln-o, ln-l-o, l-l-o, ln-o-o, l-o-o, o-o-o, o-o-s, p-o-ga, o-s-s c56 0.15 0.04 0.13 0.23 0.37 n.d. p-o-er σ 6.01 2.30 5.43 7.52 8.66 5.62 ital. j. food sci., vol 29, 2017 240 table 4. fatty acid symbols used in table 3. symbol notation fatty acid bu c4:0 butyric acid co c6:0 capronic acid cy c8:0 caprylic acid cl c10:1 decenoic acid c c10:0 capric acid la c12:0 lauric acid mo c14:1 myristoleic acid m c14:0 myristic acid po c16:1 palmitoleic acid p c16:0 palmitic acid ln c18:3 linolenic acid l c18:2 linolei acid o c18:1 oleic acid s c18:0 stearic acid me c20:3 eicosatrienoic acid ga c20:1 eicosaenoic acid er c22:2 docosaenoic acid 4. conclusions the determination of the amount of tag, free fatty acids, and phospholipids in milk from various species has been the objective of many studies (claeys et al., 2014; gantner et al., 2015; jensen et al., 1990) and the distribution of fatty acids in triglycerides and the glycerol backbone affects the assimilation and consequently the biochemical and the nutritional value (emken et al., 2004; filer et al., 1969; gastaldi et al., 2010). therefore, the influence of the tag composition is confirmed. from an analytical point of view, a simple method based on the identification of tag, expressed as cn, is very useful. the gc method described in this paper for the study of tag content, expressed as cn, is optimised for the quantitative characterisation of tag profiles and is useful for industrial quality control of donkey milk. also, the gc-oci profiles reported in this paper demonstrated there is a clear differentiation between different species of milk and the tag pattern may be considered a fundamental characteristic of milk from all species. in this paper, the tag fraction of donkey milk from the martina franca breed was compared with human, cow, sheep, and goat milk samples, and the proposed gc-oci method produced good reproducible results and permitted an easy characterisation and comparison. concerning the tag composition of donkey milk, previous studies used different methods but not always included the tag composition. therefore, this paper gives a contribute to define the extremes of natural variability for donkey milk. we determined the variability of tag composition, expressed as cn, in lyophilised donkey milk using our optimised method and we detected three degrees of variability for the different tag classes. ital. j. food sci., vol 29, 2017 241 acknowledgements the authors thanks eurolactis group sa for providing the donkey milk of its production. references barłowska j., szwajkowska m., litwińczuk z. and król j. 2011. nutritional value and technological suitability of milk from various animal species used for dairy production. compr. rev. food sci. f. 10:291-302. blasi f., d. montesano, de angelis m., maurizi a., ventura 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substitutes. j. agr. food chem. 61:7070-7080. paper received october 15, 2016 accepted november 29, 2016 ijfs#788_bozza ital. j. food sci., vol. 29, 2017 582 paper preliminary data on volatile composition of olive fruits of cv. “simona” and possible relationship to resistance to fly oviposition m. bononi* and f. tateo department of agricultural and environmental science, university of milan, via celoria, 2, milan, italy *corresponding author. tel.: tel: +39 0250316538 e-mail address: monica.bononi@unimi.it abstract by characterizing the volatile compounds of the olive cultivar “simona” from southern italy (apulia region), we identified a way to analyze characteristics possibly linked to this olive’s well-known resistance to fly oviposition. the pool of volatile compounds in the unripe and ripe fruits was identified, and even if the relative amounts of these compounds tended to vary with ripening, the fly repellent action appeared to be related to the sesquiterpenes such as α-copaene, cycloisosativene, α-muurolene, β-cubebene and hydrocarbons such as (e)-2-dodecene, undecane, tridecane, and 3-methyl undecane. in agreement with the concept that the collective pool of volatile substances can enhance olfactory pleasure/repulsion more than would the effects of a single compound, the pool of volatile compounds identified in this paper may be among the possible characteristic mixtures with repellent action against bactrocera oleae. the selection of volatile compounds made by the cuticle and responsible of the headspace quality surrounding the whole olive fruit is also demonstrated to be markedly different from the headspace volatile compounds produced by the extracted oil. keywords: cv. simona, olive fruits, olive oil, hs-spme-gc/ms, bactrocera oleae ital. j. food sci., vol. 29, 2017 583 1. introduction olive growing in the apulia region (southern italy) has led to a specific varietal population selected through centuries. about 50 varieties have been developed, most of low agronomic interest. the major crops are limited to no more than about 15 widely grown cultivars, whereas some were abandoned for low productivity, despite their specific sensory and nutritional properties. today, the promotion of local products is a common marketing trend, and the defined sensory characteristics represent the greatest attraction to consumers. in recent years, some authors have noted that temperature can influence the acidic composition of olive oils in each cultivar and that various cultivars respond differently to different environmental conditions (fiorino et al., 2003a; fiorino et al., 2003b; pannelli, 2006). recently, a very old cultivar, “simona,” long grown in southern italy, in the apulia region, and particularly in the southeast of the province of bari, in the territories of the towns of castellana grotte, conversano, monopoli, and polignano, has been reconsidered for quality characteristics. godini et al. (2002) described the essential phenological and morphological traits of this “simona” cultivar, which produces yellow oil and is slightly fruited, with fruits that are small, ellipsoidal, pruinose, and black violet at maturity. the origin is unknown, but presumably it is native to the apulia region. the main defect of this olive is the low oil output (godini et al., 2002): farmers identify the mean oil extraction value at 12%, i.e., about 20% less than the other cultivars. however, until 1960, the cv. “simona” represented about 33% of the olive cultivation in the region, probably because the resulting oil preserves its sensory characteristics for more than one year (tateo and bononi, 2016). the cv. “simona” has not been well described. lombardo et al. (2008), studying the triacylglycerol composition of 188 italian cultivars from 2001 to 2005, observed an extreme variability of percentage of each fatty acid, making each cultivar peculiar in its triacylglycerol composition. for example, in the same year 2003, the percentage of oleic acid varied from a minimum of 49.8% (cv. “orbetana”) to 78.2% (cv. “simona”), while the percentage of linoleic acid ranged from 3.9% (cv. “simona”) to 23.9% (cv. “racioppella”). the paper confirmed the great sensitivity of olives to climatic changes. the “simona” cultivar shows good resistance to the fly bactrocera oleae. studies on the influence of oviposition have been carried out for some species of pest flies (foster and harris, 1997), but for “simona” cultivars, no studies have addressed how the olive fruit affects susceptibility to b. oleae. the “simona” cultivar is particularly resistant to b. oleae and its oil is of lower acidity than other oils produced in the same growing area from different cultivars (for example, “oliastra”, “pasola”). therefore, these characteristics justify the studies of the “simona” olive fruit to discover the parameters that influence its resistance to b. oleae attack. for plants, volatile organic compounds are largely recognized as having roles in reproduction, tritrophic interactions, below ground defense, and abiotic stress (dudareva et al., 2006; parè and tumlinson, 1999; tamiru et al., 2011; unsicker et al., 2009; wu and baldwin, 2010). moreover, b. oleae appears to demonstrate cultivar preference in attacking specific olive cultivars (burrack and zalom, 2008; gonçalves et al., 2012; iannotta et al., 2007; navrozidis et al., 2007). other authors believe that oviposition is conditioned from host selection based on chemical, physical, and molecular features (corrado et al., 2012; kombargi et al., 1998; imperato et al., 2012; malheiro et al., 2016; neuenschwander et al., 1985; rizzo et al., 2012; spadafora et al., 2008). the amount of sesquiterpenes has been correlated to growth of olives; for example, in the case of cv. “serrana,” spme-gc-ms analyses have ital. j. food sci., vol. 29, 2017 584 indicated an increase in α-copaene while the green fruit grows followed by a decrease related to fruit ripening (de alfonso et al., 2014). on the other hand, a cultivar effect for sesquiterpenes and terpenes has been observed; for example, in cv. “cobrançosa”, the relative proportion of these compounds increased during olive maturation (malheiro et al., 2015). the same authors cited the cv. “verdeal transmontana” as the most susceptible and the cv. “cobrançosa” as the least susceptible to olive fly oviposition and affirmed that the susceptibility differences could be ascribed to olive volatile content and composition. thus, the volatile composition of olives may depend on the olive cultivar and be influenced by olive maturation. other authors (burrack and zalom, 2008; gonçalves et al., 2012) also support the view that a combination of several factors may affect the ovipositional preference of b. oleae. considering that cv. “simona” shows a particularly resistance to b. oleae, the aim of this work was the determination of volatile compounds released from the whole unripe and ripe olives harvested in an area of castellana grotte (bari, italy). moreover, the determination of volatile compounds in the oil obtained has been carried out in order to distinguish the selection of volatiles effected by the cuticle of whole fruits. we determined that the volatile compounds surrounding the whole olive fruits were represented by sesquiterpenes and hydrocarbons, while the volatile compound composition of the olive oil was characterized by aldehydes, alcohols, esters. the volatile compounds selected by the cuticle may represent a possible natural adaptation useful to reduce the susceptibility to olive fly attack. this preliminary study was focused on the headspace characterization of whole olive fruit of cv. simona which shows a particularly high resistant to fly attach. 2. materials and methods 2.1 description of the geographical area we evaluated the volatile compounds found in whole unripe and ripe olives harvested in an area of castellana grotte (latitude and longitude 40.86907314710559 and 17.1785811334848, respectively). in this area (2.4 ha), owned by the “agriculture” foundation, 170 “simona” olives are grown with other cultivars (e.g., “oliastra”, “coratina”, and “pasola”) in lower quantity. the olive production period covered in this study was october 2015 to january 2016. 2.2. evaluation of fly infestation level and maturity index following malheiro et al. (2016), to assess the fly infestation level of cv. “simona” in comparison with two other cv. “pasola” and “oliastra” growing in the same area, 20 random hand-picked fruits were collected in early november 2015 from five olive trees per cultivar, and the resulting 100 fruits per cultivar were inspected for signs of infestation in a stereo microscope smz-168 th – 1:6.7 zoom ratio (motic-italy) equipped with a primocam 5 hd zeiss digital camera (tiesselab-italy). for calculation of the maturation index, we followed the method described by hermoso et al. (2001), separating 100 olive fruits (20 fruits per tree) in levels from 0 to 7, and examining the epidermis and pulp color. if the epidermis was green, the fruit was classified as “0”. fruits showing epidermis of yellowish-green were classified as “1”. fruits showing red spots on less than half fruit were classified as “2”; epidermis red or purple on more than half the fruit led to classification “3”, and fruits with black epidermis and white pulp were classified as “4”. the level “5” was due to fruits with black epidermis ital. j. food sci., vol. 29, 2017 585 and purple on less than half the pulp and the level “6” was due to fruits with black epidermis and purple on more than half the pulp but not reaching the stone. the level “7” is due to fruits with black epidermis and whole pulp purple, reaching the stone. using the letters a, b, etc., to identify the number of fruits and the seven levels cited before, the maturation index is calculated as follows: mi = (a x 0 + b x 1 + c x 2 + d x 3 + e x 4 + f x 5 + g x 6 + h x 7)/100. 2.3. oil extraction process within 12 h of fruit harvest, oil was extracted using an “oliomatic 150” system, equipped with a hammer crusher, a vertical malaxator, and a two phase decanter (enoagricola rossi perugia), operated at 25 °c and with 3% of water. the oil was not filtered after the extraction. 2.4. olive oil samples oil was produced by olives of cv. “simona” at a maturation index between 3.5 and 5.5 and stored in a 5 litre dark glass bottle at 12 15 °c for 4 days before the hs-spme analysis. 2.5 hs-spme analysis of whole olive fruits and oil and gc-ms conditions for olive fruits, three aliquots of 80 ripe or unripe whole olive fruits were placed in a pyrex 250 ml round media bottle sealed with a screwcap equipped with a septum. for olive oil samples of cv. “simona”, three 100 g aliquots were weighed and placed into the same type of media bottle. for whole fruit and oil, the sealed bottle was placed in a thermostatic bath at 40 °c, with a magnetic stirrer for the oil aliquots. after 1 h, an spme fiber covered with 2 cm of dvb/car/pdms, 50/30 mm (divinylbenzene/carboxen/polydimethylsiloxane) (supelco, milan, italy) was inserted through the septum and left in the headspace for 5 h. with respect to the repeatability of the results, the headspace equilibrium, from to the cuticle of whole olive fruit, was reached in the time adopted. the volatiles absorbed by the fiber were thermally desorbed in the injection port of a gc/ms (gas chromatography/mass spectrometer) for 10 min at 220 °c in a gas chromatograph with a split/splitless injection port. the injector was operated in split mode (1:5). before sampling, the fiber was reconditioned for 15 min in the gc injection port at 240 °c, and blank runs were carried out before every analysis. the efficiency of the fiber was periodically verified by monitoring the signal of the internal standard. gas chromatography/mass spectrometry (gc/ms) analyses were performed in a shimadzu 2010 gas chromatograph coupled to a shimadzu qp-2010 msd quadrupole mass spectrometer (shimadzu, milan, italy). the gas chromatograph was equipped with a supelcowax -10 30 m × 0.25 mm column, 0.25 mm film thickness (supelco – italy). the operating conditions for the gc/ms were: helium flow 1.0 ml min-1, and oven temperature 40 °c for 5 min, increased to 240 °c at a rate of 3 °c min-1. the temperature of the ion source was 200 °c, the electron energy was 70 ev, and the interface temperature was 240 °c. mass spectra were acquired over the mass range 40–300 a.m.u. 2.6. volatile compound identification and quantification volatile compounds in the headspace of the whole olive fruits and the olive oil were identified by matching their mass spectra with the reference mass spectra of a private library (tateo-bononi oils) and the nist 147 library. ital. j. food sci., vol. 29, 2017 586 to verify that the fly repellent action produced by headspace of whole olive fruits was maintained by the same pool of volatile compounds identified in the unripe and ripe stage, we compared data expressed as normalized area counts from whole unripe and ripe olive fruits. these data were compared to the volatile composition of the oil extracted from cv. “simona”, expressed as normalized area counts. all data were normalized to the most represented volatile compound (a-copaene in unripe whole olive fruits). 2.7. statistical analysis all analyses were performed in triplicate and the results are reported as mean values. the significance of the differences between data of whole olive fruits and olive oil was confirmed using student’s t test. a p-value < 0.05 was set as a statistical threshold for significance. 3. results in the year 2015, the fly infestation measured with the method described in 2.2, and concerning our samples collected in the limited area described in 2.1, was not particularly high: the fruits of cv. “simona” showed no oviposition sites or exit holes, while the two other cv. showed 25% (“oliastra”) and 18% (“pasola”) b. oleae infestation. these data represent the mean of three replicate measurements. so, we can affirm that the values reported in this paper correspond to a meaningfully different behavior of cv. “simona” with respect of other cultivars considered for comparison from the same area. considering the headspace for whole olive fruit samples at two maturation index values 0.0 and 5.2, as shown in fig. 1 and in table 1, we deduced that the fly repellent action was maintained by the same pool of volatile compounds, even though their amounts were naturally modified by ripening. these compounds are sesquiterpenes and hydrocarbons, the two most highly represented classes of volatiles in the headspace of olive whole fruits. data reported also showed the substantially different composition between the headspace produced by whole olive fruits (ripe or unripe) and by olive oil. indeed, the olive oil headspace contained a very important fraction of more volatile oxygen-containing compounds such as alcohols ((z)-3-hexenol, (e)-2-hexenol, octanol), aldehydes (hexanal, (z)-3-hexenal, (e)-2-hexenal, nonanal), and some other typical compounds such as 5-ethyl2(5h)-furanone. in contrast, the volatile compounds released through the cuticle of the whole unripe olive fruits were almost exclusively represented by sesquiterpenes (acopaene, cycloisosativene, α-muurolene, and β-cubebene, in decreasing order of representation) and hydrocarbons ((e)-2-dodecene, undecane, tridecane, and 3-methyl undecane, in decreasing order). this result suggests that the effective fly repellent action is related to the pool of these volatile compounds. the data obtained demonstrate that gc/ms identification of the volatiles starting from whole fruits is fundamental for evaluating volatile compounds responsible for the fly repelling activity. however, the volatile composition reported in fig. 1 and derived from whole fruit of cv. “simona” appeared to be specific and not comparable to volatile composition of the olive oil, and the characterized pool of sesquiterpenes and hydrocarbons could be identified as repellent to b. oleae. ital. j. food sci., vol. 29, 2017 587 figure 1. hs-spme-gc/ms trace of unripe a) and ripe b) whole olive fruits cv. “simona.” peak identification: 1) ethyl-2-methyl butyrate; 2) (z)-3-hexenyl acetate; 3) undecane; 4) 3-methyl undecane; 5) (e)2-dodecene; 6) tridecane; 7) cycloisosativene; 8) α-copaene; 9) β-cubebene; 10) α-muurolene. ital. j. food sci., vol. 29, 2017 588 table 1. volatile compound identification and comparison of normalized for whole unripe (m.i. = 0.0) and ripe (m.i. = 5.2) olive fruits and olive oil (m.i. 3.5 5.5). values are normalized to the most represented volatile compound (α-copaene in unripe whole olive fruits). data reported demonstrate the substantially different composition between the headspace produced by whole olive fruits (ripe or unripe) and by olive oil. cv. “simona” unripe fruits cv% ripe fruits cv% oil cv% hexanal 0.2 0.01 (z)-3-hexenal 2.8 0.03 (e)-2-hexenal 33.6 0.48 (z)-3-hexenol 7.0 0.34 (e)-2-hexenol 2.9 0.03 (z)-3-hexenyl acetate 0.1 0.04 10.3 0.42 d3carene 1.8 0.02 5-ethyl-2(5h)-furanone 2.3 0.03 ethyl-2-methyl butyrate 0.4 0.02 5.3 0.62 octanol 0.2 0.01 nonanal 5.0 0.28 3-methyl undecane (h) 0.7 0.05 undecane (h) 6.7 0.92 0.2 0.07 dodecane (h) 4.6 0.25 (e)-2-dodecene (h) 15.9 0.74 4.3 0.73 26.3 0.51 tridecane (h) 2.5 0.03 phenyl ethyl alcohol 1.2 0.02 2,2-dimethyl-3-heptanone 6.7 0.13 cycloisosativene (s) 15.0 0.81 6.7 0.74 5.3 0.20 α-copaene (s) 100.0 1.20 47.0 0.82 45.8 0.40 α-muurolene (s) 8.8 0.58 4.1 0.05 17.0 0.32 α-farnesene (s) 46.4 0.42 β-cubebene (s) 2.1 0.07 0.7 0.08 total 152.1 68.4 122.4 σ sesquiterpenes (s) 125.9 58.5 114.5 σ hydrocarbons (h) 25.8 4.5 30.9 (s)= sesquiterpenes (h) = hydrocarbons 4. conclusions the resistance activity shown by cv. “simona” to fly oviposition was evidenced in a welldefined growing area of 2.4 ha as described in 2.1 where other cultivars were present but did not show the same repellent activity against b. oleae. considering that as of today the factors associated with repelling a fly attack have not been identified with certainty, we think that a concrete approach to investigate the fly repellent action could be the identification of the pool of active specific volatile compounds starting from whole fruit of other cultivars showing resistance to fly attach, following the method suggested in this paper. the analysis of whole fruit versus oil of cv. “simona” yielded two very different patterns of volatiles: the data demonstrate that the ital. j. food sci., vol. 29, 2017 589 whole fruit does not convey into the headspace the same qualitative and quantitative abundance of volatile compounds produced in the pulp, and by selective permeation, only selected volatile substances that are truly active in repelling the flies are conveyed into the headspace. this identification of a mixture of substances that can reduce the negative consequences of b. oleae attack is in accord with a fundamental principle of flavoring science and technology: no single volatile compound can always produce the best olfactory response, and often, various volatile substances collectively produce a more effective olfactory stimulus. thus, it is reasonable to suggest that a synergistic effect of a mix of various flavorings might underlie the repellence of b. oleae, and in the case of cv. “simona,” the active substances able to prevent the fly attack seem to be a pool of sesquiterpenes and hydrocarbons and no other volatile oxygen compounds. this proposed line of research is compatible with the accepted conclusion of many authors that fly repellence is related to the specificity of the cultivar. this analytical approach, based on the evidence of selective permeation of volatile compounds through the cuticle of the whole olive fruit, could be used to evaluate the composition of headspace surrounding the whole olive 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fax: +39 0874404855 e-mail address: marconi@unimol.it abstract arabinoxylans (ax) and β-glucans are the major source of soluble dietary fibre in cereals and have a significant role as functional/bioactive ingredients implicated in lowering plasma cholesterol, postprandial blood glucose and improving lipid metabolism. in this work, the variation in the content and solubility of ax and β-glucans in different cereal species and varieties were studied. different methods (phloroglucinol, orcinol-hcl, hpaec-pad) for ax analysis were tested. the results confirmed the variability in contents of both polymers (ax and β-glucans) in cereal species and varieties as well as providing additional information useful for their characterization. keywords: arabinoxylans, barley, β-glucans, emmer, spelt, wheat ital. j. food sci., vol 29, 2017 113 1. introduction the most common source of dietary fibre are the outer layers and the endosperm cell walls of cereal grains (wheat, barley, oat, rye etc.). the non-starch polysaccharides (nsp) found in mature cereal grain include the arabinoxylans (ax), which make up the pentosan portion of the insoluble fibre fraction and the β-glucans which are major components of starchy endosperm and aleurone cell walls. ax have been identified in a variety of tissues of major cereals: wheat, rye, barley, oats, rice, sorghum (fincher and stone, 1986). although these polysaccharides are minor components of the whole caryopsis, they still account for a substantial fraction of the cell walls and thus constitute a major portion of the dietary fibre of wheat flours (skendi et al., 2011). wheat varieties differ in ax amount and properties (maslen et al., 2007) such as average molecular weight and distribution, branching pattern, extractability with water (finnie et al., 2006) and interaction with other cell wall components such as lignin or cellulose (revanappa et al., 2007). ax consist mainly of a xylan chain with β-1,4-linked d-xylopyranosyl residues (xyl) to which mostly single α-l-arabinofuranose units (ara) are linked at the o-2 and/or o-3 positions of the xylose units as side residues (izydorczyk and biliaderis, 1995). in wheat ax, approximately 66% of the xylose residues, that form the backbone chain, are unsubstituted xyl (saulnier et al., 2007b). moreover, some ara units carry ferulic acid residues esterified to o-5 of ara linked to o-3 of the xylose residues (sosulski et al., 1982). extractable ax and unextractable ax structures are similar, showing only slightly differences in molecular weight and in the ara/xyl ratio (izydorczyk and biliaderis, 1995). ax represent the major polysaccharides in the aleurone fraction (65%) of the wheat caryopsis, and the arabinose to xylose ratio decreases from the pericarp to the endosperm; furthermore ax from the aleurone layer as well as ax from starchy endosperm have a lesser degree of branching than acidic ax from pericarp/testa, which may improve their solubility and their digestibility (brouns et al., 2012; saulnier et al., 2007a). β-glucans are linear polymers of high molecular weight consisting of d-glucose molecules linked by β-(1-4) and β-(1-3) linkages; the presence of β-linkages (1-3) gives to the molecule an irregular shape that makes the β-glucans flexible and partially soluble in water (papageorgiou et al., 2005; shelat et al., 2011). from a nutritional point of view, as major non-starch polysaccharides of various cereals and main constituents of cell walls of wheat, rye, barley and oat, β-glucans and soluble ax have nutritional benefits in humans (ward et al., 2008). the positive effects of ax are related to the ability to reduce postprandial blood glucose levels (garcia et al., 2007; lu et al., 2000), lowering levels of potentially toxic ammonia in the colon and the ability to reduce levels of triglycerides in the blood (garcia et al., 2006). over the last two decades β-glucans have been considered as bioactive ingredients due to their capacity in lowering plasma cholesterol, improving lipid metabolism, and reducing the glycaemic index (mӓkelӓinen et al., 2007; wood, 2007). these positive effects increased the popularity and consumption of cereal-based foods as well as of many other foods fortified with cell wall-enriched grain fractions, β-glucan concentrates and isolates (lazaridou and biliaderis, 2007). furthermore, the commission regulation (eu) no 432/2012 of 16 may 2012 (official journal of the european union 25-05-2012) included arabinoxilans and β-glucans in the list of authorized health claims. in this study, the content of ax and β-glucans in several cereal wholemeal flours was determined using different techniques, whose efficacy was compared. ital. j. food sci., vol 29, 2017 114 2. materials and methods 2.1. samples five barley varieties, acquarelle, braemar, kelibia, naturel, usa 2 (waxy variety), were provided by agroalimentare sud s.p.a. (melfi, potenza, italy). five spelt varieties, ebners rotkorn, hercule, oberkulmer, redoute, triventina, were provided by agenzia regionale per lo sviluppo agricolo, rurale e della pesca (arsarp, campobasso, italy). five emmer varieties, molise, angelo, garfagnana, molise colli, guardiaregia, were provided by arsarp (campobasso, italy). two durum wheat varieties, cappelli, saragolla, were provided by arsarp (campobasso, italy). three soft wheat varieties, roscetta, solina 1, bianchetta, were provided by arsarp (campobasso, italy). samples were milled in a refrigerated laboratory mill ika a 10 labortechnic (tanke & kunkel, gmbh & co., staufe, germany) and stored in aliquots of 100 g at 4°c in a closed containers until analysis. data reported for all parameters are the average values of three different aliquots of each sample. all results are expressed as % dry weight (d.w.). moisture content was determined according to icc method 109/1 (icc, 1995). 2.2. reagents naoh 50% (p/v) was purchased from baker (mallinckrodt baker b.v., deventer, holland), high-purity laboratory water was produced by means of a milliq-plus apparatus (millipore s.p.a., milano, italy); glucose, xylose, arabinose, fructose, standards were from sigma chemical co. (st. louis, mo, usa); all other chemicals and reagents of hplc grade were purchased from sigma chemical co. (st. louis, mo, usa). 2.3. ax analysis by phloroglucinol method total ax analysis was performed as reported by douglas (1981). flour (5.0 mg) was added to 2 ml of water followed by 10 ml of a solution of glacial acetic acid, hydrochloric acid and phloroglucinol in a stoppered tube. the tube was placed in a boiling water bath for 25 min and the absorbance of the resulting solution measured at 552 nm and 510 nm. 2.4. ax analysis by orcinol-hcl method water soluble ax and total ax analysis by orcinol-hcl method was performed as reported by hashimoto et al. (1987). water soluble ax: 100 mg of flour sample were shaken in water at 30°c for 2 h and centrifuged. aliquots of the supernatant were hydrolyzed with 4n hcl at 100°c. the ax content was estimated, after treatment in boiling water bath with fecl3 and orcinol and by reading the absorbance at 670 nm. total ax: flour (10 mg) was weighed into a glass tube, where 2 ml of 2n hcl was added, and the mixture was hydrolyzed at 100°c for 150 min. after cooling, neutralization was carried out by adding 2n sodium carbonate and fermentable sugars were removed by means of fresh compressed yeast. the mixture was then centrifuged and an aliquot of the supernatant was treated with fecl3 and orcinol in boiling water bath; the absorbance was read at 670 nm. ital. j. food sci., vol 29, 2017 115 2.5. ax analysis by hpaec-pad determination of arabinoxylans was performed as reported by messia et al. (2016). briefly, for water soluble ax: 100 mg of a flour sample were shaken in 10 ml of water at 30°c for 2 hours and centrifuged. aliquots (1 ml) of the supernatant were hydrolyzed with 1 ml of 4n hcl for 2 hours. for total ax, flour sample (10 mg) was weighed into a glass tube, 2 ml of 2n hcl were added, and the mixture was hydrolyzed at 100°c for 150 min. after cooling, neutralization was carried out by the addition of 2n sodium carbonate. diluted samples were injected in a chromatographic system equipped with a rheodyne injector (cotati, ca, usa) with a 25 μl loop. the chromatographic separation was carried out with a carbopac pa1 (250x2 mm) (dionex corporation, sunnyvale, ca, usa) analytical column. the chromatographic run (22 min) and the quantitative determination were conducted with a 0.25 ml/min flow rate, using a mobile phase of water and 200 mm sodium hydroxide (90%-10%). the control of the instrument, the data collection and the total quantification were carried out by the chromatographic software chromeleon (dionex). an hpaec-pad dionex system (dionex corporation, sunnyvale, ca, usa) composed of a gradient pump (mod gp50) with an on-line degaser and electrochemical detector (model ed40) was used. the flow-through electrochemical cell (dionex) consisted of a 1 mm diameter gold working electrode, a ph reference electrode, and a titanium body of the cell as the counter electrode. the optimized time-potential waveform used was: 0.1 v at 0-0.40 s, -2.00 v at 0.41-0.42 s, 0.60 v at 0.43 s, -0.10 v at 0.44-0.50 s. total and soluble ax were quantified on the basis of the ara and xyl content in the hydrolyzed sample: ([ara] + [xyl] x d x 0.88), where: d = dilution factor; 0.88 =adjustment for free sugar to anhydrous sugar. 2.6. β-glucans analysis total β-glucans were determined using the k-bglu assay kit (megazyme international ltd., ireland). insoluble fractions were determined after extraction of soluble β-glucans with water for 2 h at 38°c (åman and graham, 1987). soluble β-glucans were calculated as the difference between the total and insoluble components. 2.7. statistical analysis all analyses were carried out in triplicate. results were expressed as means±standard deviation (sd). a one-way analysis of variance (anova) was performed, considering species, variety or analytical method as factor. when significant differences (p < 0.05) were detected, fisher's least significant difference (lsd) was computed. all the statistical tests were performed using the software ibm spss statistics 23. 3. results 3.1. ax analysis and quantification a comparison between three different analytical methods (hpaec-pad and colorimetric methods) was carried out in order to devise a practical system for screening of different varieties of grains such as barley, wheat, spelt and emmer (table 1). ital. j. food sci., vol 29, 2017 116 table 1. total and soluble ax content in different cereal wholemeals determined by three different methods (g/100g d.w.±sd). sample phloroglucinol orcinol-hcl hpaec-pad total soluble soluble ax/ total ax total ax total ax soluble ax total ax a/x soluble ax a/x barley acquarelle 4.92±0.05b 6.42±0.06d 0.09±0.03a 6.18±0.03d 0.85 0.08±0.02a 0.65 0.013 braemar 4.30±0.04d 5.94±0.05e 0.06±0.06a 5.21±0.07e 0.75 0.07±0.03a 0.60 0.013 kelibia 5.08±0.06c 7.30±0.08b 0.08±0.04a 6.87±0.05b 0.73 0.10±0.03a 0.66 0.015 naturel 4.85±0.03b 7.01±0.03c 0.08±0.06a 6.76±0.04c 0.72 0.09±0.04a 0.62 0.013 usa 2 6.17±0.08a 9.75±0.04a 0.11±0.05a 8.96±0.03a 0.47 0.12±0.05a 0.71 0.013 mean 5.06bα 7.28aβ 0.08aβ 6.80aβ 0.70 0.09aα 0.65 0.013 wheat cappelli* 5.10±0.04c 8.78±0.05a 0.09±0.02a 8.21±0.04a 0.78 0.10±0.03a 1.25 0.012 saragolla* 5.18±0.03bc 8.17±0.04d 0.09±0.03a 7.90±0.02b 0.80 0.10±0.06a 0.80 0.013 roscetta** 5.49±0.05a 8.76±0.05a 0.11±0.03a 8.16±0.10a 0.66 0.12±0.03a 0.71 0.015 solina 1** 5.15±0.04bc 8.54±0.07b 0.10±0.04a 7.97±0.04b 0.71 0.12±0.04a 0.71 0.015 bianchetta** 5.23±0.06b 8.32±0.04c 0.11±0.03a 7.98±0.03b 0.74 0.13±0.05a 0.71 0.016 mean 5.23cα 8.51aα 0.10aα 8.04bα 0.74 0.11aα 0.84 0.014 spelt ebners rotkorn 4.18±0.05 b 5.57±0.04b 0.14±0.04a 5.41±0.05a 0.66 0.11±0.02a 0.77 0.020 hercule 4.29±0.06a 5.97±0.05a 0.14±0.03a 5.34±0.07a 0.68 0.12±0.02a 0.85 0.022 oberkulmer 3.99±0.04d 5.18±0.06c 0.12±0.03a 4.75±0.05c 0.64 0.12±0.04a 0.95 0.025 redoutè 3.89±0.07c 5.13±0.05c 0.14±0.03a 4.60±0.06d 0.61 0.10±0.02a 0.80 0.022 triventina 4.20±0.04b 5.63±0.10b 0.11±0.02a 5.03±0.03b 0.65 0.11±0.03a 0.78 0.022 mean 4.11cβ 5.50aγ 0.13aα 5.03bγ 0.65 0.11aα 0.83 0.022 emmer molise 3.65±0.04c 6.48±0.04b 0.23±0.04a 5.89±0.04b 0.67 0.18±0.05a 1.04 0.031 angelo 3.76±0.03b 5.26±0.06c 0.11±0.05b 5.03±0.05d 0.70 0.10±0.06b 1.16 0.020 garfagnana 4.12±0.09a 5.63±0.03d 0.11±0.03b 5.29±0.03c 0.62 0.11±0.05b 1.00 0.021 molise colli 4.06±0.05a 5.31±0.05c 0.09±0.05b 4.90±0.08e 0.58 0.07±0.04b 1.10 0.014 guardiaregia 3.60±0.06c 6.76±0.07a 0.12±0.04b 6.32±0.04a 0.69 0.08±0.05b 1.40 0.013 mean 3.84cγ 5.89aγ 0.13aα 5.49bγ 0.65 0.11aα 1.14 0.020 *durum wheat; **soft wheat different superscript letters (total ax=lower case, soluble ax=upper case) between species means within a row indicate statistically significant differences at p< 0.05. different superscript letters between means (species=greek font, variety=italic font) within a column indicate statistically significant differences at p< 0.05. the systematic quantification of ax in cereal wholemeal flours revealed substantial differences between the colorimetric procedures. although phloroglucinol and orcinolhcl procedures are both based on colorimetric assessments, for the measurement of total ax, the phloroglucinol method provided values that were significantly lower than those of the orcinol-hcl method. this underestimation of total ax can be attributed to the type of extraction performed for the phloroglucinol assay, that leads to a relevant release of hexoses from starch hydrolysis, which greatly exceeds pentoses, influencing their ital. j. food sci., vol 29, 2017 117 evaluation. in the orcinol-hcl method, the high glucose concentrations are removed by the action of the yeast saccharomyces cerevisiae, thus providing a better assessment of the total ax; additionally the orcinol-hcl method allows to evaluate not only the total ax but also the soluble ax, that cannot be quantified with the phloroglucinol method. a significant advantage of the hpaec-pad method is to provide more detailed information, as it offers the possibility to assess the content of individual sugars ara and xyl, which are constituents of the ax chain, and it allows to calculate the ratio ara/xyl (a/x), an important factor related to the behavior of the flour during technological processes. hpaec-pad assessments also give a more accurate estimation of the sugars present in the chain of ax without suffering problems caused by glucose interference, thus providing a more accurate and precise quantitative analysis. finally, the hpaec-pad can be applied to matrices with high contents of ax (bran) as well as to refined flour (low ax content). to sum up, the assessment made using the phloroglucinol is not reliable, whereas the values of total and soluble ax found in varieties of barley, wheat, spelt and emmer by using of either the orcinol-hcl or hpaec-pad methods are similar and comparable to the values of ax reported in the literature (berger and ducroo, 2005, henry, 1987) for these cereals. gebruers et al. (2008) have published data about the content of ax in refined flours and in bran of wheat, spelt and einkorn showing a comparable value of total ax in the different types of flour examined. instead, the results obtained in this trial show a much lower content of total ax in spelt an emmer (≈ 5.26%) compared to barley and wheat (6.80% and 8.04% respectively). this is in spite of the fact that spelt and emmer are phylogenetically close to wheat. the data obtained also show a generally close relationship between ax content and cereal varieties. among barleys, usa2, a waxy (i.e. having a low amylose content) variety has the highest content of β-glucans (9.5%) (table 2) and of total ax (6.2%). the content of ax in barley also depends on genetic and environmental factors (izydorczyk and dexter, 2008) but appears to be less variable than that of β-glucans. the hpaec-pad method allows to compute the ratio a/x which indicates the degree of branching in the polymer chain, thus permitting to deduce information about the ax structure of different species and varieties. a high a/x ratio corresponds to a higher proportion of mono-substituted xylosyl residues and a lower proportion of unsubstituted xylosyl residues. the degree of substitution of xylan backbone is relevant for predicting the cereal behavior when subjected to different technological processes. emmer varieties showed an a/x ratio of soluble ax (1.14), much higher than the ratios of barley (0.65), wheat (0.84) and also spelt (0.83). from a technological point of view, the quantification and the assessment of variations in the overall ax content is relevant because ax is generally considered to have a significant effect on wheat functionality and also to affect suitability of flours for certain applications (gebruers et al., 2008). cereal varieties rich in ax, have a strong potential for the production of healthy or even health promoting food products that contain not only a high overall dietary fiber content but also increased levels of soluble dietary fiber as well as prebiotic oligosaccharides which are produced by the in situ action of xylanases (gebruers et al., 2008). ital. j. food sci., vol 29, 2017 118 3.2. β-glucans analysis and quantification total, insoluble and soluble β-glucans were quantified in different cereals (barley, wheat, spelt, emmer) flours (table 2). results showed a different β-glucans distribution in tested species and between waxy and non waxy barley varieties. table 2. β-glucans content, ax+ β-glucans and ax/β-glucans in different cereal wholemeals (g/100g d.w.±sd). sample β-glucans total insoluble soluble soluble/ insoluble ax+βglucans ax/β-glucans barley acquarelle 4.43±0.13c 1.50±0.01e 2.94 1.97 10.61 1.40 braemar 3.89±0.11e 1.92±0.07b 1.97 1.03 9.07 1.34 kelibia 4.17±0.07d 1.61±0.02d 2.57 1.60 11.04 1.65 naturel 4.67±0.11b 1.81±0.04c 2.86 1.58 11.43 1.45 usa 2 9.49±0.09a 3.42±0.08a 6.08 1.78 18.45 0.94 mean 5.33a 2.05a 3.28 1.59 12.12 1.36 wheat cappelli * 0.52±0.03c 0.41±0.03b 0.11 0.27 8.73 15.79 saragolla * 0.42±0.02d 0.22±0.01c 0.20 0.93 8.32 18.81 roscetta ** 0.60±0.05b 0.42±0.06b 0.19 0.45 8.76 13.60 solina 1 ** 0.53±0.03c 0.37±0.03b 0.16 0.42 8.50 15.00 bianchetta ** 0.79±0.01a 0.60±0.03a 0.19 0.31 8.77 10.10 mean 0.57b 0.40b 0.17 0.47 8.62 14.66 spelt ebners rotkorn 0.68±0.02ab 0.44± 0.05ab 0.24 0.55 6.09 7.96 hercule 0.63±0.05b 0.42± 0.04ab 0.21 0.50 5.97 8.48 oberkulmer 0.70±0.02a 0.49± 0.03a 0.21 0.43 5.45 6.78 redoutè 0.65±0.03ab 0.47± 0.03a 0.18 0.38 5.25 7.08 triventina 0.57±0.02c 0.40±0.03b 0.17 0.43 5.60 8.82 mean 0.65b 0.44b 0.20 0.46 5.67 7.82 emmer molise 0.48±0.03b 0.37±0.01a 0.12 0.32 6.37 12.27 angelo 0.41±0.02c 0.29±0.05b 0.12 0.42 5.44 12.27 garfagnana 0.37±0.01c 0.28±0.03b 0.09 0.30 5.66 14.30 molise colli 0.53±0.02a 0.35±0.03ac 0.18 0.51 5.43 9.24 guardiaregia 0.48±0.03b 0.30 ±0.02bc 0.18 0.61 6.8 13.17 mean 0.45b 0.32b 0.14 0.43 5.94 12.25 *durum wheat; **soft wheat. different superscript letters between means (species=upper case, variety=lower case) within a column indicate statistically significant differences at p< 0.05. in barley, the variable β-glucans content can be influenced by genotype, culture practices and environmental growing conditions (newman and mcguire,1985; newman and ital. j. food sci., vol 29, 2017 119 newman, 2008). the presence of waxy genes can influence polysaccharides biosynthesis and their composition in the kernel. in the “normal” barley genotype, the starch is composed of about 25% of amylose and 75% amylopectin while in the waxy genotypes the starch is almost exclusively composed of amylopectin (95-100%). the reduced level of starch is usually accompanied with an increased content of β-glucans in the cell walls of the starchy endosperm (andersson et al., 2008). in fact, according to many research reports (abdel-aal et al., 2005; wood et al., 2003) a waxy barley variety (usa2) showed a β-glucans content which was twofold that of non waxy varieties. comparing the data from different species (table 2), it is evident that there is a significant difference in the average content of total β-glucans among barley and wheat, spelt and emmer (5.33% d.w. for barley, 0.57% d.w for wheat, 0.65% d.w. for spelt and 0.45% d.w. for emmer), confirming the data reported in the literature by other authors (skendi et al., 2003). with regard to soluble β-glucans content, varieties of wheat, spelt and emmer are characterized by very low levels of soluble β-glucans (average: 0.17%, 0.20% and 0.14% d.w., respectively), probably related to a higher ratio of cellotriose/cellotetraose, which generally amounts to 4.6 and 3.3 respectively in wheat and barley. statistically, although the distribution of cellotriose and cellotetraose units linked by β (1-3) is random (wood et al., 2003), the probability of a repetition of ordered cellotriose units is greater in wheat than in barley and oats (cui et al., 2000). since the structure is more ordered and more inter chain associations are favoured, the water solubility of β-glucans derived from wheat is lower than that of other cereals (lazaridou and billiaderis, 2007). the soluble β-glucans content affects the soluble/insoluble ratio, which is greater in barley (1.59) than in wheat, spelt and emmer (0.47, 0.46 and 0.43 respectively). data on total content of ax + β-glucans and their ratio (ax/β-glucans) (table 2) showed significant differences between the analyzed wholemeals. the high content of ax + β-glucans in barley corresponded to a low ax/β-glucans ratio, which is further reduced in the waxy barley variety usa 2 (0.94). while β-glucans are mainly present in barley, the ax are distributed in all the analyzed species and varieties. the relevant presence of two polymers in barley makes this cereal an excellent ingredient for the preparation of products with an increased content of total and soluble dietary fiber, capable of enhancing both the physiological effects and health benefits (verardo et al., 2011a; verardo et al., 2011b; vitaglione et al., 2010). moreover, β-glucans and ax are the chief structural constituents of cell wall in various tissues of the barley grain. in the starchy endosperm of mature barley grain, the matrixphase ax and β-glucans may represent up to 85% of total cell wall polysaccharides. the endosperm cell walls are mainly made up of β-glucans and contains a smaller amount of ax, while aleurone cell walls are composed primarily of ax (67-71%), with smaller amounts of β-glucans (26%). various studies have been carried out as to the possibility of producing barley flour enriched in β-glucans using air classification and dry milling methods to produce an enriched flour that can be used for producing different cereal products, such as bread, muffins, and pasta (marconi et al., 2000). 4. conclusions the present study revealed a wide variability in the content of ax and β-glucans in wholemeal of different cereal species and varieties. ital. j. food sci., vol 29, 2017 120 barley varieties showed a higher β-glucans and ax content compared to other cereal species/varieties. the assessment of the a/x ratio for ax and soluble/insoluble β-glucans ratio, which directly affect the behavior of cereal flours during transformation, are useful for deciding the end use of grains and evaluating the nutritional quality of the analysed grains. the hpaec-pad method proved to be advantageous compared to colorimetric methods. it allows to compute the ratio a/x which indicates the degree of branching in the polymer chain, thus permitting to deduce information about the ax structure of different species and varieties. the quantification and the assessment of variations in the overall ax content is relevant because ax is generally considered to have a significant effect on cereal flours functionality and also to affect suitability of flours for certain applications. cereal varieties rich in ax and β-glucan, have a strong potential for the production of healthy or even health promoting food products that contain not only a high overall dietary fiber but also increased levels of soluble dietary fiber, that should meet the health claims listed in the commission regulation (eu) no 432/2012. acknowledgements we would like to thank agroalimentare sud s.p.a. (melfi (pz), italy) and agenzia regionale per lo sviluppo agricolo, rurale e della pesca (arsarp), (campobasso, italy) for providing varieties of barley, wheat, spelt and emmer. this research was supported by miur (italian ministry of instruction, university and scientific research) as part of the prin project 2010 no. 2010st3amx_002. references abdel-aal e.s.m. and hucl p. 2005. hulles barley for food and feed. in “specialty grains for food and feed” e.s.m. abdel-aal, and p. hucl (ed.), p.171. aacc, st. paul, mn (usa). åman p. and graham h. 1987. analysis of total and insoluble mixed-linked (1-3), (1-4)-β-d-glucans in barley and oats. j. agr. food chem. 3:704. andersson a.a.m., lampi a.m., nyström l., piironen, v., li l., ward j.l., gebruers k., courtin c.m., delcour j.a., boros d., fraś a., dykowska w., rakszegi m., bedő z., shewry p.r. and åman p. 2008. phytochemical and dietary fiber components in barley varieties in the healthgrain diversity screen. j. agr. food chem. 56:9767. 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cereal sci. 69:138. newman r.k. and mcguire c.f. 1985. nutritional quality of barley. in “barley”. d. c .rasmusson (ed.), p. 403. american society of agronomy, madison, wi. newman r.k. and newman c.w. 2008. barley for food and health: science, technology, and products, p. 56. john wiley & sons, inc., hoboken, new jersey. papageorgeiou m., lakhdara n., lazaridou a., biliaderis c.g. and izydorczyk m.s. 2005. water extractable (1→3, 1→4) – [beta]-d-glucans from barley and oats: an intervarietal study on their structural features and rheological behavior. j. cereal sci. 42:213. revanappa s.b., bhagwat s.g. and salimath p.v. 2007. studies on pentosans in indian wheat (triticum aestivum) varieties in relation to chapatti making quality. food chem. 104:896. saulnier l., guillon f., sado p.e. and rouau x. 2007a. plant cell wall polysaccharides in storage organs: xylans (food applications). in “comprehensive glycoscience”. j. kamerling, g.j. boons, y. lee, t.a. suzuki, n. taniguchi and a.g. j. voragen (ed.), vol. 2, p. 653, oxford: elsevier science. saulnier l., sado p.e., branlard g., charmet and g. and guillon f. 2007b. wheat arabinoxylans: exploiting variation in amount and composition to develop enhanced varieties. j. cereal sci. 46:261. shelat k.j., vilaplana f., nicholson t.m., gidley m.j. and gilbert r.g. 2011. diffusion and rheology characteristics of barley mixed linkage β-glucan and possible implications for digestion. carbohyd. polym. 86:1732. skendi a., biliaderis c.g., izydorcyk m.s., zervou m. and zoumpoulakis p. 2011. structural variation and rheological properties of water-extractable arabinoxylans from six greek wheat cultivars. food chem. 126:526-536. ital. j. food sci., vol 29, 2017 122 skendi a., biliaderis c.g., lazaridou a. and izydorczyk m.s. 2003. structure and rheological properties of water soluble β-glucans from oat cultivars of avena sativa and avena byzantine. j. cereal sci. 38:15. sosulski f., krygier k. and hogge l. 1982. free esterified, and insoluble-bound phenolic-acids iii. composition of phenolic acids in cereal and potato flours. j. agr. food chem. 30(2):337. verardo v., gomez-caravaca a.m., messia m.c., marconi e. and caboni m.f. 2011a. development of functional spaghetti enriched in bioactive compounds using barley coarse fraction obtained by air classification. j. agr. food chem. 59:9127. verardo v., riciputi y., messia m.c., vallicelli m., falasca l., marconi e. and caboni m.f. 2011b. dietary fiber and flavan-3-ols in shortbread biscuits enriched with barley flours co-products. int. j. food sci. nutr. 62(3):262. vitaglione p., barone lumaga r., montagnese c., messia m.c., marconi e. and scalfi l. 2010. satiating effect of a barley beta-glucan-enriched snack. j. am. coll. nutr. 29(2):113. ward j.l, poutanen k., gebruers k, piironen v., lampi a.m., nystrom l., andersson a.a.m., åman p., boros d., rakszegi m., bedo z. and shewry p.r. 2008. the healthgrain cereal diversity screen: concept, results, and prospects. j. agr. food chem. 56(21):9699. wood p. 2007. cereal β-glucans in diet and health. j. cereal sci. 46:230. wood p.j., weisz j., beer m.u., newman c.w. and newman r.k. 2003. structure of (1,3)(1,4)-β-glucan in waxy and nonwaxy barley. cereal chem. 80(3):329. paper received august 5, 2016 accepted october 14, 2016 ijfs#68_chwastowska_siwiecka_bozza   ital. j. food sci., vol 28, 2016 391 paper gender differences in the chemical composition and selected properties of african catfish (clarias gariepinus burchell, 1822) meat i. chwastowska-siwiecka1*, n. skiepko1, j. f. pomianowski2, m.s. kubiak3, m. woźniak4 and m. baryczka1 1faculty of animal bioengineering, department of commodity science and animal raw material processing, university of warmia and mazury in olsztyn, oczapowskiego 5, pl 10-719 olsztyn, poland 2faculty of food sciences, department of commodity science and food research, university of warmia and mazury in olsztyn, pl. cieszyński 1, pl 10-950 olsztyn, poland 3the academy of hotel management and catering industry in poznań, nieszawska 19, pl 61-022 poznań, poland 4faculty of environmental sciences, department of fish biology and pisciculture, university of warmia and mazury in olsztyn, oczapowskiego 5, pl 10-719 olsztyn, poland *corresponding author. tel.: +48 0895233475; fax: +48 0895233833 e-mail address: iwona.chwastowska@uwm.edu.pl abstract the objective of this study was to analyze gender differences in the chemical composition and selected physicochemical properties of african catfish meat. the experimental material comprised fish younger than 1 year, with estimated body weight of 1 kg, cultured in an intensive fish farm. fillet acidity (ph24) was determined 24 h post mortem, and the color of external and internal fillet surfaces was described based on l*, a*, b*, c* and hº values. samples of ground meat were analyzed to determine their proximate chemical composition, energy value and tbars levels. the meat of male and female fish was characterized by high levels of total protein and fat, a low calorific value and optimal acidity. in males, internal and external fillet surfaces were darker and characterized by higher redness (a*) and lower yellowness (b*) values. in male fillets, significant (p≤0.01) correlations were noted between water content and fat content and between protein content and crude ash concentrations. the coefficients of correlation between protein content and l* values and between ash content vs. l* and b* values were also statistically significant (p≤0.01). in meat samples collected from male fish, the values of color components a* and b* were also highly correlated. high negative correlations were observed between protein concentrations and crude fat levels, and between ph24 vs. tbars values in female fillets. color lightness was also significantly (p≤0.01) positively correlated with oxidative stability. keywords: african catfish, meat and fillets, chemical composition, energy value, tbars, ph, color   ital. j. food sci., vol 28, 2016 392 1. introduction the geographic range of the african catfish (clarias gariepinus) extends from africa to south-east asia. in the course of evolution, the species has adapted to unsupportive environmental conditions. in its natural habitat, the catfish is an omnivorous predator that feeds on zooplankton, arthropods, mollusks, fish, reptiles and amphibians (vitule et al., 2006; amisah et al., 2009). in fish farms, the african catfish easily adapts to pond conditions and has relatively low requirements regarding water quality. the main goal of intensive cultures is to produce catfish with the highest body weight within a short period of time, which can only be achieved under optimal conditions. in commercial farms, fish weighing 800-1000 g are produced in 6-8 months. according to adamek (2011), fish heavier than 1200 g are in highest demand on the polish market. the meat of the african catfish is a valuable and cheap source of easily digestible protein of high quality. catfish meat is pink, almost boneless and tasty, which contributes to its popularity. the chemical composition of fish meat (in particular its protein and fat content), its energy value and sensory properties such as color, texture, taste and flavor are determined by feeding intensity, type and quality of feed, as well as natural feed intake (jankowska et al., 2007; puchała and pilarczyk, 2007). there is a general scarcity of data relating to differences in the performance traits of c. gariepinus females and males raised in pond cultures. therefore, the aim of the study was to analyze gender differences in the chemical composition and selected physicochemical properties of african catfish (c. gariepinus) meat. 2. materials and methods 2.1. experimental fish, diets and origin the experimental material comprised 60 african catfish (c. gariepinus) younger than 1 year, with estimated body weight of 1 kg, and an equal number of males and females. fish were harvested in autumn-winter of 2013 from a freshwater fish farm in northern poland. catfish were cultured in a 9000 l concrete pond (intensive system) with a closed circuit system and water temperature of 25±1°c. fish were manually fed (every 3 h) pelleted feed prepared at the farm. feed composition was as follows (per 100 kg): 17.8 kg of fish meal, 44.6 kg of extracted soybean meal, 14.9 kg of wheat grain, 7.4 kg of corn grain, 11.9 kg of rapeseed cake, 2.4 l of fish oil, and 1 kg of a vitamin-mineral premix. the nutrient content of feed was determined at the laboratory of the department of animal nutrition and feed science of the university of warmia and mazury in olsztyn (poland) according to aoac guidelines (2005). the pelleted feed mixture contained: 33.57% of total protein, 5.82% of crude fat, 6.45% of crude ash and of 3.80% crude fiber. its energy value was determined at 17.229 mj/kg. 2.2. preparation of skinned fillet samples fish were harvested 48 hours before slaughter, they were transferred to a separate pond at the farm, cleansed, stunned and slaughtered according to standard procedures (directive 2009/1099/ec). catfish were manually gutted (body cavity was slit open, viscera and blood clots were removed), decapitated (cut behind epicranium outgrowths), fins were removed (caudal, dorsal, abdominal and pectoral fins were cut off approximately 0.5 cm from the base), and fish were filleted (the entire muscle was removed and skinned). the research material comprised 60 raw fillets without skin (left   ital. j. food sci., vol 28, 2016 393 side carcass), cooled for 24 h to a temperature of 4±1°c in a frost co. chilling chamber with relative air humidity of 85%. all qualitative analyses were performed at the laboratory for meat quality assessment of the department of commodity sciences and animal raw material processing of the university of warmia and mazury in olsztyn (poland). 2.3. chemical composition, energy and tbars values in meat the fillets of male (n=30) and female (n=30) fish were ground in a laboratory grinder (three times) equipped with a 2 mm mesh, and they were thoroughly mixed to prepare samples for chemical analysis. the proximate chemical analysis involved determinations of: water content (pn-iso, 2000a), total protein content – by the kjeldahl method (pn-a, 2002) in foss tecator kieltec 2200 system i (höganäs, sweden), crude fat content accordingby soxhlet extraction (pn-iso, 2000b) in the foss tecator soxtectm avanti 2050 extractor (höganäs, sweden), and crude ash content (pn-iso, 2000c). oxidative changes in intramuscular lipids were analyzed by measuring the content of thiobarbituric acidreacting substances (tbars) in accordance with the method proposed by rak and morzyk (2002). absorbance was measured with the analityk jena ag specord 40 spectrophotometer (jena, germany) and expressed in mg of malondialdehyde per 1 kg of meat. the energy value of meat was calculated using conversion factors of 4.00 kcal (16.78 kj/g) for protein and 9.00 kcal (37.62 kj/g) for fat (jeszka, 2010). 2.4. acidity and color parameters of fillets muscle acidity was measured 24 h post mortem, immediately after carcass cooling (left side carcass) (n=60), with the 340i ph-meter and wtw tfk 150/e temperature sensor (weilheim, germany) equipped with a hamilton double pore combination glass electrode (bonaduz, switzerland). the ph-meter was calibrated against buffers with known ph before measurements (pn-iso, 2002). the color of fillets (n=60) was described based on the values of l* (lightness), a* (redness) and b* (yellowness) in the cielab system (cie, 1978) by measuring reflectance on the internal surface (abdominal side) and external surface of skinned fillets (fig. 1) at the same points relative to the surface. figure 1: color measurements on the external (a) and internal (b) surfaces of c. gariepinus fillets. measurement points 1, 2, 3 (own study).   ital. j. food sci., vol 28, 2016 394 measurements were performed in three replications in the hunterlab miniscan xe plus spectrocolorimeter (hunter associates laboratory inc., reston, va, usa). chroma (c*) and hue (h°) were calculated with the use of the respective formulas (hunt et al., 1991): c*= a! + b! and h°=tan-1(b/a). color analyses were performed with d65 light source, 10° standard observer and aperture diameter of 2.54 cm. measurements were carried out in fillets chilled at 4±1°c for 30 minutes. the spectrocolorimeter was calibrated against white and black standards before every measurement. 2.5. statistical analysis the results were processed statistically by one-way analysis of variance in the statistica v. 10.0 program (2011). they were presented in tables as mean values, standard deviation and standard error of the mean (sem). the significance of differences (p≤0.05 and p≤0.01) between the means of chemical composition, tbars values and physicochemical properties in male (n=30) and female (n=30) catfish was analyzed by the student’s t-test. the coefficients of correlation between the analyzed groups of properties were determined separately for male and female fish. data were checked for normal distribution (shapirowilk test) and equality of variances (levene's test) before statistical analysis. 3. results and discussions the average protein content of fish is determined at 16-20%, but it can be as high as 25% in tuna fish. the african catfish is an interesting species in view of its processing suitability and chemical composition. according to klasa and trzebiatowski (1992), the muscle tissue of catfish is characterized by a low fat content (3.5%) and a high total protein content (17.9%). in a study by kapeliński (2003), the fat content of farmed african catfish was estimated at 3-4%, which indicates that the species has a low calorific value and is particularly suited for cold smoking. łuczyńska et al. (2011) observed the lowest fat content (2.81%) in the dorsal muscle of carp, followed by trout (4.39%), whereas the highest fat levels were noted in salmon (11.57%). in this study, female fillets contained 17.42% of total protein and 5.76% of crude fat on a fresh weight basis. the above values were slightly higher than in males, but the observed differences were not statistically significant. crude ash content, which is the total amount of mineral compounds remaining after incineration, is also an important indicator of meat quality. according to skibniewska et al., (2012), the composition and content of those nutrients in the meat of livestock animals, including fish, is determined mainly by their availability in feed as well as by the species, physiological status and age of animals. the conducted statistical analysis also confirmed that gender had a significant influence on the ash content of african catfish meat. females were characterized by a higher (by 0.07%) crude ash content than males. in addition to higher concentrations of total protein, fat and crude ash, female fillets were also characterized by a lower water content in comparison with male fillets (75.15% in females vs. 76.03% in males). gender differences in the chemical composition of african catfish fillets have never been explored in the literature. the only relevant information contributed by other authors (polak-juszczak, 2007; skałecki et al., 2013) was that the water content of muscle tissue in various fish species is inversely proportional to the combined content of total protein, fat and crude ash.   ital. j. food sci., vol 28, 2016 395 table 1: chemical composition, energy and tbars values of meat and ph of c. gariepinus fillets. specification meat samples sem male (n=30) female (n=30) water content (%) 76.03±1.17 75.15±0.65 0.253 total protein (%) 17.32±0.69 17.42±0.26 0.248 crude fat (%) 5.15±1.35 5.76±0.65 0.298 crude ash (%) 1.06b±0.09 1.13a±0.02 0.017 energy value (kj/100 g) 484.32±60.12 508.87±22.63 10.283 tbars (mg mda/kg meat) 0.24±0.10 0.36±0.12 0.026 ph24 (left-side carcass) 6.32±0.08 6.27±0.09 0.021 the results are expressed as means±sd. the means denoted by different letters in rows differ significantly at a,b (p≤0.05); sem – standard error of the mean. polak-juszczak (2007) determined the total protein content of african catfish fillets at 17.90%, free fat content at 5.30%, crude ash content at 0.98% and water content at 75.53%, and similar results were noted in our study. in comparison with the data presented in table 1, the fresh meat of c. gariepinus evaluated by yanar (2007) was characterized by significantly lower levels of fat (3.64%) and crude ash (0.68%), but somewhat higher content of protein (17.85%) and water (77.89%). in the work of goda et al. (2007), the muscle tissue of african catfish administered standard feed contained 75.73% of water and 15.96% of protein. the cited authors also reported a significantly higher crude ash content (3.71%) and lower fat concentrations (4.60%). in studies of freshwater fish, the protein content of carp meat was determined at 17.21% by skałecki et al. (2013) and at 11.8517.74% by puchała and pilarczyk (2007). fat concentrations in carp muscles were determined at 4.44% by skałecki et al. (2013) and at 6.80-11.77% by puchała and pilarczyk (2007). significant variations in the fat content of meat from carp and other fish species can be attributed mainly to the type of administered feed which also influences the final body weight and total length of fish (goda et al., 2007; puchała, pilarczyk, 2007); skałecki et al., 2013). the energy value of meat is determined by its carbohydrate, protein and fat content. the calorific value of an average serving of fish (100 g) ranges from less than 400 kj to approximately 1225 kj. despite having higher gross energy value, fatty fish are still less calorific than other products of animal origin (jeszka, 2010). in our study (table 1), no statistical differences were determined between male and female fish, but a trend towards higher energy values (by 24.55 kj) was noted in the meat of female catfish. as a species with 7% fat content, the african catfish can be classified into a group of medium-fat fish in line with the polish standards (pn-a, 1999). the calorific value of meat from c. gariepinus males and females was relatively low at 496.59 kj/100 g on average. according to rosa et al. (2007), the energy value of 100 g of fresh muscle tissue of catfish reached 457.90 kj and was lower than the values presented in table 1 (484.32 kj/100 g for males and 508.87 kj/100 g for females). secondary products of lipid oxidation, whose presence is determined based on malondialdehyde (mda) concentrations, are a direct symptom of autoxidation processes that adversely influence meat quality (kamkar et al., 2014). in our study, chilled muscles of both male and female african catfish were characterized by low tbars values (0.24 and 0.36 mg mda/kg of meat, respectively), which was indicative of high oxidative stability of lipids. in the experiment conducted by yanar   ital. j. food sci., vol 28, 2016 396 (2007), the average tba levels of fresh c. gariepinus muscles reached 0.45 mg mda/kg. the initial mda content of fresh silver carp fillets was determined at 0.54 mg mda/kg by kamkar et al. (2014). ph value is a critical determinant of microbial growth and food spoilage. the ph of fish ranges from 6.7 to 7.0, and it fluctuates subject to season, feed, exposure to stress and activity levels (merkin et al., 2010; yanar, 2007). the data presented in table 1 indicate that african catfish males and females were characterized by similar ph 24 h post mortem (6.32 in males, 6.27 in females). according to marx et al. (1997), the boundary value of ph24 for fresh fish meat is 6.5. in a study by skałecki et al. (2008), cod meat was characterized by significantly higher ph24 (6.89) and ph48 (6.74) values than herring meat (6.67 and 6.49, respectively). the analysis of color on the internal surface of african catfish fillets (table 2) revealed that gender was significantly correlated with lightness (l*), the contribution of the red component (a*) and the yellow component (b*), and hue (hº). table 2: color parameters on the surface of c. gariepinus fillets. specification meat samples sem male (n=30) female (n=30) internal surface l* 46.65b±1.68 49.31a±0.85 0.422 a* 11.80a±1.34 8.69b±0.97 0.440 b* 15.15b±0.92 16.55a±0.41 0.220 c* 19.20±1.48 18.69±0.63 0.270 h° 52.08b±1.95 62.29a±2.10 1.268 external surface l* 41.81±1.69 43.07±1.43 0.376 a* 15.62±1.71 14.63±1.75 0.400 b* 11.20b±1.39 12.71a±1.52 0.365 c* 19.22±1.98 19.37±1.64 0.423 h° 35.64b±1.71 40.98a±2.17 0.810 the results are expressed as means±sd. the means denoted by different letters in rows differ significantly at a,b (p≤0.05) and a,b (p≤0.01); sem standard error of the mean. female muscles were characterized by higher values of l* (49.31) and b* (16.55) in comparison with male fillets, and the observed differences were statistically significant (p≤0.01). jankowska et al. (2007) evaluated the quality of the meat of the european catfish (silurus glanis) administered natural and formulated feed and did not find any differences in lightness or yellowness values. the l* values of the european catfish (47.98) were similar to those noted in the african catfish in our study (48.11). the contribution of yellow pigment in c. gariepinus fillets was determined at 15.85 on average in both sexes, and it was 5.81 higher than in s. glanis muscles. the color of fish meat is a species-specific trait which is determined by the number of red muscle fibers and pigment concentrations, including myoglobin, hemoglobin and carotenoids. wedekind (1995) reported   ital. j. food sci., vol 28, 2016 397 significant sexual dimorphism in the quality of c. gariepinus fillets. the meat of males was characterized by intense red coloration, a lower fat content and higher cohesiveness (toughness) in comparison with female fillets. in our study, the value of the a* coordinate measured on the internal surface of fillets was significantly higher (p≤0.01) in males (11.80) than in females (8.69), which is consistent with the results reported by wedekind (1995). the results presented in table 2 indicate that total chromaticity (c*) measured on the ventral side of the fillets was similar in males and females at 18.95 on average. despite an absence of statistically significant differences, chromaticity was lower (by 0.51) in female fillets. color hue differed significantly (at p≤0.01) between genders, and the value of hº measured on the internal surface of fillets was higher in females (62.29). the color profile of the external surfaces of c. gariepinus fillets is presented in table 2. the values of l* and a* were not influenced by gender. despite the above, male fillets were darker (l*=41.81) and more saturated with the red component (a*=15.62) than female fillets (43.07 and 14.63, respectively). the contribution of yellow, measured on the skinned side, was significantly higher (p≤0.05) in females at 12.71. chromaticity (c*) on the external surface of fillets was similar in both genders at 19.22 in males and 19.37 in females. statistical calculations revealed that color hue on the external side of the fillets was significantly higher in females (40.98) than in males (where it was up to 5.34 lower). the data presented in table 3 point to a highly significant (p≤0.01) negative correlation between water content and fat content (r = -0.88) in the meat of male african catfish. in male fillets, protein content was significantly (p≤0.01) correlated with crude ash levels (r = 0.84). in female meat samples, a high (p≤0.01) negative correlation was observed between protein concentrations and fat content (r = -0.85). table 3: coefficients of correlation between the chemical components of c. gariepinus fillets. parameter total protein crude fat crude ash male female male female male female water content 0.09 0.55 -0.88** -0.57 -0.13 0.26 total protein -0.50 -0.85** 0.84** 0.50 crude fat -0.23 -0.10 explanatory notes: *correlation coefficients are statistically significant at *(p≤0.05) and **(p≤0.01). the proximate chemical composition of fish meat was not significantly correlated with ph24 values in males or females (table 4). table 4: coefficients of correlation between chemical composition vs. ph24 and color parameters on the internal surface of c. gariepinus fillets. parameter ph24 l* a* b* male female male female male female male female water content 0.20 0.32 0.27 -0.16 0.29 -0.08 0.28 0.72* total protein -0.12 0.44 -0.81** -0.34 0.17 0.44 -0.45 0.31 crude fat -0.06 -0.60 0.06 0.38 -0.27 -0.48 -0.09 -0.13 crude ash -0.39 0.20 -0.76** 0.07 -0.17 -0.30 -0.77** 0.29   ital. j. food sci., vol 28, 2016 398 explanatory notes: *correlation coefficients are statistically significant at *(p≤0.05) and **(p≤0.01). a statistical analysis revealed a significant (p≤0.01) negative correlation between the protein and crude ash content of male fillets vs. color lightness measured on the internal surface of the fillets (r = -0.81 and r = -0.76, respectively). a significant (p≤0.01) negative correlation between crude ash content and the contribution of yellowness was also noted in male fillets (r = -0.77). water content was significantly (p≤0.05) correlated with the yellow component on the external surface of female fillets (table 5). in the meat of male catfish, a positive correlation (p≤0.05) was noted between fat content and the value of l* (r = 0.75). protein and crude ash content were not correlated with any color parameters on the external surface of fillets (table 5). table 5: coefficients of correlation between chemical composition vs. color parameters on the external surface of c. gariepinus fillets. parameter l* a* b* male female male female male female water content -0.62 -0.35 0.44 -0.09 0.45 0.71* total protein -0.48 -0.50 -0.00 0.60 -0.11 0.27 crude fat 0.75* 0.34 -0.36 -0.43 -0.32 -0.22 crude ash -0.30 -0.41 -0.11 0.34 -0.16 0.35 explanatory notes: *correlation coefficients are statistically significant at *(p≤0.05) and **(p≤0.01). a significant (p≤0.05) negative correlation (r = -0.68) was observed between the lightness (l*) and redness (a*) on the internal surface of female fillets (table 6). in male meat samples, parameter l* was positively correlated (p≤0.05) with the contribution of yellowness (r = 0.68). no significant correlations between the values of *a and *b were noted on the internal surface of male and female fillets. in the meat of female catfish, a positive correlation (p≤0.01) was observed between the values of l* and tbars (r = 0.78), whereas a negative correlation (r = -0.78) was noted between ph24 and tbars values (table 6). in male fillets, significant correlations (p≤0.05) were noted between the values of b* and acidity (r = 0.65) and between ph24 and oxidative stability (r = -0.67). in an analysis of parameters measured on the external surface of catfish fillets (table 7), an increase in color lightness was accompanied by a decrease in the contribution of redness and yellowness (r = -0.67 and r = -0.70, respectively; p≤0.05), and an increase in tbars values (r = 0.70, p≤0.05). in male fillets, a significant positive correlation was also noted between color parameters a* and b* (r = 0.86). table 6: coefficients of correlation between color parameters on the internal surface of fillets vs. ph24 and tbars values. parameter a* b* ph24 tbars male female male female male female male female l* -0.20 -0.68* 0.68* -0.02 0.28 -0.49 -0.19 0.78** a* 0.21 -0.01 0.50 0.05 -0.59 -0.29 b* 0.65* -0.21 -0.60 0.01 ph24 -0.67* -0.78**   ital. j. food sci., vol 28, 2016 399 explanatory notes: *correlation coefficients are statistically significant at *(p≤0.05) and **(p≤0.01). table 7: coefficients of correlation between color parameters on the external surface of fillets vs. ph24 and tbars values. parameter a* b* ph24 tbars male female male female male female male female l* -0.58 -0.67* -0.57 -0.70* -0.10 -0.58 0.59 0.70* a* 0.86** 0.01 0.17 0.34 -0.53 -0.52 b* 0.04 0.53 -0.29 -0.53 explanatory notes: *correlation coefficients are statistically significant at *(p≤0.05) and **(p≤0.01). 4. conclusions a statistical analysis revealed that the chemical composition, energy and tbars values in the meat of african catfish were not significantly influenced by gender. the analyzed fillets of male and female fish were characterized by a relatively high content of total protein, optimal concentrations of crude fat and a low calorific value. the muscle tissue of c. gariepinus was characterized by optimal acidity 24 h post mortem, and significant differences in this parameter were not observed between the genders. an analysis of color parameters revealed that the internal surface of male fillets was darker, more saturated with the red pigment (*a), less saturated with the yellow component (b*) and characterized by lower hue values (h°) in comparison with female meat samples. in female fillets, protein content was significantly correlated with fat concentrations, whereas in meat samples collected from male fish, significant correlations were noted between water content and fat content, and between total protein content and crude ash content. the value of parameter l* on the external surface of male fillets decreased with a rise in total protein and crude ash content. the contribution of yellowness (b*) was significantly correlated (p≤0.05) with water content in female fillets and with crude ash levels in male fillets (p≤0.01). in measurements performed on the external surface of male and female fillets, significant correlations were noted between fat content and color lightness, and between water content and yellowness (b*), respectively. the correlation coefficients indicate that an increase in tbars values led to a significant increase in color lightness on the internal surface of fillets and a decrease in the ph24 values of female muscles. a significant (p≤0.01) positive correlation was observed between redness and yellowness on the external surface of male fillets. in conclusion, the results of this study indicate that sexual dimorphism in the african catfish significantly differentiates the color parameters of fillets, but it has no influence on the chemical composition, ph, energy values and lipid stability of meat from males and females. references adamek j. 2011. african catfish. rearing technology. irś (pl). olsztyn, poland. amisah s., oteng m.a. and ofori j.k. 2009. growth performance of the african catfish, clarias gariepinus, fed varying inclusion levels of leucaena leucocephala leaf meal. j. appl. sci. environ. manage. 13(1):21-26. aoac. 2005. “official methods of analysis” 18th ed. association of official analytical chemists, arlington, va.   ital. j. food sci., vol 28, 2016 400 cie. 1978. recommendations on uniform color spaces-color difference equations. psychometric color terms. supplement no. 2 to 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(oncorhynchus mykiss, walbaum 1792) in the light of their own studies”. j. szarek, k.a. skibniewska, j. zakrzewski and j. guziur (ed.)., p. 22-27. elset (pl). olsztyn, poland. statsoft, inc. 2011. statistica (data analysis software system), version 10.0. tulsa, ok, usa. www.stalsoft.com. vitule j.r.s., umbria s. c. and aranha j.m.r. 2006. introduction of the african catfish clarias gariepinus (burchell, 1822) into southern brazil. biol. invasions. 8:677-681. wedekind h. 1995. dietary influences on product quality in african catfish (clarias gariepinus). j. appl. ichthyol. 11(34):347-353. yanar y. 2007. quality changes of hot smoked catfish (clarias gariepinus) during refrigerated storage. j. muscle foods. 18(4):391-400. paper received february 21, 2015 accepted december 4, 2015 paper ital. j. food sci., vol. 28 2016 73 keywords: fruit leather, antioxidant activity, phenolic content, sensory evaluation evaluation of fruit leather made from two cultivars of papaya zuhair radhi addai*1,2, aminah abdullah1, sahilah abd. mutalib1 and khalid hamid musa1 1school of chemical science and food technology, faculty of science and technology, university kebangsaan malaysia, 43600 bangi selangor, malaysia 2department of biology, faculty of education for pure science, thi-qar university *corresponding author: zuhair_2003@yahoo.com abstract two papaya cultivars were used to manufacture fruit leather. the objective of this study was to formulate papaya leather from locally grown papaya using natural ingredients like pectin, honey and citric acid. the fresh fruits were pureed and mix with natural ingredients, and dried in an oven at 60°c for 12 hours. the physicochemical properties and antioxidant activity were determined. the results showed that fruit leather made from hongkong cultivar is significantly (p<0.05) higher in sensory parameters as well as physicochemical properties and antioxidant activity. the phenolics content and antioxidant activity increased by process of drying the fruit leather compared to fresh fruits in both papaya cultivars. therefore, the consumer requirements for healthy and safe food products were respected. http://zuhair_2003%40yahoo.com 74 ital. j. food sci., vol. 28 2016 introduction like numerous fruits and vegetables, papaya is a rich source of antioxidants. antioxidants have a neutralising effect on free radicals, which are unstable molecules that can trigger a range of diseases, including cancers, cardiovascular and neurodegenerative diseases (prior et al., 1998). naturally occurring antioxidants have been examined by (prakash, 2010), who discerned that disease risk is reduced by such antioxidants as vitamin c, vitamin e, carotene, phenolic acids, phytate and phytoestrogens. similarly, epidemiological research has emphasised the important role of antioxidants derived from fruits and vegetables in preventing degenerative processes (ames et al., 1993). papaya fruit can be eaten fresh or as part of different processed foods, including baked products, beverages, cereals, confectionery, dairy snacks and sauces (ushbc, 2010). the demand for papaya as dried fruit is also high, alongside sultanas, peaches and apricots (lohachoompol, 2007). healthy products with dried papaya include breakfast cereals, energy bars and fruit snacks. in addition, there is a range of other papaya-based products, such as jam, jelly, papaya toffee, papaya bar, papaya squash, papaya soft drinks, papaya pulp powder, and others swamy and premnath (2010). fresh papaya is a seasonal fruit with a shelf life between one and two weeks. to respond to consumer demand, with fresh substitute is necessary to ensure year-round availability and drying is the most commonly used preservation method. as explained by (teshome, 2010), the drying process entails eliminating as much water as possible from the fresh fruit in order to inhibit enzyme and bacterial activity, thus halting decomposition. there are various types of drying processes, including sun drying, oven drying, cabinet drying, dehydrator drying and freeze drying. based on food type, from 2 to 30% of water is left in the dried foods. in addition to prolonging product shelf life, water content reduction ensures that the product is stable from a microbiological perspective and minimises deteriorating chemical reactions. fruit leathers are referred to the dried sheets of fruit pulp that taste sweet and have a soft, rubbery texture. their production involves the dehydration of fruit puree to a leathery sheet (raab and oehler, 1999). in this regard, the study had two goals: (a) to use locally grown papaya to make fruit leathers based by using on natural ingredients like pectin, honey and citric acid, as well as to determine the cultivar most suitable for the production of papaya fruit leather; (b) to analyse the extent to which the drying process affects fresh and processed papaya in terms of antioxidant content, physic-chemical properties and sensory evaluation. materials and methods samples collection and preparation papaya (carica papaya l. cv. hongkong and eksotika) fruits at the mature stage of ripening were collected from pusat flora cheras, jabatan pertanian, and hulu langat semenyih in selangor, malaysia. the fruits were selected to ensure uniformity in size (800 g to 1000 g) and color as well as to ensure freedom from diseases and infection. the selected fruits were transferred on the same day to the university kebangsaan malaysia food laboratory, bangi. the other three major ingredients used in the trials were honey (polleney honey, chaina), pectin (germany) and citric acid (usa). procedure for making papaya fruit leather for each cultivar, frozen papaya cultivars were thawed at 40c overnight in the fridge. six hundred grams of thawed papaya cultivars were weighed. honey 10% (v/v), 2% (v/v) of citric acid and % 6 (v/v) of pectin were weighed and mixed with papaya fruits. a cascade blender model ce071br (japan) was used to mix all these ingredients for 2 minutes to make a puree. cooking oil was lightly sprayed over trays made of stainless steel before 200 g of puree was spread uniformly over the trays with a metal spreader. the drying of the leather was done in the middle section of the cabinet dryer, which had been preheated to 60° ± 2°c. throughout the drying interval, the dryness of the leather was closely monitored. two batches were made for every cultivar each of them has three trays. the trays were dried for 12 hours for both papaya cultivars. a process flow chart of papaya leather production. ripe papaya ↓ washing ↓ pulping ↓ addition of honey + citric acid + pectin ↓ mixing ↓ cooking ↓ spreading on trays ↓ drying ↓ cutting into slabs ↓ packing ↓ labeling ital. j. food sci., vol. 28 2016 75 physiochemical properties of papaya fruits moisture content was measured by drying sample at 105°c overnight in memmert oven (germany). titratable acidity (ta) was determined from 10 ml of sample diluted with 50 ml of water, titrated with 0.1 n naoh and calculated as percent citric acid. total soluble solids (tss) were measured with an abbe refractometer at 20°c and ph was determined using ph meter using juice extracted directly from pulp. humidity content the moisture content was determined by drying samples of approximately 1 g at 105°c in an forced air oven (watson victor ltd, nz) for 24 hours. the textural of papaya leather were conducted with a stable micro system ta-eztest/ ags-hjapan). texture analyzer the procedures for operating the texture analyzer were stated in the standard operating procedure (sop). the following parameters were determined: hardness (g/f). the pulp color was longitudinally determined on four points of each flat side of the fruit using a minolta cr-300 colorimeter. the (l*) value represented the luminosity of the fruit, where 0 = black and 100 = white but the (a*) value ranged from the negative (green) to the positive (red) scale and the (b*) value ranged from negative (blue) to positive (yellow), (aoac 1998). antioxidants extraction papaya were peeled, cut into 1 cm slices and crushed in a food processor to produce uniform slurries. the mixture was prepared fresh to preserve the extracted antioxidant compounds. in the extraction process, about 1 g of papaya slurries were weighed in universal bottles and 10 ml solvent was added. solvents used were 50% aqueous methanol; samples (papaya slurries with solvents) were then homogenized using homogenizer (t 250, ika, germany) at 24,000 rpm for 1 min. all extracted samples were centrifuged by using tabletop centrifuge (mlx 210, thermo-line, china) at 4750 g for 10 min. the supernatants were collected for further analysis. total phenol content (tpc) antioxidant activity was determined using tpc based on the method of (musa et al. 2011). approximately 0.4 ml distilled water and 0.5 ml diluted folin-ciocalteu reagent were added to 100 μl papaya extracts. the samples (papaya extracts with folin-ciocalteu reagent) were set aside for 5 min before 1 ml 7.5% sodium carbonate (w/v) was added. the absorbances were taken at 765 nm wave length using a spectrophotometer after 2 h. the calibration curve of gallic acid (ga) was used for the estimation of sample activity capacity. the result was recorded in terms of mg of ga equivalents per 100 g of fresh sample (mg ga/100 g of fw). total flavonoid content (tfc) the tf content was determined by the colorimetric method as described by (abu bakar et al., 2009). a total 0.5 ml of the extract was mixed with 2.25 ml of distilled water in a test tube, followed by the addition of 0.15 ml of 5% (w/v) nano 2 solution. after 6 min, 0.3 ml of a 10% alcl 3 ·6h 2 o solution was added, and the reaction was allowed to stand for another 5 min before 1.0 ml of 1 m naoh was added. the mixture was mixed well by vortexing, and the absorbance was measured immediately at 510 nm using a spectrophotometer (epoch, biotek, usa). the results were expressed as milligrams of quercetin equivalents (qe) per 100 g of fresh sample (mg qe/100 g of fw). ferric reducing antioxidant power (frap) first, 300 mm acetate buffer frap reagent was prepared fresh as follows: ph 3.6 (3.1 g sodium acetate trihydrate plus 16 ml glacial acid made up to 1:1 with distilled water); 10 mm 2,4,6-tris (2-pyridyl)-s-triazine (tptz) in 40 mm hcl; and 20 mm fecl3·6h2o in the ratio of 10:1:1 to provide the working reagent. in addition, approximately 1 ml frap reagent was added to 100 μl papaya extracts, and the absorbances were taken at 595 nm wavelength using a spectrophotometer after 30 min. the calibration curve of trolox was established to approximate sample activity capacity. the result was recorded as mg of trolox equivalents (tes) per 100 g of fresh sample mg (te/100 g of fw) (musa et al., 2011). dpph radical scavenging activity based on the method of (musa et al. 2011) the antioxidant activity was assessed using a 2,2-diphenyl-1-picrylhydrazyl (dpph) scavenging system. the stock solution was obtained by dissolving 40 mg dpph in 100 ml methanol, which was stored at -20°c until further use. approximately 350 ml stock solution was mixed with 350 ml methanol to obtain the absorbance of 0.70±0.01 unit at 516 nm wavelength by using a spectrophotometer (epoch, biotek, usa). in the dark, approximately 100 μl papaya extracts with 1 ml prepared methanolic dpph solution was stored overnight for scavenging reaction. the percentage of dpph scavenging activity was determined based on the following equation: dpph scavenging activity (%) = [(a blank –a sample ) / a blank ] × 100, where a is the absorbance. 76 ital. j. food sci., vol. 28 2016 abts assay the abts radical cation (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) was generated by the interaction of abts (250 µm) and k 2 s 2 o 8 (40 µm). after the addition of 990 µl of abts solution to 10 ml of fruit extract, the absorbance at 734 nm was monitored. the percentage decrease of the absorbance was calculated and plotted as a function of the concentration of the extracts and trolox for the standard reference data (özgen et al. 2006). the following formula was used: percentage (%) of reduction power = [(a blank –a sample ) / a blank ] × 100, where a is the absorbance. oxygen radical absorbance capacity (orac) the orac assay was conducted according to (huang et al., 2002). the orac assay was carried out on a fluorescence microplate reader (fluostar omega, bmg labtech, multi-detection microplate reader, germany). peroxyl radicals were generated by aaph, and fluorescence microplate reader was used at an excitation wavelength of 485 nm and an emission wavelength of 525 nm. trolox was used as standard (50, 25, 12.5, 6.25, 3.12 mm). proper dilutions of papaya extracts were made with orac buffer (potassium phosphate buffer, ph 7.4). for each orac run, a micro plate was prepared containing 25uµ of trolox standards, buffer control, and sample dilutions, as well as 150ul of fluorescein (fl) solution. all orac analyses were performed at 370c with a 20 min incubation and 60 min run time. after the incubation, 25ul of aaph was added to each well for a final volume of 200 ul. the results were calculated using the differences of areas under the fl decay curves between the blank and a sample and were expressed as micromole trolox equivalents per gram of sample (µmol te/g). sensory evaluation a consumer acceptability sensory trial was conducted at university kebangsaan malaysia in the sensory evaluation laboratory. panellists comprised 30 volunteers who were staff or students at the university. each panellist was asked to taste two samples, one from both cultivar (2 x 2 cm square). attributes selected for the papaya fruit leather were colour, sweetness, sourness, flavour, texture and overall appearance. in this study, the hedonic scale was implemented; on a scale of 1 to 7 there were tabulations of scores, where 1 indicates “extremely dislike” and 7 represents “extremely like” (aminah, 2004). for reliability purposes, distilled water was given to the panelists for them to rinse the mouths between evaluations. statistical analysis data were expressed as the means values ± standard deviation. mean of minimum three measurements were compared by analysis of variance (anova). significant differences between means were determined by duncan (p<0.05). correlation analysis was performed using pearson’s. the software used was spss ver.19. (bryman and cramer, 2012). results and discussion physicochemical properties of papaya the ph, titratable acidity and tss for the two papaya cultivars are shown in table 1. the cultivars exhibited considerable differences in terms of ph (p<0.05). the hongkong cultivar had a higher level of ph (5.47), while the eksotika cultivar had a lower ph (5.34). in comparison to fresh fruit, drying caused a substantial decline in the ph of all fruit leathers (p<0.05). furthermore, there were significant discrepancies between the average ph of the hongkong cultivar (3.93) and that of the eksotika cultivar (3.82). likewise, (harsimrat, 1998) demonstrated that acidity has a positive effect on shelf life. by contrast, (babalola et al., 2002) found that papaya leather stored in a cool environment for 30 days had a higher ph compared to other samples. similar findings were obtained in the case of pineapple leather (phimpharian et al. 2011), mango leather (azeredo et al., 2006) papaya and guava leathers (babalola et al., 2006). the present study revealed that the titratable acidity differed substantially between the cultable 1 effect of processing on ph, ta and tss of two papaya cultivars. result showed mean ± standard deviation. cultivars ph ta tss fresh leather fresh leather fresh leather hongkong 5.47±0.02a 3.93±0.01b 0.15±0.01b 1.63±0.02a 11.74±0.52b 68.50±0.57a eksotika 5.34±0.03a 3.82±0.01b 0.17±0.02b 1.48±0.03a 12.46±0.14b 70.50±0.60a a-b mean with different letters within each raw are significantly different (p< 0.05). ital. j. food sci., vol. 28 2016 77 tivars of both fresh and dried papaya. hongkong and eksotika had the highest average total acid content in fresh fruit, with 0.15% and 0.17%, respectively, of citric acid (reference). in the case of all fruit leathers, drying determines a considerable increase in the titratable acidity. as shown in table 1, the highest acidity among fruit leathers was shown by the cultivar hongkong (1.63%), while the lowest was exhibited by eksotika (1.48%). in keeping with beaudry et al. (1992), the results of this study indicated that the titratable acidity (ta) of fresh papaya is between 0.3 ± 0.1 to 0.7 ± 0.1% of citric acid equivalent. the addition of 4% of citric acid to the fruit leather puree increased the ta of the two papaya leather cultivars. in addition, drying contributes to concentration in the fruit’s natural acidity, leading to a significant increase in the acidity of the fruit leather. among all cultivars, eksotika displayed the highest levels of titratable acid (0.17%). however, after drying, ta was highest in leathers with concentrations between 1.48 and 1.63 %. the high levels of acidity in fruit leather not only inhibit bacterial growth, but also protect the colour and flavour of the fruit. hence, in terms of processing or manufacturing, papaya cultivars with high acidity should be used. this study also found that hongkong cultivars is most appropriate for fruit leather production. vaidya et al., (2007) reported the acidity of fruit leather made from kiwifruit which was found to be 3.8% but the reason for the high acidity was not discussed. vega galvez et al. (2009) reported an acidity of 2.2 ± 0.12% (monohydrated citric acid), ph of 2.7 ± 0.09, and soluble solids of 15.0 ± 0.07 °brix in dried o’neil blueberries. the two cultivars also differed greatly with regard to tss (p<0.05). the tss of hongkong cultivar fresh fruit was 11.74 while eksotika fruit cultivar was 12.46. although there is a substantial increase in the °brix of all fruit leathers after drying, with a tss of 68.50 and 70.50 respectively for both papaya cultivars (hongkong and eksotika). the two cultivars did not display noticeable discrepancies. all processed papaya leathers had higher tss than fresh fruit. the higher levels of °brix in fruit leathers compared to fresh fruit, particularly sweet fruit, had already been noted in earlier research. for example, the addition of ingredients, such as pectin, glucose, syrup, and sugar, to raw pineapple puree increased the tss of the latter between 66.4 and 75.3 °brix. the pineapple fruit leather had a final tss of between 82.4 and 86.9 after drying (phimpharian et al., 2011). in the case of kiwi fruit, (vaidya et al., 2007) observed that the addition of 15% sugar increased its °brix, which became even higher (68 °brix) after the fruit was dried in a cabinet drier for 15 hours at a temperature of 45° ± 2° c. the present study used honey as an added sweetener. in this research, creamed honey (83 °brix) was added as a sweetener. the °brix found in creamed honey was simfig. 1 effect of processing on moisture of two papaya cultivars. a-b mean with different letters are significantly different (p < 0.05). ilar to that found by anupama et al. (2003). the °brix of blueberry fruit leathers increased due to the addition of 15% of honey. the high °brix of blended papaya leather was explained by (kumar et al., 2008) in terms of a high carbohydrate content, making it a good energy source. humidity content of fresh fruit as illustrated in fig. 1, the moisture content of two papaya leathers were 14.31% and 15.42%, (hongkong and eksotika) respectively. the fruit leathers from different cultivars exhibited discrepancies with respect to moisture content levels (p<0.05). in this study, given the relative reduced moisture content (22%-24%), all cultivar leathers can be classified as concentrated or intermediate moisture foods. although the product thickness may be one of the reasons for high moisture content in this product. nevertheless, the final product thickness was decreased from 4 mm to 1 mm. based on their research on hot air drying of grape leather, maskan et al. (2002) argued that drying of the product surface occurs rapidly at high temperatures, particularly in the case of thinner samples. the moisture content of jackfruit leather, papaya leather and blended papaya leather was determined to be 11-17% (che man et al., 1992), 12-13% (chan and cavaletto, 1978) and 20.80% (kumar et al., 2008), respectively. however, irwandi et al. (1998) emphasized that, despite suppressing bacterial development and extending shelf life, a low moisture content of fruit leathers may have an adverse effect on texture quality. huang and hseih (2005) found that increasing the pectin concentration (from 1 to 1.5%) affected the hardness of the sample and decreased moisture content and aw of pineapple fruit leathers had an a w value of < 0.55. similarly, phimpharian et 78 ital. j. food sci., vol. 28 2016 al. (2011) reported that both moisture content and water activity were influenced by the pectin concentration. what is more, the pectin concentration also affected aw on pear fruit leather. texture of papaya leather the papaya leather cultivars in hongkong and eksotika had a range texture (fig. 2) of 490.48 483.60 respectively. a possible cause for the high texture was the pectin, which generated a firm gel structure followed by a tough texture. the high texture due to the 6% pectin concentration used in this study was combined with the reduced moisture content of the papaya leather. reduced moisture content and harder texture are the outcome of higher temperatures and extended drying periods (che man, 1995) and (okilya et al., 2010). a comparison was difficult to achieve due not only to the different genetic structure of this fruit (babalola et al., 2002), but also to extra ingredients that influenced the texture quality. gujral and khanna (2002) found that (2005), the values obtained in this study were considerably lower. colour measurements the final fruit leather product (table 2) was lighter in colour (l* mean value of 32.10 and 30.70) less than the fresh fruit (l* mean value of 48.52 and 45.43). all cultivars decreased in brightness (l*) indicating that fresh papaya had a lighter colour compared to the fruit leathers. this was expected as the drying and addition of pectin, honey and citric acid to papaya puree can have significant effects on the colour of the papaya fruit. for example, citric acid is a strong acid and the addition of citric acid in the papaya puree may have impacted the stability of the anthocyanins. anthocyanins are highly unstable and very susceptible to degradation. anthocyanins are oxidised in the absence of oxidase enzymes and subsequent condensation reactions can lead to brown pigment formation (singleton, 1987). this reaction may have led to colour changes in the papaya fruit leather but the addition of citric acid was necessary in the production of papaya fruit leather as it protected the natural colour and helped destroy bacteria during drying. pectin concentration has also been found to affect the colour of the product as the absorbance intensity was decreased in the production of jam, which suggested a relationship between pectin and anthocyanin degradation (dervisi et al., 2001). significant increases in l* values after drying were also observed by yang and atallah (1985). the authors suggested that in both forced air and micro-convection dried papaya increased l* values indicated a higher loss of anthocyanin from thermal degradation. however, for a* values a significant decrease was found in both papaya cultivars after drying, which may be due to anthocyanin oxidation as well as heat degradation during dehydration. in this study, papaya fruit leather also showed lower a* (13. 21 and 11.61) than fresh fruit (from 27.24 and 20.34) for two papaya cultivars hongkong and eksotika respectively. the b* values in c. papaya (hongkong) were higher (10.25) compared to c. papaya (eksotika). the extra added ingredients and the drying process had an effect on the b* value. the leather cultivars in hongkong and eksotika had an average b* value of 10.25 and 8.84 respectively. similar results were also observed in previous studies with jackfruit leather (che man and sin, 1997; okilya et al., 2010) and blended papaya leather (kumar et al., 2010). after drying, these fruit leathers became darker. this was especially prevalent in light coloured fruit leather (raab and oehler, 1999). other factors that can also affect papaya anthocyanins are: ph, storage, temperature, light, light, oxygen, concentration and structure of anthocyanins, other flavonoids, protein and minerfig. 2 effect of processing on texture of two papaya cultivars. a-b mean with different letters are significantly different (p < 0.05). the tensile force in the mango leather was reduced by increased levels of sucrose (ranging from 4.5% to 9%). the texture decreased even more when skim milk powder was added, in comparison to soy protein concentrate. in this study, it is probable that the texture or extensibility of papaya leathers was affected by the pectin, honey and citric acid that were added. huang and hsieh (2005) obtained a hardness value for pear fruit leathers of between 4420 and 13200 g (18 formulations with various water, pectin and corn syrup ratios). there were also differences in terms of ingredients, while the texture of the leathers may have been influenced by water absorption and the protein content of the fruit (babalola et al., 2002). by contrast to the results of huang and hsieh ital. j. food sci., vol. 28 2016 79 oxidant activity (frap, dpph, abts and orac) were illustrated in tables 3 and.4. the fresh fruit showed different trends with regard to total phenolic content and total flavonoid content. the tpc and tfc were higher in eksotika (62.59 mg gae/100g dw and 45.40 mg qe/100g dw, respectively) than in hongkong (49.61 mg gae/100 g dw and 40.01 mg qe/100g dw, respectively). also an antioxidant activity (frap, ddph, abts and orac) was higher in eksotika (197.41 mg te/100g dw, 71.48%, 73.89% and 13.62 µmol te/g dw, respectively) than in hongkong (127.74 mg te/100g dw, 49.62%, 61.84% and 11.50 µmol te/g dw, respectively). antioxidant capacity and phenolic concentration were found to differ according to the types of papaya cultivars. the reason for this may be differences in regions, climate, as well as in the solvents employed for extraction. furthermore, antioxidant activity may also be affected by lipid composition, antioxidant concentration, temperature, ph, oxygen, and water. compared to earlier research carried out by (connor et al., 2002), (ehlenfeldt and prior, 2001) and (prior et al., 1998), this study reported higher levels of antioxidant activity. although dried fruit leathers showed comparable trends, fruit leather had higher tpc, tfc and antioxidant activity (frap, dpph, abts, and orac) than fresh fruit. drying determines increase in the levels of antioxidant activity and phenolics in both cultivars. however, eksotika exhibited a greater increase in antioxidant activity than hongkong. the fruit leathers showed different trends with regard to total phenolic content and total flavonoid content. tpc and tfc were higher in eksotika (121.41 mg gae/100g dw and 108.78 table 3 the effect of processing on the total phenolics content and total flavonoids content of two papaya cultivars. result showed mean ± standard deviation. phenolics fresh leather hongkong eksotika hongkong eksotika tpc 49.61±1.03 a 62.59±1.09 b 104.71±2.50 b 121.4±1.79 a tfc 40.01±1.26b 45.40±0.82 a 91.43±1.54 b 108.78±1.77 a a-d mean with different letters within each raw are significantly different (p< 0.05). table 2 effect of processing on colour of two papaya cultivars. result showed mean ± standard deviation. cultivars l* a* b* fresh leather fresh leather fresh leather hongkong 48.52±1.02a 32.1±0.71b 27.24±0.51a 13.21±0.20b 31.71±0.50a 10.25±0.41b eksotika 45.42±1.13a 30.7±1.01b 20.34±0.42a 11.61±0.18b 29.41±0.15a 8.84±0.65b a-b mean with different letters within each raw are significantly different (p< 0.05). table 4 the effect of processing on antioxidant activity (frap, dpph, abts and orac) of two papaya cultivars. result showed mean ± standard deviation. antioxidant activity fresh leather hongkong eksotika hongkong eksotika frap 127.74±1.88b 197.41±2.50a 231.51±3.87b 284.32±1.10a dpph 49.62±108b 71.48±0.87a 76.11±0.13b 89.47±102a abts 61.84±0.86b 73.89±1.79a 84.97±0.60b 92.12±1.52a orac 11.50±0.72b 13.62±0.96a 29.54±0.24b 34.40±1.91a a-d mean with different letters within each raw are significantly different (p< 0.05). als. these factors were associated with colour changes in papaya fruit. in this study, during the development of fruit leather the interactions between heat and the food ingredients may have significantly affected the anthocyanins’ stability and this could have resulted in the colour change of the fruit. ingredients such as honey contain antioxidants as well as hydrogen peroxide, which may cause degradation of anthocyanins by oxidation mechanism or by indirect oxidation (lohachoompol, 2007). also, it is noted that under high concentrations of oxygen and ascorbic acid increased pigmentation loss occurred which resulted in change to the colour of papaya. other major factors mentioned by irwandi et al. (1998) that influenced the colour of fruit leathers were: processing condition, storage time and temperature. phenolics content and antioxidant activity a comparison between fresh fruit and fruit leathers in terms of the total phenolic content (tpc), total flavonoid content (tfc), and anti80 ital. j. food sci., vol. 28 2016 mg qe/100g dw, respectively) than in hongkong (104.71 mg gae/100 g dw and 91.43 mg qe/100g dw, respectively). also, an antioxidant activity (frap, ddph, abts and orac) was higher in eksotika (284.32 mg te/100g dw, 89.47%, 92.12% and 34.40 µmol te/g dw, respectively) than in hongkong (231.51mg te/100g dw, 76.11%, 84.97% and 29,54 µmol te/g dw, respectively). as previously shown, in both cultivars, drying causes increase in phenolics and antioxidant activity within the range of 50% to 53%. this increase is due to loss of moisture from the samples and thus reflected in the weight, leading to an increased concentration, as well the addition of honey and lemon contributed to the increase in phenols and antioxidants. total phenolic contents assay is known to overestimate the content of phenolic compounds, because other agents present in food, such as carotenoids, amino acids, sugars and vitamin c, can interfere (bahorun et al., 2004; luximonramma et al., 2003). furthermore there may be a contribution of millard reaction products to the total phenolic and antioxidant activity (zhuang and sun, 2011). oxidation produces free radicals which are taken up by the vitamins and polyphenols. reports that the antioxidant activity of partially oxidised polyphenols is higher compared to that of non-oxidised phenols have prompted further research. as highlighted by garau et al. (2007), there are other factors that may contribute to a reduced antioxidant activity; these include extended drying intervals. despite the use of identical cultivars, it is difficult to generate a comparison between the antioxidant activity results of this study and those of earlier ones, due to differences in the assays, extraction techniques and standards (te, gae) employed. moreover, apart from fruit quality, antioxidant activity is also influenced by factors such as geography, environment, climate and harvesting practices. the analysis of the impact of drying on total phenolic content revealed that, in contrast to fresh fruit cultivars, there was a reduction in total phenolic content. thermal deterioration is the likely cause for the increase in the total phenolic content of the two cultivars. furthermore, di scala et al. (2011) specified that the total phenolic content may also decline due to dehydration, during which polyphenols bind to other compounds, such as proteins, or their chemical structure undergoes changes that extable 5 correlation coefficients of antioxidants activities of different papaya cultivars. correlation coefficient (r2) frap dpph abts orac tpc 0.95 0.80 0.87 0.98 tfc 0.92 0.86 0.85 0.98 fig. 3 the effect of processing on texture of two papaya cultivars. a-b mean with different letters are significantly different (p < 0.05) isting techniques are unable to extract or identify. in the present study, although the increase was significant, papaya fruit leathers exhibited higher antioxidant activity and phenolics content than fresh fruit. correlation of tpc and tfc with frap, dpph, abts and orac assays a correlation analysis among phenolic compounds (tpc and tfc) assays, and antioxidant activity (frap, dpph and abts) was performed regardless of the extraction cultivars. a high correlation (table 5) was found between tpc, tfc and antioxidant activity (fpap, dpph abts and orac) for both cultivars (hongkong and eksotika). thus, it can reasonably be concluded that in the extract, antioxidant activity is related to the active component. findings of researches of correlation analyses among tpc, tfc, and antioxidant activities (frap, dpph, and abts) are high (mahattanatawee et al., 2006). there have been significant effects on the antioxidant activities of papaya fruit. sensory evaluation the statistical analysis of the sensory evaluation was conducted on the basis of 30 responses. the average scores for six properties of all fruit leathers are presented in fig. 3. a score of 1 signified ‘dislike extremely’, while a score of 7 signified ‘like extremely’. the cultivars differed significantly (p<0.05) in terms of colour, sweetness, sourness, flavour, texture, and general product acceptance. of the two types of cultivars, eksotika achieved the lowest acceptability scores of colour. this implied the importance attributed to the visual appearance of the product. there was a greater preference for the papaya leather cultivar hongkong, due to its reddish colour, than for the dark coloured eksotika. surprisingly, the results for fresh papaya were different. in a study undertaken by (saftner et al., 2008), the highest scores among all cultivars ital. j. food sci., vol. 28 2016 81 were obtained by the highbush cultivars coville and hannah’s choice for the intense blue colour, acceptable appearance, colour, fruit size, sweet/tart balance, flavour and overall eating quality. in a different study, gujral and khanna (2002) increased the sucrose level in order to enhance the colour, flavour and texture of mango leather. such approaches should be applied in the case of blueberry fruit leathers as well, to improve the darker colour of certain varieties of blueberry. similarly, the colour of the papaya leather could be enhanced by adding other colourless fruit (dervisi et al., 2001). the fruit leathers obtained an average sweetness score of 6.00. this meant that the panelist ‘liked’ the product sweetness and thus it was necessary to add honey. however, as warned by kumar et al. (2008), the overall taste rating may decline due to an excessive increase in the amount of sugar. the sourness of the papaya leather was ‘moderately liked’ by a panelist, with an average score of 5.6. moisture content and duration of drying have an impact on the texture of fruit leather. the moisture content is reduced and the texture is hardened by higher temperatures and extended drying intervals (okilya et al., 2010). furthermore, the texture quality of the end-product may also be affected by the addition of flavour and colour-enhancing ingredients like pectin, honey, sugars, nuts, salt and other fruits (raab and oehler, 1999). eksotika cultivars obtained a lower flavour score (5.2) than hongkong (6.0). okilya et al. (2010) explained that the amount of sugar within the fresh pulp affects how the fruit leather tastes. the taste of papaya fruit leather was enhanced in this study by adding honey and citric acid. similarly, kumar et al. (2008) noted that papaya and guava fruit leather were affected by the addition of extra ingredients. compared to individual scoring, the overall score for sensory attributes was considerably improved by the addition of 60% papaya and 40% guava, the nutritional and textural quality of the fruit leather remained unaffected. it was necessary to make this addition in order to enhance the low scent of the papaya fruit, which constituted a major obstacle to the commercial use of this fruit. as specified by (raab and oehler 1999), the taste of fruit leather could also be improved by using additional ingredients like leaf oregano and garlic salt. the blueberry fruit leather achieved an average overall score of 5.0, indicating that the panelists ‘moderately liked’ it. theoretically, the overall reception of all sensory attributes of the papaya fruit leather was the reason for its overall acceptability. the hongkong cultivar obtained an overall acceptable score of 6.00 making it the best liked fruit leather. the colour, appearance, sweetness, sourness, texture and flavour of the cultivar determined the preference of the panelists for it. furthermore, hongkong received an overall acceptability score of 6 out of 7. on the other hand, eksotika obtained the lowest score, being ‘moderately liked’ by the panelists for its colour, general appearance and flavor. conclusions fruit leather was successfully developed from two different papaya cultivars using three additional ingredients honey, pectin and citric acid. this processed product was intended to preserve or enhance the nutritional value and sensory quality of the papaya fruit. the moisture content of the fruit leather derived from the two cultivars was reduced (14.31% and 15.42%), signifying that they were safe from a bacterial viewpoint and could be classified as an intermediate moisture food. the phenolics content and antioxidant activity were increased using the process of drying the fruit leather. substantial discrepancies in colour, sweetness, sourness, texture, flavour and overall acceptability were reflected in the consumer sensory assessment. ‘like moderately’ was the average overall acceptability score. however, according to the results of the sensory evaluation, panelists expressed a low preference for the eksotika fruit leather, which received a ‘moderately liked’ score. the results of the present study have great significance for producers of papaya leathers. among the main factors that determined the acceptability of the fruit leathers were colour, sweetness, sourness, texture and flavour. the end-product can be considered natural, as only small amounts of honey, citric acid and pectin were added in this study. hence, the consumer requirements for healthy and safe food products were respected acknowledgements this research was supported by stgl 004-2007 and bkbpfst– k004092 and dpp-2013-037. references abu bakar m.f., mohamed m., rahmat a. and fry j. 2009. phytochemicals and antioxidant activity of different parts of bambangan mangifera pajang and tarap artocarpus odoratissimus. food chemistry, 113(2): 479-483. ames b.n., shigenaga m.k. and hagen t.m. 1993. oxidants, antioxidants, and the degenerative diseases of aging. proceedings of the national academy of sciences, 90(17): 7915-7922. aminah a. 2004. prinsip penilaian sensori. bang. penerbit universiti kebangsaan malaysia. anupama d., bhat k. and sapna v. 2003. sensory and physico-chemical properties of commercial samples of honey. food research international, 36(2): 183-191. aoac. 1998. official methods of analysis. association of official analytical chemists. washington, dc. azeredo h., brito e.s., moreira g.e., farias v.l. and bruno l.m. 2006. effect of drying and storage time on the physico-chemical properties of mango leathers. international journal of food science and technology, 41(6): 635-638. 82 ital. j. food sci., vol. 28 2016 babalola s., ashaye o., babalola a. and aina j. 2002. effect of cold temperature storage on the quality attributes of pawpaw and guava leathers. african journal of biotechnology, 1(2): 61-63. bahorun, t., luximon-ramma, a., crozier, a. and aruoma, o. i. 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april 15, 2015 paper ital. j. food sci., vol. 28 2016 121 keywords: jerusalem artichoke, microwave-assisted drying, effective moisture diffusivity, response surface methodology, genetic algorithm optimization of microwave-assisted drying of jerusalem artichokes (helianthus tuberosus l.) by response surface methodology and genetic algorithm e. karacabey1,*, c. baltacioglu2, m. cevik3 and h. kalkan4 1food engineering department, engineering faculty, suleyman demirel university, isparta, turkey 2department of food engineering, faculty of engineering, niğde university, konya, turkey 3agriculture and fisheries directorate, ministry for eu affairs, ankara, turkey 4computer engineering department, engineering faculty, suleyman demirel university, isparta, turkey *corresponding author: erkankaracabey@sdu.edu.tr abstract the objective of the present study was to investigate microwave-assisted drying of jerusalem artichoke tubers to determine the effects of the processing conditions. drying time (dt) and effective moisture diffusivity (emd) were determined to evaluate the drying process in terms of dehydration performance, whereas the rehydration ratio (rhr) was considered as a significant quality index. a pretreatment of soaking in a nacl solution was applied before all trials. the output power of the microwave oven, slice thickness and nacl concentration of the pretreatment solution were the three investigated parameters. the drying process was accelerated by altering the conditions while obtaining a higher quality product. for optimization of the drying process, response surface methodology (rsm) and genetic algorithms (ga) were used. model adequacy was evaluated for each corresponding mathematical expression developed for interested responses by rsm. the residual of the model obtained by ga was compared to that of the rsm model. the ga was successful in high-performance prediction and produced results similar to those of rsm. the analysis and results of the present study show that both rsm and ga models can be used in cohesion to gain insight into the bioprocessing system. mailto:erkankaracabey%40sdu.edu.tr?subject= 122 ital. j. food sci., vol. 28 2016 1. introduction the jerusalem artichoke (helianthus tuberosus l.) has been gaining increasing attention due to the potential use of this plant as a feedstock for the synthesis of new products and the awareness of its significant health benefits. the storage form of carbon in jerusalem artichokes, inulin, makes this plant attractive compared to the majority of crops that store carbon as starch (kays and nottingham, 2008; van loo et al., 1995; watherhouse and chatterton, 1993). in spite of its high potential usage in the food industry, consumption of this plant as a raw material is limited due to changes during its postharvest period (cabezas et al., 2002; modler et al., 1993; takeuchi and nagashima, 2011). therefore, increasing the jerusalem artichoke shelf-life by processing is of prime importance, and dehydration of its tubers should also be considered in this regard. various drying technologies have been extensively used as a preservation technique in the food industry. specific technologies, such as microwave-assisted drying, for grains, crops and foods have been well documented (al-harahsheh et al., 2009; giri and prasad, 2007; sharma and prasad, 2001). the main reasons to consider the use of microwave energy are to accelerate the drying process, improve product quality, and reduce costs (al-harahsheh et al., 2009; giri and prasad, 2007; mcloughlin et al., 2003). however, additional effort is required to standardize microwave technology in the drying process. for this reason, microwave-assisted drying requires investigation in terms of the underlying physical phenomena, such as the mechanism of molecular transfer. effective moisture diffusivity is one of the parameters used to evaluate the drying of food materials from the point of view of intramolecular mass transfer, since transfer of water molecules throughout the solid matrix is generally a rate-controlling step in drying processes (dadali et al., 2007). another significant step is to optimize processing variables according to desired targets including faster and more efficient processing and improved product quality. response surface methodology (rsm) is a statistical procedure frequently used for process optimization. it uses quantitative data from an appropriate experimental design to determine and simultaneously solve multivariate problems. the equations describe the effect of the test variables on the responses, determine interrelationships among test variables and represent the combined effect of all test variables in the response. this approach enables an experimenter to efficiently explore a process or system. in recent years, other optimization techniques have also been developed and adapted to food processes. in process engineering design, genetic algorithms (gas) are considered a novel technique (goldberg, 2001). for highly complex and nonlinear processes, researchers have reported successful ga applications in analyzing the osmotic dehydration of kiwifruit (fathi et al., 2011a) and carrot slices (mohebbi et al., 2011a), and plant oil extraction from cloves by supercritical co 2 (hatami et al., 2010). to our knowledge, there are no reported studies on the microwave-assisted drying of jerusalem artichokes as well as its optimization in terms of drying performance and quality characteristics. therefore, the objective of this study was to investigate and optimize the processing conditions of microwave-assisted drying of artichoke tubers. additionally, ga was conducted to evaluate its performance in the optimization of the proposed drying technique. 2. materials and methods 2.1 preparation of samples fresh jerusalem artichoke tubers were purchased from the local market and stored at 4°c. the tubers were peeled and sliced at a specified thickness by using a lab-scale slicer on which the thickness was adjusted in the range of 1-10 mm. all slices had the same projected area (30*40 mm, wide*length) to avoid its effect on drying due to any change; the slice thicknesses for each trial were changed as presented in table 1. microwave output power was another process variable that was examined at three levels (100, 200, and 300 w), as shown in table 1. the third variable was the concentration of the pretreatment solution. the experimental design was planned such that there were some trials (run order of trials was 1, 9, 15, and 16; table 1) excluding the nacl in pretreatment, and in the remaining trials the slices were treated with nacl solution (table 1) to determine the effect of salt on the drying characteristics of interest. the pretreatment was carried out with nacl solutions of specified concentrations (table 1) at 25°c with controlled agitation for a period of 2 h. after pretreatment, the samples were removed and rinsed with distilled water to remove the solute that had adhered to the surface and then dried in a microwave oven at the output power specified in table 1. in the case of samples that were not subjected to pretreatment, aliquots of 50 g of tuber slices were directly dried in the microwave oven (details provided below), whereas pretreated samples were weighted as 50 g after immersion in nacl solution for 2 h (table 1). the initial moisture content of jerusalem artichokes was determined by placing the tubers in a conventional oven at 105°c until no further change in weight of the sample was observed. the average moisture content of fresh jerusalem artichoke tubers was 81.77 ± 0.89%. the moisture content of any pretreated j. artichoke slice did not vary significantly; even with a 2% (w/v) nacl ital. j. food sci., vol. 28 2016 123 concentration in the pretreatment solution. this may be due in part to the low temperature level and short duration of the pretreatments. 2.2 drying equipment and experimental method a programmable domestic microwave oven (samsung-mw71e, malaysia) with a maximum output power of 800 w and wavelength of 2,450 mhz was used for drying. aliquots of 50 g of pretreated or fresh tuber slices were spread on a glass dish (dried and weighed before use) as a single layer and placed on the center of the turntable of the microwave cavity. drying was performed for each trial at the microwave output power levels specified in table 1. moisture loss was measured periodically (60-s intervals) by taking out and weighing the dish on a digital balance. the drying process continued until the desired moisture content was attained (< 10%, w/w). trials were carried out according to the experimental design including the processing conditions and run order for each trial (table 1). the rehydration ratio (rhr) was also determined for j. artichoke slices dried according to each trial specified in table 1. the rhr is an important quality parameter to evaluate the drying process in terms of product quality. dried slices were immersed in warm water (50°c) and their weight gain was monitored until it stabilized. the rhr was calculated as a ratio of net weight gain to initial sample amount. 2.3 theoretical approach to effective moisture diffusivity the effective moisture diffusivity (emd) was determined to obtain information about the mechanism of moisture transfer and complexity of the drying process. it was defined by fick’s second law with the assumption that diffusion is the only physical mechanism to control the transfer of water molecules to the surface. artichoke slices prepared at different thicknesses were assumed to be an infinite slab, since other directions were large enough compared to the thickness. thus, moisture movement was only throughout thickness. fick’s second law for moisture movement was solved with the following assumptions: the particle was homogenous and isotropic the material characteristics were constant, and the shrinkage was negligible mass transfer was in one direction moisture was initially uniformly distributed throughout the mass of a sample the pressure variations were negligible evaporation occurred only at the surface surface diffusion was ended, so the moisture equilibrium arises on the surface effective moisture diffusivity was constant versus moisture content during drying resistance to mass transfer at the surface was negligible compared to the internal resistance of the sample mass transfer was represented by a diffusional mechanism the following analytical solution of fick’s second law proposed by crank (1975) was used to calculate the effective moisture diffusivity. eq. (1); where d eff is the effective moisture diffusivity (m2s-1), l is the half thickness (drying from both sides) of slab (m), mr was the fractional moistable 1 experimental design of microwave drying and corresponding responses. standard order run order power thickness nacl conc. drying time effective diffusivity*10-8 rehydration ratio (w) (mm) (g/100 ml) (min) (m2/s) 9 1 200 2 0 6 0.79 3.27 16 2 200 4 1 8 1.61 2.76 13 3 200 4 1 9 1.58 3.18 2 4 300 2 1 4 0.77 4.47 14 5 200 4 1 14 1.50 3.71 4 6 300 6 1 5 7.62 3.19 3 7 100 6 1 96 0.42 4.28 15 8 200 4 1 12 1.31 3.18 5 9 100 4 0 75 0.26 3.98 12 10 200 6 2 11 3.37 2.97 7 11 100 4 2 83 0.24 3.57 11 12 200 2 2 5 0.68 5.29 1 13 100 2 1 33 0.11 4.45 8 14 300 4 2 4 4.60 3.57 10 15 200 6 0 9 3.77 4.03 6 16 300 4 0 7 3.32 3.19 124 ital. j. food sci., vol. 28 2016 ture ratio, t was the drying time (s). m t was the moisture content of the material at any time, t; m i was the initial moisture content of the material before drying; and m e was the equilibrium moisture content of a dehydrated artichoke slice, all moisture content values were in dry basis. for long-term drying, only the first term of eq.(1) was used to explain the drying procedure. the equilibrium moisture content (m e ) was assumed to be zero for microwave-assisted drying. the final equation to calculate the emd was as follows: eq. (2); further simplification of eq. (2) resulted in a straight-line equation as eq. (3); eq. (3); the effective moisture diffusivity was calculated by fitting eq. (3) to the curve of ln(mr) vs. time (fig. 1), and the results are presented in table 1. 2.4 experimental design drying time (z 1 ), effective moisture diffusivity (z 2 ), and rehydration ratio (z 3 ) were the responses used to optimize the process variables by response surface methodology (rsm). a boxbehnken design was employed in this regard. independent process variables (x 1 , x 2 , and x 3 ) were microwave output power, slice thickness, and concentration of the pretreatment solution (nacl); each was specified at three levels with 16 runs including four replicates at the central point. the ranges and levels of independent variables are presented in table 1. minitab (minitab 15.1.0.0) was used to analyze the experimental data, which were fitted to a second-order polynomial regression model including the coefficients of linear, quadratic and two factors interaction effects. the proposed model was as follows: eq. (4) where z was the response of the equation, 0b was the constant coefficient, β i was the linear coefficient (main effect), β ii was the quadratic coefficient, and β ij was the two factors interaction coefficient. the surfaces of the predicted responses were plotted by sigma plot (v. 8.02; 2002) (spss inc. chicago, il, usa). the values of r2, adjusted-r2, and lack-of-fit of models were evaluated to check the model adequacies. 2.5 optimization by genetic algorithm the genetic algorithm (ga) is a global search algorithm, which is designed to mimic charles darwin’s principle of “survival of the fittest” to solve complex optimization problems without falling into local optima (goldberg, 2001; mohebbi et al., 2011b; morimoto, 2006). matlab version 2010b (mathworks, inc.) was used to optimize the interested responses of microwaveassisted drying of jerusalem artichoke tubers as a function of process conditions by the ga. 3. results and discussion this study was designed to evaluate microwave-assisted drying of jerusalem artichokes and to optimize the process using response fig. 1 linear relation between ln(mr) and drying time for the slice thickness of 2 mm treated with 1% nacl and dried at 100 w output power (■), the slice thickness of 2 mm without treatment and dried at 200 w output power (●), the slice thickness of 4 mm treated with 1% nacl and dried at 200 w output power (▲) and the fitted proposed model line (—). ital. j. food sci., vol. 28 2016 125 surface methodology (rsm) and genetic algorithms (ga). drying of j. artichoke tubers resulted in good performance with high quality product in terms of drying time (dt), effective moisture diffusivity (emd), and rehydration ratio (rhr). models developed by rsm and ga displayed similar performances to predict the experimental results determined for each interested response. multiple linear regression analysis of the experimental data yielded second-order polynomial models for predicting dt, emd, and rhr. analysis of variance (anova) was conducted to determine significant effects of process variables on each response and to fit second-order polynomial models to the experimental data. regression equation coefficients of the proposed models and statistical significance of all main effects calculated for each response were obtained. the effects that were not significant (p > 0.05) were stepped down from models without damaging the model hierarchy (table 2). the anova table also showed that the lack of fit was not significant for all response surface models at a 95% confidence level. on the other hand, r2 and adj-r2 were calculated to check the model adequacy as lack-offit > 0.05; r2 ≥ 0.98; and adj-r2 ≥ 0.94 (table 2). 3.1 drying time drying time (dt) is important because it is an index of the drying performance. a reduction in drying time means less energy requirement for the process. table 2 shows that both microwave power and slice thickness significantly affected dt to decrease the moisture content of slices to less than 10% (p ≤ 0.05), whereas a change in the salt (nacl) concentration of the pretreatment solution was not an important factor (p > 0.05). the microwaveassisted drying process, which reduced the moisture content of jerusalem artichoke to less than 10%, took 4-96 min varying based on the process variables. the dt decreased as microwave output power increased due to higher energy transfer for unit process time (fig. 2). a similar microwave power effect on dt was reported previously (al-harahsheh et al., 2009; soysal, 2004; sumnu et al., 2005). the favorable influence of output power on dt may be attributed to the heating mechanism of microwave technology causing high internal pressure and concentration gradients, which increases the flow of liquid throughout the food (al-harahsheh et al., 2009; sumnu et al., 2005; wang and sheng, 2006). the second factor that had a significant effect on dt values was slice thickness (fig. 2). however, an increase in dt is not desirable from an economical point of view, and there was a positive relationship between slice thickness and dt (table 2 and fig. 2). drying time to decrease moisture content under a target level (< 10%) increased with thicker slices, especially when a low output power was set (fig. 2). a similar result related to the effect of slice thickness on dt was obtained by giri and prasad (2007) studying the drying kinetics and rehydration characteristics of mushrooms that were processed in microwaves. table 2 regression coefficients of predicted models for the investigated responses of microwave assisted drying of j. artichoke. variablea coefficient drying time effective moisture diffusivity rehydration ratio β 0 130.625*** e 4.055999* 5.918046*** β 1 -1.285*** -0.02718* -0.01159* c β 2 16.562*** -1.60884** -0.79208** d β 3 0.875ns b 1.029874* β 11 0.003*** 0.0000339ns 3.22e-05** β 22 0.096813ns 0.15846*** β 33 0.114851ns β 12 -0.062*** 0.008183*** -0.00139* β 13 0.001977* β 23 -0.38475*** model *** *** *** linear *** ** ** quadratic *** ns *** cross-product *** *** *** r2 0.98 0.98 0.98 adj-r2 0.97 0.96 0.94 lack-of-fit 0.101 0.312 0.085 a polynomial model adjusted by backward elimination at the level of 0.05% with the lack-of-fit test, where β 0 is the constant coefficient, β i is the linear coefficient (main effect), β ii is the quadratic coefficient, and β ij is the two factors interaction coefficient. b ns, not significant (p > 0.05); c *, significant at p ≤ 0.05; d **, significant at p ≤ 0.01; e ***, significant at p ≤ 0.001. 126 ital. j. food sci., vol. 28 2016 3.2 effective moisture diffusivity increasing the effective moisture diffusivity (emd) is desirable in a microwave-assisted drying process, since this technique is expected to create awareness and an improvement in process performance is one of the novelties. the emd was calculated and used as an index of the rate of the drying process (table 1). the mass transfer of water molecules in potato matrix dried using different techniques has been previously studied. for microwave application on potatoes, the calculated diffusivities were reported in the range of 1.91*10-8 m2.s-1 to 3.73*10-8 fig. 2 response surface for the effects of power and slice thickness treated with 1% nacl solution on drying time of jerusalem artichoke slices. m2.s-1 (mcminn et al., 2003), which were comparable with emds (0.11*10-8 m2.s-1 to 7.62*108 m2.s-1 depending on processing conditions) of jerusalem artichoke slices dried in a microwave oven. according to the results of the anova of emd, the output power and slice thickness are two important factors affecting the emd of the drying process (p ≤ 0.05) (table 2). the emd remained almost constant with changing slice thickness (2-6 mm) at an output power of 100 w (fig. 3). similarly, changing the output power (100-300 w) did not significantly affect the emd of 2-mm thick tubers. however, there was a significant interaction between both factors (microwave output power and thickness) (p ≤ 0.05), and the emd increased when higher values of slice thickness and output power were selected (fig. 3). datta and rakesh (2013) reported that microwave heating is superior compared to conventional heating, since significant internal evaporation inside the microwave-heated material leads to additional mechanisms of moisture transport that enhance moisture loss during heating. thus, an increase in microwave power results in more energy transfer to the food material during drying and as a result more internal evaporation resulting in a higher emd. 3.3 rehydration ratio rehydration ratio (rhr) is a widely used quality index for dried products. rehydration values provide information about the changes in physical and chemical properties of a dried sample attributed to drying and treatments preceding dehydration (maskan, 2000). to investigate the effect of drying conditions on final product quality, the rhr of dried tuber slices were determined (table 1). the effects of drying conditions on rhr were analyzed by anova and showed that all processing conditions were effective on the rehydration capacity of microwave-assisted dried jerusalem artichoke slices except for the quadratic term of nacl concentration of the pretreatment solution (p ≤ 0.05) (table 2). figures 4, 5, and 6 display the change of rhr with output power, slice thickness, and nacl concentration. the rhr of dried samples at an output power around 250 w was smaller than that measured for slices dried at any other power level, when tuber slices were dried without pretreatment. on the other hand, a minimum rhr value was measured for j. artichoke pretreated slices dried at an output power of less than 250 w, and tuber slices dried at 200 w had the lowest rhr when they were treated with the highest concentration (2%) of nacl solution (fig. 4). this negative effect of increasing output power on rhr results from quick sample shrinkage due to rapid water loss depending on the internal temperature. the reason for the change in the effect of high output power with the nacl concentration of the pretreatment solution may fig. 3 response surface for the effects of power and slice thickness on emd of jerusalem artichoke slices irrespective of pretreatment. * effective moisture diffusivity ital. j. food sci., vol. 28 2016 127 result from partial water loss occurring during pretreatment, although the change in the final moisture content of dried slices pretreated with nacl solution was not significant compared to the water content of fresh tuber slices (data not shown). in other words, microwave-assisted drying finalized in a shorter period for samples with less moisture content compared to fresh ones. thus, the internal temperature of a sample never reaches to its level seen at drying of the sample without pretreatment, which means less shrinkage and high rhr. these results are consistent with the changes in rhr with microwave power also observed by wang and xi (2005). slice thickness was another factor that had a significant effect on rhr values. change in rhr was plotted as a function of slice thickness vs. nacl concentration and slice thickness vs. outfig. 4 response surface for the effects of power and slice thickness treated with 1% nacl solution on the rehydration ratio of jerusalem artichoke slices. fig. 5 response surface for the effects of power and concentration (slice thickness of 4 mm) on the rehydration ratio of jerusalem artichoke slices. put power as shown in figures 5 and 6, respectively. the rhr of the dried products decreased with an increase in slice thickness. the effect of nacl concentration on this trend was significant when low slice thickness values were conducted (fig. 5). the rhr increased with increasing nacl concentration of pretreatment solution when thinner slices were analyzed (fig. 5). a decrease in rhr was also detected with increasing thickness under the effect of power (fig. 6). thickness effects may result from greater volumetric heating, which generates higher pressure inside the jerusalem artichoke tuber, resulting in boiling and bubbling of the samples and reduced rhrs of the dried products (wang and xi, 2005). 3.4 optimal responses an optimization procedure by rsm was conducted for all responses as a function of processing conditions. the emd and rhr were maximized, since higher values of these responses means faster drying and better product quality, respectively. the dt response was minimized because a short process length is preferred due to economical considerations. as a consequence of the optimization procedures for these three drying characteristics, the following operating conditions were found to be optimal: power of 235 w; slice thickness of 5.95 mm; and nacl concentration of 0.081. 3.5 genetic algorithms the gas were used to select the best subset of variables and to build predictive regression models in order to study the relationships between the results obtained from the experimental trials (dt, emd, rhr) and the profig. 6 response surface for the effects thickness and concentration (power of 200w) on the rehydration ratio of jerusalem artichoke slices. 128 ital. j. food sci., vol. 28 2016 cess parameters. the coefficients of regression models corresponding to dt, emd, and rhr are presented in table 3. the residual is an index of model performance where a smaller residual indicates better prediction performance. thus, residuals between experimental results and predicted values by rsm and ga are shown in figures 7-9 for each response. models produced by ga display a similar performance in prediction of emd and rhr values as those produced by rsm. figure 7 shows smaller residuals of dt values predicted by models using rsm than ga. although a performance decrease was seen in the prediction of dt values by ga, this procedure presented in this work can be applied for optimization in microwave-assisted drying of food materials as a rapid and non-destructive inspection method. gas have been reported as a novtable 3 model coefficients of proposed second order polynomial modela obtained by ga. variablea coefficient drying time effective diffusivity rehydration ratio β 0 -0.500 0.522 -0.143 β 1 0.118 0.439 -0.267 β 2 0.035 0.024 0.107 β 3 0.488 0.065 0.190 β 11 0.253 0.065 0.481 β 22 -0.129 0.065 0.024 β 33 -0.335 0.439 -0.309 β 12 0.047 0.024 0.107 β 13 0.076 0.060 -0.600 β 23 -0.500 -0.558 0.600 a polynomial model , where β 0 is the constant coefficient, β i is the linear coefficient (main effect), β ii is the quadratic coefficient, and β ij is the two factors interaction coefficient. independent process variables (x 1 , x 2 , and x 3 ) were microwave power, slice thickness, and concentration of pretreatment solution (nacl). fig. 7 residuals between experimental results and predicted responses by rsm (∆) and ga (□) models calculated for each trials of drying time in experimental design. el approach in the osmotic drying of kiwifruit by fathi et al. (2011b). similarly, mohebbat et al. (2011) reported genetic algorithms as a method with a high potential for optimization in all food processes. 4. conclusions the experimental results and their analysis demonstrate the possibility of using this innovative method based on microwave technology for the drying of jerusalem artichoke tubers. to the best of our knowledge, this is the first study on the microwave-assisted drying of jerusalem artichoke tubers and optimization of process parameters using rsm and ga procedures. the results of the present work demonstrate the feasibility of the dt, emd, ital. j. food sci., vol. 28 2016 129 and rhr determinations for accurate prediction. the performance of rsm with respect to r2, adj-r2 and lack-of-fit values was acceptable. the ga and rsm methods produced similar models of performance for microwave-assisted drying of artichoke tubers. the analysis and results from this present study imply that both rsm and ga models can be used in cohesion to gain complete insight into the bioprocessing system. fig. 8 residuals between experimental results and predicted responses by rsm (∆) and ga (□) models calculated for each trials of effective moisture diffusivity in experimental design. references al-harahsheh m., al-muhtaseb a.h. and magee t.r.a. 2009. microwave drying kinetics of tomato pomace: effect of osmotic dehydration. chemical engineering and processing 48: 524. cabezas m.j., rabert c., bravo s. and shene c. 2002. inulin and sugar contents in helianthus tuberosus and cichorium intybus tubers: effect of postharvest storage temperature. journal of food science 67: 2860. crank j. 1975. “the mathematics of diffusion” 2nd ed. oxford university press, england. fig. 9 residuals between experimental results and predicted responses by rsm (∆) and ga (□) models calculated for each trials of rehydration ratio in experimental design. 130 ital. j. food sci., vol. 28 2016 dadalı g., apar d.k. and özbek b. 2007. estimation of effective moisture diffusivity of okra for microwave drying. drying technology 25(9): 1445. datta a.k. and rakesh v. 2013. principles of microwave combination heating. comprehensive reviews in food science and food safety 12: 24. fathi m., mohebbi m. and razavi s.m.a. 2011a. application of fractal theory for prediction of shrinkage of dried kiwifruit using artificial neural network and genetic algorithm. drying technology, 29:8, 918. fathi m., mohebbi m. and razavi s.m.a. 2011b. effect of osmotic dehydration and air drying on physicochemical properties of dried kiwifruit and modeling of dehydration process using neural network and genetic algorithm. food and bioprocess technology 4: 1519. giri s.k. and prasad s. 2007. drying kinetics and rehydration characteristics of microwave-vacuum and convective hot-air dried mushrooms. journal of food engineering 78: 512. goldberg d.e. 2001. genetic algorithms in search, optimization and machine learning. pearson education, singapore. hatami t., meireles m.a.a. and zahedi g. 2010. mathematical modeling and genetic algorithm optimization of clove oil extraction with supercritical carbon dioxide. the journal of supercritical fluids 51: 331. kays s.l. and nottingham s.f. 2008. “biology and chemistry of jerusalem artichoke helianthus tuberosus l.”. crc press, london. maskan m. 2000. microwave/air and microwave finish drying of banana. journal of food engineering 44: 71. mcloughlin c.m., mcminn w.a.m. and magee t.r.a. 2003. microwave drying of multicomponent powder systems. drying technology 21: 293. mcminn w.a.m., khraisheh m.a.m. and magee t.r.a. 2003. modelling the mass transfer during convective, microwave and combined microwave-convective drying of solid slabs and cylinders. food research international 36: 977. modler h.w., jones h.w. and mazza g. 1993. observations on long-term storage and processing of jerusalem artichoke tubers (helianthus tuberosus). food chemistry 48: 279. mohebbat m., mohammad-r a.t., fakhri s. and mohsen z.s. 2011. modeling and optimization of mass transfer during osmosis dehydration of carrot slices by neural networks and genetic algorithms. international journal of food engineering: 7:2, doi: 10.2202/1556-3758.1670. mohebbi m., akbarzadeh-t m.r., shahidi f. and zabihi s.m. 2011a. modeling and optimization of mass transfer during osmosis dehydration of carrot slices by neural networks and genetic algorithms. international journal of food engineering 7(2): doi:10.2202/1556-3758.1670. mohebbi m., shahidi f., fathi m., ehtiati a. and noshad m. 2011b. prediction of moisture content in pre-osmosed and ultrasounded dried banana using genetic algorithm and neural network. food and bioproducts processing 89(4): 362. morimoto t. 2006. genetic algorithm. in “handbook of food and bioprocess modeling techniques”. s.s. sablani, m.s. rahman, a.k. datta, and a.s. mujumdar (ed.),, crc press, new york. sharma g.p. and prasad s. 2001. drying of garlic (allium sativum) cloves by microwave-hot air combination. journal of food engineering 50: 99. soysal y. 2004. microwave drying characteristics of parsley. biosystems engineering 89(2): 167. sumnu g., turabi e. and oztop m. 2005. drying of carrots in microwave and halogen lamp–microwave combination ovens. lwt-food science and technology 38: 549. takeuchi j. and nagashima t. 2011. preparation of dried chips from jerusalem artichoke (helianthus tuberosus) tubers and analysis of their functional properties. food chemistry 126: 922. van loo j., coussment p., leenheer l., hoebregs h. and smits g. 1995. on the presence of inulin and oligofructose as a natural ingredients in the western diet. critical reviews in food science and nutrition 6: 525. wang j. and sheng k. 2006. far-infrared and microwave drying of peach. lwt-food science and technology 39: 247. wang j. and xi y.s. 2005. drying characteristics and drying quality of carrot using a two-stage microwave process. journal of food engineering 68: 505. watherhouse m.l. and chatterton n.j. 1993. glossary of fructan terms, in “science and technology of fructans”. m. suzuki and n.j. chatteron (ed.),, pp. 2. crc press, boca raton, fl. paper received april 3, 2014 accepted may 28, 2015 #516_ jafarzadeh_bozza ital. j. food sci., vol 29, 2017 195 opinion paper characterization of semolina biopolymer films enriched with zinc oxide nano rods shima jafarzadeh*a, fazilah ariffinb, shahrom mahmudc, abd karim aliasd, ali najafie and mehraj ahmadf aphd candidate, food biopolymer research group, food technology division, school of industrial technology, university sains malaysia, 11800 minden, penang, malaysia bassociate professor, food biopolymer research group, food technology division, school of industrial technology, school of industrial technology, university sains malaysia, 11800 minden, penang, malaysia cassociate professor, nano optoelectronic research (nor) lab, school of physics, university sains malaysia, 11800 minden, penang, malaysia dprofessor, food biopolymer research group, food technology division, school of industrial technology, university sains malaysia, 11800 minden, penang, malaysia e senior lecturer, food biopolymer research group, food science and technology department, damghan branch, islamic azad university, damghan, semnan, iran f senior lecturer, institute of nutrition (inmu), mahidol university, 999 phutthamonthon 4 rd, salaya, nakhon pathom 73170, thailand *corresponding author. shimajafar@yahoo.com abstract this research aimed to develop biopolymer-based antimicrobial films as food packaging that will consequently reduce environmental pollution caused by the accumulation of petroleum origin food packaging. zinc oxide nanorods (zno-nr) were incorporated as the antimicrobial component in nanocomposite films based on semolina, which were prepared by solvent casting. sem and xrd were used to characterize the resulting films. the mechanical, barrier, optical, physical and antimicrobial properties of the films were also analyzed. the addition of zno-nr reduced the solubility, wvp, and elongation at break while increased the tensile strength and modulus of elasticity of the nanocomposite films compared with the control film. the apparent surface color and uv transmittance of the semolina films was greatly influenced by the amount of zno-nr. the nanocomposite films exhibited 0% uva in transmittance the near infrared spectra. furthermore, the zno-nr semolina films exhibited strong antimicrobial activity against staphylococcus aureus. keywords: antimicrobial, packaging, zinc oxide nano rod, semolina ital. j. food sci., vol 29, 2017 196 1. introduction a large number of biodegradable polymers have been studied to develop edible films and finally lower the amount of waste produced through non-degradable petroleum-based food packaging activities (tharanathan, 2003; jafarzadeh et al., 2016). these edible films not only provide physical protection to foods but also prevent the mass transfer of moisture, oxygen, carbon dioxide, lipids, flavors, and aromas into and from food products (marcuzzo et al., 2010). given their nutritive value and superior properties, protein-based biopolymers have attracted considerable interest for the development of edible films (gennadios and yada, 2004). these polymers are excellent oxygen barriers and they render certain mechanical properties to the films (sothornvit et al., 2009). different types of proteins are used as components of biodegradable packaging (khwaldia et al., 2010). among these proteins, the protein from wheat is a potential component of packaging materials because of its cost effectiveness, biodegradability, renewability, and favorable film-forming and adhesive/cohesive properties (türe et al., 2013). semolina is a type of wheat whose flour contains high gluten content (quaglia, 1988). the gluten increases the nutrient value of edible films. semolina grain is extra hard, translucent, light colored, and exhibits antioxidant activities (onyeneho and hettiarachchy, 1992; jafarzadeh et al., 2017b). in addition, semolina extracts suppress radical-induced liposome lipid peroxidation and show radical cation-scavenging activity (onyeneho and hettiarachchy, 1992). however, the poor mechanical and water sensitivity of biopolymers limit their application in food packaging. nanoparticles, which reinforce biopolymers through the formation of nanocomposites, have been recently employed to overcome these limitations (dufresne, 2006). one of the most successful applications of nanotechnology in the field of packaging concerns the development of “nanocomposites”(unalan et al., 2014). the nanoparticles dispersed in the biopolymer matrix considerably reinforce the mechanical, thermal, optical, and physicochemical properties of nanocomposites compared with pristine biopolymer (petersson and oksman, 2006). given their large specific surface area and high surface energy, nanofillers exhibit excellent interfacial interactions with polymer branches and consequently enhance polymer properties significantly (kovacevic et al., 2008). zinc oxide (zno) has been widely applied as a functional filler in uv absorbers in pharmaceuticals, cosmetics, coating materials, and pigments (kumar and singh, 2008; li et al., 2009; yu et al., 2004). zno nanoparticles can potentially prevent infectious diseases through the antimicrobial effects of zno (li et al., 2009; li et al., 2010; rajendra et al., 2010; zhang et al., 2008). the size, morphology, crystallinity, composition, and shape of zno nanoparticles affect their intrinsic properties (shahrom and abdullah, 2006; jafarzadeh et al., 2017a). lin et al. (2009) reported that zno nanorods (zno-nr) exhibit optimal uv-absorption activity. semolina displays excellent properties for edible film production, and zno-nr are potent materials in reinforcing the mechanical, physicochemical, and barrier properties of semolina. despite these advantages, zno-nrreinforced semolina remains poorly understood. studies published on semolina thus far are limited. to the best of our knowledge, there is no information regarding of the preparation blend films from semolina, zno-nr as filler and glycerol and sorbitol as plasticizer. in the present study, we hypothesized that low-concentration zno-nr addition into semolina films improves the hydrophobicity of the films and that the resulting biopolymeric films exhibit uv-blocking, low wvp and antimicrobial properties. given their favorable antimicrobial activities, the proposed films can be used as food packaging especially for cheese. in this study was used zno-nr as fillers to prepare semolina film ital. j. food sci., vol 29, 2017 197 bionanocomposites and characterized the morphology, physicochemical, mechanical, and barrier properties of the prepared films. 2. materials and methods 2.1. materials semolina flour (14.2% protein, 18.5% gluten) was purchased from the local market in tehran, iran and then stored in a dry and cool place until the tests. food-grade glycerol was obtained from sim company sdn. bhd. (penang, malaysia), whereas food-grade liquid sorbitol was purchased from liangtracosdn. bhd. (penang, malaysia). the magnesium nitrate used to control humidity was purchased from sigma aldrich (kuala lumpur, malaysia), and zno-nr was synthesized through the catalyst-free combustoxidized mesh process as described by shahrom and abdullah (2007). 2.2. preparation of bionanocomposite films semolina flour (4g) was dispersed by magnetic stirring in 80 ml of distilled water (based on water or water/ethanol) at room temperature, and the ph of the dispersion was adjusted to 8 with 1m naoh. similarly, various concentrations of zno powder (1%, 2%, 3%, 4%, and 5%; w/w of total solid) and 2g of a sorbitol and glycerol (3:1) mixture were dispersed in 20 ml of distilled water for 30min followed by sonication in an ultrasonic bath (marconi model, unique usc 45 khz, piracicaba, brazil) (jafarzadeh et al., 2017c). subsequently, the dispersions of semolina flour and zno-nr plasticizer were mixed and stirred for 1 h at 90 °c. for the preparation of nanocomposite films, the homogenous mixtures were poured into plates and the solvents were allowed to evaporate at room temperature for 24h. the films were dried under controlled conditions in a humidity chamber (25 °c and 58% relative humidity (rh). a control film was prepared in a similar manner except for the addition of nanoparticles. the dried films were peeled and stored at 23 ± 2 °c and 58% rh until use. 2.3. characterization studies 2.3.1 determination of film thickness the films were equilibrated at 25 °c and 58% rh in a humidity chamber for 2 days. the thickness of the nanocomposite films was determined as the mean of measurements made at five random points. measurements were obtained using a micrometer (model no. 204608; mitutoyo tokyo, japan). 2.3.2 water solubility the solubility of the semolina/zno-nr films in deionized water was calculated as the ratio of the solubilized material in water to the initial dry weight of the film (rhim et al., 1999). the initial dry weight of films 2.5cm × 2.5cm in dimension was obtained after dehydration for 3 days at 25 ℃ in a desiccator with phosphorus pentoxide (0% rh). the samples were placed in a beaker with 80 ml of deionized water (18 mx) and then gently shaken at 40 rpm for 1 h at room temperature. the remaining pieces of the films were separated using a filter paper (whatman no. 1) and then dried to a constant weight in an oven at 60 °c for ital. j. food sci., vol 29, 2017 198 24h. finally, the weight of the dried insoluble material was determined. the weight of the water-soluble material was calculated by subtracting the weight of the insoluble dry matter from that of the initial dry matter. the film samples were weighed to the nearest 0.0001g before and after drying. the solubility of the films was determined in triplicate. 2.3.3 moisture content to measure the moisture content of the bionanocomposite films, approximately 50mg of the films were conditioned at 58% rh and 25 ℃ for 2 days. subsequently, the films were dried at 105 °c for 1 day (until equilibrium weight was attained). the moisture content was obtained using the following equation: moisture content = !"!!" !" ×100 where mi and mf are the initial and final weights (mg) of the dried samples, respectively. the weight of each sample was measured three times. 2.3.4 water vapor permeability (wvp) wvp tests for the semolina films were performed gravimetrically following the astm standard method e96-05 (astm, 2005). 2.3.5 mechanical properties a minimum of seven 100mm × 25mm films were conditioned at 25ċ and 58% rh for at least 48 h in an environmental test chamber (sang woo co., korea). a texture analyzer (ta-xt2, stable micro systems, surrey, uk) was used to measure the tensile strength [ts (mpa)], young’s modulus [ym (mpa)], and elongation at break [eb (%)] of the films in accordance with the astm standard method d882-10 (astm, 2010). the initial grip separation was set at 50mm, and the crosshead speed was set at 0.5 mm/s. 2.3.6 optical properties (color and light transmission) we studied the transmittance of the films (in triplicate) at 200 and 800 nm, by using the uv–vis spectrophotometer model uv-1650pc (shimadzu, tokyo, japan). biofilms were sectioned (60mm × 4mm) and directly placed in a spectrophotometer test cell. an empty glass plate served the reference. the color of the biofilms was determined by using a colorimeter (hunter lab system, model miniscan xe, usa). the cielab scale was applied to measure the following parameters: l* (luminosity), a* (red to green), b*(yellow to blue), chroma (c*), and hue (h*). measurements were obtained in five different points in each nanocomposite film (rhim et al., 1999). 2.3.7 film morphology the conditioned bionanocomposite samples were vacuum coated with gold for fieldemission scanning electron microscopy. the surface microstructure of the nanocomposite films was visualized using a leo supra 50 vp field-emission scanning electron microscope (carl-ziess. smt, oberkochen, germany) equipped with an oxford inca 400 energy dispersive. ital. j. food sci., vol 29, 2017 199 we used a phillips cm12 transmission electron microscope and siemens d5000 x-ray diffractometer to investigate the crystallinity of the semolina nanocomposite films. in addition, energy-dispersive x-ray spectroscopy (edx) was conducted under 15 kv incident electron energy. 2.3.8 antimicrobial assay the antimicrobial activity of the zno-nr-reinforced film was evaluated using the agar diffusion method as described by maizura et al. (2007). the test for zone of inhibition on solid media was applied to determine the antimicrobial effects of the films against common foodborne pathogens and spoilage bacteria, such as the gram-positive staphylococcusaureus. circular samples (5mm) were sterilized under uv radiation for 2h to eliminate surface contamination and were subsequently placed on nutrient agar plates that had been previously smeared with 100 μl of inoculum containing approximately 106–107 cfu/ml s. aureus. the plates containing the films were stored at 37℃ for 24h. thereafter, we measured the zone of inhibition produced with the nanocomposite films. 2.3.9 statistical analysis anova and tukey’s post-hoc tests were used to evaluate the mean values of the physical, optical, mechanical, barrier, and antimicrobial properties of the prepared semolina films at the 5% significance level. statistical analysis was conducted using spss version 22.0. 3. results and discussion 3.1. thickness table 1 shows the thickness of the control semolina film and those reinforced with various concentrations of zno-nr. the thickness of the films significantly increased with increasing zno-nr concentration (p < 0.05). this result can be attributed to the increased solid content of the films (ahmad et al., 2012). table 1. mechanical, water vapor permeability, thickness of semolina nanocomposite films. zno-nr (%w/w) ts (mpa) eb (%) ym (mpa) thickness(mm) wvp × 10 -7 [g m-1 h-1 pa-1] control 3.40± 0.105e 59.43±1.79a 63.12±2.27f 0.143±0.005e 8.61±0.304a 1% 3.53±0.098e 52.09±2.79b 73.49±3.30e 0.149±0.001cd 6.71±0.26b 2% 3.85±0.113d 46.87±2.96c 85.64±3.13d 0.152±0.005cd 5.84±0.24c 3% 4.21±0.211c 40.90±3.12d 100.03±4.99c 0.158±0.005bc 5.03±0.23d 4% 4.64±.0262b 33.53±2.52e 117.40±3.07b 0.163±0.001ab 5.03±0.23d 5% 5.13±0.151a 27.61±2.10f 143.51±4.37a 0.166±0.005a 4.40±0.19e different letters in each column represent significant difference among semolina films at the 5% level of probability. ital. j. food sci., vol 29, 2017 200 3.2. moisture content and water solubility most biopolymers are sensitive to water. however, incorporating lipids and nanoparticles, as well as the enhanced crosslinking in the biofilm, may reduce the sensitivity of the biopolymers to water (pavlath and orts, 2009). figure 1 illustrates the solubility and the moisture content of the control and nanocomposite films. compared with the control, the nanocomposite films showed lower moisture content. in addition, the solubility of the films decreased with increasing zno-nr content. this finding may be attributed to the interaction among the plasticizer, biopolymer matrix, and zno-nr, which consequently reduced the amount of hydroxyl groups that react with water, thereby creating a less hygroscopic matrix. our results were consistent with those of tunc and duman (2010) and müller et al. (2011). figure 1. effects of zno-nr on water solubility (empty bars) and moisture content (filled bars) of the semolina films. the bars represent mean (n = 5) ± sd. different letters on the bars represent significant difference at the 5% level of probability. 3.3. wvp wvp is a serious problem in the food industry; food packaging must prevent a contact between the food and the environment, and protect food products from any harmful agents. the problem with composite films in the food industry is the relatively high wvp of edible films. film permeability is controlled by the diffusivity and solubility of water within the film matrix. thus, nanoscience can be used to develop a material that prevents the migration of water in food products. table 1 shows the wvp of semolina films as a function of zno-nr content. the wvp of the control semolina film was 8.61 × 10−7, which was significantly higher than that of the nanocomposite films. the lowest wvp was found in the semolina film incorporated with 5% of zn0-nr, which is significant when compared to the control or other percentage. the enhanced water vapor barrier property of the nanocomposite films can be attributed to the impermeability of the zno-nr in the polymer matrix to water vapor and the formation of a tortuous pathway for the diffusing water molecules (yu et al., 2009). the wvp of the films reinforced with 5% zno-nr was significantly reduced by 4.40 × 10−7 compared with that of the control film (p < 0.05). these results showed that the water ital. j. food sci., vol 29, 2017 201 vapor barrier property of the bionanocomposite films was stronger than that of the biopolymer films. similar results have been reported for nanocomposite protein films (jafarzadeh et al., 2015). 3.4. mechanical properties zno-nr significantly affected the mechanical properties of the bionanocomposite films. table 1 shows the ts, eb, and ym of the bionanocomposite films. the maximum stress that the film can withstand while being stretched or pulled before failing or breaking is known as ts. eb and ym indicate the flexibility and intrinsic stiffness of the films, respectively. compared with those of the control films, the ts (5.13 mpa) and ym (143.51 mpa) of the bionanocomposite films significantly increased as the amount of zno-nr was increased from 1% to 5%. this result indicates that the bionanocomposite films had greater rigidity than the control film. this result is due to the increased surface interaction between the protein matrix and znonr with a high surface area, as well as the hydrogen bond formation between them (rhim, 2011). eab has a reverse relation to tensile strength in most cases, and ym is directly related to tensile strength. as shown in table 1, the eb decreased with increasing ts and maximum ym when the 5% zno-nr was added. the mechanical properties of the films are closely related to the distribution and density of the intra and intermolecular interactions between the polymer chains in the film matrix. moreover, the degree of chain elongation and the nature of amino acid sequence might affect the mechanical strength of the protein-based films (shellhammer and krochta et al., 1977).this finding is similar to those of sothornvit et al (2009). 3.5. color the surface color of the bionanocomposite is a critical parameter because it affects the general appearance and appeal of the food packaging to consumers (bourtoom and chinnan, 2008). table 2 presents the color properties of the semolina films and their nanocomposite counterparts. table 2. colorimetric parameters for the transparency of semolina films. zno-nr (%w/w) l* a* b* c* control 94.84±0.016a −0.794±0.017b 4.06±0.12e 4.11±0.12e 1% 84.06±0.116b −0.74±0.090ba 12.86±0.10d 12.86±0.10d 2% 82.20±0.009c −0.684±0.033ba 13.34±0.012c 13.35±0.012c 3% 78.11±0.08d −0.674±0.024ba 13.345±0.20c 13.36±0.20c 4% 74.86±0.70e −0.44±0.064ba 15.24±0.004b 15.26±0.004b 5% 71.68±0.014f −0.35±0.061a 15.82±0.020a 15.84±0.019a values represent mean (n = 5) ± sd. different letters in each column represent significant difference among semolina films at the 5% level of probability. incorporation of zno-nr had a significant effect on the l*-value, b*-value, and c*-value of the resulting film (p<0.05). evidently, the control film was colorless and transparent, ital. j. food sci., vol 29, 2017 202 whereas the semolina/zno-nr composite films became less transparent with increasing zno-nr content from 1% to 5%. this finding indicates that zno-nr addition influenced the coloring properties of the biopolymer films. as the content of zno-nr was increased, the b* (indicating blueness/yellowness) and c* values of the composite films significantly increased, whereas a* values (indicating greenness/redness) of the bionanocomposite films only slightly increased. by contrast, the l* values (indicating lightness) significantly decreased from 94.84 to 71.68 upon the addition of zno-nr into the semolina films. these results are consistent with those of nafchi et al. (2013), who found that adding zno-nr in sago starch significantly reduces the l* value and increases a* and b* values compared with the control. 3.6. light transmission the optical properties of biopolymer films are highly important in food packaging because protection against light is a basic requirement to preserve the food quality. fig. 2 shows the uv transmission in the control and nanocomposite films. the control films exhibited a relatively high transmittance within the uv range of 290-400 nm. the addition of zno-nr completely prevented uv transmission. nafchi et al. (2013) reported that adding 5% zno-nr into starch film reduces uv transmission to almost 0%. we obtained a similar result but with 3% instead of 5% zno-nr. by contrast, yu et al. (2009) have recently reported that adding 4% zno-nr into starch film allows 3.4% uv light transmission. moreover, the transmission of visible to ir (> 400 nm) spectra decreased by > 50% after adding zno-nr. the different behavior of zno in the present study can be attributed to the nanorod morphology of the particles (lin et al., 2009). these findings suggest the applicability of zno-nr-reinforced biopolymer films as uv-blocking films in the packaging industry. figure 2. uv-vis transmittance of semolina nanocomposite films at 25 °c. 3.7. xrd analysis xrd analyzes the scattered intensity of an x-ray beam on a material to reveal its crystallographic structure, chemical composition, and physical properties. this technique is widely used to characterize various materials because it is nondestructive and does not require elaborate sample preparation (espitia et al., 2013). thus, we analyzed the control 0 10 20 30 40 50 200 400 600 800 1000 t ra n sm it an ce ( % ) wavelength (nm) control zn1% zn2% zn3% zn4% zn5% ital. j. food sci., vol 29, 2017 203 film and their nanocomposite counterparts through xrd [figs. 3(a) and 3(b)]. this work presents the results obtained at the maximum amounts of zno nanoparticles, where the main characteristic peaks of the zno nanoparticles with hexagonal cross section were observed at 2𝜃 = 31.64°, 34.32°, 36.14°, 47.44°, 56.53°, 62.82°, 67.92°. moreover, the intensity of the major peaks of zno-nr increased as the zno-nr concentration in the matrix was increased. furthermore, the xrd patterns of the nanocomposite films revealed that zno-nr affected the crystallinity of the matrix. the addition of zno nanoparticles in the matrix produced sharp and strong peaks, indicating greater crystallinity of the nanocomposite films than the control film. 3.8. transmission electron microscopy (tem) fig. 3(c) shows the tem images of zno-nr. the zno-nr crystallites exhibit a rather hexagonal morphology with a diameter of 40-100 nm and a length of 200nm. 3.9. scanning electron microscopy (sem) and edx sem is the most widely applied technique to determine the shape, size, morphology, and porosity of matrices. fig. 3 shows the sem images of the semolina films and zno-nrreinforced films; the prepared zno-nr are evidently a nanostructure. tem also revealed that the nanorods are cylindrical with hexagonal cross section. the control film exhibited a smooth and compact surface morphology, whereas the nanocomposite films showed a slightly rough surface. sem images revealed that zno-nr particles were homogenously distributed throughout the film surface, which possibly rendered the surface of the nanocomposite films rough. this finding is possibly associated with the protruded film structures resulting from the increased thickness of the films (table 1). zno-nr particles were uniformly dispersed in the nanocomposite films, which triggered an effective force transfer from the protein matrix to the zno-nr reinforcing phase. this finding may be attributed to the higher ts of the semolina nanocomposite films with 5% zno-nr compared with the other films (table 1). fig 3(d) illustrates the edx spectrum of semolina/ zno-nr blend films. if zno-nr content was increased, their signals could be detected. as shown in fig. 3 (d), that c, zn, o and na elements were identified. this result agreed well with xrd analysis. 3.10. antimicrobial assay fig. 4 shows the antibacterial activity toward the gram-positive food pathogen s. aureus of the semolina films and the nanocomposite films containing various contents of zno-nr. the inhibition zone of the control and nanocomposite films significantly increased with increasing zno-nr content. the excellent antimicrobial activity of zno nanoparticles and the mechanism of action against microorganisms have already been demonstrated by other researchers (li et al., 2009; yu et al., 2004). zhang et al. (zhang et al., 2010) elucidated the mechanisms underlying the antibacterial activity of zno. in specific, zno penetrates through the cell wall of the microorganism, reacts with internal components of the cell, and finally reduces the viability of the organism. moreover, zn ions may bind to proteins and deactivate them, may interact with the microbial membrane to cause changes in the structure and permeability, and may interact with the microbial nucleic acids to prevent replication. furthermore, accumulation of zno nanoparticles in the microbial membrane causes membrane disintegration and cellular internalization (brayner et al., 2006). ital. j. food sci., vol 29, 2017 204 zhang et al. (34) also reported that nano-sized zno is more effective than micro-sized ones because the former easily penetrates through the cell wall of microorganisms. nano rods can act as needles for easy penetration through the cell wall (nafchi, 2013). (c) (d) (e) (f) figure 3. (a) xrd pattern of pure film, (b) 5% zno-nr-reinforced semolina, (c) tem micrograph of zno-nr, (d) edx spectrum (e) fesem micrograph of pure semolina film surface, and (f) zno-nr-reinforced semolina film surface. ital. j. food sci., vol 29, 2017 205 figure 4. (a) effects of zno-nr contents on the antimicrobial activity of semolina nanocomposite films against s. aureus. inhibition zone = total inhibition area − total film area. the bars represent mean (n = 5) ± sd. different letters on the bars represent significant difference at the 5% level of probability. figure 4. (b) antimicrobial assay of zno-nr supported semolina films. 4. conclusions the present research characterized and created semolina-based nanobiocomposites of zno-nr for food packaging purposes. there are some reasons why semolina was employed as a polymeric matrix including its great accessibility in nature, biodegradability, low expenditure, and great gluten content, which enhance the edible films’nutritional properties. zno-nr played an important role in enhancing the physical properties of semolina-based biocomposites. after the incorporation of low levels of zno-nr fillers, significant differences were observed in the film properties, particularly in mechanical, barrier, microbial and uv protection activities. the optical properties of bionanocomposites indicated that the uv transmission becomes almost zero with the addition of small ital. j. food sci., vol 29, 2017 206 amounts of zno-nr to the biopolymer matrix. xrd diffraction shows that the intensity of the crystal facets of (100), (101) and (002) increased with increasing zno-nr concentrations in the biocomposite matrix. moreover, the semolina-based nanocomposite films inhibited the growth of the gram-positive food pathogen s. aureus. the present findings stressed that the biopolymer-based nanocomposite films are environment friendly in films antimicrobial packaging to make an improvement in the shelf life of food as well as viable replacement to petroleum-based or synthetic packaging films. overall, this study suggests that semolina films incorporated with zno-nr show a strong potential to be used as active films. references ahmad m., benjakul s., prodpran t. and agustini t.w. 2012. physico-mechanical and antimicrobial properties of gelatin film from the skin of unicorn leatherjacket incorporated with essential oils. food hydrocolloid. 28:189-199. astm. 2005. annual book of astm standards. astm, philadelphia, pa. astm. 2010. standard test method for tensile properties of thin plastic sheeting d882-10. 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of glycerol plasticized-pea starch/znocarboxymethylcellulose sodium nanocomposites. bioresource technol. 100:2832-2841. ital. j. food sci., vol 29, 2017 208 zhang l., ding y., povey m. and york d. 2008. znonanofluids – a potential antibacterial agent. prog. nat. sci. 18:939944. zhang l., jiang y., ding y., daskalakis n., jeuken l., povey m., o’neill a. and york d. 2010. mechanistic investigation into antibacterial behaviour of suspensions of zno nanoparticles against e. coli. j. nanopart. res. 12:1625-1636. unalan i.u, cerri g., marcuzzo e., cozzolino c.a. and farris s. 2014. nanocomposite films and coatings using inorganic nanobuilding blocks (nbb): current applications and future opportunities in the food packaging sector. rsc advances 4(56):29393-29428. paper received may 23, 2016 accepted october 20, 2016 #973_ripabelli_bozza ital. j. food sci., vol. 29, 2017 667 paper dietary behaviours and awareness of seasonal food among college students in central italy m. tamburro1, g. ripabelli*1, m.b. forleo2 and m.l. sammarco1 1department of medicine and health sciences “v. tiberio”, university of molise, campobasso, italy 2department of economics, management, society and institutions, university of molise, campobasso, italy *corresponding author. tel. +39 0874 404961 e-mail address: ripab@unimol.it abstract in this study, eating behaviours and knowledge of food seasonality among college students who will become teachers were investigated. a questionnaire was administered to students to collect data on dietary habits, physical activity, and awareness of food season. two-hundred eighty students participated (95.4% female, 26±6 years old); 14% was overweight/obese, but 68% never practiced physical activity. almost 76% were aware of differences between consumption of fruit in and out of season, but a poor knowledge of foodstuffs seasonality emerged. these findings underline the need of improving awareness on proper diet and food seasonality because of the societal role of these students. keywords: university students, future teachers, mediterranean diet, eating habits, seasonal food ital. j. food sci., vol. 29, 2017 668 1. introduction the mediterranean diet (meddiet) has been identified one of the best models for health prevention as part of a “mediterranean-lifestyle” that include both physical and social activity (del balzo et al., 2012). this diet has beneficial effects due to the synergistic and interactive nutrients combination (del chierico et al., 2014), mitigates against overweight and obesity (eguaras et al., 2015), and provides dose-dependent protection against chronic diseases, such as cancers and cardiovascular diseases (grosso et al., 2014; davis et al., 2015). however, since economic, social and demographic changes have taken place, southern european countries, including italy, have deviated from this pattern toward food choices typical of a “western” diet, rich in animal-derived saturated fats, processed meat, eggs, sugars, and poor in legumes, whole cereals, fruit and vegetables (bloomfield et al., 2015; moreno et al., 2002). therefore, overweight and obesity have increased in young adults and in the areas commonly characterized by healthy diet patterns (moreno et al., 2002; de piero et al., 2015). an additional advantage of meddiet is the consumption of locally produced foodstuffs, as well as their availability during harvest season, the nutritional variety, the sustainability and environmental impact because of reduced “food miles” (edwards-jones et al., 2008; edwards-jones, 2010). the food seasonality is referred as the time-period when the harvest or flavour of a food is at its peak, usually corresponding when a food item is cheapest and freshest on the market. the best consequence of eating seasonally is that food has the best tasting carrying benefits to health, and while the costs fall down, the whole quality drives up. since food choices have been identified as key elements in diseases pathogenesis and prevention (davis et al., 2015), as well as for public health promotion (sammarco et al., 1997), the assessment of eating habits can support the design and improvement of measures for reducing the negative effects of unhealthy food patterns (de piero et al., 2015). the start of adult life is a critical period because of multiple physiological and psychological changes determining health-related habits in later adulthood. the college students can be considered as an index group reflecting general changes in lifestyles, because most at risk of poor nutrition and unsuitable dietary habits (ripabelli et al., 2001), due to a reduced food variety and scarce consumption of fruit or vegetables (rodríguez et al., 2013; shive and morris, 2006). for students, the years spent at the university represent a critical period influencing the quality of lifestyle, being characterized by freedom and independence, and referred to the first time for assuming responsibilities for food choices and preparation (teleman et al., 2015). to date, studies on dietary patterns amongst italian college students with respect of meddiet recommendations are still scarce, and previous investigations were only conducted in some metropolitan areas (steptoe et al., 2002; baldini et al., 2009; lupi et al., 2015; teleman et al., 2015). furthermore, no researches were performed to investigate knowledge on harvest season of locally produced food among college students who are studying to become teachers, and would be responsible for teaching healthy lifestyles in children attending the primary school (egeda et al., 2014). indeed, the aims of this study were to assess food choices and eating-related behaviours among college students attending the faculty of primary education sciences at university of molise, central italy, and to evaluate their awareness and knowledge on harvest season of locally produced food. ital. j. food sci., vol. 29, 2017 669 2. methods 2.1. study design this survey was conducted enrolling college students at university of molise that represents the only public higher education institution in molise region. the university counting 7,304 students during 2013-2014 academic year (http://statistica.miur.it/scripts/iu/viu1.asp) is mostly characterized by students from molise region (more than 50%) and from the neighbouring southern regions of puglia and campania. 2.2. participants and recruitment all the students attending the degree course of primary education sciences during 20132014 academic year were recruited by sending an invitation at their institutional email address. at time of the study, 304 students were attending this academic course, and were mostly females (n=281, 92.4%) (internal data referred to 2013-2014 academic year for the degree course of primary education sciences provided by university administrative offices). the enrolment was voluntary and anonymous. an informed signed consent was obtained from each student who agreed to participate to the survey, receiving verbal information by a trained interviewer. 2.3. data collection and procedures participants completed a newly developed questionnaire, which was validated in a pilot study on a small sample of randomly selected students (unpublished data). particularly, the questionnaire included 48 items ordered in four sections: a) socio-demographic characteristics; b) lifestyles, food habits and dietary intake assessment; c) knowledge and understanding of harvest season with respect to italian food production, focusing on seasonality and freshness of some fruit and seafood; d) sources of information on food quality. self-reported weight and height were used to calculate body mass index (bmi) for each student (de waure et al., 2015). a dietary index of the nutrition status was also identified, based on the meddietscore (mds) as previously described (panagiotakos et al., 2007; panagiotakos et al., 2009), with some modifications in the list of food, which included: non-refined cereals; fruit; vegetables; legumes; potatoes; fish and seafood; red meat and derived products; white meat; milk and dairy products; and olive oil. based on the reported intake, rates between 0 and 5 were used to score the food frequency for each student. for food suggested on a daily basis or more than three portions per week, the score 0 was assigned when no consumption was reported, while the scores from 1 to 5 were applied to proportional consumption rate. for food consumption deviating from meddiet pattern, a reverse scale was applied, with the score of 5 for rare or no consumption, and the score 0 for daily consumption. 2.4. statistical analysis questionnaire data were analyzed using the statistical package for social sciences software (ibm spss statistics for windows, armonk, ny: ibm corp) version 22.0. qualitative variables were reported as absolute frequencies and percentages, whilst quantitative variables expressed as means ± standard deviation (sd), median and range. differences among categorical variables were analyzed by chi-square test. p-values were calculated based on a two-tailed test, and compared to 0.05 significance level. ital. j. food sci., vol. 29, 2017 670 3. results 3.1. socio-demographic and anthropometric characteristics of enrolled students two-hundred and eighty out of 304 students agreed to participate to the study (response rate 92.1%). the sample had mean age of 25.9±6.2 years (median 23 years, range 19-50 years) and was mostly characterized by female students (n=267, 95.4%). the 77.8% (n=218) was single, and 11.8% (n=33) had children. at time of study, 32% (n=89) was already graduated, and the remaining has attended secondary high school. the 44.3% (n=124) of students lived away from parental home. particularly, 57.1% were residents in molise region, and an important proportion was from the neighbouring regions, such as puglia (19.6%), campania (16.8%), abruzzo and lazio (both 3.2%). the 57.8% (n=162) of students stated to have a paid employment, but none was food handler. familial history of diseases was as follows: hypertension (36.4%), diabetes (23.6%), high cholesterol (21.1%), cardiovascular diseases (15.4%), obesity (3.2%), and thyroid disorder (2.5%). according to bmi (mean 21.4±3.0, min 15.4, max 30.1), 71.1% (n=199) was classified within the normal range, followed by 15.0% (n=42) as underweight, and 13.9% (n=39) as overweight/obese. physical activity was only practiced by 32.1% (n=90) with a frequency of three-four times per week, mainly going to the gym, running, walking, cycling and swimming. significant differences between overweight/obese students and normal/underweight ones were found for education level (secondary school vs degree p=0.001). a greater proportion of underweight (11.1% vs 3.9%) and overweight/obese (12.9% vs 1.1%) students have attended secondary school compared to graduation. conversely, no significant differences between normal weight and overweight/obese or underweight students were observed in relation to gender, living away from home, practicing sport, and familial diseases history. 3.2. dietary patterns and behaviours concerning questionnaire items on food-related behaviours and habits, the possible answers were reported as never; sometimes or 1-4 times/week; often or 5-8 times/week; very often or 8-10 times/week; always or daily; the results are shown in table 1. the analysis of eating habits and behaviours revealed that 61.8% students eat sometimes outside home (i.e. bars, take-away, restaurants, etc.), and 84.0% and 46.0% have both a daily breakfast and a snack before lunch or in the afternoon, respectively (table 1). among students having breakfast daily or 1-4 times/week, the consumption of milk, biscuits and coffee was highly reported (fig. 1), while fruit, confectionery products/biscuits and fruit juice were the food items mostly consumed for snack before lunch or in the afternoon (fig. 2). about 50.0% (n=146) of students stated drinking at least 8 glasses (about 1.5-2l) of water per day, as recommended (efsa publication 2010). only 9.3% (n=26) indicated to drink wine during meals, with an average of 125 ml (min 50 ml, max 750 ml). for food preparation and cooking, about 60.0% of students reported to use always extra virgin olive oil, followed by 17.9% who used it often and 16.8% very often (table 1); however, 53.2% reported to use butter sometimes. salty (i.e. pretzels, chips, popcorn, fast food burgers, sandwiches, mixed nuts, etc.) and whole foodstuffs (i.e. whole-wheat and grains) were mainly consumed sometimes (38.0% and 35.0%, respectively) and half of the students do not eat these food. ital. j. food sci., vol. 29, 2017 671 table 1. eating habits and behaviours. frequency per week never sometimes (1-4 times/week) often (5-8 times/week) very often (8-10 times/week) always or daily n (%) n (%) n (%) n (%) n (%) eating outside home 2 (0.7) 173 (61.8) 60 (21.4) 35 (12.5) 10 (3.6) breakfast 14 (5.0) 32 (11.4) 234 (83.6) snack 46 (16.4) 105 (37.5) 129 (46.0) use of extra virgin olive oil 2 (0.7) 14 (5.0) 50 (17.9) 47 (16.8) 167 (59.6) use of butter 115 (41.1) 149 (53.2) 10 (3.6) 4 (1.4) 2 (0.7) salty food consumption 141 (50.4) 106 (37.9) 33 (11.8) whole food consumption 138 (49.3) 99 (35.4) 33 (11.8) 9 (3.2) 1 (0.4) fried food consumption 52 (18.6) 186 (66.0) 33 (11.8) 7 (2.5) 2 (0.7) watching television during meals 29 (10.4) 55 (19.6) 196 (70.0) fig. 1. food items consumed at breakfast by the students. the grey bar corresponds to the percentage of students consuming daily or sometimes specific food at breakfast; the black bar indicates the proportion of students who did not consume these foods at breakfast. ital. j. food sci., vol. 29, 2017 672 fig. 2. food items consumed as snacks by the students. the grey bar corresponds to the percentage of students consuming daily or sometimes specific food at snack time; the black bar indicates the proportion of students who did not consume these food at snack time. approximately 70% of students reported to eat fried food (i.e. chips, fish, chicken) sometimes and to watch television during meals (table 1). no significant differences between normal weight and overweight/obese or underweight students were found for water consumption, extra virgin olive oil and butter use, consumption of whole and fried food, and watching television, while differences were significant for salty food consumption (p=0.04). particularly, 36.8% of normal weight students did not consume salty food compared to 3.2% and 7.9% in the underweight and overweight/obese categories, respectively. furthermore, students declared to achieve most of the information on food quality from food labels (n=173, 62.0%), television programs on health topics (47.1%) and food preparation/cooking (35.4%), farmers and/or shopkeeper (19.6%). 3.3. food intake assessment the consumption frequency of selected food in terms of servings per week is reported in table 2. briefly, the consumption of both read meat and salami reported by students was very high; 60.0% and 36.0% reported 2-3 servings/week for red meat and salami, respectively; furthermore, 13.6% and 29.6% stated 4-5 servings/week for the same food. the frequency of white meat was commonly reported as 2-3 servings/week (64.3%) and less than 1 serving/week (19.6%). moreover, dairy products and cheese were largely consumed by 48.9% and 43.6% as 2-3 servings/week, respectively, and 25.7% and 21.4% as 4-5 servings/week, while milk was never consumed by 16.0% of the students. pasta and bread consumption was generally reported on daily frequency (43.2% and 62.9%, respectively), compared with rice mainly consumed as less than 1 serving/week. students reported a highly different consumption of vegetables, and a similar proportion (30.0%) was reported for 2-3 and 4-5 servings/week, whilst only 24.6% consumed daily these food items. a poor intake of legumes was also observed, as most (48.2%) students reported 1-2 servings/week, as well as for fish/shellfish, which were mostly reported as 1-2 servings/week (49.3%) and less than 1 serving/week (39.3%). fruit consumption was properly reported as 16-21 servings/week only by 42.5% of students, while about 4.0% do never consume them and 9.0% less than 1 serving/week (table 2). ital. j. food sci., vol. 29, 2017 673 table 2. frequency of weekly consumption in terms of servings per week of certain food reported by students. food frequency of consumption reported by students never ≤1 serving/week 2-3 servings/week 4-5 servings/week 6-7 servings/week n (%) n (%) n (%) n (%) white meat 10 (3.6) 55 (19.6) 180 (64.3) 35 (12.5) red meat 11 (3.9) 62 (22.1) 169 (60.4) 38 (13.6) salami 15 (5.4) 74 (26.4) 101 (36.1) 83 (29.6) 7 (2.5) eggs 12 (4.3) 126 (45.0) 119 (42.5) 19 (6.8) 4 (1.4) milk 46 (16.4) 27 (9.6) 33 (11.8) 37 (13.2) 137 (48.9) dairy products 11 (3.9) 37 (13.2) 137 (48.9) 72 (25.7) 23 (8.2) cheese 26 (9.3) 55 (19.6) 122 (43.6) 60 (21.4) 17 (6.1) bread 2 (0.7) 23 (8.2) 20 (7.1) 59 (21.1) 176 (62.9) rice 12 (4.3) 141 (50.4) 86 (30.7) 33 (11.8) 8 (2.9) pasta 8 (2.9) 20 (7.1) 40 (14.3) 91 (32.5) 121 (43.2) pizza 2 (0.7) 104 (37.1) 138 (49.3) 29 (10.4) 7 (2.5) vegetables 11 (3.9) 32 (11.4) 83 (29.6) 85 (30.4) 69 (24.6) confectionery products 11 (3.9) 54 (19.3) 95 (33.9) 65 (23.2) 55 (19.6) chocolate 22 (7.9) 86 (30.7) 74 (26.4) 57 (20.4) 41 (14.6) coffee 54 (19.3) 20 (7.1) 25 (8.9) 21 (7.5) 160 (57.1) fruit juice 38 (13.6) 86 (30.7) 75 (26.8) 53 (18.9) 28 (10.0) sparkling processed beverages 83 (29.6) 89 (31.8) 64 (22.9) 27 (9.6) 17 (6.1) never <1 1-2 3-4 5-6 fish/shellfish 16 (5.7) 110 (39.3) 138 (49.3) 16 (5.7) legumes 14 (5.0) 78 (27.9) 135 (48.2) 45 (16.1) 8 (2.9) never 1-4 5-8 9-12 13-18 potatoes 3 (1.1) 89 (31.8) 150 (53.6) 34 (12.1) 4 (1.4) never 1-4 5-8 9-15 16-21 fruit 11 (3.9) 26 (9.3) 51 (18.2) 73 (26.1) 119 (42.5) ital. j. food sci., vol. 29, 2017 674 the calculated mds was on average 29.4±3.9 (median 30, ranging between 18 and 38) compared to 50 maximum score. four mds categories were identified: score 18-22 related to the lowest adherence (5% of study population), score 23-27 to low adherence (25.7%), score 28-32 to intermediate adherence (47.1%), score 33-38 to moderate/acceptable compliance (22.1%). the majority (49.2%) of normal weight students had intermediate-moderate score compared to underweight (10.7%) and overweight/obese (9.3%) students, but differences were not significant. 3.4. awareness and knowledge on seasonality of local food almost 44.0% (n=124) of students reported that both consumption and transportation of food out of season could produce high impact on the ecosystem, followed by an intermediate and low impact for 48.6% and 7.1%, respectively. most of students (n=214, 76.4%) stated that there are remarkable differences between consumption of fruit in season and out of the harvest period, while 16.4% (n=46) did not recognize any differences. the main reasons reported by students for choosing fruit in season were limited treatment with pesticides (49.1%), great freshness (45.8%), low cost (42.5%), enhanced nutritional intake (39.2%), high quality in terms of nutritional properties (33.2%), guarantee of safety of origin and cultivation/production practice (32.7%), reduced impact on environment (29.4%), and scarce additive/preservative content (23.3%). knowledge on seasonality of selected fruit was inadequate. only 62.0% (n=174) and 48.0% (n=134) could correctly identify the seasonal period of strawberries (april-august as its best) and kiwi fruit (november-may), and differences between students who correctly and inaccurately identified these periods were significant (p<0.001). conversely, awareness of the harvest period of oranges (november-may) and peaches (june-september) resulted more satisfactory, being correctly identified by 80.7% and 82.1% (fig. 3), but differences were not significant. fig. 3. proportion of students aware of harvest period of selected food. the black bar indicates the correct answer on the natural harvest season of local food. ital. j. food sci., vol. 29, 2017 675 more than half students (n=154, 55.0%) reported to have knowledge of seasonal freshness of fish and seafood, which is closely related to their reproductive period, whereas 34.3% (n=96) did not know it, and 10.7% (n=30) said that there is no a specific period. the main reasons reported by students to prefer the consumption of fish and seafood at the natural reproductive period were great freshness (65.0%), enhanced nutritional intake (41.8%), healthy nutrition (26.4%), low cost (11.8%), and adequate size of products (10.0%). however, only 40.0% (n=112) and 35.0% (n=98) could recognize the natural period of mussels and sole, respectively (fig. 3), and differences between students who properly and incorrectly identified these periods were significant (p<0.05). 4. discussion this study aimed to evaluate eating habits and adherence to meddiet model, as well as to investigate knowledge on the seasonal period of locally produced food among college students who were studying to become teachers. the survey revealed that the majority of study population had bmi within the normal range, and only 14% was classified as overweight/obese, in agreement to a recent study conducted amongst italian university students (teleman et al., 2015). our findings differed from other studies conducted in europe and italy, in which different percentages of overweight/obese have been reported (peltzer and pengpid, 2015; organization for economic cooperation and development, 2014; osservatorio nazionale sulla salute nelle regioni italiane, 2014; epicentro, 2013); however, data are difficult to compare because of the different target population, and because the percentages were pooled and stratified by specific age groups. in our study, 68% of students reported to never practice physical activity, which is consistent with other results (rodríguez et al., 2013; osservatorio nazionale sulla salute nelle regioni italiane, 2014), indicating high prevalence of inactivity in females who represented more than 95% of whole sample. prevalence of physical inactivity was estimated of 21% in individuals aged ≥15 years from 76 countries (dumith et al., 2011) worldwide, whereas in italy percentages were of 27.2%, 27.8% and 34.8% among 18-19, 20-24 and 25-34 years old individuals, respectively (osservatorio nazionale sulla salute nelle regioni italiane, 2014), and a similar trend was reported in individuals aged 18-34 years (epicentro, 2013). these findings are of serious concerns since the world health organization recommends that adults should practice at least 150 minutes of moderate-intensity physical activity or 75 minutes of vigorous-intensity through the week. it is also well known that physical activity is associated to better anthropometric measures (zaccagni et al., 2014), whilst the inactivity is related to several health risk factors, such as smoking, unhealthy diet, and chronic diseases (moreno-gómez et al., 2012). hence, because of great benefits regardless of changes in anthropometric outcomes, especially in preventing weight gain (conn et al., 2014), college education should focus on health promotion encouraging students in regular physical activity (stanford et al., 2014). our study also revealed that 16% of students never or not every day have breakfast, which could affect performance during the rest of the day, causing fatigue and poor attention among students, as previously reported (ackuaku-dogbe and abaidoo, 2014). breakfast consumption is associated with a better nutritional profile and a lower risk of overweight/obesity (cooper et al., 2011). indeed, irregular breakfast habits are associated with poor nutrition, and skipping this meal could be an indicator of unhealthy eating habits in a population, being also linked with low vegetable intake (lazzeri et al., 2013). most college students further did not meet dietary and physical activity guidelines, suggesting the need of preventive interventions and increased understanding on overweight ital. j. food sci., vol. 29, 2017 676 related risk, in agreement with previous reports (huang et al., 2003; sánchez socarrás and aguilar martínez, 2014). hence, specific strategies involving a combination of physical activity, nutritional, and educational interventions instead of single component-based programs (shirley et al., 2015) should be improved to yield better obesity-related outcomes. in our target population, the main nutritional deviations were related to low intake of vegetables, fruit and legumes, which represent the main components of a balanced diet together with cereal grains and derived products (garcía-meseguer et al., 2014). a prevalent consumption of red meat and products compared to white meat was found, in agreement with other european countries (de piero et al., 2015). the low consumption of fruit and vegetables was reported in other studies conducted in western countries and usa (king et al., 2007; keller et al., 2008; dodd et al., 2010), showing that university students did not follow the recommended consumption because of their taste and price, which represent the most critical barriers (shive and neyman, 2003). conversely, studies conducted in south east asia reported a consumption of fruit and vegetables of five servings per day among female university students (sakamaki et al., 2005; yahia et al., 2008; perera and madhujith, 2012). significant dietary changes can occur since starting university, and students living away from home are more likely to develop unfavourable eating habits by decreasing weekly consumption of fruit, vegetables, seafood, olive oil, and increasing sugar, alcohol and fast food intake (fiore et al., 2015). in our survey, the majority of students were unmarried, and 44% were living outside from family home, suggesting that the unsatisfactory food intake could be probably related to poor education on proper food choices, limited budget and/or to lack of skills to prepare a basic healthy meal. the mds revealed a poor adherence to meddiet, in agreement with other studies among university population (cervera burriel et al., 2014; garcía-meseguer et al., 2014). the unsatisfactory adherence to meddiet was mostly associated with low consumption of food groups suggested on a daily basis or more than three portions per week, and with high intake of food suggested on rare consumption. our survey also investigated awareness on seasonal period of local food. students showed a lack of knowledge on the harvest period of many locally produced foodstuffs, indicating misperception and significant understanding gaps, as previously reported among university students and consumers (wilkins et al., 2002; brooks et al., 2011). most students associated the quality of seasonal food to inappropriate attributes, by referring their preference due to limited chemical treatment or content, guarantee of origin production and for environmental reasons. fruit have proper nutritional and sensory quality at natural harvest season, providing a good source of health-promoting compounds (voća et al., 2014). for example, in addition to beneficial compounds, the attributes of sweetness, acidity, colour and flavour are the most important characteristics of strawberries, affecting either quality, marketability, or choice of consumers. indeed, the ripening process and maturity strongly affect their nutritional composition and contribute to typical taste, indicating that strawberries should be consumed at stage of full maturity (voća et al., 2014). similar findings can be associated with seafood since in the context of human health the season could influence their compositions of both amino acids and fatty acids (çağlak and karsl, 2017). moreover, sensory properties and nutritional value are two sets of characteristics that, together with freshness, are accountable for fish quality, and are affected by many factors including seasonal changes (petrović et al., 2015). the debate on meddiet with emphasis on plant-based foodstuffs consumption and recommendations for seasonal local food consumption are key topics of global framework of sustainable eating (forleo et al., 2015). nevertheless, sustainability of seasonal food in term of environmental performance may be controversial and depends on the definition of ital. j. food sci., vol. 29, 2017 677 seasonal characteristics and environmental impact. some studies have assessed that seasonality is unlikely to deliver large environmental benefits, except for water footprint (foster et al., 2014). reduction in greenhouse gas emissions from eating seasonal vegetables is also limited, representing a minor proportion of the total emissions from food consumption (röös and karlsson, 2013). indeed, the multifaceted concept of seasonality should be considered under a nutritional-health perspective and a global approach, including environmental, socio-cultural and economic pillars of sustainability (akhatou and fernández-recamales, 2014). the study has some limitations: sample size might not be representative of the whole university population of central italy; being the sample represented mostly by females, the results could not be directly generalizable to male subjects; anthropometric measurements, as well as dietary intake were self-reported and assessed by short questions with possible under or over estimation; there were no opportunities for follow-up and verification. despite this, our survey was performed in a short data collection time and involved a priority youngadult population that could be targeted for strengthening public health strategies for eating behaviours improvement. our findings have important implications for research and practice, and provide hitherto invaluable information on a sample of italian college students recruited in a non-metropolitan area never investigated before, 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sci., vol. 28 2016 keywords: pistachio nut flour, starch-lipid complexes, optimization response surface methodology, contour plot, breaking strength, bulk density optmization of extrusion process of rice flour enriched with pistachio nut flour c. severini, t. de pilli* and a. derossi university of foggia, department of science of agriculture, food and environment (s.a.f.e.), via napoli 25, 71100 foggia, italy *corresponding author: tel. +39 881 589245, email: teresa.depilli@unifg.it abstract response surface methodology deriving by superimposing individual contour plots, was used to investigate the optimum operating conditions for extrusion-cooking of rice flour enriched with pistachio nut flour. the highest barrel temperature (128°c) produced a stiff extrudates (high values of breaking strength i.e. 100 n/mm2 and bulk density i.e. 2.2 g/ml). however, graphical optimization studies showed that the optimal operating conditions involved values of 16-17% water feed content and 70-95°c barrel temperature. this research points out the importance to study the biopolymer changes that occur during extrusion-cooking processing because of their huge effect on quality characteristics of extrudates. mailto:teresa.depilli@unifg.it ital. j. food sci., vol. 28 2016 51 1. introduction nowadays, consumers prefer foods easy and convenient to eat (schwartz, 2009). snacks and breakfast cereals are easy to carry, purchase and consume but they are essentially produced from starchy substances such as corn, rice, wheat (yaseen and shouk, 2005) and therefore they could lack some important nutrients. foods with poor nutritional value, lack in micronutrients such as vitamins, minerals, amino acids, fibers and high content of calories can be considered unhealthy. for this reasons, researches are focused on the improvement of nutritional characteristics by the addition of ingredients such as fruit, nuts, fibres, etc. among nuts, pistachios could favourably be used thanks to their ability to lower the risk of cardiovascular diseases, to improve total cholesterol to hdl-c ratios, ldl cholesterol to hdl cholesterol ratios, and hdl cholesterol levels (kocyigit et al., 2006; sheridan et al., 2007; gebauer et al., 2008). consumption of pistachios was also found to increase antioxidant activity in the body (kocygit, 2006) and to improve blood glucose levels (sari et al., 2010). a possible application of pistachio nut flour could be the production of extrudates in order to obtain snack foods with high nutritional and health value. de pilli et al., (2011; 2012) studied the processing conditions that lead to the formation of starch-lipid complexes in a model system and in real food like extrudates made up of rice starch and pistachio nut flour, by differential scanning calorimeter (dsc). in addition, they evaluated the effects of starch-lipid complexes formation on system parameters, fat loss and the breaking strength of extrudates. the results of that work showed that the barrel temperature had a huge effect on system parameters in the real foods as the extraction of lipid fraction determined a decrease of friction force and therefore a decrease of mechanical energy input of processing (de pilli et al., 2008a). moreover, the formation of starch-lipid complexes in real food, was strongly dependent on water feed content, that consequently affected starch gelatinization. the highest fat loss and the hardest texture of extrudates made up of pistachio nut flour were obtained under processing conditions that favoured the maximum formation of starch lipid complexes. the main objective of those studies was to verify the efficacy of model system to describe the biopolymers changes that occur during processing of real food. however, the relationships between process variables and characteristics of the extrudates has not been studied in detail. therefore, this study aimed to investigate the optimum operating conditions of extrusion and the effects of extrusion process variables on the characteristics of rice extrudates enriched with pistachio nut flour by using the response surface methodology (rsm). furthermore, the regression models to predict the characteristics of the extruded material as a function of the process variables were also established. 2. materials and methods 2.1 raw materials rice starch (10.9% moisture) was provided by a.d.e.a. (bursto arsizio, italy); pistachio nut flour was provided by cartellone (bronte, italy); oleic acid was provided by sigma-aldrich (milano, italy). the used pistachio nut flour had a moisture content of 4.8±0.2% and the following chemical composition (dry basis): protein (18.1±0.1%); lipid (49±0.5%); starch (3.3 ±1.5%); soluble sugars (4.5±0.2%); fiber (10.6±2%) and ash (9.7±0.1%). the fat acid composition of lipid fraction of pistachio nut flour, determined according method proposed by ratnayake et al. (2006) was: c14:0 (0.09); c16:0 (9.45); c16:17 (0.86); c17:0 (0.04); c17:1 (0.07); c18:0 (2.12); c18:1 (70.17); c18:2 (15.5); c18:3 (0.32); c20:0 (0.18); c20:1 (0.48); c22:0 (0.09); c24:0 (0.04). the chemical characteristics of tap water used for extrusion trials was: ph 7.7 + 0.1, hardness (°f) 25.1±1.5, total dissolved solids dried at 180°c 645±38.5 mg/l and chloride content 54.6±0.4 mg/l. the content of moisture, ash, protein and fat of flours were determined according to the 4415a, 08-01, 46-10, 30-25 aacc international approved methods (2003). 2.2. extrusion experiments according to previous studies (de pilli et al., 2011), the formula containing 75% rice starch and 25% pistachio nut flour was used. the extrusion experiments were carried out using a thermo prism ptw-24 (thermo haake polylab system, germany) co-rotating twinscrew extruder. the screw geometrical features were the following: diameter 24 mm and length 672 mm (l/d = 28:1) and distance between shafts 19 mm. fig. 1 reports the screw configuration used. during extrusion experiments, the screw speed was kept constant at 140 rpm, as well the flour feed rate was kept constant at 2.8 kg/h (dry weight). the flours were proportioned by volumetric gravity feeder. the extruder was divided into six zones, independent of each other for temperature control and adjustment. for all experiments, the first two zones were kept at 35 and 65°c respectively, whereas the last four zones were adjusted at the same temperature according to experimental plan (table 1). 52 ital. j. food sci., vol. 28 2016 fig. 1 screw configuration used to extrude rice starch and pistachio nut flour. table 1 coded and actual values of variables (a) and the arrangement and responses of factorial design (b). a) coded level uncoded barrel temperatures of last four zones water feed content (x1) (x2) (°c)** (%) +1.4 128 21.8 +1 120 21.0 0 100 19.0 -1 80 17.0 -1.4 72 16.2 **the first two zones were kept at 35° and 65°c respectively, whereas the last four zones were adjusted according to experimental plan. b) treatments coded level processing variables responses x1 x2 x1 (°c) x2 (%) y1 (%) y2 (nm) y3 (n/mm2) y4 (g/ml) 1 -1 -1 80 17.0 23.67±0.02 554±0.55 7.97±0.92 0,80±0.26 2 -1 1 80 21.0 23.00±0.06 556±0.80 14.21±3.12 0,81±0.45 3 1 -1 120 17.0 60.00±0.03 552±0.52 96.24±1.19 2,10±0.50 4 1 1 120 21.0 60.00±0.01 549±0.66 75.58±0.59 2,08±0.37 5 -1.4 0 72 19.0 20.00±0.07 559±1.17 7.58±1.40 0,80±0.26 6 1.4 0 128 19.0 70.00±0.01 552±0.41 99.04±0.19 2,11±0.11 7 0 -1.4 100 16.2 52.45±0.01 552±2.04 53.75±1.50 0,87±0.34 8 0 1.4 100 21.8 52.00±0.03 555±0.63 65.68±0.45 0,87±0.25 9 0 0 100 19.0 51.50±0.02 556±1.02 43.05±2.73 0,87±0.37 10 0 0 100 19.0 52.00±0.03 554±0.42 51.32±2.60 0,94±0.23 11 0 0 100 19.0 50.80±0.01 555±0.41 49.52±1.23 0,96±0.28 x1: barrel temperatures of last four zones (°c); x2: water feed content (%); y1: complex index; y2: λ max ; y3: breaking strength; y4: bulk density. the water was pumped to the first zone of the extruder and the delivery capacities of water pump were 7.5; 7; 6; 4.75; 4.25 l/h. these values were chosen to obtain the moisture feed content of dough indicated in the experimental plan (table 1). the die used had a spherical shape (diameter 300 mm) in which there was one circular hole with a diameter of 5 mm. at the exit of the die, the extrudates were manually cut into sticks (about 50 mm in length) using a knife. the extrudates were dried over night at 40°c in a vacital. j. food sci., vol. 28 2016 53 uum oven salvis vacucenter vc 50 (salvis ag, reussbühl/lucerne, switzerland). samples used to determine complexing index and iodine spectrum of the soluble fractions of the extrudates were finely ground (particles < 300 microns) using a buhler ml 1204 mill (germany). all the ground samples were defatted in a soxtec fat-extractor with petroleum ether at 37°c (bp 34.6°c) for 155 min to remove uncomplexed lipids before submit samples to chemical analyses (bhatnagar and hanna, 1994). 2.3 experimental plan the extrusion experiments were carried out at five temperature profiles (table 1a) and percentages of water feed contents (21.8%, 21.0%, 19.0%, 17.0%, 16.2% expressed as percentage of dry basis). all extrusion experiments were performed at least in triplicate. coded and actual values of variables are shown in table 1a, the factorial design of two variables (temperature profile and feed water content) and five levels of values were used according to central composite design (ccd) (box et al., 1978). this method was used to evaluate the single influences of the processing variables as well as their possible interactions. eleven tests (table 1b) with different combinations of process variable values were obtained. 2.4 complexing index complexing index (ci) was determined using the method described by guraya et al. (1997). the iodine solution used for analysis was prepared by dissolving overnight 2 g of potassium iodine and 1.3 g of i 2 in 50 ml distilled water. then the final volume was made to 100 ml using distilled water. a 5 g sample was mixed with 25 ml of distilled water in a test tube. the test tube was vortexed for 2 min and centrifuged for 15 min at 314.1 rad/s. the supernatant (500 ml) and distilled water (15 ml) were added to the iodine solution (2 ml). the tube was inverted several times and absorbance was measured at 690 nm through a uv/ vis spectrophotometer (beckman du 640, california). ci was calculated from the following equation (2): (2) the analysis was carried out in triplicate. 2.5 iodine spectra of starch samples starch samples were solubilised in l n naoh as recommended by schoch (1964). the absorbance spectra of starch-iodine complexes were measured using a spectrophotometer uv/ vis perkinelmer lambda 25 (milan, italy) from 400-700 nm, and wavelength of maximum absorption (λ max ) values were determined. 2.6. breaking strength (n/mm2) a stable dynamometer micro system tahdi texture analyser (enco s.r.l., venezia, italy) with a plunger was used for texture analysis. extrudates were placed over two supports, 1.5 cm apart, and broken in the middle by a plunger that had a shape of a cone frustum (the thickness of contact surface with extrudate was 1 mm2 and the speed was kept constant to 0.5 mm/s). results were expressed as breaking strength (n/mm2), i.e. the strength needed to break the extrudate. this index is related to microstructure of samples and it simulates the incisors impact at biting (van hecke et al., 1998). for each sample, at least ten repetitions were carried out. 2.7 bulk density (bd) bulk density was measured using a displacement method (yu et al. 2012). extrudates were cut into strands of about 25 mm long and about 10 g strands were weighed (m, grams) and put in a 100 ml cylinder; then yellow millet particles were added to fill up the cylinder. the extrudates were taken out, and the volume of the yellow millet particles was measured (v, milliliters); ten measurements were performed to calculate the average. bulk density (bd) was calculated as equation (3): (3) 2.8 statistical analysis data were submitted to statistical analysis using statsoft, vers. 5.1 (statsoft, tulsa, usa) software. the analysis was carried out in two steps. the first involved a stepwise regression analysis to identify the relevant variables, and the second used a multiple regression analysis (standard least square fitting) to fit a second order mathematical model, according to the following polynomial equation: y = b 0 + σbiχi + σbiiχii2 + σbijχiχj where y is the dependent variable (complex index, iodine spectrum of the soluble fractions of the extrudates; breaking strength and bulk density of extrudates), b 0 is a constant value, χi and χj are the independent variables (barrel temperature and water feed content) in coded values and bi, bii and bij are the regression coefficients of the model. this model allowed the effects of the linear (χi), quadratic (χi2) and combined (χiχj) terms of the independent variables to be assessed on the dependent variable. 54 ital. j. food sci., vol. 28 2016 variables with a significance lower than 95% (p>0.05) were left out of the equation. iso-response surface were developed in order to describe both individual and interactive effects of the independent variables of the extrusion-cooking process on complex index, iodine spectrum of the soluble fractions of the extrudates; breaking strength and bulk density of extrudates. extrusion processing parameters were optimized by using the design-expert version 8.07.1 (stat-ease inc., minneapolis, usa) through a conventional graphical method of rsm in order to obtain extrudates with acceptable properties. all the processing variables were kept within a range while the responses were either minimized (breaking strength and bulk density). contour plots of all the responses were then superimposed, and the optimum region appeared. the contour plots were obtained by superimposing of contour plots from which one could determine the optimum process variables range (barrel temperature and water feed content) to obtain extrudates made up of rice and pistachio nut flour with specified properties. 3. results and discussion fig. 2a shows the complexing index values as a function of barrel temperature and water feed content. the barrel temperature was the only processing variable that had a significant effect on complexing index. in particular, values of the complexing index increased with increasing of barrel temperature (fig. 2a). this means that, in this case, the highest barrel temperatures did not involve the melting of starch-lipid complexes. it is possible to suppose that the presence of other components in the dough increase the melting temperature of starch-lipid complexes, that result then more protected by heating during processing. moreover, the presence in lipid fraction of triglycerides, di-glycerides and fatty acids involves an increase of characteristic melting temperature of starcholeic complexes (de pilli et al., 2008b; 2011). to confirm the formation of starch-lipid complexes, values of λ max for native starch extruded with and without pistachio nut flour were also determined. rice flour, extruded without nut flour and with an amylose contents of 89 %, showed λ max within 592-595 nm. fig. 2b shows that the increase of barrel temperature shifted λ max from 595 nm towards the amylopectin side (520 nm), due to the decrease of available amylose that is bounded with lipids. these results are in agreement with those of complex index and confirm the formation of starch-lipid complexes (fig. 2a). in fig. 3a is reported the break strength (bs) values as a function of barrel temperature and water feed content. also in this case, the only variable that had a significant effect on mifig. 2 starch-lipid complex index (a) and λ max values (b) of samples made up of rice starch and pistachio nut flour blend as a function of barrel temperatures and water feed content. crostructure of the extrudates was the barrel temperature. in particular, the extrudates obtained at the highest values of barrel temperature (128°c) opposed the highest resistance to break, while low values of break strength were obtained at the lowest barrel temperature (70°c) (fig. 3a). the formation of starch-lipid complexes obtained with the increase of barrel temperature could explain the high compactness of extrudates (bhatnagar and hanna, 1994; de pilli et al., 2008a,b). data of bulk density (bd) are in agreement with those of the break strength. in fact, the extrudates showed high values of bulk density at the highest barrel temperature (fig. 3b). the increase of bulk density and break strength values of extrudates may be caused by an alteration in the ratio between free amylose and amylopectin. according to guy and horne (1988), the elastic character of the molten extrudates creates a swell at the die that controls the overall phenomenon of expansion of the extrudates. launay and lisch (1983) suggested that amylose–lipid complex formation was the key factor influencing the flow properties of starch pastes. when starch is extruded, expansion is dependent on the formation of a starch matrix that entraps the water vapor, resulting in the formation of bubbles (guy and horne, 1988). it ital. j. food sci., vol. 28 2016 55 is reasonable to speculate that the addition of lipids might have affected the character of this matrix (i.e., the viscoelastic properties of molten extrudate) so that it could no longer hold water vapor, resulting in lower expansion and higher break strength. the increase of bulk density and break strength caused by decrease of swelling of starch can also be compared to that one of native starch upon gelatinization. swelling is generally considered a property of amylopectin while amylose is considered a diluent. the amylose and native lipids contained in cereal starches may inhibit swelling under particular conditions when amylose–lipid complexes are likely to be formed (tester and morrison, 1990). according to krog (1973), complex formation with the linear component of starches makes the structure more rigid and stabilizes the swollen granule against breakdown, resulting in restricted swelling. these statements are in agreement with de pilli et al. (2008b). in this study, a conventional graphical method of multiresponse optimization technique was applied to obtain the combination of optimum process variable for the production of extrudates enriched with pistachio nut flour. to determine the extrudates with acceptable properties, main criteria of optimization constraints were related to bulk density (< 1.2 g/ml) and breaking strength (< 40 n/mm2). superimposing the individual contour plots for the product response variables resulted in the identification of a region (shown by the blank space area) that satisfied all constraints as shown in fig. 4. superimposed contour plots indicated the ranges of variables that could be considered as the optimum range to obtain the best characteristics of extrudates in terms of bulk density and braking strength. the optimum ranges of variables obtained from the superimposed contours were 16–17 % water feed content and 70-95°c barrel temperature. extrusion-cooking fig. 3 breaking strength (a) and bulk density values (b) of samples made up of rice starch and oleic acid blend as a function of barrel temperatures and water feed content. fig. 4 superimposed contours for the product responses affect by water feed content and barrel temperature. *bd: bulk density and bs: breaking strength. 56 ital. j. food sci., vol. 28 2016 was carried out for confirmation under the optimum process conditions and the responses were recorded (mean of five measurements). in particular, the following operating conditions were chosen: 72 °c barrel temperature and 16% water feed content. the values predicted by the software were ≤ 0.8 g/ml for bulk density and ≤ 20 n/mm2 for breaking strength. the data obtained by extrusion experiments carried out at the same operating conditions were respectively 0.78 g/ ml and 7.38 n/mm2. the veracity of values of the responses predicted by the software was assessed with the help of a two-tailed, one-sample t-test. the results of the t-test indicated that the coefficient of variation was not greater than 5 %. therefore, the developed model was suitable in representing the optimum operating conditions for this particular application. 4. conclusions the obtained results showed that the barrel temperature was the variable that has mainly affected the formation of starch-lipid complexes and structure of the extrudates. in particular, the worst characteristics of extrudates (hardness and bulk density of extruded products) were obtained at the highest temperature that corresponds to the maximum formation of starch-lipid complex. moreover, the model was found to be statistically valid and demonstrated adequate information regarding the behaviour of the responses upon variations of the process variables. optimum process conditions and the corresponding predicted responses could be obtained with the help of the models. the predicted responses at the optimum conditions were not significantly different from the experimental values. according to the optimum conditions given for the variables, the process could be referred to standardization of industrial production of snack food made up of rice and pistachio nut flours with high qualitative characteristics. 5. references aacc international. approved methods of analysis, 10th 1 ed. methods 44-15a, 08-01, 46-10, 30-25 approved 2003. aacc international: st. paul, mn. bhatnagar s. and hanna m.a. 1994. extrusion processing conditions for amylose lipid complexing. cereal chem. 71(6): 587. box g.e.p., hunter w.g. and hunter j.s. 1978. statistics for experiments. an introduction to design data analysis and model building. wiley, new york. de pilli t., carbone b.f., derossi a., fiore a.g. and severini c. 2008a. effects of operating conditions on oil loss and structure of almond snacks. int. j. food sci. technol. 43(3): 430. paper received october 29, 2014 accepted march 27, 2015 de pilli t., jouppila k., ikonen j., kansikas j., derossi a. and severini c. 2008b. study on formation of starch-lipid complexes during extrusion-cooking of almond flour. j. food eng. 87: 495. de pilli t., derossi a., talja r.a., jouppila k. and severini c. 2011. study of starch-lipid complexes in model system and real food produced using extrusion-cooking technology. innov. food sci. emerg. technol. 12(4): 610. de pilli t., derossi a., talja r.a., jouppila k. and severini c. 2012. starch-lipid complex formation during extrusion-cooking of model system (rice starch and oleic acid) and real food (rice starch and pistachio nut flour). eur. food res. technol. 234(3): 517. gebauer s.k., west s.g., kay c.d., alaupovic p., bagshaw d. and etherton p.m.k. 2008. effects of pistachios on cardiovascular disease risk factors and potential mechanisms of action: a dose-response study. am. j. clin. nutritional 88(3): 651. guraya h.s., kadam r.s. and champane e.t. 1997. effect of rice starch-lipid complexes on in vitro digestibility, complexing index and viscosity. cereal chem. 74: 561. guy r.c.e. and horne a. 1988. extrusion and co-extrusion of cereals. in “food structure-its creation and evaluation”. j.m.v. blanshard and j.r. mitchell (eds.), p. 331349, london. kocyigit a., koylu a.a. and keles h. 2006. effects of pistachio nuts consumption on plasma lipid profile and oxidative status in healthy volunteers. nutritional metabolism cardiovasc. dis. 16: 202. krog n. 1973. influence of food emulsifiers on pasting temperature and viscosity of various starches. starch/staerke, 25: 22. launay b. and lisch j.m. 1983. twin screw extrusion cooking of starches: behaviour of starch pastes, expansion and mechanical properties of extrudates. j. food eng. 2: 259. ratnayake w.m.n., hansen s.l. and kennedy m.p. 2006. evaluation of the cp-sil 88 and sp-2560 gc columns used in the recently approved aocs official method ce 1h-05: determination of cis-, trans-, saturated, monounsaturated, and polyunsaturated fatty acids in vegetable or non-ruminant animal oils and fats by capillary glc method. j. am. oil chem. soc. 83: 475. sari i., baltaci y., bagci c., davutoglu v., erel o., celik h., ozer o., aksoy n. and aksoy m. 2010. effect of pistachio diet on lipid parameters, endothelial function, inflammation, and oxidative status: a prospective study. nutrition 26(4): 399. schoch t.j. 1964. iodimetric determination of amylose. potentiometric titration: standard method. in: “methods in carbohydrate chemistry”. r. whistler (ed.), p. 157-160. academic press, new york. schwartz c. 2009. the impact of removing snacks of low nutritional value from middle schools. health education and behav. 36 (36): 999. sheridan m.j., cooper j.n., erario m. and cheifetz c.e. 2007. pistachio nut consumption and serum lipid levels. j. am. coll. nutrition 26(2): 141. tester r.f. and morrison w.r. 1990. swelling and gelatinization of cereal starches. i. effects of amylopectin, amylose, and lipids. cereal chem. 67: 551. van hecke e., allaf k. and bouvier j.m. 1998. texture and structure of crispy-puffed food products ii: mechanical properties in puncture. j. texture stud. 29: 617. yaseen a.a.e. and shouk a.a. 2005. effect of extrusion variables on physical, structure and sensory properties of wheat germ-corn grits extrudates. egypt. j. food sci. 33 (1): 57. yu l., ramaswamy h.s. and boye, j. 2012. twin-screw extrusion of corn flour and soy protein isolate (spi) blends: a response surface analysis. food and bioprocess technol. 2(5): 485. http://www.springerlink.com/content/0003-021x/ ital. j. food sci., vol. 30, 2018 428 paper bioactive and pharmacokinetic characteristics of pre-matured black raspberry, rubus occidentalis d. shin1, k.s. chae1, h.r. choi1, s.j. lee1, s.w. gim1, g.t. kwon1, h.t. lee3, y.c. song3, k.j. kim3, h.s. kong*2 and j.w. kwon*1 1berry & biofood research institute, gochanggun, jeonbuk, republic of korea 2college of animal biotechnology & resource, sahmyook university, seoul, republic of korea 3college of pharmacy, sahmyook university, seoul, republic of korea *corresponding author: tel.: +82 635605190 *e-mail address: hskong0813@syu.ac.kr, kjwung@hanmail.net abstract black raspberry (br, rubus occidentalis) is a berry originating from north america. it has a high anthocyanin and flavonoid content that seems to be dependent on ripening. therefore, the bioactive and pharmacokinetic effects of br were evaluated using prematured br(pbr) collected from may to june. the total polyphenol, flavonoid and vitamin c content in pbr decreased while anthocyanin increased from 0.13 to 10.70 mg/g at 35 days after fruit set. the antioxidant activities due to dpph and abts determination decreased as brs were matured. moreover, it is clear that the content of many phenolic compounds including gallic acid, caffeic acid, p-coumaric acid, ferulic acid, rutin, myricetin, luteolin and kaempferol was diminished during maturing. both ferulic acid and rutin of br generated the highest decrease as compared to other phenolic compounds. the pharmacokinetic tmax and cmax of pbr were 0.6 h and 0.264 µg/ml, respectively. based on these results, pbr can generate qualified functionality as food and/or as a raw medicinal material. keywords: black raspberry, anthocyanin, flavonoid, polyphenol, pharmacokinetic ital. j. food sci., vol. 30, 2018 429 1. introduction due to concerns for wellbeing and lifestyles of health and sustainability (lohas) resulting from a sharply increasing income, the consumption for functional foods is also growing very fast. many studies have been conducted to develop functional food products containing natural, bioactive compounds to meet consumer demand. berries – such as blueberry, strawberry, and black raspberry – are used as common functional food additives to meet demand from health-conscious consumers. black raspberry (br, rubus occidentalis), which originated from north america, is one of the main functional food additives due to its pharmacological effects that were written about in donguibogam, an ancient korean medicinal textbook written by j. hur (cho et al., 2005). most of the br cultivated in gochang, korea belongs to rubus occidentalis. however, the br written of in donguibogam, korea is rubus coreanus miquel. br is normally cultivated in soil with ph 6.0–7.2, and it shows medium water usage (ozgen et al., 2008). since br is generally cultivated in moderate to low incident light on coarsely textured soil with average to low levels of organic matter and inorganic nitrogen, brs form in june in korea (bajcz, 2014). a morphological study indicates br is a multi-stemmed shrub that forms broad colonies. br contains thorns with three (ternate) or five palmately arranged leaflets (lee et al., 2014). br seems to generate a bright red flower in may, and it tends to breed a floral leaf that is shorter than its sepal. it has semi-spherical shape with naps. br berry will turn to wine in color when the berry has matured. normally, edible br berries are harvested at the early stage of summer. however, some pre-matured br berries are also gathered as medicines to improve sexual dysfunction of males, as mentioned in donguibogam (zhao et al., 2011). approximately 1.7% of anthocyanin in br tends to be responsible for most br functions. the main anthocyanins of br are cyanidin 3-glucoside, cyanidin 3-rutinoside, cyanidin 3sambubioside, and cyanidin 3-xylosylrutinoside (tian et al., 2006; tulio et al., 2008; lee et al., 2013). torre and barritt (1977) have indicated that both cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside of br anthocyanin seem to contribute to the red or black color and antioxidant effect. in addition, as mentioned above, j. hur during the joseon dynasty indicated that br (rubus coreanus miquel) has anti-biotic, anti-cancer, and anaphylaxis effects (baek et al., 2005). such effects have been confirmed by many current scientists (baek et al., 2005; liu et al., 2005; duncan et al., 2009; jeong et al., 2010; lee et al., 2011). a study conducted by cho et al. (2005) showed that br can increase antioxidant activities, and it was confirmed through abts, dpph and tbars when 1 g br is added to 100 ml of 10 or 100% ethanol. the study also showed that protocatecuic acid is the most abundant phenolic compound in br when analyzed via hplc (cho et al., 2005). chae et al. (2014) and bobinaite et al. (2012) demonstrated that br contains a high amount of ellagic acid. they also reported that ellagic acid of br possesses strong antioxidant effects (bobinaite et al., 2012; chae et al., 2014). moreover, since ellagic acid is not easily metabolized by microflora and/or broken down by stomach acids, it is commonly selected as standard index material for berries. to increase the use of br as a functional food additive in korea, br was collected and its individual phenolic content was determined in this study. based on the phenolic content of br, its antioxidant effects were examined for br depending on the stage of maturation. in addition, to evaluate the pre-matured br (pbr, <28 days after fruit set) value and its quality as a functional food additive, the ellagic acid content, which is the standard material index for berries, was evaluated to determine the pharmacokinetic index of pbr. ital. j. food sci., vol. 30, 2018 430 2. materials and methods 2.1. sample preparation according to research conducted by chung et al. (2008), br planting soil in gochang, korea mostly consisted of silt loam (64%), loam (35%) and clay (1%). brs were cultivated in an open field with soil at ph 5.3∼6.7 and harvested from 4-year-old br plants. all brs were sampled from may to june of 2014 with an interval of 3-4 days (fig. 1). the average temperature and humidity of gochang, korea during harvest, was 17.4∼21.7°c and 72.2∼81.0 %, respectively (kma, 2017). all brs collected were similar to the shuttleworth, which was confirmed by the random amplified polymorphic dna (rapd). after harvest of the br, they were dried at 60°c for 36 h and then powered for storage. 10 g of br powder were sampled and mixed with 90 ml double distilled water (ddw). the mixture was distilled twice at 80°c for 2 h. all br extracts were filtered, freeze dried, and stored at -70°c until use. 15 days after fruit set 22 days after fruit set 25 days after fruit set 28 days after fruit set 32 days after fruit set 38 days after fruit set 41 days after fruit set figure 1. pre-matured black raspberry (pbr, rubus occidentalis) and matured black raspberry fruits harvested from may to june with an interval of 2-3 days. 2.2. determination of the total phenolics, flavonoids, anthocyanins, and ascorbic acid content the total phenolics of br were measured using a modified folin-ciocalteu colorimetric method (singleton and rossi, 1965). 20 µl of filtered br (1 mg/ml) was mixed with water and folin-ciocalteu reagent. after 3 min of incubation at room temperature, 300 µl of 20% na2co3 were added. the mixture was then incubated in the dark for 30 min at ital. j. food sci., vol. 30, 2018 431 40°c. the absorbance was measured at a wavelength of 725 nm (beckman du 730, beckman coulter inc., fullerton, ca, usa) and expressed as its gallic acid equivalent. the flavonoid content was determined using the method described by meda et al. (2005) with slight modifications. briefly, 0.25 ml of br (1 mg/ml) were mixed with 1 ml ddw, 0.075 ml of 5% nano2, 0.075 ml of 10% alcl3, and 0.5 ml of 1 m naoh. the final volume was then adjusted to 2.5 ml with ddw. the absorbance of each sample was then measured at a wavelength of 410 nm (beckman du 730, beckman coulter inc., fullerton, ca, usa). quercetin was used as a standard. all readings were expressed as micrograms of quercetin equivalent. monomeric anthocyanin was evaluated using the modified ph differential method (chaovanalikit and wrolstad, 2004). optimum dilution for br filtrate was defined using 0.025 m of potassium chloride buffer. individual br was diluted with either potassium chloride buffer or sodium acetate buffer to reach an optimum state. after 15 min of incubation, the absorbance of each diluted br was measured at a wavelength of 700 nm. the amount of anthocyanin was calculated as follows: anthocyanin (mg/g) = (a × mw × df × 1000)/(ε × 1) a: absorbance of diluted br = (aλvis-max – a700nm) ph1.0 – (aλvis-max – a700nm) ph 4.5; mw: expressed as cyanidin 3-glucoside (450); df: dilution factor; ε: molar absorptivity (26,900). the ascorbic acid content was determined using the modified method described by kampfernkel et al. (1995). briefly, 0.8 ml of 10 % (w/v) trichloroacetic acid were added to 200 µl of br filtrate (1 mg/ml). each mixture was then pre-cooled and centrifuged at 3,000 rpm (4°c) for 5 min. then, 1.5 ml water and 0.2 ml 0.2 n folinciocalteu were added to 0.5 ml of br supernatant. after 10 min of incubation at room temperature, the absorbance was recorded at a wavelength of 765 nm. l-ascorbic acid was used as a standard. 2.3. dpph and abts radical scavenging activities the 1,1-dipenyl-2-picryl-hydrazyl (dpph) free radical scavenging activity of each extract sample was determined using the method described by brand-williams et al. (1995). briefly, freeze-dried brs were diluted with methanol to obtain five different concentrations. each diluted br (10 µl from 1 mg/ml of br) was mixed with 0.2 mm of dpph in dimethyl sulfoxide. after 30 min of incubation at room temperature in the dark, the absorbance was measured at a wavelength of 517 nm using a spectrophotometer. the inhibition percentage was calculated from the equation below: dpph radical scavenging activity (%) = [(control absorbance – br absorbance)/control absorbance] × 100 the 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (abts) radical scavenging activities of the br extract were determined using the method described by re et al. (1999). 2.45 mm of potassium persulfate was added to 7 mm of abts and the mixture was kept in the dark at room temperature for 12-16 h. the abts radical cation solution was diluted with phosphate buffer saline to obtain an absorbance value of less than 0.70 at a wavelength of 734 nm before the analysis. after adding 2.0 ml of diluted abts radical cation solution to 20 µl of br sample (1 mg/ml), the reaction mixture was incubated in a cuvette at 30°c for 6 min. trolox, an analog of vitamin e antioxidant, was used as control. ital. j. food sci., vol. 30, 2018 432 the abts radical scavenging capacity of the br was calculated using the following equation: abts free radical scavenging activity (%) = [(control absorbance – br absorbance)/control absorbance] × 100 all dpph and abts values calculated using graphpad prism 5.0 (graphpad prism version 5.0, graphpad software inc., san diego, ca, usa), were generated as the 50% inhibition concentration (ic50). 2.4. liquid chromatography analysis for the phenolic quantification and ellagic acid identification five g of br were homogenized in 20 ml of 80 % acetone containing 0.2 % formic acid for 1 min. br was then concentrated, mixed with 10 ml of acidified water, and passed through an activated c18 sep-pak cartridge (waters corp., milford, ma). all adsorbed phenolics onto the c18 column were then recovered with 2 ml of acidified methanol containing 3% formic acid. individually recovered br in methanol was filtered, and 10 µl of br methanol extract was used to analyze the phenolics using liquid chromatography (lc) (acquity h-class, waters) equipped with an autosampler/injector and photodiode array (pda) detector. a shiseido capcellpak c18 ug (5 um, 4.6 × 250 mm) was used for separation. both mobile phase a (0.2 m ortho-phosphoric acid, ph1.57) and b (20% 50 mm ammonium dihydrogen phosphate, ph2.6 in 80% acetonitrile) were used for elution. the flow rate was 1.0 ml/min, and detection was performed at 280 nm (gallic acid), 320 nm (caffeic acid, p-coumaric acid, ferulic acid) and 360 nm (rutin, myricetin, luteolin, kaempferol). a gradient was employed as follows: 0-10 min, 80% a; 10-15 min, 70% a; 1520 min, 60% a; 20-25 min, 10% a; 25-30 min, 10 % a; 30-40 min, 95% a. all phenolics were identified and quantified using external standards (gallic acid, caffeic acid, p-coumaric acid, ferulic acid, rutin, myricetin, luteolin, and kaempferol). data scanned at 280–360 nm were collected. all lc handling steps were followed as mentioned in a previous study (chae et al. 2014). 2.5. animal model and blood sample preparation male sprague dawley rats (7 wks, 220 g) were obtained from kosa bio, korea. a total of six rats were acclimated in an environmentally controlled breeding room at a temperature of 22±2°c and relative humidity of 50±10 % with a 12 h dark/light cycle for one week before being used for the experiments. they were provided ad libitum access to commercial chow and water. all rats were handled in accordance with the recommendation of the regulations for the administration of affairs concerning experimental animals (bbriiacuc-16001). pbr at a dose of 150 mg/kg was orally administered to each rat. a serial of blood samples was collected at 0, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h post oral administration. all blood samples were immediately transferred to a bd vacutainer lithium heparin tube and centrifuged at 3,000 rpm for 10 min at 4°c. the ph of rat plasma (100 µl) was adjusted to ph 2.5 using 1 m potassium dihydrogen phosphate solution. serial addition of 50% phosphoric acid and acetonitrile was then performed. the plasma samples were centrifuged at 12,000 rpm for 8 min under refrigerated conditions. the supernatant was taken, dried, and re-dissolved in 0.1 ml methanol. each supernatant sample was then used for ellagic acid analysis under the lc/ms/ms conditions summarized in tables 1 and 2. ital. j. food sci., vol. 30, 2018 433 table 1. liquid chromatography (lc)/ms/ms1 analytical conditions used for analysis of ellagic acid in sprague dawley rat serum. parameter condition hplc system agilent 1290 infinity binary hplc system with 6420 triple quadrupole lc/ms system column shiseido capcellpak c18 ug (5 μm, 4.6×250 mm) column temperature 40oc flow rate 1.0 ml/min injection volume 10 μl mobile phase a : 0.1% formic acid b : methanol gradient step time (min) a (%) b (%) 0 70 30 7.0 50 50 13.0 10 90 15.0 70 30 18.0 70 30 1ionization type: esi (electospray ionization) negative; gas temperature (°c): 320; gas flow (l/min): 9; nebulizer (psi): 15; capillary (v): 4,000; scan mode: multiple reaction monitoring (mrm). table 2. liquid chromatography (lc)/ms analytical conditions used for analysis of ellagic acid in prematured black raspberry (pbr, rubus occidentalis) extract. compound rt 1 (min) mw 2 precursor ion (ms m/z) product ion (ms, m/z) confirm ion frag (v) collision energy (v) ellagic acid 12.1∼12.3 302 30[m-h]229 257 185 170 20/16/15 1rt: retention time; 2mw: molecular weight. 2.6. pharmacokinetic analysis 5 µl of different concentrations (0.625, 1.25, 2.5, 5.0, and 10.0 µg/ml) of ellagic acid in plasma were used to obtain the ellagic acid calibration curve (final concentrations of 31.25, 62.50, 125, 250, 500 ng/ml). as a result of ellagic acid calibration (r2 = 0.998), a linear regression equation was obtained. each plasma sample was then injected to lc/ms/ms. the pharmacokinetic parameters of ellagic acid were calculated using winnonlin software version 6.3 on non-compartmental analysis (pharsight cor., mountain view, usa). 2.7. statistical analysis all experiments were carried out in triplicates or quadruplicates and expressed as mean ± standard deviation. statistical analyses were performed using spss program (spss version 12.0, spss chicago, il, usa). unpaired t-tests or one-way repeated measures anova were performed when appropriate. if significant in the anova test, differences in the ital. j. food sci., vol. 30, 2018 434 means were determined using duncan’s multiple range tests. statistical significance was considered when the p-value was less than 0.05. 3. results 3.1. quality properties and phenolic compound of rubus occidentalis the anthocyanin content in br increased from 0.13 to 10.70 mg/g, although the total polyphenols, total flavonoids, and vitamin c content in br decreased during maturing (table 3). the dpph and abts radical scavenging activities of br collected on june 19th were 8.44 and 4.53 times higher, respectively, compared to those of br collected on may 23. this indicates that antioxidant activities decreased while maturing. gallic acid, caffeic acid, p-coumaric acid, ferulic acid, rutin, myricetin, luteolin, and kaempferol were detected in br (table 4). both gallic acid and ferulic acid were found to be its major phenolics. depending on the br harvest day, the amount of eight different phenolics decreased (p < 0.05) when collected on june 19th, the last day of harvest. five phenolics (caffeic acid, pcoumaric acid, rutin, myricetin, and kaempferol) had the maximum contents on may 26 or may 30. however, the luteolin content was the highest on june 9th. for this reason, luteolin generated the least disappearance, and only 42.5% of luteolin disappeared when br was collected on june 19. however, only 1% rutin was left at the end of harvest. the rutin and ferulic acid contents at the end of the harvest were 1 and 3%, respectively. the content decreases for both were the highest compared to the other six phenolics. 3.2. pharmacokinetic and kinetic analysis pbr was orally administrated to male sprague dawley rats. the plasma concentrationtime profile of ellagic acid in sprague dawley rats (n = 5) is shown in fig. 2. ellagic acid was confirmed and quantified when plasma was sampled at 0.25, 0.5, 1, 2, and 4 h after oral administration of pbr. figure 2. the mean plasma concentration-time profile of ellagic acid in rats after oral administration of prematured black raspberry (pbr, rubus occidentalis) at 150 mg/kg (mean±sd, n = 5). time (h) 0 2 4 6 8 10 co nc en tra tio n (u g/ m l) 0.0 0.1 0.2 0.3 0.4 0.5 ital. j. food sci., vol. 30, 2018 435 table 3. chemical quality properties of pre-matured black raspberry (pbr, rubus occidentalis)1 harvested from late may to early june. days after fruit set (date) total polyphenol (mg/g) total flavonoid (mg/g) anthocyanin (mg/g) ascorbic acid (mg/100g) dpph2 ic50 (ug/ml) abts3 ic50 (ug/ml) 15 (may 23) 170.75±4.65a 1.21±0.02a 0.13±0.01e 24.30±0.38a 95.9±4.52e 261.87±10.25d 18 (may 28) 162.16±4.06a 1.10±0.02b 0.18±0.01e 21.86±0.51b 114.57±4.85de 278.13±15.39d 22 (may 30) 148.64±7.78b 1.07±0.04b 0.08±0.01e 20.34±0.14c 126.57±5.69de 266.23±9.58d 25 (june 2) 126.52±8.37c 0.80±0.04c 0.12±0.01e 17.03±0.12d 160.67±5.91de 326.60±25.54d 28 (june 5) 111.41±2.94d 0.68±0.01d 0.05±0.01e 15.25±0.03e 193.97±1.36d 406.13±31.42d 32 (june 9) 79.04±2.87e 0.51±0.05f 0.81±0.05d 16.79±0.94f 418.83±18.75c 677.73±25.28c 35 (june 13) 60.44±2.62f 0.60±0.01e 3.75±0.12c 10.78±0.03f 824.93±59.99a 912.50±4.90b 38 (june 16) 61.16±7.37f 0.45±0.01f 6.08±0.05b 14.42±0.03g 511.13±57.92b 771.53±99.54bc 41 (june 19) 45.15±0.71g 0.52±0.02g 10.70±0.1a 10.62±0.03h 809.77±99.82a 1186.17±294.46a 1values are the mean±sd (n = 3) based on dry basis. 22,2-diphenyl-1-picrylhydrazyl. 32,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid. a-hvalues with different superscripts within the same columns are significantly different (p < 0.05). table 4. phenolic compositions of pre-matured black raspberry (pbr, rubus occidentalis)1 harvested from late may to early june. days after fruit set (date) phenolic compound (μg/g) gallic acid caffeic acid p-coumaric acid ferulic acid rutin myricetin luteolin kaempfrol 15 (may23) 3321.72±1.25a 381.71±52.17ab 48.38±46.51ab 1733.63±53.32a 326.11±131.49a 63.66±5.18ab 2.52±0.13b 11.40±0.31b 18 (may26) 2638.79±6.67b 390.88±72.29a 52.40±22.98a 1260.60±19.55b 398.79±100.58a 60.59±4.34b 2.73±0.05b 13.56±0.38a 22 (may30) 3040.08±0.01c 325.25±65.51ab 31.54±13.53abc 1241.73±15.96b 324.48±126.41a 70.12±5.70a 2.33±0.23b 11.30±2.26b 25 (june2) 1826.81±0.14d 330.65±53.15ab 52.44±5.79a 703.88±6.22c 106.96±77.03b 23.05±5.90d 3.08±0.12b 9.55±0.60cd 28 (june5) 1225.55±2.72e 310.03±33.78b 23.17±14.66abc 414.35±1.26d 37.89±45.01b 30.16±1.10c 3.44±0.07b 10.18±0.10bc 32 (june9) 489.50±1.06f 147.24±9.90c 18.05±0.49abc 119.31±5.28e 38.89±14.71b 12.63±2.46e 6.35±3.39a 8.24±0.17d 35 (june13) 373.67±1.23g 21.21±1.50d 15.34±3.63bc 77.24±0.32f 46.04±53.14b 13.19±4.02e 3.35±0.15b 4.93±0.43e 38 (june16) 303.98±0.19h 46.84±11.87d 12.64±1.32bc 53.15±3.08f 73.47±63.69b 8.79±0.24e 3.22±0.03b 4.47±0.10e 41 (june19) 278.71±3.39i 26.74±1.17d 5.98±0.20c 48.30±0.44f 3.68±3.19b 8.66±0.69e 1.45±1.17b 2.08±0.35f 1values are the means ± sd (n = 3) based on dry basis. a-ivalues with different superscripts within the same columns are significantly different (p < 0.05). ital. j. food sci., vol. 30, 2018 436 ellagic acid was below the detectable level at 8 h after oral administration of pbr. it had the maximum plasma concentration (250 ng/ml) at 0.5 h post oral administration of pbr. the plasma level of ellagic acid decreased as time went by, and its half life (t1/2) was 1.018 h. the results of other pharmacokinetic parameters of ellagic acid are summarized in table 5. table 5. main pharmacokinetic parameters1 of ellagic acid in rats plasma after oral administration of prematured black raspberry (pbr, rubus occidentalis) at 150 mg/kg (mean ± sd, n = 5). unit mean2 auc0-t ug h ml -1 0.330±0.109 auc0-∞ ug h ml -1 0.363±0.118 t1/2 h 1.018±0.195 tmax h 0.600±0.223 cmax ug ml -1 0.264±0.109 mrt h 1.494±0.309 1auc0-t: area under the curve, auc0-∞: area under the curve, t1/2: half life time, tmax: maximum time, cmax: maximum concentration, mrt: mean residence time. 2each value represents the mean±sd. 4. discussion br was collected during its period of maturation at an interval of 2-3 days. the results for the amount of anthocyanin indicated that anthocyanin may not be a major component in br contributing to its antioxidant effects before its complete maturation (table 3). floegel et al. (2011) reported that high-pigmented and hydrophilic br antioxidants seem to have both dpph and abts scavenging activities, with higher activities toward abts than that for dpph. in a study conducted by lee et al. (2013), seven different anthocyanins were detectable in br. only cyanidin 3-glucoside, which has a lower amount compared to cyanidin 3-rutinoside and/or cyanidin 3-xylosylrutinoside, is found to have stronger antioxidant effect (dpph scavenging activity) than ascorbic acid, but not trolox (kähkönen and heinonen, 2003). however, borges et al. (2010) suggested that the antioxidant activity of raspberries is influenced by ascorbic acid content rather than cyanidin 3-glucoside in this case, they showed 10.5 and 8.1 % antioxidant activity, respectively. in this study, pbr polyphenol and flavonoid contents were found to have significantly diminished with increase in maturation days. the study also indicated that the amount of ascorbic acid also showed a similar trend as evidenced by both polyphenol and flavonoid content. therefore, in pbr, both dpph and abts seem to be influenced by the amount of polyphenol, flavonoid and ascorbic acid rather than anthocyanin. this is similar to results obtained in a study conducted by ogawa et al. (2008), which suggests that ascorbic acid and flavonols contribute to the antioxidant properties of berries. in pbr, eight major phenolic compounds were detected. each phenolic compound showed the least amount when br was harvested on june 19th (table 4). a study conducted by kang et al. (2015), showed that whole black raspberry did not contain luteolin and myricetin but catechin, epicatechin, rutin, gallic acid and quercetin were detectable. although our study did not trace any catechin, epicatechin and quercetin due to different analysis conditions, the amount of rutin was similar to that of fully matured black raspberry. in addition to that, many phenolic compounds of pbr were noted to decrease ital. j. food sci., vol. 30, 2018 437 during maturing periods. chirinos et al. (2007) indicated that the contents of both phenolic compounds and flavan 3-ols during maturity stage of 10 mashua cultivars were different, and diminution of both phenolic compound and flavan 3-ols were attributed due to genotypic difference of mashua. therefore, among brs, the shuttleworth at gochang, korea is a cultivar that may decrease some phenolic compounds in maturing stag, therefore, ascorbic acid and flavonols may be major factors that contributed to the dpph and abts scavenging activities of br at the early stage of the harvest. to determine the lc protocol accuracy (recovery) and repeatability (precision) for ellagic acid determination in both pbr and rat plasma, ellagic acid was used. the recovery (103.01–104.23 %) and relative standard deviation (rsd, ≤ 3.35%) implied that the protocol had satisfactory accuracy and high recovery for ellagic acid. in addition, the rsd of intra (1.92 %) and inter-day (0.61–2.14 %) differences indicated that the lc protocol was precise. hence, the lc method seemed to be accurate and precise to quantify ellagic acid in pbr (32.13 ± 0.62 mg/g) (data are not shown). based on the accuracy and repeatability of lc, ellagic acid plasma concentration-time profile in rats and six major pharmacokinetic parameters were analyzed. the pharmacokinetic results of pbr ellagic acid indicated that pbr ellagic acid in rats seemed to increase rapidly (table 5), and the cmax reached 0.264 µg/ml. the half time of ellagic acid was found to be 1.018 h after oral administration. similar results were reported in a pharmacokinetic study by lei et al. (2003), in which the maximum concentration of pomegranate leaf ellagic acid in rat plasma is found to be 213 ng/ml after 0.55 h of oral administration. the pharmacokinetic characteristics revealed that ellagic acid seems to have poor absorption and rapid elimination after oral administration (seeram et al., 2004; bala et al., 2006). the rapid elimination of ellagic acid has been confirmed via urine and feces analysis by smart et al. (1986), with up to 70 % ellagic acid being detected in urine and feces. 5. conclusions this is the first study to characterize pre-matured black raspberry (pbr, rubus occidentalis). pbrs are pre-matured black raspberry when brs are collected from 15 to 28 days after fruit set. the results showed that pbr under 28 days after the fruit set seemed to produce a high content of phenolic compounds, flavonoids, and vitamin c, thereby possessing excellent antioxidant activities. based on the pbr pharmacokinetic results and chemical analysis along with antioxidant results, pbr can be used as a functional food or medicinal material derived from the nature. our data could be used us a baseline to develop nutraceuticals, natural colorants, and other related products. acknowledgements this study was supported by a project (#314044-3) funded by the korea institute of planning and evaluation for technology in food, agriculture, forestry & fisheries, republic of korea and by a research funded by the 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erection? phytother. res. 25:1046. paper received august 20, 2017 accepted february 7, 2018 #479_nicolini_bozza ! ital. j. food sci., vol 28, 2016 744 short communication clarifying agents and 3-sulfanylhexanol precursors in grape juice t. román, r. larcher, d. slaghenaufi, l. tonidandel, s. moser and g. nicolini* unità chimica vitienologica e agroalimentare, centro trasferimento tecnologico fond. edmund mach, via e. mach 1, 38010 san michele all’adige, tn, italy *corresponding author. giorgio.nicolini@fmach.it abstract we evaluated the impact of a number of clarifying agents on the concentration of s-3(hexan-1-ol)-l-cysteine (cys-3sh) and s-3-(hexan-1-ol)-l-glutathione (gsh-3sh). 19 clear grape juices were spiked with a grape skin tannin rich in cys-3sh and gsh-3sh. juices were then treated with na-bentonite, pvpp or charcoal (1 g/l) and cold settled. the concentration of precursors was measured and compared to the corresponding untreated control juices in the devatted samples. cys-3sh and gsh-3sh were analysed using uhplc-ms/ms and accuracy was guaranteed with deuterated internal standards. only charcoal caused a statistically significant depletion of both precursors, quantitatively limited even at the highest dose adopted. technologically, the clarifiers used in juice affected the thiol precursors in a marginal manner. keywords: bentonite, charcoal, grape juice, polyvinylpolypyrrolidone, varietal thiols ! ital. j. food sci., vol 28, 2016 745 1. introduction s-3-(hexan-1-ol)-l-cysteine (cys-3sh) and s-3-(hexan-1-ol)-l-glutathione (gsh-3sh) are precursors, present in grapes and/or formed in juice (tominaga and dubourdieu, 2000; peyrot des gachons et al., 2002; schneider et al., 2006; fedrizzi et al., 2009; roland et al., 2011a), of 3-sulfanylhexanol (3sh), responsible together with its acetate for the tropical and grapefruit-like fruity notes produced during fermentation by some yeast strains having lyase activity (roncoroni et al., 2011; winter et al., 2011). the grape variety, as well as the processing conditions of grape, pomace and juice, are very important in saving/producing a high level of precursors (roland et al., 2011a; cerreti et al., 2015; román villegas et al. 2016). technologically speaking, for example, 3sh precursors increase with longer skin-contact and stronger pressing conditions (mattivi et al., 2012) and gsh-3sh in particular increases when oxidative pre-fermentative maceration is adopted (larcher et al., 2013a). nevertheless, the effects of the main clarifying agents on the content of the precursors cited are little known to date. for this reason, the aim of the experiment reported in this paper was to investigate whether certain common clarifiers used in juice can modify the concentration of cys-3sh and gsh-3sh. 2. materials and methods 2.1. juice preparation nineteen lots of must (20 l) were produced from sound white and red grapes coming from different varieties and plots in trentino (northern italy), selected in order to include a wide compositional variability. grape lots (300 kg) were destemmed, crushed and pressed (3.5 bar; press mod. up600, willmes, lorsch, germany) on a semi-industrial scale at the edmund mach foundation experimental winery (san michele all'adige, italy). to ensure a high concentration of thiol precursors, only the juice fraction over 65% w/v yield was used in the experiment (maggu et al., 2007; allen et al., 2011; rolland et al., 2011b). moreover, the juices were indirectly enriched randomly with 500-1000 mg/l of grape skin tannin containing 224.2 mg/kg gsh-3sh and 25.5 mg/kg cys-3sh, quantified according to larcher et al., (2013b), and supplemented with a volume of 15-25% of sauvignon blanc juice, a variety well-known for its richness in thiol precursors (capone et al., 2010; larcher et al., 2013a). after sulfiting (20 mg/l so2), all juices were cold settled (< 20 nephelometric turbidity units), well beyond normal winemaking practice, in order to minimise the effect of solids suspended in the turbid juice. clear juices were then devatted, divided into 4 fractions of 5 l each and supplemented with activated na bentonite (pentagel, 1 g/l; perdomini-ioc s.p.a., s. martino buon albergo, italy), charcoal (eno anticromos, 1 g/l; dal cin s.p.a., concorezzo, italy) or polyvinylpolypyrrolidone (pvpp v, 1 g/l; perdomini-ioc) in comparison with the unspiked fraction respectively. after treatment, all samples were cold settled again for 48h at 4°c. 2.2. sampling the settled juice was sampled (25 ml), supplemented with methanol (25 ml, -20°c) and stored at -20°c until analysis. the methanol solution was spiked with d3-gsh-3sh and d3cys-3sh as labelled internal standards and filtered through a 0.22 µm filter (millex-gv, millipore, ireland) before analysis. ! ital. j. food sci., vol 28, 2016 746 2.3. chemical analysis the juice composition was analysed using a winescan ft 120 type 77310 (foss, hillerød, denmark), accurately aligned according to the official methods (oiv 2012). an uplc acquity system coupled with a xevo tq ms mass spectrometer (waters corporation, milford, usa) was used for lc-ms/ms quantification of thiol precursors. a 5 !l sample was injected into an acquity uplc hss t3 c18 column (1.8 µm film thickness, 2.1 mm × 100 mm; waters) set with a flow rate of 0.45 ml/min and a temperature of 40°c. ms isotopic dilution analysis was performed in positive ion mode (capillary voltage, 2.5 kv), using argon (0.20 ml/min) and nitrogen (1,000 l/h) as collision and desolvation gas respectively. other characteristics of the method are specified in larcher et al. (2013a). 2.4. statistical analysis anova (main effects: juice, clarifier) and tukey's hsd test were carried out using statistica v. 8.0 (statsoft inc., tulsa, ok). 3. results and conclusions the clarifiers were chosen because they are extensively used in winemaking during prefermentation manipulation of white grape must, due to their depletion features in relation to specific classes of compounds (bentonite vs. proteins; pvpp vs. polyphenols) or to their high but non-selective adsorption capacity (charcoal). to our knowledge, there are no reports that specifically link fining agents and thiol precursor content, while their depletion capacity has been previously reported in relation to free and bound primary aromas (moio et al., 2004) and other odour active compounds in juice (lambri et al., 2010). the juices were chemically characterised by their base composition (mean ± st. dev.; min max) for total soluble solids (21.2±2.0 °brix; 18.6-26.0), ph (3.23±0.11; 3.01-3.45), titratable acidity (6.63±1.23 g/l; 4.70-10.00), tartaric acid (5.76±0.69 g/l; 4.93-7.62), malic acid (3.25±0.76 g/l; 1.93-4.68), potassium (1348±158 mg/l; 1086-1618). these data highlight the considerable compositional variability used to ensure the robustness of the results, since the grape cultivar and ripeness not only affect the precursor content (kobayashi et al., 2010; cerreti et al., 2015) but also influence either the composition (pirie and mullins, 1977; pocock et al., 2000) or the haze (mesquita et al., 2001) of the most usual target molecules for these clarifiers and hence the clarifying activity. the ranges obtained for gsh-3sh (min-max: 240 564 !g/l) and cys-3sh (36,5 244 !g/l) in the control juices match the literature (peña gallego et al., 2012; larcher et al., 2013a;). comparison of the results of the corresponding control juices and treated samples showed that bentonite and pvpp had a limited and not statistically significant effect (table 1) on the concentration of gsh-3sh and cys-3sh. on the contrary, charcoal treatment significantly reduced (p<0.05) the two thiol precursors, however this reduction was limited, being roughly 20% for gsh-3sh and 10% for cys-3sh. the significance of these results is not limited to winemaking, but could also be of interest for the grape juice industry, where there is the possibility of using hybrid varieties, resistant to mold diseases and consequently with lower operating costs. precursors are also present in their juices (larcher et al., 2014), and the release of 3sh through specific commercial enzymes could contribute to overall aroma. ! ital. j. food sci., vol 28, 2016 747 table 1: thiol precursors in juice in relation to the clarifying agent used. (values with the same letter are not statistically different in tukey's hsd test, p<0.05; n.s. = non significant). gsh-3sh (µg/l) cys-3sh (µg/l) treatment mean (n=19) s.d. sign. mean (n=19) s.d. sign. control 344 73 a 80 53 a bentonite (1 g/l) 336 75 a 79 53 a charcoal (1 g/l) 276 66 b 73 46 b pvpp (1 g/l) 344 74 a 80 53 a in conclusion, of the clarifying agents used in this experiment, only charcoal proved able to significantly reduce 3-sulfanylhexanol precursors in juice. nevertheless, in the light of the usually lower doses of these products adopted for juice in modern white winemaking, the low conversion ratios of the precursors to the corresponding free thiols (roland et al., 2011a), and the limited percentage changes observed in this experiment, it can be deduced that the clarifying agents used affect the content of thiol precursors in a technologically and sensorially negligible manner, despite the low sensory threshold of the relative derivatives in free and acetate form. acknowledgements the authors wish to thank cavit s.c. and d. zatelli for their cooperation. references allen t., herbst-johnstone m., girault m., butler p., logan g., jouanneau s. and kilmartin p.a. 2011. influence of grapeharvesting steps on varietal thiol aromas in sauvignon blanc wines. journal of agricultural and food chemistry 59:10641. cerreti m., esti m., benucci i., liburdi k., de simone c. and ferranti p. 2015. evolution of s-cysteinylated and sglutathionylated thiol precursors during grape ripening of vitis vinifera l. cvs. grechetto, malvasia del lazio and sauvignon blanc. australian journal of grape and wine research 21:411. capone d.l., sefton m.a., hayasaka y. and jeffery d.w. 2010. analysis of precursors to wine odorant 3-mercaptohexan1-ol using hplc-ms/ms: resolution and quantitation of diastereomers of 3-s-cysteinylhexan-1-ol and 3-sglutathionylhexan-1-ol. journal of agricultural and food chemistry 58:1390. fedrizzi b., pardon k.h., sefton m.a., elsey g.m. and jeffery 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3-mercaptohexan-1-ol (3mh) and 3-mercaptohexyl acetate (3mha) during fermentation by saccharomyces cerevisiae. australian journal of grape and wine research 17:285. paper received march 17, 2016 accepted may 20, 2016 paper ital. j. food sci., vol. 28 2016 15 keywords: consumption, neophilia, vitamins beyond the use of food supplements: an empirical analysis in italy f. caracciolo, a. lombardi*, f. verneau and p. lombardi department of agricultural sciences, agricultural economics and policy group, university of naples federico ii, via università 96, 80055 portici, italy *corresponding author: tel. +39 081 2539083, fax +39 081 7755143, email: alessialom@gmail.com abstract this paper aims to profile italian food supplements used by consumers based upon their psychometric patterns and demographic characteristics. the ftns scale is used to assess empirically and evaluate the role of technophobic/technophilic consumer traits in determining the decision whether or not to consume supplements and vitamins and the frequency of their consumption. an ad-hoc survey was carried out in 2012 involving 400 residents of a metropolitan area in southern italy. our results show that women have a higher consumption frequency of dietary supplements, while age, bmi and education influence the propensity to consume. as regards food habits, the propensity to use dietary supplements is positively associated to the consumption of bread and pasta, red meat and pulses, and negatively with the consumption of fruit and cheese. finally, the research supports the role of technophobic traits as consistent and significant determinants of the consumption frequency of dietary supplements. mailto:alessialom%40gmail.com?subject= 16 ital. j. food sci., vol. 28 2016 1. introduction food supplements are edible products that include components proposed as a dietary enhancement (us food and drug administration, 1994), regulated as food rather than drugs at least in the majority of developed countries. the dietary components might include vitamins, minerals, proteins (metabolites, enzymes and amino acids) and energy concentrate (energy bars). although by the end of the 1990s the use of dietary supplements was relatively frequent in industrialised countries, the consumption of such supplements has been further boosted in all the more affluent western countries by new motivations associated to ageing populations and to continually changing lifestyles (blendon et al. 2001; gragar, 2001; babgaleh et al., 2006; feldman, 2014). in recent years, as regards general food consumption, ever more consumers have shifted from mere satisfaction of energy requirements to an attitude dictated by the need to promote well-being and reduce the risk of disease (marques-vidal, 2004). in the us, the market for dietary supplements has grown dramatically in recent decades, recording an almost 80% increase from 1994 to 2000 (greger, 2001; balluz et al., 2005). in italy, according to a recent survey by gfk eurisko and federsalus, for example, three out of every four individuals stated that they used at least one supplement for personal well-being in 2012. wellness trends are generating new opportunities and challenges for companies in the vitamin and dietary supplement sector, and the producers of dietary supplements have substantially increased their investments and studies to ascertain the behaviour of consumers vis-à-vis food supplements. this analytical need is also emerging at scientific level with a view to understanding not only the consumption dynamics of such products but also the various underlying motivations (nichter and thompson, 2006; o’connor and white, 2010; tavani et al., 2014). in this regard, medical research and economic research are proceeding apace. the former aims to gain insights into the effects that food supplements have on the wellbeing of the individual, or the harmful effect of their excessive use to be able to better satisfy the demand for information and steer consumption of these products (greger, 2001); by contrast, economic research aims to analyze markets opportunities and challenges for new or existing products and the regulations for consumer information (russo france and fitzgerald bone, 2005). furthermore, a large strand of research aims to profile consumers of supplements. in the uk, users of supplements are primarily female, vegetarian, less likely to drink alcohol, non-smoking, and more likely to engage in physical activities (kirk et al., 1999). similar results were confirmed in the us (lyle et al., 1998; greger, 2001; rock, 2007). the increasing number of users is dictated by a strong aspiration towards better health (greger, 2001), by the need to protect one’s state of health and at the same time lower the risk of disease, rather than only satisfy metabolic needs (roberfroid, 2000; greger, 2001; cox et al., 2004; marques-vidal, 2004). although health considerations appear to be a predominant incentive in choosing to use dietary supplements, the reasons for consuming such supplements are complex, combining social, psychological, educational and economic factors. a paradox has been pointed out (conner et al., 2001): consumers of dietary supplements generally have higher recorded nutrient intakes from food sources alone than those who do not consume supplements. although only a small percentage of individuals go beyond the line of reasonable consumption, those consuming excessively may sustain harmful effects (medeiros et al., 1999). this seemingly irrational behaviour provides the motivation to discard the neo-classical approach which analyses the consumer’s rational choice as a utility maximisation process under budget constraint and graduate towards the use of analytical instruments that investigate individuals’ cognitive and affective factors and their relations with consumption behaviour (verneau et al., 2014). for instance, using a psychosocial model, the protection motivation theory, cox et al. (2004) analysed which characteristics of the product/ message would impact on the motivations to purchase dietary supplements to prevent shortterm memory loss. russo france and fitzgerald bone (2005) examined the information environment in the dietary supplement industry. they analysed consumer product-specific as well as general beliefs about health, the supplement industry and the government. their results suggest that information regarding a particular product can be overridden by the consumer’s existing and distantly related beliefs. dietary supplements and vitamins represent a product category that falls between and links – food and medicine, and therefore the perception of these products might also be influenced by the effect of risk. on this field, o’connor and white (2008) analysed consumers’ willingness to trial functional foods and vitamin supplements. they found support for the theory of planned behaviour (tpb) model in predicting people’s willingness to trial functional food and vitamin supplement. the authors also suggested that non-users are influenced by the high-perceived risk associated with their use. according to the psychometric paradigm proposed by slovic (1987), the more people are familiar and well informed about specific hazards, the lower is the perception of risk towards emerging technologies. in other words, the risk perception is affected by the knowledge of both ital. j. food sci., vol. 28 2016 17 risks and benefits related to novel food technologies (fife-schaw and rowe, 1996, siegrist et al., 2006). thus, when knowledge is lacking, consumers’ assessment of risks and benefits related to novel food products and emerging technologies is driven by heuristics (siegrist et al., 2008) and among them, trust and perceived naturalness have been identified as powerful factors (frewer et al., 2003; bronfman et al., 2008; chryssochoidis et al., 2009; earle and siegrist, 2008; kjærnes, 2006 rozin et al., 2004; steptoe et al., 1995). siegrist et al. (2008) have shown, for instance, that food products perceived as natural and healthy are more likely to be accepted by consumers. various psychometric scales have been developed and tested to study consumer acceptance towards new technology and, more generally, new food (goldsmith and hofacker, 1991; pliner and hobden, 1992; eiser et al., 2002; kirk et al., 2002; cox et al., 2007; cox and evans, 2008). among them, the food technology neophobia scale (ftns) (cox and evans, 2008) has been judged to be a more suitable tool for assessing consumer fears of food technologies than an earlier food neophobia scale (fns) (pliner and hobden, 1992) because of its specific focus on technology rather than food (matin et al., 2012). the ftns is a multidimensional scale which integrates the main drivers previously discussed, including naturalness, trust and perception of both risks and benefits of novel food technologies (coppola et al., 2014; verneau et al., 2014; cox et al., 2010). in this study, we adopted the ftns to assess empirically and to evaluate the role of technophobic/technophilic traits in determining whether or not to consume supplements and vitamins and their consumption frequency. moreover, to the best of our knowledge, the potential effect of the technophobia and technophilia traits in affecting this decision is still underexplored and this work is probably the first quantitative attempt to ascertain the determinants of supplement consumption in italy. while an empirical analysis of the motivations behind their use or non-use can be considered strategic for the industry sector to improve product penetration, assessment of the determinants of their consumption frequency is even more important in the policy debate on public health. ascertaining the profile of consumers who are likely to use them excessively is an important step towards prevention. the market for dietary supplements in italy is one of the largest in europe: it reached nearly 2 billion euros in 2013, showing an annual increase of around 3% (cousyn et al., 2013). the results show whether, and to what extent, the degree of consumer technophilia and hence the perception of risk for novelty and neophilia are associated, if at all, with dietary supplement consumption. 2. material and methods 2.1 the survey an ad-hoc, face-to-face survey was conducted in 2012 with a convenient sample of 400 residents of the naples metropolitan area (southern italy). the questionnaire used for data collection comprised three sections. section a) includes socio-demographic characteristics and lifestyle factors including physical activities (such as gym and sport activities). section b) includes the consumption frequency scale for five categories of dietary supplements, namely mineral supplements, amino acid and/or protein supplement, vitamin supplement, beverages enriched with vitamins or minerals, and energy and protein bars. several classifications of food supplements are proposed by the scientific literature, varying among those focusing on the product’s functionality (radimer et al., 2000), ingredients (skeie et al., 2009), disease perspectives (millen et al., 2004) or consumer’s point of view (tavani et al., 2014). this paper adopts a mar ket-driven classification, coherently with the european commission directive (2002/46/ec) and with the italian legislation (legislation decree 169/2004), which characterizes food supplements as concentrated sources of nutrients for supplementing the intake in a normal diet (primarily vitamin, mineral salts and amino acids). for each product, respondents were asked to select one of the following five options, labelled 0 “i do not consume the product”, 1 “i seldom consume the product (no more than once a month)”, 2 “i occasionally consume the product (no more than once a week), 3 “i consume the product frequently (more than once a week)” and 4 ‘i regularly eat the product (almost every day)”. the above frequency scale of consumption can be considered an easily quantifiable measure of the phenomenon under observation in this study, namely individual consumption of dietary supplements. furthermore, section b) comprises consumption frequency of all the other food categories in order to represent the whole consumption habits of the respondents. section c) comprises the food technology neophobia scale for investigating consumer attitudes to technology using the 13 items provided by cox and evans (2008). the ftns translated into italian were provided to the respondents who were called upon to express their degree of agreement-disagreement by using a likert 7-point agree/disagree scale on their perception of new food technology, its uses, benefits and associated risks; the way they feel in new situations and behave when facing unknown circumstances; their food habits and the propensity to taste new food products. 18 ital. j. food sci., vol. 28 2016 2.2 the analytical framework from an empirical point of view, individual consumption decisions on dietary supplements may be modelled through a two-stage process. in the first stage, individuals decide whether or not to consume supplements. in the second stage, the individuals shape their consumption habit by consumption decisions over time (cembalo et al., 2014). the second stage decision is approximated here through a consumption frequency scale. if the first stage could be of interest for marketing purposes, for identifying segments ready or “nearly” ready for the use of supplements, the second stage is particularly important for public health policy reasons, since it investigates the determinants of the use (including the excessive use) of dietary supplements. both stages may depend on several individual factors involving interaction among the cognitive, social and cultural dimensions of consumption. in this paper we formally assess the extent to which neophobia-neophilia forces may influence such decisions. this study implements the food technology neophobia scale (ftns) proposed by cox and evans (2008), allowing consumers with greater neophilia attitudes to be identified, potential early adopters of dietary supplements. moreover, dietary supplement consumption decisions could depend on individual socio-characteristics and overall diet. therefore other variables capturing these consumer characteristics and described in the previous section are included in the model. analytically, a two-step heckman procedure (1979) can be used to analyse both stages of consumption decisions. as concerns the first stage, we assumed that observable characteristics of the consumers influence their consumption choices in terms of the probability that the consumer will use dietary supplements. considering a sample of n observations indexed by i, the outcome { y i } of whether or not to consume is a qualitative random variable taking in the presented case two levels: 0, 1. where c i * indicates consumption over time. empirically, this relation can be analysed through probit specification as (1) where π i identifies the probability that the i-th respondent consumes the dietary supplement, u 1i , is the error term, (i.i.d.) ∼ n(0,1), φ is the cumulative density function of a standardized normal distribution, x’ 1i is the set of k1 consumer characteristics influencing the probability of consuming dietary supplements while β 1 are the respective parameters to be estimated. as regards the second stage, consumption frequency, we may focus only on the “consumers” or the individuals who have decided to consume a dietary supplement at least once, (y 1 = 1). more specifically, a positive consumption over time, (ci *), is observed only if a consumer chooses to consume dietary supplements: c* i > 0. formally, we can write the selection equation and the resultant outcome equation for c* as follows: (1) where assuming that consumption frequency is influenced by a set of k2 explanatory variables x2, we wish to estimate β 2 parameters, under sample selection, with a potential source of inconsistency as: because error terms have a bivariate normal distribution, the expectation e(u2i|c*i>0) is equal to σ 12 λ i (x' 1 iβ 1 ) where λ i is known as the inverse of the mills’ ratio: where ϕ(·) is the probability density function of the standard normal distribution. following heckman (1979) a consistent estimation of β 2 and σ 12 can be obtained by augmenting the outcome equation with the inverse of the mills’ ratio obtained from the estimates of the selection equation. in order to obtain a better identification of the heckman model, we also impose exclusion restrictions (exclusion of at least one regressor being significant in the selection part, but not in explaining the outcome). the augmented equation was estimated by ols using a linear functional form, while test statistics are based on huber–white sandwich estimation of variance. the dependent variable of the outcome equation c i * is based on the stated frequency of consumption of the five categories of dietary supplements, and more precisely is defined as a linear additive aggregation of their stated frequency: where s d,i represents the stated frequency consumption score of the d-th dietary supplement for the i-th individual. ital. j. food sci., vol. 28 2016 19 3. results 3.1 descriptive results of the 400 respondents 9% failed to complete the survey or to answer key questions fully and thus, the final sample is based on 368 individuals. socio-demographic information shows that the interviewees (165 male and 203 female; italian frequency of female) were in the age range 17–70 years (32 ± 11 years; italian average 43.0, italian national institute of statistics 2011). almost one third of consumers (30.7 %) fail to do any physical activity, while the others spend one hour per day on average. just over half the interviewees have university degrees (italian average 11.7%, italian national institute of statistics 2011), while 7% had achieved minimum education levels (italian average 21.7%, italian national institute of statistics 2011). as regards the body mass index (bmi), 66% of respondents were normal weight (bmi 18.5–24.9 kg/m2) (italian aver age 52.6%, world health organization 2005). these differences with the italian population could be due to the specific context in which survey was carried out (residents of naples metropolitan area). table 1 reports the stated frequency of consumption for the various categories of supplements: enriched drinks and vitamin supplements appear to be the supplements with a highest penetration rate in our sample, while energy bars and protein supplements report the lowest penetration rate. around 50 % of respondents stated they used at least one vitamin supplement in the past; this percentage rises to 60 % when considering enriched drinks. only 25 % and 15% of the respondents stated they had made previous use of energy bars and protein supplements, respectively. overall, about 75% of the respondents declared to have consumed at least one category of food supplements in the past. this result is in agreement with data reported by gfk-eurisko and federsalus. table 2 summarises the main characteristics of the respondents divided into two groups: consumers of dietary supplements and nonconsumers. from a preliminary analysis of the average values between the two groups, we found that consumers of dietary supplements seem younger than those not consuming dietary supplements. among the different types of dietary supplements, the beverages enriched with vitamins or minerals are those most commonly consumed while amino acid and/or protein supplements are those least consumed (table 2). as regards dietary habits, consumers of dietary supplements compared to non-consumers show a higher consumption frequency for meats (both red and white meat), snacks and sugary drinks, while they show a lower consumption frequency for cheese, fruit and salad (fig. 1). for consumers of dietary supplements the consumption index (fig. 2) assumes a mean value of 3.57 ± 2.5 in the range (1-13), while it necessarily assumes a value of zero for nonconsumers. respondents general attitudes towards novel technology and how its benefits and risks are perceived are assessed by means of the 13 psychometric items of the ftns (table 3). consumers of dietary supplements compared to non-consumers show a higher ftns score. cronbach’s α of the scale is 0.83, indicating very good internal reliability. the mean level of agreement stated on a scale from 1 to 7 shows in the sample that the statements with the highest rates are “there is no sense trying out high-tech food products because the ones i eat are already good enough” together with “new foods are no healthier than traditional foods” and “the benefits of new food technologies are often grossly overstated”. these results seem in agreement with those of verneau et al. (2014), highlighting that there is a great belief in italian society in supporting natural foods, the mediterranean diet along with the promotion of local and typical products. in turn, this outcome might reflect the opinion that innovation and manipulation in the food industry is somewhat futile, since traditional food products are often more highly appreciated and healthier. by comparing the mean ftns score for the entire sample (mean = 55.2, sd = 13.7, range 1685) with those evaluated from other studies, it may be stated that our sample from the naples metropolitan area presents less fear of food compared to the whole italian population (mean = table 1 stated frequency of consumption/use of dietary supplements. mineral supplements protein supplements vitamin supplements enriched drinks energy bars never 56.25 83.7 53.53 39.95 75 seldom 30.43 10.05 30.43 39.95 15.76 monthly 9.24 2.72 10.05 14.4 7.34 weekly 4.08 3.53 5.98 5.71 1.9 daily 0 0 0 0 0 20 ital. j. food sci., vol. 28 2016 table 2 sample descriptive statistics according to consumption of dietary supplements. variable average std. dev. min max average std. dev. min max non-consumers of dietary supplements dietary supplement consumers socio-demographic characteristics age (years) 38.94 13.85 20 71 30.05 9.92 17 70 gender (2 male; 1 female) 1.61 0.49 1 2 1.53 0.50 1 2 bmi 24.04 3.23 18.14 34.29 23.06 3.72 17.02 40.14 education classesa 3.51 0.69 1 4 3.44 0.62 1 4 presence of children (1 yes; 0 no) 0.88 0.33 0 1 0.91 0.28 0 1 physical activity (hours per week) 7.50 10.46 0 45 6.85 8.31 0 45 income classesb 2.41 0.71 1 4 2.35 0.83 1 4 dietary habitc salads 2.41 0.78 0 4 2.29 0.89 0 4 other vegetables 2.40 0.78 0 4 2.33 0.80 0 4 fruit 3.16 0.97 0 4 2.85 1.09 0 4 pulses 1.78 0.50 0 3 1.93 0.62 0 4 milk & yogurt 2.50 1.11 0 4 2.51 1.11 0 4 cheese 2.11 0.70 0 4 1.91 0.86 0 4 red meat 1.83 0.54 0 3 1.98 0.48 0 4 white meat 2.00 0.54 0 3 2.09 0.56 0 4 eggs 1.62 0.62 0 3 1.69 0.71 0 4 bread and pasta 2.87 0.72 0 4 2.99 0.75 0 4 sugary drinks 1.23 1.03 0 4 1.48 1.14 0 4 snacks 1.56 1.07 0 4 1.94 1.13 0 4 wine & beer 1.23 1.06 0 4 1.30 1.05 0 4 dietary supplement consumptionc mineral supplements 0 0 0 0 0.79 0.85 0 3 amino acid and/or protein supplement 0 0 0 0 0.34 0.75 0 3 vitamin supplements 0 0 0 0 0.88 0.91 0 3 beverages enriched with vitamins or minerals 0 0 0 0 1.10 0.84 0 3 energy and protein bars 0 0 0 0 0.47 0.77 0 3 a(1 primary school; 2 middle school; 3 high school; 4 university and higher); b(1 less than €1,000 per month; 2 €1,000-2,000; 3 €2,000-3,000; 4 more than €3,000); c(0 no consumption; 1 rare consumption, once a month; 2 frequent consumption, once a week; 3 very frequent consumption, more than once a week; 4 addictive consumption, every day). fig. 1 comparison of dietary habits of respondents consuming dietary supplements and those not consuming. note: the scale range from 0 “i do not consume the product”, to 4 ‘i regularly eat the product (almost every day)”. fig. 2 frequency distribution of the dietary supplement index of consumption. ital. j. food sci., vol. 28 2016 21 61, sd = 11.3, verneau et al., 2014). that said, our results are comparable with those obtained with canadians (mean = 58, matin et al., 2012) and australians (mean = 54, evans et al., 2010). 3.2 propensity to consume dietary supplements the two-stage process of individual consumption decisions on dietary supplements is shown in table 4. table 3 descriptive statistics cox psychometric questions: item score on a 1 to 7 scale (1= strongly disagree; 7= strongly agree). ftns statements mean std. dev. mean mean (total sample) (total sample) (non-consumers (dietary supplement of dietary supplements) consumers) there is no sense trying out high-tech food products 4.8 1.8 5.38*** 4.64 because the ones i eat are already good enough new food technologies are something i am uncertain about 4.2 2.0 4.48* 4.14 new foods are no healthier than traditional foods 4.6 2.0 4.73 4.53 the benefits of new food technologies are often grossly overstated 4.6 1.8 4.70 4.57 there are plenty of tasty foods around, so we do not need to use 4.2 2.1 4.46* 4.14 new food technologies to produce more new food technologies decrease the natural quality of food 4.3 1.9 4.51 4.22 new food technologies are unlikely to have long term negative 4.1 1.7 4.19 4.10 health effects (r) new food technologies give people more control over their 3.9 1.7 4.07* 3.80 food choices (r) new products using new food technologies can help people 4.2 1.4 4.28 4.11 have a balanced diet (r) new food technologies may have long-term negative environmental effects 4.0 1.8 4.21* 3.88 it can be risky to switch to new food technologies too quickly 4.2 1.8 4.45* 4.10 society should not depend heavily on technologies to solve its food problems 4.6 1.9 4.77 4.56 the media usually provides a balanced and unbiased view 3.7 1.9 3.75* 3.45 of new food technologies (r) note: cronbach’s α: 0.832; (r) means the item is reverse-coded. *difference across mean significant at the 10% level; ** at the 5% level; *** at the 1% level. variables not significant at the p < .10 level in explaining any of the two stages are eliminated from the final models, starting with the least significant variable. on the left are the results of the first stage, indicating the individual determinants of the decision whether or not to consume dietary supplements. the results show that the propensity to consume dietary supplements depends on the age of the respondents (the latter is significantly associated with a lower propensity to consume dietary supplements) table 4 i stage and ii stage estimates. i stage: propensity to consume dietary supplements ii stage: consumption frequency decision coef. β1 elasticity std. dev. p-value coef. β2 elasticity std. dev. p-value socio-demographic cons. 3.389 1.264 0.007 7.102 2.913 0.015 age -0.034 -0.353 0.007 0.000 -0.003 -0.044 0.036 0.924 sex -0.101 -0.051 0.182 0.579 -0.889 -0.541 0.370 0.016 edu. -0.284 -0.319 0.132 0.031 -0.284 -0.385 0.354 0.422 bmi -0.043 -0.329 0.026 0.095 -0.118 -1.081 0.062 0.058 food habits fruit -0.136 -0.129 0.083 0.103 0.010 0.011 0.178 0.956 pulses 0.274 0.170 0.148 0.063 0.436 0.325 0.330 0.186 cheese -0.263 -0.168 0.108 0.015 -0.451 -0.345 0.282 0.110 r. meat 0.346 0.219 0.179 0.053 0.308 0.235 0.470 0.512 w. meat 0.085 0.057 0.163 0.603 1.093 0.889 0.329 0.001 bread& pa 0.186 0.180 0.111 0.094 0.261 0.304 0.268 0.330 attitudes ftns -0.191 -0.088 0.383 0.619 -2.083 -1.161 0.753 0.006 22 ital. j. food sci., vol. 28 2016 and on their bmi values (higher consumption of dietary supplements is associated with lower bmi levels). respondent education also plays a major role in determining consumption decisions (diamantopoulos et al., 2003). our estimate shows that less educated respondents are associated with a higher propensity to consume dietary supplements. daily eating habits are a particularly significant factor affecting propensity to use supplements (kim and keen, 2002). our estimates show that intakes of carbohydrate and protein from the diet differ between those using or not using dietary supplements. respondents who often consume refined cereals (bread and pasta), red meat and pulses show a higher propensity to use dietary supplements compared to those frequently consuming fruit and cheese. finally, the attitudes of individuals to food technology measured through the ftns do not appear to affect this propensity significantly. 3.3 frequency of dietary supplement consumption the results of the second stage on the determinants of the frequency of the supplements use is also reported on table 4. studies in the us and europe have shown that females, individuals in high socioeconomic categories, and individuals living in large cities are likely to use dietary supplements more often than others (slesinski et al., 1995; schellhorn et al., 1998). our results confirm that women show a higher consumption frequency of dietary supplements. as regards the remaining sociodemographic characteristics, only the bmi value seems to influence (in inverse relation) the use of supplements. although daily eating habits proved clearly associated to the propensity to use supplements, such habits almost entirely fail to explain respondents’ consumption frequency. only the consumption frequency of one food category over the 13 tested (white meat) is significantly associated to a higher use of supplements. estimates show an elastic complementary relationship between the consumption of white meat and supplements intake. however, with the exception of age, bmi and the frequency of white meat consumption, the great majority of the socio-demographic variables collected and used in the analysis are unable to explain the consumption frequency of dietary supplements. in this case, analytical instruments that investigate individuals’ cognitive and affective factors might help to profile the consumers of supplements. the attitude of consumers to food technologies, as measured by the ftns, effectively contributes to meeting this requirement. specifically, respondents characterised by neophobia patterns and consequently showing low demand for novelty and neophilia are associated with a low consumption of dietary supplements. furthermore, the estimated association between the ftns and the index of supplements’ frequency of consumption is quite strong (elasticity -1.161). 4. discussion and conclusions dietary supplements are a relatively new class of product that has gradually become established on the markets, especially in the us, but that is rapidly increasing its penetration also in european and italian markets. however, this trend has generated several major issues and posed a number of challenges. for example, it has been shown that consumers have a certain difficulty in interpreting the claims of such products, both as regards functions and disease prevention. moreover, general and specific beliefs systematically bias product-specific judgments regarding efficacy as well as scientific certainty. this leads to questions as to how such labels are interpreted by those at risk or affected by specific diseases. another important issue stems from the evidence that the use of dietary supplements is at least partially motivated by self-control of health (eisenberg et al., 1998; greger, 2001). this can lead many people to make inappropriate choices, for example, favouring the use of dietary supplements compared to proper varied nutrition, especially among those who are more vulnerable to pressure to use dietary supplements unnecessarily, despite the lack of evidence to suggest they are needed to meet dietary deficiency. it is this group, namely the more vulnerable, that needs to be able to make an informed choice so that their use of dietary supplements is connected to real rather than perceived need. this presents a paradox, because dietary supplements, which are used to enhance human health, have the potential to create distortions in eating habits, keep people from the objective of a healthy and complete diet, and may cause adverse reactions when used inappropriately and taken in excessive amounts. for these reasons, the study of consumer behaviour and analysis of the motivations that cause them to consume dietary supplements or otherwise is particularly important for both policy makers and industry. while the major studies that have analysed supplement-taking behaviour are focused on motivational systems, primarily resorting to protection motivation theory (pmt) and theory of planned behaviour (tpb) (conner et al., 2001; cox et al., 2004; o’connor and white, 2010), in our research we tested the role of attitude to food technologies as a predictor of the intention to consume dietary supplements. in particular, we analysed the role of food technology neophobia/neophilia, which has been extensively researched with reference to a great number of food products, technologies and attributes (arvola et ital. j. food sci., vol. 28 2016 23 al., 1999; henriques et al., 2009; tourila et al., 2001). testing food technology neophobia/neophilia in the case of dietary supplements seems to be particularly useful because, at least from a marketing point of view, this class of product is difficult to classify, lying at the crossroads of food and drugs. furthermore, this paper provides the first empirical attempt to profile dietary supplement consumers in italy. the study outlined three main results: first of all, our analysis shows two different patterns for users and non-users. secondly, results show that being young, female, less educated and having a low bmi are factors that are associated to higher propensity to consume dietary supplements. as regards food habits, the propensity to use dietary supplements is positively associated to the consumption of refined cereals (bread and pasta), red meat and pulses. on the contrary, propensity to use dietary supplements is negatively related to consumption of fruit and cheese. the investigation of whether these factors are similar to those associated with dietary deficiency/excess goes beyond the scope of the current analysis and would require another study with that specific objective. finally, the research supports the role of technophobic traits as important determinants of the consumption frequency of dietary supplements: consumers of dietary supplements compared to nonconsumers show a higher ftns score. in particular, non-consumers of dietary supplements endorse the notions that “there is no sense trying out high-tech food products because the ones i eat are already good enough”, “new food technologies are something i am uncertain about” and “it can be risky to switch to new food technologies too quickly”. on the other side, consumers of dietary supplements show higher trust levels than non-consumers, and they highlight the benefits of the new technologies, agreeing with the statements “ new food technologies give people more control over their food choices” and “the media usually provides a balanced and unbiased view of new food technologies”. it would appear that a negative attitude to food technologies has the capacity to contain the consumption of dietary supplements within lower levels. bearing in mind the potential challenges linked to the increasing consumption of dietary supplements, this finding may steer communication policies and information towards the specific group of people less affected by food technophobia. more generally, the results confirm the ftns as a powerful tool to capture technophobic traits. the findings of this study suggest that the introduction of some risk-based construct, namely the technophobic traits measured by means of the ftns, might strengthen standard tpb models when health-related products like vitamins and dietary supplements are considered. however, surveying residents from a single metropolitan area may limit generalisability of the results to different areas. further studies could follow two different avenues. on the one hand, it could be useful to build up a modified structure of the tpb model, incorporating risk perception and food technophobia among general and specific-product attitudes in order to gain insights into supplement-taking behaviour. on the other, a greater effort is needed to make more thorough information available on the proper use of supplements. indeed, it has observed that the propensity to consume dietary supplements is more commonly found among young people who are presumably also the category that has less need of supplements. this paradox, that people who least seem to need supplements are most likely to use them, has been called in the literature “the inverse supplement hypothesis” (kirk et al., 1999). it seems particularly true in the more affluent western countries, like italy, where a healthy and balanced diet should be adequate to ensure intake of all the main nutrients. 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information provided by the ftns. appetite, 73(1):140-146. yetley e. a. 2007. multivitamin and multimineral dietary supplements: definitions, characterization, bioavailability, and drug interactions. am. j clin nutr. 85(1): 269s-276s. paper received october 1, 2014 accepted february 19, 2015 ijfs#945_bozza ital. j. food sci., vol. 30, 2018 280 paper the influence of quinoa (chenopodium quinoa willd.) flour on the physicochemical, textural and sensorial properties of beef meatball a. bağdatli department of food engineering, manisa celal bayar university, yunusemre, 45140 manisa, turkey corresponding author: +90 2362012266; fax: +90 2362412143 e-mail address: aytunga.bagdatli@cbu.edu.tr abstract in this study, beef meatballs were produced by using different percentages of quinoa flour as functional ingredient. the effects of quinoa flour levels on physicochemical, textural, sensorial properties of meatballs were examined. quinoa level had significant effect on fat and moisture contents (p<0.0001). the protein content was improved by adding quinoa flour. cohesiveness, springiness, gumminess, redness(a*), yellowness(b*) values were significantly affected by addition of quinoa flour. according to sensorial analysis, meatball samples had high acceptability and favorable scores. consequently, quinoa flour has high potential as gluten-free ingredient for use in meatball production in addition to nutritional value and health benefits. keywords: beef, meatball, quinoa flour ital. j. food sci., vol. 30, 2018 281 1. introduction celiac disease is one of the most common lifelong disorders worldwide with as estimated mean prevalence of 1% of the general population. the only acceptable treatment to date for celiac disease is the strict elimination of gluten from the diet. gluten-containing wheat proteins and/or starches are often added to many commercial products such as ready meals, convenience food products (meatball etc.), some medicines for technological reasons, to act as fillers, thickeners, binders and stabilizers (alvarezjubete et al., 2010). the increasing consumer demand for foods that combine extra benefits in addition to common nutrients imposes on the food industry. therefore, it is needed to advance the new ingredients and formulations, particularly for the production of functional foods. quinoa (chenopodium quinoa willd) is a gluten-free pseudo-cereal that contains a high amount of fibre, high biological-value proteins and essential fatty acids (ω-3 and ω-6). it is consumed in the raw or processed as flakes and flour (brito et al., 2015). quinoa, is a good source of minerals, vitamins and natural antioxidants like vitamin e. the most important characteristic of this pseudocereal is the high amount and quality of its protein. studies have been carried out to investigate the use of quinoa as a food ingredient to increase the protein level and for taste improvement (schumacher et al., 2010). currently, natural extracts, vegetable and fish oils can be used in order to develop the functional properties of meat products (bilek and turhan, 2009). non-meat ingredients such as bean flour, corn flour, oat flour have been used to binding and extending in comminuted meat products in previous studies. however, quinoa flour have not been used in meatballs before. in this study, the effects of quinoa flour on the properties of beef meatballs were presented. the results of chemical composition, ph, cooking yield, texture profile analysis, sensory analysis and hunter (l*, a*, b*) were obtained. 2. materials and methods 2.1. preparation of meatballs meatballs were prepared in duplicate according to the following traditional recipe. quinoa flour was provided from a local market in manisa. medium-fat (max. 15% fat) ground beef meat were purchased from a local butcher shop in manisa 20 kg of ground beef were used in each batch. ingredients were as follows; 2% salt, 3% ground onion, 2% red pepper, 0.3% black pepper, 3% cumin, 0.8% garlic powder. in the first batch (control) 5% bread crumbs was added; in the second batch 2.5% quinoa flour was added; in the third batch 5% quinoa flour was added, in the fourth batch 7.5% quinoa flour was added to the formulation. therefore, four different quinoa flour levels (0%, 2.5%, 5% and 7.5%) were used in meatball preparation. all ingredients were mixed at research laboratories of manisa celal bayar university-food engineering department. each batch was kneaded for 15 min by hand to obtain homogenous dough. the doughs were stored in a refrigerator (+4˚c) for 12 hours and then shaped in to ball with a diameter of 3 cm and a weight of 20 g. the meatballs were cooked for 20 min in a preheated hot air oven at 180˚c. for every treatment two replicates were maintained. ital. j. food sci., vol. 30, 2018 282 2.2. cooking yield cooking yield was determined by measuring the difference in the sample weight before and after cooking and was calculated according to following equation (ulu, 2006). 𝐶𝑜𝑜𝑘𝑖𝑛𝑔 𝑦𝑖𝑒𝑙𝑑 % = 𝐶𝑜𝑜𝑘𝑒𝑑 𝑚𝑒𝑎𝑡𝑏𝑎𝑙𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 (%) 𝑈𝑛𝑐𝑜𝑜𝑘𝑒𝑑 𝑚𝑒𝑎𝑡𝑏𝑎𝑙𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 (%) 𝑥 100 2.3. proximate analysis ash, moisture, protein and fat contents were determined according to aoac methods (aoac, 2000). protein content was measured by the kjeldahl method (nx6.25). fat was determined extracting samples in a soxhlet apparatus using diethyl ether as a solvent. moisture content was measured by the weight difference before and after oven drying at 105°c. ash was determined after incineration in a furnace at 500°c. carbohydrate content was calculated by computing the difference. 2.4. ph 10 g meatball and 100 ml distilled water were blended for two minutes to obtain ph value by using ph meter (hanna instruments model hi 221, usa) (aoac, 1984). 2.5. color the color of the surface of meatball samples was measured with a colorimeter (minolta cr-300) on three different points using d-65 illuminant. the l*, a*, b* values were recorded. 2.6. tpa (texture profile analysis) texture profile analyses (tpa) of cooked beef meatballs were determined by using ta.xt plus texture analyzer (godalming, england). six cores (diameter 25 mm) were taken from random cooked beef meatballs per treatment. 50 kg of load cell was applied. p/25 cylindrical probe was used. the pretest speed was considered as 1mm/s with 2 mm/s of test and posttest speed. double compression was applied with 50% of compression rate. the results of hardness, cohesiveness, springiness, chewiness and gumminess were achieved (bruna et al. 2000). 2.7. sensory analysis the sensory attributes (color, taste, odor, texture, appearance and overall acceptability) of cooked meatball samples were evaluated by 12 well trained panelists from the staff members of manisa celal bayar university using a 9-point hedonic scale. the analysis was performed in the food engineering research laboratory under white fluorescent lights. the samples were scored on scale of 1-9. the results between 1-3 are considered as unacceptable; 4-5 are acceptable; 6-7 are good; 8-9 are excellent. ital. j. food sci., vol. 30, 2018 283 2.8. trial plan and statistical analysis the data obtained from two replications were processed by analysis of variance (anova) using statistical analysis system (sas) (sas institute, 2001). proc glm procedure was done. the level of statistical significance is p≤0.05. 3. results and discussion table 1 presents the results of moisture, ash, fat, protein and carbohydrate contents of quinoa flour and bread crumbs. the moisture content for quinoa flour and bread crumbs were found as 13.77 and 7.73, respectively. the results show that quinoa flour is good source of protein and carbohydrate. the similar results of quinoa flour composition were obtained in previous studies by oshodi et al. (1999), alvarez-jubete et al. (2009), ogunbelle (2003). table 1. chemical composition of quinoa flour and bread crumbs (%). component quinoa flour bread crumbs moisture (%) 13.77±0.80 7.73±0.15 ash (%) 2.46±0.06 1.28±0.04 fat (%) 4.93±0.15 2.69±1.09 protein (%) 13.60±0.22 10.75±0.71 carbohydrate (by difference) 65.26±0.67 77.56±0.57 all the values indicate mean ± sd. mean percent of moisture, fat, protein, ash, cooking yield and ph values of raw meatballs with quinoa flour are given in table 2. meatballs had moisture contents ranging from 44.06% to 52.50%. the maximum content moisture content was determined in meatballs with the addition of 7.5% quinoa. the quinoa level used in meatball production had very significant effect on moisture content of meatball samples (p<0.05). the moisture contents increased by the percentage of quinoa flour was increased. in fact, the increase of the moisture content with the increase of quinoa flour percentage is related to the water holding capacity of quinoa flour. oshodi et al. (1999) reported that the water absorption capacity for quinoa is 147%. according to ogungbenle (2003), the water absorption capacity for quinoa seed is 14%. it is higher than soy flour, pumpkin seed and pigeon pea flour. the present findings of moisture contents are agreeing with the findings of bilek and turhan (2009) and turhan et al. (2005). fat contents of meatballs having 0% (control), 2.5%, 5% and 7.5% quinoa flour were found as 12.66%, 12.09%, 9.86% and 9.80%, respectively. the amount of quinoa flour affected the fat content of meatball samples significantly (p<0.05). when the quinoa flour was increased, the fat content was decreased. the decrease of fat content with the increase of quinoa flour percentage is related to the composition of the flour. and also, ogungbenle (2003) was determined that the oil absorption capacity of the quinoa flour (46.0%) was lower than wheat flour (84.2%). this is consistent with the findings of yılmaz (2005) who reported wheat bran addition at the level of 20% resulted in a significant (p<0.05) reduction in the fat content of meatballs. on the contrary, modi et al. ital. j. food sci., vol. 30, 2018 284 (2009) reported that the fat content of uncooked kofta did not affected by different levels of carrageenan and out flour (p>0.05). protein contents of meatballs with 0% (control), 2.5%, 5% and 7.5% quinoa flour were found as 33.38%, 33.81%, 34.36% and 38.49%, respectively. analysis of variance showed that the differences of protein amounts of the meatballs were non significant (p>0.05). similarly, serdaroğlu and değirmencioğlu (2004) determined that the protein content of uncooked meatballs did not affected by corn flour addition (p>0.05). also, aukkanit et al. (2015) found that protein contents in corn silk added low fat meatballs have not significant difference(p>0.05). table 2. moisture, fat, protein, ash contents and ph, cooking yield (%) and lab values of beef meatballs formulated with different percentages of quinoa flour. a-dmean in the same row with different letters are significantly different (p<0.05). the ash contents of meatball samples formulated with quinoa flour were presented in table 2. the highest ash content was observed in 7.5% quinoa flour added meatballs as 4.39%. the percentage of quinoa affected the ash content of meatball samples significantly (p<0.05). similar results of ash contents were obtained by several researchers (bilek and turhan, 2009; aukkanit et al., 2015; yilmaz, 2005). utilization of quinoa flour affected the ph values of the samples non-significantly (p>0.05). the ph values ranging from 5.57 to 5.59. this is similar with the data of baugreet et al. (2016) who reported that there was no treatment effect or interaction between treatments among ph values. also, bilek and turhan (2009) found that the ph values of raw and cooked beef patties enhanced with flaxseed flour at different levels (3%, 6%, 9%, 12% and 15%) were not significantly different between treatments. from the perspective of meatball production process, cooking yield is the most important factor to guess the characteristic of final products during cooking considering non-meat ingredients (aukkanit et al., 2015). it was determined that cooking yield values did not affected by the quinoa flour percentage significantly (p<0.05). cooking yield value decreased with the increase of quinoa flour percentage. the maximum value was obtained in control samples and the minimum value was found in 7.5% quinoa flour added samples. similarly, aukkanit et al. (2015) found that cooking yields of low fat meatballs decreased with the addition of corn silk powder (1-4%). also, serdaroğlu et al. (2005) observed that cooking yields ranged between 85.2% and 93.2% for meatballs having parameters control (0) quinoa flour level (%) 2.5 5 7.5 moisture (%) 44.06 d 46.45 c 48.93 b 52.50 a fat (%) 12.66 a 12.09 b 9.86 c 9.80 c protein (%) 33.38 b 33.81 ab 34.36 ab 38.49 a ash (%) 2.60 d 3.23 c 3.89 b 4.39 a ph 5.57 a 5.59 a 5.59 a 5.59 a cooking yield (%) 70.49 a 68.44 b 66.39 c 66.32 c l* 44.32 a 43.06 a 43.87 a 45.38 a a* 9.91 c 14.00 a 10.94 b 11.50 b b* 11.03 a 10.20 b 9.94 b 10.44 ab ital. j. food sci., vol. 30, 2018 285 blackeye bean flour and lentil flour resulted in the maximum cooking yield values (p<0.05). the color (l*, a*, b*) values were given in table 2. the redness and yellowness values were significantly (p<0.05) affected by quinoa flour percentage. the lightness of meatballs was measured by hunter-l. amount of quinoa had no significant effect on lightness (l*) values (p>0.05). the maximum l* values were displayed for 7.5% addition of quinoa flour, which means that the addition of quinoa flour resulted in a lighter-colored product. yılmaz and dağlıoğlu (2003) found similar results with oat bran added meatballs. a* (redness) values were also different (p<0.05) for different amount of quinoa flour. therefore, a* values were higher in the samples with quinoa flour than in the control. the highest a* value was for the samples with 2.5% quinoa. also, the lowest redness value was determined in control group samples. similarly, bilek and turhan (2009) reported that redness values were the lowest in the uncooked control beef patties (20% fat) when compared with different amounts of flaxseed flour added samples (p<0.05). all values for yellowness were higher in control samples than in the samples formulated with quinoa flour. quinoa addition appears to decrease product yellowness. turhan et al. (2005) obtained similar results of yellowness values in low-fat beef burgers produced with hazelnut pellicle. table 3. texture profile analysis (tpa) of cooked beef meatballs formulated with different levels of quinoa flour. parameters control (0) quinoa flour level (%) 2.5 5 7.5 hardness (n) 46.382 a 46.460 a 51.356 a 56.359 a cohesiveness 0.466 c 0.494 bc 0.499 b 0.638 a springiness 75.143 b 78.512 a 72.795 c 68.016 d chewiness (n) 15.853 a 20.130 a 23.632 a 20.768 a gumminess (n) 21.614 b 22.951 b 25.627 b 35.957 a a-dmean in the same row with different letters are significantly different (p<0.05). tpa parameters of meatball samples were given in table 3. the maximum hardness value was detected in samples of 7.5% quinoa flour and the minimum value was obtained in control samples. the differences of hardness values of the meatballs were nonsignificant (p>0.05). table 3 indicates that increasing quinoa flour level increased hardness. similar results of hardness values were found by sarıçoban et al. (2009) and ulu (2006). also, aukkanit et al. (2015) observed that corn silk powder did not affect the hardness values significantly. cohesiveness is defined as the degree to which the sample can be deformed before it breaks. results of statistical analysis demonstrated that the amount of quinoa flour affected the cohesiveness values significantly (p<0.05). when the quinoa flour was increased, the cohesiveness values were increased. springiness can be defined as the rate at which the deformed beef meatball springs back after the compression (baugreet et al., 2016) quinoa level had a very significant (p<0.05) effect on springiness of meatballs. the minimum springiness value was determined in the samples with 7.5% quinoa flour. on the other hand, the maximum springiness value was found in the samples with 2.5% quinoa flour. similarly, ulu (2006) ital. j. food sci., vol. 30, 2018 286 determined that guar gum significantly affected the springiness of cooked meatballs produced with 15% and 10% fat levels. in this study, chewiness values of meatball samples varied between 15.853 n to 23.632 n. quinoa flour level did not affect the chewiness values significantly (p>0.05). meatballs with 5% quinoa flour had the maximum chewiness value and the control meatballs had the lowest chewiness value. al-juahimi et al. (2016) reported that the chewiness of uncooked meatballs increased with increment of moringa seed flour. the amount of quinoa flour displayed a significant (p<0.05) effect on the gumminess values of meatball samples. when the amount of quinoa flour in meatball formulation increased, the gumminess values were increased. all gumminess values were higher in the samples with quinoa flour than in the control groups. the highest gumminess value was found in the samples with 7.5% quinoa flour. consistent results of gumminess values were determined by aukkanit et al. (2015). they observed that gumminess increased as the increment of corn silk powder amount. duncan’s multiple range test results for sensory scores of meatball samples were given in table 4. all values for color (sensorial) were lower (p<0.05) in the samples with quinoa flour than the control samples. table 4. sensorial characteristics of cooked beef meatballs formulated with different levels of quinoa flour. sensory scores control (0) quinoa flour level (%) 2.5 5 7.5 color 7.85 a 7.13 b 7.07 b 6.83 c taste 7.78 b 8.28 a 8.40 a 7.07 c odor 8.00 a 8.34 a 8.00 a 5.36 b appearance 8.58 a 7.46 c 7.83b 6.69 d texture 7.61 a 7.57 a 7.93 a 6.75 b overall acceptability 8.00 a 8.00 a 7.93 a 6.75 b a-dmean in the same row with different letters are significantly different (p<0.05). quinoa flour level had a significant effect on scores of taste (p<0.05). the highest score of taste was determined in meatball samples with 5% quinoa flour. a significant correlation (r =+ 0.88, p<0.05) was found between taste and odor scores. the quinoa flour amount affected the odor significantly (p<0.05). the lowest odor score was found in meatball samples with 7.5% quinoa flour. on the contrary, serdaroğlu et al. (2005) reported that no differences in flavor scores were observed among treatments containing legume flours (p>0.05). a significant correlation (r =+ 0.74, p<0.05) was found between odor and appearance scores. the mean values of appearance scores were shown in table 4. the amount of quinoa flour affected the appearance scores significantly (p<0.05). the highest score was determined in the control samples and the lowest appearance score was found in samples with 7.5% quinoa flour. the present findings are agreeing with the findings of turhan et al. (2005) who reported that increasing the pellicle level resulted in beef burgers with decreased appearance scores. also, serdaroğlu and değirmencioğlu (2004) determined that adding corn flour (4%), affected the appearance scores (p<0.05) significantly; meatballs with 4% corn flour had lower appearance scores. in this study, a significant correlation (r =+ 0.91, p<0.05) was found between appearance and color scores. ital. j. food sci., vol. 30, 2018 287 quinoa flour level had significant effect on the texture scores (p<0.05). the meatball samples with 5% quinoa flour had the highest texture score and the lowest score was determined in the samples with 7.5% quinoa flour. similarly, aukkanit et al. (2015) concluded that meatballs with 4% corn silk powder had the lowest texture scores compared with control and the meatballs with 1%, 2%, 3% of corn silk powder. also, a significant correlation (r =+ 0.90, p<0.05) was determined between texture and taste scores. on the other hand, a significant correlation (r =+ 0.81, p<0.05) was found between texture and odor scores. the overall acceptability score was shown in table 4. the quinoa level affected the overall acceptance scores significantly (p<0.05). the maximum score was observed in the control and samples with 2.5% quinoa flour (8.00). on the other hand, the lowest score was found in the samples with 7.5% quinoa flour (6.75). the scores of overall acceptability decreased as the quinoa flour was increased. similar results were found by turhan et al. (2005). a significant correlation (r =+ 0.92, p<0.05) was found between acceptability and odor scores. also, a significant correlation (r =+ 0.86, p<0.05) was obtained between acceptability and taste scores. in this study, 2.5% and 5% of quinoa flour is considered optimum for use an enhancer to the functional properties in beef meatballs. similarly, sarıçoban et al. (2009) concluded that patties enriched with <7.55% wheat bran were determined as more suitable with respect to sensorial overall quality. 4. conclusions in conclusion, the addition of quinoa flour had significant and variable effects on moisture, fat, ash, cooking yield, cohesiveness, springiness, gumminess values and all sensorial characteristics (color, taste, odor, appearance, texture and overall acceptability) of beef meatballs. the addition of quinoa flour improve the protein content compared to control. considering sensorial analysis, all meatball samples had high acceptability and favorable scores (5.36 and above). in this study, it is found that when the functional properties of beef meatballs are considered, the samples with 2.5% and 5% of quinoa flour give the best results. it is concluded that, in addition to its nutritional value and health benefits of quinoa flour, it has high potential as gluten-free ingredient to use in meatball production instead of bread crumbs. 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of different fish species farmed in the adriatic sea j. pleadina, t. lešića, g. krešić*b, r. barićc, t. bogdanovićd, d. oraiće, a. vulića, a. legacc and s. zrnčiće acroatian veterinary institute, laboratory for analytical chemistry, savska cesta 143, 10000 zagreb, croatia bfaculty of tourism and hospitality management, university of rijeka, department of food and nutrition, primorska 42, 51410 opatija, croatia ccromaris d.d., gaženička cesta 4b, 23000 zadar, croatia dcroatian veterinary institute, veterinary institute split, poljička cesta 33, 21000 split, croatia ecroatian veterinary institute, laboratory for fish pathology, savska cesta 143, 10000 zagreb, croatia *corresponding author. tel.: +38 551294714; fax: +38 551291965 e-mail address: greta.kresic@fthm.hr abstract we investigated the nutritional quality of commercially important farmed fish species: sea bream (sparus aurata), sea bass (dicentrarchus labrax), dentex (dentex dentex), and turbot (scophthalmus maximus). the omega-3 fatty acid content of dentex was twice as high as that of any other fish species, and its eicosapentaenoic (epa) and docosahexaenoic (dha) acid contents were 2-4 times higher. the recommended n-3/n-6 ratio was present in all the fish species, but the recommended polyunsaturated/saturated fatty acid ratio was not present in the turbot. all the fish species, except turbot, met the recommended atherogenic index, thrombogenic index, and ratio of hypocholesterolaemic to hypercholesterolaemic fatty acids, whereas the highest flesh lipid quality value was observed in the dentex. keywords: nutritional quality, farmed fish, adriatic sea, basic chemical composition, mineral, fatty acid ital. j. food sci., vol 29, 2017 538 1. introduction since 1970, fish farming (aquaculture) has been the world’s fastest growing sector in the production of foods of animal origin. because wild fisheries are stagnating and the human population is growing, aquaculture is expected to fill the gap in supplying fish intended for human consumption, because the demand has continued to increase (fao, 2016). consumer habits are also changing continuously, and issues such as convenience, health, ethics, variety, value for money, sustainability, and safety are becoming more important. health and well-being increasingly influence our decisions about what we consume. fish have a particularly prominent role in this context because mounting evidence confirms the health benefits of consuming fish. a moderate-to-high fish intake is associated with a reduced prevalence of the chronic diseases that go hand in hand with obesity, such as cardiovascular diseases, diabetes, and some cancers (oehlenschläger, 2012). fish is also considered an integral component of a well-balanced diet, providing a healthy source of energy, high-quality proteins, vitamins (d, a, e and b12), essential minerals and particularly n-3 long-chain polyunsaturated fatty acids (lc pufa), mainly eicosapentaenoic acid (20:5 n-3 epa) and docosahexaenoic acid, (22:6 n-3 dha), whose pleiotropic effects on health promotion and disease prevention are well recognized (gil and gil, 2015). in fresh fish, the muscle composition is considered to be the most important aspect of quality, whereas its sensory properties and nutritional value, together with freshness, are also important quality parameters in terms of consumer acceptability (grigorakis, 2007). fish proteins are easily digested (because the proportion of collagen is low). they are also a good source of essential amino acids. the nutritional value of fish meat is also linked to its lipid composition, as well as its constituent proteins (oehlenschläger, 2012). lipid contents are highly variable both between and within fish species. many factors contribute to this variability, including feed, farm location, fish size and stage of maturity, biological variations, tissue sampled, and starvation (rueda et al., 2001). fish lipids contain a high proportion of unsaturated fatty acids (60%-84%), especially long-chain n-3 fatty acids. the fatty acids (fas) in fish, especially epa and dha, have important effects on the health of the human body and differ from those of meat. the well-known hypotriglyceridaemic effect of n-3 lc pufa may be beneficial in terms of reducing the percentage of pro-atherogenic small low-density lipoprotein (ldl) particles, and perhaps by ameliorating the inflammatory processes associated with the metabolic syndrome seen in patients with diabetes mellitus or cardiovascular disease (lopez-huertas, 2012). in recent decades, fish nutrition research has devoted much effort to the development of sustainable fish feeds with an optimal fa composition so that the fish provide adequate levels of n-3 fas for human nutrition (izquierdo et al., 2005; kris-etherton et al., 2003; pratoomyot et al., 2010). the minerals in raw farmed fish meat, which occur at overall levels of 0.6-1.5 g/100 g, are also very important in the human diet (erkan and özden, 2007). according to the literature, the origins of fish and their feeding patterns have no effect on their mineral composition, other than their calcium content (fuentes et al., 2010). the nutritional value of fish is affected by a low-energy value and high levels of fat-soluble vitamins, especially a and d (mahan and escott-stump, 2004). because the world’s fish stocks are limited, it is now suggested that farmed fish provide an alternative for consumers. farmed sea-foods have an advantage over captured fishery products because they are produced and harvested under controlled conditions, which allow consumption-related risks to be minimized. in recent years, the mediterranean aquaculture industry has become interested in farming new species to overcome the problems arising from the overproduction of two main species, the gilthead sea bream (sparus aurata) and the european sea bass (dicentrarchus labrax) and to diversify their ital. j. food sci., vol 29, 2017 539 products. the common dentex (dentex dentex) is a fast-growing sparid, and a candidate species for fish farming in the mediterranean (rigos et al., 2012). alternatively, the commercial farming of turbot (scophthalmus maximus), a high-value flat fish, along the adriatic coast is still in its infancy, and the turbot is mainly farmed along the atlantic coasts of france and spain (sérot et al., 1998; manthey-karl et al., 2016). although the production of this species has achieved high quality and efficiency, very little is known about its nutritional quality relative to our knowledge of other fish species. given that the demand for fish is increasing globally and fish consumption is generally recommended by dieticians, it is surprising that there are no data on the fa profiles and fa contents of the different fish species farmed in the adriatic sea, especially the turbot and dentex. therefore, in this study, we investigated the quality parameters of four farmed white fish species sampled from different farms along the adriatic coast: the european sea bass (d. labrax), gilthead sea bream (sp. aurata), turbot (s. maximus), and common dentex (d. dentex). the basic chemical parameters, minerals, fa compositions, and health-related lipid indices: atherogenic index [ai], thrombogenic index [ti], the ratio of hypocholesterolaemic to hypercholesterolaemic fas [hh], and flesh lipid quality [flq]) were determined and compared across the four species analyzed. 2. materials and methods 2.1. fish farming and sampling conditions sea bass and sea bream are the main marine fish species commercially farmed in croatian sea farms, whereas the cultivation of dentex, and especially turbot, is far less common in these regions. fish are cultivated in a similar manner at each farming location, consistent with the rules of mediterranean fish farming, which are as follows: cultivation density of 5-12 kg per cubic meter (for turbot > 20 kg per cubic meter) in 22-28 months; the use of cages; and feeding regimens adjusted to bodyweight, sea temperature, photoperiod, oxygen saturation, etc. the main difference between the northern part of the adriatic and its middle part is the greater temperature differential between the winter and summer months in the north, whereas in the middle part, the temperature never falls below 12 °c in winter or exceeds 25 °c in summer. the turbot-farming technology has been sufficiently mastered in some geographic areas, but is still in the experimental stage in the adriatic sea. the biggest challenge is the extremely high temperatures in the adriatic sea, and the most effective modes of manipulation are yet to be determined. fry are released from different commercial hatcheries in france, italy, and greece to on-grow, are cultivated in floating net cages of different sizes, and are fed a commercially available fish feed with different nutritional contents, as shown in table 1. table 1. declared compositions of feeds used in the production of cultivated fish species. parameter (%) commercial name of the food/fish species efico sigma 870/turbot/dentex efico ym 854/sea bream efico ym 868/sea bass crude proteins 54.0 41.0 40.0 crude lipids 20.0 18.0 23.0 crude cellulose 0.4 4.0 3.0 ash 10.2 6.5 6.6 ital. j. food sci., vol 29, 2017 540 in this study, samples of sea bass, sea bream, and dentex were collected from different farms situated in the northern (istria) and middle parts of the adriatic sea, whereas turbot was only sampled from the northern part of the adriatic sea (istria). the fish were sampled in march-may 2016 (three groups of samples were collected per species). in total, 72 pieces of fish (18 pieces of each species) were transported to our laboratory in styrofoam boxes on ice. before the edible part of the fish flesh was eviscerated and filleted for analysis, the bodyweight and length were measured to the closest g (280-300 g for sea bass, sea bream, and dentex) and cm (28.5-30.5 g for sea bass, sea bream, and dentex), respectively. for the turbot, these parameters were 260-320 g and 24.5-31.0 cm, respectively. sample preparation involved cleaning the body surfaces, descaling and beheading each specimen, and removing the vertebrae and viscera (not the skin). the fillets and skin of each of the 72 fish samples were homogenized with a laboratory homogenizer (grindomix gm 200, retsch, haam, germany). the samples were analyzed immediately, first for their basic chemical parameters and then for their fa profiles. 2.2. determination of basic chemical composition and mineral content the water content was determined with a gravimetric analysis (iso 1442:1997) using an epsa 2000 thermostat (ba-ri, velika gorica, croatia). the total protein content was determined by the kjeldahl method given by international organization for standardization (iso) (iso 937:1978) using a digestion unit 8-basic (foss, höganäs, sweden) and a kjeltec 8400 automated distillation and titration device (foss). the total fat content was determined with the soxhlet method (iso 1443:1973), in which the samples are digested with acid hydrolysis and the fats are then extracted with petroleum ether using a soxtherm 2000 automated device (gerhardt, munich, germany). the ash content was determined according to iso 936:1998 using a nobertherm lv9/11/p320 furnace (lilienthal, germany). the phosphorus content was determined according to iso 13730:1996 with a dr/4000u spectrophotometer (hach, düsseldorf, germany). we used the procedures described by petrović et al. (2015) to determine the calcium and sodium contents. for the basic chemical composition and mineral content analyses, two samples of each of the 72 fish samples (18 samples for each species) were tested in parallel, and the mean values were analysed statistically for percentage weight (%), with an accuracy of 0.01%. 2.3. fatty acid analysis the preparation of samples for the analysis of fa methyl esters has been described previously by pleadin et al. (2015). the fa methyl esters were analysed with gas chromatography (gc) according to iso 12966-4:2015 and en iso 12966-4:2015. a 7890ba gas chromatographer, equipped with a flame ionization detector (fid) and a 60 m db-23 capillary column with an internal capillary diameter of 0.25 mm and a stationary phase thickness of 0.25 μm (agilent technologies, santa clara, ca< usa), was used. the components were detected with a fid at a temperature of 280 °c, a hydrogen flow of 40 ml/min, and an airflow of 450 ml/min. nitrogen was used as the make-up gas at a flow rate of 25 ml/min. the initial column temperature was 130 °c; after 1 min, it was increased by 6.5 °c/min until it reached 170 °c. the temperature was further increased by 2.75 °c/min until it reached 215 °c, where it was maintained for 12 min. the temperature was then increased further by 40 °c/min until the final column temperature reached 230 °c, which was maintained for 3 min. each sample (1 μl) was injected into a splitsplitless injector at a temperature of 270 °c with a split ratio of 1:50. the carrier gas was helium (99.9999%), flowing at the constant rate of 43 cm/s. the fa methyl esters were ital. j. food sci., vol 29, 2017 541 identified by comparing their retention times with those of fa methyl esters in the standard mixture, as described previously by pleadin et al. (2015). the fa composition was determined for each of the 72 fish samples (18 pieces of each species). the mean values per species are expressed as the percentage (%) of a particular fa in the total fas, with an accuracy of 0.01%. the fa methyl ester values were converted into fa values per 100 g of edible part (ep), according to the fao/infoods guidelines for converting units, denominators and expressions (2012). 2.4. determination of lipid quality the lipid quality indices ai, ti, hh, and flq were calculated from the fa compositions. ai indicates the relationship between the total major saturated fats, which are considered pro-atherogenic agents (because they facilitate the adhesion of lipids to the cells of the immune and circulatory systems), and the total major unsaturated fats, which are considered anti-atherogenic agents (because they inhibit the formation of plaques and reduce the levels of esterified fas, cholesterol, and phospholipids, and therefore prevent microand macro-coronary diseases) (ulbritcth and southgate, 1991). this parameter was calculated as: ai = ([12:0 + (4 × 14:0) + 16:0])/(total monounsaturated fatty acids [mufa] + + pufa n-6 + pufa n-3) ti indicates the tendency for blood to clot in blood vessels. it is defined as the relationship between pro-thrombogenic (saturated) and anti-thrombogenic fas (mufa, pufa n-6, and pufa n-3) (ulbritcth and southgate, 1991). the index is calculated as: ti = (14:0 + 16:0 + 18:0)/([0.5 × total mufa + 0.5 × pufa n-6 + 3 × pufa n-3] + + [pufa n-3/pufa n-6]) the ratio between the hypocholesterolaemic and hypercholesterolaemic fas (hh) takes into account the known effects of certain fas on cholesterol metabolism (santos-silva et al., 2002). it is calculated as: hh = (c18:1n-9 + c18:2n-6 + c20:4n-6 + c18:3n-3 + c20:5n-3 + c22:5n-3 + + c22:6n-3)/(c14:0 + c16:0) flq indicates the proportion of the main pufas n-3 (epa and dha) in the muscles relative to the total lipid content. the higher this index, the higher the quality of the dietary lipid source (abrami et al., 1992; senso et al., 2007). the index is calculated as: flq = (epa + dha in g fas/100 g ep)/(total lipids in g/100 g ep) × 100 2.5. statistical analysis the statistical analysis was performed with spss statistics software 22.0 (ibm spss statistics 2013, ny, usa). the results were tested for the normality of their distribution (p > 0.05) using the shapiro-wilks test. to determine the statistical significance of the differences in the chemical and fa compositions between the fish species analysed, oneway anova and the robust brown-forsythe test were used. to establish the homogeneity of variance, the scheffe post hoc test or tamhane’s t2 post hoc were used. decisions on statistical relevance were made at the significance level of p < 0.05. ital. j. food sci., vol 29, 2017 542 3. results and discussions the results presented in this study provide nutritional profiles of four fish species (sea bream, sea bass, turbot, and dentex), with particular emphasis on their fa components and health-related lipid indices. all the fish species are farmed in the adriatic sea. the basic chemical and mineral compositions of these fish species are shown in table 2. table 2. basic chemical and mineral compositions of four species of farmed fish. parameter sea bass dentex turbot sea bream water (%) 70.81±3.28c 69.12±0.39c 77.88±1.31a,b,d 70.16±2.50c ash (%) 1.21±0.02b 1.31±0.03a,c 1.16±0.05b,d 1.24±0.07c fat (%) 9.11±3.06 c 10.46±0.40c 4.00±1.20a,b,d 10.48±3.08 c protein (%) 19.22±1.46c 18.92±0.43c 17.59±1.15a,b,d 19.09±0.33 c na (mg/kg) 706±277c 465±43.9c,d 1212±292a,b 968±184b ca (mg/kg) 729±189b 1300±128a,c,d 656±151b 540±152b p (mg/kg) 2214±122b,c 2740±195a,c,d 1811±127a,b,d 2180±164b,c results are expressed as mean values (18 samples per species, each of which was analysed in duplicate) ± standard deviations. significant differences (p < 0.05): avs. sea bass; bvs. dentex; cvs. turbot; dvs. sea bream. analysis of the basic chemical and mineral parameters showed that the turbot had a statistically significantly (p < 0.05) higher water content and lower fat and protein contents than the other fish analysed. in the remaining three fish species, the basic quality parameters were similar to those presented in earlier studies (özden and erkan, 2008; erkan and özden, 2007; kyrana and lougovois, 2002). the descriptive data for the basic chemical fish composition did not clearly correlate with the composition of the feed with which the fish were fed (table 1), because it is not the only parameter that affects the nutritional composition of a fish species. nutritional composition may be affected by a number of factors, including age, sex, and environmental factors, such as temperature, salinity, etc. (grigorakis, 2007). therefore, although the dentex and turbot were fed the same type of feed, their basic chemical and mineral contents differed significantly. in a study by özden and erkan (2008) that compared the properties of sea bream, sea brass, and dentex, the water, ash, protein, and fat contents were in the ranges 69.68%76.42%, 1.49%-1.95%, 18.21%-21.70%, and 2.29%-8.10%, respectively. in a study of the composition of turbot, these parameters were in the ranges 78.7%-80.2%, 1.0%-1.3%, 18.9%-20.3%, and 1.0%-2.0%, respectively (manthey-karl et al., 2016). in general, research has shown that the basic chemical compositions of fish vary with the season of cultivation. because fish are ectothermic poikilotherms, the fat content in a certain period of the year can be attributed to the ongoing physiological process of conserving the fat stock, which occurs in autumn after intense feeding, or the process of spending the fat stock, which occurs during winter. in early summer, when environmental conditions change, primarily as an increase in temperature, the fish metabolism accelerates and the energy taken from food is used for fish growth. petrović et al. (2015) showed similar proportions of fat and moisture in the sea bass and sea bream, with fat contents of 3.2%12.3% in the sea bass and 4.2%-15.0% in the sea bream, which depended on and varied strongly across the farming seasons, as explained previously. the fat content is inversely ital. j. food sci., vol 29, 2017 543 related to the water content, and these parameters together account for approximately 80% of the total fish meat composition. the dentex had a significantly higher (p < 0.05) proportion of calcium and phosphorus than the other fish species analysed, whereas the turbot had the lowest proportion of phosphorus (significantly lower than in the other fish species, p < 0.05). the dentex had a significantly lower sodium content than the turbot and sea bream, and the sodium content of the turbot was significantly higher than that of the common dentex or sea bass. as in earlier studies, the calcium and phosphorus contents were higher in the sea bass than in the sea bream (erkan and özden, 2007; petrović et al., 2015). however, the calcium content determined in this study was significantly higher than the results previously published for the sea bream (orban et al., 2003; petrović et al., 2015) and turbot (manthey-karl et al., 2016), whereas our results for the sea bass were similar to previous results (petrović et al., 2015). the fa compositions, expressed as mean values and standard deviations relative to the total fas for each species, are shown in table 3. the results show that the most strongly represented fa in all four species analysed was oleic acid (c18:1n-9, oa), followed by linoleic acid (c18:2n-6, la) and palmitic acid (c16:0, pa). the results of this study are consistent with earlier research that demonstrated an increase in c18 fas, such as oa, la, and ala, in farmed fish, in response to the use of vegetable oils in their feed (strobel et al., 2012). among the unsaturated fas, oa and la contribute most significantly to the enrichment of aromatic components (elmore et al., 1999) and are considered to be of high nutritional value because they protect against cardiovascular disease (hornstra, 1999). statistically significant differences in the fa profiles were observed among the fish species analysed (p < 0.05). no fa analysis of the commercial feed used was available. the proportion of saturated fas (sfas; c14:0, c15:0, and c16:0) was the highest in the turbot. the sea bass and sea bream had significantly higher proportions of mufa than the dentex or turbot, which is attributable to their oleic acid contents. a significantly lower proportion of pufa (c18:2n-6c, c18:3n-3) was found in the turbot. in the study by özden and erkan (2008), the fas detected in the sea bass, sea bream, and dentex were 28.01%-32.41% sfa, 25.88%-28.62% mufa, and 24.75%-27.42% pufa. compared with these values, our study found significantly higher proportions of mufa in all the fish species investigated, whereas our sfa and pufa values were similar to previously reported values, except in the turbot. the samples of turbot analysed in this study contained more sfa and mufa, and at the same time, less n-3 and less pufa than previously reported levels, resulting in a less favorable n-3/n-6 ratio than obtained by other researchers (serot et al., 1998; manthey-karl et al., 2016). the n-3/n-6 ratios obtained for turbot in these studies were 3-7-fold higher than that obtained in the present study. the protective role of fish consumption against coronary heart disease has been widely demonstrated and is mainly attributed to the effects of n-3 fas and their cardioprotective action (kris-etherton et al., 2003; psota et al., 2006). earlier studies suggested that the n-3/n-6 ratio is a reliable index for interspecies comparisons of relative nutritional values (piggot and tucker, 1990). according to sargent (1997), the optimum n-3/n-6 pufa ratio should be 1:5 (0.2). the amounts of the fas (ala, epa, dha, aa, and la,) present in the four fish species in our study, as determined by converting the proportion of an individual fa in the total fas to the amount of the individual fa per 100 g of fish ep, are shown in table 4. ital. j. food sci., vol 29, 2017 544 table 3. fatty acid compositions (% of total fas) of four species of farmed fish. fatty acids relative fa amount (% of fa) mean±standard deviation sea bass dentex turbot sea bream c12:0 nd 0.06±0.00 0.17±0.09 0.06±0.05 c14:0 2.65±0.52b,c 4.13±0.18a,c,d 9.66±0.81a,b,d 2.69±0.78b,c c15:0 0.33±0.05b 0.45±0.02c 0.75±0.11a,b,d 0.28±0.11c c16:0(pa) 16.17±1.43c 17.95±0.39c,d 25.36±2.02a,b,d 14.18±1.74b,c c17:0 0.39±0.08 0.51±0.01 0.49±0.25 0.26±0.11 c18:0 3.97±0.41b 5.50±0.12a,d 4.72±1.37 3.30±0.59b c20:0 0.33±0.05 0.30±0.15 0.36±0.04 0.35±0.07 c22:0 0.08±0.08 0.10±0.01 nd 0.13±0.11 c14:1 nd 0.08±0.00 0.08±0.09 nd c16:1n-7t 0.45±0.03 0.44±0.01 0.51±0.07 0.45±0.03 c16:1n-7c 3.46±0.58b,c 5.82±0.11a,c 11.37±1.53a,b,d 4.11±1.25c c17:1 0.24±0.06 0.36±0.01 0.17±0.19 0.20±0.07 c18:1n-9t 0.34±0.25 0.07±0.07 nd 0.27±0.11 c18:1n-9c (oa) 38.85±3.98b,c 28.10±1.16a,c,d 20.63±2.18a,b,d 42.07±6.23b,c c18:1n-7 3.16±0.22 c 3.41±0.06 c 4.66±0.15a,b,d 3.07±0.18 c c20:1n-9 3.10±0.45 c 2.53±0.05 c 3.87±0.37a,b,d 2.41±0.71 c c22:1n-11 1.42±0.77c 1.15±0.04c 3.35±0.47a,b,d 1.01±0.42c c22:1n-9 0.49±0.09 0.49±0.02 0.89±0.45 0.62±0.18 c24:1n-9 0.31±0.06 0.40±0.23 0.89±0.86 0.46±0.16 c18:2n-6t 0.08±0.06 0.13±0.01 0.09±0.10 0.05±0.05 c18:2n-6c (la) 14.60±2.00b,c 10.98±0.02a,c,d 6.08±0.84a,b,d 15.71±0.62b,c c18:3n-6 0.11±0.06 0.13±0.00 nd 0.12±0.13 c20:2n-6 0.68±0.23b 0.40±0.02a 0.37±0.23 0.68±0.21 c20:3n-6 nd 0.13±0.01 nd 0.11±0.10 c20:4n-6 (aa) 0.26±0.05b 0.57±0.05a,c,d 0.27±0.15b 0.20±0.10b c18:3n-3 (ala) 2.92±0.46b,c 1.78±0.04a,c 0.52±0.31a,b,d 3.06±0.99c c18:4n-3 0.39±0.13b 0.82±0.06a 0.50±0.30 0.33±0.11 c20:3n-3 0.07±0.07d 0.14±0.01 nd 0.22±0.05a c20:4n-3 0.23±0.07b 0.46±0.02a 0.23±0.19 0.32±0.15 c20:5n-3 (epa) 1.89±0.53 b 4.23±0.42a,c,d 2.01±0.87 b 1.12±0.58 b c22:6n-3 (dha) 3.01±0.89b 8.32±1.12a,c,d 2.06±1.47b 2.19±1.15b sfa 23.93±2.13b,c 29.06±0.60a,c 41.45±3.04a,b,d 21.24±3.25c mufa 51.82±2.45b,c 42.85±1.38a,d 46.43±2.64a,d 54.66±3.74b,c pufa 24.25±2.30c 28.09±1.73c 12.12±3.72a,b,d 24.10±1.38c total n-6 15.74±2.06b,c 12.34±0.07a,c,d 6.81±1.12a,b,d 16.87±0.37b,c total n-3 8.51±1.29b 15.75±1.66a,b,c 5.31±2.61b 7.23±1.48b results are expressed as mean values (18 samples per species) ± standard deviations; nd, not detected; lod, the limit of detection of 0.05%; pa, palmitic acid; oa, oleic acid; la, linoleic acid; ala, α-linolenic acid; aa, arachidonic acid; epa, eicosapentaenoic acid; dha, docosahexaenoic acid; sfa, saturated fatty acids; mufa, monounsaturated fatty acids; pufa, polyunsaturated fatty acids. significant differences (p < 0.05): avs. sea bass; bvs. dentex¸ cvs. turbot; dvs. sea bream. ital. j. food sci., vol 29, 2017 545 table 4. absolute quantification of fatty acids (mg fa/100 g edible muscle part) ala, epa, dha, aa, and la. fatty acids (mg/100 g) sea bass dentex turbot sea bream ala 245.01±38.71b,c 172.28±4.34a,c 18.59±11.01a,b,d 295.29±95.63c epa 158.79±44.66b,c 410.40±40.68a,c,d 72.74±31.67a,b 108.27±56.02b dha 254.06±75.55b,c 810.32±109.05a,c,d 74.99±53.53a,b 219.90±112.20b epa + dha 418.25±113.42b,c 1220.72±149.60a,c,d 147.73±69.83a,b 321.16±162.62b aa 22.20±4.29b 55.54±4.48a,c,d 9.69±5.37b 19.57±9.43b la 1223.79±167.09c,d 1062.69±2.47c,d 219.33±30.40a,b,d 1517.76±57.98a,b,c results are expressed as mean values (18 samples per species) ± standard deviations; ala, α-linolenic acid; epa, eicosapentaenoic acid; dha, docosahexaenoic acid; aa, arachidonic acid; la, linoleic acid. the contents of la and ala should depend on the fish’s dietary intake because fish lack the delta 12 and delta 15 desaturase enzymes responsible for the conversion of oa to la and ala (ruiz-lopez et al., 2012). the higher la and ala contents presumably indicate the presence of vegetable oils in the feed, such as sunflower, soybean, rapeseed, and linseed oil. la and ala were significantly (p < 0.05) lower in the turbot, whereas la was significantly higher (p < 0.05) in the sea bream than in the other species (table 4). there are various recommendations for both the consumption of fish and the intake of n-3 lcpufa (primarily epa and dha). to meet the recommended epa and dha dietary requirements for human nutrition, the american heart association (2015) recommends the consumption of two servings of fish (particularly oily fish) twice a week. in terms of the fat content, the german nutrition society recommends a smaller serving of oily fish (i.e., 70 g) and a larger serving of lean fish (80-150 g) once a week (berglaiter, 2012). the majority of organizations recommend two servings of fish (approximately 140 g per meal) per week. this would provide approximately 500 mg of epa and dha combine a day, which is the intake most countries and organizations recommend (who, 2003; krisetherton and innis, 2007) for optimal overall health and a reduced cardiovascular risk. however, the lowest daily value set for adults by the european food safety authority (efsa) is 250 mg of epa plus dha (efsa, 2009). the absolute epa and dha contents were significantly higher (p < 0.05) in the dentex than in other species analysed, with values of 1220 mg/100 g ep (table 4). therefore, to fulfil the lowest-level recommendation set by the efsa, the amounts of sea bass, dentex, turbot, and sea bream that must be consumed on a daily basis are 61 g, 21 g, 167 g, and 78 g, respectively. the dietetic value of fish meat is also determined by its lipid quality indices, which reflect the relative proportions of constituent saturated and unsaturated fas. these indices indicate the global dietetic quality of the lipids and their potential impact on the development of coronary heart disease (ulbritcth and southgate, 1991). the nutritional quality indices determined for different fish species in this study are shown in table 5. the sea bass, sea bream, and turbot had significantly higher proportions of n-6 fas than the dentex, whereas the dentex had a significantly higher proportion of n-3 fas (c18:4n-3, c20:4n-3, epa, dha) (table 2). consequently, the n-3/n-6 ratio was significantly higher for the dentex (table 5). rigos et al. (2012) also confirmed the high proportion of n-3 fas in farmed dentex. they also found a significantly higher n-3/n-6 ratio in farmed dentex than in wild dentex. the n-3 fas are lower in modern aquaculture products than in wild, naturally caught fish, and the high levels of terrestrial-plant-originating c18:2 n-6 present in feedstuff affect the n-3/n-6 ratio in the ep of farmed fish (grigorakis, 2007). ital. j. food sci., vol 29, 2017 546 table 5. nutritional quality indices determined for four species of farmed fish. parameter sea bass dentex turbot sea bream n-3/n-6 0.55±0.11b 1.28±0.13a,b,c 0.74±0.30b 0.43±0.09b pufa/sfa 1.02±0.17c 0.97±0.07c 0.30±0.10a,b,d 1.15±0.19c ai 0.35±0.05c 0.49±0.02c 1.10±0.13a,b,d 0.32±0.07c ti 0.38±0.04c 0.36±0.03c 0.97±0.33a,b,d 0.35±0.06c hh 3.34±0.55b,c 2.46±0.08a,c,d 0.91±0.13a,b,d 3.94±1.03b,c flq 4.53±1.25b 11.67±1.43a,c,d 3.69±1.75b 3.06±1.55b results are expressed as mean values (18 samples per species) ± standard deviations; n-3, omega-3 fatty acids; n-6, omega-6 fatty acids; sfa, saturated fatty acids; pufa, polyunsaturated fatty acids; epa, eicosapentaenoic acid; dha, docosahexaenoic acid; ai, atherogenic index; ti thrombogenic index; hh hypocholesterolaemic/hypercholesterolaemic ratio; flq, flesh lipid quality. significant differences (p < 0.05): avs. sea bass; bvs. dentex; cvs. turbot; dvs. sea bream. pufa/sfa values above 0.4-0.5 and n-3/n-6 < 0.25 are required if a diet is to combat various ‘lifestyle diseases’, such as coronary heart disease and cancer (simopoulos, 2002). the higher the n-3/n-6 ratio, the more the body is able to use n-3 fats (wood et al., 2008). in this study, the recommended n-3/n-6 ratio was met by all the fish species, but the recommended pufa/sfa ratio was not met by the turbot, which had the lowest pufa/sfa ratio (0.30 ± 0.10). the n-3/n-6 ratio is suggested to be a good parameter for comparing the relative nutritional values of different species (piggot and tucker, 1990). however, this index is of limited value if the fa composition is unknown. generally, c20 and c22 fas are more valuable from a nutritional standpoint than c18 fas (arts et al., 2001). because they are quantitatively predominant, the two fas epa and dha are largely responsible for differences in the n-3/n-6 ratio, a reliable indicator of relative nutritive value of lipids, as was also confirmed in our study (table 5). however, some researchers consider that an index such as pufa/sfa may be inadequate for evaluating the nutritional value of fats, because some sfas do not increase plasma cholesterol and because the ratio ignores the effects of mufa (orellana et al., 2009). a recent study suggested that c12:0 and c14:0 more effectively increase total cholesterol than c16:0, whereas c18:0 has no effect on the concentration of total serum cholesterol, and no apparent effect on either ldls or high-density lipoproteins (mensink and katan, 1992; daley et al., 2010). therefore, the c12:0, c14:0, and c16:0 fas present in human diets are associated with increased plasma cholesterol. this association is strongest for c14:0, which has a potentially 4-6 times higher capacity to increase cholesterol concentrations than c16:0 (ulbritcth and southgate, 1991; mensink and katan, 1992; bressan et al., 2011). based on the discussion presented above, another indicator of nutritional quality, hh, was determined to gain insight into the effects of fas on blood cholesterol levels. a higher value for the hh index is preferable (santos-silva et al., 2002; testi et al., 2006). in this study, the hh values ranged from 0.91 ± 0.13 in the turbot to 3.94 ± 1.03 in the sea bream. the hh ratios were significantly higher for the sea bass and sea bream than for the turbot because these two species contain significantly lower proportions of sfa. two other indices, ai and ti, were investigated because their effects on the incidence of pathogenic phenomena, such as atheroma and/or thrombus formation, differ from those of single fas. as expected, ai and ti were always highest for the most atherogenic and thrombogenic dietary components. it is assumed that lipids with ai < 1 and ti < 1 are beneficial to human health. tonial et al. (2014) suggested that mufa and pufa have more profound health benefits because they prevent coronary disease. the ai and ti ital. j. food sci., vol 29, 2017 547 values determined in this study were lower than 1, except those for turbot, which had significantly higher (p < 0.05) ai and ti values than the other fish species because it has a higher proportion of sfa. in the study conducted by valfrė and co-authors (2003), an ai of 0.45 and a ti of 0.25 were determined for the sea bass, whereas de francesco et al. (2007), in a study of the sea bream, found ais of 1.545-1.565 and tis of 0.196-0.430. our results are consistent with previously published data. flq was significantly higher in the dentex (p < 0.05) than in the other three species. because this index is related to the proportions of dha and epa in the total fish lipid content, it is expected to be highest in the dentex, because this species is the richest source of these two n-3 fas. however, the lipid quality indices ai and flq for the dentex were lower in this study than in another study (suarez and cervera, 2010), whereas the ti values were quite similar. however, flq for the other species in this study were lower than those reported previously for the gilthead sea bass (senso et al., 2007). 4. conclusions this study demonstrates the high variability in the basic chemical and mineral contents of the species studied. the proportion of omega-3 fas was twice as high in the dentex than in the sea bream, sea bass, or turbot, and the proportions of epa and dha are 2-4 times higher in the dentex. the recommended n-3/n-6 ratio was present in all the fish species, but the recommended pufa/sfa ratio was not present in the turbot, which had the lowest pufa/sfa ratio. the hh ratio was significantly higher for the sea bass and sea bream than for the turbot. because the fish species analysed vary in their nutritional quality, the consumption of diverse fish species is advisable. based on established lipid quality indicators, the dentex, a fish species underexploited in aquaculture, is a highly recommended and important source of the fas required for human health. abbreviations aa, arachidonic acid (c20:4 n-6); ai, atherogenic index; dha, docosahexaenoic acid (c22:6 n-3); ep, edible part; epa, eicosapentaenoic acid (c20:5 n-3); fas, fatty acids; flq, flesh lipid quality; hdl, high-density lipoprotein; hh, ratio of hypocholesterolaemic to hypercholesterolaemic fatty acids; la, linoleic acid, c18:2 n-6; lc pufa, long-chain 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and southgate d.a.t. 1991. coronary heart disease: seven dietary factors. lancet 338: 985. valfrė f., caprino f. and turchini g.m. 2003. the health benefit of seafood. vet. res. commun. 27(suppl. i): 507. who 2003. joint who/fao expert consultation on diet, nutrition and the prevention of chronic diseases. who technical report series 916. geneva, switzerland: world health organization. wood j.d., enser m., fisher a.v., nute g.r., sheard p.r., richardson r.i., hughes s.i. and whittington f.m. 2008. fat deposition, fatty acid composition and meat quality: a review. meat sci. 78: 343. paper received december 3, 2016 accepted march 14, 2017 ijfs#433_raposo_bozza   ital. j. food sci., vol 28, 2016 448 paper vending machine foods: evaluation of nutritional composition a. raposo1*, c. carrascosa2, e. pérez2, a. tavares3, e. sanjuán2, p. saavedra4 and r. millán2 1centro de investigação interdisciplinar egas moniz, ciiem, instituto superior de ciências da saúde egas moniz, iscsem, quinta da granja, monte de caparica, 2829-511 caparica, portugal 2department of animal pathology and production, bromatology and food technology, faculty of veterinary, universidad de las palmas de gran canaria, trasmontaña s/n, 35413 arucas, spain 3escola de ciências e tecnologias da saúde, universidade lusófona de humanidades e tecnologias, campo grande, 376, 1749-024 lisboa, portugal 4department of mathematics, universidad de las palmas de gran canaria, mathematics building, campus universitario de tafira, 35018 las palmas de gran canaria, spain *corresponding author. tel. +351 918376093 e-mail address: araposo@egasmoniz.edu.pt abstract the nutritional quality of vending machine foods may be a factor that contributes to significantly increase obesity and associated diseases, and the vending industry is significantly growing worldwide. this study aims to evaluate the nutritional composition of vending machine foods and to compare it with the consumption of the gran canaria population. food products from 74 snack and 71 refrigerated vending machines located in las palmas (gran canaria), and on university campuses, were nutritionally assessed. the percentages of sales per food type were accessed during a 12 month-period to verify user preferences. significant differences (p <0.05) were found in the content of nutrients compared with the kruskal-wallis test with all the food groups. sandwiches (wholemeal and white bread) had the lowest energy levels, while croissants had the highest. we highlight the increased sodium content in baguettes compared to the other foods. the findings suggest that vending machine foods contain more fat/saturated fat, calories and sodium than recommended. further studies on the nutritional assessment of vending machine foods, governments’ awareness and policies that promote the intake of healthy foods are essential to increase the amount of foods with an appropriate nutritional profile according to recommendations in vending machines. keywords: consumer, food choices, nutritional assessment, vending machines   ital. j. food sci., vol 28, 2016 449 1. introduction recent decades have witnessed a significant increase in industrial vending machine development. japan is the world leader in this sector, the usa makes 30 billion american dollars per year (lin et al., 2011), and the uk makes approximately 1,700 million pounds sterling (mintel, 2009). spain is a european power in using vending machines, with a consolidated industry and highly integrated use (raposo et al., 2015). there are 560,000 vending machines across spain; that is, one machine for every 80 inhabitants. japan, the industry leader, has 5.5 million vending machines, which is one for every 23 people (mtv, 2008). for decades, the nutritional value of foods present in vending machines has been consistently documented (cheney, 1974; ezell et al., 1985; hruban, 1977; koehler et al., 1977; shearer et al., 1980). in general, authors have voiced concern about the relatively low value of nutrients, and the high sugar and fat contents in many of the most frequently chosen items (hunter, 1992; kubik et al., 2013; pasch et al., 2011). nowadays, such concern has led to some governments, as in spain, to legislate the products sold in vending machines in schools and colleges (spanish law 17/2011, of july 5, of food safety and nutrition, especially article 41), and also because of the high childhood obesity rates and unhealthy food habits detected among consumers (cavaliere et al., 2014; ebenegger et al., 2010; mackay, 2011). although the vending machine policy has been effective in changing snack behaviour in a wide variety of settings, no published research has investigated vending snack behaviour in a university campus community (caruso et al. 2014). despite the potential risks or health benefits, very little is known about the variety of food and beverages sold in vending machines on university campuses. a study carried out by byrd-bredbenner et al. (2012), which aimed to assess the drinks and snacks sold in vending machines in different universities, reported that snacks and drinks offered poor nutrient quality. most snacks were low in fibre and had high calorie and fat contents, and almost half contained too much sugar. most drinks also contained high levels of sugar and calories. the findings of this study suggested that vending machines offer limited healthy options. other studies have demonstrated that the sale and consumption of healthy foods can be influenced by increased availability of healthy foods (french et al., 2004; lytle et al., 2006; muckelbauer et al., 2009; perry et al., 2004). similarly, purchase behaviour can be positively influenced by driving customers to healthier choices through strategies such as labelling, and by providing information, reminders and reinforcement (gerend, 2009; harnack et al., 2008; holdsworth et al., 2004). as the effect of labelling itself is slight, it is likely to be more effective if combined with other methods (french et al., 2001; grunert et al., 2010; sacks et al., 2009); e.g. strategies that influence purchasing behaviour by reducing low-calorie food prices (french et al., 2001). the results of another study (kocken et al., 2012) indicated that when the availability of low-calorie foods increased and was combined with labelling that highlighted nutritional properties, and prices lowered at the same time, students made healthier choices without buying more or fewer products from vending machines. vending machines reflect the good availability and suitability of food and beverages in western society, and also in many environments, including schools (rideout et al., 2007; van der horst et al., 2008), workplaces (french et al., 2010) and health centres (lawrence et al., 2009). in some places, availability of alternative foods and beverages from vending machines may be limited (farley et al., 2010). based on these data, providing healthy varied foods in such machines is extremely important to counteract the facts that some studies have reported (finkelstein et al., 2008; french et al., 2003;   ital. j. food sci., vol 28, 2016 450 lytle et al., 2006), which have documented that most vending machines are typically stocked with food and beverages that are rich in energy, but low in nutrients. some studies have shown the relationship between students who use vending machines regularly and excessive consumption of sugary drinks (wiecha et al., 2006). however, not only sugary drinks from vending machines contribute to a significant increase in obesity and associated diseases, but snack vending machines form part of an environment that may lead to obesity, which encourages easy access to energy-dense and nutrient-poor food (kubik et al., 2015; park and papadaki, 2016). typically vending machines offer few healthy options (lawrence et al., 2009). we must take into account that vending machines increasingly grow in number, become more prevalent and available, thus they supply daily energy intake to more and more individuals. so it is important to accurately assess and monitor the nutritive value of vending machine products (matthews and horacek, 2015). for all these reasons, and taking into account the implementation of law 17/2011 in spain on food safety and nutrition, we believe that it is necessary to determine the nutritional composition of the food dispensed in vending machines to compare not only the nutritional profiles of various foods, but also the consumption of such foods in the gran canaria population (canary islands, spain). 2. materials and methods for this work, we comprehensively considered the gran canaria island (spain) and placed particular emphasis on the ulpgc (universidad de las palmas de gran canaria) university campuses located on it. the ulpgc has 25,172 students enrolled in various degree programmes distributed over four campuses on gran canaria (ulpgc, 2015). this study was carried out with 108 vending machines located in the town of las palmas de gran canaria (50 snack vending machines and 58 refrigerated vending machines that dispensed solid food products), and all 37 vending machines of snacks/refrigerated solid food products in ulpgc campus buildings (24 snack vending machines and 13 refrigerated vending machines that dispensed solid food products). only the vending machines (145) of a single company in the sector were considered, which has 65% of the volume of business in las palmas de gran canaria, spain. regarding the 37 machines located at the ulpgc, the company has been awarded the exclusive installation of these machines throughout the campus. 2.1. nutritional assessment over 3 months we accessed the cuisine of the vending company involved, which collaborated in this study and calculated all the products it made. while food handlers were preparing food, ten samples of each product were taken to be weighed on a balance (model 2200c, precisa, dietikon, switzerland). finally the mean weight of each ingredient was used to calculate nutrients (proteins, total and saturated fats, carbohydrates and fibre), the total energy value and sodium present in each product item. it should be noted that the nutritional information of 61% of the ingredients used in all the products was supplied by the vending company’s providers. for the remaining 39% for which it was not possible to obtain such information, we used the food table of the dial program (2011), developed by a group of professors from the department of nutrition and bromatology i in the faculty of pharmacy at the universidad complutense de madrid (spain).   ital. j. food sci., vol 28, 2016 451 among the processed products, the following categories appeared: 9 different types of wholemeal baguettes (85 g); 12 different types of croissants (100 g); 11 different types of white bread baguettes (85 g); 14 different types of white bread sandwiches (50 g); 5 different kinds of wholemeal sandwiches (50 g). all these products were composed only of the ingredients described in tables 1-5, except those prepared with tuna, chicken, crab and watercress, which also contained mayonnaise. we randomly took ten different products marketed in vending machine foods: five chocolate bars and five industrial pastry products. the purpose of this procedure was to establish the nutritional, total energy and sodium values of the above food categories by collecting the information provided by these foods. finally from the vending company, we requested the percentage of sales for each food type during a 12-month period to verify what user preferences were. table 1: nutritional assessment wholemeal baguettes. wholemeal baguette (85 g) energy value (kcal) protein (g) total fat (g) saturated fat (g) total carbohydrate (g) dietary fibre (g) sodium (mg) salami and cheese 547.6 23.8 33.6 13.4 34.1 6.5 1732.5 chicken and cheese 537.2 52.0 20.7 12.3 32.3 6.4 516.8 salami 437.3 14.9 25.4 9.1 33.9 6.6 1559.5 tuna and corn vegetable 432.9 14.5 20.4 3.1 42.9 9.9 723.3 tuna and corn 427.9 13.9 23.6 3.6 36.5 7.0 737.5 pork loin and cheese 424.9 41.2 12.8 5.9 33.0 6.4 1450.9 ham and cheese 396.7 32.4 13.1 6.8 33.5 6.4 1198.3 goat cheese 349.4 15.9 16.1 8.6 32.3 6.4 792.4 tortilla 291.5 10.4 8.6 1.8 39.5 7.5 885.7 427.3 24.3 19.4 7.2 35.3 7.0 1066.3 σ 80.7 14.5 7.7 4.0 3.7 1.2 431.2 : mean of all types of wholemeal baguette. σ : standard deviation of all types of wholemeal baguette. 2.2. statistical analysis the data analysis of this work was carried out with the statistical software package spss 20.0 (spss, chicago, il, usa) for mac os x (apple computers, cupertino, ca, usa). for the nutritional evaluation, different products were grouped by category, wholemeal baguette; croissant; white bread baguette; white bread sandwich; wholemeal sandwich, and the means and standard deviations of the nutrients (markers), sodium and total energy values per category were calculated. variables (markers) were summarised as medians and interquartile ranges (iqr). they were compared by the kruskal-wallis test. multiple comparisons were made by the wilcoxon test. statistical significance was set at p<0.05.   ital. j. food sci., vol 28, 2016 452 table 2: nutritional assessment croissants. croissant (100 g) energy value (kcal) protein (g) total fat (g) saturated fat (g) total carbohydrate (g) dietary fibre (g) sodium (mg) tomato, salad, turkey, tuna and corn 758.8 40.4 34.5 10.9 68.3 6.0 2676.3 tuna and corn 686.8 18.9 42.5 12.2 55.2 3.2 763.3 tuna, corn and vegetables 675.4 19.1 36.7 11.3 63.4 7.0 696.5 tuna and peppers 671.2 21.5 40.3 11.8 54.0 4.4 690.2 corn, crab and pineapple 668.6 13.3 40.8 11.8 59.1 4.9 685.8 crab 658.5 13.2 43.1 12.3 52.6 2.5 658.3 salami and cheese 647.7 37.1 35.8 19.1 50.3 2.3 998.5 chicken 622.4 14.7 39.6 11.7 50.2 2.5 595.6 watercress 597.3 8.4 39.4 11.6 50.5 2.6 580.1 tuna 571.9 18.3 32.6 10.6 49.8 2.4 612.7 ham and cheese 570.3 32.8 25.8 15.8 50.0 2.3 721.2 tortilla 474.0 13.1 20.4 9.2 57.7 3.5 870.6 633.6 20.9 36.0 12.4 55.1 3.6 879.1 σ 72.9 10.3 6.9 2.6 6.0 1.6 578.3 : mean of all types of croissant. σ : standard deviation of all types of croissant. table 3: nutritional assessment – white bread baguettes. white bread baguette (85 g) energy value (kcal) protein (g) total fat (g) saturated fat (g) total carbohydrate (g) dietary fibre (g) sodium (mg) sobrasada and cheese 570.4 20.0 34.5 12.6 43.0 3.0 947.9 salami and cheese 555.4 25.6 29.6 10.3 44.5 3.0 1622.4 pork loin and cheese 468.3 45.2 11.7 5.8 43.7 3.0 1519.4 tuna and corn 404.4 14.3 17.1 2.6 46.2 3.5 692.7 chicken and cheese 403.5 40.3 7.2 3.6 43.0 3.0 552.8 ham and cheese 396.3 28.7 11.1 6.8 43.5 3.0 766.2 tuna, corn, tomato and vegetables 393.3 14.2 13.8 2.1 50.3 5.5 666.0 salami 383.5 14.1 16.1 5.8 44.0 3.0 1203.5 goat cheese 372.5 17.6 13.8 7.9 43.0 3.0 792.6 tortilla 304.8 11.8 5.9 1.2 49.0 3.9 829.9 serrano ham and tomato 285.1 16.9 3.1 0.9 45.2 3.9 1381.7 412.6 22.6 15.0 5.4 45.0 3.4 997.7 σ 89.2 11.2 9.7 3.8 2.5 0.8 371.4 : mean of all types of white bread baguette. σ : standard deviation of all types of white bread baguette.   ital. j. food sci., vol 28, 2016 453 table 4: nutritional assessment – white bread sandwiches. white bread sandwich (50 g) energy value (kcal) protein (g) total fat (g) saturated fat (g) total carbohydrate (g) dietary fibre (g) sodium (mg) tortilla, ham and cheese 363.3 23.7 15.9 5.4 31.6 3.5 1272.8 sobrasada and cheese 337.5 17.0 21.0 8.9 21.2 2.1 490.1 salami and cheese 336.9 19.6 19.5 9.0 21.9 2.1 785.6 chicken 326.4 8.9 23.1 3.6 21.8 2.3 464.1 tuna, corn, tomato and vegetables 319.9 10.7 17.4 2.7 29.8 4.9 515.9 corn, crab and pineapple 313.5 7.0 19.7 3.0 27.3 3.7 489.1 two cheeses 312.6 18.0 17.3 10.2 22.1 2.1 652.8 tuna 310.6 12.1 20.1 3.1 21.4 2.2 498.0 watercress 308.6 4.0 23.2 3.6 22.0 2.4 458.5 tuna and peppers 290.1 11.1 17.3 2.7 23.9 3.3 467.7 turkey and cheese 283.0 26.9 8.5 4.5 25.6 2.1 1446.6 ham and cheese 276.1 25.1 10.1 4.9 22.4 2.1 996.6 crab 266.8 6.2 17.3 2.7 22.9 2.2 436.0 tuna and corn 257.6 9.8 14.4 2.2 23.5 2.4 467.1 307.4 14.3 17.5 4.8 24.1 2.7 674.4 σ 29.7 7.4 4.3 2.7 3.3 0.9 331.5 : mean of all types of white bread sandwich. σ : standard deviation of all types of white bread sandwich. table 5: nutritional assessment – wholemeal sandwiches. wholemeal sandwich (50 g) energy value (kcal) protein (g) total fat (g) saturated fat (g) total carbohydrate (g) dietary fibre (g) sodium (mg) salami and cheese 516.6 24.7 35.0 14.4 24.0 3.1 1623.8 watercress 294.4 5.8 19.2 3.1 22.7 3.2 394.3 tuna, corn and vegetables 286.3 11.0 13.0 2.1 28.7 5.4 428.0 crab 285.4 8.2 16.8 2.7 23.7 3.1 402.8 tuna and corn 275.5 10.3 14.4 2.3 24.5 3.4 429.3 331.6 12.0 19.7 4.9 24.7 3.6 655.6 σ 103.6 7.4 8.9 5.3 2.3 1.0 541.4 : mean of all types of wholemeal sandwich. σ : standard deviation of all types of wholemeal sandwich.   ital. j. food sci., vol 28, 2016 454 3. results and discussion by means of box plots, fig. 1 shows the seven categories of processed food products against markers by their quartiles, where data are medians and iqr.   ital. j. food sci., vol 28, 2016 455 figure 1: categories of processed food products against markers by box plots graphics. 3.1. processed foods in the vending company tables 1-5 show the nutritional results calculated from the foods processed in the vending company, which were subsequently sold on the market in vending machines. note that the products in these tables prepared with tuna, chicken, crab and watercress also contained mayonnaise.   ital. j. food sci., vol 28, 2016 456 table 6 shows the nutritional value of each bread/croissant type (with no added ingredients) used to make various types of processed products. table 6: nutritional assessment – types of bread/croissant. product energy value (kcal) protein (g) total fat (g) saturated fat (g) total carbohydrate (g) dietary fibre (g) sodium (mg) croissant (100 g) 355.0 8.0 13.4 7.7 49.4 2.3 390.0 white bread baguette (85 g) 219.3 8.2 0.9 0.2 43.0 3.0 484.5 wholemeal baguette (85 g) 187.9 6.0 2.5 0.5 32.3 6.4 467.5 wholemeal sandwich (50 g) 129.5 5.5 1.5 0.4 22.0 3.0 265.0 white bread sandwich (50 g) 113.0 3.6 2.2 0.4 21.2 2.1 300.0 200.9 6.3 6.8 1.8 33.6 3.4 381.4 σ 96.3 1.9 6.8 3.3 12.5 1.7 97.8 : mean of all types of bread/croissant. σ : standard deviation of all types of bread/croissant. to analyse the results presented in tables 1-6, it was necessary to compare them to the reference daily value (dv) values obtained from the food and drug administration (fda) (fda, 2013), which are provided in table 7. for sodium, it should be noted that on january 31, 2013, the world health organization (who) considered that its reference value for daily intake for adults should be < 2000 mg (2013). table 7: reference daily intake values (fda, 2013). in the analysis of the results shown in table 6, the high calorie intake (355 kcal) in croissants was checked and compared to the other products. their energy value more than tripled that of white bread sandwiches. a large quantity of total and saturated fats in croissants was also noted compared with other products. a croissant had about 20 times more saturated fat than a white bread sandwich. sodium levels were generally high: a white bread baguette contained 484.5 mg of sodium, which means that according to the who (2013), the consumers who ate this product with no added ingredients had already eaten 24.2% of their daily sodium needs. when interpreting the results of the nutritional calculations made with all the bread/croissant types with added ingredients, those foods with a high sodium content and lots of calories also presented high levels of saturated fat. when we focused on the different wholemeal baguette types (table 1), we saw that the average sodium levels surpassed almost half the daily who recommendations (2013) ( = caloric intake (kcal) protein (g) total fat (g) saturated fat (g) total carbohydrate (g) dietary fibre (g) sodium (mg) 2000 50 65 20 300 25 2400   ital. j. food sci., vol 28, 2016 457 1,066.3 mg), while the baguettes that contained salami contained more than one third of the sodium level according to the same recommendations. another point was that two baguette types (salami and cheese / chicken and cheese) contained more than 50% of the dv (fda, 2013) for saturated fat. two other types of wholemeal baguettes had a high protein content: the chicken and cheese baguette (52 g) and the pork loin and cheese baguette (41.2 g). when choosing a wholemeal baguette type, e.g. for a mid-morning snack, tortilla was the best option since it contained the least calories (291.5 kcal), less saturated fat (1.8 g) and its sodium content (885.7 mg) was below the average value (1,066.3 mg) of other types of wholemeal baguettes. the sodium levels of the white bread baguettes (table 3) were high ( = 997.7 mg), which also occurred with the wholemeal baguettes. two types of white bread baguettes (sobrasada and cheese / salami and cheese) surpassed 50% of the dv (fda, 2013) for saturated fat. protein content was high in the pork loin and cheese (45.2 g) and the chicken and cheese (40.3 g) sandwiches. when it comes to choosing a white bread baguette, it is important to avoid the sobrasada and cheese and salami and the cheese kinds because, apart from having a high saturated fat content, they contained a large amount of sodium which, for the salami and cheese baguette, represented 81.12% of the who’s daily recommendations (2013). no major differences sandwiches were found between wholemeal and white bread and their average nutritional values were similar (tables 4 and 5). perhaps wholemeal sandwiches make consumers feel fuller given their high fibre content. the high energy value of the wholemeal salami and cheese sandwich was noteworthy (516.6 kcal), as was its sodium content (1,623.8 mg), compared to the other wholemeal and non-wholemeal sandwiches. sandwiches that were not made with wholemeal bread stood out for their high sodium content: turkey and cheese (1,446.6 mg) and tortilla with ham and cheese (1,272.8 mg). croissants (table 2) had very high values of sodium, saturated fat and calories. only one (tortilla) of the twelve available croissant types contained less than 50% of the dv (fda, 2013) for saturated fat. its average energy intake value was 633.6 kcal. the croissant that contained tomato, salad, turkey, tuna and corn should be noted as its sodium content was 2,676.3 mg, which surpassed the who’s 676.3 mg daily recommendation (2013), and its calorie content was 758.8 kcal, which represented 37.94% of the recommended daily intake (fda, 2013). regarding the statistical relationship among the different products grouped by category, table 8 summarises the seven markers analysed as medians and iqr in each food group. all the markers (energy, proteins, total fat, saturated fat, carbohydrates, fibre and sodium) showed significant differences among the five food groups (p<0.05). sandwiches (wholemeal and white bread) had the lowest energy levels, while croissants had the highest. the croissants showed a significantly larger amount of total and saturated fats and carbohydrates than the other studied food groups, and also protein when compared with both sandwich types (white bread and wholemeal) (table 8). the carbohydrate values in both sandwich types were significantly lower (table 8). the larger amount of fibre content detected in the wholemeal baguettes stood out from the other foods, while the sodium content levels in the white bread and wholemeal sandwiches were significantly lower compared to the other food groups analysed herein (table 8). we highlight the high sodium content in the baguettes compared to the other foods (table 8).   ital. j. food sci., vol 28, 2016 458 table 8: comparison of nutritional markers between groups of processed foods. data are medians (iqr). different superscript letters a, b, c, d indicate significant differences (p < 0.05). (*) kruskal-wallis test. based on all these findings, we once again stress the very high content in sodium and saturated fat of many processed foods, and their high calorie intake. croissants are the least suitable choice because they contain high saturated fat compared to other foods, and they also contain many calories and a lot of sodium. sandwiches are the most suitable snack choice. choosing a food type by considering the ingredients that it contains is important to help avoid eating the foods that contain sausage meat because its sodium, saturated fat and energy values are very high. 3.2. chocolate and industrial pastry products the nutritional values that correspond to chocolate and industrial pastry products on sale in vending machines are included in tables 9 and 10. table 9. nutritional assessment – chocolates. chocolate energy value (kcal) protein (g) total fat (g) saturated fat (g) total carbohydrate (g) dietary fibre (g) sodium (mg) 1 280.0 4.0 14.0 5.0 35.0 1.0 140.0 2 245.0 4.9 12.7 4.9 29.4 8.6 24.5 3 233.0 3.0 12.0 0.0 29.0 0.0 0.0 4 213.0 3.0 11.0 4.1 26.0 0.8 118.0 5 134.0 1.1 6.8 0.0 18.2 0.0 0.0 221.0 3.2 11.3 2.8 27.5 2.1 56.5 σ 54.4 1.4 2.7 2.6 6.1 3.7 67.4 : mean of all samples of chocolate. σ : standard deviation of all samples of chocolate. wholemeal baguettes croissants white bread baguettes white bread sandwiches wholemeal sandwiches p* energy 427.9 a (369 ; 437) 653.1 b (591 ; 672) 396.3 a (378 ; 436) 311.6 c (285 ; 325) 286.3 c (285 ; 294) < .001 protein 15.9 a,b (14.5 ; 32.4) 18.6 a,b (13.3 ; 24.3) 17.6 a (14.2 ; 27.1) 11.6 b,c (9.1 ; 19.2) 10.3 c (8.2 ; 11.0) .044 total fat 20.4 a (13.1 ; 23.6) 38.0 b (34.0 ; 40.4) 13.8 a (9.1 ; 16.6) 17.4 a (16.2 ; 20.0) 16.8 a (14.4 ; 19.2) < .001 satured fat 6.8 a (3.6 ; 9.1) 11.8 b (11.2 ; 12.2) 5.8 a (2.3 ; 7.3) 3.6 a (2.8 ; 5.3) 2.7 a (2.3 ; 3.1) < .001 carbohydrates 33.9 a (33.0 ; 36.5) 53.3 b (50.3 ; 58.1) 44.0 c (43.2 ; 45.7) 22.6 d (21.9 ; 25.2) 24.0 d (23.7 ; 24.5) < .001 fibre 6.5 a (6.4 ; 7.0) 2.9 b (2.5 ; 4.5) 3.0 b (3.0 ; 3.7) 2.2 c (2.1 ; 3.1) 3.2 b,c (3.1 ; 3.4) < .001 sodium 885.7 a (737 ; 1451) 693.4 a (647 ; 790) 829.9 a,c (729 ; 1293) 494.1 b (467 ; 752) 428.0 c (402 ; 429) .004   ital. j. food sci., vol 28, 2016 459 table 10. nutritional assessment – industrial pastry products. industrial pastry product energy value (kcal) protein (g) total fat (g) saturated fat (g) total carbohydrate (g) dietary fibre (g) sodium (mg) 1 240.0 3.5 16.0 11.5 19.0 3.0 105.0 2 237.0 5.0 13.8 7.8 25.2 2.3 383.0 3 223.0 4.8 8.4 1.7 31.5 1.3 0.1 4 175.0 2.1 7.2 1.3 26.3 1.4 172.0 5 132.0 1.5 7.0 1.1 15.4 0.3 0.1 201.4 3.4 10.5 4.7 23.5 1.7 132.0 σ 46.7 1.6 4.1 4.7 6.3 1.0 158.2 : mean of all samples of industrial pastry products. σ : standard deviation of all samples of industrial pastry products. all these foods obtained similar nutritional composition values. it should be noted that most of the carbohydrates in these foods were sugars. back in the 1990s, hunter (1992) voiced concern about the relatively low value of the nutrients and the high sugar content of the foods most frequently chosen from vending machines, including snacks like those cited in this section of our study. table 11 shows the relationship of the consumption percentages for all the foods sold in vending machines over a 12-month period. these results reflect a consumer preference for snacks, i.e. chocolate (29.04%) and industrial pastry products (33.84%), and a very low consumption of baguettes (4.18%). a recent study (byrd-bredbenner et al., 2012) detected snacks in vending machines with a high content of fat and sugar, lots of calories and very little fibre. these results coincide with the products studied herein. in fact since the 1970s, the reduced nutritional value of most products sold in vending machines (cheney, 1974; ezell et al., 1985; french et al., 2010; kubik et al., 2011; lawrence et al., 2009), their high sugar content (ezell et al., 1985; hruban, 1977; koehler et al., 1977; shearer et al., 1980) and their high energy contribution (finkelstein et al., 2008; french et al., 2010; kibblewhite et al., 2010; naylor et al., 2010; pasch et al., 2011) have been known. table 11: percentage of consumption from vending. consumption during a 12-month period category total baguettes 4.18% wholemeal baguettes 22.83%* white bread baguettes 77.17%* chocolate 29.04% croissants 10.05% industrial pastry products 33.84% sandwiches (white bread and wholemeal) 13.96%   ital. j. food sci., vol 28, 2016 460 these values correspond to the breakdown in the category baguettes. so the total % of baguettes consumption was 4.18%, and 22.83% for baguettes made with wholemeal bread and 77.17% for white bread baguettes. 3.3. future perspectives and recommended strategies based on the similarity between the results of the above-cited recent studies and those obtained in this paper, the implementation of strategies to promote the sale and consumption of healthier foods is believed necessary. to go about this, it is advisable to adopt measures like those proposed by kocken et al. (2012). their study increased the availability of foods in vending machines with a lower calorie intake, included labelling that highlighted nutritional properties, and also lowered the price of these foods. another measure to adopt could be to lower the prices of foods with a low fat content, as demonstrated in the study conducted by french et al. (2001). the impact of implementing these strategies, along with policies to encourage eating healthier foods, and the possibilities of improving the diet of a high percentage of the population would be strong, and the costs associated with treating a group of diseases caused by inadequate diet could lower. law 17/2011, of july 5, on food safety and nutrition already imposes new legislative terms to this problem by preventing the sale of foods with high contents of saturated fat, sodium and sugar in schools. this has been the first step to ensure that the general population becomes more aware of healthier food choices when choosing food from vending machines. 4. conclusions plenty of additional work is needed to enhance the nutritional quality and appropriate portion size of the foods offered in vending machines. to improve vending machine selections on university campuses, benchmark data like those reported herein can help stakeholders set priorities and work with decision makers on campuses to advocate healthy campus food environments, which include all food retail outlets. the ultimate goal of studies like the present one is to compel environmental changes that make healthy choices possible and easier. future research should also investigate the behavioural and financial impact of eliminating unhealthy snacks from vending machines. thus we propose that vending companies take steps to change the processed foods sold in their machines. it would be much more appropriate to provide these machines with foods that contain fewer calories, especially with much lower levels of saturated fat and sodium, compared to those currently available to vending users. in this way, the intake of these foods would be more in line with who recommendations (2013) and with the dv set by the fda (2013). we believe that awareness of governments and the adoption of policies to promote consumer intake of healthy food choices are essential measures to increase the amount of foods with an appropriate nutritional profile that meets these recommendations (who, 2013; fda, 2013). therefore, conducting further studies would help support and implement public health policies and environmental changes, which could improve healthy food access and availability in vending machines. acknowledgements the authors are very grateful to their families and friends and also to egas moniz – cooperativa de ensino superior, crl and universidad de las palmas de gran canaria for all the support provided.   ital. j. food sci., vol 28, 2016 461 references byrd-bredbenner c., johnson m., quick v.m., walsh j., greene g.w., hoerr s., colby s.m., kattelmann k.k., phillips b.w., kidd t. and horacek t.m. 2012. sweet and salty. an assessment of the snacks and beverages sold in vending machines on us post-secondary institution campuses. appetite 58(3):1143-51. cavaliere, a., de marchi, e. and banterle, a. 2014. healthy-unhealthy weight and time preference. is there an association? an analysis through a consumer survey, appetite 83:135-143. caruso m.l., klein e.g. and kaye g. 2014. campus-based snack food vending consumption. journal of nutrition education and behavior 46(5):401-405. cheney h.g. 1974. survey of foods and beverages in vending machines at dental schools. journal of dental education 38:392. dial (programa dial – programa de uso general y profesional para valoración de dietas y cálculos de alimentación). 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machine use and fast-food restaurant use are associated with sugar-sweetened beverage intake in youth. journal of the american dietetic association 106(10):1624-16. paper received february 11, 2016 accepted april 7, 2016 paper ital. j. food sci., vol. 27 2015 1 keywords: bioavailability, calcium salts, hamburgers technological and sensory properties of hamburgers enriched with calcium study of the in vitro bioavailability ana m. soto, m. luisa garcía and m. dolores selgas* dpto. nutrición, bromatología y tecnología de los alimentos, faculty of veterinary, university complutense, avda. puerta de hierro s/n. 28040 madrid, spain *corresponding author: tel. +34 91 3943745; fax: +34 91 3943743, email: selgar@vet.ucm.es abstract hamburgers were supplemented with three calcium salts (calcium gluconate cg, calcium lactate cl and calcium citrate-malate ccm). they were added in sufficient amount to that 100 g of hamburger gives 20 or 30% of the ca rda (1000 mg). their technological and sensory properties were studied. cg 30% gave the worst sensory properties and it was discarded. bioavailability of calcium depends on the type of salt used and the highest value was obtained with ccm (14.5%). for that, this salt is proposed as the most adequate for the enrichment of fresh meat products. 2 ital. j. food sci., vol. 27 2015 introduction meat and meat products are important to the human diet; they contain proteins with all nine essential amino acids of high biological value, accounting for 40% of total amino acids. they are an excellent source of bioactive compounds, including vitamins (b-complex), iron, zinc, phosphorus (fernández et al., 2005; weiss et al., 2010). the interest on the human health and the actual consumer tendencies, who prefer more nutritious food, have stimulated interest in developing meat products with bioactive compounds with attractive physiological activities (griguelmo et al., 1999; cengiz and gokoglu, 2005; decker and park, 2010). greater emphasis has been placed on strategies involving the addition of bioactive compounds with recognized health benefits, such as proteins, fibre, polyphenols, unsaturated fatty acids, probiotics or minerals (roberfroid, 2002; saiga et al., 2003; caceres et al., 2006; arhiara, 2006; jiménez-colmenero et al., 2006; decker and park 2010; alonso et al., 2010; zhang et al., 2010; khan et al., 2011). dietary minerals are essential for various physiological functions and they have been associated with the prevention of several diseases (menéndez-carreño et al., 2008; decker and park, 2010; zhang et al., 2010). calcium (ca) is one of the most important. it gives structural integrity to mineralized tissue preventing osteoporosis and contributing to the “bone health”. although interest in ca primarily derives from this role, it also plays other essential physiological roles in arterial hypertension, cellular function, skeletal muscle contraction, blood coagulation and enzymatic reactions as a co-factor (prince et al., 2006; straub, 2007, adluri et al., 2010). health authorities have recommended a ca daily allowance (rda) of 1000 mg for adults aged 19-50 (institute of medicine, iom, 2004) or 800 mg (directive 2008/100/ec), without concern for age. milk and dairy products account for much of the ca in the human diet, (75% of ca intake, aprox); only 16% comes from fish and vegetables and a 6-7% from mineral water (guéguen and pointillart, 2000; charoenkiatkiu et al., 2008). since meat and meat products are poor source of ca, supplementing them with ca salts could be a good option to increase its intake particularly those in which the consumption of milk can be a health problem. previous studies have examined the addition of ca to meat products, but mainly for reducing sodium levels (gimeno et al., 1998, 1999) and the final ca content was not sufficient to consider them as a source of this mineral. studies previously performed in our laboratory (cáceres et al., 2006), it was reported that calcium could be successfully added to both cooked and dry-fermented sausages, but until now, fresh meat products have been not assayed. in this way, the present work deals with the manufacture of fresh meat products (hamburgers) enriched with calcium. for that, three different calcium salts were assayed: ca gluconate (cg), ca lactate (cl) and ca citrate-malate (ccm). they are permitted in food (regulation ec no 1907/2006) and characterized because of their high bioavailability (korstanje and hoek, 2001). the technological and sensory properties of the hamburgers manufactured were studied and finally, a study of the ca bioavailability have been performed, using an in vitro static method to simulate the passage through the intestinal cell-membrane (glahn et al., 2002; perales et al., 2005; shiowatana et al., 2006). material and methods hamburger manufacture beef meat was obtained from a local abattoir and chopped in a grinder using a 3 mm plate (grinder c10, falsf co., spain).the ca salts (panreac, castellar del vallés, spain) were added separately to the ground meat in sufficient amount to give a final ca content of 20 or 30% of the rda (1000 mg/day) (iom, 2004). these calculations took into account the calcium content of the molecules and the purity of the ca salts, which was >98% based on the anhydrous formula (table 1). the ca salts were homogeneously distributed into the ground meat in a mixer (mainca, pamplona, españa). then, hamburgers were moulded into plates (10 cm diameter, 1 cm height) and kept under refrigeration (2 ºc) until analysis, less than 24 h. seven batches were manufactured: a control batch without ca and 6 batches added with cg, cl or ccm at two concentrations (20 and 30% rda). according to the type and ca salt amount, the batches were named as cg20, cg30, cl20, table 1 ital. j. food sci., vol. 27 2015 3 cl30, ccm20 and ccm30. the hamburgers manufacture was made in triplicate. hamburgers were cooked on an electric grill preheated to 180 ºc. they were placed for 2 min on each side, sufficient time to achieve a temperature of 60 ºc in the inner of the hamburger and a good final degree of doneness (thornberg, 2005). temperature was controlled using a digital thermometer (testo mod 735, barcelona, spain). physico-chemical analyses water activity was determined with a decagon cx1 dew point hygrometer (decagon devices, pullman, wa, usa). the ph was measured using a crison 2001 ph meter using a glass electrode and according to the aoac (2011). waterholding capacity (whc) was tested according to zamorano and gambaruto (1997). for that, a sample of 0.1 g was placed on a filter paper (whatman no. 2), sandwiched between translucent plastic plates, and pressed for 1 min. the meat area and liquid area on the filter paper were measured with a planimeter. the following formulae were applied for whc: whc= [area of liquid (cm2) area of meat (cm2)] x [9.47/moisture in sample (mg)] * 100 the measurements were made in quintuplicate and the final result was the average value. colour analysis colour was measured at room temperature on the surface of raw hamburgers, using a chroma meter cr-200 colorimeter (minolta co., osaka, japan) according to the space colour cie l*a*b* system and calibrated with a rose tile (l* 44.88, a* 25.99, b* 6.67). a d-65 illumination source was used. l*, a*, b*, hue angle (tonality) and saturation index (vivacity) were estimated according to artés and mínguez, (2002). for each batch, twenty five measurements were taken. texture analysis textural properties were determined using a texturometer stable micro system mod. ta.xt 2i/25 (surrey, uk). texture profile analysis (tpa) was performed on central cores of cooked hamburgers which were compressed twice to 50% of their original height. a cylindrical probe (2.5 cm diameter) of aluminium was used for the assay. the following parameters were determined: hardness (n), springiness (cm), cohesiveness (ratio), adhesiveness (n s), gumminess (n) and chewiness (n cm) (bourne, 1978). shear force (n) and work of shearing (n s) were estimated using a warner-bratzler blade. in both tests, the samples were 1 cm high and 2.5 cm in diameter; the crosshead speed was 2 mm/s. all determinations were carried out in quintuplicate for each batch. sensory analyses the taste panel consisted of forty untrained assessors selected according to their eating habits, acquaintance with the product to be analyzed and sensitivity, as well as the reproducibility of their evaluations. first, an anchored descriptive analysis was performed in which the assessors evaluated the similarity between the external appearance of the enriched raw hamburgers and the control batch. this test was performed under a d-65 illumination source using a 5-point descriptive scale, in which the value of 3 points corresponded to the control batch. the value of 1 point meant much worse than the reference; 5 points, much better than the reference; 2 and 4 points were intermediate values. three series were prepared: control-cg20; control-cl20-cl30 and control-ccm20-ccm30. the series were presented to the panellists with 30 min of difference to avoid subjectivities. after this test, a hedonic test was performed with cooked hamburgers. in this case the taste panel consisted by 15 trained panellists. they were in individual booths constructed according to iso dp 6658 (iso, 1985), under white fluorescent light. the assessors evaluated different attributes (odour, colour, texture, taste and overall acceptability) using a 10 cm non-structured scale (0 = extremely dislike and 10 = extremely like). two sessions per day were carried out with an interval of at least 1 h between them to avoid panellist fatigue. unsalted crackers and room-temperature water were provided to clean the palate between samples. the hamburgers were cooked and served in transparent petri dishes. in each session three randomly selected hamburgers were served. bioavailability calcium bioavailability was studied using a static in vitro test that simulates the gastric and intestinal phases of the digestion process according to the methodology of shiowatana et al., (2006). all enzymes were from sigma-aldrich (steinheim, germany) and the reactives from panreac (barcelona, spain). the samples were 10 g of cooked hamburgers which were homogenised (polytron®, littau-luzerne, switzerland) with 50 ml of 0.2 m phosphate buffer. the ph was adjusted to 2.0 with 5 n hcl. to simulate the gastric phase, 0.33 ml of suspension of pepsin (0.16 g pepsin (ec 232-629-3) per ml 0.1 n hcl) was added. then, the samples were incubated in a shaker (130 rpm) at 37 ºc for 2 h (thermo scientific maxq4000, iowa, usa). to simulate the intestinal phase, 20 g of the gastric digest was mixed with 5 ml of pancreatin-bile conjugate, [0.2 g pancreatin from porcine pancreas (ec 232-468-9) and 1.25 g porcine bile extract (ec 232-369-0) in 50 ml 1 m 4 ital. j. food sci., vol. 27 2015 nahco 3 ]. the ph was adjusted to 7-7.5 with 2 m nahco 3 and the total volume of the digest was measured. distilled water (25 ml) and the same volume of 2 m nahco 3 determined before were added inside to a cellulose dialysis tube with a molecular weight cut-off of 12000-14000 da and a diameter of 25 mm (sigma aldrich, steinheim, germany). the tube was introduced into a flask containing other 20 g aliquots of the gastric digest and it was incubated at 37 ºc in a shaker (130 rpm) until the ph reached a value of 5. then, 5 ml of pancreatin-bile conjugate was added to the gastric phase and it was incubated again during 2 h at 37 ºc. after the dialysis process, the dialyzed calcium (inside the dialysis tube) and the non-dialyzed calcium (outside the dialysis tube) were determined. this last one, beside with the calcium in the solid delivery, corresponds to the calcium that would be eliminated. the difference between the amount of calcium detected in these two phases and that determined in the gastric phase was considered as the calcium remaining in the solid residue (solid delivery) and would be eliminated with the feces. it was calculated by difference. the percentage of bioavailability was expressed as follows: bioavailability (%) = [dialyzed ca (mg)/ ca sample (mg)] *100 calcium determination calcium levels were determined according to ikem et al., (2002). for microwave digestion, 1.0 g of each sample was subjected to an acid digestion with 6 ml of hno 3 and 2 ml of h 2 o 2 (suprapure, merck) in a microwave digestion system and diluted to 10 ml with deionised water (milli-q, millipore). blank digestion was carried out in the same way. digestion conditions for the microwave system were the following: 2 min 250 w, 2 min 0 w, 6 min 250 w, 5 min 400 w and 8 min 550 w. a perkin elmer dv 3300 inductively coupled plasma-optical emission spectrometry (icp-oes) was used to analyze the calcium level in the digested samples. bovine muscle bcr no 184 was used as reference (cáceres et al., 2006). experimental values obtained were in the range of the certificate value with its uncertainty. all determinations were carried out in duplicate. statistical analyses results were statistically analysed by twoway anova, which factors were represented by type and concentration of calcium salt. values of p<0.05 were considered to be significant. the effect of the panellists in the sensorial responses was removed by rescaling all the scores given by each assessor from 0, which represents the minimum score used by a given assessor, to 10, which is the maximum score used by that assessor. after rescaling, the effect of the assessors in the sensorial responses was analysed by a two factor analysis of variance according to a randomised balanced block experimental design. the factor represented the type and percentage of calcium salt, while the block variable had 15 levels representing each of the panellists that collaborated in the trials.  the f test showed that the effect of the panellists was not significant (p > 0.05).  in consequence, the final model considered of only 1 factor (one-way anova) representing the calcium salt content. statistical analyses were performed using the statgraphics centurion xvi.i (statistical graphics corporation, herndon, va, usa). results and discussion physico-chemical parameters table 2 showed the results of the a w and ph of the batches manufactured. the a w of the reminded batches ranged between 0.945 and 0.989 and no significant differences were observed between control and calcium added batches. cl batches showed the lower values. the lower ph values corresponded to the cg20, cl20 and cl30 batches, which showed a ph of 5.70, 5.55 and 5.50, respectively. they differ significantly to the reminder batches. according to the fda (fda, 2014) these values assure the safety of these meat products. gc30 values are not showed due to its technological attitude like it was explained in the following section (colour parameters). whc showed similar values in all the batches and ranged between 36.0-39.0%. the addition of whichever calcium salts supposed no significant changes in the whc (p>0.05). the values of all these parameters are according to those reported for several authors in raw table 2 ital. j. food sci., vol. 27 2015 5 or ground meat (lawrie, 1998; lee et al., 1998; poulanne and halonen, 2010). colour analysis the worse results were obtained with the cg batches. the cg crystallized in contact with the water of the meat and formed visible small white crystals that appeared homogeneously distributed in the hamburger. these crystals gave an external appearance different to that usually expected by the consumers in a commercial hamburger. it was especially striking at the maximum concentrations (cg30) because the crystals were larger and numerous. for that, it was considered that this batch didn’t have enough quality and it was discarded for this and the following tests. batch with cg20 also had crystals, but they were smaller, as white dots. therefore, it was considered adequate for continuing the experiment, although its visual appearance would be compromised. colour parameters (l*, a* and b*) were determined in raw hamburgers (table 3). the differences observed were related to the type of salt added. lightness was very similar in all the batches without significant differences (p<0.05). however, it can be observed as the l* parameter was slightly higher in batches manufactured with cl, while, the lower values corresponded to those manufactured with ccm. it is important to remember that cl batches were those that showed the lower ph values. the relationship between color and ph is widely accepted (mancini and hunt, 2005) and several authors (kim et al., 2006; mancini and ramanathan, 2008; nair et al., 2014) have reported specifically the effects of lactate on meat color. these authors proposed that lactate plays an indirect role in color stability by generating nadh, which is subsequently used to maintain reduced forms of myoglobin, increasing in this way, the stability of meat colour. the values of redness (a*) and yellowness (b*) were similar between the control and calcium added batches. in relation to the redness, the lower values were observed in the ccm batches and the same occurs in the yellowness (b*). the higher value was recorded in the batch cl20 but it seems to be an inconsistent data. it was described that the haem pigment contents are mainly related to a* and, consequently, it is considered as the most important parameter in meat, while redox state influenced b* (mancini and hunt, 2005). this parameter reach less importance because colours represented (blue and yellow) are not typical or intuitively related to meat. according to these authors, and giving greater relevance to the a* parameter, it can be concluded that the colour is very similar in all the batches, although cl20 and ccm30 batches were the only ones that showed any difference. hue angle and saturation index behaved in a similar way and these batches showed again, the only differences in relation to the remainder ones. the colour lecture of hamburgers did not reflect the presence of the small white crystals due to their size. it is important to take into account, firstly, that the colorimeter project the light from the probe, which has 1 cm of diameter and secondly, that the crystals had a much smaller size. for that, the colorimeter read the colour of the meat and thus the data of cg20 and control batches (without calcium) were similar, without significant differences. the colour of cooked samples (data not shown) was very similar in all batches without exceptions. it has been described that during heat treatment, the meat colour changes from red to grey-brown (maillard reaction) and increase table 3 6 ital. j. food sci., vol. 27 2015 the opacity when the internal meat temperature is between 45 °c and 67 °c ( pakula and stamminger, 2012). in this way, tornberg (2005) reported that the increase in meat opacity is related to the myosin desnaturation, which starts at about 35 °c. above 50 °c, the myosin molecules are completely coagulated and the meat appears opaque. textural analyses textural parameters were determined in cooked hamburgers (table 4). it was observed that in both cl and ccm batches, the higher the calcium amount, the greater the hardness was. this is probably because calcium, which is a divalent cation, establishes bonds between meat proteins, mainly with myosin. this favors the formation of a stronger network which leads to the highest firmness (damodaran, 2008). according to tornberg (2005) it could be also due to the relationships stabilized between calcium and meat proteins, partially denatured by the cooking. this last one favors the formation of a more compact network that increases the hardness. springiness was quite similar in all the calcium enriched batches and shown higher values to batch control. according to belitz et al., (2009), the increase of the ionic strength leads the extraction to the surface of the particles of minced meat yielding sticky exudates. during heating, the proteins interact between them, yielding a structure consist on a protein gel that can modify the springiness of the cooked products. (tornberg, 2005). adhesiveness and cohesiveness parameters did not change significantly (p>0.05). gumminess and chewiness behaved as the hardness because they are secondary parameters dependent on it. so, the lower values corresponded to the cg which was the batch which showed the lower harness the results obtained in the shear test showed the same tendency than in the tpa and so, table 4 table 5 ital. j. food sci., vol. 27 2015 7 the principal differences were an increase of the work of shearing related with the calcium amount added, independently to the salt added (data not shown). sensory analyses table 5 shows the results of the anchored descriptive analysis, performed with raw hamburgers. control batch was the reference and it was awarded a score of 3 in the 5 points scale used. the panellists evaluated the visual appearance of enriched hamburgers in comparison to the control. the worse results were obtained with the cg20 batch, mainly due to the presence of small white crystals (see colour section). in this case, the number and the size of the crystal were lower than in the cg30, but they were sufficient to modify negatively the appearance of the hamburgers. cl30 batch was the best evaluated, even more than the reference (p<0.05). this result is according to the instrumental colour measurement (table 3) in which this batch was the one that had the greatest the higher l* and hue angle. table 5 also shows the results of the hedonic test performed with cooked hamburgers. odour and colour were well-evaluated in all the cases obtaining similar values in all batches (p>0.05) (data not shown). the texture achieves punctuations higher than 6 except the batches enriched with ccm20 and ccm30 which achieved scores of 5.79±1.78 and 5.90±1.30, respectively. significant differences were observed only between cl20 and ccm20, but, in general terms it can be observed a great similarity between the enriched batched and the control batch. however, the presence of calcium salts influenced negatively on the taste and so, independently of the type of salt or the amount added, all the enriched batches achieved punctuation lowers than the control batch. significant differences were observed between control and cl30 batches; the panellists described this last batch as slightly more acidic. the overall acceptability values behaved similar than the taste ones and, consequently, the taste seemed to be the most influential parameter. so, the punctuations obtained by the acceptability were very similar in all the enriched batches, although it was observed that the lowest values were those for batches cg20 and cl30. the control batch reached the best score although the difference was not significant with cl20 and ccm30 batches. in any case none of the batches were discarded because all of them exceeded the value of 5 points. bioavailability in order to facilitate the calcium analysis, it was decided to perform the bioavailability study using the batches manufactured with the higher concentration: cl30 and ccm30. the cg batches were discarded for this study because of the low sensory quality. table 6 shows the results obtained. the bioavailability was determined in the cooked hamburgers. the calcium amount determined was slightly higher than the expected, a 20% approximately. this increase could be due to the loose of water during the cooking as it has been reported by pan and shing (2001). these authors established that the water losses during ground beef cooking (60 ºc, 2 min) is closed to 15-20%. the final calcium amount determined was sufficient to give the calcium levels proposed in our objective (30% rda). in the gastric phase was detected all the calcium presented in the cooked hamburgers. the dialyzed calcium was close two fold higher in the hamburgers enriched with ccm30 than those with cl30. the solid deliveries calculated were 171.29 mg in the cl batch and 129.12 mg in the ccm one. this difference represents a calcium loss close to 47% and 34%, respectively. it could be due to that the ccm interferes with proteins during the cooking and contributes, as it has been described above, to the formation of a more compact protein network that could difficult the activity of pancreatin-bile complex, and to favor the retention of a higher calcium amount. applying the formula (see material and methods section), the bioavailability percentage was 7.73% for cl and 14.55% for ccm. the variability in calcium bioavailability is table 6 8 ital. j. food sci., vol. 27 2015 consistent with numerous studies. one of the first bioavailability assays performed (morrisey and flynn, 1972) reported that the calcium absorption from cow’s milk ranged from 21 to 45% in healthy human adults. recker et al., (1988) described that the absorption of calcium carbonate from enriched whole milk, chocolate milk, yogurt, imitation milk and cheese ranged between 21-26% in postmenopausal women. kruger et al., (2003) concluded that calcium bioavailability was similar in milk fortified with calcium carbonate or milk calcium. in a study performed by sittikulwitit et al., (2004) with different calcium salts and milk powder, bioavailable calcium ranged between 28.5 and 58.7%. other authors determined calcium absorption from infant milk formula of 39% (nelson et al., 1996) and 29% from enriched orange juices (gonelli et al., 2007). cilla et al., (2011) reported calcium efficiency uptake values from 10.47-19.82% for different milk-based fruit beverages and perales et al., (2005) found calcium bioavailability to be 5.0-31% for infant and adapted milks. the differences reported between the values of calcium dialyzable have be also attributed to the methodology applied, particularly with regard to the amount and activity degree of the enzymes used, the ph values and incubation times during the gastric and intestinal phases. in this way, van der hee et al., (2009) reported that calcium absorption from food depends on numerous factors, causing a range broadly from 15 to 44%. so, bioavailability described in this paper is according to the data collected by other authors. furthermore, our results showed that ccm would be the most bioavailable salt from hamburgers, which suggests that is the most appropriate for the enrichment of fresh meat products. sittikulwitit et al., (2004) reported that the dialysis rate of calcium (calcium bioavailable) in milk powder is 28%. taking into account that 200 ml of milk would give 240 mg of calcium, the bioavailable amount would correspond to 67.2 mg. according to our data, if 100 g of hamburger enriched with ccm at the concentration of 30% idr leaves bioavailable 52.66 mg (table 6), one commercial hamburger of 120 g, would leave 63.2 mg calcium, a similar quantity to that reported for milk. conclusions ccm and cl can be adequate for enriching fresh meat products (hamburgers). they can be added in sufficient amount to give a theoretical calcium level of 30% of rda. gc crystallizes and it is not recommended for its use. the calcium bioavailability from ccm enriched hamburgers was the highest, close to 15%. this allows that one hamburger of 120 g enriched with ccm 30% rda, would leave bioavailable 63.2 mg calcium, a similar calcium amount to 200 ml of milk. consequently, ccm is proposed as the most adequate salt and the hamburger manufactured with it could be considered as a source of calcium, according to the regulation nº 1907/2006 of the european parliament. acknowledgments this work was supported by the projects consolider-ingenio 2010 (ref. csd2007-00016), and group of investigation bsch-ucm no. 920276 (ref. gr35/10a). thanks to the cai of geocronología y geoquímica isotópicas (ucm) for technical support in calcium determination. references adluri r.s., zhan l, bagchi m., maulik n. and maulik g. 2010. comparative effects of a novel plant-based ca supplement with two common ca salts on proliferation and mineralization in human osteoblast cells. molec. cell. biochem. 340 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fax: +39 0651494550 e-mail address: alessandra.durazzo@crea.gov.it abstract this study aims to characterize grains and derivatives of solina, an italian traditional winter soft wheat (triticum aestivum l.). total polyphenol content and antioxidant properties were investigated in grains, whole flours and bread, both in aqueous-organic extracts and residues. results showed the important contribution of hydrolysable polyphenols, isolated in the residue of the aqueous-organic extract, to antioxidant properties (over 90% in grains and derivatives), and the key role played by milling and baking in antioxidant properties. moreover, the analysis of potentially bioactive antioxidants remaining in the residues showed to be required for a comprehensive determination of antioxidant capacity in cereals. the study highlights that solina represents a valuable italian traditional wheat cultivar with proved antioxidant properties. keywords: antioxidants, total polyphenol content (tpc), traditional foods, soft wheat solina, whole wheat flours, bread   ital. j. food sci., vol 28, 2016 222 1. introduction nowadays the traditional character of foodstuffs is a value-adding characteristic which contributes to defining quality thereof and is also often associated with positive health benefits. traditional foods, in fact, not only play an important role in the cultural and nutritional food patterns of every country because their cultivation or preparation have been passed down from generation to generation, and/or because they have been consumed locally for years and become part of daily life in communities; they are often considered healthy and wholesome and are also often associated with positive health benefits because of their chemical profile. solina is a traditional italian winter soft wheat (triticum aestivum l.), typical of the province of l’aquila, in the abruzzo region, within the national park of gran sasso. its cultivation in abruzzo dates back to 1500 a.c. and there is evidence of a strong connection between this variety and the life of people from abruzzo who keep appreciating it for the sensory properties it provides to bakery products. it is grown in mountainous areas and shows a high adaptability to marginal areas, as well as resistance to cold. that ancient cultivar was, in fact, defined by last century agronomists “wheat hybernum”. solina also shows a high stability in yields, and according to agronomists it is also compatible with organic farming methods because it does not require a large amount of nitrogen; moreover it is able to tolerate and compete strongly with weeds for its size and tillering capacity (porfiri et al., 2001). in this study, grains of the solina cultivar, whole flours and bread samples thereof were examined in order to evaluate the phenolic components and the antioxidant properties of this traditional crop. some technological and chemical parameters of grains were also considered, so as to identify a comprehensive portrait of this italian wheat cultivar. according to literature, grains, in particular whole grains, provide a wide range of nutrients and phytochemicals that work in synergy to maintain human health (liu, 2007; okarter and liu, 2010). whole grain consumption has been associated with reduced risk of chronic diseases, such as cardiovascular diseases and cancer (schatzkin et al., 2007; mellen et al., 2008; aune et al., 2013). these beneficial properties have been rediscovered by consumers and producers (arvola et al., 2007). as the content of bioactive molecules is affected by genetic and growing conditions in grains as well as by processing conditions in foods (slavin et al., 2000), samples of solina grains, flours and bread formulated with this soft wheat cultivar were studied. 2. materials and methods 2.1. sampling analysis were performed on grain, whole wheat flour and bread samples. grains were supplied by three farms located in three different spots of the same area: castelvecchio subequo, capestrano and introdacqua. according to sample location they were labeled as solina 1, solina 2 and solina 3, collected respectively at castelvecchio subequo, capestrano and introdacqua. the grains of each collected sample were divided in two aliquots. an aliquot was milled by a water cooled mill (janke & kunkel ika labortechnik, staufen, germany) and stored at 4°c until chemical analyses were performed. an aliquot was tempered for 24 h to 15.5% moisture content, then milled in an experimental mill mlu-202 bühler (switzerland)   ital. j. food sci., vol 28, 2016 223 equipped with three breaks and three reduction rolls and six screens to obtain whole mill flours. solina bread was also studied. five loaves of bread (four long loaves and one round loaf), produced according to a traditional bread making process, were purchased in bakeries located in the same area of grain collection. they were coded as reported in table 1. table 1. code, description and some chemical parameters of bread samples*. code description composition moisture (%) protein (%) ash (%) a bread long loaf (900 g) 9.9 11.5 1.90 b bread long loaf (1660 g) 7.8 11.2 2.74 c bread round loaf (900 g) 10.8 12.5 2.96 d bread long loaf (810 g) 11.0 11.6 2.65 e bread long loaf (1700 g) 10.4 11.6 1.81 *data are expressed as mean of replicate measurements (n=3); differences within measurements were in the range reported in the method. 2.2. chemicals and standards common reagents and standards were purchased from sigma–aldrich srl (milan, italy), extrasynthèse (genay, france), carlo erba (milan, italy) and bdh laboratory supplies (poole, uk) and their purity degree was chosen according to the analysis to be performed. double-distilled water (millipore, milan, italy) was used throughout the study. 2.3 general chemical analysis grain quality was determined after removing impurities, i.e., broken grains, heatdamaged grains, shriveled grains, vegetable impurities, vitreous grains, on 100 g seeds by visual evaluation. test weight (tw), thousand kernel weight (1,000 kw) and kernel hardness (kh) were assessed. tw was determined by a shopper chondrometer equipped with 250 ml cylinder. 1,000 kw was determined by counting and weighing 1,000 kernels, while kernel hardness (kh), diameter and moisture of grains were determined by the skcs 4100 apparatus (perten instruments, sweden) according to the standard method aacc 55-31 (2003). total proteins were determined by kjeldahl method according to icc standard method no 105/2 (2003) using 5.70 as specific conversion factor. ash content was determined on the inorganic residue remaining after the incineration of the sample in a muffle furnace at the temperature of 900°c according to the icc method no 104/1 (2003), while moisture was determined according to icc standard method no 110/1 (2003). moisture is reported as g/100 g fresh weight (f. w.), while both protein and ash content are reported as g/100 g dry matter (d. m.).   ital. j. food sci., vol 28, 2016 224 2.4. sample extraction for evaluation of total polyphenol content (tpc) and antioxidant properties total polyphenols were extracted as described by durazzo et al. (2013), with some modifications. extractable polyphenols were determined on aqueous-organic extracts, while non extractable polyphenols were determined on solid residues of aqueous-organic extraction. in details, some non-extractable polyphenols, i.e. hydrolysable polyphenols, were isolated and determined following a specific and suitable acid hydrolysis as reported below. extractable polyphenols about 3.0 g, 3.5 g, 4.5 g of grains, flours and bread, respectively, were placed in a test tube and added with 20 ml of acid methanol/water (50:50 v/v, ph= 2). tubes were swirled at room temperature for 3 min, then mildly shaken for 1 h in a water bath at room temperature as well. tubes were centrifuged at 2500 x g for 10 min, and the supernatant was recovered. then, 20 ml acetone/water (70:30, v/v) were added to residue and the extraction repeated as reported above. methanolic and acetonic extracts were combined and centrifuged at 2800 x g for 15 min. the resulting mixture was then used for the determination of total polyphenol content and antioxidant properties. hydrolysable polyphenols the residue left after the above described extraction was dried in a ventilated oven at 25°c. 250 mg, 300 mg, 400 mg of grain, flour and bread residue, respectively, were mixed with 20 ml of methanol and 2 ml of sulfuric acid (18 m). samples were gently stirred for 1 min and shaked at 85°c for 20 h in a water bath. samples were then centrifuged (2500 x g for 10 min), and the supernatant was recovered. after two washings with minimum volume of distilled water and re-centrifuging as necessary, the final volume was taken up to 50 ml. the tube was centrifuged at 2800 x g for 20 min and the resulting supernatant was used for the determination of total polyphenol content and antioxidant properties. 2.5. determination of total polyphenol content (tpc) the tpc was determined using the folin-ciocalteau procedure (singleton et al., 1999). briefly, appropriate dilutions of extracts were oxidised with folin-ciocalteau reagent, and the reaction was neutralised with sodium carbonate. the absorbance of the resulting blue colour solution was measured at 760 nm against an appropriate blank after 2 h of reaction at room temperature at dark. gallic acid was used as standard. 2.6. antioxidant properties evaluation the determination of antioxidant properties was carried out by frap (ferric reducing antioxidant power) assay according to methods of benzie and strain (1996) and pulido et al. (2000) and by using a tecan sunrise® plate reader spectrophotometer. the method is based on the reduction of fe3+-tptz (2,4,6-tripyridyl-s-triazine) complex to ferrous at low ph.   ital. j. food sci., vol 28, 2016 225 2.7. statistical analysis all analyses were performed in triplicate. data are presented as mean±standard deviation (s.d.). data obtained by official methods are presented as mean. statistica for windows (statistical package; release 4.5; statsoft inc., vigonza pd, italy) was used to perform oneway analysis of variance (anova). significant differences between grain and flours of the same origin were evaluated by the student’s t test. 3. results and discussions the inspection of grain impurities in the samples is an important control procedure necessary to verifying wheat quality and it is a preliminary step prior to the milling phase. the percentage of ripe grains were higher than 95% in all samples. this confirms the good quality of analysed grains. the values obtained for the main physical parameters (kernel hardness, test weight, thousand kernel weight, diameter) and the chemical composition (moisture, protein, ash) of grain samples are reported in table 2. as concerns physical parameters, no significant differences were found among grain samples. the values obtained for tw (74.1 80.2 kg/hl), 1,000 kw (>43 g) and grain diameter (2.11-2.47 mm) show that kernels are wholesome, sound, not sprout damaged. table 2. physical and chemical parameters of grains*. sample kh tw 1,000 kw diameter moisture protein ash (kg/hl) (g) (mm) (g/100 g) (g/100 g d.m.) (g/100 g d.m.) solina 1 19.70 74.1 44.6 2.47 12.4 13.1 2.04 solina 2 16.69 80.2 54.4 2.46 12.6 11.9 1.75 solina 3 33.70 79.0 43.4 2.11 12.4 13.7 2.00 *data are expressed as mean of replicate measurements (n=3); differences within measurements were in the range reported in the method. as regards kernel hardness, the data reported in table 2 confirm our samples belong to the “soft” species and their variation may depend on the different growing locations. according to pomeranz and williams (1990), in fact, the main factors that affect the grain hardness are growing location (soil type, elevation, planting type, irrigation, fertilizers and cultivation practice), growing season (precipitation and temperature during maturation and post ripening), storage conditions, protein content, moisture and kernel size. regarding the main chemical parameters, significant differences were observed: solina 2 showed the lowest protein and ash content. those data show, therefore, that environmental and growing factors not only influence hardness, but also chemical parameters such as protein and ash content. in table 3, total polyphenol content (tpc) and antioxidant properties (frap) are reported for grains and whole wheat flours. values refer to aqueous-organic extracts and residues. in grains, tpc values ranged between 165.57 and 183.75 mg/100g d.m. in aqueous-organic   ital. j. food sci., vol 28, 2016 226 extracts, and between 1084.42 and 1325.32 mg/100g d.m. in residues, whereas the range for frap values was 6.34-6.60 !mol/g d.m. in aqueous-organic extracts and 85.06-109.12 !mol/g d.m. in residues. tpc and frap values in aqueous-organic extracts showed no differences between the grain samples supplied by the three farmers. on the other hand, as to the residue, the tpc resulted significantly different in the grain samples and the highest value was obtained for solina 1; the values for antioxidant properties in solina 1 were higher than those obtained for solina 2 and solina 3 that were, at their turn, comparable. as reported by several authors (adom et al., 2003; carcea et al., 2009) differences in the bioactive molecule distribution and antioxidant properties in wheat, as well as in foodstuffs in general, could be related to genetic factors, agronomic practices and environmental conditions. it is interesting to notice that in grains the frap values in residues are about 15 fold higher than in aqueous-organic extracts. this trend was also confirmed by the data obtained for tpc. the hydrolysable polyphenols, isolated in residue, belong to several classes of phenolic compounds, i.e. hydrolysable tannins, phenolic acids and hydroxycinnamic acids, and consist on ferulic acid, caffeic acid, sinapic acid, etc. these phenols are linked to carbohydrates and protein by covalent bonds, hydrogen bonds and/or hydrophobic interactions and they are released from food matrix after a strong acid hydrolysis (pérez jiménez and torres, 2011; pérez-jiménez et al., 2013). these compounds represent a significant fraction in some groups of foods, among which cereals and have appreciable antioxidant properties and possibly specific health properties. however, they have been generally underestimated and studies are still required to tackle this issue (saura-calixto, 2012). in addition, recent studies have shown that non-extractable polyphenols significantly contribute to the beneficial properties associated to dietary fibre (vitaglione et al., 2008; saura-calixto, 2011). table 3: tpc and frap values in aqueous-organic extracts and their residues in grains and flours*. products aqueous-organic extract (extractable polyphenols) tpc (mg/100g d.m.) frap (μmol/g d.m.) grain flour p valueω grain flour p valueω solina 1 170.43±28.61 129.28±13.64a 0.01 6.60±0.47 2.75±0.17b 0.0001 solina 2 165.57±26.11 130.87±15.85a 0.001 6.34±0.27 2.35±0.14a 0.0001 solina 3 183.75±14.07 155.24±12.74b 0.01 6.47±0.60 2.31±0.07a 0.0001 mean 173.25±24.05 138.46±18.16 0.0001 6.47±0.46 2.48±0.24 0.0001 products residue (hydrolysable polyphenols) tpc (mg/100g d.m.) frap (μmol/g d.m.) grain flour p valueω grain flour p valueω solina 1 1325.32±57.99c 981.01±50.27b 0.0001 109.12±10.54b 114.25±3.24b n.s. solina 2 1084.42±66.99a 811.65±38.57a 0.0001 85.06±9.90a 99.20±11.14a 0.01 solina 3 1198.73±37.49b 791.50±80.14a 0.0001 90.40±4.30a 118.29±4.26b 0.0001 mean 1202.82±114.39 866.82±104.77 0.0001 92.07±12.22 111.61±10.13 0.0001 *mean ± s.d.; anova, tukey hsd test: by column, means followed by different letters are significantly different (p < 0.05). " student’s t test ; n.s.=not significant   ital. j. food sci., vol 28, 2016 227 in our study, the determined hydrolysable polyphenols (isolated in the residue of the aqueous-organic extract of grains) showed to account for 87-89% of total polyphenols and contribute to the total antioxidant properties for about 93%. these data match those obtained in the other studies on cereals (perez-jimenez and saura-calixto, 2005; chanrasekara and shahidi, 2010). regarding the effect of milling process on tpc content, whole flours exhibited a more significant reduction than in grains, both in the aqueous-organic extract and in the residue. results showed that antioxidant properties also appear to be affected by the milling process: a decrease in frap values was, in fact, observed in aqueous-organic extract (mean value: 6.47±0.46 vs 2.48±0.24 !mol/g d.m.; p<0.0001), whereas an increase in frap values was reported in residues (mean value: 92.07±12.22 vs 111.61±10.13 !mol/g d.m.; p<0.0001). the increase of frap values in residue after milling process could be due to a better efficiency of extraction of bound compounds with antioxidant properties, related to improvement of solvent penetration for the enlargement of the particle surface. these data confirm the importance of milling process in retention of components contributing to antioxidant properties (duodu, 2011). nowadays, several investigations have been carried out to study the effects of cereal processing technologies, such as milling, baking, extrusion, etc., on antioxidant properties (liyana-pathirana and shahidi, 2007; ragaee et al., 2014). as a consequence, tpc (mg/100g d.m.) and frap (!mol/g d.m.) in aqueous-organic extracts and their residues were also studied in solina bread samples, as reported in table 4. it is worth mentioning that whole wheat bread represents a rich source of bioactive compounds as reported in literature (jensen et al., 2011; abdel-aal and rabalski, 2013). table 4. tpc and frap values in aqueous-organic extracts and their residues in bread*. loaves tpc (mg/100g d.m.) frap (μmol/g d.m.) aqueous-organic extract residue aqueous-organic extract residue code a 63.13±6.03cd 832.91±46.21a 8.57±0.34d 106.21±16.96a code b 49.44±6.98b 894.07±62.64a 3.93±0.17a 120.27±30.77ab code c 52.87±6.93bc 1326.90±48.00b 6.14±0.34b 141.90±8.33b code d 36.96±5.59a 1237.41±69.35b 4.14±0.37a 114.19±3.52a code e 71.23±6.04d 877.73±88.77a 7.80±0.26c 126.60±7.80ab *mean ± s.d.; anova, tukey hsd test: by column, means followed by different letters are significantly different (p < 0.05). in bread samples, as it was observed in raw materials, the major contribution to antioxidant properties was given by hydrolysable polyphenols (93-97%). moreover, the comparison of the tpc and frap values of the residue in bread samples and the corresponding values in grains showed that a decrease of tpc, from grains to bread, corresponded to an increase of frap values. several authors have observed that some antioxidants can be generated during non-enzymatic browning reactions, such as the maillard reaction (manzocco et al., 2001).   ital. j. food sci., vol 28, 2016 228 4. conclusions our results showed that in grains and derivatives thereof, hydrolysable polyphenols (isolated in the residue of the aqueous-organic extract) represent an important fraction, with appreciable antioxidant properties and possibly health properties. in particular, this research highlighted that the analysis of potentially bioactive antioxidants, remaining in the residues, is required for an adequate and comprehensive determination of antioxidant capacity in cereals. results also pointed out the key role played by the milling and baking process in antioxidant properties. so, in conclusion, solina wheat represents a valuable italian traditional wheat cultivar with proved antioxidant properties. it also contributes to highlighting the benefits that traditional foodstuffs may have europe-wide: they represent foods with an increased nutritional value and bring a strong contribution to land protection and conservation of biodiversity. acknowledgements this work was undertaken within the project terravita, funded by the italian ministry of agriculture, food and forestry policies. the authors wish to thank dr. d. silveri, dr. v. galli and mr. l. bartoli for the technical assistance and dr. francesca melini for the technical and linguistic assistance in the revision of this paper. references abdel-aal e.m. and rabalski i. 2013. effect of baking on free and bound phenolic acids in wholegrain bakery products. journal of cereal science 3:312. adom k.k., sorrells m.e. and liu r.h. 2003. phytochemical profiles and antioxidant activity of wheat varieties. journal of agricultural and food chemistry 51: 7825. american association of cereal chemists. 2003. approved methods of the aacc, 10th edn. methods 236 55-31, st. paul, mn, the association. arvola a., lähteenmäki l., dean m., vassallo m., winkelmann m., claupein e., saba a. and shepherd r. 2007. consumers’ beliefs about whole and refined grain products in uk, italy and finland. journal of cereal science 46: 197. aune d., norat t., romundstad p. and vatten l.j. 2013. whole grain and refined grain consumption and the risk of type 2 diabetes: a systematic review and dose-response meta-analysis of cohort studies. european journal of epidemiology 28: 845. benzie i.f.f. and strain j.j. 1996. the ferric reducing ability of plasma (frap) as a measure of ‘‘antioxidant power’’: the assay. analytical biochemistry 239: 70. carcea m., durazzo a., raguzzini a., azzini e., foddai m.s. and maiani g. 2009. bioactive components in wheat grains and their products. international wheat quality conference-iv, wheat science dynamics: challenges & opportunites, saskatoon, 2-6 giugno 2009. agrobios (international), 437-448. chandrasekara a. and shahidi f. 2010. content of insoluble bound phenolics in millets and their contribution to antioxidant capacity. journal of agricultural and food chemistry 58: 6706. duodu k.g. 2011. effects of processing on antioxidant phenolics of cereal and legume grains. in: advances in cereal science: implications to food processing and health promotion. chapter 3, 31-54, 2011 american chemical society. durazzo a., turfani v., azzini e., maiani g. and carcea m. 2013. phenols, lignans and antioxidant properties of legume and sweet chestnut flours. food chemistry 140: 666. international association for cereal science and technology. 2003. standard methods of icc. methods no. 104/1, 105/2, 110/1. vienna, austria: the association. jensen s., ostdal h., skibsted l.h. and thybo a.k. 2011. antioxidants and shelf life of whole wheat bread. journal of cereal science 53: 291.   ital. j. food sci., vol 28, 2016 229 liyana-pathirana c.m. and shahidi f. 2007. the antioxidant potential of milling fractions from bread wheat and durum. journal of cereal science 45: 238. liu r.h. 2007. whole grain phytochemicals and health. 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antioxidant: occurrence, metabolic fate and health effects. nutrition research reviews 26: 118. pomeranz y. and williams p.c. 1990. wheat hardness: its genetic,structural, and biochemical background, measurement, and significance. pages 471-548 in: advances in cereal science and technology, vol. x. y. pomeran, ed. aacc international: st. paul, mn. porfiri o., torricelli r., silveri d.d., papa r., barbaccia g. and negri v. 2001. the triticeae genetic resources of central italy: collection, evaluation and conservation. hereditas 135: 187. pulido r., bravo l. and saura-calixto f. 2000. antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/antioxidant power assay. journal of agricultural and food chemistry 48: 3396. ragaee s., seetharaman k. and abdel-aal el-sm. 2014. the impact of milling and thermal processing on phenolic compounds in cereal grains. critical reviews in food science and nutrition 54: 837. schatzkin a., mouw t., park y., subar a.f., kipnis v., hollenbeck a., leitzmann m.f. and thompson f.e. 2007. dietary fiber and whole-grain consumption in relation to colorectal cancer in the nih-aarp diet and health study. american journal of clinical nutrition 85:1353. saura-calixto f. 2011. dietary fibre as a carrier of dietary antioxidants: an essential physiological function. journal of agricultural and food chemistry 59: 43-49. saura-calixto f. 2012. concept and health-related properties of nonextractable polyphenols: the missing dietary polyphenols. journal of agricultural and food chemistry 60: 11195. singleton v. l., orthofer, r. and lamuela-raventos r. m. 1999. analysis of total phenols and other oxidation substrates and antioxidants by means of folinciocalteau reagent. methods in enzymology 299: 152. slavin j.l., jacobs d. and marquart l. 2000. grain processing and nutrition. critical reviews in food science and nutrition 40: 309. vitaglione p., napolitano a. and fogliano v. 2008. cereal dietary fibre: a natural functional ingredient to deliver phenolic compounds into the gut. trends foods science and technology 19: 451. paper received february 13, 2015 accepted june 20, 2015 ijfs#95_santonicola_bozza   ital. j. food sci., vol 28, 2016 155 review occurrence and production of furan in commercial foods s. santonicola and r. mercogliano* department of veterinary medicine and animal production, university federico ii, via f. delpino 1, 80137 napoli, italy *corresponding author. tel.: +39 0812536062; fax: +39 081458683 e-mail address: raffaella.mercogliano@unina.it abstract furan (c4h4o) is a compound classified as "possibly carcinogenic to humans" by international agency for research on cancer. as precursors, ascorbic acid, unsaturated fatty acids, amino acids and carbohydrates have been suggested to induce furan formation. human exposure occurs mainly through consume of coffee, canned foods and baby food. average intake of furan is 1.5 µg/day for children aged 4-6 years and 27 µg/day for adults. currently no limits for furan in food were fixed by european legislation. since the carcinogenicity of furan, levels in food should be kept as low as reasonably achievable. keywords: baby food, food safety, furan   ital. j. food sci., vol 28, 2016 156 1. introduction furan (c4h4o) is a small cyclic ether with aromatic character. it is a lipophilic coumpond with low molecular weight, high volatility, and 31°c as boiling point (ntp 1993) (fig. 1). furan and its derivatives (2-methylfuran, 2-ethylfuran, 2-pentylfuran, 2,5-dimethylfuran, 2-butylfuran, 2,3-benzofuran) have known to occur in heat-treated foods and drinks, and to contribute to the sensory property of foods (maga, 1979; merrit et al., 1963; efsa 2010). figure 1: chemical structure of furan. classified as possibly carcinogen to humans (group 2-b) by the international agency for research on cancer (iarc, 1995), furan is carcinogenic for rats and mice. in the opinion of european food safety authority (efsa) the weight of evidence indicates that furaninduced carcinogenicity is probably attributable to a genotoxic mechanism (efsa, 2004). furan is formed during heating process used for the manufacture of foods, and has been detected in hot-air dried, baked, fried and roasted food items, such as cereal products and coffee, as well as canned or jarred prepared foods (us fda, 2004a; efsa, 2005; zoller et al., 2007). there appear to be multiple precursors (sugars, amino acids, ascorbic acid, polyunsaturated fatty acids), and many pathways to the furan production (fig. 2). the principal are: thermal degradation of certain amino acids; thermal degradation/maillard reaction of reducing sugars; thermal oxidation of ascorbic acid, poly-unsaturated fatty acids and carotenoids (yaylayan, 2006); thermal degradation of the common precursors in the process of roasting. the primary source of furan in food is the thermal degradation of carbohydrates such as glucose, lactose, and fructose (maga, 1979). figure 2: precursors and sources of furan in food.   ital. j. food sci., vol 28, 2016 157 amino acids serine and cysteine are able to metabolize acetaldehyde and glycolaldehyde, which react by aldol-condensation, then produce aldotetrose derivatives and furan. alanine, threonine, and aspartic acid can generate only acetaldehyde; they require the presence of serine, or cysteine and reducing sugars, also, to produce glycolaldehyde and then furan (perez and yaylayan, 2004). the reactions between several amino acids and carbohydrates on heating form aldotetrose derivatives, then after cyclisation can form furan (perez and yaylayan, 2004; limacher et al., 2008). when ascorbic acid was mixed in model systems with single amino acids (glycine or serine), sugar (erythrose) or unsaturated fatty acids (e.g. linoleic), the mixtures produced far less furan on heating than ascorbic acid alone did (mark et al., 2006). experimentally monounsaturated acid (oleic) did not form furan (becalski and seaman, 2005), and if furan is formed from unsaturated fatty acids the yield increases as the degree of unsaturation increases. furan production has been linked with free radicals autoxidation process (perez and yaylayan, 2004). so, ferric ions increased furan formation in linoleic acid by 79%, and in trilinolein by 29% (mark et al., 2006), addition of commercially available antioxidants (such as tocopherol acetate) reduced the formation of furan (perez and yaylayan, 2004). moreover the effects of lipid oxidation, catalysts such as fe(ii) or antioxidants are sometime contradictory. given the complicated and competing reaction pathways available in autoxidation processes (some of which may lead to furan, and some not) limiting autoxidation may not always lead to a corresponding reduction in furan formation (fig. 3).   ital. j. food sci., vol 28, 2016 158 figure 3: proposed pathways of formation of parent furan from three main groups of sources: amino acids, carbohydrates, and polyunsaturated fatty acid (vranovà and ciesarovà, 2009). due to its low polarity, furan can pass trough biological membranes and enter various organs, it is rapidly and extensively absorbed from the intestine, and the lung. repeated doses accumulate in the liver (efsa, 2004a) and kidney, and in fewer quantities in intestine, stomach, blood and lung (burka et al., 1991). absorbed furan is metabolized rapidly by cytochrome p-450 (cyp) enzymes via ring opening to form (z)-2-butene-1,4-dialdehyde. the rapid hepatic metabolism seems to limit, however, its systemic delivery. the capacity to furan’s bioactivity is also of relevance for the distribution, since it can result in an irreversible binding to the respective tissue. within the first 24 hours after a single oral application of furan (8 mg/kg bw), 80% of the total radioactivity was eliminated via the lung, urine and feces (burka et al., 1991).   ital. j. food sci., vol 28, 2016 159 in the 2-year rat study, animals of each sex (n=70) were administered furan at 2, 4, or 8 mg/kg bw 5 days per week. mean body weights of male rats that received 8 mg/kg furan were lower than controls. increased incidences of numerous non-neoplastic liver lesions (biliary tract fibrosis, hyperplasia, chronic inflammation, and proliferation and hepatocyte cytomegaly, cytoplasmic vacuolization, degeneration, nodular hyperplasia, and necrosis) were present in treated rats. cholangiocarcinoma of the liver occurred in all groups of dosed rats. a separate 2-year study was conducted in which 50 male rats were administered 30 mg/kg furan 5 days per week. cholangiocarcinoma of the liver occurred with an overall incidence of 100% (40/40) and hepatocellular carcinoma occurred with an overall incidence of 15 % (6/40) (burka et al., 1991; ntp, 1993). both in vitro and in vivo studies show that metabolic activation by cytochrome p-450 (cyp) enzymes is involved in furan-induced toxicity (kedderis et al., 1993). it is likely that furan or (z)-2-butene-1,4-dial reacts with dna in target cells and can play a role in furan induced tumors. furan causes loss of atp after bioactivation to metabolites which cause an irreversible uncoupling of hepatic mitochondrial oxidative phosphorylation, this activates cytotoxic enzymes, including endonucleases, that produce dna double-strand breaks prior to cell death (mugford et al., 1997; kedderis and ploch, 1999). furan was able to induce: a) gene mutations, chromosome aberrations and sister chromatid exchanges (sce) in cultured mammalian cells, and chromosomal aberrations in mice bone marrow cells; b) hepatocellular tumours after ras oncogene activation, suggesting that furan, or a reactive metabolite, can directly activate protooncogenes (reynolds et al., 1987); c) chronic hepatic cytotoxicity in the genesis of liver tumours in infant male mice (johansson et al., 1997); d) monocytic cell leukemias with minimal hyperplasia of bone marrow. hepatocytes from human livers donors oxidized furan at rates equal to, or greater than, those of rat hepatocytes in vitro (kedderis et al., 1993) (fig. 4). figure 4: blood flow limitation of furan biotransformationa (kedderis et al., 1993). in alcohol consumption the furan metabolism is higher due induction of cytochrome p450 2e1 in humans by ethanol (perrot et al., 1989). however there is small difference   ital. j. food sci., vol 28, 2016 160 between the levels of human exposure and doses that induce carcinogenic effects in experimental animals. 1.1. hydroxymethyl-2-furfural heat-induced formation and occurrence in food furan and 5-hydroxymethylfurfural (hmf) are compounds that are formed in a variety of heat-treated commercial foods. such a widespread occurrence of furan and hmf in many types of food is due to the fact that they are products of different reactions following multiple routes and involving different precursors and intermediate (anese and suman, 2013). in particular, hmf can be formed as an intermediate in the maillard reaction, which occurs when carbohydrates are heated in the presence of amino acids or proteins (mauron, 1981), or, alternatively, by thermal dehydration of a sugar under acidic conditions (kroh, 1994). at low ph, glucose or fructose may undergo 1,2 enolization and dehydration to form 3-deoxyosone, which is the key intermediate in hmf formation. fructose is more reactive than glucose in the formation of hmf (lee and nagy, 1990). according to perez locas and yaylayan (2008), hmf can form from fructose or sucrose via the generation of a highly reactive fructofuranosyl cation. at high temperatures and in dry systems this cation can quickly be converted into hmf. hmf formation in foods has been found to be affected by sugar type, ph, water activity and the presence of divalent cations (gokmen et al., 2008). as furan and hmf formation is concomitant to that of color and flavor of heated foods, it is very difficult to mitigate their formation without compromising the food sensory acceptability. changes in process parameters, i.e. heating regime modification, and formulation can be regarded as strategies that can be applied to prevent furan and hmf formation (anese and suman, 2013). particularly breakfast cereals, coffee, honey as well as pasteurized juices or pulps etc. are subjected to intensive hmf formation. cereal products: rufían-henares et al. (2006) revealed that the hmf concentration varied between 6.59 and 240.51 mg/kg (w/w). the highest average concentration of hmf was found in maize-based breakfast cereal (42.81±7.92 mg/kg), followed by wheat (40.79±8.57 mg/kg) and rice (32.14±10.79 mg/kg) products. authors have also compared products with and without the addition of honey and stated that hmf concentration was higher in the former group, 43.44±10.35 versus 34.24±6.17 mg/kg, respectively. hmf formation was also studied as one of the factors influencing browning of infant cereals. increases in hmf concentration were investigated at different stages of cereals processing (toasting, hydrolysis, drying) in model systems (fernandez-artigas et al., 1999). the hydrolysis process was connected with increases in hmf concentration. the drying stage, however, did not contribute to overall hmf synthesis probably due to short processing times. coffee: on the basis of analysis of 22 coffee samples murkovic and pichler (2006) stated that hmf concentration in the investigated products ranged from 300 to 1900 mg/kg. they found that roasting coffee at 240°c caused a rapid increases in hmf (up to 900 mg/kg) in the first 3 min. further roasting was connected with decreases in hmf contents probably because of the occurrence of consequent degradation reactions. arribas-lorenzo and morales (2010) analysed 35 commercial roasted coffee brands as well as 19 soluble coffee brands. they estimated four levels of hmf: 110, 625, 1734, and 2480 mg/kg for natural, blend (mixture of torrefacto and natural coffee), torrefacto (coffee roasted with sugar addition) and soluble coffee, respectively. the largest differentiation in hmf level was found in soluble coffee clusters (min. 691, max. 4023 mg/kg). the authors   ital. j. food sci., vol 28, 2016 161 established that the different modes of coffee brewing (espresso, filtered, italian, soluble) influence potential content of hmf. a possible mitigation strategy is represented by the physical removal of furan and hmf by means of vacuum treatments. the vacuum technology has already been studied as a tool to remove hmf from roasted coffee, by exploiting the chemical and physical properties of these molecules (quarta and anese, 2012). in this case, thinking of a possible industrial exploitation of this technology for coffee, the product coming from the tunnel oven could be moved to a hydration step (e.g. carried out by means of a spray of pressurized water) followed by a vacuum step. the vacuum-treated food with reduced furan and/or hmf and water contents can be then subjected to eventual flavor enrichment and finally packaged (anese and suman, 2013). fruit and vegetable products: these products are usually rich sources of sugars and organic acids as well as amino acids. processing of this group of foodstuffs thus leads to the formation of significant amounts of hmf. burdurlu and karadeniz (2003) investigated the influence of extract, storage time and temperature on non-enzymatic browning of apple juice concentrates. browning index was correlated with hmf concentration. juice samples were stored at different temperatures (5, 20, 37°c) for four months. for juices stored at 5°c as well as 20°c, increases in hmf level were minor (increase from 0.62 up to 4.37 mg/kg). hmf formation was much more significant at 37°c reaching 190 and 963 mg/kg. on the basis of the results, the authors confirmed the usefulness of hmf both as a heat processing index and an indicator of storage conditions. recently, saldo et al. (2009) demonstrated that processing apple juice by means of ultrahigh-pressure homogenization could represent an alternative to conventional pasteurization. ultrahigh-pressure homogenization, while causing a significant decrease in microbial counts, allowed hmf formation to be reduced. hmf concentration in pasteurized juice was indeed 100-fold higher than in ultra-high-pressure homogenized and raw counterparts. honey: in case of honey, the level of hmf is strictly normalized (council directive, 2001). according to normalization, hmf concentration should not exceed 40 mg/kg with exception of honeys from tropical climate (not more than 80 mg/kg). increased amounts of hmf can result from improper processing or prolonged storage (tosi et al., 2002; 2004; 2008; fallico et al., 2004). an increased of hmf level in honey was also found to be connected with initial ph (acidity) (fallico et al., 2004). tosi et al. (2002; 2004; 2008) investigated the kinetics of hmf formation and changes in enzymatic activity during honey heating. it was shown that the initial hmf concentration did not influence the kinetics of its formation. even after intensive heating (90°c for 20 min) hmf concentration did not reach 40 mg/kg. fallico et al. (2008) however, pointed out that under different storage conditions degradation hmf can occur in honey samples. rate constants of degradation process at temperatures between 25 and 50°c (for citric as well as chestnut honey) were higher than the corresponding rate constant of formation. these findings should be taken under consideration in proper legislation process (fallico et al., 2008). dairy products: sterilization processes are the origin of hmf in dairy products and may be connected with their colour change (browning). the total hmf-value has generally been applied to distinguish heat-treated milk (pasteurized, uht, concentrated and powdered). albala-hurtado et al. (1998) studied changes of hmf concentration during storage of infant milk. powdered infant milk had more hmf than corresponding liquid milks (34.7 and 12.2 μg/kg (w/v), respectively, after 9 month, 37°c). the influence of different temperatures on hmf formation   ital. j. food sci., vol 28, 2016 162 during the storage of uht milk was studied by cais-sokolinska et al. (2004). there were no significant differences in hmf concentration in milk stored at 4 and 8°c, but storage at room temperature caused a two fold increase in its amount when compared with freshly sterilized product (cais-sokolinska et al., 2004). oral et al. (2011) compared changes in total hmf fraction in sweet whey powder (swp) and skim milk powder (smp) at 3 different temperatures (25, 35 and 45°c) and moisture (2.5, 5, 7.5% for swp and 5.5, 7.7, 10% for smp) during eight month storage period. small formation of hmf was observed during storage at 25°c in all studied samples but a great increase in hmf during storage at 45°c was observed, which indicates a large dependence of the formation of hmf on time and temperature of storage and moisture content. since milk powder is used in the formulation of infant formula, instant beverage, bakery products, chocolate, its hmf content should be under consideration for end product quality and nutritional value. several techniques have been used for hmf detection in foods. liquid chromatography is the most widely method used. these techniques utilize ultraviolet (uv) detection because of the strong absorption of hmf at 280-285 nm. however, many compounds naturally present or formed in foods during processing may also absorb at this wavelength. poor chromatographic resolution of these compounds may adversely affect the quantification of hmf during uv detection (gokmen et al., 2006). for this reason several techniques involve the protein hydrolysation step process. oral et al. (2014) studied the hmf binding capacity of the most common proteins (e.g. caseine) in infant formulas and determined separability from them by acid digestion. consequently, the hmf levels of the samples were evaluated separately either by no treatment or by the acid-heat treated method. the hmf values of the 10%, 5% and 2.5% casein solutions prepared with hmf stock solution (131.89 mg/l) were found as 116.31, 122.31 and 127.48 mg/l respectively, without acid hydrolysis. after acid hydrolysis, the hmf levels of the casein solutions were determined as 115.52, 121.10 and 124.51 mg/l in 10%, 5% and 2.5% respectively. acid application have not got any statistical importance at all concentration for making free of bounded hmf (p < 0.05). also, hmf was produced by the chemical reaction between acid and carbohydrates (certain food ingredients) during the acid hydrolysis stage. to avoid this situation, the acid hydrolysis phase of the analysis should be omitted. in the samples prepared without acid hydrolysis stage, the level of hmf did not change with varying of sample concentration. also, during the acid hydrolysis stage the hmf levels in samples are thought to be dependent on the amount of sample. this is because, during the degradation of proteins with acid, acid is consumed more due to the solution having a high concentration. therefore, in this study, hmf level was lower in 10% than in 2.5% concentrations due to the decrease in the amount of acid per food compound (carrageenan, sucrose, inulin and fructose). as a result, it was concluded that hmf cannot be evaluated by the traditional method which is based on the principle of separating it from proteins for measurement (oral et al., 2014). although hmf has been used for years as a quality indicator of thermally processed foods, recently some toxicological concerns are raised. hmf has a number of structural alerts (furan ring, α,β-unsaturated carbonyl group, and allylic hydroxyl group) that pose possible genotoxic and carcinogenic risks (anese and suman, 2013). however, further studies suggest that hmf does not pose a serious health risk, but the subject is still a matter of debate (gokmen et al., 2006). hmf can initiate and promote the growth of aberrant crypt foci (acf) in rat colons in a dose-dependent manner (zhang et al., 1993). hmf induced a significant number of chromosome aberrations and a significant lowering of mitotic activity in cultured chinese hamster v79 cells (nishi et al., 1989). in a two years study conducted by the national   ital. j. food sci., vol 28, 2016 163 toxicology program, hmf was found to increase the incidence of hepatocellular adenomas in female b6c3f1 mice, whereas no carcinogenic activity was observed in male or female f344/n rats as well as in male b6c3f1 mice (ntp, 2010). in addition, hmf was associated with increased lesions of the olfactory and respiratory epithelium of the nose in male and female rats and mice (ntp technical report, 2010). its derivative 5sulfidemethylfurfural (smf), exhibited direct mutagenicity in human lymphoblasts and induced 8-azaguanine-resistant mutants in salmonella typhimurium tm677 in a dosedependent manner (surh et al., 1994). histopathological analyses revealed that smf induced moderate damage to liver tissue and notable damage to the kidneys (nearly all proximal tubules in smf exposed animals were destroyed). the molecular mechanism underlying this selective toxicity of smf for proximal tubules is unknown (bakhiya et al., 2009). on the basis of kinetic data (monien et al., 2009), it was estimated that between 452 and 551 mg/kg of the initial hmf dose (500 mg/kg) was converted in mice into smf which was subsequently circulated. the sulfotransferases (sult) are the enzymes that converted hmf to smf. human sult isoforms have a widespread tissue distribution and are expressed in many tissues including liver, lung, brain, skin, platelets, breast, kidney, and gastrointestinal tract (salman et al., 2009). moreover, humans express sult in extrahepatic tissues more extensively than rodents do and may therefore be more sensitive to hmf (teubner et al., 2007). some studies on mutagenicity or carcinogenicity of other hmf derivatives showed that furfuryl alcohol and furfural were not observed to be mutagenic in salmonella typhimurium strains ta98, ta100, ta102, ta1535, or ta1537 (ntp, 1999). it should be noted that studies which demonstrate the positive and protective role of hmf are also available. wang et al. (2010) revealed that hmf protects lo2 hepatocytes cell from oxidative damage. yamada et al. (2011) have been suggested that hmf could be useful for the treatment or prevention of type i allergic diseases. due to the fact that there is inconclusive evidence regarding hmf’s potential toxicity to human health, it cannot be determined whether hmf should be considered unsafe or whether the benefits of its use in industry outweighs the risks it may pose. additional studies are needed to elucidate the potential effects that long-term exposure to hmf could have on human health. 1.2. formation of furan in food different factors, such as temperature, ph, water activity, storage and time conditions, presence/absence of inhibitors and activators may influence the furan levels in food. in industrial field, at temperature around 200°c, the furan levels increase with the temperature increasing. however when it exceeds the value of 200°c the concentration of furan is independent from the temperature values. in domestic cooking furan formation depends on the type of cooking. frying (150°-200°c) produces higher levels, if compared with the baking. furan has been associated with the flavor of foods. since it is a highly volatile compound, an important part of the furan formed during thermal treatment will be lost during food handling and food preparation by evaporation, depending on the food matrix. the combination of higher temperatures and lower water content leads to a higher furan content. furan seems likely to form in browned products, especially if the water activity is low. in particular high levels of furan were founded in bread toasted from a brown to very dark colour. furan levels may formed at various ph and temperatures in experimental model systems (nie et al., 2013), and ph has a significant effect on thermally induced furan formation, if temperature is greater than 110°c. at ph 7.00 higher levels of furan were observed than at ph 9.40 and 4.18, suggesting that ph is an important factor influencing furan formation as   ital. j. food sci., vol 28, 2016 164 a function of thermal treatment and temperature. for instance, after heating for 30 minutes at 150°c in experimental model at ph 7.00, 9.40, and 4.18 the furan concentration was founded at 304, 238, and 40 ng/ml, respectively. also it is reported (fan, 2005a) that less furan is formed at ph 3.00 than at ph 7.00 for glucose solution. at ph 7.00 furan content increase rapidly from 34 to 304 ng/ml with temperature increasing from 120° to 150°c. these data suggest that temperature is also a major factor affecting furan formation in model system (nie et al., 2013). under sterilization conditions, fructose-glycine system produces high level of furan, while less furan significantly is formed in glucose-glycine system (nie et al., 2013) (fig. 5). it seems that formation of furan by maillard reactions is dependent, as activators, on the amino acids and sugars used. the addiction of phenylalanine to glucose results in an increase of about 50% of furan. moreover the presence of the amino acids alanine, threonine, and serine results in higher furan amounts. in contrast, the furan amount decreases by 20% in a binary mixture of fructose and phenylalanine (limacher et al., 2008). carbohydrates and amino acids are also commonly used as additives in food products, thus if carbohydrates and amino acids are required together in formulation, sucrose would be a better choice than glucose and fructose to reduce the accumulation of furan by maillard reactions during the heating (limacher et al., 2008). figure 5: furan formation in a glucose-glycine, fructose-glycine and sucrose-glycine heating model system at 120° c for 30 minutes, simulating sterilization conditions (nie et al., 2013). furan may be produced from polyunsaturated fatty acids during thermal and uv-c treatment. (fan, 2015). furan is also produced from linolenic acid emulsion during storage at 25 °c. at ph 9 more furan was formed than at phs 3 or 6 during longer storage (fan, 2015). part of the furan concentrations founded in commercially available food products might originate from chemical deterioration reactions during storage (palmers et al., 2015). a range of individual vegetable purées was stored at two different temperatures to investigate the effects of storage on the furan concentrations of shelf-stable, vegetablebased foods. after 5 months of storage at 35°c (temperature-abuse conditions), a general   ital. j. food sci., vol 28, 2016 165 increase in furan concentrations was observed. the furan formation during storage could be reduced by storing the vegetable purées at a refrigerated temperature of 4°c, at which the furan concentrations remained approximately constant for at least 5 months. following storage, the vegetable purées were briefly reheated to 90°c to simulate the effect of the final preparation step before consumption. contrary to storage, furan concentrations decreased as a result of evaporative losses. consequently, both refrigerated storage and the reheating step prior to consumption showed the potential of mitigation measures for furan formation in vegetable-based foods (e.g. canned vegetables, ready-to-eat soups, sauces or baby foods). on the contrary methylfuran concentrations rapidly decreased during storage (palmers et al., 2015). therefore, the retention or release of furan by different food constituents was systematically evaluated. the presence of oils in foods decreases the volatilization of furan and may as such increase the actual intake of furan to a large extent (becalski and seaman, 2005). for oils, furan concentrations are similar in olive oil and in corn oil. palm oil contains significantly more furan. it is believed that the high amounts of carotenoids in crude palm oil are responsible for this difference (shahidi, 2005), since carotenoids were identified as important precursors of furan by becalski and seaman (2005). it is possible that furan have been formed by lipid oxidation and has accumulated over time during oils storage. fromberg et al. (2009) evaluated the influence of the browning process in fried meat and fish meat balls using either vegetable oil or butter. furan was found at low level in the heavily fried fish meat balls using butter (3.1 ng/g) as frying agent and in medium fried fish meat balls using vegetable oil (2.5 ng/g) as frying agent fried. however furan was not found above the limit of quantification of 2.4 ng/g in the crust of the medium fried meat balls using vegetable oil as frying oil. on the other hand furan was found in the crust (2.4 ng/g) of the heavily fried meat balls using butter, but not in the heavily fried meat balls when the whole meat balls were analysed. surprisingly, fromberg et al. (2009) founded furan in the minced ingredients before frying, even if they were not able to find the explanation for the context (fig. 6). figure 6: levels of furan [ng/g] in home fried meat balls and fish balls (fromberg et al., 2009). some conservation treatments such as irradiation may facilitate the production of high concentrations of the contaminant. fan (2005a) reported that ionising radiation induced the formation of furan in apple and orange juices. furan levels increased linearly as the radiation dose increased from 0 to 5 kgy. furthermore, in the first 3 days of storage after the irradiation treatment, the furan levels continued to increase in both apple and orange   ital. j. food sci., vol 28, 2016 166 juices. according to fan (2005ab), the increase in furan during the earlier storage period may be due to the residual effect of irradiation. to monitor the presence of furan in food, the commission recommendation 2007/196/ec requests the member states to collect data on heat-treated commercial food products. in response to the commission request, a total of eighteen member states have so far submitted analytical results for furan content in food to the european food safety authority (efsa). a total of 4186 complete results were reported for foods sampled between 2004 and 2009 (table 1). data were sorted into 21 different food categories (5 coffee and 16 non coffee categories). the ‘baby food’ category was subcategorised into 6 groups according to the ingredient combination and the category ‘others’ was subcategorised into more homogenous subgroups in order to extract further information (efsa, 2010). table 1: number of samples collected by member states (indicated by iso country code) between 2004 and 2009 for the analysis of furan content in food (efsa, 2010) the report data have identified as types of foods most responsible of dietary exposure (content> 100 mg / kg) (table 2): coffee, baby foods, sauces and soups.   ital. j. food sci., vol 28, 2016 167 table 2: furan content in food per main food category (efsa, 2010). among all products tested, the highest furan content was reported in roasted coffee beans with an average of 3.611 μg/kg. furan and furan derivatives have long been known as intrinsic components of coffee flavours. green coffee beans contain only traces of furan. the furan levels in the roasted coffee are correlated with the roast colour (guenther et al., 2010). furan retention studies were also conducted with coffee, since it is believed to be the major source of furan in adults diet. in coffee, furan retention was mainly caused by the lipophilic fraction. defatted coffee brew showed a significantly lower retention of furan than coffee brew: the furan response increased significantly from 78 to 89% after defatting. on the one hand, oils are precursors of furan. palm oil contains significantly more furan and it is responsible for the high content of furan in roasted coffee (lachenmier et al., 2009). automatic coffee machines produced brews with the highest levels of furan, because a higher ratio of coffee powder to water is used giving a lower dilution factor, and because the closed system favors retention of furan. much lower levels were produced by standard home coffee-making machines and by manual brewing (goldman et al., 2005; zoller et al., 2007). furan is found in a wide assortment of foods including potato chips, tortilla chips (fda, 2004a), dried fruits, popcorn, corn crisp, cereal product (puffed rise) might be consumed by children and findings of furan in these products may cause food safety concern (fromberg et al., 2009). milk based processed food showed low mean furan content (6 μg/kg), but interestingly a maximum furan content of 80 μg/kg was found in sweetened condensed milk (efsa, 2006). maximum values exceeding a level of 100 μg/kg were found in fish products such as mackerels and sardines in tomato sauce, in meat products like canned duck with lentils or rabbit with prunes, in soups such as tomato soup and in gravy (fromberg et al., 2009). department of nutrition, national food institute has developed a recipe database with potential furan containing dishes which was used as a platform for analyzing furan in   ital. j. food sci., vol 28, 2016 168 commonly eaten freshly prepared home cooked dishes. the recipes have been chosen on basis of knowledge upon potential furan containing ingredients after heating (zoller et al., 2007) as well as knowledge on dietary habits in the european region (männistö et al., 2003; elmadfa and weichselbaum, 2005; debacker et al., 2007; fagt et al., 2008). as worst-case scenarios, foods were home cooked using canned ingredients which contained furan. however, this did not lead to elevated levels of furan in the prepared home cooked foods. for ready-to-eat foods with an initial level of furan, cooking reduced the level of furan in the food to about half the original content probably due to evaporation of furan during heating. nevertheless furan is relatively stable in heated foods left for cooling where the losses of furan were insignificant (zoller et al., 2007; fromberg et al., 2009) (figg. 7, 8). figure 7: main ingredients and preparation procedure in selected recipes (fogt et al., 2007). figure 8: levels of furan in homemade meat sauce including ingredients [ng/g] (fromberg et al., 2009). high levels of furan were found in toasted bread slices (e.g. 83 μg/kg furan for dark toasted bread) and this was correlated to the browning level (fromberg et al., 2009). the dark and black toasted bread had high to very high levels of furan and the degree of browning of the toasted bread has very high influence on the amount of furan in the food item and therefore influence on the amount of furan consumed. the furan level might be   ital. j. food sci., vol 28, 2016 169 associated with the use of ascorbic acid in the flour used for the bread combined with a baking process leaving low levels of water in the final products. the untoasted bread analysed did not contain furan above 2.4 ng/g (fromberg et al., 2009). the crust always contained more furan than the entire bread, with a 3to 20-fold difference depending on the surface-to-volume ratio (zoller et al., 2007). when an initial level of furan is present in the food item heated, changes in furan levels appear when the food item is heated. heating the food item almost reduced the furan level in the food by 50%, however the furan level in the foods do not change when left for cool for one hour, and furan therefore seems to be stable in the food when it is not heated (fromberg et al., 2009) (fig. 9). figure 9: development in the furan concentrations over time in homemade soups [ng/g] (fromberg et al., 2009). the exceptions are vigorous boiling or cooking where furan can be lost, presumably by evaporation and by entrainment in the large volumes of steam that are released. on the other hand, warming the food even in lightly lidded containers can increase furan levels, so any additional formation appears to be balanced by evaporative losses (hansip et al., 2006). 1.3. presence of furan in baby food children are sensible consumers, for this reason food safety and quality are essential. in efsa report (efsa, 2010), the highest maximum concentrations of furan for the non-coffee categories were found in baby food with 224 μg/kg. children have high capacity of absorption nutrients and non-nutrients (fimp, 2011) but they have a reduced capacity of detoxification compared to an adult organism (ginsberg, 2004). the metabolic differences baby\adult decrease with increasing age, but still have an influence through adolescence, for which there is still a high degree of risk of exposure to toxic agents (madhavan and naidu, 1995; ginsberg, 2004). with the exception of coffee products, commercial complementary foods (6-24 months) were the food group with the highest furan concentrations. the problem is restricted only to commercially sterilized baby foods, while freshly cooked home-made complementary food was found to be furan-free (lachenmeier et al., 2009). the exposure assessment for babies is therefore challenging as it is not the total consumption that has to be evaluated, but more specifically, only the consumption of commercial products. in efsa report (2010) were analyzed, between 2004 and 2009, 11 samples of infant food (infant formula) and 1322 samples of baby food, divided in 6 sub-categories. jarred baby food and infant formulae are of particular interest as they may form the sole diet for many infants and furan has been reported in those products (efsa, 2010) (fig. 10).   ital. j. food sci., vol 28, 2016 170 figure 10: number of samples collected by member states between 2004 and 2009 for the analysis of furan content in food (efsa, 2010). the mean furan content in infant formulae was 3 μg/kg, the furan content of jarred commercial baby food with an overall mean content of 29 μg/kg and a maximum value of 224 μg/kg was similar to previously reported data (crews and castle, 2007). mean furan content in baby food containing only fruits is 5 μg/kg and 40 μg/kg in baby food containing only vegetables. bianchi et al. (2006) assumed that this difference in furan content could be due to different heating treatment as fruit samples are generally pasteurised whereas the vegetables are generally sterilized (efsa, 2010) (fig. 11). figure 11: furan content in baby food sub categories (efsa, 2010). baby food containing mainly vegetables and meat show mean furan concentrations of 40 μg/kg whereas cereal based baby food show lower mean values (19 μg/kg). furan was analysed in 21 different baby-food samples purchased from the finnish markets. the mean levels of furan varied between 4.7 and 90.3 μg/kg (jestoi et al., 2009). us fda (2004a) internet database is rather extensive, and data from years 2004-2005 show furan concentrations in fruit-based baby-foods below 8 μg/kg, vegetables and mixed vegetables up to 112 μg/kg and meat containing mixed baby and toddler foods up to 90 μg/kg. in another study the retention of furan was significantly higher in baby food ‘‘beef and vegetables’’ and even more in baby food ‘‘spinach’’. baby food ‘‘spinach’’ and baby food ‘‘beef and vegetables’’ contained 2 and 1% corn oil, respectively. although the total fat content of baby food ‘‘beef and vegetables’’ (3.4%) was higher than the total fat content of baby food ‘‘spinach’’ (1.9%), the retention of furan by baby food ‘‘spinach’’ was significantly higher than the retention by baby food ‘‘beef and vegetables’’. these results lead to the assumption that the addition of a low amount of oil significantly increased the retention of furan, and this to a much higher extent than the total fat content. this implies that the presence of oils influences the actual intake of furan. as, from a nutritional point of view, elimination of oils from baby food is not an option, it would be better to add the oils after heat-processing, right before consumption of the baby food (lachenmeier et al., 2009; jestoi et al., 2009). the problem is restricted only to commercially sterilized   ital. j. food sci., vol 28, 2016 171 baby foods, while freshly cooked home-made baby food was found to be furan-free (lachenmeier et al., 2009). therefore, the differences in furan retention caused by various food constituents are an important problem to be systematically studied. 1.4. dietary furan intake the danish national survey of dietary habits and physical activity 2000-2004, calculated the exposure of furan. the dietary survey comprised a random sample of 4120 individuals aged 4-75 years. dietary intake was obtained using a 7 d pre-coded food diary. the amounts of food consumed were given in household measures (cups, spoons, slices, etc.) or estimated from photos of different portion sizes showing four to six different portions. the mean food intakes were calculated for each individual using the general intake estimation system (gies) version 0.995a (danish institute for food and veterinary research, søborg, denmark). in this analysis were used data from children 4-6 years (n=335) and adults 15-75 years (n=4692). calculations of the furan exposures showed a median intake of 1.1 μg/day for children (mean 1.5 μg/day) and a median intake of 33.5 μg/day for adults (mean 27 μg/day) (fromberg et al., 2009). figure 12: foods contributing to the median intake of furan for adults of 34 μg/day (fromberg et al., 2009). for adults the main contributor (95%) to the exposure of furan is coffee with an average daily intake of more than 0.6 l of coffee (the coffee is mostly made of 40 g medium roasted ground coffee per litre water) (johansson et al., 1998; fagt et al., 2008) (fig. 12). as children do not have a high intake of coffee, the foods contributing to the intake are from other sources. the main food group contributing to furan is the breakfast cereal as high levels of furan was found in the breakfast cereals combined with children’s high consumption (fromberg et al., 2009) (fig. 13). 97%   1%  1%   1%   coffee   beer   fruit   sauces     ital. j. food sci., vol 28, 2016 172 figure 13: foods contributing to the median intake of furan for children of total 1.1 μg/day (fromberg et al., 2009) the donald data show that the consumption of jarred baby foods in children 3 months aged is rather low. the highest exposure occurs in the children 9 months aged, and may reach a daily intake over 1 µg /kg bw. afterwards, the exposure again decreases, due to the fact that both bodyweight and the use of non-jarred foods increase. the efsa assumed a daily mean exposure of 1.01 µg /kg bw for children 9 months aged (bfr, 2009; kim et al., 2009; bakhija and appel, 2010; lachenmeier et al., 2012). the only exception is the study of kim et al. (2009a) from korea, which reported a considerably lower exposure (0.017-0.084 µg/kg). the difference between korea and the high exposures in europe might be explained by cultural differences (e.g. less consumption of commercially jarred foods) or that major sources were overlooked in the study. the data show a potential public health concern for this contaminant mostly for consumers 9 months aged (efsa, 2005). the exposure data were then used to characterize risk using the margin of exposure method based on a benchmark dose lower confidence limit for a 10% response (bmdl10) of 1.28 mg/ kg bw/ day for hepatocellular tumours in rats (carthew et al., 2010). the margin of exposures (moes) was below the threshold of 10000, which is often used to define public health risks (lachenmeier et al., 2012). the foremost question that should be considered in the exposure estimation of furan in complementary foods is the treatment of the different subgroups of complementary foods, as beverages in particular contain lower concentrations than other groups. in the past, this differentiation was not made. therefore, depending on the proportion of analysed beverages, the average furan content might be underestimated mostly in children 9 months aged. 2. conclusions we can conclude that furan is present in a variety of heat-treated commercial foods for adults (coffee, sauce and soup) and infants (jarred and canned baby food, breakfast cereal). preliminary studies indicate that home-prepared food, with the exception of coffee, hardly contain any furan above the limit of detection (efsa, 2010). for the home cooked foods, foods rich in carbohydrates are most likely to form furan, probably due to a maillard browning reaction of the food. high levels were found in 40%   3%  6%   25%   13%   breakfast  cereals   bread   vegetables  and  fruit   stew  and  sauces   cake     ital. j. food sci., vol 28, 2016 173 toasted bread and the content was correlated to the browning level, therefore not to toast the bread to a dark brown color might reduce the intake of furan. even the worst case scenarios using ingredients containing furan for the home cooked foods did not lead to evaluated levels of furan during cooking. for ready-to-eat foods with initial occurrences of furan, cooking reduces the level of furan in the food to about half the initial concentration. nevertheless, furan is stable in hot food items and the loss of furan present in the food before heating compared to the content after boiling the food is negligible. the furan level remained stable for one hour after heating and it can therefore be concluded that furan is stable in the food items. it was not until the food was reheated that the level of furan decreased (fromberg et al., 2009). it appears to be possible to reduce the furan content in some food by volatilisation through heating and stirring of canned or jarred foods in an open saucepan. recent studies have suggested that a simple approach to avoiding furan would be to heat infant foods in an open can while applying stirring (jestoi et al., 2009; liu and tsai, 2010). this would really result in a considerable evaporation of furan, if parents would adhere to this practice. the first studies regarding this phenomenon reported losses of 29-55% in vegetable purees during different warming procedures in microwave ovens (zoller et al., 2007), or even losses of up to 85% reported during heating opened jars over a period of 5.5 h in boiling water, and a reduction of ca.50% if the baby food jar was opened but not heated (goldmann et al., 2005). reduction of furan in foods is likely to be more challenging compared to other process contaminants, for two reasons. first, there may be little room for maneuver to lower heating times and temperatures because the processes of pasteurisation and sterilisation are indispensable for the microbiological safety of foods. second, furan has a wide range of precursors. ascorbic acid shows the highest potential to form furan, followed by polyunsaturated fatty acids and then sugars (stadler, 2006). ascorbic acid and polyunsaturated fatty acids are regarded as desirable food components because of their health benefits. the best approaches appear so far to involve intervention in the reaction mechanisms. for example, formation of furfural from ascorbic acid in model orange juice was repressed by the presence of ethanol and mannitol acting as free radical scavengers (shinoda et al., 2005). reduction of atmospheric oxygen reduces the autoxidation of unsaturated fatty acids and also reduces furan formation from several precursors, notably ascorbic acid, as the addition of sulphite does (mark et al., 2006). therefore, modification of the atmospheres within heating systems might be effective in reducing furan in foods. since the carcinogenicity of furan is probably attributable to a genotoxic mechanism (efsa, 2004) levels in food should be kept alara as low as reasonably achievable. currently, the limits for furan in food have not been established yet by european legislation. fda recommends that consumers eat a balanced diet, choosing a variety of foods that are low in trans-fat and saturated fat, and rich in high-fibre grains, fruits, and vegetables (fda 2004c). under the circumstances described previously, the continuation of the research is desirable for achieving safer and healthier foods. references albala-hurtado s., veciana-nogues m.t., marine-font a. and vidal-carou m.c. 1998. changes in furfural compounds during storage of infant milks. j. agric. food chem. 46: 29983003. anese m. and suman m. 2013. mitigation strategies of furan and 5-hydroxymethylfurfural in food. food res. int. 51: 257264.   ital. j. food sci., vol 28, 2016 174 arribas-lorenzo g. and morales f.j. 2010. estimation of dietary intake of 5-hydroxymethylfurfural and related substances from coffee to spanish population. food chem. toxicol. 48: 644-649. bakhiya n. and appel k.e. 2010. toxicity and 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however, the tpg understood it as “healthy life” and “food terror”. individuals with higher education showed a high interest in the food package. halal certification was highly appreciated by tg and tpg. 2 ital. j. food sci., vol. 27 2015 introduction turkey is a developing country and a recent official report on the growth rate in the agricultural and food sector has clearly demonstrated that turkey has gradually increased its yearly food production (mfal, 2012). in 2013 in turkey, cheese production is set to register 9% retail value growth and drinking milk products will likely register a growth of 6%. the market of both meat and meat products and of fish and fish products has recorded an average annual growth of 11% and 13% over 2007-2012, respectively. in the same period, turkey’s market for vegetable, potato and fruit products recorded average growth of 8% (euromonitor, 2014). the issue of quality control and of an efficient quality assurance is essential and inevitable in every single food production line. any risk, hazard and potentially undesirable substance entering the food chain must be monitored by the authority (yasar, 2011). turkey is a candidate country of the european union (eu) and its progress is very appreciated in terms of implementation and reinforcement of the common agricultural policy (cap) and of the eu regulations on food safety since the start of the eu accession negotiation (october 2005). turkey has now changed its national food safety policy to align with the eu rules. as a consequence, the turkish public opinion has become more aware on important issues of food safety, such as genetically modified organisms (gmos), the ban of unofficial food production, food traceability and the rapid alert systems (mfal, 2012). the effects of such publicity should be monitored in terms of consumer perception, habits and response to these issues in order to take the necessary actions in view of the eu accession. public perception and awareness on food safety are not only about human health but also about agricultural diversity, ecology, environmental protection and food culture (holm and kildevang, 1999). the effects of the reinforcement of new food safety regulations in the public mind of a given country are under the influence of an efficient publicity by the media as well as by the public authorities. any innovation and technological change associated with food production (e.g., gmo or residues of pesticides) is perceived as somehow important to food consumers (holm and kildevang, 1999). policy makers are primarily interested in consumers’ attitudes towards food safety and their related practices. moreover, food producers and retailers, public authorities and health educators shall know the reflection of food safety in the public mind not only in turkey, but also in wider communities, which mainly determine the direction of food production to a better quality of life (francis, 1979; rozin et al., 1999). the effect of the media in the food risk communication has been widely discussed. it was recently concluded that the mass publicity on food safety risk management may have induced long-lasting effects on the perception by the europeans, particularly in the case of gmo derived food products (swinnen and vandemoortele, 2010). therefore, mislead or exaggerated information created by the media in the risk communication between the authority and public can be corrected by the evidences based on scientific data. the authority should pay more attention to publish the information supported by scientific data and literature, since such scientific evidences are much more appreciated by the consumer and are seriously taken into account, as already reported (frewer et al., 1997). this attitude leads to an efficient risk communication. similar implications were reported earlier from a study conducted on turkish subjects (aygen et al., 2012). on the other hand, earlier reports, for instances, on the cases of dioxin, bse or foot and mouth disease, were published more extensively than the corrective scientific actions adopted later (swinnen et al., 2005). this indicated that the policy makers should communicate with the public to measure their level of understanding of any modifications and changes in the risk management and how the quality of life would have been enhanced by implementing the new changes. the way in which information is received possibly determines the perception by the public on the food risks. it was reported that most of consumers are rationally ignorant at first glance (mccluskey and swinnen, 2004). for instance, the public opinion think that organically grown products which naturally could bear a high risk of mycotoxins are safer than conventional food products where such risks are easily manageable (loureiro et al., 2001). gender and education are consistent demographic predictors of food-risk perceptions. furthermore, non-demographic predictors are also important, and these include the nature of the perceived threat, the public’s trust in regulatory authorities, the source of the information and the way in which it is distributed, and health and environmental concerns (ellis and tucker, 2009). there are some studies undertaken in turkey which partially determined the degree of perception and awareness of food safety in the country (sanlier and konaklioglu, 2012; bektas et al., 2011; demirbas et al., 2012; aygen, 2012; unusan, 2007; celile, 2012). the credibility of the source of information was shown to highly influence the attitude-formation condition, but its impact on changing the present attitude is low (kumkale et al., 2010). strong correlation was reported between the food safety knowledge, attitude and practices (sanlier and konaklioglu, 2012). the females were found to be more knowledgeable than males, in particular for household safety (celile et al., 2012). ital. j. food sci., vol. 27 2015 3 a high income and education level as well as the increased age increases the probability of having knowledge on food safety (bektas et al., 2011). male consumers are more attached to the attribute “safety” compared to female consumers (verbeke and viaene, 1999). furthermore, males below the age of 30 attached significantly less to the absence of hormones and harmful substances in food than did older consumer categories (verbeke and viaene, 1999; celile et al., 2012). total food safety knowledge was also found to be statistically higher in female than male students (sanlier and konaklioglu, 2012; aygen, 2012; unusan, 2007). previous technical knowledge on a given food production line inevitably affects the awareness of food safety, particularly in the house members in turkey (celile et al., 2012). however, debirmas et al. (2012) indicated that the milk producers did not demonstrate a good level of food safety awareness. moreover, two surveys regarding the awareness of household food safety (unusan, 2007; celile, 2012) revealed that the gender, age and education level are important attributes for such awareness. in the present survey, we measured the response of different social groups of the turkish public opinion to specific parameters of food safety issues and we compared them with the response of a group of eu citizens. the aim of the study was to register the diversity of the consumer attitudes towards the safety of food and to establish a relationship between consumer attitudes, knowledge and behaviour of the different groups regarding food safety. thus, this will be the first study to extensively reveal the degree of perception and awareness of consumers on recently introduced aspects of food safety during the eu accession negotiation of turkey. materials and methods selection and participation of the subjects a questionnaire consisting of ten specific questions related to the perception and awareness of food safety was developed (table 1). three consecutive surveys, each carried out on a socio-demographically different group were carried out using the questionnaire of table 1. three groups of subjects were interviewed. the first group was from national academic and administrative staff employed at the suleyman demirel university (sdu) located in isparta (turkey). this group (identified in the manuscript as turkish educated group tg) consisted of 242 persons who answered the online available questionnaire. the europeans attending an erasmus intensive programme on food and feed safety (iprasaff, 2012) formed the second group. the number of subjects (university students and professors) in the second group (identified as european educated class eg) was 73 and was also asked to answer the online survey too. the number of respondents was 47. the third group consisted of randomly selected subjects living all over turkey. the total number of participants in the third group was 250; the persons were randomly selected on the street in several towns in turkey. this group was identified as “turkish public group tpg”. preparation of the questionnaire the survey team prepared several questions during the lecture courses of the third year class of undergraduate students at sdu. the academic lecturers who were specialised table 1 questionnaire on the perception and awareness of food safety. no. question (with options) possible answers 1 your age 15-20 20-30 30-50 over 50 2 education level primary secondary university 3 country of origin turkey poland non-european belgium the netherlands italy continued table 1. 4 ital. j. food sci., vol. 27 2015 4 gender female male 5 which ministry regulates food safety health environment agriculture/food energy tourism 6 what is the degree of importance producer name of the following item when you buy a product? (no, low, medium, high) price food label price and producer 7 what is the degree of importance quality control of the following item in respect of food safety? healthy life and nutrition (no, low, medium, high) gmo-food terror etc. natural/organic foods high cost of living 8 how do you regulate your daily life consumption of foods sold in open-air market according the concept of food safety sensitive to food-packing materials (no, very low, low, normal, high, very high) sensitive to “expiring date” complaining on “food-fraud” buying foods packed in syntetic-plastic material eating in the restaurants/places you have no idea of eating ready-made foods preference for organic/natural foods do you trace the origin of the food you buy? buying foods containing additives eating “fast-foods” 9 how often and where do you call when you call emergency (e.g., 112) have serious complaints on the foods you buy? call police number (no, seldom, normally, always) call a specific number (such as 174 aloo food in turkey) call food inspector warning the seller search the internet tell friends 10 if you think the publicity on food safety what needed to be improved is not sufficient, what do you recommend the label shall contain “gmo” or “gmo-free” to be done by the authority? “public awareness” shall be improved by the ministry the names of the food firms making frauds shall be made “public” “unofficial” food production shall be banned the results of “official controls and food analysis” shall be made “public” all the food-producing-locations shall be certified all kind of un-official animal slaughering and marketting of such meat shall be banned animal farms shall be “officially controlled” the food label shall contain “the information on undesirable substances and their legal limits” the label shall contain “the names of allergenes” synthetic food additives should be indicated on the label food tracebility should be extended the open-air market shall be “officially controlled” green-houses and fields of plant production shall be “officially controlled” fresh vegetable and fruit wholesalers and retailers shall be “officially controlled” the imported food shall be known by the consumer the food with no label shall be banned “food terror law” must be reinforced halal certificate shall be issued for “suspicious foods” for muslims no. question (with options) possible answers continued table 1. ital. j. food sci., vol. 27 2015 5 in food safety filtered the questions. the criteria for the final selection of the questions were as follows: simple to answer; demographically representative; relevance to daily life and nutrition; must test the general knowledge on food safety, food authority and current food safety issues and finally they should reveal the public concerns on the up-to-date problems and solutions. ten questions were selected and categorized. the questions were created and published online, and a direct link was sent to the tg and eg participants by email. the subjects of tpg were face-to-face interviewed by the survey team of students during the holiday at their hometown. data analysis data from the groups of tg and eg was collected, stored and statistically analysed by using a commercial survey website where the questionnaire was posted (surveymonkey, 2014). a cross-check for the compliance of analysed data was carried out through minit ab statistical package programme (minitab inc., coventry, uk). all the results were then presented for each of the survey groups. the number of responses by the participants to the questions in the form of yes/no was subjected to the estimation of frequency. this was calculated by dividing the number of positive/negative responses by the number of participants who answered the questions. in the multiple choice questions, the percentage or frequency of participants who ticked each of choices was similarly calculated as mentioned above. thus, the number of non-respondents was excluded from the frequency calculation within each question. additional descriptive statistical elaboration of data was achieved by using instat software ver. 3.05 (graphpad software inc., la jolla, usa). results and discussions socio-demographical parameters (q1 to q4) in table 2, the socio-demographic data of the participants are reported. the frequency of age, gender, education level and type of occupation is reported as percentage for the groups of tg, eg and tpg, respectively. the participating subjects have similar age profile irrespective of various social-demographical groups in the present research; in fact the age composition did not differ statistically among the three groups according to the non parametric repeated measures anova (friedman test) performed with instat software (p=0.6271). the percentage of subjects aged between 15 and 20 years was around 6%; the percentage of subjects aged between 21 and 50 years was around 85% and table 2 socio-demographic data of the participants. age tg %1 eg % tpg % 15-20 5 6 6 21-30 37 72 31 31-50 49 17 50 over 50 9 4 12 n2 242 47 250 gender tg % eg % tpg % male 66 32 70 female 34 68 30 n 240 47 250 education tg % eg % tpg % primary 0 0 20 high school 4 0 20 university 60 64 60 msc or phd diploma 36 36 0 n 242 47 249 occupation tg % eg % tpg % students 28 64 14 academics 41 36 0 administrators 42 0 0 contractual staffs 2 0 0 public servants 0 0 42 drivers 0 0 2 farmers 0 0 10 running own business 0 0 32 n 238 47 246 1tg: turkish educated group; eg: european educated group; tpg: turkish public group. 2n is the number of participants who answered to the question. the frequency (%) is calculated from the total number of participants who provided an answer. those aged over 51 years was only 7%. so, the majority of the participants in the survey aged from 20 to 50 (statistically higher number of persons with an age ranging 21-50 years with respect to the other age groups, according to the friedman test, p=0.0330). the gender ratio (m:f) was 65 or 70 male versus 35 or 30 females for tg and tpg groups, respectively as compared to m:f ratio of 32:68 in the international group of eg. the level of education differed significantly between the studied groups: tpg had 20% subjects with no higher education while the majority (100%) in tg and eg groups was graduated from higher education institutions (tpg was statistically different with two-tailed p=0.0215). the same pattern well reflected the type of occupation in the groups: the majority of tg and eg groups were students and lecturers with less number of administration staff, while the majority in the tpg group was either of public servants or persons engaged in private business. 6 ital. j. food sci., vol. 27 2015 table 3 the level of awareness by the participants on the ministerial authority of food safety in their respective countries. ministry tg % eg % tpg % health 18 21 15 environment and forestry 1 0 0 energy and natural sources 0 0 0 food, agricultural and livestock 81 79 85 awareness on food safety management authority (q5) it is very important for the public opinion to be aware of the public authority regulating the food safety aspects in a given country. the majority (over 80%) of the participants irrespective to the national and international group indicated that food safety management is regulated by the ministry of agriculture, food and livestock (table 3). in second place, the participants chose the ministry of health. the friedman test showed no significant differences (p=0.7613) among the three groups of participants. this is, in fact, a good result, indicating that the public is highly aware of the activities of the authority. food safety is mostly perceived by the public as health-related issue. however it is well known that food safety is an integrated issue which covers not only the health of human subjects, but also the health of animal and plants as well as mark and price of food products are of high importance for all the subjects (70%), especially to the subjects of tg and tpg (97%), whereas the food label became another important parameter (53% of all the subjects) in addition to the producer’s name and price of the food product especially in the eg group. the price per se was not very important to any of the groups (32% of all the subjects). perception of the food safety (q7) considering the totality of the subjects answering question no. 7 (table 1) (456 persons), the highest level of perception was assigned to “healthy life and nutrition” (81.5%), “quality control” (77%), “gmo-food terror etc” (63.5%) and “natural/organic foods” (54.5%). less importance was given to the “high cost of living” (21.2%), thus to the economic cost of high quality food products. considering each group of subjects (table 4), tg had a high level of perception for “quality control”, “healthy life and nutrition”, “gmo etc.,” and natural/organic foods” in regard to food safety. the subjects of eg have a high level of perception for “quality control”, “healthy life and nutrition” and “high cost of living” regarding food safety. gmo issue for this international group was not of high importance as compared to tpg. highly perceived parameters of food safety in the tpg were the gmo issue and “natural food”, lastly “healthy life and nutrition”. this may be due to the fact that the gmo issue is very well regulated by the european union compared to the actual turkish legislation. it is easy to understand that the turkish public opinion could be highly manipulated by the media on the issue of gmo, since this matter has not been yet fully addressed by the national authority. application of the concept of food safety into daily life (q8) the public opinion can only make changes in the daily life style upon the scientific evidences and information found in the media (mccluskey and swinnen, 2011). we determined what these changes are like (table 5) in the three social groups. the international group (eg) felt safe enough towards the food products sold in open markets (49% in eg versus 25% in tg and 22% in tpg). this is simply due to routinely controls operated in open markets in the eu. sensitivity to food packing materials received 91% preference of tg, 70% of eg and only 52% of tpg. this was found highly related with the education level. similar trend between the subject groups was found for the sensitivity to “expiring date” of the food products. in all groups, the “food complaints”, the protection of the environment. therefore, an authority which deals with a wide range of aspects related to food safety should be extremely efficient and collaborate quickly with all the involved stakeholders (private companies and/or other public bodies). it should be also mentioned that in most cases food safety may be managed by different public bodies in different countries, however the answer of all the groups participating to the survey was the same. in italy for example (unlike turkey), food safety is jointly administered by the ministry of health and by the ministry of agriculture, food and forestry; the latest is responsible for the food policies. also other ministries can be involved in specific aspects of food safety, such as the prevention of food frauds (e.g., defense in italy) and financial irregularities related to the food sector (economy). consumer buying behaviour (q6) when buying a food product there are many criteria taken into account by the consumers. of the eligible answers proposed in the survey (table 1, question 6) the combination of trade ital. j. food sci., vol. 27 2015 7 table 4 perception level of “food safety” by the consumers of different socio-demographic classes (n= 242, 47, 250 in tg, eg and tpg, respectively). tg degree of importance (in %) quality control healthy life gmo-food natural/organic high cost and nutrition terror etc. foods of living none 2 2 3 3 9 low 3 3 10 9 23 medium 17 16 17 28 43 high 78 79 71 60 25 total1 100 100 100 100 100 eg none 5 0 11 9 13 low 0 11 30 28 33 medium 27 34 34 28 42 high 73 55 26 35 11 total 100 100 100 100 100 tpg none 0 0 2 6 8 low 4 1 2 8 12 medium 47 44 59 57 40 high 49 55 37 30 40 total 100 100 100 100 100 1the total percentage is referred to the total number of participants (n) who answered the questions. “buying food in synthetic materials”, “eating at the places having no idea before”, “tracing the origin of foods”, “buying food containing additives” and “eating fast foods” received less frequency, while the preferences towards organic or natural food received moderate frequency by all the groups. the friedman test did not evidence statistical differences among the three groups of subjects (p=0.2557) due to the high standard deviation of the answers. this aspect can be encouraging because it shows that the perception of food safety into daily life is very similar in european and turkish citizens of different extraction. reactions to food complaints (q9) subjects were asked to indicate how they react against any food complaint they face to in their daily life (table 1, question 9). in all groups, the frequency of calling a specific number for help or calling for a police was low (72.9% of the participants has never called any emergency, police, or specific phone number to complain). twenty percents of turkish participants indicated that they called “the national food line”, alo 174. this is a very promising result because this specific public phone line has been activated only recently and is already quite well-known by the table 5 application of the concept of food safety into daily life (n= 242, 47, 250 in tg, eg and tpg, respectively). preventive reactions tg frequency1 % eg frequency % tpg frequency% consumption of foods sold in open-air market 25 49 22 complaining on “food-fraud” 55 79 41 buying “foods packed in synthetic-plastic material 50 60 49 eating in the restaurants/places you have no idea on 26 46 21 eating “ready-made foods” 50 65 68 positive reactions sensitive to food-packing materials 91 70 52 sensitive to “expiring date” 95 87 63 preference towards “organic/natural” foods 79 68 77 do you trace the origin of the food you buy? 59 64 60 buying “foods” containing additives 29 62 38 eating “fast foods” 34 30 60 1the total number of participants (n) who answered the questions was reported as “total percentage”. note that the answers were pooled from upper degree of satisfaction (high, very high answers of table 1, question 8). 8 ital. j. food sci., vol. 27 2015 participating subjects. the most frequently actions amongst all groups were “warning the seller” (34.3%) and “tell the friends” (30.7%). the answers “search on the net” (28.7%) and “call for a food inspector” (21.2%) occurred with moderate frequency. priority list of the subjects for possible changes in food safety management (q10) question 10 of the survey had the aim to establish a list of priorities stated by the public opinion regarding the policy of the public authority on the issue of food safety. the results are reported in table 6. the turkish educated class (tg) highly demanded for the following aspects to be improved: “the food should be labelled if gmo is included”, “the ministry should improve public awareness on food safety” and “the names of firms involved in frauds should be made public and falsified foods should be banned”. the turkish public group (tpg) found the latter two options very important as well. tpg group would also like to see: “the food with no label being banned”, “all kind of unofficial animal slaughtering and marketing of such meat shall be banned”. however, these options were not very prioritized by the international group (eg). what the eg group mostly preferred were: “public awareness be improved”; “more official controls of foods”; “food labelling for allergens” and “banning food with no label”. these differences between national and international social classes were inevitable because europeans have new priorities such as the issue of allergens and more official control than turkish citizens who are still facing problems which have been already solved in europe, such as “illegal food production” and “gmo containing foods”. halal food aspects received 50-80% attention by turkish subjects, unlike eg. conclusions the results of this survey revealed important information which is very valuable not only for food researchers, but also for policy makers. the majority of the respondents was aware which authority is responsible for food safety at national level but did not clearly understand how to make food complains (mostly made to food companies instead of public institutions). in addition, the concept of food safety and subsequent behaviours were greatly different among socialdemographically different classes at both national and international level (question 7 to 10). altekrause et al. (1999) found that men are more likely to report risky practices than women. in our survey, the tpg was mostly formed by males belonging to the working class. they showed a higher concern (compared to the students of the tg and eg groups) towards the intrinsic safety of the food products and probably to the family safety (i.e., food terror law, ban of unauthorized slaughters, less trust in open-air markets, severe control of wholesalers, publication of the producers implicated in frauds, publication of the results of the public controls). social and individual factors could dampen the perception of the risks (flynn et al., 1998). the risk perception was reported to be influtable 6 consumer priorities on the actions requested to the authority (n= 242, 47, 250 in tg, eg and tpg, respectively). national and international social groups1 tg group eg group tpg group priority list of food issues % % % the label shall contain “gmo” or “gmo-free” 79 46 92 “public awareness” shall be improved by the ministry 78 56 91 the names of the food firms making frauds shall be made “public” 78 1 94 “under the cover” food production shall be banned 77 1 93 the results of “official controls and food analysis” shall be made “public” 73 52 90 all the food-producing-locations shall be certified 72 41 92 all kind of un-official animal slaughtering and marketing of such meat shall be banned 69 35 94 animal farms shall be “officially controlled” 69 43 91 the food label shall contain “the information on undesirable substances and their legal limits” 67 33 92 the label shall contain “the names of allergens” 67 57 91 synthetic food additives should be indicated on the label 66 48 92 food traceability should be extended 66 43 92 the open-air market shall be “officially controlled” 65 37 83 green-houses and fields of plant production shall be “officially controlled” 65 30 91 fresh vegetable and fruit wholesalers and trailers shall be “officially controlled” 63 41 92 the imported food shall be known by the consumer 61 46 92 the food with no label shall be banned 61 54 93 “food terror law” must be reinforced 55 30 87 halal certificate shall be issued for “suspicious foods” for muslims 53 28 76 1the results are the percentage of consumers (n) who are in favour of the requests. ital. j. food sci., vol. 27 2015 9 enced by two factors: the dread or the unknown risks (slovic, 1987). food scares are highly considered as the dread risks, whereas gmos, due to their unknown consequences, are rated as highly unknown risks despite the fact that the educated consumers do not think that gmos are risky (slovic, 1987; sanlier and konaklioglu, 2012; aygen, 2012; unusan, 2007). this evidence was also validated by the present study; in fact, both turkish educated and public group highly rated the gmos as risky, while the european educated group did not. on the other hand, initial perception is also important to determine the current perception of public (aygen, 2012). for instance, the intensive publicity on gmos could also affect the present perception (frewer et al., 1997), and the consumers sometimes give more weights to the negative than the positive information (swinnen et al., 2005; mccluskey and swinnen, 2004). this proves that the perception is a complex issue mediated by individual and social factors. thus, the public awareness of turkish public about recently introduced aspects of food safety related to the eu accession negotiation must be improved. we propose the public authority to validate our data and previously data reported for turkey from a nation-wide survey (sanlier and konaklioglu, 2012; bektas et al., 2011; demirbas et al., 2012; aygen, 2012; unusan, 2007). acknowledgements the authors are extremely grateful to veli peksüslü, a thirdyear student, and other students of department of animal science at suleyman demirel university, isparta, turkey for their help in organising the survey questions. the authors would like to give a special thank to the consortium partners and their students of the intensive programme on “regulatory aspects and scientific risk assessment of food and feed safety rasaff-safety”, funded by erasmus llpprogramme (http://projeler.sdu.edu.tr/rasaff-safety/) for their participation in the survey. references altekruse s.f., yang s., timbo b.b. and angulo f.j. 1999. a multi-state survey of consumer food-handling and foodconsumption practices. american journal of preventive medicine, 16: 216-221. aygen f.g. 2012. safe food handling: knowledge, perceptions, and self-reported practices of turkish consumers. international journal of business and management, 7(24): 1-11. bektas z.k., miran b., uysal o.k. and gunden c. 2011. consumer awareness for food safety in turkey. bulgarian journal of agricultural science, 17(4): 470-483. bignebat c., koc a. and lemelilleur s. 2009. small producers, supermarkets, and the role of intermediaries in turkey’s fresh fruit and vegetable market. agricultural economics, 40(s1): 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(in turkish). isparta valiliği avrupa birliği çalışmaları dergisi, aralık, 34-37. paper received january 13, 2014 accepted september 2, 2014 ijfs#713_bozza ital. j. food sci., vol 29, 2017 443 paper effectiveness of a temperature control system in home induction hobs to reduce acrylamide formation during pan frying s. guillén*1, r. oria2, m.l. salvador2, i. martorell1, a. corrales1 and k. granby3 1european atlantic university, parque científico y tecnológico de cantabria, c/isabel torres, 21, 39011, santander, spain 2plant foods research group, instituto agroalimentario de aragón ia2 universidad de zaragoza cita, miguel servet 177, 50013 zaragoza, spain 3technical university of denmark, national food institute, mørkhøj bygade 19, 2860 søborg, denmark *corresponding author. tel.: +34 942244244 e-mail address: sofia.guillen@uneatlantico.es abstract three trials were conducted to determine the influence of the use of temperature control systems on physico-chemical characteristics and acrylamide formation in the domestic preparation of potatoes. french fries were pre-treated by soaking in water or acidified water, and then they were cooked using a range of home-cooking procedures. soaking raw potatoes in acidified water (ph=3.17) before frying at a controlled temperature (180 °c) was the most efficient pretreatment for reducing acrylamide formation (76%). for the same temperature, roasted frozen par-fried potatoes contained less fat and acrylamide than similar pan-fried potatoes. potatoes butter fried at 140 °c had an acrylamide concentration similar to that of potatoes fried in oil at 180 °c, but this value was reduced by 71% when the frying was carried out using a temperature control system. controlling the frying temperature reduced acrylamide formation at all the temperatures studied. keywords: acrylamide, frying, induction hob, temperature control, potatoes, home-cooking, pretreatments, roasting ital. j. food sci., vol 29, 2017 444 1. introduction in 2002, tareke et al. detected high concentrations of acrylamide in heat-processed foodstuffs. this compound is classified as being likely carcinogenic to humans (rosén and hellenäs, 2002). since then, scientists have identified different factors affecting the formation of acrylamide in food. stadler et al. (2002) have shown that acrylamide can be released by the thermal treatment of certain amino acids such as asparagine, particularly in combination with reducing sugars, and by the thermal treatment of early maillard reaction products. because potato products are especially high in asparagine, it is currently thought that this amino acid is responsible for the majority of the acrylamide formation in potato crisps and french fries. considerable research has been carried out to develop strategies to reduce acrylamide formation (gökmen, 2006; friedman and levin, 2008; kumar et al., 2014; urbancic et al., 2014; kahkeshani et al., 2015; zuo et al., 2015). the main problem in developing such strategies is that the main pathway is the same as that for the formation of compounds with desirable aromas during frying (matthäus, 2009). therefore, the use of reliable temperature control systems during frying to avoid overheating is necessary to prevent further acrylamide formation (gertz and klostermann, 2002; majcher and jelen, 2007; arias-mendez, 2013). some authors have studied the effect of different pretreatments on acrylamide formation during frying. for example, lowering the ph by adding acids before frying has been found to be an efficient way of considerably diminishing acrylamide formation for french fries (jung et al., 2003; pedreschi et al., 2007). roasting produces lower acrylamide contents than microwaving. hence, the important factors influencing acrylamide formation are not only the heating temperature and time but also the modality of the heat transfer process (claeys et al., 2005; yuan et al., 2007). the heating source is one of the most sensitive parts of the equipment for the formation of acrylamide in the product. it is important to ensure a consistent transfer of heat from the heating source to the product (matthäus, 2009). with most household appliances, the temperature settings are uncertain. recently, induction hobs have been introduced on the market. the hob is equipped with infrared sensors that measure the heat emitted by the pan and accordingly adjust the temperature of the food being fried so that the desired temperatures are maintained within ± 2 °c. the main objective of the present study is to evaluate the possible effect of the use of temperature control systems on the formation of acrylamide, taking into account the organoleptic characteristics of the fried product together with color, water loss and oil uptake. three trials were conducted for this purpose. the aim of the first test was to determine the effect of using induction hobs with temperature control systems on the abovementioned factors. in addition, the effect of preparation by par-frying and freezing of fresh potatoes was studied. in the second trial, the possible additional effects of two pretreatments (water immersion or immersion in water acidified with vinegar) on the temperature-controlled frying were studied. finally, in the third trial the aim was to establish a comparison between pan-frying and other cooking methods for potatoes (oven roasting and pan frying in butter at medium temperature), given the lack of data in the literature on the presence of acrylamide in home-cooked potato dishes. ital. j. food sci., vol 29, 2017 445 2. materials and methods 2.1. materials fresh potatoes (variety bintje) and frozen par-fried potatoes (variety fontana) were provided by flensted a/s (ansager, denmark). high oleic sunflower oil (riquísimo koipesol, deoleo, madrid, spain), vinegar (x-tra vinegar, fdb, copenhagen, denmark) and butter (x-tra butter, fdb, copenhagen, denmark) were obtained from local supermarkets. 2.2. cooking procedures fresh potatoes were stored at 4 °c and subsequently washed, peeled and cut into strips (5 cm x 1 cm x 1 cm) or slices of 3 mm thickness before frying. the samples were fried on an induction hob provided with an automatic control system to maintain a specific temperature once it was reached (power induction, bosch pib675l34e, bsh, munich, germany). pan frying was done in a saucepan (18 cm diameter) using a ratio of 100 g of potatoes per 100 ml of oil. to evaluate the effect of the temperature control (trial 1), fresh and frozen par-fried potato strips were fried for 10 min at initial oil temperatures of 160 °c, 180 °c and 200 °c with and without temperature control. the aim of trial 2 was to test if soaking pretreatment of the potato strips could mitigate the amount of acrylamide even in the potatoes fried using the temperature control. fresh potato strips (100 g) were soaked for 20 min in a tap water bath (1 l) at room temperature or in an acidified water bath with vinegar (ph= 3.17). once the superficial water retained in the samples was eliminated with paper towels, the potatoes were then fried following the same frying conditions as used in trial 1. in trial 3 alternative domestic preparations to frying were carried out: a) fresh potato slices (100 g) were fried for 7 min on each side in butter (5 g) at lower than usual initial frying temperatures (140 °c), with and without temperature control, and b) frozen par-fried potatoes (100 g) were placed in a universal pan and roasted for 25 min at 170, 175 and 180 °c in a pre-heated electric oven with forced-air circulation (iq500 siemens, bsh, munich, germany). during frying the temperature profiles of the oil were monitored by a type k thermocouple probe and recorded by a data-logger (testo loger 177.t4). three batches were made for each of the experimental conditions. superficial oil retained in the potatoes was eliminated with paper towels and the samples were analyzed. 2.3. acrylamide determination the liquid chromatography-tandem mass spectrometry method described by nielsen et al. (2006) was used for the determinations, with slight modifications. thirty ml of milliq water was added to a 0.3 g aliquot homogenate. acrylamide (2-propene amide) [cas no. 79-06-1] (>99.5%) was obtained from sigma-aldrich (st. louis, mo, usa). labelled d3acrylamide (> 98%) was supplied by polymer source inc. (dorval, quebec, canada). the potato strips were homogenized using a mixer (model 4169/4297, braun ag, kronberg, germany). the acrylamide analysis was performed on 3 g aliquots of homogenized fried potato samples with an added internal standard comprised of 150 μl of 10 μg ml-1 d3acrylamide and 30 ml of deionized water. the sample was extracted by a homogenizer (model ultra turrax t25, janke and kunkel, staufen, germany) at 1,000-1,200 rpm for 2 min. the sample was then centrifuged at 500 g for 20 min (heraeus multifuge, osterode, germany) and an aliquot of 2 ml was transferred to an eppendorf vial, frozen to -18 °c for at least 30 min and subsequently centrifuged in an eppendorf centrifuge at 12,100 g for 10 min (minispin centrifuge, eppendorf ag, hamburg, germany). the sample was ital. j. food sci., vol 29, 2017 446 thawed during centrifuging, and starch precipitated from the supernatant at this low temperature. the spe (solid phase extraction) cleanup was performed by an automated sampler (gilson aspec xl, gilson company inc., lewis center, oh, usa) using licrholut rp-c18 spe-cartridges (500 mg) from merck (darmstadt, germany). the spe columns were conditioned with 2 ml of methanol, 2×2 ml of water, and 0.5 ml of sample lead to waste. subsequently, 1.75 ml of sample was loaded onto the cartridge and the eluate transferred to miniprep ptfe filter hplc vials with a pore diameter of 0.45 µm (whatman inc., little chalfont, uk). the lc system consisted of a liquid chromatograph (model hp1100, agilent technologies, santa clara, ca, usa). separation was performed with 0.1% formic acid in water, with a flow of 0.2 ml min-1 on a hypercarb column (dimensions 2.1 mm x 100 mm, particle size 5 µm). the ms-ms detection was performed using a micromass quattro ultima triple quadrupole instrument (waters corporation, milford, ma, usa). the source was maintained at 120 °c and the desolvation gas at 400 °c. nitrogen was used as the cone and desolvation gas with flow rates of 150 and 500 l h-1, respectively. argon was used as the collision gas and maintained at a pressure of 0.24 pa. the detection was performed by multiple reactions monitoring (mrm). acrylamide was detected in positive ion mode. quantification of the fragmentations was done using the masslynx software version 4.1 including quanlynx. each determination was performed in triplicate. 2.4. color determination the potato strip color was measured using digital image analysis. for image acquisition an hp scanjet g4010 scanner (hewlett-packard, palo alto, ca, usa) delivering 4800 x 9600 dpi hardware resolution was used. the image resolution was set at 200 ppp. digital image processing was performed using the matrox inspector 8.0 software (matrox electronic systems ltd., quebec, canada). calibration of the digital system was done using the une 48-103-94 spanish color norm (aenor). the correlation with cielab values was calculated using a quadratic model (león et al., 2006). once tempered, the surfaces of 5 potatoes for each batch (15 potatoes for each condition) were scanned. the corresponding rgb (red, green and blue) coordinates were obtained and, applying the transformation model, the corresponding cielab coordinates were calculated. 2.5. fat content samples previously finely ground in a cooled mill homogenizer ika a10 (janke and kunkel, staufen, germany) were extracted with 80 ml of petroleum ether to a 2055 soxtec (foss, hillerød, denmark). the extraction program was operated at 115 °c and included 30 min of immersion and 1 h 20 min of extraction and draining. the fat content was determined by weight difference, method 30-25(aacc, 2000). each determination was performed in triplicate. 2.6. statistical analysis statistical analysis was performed using xlstat 2014 software (addinsoft, new york, usa). one-way analysis of variance (anova) followed by tukey’s multiple range test for comparisons of means and least significant differences (p<0.05) were performed with the data. all the data were expressed as the mean±standard deviation. the standard deviation was calculated from 3 (for acrylamide and fat content) or 5 (for color) determinations for each of the three independent experiments. ital. j. food sci., vol 29, 2017 447 3. results and discussion 3.1. temperature monitoring the oil temperature evolution recorded at different initial temperatures with and without activating the temperature control system is shown in fig. 1 for fresh potatoes. during the frying, the oil temperature undergoes sharp changes, even in the case of frying with temperature control, due to the abrupt temperature decrease caused by the addition of the potatoes (up to 45 °c). the oil temperature evolution in the frying of frozen par-fried potatoes follows a similar trend, but the initial temperature drop is more marked (up to 49 °c). for the same initial temperature, the oil is hotter if frying is carried out without control. after a decreasing stage, when the control of temperature is switched off, the temperature begins to increase in an almost linear fashion overshooting the initial value by 36.8 °c when the initial temperature of the oil is 200 °c, by 23.5 °c when the oil is at 180 °c, by 17.2 °c when the oil is at 160 °c and by 11.8 °c when the oil temperature is at 140 °c. however, in the event that temperature control is switched on, the temperature trends asymptotically to its initial value ±2 °c. the temperature differences between frying with or without temperature control decrease as the initial oil temperature decreases. figure 1. oil temperature during the frying of raw potatoes. filled symbols correspond to frying with temperature control and hollow symbols to frying without temperature control. initial temperature: squares, 200 °c; circles, 180 °c; triangles, 160 °c and inverted triangles, 140 °c. 3.2. effect of par-frying-freezing and temperature controlled frying table 1 shows the weight loss, color, fat and acrylamide concentration of fresh and frozen par-fried potatoes of trial 1. the weight loss increased with the temperature, and there were no differences in this respect between samples that were fried at the same initial temperature. the oil uptake in fresh potatoes during temperature-controlled frying was unchanged with increasing temperature. however, it increased in the absence of temperature control. furthermore, these samples had a higher oil content than the corresponding samples that were fried with temperature control. in the case of frozen parfried potatoes, the oil concentration increased as the temperature increased, and again the ital. j. food sci., vol 29, 2017 448 use of the temperature control had a positive effect by reducing the uptake of oil at a higher frying temperature (200 °c). temperature is a factor that directly affects the three main mechanisms of absorption (dana and saguy, 2006; huang and fu, 2014). table 1. effect of par-frying-freezing and temperature controlled frying on the physico-chemical data, fat and acrylamide contents of potatoes. potatoes ti (°c) temperature control weight loss (%) color fat content (g/100 g) acrylamide content (mg/kg) l* a* fresh 160 with 32.6±2.9 b 69.3±1.3 f -1.7±0.8 a 6.4±1.7 a 403±40 a 180 without with 45.3±2.2 cde 44.0±1.6 cde 60.8±2.5 c 68.7±0.3 ef 5.3±0.7 d 2.6±0.7 c 9.4±0.4 b 6.5±1.3 a 1547±120 d 1154±150 c 200 without with 52.1±0.7 e 50.8±0.6 e 59.5±0.8 c 64.1±0.6 cde 6.6±0.1 e 4.9±0.9 d 12.3±0.6 c 6.9±0.8 a 5600±560 h 3030±180 f par-friedfrozen 160 with 28.7±1.1 a 65.7±2.8 def 0.9±0.2 b 16.4±1.1 cd 450±30 a 180 without with 38.2±3.9 bc 41.2±1.5 cd 61.7±2.5 cd 64.3±0.6 cde 5.5±0.2 d 3.2±0.6 c 18.9±1.3 d 18.0±1.3 d 1905±97 e 1632±121 d 200 without with 47.6±0.2 de 46.7±3.3 de 47.0±1.7 a 53.1±1.2 b 10.7±0.3 f 10.0±1.0 f 36.6±1.9 f 24.9±1.3 e 5247±260 h 4267±304 g significant differences (p<0.05) between potato types and temperatures are indicated by different letters. a higher frying temperature causes further degradation of the surface of the potato structure, favoring oil absorption. it also favors a more rapid removal of water, enhances the absorption of oil during cooling, and leads to a faster degradation of the frying medium producing the formation of surface-active substances promoting an increase in the oil uptake. the frozen par-fried potatoes had an initial fat content of 6.0 g/100 g, and showed a markedly higher final fat content than fresh potatoes under the same frying conditions. color changes in french fries during frying caused by the maillard reaction depend on factors such as the content of reducing sugars, the temperature and the frying time (márquez and añón, 1986). the l* and a* coordinates are color parameters that best reflect this darkening (pedreschi et al., 2006) and therefore show greater differences between samples. the l* decreases when increasing the frying temperature of fresh and frozen par-fried potatoes. for the same initial temperature, fresh potatoes are lighter than frozen par-fried potatoes. as a result of frying, the a* coordinate value increases from negative values in fresh potatoes at the lowest initial temperature, 160 °c, (-1.7±0.8), to values which become greater when increasing the frying temperature. these changes in the l* and a* coordinates occur as a result of the maillard reaction, generating compounds that give brownish tones. the rate at which the maillard reaction occurs is strongly dependent on temperature and the concentration of the compounds involved (pedreschi et al., 2006). the frozen par-fried potatoes previously acquire some color in the industrial pre-frying process which could explain the lower value of l* and higher a* for the same frying conditions. french fries fried with the temperature control activated had a lower a* and a higher l* than those fried without controlled temperature. numerous studies suggest a link between acrylamide formation and the development of nonenzymatic browning during frying (pedreschi and moyano, 2005; pedreschi et al., 2006; romani et al., 2009). this is a result of the good correlations between changes in the color parameters l* and a* and the concentration of acrylamide. the concentrations of ital. j. food sci., vol 29, 2017 449 acrylamide in french fries under different conditions are shown in table 1. the color changes show that the acrylamide content increases with temperature. this is consistent with data found in the literature (gertz and klostermann, 2002; rydberg et al., 2003; pedreschi et al., 2006; miao et al., 2014). furthermore, it can be observed that the temperature control reduces the formation of acrylamide. gertz and klostermann (2002), fiselier et al. (2006), and matthäus (2009) noted the importance of using efficient temperature control fryers to ensure the recovery of the initial oil temperature after adding the food to achieve the formation of a crispy crust on the fried product and to avoid overheating during frying that could produce an increase in the formation of acrylamide. in all cases, frozen par-fried potatoes had higher acrylamide contents than the corresponding fresh potatoes. 3.3. pre-treatments pre-treatment by soaking 20 min in tap water or in acidified water (vinegar ph= 3.17) had no impact on the weight loss or oil uptake, but it had an effect on the color of the potatoes; they were paler and less brown (higher l* and lower a*), especially when the initial frying temperature was 200 °c, as shown in table 2. acidification of the soaking water exerted a positive effect with respect to the coordinate l*; therefore, the french fries were slightly brighter. in the control and the pre-treated samples, the a* increased with the temperature. however, soaking in acidified water reduced the development of brownish tones as the a* color coordinate was lower than in the control sample. other authors have found the same trend after soaking in an aqueous nacl solution (pedreschi et al., 2007; santis et al., 2007). this reduction of the coordinate a* could be due to a more limited development of brownish compounds caused by the lixiviation of the precursors of the maillard reaction, reducing sugars and asparagine in the soaking water (márquez and añón, 1986). the pretreatments influenced the concentration of acrylamide in the french fries (table 2). the pre-soaked samples had a lower concentration of acrylamide at all frying temperatures. the results also indicate that the addition of vinegar to the soaking water makes this pretreatment more effective when potatoes are fried at the initial temperatures of 160 and 180 °c. at 200 °c, acidification of the soaking water did not reduce the concentration of acrylamide compared to the sample soaked in water at a higher ph. soaking produced a reduction in the acrylamide content of between 23% and 50%. the lower the temperature, the greater the relative reduction. with the pretreatment with acidified water, the acrylamide content was reduced up to 76%. table 2. influence of soaking on the physico-chemical data, fat and acrylamide contents (μg kg−1) of potatoes fried with temperature control. ti (°c) pre-treatment weight loss (%) color fat content (g/100 g) acrylamide content (mg/kg) l* a* 160 without soaking water bath acidified water bath 32.6±2.9 a 33.6±2.3 a 33.1±3.4 a 68.8± 0.5 d 72.2±3.7 d 73.1±0.7 d -2.1±0.5 a -2.2±0.3 a -2.2±1.0 a 6.3±1.5 ab 5.1±0.6 a 5.2±1.2 a 412±34 c 206±23 b 160±12 a 180 without soaking water bath acidified water bath 42.0±0.3 b 42.6±3.0 b 45.2±2.7 bcd 60.0±3.1 b 64.0±3.0 bc 66.0±2.0 c 2.3±0.3 b 1.9±0.4 b 1.8±0.5 b 6.4±1.4 ab 7.1±0.1 bc 7.2±0.9 bc 1109±134 e 734±58 d 267±43 b 200 without soaking water bath acidified water bath 52.4±4.1 de 50.0±2.3 cd 50.±2.0 cd 54.6±0.9 a 58.9±1.7 b 61.3±3.0 bc 5.5±0.4 d 5.6±0.9 d 4.0±0.7 c 6.9±0.7 bc 7.1±0.5 bc 6.9±0.2 bc 3056±155 h 2348±134 f 2457±113 fg there are significant differences (p <0.05) between values with different letters within the same column. ital. j. food sci., vol 29, 2017 450 some authors, such as pedreschi et al. (2004) and mestdagh et al. (2008), have noted that reducing sugars by blanching or soaking prior to frying reduces acrylamide levels up to 60% in potatoes. furthermore, pedreschi et al. (2007) found that the reduction in the acrylamide concentration was related to the soaking time. compared to the control, they found a reduction of 15% after 60 min and 30% after 120 min of soaking. acidifying the soaking water with citric acid further reduced the acrylamide in the potato slices, especially at lower temperature frying (pedreschi et al., 2004). this is consistent with the results of the vinegar addition in the present study. jung et al. (2003) found the same trend in potatoes pretreated in a solution of citric acid, achieving a 25% reduction in the acrylamide concentration in french fries. they attributed this decline to the decrease in the ph as well as the leaching of free asparagine and reducing sugars from the surface of the potatoes into the solution. the formation of acrylamide is strongly dependent on the ph (rydberg et al., 2003). 3.4. alternative cooking methods table 3 shows the physico-chemical parameters such as weight loss and color as well as the fat and acrylamide contents of roasted or butter fried potatoes. no significant differences between oven temperatures were obtained (p>0.05) for the studied parameters except the acrylamide content. comparing roasted potatoes with those fried in oil (trial 1), it can be concluded that the roasted potatoes had lower levels of fat than the fried ones and they were less brown. the roasted potatoes had acrylamide values between 213 and 742 mg/kg. the temperature strongly influences the acrylamide concentration, as the concentration increased with the oven temperature. however, in this case there was no correlation with the color development, as there was no significant difference in the l* or a* coordinates with the temperature. even at the highest roasting temperature (180 °c), the acrylamide formation was lower than that generated when frying with oil temperature control at the same initial temperature (1632 mg/kg). butter fried potatoes showed significant differences in the color coordinate a* and in the acrylamide content depending on whether or not the temperature control was used. thus, good temperature control markedly diminished the acrylamide content (71% reduction) and reduced the appearance of the brown tones while maintaining the other parameters such as weight loss and fat content. the high concentration of acrylamide in butter fried potatoes, even at a lower temperature than usual for frying (140 °c), could be explained by the reaction between butter, sugars and amino acids such as lysine (uribarri et al., 2010; niquet-leridon et al., 2015). table 3. physico-chemical data, fat and acrylamide contents of roasted and butter fried potatoes. cooking condition weight loss (%) color fat content (g/100 g) acrylamide content (mg/kg) l* a* roasted 170 °c 175 °c 180 °c 47.0±2.4 b 47.6±1.4 b 48.4±1.4 b 63.0±1.3 a 63.1±4.4 a 64.9±2.0 a 2.1±0.6 ab 2.6±1.4 bc 1.9±0.7 ab 11.2±0.5 c 10.2±1.4 bc 11.4±1.1 abc 213±60 a 625±45 c 742±47 d butter fried at ti=140 °c without control with control 44.9±1.1 a 45.3±2.0 ab 63.3±4.0 a 64.7±6.1a 3.3±1.2 c 2.3±0.6 ab 10.4±1.3 bc 9.4±1.1 ab 1123±80 e 323±43 b significant differences (p<0.05) between cooking conditions and temperatures are indicated by different letters. ital. j. food sci., vol 29, 2017 451 4. conclusions the results of this study indicate that it is important to include a system of temperature control in domestic cooking equipment to optimize culinary quality while at the same time mitigating the acrylamide content. such control prevents overheating during frying and enables the formation of acrylamide to be limited in domestic preparations. during frying, potatoes lose more water and capture more oil under higher temperature conditions. potatoes prepared at higher temperatures become darker as they develop more brown colors. frying at controlled and moderate temperatures (180 °c or less) reduces the acrylamide concentration of potatoes up to 46% with respect to potatoes fried without temperature control. the reduction improves to 76% if the potatoes are previously soaked in acidified water (ph = 3.17 for 20 min). the formation of acrylamide varies greatly depending on the method of cooking the potatoes. for the same temperature, roasted potatoes contained less acrylamide than fried potatoes. not only is the temperature control an important factor in acrylamide formation during frying, but the type of frying fat is also critical. the dissociation of the lactose present in butter into reduced sugars is suggested as an explanation for this high concentration in butter fried potatoes. in addition, frying with butter must be performed at moderate temperatures because of its lower smoke point. the substitution of butter with a mixture of sunflower oil and butter could be a solution to obtain a higher smoke point. frozen par-fried potatoes gained more fat, developed a darker color and contained more acrylamide than fresh potatoes when prepared under the same conditions; therefore, frozen par-fried potatoes should be prepared at lower heating conditions than those selected for fresh potatoes. acknowledgements the project: application of culinary technology for the improvement and development of automatic systems for controlling temperature (2012/0138) was financially supported by bsh electrodomésticos españa, s.a. references aacc. 2000. “approved methods of the aacc” 10th ed. american association of cereal chemistry, st. paul, mn. 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extremely thermostable l-asparaginase during french fries processing. extremophiles. 19:841. paper received december 12, 2016 accepted february 22, 2017 ijfs#814_bozza ital. j. food sci., vol. 30, 2018 249 paper descriptive sensory properties of cecina de león d. rodríguez-lázaroa, m. hernández-pérezb, r. capitac,d and c. alonso-calleja*c,d adepartment of biotechnology and food science, university of burgos, e-09001, burgos, spain bagricultural technological institute (ita), regional government of castilla y león e-47071, valladolid, spain cdepartment of food hygiene and technology, university of león, e-24071 león, spain dinstitute of food science and technology, university of león, e-24071 león, spain *corresponding author: tel.: +34 987291000 x 5633; fax: +34 987293073 e-mail address: carlos.alonso.calleja@unileon.es abstract cecina de león is the protected geographical indication of a dry-cured beef produced in northwest spain. a quantitative descriptive analysis (qda)® of three types of cecina de león pieces (thick flank or babilla, silverside or contra, and topside or tapa) was performed by a trained 10-member sensory panel using an intensity non-structured 10-cm length scale. average sensory scores varied between 3.14±1.54 (beef flavour) and 6.95±1.26 (brightness of lean). contra pieces showed lower percentage of unacceptable scores (9.57%) than babilla (10.24%) or tapa (13.09%). frequency of unacceptable values was lower for appearance (4.09%) than for flavour (15.32%) or texture (8.79%) attributes. keywords: cecina, dry-cured meat, quantitative descriptive analysis ital. j. food sci., vol. 30, 2018 250 1. introduction cecina de león is a high value intermediate moisture meat (approximately 50% humidity) produced exclusively in the province of león (northwest spain) from hind leg pieces (babilla or thick flank, cadera or rump, contra or silverside, and tapa or topside) of beef cattle, with a minimum age of 5 years old and weight of 400 kg. this food product has the quality label, protected geographical indication (pgi; ojec, 1996). cecina de león is manufactured following a processing scheme based on the preparation of pieces and profiling (excision from the carcass and rubbing in order to eliminate any remaining blood, and shaping of the pieces for adjusting); salting (with common salt at 35ºc for 0.3-0.6 days per kg weight); washing (with lukewarm water in order to eliminate any remaining salt); settling or post-salting (for 30-45 days in a cold room to allow for a homogeneous distribution of salt within the meat mass); smoking (optional, with oak or holm-oak wood, between 12 and 16 days), and drying (in natural drying kilns with adjustable windows to control the temperature and humidity using the traditional system of “opening and closing windows”, or in industrial drying installations). the whole process takes a minimum of seven months after salting. the production of cecina de león has increased over the last few years from 1,500 manufactured pieces in 1994 to more than 100,000 pieces in 2012. cecina from babilla, contra and tapa make up more than 95% of the production (supervisory council of protected geographical indication cecina de león, private communication). the supervisory council of pgi cecina de león has to control the sensory quality of the cecina pieces in order to detect the presence of defects in the product as well as to certify its typicality in such a way that it can be differentiated in comparison with non-labeled products. most reports on cecina de león refer to physicochemical and microbiological characteristic (garcía et al. 1998; menéndez et al., 2015; molinero et al., 2008). the hedonic and descriptive sensory properties have been scarcely studied (rubio et al., 2007; molinero et al., 2008). to the best to our knowledge, the influence of the type of piece used for manufacture on the descriptive sensory attributes of cecina de león has yet to be reported. this study was designed to describe the sensory properties of cecina de león; to investigate whether the type of meat used for manufacturing has a significant influence on sensory attributes of this foodstuff, and to determine the frequency of intensity scores outside specifications for each attribute and type of meat piece tested. 2. materials and methods 2.1. samples eleven cecina de león pieces (three babilla, five contra and three tapa pieces) were randomly obtained from normal production in different processing plants in the province of león (northwest spain). babilla or thick flank is made up of vastus lateralis, vastus intermedius, vastus medialis and rectus femoris muscles, contra or silverside is composed by semitendinosus and gluteobiceps muscles, and tapa or topside contains the quadratus femoris, semimembranosus, adductor, gracilis, pectineus and sartorius muscles and a fragment of obturatorius externus muscle. 2.2. sensory evaluation a trained 10-member sensory panel (eight males and two females, ranging in age from 23 to 47 years, with experience in sensory evaluations) was used to evaluate attributes of each ital. j. food sci., vol. 30, 2018 251 sample. the trained assessors were selected and trained for two years according to international organization for standardization regulations (iso 6658:2005, iso 8586:2012, iso 11132:2012). a quantitative descriptive analysis (qda; iso 13299:2016) was used to describe cecina pieces, which were evaluated in a tasting room equipped with white fluorescent lighting (iso 8589:2007). scores were given for appearance (cherry colour, brightness of lean, marbling and fat colour), flavour (odour characteristic, flavour characteristic, persistence of flavour, taste characteristic, saltiness, beef flavour and smokiness) and texture (tenderness, juiciness and fibrousness) attributes on a non-structured 10-cm length scale with anchor points one cm from each end, where 0 means absence (white for fat colour attribute) and 10 means great intensity (yellow for fat colour attribute). scores were the distances (cm) from the left extreme. the panelists were also asked to indicate the heterogeneity of the colour (table 1). cecina pieces showing different intensities were used to define the scale for the descriptors (reference standards). table 1. description of the sensory attributes considered in this work. attribute definition cherry colour visual assessment relating to the hue and the lightness (intensity) of the typical red colour of cecina brightness of lean brightness intensity (attribute of a glossy surface showing bright reflection) of the lean surface marbling level of visible intramuscular fat fat colour colour intensity of subcutaneous fat odour characteristic assessment relating to the odour before eating the sample, associated with the ripening and smoking process flavour characteristic assessment relating to the olfactory/gustatory sensation caused by salt, ripening and smoking process persistence of flavour the time during the olfactory/gustatory sensation is perceptible after the bolus has been swallowed or ejected taste characteristic assessment relating to the taste associated with the salt, ripening and smoking process saltiness basic taste sensation elicited by nacl beef flavour flavour after cooking/heating of beef smokiness assessment relating to the olfactory/gustatory perception caused by the smoking of these products with smoke obtained from wood burning tenderness softness and ease of chewing before swallowing juiciness perception of the amount of water released by the product during the first chews fibrousness perception of the amount of muscle fibers detected during chewing heterogeneity of the colour assessment of the uniform distribution of colour on the slice a portion of each piece of cecina was presented to the panelists at room temperature (21±1ºc) for evaluation of visual attributes and odour characteristic, and slices of approximately 2 mm thick were presented for evaluation of the remaining sensorial attributes. samples were randomly labeled with three digit codes and panelists were asked to evaluate each sample in randomized order. mineral water at room temperature was used to cleanse the palate between successive samples. the testing of the eleven cecina pieces was carried out in four sessions (four sets of two or three samples, randomly chosen) at daily intervals. each sample was evaluated by all panelists in the same session. a replication for each cecina piece was carried out in a different session; each session lasting approximately 2 hours. the performance of panel ital. j. food sci., vol. 30, 2018 252 and panelists was confirmed by their reliability, reproducibility and discrimination in sensory descriptive tests (rossi, 2001; rodríguez-lázaro et al., 2002b, c). sensory specifications (represented by the range of intensities tolerated for each attribute) were established by correlating descriptive data with scores from consumer hedonic evaluation (nine-point hedonic scale). intensities of attributes in the qda were considered acceptable when they were associated with scores ≥ 5 in the hedonic evaluation. attributes of positive evaluation (the better the intensity, the better the quality): cherry colour, brightness of lean, marbling, odour characteristic, flavour characteristic, persistence of flavour, taste characteristic, tenderness and juiciness, were deemed as unacceptable if a score lower than 5 was given by the panelist, according to the 10-cm scale. the beef flavour (attribute of negative evaluation) was considered unacceptable when scores were higher than 5. for the remaining attributes (fat colour, saltiness, smokiness and fibrousness), scores lower than 3 and higher than 8 were considered as unacceptable values (rodríguez-lázaro et al., 2002d). statistical analysis. mean and standard deviations for all cecina samples data were calculated. sensory panel evaluation averages were analyzed by an analysis of variance (anova). mean separation was carried out using the duncan’s multiple range test. pearson’s correlation coefficients were calculated. the statistica® 8.0 (statsoft ltd., tulsa, ok, usa) software package was used. 3. results and discussion analysis of variance of the four factors (replication, attribute, type of meat piece and panelist) showed statistical differences (p < 0.001) between scores from different attributes. replication, panelist, type of meat piece or their interactions did not influence (p > 0.05) descriptive scores. the score values obtained by the attributes tested (the mean data of replications were considered) are given in table 2. values differed markedly between samples, as indicated by the standard deviations (std) calculated on all groups of samples, which were much greater than the std obtained from replicate analysis (data not shown). the absence of significant differences between babilla, contra and tapa pieces are probably due to the relatively high standard deviations found, which are mainly due to the heterogeneity of the samples. according to reyes-cano et al. (1994), there are differences between animals (age, breed, sex) that may have an influence on the sensory properties of cecina pieces. even though significant differences were not found between types of piece, contra pieces showed the best behaviour because they scored higher (p > 0.05) than babilla and tapa pieces in five (55.6%) of the nine attributes of positive evaluation (cherry colour, odour characteristic, flavour characteristic, persistence of flavour and taste characteristic), and lower (p>0.05) in beef flavour (attribute of negative evaluation). moreover, contra pieces showed the lowest mean percentage of unacceptable scores: 9.57%, as opposed to 10.24% and 13.09% for babilla and tapa pieces, respectively (fig. 1). the only cecina without any unacceptable (outside specifications) score was a contra piece. no substantial differences were found for average scores between babilla and tapa. however, tapa pieces showed a higher percentage of unacceptable scores, and heterogeneity in colour was detected in 10% of tests. this attribute is an important and desirable sensory property of dry-cured meats when they are sliced (arnau et al., 1998). ital. j. food sci., vol. 30, 2018 253 table 2. average values for the sensory properties of three different types of cecina de león pieces. attribute sensory modality for the attributes meat piece average babilla contra tapa cherry colour appearance attributes 6.70±1.56a 6.94±1.35a 6.87±1.33ab 6.85±1.39ab brightness of lean 7.00±1.39a 6.96±1.19a 6.90±1.27b 6.95±1.26ab marbling 6.63±1.88a 6.64±1.59ab 6.90±1.24ab 6.71±1.58abc fat colour 5.47±1.09bc 5.49±1.14cd 5.49±1.02cd 5.48±1.09d odour characteristic flavour attributes 6.57±1.59ad 6.66±1.36ab 6.57±1.72ab 6.61±1.51bc flavour characteristic 6.07±1.66bd 6.24±1.49ac 5.97±1.79ace 6.12±1.61ef persistence of flavour 6.00±1.55bd 6.34±1.57ab 5.87±1.81ace 6.12±1.63ef taste characteristic 6.07±1.64bce 6.10±1.70bc 5.67±1.88ace 5.97±1.73e saltiness 3.68±0.83f 3.70±0.95ef 3.48±1.15f 3.63±0.97g beef flavour 3.30±1.64f 3.04±1.54e 3.13±1.48f 3.14±1.54h smokiness 4.04±0.85f 4.04±1.01f 3.76±1.11f 3.96±1.00g tenderness texture attributes 6.26±1.48ad 6.30±1.50ab 6.50±1.41abc 6.34±1.46ce juiciness 6.63±1.27ade 6.36±1.53ab 6.37±1.45be 6.44±1.44cf fibrousness 5.04±1.25c 4.83±1.26d 5.01±1.24d 4.94±1.24i grouping attributes1 appearance attributes 6.45±1.60a 6.51±1.45a 6.54±1.35a 6.50±1.46a flavour attributes 5.10±1.90b 5.16±1.97b 4.92±2.04b 5.08±1.97b texture attributes 5.98±1.49c 5.83±1.59c 5.96±1.51c 5.91±1.54c 1,mean score for the attributes in each modality. a 10-cm non-structured scale was used by trained assessors. average values within a column (for single attributes or for grouping attributes) that are not followed by the same letter are significantly different (p<0.05). no significant differences were found between means in the same row. data are the means of 60, 100, 60 and 220 determinations for the first (3 cecina pieces x 10 panelists x 2 replications), second (5 cecina pieces x 10 panelists x 2 replications), third (3 cecina pieces x 10 panelists x 2 replications) and fourth columns, respectively. the lower number of unacceptable scores was obtained by a contra piece (0%) and the higher by a tapa piece, which showed a total of 24 of 140 (14 attributes x 10 panelists) unacceptable values (17.14%). it must be noted that scores considered as unacceptable were close to the intensity range tolerated for each attribute. average scores of appearance attributes were significantly (p<0.05) higher than those of flavour and texture (table 2). moreover, unacceptable values were substantially lower for appearance (4.09%) than for flavour (15.32%) and texture (8.79%) attributes. martín et al. (1999) also observed the best scores for appearance attributes in cecina de maestrazgo pieces. significant (p<0.001) pearson’s correlations were found between marbling and odour characteristic (0.542), flavour characteristic (0.431), persistence of flavour (0.407) and taste characteristic (0.399). these results coincide with findings of de anda-serrano et al. (1999) in ham samples, and may be explained by taking into account the fact that fat compounds are important components of flavour in meat products. according to kauffman (1993), marbling is required to adequately provide flavour attributes. the high correlation coefficients detected between smokiness and characteristic flavour and taste attributes (p<0.001; r>0.7) also agrees with previous findings in smoked meat products (sink and hsu, 1979). ital. j. food sci., vol. 30, 2018 254 for cherry colour, brightness of lean, marbling, odour characteristic, flavour characteristic, persistence of flavour, taste characteristic, tenderness and juiciness, a value was considered as unacceptable when a score lower than 5 was given by the panelists, according to the 10-cm scale. the beef flavour was considered unacceptable for scores higher than 5. for fat colour, saltiness, smokiness and fibrousness, scores lower than 3 and higher than 8 were considered as unacceptable values. figure 1. percentage of unacceptable scores for each attribute tested in three different types of cecina de león pieces. the negative correlation coefficient found between saltiness and brightness (p<0.01; r=0.270) and tenderness (p<0.05; r=-0.197) may be explained by considering the influence of salt on proteolysis activity (guerrero et al., 1996). lower salt levels are related to a higher proteolytic activity and consequently with a higher tenderness and brightness. it should be noted that the normal salt concentration in cecina de león (5.6%) is generally lower than that of ham (rodríguez-lázaro et al., 2002a). finally, a significant (p<0.001) correlation was found between fibrousness and flavour, persistence of flavour and taste attributes (r=0.428 to 0.467). these results are similar to previous findings by buscailhon et al. (1994) in dry-cured ham. according to these authors, higher fibrousness induces longer chewing time, which allows for better extraction and stronger perception of some compounds responsible for taste and flavour. to summarize, sensory properties of cecina de león are not significantly influenced by the type of meat piece used for manufacturing, which is a positive aspect for producers. however, contra pieces showed the best behaviour, with the lowest percentage of outside specifications (unacceptable) scores. flavour attributes showed the highest, and appearance attributes the lowest, percentage of unacceptable scores for all cecina pieces examined. on average, one tenth of the scores for each piece were outside (although close to) the range of intensities tolerated. 0 5 10 15 20 25 30 p er ce nt ag e of u na cc ep ta bl e sc or es attribute babilla contra tapa average ital. j. food sci., vol. 30, 2018 255 acknowledgements the authors wish to thank the supervisory council of pgi cecina de león for technical assistance. the authors have no conflict of interest to declare. references arnau j., guerrero l. and sárraga c. 1998. the effect of green ham ph and nacl concentration on cathepsin activities and the sensory characteristics of dry-cured hams. j. sci. food agric. 77:387. buscailhon s., berdagué j.l., bousset j., cornet m., gandemer g., touraille c. and monin g. 1994. relations between compositional traits and sensory qualities of french dry-cured ham. meat sci. 37:229. de anda-serrano a., rubio-lozano m.s., santillán-valverde m.c. and méndez-medina, d. 1999. análisis descriptivo cuantitativo del jamón tipo “serrano” elaborado a partir del cerdo pelón mexicano [quantitative descriptive analysis of serrano ham from mexican pelón pig]. alimentaria 306:29. garcía i., díez v. and zumalacárregui j.m. 1998. changes in nitrogen fractions and free amino acids during ripening of spanish dried beef “cecina”. j. muscle foods 9:257. guerrero l., gou p., alonso p. and arnau j. 1996. study of the physicochemical and sensorial characteristics of dry-cured hams in three pig genetic types. j. sci. food agric. 70:526. kauffman, r.g. 1993. opportunities for the meat industry in consumer satisfaction. food technol. 47:132. martín m.l., marquina p. and roncalés, p. 1999. bull “cecina” of maestrazgo: processing, characterization and shelf-life. in: “proceedings of the international congress improved traditional foods for the next century, 28-29 october, 1999”, q68-q71. valencia, spain. menéndez r.a., rendueles e., sanz j.j., capita r. and garcía-fernández c. 2015. behavior of listeria monocytogenes in sliced ready-to-eat meat products packaged under vacuum or modified atmosphere conditions. j. food prot. 78:1891. molinero c., martínez b., rubio b., rovira j. and jaime i. 2008. the effects of extended curing on the microbiological, physicochemical and sensorial characteristics of cecina de león. meat sci. 80:370. ojec. 1996. commission regulation (ec) no 1107/96 of 12 june 1996 on the registration of geographical indications and designations of origin under the procedure laid down in article 17 of council regulation (eec) no 2081/92. off. j. eur. communities, l148:1. reyes-cano r., dorantes-álvarez l., hernández-sánchez h. and gutiérrez-lópez g.f. 1994. a traditional intermediate moisture meat: beef cecina. meat sci. 36:365. rodríguez-lázaro d., capita r., garcía-arias m.t., hernández-pérez m. and alonso-calleja, c. 2002a. cecina de león: un alimento nutritivo y saludable [cecina de león: a nutritious and healthy food]. alimentación, equipos y tecnología, xxi(167):59. rodríguez-lázaro, d., hernández-pérez, m., capita, r. and alonso-calleja c. 2002b. “cecina de león”: selección y entrenamiento de catadores (i) [cecina de león: selection and training of panelists (i)]. alimentaria junio 2002:55. rodríguez-lázaro, d., hernández-pérez, m., capita, r. and alonso-calleja c. 2002c. “cecina de león”: selección y entrenamiento de catadores (ii) [cecina de león: selection and training of panelists (ii)]. alimentaria junio 2002: 65. rodríguez-lázaro d., hernández-pérez m., capita r. and alonso-calleja c. 2002d. diseño y propuesta de hojas de cata para la cecina de león [design and proposal of tasting sheets for cecina de león]. eurocarne 105:99. rossi, f. 2001. assessing sensory panelist performance using repeatability and reproducibility measures. food quality pref. 12:467. rubio b., martínez b., garcía-cachán m.d., rovira j. and jaime, i. 2007. effect of high pressure preservation on the quality of dry cured beef “cecina de leon”. innov. food sci. emerg. technol. 8:102. sink, j.d. and hsu, l.a. 1979. chemical effects of smoke processing on frankfurter quality and palatability characteristics. meat sci. 3:247. paper received march 20, 2017 accepted november 20, 2017 #750_manzocco_bozza ital. j. food sci., vol 29, 2017 317 paper effect of pulsed light on selected properties of cut apple l. manzocco, p. comuzzo, m. scampicchioa and m.c. nicoli dipartimento di scienze agro-alimentari, ambientali e animali, università degli studi di udine,via sondrio 2/a, 33100, udine, italy a faculty of science and technology, libera università di bolzano, piazza università 1, 39100, bolzano, italy *corresponding author. lara.manzocco@uniud.it abstract the effect of pulsed light (0, 8.8 and 17.5 j cm-2) on selected properties of cut apple (thermal power, oxygen consumption, volatile compounds, colour and firmness) was investigated during storage at 30 °c for up to 6 days. samples exposed to pulsed light showed lower heat production due to a decrease in tissue respiration. pulsed light treated samples also showed different evolution of volatile compounds (ethanol, acetaldehyde and ethyl acetate), browning and tissue softening. modification of tissue metabolisms by pulsed light could be exploited in the processing of fresh-cut fruit and vegetables leading to advantages well beyond its germicidal activity. keywords: fresh-cut, isothermal microcalorimetry, light, respiration, volatile ital. j. food sci., vol 29, 2017 318 1. introduction pulsed light processing consists in exposing food to consecutive intense flashes of a radiation with a spectrum similar to that of the sun, including not only ultraviolet light but also visible and infrared radiation (170-2600 nm) (moraru and uesugi, 2009; falguera et al., 2011). because of its germicidal effect, pulsed light has been investigated as an interesting technological approach to decontaminate the surface of fresh-cut fruits and extend their shelf life (gòmez-lòpez et al., 2007; oms-oliu et al., 2010; ramos-villarroel et al., 2011; gòmez et al., 2012). the antimicrobial efficacy of pulsed light has been attributed to localized photothermal and photophysical effects but especially to the capability of its uv light component to modify the structure of biomolecules (wekhof, 2000; takeshita et al., 2003; wang et al., 2005; krishnamurthy et al., 2008). pulsed light has actually the potential of modifying protein conformation and configuration, leading to significant modification in their biologic activity (manzocco, 2015). these changes promote the death of microorganisms but also the inhibition of oxidative enzymes (gòmez et al., 2012; manzocco et al., 2013a; 2013b; ignat et al., 2014). it is thus likely that pulsed light could modulate the metabolic activity of vegetable tissues following adaptation mechanisms to the light stressor (luckey, 1980). a circumstantial evidence supporting this hypothesis is the well-known physiological response of plant tissue to uv-c light, which is part of the pulsed light spectra. common response to uv-c light in plants actually involves: (i) enhancement of biosynthesis of phenols toxic to pathogens; (ii) reduced ethylene production, and (iii) development of antisenescent putrescine exerting opposite physiological effects to ethylene (stevens et al., 1998; maharaj et al., 1999). physical effects could also be expected since uv-c light actually promotes moisture redistribution in the plant tissue due to local thermal effects deriving from electron excitation of food components (manzocco et al., 2011a). it has been hypothesised that modification of water availability could further modify tissue metabolic activity (manzocco et al., 2011b). nevertheless, limited information is available about effects of pulsed light other than the germicidal one. the aim of the present work was to evaluate the effect of pulsed light on some metabolism-related properties of cut apple. in particular, golden delicious apple tissues were submitted to pulsed light in a fluence range typically applied to inactivate microorganisms and inhibit enzymatic browning (up to 17.5 j cm-2). samples were then analysed for heat production, oxygen consumption, carbon dioxide formation, evolution of volatile compounds, colour and firmness during storage at 30 °c. 2. materials and methods 2.1. sample preparation golden delicious apples were purchased at the local market. fruits were washed with water and rinsed. apples were cored using a perforated cylinder having 4 mm internal diameter and 50 mm length. the extremities of the cored apple tissue were discarded to get a 35 mm long cylinder of apple mesocarp. a cylindrical shape was chosen to guarantee homogeneous exposure of sample surface to pulsed light and allow sample introduction in the microcalorimetry vials. ital. j. food sci., vol 29, 2017 319 2.2 pulsed light treatments pulsed light treatments were carried out at room temperature by using a pulsed light mobile decontamination unit (claranor, avignon, france) equipped with 4 xenon lamps with maximum emission in the range 200-1000 nm (200-400 nm: 41%; 400-700 nm: 51%; 700-1000 nm: 8%). lamps were positioned at each side of a quartz plaque held in the centre of the cube shaped chamber. two lamps were symmetrically positioned above and below the apple cylinder at a distance of 1 cm. two lamps were symmetrically positioned at the lateral side of the apple cylinder at 1 cm distance from the quartz plaque, which corresponded to 3 cm from the sample. apple cylinders were thus individually exposed to increasing light fluence up to 17.50 j cm-2, by means of increasing number of light pulses. according to the manufacturer’s instructions, each pulse delivered to the sample a light fluence of 1.75 j cm-2. pulse duration was 0.50 !s and repetition rate was 0.50 hz. treated apple cylinders were individually introduced in 1.2 ml capacity vials (lab logistic group gmbh, meckenheim, germany), hermetically sealed in the presence of air with butyl septa and metallic caps (lab logistic group gmbh, meckenheim, germany), and stored for increasing time up to 6 days at 30 °c (climacell 222, mmm group, grӓfelfing, germany). this temperature was chosen to emphasise metabolic activity of apple tissue. analogous sample not exposed to pulsed light were prepared as control. 2.3. temperature temperature was measured by a testo 805 pyrometer (testo, settimo milanese, italy). sample temperature was measured immediately after the pulsed light treatment. interval time between the end of the pulsed light treatment and sample temperature measurement was less than 10 s. 2.4. isothermal microcalorimetry isothermal calorimetry was performed by a multichannel microcalorimeter (tam iii air isothermal calorimetry, ta instruments, new castle, delaware, usa) equipped with an oil bath thermostat (accuracy 0.0001 °c) operating through a peltier element. the instrument has a sensitivity of ± 100 nw and allows accurate maintenance of temperature in the 25 ± 5 °c temperature range. vials containing the apple cylinders were placed in the calorimeter and isothermal traces recorded at 30 °c for 5 days. the thermal profile was expressed as w g-1 by normalising the heat flux (w) of each sample based on the sample weight. 2.5 firmness firmness was measured by warner-blatzler shear test using an instron 4301 (instron ltd. high wycombe, united kingdom). the instrumental settings and operations were accomplished using the software automated materials testing system (version 5, series ix, instron ltd, high wycombe, united kingdom). the blade was lowered into the apple sample perpendicularly to the cylinders at a speed of 5 cm/min. force was measured over time and sample firmness was taken as the force (kn) required to shear apple cylinders. ital. j. food sci., vol 29, 2017 320 2.6 image analysis images of apple cylinders were acquired by using an image acquisition cabinet (immagini & computer, bareggio, italy) equipped with a digital camera (eos 550d, canon, milan, italy). in particular, the digital camera was placed on an adjustable stand positioned 60 cm above a black cardboard base where the apple sample was placed. light was provided by 4 100 w frosted photographic floodlights, in a position allowing minimum shadow and glare. other camera settings were: shutter time 1/125 s, f-number f/6.0, focal length 60 mm. images were saved in jpeg format. image-pro® plus (ver. 6.3, media cybernetics, inc., bethesda, md, usa) was used to analyzed apple browning. brown pixels in the apple images presented 103 < r < 194, 76 < g < 188, 5 < b < 100. browning was defined as the percentage ratio between brown pixels and pixels corresponding to the apple area. 2.7 gas chromatographic analyses a fisons 8000 series gas chromatogram, equipped with a thermal conductivity detector fisons hwd (both from fisons instruments, milan, italy), was used for analysis of oxygen and carbon dioxide in the headspace of vials containing apple cylinders. compounds were separated on two glass columns (2 m x 2 mm i.d.), packed with porapaqs (80/100 mesh), in isothermal conditions (column temperature 70 °c). carrier gas was nitrogen, at a flow rate of 27 ml min-1; injector and detector temperatures were 180 and 120 °c respectively. the temperature of the filament was 170 °c. samples were equilibrated at 25 °c before injection; then, 200 µl of headspace was sampled by a 500 µl gastight manual syringe (dynatech, batonrouge, lousiana, usa) and immediately injected in the gc system. capillary gas chromatography (gc) and solid-phase microextraction (spme) were used for the analysis of low-boiling volatile compounds in the headspace of the vials containing apple cylinders. the fiber used was a 1 cm 85 µm carboxen/pdms (supelco, bellefonte, pa, usa). spme was run for 2 min at 25 °c; vials were preliminary equilibrated at the operating temperature for 15 min before microextraction. gc injection was carried out in split mode (split ratio 1:10) and the fiber remained in the injector for 1 min. the gc system was a hrgc 8560 mega series 2 gas chromatograph (carlo erba, milan, italy), equipped with a flame ionisation detector (fid); carrier gas was helium, at a linear flow rate of 35 cm s-1. compounds were separated on a j&w db-wax capillary column (30 m x 0.25 mm i.d., 0.25 µm film thickness), purchased from agilent technologies inc. (santa clara, ca, usa). the column temperature was programmed as follows: 35 °c for 2 min, then at 4 °c min-1 up to 44 °c; temperature was then further increased at 20 °c min-1 up to 240 °c, held for 15 min. injector and detector temperature were set at 250 and 240 °c respectively. the concentration of the volatile compounds was expressed in absolute area units. the identity of the peaks was confirmed by comparing their retention time with those of standard compounds; all the standards were from sigma-aldrich (st. louis, mo, usa). 2.8 statistical analysis analyses were performed on at least duplicated samples. colour and firmness analyses were performed on at least 8 duplicated samples. results are reported as mean value ± sd. pearson correlation analysis was performed by using statistica for windows (ver. 5.1, statsoft inc., tulsa, usa, 1997). a p-value >0.05 was set as a statistical threshold for significance. ital. j. food sci., vol 29, 2017 321 3. results and discussion apple cylinders were submitted to pulse light treatments with increasing fluence at environmental temperature. after the treatment, the temperature of the apple cylinder surface never exceeded 30 °c. the effect of pulsed light was initially monitored by isothermal microcalorimetry. this technique was chosen since the evaluation of heat production provides a direct indication of the metabolic responses of raw materials, such as respiration and reaction to wounding stress (criddle et al., 1991; gòmez galindo et al., 2005; wadsö and gòmez galindo, 2009; rocculi et al., 2012). apple tissue was thus exposed to increasing fluence of pulsed light and evaluated for heat production under isothermal conditions at 30 °c for up to 6 days (fig. 1). figure 1. calorimetric traces at 30 °c of apple tissue exposed to pulsed light with increasing fluence. independently on the fluence of the pulsed light treatment, calorimetric traces of all samples showed a main exothermal peak within 1-2 days of observation. prolonging observation time, no further changes in calorimetric traces were detected (data not shown). according to the literature, the exothermal peak (fig. 1) should be related to the thermal effects of microbial growth and/or metabolic activity of the apple tissue (riva et al., 2001; gómez galindo et al., 2005). however, calorimetric peaks due to microbial growth are generally preceded by a plateau signal corresponding to the lag phase of micro-organisms. as clearly shown in figure 1, no initial plateau signal was detected, suggesting the peak to be mainly attributable to heat generated by the respiration process of wounded apple tissue. it can be inferred that the hypoxic conditions that are quickly generated inside the vial upon apple tissue respiration may inhibit microbial growth making its contribution negligible as compared to that of tissue metabolism. the calorimetric trace of the control untreated apple showed the occurrence of a sharp and narrow peak (fig. 1). the peak shoulder could be explained considering the occurrence of physiologic activities with different metabolic rate depending on the storage period. after 1 day of storage at 30 °c, the calorimetric signal reached a plateau value. the latter was above the starting base line and indicated that beyond this storage time, there was still a slight but constant residual metabolic activity. when apple samples were exposed to 0,00000 0,00004 0,00008 0,00012 0,00016 0,00020 0 0,5 1 1,5 2 t he rm al p ow er (w g1 ) time (day) 0.0 j cm-2 8.8 j cm-2 17.5 j cm-2 e xo ital. j. food sci., vol 29, 2017 322 pulsed light with increasing fluence, the exothermal peak showed a progressively lower intensity and appeared broader. in addition, the plateau was reached in longer times and its level was lower than in the control sample. these results indicate that pulsed light treatment modifies tissue metabolism. to this regard, the uv-c light component of pulsed light was reported to reduce ethylene production in fresh tomato, favouring the accumulation of putrescine, an anti-senescence agent with an opposite physiological effect with respect to ethylene and delayed the appearance of the climacteric peak (maharaj et al., 1999). a similar effect of uv-c radiation has been also reported for other climacteric fruits, such as apples and peaches (lu et al., 1991). to verify the effect of pulsed light on apple tissue respiration, samples were stored for increasing time under the same temperature adopted during the calorimetric analysis (30 °c) and evaluated for oxygen consumption and carbon dioxide formation (fig. 2). figure 2. headspace oxygen and carbon dioxide during storage at 30 °c of apple tissue exposed to increasing fluence of pulsed light. headspace oxygen was rapidly consumed during the first days of cut apple storage while carbon dioxide concomitantly accumulated following the typical gas evolution of plant ital. j. food sci., vol 29, 2017 323 tissues performing a respiration metabolism. the respiration-driven oxygen consumption and formation of carbon dioxide resulted progressively less intense as the fluence of the pulsed light treatment was increased. this result apparently contradicts with the negligible effect of pulsed light on respiration of endive salad and mung bean sprouts (kramer et al., 2015). contradictory results were also reported by ramosvillarroel et al. (2011) with reference to fresh-cut avocado. these authors showed that pulsed light treatment reduced oxygen consumption but increased respiration products such as carbon dioxide and ethanol. additional information can be obtained considering the plant respiration effects of uv-c light, which is an important component of the pulsed light radiation. uv-c light was reported to reduce respiration rates of fresh cut cantaloupe melon and intact fuji apples (lamikanra et al., 2005; yujuan et al., 2015). it can be hypothesised that the different effects of pulsed light on respiration rate may be strongly dependent not only on the nature of the plant matrix and its peculiar respiration metabolism but also on the spectral composition of the light radiation. the progressive decrease of oxygen partial pressure in the sample headspace (fig. 2) is well known to be associated to hypoxic conditions, leading to accumulation of ethanol and acetaldehyde involved in post harvest maturation and flavour development (dixon and hewett, 2000; pesis, 2005). samples were thus analysed for ethanol, acetaldehyde and ethyl acetate (fig. 3). the latter was taken as an example of volatile compound that is expected to play a role in the flavour of fresh-cut apple derivatives (song and bangerth, 1996; dixon and hewett, 2000). gas chromatographic analyses were only performed on samples stored up to 2 days of storage due to the minimum metabolic activity on further storage (figure 1 and 2). pulsed light treated apple tissues showed significantly lower amounts of ethanol and higher values of acetaldehyde than the control sample (fig. 3). in addition, when pulsed light was applied at the highest fluence (17.5 j cm-2), it also modified the evolution of ethyl acetate (fig. 3). as reported in the literature, piruvate is converted to acetaldehyde and co2 by the enzyme pyruvate decarboxilase, and acetaldehyde is reduced to ethanol by the enzyme alcohol dehydrogenase (mathews and van holde, 1996). esterification of alcohols, although not fully understood in its biosynthetic pathway, is then responsible for the formation of esters contributing to apple flavour during maturation (dixon and hewett, 2000). data shown in figure 3 suggest that exposure to pulsed light radiation could modulate the activity of the enzymes of the anaerobic biosynthetic pathway for the formation of acetaldehyde, ethanol and esters. to this regard, light radiation is known to be absorbed by enzyme proteins due to the presence of endogenous chromophores within their structure (davies and truscott, 2001). as a consequence, light would modify the structure of enzymes leading both to their activation or inactivation (manzocco et al., 2009). results acquired in this experimentation indicate that pulsed light is able to modify the metabolic response of apple tissue to the wounding stress, modulating the kinetics of respiration and volatile formation (figs. 2 and 3). these phenomena would be probably the result of a combination of different biological activities that are concomitantly monitored when measuring the thermal power of the sample (fig. 1). in order to verify the capability of isothermal calorimetry to study the metabolic consequences of pulsed light treatment, correlation analysis was performed. table 1 shows the correlation coefficients between thermal power and analytical parameters used to monitor respiration and volatile formation, during storage at 30 °c of cut apple exposed to increasing fluence of pulsed light. ital. j. food sci., vol 29, 2017 324 figure 3. headspace ethanol, acetaldehyde and ethyl acetate during storage at 30 °c of apple tissue exposed to increasing fluence of pulsed light. ital. j. food sci., vol 29, 2017 325 table 1. correlation coefficients between thermal power and indices of respiration and volatile formation of cut apple exposed to increasing fluence of pulsed light and stored for increasing time at 30 °c. thermal power oxygen 0.77a carbon dioxide -0.73 a ethanol -0.77 a acetaldehyde 0.05 ethyl acetate -0.32 a significant at p < 0.05 a very low correlation of thermal power with acetaldehyde and ethyl acetate formation was observed. this can be attributed to the fact that the evolution of these parameters is quite complex, being affected by a number of different concomitant and consequent reaction pathways. by contrast, a high correlation between changes in thermal power and oxygen, carbon dioxide and ethanol was detected. this result definitely indicates that the evolution of the calorimetric signal (fig. 1) mainly accounts for the metabolic phenomena associated with the respiration process of apple tissue (gómez galindo et al., 2005). the effects of pulsed light on respiration rate could be associated to modification of the metabolism pathways leading to tissue browning and softening. to verify this hypothesis, apple samples exposed to pulsed light were evaluated for the development of browning and the change in firmness. the effect of increasing pulsed light fluence on the percentage of brown colour on the surface of cut apple during storage is shown in fig. 4. figure 4. browning of cut apple exposed to increasing fluence of pulsed light and stored at 30 °c. the control sample showed a quick development of enzymatic browning during the first hours after cutting (sapers and douglas, 1987). no significant further colour changes were detected when the sample was maintained at 30 °c for up to 6 days. apple samples exposed to pulsed light appeared browner just after their preparation (0 days). this is in ital. j. food sci., vol 29, 2017 326 agreement with the enhancement of phenol biosynthesis as a common response of plant tissue to uv-c light (stevens et al., 1998; maharaj et al., 1999). after 16 hours of storage, pulsed light treated samples showed a browning level analogous to that of the control sample. a significant decrease in browning was observed when pulsed light samples were stored beyond one day. this sample whitening could be attributed to the modification of the phenolic metabolism controlling the formation of brown polyphenols in apple tissue. however, it is not excluded that brown polyphenols could be further degraded to uncoloured compounds. a similar effect was reported as a consequence of pulsed light treatment of protein rich ingredients, such as egg white (manzocco et al., 2013a). in that case, sample bleaching was attributed to melanoidin electronic transitions following light radiation absorption. colour changes could also be accounted for by changes in physical structure of apple tissue. water distribution could actually play a role in determining the overall sample colour as well as changes in apple tissue firmness. to this regard, fig. 5 shows the evolution of firmness during storage at 30 °c of apple samples exposed to increasing fluence of pulsed light. figure 5. firmness of cut apple exposed to increasing fluence of pulsed light and stored at 30 °c. all samples showed an initial increase in firmness, reaching a maximum value after 16 hours of storage. beyond this storage time, firmness decreased so that a maximum value was identified, followed by a plateau. the increase in firmness could be attributed to sample dehydration while the following decrease could account for progressive pectolityc activity (oms-oliu et al., 2010). the maximum value of firmness resulted lower in the case of the samples exposed to pulsed light. these result is in agreement with the fact that radiation can promote dehydration of a thin surface layer of the wounded plant tissue, begetting a protective film that hinders moisture migration from the inside to the environment (manzocco and nicoli, 2015). 4. conclusions pulsed light has been investigated as an interesting technological approach to decontaminate the surface of fresh-cut fruits and extend their shelf life. results reported in this work demonstrate that pulsed light exerts additional complex effects of different ital. j. food sci., vol 29, 2017 327 metabolism-related properties of fresh-cut vegetables. in the case of apple, exposure to pulsed light modified the kinetics of respiration, volatile formation, browning and tissue softening. the possibility of pulsed light implementation in fresh-cut processing will be strictly dependent on the availability of detailed information about its consequences on tissue metabolisms, especially in relation to different temperature and atmosphere conditions during storage. these data should be merged with those relevant to the antimicrobial activity of pulsed light in order to select optimal fluence to be adopted during fruit treatment. in this context, isothermal calorimetry could represent a very useful methodological tool since potentially allowing to concomitantly quantify both metabolic and microbial activity. acknowledgements this research was supported by ministero dell’istruzione, dell’università e della ricerca (prot. 957/ric, 28/12/2012), through the project 2012zn3kjl “long life, high sustainability”. references criddle r.s., breindenbach, r.w. and hansen, l.d. 1991. plant calorimetry:how to quantitatively compare apples and oranges. thermochim. acta 193:67. davies m.j. and truscott r.j. 2001. photo-oxidation of proteins and its role in cataractogenesis. j. photochem. photobiol. b 63:114. dixon j. and hewett e.w. 2000. factors affecting apple aroma/flavour volatile concentration:a review. new zealand j. crop horticultural sci. 28:155. falguera v., pagán j., garza s., garvín a. and ibarz a. 2011. ultraviolet processing of liquid food:a review. food res. int. 44:1580. gòmez galindo f., rocculi p., wadsö l. and sjöholm i. 2005. the potential role of isothermal calorimetry in monitoring and predicting quality changes during processing and storage of minimally processed fruits and vegetables. trends food sci. technol. 16:325. gòmez p.l., salvatori d.m., garcìa-loredo a. and alzamora s. m. 2012. pulsed light treatment of cut apple: dose effect on color, structure and microbiological stability. food bioprocess technol. 5:2311. gòmez-lòpez v.m., devlieghere f., bonduelle v. and debevere j. 2005. intense light pulses decontamination of minimally processed vegetables and their shelf-life. int. j. food microbiol. 103:79. gòmez-lòpez v.m., ragaert p., debevere j. and devlieghere f. 2007. pulsed light for food decontamination:a review. trends food sci. technol. 18:464. ignat a., manzocco l., maifreni m., bartolomeoli i. and nicoli m.c. 2014. surface decontamination of fresh-cut apple by pulsed light:effects on structure, colour and sensory properties. postharvest biol. technol. 91:122. kramer b., wunderlich j. and muranyi p. 2015. pulsed light decontamination of endive salad and mung bean sprouts and impact on color and respiration activity. j. food protection 78:340. krishnamurthy k., tewari j.c., irudayaraj j. and demirci a. 2008. microscopic and spectroscopic evaluation of inactivation of staphylococcus aureus 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(ed.). 1980. “hormesis with ionizing radiation” p. 222. crc press, boca raton, fl. manzocco l. 2015. photo-induced modification of food protein structure and functionality. food eng. rev. 7:346. ital. j. food sci., vol 29, 2017 328 manzocco l. and nicoli m.c. 2015. surface processing:existing and potential applications of ultraviolet light. crit. rev. food sci. nutr. 55:469. manzocco l., panozzo a. and nicoli m.c. 2013a. effect of pulsed light on selected properties of egg white. innov. food sci. emerg. technol. 18:183. manzocco l., panozzo a. and nicoli m.c. 2013b. inactivation of polyphenoloxidase by pulsed light. j. food sci. 78:e1183. manzocco l., da pieve s. and maifreni m. 2011a. impact of uv-c light on safety and quality of fresh-cut melon. innov. food sci. emerg. technol. 12:13. manzocco l., da pieve s., bertolini a., bartolomeoli i., maifreni m., vianello a. and nicoli m.c. 2011b. surface decontamination of fresh-cut apple by uv-c light exposure:effects on structure, colour and sensory properties. postharv. biol. technol. 61:165. manzocco l., quarta b. and dri a. 2009. polyphenoloxidase inactivation by light exposure in model systems and apple derivatives. innov. food sci. emerg. technol. 10:506. maharaj r., arul j. and nadeau p. 1999. effect of photochemical treatment in the preservation of fresh tomato (lycopersicon esculentum mill cv. capello) by delaying senescence. postharv. biol. technol. 15:13. mathews c.k. and van holde k.e. (eds) 1996. “biochemistry”. 2nd edition (p. 1159). the benjamin/cummings publishing company, inc., redwood city, ca. moraru c. and uesugi a. 2009. pulsed-light treatments:principles and applications. in t. koutchma, l. j. forney, and c. i. moraru (eds.) 2011. “ultraviolet light in food technology. principles and applications” (pp. 235–265). crc press, boca raton, fl. oms-oliu g., aguilò-aguayo i., martin-belloso o. and soliva-fortuny r. 2010. effects of pulsed light treatments on quality and antioxidant properties of fresh-cut mushrooms (agaricus bisporus). postharv. biol. technol. 56:216. pesis e. 2005. the role of the anaerobic metabolites, acetaldehyde and ethanol, in fruit ripening, enhancement of fruit quality and fruit deterioration. postharv. biol. technol. 37:1. ramos-villarroel a.y., martín-belloso o. and soliva-fortuny r. 2011. bacterial inactivation and quality changes in freshcut avocado treated with intense light pulses. eur. food res. technol. 233:395. riva m., fessas d. and schiraldi a. 2001. isothermal calorimetry approach to evaluate shelf life of foods. thermochimica acta, 370:73. rocculi p., panarese v., tylewicz u., santagapita p., cocci e., gòmez galindo f., romani s. and dalla rosa m. 2012. the potential role of isothermal calorimetry in studies of the stability of fresh-cut fruits. food sci. technol. 49:320. sapers g.m. and douglas f.w. 1987. measurement of enzymatic browning at cut surfaces and in juice of raw apple and pear fruit. j. food sci. 52:1258. song j. and bangerth f. 1996. the effect of harvest date on aroma compound production from ‘golden delicious’ apple fruit and relationship to respiration and ethylene production. postharv. biol. technol. 8:259. stevens c., liu j., khan v.a., lu j.y., wilson c.l., igwegbe e.c.k., kabwe m.k., chalutz e. and droby s. 1998. application of hormetic uv-c for delaying ripening and reduction of rhizopus soft rot in tomatoes:the effect of tomatine on storage rot development. j phytopathol. 146:211. takeshita k., shibato j., sameshima t., fukunaga s., isobe s., arihara k. and itoh m. 2003. damage of yeast cells induced by pulsed light irradiation. int. j. food microbiol. 85:151. wadsö l. and gòmez galindo f. 2009. isothermal calorimetry for biological applications in food science and technology. food control 20:956. wang t., macgregor s.j., anderson j.g. and woolsey g.a. 2005. pulsed ultra-violet inactivation spectrum of escherichia coli. water res. 39:2921. wekhof a. 2000. disinfection with flash lamps. pda j. pharm. sci. technol. 54:264. yujuan l., ming’an y. and xiaolin r. 2015. effects of uv-c radiation on storability and quality in harvested apple. food sci. china 36:244. paper received january 12, 2016 accepted january 25, 2017 #582_uzmai_bozza ital. j. food sci., vol 29, 2017 209 paper the likelihood of sheep meat consumption in turkey a. uzmay*a and g. çinar b adepartment of agricultural economics, faculty of agriculture, ege university, bornova izmir 35100, turkey bdepartment of agricultural economics, faculty of agriculture, adnan menderes university, aydin 09100, turkey *corresponding author. tel.: +90 2323111437; fax: +90 2323881862 e-mail address: ayse.uzmay@ege.edu.tr abstract the aims of this study are threefold: first, to determine the factors affecting the likelihood of sheep meat consumption; second, to determine the reasons for consumers’ preferences; and third, to determine consumers’ willingness to pay for quality-based labelling. the study conducted interviews with 300 households in izmir province. according to the results of logistic regression analysis, gender, level of income, number of people in the household, beef consumption, and whether the interviewed individual has the highest income in his or her household affect the probability of sheep meat consumption. while the personal preferences of regular sheep meat consumers vary according to their red meat consumption, willingness to pay based on labelling is $1.62/kg. keywords: consumer preferences, logistic regression, sheep meat consumption, willingness to pay ital. j. food sci., vol 29, 2017 210 1. introduction according to oecd reports, worldwide consumption of sheep meat is increasing. in the year 2023, average world consumption of sheep meat per capita is expected to reach 1.91 kg, an increase of 12.3% compared to 2014 (oecd, 2015). recently conducted studies also indicate that consumer demands for different types of meat are changing (bernués et al., 2012, montossi et al., 2013). in particular, factors such as the relationship between red meat and cancer (norat et al., 2002; chao et al., 2005), an increase in other healthrelated concerns, changes in demographic structure, economic growth, and changes in meat’s, price, quality and image have been effective in driving this change (bernabéu and tendero, 2005; grunert, 2006). the negative effect of red meat consumption on cardiovascular health and the recommended diet to maintain a healthy lifestyle are among the important factors affecting the preferences of consumers, especially in developed countries (lichtenstein et al., 2006, daniel et al., 2010). there have been several studies of the factors that affect consumer preferences and willingness to pay for sheep meat (gracia et al. 2011). while some studies examine factors such as price, origin, certification, meat type and size, and feeding method (grainfed, grass-fed and grain + grass fed) (sañudo et al., 2007; font i furnols et al., 2011; joy et al., 2012; bernués et al., 2012), others primarily focus on the effects of factors such as the environment, animal welfare and food safety (dickinson et al., 2003; napolitano et al., 2007; napolitano, 2009; sepúlveda et al., 2011). the results of these studies indicate that origin is a significant factor in meat consumption preferences; in particular, local meat is preferred (kaur, 2010; foint i furnols et al., 2011; meas, 2014). in addition, studies concerning sheep meat consumption generally observe that origin is an essential factor in preferences and that consumers' willingness to pay is highly affected by the meat’s origin (imami et al., 2011; gracia, et al., 2011; hersleth et al., 2012; montossi et al., 2013). however, berneues et al. (2003) determined that, with regard to lamb meat preferences in france and spain, the attention paid to animal feeding systems may be a stronger factor than the origin of the meat. on the other hand, prescott et al. (2004) report that the peculiar fat and smell of sheep meat are important reasons why it is rarely or never preferred in some countries or regions. it should be further noted that the culture, habits and beliefs of some countries may also affect sheep meat consumption (bonne and verbeke, 2006; font i furnols et al., 2006; nakyinsige 2012; montossi et al., 2013). because studies of consumer behaviours related to sheep meat – and the factors that affect these behaviours – are closely related to several disciplines, including psychology, sociology, agronomics, food science and medicine, several relevant studies can be found in the literature. nevertheless, in comparison to the number of studies conducted on other types of meat, the number of studies focused on lamb/mutton is still relatively low. although studies concerning meat consumption in turkey are available, a robust database containing the production and consumption figures of animal products at the national level is missing (mfal, 2015). nevertheless, it was announced by the meat and milk foundation (ukon, 2013) that meat consumption per capita in 2013 was 32.5 kg, with 60% (19.4 kg) of that being poultry meat consumption, 35% (11.4 kg) being bovine consumption and 5% (1.7 kg) being ovine consumption. in turkey, anaemia is observed in 29% of women and 30% of children younger than 5 years. annual per capita consumption of red meat in turkey (29 g) is below the world average (31 g) (fao, 2014). there have been several studies of red meat consumption in turkey and the factors that affect it (atay et al., 2004; karakuş et al., 2008; kaya et al., 2011; tüzemen, 2012; erdoğan, 2013). however, no study could be found that specifically examines the factors directly affecting sheep meat consumption and consumer preferences. as ital. j. food sci., vol 29, 2017 211 mentioned earlier, off-the-book ovine breeding in turkey and the fact that agricultural policy implementers are discussing whether the consumption gap in red meat can be closed with ovine meat not only require studies of production potential but also require that meat consumption amounts and consumer preferences be researched at both the regional and provincial levels. in this context, the main objectives of this study fall into two categories: first, to determine meat consumption patterns in the province of izmir and to identify the socio-economic factors that affect the possibility of households’ sheep meat consumption, and second, to identify personal reasons for preferring sheep meat among those who consume lamb regularly and to determine their willingness-to-pay based on quality-related labelling. 2. materials and methods the metropolis of izmir is turkey's third largest province, with a population of 4,061,000. the average household has three members (tsi, 2014). in this context, the population of the study was set to 1,353,667 households. the number of households to be included within the scope of the study was calculated as 296 according to the proportional sampling method (newbold, 1995, equation 1). considering proportional losses based on the population’s distribution among districts, interviews with a total of 300 respondents were completed. )1()1( )1( 2 ppn pnp n p −+− − = σ (1) where n is the sample size, n is the population size (1,353,667), and p is the prediction rate (0.5 for the maximum sample size) and the probability level confidence interval (99% confidence interval, 𝜎p: 0.02960 for 0.075 margin of error from the equation of 2.58𝜎p: 0.075). a total of four districts in izmir with high population densities were included within the scope. accordingly, the sample size, determined as 300 people, was distributed per the respective populations of the districts. thus, 151 people from the balcova district, 76 people from the karsiyaka district, 46 people from the konak district and 27 people from the menemen district were included in the sample. because meat variety in hypermarkets is high, the survey was conducted among consumers who shop at hypermarkets in the research area. moreover, the consumers participating in the survey were always responsible for shopping for their households. the questionnaire used for data collection included four main sections: (a) socio-economical characteristics of respondents (b) meat consumption of the households c) the reasons for consumers’ preferences (the reasons why consumers prefer sheep meat / the reasons for do not regularly consume sheep meat) d) willingness to pay for a labelled product. in sections a and b, respondents were asked their age as well as open-ended questions regarding their education level, household, district, occupation, marital status, and amount of meat consumed in their household. however, income groups were divided into three categories to encourage the respondents to answer without hesitation. the scale was determined by the turkish statistical institute’s classifications. therefore, the lowest income category included families of four with incomes at or below the poverty line ($2270). queries regarding consumer preferences consisted of multiple choice questions. respondents were asked to select the most appropriate option. finally, because there are no labelling or certification systems used for ital. j. food sci., vol 29, 2017 212 sheep meat in turkey, we attempted to determine whether consumers would be willing to pay a higher price for labelled (thus quality-assured) sheep meat. in this respect, sheep meat consumers were informed of the importance of labelling, and their willingness to pay for labelled meat was assessed. in this context, consumers in i̇zmir were told the average current price of sheep meat ($15.71) and were asked whether they would pay extra for labelling. if the answer was positive, then they were asked to indicate the final price they were willing to pay. in the present study, the factors that affect sheep meat consumption were determined by means of logistic regression analysis. as the dependent variable, while the respondents regularly consuming sheep meat were set as (1), those who do not regularly consume sheep meat were set as (0). in the logistic regression model, where the dependent variable has two categories, independent variables can be discrete, continuous and qualitative. the logistic regression model employed in this study is presented in equation 2. ( ) ( ) 0 1 1 2 2 0 1 1 2 2 0 1 1 2 2 ... ... ... 1 ( 1/ ) 1 1 p p p p p p x x x x x x x x x e x p y x x e e β β β β β β β β β β β β π + + + + + + + + − + + + + = = = = = + + (2) where x is the data matrix with regard to the independent variable, and when x = x (when the value x is known), the probability of the occurrence is (y=1) p. β is the constant, βi is the parameter to be predicted for each explanatory (independent) variable, and xi indicates the ith independent variable. the nonlinear logistic regression function given in the equation was subjected to logit conversion and linearized. designing the model as per the data of the study produced the following equation, ( ) ( ) ( ) 0 1 0 1 ...(1 ) xxg x in ine x x β βπ β β π + ⎡ ⎤ = = = +⎢ ⎥ −⎣ ⎦ (3) where b1 indicates the variation in the dependent variable caused by 1 unit of change in the independent variable x, demonstrating how much change is caused by 1 unit of change in the x in the logistic model (aldrich and nelson, 1984). within the scope of the study, the respondents' age, education, gender, marital status, employment status, household income, number of people in the household, whether the respondent contributes the highest income to the household, beef consumption of the household, chicken consumption of the household and whether there are any cardiac patients (family members taking medication) in the household were accepted as the independent variables (table 1). the -2llr value obtained for the model was 256.057, significant at a 5% margin of error (table 2). with this value, the significance of the coefficients pertaining to the variable levels was tested. in this study, the conditional valuation method is used. to apply this method, an imaginary market is created for a good or a service and people are asked how much they would pay in return for that good or service (carson, 2000). the price that consumers were willing to pay for labelled lamb/mutton products was determined by means of the lower bound mean method initially implemented by blaine et al. (2003). lbm= )()( 1 1 0 0 − = −+∑∏∏ i k i i i ppp (4) ital. j. food sci., vol 29, 2017 213 where π0 is the cumulative percentage of willingness to pay, p0 is the lowest payment boundary and k is the number of boundaries. table 1. variables used in logit model and descriptive statistics of the variables. dependent variable (y) type of variable description frequency percent(%) dichotomus 0 1 221 79 73.67 26.33 independent variables (x) mean standard d. the respondents' age (age) 41.39 11.2490 number of people in the household (hs) 3.11 1.193 beef consumption of the household kg (monthly) (bc) 4.22 1.010 chicken consumption of the household kg (monthly) (cc) 6.20 1.10 gender of the respondent (gen) dichotomus 0: male 1 :female 142 158 47.3 52.7 education of the respondent (edu) ordinal categorical 0: otherwise 1: university 161 139 53.7 46.3 income level of the household (incm) ordinal categorical 1: x≤ $2270 2: $2271– $3974 3: x ≥$3975 83 175 42 27.7 58.3 14.0 employment status of the respondent (es) dichotomus 0: no 1: yes 94 206 31.3 68.7 marital status of the respondent (ms) dichotomus 0: other 1: yes 64 236 21.3 78.7 whether the respondent brings the highest income to the household (hincm) dichotomus 0: no 1: yes 138 162 46.0 54.0 whether there are any cardiac patients (family members taking medication) in the household (pih) dichotomus 0: no 1: yes 239 61 79.2 20.3 3. results and discussions 3.1. factors affecting the likelihood of sheep meat (lamb/mutton) consumption among all households included in the study, 26.3% consume sheep meat (lamb/mutton). the average age of the respondents is 41, 78.7% are married, and the total income of 58.3% of the households is between $2,271 and $3,974 per month. a total of 46% of the participating consumers have bachelor's degrees, approximately 53% are women, 54% contribute the highest income to their households, and 69% are employed. according to the results of the study, among the meat preferences of the households in izmir, poultry, beef and sheep meat have shares of 55% (6.22 kg/month), 38% (4.22 ital. j. food sci., vol 29, 2017 214 kg/month) and 7% (0.78 kg/month), respectively. none of the families consumed goat meat. on the basis of the types of meat in question, the consumption average of the households is 43.21 kg/year, or 3.60 kg/month. the annual average consumption of red meat, on the other hand, is 27 kg. while beef constitutes 89% of red meat consumption, sheep meat constitutes 11%. the average consumption of the 79 households that regularly consume sheep meat is 1.48 kg/month (while the same average among all households included in the sample is 0.78 kg/month). while 36.7% of the families that regularly consume sheep meat prefer sheep meat to beef, 20.3% of them prefer to consume solely sheep meat. on the other hand, among the whole population included in the study, 5.3% prefer to consume only sheep meat. examining studies conducted in other provinces of turkey demonstrates that consumption patterns in aydin province, which is also within the aegean region (atay et al., 2004), and erzurum province, located in the eastern anatolia region (kaya et al., 2011), are similar to the pattern in izmir province. however, it is also noteworthy that sheep meat consumption increases toward the regions of central anatolia and southeastern anatolia (karakus et al., 2008; tuzemen, 2012). according to the findings of the present study conducted in the izmir region, average annual sheep meat consumption per capita is approximately 3 kg in this province. although above the world average according to oecd data, this level of consumption is still below the average for turkey. furthermore, while consumers in izmir mostly prefer spring lamb meat, towards southeastern anatolia, mutton is preferred (önenç and özşenoğullari, 2009). indeed, in the present study, 58% of sheep meat consumers in izmir reported that they prefer lamb. comparing – in terms of income groups – the rate of households that regularly consume sheep meat to those that do not demonstrates that while the rate is 10.84% in the lowest income group (x≤ $2,270), in the middle-income group ($2,271-$3,974) and the highest income group ($3,975≤x) the rates are 33.14% and 28.4%, respectively. quantitatively examining the relationship between income groups and consumption amounts demonstrates that 100% of the consumption in the lowest income group is 2 kg or lower. the rates of the households consuming 4 kg and less in the middle and the highest income groups, on the other hand, are 88% and 75%, respectively. the households that consume more than 4 kg have shares of 12% in the medium income group and 25% in the high income group. according to the results of pearson’s chi-squared test (37.329), the income groups and consumption amounts of the households were significant at the level of 0.01. in a study conducted by pearce (2013), the findings of previous studies concerning australia, the usa, eu and uk were evaluated, and it was suggested that the number of lamb meat consumers is higher in the medium income group. the highest rate of regular sheep meat consumers, compared to those who do not consume sheep meat in turkey (33.14%), is also found in the medium income group. the rate of households consuming sheep meat in the districts of bornova, karsiyaka, konak and menemen, compared to the total number of households, varies between 25% and 28%. the results of pearson’s chi-squared test (0.126) indicate that there is no significant difference between the districts of i̇zmir and sheep meat consumption at the level of 0.05. according to the results of the logistic regression model (table 2), the respondent's gender, whether he or she contributes the highest income to the household, total income of the household, amount of beef consumption, and number of people in the household were determined to affect the likelihood of sheep meat consumption. ital. j. food sci., vol 29, 2017 215 table 2. statistical results of logit model. b s.e. wald df sig. exp(b) age 0.004 0.020 0.034 1 0.854 1.004 gen -1.648 0.619 7.088 1 0.008* 0.192 edu -0.153 0.402 0.144 1 0.704 0.859 ms 0.086 0.522 0.027 1 0.870 1.090 es 0.287 0.391 0.537 1 0.464 1.332 hincm -2.005 0.646 9.624 1 0.002* 0.135 pih -0.471 0.459 1.053 1 0.305 0.625 incm 1.505 0.365 16.969 1 0.000* 4.503 cc -0.116 0.162 0.514 1 0.474 0.890 bc -1.316 0.233 31.770 1 0.000* 0.268 hs 0.701 0.178 15.571 1 0.000* 2.016 constant 1.063 1.616 0.433 1 .511 2.895 b s.e. wald df sig. exp(b) variables in the equation model summary -1.001 0.132 57.910 1 0.000* 0.367 model summary -2 log likelihood 256.057(a) cox & snell r square 0.254 nagelkerke r square 0.369 hosmer and lemeshow test: chi-square 13,801, df 8, p(0.087>0.05). women's probability of consuming sheep meat is 80.8% (0.192-1) less than that of men. as a matter of fact, according to the conclusions of the study conducted by prättälä et al. (2006) in countries with different cultures and economies (finland and baltic countries), it was reported that, in general, women consume less meat than men, and they usually prefer to consume vegetables and fruits. in addition, other studies (kubberad et al., 2002; santos and booth, 1996; hughes, 1995) report that, in particular, younger women tend to consume mostly white meat. ureña et al. (2008) reported that women and men display different attitudes and behaviours in buying food; in terms of lifestyle, women have a more positive attitude toward buying organic food and men tend to pay higher prices for organic food. households with total incomes above $3,975 are 350% (14.503) more likely to consume sheep meat than households with total incomes of less than $2,271. the likelihood of consuming sheep meat increases in line with the increasing income of the household. however, it is noteworthy that the likelihood decreases by 86.5% (0.135-1) when considering sheep meat consumption in terms of the person who contributes the highest income to the household (table 2). furthermore, shiflett et al. (2007) reported that in the usa, income has a positive effect on lamb meat consumption per capita. in may 2015, beef carcass and lamb carcass prices in izmir were $8.40/kg and $8.22/kg, respectively (ukon, 2015). the average prices in hypermarkets, on the other hand, are $15.35/kg for beef meat and $15.72/kg for lamb meat, indicating that because the recent prices of beef and lamb/mutton are quite close to each other, factors other than price affect ital. j. food sci., vol 29, 2017 216 consumers' preferences for red meat. a one-kilo decrease in beef consumption in a household in izmir increases the likelihood of sheep meat consumption by 73% (0.268-1). in a study by byrne et al. (1993), pork prices were found to affect demand for lamb meat, while the prices of chicken and beef were determined to have no effect on sheep meat demand. pork is not consumed in turkey because of religious beliefs. because there is no habit of consuming goat meat in izmir, this increases the chance of beef and lamb/mutton being consumed as each other's substitutes. the presence of one additional person in a household increases the likelihood of consuming sheep meat by 101.6% (1-2.016). educational status, age, whether there are any cardiac patients in the household, employment status and poultry consumption of the household do not affect sheep meat consumption in a statistically significant way. on the other hand, while chicken consumption, the existence of a cardiac patient in the household and having a bachelor's degree are negatively correlated with sheep meat consumption, the age of the consumer and his or her marital status are positively correlated with sheep meat consumption (table 2). according to the findings of a study conducted by russel and cox (2004), the meat preferences of middle-aged and older individuals differ; in comparison to younger individuals, elders' perceptions of processed food, roasted chicken and lamb and pork chops are more positive. in addition, pearce (2013) examined the results of studies conducted in australia and the usa, eu and uk and reached the generalization that consumers older than 35 consume more lamb meat. in the present study, grouping the respondents into two groups (those that are younger and older than 35) produced no statistically significant difference in terms of lamb/mutton consumption. the rates of sheep meat consumption in groups of people both younger than and older than 35 are fairly similar and are between 26% and 27%. according to the results of pearson’s chisquared test (0.034), there is no significant difference between age and sheep meat consumption at 0.05. on the other hand, the total rate of sheep meat consumption among families that have members 25 years of age or younger is 64%; lamb/mutton is consumed in 30.7% of these households. in families in which all members are older than 25, the rate of sheep meat consumption is 18.5%. thus, the results of pearson’s chi-squared test (5.313) were significant at 0.05. while the rate of families with members 55 years of age and older is 18% among all families, 26% of them regularly consume sheep meat. the results of pearson’s chi-squared test (0.006) were not significant at 0.05. accordingly, it was determined that in izmir, the rate of families that have young members and regularly consume sheep meat is 12.2% higher than families that also regularly consume sheep meat but do not have young members. the marital status of consumers in izmir was determined to have no statistically significant effect on sheep meat consumption (table 2). while prättälä et al. (2006), similarly, could find no effect of marital status in finland and latvia on meat consumption, the authors determined that married people in estonia and lithuania consume meat more frequently. 3.2. individual reasons for sheep meat consumption as for the reasons why consumers prefer sheep meat, they were under the impression that lamb/mutton is healthier (29.1%) because sheep in turkey are mostly fed in pastures, and it is believed that sheep breeding requires less medicine than bovine breeding. they also noted that sheep meat is considered tastier (41.8%) and is preferred for reasons of habit (29.1%) however, when grouping sheep meat consumers on the basis of their red meat consumption (as beef-heavy red meat consumption, lamb/mutton-heavy red meat consumption and exclusively lamb/mutton consumption) significant differences among ital. j. food sci., vol 29, 2017 217 the groups were determined. thus, the results of pearson’s chi-squared test (20.453) were significant at 0.01. while the group that exclusively consumes sheep meat as its red meat prefers sheep meat because of the belief that it is healthier (62.5%), in the beef-heavy consumption group, the preference for consuming sheep meat is less related to the perception of it being healthier (14%). in a study conducted by font i furnols et al. (2011), it was reported that the belief that lamb/mutton is healthier is based on the fact that feeding with fresh grass is healthier, more natural and more environmentally friendly than intensive pellet feeding. in addition, consumers’ perception that sheep meat is healthier is based on the fact that sheep are fed in pastures. it is also reported that consumers prefer animals bred in highland pastures to those bred in lowland pastures (imami et al., 2011; hersleth et al., 2012; montossi et al., 2013). approximately 77% of the participating sheep meat consumers stated that choosing meat of domestic origin is important to them. in some studies, consumers that considered meat origin to be important were older; in addition, gender has a distinctive effect on purchasing decisions (verbeke et al., 2000; font i furnols et al. 2011). however, no significant difference among consumers' age group (young, middle-aged or older groups) in izmir was found in terms of regular consumers' preferences for meat origin. the results of pearson’s chi-squared test (2.170) were significant at 0.05. 3.3. the respondents who do not regularly consume sheep meat consumers’ reasons for not consuming sheep meat include the lack of appeal of the specific smell and taste of sheep meat (53%), the consideration that it is fattier than beef and therefore unhealthy (35%), and not having the habit of consuming it because their families did not consume sheep meat (12%). in addition, according to the findings of several studies (prescott et al, 2001, prescott et al., 2004), the lack of the habit of consuming sheep meat and mutton and the factor of taste are important in terms of consumer preferences; factors such as how the animals are fed and deodorization by using spices are also worth considering. 3.4. willingness to pay for a labelled product when the consumers who prefer sheep meat were asked how much their willingness to pay would increase if the lamb/mutton were sold with labels indicating its quality, some of the consumers (13 respondents, 16.45%) stated that the price of sheep meat was already too high and that they could not pay more. as for the extra amount the willing respondents would pay for labelled products, this was calculated as $1.62/kg by means of blaine's method, as presented in equation 3 (table 3). dickinson et al. (2003) determined that 35% of consumers in the usa and 37% of consumers in canada would be willing to pay $1.35 and $1.85, respectively, for certification of meat traceability, animal welfare and advanced food safety and that the demographic characteristics of the consumers affect this willingness. lyford et al. (2010), on the other hand, reported that among consumers from australia, japan, the usa and ireland, the willingness to pay for quality is highest in japan; the willingness to pay is higher among consumers aged 25 to 35 in all four countries; and the effects of the other demographic variables are relatively lower. in a study conducted by sánchez et al. (2001) in spain, it was determined that while willingness to pay more for lamb is based on meat origin, for beef it depends on food quality. ital. j. food sci., vol 29, 2017 218 table 3. willingness to pay and the lower bound mean (lbm)* consumers (number) payment willingness tl/ kg % cumulative percentage % 1 8 1.52 1.52 2 7 3.03 4.55 6 6 9.09 13.64 9 5 13.64 27.28 13 4 19.70 46.98 15 3 22.73 69.71 15 2 22.73 92.42 5 1 7.58 100.00 66 100.00 lbm= tl3.56/kg ($1.62/kg) *1 us$ = 2.192 tl. 4. conclusions the present study addressed preferences and willingness-to-pay among sheep meat consumers. according to the findings of the study, the likelihood of consuming sheep meat in the izmir province is affected by gender, level of income, number of people in the household, beef consumption and whether the respondent is the member who contributes the highest income to the household. according to the model results, household income positively affects the possibility of sheep meat consumption. on the other hand, when the three income groups were compared, it was determined that the highest sheep meat consumption ratio (33%) was in the middle-income group ($2271– $3974). in this context, we can recognize the importance of developing marketing strategies aimed specifically at high-income groups. as goat meat and pork are not consumed in izmir, and the prices of sheep meat and beef have recently become very similar, it was determined that factors other than price affect consumer preferences related to red meat. however, if, from the perspective of consumers, parity between cattle meat and sheep meat develops on behalf of sheep meat, the demand for sheep meat among low-income groups will also increase. in this context, analyses of the effects of supply-increasing policies in sheep breeding can be tested by new studies. one of the important findings of this study is that women negatively affect the possibility of sheep meat consumption. if women are to prefer sheep meat, it is important to disseminate information about the change in fat ratio in the meat according to different carcass sizes and different rations, and about the frequencies with which sheep are allowed to forage. one of the most prevalent reasons for not consuming sheep meat regularly is dislike of its smell and taste. companies should test different meat types in the market according to consumer preferences (spiced and sauced meat, different methods of feeding, etc.) the results of this study showed that the majority of consumers are willing to pay more for meat that uses quality-based labelling. in this context, if companies develop standards according to quality and especially if this becomes a legal obligation consumption of sheep meat will increase. in conclusion, the possibility of sheep meat consumption may increase in izmir, which represents the aegean region. however, it is also understood from these results that the ital. j. food sci., vol 29, 2017 219 increase in the possibility of 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valencia-chamorro et al., 2011). a dip treatment of fresh-cut fruit in organic acids (such as citric acid and ascorbic acids) and calcium salts as an alternative to sulphites were used to prevent enzymatic browning after fruits peeling and/or cutting (oms-oliu et al., 2010). also, calcium treatments can maintain or improve the tissue firmness and crispness (oms-oliu et al., 2010). in this regards, edible coatings containing aloe vera and green tea extracts are well documented in the literature. aloe vera gel and gelatin have been used as edible coatings in fruit storage technology (andrade et al., 2014; dang et al., 2008). the barrier properties of aloe gel coatings towards respiratory gases (chauhan et al., 2011), as well as its antimicrobial functions (martínezromero et al., 2006) in coated fruit and fresh-cut fruit are reported. besides, gelatin coatings show good barrier characteristics against oxygen and aroma transfers at low and intermediate relative humidity. however, gelatin has poor barrier properties against water vapour transfer due to its hydrophilic nature (andrade et al., 2014). in recent years, the aloe vera gel has been used as an edible coating for sweet cherries (martínez-romero et al., 2006), mangoes (dang et al., 2008), apples (chauhan et al., 2011; song et al., 2013), papayas (marpudi et al., 2011), fresh-cut kiwifruit (benítez et al., 2015; benítez et al., 2013), and fresh-cut orange (radi et al., 2017). besides, the effect of aloe vera coating, containing anti-browning solution, on apples slices (song et al., 2013) has been published in literature. furthermore, tea (camellia sinensis), is a good source of polyphenolic compounds, which have strong antioxidant properties. the high antioxidant capacity and overall antimicrobial activity of green tea have been attributed to catechins and their oxidized condensation products (martín-diana et al., 2008; matan et al., 2015). coating with gelatin incorporated with green tea extract successfully retarded the microbial growth and therefore extended the shelf life of fresh-cut orange during cold storage (radi et al., 2017). such properties made us use green tea as our coating alternative. the aim of the present study was to investigate the combined effects of edible coatings containing gelatin, calcium chloride, ascorbic acid, and citric acid as well as various concentrations of aloe vera and green tea extracts on physicochemical and microbial characteristics of fresh-cut apples during storage. 2. materials and methods 2.1. materials gelatin, sodium hydroxide, calcium chloride, citric acid, ascorbic acid, and plate count agars (pca) were purchased from merck (darmstadt, germany). aloe vera leaves and green tea were purchased from a local wholesale market (yasooj, iran). red apples (red delicious) grown at semirom orchard (isfahan, iran) were freshly harvested at a commercially mature stage, sorted to eliminate the damaged ones, and selected for uniform size and colour. ital. j. food sci., vol. 30, 2018 63 2.2. preparation of the film-forming solutions for coating the apple slices aloe vera extract was obtained from fresh aloe vera leaves according to the method described by navarro et al. (2011). the extract was used intact (for aloe vera 100% treatment) or was diluted 50:50 with distilled water (for aloe vera 50% treatment). moreover, the aloe vera gel was concentrated to 150% using a rotary evaporator (heidolph, germany) at 45°c. moreover, green tea extract was prepared, based on the siripatrawan and harte (2010) method. the total solid content (tsc) of tea extract was determined by the air oven method at 105°c. according to the tsc of tea, the final concentration of extracts was adjusted at 5, 10, and 15% tsc using a rotary evaporator (heidolph, germany) at 45°c. gelatin powder was dissolved in distilled water or concentrated-adjusted aloe vera and tea extracts by stirring and heating to 50°c under nitrogen gas atmosphere to form 1% gelatin solution. the following coating solutions were assigned: (a) basic formula 1 (bf1): gelatin (1%), citric acid (0.1%), and calcium chloride (0.5%); (b) basic formula 2 (bf2): gelatin (1.0%), citric acid (0.1%), calcium chloride (0.5%), and ascorbic acid (0.5%); (c) the basic formula 1 and 2 with aloe vera extract at three levels (50, 100, and 150%) that was abbreviated as bf1 or bf2+50, 100, and 150% aloe; (d) the basic formula 1 and 2 with green tea extract at three levels (5, 10, and 15%) that was abbreviated as bf1 or bf2+5, 10, and 15% gt; and (e) coated with water which served as control. 2.3. coating the apple slices apples of uniform size and shape, and without any signs of mechanical damage, were selected, washed with chlorinated water (50 mg cl2/kg h2o) and manually sliced in chilled water (5–6°c). apple slices were dipped in the above-mentioned coating solutions for 1 min. and then drained for 30 min. the prepared apple slices were placed in polyethylene terephthalate (pet) clamshells (140 × 128 × 30 mm3) (pars plastic khuzestan, ahwaz, iran), and stored at 4°c for 16 days. 2.4. measurement of titratable acidity (ta) and total soluble solids (tss) the apple slices were homogenized in a blender (moulinex, barcelona, spain) and centrifuged at 2000 rpm for 1 min. to obtain a clear juice. the titratable acidity and total soluble solid of clear juice were measured (radi et al., 2010). 2.5. weight loss determination the weight loss in the samples was calculated as loss in weight of the apple slices in each container during storage and the values were reported on a percentage basis (radi et al., 2010). 2.6. firmness measurement the firmness of the apple slices was measured using a texture analyzer (ct3, brookfield engineering laboratories, stoughton, ma, usa) with a uniaxial penetration test. a stainless steel flat-end probe of 4 mm diameter was used to evaluate the firmness of the apple slices. the test conditions used for the measurement were pre-test speed 2mm/s; test speed 1 mm/s; post-test speed 10 mm/s; penetrating distance of 10 mm into the fruit, and a trigger force of 5 g (benítez et al., 2013). ital. j. food sci., vol. 30, 2018 64 2.7. measurement of colour the surface colour of the samples was measured using a hunter colorimeter (colorflex, virginia, usa). hunter cie l* for lightness, a* for redness, and b* for yellowness were determined (radi et al., 2017). 2.7. microbiological evaluation the microbiological analysis of the apple slices was carried out for standard plate counts in accordance with chauhan et al. (2011) procedures. the results were expressed as log cfu/g of sample. 2.8. sensory analysis sensory evaluation was performed immediately after the apple slices were prepared at storage times of 0, 8, and 16 days. twelve panellists were asked about the different quality attributes (colour, aroma and flavour, texture or firmness, and overall acceptance) of the apple slices using a scale with anchors at 0 and 5 as follows: colour, ranging from dark (0) to colour normal (5); aroma and flavour of apple, from weak (0) to strong (5), texture from soft (0) to hard (5). a final, overall preference test was also performed with a hedonic scale from dislike extremely (0) to like extremely (5). scores from 2.5 to 5 were considered acceptable. 2.9. statistical analysis all the experiments were run in triplicate. statistics on a completely randomized design were performed with the analysis of variance (anova) procedure in sas (release 9.1, sas institute inc., cary, nc) software and mean comparisons were carried out by duncan’s multiple range test (p<0.05). 3. results and discussions 3.1. titratable acidity (ta) and total soluble solids (tss) the effects of coating treatments on the ta and tss parameters during cold storage are shown in tables 1 and 2. the ta levels in the control and coated samples gradually decreased during the storage period, and the difference was significant in the control sample only on day 16. but, the decreasing trends of ta in coated samples were not significant during the storage period (data not shown for apple slices coated with bf1 and bf2 containing aloe vera and green tea extracts). a further reduction of acidity in the control sample in comparison with trehalose/nacl/sucrose-coated apple slices on the eighth day (albanese et al., 2007) and gel-coated apple slices with cysteine, citric acid, ascorbic acid, and aloe vera during the 16th day (song et al., 2013) were also reported. this phenomenon was linked to the malic acid decrease due to an increase in the respiration rate following peeling and cutting (albanese et al., 2007). the higher acidity of coated apple slices could be attributed to the barrier properties of aloe gel coatings towards respiratory gases (chauhan et al., 2011; radi et al., 2017). it seems that during storage, organic acids are used as substrates in respiration metabolism, thereby decreasing the ta and increasing the tss (benítez et al., 2013). ital. j. food sci., vol. 30, 2018 65 table 1. titratable acidity changes in the control and coated apple slices with basic formulas (bf1 and bf2) during the 16 days of storage at 4°c. treatment storage time (day) 0 4 8 12 16 control 0.37±0.02aa* 0.35±0.03aa 0.34±0.03aa 0.35±0.03aa 0.27±0.02bb bf1 0.37±0.05aa 0.36±0.02aa 0.33±0.04aa 0.31±0.01aa 0.31±0.03aa bf2 0.34±0.04aa 0.38±0.05aa 0.35±0.03aa 0.35±0.03aa 0.32±0.02aa *mean ± standard deviation (n = 3); means followed by the different small letter within the same row or by the different capital letter within the same column are statistically different (p<0.05). table 2. tss changes in the control and coated apple slices with basic formulas (bf1 and bf2) incorporated aloe vera and green tea extracts during the 16 days of storage at 4°c. group treatment storage time (day) 0 4 8 12 16 1 control 16.03±0.02ae* 16.08±0.01ad 16.18±0.02ac 16.31±0.02ab 16.36±0.02aa bf1 16.01±0.02ad 16.03±0.01bd 16.08±0.02bc 16.17±0.02bb 16.22±0.02ba bf2 16.02±0.01ad 16.02±0.02bd 16.08±0.01bc 16.15±0.02bb 16.23±0.02ba 2 bf1 16.01±0.02ad 16.03±0.01ad 16.08±0.02ac 16.17±0.02ab 16.22±0.02aa bf1+50% aloe 16.01±0.01ae 16.04±0.01ad 16.07±0.02ac 16.15±0.02ab 16.23±0.02aba bf1+100% aloe 16.02±0.01ad 16.04±0.01ad 16.06±0.01ac 16.13±0.01bb 16.19±0.02bca bf1+150% aloe 16.02±0.02ad 16.03±0.02ad 16.05±0.01ac 16.11±0.01cb 16.17±0.02ca 3 bf2 16.02±0.01ad 16.02±0.02ad 16.08±0.01abc 16.15±0.02abb 16.23±0.02aa bf2+50% aloe 16.02±0.01ad 16.03±0.02ad 16.09±0.02ac 16.16±0.02ab 16.20±0.01aa bf2+100% aloe 16.01±0.01ad 16.03±0.01ad 16.06±0.02bc 16.13±0.01bcb 16.18±0.02ba bf2+150% aloe 16.01±0.01ae 16.04±0.01ad 16.05±0.01bc 16.11±0.02cb 16.15±0.01ca 4 bf1 16.01±0.02ad 16.03±0.01ad 16.08±0.02ac 16.17±0.02ab 16.22±0.02aa bf1+5% gt 15.99±0.02ae 16.03±0.02ad 16.08±0.02ac 16.18±0.01ab 16.21±0.01aba bf1+10% gt 16.02±0.01ad 16.04±0.01ad 16.07±0.02abc 16.16±0.02abb 16.20±0.02aba bf1+15% gt 16.00±0.05ad 16.02±0.01adc 16.05±0.01bc 16.14±0.02bb 16.19±0.01aa 5 bf2 16.02±0.01ad 16.02±0.02ad 16.08±0.01abc 16.15±0.02abb 16.23±0.02aa bf2+5% gt 15.99±0.02ae 16.03±0.01ad 16.08±0.01ac 16.15±0.02ab 16.20±0.01ba bf2+10% gt 15.98±0.03ad 16.03±0.02ac 16.06±0.01bc 16.13±0.02ab 16.18±0.01ca bf2+15% gt 15.99±0.03ad 16.01±0.01ad 16.05±0.02bc 16.12±0.02ab 16.18±0.01ca *mean ± standard deviation (n = 3); means followed by the different small letter within the same row or by the different capital letter within the same column of each group are statistically different (p<0.05). the tss of the control and coated samples significantly increased with storage time, while the coated samples showed a slight increase compared to the control sample (table 2). in this regard, there was a significant difference between the control and the coated samples with the basic formulas (bf1 and bf2 without aloe vera and green tea extracts) only after four days of storage. but, no difference was observed between the bf1 and bf2, which indicated the same effect of bf1 and bf2 treatments on apple slices during storage time. increasing the concentration of aloe vera and green tea extracts in the basic formulas (bf1 and bf2) increased the tss significantly only after eight days of storage, and especially at ital. j. food sci., vol. 30, 2018 66 the end of the storage period. furthermore, no significant differences were found between bf1+aloe and bf2+aloe treatments in similar concentrations of the aloe vera extract (50, 100, and 150%). without considering the bf1 and bf2 coatings, samples coated with higher concentrations of aloe vera and green tea extracts showed a lower increase in tss at the end of the storage periods. the highest increase of tss was observed in the control (~ 2.5%, tss increased from 16.0 to 16.4 after 16 days of storage at 4°c), while the lowest increase was observed in samples coated with bf2+150% aloe (~ 1.3%, tss increased from 16.0 to 16.2). the findings of this study were similar to the results of ahmed et al. (2009), marpudi et al. (2011), and radi et al. (2017), who reported that ta decreased and tss increased with increasing storage time in nectarines, papaya and fresh-cut orange, respectively. during ripening, organic acids are used as substrates in respiration metabolism, thereby resulting in an increase in ta and decrease in tss. in general, it seems that the total soluble solid content tends to increase over the storage period as a consequence of the ripening process (benítez et al., 2013). a reduction in the respiration rate has been observed in sweet cherries (martínez-romero et al., 2006) and kiwifruits (benítez et al., 2013) coated with aloe vera gel. furthermore, softening occurs primarily because of an enzymatic degradation (pectin methylesterase and polygalacturonase) of the cell wall, which is mainly composed of cellulose, hemicelluloses, and pectins (oms-oliu et al., 2010). this may affect some physicochemical characteristics such as ph, ta, tss, etc., of fresh-cut fruit. in this regard, the increasing trend of tss in apple slices can be attributed to the softening and may, therefore, be associated with ripening (benítez et al., 2013). 3.2. weight loss the weight loss is mainly associated with moisture evaporation through the surface of fruit slices (olivas et al., 2007). all samples demonstrated a gradual weight loss during storage (table 3). the weight loss of uncoated fruit (25.10 %) was significantly greater than those of coated fruits during storage time. the weight loss of apple slices coated with bf1 and bf2 treatments was significantly lower than the control (p<0.05). in this regard, no significant difference was observed between bf1 and bf2 treatments until the eighth day, but the difference was significant on the 12th and the 16th days, and also the weight loss of bf1was significantly lower than bf2 at the end of the storage time. the least rate of weight loss (11.18 % and 11.52 %) was observed, respectively, in the samples coated with bf1+150% aloe and bf2+150% aloe treatments. consequently, the weight loss of apple slices coated with bf+aloe was significantly lower than other samples (p<0.05). accordingly, the bf+aloe coating was more effective than the bf+green tea coatings. in the case of bf+15% gt weight loss was ~18.5%. furthermore, weight loss of samples coated with both basic coatings (bf1 and bf2) containing 150% aloe and 15% gt was significantly lower than of those coated with a lower level of extracts at the end of the storage periods. similar results were obtained by song et al. (2013) and rdai et al. (2017). these authors reported that the weight loss increased during storage, but the weight loss of the aloe vera gel-coated apple slices was significantly (p<0.05) reduced compared to the control during storage. the binding of aloe vera gel molecules to the surface of apple slices may have reduced the porosity of apple slices, resulting in lower water loss (song et al., 2013). it is reported that aloe vera gel reduces the respiration rate, ethylene production, weight loss and, therefore, the softening of fresh-cut fruit textures (benítez et al., 2013). ital. j. food sci., vol. 30, 2018 67 table 3. weight loss changes in the control and coated apple slices with basic formulas (bf1 and bf2) incorporated aloe vera and green tea extracts during the 16 days of storage at 4°c. group treatment storage time (day) 0 4 8 12 16 1 control 0.5±0.2ae* 12.13±0.26ad 19.68±0.14ac 23.32±0.26ab 25.10±0.37aa bf1 0.3±0.1ae 8.34±0.36bd 12.19±0.29bc 16.5±0.7bb 18.41±0.19ba bf2 0.3±0.2ae 7.7±0.43 bd 12.63±0.23bc 15.59±0.26cb 19.55±0.3ca 2 bf1 0.3±0.1ae 8.34±0.36ad 12.19±0.29ac 16.5±0.7ab 18.41±0.19aa bf1+50% aloe 0.43±0.15ae 7.9±0.58abd 11.69±0.17ac 14.90±0.28bb 17.20±0.2ba bf1+100% aloe 0.23±0.15ae 7.23±0.32bd 10.3±0.35bc 13.09±0.24cb 15.41±0.2ca bf1+150% aloe 0.2±0.10ae 6.2±0.2cd 8.15±0.39cc 9.50±0.22db 11.18±0.19da 3 bf2 0.3±0.2ae 7.7±0.43ad 12.63±0.23ac 15.59±0.26ab 19.55±0.3aa bf2+50% aloe 0.27±0.15ae 7.78±0.14ad 12.40±0.25ac 14.59±0.27bb 17.51±0.29ba bf2+100% aloe 0.23±0.15ae 7.24±0.39ad 10.69±0.17bc 12.50±0.17cb 15.51±0.34ca bf2+150% aloe 0.37±0.21ae 5.99±0.32bd 8.72±0.21cc 10.20±0.17db 11.52±0.23da 4 bf1 0.3±0.1ae 8.34±0.36bcd 12.19±29bc 16.5±0.7ab 18.41±0.19aa bf1+5% gt 0.27±0.15ae 8.10±0.14cd 11.72±0.20cc 14.87±0.21bb 17.23±0.25ba bf1+10% gt 0.37±0.15ae 8.84±0.33abd 11.81±0.25bcc 13.27±0.36cb 17.50±0.32ba bf1+15% gt 0.17±0.12ae 9.2±0.36ad 12.68±0.18ac 14.70±0.23bb 18.4±0.22aa 5 bf2 0.3±0.2ae 7.7±0.43bd 12.63±0.23bc 15.59±0.26bb 19.55±0.3aa bf2+5% gt 0.23±0.15ae 10.09±0.29ad 12.19±0.4bc 14.56±0.21cb 17.51±0.40ca bf2+10% gt 0.17±0.12ae 9.65±0.34ad 12.69±0.18bc 15.84±0.24abb 18.11±0.21ba bf2+15% gt 0.27±0.15ae 9.81±0.28ad 13.31±0.28ac 16.09±0.24ab 18.50±0.20ba *mean ± standard deviation (n = 3); means followed by the different small letter within the same row or by the different capital letter within the same column of each group are statistically different (p<0.05). 3.3. texture evaluation the texture degradation and softening trend continued through the storage time, but its rate was slowed down by the bf1 and bf2 coating compared to the control sample (table 4). the maximum firmness was observed in bf1+150% aloe (firmness decreased from 10.51 to 9.82 n after 16 days of storage at 4°c), while the least firmness was observed in control (firmness decreased from 10.44 to 5.62 n after 16 days of storage at 4°c). in addition, similar concentrations of aloe vera (50, 100, and 150%) and green tea (5, 10, and 15%) extracts used in the bf1 and bf2 coatings had no significant effect on firmness, which indicated the same effects of bf coatings on apple slices during storage. regardless of the bf1 and bf2 coatings, samples coated with the higher concentration of aloe vera and green tea extracts had higher firmness. there was also no significant difference between coated samples with green tea and aloe vera extracts. the lower firmness of the control sample than the coated samples was probably due to the growth of spoilage microorganisms in the sliced apple, but which was limited in coated samples due to the antimicrobial properties of aloe vera and green tea extracts (benítez et al., 2013; matan et al., 2015). softening occurred primarily due to the enzymatic degradation (pectin methylesterase and polygalacturonase) of the cell wall. calcium is reported to maintain firmness by cross-linking with pectins to form insoluble calcium pectates, which strengthen the structure of the cell wall (oms-oliu et al., 2010). in this ital. j. food sci., vol. 30, 2018 68 regard, the fresh-cut apples, treated with calcium, showed no significant differences throughout the three weeks of storage (alandes et al., 2006). table 4. apple slices firmness changes (n) in the control and coated apple slices with basic formulas (bf1 and bf2) incorporated aloe vera and green tea extracts during the 16 days of storage at 4°c. group treatment storage time (day) 0 4 8 12 16 1 control 10.44±0.25aa* 8.70±0.13cb 6.34±0.18cc 6.02±0.08bd 5.62±0.11be bf1 10.48±0.24ab 10.88±0.09aa 9.68±0.20ac 9.13±0.08ad 8.87±0.06ad bf2 10.24±0.08aa 10.23±0.06ba 9.33±0.11bb 9.10±0.07ac 8.86±0.11ad 2 bf1 10.48±0.24ab 10.88±0.09aa 9.68±0.20bc 9.13±0.08cd 8.87±0.06cd bf1+50% aloe 10.43±0.09aa 10.50±0.12ba 9.91±0.08abb 9.52±0.11bc 8.98±0.08cd bf1+100% aloe 10.34±0.11aa 10.26±0.06ca 9.96±0.06ab 9.73±0.09ac 9.25±0.11bd bf1+150% aloe 10.51±0.13aa 10.41±0.08bca 10.05±0.10ab 9.86±0.06ac 9.82±0.10ac 3 bf2 10.24±0.08ca 10.23±0.06ba 9.33±0.11cb 9.10±0.07cc 8.86±0.11cd bf2+50% aloe 10.35±0.13bca 10.16±0.05bb 9.87±0.08bc 9.45±0.10bd 9.09±0.14bce bf2+100% aloe 10.52±0.09aba 10.27±0.09abb 10.06±0.09ab 9.71±0.17ac 9.32±0.12abd bf2+150% aloe 10.55±0.08aa 10.43±0.12aa 10.04±0.07ab 9.87±0.06ab 9.47±0.14ac 4 bf1 10.48±0.24ab 10.88±0.09aa 9.68±0.20bc 9.13±0.08cd 8.87±0.06dd bf1+5% gt 10.27±0.08aa 10.21±0.02ba 9.99±0.04ab 9.46±0.12bc 9.05±0.10cd bf1+10% gt 10.34±0.14aa 10.22±0.04bab 10.03±0.15ab 9.63±0.12bc 9.23±0.08bd bf1+15% gt 10.47±0.03aa 10.34±0.03ba 10.13±0.06ab 9.83±0.07ac 9.50±0.07ad 5 bf2 10.24±0.08ba 10.23±0.06aa 9.33±0.11bb 9.10±0.07cc 8.86±0.11cd bf2+5% gt 10.33±0.09aba 10.27±0.05aa 9.93±0.07ab 9.64±0.12bc 9.34±0.07bd bf2+10% gt 10.34±0.08aba 10.23±0.08aa 9.96±0.09ab 9.85±0.10ab 9.46±0.11abc bf2+15% gt 10.44±0.06aa 10.24±0.14aab 10.08±0.14ab 9.86±0.12ac 9.53±0.10ad *mean ± standard deviation (n = 3); means followed by the different small letter within the same row or by the different capital letter within the same column of each group are statistically different (p<0.05). the results of studies have indicated that aloe vera reduces the respiration rate and ethylene production, weight loss, and softening (benítez et al., 2013). in this regard, chauhan et al. (2011) showed that aloe gel coating alone or in combination with shellac, preserves the firmness in apple slices. further, the aloe vera edible coating application generally resulted in harder kiwifruit slices (benítez et al., 2013). in addition to the antimicrobial effects, the improvement in mechanical properties of the films incorporating green tea extracts may be responsible for the interaction between polymeric matrix and polyphenolic compounds from green tea extracts (siripatrawan and harte, 2010). 3.4. colour change colour is an important factor in the perception of the quality of fresh-cut fruit during their shelf-life. the colour indices (l*, a*, and b*) of apple slices stored at 4°c for 16 days were measured and only l* is reported in table 5. statistical analysis showed that the l*, a*, and ital. j. food sci., vol. 30, 2018 69 b* colour indices significantly changed during storage. a significant increase in colorimetric a* and b* values, and a significant decrease in the l* value were observed in apple slices during storage time. the colour indices of apple slices showed a significant difference (p<0.05) between the uncoated and coated samples. table 5. l* changes in the control and coated apple slices with basic formulas (bf1 and bf2) incorporated aloe vera and green tea extracts during the 16 days of storage at 4°c. group treatment storage time (day) 0 4 8 12 16 1 control 76.00±2.00aa* 72.33±0.58ab 69.00±1.00ac 66.33±1.53cd 64.67±1.53bd bf1 74.00±1.00aa 71.66±0.58ab 70.00±1.00ac 68.66±0.58bcd 68.00±1.00ad bf2 75.66±1.15aa 73.00±1.00ab 71.00±1.00abc 71.33±1.14abc 70.00±1.00ac 2 bf1 74.00±1.00aa 71.66±0.58ab 70.00±1.00bc 68.66±0.58acd 68.00±1.00cd bf1+50% aloe 74.33±1.53aa 71.66±0.58ab 70.33±0.56abbc 68.66±0.55acd 68.00±1.00cd bf1+100% aloe 74.66±1.53aa 72.33±1.50aab 71.33±0.57abcb 70.33±1.00aa 70.33±0.58ba bf1+150% aloe 74.67±0.56aa 72.67±1.53aab 72.00±1.00acb 70.33±1.53ac 70.67±0.55ac 3 bf2 75.66±1.15aa 73.00±1.00ab 71.00±1.00abc 71.33±1.14abc 70.00±1.00ac bf2+50% aloe 75.33±1.50aa 73.00±2.00aab 70.67±1.15acb 69.66±1.12ac 69.00±1.00abc bf2+100% aloe 75.34±1.14aa 73.33±1.50ab 72.33±0.54ab 70.33±0.59ac 70.00±1.00abc bf2+150%aloe 76.67±1.55aa 74.66±1.12aa 72.00±1.00ab 71.67.00±1.50ab 71.33±1.10bb 4 bf1 74.00±1.00aa 71.66±0.58bb 70.00±1.00bc 68.66±0.58ccd 68.00±1.00bd bf1+5% gt 75.00±0.95aa 72.00±1.00bb 71.00±1.00abb 69.00±1.00bcc 68.66±1.12abc bf1+10% gt 75.33±1.49aa 74.33±1.50aa 71.67±1.45abb 70.66±0.52abb 69.33±1.50abb bf1+15% gt 76.33±1.44aa 74.67±1.10aab 73.00±1.00abc 71.66±1.50acd 70.66±0.51ad 5 bf2 75.66±1.15ba 73.00±1.00bb 71.00±1.00abc 71.33±1.14abbc 70.00±1.00ac bf2+5% gt 75.00±1.00aba 72.67±1.60bb 71.00±1.05acb 69.66±0.62bc 69.33±0.60ac bf2+10% gt 76.33±1.61aba 75.33±1.55aa 72.66±1.49ab 71.00±1.00abb 70.66±1.09ab bf2+15% gt 77.66±1.55aa 76.33±0.63aa 73.34±1.60ab 72.33±1.17abc 71.00±1.00ac *mean ± standard deviation (n = 3); means followed by the different small letter within the same row or by the different capital letter within the same column of each group are statistically different (p<0.05). the reduction trend of l* values in coated and uncoated samples occurred at different rates during the storage (p<0.05), showing a darkening tendency in the surface colour of the apple slices. the least reduction trend for l* was observed in the coated samples with both basic coatings (bf1 and bf2) containing 150% aloe and 15% gt (~ 7.0%), in contrast to the control, which had the highest l* reduction (14.9%). the l* reduction during storage may be related to the occurrence of browning (martín-diana et al., 2008). the least increasing trend for a* and b* was observed in the coated samples with both basic coatings (bf1 and bf2) containing 150% aloe and 15% gt in contrast to control, which had the highest a* and b* increasing (a* and b* respectively increased from -5.33 and 22.0 to 4.33 and 33.67 after 16 days of storage at 4°c). the variations of l*, a*, and b* in the coated samples were significantly low at the highest concentrations of aloe vera and green tea extracts at the end of the storage periods, confirming the effect of these coatings in preventing the darkening and browning of apple slices. but at the same concentrations (50, 100, and 150%) of aloe vera and green tea (5, 10, ital. j. food sci., vol. 30, 2018 70 and 15%) extracts, there was no significant difference between bf1 and bf2 treatments in any of the measured colour parameters. colour is a critical quality property of fresh-cut fruit, since the slicing of fruit may often lead to enzymatic browning by polyphenol oxidases and peroxidases, which react with phenolic compounds and cause surface browning (albanese et al., 2007; oms-oliu et al., 2010). furthermore, the oxidative degradation of ascorbic acid and non-enzymatic browning are reported to be a major deteriorative reactions occurring during storage (wibowo et al., 2015). thus, anti-browning agents such as ascorbic acid, thiol-containing substances, carboxylic acids, and certain phenolic acids have been studied (oms-oliu et al., 2010). in this study, citric and ascorbic acids were used as anti-browning agents to inhibit enzymatic browning. cut-surface colour of apple slices that had been treated with ascorbic acid (in bf2) was well maintained than bf1-coated samples, but this effect was not significant. an increase in the browning reactions in fresh-cut apples during storage was observed after increases in hunter a* and b* values and a decrease in l* (perez-gago et al., 2006; song et al., 2013). the findings of this study indicated that the variations of colour in aloe-treated apple slices, especially at higher concentration (150%), were significantly lower than bf1and bf2-coated apple slices. similarly, chauhan et al. (2011) reported that the l*, a*, and b* values of the aloe vera gel-coated apple slices showed fewer changes compared to the control during storage for 30 days at 6°c, suggesting the anti-browning functionality of the aloe vera coating. it is reported that the application of aloe vera gel coating is an effective method for maintaining the colour of fresh-cut apple slices, as the aloe vera gel coating can act as an oxygen barrier film, thus reducing enzymatic browning. however, the coating does not completely prevent oxidative browning (song et al., 2013). therefore, to inhibit enzymatic browning, anti-browning agents were added to the aloe vera gel coating solution (song et al., 2013). in this regard, song et al. (2013) reported that aloe vera gel, containing 0.5% cysteine, was most effective in delaying the browning of apple slices during storage. the l* levels of coated apple slices increased with green tea extract concentrations. but the increase was not significant, indicating the anti-browning effects of green tea extract. conversely, martín-diana et al. (2008) reported that an increase in green tea concentrations decreased the l* values of coated lettuce. 3.5. microbial analysis fresh-cut fruit is highly susceptible to pathogenic and spoilage microorganisms during the preparatory steps as a consequence of cross-contamination, the presence of a large area of cut surfaces, and juice and sugar leakage from damaged tissues (oms-oliu et al., 2010). the microbial growth on the surface of coated apple slices showed significant differences between the coated and uncoated samples (table 6). a significant difference was found between the bf1and bf2-coated samples after 12 days of storage. the total viable counts gradually and significantly increased with storage time in all treatments (table 6). the microbial population of the control sample was higher than in the other treatments (2.63 and 6.78 log cfu/g at 0 and 16 days of storage, respectively), while samples coated with bf1+15% gt had the lowest microbial count compared to other treatments (2.42 and 3.17 log cfu/g at day 0 and 16 of storage period, respectively). the microbial counts in the aloe veraand green tea-coated samples did not exceeded 4.0 log cfu/g during storage time and were significantly lower than the coated samples with bf1 and bf2 (p<0.05). therefore, the addition of aloe vera and green tea extracts in bf1 and bf2 coatings reduced the microbial population significantly, and this effect was ital. j. food sci., vol. 30, 2018 71 enhanced at higher concentrations of these extracts. in both the bf1 and bf2 coatings, the inhibition of microbial growth was a function of the aloe vera and green tea extracts. significant differences were also found between bf1 and bf2 treatments in similar concentrations of aloe vera (100 and 150%) and also green tea extract (10% and 15%). the results showed that the antimicrobial effect of green tea was more than that of aloe vera, especially in the higher concentrations of these extracts. table 6. microbial growth (log cfu/g) in the control and coated apple slices with basic formulas (bf1 and bf2) incorporated aloe vera and green tea extracts during the 16 days of storage at 4ºc. group treatment storage time (day) 0 4 8 12 16 1 control 2.48±0.06ae* 3.16±0.07ad 5.25±0.13ac 6.10±0.04ab 6.78±0.04aa bf1 2.63±0.09ad 2.73±0.09cd 3.22±0.05bc 4.02±0.04bb 4.94±0.07ba bf2 2.52±0.11ae 2.92±0.05bd 3.17±0.06bc 3.73±0.08cb 4.65±0.11ca 2 bf1 2.63±0.09ad 2.73±0.09ad 3.22±0.05ac 4.02±0.04ab 4.94±0.07aa bf1+50% aloe 2.47±0.06bd 2.56±0.05bd 3.03±0.08bc 3.32±0.07bb 3.91±0.09aa bf1+100% aloe 2.45±0.09bd 2.56±0.07bd 2.96±0.07bc 3.22±0.01bcb 3.92±0.05aa bf1+150% aloe 2.43±0.05be 2.56±0.08bd 2.92±0.06bc 3.17±0.08cb 3.95±0.07aa 3 bf2 2.52±0.11ae 2.92±0.05ad 3.17±0.06ac 3.73±0.08ab 4.65±0.11aa bf2+50% aloe 2.47±0.06ae 2.62±0.06bd 2.95±0.09bc 3.51±0.09bb 3.99±0.06ba bf2+100% aloe 2.49±0.06ad 2.59±0.02bd 2.86±0.11bc 3.19±0.07cb 3.65±0.10ca bf2+150% aloe 2.43±0.05ad 2.54±0.05bd 2.80±0.12bc 3.14±0.11cb 3.56±0.08ca 4 bf1 2.63±0.09ad 2.73±0.09ad 3.22±0.05ac 4.02±0.04ab 4.94±0.07aa bf1+5% gt 2.47±0.04abd 2.57±0.04bd 2.96±0.06bc 3.29±0.09bb 4.14±0.10ba bf1+10% gt 2.43±0.09bd 2.49±0.02bccd 2.62±0.04cc 3.08±0.13cb 3.64±0.09ca bf1+15% gt 2.42±0.12abc 2.47±0.03cc 2.55±0.06cc 2.85±0.06db 3.17±0.06da 5 bf2 2.52±0.11ae 2.92±0.05ad 3.17±0.06ac 3.73±0.08ab 4.65±0.11aa bf2+5% gt 2.51±0.05ad 2.59±0.05bd 2.89±0.08bc 3.26±0.10bb 4.10±0.08ba bf2+10% gt 2.42±0.03ad 2.46±0.06cd 2.58±0.02cc 3.11±0.09bb 3.85±0.10ca bf2+15% gt 2.48±0.03ac 2.44±0.03cc 2.52±0.03cc 2.86±0.09cb 3.30±0.07da *mean ± standard deviation (n = 3); means followed by the different small letter within the same row or by the different capital letter within the same column of each group are statistically different (p<0.05). according to table 6, lower microbial populations in samples coated with aloe vera and green tea extracts can be attributed to antimicrobial properties of the coated compounds (benítez et al., 2013; matan et al., 2015; radi et al., 2017)). aloe vera extract was reported to have antimicrobial functions, significantly reducing mesophilic bacteria, and especially showing antifungal activity (martínez-romero et al., 2006; valverde et al., 2005). some individual components found in aloe vera gel, such as saponins, acemannan, and anthraquinone derivatives, are known to have antibiotic activity and could be responsible for its antibacterial activity (valverde et al., 2005). green tea, too, is a rich source of polyphenols (mainly catechins and catechin derivatives) and the antimicrobial activity of green tea has been attributed to these compounds (martíndiana et al., 2008; matan et al., 2015). matan et al. (2015) and radi et al. (2017) ital. j. food sci., vol. 30, 2018 72 confirmed the antimicrobial activity in green tea extracts on fresh-cut dragon and fresh-cut orange, respectively. the reduction of microbial populations presented in this study was in good agreement with the antimicrobial effects of aloe vera coating on table grape ((valverde et al., 2005), sweet cherry (martínez-romero et al., 2006), apple slices (chauhan et al., 2011; song et al., 2013), kiwifruit slices (benítez et al., 2015), raspberry fruit (hassanpour, 2015), and fresh-cut orange (radi et al., 2017) which reduced the aerobic bacteria, as well as yeast and mould counts during storage. 3.6. sensory analysis the quality attribute scores (colour, aroma and flavour, texture or firmness, and overall acceptance) for the control and coated samples were studied on days 0, 8, and 16 (fig. 1). figure 1: sensory attributes of apple slices coated with basic formulas (bf1 and bf2) incorporated with aloe vera extracts during the 16 days of storage at 4ºc. the panellists gave greater sensory scores to coated slices than uncoated slices at the three stages of the experiment (days 0, 8, and 16). thus, the sensory analyses revealed the beneficial effects of coating in terms of delaying browning and maintaining the sensory quality of the apple slices. all edible coating treatments resulted in higher sensory scores than uncoated apple slices for all quality factors tested. but, except for colour, the other sensory characteristics were not significantly different in control and bf1 and bf2. in this ital. j. food sci., vol. 30, 2018 73 regard, the colour score of the bf1 was significantly higher than those of bf2 and control samples. although increasing the concentration of aloe vera and green tea extract in the basic formulas led to higher sensory scores, no significant difference was observed between the apple slices coated with different concentrations of aloe vera and green tea extracts. but, at the end of the storage, the panellists gave greater sensory scores (colour and overall acceptance) to bf2+150% aloe-coated slices than the other treatments. unexpectedly, aloe gel-coated samples showed the lowest firmness at the end of storage even at high concentrations. the higher scores of coated slices compared to the uncoated ones were reported in the aloe vera gel-coated apple slices (chauhan et al., 2011; song et al., 2013) and aloe-coated orange slices (radi et al., 2017). 4. conclusions in this study, an attempt was made to use aloe vera and green tea extracts in gelatin-based coating to maintain the freshness of apple slices. although gelatin-based coatings obtained higher quality attributes than those of control during storage time, the coatings did not completely prevent chemical and biochemical reactions. the addition of aloe vera and green tea extracts in the gelatin-based coatings maintained the quality parameters of apple slices for a longer time during the storage period. in this regard, the least increasing trend for a* and b* was observed in samples coated with both gelatin-based coatings (bf1 and bf2) containing 150% aloe vera and 15% green tea extracts. the samples coated with higher concentrations of aloe vera and green tea extracts had lower increases in tss at the end of storage periods. in terms of microbial count, the total count gradually and significantly increased with storage time in all treatments. the antimicrobial compounds aloe vera and green tea extracts contributed to the lower microbial populations in samples coated with them. slices coated with 150% aloe vera and 15% green tea extracts obtained higher values for firmness. moreover, the panellists gave greater sensory scores to coated apple slices than uncoated samples during the storage period. in this regard, the bf2+150% aloe vera sample achieved higher sensory attributes than those of other treatments at the end of storage time. acknowledgements the authors would like to acknowledge the islamic azad 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of specific parameters and their relation to colour instability. food chem. 187:140. paper received december 1, 2016 accepted may 20, 2017 ijfs#626_bozza ital. j. food sci., vol 29, 2017 377 paper profiling wine consumers by price segment: a case study in beijing, china w. mu, h. zhu, d. tian and j. feng * college of information and electrical engineering, china agriculture university, 209# no.17 qinghuadonglu, haidian district, beijing, 100083, p.r. china *corresponding author. tel.: +86 1062736717; fax: 13581585962 e-mail address: fjying0306@163.com abstract this study aimed to analyze and identify the purchase behavior and preferences of 976 chinese wine consumers, particularly the differences in their demographics, purchase behaviors and recognition about wine based on a price segmentation categorized as high, moderate, and low spenders. price segmentation was defined as the price typically paid for a bottle (750 ml) of wine for daily drinking at home. significant differences among the three segments were discovered based on income, educational background, job category, purchasing places, favorite brands, recognition of the importance of wine attributes and acceptable price for gift-oriented wine. research on market segmentation based on price about chinese wine market is limited. the current study provides insight into market segmentation and may serve as basis for future research. keywords: consumer behavior, consumer preferences, wine market, market segmentation ital. j. food sci., vol 29, 2017 378 1. introduction the chinese wine market has expanded in recent years, china produced 1.12 billion liters of wine and consumed 1.6 billion liters of wine in 2015, and china ranked ninth in terms of wine production and fifth in terms of wine consumption in the world (oiv, 2015), thus indicating that china has been a major player in the international market (vinexpo, 2013). trade reports show that the red wine sector is growing at a significant rate (euromonitor international, 2013). the total volume of wine that china imported in 2015 reached 554 million liters, which is approximately 45% higher than that in 2014 (un trade, 2015). the annual growth rate of the chinese wine market is 25%30% (china daily, 2012). however, the per capita wine consumption in china is only 1.17 liters a year; therefore, the wine consumption of each chinese consumer is not more than two bottles (750 ml/bottle) each year, whereas the world average is 3.32 liters (wine institute, 2015). the chinese wine consumption per capita is far lower than that in western countries, and thus opportunities for growth are vast. undoubtedly, as one of the emerging and new wine markets in the world, it can be forecasted that chinese wine market will attract more and more domestic and international wine producers, marketers and researchers. it is widely accepted that firms need to understand their consumers, which in turn requires analysis of all the dynamics and factors influencing consumers’ buying behavior so as to improve their competitive advantages and business performance (gunay and baker, 2011). understanding the purchase behavior of chinese wine consumers is crucial because preferences and behaviors of these consumers significantly affect the development direction of wine markets (christodoulidou and james, 2011). consumer behavior is a cornerstone in marketing strategy and it involves conceptual dimensions such as decision-making, values, motivations, self-concept and personality, expectations, attitudes, perceptions, satisfaction, trust and loyalty (cohen et al., 2014). consumers do not have homogeneous taste preferences (king et al., 2012). thus, segmenting the market is useful because different people have various needs (dolnicar, 2002). consumers within a segment are relatively similar, and consumers between segments are comparatively different (brunner and siegrist, 2011). thus, segmentation is a relevant tool in strategic marketing, which helps researchers understand consumers (dolnicar, 2002). market segmentation is defined as the process of dividing the total market into homogenous segments of consumers with similar needs and wants and the segmentation process addresses the needs of each subgroup efficiently (marshall and johnston, 2010). market segmentation is widely used in marketing products (rondancataluña and rosadiaz, 2014). guillet and kucukusta (2016) provide a method for segmenting spa consumers according to their preferences in the selected spa attributes. chen et al. (2013) use the market segmentation based on consumer motivation to analyze the bed and breakfast industry in taiwan. prayag et al. (2014) identify three clusters of travelers based on their service expectations, and determine that cultural differences are crucial for heterogeneity in travel behavior. cirer costa (2013) investigates price formation and market segmentation in seaside accommodations. bruwer and buller (2012) provide insights into the consumer behavior characteristics and consumption dynamics of japanese wine consumers, explore the validity of existing generic consumer segmentation of the japanese market, and consider its suitability to predict wine-purchasing behavior. voorhees et al. (2011) propose a novel perspective to improve segmentation in the hotel industry. simpson and bretherton (2004) utilize market segmentation based on lifestyle of customers to explore the wine tourism context. ital. j. food sci., vol 29, 2017 379 various studies on wine consumption have been conducted in different countries, but those on market segmentation in the chinese wine market are scarce. olsen et al. (2015) divide wine consumers in the united states into three segments based on consumer variety-seeking behavior, namely, high variety-seeking, moderate variety-seeking and variety avoiders and find significant differences in high variety-seeking consumers compared with moderate variety-seeking and variety avoiders. molina et al. (2015) identify the different segments of wine tourists who visit spanish wineries. cohen (2015) tests a segmentation of hong kong wine consumers based on the perception of wine labels and finds that approximately 95% of young hong kong chinese wine consumers prefer “elegant contemporary” labels with red as the dominant color. gergely and dieter (2014) test the segmentation of wine consumers based on the usage of sales channels and define six segments that are aggregated into two groups, namely, basic (discount, supermarket and food-retail consumers) and premium (cellar-door, wine-shop and multichannel consumers). tang et al. (2011) use cluster analysis to divide wine consumers into six segments, namely, price-conscious wine consumers, involved consumers, knowledgeable consumers, image-oriented consumers, indifferent consumers, basic consumers, enjoyment-oriented consumers, and social consumers. bruwer et al. (2002) identify nine different segmentation variables in wine market studies in australia: quality, consumption, risk reduction, occasion based, cross-cultural, behavioral, involvement, geography and wine-related lifestyle. wine knowledge is used in several segmentation studies on wine consumers, such as works of vigarellis (2015), bruwer and buller (2012), and johnson and bastian (2009). the main segmentation variables used in existing studies are mainly related to consumers (interest in wine, knowledge of wine, motivations, sensation-seeking attitude and behavior, cultural variables, values and lifestyle) (molina et al., 2015). however, studies that conduct segmentation based on consumers’ perception of wine by price paid for a bottle (750 ml) of wine for daily drinking in chinese wine market are non-existent. and according to the wine intelligence china portraits (2015), developing drinkers who enjoy wine flavor, regard wine as a major part of their lives, and thereby often drink at home occupied an increasingly large proportion of chinese wine consumers. therefore, this topic is a new area for chinese wine market research. the current study aims to provide insight into the consumption characteristics and preferences of chinese consumers in wine. further the study categorizes consumers into three segments, namely, high, moderate, and low spenders based on the preferred wine price for daily drinking, explores the difference in the three groups in beijing and provides valuable information and marketing suggestions on the chinese wine market for wine marketers, wine business owners and other stakeholders. 2. materials and methods 2.1. conceptual framework various studies focused on wine market segmentation (see literature review). some studies highlighted consumer demographics, such as sex, age, income. some studies focused on consumer consumption habits, such as consumption occasions, purchase places, channels of obtaining wine information. other studies focused on consumer preferences about wine attributes, such as quality, brand, flavor and wine labels, and some examined consumer recognition of wine, such as knowledge in wine. the variables that can be used for segmentation can include consumption habits, cognitions, perceptions and acceptability. therefore, a conceptual model was developed to show and examine the ital. j. food sci., vol 29, 2017 380 potential segmentation variables that could be used for the segmentation of the wine market. the conceptual model is presented in fig. 1. in the current study, price segmentation is defined as the price typically paid for a bottle of wine for home consumption, provides a chance to understand differences among the segments. figure 1. the conceptual model. 2.2. questionnaire the questionnaire on the consumption characteristics and preferences of consumers in wine was designed on the basis of the literature to collect information on consumer behavior. the questionnaire consisted of 20 questions, organized into four sections: consumer demographics (gender, age, level of wage, educational background and job category), consumers’ purchase behavior (frequency of wine consumption, occasions for wine consumption and purchasing places), consumer awareness and preference in wine (consumers’ recognition of wine attributes, cognition of the best or worst wine origins and awareness and preference in wine brands), consumers’ acceptable price for wine (acceptable price for daily drinking and gift-oriented consumption). 2.3. survey in this research, beijing was selected as the case because it is one of the most developed areas in china, and it is one of the cities with high wine consumption in china (wang, 2006). consumers’ demand for wine in beijing may indicate the future demand trends in china. a cross-sectional survey was conducted in beijing, from september to october 2014. consumers’ characteristics were investigated on the basis of consumption behavior and wine preferences. questionnaire surveys are usually conducted via telephone, online, or face-to-face interviews. the face-to-face interview and online survey were employed in this study to ital. j. food sci., vol 29, 2017 381 ensure sufficient sampling, given that respondents’ cooperation is difficult to guarantee through telephone interviews. the process of the two research methods is as follows. face-to-face interview: ü first of all, six representative wine retailers in beijing were selected to participate in the survey. three supermarkets (merry mart, carrefour and wu mart) and three wine stores (guijie branch, dongzhimen branch and donglu branch of cheers, which is a popular wine store in beijing) were identified. ü secondly, five undergraduate students from china agricultural university were chosen as investigators. the students were recruited and trained before the survey. ü thirdly, the face-to-face survey was conducted by randomly selecting the interviewees. the surveys were conducted between 9:00 and 17:00 and a typical respondent who purchased wine was given approximately 10 minutes to complete the questionnaire. after the five-day survey, 410 questionnaires were obtained. the questionnaire was reasonable and concise, and the survey was supervised by trained investigators. the completion of the questionnaires was preliminarily ideal. all the answered questionnaires were valid. online survey: ü firstly, a famous professional questionnaire survey website in china called sojump was used to ensure that random consumers in different areas could participate in the survey. ü secondly, investigators released the corresponding website through different social media, such as wechat and qq to ensure the high respondent turnout of wine consumers in different regions in the survey. the online survey lasted one week and 604 questionnaires were obtained. after disregarding the incomplete or the logically paradoxical questionnaires, 566 valid questionnaires were acquired. the questionnaire used in the online survey was same as that in the face-to-face survey. however, in the online survey, respondents could fill out the questionnaire at a convenient time and without a time limit. and the questionnaire survey website provided storage and downloading functions, so that analysts could examine the results at any time. a total of 1,014 questionnaires were distributed, and 976 valid ones were obtained. the recovery efficiency reached a high 96.3%. 2.4. statistical methods the collected information from the survey was transformed into statistical variables and processed by spss software 19.0. descriptive statistical method and chi-square analysis testing were adopted to analyze the relation between consumers’ demographics and purchase behavior which includes the frequency and occasion of wine purchase and the demographic differences among price segments. the mean responses with the standard deviation, frequencies and percentages of responses in each category were calculated and presented in tables and graphs. ital. j. food sci., vol 29, 2017 382 3. results and discussions 3.1. consumers’ demographics table 1 shows the consumer demographics by gender, age, wage level, educational background and job category. the sample consisted of 455 women and 521 men. the most common age group was under 25, followed by 26-35. per capita monthly income was nearly evenly distributed from 2,000 to 10,000. well educated respondents (undergraduate and above) dominated the sample (74.9%). staff members in enterprises (44.5%) represent the most common occupation in the job category, and they are followed by students, thus explaining the high educational level and young age of most of the respondents. table 1. statistical characteristics of the respondents. demographics category %,n=976 demographics category %,n=976 gender male 53.4 educational background junior high school and below senior high school junior college undergraduate and above government official staff in enterprises liberal profession farmers education departments students unemployed/retired else 1.7 female 46.6 age under 25 26-35 45.9 36.4 8.6 14.8 74.9 per capita monthly income (cny) 36-45 11.8 46-55 4.9 job category 7.4 above 55 under 2000 2001-3000 3001-4000 4001-5000 5001-7000 7001-10000 above10000 1.0 23.4 11.6 15.7 17.7 15.3 12.0 4.3 44.5 11.9 0.3 4.4 25.0 2.2 4.4 notes: n, the total number of respondents. 3.2. purchasing behavior some studies considered wine as a female product (li et al., 2011). however, a qualitative study (liu and murphy, 2007) determined that wine buying is a male interest in future asian markets. age was also found to be correlated with wine involvement (bruwer, 2013). therefore, a chi-square test was used to examine the difference of gender and age on the frequency of wine purchase. a significant difference caused by age was identified (table 2). table 2 shows that approximately 18.0% of the men and 15.4% of the women often drank wine. while considering the difference of gender on wine drink, the chi-square test was insignificant, thus indicating that the frequency of wine consumption among men and women does not significantly differ. this result, which is consistent with that of forbes (2012), suggests that further research could be conducted to explore the effect of gender on the frequency of wine purchases in china. the 26-35 age group dominated the cluster of often purchased wine. the under 25 age group mostly purchased wine occasionally, thereby indicating that young and middle-aged consumers preferred wine. young people appeared to be the target of wine marketing in china (jenster and cheng, 2008; liu and murphy, 2007). ital. j. food sci., vol 29, 2017 383 table 2. difference of gender and age on the frequency of wine purchase. variables wine purchase frequency (%) (n=976) chi-square test often occasionally never f sig. gender male 18.0 73.3 8.7 3.531 0.171 female 15.4 72.7 11.9 age under 25 7.8 75.9 16.3 26-35 21.4 72.4 6.2 36-45 31.3 67.8 0.9 81.735 0.000*** 46-55 27.1 66.7 6.2 above 55 40.0 60.0 0.0 notes: n, number of respondents, 3-never, *** significant at 1% level respectively. wine is a social product and is consumed in various occasions. however, few chinese consumers buy wine for home entertainment (liu et al., 2014). in china, wine consumption is mainly related to gift-giving, business banquets and special occasions such as the traditional holidays like the middle festival, and so on. income and status-seeking are the main factors affecting wine consumption occasions (sun et al., 2009; richie, 2007). in this research, a multi topic was designed to investigate the occasions on which consumers consume wine. occasions on which wine is consumed vary (table 3). the result showed that 50.8% respondents drank wine during holidays, 41.3% consumers drank wine daily at home, and 30.0% purchased wine as gifts. the results are consistent with those of previous studies (sun et al., 2009; jenster and cheng, 2008; liu and murphy, 2007). the results of the chi-square analysis (table 3) indicated that the occasions on which the respondents consumed wine were significantly different among consumers with various income levels. respondents with an income of below 2000 (23.7%) mostly comprised those who consumed wine during holidays. this result is consistent with that of a previous research, which determined that consumers with low income tended to drink wine on special occasions such as holidays (sun et al., 2009). for business banquets, consumers whose income were cny 7001cny 10000 (28.8%) seemed to have the money and opportunity to consume wine. the survey determined that consumers most frequently purchased wine is supermarkets (61.5%), followed by wine stores (20.4%). the proportion of consumers who purchased wine online was relatively high (9.8%). the result is consistent with that of liu et al., (2014), who determined that chinese wine consumers mostly purchased wine in supermarkets. this result is attributed to two reasons. first, supermarkets are convenient for consumers (lockshin et al., 2012). second, price discounts in supermarkets attract consumers who want to save (flynn et al., 2010). the result also showed that online shopping for wine was still weak despite the prosperous electronic commerce in china. many factors contribute to this result, such as the fragileness of wine bottles and false wine information. ital. j. food sci., vol 29, 2017 384 table 3. influence of income on occasions on which wine is consumed. variables consumption occasiona (%) (n=976) chi-square test business banquet holidays as gifts daily others f sig. incomeb 0.000*** below 2000 14 117 60 85 8 2001-3000 5 64 28 35 6 3001-4000 6 84 52 54 2 56.835 4001-5000 14 86 51 84 0 5001-7000 11 75 54 70 1 7001-10000 21 46 33 51 1 above10000 2 21 14 23 3 notes: a the question is multiple choice, b average monthly income per capita. n, the total sample number of respondents, *** significant at 1% level respectively. 3.3. awareness and preference in wine 3.3.1. consumers’ cognition on the origins of wine the cognition of consumers on the origin of wine is presented in fig. 2. the result showed that consumers considered xinjiang province and the ningxia hui autonomous region as the best origins of wine, and beijing city, tianjin city and hebei province were deemed as the worst wine origins in china. the northwest areas are known to be famous wine origins in china (mu and feng, 2010), this is consistent with the survey results. however, 15% of the consumers deemed hebei province as the best wine origin. more than 10% of the respondents determined gansu, shandong and jilin provinces as the best and worst wine origins. these results indicate that consumers’ limited knowledge or lack of professional knowledge about wine origins. figure 2. consumers’ cognition on wine origins in china. ital. j. food sci., vol 29, 2017 385 3.4. consumer awareness and preference in wine brands figure 3 shows consumers’ familiarity with wine brands. brand effect is very important to wine. although the cognitive value of consumers in the existing brands of wine in china does not represent the actual value of the product, cognitive value is closely related to actual value. to some degree, cognitive value reflects the actual value of products (mu and feng, 2010). thus, 10 wine brands are considered in the current study and the respondents were requested to choose three brands that they were most familiar with and the three brands they were willing to buy. the results showed that greatwall (71.6%), changyu (70.8%), and dybasty (35.7%) were the three highly cognitive wine brands for chinese consumers. this result is related to brand history, advertising, and other factors (mu and feng, 2010). figure 3. consumers’ familiarity with wine brands. as shown in fig. 4, in terms of buying, 39.8% of the respondents considered changyu as their first choice, followed by greatwall (26.3%). forbes et al. (2014) verify that brand name, in the absence of other product information, influences consumer perception of quality and price, and purchase intentions, and that some categories of brand names perform better than others. it is notable that foreign brands (21.1%) is also coming into consumers’ eyes. these consumers consider foreign wine may be purer than that in china. the wine in china culture is underdeveloped, and consumers have limited knowledge about wine, including origins and brands. most chinese consumers are novice wine buyers, and tend to buy wines from the famous wine-producing regions they only knows. these consumers believe that wine imported from foreign countries is better than domestic wine (bruwer, 2002), and they seek little information about other brands. ital. j. food sci., vol 29, 2017 386 figure 4. consumers preferred wine brands when buying. 3.5. consumers’ recognition of wine attributes eight kinds of wine attributes, namely, price, quality, advertisements, recommendation of others, flavor, package, origins and productive years were selected in the current study. these scale items were based on the literature (hall et al., 2013; king et al., 2012; goodman, 2009; geraghty and torres, 2009). the respondents were asked to rank them according to importance. for the each attribute i (i =1, 2, ..,8, representing the 8 kinds of wine attributes), its final score si can be mathematically formulized as follows: where valuej is the importance for attribute i and ranges from 1 to 8 (8-the most important, 7the second important, 6the third important, 5the fourth important, 4the fifth important, 3the sixth important, 2the seventh important, 1the eighth important), and frequencyj is the times for valuej occurence. n is the number of respondents. the responses indicated that quality obtained the highest score of 5.78, followed by price (5.43), as shown in fig. 5. the third highest score (5.05) pertained to taste, followed by source (2.72). the result indicated that consumers prioritized wine quality in purchasing wine. this result may indicate the need for the chinese wine industry to improving wine quality. 3.6. market segments before designing the questionnaire, the research staff surveyed two supermarkets and three wine shops in beijing to acquire information on wine price. after analyzing the price data, the wine price ranges for daily drinking wine and wine for gifts were set in the questionnaire. however, the current study focused only on daily drinking of wine. in the analysis of market segmentation, 121 questionnaire were abandoned for missing the information about wine price for daily drinking. fig. 6 shows that for daily drinking wine ital. j. food sci., vol 29, 2017 387 (bottle/750 ml), most consumers accepted the price range of cny 75-cny 100, and few samples were willing to purchase wine higher than cny 175. based on consumers’ preference in wine price for daily drinking, chinese wine consumers in the current study were categorized into 3 groups (table 4). figure 5. consumers’ recognition of wine attributes. notes: 8-most important, 7-second important, 6-third important, 5-fouth important, 4-fifth important, 3-sixth important, 2-seventh important, 1-eigth important. figure 6. consumers’ acceptable price for wine for daily drinking. these groups included low spenders, moderate spenders and high spenders. after the groups were identified, chi-square analysis was employed to identify the significant difference between the groups. the variables in the analyses were demographics, which include sex, age, income, level of education, and job category. the statistic results are shown in table 5. ital. j. food sci., vol 29, 2017 388 table 4. clustering chinese wine consumers according to consumer preference in wine price for daily drinking segments wine price for daily drinking low spenders below cny 75 (16.6%) moderate spenders between cny 75-100 (24.2%) high spenders above cny 100 (59.2%) table 5. demographic differences among price segments. variables groups (n=825) chi-square test low moderate high f sig. gender male 69 98 259 1.049 0.592 female 68 102 229 age under 25 70 87 211 26-35 42 77 176 6.492 0.592 36-45 13 22 70 46-55 9 11 27 above 55 3 3 6 income a below 2000 41 39 98 2001-3000 22 29 54 3001-4000 29 47 69 4001-5000 16 30 80 35.297 0.000*** 5001-7000 18 24 85 7001-10000 9 25 69 above 10000 2 6 35 educational background junior high school and below senior high school junior college undergraduate and above 3 23 30 81 1 29 32 138 13 29 79 369 28.469 0.000*** job category government official 5 12 39 staff in enterprises liberal profession 52 18 49 32 126 147 farmers 1 1 2 education departments 4 50 26 114.014 0.000*** students 39 48 110 unemployed/retired 6 5 10 else 12 3 10 notes: aaverage monthly income per capita. n, the total sample number of respondents, *** significant at 1% level respectively. ital. j. food sci., vol 29, 2017 389 3.7. demographic differences among the three segments chi-square test was conducted to distinguish the significant consumer demographics, as table 5 shows, three variables of income, educational background and job category are significant. low spenders have a significantly lower income than the other two groups. therefore, this may explain why more people prefer to middle-priced wine, after all, wine is also only a decoration in consumers’ life. wine is not like food, consumers are conscious about the price of wine. when wine price exceeds their psychological expectation, consumers are bound to reduce their wine consumption (mu and feng, 2010). income is an important predictor of wine involvement bacause consumers need to have the financial resources to support their interest in wine (bruwer, 2013; barber et al., 2006). the group composed of undergraduate and above dominated among the three groups. the education standard of high spenders was higher than that of the two other groups. the number of undergraduate and above respondents accounted for 75.3% of the high spenders, 59.1% of the moderate spenders and 69.0% of the low spenders. according to liu et al. (2014), consumers are frequently exposed to information about wine because of the rapid development of the wine market in china, thus resulting in more mature consumers. thatch (2008) argues that the actual tasting of wine helps consumers develop an educated palate. job category did significantly differ among the three groups. high spenders were likely to be liberal professions. consumers who worked at education departments were likely to be moderate spenders, and staffs in enterprises mainly comprised the low spender group. occupational difference reflects the status of consumers in society somehow. researchers have verified that wine is considered as a symbol of social status and sophistication (fan, 2007; liu and murphy, 2007). no significant difference was observed among the three groups in terms of gender and age. previous research indicated that wine is not a luxury goods for consumers with improved standard of living, and that people can significantly benefit from wine (pettigrew and charters, 2010). thus, people prefer wine to other drinks, such as beer and liquor (zhou et al., 2011). income, education background and job category may influence the three segments. to distinguish the actual factors that influence the three segments, a principal component analysis was conducted. table 6 shows that the data passed the kmo and bartlett’s test of sphericity. the eigenvalues of income and educational background were all above one, and the overall contribution rate of the three segments accounted for 82.829%. these findings indicate that income and educational background can be regarded as main factors affecting the preferred wine price of consumers for daily drinking. table 6. principal component analysis of income, educational background and job category. component total % of variance cumulative kmo and bartlett’s test of sphericity (%) f sig. income a 1.484 49.482 49.482 educational background 1.000 33.347 82.829 0.499 0.000*** job category 0.515 17.171 100.000 notes: aaverage monthly income per capita, ***significant at 1% level respectively. ital. j. food sci., vol 29, 2017 390 3.8. behavior and recognition differences in the three segments for behavior, preferred purchase places and favorite brands were examined. first, the places where the three segments purchased wine were observed. five common wine selling places were selected in the current study, and the respondents were asked to choose the places they usually purchase wine. the results were listed in table 7. table 7. behavior and preferences differences among the three segments. variables groups (n=825) chi-square test low moderate high f. sig. purchase places supermarkets 94(68.6%) 135(67.5%) 278(57.0%) exclusive shop 21(15.3%) 37(18.5%) 110(22.5%) online shopping 17(12.4%) 15(7.5%) 49(10.0%) 20.287 0.009*** wine stores 1(0.7%) 11(5.5%) 39(8.0%) else 4(2.9%) 2(1.0%) 12(2.5%) favorite bands changyu 53(38.7%) 76(38.0%) 194(39.8%) greatwall 41(29.9%) 70(35.0%) 112(23.0%) dynasty 6(4.4%) 3(1.5%) 9(1.8%) wilon 3(2.2%) 2(1.0%) 15(3.1%) 25.459 0.013** suntime 4(2.9%) 3(1.5%) 7(1.4%) foreign brands 24(17.5%) 38(19.0%) 138(28.3%) else 6(4.4%) 8(4.0%) 13(2.7%) notes: n, the total sample number of respondents, *** , ** significant at 1% and 5% level respectively. consumers most frequently purchase wine in supermarkets among the three groups. the main factors affecting consumers to select the place to purchase wine are convenience and price (feng et al., 2012). as various supermarkets, which conveniently supply various and conveniently priced wines for consumers can be found in residential areas in china. supermarkets are important in making wine accessible to the mainstream market (richie, 2007). changyu, greatwall, and foreign bands were preferred by the three segments. the most popular wine brand in china is changyu, followed by greatwall (mu and feng, 2010). and high spenders and low spenders also preferred dynasty, wilon, sumtine and other brands. the results of chi-square test indicated that consumer behavior and recognition of wine showed some statistical significantly difference. it was found that the number of high spenders was higher in other places where wine could be purchased, such as exclusive shops and wineries, than those of the other two groups. low spenders were more likely to buy wine online shopping and other places, after all, products that sold online were cheaper than other places. for favorite brands, such as foreign brands, there was a great possibility that consumers were high spenders. ital. j. food sci., vol 29, 2017 391 3.9. knowledge and attitude differences among the three segments to examine wine knowledge, consumers’ cognition towards wine origins were analyzed. table 8 shows that xinjiang province and the ningxia hui autonomous region were considered as the best wine origins by the three groups. high spenders considered yunnan province to be the best wine origin. to hebei province, which is one of the better wine origins, the number of low spenders is greater than those of the other two groups. beijing city, tianjin city and hebei province were considered as the worst wine origins in china by the three segments. northwest areas are the best wine origins (mu and feng, 2010), the survey result is consistent with this outcome. however, more than 5.0% of the respondents in each group considered ganus, shandong, and jilin provinces as the best and worst wine origins, thus indicating that consumers’ limited knowledge about wine origins (mu and feng, 2010). table 8. knowledge differences among the three segments. groups (n=825) chi-square test low moderate high f sig. best originsa beijing city 19 26 55 tianjin city 12 10 36 hebei province 27 26 71 jilin province 21 30 64 gansu province 16 39 77 ningxia 49 85 165 22.076 0.229 xinjiang province 82 128 324 shandong province 24 24 81 yunnan province 13 18 78 liaoning province 11 14 35 worst originsa beijing city 49 66 169 tianjin city 45 61 172 hebei province 39 59 149 jilin province 21 32 74 gansu province 15 32 72 12.179 0.838 ningxia 12 17 41 xinjiang province 5 3 19 shandong province yunnan province liaoning province 23 25 39 24 40 56 85 91 104 notes: n, the total sample number of respondents, a the question is multiple choice. for deeper statistical analysis of consumers’ origins knowledge above findings, it is found that consumers presented no significant difference in wine origin knowledge. this finding is not surprising. according to liu et al. (2014) confirmed that a wine culture is lacking in china and many chinese wine consumers were novice wine buyers. chinese even have low awareness of grape varietals or food matching (jenster and cheng, 2008). ital. j. food sci., vol 29, 2017 392 consumers’ recognition of wine attributes was examined to determine the differences in attitude of the three segments. table 9 shows that significant difference exists among the respondents in terms of the importance of wine attribute. table 9. attitude differences among the three segments. attributes groups (n=825) chi-square test low moderate high f. sig. most important price 64(46.7%) 68(34.0%) 134(27.5%) quality 35(25.5%) 57(28.5%) 140(28.7%) advertisement 3(2.2%) 8(4.0%) 13(2.7%) recommended by friends 6(4.4%) 5(2.5%) 36(7.4%) flavor 16(11.7%) 38(19.0%) 93(19.1%) package 2(1.5%) 5(2.5%) 6(1.2%) 29.534 0.009*** origins 6(4.4%) 12(6.0%) 42(8.6%) productive year 5(3.6%) 7(3.5%) 24(4.9%) second important price quality 30(21.9%) 47(34.3%) 33(16.5%) 68(34.0%) 52(10.7%) 159(32.7%) advertisement 13(9.5%) 7(3.5%) 23(4.7%) recommended by friends 7(5.1%) 22(11.0%) 46(9.5%) 30.602 0.006*** flavor 30(21.9%) 38(19.0%) 118(24.3%) package 4(2.9%) 11(5.5%) 26(5.3%) origins 3(2.2%) 13(6.5%) 38(7.8%) productive year 3(2.2%) 8(4.0%) 24(4.9%) third important price 19(13.9%) 36(18.0%) 78(16.0%) quality advertisement recommended by friends 18(13.1%) 12(8.8%) 20(14.6%) 23(11.5%) 14(7.0%) 25(12.5%) 58(11.9%) 23(4.7%) 42(8.6%) flavor 36(26.3%) 49(24.5%) 121(24.8%) 16.144 0.305 package 9(6.6%) 15(7.5%) 42(8.6%) origins 16(11.7%) 26(13.0%) 74(15.2%) productive year 7(5.1%) 12(6.0%) 50(10.2%) forth important price 9(6.6%) 26(13.0%) 82(16.8%) quality 6(4.4%) 14(7.0%) 33(6.8%) advertisement 7(5.1%) 11(5.5%) 24(4.9%) 25.874 0.027** recommended by friends 23(16.8%) 21(10.5%) 39(8.0%) flavor 14(10.2%) 22(11.0%) 41(8.4%) package 21(15.3%) 18(9.0%) 44(9.0%) origins 24(17.5%) 48(24.0%) 111(22.7%) productive year 33(24.1%) 40(20.0%) 114(23.4%) notes: n, the total sample number of respondents, ***, ** significant at 1% and 5% level respectively. ital. j. food sci., vol 29, 2017 393 low spenders (46.7%) and moderate spenders (34.0%) considered price as the most important attribute, followed by quality, flavor, and origins. high spenders (28.7%) prioritized quality, followed by price, flavor, and source. the results of the three groups were also inconsistent regarding the second most important wine attribute. high spenders focused on the intrinsic attributes (quality, flavor, and source). the three segments did not significantly differ in the third most important attribute of wine, as flavor dominated all the three segments. low and high spenders chose productive year as forth most important attribute, and moderate spenders favored source. this segment result was similar to that of liu et al. (2014), in which consumers were categorized to intrinsic and extrinsic cluster. the intrinsic cluster mainly sought information about wine quality, price, flavor, and brand name, which are considered as the intrinsic attributes of wine. the extrinsic cluster pertained to wine package, and foreign wine, which are regarded as the extrinsic attributes of wine. according to liu et al. (2014), chinese wine consumers with a high income were likely to belong to the intrinsic cluster, whereas consumers with a low income belonged to the extrinsic cluster. the previous analysis indicated that high spenders have a higher income than the other two groups. therefore it was persuasive that why high spenders prioritized quality and low spenders and moderate spenders considered price as the most important factor when purchase. attitude positively affects the identification of factors influencing the purchasing behaviors of consumers. the result indicates that quality, price, flavor and source are four most important wine attributes. batt (2000) verifies that price is the most important factor affecting the purchasing behavior of consumers. gunay and baker (2011) demonstrate the importance of price, origin, and quality, and finds that quality and price are the most important factors affecting the purchase decision of consumers. 3.10. gift-oriented acceptable price of wine difference among the three segments the previous analysis showed that a relatively high number of consumers purchased wine for daily drinking and gifts, which were quite different in terms of motivation, preferred price and brand preference (pan, 2012). consumers are willing to pay a premium for wine as gifts because wine is a status symbol (liu and murphy, 2007). thus, the accepted price of wine as gifts may differ. therefore, this study separately analyzed consumers’ price acceptability of wine for daily drinking and that of wine for gifts. before designing the questionnaire, the research staff surveyed two supermarkets and three wine shops in beijing to acquire information about wine price. the price data and wine price ranges were set for wine as gifts in the questionnaire. table 10 shows that the acceptable prices for gift-oriented wine differed among the three segments. high spenders tend to buy high priced wine. respondents with an income of cny 100cny 150(30.7%) dominated the low spenders. respondents with an income of cny 150cny 200(26.0%) comprised the largest proportion in the moderate spenders group, followed by those with an income of cny 200-cny 250(21.0%). the price of the overall tendency of high spenders was higher than that of the other two groups. a consumer who buys a bottle (750 ml) of wine with a price above cny 250 may be to be a high spender (77.7%). a chi-square test was conducted to identity statistical significant. the results verify that there is great significant difference in preferred price of wine as gifts. income is an important predictor of wine involvement because consumers need to have the financial resources to support their choice (barber et al., 2006). ital. j. food sci., vol 29, 2017 394 table 10. gift-oriented wine price preference difference among the three segments. variables groups (n=825) chi-square test low moderate high f. sign. price for gift-oriented wine(cny) under 50 3(2.2%) 0(0.0%) 2(0.4%) 50-100 23(16.8%) 5(2.5%) 1(0.2%) 100-150 42(30.7%) 28(14.0%) 19(3.9%) 150-200 30(21.9%) 52(26.0%) 63(12.9%) 200-250 16(11.7%) 42(21.0%) 69(14.1%) 250-300 3(2.2%) 17(8.5%) 55(11.3%) 262.813 0.000*** 300-350 6(4.4%) 16(8.0%) 73(15.0%) 350-400 4(2.9%) 18(9.0%) 45(9.2%) 400-450 4(2.9%) 9(4.5%) 57(11.7%) above 450 6(4.4%) 13(6.5%) 104(21.3%) notes: n, the total sample number of respondents, ***significant at 1% and 5% level respectively. the previous analysis shows that income and education background significantly differ among the three segments. high spenders are likely to have a high income and educational level. sending expensive wine is an act of politeness and respect (sun et al., 2009). therefore, high spenders had sufficient money and were more likely to choose expensive wine than other types of spenders. 3.11. profiles of the three consumers segments creating profiles of the three different price segments based on the significant variables identified in this research is required. table 11 illustrates the major differences among the segments. table 11. profiles of chinese consumers by price. significant variables groups low moderate high demographics income below 2000 3001-4000 5001-7000 educational background undergraduate and above undergraduate and above undergraduate and above job category staff enterprises education department liberal profession behaviors supermarkets, supermarkets, wine stores, purchase places exclusive shops, online shopping exclusive shops, online shopping supermarkets, exclusive shop, online shopping preferred brands changyu,greatwall changyu, greatwall changyu, foreign brands attitude to wine attributes price, quality, flavor, productive year price, quality, flavor, origins quality, price, flavor, productive year accepted price for giftoriented wine cny 100-150 cny 150-200 above cny 250 ital. j. food sci., vol 29, 2017 395 4. conclusions the frequency of wine consumption is affected by age. the 26-35 age group dominates the often purchase cluster, and under 25 age group is mostly in the purchase occasionally. these results show that more young are involved in wine consumption. wine consumption occurs in many occasions, mainly at home, during holidays; most people also send wine as gifts. a significant difference is found between consumers’ income and consumption occasion. consumers with low income tend to consume wine on special occasions, such as the chinese traditional holiday. and on occasions that require the consumption of wine such as business banquets, consumers whose income is cny 7001cny 10000 (28.8%) seem to have much money and more chances to consume wine. consumers are familiar with three brands, namely, greatwall, changyu, and dynasty. this research introduces the chinese wine consumer price segments, namely, low spenders, moderate spenders, high spenders, and explores their differences in demographics, behaviors and preferences, and attitudes. high spenders have incomes higher than those of the other two groups, and they may be liberal professions. conversely, lower spenders may work in enterprises and moderate spenders can be in the education department. all three groups choose supermarket as their first wine purchase place, and a considerable number of high spenders purchase wine in wine stores. excluding changyu, the favorite wine brands of high spenders are foreign brands, and the favorite wine of the other two groups is greatwall. knowledge about wine origins of the three segments is limited, and their cognition of the importance of wine attributes has a significant difference. high spenders look for quality, price, flavor, productive year; moderate spenders look for price, quality, flavor, source; and low spenders look for price, quality, flavor, and productive year. a significant difference is observed in the acceptable wine price for gift-oriented. low spenders prefer wines priced at cny 100-cny 150, and moderate spenders are inclined to buy wine priced at cny 150-cny 200. high spenders prefer wines 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34 (1): 86–113 p u b l i c a t i o n s codon environmental impact of the main household cooking systems—a survey alessio cimini and mauro moresi* department of innovation in biological, agrofood and forestry systems, university of tuscia, viterbo, italy *corresponding author: mauro moresi, department of innovation in biological, agrofood and forestry systems, university of tuscia, viterbo, italy. email: mmoresi@unitus.it received: 9 january 2022; accepted: 4 february 2022; published: 23 february 2022 © 2022 codon publications open access review article abstract the food cooking energy may represent the primary hotspot in the cradle-to-grave life cycle of several foods and drinks. it is mainly affected by the type of food and its cookery method, cooking appliance and the fuel selected as well as the number of portions to be cooked. the primary aim of this survey was to demonstrate the basic characteristics of the main cooking methods, appliances, and fuels as well as energy required for some key foods. the secondary aim was to assess the environmental impacts of a generic cooking system as a function of few household cookers fueled by different fuels (i.e., firewood, charcoal, coal, natural gas, liquefied petroleum gas, kerosene and biogas) and electricity in the italian scenario by using the recipe 2016 and product environmental footprint (pef) standard methods and ecoinvent v. 3.7 database. a functional unit equal to per capita useful energy delivered to the pot for cooking (1.41 gigajoule [gj]) in 27 european union countries in 2019 was used as the basis of comparison. the use of natural gas resulted in minimum impact in nine of the 18 mid-point impact categories of recipe 2016 method and two damage categories (human health and ecosystem quality) with a minimum overall weighted damage score (owdsr) of ~5 pt. thus, such a cookstove appeared to be more apt to minimize both indoor and outdoor air pollution. even if the electric cookstove yielded a greater owdsr (8.6 pt) because the italian electricity grid mix was mainly based on fossil sources, it was possible to forecast that new-generation, smart cooktops driven by hydroand wind-power electricity would minimize owdsr to as low as 0.9 and 1.4  pt, respectively, thus not only avoiding the consumption of any fossil energy source but also improving people’s health. keywords: clean cooking, cooking appliances, cooking fuels, cooking systems, environmental impacts, life cycle assessment, pef standard method, recipe 2016 standard method introduction nowadays, cooking of food has become mandatory for humans (wrangham and conklin-brittain, 2003). its associated energy requirements represent the preponderant share of energy used in the cradle-to-grave life cycle of several foods and drinks, as in the case of vegetal products with low to medium degree of processing (carlsson-kanyama and boström-carlsson, 2001), dry pasta (bevilacqua et al., 2007; cimini et al., 2019, 2021a, 2021b) and coffee brewing (cibelli et al., 2021). energy use for cooking is largely affected by the food type and its cookery method, and the cooking appliance selected. energy utilization reduces as the number of portions cooked increases (carlsson-kanyama and boströmcarlsson, 2001). about one-third of the human population (that is 2.5billion people), generally living in lowand middle-income countries, still relies on solid biomass fuels (i.e., firewood, crop residue, charcoal and dung) for cooking, while the remaining 5-billion people rely on fossil energy, such as coal, natural gas (ng), kerosene, liquefied petroleum gas (lpg) and electricity (wright et al., 2020). mailto:mmoresi@unitus.it italian journal of food science, 2022; 34 (1) 87 environmental impact of the main household cooking systems fuels as well as energy requirements for some key foods. the secondary aim was to account for a generic cooking system capable of delivering the useful per capita energy transferred to the cooking pot in 27 european union  (eu) countries in 2019 (eurostat, 2021a). the final goal of this study was to assess its life cycle environmental impacts by using the well-known recipe 2016 (huijbregts et al., 2016) and product environmental footprint (pef) (european commission [ec], 2018b) lcia methods and ecoinvent v. 3.7 database (ecoinvent, 2020). by using different fuels (i.e., firewood, charcoal, coal, ng, lpg, kerosene, and biogas) and electricity used in the italian scenario, it was possible to identify the cooking system with overall minimum environmental impact and, thus, prospect promising new eco-smart household cooktops. basic characteristics of main cooking methods cooking makes food as more edible, easily digestible and relishing. in this way, cooked foods exhibit quite significant variations in their physical aspect, structure, composition and nutritional value. any food-cooking operation is characterized by a heating element (i.e., open flame etc.) and a heat transfer medium that allows cooking by: 1. expansion if the hot medium is made of water or oil, and 2. concentration if the heating element transmits heat to food through a pan, plate or grill. mixed cooking methods include both modes. generally, the first part of cooking is carried out by concentration, while the second one by expansion, thanks to the addition of a liquid. alternatively, the dry heat cookery methods (i.e., baking, steaming, grilling and roasting) can be discriminated from the moist heat cookery ones (i.e., boiling, stewing, shallow frying, deep frying and basting). in the former, the heating element conveys heat directly to the food (which in turn is cooked in its own juice and the steam generated by the evaporation of water added to the food during its preparation) mainly by means of convection and/or irradiation. on the contrary, in the moist heat methods a liquid medium (i.e., water, milk, coconut cream, molted butter, oil etc.) is heated before or after the food to be cooked is placed in the cooking pan. table 1 briefly describes both basic cooking methods and some combined and microwave cooking methods by pointing out heat propagation media, range of cooking temperature and main heat transfer modes. the combustion products emitted by solid fuels give rise to by far higher levels of indoor air pollution than those recommended by the world health organization (who, 2018), especially in poorly ventilated dwellings. these harmful emissions have been associated with respiratory diseases and other health problems (i.e., lung cancer, chronic obstructive pulmonary disease [copd], pneumonia, tuberculosis, cardiovascular events, low birth weight and cataracts; fullerton et al., 2008), and are responsible for as many as 4 million premature deaths per year globally (who, 2021). who is thus committed to attaining the so-called ‘sustainable development goals’ (sdg) on health (sdg 3) and energy (sdg 7) as scheduled by the united nations development program (undp, 2021) in order to improve the health and well-being of the people still using polluting technologies and fuels not only for cooking but also for heating and lighting. so far, numerous programs have been implemented globally to introduce cleaner and more efficient cooking technologies. all the attempts to improve the performance of biomass-burning stoves have until now caused limited health benefits, while the use of other cleaner fuels (i.e., lpg, ethanol and biogas) has offered not only greater health benefits but also smaller greenhouse gas (ghg) emissions (rosenthal et al., 2018). in particular, the replacement of solid biofuels with lpg was quite successful in several countries, such as brazil (wright et al., 2020), ecuador (martínez-gómez et al., 2016), ghana (afrane and ntiamoah, 2011, 2012), indonesia (thoday et al., 2018), india (gould and urpelainen, 2018; jungbluth et al., 1997; singh et al., 2014), and a few countries of the indo-chinese peninsula (aberilla et al., 2020) for at least two reasons: (i) lower ghg emissions compared to burning solid fuels, and (ii) lower infrastructure requirement compared to ng and electricity (wright et al., 2020). unfortunately, although use of such a clean cooking technology improves people’s health conditions, it still relies on fossil energy sources. a long-term sustainable cooking should alternatively rely on renewable energy sources only (i.e., solar or wind energy, biogas and bioethanol; aro, 2016). several life cycle assessment (lca) studies have so far dealt with cooking appliances, such as cookstoves fired by different fuels (aberilla et al., 2020; afrane and ntiamoah, 2011, 2012; jungbluth et al., 1997; singh et al., 2014), induction and gas hobs (favi et al., 2018) and electric and gas ovens (landi et al., 2019) in the italian context as well as induction hobs with different electronic boards (elduque et al., 2014), using diverse life cycle impact assessment (lcia) methods, some of which, unfortunately, use old databases. the primary aim of this survey was to review the basic characteristics of main cooking methods, appliances and 88 italian journal of food science, 2022; 34 (1) cimini a and moresi m ta bl e 1. m ai n fo od c oo ki ng m et ho ds (h ag er a nd m or aw ic ki , 2 01 3; m cg ee , 2 00 4) . m ai n co ok in g m et ho ds c oo ki ng sy st em d es cr ip tio n h ea t p ro pa ga tio n m ed iu m c oo ki ng te m pe ra tu re (° c ) h ea t t ra ns fe r m od e u se s d ry h ea t c oo ki ng b ak in g fo od is p ut in a n ov en . a ir 14 0– 25 0 a ir co nv ec tio n + w al l r ad ia tio n + pa n co nd uc tio n c er ea l-b as ed fo od s g ril lin g fo od is p la ce d on a g ril l t ra y, w hi ch is h ea te d by b ur ni ng ch ar co al a nd g as , o r b y el ec tri ci ty . a ir 22 0– 25 0 a ir co nv ec tio n + gr ill c on du ct io n + gr ill o r fl am e ra di at io n m ea t, ve ge ta bl es , c he es es , an d m ar sh m al lo w s te am in g fo od is c oo ke d by th e ho t s te am ri si ng fr om a n un de rly in g po t fi lle d w ith w at er . s te am a t a m bi en t or s te am p re ss ur e 10 0– 12 0 va po r c on ve ct io n an d co nd en sa tio n m ea t a nd fi lle d pa st as r oa st in g in an o ve n fo od is fl av or ed , t ie d w ith s tri ng s an d pl ac ed in a s au ce pa n co nt ai ni ng a v er y ho t f at ty s ub st an ce to w et th e ex te rn al su rfa ce o f th e fo od a nd a vo id it s dr yi ng . a ir an d fa ts 20 0– 22 0 a ir co nv ec tio n + w al l r ad ia tio n + pa n co nd uc tio n m ea t a nd n ut s r oa st in g on a sp it p ie ce s of fo od a re s ke w er ed , s al te d an d fla vo re d to b e he at ed b y w oo d, c ha rc oa l, co al , e le ct ric ity a nd g as . a ir 25 0 a ir co nv ec tio n + bu rn er ra di at io n + sp it co nd uc tio n po ul tr y an d m ea t c oo ki ng a u gr at in fo od is p la ce d in a s ha llo w o ve npr oo f co nt ai ne r a nd b ak ed or c oo ke d un de r a n ov er he ad g ril l w ith a to pp in g of s ea so ne d br ea dc ru m bs , g ra te d ch ee se , e gg , o r b ut te r. a ir 25 0– 30 0 a ir co nv ec tio n + gr ill ra di at io n + pa n co nd uc tio n ve ge ta bl es a nd p as ta s b ar be qu in g fo od is v ar io us ly c oo ke d ov er li ve fi re fr om g lo w in g fir ew oo d, ch ar co al o r c oa l a nd s m ok e us in g di re ct o r i nd ire ct h ea tin g w ith s ev er al n at io na l a nd re gi on al d iff er en ce s. s m ok e 20 0– 25 0 s m ok e co nv en tio n + fla m e ra di at io n m ea t, ve ge ta bl es a nd br ea ds m oi st h ea t co ok in g b oi lin g a w at er -fi lle d po t i s us ed to c oo k fo od o ve r t he fi re . e xc ep t fo r r ic e an d m ea t, th e co ok in g w at er is th ro w n aw ay a t t he en d of c oo ki ng . w at er ≤1 00 w at er c on ve ct io n p as ta s, ri ce , m ea t a nd ve ge ta bl es s te w in g fo od is c ho pp ed , d ic ed a nd c ub ed , a nd a dd ed to a p ot pa rt ia lly fi lle d w ith a li qu id . s te w ed fo od is s er ve d w ith th e th ic ke ne d liq ui d. w at er a nd o il ≤1 00 va po r c on de ns at io n + liq ui d co nv ec tio n + pa n co nd uc tio n m ea t, ve ge ta bl es a nd gr ai ns d ee p fr yi ng fo od is s ub m er ge d in h ot b oi lin g oi l c on ta in ed in a d ee p pa n til l i t t ur ns b ro w n on th e ou ts id e. a p ie ce o f br ow n pa pe r he lp s so ak in g up a ny o il fro m th e fo od b ef or e it is s er ve d. o ils a nd fa ts 15 0– 20 0 c on ve nt io n + pa n co nd uc tio n m ea t, ve ge ta bl es a nd do ug h p an o r sh al lo w fr yi ng fo od p ie ce s ar e st irr ed a ro un d a fe w ti m es in a fr yi ng p an co at ed w ith a s m al l a m ou nt o f oi l a nd fa t, an d m us t b e pr op er ly h ea te d to p re ve nt c oo ke d fo od s fro m b ec om in g to o oi ly, g re as y or b ur nt o ut si de a nd u nc oo ke d in si de . o ils a nd fa ts 15 0– 20 0 o il an d pa n co nd uc tio n m ea t, ve ge ta bl es a nd do ug h italian journal of food science, 2022; 34 (1) 89 environmental impact of the main household cooking systems b as tin g m ea t i s co ok ed in it s ow n ju ic es , o r a s au ce o r m ar in ad e in a n ov en u si ng a li dd ed o ve npa n. a ny ju ic e is o fte n sp oo ne d ov er th e ou te r p ar t o f th e m ea t t o av oi d its d ry in g du rin g co ok in g. u se o f a cl os ed o ve nba g tra ps e va po ra tin g m oi st ur e, th e co nd en sa tio n of w hi ch fo rm in g th e re al c oo ki ng ju ic e of fo od . a ir, s te am o ils , an d fa ts 10 0– 12 0 p an c on du ct io n + liq ui d co nv ec tio n + w al l r ad ia tio n m ea t m ix ed c oo ki ng b ra is in g fo od is fi rs t b ro w ne d at a h ig h te m pe ra tu re , a nd th en si m m er ed in a li dd ed p ot in a c oo ki ng li qu id (i .e ., w in e, b ro th , co co nu t m ilk a nd b ee r) . i t d iff er s fro m s te w in g fo r a bo ut 3 /4 of th e pr od uc t b ei ng s ub m er ge d by th e co ok in g liq ui d. a ir, s te am , o ils an d fa ts ~1 50 p an c on du ct io n + liq ui d co nv en tio n m ea t, ve ge ta bl es a nd gr ai ns c oo ki ng in a ca ss er ol e s m al l a nd te nd er p ie ce s of m ea t a re s al te d, p la ce d ov er fin el y ch op pe d ve ge ta bl es s pr in kl ed w ith m el te d bu tte r i n a lid de d ov en -p ot , a nd c oo ke d in th e ov en o n a ve ry lo w fl am e. a ir, o ils a nd fa ts 15 0– 22 0 p an c on du ct io n + liq ui d co nv ec tio n + w al l r ad ia tio n m ea t a nd v eg et ab le s m ic ro w av e co ok in g e le ct ro m ag ne tic ra di at io n at a fr eq ue nc y of 9 15 o r 2, 45 0  m h z is u se d to h ea t d ire ct ly a nd ra pi dl y th e on ly po la r m ol ec ul es o f fo od s, w hi le th e ov en a ir an d no np ol ar co nt ai ne r m at er ia ls (i .e ., gl as s, s to ne w ar e an d pl as tic ) a re he at ed b y th e fo od it se lf as it h ea ts u p. w hi le in fra re d en er gy is a lm os t e nt ire ly a bs or be d at th e fo od s ur fa ce , m ic ro w av es ca n pe ne tra te fo od to a d ep th o f ab ou t 2 .5 c m . po la r m ol ec ul es va ria bl e m ic ro w av e ra di at io n m ea t a nd v eg et ab le s main cooking appliances open wood fires were used for cooking by humans for approximately two million years (wrangham, 2009). the energy performance and resulting emissions from biomass cookstoves depend on various factors, such as the stove design, fuel feeding practice, lighting, and combustion temperature (okino et al., 2021; rasoulkhani et al., 2018). generally, traditional biomass stoves, largely used indoor in developing nations, are improperly ventilated, which leads to a significant increase in indoor levels of particulate matters (pm2.5) and carbon monoxide that cause lung inflammation and lead to  chronic obstructive pulmonary disease. excessive consumption of biomass fuels has led to natural forest degradation and deforestation, as already observed in south italy during period of roman empire, as well as shortage of firewood for cooking in some areas of africa even today (okino et al., 2021). in order to improve the efficiency of cooking energy and thus reduce the biomass consumption of the so-called three-stone fire stove, quite numerous improved stoves have been developed (okoko et al., 2018; wikipedia, 2021a). for instance, darlami et  al. (2019) reported that the thermal efficiency (ηcs) of a traditional nepalese cookstove increased from 18.0% to 25.6% when it was modified with mud, while okino et al. (2021) improved the thermal efficiency (ηcs) of a cooking stove insulated with sawdust from 13–21% to 19–35% with the use of a few indigenous wood fuels available in uganda. nevertheless, a recent review has found that the use of such improved cookstoves has so far had a small mitigating effect on the health results of household air pollution and called for more initiatives and policies to favor the adoption of cleaner fuels and improved cookstoves in households (pratiti et al., 2020). in fact, more than 2.5-billion people in developing countries are still relying on quite polluting cooking fuels, such as wood, crop residues, animal dung, charcoal, coal and kerosene. for instance, wood and coal burning cookstoves are still manufactured by the amish in lancaster county (pa, usa) to satisfy not only cooking and baking requirements but also heating and hot water requirements. in india  (jungbluth et al., 1997) and nigeria (anozie et al., 2007), kerosene stoves replaced most of the traditional biomass cookstoves, thanks to governmental fuel subsidies to prevent deforestation for cooking fuels. cheaper and more efficient gas stoves fueled by ng and lpg started to disseminate once a distribution network for gas pipeline and bottled lpg transport was available. this allowed their increasing diffusion either in europe or the united states since the beginning of the 20th century. gas ignition was originally done by matchsticks. then it became possible with a pilot light, a continuously burning gas flame under the cooktop to immediately light the gas leaving the burner if the stove was 90 italian journal of food science, 2022; 34 (1) cimini a and moresi m combustion (international renewable energy agency [irena], 2017). alcohol burning stoves are like existing kerosene stoves, their main differences being related to the use of stainless steel to minimize corrosion and fuel type used. interest for such stoves is mainly due to the use of bioethanol, its combustion practically providing pollutant-free emissions. unfortunately, such stoves are expensive and suffer from the costs of bioethanol supply chain. in the eu, such stoves are used for marine and mobile leisure applications in conjunction with an aqueous mixture of 85% (v/v) ethanol. their use was extended to a few developing countries, especially in brazil, where the above mixture is available as a biofuel (benka-coker et al., 2018; stokes and ebbeson, 2005; zuzarte, 2007). in order to avoid using such an inflammable mixture, other safer prototypes were developed in india and south africa to utilize diluted ethanol mixtures at 50% (v/v) (rajvanshi et al., 2004) or enriched with colorants and thickeners (i.e., calcium acetate) and flavoring agents (greengel; climate technology center & network [ctcn], 2017; okusanya et al., 2019), respectively. solar cooking appliances started to be commercialized in the 1980s as 100% emission-free devices capable of concentrating solar thermal energy to cook foods. each one consists of three components: concentrator, absorber and retainer. the first one, being made of shiny materials, such as silver, chromium or aluminum, allows the sunlight to be concentrated at a fixed point, where it is absorbed by a black-painted cookware to cook food. to minimize heat loss, the cookware is to be properly insulated and lidded. four types of solar cookers are currently available (pandey et al., 2021), as shown in table 2. the box cooker simply consists of a box inside another one and is the cheapest type of solar cooker. the transparent cover on the top of the outer box allows entrance of the sunlight, where it is absorbed by the black-painted inner box. a mirror in the inner side of the outer box helps in reflecting the heat energy radiated from the inner black box. the panel solar cooker has a large flat panel, which mainly reflects and focuses the sunlight falling vertically on the cooker for cooking. owing to the instability of panel to high wind, such a solar cooker is not used frequently. the parabolic solar cooker collects the solar radiation in the central focus point of a collector dish, where a pressure cooker with black-painted bottom is placed. in this way, temperatures as high as those causing the burning of food can be achieved. finally, the vacuum-tube solar cooker entails two tubes, one inside the other. the inner tube contains food to be cooked and is black painted to maximize heat absorption, while the outer one is transparent. vacuum is created in the space between these tubes to minimize heat loss and trap the heat absorbed for a longer period. such type of cooker is highly efficient (pandey et al., 2021). turned on. nowadays, gas stoves have electronic ignition to avoid any gas consumption when the stove is not used, a flame failure device to stop gas flowing without igniting and prevent from accidental explosion, and an extractor hood to evacuate fumes and minimize indoor air pollution (wikipedia, 2021b). by the end of the 19th century, several electric stoves were patented in canada, the united states and australia, even though their diffusion in household kitchens was conditioned by the extension of urban and rural electrification. whereas gas cooktops heat food with flame and disperse much of the heat in the air, electric cooktops mainly transfer heat to the cooking surface and thus are more efficient than gas cooktops. electric cooktops include both open coil and smooth (or radiant) cooktop types. the former is made of resistive wires encased in hollow metal tubes arranged in a spiral to directly support the cookware, and thus is quite affordable and durable. on contrary, smooth and radiant types consist of a hotplate surface or a smooth glass-ceramic surface heated locally via electrical heating coils or  halogen  lamps. the latter guarantees less heat loss with easier cleanability but, unfortunately, higher stretchability and breakability. nowadays, in such cooktops, the maximum temperature of the heating element is controlled thermostatically while power supply is regulated either discretely or continuously between minimum and maximum heat settings (wikipedia, 2021c). different studies have indicated that hotplates are least efficient among electric stoves (hager and morawicki, 2013), even if the glass–ceramic cooktops are found to be up to 20% less efficient than hotplate surfaces (carlsson-kanyama and böstrom-carlsson, 2001). moreover, reflective trays beneath electric coils in coiled cooktops appeared to increase the energy efficiency and reduce heating period by 20% (hager and morawicki, 2013). induction stoves are de facto the latest version of electric stoves. in fact, these use electricity to generate electromagnetic induction in just ferromagnetic cookware, which is thus directly heated instead of being heated by beneath smooth surface of glass– ceramic. such cooktops are not only highly safe but also the most energy-efficient appliances. their accurate control of cooking temperature could be improved by using a low-cost, open-source electronic platform pilotable via smartphone, as in the case of the novel home eco sustainable pasta cooker previously developed by cimini et al. (2020). among the diverse renewable fuel stoves, it is worth citing the biogas, ethanol and solar stoves. biogas stoves resemble the conventional ng and lpg stoves with just some modifications in burner design to maximize their combustion effectiveness and reduce unburned methane and soot from incomplete italian journal of food science, 2022; 34 (1) 91 environmental impact of the main household cooking systems table 2. main types of cookstoves and performances together with cooking fuels used, some typical models, fuel availability and cookstove efficiency (𝝶 cs ). category fuel type used some typical models fuel availability 𝝶 cs (%) references traditional biomass stove firewood usually, easily available in situ 11 13.5 14 17 20 13–21 aberilla et al., 2020 singh et al., 2014 afrane and ntiamoah, 2011, 2012 hager and morawicki, 2013 benka-coker et al., 2018 okino et al., 2021 crop residues usually easily available in situ 11 singh et al., 2014 aberilla et al., 2020 charcoal supply chain required 14 17.5 18 23 24–32 aberilla et al., 2020 singh et al., 2014 afrane and ntiamoah, 2011, 2012 benka-coker et al., 2018 okoko et al., 2018 coal stove coal supply chain required 15.5 singh et al., 2014 improved biomass stove firewood usually, easily available in situ 25.0–42.8 mehetre et al., 2017 modern fossil fuel stove kerosene supply chain required 35 40 45 46 47 42–64 afrane and ntiamoah, 2012 benka-coker et al., 2018 hager and morawicki, 2013 aberilla et al., 2020 singh et al., 2014 jungbluth et al., 1997 lpg supply chain required 24–34 45 46* 49 50 56 57 60–72 cimini and moresi, 2017 afrane and ntiamoah, 2012 cimini and moresi, 2017 aberilla et al., 2020 hager and morawicki, 2013 benka-coker et al., 2018 singh et al., 2014 afrane and ntiamoah, 2011 jungbluth et al., 1997 natural gas supply infrastructure required 50 hager and morawicki, 2013 electric stove electricity generic electric stove supply infrastructure required 55 59 65 70 80 benka-coker et al., 2018 aberilla et al., 2020 afrane and ntiamoah, 2012 singh et al., 2014 hager and morawicki, 2013 (a) hotplate hob 42–50 57* cimini and moresi, 2017 (b) coil stove 49 wollele, 2020 (c) induction cooktop 27–39 65* 45–59§ 66–68# cimini and moresi, 2017 cimini et al., 2020 renewable fuel stove biogas supply chain required 55 50 singh et al., 2014 aberilla et al., 2020 afrane and ntiamoah, 2011, 2012 ethanol (a) low-grade ethanol stove (b) ethanol-gel stove supply chain required 43–45 55 43 rajvanshi et al., 2004 benka-coker et al., 2018 okusanya et al., 2019 solar (a) box cooker (b) panel cooker (c) parabolic cooker (d) vacuum tube cooker weather-dependent 20 26.6 hager and morawicki, 2013 arenas, 2007 xu et al., 2015 * as referred to dry pasta cooking with a water-to-pasta ratio (wpr) of 10 l/kg when using the environmentally sustainable cooking practice set up by cimini and moresi (2017). # as referred to dry pasta cooking with wpr = 10 l/kg when using the home eco-sustainable pasta cooker (cimini et al., 2020). § as referred to dry pasta cooking with the minimum wpr of 2–4 l/kg when using the home eco-sustainable pasta cooker (cimini et al., 2020). 92 italian journal of food science, 2022; 34 (1) cimini a and moresi m table 2 provides an overview of main cooking stoves in use together with the cooking fuels used, fuel availability and cookstove efficiency (ηcs). besides the cooking stoves mentioned above, useable for the so-called surface (or stovetop or cooktop) cooking, oven cooking, must also be considered, since it is essential for cooking quite numerous food products (i.e., bread, cakes, biscuits and various meat products) requiring diverse cooking methods, such as baking, grilling, roasting etc. (table 1). an oven is a device liable to expose foods to a hot environment. it consists of a hollow chamber that can be heated in a controlled manner using a burning gas, electricity or microwaves. when an oven is combined with cook-tops (range), the fuel used for the oven may be the same as, or different from, that used for the burners on the stovetops. generally, the food placed in an oven is heated from below, as in the case of baking and roasting. it can be also heated from the top, as in the case of broiling and grilling. in a conventional oven, the air is naturally circulated in the oven chamber, while in a convection oven, air recirculation is assisted by a small fan, thus resulting in faster and more energy-efficient cooking of food. such ovens make use of a thermostat for on and off mode in order to maintain about constant the temperature selected, and a timer to turn off the oven automatically after selected period. the so-called smart ovens may use computer-based controls to program quite different cooking modes and even the possibility of automatically shutting it off when the minimum core temperature of the food has been attained. moreover, self-cleaning ovens are earning an increasing popularity among consumers, their manual cleaning being complicated and asking for critical cleaning chemicals. there are two types of self-cleaning ovens (barratt, 2021; hager and morawicki, 2013): (i) pyrolytic ovens, which feature a self-cleaning mode that heats the oven to about 500°c for as long as 2 h to convert food and fat residues into a white ash that can be wiped away easily. (ii) catalytic ovens, their porous surfaces being embedded with catalysts, which oxidize residual food by converting it into ash during cooking of food. currently, the pyrolytic version of self-cleaning ovens is not only that most widespread since it needs no expensive catalyst (hager and morawicki, 2013), but also because it is more thermally efficient due to greater wall insulation density required to withstand the high self-cleaning temperatures used. generally, the thermal efficiency (ηcs) of well-insulated conventional electric ovens ranges from 10% to 15%, while that of gaseous counterparts ranges from 6% to 7% because of the higher air flows and electric glow-bar that run continuously to reignite the gas flame should it blow out (barratt, 2021; hager and morawicki, 2013). the energy requirements of convection ovens are less than those of conventional ovens by 20–30% (barratt, 2021). finally, the mean efficiency of a microwave oven was reported to range between 56% and 60% depending on the class of microwave (hager and morawicki, 2013), although efficiency of as low as 35% was reported by probert and newboroug (1985). in addition to the above oven types, it is necessary to acknowledge wood-fired ovens, which are used globally in restaurants, rotisserie shops and bakeries. for instance, about 6,400 pizza restaurants are operating in the city of são paulo in brazil and using about 48 megagram (mg)/year of wood as fuel in their pizza ovens. these are responsible for an average emission factor of pm2.5 = 0.38 g per kg of wood burned (lima et al., 2020). the average pm2.5 concentration at the exit of their chimneys was quite high (6,171 μg/m3), while indoor, it was about two orders smaller in magnitude (68 μg/m3) (lima et al., 2020), although it definitively exceeded the indoor 24-h mean level of 15 μg/m3 of pm2.5 recommended by who (2018). in order to limit such a high pm2.5 emission in delhi (india), it was proposed to replace coal-fired with electricand gas-fired appliances in all restaurants with a seating capacity of more than 10 persons (apurva, 2016). also, san vitaliano, a town with a population of 5,000 people located near naples (italy), banned the use of wood-fired ovens in restaurants and bakeries during the cold season unless their chimneys were equipped with pollution-reducing filters (singh and highway, 2016). however, wood-fired ovens are specifically used to bake the well-known pizza napoletana (tsg), registered as a traditional specialty guaranteed by the ec (2010) regulation no. 97/2010. such ovens consist of a base of tuff bricks covered with a circular cooking floor over which is built a dome made of refractory materials to minimize heat dispersion. their appropriate geometric dimensions (i.e., mouth of an having a width of 45–50 cm and a height of 22–25 cm, a cooking floor with a diameter of 105–140 cm and a vault height of 40–45 cm) allow the temperature of the dome and cooking floor maintained at about 485 °c and 430 °c, respectively; this ensures the baking quality of the pizza napoletana tsg (ec, 2010). the thermal efficiency (ηcs) of such ovens should be like that of conventional gas ovens. according to igo et  al. (2020), the thermal efficiency of a metal fired-wood oven to heat 20 liters of water from 35 to 90°c was found to be of ~19%, about 55% of the energy consumed being lost by hot fumes and 26% dispersed through the oven walls. alternatively, the specific consumption of two types (indirect and semi-direct) of bakery ovens resulted in 0.55 and 0.90 kg of wood used per kg of wheat flour baked, respectively (manhiça et al. 2012). this was equivalent to an estimated oven efficiency of 3–5% when assuming an increment in temperature from 25 to 150°c italian journal of food science, 2022; 34 (1) 93 environmental impact of the main household cooking systems for dough having a moisture content of 36.5% (w/w) and the lower heating value of firewood as shown in table 3. cooking fuels according to eurostat (2021a), the final energy consumption in households in eu-27 in 2019 amounted to about 10.3 × 1018 j, 63.6% of which was used for space heating, 14.8% for water heating, 14.1% for lighting and appliances, 6.1% for cooking devices and 0.4% for space cooling. the share of energy consumption for cooking ranged from 1% in finland to as much as 36% in portugal, while it was 6.5% in italy. the cooking energy consumption in eu-27 was mainly supplied by electricity (49.8%), gas (31%), oil and petroleum products (13%), renewables and wastes (5.7%) and solid fuels (0.6%). in italy, it was primarily supplied by gas (69.2%), then by electricity (15.8%), oil and petroleum products (10.2%) and renewables and wastes (4.8%). the specific energy consumption for cooking was thus estimated by referring the overall cooking energy consumed in 2019 (628.67 petajoule [pj]) by eu-27 population (~447 million) (eurostat, 2021b), and it amounted to about 1.41 gigajoule [gj] (i.e., 391 kwh) per capita/year. the main characteristics of the cooking fuels used globally are shown in table 3. biomass fuel the use of biomass as a fuel in thermal and electrical applications is related to the fact that its combustion is co2 neutral that is, co2 released into the air equals to that absorbed during photosynthesis. the ultimate composition of wood biomass is slightly dependent on species. roughly, it is made of 50% carbon, 6% hydrogen, 44% oxygen and 0.1–0.5% nitrogen (vassilev et al., 2010). such composition may vary in other agricultural residues (i.e., rice husk, straw, cotton stalk and grasses), mainly because of higher hemicellulose and ash contents (shen et al., 2010). ash is an inorganic fraction of biomass fuel that remains after its burning, and it includes calcium, potassium, sodium, magnesium and other elements. the heating value of biomass is often expressed as the higher (hhv) and lower (lhv) heating values, provided the heat released by its complete combustion leading to the production of water vapors includes or does not include the latent heat of water condensation. such values decrease as the initial moisture content of biomass increases. these can be determined experimentally via an adiabatic bomb calorimeter or predicted based on the weight fraction of carbon (x’c), hydrogen (x’h), oxygen (x’o) and moisture content (xm) of the biomass under t ab le 3 . c ha ra ct er is tic s of th e m ai n co ok in g fu el s: d en si ty , m oi st ur e (x m ), el em en ta l c om po si tio n (c , h , o , n , s ) a nd a sh (x a ) c on te nt o n a dr y m at te r (d m ) b as is ; r aw fo rm ul a; c o 2 g en er at ed p er k g of bu rn ed fu el (c fr ); a nd h ig he r he at in g va lu e (h h v ) a nd lo w er h ea tin g va lu e (l h v ) ( e pa , 1 99 8; s in gh e t a l., 2 01 4) . fu el ty pe d en si ty (k g m –3 ) x m (g /1 00 g ) x′ c x′ h x′ o x′ n x′ s x′ a r aw fo rm ul a c fr (k g/ kg ) h h v (m j/ kg ) lh v (m j/ kg ) (g /1 00 d m ) fi re w oo d 45 0– 98 01 30 0– 45 02 22 .4 46 .0 0 5. 80 44 .8 7 0. 30 0. 01 3. 02 c h 1. 51 3o 0. 73 2n 0. 00 6s 0. 00 2 1. 74 15 .8 4 13 .9 5 c ro p re si du es 20 0– 40 03 14 .0 42 .1 0 6. 30 48 .3 7 0. 36 0. 17 2. 70 c h 1. 79 6o 0. 86 2n 0. 00 7s 0. 00 2 1. 59 14 .6 2 12 .8 4 c ha rc oa l 18 0– 22 0 1. 7 80 .0 0 1. 80 10 .0 0 0. 74 0. 06 7. 40 c h 1. 51 3o 0. 27 0n 0. 09 4s 0. 00 0 3. 17 27 .8 6 27 .4 1 c oa l 64 0– 93 04   10 .0 39 .0 0 4. 00 15 .0 0 1. 50 0. 50 40 .0 0 c h 1. 23 1o 0. 28 8n 0. 03 3s 0. 00 5 2. 38 16 .3 0 15 .1 4 n at ur al g as 0. 57 –0 .7 2 0. 0 72 .3 8 23 .5 8 1. 37 2. 66 0. 01 0 c h 3. 90 9o 0. 01 4n 0. 03 2s 0. 00 05 2. 65 58 .2 5 52 .9 2 lp g 50 8– 57 3 0. 0 82 .2 9 17 .7 0 0. 00 0. 00 0. 01 0. 00 c h 2. 58 1 3. 02 53 .3 7 49 .3 7 k er os en e 78 0– 81 0  0. 0 85 .9 0 13 .8 0 0. 05 0. 05 0. 20 0. 00 c h 1. 92 8s 0. 00 1 3. 15 48 .9 7 45 .8 5 b io ga s 0. 85 –0 .9 3 0 56 .5 0 11 .9 0 27 .8 6 3. 47 0. 27 0. 00 c h 2. 52 7o 0. 37 n 0. 05 3s 0. 00 2 2. 07 31 .2 8 28 .5 9 e th an ol 78 5– 79 4 45 52 .1 7 13 .0 4 34 .7 8 0 0 0 c h 3o 0. 5 1. 91 26 .8 –2 9. 7 21 .4 –2 6. 9 1 d en si ty a t 1 3% m oi st ur e co nt en t ( fr an ce sc at o et a l., 2 00 8) . 2 b ul k de ns ity o f w oo d lo gs a t 1 5% m oi st ur e co nt en t ( fr an ce sc at o et a l., 2 00 8) . 3 b ul k de ns ity o f ag ric ul tu ra l r es id ue s at 7 –1 1% m oi st ur e co nt en t ( m ak av an a et a l., 2 01 8) . 4 b ul k de ns ity (h ttp s: //w w w. ta pc oi nc .c om /im ag es /u pl oa ds /t ap co _c at al og _0 9_ p8 894 .p df ). 5 w at er c on te nt o f th e et ha no l m ix tu re u se d by c le an c oo k et ha no l s to ve (a pp ro pe di a, 2 00 8; b en ka -c ok er e t a l., 2 01 8) . https://www.tapcoinc.com/images/uploads/tapco_catalog_09_p88-94.pdf� 94 italian journal of food science, 2022; 34 (1) cimini a and moresi m several crop residues (i.e., rice husk, wheat straw, cotton stalk, corn stover etc.), which are mostly left on fields after harvesting, and forestry residues (i.e., branches, leaves, bark etc.) are usually collected and burned by households. these are characterized by low bulk density and heating values in the range of 12–20 mj/kg, depending on their ash and moisture contents. most of the woody biomass free from leaves and needles has an ash content of less than 2%, while in some agricultural residues, it could be as high as 21% as in the case of rice husk (shen et al., 2010). table 3 shows the ultimate composition of a typical mixture of crop residues used in india (singh et al., 2014). emissions in the air resulting from the combustion of typical firewood and crop residues as shown in table 4 are derived from singh et al. (2014). the resulting ash contents are generally disposed of in landfills. the theoretical co2 emissions shown in table 3 were greater than those obtained under conditions of real combustion, probably because of an inappropriate mass ratio of air to the solid fuel used. charcoal charcoal (chc) is a high-carbon solid fuel obtained from the carbonization of wood and wood wastes, which study on dry basis using one of the mathematical models available in literature (vargas-moreno et al., 2012). in this work, hhv and lhv (expressed in mj/kg) were calculated as follows (mukunda, 2009): hhv = 33.823 x′c + 144.249 (x′h – x′o/8) + 9.418 x′s, (1) lhv = hhv – 22.604 x′h – 2.581 xm. (2) table 3 shows the elemental composition and moisture content of typical firewood and crop residues (singh et  al., 2014), together with their estimated raw formula, theoretical amount of co2 produced by the overall combustion of a unitary mass of biofuel, and higher and lower heating values calculated using equations (1) and (2), respectively. the heating value of anhydrous wood biomass varies between 18.5 mj/kg and 19 mj/kg, whatever be the wood species examined. owing to their higher lignin, resin, wax and oil contents, the heating value of conifers is about 2% higher than that of broad leave trees. the calorific value of anhydrous lignin (26–27 mj/kg) is higher than that of cellulose (17.2–17.5 mj/kg) or hemicellulose (16 mj/kg). further variability in the heating value is due to slight variation in hydrogen content and especially to diverseness in ash and moisture contents (francescato et al., 2008). table 4. emissions to air and waste generated by the combustion of typical cooking fuels (epa, 1998; singh et al., 2014). emissions to air/waste   firewood (g/kg) crop residues (g/kg) charcoal (g/kg) coal (g/kg) natural gas (g/stpm3) kerosene (g/kg) lpg (g/kg) biogas (g/kg) co 2 (biogenic) 326 1,302 625  0 0  0  0 1,450 co 2 1,032 0 1,979 1,559 1,918.5 2,943 3,085 0  co 69 65.6 275 49 1.3 62 14.9 1.88 ch 4 4.2 6.8 7.9 4.69 0.04 0.74 0.074 0.43 no 0.41 0.54 0.62 0.55 1.60 0.58 0.98 0.38 no 2 0.35 0.54 0.51 0.45 0.00 0.52 0.78 0.24 n 2 o 0.09 0.05 0.08 0.08 0.04 0.09 0.015 0.009 non-methane volatile organic compounds (nmvoc) 7.35 8.2 10.3 10.5 0.09 13.2 10.59 0.56 pm 2.5 3.3 7.5 0.4 12.2 0.12 1.9 0.32 0.66 pm 10 4.34 7.54 0.43 17.9 0.00 0.52   0.66 total suspended particulate (tsp) 1.04 0.63 2.19 1.3 0.00 0.7 0.51 0.52 black carbon 0.6 0.51 0.2 5.42 0.00 0.16 0.01 0.01 organic carbon 0.95 1.46 1.18 6.75 0.18 0.12 0.02 0.02 so 2 0.32 0.27 0.34 2.67 0.01 2.56 2.12 0.85 formaldehyde  0 0  0.03  0 0 0   0  0 ash 30.2 27 74 400.0 0 0 0 0  digested slurry  0 0   0  0 0 0   0 1060.0 italian journal of food science, 2022; 34 (1) 95 environmental impact of the main household cooking systems are heated under limited aeration to remove water and volatile components. such a process can be carried out in traditional earth mound kilns with a yield of about 14%, or in closed retorts with yields as high as 25–40%, thanks to the heat recovered from the combustion of volatile components baked off (singh et al., 2014; wikipedia, 2021d). generally, the carbon content of charcoal ranges from 0.68 to 0.82 kg per kg of charcoal, which upon combustion gives rise to 2.5–3 kg of biogenic co2 (table 3). the main disadvantage of this process is the emission of unburnt methane, combustion gases, and particulates harmful to human health and the environment (table 4). coal coal (co) is a common resource of energy and chemicals. it is a complex heterogeneous solid composed of organic and inorganic matter with quite different chemico-physical properties. it is generally ranked on the basis of its carbon content into four types, namely, (1) anthracite with 86–97% carbon content (x’c) and the highest heating value, (2) bituminous coal with x’c = 45–86%, (3) sub-bituminous coal with x’c = 35–45% and (4) lignite with x’c = 25–35%. table 3 shows the ultimate composition of a typical sub-bituminous coal generally used for cooking purposes. its combustion gives rise to the emissions to air as listed in table 4, and to coal ash usually disposed in landfills like that resulting from biomass fuels. kerosene kerosene (ker) is a combustible hydrocarbon liquid obtained from the fractional distillation of petroleum between 150 °c and 275 °c. it is mainly used as aviation fuel and indoor cooking fuel. its typical physico-chemical properties are shown in table 3, while main emissions after its combustion are shown in table 4. even in this case, the theoretical co2 emissions (table 3) were greater than those obtained under the real combustion conditions of this liquid fuel (table 4). natural gas natural gas is a nonrenewable mixture consisting of methane (85–96% mol/mol), other alkanes (1.9–7.4% mol/mol) and inert compounds (i.e., carbon dioxide,  nitrogen  and hydrogen sulfide) (florida power & light co., 2003). table 3 shows typical properties and composition of ng. it is used as a fuel for generating electric and thermal energy, household heating and cooking, in vehicles as well as a chemical feedstock for manufacturing plastics and organic chemicals. it is primarily transported in its gaseous form using specific gas  transmission  network  in industrialized countries. it can also be compressed and cooled into a liquid form and transported by sea. emissions to the air resulting from its combustion are listed in table 4 (us environmental protection agency [epa], 1998). liquified petroleum gas liquified petroleum gas is a fossil fuel mixture consisting of propane (c3h8) and butane (c4h10) with smaller percentage of isobutene and propylene. its composition may range from 100% propane to 20% propane and 80% butane depending on the local winter and summer weather conditions, respectively. lpg can be manufactured during the refining of crude oil or extracted from petroleum or ng streams. it can be used as a fuel gas for heating and cooking, and in vehicles. to this end, it can be stored in portable steel cylinders, barbecue gas bottles and larger tanks. it is considered a clean cooking fuel because it gives rise to by far smaller indoor air pollution than biomass fuels. for this reason, lpg supply chains have been developed in several countries (e.g., brazil, india, indonesia, bangladesh, ethiopia, haiti, burundi, mozambique etc.) to convert people in rural and urban areas to cleaner and healthier cooking solutions because its supply chain requires no investment in infrastructure as in the case of electricity grid and ng network (rosenthal et al., 2018; wright et al., 2020). table 3 shows the typical properties and composition of lpg, while table 4 reports the main emissions to air as resulting from lpg cookstoves. beyond the fact that lpg is ideal for users living in areas not accessible to ng lines, it has the advantage of a greater calorific value of 93.2 mj/m3 against 38.7 mj/m3 for ng. moreover, because of the easier regulation of mass ratio of air  to lpg, the co2 emitted during real burning is near to the theoretical amount shown in table 3. biogas biogas (bg) is obtained from the process of anaerobic digestion of organic wastes, such as  agricultural waste, manure, municipal waste, sewage, green waste and food waste, in anaerobic digesters (bedoić et al., 2020). it mainly comprises methane (50–75% v/v), carbon dioxide (25–45% v/v) and nitrogen (0–10% v/v). it also contains small amounts of oxygen, ammonia, hydrogen and hydrogen sulfide, each being less than 1% v/v, and siloxanes. its moisture varies from 2% v/v at 20 °c to 7% v/v at 40 °c. it is regarded as a renewable energy  source, since its combustion practically releases the co2 absorbed from 96 italian journal of food science, 2022; 34 (1) cimini a and moresi m the atmosphere in the growth of primary bio-resource. it can be used for different purposes, such as electricity and heat generation, cooking etc. a greater percentage of household-scale biogas digesters are installed in china and india. economic governmental subsidies are provided to encourage rural population in asia, africa and south america to produce and use biogas at household levels, and thus avoid health problems associated with the use of biomass cookstoves (wright et al., 2020). table 3 shows the typical properties and composition of biogas, while table 4 reports the main emissions to air resulting from biogas cookstoves. bioethanol bioethanol is a renewable fuel used as a low-carbon alternative to fossil-derived fuels. it is the main fermentation product of yeast (e.g., saccharomyces cerevisiae) or bacteria (e.g., zymomonas mobilis) cultured on the media rich in simple sugars under anaerobic conditions. the so-called first-generation bioethanol stems from sugar-based raw materials (i.e., sugarcane, corn, sweet sorghum and cassava), while the second-generation bioethanol is derived from lignocellulose raw materials (such as straw, corn stover, wood trimmings, sawdust, bamboo, citrus peels etc.), which are presented for preliminary enzymatic treatment to hydrolyze cellulose (kang et al., 2014). brazil and the united states currently cover ~85% of global supply of bioethanol by utilizing sugarcane and corn as substrates, respectively (bertrand et al., 2016). numerous life cycle assessment studies attempted to estimate the environmental impact of bioethanol from different substrates with contradictory results. according to jeswani et al. (2020), if no land-use change is involved, only bioethanol from sugarcane can meet the eu renewable energy directive (ec, 2018a) of 60% reduction in ghg emissions relative to petrol, while lignocellulosic bioethanol from agricultural and forest residues appears to have a greater mitigation effect. among the several initiatives aiming at testing the use of ethanol cookstoves, it is worth citing the case of gaia association in addis ababa (ethiopia), where the stoves are fed with 96% (v/v) technical ethanol, which cannot be used as power ethanol for vehicles and is denatured with a bitter additive and dyed blue to make it unpalatable for drinking and unmistakable for water (appropedia, 2008). table 3 shows the typical properties and composition of bioethanol. no information was found in the literature about the emissions in the air generated by ethanol cookstoves, although several studies monitored emissions to air resulting from the combustion of different ethanol-gasoline mixtures in spark-ignition engines (iodice et al., 2018; manzetti and andersen, 2015). the ethanol blend of 85% (v/v), generally used as a vehicle fuel in brazil, generated 90%, 15% and 50% less particulates, co and nox, respectively, or benzene and 1,3butadiene with respect to 100% gasoline, but almost 3.5 times higher carbonylic compound emissions were noted, mainly acetaldehyde (costagliola et al., 2013). electricity the use of electric energy (el) for cooking results in no indoor emissions and smoke, and thus is a minimum health risk source. nevertheless, such use affects ambient air pollution and climate change in a smaller or greater manner, especially if the electricity is made from wind turbines and solar photovoltaic panels or coal, respectively. globally, about 26,603 twh of electricity was generated in 2018, about 38% of which being made from coal, 23% from ng, 10% from nuclear power plants and 25.5% from renewables (international energy agency [iea], 2019). in industrialized countries, where electrification rates are very high, about 50% and 61% of the cooking energy consumption in the eu-27 (eurostat, 2021a) and the united states (iea, 2018) is supplied by electricity whereas 31% and 33% by ng, respectively. nevertheless, cooking devices consume about 6% of the overall energy consumption in the eu-27 and the us households, while the energy consumption for noncooking purposes (e.g., space and water heating, lighting and air conditioning) is by far dominating (eurostat, 2021a). in lowand middle income countries, use of electricity for cooking is limited, being even lower than the electricity access rate, for its high specific cost (wright et al., 2020). food cooking energy requirements several studies have attempted to measure the energy required to cook some food items in single or multiple portions using different cookstove or oven types, cooking fuels, and methods, and in some cases to assess the resulting ghg emissions (carlsson-kanyama and boström-carlsson, 2001; foster et al., 2006; frankowska et al., 2020; martinez-gómez et al., 2016; nielsen, 2003). many of these studies pointed out different thermal energy efficiencies of the main cooking systems used. as an example, table 5 shows the specific life cycle energy use (lceu) and cooking time (tc) of a few food items as a function of different appliances and cooking modes, number of portions, including mass of food and water used, as interpreted by carlsson-kanyama and boström-carlsson (2001). for instance, these authors demonstrated that boiling water in an electric kettle or italian journal of food science, 2022; 34 (1) 97 environmental impact of the main household cooking systems baking a single portion of potatoes in a microwave oven was by far more energy-efficient than a hotplate or conventional oven. moreover, lakshmi et al. (2007) observed that an electric rice cooker was more energy-efficient than a pressure cooker or microwave cooker. similarly, martinez-gómez et al. (2016) analyzed several cooking parameters for eight typical ecuadorian meals when cooked using lpg-, coilor induction-stoves. for example, the energy requirements to cook four hard-boiled eggs or grill 400-g chicken reduced from about 0.44 to table 5. specific life cycle energy use (lceu) and cooking time (t c ) of a few food items as a function of different appliances and cooking modes used, number of portions, including the overall masses of food and water used (carlsson-kanyama and boström-carlsson, 2001). no. food item appliance & cooking mode no. of portions food mass (g) water mass (g) t c (min) lceu (mj/kg) 1 wheat hotplate 4 180 350 17 1.8 2 wheat hotplate 1 45 88 15 11.3 3 wheat microwave oven 4 180 350 15 2.1 4 wheat microwave oven 1 45 85 12 14.9 5 barley hotplate 4 160 700 23 2.3 6 barley hotplate 1 40 175 30 16.5 7 barley microwave oven 4 160 700 26 2.9 8 barley microwave oven 1 40 160 23 23.8 9 couscous electric kettle 4 240 300 1.00 4.0 11 couscous hotplate 4 240 300 2.42 4.2 12 couscous hotplate 1 80 100 2.00 13.8 13 boiled potatoes hotplate 4 800 1,000 32.4 1.1 14 boiled potatoes hotplate 1 190 600 28.3 6.8 15 boiled potatoes hotplate1 4 800 1,000 32.4 1.1 16 boiled potatoes hotplate1 method 1 190 600 28.3 6.3 17 baked potatoes microwave oven 4 1,200 28.0 1.2 18 baked potatoes microwave oven 1 300 7.0 5.0 19 baked potatoes conventional oven 4 1,200 65.0 1.8 20 baked potatoes conventional oven 1 300 65.0 20.0 21 mashed potatoes electric kettle 4 140 650 4.25 7.1 22 mashed potatoes electric kettle 1 35 175 2.25 28.6 23 mashed potatoes hotplate 4 140 650 1.83 7.9 24 mashed potatoes hotplate 1 35 175 0.83 34.3 25 swedish-made pasta hotplate 4 280 2,500 18 4.3 26 italian-made pasta hotplate 4 280 2,500 18 4.6 27 swedish-made pasta hotplate 1 70 1,000 14 21.4 28 italian-made pasta microwave oven 1 70 1,000 14 22.9 29 fresh pasta microwave oven 4 520 2,500 12 3.5 30 fresh pasta microwave oven 1 130 1,000 8 16.2 31 rice hotplate 4 240 600 20 4.2 32 rice hotplate 1 60 150 20 21.7 33 rice microwave oven 4 240 600 17 5.0 34 rice microwave oven 1 60 150 17 25.0 1 energy-saving method. 0.29 or 0.2 kwh. this clearly indicated that induction stove was more efficient thermally than other stoves examined. according to frankowska et al. (2020), the cooking of vegetables (e.g., cabbage, carrots, cauliflower, onions and potatoes) and meat and fish accounts for around 61% and 8–27% of total ghgs emitted during their overall life cycle, respectively. similarly, home cooking of 1 kg of conventional dry pasta in 10 l of boiling water laced 98 italian journal of food science, 2022; 34 (1) cimini a and moresi m waste disposal. its system boundary is sketched in figure 1. if the per capita cooking energy consumption per year is known as (ec), the mass of cooking fuels consumed (mcf) and the electric energy absorbed from the national grid (eee) can be estimated as follows: η = ccf cs e m , lhv (3) and cee cs eg e e , (1 )η η = − (4) where lhv is the lower heating value of each cooking fuel (see table 3), ηcs is the average thermal efficiency of cookstove, and ηeg is the average loss of electric grid. while the range of values for ηcs is shown in table 2, the latter was about 5.8% for the italian grid in 2020 (terna, 2020). specific emissions into the air, water and solid wastes resulting from the use of different cooking fuels are reported in table 4. in italy, electricity is produced mainly from fossil fuels (52% of total, that is, 43% from ng, 4.3% from coal and 1.0% from petroleum products etc.), and from renewable energy sources (37.6% of total, that is, 15.3% from hydroelectric sources, 8.3% from solar, 6.0% from wind and 1.94% from geothermic power, and 6.3% from biofuels) (terna, 2020). methodology the life-cycle analysis (lca) was carried out according to specific international standards (international with 70 g of table salt consumed as much as 2.8 kwh/kg, which represented about 50% of the cradle-to-grave carbon footprint, while wheat-milling and pasta-making and packaging or durum wheat cultivation covered 24.8% and 21.7% of total ghg emissions respectively (cimini et al., 2020). by using the innovative arduino®-based eco-sustainable pasta cooker, operating with a water-to-dry pasta ratio of 3 ± 1 l/kg and consuming just 0.6 ± 0.1 kwh/kg (cimini et al., 2020), the cradle-to-grave carbon footprint of dry pasta reduced by 27% (cimini et al., 2020). in the case of brewing of a cup of coffee using different coffee makers, the use phase represented the secondary hotspot (12.5–18.2% of cradle-to-grave carbon footprint), coffee bean cultivation and green coffee production phase embodying 59–70% of total ghg emissions (cibelli et al., 2021). the cooking of lamb and beef is highly energy-intensive in consequences of their long cooking period (>1 h) as in the case of roasting in an oven. nevertheless, the contribution of their cooking to the total ghgs emitted was found to be lower than 10% because their cradle-to-grave carbon footprint was by far higher than that of vegetables. under these circumstances, it would be much more environment-friendly to reduce the consumption of lamb and beef than to improve the energy efficiency of cooking method of choice (frankowska et al., 2020). table 6 summarizes the specific energy requirements for cooking different food items using either a few ordinary moist (e.g., boiling, and frying), dry (grilling) and combined (microwave) heat cookery methods, as interpreted by foster et al. (2006). description of the cooking systems studied a cooking system does not entail just the cookstove and oven but it accounts for the stove technology, cooking fuels and their supply chains, cookware, food materials as well as all the stages involved in the process of cooking from collection, handling, transportation and use of raw materials, extraction and/or refining, transportation to consumers, cooking, as well as post-consumer table 6. specific energy requirements for cooking different food items using a few ordinary cookery methods (foster et al., 2006). cookery methods specific energy required (mj/kg of raw food) boiling 3.5 frying 7.5 grilling 8.5 microwave cooking 0.34 material cooking fuels/ electricity cookwares cooked foods wastes emissions to air emissions to water cooking appliance tr figure 1. schematic of a generic cooking system, including the transportation stage (tr) of cooking fuels. italian journal of food science, 2022; 34 (1) 99 environmental impact of the main household cooking systems solid (i.e., firewood, charcoal and coal) and liquid (i.e., kerosene and lpg) cooking fuels were distributed by road using euro5 lorries with a load capacity of 3.5–7.5 mg for an average distance of 50 km. kerosene was packed in 20-l high-density polyethylene tanks weighing 0.75 kg each and lpg was filled into 10-kg steel bottles, weighing 11 kg each. finally, gaseous fuels (i.e., ng and biogas) were distributed by 50-km pipelines, while electricity was drawn from the italian grid mix. pollutants from cookstoves are mainly derived from incomplete combustion processes. they included biogenic and/or fossil co2, carbon monoxide, methane, oxides of nitrogen (nox), non-methane volatile organic compounds (nmvoc), particulate matter (pm), black carbon, organic carbon compounds and sulfur dioxide. these pollutants are of great concern because of their harmful effects on human health. electric cookstoves have no direct indoor emissions but a more or less severe indirect environmental impact depending on the electric power supply. emissions and solid wastes resulting from the combustion of fuels (singh et al., 2014) are shown in table 4, as referred to 1 kg of cooking fuel or 1 standard temperature and pressure (stp) m3 of ng. the effective mass of cooking fuels burnt and electricity drawn from the electric grid were calculated by using equations (3) and (4), respectively. finally, solid wastes (wood and coal ash) from cookstoves were disposed of in landfills. impact assessment the impact assessment was carried out using the recipe 2016 (huijbregts et al., 2016), and pef (ec, 2018b; manfredi et al., 2012; sala et al., 2017, 2018) standard methods, all these methods being embedded in the software simapro 9.2.0.2. any generic impact category (icj) was estimated by summing up release into the air, water and soil (ψi, expressed in mass, energy, and mass-km basis) associated to the system boundaries times its corresponding characterization factor (fi,j) as: i ,j i ,j i ic ( f )= ψ∑ (5) the updated recipe 2016 method (huijbregts et al., 2016) included the following 18 midpoint impact categories; the reference substance of each is indicated in parentheses: global warming (kg co2e); stratospheric organization for standardization [iso], 2006a, 2006b), and included the following stages: goal and scope definition, inventory analysis, impact assessment and interpretation of results. goal and scope definition the goal of this study was to determine the potential life cycle environmental impacts of different cooking fuels (i.e., firewood, charcoal, kerosene, ng, lpg, biogas and electricity) in italy using the lca software simapro 9.2.0.2 (prè consultants, amersfoort, nl) with embedded background ecoinvent v. 3.7 database. the cooking energy requirements depend not only on the thermal efficiency (𝜂cs) of the cookstove used but also on the type and energy (lhv) value of each fuel, as shown in tables 2 and 3. for a valid comparison between different cooking systems, the production of useful eu-27 per capita cooking energy consumption of 1.41 gj/yr, as transferred to the cooking pot after the combustion of each fuel in the cookstove, was used as the functional unit. in this way, differences in the fuel energy values and efficiencies of end-use cookstoves were accounted for. as suggested by the guidelines established by the publicly available specification (pas) 2050 standard method (british standards institution [bsi], 2011), the production of capital goods (cookstoves, cookware etc.) as well as their cleaning and disposal (section 6.4.4; bsi, 2011), was not included in the system boundary. such assumption was also corroborated by a few lca analyses which confirmed that the use phase was responsible for the greatest environmental impact in several impact categories in the case of mud stoves for firewood (afrane and ntiamoah, 2012), gas and induction hobs (favi et al., 2018), electric and gas ovens (landi et  al., 2019) as well as domestic induction hobs equipped with different electronic boards (elduque et al., 2014). additionally, the lifespan of each cookstove was, in general, more than 10 years, this being the average life of the appliances installed in italy (favi et al., 2018). in addition, the production and transportation of food materials, as well as the transportation and disposal of food wastes, were excluded from the system boundaries, as they were assumed to be the same for all the cookstoves examined. inventory analysis the production processes of all cooking fuels, as well as electricity drawn from the italian grid mix, were extracted from the ecoinvent v. 3.7 database (table 7). 100 italian journal of food science, 2022; 34 (1) cimini a and moresi m table 7. production processes for the cooking fuels used in this work (extracted from the ecoinvent v. 3.7 database). cooking fuels ecoinvent v. 3.7 database description firewood wood pellet, measured as dry mass (row)| wood pellet production| cut-off, s wood pellets are produced in a wood pellets factory, which uses wood residue from sawmills and woodchips as raw materials. the raw materials are first pre-treated and dried; then comminuted, mixed, pelletized, cooled and bagged. 20% of the production was packed in 15-kg bags, while the remaining 80% was sold unpacked. charcoal charcoal (glo)| production| cut-off, s charcoal with an average carbon content of 80% (w/w) is produced from hardwood from forest plantations. coal hard coal (europe, without russia and turkey)| market for hard coal| cut-off, s this activity starts at the hard coal preparation plant with coal ready to be loaded on rail, truck, barge or conveyor. the activity ends with the unloading of hard coal at domestic consumers or export hubs. the inventory refers to the average transport distance specific to the domestic market of hard coal in europe without russia and turkey. natural gas natural gas, high pressure (it)| import from ru| cut-off, s this dataset represents the extraction of ng in the russian federation and includes the following activities: exploration, production, processing, underground storage of ng, and feeding of produced gas in the pipeline for transport to the country where it is consumed. the leakages of production and processing of the raw gas are included. this dataset describes the transport required for the export of russian ng to italy (expressed in mg km). gas losses and emissions during seasonal storage are included. an average distance of 6,400 km is estimated for the export. lpg liquefied petroleum gas (europe without switzerland)| liquefied petroleum gas production, petroleum refinery operation| cut-off, s this dataset describes the operation of a representative average petroleum oil refinery in europe without switzerland. it includes the following activities: crude oil and product storage on refinery grounds and energy provision, refinery infrastructure, wastewater treatment, freshwater supply (from nature), refined petroleum products leaving the refinery. its sulfur content was 1.03%. kerosene kerosene (europe without switzerland) | kerosene production, petroleum refinery operation| cut-off, s this dataset describes the operation of a representative average petroleum oil refinery in europe without switzerland. activity starts with crude oil entering the petroleum refinery. wastewater treatment, freshwater supply (from nature), refinery infrastructure, crude oil and product storage on refinery grounds and energy provision are included. electricity requirements are met by the on-site generation mix. activity ends with refined petroleum products leaving the refinery. biogas biogas (row)| anaerobic digestion of manure| cut-off, s this activity produces biogas and digestate from manure and includes the following activities: input of livestock manure (cattle slurry, pig slurry and cattle manure) to incoming storage at the biogas plant, storage of the substrates, anaerobic fermentation, and storage of digestate after fermentation. the activity ends with the biogas and digestate being available at the biogas plant. the calorific value of the biogas only accounts for the methane content excluding the presence of h 2 s in it. electricity electricity, low voltage (it)| market for| cut-off, s this dataset describes the electricity available on the low-voltage level in italy in 2017 as well as grid losses. it includes electricity inputs produced in this country and from imports and transformed to low voltage, the transmission network over aerial lines and cables, direct emissions to air (sf 6 from the insulation gas in the high-voltage level switchgear are allocated to the electricity demand on medium voltage), and electricity losses during transmission. electricity, low voltage (fr)| market for| cut-off, s this dataset describes the electricity available on low-voltage level in france in 2017. it includes electricity inputs produced in france from imports and transformed to low voltage, the transmission network, direct emissions to air (sf 6 from the insulation gas in the high-voltage level switchgear are allocated to the electricity demand on medium voltage), and electricity losses during transmission. this dataset excludes electricity losses during transformation from high to medium voltage or medium to low, as these are included in the dataset for transformation, leakage of insulation oil from cables and electro technical equipment, sf 6 emissions during production and deconstruction of the switchgear, as these are accounted for in the transmission network dataset. electricity, low voltage (pl)| market for| cut-off, s this dataset describes the electricity available on low-voltage level in poland in 2017. it includes electricity inputs produced in poland and from imports and transformed to low voltage, the transmission network, direct emissions to air (sf 6 from the insulation gas in the high-voltage level switchgear are allocated to the electricity demand on medium voltage level), electricity losses during transmission. this dataset excludes electricity losses during transformation from high to medium voltage or medium to low, as these are included in the dataset for transformation, leakage of insulation oil from cables and electro technical equipment (transformers, switchgear and circuit breakers) because this only happens in case of accidental release, sf 6 emissions during production and deconstruction of the switchgear, as these are accounted for in the transmission network dataset. (continues) italian journal of food science, 2022; 34 (1) 101 environmental impact of the main household cooking systems table 7. continued cooking fuels ecoinvent v. 3.7 database description electricity, high-voltage (it)| electricity production, hydro, reservoir, alpine region| cut-off, s this dataset represents the production of high-voltage electricity at grid-connected reservoir hydropower plants in italy in 2012. net average electrical efficiency, including pipe losses, is 78%. this dataset starts from the power plant ready to produce electricity, i.e., the reservoir filled with water, and ends with 1 kwh of high-voltage electricity produced at the power plant and arrived at the busbar. this dataset doesn’t include land use for access roads to the reservoir, emissions of carbon dioxide, raw materials extraction, decommissioning and waste treatment as these activities are already included in the infrastructure datasets, transformation of the electricity produced. electricity, high-voltage (it)| electricity production, wind, 1–3-mw turbine, onshore| cut-off, s this dataset represents the production of high-voltage electricity at on-shore grid-connected wind power plants with a capacity between 1 mw and 3 mw in italy in 2005–2020. it includes operation and maintenance expenditures as well as infrastructure inputs. electricity, low-voltage (it)| electricity production, photovoltaic, 570-kwp open ground installation, multi-si | cut-off, s this dataset represents the production of grid-connected low-voltage electricity with a 570-kwp open ground photovoltaic plant in italy in 2008–2020. an inverter is used to convert the lowvoltage dc power into ac power. use of tap water for cleaning the module and its treatment is included. fr france; glo, global; it italy; pl poland; row rest of world; ru russia, s system. ozone depletion (kg trichlorofluoromethane or freon11, cfc-11e); ionizing radiation (kbq 60coe); fine pm formation (kg pm2.5e); ozone formation-human health, and ozone formation-terrestrial ecosystems (kg noxe); terrestrial acidification (kg so2e); freshwater (kg pe) and marine (kg ne) eutrophication; terrestrial, freshwater, and marine ecotoxicity (kg 1,4-dichlorobenzene [dcb]); human carcinogenic and noncarcinogenic toxicity (kg 1,4-dcb); land use (m2 annual crope); mineral (kg cue) and fossil (kg oile) resource scarcity; and water consumption (m3). finally, the pef method accounted for the following 16 impact categories, the reference substance of each is indicated in parentheses: climate change (kg co2e), ozone depletion (kg cfc-11e), ionizing radiation-human health (kbq 235ue), photochemical ozone formation (kg nmvoce), pm (diseases included), human toxicity, noncancer (human comparative toxic unit, ctuh); human toxicity, cancer (ctuh), acidification (mol h+e), freshwater eutrophication (kg pe), marine eutrophication (kg ne); terrestrial eutrophication (mol ne), freshwater ecotoxicity (ecotoxicity comparative toxic unit, ctue), land use (point [pt]), water scarcity (m 3 depriv.), resource usefossils (mj), and resource use-mineral and metals (kg sbe). both standard methods combine the above-mentioned environmental impacts into one point value. more specifically, the recipe 2016 method groups the aforementioned impact categories into the following three endpoint indicators: (i) damage to human health (hh), expressed in daly, that is, the number of years of life lost as a result of premature mortality and/or disability after an exposure to toxic chemicals; (ii) damage to ecosystem quality (eq), expressed in loss of species during a year; and (iii) damage to resource availability (ra), expressed in us$ 2013 to quantify the extra costs involved for future mineral and fossil resource extraction. such damage categories are then normalized with respect to the global population and aggregated using specific weights. finally, the three damage categories may be grouped into individualistic, hierarchic, or egalitarian perspective, according to the ‘cultural theory’ (thompson et al., 1990). in this study, hierarchic perspective was used to estimate the overall weighted damage score (owdsr), since such a perspective is regarded as the most balanced one between future and present impacts, and risks and benefits (huijbregts et al., 2016). thus, the midpoint recipe-hierarchic (h) version-europe was used to characterize the results of lcia, while the environmental impacts were calculated according to the recipe endpoint hierarchic (h) version-europe h/a with the average weighting set (a), both methods being encoded in the lca software simapro 9.2.0.2. concerning the pef method, any impact category was normalized with respect to its corresponding global impact as recommended by sala et al. (2017), weighted as suggested by sala et al. (2018), and finally summed up to yield another overall weighted score (owsp). sensitivity analysis uncertainty in the outputs of the above-mentioned lca models was mainly apportioned to the range of variations in thermal efficiency (ηcs) of the cooking fuels used (see table 2) and to the electric power supply (i.e., the french or polish grid mix, hydro, solar photovoltaic and 102 italian journal of food science, 2022; 34 (1) cimini a and moresi m the other lcia method, the use of ng led to minimum impact in 10 of the 16 categories (i.e., ionizing radiation, photochemical ozone formation, pm, noncancer and cancer human toxicity, acidification, eutrophication freshwater, ecotoxicity freshwater, land use, and resource use, that is, -mineral and metals). the use of kerosene and biogas exerted minimum impact on the remaining three (i.e., marine, terrestrial eutrophication, and water scarcity) and three (namely, climate change, ozone depletion, and resource use, that is, fossils) impact categories, respectively. whereas the pef method refers to the 100-year time horizon global warming potentials (myhre et al., 2013), in the recipe 2016 method, the characterization factors for global warming differ from the former because climate–carbon feedback for non-co2 ghgs is included (huijbregts et al., 2017). therefore, scores of the global warming category, as estimated using both methods, resulted to be slightly different. these ranged from as high as 1,210 kg co2e in the case of coal cookstoves to as low as 153 kg co2e in the case of biogas cookstoves. except for charcoal cookstoves (which emitted about 607 kg co2e), all other cookstoves emitted 188–256 kg co2e per person per year. by contrast, thanks to the model developed by van zelm et al. (2016), it was possible to assess maximum formation of fine pm if using coal, firewood and charcoal cookstoves (i.e., 7.5, 2.3 and 0.46 kg pm2.5e per person per year, respectively), and minimum formation of fine pm if using ng cookstoves (~0.1-kg pm2.5e). lpg and kerosene cookstoves emitted about 0.15–0.27 kg pm2.5e per person per year. these results were similar to those obtained with the pef method, despite this method estimates the impact of such a category in terms of disease incidence using the united nation environment program (unep) model (fantke et al., 2016). end-point environmental profile of the cooking systems examined the recipe 2016 standard method groups its 18 midpoint impact categories into three damage categories (dc) to highlight the environmental compartments damaged by any cooking system during its life cycle. in particular, the impact categories of global warming and water consumption exerted their damage to both human health and ecosystem quality compartments. by contrast, the categories of stratospheric ozone depletion, ionizing radiation, fine pm and ozone formation affecting human health, and human carcinogenic and noncarcinogenic toxicity affected the human health compartment only, while categories of ozone formation affecting terrestrial ecosystems, terrestrial acidification, freshwater and marine eutrophication, terrestrial, freshwater and marine wind power). in particular, the french electricity mix is largely dominated by the nuclear power, while coal governs the power sector of poland (www.iea.org/countries). once the default triangular and/or normal distribution uncertainty range for ηcs was accounted for, it was possible to resort to the well-known monte carlo analysis (theodoridis, 2015). results and discussion mid-point environmental profile of the cooking systems examined environmental impacts of the cooking fuels examined in this work at the first stage of cause–effect chain are shown in table 8, depicted according to the recipe 2016 and pef standard methods. according to the recipe 2016 method, the use of coal appeared to have the maximum impact in 12 of the 18 categories (i.e., global warming, fine pm formation, ozone formation affecting human health and terrestrial ecosystems, terrestrial acidification, freshwater and marine eutrophication and ecotoxicity, human carcinogenic and noncarcinogenic toxicity, and fossil resource scarcity). the use of charcoal, electricity and firewood largely affected three impact categories (namely, stratospheric ozone depletion, land use and water consumption), two impact categories (e.g., ionizing radiation and mineral resource scarcity), and one (terrestrial ecotoxicity) impact category, respectively. in contrast, the use of ng gave rise to the minimum impact in 9 of the 18 categories (i.e., ionizing radiation, fine pm formation, terrestrial acidification, freshwater eutrophication, terrestrial, freshwater and marine ecotoxicity, human noncarcinogenic toxicity, and land use). the use of biogas, lpg and kerosene minimized the impact of four impact categories (i.e., global warming, ozone formation affecting human health and terrestrial ecosystems, and fossil resource scarcity), four impact categories (namely, stratospheric ozone depletion, marine eutrophication, human carcinogenic toxicity, and mineral resource scarcity) and one impact category (water consumption), respectively. in addition, with the pef method, the use of coal appeared to have the maximum impact in 10 of the 16 categories (i.e., climate change, photochemical ozone formation, pm, acidification, freshwater, marine and terrestrial eutrophication, freshwater ecotoxicity, resource use, that is, fossils and mineral and metals). the use of charcoal, ng and electricity had the maximum impact on four impact categories (namely, human noncancer and cancer toxicity, land use, and water scarcity), one impact category (ozone depletion), and one impact category (ionizing radiation), respectively. in agreement with www.iea.org/countries� italian journal of food science, 2022; 34 (1) 103 environmental impact of the main household cooking systems ta bl e 8. e nv ir on m en ta l p ro fil e at th e fir st s ta ge s of th e ca us e– ef fe ct c ha in o f di ff er en t c oo ki ng s ys te m s us ed , a s pr ov id ed b y th e m id -p oi nt im pa ct c at eg or ie s of th e r ec ip e 20 16 a nd p ro du ct en vi ro nm en ta l f oo tp ri nt (p e f) s ta nd ar d m et ho ds p er p er so n pe r ye ar . im pa ct c at eg or y c oo ki ng fu el s u ni t fw c h c c o n g lp g k e r b g e l r ec ip e 2 01 6 g lo ba l w ar m in g (g w 10 0) 2. 28 e +0 2 6. 07 e +0 2 1. 21 e +0 3 2. 35 e +0 2 1. 88 e +0 2 2. 27 e +0 2 1. 53 e +0 2 2. 56 e +0 2 kg c o 2e s tra to sp he ric o zo ne d ep le tio n 6. 57 e -0 4 1. 75 e -0 3 5. 89 e -0 4 1. 27 e -0 4 6. 11 e -0 5 1. 24 e -0 4 3. 34 e -0 4 1. 59 e -0 4 kg c fc -1 1 e io ni zi ng ra di at io n 8. 22 e +0 0 2. 82 e +0 0 9. 71 e +0 0 2. 71 e -0 1 1. 77 e +0 0 2. 07 e +0 0 1. 87 e +0 0 2. 75 e +0 1 kb q 60 c o e fi ne p ar tic ul at e m at te r f or m at io n 2. 30 e +0 0 4. 63 e -0 1 7. 50 e +0 0 9. 73 e -0 2 1. 46 e -0 1 2. 73 e -0 1 2. 53 e -0 1 2. 67 e -0 1 kg p m 2. 5e o zo ne fo rm at io n, h um an h ea lth 1. 75 e +0 0 1. 58 e +0 0 3. 07 e +0 0 3. 44 e -0 1 3. 31 e -0 1 3. 79 e -0 1 1. 91 e -0 1 4. 37 e -0 1 kg n o xe o zo ne fo rm at io n, te rr es tri al e co sy st em s 2. 23 e +0 0 2. 12 e +0 0 3. 72 e +0 0 3. 56 e -0 1 3. 97 e -0 1 4. 82 e -0 1 2. 00 e -0 1 4. 45 e -0 1 kg n o xe te rr es tri al a ci di fic at io n 8. 49 e -0 1 5. 94 e -0 1 2. 81 e +0 0 2. 79 e -0 1 4. 01 e -0 1 4. 67 e -0 1 7. 48 e -0 1 7. 77 e -0 1 kg s o 2e fr es hw at er e ut ro ph ic at io n 4. 70 e -0 2 2. 80 e -0 2 7. 89 e -0 1 1. 55 e -0 3 1. 83 e -0 3 2. 08 e -0 3 2. 41 e -0 2 6. 25 e -0 2 kg p e m ar in e eu tro ph ic at io n 8. 64 e -0 3 1. 08 e -0 2 4. 90 e -0 2 2. 06 e -0 4 1. 87 e -0 4 2. 26 e -0 4 1. 68 e -0 3 4. 57 e -0 3 kg n e te rr es tri al e co to xi ci ty 4. 75 e +0 2 3. 02 e +0 2 3. 64 e +0 2 7. 32 e +0 0 1. 41 e +0 2 1. 14 e +0 2 9. 60 e +0 1 2. 88 e +0 2 kg 1 ,4 -d c b fr es hw at er e co to xi ci ty 9. 70 e +0 0 8. 44 e +0 0 5. 75 e +0 1 2. 02 e -0 1 3. 12 e -0 1 3. 44 e -0 1 1. 86 e +0 0 2. 18 e +0 1 kg 1 ,4 -d c b m ar in e ec ot ox ic ity 1. 34 e +0 1 1. 19 e +0 1 7. 90 e +0 1 2. 83 e -0 1 5. 85 e -0 1 6. 24 e -0 1 2. 47 e +0 0 2. 69 e +0 1 kg 1 ,4 -d c b h um an c ar ci no ge ni c to xi ci ty 1. 04 e +0 1 5. 53 e +0 0 7. 50 e +0 1 2. 41 e +0 0 1. 51 e +0 0 1. 66 e +0 0 4. 06 e +0 0 1. 16 e +0 1 kg 1 ,4 -d c b h um an n on ca rc in og en ic to xi ci ty 3. 11 e +0 2 3. 10 e +0 2 3. 89 e +0 3 3. 81 e +0 0 1. 03 e +0 1 1. 13 e +0 1 5. 33 e +0 1 1. 69 e +0 2 kg 1 ,4 -d c b la nd u se 2. 65 e +0 2 4. 11 e +0 2 1. 49 e +0 1 9. 66 e -0 2 3. 14 e -0 1 3. 32 e -0 1 5. 68 e +0 0 6. 40 e +0 0 m 2 an nu al c ro p e m in er al re so ur ce s ca rc ity 2. 54 e -0 1 1. 18 e -0 1 3. 57 e -0 1 5. 00 e -0 2 4. 28 e -0 2 5. 19 e -0 2 9. 00 e -0 2 4. 51 e -0 1 kg c u e (c on tin ue d) 104 italian journal of food science, 2022; 34 (1) cimini a and moresi m ta bl e 8. c on tin ue d im pa ct c at eg or y c oo ki ng fu el s u ni t fw c h c c o n g lp g k e r b g e l fo ss il re so ur ce s ca rc ity 3. 67 e +0 1 2. 05 e +0 1 3. 79 e +0 2 8. 54 e +0 1 6. 43 e +0 1 7. 62 e +0 1 1. 38 e +0 1 7. 78 e +0 1 kg o il e w at er c on su m pt io n 7. 64 e -0 1 5. 99 e +0 0 8. 57 e -0 1 3. 82 e -0 2 3. 85 e -0 2 2. 55 e -0 2 1. 67 e -0 1 3. 62 e +0 0 m 3 p e f c lim at e ch an ge (g w 10 0) 2. 36 e +0 2 6. 17 e +0 2 1. 26 e +0 3 2. 36 e +0 2 1. 89 e +0 2 2. 34 e +0 2 1. 59 e +0 2 2. 56 e +0 2 kg c o 2e o zo ne d ep le tio n 1. 03 e -0 5 8. 05 e -0 6 1. 87 e -0 5 6. 64 e -0 5 4. 52 e -0 5 5. 36 e -0 5 2. 96 e -0 6 3. 47 e -0 5 kg c fc -1 1 e io ni zi ng ra di at io n, h um an h ea lth (h h ) 1. 18 e +0 1 5. 01 e +0 0 1. 51 e +0 1 3. 82 e -0 1 1. 25 e +0 1 1. 48 e +0 1 2. 61 e +0 0 3. 01 e +0 1 kb q 23 5 u e p ho to ch em ic al o zo ne fo rm at io n, h h 5. 25 e +0 0 5. 56 e +0 0 9. 14 e +0 0 3. 94 e -0 1 8. 62 e -0 1 1. 35 e +0 0 4. 41 e -0 1 5. 65 e -0 1 kg n m vo c e p ar tic ul at e m at te r ( p m ) 6. 08 e -0 4 4. 14 e -0 5 2. 13 e -0 3 3. 22 e -0 6 7. 12 e -0 6 3. 49 e -0 5 2. 90 e -0 5 4. 50 e -0 6 di se as e in c. h um an to xi ci ty , n on ca nc er 1. 84 e -0 6 6. 46 e -0 5 3. 56 e -0 5 2. 59 e -0 7 1. 18 e -0 6 4. 81 e -0 6 4. 35 e -0 6 1. 79 e -0 6 c tu h h um an to xi ci ty , c an ce r 6. 85 e -0 8 2. 25 e -0 7 1. 77 e -0 7 1. 39 e -0 8 1. 45 e -0 8 1. 43 e -0 8 4. 09 e -0 8 8. 66 e -0 8 c tu h a ci di fic at io n 1. 38 e +0 0 9. 79 e -0 1 4. 26 e +0 0 4. 52 e -0 1 5. 85 e -0 1 6. 69 e -0 1 1. 12 e +0 0 1. 14 e +0 0 m ol h + e e ut ro ph ic at io n fre sh w at er 4. 70 e -0 2 2. 80 e -0 2 7. 89 e -0 1 1. 55 e -0 3 1. 82 e -0 3 2. 07 e -0 3 2. 41 e -0 2 6. 24 e -0 2 kg p e e ut ro ph ic at io n m ar in e 4. 05 e -0 1 3. 16 e -0 1 9. 57 e -0 1 1. 27 e -0 1 8. 71 e -0 2 8. 26 e -0 2 9. 18 e -0 2 1. 81 e -0 1 kg n e e ut ro ph ic at io n te rr es tri al 4. 36 e +0 0 3. 28 e +0 0 9. 39 e +0 0 1. 38 e +0 0 9. 53 e -0 1 9. 03 e -0 1 3. 38 e +0 0 2. 01 e +0 0 m ol n e e co to xi ci ty fr es hw at er 2. 78 e +0 3 2. 72 e +0 3 2. 92 e +0 4 3. 77 e +0 2 1. 42 e +0 3 1. 71 e +0 3 2. 39 e +0 3 3. 04 e +0 3 c tu e la nd u se 3. 07 e +0 4 4. 66 8e +0 4 2. 54 e +0 3 6. 26 e +0 1 3. 36 e +0 2 3. 91 e +0 2 7. 93 e +0 2 1. 21 e +0 3 p t w at er s ca rc ity 2. 20 e +0 1 2. 53 0e +0 2 1. 71 e +0 1 1. 23 e +0 0 9. 78 e -0 1 3. 46 e -0 1 4. 41 e +0 0 1. 32 e +0 2 m 3 d ep riv . r es ou rc e us e, fo ss ils 1. 72 e +0 3 9. 33 7e +0 2 1. 69 e +0 4 3. 51 e +0 3 2. 78 e +0 3 3. 29 e +0 3 6. 31 e +0 2 3. 78 e +0 3 m j r es ou rc e us e, m in er al a nd m et al s 6. 75 e -0 4 2. 05 0e -0 4 1. 26 e +0 3 1. 61 e -0 5 3. 93 e -0 5 4. 08 e -0 5 2. 14 e -0 4 2. 38 e -0 3 kg s b e fw : fi re w oo d; c h c : c ha rc oa l; c o : c oa l; n g : n at ur al g as ; l p g : l iq ui fie d pe tro le um g as ; k e r : k er os en e; b g : b io ga s; e l: e le ct ric ity ; c tu e: co m pa ra tiv e to xi c un it of e co to xi ci ty ; n m vo c : n on -m et ha ne v ol at ile o rg an ic co m po un ds . italian journal of food science, 2022; 34 (1) 105 environmental impact of the main household cooking systems from the monte carlo analysis (using a fixed number of 2,000 runs) and characterized by standard deviations ranging from ~3% (in the case of biogas cookstove) to 19% (in the case of charcoal cookstove). moreover, at a probability level of 0.05, there was no statistically significant difference between the overall scores for ng and lpg cookstoves. under these circumstances, both cooking fuels appeared to be less damaging to the compartments of human health and ecosystem quality, and thus more apt to minimize both indoor and outdoor air pollution. as shown in figure 2, owdsr increased from a minimum value of ~5 pt to 6 pt, 7 pt and 9 pt if biogas, kerosene and electric cookstoves are used, respectively. quite similar results were obtained by comparing the overall weighted scores owsp according to the pef method (figure 2). in the case of electric cookstoves, both single scores were related to different primary energy sources producing electricity in italy. to point out the effect of different nonrenewable and/or renewable sources used to generate electricity, it was assumed to draw electricity from the french and polish grid mix, or from hydro, solar photovoltaic and wind power plants. table 10 shows the mean values and standard deviations for damage categories of human health, ecosystem quality and resource availability, as such or normalized, as well as the overall weighted score owdsr and owsp for the electric cooking system examined. if using the italian grid mix, which uses about 52% fossil sources (mainly ng) and 37.6% renewable ones (mainly hydroelectric and wind power) (terna, 2020), owdsr was equal to about 8.7 pt and was practically controlled by damage to ecosystem quality. by contrast, large use of electricity from nuclear power in france had positive effects of reducing owdsr to about one-third (3.1 pt); however, this being predominantly affected by damage to human health. such a damage enhanced up to 28.8 pt when using the electricity produced by coal power plants as in poland, while the total environmental score owdsr amounted to ~30 pt (table 10). by using renewable energy sources only, owdsr reduced to as low as ~0.9 pt if hydropower electricity was used. it slightly increased to 1.4 and 2.8 pt if electricity was generated through wind and solar photovoltaic plants, respectively (table 10). according to the pef method, the single environmental score owsp was maximum in the case of polish grid mix (59 mpt) but almost indifferent in the case of italian and french grid mix (21–22 mpt). it reached a minimum value if electricity from wind was used (1.8 mpt) but increased to 6.3 mpt and 8.4 mpt if electricity was used from hydropower and solar energy, respectively. ecotoxicity, and especially land use distressed the ecosystem quality compartment. finally, the categories of mineral and fossil resource scarcities limited the resource availability compartment. table 9 shows single scores of the three damage categories for any cooking system examined, which were first normalized and then aggregated to complete assessment to the end-point approach. the use of coal appeared to have maximum impact on two of the three damage categories (i.e., human health and resource availability). the use of charcoal affected maximum the other damage category (ecosystem quality). in contrast, the use of ng exerted the least impact on human health and ecosystem quality, while the use of biogas had a minimum impact on resource availability. the resulting single score (owdsr) was maximum in the case of coal cookstoves (118 pt) and minimum in the case of lpg cookstoves (~5 pt). the contribution of damage to human health ranged from 91% to 98% of owdsr in the case of charcoal and coal cookstoves, respectively. moreover, the overall weighted damage score for ng, biogas, kerosene and electricity cookstoves were 5.2, 5.7, 7.0 and 8.6 pt, respectively; these scores confirmed their suitability for being included in the category of the so-called clean cooking fuels (iea and the world bank, 2014). as suggested by the pef method, all mid-point impact categories were normalized with respect to their corresponding global impact and weighted to obtain another single score (owsp). even with this method, the overall environmental impact of coal cookstoves was maximum (425 millipoint [mpt]), while that of lpg cookstoves was minimum (12.6 mpt). higher scores characterized the ng (12.9 mpt), biogas (14.2 mpt), kerosene (19.7 mpt) and electricity (21.8 mpt) cookstoves, while the overall environmental impact of firewood and charcoal cookstoves represented just 27% and 11% of owsp of coal cookstoves (table 9). it also confirmed the predominant contribution of pm on owsp for coal (75%) and firewood (79%) cookstoves; it was minimum in the case of electric (3%) and ng (4%) cookstoves. it is worth pointing out that such a contribution grew to 13%, 27% and 31% in the case of charcoal, kerosene and biogas cookstoves (table 9). sensitivity analysis figure 2 shows how the environmental single scores, owdsr and owsp, of cooking systems under study were affected by uncertainty of thermal efficiency (ηcs) of the cooktops used (table 2). such mean values were derived 106 italian journal of food science, 2022; 34 (1) cimini a and moresi m ta bl e 9. e nd po in t c ha ra ct er iz at io n of th e en vi ro nm en ta l p ro fil e of c oo ki ng s ys te m s ex am in ed a cc or di ng to th e r ec ip e 20 16 a nd p e f st an da rd m et ho ds : s in gl e an d w ei gh te d da m ag e sc or es o f ea ch d am ag e ca te go ry (d c ), an d ov er al l w ei gh te d sc or es (o w d s r , a nd o w s p ). p ar am et er c oo ki ng fu el s u ni t fw c h c c o n g lp g k e r b g e l r ec ip e 20 16 s in gl e d c s co re h um an h ea lth (h h ) 1. 77 e -0 3 9. 59 e -0 4 6. 98 e -0 3 2. 88 e -0 4 2. 73 e -0 4 3. 91 e -0 4 3. 27 e -0 4 4. 89 e -0 4 d a ly e co sy st em q ua lit y (e q ) 3. 51 e -0 6 5. 85 e -0 6 5. 18 e -0 6 7. 65 e -0 7 6. 67 e -0 7 8. 04 e -0 7 6. 84 e -0 7 1. 07 e -0 6 lo ss o f s pe ci es y r r es ou rc e av ai la bi lit y (r a ) 1. 01 e +0 1 6. 11 e +0 0 4. 11 e +0 1 3. 05 e +0 1 2. 89 e +0 1 3. 43 e +0 1 2. 42 e +0 0 2. 32 e +0 1 u s $2 01 3 w ei gh te d d c s co re h um an h ea lth (h h ) 2. 95 e +0 1 1. 60 e +0 1 1. 16 e +0 2 4. 81 e +0 0 4. 56 e +0 0 6. 52 e +0 0 5. 46 e +0 0 8. 15 e +0 0 p t e co sy st em q ua lit y (e q ) 9. 49 e -0 1 1. 58 e +0 0 1. 40 e +0 0 2. 07 e -0 1 1. 80 e -0 1 2. 17 e -0 1 1. 85 e -0 1 2. 91 e -0 1 p t r es ou rc e av ai la bi lit y (r a ) 7. 24 e -0 2 4. 36 e -0 2 2. 93 e -0 1 2. 18 e -0 1 2. 07 e -0 1 2. 45 e -0 1 1. 73 e -0 2 1. 66 e -0 1 p t o w d s r 30 .5 17 .6 11 8. 0 5. 2 5. 0 7. 0 5. 7 8. 6 p t p e f o w s p 11 6. 0 46 .2 42 5 12 .9 12 .6 19 .7 14 .2 21 .8 m p t p ar tic ul at e m at te r s co re 91 .6 6. 23 32 0 0. 49 1. 07 5. 25 4. 36 0. 68 m p t fw : fi re w oo d; c h c : c ha rc oa l; c o : c oa l; n g : n at ur al g as ; l p g : l iq ui fie d pe tro le um g as ; k e r : k er os en e; b g : b io ga s; e l: e le ct ric ity . o w d s r : o ve ra ll w ei gh te d da m ag e sc or e; o w s p : o ve ra ll w ei gh te d sc or e. italian journal of food science, 2022; 34 (1) 107 environmental impact of the main household cooking systems 1000 100 28 15 39 112 402 5 5 7 6 9 22 1413 13 19 107 o w d s r (p t) o r o w s p (m p t) 10 1 fw chc co ng cooking fuels recipe2016 pef lpg ker bg el figure 2. comparison of environmental impact of different cooking systems examined using the overall weighted scores owds r and ows p as estimated according to the recipe 2016 end-point—hierarchic (h) version—europe h/a and pef standard methods, respectively: fw, firewood; chc, charcoal; co, coal; ng, natural gas; lpg, liquified petroleum gas; ker, kerosene; bg, biogas, and el, electricity. discussion of results and the future perspectives the main results of this lca study pointed out quite similar environmental effects despite the different life cycle impact assessment methods used, and can be summarized as follows: 1. among the cooking fuels used in the italian context, the lowest environmental impact was associated with the use of ng and lpg if using the recipe 2016 method, their overall weighted damaged scores being not statistically different at 5% probability level. such impact was, in turn, significantly smaller than that induced by burning biogas at 95% confidence level (cl) despite the fact that use of such a biofuel exerted just 8% of the damage to resource availability induced by other fossil fuels accounted for (table 10). this was further corroborated by the fact that the database used excluded the infrastructure required to distribute biogas to final users (table 7). according to the pef method, lpg cookstoves exerted a lesser impact than that derived from ng and biogas cookstoves, probably because the average distance travelled by lpg bottles was assumed as short as 50 km. 2. when using solid fuels derived from fossils (coal) and renewable (firewood and charcoal) sources, damage to the human health compartment increased by approximately 25 or six and three times, respectively, with respect to that caused by the cooking fuels mentioned above. this was similar to the previous findings from a few lca studies relative to other countries, such as india (singh et al., 2014), ghana (afrane and ntiamoah, 2011, 2012) and remote communities in the southeast asia pacific region (aberilla et al., 2020) despite different lcia methods used. likewise, a recent meta-analysis of 50 studies from africa, asia, south and latin america confirmed that indoor pm2.5 concentrations were very low if cooking is done with lpg and electricity (pope et al., 2021). finally, the lca study of cooking fuel systems in india, china, kenya and ghana pointed out the primary requirement to reduce indoor pm formation, especially in china (its cooking fuel mix includes 31% lpg, 29% coal, 15% firewood and 12% crop residues) and india (its cooking fuel mix being 108 italian journal of food science, 2022; 34 (1) cimini a and moresi m ta bl e 10 . e nd po in t c ha ra ct er iz at io n of th e en vi ro nm en ta l p ro fil e of c oo ki ng s ys te m s su pp lie d w ith d if fe re nt e le ct ri ci ty g ri d m ix es a cc or di ng to th e r ec ip e 20 16 a nd p e f st an da rd m et ho ds : si ng le a nd w ei gh te d da m ag e sc or es o f ea ch d am ag e ca te go ry (d c ), an d ov er al l w ei gh te d sc or es (o w d s r , a nd o w s p ). p ar am et er e lit e lfr e lp l e lh y e ls p e lw i u ni t r ec ip e 20 16 s in gl e d c s co re h um an h ea lth (h h ) (4 .9 4 ± 0. 49 ) × 1 04 (1 .7 8 ± 0. 18 ) × 1 04 (1 .7 2 ± 0. 17 ) × 1 03 (5 .1 3 ± 0. 51 ) × 1 05 (1 .6 1 ± 0. 16 ) × 1 04 (7 .9 8 ± 0. 80 ) × 10 -5 d a ly e co sy st em q ua lit y (e q ) (1 .0 9 ± 0. 11 ) × 1 06 (3 .2 5 ± 0. 32 ) × 1 07 (3 .4 0 ± 0. 33 ) × 10 -6 (6 .4 8 ± 0. 64 ) × 1 08 (3 .7 0 ± 0. 37 ) × 1 07 (5 .7 3 ± 0. 57 ) × 10 -8 s pe ci es y r r es ou rc e av ai la bi lit y (r a ) 23 .5 ± 2 .3 5. 97 ± 0 .5 9 12 .7 ± 1 .2 4 (1 .8 6 ± 0. 18 ) × 1 01 2. 82 ± 0 .2 8 (8 .7 1 ± 0. 87 ) × 1 01 u s $2 01 3 w ei gh te d d c s co re h um an h ea lth (h h ) 0. 29 ± 0 .0 3 2. 97 ± 0 .2 9 28 .8 ± 2 .8 (8 .5 6 ± 0. 85 ) × 1 01 2. 69 ± 0 .2 7 1. 33 ± 0 .1 3 p t e co sy st em q ua lit y (e q ) 8. 24 ± 0 .8 1 (8 .8 0 ± 0. 86 ) × 1 02 (9 .1 9 ± 0. 90 ) × 1 01 (1 .7 5 ± 0. 17 ) × 1 02 (1 .0 0 ± 0. 10 ) × 1 01 (1 .5 5 ± 0. 16 ) × 1 02 p t r es ou rc e av ai la bi lit y (r a ) 0. 17 ± 0 .0 2 (4 .2 7 ± 0. 42 ) × 1 02 (9 .0 9 ± 0. 89 ) × 1 02 (1 .3 3 ± 0. 13 ) × 1 03 (2 .0 1 ± 0. 20 ) × 1 02 (6 .2 2 ± 0. 62 ) × 1 03 p t o w d s r 8. 71 ± 0 .8 6 3. 10 ± 0 .3 1 29 .8 ± 2 .9 0 0. 88 ± 0 .0 9 2. 81 ± 0 .2 8 1. 35 ± 0 .1 4 p t p e f o w s p 22 ± 2 21 ± 2 59 ± 6 6. 3 ± 0. 6 8. 4 ± 0. 8 1. 8 ± 0. 2 m p t e lit : i ta lia n el ec tri ci ty g rid m ix ; e lfr , f re nc h el ec tri ci ty g rid m ix ; e lp l, p ol is h el ec tri ci ty g rid m ix ; e lh y, e le ct ric ity fr om h yd ro po w er p la nt s; e ls p, e le ct ric ity fr om s ol ar p ho to vo lta ic p ow er p la nt s; e lw i, el ec tri ci ty fr om w in d po w er p la nt s. o w d s r : o ve ra ll w ei gh te d da m ag e sc or e; o w s p : o ve ra ll w ei gh te d sc or e. formed by 49% firewood, 25% lpg, 11% dung and 9% crop residues), and secondarily to replace traditional stoves with improved ones so as to allow the use of traditional cooking fuels in more appropriate forms and thus enhance their thermal efficiency and limit their environmental burden (morelli et al., 2017). for instance, in china, substituting conventional honeycomb coal briquettes with coal powder would reduce the impact of climate change and pm formation by about 70% and 97%, respectively (morelli et al., 2017). 3. electric cookstove accounted for more than 174% of overall weighted damage score of ng hob. this reflects the environmental impact of two typical cooking appliances, induction hob vs. gas hob and electric oven vs. gas oven, in italian kitchens, as assessed by favi et al. (2018) and landi et al. (2019), respectively. obviously, such unfavorable comparison stems from the nature of electricity grid mix in the italian scenario, which is mainly based on fossil sources (terna, 2020). alternatively, use of french electricity grid mix, mainly established from nuclear energy, had the advantage of reducing damage to human health, ecosystem quality and resource availability by about 66%, 49% and 21%, respectively. more favorable environmental impact would arise by using renewable electricity from hydro and wind plants. in this way, electric cooking would not only improve people’s health but also avoid the consumption of any fossil energy source. 4. electricity is currently the only means of cooking suitable for developing new-generation, energy-efficient home smart cookers. in fact, the prospective diffusion of low-cost, open-source platforms would in all probability reduce their selling price and thus promote their market penetration. such platforms would, for instance, keep the selected cooking temperature practically constant or variable according to specific cooking programs, and shut the cooktop after preset period or as soon as the minimum core temperature is achieved. in this way, it would be possible to minimize both food cooking period and energy requirements. as an example, it is worth citing the eco-sustainable pasta cooking system developed by cimini et al. (2021b). it consisted of a commercial 2-kw induction-plate hob, an induction stainless steel cooking pot, a stainless steel rod mixer piloted by a direct current electric motor welded to pot lid, a digital temperature sensor to monitor temperature of cooking water and a current sensor to register consumption of electric energy. all operations were accomplished via arduino® platform and an application installed in a smartphone with android system. it allowed short and long dry pastas to be cooked even at lower temperatures than the water boiling point at ambient pressure with as low as 2.7–3.2 l of cooking water per kg of dry pasta. although the cooked pasta italian journal of food science, 2022; 34 (1) 109 environmental impact of the main household cooking systems afrane g. and ntiamoah a. 2011. comparative life cycle assessment of charcoal, biogas, and liquefied petroleum gas as cooking fuels in ghana. j indus ecol. 15(4):539–549. https://doi. org/10.1111/j.1530-9290.2011.00350.x afrane g. and ntiamoah a. 2012. analysis of the life-cycle costs and environmental impacts of cooking fuels used in ghana. appl energy. 98:301–306. https://doi.org/10.1016/j. apenergy.2012.03.04 anozie a.n., bakare a.r., sonibare j.a., and oyebisi t.o. 2007. evaluation of cooking energy cost, efficiency, impact on air pollution and policy in nigeria. energy. 32:1283–1290. https://doi. org/10.1016/j.energy.2006.07.004 appropedia. 2008. ashden awards.  cleancook ethanol stove. available at: https://www.appropedia.org/cleancook_ethanol_ stove (accessed: 19 jan 2022). apurva. 2016. tandoors, burning of solid waste adding to dirty delhi air: iit study. indian express. available at: https://indianexpress.com/article/india/india-news-india/tandoors-burningof-solid-waste-adding-to-dirty-delhi-air-iit-study/ (accessed: 13 dec 2021). arenas j.m. 2007. design, development and testing of a portable parabolic solar kitchen. renew energy. 32:257–266. https://doi. org/10.1016/j.renene.2006.01.013 aro e.m. 2016. from first generation biofuels to advanced solar biofuels. ambio. 45(suppl 1):s24–s31. https://doi.org/10.1007/ s13280-015-0730-0 barratt n. 2021. different oven types explained! available at: https:// www.canstarblue.co.nz/appliances/ovens/different-types-of ovens-explained/ (accessed: 2 dec 2021). bedoić r., ćosić b., pukšec t., and duić n. 2020. anaerobic digestion of agri-food by-products. in holden n.m., wolfe  m.l., ogejo j.a., and cummins e.j. 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(eds.) green fuels technology. green energy and technology. springer, cham, denmark. https://doi.org/10.1007/978-3-319-30205-8_8 bevilacqua m., braglia m., carmignani g., and zammori f.a. 2007. life cycle assessment of pasta production in italy. j food qual. 30:932–952. https://doi.org/10.1111/j.1745-4557.2007.00170.x british standards institution (bsi). 2011. pas 2050:2011. specification for the assessment of the life cycle greenhouse gas emissions of goods and services. british standards institution, london. carlsson-kanyama a. and boström-carlsson k. 2001. energy use for cooking and other stages in the life cycle of food. a study did not differ from that conventionally cooked at 100 °c with 10-l water per kg of dry pasta in terms of chemico-physical quality parameters and ultrastructure, the pasta cooking energy requirement reduced from the default value of 2.8 kwh/kg to 0.45 kwh/ kg, and the overall ghg emissions reduced to about one-sixth of those resulting from the use of average european home appliances. 5. finally, even if the energy used for cooking represents just 6% of a household’s typical energy consumption in the united states and eu-27 countries, such a percentage is quite high in some european, asian and african countries. above all, reduction of indoor and outdoor household air pollution and personal exposure to health damaging pollutants, such as pm and carbon monoxide, is a top-priority environmental goal. conclusions after reviewing the basic characteristics of main cooking methods, appliances and fuels, and assessing environmental impact of a few household cookers  fired  by different fuels and electricity in the italian scenario in compliance with the recipe 2016 and pef standard methods, it is pointed out that ng cookstove generates minimum indoor and outdoor air pollution. this finding was comforting, because the italian cooking energy requirements are predominantly fulfilled by gas (69.2%), although such cookers still rely on fossil energy sources. to avoid finishing such sources and concurrently improving people’s health, household cooking appliances must be replaced by new-generation smart-cooktops driven by hydropower or wind-power electricity, of course, on condition that global electricity generation from  renewable  energy sources is accelerated faster than done ever before. acknowledgements this research was supported by the italian ministry of instruction, university and research within the research project entitled ‘the neapolitan pizza: processing, distribution, innovation and environmental aspects’; 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fax: +39 0712204988 e-mail address: a.osimani@univpm.it abstract a challenge test based on the inoculum of a multi-strain cocktail of listeria innocua and salmonella enterica viable cells was carried out to evaluate the capacity of an accelerated manufacturing technique (including conventional fermentation of the meat batter followed by freezing, slicing and drying) to guarantee the safety of milano-type fermented sausages. the counts of s. enterica decreased by 1.5 and 1.6 log cfu/g in the sausages inoculated with 2 and 4 log cfu/g, respectively, while a less notable reduction (0.4 log cfu/g) was recorded for l. innocua, independently from the inoculum load. the comparison between the main microbiological and physico-chemical features of noninoculated fermented sausages, produced through either the accelerated or the traditional process, highlighted significant differences in the percent r.h. and aw values, as well as ph in both cases, the absence of salmonella spp. and listeria monocytogenes was ascertained. these outcomes encourage further investigation on the fate of these foodborne pathogens during a shelf-life challenge test. no differences were highlighted for the main sensory parameters analyzed. keywords: accelerated drying, challenge test, dry fermented sausages, food safety, milano-type fermented sausages ital. j. food sci., vol 29, 2017 551 1. introduction meat fermentation combined with salting, drying, and sometimes smoking dates back to very ancient times; therefore, in most european countries the production of fermented dry sausages is largely carried out by following traditional procedures (aquilanti et al., 2016). as ready-to-eat (rte) meat products, dry (fermented) sausages must be stable and safe at the end of the production process; therefore, particular attention must be devoted to the control of pathogenic microorganisms such as listeria monocytogenes and salmonella spp. that are more likely to survive during manufacturing (petruzzelli et al., 2010), thus constituting a risk for consumer health. safety of fermented dry sausages, as well as stability towards alterations caused by spoilage microorganisms are generally assured by the interaction of various hurdles that include water activity (aw), acidity (ph), redox potential (eh), preservatives (e.g., nitrate/nitrite, sorbate, sulfite), and competitive microorganisms, such as lactic acid bacteria. among these hurdles, low values of aw and ph are considered the most important for the inhibition and inactivation of pathogenic bacteria, moreover, ph decrease and the water loss occurring during ripening also exerts a fundamental effect on the textural properties (feiner, 2006; dalzini et al., 2015). the drying/ripening phase is therefore regarded as crucial in fermented dry sausage production, where it also represents the most time-consuming step. for this reason, in 2004, comaposada et al. (comaposada et al., 2004) proposed a new technique called quick-dry-slice “qds process ®”, which is aimed at accelerating the drying/ripening phase of dry sausages. with such a method, sausages are fermented to the desired ph and then frozen, sliced, and dried using a continuous system based on the application of convective air (comaposada et al., 2008; stollewerk et al., 2011). in the present study a challenge test based on the inoculum of listeria and salmonella viable cells was carried out in order to evaluate the capacity of an accelerated manufacturing technique, similar to the qds process®, to guarantee the safety of milanotype sausages. the main microbiological and physico-chemical features of the fermented sausages produced through either the accelerated or the traditional process were compared as well. 2. materials and methods 2.1. experimental manufacturing two independent manufacturing trials were performed according to the traditional procedure originally described for the production of salame milano and the modified procedure, including accelerated drying (fig. 1). in both cases the same recipe was used, including pork shoulder (75%) and belly (25%), minced with a 4-mm plate, and the following ingredients (expressed as g/100 kg of meat): nacl (2,300), black pepper powder (200), white pepper powder (100), and granulated garlic (20). the recipe also included 100 g of nitritec 10/90 sa, 1,000 g of saltec whitec-16+niko sa, and 400 g of protec pork pr 70 nat sa (all purchased by tec-al, traversetolo, italy) that supplied a defined amount of nacl (1,500 g/100 kg of meat), sodium nitrite (e252), sodium ascorbate (e301), dextrose, and saccharose. the addition of lactobacillus sakei starter cultures (lyocarni bom-13clerici-sacco group, cadorago, italy) was carried out according to the manufacturer’s directions. the meat batter destined for the production of milano-type sausages through the accelerated process was divided into three separate batches (1a, 1b, and 1c) in order to carry out the challenge test. two batches (1a and 1b) were inoculated with the pathogens ital. j. food sci., vol 29, 2017 552 under study at two different contamination levels, whereas the third batch (1c) was processed without pathogens. this last batch was taken as the negative control of the challenge test and the final product obtained from it was compared with milano-type fermented sausages produced through the traditional process. all three meat batter batches were stuffed into 100-mm-diameter collagen casings and subjected to fermentation at 20-25 °c and 60-70% relative humidity (r.h.) for 48 hours. subsequently, the sausages were placed at -20 °c, and the frozen sausages were sliced. the slides were placed on a grid and a final drying was achieved in a ripening room with forced ventilation at a temperature of 20-25°c and 50-60% r.h., for approximately 50 minutes; a decrease in humidity of approximately 40% in the product was obtained. for each batch, six samples weighing 500 g each were produced. figure 1. flow chart of the traditional (a) and accelerated (b) process for manufacturing of milano-type fermented sausages. *the frozen product is further processed upon market request. stuffing into collagen casings fermentation at 20-25 °c for 48 h preparation of the meat batter day 1 day 2 day 4 day 3 * slicing drying at 20-25 °c for 50 min. freezing at -20 °c milano-type fermented sausages accelerated process day 1 day 2 day 4 day 3 day 5 day 6 days 21-63 ripening at 9-13 °c for 3-9 weeks milano-type fermented sausages drying at 15-25 °c for 4-5 days traditional process ba stuffing into collagen casings fermentation at 20-25 °c for 48 h preparation of the meat batter day 1 day 2 day 4 day 3 * slicing drying at 20-25 °c for 50 min. freezing at -20 °c milano-type fermented sausages accelerated process slicing drying at 20-25 °c for 50 min. freezing at -20 °c milano-type fermented sausages accelerated process day 1 day 2 day 4 day 3 day 5 day 6 days 21-63 ripening at 9-13 °c for 3-9 weeks milano-type fermented sausages drying at 15-25 °c for 4-5 days traditional process ripening at 9-13 °c for 3-9 weeks milano-type fermented sausages drying at 15-25 °c for 4-5 days traditional process ba ital. j. food sci., vol 29, 2017 553 2.2. challenge test four strains were used in the challenge test: the listeria innocua atcc 33090 reference strain was purchased from the american type culture collection (atcc, manassas, virginia, usa), whereas the three salmonella enterica serovars, namely, salmonella pomona izsum 76/13, salmonella derby izsum 31/13, and salmonella agona izsum 39/13, previously isolated from meat products, were obtained from the culture collection of the istituto zooprofilattico sperimentale dell’umbria e delle marche (perugia, italy). all of the strains were stored at -80 °c. the strain of listeria innocua (as a surrogate of l. monocytogenes) and the three lowpathogenicity serovars of salmonella enterica were chosen to allow the challenge test to be safely carried out at the factory. before being inoculated into the meat batter, each strain was individually subcultured twice in brain heart infusion (bhi, biokar, beauvais, france) at 37 °c for 24 h and grown overnight in the same substrate, harvested by centrifugation, washed twice, and resuspended in 0.85% nacl. two batches (1a and 1b) were inoculated with the strain cocktail at 2 and 4 log colony forming units (cfu)/g, respectively. the third non-inoculated batch (1c) was taken as a negative control. triplicate samples were withdrawn from the three batches along the production timeline: after meat batter preparation (t0), after fermentation and freezing (t1), and after slicing and drying (t2). counting of listeria innocua was carried out in accordance with the uni en iso 11290-2: 2005 standard method, whereas salmonella spp. viable counts were carried out in chromagar salmonella plus (sharlab, barcelona, spain) with incubation at 37 °c for 48 h. the ph values were determined according to iso 2917:1999 using a portable ph meter (seven multi, mettler toledo); aw was determined in accordance with the iso 21807:2004 standard method using an aqualab 3te device (decagon devices, inc. pullaman, washington, usa). 2.3. microbiological analyses viable counts were carried out on triplicate samples as follows: total mesophilic aerobes in accordance with uni en iso 4833-1:2013; lactic acid bacteria in accordance with iso 15214:1998; enterobacteriaceace in accordance with afnor bio 12/21–12/06 (tempo, biomérieux); yeasts on wallerstein laboratory nutrient (wln) agar (osimani et al., 2009); moulds on rose-bengal agar with chloramphenicol selective supplement (oxoid) at 22 °c for 3-5 days. the presence of listeria monocytogenes and salmonella spp. was assessed in accordance with afnor bio 12/11-03/04 and afnor bio 12/16-09/05 standard methods, respectively. 2.4. physico-chemical analyses physico-chemical analyses were carried out on triplicate samples, as follows: moisture as percent r.h., in accordance with aoac official method 950.46; percent ashes, in accordance with aoac official method 935.42; nitrates and nitrites, via high-performance anion exchange chromatography, using dionex dx-500 chromatography system (dionex, sunnyvale, ca, usa). 2.5. sensory analyses a preliminary acceptance test to compare the end products obtained through the accelerated technique and the traditional process was carried out. to this aim, eight panelists familiar with the taste/consumption of fermented dry sausages were chosen ital. j. food sci., vol 29, 2017 554 among the employees of the istituto zooprofilattico sperimentale dell’umbria e delle marche of perugia (italy). the following sensory parameters were assessed: odour (meat, animal, spicy, other); aroma (meat, animal, spicy, other); salinity (form low to high); sourness (from low to high); bitterness (from low to high); spicy (from low to high); colour intensity (from pink to dark red); colour uniformity; consistency (from low to high); elasticity (from low to high); hardness (from low to high); moisture (from low to high); chewiness (from low to high). the parameters ranked from low to high were assessed using a 8-point hedonic scale, ranging from 0 (low) to 7 (high). 2.6. statistical analyses the data collected were subjected to one-way analysis of variance (anova) using jmp statistical software version 11.0.0 (sas institute inc., cary, nc, usa), and differences were considered non-significant at p > 0.05. 3. results and discussion salame milano is a dry fermented sausage originally produced in the geographical area around the city of milan (italy). this denomination is included on the official list of italian traditional products published yearly by the italian ministry of agriculture and forestry (g.u. repubblica italiana no. 147, 27/06/2013 suppl. ord. no. 52). today, milano-type fermented sausages are produced in various italian areas according to the traditional process for salame milano. both products are characterized by a bright red colour, a homogeneous “grain of rice” (compact but not elastic) texture, and a sweet-delicate flavour. all of these features are obtained after a long ripening period that varies from 3 to 9 weeks, depending on the product size (20-60 cm of length and 6-11 cm of width). however, recent studies (arnau et al., 2007; stollewerk et al., 2011) suggested the possibility of shortening the drying period of fermented sausages, thus reducing the cost of drying facilities (capital and labour) and increasing both profit margin and product competitiveness. conversely, safety concerns are always to be faced when food processes are shortened. accordingly, in this study, a challenge test was carried out to investigate the capacity of pathogens to survive during an accelerated production of milano-type dry fermented sausages. a comparison between the main microbiological and physicochemical features of the dry fermented sausages produced through the traditional protocol for salame milano and the accelerated technique was carried out as well. the results of the challenge test during the production of the milano-type fermented sausages are reported in fig. 2. it is worth noticing that the samples withdrawn after fermentation and freezing (t1) had microbial counts of salmonella spp. that showed a significant decrease of 1.5 or 1.6 log cfu/g in the sausages inoculated with 2 and 4 log cfu/g, respectively. afterwards, in both cases, the viable counts did not change significantly, and values of 0.7 ± 0.04 and 2.7 ± 0.04 log cfu/g were recorded in the final samples withdrawn after slicing and drying (t2). a similar behaviour was highlighted with regard to the inoculated l. innocua cells, although the decrease recorded from the t0 to the t1 samples (0.4 log cfu/g) was less notable and the final (t2) counts (1.4 ± 0.60 and 3.7 ± 0.01 log cfu/g) were higher than those measured for salmonella. the non-inoculated control batch (1c) was always negative for the presence of both salmonella and listeria viable cells. ital. j. food sci., vol 29, 2017 555 figure 2. viable counts of inoculated listeria innocua and salmonella spp. strains and ph values measured during the challenge test carried out along the accelerated process for manufacturing of milano-type fermented sausages. process steps: after meat batter preparation (t0), after fermentation and freezing (t1, from day 1 to day 3), after slicing and drying (t2, day 4). two batches (1a and 1b) were inoculated with the strain cocktail (listeria innocua and salmonella spp.) at 2 and 4 log colony forming units (cfu) g-1, respectively, at t0. within each curve, means with different letters are significantly different p < 0.05, data observed are represented with standard deviation as error bar. as reported by stollewerk et al. (2011), the persistence of salmonella could be related to its adaptation towards acidic environments. indeed, it is well known that salmonella acidadapted cells may show increased resistance to organic acids such as those produced by lactic acid bacteria in fermented dairy products. these cells are capable of surviving better than non-adapted ones during fermentation of dairy products stored at 5 °c, possibly as a consequence of adaptive responses to other stresses, including heat shock, oxidative stress, and osmotic stress (leyer et al., 1992). conversely, different authors (cole et al., 1990; shabala et al., 2001) have reported that l. monocytogenes can survive in stressful environments, such as those characterized by low temperature and high acidity and salt contents. on this topic, mataragas et al. (2015) recently found that environmental conditions that prevail during sausage manufacturing may stimulate the expression of generaland/or specific-stress response genes and the intensiveness of these stresses may have an impact on their expression. according to the previous statements, the results of the present challenge test revealed the capacity of both inoculated salmonella enterica serovars and l. innocua cells to survive during the accelerated process for the manufacturing of the milano-type fermented sausages. however, the results of our test were more encouraging (especially for listeria) if compared to those obtained by dalzini et al. (2015) in a challenge test during a conventional process for the production of semidry low-fat salami. this finding was likely due to the ph (4.91 ± 0.02 and 4.93 ± 0.05 at 2 and 4 log cfu/g, respectively) and the aw (0.90 ± 0.0) values reached in our experimental conditions, which were much lower when compared to those attained by dalzini et al. (2015). conversely, the final aw values obtained through the accelerated process were similar to those reported by zanardi et al. (2002) for milano-type sausage (0.89) produced in a traditional way. 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 t0 t1 t2 0 1 2 3 4 5 6 7 salmonella 2 log listeria 2 log salmonella 4 log listeria 4 log ph 2 ph 2 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 t0 t1 t2 0 1 2 3 4 5 6 7 salmonella 2 log listeria 2 log salmonella 4 log listeria 4 log ph 2 ph 2 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 t0 t1 t2 0 1 2 3 4 5 6 7 salmonella 2 log listeria 2 log salmonella 4 log listeria 4 log ph 2 ph 2 batch 1a batch 1b0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 t0 t1 t2 0 1 2 3 4 5 6 7 salmonella 2 log listeria 2 log salmonella 4 log listeria 4 log ph 2 ph 2 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 t0 t1 t2 0 1 2 3 4 5 6 7 salmonella 2 log listeria 2 log salmonella 4 log listeria 4 log ph 2 ph 2 l og c fu g -1 ph a a a b b b b b ba a b b b b a b b 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 t0 t1 t2 0 1 2 3 4 5 6 7 salmonella 2 log listeria 2 log salmonella 4 log listeria 4 log ph 2 ph 2 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 t0 t1 t2 0 1 2 3 4 5 6 7 salmonella 2 log listeria 2 log salmonella 4 log listeria 4 log ph 2 ph 2 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 t0 t1 t2 0 1 2 3 4 5 6 7 salmonella 2 log listeria 2 log salmonella 4 log listeria 4 log ph 2 ph 2 batch 1a batch 1b0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 t0 t1 t2 0 1 2 3 4 5 6 7 salmonella 2 log listeria 2 log salmonella 4 log listeria 4 log ph 2 ph 2 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 t0 t1 t2 0 1 2 3 4 5 6 7 salmonella 2 log listeria 2 log salmonella 4 log listeria 4 log ph 2 ph 2 l og c fu g -1 ph 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 t0 t1 t2 0 1 2 3 4 5 6 7 salmonella 2 log listeria 2 log salmonella 4 log listeria 4 log ph 2 ph 2 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 t0 t1 t2 0 1 2 3 4 5 6 7 salmonella 2 log listeria 2 log salmonella 4 log listeria 4 log ph 2 ph 2 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 t0 t1 t2 0 1 2 3 4 5 6 7 salmonella 2 log listeria 2 log salmonella 4 log listeria 4 log ph 2 ph 2 batch 1a batch 1b0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 t0 t1 t2 0 1 2 3 4 5 6 7 salmonella 2 log listeria 2 log salmonella 4 log listeria 4 log ph 2 ph 2 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 t0 t1 t2 0 1 2 3 4 5 6 7 salmonella 2 log listeria 2 log salmonella 4 log listeria 4 log ph 2 ph 2 l og c fu g -1 ph a a a b b b b b ba a b b b b a b b ital. j. food sci., vol 29, 2017 556 the main microbiological and physico-chemical features of the milano-type sausages produced through the accelerated technique (batch 1c) were analysed in comparison with the corresponding product obtained (after 3 weeks of ripening) through the traditional process, and the results are reported in table 1. the counts of total mesophilic aerobes and lactic acid bacteria were similar in both types of products, independently of the manufacturing process adopted. because lactic acid bacteria possibly represent a significant fraction of the total mesophilic population, this finding suggests that the accelerated technique does not interfere with the growth of the pro-technological bacteria that was added as starter cultures in either the traditional or the accelerated process. in both cases, the values recorded for lab counts were in line with those (8 log cfu/g) reported by rebecchi et al. (1998) for the same type of fermented dry sausages obtained through a traditional process. by contrast, the absence of a conventional ripening phase was responsible for the significantly lower values found for yeasts and moulds in the samples of sausages obtained through the accelerated process because a conventional, long-lasting ripening is required to allow these “cosmetic” microorganisms to grow on the sausages surface (aquilanti et al., 2007). similarly, when the milano-type sausages were produced through the accelerated technique, the counts of enterobacteriaceae were significantly higher because the conventional ripening is usually responsible for the abatement of such an acid-sensitive microbial population. as is well known, the load of enterobacteriaeceae is currently used as an index of enteric contamination in meat (petruzzelli et al., 2016) and may also imply the presence of pathogens (brown et al., 2000). however, neither the traditional products nor those obtained through the accelerated technique were found to be positive for salmonella spp. or listeria monocytogenes. table 1. microbiological and physico-chemical features of the milano-type fermented sausages produced through either the traditional or the accelerated process. traditional process accelerated process microbiological parameters (log cfu g-1) total mesophilic aerobes 8.4±0.46 a 8.4±0.02 a enterobacteriaceae < 1 1.4±0.02 a lactic acid bacteria 8.4±0.18 a 8.4±0.01 a yeasts 4.2±0.08 a 2.2±0.46 b moulds 4.8±0.03 a 1.7±0.05 b salmonella spp. absent in 25 g absent in 25 g listeria monocytogenes absent in 25 g absent in 25 g physico-chemical parameters r.h. (%) 31.2±0.62 b 33.0±0.21 a ashes (%) 5.6±0.06 a 5.0±0.15 b nitrates (mg/kg) 178.0±6.00 a 174.0±9.05 a nitrites (mg/kg) n.d. n.d. ph 5.6±0.03 a 5.0±0.05 b aw 0.86±0.01 b 0.89±0.01 a values are expressed as means ± standard deviation of triplicate samples. within each raw, means with different letters are significantly different p < 0.05 cfu = colony forming units n.d. = not detectable d.m. = dry matter ital. j. food sci., vol 29, 2017 557 with regard to the results of the physico-chemical analyses (table 1), as expected, the milano-type fermented sausages produced through the accelerated drying process showed percent r.h. and aw values higher (and percent ash values lower) than those of the products obtained after a traditional ripening/drying phase. however, the recorded value of aw (0.89 ± 0.01) was considerably lower than that of chorizo obtained through the qds process ® (stollewerk et al., 2011) and was consistent with the values (< 0.90) reported for dry sausages manufactured in the mediterranean area through a ripening period of at least 4 weeks (aquilanti et al., 2016). the other significant difference shown by the anova concerns the ph that was higher in the traditionally produced milano-type sausages, most likely due to the more intense oxidative activity of the mould population on lactic acid produced by lab during carbohydrate fermentation (paulsen et al., 2011). regarding the preliminary sensory analyses carried out to compare the end products obtained through the accelerated technique and the traditional process, no differences were highlighted for odour, texture, aroma, appearance, salinity, bitterness and sourness (data not shown). it is worth noting that, for the meat descriptor, fermented sausages produced through the traditional technique were characterized by a more intense flavour and aroma of ripened meat, whereas a flavour of sour meat was mainly perceived in fermented sausages produced through the accelerated technique. 4. conclusions safety concerns need always to be faced when food processes are supposed to be shortened or modified. as ready-to-eat (rte) meat products, dry sausages must be stable and safe at the end of the production process; therefore, particular attention must be devoted to the control of pathogenic microorganisms such as listeria monocytogenes and salmonella spp. that are more likely able to survive during manufacturing, thus constituting a risk for consumer health. execution of challenge tests represents a key step to evaluate the behaviour of foodborne pathogens when innovative products or processes are implemented, and they are especially required when scarce literature is available on the innovation applied, as in the present case. overall, the reduction of viable cells of salmonella and listeria recorded in the challenge test carried out in our study and the microbiological and physico-chemical characterization of the products obtained were quite encouraging. further investigation on the fate of these foodborne pathogens during the shelf-life of the product must be envisaged in order to provide more complete information to food business operators who would manufacture fermented dry sausages using an accelerated production process. acknowledgements the authors wish to thank frigoimpianti, via dei lecci, 18, bastia umbra (pg), italy, for the production of the fast drying salami manufactures studied. this project was supported by the italian ministry of health, department of veterinary public health, nutrition and food safety. references aquilanti l., garofalo c., osimani a. and clementi f. 2016. ecology of lactic acid bacteria and coagulase negative cocci in fermented dry sausages manufactured in italy and other mediterranean countries: an overview. int. food res. j. 23:429. ital. j. food sci., vol 29, 2017 558 aquilanti l., santarelli s., silvestri g., osimani a., petruzzelli a. and clementi f. 2007. the microbial ecology of a typical italian salami during its natural fermentation. int. j. food microbiol. 120:136. arnau j., serra x., comaposada j., gou p. and garriga m. 2007. technologies to shorten the drying period of dry-cured meat products. meat sci. 77:81. brown m.h., gill c.o., hollingsworth j., nickelson r., seward s., sheridan j.j., stevenson t., sumner j.l., theno d.m., usborne w.r. and zink d. 2000. the role of microbiological testing in systems for assuring the safety of beef. int. j. food microbiol. 62: 7. cole m.b., jones m.v. and holyoak c. 1990. the effect of ph, salt concentration and temperature on the survival and growth of listeria monoytogenes. j. appl. bact. 69:63. comaposada j., arnau j., gou p. and monfort j.m. 2004. accelerated method for drying and maturing sliced food products. patent number wo2004ib00661. comaposada j., arnau j., garriga m., xargayó m., freixanet j., bernardo j., corominas m. 2008. development of new formats and products fast drying of dry-cured meat products applying the quick-dry-slice (qds) process. fleischwirtschaft int. 23:51. dalzini e., cosciani-cunico e., bernini v., bertasi b., losio m-n., daminelli p. and varisco g. 2015. behaviour of escherichia coli o157 (vtec), salmonella typhimurium and listeria monocytogenes during the manufacture, ripening and shelf life of low fat salami. food control 47:306. feiner g. 2006. raw fermented salami. meat products handbook: practical science and technology, cambridge lk: woodhead publishing limited. 314-375. leyer g.j. and johnson e.a. 1992. acid adaptation promotes survival of salmonella spp. in cheese. appl. environ. microbiol. 58:2075. mataragas m., rovetto f., bellio a., alessandria v., rantsiou k., decastelli l. and cocolin l. 2015. differential gene expression profiling of listeria monocytogenes in cacciatore and felino salami to reveal potential stress resistance biomarkers. food microbiol. 46:408. osimani a., zannini e., aquilanti l., mannazzu i., comitini f. and clementi f. 2009. lactic acid bacteria and yeasts from wheat sourdoughs of the marche region. ital. j. food sci. 21:269. paulsen p., vali s. and bauer f. 2011. quality traits of wild boar mould-ripened salami manufactured with different selections of meat and fat tissue, and with and without bacterial starter cultures. meat sci. 89:486. petruzzelli a., blasi g., masini l., calza l., duranti a., santarelli s., fisichella s., pezzotti g., aquilanti l., osimani a. and tonucci f. 2010. occurrence of listeria monocytogenes in salami manufactured in the marche region (central italy). j. vet. med. sci. 72:499. petruzzelli a., osimani a., pasquini m., clementi f., vetrano v., paolini f., foglini m., micci e., paoloni a. and tonucci f. 2016. trends in the microbial contamination of bovine, ovine and swine carcasses in three small-scale abattoirs in central italy: a four-year monitoring. meat sci. 111:53. rebecchi a., crivori s., sarra p.g. and cocconcelli p.s. 1998. physiological and molecular techniques for the study of bacterial community development in sausage fermentation. j. appl. microbiol. 84:1043. shabala l., budde b., ross t., siegumfeldt h. and mcmeekin t. 2001. responses of listeria monocytogenes to acid stress and glucose availability monitored by measurements of intracellular ph and viable counts. int. j. food microbiol. 75:89. stollewerk k., jofré a., comaposada j., ferrini g. and garriga m. 2011. ensuring food safety by an innovative fermented sausage manufacturing system. food control 22:1984. zanardi e., dorigoni v., badiani a. and chizzolini r. 2002. lipid and colour stability of milano-type. meat sci. 61:7. paper received september 19, 2016 accepted november 30, 2016 paper ital. j. food sci., vol. 27 2015 1 keywords: ice cream attributes, manufacturer/national brand, principal component, cluster and multiple regression analyses, turkish consumers’ purchase decisions turkish consumer decisions affecting ice cream consumption yavuz topcu department of agricultural economics, collage of agriculture, ataturk university 25240-erzurum, turkey tel. 90 4422311393, email: yavuztopcu@atauni.edu.tr abstract the aim of the study is to determine the main factors affecting the national-branded ice cream preferences of turkish consumers, and to analyze the relationships between their preferences and consumption amounts. the data obtained from 400 households in kahramanmaras, turkey were used for principal component, k-means cluster and multiple regression analyses. the results of the study highlighted clearly that the consumers of the high (c1), middle (c2) and low-income users (c3) satisfied with the manufacturer brand, the individual private label, and the retailer brands on the ice cream purchase decision and consumption amounts, respectively. the manufacturers, retailers and marketers of the ice cream, therefore, should implement effectively the manufacturer brand, the private label and the retailer brand for c1, c2 and c3, respectively, and then they could also increase the demand trends of the target consumers segments satisfied. 2 ital. j. food sci., vol. 27 2015 introduction consumers purchase decisions towards the food products are a complex phenomenon influenced by a numerous factors classified as product-related (intrinsic and extrinsic food attributes), consumer-related (demographic, personal, psychological and physiological characteristics) and marketing environmental-related (economic, cultural, natural, technological, political and social environments) (realini et al., 2013; topcu and uzundumlu, 2012; troy and kerry, 2010; topcu et al., 2009). traditional sensory analyses focused on the intrinsic product attributes are not sufficient to meet not only the requirements of today’s fast developing food markets but also the food choices of the consumers at the sale points. in order to be able to reply the consumers’ need and willingness to buy, therefore, the suppliers must evaluate the food products with integrated approaches covering their intrinsic and extrinsic attributes by thinking of the consumers’ socioeconomics characteristics. in order to design the actual food product image optimized, the suppliers have focused on not only the intrinsic ice cream1 attributes such as the sensory, structural, visual, nutritional, chemical and confidential properties including in aroma, taste, flavor, viscosity, color, texture and its content (realini et al., 2013; menichelli et al., 2012; topcu and uzundumlu, 2012; soukoulis et al., 2010; simeone and marotta, 2010; topcu et al., 2009) but also the extrinsic ones consisting of hedonic quality attributes such as the price, country of origin, actual product image and quality, brand, labelling, packaging, promotion, advertising, etc. (topcu and uzundumlu, 2012; troy and kerry, 2010; topcu et al., 2009; siro et al., 2008; mccarty et al., 2003; orth and firbasova, 2003) along with the consumers’ socioeconomic characteristics such as income, food expenditure, education, age and lifecycle, occupation, etc. (topcu, 2012; topcu and uzundumlu, 2012; topcu et al., 2009). while the consumers have considered the hedonic and visual quality attributes of the ice creams at point of sale before purchase and the sensory ones after consumption; the manufacturer, retailer and marketers have also interested with their various attributes to develop, improve and design the innovative ice creams based on their need and willingness to buy. providing an important communication among the manufacturer, retailer and the consumers, and then establishing a strong bridge between each group; the brands, one of the utmost important pieces of the information read a foodstuff, play an important role on the consumers’ purchase decision making processes (topcu et al., 2008; wulf et al., 2005; guerrero et al., 2000). their effects on the purchase decisions, therefore, could be explained by several functions including in their identification and attributes, the reference function formalizing their purchase models, the guarantee function providing the quality image by reducing the feeling of risk, the personalization function allowing the consumers to locate themselves in their social environment, the entertainment function getting the consumers to motivate into the different brand choices, and the practical function allowing the consumers to learn and evaluate the results of different shopping experiences (topcu and uzundumlu, 2012; ailawadi et al., 2011; aldazabal et al., 2006; guerrero et al., 2000). not only has been consumed increasingly the ice cream by many consumer segments such as children, adolescents, adults and elder people during all the lifecycle due to their refreshing, sensational, nutritional and sanitarian attributes, on the other hand, but also the ice cream industry becoming a much profitable sub-sector owing to rapidly developing technological progresses has introduced 240 different types of ice cream resulting from the diverse ingredients and methods of freezing to the domestic markets under a strict competition between manufacturer and retailer in recent years (topcu and uzundumlu, 2012; turgut and cakmakci, 2009). therefore, the upmost motivation drives of the fundamental selection criteria in the ice cream purchase decision making process of all the consumers under various product depths and varieties are the brands. therefore, it makes possible to distinguish among the consumers in terms of their sensitivity and loyalty to the brand highlighting the persistent purchase of a specific brand within a well-defined context and having a positive attitude towards it. in other word, there is a strong relationship between their varieties and brand types impacting on their purchase decisions (topcu, 2012a; enneking et al., 2007; guerrero et al., 2000). the brand type or names of the food products could be generally explained in two categories as the manufacturer brands created by manufacturers and bearing their chosen brand name (the local, national, international and global brands) and private label products derived and owned by retailers functioning at a distribution channel (the store/retailer, store sub-brand, generic and individual product brands) (topcu et al., 2008). the effects of the brand types associated with the food choice and acceptability, and the consumers purchase attitude and behaviors were widely studied in marketing researches (fornerino and hauteville, 2010; zhou et al., 2010; gehlhar et al., 2009; kumar et al., 2009; topcu et al., 2009; dimofte et al., 2008; enneking et al., 2007). 1 ice cream has a complex food colloidal system including in air bubbles, ice crystals and partially destabilized fat globules dispersed in a continuous aqueous phase. therefore, its manufacturing as the most popular frozen dairy dessert has fairly complex processes and follows the subsequent steps such as preparation of the mixture, pasteurization, homogenization, cooling, aging of the mixture, addition of fermented milk, freezing, packaging and storage of the final mixture (soukoulis et al. 2010). ital. j. food sci., vol. 27 2015 3 the brands of the leader manufacturer manufacturing the ice cream with the national and international brands in turkey consist of unilever’s algida, ulker’s natura, has food’s panda, dinamik food’s alaska, izmir dairy products’ memo, nestlé’s nestle, yasar food’s mado and ferah food’s edo (food, 2009). their annual production, export, import, consumption and per capita consumption amounts of the ice cream along with some macroeconomic indicators in turkey are indicated in table 1. according to table 1, as considered the annual increasing production (7.1%), consumption (7.5%) and export (8.3%) trends between 2005 and 2012; it should be increased dramatically the domestic production amount responding to the needs and willingness to buy of turkish consumers. additionally, the annual increase rate in the consumer population (0.6%) and gross domestic product (gdp) per capita (1.9%) between 2005 and 2012 in turkey could increase considerably its consumption trends. furthermore, it has been estimated that the ice cream consumption at selected european markets reported by mgmn for turkey will also increase about 64% between 2012 and 2016. annual ice cream consumption amounts per capita of the leader countries such as new-zealand, us, finland and sweden in the ice cream consumption in 2012, on the other hand; were calculated as 25, 21, 14 and 12 liters, respectively (fiind, 2013). furthermore, those per capita in turkey, eu and the world in 2012 were calculated as 4, 12 and 6.0 liters, respectively (eica, 2013; faostat, 2013; tuik, 2013). as a result of all this, compared with the consumers of the leader countries in the ice cream consumption, turkish consumers have consumed about 3-6 times less ice cream than them, but this difference has continued to increase steadily. according to all the indicators, the ice cream production should be increased to be able to meet the increasing demands of turkish consumers at domestic food markets. in order to increase considerably the annual ice cream consumption amounts of turkish consumers, traditional sensory analysis should be combined with integrated marketing approaches focused on the intrinsic and extrinsic ice cream attributes, and then they should develop/design the marketing tactic and strategies under homogenous consumer segments based on their socioeconomic characteristics. there is not any integrated scientific research taking into consideration all the conceptual frameworks with regard to the ice cream purchase decisions for the target consumers segments in the scientific literature. with the present study focused on integrated marketing approaches combined with all the factors influencing on the ice cream purchase decision making process of turkish consumers, it could be filled an important gap in the literature by contributing considerably to the scientific literature. this study, therefore, was designed to reach all the objectives mentioned above. in this scope, the main aims of the study are to explore the core factors related to the intrinsic and extrinsic product attributes impacting on the national-branded ice cream purchase decisions of turkish consumers; and then to determine the target homogenous consumer segments based on their socioeconomic characteristics, and finally to analyze the effectiveness of the factors effecting on their consumption amounts. material and methods material the primary data used in this study which include in the variables based on the national-branded ice cream attributes and turkish consumers’ socioeconomic characteristics influencing on their consumption decisions and amounts were obtained from a face-to face questionnaire in kahramanmaras2 from autumn of 2012 until winter of 2013. the questionnaire was conducted with the heads of the households in their houses. the participants consuming the national-branded ice 2 kahramanmaras is located in the southeastern part of turkey. the province lies on plain at the foot of the taurus mountains and has a total population of 1.063.174 in 2013, and about 42% of those live in the city centre. table 1 annual production, export, import, consumption (million liters) and per capita consumption amounts (liter) of ice cream, and macroeconomic indicators in turkey. items 2000 2005 2010 2012 annual change(%) and macroeconomic indicators 2005-2012 production 60.0 113.0 324.0 260.0 7.1 export 4.0 8.0 12.0 8.3 import 0.8 0.2 1.5 5.8 consumption 99.0 229.0 247.0 7.5 per capita consumption 1.0 1.5 3.0 4.0 7.8 population (million) 67.8 72.1 73.7 75.6 0.6 grow rate of gdp (%) 6.8 8.4 9.2 2.1 2.9 grow rate of gdp per capita (%) 5.3 7.1 7.5 0.8 1.9 sources: (tuik, 2013 and 2013a; asud, 2013). 4 ital. j. food sci., vol. 27 2015 cream, and accepting the voluntary contribution were selected randomly. they have demographic and socioeconomic characteristics such as average 46 years old, $1282 income, 3.7 liters ice cream consumption, 4.3 family sizes 51% male population and 61% high school education. methods method used in determination of the sample size in order to determine the sample size, while minimizing sample bias and representing correctly the population; the city center was divided into four parts covering the west, east, south and north-sides of kahramanmaras with 73.929 households consisting of about six family members (apdk 2013; tuik 2013). in order to calculate the sample size for each district, the following formula was used (topcu et al. 2010). where n = sample size z = z value, (1.96 for 95% confidence level) p = percentage making a choice, (0.5 used for sample size needed) c = confidence interval, (used 0.05 = ±5) the minimum sample size having the capability of representing the main population was calculated as 385 households (but the study was conducted with 400 households). by considering the information obtained from the food science and marketing literature under the expert consultancy and the prior experiences of the researcher, a draft questionnaire was prepared (topcu, 2012). in order to check out non-sampling error which occurs due to ambiguous definitions, unclear instructions, questionnaire wording, format and length, a pre-test was carried out 15 consumers selected randomly in the target regions. the flow and nature of the questionnaire were tested, and the order and timing of the questions were re-arranged. the questionnaire was then modified and refined before starting the fieldwork. methods used in the preparation of the questionnaires it was asked the participants in the survey to respond to each statement indicating the significance levels of the ice cream attributes by using a likert-format with 1-5 scale (where 1 refers to the least important and 5 refer to the most important attribute). of eighteen ice cream attributes, five are related to the intrinsic ice cream attributes (ingredient quality, texture and aroma, taste and flavor, hardness and viscosity and organic content); six are covered by the extrinsic food attribute referring the marketing mix (the region of origin, advertisement and promotion, package material and appearance, product assortment, quality-price relation and price); two are determined by manufacturing process (the reliability of manufacturing process and food safety and hygiene); five are stated by the relationships between the consumer and marketing environments (brand recognition, store satisfaction and confidence, manufacturer brand satisfaction and image). the sources in the literature related to the intrinsic and extrinsic ice cream attributes and their items were showed in table 2. on the other hand, of the two string and three numeric variables referring to the consumer demographic and socioeconomic characteristics, three referred to the consumer demographic characteristics (age as a numeric variable; consumer education (0: primary and 1: high school and college graduate) and the occupation (0: others and 1: officer) as string variables); two included in the consumer socioeconomic characteristics (monthly consumer income ($) and the share of the ice cream expenditure within total food one as numeric variable). methods used in the statistics analyses after editing and coding, the primary data were first used in principal component analysis (pca)3 to determine the main factors related to the product attitudes influencing on the national ice cream purchase patterns of turkish consumers. pca is a data reduction technique that reduces the number of variables used in an analysis by creating new variables (called factors) that combine redundancy in the data (spss 15.0 2006). the first step in pca is to determine the number of relevant factors. this was conducted by pca using varimax rotation method (vrm)4. pca was used initially to identify underlying aspects explaining a correlation among a set of the food product attributes. the purpose of pca was to identify those attributes accounting for a relatively large proportion of the variance in the sample. in the second and final steps of the statistics analyses, the main factors obtained from pca were used for k-means cluster and multiple regression/correlation mrc analyses, respectively. in the second step, according to 3 a factor extraction method used to form uncorrelated linear combinations of the observed variables. the first component has the maximum variance. successive components explain progressively smaller portions of the variance and are all uncorrelated with each other. pca is used to obtain the initial factor solution. it can be used when there is a single correlation matrix. 4 this method is an orthogonal rotation method that minimizes the number of variables that have high loading on each factor. it simplifies the interpretation of the factors. ital. j. food sci., vol. 27 2015 5 table 2 the sources in literature related to the macro variables and their items. macro variables items sources texture and aroma soukoulis et al., 2010; cruz et al., 2009 ozdemir et al., 2005; aime et al., 2001 taste and flavor topcu and uzundumlu 2012; soukoulis et al., 2010; turgut and cakmakci, 2009; enneking et al., 2007 ice cream content soukoulis et al., 2010; turgut and cakmakci, 2009; alvarez et al., 2005 hardness and viscosity atsan and caglar, 2008; kayacier and dogan, 2006; granger et al., 2005 ingredient quality karaman et al., 2011; farhoosh and riazi, 2007; granger et al., 2005 food safety and hygiene topcu and uzundumlu, 2012; goff, 2008; roininen et al., 1999 actual image quality topcu and uzundumlu, 2012; siro et al., 2008; wildmoser et al., 2004 ice cream assortment soukoulis et al., 2010; lange et al., 1999; goff, 2008 brand recognition ailawadi et al., 2011; gehlhar et al., 2009; wulf et al., 2005 store satisfaction topcu and uzundumlu, 2009; topcu et al., 2008; wulf et al., 2005; guerrero et al., 2000 store confidence topcu and uzundumlu, 2009; soberman and parker, 2006; guerrero et al., 2000 brand satisfaction ailawadi et al., 2011; dimofte et al., 2008; strizhakova et al., 2008 brand image topcu, 2012; kumar et al., 2009; brakus et al., 2009; li and housten, 2001 package appearance topcu and uzundumlu, 2012; topcu and isik, 2008; enneking et al., 2007 promotion mix karray and martin-harran, 2009; topcu and isik, 2008; li and housten, 2001 price and quality relation topcu and uzundumlu, 2012; amrouche and zaccour, 2009; kumar et al., 2009; wolk and spann, 2008; enneking et al., 2007 region/country of origin realini et al., 2013; batra et al., 2010; topcu et al., 2010; orth and firbasova, 2003 in tri ns ic pr od uc t a ttr ib ut es ex tri ns ic pr od uc t a ttr ib ut es monthly income levels of turkish consumers, therefore, the target consumers were separated to three homogeneous clusters including in low-income users (less than $500 per month), middle-income users ($500-1250 per month) and high-income users (more than $1250 per month) (topcu 2012), and then the main factors were allocated to the homogeneous consumer clusters based on monthly income levels of the target consumers by k-means cluster analysis. in the final step, the main factors obtained from pca were used multiple regression/correlation (mrc) analysis. mrc analysis was used to measure the effects of variable factors delivered from the ice cream attributes and turkish consumers’ socioeconomic characteristics effecting on their ice cream amounts. in order to test whether the normal distribution of the main factors delivered from pca and the socioeconomic variables collected from the consumers exhibited or not was applied the various transformations techniques, and it was tested that the closest distribution to the normal of all the factors provided. on the other hand, the coefficient estimations were estimated by using ordinary least squares (ols). individual and group significance of these coefficients were tested using t and f tests, respectively. in order to evaluate whether to be any econometrical problem among the variables, it was tested the overall multicollinarity and auto-correlation problems by considering variance-inflating factor (vif) and durbin-watson d statistics, respectively. multicollinearity among variables was detected by calculating (vif) (gujarati 2005; spss 15.0 2006). the analysis techniques have been widely used in many marketing researches with regard to the dairy food product attributes (davies and cline, 2005; ishii et al., 2007; batra et al., 2010; cadena and bolini, 2011). spss 15.0 statistical software program was used to run the pca and mrc analyses. mrc model could be written as follows: icc = f (rprs, tmnf, mbst, hqlt, sqlt, eth, inc, exp,ocu, edu, age, ) dependent variable icc: monthly national ice cream consumption amount per household (l/month) independent variables rprs: retailer prestige tmnf: trust to manufacturer mbst: manufacturer brand satisfaction hqlt: hedonic quality sqlt: sensory quality eth: ethnocentrism inc: monthly consumer income ($) exp: the shares of the ice cream expenditures within total food ones ocu: consumer occupation edu: consumer education age: consumer age results and discussion demographic and socioeconomic profiles of the participants the results of descriptive statistics related to the gender, marital status, tasks in the family, education levels, occupation and age of the household heads, and monthly income, national-branded ice cream consumption and expenditure of the households attaining in questionnaire were indicated in table 3. the results of the statistics showed that 51.0, 90.2, 47.1, 61.3 and 28.0% and 46.2 years 6 ital. j. food sci., vol. 27 2015 of the participant household heads consisted of the male, married, husband, high school graduate and retailer, respectively. on the other hand, monthly average income, ice cream expenditure, ice cream consumption amount and family size of the households were calculated as $1281.9, $48.6, 3.9 l and 4.3 individuals, respectively. the results of pca related to the ice cream consumption satisfaction kaiser normalization (kmo) which compares partial correlation coefficients with observed ones was calculated as 0.87 for the ice cream attributes, and this means that the data set was at a perfect level for the factor analysis since the test score was greater than 0.50 (table 4). the principal component analysis using vrm grouped the eighteen variables related to the ice cream attributes into the six factors with eigenvalues greater than 1.0, which these factors explained the 71.96% of the total variance. f1 being the first of these factors was explained by 19.00% of the total variance, and consisted of the retailer/ store positioning. f1, therefore, could be called by retailer prestige (rprs) (topcu et al., 2009). explaining the 12.15% of the total variance, f2 gave us information about the manufacturing process and the used material quality, and thus this factor could be determined by trust to manufacturer (tmnf) (topcu, 2012). reporting the 10.99% of the total variance, f3 gathered together the variables related to the brand satisfaction, and it could be named as manufacturer brand satisfaction (mbst) (topcu and uzundumlu, 2012). referring to the 10.28% of the total variance, f4 could be represented hedonic quality (hqlt) covering the relationship between the product quality and its price (topcu, 2012, 2012a). considering the 10.08% of the total variance, f5 was constituted by the sensory quality attributes of the ice cream, and could be entitled by sensory quality (sqlt) (topcu, 2012). finally, referring the 7.20% of total variance, f6 stated the relationship between the country of origin and the ice cream consumption with manufacturer brand, and thus it could be denominated by ethnocentrism (eth) (orth and firbasowa, 2003). the results of cluster analysis related to the ice cream consumption satisfaction the main factors derived from the pca and effecting on the ice cream purchase decisions of turkish consumers were separated into three homogeneous consumers segments through k-means cluster according to their income levels including in the low, middle and high-income users (table 5). table 3 demographic and socioeconomic characteristics of the participants. demographic characteristics frequency percent cumulative gender 400 100.0 male 204 51.0 51.0 female 196 49.0 100.0 marital status 400 100.0 married 360 90.2 90.2 not married 40 9.8 100.0 tasks in the family 400 100.0 husband 188 47.1 47.1 wife 212 52.9 100.0 education level 400 100.0 first school 40 10.0 10.0 high school 245 61.3 71.3 college 115 28.7 100.0 occupation 400 100.0 white-collar state employ 107 26.8 26.8 blue-collar state workers 50 12.5 39.3 businessman 40 10.0 49.3 retailer 112 28.0 77.3 pensioner 67 16.8 94.1 others 24 5.9 100.0 socioeconomic characteristics minimum maximum mean std. dev. age 23.0 77.0 46.2 12.4 monthly average income ($)* 190.0 3810.0 1281.9 1448.4 ice cream consumption amount (l) 1.0 15.0 3.7 2.1 monthly ice cream expenditure ($)* 2.0 200.0 48.6 36.5 average family size 1.0 9.0 4.3 2.1 *the prices of the products were converted from turkish lira (tl) to us dollar ($) using the exchange rate on february 25, 2014. the conversion rate used was 2.15 tl/$. ital. j. food sci., vol. 27 2015 7 table 4 factors and correlated variable loadings related to the ice cream attributes. variables factor loadings* f1 f2 f3 f4 f5 f6 retailer prestige (f1:rprs ) the ice cream assortment 0.845 0.048 0.032 -0.205 -0.061 0.249 brand recognition 0.727 -0.343 -0.304 0.198 -0.005 -0.151 store satisfaction 0.640 0.273 -0.086 -0.362 -0.222 -0.075 store confidence 0.558 0.121 -0.214 -0.370 -0.051 -0.435 advertisement and promotion 0.532 0.258 0.340 -0.210 0.070 -0.031 trust to manufacturer (f2: tmnf) ingredient quality of the ice cream -0.056 0.879 0.126 0.054 -0.035 0.134 food safety and hygiene -0.009 0.741 -0.189 0.091 0.080 -0.210 reliability of manufacturing process -0.284 0.729 0.169 -0.052 -0.092 0.017 manufacturer brand satisfaction (f3: mbst) manufacturer brand satisfaction -0.021 0.090 0.828 -0.053 0.058 -0.021 package material and appearance -0.090 -0.051 0.790 0.021 -0.200 0.091 manufacturer brand image 0.368 0.220 0.581 0.230 0.450 0.098 hedonic quality (f4:hqlt) quality-price relation -0.096 0.177 -0.186 0.765 0.031 -0.081 product price 0.058 -0.088 0.196 0.745 -0.145 0.139 sensory quality (f5: sqlt) texture and aroma 0.112 0.035 0.094 -0.157 0.866 -0.097 taste and flavor -0.166 0.028 0.038 -0.097 0.735 -0.187 organic content of the ice cream mix -0.160 -0.172 -0.358 0.024 0.731 0.311 hardness and viscosity 0.220 -0.202 0.220 -0.409 0.516 0.209 ethnocentrism (f6: eth) the region of the origin 0.025 0.070 -0.027 0.098 -0.002 0.932 eigen-value 3.230 2.449 1.868 1.748 1.713 1.223 share of explained variance (%) 19.00 14.41 10.99 10.28 10.08 7.20 cumulative share of explained variance (%) 19.00 33.41 44.40 54.68 64.76 71.96 kmo (kaiser-meyer-olkin) statistic 0.873 bartlett’s test of sphericity (chi-square, df: 136): 2552.21 (p: 0.000) *bold numbers indicate the largest loading for each variable. table 5 final cluster centres and the number of cases in each cluster. main factors clusters* high-income middle-income low-income users (c1)** users (c2)** users (c3)** rprs (retailer prestige) -0.266 -0.586 0.991 tmnf (trust to manufacturer) 0.276 0.256 -0.109 mbst (manufacturer brand satisfaction) 0.813 -0.375 -0.599 hqlt (hedonic quality) 0.301 -0.375 0.381 sqlt (sensory quality) -0.408 0.514 0.105 eth (ethnocentrism) -0.332 0.380 -0.414 number of total cases in each cluster *** 140 187 73 % of total cases in each cluster 35% 47% 18% * bold numbers indicate the largest final cluster centre scores for each factor. ** according to f statistics, the final cluster center scores were found very importance (p<0.01) *** the total number of the cases (n): 400 8 ital. j. food sci., vol. 27 2015 the results of the study showed that high-income users of the national-branded ice cream (c1) formed their consumption satisfaction according to trust to manufacturer (tmnf) and manufacturer brand satisfaction (mbst) by taking into consideration the manufacturer-branded ice cream willingness to buy as an indicator of the main component of the hedonic quality attributes. the target homogeneous consumers in the c1, therefore, gave a much more attention to the manufacturer brand satisfaction on their purchase decision and satisfactions, and thus it could be designed/developed the manufacturerbranded ice creams for the target consumers at this segment. the results of the study also indicated that the middle-income users of the national-branded ice cream (c2) focused on the purchase patterns constructed by a combination of the factors such as the sensory quality (sqlt), trust to manufacturer (tmnf) and ethnocentrism (eth). the consumers in c2 provided much stronger purchase motivations for the private labels under trust to local manufacturers by emphasizing the core benefits of the ice cream based on the sensory quality attributes. it could be introduced the private labeled local ice creams for the target segments to the region retailers. the results of the study explained that the low-income users of the national-branded ice cream (c3) tended to buy the ice cream with retailer prestige (rprs) under hedonic quality attributes (hqlt). the target consumers in c3 focused on the actual ice cream-imaged purchase decision and satisfactions based on the retailer/ store brands. it could be presented, therefore, the retailer branded ice creams for this segment. the results of mrc analysis with regard to the ice cream consumption amounts vif values calculated as 1.028 and 1.582 indicating the scores between 1.00 and 2.50 determining the acceptable reference range for multicollinearity problem showed that there was not it. durbin-watson d statistics, on the other hand, computed as 2.09 was not located between d u (1.89) and 4-d u (1.65). there, therefore, was no problem related to auto-correlation in the mrc model (kalayci, 2005). according to these statistical test results diagnosing the econometrics problems, we could directly use this data set for the mrc model. the determination coefficient (adj.r2) was calculated as 0.80 in the mrc model, this means that all the independent variables explained the 80% of the dependent variable. the ols estimates of the model coefficients and other statistical measurements were presented in table 6. the results of statistical measurements highlighted that the fstatistic rejecting the null hypothesis that makes all the coefficients equal to zero was calculated as 153.76 (p<0.01). on the other hand, the partial regression coefficients of all the independent variables, except for those of rprs, were statistically found to be meaningful (p<0.00 and p<0.05). their signs, moreover, were also found in conformity with economic theory. however, rprs and eth were not important statistically (p=0.333 and p=0.384), and thus they were not evaluated for compliance with the economic theory. additionally, they had no impact on the manufacturer/national-branded ice cream consumption amounts, and thus there was an inverse (negative) relationship between eth and the consumption table 6 the results of multiple linear regression (mrc) analysis. n: 400 r2: 0.82 adj r2: 0.80 f (11, 388): 153.76* dl=1.65 du=1.89 dw dh=2.09 variables multiple linear regression model collinearity statistics correlations coefficients a sd.error t h -value p-value tolerance vif zero-order partial part constant 0.672 1.729 4.437 0.000* rprs 0.023 0.099 0.970 0.333 0.866 1.154 0.196 0.049 0.021 tmnf 0.072 0.102 3.218 0.001* 0.973 1.028 0.011 0.161 0.075 mbst 0.045 0.095 1.996 0.047** 0.893 1.120 0.103 0.098 0.042 hqlt 0.066 0.077 2.503 0.010* 0.958 1.043 0.080 0.126 0.066 sqlt 0.090 0.091 4.045 0.000* 0.975 1.026 0.126 0.201 0.089 eth -0.019 0.089 -0.872 -0.384 0.968 1.033 -0.004 -0.044 -0.019 inc 0.080 0.511 2.944 0.003* 0.648 1.544 0.331 0.148 0.065 exp 0.736 0.347 26.689 0.000* 0.632 1.582 0.358 0.305 0.385 ocu 0.062 0.379 2.281 0.018** 0.703 1.422 0.023 0.120 0.052 edu 0.180 0.395 4.738 0.000* 0.648 1.543 0.081 0.106 0.048 age 0.424 0.018 18.958 0.000* 0.690 1.448 0.294 0.101 0.046 a coefficients consist of the standardized coefficients. *p<0.01 **p<0.05 ital. j. food sci., vol. 27 2015 9 amounts of the ice cream. the findings were supported by the similar results of some studies (brakus et al., 2009; topcu et al., 2009; dimofte et al., 2008; strizhakova et al., 2008; enneking et al., 2007). the results of the study indicated that of the six main factors, the four ones such as sqlt, tmnf, hqlt and mbst had an important effect on the national-branded ice cream consumption amount per household in c2; c1 and c2; c1and c3; and c1, respectively. sqlt was regarded as their main determinant on their satisfaction after consumed, and thus it played an important role in this process for c2 by providing much important information about the consumer satisfaction associating with the intrinsic ice cream attributes. the results were similar with respect to those of the studies carried out by topcu (2012); topcu (2012a); topcu and uzundumlu (2009); enneking et al. (2007); ishii et al (2007); guerrero et al. (2000). the results of the study also showed that there was a positive relationship between tmnf giving much important information about the manufacturing process to turkish consumers, reducing their health concern related to the ice cream based on the manufacturer confidence constructed with its ingredient quality at the manufacturing process under hygienic conditions and their consumption decision and amounts. this caused the consumers to increase dramatically the ice cream consumption trends in c1 and c2 due to the safe and hygienic ice cream manufactured by the manufacturers. this findings were quite similar to the results reported by several researchers (topcu, 2012; topcu and uzundumlu, 2012; topcu et al., 2010; soberman and parker, 2006; schuiling and kapferer, 2004). the results of the present study also reported that there was a string relationship between hqlt having a bigger impact effect on the consumer demands making possible the product differentiation through the quality and price rating in c1 and c3, and their purchase decisions. the marketers could, therefore, separate target turkish consumer masses into two homogenous segments, and then they could also stimulate much more effective marketing tactic and strategies for each segments (wolk and spann, 2008; kumar et al., 2009; topcu et al., 2010; zhou et al., 2010). the results of the study also revealed that mbst provided attitudinally a positive motivation on the national-branded ice cream consumption amounts in c1, and it had a string relationship between tmnf and hqlt of the manufacturer-branded ice cream. as a result, mbst was of a strong linear relationship with these factors influencing on not only the ice cream purchase decision at the sale points but also its consumption satisfaction and amounts after consumed. there were a lot of the studies referring to the relationship between mbst and the food consumption amounts (topcu, 2012, topcu, 2012a; topcu and uzundumlu, 2012; zhou et al., 2010; amrouche and zaccour, 2009; strizhakova et al., 2008). the results of this study also provided the important information about how the demographic and socioeconomic characteristics of turkish consumers affected their purchase decision and attitudes towards the national-branded ice cream. in this research considering their distinctive characteristics, the results of the study also referred that the share of the ice cream expenditure within total food ones (exp), age (age), education (edu), income (inc) and occupation (ocu) of turkish consumers had a much bigger effect on the ice cream consumption amounts than the other preference factors, especially, exp, age, edu. these findings were supported by results of previous researches based on the consumers purchase attitude and behaviors towards the food products (topcu, 2012; topcu and uzundumlu, 2012; topcu et al., 2010; zhou et al., 2010; topcu et al., 2009; dimofte et al., 2008; enneking et al., 2007; li and houston, 2001). conclusions in this study, the integrated approaches patterns based on not only the intrinsic and extrinsic attributes of the national-branded ice cream but also the socioeconomic characteristics of turkish consumers impacting on their ice cream purchase decisions and consumption amounts were evaluated. the measurement results of the study highlighted clearly that the consumers in c1, c2 and c3 satisfied with the actual manufacturer brands linking between trust to manufacturer and the hedonic quality attributes, the individual private brands under trust to local manufacturers and the sensory ones, and the retailer brands emphasizing the hedonic ones on the ice cream purchase decision and consumption amounts, respectively. the manufacturers, retailers and marketers of the ice cream, therefore, should widely implement the manufacturer brand, the individual private label and the retailer brand for c1, c2 and c3, respectively in order to be able to create the demand trend increases under the integrated marketing tactic and strategies affecting positively the ice cream purchase decisions and consumption amounts of the target consumer segments. although this study has some scientific merit for the academic and food manufacturing communities, there are some limitations. the results of this study have a limited generalizability since the data were obtained from only one city. if the survey is conducted nationally, more data will give more objective results about the purchase decisions of all the population. in future studies, furthermore, this model could be expanded to incorporate more factors and factor levels into the model. 10 ital. j. food sci., vol. 27 2015 references ailawadi k.l., neslin s.a. and gedenk k. 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(2010). non-local or local brands? a multi-level investigation into confidence in brand origin identification and its strategic implications. journal of the academic marketing science 38: 202-218. paper 96 ital. j. food sci., vol. 28 2016 keywords: cie l*, a*, b*, horse salami, microbiological aspect, physicochemical properties, sensory attributes, texture profile analysis physicochemical, microbiological and colour attributes of horse salami established during the ripening period d. kovačevića, k. mastanjevića *, j. pleadinb and j. frecec adepartment of food technology, faculty of food technology university of osijek, f. kuhača 20, hr-31000 osijek, croatia blaboratory for analytical chemistry, croatian veterinary institute, savska 143, hr-10000 zagreb, croatia cdepartment of biochemical engineering, faculty of food and biotechnology university of zagreb, pierrotijeva 6, hr-10000 zagreb, croatia *corresponding author: tel. +385 31 224 298 fax +385 31 207 115, email: kresimir.mastanjevic@ptfos.hr abstract changes in physicochemical, colour, textural, microbiological and sensory attributes occurring during the processing of horse salami and established on manufacturing days 0, 7, 14, 21, 28, 42, 60, 90 were studied. significant changes (p<0.05) in physicochemical parameters attributable to moisture loss, as well as changes in colour and textural properties were observed during the fermentation and ripening stage. proteolysis and lipolysis, coming as a result of endogenous enzymatic activity and high lactic acid bacteria and staphylococci counts, contributed to specific organoleptic properties of the final product. sensorial profiling showed a significant (p<0.05) acid taste, lactic acid odour and flavour intensity, and low fat/lean ratio and smokiness and saltiness values. final horse salami products were microbiologically safe, the dominant microbial population thereby being lactobacillus plantarum, lactococcus lactis ssp. lactis, enterococcus faecium and staphylococcus xylosus. mailto:kresimir.mastanjevic%40ptfos.hr?subject= ital. j. food sci., vol. 28 2016 97 introduction horse salami, an indigenous croatian meat product, is a dry fermented sausage made of horse meat supplemented with pork fatback, salt and spices. in croatia, the tradition of horse salami production is kept by the italian minority populating the eastern part of the country (in specific, the western slavonian region). in the past, this product had been known as “the dish of the poor”; nowadays, it represents a highly appreciated autochthonous croatian meat product having a great potential to become a pgi (protected geographical indications) & pdo (protected designation of origin). although horse meat has a high nutrition, as well as a high mineral value (due to its vitamin b and iron content, respectively) (badiani et al., 1997; franco et al.,, 2011), human consumption is negligible in comparison with other conventional types of meat like pork, beef or chicken (lombardi et al., 2005). horse meat used for the production of horse salami is obtained from horses slaughtered at the end of their (5-year or longer) lifecycle. the meat has no appreciable organoleptic qualities. its original colour is deep red larded with yellow fat, while the meat is tough to chew due to the connective tissue maturation (litwinczuk et al., 2008; tateo et al., 2008). horse salami has specific sensorial properties (smell and taste) attributable mainly to drying and smoking, but also to ripening, as well as to enzymatic, lactic acid bacteria and mould activity. the recipe is 130 years old and the sole difference in final products coming from various producers boils down to the difference in mass fraction of fatback used in the salami preparation (ranging from 12 to 15%). the production of the traditional horse salami mainly takes place on small farms; we are therefore talking a small-scale production seasonal in its nature, fluctuating on a year-by-year basis dependent on weather conditions. in light of the foregoing, standardization of the horse salami production becomes imperative. dry sausages produced in various european countries, mainly spain and italy, have been extensively studied for their physicochemical composition, colour and textural properties (casiraghi, et al., 1996; gimeno et al., 2000; bruna et al., 2001; spaziani et al., 2009). however, scientific information on this croatian indigenous dry sausage, which would efficiently contribute to its characterization and production standardisation, is virtually non-existent. therefore, the aim of this study was to investigate, for the first time ever, physicochemical composition, microbiological and sensorial attributes of the dry-fermented sausage known as horse salami and the changes occurring during 90 days of its manufacturing. investigations also included instrumental measurements of colour and texture of the studied salami on certain processing days, as well as the isolation and identification of autochthonous microbial population and gathering of other data needed for microbiological safety evaluation of the final product. material and methods the manufacturing process samples of traditional horse salami (24 units) were manufactured in a small-scale facility in the western slavonian region (the eastern croatia). all samples were prepared using traditional procedures that made no use of additives such as starter cultures supplemented with nitrites, nitrates or ascorbic acid (namely, the production of traditional croatian meat products does not involve the use of additives). such a traditional production takes about 3 months (90 days). horse salami is made of meat of older (5+ years), worn-out horses, mainly of the hrvatski posavac breed. after slaughtering, fat and connective tissue are carefully removed from the horse meat. this is especially important when it comes to fat, because horse fat has a particularly unpleasant smell and taste. the meat is then grinded using a grinding plate having holes measuring 6 mm in their diameter and left to rest overnight (12 hours at the minimum) in a special container equipped with a decantation hole. grinded horse meat is then mixed with pig fatback represented in the amount of 12%. before its mixing with the horse meat, the fatback is grinded using a grinding plate having holes measuring 10 mm in their diameter. the mixture of meat and fat is then mixed with salt added in the amount of 2.2%, red paprika powder added in the amount of 0.2%, hot red paprika powder added in the amount of 0.3%, garlic added in the amount of 0.2%, and black pepper added in the amount of 0.3%. in the subsequent course, the mixture gets to be stuffed into a horse small intestine (roughly 50 cm long and 50 mm wide in diameter) or into collagen casings (of the same dimensions). thereafter, the horse salami is smoked on a dry hard wood (hornbeam, beech and its sawdust) every few days (for 2-3 hours) for the total of four weeks. at this stage, the temperature and relative humidity should be kept at 18 to 20ºc and 70 to 90%, respectively. after smoking, the horse salami is left to ripen. this stage is the longest and should take about two months, throughout which period the salami should be kept in a dark room at the temperature ranging from 14° to 17ºc, with the relative humidity ranging from 70 to 80%. after that, horse salami is ready for consumption. within this study frame, samples of horse salami were taken on the processing days 0, 7, 14, 21, 28, 42, 60 and 90. in total, 24 samples were produced; at each processing stage, three samples were taken for the analyses. 98 ital. j. food sci., vol. 28 2016 analytical methods physicochemical parameters before the analysis, the sausage samples were homogenised using a knife mill gridomix gm 200 (retsh, germany) and prepared according to iso 3100-1:1975. water content was determined gravimetrically (iso 1442:1997) at 103°c (epsa 2000 bari, croatia), while the ash content was established according to iso 936:1998, by virtue of burning the samples at 550 °c (lv9/11/p320 nobertherm, germany). total protein content was determined using the kjeldahl method (iso 937:1978) that made use of an unit 8 basic digestion block (foss, sweden) and a kjeltec 8400 automated distillation & titration device (foss, sweden). the total fat content was determined using the soxhlet method (iso 1443:1973), which involves digestion of a sample in acidic environment followed by fat extraction with petroleum ether using a soxtherm 2000 automatic device (gerhardt, germany). the determination of collagen content was performed through the analysis of hydroxyproline according to iso 3496:1994 that made use of a spectrophotometer (hach dr/4000u, germany). sodium chloride content was determined using the internal titration method (trajković et al., 1983). in this analysis, 2 g of each sample were homogenized with sand and 3 ml of water. the content was transferred into a 100 ml-volumetric flask, stirred and placed for 15 min into a water bath at 100 °c. after cooling, the flask was filled with water up to the mark and filtered. an aliquot (25 ml) of the filtrate was transferred into an erlenmeyer flask containing a few drops of k 2 cro 4 indicator (62 g/100 ml of water) and titrated with 0.1 m-agno 3 until a persistent reddish colour was obtained. sodium chloride content was calculated based on the expenditure of titration reagent and its concentration. ph values were determined in a homogenate diluted with distilled water (1:10, p/v) using ph/ ion 510 – bench ph/ion/mv meter (eutech instruments pte ltd/ oakton instruments, usa) according to the ph/ion 510 instruction manual. water activity (a w ) was determined at the room temperature (20°±2ºc) using a rotronic hygrolab 3 (rotronic ag, bassersdorf, switzerland). all chemicals used for analyses of physicochemical parameters were of an analytical grade. for each sample, three independent measurements were made. instrumental determination of colour instrumental colour measurements (those of l*, a*, and b* values) were performed using a hunter-lab mini scanxe (a60-1010-615 model colorimeter, hunter-lab, reston, va, usa). the instrument was standardized on each occasion using a white ceramic plate (l 0 = 93.01, a 0 = -1.11, and b 0 = 1.30). the cielab space values (l*, a* and b*) (cie, 1976) correspond to lightness, greenness (-a*), redness (a*), blueness (-b*) or yellowness (b*). the colour measurements performed on the horse salami took place at the room temperature (20°±2ºc). each sample was cut in slices and colour-measured at ten different spots. texture profile analysis texture profile analysis (tpa) was performed using a ta.xt2i sms stable micro systems texture analyzer (stable microsystems ltd, surrey, england) equipped with a p/75 aluminium cylindrical probe. this involved cutting the samples into 1.5 cm-thick slices and their double compression so as to downsize them to 40% of their original thickness. force-time curves were recorded at the across-head speed of 5 mms-1 and at the same recording speed. the following parameters were quantified (bourne, 1978): hardness (kg), i.e. the maximum force required to compress the sample; springiness (ratio), i.e. the ability of the sample to recover its original form after the cessation of the deforming force; cohesiveness (ratio), i.e. the extent to which the sample could be deformed prior to rupture; chewiness (kg), i.e. labour required to masticate the sample before swallowing, which represents the product of hardness multiplied by cohesiveness and springiness; and finally resilience (ratio), so as to determine how well the product “fights to regain its original position”. these parameters were obtained using the texture expert for windows (version 1.0) stable micro systems. with each sample, eight determinations of texture parameters were made. microbiological analysis after aseptically removing and discarding the casing, 10 g of the product were recovered in an aseptic manner, homogenized in 90 ml of the sterile 0.5%-saline solution and serially diluted before their planting on a non-selective (peptone yeast extract glucose agar, biolife, milano, italy), pca-agar (standard plate count agar) (biolife, milano, italy) and the following selective media: mrs-agar (biolife, milano, italy) intended for lactic acid bacteria growth and baird-parker agar (merck, darmstadt, germany) intended for staphylococci growth. the plates were incubated under conditions specified in table 1. isolation and identification of microbial population in the final product classical microbiological and biochemical (api) methods (table 1) were used for the isolation and identification of the natural microbial population in the traditionally produced horse ital. j. food sci., vol. 28 2016 99 salami (i.e. in the final product obtained after 90 production days). ten grams of the sample were homogenized in 90 ml of sterile 0.5% saline solution and serially diluted before planting on a non-selective medium (peptone yeast extract glucose agar, biolife, milano, italy) and selective media under conditions specified in table 1. colonies randomly taken from selected plates were identified on the basis of their morphology, gram-staining, cell morphology and catalase reaction. the identity of bacteria species was further confirmed using the api identification kits (biomérieux, france). sensorial analysis the final horse salami product (obtained after 90 days) was subjected to a quantitative descriptive analysis performed by a panel of seven (3 male and 4 female) trained experts according to iso 6658:2005 standard. the panellists had completed a preliminary three session-training in order to familiarize themselves with the samples under investigation. fourteen attributes were examined and rated on a 5-point scale, “1” thereby standing for “poorly perceived or absent” and “5” standing for “intensely perceived”. during these three training sessions, the descriptors to be targeted by the analysis were agreed table 1 classical microbiological and biochemical (api) methods of isolation and identification of microbial population applied in the horse salami analyses. microorganism nutrient media incubation conditions api test salmonella sp. rp-broth, xld 37°c api 20 e (biolife, italy) 24-48 h v4.1 enterobacteriaceae vrbg 37°c api 20 e (biolife, italy) 24 h v4.1 staphylococcus aureus bp 37°c api staph (biolife, italy) 48 h v4.1 coagulase negative staphylococci (cns) bp 37°c api staph (biolife, italy) 48 h v4.1 sulphite reducing clostridia sulphite agar 37°c (biolife, italy) 72 h listeria monocytogenes fraser broth 37°c api listeria palcam agar 24 h v1.2 (biolife, italy) lactic acid bacteria mrs agar 30°c api 50 chl (biolife, italy) 48-72 h v5.1 api 20 strep v7.0 yeasts sabouraud agar 25°c api 20 c (biolife, italy) 48-72 h aux v4.0 yeasts upon. the latter included as follows: 2 external attributes (appearance, hardness), 4 attributes descriptive of a slice (fat/lean ratio, easy peeling capability, colour intensity, sliceability), 5 attributes descriptive of perceptions during mastication (flavour intensity, juiciness, smokiness, acid taste, saltiness) and 3 attributes descriptive of the product smell (spice odour, lactic acid odour, mould odour). the sausage samples were coded using a three-digit code and presented in form of oblique slices approximately 0.4 cm thick. water was provided to clean the panellists’ palate between analyses. data analysis differences between the average values of the same physicochemical, colour, texture, microbiological and sensory parameters were analyzed using the analysis of variance (anova) and the fisher’s least significant difference test (lsd), with statistical significance being set at p<0.05. moisture, fat, protein, collagen and nacl content, ph, a w, colour and textural parameters were subjected to correlation analysis (pearson´s correlation test) so as to determine their possible statistically meaningful relationships. statistical analysis was carried out using statistica ver. 8.0 statsoft inc. tulsa, ok, usa. 100 ital. j. food sci., vol. 28 2016 results and discussion physicochemical parameters basic chemical composition, salt (nacl) content, ph values and water activity (a w ) of the horse salami, established at various processing stages, are given in table 2. the average initial moisture content of the horse salami found to be 61.91% had significantly decreased (p<0.05) as the processing went on due to smoking and dry-ripening typical of dry fermented sausages (lizaso et al., 1999; perez-alvarez et al., 1999; salgado et al., 2005; salgado et al., 2006; lorenzo et al., 2012). higher moisture losses were observed in the first 21 processing days and on day 28, which is characteristic for this type of product (< 40%) and dry sausages in general (pleadin et al., 2014). further ripening leads to additional moisture content reduction, so that the lowest value (28.51%) was determined on manufacturing day 90. in 2012, lorenzo and co-workers reported higher initial and final moisture values for the foal salchichon. this can be explained by the fact that horse meat has a lower water content as compared to foal meat (litwinczuk et al., 2008; lanza et al., 2009; tateo et al., 2008), as well as by the longer ripening period of the horse salami. the final moisture content was also lower than in similar dry sausages coming from spain (gimeno et al., 2000; rubio et al., 2007; lorenzo et al., 2012), which can also be attributed to a longer ripening period of the horse salami. the highest amount of proteins (30.53%) was determined on day 90. the results are consistent with the published literature data, which show that due to prolonged drying and ripening (weight loss of up to 50%) and a high share of lean meat used in stuffing preparation, moisture and protein content in ripened dry-fermented sausages tend to be similar (30-40%), indicating a high nutritional value of the final product (pleadin et al., 2014). the average fat content of the horse salami had increased significantly (p<0.05) from day 1 to day 90 (from 13.84 to 28.54%), in proportion to the duration of the horse salami ripening process and dehydration, i.e. the continuous reduction of water content in the product; the same goes for the protein and collagen content (table 2). fat as a substantial component of fermented sausages has multiple functions; it represents a concentrated energy source (9 kcal/g) and the source of essential fatty acids and fat-soluble vitamins (mela, 1990). furthermore, it is contributing to the fullness of flavour, texture and softness of the product, all of the aforementioned being relevant for the quality and acceptability of the product in question (olivares et al., 2010). hydrolysis and oxidation of fatty acids that occur during the ripening process largely contribute to the taste of fermented sausages (ordonez et al., 1999). the final fat content was lower, while the final protein content turned out to be higher than in spanish and italian dry fermented sausages (dellaglio et al., 1996; rubio et al., 2008). the average initial ash content was 3.13% and had increased significantly (p<0.05), reaching the ultimate value of 5.72%, whereas water activity (a w ) (table 2) had decreased significantly (p<0.05) during the smoking and dry-ripening period (from 0.96 to 0.78). changes in mass fraction of individual basic constituents and water activity decrease seen after 90 days of horse salami production (table 2) are mostly caused by the drying process, i.e. the loss of water occurring during ripening. changes in ph values seen during the processing of the horse salami are presented in table 2. ph value had decreased during the first 21 days of processing (from 5.58 to 4.71), possibly as a result of the presence of organic acid produced by bacteria (lucke, 1994). this ph drop is typical of most dry fermented sausage (perezalvarez et al., 1999; gimeno et al., 2000; lizaso et al., 1999; muguerza et al., 2002; bozkurt and bayram, 2006; van schalkwyk et al., 2011). at the final processing stage, ph values increased to 4.94, possibly due to the liberation of peptides, amino acid and ammonia retable 2 basic chemical composition, salt content, a w and ph of the horse salami established during the manufacturing process. processing time (days) 0 7 14 21 28 42 60 90 moisture (%) 61.91a±0.06 55.67b±0.01 48.92c±0.09 43.22d±0.06 37.57e±0.01 35.16f±0.04 31.61g±0.06 28.51h±0.02 fat (%) 13.84h±0.03 15.83f±0.03 17.71e±0.01 18.55d±0.02 18.59d±0.04 20.59c±0.02 25.45b±0.06 28.54a±0.12 protein (%) 17.05h±0.04 22.48g±0.01 23.73f±0.01 24.36e±0.08 27.62d±0.04 27.95c±0.05 29.34b±0.23 30.53a±0.05 collagen (%) 0.63e±0.11 1.19d ±0.11 1.56cd±0.07 2.05c±0.21 2.06c±0.09 2.82b±0.15 2.84b±0.12 3.93a±0.10 ash (%) 3.13g±0.02 3.73f±0.06 4.56e±0.04 4.87d±0.01 4.94d±0.01 5.36c±0.05 5.45b±0.06 5.72a±0.01 salt (nacl) (%) 2.30g±0.04 2.71f±0.05 3.29e±0.03 3.63d±0.05 3.75c±0.05 3.81c±0.05 4.24b±0.02 4.51a±0.03 aw 0.96a±0.01 0.93ab±0.02 0.91b±0.01 0.88c±0.01 0.87c±0.03 0.86c±0.04 0.86c±0.01 0.78d±0.01 ph 5.58a±0.03 4.99b±0.05 4.74f±0.10 4.71g±0.16 4.72fg±0.08 4.76f±0.06 4.81d±0.10 4.93c±0.07 values are means ±sd obtained with three measurements. values displayed in the same row and tagged with different letters (a-h) are significantly different (p<0.05). ital. j. food sci., vol. 28 2016 101 sulting from a proteolityc reaction (spaziani et al, 2009). the final ph was lower than in most dry fermented sausages (5.2 to 5.8) (bover-cid et al., 2001; rubio et al., 2007; roserio et al., 2010), which can be explained by horse meat properties in terms of higher glycogen content as compared to pork, beef and foal meat (lawrie and ledward, 2006). the salt content of the horse salami had significantly increased during processing (p<0.05) (table 2). literature sources have reported the average mass fraction of salt in dry sausage stuffing to range from 2.0% to 2.6%, and that in final products to range from 3.3% to 4.3% (ockerman and basu, 2007; stahnke and tjener, 2007). in this study, mass fraction of salt (nacl) established during the horse salami manufacturing process ranged from 2.31% to 4.51%. instrumental colour properties the cielab space (l*, a* and b*) values of the horse salami were significantly affected (p<0.05) by the length of smoking and ripening period (table 3). lower lightness l* values seen with an increased length of processing are probably related to the dark colour of the horse salami coming as a consequence of browning. a similar decrease in l* values during ripening was reported by bozkurt and bayram (2006) for turkish sucuk, and by lorenzo et al. (2012) for foal salchichon. redness (a*) had significantly (p<0.05) decreased at all processing stages. similar lower a* values were seen during the ripening of spantable 3 colour parameters of the horse salami established during the manufacturing process. processing time (days) 0 7 14 21 28 42 60 90 l* 46.67a±1.08 43.01b±1.05 41.81c±0.37 40.54d±0.30 38.75e±0.35 33.77f±1.27 33.28f±0.92 31.28g±0.89 a* 17.71ab±0.40 17.29ab±2.03 18.54a±3.44 16.16ab±2.65 15.49b±0.39 12.07c±0.62 10.86cd±01.06 8.15d±0.69 b* 20.32a±1.49 18.01bc±2.52 17.98bc±2.63 16.51c±2.22 13.39d±0.43 13.14d±0.80 12.14d±2.04 9.11e±0.58 values are means ±sd obtained with ten measurements. values displayed in the same row and tagged with different letters (a-g) are significantly different (p<0.05). ish pork dry sausages and foal salchichon, as reported by perez-alvarez et al. (1999) and lorenzo et al. (2012). lowering of a* values can possibly be explained by total or partial denaturation of nitrosomyoglobin coming as a result of lactic acid production. l* and a* values lower than those reported by lorenzo et al. (2012) can probably be related to the nature of horse meat, which is darker and redder than foal (tateo et al., 2008) yellowness (b*) had decreased from 20.32 to 9.11 and had varied significantly (p<0.05) during the production process. the decrease in b* values seen with the prolongation of the processing time was also reported by other authors (perezalvarez et al., 1999; lorenzo et al., 2012) and explained by the decrease in concentration of oxymyoglobin coming as a result of oxygen consumption executed by microorganisms. texture profile analysis texture profile analysis (tpa) parameters of the horse salami established during the smoking and dry ripening period are presented in table 4. average hardness values had significantly increased (p<0.05) from 0.32 to 20.54 kg as the processing went by. this can be related to the coagulation of muscle protein coming as a result of low ph values and sausage drying (bozkurt and bayram, 2006). springiness and cohesiveness had significantly decreased (p<0.05) during the processing (from 0.76 to 0.64 and from 0.67 to 0.43, respectively). springiness is related to elastic propertable 4 parameters obtained by virtue of textural profile analysis (tpa) of the horse salami during the manufacturing process. processing time (days) 0 7 14 21 28 42 60 90 hardness (kg) 0.32h±0.01 3.67g±0.19 4.61f±0.09 6.19e±0.21 9.91d±0.11 14.94c±0.25 17.58b±0.81 20.54a±0.92 springiness 0.76a±0.03 0.62def±0.03 0.68bc±0.01 0.73ab±0.02 0.58f±0.01 0.61ef±0.02 0.66cd±0.01 0.64cde±0.03 cohesiveness 0.67a±0.04 0.51c±0.04 0.48cd±0.01 0.65ab±0.12 0.46cd±0.01 0.46cd±0.01 0.43d±0.03 0.43d±0.03 gumminess (kg) 0.26f±0.01 1.87e ±0.15 2.26e±0.07 4.02d±0.50 4.56d±0.06 6.87c±0.13 7.73b±0.50 8.83a±0.54 chewiness (kg) 0.20g±0.02 1.16f±0.09 1.54e±0.04 2.94d±0.40 2.64d±0.04 4.19c±0.10 5.10b±0.49 5.65a±0.55 resilience 0.19a±0.04 0.15bcd±0.02 0.16bc±0.03 0.18ab±0.05 0.15bcd±0.01 0.12e±0.01 0.13de±0.02 0.14cde±0.01 values are means ±sd obtained with eight measurements. values displayed in the same row and tagged with different letters (a-h) are significantly different (p<0.05). 102 ital. j. food sci., vol. 28 2016 ties, so that the decrease in this textural property of the horse salami is most likely to be related to the removal of water (bozkurt and bayram, 2006). increases in gumminess and chewiness values (from 0.26 to 8.83 and from 0.20 to 5.65, respectively) seen during the horse salami processing were statistically significant (p<0.05). increase in chewiness values indicates that the horse salami becomes tougher during the ripening period (szczesniak, 2002), possibly due to moisture loss. resilience values established at the beginning and at the end of the processing were 0.19 and 0.14, respectively. significant changes in resilience during smoking and ripening failed to be observed (p>0.05) (table 4). microbial counts microbial flora changes seen during manufacturing are shown in fig. 1. the initial bacterial counts were 6.09 log cfu g-1 for total viable count (tvc), 5.29 log cfu g-1 for lactic bacteria (lab), and 3.88 log cfu g-1 for staphylococcus spp, respectively. relatively low bacterial counts in the salami stuffing indicate a good hygienic quality of the raw materials. tvc, lab and staphylococcus spp counts had significantly increased during the ripening period (p<0.05). this increase in bacterial count is typical of most naturally dry fermented european sausages (kozačinski et al., 2008). at the end of the horse salami production process, the mean values were 9.10, 7.79 and 5.10 log cfu g-1, respectively. as reported by many studies, microorganisms most represented during the ripening of cured sausages and meat products are lab (lizaso et al., 1999; samelis and georgiadou, 2000), whose counts tend to remain stable throughout the ripening period. within the frame of this study, high lab counts had been found during the first 28 ripening days, which can be related to a substantial ph drop witnessed during that period (table 2). lab inhibit the growth of pathogenic and spoilage bacteria by virtue of formation of lactic acid, acetic acid and possibly bacteriocins (lucke, 2000). isolation and identification of microbial population native sausage products are of a higher quality than those obtained by virtue of controlled fermentation with the addition of industrial starters (lebert et al., 2007). many authors support the view that indigenous microflora or microorganisms present in traditional sausages originate from raw materials or the manufacturing environment (mauriello et al., 2004; rantsiou et al., 2005). this microbiota is commonly referred to as “the house flora” (garcia-varona et al., 2000). therefore, in this study, the isolation and identification of autochthonous microbial population inhabiting the horse salami was performed. the results of a microbiological analysis (table 5) showed the dominant microflora to be the lactic acid bacteria strain termed lactobacillus plantarum, lactococcus lactis ssp. lactis, and enterococcus faecium while the most represented coagulase-negative staphylococci strain was s. xylosus. the yeast candida famata/debaryomyces hansenii was found as well, which is in agreement with the results of nielsen et al. (2008), who stated that halophilic yeasts most frequently isolated from fermented meat prodfig. 1 changes in microbial counts seen during the processing of the horse salami (mean±standard deviation obtained with three samples). ital. j. food sci., vol. 28 2016 103 ucts are debaromyces hansenii, candida famata, candida zeylanoides, trichosporon sp., cryptococcus sp. and rhodotorula sp. yeasts also play an important role in the maturation of sausages, since their lipolytic and proteolytic activity contributes to the development of sensory characteristics of fermented sausages (kovačević, 2001; alagić et al., 2008). in the horse salami samples, bacteria of the salmonella genus, enterobacteriaceae, sulphitereducing clostridia, l. monocytogenes or s. aureus were not found; however, api biochemical tests uncovered the presence the listeria grayi bacterium which is non-pathogenic (table 5). issues sometimes emerging with this type of fermented meat product are short shelf-life and poor hygienic surroundings, but the sausages produced in this investigation were proven to be microbiologically safe. it should be pointed out that biochemical (api) tests gave very good results (identification of one species with id > 98,2-99.9 %). the isolated lactic acid bacteria l. lactis ssp. lactis. l. plantarum and e. faecium could be used as starter cultures for meat products. l. plantarum as an autochthonous meat microflora is widely spread in nature (salama et al., 1995; ayad et al., 2001), l. lactis ssp. lactis in fermented sausage has rarely been reported so far and therefore further studies must to include detail molecular identification of isolated strains because api identification is not 100% precisely. the interest in exploring the potential of new strains isolated from different natural ecosystems to the effect of aroma compounds production has recently increased (ayad et al., 2001; frece et al., 2009; babić et al., 2011, frece et al., 2014). metabolic properties of the l. plantarum, e. faecium and l. lactis species have both direct and indirect influence on organoleptic, nutritional and hygienic quality of fermented products. more and more research is focused on the isolation and identification of autochthonous functional starter cultures with the aim of developing new functional meat products that will be recognised and labelled as autochthonous to the region in which they are produced (babić et al., 2011, frece et al., 2014, frece et al., 2014 a,b). therefore, l. plantarum, e. faecium, l. lactis and s. xylosus as potential functional autochthonous starter cultures will be thoroughly investigated in the future. further studies will be carried out to detail phenotypic, genotypic and physiological characterization of isolated strains of staphylococci and lab. sensory characteristics complex interaction between physicochemical, biochemical and microbiological processes, playing a role in formation of chemical compounds, and the modification of molecules responsible for the texture and appearance of the final product also determine its sensory characteristics. average scores given by the panellists at the end of the horse salami manufacturing process are shown in fig. 2. as for the external attributes, the horse salami scored highly when it comes to hardness (4.10±0.71) and low when it comes to appearance (3.60±0.43). it was highly rated for its sliceability, but low-rated when it comes to its colour intensity, fat/ lean ratio and easy peeling capacity. after slicing, the highest scores were obtained for the fat distribution (4.78±0.67), while the fat/lean ratio scored low (2.22±0.44). table 5 biochemical (api) results of the final product obtained after 90 days of manufacturing. microorganism values log cfu g-1±sd api test salmonella sp. enterobacteriaceae staphylococcus aureus cns (coagulase negative staphylococci) 5.10±1.5 s. xylosus sulphite reducing clostridia listeria sp. listeria grayi lactic acid bacteria 7.79±1.3 l. lactis ssp. lactis, lactobacillus plantarum, enterococcus faecium yeasts 3.25±1.2 candida famata/debaryomyces hansenii fig. 2 mean values of sensory properties of the final horse salami. 104 ital. j. food sci., vol. 28 2016 table 6 pearson´s correlation coefficients established between basic chemical composition, salt content, aw, texture and instrumental colour parameters. hardness springiness cohesiveness gumminess (kg) chewiness (kg) resilience l* a* b* (kg) moisture (%) -0.95** 0.19 0.68** -0.96** -0.94** 0.48 0.95** 0.89** 0.95** fat (%) 0.95** 0.058 -0.64 0.94** 0.95** -0.35 -0.91** -0.95** -0.93** protein (%) 0.96** -0.28 -0.77** 0.95** 0.93** -0.51 -0.95** -0.90** -0.97** collagen (%) 0.95** -0.09 -0.59 0.95** 0.94** -0.37 -0.94** -0.96** -0.96** ash (%) 0.92** -0.10 -0.61 0.94** 0.93** -0.44 -0.95** -0.87** -0.91** salt (nacl) (%) 0.94** -0.03 -0.62 0.94** 0.95** -0.33 -0.92** -0.90** -0.95** a w -0.86** 0.12 0.51 -0.86** -0.84** 0.20 0.84** 0.89** 0.94** values marked with ** are statistically significant (p<0.05). regarding the attributes that describe perceptions during mastication, horse salami was highly rated for its flavour intensity (4.10±0.44), juiciness (4.24±0.21) and acid taste (3.79±0.17), and low-rated for its saltiness and smokiness (2.41±0.31 and 2.20±0.19). during the fermentation of dry sausages, lab produce lactic acid (mateo et al., 1996) responsible for the sour taste (lotong et al., 2000) and odour of the product, while mould odour is to be associated with 1-octen-3-ol, which spreads a typical mushroom odour (meynier et al., 1998). in the present study, all three attributes scored highly (lactic acid taste 4.24±0.18; lactic acid odour 4.31±0.22; mould odour 3.82±0.15). as for the smell descriptors, lactic acid (4.3±0.22) and mould odour (3.8±0.15) were dominant, while the spice odour scored low (3.00±0.28). correlation between the parameters instrumental colour parameters of the horse salami, established during its processing, were significantly inversely correlated (p<0.05) to the protein, fat, ash, collagen and salt content. moisture content and a w values exhibited a significant direct correlation (p<0.05) to the instrumental colour parameters (table 6). relationships between the moisture, protein, fat, ash, collagen and salt content and a w on one hand, and hardness, gumminess and chewiness on the other, were also significant (p<0.05) (that between moisture and a w being an inverse one). pearson’s correlation coefficients indicated that springiness and resilience are not significantly (p>0.05) correlated to the basic chemical composition, salt content and a w (table 6). conclusions this study investigated into the changes in physicochemical, colour, textural, microbiological and sensorial properties of the horse salami as an indigenous croatian dry fermented sausage. during 90 days of manufacturing, major changes in physicochemical, colour and textural properties took place during the fermentation and ripening stage, pointing to proteolysis and lipolysis phenomena coming as a result of endogenous enzymatic activity, as well as to high lactic acid bacteria and staphylococci counts contributing to the specific organoleptic attributes of the final product. sensorial profiling of the final horse salami showed a significant acid taste, lactic acid odour and flavour intensity, and low fat/lean ratio, smokiness and saltiness values. the final product was proven to be microbiologically safe, the dominant microbial population being l. lactis ssp. lactis, l. plantarum, e. faecium and s. xylosus. references alagić d., kozačinski l., filipović i., zdolec n., hadžiosmanović m., njari b., kozačinski z. and uhitil s. 2008. microbiological changes during ripening of fermented sausages of horsemeat. meso. 10: 200. ayad e.h.e., verheul a., engels w.j.m., wouters j.t.m. and smit g. 2001. enhanced flavour formation by combination of selected lactococci from industrial and artisanal origin with focus on completion of a metabolic pathway. j. appl. microb. 90: 59. babić i., markov k., kovačević d., trontel a., slavica a., đugum j., čvek d., svetec i. k., posavec s. and frece j. 2011. identification and characterization of potential autochthonous starter cultures from a croatian “brand” product “slavonski kulen”. meat sci. 88: 517. badiani a., nanni n., gatta p., tolomelli b. and manfredini m. 1997. nutrient profile of horsemeat. j. food compos. anal. 10: 254. bourne m.c. 1978. texture profile analysis. food technol.chicago. 32: 62. bover-cid s., izquierdo-pulido m. and vidal-carou m.c. 2001. effectiveness of a lactobacillus sakei starter culture in the reduction of biogenic amine accumulation as a function of the raw material quality. j. food protect. 64: 367. bozkurt h. and bayram m. 2006. colour and textural attributes of sucuk during ripening. meat sci. 73: 344. bruna j.m., ordonez j.a., fernandez m., herranz b. and de la hoz l. 2001. microbial and physico-chemical changes during the ripening of dry fermented sausages superficially inoculated with or having added an intracellular cell-free extract of penicillium aurantiogriseum. meat sci. 59: 87. ital. j. food sci., vol. 28 2016 105 casiraghi e., pompei c., dellaglio s., parolari g. and virgili r. 1996. quality attributes of milano salami, an italian dry-cured sausage. j. agr. food chem. 44: 1248. cie. 1976. “colorimetry: official recommendations of the international commission on illumination”. comisión internationale de l’èclairage [international commission on illumination], cie no. 15 (e-1.3.1). paris, fr. dellaglio s., casiraghi e. and pompei c. 1996. chemical, physical and sensory attributes for the characterization of an italian dry-cured sausage. meat sci. 42: 25. franco d., rodríguez e., purriños l., bermúdez r. and lorenzo j.m. 2011. meat quality of “galician mountain” foals breed. effect of sex, slaughter age and livestock production system. meat sci. 88:292. frece j., kovačević d. and markov k. 2014.a national patent p20130089a: “formulation of bacterial starter cultures for the production of dry sausage and its use„ (15.8. 2014.) hrvatski glasnik intelektualnog vlasništva 17/2014. frece j., kovačević d. and markov k. 2014.b national patent p20130569a: “use of probiotic bacterial cultures lactobacillus plantarum 1k for the production of functional foods„ (9.10.2014.) hrvatski glasnik intelektualnog vlasništva 1/2015. 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italian heavy draft horses slaughtered at the age of eleven months. j. anim. sci. 86: 1205. trajković j., mirić m., baras j. and šiler s. 1983. analize životnih namirnica. tehnološko metalurški fakultet. beograd, yu. van schalkwyk d.l., mcmillin k.w., booyse m., witthuhn r.c. and hoffman l.c. 2011. physico-chemical, microbiological, textural and sensory attributes of natured game salami produced from springbok (antidorcas marsupialis), gemsbok (oryx gazella), kudu (tragelaphus strepsiceros) and zebra (equs burchelli) harvested in namibia. meat sci. 88: 36. #560_beghi_bozza ital. j. food sci., vol 29, 2017 357 paper rapid determination of crucial parameters for the optimization of milling process by using visible/near infrared spectroscopy on intact olives and olive paste v. giovenzanaa, r. beghi*a, r. civellia, s. trapanib, m. migliorinic, e. cinid, b. zanonib and r. guidettia adepartment of agricultural and environmental sciences production, landscape, agroenergy, università degli studi di milano, via celoria 2, 20133 milano, italy bdepartment of agricultural biotechnology, food technology section, university of florence, via donizetti 6, 50144 firenze, italy cpromofirenze, special agency of the florence chamber of commerce, laboratorio chimico merceologico unit, via orcagna 70, 50121 firenze, italy ddepartment of economics, engineering, science and technology agriculture and forestry, university of florence, via san bonaventura 13, 50145 firenze, italy *corresponding author. tel.: +39 0250316843; fax: +39 0250316845 e-mail address: roberto.beghi@unimi.it abstract the aim of this work is the application of vis/nir spectroscopy in order to correlate spectral data acquired on intact olives just before or in pastes during the milling process, to the crucial parameters for the optimization of the process. physical measurements (i.e. yield point force and total deformation energy) were performed on the olive samples; after the fruit were crushed for olive paste production, chemical analysis (moisture, oil and sugars content) and maturity index (mi) were measured and correlated to the spectral data. the obtained results were encouraging for chemical, texture and mi parameters, demonstrating the feasibility of real-time prediction of important indices for the milling plant settings. keywords: chemometrics, milling plant, olive pastes, optical analysis, ripening, texture ital. j. food sci., vol 29, 2017 358 1. introduction in olive fruits, physical parameters such as weight, color, pulp-to-stone ratio, and texture, and chemical parameters such as oil content, composition of fatty acids, levels of polyphenols, tocopherols, and sterols, change during the ripening process (beltrán et al., 2004; yousfi and garcía, 2005). these features are of high commercial importance as they influence the olive oil shelf life. in fact, olive oils derived from ripe fruit results in a less stable shelf life due to an increase in polyunsaturated fatty acids and a decrease in total polyphenol content (caponio et al., 2001; marsilio et al., 2001; morello et al., 2004; beltrán et al., 2005). phenolic compound content is considered an important parameter in the evaluation of virgin olive oil quality because of phenols contribute to oil flavour and aroma. phenols also, protect oil from autoxidation. in addition, olives processed at an over ripened stage may result in unstable oil during shelf-life owing to the low phenolic compound content (cherubini et al. 2009). early-harvested fruits produce oil with high polyphenol concentrations and a degradation may occur during the processing and shelf-life. degradation may result in variations in nutritional quality of the product since antioxidant content decreases and free radical content increases. this can lead to sensory modification and to an appreciation reduction of the product, since aroma, colour, taste and flavour attributes change and some unpleasant sensory attributes may occur (zanoni et al., 2005; diraman and dibeklioglu, 2009). for these reasons, the influence of harvesting time on the quality stability and sensory characteristics of olive oils is of crucial interest for the growers. an investigation of the olives’ characteristics before the milling process could allow the quality of the oil output to be controlled. better monitoring of the oil production process also depends on controlling the paste, the intermediate product between the olives inlet in the process and the oil outlet from the mill, to establish correlations among olives, paste and oil. nowadays, established methods for quality assessment are generally based on tedious and timeconsuming techniques that are impractical for processing a large number of samples. therefore, there is a lack of real-time information during the milling process in order to monitor the operating parameters continuously. hence, there is a strong need in the modern oil industry for a simple, rapid, and easy-to-use method for (i) objectively evaluating the level of olive ripening and the characteristics of the paste, (ii) early detection of possible failure, (iii) permanent monitoring of the production process, and (iv) assessment of oil process at any desired time in order to control the oil quality deriving from the process. the rapid analysis of olives during consignment and paste during the process would allow preliminary separation of homogeneous classes and a more efficient decision-making process about the destination of lots. therefore, the sector could be helped by optical non-destructive and rapid applications for olive oil chain optimization. despite there being some works regarding the application of nir spectroscopy for olive oil analysis (armenta et al., 2010), few works about the characterization of intact olives and olive pastes using spectroscopy can be found. fernández-espinosa (2016) combined chemometric analysis with nir spectroscopy to monitor quality parameters in intact olives to determine the optimal harvesting time; salguero-chaparro et al. (2013) used nir spectroscopy for the online determination of the oil content, moisture and free acidity parameters in intact olive fruits; and bellincontro et al. (2012) studied the application of a portable nir for on-field prediction of phenolic compounds during olive ripening. cayuela et al. (2009) determined the effectiveness of a portable nir spectrometer for the prediction of oil free acidity, oil yield, oil content in fresh fruit, oil content in fruit dry matter and fruit moisture content, analyzing intact fruits. for olive oil fruits, textural properties could be used as indices of ripeness to meet requirements for the technological processes and oil characterization. texture-measuring ital. j. food sci., vol 29, 2017 359 instruments are time-consuming, and there are high costs for the devices. the parameter setting of the process operations (i.e. crushing, malaxation, and extraction using decanter) is highly influenced by ripeness degree and olive texture. therefore, there is nowadays a lack of available information that could allow feedback-based real-time control of the plant, for better oil quality and the reduction of process wastes. beghi et al. (2013) and giovenzana et al. (2015) conducted preliminary studies on the laboratory scale of the capability of portable visible/near infrared (vis/nir) and nir spectrophotometers to investigate different textural indices for the characterization of olive fruits entering the milling process. on olive paste, garcía sánchez et al. (2005) tested the suitability of nir and nmr spectroscopy for the determination of moisture and fat contents, while gallardo et al. (2005) used near-infrared spectroscopy for the real-time determination of moisture and fat content in olive pastes and solid-liquid wastes. hermoso et al. (1999) examined the applicability of nir spectroscopy for the measurement of oil content and humidity in olive pomace. the challenge of producing high-quality olive oil is of great concern, and the selection of olive fruit with defined properties that ensure positive attributes in olive oil is foreseeable using vis/nir and nir spectroscopy in olive oil production (armenta et al., 2010). the olive oil sector is increasingly becoming more interested in the implementation of quality control systems in a mill industry context. however, limited work has been undertaken about the implementation of vis/nir and nir spectroscopy directly in the mill. research regarding the application at the factory level is desirable, mainly concerning the definition of parameters related to the on-line spectrum acquisition (salguero-chaparro et al., 2012) and the testing of compact and low-cost devices also usable for the sme of the sector. hence, in this study, the applicability of a vis/nir low-cost and compact system was tested on intact olives, acquired just before the milling process, and on olive pastes, in order to correlate spectral data to the crucial parameters (yield point force, total deformation energy, moisture, oil and sugars content, and maturity index) for the optimization of the milling process. the predictive models calculated here could be applied in future on-line for the rapid monitoring of crucial parameters for the enhancement of extraction oil yield and the control of semi-finished products of the process. 2. materials and methods two olive varieties were considered: frantoio and moraiolo (~50% of each). these varieties are typical of the tuscan hills, in the province of florence, italy, and are cultivated in several european olive growing areas. 2.1. sampling sampling was performed in 2013, from september to december, to obtain a wide sample variability. the sampling was conducted by hand once a week at 08:00 a.m. on a selected number of plants (about 10), belonging to the two different cultivars selected for the experiment. the olives were picked along plant circumference at approx. 1.7 m from the soil. a total of 54 olive samples (400-500 g), which presented no infection or physical damage, were quickly transported to the laboratory to be analysed and for each sample a homogeneous batch of olives (i.e. approx. 300 g) was selected. for each sample, 30 olive fruit vis/nir spectra were acquired, for a total of 1620 optical measurements. using a portable spectrophotometer, two spectral measurements were taken in reflectance mode ital. j. food sci., vol 29, 2017 360 on individual fruit along their equatorial region and averaged. all the olives composing a sample were crushed using a laboratory crusher (zeutec, rendsburg, germany), obtaining olive paste. five acquisitions were performed for each paste sample through disposable laboratory cuvettes (12.5 mm x 12.5 mm x 45.0 mm). 2.2. texture analysis the firmness was assessed using a laboratory dynamometer (mts criterion® systems, eden prairie, mn, usa) providing time-series data of product compression, allowing the calculation of textural attributes from load-extension traces. table 1 shows the settings for the compression test. the measurements were carried out using a 5 cm diameter plate coupled with a load cell of 100 n. the sample was placed under the plate, without holder, with the major axis perpendicular to the direction of the compression test. table 1. settings for the compression test. settings units gage adjustment speed mm s-1 0.2 gage adjustment load n 0.6 experimental speed mm s-1 1.0 date acquisition rate hz 400.0 break threshold n 5.0 break sensibility % 50.0 strain end point % 80.0 the textural parameters obtained from the elaboration of the load-extension curve were as follows: yield point force (n): the maximum force recorded during the elastic deformation phase; -total deformation energy (mj): the work required for complete compression of the olive pulp. in figure 1 is shown an example of load-extension curve obtained from the compression test for the identification of yield point force (n) and total deformation energy (mj). figure 1. example of load-extension curve obtained from the compression test. ital. j. food sci., vol 29, 2017 361 2.3. chemical analyses the olive paste was used for the following chemical analyses. 2.3.1 water content the water content was measured on olive paste by heating 60 g of sample in an oven at 105°c until reaching constant weight (cecchi et al. 2013). the results were expressed as moisture content (%). 2.3.2 oil content the total oil content was determined on 5 g of dried olive paste (see the oven method above). samples were extracted with hexane in an automatic extractor (randall mod. 148, velp scientifica, milan, italy), following the method of cherubini et al. (2009). the results were expressed as oil content on dry matter basis (g kg−1). 2.3.3 sugar content 8 g of olive paste was cold extracted (6 ± 2°c) with distilled water in a 200 ml flask for 2 hours. the content of the flask was filtered on paper, and 10 ml of the obtained solution was diluted with water in a 20 ml flask. the measurements were performed by analyzing the obtained solution with an enzymatic method using an automatic chemwell analyzer (awareness technology, chemwell 9210, palm city, fl). three enzymatic kits were used to measure, respectively, (i) the sum of two monosaccharides contents, namely glucose and fructose; (ii) the sum of disaccharide sucrose content with glucose and fructose contents; (iii) the mannitol content. all kits were purchased from r-biopharm (darmstadt, germany). the measurements were performed by means of external calibration standards: fructose and glucose (purity > 99%, sigma aldrich srl, milano, italy), and mannitol (purity > 98%, sigma aldrich srl, milan, italy). the results provided by the instrument were expressed in g l-1; they were also converted in sugar content on dry matter basis (g kg-1) as the average of two readings, carried out for each sample. the sucrose contents were determined by multiplying by 0.95 the difference between the sum of glucose, fructose, sucrose contents and the sum of glucose and fructose contents. 2.4. maturity index mi was based on visual assessment according to uceda and frias (1975). a sample of 100 drupes was classified into eight different classes according to pulp and skin colors. the values ranged from 0 (skin color deep green) to 7 (skin color black with all the flesh purple to the stone). 2.5. visible/near infrared device spectral acquisitions were performed on samples (olives and pastes) using an optical portable system (jaz vis/nir spectrophotometer, oceanoptics, inc., dunedin, fl, usa) operating in the 400-1000 nm wavelength range. the system is composed of five components: 1) vis/nir lighting system; 2) fiber-optic probe for reflection measurement; 3) spectrophotometer; 4) hardware for data acquisition and instrument control; 5) power battery. ital. j. food sci., vol 29, 2017 362 spectra were acquired in reflectance mode: light radiation was guided to the sample through a y-shaped, bidirectional fiber optic probe (oceanoptics, inc., dunedin, fl, usa). a y-shaped fiber allowed light from a halogen lamp to be guided to illuminate the sample while simultaneously collecting the radiation coming from the berry and guiding it back to the spectrophotometer. the tip of the optical probe was equipped with a soft plastic cap to ensure contact with sample skin during measurements, minimizing environmental light interference. white background and black background were acquired before each acquisition session. the integrated spectrophotometer was equipped with a diffractive grating for spectral measurements, optimized in the range 400-1000 nm, and a ccd sensor with a 2048 pixel matrix, corresponding to a nominal resolution of 0.3 nm. each spectrum corresponds to the average of five spectral acquisitions. 2.6. data analysis the data acquired were processed using chemometric techniques to extract maximum data information. chemometric analysis was performed using the unscrambler software package (version 9.8, camo asa, oslo, norway). different pre-treatments were applied to the vis/nir spectra in order to maximize the model accuracy. moving-averaged smoothed spectra (15 point-wide window corresponding to a window of 4.5) and multiplicative scatter correction (msc) were applied before building the calibration models. these pre-treatments were applied to improve the signal-to-noise ratio in order to reduce the effects caused by the physiological variability of olive and paste samples. the olive samples available were used for the calculation of a chemometric regression model for reference parameters by using partial least squares (pls) regression analysis. the vis/nir spectra acquired on the single olives were correlated to the textural parameters (one-to-one correlation) using the pls regression algorithm, while the 30 olive spectra representing each experimental sample were averaged, and the resulting mean spectrum was correlated to the chemical indices and mi. similarly, the olive paste samples were also correlated to chemical reference data and to mi to create pls models. to evaluate model accuracy, the statistics used were the coefficient of determination in calibration (r2cal), coefficient of determination in cross-validation (r2cv), root mean square error of calibration (rmsec), and root mean square error of cross-validation (rmsecv). calibration models were evaluated using a cross-validation leave-more-out procedure using five cancellation groups randomly selected. with a small number of cancellation groups, the resulting training sets are very different, and the measure of the predictive ability is not optimistic, possibly pessimistic (casale et al., 2008). moreover, the ratio performance deviation (rpd) value was calculated. rpd is defined as the ratio between the standard deviation of the response variable and rmsecv. rpd values below 1.5 indicate that the calibration is not useful. when the rpd value is higher than 2, quantitative predictions are possible. between 1.5-2.0, the algorithm has the possibility to distinguish between high and low values (williams and norris, 1987). the best model calibrations were selected based on minimizing the rmsecv and maximizing the rpd. 3. results and discussions the average spectra of both olives and pastes showed three main peaks: around the 670 nm band, corresponding to the chlorophyll absorption peak (mcglone et al., 2002); around the 730 nm band, equal to the maximum reflectance peak; and the 780 nm band, representing the third overtone of oh bond stretching (clement et al., 2008). ital. j. food sci., vol 29, 2017 363 changes in spectra reflected modifications in chemical parameters. for a better visualization, two arbitrary classes based on oil content (a, level 1 ≤ 358.3 g kg-1; and level 2 > 358.3 g kg-1) and on moisture content (b, level 1_m ≤ 53.5 %; and level 2_m > 53.5 %) were created to show changes in the optical data. the average vis/nir spectra acquired on intact olives are grouped into two classes by oil and moisture content, and are shown in figures 2a and 2b, respectively. figure 2. average vis/nir smoothed spectra of intact olives, frantoio (f) and moraiolo (m) cultivar, grouped in two classes of oil content (a, level 1 ≤ 358.3 g kg-1; level 2 > 358.3 g kg-1) and in two classes of moisture content (b, level 1_m ≤ 53.5 %; level 2_m > 53.5 %). vis/nir spectra exhibited differences for both the cultivars among the two classes, with relevant changes particularly in the visible range occurring from level 1 to level 2 for moraiolo cultivar. the spectra of moraiolo cultivar showed higher absorption in the visible range compared to frantoio cultivar. this is linked to anthocyanin pigmentation during ripening from green berries to the completely black-pigmented olives, which leads to a strong decrease in reflectance in the visible band associated with the anthocyanin absorption peak centered on 540 nm. this different behavior in the spectral reflectance is confirmed by the maturity index (uceda and frias, 1975). this parameter is based on the subjective evaluation of the progressive pigmentation of olive skin and flesh. the index is the main reference utilized by the olive oil chain to characterize the olive ripeness degree at the mill or on the tree (garcía et al., 1996), and by scientists to identify the ripeness levels of olives for harvesting all over the world, e.g. in israel (dag et al., 2011) in spain (gutiérrez et al., 1999); beltrán et al., 2005) in tunisia (mraicha et al., 2010), and in italy (sinelli et al., 2008). when the olives are fully ripe, the mi reached values equal to 6 on barnea and souri cultivars (dag et al., 2011) and 7 on chemlali cultivar (mraicha et al., 2010). instead, for the two analyzed cultivars, the mi achieved at harvest maximum values of 3.39 for moraiolo cv and 2.31 for frantoio cv. frantoio remains substantially green even when fully ripe, hence the spectra in the visible range not showing evident changes between the two classes considered. in figure 2, as expected, an opposite trend can be noticed between spectra grouped by oil content and by moisture content; the oil accumulation in the fruit caused greater absorption in the spectra of both cultivars, which results in lower average values of reflectance in the whole spectra of the class level 2. in particular, this behavior is evident for the moraiolo cv, due to an increase of oil content and a simultaneously external pigmentation of the berries. this leads to a decrease in reflectance in the visible band associated with the anthocyanin absorption peak centered around 540 nm. ital. j. food sci., vol 29, 2017 364 conversely, the spectra of the berries richer in water (level 2_m) showed slightly higher values of reflectance, especially in the visible spectral range for the moraiolo cv. similar behavior, as shown in figure 3, can be noticed for the average spectra of olive pastes grouped by the same two classes of oil and moisture content. in particular, the spectra of pastes richer in water (level 2_m) showed the highest values of reflectance. also, in this case, the behavior is more evident for the moraiolo cv. figure 3. average vis/nir smoothed spectra of olive pastes, frantoio (f) and moraiolo (m) cultivar, grouped in two classes of oil content (a, level 1 ≤ 358.3 g kg-1; level 2 > 358.3 g kg-1) and in two classes of moisture content (b, level 1_m ≤ 53.5 %; level 2_m > 53.5 %). pls regression models were built for each parameter measured. in table 2, the results of the pls regression models arising from spectra on 30 intact olives for the predictions of moisture, oil and sugars content, of yield point force, total deformation energy, and mi are shown. regarding textural parameters the possibility to use the reference data on a single berry allowed us to obtain acceptable results for the prediction of indices usually difficult to predict in an optical non-destructive way. interesting results were obtained for the prediction of the yield point force. similar results were obtained for firmness prediction on intact olives by kavdir et al. (2009) and by beghi et al. (2013), using ft-nir spectroscopy in the wavelength range 780-2500 nm in reflectance mode and vis/nir spectroscopy (400-1000 nm), respectively. instead, regarding the prediction of the total deformation energy, results are not satisfactory and similar to those obtained in a previous study by giovenzana et al. (2015): r2 in cross-validation equal to 0.58 for vis/nir range and equal to 0.33 for nir range. the application of vis/nir and nir spectroscopy for the analysis of textural parameters often encounters considerable difficulties, which was highlighted elsewhere (zude et al., 2006; nicolaï et al., 2008). this difficulty is usually due to several factors: first, the extreme variability of this parameter among berries; the high instrumental error of the penetrometer; and the difficulty of calibrating a model for the estimation of an index not directly associable with a chemical species (and consequently the absorption bands of those chemical bonds). encouraging results were also obtained for chemical parameters, in particular for the prediction of moisture and oil content, with rpd values of about 2. ital. j. food sci., vol 29, 2017 365 table 2. descriptive statistics and statistics of the pls models elaborated on vis/nir spectra of intact olives for the prediction of chemical, maturity and textural parameters calibration models cross-validation models chemical parameters sample n media ds lvs r2cal rmsec r 2 cv rmsecv rpd moisture (%) all samples 48 53.32 3.97 8 0.87 1.39 0.75 1.89 2.10 frantoio cv 28 51.81 3.33 7 0.89 1.05 0.79 1.43 2.33 moraiolo cv 18 55.42 4.07 5 0.91 1.13 0.74 2.12 1.92 oil content (g kg -1) all samples 44 371 110 49.93 9 0.85 18.67 0.74 25.85 1.93 frantoio cv 28 361.34 49.88 2 0.67 28.01 0.67 31.12 1.60 moraiolo cv 18 390.71 50.87 3 0.88 16.97 0.81 22.3 2.28 sugar content (g kg -1) all samples 46 38.87 5.69 7 0.65 3.31 0.42 4.35 1.31 frantoio cv 24 35.91 3.76 10 0.95 0.77 0.75 2.03 1.85 moraiolo cv 18 44.18 4.2 10 0.95 0.88 0.38 3.38 1.24 maturity index sample n media ds lvs r2cal rmsec r 2 cv rmsecv rpd mi all samples 47 0.85 1.02 3 0.93 0.24 0.92 0.26 3.92 frantoio cv 28 0.32 0.46 5 0.87 0.15 0.7 0.22 2.09 moraiolo cv 18 1.71 1.08 3 0.96 0.21 0.94 0.28 3.86 textural parameters sample n media ds lvs r2cal rmsec r 2 cv rmsecv rpd yield point force (n) all samples 1410 41.26 17.26 9 0.63 12.32 0.62 12.44 1.39 frantoio cv 918 52.83 22.29 10 0.63 13.51 0.61 13.82 1.61 moraiolo cv 486 43.6 17.81 9 0.74 8.04 0.72 8.31 2.14 total deformation energy (mj) all samples 1373 450.35 103.69 10 0.42 78.83 0.4 79.96 1.30 frantoio cv 876 467.11 106.65 10 0.54 71.96 0.52 73.59 1.45 moraiolo cv 408 426.98 81.63 3 0.33 66.67 0.31 67.93 1.20 a similar study was performed by salguero-chaparro et al. (2013) for the evaluation of moisture and fat content in intact olive fruits from 50 varieties using an nir instrument in the range 400-2500 nm. they achieved for pls models on moisture, fat content and acidity rpd values of 2.32, 2.08 and 1.70, respectively. cayuela et al. (2009) obtained slightly worse results using an aotf-nir on intact olives in the range 1100-2300 nm for the prediction of oil content (r2 equal to 0.65) and moisture (r2 ranged 0.35-0.78) compared with those obtained by the authors of this study for the same parameters. moreover, similar results were achieved by fernández-espinosa (2016) using an online aotf-nir system (1000-2300 nm) on intact olives for the estimation of the oil content (r2 0.76), while better results were obtained by the same author for the prediction of the moisture content (r2 0.88). excellent results were obtained for the estimation of the mi with rpd about 4. this result may have interesting applicative implications, since the mi requires time for measuring ital. j. food sci., vol 29, 2017 366 and sample preparation. guzmán et al. (2015) classified intact olives based on mi using image analysis, obtaining positive predictive values of about 90%. in table 3, the results for the pls regression models arising from spectra on olive pastes are shown. in this case, the considered parameters were obviously only the chemical ones and the mi. slightly better results were obtained compared to those arising from the models calculated using spectra on intact olives. also in this case, better results were achieved for the prediction of mi, in particular for the moraiolo cultivar (rpd = 4.15). bendini et al. (2007) applied ft-nir for in-process monitoring of different cultivar pastes in diffuse reflectance mode, obtaining models with r2 equal to 0.92 and 0.91 for the prediction of oil content and moisture, respectively. table 3. descriptive statistics and statistics of the pls models elaborated on vis/nir spectra of olive pastes for the prediction of chemical and maturity parameters. calibration models cross-validation models chemical parameters sample n media ds lvs r2cal rmsec r 2 cv rmsecv rpd moisture (%) all samples 45 53.4 3.99 9 0.86 1.44 0.75 2 2.00 frantoio cv 26 51.79 3.16 4 0.74 1.56 0.55 2.11 1.50 moraiolo cv 18 55.42 4.07 4 0.86 1.42 0.79 1.91 2.13 oil content (g kg -1) all samples 46 378.23 50.98 5 0.78 23.46 0.69 28.64 1.78 frantoio cv 26 372.19 44.83 2 0.68 24.56 0.7 27.32 1.64 moraiolo cv 18 390.71 50.87 3 0.88 16.66 0.85 19.76 2.57 sugar content (g kg -1) all samples 45 51.68 7.3 4 0.61 4.5 0.51 5.2 1.40 frantoio cv 28 48.67 5.72 4 0.6 3.53 0.51 4.42 1.29 moraiolo cv 13 55.83 4.4 6 0.98 0.59 0.83 1.86 2.37 maturity index sample n media ds lvs r2cal rmsec r 2 cv rmsecv rpd mi all samples 46 0.96 1.04 4 0.88 0.33 0.85 0.39 2.67 frantoio cv 25 0.4 0.51 4 0.79 0.21 0.58 0.31 1.65 moraiolo cv 18 1.71 1.08 7 0.98 0.12 0.95 0.26 4.15 similar prediction performances were obtained starting from the spectra acquired on intact olives or on pastes. rpd values for the considered chemical parameters were 1.31– 2.10 and 1.40–2.00 for the models calculated for intact olives and pastes, respectively. this result is very interesting with a view to future applications, as the possibility to perform optical analysis directly on the fruits before the process could be envisaged, without any sample preparation. regarding the mi, better results were obtained as expected starting from intact olives, due to the high correlation between ripeness and peel pigmentation; the evolution of mi values is driven by color evolution during ripening, which is mainly influenced by the external color of the fruits. the moraiolo cultivar gave the best results overall. this is probably due to the evident evolution of external pigmentation of the fruits during the ripening process ital. j. food sci., vol 29, 2017 367 that helps the correlation with the vis/nir spectra, especially due to the contribution of the visible range. predictive models are usable for the monitoring of operative parameters in different steps of the milling process, i.e. crushing, malaxation, and extraction using decanter, for the enhancement of extraction oil yield and the control of semi-finished products of the process. 4. conclusions the olive oil sector is interested in new user-friendly systems for rapid analysis that can be performed directly on-line on the milling plant with the objective of using information from sensors to manage the product better, and to preserve consumers' expectations of high-quality extra virgin olive oil, closely related to the composition of phenols and of volatile compounds. increasing demand for rapid, cost-effective and non-invasive measurement of texture remains a challenge for the oil extraction process. this study has interrogated the applicability of vis/nir spectroscopy as a rapid technique for the analysis of olives directly on the tree or at the mill just before the oil extraction process, and the olive paste, for the monitoring of crucial parameters for the enhancement of extraction oil yield (moisture, oil, sugar content, mi and textural indices). our results were encouraging for chemical, texture and mi parameters. regression models could be used for real-time prediction of crucial indices to support specific requirements of the process, considering the technological characteristics of the different olives or olive pastes, in order to diversify quickly the oil production. investigation of wavelength bands in order to highlight and select the most informative ones is desirable in order to design a simple and inexpensive device to 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proceedings of the segundo seminario oleicola internacional 125-128. williams p. and norris k. 1987. near-infrared technology in the agricultural and food industries. st paul, minn, usa, american association of cereal chemists. yousfi k., cert r. and garcía j. 2005. changes in quality and phenolic compounds of virgin olive oils during objectively described fruit maturation. eur. food res. technol. 223:117-124. zanoni b., bertuccioli m., rovellini p., marotta f. and mattei a. 2005. a preliminary approach to predictive modelling of extra virgin olive oil stability. j. sci. food agric. 85:1492-1498. zude m., herold b., roger j.m., bellon-maurel v. and landahl s. 2006. non-destructive tests on the prediction of apple fruit flesh firmness and soluble solids content on tree and in shelf life. j. food eng. 77:254-260. paper received july 20, 2016 accepted february 6, 2017 #483_toschi_bozza ital. j. food sci., vol 29, 2017 38 paper simulating international shipments of vegetable oils: focus on quality changes z. ayyad1,2, e. valli2,3, a. bendini2,3, r. accorsi4, r. manzini4, m. bortolini4, m. gamberi4 and t. gallina toschi*2,3 1 department of food technology, college of science and technology, al-quds university, abu dies, p.o. box 20002, jerusalem, palestine 2 department of agricultural and food sciences, alma mater studiorum university of bologna, cesena (fc), italy 3 interdepartmental centre for agri-food industrial research, alma mater studiorum university of bologna, cesena (fc), italy 4 department of industrial engineering, alma mater studiorum university of bologna, italy *corresponding author. tel.: +39 0512096010 e-mail address: tullia.gallinatoschi@unibo.it abstract this investigation evaluated the quality changes of commercial vegetable oils after different simulated shipments. in particular, the oils were placed in containers with or without thermal insulation and subjected to two simulated shipments, from italy to los angeles and to quebec. the temperature profiles were monitored to simulate the real shipments conditions in laboratory through properly developed climate chambers. different quality parameters were evaluated before and after the simulations, showing a high degree of oxidation for samples shipped to los angeles in standard containers. in this study, the thermal insulation container was effective in protecting samples from potential oxidative damage during simulated shipping. keywords: edible vegetable oils, food quality, oxidation, simulated shipment; thermal insulation ital. j. food sci., vol 29, 2017 39 1. introduction vegetable oils such as sunflower, palm kernel, and soybean oils are extensively used for cooking purposes. these types of fatty food products are more susceptible to oxidation than animal fat because of their content of unsaturated fatty acids (parker et al., 2003). in 2014, about 168 million tons of vegetable oil was produced worldwide (usda, 2014). among vegetable oils, italy is considered as the dominant supplier of olive oils to canada and usa, and about 72 % and 60 % of olive oil imported in 2014 to canada and usa, respectively, was from italy. furthermore, in 2014, italy exported around 230,000 metric tons of virgin olive oil (ioc, 2014b). during transportation by sea, the desired temperature for most edible oils is ambient temperature (cac, 2013b). considering that solidification and crystallization of the product occurs at 3-4°c (piscopo and poiana, 2012), edible oils may suffer from deterioration in quality, which involve hydrolytic and oxidative modifications promoted by several factors, such as temperature and humidity in the stages of pumping and tank filling, in addition to the effect of light exposure for samples transported in clear bottles (btm, 2013). raw edible oils, even after soft refining, as well as virgin olive oils, contain a range of minor compounds such as chlorophylls, tocopherols, carotenoids, and phenolic compounds that function as natural antioxidants by enhancing the stability of the oil during storage (kristott, 2000). the monounsaturated/polyunsaturated fatty acid ratio, as well as the presence of phenolic compounds, make virgin olive oil more stable towards heat induced oxidation (bendini et al., 2004). moreover, the hydrolysis of acylglycerols, catalyzed mainly by an increase in temperature during storage, as well as the presence of moisture, oxygen, or light (frankel, 1991), plays an important role in development of off-flavors, thus making edible oils unpalatable and shortening their shelf-life (kristott, 2000). high temperatures increase the rate of oxidation, while very low freezing temperatures may also change the availability of some micro components, such as phenolic compounds, water distribution around crystals, and the physical characteristics of olive oil (bendini et al., 2007). several studies have been carried out on the simulated transportation of foodstuffs. for example, an interesting report done by burger (1985) studied the effect of bulk storage and transportation on the quality of palm oil, and found that during the 25 days of an actual journey at temperatures ranging between 37-55°c, there was a slight increase in free acidity, while peroxide values were doubled at the final stage of the voyage. the effect of different thermal conditions registered in the food supply chain during transportation of edible oils was recently studied by our group (valli et al., 2013). in that study, we investigated the effect of simulated shipment on the quality of different types of edible oils from italy to taiwan, starting from the stage of truck loading and ending at the truck delivery phase. it was found that vegetable oils underwent a loss of quality and deterioration after the journey, especially in terms of primary and secondary oxidation products. the simulation runs were conducted using ad-hoc closed-loop controlled chambers (manzini and accorsi, 2013), in order to measure and control the effects of transportation on the quality of edible oil. moreover, we have also compared the performance of these containers (accorsi et al., 2014; manzini et al., 2014). in the present study, changes in the quality of three kinds of vegetable oils (extra virgin olive oil, rice oil, and grape seed oil) after two simulated shipments were investigated. the first journey was characterized by high temperatures during 37 days of shipment from italy to los angeles (usa), and the latter by lower temperatures during 30 days of shipment from italy to quebec (canada). such temperatures were monitored using a thermal data logger during actual shipping and then reproduced in the laboratory. in particular, both the shipping profiles experienced by the bottles were tracked within a standard container (sc), i.e., a general-purpose one or dry container, and a thermal liner ital. j. food sci., vol 29, 2017 40 containers (tlc), i.e., a basic dry containers equipped with a thermal liner that can partially or completely insulate cargo from climate stresses. this study evaluated the ability of the thermal insulated container to protect the quality of the oils in both shipments. with this aim, quality parameters such as free acidity, oxidation indexes (peroxide value, thiobarbituric acid content, and oxidative stability index) as well as sensory analysis and other physicochemical parameters (water amount, turbidity, and cielab color indexes) were evaluated before and after the simulated shipments. 2. materials and methods 2.1. samples the two simulated shipments were carried out using three different kinds of commercial vegetable oils: extra virgin olive oil (evoo), grape seed oil (gso), and rice oil (ro). in particular, two bottles (1 liter each) of oil were subjected to the simulated shipments. the two bottles of each oil for each destination (quebec, coded as “q” or los angeles, coded as “la”) contained edible oil coming from the same production line batch. for both bottles, the primary packaging is a glass bottle. the cork is made of aluminum with a pet pourer. the bottles are contained within a secondary package made by corrugated carton (i.e., wrap package). the shipping profiles experienced by the two sets of bottles are tracked respectively within a standard container (sc), i.e., a general-purpose 40-foot equivalent units (feu) container or dry container, and thermal liner containers (tlc), i.e., a basic dry containers equipped with a thermal liner that can partially or completely insulate cargo from climate stresses. a more detailed definition of these two containers is given in accorsi et al. (2014), while for the primary (i.e., bottle) and secondary packaging (i.e., carton) (accorsi et al., 2015). 2.2. simulation process the temperature profiles were reproduced using closed-loop climate-controlled chambers placed in standard or thermally insulated containers (fig. 1). the two container solutions have been previously described in a paper by the same research group (manzini et al., 2014). the simulation chambers reproduced temperature cycles to fit the monitored temperatures registered during actual shipments. the temperature inside the chambers covers the possible range of -20°c to 65°c. the integrated cooling system consists of an evaporator utilizing 21 g of r600a iso-butane as a refrigerant. a closed-loop algorithm, developed with labview national instrument software, controls the actuators so that the chamber temperature reaches a defined set point. the first international simulated shipment (coded as “q”) from italy to quebec started on january 30 from the port of origin (livorno) and ended on march 1 at the port of final destination (quebec); the temperature profile of this shipment is illustrated in fig. 2. the second international shipment (coded as “la”) from italy to los angeles started on june 26 from the port of origin (livorno) and ended on august 2 at the port of final destination; the temperature profile of this shipment is shown in fig. 3. ital. j. food sci., vol 29, 2017 41 figure 1. closed-loop protocol system for simulation of shipping. 2.3. chemical, physical and sensory analyses 2.3.1 free acidity and thiobarbituric acid reactant substance content (tbars) free acidity (fa) expressed as g oleic acid 100 g-1 oil and peroxide value (pv) expressed as milliequivalent o2 kg-1 oil were determined for evoo according to the official methods described in eec. reg. 2568/91 and successive amendments (eec reg. 1348/2013). for the two other edible oils, free acidity values (av) were obtained by the codex alimentarius official method (cac 2013a), and expressed in mg koh g-1 oil. thiobarbituric acid reactant substance content (tbars) was determined in triplicates according to the aocs official method cd 19-90 (aocs, 2006) and expressed as tba value (milligram of malonaldehyde equivalent per kilogram of oil). oil sample (50-200 mg) was weighted into 25 ml volumetric flask and dissolved with a small portion of 1butanol. the solution volume was then filled by using 1-butanol. a portion (5 ml) of the dissolved sample was transferred into a screw-capped test tube. the reagent solution (200 mg of 2-thiobarbituric acid dissolved in 100 ml of 1-butanol) was added, and the mixture was thoroughly mixed. the tubes were then placed in a water bath at 95°c for 2 h. after cooling at room temperature, absorbance was determined at 530 nm by using 1 ml glass ital. j. food sci., vol 29, 2017 42 cuvettes with a uv-vis 1800 spectrophotometer (shimadzu co., kyoto, japan). the reagent blank was prepared simultaneous to sample preparation. tba value was obtained using the following equation: tba = 50 × (abs of the sample abs of the blank) /weight of the sample (mg) 2.3.2. spectrophotometric determination of total phenolic content (tp) phenolic compounds were extracted according to the method of pirisi et al. (2000). absorbance was determined at 750 nm by using a uv-vis 6705 spectrophotometer (jenway, united kingdom) through the method reported by singleton and rossi (1965). briefly, each sample (2 g) was dissolved in 1 ml of n-hexane and extracted three times with 2 ml of methanol–water solution (60:40 v/v). in each extraction, the mixture was shaken with a vortex mixer for 1 min and then centrifuged for 5 min at 3,000 rpm. the aqueous phase was collected and transferred into another test tube after each centrifugation cycle. n-hexane (2 ml) was added to the collected phenolic extract, mixed on the vortex, and then centrifuged for 5 min at 3,000 rpm. after the n-hexane phase was removed, the extract was evaporated using a rotary evaporator at 35°c. the residue was dissolved with 5 ml of methanol-water solution (50:50 v/v). absorption was determined with a spectrophotometer, and a standard calibration curve was prepared using different concentrations of gallic acid. the results were calculated and expressed as milligram of gallic acid per kilogram of oil. 2.3.3 evaluation of the colour (cielab) cielab color for evoo samples was determined (gomez-caravaca et al., 2007), using a hunterlab (reston, va, usa) colorflex instrument and expressed as l*, a*, b* chromatic coordinates. turbidity (td) of samples was determined using a ratio turbidimeter model 18900 (hack, colorado, usa) and expressed as nephelometric turbidity units (ntu). 2.3.4 determination of the water content water amount was determined at 103°c through air drying technique (iso 662:1988). oil sample (10 g) was weighed in an empty aluminum moisture dish (approximately 50 mm in diameter and 30 mm height, with a flat bottom). the samples were heated for 1 h in a drying oven at 103±2°c, and the dish was cooled in the desiccator and weighed. the sample was reheated for another 0.5 h, cooled, and then weighed again. the half-hour reheating, cooling, and weighing cycle may be repeated until the difference between the final successive weights was lower than 2 mg. the water amount was calculated with the following equation: weight of sample − weight of dried sample / weight of sample. 2.3.5 sensory analysis sensory analysis of evoo samples was performed according to the procedure outlined in eec reg. 640/2008 by a fully trained panel of 8 expert and trained tasters of the department of agricultural and food sciences of the university of bologna. ital. j. food sci., vol 29, 2017 43 2.3.6 statistical analysis all analyses were run in triplicate and expressed as mean ± standard deviation (sd). analysis of variance (anova) was performed using xlstat 7.5.2 software (addinsoft, ny, usa) at a 95% confidence level (fisher lsd, p < 0.05) to evaluate significant differences between means. 3. results and discussion 3.1 effect of simulated shipment on hydrolytic degradation free acidity is considered as an important parameter to determine the hydrolysis of triacylglycerol in olive oil. moreover, acidity values are considered as a basic criterion to classify the different categories of olive oil. the results in table 1 show that fa increased slightly during shipments to both destinations. in addition, there was a slight increasing trend in fa for evoo la shipped in a standard container compared with that before shipping, which was influenced by the increase in temperature during the simulated journey (paradiso et al., 2010). however, none of the shipped evoo samples reached the limit of 0.8% accepted for the extra virgin olive oil category (eec reg. 1348/2013). table 1. fa, free acidity (g oleic acid 100 g-1 oil); pv, peroxide values (meq o2 kg-1 oil); tbars, thiobarbituric acid reactive substances value (mg of malonaldehyde equivalent kg-1 oil); tp, total phenols (mg gallic acid kg-1 oil) tested before simulation and after simulation of shipping in insulated and standard containers for evoo samples to the two final destinations (evoo q, quebec and evoo la, los angeles). values (mean ± standard deviation) with different superscript capital letters in each column and for each sample were significantly different between the simulated shipping conditions (p < 0.05; fisher’s test). acid value results (table 2) of gso stored in the standard container for both simulated shipments were significantly higher in comparison with the thermally insulated samples and that before shipping. considering the ro samples shipped to quebec which, before starting the simulation, had an av higher than the accepted limit of 0.6% for edible oils (cac 2013a), the av registered for the sample stored in the standard container was significantly higher than both the respective values for samples with and without thermal insulation. sample experimental condition fa (g oleic acid 100 g-1) pv (meq o2 kg-1) tbars (mg of malonaldehyde equivalent kg-1) tp (mg gallic acid kg-1) evoo q before shipping 0.52b±0.04 11.7c ±0.7 0.013 b ± 0.001 353 b ± 25 insulated container 0.59a±0.01 13.1b ±0.3 0.012 b ± 0.001 372 b ± 38 standard container 0.60a±0.01 17.0a ±0.8 0.016 a ± 0.001 478 a ± 30 evoo la before shipping 0.45b±0.01 8.8c ± 0.2 0.015 c ± 0.001 259 a ± 2 insulated container 0.45b±0.01 9.2b ± 0.1 0.028 b ± 0.001 257 a ± 8 standard container 0.48a±0.01 10.4a ± 0.1 0.040 a ± 0.001 222 b ± 3 ital. j. food sci., vol 29, 2017 44 table 2. av, acid values (mg koh g-1); pv, peroxide values (meq o2 kg-1 oil); tbars, thiobarbituric acid reactive substance values (mg of malonaldehyde equivalent kg-1 oil) of vegetable oil samples [grape seed oil (gso) and rice oil (ro)] tested before and after simulation of shipping in insulated or standard containers to the two final destinations (coded as “q” to quebec and as “la” to los angeles). values (mean ± standard deviation) with different superscript capital letters in each column and for each sample were significantly different between the simulated shipping conditions (p < 0.05; fisher’s test). sample experimental conditions av (mg koh g-1) pv (meq o2 kg-1) tbars (mg of malonaldehyde equivalent kg-1) gso q before shipping 0.27c ± 0.00 4.2b ± 0.1 0.018a ± 0.001 insulated container 0.36b ± 0.03 6.3a ± 0.9 0.020a ± 0.003 standard container 0.43a ± 0.00 6.2a ± 0.1 0.017a ± 0.002 ro q before shipping 0.74c ± 0.01 4.4b ± 0.2 0.017b ± 0.001 insulated container 0.86b ± 0.03 4.8a ± 0.1 0.016b ± 0.002 standard container 0.98a ± 0.08 4.1b ± 0.1 0.022a ± 0.003 gso la before shipping 0.24b ± 0.04 1.6b ± 0.0 0.018c ± 0.001 insulated container 0.24b ± 0.03 3.3a ± 0.5 0.020b ± 0.001 standard container 0.35a ± 0.02 3.0a ± 0.2 0.043a ± 0.001 ro la before shipping 0.46a ± 0.01 3.3b ± 0.3 0.014b ± 0.001 insulated container 0.45a ± 0.03 3.5b ± 0.4 0.020a ± 0.001 standard container 0.51a ± 0.03 4.9a ± 0.4 0.020a ± 0.001 the results for the ro sample to quebec revealed a drastic effect of temperature variation, and in particular for low quality edible oils. in fact, as recorded during the simulation in a standard container to quebec, the temperature decreased to -10°c (fig. 2). such low temperatures probably facilitate hydrolytic processes due to water droplets in the liquid phase that surrounds the lipid crystals (kristott 2000). in the case of ro in the simulated shipment to los angeles, on the other hand, the change in av after simulation in both the standard and thermally insulated containers was not significant; in this case, the samples experienced a slight temperature fluctuation during 13 days of simulated shipment before reaching the final destination. 3.2. influence of simulated shipment on oxidation stability in order to estimate the effect of shipment on evoo and other vegetable oils, oxidation quality was tracked by evaluating i) pvs, which indicate the increase in primary oxidation products, such as hydroperoxides, and ii) tbar values, which detect the formation of malondialdehyde from fatty chains with three or more double bonds (frankel, 1991), and indicate the trend in secondary oxidation products in edible oil. as seen in table 1, the pv was significantly higher in the evoo sample for which the simulated shipment was conducted in a standard container compared to that shipped in a thermally insulated container for both destinations. tbars values were also significantly higher when a standard container was used to transport evoo samples compared with those subjected to simulation in a thermally insulated container for both destinations. these results suggest that thermally insulated containers have a beneficial effect, compared with a standard container, in terms of protecting evoo samples against oxidative stress. moreover, starting from similar values for both samples before shipping, higher tbars ital. j. food sci., vol 29, 2017 45 values were reported for evoo sent to los angeles compared with the sample sent to quebec; this may be related to the higher temperature stress applied in the los angeles simulation (figs. 2 and 3). figure 2. temperature profile monitored using data loggers for the quebec simulation (in the world map, 1: livorno port; 2: quebec port). a: inside standard container; duration: 30 days; highest temperature: 19°c; lowest temperature: -11.5°c. b. inside thermal insulated container; duration: 30 days; highest temperature: 11°c; lowest temperature: 6.5°c. regarding the other vegetable oils, the pvs (table 2) had higher values after simulation compared with those before shipping, for both destinations, except for ro shipped in a standard container to quebec. considering ro to los angeles, a higher increase was observed in pvs in a standard container compared with thermally insulated samples, which indicate more advanced formation of peroxides in the standard container. on the other hand, the lower pv values seen in ro to quebec in a standard container compared with samples shipped in an insulated container reveals possible additional transformation of peroxides to secondary oxidation products, which was also confirmed by the increase in tbar observed in the same sample (table 2). the higher impact on oxidative status on all ital. j. food sci., vol 29, 2017 46 edible oils by the los angeles simulation is also demonstrated by considering the changes in total phenols in evoo (table 1): these minor components, in addition to their nutritional role, act as antioxidants in evoo (bendini et al., 2007). before simulation, evoo samples contained about 353 and 259 mg gallic acid kg-1 oil, respectively, for samples sent to quebec and los angeles (table 1); after shipping, these values tended to decrease in standard container for the samples sent to los angeles. this reduction was more pronounced for samples stored in the standard container than after the nonthermally insulated journey due to the effect of higher temperature stress (fig. 3). the anomalous increase in total phenolic content registered for the evoo shipped to quebec after simulation in standard container could be attributed to the higher extractability of phenolic molecules after a crystallization and subsequent thawing out caused by the low temperatures reached during the simulation (fig. 2). on the other hand, for the sample evoo la shipped at higher temperature, this effect was not observed. figure 3. temperature profile monitored using data loggers for the los angeles simulation (in the world map: 1, genoa port; 2, panama canal ; 3, los angeles port). duration: 37 days, highest temperature: 58°c, lowest temperature: 11.5°c. ital. j. food sci., vol 29, 2017 47 3.3. influence of simulated shipment on physical and sensory properties color changes in evoo reflect the visual color appearance that is considered to be an important factor in consumer satisfaction (moyano et al., 2010). the color of olive oils, in general, is principally affected by two classes of minor compounds, namely chlorophylls and carotenoids. the degradation of these compounds is due to different conditions of stress, such as temperature and light, which may alter color in addition to clarity and transmittance (sikorska, et al., 2007). color indexes were expressed as chromatic coordinates: l* corresponds to brightness and positive b* to yellowish color, while negative a* corresponds to light green color (minguez-mosquera et al., 1991). as seen in table 3, there were significant changes in the brightness (l*) and b* indices for evoo samples sent to quebec after simulation in the standard and insulated containers (more bright and more yellowish). however, a reduction in l* values (meaning less bright oils) was seen in both shipping conditions for the simulated shipment to los angeles. a reduction was also observed for b* values (less yellow toward light blue) of samples shipped to los angeles, corresponding to the degradation of yellow chromophores (pigments), that function as natural antioxidants, such as carotenoids and pheophytins (psomiadou and tsimidou 2002), since oxidation is promoted by the increased temperature (morello et al., 2004) during the simulation to los angeles (fig. 3). as previously reported, degradation of natural pigments such as carotenoids occurs at around 40°c (thakkar et al., 2009). moreover, an increase in a* values (partial loss of green color toward redness) was recorded for samples sent to los angeles: such a partial loss of green color, in general, may correspond to partial degradation of chlorophylls, which are partially converted into other gray/brown compounds, and specifically to pyropheophytin a which is formed from pheophytin a due to degradation triggered by inadequate temperatures during the storage of oil (aparicio-ruiz et al., 2014). consequently, the increased degradation of chlorophyll and carotenoid pigments is likely related to the increased temperature (up to 58°c) in the final stages of the los angeles simulation (fig. 3). in addition, variations in water amount and turbidity were not significant (table 3) in either simulation. sensory analysis, realized according to the eu reg. 640/2008, is an essential technique for the assessment of the quality of evoo. the sensory evaluation (results not shown) indicated that no sensory defects developed after simulated shipment to quebec or los angeles, and all samples remained within the “extra virgin” category in both thermally insulated and standard containers. table 3. color coordinates (l*, a*, b*); td, turbidity (ntu); wa, water amount (mg kg-1 oil) before and after simulated shipping in an insulated and standard container for evoo samples to the two final destinations (evoo q, quebec and evoo la, los angeles). values (mean ± standard deviation) with different superscript capital letters in each column and for each sample were significantly different between the simulated shipping conditions (p < 0.05; fisher’s test). samples experimental conditions l* a* b* td (ntu) wa (mg kg-1 oil) evoo q before shipping 54b ± 0.1 4.9a ± 0.0 80b ± 0 11.7a ± 0.2 719a ± 98 insulated container 55a ± 0.1 4.8b ± 0.0 84a ± 0 11.3a ± 0.1 621a ± 6 standard container 55a ± 0.1 4.6c ± 0.0 84a ± 0 11.5a ± 0.2 708a ± 92 evoo la before shipping 63a ± 0.0 4.3b ± 0.1 89a ± 0 11.6a ± 0.2 650a ± 30 insulated container 50b ± 1.4 5.8a ± 0.2 71c ± 1 11.5a ± 0.2 607a ± 64 standard container 52b ± 1.5 5.5a ± 0.2 79b ± 2 11.4a ± 0.1 562a ± 72 ital. j. food sci., vol 29, 2017 48 4. conclusions it is important to point out that this study is related to two specific simulations, and thus the results cannot be generalized to all shipments of vegetable oils to los angeles or quebec. from parallel study of two simulated shipments to different destinations with different thermal conditions, it was found that thermal isolation is associated with significant benefits in terms of avoiding an increase in degradative reactions for edible oils, and especially on oxidative status. considering the different parameters evaluated, the quality of the edible oils subjected to the simulation to quebec was higher than those shipped to los angeles, which was due to the different thermal profiles of the two journeys. the aim of future studies is the adoption of a proposed ex-post simulation analysis on edible oils having different ages, shipped in different periods of the year and to different destinations, in agreement with specific logistic decisions (storage, material handling, transportation modes, etc.) and packaging solutions including primary, secondary, tertiary packaging, and containment equipment. acknowledgements the authors would like to thank enhancement of the palestinian university system (eplus) for phd scholarship grants financed by the italian ministry of foreign affairs-directorate general for cooperation and development (coordinated by the university of pavia). references accorsi r., manzini r. and ferrari e. 2014. a comparison of shipping containers from technical, economic and environmental perspectives. transport. res. d-tr e. 26: 52-59. accorsi, r., manzini, r., versari, l. 2015. glass vs. plastic: life cycle assessment of extra-virgin olive oil bottles across global supply chains. sustainability, a journal, v 7(3), 2818-2840, doi:10.3390/su7032818 aocs. 2006. thiobarbituric acid reactive substances. in official method and recommended practices of the aocs, (aocs) pp. 1-6, champaign, illinois, usa. aparicio-ruiz r., aparicio r. and garcía-gonzalez d.l. 2014. does “best before” date embody extra-virgin olive oil freshness?. j. agric. food chem. 62:554-556. bendini a., cerretani, l., carrasco-pancorbo a., gomez-caravaca a.m., segura-carretero a., fernández-gutiérrez a. and lercker g. 2007. phenolic molecules in virgin olive oils: a survey of their sensory properties, health effects, antioxidant activity and analytical methods. an overview of the last decade. molecules 12:1679-1719. bendini a., cerretani l., salvador m.d., fregapane g. and lercker g. 2004. stability of the sensory quality of virgin olive oil during storage: an overview. ital. j. food sci. 21:389-406. bmt survey. 2013. the world largest cargo information on line. http://www.cargohandbook.com/index.php/bulk_oils_and_fats. 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http://www.usda.gov/oce/commodity/wasde/latest.pdf, [accessed 5 may. 2014]. valli e., manzini r., accorsi r., bortolini m., gamberi m., bendini a., lercker g. and gallina-toschi t. 2013. quality at destination: simulating shipment of three bottled edible oils from italy to taiwan. riv. ital. sostanze grasse. 90:163-169. paper received march 23, 2016 accepted june 22, 2016 ijfs#836_bozza ital. j. food sci., vol. 30, 2018 50 paper effect of degree of rice milling on antioxidant components and capacities h-y. park1, j. sung2, b-s. kim3, s.k. ha1 and y. kim*1 1 division of functional food research, korea food research institute, gyeonggi 13539, republic of korea 2 division of food and animal sciences, chungbuk national university, chungbuk 28644, republic of korea 3 division of food safety, distribution and standard, korea food research institute, gyeonggi 13539, republic of korea *corresponding author. tel.: +82 317809347; fax: +82 317099876 e-mail address: kimyus@kfri.re.kr abstract de-husking and milling-induced changes in the content of antioxidant compounds and the antioxidant capacities of rice fractions were investigated in this study. six fractions rice husk, brown rice, and milled rice (mr) after four different degrees of milling (mr-3.5, mr-5.3, mr-7.1, and mr-9.9) were extracted with 70% aqueous ethanol or water. total phenolic and flavonoid contents decreased significantly (p < 0.05) as the degree of milling increased. rice husk and brown rice fractions showed higher 1,1-diphenyl-2picrylhydrazyl (dpph) and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (abts) radical scavenging activities than those of the mr, as well as higher levels of antioxidant components (total phenolics and total flavonoids). phytochemicals such as phenolic compounds and vitamin e are mainly concentrated in the outer layers of the grains rather than in the endosperm. these findings suggest that consuming rice milled to a lesser degree may have certain health benefits. keywords: rice, degree of milling, antioxidant, phenolics, vitamin e ital. j. food sci., vol. 30, 2018 51 1. introduction rice (oryza sativa) is one of the world’s most important cereal crops and a main staple food in korea and many other asian countries. as much as 75% of the daily caloric intake of the population of some asian countries is derived from rice (huang et al., 2015). the rice grain has a hard husk that protects the kernel within. brown rice is obtained by removing the husk but leaving the bran, germ, and endosperm. milled rice is produced by removing the bran layers of the rough rice kernel by milling (butsat and siriamornpun, 2010). although brown rice and under-milled rice are considered excellent sources of calories and nutrients such as vitamins and minerals (zhang et al., 2014), white rice is preferred by consumers because of its good eating quality. however, because of growing health consciousness, some consumers in asia have recently started consuming rice milled to a lesser degree or even brown rice. plant-derived phytochemicals, which are potential sources of natural antioxidants, may combat oxidative stress in the human body by maintaining the balance between oxidants and antioxidants (temple, 2000). many naturally occurring phytochemicals in plantderived products contain a complex mixture of phenolic compounds that can exert several biological effects including antioxidant activity (pandey and rizvi, 2009). it has been reported that phenolic compounds may function as free radical scavengers and quenchers of singlet oxygen. their antioxidant activities have been attributed to their redox properties (patel et al., 2011). antioxidant phytochemicals that quench free radicals may play a significant role in human health. a wide variety of biologically active phytochemicals are concentrated mainly in the pericarp and aleurone layers in cereal grains such as rice, wheat, and oats (butsat and siriamornpun, 2010; ha et al., 2006). several studies have demonstrated that the aleurone layer has high nutritive value and has beneficial health effects, such as decreasing the incidence of atherosclerotic disease (daniel et al., 1999; morton et al., 2000), lowering blood cholesterol (okarter and liu, 2010), and preventing cardiovascular disease (martinezvalverde et al., 2000). it has also been reported to have an anticancer effect (newmark, 1996). some of these protective effects may be attributed to polyphenols, which comprise several classes of flavonoids and vitamin e as well as other phenolic constituents and are present in the aleurone layer. however, dehusking and milling decreases the content of antioxidant compounds in the grain. thus, the degree of milling is an important factor in the nutritional value of milled rice. previous studies have reported the effect of milling on the physicochemical properties of rice and the cooking and textural properties of milled rice (singh et al., 2005; liu et al., 2015). several studies have also reported the content of antioxidant compounds and the antioxidant activity of white or black rice and the contents of various beneficial components (several phenolic compounds, tocopherols, tocotrienols, and γ-oryzanol) of rice bran or husks (adom and liu, 2002; zhang et al., 2010; butsat et al., 2009). however, limited information is available on the correlation between the antioxidant compound content and activity of rice husks, brown rice, and rice milled to different degrees. therefore, the aim of this study was to determine the antioxidant compound content and antioxidant activity of milled rice fractions and to evaluate husks as a source of natural antioxidants. ital. j. food sci., vol. 30, 2018 52 2. materials and methods 2.1. materials 1,1-diphenyl-2-picrylhydrazyl radical (dpph), the diammonium salt of 2,2-azino-bis-(3ethylbenzothiazoline-6-sulfonic acid) (abts), folin-ciocalteu’s (fc) phenol reagent, tocopherol (α-, β-, γ-, and δ-t), tocotrienol (α-, β-, γ-, and δ-t3), ascorbic acid, gallic acid, (+)-catechin, butylated hydroxytoluene (bht), potassium hydroxide, and sodium chloride were purchased from sigma-aldrich (st. louis, usa). all other chemicals and solvents used were of analytical or hplc-grade. 2.2. dehusking and milling of rice the paddy rice sample (o. sativa l. chucheongbyeo, short-grain rice) used in this study was obtained from the korea food research institute. the paddy sample was harvested in 2014. the rice variety used in the study is commercially available in korea. rice was threshed to separate the rice husk (rh) and brown rice (br) using an automatic rice husker (kett tr200, kokyo, japan). the brown rice samples were polished in a mcgrill no. 2 mill (ricepal32, yamamoto co., ltd., tendo, japan) to obtain rice grains milled to different degrees (3.5% [mr-3.5], 5.3% [mr-5.3], 7.1% [mr-7.1], and 9.9% [mr-9.9]). the degree of milling was determined using the following relationship: [1 − (1000 − kernel weight of milled rice)/(1000 − kernel weight of brown rice)] × 100. all fractions, except the bran, were stored in double-sealed polythene bags at -25°c prior to analysis. 2.3. sample preparation all fractions were ground using a blender. samples of approximately 200 g were extracted using 1 l of 70% aqueous ethanol or distilled water in a shaker (jssi-100t, js research inc., gongju, korea) at 25±3°c for 24 h. the extracts were filtered using whatman no. 2 filter paper, and the residues were discarded. the filtrate was concentrated at 40°c using a vacuum rotary evaporator (r-205, büchi, flawil, switzerland), lyophilized, and then stored at -20°c until further analysis. 2.4. determination of total phenolic and flavonoid contents the concentrations of total phenolic and flavonoid compounds in the extracts were measured spectrophotometrically. total phenolic content (tpc) was determined using the fc-method (dewanto et al., 2002) with some modifications. standard solution or sample extracts were mixed with 2 ml of 2% sodium carbonate solution and 100 µl of 1 n folin-ciocalteu reagent. after incubating for 30 min at 25±3°c, the absorbance at 750 nm was measured using a spectrophotometer. the results are expressed as mg of gallic acid equivalents per 100 g of sample (the equation of the standard curve was y = 0.0024x – 0.0897, r2 = 0.998). total flavonoid content (tfc) in the samples was determined by a colourimetric method (tian et al., 2011). briefly, either standard solution or sample extracts were mixed with 1.25 ml of distilled water and 75 μl of 5% nano2. after incubating for 30 min at 25±3°c, 150 μl of 10% alcl3·6h2o was added, and the mixture was allowed to stand for 5 min before the addition of 0.5 ml of 1 m naoh. the absorbance at 510 nm was then measured. the results are expressed as (+)-catechin equivalents per 100 g of sample (the equation of the standard curve was y = 0.0024x +0.0275, r2 = 0.998). ital. j. food sci., vol. 30, 2018 53 2.5. determination of the vitamin e composition total vitamin e content and the content of each of its isomers in the samples were determined using hplc with a fluorescence detector (wie et al., 2009). a 10 ml aliquot of ethanol containing pyrogallol (6%, w/v) was added to 1 g of sample in a saponification vessel. after sonication for 5 min, 5 ml of 60% potassium hydroxide was added, and the vessel was flushed with nitrogen gas for 1 min. an air condenser was attached to the vessel, and the contents were digested at 70°c for 50 min in a shaking water bath. the contents were cooled in an ice bath, 20 ml of 2% sodium chloride was added, and the mixture was extracted three times with 20 ml of hexane/ethyl acetate (85:15, v/v) containing 0.001% bht. the extracts were pooled and diluted to 50 ml and filtered through a 0.45 μm nylon membrane filter. an aliquot of the filtered extract was analysed using a normal-phase hplc system (pu-1580; jasco, tokyo, japan). analysis of tocopherol and tocotrienol isomers was performed on a lichrospher® diol column (250 × 4 mm, 5 μm; merck, darmstadt, germany) using a mobile phase of hexane/isopropanol (98.7:1.3, v/v) at a flow rate of 1.0 ml/min. peaks were detected by fluorescence (fp-1520 l jasco) using an excitation wavelength of 290 nm and an emission wavelength of 330 nm. tocopherol and tocotrienol peaks were identified by comparing their retention times to those of standards (the standard curve equations and r2 values were α-t, y = 0.000001x + 0.021928, r2 = 0.998; α-t3, y = 0.000001x + 0.018294, r2 = 0.997; β-t, y = 0.000001x + 0.005295, r2 = 0.997; γ-t, y = 0.000001x + 0.024276, r2 = 0.998; γ-t3, y = 0.000001x – 0.000236, r2 = 0.998; δt, y = 0.0000005x + 0.0036275, r2 = 0.998; δ-t3, y = 0.0000003 + 0.0110694, r2 = 0.999). all analyses, except for that of vitamin e, were conducted in triplicate. 2.6. determination of dpph radical scavenging activity dpph radical scavenging activity was assayed by the method described by choi and lee (2009) with some modifications. briefly, 0.1 ml of 0.2 mm dpph radical solution (1 ml) was added to 20 µl of sample extract or a standard solution of ascorbic acid, and the mixture was allowed to stand for 30 min. the absorbance of the mixture at 520 nm was measured against a blank of distilled water and an ascorbic acid standard calibration curve was constructed. the dpph radical scavenging activity was expressed as ascorbic acid equivalent antioxidant activity (aeac) and defined as mg of ascorbic acid equivalents per 100 g of sample (the equation of the standard curve was y = -0.0268x + 1.4586, r2 = 0.991). 2.7. determination of abts radical scavenging activity total antioxidant capacity of each sample extract was determined with an improved cationic abts radical method using a spectrophotometer (gong et al., 2013). the abts radical was generated by adding 7 mm abts to a 2.45 mm potassium persulfate solution and letting the mixture stand overnight in the dark at 25±3 °c. the solution of positively charged abts radicals was diluted with distilled water to obtain an absorbance of 1.0 at 734 nm. diluted atbs radical solution (1 ml) was then added to 50 of μl sample extract or ascorbic acid standard solution. after 90 min, the absorbance of the mixture was measured at 734 nm. the antioxidant activity of the extracts was expressed as mg of aeac in 100 g of sample (the equation of the standard curve was y = -0.0055x + 1.1115, r2 = 0.995). all analyses were performed in triplicate. ital. j. food sci., vol. 30, 2018 54 2.8. statistical analysis data from the analyses of each of the samples in triplicate were reported as the mean±sd. different samples were compared using one-way analysis of variance using sas, version 8.1 (sas institute, cary, usa). a value of p < 0.05 was considered to be statistically significant. 3. results and discussions antioxidants can be classified into two groups according to their solubility; hydrophilic antioxidants, such as phenolic and flavonoid compounds, and lipophilic antioxidants (fatsoluble), such as vitamin e (dhibi et al., 2012). these compounds exhibit antioxidant properties in many in vitro model systems and have the potential to reduce the risk of chronic diseases associated with oxidative stress (liu, 2003). 3.1. total phenolic content phenols are one of the most effective antioxidative components in plant-derived foods, including fruits, vegetables, and grains (choi and lee, 2009). these compounds are effective antioxidants because of their ability to donate a hydrogen atom or an electron to form stable radical intermediates. therefore, it is important to quantify the tpcs to determine their contribution to the antioxidant activities of the test samples. the results of tpc analysis are expressed as mg of gallic acid equivalent/100 g of sample (table 1). table 1. total phenolic and flavonoid contents of milled fractions obtained from paddy rice. means with different letters in a column are significantly different (p < 0.05). solvent rice fraction total phenolic content 1) total flavonoid content 2) 70% ethanol rh 166.02±4.48a3) 18.42±0.84a br 45.94±0.51b 9.53±0.91b mr-3.5 20.23±0.13c 5.84±0.34c mr-5.3 16.94±0.14c 4.84±0.20d mr-7.1 10.61±0.11d 2.77±0.33e mr-9.9 7.64±0.08d 1.57±0.05f water rh 32.39±1.95a 5.71±0.23a br 26.15±0.26b 5.44±0.54a mr-3.5 17.61±1.06c 4.77±0.42b mr-5.3 10.42±0.21d 2.67±0.29c mr-7.1 10.38±0.07d 2.45±0.10cd mr-9.9 6.69±0.15e 1.89±0.04d 1) means±standard deviation (sd) for triplicate determinations expressed as mg of gallic acid equivalents/100 g of sample. 2) means±sd for triplicate determinations expressed as mg of catechin equivalents/100 g of sample. ital. j. food sci., vol. 30, 2018 55 tpcs in the 70% ethanol and water extracts were 7.64-166.02 and 6.69-32.39 mg/100 g of sample, respectively. the determination of tpc was affected by the extraction solvent; 70% ethanol was more effective than water. solvent extraction is frequently used to isolate antioxidants, and both the yield and antioxidant activity of the extracts are strongly dependent on the solvent; this is because compounds with different polarities often have different antioxidant potentials (marinova and yanishlieva, 1997). many studies have indicated that using 70% ethanol in water as the extraction solvent affords significantly higher quantities of phenolic compounds than other solvents, such as methanol and water (ajila et al., 2011). the ethanol extracts show the highest antioxidant activities, which is consistent with our results. ethanol-water extraction systems were used in the present study since they are the most widely employed solvents for reasons of chemical hygiene and ease of availability. more importantly, these solvents are compatible with the production of food-grade materials (soong and barlow, 2004). tpcs in the rice fractions for all samples were found to be in the following order: rh > br > mr-3.5 > mr-5.3 > mr-7.1 > mr-9.9. the ethanol extract of the husk fraction exhibited the highest tpc (166.02±4.48 mg/100 g of sample), and the tpc decreased significantly (p < 0.01) as the degree of milling increased. phenolic compounds are located predominantly in the bran layer, which is progressively removed during the milling process (butsat et al., 2009). rice husk and rice bran are also rich in tpc (vijayalaxmi et al., 2015). the outer layers of the cereal grains, such as husk, pericarp, testa and aleurone cells, contain the highest concentrations of tpc, whereas its concentration is considerably lower in the endosperm (kahkonen et al., 1999). the effect of dehusking and milling on tpc could be due to the variable distribution of phenolic compounds in the husk and bran. 3.2. total flavonoid content flavonoids are a group of phenolic compounds that contain two aromatic rings linked by three carbons that are usually part of an oxygenated heterocycle. these compounds have potent antioxidant and anticancer activities (shen et al., 2009). the tfc, expressed as mg of catechin equivalents per 100 g of sample, was lower than the tpc in all the samples tested (table 1). the tfcs in the milled fractions were 1.57-18.42 and 1.89-5.71 mg/100 g of sample in the 70% ethanol and water extracts, respectively. rh had the highest tfc among the six rice fractions, followed by br, mr-3.5, mr-5.3, mr-7.1, and mr-9.9 in that order. the order of abundance of tfc was similar to that of tpc in all samples. this result indicates that the concentration of flavonoids increases from the endosperm to the aleurone layer. 3.3. total content of vitamin e and its isomers vitamin e is another antioxidant present in grains that protects polyunsaturated fatty acids in cell membranes from oxidative damage (slavin et al., 1999). the antioxidant activity of the tocopherols and tocotrienols (collectively known as chromanols) is mainly due to their ability to donate their phenolic hydrogens to lipid free radicals. vitamin e is synthesized only by plants. therefore, it is a vital nutrient for humans and animals that can be obtained only from dietary sources (kahkonen et al., 1999). the individual concentrations of eight vitamin e isomers (α, β, γ, and δ-t and α, β, γ, and δ-t3) and their total contents in different parts of the rice grain are presented in table 2. four tocopherol isomers, α, β, γ, and δ-tocopherol, and three tocotrienol isomers, α, γ, and δ-tocotrienol, were identified. β-tocotrienol was not detected. ital. j. food sci., vol. 30, 2018 56 table 2. composition of eight vitamin e isomers in the milled fractions obtained from paddy rice. means with different letters in the columns for each fraction are significantly different (p < 0.05). rice fractions tocopherol (t) tocotrienol (t3) total vitamin e α-t β-t γ-t δ-t α-t3 β-t3 γ-t3 δ-t3 rh 1.15±0.05b 0.04±0.00bc 0.18±0.00c 0.51±0.00c 0.35±0.01b nd 0.51±0.00c 0.02±0.00d 2.27±0.08c br 1.25±0.09b 0.04±0.01b 0.21±0.01b 1.19±0.02bc 0.76±0.05a nd 1.19±0.02a 0.05±0.00a 3.53±0.15b mr-3.5 1.61±0.03a 0.06±0.00a 0.28±0.01a 1.23±0.05a 0.70±0.04a nd 1.23±0.05a 0.05±0.00a 3.98±0.11a mr-5.3 0.88±0.04c 0.03±0.00c 0.16±0.02c 0.66±0.03bc 0.32±0.01b nd 0.66±0.03b 0.04±0.00b 2.12±0.11cd mr-7.1 0.72±0.14c 0.02±0.00d 0.13±0.02d 0.56±0.06bc 0.26±0.04b nd 0.56±0.06bc 0.03±0.00bc 1.74±0.26d mr-9.9 0.30±0.06d 0.01±0.00e 0.06±0.00e 0.47±0.10b 0.15±0.03c nd 0.47±0.10c 0.03±0.00c 1.06±0.20e means±standard deviation (sd) of duplicates (mg/100 g of sample). nd, not detected. the vitamin e level was 0.86-4.09 mg/100 g of sample in all the extracts of the six fractions. mr-3.5 was the best source of vitamin e (3.98±0.11 mg/100 g), followed by br (3.53±0.15 mg/100 g), rb (2.27±0.08 mg/100 g), mr-5.3(2.12±0.11 mg/100 g), mr7.1(1.74±0.26 mg/100 g), and finally mr-9.9 (1.06±0.20 mg/100 g). tocopherols are known to be the principal antioxidants present in rice bran. the average content of total vitamin e in br was higher than that in rh (huang and ng, 2011). the levels of vitamin e detected in the br and rh fractions in our study were similar to previously reported values of 1.04-3.25 mg/100 g of sample (kahkonen et al., 1999), 3.62–3.82 mg/100 g of sample and 1.81-4.08 mg/100 g of br, and 0.41–5.50 µg/100 g of rh (soong and barlow, 2004; okarter and liu, 2010). the major forms of vitamin e were α-t and γt3, whereas β-t and δ-t3 were present in trace amounts and β-t3 was not detected. these results were in agreement with the finding that the major isomers of vitamin e in rice samples were γ-t3 and α-t, whereas the δ-t and β-t3 contents were the lowest (ha et al., 2006). similar to the present study, previous studies have shown that vitamin e was more concentrated in regions close to the bran and husk layers than in the endosperm (soong and barlow, 2004). the removal of the husk, the aleurone layer, and the germ during milling reduces the vitamin e content of the final milled products (okarter and liu, 2010). lipid fractions are distributed mainly in the outer layers of rice grains (adom and liu, 2002). these findings are in good agreement with our data in which the vitamin e content decreased as the degree of milling increased. therefore, our results indicate that antioxidant components comprising total phenolics, total flavonoids, and vitamin e are primarily concentrated in the outer layers rather than in the endosperm. 3.4. dpph radical scavenging activity the dpph and abts radical scavenging activities are related to the nature of the phenolic compounds as that affects their electron transfer/hydrogen donating ability (wettasinghe and shahidi, 2000; choi et al., 2007). the increase in free radicals can accelerate the oxidation of foods and decrease their quality. the radical scavenging activity is very important due to the deleterious role of free radicals in foods and in biological systems. therefore, this study investigated the free radical scavenging activity of the 70% ethanol and water extracts of different rice fractions using dpph and abts ital. j. food sci., vol. 30, 2018 57 assays. both these radicals are commonly used for in vitro assessment of antioxidant activity. the dpph radical is stable and is widely used to evaluate the free radical scavenging activities of many plant extracts. the capacity of the six rice fractions to act as hydrogen donors in the transformation of the dpph radical to its reduced form was examined. aeac values are often used to rank the antioxidant activity of unknown mixtures (kong and lee, 2010). the aeac of a sample determines its antioxidant activity relative to that of ascorbic acid. the dpph radical scavenging activities of the milled rice fractions, expressed as mg of aeac per 100 g, are shown in fig. 1. the colour of the dpph reagent changes from purple to yellow as a result of the antioxidant activity of the test sample. similar to the antioxidant content, the free radical scavenging activity was affected by the extraction solvent; 70% ethanol was more effective than water. the dpph radical scavenging activities of the extracts of the six samples were in the order rh > br > mr-3.5 > mr-5.3 > mr-7.1 > mr-9.9 for both solvents. the results show that the antioxidant capacity of all extracts decreased during the milling process. the high tpc and tfc in the husks may account for the strong dpph radical scavenging activity. this finding is consistent with the finding of a previous study that found that the antioxidant activity was dependent on the composition of the milled fractions (shen et al., 2009; butsat and siriamornpun, 2010). the dpph radical scavenging activity correlated directly with the tpc and tfc in the extracts of the six samples with correlation coefficients (r2) of 0.880 and 0.942, respectively. however, there was no correlation between dpph radical scavenging activity and total vitamin e content (r2 = 0.274). thus, there is a strong association between the tpc and tfc of different milled fractions and their respective dpph radical scavenging activities. rh br mr-3.5 mr-5.3 mr-7.1 mr-9.9 d p p h r ad ic al s ca ve ng in g ac tiv ity (m g a e a c /1 00 g o f s am pl e) 0 10 20 30 40 50 70% ethanol water a b c d e f a' a' b' c' c' c' figure 1. dpph radical scavenging activity of milled fractions obtained from paddy rice. data are expressed as the mean±sd of the results for the six rice fractions. a–f, a’–c’ means with different letters are significantly different (p < 0.05) by one-way analysis of variance. ital. j. food sci., vol. 30, 2018 58 3.5. abts radical scavenging activity total antioxidant content (tac) was measured using the abts assay. the abts radical scavenging test is widely used to determine the antioxidant activity of both hydrophilic and lipophilic compounds and to measure the relative radical scavenging activity of hydrogen-donating and chain-breaking antioxidants in many plant extracts (apak et al., 2007). the cationic abts radical assay can be used over a wide ph range, is inexpensive, and is more rapid than the dpph radical assay (slavin et al., 1999). fig. 2 shows the antioxidant activities of the extracts expressed as mg of aeac per 100 g of sample. the extraction solvent influenced the antioxidant activity of the extracts of all the fractions. of the two methods used in this study, extraction with 70% ethanol afforded higher antioxidant activities. tac varied among the different fractions obtained from different degrees of milling. the tac of 70% ethanol and water extracts ranged from 5.20-48.74 and 1.88-4.13 mg of aeac per 100 g sample, respectively. the tac of the samples tested in decreasing order was rh > br > mr-3.5 > mr-5.3 > mr-7.1 > mr-9.9. thus, the tac in rice decreased significantly (p < 0.01) as the degree of milling increased. many bioactive compounds including phenolic antioxidants, tocopherol, and tocotrienol were present in higher quantities in the removed husk and bran than in the remaining endosperm (iqbal et al., 2005; ha et al., 2006). the highest tac was detected in rh, which was consistent with the results of the analyses of tpc, tfc, vitamin e and dpph radical scavenging ability. the lowest tac was observed for mr-9.9, which exhibited markedly lower tpc, tfc, and vitamin e levels, and lower dpph radical scavenging capacity than those of the other samples. there was a positive correlation (r2 = 0.996) between abts and dpph radical cation scavenging activities. the decrease in tac might have been the result of decreased levels of antioxidant compounds. tac was positively correlated with tpc and tfc. the correlation coefficients (r2) between the abts assay and tpc and between the abts assay and tfc were 0.800 and 0.873, respectively. there was no correlation between the vitamin e and tac. these results confirm that the phenolic and flavonoid compounds may be the major contributors to the antioxidant activity of the grains, which is consistent with the findings of earlier studies (butsat and siriamornpun, 2010; adom and liu, 2002). rh br mr-3.5 mr-5.3 mr-7.1 mr-9.9 a b t s r ad ic al s ca rv en gi ng a ct iv ity (m g a e a c /1 00 g o f s am pl e) 0 10 20 30 40 50 60 70% ethanol water a b c d e fa' a' a' ab' ab' b' figure 2. abts radical scavenging activity of milled fractions obtained from paddy rice. data are expressed as the mean±sd of the results for the six rice fractions. a–f, a’–b’ means with different letters are significantly different (p < 0.05) by one-way analysis of variance. ital. j. food sci., vol. 30, 2018 59 4. conclusions this study shows that the antioxidant strength of rice depends on the degree of milling. br contains more components that have health benefits than mr, such as polyphenolics, flavonoids, and isomers of vitamin e. these results demonstrate that brown rice is a good dietary source of antioxidants and has health benefits. thus, it is important to modulate the milling process to preserve bioactive compounds. acknowledgements this study was supported by a research grant from korea food research institute. references adom k.k. and liu r.h. 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palermo, italy 2food processing department, research institute of food science and technology (rifst), mashhad, iran 3department of food science and agricultural chemistry, macdonald campus, mcgill university, ste anne de bellevue, quebec, h9x 3v9, canada 4department of food science and technology, gorgan university of agricultural sciences and natural resources, gorgan, iran *corresponding author. tel. +39 3928427272 e-mail address: mansour.rabieashkezary@unipa.it abstract the purpose of this study was to investigate the possibility of producing reduced fat dark compound chocolate in the ball mill refiner and using some selected emulsifiers. the effects of selected emulsifiers including lecithin, polyglycerol polyricinoleate (pgpr) and citrem in two levels and two refining times on the characteristics such as moisture, particle size, hardness and rheological properties of the samples was examined. data analysis revealed that the casson model was appropriate to describe the rheological behavior of the samples containing lecithin and citrem; however, power law model was appropriate for the samples containing pgpr. the results showed that citrem is the most effective emulsifier to reduce hardness and rheological parameters such as apparent viscosity; casson viscosity and casson yield value and using citrem as a part of formulation in the production of reduced fat dark compound chocolate can solve many technological problems. keywords: citric acid ester, casson model, power law, pgpr, lecithin, reduced fat compound chocolate ital. j. food sci., vol. 30, 2018 27 1. introduction production of chocolate and chocolate products in ball mill refiner is currently spreading worldwide to due to lower costs and easier operational systems. the increase of diseases caused by dietary misbehaviors in industrialized countries, leads to larger knowledge for nutritional requirements by the consumer and, therefore, by food industry (dias et al. 2015). as a result, the possibility of developing a formulation for chocolate models by reducing its fat content while maintaining its richness and flavor will give the consumer a new and healthy food option to enjoy. chocolate is a fat-based suspension with about 30%wt fat. reducing fat content causes an increase in hardness and molten chocolate viscosity that leads to difficulties in the process and a loss of eating quality in the final product. there are, however, some technical issues that must be scrutinized to achieve successful ball mill processing. generally, several methods are introduced to reduce the fat content of chocolate with acceptable viscosity and hardness such as increasing the emulsifier levels and/or using emulsifier blends (kaiser et al., 1998), using fat replacers (beckett, 2009), and substituting fat phase with a water-in-oil emulsion (hugelshofer, 2000). optimizing the particle size distribution is another method of decreasing the fat content (mongia and ziegler, 2000; do et al., 2007). the optimization of the particle size distribution (psd) method has a significant effect on the rheological and textural properties of chocolate samples such as reduction of the apparent viscosity, decrease of hardness and an increase of melting rate in the mouth (mongia and ziegler, 2000). the non-newtonian flow behavior of molten chocolate is generally studied by some well-known models for shear thinning fluids such as power law, bingham, herschel-bulkley and casson (sokmen and gunes, 2006). in terms of utilization of the latter model, comparing the rheological methods proposed by international confectionary association (ica, 2000) and chocolate manufacturers association (cma, 1997) revealed a high correlation between: і) casson plastic viscosity and apparent viscosity; іі) between casson yield value and yield stress; ііі) casson plastic viscosity and casson yield value and іv) yield stress and apparent viscosity (afoakwa et al., 2009). in order to have a quality product, investigating the changes which occur in the product matrix at every manufacturing stage could be very useful (glicerina et al. 2013). structurally, chocolate is made from fat phase (cocoa butter and emulsifier), in which solid material (crystal sugar, milk powder and cocoa powder) are spread (beckett, 2000). the composition of chocolates in terms of fat and nonfat cocoa solids, and sugar content affect their rheological characteristics (fernandes et al., 2013). in addition to cocoa butter, emulsifier also forms one of the constituents of chocolate fat phase. in the chocolate matrix, emulsifiers cover sugar particles to develop the flow in cocoa butter. this assists in the equal distribution of particles in emulsion and prevents agglomeration. some emulsifiers decrease viscosity and yield stress significantly, so they will be very useful additives in production of chocolates with reduced fat. lecithin and pgpr are emulsifiers usually used in chocolate (schantz and rhom, 2005). both lecithin and pgpr work synergistically with other emulsifiers, such as ammonium phosphatide and citric acid esters (stier, 2009). citric acid ester has the attributes of the lecithin and pgpr combination (beckett, 2009). emulsifiers have ability of changing viscosity in certain foods (walter and cornillon, 2001). this feature is extremely important in producing chocolate, for example in chocolate coating, pumping and molding, etc. (rector, 2000). emulsifiers have been used in chocolate to modify and improve the flow characteristics of chocolate since chocolate was first processed. however, the most important of emulsifier applications in chocolate industry is improving flow parameters and minimizing consumption of cocoa butter and its costs of production (schantz and rhom, 2005). achieving desirable functional properties is not only ital. j. food sci., vol. 30, 2018 28 related to providing a basic level of knowledge about ingredients, but also understanding each ingredient’s effect in the combinational form will help the manufacturers to satisfy the consumers expectations (manzocco et al., 2014). the purpose of this study was to investigate the possibility of producing reduced fat compound chocolate in ball mill refiner and using some selected emulsifiers in the manufacturing process including lecithin, polyglycerol polyricinoleate and citrem (citric acid ester). 2. material and methods 2.1. materials cocoa powder (guan chong cocoa manufacture sdn bhd, malaysia), refined sugar, cocoa butter substitute (cbs) (cargill, malaysia), lecithin, polyglycerol polyricinoleate (pgpr) and citrem (palsgard, juelsminde, denmark). 2.1.1 preparation of compound chocolate samples the basic formulation of the dark compound chocolate contained 46.5% cocoa powder, 30% sugar, 23% cbs, and 0.5% lecithin. the method for producing compound chocolate was as follows: first, all raw materials, including cocoa powder, refined sugar, palm kernel oil and lecithin, were weighed and poured into semi-industrial ball mill device (sepehr machine company, tehran, iran). eventually, fourteen formulas were produced (twelve formula in addition to two basic formula with 60 and 90 refining times as the control samples). mixing, refining (in two groups, one for 60 and the other one for 90 minutes) and conching were done simultaneously in this device for 30 min at 60 ℃ and speed of 100 rpm. each sample was then divided into seven portions. afterward, emulsifiers were added to the samples (lecithin and citrem at two levels of 0.5 and 1 % and pgpr at two levels of 0.25 and 0.5%). the conching process was performed (heidolph mixer) at a speed of 60 rpm for 30 minutes. next, the mixture was refrigerated at 4°c for 30 min in silicon containers. finally the samples were kept in aluminum foils and stored at room temperature for analysis. 2.1.2 moisture content measurements the moisture content of chocolate samples was determined using oven method (ioccc, 1952). 2.1.3 particle size distribution measurements particle size distribution was determined through laser diffraction method by particle analysis machine (shimadzu sald-2101), according to mcfarlane (1999). before analysis, the compound chocolate samples dissolved in acetone solvent and stirred vigorously under ultrasonic waves of 50 hz, 200 w for 5 minutes. low-intensity ultrasound produced optimal component emission. after the initial preparation, samples were transferred to the laser chamber. results obtained from the laser chamber of the parameters of the largest particle size (d90), the mean particle volume (d50) and the smallest particle size (d10) in micrometer scale were determined (alamprese et al., 2007) with three replicates. ital. j. food sci., vol. 30, 2018 29 2.1.4 hardness measurements hardness of samples was measured using a texture analyzer (ta-xt plus, stable micro systems ltd, surrey, uk), connected to the computer with the software texture expert 1.05. the flow bottom steel probe with 2 mm diameter was utilized for measurements. the maximum force of penetration to samples (45×20×10 mm) was determined with a depth of 5 mm at a speed of 1 mm/s at room temperature. loading force was set to 0.05 n direction of the sample, and kept constant for all samples. hardness was taken as the maximum peak force in newton. results for hardness are expressed as the mean value of three replicates conducted on each sample. 2.1.5 rheological measurements samples were prepared according to the proposed methods of the international confectionery association (ica, 2000); the compound chocolate sample was and melted in an incubator at a temperature of 50°c for 75 minutes and then, transferred to the rheometer cub. after a pre-shear period of 15 min at 5/s, shear rate was applied from 5 to 50 (ramp up) within 120 s and then shear rate was reduced from 50 to 5 (ramp down), and in each ramp 50 measurements were taken. the temperature was kept constant at 40℃. an anton paar rheometer (rheolabqc sn80677512, austria) was used for all rheological measurements and the data were collected by use of the rheoplus/32 service v3.10 software. the apparent viscosity of the samples was measured at 40/s and results are reported as the mean value of two replicates. servais et al. (2003) reported that the apparent viscosity can be measured at 30, 40 or 50/s depending on the type of product, but recommended the measurement at 40/s for the chocolate regarding to its repeatability. in this study, a locally designed model for analysis of flow time independent characteristics was utilized to analyze the flow properties of compound chocolate. due to the decrease in viscosity by increasing the shear rate for all rheological behavior applied and nonnewtonian actions of compound chocolate samples, 4 non-newtonian models (dependent on shear rate) were fitted on the test data (shear stress – shear rate). these four models include (should be in the sequence that is in the table 2): power law (𝜏 = 𝑘(�̇�)()، bingham (𝜏 − 𝜏* = 𝜂,-�̇�), herschel-bulkley (𝜏 − 𝜏* = 𝜂,-(�̇�)() and casson (𝜏*./ = 𝜏* + (𝜂,-)*./.(�̇�)*./); where, τ is shear stress, τ0 is yield stress, ηpl is plastic viscosity, γ˙ is shear rate, n is flow behavior index and k is consistency index. molten chocolate is a non-newtonian fluid with a yield stress, which can be characterized using a number of mathematical models, including the bingham, herschel-bulkley and casson models (ica, 2000; servais et al., 2003; konar, 2013). to select the best model for describing time-independent rheological behavior of compound chocolate samples, three statistical parameters of correlation coefficient (r), root mean square error (rmse) and standard error (se), were utilized. 2.1.6 statistical analysis the spss version 21, curve expert softwares and analysis of variance (anova) were used for statistical analysis of experimental data. due to unequal levels of used emulsifiers in the formulae, the significance of difference among samples was examined by nested following duncan’s multiple range tests for mean comparisons. ital. j. food sci., vol. 30, 2018 30 3. results and discussions 3.1. moisture content moisture content of all samples ranged from 0.39 to 0.52. moisture contents of all samples were within an acceptable range for chocolate (below 1.5 percent). afoakwa et al. (2007) reported that a moisture content of the chocolate samples over 1.5 percent would have a negative impact on the rheological properties. 3.2. particle size distribution results for the d90, d50 and d10 of the samples are shown in table1. since increase in emulsifier level and conching did not lead in change of particle size, only base formula was studied. the mean particle size in the d90, d50, and d10 was 10%, 50% and 90%; the particles were finer than this size, respectively. in this study, as expected, by increasing the refining time, all parameters in the particle size distribution were reduced. observations in this study determined the particle size of both samples to be below 30 μm. beckett (2009) reported that the size of the largest particle is a key parameter for chocolate production and plays a critical role in the hardness, sensory properties, and other properties of chocolates. the largest particle size (d90) plays an important role in the creation of grittiness and mouth feel, however smaller particles affect the flow properties (beckett, 2000; mongia and ziegler, 2000). particle size in chocolate roughly ranges between 1 and 50 μm, whereby particles larger than 30 μm cause a gritty perception in the mouth. kruger (1999) reported that minimum d90 size for optimal rheological properties was 6 μm. however, in this study, the minimum size of d90 in both of samples was greater than 6 μm. particle size and flow properties of chocolate are very important factors in determining the viscosity and also texture of final product (minifie, 2012). table 1. d90, d50, and d10 values in control and basic formulae. sample d90 (μm) d50 (μm) d10 (μm) chb1 26.87±0.59a 7.67±0.04a 1.71±0.04a chb2 22.21±0.34b 6.91±0.05b 1.59±0.03b (chb: basic formula, 1: refining time in 60 min, 2: refining time in 90 min). 3.3. hardness hardness of samples ranged from 32.09 to 53.25 n. as expected, hardness decreased by increasing the levels of emulsifiers in the samples (fig. 1). hardness showed inverse relationships with ps, fat and lecithin contents specially in low fat (25%) chocolate samples (afoakwa, 2009). at both 60 and 90 minutes of refining time, citrem 1% was the softest and the pgpr 0.25% was the hardest sample (fig. 1). there was no significant difference between samples containing 0.25% pgpr at 60 min refining time and 0.5% pgpr and citrem at 90 min refining time (p<0.05) and also the results showed that, there was no significant differences between samples containing 0.5% pgpr, 0.5% and 1% lecithin at the first refining time (p<0.05). previously, tisoncik (2010) claimed that increasing concentrations of lecithin and pgpr led in decrease of hardness characteristics of dark chocolate. by increasing the refining time from 60 to 90 minutes and reducing the particle ital. j. food sci., vol. 30, 2018 31 size from 26.87 μm to 22.211 μm, the hardness of the samples containing lecithin, pgpr and citrem increased due to the interaction between the particles of the compound chocolate. reducing the particle size leads to increase in resistance of chocolates to break and gives a harder texture (afoakwa et al., 2009). in similar results, afoakwa et al. (2008) reported that by reducing the particle size from 50 microns to 18 microns, the hardness of chocolate samples increased. do et al. (2007) concluded that by selecting a specific range of particle sizes, hardness of chocolate samples could be reduced and controlled. beckett (2009) reported different factors like formulation, production method, tempering, polymorphism and cooling temperatures determine the hardness of the chocolate samples. in this study no tempering was required since cbs had been used. in addition, the cooling temperature and production method of all samples were the same. so it can be concluded that desired hardness was achieved by changing the emulsifier or by combination of emulsifiers. there is direct correlation between sensory properties during consumption and hardness; therefore, measuring the hardness parameter is an important indicator for assessing qualitative changes of chocolates with different formulations. figure 1. hardness of the all samples with different emulsifiers at two refining times. different letters indicate statistically significant differences (p<0.05). 3.4. rheological parameters 3.4.1 evaluation of fitted models table 2 illustrates the results of the three statistical parameters of r, rmse and se. by fitting the data of the shear rate shear stress on the four rheological models of power law, bingham, herschel-bulkley and casson, the casson model showed the highest r, low rmse and the lowest se. therefore it was the best model to analyze the samples containing lecithin and citrem. the ica (2000) has proposed casson model as an appropriate model to analyze the rheological properties of chocolates. in the samples containing pgpr, due to a low yield stress and close to zero, a negative intercept was obtained in the casson, bingham and herschel-bulkley models. therefore, the casson model could not be used for analysis, while the power law model was successful in analyzing the samples containing pgpr. b c e e e d f a b c cd d c ef 0 10 20 30 40 50 60 0.25 0.5 0.5 1 0.5 1 base pgpr lecitin citrem h ar dn es s (n ) emulsifier (%) 60 min ital. j. food sci., vol. 30, 2018 32 table 2. the measured values of the three statistical parameters of r, rmse and se. se rmse r model sample 18.11 4.263 0.996 power law 1bch 30.57 4.206 0.989 bingham 12.97 3.602 0.998 herschel-bulkley 0.48 3.575 0.993 casson 10.49 3.627 0.999 power law 2bch 25.37 2.854 0.995 bingham 14.63 3.754 0.998 herschel-bulkley 0.29 2.105 0.998 casson 2.53 3.672 0.999 power law 11lch 14.03 1.706 0.998 bingham 4.24 2.961 0.999 herschel-bulkley 0.13 0.894 0.999 casson 8.66 2.333 0.999 power law 12lch 11.46 3.216 0.993 bingham 7.93 2.143 0.999 herschel-bulkley 0.27 2.274 0.999 casson 4.28 1.575 0.999 power law 21lch 11.13 2.057 0.999 bingham 8.60 0.998 0.999 herschel-bulkley 0.19 1.072 0.999 casson 3.60 3.015 0.999 power law 22lch 5.66 2. 706 0.994 bingham 3.43 0.589 0.999 herschel-bulkley 0.23 1.421 0.998 casson 1.96 3.787 0.999 power law 11pch bingham herschel-bulkley casson 7.63 1.673 0.999 power law 12pch bingham herschel-bulkley casson 6.54 3.212 0.999 power law chp21 bingham herschel-bulkley casson 9.18 2.229 0.999 power law chp22 bingham herschel-bulkley casson 0.11 1.050 0.999 casson 5.72 1.469 0.999 power law chc11 2.83 0.464 0.999 bingham 1.29 0.712 0.999 herschel-bulkley ital. j. food sci., vol. 30, 2018 33 0.06 0.420 0.999 casson 14.70 2. 370 0.995 power law chc12 27.60 4.451 0.985 bingham 12.68 3.303 0.997 herschel-bulkley 0.40 3.497 0.993 casson 3.33 0.812 0.999 power law chc21 6.03 1.169 0.999 bingham 1.53 1.004 0.999 herschel-bulkley 0.04 0.410 0.999 casson 5.36 2.194 0.999 power law chc22 5.02 0.875 0.999 bingham 0.95 2.830 0.999 herschel-bulkley 0.03 0.395 0.999 casson (chb: the sample containing base formulation, chl: the sample containing lecithin, chp: the sample containing pgpr, chc: the sample containing citrem; the first number: refining time (1: 60 min, 2: 90 min), the second number: emulsifier level (1: 0.5% or 0.25% (the sample containing pgpr), 2: 1% or 0.5% (the sample containing pgpr)). 3.4.2 apparent viscosity the apparent viscosities of the samples are shown in table 3. in this study, no significant differences between samples containing lecithin and basic formula in second refining time (p<0.05) were found. a general trend regardless to fat content was seen as consistent decreases in apparent viscosity while increasing particle size (afoakwa 2009); increase in particle size from 18 to 50 μ m caused noticeable decrease in apparent viscositywhich was similar with casson plastic viscosityspecially at low fat (25%). in addition, it was reported that by increasing lecithin from 0.3 to 0.5%, the apparent viscosity decreased regardless to particle size and fat content. this study proves that different refining times have no effect on the specified levels of samples containing citrem, whereas, amount of citrem is effective on apparent viscosity. table 3. the measured values of casson viscosity, casson yield, and apparent viscosity for different formula. apparent viscosity (pa.s) casson yield value (pa) casson viscosity (pa.s) sample 24.7 b 16.92 d 18.74 b chb1 30.4 a 22.18 c 22.56 a chb2 28 a 18.66 d 21.25 a chl11 22.1 c 29.37 b 14.89 c chl12 28.9 a 31.92 b 20.94 a chl21 29.6 a 66.09 a 17.22 b chl22 21.7 c 10.23 e 15.13 c chc11 18.6 d 11.15 e 13.46 c chc12 20.9 c 21.23 c 14.97 c chc21 19.3 d 15.88 d 14.59 c chc22 (chl: the sample containing lecithin, chc: the sample containing citrem; the first number: refining time (1: 60 min, 2: 90 min), the second number: emulsifier level (1: 0.5%, 2: 1%). different letters indicate statistically significant differences (p<0.05). ital. j. food sci., vol. 30, 2018 34 casson model casson viscosity values for casson viscosity and casson yield value were determined through the casson model fit on the data (shear stress shear rate). casson viscosity values ranged between 13.46 and 22.56 pa.s (table 3). in this study, there were no significant differences between samples containing citrem at both refining times and different levels (p<0.05). the results showed that different refining time have no effect on samples containing 0.5% lecithin, whereas, in the samples containing 1% lecithin, by increasing refining time, casson viscosity increased and also by increasing lecithin, casson viscosity decreased at both refining time. afoakwa (2009) reported increase in casson viscosity while increasing refining time and reducing particle size. moreover, it was seen that, especially at lower fat and lecithin levels, casson plastic viscosity, casson yield value, yield stress and apparent viscosity decreased in higher particle sizes. fat reduction up to 30% has little effect on the casson parameters; however, in chocolates with a fat content of less than 30%, by reducing the fat content, the casson parameters, particularly the casson viscosity, will increase (beckett, 2000). casson yield value the casson yield values are shown in table 3. the yield stress or yield value relates to shape retention, pattern holding, feet and tails, inclined surface coating and presence of air bubbles (seguine, 1988). the casson yield values ranged 10.23 to 66.09. the sample having citrem in the initial refining time and level of 0.5% had the least yield value and lecithin in second refining time and level of 1% had the highest yield stress. in all samples, by increasing refining time and reducing particle size, the casson yield stress increased. evaluation of rheological characteristics revealed that increasing particle size, fat percentage (more specifically in low fat samples (25%)) and lecithin concentration play as a reduction agent for casson yield values (afoakwa, 2009). it was observed that casson yield value of the samples containing lecithin was more than samples with base formulations. the reason is that if the amount of lecithin rises above 0.3%, casson yield stress increases (fincke, 2013). the samples containing citrem at initial refining time was the most effectives in reducing casson yield value (p <0.05). yield value is affected largely by interparticle contacts and consequently shows a linear dependence on the mean particle size, or more accurately, on the specific surface area (mongia, 1997; mongia and ziegler, 2000). by decreasing the particle size there are more particles for intermolecular contact, thus the casson yield value increases. prentice (1984) reported that when particle size decreases, interactions and subsequent friction constants between the particles increase, thus the casson yield stress increases. power law model as described before, power law model was chosen as an appropriate flow model for pgpr containing samples (table 4). the shear stress-shear rate tests (in the mentioned range) showed a consistency coefficient range of 17.49 to 26.05 for the four formulations. however, the estimated flow behavior indices showed to be close to n=1 for all formulae. it can be concluded that adding pgpr emulsifier to the compound chocolate caused change in consistency index but it did not affect the flow behavior index. it is also worthy to note that, in samples containing 0.25% pgpr, increasing refining time was affective and ital. j. food sci., vol. 30, 2018 35 led to increased consistency index, whereas, there was no significant deference between samples having 0.5% pgpr (p<0.05). table 4. the values of consistency index, flow behavior index, and apparent viscosity for the samples containing pgpr. apparent viscosity (pa.s) flow behavior index consistency index (pa.s) sample 19.5 c 0.99 a 17.49 c chp11 22.6 b 0.99 a 19.72 b chp12 25.5 a 0.99 a 26.05 a chp21 22.4 b 0.99 a 20.40 b chp22 (chp: the sample containing pgpr, the first number: refining time (1: 60 min, 2: 90 min), the second number: emulsifier level (1: 0.25%, 2: 0.5%). different letters indicate statistically significant differences (p<0.05). 4. conclusions to conclude, the refining time as a main factor affecting particle size distribution, emulsifier types and their levels are two important factors in optimization of compound chocolate with reduced fat content. reduction of particle size increased the casson yield value, although the rheological properties were related to type of emulsifiers and refining times, too. in addition, the hardness of the samples decreased by increasing emulsifier content and decreasing refining time. the casson model was selected as an appropriate rheological model to illustrate the rheological parameters of the samples containing the citrem and lecithin as emulsifiers. nevertheless, chocolate models with reduced fat content containing the pgpr were not in a good agreement with the casson model. on the contrary, the power law model showed the highest correlation to their flow behavior. finally, reduction of fat content leads to an increase in the molten compound chocolate viscosity and hardness, therefore, using citrem emulsifier because of significant reduction in the hardness and rheological parameters such as apparent viscosity, casson viscosity and casson yield value can be effective and useful for production of reduced fat dark compound chocolate. references afoakwa e.o., paterson a. and fowler m. 2008. effects of particle size distributionand composition on rheological properties of dark chocolate. eur. food res. technol. 226:1259-1268. afoakwa e.o., paterson a. and fowler m. 2007. factors influencing rheological and textural qualities in chocolate – a review. trends food sci. technol 18:290-298. afoakwa e. o, paterson a., fowler m. and vieira j. 2009. microstructure and mechanical properties related to particle size distribution and composition in dark chocolate. int. j. food sci. technol. 44:111-119. alamprese c., datei l. and semeraro q. 2007. optimization of processing parameters of a ball mill refiner for chocolate. j food eng. 83:629-636. beckett s.t. 2009. “industrial chocolate manufacture and use”. 4th ed. west sussex: wiley-blackwell. beckett s.t. 2000. “the science of chocolate”. 2nd ed. london: royal society of chemistry paperbacks. 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karre et al., 2013; shah et al., 2014). onion (allium cepa l.), garlic (allium sativum l.) and marjoram (origanum majorana l.) possess both antioxidant and antimicrobial activity (busatta et al., 2008; roby et al., 2013; sallam et al., 2004; ye et al., 2013). onion and garlic bulbs, as well as dried marjoram, are three major meat additives widely used in food preparation. park and chin (2010) used onion extracts to control the development of lipid oxidation in fresh pork patties. el-alim et al. (1999) investigated the use of ground marjoram as an antioxidant in raw ground chicken. cao et al. (2013) proved the antimicrobial and antioxidant properties of onion and garlic for extending the shelf life of stewed pork during refrigerated storage. moreover, park et al. (2008) demonstrated the antimicrobial and antioxidant activity of garlic and onion powder in fresh pork belly and loin during storage. fresh garlic blended with paprika was also successfully used as an antioxidant in dry sausages (aguirrezábal et al., 2000). however, due to the fact that the rate and extent of lipid oxidation are influenced by a number of factors, which include iron content, distribution of unsaturated fatty acids, ph, the form of natural additives (fresh, dried or extract) and various processing technologies, further research is still needed, particularly with reference to the meat system. the aim of the present study was to evaluate the effect of natural antioxidants contained in onion, garlic and marjoram on the oxidant and microbial stability of refrigerated minced pork blade meat during refrigerated storage. 2. materials and methods 2.1. samples preparation samples of dried marjoram leaves, fresh garlic and onion bulbs were purchased in a local market. all the products were produced in poland. three pork blades (each from different animals) were obtained from a local market (where the process of cutting and deboning is conducted locally) and transported to the laboratory under chilled conditions in iceboxes. blade muscles containing approximately 4% intramuscular fat were used. the blades were chopped and minced separately in a meat grinder, after the removal of excessive fat and connective tissue. dried marjoram leaves and freshly crushed garlic and onion bulbs were ital. j. food sci., vol. 29, 2017 646 mixed with the meat. the following four samples were prepared from each blade: 1 control (meat without any addition) and 3 treatments, namely, with garlic 0.1% (m/m), onion 0.5% (m/m) and marjoram 0.5% (m/m). the mixing process for each sample was conducted separately for each blade. the concentrations of the additives were established during preliminary studies and were accepted by a sensory panel (data not presented). meat samples were placed in plastic foam meat trays, wrapped with polyethylene and kept at 4°c for 9 days. a strict sanitation procedure was followed during the preparation of the meat samples to avoid microbial contamination. 2.2. antioxidant activity of plant extract the antioxidant activities of marjoram leaves, garlic and onion were determined by methods, which reflected the various mechanisms of antioxidant action, such as the scavenging of free radicals or chelation of transition metal ions (methods such as dpph and teac or frap, respectively). each method has its advantages and limitations, thus a combination of these methods was implemented for assessing antioxidant activity to get more insight into the antioxidative potential of the extracts studied. to investigate the antioxidant activity of the plant material, extracts were prepared in methanol. for the extraction, 2 g of dried marjoram leaves, onion or garlic were mixed with 30 ml of pure ethanol. the procedure was conducted in the dark at room temperature. after 30 min of extraction, each extract was put through a filter (type 388) and the solutions of the corresponding concentrations were prepared in methanol for antioxidant activity measurements. 2.3. dpph method the antioxidant activity of marjoram leaves, garlic and onion were evaluated by the dpph method according to the procedure described by sánchez-moreno et al. (1998) with some modifications. briefly, 10 µl of the plant extract samples was added to 990 µl dpph in methanol (giving a final concentration 0.1 mm) and mixed in a vortex. the reaction mixtures were incubated in the dark at room temperature for 30 min, and the decrease in absorbance caused by the plant extract was measured at 515 nm using a cary 1e spectrophotometer (varian, berlose, australia). for each sample, three separate determinations were conducted. the corresponding solvent blank readings were also recorded (methanol). the dpph• radical scavenging activities of the plant extracts were expressed in teac(dpph) values trolox equivalents antioxidant capacity (mm of trolox/g of dry weight). teac(dpph) values were calculated as the ratio of the slope of the linear plot for the scavenging of dpph• radicals by the plant extract tested to the slope of the plot for dpph• radicals scavenging by the water-soluble vitamin e analogue trolox, used as an antioxidant standard. 2.4. teac assay the teac (trolox equivalent antioxidant capacity) assay is based on the inhibition of the absorbance of the blue-green coloured abts•+ radical cation by antioxidants (miller et al. 1993). in the study, the abts•+ radical cation was generated with potassium disulphate according to the modifications of re et al. (1999). briefly, abts was dissolved in water to a 7 mm concentration and mixed with 2.45 mm potassium disulphate at a ratio of 2:1 (stoichiometrical reaction). to produce abts•+ radical cation, the mixture was allowed to stand in the dark at room temperature for 12-16 h. the radical was stable in this form for more than two days (i.e. stored in the dark at room temperature). the abts•+ solution was ital. j. food sci., vol. 29, 2017 647 diluted with pbs (phosphate buffer saline), ph 7.4 to an absorbance of 0.7 (±0.02). 10 µl of plant extract was added to 990 µl abts•+ solution in pbs, ph 7.4 and the decrease in the absorbance of the mixture after 6 min of incubation in the dark was monitored spectrophotometrically at 734 nm. the corresponding solvent blank readings were run (abts in pbs, ph 7.4 with methanol). all determinations were conducted in triplicate, and the results were expressed as teac values. the teac value represents the ratio of the angle of the plot for the scavenging of abts•+ by the particular extract under investigation to the slope of the plot for abts•+ scavenging by trolox, used as an antioxidant standard (miller et al., 1993). the teac value is expressed in milimolar concentrations (mm), according to the definition of the teac value introduced by miller et al. (1993). the teac value is defined as the concentration of trolox solution with an equivalent antioxidant potential to a 1 mm concentration of the compound under investigation (miller et al., 1993). 2.5. frap assay the frap (ferric reducing antioxidant power) assay directly measures the ability of antioxidants to reduce a ferric tripyridyltriazine complex (fe+3-tptz) to a ferrous complex (fe+2-tptz) at a low ph, with an intense blue color and an absorption of 593 nm (benzie and strain 1996). the frap solution was prepared by mixing an acetate buffer (300 mm, ph 3.6), 10mm tptz (2,4,6-tripyridyl-s-triazine) and fecl3•6h2o (20 mm in 40 mm hcl) at a ratio of 10:1:1. the mixture was allowed to stand in the dark at 30°c for 30 min directly before use. then, 50 µl of an increasing concentration of plant extract was added to the frap solution in a 1ml total volume and allowed to react for 10 min in the dark. readings were taken at 593 nm and results were expressed as mm trolox equivalent (te) per g of dry weight. all determinations were conducted in triplicate. 2.6. total polyphenol content of plant extracts the total polyphenol content (tpc) of the plant materials was determined using the folinciocalteu reagent (fcr) as described by singleton and rossi (1995). an aliquot of 20 µl of plant extract (onion, garlic or marjoram), prepared in the same way as for antioxidant activity measurements, was added to 100 µl of fcr, mixed and incubated for 3 min at room temperature in a dark place. then, 300 µl of sodium carbonate (20% m/v) was added and filled to 2 ml with distilled water. after 2h of incubation in the dark at room temperature the absorbance was read at 765 nm against the blank sample (solvent instead of extract). the results were presented as mg gallic acid equivalent (gae) per g of dry weight (dw). all determinations were conducted in triplicate. 2.7. total flavonoid content the total flavonoid content was determined by using the aluminum chloride colorimetric method as described by medaa et al. (2005). the basis of this assay is that aluminium chloride forms acid stable complexes with the c-4 keto group and either the c-3 or c-5 hydroxyl group of flavones and flavonols. in addition, it also forms acid labile complexes with the ortho-dihydroxyl groups in the a or b ring of flavonoids. 100 µl of plant extracts were mixed with aluminium chloride in methanol (2% m/v) to a final volume of 1 ml. the mixtures were incubated for 15 min in the dark at room temperature. then, the absorbance was monitored at 410 nm. the results were expressed in mg of quercetin per g of dry weight. ital. j. food sci., vol. 29, 2017 648 2.8. measurement of lipid oxidation the extent of lipid oxidation was monitored by the formation of thiobarbituric acidreactive substances (tbars). the tbars index was determined, in triplicate samples, by the extraction method of sørensen and jørgensen (1996) with some modification. for extraction, 10 g meat was homogenized (15,000 rpm, 30 s, 20°c) together with 30 ml of a 7.5% aqueous solution of trichloroacetic acid (tca). after filtration and centrifugation, (5 min, 5000 rpm), a 5.0 ml extract was mixed with 5.0 ml of 0.02 mol l-1 aqueous thiobarbituric acid (tba) in a stoppered test tube. the samples were incubated at 100c for 35 min in a water-bath and subsequently cooled for 10 min in cold water. absorbance was measured at 532nm by a carry 1e uv/vis spectrophotometer against a blank containing 5ml distilled water and 5ml tba reagent. the results, expressed as milligrams malondialdehyde per kg meat, were calculated from the standard curve of tep (1,1,3,3tetraethyoxypropane) standards. the antioxidant potential, which is expressed in terms of the percentage of antioxidant activity (%aoa), were calculated using the following equation (wijewickreme and kitts 1998): %aoa= ((x value of the control – x value of the test sample) × 100)/ x value of the control where x is the tbars value. 2.9. fatty acids composition analysis the meat samples were minced in a blender. lipids were extracted from the samples with chloroform:methanol (2:1 v/v) according to folch et al. (1957). the extracts were dried in a vacuum on a rotary evaporator and under a nitrogen flow. the fatty acid content was determined as fatty acid methyl ester (fame) derivatives in a hp 5980 series ii gas chromatographer (hewlett packard, oalo alto, usa) using the aocs official method ce 1k-07. the gc system consisted of an innowax capillary column (30 m x 0.2 mm x 0.2 mm) and fid detector. hydrogen was used as the carrier gas, at a flow rate of 1.5 ml/min. the temperature of both the injector and detector was set at 240°c. the column temperature was maintained at 220°c isotherm. peak identification was based on the fatty acid ester standards, and fatty acid compositions were estimated from the chromatogram peak areas and were expressed as mg/1 g meat sample. 2.10. microbial analysis an amount of 10 g of meat was collected from prepared samples and introduced, under sterile conditions, to a flask containing 90 ml of 0.1% sterile peptone diluents (bacteriological peptone, oxoid, england). the samples were homogenized using an ultra-turrax t25 homogeniser (ika, germany), producing an initial suspension of 1:10. the microbiological analyses were conducted in line with iso reference methods (according to the polish standard of pn-iso). bacterial counts were given in cfu g-1. the total bacterial count was determined on a standard plate count agar (cm 463, oxoid, england). incubation was run at 30°c for 72 h. for counts of the enterobacteriaceae rods, a 1 ml sample was inoculated into 15 ml of a molten selective vrbg medium (p-0256, btl, poland). after setting, a 10 ml overlay of molten medium was added and incubation was conducted at 37°c for 24-48 h. the counts of pseudomonas were determined on a solid pseudomonas agar medium (cm 0559, oxoid, england) supplemented with pseudomonas cfc selective agar suplement (sr 0103, oxoid, england) after incubation ital. j. food sci., vol. 29, 2017 649 at 30°c for 48 h. de man, rogosa and sharpe (mrs) agar (cm 0361, oxoid, england) was used for determining lactic acid bacteria counts. mrs agar was also overlayed with a molten medium and incubation was conducted at 30°c for 48-72 h. an oxidase test was used to confirm lactic acid bacteria (mbo 266, oxoid, england). dichloran rose bengal chloramphenicol drbc agar (cm 0727b, oxoid, england) was used in the determination of moulds and yeasts. incubation was conducted at 20°c for 5 days. total viable aerobic bacteria counts (tvc) were conducted as an indicator of microbial spoilage in the pork meat samples. the samples (10 g) were homogenized with 90 ml with sterile peptone water (1g/l) using an ultra-turrax t25 homogenizer (ika, germany). serial decimal dilution was performed and plated onto a standard plate count agar (cm 463, oxoid, basingstoke, england). incubation was performed at 30°c for 72 h. bacterial counts were enumerated and expressed as log10 cfu/g. 2.11. statistical analysis all tests were run in triplicate. the statistical package statistic 13.1 was used for analysis of covariance (ancova). the influence of various plant additions on oxidative and microbial raw meat stability was assessed using analysis of covariance. this analysis procedure is applied when looking at group effects on a continuous outcome (tbars value, fatty acid content and microbial count) when another continuous explanatory variable (storage time) may also have an effect on the outcome. comparison of the treatment means was based on tukey’s honest significant difference (hsd) test. this test is the most useful for multiple comparisons and avoids type ii errors. in addition, dunnett’s t-test was performed for a comparison of the treatments to the control. to compare the rates (slope of regression equation) of fatty acids' degradation, a linear regression analysis between the storage time and the fatty acid content was calculated. to compare the regression coefficients (slope) of the control with the other treatments, the null hypothesis h0: bc=bt, (where bc is the regression coefficient for the control and bt is the regression coefficient of the sample with additives) was tested. the comparisons between the coefficients were performed introducing two dummy variables as predictors to regression analysis. the first dummy variable was coded “0” for the control and “1” for the meat sample with plant additive, and the second, which was the product of the first dummy variable and storage time. the significant differences between the regression coefficients were based on the result of the t-test (p ≤ 0.05) for the second dummy variable. bacterial counts were converted to their equivalent log cfu for data uniformity. differences were considered significant at the p ≤ 0.05 level. 3. results and discussions 3.1. antioxidant activity and total polyphenol content of the plant extracts all results of the antioxidant activity and phenolic content (total polyphenol content tpc and total flavonoid content tfc) measurements are presented in table 1. according to radical scavenging methods like dpph and abts and ion chelating methods such as frap, the most antioxidant active plant extract was marjoram. there were no statistically significant differences between the antioxidant activities of garlic and onion. gorinstein et al. (2008) showed higher antioxidant activity of garlic in comparison to onion in the dpph method but significantly lower antioxidant activity of garlic than onion in the abts and frap methods. mariutti et al. (2008) reported that marjoram is the most antioxidant active among the tested extracts in the dpph and abts methods, which ital. j. food sci., vol. 29, 2017 650 is in accordance with the results presented in this paper. however, the authors showed that garlic is more antioxidant active than onion as a dpph• and abts•+ radical scavenger table 1. phenolic content and the antioxidant activities of marjoram, garlic and onion. plant extract marjoram garlic onion phenolic content mean±sd mean±sd mean±sd tpc (mg gae/g) 24.48 ± 2.66 a 0.79 ± 0.23 b 2.74 ± 0.65 b tfc(mg qe/g) 15.72 ± 1.26 a 0.22 ± 0.00 b 0.27 ± 0.01 b antioxidant activity teac(dpph)(mm te/g) 175.11 ± 11.43 a 3.05 ± 0.46b 3.77 ± 0.51b teac(abts)(mm te/g) 261.84 ± 15.04 a 7.68 ± 0.25b 19.09 ± 1.15b frap(mm te/g) 186.9 ± 10.60a 4.16 ± 0.06b 3.86 ± 0.19b (a-b)means with the same superscript within the same row are not different (p > 0.05). in this study, the tpc and tfc values correspond to the antioxidant activity (table 1). rpearson coefficients for the linear correlations between the antioxidant activity and tpc or tfc values are equal to 0.99. the higher the tpc (and tfc) value, the higher the antioxidant activity is. in literature, data on the tpc values of onion, garlic and marjoram vary. generally, it was shown that onion possesses a higher tpc value compared to garlic (gorinstein et al. 2008; nuutila, et al. 2002). it is also hard to compare the tpc values of marjoram to reference results since various modifications to the methods and units were implemented by the authors. 3.2. measurement of lipid oxidation the lipid oxidation in the fresh minced pork meat subjected to refrigerated storage was determined using the tbars method. this method is widely used to estimate the degree of lipid oxidation because of its relatively simple measurement and good correlation with the sensory quality of foods (mottram 1998). tbars are formed through the second stage of lipid oxidation, during which peroxides are oxidized to aldehydes and ketones such as malondialdehyde (mda). fig. 1 presents the tbars values of the samples under study. the results showed that both the storage period and the type of additive affected the tbars values (with p < 0.05). the tbars values of all the tested samples (control and treatment) increase constantly with the time of storage (fig. 1). this increase was the most pronounced for the control sample, whose tbars values rose from 0.11 mg mda/kg meat on the first day of analysis to 0.352 mg mda/kg meat at the end of storage (9th day). the tbars values in the minced meat were influenced by onion, garlic and marjoram, resulting in significantly lower values than those without treatment. as shown in fig. 1, the most effective treatments in inhibiting the lipid oxidation were the onion and garlic ones. there were no statistically significant differences between these two samples (p >0.05). samples treated with onion and garlic reached tbars values of 0.22 and 0.186 mg mda/kg respectively on the last day of storage, compared to the control samples which exhibited a tbars value equal to 3.5 mda/kg. significant differences in the tbars were found between the treated samples and the control from day 3 to the end of storage. yin and cheng (1998) found that garlic had a stronger antioxidant effect than onion in their ital. j. food sci., vol. 29, 2017 651 liposome model system. in this study, due to sensory acceptability, different levels of additives were tested (0.15 m/m for garlic and 0.5% m/m for onion), which may have caused the lack of difference between the onion and garlic inhibition of lipid oxidation. a statistically significant protective effect for lipid oxidation in meat was also noted for samples with the addition of marjoram. the tbars value for the treatment with marjoram at the end of storage was 0.28 mg mda/kg and was significantly lower than the control one. however, this effect was less pronounced when compared to the samples with garlic and onion. in conclusion, the highest inhibition of secondary oxidation products was observed in the samples with garlic (47%). in the samples with onion and marjoram, the percentage of inhibition was 37% and 25% respectively. these results were consistent with those of previous studies in which fresh onion and garlic bulbs or their extracts showed antioxidant effects in the meat matrix (cao et al., 2013; kim et al., 2010; park et al., 2008; sallam et al., 2004). figure 1. changes in the tbars values of fresh minced pork with different treatments during refrigerated storage at 4°c. 3.3. fatty acid composition analysis the fatty acid composition of minced pork muscle fat is shown in table 2. both the type of additive and storage time influenced the fatty acid composition. the predominant fatty acids in all meat samples were oleic (c18:1n-9), palmitic (c16:0), stearic (18:0) and linoleic (c18:2n-6) acids, in order of the increasing concentration in the samples. the amount of these acids accounted for more than 91% of the total fatty acid contents in all samples. park et al. (2008) obtained similar results in fresh pork belly and loin. mufas were the major part followed by sfas. generally, a decrease in the fatty acid content in the ital. j. food sci., vol. 29, 2017 652 intramuscular lipid was noted during storage. this effect was expected because, due to cellular structure damage and exposure to the oxygen, minced muscle is very prone to lipid and protein oxidation (min and ahn, 2005). therefore, to evaluate whether the fatty acid contents were equal in the groups being studied (type of additive) analysis of covariance was performed. this analysis allows for comparisons of the effects of additives and control the influence of storage time simultaneously. comparison of the additives showed that a stronger inhibition of lipid oxidation was exhibited by garlic (table 2). statistically significantly higher contents of stearic (56.82 mg/g) palmitileic (15.28 mg/g), linoleic (41.1 mg/g), cis-10-heptadanoic (1.62 mg/g) and arachidonic acids (5.14 mg/g) were observed in this treatment when compared to the control. the lowest content of fatty acids was noted in the samples with the addition of fresh onion, which makes it the least effective in the retardation of lipid oxidation. the marjoram additive was the most effective in inhibiting the decomposition of linoleic (but only until the 7th day of storage) and cis-10-heptadanoic acids. furthermore, to compare the effect of the added natural ingredients on the change rates of fatty acids, linear regression analysis was performed. table 2. effect of onion, garlic and marjoram on the fatty acid profile of fresh minced pork during storage at 4°c (mg/g). fatty acid treatment control onion garlic marjoram se c14:0 4.90a 4.62a 6.08a 5.01a 0.39 c16:0 84.31ab 79.95a 106.26b 89.22ab 6.86 c18:0 41.41a 39.89a 56.82b* 47.02ab 3.40 c16:1 12.42a 11.56a 15.28b* 12.64a 0.62 c17:1 1.26a 1.18a 1.62b* 1.85c* 0.05 c18:1 176.59ab 166.92a 198.84b 159.50a 12.06 c20:1 3.35a 3.33a 3.20a 2.74a 0.15 c18:2 35.53ab 33.27a 41.10bc* 42.75c* 1.79 c20:2 1.52a 1.52a 1.47a 1.52a 0.08 c18:3 4.91a 4.56a 2.38b 2.52b 0.21 c20:4 3.33a 3.31a 5.14b* 3.92a* 0.17 sfa 130.63ab 124.46a 169.15b* 141.26ab 10.62 mufa 193.62ab 182.99a 218.95b 176.73a 8.94 pufa 45.29ab 42.66a 50.08ab 50.70b 3.19 a-c)means with the same superscript within the same row are not different (p > 0.05). (*) paired comparison (control compared with other natural additives) significantly greater than control at the p < 0.05 level using dunnett’s-t test. results showing the regression coefficients from the linear models of the decay of fatty acids are presented in table 3. the linear model was statistically significant in almost all cases (p ≤ 0.05). only for saturated fatty acids in the meat samples with garlic and marjoram, was the linear model not significant (p > 0.05). in the samples with garlic, the level of saturated fatty acids was stable during storage. however, in the samples with marjoram the level of saturated fatty acids increased till the 7th day of storage and then rapidly decreased, reaching the lowest values of all the meat samples. the values of the determination coefficients ranged from 74% to 96%. ital. j. food sci., vol. 29, 2017 653 table 3. effect of onion, garlic and marjoram on the fatty acids content changes of fresh minced pork during storage at 4°c (expressed as slope (mg/g/day)). fatty acid treatment control onion garlic marjoram c14:0 -0.568 -0.472 -0.057n -0.340n c16:0 -0.500 -7.801 -1.282n -6.130n c18:0 -4.357 -3.713 -0.831n -3.040n c16:1 -1.391 -1.298 -0.762* -1.865 c17:1 -0.123 -0.098 -0.076* -0.173 c18:1 -19.89 -18.97 -9.381* -23.94 c20:1 -0.401 -0.422 -0.144* -0.346 c18:2 -3.299 -3.169 -1.613* -6.089* c20:2 -0.162 -0.148 -0.068* -0.133 c18:3 -0.652 -0531 -0.214* -0.256 c20:4 -0.282 -0.200 -0.165* -0.621* sfa -14.42 -11.99 -2.165* -9.512 mufa -21.81 -20.79 -10.36* -26.32 pufa -4.396 -4.051 -2.060* -7.101* (n)regression coefficients are not significant (p > 0.05). (*) paired comparison (control compared with other natural additives) were significantly different to the control at p < 0.05. all the slopes have a negative value indicating the inverse relationship between the storage time and fatty acid content. the lowest rate of fatty acid degradation was recorded in the samples with the fresh garlic addition. this dependency was noted for all fatty acids, which confirms the highest inhibiting effect of garlic on lipid degradation. in addition, discriminant analysis corroborates these outcomes. as shown in fig. 2, the first discriminant function extracts the sample of meat with garlic, while the second extracts the meat samples with marjoram. the meat samples with onion exhibit the same fatty acid composition during storage as the meat samples without any addition (the points are located in the same place of the graph). the lda model allows meat samples enriched with garlic and marjoram to be distinguished with 100% accuracy. the highest decomposition rate was noted in the meat samples with marjoram, namely, arachidonic and linoleic acid. the contents of these acids decreased from 6.24 to 1.63 and from 60.2 to 9.2 mg/g respectively. the level of linoleic acid in the samples with marjoram was significantly the lowest among the tested samples at the end of storage. 3.4. microbial changes the bacteria selected in this study such as pseudomonas and enterobacteriaceae belong to the genera of bacteria constituting microflora typical for spoilage processes in meat during storage. to assess the effect of the addition of the plants on bacterial growth in meat samples covariance analysis was performed. throughout the storage period, the inhibition effect of the plant additives on bacterial growth was observed. only for lactic acid bacteria was this effect not statistically significant. ital. j. food sci., vol. 29, 2017 654 figure 2. discrimination of minced meat samples based on the fatty acid composition during cold storage (4°c); c-control, g-meat with garlic (0,1%), mmeat with marjoram (0.5%) and omeat with onion (0.5%). as shown in table 4, the highest numbers of all the tested bacteria were noted in the control samples. the results showed that the addition of onion and marjoram to fresh meat exerted a statistically significant bacteriostatic effect against tvc, enterobacteriaceae and pseudomonas bacteria. the highest inhibitory effect against enterobacteriaceae and pseudomonas bacteria was noted in the meat samples with the addition of fresh onion. the results of our study supported the findings of other authors. grohs et al., (2000) reported that spice mixtures might delay bacteria growth in fresh pork and beef. park et al., (2008) showed that the addition of garlic and onion powder to pork loin and belly inhibited the growth of the total amount of bacteria and enterobacteriaceae. sallam et al., (2004) noted that fresh garlic and garlic powder, through their antioxidant and antimicrobial effects, are potentially useful in preserving meat products. however, murray (1997) claimed that only fresh garlic preparations provide the full range of beneficial compounds. table 4. effect of onion, garlic and marjoram additions on the microbial changes (cfu/g) of fresh minced pork stored at 4°c. type of bacteria treatment control onion garlic marjoram tvc 8.02a 7.54c* 7.92ab 7.8b* enterobacteriaceae 6.16a 5.65b* 6.09a 5.70b* pseudomonas 8.00a 7.52c* 7.84ba 7.74bc* lactic acid bacteria 2.84 2.64 2.71 2.65 (a-c)means with the same superscript within the same row are not different (p > 0.05). (*) paired comparison (control compared with other natural additives) is significant at the p < 0.05 level using dunnett’s ttest. ital. j. food sci., vol. 29, 2017 655 4. conclusions due to the natural potential health benefits and safety of the plant species when compared to synthetic derivatives, natural species have gained appreciable interest among the research and industry community. the highest antioxidant activity is demonstrated by fresh garlic, whereas fresh onion in minced pork meat reveals the highest antibacterial activity. the synergistic behaviour of these plants would require further research. although marjoram was characterised by the highest antioxidant activity, its protective effect on lipids in meat was less significant than other treatments. this result confirms the necessity to examine the plant antioxidant effect in particular food products. our data indicated that the addition of fresh garlic and onion, as well as dried marjoram, enhances raw minced meat safety and shelf life. in addition to their economical and health promoting benefits, the application of plants, as natural and safe bio-preservatives, could be highly recommended for the improvement in the quality of ground pork. acknowledgements this research project was partly supported by the polish state committee for scientific research (no. 2014/15/d/nz9/04261). references aguirrezábal m., mateo j., domı ́nguez m. and zumalacárregui j. 2000. the effect of paprika, garlic and salt on rancidity in dry sausages. meat sci. 54:77-81. american oil chemist's society (aocs) official method ce 1k-07 (2007) direct methylation of lipids for the determination of total fat, saturated, cis-monosaturated, cis-polyunsaturated and trans fatty acids by chromatography. busatta c., vidal r.s., popiolski a.s., mossi a.j., dariva c., rodrigues and m.r. cansian r.l. 2008. application of origanum majorana l. essential oil as an antimicrobial agent in sausage. food microbiol. 25:207-211. cao y., gu w., zhang j., chu y., ye x., hu y. and chen j. 2013. effects of chitosan, aqueous extract of ginger, onion and garlic on quality and shelf life of stewed-pork during refrigerated storage. food chem. 141:1655-1660. el-alim s.s.l.a., lugasi a., hóvári j. an dworschák e. 1999. culinary herbs inhibit lipid oxidation in raw and cooked minced meat patties during storage. j. sci. food agr. 79:277-285. falowo a.b., fayemi p.o. and muchenje v. 2014. natural antioxidants against lipid-protein oxidative deterioration in meat and meat products: a review. food res. int. 64:171-181. folch j., lees m. and sloane-stanley g.r. 1957. a simple method for the isolation and purification of total lipids from animal tissue. j. biol. chem. 226:497-509. gorinstein s.. leontowicz h.. leontowicz m.. namiesnik j.. najman k.. drzewiecki j. and trakhtenberg s. 2008. comparison of the main bioactive compounds and antioxidant activities in garlic and white and red onions after treatment protocols. j. agr. food chem. 56:4418-4426. karre l., lopez k. and getty k.j.k. 2013. natural antioxidants in meat and poultry products. meat sci. 94:220-227. kim y.j., jin s.k., park w.y., kim b.w., joo s.t. and yang h.s. 2010. the effect of garlic or onion marinade on the lipid oxidation and meat quality of pork during cold storage. j. food quality 33(suppl. 1):171-185. mariutti l.r.b., barreto g.p.d.m., bragagnolo n. and mercadante a. z. 2008. free radical scavenging activity of ethanolic extracts from herbs and spices commercialized in brazil. braz. arch. biol. techn. 51:1225-1232. min b. and ahn d.u. 2005. mechanism of lipid peroxidation in meat and meat products -a review. food sci. biotechnol. 14:152-163. mottram d.s. 1998. flavour formation in meat and meat products : a review. food chem. 62:415-424. ital. j. food sci., vol. 29, 2017 656 murray m. 1997. which is better. aged versus fresh garlic; glucosamine sulfate versus chondroitin sulfate. am. j. med. 4:5-8. nuutila a.m., kammiovirta k. and oksman-caldentey k.m. 2002. comparison of methods for the hydrolysis of flavonoids and phenolic acids from onion and spinach for hplc analysis. food chem. 76:519-525. park s.y. and chin k.b. 2010. effects of onion on physicochemical properties, lipid oxidation and microbial growth of fresh pork patties. int. j food sci. tech. 45:1153-1160. park s.y., yoo s.s., shim j.h. and chin kb 2008. physicochemical properties, and antioxidant and antimicrobial effects of garlic and onion powder in fresh pork belly and loin during refrigerated storage. j. food sci. 73:577-584. roby m.h.h., sarhan m.a., selim k.a.h. and khalel k.i. 2013 evaluation of antioxidant activity, total phenols and phenolic compounds in thyme (thymus vulgaris l.), sage (salvia officinalis l.), and marjoram (origanum majorana l.) extracts. ind. crop prod. 43:827-831. sallam k.i., ishioroshi m. and samejima k. 2004. antioxidant and antimicrobial effects of garlic in chicken sausage. food sci technol-leb 37:849-855. sánchez-moreno c., larrauri j.a. and saura-calixto f. 1998. a procedure to measure the antiradical efficienc y of polyphenols. j. sci. food agr. 270:270-276. shah m.a., bosco s.j.d. and mir s.a. 2014. plant extracts as natural antioxidants in meat and meat products. meat sci 98:21-33. singleton v.l. rossi j.a. 1965. colorimetry of total phenolics with phosphomolybdic phosphotungstic acid reagents. am. j. enol. viticult. 16:144-158. sørensen g. and jørgensen s.s. (1996). a critical examination of some experimantal variables in the 2thiobarbituric acid (tbars) test for lipid oxidation in meat products. z lebensm unters for 202:205-210. wijewickreme a.n. nd kitts d.d. 1998. modulation of metal-induced genotoxicity by maillard reaction products isolated from coffee. food chem. toxicol. 36:543-553. ye c.l. dai d.h. and hu w.l. 2013. antimicrobial and antioxidant activities of the essential oil from onion (allium cepa l.). food control 30:48-53. yin m.c. and cheng w.s. 1998. antioxidant activity of several allium members. j agric food chem 46:4097-4101. paper received march 24, 2017 accepted july 10, 2017 ijfs#676_bozza ital. j. food sci., vol. 29, 2017 657 paper fatty acid composition of different adipose tissues in heavy pigs a. dazaa, a. olivares*b, m.a. latorrec, a.i. reyb, a. callejoa and c.j. lópez boteb adepartamento de producción animal, etsi agrónomos, universidad politécnica. 28040, madrid, spain bdepartamento de producción animal, facultad de veterinaria, universidad complutense. 28040, madrid, spain cdepartamento de producción animal y ciencia de los alimentos, facultad de veterinaria, universidad de zaragoza. 50013, zaragoza, spain *e-mail address: alolivares@vet.ucm.es abstract forty-seven castrated male duroc x (landrace x large white) pigs were used to determine fatty acids compositions from different adipose tissues. the outer subcutaneous backfat layer had a lower proportion of saturated and higher monounsaturated and polyunsaturated fatty acids than the inner layer. liver fat had the highest proportion of polyunsaturated fatty acids. intramuscular fat followed by subcutaneous backfat had the highest monounsaturation indexes. moreover, omental and hepatic fat had the highest amount of n-3 fatty acids. in conclusion, the fatty acid profile was depended on fat location, with intramuscular and outer backfat the most beneficial from the point of view of nutrition and health. keywords: fatty acid profile, fatty tissues, heavy pigs ital. j. food sci., vol. 29, 2017 658 1. introduction the fatty acid profile of adipose tissue in pigs is especially important since it relates to quality aspects in the process of preparing dry-cured products such as consistency, lipid oxidation, salt and water migration, curing duration, etc. (lopez-bote, 2000; lopez bote et al., 2004) and the production of volatile compounds that are responsible for odor and flavor (belitz and grosch, 1997). moreover, much attention is currently being paid to the fatty acid profile in animal products because of its association with consumer health (wood and enser, 1997; fao, 2012). hence a correlation has been found between saturated fatty acid consumption and cardiovascular diseases (fao, 2012), whereas polyunsaturated fatty acids (mainly n-3 fatty acids) have been found to improve the status of the cardiovascular system and the immune response (wood and enser, 1997). however, high proportions of n-3 fatty acids in tissues reduce oxidative stability of meat (rey et al., 2001). in pigs, fatty acid composition of fat tissue is influenced by a wide range of factors such as genetics, sex, weight and age at slaughter, livestock production system, feed, environmental conditions, pre-slaughter management, etc. (mourot and hermier 2001; de smet et al., 2004; lopez-bote et al., 2004). the effect of adipose tissue location on fatty acid profile has recently been studied in the celta pig breed, which is a rustic breed from spain (dominguez et al. 2014; dominguez & lorenzo 2014); however, there is a lack of informations in pigs of improved genotypes at heavy weights. therefore, given the importance of the topic, the aim of this study was to investigate the fatty acid profile of fat from different locations (hepatic, intramuscular and omental) in heavy pigs. 2. materials and methods 2.1 animals, diet and sampling all the experimental procedures used in the study were in compliance with the spanish guidelines for the care and use of animals in research (boletín oficial del estado, 2007). forty-seven castrated male duroc x (landrace x large white) pigs were used. all the animals received the same diet during the finishing period and before the beginning of the experiment all pigs were subjected to the same management and feeding conditions. the feedstuff consisted of a commercial diet based on barley, wheat and various vegetable protein sources (soybean, rapeseed, sunflower) that was administered ad libitum. the calculated energy (fedna, 2010) and composition (aoac, 2000) of the diet are shown in table 1. the pigs were slaughtered at 126±2.8 kg of live weight. on each carcass, fat thickness was measured by means of a graduated rule at the level of the gluteus medius muscle. individual samples of inner and outer subcutaneous backfat layers and longissimus dorsi muscle at the level of the last rib from each left loin, and omental and hepatic fat samples were taken. samples were vacuum-packaged in individual bags and stored at -20ºc for 3 weeks until subsequent analysis. 2.2 fatty acid analysis of diet and fat fatty acids of the diet were extracted and quantified using the one-step procedure described by sukhija and palmquist (1988) in lyophilized samples. pentadecenoic acid (c15:1) (sigma, alcobendas, madrid, spain) was used as internal standard. previously, methylated fatty acids samples were identified according to rey et al. (1997) ital. j. food sci., vol. 29, 2017 659 using a gas chromatograph (model hp6890; hewlett packard co., avondale, pa, usa) and a 30 m x 0.32 mm x 0.25 μm cross-linked polyethylene glycol capillary column (hewlett packard innowax). a temperature program of 170ºc to 245ºc was used. the injector and detector were maintained at 250 ºc. the carrier gas (helium) flow rate was 3 ml/min. lipids from subcutaneous and omental fat were extracted using the procedure proposed by bligh and dyer (1959), whereas lipids from longissimus dorsi and liver fat samples were extracted according to method developed by marmer and maxwell (1981). fat extracts were methylated in the presence of sulphuric acid and analysed as described above. fatty acid percentages of the saturated fatty acids (sfa), monounsaturated fatty acids (mufa) and polyunsaturated fatty acids (pufa) were calculated. the indexes used to estimate thioesterase (zhang et al., 2007) and elongase enzyme activity were c16:0/c14:0 and c18:0/c16:0 respectively. the ∆-9 desaturase activity was estimated by means of the following ratios: c16:1n-7/c16:0, c18:1n-9/c18:0 and mufa/sfa. table 1. calculated and determined analyses of the diet. calculated analysis g/kg digestible energy (mj/kg) 13.62 determined analysis (1) 886.2 crude protein (n x 6.25) 145.2 crude fat 56.2 crude fibre 35.1 fatty acids (%) c14:0 0.29 c16:0 4.71 c18:0 1.61 c18:1 n-9 5.68 c18:2 n-6 16.23 c18:3 n-3 3.49 the ingredients composition of the diet in g/kg was: barley 430, wheat 301, full fat soybean toasted 65.2, rapeseed meal 84.0, sunflower meal 54.2, fat (lard) 31.0, calcium carbonate 11.0, sepiolite, 10.0, l lysine 50% 5.5, sodium chloride 4.1, vitamin and mineral premix 4.0. (1)according to association of official analytical chemists (2000). 2.3 statistical analysis the pig was the experimental unit for statistical analysis. data were analyzed by means of variance analysis that included the adipose tissue location as the main effect. the newman-keuls test was used to assess differences between means. the relations between c18:2n-6 and c16:0 proportions in subcutaneous backfat layers were calculated by simple linear regression, comparing intercepts and slopes using “t” student test. correlation coefficients between gluteus medius fat thickness and major fatty acid proportions from subcutaneous backfat, and correlation coefficients between intramuscular fat percentage from longissimus dorsi and major fatty acid proportions of such tissue were calculated. all analyses were carried out using the glm and corr procedures of sas (1999). ital. j. food sci., vol. 29, 2017 660 3. results the omental fat had the highest proportion of sfa followed by the liver fat due to the high c18:0 proportion (table 2). the outer subcutaneous backfat layer showed a lower saturation and higher mono and polyunsaturation than the inner layer. the c16:0 and c18:0 proportions were lower (p<0.0001) in the outer subcutaneous backfat layer than in the inner layer, whereas c16:1n-7, c18:1n-9, c18:1n-7, c18:2n-6 and c18:3n-3 were higher (p<0.0001) (table 2). table 2. major fatty acid composition (%) of different adipose tissues from heavy pigs. tissue sfol sfil ldif of hf sem p < n 47 47 47 47 46 c16:0 23.78c 24.84b 24.43b 27.38a 21.97d 0.22 0.0001 c16:1 n-7 1.86c 1.67d 3.49a 1.58d 2.43b 0.061 0.0001 c18:0 14.56d 16.74c 13.29e 20.37b 23.18a 0.45 0.0001 c18:1 n-9 41.84ab 40.53b 43.05a 35.72c 30.72d 0.48 0.0001 c18:1 n-7 2.41b 2.16c 3.54a 1.50d 0.036 0.0001 c18:2 n-6 10.56a 9.36b 6.70c 8.98b 11.16a 0.24 0.0001 c18:3 n-3 0.79a 0.70b 0.044d 0.72b 0.44c 0.020 0.0001 c20:4 n-6 0.63b 0.58b 0.14b 0.42b 5.96a 0.18 0.0001 sfa 40.06d 43.26c 39.64d 49.66a 47.03b 0.37 0.0001 mufa 47.81b 45.96c 51.57a 40.07d 34.14e 0.050 0.0001 pufa 12.13b 10.77c 8.77d 10.27c 18.83a 0.47 0.0001 mufa+pufa 59.94a 56.73b 60.34a 50.34d 52.97c 0.47 0.0001 n-6 11.19b 9.93c 6.84d 9.41c 16.84a 0.54 0.0001 n-3 0.93b 0.83b 0.37c 0.86b 1.74a 0.42 0.0001 n = number of observations, sfol= subcutaneous backfat outer layer, sfil = subcutaneous backfat inner layer, ldif = longissimus dorsi intramuscular fat, of = omental fat, hf = hepatic fat. sem = error standard of the mean. sfa, mufa, pufa, n-6, n-3 = sum of all saturated (sfa), monounsaturated (mufa), polyunsaturated (pufa), n-6 and n-3 fatty acids. in the current experiment, the relations obtained between c18:2n-6 and c16:0 proportions in the outer and inner subcutaneous backfat layers were described by the following simple regression equations: outer layer: c18:2n-6 (%) = (28.40a±2.58) – (0.750b±0.11) c16:0 (%) r2 = 0.53, rsd = 0.98, p <0.0001 n = 47. inner layer: c18:2n-6 (%) = (29.76a±2.48) – (0.823a±0.10) c16:0 (%) r2 = 0.62, rsd = 0.88, p <0.0001, n = 47. table 2 also shows that c16:1n-7, c18:1n-9, c18:1n-7 and mufa proportions were higher in the intramuscular fat of longissimus dorsi muscle than in the other adipose tissues (p<0.0001). ital. j. food sci., vol. 29, 2017 661 the omental fat had lower monounsaturated and polyunsaturated fatty acids than subcutaneous backfat. the 18:2n-6, c20:4n-6, n-6, n-3 and pufa proportions were higher in hepatic fat than in the other adipose tissues. in this study, the highest unsaturation ranges (sum of mufa and pufa fatty acids) were obtained in intramuscular fat from longissimus dorsi muscle and outer subcutaneous backfat layer followed by inner subcutaneous backfat layer, liver fat and omental fat. the enzymatic activity indexes of different adipose tissues are shown in table 3. the thioesterase index (c16:0/c14:0), that indicates catalysis of c16:0 synthesis from c14:0, and the elongase index, as an indicator of c18:0 synthesis from c16:0 (c18:0/c16:0), were higher (p<0.0001) in liver fat than in the other adipose tissues. the c16:0/c14:0 and c18:0/c16:0 indexes in omental fat were higher than those in longissimus dorsi intramuscular fat (p<0.0001). however, c16:0/c14:0 indexes were of similar magnitude in omental fat and inner backfat layer. both indexes were higher in subcutaneous backfat than in intramuscular fat. the highest monounsaturation indexes c18:1 n-9/c18:0 and mufa/sfa were found in intramuscular fat followed by outer the subcutaneous backfat layer, inner subcutaneous backfat layer, omental fat and liver fat. however, the c16:1 n7/c16:0 index was higher in hepatic fat than in subcutaneous and omental fat. the highest pufa/sfa ratios were found in hepatic fat and the outer subcutaneous backfat layer and the lowest in intramuscular and omental fat. the lowest n-6/n-3 ratios were found in hepatic and omental fat and the highest in intramuscular fat from longissimus dorsi muscle. the highest mufa/sfa ratio was observed in intramuscular fat. the subcutaneous backfat thickness at the level of ham gluteus medius muscle was 23.5±5.93 mm. this variable was positively correlated with c16:0 and sfa and negatively correlated with c18:1n-9, mufa, c18:2n-6 and pufa proportions of the outer and inner subcutaneous backfat layers (table 4). the intramuscular fat percentage from longissimus dorsi muscle was 4.02±0.90%. this variable was positively correlated with c16:0 (r = 0.47 p<0.001) and sfa (r = 0.30 p<0.01), and negatively correlated with c18:2n-6 (r = -0.38, p<0.01) and pufa (r = –0.39 p<0.01) proportions found in longissimus dorsi intramuscular fat. however, correlations were not found between longissimus dorsi intramuscular fat and c18:1n-9 and mufa proportions. table 3. saturation and monounsaturation and quality index of different tissues from heavy pigs. tissue sfol sfil ldif of hf sem p < n 47 47 47 47 46 c16:0/c14:0 20.15c 22.04b 16.59d 21.38bc 27.26a 0.57 0.0001 c18:0/c16:0 0.61c 0.68bc 0.54d 0.74b 1.08a 0.025 0.0001 c16:1 n-7/c16:0 0.078c 0.067d 0.14a 0.057e 0.11b 0.0024 0.0001 c18:1n-/c18:0 2.92b 2.41c 3.28a 1.77d 1.51e 0.072 0.0001 mufa/sfa 1.20b 1.07c 1.31a 0.81d 0.75e 0.019 0.0001 pufa/sfa 0.31b 0.25c 0.22cd 0.21d 0.41a 0.011 0.0001 n-6/n-3 12.52b 12.03bc 19.08a 11.00cd 10.32d 0.44 0.0001 n = number of observations, sfol= subcutaneous backfat outer layer, sfil = subcutaneous backfat inner layer, ldif = longissimus dorsi intramuscular fat, of = omental fat, hf = hepatic fat. sem = error standard of the mean. sfa, mufa, pufa, n-6, n-3 = sum of all saturated (sfa), monounsaturated (mufa), polyunsaturated (pufa), n-6 and n-3 fatty acids. ital. j. food sci., vol. 29, 2017 662 table 4. correlation coefficients (r) between fat thickness (mm), at the level of gluteus medius muscle, and major fatty acid proportions (%) in outer and inner backfat layers. fatty acid (outer layer) r fatty acid (inner layer) r c16:0 0.64**** c16:0 0.55*** sfa 0.61**** sfa 0.58*** c18:1 n-9 0.41** c18:1 n-9 0.40** mufa 0.44** mufa 0.45** c18:2 n-6 0.53*** c18:2 n-6 -0.52*** pufa 0.54*** pufa -0.52*** n = 47, ** p<0.01, *** p<0.001, **** p<0.0001. sfa, mufa, pufa = sum of all saturated (sfa), monounsaturated (mufa) and polyunsaturated (pufa) fatty acids. 4. discussions the fact that omental and liver fat were more saturated than subcutaneous and intramuscular fat, is explained by the activity of lipogenic enzymes involved in de novo synthesis, which vary with tissue location (narvaez-rivas et al., 2009). thus, in our experiment, the highest elongase index values (c18:0/c16:0) were detected in liver and omental fat whereas the thioesterase index (c16:0/c14:0) was higher in the liver, omental and inner subcutaneous backfat layer compared to the other fatty tissues. these results are in agreement with dominguez et al. (2014), who observed in the celta breed pigs higher values of elongase and thioesterase indexes in internal perirenal fat than in subcutaneous and intramuscular fat. according to bee (2001), lipogenic activity of malic enzyme was higher in omental fat than inner and outer subcutaneous backfat layers, but fatty acid synthase activity was similar in these tissues, for different dietary fat types. on the other hand, the activities of both enzymes were similar in the inner and outer subcutaneous backfat layers when pigs received the same energy and fat content in feed as in this experiment. moreover, benitez et al. (2012) found in iberian pigs fed saturated (5% hydrogenated lard), monounsaturated (5% high oleic sunflower) or polyunsaturated (5% sunflower) enriched diets that liver fat was more saturated than inner and outer subcutaneous backfat layers and longissimus dorsi intramuscular fat. in our study, the outer subcutaneous backfat layer was more monounsaturated and polyunsaturated and less saturated than the inner layer, which is consistent with the results observed by irie and sakimoto (1992), bee et al. (2002), daza et al. (2007) and monziols et al. (2007). in a later study, bee et al., (2002) observed that the activity of the different lipogenic enzymes was not uniform in the outer and inner subcutaneous backfat layers. hence, these authors reported that the activity of malic enzyme was lower in the inner than outer subcutaneous backfat layer, the activity of glucose 6-phosphate dehydrogenase was similar in both layers, whereas the fatty acid synthase enzyme activity was higher in the inner than in the outer layer. the metabolic principles, on which the deposition of linoleic acid is higher in the outer than inner subcutaneous backfat layers in pigs, have not been sufficiently explained (monziols et al., 2007). the higher de novo synthesis of sfa (especially c16:0) in the inner subcutaneous backfat layer resulted in a higher dilution of c18:2n-6 in the inner than outer subcutaneous backfat layer, in agreement with the results of the regression equations obtained between the proportions of c18:2n-6 and c16:0. the slope of the regression equation corresponding to the inner subcutaneous backfat layer was higher ital. j. food sci., vol. 29, 2017 663 than in the outer layer (0.823 vs 0.750, p<0.01). this means that as the c16:0 proportion increased in subcutaneous backfat, the decrease in the proportion of c18:2n-6 was higher in the inner than in the outer layer. these results are in agreement with those obtained by christie et al. (1972) and monziols et al. (2007). according to the earlier study of thompson and allen (1968), the degree of unsaturation of the fatty tissue in swine decreases from external to internal tissues, so that the high-to-low unsaturation gradation follows the series: outer subcutaneous layer, middle subcutaneous layer, inner subcutaneous layer, intramuscular fat and internal fat (omental, perirenal, etc.) (villegas et al., 1973; monziols et al., 2007). dean and hilditch (1933) reported that this gradation can be explained by a possible adaptation of adipose tissue to temperature in order to maintain adequate physical fluidity of lipids in different adipose tissues, so that the melting point of the fat would increase from subcutaneous fat to internal locations. in this study, the unsaturation of longissimus dorsi intramuscular fat was similar to the outer subcutaneous backfat layer and higher than the internal and omental fat, due to high proportions of c16:1n-7, c18:1n-9 and c18:1n-7 in intramuscular fat. unsaturation indexes such as c16:1n-7/c16:0, c18:1n-9/c18: 0 and mufa/sat, which estimate the activity of the enzyme delta-9 desaturase (responsible for the formation of c18:1n-9 from c18:0), were higher in the longissimus dorsi intramuscular fat than subcutaneous backfat, omental and hepatic fat. also, lopez-bote et al. (2002), bee (2001), bee et al. (2002) and bee et al. (2008) in improved white pigs, daza et al. (2014) in iberian pigs and dominguez et al. (2014) and dominguez and lorenzo (2014) in celta breed pigs found that intramuscular fat was more monounsaturated than outer and inner subcutaneous backfat and omental fat. however, lluch et al. (1993) found that the intramuscular fat was less monounsaturated than subcutaneous fat, although this result was obtained from male and female heterogeneous samples. franco et al. (2006) did not detect significant differences between the ratio of mufa/sfa in intramuscular fat and subcutaneous backfat in celta breed pigs, but observed lower concentrations of c18:1n-9 in intramuscular fat than subcutaneous backfat. monziols et al. (2007) found higher proportions of c18:1n-9 and mufa in subcutaneous backfat than intermuscular fat from the loin, but these authors did not provide data on fatty acid profile of intramuscular fat. omental fat showed lower (p <0.05) proportions of c18:1n-9 and mufa fatty acids than outer and inner subcutaneous backfat layers and intramuscular fat of longissimus dorsi. these results agree with those obtained by bee (2001) and bee et al. (2002). liver fat was the most polyunsaturated because the fatty acid profile in liver is largely influenced by diet. pig liver has little ability to synthesize fatty acids, hence fatty acids used in the synthesis of lipids are mainly derived from circulating fatty acids provided by adipose tissue or feed (mainly c18:2n-6). most triglycerides synthesized in the liver are esterified or stored, so that the composition is a reflection of the composition of plasma fatty acids, mainly, from the feed consumed (otten et al. 1993). also, dominguez et al. (2014) found in celta breed pigs, that liver fat was the most polyunsaturated when compared with the dorsal and ventral subcutaneous fat and longissimus dorsi intramuscular fat. the nutritional value of adipose tissues is related to high values of pufa/sat, mufa/sat ratios and low values for n-6/n-3 ratio. pufa/sat and n-6/n-3 values of the present study were similar to those obtained by dominguez and lorenzo (2014) and dominguez et al. (2014) in celta breed pigs. in turn, in agreement with the present study, dominguez et al. (2014) observed the highest value of pufa/sfa in liver fat and the lowest in longissimus dorsi intramuscular fat. wood and enser (1997), recommended values for pufa/sat higher than 0.45 and n-6:n-3 lower than 4.0 in contrast to the results found in the present study. the high n-6/n-3 fatty acid ratio of the present study is explained by the fact that pigs were fed a concentrate rich in ital. j. food sci., vol. 29, 2017 664 carbohydrates and c18:2 n-6. according to the results of the present study, the highest n-3 fatty acid proportion was found in omental and hepatic fat. these tissues would have lower lipid stability than the others (rey et al. 2001) and this may lead to more production of undesirable substances for consumers than potential benefits. regarding the curing process of meat and in order to obtain adequate fat quality, mourot and hermier (2001) recommended that pig adipose tissue should contain no more than 12% of c18:2n-6 and exceed 12% of c18:0. all adipose tissues studied in the present research were within those ranges. in the present study, we found positive correlations between backfat thickness and c16:0 and sfa proportions, whereas correlations were negative between thickness and c18:1n-9, mufa, c18:2n-6 and pufa proportions. monziols et al. (2007) found positive and negative correlation coefficients among pufa and sfa ratios and muscle percentage of carcass, which is negatively correlated with backfat thickness at gluteus medius level (irta, 2013). according to mourot and hermier (2001), a reduction of backfat thickness in pigs leads to an increase in c18:2n-6 proportion and a decrease in endogenous synthesis. in the present experiment, saturated and polyunsaturated fatty acids proportions and longissimus dorsi intramuscular fat percentages were positively and negatively correlated, respectively. these results explain that de novo synthesis of fatty acids is reduced in the leanest pigs and, consequently, c18:2n-6 is less diluted which is related to a higher content of c18:2n -6 in adipose tissue. 5. conclusions fatty acid profile varies according to fat tissue location probably due to variability of enzyme activity. in heavy pigs fed conventional feed, the omental and liver fats are the most saturated followed by the inner, outer subcutaneous backfat and intramuscular fat. the highest unsaturation corresponded to intramuscular fat, due to increased activity of the delta-9 desaturase enzyme, followed by outer, inner subcutaneous backfat, omental and liver. negative correlations were detected between the backfat thickness and the percentage of intramuscular fat with respect to the proportion of pufa, which is important given the effect of pufa content on susceptibility to oxidation. the fatty acid profile of adipose tissue from heavy pigs is suitable for the preparation of cured products, and intramuscular and outer backfat are the most suitable from the point of view of nutrition and health. acknowledgements this research was supported by inia (instituto nacional de investigación y tecnología agraria y alimentaria, spain) project (pet-2007-08-c11-04). references aoac. 2000. official methods of analysis. aoac , arlington, va. belitz h.d. and grosch w. 1997. química de los alimentos. ed acribia, 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fax 39 0512096017 e-mail address: maria.rodriguez@unibo.it abstract the content of ubiquinone (ubn) was evaluated in italian high-quality (hq) raw cow milk. samples were collected from four cowsheds in two different days during summer and winter. the fat content in hq raw cow milk ranged between 2.86% and 3.46%, while ubn content varied between 0.15 and 0.45 µg/g milk. the fat content was significantly influenced by the cowshed only, whereas the ubn content was significantly more affected by both season and sampling days. although ubn is a lipophilic antioxidant, no statistically significant correlation was found between ubn and fat content in hq raw cow milk. keywords: ubiquinone, coenzyme q10, lipophilic antioxidant, holstein-friesian cows, high-quality raw cow milk, season, cowshed ital. j. food sci., vol. 30, 2018 145 1. introduction cow milk is undoubtedly the most frequently consumed dairy product (merdji et al., 2015), due to its nutritional composition and properties (assolatte, 2006). the dairy industry offers many product categories with diverse characteristics, based on consumers’ requests and nutritional requirements. the quality of raw milk is greatly affected not only by the technology used for the preservation and diversification of milk products, but also by the characteristics of raw milk itself. the ec regulation 853/2004 defines raw milk as the product of the mammary gland secretion of farmed animals that has not been heated to more than 40 °c and has not been subjected to any treatment; the same regulation also states the health requirements for raw milk production and its standards of quality. raw drinking milk can contain pathogenic microflora, so consuming it can pose a significant public health risk (efsa, 2015). in some eu countries (ec regulation 853/2004; intesa stato-regioni 25/01/2007), the sale of raw milk is allowed through vending machines, but current law clearly states that it must be heat-treated before being consumed (ec regulations 852/2004 and 853/2004). the overall quality of raw milk will thus be highly dependent on the breeding conditions and hygienic controls, which will give rise to diverse quality-labelling categories among which that labelled ‘high-quality’ (hq) milk is considered the best commercialised one; in fact, the compositional profile of hq milk is most similar to that of raw milk. the italian ministerial decree 185/1991 imposes rigorous breeder management and hygienic controls aimed to obtain hq milk, which should fulfil the following stringent sanitary and quality requirements: fat content and protein content > 3.50% and 32.0 g/l, respectively; bacterial load < 100.000/ml at 30 °c; somatic cells < 300.000/ml; lactic acid content < 30 ppm; level of non-denatured soluble serum proteins > 15.50% of the total protein (when ready for consumption). the italian legislation n.169/1989 actually defines hq fresh pasteurised milk, stating that to further preserve the quality of hq milk, pasteurization is always necessary and must be performed within 48 h after milking. cow milk contains many health promoting compounds, such as vitamins (claeys et al., 2014) and ubiquinone (ubn), also known as coenzyme q10 (mattila and kumpulainen, 2001). ubn is present in all cells and membranes, and it has been reported to increase the energy level, to augment the immune system, to act as an antioxidant, to exert a protective effect on the cardiovascular system, and to guard against skin aging and neurodegenerative diseases (hemat, 2004; prahl et al., 2008; kewal, 2011; saini, 2011, hechtman, 2011; amar-yuli et al., 2009; quinzii and hirano, 2010). as a lipophilic substance, ubn is absorbed following the same process as that of lipids in the gastrointestinal tract, being first incorporated into chylomicrons, followed by absorption and transportation via the lymphatics to the circulatory system (bhagavan and chopra, 2006). due to its high molecular weight and low water solubility, ubn is poorly and slowly absorbed (tmax 2-10 h) from the gastrointestinal tract (seo et al., 2009). pakamula et al. (2005) in fact observed different regional permeability of ubn in isolated rat gastrointestinal tracts, suggesting that ubn formulations should target the duodenum to get maximum dosage effect; to compensate for its low absorption rate, diverse strategies can be adopted to enhance ubn bioavailability, such as particle size reduction, solubility improvement (i.e. solid dispersion, complexation, ionization), and use of carriers (i.e. liposomes, microspheres, nanoemulsions, nanoparticles, self-emulsifying systems) (beg et al., 2010). once ubn is slowly absorbed from the small intestine, it passes into the lymphatics, and finally to the blood and tissues (garrido-maraver et al., 2014). once ubn reaches the tissues, it is quickly broken down (short half-life of 49-125 h) and degraded by ω-oxidation and β-oxidation of its side-chain (thelin et al., 1992). the main breakdown product found in tissues, urine and faeces has an intact, fully substituted ital. j. food sci., vol. 30, 2018 146 ring, a short side-chain (5-7 carbon atoms) and a carboxylated ω-terminus (nakamura et al., 1999). by using labelled ubn, it has been demonstrated that ubn is metabolised in all tissues. the metabolites are converted into more hydrophilic compounds (mostly in their phosphorylated form) in the cells, transported in the blood to the kidney, and excreted into the urine (bentinger et al., 2003); however, other minor metabolites were also detected in the faeces, which contained non-metabolised labelled ubn, excreted through the bile. under certain physical conditions (such as aging, cardiomyophathies, degenerative muscle diseases, and carcinogenesis), ubn concentration can diminish greatly (bentinger et al., 2003). ubiquinone deficiency may be due to insufficient dietary intake, impairment in ubn biosynthesis, excessive utilization by the body or a combination of any of these three (fedacko et al., 2011). ubn deficiencies are clinically and genetically heterogeneous. this syndrome has been associated with five major clinical phenotypes: (1) encephalomyopathy, (2) severe infantile multisystemic disease, (3) cerebellar ataxia, (4) isolated myopathy, and (5) nephrotic syndrome (quinzii and hirano, 2011). several studies show that milk is a natural source of ubn, whose concentration level varies from species to species and is influenced by the lactation stage and the heat treatment during processing (mattila and kumpulainen, 2001; strazisar et al., 2005; niklowitz et al., 2005; tang et al., 2006; quiles et al., 2006). mattila and kumpulainen (2001) assessed the levels of coenzyme q9 (coq9) and ubn in milk purchased from major dairies, finding only ubn at a concentration level equal to 0.1 µg/g in milk (1.5% fat). strazisar et al. (2005) also evaluated the ubn content of fresh cow milk produced in a slovenian farm (3.6% fat), cow milk from the alpine region (3.5% fat), and ultra-heat-treated homogenised milk (3.5% fat), finding a ubn content equal to 1.90 µg/g, 1.57 µg/g, and 1.70 µg/g, respectively. as reported earlier, most studies have been carried out on pasteurised milk, but to the best of our knowledge, there is no report available in the literature about the ubn content in hq raw cow milk. the aim of this survey was to evaluate the level of ubn in italian hq (“alta qualità”) raw cow milk. to this purpose, milk samples were collected from four different italian cowsheds producing hq milk, in two different days during both summer and winter. raw cow milk samples were analysed for both fat and ubn content. 2. materials and methods 2.1. chemicals all reagents and solvents used were analytical grade chemicals. potassium hydroxide pellets (≥ 85%) and pyrogallol (≥ 98%), were purchased from carlo erba (milan, italy). commercial standards of ubn (≥ 98%, hplc) and coq9 (≥ 96%, hplc) were supplied by sigma-aldrich co. (st. louis, mo, usa), whereas bidistilled water (100%) was purchased from panreac (barcelona, spain). petroleum ether with a boiling range 40-60°c (≥ 95.0%), diethyl ether (≥ 99.5%), ethanol (≥ 99.9%), acetonitrile (≥ 99.9%, hplc), n-pentane (≥ 99.0%), anhydrous sodium sulphate (≥ 99.9%), ammonia solution 14 m, and 2-propanol (≥ 99.8%, hplc), were supplied by merck kgaa (darmstadt, germany). acetonitrile and 2propanol were degassed before use by filtering under vacuum through a 0.20 µm nylon membrane filter (phenomenex, westboro, ma, usa). ital. j. food sci., vol. 30, 2018 147 2.2. sampling and experimental design milk samples were obtained from holstein-friesian cows that were bred in four different cowsheds. the latter belonged to medium/large-sized italian farms focusing on the production of hq milk and located in the valley area between the provinces of bologna, mantova, and modena (italy). the management of these farms is characterised by a particular attention to hygienic conditions, nutrition and animal welfare, which are the basic requirements for obtaining hq milk as regulated by the italian d.m. 185/1991. in these farms, unifeed or "single pot" is used for feeding, with the objective of providing a food ration that is homogeneously mixed, properly formulated, and nutritionally balanced. the daily dose administered consisted of 20-25 kg of unifeed per milk cow. ubn in the food ration is provided mainly by the forage, which is almost exclusively hay from medicago sativa l. ubn was evaluated in hay specimens at different mowing times and was found to be present in an average content of 11 mg/kg; therefore, the amount of presumed ubn taken in the daily ration could be estimated around 46-58 mg. two milk samples were collected from each cowshed (cs) in two different sampling days (sd, i and ii) and two diverse seasons (s, summer and winter), thus giving a total of 16 (4x2x2) samples that were analysed in duplicate (32 analysis in total). raw cow milk samples were collected from bulk tanks containing the morning and evening milk of the entire herd. the collected milk was placed in 1-l pet bottles and kept at 2-6°c during sample delivery. the milk samples were then divided into 100-ml pet bottles, frozen and stored at -20°c until subsequent analysis. all milk samples were analysed for fat content (by cold extraction), as well as for ubn content (by direct cold saponification followed by extraction of the unsaponifiable matter and high-performance liquid chromatographyultraviolet diode-array (hplc-uv/dad) analysis. 2.3. lipid extraction lipids were extracted according to the iso 14156:2001 method (idf 172:2001). an aliquot of 100 ml (at 20 °c) milk was introduced into a 500-ml separatory funnel. eighty ml of ethanol, 20 ml of 14 m ammonia aqueous solution and 100 ml diethyl ether were added, and the funnel was shaken vigorously for 1 min. thereafter, 100 ml of n-pentane were added and the funnel was gently shaken. after phase separation, the aqueous phase was discarded. the organic phase (lipid-containing one) was washed twice with 100 ml of 10% (w/v) sodium sulphate aqueous solution. the organic phase was transferred into a 250ml erlenmeyer flask fitted with a ground glass stopper and approximately 10 g of anhydrous sodium sulphate were added. the flask was stoppered, well shaken, and allowed to stand for 10 min. the organic phase was then filtered into a 100-ml roundbottom flask through a whatman no. 1 filter paper, dried at 40°c using a vacuum rotary evaporator and at the end dried under a gentle stream of nitrogen. the fat content was gravimetrically determined. two replicates for each sample were performed. 2.4. saponification and extraction of the unsaponifiable matter the sample preparation method for the quantification of ubn in milk consisted of a direct cold saponification of milk followed by the extraction of the unsaponifiable matter. the direct cold saponification was performed according to the modified method of renken and warthesen (1993). about 35 g of milk were weighed into a 100-ml glass bottle with screw cap. fifteen micrograms of coq9 (internal standard), 35 ml of 2 n potassium hydroxide in 85% ethanol, and 20 ml of 1% (w/v) pyrogallol in ethanol were added. the headspace of the bottle was flushed with a nitrogen stream to remove the oxygen, ital. j. food sci., vol. 30, 2018 148 stoppered, and kept at room temperature for 18-20 h, in the dark and under continuous agitation (180 oscillations/min). after the saponification had taken place, the alcoholic soap solution was transferred into a 250-ml separatory funnel to extract the unsaponifiable matter containing ubn. the unsaponifiable portion was extracted by adding consecutively 15 ml of bidistilled water, 5 ml of ethanol, and 35 ml of a petroleum ether:diethyl ether mixture (9:1, v/v), under shaking. after phase separation, the aqueous phase was transferred into a 150-ml separatory funnel. five ml of ethanol and 35 ml of a petroleum ether:diethyl ether mixture (9:1, v/v) were added, shaken, and allowed to stand until phase separation. the two ethereal fractions were combined in the 250-ml separatory funnel and washed until neutrality was reached by using cold bidistilled water (approximately 3 x 30 ml). the ethereal extract was transferred into a 100-ml erlenmeyer flask fitted with a ground glass stopper; next, approximately 5 g of anhydrous sodium sulphate were added. the flask was stoppered, shaken well, and allowed to stand for 60 min. the ethereal extract was then filtered into a 100-ml roundbottom flask through a whatman no. 1 filter paper and dried at 40 °c using a vacuum rotary evaporator. the unsaponifiable matter was dissolved in 1 ml of 2-propanol and filtered through a 0.45 µm nylon syringe-type filter (econofilter, 25-mm diameter, agilent technologies, wilmington, de, usa), before injection into a hplc system. two replicates for each sample were performed. 2.5. hplc-uv/dad determination of ubn the separation of the compounds of interest (ubn and coq9) was performed as suggested by rao et al. (2008), with minor modifications. a hplc system (hp 1050 series; hewlettpackard, palo alto, ca, usa) consisting of an autosampler (series 1100), a quaternary pump, a uv/dad detector and a poroshell 120 ec-c18 (3.0 x 50 mm x 2.7 μm particle size) analytical column (agilent technologies, santa clara, ca, usa), was used. ten microliters of the sample solution were injected in isocratic mode, using a mixture of acetonitrile:2-propanol (70:30, v/v) at a flow rate of 1.2 ml/min. the column temperature was maintained at room temperature (25°c). the detection wavelength of the uv/dad detector was set at 275 nm as suggested by kommuru et al. (1998). data were acquired using chemstation for lc3d software (agilent technologies, palo alto, ca, usa). standard solutions (coq9 and ubn) were prepared in 2-propanol and stored at -20°c in amber vials until further analysis. their concentrations were periodically checked by measuring the absorbance at 275 nm using a uv-visible spectrophotometer (v-550; jasco, tokyo, japan) and using the known molar extinction coefficients for coq9 (e1%1cm 185) and ubn (e1%1cm 165) as reference (hatefi, 1963; souchet and laplante, 2007). two replicates were performed for each sample. the limits of detection (lod) and quantification (loq) were determined according to vial and jardy (1999), with a signal-to-noise ratio equal to 3 and 10, respectively. lod of ubn was 0.35 µg/ml, while its loq was equal to 1.18 µg/ml. the content of ubn was calculated using the following equation [1]: ubn (µg/g milk) mis isa wa ca 1 × × = (1) where: aa is the peak area of the analyte; ais is the peak area of the internal standard; cis is the concentration of the internal standard, in µg; wm is the weight of the milk sample, in g. ital. j. food sci., vol. 30, 2018 149 to identify coq9 and ubn in milk samples, commercial standards of both compounds were individually injected into the hplc system and their corresponding chromatographic retention times were compared with those of the unknown peaks in milk samples. the identification of ubn in milk samples was further confirmed by lc-ms analysis. an analytical column kinetex 5 µm (c18 100a) (phenomenex, torrance, ca, usa) was used. fifty microliters of the sample solution were injected in isocratic mode. the eluent mixture was made up of methanol:2-propanol (70:30, v/v) at a flow rate of 1.0 ml/min. the hplc flow was split in two detectors in parallel, dad and electrospray interface (esi), through a three-way valve. esi was used in positive mode at a voltage of 4.4 kv (lcq duo mass spectrometer, thermo finnigan, san josé, ca, usa). the flow was 0.1 ml/min and the temperature was 200°c. the presence of ubn was monitored at m/z 880, which corresponds to the molecular weight of ubn (863.3) + nh4–h. the dad detector (varian mod. 330) was set at 275 nm. 2.6. statistical analysis to perform the statistical tests, minitab software (version 16.1.0; lead technologies, inc., charlotte, nc, usa) was used. the analysis of variance (anova) on the whole set of sample data was assessed, as well as the effects of season (s), cowshed (cs), sampling day (sd), their first-degree interactions (s × cs, s × sd, and cs× sd), and second-degree interaction (s × cs × sd) on fat and ubn contents. tukey’s honest significance test was carried out at a 95% confidence level (p < 0.05). the percentage contribution of each factor and interaction was calculated using eta-squared values from the anova summary table. the pearson’s correlation (α= 0.05) with two-tailed probability value was used to estimate the strength of association between fat content and ubn content. 3. results the fat and ubn contents of sixteen samples of hq raw cow milk were analysed in duplicate. table 1 reports the fat content (%) and the ubn content (expressed as µg/g milk) of hq raw cow milk samples. data correspond to the mean of two analytical determinations. a three-factor experimental design was used. each factor was set at different levels (season (s)-2 levels, cowshed (cs)-4 levels, and sampling day (sd)-2 levels). the fat content ranged from 2.9% to 3.5%, whereas the ubn content varied from 0.15 to 0.45 µg/g milk. table 1. contents of fat (%) and ubn (µg/g milk) in raw cow milk. season (s) cowshed (cs) sampling day (sd) fat ubn summer 1 i 2.89±0.08d 0.177±0.004hi ii 3.22±0.00bc 0.149±0.001i 2 i 3.34±0.02ab 0.198±0.013gh ii 2.95±0.02d 0.451±0.000a 3 i 2.97±0.03d 0.236±0.019fg ii 3.43±0.02a 0.274±0.015ef 4 i 3.01±0.05d 0.192±0.009ghi ii 2.94±0.02d 0.312±0.002de ital. j. food sci., vol. 30, 2018 150 winter 1 i 3.00±0.10d 0.351±0.007cd ii 2.98±0.06d 0.378±0.010bc 2 i 3.46±0.03a 0.355±0.020cd ii 3.31±0.00abc 0.413±0.003ab 3 i 2.93±0.00d 0.294±0.015e ii 2.92±0.01d 0.236±0.006fg 4 i 3.18±0.03c 0.421±0.019ab ii 2.86±0.01d 0.420±0.003ab values are expressed as mean±standard deviation of two replicates. i and ii correspond to 2 independent sampling days. different letters within the same column denote statistically significant differences at p < 0.05 (tukey’s test) between the milk samples; the letter "a" indicates the highest value and "e" the lowest one. figure 1 shows the hplc-uv/dad chromatograms (at 275 nm) of coq9 standard solution, the unspiked and spiked (with ubn) unsaponifiable fraction of hq raw cow milk. since coq9 was absent in hq milk, it was therefore used as internal standard (is) in the quantitative determination of ubn. figure 1. overlaid hplc-uv/dad chromatograms (at 275 nm) of the unspiked (a) and the spiked (with ubn) unsaponifiable fraction of raw cow milk (b), as well as the coq9 standard solution (c). peak identification: 1, coq9 internal standard; 2, ubn. ital. j. food sci., vol. 30, 2018 151 4. discussion the fat content was determined to find out whether there was a correlation with the level of ubn in the hq milk samples. data analysis (table 2) was carried out to emphasise the effect of each individual factor and their first-and second-degree interactions on fat and ubn contents of hq raw cow milk. regarding milk fat content, data processing showed that it was significantly influenced by cowshed (cs) (28.1%) and by all interactions. however, season (s) and sampling day (sd) factors had no significant effects on the hq raw cow milk fat content. the fat content of hq milk significantly varied among cowsheds; in general, the highest milk fat content was found at cs no. 2, followed by 3, 1, and 4. this variation may be due to feeding with different fodders and concentrates, the lactation stage of individual animals from herds, the variability among animals and/or the cs location. the average fat content of milk varies considerably through lactation, from approximately 3% in early lactation to more than 4.5% in late lactation, and among individuals (fox and kelly, 2012). concerning the ubn content of hq milk, it was influenced by all factors and their interactions (table 2); however, the factors that exerted the greatest influence were s (32.9%) and cs (19.5%). the highest content of ubn was found in milk from cowshed no. 2, in which the cs x sd interaction indicated that the ubn milk content varied according to the sampling day. table 2. effects of season, cowshed, sampling day, and their firstand second-degree interactions on fat (%) and ubn contents (µg/g milk). factor fat ubn season (s) summer 3.09 0.249b winter 3.08 0.358a p/contribution (%) 0.353 n.s./0.1 < 0.001***/32.9 cowshed (cs) cowshed 1 3.02bc 0.264c cowshed 2 3.27a 0.354a cowshed 3 3.07b 0.260c cowshed 4 3.00c 0.336b p/contribution (%) < 0.001***/28.1 < 0.001***/19.5 sampling day (sd) i 3.10 0.278b ii 3.08 0.329a p/contribution (%) 0.148 n.s./0.3 < 0.001***/7.1 s x cs summer x cowshed 1 3.06cd 0.163e summer x cowshed 2 3.15bc 0.324c summer x cowshed 3 3.20b 0.255d summer x cowshed 4 2.98de 0.252d winter x cowshed 1 2.99de 0.364b winter x cowshed 2 3.38a 0.384b winter x cowshed 3 2.93e 0.265d winter x cowshed 4 3.02de 0.420a p/contribution (%) < 0.001***/21.6 < 0.001***/16.7 ital. j. food sci., vol. 30, 2018 152 s x sd summer x i 3.06b 0.201c summer x ii 3.13a 0.297b winter x i 3.14a 0.355a winter x ii 3.02b 0.361a p/contribution (%) < 0.001***/6.5 < 0.001***/5.5 cs x sd cowshed 1 x i 2.94c 0.264d cowshed 1 x ii 3.10b 0.263d cowshed 2 x i 3.40a 0.276d cowshed 2 x ii 3.13b 0.432a cowshed 3 x i 2.95c 0.265d cowshed 3 x ii 3.18b 0.255d cowshed 4 x i 3.10b 0.306c cowshed 4 x ii 2.90c 0.366b p/contribution (%) < 0.001***/29.6 < 0.001***/11.9 s x cs x sd p/contribution (%) < 0.001***/11.7 < 0.001***/5.6 values are expressed as mean of two replicates. abbreviations: cs, cowshead; s, season; sd; sampling day; i and ii correspond to 2 independent sampling days. different letters within the same column denote statistically significant differences (tukey’s test p < 0.05) between the milk samples; the letter "a" indicates the highest value and "e" the lowest one. significant differences are denoted by asterisks: *p < 0.05; **p < 0.01; ***p < 0.001; p ≥ 0.05, non-significant. the level of ubn in milk may be influenced by genetic characteristics, diet, duration of lactation, length of gestation (tang et al., 2006), and metabolic changes (souchet and laplante, 2007). previous studies on the quantification of ubn were carried out using various dairy product categories that contained different fat percentages and that underwent diverse heat treatments (mattila and kumpulainen, 2001; strazisar et al., 2005). thus, the comparison with our results is difficult. strazisar et al. (2005) reported a higher ubn content (1.90 µg/g milk) in fresh cow milk (3.6% fat) than that detected in the present study (0.15-0.45 µg/g milk). the same authors noticed that ubn levels were lower in uht milk having diverse fat content (0.46 µg/g milk in skimmed milk (0.5% fat), 1.16 µg/g milk in semi-skimmed milk (1.6% fat), and 1.70 µg/g milk in whole milk (3.5% fat)). however, mattila and kumpulainen (2001) found a significantly lower ubn content (0.1 µg/g milk) in commercially available semi-skimmed milk (1.5% fat), probably due to the heat treatment. as already mentioned, ubn is known to be a temperature sensitive molecule; milivojevic fir et al. (2009) in fact showed that pure ubn is degraded by 72.3% after being exposed at 80°c for 120 min in the presence of uv light. regarding the correlation of the fat and ubn contents in hq raw cow milk samples, the results of the current study did not show any significant relationship (r= 0.034, p = 0.855). this lack of correlation is further confirmed by the scatter plot (figure 2) developed with the fat and ubn contents (linear regression and no fit intercept). this is in contrast to the data of strazisar et al. (2005), who noticed a positive correlation trend between the ubn and the fat contents of milk, even though it was not statistically confirmed. on the other hand, niklowitz et al. (2005) found a high level of ubn in human colostrum, even though its fat content was low. ital. j. food sci., vol. 30, 2018 153 figure 2. scatter plot of fat content vs. ubn content. abbreviations: s, summer; w, winter; 1-4, number of single cowsheds; i, sampling day 1; ii, sampling day 2. the scatter plot of fig. 2 also displays the distribution of the samples according to both variables (ubn and fat content). only two groups can be distinguished in terms of fat content: one below 3.02% (10 samples) and the other above 3.17% (6 samples). no more clustering was evident as related to the rest of variables (cowshed, season, sampling day). 5. conclusions the content of ubn in italian hq (“alta qualità”) raw cow milk was for the first time determined in the present study, ranging from 0.15 to 0.45 µg/g milk. although health authorities have not yet established specific dietary intake recommendations for ubn, some researchers suggest a daily dose of 30-200 mg ubn for 19-year adult and older (efsa, 2010). considering that the guidelines for healthful italian food habits published by the italian national institute for research on food and nutrition (inran, 2003) recommend an average consumption of three 125-ml portions of milk and/or yogurt a day, a consumption of 250 ml of hq raw milk would potentially provide an intake of 0.04-0.11 mg ubn. however, ubn content in pasteurised hq milk may suffer a decline due to the heat treatment (especially uht) and storage, even though modern pasteurization technologies applied to hq milk with relatively low temperatures and short times (72°c for 15 sec) should limit significant thermal degradation of ubn. the results of this study indicate that the ubn content in hq raw cow milk was significantly affected by both seasons and sampling days within the same cowshed. however, no significant correlation was found between ubn and fat content in hq raw cow milk, even though ubn is a lipophilic antioxidant. although an investigation involving a greater number of different cowshed types from diverse locations and product categories would be necessary to better understand the contribution level of the various factors on the increase of ubn content in milk, the adoption of the italian hq regulation by the eu could be a way to further improve the nutritional quality of european milk, including its ubn content. ital. j. food sci., vol. 30, 2018 154 acknowledgements the authors would like to thank dr. andrea borsari from granarolo s.p.a. 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fax: +52 444842435 e-mail address: iizd@uaslp.mx abstract the prickly pear (opuntia spp.) usually is consumed as fresh fruit. in this study of prickly pear juice in vitro we characterized and quantified secondary metabolites including antioxidant capacity of ten opuntia spp. variants. gallic acid was abundant in most variants. catechin and epicatechin isomers, and procyanidins b1 and b2 were present in most variants. ascorbic acid content was higher than 84 mg. betacyanins stand out in redcolored juices, betaxanthins in the yellow ones; this caused lack of relationship between antioxidant capacity and total phenolic content. the soluble fiber content, sugars, betalains and ascorbic acid position this juice as a functional food. keywords: abts, antioxidant, betalains, frap, juice, prickly pears ital. j. food sci., vol. 30, 2018 615 1. introduction normal metabolic processes produce free radicals, which cause oxidative damage. the human body possesses endogenous antioxidant mechanisms using substances that significantly delay or prevent oxidation. these substances include the cellular enzymes superoxide dismutase, glutathione peroxidase and catalase (wang and quinn, 2000). there are also non-enzymatic defense substances against oxidation stress, including vitamin e, an effective antioxidant of polyunsaturated membrane lipids, and vitamin c which, as a reducing agent or electron donor, reacts rapidly with the hoand the superoxide anion and also prevents the oxidation of membrane lipids (wang and quinn, 2000). when endogenous antioxidant mechanisms are insufficient to offset the imbalance resulting from oxidative stress, physiological and biochemical changes take place such as protein glycosylation, lipid peroxidation, and glucose auto-oxidation (opera, 2004). diseases associated with oxidative stress include type-2 diabetes mellitus (dm2), hypertension, renal and hepatic impairment, cancer, and neurodegenerative diseases such as amyotrophic lateral sclerosis, alzheimer's, parkinson's and huntington (jellinger, 2003; d’amico et al., 2013). the intake of natural or synthetic antioxidants can reinforce the antioxidant capacity of the organism (hidalgo et al., 2006). recent research has shown that certain compounds present in plants, such as terpenes, flavonoids, betalains and anthocyanins, possess antioxidant properties that are more powerful than those of vitamins (harasym and oledzki, 2014). global trends in food and nutrition indicate a growing interest in the consumption of fruits and vegetables, given their nutritional value and benefits for the functions of the human body. these trends in eating patterns have led to a new area of research and development in nutrition related to the so-called "functional foods", defined as any food, either natural or processed, which in addition to its nutritional components contains substances that boost a person’s health, physical ability and mental state (konigsbergfainstein, 2008). functional compounds include exogenous antioxidants, which safely interact with free radicals and disrupt their chain reaction before they damage vital molecules (oroian and escriche, 2015). the prickly pear, the fruit of cacti of the genus opuntia is widely available throughout mexico’s south highland. more than 189 species of wild prickly pear cacti are known, 83 of which are mexican; of these, 29 are distributed in the north-central region of mexico, in an area of approximately 300 000 km2 that stretches across part of the states of aguascalientes, guanajuato, hidalgo, jalisco, queretaro, san luis potosí, zacatecas, and around méxico city. in this area, a number of variants with different degrees of humanization can be found, from the wild o. streptacantha and the cultivated o. hyptiacantha, o. megacantha and o. albicarpa, to o. ficus-indica, the species considered as the one with the highest degree of domestication (reyes-agüero et al., 2005). prickly pears are consumed mainly as fresh fruit and display marked differences in size, shape, color and flavor, as well as in seed quantity, size and hardness; prickly pear is also processed to produce jelly, jam and paste. chemical compounds found in prickly pear include polyphenols and betalains (figueroa-cares et al., 2010; yeddes et al., 2013). these antioxidant metabolites either prevent or control the excessive production of highly unstable free radicals and reactive molecules that have the ability to disrupt the functions of various biomolecules, i.e., oxidative stress (rodríguez et al., 2001; soobrattee et al., 2005). evaluations of the prickly pear fruit indicate its potential to be considered as a functional food due to its content of ascorbic acid, phenols, carotenoids and betalains at levels that ital. j. food sci., vol. 30, 2018 616 exceed those in plums, nectarines or peaches (fernández-lópez et al., 2010). these phytochemicals may contribute to mitigation of the effects of prolonged hyperglycemia and reinforcement of the antioxidant system in normal glycemic patients. antioxidants have been shown to increase the sensitivity of insulin receptors or may moderate the rise in blood glucose concentration after the ingestion of carbohydrates by inhibiting the action of digestive enzymes and glucose transporters sglt-1 (bryans et al., 2007). in addition, these phytochemicals have been associated with anti-inflammatory, antioxidant, immunomodulatory and apoptotic properties (kaulmann and bohn, 2016). phytochemicals, which locally reduce oxidative stress, are widely studied as cancerprotective agents (moore et al., 2016). indeed, animal assays indicate that supplementation with green and black tea (rich in polyphenolic compounds) led to a decrease in postprandial blood glucose levels in sprague-dawley rats (zeyuan et al., 1998). furthermore, in vivo studies showed a drop in glycosylated hemoglobin (fukino et al., 2008) and increased insulin activity after consumption of tea extracts (richarda and dolansky, 2002). based on the above, the objective of this study was to supplement existing assessments of prickly pear juice as a functional food by identifying and quantifying antioxidant compounds in the juice of fruits of opuntia and to investigate their antioxidant capacity in vitro. 2. materials and methods 2.1. selection of variants and sample preparation ten prickly pear variants, six of them cultivated, were evaluated as ripe fruits: rojo pelón (opuntia ficus-indica), blanca (o. albicarpa), amarilla monteza, pico chulo, torreoja and sangre de toro (o. megacantha), and four wild variants: cardona (o. streptacantha), charola (o. streptacantha ssp. aguirrana), tapona and tapón rojo (o. robusta). fruits were collected in the municipality of villa de arriaga, state of san luis potosí, méxico. opuntia variants were selected based on: (a) degree of humanization, (b) abundance and economic potential in the state of san luis potosí, and (c) fruit color. the skin of prickly pears was removed, then the juice was extracted from the pulp with a stainless-steel blender (international li-12-106), and seeds were separated with an 8 mesh filter; the juice was stored in sterile containers at -20°c until use. 2.2. total phenolic compounds total phenolic compounds in prickly pear juice was quantified using the folin-ciocalteu method modified by yeddes et al. (2013), and expressed as gallic acid equivalents (mg gae g-1). to extract phenols, cool absolute ethanol was added to 0.15 g of lyophilized juice stored at -50°c (frezer dryers iishin, corea), the mixture was sonicated for 10 min and then maintained under constant stirring for 2 h at 4°c. the solution was filtered through whatman grade 42 filter paper. extracts were brought to 15 ml with ethanol and stored protected from light at -20°c. total phenols were measured in triplicate; to this end, 437.5 µl of 1n folin-ciocalteu reagent (sigma) were added to 35 µl of the ethanol extract and were left to react at room temperature for 3 min. afterwards, 2187.5 µl of a 20% na2co3 solution were added and the volume was brought to 3500 µl. the mixture was left to stand at room temperature in the dark for 2 h for the development of color. absorbance was read at 760 nm in a spectrophotometer (agilent technologies, germany), using blank samples made of distilled water and the reagents used. the amount of phenolic ital. j. food sci., vol. 30, 2018 617 compounds was estimated by comparing the absorbance values of samples with those of the gallic acid standards. 2.3. phenolic acids and flavan-3-ols phenolic compounds were extracted with the method used by rodarte et al. (2007). two grams of lyophilized prickly pear juice were mixed with 3 ml of acidified methanol (0.1% hydrochloric acid), and sonicated in a water bath for 10 min; then, the supernatant was collected and the previous procedure was repeated five times with the precipitate to obtain six extractions. supernatants were collected and centrifuged for 10 min at 5000 rpm; the centrifuged fraction was concentrated under vacuum on a rotary evaporator at 30°c (heidolph, alemania) followed by reconstitution with 2 ml of methanol. all samples were filtered through 0.45 µm nylon filters. phenolic acids were identified and quantified in a liquid chromatograph (spectra-physics uv6000lp), with a lichrospher® 100 rp-18 column (250 mm x 4.6 mm, 5 μm particle size). formic acid (10% in water) (a) and acetonitrile/water/formic acid (45:45:10) (b) were used as mobile phase, with a flow rate of 1 ml/min. the identification was made by comparing the retention times and uv-vis spectra obtained using a diode array detection system (thermo scientific, usa), with reference standards. hydroxybenzoic acids were quantified at 280 nm; hydroxycinnamic acid esters, at 315 nm. flavan-3-ols were identified and quantified in a liquid chromatograph (thermo spectra physic series p100, usa), coupled to a fluorescence detector (perkin elmer series 200ª, usa), with a lichrospher® 100 rp-18 column (250 mm x 4.6 mm and 5 μm particle size). acetonitrile (a) and acetic acid (b) were used as mobile phase, with a flow rate of 1.4 ml/min. flavanols were identified using the wavelengths λexc = 280 nm and λem = 320 nm. in this study, procyanidins were quantified as catechins. 2.4. identification and quantification of betalains betalains were measured using the method of castellanos-santiago and yahia (2008). to do this, 100 mg of lyophilized prickly pear juice were weighed and 10 ml of 80% methanol acidified with 0.5% hcl were added; this mixture was sonicated for 15 min and filtered through a 0.45 µm nylon filter (agilent technologies, alemania). an electronic scan was run between 400 and 700 nm in an agilent 8453 uv-visible spectrophotometer (agilent technologies, alemania), which identified the absorption peaks of betalains at 547 nm and 490 nm. absorbance units were converted to concentration units. 2.5. ascorbic acid quantification ascorbic acid was quantified using the method of sdiri et al. (2012). to this end, 2 ml of 4.5% meta-phosphoric acid were added to 0.1 g of lyophilized juice; this mixture was sonicated in a water bath for 2 min, then centrifuged for 10 min at 5000 rpm, and finally filtered through 0.45 µm nylon filters. ascorbic acid was quantified in a hplc chromatograph (thermo spectra physic series p100), coupled to a uv detector (thermo finnigan spectra system uv2000), with a lichrospher® 100 rp-18 column (250 mm x 4.6 mm and 5 μm particle size), and kh2po4 (0.2 m at ph=2.3-2.4) was used as mobile phase, with a flow rate of 1.0 ml/min for 15 min at λ = 243 nm and an injection volume of 20 µl. this compound was estimated using the following calibration equation y = 76165x 161251, r2= 0.9993. ital. j. food sci., vol. 30, 2018 618 2.6. frap (ferric reducing antioxidant power) method the frap assay assessed the capacity of juice samples to reduce the ferric ion (fe+3) in a complex with 2,4,6-tri(2-pyridyl)-s-triazine (tptz), to the ferrous ion (fe+2), in accordance with firuzi et al. (2005). the frap reagent was prepared daily by mixing 10 ml of 300 mm sodium acetate buffer solution (ph 3.6) with 1 ml of 20 mm ferric chloride hexahydrate and 1 ml of 10 mm tptz dissolved in 40 mm hydrochloric acid. twenty five microliters of prickly pear juice diluted 1/20 with methanol were added to 96-well flat bottom microplates in triplicate, followed by the addition of 175 µl frap solution. a control treatment was prepared with 200 µl methanol; another control was prepared by mixing 25 µl methanol with 175 µl frap; for a third control, 25 µl ferric sulphate and 175 µl sodium acetate buffer were added; finally, 25 µl of juice sample were added to 175 µl sodium acetate buffer solution. readings were recorded with a multiskan ascent reader (thermo electron corporation 100-240 vac type: 354) at 595 nm. the first reading was taken at time 0, and the plate was incubated at 37°c immediately afterwards. the second reading was taken after 60 min. the calibration curve was constructed with ferric sulphate heptahydrate (7.194 mm) dissolved in methanol at concentrations of 108 µm to 864 µm. the resulting calibration equation was y = 0.0011x 0.069, r2= 0.9971. the frap value for the curve was calculated according to the following equation: 𝐹𝑅𝐴𝑃 𝑀 = ∆𝑎!𝐹𝐼 ∆𝑎!𝐹𝑒!! 𝑥10!! where ∆𝑎!𝐹𝐼 = change in absorbance of the analyte after the time interval. ∆𝑎!𝐹𝑒!!= change in absorbance of iron sulfate at the same concentration and after the time interval. the results of each sample are expressed as µm feso4 eq. 2.7. estimate of the trolox equivalent antioxidant capacity (teac) with the chemical mediator abts•⁺ this estimate was made in accordance with the methodology of nenadis et al. (2004). the teac of samples was based on 2,2'-azino-bis(3-ethylenebenzothiazoline-6-sulfonic acid) (abts), which produces the radical abts•⁺ and is compared with an antioxidant (trolox). for each evaluation, the abts•⁺ solution was prepared by mixing 5 ml of 7 mm abts and 88 µl of 140 mm potassium persulphate; the mixture was stored in the dark covered with aluminum foil and left to stand for 12 h at room temperature to produce the radical; afterwards, 500 µl of the solution were mixed with 25 ml of ethanol and its absorbance was read in an agilent 8453 uv-visible spectrophotometer (agilent technologies, alemania) to confirm that it was between 0.7 and 1 at 734 nm. samples and controls were measured in triplicate (20 µl sample plus 230 µl abts•⁺) and placed in a 96-well flat bottom plate; after adding the radical ion, the plate was covered with aluminum foil and after 6 min the reading was recorded at 734 nm in a multiskan ascent reader (thermo electron corporation 100-240 vac tip: 354). the percent inhibition of the standard was obtained with the following equation: ital. j. food sci., vol. 30, 2018 619 % inhibition = !"# !"#$%"&!!"# !"#$%& !"# 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 ∗ 100 the absorbance of the sample was subtracted from the absorbance of the control to obtain the true absorbance. the calibration curve was constructed with 50 µm, 100 µm, 200 µm, 300 µm, 400 µm and 500 µm trolox standards, adding 20 µl of the standard solution and 230 µl abts•⁺; the resulting regression equation was y = 0.2263x + 7.5033; r2= 0.9979. the results were expressed as µg·mol trolox equivalent (te) per gram of juice (dry weight). 2.8. experimental design and statistical analysis the experiment was performed according to a completely randomized experimental design. treatments were the juices from the 10 prickly pear variants, which were tested for content of phenolic compounds, betalains and ascorbic acid, as well as their antioxidant capacity through frap and abts. three replicates were used for each of these measurements. the data were subjected to an analysis of variance and tukey’s multiple comparison test. a pearson correlation was carried out between frap and abts variables (sas, version 8.0; sas institute, cary, north carolina). 3. results and discussion the potential of these prickly pear juices as functional foods is high due to outstanding content of soluble fiber and the adequate content and proportion of glucose to fructose (zenteno-ramírez et al., 2015). in order to determine whether consumption of prickly pear juices can help prevent or cure diseases associated with the excess of free radicals it is essential to identify and quantify the compounds with antioxidant capacity contained in prickly pear juice. phenols in fruits, flowers and vegetables have attracted the attention due to their antioxidant potential. it has been shown that various parts of opuntia (pulp, fruit skin, seeds and cladodes) are rich in polyphenols (galati et al., 2003; valente et al., 2010; tounsi-saidani et al., 2011). in addition, various studies have demonstrated their antioxidant effects (dok-go et al., 2003; tesoriere et al., 2004; siriwardhana and jeon, 2004; osorio-esquivel et al., 2011). 3.1. total phenol content due to their ubiquitous presence in plant foods, phenolic compounds are normally included in the daily human diet. the daily intake ranges between 25 mg and 1 g, depending on the amount of fruits, vegetables, pulses, tea and spices consumed (hagerman et al., 1998). raw extracts of phenol-rich plant products are attracting interest in the food industry, since these slow down the oxidative degradation of lipids, and hence improve the quality and nutritional value of food; their antioxidant power protects against heart disease and cancer, in addition to other chronic degenerative diseases (käkhönen et al., 1999). the protection against ldl oxidation is not due to a single compound, but results from the effect of several phenolic compounds (ricchelle et al., 2001). the total content of polyphenols was estimated in the ethanol extracts of lyophilized juice samples (table 1). the statistical differences between prickly pear variants seem to be unrelated to fruit color and degree of humanization, and it should be noted that the four ital. j. food sci., vol. 30, 2018 620 o. megacantha variants evaluated showed the highest total content of phenolic compounds. among the variants evaluated by mabrouki et al. (2015), the highest concentration was observed in the pulp of o. streptacantha, followed by o. ficus-indica, with 104.6 gae per 100 g of juice. in general, it has been pointed out that the concentration of phenolic compounds in prickly pears range from 54 mg/100 g to 104 mg/100 g fresh weight (katabi el al., 2013; figueroa-cares, et al., 2010). thus, the concentration of phenolic compounds in prickly pear juice is similar or higher than in pineapple, tomato, banana, mango and cucumber (1.7, 2.0, 2.3, 2.6, and 3.8, all in mg/g dry weight, respectively) (muñóz-jáuregui and ramos-escudero, 2007). 3.2. quantification of phenols by high performance liquid chromatography (hplc) table 1 shows the concentration of phenolic acids in the studied prickly pear variants, which show significant differences (p < 0.0001). gallic acid was recorded in all variants except tapona, and was the main phenolic compound in most of them, with varying concentrations between 32.6 µg/g and 81.2 µg/g. syringic acid was absent only in torreoja and cardona, and ellagic acid in blanca, sangre de toro and tapón rojo. protocatechic acid was recorded only in pico chulo (41.6 µg/g). pico chulo showed the four phenolic acids and recorded the highest total phenolic acid concentration (176 µg/g). by contrast, blanca showed the lowest total phenolic acid content (79.4 µg/g). table 1. average concentration (µg/g) of total phenols and phenolic acids in lyophilized juices of 10 prickly pear variants. variant* total phenols gallic acid syringic acid ellagic acid total phenolics acids rojo pelón 1.92±0.11de 32.6±0.6g 29.2±0.9d 25.0±0.9e 86.9±2.3e blanca 1.93±0.25de 53.7±0.6e 25.6±0.4e n.d. 79.4±0.8e amarilla monteza 3.81±0.75bc 74.8±3.6bc 13.6±0.3h 33.5±0.1d 122.0±3.8b pico chulo 2.81±0.39bcd 63.6±0.6d 20.0±2.7f 50.5±2.3b 176±7.9a torreoja 3.90±0.06ab 49.7±1.8e n.d. 41.9±1.9c 91.6±3.6d sangre de toro 5.21±0.83a 42.4±0.5 f 66.5±0.1a n.d. 109±1.2c cardona 1.67±0.36de 81.2±0.7a n.d. 26.7±0.4e 108.0±1.0c charola 1.69±0.48de 78.3±1.0ab 16.9±0.3 g 73.2±1.5a 168±2.2a tapona 2.52±0.42cde n.d. 45.3±1.1b 68.3±4.1a 114.0±5.2bc tapón rojo 1.45±0.14e 71.5±0.1c 38.4±0.3c n.d. 110.0±1.2c p value ˂0.0001 <0.0001 <0.0001 <0.0001 <0.0001 *variants are sorted from highest to lowest degree of humanization. n = 3. treatments with different letters in the same column are statistically different (<0.05). n.d.= not detected flavonoids are the dominant class of phenols in food, accounting for approximately two thirds of the phenols consumed in the human diet (lotito and frei, 2006). table 2 shows the concentrations of the flavan-3-ol derivatives found in the juice of all prickly pear variants studied, with significant differences (p < 0.0001) between them. these four derivatives were recorded in the juice of all variants; however, catechin was not found in blanca, being the derivative found at the lowest concentration in all variants except charola, where epicatechin attained the lowest concentration. the derivative registered at the highest concentration in these prickly pear juices was either epicatechin or procyanidin ital. j. food sci., vol. 30, 2018 621 b2, according to the variant. the highest epicatechin concentrations were found in tapona juice, and the lowest in cardona, with 90.8 µg/g and 17.2 µg/g, respectively. with regard to the total content of flavan-3-ol derivatives, the tapona juice showed the highest concentration (223±6.09 µg/g), and also the highest levels of each individual derivative; in contrast, the cardona juice showed the lowest concentration of these derivatives (73.7 µg/g). table 2. average concentration (µg/g) of flavan-3-oles in lyophilized juices of 10 prickly pear variants. species variant* catechin epicatechin procyanidin b1 procyanidin b2 total flavan 3-oles o. ficus-indica rojo pelón 13.30±0.63de 19.26±1.68f 16.71±0.13f 28.36±0.38e 77.6±1.30f o. albicarpa blanca n.d. 60.94±1.26b 32.50±1.49d 38.76±0.43c 132±3.18c o. megacantha amarilla monteza 14.23±0.69 cd 37.4±0.16c 21.03±0.74ef 33.93±0.55d 107±4.82d pico chulo 14.87±0.6cd 24.58±0.02e 23.59±1.25e 29.51±1.19de 92.5±1.86e torreoja 19.61±0.94b 32.10±0.39d 25.40±0.87e 20.37±0.40f 97.5±1.80de sangre de toro 19.61±1.60 b 61.93±0.77b 40.67±1.40c 51.62±1.31a 174±1.88b o. streptacantha cardona 10.44±0.18e 17.15±0.45f 22.41±0.51e 23.68±1.08f 73.7±2.21f o. streptacantha ssp. aguirrana charola 27.25±0.95 a 17.97±0.98f 47.06±2.19b 44.45±0.28b 137±4.40c o. robusta tapona 27.89±0.97a 90.81±2.18a 59.47±0.90a 45.20±2.05b 223±6.09a tapón rojo 17.56±1.14bc 19.16±0.30f 24.67±1.56e 23.63±1.81f 85.1±4.21ef p value ˂0.0001 ˂0.0001 ˂0.0001 ˂0.0001 ˂0.0001 *variants are sorted from highest to lowest degree of humanization. n = 3. treatments with different letters in the same column are statistically different (<0.05). n.d.= not detected 3.3. concentration and identification of betalains according to stintzing et al. (2005), prickly pear color is due to betalains, since these authors recorded indicaxanthin and betaxanthins (84 mg/kg and 100 mg/kg, respectively) in yellow-orange prickly pears, while red prickly pears contained betacyanins at concentrations of 400 mg/kg, as the chemicals responsible for this color. as shown in table 3, the juice of red-colored prickly pears have a higher betacyanin content, while betaxanthins predominate in the yellow variants (amarilla monteza and pico chulo), a finding that is consistent with other studies (stintzing et al., 2005; chávez et al., 2009; yahia and mondragón, 2011). the tapona juice showed the highest content of betacyanins and betaxanthins, but its purple-red color derives from the prevalence of betacyanins. the intermediate values of both compounds in the juice of the red o. megacantha variants is worth noting, as well as the minimum content of them in blanca juice; this finding coincides with the results of castellanos-santiago and yahia (2008) for the same species. ital. j. food sci., vol. 30, 2018 622 table 3. average content of betaxanthins and betacyanins (mg/g dry weight) in lyophilized juices of 10 prickly pear variants. species variant* betaxanthins betacyanins total betalalains o. ficus-indica rojo pelón 0.148±0.005f 0.149±0.010g 0.298g o. albicarpa blanca 0.018±0.003 g 0.021±0.004h 0.044h o. megacantha amarilla monteza 0.120±0.007 f 0.011±0.001h 0.130h pico chulo 0.085±0.017 f 0.019±0.004h 0.105f torreoja 0.313±0.029 e 0.358±0.030f 0.671ef sangre de toro 0.810±0.007 c 1.580±0.030c 2.390c o. streptacantha cardona 0.423±0.030d 0.800±0.008d 1.213d o. streptacantha ssp. aguirrana charola 0.290±0.007e 0.660±0.020e 0.945e o. robusta tapona 1.450±0.017a 2.610±0.030a 4.074a tapón rojo 1.230±0.04 b 2.380±0.060b 3.610b p value ˂0.0001 ˂0.0001 ˂0.0001 *variants are sorted from highest to lowest degree of humanization. n = 3. treatments with different letters in the same column are statistically different (<0.05). 3.4. quantification of ascorbic acid by hplc ascorbic acid is one of the most effective and abundant antioxidants in fruits and vegetables (loganaki and manian 2010), participating in various biological functions that include the synthesis of collagen, hormones and neurotransmitters. the increase in the consumption of ascorbic acid is associated with a lower risk of chronic diseases such as cancer, cardiovascular disease and cataracts. this may be due to its ability to eliminate free radicals in biological systems. this study only measured ascorbic acid content (table 4) without performing the reduction of dehydroascorbic acid (dhaa), necessary to obtain the total vitamin c content (sdiri et al., 2012). table 4. average concentration of ascorbic acid (mg/g dry weight) in lyophilized juices of 10 prickly pear variants. species variant* ascorbic acid o. ficus-indica rojo pelón 1.328±0.003a o. albicarpa blanca 0.316±0.003d o. megacantha amarilla monteza 0.327±0.016d pico chulo 0.542±0.004 c torreoja 0.327±0.004 d sangre de toro 0.652±0.041 b o. streptacanta cardona 0.325±0.006d o. streptacanta ssp. aguirrana charola 0.191±0.000e o. robusta tapona 0.527±0.029c tapón rojo 0.691±0.006 b p value ˂0.0001 n=3. treatments with different letters in the same column are statistically different (<0.05). *variants are sorted from highest to lowest degree of humanization. ital. j. food sci., vol. 30, 2018 623 ascorbic acid concentration showed significant differences between the prickly pear juices evaluated (p < 0.0001). the highest ascorbic acid content was recorded in rojo pelón, followed by sangre de toro and tapón rojo; charola was the variant with the lowest ascorbic acid concentration in juice. however, all concentrations measured were sufficient to meet easily the minimum daily intake (84 mg) of ascorbic acid in the human diet (sáenz et al., 2007). among the variants evaluated by yahia and mondragón (2011), the highest ascorbic acid concentration was recorded in the juice of the camuesa prickly pear (o. robusta), followed by cardona (o. streptacantha), with 4.0 mg/100 g and 2.1 mg/100 g fresh weight, respectively, while the lowest concentration was observed in the juice of naranjona (o. megacantha), ranging between 1.2 mg/100 g and 1.4 mg/100 g fresh weight; according to these authors, dhaa showed a pattern similar to that of ascorbic acid. in general, it has been pointed out that the concentration of ascorbic acid in prickly pear (opuntia spp.) ranges from 12 mg/100 g to 81 mg/100 g fresh weight (feugang et al., 2006). thus, the concentration of ascorbic acid in prickly pear juice is similar to or higher than in grapes, apple and pear (0.5 mg/g, 0.3 mg/g and 0.2 mg/g edible dry weight, respectively), but lower than in guava and kiwi fruit (9.4 mg/g and 4.9 mg/g edible dry weight, respectively) (lotito and frei, 2006). to note, the variants with the highest and lowest degree of humanization showed the highest concentrations of this antioxidant, suggesting that this process is unrelated to the concentration of this antioxidant. 3.5. antioxidant capacity of prickly pear juice in vitro the antioxidant capacity of prickly pear juice was estimated through abts and frap, since both are the assays most frequently used and they measure most antioxidants present. abts is typically used for mixtures or complex beverages, and measures mainly set (single electron transfer) antioxidants, without excluding hat (hydrogen atom transfer) antioxidants, in both water-soluble and fat-soluble media. in contrast, frap is applicable mostly to vegetables with set and hat antioxidants, mainly phenols and ascorbic acid (surveswaran et al., 2007; gülçin, 2012). these methods were considered as mutually complementary and were contrasted through the correlation between their respective results. surveswaran et al. (2007) point out that various herbs, fruits and vegetables show a direct relationship between antioxidant capacity and total phenolic content. in the prickly pear juices evaluated, this trend was not observed due to their contrasting differences in color, related to the presence of antioxidants such as ascorbic acid and betalains (table 5). the data obtained with abts were normally distributed, but those with frap had to be log-transformed before being analyzed. the estimates of antioxidant capacity obtained with both methods (frap and abts) for total phenols, betalains and ascorbic acid in the juice of the 10 prickly pear variants were compared through a simple linear correlation analysis (table 6). all correlation values for each comparison had the same sign, which evidences a consistent general trend in the estimates obtained with both methods. the results show that the estimates of the reduction ability of betalains with frap and abts were positively and significantly correlated (p < 0.0001). on the other hand, the estimates for ascorbic acid and phenolic compounds were not significantly correlated. the significant correlation of the total antioxidant capacity between both methods is explained by the abundance of betalains and because both methods produced similar estimates of the antioxidant capacity for all other compounds tested. ital. j. food sci., vol. 30, 2018 624 table 5. antioxidant total capacity of juices of 10 prickly pear variants. species variant* abts •+ teac (µm/g dry weight) frap (µm eq. feso4/g dry weight) o. ficus-indica rojo pelón 43837±2601abc 49102±4280cd o. albicarpa blanca 39570±8473 c 45202±4098cd o. megacantha amarilla monteza 37504±6726c 38490±2591d pico chulo 38307±6833 c 39157±2583d torreoja 46264±8198 abc 42378±9272cd sangre de toro 51422±400 abc 79066±7562b o. streptacantha cardona 45501±3565abc 60141±4645bc o. streptacantha spp. aguirrana charola 40778±3741bc 57543±4843cd o. robusta tapona 62117±10439a 112651±15066a tapón rojo 59968±12243 ab 118790±16262a p value ˂0.0016 ˂0.0001 *variants are sorted from highest to lowest degree of humanization. n=3. treatments with different letters in the same column are statistically different (<0.05). table 6. correlation (r) between estimates of antioxidant capacity generated by abts and frap methods in the juices of 10 prickly pear variants. antioxidant abts frap total 0.7845 *** 0.9436 *** total phenolic compounds 0.0848 -0.11811 phenolic acids -0.2033 -0.0943 flavan-3 ols 0.3943 0.4781 betalains 0.7828*** 0.9511*** ascorbic acid 0.1829 0.1635 significance *** p < 0.0001. 4. conclusions the higher content of betalains in the red prickly pear variants tapona, tapón rojo and sangre de toro, and of ascorbic acid in rojo pelón, tapón rojo and sangre de toro, resulted in their total antioxidant capacity being higher than in the other color variants, and explains the lack of a significant correlation between the estimates of the antioxidant capacity of phenols. of the variants evaluated, pico chulo was the richest in phenolic acids, and tapona in flavan-3-ols. unlike other table fruits, prickly pear is an important source of phenols, in addition to having the most common antioxidant phytochemicals, such as betalains and ascorbic acid. therefore, this study of prickly pear juice in vitro provides further support for recommending consumption of prickly pear juice as a functional food due to its antioxidant properties similar or superior to the juice of various marketed fruits. the study confirms the antioxidant capacity of the analyzed fruit, however before prickly pear fruit can be considered a potential functional food it is important to highlight the importance of running in vivo studies (animals and humans) in order to confirm ital. j. food sci., vol. 30, 2018 625 bioavailability of the compounds analyzed in the study and to find whether consumption of prickly pear fruit actually induces positive effect in promoting a healthy status. acknowledgements this study was supported by fundación produce de san luis. ing. roberto canovas garfias, president of sistema producto nopal san luis potosí, promoted and supported this project and provided all raw materials (prickly pears) required. gabriela zenteno-ramírez got a doctoral degree and monserrat monreal-montes got a master degree at programa multidisciplinario de posgrado en ciencias ambientales of universidad autónoma de san luis potosí in mexico, with conacyt scholarships. the authors thank josefina acosta and ma. del socorro jasso-espino for technical assistance. references bryans j.a., judd p.a. and ellis p.r. 2007. the effect of consuming instant black tea on postprandial plasma glucose and insulin concentrations in healthy humans. j. am. col. nut. 26(5):471-477. castellanos-santiago e. and yahia m. 2008. identification and quantification of betalains from the fruit of 10 mexican prickly pear cultivars by high-performance liquid chromatography and electrospray 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*corresponding author. tel.: +90464 2233385; fax: +90464 2234118 e-mail address: emre.caglak@erdogan.edu.tr abstract in this study, the seasonal fatty acid and amino acid amounts in the muscles of the zander from beyşehir lake, turkey, and their important indices for human health were evaluated. it was found that aspartic acid, glutamic acid and lysine levels in zander were dominant among the amino acids. the ratio of essential amino acids (eaa) to non-essential amino acids (neaa) was between 0.69 and 0.78. in all seasons, the polyunsaturated fatty acid (pufa, 89.85-109.11 mg/100g) amount in zanders was higher than saturated fatty acids (sfa, 55.08-81.89 mg/100g) and monounsaturated fatty acids (mufa, 29.16-78.89 mg/100g). it was determined that epa, dha and omega-3 rates were high. the fatty acid quality indices (ai, ti, flq, w6/w3, h/h) were found at proper levels for human health. summing up the results, it was found that seasons influenced both the compositions of amino acids and fatty acids of zander. keywords: amino acids, beyşehir lake, fatty acids, seasons, zander ital. j. food sci., vol 29, 2017 267 1. introduction seafood includes healthy nutrients being rich in protein, unsaturated essential fatty acids, minerals and vitamins they contain (sidhu, 2003). fish and shellfish play important roles for human health because of their fatty acid and amino acid varieties (guler et al., 2008). fish, as a source of food, have protein with high biological value. the building stones of the proteins consist of amino acids (who, 2007). amino acids exist in seafood in important amounts and are classified as essential, non-essential and semi-essential amino acids according to their biological status (wu, 2010). these amino acids are the start-up material of many important substances for organisms, and have an important role, especially in energy metabolism. in addition, fish contains essential amino acids (threonine, valine, leucine, isoleucine, lysine, methionine, phenylalanine, tryptophan, histidine, and arginine) in proper amounts in their bodies (polat, 1999; varlik et al., 2011). the polyunsaturated fatty acids in fish oil are a vital importance for human health. consuming fish and fish oil decreases the risk of coronary heart diseases. the nutritional importance of the fish consumption is closely related to the ω-3 fatty acid content of each species. these health benefits are also in a close relationship with ω-3 pufas. long-chain ω-3 pufas cannot be synthesized by humans, and therefore they have to be taken with food. it was demonstrated with clinical and epidemiological studies that the major source of epa and dha, which constitute most ω-3 pufas, is the seafood (guler et al., 2008; cengiz et al., 2012). dha and epa fatty acids are significant for the body because they could prevent coronary artery diseases (connor, 2000; mozaffarian et al., 2005). since dha is the main component of the brain, eye retina and heart muscles, its importance for human health is undeniable. it was reported that epa is beneficial in brain diseases and cancer treatment (cengiz et al., 2012). fish are a good source of epa and dha. some countries (canada, sweden, united kingdom, australia, japan), world health organization (who) and north atlantic treaty organization (nato) declared the daily ω-3 need as 1.1.-1.6 g; and suggested that the intake should be as 0.3-0.5 g epa+dha and 0.8-1.1 g α-linoleic acid (erkan, 2013). when compared to sea fish, freshwater fish have higher c18 pufa and lower epa and dha levels. freshwater fish are generally characterized with high n6 pufa (especially linoleic acid (18:2n6) and arachidonic acid (20:4n6)). for this reason, freshwater fish contain lower n3 pufa and n3/n6 levels than sea fish (özoğul et al., 2007; çelik et al., 2005). in addition, the criteria such as atherogenic index (ai), thrombogenic index (ti), flesh lipid quality (flq) and hypocholesterolemic/hypercholesterolemic ratio (h/h) provide information on the lipid quality of fish (ulbricht and southgate, 1991; abrami et al., 1992; santos-silva et al., 2002). the fish oil and fatty acid compounds show biochemical changes depending on ecological factors and the physiological status of the fish. even among the same species, the fatty acid component may vary according to the nutrition, region, season, gender and environmental conditions (uysal, 2004; özoğul et al., 2007; guler et al., 2007). the rich amino acid and fatty acid contents in the bodies of freshwater fish make them nutritious, and therefore they are used as animal protein source all over the world (steffens, 2006). the zander is a predatory freshwater species from the percidae family, and is an important nutrient because of its high protein, low lipid rate, and essential ω-3 fatty acids. the zander is a lean carnivorous fish with high economic value spreading in inland waters in turkey (uysal, 2004; çelik et al., 2005). ital. j. food sci., vol 29, 2017 268 the aim of this study was to determine the seasonal fatty acids and amino acid amounts in the muscles of the zander, and evaluate their important indices (ai, ti, flq, w6/w3, h/h) for human health. 2. materials and methods 2.1. the study area and period this study was conducted in 2012-2013 seasonally, in beyşehir lake located within the borders of konya and isparta in 37°47′0″n, 31°33′0″e coordinates. 2.2. fish material the zander (sander lucioperca linnaeus, 1758) whose height was between 34.85±1.33 cm and weight was between 395±44.76 g in 2-3 years of age were obtained from the fishermen in the beyşehir side of the lake. the fishermen stated that they used stretching nets for fishing. a total of 32 fishes were examined throughout the study. the head, tail, fins, and viscera of zander were removed and muscle tissues of zander were kept at -70 oc until analysis of fatty acid and amino acid composition. 2.3. amino acid analysis zander samples were sent to the scientific and technological research council of turkey (tubitak) marmara research center (mam) food institute for analysis of amino acid. in amino acid analysis, an in-house method was created by modifying those of dimova (2003) and gheshlaghi et al. (2008), and the sample analysis was carried out. the analysis process was performed using a uflc (ultra-fast liquid chromatography) device and a uv detector. the amino acid analyses were conducted in triplicate. 2.4. analysis of fatty acid methyl esters analysis of fatty acid methyl esters (fame%) was carried out according to tufan et al. (2013). lipid extraction of the samples was carried out in triplicate based on the method of bligh and dyer (1959), using chloroform:methanol (2:1, v/v). methyl esters were prepared by transesterification using 2m potassium hydroxide (koh; merck, darmstadt, germany) in methanol and n-hexane (sigma-aldrich, steinhein, germany) according to the method described by ichihara et al. (1996) with minor modification; 10 mg of extracted oil were dissolved in 2 ml n-hexane, followed by 4 ml of 2m methanolic koh. the tube was then vortexed for 2 min at room temperature. after centrifugation at 4,000 rpm for 10 min, the hexane layer was taken for gas chromatography (gc) mass spectrometry analyses. 2.5. gas chromatography-mass spectrometry conditions the identification of fatty acids was conducted on gas chromatography-mass spectrometry (gc-ms) device (qp2010 ultra with aoc-20i+s model auto sampler) using a mass selective detector (gc-ms qp 2010 plus) equipped with gc/ms solutions software (shimadzu, kyoto, japan). fame mix standards were separated on a restek rt-2560 column (usa cat no: 13199 serial no: 47623-07; 100 m × 0.25 mm internal diameter, thickness: 0,20μm) with helium (1.0 ml/min) as the carrier gas. the injection temperature ital. j. food sci., vol 29, 2017 269 was 240 ºc, and split ratio 50 injection mode was used. the oven temperature was programmed as follows: column oven temperature was started as 140 ºc, then at 4th min, the temperature increased to 240 ºc and held at this temperature for 20 min, and then held at this temperature for further 50 min starting at 25th min. the ms was scanned from m/z of 45 to 550. the ion source and interface temperatures were 200 ºc and 240 ºc, respectively. fatty acids were identified by comparing the retention times of fame with supelco (tm) 37 component fame mixture (cat. no. 47885-u) and the results were confirmed by using wiley/nist 2011 library. quantification of fame was carried out using the area normalization method. according to the area value of each compound, area compositions were detected and results were shown as fame%. the fatty acid content in the zander was calculated according to weihrauch et al. (1977). 2.6. lipid quality indices lipid quality indices as atherogenicity index (ai), thrombogenicity index (ti), fish lipid quality (flq) and hypocholesterolemic/hypercholesterolemic ratio (h/h) were calculated with the formulas below (ulbricht and southgate, 1991; abrami et al. 1992; fernández et al., 2007). ai= [(12:0+(4x14:0)+16:0)]/[(n-6 pufa+n-3 pufa)+∑ mufa] it= [14:0+16:0+18:0]/[(0.5x∑ mufa)+0.5(n-6 pufa)+3(n-3 pufa)+(n-3 pufa/n-6 pufa)] flq= (epa+dha)/total lipids h/h=(c18:1+c18:2+c18:3+c20:3+c20:4+c20:5+c22:4+c22:5+c22:6)/(c14:0+c16:0 2.7. statistical analysis statistical analysis was performed using the jmp 5.0.1 (sas) package program. analysis of variance (anova) was used to compare the results the among seasons, and the tukey's test was applied to the groups demonstrating difference (p<0.05) (sokal and rohlf, 1987). 3. results and discussions the amino acid contents of the zander in seasonal periods are shown in table 1. the protein contents of the zander are the lowest in spring (17.75%) and the highest in autumn (19.35%). while the protein amount in summer and winter seasons showed statistical similarities to each other (p>0.05), they were found to be different from the other seasons (p<0.05). when the amount of the amino acids of the zander, which contain 16 types of amino acids, are examined, the methionine, which is one of the essential amino acids, had the lowest value with 465.5 mg/100g and aspartic acid, which is one of the non-essential amino acids had the highest value with 3202.5 mg/100g. it was determined that ten of the amino acids (phenylalanine, lysine, valine, leucine, isoleucine, tyrosine, glycine, proline, arginine and alanine) were at maximum level in autumn; five of them (histidine, threonine, aspartic acid, glutamic acid and serine) were at maximum level in summer; and one of them (methionine) was highest in winter. it was also seen that in spring, when the zander reproduce, all the amino acids except for lysine were at the lowest level. lysine, leucine, threonine and valine were dominant in eaa. the aspartic acids, glutamic acid, alanine and serine were at the highest level in neaa. similarly, mohanty et al. (2012) ital. j. food sci., vol 29, 2017 270 found that in giant river-catfish (sperata seenghala) histidine, threonine and leucine from eaa; and glutamic acid, aspartic acid and serine amino acids from neaa were dominant. it was observed that the highest values of the total essential amino acids (σeaa) were in autumn; the highest values of the total non-essential amino acids (σneaa) were in summer. the σeaa/σneaa ratio is an index that shows the protein quality (swendseid et al., 1963). the σeaa/σneaa ratio, which was obtained seasonally, changed between 0.69-0.78. iwasaki and harada (1985) reported the eaa/neaa ratio as being 0.70 in average in seawater fish species. mohanty et al. (2012) noted the σeaa/σneaa ratio of giant river-catfish as being 0.89. in another study, the σeaa/σneaa ratio (1.08) of puntius sophore, which are among freshwater fish, was found to be higher than those reported in other studies (mahanty et al., 2014). it is considered that the differences between the σeaa/σneaa ratios are resulted from the fish species, seasons, nutrition and location. table 1. seasonal amino acid contents of zander samples. eaa*: essential amino acid, neaa: non-essential amino acid, aa: amino acid. different letters (a,b,c,d) in the same line indicates statistical differences among seasons (p<0.05). amino acids (mg/100g) spring summer autumn winter protein (%) 17.75±0.01a 18.75±0.11b 19.35±0.05c 18.73±0.26b methionine* 465.5±7.78a 509±1.41b 499±0.00b 513±5.66b phenylalanine* 661±9.90a 687.5±6.36b 812±1.41c 749±2.83d lysine* 2052.5±17.68a 1975±2.83b 2544.5±12.02c 1969.5±6.36b histidine* 492±12.73a 643±1.41b 628±28.28b 632.5±26.16b valine* 790.5±13.44a 861±1.41b 928±7.07c 887±11.31d leucine* 1119.5±19.09a 1181±5.66b 1317.5±7.78c 1198±9.90b isoleucine* 701.5±12.02a 744±2.83b 839.5±3.54c 809±5.66d threonine* 808±12.73a 933±1.41b 878.5±0.71c 836.5±2.12d tyrosine 579±0.00a 603±1.41b 682.5±2.12c 630±1.41d glycine 613±8.49a 706.5±4.95b 764±1.41c 682±2.83d proline 496±8.49a 543±24.04ab 604.5±0.71c 579.5±2.12bc arginine 724.5±10.61a 744±1.41a 851.5±0.71b 806±2.83c alanine 1034.5±16.26a 1116±31.11b 1194±1.41c 1125±2.83bc aspartic acid 2779.5±47.38a 3202.5±31.82b 2783.5±3.54a 3079±16.97c glutamic acid 2781±42.43a 3174±2.83b 3097.5±4.95b 2985±4.24c serine 809±14.14a 899±2.83b 851.5±4.95c 866±7.07bc σeaa 7090.5±70.00a 7533.5±23.33b 8447±9.90c 7594.5±4.95b σneaa 9816.5±147.79a 10988±79.20b 10829±7.07b 10752.5±14.85b σaa 16907±217.79a 18521.5±55.86b 19276±2.83c 18347±9.90b σeaa/σneaa 0.72 0.69 0.78 0.71 ital. j. food sci., vol 29, 2017 271 the world health organization recommended methionine, phenylalanine, lysine, histidine, valine, leucine, isoleucine and threonine requirements for adults of 10, 25, 30, 10, 26, 39, 20, respectively and 15 mg amino acid/kg body weight per day (who, 2007). when the results of our study are evaluated according to the reports of who (2007), it is clear that if a person whose weight is 70 kg consumes 200 g meat of zander, he or she receives methionine, lysine, histidine, isoleucine and threonine amino acids; and if one consumes 300 g zander, all his or her amino acid needs could be met. the fatty acid contents of the zander in each season are shown in table 2. table 2. seasonal fatty acid contents of zander samples. fatty acids mg/100g spring summer autumn winter c14:0 1.79±0.32a 2.38±0.13a 3.10±0.25a 5.80±0.79b c15:0 0.74±0.01a 0.72±0.12a 0.87±0.19a 1.65±0.14b c16:0 38.05±0.60a 51.87±0.93b 54.05±0.90b 52.99±3.70b c17:0 0.96±0.09a 1.14±0.09ab 1.14±0.02ab 1.76±0.34b c18:0 10.65±0.18a 18.99±0.25b 15.35±0.10bc 11.83±1.96ac c20:0 0.22±0.03a 0.31±0.01a 0.51±0.02b 0.56±0.06b c21:0 1.84±0.10a 2.03±0.15a 3.49±0.36b 5.67±0.45c c24:0 0.84±0.04a 0.80±0.00a 0.77±0.07a 1.65±0.10b σsfa 55.08±0.93a 78.24±1.42b 79.28±0.14b 81.89±3.95b c16:1 7.51±0.16a 5.50±0.27a 12.65±1.02a 25.31±3.95b c17:1 0.75±0.00a 0.47±0.00a 0.81±0.10a 2.02±0.22b c18:1n9t 0.13±0.00a 0.24±0.01ab 0.31±0.00b 0.57±0.08c c18:1n9c 20.51±0.53a 22.42±0.99a 36.17±2.00b 49.48±0.77c c20:1 0.59±0.10a 0.53±0.00a 0.63±0.12a 1.50±0.18b σmufa 29.49±0.58a 29.16±1.27a 50.58±3.24b 78.89±5.20c c18:2n6c 4.68±0.05a 5.01±0.45ab 8.46±0.47b 12.05±0.57c c18:3n6 0.30±0.19a 0.55±0.00a 0.57±0.17a 1.26±0.00b c20:2n6 0.17±0.00a 0.42±0.00b 0.69±0.03c 0.63±0.00c c20:3n6 0.60±0.01a 0.42±0.03a 0.55±0.07a 0.90±0.06b c20:3n3 0.28±0.00ab 0.13±0.03a 0.34±0.07b 0.86±0.04c c20:4n6 18.33±0.30ab 19.01±0.16a 15.56±0.80b 18.06±1.06ab c20:5n3 9.19±0.22a 11.08±0.15a 23.04±1.32b 15.47±1.19c 22:5n3 8.14±0.38bc 5.35±0.03a 6.69±0.02ab 9.29±0.63c c22:6n3 48.17±1.23ab 53.28±1.72a 42.76±3.29b 50.60±1.58ab σpufa 89.85±1.06a 95.26±1.47ab 98.66±4.81ab 109.11±4.71b different letters (a,b,c,d) in the same line indicates statistical differences among seasons (p<0.05). ital. j. food sci., vol 29, 2017 272 it was observed that in all seasons, the pufas are the highest (ave. 43.32%), mufa’s are the lowest (ave. 19.64%). the palmitic acid (c16:0) has the highest amount among saturated fatty acids. the 16:0 amount was detected between 18.52%-24.52%. similarly, in a study conducted by jankowska et al. (2003), c16:0 was determined as 19.91% in wild zander, 20.24% in cultured zander (fed artificial feed) and 20.33% in cultured zander (fed natural food), respectively. the total saturated fatty acid (∑sfa) were found in spring, summer, autumn and winter as 55.08 mg/100g, 78.24 mg/100g, 79.28 mg/100g and 81.89 mg/100g, respectively; which shows the values in spring were different from the other seasons (p<0.05). among the monounsaturated fatty acids (mufa), palmitoleic acid (c16:1), cis-10-heptadecanoic acid (c17:1) and cis-11-eicosenoic acid (c20:1) values were observed minimum in summer; while the lowest values of elaidic acid (c18:1n9t) and oleic acid (c18:1n9c) were found in spring. the values of c16:1, c17:1, c20:1, c18:1n9t and c18:1n9c in winter was found different from other seasons (p<0.05). the ∑mufa amounts were determined between 29.49-78.89 mg/100g and there were significant differences among the seasons (p<0.05). polyunsaturated fatty acids (∑pufa) showed constant increase from spring till winter. the majority of ∑pufa consisted of docosahexaenoic acid (c22:6n3) dha, arachidonic acid (c20:4n6) and eicosapentaenoic acid (c20:5n3) epa. in all seasons, the dha amount was found higher than the other pufa amounts at a significant level (p<0.05) and was determined as 48.70 mg/kg in average. the sfa (28.62%-30.01%), mufa (13.79%-27.57%) and pufa (38.19%-48.95%) amounts of zander in this study were found similar to the results reported in zander as 31.8%, 13.8% and 42.4%, respectively in the study conducted on seawater and freshwater fish species of turkey by özoğul et al. (2007). in another study, the sfa, mufa and pufa contents of the zander were determined in seyhan lake as 32.9%, 28.0%, 20.8%, in eğirdir lake as 30.5%, 20.3%, 30.5% (çelik et al., 2005). the fatty acid contents of the zander examined in this study were more consistent with the results of the zander of eğirdir lake than those from seyhan lake. however, the pufa rates were found to be lower than our results. it is considered that this difference occurs from the undetected % fatty acid rates being high in the study conducted by çelik et al. (2005). in another study, the sfa%, mufa% and pufa% rates of the wild zander were found as 27.84%, 21.36% and 50.80, respectively (jankowska et al., 2003). the pufa/sfa ratio must be minimum 0.45 (justi et al., 2003; tufan et al., 2011). the data of this study were 1.22-1.63, which is consistent with the values recommended in terms of health (table 3). the pufa/sfa ratio determined in spring (1.63) was statistically different from those determined in other seasons (p<0.05). özoğul et al. (2007) reported that the pufa/sfa ratio changed between 0.78-1.56 is freshwater fish, and in zander this ratio was 1.33. this pufa/sfa ratio was found to be similar with our results especially recorded in winter. it is reported that the atherogenic (ai) and thrombogenic (ti) indices that are higher than (>1.0) is harmful for human health (ouraji et al., 2009). if this value gets lower, the risk of coronary heart diseases decreases (cutrignelli et al., 2008). the ai (0.38-0.49) and ti (0.22-0.31) values obtained in this study were found lower than this value in all seasons (table 3), and it was also determined that there were no risks for human health. sousa bentes et al. (2009) reported that the h/h ratio of fatty acids is the indicator of whether the fat in the product is nutritionally adequate. the h/h ratio was found between 2.17-2.77 in this study, and no differences between spring and winter, also between summer and autumn were determined (p<0.05). ital. j. food sci., vol 29, 2017 273 table 3. fatty acid ratios and lipid quality indexes. fatty acid spring summer autumn winter ∑pufa/∑sfa 1.63±0.05a 1.22±0.04b 1.24±0.06b 1.33±0.01b ∑pufa/∑mufa 3.05±0.10a 3.27±0.19a 1.96±0.22b 1.39±0.15b epa+dha 57.36±1.01a 64.36±1.57a 65.8±4.61a 66.06±2.77a ∑n3 pufa 65.78±0.64a 69.84±1.57a 72.83±4.56a 76.21±4.57a ∑n6 pufa 24.07±0.43a 25.42±0.1b 25.83±0.25b 32.9±0.14c ∑n3/∑n6 2.73±0.02ab 2.75±0.07a 2.82±0.15a 2.32±0.13b ∑n6/∑n3 0.37±0.00a 0.36±0.01a 0.36±0.02a 0.43±0.02b ai 0.38±0.02a 0.49±0.01b 0.45±0.01bc 0.41±0.00ac ti 0.22±0.01a 0.31±0.01b 0.28±0.01bc 0.25±0.01ac flq 32.89±0.67a 31.76±0.97a 28.79±1.80ab 24.47±0.71b h/h 2.77±0.08a 2.17±0.05b 2.35±0.02b 2.70±0.07a ai: atherogenic index, ti, thrombogenic index, flq: flesh-lipid quality, h/h: hypocholesterolemic/hypercholesterolemic ratio. different letters (a,b,c,d) in the same line indicates statistical differences among seasons (p<0.05). if the flq value is high, this indicates that there are nutrient lipids with good quality (abrami et al., 1992). the highest flq values were seen in spring (32.89), and the lowest in winter (24.47). the difference between them was significant (p<0.05) (table 3). the flq values in winter were found to be similar only to those in autumn (p>0.05). the epa+dha amounts in zander were determined between 57.36-66.06 mg/100g (table 3). there were differences between the seasons (p>0.05). the epa+dha amounts in the zander caught in the same lake were determined as 29.23%, 21.32%, 28.27% and 24.24% for spring, summer, autumn and winter, respectively (guler et al., 2007). these results show similarity with this study except for the summer season. the n6/n3 ratio is recommended max. 4 by the uk department of health (justi et al., 2003). the ∑n6/∑n3 ratios in this study were found in the recommended limit value. the ∑n6/∑n3 ratio in winter (0.43) was found higher (p<0.05) than the other seasons (0.36-0.37) (table 3). jankowska et al. (2003) found the ∑n6/∑n3 ratio of the wild zander as 0.31. özoğul et al. (2007) determined that the n6/n3 ratio of zander is 0.46 similarly with our winter data. on the other hand, guler et al. (2007) reported that n6/n3 ratio of zander is between 0.67-1.39 in all seasons. 4. conclusions as a conclusion, the zander, which are also called freshwater seabass, have an important fatty acid composition. it was determined that the epa, dha and omega-3 rates of the zander, which played an important role in human nutrition are high. in addition, the fatty acids quality indexes were found at proper levels for human health. moreover, it was observed that the zander have high protein and rich amino acid content although the rate of amino acids changes according to the seasons. the lowest amino acid rates were found in spring, the period of reproduction. at the end of the seasonal examination of amino ital. j. food sci., vol 29, 2017 274 acid and fatty acid contents of the zander, it has been observed that these contents have a nutrient function for a healthy and balanced diet. references abrami g., natiello f., bronzi p., mckenzie d., bolis l. and aggrade e. 1992. a comparison of highly 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sirnak university, engineering faculty, department of energy systems engineering, sirnak, turkey ekinihsan@gmail.com abstract in the present work, main organs (digestive gland, cephalopedal, gonad and mantle) and lipid classes (total, neutral and phospholipid) of land snails assyriella escheriana and assyriella guttata from southeastern anatolia were examined for their fatty acids. the major components detected in both of the species were c16:0, c18:0, c18:1ω9, c18:2ω6, c18:3ω3, c20:2ω6 and c20:4ω6. c18:2ω6 was identified as the primary fatty acid ranging from17.07% to 28.12% in a. guttata and 18.02% to 27.43% in a. escheriana. the proportions of c20:4ω6 modified to form prostaglandins that are directly involved in regulation of reproduction, ranged from10.01% to 20.30% in a. escheriana and 11.05 % to 16.58% in a. guttata. taking into consideration that σpufa levels were always higher than σsfa and σmufa levels in all treatments of both species. this was an expected finding for the snails collected during the breeding season because pufa plays an important role as precursors for signal-transduction involved in the regulation of mating and reproduction. a significant amount of c20:2ω6 was concentrated in the cephalopedal of a. guttata (13.42%) and a. escheriana (14.93%). probably, cephalopedal serves as a storage organ of this component. consequently, the findings revealed that the snail’s fatty acid profiles were qualitatively similar, but quantitatively there were some differences. most important of all, tissues of the snails were good source of essential fatty acids (c18:2ω6 and c18:3ω3) and pufa, particularly omega 6 fatty acids. 2 ital. j. food sci., vol. 27 2015 introduction assyriella escheriana and assyriella guttata (endemic to southeastern turkey and north iraq) species are common in moist and calciferous habitats of southeastern turkey. although they are not eaten and commercially not exported, they are big enough and fleshy as much as edible relatives helix lucorum, eobania vermiculata dwelling in the same region. the fatty acid distribution of a large number of commercially important marine and freshwater molluscs have been reported and reviewed in varying degrees of details (ackman, 2000; karakoltsidis et al., 1995). lipids from marine, freshwater and edible land molluscs are more extensively studied (özoğul et al., 2005; milinsk et al., 2006; miletic et al., 1991; rakshit et al., 1997; eki̇n and başhan, 2010; eki̇n et al., 2012, 2014) than those from nonedible terrestrial members. nevertheless, nonedible land snails deserve special attention from the point of their evolutional relationship, roles in food chain, nutritional value, taxonomic and possible benefits in cosmetic, medicine and biochemistry. nutritional, food chain and taxonomic studies are important in understanding interrelationship in marine, freshwater and terrestrial environment, however for the southeastern anatolia; quite little data were available in the literature on edible and nonedible land snails. a. escheriana and a. guttata species whose edible relatives including helix aspersa, h. asemnis, h. cincta and h. lucorum, theba pisana, eobania vermiculata and cantareus apertus, living in turkey (yildirim, 2004), can deserve more detailed studies. many snail farms are being established in some countries in order to produce good quality snails for consumption and export. so, it seemed useful to make a comparative study of similarity and differences in biochemical and nutritional composition of edible and nonedible snail species. a comparative biochemical study on fatty acid composition of snails belonging to same classes but living in different habitats is hoped to provide an insight into the adaptive capabilities and influence of the environment on their fatty acid distribution. this paper discusses the fatty acid distribution of main organs and lipid classes in two same genus gastropods from southeastern anatolia, as data which is hoped to be basic to further comparative biochemical, nutritional, taxonomic and evolutionary studies. materials and methods sample collection and preparation fifteen adult a. escheriana species were collected from a woodland near tizyan (elmabahçe) village, 20 km north of mardin (n 37° 49’ / e 40° 68’) at an altitude of 985 m and fifteen adult endemic assyriella guttata species were collected from stony and rocky region of diyarbakır city walls (n 37° 55.2’ / e 40° 13.8’) at an altitude 675 m. both species were collected in april 2012. similar size (length: 4 ± 0.60 cm, wet flesh weight: 13 ± 0.50 g) snail species were sampled for lipid analyses. the snails’ shells were removed and divided into seven groups (digestive gland, cephalopedal, gonad, mantle, total lipid, neutral lipid and phospholipid) and their organs were dissected out. then, tissues of each experimental set were conditioned in polyethylene bags and kept at -80°c until chemical analysis. extraction of fatty acids and gc analysis digestive gland, gonad, mantle, cephalopedal and whole body samples were homogenized in chloroform/ methanol (2:1, v/v) solution in order to extract total body lipids (bligh and dyer, 1959). organ’s lipid, total lipid, phospholipid and neutral lipid fractions were obtained according to the method of stanley-samuelson and dadd, 1983. fatty acids methyl esters (fames) were provided by capillary gas chromatography (gc) using hewlett packard (wilmington, de) gas chromatograph (model 6890), a db-23 capillary column (60 m × 0.25 mm i.d. × 0.250 μm film thickness and bonded 50% cyanopropyl) (j & w scientific, folsom, ca), a flame ionization detector, and hewlett-packard chemstation software. the injection port and the detector temperatures were 270°c and 280°c, respectively. the split ratio was 1:20. the flow rates of compressed air and hydrogen were 300 ml/min, 30 ml/min, respectively. carrier gas was helium (2.8 ml/ min). the oven temperature was programmed at a rate of 6.5°c/min from 130°c (1 min hold) to 170°c, then increased at a rate of 2.75°c/ min to a 215°c, then again increased at a rate of 40°c/min to 230°c, was held for 12 minutes. each tissue fatty acids percentages and spectra of fames are obtained by hp 3365 chemstation computer program. fames existence and retention times were determined by comparing the spectra of authentic standards (sigma-aldrich chemicals). individual fame was identified by comparisons with the chromatographic behaviors of authentic standards. statistical analyses the results were expressed as mean values ± sd (standard deviation). all analytical determinations were performed in triplicate and the mean values were reported. the analyses were performed using a commercial statistical program (spss 20). the percentages of fatty acid were compared by anova variance analysis with 5% significance level. tukey’s test was used for cooperation of average values. ital. j. food sci., vol. 27 2015 3 results in both snails, c16:0, c18:0, c18:1ω9, c18:2ω6, c18:3ω3, c20:2ω6 and c20:4ω6 were presented as predominant fatty acids. of the detected fatty acids, amount of c18:2ω6 was the highest in all analyses from both a. escheriana and a. guttata (tables 1 and 2). compared to the fatty acids of the species’ organs, in a. escheriana, highest level of c16:0 (11.81%) and c18:1ω9 (20.56%) were presented in the gonad; c18:0 (15.18%) and c20:2ω6 (14.93%) were in the cephalopedal; c18:2ω6 (26.67%) and c18:3ω3 (7.36%) were in the digestive gland and c20:4ω6 (17.86%) was in the mantle (table 1). in a. guttata highest level of c16:0 (9.72%), c18:1ω9 (19.65%) and c18:3ω3 (4.76%) were found in the gonad; c18:0 (15.21%) and c20:4ω6 (15.68%) were in the mantle; c18:2ω6 (28.12%) was in the digestive gland and c20:2ω6 (13.42%) was in the cephalopedal (table 2). on the other hand, the fatty acids from lipid classes of the species showed some differences, in a. escheriana, highest level of c16:0 (10.16%) and c18:3ω3 (8.42%) were identified in the total lipid; c18:0 (11.21%), c18:1ω9 (13.33%) and c20:2ω6 (11.79%) were in the neutral lipid; c18:2ω6 (27.43%) and c20:4ω6 (20.30%) were in the phospholipid (table 1). in a. guttata, highest level of c16:0 (9.61%) and c20:4ω6 (16.58%) were presented in the total lipid; c18:0 (13.01%), c20:2ω6 (10.99%) and c18:1ω9 (16.03%) were in the neutral lipid; c18:2ω6 (22.34%) and c18:3ω3 (10.70%) were in the phospholipid (table 2). in all treatments, results showed that concentration of ω6 (omega 6) always higher than concentration of ω3 (omega 3) family fatty acids. in a. escheriana, the ratio of σω6 / σω3 was 4.86, 6.71, 5.44 and 7.66 in the digestive gland, cephalopedal, gonad and mantle, respectively (table 1). in a. guttata it was 5.13, 8.49, 5.29 and 4.84 in the digestive gland, cephalopedal, gonad and mantle, respectively (table 2). additionally, σω6 / σω3 ratio were 5.19 in the total lipid, 4.86 in the neutral lipid and 6.59 in the phospholipid of a. escheriana (table 1). this ratio was observed 4.00 in the total lipid, 3.91 in the neutral lipid table 1 fatty acid profile of main organs total lipid and lipid classes from assyriella escheriana. fatty acid compositions of total lipid fatty acid compositions from a. escheriana organs of lipid classes from a. escheriana fatty acids digestive gland cephalopedal gonad mantle total lipid neutral lipid phospholipid (mean*±s.d.)** (mean*±s.d.)** (mean*±s.d.)** (mean*±s.d.)** (mean*±s.d.)** (mean*±s.d.)** (mean*±s.d.)** c10:0 0.12±0.02a 0.05±0.01b 0.08±0.02ab 0.02±0.01c 0.04±0.01b 0.10±0.02a c12:0 0.03±0.01a 0.07±0.02b 0.04±0.01a 0.05±0.01ab 0.06±0.01ab 0.07±0.01b 0.09±0.02b c13:0 0.08±0.01a 0.09±0.02a 0.03±0.01b 0.10±0.02a 0.05±0.01b c14:0 1.31±0.11a 0.29±0.04b 0.49±0.06c 0.81±0.09d 0.58±0.07cd 0.73±0.08d 0.44±0.06c c15:0 0.39±0.04a 0.19±0.03b 0.23±0.04b 0.30±0.04a 0.23±0.04b 0.68±0.06c 0.37±0.04a c16:0 8.41±0.67a 9.33±0.83a 11.81±1.03b 9.72±0.80a 10.16±0.91ab 9.99±0.82a 7.05±0.61c c17:0 1.63±0.14a 2.03±0.15b 0.87±0.09c 2.48±0.25b 1.60±0.14a 1.11±0.10d 1.08±0.09d c18:0 10.66±0.84a 15.18±1.05b 10.11±0.79a 11.06±0.87a 10.30±0.73a 11.21±0.91a 9.67±0.69a c14:1ω9 0.45±0.05a 0.90±0.10b 0.32±0.04a 0.15±0.03c 0.07±0.02c c16:1ω7 0.88±0.09a 0.63±0.07b 1.07±0.09c 0.99±0.08c 1.80±0.16d 1.02±0.12c 0.98±0.09c c18:1ω9 19.50±1.41a 15.20±1.30b 20.56±1.52a 14.53±1.21b 12.68±1.10c 13.33±1.11c 13.04±1.10c c20:1ω9 0.60±0.05a 0.75±0.07a 0.76±0.06a 0.46±0.04b 0.34±0.04b 0.64±0.05a 0.96±0.08c c22:1ω9 0.03±0.01a 0.05±0.01a 0.12±0.03b 0.14±0.03b 0.06±0.01a 0.18±0.03b c18:2ω6 26.67±1.72a 18.02±1.41b 24.42±1.55c 22.32±1.49d 20.45±1.39e 19.77±1.43e 27.43±1.66a c18:3ω3 7.36±0.69a 1.74±0.15b 5.66±0.44c 3.96±0.29d 8.42±0.71a 8.00±0.70a 6.60±0.51e c20:2ω6 7.35±0.68a 14.93±1.26b 8.51±0.75a 10.91±0.96c 10.72±0.94c 11.79±1.01d 7.25±0.64a c20:3ω6 1.41±0.12a 0.66±0.05b 1.42±0.13a 0.88±0.07b 0.73±0.06b 0.88±0.09b 1.30±0.10a c20:4ω6 11.14±1.04a 14.37±1.12b 10.01±0.94a 17.86±1.27c 19.51±1.39d 17.12±1.25c 20.30±1.44d c20:5ω3 1.07±0.09a 4.11±0.31b 1.90±0.13a 1.91±0.15a 1.03±0.14a 1.75±0.15a 1.00±0.09a c22:2ω6 0.09±0.02a 0.22±0.03b 0.06±0.01a 0.05±0.01a c22:5ω6 0.21±0.03a 0.45±0.04b 1.05±0.08c 0.73±0.05d 0.89±0.08d 0.78±0.06d 1.02±0.10c c22:6ω3 1.22±0.12a 1.40±0.13a 0.78±0.06b 1.02±0.09a 0.63±0.05b 0.60±0.05b 1.10±0.10a σω6 / σω3 4.86 6.71 5.44 7.66 5.19 4.86 6.59 σsfa 22.63±1.50a 27.14±1.63b 23.72±1.56a 24.45±1.58c 23.05±1.49a 23.83±1.51a 18.85±1.41d σmufa 21.46±1.47a 17.53±1.25b 22.83±1.44a 16.12±1.23b 14.97±1.17c 15.05±1.19c 15.23±1.18c σpufa 56.52±2.28a 55.90±2.27a 53.75±2.20b 59.65±2.48c 62.38±2.55d 60.69±2.52c 66.05±2.61e results expressed as percentage of total fatty acids methyl esters. *values are means ± s.d (standard deviation) for three samples of triplicate analysis. **means followed by different letters in the same line are significantly different (p < 0.05) by tukey’s test. sfa: saturated fatty acids, mufa: monounsaturated fatty acids, pufa: polyunsaturated fatty acids, σω6: total of omega 6 fatty acids, σω3: total of omega 3 fatty acid. 4 ital. j. food sci., vol. 27 2015 and 2.28 in the phospholipid of a. guttata (table 2). these high levels of σω6/σω3 were mostly on account of higher concentration of c18:2ω6 and c20:4ω6. the most notable result was significantly high level of σpufa (total polyunsaturated fatty acids) and low level of σsfa (total saturated fatty acids) and σmufa (total monounsaturated fatty acids) in all organs and lipid classes. among the organs, the maximum level of σpufa was obtained in the mantle (59.65%) of a. escheriana (table 1) and in the digestive gland (60.17%) of a. guttata (table 2). on the other hand, maximum level of σsfa was detected in the cephalopedal (27.14%) of a. escheriana (table 1) and in the mantle (28.76%) of a. guttata (table 2). the level of σmufa in all treatments was found significantly lower than σpufa and σsfa. it ranged from 16.12% to 21.46% in a. escheriana and 16.44% to 21.63% in a. guttata. it was noteworthy that, the amount of σpufa was significantly high; 66.05% in the phospholipid, 62.38% in the total lipid and 60.69% in the neutral lipid of a. escheriana (table 1) and 66.23% in the phostable 2 fatty acid profile of main organs total lipid and lipid classes from assyriella guttata. fatty acid compositions fatty acid compositions of total lipid from a. guttata organs of lipid classes from a. guttata fatty acids digestive gland cephalopedal gonad mantle total lipid neutral lipid phospholipid (mean*±s.d.)** (mean*±s.d.)** (mean*±s.d.)** (mean*±s.d.)** (mean*±s.d.)** (mean*±s.d.)** (mean*±s.d.)** c10:0 0.09±0.02a 0.12±0.03b 0.08±0.02a 0.07±0.02a 0.24±0.04c c12:0 0.70±0.08a 0.87±0.10b 0.95±0.10b 0.60±0.07a 0.77±0.08ab c13:0 1.35±0.13a 1.16±0.12a 1.09±0.10a 1.30±0.12a 1.11±0.12a 1.05±0.11a 0.09±0.02b c14:0 2.22±0.21a 1.47±0.16b 1.10±0.12a 1.78±0.19ab 1.85±0.20ab 2.03±0.21a 0.64±0.08c c15:0 0.99±0.10a 1.02±0.11a 1.32±0.14a 1.44±0.15a 1.32±0.14a 1.82±0.20b 0.47±0.07c c16:0 7.01±0.64a 9.01±0.72b 9.72±0.78b 7.82±0.66ab 9.61±0.73b 7.78±0.58ab 5.92±0.41c c17:0 0.13±0.03a 0.33±0.06b 0.16±0.03a 0.18±0.04a 0.06±0.02c 0.09±0.02ac 0.08±0.02ac c18:0 8.62±0.64a 13.58±0.91b 10.11±0.74c 15.21±1.04d 12.33±0.86bc 13.01±0.94b 8.76±0.59a c14:1ω9 0.35 ±0.05a 0.21±0.04a 0.23 ±0.04a 0.25±0.04a 0.67±0.06b c16:1ω7 1.34±0.12a 0.77±0.09b 0.87±0.10b 0.89±0.16b 0.40±0.03c 0.44±0.03c 0.68±0.07bc c18:1ω9 17.42±1.32a 16.32±1.25a 19.65±1.49b 15.35±1.21c 15.06±1.21c 16.03±1.30a 16.02±1.29a c20:1ω9 0.60±0.07a 0.75±0.08a 0.67±0.06a 0.16±0.03b 0.43±0.05c 0.31±0.05c 0.69±0.08a c22:1ω9 0.21±0.04a 0.04±0.01b 0.09±0.02b 0.08±0.02b c18:2ω6 28.12±1.75a 21.60±1.48b 24.24±1.55c 20.23±1.45b 18.54±1.38d 17.07±1.35d 22.34±1.65a c18:3ω3 4.06±0.31a 2.43±0.19b 4.76±0.44a 2.90±0.32a 6.24±0.51c 7.01±0.57c 10.70±0.41ac c20:2ω6 6.14±0.53a 13.42±1.16b 9.15±1.02c 8.81±0.96c 9.22±1.04c 10.99±1.16bc 8.52±0.84c c20:3ω6 0.71±0.62a 0.61±0.56a 0.24±0.03b 0.19±0.03b 0.13±0.03c 0.08±0.02c 0.80±0.09a c20:4ω6 15.09±1.29a 13.73±1.12b 11.05±1.10c 15.68±1.25a 16.58±1.30d 15.71±1.28a 16.13±1.33ad c20:5ω3 4.53±0.39a 2.16±0.21b 2.45±0.23b 4.85±0.35c 3.75±0.38c 3.54±0.34c 4.76±0.41a c22:2ω6 0.14±0.03a 0.02±0.01b 0.04±0.01b 0.59 ±0.05c 0.08±0.02ab c22:5ω6 0.15±0.03a 0.13±0.03a 0.55±0.06b 0.37±0.04c 0.98±0.10d 0.67±0.08b 0.82±0.09d c22:6ω3 1.23±0.13a 1.24±0.12a 1.38±0.14a 1.60±0.16a 1.36±0.15a 1.00±0.10a 2.08±0.24b σω6 / σω3 5.13 8.49 5.29 4.84 4.00 3.91 2.28 σsfa 21.02±1.51a 26.66±1.62b 24.49±1.56ab 28.76±1.74c 26.95±1.67b 26.79±1.59b 15.96±1.32c σmufa 19.71±1.37a 18.05±1.33a 21.63±1.42b 16.44±1.28c 16.14±1.22c 16.87±1.30c 18.14±1.33a σpufa 60.17±2.49a 55.34±2.25b 53.86±2.15c 54.63±2.18b 56.80±2.39d 56.66±2.36d 66.23±2.51e results expressed as percentage of total fatty acids methyl esters. *values are means ± s.d (standard deviation) for three samples of triplicate analysis. **means followed by different letters in the same line are significantly different (p < 0.05) by tukey’s test. sfa: saturated fatty acids, mufa: monounsaturated fatty acids, pufa: polyunsaturated fatty acids, σω6: total of omega 6 fatty acids, σω3: total of omega 3 fatty acids. pholipid, 56.80% in the total lipid and 56.66% in the neutral lipid of a. guttata (table 2). discussion the significance of fatty acids drives from their role as fuel to provide metabolic energy, their usage for storage products, eicosanoids, physiological activities, structural components such as membrane lipids particularly phospholipids and sterol esters. most importantly, they fulfill a structural role and are also very important intermediates in cell physiology, formation of prostaglandins and other eicosanoids from ω3 and ω6 fatty acids (stanley-samuelson, 1994). the importance of specific fatty acids as dilatory compounds for animals is partly because of almost all animals to introduce second or third double bond into fatty acids to synthesize polyunsaturated fatty acids (beenakkers et al., 1985). the seriousness of fatty acids was mostly emphasized for freshwater molluscs. however, lipid data correlated with nutritional, physiologiital. j. food sci., vol. 27 2015 5 cal, structural and environmental factors in terrestrial snails is notably limited in literatures and relatively little is known about terrestrial snails’ fatty acid composition, particularly on their organs. there are only a few studies regarding of fatty acids of edible, nonedible snails and land slugs such as h. aspersa (çağiltay et al., 2011), h. aspersa maxima (milinsk et al., 2006), h. pomatia (özoğul et al., 2005), e. vermiculata (stavrakakis et al., 1989), helix sp., haplotrema sportella, vespericola columbiana, arion ater, limax maximus, prophysaon andersoni (zhu et al., 1994). furthermore, only few studies are present on fatty acid distribution of mollusc organs and tissues. macoma balthica (wenne and polak, 1989), telescopium telescopium (rakshit et al., 1997), argopecten purpuratus (caers et al., 1999), bellamya bengalensis, pila globosa (misra et al., 2002), unio elongatulus (eki̇n and başhan, 2010), corbicula fluminalis (eki̇n, 2012), h. lucorum (eki̇n 2014) are some of known mollusc species, tissue and organs studied. qualitatively, fatty acid profiles of two species were similar. similarity of the fatty acid content of both species is not surprising. because they are close relatives and derived from the same origin. however, quantitative differences in the fatty acid profile were likely due to environmental, nutritional and physiological effects. generally, molluscs are well known to contain c16:0, c18:0, c18:1ω9, c18:2ω6, c18:3ω3 and c20:4ω6 as major fatty acids. these fatty acids have previously reported in most of the mollusc species and explored for their potential use in food chain studies. they were identified as predominant components in theodoxus jordani, melanoides tuberculata, pyrigula barroisi, melanopsis praemorsum freshwater snails (go et al., 2002); in helix sp. h. sportella, v. columbiana (zhu et al., 1994), h. aspersa (çağiltay et al., 2011), h. pomatia (özoğul et al., 2004), h. lucorum (eki̇n, 2014) land snails; in t. telescopium marine snail (rakshit et al., 1997); in u. elongatulus (eki̇n and başhan, 2010), c. fluminalis (eki̇n, 2012), b. bengalensis, p. globosa (misra et al., 2002), a. purpuratus mussels (caers et al., 1999); in p. andersoni, a. ater, l. maximus (zhu et al., 1994) slugs. as highlighted above, a. escheriana and a. guttata also contained high amount of c16:0, c18:0, c18:1ω9, c18:2ω6, c18:3ω3 and c20:4ω6, concentrated in the organs and fractions. in previous studies, it was stated that c20:4ω6 is more characteristic of sea urchins and starfish, c20:5ω3 is characteristic of invertebrates that feed on single-celled algae and occurs in almost all classes, additionaly c22:6ω3 is more characteristic of fish and crustacea (sinanoglou and miniadismeimaroglou, 1998). notably, c16:0, c18:0 and c18:1ω9 can be found in most of the animal tissues and very common among fatty acids. pufas may be further modified to form prostaglandins that are directly involved in regulation of reproduction, renal function, ion regulation as known from mollusc species, (stanleysamuelson, 1987). it is stated that egg production in freshwater snail helisoma durgi was stimulated by prostaglandins (kunigelis and saleuddin, 1986). c20:4ω6 precursors of prostaglandins was found to be 15.09% in the digestive gland, 13.73% in the cephalopedal, 11.05% in the gonad, 15.68% in the mantle of a. guttata and 11.14% in the digestive gland, 14.37% in the cephalopedal, 10.01% in the gonad, 17.86% in the mantle of a. escheriana. probably, this high value is related to reproduction and other physiological activities of the snails. in animal cells prostaglandins precursor c20:4ω6 is mostly obtained from phospholipid main source of pufa. to remember, the phospholipids of the snails contained high level of c20:4ω6, 20.30% in a. escheriana and 16.13% in a. guttata. as a matter of fact, σpufa levels in the phospholipid were detected much higher than other lipid fractions and organs both in a. escheriana and a. guttata. in the phospholipid of a. escheriana and a. guttata, σpufa levels were presented to be 66.05% and 66.23%, respectively. recognizing that snail species in this study are herbivores, therefore containing high proportion of pufa was expected result. because plant based diet is containing much more pufa than flesh based diet. in the present study, σpufa levels were always found to be higher than σsfa and σmufa. this finding was in agreement with garden snail h. aspersa stating that pufa was most abundant fatty acids (çağiltay et al., 2011). it was also declared that c18:2ω6, c20:4ω6, c18:3ω3 and c20:5ω3 were the dominant fatty acids. snails frequently feed on decaying plant materials to avoid high concentration of deterrent or toxic plant metabolites (speiser et al., 1992). aging of plant material results in a decrease of its pufa content (kis et al., 1998), suggesting that snails eating old plant material may suffer from a shortage of pufa. therefore, it can be said that natural food sources vary seasonally in the composition of ingredients (wacker, 2005). in the present study, the snails were collected in spring season and they mostly fed on fresh plant materials. maximization of pufa levels in all organs and fractions were probably because of fresh plant diets. the snails’ mating activities are significantly reduced when snails were fed the pufa-deficient diet. it is stated that pufa played important role in reproductive allocation (wacker, 2005). a. escheriana species were collected from woodland, whereas a. guttata species were collected from stony and rocky region of city walls which is containing decaying organic matter, garbage, sediments, grass, shrubs and etc. decaying organic matter containing places mostly shelters bacteria, protozoa, mold, invertebrates 6 ital. j. food sci., vol. 27 2015 and other microorganisms. c13:0, c14:0, c15:0, c17:0 and other short-chain saturated fatty acids are common in bacteria (wacker, 2005). in the analyses, it was observed that a. guttata contained slightly higher amount of short-chain fatty acids than a. escheriana. c14:0 ranged from 1.10% to 2.22% in a. guttata organs and 0.49% to 1.31% in a. escheriana organs. c15:0 varied from 0.99% to 1.44% in a. guttata and from 0.19% to 0.39% in a. escheriana. probably, it was stem from habitats of a. guttata which is suitable for living for microorganisms. in all fractions and organs, c18:2ω6 essential fatty acid was the main components followed by c18:1ω9 and c20:4ω6. the highest concentration of the fatty acid was found to be 28.12% and 26.67% in the digestive gland of a. guttata and a. escheriana, respectively. among the snails’ lipid fractions, the phospholipid contained 27.43% in a. escheriana and 22.34% in a. guttata of c18:2ω6 (table 1, 2). on the contrary, this fatty acid was found in low level in t. telescopium freshwater snail’s organs; 2.5% in the digestive gland, 4.3% in mantle and 4.95% in the cephalopedal (rakshit et al., 1997). on the other hand, in edible snail h. aspersa maxima, c18:2ω6 was found rather a lot, between 44.79%-51.19% (milinsk et al., 2006) and this fatty acid was also found good amount in edible snail h. lucorum (eki̇n, 2014). most likely, these different data stem from the requirement of the fatty acid for snail species. this essential fatty acid plays central role in production of other pufa and most animals cannot synthesize it, for this reason they are dependent on taking it from their diets. an interesting fact was that snails had c20:2ω6 with high concentrations varying from 6.14% to 13.42% in a. guttata and from 7.25% to 14.93% in a. escheriana. in particular, the highest level of the fatty acid was detected in the cephalopedal of both species. in some studies, it is stated that snail cephalopedal served as a storage organ (johns et al., 1979), probably, the cephalopedal stored this fatty acid for further metabolic activities. in comparison with a. escheriana and a. guttata, it was observed some strange results in t. telescopium snail, for instance c18:3ω3 was not detected in the digestive gland and mantle, but it was found 10.7% in the cephalopedal tissue as well as c16:1ω7 was found to be 11.3% in the digestive gland, 6.1% in the mantle, 4.9% in the cephalopedal (rakshit et al., 1997). in a. guttata and a. escheriana, c16:1ω7 was found at low concentrations, did not exceed 1.80%. however, c18:3ω3 was presented 7.36% in the digestive gland, 3.96% in the mantle of a. escheriana and 4.06% in the digestive gland, 2.90% in the mantle of a. guttata. for a. escheriana, the highest proportion of c18:3ω3 was found to be 8.42% in the total lipid in comparison with 10.70% in the phospholipid of a. guttata. c18:3ω3 was another essential fatty acid and its content was expected to be high in the phospholipid fractions, because phospholipid contains much more pufa than mufa and sfa. it is also noteworthy that, the content of fatty acids may differ from year to year, season to season, and depend on the nutrition of organism. above all, the distribution of an organism is mostly influenced by many factors including temperature, reproduction season, growth, nutrient availability, genetic, physiology and etc. in the treatments, neutral lipid and total lipid fatty acid distribution are more similar to each other than phospholipids. in particular, σsfa, σmufa and σpufa contents in neutral lipid and total lipid were detected so close to each other in both species. this kind of determination is very normal, because neutral lipids and total lipids are structurally and contently similar. both ω3 and ω6 fatty acids are important components of biomembranes and are precursors to many other substances in organisms. researches indicate that omega fatty acids especially ω3 fatty acids reduce inflammation and may help lower risk of chronic diseases such as heart disease, cancer, and arthritis. the ratio of σω6/σω3 is usually received to be useful indicator for comparing nutritional values of the samples. in a. escheriana, the highest value of σω6/σω3 was in the phospholipid (6.59), whereas the lowest value was in the neutral lipid and digestive gland (4.86). on the other hand, in a. guttata the highest value of σω6/σω3 was in the cephalopedal (8.49), the lowest value was in the phospholipid (2.28). this wide difference between snails’ phospholipids fractions was due to the high proportion of c18:2ω6 in a. escheriana. in agreement with our findings, σω6/σω3 ratio was also found to be high in a. ater, l. maximus, p. andersoni, slugs and v. columbiana, h. sp. h. sportella (zhu et al., 1994) and in h. lucorum land snails (eki̇n, 2014). but, in marine molluscs, percentage of σω6 was found to be lower than σω3 (abad et al., 1995; pazos et al., 2003). eventually, the results showed that species were rich in pufa, totally always over 50% in all analyses and maximization of c16:0, c18:0, c18:1ω9, c18:2ω6, c18:3ω3, c20:2ω6 and c20:4ω6 were observed. particularly, the organs and lipid fractions of both snails contained good amount of essential fatty acid, c18:2ω6 taking role in the synthesis of other fatty acids. moreover, σω6/σω3 and σpufa/σsfa+ σmufa ratios were found in good range. herewith, the results can be important guide for further investigation on nutritional, physiological, biochemical and taxonomic studies of molluscs. commercially some important edible snails cryptomphalus aspersus (h. aspersa), h. asemnis, h. cincta, h. lucorum, t. pisana, e. vermiculata and c. apertus dwell in turkish territories (yildirim, 2004). although a. escheriana and a. guttata are not edible snails; however they are very common in ital. j. food sci., vol. 27 2015 7 the southeastern anatolia region. it should not be forgotten, snails collected from the wild environment may accommodate poisonous chemicals, heavy metals, drugs, alkaloids and agricultural chemicals. perhaps, a. escheriana and a. guttata land snails will be used as edible after the pathological and biochemical detailed studies in the future; however we can offer no adequate explanation about edibility at present. references abad m., ruiz c., martinez d., mosquera g. and sanchez j.l. 1995. seasonal variation of lipid classes and fatty acids in flat oyster, ostrea edulis, from san cibrian (galicia, spain). comp. biochem. physiol. 110c (2): 109. ackman r.g. 2000. fatty acids in fish and shellfish. in: “fatty acids in foods and their health implications”. c.k. chow (ed.), pp. 153-172, m. dekker, inc, new york and basel beenakkers a.m.t., van der host d.j. and van marrewijk w.j.a. 1985. insect lipids and lipoproteins and their physiological processes. prog. lipid res. 24:16. bligh e.g. and dyer w.j. 1959. a rapid method of total lipid extraction and purification. can. j. biochem. physiol. 37: 911. caers m., coutteau p., cure k., morales v., gajardo g. and 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wacker a. 2005. lipids in the food of a terrestrial snail. invertebr. reprod. dev. 47(3): 205. wenne r. and polak l. 1989. lipid composition and storage in the tissues of the macoma balthica. biochem. syst. ecol. 17: 583. yıldırım m.z. and kebapçı ü. 2004. slugs (gastropoda: pulmonata) of the lakes region (göller bölgesi) in turkey. turk. j. zool. 28: 155. zhu n., dai x., lin d.s. and cornor w.e. 1994. the lipids of slugs and snails: evolution, diet and biosynthesis. lipids. 29: 869. paper received april 24, 2014 accepted june 23, 2014 ijfs#649_bozza ital. j. food sci., vol 29, 2017 398 paper nutritional evaluation of fresh and dried goji berries cultivated in italy s. niro, a. fratianni*, g. panfili, l. falasca, l. cinquanta and md rizvi alam department of agricultural, environmental and food sciences, university of molise, via f. de sanctis, 86100 campobasso, italy *corresponding author. fratianni@unimol.it abstract the nutritional profile of fresh and dried goji berries cultivated in italy was investigated. the obtained data confirm goji berries as a source of nutritional and healthy components, such as vitamin e, minerals and fibre. taking into account the recommended daily allowance (rda) for minerals and vitamins established by the commission of the european communities, goji berries provide significant amounts of dietary fibre and zeaxanthin and can be declared on the label as a potential source of vitamins e and c. moreover, dried goji berries can be declared as a source of k, p, cu, fe mn, zn. keywords: goji berries, lycium barbarum, superfruit, wolfberries ital. j. food sci., vol 29, 2017 399 1. introduction fruits of lycium barbarum l., belonging to solanaceae family, commonly known as goji berries or wolfberries, have been used in chinese traditional medicine for centuries. lycium barbarum grows in china, tibet and other parts of asia and its fruits are 1-2 cmlong, bright orange-red ellipsoid berries. the native area of lycium is not definitively established but it is likely found in the mediterranean basin (potterat, 2010). traditionally, goji berries are collected in summer and autumn. the fruits can be eaten fresh or dried, and they are also found in conventional food products, such as yoghurt, fruit juices, bakery foods, chocolate and others (mikulic-petkovsek et al., 2012a, 2012b). the drying process is intended to remove water from foodstuff in order to prevent microbial spoilage and chemical alterations, thus prolonging shelf life, while realizing space and weight saving (cinquanta et al., 2010; cuccurullo et al., 2012; fratianni et al, 2013). traditionally, the berries are dried in the shade until the skin shrinks and then exposed to the sun until the outer skin becomes dry and hard but the pulp is still soft (amagase and farnsworth, 2011). the sun drying method is cheap, but there is a risk of damage due to dust and insect infestation. an alternative is hot air drying. today the goji fruit market is significantly expanding because of an increased awareness of the possible health benefits, as fruits contain different nutrients, such as polysaccharide complexes, organic acids, phenolic compounds and antioxidants with high biological activity. dietary fibre provides several health benefits, including the reduction of the risk of coronary heart disease, of diabetes, hypertension, obesity, stroke and some gastrointestinal disorders (efsa, 2010). recent studies indicate that polysaccharides from lycium barbarum possess a range of biological activities, including antioxidant properties (amagase and nance, 2008; chang and so, 2008). goji fruits constitute a variety of antioxidants such as ascorbic acid, different carotenoids (kulczyński and gramzamichałowska, 2016) and high levels of phenolic compounds (zhang et al., 2016). carotenoids are a significant group of biologically-active constituents with health promoting properties (amagase et al., 2009; donno et al., 2014) responsible for the colour of a wide variety of foods (fratianni et al., 2005). the reddish-orange colour of l. barbarum fruits derives from a group of carotenoids, which make up only 0.03–0.5 % of the dried fruit. zeaxanthin is the major carotenoid found in goji. this is a yellow pigment, an isomer of lutein and a derivative of β-carotene. when ingested, zeaxanthin accumulates in fatty tissues, but especially in the macula, a region of the retina, helping in protecting the macula from degeneration, which can be induced by excessive sun exposure (uv light) and by other oxidative processes. in goji, zeaxanthin is present as an ester of dipalmitate. studies focusing on carotenoid goji berries are few and mainly aimed at the identification and quantification of ester-form carotenoids. inbaraj et al. (2008) and zhao et al. (2013), in particular, identified free-forms and ester-forms of carotenoids. beta-carotene, neoxanthin, and cryptoxanthin are also present at low concentrations (peng et al., 2005; wang et al., 2010). regarding other antioxidants, studies made on lycium chinense miller reported high amounts of α-tocopherol, together with other vitamin e compounds (isabelle et al., 2010). vitamin e is a generic term indicating structurally related compounds, namely tocols, comprising two groups of vitamers, i.e. tocopherols and tocotrienols, which occur in eight forms: α-tocopherol (α-t), β-tocopherol (β-t), γ-tocopherol (γ-t), and δ-tocopherol (δ-t) and α-tocotrienol (α-t3), βtocotrienol (β-t3), γ-tocotrienol (γ-t3), and δ-tocotrienol (δ-t3). the potential health benefits of tocols have been the subject of several reviews (tiwari and cummins, 2009). vegetable oils are the main tocol source; however, substantial amounts of these compounds are also reported in most cereal grains (fratianni et al., 2013; mignogna ital. j. food sci., vol 29, 2017 400 et al., 2015; panfili et al., 2003). to our knowledge, no literature data are available on the composition and content of tocols in l. barbarum fruits. goji is also an extremely rich source of many essential minerals, which are essential for many actions in the body, like muscle contraction, normal heart rhythm, nerve impulse conduction, oxygen transport, oxidative phosphorylation, enzyme activation, immune functions, antioxidant activity, bone health, and acid-base balance of the blood (williams, 2005; saldaml and sağlam, 2007). an adequate daily amount of minerals is necessary for an optimal functioning of the body. for the above reported reasons, goji berries are often proposed as functional foods and have been included in the novel category of “superfruits” or “superfoods”. superfruits, a subcategory of superfoods, is a relatively recent word and is considered a new marketing approach to promoting common or rare fruits which can be consumed as foodstuffs or used as ingredients by manufacturers of functional foods, beverages and nutraceuticals. superfruits have a high nutritional value due to their richness in nutrients, antioxidants, proven or potential health benefits and taste appeal (felzenszwalb, 2013). in the functional foods market, the products targeting health and mental well-being have prompted the food industry to increase the research and the development of these new foods, outlining a rapid expansion market in several countries (vicentini et al., 2016). in the last years, goji berries have been cultivated in italy and are available both as fresh and dried fruits. while several papers on the medical effect of goji berries have been published, little information is available on the nutritional composition of dried and, above all, fresh goji berries. the aim of the present study is therefore to determine the compositional and nutritional value of fresh and dried goji berries cultivated in italy, with a particular focus on minerals and some antioxidant compounds, such as carotenoids, tocols and vitamin c, to increase the awareness about their nutritional profile. 2. material and methods 2.1. sample collection and preparation fresh goji berries (l. barbarum l.) were provided by favella spa farm (sibari, southern italy). the farm has 21000 plants in 5 ha (2.5 m x 1 m), with a drip irrigation system. goji berries were cultivated in two consecutively growing seasons (2014 and 2015) and were collected in july. all harvested fruits were randomly collected in the orchard from different plants and analysed fresh and air-dried. fresh goji berries (about 1 cm size) were subjected to freeze-drying before analyses, as reported by fratianni et al., 2013 (fresh fruits). one-half of collected goji berries were air-dried in a convective dryer (b80fcv/e6l3, termaks, norway), at 60 °c, with an air velocity of 2.1 m/s, until a constant weight was reached (dried fruits). the drying time was about 21 h. results are reported as the average of the two growing seasons (2014-2015). 2.3 proximate composition analysis fresh and dried fruits were analysed for moisture, ash, fat, and protein (n×6.25) contents, according to standard methods of aoac (2000). dietary fibre content was determined according to aoac method 991.43 (1995) and aacc method 32-07 (1995). total dietary fibre content was the sum of insoluble and soluble dietary fibre content. vitamin c was determined by using an enzymatic kit (megazyme international, ireland), following the manufacturer instructions. ital. j. food sci., vol 29, 2017 401 2.4. mineral analysis ultrapure nitric acid for trace analysis, sulfuric acid (96 %) and standard mono elements in nitric acid 2 % were purchased from sigma-aldrich (20151 milan, italy). the determination of metals (k, ca, co, cu, fe, mg, mn, mo, na, p, se, zn) in goji samples was carried out by using the technique of nitric mineralization and the analysis by spectrophotometry plasma emission (varian icp 710, oes inductively coupled plasmaoptical emission spectrometers, palo alto, ca 94304-1038). samples were ground and 0.5 g was digested with 10 ml of nitric acid with a mineralizer (scp science digiprep, quebec h9x 4b6, canada), with the following instrumental conditions: start at 40 °c for 15 minutes; heating at 60 °c for 15 minutes; stay at 60 °c for 15 minutes; heating to 90 °c for 20 minutes. the digested samples were cooled and brought to a volume of 50 ml with bidistilled water and analysed with the optical icp. the precision was calculated as a mean deviation of three measurements. 2.5. carotenoid extraction and determination carotenoid extraction was carried out using the direct solvent extraction method reported in fratianni et al. (2013) with slight modifications due to the complex structure of goji berries. about 0.1 g of milled freeze-dried samples (fresh fruit) and air-dried samples (dried fruit) was weighed and placed in a screw-capped tube. then, 5 ml of ethanolic pyrogallol (60 g/l) was added as an antioxidant. the sample was stirred for 10 minutes. after that, 2 ml of absolute ethanol was added and the sample was stirred again for a few minutes. the suspension was then extracted with 15 ml of n-hexane/ethyl acetate (9:1 v/v) and stirred; after that 15 ml of sodium chloride (10 g/l) was added. further extractions with n-hexane/ethyl acetate (9:1 v/v) were made until the organic layer was colourless. finally, the organic layer was collected and evaporated to dryness, and the dry residue was dissolved in methanol: mtbe 50:50 (v/v). this sample was used to determine the free carotenoids not esterified with the lipid components and carotenoids esterified with fatty acids (unsaponified). a volume of 2 ml of this extract was evaporated to dryness and subjected to alkaline hydrolysis under a nitrogen flux for 1 minute in a screw-capped tube with 1 ml of ethanolic pyrogallol (60 g/l), 10 ml of solvent heat (hexane: ethanol: acetone: toluene 10: 6: 7: 7 v/v/v/v), 2 ml of methanolic koh (40 %) and glass balls. the tubes were placed in a 56 °c water bath and mixed every 5 to 10 min. after alkaline digestion at 56 °c for 20 minutes, the tubes were cooled in an ice bath, and 15 ml of sodium chloride (10 g/l) were added. the suspension was then extracted with 15 ml of n-hexane/ethyl acetate (9:1 v/v) until the organic layer was colourless. the organic layer was collected and evaporated to dryness, and the dry residue was dissolved in methanol: mtbe 50:50 (v/v). this sample was used to determine carotenoids esterified with lipid components (saponified). an aliquot of the carotenoid extract was separated, as in mouly et al. (1999), by a reverse-phase hplc system. an hplc dionex (sunnyvale, ca) analytical system, consisting of u3000 pumps, and an injector loop (rheodyne, cotati) were used. separation was performed as in fratianni et al. (2013) by using a ymc (hampsted, nc, usa) stainless steel column (250×4.6 mm i.d.) packed with 5 μm silica spheres that were chemically bonded with a c30 material at a flow rate of 1 ml/min. the mobile phase was methanol: mtbe (v/v). the eluted compounds were monitored by a photo-diode array detector (dionex, sunnyvale) set at 430 nm. ital. j. food sci., vol 29, 2017 402 2.6. carotenoid identification and quantification carotenoids were identified on the basis of their diode array spectral characteristics, retention times, and relative elution order, compared with known commercially available standards. all-trans-β-carotene and lutein were from sigma chemicals (st. luis, mo, usa); zeaxanthin and β-cryptoxanthin were obtained from extrasynthese (z.i. lyonnord, genay, france). zeaxanthin dipalmitate was identified by means of its spectral characteristics found in literature (inbaraj et al., 2008). compounds were identified by comparison of their retention times with those of known available standard solutions and quantified on the basis of the calibration curves of standard solutions. zeaxanthin dipalmitate was quantified as zeaxanthin. 2.7. tocol analysis tocols were determined after the saponification method of the extract described for carotenoids. an aliquot of the carotenoid extract was collected and evaporated to dryness, and the dry residue was dissolved in 2 ml of isopropyl alcohol (1 %) in n-hexane and was analysed by hplc under normal phase conditions, using a 250 x 4.6 mm i.d., 5 mm particle size, and kromasil phenomenex si column (torrance, ca, usa) (panfili et al., 2003). fluorometric detection of all compounds was performed at an excitation wavelength of 290 nm and an emission wavelength of 330 nm by means of an rf 2000 spectrofluorimeter (dionex, sunnyvale, usa). the mobile phase was n-hexane/ethyl acetate/acetic acid (97.3:1.8:0.9 v/v/v) at a flow rate of 1.6 ml/min (fratianni et al., 2002; panfili et al., 2003). compounds were identified by comparison of their retention times with those of known available standard solutions and quantified on the basis of the calibration curves of standard solutions. the concentration range was 5-25 µg/ml for every tocol standard. vitamin e activity was expressed as tocopherol equivalent (t.e.) (mg/100 g product), calculated as reported by sheppard et al. (1993). 3. results and discussion 3.1. nutritional composition the nutritional composition of fresh and dried goji berries is shown in table 1. fresh goji berries have 77.4 % moisture, 1.1 % fats, 2.5 % proteins, 15.3 % carbohydrates and 2.9 % fibre. in dried goji berries, 4.4 % fats, 10.2% proteins, 61.3 % carbohydrates and 11.4 % fibre were found. similar results on dried goji were reported by endes et al. (2015). our data suggest that dried fruits contain notable levels of dietary fibre, either as water-soluble form (2.6 %) or as insoluble form (8.8 %). the ratio between insoluble and soluble fibre is about 3:1. dietary fibre intake recommendation for adults is 25 g/day (larn, 2014). with the consumption of a portion of 30 g of dried fruits, dietary fibre intake for the adults is about 14 % of its daily recommended intake. taking into account the european law (regulation ce 1924/2006), dried goji can be declared in label with the claim “high fibre content”, since it contains at least 6 g of fibre per 100 g. finally, fresh and dried goji berries provide about 87 and 348 kcal/100 g, respectively. ital. j. food sci., vol 29, 2017 403 table 1. nutritional composition of fresh and dried goji berries (g/100 g) (mean±standard deviation). moisture fats proteins carbohydrates* fibre ash soluble insoluble total fresh 77.4±0.4 1.1±0.02 2.5±0.12 15.3 0.7±0.17 2.2±0.02 2.9 0.84±0.11 dried 9.3±0.02 4.4±0.45 10.2±0.22 61.3 2.6±0.06 8.8±0.01 11.4 3.4±0.16 * calculated by difference. 3.2. mineral composition the content of both macro and microelements in goji berries is reported in table 2. potassium (k) is the predominant element (276.2 mg/100 g and 881.9 mg/100 g for fresh and dried fruits, respectively), followed by sodium (na). potassium and sodium play an important role in regulating blood pressure and the body’s acid-base balance (clausen et al., 2013). goji could also be a good source of phosphorus (p) and calcium (ca), with an appreciable concentration of magnesium (mg), which is needed to prevent heart disease and growth retardation (chaturvedi et al., 2004). a discrete amount of copper (cu), iron (fe) and manganese (mn) were also found. bellaio et al. (2016), endes et al. (2015) and llorent-martínez et al. (2013) reported slightly different results. as in any other plant food, the mineral content of berries reflects the soil in which they are grown. it is important to highlight that essential and nonessential element concentration is dependent on the soil characteristics, the physiology of the plant, the water source composition, and fertilizers, insecticides, pesticides, and fungicides used in the plantations. plants can absorb, carry, and accumulate chemical elements. each species has its own requirements and differing levels of tolerance when absorbing and accumulating an element. the movement of the inorganic constituents is selectively controlled by the plant, with some being easily absorbed and others impeded to a different degree (naozuka et al., 2011). table 2. average values of mineral elements in fresh and dried goji berries (mg/100 g) (mean±standard deviation). fresh dried ca 26.6±4.90 101.3±22.60 k 276.2±41.00 881.9±239.70 mg 12.7±2.80 45.9±9.20 na 57.3±8.70 209.8±72.30 p 48.4±9.26 174.3±32.10 co 0.001 0.001 cu 0.3±0.04 0.8±0.25 fe 0.9±0.22 3.4±1.57 mn 0.2±0.03 0.5±0.18 zn 0.5±0.12 1.5±0.62 se (µg/100g) 0.1±0.01 0.17±0.03 mo (µg/100g) 0.00 0.00 ital. j. food sci., vol 29, 2017 404 table 3 reports the percentage contribution to the rda of 100 g of fresh and dried goji berries, according to reg. eu 1169/2011. for dried goji berries, the percentage of rda per portion (30 g) is also reported. from data, fresh goji berries can be declared on the label as a source of cu; in fact, 100 g of fresh goji berries contributed to about 25% of the rda. the contribution of other minerals from fresh goji is low. dried goji berries can be declared as a source of k, p, cu, fe mn, zn. a consumption of 30 g of dried goji per day contributes to the rda approximately of 25 % for cu, 13 % for k and less than 10 % for other elements. table 3. percentage contribution to the rda of minerals in fresh and dried goji berries. 3.3. carotenoid, tocol and ascorbic acid amounts table 4 shows hplc carotenoid analysis of fresh and dried fruits. drying of fruits did not cause significant changes in carotenoid amounts (data not shown). unsaponified carotenoids, determined after solvent extraction, and saponified carotenoids, determined after saponification of the extract, are reported. table 4. average carotenoid amounts in fresh and dried goji berries (mg/100 g) (mean±standard deviation). fresh dried unsaponified saponified unsaponified saponified lutein 0.0 1.1±0.02 0.0 5.7±0.44 zeaxanthin 0.0 53.8± 0.82 0.0 186.0±3.80 β-cryptoxanthin 0.0 2.3± 0.25 0.0 6.1±0.14 zeaxanthin dipalmitate 47.8±2.32 0.0 158.8±1.53 0.0 esters 8.5±1.24 0.0 24.5±2.30 0.0 β-carotene 0.2±0.01 0.1±0.01 0.9±0.09 1.0±0.20 total carotenoids 56.4±1.23 57.3±0.80 184.2±1.52 198.8±3.80 unsaponified carotenoids are characterized by a significant peak, identified as zeaxanthin dipalmitate, the dominant ester of goji berries (weller and breithaupt, 2003; inbaraj et al., 2008). beta-carotene is also present. before zeaxanthin dipalmitate peak, other unidentified peaks, probably carotenoid esters (inbaraj et al., 2008), were also reg. rda fresh dried dried mg/day % rda % rda % rda x 30g ca 800 3 13 4 k 2000 14 44 13 mg 375 3 12 4 p 700 7 25 7 cu 1 25 84 25 fe 14 6 24 7 mn 2 8 26 8 zn 10 5 15 5 se (µg) 55 0 0 0 ital. j. food sci., vol 29, 2017 405 detected. the amount of zeaxanthin dipalmitate in dried fruits is about 159 mg/100 g and of β-carotene is about 1 mg/100 g. inbaraj et al. (2008) found values of zeaxanthin dipalmitate and of β-carotene of 114.3 mg/100 g and 2.4 mg/100 g, respectively. this difference is probably due to the fact that carotenoid levels can be influenced by different harvest stage fruits, geographical origin, and seasonality (wen-ping at al., 2008). saponification of the extract is necessary to convert esters to free-compounds and it is often used to remove chlorophylls, lipids and other analytical interferences (fratianni et al., 2015). the saponified extract of dried fruits shows high zeaxanthin contents (about 190 mg/100 g) and small lutein and β-cryptoxanthin amounts (about 6 mg/100). small amounts of lutein were also found, after saponification, in a work of zao et al., 2013. as a dietary supplement for eye health (cheng et al., 2005), a dose of 15 g per day was deemed beneficial in supplying adequate zeaxanthin (estimated at 3 mg/day). thirty g of our goji samples provide a zeaxanthin amount of 14 mg/day (fresh fruit) and 48 mg/day (dried fruit). table 5 shows the tocol amounts in fresh and dried goji berries. as for carotenoids, the drying treatment did not cause significant changes in tocol contents (data not shown). table 5. average tocopherol amounts in fresh and dried goji berries (mg/100 g) (mean±standard deviation). fresh dried α-tocopherol 1.4±0.10 5.5±0.48 β -tocopherol 1.0±0.01 4.2±0.04 total tocopherols 2.4±0.04 9.7±0.20 tocopherol equivalent (te) § 2 8 § calculated as in sheppard et al., 1993. goji berries were found as a source of αand β-tocopherol (about 1.4 and 1.0 mg/100 g, respectively, in fresh fruits, and 5.5 and 4.2 mg/100 g, respectively, in dried fruits). a paper by isabelle et al. (2015) reports, in lycium chinenses, belonging to the same lycium species, the presence of α-tocopherol (3.9 mg/100 g), together with γ-tocopherol (0.46 mg/100 g), δ-tocopherol (0.12 mg/100 g), and traces of α-γ-δ tocotrienol (< 0.1 mg/100 g). table 5 also reports values of vitamin e activity provided by 100 g of product, expressed as tocopherol equivalent (te) (mg/100 g product) (sheppard et al., 1993). taking into account the recommended daily allowance (rda) for vitamin e, which is of 12 mg/day (regulation eu 1169/2011), 100 g of fresh goji berries contribute approximately 16 % of the rda, while 100 g of dried fruits contribute approximately 66 % of the rda, so that to be declared in label as a source of vitamin e. a portion of dried goji berries (30 g) contributes approximately 20 % of the rda. the concentration of vitamin c was about 40 mg/100 g in fresh fruits and 38 mg/100 g in dried fruits. donno et al. (2015) report an amount of about 42 mg/100 g in dried goji berries. taking into account the recommended daily allowance (rda) for vitamin c of 80 mg/day (regulation eu 1169/2011), 100 g of fresh or dried goji berries contribute approximately 50 % of the rda, so that they can be declared on the label as a source of vitamin c. a portion of dried goji berries (30 g) contributes about 16 % of the rda. ital. j. food sci., vol 29, 2017 406 4. conclusions goji berries cultivated in italy were confirmed as an important source of healthy compounds, providing a significant contribution to the diet, in terms of some inorganic nutrients, and of dietary fibre, zeaxanthin, vitamins e and c. in particular, taking into account the recommended daily allowance (rda) for minerals and vitamins established by the commission of the european communities, dried goji berries can be declared as a source of k, p, cu, fe mn, zn. moreover, both fresh and dried berries can be declared on the label as a potential 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fax: +39 0116708658 e-mail address: valentina.chiabrando@unito.it abstract the study investigated the effects of chlorine dioxide (clo2) gas on the postharvest quality of raspberries (cv grandeur) during storage. weight loss, color, total soluble solids content (tssc), titratable acidity (ta), ph, vitamin c, total phenols, anthocyanins and antioxidant capacity were evaluated. the clo2 positively influenced weight loss, color, ta, tssc and antioxidant capacity. moreover, clo2 treatment decreased the total yeast and mold count. in contrast, the vitamin c, anthocyanins and total phenolics were not influenced by the clo2 treatment. keywords: chlorine dioxide, fruit quality, pad, postharvest, red raspberries, storage ital. j. food sci., vol 29, 2017 477 1. introduction red raspberries (rubus idaeus l.) are rich in ascorbic acid, total phenols and anthocyanins. therefore, consumption of these berries is an important source of antioxidant compounds with a significant role in the preservation and promotion of health, such as preventing diabetes, cardiovascular risk factors and oxidative stress (atienza et al., 2015; sen and chakraborty, 2016). red raspberry is a highly perishable product in which modification of quality compounds are related to the growth of microorganisms, and natural decay (giacalone and chiabrando, 2012). raspberry harvest and storage are complex operations and can affect the quality and aroma compounds of the fruit (giuggioli et al., 2014). thus, the raspberry postharvest control strategies are critical to preserving the fruit quality, particularly on a long distribution chain (briano et al., 2015a; haffner et al., 2002). many technologies, like active packaging, modified atmosphere packaging, edible coating, difolatan, hexanal, vapor essential oils or sanitizer treatments, such as chlorine dioxide (clo2) and ozone have been studied to reduce the growth of microorganism, control postharvest decay and preserve the quality and freshness of berries (appendini and hotchkiss, 2002; briano et al., 2015b; chiabrando and giacalone, 2008; chiabrando and giacalone, 2015a; hajizadeh and kazemi, 2012; sun et al., 2014). chlorine dioxide is an alternative sanitizer, approved by the u.s. food and drug administration (fda) and by the u.s. environmental protection agency (epa). clo2 is legally used in china and usa for sanitizing fruit and vegetables (ministry of health of the people's republic of china, 2008 and usfda, 2010). particularly, clo2 is authorized in the us for use in washing, whole fresh fruit, vegetables, shelled beans and peas with intact cuticles at a concentration not exceeding 5ppm. in eu, however, there are no clear regulations regarding the use of clo2 for fresh produce. therefore, as a provisional solution, it was agreed that the individual member states will be given the ability to establish enforcement levels at the national level until risk management can take place based on european food safety authority (efsa) scientific opinion and monitoring data (banach et al., 2015). clo2 has been postulated as an alternative to sodium hypochlorite (naclo) for fresh and fresh-cut produce sanitization. cordis (community research and development information service), designing new decontamination approaches for freshcut food and sanitation strategies, include clo2 among the most promising sanitation methods. in the european union, clo2 can be used in other forms, like active packaging, as a gasgenerating pad (dukan et al., 1999). the active substances of the pad are molecules of hclo (hypochlorous acid), found in silica gel and toxic to microorganisms. chlorine dioxide gas is also a great sanitizer for food, at low concentration (chen et al., 2010; chang et al., 2000). the principal advantage of clo2 gas is its high penetrability (han et al., 2001). different studies (chen et al., 2010; chang et al., 2000) showed the positive effects of gaseous clo2 on the storage quality of lettuce, mulberries, plums and strawberries. the objective of this study was to evaluate the impact of clo2 treatment as a pad applied on the top lids of the clamshells, on the postharvest quality, nutraceutical aspects and microbiological decay of raspberries during four different storage conditions. ital. j. food sci., vol 29, 2017 478 2. materials and methods 2.1. samples and treatment red raspberries (r. idaeus l.) cv. grandeur were handpicked from a commercial orchard of the agrifrutta soc. coop. srl (piedmont, italy) at full ripeness and directly placed in commercial plastic boxes, clamshell type (13.5 x 9.0 x 2.5 cm, perforated, polyethylene terephthalate, 120 g of fruits). the samples were transported immediately to the laboratory of the disafa, university of turin, and only sound fruit was selected for the experiment. the boxes were randomly divided into two groups. on one group, a chlorine dioxide gasgenerating pad (cdp) (oplon pure science, ltd., ness tsiyona, israel) was placed on the top lids of the clamshells (u.s. food and drug administration (fda)-approved technology). the second group was used as the control. the effect of the cdp on the berries quality and antimicrobial activities during storage were measured. each treatment included three replicates. 2.2. storage treatments samples were stored in the dark in a controlled temperature room, and four different storage conditions were evaluated: s1: 4 days at 1 °c (short storage) s2: 8 days at 1 °c (long storage) s3: 4 days at 1 °c and 3 days at 4 °c (short storage + short-range international transport) s4: 8 days at 1 °c and 3 days at 4 °c (long storage + short-range international transport) the hypothesized transport time was 3 days, simulating a refrigerated transport from italy to northern europe. three repetitions were performed for all chemical analyses each time. 2.3. weight losses weight loss was determined by weighing the numbered samples boxes at the beginning of the experiment (time 0) and at the end of the four different storage conditions. the values were reported as the percentage of weight loss per initial boxes weight, as shown in equation (1). % 𝑤𝑒𝑖𝑔ℎ𝑡 𝑙𝑜𝑠𝑠𝑒𝑠 = !"!#!$% !"#$!!!!"#$% !"#$!! !"!#!$% !"#$!! ∗ 100 (1) 2.4. quality measurements the physicochemical quality attributes of the berries were measured at the beginning of the testing (time 0) and at the end of the four different storage conditions. 2.4.1 color the color of the berries was measured using a tristimulus cr-400 chroma meter (konica minolta sensing, inc. osaka, japan) with the d65 lamp and 2° observation angle. the instrument was calibrated against a standard white plate (y = 93.7, x = 0.3158, y = 0.3321) before the analysis. thirty measurements (30 berries) per treatment and sampling time ital. j. food sci., vol 29, 2017 479 were made. the results were express as cielab (l*a*b*) color space. the l* values describe the lightness, and a* and b* values express the red-greenness and blueyellowness, respectively. the color of berries was also expressed as c* (chroma or saturation). this parameter indicates the color variation c* = [(a*)2 + (b*)2]1/2) (francis, 1980). the reported values were the mean ± sd of 30 determinations. 2.4.2 total soluble solid content (tssc), titratable acidity (ta) and ph for each treatment, a digital refractometer (atago refractometer model pr-32; atago italia, milan, italy) was used to determine the tssc (°brix) in three undiluted filtered juice samples, each extracted from 30 berries. the instrument was calibrated against distilled water. the ta and ph were determined by adding 50 ml of distilled water into 10 ml of filtered juice and titrated with 0.1 n naoh to ph 8.2 with an automatic titrator (titration workstation titralab at1000 series, hach, milan, italy). titration data were expressed as meq l-1. 2.5. extraction and evaluation of total anthocyanins content, total phenolic contents, and total antioxidant capacity the anthocyanins, phenolics and antioxidant capacity were determined on the fruit extracts, obtained using 12.5 ml of extraction solvent (500 ml of methanol, 28.3 ml nanopure water and 1.4 ml 37% hcl) and 5 g of fresh fruit. after 60 min at 25 °c under reduced light conditions, the extracts were homogenized at 24000 rpm for 1 min, with an ultra-turrax t-25 tissue homogenizer (janke and kunkel, ika®-labortechnik, saufen, germany) and centrifuged at 3000 rpm for 15 min (centrifuge avantitm j-25, beckman instruments inc.). the supernatant was recovered and stored at -26 °c. three replicates for each treatment were performed at day 0 and at the end of the four different storage conditions. anthocyanins were determined using the ph differential method of cheng and breen (1991) by measuring the absorbance of the aqueous phase at 515 and 700 nm using a uvvisible spectrophotometer (u-5100, hitachi, tokyo, japan). anthocyanins were estimated by the difference in absorbance at 515 and at 700 nm in buffer at ph 1.0 and 4.5, where a = (a515 – a700)ph1 – (a515 – a700)ph4.5. results were expressed as mg cyanidin-3-glucoside per 100 g fresh berries. total phenolics contents were quantified using the slinkard and singleton protocol (1977) with folin-ciocalteu reagent. absorbance was measured at 765 nm. the results were calculated as gallic acid equivalents (gae) (mg gae 100 g-1 of fresh berries). the antioxidant activities of the berries were measured by the ferric reducing antioxidant power assay, as described by benzie and strain (1996), with some modifications (pellegrini et al., 2003). results were expressed as mmol fe2+ kg-1 fresh berries. 2.6. extraction and evaluation of vitamin c the vitamin c content was performed in agreement with sanchez-moreno et al. (2003) and gonzalez molina et al. (2008) at day 0 and after 4 and 8 days of storage. fruit flesh (10 g) was homogenized in 10 ml of methanol/water (5:95 v/v) using an ultraturrax t-25 for 3 min. then, the ph was adjusted to 2.2–2.4, and the extract was filtered through a c18 sep-pak cartridge (waters associates, milford, ma, usa). the resultant solution was combined with 1,2-phenylenediamine dihydrochloride (fluka chemika, neu-ulm, switzerland) for 37 min before hplc analysis. three replicate analyses of 10 fruits were performed for each treatment. the chromatographic system (agilent) was ital. j. food sci., vol 29, 2017 480 equipped with a diode array detector and kinetex-c18 column (4.6 x 150 mm, 5 µm, phenomenex., torrance, ca, usa) and controlled through hplc online software (agilent). the mobile phase (isocratic) consisted of 50 mm monobasic potassium phosphate and 5 mm cetrimide (sigma-aldrich corporation, saint louis, usa) in methanol:water (v/v) 5:95. the flow rate of 0.9 ml/min. the temperature was 40 °c, and the detector was set at 261 nm for ascorbic acid (aa) and 348 nm for dehydroascorbic acid (dhaa). the vitamin c content (aa and dhaa contents) was expressed as mg 100 g-1 of fresh weight. all standards and reagents were of analytical purity and were purchased from sigma italiana srl (ozzano emilia, italy). 2.7. yeast and mold evaluation the yeasts and molds content was evaluated at day 0 and after 4 and 8 days of storage as described by the compendium of methods for the microbiological examination of foods (vanderzant and splettstoesser, 1992). a 30 g sample of fresh berries was blended with 270 ml of peptone buffered water (sigma italiana srl, italy) for 1 min in a stomacher® bag using a blender (stomacher®400 circulator, seward, worthing, uk). rose bengal agar (sigma italiana srl, italy) was used for the yeast and molds evaluations. all the plates were incubated at 30 °c for 5 days. microbial counts were expressed as log colony forming units (cfu) g−1. 2.8. statistical analysis analysis of variance (anova) was performed on the data, and the means were compared by tukey’s honestly significant differences test. the source of variation was the treatments (control and cdp) and the storage time. differences between mean values were considered significant when p ≤ 0.05. spss software was used for all data analyses (spss statistics version 22 ibm). 3. results and discussions 3.1. weight loss in general, raspberries are affected by considerable weight loss because the tissues are exposed to a high level of respiration and transpiration (krüger et al., 2011). in the present study, low weight losses were observed in all the samples compared to previous studies (krüger et al., 2011; haffner et al., 2009). after 4 days of storage (s1), the weight loss of the cdp samples was 0.71%, which was significantly lower than the control (0.98%). therefore, in this postharvest storage condition, the cdp positively influenced the weight loss of raspberries, maintaining lower values compared to the control, according to aday and caner (2011). the same result was also obtained in the s3 and s4 storage conditions, with 1.16 and 2.13% weight loss, respectively of the cdp treated fruit, while the corresponding control values were 1.52 and 2.61%. the effect of clo2 on the weight loss depends on the inactivity of the microbial population, the inhibition of enzyme activity, such as polyphenol oxidase, and the inhibition of respiration rate and ethylene biosynthesis (aday and caner 2011; guo et al., 2013; wang et al., 2011; sun et al., 2014). ital. j. food sci., vol 29, 2017 481 3.2. quality measurements 3.2.1 color color change is an important factor that affects the visual appearance and the postharvest quality of fresh raspberry fruit. the l* and c* values of raspberry after 4 and 8 days of storage are reported in table 1. the l* values after 4 and 8 days of storage showed a significant decrease in both treatments (control and cdp treatment), compared to day 0. significant differences were observed between treatments after 4 days of storage at 1 °c, with higher values of l* in the cdp treated berries (fruit with a higher luminosity). as described by krüger et al. (2011) and shin et al. (2008), a decrease in lightness values during storage, indicates that the fruit became darker, less red and bluer with advance ripening. in the present work, samples treated with the cdp maintained the original color of the berries, in concurrence with the study of aday and caner (2011) on strawberries. the clo2 gas did not influence the lightness during the s3 and s4 storage conditions. in these instances, there were no significant differences (p ≥ 0.05) in l* value between the cdp treatment (l* 27.9 and 29.4 in s3 and s4, respectively) and the control (l* 28.4 and 29.2 in s3 and s4, respectively). the c* values initially increased and then decreased during storage as shown in table 1. considering the short period of storage (s1), results showed significantly higher values for the cdp samples compared to the control. in this instance, the cdp treatment significantly improved the redness intensity of berries during storage. in previous studies, the cdp treatment caused no pigment degradation and consequently, no changes in external color of berries (zheng et al., 2008; gomez lopez et al., 2009). the same result was also obtained under the s4 storage condition, where the cdp samples showed c* values of 23.3, which was significantly higher compared to the control (c* 20.8). table 1. effect of clo2 on color parameters (lightness and chroma) of raspberries after 4(s1) and 8(s2) days of storage at + 1°c. the data are average of 30 replicates ± sd. color parameters storage time (days) treatments 0 4 8 lightness control 35.1±1.9 aa 29.1±3.0 bb 29.8±1.74 ab (l*) chlorine dioxide 35.1±1.9 aa 30.4±2.2 ab 30.0±2.24 ab chroma control 22.7±4.0 ab 25.9±4.5 ba 25.4±4.1 aa (c*) chlorine dioxide 22.7±4.0 ab 28.9±5.3 aa 25.9±5.7 aa 3.2.2 tssc, ta and ph tssc, ta and ph values after 4 (s1) and 8 (s2) days of storage are shown in table 2. the tssc values were significantly different between treatments after 4 and 8 days of storage at +1°c, while the differences were not significant under s3 and s4 conditions (data not shown). the higher tssc levels in the control than cdp treated berries may be due to the higher respiration rate and weight losses and, consequently, more concentrated juice of the control berries. wu et al. (2011) showed that clo2 treatment maintained the tssc similar to the value recorded at harvest, which was better than untreated fruit, probably due to the reduction of postharvest infections. the ta decreased significantly during storage for both treatments, but without significant differences. the ta values decreased significantly for all samples during storage. in the ital. j. food sci., vol 29, 2017 482 control samples, the lowest recorded ta value was under s4 conditions, and in the cdp sample, it was under s3 conditions. the decrease in ta and increase in tssc during storage were associated with the enhancement of temperature from 1 to 4 °c in s3 and s4 that caused an increase in respiration rate and the natural consumption of organic acids by the metabolism (haffner et al., 2009). the changes in ta probably depended more on temperature than the effect of clo2, according to the study of aday and caner (2011) on strawberries (table 2). the ph level increased in the control samples in agreement with the ta result, while in the cdp treated samples no significant differences were observed during storage. aday and caner (2011) reported that clo2 treatment maintained stable ph levels in samples during storage probably due to the antimicrobial activity of clo2 on yeast and mold, and consequently, to the inhibition of natural decay. table 2. effect of clo2 on quality parameters of raspberries after 4(s1) and 8(s2) days of storage at + 1°c. total soluble solid content (tssc), titratable acidity (ta) and ph. the data are average of 3 replicates ± sd. storage time (days) treatments 0 4 8 tssc control 8.4±0.1 ab 9.0±0.3 aa 8.6±0.0 ab °brix chlorine dioxide 8.4±0.1 aa 8.3±0.2 ba 8.1±0.1 ba ta control 447.65±17.1 aa 450.98±5.6 aa 390.83±17.6 ab meq/l chlorine dioxide 447.65±17.1 aa 447.06±12.0 aa 402.03±8.1 ab ph control 3.04±0.01 ab 3.07±0.01 aab 3.10±0.04 aa chlorine dioxide 3.04±0.01 aa 3.02±0.01 ba 3.16±0.12 aa means sharing the same letters in rows (a, b) and in column (a, b) are not significantly different from each other (tukey’s hsd test, p ≤ 0.05). 3.3. total anthocyanin content total anthocyanin content is one of the functional constituents in raspberries that are associated with their bright red color. the content of anthocyanins increased during the storage at 1 °c (s1 and s2) (table 3) and then decreased during storage at 4°c (s3 and s4). this trend may be due to the decomposition of procyanidins at the beginning of storage (chun et al., 2013). the results of the present study agree with the research of haffner et al. (2002) that reported a similar increase in anthocyanin content after 7 days of cold storage (1.7°c), but contrasts with the study of mullen et al. (2002) that did not show a significant increase during cold storage. moreover, the results indicated that the content of anthocyanins increased in the same manner as the ph after 8 days of cold storage at 1 °c. according to orak (2007), a correlation exists between anthocyanins and ph in grapes. hence, the current results suggest a similar trend in raspberries. considering the treatment, clo2 does not seem to directly influence the anthocyanins level because no significant differences between treatments were found. 3.4. total phenolics content as shown in table 3, according to the total anthocyanin content results, no significant differences in total phenolic content were observed during storage time or between ital. j. food sci., vol 29, 2017 483 treatments. in general, an increase in total phenols was observed throughout the storage. a similar result was also noted previously in raspberries (krüger et al., 2011) in strawberries (cheng and breen, 1991) and in blueberries (chiabrando and giacalone, 2015b) and it seems to depend on the weight loss. in this work, the lack of significant difference between the cdp treated and control samples was probably due to the low level of clo2. gomez lopez et al. (2009) and napolitano et al. (2005) found that a high level of clo2 can react with phenolic compounds, decreasing their content in the food. considering the storage at 4 °c (s3 and s4), the clo2 gas did not influence the total phenolic content. indeed, there were no significant differences (p ≥ 0.05) between the cdp treatment (202.24 and 232.26 mg gae 100 g-1 in s3 and s4, respectively) and the control (216.31 and 240.95 mg gae 100 g-1 in s3 and s4, respectively). table 3. effect of clo2 on nutraceutical parameters of raspberries after 4(s1) and 8(s2) days of storage at +1 °c. the data are average of 3 replicates ± sd. storage times (days) treatments 0 4 8 total anthocyanin content control 43.62±6.65 ab 54.5±5.24 aab 78.11±7.66 aa mg cyanidin 3-gluc100 g-1 fw chlorine dioxide 43.62±6.65 ab 52.24±5.71 aab 68.22±7.47 aa total phenolics content control 198.3±16.55 aa 235.66±19.44 aa 219.45±23.41 aa mg gallic acid equivalent (gae)100 g-1 fw chlorine dioxide 198.3±16.55 aa 217.65±17.33 aa 204.21±9.87 aa total antioxidant capacity control 27.81±2.35 aa 28.93±0.60 aa 30.96±1.25 aa mmol fe2+kg-1 fw chlorine dioxide 27.8±2.35 aa 28.11±1.42 aa 28.11±0.80 ba vitamin c content control 12.45±0.40 aa 12.97±1.09 aa 9.39±0.85 ab mg100 g−1 fw chlorine dioxide 12.45±0.40 aa 12.87±0.57 aa 8.76±1.30 ab means sharing the same letters in rows (a, b) and in column (a, b) are not significantly different from each other (tukey’s hsd test, p ≤ 0.05). 3.5. total antioxidant capacity as shown in table 3, the total antioxidant capacity increased slowly during storage. statistical analysis showed significant differences between samples after 8 days of storage, with a higher value in the control than cdp treated sample. this result is in accordance with mullen et al. (2002) and kalt et al. (1999). considering the storage at 4 °c (s3 and s4), the clo2 did not influence the antioxidant capacity and there was no significant difference (p ≥ 0.05) between the cdp treatment (28.54 and 28.27 mmol fe2+ kg-1 in s3 and s4, respectively) and the control (29.68 and 31.03 mmol fe2+ kg-1 in s3 and s4, respectively). 3.6. vitamin c content table 3 presents the vitamin c values of the untreated and treated samples after 4 and 8 days of storage. the vitamin c content decreased over time in both treatments, but without statistical differences between treatments. a similar trend was observed in the study of haffner et al. (2002) and kalt et al. (1999) during storage at 0 °c. according to krüger et al. (2011), the aa content depends particularly on the storage conditions and the genotype of the plant. ital. j. food sci., vol 29, 2017 484 3.7. yeast and mold evaluation raspberries are highly perishable and susceptible to microbial decay during postharvest storage. therefore, decay is a primary factor of postharvest quality loss of raspberries, and a strategy is necessary to improve their shelf-life quality. no mold growth was visually observed during the present study. at day 0, the yeast and mold were present in relatively low amounts, at 2.9 and 4.2 log cfu/g-1, respectively (table 4). after 4 and 8 days of storage at 1 °c, a reduced yeast and mold count was observed in the cdp treated samples compared to the control. the decay incidence of the raspberries gradually increased with time of storage only in the control samples. hence, in this study, the cdp treatment was effective against the growth of yeast and mold throughout the storage period. this result was in agreement with the study of sun et al. (2014) on blueberries. the same trend has also been found in lettuce, carrot, apples, peaches, tomatoes and fresh-cut produce (sy et al., 2005). table 4. effect of clo2 on nutraceutical parameters of raspberries after 4(s1) and 8(s2) days of storage at +1°c. the data are average of 3 replicates ± sd. storage time (days) treatments 0 4 8 yeast control 2.85 ab 4.88 aa 3.61 aa log cfu g-1 chlorine dioxide 2.85 aa 2.56 ba 2.60 ba mold control 4.20 aa 4.79 aa 4.41 aa log cfu g-1 chlorine dioxide 4.20 aa 2.38 bb 2.34 bb means sharing the same letters in rows (a, b) and in column (a, b) are not significantly different from each other (tukey’s hsd test, p ≤ 0.05). 4. conclusions the present study showed the action of clo2 against the natural decay of raspberries and its efficacy in preserving berry quality under various postharvest storage conditions. results suggest that clo2 treatment in active packaging is useful to reduce decay and maintaining raspberry quality during storage. in particular, clo2 slowed down the tissue metabolism and consequently, lower weight losses were found compared to the untreated fruit. moreover, clo2 treatment significantly improved the redness intensity of berries during storage, but no significant effect on maintaining the stability of nutraceutical components was recorded. in summary, the cdp can be a valuable alternative sanitizer with a beneficial action against yeast and mold without reducing the quality of the final produce. this treatment also improved the shelf-life quality by inhibiting the weight loss and the color changes of raspberries during short period storage time. acknowledgements the study was supported by the university of turin local research. references aday m.s. and caner c. 2011. the applications of ‘active packaging and chlorine dioxide’ for extended shelf life of fresh strawberries. pack. technol. sci. 24:123. ital. j. food sci., vol 29, 2017 485 appendini p. and hotchkiss j.h. 2002. review of antimicrobial food packaging. innov. food sci. emerg. technol. 3:113. atienza l., perez e.g. and noratto g. 2015. effects of raspberry on biomarkers of diabetes, cardiovascular disease (cvd) and oxidative stress in obese diabetic (db/db) mice. faseb j. 29:1. banach, j. l., sampers, i., van haute, s. and van der fels-klerx, h. j. 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for the microbiological examination of food (splittstoesser, eds.) washington, dc. wang y., wu j., ma d. and ding j. 2011. preparation of a cross-linked gelatin/bacteriorhodopsin film and its photochromic properties. sci. china chem. 54(2):405. wu b., li x., hu h., liu a. and chen w. 2011. effect of chlorine dioxide on the control of postharvest diseases and quality of litchi fruit. afr. j. biotechnol. 10:6030. zheng y., yang z. and chen x. 2008. effect of high oxygen atmospheres on fruit decay and quality in chinese bayberries, strawberries and blueberries. food control 19(5):470. paper received november 28, 2016 accepted march 6, 2017 #528_riciputi_bozza ital. j. food sci., vol 29, 2017 63 paper assessing oil oxidative stability in tarallini by oxitest® y. riciputi*1 and m.f.caboni1,2 1department of agro-food sciences and technologies, alma mater studiorum, university of bologna, piazza goidanich 60, 47521 cesena, fc, italy 2inter-departmental centre for agri-food industrial research (ciri agroalimentare), university of bologna, piazza goidanich 60, 47521 cesena, fc, italy *corresponding author. tel.: +39 0547338117; fax: +39 0547382348 e-mail address: ylenia.riciputi@unibo.it abstract the shelf life of the typical italian bakery snack “tarallini” depends on the recipe and on the cooking and storage conditions. in this work, the lipid oxidative stability of tarallini was measured using an oxitest® instrument, an accelerated oxidation test. the oxitest® methodology was optimised taking into account sample amount and the sample particle size. homemade tarallini prepared using sunflower oil, extra virgin olive oil and a blend of extra virgin olive oil and lard were cooked for two different cooking times. the results showed a good ability of oxitest® to discriminate between lipid unsaturation and cooking time, providing information on the lipid shelf life of complex food matrices, such as tarallini. keywords: accelerated oxidation, bakery products, oxitest®, shelf life, tarallini ital. j. food sci., vol 29, 2017 64 1. introduction lipid oxidation is one of the main deteriorating reactions in food chemistry; food quality is deeply affected by lipid oxidation (calligaris et al., 2008). in particular, it damages lipids, especially essential fatty acids (choe and min, 2006), in fat-rich foods, such as bakery products, biscuits and snacks. moreover, lipid oxidation is a promoter of offflavours, producing the worst sensorial properties, reducing nutritional value and increasing the production of potentially toxic compounds (verleyen et al., 2005). thus, bakery product formulation must consider the characteristics of the used lipids, the matrix effect that influences their contact with oxygen and the technological treatment (cooking) that the products undergo. due to influence of food composition on shelf life, formulation can play an important role in food quality, even in terms of lipid oxidation. in fact, the interaction of lipid oxidation products with sugar, proteins and maillard reaction products greatly affect the development of lipid rancidity in complex food (frenkel, 1984). moreover, different other factors (temperature, light, oxygen partial pressure, etc.) could affect and enhance lipid oxidation during storage. in particular, among them, partial oxygen pressure affect oxidation rate (kacyn et al. 1983): higher is the headspace partial oxygen pressure and higher is the amount of oxygen dissolved in food. consequently, the oxygen available for lipid oxidation is increased and, in the dark, it will be enough to reach a value of peroxide value of 10meqo2/kg of fat (przybylski and eskin, 1988). in addition, fatty acid composition, in particular fatty acid unsaturation degree, storage time and proand antioxidant compounds significantly affect the auto-oxidation rate (calligaris et al., 2008). briefly, when the oxidation occurs, fatty acids are converted at first into hydroperoxide, can then decompose to form volatile molecules, hydroxylated, ketoor epoxycompounds or react with other oxidised fatty acids to form dimers or polymers. many different methods have been developed to assess these oxidation compounds in various food ingredients and products. gc analyses of volatiles compounds, free fatty acid or mono and diglicerides, hplc evaluation of oxidised fatty acid or spectrophotometric determination of peroxide value, conjugated diene and trienes are the main traditional methods applied to asses lipid oxidation in foods (verardo et al., 2010; verardo et al., 2011) however, the lipid oxidation rate is usually slow at room temperature and the rancidity threshold, which is strictly related to the consumer rejection of foods, could take months. moreover, oxidation products analyses are often time consuming and for the food industry it is very important to check as quickly as possible the food stability (màrquezruis et al., 2003). thus, excessively time-consuming shelf-life tests are useless for industry needs, and it is essential to apply methods that give quick answers (gómez-alonso et al., 2004). an interesting way to reduce the time of analysis of lipid oxidation compounds is using accelerated oxidation tests, allowing foods lipid stability assessment in a significantly shorter time than under real storage conditions (przybylski and eskin, 1988). usually, in this type of test, one or more parameters (temperature, oxygen pressure, light, etc.) (wan et al., 2000) that can increase the lipid oxidation rate are modulated, but the temperature is the most critical factor affecting the oxidation rate; thus, it is the most commonly considered (ragnarsson and labuza, 1977; labuza and schmidt, 1985; waterman and adami, 2005). several tests were developed to evaluate accelerated lipid oxidation. in particular, most of them like rancimat or oxidative stability instrument (osi) tests are suitable only for oils ital. j. food sci., vol 29, 2017 65 or fat extracted from foods, but are not applicable to the whole food. in this way, however, the effect of food matrix on lipid oxidation onset cannot be considered. a newer instrument, the oxitest® reactor, has been used to asses oils lipid oxidation in several studies (amato et al., 2015; cavazza et al., 2015; claus et al., 2015; claus et al., 2015 b; mora et al., 2009; mora et al., 2011) but it is particularly fitted to investigate the oxidation sensitivity also in solid foods (verardo et al., 2013; kwon et al. 2015), such as bakery products, with limited preparation and without any fat extraction (velp scientifica, 2006; maruyama et al., 2014; ferreira silva et al., 2015). the oxitest® reactor subjects the sample to an oxidative stress environment at high temperature and high oxygen pressure; the drop in oxygen pressure inside the oxidation chambers is monitored according to ability of the food to oxidise and is expressed as the induction period (ip) which is theoretically defined as the time required to obtain a continuous oxidation cycle in the oxidation process; it is measured as the time required for a sudden and rapid change in the oxidation rate (frenkel, 1998). considering the importance of the rapid determination of lipid oxidation in foods as a marker of quality during shelf life, the aim of this work was to evaluate the performance of oxitest® as a new screening instrument to assess lipid stability directly on bakery products, tarallini, a typical italian snack. in particular, the sample amount, particle size, and formulations were setted in order to enhance the discriminating power of the technique. traditionally, tarallini are a typical southern italian salted snack formulated with wheat flour, oil, water, salt and white wine. the oil used during its preparation plays the main role in the oxidative stability and thus shelf life of this snack; homemade tarallini is generally prepared using extra virgin olive oil (evoo), but the industrial one could be formulated with sunflower oil (cheaper than evoo) or with a blend of evoo and lard, more expensive but with longer shelf life (caponio et al., 2009). the longer shelf life is mainly due lower susceptibility of fat blend to lipid oxidation, related to the fatty acids composition of fats and the presence of natural antioxidants in evoo. in addition, cooking time is critical for tarallini quality. in particular, cooking time should be optimised for a golden colour without negatively altering the crunchiness and the texture of the product. in fact, a cooking time or a cooking temperature too high could led to a product extremely dry and with a darker colour compared to the typical one. moreover, a longer cooking enhance the heat stress promoting lipid oxidation onset and reducing the shelf life of the snack. 2. materials and methods 2.1. solvents and reagents all of the solvents were purchased from vwr international (radnor, pennsylvania, usa). the reagents were provided by sigma aldrich (st. louis, mo, usa). 2.2. samples the analysed samples were italian typical salted snacks (“tarallini”) prepared following three different recipes (reported in table 1): recipe i: wheat flour, water, salt and sunflower oil. recipe ii: sunflower oil was replaced with extra virgin olive oil (evoo). recipe iii: a blend of evoo and lard (4:1 w/w) was used as a fat source. ital. j. food sci., vol 29, 2017 66 table 1: recipes of formulated tarallini. ingredients so tarallini evo tarallini evo/la tarallini wheat flour (g) 1000 1000 1000 water (ml) 350 350 350 salt (g) 20 20 20 oil (g) * sunflower oil 200 0 0 * extravirgin olive oil 0 200 160 * lard 0 0 40 in all of the recipes, the dough was kneaded for 15 minutes, manually formed (~4 cm diameter, 0.7 cm thickness) and then oven cooked at 220°c for different times: 8 and 11 minutes to evaluate the ability of the instrument to detect minimal differences in thermalinduced oxidation of the products. the cooking times were chosen based on the typical commercial golden colour of the products (8 minutes), and giving more heat stress, compatibly with the snack’s crunchiness (11 minutes). the samples that were used as control to optimise the oxitest® analytical conditions were commercial tarallini snacks purchased from a local supermarket in cesena (italy). the commercial products that were used for the analytical optimisation conditions were formulated with wheat flour, water, salt and a mixture of evoo and lard. the oxidation tests evaluation with oxitest® was carried immediately after sample production. 2.3. fatty acid analysis of fats used for the formulation of tarallini snacks the fatty acid methyl esters (fames) of oils used for the lab scale tarallini productions were identified after alkaline treatment, as described by christie (1989), on a gc-2010 plus gas chromatograph (shimadzu corporation, kyoto, japan) equipped with a flame ionisation detector (fid) according to the method of verardo et al. (2013a). peaks were identified by comparing peak retention times with glc-463 from nu-check (elysian, mn, usa) and fame 189-19 standard mixtures from sigma aldrich chemicals (st. louis, mo, usa). the fatty acids were expressed as weight percentages of total fame. 2.4. oxitest® analysis optimisation the method of analysis was optimised using commercial samples. the oxitest® reactor (velp scientifica, usmate, milan, italy) was fitted with two separate oxidation chambers at 90°c with an oxygen pressure of 6 bar, as indicated by velp scientifica (2006). at first, the ground commercial sample, milled mechanically using a water-cooled mill (ika-werke m20 mill, speed 20000 rpm, maximum particle size 6-7 mm) (tfg), was analysed in different amounts (10, 20 and 30 g) to determine the best compromises between quantity, time of analysis and repeatability. each sample was analysed three times and monitored twice during each test (in chambers a and b). the results, expressed as ips, were obtained using the two-tangent method. after establishing the sample amount, an oxitest® analysis was performed on commercial samples with different particle sizes: whole product (wt) and after mechanical (tfg) (using ika-werke m20 mill) and manual (tcg) milling. briefly, 10 g ital. j. food sci., vol 29, 2017 67 of tarallini was oxidised as whole (wt) after milling five times for 30 seconds using a water-cooled mill (tfg) and after grinding manually in a mortar (tcg) to obtain bigger particles. inter-days tests were performed to evaluate the instrument repeatability with different types of samples. thus, each type of sample was monitored twice by the oxitest® reactor over five days. intra-day tests were not allowed by the experiment duration (greater than ten hours). 2.5. oxitest® analytical conditions all of the analyses were carried out under the same conditions of temperature (90°c) and oxygen pressure (6 bar). after optimisation of analytical conditions, the amount of handmade tarallini that was analysed was 10 g. the samples were analysed immediately after production. 2.6. statistical analysis one-way (tukey’s honest significant difference multiple comparison) and multifactorial analyses of variance (anova), were carried out to establish significant differences. all of the statistical tests were evaluated using statistica 8.0 software (statsoft, tulsa, ok, usa), and p values less than 0.05 were considered statistically significant. 3. results and discussions 3.1. chemical composition of so, evoo and evoo/lard the analysis of the oils used as raw materials showed a high amount of unsaturated fatty acids, as expected. in evoo, the most abundant fatty acid was oleic acid (c18:1), 70.0%, followed by palmitic acid (c16:0, 13.7%) and linoleic acid (c18:2 9.2%). linoleic (c18:2, 59.0%), oleic (c18:1, 28.9%) and palmitic (c16:0, 6.4%) acids were also the main fatty acids in sunflower oil. the fatty acid composition of the evoo/lard (4:1 w/w) blend closely reflected the composition of evoo, with some changes in the percent distribution of each fatty acid. oleic acid (c18:1) accounted for 63.4%, palmitic acid (c16:0) 15.4%, and linoleic (c18:2) and stearic (c18:0) acids 9.7% and 4.3%, respectively, of the total fatty acids. in small quantities, this blend also contained saturated medium chain fatty acids, such as lauric acid (c12:0, 0.02%) and myristic acid (c14:0, 0.28%), typical of lard, as shown in table 2. the oxidative status of the fats used during formulation was assessed by an oxitest® reactor, at the same condition applied for tarallini analyses (90°c, 6 bar oxygen pressure) but weighting 5 g of products. the results showed that evoo was characterised by a similar and not significantly different oxidative stability compared to evoo/lard blend (1371 vs 1408 minutes, respectively) but by a greater oxidative stability than the one of sunflower oil (699 minutes), confirming the results reported by comandini et al. (2009). 3.2. optimisation of the analytical parameters of the oxitest® reactor preliminary tests performed on commercial tarallini were aimed to to optimise the conditions of analysis of bakery products by oxitest®; in particular they were aimed to determine the amount of sample and particle size that allow the best repeatability. thus, different quantities loaded into the reactor chambers were tested. moreover, once decide ital. j. food sci., vol 29, 2017 68 the best amount to load, trying to reach the most homogenous and repeatable contact with oxygen, the samples were analysed as whole (wt) or ground mechanically with a mill (tfg) or manually with a mortar (tcg). table 2: fatty acid composition (%) of fats used in tarallini formulation. so evoo evoo/la c12:0 0.00±0.00 0.00±0.00 0.02±0.00 c14:0 0.07±0.00 0.00±0.00 0.28±0.02 c16:0 6.41±0.01 13.70±0.09 15.38±0.01 c16:1t 0.02±0.00 0.13±0.04 0.16±0.01 c16:1c 0.11±0.00 1.13±0.03 1.30±0.00 c17:0 0.06±0.00 0.13±0.00 0.20±0.01 c17:1c 0.03±0.00 0.21±0.01 0.22±0.01 c18:0 3.25±0.02 2.03±0.02 4.32±0.07 c18:1 28.90±0.22 70.01±0.05 63.36±0.21 c18:2 tt 0.23±0.05 0.00±0.00 0.00±0.00 c18:2 n6 58.97±0.24 9.22±0.09 9.73±0.00 c18:3n6 0.06±0.01 0.07±0.01 0.10±0.03 c18:3n3 0.09±0.02 0.57±0.01 0.58±0.01 c20:0 0.24±0.02 0.39±0.01 0.37±0.00 c20:1 0.15±0.04 0.30±0.02 0.42±0.02 c20:2 0.00±0.00 0.00±0.00 0.13±0.01 c20:3n6 0.10±0.06 0.37±0.14 0.90±0.13 c22:0 0.71±0.01 0.14±0.00 0.10±0.00 c20:5+c22:1 0.00±0.00 0.06±0.03 0.11±0.02 c22:2 0.06±0.00 0.52±0.02 0.44±0.02 c22:3+c22:4 0.00±0.00 0.16±0.06 0.39±0.09 c24:0 0.33±0.03 0.39±0.15 0.69±0.02 c24:1 0.10±0.00 0.15±0.01 0.33±0.08 c22:5 0.14±0.01 0.33±0.02 0.46±0.05 oxidising 30, 20 and 10 g of tfg, the ip values were 757, 873 and 788 minutes, respectively, and there were no significant differences in term of ips between the three amounts tested (table 3). thus, in order to save the sample to use, 10 g was chose as the amount to use in the following trials. thirty grams of sample was the maximum quantity per plate in a single oxidation chamber; a higher amount, in fact, needed to be placed on more than one, causing a not-reliable comparison between trials with smaller amounts. lower amounts were not chosen because quantities below 10 g did not allow the total covering of the steel plate in the oxidation chamber, leading to a possible irregular oxygen distribution on the sample and showing a non-linearity of response. after choosing the more repeatable amount of sample and testing the instrument repeatability, the instrument capacity of discrimination for different sample particles size was tested. ital. j. food sci., vol 29, 2017 69 table 3: ip values of different amounts of tarallini. tfg: tarallini fine ground, ground mechanically; tcg: tarallini coarse ground, ground by mortar; wt, whole tarallini). different letters indicate significantly differences between values (p < 0.05). as reported in table 4, statistically significantly differences in the ips were observed between the two different types of milled products (tcg and tfg) and between wt and the tcg. these differences could be due to the initial heating effects that, related to the milling process, increase the susceptibility to oxidation in tfg with a decrease in the ip value compared to tgc. moreover, for ground products, as smaller was the particles size (tfg respect to tcg) as higher was the susceptibility of the product to lipid oxidation, as reported by tan et al. (2002) as concerning wt, the ip value was not significantly different to the one of tfg but significantly different from tgc. however should be noticed that wt ip showed a high standard deviation suggesting that oxitest® were less repeatable when used on whole product compared to the ground one; this can be probably connected to the lower sample homogeneity (e.g slightly different shape and thickness) of the whole product, that could affect interaction between oxygen an oil fraction. table 4: ip values of whole tarallini and tarallini ground differently. samples ip values (minutes) tfg 10 g 788±37a tcg 10 g 902±54b wt 10 g 788±168a (tfg: tarallini fine ground, ground mechanically; tcg: tarallini coarse ground, ground by mortar; wt, whole tarallini). different letters indicate significantly differences between values (p < 0.05). the results showed that the larger the particles (tcg compared to the same quantity of tfg saqmple) and thus the smaller the surface area-to-volume ratio exposed to oxygen, the higher the ip. however, the above hypothesis is confirmed only by ground products because the results obtained by the oxidation of the whole sample are very changeable, probably due to the shape of tarallini, which could lead to a different exposure to oxygen and different oxidation rates among trial tests. the whole product showed an induction period very close to that milled by grinder (tfg) but was characterised by a higher variation between replicates. the results of inter-day experiment showed a lower cv (4.1%) for tfg compared to tcf (7.5%) and wt (23.2%). these results suggest the product ground mechanically (tfg) as the one to choose for oxitest® analysis; thus, handmade tarallini was ground by a watercooled mill before analysis (tfg). samples ip values (minutes) tfg 30 g 757±38a tfg 20 g 873±48a tfg 10 g 788±37a ital. j. food sci., vol 29, 2017 70 3.3. evaluation of the oxidative stability of handmade tarallini to assess the effect of food composition (“matrix effect”) on lipid rancidity, handmade tarallini formulated with three different fat sources were oxidised by the oxitest® reactor, testing the reliability of this instrument for the screening of the oxidative stability of baked snacks. comparing the ip values of tarallini formulated with different recipes, it can be noticed that the most oxidable products were sunflower oil tarallini snacks, showing the lowest ips (274 and 599 minutes). a low ip means high oxidation susceptibility. increasing lipid saturation (evoo and evoo/lard as fatty sources), the ips increased, reaching values more than double (1254 and 1150 minutes for evoo snacks and 1192 and 1100 minutes for evoo/la tarallini) compared to those of sunflower snacks. this trend may be associated with the fatty acid composition of the fat used: the higher degree of unsaturation of sunflower oil reduces the oxidative stability of the produced snacks (table 5). table 5: induction period (ips) of handmade tarallini snacks cooked for 8 and 11 minutes and reduction of ips by increasing cooking time. samples 8 minutes cooking 11 minutes cooking reduction of ips evoo 1254 a 1150 a,b 8.3% evoo/la 1192 a,b 1100 b 7.7% so 599 c 274 d 54.3% different letters mean significantly differences between values (p < 0.05) to better explain these results, the unsaturation degree was expressed as the “unsaturation point” and calculated as the sum of the main fatty acids weighted by the number of double bounds of the fatty acid. sunflower oil had the highest unsaturation point (147), followed by those of evoo (88.5) and the evoo/lard blend (82.8). although the evoo/lard blend had a lower unsaturation point compared to that of evoo, it showed greater susceptibility to oxidation. this is probably due to the blend’s lower content of natural antioxidants, typical of evoo, which protects the fat against oxidation. in addition to lipid unsaturation degree, cooking time also played a significant role in lipid oxidation onset in tarallini snacks as reported in tables 5 and 6. cooking for a longer time decreases the ips of the samples analysed due to an increase in the lipid oxidation rate. the effect of cooking time was emphasised in the products characterised by the higher unsaturation degree (so tarallini), with a significant reduction of ip values (274 vs 599 minutes). even if the ip values decreased with 11 minutes of cooking, significant differences were not observed in tarallini made with evoo or evoo/la when cooking time was changed. anyway, the highest ip value was shown by tarallini made of evoo and cooked for 8 minutes (ip=1254 minutes). this result may indicate that the phenolic compounds naturally present in evoo have a strong antioxidant activity that can help to prevent lipid oxidation. the ip values of evoo tarallini were higher, even if not significant, also ital. j. food sci., vol 29, 2017 71 compared to that of a more saturated lipid (evoo/la tarallini), confirming the report of hrncirik and fritsche (2005) in different evoo samples. table 6: multifactorial anova (univariate results). source of variation probability oil *** baking time *** oil x baking time * p < 0.05; *** p < 0.001 thus, the cooking heating effect caused a significant increase in the oxidation onset only in snacks formulated with highly oxidable oils, such as sunflower oil. in this case, comparing snacks formulated with the same fat but cooked for different times, it can be seen that even only 3 minutes of cooking changes the oxidation stability of the products, suggesting that heat effect plays a dominant role in the high unsaturated lipid oxidation onset. the cv (all values below 5%, except for tarallini made with evoo and cooked for 11 minutes, where the cv was 9%) for handmade snacks confirms the good repeatability of the oxitest® reactor even when fats with different susceptibilities to oxidation were used for formulations. 4. conclusions tarallini ground in different ways and with different particle sizes showed different behaviour during the accelerated oxidation process due to the different surface/volume ratio and, consequently, different contact with oxygen. these results highlight the necessity to perform an analysis on ground foods, especially when their shape is not homogeneous. the oxitest® instrument also showed good performance for the discrimination of different fat used in snacks formulations. moreover, baking tarallini for different cooking times led to an ~50% reduction of ips, highlighting the power of the oxitest® to discriminate between products under different thermal stresses. thus, oxitest®, different from other accelerated shelf life tests, is suitable for solid, liquid and doughy foods (velp scientifica, 2006). the possibility of screening lipid stability to oxidation with good repeatability of results in complex foods, such as bakery products, avoiding lipid extractions and with the minimal preparation of the sample, suggest the oxitest® instrument as a good option to save time in the preliminary evaluation of lipid oxidation. moreover, this instrument considers the complexity of formulated foods giving more reliable results due to interactions between compounds that can be considered. nevertheless, more investigations are needed to study this accelerated oxidation test with other foodstuffs. acknowledgements the authors gratefully thank velp scientifica (usmate, milano) for the helpful advice, assistance and support in performing this work. ital. j. food sci., vol 29, 2017 72 abbreviations ip, induction period; 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(ed.), p. 210, aocs publishing, urbana, il. wan p.j. 2000. accelerated stability tests. in “methods to assess quality and stability of oils and fat-containing foods”. k. warner and n.a.m eskin (ed.), p. 179. aoac press, champaign, il. waterman k.c. and adami r.c. 2005. accelerating testing: prediction of chemical stability of pharmaceuticals. int. j. pharmacol 293:101. paper received june 6, 2016 accepted september 3, 2016 ijfs#954_bozza ital. j. food sci., vol. 29, 2017 707 paper comparison of black tea types with grades and blends a. ciftaslana and a.l. inanc*b adepartment of food engineering, graduate school of natural and applied sciences, ksu, kahramanmaras, turkey bdepartment of food engineering, faculty of engineering and architecture., ksu, kahramanmaras, turkey *corresponding author. fax: +90 3443002084 e-mail address: linanc@ksu.edu.tr abstract the chemical and sensory properties of conventional and organic black tea kinds (camellia sinensis var. sinensis) regarding of blend and grade were compared. organic teas were produced from teas harvested from hemsin region, rize, turkey where has a latitude of 41°2'53.53"n and a longitude of 40°53'56.61"e. conventional black teas were produced from teas harvested from tirebolu region, giresun, turkey where has a latitude of 41°0'26.85"n and a longitude of 38°48'52.54"e. the water extract, cellulose, polyphenol, mineral, and caffeine contents; tf/tr ratio; and a sensory evaluation were used as parameters to compare conventional and organic black tea blends and grades. the polyphenol contents (12.53-8.57%) of conventional teas were higher than that (9.86-7.60%) of organic teas, and the cellulose contents (17.86-13.45%) of organic teas were higher than that (17.55-11.60%) of conventional tea samples. the highest caffeine contents were found in first grades of first blends of tea samples. the amount of caffeine in blend 1 of grade 1 of conventional tea was 2.67% as it was 1.86% for organic tea in the same blend and grade. regarding the tf/tr ratios and the sensory evaluation scores, both the conventional and organic teas were similar. the grade and blend affected significantly the quality of black tea. keywords: black tea, blend, chemical component, grade, sensory ital. j. food sci., vol. 29, 2017 708 1. introduction tea (camellia sinensis), which is one of the oldest beverages, is the most consumed, hot or cold, manufactured drink in the world. it is the second most popular non-alcoholic beverage, after water, consumed by approximately half of the world’s population. it is available for consumption in different varieties, which are mainly based on the oxidization and fermentation technique used. there are specific climatic requirements for tea crops. tea can only be grown in tropical and subtropical climates. the tea plant requires temperatures between 10-30°c, an annual rainfall of at least 1250 mm, acidic soils, ideally 0.5-10° slopes and elevations up to 2000 meters. thus, tea production is geographically limited to a few areas around the world, and the growing conditions are highly sensitive (cai et al., 2016; chen, 2016). the secondary metabolite compounds in plants serve as defense compounds and vary in the amounts depending on the parameters such as environmental conditions, agro-techniques, and producing processes. the amounts of the compounds like polyphenolic catechin compounds in tea plants vary with geographic location, cultivar, herbivory, season, shade, soil, slope, water availability, and management. it can be perceived changes in the amounts of tea functional compounds by their sensory characteristics such as astringency, bitterness or sweetness. thus, tea quality influences the purchasing decisions, farmer livelihoods, and functional benefits derived from crops (ahmed et al., 2014a; ahmed et al., 2014b; ahmed et al., 2012; ahmed et al., 2010; lin et al., 2003). in general, tea quality is determined via sensory testing and by examining the significant correlations between some of the chemical compounds and the sensory tests. the chemical compounds in tea affect the sensory properties such as color, taste, odor, and flavor in addition to the nutritional and pharmacological benefits of tea (shekhar et al., 2016). tea contains many chemical compounds but theaflavin (tf), thearubigin (tr), phenolics, caffeine and minerals are the most important compounds for tea quality. moreover, the crude fiber content of tea is an important parameter to determine the tea quality (marbaniang, 2011), and the presence of water-soluble ingredients in tea is very important for the crude fiber content. the water-soluble substances in tea are flavonols, acids, caffeine, amino acids, carbohydrates and organic acids. in hot water, the low solubility substances are starches, pectins, ashes and pentoses. the insoluble substances are cellulose, lipids, some pigments and volatiles. the fresher the tea leaves are, the higher the water extract is, and this depends on the environmental conditions, ecological impacts and production procedures. cellulose indirectly affects the tea quality and is an undesirable compound because it reduces the proportions of the other compounds in the total solid. the amount of cellulose in the tea leaves increases as the length of the sprouts increases, which occurs when a standard harvest is not performed (ozdemir and karkacier, 1997). tea leaves contain large quantities of polyphenols, especially catechin, and the catechin amount is related to the black tea quality (owuor and obanda, 2011). oxidoreductase enzymes such as polyphenol oxidase (ppo) and peroxidase (po) interact with the phenolic compounds in tea leaves and react to produce the well-known, golden-yellow color in fermented teas. golden-yellow theaflavin, a product of the condensation reaction between two molecules of o-quinone (one derived from epicatechin (dihydroxy) and the other derived from epigallocatechin (trihydroxy), is probably generated by ppo due to the exposure of the tea leave surfaces to air. additionally, thearubigins, which are more intensely colored products with diverse structures, form because of the reactions of oquinones with amines, phenols, amino acids, peptides, and proteins (mahanta and baruah, 1992). ital. j. food sci., vol. 29, 2017 709 the mineral content is 4-5% for fresh tea leaves and 5-6% for processed tea. mineral substances have an important role in plant physiology and in their chemical and biochemical functions in addition to the growth of the tea plant. some of the minerals are absorbed by the human body from drinking tea. minerals are essential for the proper functioning and maintenance of the human body and metabolic events (kacar, 1997). the amount of minerals in tea leaf shoots vary depending upon the soil type and husbandry of the bush. additionally, the genetic characteristics, growing locations, and tea production methods contribute to the quality of black tea. among the minerals and essential trace elements, ca, na, k, mg, and mn are present in tea leaves at g/kg levels, and cr, fe, co, ni, cu, zn are present at mg/kg levels (street et al., 2006). a previous study reported that there is a wide variation in the percent transfer for the examined elements from the black tea leaves to the tea infusion. the solubilities of ca and k are the highest among the elements studied. the extraction of trace metals, such as mn, zn and al, is also relatively high. only fe is insoluble and remains in the solid particles during beverage preparation (dambiec et al., 2013). conventional and organic agriculture are two of the primary cultural agricultures used in the production of food. one of the clearest distinctions between organic tea and conventional tea is that organic tea is grown without the use of chemical fertilizers, pesticides, fungicides, or herbicides. these chemicals have well-documented harmful effects on the environment and farmers and consumers who may ingest the residues. conventional tea growing methods may maximize production in the short term but with serious environmental consequences and human costs. additionally, organic farm land is prohibited from being treated with synthetic pesticides and herbicides for at least 3 years prior to harvest (asami et al., 2003). camellia plants usually have a rapid growth rate. typically, they will grow about 30 cm per year until mature, although this does vary depending on their variety and geographical location. when the plant is harvested for tea, the shoot and two to three leaves are harvested every 8 to 10 days. these buds/shoot and leaves are called ‘flushes’. a plant will grow a new flush every 7 to 15 days during the growing season. the tea spring leaf tip is valued the most. camellia sinensis usually will produce an abundant crop twice a year, once in the spring and again in the summer. harvesting can be done every 7 to 15 days during these periods, until the plant no longer produces new growth (daff, 2016). in this study, the aim was to investigate effects of blend (blend of tea leave harvested in the first flush period) and grade on the quality of black tea, and to determine chemical and sensory differences among the black teas produced via organic and conventional production techniques depending on blend and grade. 2. materials and methods 2.1. materials the first flush organic and conventional black teas (camellia sinensis var. sinensis) that were harvested at the end of the first growth period during the growing season in 2013 were chosen for this study. organic black tea was obtained from hemsin, rize, turkey tea factory in the caykur general directorate, and conventional black tea was obtained from the tirebolu, giresun, turkey factory in the same directorate. the climate in hemsin (a latitude of 41°2'53.53"n and a longitude of 40°53'56.61"e) is warm and temperate. the rainfall in hemsin is significant, with precipitation even during the driest month. according to köppen and geiger, this climate is classified as cfb (oceanic climate). the average annual temperature, rainfall and relative humidity are respectively 12.5°c, 1423 ital. j. food sci., vol. 29, 2017 710 mm and 75% in hemsin (climate-data 2017a; distancesto 2017a). the climate in tirebolu (a latitude of 41°0'26.85"n and a longitude of 38°48'52.54"e) is mild, and generally warm and temperate. the rainfall in tirebolu is significant, with precipitation even during the driest month. the climate here is classified as cfa by the köppen-geiger system. the average annual temperature, rainfall and relative humidity in tirebolu are respectively 14.5°c, 1002 mm and 67.5%. the average field slopes and altitues of both regions are 1020% and 100-300 m (climate-data 2017b; distancesto 2017b). 2.2. preparation of the black tea samples samples were collected over 30 days from the sorting stages, which are after the drying stage, at the production lines in the factories based on the diameter of the teas. the sorting process was performed at two levels. in the first level, the graded teas were passed through three different sieves with diameters of 1.405, 0.776 and 0.505 mm (12, 20 and 30 mesh), and the teas were coded as grade 1, grade 2 and grade 3. in the second level, the teas that did not pass through the sieves and had diameters of 2.057 and 1.676 mm (8 and 10 mesh) were passed through the sieves (12, 20 and 30 mesh). these teas were coded as grade 4, grade 5 and grade 6. the sorting process was performed five times a day. after the daily sorting process, 100 g of tea were taken for each grade, and a sample of 500 g of tea was taken for one grade in one day. the sorting process was divided into three different periods of 10 consecutive days. these three periods were called blend 1, blend 2 and blend 3. a total of 5,000 g of tea was sampled by mixing the teas of the same grade into one blend. the samples were stored in sealed glass jars in the dark at room temperature until analysis. 2.3. analysis all analyses of the tea samples outlined below were replicated three times. 2.3.1. water extract the water extract analysis was conducted using the method described by iso (1994). distilled boiling water (200 ml) was added to the tea leaf (2±0.001 g) in a balloon and was boiled for 1 h using a reflux condenser. tea liquor was filtered through cotton wool, and the residue (extract) was washed with distilled water three times. the tea liquor was cooled to room temperature, and the washings were diluted to 200 ml with distilled water. the tea liquor (75 ml) was placed in a weighed evaporating dish and evaporated to dryness over a water bath. the tea residue in the dish was completely dried in a vacuum oven at 103°c for 16 h until the weight of the dish with the residue was constant. the water extract of the black tea was expressed as a percentage of the mass of the dry tea leaf. 2.3.2. crude fiber content the tea leaf was ground using a mill and passed through a 1 mm screen. the tea (2±0.001 g) was then weighed into a 1 l conical flask. a 0.255 n sulfuric acid solution (200 ml) was measured at room temperature, boiled, and added to the sample. a reflux condenser was inserted into the neck of the flask, and the solution was boiled gently for 30 min. a buchner flask with a hartley funnel and wet filter paper (whatman no. 541) were used for filtration. after boiling, the acid digest was poured into a shallow layer of hot water in the funnel under gentle suction, and the flask was rinsed with two aliquots of approximately 50 ml of boiling water poured through the filter funnel. using a dispenser capable of ital. j. food sci., vol. 29, 2017 711 dispensing 200 ml of hot liquid, the insoluble matter was washed from the filter paper into the original 1 l conical flask using 200 ml of a 0.313 n sodium hydroxide solution and boiled for 30 min. using boiling water, all the insoluble matter was transferred into a sintered glass crucible (porosity no. 1, 40 mm plate diameter and 70 ml capacity) fitted to the buchner flask via an adaptor by applying gentle suction. the residue was washed with approximately 50 ml aliquots of boiling water, hcl solution (1%; v/v) and boiling water. finally, the residue was washed twice with ethanol (95%; v/v) and three times with acetone. the crucible and residue were heated in an oven at 103°c for 2 h. the crucible was cooled in a desiccator, weighed to the nearest 0.001 g, returned to the oven and heated again for 1 h. finally, the crucible was cooled in a desiccator and weighed. the crude fiber content is expressed as a mass fraction, in percent, of the sample on a dry basis (iso, 2012a). 2.3.3. total phenolic content the extraction tube (10 ml) containing the ground tea leaf (0.2±0.001 g) was placed in a water bath set at 70°c. hot (70°c) 70% methanol (5 ml) was dispensed into the extraction tube, which was stoppered and mixed on the vortex mixer. the extraction tube was heated in the water bath for 10 min with vortex mixing after 5 and 10 min. the extraction tube was removed from the water bath and allowed to cool to room temperature. the stopper was removed, and the tube was placed in a centrifuge at 3500 r/min for 10 min. the supernatant was carefully decanted into a graduated tube. the extraction steps were repeated, and the extracts were combined, diluted to 10 ml with cold 70 % methanol and mixed. the leaf tea extract (1 ml) was diluted to 1/100 (v/v), transferred into a tube and 5.0 ml of dilute folin-ciocalteu phenol reagent was added. within 3 to 8 min after the addition of the folin-ciocalteu phenol reagent, 4.0 ml of a sodium carbonate solution were pipetted into the tube, which was stoppered and mixed. the tube stood at room temperature for 60 min, and the optical densities were measured in 10 mm path length cells against water on a spectrophotometer (uv-160 shimadzu) set at 765 nm. gallic acid standard solutions were used for the standard curve. the total phenolic content was calculated as a mass percentage of the dry tea leaf using the following formula (gallic acid equivalent; mg gae). the concentration of gallic acid was established in mg/ml using the calibration curve (iso, 2005). wt=((dsample-dintercept )*vsample*d*100)/(sstd*msample*10000*w(dm,sample) ) where dsample is the optical density obtained for the sample solution; dintercept is the optical density at the point; sstd is the slope obtained from the best-fit linear calibration; msample is the mass in grams of the sample; vsample is the sample extraction in ml; d is the dilution factor used prior to the colorimetric determination; wdm, sample is the dry matter content expressed as a mass fraction percent. 2.3.4. caffeine the tea liquor (50 ml) obtained from the water extract analysis method was poured into a separatory funnel and 5 ml of an ammonia solution (70 g/l) and 50 ml of chloroform were added. after careful mixing, the water phase and the chloroform phase were separated. the water phase was washed twice with chloroform. the chloroform phases passed through a glass cotton filter and were collected in a volumetric flask. the phases were diluted to the mark with chloroform and mixed. seven different caffeine standard solutions were prepared (0.5, 1, 2, 3, 4, 5 and 8 g/ml) in chloroform. the absorbance of the ital. j. food sci., vol. 29, 2017 712 samples and the standard caffeine solutions in chloroform were measured against chloroform blank at 276 nm using a uv-visible spectrometer (uv-160 shimadzu). the caffeine content of the sample is expressed as a mass percent of the dry tea leaf (iso, 2012b). 2.3.5. theaflavin (tf) and thearubigin (tr) the tf and tr analysis was conducted using the method described by kumar et al. (2011). water (125 ml) was added to 3±0.001 g of ground tea leaf and boiled for 10 min. the black tea extract was obtained by filtering the black tea through a cloth filter. after mixing the extract (10 ml) with ethyl acetate (10 ml), the mixture separated into two liquid phases, the water phase (wp) and the organic phase (op). five different solutions, s0, s1, s2, s3 and s4, were prepared from the wp and op as indicated below. s0 = op (10 ml) + 2.5% nahco3 (10 ml) s1 = op (4 ml) + methanol (21 ml) s2 = wp (2 ml) + distilled water (10 ml) + methanol (13 ml) s3 = wp (2 ml) + oxalic acid (2 ml) + distilled water (6 ml) + methanol (15 ml) s4 = op (4 ml) of s0 + methanol (21 ml) the optical densities of solutions 1, 2, 3 and 4 (e1, e2, e3 and e4) were measured at 380 nm using a spectrophotometer (optimum-one, chebios, roma, italy). the percentages of tf and tr were calculated using the followings formulas: tf (%)=2.25*e3 tr (%)=(1.77*e4+e1-e3)*7.06 tf/tr ratio=(tf(%))/(tr(%)) 2.3.6. minerals a standard method (nmkl, 1998) based on atomic absorption spectroscopy was used to determine the mineral content. pure hno3 (10 ml) was added to the ground tea leaf (0.2±0.001 g), and the mixture equilibrated for 30 min. after 30 min, the mixture was combusted in a microwave oven (speedwave four, berghof, eningen, germany) at 190°c. the sample solution was then transferred to a 50 ml volumetric flask and diluted to the mark with ultra-distilled water. a standard solution was prepared for each mineral (cu, fe, zn, mn, mg, ca, k). the samples were analyzed using an atomic absorption spectrometer with an inserted hollow cathode lamp (gbc, avanta p, australia). the mineral content in the samples is expressed in g/kg. 2.3.7. sensory test the tea liquor used in the sensory test was prepared by infusing the tea leaf according to iso (1980). the tea was weighed (2.8±0.05 g) and transferred to a pot. the pot was filled with approximately 140 ml of fresh, boiling water. the tea was allowed to brew for 6 min, and the liquid was poured through the serrations into a bowl to separate the liquid from the solid tea. the lid was removed and inverted, and the infused leaf was placed on the inverted lid to allow the infused tea leaf to be inspected. black teas were assessed using sensory test method of ts en iso 13299 (tse, 2016). eight tea sensory experts (four males ital. j. food sci., vol. 29, 2017 713 and four females, aged 25-40 years) from the sensory test and chemical analysis laboratory at caykur fabric, rize, turkey served as the panel. the experts completed 200 h of sensory testing for all samples. the experts evaluated the sensory attributes of the samples by using the sensory evaluation chart from the turkish standard institute (table 1). the sensory evaluation chart includes 5 disciplines (properties) having different maximum point, totally 100 points. the experts gave a point for each discipline between 0its maximum point. it is rated as good tea if a tea sample collects 50 points or above on the basis of the total 100 points. table 1. sensory evaluation chart of turkish black tea. sensory properties of black tea description point appearance of the dried tea leaf should be good appearance, black or dark copper color, and no fiber and stalk 10 color of tea liquor should be bright dark red or reddish color. should not be dull, fuzzy and a residual or brownish color 25 astringency and body should be a lively puckery sensation on the tongue and gums, and also the good impression of a tea’s weight in the mouth, its viscosity and mouth feel 30 color and odor of the infused leaf should be bright copper red color, no excess green leaf and no brownish color 15 aroma of liquor should be unique and pleasant for good tea 20 total 100 2.3.8 statistical analysis two types of tea, three blends and six grades were compared. differences were considered to be significant at p ≤ 0.05. the data, collected from organic and conventional black tea samples in triplicate, were subjected to a three-way analysis of variance (anova) using the spss software (spss for win, release 19.0, 2012). the means were compared using duncan's multiple range test for multiple comparisons, and the “student” t-test was applied to the two sets of data that were significantly different. 3. results and discussion 3.1. water soluble extract the water-soluble extract amounts and their statistical results for the samples are presented in fig. 1. the extract amounts for the conventional teas were in the range of 30.83-35.69% and 31.73-35.26% for the organic teas depending on the grades and blends. in the comparison of teas in the same grade and blend, the extracts amount in the conventional teas were higher than those in the organic teas. the highest values were from the first blends, and the lowest values were from the third blends in both tea blends. the extract amount decreased from grade 1 to grade 6 for all tea blends. the triplet interaction among the tea type-grade-blend was not significant (p>0.05), but the double interaction among them was very significant (p≤0.05). the differences between grade 3 and grade 4 in ital. j. food sci., vol. 29, 2017 714 blend 2 for conventional tea and between grade 4 and grade 5 in blend 1 and grade 2 and grade 3 in blend 3 for organic tea were less. additionally, compared to the blends, there was no difference between grade 2 and grade 3 in blend 3 of the conventional tea, but this was not seen in the organic tea. the extract amounts for the tea leaves at the beginning of the first flush period are higher than that seen in the other periods. a difference among the tea extracts was not observed for the midand end-first flush periods. it was reported that the maximum extract amount for black tea produced in turkey is from the first harvest season, and the second and third harvest season have less. additionally, it was reported that the extract amount is higher in fresh tea leaves. similarly, the extract amounts in the tea produced by different methods decreased from grade 1 to grade 6 (gokalp et al., 1991; kacar, 1997). 3.2. crude fiber content the crude fiber contents of the conventional teas were in the range of 11.60 to 17.55% and 13.45 to 17.86% for the organic teas with respect to the grades and blends (fig. 2). the crude fiber contents of almost all the organic teas were higher than that of the conventional teas with the same grade and blend. the highest crude fiber contents among the blends of both tea types were found in the third blend, and the lowest values were in the first blend. the values increase from the first grade to the final grade, which is the inverse of the extract behavior. the contents of grade 2 and grade 3 teas in blend 2 of the conventional samples were very close, and the contents of grade 1 and grade 2 teas in blend 3 of the organic samples were almost the same. otherwise, the contents of the tea types with the same grade and blend were different (p≤0.05). the crude fiber contents in the study were similar to those noted by venkatesan and ganapathy (2004) for indian teas. 3.3. total phenolic content and theaflavin (tf)/thearubigin (tr) ratio the total phenolic content of the tea samples and the statistical results are given in fig 3a. the values in the conventional teas were higher than that in the organic teas compared to the polyphenols in the two tea types with the same grade and blend. the highest and lowest polyphenol contents among the blends in the conventional tea were in blend 1 and blend 3 and in blend 2 and blend 3 in the organic tea. neither a negative or positive trend was found in the comparison of the grades. the contents of the second, third, fifth and sixth grade teas in blend 1 of the conventional samples and in grade 2, 3 and 5 teas in blend 1 of the organic samples were close to each other. compared to the blends of one grade, there was very little difference between blend 1 and blend 2 teas in grade 2 of the conventional sample and between blend 1 and blend 2 teas in grade 4 of the organic tea (p≤0.05). the values (3.79-8.36%) of the tea samples reported by ozdemir et al. (2008) were lower than those in both the conventional and organic samples. the tf/tr ratios are shown in fig 3b. in the conventional teas, the tf/tr ratios of the blends were identified in the range from 0.037 to 0.040 and from 0.034 to 0.044 for the grades. in the organic teas, the ratios were 0.036 to 0.040 for the blends and 0.032 to 0.043 for the grades. the lowest tf/tr was found in blend 3 in the conventional teas, but the ratios in blend 1 and 2 were very close. the lowest and the highest tf/tr ratios were in blend 2 and 1 in the organic teas. an increasing or decreasing trend was not found for the tf/tr ratios of the grades from the blends for all the tea samples. ital. j. food sci., vol. 29, 2017 715 figure 1. the water soluble extract amounts and their statistical results for the teas. values followed by the same letter are not significantly different at the level of 5% (series ‘a-f’ for grades in a blend of a tea type, series ‘a-c’ for the same grades in blends of a tea kinds, and series ‘x-y’ for teas in the same blend and the same grade). f c x e b y f a y e c x f b x e a x e c y d b x e a y d c x e b x d a x d b x c a x d a x c c x d b x d a x c c y c b x c a x b c x c b x c a x b c x b b x b a x b c x b b x b a y a c x a b x a a x a c x a b y a a y 0 10 20 30 40 blend 1 blend 2 blend 3 blend 1 blend 2 blend 3 convectional black tea organic black tea w at er e xt ra ct % grade 1 grade 2 grade 3 grade 4 grade 5 grade 6 ital. j. food sci., vol. 29, 2017 716 figure 2. the crude fiber contents of the teas. values followed by the same letter are not significantly different at the level of 5% (series ‘a-f’ for grades in a blend of a tea type, series ‘a-c’ for the same grades in blends of a tea kinds, and series ‘x-y’ for teas in the same blend and the same grade). a a x a b x a c x a a y a b y a c y b a x b b x b c x b a y b b y a c y c a x b b x c c x c a y c b y b c y d a x c b x d c x d a y d b y c c y e a x d b x e c x e a y e b y d c y f a x e b x f c x f a y f b y e c y 0 4 8 12 16 20 blend 1 blend 2 blend 3 blend 1 blend 2 blend 3 convectional black tea organic black tea cr ud e fi be r c on te nt (% d ry b as is ) grade 1 grade 2 grade 3 grade 4 grade 5 grade 6 ital. j. food sci., vol. 29, 2017 717 figure 3. total phenolic content and theaflavin (tf)/thearubigin (tr) ratio of the teas. values followed by the same letter are not significantly different at the level of 5% (series ‘a-f’ for grades in a blend of a tea type, series ‘a-c’ for the same grades in blends of a tea kinds, and series ‘x-y’ for teas in the same blend and the same grade). a c y a b y c a y c b x cd a b d a x b c y a b y b a y b b x a b x a a x a c y a b y b a y c c x b b x b a x a b y b b y a a y b b x d c x aa x 0 3 6 9 12 15 blend 1 blend 2 blend 3 blend 1 blend 2 blend 3 convectional black tea organic black tea to ta l p he no lic c on te nt (% ) fig. 3a e b x d c y dc a x dc c x a a x c b x de a x c a y d a x e c y b a x e b x bc a x c a x e b y d b y b a x d a x dc b x b b x a a x e c x b b x b a y a a y a a x b a x a a x a b x a c x ab a x a a x c a x b b x a a x cd b x 0,000 0,020 0,040 0,060 blend 1 blend 2 blend 3 blend 1 blend 2 blend 3 convectional black tea organic black tea t f/ t r grade 1 grade 2 grade 3 grade 4 grade 5 grade 6 fig. 3b ital. j. food sci., vol. 29, 2017 718 for the statistical evaluation of the tea types at the 5% level, the difference among the blends of grade 2, 5 and 6 in the conventional teas was less, and there was a similarity between blend 2 and blend 3 in grade 3 and blend 1 and blend 3 in grade 6 for the organic teas (fig. 3b). a decrease in the tf/tr ratio was observed from the beginning of the flush period until the end. the reason for this could be the aging of the tea leaves and a decrease in the oxidative compounds as the flush period continues (gokalp et al., 1991). ozdemir and karkacier (1997) reported that black and green teas had a mean tf/tr ratio of 0.032. the tf and tr contents and their ratio are components of the tea quality index (yao et al., 2006). kumar et al. (2011) reported that the tf/tr ratio should be in the range of 1:10-1:12 to achieve a taste-water extract balance for a quality tea. 3.4. caffeine the ranges and statistical results for the caffeine content in dry tea samples are shown in fig 4. the results show that the caffeine content in conventional teas was in the range of 2.31-2.67% while organic teas contained 1.50-1.89% caffeine. the caffeine content in conventional tea was higher than that in organic tea for the same blend and grade. no relationship was found between the caffeine values of the tea types depending on the blend and grade. no similarity was found among tea types belonging to the same blend and grade (p≤ 0.05). one of the reasons that conventional teas contain high caffeine and water extract amounts is the usage of nitrogen fertilizer (chen et al., 2015). ozdemir et al. (2008) reported that the caffeine content in black teas was in the range of 1-5% and 1.5-2.49% in seven different tea types within a third flush period. the results in the study are similar to the results (2.21-2.80%) reported by khokar and magnusdottir (2002). 3.5. mineral elements the ranges and statistical evaluations of the total element contents in the tea samples are summarized in table 2. the mean concentrations of the elements in both tea leaf types differed significantly with the grades and blends (p≤ 0.05). copper (cu): the cu concentrations in the organic teas were higher than those in the conventional teas for the same blend and grade. the lowest and highest cu concentrations were found in blend 1 and blend 2, respectively, in the conventional teas. the lowest cu concentration was found in blend 3 among the organic tea blends, the cu values in blend 1 and blend 2 were very close. during the black tea manufacturing process, one of the important chemical changes in the leaves is the oxidation of polyphenols via polyphenol oxidases. a polyphenol oxidase is a tetramer that contains four atoms of copper per molecule (sullivan, 2015). therefore, the copper content may vary in blends. marbaniang et al. (2011) reported that the cu content varied from 0.072 to 0.105 g/kg. however, street et al. (2006) reported that different black teas sold in the czech republic contained 0.103 to 0.405 g/kg cu, and these values are similar to the values in the present work. ital. j. food sci., vol. 29, 2017 719 figure 4. the caffeine content in dry tea samples values followed by the same letter are not significantly different at the level of 5% (series ‘a-f’ for grades in a blend of a tea type, series ‘a-c’ for the same grades in blends of a tea kinds, and series ‘x-y’ for teas in the same blend and the same grade). d b y c a y c a y b b x b b x c a x c b y b a y b a y b a x b a x d a x ab a y b a y bc a y ab b x ab a b x b a x bc b y ab a b y a a y a c x a b x a a x bc b y ab a y bb y a b x a a x b a x a a y a a y a a y ab c x a b x a a x 0 1 2 3 blend 1 blend 2 blend 3 blend 1 blend 2 blend 3 convectional black tea organic black tea ca ff ei ne (% ) grade 1 grade 2 grade 3 grade 4 grade 5 grade 6 ital. j. food sci., vol. 29, 2017 720 iron (fe): the fe concentrations were in the range of 0.145-0.377 g/kg in the conventional teas and 0.049-0.223 g/kg in the organic teas. the iron amounts in the organic teas were higher than those in the conventional teas for the same blend and grade. while the lowest iron values were found in the grades of the third blend for the conventional tea types, the values for the grades of blend 2 and 3 were close. in the organic tea types, the highest values were for the grades of blend 1, and the lowest values were for the grades of blend 3. table 2. mineral contents in the tea samples. m in er al b le nd grade 1 2 3 4 5 6 na c1 0.055 cby 0.047 bbx 0.047 bby 0.059 dcy 0.046 abx 0.047 bax c2 0.056 eby 0.058 fcy 0.045 aay 0.048 day 0.046 bcx 0.047 cay c3 0.045 bay 0.047 cay 0.052 ecy 0.050 dby 0.043 aax 0.057 fby o1 0.050 ecx 0.048 dcx 0.041 bcx 0.038 abx 0.046 cax 0.047 cbx o2 0.046 dbx 0.042 cbx 0.037 aax 0.036 aax 0.048 eby 0.038 bax o3 0.038 bax 0.036 aax 0.038 bbx 0.042 ccx 0.060 ecy 0.054 dcx k c1 10.88 6 fcx 10.759 ecx 10.691 dcx 10.496 ccx 10.320 acx 10.377 bcx c2 10.70 2 fbx 9.82 cbx 9.914 dbx 9.964 ebx 9.408 aax 9.797 bbx c3 9.79 eax 9.39 cax 9.869 fax 9.127 bax 9.641 dbx 8.916 aax o1 14.00 5 fby 13.449 ecy 13.394 dby 13.091 bby 13.200 cby 13.038 aby o2 14.00 2 eby 13.368 bby 13.525 ccy 13.707 dcy 13.222 acy 14.213 fcy o3 12.63 2 fay 12.222 day 11.629 bay 12.334 eay 11.925 cay 11.510 aay ca c1 4.168 bax 4.097 aax 4.244 cax 4.307 dbx 4.312 dbx 4.333 eax c2 4.310 cbx 4.316 cbx 4.350 ebx 4.220 aax 4.231 bax 4.333 dax c3 4.434 bcx 4.568 ecx 4.379 acx 4.458 ccx 4.559 ecx 4.506 dbx o1 4.691 bay 4.619 aby 4.774 dby 4.809 fby 4.724 cay 4.789 eay o2 4.871 bcby 4.864 bcy 4.796 acy 4.868 bcy 4.875 cby 4.925 dcy o3 4.880 dby 4.561 aax 4.739 bay 4.553 aay 4.944 ecy 4.818 bcy mg c1 1.220 dax 1.097 aax 1.220 dax 1.219 dax 1.167 cax 1.126 bax c2 1.339 ebx 1.277 cbx 1.265 bbx 1.309 dbx 1.195 abx 1.192 abx c3 1.627 dcx 1.659 ecx 1.602 ccx 1.714 fcx 1.596 bcx 1.588 acx o1 1.226 bay 1.168 aay 1.241 cay 1.259 day 1.265 day 1.231 bay o2 1.388 bby 1.438 dby 1.398 cby 1.473 eby 1.367 aby 1.534 fby o3 1.842 ecy 1.711 ccy 1.656 bcy 1.723 dcy 1.717 cdcy 1.624 acy ital. j. food sci., vol. 29, 2017 721 table 2. continues. cu c1 0.015 dax 0.014 aax 0.015 dax 0.015 cax 0.015 dax 0.014 bax c2 0.017 ccx 0.017 ccx 0.017 cbx 0.015 bbx 0.015 bax 0.015 abx c3 0.016 cbx 0.015 abx 0.015 aax 0.015 abx 0.016 bbx 0.016 bcx o1 0.019 cby 0.018 bby 0.018 bby 0.018 aby 0.018 bby 0.018 bby o2 0.019 dcy 0.019 ccy 0.018 bby 0.018 aby 0.018 aay 0.018 bby o3 0.018 cay 0.017 bay 0.017 bay 0.017 bay 0.018 cay 0.017 aay zn c1 0.022 bcdcy 0.024 dby 0.023 cdby 0.022 bccy 0.021 bcx 0.018 acx c2 0.020 cbxx 0.017 bax 0.016 abax 0.015 abx 0.016 abbx 0.016 abbx c3 0.017 day 0.017 day 0.016 cay 0.013 bax 0.012 bax 0.011 aax o1 0.020 bbx 0.018 abx 0.020 bcx 0.019 bcx 0.021 cbx 0.020 bcx o2 0.020 dbx 0.019 ccy 0.018 bcby 0.018 bcby 0.016 aax 0.017 bbx o3 0.014 dax 0.013 cax 0.012 aax 0.013 bax 0.015 eay 0.015 fay fe c1 0.327 fay 0.230 day 0.236 ecy 0.209 ccy 0.203 bcy 0.177 aby c2 0.362 eby 0.308 dby 0.206 cby 0.182 bay 0.179 bby 0.161 aay c3 0.377 fcy 0.236 eay 0.167 bay 0.202 dby 0.145 aay 0.177 cby o1 0.166 dbx 0.122 cax 0.163 dcx 0.091 bbx 0.0 83 acx 0.125 ccx o2 0.223 ecx 0.137 dcx 0.093 cax 0.062 bax 0.0 55 aax 0.060 bbx o3 0.155 eax 0.130 dbx 0.098 cbx 0.098 ccx 0.0 78 bbx 0.049 aax mn c1 1.049 fay 0.890 aax 0.947 cay 0.991 eay 0.9 55 dax 0.924 bax c2 1.159 eby 1.061 cbx 1.040 aby 1.140 dby 1.0 42 aby 1.054 bbx c3 1.324 dcx 1.170 acx 1.164 acx 1.170 acx 1.2 10 ccx 1.187 bcx o1111 0.986 fax 0.908 bay 0.900 aax 0.978 eax 0.9 66 day 0.922 cax o2 1.093 ebx 1.086 dby 1.012 bbx 1.057 cbx 0.9 68 aax 1.126 fby o3 1.346 fcy 1.246 bcy 1.258 ccy 1.294 ecy 1.2 74 dby 1.193 acy values followed by the same letter are not significantly different at the level of 5% (series 'a-f' for grades in a blend of a tea type, series 'a-c' for the same grades in blends of a tea kinds, and series 'x-y' for teas in the same blend and the same grade). o: organic blend, c: convectional blend. when comparing the fe values for the grades belonging to blends, a decrease in fe from grade 1 through grade 6 was observed. the differences between grade 4 and grade 5 in blend 2 of the conventional tea, and the differences between grade 1 and grade 3 in blend 1, grade 4 and grade 6 in blend 2 and grade 3 and grade 4 in blend 3 of the organic teas were very low (p>0.05). no such similarity was found among the blends of the organic teas, and the difference between blend 1 and blend 3 in grade 2 and grade 6 of the conventional teas was very low. however, the tea types with the same grade and blend had significantly different values (p≤ 0.05) tascioglu and kok (1998) reported that ital. j. food sci., vol. 29, 2017 722 seven different teas produced in turkey contained 0.130-0.171 g/kg fe, and aksuner et al. (2012) found 0.235 g/kg fe in black tea. zinc (zn): the zn concentrations of the conventional teas were higher than that in the organic teas for the same blend and grade. the highest zn concentration among the blends was in the first blend for both tea types, and the lowest was in the third blends. a linear increase or decrease in the zn concentration was not seen among the grades belonging to the blends of teas. there was a small difference among the zn concentration for grades 1, 2, 3, 4 and 5 in blend 1 of the conventional tea and for grades 1, 3, 4 and 6 in blend 1 of the organic tea. moreover, there were small differences between blend 2 and 3 in grade 2 of the conventional tea and between blend 1 and 2 in grade 1 and blend 2 and 3 in grade 5 of the organic tea (p≤0.05). the zn concentrations in 10 commercial turkish black blend teas in a previous work (arslan and togrul, 1995) were found in the range of 0.033-0.052 g/kg. the zn concentration in fresh cells is higher than that found in aged tea plant cells (kacar, 1997). sodium (na): the na concentrations in conventional and organic teas were determined to be in the range of 0.043-0.060 and 0.036-0.059 g/kg, respectively. the na concentration among grades 6, 2 and 3 in blend 1 of the conventional teas were close to each other (p≤ 0.05). a similar situation existed between blend 1 and blend 2 for grade 1 of the conventional teas. however, this was not found for the organic teas. the na concentrations in teas produced in different regions of china were 0.026-0.079 g/kg (zhang et al., 2011), and mckenzie et al. (2010) reported concentration in the range of 0.011-0.86 g/kg. potassium (k): k was the most abundant macro-mineral in all the tea samples analyzed. the k values in conventional teas and organic teas were in the range of 8.916-10.886 g/kg and 11.510-14.213 g/kg, respectively, depending on the blends and grades. interaction among the tea type-blend-grade was found to be significant according to the results of the anova of the k values (p≤ 0.05). in organic tea, there was an important difference among the blends except for blend 1 and 2 in grade 1, but all the k values were significantly different in the blends of the conventional teas. the present results correspond to the data of mckenzie et al. (2010). calcium (ca): the ca concentrations in the conventional and organic teas were in the range of 4.097-4.568 and 4.553-4.944 g/kg, respectively. the ca concentrations in the organic teas were higher than those in the conventional teas for the same blend and grade. the highest ca concentration among the blends in the conventional teas was in blend 3, and the lowest concentration was in blend 1. the highest and lowest ca concentrations in the blends of organic teas were in blend 2 and blend 1, respectively (p≤ 0.05). the ca values in a study by malik et al. (2008) were 4.33-6.68 g/kg, but pereira et al. (2006) reported values in the range of 3.13-9.72 g/kg. manganese (mn): the maximum and minimum values for mn were in the third blends and first blends of the conventional and organic teas, respectively. a linear relationship between the samples was not observed when comparing the grades of the blends of the teas. the differences between the samples were significant, except for the relationship among grades 2, 3 and 4 of blend 3 in the conventional tea (p≤0.05). the mn values in the study are similar to the values in studies by ozdemir et al. (1999), narin et al. (2004), pereira et al. (2006), and mehra and baker (2007). ital. j. food sci., vol. 29, 2017 723 magnesium (mg): the magnesium concentration in all the tea samples varied in the range of 1.097-1.842 g/kg. the mg concentrations of the organic teas were higher than those in the conventional teas for the same blend and grade. it was determined that the triple interaction among the tea samples was significant at the 5% level. the mg values in a study (horuz and korkmaz, 2006) were found to be 3.3 g/kg for the first harvest tea, 4.7 g/kg for the second harvest tea and 3.9 g/kg for the third harvest tea. 3.6. sensory test sensory evaluation of tea types is presented in fig. 5. the conventional teas collected 76-89 points from the panelists. in the evaluation of the conventional tea blends, the mean points for blend 1, 2 and 3 were 85, 83.16 and 81.16, respectively. the evaluation points for the grades between 1 and 6 were in the range of 8779. otherwise, the organic teas collected 75-90 points, and the points for blends 1, 2 and 3 of the organic teas were 86.66, 84.50 and 80.83, respectively. the points for the grades were in the range of 88.33-80.00. the evaluation points for the organic teas were higher than those of the conventional teas depending on the blends and grades. compared to the grades of blends in both tea types, a decreasing trend in the points from grade 1 to grade 6 was observed. the triplet interaction among the tea type-grade-blend was not significant (p>0.05), but the two-way interactions between the tea kind-grade and grade-blend were significant (p≤ 0.05). there is a reverse relation between the score of overall acceptability and total catechins amounts (xu et al., 2017). besides a high amounts of minerals, especially calcium and magnesium (mossion et al., 2008; ananingsih et al., 2013) such as high ph (zhou et al., 2009) influence the extraction yield and stability of catechins and other chemicals in tea infusions. therefore, these chemicals may change sensory quality of tea. in a study investigating the relationship between theaflavin and tea quality, it was reported that the scores should be among 18.2-78 for good quality teas and 14.4-53 for poor quality teas (wright et al., 2002). the astringency of tea infusions increases with increasing ca2+ ion while bitterness and umami intensity decreased (yin et al 2014). xu et al. (2017) reported that total scores (overall acceptability) for different taste attributes, including bitter, astringent and umami tastes of black tea brewing with different water types were in range of 7.6-6.4. another study reported that the sensory property of organic food was better than that of conventional food. it was stated that the shelf-life of organic food was too long, and there was a good correlation between a low nitrate level and good taste perception (oco, 2016). ital. j. food sci., vol. 29, 2017 724 figure 5. sensory evaluation of tea types. values followed by the same letter are not significantly different at the level of 5% (series ‘a-f’ for grades in a blend of a tea type, series ‘a-c’ for the same grades in blends of a tea kinds, and series ‘x-y’ for teas in the same blend and the same grade). de b x dc b x de a x d c x c b x c a x e b x da b x e a x e b x d b x d a x dc b x c b x d a x cd c x bc b x c a x bc b x b a x c a x bc c x b b x b a x ab b x a a x b a x ab c x a b y b a x a b x a b x a a x a c y a b x a a x 0 25 50 75 100 blend 1 blend 2 blend 3 blend 1 blend 2 blend 3 convectional black tea organic black tea se ns or y po in t grade 1 grade 2 grade 3 grade 4 grade 5 grade 6 ital. j. food sci., vol. 29, 2017 725 4. conclusions some chemicals and sensory properties of conventional and organic black turkish teas were investigated according to their grades and blends. the extract, polyphenol and caffeine contents in both teas decreased from blend 1 to blend 3, and the cellulose contents increased from blend 1 to blend 3. similar trends were observed for the grades. the extract and tf/tr values were close to each other among the tea types. the cellulose values in organic teas and caffeine and polyphenol values in conventional teas were higher than those of the other tea type. for the tf/tr ratios, there was not a linear increase or decrease among the blends and grades. whereas the organic black tea was rich in cu, k, ca, mn and mg, the 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university, fasa, iran bdepartment of food science and technology, college of agriculture, urmia university, urmia, iran cdipartimento di scienze degli alimenti e del farmaco, università di parma, parco area delle scienze 47/a, 43124 parma, italy *e-mail address: hashemi@fasau.ac.ir; hasshemii@yahoo.com emma.chiavaro@unipr.it abstract the effect of fermentation by lactobacillus fermentum ptcc 1638, lactobacillus plantarum subsp. plantarum ptcc 1745 and lactobacillus sakei subsp. sakei ptcc 1712 on antimicrobial activity against alternaria alternate ptcc 5224, aspergillus parasiticus ptcc 5018, staphylococcus aureus atcc 25923, escherichia coli o157 h7 atcc 35150 and salmonella typhimurium atcc 14028 as well as antioxidant properties (carbonyl assay, peroxide and anisidine value) in a beef patty during 24 h of fermentation and further storage at 4°c for 8 days were investigated. results indicated that l. plantarum subsp. plantarum had the highest radical scavenging activity (54.3±1.7%) before fermentation. during the fermentation process, dpph and abts activities of the meat patty were improved in comparison to the control. the highest antioxidative value was observed for l. plantarum subsp. plantarum. all of three strains had a strong antimicrobial effect against pathogenic bacteria and fungi. oxidation products were enhanced in fermented and non-fermented samples. however, the increasing trend of the oxidation process was mitigated in all fermented samples. in particular, the lowest protein and lipid oxidation values were observed in the samples treated by l. plantarum subsp. plantarum. generally speaking, fermentation improves the antioxidative and antimicrobial effect of meat patty and lengthens its storage period. keywords: antimicrobial activity, antioxidant activity, fermentation, lactobacillus strains, meat product ital. j. food sci., vol. 30, 2018 269 1. introduction lactic acid bacteria (lab) are the primary microorganisms involved in fermentation (salminen et al., 2004) by consuming simple sugars such as glucose as the substrate (yadav, 2017). lab possess antimicrobial and anticancer activities and play a critical role in the balance of gut microbial flora, synthesis of vitamins, improvement of immunes system, reduction of cholesterol level, prevention of food allergy, improvement of lactose absorption and so forth (mansouripour et al., 2013). moreover, as antioxidant and anti-inflammatory agents are used for treatment of various diseases such as diabetes, alzheimer's, parkinson's, high blood pressure, liver disorders (woo et al., 2014). also, application of lab cultures to promote antibacterial and antioxidant properties of foods has been recommended by many investigations (li et al., 2012; pisano et al., 2014). fermentation was a cheap and simple method to conserve meat since ancient time. production of acid (ph reduction), h2o2 and bacteriocins alone or in combination with starter cultures prevents from the growth of meat deteriorating microorganisms. improving in aroma is another advantage of fermentation. considering limitation of chemical additives, application of fermentation techniques in meat has been increased (sakhare and rao narasimha, 2003). one of oldest meat products created to increase meat conservation is fermented sausage in which fermenting microorganisms especially lab are used. the mentioned microorganisms (single species or a mix of different microorganisms) are added into meat paste as starter cultures (yilmaz and velioglu, 2009). leroy et al. (2005) demonstrated that the application of functional meat starter cultures in fermented sausages promotes product's safety via production of bacteriocins and other antimicrobial compounds (jafari et al., 2017). lab species used in the production of dry fermented sausages possess antibacterial properties against listeria monocytogenes, staphylococcus aureus (papamanoli et al., 2002,2003). various strains of lactobacillus plantarum show strong antioxidant and antibacterial properties during the fermentation process (hashemi et al., 2017). furthermore, use of lactobacillus in fermented pork prevents the growth of different clostridium species (di gioia et al., 2016). however, based on the some investigations, other microorganism such as probiotic bacillus could pose similar effects (jafari et al., 2017). considering above mentioned issues, the present study was conducted to investigate the antioxidant and antimicrobial effect of meat patty fermented by lactobacillus fermentum ptcc 1638, lactobacillus plantarum subsp. plantarum ptcc 1745, lactobacillus sakei subsp. sakei ptcc 1712 during fermentation process and storage period. 2. materials and methods 2.1. microbial culture lactobacillus fermentum ptcc 1638, lactobacillus plantarum subsp. plantarum ptcc 1745, lactobacillus sakei subsp. sakei ptcc 1712, alternaria alternate ptcc 5224, and aspergillus parasiticus ptcc 5018 were purchased from the culture collection at iran institute of industrial and scientific research. reactivation of the lactobacillus strains was done in the mrs broth (oxoid, uk) at 37°c for 48 h. the mold cultures were cultivated on yeast extract dextrose chloramphenicol agar (lab m, uk) slants for 9 days at 25°c. staphylococcus aureus atcc 25923, escherichia coli o157 h7 atcc 35150 and salmonella typhimurium atcc 14028 were obtained from microbial culture stock of veterinary ital. j. food sci., vol. 30, 2018 270 school, shiraz university. the strains were reactivated in defined mueller hinton broth (oxoid, uk) and left for incubation at ~37°c. 2.2. fermented meat patty preparation and storage fresh ground beef was obtained from a local supermarket in shiraz city (fars, iran). ground beef and irradiated herb spice were pasteurized at 80°c for 15 min. fermented meat patty was prepared by mixing ground beef (2 kg), herb spice (10 g), pasteurized brine (8 ml, 10% w/v) and each lactobacillus strain culture (~105 cfu/g). this preparation was subsequently placed into glass vessels (3 l) and kept in an incubator (shimazu, shi1 55 al, iran) to ferment at 35°c for 24 h. a time course analysis was carried out prior to fermentation and 4, 8, 16, 20 and 24 h during the fermentation process. a control (nonfermented) sample was also prepared. after fermentation, fermented and non-fermented meat patty samples were kept at 4°c in refrigerator for 8 days. approximately, 120 samples were prepared and all of the experiments were carried out in triplicate. 2.3. dpph free radical scavenging activity of lactobacillus strains the dpph content was determined using the described method of kao and chen (2006). after centrifugation at 3500 × g (hettich, eba21, germany) for 20 min, the absorbance of cell samples was determined using spectrophotometer (uv/visible philips cambridge, uk) at 517 nm. the blank sample corresponded only to the cells emerged in methanol. 2.4. meat patty analysis during fermentation the ph of fermented and non-fermented samples was determined during the 24 h period of fermentation using a ph-meter (model 520a, orion research inc., ma, usa). the enumeration of l. fermentum, l. plantarum subsp. plantarum, and l. sakei subsp. sakei was done during 24 h period of meat patty fermentation. enumeration of lactobacillus strains was carried out using mrs agar (oxoid, uk) after incubation under anaerobic conditions (35°c, 72 h). dpph radical scavenging activity of fermented meat sauce samples was performed according to the method of kato et al. (1988). about 1 ml of the filtrated sample was mixed with 1ml of dpph reagent (250 mm) and 1ml of 0.1mtris-hcl buffer (ph 7.4) in test tubes. after incubation at room temperature, the absorbance of sample was assessed at 517 nm. ethanol was used as a blank. abts+ activity was measured according to the method of shirwaikar et al. (2006). about 100 ml of sample extract was blended with 4.9 ml of abts+ working standard solution and absorbance was determined after 20 min at 734 nm. the abts+ activity was evaluated by using equation: abts+ activity (%) = [!"#$%"&'() ! !!"#$%"&'() (!") !"#$%"&'() (!) ]×100 the antimicrobial activity of fermented samples against alternaria alternata, aspergillus parasiticus, staphylococcus aureus, escherichia coli o157 h7 and salmonella typhimurium was measured at the end of fermentation, by means of the well diffusion method. for bacterial cells, the inoculum (106 cfu/ ml) was spread on plates containing mueller-hinton agar (oxoid, uk). for molds, well was created on yeast extract dextrose chloramphenicol agar (lab m, uk) plates, which had been previously inoculated by 0.1 ml of inoculums ital. j. food sci., vol. 30, 2018 271 containing indicator molds in the range of 104-105 spores/ml. subsequently, aliquot solutions (80 µl) from meat patty samples were forwarded into the wells. the agar plates were incubated at 37°c for 24 h and at 25°c for 48 h for pathogenic bacteria and molds, respectively. then, the disk diameter of inhibition zones was measured. 2.5. meat patty analysis during storage protein and lipid oxidation of samples were measured during storage. protein carbonyls of fermented and non-fermented meat patty samples were measured according to the described method of levine et al. (1994). carbonyl groups were measured by precipitation of 100 !l of sample with 50 !l of trichloroacetic acid (100%, w/v). after preparation of samples, the absorbance was determined at 280 and 370 nm for measurement of carbonyl content of samples. lipids were extracted according to the described method of bligh and dyer (1959) using chloroform/methanol (1:1, v/v). the ferric-thiocyanate technique described by shanta and decker (1994) was conducted for the measurement of peroxide value (pv). anisidine value (anv) of the samples was measured according to the aocs method (1998). 2.6. statistical analysis statistical analysis was conducted with one-way anova and duncan’s multiple range tests (spss package program; v. 20.0 for windows, spss inc., chicago, il, usa). differences were considered significant at p<0.05. 3. results and discussion 3.1. lactobacillus growth and ph changes during fermentation the reduction of ph, production of lactic acid and antimicrobial compounds including bacteriocins, non-bacteriocins and non-lactic substances can be considered as the major mechanisms of lab activities include (fayol-messaoudi et al., 2005). in current study, ph variation of meat patty during 24h fermentation was investigated as depicted in fig.1a. ph variation trend was similar in the three species l. fermentum, l. plantarum subsp. plantarum and l. sakei subsp. sakei. initial ph was 6.1 in all of the three species and decreased to 4.1, 4.3 and 4.4 at the end of the fermentation. significant variation (p<0.05) occurred between 4 and 20 hours of the fermentation process as shown in the fig. 1a. ph reduction after fermentation is due to the growth of fermenting microorganisms and production of lactic acid from available carbohydrates by these strains (drosinos et al., 2007). yadav (2017) reported that application of l. plantarum significantly (p<0.05) reduced ph during fermentation of chicken sausages. similar results also have been reported the direct correlation between ph reduction and lab growth during meat fermentation (cocolin et al., 2001a, 2001b). according to the united states department of agriculture, ph should be lower than 5 in highly stable fermented meat products (leistner and rodel, 1975). ital. j. food sci., vol. 30, 2018 272 figure 1. ph changes (a) and changes in cell viability of lactobacillus strains (b)of meat patty samples during fermentation. means in each hour with the same superscript lowercase letters are not significantly different at p<0.05. the growth of lab in current investigation was promoted during fermentation (fig.1b). an initial number of the three strains in fermented meat patty was about 5.2 cfu/g that reached to 8.5 cfu/g at the end of fermentation (24 h). all strains showed similar logarithmic growth during the first 16 hours and a constant and slow growth afterward. increased lab growth coinciding with ph reduction suggests resistance and compatibility of them against ph variation (cebeci and gürakan, 2003). indeed, there is a direct correlation between microbial cells population and ph (johnson and steele, 2013). according to comi et al. (2005), lab population increased at early stages of production of dry fermented sausages and then remained at a constant value of 7-9log cfu/g. moreover, in french fermented sausages, the population of fermenting strains increased and reached the same value at the end of the fermentation process (rebecchi et al., ital. j. food sci., vol. 30, 2018 273 1998). increased lab growth is attributed to ph reduction during early stages of fermentation (cocolin et al., 2001a, 2001b). 3.2. radical scavenging activity of lactobacillus strains and fermented meat patty radical scavenging activity was measured, and the corresponding results are presented in table 1. as seen, there is a significant difference (p<0.05) among the bacterial species, and the highest value was observed in l. plantarum subsp. plantarum (54.3±1.7%); followed by l. sakei subsp. sakei and l. fermentum. table 1. radical scavenging activity of lactobacillus strains against dpph radicals. lactobacillus strains radical scavenging activity (%) l. fermentum 38.2±1.1c l. sakei subsp. sakei 44.6±1.5b l. plantarum subsp. plantarum 54.3±1.7a ameans in the column with different superscript letters differ significantly (p < 0.05). moreover, radical scavenging activity (rsa) was measured in fermented meat patty during the 24h period (fig. 2a). the results indicated that rsa was increased through the time. by progress and completion of fermentation, rsa value was elevated. the highest rsa value was observed in the sample treated by l. plantarum subsp. plantarum that increased from 12.1% to 48.2% at the end of fermentation, followed by l. sakei subsp. sakei (increase from 12.1% to 41.3%) and l. fermentum (from 12.1% to 33.5%). thus, l. plantarum had better antiradical properties. for example, rsa value obtained in fermentation by l. sakei subsp. sakei can be achieved by fermentation with l. plantarum subsp. plantarum for 12 hours. li et al. (2012) investigated antioxidant activity of traditional chinese fermented food and concluded that l. plantarum had the highest hydroxyl radical and dpph scavenging activities (44.31% and 53.05%; respectively). moreover, it was found out that proteins and polysaccharides residing at the surface of l. plantarum provide the species with antioxidant power; hence, degradation of these compounds reduces dpph free radical scavenging capacity of l. plantarum. evaluation of antioxidant activity of l. plantarum isolated from marcha of sikkim indicated that this strain possesses high dpph scavenging activities (das and goyal, 2015). similar investigations support our findings regarding application of l. plantarum in the fermentation of various foods (kullisaar et al., 2002; wang et al., 2009; hashemi et al., 2017). evaluation of abts + activity in meat sauce during fermentation revealed that abts + value was enhanced by an increase in time and the highest value of this parameter was achieved by l. plantarum subsp. plantarum that increased from 26.3% at the first hours to 64.24% at the 24th hour (fig. 2b). the last two assays suggest that lactobacillus strains especially l. plantarum had high antioxidant power because the results show that the highest value of this parameter obtained by 24h fermentation with l. sakei subsp. sakei and l. fermentum can be achieved by 14 h and 10 h fermentation with l. plantarum subsp. plantarum. these findings accord with those reported by yadav (2017) who found out that application of l. plantarum in sausage fermentation promotes abts + activity and dpph activity. ital. j. food sci., vol. 30, 2018 274 figure 2. changes in dpph activity (a) and abts+ activity (b) of meat patty samples during fermentation with lactobacillus strains. means in each hour with the same superscript lowercase letters are not significantly different at p<0.05. 3.3. antimicrobial activity of fermented meat patty antimicrobial activity of meat patty during fermentation was investigated against a. alternate, a. parasiticus, s. aureus, e. coli o157 h7 and s. typhimurium, and the result were represented in table 2. the highest antimicrobial activity was corresponded to sample fermented with l. plantarum whose inhibition zone diameters (table 2). inhibition zone diameters of l. plantarum against e. coli o157:h7, s. typhimurium, s. aureus, a. parasiticus and a. alternate were 22.6±0.3mm, 20.9±0.4mm, 25.9±0.4mm, 28.5±0.5mm, and 27.9±0.8mm; respectively. the highest inhibition zone diameter of l. plantarum was 28.5±0.5mm that obtained for a. parasiticus; followed by l. sakei subsp. sakei and l. fermentum. inhibition zone of l. plantarum often had significant difference (p<0.05) with that of the two other strains. labs prevent microbial growth and meat deterioration by acid production (ph reduction), h2o2 and bacteriocins production. yadav (2017) ital. j. food sci., vol. 30, 2018 275 reported that l. plantarum plays a critical role in the prevention of microbial growth during sausages chicken fermentation. yilmaz and velioglu (2009) found out that lab was the leading cause of microbial growth inhibition in fermented products. the author observed that the number of bacillus strains was significantly (p<0.05) lower in the samples treated by 24h fermentation, while the number was increased in control group. rea et al. (2013) investigated antimicrobial properties of species used in fermented sausages and concluded that the main reason behind the reduction of bacillus in these products was ph reduction and bacteriocins production by the fermenting bacteria. application of l. plantarum in fermentation has antimicrobial activity against e. coli o157: h7 and s. aureus (hashemi et al., 2017). table 2. antimicrobial activity of meat patty samples with different lactobacillus strains. meat samples inhibition zone diameters (mm) e. coli o157:h7 s. typhimurium s. aureus a. parasiticus a. alternata l. fermentum 18.4±0.5cc 15.7±1.1bd 20.5±0.6cb 23.7±0.6ba 24.1±0.7ca l. sakei subsp. sakei 20.3±0.9 bd 16.3±0.8be 23.1±1bc 27.4±1.2aa 25.5±0.4bb l. plantarum subsp. plantarum 22.6±0.3 ac 20.9±0.4ad 25.9±0.4ab 28.5±0. 5aa 27.9±0. 8aa control (nonfermented) 0±0 d 0±0d 0±0d 0±0d 0±0d avalues represent means ± standard deviations of inhibition zones. means within a column with the same superscript lowercase letters are not significantly different at p<0.05 and means within a row with the same superscript uppercase letters are not significantly different at p<0.05. 3.4. lipid and protein oxidation of meat sauce samples during storage pv and anv were measured to evaluate oxidative stability in fermented meat patty at 4°c for 8 days. lipid oxidation includes continuous formation of hydroperoxide as primary oxidation products that can be degraded to various volatile substances as secondary oxidation products (adegoke et al., 1998). pv indicates primary oxidation products that are odorless materials that are degraded during the reaction and converted to a wide range of substances including carbonyl, hydrocarbons, furans and other products creating an unsuitable taste of the foods (yanishlieva and marinova, 2001). pv was measured during 8-day period after fermentation. as depicted in fig.3a, the samples fermented with three lab strains had lower pv than the control group (p<0.05). among fermented samples, the sample fermented with l. plantarum subsp. plantarum had the lowest pv (p<0.05), suggesting antioxidant properties of the lab. in this research, the highest antioxidant activity was observed for l. plantarum subsp. plantarum. pv increased with time, but this increasing trend for l. plantarum was at the lowest rate compared to other fermented and control samples (p<0.05). as an indicator of secondary oxidation, anv was measured during the 8day period. a similar trend was observed for this parameter (fig. 3b), meaning that the parameter was increased in all fermented and control samples, but l. plantarum was the best species considering resistance against formation of secondary oxidation products. anv was increased by the time but the lowest value on the first and eighth days was observed in the sample treated by l. plantarum subsp. plantarum (p<0.05). these results accord with those reported by other authors indicating that food fermentation by lab especially by l. ital. j. food sci., vol. 30, 2018 276 plantarum improves the oxidative stability of the products (tseng and zhao, 2013; hashemi et al., 2017; yadav, 2017). figure 3. peroxide value (a) and anisidine value (b) of meat patty samples during storage at 4°c for 8 days storage. means in each day with the same superscript lowercase letters are not significantly different at p<0.05. carbonyl value was measured during 8 days of storage to determine protein oxidation. as depicted in fig. 4, carbonyl formation was increased by the progress of storage time. the highest value of this parameter was observed in non-fermented (control) sample, and the lowest value was obtained in meat patty fermented with l. plantarum subsp. plantarum as 0.9 and 2.1nmol/mg on the first and eighth days, respectively. indeed, protein oxidation rate was much more decreased in this sample. protein oxidation may occur naturally during cold storage of foods. indeed, protein oxidation occurs in side chains of amino acids including thiol and aromatic hydroxyl that results in the formation of carbonyl groups (stadtman, 1990). the concentration of carbonyl groups can be considered as ital. j. food sci., vol. 30, 2018 277 an index of oxidative activities (hashemi et al., 2015). hashemi et al. (2017) concluded that application various strains of l. plantarum in fermentation reduced protein oxidation rate, which accords with the results obtained in the present study. figure 4. carbonyl value of meat patty samples during storage at 4°c for 8 days storage. means in each day with the same superscript lowercase letters are not significantly different at p<0.05. 4. conclusions the results obtained in this research revealed that meat patty fermentation using lactobacillus strains improved its antimicrobial and antioxidative properties. antiradical activity was enhanced during fermentation, and the fermented product had antimicrobial activity against pathogenic bacteria and fungi. moreover, during storage of meat patty, lipid and protein oxidation was lower in fermented samples compared to non-fermented ones. future studies can focus on the antioxidant mechanism of lactobacillus strains. acknowledgements s.m.b. hashemi would like to express his appreciation to fasa university 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ying and gui, 2012). although, data exist on the differences in composition of some bas in chicory leaves (sahan et al., 2017; d’acunzo et al., 2017), only data on nutrient composition and its difference between wild and cultivated plants from montenegro is available (jancic et al., 2016). bas include a group of nutritive components, which are naturally present in plants, fruits, and vegetables. conducting a research on bas is important due to their numerous health benefits. biologically active substances can reduce the risks of vascular and renal diseases, lower glycemic index in diabetics, reduce risks of cancer and increase bifidobacteria population in the colon (guhr and lachance, 1997; hasler, 1998). the aim of this study was to estimate the profile of biologically active ingredients in chicory leaves, growing in different locations throughout montenegro. within the category of bas, the profile of dietary fiber (total, soluble and insoluble dietary fibers, hemicelluloses, cellulose, lignin, and fructan content), essential fatty acid profile, pigments and major antioxidant compounds were determined. this study is the first comprehensive study on the above named compounds in chicory leaves that are growing in montenegro. 2. materials and methods 2.1 sample collection fresh materials (leaves) of the same chicory variety were collected from different locations in montenegro. out of the nine samples examined, seven were samples of wild plants (zoganje, risan, podgor, tivat, pricelje, plavnica and pljevlja locations) and two were samples of greenhouse cultivated plants (komani and susanj). all plant leaves (ca. 2 kg) were sampled in their vegetative stage (before flowering) of growth between may and july, 2015. the leaves were collected in the morning. 2.2. sample preparation fresh leaves were separated and all dirt was removed. a part of the leaves were immediately wrapped in aluminum foil to avoid degradation of pigments by light. ital. j. food sci., vol. 29, 2017 629 one part of the fresh plant material (ca. 1 kg) was milled using an electric grinder (ika a11, staufen, germany) and stored in well-labeled air tight polyethylene bottles at -18 °c until the time for chemical testing in the laboratory. the remaining samples (for the purpose of polyphenol, pigment, and antioxidative capacity determinations) were lyophilized (alpha 1-4ld, christ, germany) and grounded to a fine powder using a planetary ball mill (s100, retsch, germany) and stored at room temperature in tightly closed humidity-proof plastic containers until analysis. before and after lyophilisation, samples were weighed in order to recalculate the data obtained from dry weight (dw) to fresh weight (fw). 2.3. determination of content of total, soluble and insoluble dietary fibers, hemicelluloses, cellulose, lignin and fructan for the determination of total (tdf), soluble (sdf) and insoluble (idf) fractions of dietary fibers, samples were analyzed in accordance with aoac 991.43 following the enzymatic-gravimetric procedure (aoac, 1995) as described by lee et al. (1992). determination of neutral detergent fibers (ndf), acid detergent fibers (adf) and acid detergent lignin (adl) was performed in order to obtain the content of hemicellulose, cellulose and lignin. ndf was determined gravimetrically as a fibrous residue (primarily cell wall components of plants as cellulose, hemicellulose, and lignin), which was formed after refluxing with a neutral detergent solution and heat-stable amylase. adf was determined gravimetrically as the residue of cellulose, lignin, and heat damaged protein and a portion of cell wall protein and minerals (ash) remaining after extraction with an acidified quaternary detergent solution. adl was determined gravimetrically upon treatment with an acid detergent solution, which includes cooking, filtering and drying. hemicellulose, cellulose and lignin content were calculated as follow: a) hemicellulose=ndf-adf; b) lignin=adl; c) cellulose=ndf-hemicelluloselignin (goering and van soest, 1970; van soest et al., 1991; aoac, 1990). fructan content was measured in accordance with the enzymatic/spectrophotometric aoac 999.03 and aacc 32.32 methods using the enzyme assay kit k-fruc (megazyme, bray, ireland) (mccleary and blakeney, 1999; aoac, 2002; aacc international, 2000). in the process of fructan analysis, raffinose oligosaccharides were removed with the α-galactosidase treatment (megazyme, bray, ireland) before degradation of starch, maltosaccharides and sucrose, as described in the kit. 2.4. determination of fatty acids profile the method recommended by the association of official analytical chemists aoac 930.09 was used for the determination of crude lipid content (aoac, 1990). fatty acids were determined by gas chromatography (gc) after the following transesterification procedure: fatty acids in crude fat with added hexane were methylated by shaking for 20 s with 5 ml of 2 m koh. sample was heated for 60 s on water bath (60°c) and additionally shaken up for 20 s. after addition of 10 ml of 1 n hcl, the mixture was shaken up well again and another portion of hexane was added. in the separated phases, fatty acid methyl esters (fame) were in the upper hexane layer. fames were analyzed using gas chromatograph with fid detector (shimadzu gc-17a, japan) and supelco sp-2560 fused silica capillary column (100 m, id 0.25 mm, deb. ital. j. food sci., vol. 29, 2017 630 phase: 0.20 µm, sigma-aldrich, germany). fatty acid identification was performed by comparing the relative retention times of fame peaks from samples with the standards. fame standard (supelco 37 component fame mix, sigma co, st louis, mo, usa) was used for the identification process. all solvents and chemicals were of analytical grade. 2.5. determination of pigments extraction of pigments was carried out according to znidarcic et al. (2011): 100 mg of the dry leaf powder with 5 ml of ice-cold acetone on an ice bath, using t-25 ultraturrax (ika-labortechnik, staufen, germany) homogenizer for 25 s. all extraction procedures were performed in dim light. acetone extracts were filtered through 0.2 µm minisart srp 15 filter (sartorius stedim biotech gmbh, goettingen, germany) and then subjected to hplc gradient analysis (a spherisorb s5 ods-2 250x4.6 mm column with an s5 ods-2 50x4.6 mm precolumn, alltech associaties, inc., deerfield, usa), using the following solvents: solvent a: acetonitrile/methanol/water (100/10/5, v/v/v); solvent b: acetone/ethylacetate (2/1, v/v), at a flow rate of 1 ml/min, employing linear gradient from 10% solvent b to 70% solvent b in 18 min, with a run time of 30 min, and photometric detection at 440 nm. the hplc analysis was performed on a spectraphysics hplc system with spectra focus uv-vis detector (fremont, usa). pigments were quantified by determining peak areas under the curve in the high-performance liquid chromatograms calibrated against known amounts of standards. each peak was confirmed by the retention time and characteristic spectra of the standards. the following standards were used for the determination of photosynthetic pigments: neoxanthin, violaxanthin, antheraxanthin, zeaxanthin, lutein, chlorophyll a and b, pheophytin a and b, and α-, β-carotene, all from dhi lab products (hoersholm, denmark). all standards were highly purified. the solvents acetone, ethylacetate, methanol, and acetonitrile were from merck and hplc grade. 2.6. determination of total polyphenol, total flavonoid, chlorogenic and caffeic acid content plant extracts were prepared as described by wen et al., 2005. one gram of each lyophilized sample was taken in a measuring flask and dissolved in methanol/water/trifluoroacetic acid (50/50/0.1, v/v/v) mixed solvent, and then the volume of the turbid fluid was adjusted to 10 ml accurately. the mixture was sonicated for 30 min at room temperature, centrifuged at 3000 rpm for 5 min, and finally filtered through a 0.45 µm nylon filter. total polyphenol content (tpc) of plant extracts was determined spectrophotometrically according to the follin-ciocalteau method as described by todorovic et al. (2015). briefly, to a 0.5 ml aliquot of samples, 2.5 ml folinciocalteu’s reagent, 30 ml distilled water and 7.5 ml of 20% na2co3 were added and filled up to 50 ml with distilled water. the absorbance of blue coloration was measured at 765 nm against a blank sample, after 2 h storage in the dark. gallic acid (sigma aldrich, germany) was used as the standard and the results were expressed as mg gallic acid equivalents (gae) per gram of fresh sample. a calibration curve was developed for the working solutions of gallic acid in the concentration range of 0-80 mg/ml and it showed good linearity. ital. j. food sci., vol. 29, 2017 631 for the purpose of determination of total flavonoid content (tfc), the following procedure was followed (todorovic et al., 2015): 0.5 ml of each extract was transferred into a 5 ml volumetric flask and 0.15 ml of the 5% nano2 was added for 6 min and evenly mixed. after that 0.15 ml of the 10% alcl3 solution was added and shaken up. six minutes later 1 ml of the 1 m naoh solution was added. the mixture was diluted with distilled water up to 5 ml. absorbance was measured on a uv-vis spectrophotometer (j.p. selecta, barcelona, spain) at 510 nm and compared to the blank solution. a standard curve was prepared using a 1000 mm solution of catechin (sigma aldrich, germany) at intervals of 200 mm catechin concentration. the results were expressed as µmol catechin equivalents (ce)/g fw. the separation of chlorogenic and caffeic acid was performed using lc-ms/ms quattro microtm api tandem quadrupole mass spectrometer (waters, milford, ma, usa) equipped with sunfire c18 column (3.5 µm; 3.0 x 100.0 mm, waters, milford, ma, usa). the elution was carried out at a flow rate of 0.5 ml/min with 0.1% formic acid in water (eluent a) and acetonitrile (eluent b). the gradient started with 5% b, reached 31% b in 28 minutes, and 71% b after 35 minutes; while 76% b was kept for 2 minutes. the temperature of column was controlled at 40°c. injection volume was 5 µl. the components were detected by negative electrospray ionization (esi-): capillary voltage, 3.2 kv; ion source temperature, 120°c; desolvation gas temperature, 450°c; desolvation gas flow rate, 600 l/h; and cone gas flow rate, 50 l/h. the multiple reactions monitoring (mrm) was used to confirm the detected substances. characteristic masses for component identification and their retention times (mrm parameters) are presented in table 1. calibration curves were developed using standard solution prepared from chlorogenic (≥95%, sigma aldrich, germany) and caffeic acid (≥98%, sigma aldrich, germany). table 1. characteristic mass for chlorogenic and caffeic acid identification and their retention times (mrm parameters). component parent ion (m/z) daughter ion (m/z) dwell (s) cone voltage (v) collision energy (v) chlorogenic acid 353.00 191.00 0.2 25 22 caffeic acid 179.00 135.00 0.2 18 19 2.7. antioxidant activity determination antioxidant capacity of extracts was determined by running 3 tests. in dpph (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay, every diluted sample (0.2 ml) was added to the dpph working solution (2.8 ml). dpph solution was prepared as a mixture of 1.86×10−4 mol/l dpph in ethanol and 0.1 m acetate buffer (ph 4.3) in volume ratio 2:1. the free radical scavenging capacity was evaluated at room temperature by measuring the absorbance at 525 nm after 1 h of reaction in the dark. calibration curve, in the range of 0.2–0.7 mmol trolox l-1 was used for the quantification of antioxidant activity. the results are expressed as µm trolox equivalents (te)/g fw (brand-williams et al., 1995). ital. j. food sci., vol. 29, 2017 632 in frap (ferric ion reducing antioxidant power) assay, stock solutions were prepared by mixing 300 mm of acetate buffer (ph 3.6), 10 mm tptz (2,4,6-tripyridyl-s-triazine) solution in 40 mm hcl, and 20 mm fecl3×6h2o solution. the fresh working solution was made using 25 ml of acetate buffer, 2.5 ml tptz solution, and 2.5 ml fecl3×6h2o solution and then warmed at 37°c before using. methanol/water/trifluoroacetic acid diluted samples (300 µl) were allowed to react with 3 ml of the frap solution for 40 minutes under dark conditions. readings of the colored product (ferrous tripyridyltriazine complex) were then taken at 593 nm. the antioxidant activity was calculated from the calibration curve using the range 0.1-0.8 mmol trolox l-1. results are expressed in µm trolox equivalents (te)/g of fw (benzie and strain, 1996). the trolox equivalent antioxidant capacity (teac or abts assay) of chicory extracts was estimated by the abts radical action decolorization assay. stock solutions of abts (14 mm) and potassium peroxodisulfate (4.9 mm) in phosphate buffer (ph 7.4) were prepared, and mixed together in equal volumes. the mixture was left to react overnight (12-16 h) in the dark, at room temperature. on the day of analysis, the abts radical solution was diluted with phosphate buffer to an absorbance of 0.70 (±0.02) at 734 nm. exactly 30 µl of aliquoted samples were added to 3.0 ml of the abts radical solution, and after 6 min at 30°c the absorbance readings were taken. instead of the sample, the reagent blank used was 30 µl of phosphate buffer. calibration curve was developed using a range of 0.2-1.5 mmol trolox l-1. the results were expressed as µm trolox equivalents (te)/g of fresh weight (re et al., 1999). an overall antioxidant potency composite index was determined according to seeram et al. (2008). an index value of 100 was assigned to the best score for each test and an index score was calculated for all other samples within the test as follows: antioxidant index score = [(sample score/best score) × 100]. the overall mean index value was determined by dividing the sum of the individual index by the number of tests (three assays in total: dpph, frap and abts). 2.8. statistical analyses analyses were performed in triplicate. results are expressed as mean values with the corresponding standard deviation (sd). statistical difference between means of the two groups (wild and cultivated plants) was determined using student’s t-test, two sample assuming unequal variances, and a p value <0.05 was considered statistically significant. analysis of variance, one-way anova was performed to test the significance of the observed differences between wild plants locations. when the observed differences were significant (p<0.05) the mean values were compared by the least significant difference (lsd) post hoc multiple comparison test. correlation analysis was performed using pearson’s. all statistical analyses were performed using the software spss ver. 19 (bryman and cramer, 2012). ital. j. food sci., vol. 29, 2017 633 3. results and discussions 3.1. fiber profile the results for total, soluble and insoluble dietary fibers, hemicelluloses, cellulose, lignin and fructan in analyzed samples are presented in table 2. the results are expressed on fresh weight (fw) basis. table 2. fiber profile in leaves of wild and cultivated chicory*. location content (g/100 g) total fiber insoluble fiber soluble fiber hemicelluloses lignin cellulose fructan wild chicory (cichorium intybus l.) zoganje 5.0±0.1b 3.7±0.1a 1.26±0.15b 1.14±0.11bc 0.24±0.10 1.60±0.25a 0.23±0.05a risan 4.3±0.2c 3.3±0.3b 1.10±0.09c 1.10±0.20bc 0.20±0.12 1.49±0.17a 0.06±0.03cf podgor 4.1±0.2c 2.9±0.2c 1.17±0.14bc 1.31±0.16ac 0.17±0.08 1.31±0.22ab 0.15±0.07b tivat 3.0±0.2e 2.1±0.2d 0.93±0.13d 1.07±0.18bc 0.09±0.14 0.77±0.26cd 0.07±0.02cd pricelje 3.4±0.1d 2.8±0.2c 0.64±0.12e 1.51±0.19a 0.11±0.06 1.06±0.38bc 0.12±0.04bc plavnica 3.3±0.1de 2.1±0.2d 1.14±0.12bc 1.34±0.22ab 0.19±0.06 0.41±0.14d 0.07±0.01ce pljevlja 6.2±0.2a 3.8±0.3a 2.35±0.11a 1.56±0.19a 0.21±0.08 1.56±0.25a 0.07±0.02bdef average 4.2±0.1 3.0±0.1 1.23±0.02 1.29±0.04 0.17±0.03 1.17±0.08 0.11±0.02 cultivated chicory (cichorium intybus l.) komani 4.3±0.2 3.2±0.2 1.08±0.10 0.80±0.16 0.19±0.08 1.11±0.19 0.10±0.03 susanj 2.9±0.1 2.2±0.1 0.67±0.08 0.58±0.12 0.18±0.11 1.30±0.16 0.06±0.02 average 3.6±0.1 2.7±0.1 0.88±0.01 0.69±0.03# 0.19±0.02 1.21±0.02 0.08±0.01 *data are expressed on 100 g fresh weight and presented as mean±sd of three independent determinations. #statistically significant difference between wild and cultivated chicory, p<0.05. data sharing the same letter (a, b, c, d, e, f) in the same column are not significantly different, p>0.05. determined average values for total fiber were 4.2 g/100 g for the leaves of wild plants and 3.6 g/100 g of the cultivated plant leaves. results indicate that the amount of soluble fiber was 1.6 to 4.4 times higher than the content of soluble fiber (dodevska et al., 2015). hemicellulose and cellulose were the main insoluble fiber in chicory leaves (average values of 1% and 1.19%, respectively), while lignin was present in small amounts (ca. 0.2%). fructan is an important fiber in chicory plant, but its content in leaves is quite low (on average around 0.1%). fructan is a fructose polymer that stores carbohydrate in a large number of plant species. the content of inulin and other fructan in chicory root is 15-20% and these compounds comprise more than 70% of total carbohydrates in fresh chicory roots (gupta et al., 2003). chicory fructan is known as inulin and the chicory root, together with jerusalem artichoke root, are the most important sources for industrial inulin production. since fructan is not digested in the small intestine because of the b (2-1) bonds between fructose molecules, it belongs to the ital. j. food sci., vol. 29, 2017 634 soluble dietary fiber fraction. there are several known positive nutritional effects of fructan and inulin: when consumed in adequate quantities they increase stool frequency, have beneficial effects on blood lipids, and have prebiotic effects (stimulate growth of “good” bifidobacteria in the intestine) (roberforid, 1999). unfortunately, although the root of chicory plants is rich in fructan, the leaves are very poor in this prebiotic carbohydrate and the result of our investigation showed that fructans represented only ca. 4% of insoluble and ca. 2% of total fiber fraction. milala et al. (2009) also detected low amounts of fructan in chicory leaves, although their results were slightly higher. results for dietary fiber profile showed that wild plants had higher amounts of almost all fiber fractions in comparison to cultivated plants, but significant difference was identified only in the hemicellulose content (p<0.05). the location influenced the total fiber content both in wild and cultivated plants, which was obvious from the wide range of results (3.0-6.2% of total fiber in wild leaf samples, and 2.9-4.3% in cultivated ones). after statistical analysis, it was seen from the results obtained that there was a significant statistical difference in the content of tdf, idf, sdf, cellulose, and fructan depending on the wild plants’ sampling locations. in comparison with other leafy vegetables, chicory leaves are significantly better fiber sources. our results for cellulose content were about twenty times higher than results published for chard and even forty times higher than those published for lettuce. as far as hemicellulose content is concerned, wild chicory was twice richer than spinach, 3.5 times than chard, and 7.5 times richer than lettuce (herranz et al., 1981). 3.2. total fat content and fatty acid composition composition of fatty acids (fa) and total fat content of analyzed samples of wild and cultivated chicory are presented in table 3. the results for total fat content are presented on fw basis, while results for fa composition are expressed as percentage of total fatty acids. all studied samples showed fat content to be lower than 0.5 g/100 g, ranging between 0.22-0.49g/100 g, with no difference between cultivated and wild plants. low level of fat is common in leafy vegetables. ten different fatty acids were identified and quantified. the main fatty acids found were α-linolenic (c18:3n-3), linoleic (c18:2n-6c), and palmitic acid (c16:0) and they comprised more than 95% of all fatty acids. these results are in good correlation with the data obtained for cultivated chicory leaves in slovenia and holland (sinkovic et al., 2015(a); warner et al., 2010). alpha-linolenic acid was the most abundant fatty acid in chicory leaves and its content varied between 60.0 and 75.3% for the wild plants leaves, while the average content in cultivated plants was 75.8%. the linoleic acid content ranged from 11.0% in the samples from komani to 17.4% in the samples from podgor. oleic acid (c18:1n-9c) was the only determined unsaturated fatty acid in all chicory samples. the difference between the contents of linoleic acid and α-linolenic acid in the leaves of wild and cultivated plants was significant (p<0.05). wild plants were richer in linoleic, while cultivated ones had higher content of α-linolenic acid. furthermore, obvious differences in the content of palmitic, linoleic and α-linolenic acid was noticed between wild plants sampled from different locations. ital. j. food sci., vol. 29, 2017 635 table 3. total fat content* and fatty acid composition** in wild and cultivated chicory leaves. wild chicory (cichorium intybus l.) cultivated chicory (cichorium intybus l.) % zoganje risan podgor tivat pricelje plavnica pljevlja average komani susanj average c14:0 0.5±0.1 0.2±0.1 0.4±0.1 0.2±0.1 0.3±0.1 0.3±0.1 0.2±0.1 0.3±0.1 0.4±0.1 0.2±0.1 0.3±0.1 c15:0 nd nd 0.2±0.1 nd nd 0.2±0.1 nd nd nd c16:0 17.5±0.6a 9.6±0.5d 13.6±0.7b 10.0±0.3d 12.6±0.4c 12.2±0.7c 10.2±0.4d 12.2±0.5 10.2±0.5 10.4±0.1 10.3±0.3 c18:0 2.0±0.1 0.7±0.1 1.0±0.1 0.8±0.2 0.8±0.2 1.1±0.2 1.4±0.3 1.1±0.2 0.9±0.2 0.6±0.2 0.8±0.2 c18:1n-9c 2.8±0.2 0.8±0.2 1.1±0.2 0.7±0.2 1.4±0.4 1.1±0.2 1.2±0.4 1.3±0.3 0.7±0.1 0.6±0.1 0.7±0.1 c18:2n-6c 15.7±0.4b 12.1±0.6d 17.4±0.8a 14.4±0.5c 15.5±0.8b 15.5±0.5b 13.8±0.8c 14.9±0.6 11.0±0.6 11.2±0.8 11.1±0.7# c18:3n-3 60.0±0.8e 75.3±0.9c 64.8±1.0d 72.5±1.1b 68.1±1.2c 67.7±0.9c 71.9±0.8b 68.6±1.0 75.5±1.0 76.0±1.2 75.8±1.1# c20:0 0.2±0.1 0.2±0.1 0.2±0.1 0.2±0.1 0.2±0.1 0.2±0.1 0.2±0.1 0.2±0.1 0.2±0.1 0.2±0.1 0.2±0.1 c22:0 0.5±0.1 0.4±0.1 0.5±0.1 0.5±0.1 0.5±0.1 0.7±0.2 0.3±0.1 0.5±0.1 0.5±0.1 0.3±0.1 0.4±0.1 c24:0 0.8±0.2 0.7±0.2 0.8±0.2 0.7±0.1 0.7±0.1 0.9±0.1 0.8±0.2 0.8±0.1 0.6±0.1 0.5±0.1 0.6±0.1 total fat 0.45±0.09 0.49±0.10 0.35±0.05 0.41±0.11 0.45±0.10 0.33±0.14 0.22±0.07 0.39±0.09 0.48±0.06 0.39±0.10 0.44±0.06 *data are expressed on 100 g fresh weight and presented as mean±sd of three independent determinations. **expressed as relative percentage of total fatty acids. #statistically significant difference between wild and cultivated chicory, p<0.05. data sharing the same letter (a, b, c, d) in the same row are not significantly different, p>0.05. myristic acid (c14:0); pentadecanoic acid (c15:0); palmitic acid (c16:0); stearic acid (c18:0); oleic acid (c18:1n-9c); linoleic acid (c18:2n-6c); αlinolenic acid (c18:3n-3); arachidic acid (c20:0); behenic acid (c22:0); lignoceric acid (c24:0); nd: not detected. ital. j. food sci., vol. 29, 2017 636 the ratio of unsaturated and saturated acids in wild plants was 6:1, while in cultivated ones it was 7:1. alpha-linolenic acid as a ω-3 polyunsaturated fatty acid has many nutritional and health benefits (it contributes to lowering the level of ldl cholesterol, reducing triglyceride levels and platelet aggregation, vasoconstriction and ventricular arrhythmia) and increasing its intake in diet have been widely suggested (bradberry and hilleman, 2013). it is not unusual that plant leaf lipids contain significant amounts of c18:3n-3, which is a component of chloroplast membrane lipids. through history, it has been observed that wild plants are very important sources of this essential omega-3 fatty acid (simopoulos, 2004). compared to data from literature on fatty acid composition in the most-commonly consumed leafy vegetable species, chicory from montenegro was richer in α-linolenic acid than lettuce by almost 20% (vidrih et al., 2009) and by 40% than spinach (narsing rao et al., 2015). 3.3. pigments three classes of pigments namely: xanthophylls, chlorophylls and carotenes were identified and quantified (table 4). the results are presented on fw basis. table 4. pigments in wild and cultivated chicory leaves*. location content (mg/100 g) lutein violaxanthin antheraxanthin vaz** neoxanthin wild chicory (cichorium intybus l.) zoganje 7.0±0.5f 3.0±0.4c 0.25±0.04a 3.2±0.2b 3.3±0.2d risan 10.7±0.9a 5.2±0.5b 0.14±0.02bc 5.4±0.4b 5.4±0.5a podgor 9.5±0.6bd 3.1±0.4c 0.11±0.02bc 3.2±0.3b 3.5±0.2cd tivat 10.3±0.6ab 3.5±0.3c 0.10±0.02c 3.6±0.2b 4.9±0.4a pricelje 9.6±0.6bc 6.5±0.6a 0.29±0.05a 6.8±0.4a 4.0±0.3bc plavnica 8.9±0.6cde 6.3±0.6a 0.15±0.04bc 6.5±0.4a 4.0±0.3bc pljevlja 9.5±0.6b,e 5.5±0.5b 0.16±0.05b 5.7±0.3b 4.2±0.4b average 9.4±0.6 4.7±0.5 0.17±0.03 4.9±0.3 4.2±0.3 cultivated chicory (cichorium intybus l.) komani 13.1±0.2 5.8±0.5 0.05±0.01 5.8±0.3 6.6±0.6 susanj 11.9±0.2 3.3±0.3 0.06±0.02 3.3±0.2 6.5±0.7 average 12.5±0.2 4.6±0.4 0.05±0.01# 4.6±0.2 6.6±0.7# location content (mg/100 g) chlorophyll a chlorophyll b pheophytin a pheophytin b β-carotene wild chicory (cichorium intybus l.) zoganje 45.0±0.9f 13.7±0.9f 1.6±0.2c 20.1±0.4b 3.7±0.5c risan 97.2±1.3a 30.2±1.0a 2.1±0.1b 13.4±0.3c 6.1±0.7a podgor 74.0±1.1e 23.4±1.0de 2.1±0.2b 19.8±0.2b 6.2±0.6a tivat 77.6±1.0d 28.2±1.0b 2.4±0.4a 22.9±0.4a 6.1±0.7a pricelje 92.5±1.2b 25.5±1.0c 1.7±0.1c 10.4±0.2d 5.8±0.4ab plavnica 84.3±1.0c 24.1±1.0ce 0.1±0.1d 8.7±0.2e 6.2±0.5a pljevlja 77.9±1.2d 24.2±1.1cd 1.7±0.2c 10.4±0.3d 4.8±0.5b average 78.4±0.1 24.2±1.0 1.7±0.2 15.1±0.3 5.6±0.6 cultivated chicory (cichorium intybus l.) komani 115.0±1.3 36.9±0.9 2.2±0.2 13.9±0.9 8.2±0.5 susanj 107.9±1.5 36.0±0.9 0.6±0.1 3.5±0.7 6.5±0.6 average 111.5±1.4# 36.4±0.9# 1.4±0.2 8.7±0.8 7.4±0.6 *data are expressed on the original weight basis and presented as mean±sd of three independent determinations. **content of xanthophyll cycle pigments (violaxanthin, antheraxanthin, zeaxanthin). #statistically significant difference between wild and cultivated chicory, p<0.05. data sharing the same letter (a, b, c, d, e, f) in the same column are not significantly different, p>0.05. ital. j. food sci., vol. 29, 2017 637 four xanthophyll pigments − lutein, violaxanthin, antheraxanthin, and neoxanthin were identified and quantified in the leaves of chicory. zeaxantin was not found in any of the samples. based on concentration, the major xanthophyll was lutein representing on average 50% of the total xanthophylls, which is in agreement with the results of znidarcic et al. (2011). the lowest lutein content was measured in chicory from zoganje (7.0 mg/100 g), while the highest was measured in chicory from komani (13.1 mg/100 g). lutein was confirmed as the main xanthophyll in the edible portion of wild and cultivated chicory varieties grown in the south of italy as supported by montefusco et al. (2015). although, the reported concentrations in the tissues were much lower (0.8-3.0 mg/100 g fw). the content of xanthophyll cycle pigments vaz (violaxanthin, antheraxanthin and zeaxanthin) in analyzed chicory varied at intervals 3.2-6.8 mg/100 g fw. the major cycle pigment was violaxanthin, with antheraxanthin representing only 0.9-7.8% of the vaz pool. the difference between wild and cultivated chicory was significant in the case of antheraxanthin (p<0.05). the average values for the only non-vaz pigment neoxantin content in chicory leaves were 4.2 mg/100 g in wild plants and 6.6 mg/100 g fw in cultivated plants, and there was a significant difference between them (p<0.05). a significant difference for all pigments in wild samples from different locations was noticed. dietary carotenoids, especially xanthophylls, enjoyed significant scientific attention because of their characteristic biological activities, including anti-allergic, anti-cancer, and anti-obese actions. lutein is one of the major xanthophylls present in green leafy vegetables and it is known to selectively accumulate in the macula of the human retina (kotake-nara and nagao, 2011). as an antioxidant (miller et al., 1996; di mascio et al., 1989) and as a blue light filter (junghans et al., 2001), lutein can protect the eyes from oxidative stress, of which a non-protectioncan lead to age-related macular degeneration and cataracts. when compared with data from literature, our results for lutein content in chicory leaves were twice higher than in spinach and even sixty times higher than in lettuce (perry et al., 2009). the chlorophyll a/b ratio was found to be similar in all analyzed samples, although the chlorophyll a and b contents varied greatly (chlorophyll a 45.0-115.0 mg/100 g and chlorophyll b 13.7−36.9 mg/100 g). there was a statistical difference in chlorophyll a and b contents between leaves of cultivated and wild plants, with cultivated ones being richer than the wild ones. the results also indicated that significant amounts of chlorophyll a and b were converted into pheophytin a and b, probably during the lyophilization process. chlorophyll gives a characteristic coloration to the green leafy plants and its content correlate with the photosynthetic potential, giving some indication of the plant physiological status. there are indications that chlorophyll can play an important role in the prevention of various diseases associated with oxidative stress and certain environmental contaminants, such as cancer, cardiovascular diseases and other chronic diseases (gamon and surfus, 1999; sangeetha and baskaran, 2010). the mechanism underlying the supposed chlorophyll suppression of in vitro mutagenicity of certain environmental contaminants could be the trapping of the carcinogenic molecules (sarkar et al., 1994). in comparison with lettuce, chicory contained about 7 times more chlorophyll a and chlorophyll b. the obtained results confirm the similarity between chicory and spinach in chlorophyll a and chlorophyll b content (duma et al., 2014). beta-carotene was found in all samples (3.7–8.2 mg/100 g), while α-carotene was below detection limit. these results were similar to the results obtained by montefusco et al. (2015). compared to spinach and chard, chicory leaves contained 10-30% and 500%, respectively, more β-carotene (perry et al., 2009; raju et al., 2007). an increased intake of β-carotene rich food in daily diet may be one of the strategies for improving vitamin a status instead of synthetic vitamin a (gopalan, 1992). obtained results for β-carotene ital. j. food sci., vol. 29, 2017 638 showed that consumption of 100 g of chicory leaves satisfied up to 135% of the referenced daily intake values (rdi) of vitamin a. 3.4. total polyphenols, total flavonoids, chlorogenic and caffeic acid the total amount of polyphenols and flavonoids in different chicory samples ranged between 0.65 and 3.73 mg gae/g fw and 1.57-4.42 µmol ce/g fw, respectively (table 5). the flavonoids content followed the content of total polyphenols in all samples. our results showed that plants grown on different locations had different tpc and tfc. table 5. content of polyphenols (tpc), flavonoids (tfc), chlorogenic and caffeic acid in wild and cultivated chicory leaves*. location tpc (mg gae/g) tfc (µm ce/g) chlorogenic acid (µg/100 g) caffeic acid (µg/100 g) wild chicory (cichorium intybus l.) zoganje 3.73±0.04a 4.42±0.01a 1034±18a 7.0±0.6a risan 1.17±0.02d 2.54±0.12e 251±9f 3.2±0.1d podgor 2.90±0.08b 3.81±0.01b 908±14b 6.6±0.1a tivat 1.45±0.02c 2.67±0.03d 437±9d 4.6±0.8bc pricelje 1.45±0.03c 3.00±0.09c 440±10d 4.4±0.6bc plavnica 1.15±0.02d 2.50±0.02e 378±10e 3.1±0.8d pljevlja 1.05±0.02e 2.29±0.04f 526±8c 3.8±0.5cd average 1.84±0.03 3.03±0.05 568±4 4.7±0.3 cultivated chicory (cichorium intybus l.) komani 0.82±0.01 2.01±0.03 104±4 2.1±0.5 susanj 0.65±0.01 1.57±0.02 104±6 1.4±0.6 average 0.74±0.01# 1.79±0.01# 104±1# 1.7±0.5# *data are expressed on the original weight basis and presented as mean±sd of three independent determinations. #statistically significant difference between wild and cultivated chicory, p<0.05. data sharing the same letter (a, b, c, d, e, f) in the same column are not significantly different, p>0.05. tpc total polyphenol content; tfc total flavonoid content; gae galic acid equivalents; ce catechin equivalent. similar range of tpc was obtained for “catalogna” landraces (c. intybus) in the study carried out by d’acunzo et al. (2017). the average value for content of total polyphenols was lower than in the work of sahan et al. (2017) and milala et al. (2009), but at the same time was significantly higher than in all chicory cultivars analyzed in the study of sinkovic et al. (2014) also, our results for tfc were higher than those obtained by montefusco et al. (2015). the most important phenolic compounds in chicory leaves are hydroxycinnamic acid derivatives, such as chlorogenic and chicoric acids (milala et al., 2009; sinkovic et al., 2015(b)). the chlorogenic acid content (sum of isomers) in our study was the highest in the sample from zoganje (1034 µg/100 g fw) and the lowest in those samples cultivated in komani and susanj (104 µg/100 g fw). overall range for caffeic acid content was 1.4-7.0 µg/100 g fw. ital. j. food sci., vol. 29, 2017 639 difference in tpc, tfc and analyzed polyphenol acids between wild and cultivated plants, as well as between wild samples from different locations, was significant (p<0.05). the wild plants were 2.5 times richer in tpc and 1.7 times in tfc than the cultivated ones. 3.5. antioxidant capacity antioxidant activities of obtained extracts from chicory leaves, their abilities to scavenge the synthetic dpph and abts radicals, as well as their power to reduce ferric (frap) ions were examined. the analysis were performed in triplicates and expressed as µm te/g of fresh weight (table 6). the frap assay is quick and simple to perform, the reaction is reproducible and the reducing power that is measured in this assay is linearly related to the molar concentration of the antioxidants (müller et al., 2010). therefore, total phenolic content represents a reliable indicator of the antioxidant activity of analyzed plant. the antioxidant activity measured with frap test and total polyphenol content was almost 3 times lower in cultivated than in wild chicory samples. table 6. antioxidant capacities of wild and cultivated chicory leaves*. location frap (µm te/ 1g) dpph (µm te/1 g) abts (µm te/ 1g) wild chicory (cichorium intybus l.) zoganje 46.90±1.08a 11.66±0.02a 32.81±0.15a risan 13.31±0.42e 6.85±0.26e 16.82±0.09c podgor 36.45±0.56b 10.29±0.03b 26.32±0.15b tivat 19.44±0.36c 7.64±0.08d 16.20±0.11d pricelje 17.43±0.50d 8.41±0.27c 15.12±0.17e plavnica 13.65±0.09e 6.90±0.29e 10.61±0.08f pljevlja 13.33±0.27e 6.19±0.22f 10.80±0.09f average 22.93±0.47 8.28±0.17 18.38±0.12 cultivated chicory (cichorium intybus l.) komani 8.80±0.52 5.05±0.27 11.07±0.07 susanj 6.75±0.13 3.76±0.17 10.46±0.09 average 7.77±0.33# 4.40±0.22# 10.77±0.08 *data are expressed on the original weight basis and presented as mean±sd of three independent determinations. #statistically significant difference between wild and cultivated chicory, p<0.05. data sharing the same letter (a, b, c, d, e, f) in the same column are not significantly different, p>0.05. dpph 2,2-diphenyl-1-picrylhydrazyl; frap ferric ion reducing antioxidant power; abts (teac) trolox equivalent antioxidant capacity; te trolox equivalent. for evaluation of free radical scavenging properties of the extracts, we used two assays: the dpph radical and the abts radical cation assay. the relatively stable organic radical dpph has been widely used in the determination of antioxidant activity of different plant extracts (costa et al., 2009). the free radical scavenging ability varied between 3.76 µm te/g in cultivated sample from susanj and 11.66 µm te/g in wild sample from zoganje, using the dpph antioxidant method. ital. j. food sci., vol. 29, 2017 640 in accordance with the obtained data for the abts radical cation, it is interesting to note that the reduced antioxidant activity of cultivated samples is in agreement with the lower total flavonoid content of the same samples compared to the wild ones (40% lower on average). the reason for this pattern could be that flavonoids are the phenol class that specifically reflect and are better indicators of the segment of antioxidant activity obtained in abts test. obtained results for all three assays have shown that the antioxidant capacity of analyzed extracts, as presumed, followed the pattern of tp and tf contents. frap and dpph assays showed significantly different results for wild and cultivated plants (p<0.05), as well as for wild plants sampled from different locations. correlations among results obtained with all three antioxidant assays were positively high as well as correlation between antioxidant activity with tp and tf contents (r~0.99, p<0.05). when compared with data of sahan et al. (2017), the obtained results for abts and dpph assay were lower. the overall antioxidant activity of analyzed samples is expressed as antioxidant composite index (aci). aci value is followed by one statistical-mathematical model, which is a modern ranking tool for the representation of antioxidant activity of various plants. one basic advantage of using aci is the value being expressed in percent, which covers all segments of antioxidant action obtained in different antioxidant assays. another advantage of using aci index instead of three assays is because it simplifies the process of comparison between antioxidant properties of different foods. the antioxidant composite index (aci) of chicory samples (table 7) was in the following decreasing order: zoganje > podgor > tivat > pricelje > risan > plavnica > pljevlja > komani > susanj. all samples of wild plants had higher aci than the cultivated plants. table 7. antioxidant composite index (aci) of wild and cultivated chicory leaves. location frap index dpph index abts index aci (%) wild chicory (cichorium intybus l.) zoganje 100.0 100.0 100.0 100.0 risan 28.4 58.7 51.3 46.1 podgor 77.7 88.2 80.2 82.1 tivat 41.4 65.5 49.2 52.1 pricelje 37.2 72.1 46.1 51.8 plavnica 29.1 59.2 32.3 40.2 pljevlja 28.4 53.1 32.9 38.1 cultivated chicory (cichorium intybus l.) komani 18.8 43.3 33.7 31.9 susanj 14.4 32.2 31.9 26.2 frap ferric ion reducing antioxidant power; dpph 2,2-diphenyl-1-picrylhydrazyl; abts (teac) trolox equivalent antioxidant capacity; aci antioxidant composite index. 4. conclusions this study is the first comprehensive study offering detailed information on fibers, fatty acids, pigments, phenolics, flavonoids, and antioxidant activity of chicory (cichorium intybus l.) leaves grown in montenegro. chicory leaves are rich sources of fiber, polyphenols, flavonoids, and almost all analyzed pigments. the lipid content in chicory ital. j. food sci., vol. 29, 2017 641 leaves is quite low, but they have high nutritional 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medicines. j. agric. food chem. 53:6624. znidarcic d., ban d. and sircelj h. 2011. carotenoid and chlorophyll composition of commonly consumed leafy vegetables in mediterranean countries. food chem. 129:1164. paper received march 2, 2017 accepted june 25, 2017 ijfs#743_bozza ital. j. food sci., vol. 29, 2017 697 paper yoghurt drink for lactose and galactose intolerant patients i̇.e. tonguç and c. karagözlü* ege university faculty of agriculture dairy technology department, bornova, izmir, turkey *corresponding author. tel.: +90 2323112902; fax: +90 2323881864 e-mail address: cem.karagozlu@ege.edu.tr abstract in this study, yoghurt drink with lactose free and low galactose content for patients with galactose intolerance were produced using a 1:1 mixture of lactose free milk and two different types of infant formula, fortified with strawberry flavor. the results indicated that galactose content of yoghurt drinks produced from lactose free raw materials were declined to a level that is suitable for the diets of patients with galactosemia. chemical, microbiological and sensory properties of these products were found to match the common quality characteristics of a commercial fermented dairy product. keywords: yoghurt drink, food allergies, galactosemia, lactose intolerance ital. j. food sci., vol. 29, 2017 698 1. introduction classic galactosemia is an autosomal recessive disorder of carbohydrate metabolism (omim 230400), due to a severe deficiency of the enzyme, galactose-1-phosphate uridyltransferase (galt, ec 2.7.7.12), which catalyzes the conversion of galactose-1phosphate and uridine diphosphate glucose (udpglucose) to uridine diphosphate galactose (udpgalactose) and glucose-1-phosphate. upon consumption of lactose in the neonatal period, the affected infants develop a potentially lethal disease process with multiorgan involvement. however, since the advent of newborn screening (nbs) for galactosemia, we rarely encounter such overwhelmingly ill newborns. the following case report illustrates the acute neonatal toxicity that may be seen in infants with severe galt deficiency, marked elevation of galactose-1-phosphate in target tissues and severe hypergalactosemia due to lactose ingestion (riedel et al., 2005; berry, 2012). its estimated incidence is 1/40,000 60,000 live births. this form is called the classical galactosemia. patients with galt deficiency appear normal at birth but soon develop severe hepatic, renal and gastro-intestinal manifestations that, if not treated, mostly lead to death. removal of dietary lactose and galactose is essential as this will prevent or decrease the severity of the initial metabolic crisis in the neonate (kerckhove et al., 2015). individuals with galactosemia are intolerant of dietary lactose and galactose, primarily found in milk and milk products. if untreated, the disorder can cause liver failure, kidney dysfunction, sepsis, and death. if it is diagnosed soon after birth and treated by removal of lactose and galactose from the diet, the symptoms will resolve and many of the long-term complications, including cataracts and mental retardation, can be prevented (grange, 2004). individuals suffering from galactosemia cannot consume products containing galactose, which means that they cannot consume dairy products, which have a critical role in healthy growth and nutrition. a strategy, similar to the introduction of the gluten free products to the diet of individuals with celiac disease, may be adopted for galactosemia suffering individuals by developing galactose free dairy products. previous studies have reported the following galactose consumption limit values that were verified by doctors and dieticians based on many years of experience: babies 50 (–200 mg), infants 150 (–200 mg), schoolchildren 200 (–300 mg), youth 250 (–400 mg), adults 300 (-500 mg) galactose/day (varga et al., 2006, schweitzer et al., 1998). depending on these limit values, the galactose content of the yoghurt-like product developed by szigeti and krász (1992) was found to be higher than the required for children suffering from galactosemia, where varga et al., (2006) found the galactose levels of kefir-like products suitable for galactosemia patients from all ages. yoghurt is a fermented dairy product commonly produced in the world, produced by the fermentation of milk by lb. bulgaricus and s. thermophilus bacteria. yoghurt drink also carries the health and nutritional value of yoghurt and it is produced by the addition of water to yoghurt. it is called in different ways in different regions such as ayran in turkey, dough in iran, tan in armenia, laban in syria and lebanon. as well its many health benefits, yoghurt drink has positive effects on health including the regulation of lactose intolerance and supporting the immune system (erkaya et al., 2015). the aim of this study was to develop yoghurt drink for individuals with galactosemia and/or lactose intolerance by full hydrolization of lactose content and lowering the galactose levels suitable for the safe consumption of these products by galactosemic individuals. accordingly, yoghurt drink with galactose levels lower than 200 mg/100 c m3 for galactosemic patients from all ages by using by using a 1:1 mixture of lactose free milk and two different types of infant formula, fortified with strawberry flavor. ital. j. food sci., vol. 29, 2017 699 2. materials and methods 2.1. milk samples and fermented dairy production, experimental design uht cow’s milk and lactose hydrolyzed uht cow’s milk used in the studies were obtained from pinar sut co. (izmir, turkey). in order to lower the galactose content before the fermentation, lactose hydrolyzed uht cow’s milk was mixed with galactose free infant formulas. the ratios in the mixtures were one part of lactose hydrolyzed milk and one part of galactose free infant formula (1:1). two different galactose free infant formulas were used as supplements of lactose hydrolyzed milk: neocate, a maltose based, galactose free infant formula (milupa/numico, netherlands) and galactomin 19, fructose based, galactose free complete infant formula (shs, uk). the sensory properties of the two formulas were different, possibly influencing the sensory properties of both raw material mixtures and fermented products. uht cow's milk as the control group, lactose hydrolyzed milk and the two types of mixtures were inoculated with yoghurt drink cultures respectively (table 1). table 1. raw material properties of fermented dairy drinks for the individuals with galactosemia. c: conventional uht milk, l: lactose-free uht milk, ln: lactose-free uht milk + neocate, lg19: lactose-free uht milk + galactomin 19. commercial freeze-dried yoghurt drink starter culture lb340, containing lactobacillus delbrueckii spp. bulgaricus and streptococcus thermophilus, were obtained from ezal (texel, france). the strawberry sauce and flavor used for enhancing the sensory properties of the products were obtained from aromsa co. (kocaeli, turkey). skim milk powder used for the preparation of starter cultures were obtained from pinar sut co. (pinarbasi, izmir). 500 ml of reconstituted skim milk with 12 % non-fat dry matter were inoculated with freeze-dried yoghurt drink (2 %, in 42° c) cultures. the inoculations ended when the ph levels of the inocula dropped to 4.6. raw materials prepared for the production of fermented drinks were inoculated with 3.25 % culture in all cases. incubation parameters for the products were, 3 hours in 42° c. fermentation were run in duplicate and repeated twice in bottles containing 500 ml of raw materials and 3.25 % inoculum. in order to enhance the sensory properties of products, fermented drinks were fortified with galactose free strawberry sauce (1.8 %) and strawberry aroma (0.1 %). manufacture of the products were run in duplicate and repeated twice in all cases. 4 different raw materials were coded as follows; cay: control yoghurt drink, lay: lactose free milk yoghurt drink, lnay: lactose free milk + neocate yoghurt drink, lg19ay: lactose free milk + galactomin 19 yoghurt drink. raw material dry matter (%) fat (%) protein (%) ph lactose (mg/100cm3) galactose (mg/100cm3) c 10.31±0.50 1.50±0.06 3.10±0.00 6.7±0.03 4208.35±23.35 0.00±0.00 l 10.19±0.18 1.45±0.05 3.10±0.04 6.6±0.11 0.00±0.00 2160.40±34.21 ln 10.54±0.24 2.50±0.07 2.97±0.02 6.4±0.14 0.00±0.00 1068.11±12.30 lg19 10.42±0.08 2.80±0.01 2.98±0.06 6.5±0.08 0.00±0.00 1080.07±14.10 ital. j. food sci., vol. 29, 2017 700 2.2. chemical, microbiological and sensory analyses the ph was determined using a ph meter (hanna ph 211 microprocessor, portugal). dry matter, protein and fat contents were determined according to a.o.a.c (2006). tyrosine levels were measured according to hull (1947). acetaldehyde contents of the samples were determined using spectrophotometric method according to robinson et al., (1977). for the determination of lactose and galactose levels, megazyme k-lacgar 12/05 enzymatic kit obtained from megazyme international ireland limited (co.wicklow, ireland) was used. bacterial enumerations were carried out at the 1st, 10th, 20th, 30th days of the storage period. samples (1 ml) were diluted with ringer solution (9 ml). serial dilutions were carried out, and bacteria were counted, applying the pour plate method. l. bulgaricus counts in yoghurt drink samples were enumerated in mrs agar (ph 5.8) (merck / 1.10660, darmstadt, germany) anaerobically at 42°c for 48 h, whereas s. thermophiles in yoghurt drink samples were counted in m17 agar (ph 6.9) aerobically at 37°c for 48 h (bracquart, 1981). samples were evaluated for their sensory properties (taste-aroma, consistency, overall). the evaluation cards were prepared according to bodyfelt et al., (1998). the evaluation was performed by the academicians from the dairy technology department of ege university. 2.3. statistical analyses the experiments were performed in two repetitons with three parallels. the mean value of the six values for each sample was calculated (n=6). the obtained data was statistically analyzed by one-way anova using the general linear model. the constant effects (different production process and storage period and the effects of the interactions between these effects were analyzed by analysis of variance (anova) using spss© v.15.00 (spss inc., chicago, illınois usa). the significant data as a result of anova were tested according to the duncan multiple comparison test at p <0.05 level. 3. results and discussions 3.1. chemical properties in terms of obtaining the desired structural and sensory properties of yoghurt drink dry matter content and its properties are the important basic parameters. many studies have reported that dry matter contents had a direct effect on the structural, microbiological and sensory properties of the products. in the production of fermented dairy products, it is required to comply with the legally prescribed minimum dry matter levels. dry matter, fat and protein contents of yoghurt drink samples were analyzed on the 1st day of the storage (table 2). the results showed that dry matter, fat and protein contents of all yoghurt drink and kefir samples were in accordance with the nutrient contents specified in fermented dairy products communiqué (communiqué no: 2009/25) in turkish food codex (2009). in accordance with the aim of our study, lactose-free milk and milk-formula mixtures were used in productions. additionally, no lactose hydrolization process was done in control group that is conventional semi-skimmed uht drinking milk. therefore, lactose was not detected in the study, except the control samples coded as cay (table 2). ital. j. food sci., vol. 29, 2017 701 table 2. the results for the compositional analysis of yoghurt drinks for the individuals with galactosemia. dry matter (%) fat (%) protein (%) acetaldehyde (ppm) lactose (mg / 100 cm3) galactose (mg / 100 cm3) cay 10.94±0.08b 1.50±0.00a 2.71±0.19b 6,97±0,01c 2129.20±23.81 120.67±.072b lay 10.82±0.47b 1.55±0.00b 3.01±0.36c 7,04±0,01c ≤0.01±0.00 225.51±2.78d lnay 11.05±0.50c 2.56±0.00c 2.69±0.10a 6,00 ±0,01a ≤0.01±0.00 153.79±2.05c lg19ay 10.62±0.21a 2.76±0.02d 2.66±0.84a 6,30±0,01b ≤0.01±0.00 63.70±6.39a a, b, c, d: values with different lower-case letters in the same column differ significantly (p < 0.05). examining the galactose levels of other three lactose free samples, it was found that galactose level in las sample was higher than the level reported by varga et al., (2006), with 212.46 mg/100 cm3. different raw material contents had a significant effect on the galactose contents in all kefir samples (p<0,05). it was found that lkf, lnkf and lg19kf samples contained lower galactose than the threshold reported by varga et al., (2006), with 161.95, 132.74 and 106.54 mg/100 cm3, respectively. varga et al., (2006), in their study, determined the galactose level of kefir sample pre-determined as the control sample produced from lactose free milk as 270 mg/100 cm3, the galactose level of kefir produced from milk-formula mixture containing pregomin as 169 mg/100 cm3, and the galactose level of kefir produced from milk-formula mixture containing nutrilon as 171.5 mg/100 cm3. in their study, the researchers determined the milk-formula ratio as 2 parts milk and 1 part formula (2:1). comparing those results with our study, the galactose levels obtained in our study appear to be lower than those by varga et al., (2006). the most likely reason for this difference is the 1:1 milk-formula ratio we adopted for our method. in all samples ph decreased during storage (fig. 1). figure 1. ph values of yoghurt drinks for the individuals with galactosemia. the differences between the ph values in acidophilus milk samples at the 10th, 20th and 30th day of the storage were found to be statistically not significant (p>0.05). in yoghurt ital. j. food sci., vol. 29, 2017 702 drink samples, different raw material content had no effect on the ph values at the 1st and the 20th days of the storage (p<0.05). ph values of yoghurt drink samples in our study were similar to those in similar studies by ozer et al., (2005), martini et al., (1991), tonguc et al., (2013), yerlikaya et al., (2013) and erkaya et al., (2015). in fermented dairy products, as a result of the hydrolization of lactose by culture bacteria and the formation of lactic acid during incubation, ph reaches to a certain level and coagulates and maintains the gel formation. during ripening and storage, acidity increases and the decrease in ph value continues. the type of the bacteria used in the incubation is largely responsible for the speed of the decrease in ph. it was reported that the amount of acetaldehyde required for the formation of characteristic aroma in fermented dairy products varied between 13 and 48 ppm (sahan et al., 2008; cheng, 2010). different raw material compositions had a statistically significant effect on the acetaldehyde contents in all samples (p<0.05). this result was supported by the panelists' comments in taste-aroma evaluations in sensory analyses reporting that they perceived acetaldehyde aroma in products. tyrosine values of yoghurt drink samples and the changes in these values during storage are given in fig. 2. the differences between tyrosine values of yoghurt drink samples were statistically significant (p<0.05). also, storage had a statistically significant effect on the tyrosine values of the samples (p<0.05). tyrosine levels obtained in our study was compatible with those reported in studies on various fermented dairy products (ozer et al., 2005, yerlikaya et al., 2013). however, tyrosine levels of sample ln were higher than those values. in this perspective, tyrosine values of all the samples and the scores obtained in sensory evaluations were substantially parallel. high tyrosine values determined in sample lnay supports the claim by de mann (2013) that bitter taste forms as tyrosine content increases. sample lnay received the lowest taste-flavor scores in sensory evaluations throughout the whole storage. panelists reported “bitter tasteflavor formations” in the sensory evaluation sheets many times throughout the sensory evaluation process. figure 2. tyrosine values of yoghurt drinks. ital. j. food sci., vol. 29, 2017 703 3.2. microbiological properties examining the microbiological properties of yoghurt drink samples, it was found that different raw material contents had a significant effect on the l. bulgaricus counts in yoghurt drink samples at the 10th and 20th day of the storage (table 3). the effect of storage period on the l. bulgaricus counts in all yoghurt drink samples was significant (p<0.05). using different raw materials had a significant effect on the s. thermophilus counts of yoghurt drink samples (p<0.05). in addition, the effect of storage on s. thermophilus counts of yoghurt drink samples was statistically significant (p<0.05). s. thermophilus counts of yoghurt drink samples were above log 7 cfu/ml and the lowest value was determined in lay with log 6.97 cfu/ml on the 10th day of the storage (table 4). lb. bulgaricus and s. thermophilus counts in lnay and lg19ay samples maintained their viability during 30-day storage period. lb. bulgaricus and s. thermophilus counts in our samples were higher than the results found in previous studies by akalin and unal (2010) and were similar to those obtained by varga et al., (2003), yerlikaya et al., (2012), tonguc et al., (2013) and erkaya et al., (2015). table 3. microbiological contents of yoghurt drinks for the individuals with galactosemia. helv storage (day) 1st 10th 20th 30th lb. bulgaricus (log cfu/ml) cay 8.07±0.26yb 8.12±0.10yb 7.42±0.04xa 7.57±0.08x lay 7.85±0.34xyab 8.02±0.01yb 7.58±0.07xyb 7.40±0.01x lnay 7.50±0.07xa 8.03± 0.06wb 7.61±0.01xyb 7.71±0.02y lg19ay 7.46 ±0.03xya 7.79±0.01ya 7.32±0.07xa 7.54±0.25xy s. thermophilus (log cfu/ml) cay 7.37±0.04ya 7.12±0.10xb 7.63±0.03wb 7.16±0.01xa lay 7.48±0.01wab 6.97±0.04xa 7.85±0.04zb 7.22±0.02ya lnay 7.41±0.00yab 7.30±0.01xc 7.39±0.00ya 7.53±0.03wb lg19ay 7.54±0.08yb 7.27±0.02xbc 7.66±0.07yb 7.66±0.08yb a, b, c: values with different lower-case letters in the same column differ significantly (p<0.05). x, y, w, z: values with different capital letters in the same row for each analysis differ significantly (p <0.05). 3.3. sensory properties in yoghurt drink samples, using different raw material formulations had a significant effect on the taste-aroma properties on the 1st, 10th and the 20th day of the storage (p<0,05). lnay received considerably lower points compared to the other samples (table 4). the difference between taste-aroma scores of the samples was found to be statistically not significant on the 30th day of the storage (p>0.05). also, it was found that storage had no significant effect on the taste-aroma properties of the samples (p>0.05). the panelists reported that taste-aroma characteristics of yoghurt drink samples, including lnay sample, which received the lowest points, were very stable throughout the storage, and no negative developments occurred such as increase in acidity or souring. as a result of anova and duncan tests, it was found that different raw material composition of the yoghurt drink samples had a significant effect on their consistency properties (p<0.05). ital. j. food sci., vol. 29, 2017 704 among the yoghurt drink samples, lg19ay received the highest consistency points and much better and more tasteful product compared to the control sample. the difference between general scores of the yoghurt drink samples was found to be statistically not significant at the 30th day of the storage (p>0.05). cay, lay and lg19ay samples had no significant differences in terms of general sensory analysis scores at the 1st, 10th and 20th days of the storage (p>0.05), and lnay was statistically different than the other three yoghurt drink samples during the storage (p<0.05). table 4. sensory evaluation of yoghurt drinks for the individuals with galactosemia. storage (day) 1st 10th 20th 30th tastearoma cay 6.87±0.17b 6.75±1.06ab 6.80±0.28ab 7.40±0.84b lay 6.65±0.21b 7.55±0.07b 6.65±0.49ab 7.01±0.55b lnay 4.10±0.14a 5.15±0.49a 4.57±1.80a 5.80±0.28a lg19ay 7.10±0.14b 8.00±0.00b 7.51±0.12b 7.30±0.98b consistency cay 7.02±1.10ab 7.50±0.70ab 7.37±0.32b 7.50±0.14b lay 7.35±0.77b 7.95±0.77b 7.64±0.50b 7.60±0.00b lnay 4.72±0.67a 6.10±0.14a 5.22±0.88a 5.47±0.38a lg19ay 7.87±0.17b 8.35±0.21b 8.00±0.56b 8.10±0.14b general cay 6.75±0.35b 7.05±0.63b 6.80±0.28b 7.50±0.70b lay 6.87±0.17b 7.45±0.07b 6.94±0.48b 7.01±0.55b lnay 4.52±0.38a 5.60±0.56a 4.71±1.00a 5.88±0.68a lg19ay 7.20±0.28b 8.25±0.35b 7.80±0.28b 7.76±0.90b a, b, c: values with different lower-case letters in the same column differ significantly (p < 0.05). 4. conclusions consumption of dairy products by patients with galactosemia in their daily diet leads to consequences physiologically far more different and serious than those in lactose intolerance cases. therefore, galactosemia patients have to eliminate dairy products from their daily diet completely in order not to experience these serious adverse effects and physiological damages. in our study, it was found that galactose levels in yoghurt drink produced from lactose free milk and infant formula mixtures for patients with galactosemia were lower than the galactose threshold values reported in the referred studies (varga et al., 2006) yoghurt drink samples exhibited good acidity development, microbiological content, and the stability of these contents. in our study, lg19ay sample was possibly the most successful sample among the yoghurt drinks fulfilling the aim and purpose of our study. strawberry flavor fortification, comparing with the previous studies, improved the sensory properties and positive results were obtained in the sensory analysis. however, it is necessary to confirm these results with further studies and subsequently these products can be presented to the consumption of the patients. ital. j. food sci., vol. 29, 2017 705 acknowledgements the authors thank the ege university scientific research fund (project no: 2009-zrf-018) council for financial support to this study. the authors also would like to thank pinar sut inc. and aromsa inc. for their lactose free milk and flavor procurements. references akalin a. s. and unal g. 2010. the influence of milk supplementation on the microbiological stability and textural characteristics of fermented milk. milchwissenschaft 65: 291294. aoac. 2006. official methods of analysis of aoac international, 18th ed. association of official analytical chemists, arlington, virginia. usa. berry, g. t. 2012. galactosemia: when is it a newborn screening emergency? molecular genetics and metabolism 106: 711). bodyfelt f.w., tıbias j. and trout g. 1988. the sensory evaluation of dairy products, van nostrand reinhold, new york, usa. bracquart p. 1981. an agar medium for the differential enumeration of streptococcus thermophilus and lactobacillus bulgaricus, journal of applied bacteriology 51:303–305. cheng h.f. 2010. volatile flavor compounds in yogurt: a review. critical review food science. nutrition, 50: 938-950. de mann j.m. 2013. principles of food chemistry. springer inc. isbn 978-1-4614-6390-0. new york, usa. erkaya t., başlar m., şengül m. and ertugay m.f. 2015. effect of thermosonication on physicochemical, microbiological and sensorial characteristics of ayran during storage, ultrasonics sonochemistry 23:406-412. grange d.k. 2004. galactosemia in encyclopedia of gastroenterology, edited by leonard r. johnson, university of tennessee college of medicine, memphis, tennessee, usa. hull m.e. 1947, studies on milk proteins, ii. colorimetric determination of the partial hydrolysis of the proteins in milk, journal of dairy science, 30:881 kerckhove k.v., diels m., vanhaesebrouck s., luyten k., pyck n., de meyer a., van driessche m., robert m., corthouts k., caris a., duchateau e., dassy m. and bihet g. 2015. consensus on the guidelines for the dietary 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storage. food hydrocolloids 22: 1291-1297. schweitzer s., pryzembel h., ullrich k. and wendel u. 1998. empfehlung der arbeitsgemeinschaft für pädiatrischestoffwechselstörungen (aps) zurbehandlung der galaktosämie (recommendations of team of metabolic disease in childhood for treatment of galactosemia). in: thauer e. and bake g. (eds.) galaktosämie. jubileumsausgabe elterninitiative galaktosämie e. v. (proceedings of parent initiative registered association on galactosemia), düsseldorf, 21-24. szigeti j. and krász á. 1992. dietetikusigényeketkielégít savanyútejkészítményekel állítása” (production of fermented dairy products for diet). tejipar 42:25-29. ital. j. food sci., vol. 29, 2017 706 tonguç i.e., kinik ö. kesenkaş h.and acu m. 2013. physicochemical, microbiological and sensory characteristics of using different probiotic fermented milk. pakistan journal of nutrition 12:549-554. turkish food codex. 2009. fermented dairy products communiqué (communiqué no: 2009/25) ankara, turkey. varga l., szieti j. and csengeri é. 2003. effect of oligofructose on the microflora of an abt-type fermented milk during refrigerated storage. milchwissenschaft 58: 55-58. varga z., palvolgyi m., juhasz-roman m. and toth-markus m. 2006. development of therapeutic kefir-like products with low galactose content for patients with galactose intolerance. acta alimentaria 35:295-304. yerlikaya o., akpınar a., torunoglu a., kınık o., akbulut n. and uysal, h. 2012. effect of some prebiotic combination on viability of probiotic bacteria in reconstituted whey and milk beverages. agrofood industry hi-tech, monographic supplement series: dietary fibers and pre/probiotics 23:27-29. yerlikaya o., ender g., torunoglu, a. and akbulut, n. 2013. production of probiotic milk drink containing lactobacillus acidophilus, bifidobacterium animalis ssp. lactis and lactobacillus casei. agro food industry hi-tech 22:49-52. paper received january 9, 2017 accepted july 10, 2017 p u b l i c a t i o n s codon issn 1120-1770 online, doi 10.15586/ijfs.v33i2.2009 63 functionality that could be used as ingredients for food, food supplements or active bioactive compounds in pharmaceutical products (rombaut et al., 2014). in food industry, citrus by-products and their value-added compounds, including polyphenols, vitamins, microelements and fiber are utilized as natural additives with the following properties: antimicrobials, antioxidants, colorants and flavoring agents (mahato et al., 2019). these wastes have gained increasing interest for further exploitation on the production of food additives, supplements with high nutritional value and pharmaceutical products. the recent data were collected from several scientific databases (pubmed, science direct, scifinder, scopus, elsevier, springerlink, reserchgate and google scholar) from 2015 to 2020 using the following keywords: citrus, citrus by-products, juice, seed, peels, leaves, and p u b l i c a t i o n s codon citrus species: modern functional food and nutraceutical-based product ingredient mariarosaria leporini1, rosa tundis1, vincenzo sicari2, monica rosa loizzo1* 1department of pharmacy, health and nutritional sciences, university of calabria, 87036 rende (cs), italy; 2department of agraria, “mediterranea” university of reggio calabria, cittadella universitaria, località feo di vito 89124 reggio calabria (rc), italy *corresponding author: monica rosa loizzo, department of pharmacy, health and nutrition science, university of calabria, via p. bucci, edificio polifunzionale, 87036 rende (cs), italy. email: monica_rosa.loizzo@unical.it received: 25 january 2021; accepted: 22 april 2021; published: 27 may 2021 © 2021 codon publications open access review abstract citrus is the most cultivated fruit crop in the world and occupies a place of considerable importance in the country’s economy. almost 33% of the citrus fruits are processed for juice production; however, a great amount of wastes, including peels, segment membranes, and seeds are also produced. indeed, citrus fruits consist of 45% juice, 26% pulp, 27% peels, and 2% seeds. pruning, a cultural practice involving the removal of tree branches and limbs, was applied to improve fruit’s quality. a large amount of leaves are produced through pruning. these agrifood matrices contain a wide range of bioactive phytochemicals compared to fruits. the present review covers the past 5 years of research carried out in chemistry, health properties, and applications in food and nutraceutical industries of all portions of citrus fruit and its major bioactive compounds. additionally, patents are also included. keywords: bioactive compounds, by-products, citrus, health properties, juice, peels, pulp, seeds introduction citrus is the most cultivated fruit tree in the world and occupies a place of considerable importance in country’s economy. the citrus fruits are processed for juice production (45%), and a great amount of waste, including peels (27%), pulp (26%) and seeds (2%), is produced (mahato et al., 2018). food waste is defined as the by-product obtained from various industrial, agricultural and other activities of food sector. especially, food-processing industries produce large quantities of by-products, which are difficult to dispose of as they have a high demand for biological oxygen. indeed, waste disposal has high costs and, also a potential negative impact on the environment (kumar et al., 2017). these agri-food matrices contain a wide range of bioactive phytochemicals with different structures and italian journal of food science, 2021; 33 (2): 63–107 mailto:monica_rosa.loizzo@unical.it 64 italian journal of food science, 2021; 33 (2) m. leporini et al. the antioxidant ability of fresh squeezed citrus limon l. burm. cv. femminello comune juice was analyzed by loizzo et al. (2019), founding the ic50 values of 40.3 and 46.5 g/ml in dpph and abts test, respectively, and 49.7 mg fe (ii)/g in the ferric-reducing ability power (frap) test. more recently, the antioxidant ability of two citrus sinensis cultivars (sanguinelli and salustiana) was demonstrated by applying abts, dpph, and orac assays (ordóñez-díaz et al., 2020). citrus sinensis cv. sanguinelli extracts were 40% more active than citrus sinensis salustiana samples. recently, ali et al. (2020) reported that animals treated with 0.75% hydrogen peroxide in drinking water with daily drenching with 1 ml lemon juice, exhibited enhancement in hemoglobin concentration, red blood cells count, white blood cells count, and total proteins, and reduction in the level of aspartate aminotransferase and alanine aminotransferase. these findings clearly confirmed the protective and antioxidant features of lemon juice on hematological and biochemical parameters of the oxidatively stressed female mice. metabolic syndrome metabolic syndrome (ms) is a clustering characterized by abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides (tg), low serum, and high-density lipoprotein (hdl) that directly increase the risk of cardiovascular disease, type 2 diabetes mellitus (t2dm), and all-cause mortality (kaur et al., 2014). recently, the beneficial effects of citrus bergamia juice were evaluated using an experimental animal model of ms and cardiovascular risk (de leo et al., 2020). results demonstrated that daily oral treatment reduced tg levels, cardiovascular risk, and showed protective effects on hepatic steatosis, probably due to the reduction of oxidative stress and inflammation. previously, impellizzeri et al. (2015) tested the in vivo anti-inflammatory activity of bergamot juice extract. mice treated with this extract were more resistant to induction of colitis and reduction in the expression of important inflammatory mediators, tumor necrosis factor-alpha (tnf-α) and interleukin-1 β (il-1β), was observed. the effects of a bergamot phytocomplex (patent no. ep3116520a1) was investigated by di folco et al. (2018). each tablet provided 200-mg bergamot juice dry extract, 120-mg phytosterols, 80-mg artichoke leaf extract, and 20-mg vitamin citrus. after 6 months of administration, patients in the intervention group showed a significant reduction in fasting blood glucose compared to the simple dietary intervention alone. essential oils. this work is structured by dividing the text in citrus portion and discussions on the phytochemical profile and biological activities of each. additionally, patents are also included. the information collected would be useful for research on citrus species and for food and nutraceutical industries interested in using ingredients with health potential. juice antioxidant activity reactive oxygen species (ros) and reactive nitrogen species (rns) are involved in the pathogenesis of many human diseases, and antioxidants play a crucial role in restoring the physiological oxidative balance and modulating biological pathways and membrane functions (smeriglio et al., 2018). the citrus genus is recognized for its protective effects against free radical-induced damage. barberis et al. (2020) analyzed the antioxidant potential of fresh squeezed pompia, lemon (cv. lisbon) and orange juices (cv. hamlin, sanguinello, and moro). among these, pompia juice had a marked effect against ros, and a moderate capacity to reduce ros damages on cell membrane. on the contrary, orange juices resulted much less effective. the in vitro antioxidant potential (table 1) of fresh squeezed citrus × clementina juices, collected from different areas of calabria, was recently evaluated (leporini et al., 2020a). results revealed that juice obtained from fruits collected in corigliano calabro exhibited the highest radical activity, with the concentration giving 50% inhibition (ic50) values of 81.13 and 27.82 mg/ml for 2,2-diphenyl-1-picrylhydrazyl (dpph) and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (abts) tests, respectively. the same juice exhibited the highest protection of lipid peroxidation. these results are similar to the previous study conducted by loizzo et al. (2018) that reported the antioxidant activity of fresh squeezed citrus × clementina juices from fruits collected in flood plains, hills, and coastal plains of sibari (calabria, italy). the following trend of radical potency was found: flood plain > coastal plain > hill. haraoui et al. (2020) compared the antioxidant activity of fresh squeezed juices derived from different citrus fruit varieties. all investigated samples possessed radical scavenging activity with ic50 values comparable to positive control ascorbic acid and butylated hydroxytoluene (bht). among them, citrus maxima and citrus aurantium juices showed the highest dpph radical scavenging activity (ic50 = 0.42 and 0.44 mg/ml, respectively). the same trend was observed in β-carotene bleaching test with percentage exceeding to 80.55%, followed by citrus sinensis cv. sanguinelli and citrus limon. italian journal of food science, 2021; 33 (2) 65 citrus species: modern functional food and nutraceutical-based product ingredient table 1. biological effects of citrus juices. juice biological activity mechanism tpc, tfc, tcc, and/or main abundant identified compounds references in vitro citrus aurantifolia antioxidant radical scavenging ferric-reducing power tpc = 52 mg gae/l; tfc = 29.5 mg qe/l oboh et al., 2015b antibacterial s. aureus, s. epidermis, m. luteus, e. faecalis, b. subtillis, p. aeruginosa, k. pneumonia s. thypii, and c. diphtheriae inhibition nr nr nr abdallah, 2020 fadillah et al., 2020 azhara et al., 2020 citrus aurantium antioxidant radical scavenging tpc = 295.37 mg gae/g; fc = 26.08 mg qe/g tpc = 0.58 mg gae /ml; tfc = 0.43 mg re/ml; neohesperidin = 144.85 mg/ml; naringin = 79.19 mg/ml; hesperidin = 4.68 mg/ml. haraoui et al., 2020 chen et al., 2020 antibacterial s. aureus inhibition haraoui et al. 2020 citrus grandis antioxidant radical scavenging tpc = 0.49–1.27 mg gae /ml; tfc = 0.35–1.17 mg re/ml; naringin = 40.82–419.28 mg/ml; neohesperidin = 37.52–42.54 mg/ml; hesperidin = 3.14–12.17 mg/ml; diosmin = 14.79–21.38 mg/ml; tangeretin = 3.09–3.64mg/ml. chen et al., 2020 citrus hystrix antibacterial s. aureus inhibition nr kusumawardhani et al., 2020 citrus limon antioxidant radical scavenging tpc = 151.7 mg gae/l; tfc = 30.8 mg qe/l; eriocitrin = 16.7 mg/100 ml; hesperidin = 14.1 mg/100 ml. loizzo et al., 2019 metabolic syndrome inhibition of α-amylase and β-glucosidase enzymes loizzo et al., 2019 antibacterial s. aureus, s. epidermis, m. luteus, e. faecalis and b. subtillis inhibition. tpc = 231.16 mg gae/g; tfc = 25.04 mg qe/g. haraoui et al. 2020 neuroprotective ache and bche inhibition diosmetin 6,8-di-c-glucoside = 5.35 mg/100 ml; hesperetin 7-o-rutinoside = 3.11 mg/100 ml. gironés-vilaplana et al., 2015 citrus maxima antioxidant radical scavenging tpc = 350.05 mg gae/g; tfc = 55.38 mg qe/g. haraoui et al., 2020 antibacterial s. aureus, s. epidermis, m. luteus, e. faecalis and b. subtillis inhibition. haraoui et al. 2020 citrus medica antioxidant radical scavenging tpc = 0.30–1.37 mg gae /ml; tfc = 0.19–0.68 mg re/ml; hesperidin = 42.13 mg/ml; eriocitrin = 28.46 mg/ml; narirutin = 28.46 mg/ml; didymin = 12.50 mg/ml; tangeretin = 3.76 mgml. chen et al., 2020 citrus paradisi antioxidant radical scavenging tpc = 153.08 mg/ml; tfc = 390.21 mg/ml; naringin = 287.15 mg/ml; narirutin = 37.07 mg/ml; naringenin = 31.25 mg/ml; poncirin = 17.32 mg/ml; neohesperidin = 13.48 mg/ml. sicari et al., 2018 (continues) 66 italian journal of food science, 2021; 33 (2) m. leporini et al. table 1. continued juice biological activity mechanism tpc, tfc, tcc, and/or main abundant identified compounds references citrus reticulata antioxidant radical scavenging tpc = 0.30–1.37 mg gae /ml; tfc = 0.19–0.68 mg re/ml; hesperidin = 50.53–141.85 mg/ml; naringin = 26.57–78.39 mg/ml; neohesperidin = 69.19 mg/ml; didymin = 1.73–10.33 mgml; eriocitrin = 27.17–78.39 mg/ml; tangeretin = 3.43–4.32 mg/ml. chen et al., 2020 citrus sinensis antioxidant radical scavenging tpc = 0.47–0.67 mg gae /ml; tfc = 0.23–0.92 mg re/ml; hesperidin = 94.98–173.11 mg/ml; eriocitrin = 28.38–46.95 mg/ml; narirutin = 28.38–46.95 mg/ml; didymin = 1.73–10.33 mg/ml; tangeretin = 3.52–3.67 μg/ml. chen et al., 2020 citrus × clementina antioxidant radical scavenging ferric-reducing power inhibition of lipid peroxidation tpc = 17.58–54.65 mg cae/100 ml; tfc = 18.16–51.48 mg qe/100 ml; tcc = 18.23–53.54 mg β-carotene e/100 ml; neohesperidin = 80.26–110 mg/ 100 ml; hesperidin = 40–81.08 mg/100 ml; naritutin = 6.25–8.50 mg/100 ml. tpc = 29.74–44.20 mg gae/100 ml; tcc = 42.89–75.45 mg β-carotene e/100 ml; neohesperidin = 72.96–116.50 mg/100 ml; hesperidin = 55.24–69.52 mg/100 ml; didymin = 3.65–5.65 mg/100 ml leporini et al., 2020a loizzo et al., 2018 metabolic syndrome inhibition of α-amylase, β-glucosidase and lipase enzymes leporini et al., 2020a loizzo et al., 2018 antibacterial m. luteus and b. subtillis inhibition tpc = 75.60 mg gae/g; tfc = 20.51 mg qe/g haraoui et al. 2020 in vivo citrus aurantifolia metabolic syndrome reduction in plasma tc, tg, and ldl-c levels and increase in plasma hdl-cholesterol levels. oboh et al., 2015b citrus bergamia metabolic syndrome reduction tg levels, cardiovascular risk, oxidative stress and inflammation, protective effects on hepatic steatosis. neohesperidin 182.3 mg/ml; neoeriocitrin = 165.0 mg/ml; naringin =160.1 mg/ml not reported de leo et al., 2020 impellizzeri et al., 2015 citrus lemon antioxidant reduction in ros levels hesperidin = 77.1 mg/l; isorhamnetin 3-o-rutinoside = 44.9 mg/l; rhoifolin = 31.4 mg/l; eriocitrin = 29.9 mg/l; diosmin = 25.7 mg/l. barberis et al., 2020 citrus sinensis antioxidant ros scavenger hesperidin = 422.8 mg/l; naringin = 132.6 mg/l; narirutin = 100.1 mg/l barberis et al., 2020 metabolic syndrome reduction in body mass index 1 tablet/die [day] containing 400 mg of morosil® cardile et al., 2015 nr: not reported; tpc: total phenolics content; tfc: total flavonoids content; tcc: total carotenoids content; ros: reactive oxygen species. italian journal of food science, 2021; 33 (2) 67 citrus species: modern functional food and nutraceutical-based product ingredient abdallah (2020) suggested citrus aurantifolia as natural antibacterial agent against s. aureus, s. epidermis, pseudomonas aeruginosa, and klebsiella pneumonia. low antibacterial activity was found for citrus sinensis. prebiotic effects the prebiotic effect of orange juice could be due to its positive effect on the intestinal microbiota and metabolic biomarkers of young women (aged 28.5 years) (lima et al., 2019). indeed, daily intake of orange juice (300 ml/ day for 2 months) did not change women’s body composition, but improved blood biochemical parameters, such as low-density lipoprotein (ldl)-cholesterol, glucose, and insulin sensitivity. additionally, orange juice positively modulated the composition and metabolic activity of microbiota, increasing the population of bifidobacterium spp. and lactobacillus spp and reduction of enterobacteria. reduction in ammonium (nh4 +) and increase in the production of short-chain fatty acids were also demonstrated. more recently, this prebiotic effect in healthy female volunteers after intervention with 300-ml/day orange juice for 60 days was confirmed (fidélix et al., 2020). orange juice stimulated the growth of lactobacillus spp. in the intestinal microbiota and improved glucose metabolism due to the probiotic effect of these bacteria. daily supplementation of juices of two oranges (cv. cara cara and cv. bahia) with different flavanone content for 7 days in healthy volunteers resulted in increase in the abundance of lachnospiraceae and ruminococcaceae that represented the two most abundant phylum firmicutes’ families present in the gut environment (brasili et al., 2019). interestingly, after intake of cara cara juice positive correlations were found between lachnospiraceae and butyrate, as well as between the most abundant short-chain fatty acids present in the colon, including acetate, butyrate, and propionate (brasili et al., 2019). pulp antioxidant activity the radical scavenging ability of eight citrus pulp methanol extracts, namely citrus sinensis cv. hamlin, cv. red blood, cv. succuri, citrus limetta mosambi, citrus reticulata tangerine, citrus paradise macfed, citrus aurantium l., and citrus jambhiri lush (table 2), were investigated (rehman et al., 2020a). among these, citrus sinensis cv. succuri had the highest values (65.3%). costanzo et al. (2020) compared the antioxidant potential of powdered citrus reticulata, citrus japonica, and the edible portion of hybrid tacle®, a crossbreeding of citrus × clementina and tarocco tetraploids, was able to influence anthropometric values and lipid and glucose metabolism in a rat model having obesity and ms. for this reason, it could be included in dietary supplements for the management of metabolic disorders (casacchia et al., 2019). a promising anti-obesity potential of fresh squeezed citrus × clementina juices against lipase enzymes with ic50 values in the range of 179.32–197.69 mg/ml was recently confirmed (leporini et al., 2020a). moro juice (citrus sinensis) extract (morosil®, 400 mg/die [day]) was able to induce a significant reduction in body mass index (bmi) after 4 weeks of treatment (cardile et al., 2015). one important therapeutic approach for suppressing postprandial hyperglycemia is to reduce or bring down dietary carbohydrate digestion and absorption. the inhibition of carbohydrate-hydrolyzing enzymes, α-glucosidase and α-amylase, in the digestive tract also determined reduction in the rate of glucose absorption and consequently blunting the post-prandial plasma glucose rise (tundis et al., 2010). the fresh squeezed citrus lemon exhibited a promising hypoglycemic inhibitory potential with the ic50 values of 31.1 and 35.3 mg/ml against α-amylase and α-glucosidase enzymes, respectively (loizzo et al., 2019) whereas values in the range of 67.19–103.43 μg/ml against α-glucosidase were found for fresh squeezed citrus × clementina juices from different areas of collection (leporini et al., 2020a). the hypoglycemic ability of poncirus trifoliata juice, related to the genus citrus, was investigated against α-amylase and α-glucosidase enzymes (tundis et al., 2016), with the ic50 values of 138.14 and 81.27 μg/ml, respectively. 2.3. antibacterial activity the development of antibiotic resistance by pathogenic microorganisms necessitated the quest for alternative drug therapy. medicinal plants are traditionally recognized as conventional medicines, and numerous studies have confirmed their antibacterial activity (gavarić et  al., 2015). haraoui et al. (2020) investigated the bacteriostatic action of citrus variety juices. fresh squeezed citrus limon juice exhibited inhibition zone of 27.66 mm on micrococcus luteus, followed by citrus aurantium with an area of 24.66 mm against staphylococcus aureus. interesting results were observed also for citrus maxima and citrus × clementina with inhibition zones of 23.00 and 17.66 mm, respectively, against m. luteus. the lime fresh squeezed juice as antibacterial agent was confirmed against salmonella thypii (fadillah et al., 2020) and corynebacterium diphtheriae (azhara et al., 2020). growth of s. aureus was inhibited by fresh squeezed citrus hystrix juice (kusumawardhani et al., 2020). 68 italian journal of food science, 2021; 33 (2) m. leporini et al. ta bl e 2. . c itr us p ul p an d bi ol og ic al p ot en tia l. p ul p e xt ra ct b io lo gi ca l ac tiv ity m ec ha ni sm tp c , t fc , t c c , a nd /o r m ai n ab un da nt id en tifi ed c om po un ds r ef er en ce s c itr us au ra nt ifo lia 50 % e th an ol m et ha no l 10 % (k o h , sa po ni fic at io n) a nt io xi da nt li po ph ili c an tio xi da nt c ap ac ity h es pe rid in = 2 3– 33 8 m g/ 10 0 g fw ; n ar in gi n = 0– 27 1 m g/ 10 0 g fw ; n eo he sp er id in = 51 –1 58 m g/ 10 0 g fw . lu te ol in = 0 .0 2– 0. 21 m g/ 10 0 g fw ; (a lle )ze ax an th in = 0 .0 2– 0. 03 f w . e rn aw ita e t a l., 2 01 7 e rn aw ita e t a l., 2 01 6 m et ab ol ic sy nd ro m e in hi bi tio n of α -a m yl as e an d βgl uc os id as e en zy m es e rn aw ita e t a l., 2 01 7 e th an ol , m et ha no l, an d ac et on e a nt ib ac te ria l k . p ne um on ia e, s . a ur eu s in hi bi tio n e . c ol i, k le bs ie lla , p se ud om on as , s al m on el la in hi bi tio n n r e rn aw ita e t a l., 2 01 7 b hu iy an e t a l., 2 01 9 c itr us a ur an tiu m e th an ol a nt io xi da nt r ad ic al s ca ve ng in g tp c = 1 0. 45 m g g a e /g d w ; tf c = 7 .1 4 m g r e /g d w ; n eo he sp er id in = 5 17 .1 0 m g/ 10 0 g d w ; n ar in gi n = 32 6. 44 m g/ 10 0 g d w ; d io sm in = 1 89 .1 6 m g/ 10 0 g d w ; h es pe rid in = 5 3. 05 m g/ 10 0 g d w . c he n et a l., 2 02 0 c itr us b er ga m ia e th an ol a nt io xi da nt r ad ic al s ca ve ng in g tp c = 2 08 .0 2 m g g a e /g f w . fr at ia nn i e t a l., 2 01 9 a nt ib ac te ria l e . c ol i, l. m on oc yt og en es , p. a er ug in os a, s . a ur eu s, a nd p. c ar ot ov or um . i nh ib iti on fr at ia nn i e t a l., 2 01 9 c itr us g ra nd is e th an ol a nt io xi da nt r ad ic al s ca ve ng in g tp c = 4 .5 2– 7. 65 m g g a e /g d w ; tf c = 3 .6 7– 8. 25 m g r e /g d w ; n ar in gi n = 19 9. 95 –7 77 .6 5 m g/ 10 0 g d w ; n eo he sp er id in = 3 4. 29 –5 2. 20 m g/ 10 0 g d w ; h es pe rid in = 2 .7 3– 13 .0 0 m g/ 10 0 g d w ; d io sm in = 2 5. 52 –6 2. 6 m g/ 10 0 g d w ; e rio ci tri n = 15 .9 5– 19 .7 7 m g/ 10 0 g d w . c he n et a l., 2 02 0 c itr us h ys tri x 50 % e th an ol m et ha no l 10 % (k o h , sa po ni fic at io n) a nt io xi da nt li po ph ili c an tio xi da nt c ap ac ity h es pe rid in = 7 4 m g/ 10 0 g fw ; n eo he sp er id in = 7 5 m g/ 10 0 g fw . lu te ol in = 0 .2 1 m g/ 10 0 g fw ; (a lle )α -c ar ot en e = 0. 04 m g/ 10 0 g fw ; (a lle )ze ax an th in = 0 .0 2 m g/ 10 0 g fw . e rn aw ita e t a l., 2 01 7 e rn aw ita e t a l., 2 01 6 m et ab ol ic sy nd ro m e in hi bi tio n of α -a m yl as e an d βgl uc os id as e en zy m es e rn aw ita e t a l., 2 01 7 a nt ib ac te ria l k . p ne um on ia e, a nd s . a ur eu s in hi bi tio n e rn aw ita e t a l., 2 01 7 c itr us ja po ni ca 80 % m et ha no l a nt io xi da nt r ad ic al s ca ve ng in g n r c os ta nz o et a l., 2 02 0 italian journal of food science, 2021; 33 (2) 69 citrus species: modern functional food and nutraceutical-based product ingredient c itr us li m on e th an ol , m et ha no l, an d ac et on e a nt ib ac te ria l s . a ur eu s, e . c ol i, k le bs ie lla , p se ud om on as , s al m on el l i nh ib iti on n r b hu iy an e t a l., 2 01 9 c itr us m ac ro pt er a e th an ol , m et ha no l, an d ac et on e a nt ib ac te ria l k . p ne um on ia e, a nd s al m on el la in hi bi tio n n r b hu iy an e t a l., 2 01 9 c itr us m ed ic a e th an ol a nt io xi da nt r ad ic al s ca ve ng in g tp c = 1 48 .0 2 m g g a e /g f w . fr at ia nn i e t a l., 2 01 9 a nt ib ac te ria l e . c ol i, l. m on oc yt og en es , p. a er ug in os a, s . a ur eu s, a nd p. c ar ot ov or um in hi bi tio n tp c = 3 .8 9– 8. 07 m g g a e /g f w ; tf c = 1 .8 9– 5. 08 m g r e /g f w ; n ar in gi n = 10 .6 3 m g/ 10 0 g fw ; n eo he sp er id in = 2 2. 96 m g/ 10 0 g fw ; h es pe rid in = 1 8. 55 –1 36 .3 9 m g/ 10 0 g fw ; d io sm in = 1 7. 36 –2 1. 64 m g/ 10 0 g fw ; e rio ci tri n = 23 .7 6– 24 .1 1 m g/ 10 0 g fw ; n ar iru tin = 2 6. 85 m g/ 10 0 g fw . c he n et a l., 2 02 0 fr at ia nn i e t a l., 2 01 9 c itr us n ob ili s m et ha no l 10 % (k o h , sa po ni fic at io n) a nt io xi da nt li po ph ili c an tio xi da nt c ap ac ity (9 z) -v io la xa nt hi n = 2. 76 m g/ 10 0 g fw ; (a lle )v io la xa nt hi n = 1. 93 m g/ 10 0 g fw ; (a lle )a nt he ra xa nt hi n = 0. 64 m g/ 10 0 g fw . e rn aw ita e t a l., 2 01 6 50 % e th an ol m et ab ol ic sy nd ro m e in hi bi tio n of α -a m yl as e an d βgl uc os id as e en zy m es e rn aw ita e t a l., 2 01 7 a nt ib ac te ria l k . p ne um on ia e, a nd s . a ur eu s in hi bi tio n e rn aw ita e t a l., 2 01 7 b en ze ne , e th an ol , an d m et ha no l b . c er us , s . a ur eu s, s . e pi de rm id is , p. v ul ga ris , s . t yp hi m ur iu m , p . ae ru gi no sa , c . a lb ic an s an d t. v iri de in hi bi tio n n r s ha rm a an d ty ag i, 20 19 c itr us r et ic ul at a e th an ol 80 % e th an ol a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er tp c = 4 .2 5– 8. 55 m g g a e /g d w ; tf c = 5 .2 1– 10 .9 0 m g r e /g d w ; n ar in gi n = 7. 68 –1 60 .0 3 m g/ 10 0 g; d w n eo he sp er id in = 1 9. 86 –2 87 .4 m g/ 10 0 g d w ; h es pe rid in = 1 3. 83 –4 15 .5 9 m g/ 10 0 g d w ; n ar in gi n = 7. 68 –1 60 .0 3 m g/ 10 0 g d w ; e rio ci tri n = 23 .9 2– 45 .5 0 m g/ 10 0 g d w ; n ar iru tin = 2 3. 91 –1 20 .0 3 m g/ 10 0 g d w ; ta ng er et in = 1 .6 8– 3. 84 m g/ 10 0 g d w ; tp c = 1 27 .3 3 m g g a e /g e xt ra ct ; tf c = 0 .8 7 m g q e /g e xt ra ct . c he n et a l., 2 02 0 b en ta ha r e t a l., 2 02 0 (c on tin ue s) 70 italian journal of food science, 2021; 33 (2) m. leporini et al. ta bl e 2. . c on tin ue d p ul p e xt ra ct b io lo gi ca l ac tiv ity m ec ha ni sm tp c , t fc , t c c , a nd /o r m ai n ab un da nt id en tifi ed c om po un ds r ef er en ce s c itr us s in en si s e th an ol 80 % e th an ol 50 % e th an ol a nt io xi da nt m et ab ol ic sy nd ro m e r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in hi bi tio n of α -a m yl as e tp c = 4 .9 8– 9. 49 m g g a e /g d w ; tf c = 5 .3 2– 9. 81 m g r e /g d w ; h es pe rid in = 1 03 .1 4– 38 5. 37 m g/ 10 0 g d w ; e rio ci tri n = 13 .8 5– 24 .5 5 m g/ 10 0 g d w ; n ar iru tin = 4 1. 62 –1 53 .4 8 m g/ 10 0 g d w ; d id ym in = 4 .1 9– 13 .1 5 m g/ 10 0 g d w ; tp c = 1 59 .6 6 m g g a e /g e xt ra ct ; tf c = 0 .8 5 m g q e /g e xt ra ct . tp c = 2 07 .6 9 m g c a e /g f w ; tf c = 8 5. 9 m g q e /g f w c he n et a l., 2 02 0 b en ta ha r e t a l., 2 02 0 c as ac ch ia e t a l., 2 01 9 b en ze ne , e th an ol , an d m et ha no l a nt ib ac te ria l b . c er us , s . a ur eu s, s . e pi de rm id is , p . vu lg ar is , s . t yp hi m ur iu m , p. a er ug in os a, c . a lb ic an s an d t. v iri de in hi bi tio n s ha rm a an d ty ag i, 20 19 c itr us × cl em en tin a 80 % m et ha no l 50 % e th an ol a nt io xi da nt r ad ic al s ca ve ng in g li pi d pe ro xi da tio n in hi bi tio n n r tp c = 9 0. 01 m g c a e /g ; f w tf c = 4 1. 30 m g q e /g f w . βcr yp to xa nt hi n = 23 2. 4 µm p hy to en e = 62 .7 µ m 9zv io la xa nt hi n = 60 .5 µ m a nt he ra xa nt hi n = 39 .0 µ m h es pe rid in = 3 .3 µ m n ar in ge ni n7o -g lu co si de = 1 .4 µ m n ar iru tin = 1 .0 µ m c os ta nz o et a l., 2 02 0 c as ac ch ia e t a l., 2 01 9 c ill a et a l., 2 01 8 50 % e th an ol m et ab ol ic sy nd ro m e in hi bi tio n of α -a m yl as e an d lip as e en zy m e c as ac ch ia e t a l., 2 01 9 n r : n ot re po rt ed ; t p c : t ot al p he no lic s co nt en t; tf c : t ot al fl av on oi ds c on te nt ; t c c : t ot al c ar ot en oi ds c on te nt . italian journal of food science, 2021; 33 (2) 71 citrus species: modern functional food and nutraceutical-based product ingredient the hdl-cholesterol level was found to improve (arumugam et al., 2019). the lipase inhibitor activity of citrus pulp extracts was reported by casacchia et al. (2019), founding the ic50 values of 86.30, 105.90, and 67.20 mg/ml, respectively, for citrus clementina, citrus sinensis, and their hybrid called tacle®. antibacterial activity citrus bergamia, citrus medica, and citrus medica cv. salò pulp extracts were described as antibacterial agents against escherichia coli, listeria monocytogenes, pseudomonas aeruginosa, s. aureus, and pectobacterium carotovorum (fratianni et al., 2019). the antibacterial activity of various citrus pulp extracts was also reported by ernawita et al. (2017). among them, jeruk makin and jeruk nipis showed the highest inhibitor capacity against klebsiella pneumoniae (ic50 = 3.3 and 4.1 mg/ml) and s. aureus (ic50 = 2.6 and 3.1 mg/ml). in addition, citrus limon, citrus aurantifolia, and citrus macroptera pulp extracts were screened for antimicrobial activity against s. aureus, e. coli, klebsiella sp., pseudomonas sp., and salmonella sp. (bhuiyan et al., 2019). citrus macroptera, a taxonomic synonym of citrus hystrix (kaffir lime) known for its antioxidant, nutritious, and  therapeutic uses (paul et al., 2017) ethanol extracts exhibited the highest zone of inhibition (14 mm) against klebsiella sp. while citrus aurantifolia methanol extract showed the highest zone of inhibition (8 mm) against salmonella sp. citrus seed antioxidant potential bitter orange, blonde orange, sweet orange, lemon, and mandarin seed ultrasound methanol extracts (table  3) were investigated for their antioxidant ability but no differences were found in the radical scavenging activity (falcinelli et al., 2020). conversely, costanzo et al. (2020) demonstrated that powered citrus reticulata seed extract tissue showed the highest antioxidant capacity (55.6 mmol te/mg fw) compared to citrus japonica seed extract tissue (3.2 mmol te/mg fw). the radical scavenging ability of eight citrus seed extracts were investigated (rehman et al., 2020a). among them, citrus jambhiri lush, citrus sinensis cv. red blood, and citrus reticulata tangerine possessed the highest activity (54.3, 53.6, and 53.3%, respectively). previously, the following ranking of radical scavenging effect was demonstrated: lemon seeds extract > orange seeds extract > mandarin seeds extract (i̇nan et al., 2018). citrus × clementina tissue. in the citrus × clementina pulp extracts, the total antioxidant capacity (tac) was found to be three-fold higher (7.1 mmol te/mg fresh weight [fw]) compared to citrus reticulata (2.6 mmol te/mg fw) and six-fold higher compared to citrus japonica (1.2 mmol te/mg fw). similarly, citrus sinensis and citrus reticulata fruits possessed good antioxidant activity studied by the hydroxyl radical scavenging activity and reducing power capacity methods (bentahar et al., 2020). fratianni et al. (2019) indicated that citrus bergamia and citrus medica cv. salò homogenized  pulp extracts exhibited the highest antioxidant potential compared to citrus medica. previously, the in vitro lipophilic antioxidant capacity of seven citrus pulp extracts was reported (ernawita et al., 2017). among them, jeruk makin (citrus aurantium) showed the highest antioxidant capacity (19.5  µmol te/100 g), followed by jeruk calung pulp extracts (citrus aurantium) and jeruk nipis (citrus aurantiifolia) (10.7 and 10.6 µmol α-te/100 g, respectively). previously, in vivo studies have reported that citrus macroptera ethanol pulp extract possessed a significant lipid-lowering activity and a significant diminution of lipid peroxidation in liver and kidney tissues was observed (paul et  al., 2015). the protective effect against oxidative stress of pulp bio-accessible fractions of oranges from navel and cara oranges cultivars as well as clementine was also demonstrated (cilla et al., 2018). these fractions act by pre-serving cell viability, correct cell cycle progression, mitochondrial membrane potential, and diminishing ros level and lipid peroxidation. recently, the effect of dried orange pulp on antioxidants level in the plasma was evaluated (allam sabbah et al., 2020). results demonstrated that the value of total antioxidant capacity, as a biomarker of oxidative stress, was gradually increased (ranged from 0.420 to 0.433 mm/l) by increasing the level of dried orange pulp supplementation (25, 50, and 75%). additionally, a reduction of total lipids values was observed. metabolic syndrome the α-amylase inhibition activity of seven citrus pulp extracts was analyzed by ernawita et al. (2017). makin and jeruk nipis pulp extracts exhibited the lowest ic50 values (18.8 and 19.4 mg/ml, respectively), while calung extract possessed less activity (ic50 = 56.2 mg/ml). oral administration of citrus hystrix and citrus maxima pulp extracts (5, 50, 300, and 2,000 mg/kg body weight (bw) in streptozotocin (stz)-induced diabetic rats for 14 days was able to reduce blood glucose, tg level, and serum cholesterol. additionally, 72 italian journal of food science, 2021; 33 (2) m. leporini et al. table 3. bioactivity of citrus seed. seed extract biological activity mechanism tpc, tfc, tcc, and/or main abundant identified compounds references citrus aurantium methanol antioxidant radical scavenging tpc = 2.5 gae/g dw falcinelli et al., 2020 anti-inflammatory anti-edematogenic effects antibacterial proteus and pseudomonas inhibition nr aladekoyi et al., 2016 citrus aurantium methanol antioxidant radical scavenging tpc = 107 mg/100 g; tfc = 20.8 mg/100 g. rehman et al., 2020c citrus jambhiri methanol antioxidant radical scavenging tcc = –25 mg/g fw costanzo et al., 2020 citrus jambhiri methanol antioxidant radical scavenging tpc = 129 mg/100 g; tfc = 22.8 mg/100 g. rehman et al., 2020c citrus junos anti-inflammatory inhibition of no production not reported ko et al., 2020 citrus limon methanol antioxidant radical scavenging restoration of antioxidant defense system tpc = 1.2 gae/g dw tpc = 152.70–212.30 mg gae/kg falcinelli et al., 2020 i̇nan et al., 2017 metabolic syndrome reduction of glucose and lipid levels nr demir and celik, 2019 antibacterial klebsiella, proteus and pseudomonas inhibition nr aladekoyi et al., 2016 citrus limetta methanol antioxidant radical scavenging tpc = 99.1 mg/100 g; tfc = 19.37 mg/100 g. rehman et al., 2020c citrus maxima ethanol antibacterial s. aureus, e. coli, and b. subtilis inhibition tfc = 1602.740 mg/kg. sahlan et al., 2018 citrus paradise ethanol antibacterial s. aureus, e. coli, s. typhimurium, s. enteritidis, p. aeruginosa, k. pneumoniae, citrus utilis, and b. cereus inhibition tfc = 483.562 mg/kg. sahlan et al., 2018 citrus reticulata methanol antioxidant radical scavenging tpc = 112 mg/100 g; tfc = 21.5 mg/100 g. rehman et al., 2020c methanol tpc = 2.4 gae/g dw. tcc = –10 mg/g fw. tpc = 152.70–212.30 mg gae/kg. falcinelli et al., 2020 costanzo et al., 2020 i̇nan et al., 2017 citrus sinensis methanol antioxidant radical scavenging tpc = 101–118 mg/100 g; tfc = 20.1–22.60 mg/100 g. rehman et al., 2020c citrus sinensis methanol tpc = 1.3 gae/g dw falcinelli et al., 2020 n-hexane metabolic syndrome reduction of fasting blood glucose, serum tg, serum cholesterol, hdl nr chilaka et al., 2015 ethanol antibacterial s. aureus, enterococcus faecalis, p. aeruginosa, e. coli and citrus albicans inhibition nr oikeh et al., 2020 nr: not reported; tpc: total phenolics content; tfc: total flavonoids content; tcc: total carotenoids content. metabolic syndrome it has been reported recently that lemon seed extract could prevent diabetic complications due to reduction in glucose and lipid profile levels and restoration of antioxidant defense system (demir and celik, 2019). previously, a reduction of blood glucose in alloxan-induced diabetic rats was observed after treatment with emulsified  sweet orange seed oil (1000 mg/kg bw). in addition, this seed oil improved sugar and lipid profile with reduction of serum tg, cholesterol, and increased hdl-cholesterol in diabetic rats (chilaka et al., 2015). italian journal of food science, 2021; 33 (2) 73 citrus species: modern functional food and nutraceutical-based product ingredient citrus peels antioxidant effects recently, the antioxidant potential of citrus × clementina peel extracts, collected from different areas of calabria and obtained by using different methodologies, was studied (leporini et al., 2020a). results demonstrated that sample from cetraro obtained by ultrasound extraction in ethanol possessed the highest antioxidant activity (table 4). interestingly, the citrus × clementina juice enriched with this extract (20% v/w) increased its antioxidant potential. similarly, pereira et al. (2020) reported the increase of beer antioxidant activity after addition of orange peels extract. huang et al. (2020) compared the antioxidant ability of eight citrus peel extracts: grapefruit, pomelo, kumquat, mandarin, ponkan, tangerine, lemon, and sweet orange. the most active samples were ponkan extract in dpph and frap assays (386.25 and 466.14 μmol te/g of extract, respectively), tangerine extract in abts assay (689.43 μmol te/g), and pomelo in orac assay (1964.0 μmol te/g). an inhibition of 92.87% was reported for citrus hystrix peels extract against dpph radical (ramli et al., 2020). citrus sinensis and citrus aurantium peels’ extracts were investigated as potent antioxidant agents against lipid peroxidation (rafiq et al., 2018). furthermore, the bergamot extract showed a higher abts radical inhibition with a value of 136.3 mmol te/g dry weight (dw). the capacity of citrus medica diamante hydroalcoholic peels extract to inhibit both dpph and abts radicals (ic50  = 0.81 and 3.48 mg/ml, respectively) was also demonstrated by menichini et al. (2016). in β-carotene, this extract exhibited an ic50 value of 0.23 mg/ml. da silva et al. (2018) studied the antioxidant potential of pomelo peels cv. toranja buraram in n-hexane, ethyl acetate, acetone, ethanol, methanol, and methanol:water (80:20). the ethyl acetate and methanolic extracts presented the highest antioxidant activity in vitro by dpph (ic50 = 298.3 and 303.8 μg/ml, respectively), abts assay (ic50 = 298.2 and 296.4 μg/ml, respectively), and frap (ic50 = 234.6 and 398.1 μg/ml, respectively). long et al. (2021) evaluated the antioxidant effects of ethanol extract and its three subfractions—petroleum ether, ethyl acetate, and water extracts—of citrus sinensis cv. gannanzao peels. the ethyl acetate extract exhibited the best antioxidant potential compared to four extracts in all antioxidant assays with the ic50 values of 38.33 mg/ml and 8.47 mg/ml in dpph and abts tests, respectively, and value of 21.54 mm trolox equivalents (te)/mg dw in frap assay. the results correspond to those reported by guo et al. (2020) for citrus sinensis cv. newhall peels extract. anti-inflammatory effects nitric oxide (no) is recognized as a mediator and regulator in pathological reactions, especially in acute inflammatory responses (terao, 2009). the development of substances to prevent the overproduction of no has become a new research target to treat chronic inflammatory diseases. ko et al. (2020) reported the anti-inflammatory effect of citrus junos seed oil. no production was suppressed by 53% at a concentration of 0.05% that does not show cytotoxicity. the possible anti-inflammatory and antinociceptive activity of citrus aurantium seed oil, obtained by using soxhlet apparatus with n-hexane, was evaluated by using formalin-induced paw licking, edema, and myeloperoxidase activity assessment (azadeh et al., 2019). the results showed that seed oil exhibited anti-inflammatory properties in the first and second phases of formalin test, antiedematogenic effects but exerted no effects on myeloperoxidase activity. antibacterial potential recently, the antibacterial activities of citrus sinensis seed oil, obtained by soxhlet apparatus using n-hexane as solvent and ethanol extract, was studied (oikeh et al., 2020). the results showed that the non-oil extract had better antibacterial activity against s.  aureus, enterococcus faecalis, and e. coli. on the contrary, the seed oil had better activity against salmonella spp. similar susceptibility was found for p. aeruginosa. previously, it was demonstrated that citrus sinensis seed oil obtained by soxhlet apparatus using n-hexane as solvent possessed antibacterial activity against s. aureus and candida albicans (olabanji et al., 2016). buket et al. (2018) also reported that the lemon, orange, and grapefruit cold-pressed seed oil had inhibition zones ranging from 6.62 to 11.00 mm against pathogenic bacteria such as s. aureus, e. coli, s. typhimurium, salmonella enteritidis, p. aeruginosa, k. pneumoniae, candida utilis, and bacillus cereus holl. the antimicrobial and antifungal activities of aqueous and ethanolic grapefruit seed extracts were confirmed against s. aureus, e. faecalis, bacillus subtilis, e. coli, p. aeruginosa, k. pneumoniae, and c. albicans (eryilmaz et al., 2018). the ethanolic extract of pomelo seeds also gives positive results with growth-inhibition effect on bacillus subtilis, s. aureus, and e. coli (sahlan et al., 2018). oil extracted from lemon, lime, and bitter orange seeds possessed different antimicrobial potential. similar activity was found for staphylococcus, but only lemon seed oil has activity against klebsiella and the highest zone of inhibition against proteus, while bitter orange has a maximum zone of inhibition (0.25 mm) against pseudomonas (aladekoyi et al., 2016). 74 italian journal of food science, 2021; 33 (2) m. leporini et al. ta bl e 4. b io lo gi ca l a ct iv ity o f c itr us p ee ls . p ee ls e xt ra ct /e ss en tia l o il b io lo gi ca l ac tiv ity m ec ha ni sm tp c , t fc , t c c , a nd /o r m ai n ab un da nt id en tifi ed c om po un ds r ef er en ce s c itr us a ur an tif ol ia 96 % e th an ol a nt ib ac te ria l a nt i-i nfl am m at or y s . t yp hi in hi bi tio n r ed uc tio n of il -6 le ve ls n r k as im e t a l., 2 02 0 k as im e t a l., 2 02 0 70 % e th an ol in hi bi tio n of p aw e de m a. n r p al la vi e t a l., 2 01 8 c itr us a ur an tiu m e th an ol a nt io xi da nt m et ab ol ic sy nd ro m e r ad ic al s ca ve ng in g in hi bi tio n of li pi d pe ro xi da tio n tp c = 1 8. 15 m g g a e /g f w ; tf c = 1 7. 09 m g r e /g f w ; n eo he sp er id in = 1 62 0. 77 m g/ 10 0 g d w ; n ar in gi n = 87 9. 33 m g/ 10 0 g d w ; h es pe rid in = 3 5. 88 m g/ 10 0 g d w ; n ar ur itu n = 18 .7 2 m g/ 10 0 g d w . n r c he n et a l., 2 02 0 r afi q et a l., 2 01 8 80 % e th an ol r ed uc tio n of b w , l ip id d ro pl et s re gu la tin g ad ip og en es is a nd th er m og en es is . n ar in gi n = 0. 91 6 m g/ m l; n eo he sp er id in = 0 .6 57 m g/ m l p ar k et a l., 2 01 9 70 % e th an ol a nt i-i nfl am m at or y in hi bi tio n of p aw e de m a. p al la vi e t a l., 2 01 8 c itr us g ra nd is e th an ol a nt io xi da nt r ad ic al s ca ve ng in g tp c = 8 .7 9– 14 .9 3 m g g a e /g f w ; tf c = 8 .2 5– 14 .0 3 m g r e /g f w ; n ar in gi n = 69 4. 15 –1 67 6. 31 m g/ 10 0 g d w ; n eo he sp er id in = 2 8. 42 –7 3. 04 m g/ 10 0 g d w ; h es pe rid in = 7 .3 9– 33 .3 9 m g/ 10 0 g d w ; n ar ur itu n = 2. 72 –6 .3 7 m g/ 10 0 g d w ; e rio ci tri n = 20 .4 2– 34 .4 9 m g/ 10 0 g d w ; d io sm in = 6 .0 2– 1. 29 m g/ 10 0 g d w . c he n et a l., 2 02 0 70 % e th an ol a nt i-i nfl am m at or y in hi bi tio n of p aw e de m a. p al la vi e t a l., 2 01 8 c itr us li m on 70 % e th an ol a nt io xi da nt r ad ic al s ca ve ng in g tp c = 1 98 .5 2 m g g a /g e xt ra ct ; tf c = 1 83 .1 3 m g/ g ex tra ct ; h es pe rid in = 8 4. 24 m g/ g ex tra ct ; e rio ci tri n = 84 .8 0 m g/ g ex tra ct ; n ar ru tin = 1 3. 56 m g/ g ex tra ct . h ua ng e t a l., 2 02 0 c itr us m ax im a 70 % e th an ol a nt io xi da nt r ad ic al s ca ve ng in g tp c = 1 38 .9 3 m g g a /g e xt ra ct ; tf c = 4 16 .5 4 m g/ g ex tra ct ; n ar in ge ni n = 38 6. 37 m g/ g ex tra ct ; r ho ifo lin = 2 8. 54 m g/ g ex tra ct . h ua ng e t a l., 2 02 0 95 % e th an ol m et ab ol ic sy nd ro m e r ed uc tio n th e bl oo d gl uc os e le ve l, to ta l c ho le st er ol , t g , a nd l d lc . in hi bi tio n of li pa se e nz ym e n r a ni a nd o ch u, 2 02 0 h ua ng e t a l., 2 02 0 c itr us m ed ic a d ia m an te 70 % e th an ol a nt io xi da nt r ad ic al s ca ve ng in g in hi bi tio n of li pi d pe ro xi da tio n a pi ge ni n = 62 .8 m g/ k g fw ; h es pe rit in = 3 0. 4 m g/ k g fw ; n er in ge ni n = 18 .6 m g/ k g fw ; q ue rc et in = 1 8. 2 m g/ k g fw . m en ic hi ni e t a l., 2 01 6 italian journal of food science, 2021; 33 (2) 75 citrus species: modern functional food and nutraceutical-based product ingredient m et ab ol ic sy nd ro m e r ed uc tio n of s er um g lu co se , ch ol es te ro l a nd t g . m en ic hi ni e t a l., 2 01 6 c itr us p ar ad is i 70 % e th an ol a nt io xi da nt r ad ic al s ca ve ng in g tp c = 1 79 .1 3 m g g a /g e xt ra ct ; tf c = 4 74 .5 5 m g/ g ex tra ct ; n ar in ge ni n = 25 2. 13 m g/ g ex tra ct ; n eo he sp er id in = 1 82 .3 2 m g/ g ex tra ct ; n ar iru tin = 1 4. 80 m g/ g ex tra ct ; h es pe rid in = 6 .5 5 m g/ g ex tra ct . h ua ng e t a l., 2 02 0 w at er , 8 0% e th an ol , a nd nhe xa ne m et ab ol ic sy nd ro m e r ed uc tio n in th e ch ol es te ro l a nd tg le ve ls n ob ile tin = 1 8. 13 µ g/ m l. fa ye k et a l., 2 01 7 c itr us r et ic ul at a 70 % e th an ol a nt io xi da nt r ad ic al s ca ve ng in g tp c = 2 15 .1 1 m g g a /g e xt ra ct ; tf c = 1 92 .2 2 m g/ g ex tra ct ; h es pe rid in = 1 50 .9 6 m g/ g ex tra ct ; n ar iru tin = 1 6. 79 m g/ g ex tra ct ; e rio ci tri n = 11 .3 3 m g/ g ex tra ct . h ua ng e t a l., 2 02 0 e th an ol fe rr ic -r ed uc in g po w er tp c = 1 0. 58 –2 3. 46 m g g a e /g f w ; tf c = 7 .5 7– 21 .3 7 m g r e /g f w ; n ar in gi n = 6. 31 –7 7. 99 m g/ 10 0 g d w ; n eo he sp er id in = 0 –7 45 m g/ 10 0 g d w ; h es pe rid in = 3 9. 98 –1 89 3. 73 m g/ 10 0 g d w ; n ar ur itu n = 16 .2 1– 14 5. 56 m g/ 10 0 g d w ; e rio ci tri n = 10 .3 2– 26 8. 69 m g/ 10 0 g d w ; d io sm in = 3 .2 9– 38 .7 3 m g/ 10 0 g d w . c he n et a l., 2 02 0 w at er nbu ta ne m et ab ol ic sy nd ro m e r ed uc tio n in b od y m as s in de x, bo dy fa t p er ce nt ag e an d in w ai st ci rc um fe re nc e, t c a nd t g le ve ls . r ed uc tio n of b lo od g lu co se le ve l an d pl as m a in su lin le ve l. in hi bi tio n of li pa se e nz ym e h es pe rid in = 4 0 m g n ar in ge ni n = 28 m g q ue rc et in = 2 6 m g r ut in = 2 5 m g n ob ile tin = 3 2. 28 % ta ng er iti n = 22 .8 2% k am el e t a l., 2 01 9 g uo e t a l., 2 01 6 h ua ng e t a l., 2 02 0 a lk al in e ho t w at er d ic hl or om et ha ne a nd et hy l a ce ta te a nt i-i nfl am m at or y in hi bi tio n of n o in hi bi tio n of c o x 1 an d c o x 2 n ar iru tin = 0 .2 6– 4. 52 m g/ g ex tra ct ; h es pe rid in = 7 .0 2– 26 .8 1 m g/ g ex tra ct ; n ob ile tin = 0 .3 9– 7. 79 m g/ g ex tra ct ; ta ng er et in = 0 .1 9– 3. 37 m g/ g ex tra ct . n r c he n et a l., 2 01 7 h am da n et a l., 2 02 0 c itr us s in en si s 70 % e th an ol 95 % e th an ol , p et ro le um et he r, et hy l a ce ta te , a nd w at er 95 % e th an ol , p et ro le um et he r, et hy l a ce ta te , a nd w at er a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er tp c = 1 49 .4 2 m g g a /g e xt ra ct ; tf c = 1 86 .8 1 m g/ g ex tra ct ; h es pe rid in = 1 48 .6 3 m g/ g ex tra ct ; n ar iru tin = 2 1. 49 m g/ g ex tra ct ; d id ym in = 7 .1 8 m g/ g ex tra ct . tp c = 0 .1 2– 0. 49 m m g a e / m g d w ; tf c = 1 .2 9– 4. 20 m m h e / m g d w ; s in en se tin = 0 –1 21 .3 m g/ m g; h es pe rid in = 1 .5 6– 21 .2 3 µg /m g; e rio ci tri n = 0– 4. 20 m g/ m g; h ua ng e t a l., 2 02 0 lo ng e t a l., 2 02 1 (c on tin ue s) 76 italian journal of food science, 2021; 33 (2) m. leporini et al. ta bl e 4. c on tin ue d p ee ls e xt ra ct /e ss en tia l o il b io lo gi ca l ac tiv ity m ec ha ni sm tp c , t fc , t c c , a nd /o r m ai n ab un da nt id en tifi ed c om po un ds r ef er en ce s n ar iru tin = 0 .9 4– 6. 27 m g/ m g; ta ng er et in = 0 .1 8– 2. 72 m g/ m g. tp c = 1 8. 71 –9 1. 55 m g g a e /g d w ; tf c = 3 .6 2– 86 .9 1 m g q e /g d w ; s in en se tin = 0 –3 6. 92 m g/ m g d w ; n ob ile tin = 0 –3 5. 54 m g/ m g d w ; n ar iru tin = 1 .2 7– 20 .2 1 m g/ m g d w ; h es pe rid in = 1 .6 5– 42 .5 6 m g/ m g d w . g uo e t a l., 2 02 0 0. 5 g an d 1 g of c itr us im ® m et ha no l w at er , 8 0% e th an ol , a nd nhe xa ne 50 % e th an ol m et ab ol ic sy nd ro m e r ed uc tio n of fa t, an d in cr ea se le an m as s re du ci ng w ai st ci rc um fe re nc e. r ed uc ed b lo od g lu co se a nd pl as m a in su lin . r ed uc tio n in th e ch ol es te ro l a nd tg le ve ls . in hi bi tio n of α -a m yl as e an d lip as e en zy m es n r r ut in = 1 24 8. 3 m g/ g d w ;p -c ou m ar ic a ci d = 95 7. 4 m g/ g d w ; p ro to ca te ch ui c ac id = 3 26 .3 m g/ g d w ; fe ru lic a ci d = 31 6. 0 m g/ g d w ; n ar in ge ni n = 22 0. 7 m g/ g d w ; va ni lli c ac id = 1 12 .2 m g/ g d w . n ob ile tin = 7 3. 15 m g/ m l. tp c = 1 77 .1 6 m g c a e /g f w ; tf c = 6 5. 9 m g q e /g f w ; k eg el e et a l., 2 01 9 s at hi ya ba m a et a l, 20 18 fa ye k et a l., 2 01 7 c as ac ch ia e t a l., 2 01 9 m et ha no l a nd e th an ol a nt i-i nfl am m at or y in hi bi tio n of e de m a. n ot re po rt ed o sa ru m w en se e t a l., 20 17 b en ze ne , e th an ol , a nd m et ha no l a nt ib ac te ria l b . c er us , s . a ur eu s, s . e pi de rm id is , p . v ul ga ris , s . t yp hi m ur iu m , p . a er ug in os a, c . a lb ic an s an d t. v iri de in hi bi tio n e . c ol i a nd b . s ub til lis in hi bi tio n n r s ha rm a et t ya gi , 20 19 g uo e t a l., 2 02 0 c itr us tu m id a h d f+ 5 % p ee l p ow de r m et ab ol ic sy nd ro m e s up pr es si on b w g ai n n r s at o et a l., 2 01 9 c itr us u ns hi u m et ha no l a nt io xi da nt m et ab ol ic sy nd ro m e r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in hi bi tio n of α -g lu co si da se a nd lip as e en zy m es h es pe rid in = 5 00 27 m g/ g d w ; n ar iru tin = 9 28 4 m g/ g d w ; n ob ile tin = 1 03 .8 m g/ g d w ; ta ng er et in = 5 5. 5 m g/ g d w . k im e t a l., 2 02 0 k im e t a l., 2 02 0 fe rm en te d dr ie d a nt i-i nfl am m at or y in hi bi tio n of l p s -in du ce d n o , in o s , c o x -2 p ro te in , t n fγ an d il -6 n r k im e t a l., 2 01 9a c itr us × cl em en tin a e th an ol a nd 8 0% e th an ol a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in hi bi tio n of li pi d pe ro xi da tio n tp c = 3 .4 5– 8. 75 m g c a e /g f w ; tf c = 2 .4 7– 6. 05 m g q e /g f w ; tc c = 9 .6 6– 39 .8 4 m g bca ro te ne e /g f w ; h es pe rid in = 1 55 .2 8– 10 93 .3 6 m g/ 10 0 g fw ; s in en se tin = 1 9. 56 -3 7. 99 m g/ 10 0 g fw ; ta ng er et in = 5 .4 3– 9. 60 m g/ 10 0 g fw ; lu te ol in = 3 .0 2– 8. 58 m g/ 10 0 g fw . le po rin i e t a l., 2 02 0a italian journal of food science, 2021; 33 (2) 77 citrus species: modern functional food and nutraceutical-based product ingredient 50 % e th an ol m et ab ol ic sy nd ro m e in hi bi tio n of α -a m yl as e, βgl uc os id as e an d lip as e en zy m es tp c = 1 09 .8 6 m g c a e /g f w ; tf c = 6 1. 3 m g q e /g f w ; le po rin i e t a l., 2 02 0a c as ac ch ia e t a l., 2 01 9 c itr us a ur an tif ol ia e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g li m on en e = 42 .3 5% γte rp in en e = 15 .4 4% βpi ne ne = 1 2. 57 % li n et a l., 2 01 9 m et ab ol ic sy nd ro m e im pr ov e th e se ru m t c , t g , l d lc, al an in e am in ot ra ns fe ra se , an d as pa rt at e tra ns am in as e le ve ls li n et a l., 2 01 9 c itr us a ur an tiu m e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in cr ea se in m r n a g en e ex pr es si on o f c uzn s o d , c at , an d g p x li m on en e = 85 .2 2% ; βm yr ce ne = 4 .3 0; α -p in en e = 1. 28 % . li m on en e = 81 .1 9% ; li na lo ol = 4 .0 6% ; βm yr ce ne = 3 .0 7. li m on en e = 48 .7 % ; li na lo ol = 3 2. 4% ; βm yr ce ne = 1 .2 % . ta ne va e t a l., 2 01 9 fa ra hm an df ar e t a l., 20 20 h so un a et a l., 2 01 8 a nt ib ac te ria l e . c ol i, p. a er ug in os a, l . m on oc yt og en es , s . a ur eu s, b . s ub til is , c . a lb ic an s an d s. pa ra ty ph i b in hi bi tio n li m on en e = 61 .8 5% ; γte rm in en e = 9. 15 % ; o ct an al = 5 .2 8% ; α -p in en e = 3. 02 % . g uo e t a l., 2 01 8 a nt i-i nfl am m at or y r ed uc tio n of n o p ro du ct io n h so un a et a l., 2 01 8 r ed uc tio n of c el l m ig ra tio n, cy to ki ne p ro du ct io n an d pr ot ei n ex tra va sa tio n li m on en e = 31 .1 % ; γte rp in en e = 10 .8 % ; βpi ne ne = 8 .5 % ; n er al = 7 .1 % . a m or im e t a l., 2 01 6 c itr us b er ga m ia e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er c he la te p ro -o xi da nt m et al n r lo m ba rd o et a l., 2 02 0 a nt i-i nfl am m at or y r ed uc tio n of il -1 α , i l6, t n fγ ni tri te /n itr at e an d p g e 2 lo m ba rd o et a l., 2 02 0 c itr us li m on e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g li m on en e = 61 .7 2% ; 3c ar en e = 13 .6 7% ; α -p in en e = 13 .9 7% . g uo e t a l., 2 01 8 m et ab ol ic sy nd ro m e in hi bi tio n of α -a m yl as e an d βgl uc os id as e en zy m es li m on en e = 53 .0 7% ; βpi ne ne = 9 .5 3% ; b or ne ol = 5 .5 7% : o bo h et a l., 2 01 7 a nt i-i nfl am m at or y r ed uc tio n of c el l m ig ra tio n, cy to ki ne p ro du ct io n an d pr ot ei n ex tra va sa tio n li m on en e = 53 .9 % ; βp in en e = 13 .1 % ; s ab in en e = 3. 4% . a m or im e t a l., 2 01 6 (c on tin ue s) 78 italian journal of food science, 2021; 33 (2) m. leporini et al. ta bl e 4. c on tin ue d p ee ls e xt ra ct /e ss en tia l o il b io lo gi ca l ac tiv ity m ec ha ni sm tp c , t fc , t c c , a nd /o r m ai n ab un da nt id en tifi ed c om po un ds r ef er en ce s a nt ib ac te ria l e . c ol i, fu so ba ct er iu m ne cr op ho ru m , tr ue pe re lla p yo ge ne s, s . a re us in ib iti on p. a er ug in os a, l . m on oc yt og en es , b . s ub til is , c . a lb ic an s an d s . p ar at yp hi b in hi bi tio n li m on en e = 65 .5 9% βp in en e = 15 .0 6% ; γte rp in en e = 7. 93 . b ra ga e t a l., 2 02 0 g uo e t a l., 2 01 8 c itr us lu m ia e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in hi bi tio n of li pi d pe ro xi da tio n li m on en e = 48 .9 0% ; li na lo ol = 1 8. 24 % . li na ly l a nt hr an ila te = 1 0. 96 % . s m er ig lio e t a l., 2 01 8 c itr us m ed ic a e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g li m on en e = 48 .9 4% ; α -p in en e = 2. 88 % ; m yr ce ne = 2 .2 9% . g uo e t a l., 2 01 8 a nt ib ac te ria l e . c ol i, p. a er ug in os a, l. m on oc yt og en es , s . a re us , b . s ub til is , c . a lb ic an s an d s . p ar at yp hi b in hi bi tio n g uo e t a l., 2 01 8 c itr us p ar ad is i e ss en tia l o il a nt io xi da nt a nt ib ac te ria l r ad ic al s ca ve ng in g e . c ol i, s . a ur eu s, p . a er ug in os a an d c itr us a lb ic an s in hi bi tio n li m on en e = 91 .7 8% ; δ3ca re ne = 2 .0 7% . d en ko va -k os to va et a l., 2 02 0 c itr us r et ic ul at a e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g li m on en e = 61 .7 2% ; 3c ar en e = 13 .6 7% ; α -p in en e = 13 .9 7% . g uo e t a l., 2 01 8 m et ab ol ic sy nd ro m e im pr ov e th e hy pe rc ho le st er ol em ia , an d he pa tic s te at os is . r ed uc tio n in s er um to ta l c ho le st er ol , l d lc , he pa tic t c a nd t g le ve ls . li m on en e = 84 .8 9% ; δ3ca re ne = 3 .1 4% . li m on en e = 76 .5 8% ; γte rp in en e = 12 .8 8% . βm yr ce ne = 2 .4 5% . d en ko va -k os to va et a l., 2 02 0 k on gl on g et a l., 2 02 0 italian journal of food science, 2021; 33 (2) 79 citrus species: modern functional food and nutraceutical-based product ingredient a nt ib ac te ria l e . c ol i, p. a er ug in os a, l. m on oc yt og en es , s . a re us , b . s ub til is , c itr us a lb ic an s an d s. p ar at yp hi b in hi bi tio n g uo e t a l., 2 01 8 c itr us s in en si s e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g in hi bi tio n of li pi d pe ro xi da tio n li m on en e = 95 .1 1% ; m yr ce ne = 1 .0 7% . m ag al hã es e t a l., 20 19 m et ab ol ic sy nd ro m e in hi bi tio n of α -a m yl as e an d βgl uc os id as e en zy m es li m on en e = 92 .1 4% ; βm yr ce ne = 2 .7 0% . o bo h et a l., 2 01 7 a nt ib ac te ria l e . c ol i, p. a er ug in os a, l. m on oc yt og en es , s . a re us , b . s ub til is , c . a lb ic an s an d s . p ar at yp hi b in hi bi tio n li m on en e = 79 .2 8% ; 3ca re ne = 7 .7 6% ; βp in en e = 2. 28 % . g uo e t a l., 2 01 8 a nt i-i nfl am m at or y r ed uc tio n of e de m a li m on en e = 80 .5 % ; tra ns -b -o ci m en e = 6. 5% ; li na lo ol = 2 .7 % . th an di sw a et a l., 20 20 c itr us u ns hi u e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g li m on en e = 64 .2 1% ; gte rp in en e = 9. 44 % ; m yr ce ne = 8 .3 7% ; ap in en e = 4. 98 % . g uo e t a l., 2 01 8 a nt ib ac te ria l e . c ol i, p. a er ug in os a, l . m on oc yt og en es , s . a re us , b . su bt ili s, c . a lb ic an s an d s . pa ra ty ph i b in hi bi tio n g uo e t a l., 2 01 8 c itr us × cl em en tin a e ss en tia l o il a nt io xi da nt m et ab ol ic sy nd ro m e r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in hi bi tio n of li pi d pe ro xi da tio n in hi bi tio n of α -a m yl as e an d βgl uc os id as e an d lip as e en zy m es . li m on en e = 61 .3 1% ; li na lo ol = 3 .2 9– 6. 64 % ; m yr ce ne = 3 .5 6– 9. 10 % . le po rin i e t a l., 2 02 0a n r : n ot re po rt ed ; t p c : t ot al p he no lic s co nt en t; tf c : t ot al fl av on oi ds c on te nt ; t c c : t ot al c ar ot en oi ds c on te nt . 80 italian journal of food science, 2021; 33 (2) m. leporini et al. the effectiveness of citrus maxima peels ethanol extract was suggested recently in the management of diabetes (ani and ochu, 2020). indeed, the administration of this extract (600 mg/kg bw/day) for 14 days decreased the blood glucose level (70.17%), ta (30.86%), tg (10.58%), and ldl-cholesterol (10.20%). additionally, an increase of hdl-cholesterol (4.43%) was observed. dietary ingestion of citrus tumida hort. ex tanaka peels powder (5% w/w) suppressed bw gain by decreasing epidydimal, perirenal, and subcutaneous fat weights (sato et al., 2019). a similar effect, that is a significant decrease of bw, was observed for citrus aurantium extract (100 mg/kg/day) administered for 8 weeks. additionally, the same treatment in 3t3-l1 adipocytes determined a reduction of lipid droplets regulating adipogenesis and thermogenesis via amp-activated protein kinase alpha (ampkα) pathway (park et al., 2019). kegele et al. (2019) investigated the effects of citrusim® (citrus sinensis dried extract) on body composition: percentage of lean mass and percentage of fat mass. this extract determined a significant reduction of fat, and increase in lean mass reducing waist circumference after a dose of 0.5 or 1 g/day. similarly, the obese mice treated with supplementation of 0.25% and 0.5% of citrus reticulata extract in food for 12 weeks exhibited a reduction of 21% and 34% in bw, respectively (guo et al., 2016). this effect was probably due to the action of citrus phytochemicals on metabolism of glucose and fatty acids. administration of citrus sinensis methanol peels extract at doses of 50 and 100 mg/kg in diabetic rats reduced fasting blood glucose by 56.1% and 55.7%, respectively, and plasma insulin levels by 22.9% and 32.7%, respectively (sathiyabama et al., 2018). citrus medica diamante hydroalcoholic peels extract was tested in db/db mouse model for leptin deficiency. this mutation confers susceptibility to obesity, insulin resistance, and t2dm. administration of 600 mg/kg of diamante peels extract significantly decreased the serum glucose level (menichini et al., 2016). this extract was rich in phenolic compounds that are known to posses several actions to improve glucose tolerance as reported below. the in vivo reduction of blood glucose and plasma insulin levels was demonstrated for both citrus reticulata and citrus sudachi peels extract (guo et al., 2016; kobayashi et al., 2017). in particular, citrus sudachi exerted its effect via reduction of tnf-α mrna expression. literature showed that citrus genus was able to counteract the effect of high cholesterol level (favela-hernández et al., 2016). recently, the hypocholesterolemic effects of mandarin peels’ aqueous and n-hexane extracts was demonstrated (fayek et al., 2017). the results showed that these extracts decrease the cholesterol level by 59.3% and 56.8%, respectively. a reduction in cholesterol and tg levels was also observed with citrus medica cv. recently, the antioxidant activities of citrus reticulata, citrus paradise, and citrus lemon peels’ essential oils were reported (denkova-kostova et al., 2020). the radical scavenging potential on dpph radical revealed that the highest percentage of inhibition was found in the grapefruit (87.5%), followed by lemon and tangeretine with values of 86.1% and 78.0%, respectively. the antioxidant potential of grapefruit, lemon, mandarin, and orange essential oils was also investigated by raspo et al. (2020). mandarin exhibited the highest activity in abts test, grapefruits exhibited the highest activity in frap test, and lemon exhibited the highest activity in dpph test. citrus lumia essential oil showed a strong antioxidant activity in different assays, with the following order of potency (expressed as ic50): β-carotene (22 μg/ml) > orac(46 μg/ml) > dpph (104 μg/ml) > folin-ciocalteu (181 μg/ml) > frap (202 μg/ml) > trolox equivalent antioxidant capacity (teac) (233 μg/ml) (smeriglio et al., 2018). for citrus aurantium peels’ essential oil, an inhibition percentage of 88.1% against dpph radical was observed (tevena et al., 2019). a lower activity was reported for bitter orange with an inhibition percentage of 31.33% (farahmandfar et al., 2020). metabolic syndrome recently, the effects of citrus reticulata peels’ water extract (800 mg) administered to obese adolescents were analyzed (kamel et al., 2019). in this clinical trial, the extract showed a reduction in bmi, body fat percentage, and waist circumference after 4 and 8 weeks of supplementation. additionally, a reduction of total cholesterol (tc) and tg levels was observed. huang et al. (2020) compared the in vitro anti-obesity ability of grapefruit, pomelo, kumquat, mandarin, ponkan, tangerine, lemon, and sweet orange peels’ extracts. among them, the most active sample was sweet orange, followed by tangerine and ponkan with the ic50 values of 87.25, 109.44, and 126.62 mg/ml, respectively, against lipase enzymes. also, for citrus unshiu peels’ water extract, an inhibitor effect was reported for lipase activity (ic50 = 507.01 μg/ml) (kim et al., 2016). better results were observed for citrus × clementina peels extract with values in the range of 112.06–191.91 mg/ml (leporini et al., 2020a). in particular, peels extract from cetraro, obtained by ultrasound extraction etoh, exhibited the strongest hypolipidemic activity. additionally, this extract increased the hypolipidemic activity of citrus × clementina juice when added at a concentration of 20% (w/v). oboh et al. (2017) reported that the lemon peels’ essential oil exhibited stronger inhibitory activity on α-amylase and α-glucosidase activities (ic50 values of 8.16 and 7.56 µg/ml, respectively) compared to orange peels’ essential oil (ic50 values of 11.51 and 11.53 µg/ml, respectively). italian journal of food science, 2021; 33 (2) 81 citrus species: modern functional food and nutraceutical-based product ingredient essential oil, without furanocoumarins fraction, reduced levels of il-1β, il-6, and tnf-α in the paw homogenates, nitrite/nitrate, and prostaglandin e2 (pge2) contents in exudates, and possesses antioxidant properties (lombardo et al., 2020). antiproliferative activity selim et al. (2019) investigated the cytotoxicity activity of citrus reticulata peels 70% ethanolic extract against human breast carcinoma, hepatocellular liver carcinoma (hepg2), and colon carcinoma and determined the ic50 values of 34, 9.9, and 30 mg/ml, respectively. previously, the anti-cancer effects of citrus medica (2 morphotypes), citrus sinensis, citrus maxima, citrus limon, and citrus reticolata peels’ water extracts were studied (nair et al., 2018). among these, citrus reticolata had significant activity against dalton’s lymphoma ascites (dla) cell-inducing cell cycle arrest of dla in g0/g1 phase. antibacterial potential the in vivo antibacterial activity of citrus hystrix ethanol peels extract against s. typhimurium was demonstrated by zulvikar et al. (2020). in particular, the bacterial loads of this pathogen in the ileum, liver, and spleen decreased after 24 h of administration of the extract (16 mg daily for 3 days in a mouse). lime peels extract was used to inhibit the colonization and growth of bacteria s. typhi in balb/c mice. doses of 510 and 750 mg/kg bw decreased the number of s. typhi colonies; even maintenance for 20 days after the intervention showed no bacterial growth (kasim et al., 2020). sharma and tyagi (2019) analyzed benzene, ethanol, and methanol peels’ extracts of citrus nobilis and citrus sinensis against four gram-positive and four gram-negative bacteria and two fungal pathogens. the minimum inhibitory concentration (mic) values in the range of 18–40 μg/ml were found against bacillus cerus, s. aureus, s. epidermidis, proteus vulgaris, s. typhimurium, p. aeruginosa, c. albicans, and trichoderma viride for citrus nobilis ethanolic extract, while less activity was reported for methanol and benzene extracts. the same observation was made for citrus sinensis extracts, and, in particular, the mic values in the range of 20–50 μg/ml were observed for ethanolic extract. the results were in accordance with rehab et al. (2018) that reported antibacterial and antifungal effects of citrus sinensis peels’ hot, cold, and ethanol extracts against s. aureus, e. coli, p. aerogenes, b. cereus, and c. albicans. interestingly, the green synthesis of zinc oxide nanoparticles using citrus sinensis peel extract was proposed by gao et al. (2020) in food packaging application as nanocoatings on fresh strawberries with similar antibacterial characteristic of commercial zinc oxide nanoparticles. diamante peels’ hydroalcoholic extract of (300 and 600 mg/kg/day) administered in zucker diabetic rats for 4 weeks (menichini et al., 2016). successively, konglong et al. (2020) demonstrated that citrus reticulata peels’ essential oil was able to ameliorate hypercholesterolemia and hepatic steatosis. in addition, a reduction in serum tc, ldl-cholesterol, and hepatic tc and tg levels was observed after supplementation (0.5% and 0.75%). anti-inflammatory activity fermented dried citrus unshiu peel extracts were investigated for its anti-inflammatory activities in murine macrophages and moisturizing effects in human keratinocytes (kim et al., 2019a). results evidenced that citrus unshiu peels extract, rich in polyphenolic compounds, was able to suppress lipopolysaccharide (lps)-induced no without exerting cytotoxic effects on raw 264.7 cells. moreover, extracts inhibited the expression of inducible nitric oxide synthase (inos), cyclooxygenase-2 (cox-2) protein, tnf-α, and il-6. the inhibition of no without compromising cell viability was also reported for citrus reticulata peels alkaline hot water extract (ic50 1.04–2.74 mg/ml) (chen et al., 2017). recently, the anti-inflammatory effect of citrus sinensis peels’ hydroalcoholic and methanol extracts was confirmed by osarumwense et al. (2017). interestingly, methanol extract was more active than hydroalcoholic extract, and a positive control drug (indomethacin) with an inhibition of 95% on carrageenan induced rat paw edema at a concentration of 40 mg/kg. similarly, after 4 h of edema induction, the oral administration (300 and 500 mg/kg bw) of pomelo peels methanol extract determined an inhibition of paw edema by 34.47% and 38.68%, respectively (ibrahim et al., 2019). the same model of paw was used by pallavi et al. (2018), establishing that intraperitoneal (i.p.) doses (250 and 500 mg/kg) of pomelo peels extract inhibited paw edema (17% and 48%, respectively). the mandarin dichloromethane and ethyl acetate peels’ extracts against cox-1 and cox-2 were tested (hamdan et al., 2020). the dichloromethane extract was more active against cox-1 (ic50 = 25.5 mg/ml) than ethyl acetate extract (ic50 = 28.79 mg/ml); conversely against cox-2, the ethyl acetate extract had the highest activity (ic50 = 3.55 mg/ml). citrus limon essential oil exhibited anti-inflammatory activity (30 or 10 mg/kg oral [p.o.]) by reducing cell migration, cytokine production, and protein extravasation induced by carrageenan (amorim et al., 2016). treatment (200 and 50 mg/kg) with sweet orange dried peels essential oil evidenced a significant reduction of edema in rats (thandiswa et al., 2020). citrus bergamia 82 italian journal of food science, 2021; 33 (2) m. leporini et al. et al. (2020). all investigated samples exhibited radical scavenging activity with ic50 values in the same order of positive controls such as ascorbic acid and bht. among them, citrus maxima and citrus aurantium leaves showed the highest dpph radical scavenging activity with the ic50 values of 0.51 and 0.57 mg/ml, respectively (table 5). more recently, the antioxidant activity of methanol leaves extract and ethyl acetate fraction of citrus pseudolimon was examined (kumar et al., 2019). the ethyl acetate fraction displayed greater dpph radical scavenging activity than the methanol leaves extract with the ic50 values of 278.60 and 313.20 μg/ml, respectively. the ic50 values of 476.39 and 498.26 μg/ml were also found in h2o2 scavenging assay. the methanol extract of citrus medica leaves was also investigated for its capacity to inhibit dpph radical (shojaemehr et al., 2020). similar values were observed in extracts (ic50 = 0.111 mg/ml) and ascorbic acid (ic50 = 0.109 mg/ml) used as control. the citrus × clementina leaves subjected to different extractions were investigated for their antioxidant potential (leporini et al., 2020b). the hydroalcoholic extract obtained by using ultrasound-assisted maceration had the highest antioxidant dpph, abts, frap, and βcarotene bleaching values. previously, methanol and aqueous leave extracts of citrus clementina, citrus limon, citrus hamlin, citrus navel, citrus aurantifolia, citrus aurantium, and citrus grandi were investigated for their antioxidant activity (khettal et al., 2017). among aqueous extracts, citrus limon had an important dpph radical scavenging activity (ic50 = 35.35 µg/ml), while citrus clementina exhibited the highest abts radical scavenging activity (ic50 = 1,174.43 µm te/g) and ferric reducing potential (ic50 = 30.60 mg butylhydroxyanisole equivalents (bhae)/g). regarding methanolic extracts, citrus clementina showed the highest antioxidant activity in all assays with the ic50 values of 41.85 µg/ml, 378.63 µm te/g dm, and 13.85 mg bhae/g dm for dpph, abts radicals scavenging activities, and ferric reducing potential, respectively. the antioxidant potential of citrus macroptera leaf methanol extract has been recently demonstrated by lala et al. (2020) that reported the capacity of this extract to reduce ros, which was generated on hepg2 cell line. previously, bonesi et al. (2018) investigated six citrus petitgrain essential oils for their antioxidant properties. in this study, citrus aurantium petitgrain oil demonstrated the strongest radical scavenging activity in dpph assay with an ic50 value of 27.2 μg/ml, followed by citrus  × clementina oil with an ic50 value of 39.0 μg/ml, while in β-carotene bleaching test, the highest antioxidant capacity was observed with citrus sinensis oil with the ic50 values of 176.3 and 51.3 μg/ml after 30 and 60 min of incubation, respectively. less activity was reported for clementine essential oils by leporini et al. (2020b). the antimicrobial potential of citrus sinensis l. and citrus limonia osbeck methanol, ethyl acetate, ethanol, and distilled water peels extracts was also evaluated by saleem and saeed (2020) against six gram-positive (s.  aureus, aeromonas hydrophila, enterococcus faecalis, streptococcus pyogenes, listeria monocytogenes, and lactobacillus casei), six gram-negative (p. aeruginosa, k.  pneumoniae, serratia marcescens, e. coli, p.  vulgaris, and s. typhi), two microscopic filamentous fungi (aspergillus niger and penicillium citrinum), and two yeasts (c. albicans and saccharomyces cerevisiae). interestingly, the zone of inhibition is well comparable with amoxicillin, used as a positive control. in addition, the yellow lemon extract exhibited the highest antimicrobial activity compared to orange peels, and resulted more effectively on gram-negative bacteria as compared to gram-positive bacteria. strawberries treated with citrus limon, citrus sinensis, and citrus reticulata essential oils showed the highest tac and physicochemical parameters compared to untreated fruits. this effect extends the shelflife and delays the fruit senescence (shehata et al. 2020). the antimicrobial effects of tangerine, grapefruit, and lemon peels’ essential oils on the growth of saprophytic and pathogenic microorganisms were compared by denkova-kostova et al. (2020). the highest inhibitory activity was observed for grapefruit, followed by tangerine and lemon essential oil, with mic values in the range of 60–60 ppm against e. coli, s. aureus, p. aeruginosa, and citrus albicans. similarly, grapefruit and lemon have respective mic values of 0.35 mg/ml and 0.33 mg/ml against e. coli (raspo et al., 2020). the antimicrobial effect of bitter orange essential oil against gram-positive and gram-negative selected bacterial strains was studied by farahmandfar et al. (2020). mic values of 20, 40, and 10 mg/ml were found, respectively, for e. coli, p. aeruginosa, s. aureus, and l. monocytogenes. the addition of citrus medica essential oil to the wines (0.010%) determined reduction in microbial counts compared to untreated wine, and is thus proposed as bio-preservative. in particular, it the antimicrobial activity of enriched wine against the common spoilage bacteria and yeasts/molds such as gluconobacter cerinus, oenococcus oeni, pediococcus pentosaceus, dekkera bruxellensis, candida zemplinina, hanseniaspora uvarum, pichia guilliermondii, or zygosaccharomyces bailii was studied and inoculated (mitropoulou et al., 2020). leaves antioxidant effects the antioxidant activity of leaf methanol–water extracts of 10 varieties of citrus fruits was reported by haraoui italian journal of food science, 2021; 33 (2) 83 citrus species: modern functional food and nutraceutical-based product ingredient ta bl e 5. b io lo gi ca l p ro pe rt ie s of c itr us le av es le av es e xt ra ct /e ss en tia l o il b io lo gi ca l a ct iv ity m ec ha ni sm tp c , t fc , t c c , a nd /o r m ai n ab un da nt id en tifi ed co m po un ds r ef er en ce s c itr us a ur an tif ol ia w at er a nd m et ha no l a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er tp c = 5 .7 7– 10 6. 05 m g g a e /g d w ; tf c = 2 .7 2– 38 .3 6 m g q e /g d w . k he tta l e t a l., 2 01 7 e th an ol m et ab ol ic s yn dr om e r ed uc tio n in th e to ta l s er um c ho le st er ol n r c yn di e t a l., 2 01 6 c itr us a ur an tiu m w at er a nd m et ha no l a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er tp c = 7 .7 7– 69 .9 7 m g g a e /g d w ; tf c = 5 .0 8– 11 .9 9 m g q e /g d w . k he tta l e t a l., 2 01 7 c itr us g ra nd is w at er a nd m et ha no l a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er tp c = 2 .4 8– 68 .2 3 m g g a e /g d w ; tf c = 1 .0 4– 13 .0 6 m g q e /g d w . k he tta l e t a l., 2 01 7 c itr us li m on w at er a nd m et ha no l 80 % m et ha no l a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in hi bi tio n of li pi d pe ro xi da tio n tp c = 3 .8 3– 98 .0 6 m g g a e /g d w ; tf c = 2 .8 3– 38 .7 3 m g q e /g d w . tp c = 3 0. 51 m g g a e /g ; tf c = 1 4. 64 m g q e /g . k he tta l e t a l., 2 01 7 h ar ao ui e t a l., 2 02 0 w at er m et ab ol ic s yn dr om e r ed uc tio n of th e b w a nd p la sm a in su lin le ve ls n r th om as e t k am at h, 2 01 7 c itr us m ac ro pt er a m et ha no l a nt io xi da nt r ed uc tio n of r o s tp c = 2 4. 55 m g g a e /g e xt ra ct ; la la e t a l., 2 02 0 a nt i-i nfl am m at or y r ed uc tio n of e de m a tf c = 1 0. 76 m g q e / g e xt ra ct . a nt ib ac te ria l s ta ph yl oc oc cu s sp . a nd k le bs ie lla s p. in hi bi tio n c itr us m ax im a 80 % m et ha no l a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in hi bi tio n of li pi d pe ro xi da tio n tp c = 9 1. 76 m g g a e /g ; tf c = 1 6. 98 m g q e /g . h ar ao ui e t a l., 2 02 0 e th an ol m et ab ol ic s yn dr om e r ed uc tio n of t g , t c , h d l, l d l, v ld l se ru m le ve l a nd b w n r d in es h an d h eg de , 2 01 6 a nt ib ac te ria l m . l ut eu s, s . e pi de rm is , b . s ub til is , a nd e . f ec al is in hi bi tio n h ar ao ui e t a l., 2 02 0 c itr us m ed ic a m et ha no l a nt io xi da nt r ad ic al s ca ve ng in g tp c = 1 02 .7 m g g a e /g e xt ra ct ; s ho ja em eh r e t a l., 2 02 0 a nt ib ac te ria l b . s ub til is , b . c er eu s, s . a ur eu s, m . l ut eu s, e . f ae ca lis , p . a er ug in os a, k . p ne um on ia e, s . t yp hi a nd e . c ol i in hi bi tio n tf c = 3 .9 5 m g g a e /g e xt ra ct . c itr us p se ud ol im on m et ha no l a nt io xi da nt r ad ic al s ca ve ng in g tp c = 1 0 m g g a e /g e xt ra ct tf c = 7 .9 m g g a e /g e xt ra ct k um ar e t a l., 2 01 9 m et ab ol ic s yn dr om e in hi bi tio n of α -a m yl as e an d βgl uc os id as e en zy m es . r ed uc tio n of b lo od g lu co se le ve l c itr us s in en si s 80 % m et ha no l a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in hi bi tio n of li pi d pe ro xi da tio n tp c = 3 509 -6 2. 59 m g g a e /g ; tf c = 3 .6 7– 6. 39 m g q e /g . h ar ao ui e t a l., 2 02 0 (c on tin ue s) 84 italian journal of food science, 2021; 33 (2) m. leporini et al. ta bl e 5. co nt in ue d le av es e xt ra ct /e ss en tia l o il b io lo gi ca l a ct iv ity m ec ha ni sm tp c , t fc , t c c , a nd /o r m ai n ab un da nt id en tifi ed co m po un ds r ef er en ce s m et ab ol ic s yn dr om e in hi bi tio n of li pa se e nz ym e tp c = 2 09 .2 7 m g g a e /g f w ; tf c = 6 5. 02 m g q e / g f w . a nt ib ac te ria l m . l ut eu s, s . e pi de rm is , b . s ub til is , a nd e . f ec al is in hi bi tio n h ar ao ui e t a l., 2 02 0 c itr us u ns hi u m et ha no l m et ab ol ic s yn dr om e in hi bi tio n of li pa se e nz ym es n r ito h et a l., 2 01 9 c itr us × c le m en tin a 80 % m et ha no l e th an ol a nd 8 0% e th an ol a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in hi bi tio n of li pi d pe ro xi da tio n tp c = 3 1. 43 m g g a e /g ; tf c = 4 .8 4 m g q e /g . tp c = 1 33 13 –4 5. 54 m g g a e /g f w ; tf c = 5 .5 0– 29 .1 6 m g q e /g f w ; h es pe rid in = 1 74 .9 1– 65 6. 66 m g/ 10 0 g fw ; r ut in = 4 .2 4– 68 .5 2 m g/ 10 0 g fw ; is oq ue rc itr in = 7 .7 3– 52 .0 1 m g/ 10 0 g fw ; s in en se tin = 7 .2 4– 35 .6 9 m g/ 10 0 g fw ; ta ng er et in =7 .4 6– 41 .7 6 m g/ 10 0 g fw ; h ar ao ui e t a l., 2 02 0 le po rin i e t a l., 2 02 0a m et ab ol ic s yn dr om e in hi bi tio n of α -a m yl as e, β -g lu co si da se an d lip as e en zy m es le po rin i e t a l., 2 02 0a a nt ib ac te ria l m . l ut eu s, b . s ub til is , a nd e . f ec al is in hi bi tio n h ar ao ui e t a l., 2 02 0 c itr us a ur an tif ol ia e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g li m on en e = 63 .3 5% ; g er an io l = 6 .2 3% ; c itr al = 4 .3 5% . a l-a am ri et a l., 2 01 8 m et ab ol ic s yn dr om e r ed uc tio n in fa st in g bl oo d, h ep at ic gl uc os e, t c , t ria cy lg ly ce ro l a nd l d lc li m on en e = 57 .8 4% ; n er al = 7 .8 1% ; li na lo ol = 4 .7 4% . ib ra hi m e t a l., 2 01 8 a nt ib ac te ria l s . a ur eu s, a nd p . a er ug in os a in hi bi tio n e . c ol i, s . t yp hi , a nd b . c er eu s in hi bi tio n li m on en e = 30 .1 1% ; bpi ne ne =1 9. 27 % ; bo ci m en e = 3. 48 % : c hi e t a l., 2 02 0 a l-a am ri et a l., 2 01 8 c itr us a ur an tiu m e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in hi bi tio n of li pi d pe ro xi da tio n s ab in en e= 39 .8 1% ; li na lo ol = 1 3. 75 % ; gte rp in en = 7 .4 3% ; d3ca re ne =6 .5 5% b on es i e t a l., 2 01 8 a nt ifu ng al c . a lb ic an s in hi bi tio n n id hi e t a l., 2 02 0 c itr us b er ga m ia e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in hi bi tio n of li pi d pe ro xi da tio n li na ly l a ce ta te = 7 0. 51 % ; li na lo ol =1 0. 25 % ; g er an yl a ce ta te = 3 .0 4% . b on es i e t a l., 2 01 8 c itr us g ra nd is e ss en tia l o il a nt io xi da nt a nt ib ac te ria l r ad ic al s ca ve ng in g s . a re us , s . t yp hi , a nd b . c er eu s in hi bi tio n li m on en e = 21 .8 7% ; α -c ar yo ph yl le ne = 6 .7 5% ; βoc im en e = 6. 35 % . c hi e t a l., 2 02 0 italian journal of food science, 2021; 33 (2) 85 citrus species: modern functional food and nutraceutical-based product ingredient c itr us li m on e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in hi bi tio n of li pi d pe ro xi da tio n li m on en e = 30 .5 7% ; g er an ia l = 1 4. 44 % ; βpi ne ne = 1 4. 98 % : b on es i e t a l., 2 01 8 a nt ib ac te ria l li st er ia in hi bi tio n s . a ur eu s, e . c ol i, an d b . s ub til is in hi bi tio n g er an io l = 2 98 .6 5 m g/ m l; li m on en e= 25 6. 87 m g/ m l; g er an ia l = 9 8. 39 m g/ m l; n er al = 8 6. 81 m g/ m l. n r fa nc el lo e t a l., 2 02 0 s ae b et a l., 2 01 6 c itr us r et ic ul at a e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in hi bi tio n of li pi d pe ro xi da tio n s ab in en e = 39 .8 1% ; li na lo ol = 1 3. 75 % ; γte rp in en e = 7. 43 % ; δ3ca re ne = 6 .5 5% : b on es i e t a l., 2 01 8 a nt ib ac te ria l s . a ur eu s, e . c ol i, an d b . s ub til is in hi bi tio n n r s ae b et a l., 2 01 6 c itr us s in en si s e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in hi bi tio n of li pi d pe ro xi da tio n li m on en e = 13 .7 7% ; βpi ne ne = 1 6. 93 % ; βoc im en e = 7. 48 % . s ab in en e = 49 .6 7% ; βoc im en e = 9. 25 % ; li m on en e = 5. 48 % c hi e t a l., 2 02 0 b on es i e t a l., 2 01 8 a nt i-i nfl am m at or y r ed uc tio n of e de m a s ab in en e = 20 .4 % ; te rp in en -4 -o lo = 1 3. 2% ; li m on en e = 7. 5% ; δ3ca re ne = 6 .5 5% . th an di sw a et a l., 2 02 0 a nt ib ac te ria l s . a re us , s . t yp hi , a nd b . c er eu s in hi bi tio n c hi e t a l., 2 02 0 c itr us × c le m en tin a e ss en tia l o il a nt io xi da nt r ad ic al s ca ve ng in g fe rr ic -r ed uc in g po w er in hi bi tio n of li pi d pe ro xi da tio n s ab in en e = 27 .8 4% ; li na lo ol = 1 9. 76 % ; li m on en e = 6. 44 % : b on es i e t a l., 2 01 8 m et ab ol ic s yn dr om e in hi bi tio n of α -a m yl as e, α -g lu co si da se a nd li pa se e nz ym es li na lo ol = 1 5. 80 % ; li m on en e = 6. 41 % ; βo ci m en e = 6. 52 % ; δ3ca re ne = 6 .3 3% . le po rin i e t a l., 2 02 0b le po rin i e t a l., 2 02 0b n r : n ot re po rt ed ; t p c : t ot al p he no lic s co nt en t; tf c : t ot al fl av on oi ds c on te nt ; t c c : t ot al c ar ot en oi ds c on te nt ; r o s : r ea ct iv e ox yg en s pe ci es . 86 italian journal of food science, 2021; 33 (2) m. leporini et al. and citrus sinensis cv. jaffa leave extracts with an inhibition area of 20.00 mm and 16.00 mm, respectively. for gram-negative bacteria, the best results were observed for the citrus lemon extract with an inhibition area of 15.66 mm (p. aeruginosa) and 15.33 mm (e. coli). the antibacterial effects of different extracts obtained from citrus medica leaves were tested. interestingly, the inhibitory activity of methanol extract on b. cereus, e. coli, and e. aerogenes was more potent than the gentamicin used as a positive control (shojaemehr et al., 2020). citrus aurantium leaves essential oil demonstrated strong antifungal activity against two strains of citrus albicans with mic values of 0.15–0.31% (v/v) (nidhi et  al., 2020). interestingly, citrus limon var pompia leaves essential oil showed specific anti-listeria activity on ricotta salata cheese (fancello et al., 2020). recently, de oliveira filho et al., (2020) proposed a chitosan films enriched with citrus limonia leaves essential oil as an active packaging material for food preservation for its capacity to: (a)  reduce the moisture content and water vapor permeability; (b) decrease the visible light transmission rate values; (c) change the color of bioactive films significantly, remaining darker and yellowish; and (d) inhibit s. aureus. similarly, the addition of lemon essential oil to chitosan coatings enhanced fermentative process during storage, with modification of strawberry fruit aroma composition notably appreciated (perdones et al., 2015). bioactive compounds polyphenolic compounds are a wide group of metabolites that originate from the secondary metabolism of plants. these are considered as potent antioxidants for their capacity to increase catalase activity, trap reactive oxygen species, and to act as a metal chelator. additionally, they determined the inhibition of chain lipid peroxidation by trapping peroxyl radical and quickly reacted with peroxy nitrite (pisoschi and pop, 2015). flavonoids and phenolic acid (figure 1) are dominant bioactive compounds found in citrus. in particular, peels are rich in flavone aglycons and polymethoxy flavones, rarely found in other plants. polyphenols are present in both edible and nonedible parts of the fruits (singh et al., 2020). in addition, citrus fruit is a good source of carotenoids (figure 2) compounds recognized for their beneficial effects on human health (ikoma et al., 2016). citrus by-products represented a rich source of essential oils that possessed a wide range of antioxidant, antimicrobial, and antidiabetic properties, and thus used in pharmaceutical and food industries (bora et al., 2020). metabolic syndrome in recent decades, numerous in vitro and in vivo studies have demonstrated the importance of genus citrus in the prevention of t2dm. recently, the hypoglycemic effects of citrus × clementina leaves extract has been reported by leporini et al. (2020b), who found the ic50 values of 64.37–247.61 mg/ml in α-amylase enzyme and the ic50 values of 51.61–282.65 mg/ml against α-glucosidase. in particular, hydroalcoholic extract obtained by ultrasound-assisted maceration from corigliano calabro leaves was found to be the most active. the addition of this extract to the juice increased its hypoglycemic (+37% and +25% against α-glucosidase and α-amylase, respectively) and hypolipidemic (+17% against lipase) potential. the inhibitory activity of citrus unshiu leaf methanol extract on pancreatic lipase enzyme was reported by itoh et al. (2019) that showed an ic50 value of 44 µg/ml. citrus pseudolimon methanol leave extracts and ethyl acetate fraction possessed a hypoglycemic potential (kumar et al., 2019). the ethyl acetate fraction displayed a greater inhibition against α-glucoside (84.18%) in comparison to the methanol extract (82.94%). the ic50 values of 83.66% and 78.52% for ethyl acetate and methanol extract, respectively, were found against α-amylase. in addition, the authors indicated that oral administration of methanol leaves extract (200 mg/kg) and ethyl acetate fraction (100 mg/kg) for 21 days decreased the fasting blood glucose level in diabetic rats. aqueous extract of citrus limon leaves was tested against stz-induced diabetic rats. this extract, orally administered at doses of 50 mg/kg bw and 100 mg/kg bw for 28 days, decreased bw and plasma insulin levels and increased blood glucose levels (thomas and kamath, 2017). the hypocholesterolemic effects of citrus aurantifolia was reported by cyndi et al. (2016). indeed, the ethanol extract of leaves determined reduction in tc serum in mice, with the most significant reduction at a dosage of 3.5 g/kg bw. similarly, the oral administration of citrus maxima leaves extract (200 and 400 mg/kg bw) in obese rats determined reduction in tg, tc, hdl, ldl, and very low-density lipoprotein (vldl) serum levels and bw (dinesh and hegde, 2016). antibacterial effect more recently, haraoui et al. (2020) investigated the antibacterial activity of leaves methanol water extracts obtained from citrus aurantium, citrus maxima, citrus lemon, citrus clementine, and citrus sinensis cv. sanguinelli, thomson, washington, portuguese, double fine, and jafa. m. luteus resulted in the most sensitive gram-positive bacteria to the action of citrus aurantium italian journal of food science, 2021; 33 (2) 87 citrus species: modern functional food and nutraceutical-based product ingredient in an in vivo study, hesperidin and naringin increased the production and release of insulin from the islet cells and decreased intestinal glucose absorption (mahmoud et al., 2015). in addition, hesperidin and hesperetin inhibited two gluconeogenesis enzymes, alanine aminotransferase and aspartate aminotransferase, indicating their effectiveness in treating diabetes mellitus (zareei et al., 2017). the therapeutic potential of hesperidin has been confirmed recently (rehman et al., 2020b). this flavanone  improved leptin and insulin resistance, il-6 and tnf-α more significantly compared to the reference drug orlistat used in high fat diet (hfd)-induced obese rats. in addition, the treatment with 500-mg hesperidin significantly reduced the plasma levels of c-reactive protein and serum amyloid a in individuals with ms (homayouni et al., 2017). moreover, hesperidin reduced symptoms of ms and improved cardiac function in hfdinduced ms in rats (prasatthong et al., 2021). indeed, treatment with hesperidin (15 or 30 mg/kg) ameliorated cardiac dysfunction and hypertrophy in rats, restored the insulin signaling pathway, and irs/akt/glut4 protein expression. the consummation (500 ml) of orange juice enriched with hesperidin had positive effects on blood and pulse pressures in mildly hypertensive individuals (valls et al., 2021). the results are in accordance with a recent study in which high blood pressure was attenuated by hesperidin (50 mg/kg bw). regulation in the expressions of tnf-α, cox-2, and pge2 with improvement of oxidative stress by increasing glutathione reductase and decreasing malondialdehyde (mad) was also observed (khidr et al., 2020). previously, the cardioprotective effect of hesperidin was investigated by haidari et al. (2015). administration of 600 mg/day of hesperidin decreases levels of adiponectin and hdl-cholesterol and increases e-selectin in patients with myocardial infarction. the neuroprotective activity of hesperidin was evaluated by thenmozhi et al. (2015). in this study, administration of 100 mg/kg of hesperidin along with aluminum chloride (alcl3) injection for 60 days significantly reduced the concentration of ros in hippocampus and cortex, the ache activity, the protein expressions of amyloid precursor protein, the levels of both ab1–42 and b and g secretases. recently, li and schluesener (2017) demonstrated that administration of 100 mg/kg of hesperidin for 10 days significantly attenuated α-amyloid deposition and microglial activation in brain of transgenic mice. the combination of diosmin and hesperidin exerted analgesic and/or anti-inflammatory effects (patent no. flavonoids flavonoids are secondary metabolites in plants, with a multitude of functions: they regulate the development of plants, their pigmentation, and protect them from uv-light. furthermore, they act as defense and signaling between plants and microorganisms (mathesius, 2018). hesperidin hesperidin is one of the main flavanone glycosides know in citrus fruits. great attention has been focused on hesperidin and its aglycone form, hesperetin, which plays an important role in the prevention of diseases associated with oxidative stress such as obesity, diabetes, inflammation, and cancer (barreca et al., 2017). its antioxidant mechanism was correlated to direct ros scavenging, transition metal ion chelation, and its ability to increase cellular glutathione content. de souza et al. (2016) compared the antioxidant activity of hesperidin, hesperetin, and g-hesperidin in vitro and in vivo, administrating each of these for 30 days at 1 mmol/kg body mass to wistar male rats. the aglycone form has the greatest inhibitory activity of xanthine oxidase by increasing superoxide dismutase (sod) activity in the liver of animals. recently, the antioxidant activity of hesperidin, and its ability to inhibit pancreatic lipase enzyme, was studied (huang et al., 2020). results demonstrated that hydrogen bonds and van der waals forces played major roles in the interaction of hesperidin and lipase. the metabolic effects of hesperidin were also demonstrated by sahnoun et al. (2017) and zeng et al. (2018), who reported its ability to inhibit α-amylase, α-glucosidase, and lipase enzyme with the ic50 values of 111 and 1 μm, and 688.25 μg/ml, respectively. in a randomized double-blind controlled clinical trial design, 23 subjects with t2dm consumed 500 mg/day hesperidin supplement for 8 weeks. hesperidin supplementation led to significant decrease in fasting blood glucose and glycosylated hemoglobin (hba1c). a significant increase in serum insulin and decrease in tg were also observed in the hesperidin-treated group (eghtesadi et al., 2016). similarly, the supplementation with hesperidin (500 mg/ day for 8 weeks) in t2dm patients resulted in reduction of fasting blood glucose, tc, and hba1c, and at the same time a significant increase in serum insulin (mohammadi et al., 2016). a dose of 100 mg or 500 mg of hesperidin for 6 weeks in subjects with hypercholesterolemia decreased serum tg and ldl levels (li and schluesener, 2017). in addition, intra-gastric hesperidin attenuates the increased level of plasma cholesterol, ldl-cholesterol vldl-cholesterol, tg, free fatty acids, and phospholipids, and decreased levels of highdensity lipoprotein-cholesterol (hdl-c) (homayouni et al., 2017). 88 italian journal of food science, 2021; 33 (2) m. leporini et al. (a) structure of flavones (b) structure of flavones (c) structure of flavonols (c)(b)(a) (d) chlorogenic (a), gallic (b) and caffeic acid (c) name r1 r2 r3 r4 r5 r6 r7 naringenin oh h oh h h oh h hesperetin oh h oh h oh och3 h narirutin oh h o-rut h h oh h naringenin oh h o-neo h h oh h poncirin oh h o-neo h h och3 h eriocitrin oh h o-rut h oh oh h neoeriocitrin oh h o-neo h oh oh h hesperidin oh h o-rut h oh och3 h neohesperidin oh h o-neo h oh och3 h didymin oh h o-rut h h och3 h name r1 r2 r3 r5 r5 r6 r7 apigenin oh h oh h h oh h luteolin oh h oh h oh oh h sinensetin och3 och3 och3 h och3 och3 h tangeretin och3 och3 och3 och3 h och3 h nobiletin och3 och3 och3 och3 och3 och3 h name r1 quercetin oh kaempferol oh rutin och3 r5 r6 r7 r4 r3 r2 r1 o o r5 r1 oh oh oh o oho ho ho ho ho cooh oh oh oh oh oh oh ho o o o co2h r6 r7 r4 r3 r2 r1 o o figure 1. the main phenolic constituents of citrus species. wo2015019334) as reported by lópez muñozmaría et  al. (2015). this application was used for the treatment of different kinds of pain: moderate to severe pain, chronic pain, and/or neuropathic pain. no occurrence of adverse effects was observed. supplementation of a mixture of imperata cylindrical, citrus unshiu markovich-hesperidin, and evodia officinalis dode-evodiamine for 12 weeks significantly reduced the bw, body fat mass, and waist circumference in overweight subjects (cho et al., 2017). recently, it was italian journal of food science, 2021; 33 (2) 89 citrus species: modern functional food and nutraceutical-based product ingredient r1 ho ho ho o oh oh β-cryptoxanthin β-carotene lutein violaxanthino r1 r2 r2 name figure 2. the most abundant citrus carotenoids. reported that hesperidin ameliorates hepatic dysfunction and dyslipidemia in male wistar rats exposed to cadmium chloride (aja et al., 2020). hesperetin recently, hesperetin showed cellular antioxidant activity with a value of 23.57 μmol of qe/100 μmol (huang et al., 2020). both hesperidin and hesperetin were able to reduce oxidative stress directly by scavenging intracellular ros and increase natural antioxidant defense system with particular reference to glutathione (dhanya and jayamurthy, 2020). in addition, these flavonoids inhibited the non-enzymatic glycation of proteins involved in the formation of advanced glycation end-products which have an important role in developing diabetes. previously, jayaraman et al. (2018) investigated the anti-hyperglycemic, antioxidant, and anti-hyperlipidemic effects of hesperetin against stz-induced experimental rats. supplementation with 40 mg/kg of hesperetin for 45 days determined a significant decline in plasma glucose level and a marked improvement in insulin and glycogen secretions. hesperetin is also known to induce apoptosis in cancer cells primarily through activation of caspase-9 (farooqi et al., 2015). this compound revealed significant cytotoxicity for hela cell line, and its anticancer ability was revalidated by in silico molecular docking study, which exhibited strong interaction with e6 protein of hpv16 cervical carcinoma with significant binding energy (prakash et al., 2020). the capacity of hesperetin to attenuate testicular alteration in wistar rats was also reported through inhibition of inflammation, oxidative stress, and apoptosis (samie et al., 2018). interestingly, li et al. (2018; patent no. cn108815154a) investigated the ability of hesperetin to inhibit chloride channel and propose its use for the treatment of diarrhea, heart disease, pulmonary disease, stomach, brain, and mental diseases, rhinitis, ontological disease, and eye disease drug development. hesperetin administered orally (50 mg/kg/day for 46  days) reduced ros, dna fragmentation, serum glucose, mda levels, and caspase 3 activity. in addition, this compound potentiated testicular antioxidant system with consequent increase in glutathione levels, ferric-reducing antioxidant power, catalase (cat), sod, and glutathione peroxidase (gpx) activity in diabetic rats (samie et al., 2018). shagirtha et al. (2017) has recently demonstrated the neuroprotective properties of hesperetin. the oral administration of this flavanone (40 mg/kg bw for 21 days) protected the brain of wistar rats by increasing the levels of enzymatic antioxidants such as cat, sod, gpx, and glutathione-s-transferase (gsts). in addition, hesperetin reduced oxidative stress, neuroinflammation, and motor dysfunction as well as amyloidogenesis and cognitive dysfunction in mice with positive effect against parkinson’s and alzheimer’s diseases (khan et al., 2020). neohesperidin the in vivo hypoglycemic and hypolipidemic effects of neohesperidin on kk-a(y) mice were studied (jia et  al., 2015). treatment with neohesperidin significantly 90 italian journal of food science, 2021; 33 (2) m. leporini et al. moreover, naringenin decreased blood glucose, serum lipid, and ameliorated glucose tolerance through downregulating oxidative stress and inflammation in stzinduced rats (jia et al., 2015). liang et al. (2015; patent no. cn104940932a) reported the protective effects of naringenin and naringin during radiotherapy. additionally, the use of naringenin and its derivative in preventing alzheimer’s disease and other cognitive disorders was reported (liao, 2018; patent no. cn108785301a). the administration of naringenin (50 mg/kg/day) increased the serum level of insulin and consequently glucose uptake, improved lipid profile, tnf-α, il-6, normalized level of no, and increased sod level (rehman et al., 2020c). these effects were confirmed by wu et al. (2016); they showed how this compound inhibited the expression of cytokine signaling, inos, cox-2, and release of no and pro-inflammatory cytokines in microglial cells. a direct effect of this flavanone determined downregulation of genes involved in de novo lipogenesis, lipolysis, and triglyceride synthesis/storage. moreover, narirutin and didymin are able to inhibit lipase enzyme with the ic50 values of 58.98 and 67.30 μg/ml, respectively (zeng et al., 2018). didymin didymin acted as an anticancer agent by inhibiting phthalate-mediated invasion, migration, and proliferation of breast cancer cells (hsu et al., 2016), and as a scavenger of free radicals (lin et al., 2016). more recently, ali et al. (2019) demonstrated that didymin was also able to inhibit α-glucosidase and α-amylase enzymes and increase glucose uptake. in addition, didymin reduced the expression of two key enzymes involved in the gluconeogenesis such as glucose 6-phosphatase and phosphoenolpyruvate carboxy-kinase with a consequent decrease of glucose production. recently, it was found that didymin prevented hyperglycemia-induced ros, production of lipid peroxidation product mad, hyperglycemia induced monocyte-endothelial cell adhesion, and nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kb) activation. in addition, this compound inhibited the release of various inflammatory cytokines and chemokines (kirtikar et al., 2018). eriocitrin eriocitrin is known as a strong antioxidant agent (smeriglio et al., 2019). it has been shown that a major role is played by its two hydroxy groups that are bound to the b ring in ortho position with respect to each other (diab et al., 2015). this flavanone (200 mg/kg) showed protective effects against inflammation and oxidative stress in c57bl/6j mice, and may therefore prevent metabolic alterations associated with the development of cardiovascular diseases (ferreira et al., 2016). decreased serum glucose, fasting glucose, glycosylated serum protein, and insulin resistance. moreover, this bioactive compound significantly decreased tc, serum tg, leptin level, and inhibited lipid accumulation. lv et al. (2015) also noted that naringin and neohesperidin mainly inhibited amylose digestion. in addition, the neohesperidin administration (50 mg/kg/day) attenuates weight gain, low-grade inflammation, and insulin resistance in mice, as well as restored gut barrier damage and metabolic endotoxemia (lu et al., 2020). a novel pharmaceutical use of neohesperidin in the preparation of drug for treating bronchial asthma or diseases caused by th1/th2 cell immune imbalance was disclosed (shi and yang, 2018; patent no. cn108478586). the efficacy of citrus flavonoids on ms resulted in the commercialization of bergavit®, a standardized extract containing 150 mg of main active flavonoids of bergamot juice (16% of neoeriocitrin, 47% of neohesperidin, and 37% of naringin). this supplement was administrated at a fixed daily dose for 6 months in patients with moderate hypercholesterolemia. results revealed reduction in tg, tc, and ldl-cholesterol (toth et al., 2016). it has been demonstrated recently that the neohesperidin inhibited angiotensin ii-induced myocardial contractile dysfunction, and reduced hypertension, myocardial hypertrophy, fibrosis, sod production, and inflammation (zhang et al., 2020). naringenin naringin, as reported by sahnoun et al. (2017), showed an excellent inhibition for α-amylase and α-glucosidase enzyme, with ic50 values of 8.0 μm and 0.55 μm, respectively. lim et al. (2018) studied the protective effects and molecular mechanisms of naringin in diabetic mice. the results showed that this flavanone ameliorated hyperglycemia and protected stz-induced β-cell death by inhibiting both intrinsic and extrinsic apoptotic pathways. these protective effects have been related to the ability of naringin to reduce ros and pro-inflammatory cytokines accumulation. it was suggested recently that antioxidant and anti-inflammatory properties of naringenin could confer hepatoprotective effects after oral treatment with 60 mg/kg bw (kometsi et al., 2020). in a clinical study, administration of naringin (400 mg/ capsule/day) for 8 weeks in hypercholesterolemic individuals resulted in reduced concentration of plasma tc and ldl-cholesterol. naringin exerted its effect by inhibiting gluconeogenesis and upregulation of ampk, hence metformin-like effects. in addition, it increased glucose uptake in skeletal muscles, ameliorated pro inflammatory reactions, and prevented metabolic dysregulation and atherosclerosis (nyane et al., 2017). italian journal of food science, 2021; 33 (2) 91 citrus species: modern functional food and nutraceutical-based product ingredient (100  mg/kg/day for 6 weeks) ameliorated lps-triggered memory deficit regarding synaptic dysfunctions and neuronal loss, and inhibited the microglial activation and pro-inflammatory cytokine secretion (il-1β, cox-2, tnf-α, and inos). in addition, in bv-2 microglia cells, the action of this flavone decreased pro-inflammatory cytokines secretion, and channeled modulation of mitogen-activated protein kinase (mapks), phosphatidylinositol 3-kinase/phosphorylated protein kinase b (pi3k/akt), and nf-κb signaling pathways. interestingly, nobiletin promotes antioxidant and anti-inflammatory responses and elicits protection against ischemic stroke in vivo with increase in the expression of sod and glutathione (gsh) which are responsible of antioxidant endogenous defense systems. moreover, a reduction in the levels of nf-κb and mda was also observed (zhang et al., 2016). wen-zhe et al. (2015; patent no. us9808477b2) detected a pharmaceutical composition for multidrug-resistant cancer treatment comprising citrus methoxyflavone (nobiletin) and chemotherapeutic drug. in addition, chen and wang (2015; patent no. cn105030559a) proposed application of nobiletin in preparation of health products or medicines for prevention and/or treatment of oral cancer. the experiments showed that these compounds possessed an obvious effect on inhibiting proliferation of human oral epidermoid carcinoma cells through the anti-proliferation effects of hesperetin, naringenin, and nobiletin on human oral epidermoid carcinoma cells. tangeretin both nobiletin and tangeretin ameliorated ros production and lipid peroxidation in mutant saccharomyces cerevisiae deficient in glutathione synthase, sod, or cat (wang et al., 2018). similarly, a significant decrease in ros content, with increase in the activities of sod, cat, and gpx through inhibition of nf-κb pathway in rats’ insulinoma cell line (ins-1) pre-treated with tangeretin (0, 10, or 20 μm) for 12 h was also observed (liu et al., 2019b). recent report has elucidated the anti-obesity capacity of tangeretin via inhibition of pancreatic lipase. this compound inhibited the enzyme with an ic50 value of 57.31 mg/ml (zeng et al., 2018). moreover, tangeretin ameliorated insulin resistance and increased glucose uptake by attenuating obesity-induced inflammation in adipose tissue through reduction of no production, the expression of il-6, il-1β, tnf-α, inos, and cox-2 in 3t3-l1 adipocytes and macrophage cell line (shin et al., 2017). sahnoun et al. (2017) evaluated the inhibitory activities of tangeretin on carbohydrate metabolism key enzymes. this pentamethoxy flavone showed the ic50 values of 141.0 μm and 14.8 μm against α-amylase and α-glucosidase, respectively. recently, the neuroprotective effect of tangeretin against cerebral ischemia-reperfusion injury was demonstrated more recently, kwon and choi (2020) proposed a possible eriocitrin mechanism of action. in this study, dietary supplementation with eriocitrin (0.005%) in c57bl/6n mice for 16 weeks improved adiposity by increasing adipocyte fatty acid oxidation, energy expenditure, mrna expression of thermogenesis-related genes in brown adipose tissue and skeletal muscle, and decreasing the expression of lipogenesis-related genes in white adipose tissue. the supplementation with eriocitrin also decreased hepatic lipogenesis and prevented hyperlipidemia whereas increased hepatic fatty acid (fa) oxidation and fecal lipid excretion. moreover, eriocitrin supplementation improved insulin resistance, glucose tolerance, and decreased hepatic gluconeogenesis and pro-inflammatory responses. previously, liu et al. (2019a; patent no. cn109806272a) proposed eriocitrin as potential α-glucosidase inhibitor. nobiletin nobiletin is one of the most abundant polymethoxylated flavones. this compound was investigated for its capacity to improve and prevent obesity and metabolic diseases. recently, the application of nobiletin in preparation treatment of gastric accommodation disorder remedies was reported (li, 2019; patent no. cn108619130b). this compound selectively relaxes stomach smooth muscles, promotes the recovery of physiological gastrointestinal motility, and calms stomach upset. as a new therapeutic agent, it has provided and presented great market prospects and economic value. sahnoun et al. (2017) reported the carbohydrate hydrolyzing enzymes inhibitory activity of nobiletin with the ic50 values of 42.0 μm and 50.0 μm against α-amylase and α-glucosidase, respectively. this flavone was also able to inhibit lipase with an ic50 value of 26.28 mg/ml (zeng et al., 2018), with ic50 value being better than that those reported for the positive control. in db/db diabetic mice, oral administration of nobiletin (200 mg/kg bw for 10 weeks) significantly attenuated bw gain, decreased fasting glucose levels, improved glucose tolerance and insulin sensitivity, and diminished serum tg levels (he et al., 2016). moreover, nobiletin was able to reduce the protein peroxisomal acyl-coenzyme a oxidase 1, carnitine palmitoyltransferase-1, and ameliorated fatty acids β-oxidation via ampk (lone et al., 2018). in addition, treatment with this compound at 10–100 mg/kg bw for 8 weeks in obese mice accelerated lipid catabolism in adipose tissues. recently, it was found that nobiletin improved cognitive deficits and the pathological features of alzheimer’s disease, such as aβ pathology, hyperphosphorylation of tau, and oxidative stress (nakajima and ohizumi, 2019). in addition, nobiletin ameliorated motor and cognitive deficits in parkinson’s disease models. qi et al. (2019) also demonstrated that oral administration of nobiletin 92 italian journal of food science, 2021; 33 (2) m. leporini et al. quercetin quercetin has been used as a nutritional supplement and may have beneficial effects against a variety of diseases. several in vitro and in vivo studies have evidenced its biological functions. recently, doustimotlagh et al. (2020) suggested the ability of quercetin (50 mg/kg/ day for 10 days) to cause a significant decrease in protein carbonyl, hydroxyproline, and to regulate the gpx activity. therefore, quercetin acted as an enzyme inducer by renewing the glutathione peroxidase activity and inhibiting the oxidation of proteins, and hence decreases ros production. these results confirmed the positive role of quercetin in attenuating the liver damage and degeneration. milanezi et al. (2019) analyzed the antioxidant activity of quercetin-capped gold nanoparticles. quercetin-capped gold nanoparticles (ir50 0.37 μg/ml) exhibited greater activity than free quercetin (ir50 0.57 μg/ml) by no free radical scavenging assay. similarly, quercetin vesicular formulations (eudragitcoated liposomes) were capable of ensuring optimal protection against oxidative stress in human intestinal cells by reducing ros production, as reported by caddeo et al. (2019). its antioxidant capacities were correlated to the presence of two antioxidant pharmacophores in the molecule that had optimal configuration for free radical scavenging. the high antioxidant potential of quercetin was also confirmed in superoxide test with the ic50 values of 0.025 mm versus 0.243 mm, for quercetin and kaempferol, respectively. increasing in vivo studies have proved that quercetin acted as an antioxidant because of its ability to ameliorate antioxidant defenses, decrease free radical formation, and inhibit xanthine oxidase and lipid peroxidation (shi et al., 2019). literatures data show that quercetin was able to reduce glucose levels when it was administered at a minimum dose of 30 mg/kg bw for 14 days (yang and kang, 2018). additionally, this compound potentiated insulin secretion induced by glucose and glibenclamide and protected β-cells against oxidative damages (shi et al., 2019). it was reported recently that the oral administration of quercetin (25 and 50 mg/kg) for 28 days remarkably reduced the level of blood glucose, hba1c, hepatic glycogen, and restored the activity of glucose-6-phosphatase and hexokinase in diabetic rats (oyedemi et al., 2019). eid et al. (2015) proposed the use of quercetin as an antidiabetic compound, since this flavonoid could act through the stimulation of glut4 translocation in the skeletal muscle and the inhibition of glucose-6-phosphatase in hepatocytes. in a human study of 12-week, lee et al. (2016) used 100 mg/day/subject of quercetin to treat obesity and showed that this compound diminished the total body fat, and decreased the bmi of overweight or obese subjects. in (yang et al., 2020). this compound downregulated the inflammatory and pro-inflammatory cytokines and oxidative stress parameters in the serum and brain tissues of rats with suppression of il-1β, tnf-α, and il-6. lee et al. (2018; patent no. kr102015221b1) proposed the application of tangeretin for the prevention and treatment of post-traumatic stress disorder. this compound showed an excellent anti-anxiety effect, and was consequently included in the pharmaceutical composition of foods as an active ingredient. moreover, tangeretin was an active ingredient for alleviating, preventing, or treating renal fibrosis or cirrhosis of kidney glomerulus or albuminuria (young-hee and min-kyung, 2018; patent no. kr101949471b1). sinensetin the effects of sinensetin on lipid metabolism in mature 3t3-l1 adipocytes without causing cytotoxicity were reported by kang et al. (2015). this compound showed anti-adipogenic property by downregulation of sterol regulatory element-binding protein 1c, and lipolytic property with increase of lipase enzyme. moreover, sinensetin inhibited insulin-stimulated glucose uptake by decreasing the phosphorylation of insulin receptor substrate, and increased the phosphorylation of ampk and acetyl-coa carboxylase. it also upregulated mrna expression of carnitine palmitoyltransferase-1a, suggesting that sinensetin enhances fatty acid β-oxidation through ampk pathway. in addition, it was found that sinensetin quenched the fluorescence of α-glucosidase, and inhibited α-glucosidase and non-enzymatic glycation (liu et al., 2020). kim et al. (2019b) reported the anti-inflammatory activities of sinensetin on lpsstimulated l6 skeletal muscle by regulating nf-κb. recently, the application of sinensetin as an active ingredient for preventing, ameliorating, or treating liver cancer or gastric cancer has been proposed (kim and lee, 2018; patent no. kr20190050535a). luteolin sangeetha (2019) reported the antioxidant activity of luteolin and demonstrated how this polymethoxyflavone protects the pancreas and promotes insulin secretion. in addition, luteolin suppressed oxidative damage, lipid peroxidation, and increased antioxidant enzymes such as cat and sod (xu et al., 2019). antioxidant properties of luteolin are also proved in the central nervous system (cns). the inhibition of gastric secretion and reduction of pepsin activity by luteolin was reported by dai and li (2018; patent no. cn108309971b). in particular, the preparation includes 3–5 parts of luteolin and 1–2 parts of schisandrin b as active ingredients, and the dosage form of the compound preparation was preferably tablets, capsules, injections, and granules. italian journal of food science, 2021; 33 (2) 93 citrus species: modern functional food and nutraceutical-based product ingredient levels of tg and vldl, and increased the level of hdl. additionally, rutin decreased ros formation, advanced glycation end-product precursors, and production of inflammatory cytokines. the anti-inflammatory activity of rutin was recently confirmed by su et al. (2019). authors evidenced the inhibition of nf-κb pathway and understatement of endoplasmic reticulum stress. phenolic acids phenolic acids are a diverse class of phenolic compounds made by plants. they act as agents of plant defense, and are, indeed, immensely important in plant–microbe interactions/symbiosis (mandal et al., 2010). chlorogenic acid chlorogenic acid is an important bioactive dietary polyphenol. several studies have reported the ability of chlorogenic acid to act in metabolic disease through different mechanisms of action. recently, use of chlorogenic acid in the treatment of metabolic disorders was proposed (kodimule, 2018; patent no. us20190111015a1). chlorogenic acid supplementation in hypercholesterolemic rats at a dose of 20 or 90 mg/kg bw for 12 weeks suppressed serum lipid levels, while a dosage of 10 mg/ kg significantly reduced total ldl-cholesterol and increased hdl-cholesterol by upregulating the expression of ppar-γ gene (huang et al., 2015). additionally, administration of chlorogenic acid at a dose of 80 mg/kg bw for 12 weeks decreased percentage of body fat, fasting plasma glucose, and hba1c level via modulation of adiponectin receptor signaling pathways (jin et al., 2015). recently, di wang et al. (2019) reported that chlorogenic acid (100 mg/kg/day bw) taken for 4 weeks ameliorated the survival rate after myocardial infarction and demonstrated that this compound showed a protective effect on myocardial infarction by reducing inflammatory response, exerting antioxidant activity, and minimizing weight gain. similarly, chlorogenic acid (100 or 150 mg/ day) reduced oxidative-induced damage and increased antioxidant protection in the inflamed paw skin, and reduced lipid peroxidation in serum (mitrea et al., 2020). the effect of chlorogenic acid (100 mg/kg bw for 13 weeks) on energy balance in obese mice has been studied recently (he et al., 2020). this compound reduced food intake, increased body temperature, thermal dissipation, brown adipose tissue activity, and improved glucose tolerance. the anti-obesity effect of chlorogenic acid was also observed in male sprague–dawley rats at a dose of 20 or 90 mg/kg bw for 12 weeks (huang et al., 2015). oboh et al. (2015a) evaluated the inhibitory effects of chlorogenic acid on α-amylase and α-glucosidase enzymes. this compound showed the ic50 values of 9.10 μg/ml and 9.24 μg/ml for α-amylase and α-glucosidase, addition, quercetin ameliorated mitochondrial functions in adipose tissue of hfd-induced obese mice by increasing the levels of oxidative stress-sensitive transcription factor and antioxidant enzymes (kobori et al., 2016). kaempferol the protective effect of kaempferol against oxidative stress in stz-induced diabetic rats was evaluated by al-numair et al. (2015). kaempferol administration (100 mg/kg bw) to diabetic rats reduced plasma glucose, insulin, and lipid peroxidation products enzymatic such as sod, cat, gpx, and gsts. another study (alkhalidy et al., 2018) demonstrated that oral administration of kaempferol (50 mg/kg/day than corresponding human equivalent dose of 240 mg/ day for 60 kg) ameliorated blood glucose control in obese mice as well as reduced hepatic glucose production and improved insulin sensitivity. additionally, these authors have found that kaempferol was a direct inhibitor of pyruvate carboxylase and suppressed gluconeogenesis in hepg2 cells. torres-villarreal et al. (2019) studied the kaempferol effects (60 μm for 21 days) in order to evaluate its lipolytic and anti-adipogenic potential. the results of anti-obesity effects showed that kaempferol modulated adipogenic differentiation in 3t3-l1 cells through promoting downregulation of cebpa gene expression and decreased lipid accumulation in mature adipocytes for its positive effects on pnpla2 and lipe mrna levels. rutin rutin is considered a strong antioxidant agent; in fact, it acts as free radical scavenger, metal ions chelator, and reducing agent (kaurinovic et al., 2019). in stz-induced diabetic rats, oral administration of 50 or 100 mg/kg bw of this compound decreased fasting blood glucose as well as hba1c levels. moreover, chronic administration of 200 mg/kg bw of rutin reduced (30– 40%) the prevalence of diabetes in stz-treated mice (ghorbani, 2017). in addition, rutin treatment (50 mg/ kg) for 24 weeks arrested the biochemical disturbances of diabetic retinopathy, lowering vascular endothelial growth factor (vegf), tnf-α, and increasing tac in the retina (gupta et al., 2019). this compound also acted in reducing adiposity, increasing energy expenditure, and improving glucose homeostasis in obese mice (yuan et al., 2017). the positive effects of rutin on lipid profile was also proved (wang et al., 2015). glucose and lipid metabolism are strictly correlated. the most important clinical manifestation of this interaction is diabetic dyslipidemia characterized by high level of tg, ldl, and vldl. rutin, among its antidiabetic effects, decreased serum 94 italian journal of food science, 2021; 33 (2) m. leporini et al. contains lactobacillus sporogenes (1×107 cfu), 0.1-g inulin, and 0.05-g β-carotene. results showed that this synbiotic food had favorable effects on homeostatic model assessment of insulin resistance, insulin, tg, vldlcholesterol, and tc/hdl-cholesterol ratio, and no and glutathione levels. antioxidant immune response, and anti-inflammatory, anti-diabetic, and antitumor activities of β-carotene are also reported (torregrosa-crespo et al., 2018). in addition, existence of a positive effect of β-carotene on insulin sensitivity in obese patients through a positive regulation of adiponectin, either directly or via its pro-vitamin, was also suggested (ben amara et al., 2015). lutein (β, ε-carotene-3,30-diol) acted as a powerful antioxidant, prevented hfd-induced atherosclerosis in apoe-deficient mice by inhibiting nadph oxidase and increasing ppar-γ gene expression (han et al., 2015). additionally, it protects dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (mptp)induced apoptotic death and motor dysfunction by ameliorating mitochondrial disruption and oxidative stress (nataraj et al., 2016). β-cryptoxanthin is used as a coloring agent for food products in certain countries. it is associated with the e number, e161c β-cryptoxanthin, obtained from its common food sources. it exhibits high bioavailability, and β-cryptoxanthin-rich foods might be considered equivalent to β-carotene-rich foods as a source of retinol (burri et al., 2016). recently, dhuique-mayer et al. (2020) suggested that citrus × clementina juice enriched in β-cryptoxanthin (43 μg/g), hesperidin (2,850 μg/g), and pectin (376 mg/100 g) can be used for prevention of ms/t2dm. moreover, the cancer preventive effects of β-cryptoxanthin have been described (leoncini et al., 2016). the study included over 6,000 subjects with oral, laryngeal, and pharyngeal cancers. the treatment with β-cryptoxanthin determined a reduction of at least 18% in the rate of oral and pharyngeal cancers and a reduction of 17% in the rate of laryngeal cancer. lycopene, one of most potent oxygen-quenching reagents among carotenoids, possessed the ability to inhibit the reactions initiated by free radicals, such as peroxy radicals or hydroxyl radicals. indeed, cellular enzymes glutathione s-transferase, superoxide dismutase, and quinone reductase were activated by lycopene with consequent protection cells against ros (supatra, 2019). owing to its antioxidant potential, lycopene (a) facilitated cell-to-cell communication at sites called “gap junctions” and consequently prevent cancer from developing; (b) stimulated the immune system; (c) regulated the endocrine communication pathways; and (d) regulated the cell reproductive cycle, preventing development of cancer (supatra, 2019). caseiro et al. (2020) also reported the ability of lycopene respectively. additionally, the same authors suggested its antioxidant properties with an ic50 value of 38.83 μg/ml. caffeic acid the antioxidant protection of caffeic acid and chlorogenic acid against oxidative stress was studied in vivo using by4741 strain and sod and glutathione-deficient mutants of s. cerevisiae (prudêncio et al., 2019). in the cell viability tests, caffeic acid showed higher stress tolerance, with a 106% increase in s. cerevisiae by4741. however, in the sod mutant, the effect of chlorogenic acid was stronger than caffeic acid, with a 3.3-fold increase. conversely, in the glutathione-deficient mutant both treatments showed a similar level of protection. arriagada et al. (2019) proposed the use of a hybrid nano-carrier consisting of core-shell silica nano-spheres linked to the surface with caffeic acid. these nano-spheres characterized by a potentiated antioxidant property accept the caffeic acid alone. gallic acid gallic acid was able to restore vitamin c and gsh levels in the pancreas of stz-treated rats (kahkeshani et  al., 2019). yang (2018; patent no. cn108464949a) disclosed a kind of antioxidant lightening compositions and its applications. the antioxidant lightening compositions include element of orange peels (tangeretin) and gallic acid. carotenoids carotenoids are a group of natural tetraterpenoid pigments distributed widely in plants. they play essential roles: (a) in photosynthesis and photoprotection; (b) as precursors for the biosynthesis of phytohormones; and (c) as signaling molecules to mediate plant development and responses to environmental cues (sun et al., 2018). in humans, carotenoids were recognized for their biological activities associated with the reduction of risk of developing chronic diseases such as cancer, cardiovascular and neurodegenerative diseases as well as metabolic disease. additionally, these compounds acted as antioxidants and protected the cells against free radicals formed in the tissues. some of these compounds are vitamin a precursors (cardoso et al., 2017). β-carotene (figure 2) is an intense orange-colored pigment used as a food coloring agent (milne, 2005). in nature, β-carotene is a vitamin a precursor, which is synthesized from carotenoids via the action of enzyme β-carotene 15,150-monooxygenase. the beneficial effects of β-carotene-fortified synbiotic food intake on metabolic status were studied in t2dm patients (asemi et al., 2016). the β-carotene-fortified synbiotic food also italian journal of food science, 2021; 33 (2) 95 citrus species: modern functional food and nutraceutical-based product ingredient (figure  3), and d-limonene is the most abundant element. this monocyclic terpene is consumed by humans as an ingredient of traditional foods and is listed in the code of federal regulations, as generally recognized as a safe (gras) and used as a flavoring agent (roberto et al., 2010). the ameliorative effects of limonene on cadmium-induced genotoxicity in cultured human peripheral blood lymphocytes has been demonstrated recently (verma et al., 2019). in this in vitro study, at concentrations of 20 and 100 μm, it reduced the sister chromatid exchange frequency and peroxidation of lipids. d-limonene reduced weight gain percentage, tc, ldl, and vldl, and increased the level of hdl-cholesterol (khan et al., 2019). in addition, the monoterpene (400 mg/kg) increased the levels of thiobarbituric acid (tbars), sod, cat, and (gsh) in the liver tissue after treatment for 28 days. these results agreed with those reported by yu et al. (2017); the authors observed how treatment with 50 or 100 mg/kg of d-limonene increased the levels of endogenous antioxidant enzymes. the treatment with limonene (50 mg/kg) displayed anti-inflammatory activity through decreasing tnf-α, il-6, and il-1β levels and increasing the level of il-10 (de souza et al., 2019). additionally, this compound determined reduction in gastric ulcer area (93%) and myeloperoxidase activity. increase in gpx activity was also observed. limonene has also reported its ability to protect pc12 cells against corticosterone-induced neurotoxicity by activating the ampk pathway (tang et al., 2019). in fact, reductions were observed in mda to protect lipids, proteins, and dna from oxidative damage, and stimulate the modulation of cell growth and the expression of connexin 43, insulin-like growth factor-1 and/or blood levels of insulin-like growth factor-binding proteins, as well as intermediate levels in the immune system and inflammatory processes. in addition, lycopene improved insulin sensitivity through inhibition of signal transducer and activator of transcription 3/srebp1c-mediated lipid accumulation and inflammation in mice fed with hfd (zeng et al., 2017). terpenes terpenes are the largest class of natural products applied in industrial sector as flavors, fragrances, and spices as well as used in perfumery and cosmetics.  in plants, these act as defense against biotic and abiotic stresses, or they are treated as signal molecules to attract insects for pollination (singh and sharma, 2015).  ameh and obodozie-ofoegbu (2016) reported the utilization of citrus essential oil as flavorings in carbonated cola and citrus soft drinks. in particular, lemon–lime sodas contain citrus limon, citrus aurantifolia, and citrus aurantium essential oils as main flavorings, while orange sodas contain citrus aurantium oil as the main flavoring constituent. the chemical variation of each component in citrus essential oil is based on variety, season, and geographical position as well as the ripening phase of the fruit (bora et al., 2020). the major components are monoterpenes ch3 ch3 ch3 h (a) (b) (c) (d) (e) h2c h figure 3. chemical structure of main monoterpenes of citrus essential oils: (a) sabinene, (b) limonene, (c) δ-3-carene, (d) linalool, (e) β-caryophyllene. 96 italian journal of food science, 2021; 33 (2) m. leporini et al. and no levels, nadph oxidase activity, inos, cox-2, il-6, il-1β, tnf-α, and expressions of pro-apoptotic proteins. another monoterpene found particularly abundant in citrus essential oil is sabinene which acts as a potential modulator of bacterial resistance. it could act in synergism with antibiotics to reduce mic values against bacterial strains of pa03 and sa358 (matias et al., 2016). linalool is an acyclic monoterpene tertiary alcohol (figure 3) and is one of the most investigated aroma compounds. currently, linalool and citral are mainly used as flavoring and natural preservatives due to their antimicrobial and antifungal ability. indeed, they were used to extend the short shelf-life of seafood products and cheese because of their capacity to reduce populations of microorganisms, especially enterobacteriaceae (bora et al., 2020). at a concentrations of 0.1%, linalool exhibited antimicrobial activity against different strains such as s. aureus, e. coli, b. subtilis, and pasteurella multocida, with major activity against gram-positive bacteria than gram-negative bacteria. baldissera et al. (2017) evaluated the effect of β-caryophyllene on hypercholesterolemia in rats and the possible effect on hepatic antioxidant enzymes. administration of β-caryophyllene at a dose of 1.0 ml/kg for 3 days reduced the levels of tc, ldl-cholesterol, and tg, inhibited the hmg-coa reductase activity, and increased the antioxidant system of ros and tbars levels. these results agree with those reported by basha and sankaranarayanan (2016), who investigated the effect of β-caryophyllene on hyperglycemia. oral administration of this compound (200 mg/kg bw) for 45 days reduced the level of glucose and increased the level of insulin, with restored antioxidant status enhancing the activity of cat, sod, and gpx as well as inhibition of pro-inflammatory cytokines, tnf-α and il-6. it has been recently demonstrated that β-caryophyllene reduced pge2 and inos production and cox-2 expression (hu et al., 2017). varga et al. (2018) have successively evidenced that at a dose of 10 mg/kg bw, this compound improved the chronic and binge alcohol-induced liver injury and inflammation by attenuating the pro-inflammatory phenotypic “m1” switch of kupffer cells and diminishing the expression of e-selectin, p-selectin, and neutrophil infiltration. additionally, it ameliorated the hepatic metabolic dysregulation, such as protein hyperacetylation, steatosis, and ppar-γ – gene signaling. these protective effects were correlated to activation of type-2 cannabinoid receptor. interaction with this receptor causes the expression of vascular cell adhesion molecule-1 mediated by the jak2/stat1/irf-1 pathway (zhang et al., 2017). conclusion a critical review of recent studies on the health properties of different portions of citrus fruits and their major bioactive compounds was reported. it was interesting to observe that not only the edible portion but also its by-products are characterized by high biological value. a large 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_hlk57974142 _hlk68158860 _hlk59615671 _hlk57792959 _hlk59541856 _hlk57792847 _hlk59551210 paper ital. j. food sci., vol. 27 2015 1 keywords: trans fatty acids, consumer awareness, polish students, dutch students, eating habits comparison of knowledge in the field of nutritional fats among students s. onacik-gür a, a. żbikowska a and m. kowalska b* a. faculty of food science, warsaw university of life sciences (sggw), poland b faculty of materials science, technology and design, kazimierz pulaski university of technology and humanities in radom, poland, *corresponding author: tel./fax: 48 48 3617547, email: mkowalska7@vp.pl abstract the aim of this work was to analyze the knowledge in the field of trans fatty acids (tfas) and nutritional recommendations related to fats among students at the university of life sciences in warsaw (sggw) and wageningen (wur). the research was done using a questionnaire composed of 16 questions among 194 students from sggw and wur in 2012. in poland 96% and in holland 89% of students had heard the name “trans fat”. more than half of the questionnaire respondents knew industrial sources of tfas. after comparing the results of the research, in which students succeeded, it was concluded that differences in the level of knowledge were statistically not significant (p<0.05). eating habits for fatty pastry products representing a potential source of tfas were similar. wur students’ purchase of pastry products was dependent on price, whereas that of polish students depended on information and ingredients listed on the package. 2 ital. j. food sci., vol. 27 2015 introduction fats are one of the fundamental food ingredients, which play a significant role in the human body. their nutritional value depends on the composition of fatty acids (fas) and content of diluted vitamins. the most important for the organism are fas, from the group of efas (essential fatty acids) (gurr, 2000; bayir et al., 2011), and they are easily oxidized. fats used for technological purposes should be resistant to high temperatures and storage conditions. for pastry products and frying fats, mainly fats rich in saturated fatty acids (sfas) and sometimes trans fatty acids (tfas) are used. in the manufacture of many food products it is necessary to use fats with solid consistency. it could be a natural solid fat or a modified fat (mcdonald and mossoba, 1996; matthäus, 2007). it is possible to alter the characteristics of fats by using hydrogenation, transesterification, mixing and fractionation. hydrogenation is one of the oldest methods, where double bonds in triacylglycerols (tag) are saturated. as a result of this treatment unsaturated fatty acids (ufas) become sfas. moreover, trans isomerization of fas occurs within the process of hydrogenation, which results in tfas. the modified fat has a higher melting point and increased resistance to oxidation (flaczyk and korczak, 2002; matthäus, 2007; downes et al., 2013). tfas may occur in natural products originating from ruminants. as a result of enzyme activity in ruminants’ bodies there occurs trans isomerization from cis bonds to trans (goodman et al., 2001; efsa, 2004; gebauer et al., 2007). the content of tfas originating from natural sources (from meat and milk) may range from 2 to 8 g/100 g fat depending on kind, breed, way of feeding and season. in europeans’ diet on average 30% of consumed tfa comes from milk and 10% from meat. fats developed with industrial hydrogenation way may include even 70% of tfa in the whole fas (matthäus, 2007; jasti and kovacs, 2010; krasnowska and salejda, 2011). cis-trans isomerization may also happen during high temperature treatment, for example during deodorization of oils or frying (krasnowska and salejda, 2011). according to most researchers, trans fatty acid isomers have a negative impact on human health. because of the hazards related to excessive consumption of tfas, international organizations such as efsa and who have stated that daily intake for adults and children should be reduced as much as possible (efsa, 2004; downes et al., 2013). fats rich in tfas and sfas have a negative impact on the cardiovascular system. they contribute to the cholesterol rise in the blood and the higher risk of cardiovascular diseases. there are also reports indicating negative influences of tfa on other human body organs. consumption of 5 g of sfas a day may increase the risk of heart diseases by about 2% and of tfas by even 25% (martin, 2007). due to the unfavorable effect of tfas on the human body, legal restrictions of their content in food products have been implemented (denmark and canada). a different solution to reduce the consumption of tfas is to require labeling of food products with information about the content of tfas (canada, usa) (żbikowska, 2010; downes et al., 2013). denmark is the first country in the world to introduce a tax on food products containing sfas. the aim of this tax is to reduce the consumption of products rich in saturated fas and increase the consumption of fruits and vegetables (simopoulos, 1996). due to the negative impact of tfa on the body and any actions concerning with reduction of their consumption, it is important to follow the level of consumer’s education in this area. it can be assumed that people with higher education related to food and nutrition sciences, interested in the subject of nutrition, should avoid consumption of products that may adversely affect their health, eg: products with trans fatty acids. the aim of this study was to verify how the education of students at both universities influenced the knowledge in the field of tfas, purchases of food products and eating habits. therefore students’ scores from the test were compared among students from studies related to food and health sciences with different studies (economic, marketing, agriculture, etc.). in addition, it was hypothesized that students of a master’s degree course, especially from majors related to food and nutrition, are characterized by a high level of knowledge in the field of tfas and fats. materials and methods the analysis was based on the results of a survey carried out in 2012 among students from the university of life sciences in warsaw (sggw) (in poland) and the university of life sciences in wageningen (wur) (in the netherlands). before the questionnaire was distributed to respondents, a pilot test had been done. 215 people participated in the survey, but 194 of these questionnaires were qualified. 90 questionnaires were completed by students with different nationalities from wageningen university (table 1), while the rest of the respondents were polish students from sggw. the survey was addressed to students finishing their education at universities, from fields of studies related to food and nutrition, and also from other majors, but declaring an interest in the issues of food and nutrition. at first the study was conducted in the netherlands. participants of courses related to food sciences, which are in the master’s degree program, were invited to take part in the surital. j. food sci., vol. 27 2015 3 vey. students taking the bachelor’s degree were allowed to participate in these classes (table 1) only if they had finished specialized courses. participants of these elective classes were students representing different majors. therefore people who took part in the study were divided into two groups: respondents studying subjects related (fs) and not related to food and nutrition sciences (nfs). students from wur came from different countries, mainly of the european union (table 1). master’s degree studies at this university are open, which means that students are recruited from all over the world and classes are taught in english. then, the study was conducted on respondents from sggw in poland. they were mainly students taking a master’s degree, from majors related and not related to food and nutrition science. students not attending any food faculties declared that they were interested in food related topics. in this connection, although there was a small population size it is assumed that the obtained results will answer the question whether it is necessary to intensify the education of students (including those that theoretically should have a high nutritional knowledge) in the subject of food fats especially those that contain trans isomers. the survey was held in the classrooms and the questionnaire was distributed in a paper version to respondents. the interviewer was present with the students. surveys that were distributed to students in the netherlands were written in english, while in poland they were in polish. questions in terms of content were divided into those which were a test of the respondents’ knowledge (10 questions) and observations of the students’ eating habits. in the questionnaire there were also basic questions – legal information (tables 1 and 2). most of the respondents were women (table 2). most of the respondents were aged 24. in the case of wur they constituted 24.4% of the whole population, while in sggw 55.8%. the average respondents’ age in holland was around 25 and in poland 23. in the netherlands the age range was much wider than in poland. the eldest respondent of the survey was 58 years old. in the discussion results of students from studies related to food and human nutrition and students from studies not related to these fields were analyzed separately. with regard to questions concerning the knowledge test a scoring system was used in which each respondent received one point per correct answer. the maximum score was 13 points. such presentation of responses helped to interpret the results and assess the level of knowledge of the students. survey results were analyzed in excel 2011 and the statistical program statistica v10.0. one-way analysis of variance and χ2 test were applied, with the significance level p<0.05. in order to conduct statistical analysis the responses were expressed as a percentage of answers. results and discussion analysis of the answers given by respondents concerning knowledge of trans fatty acids over 90% of the examined populations were students from studies related to food or health and respondents interested in this topic. all students studying in the field related to food and nutrition had heard earlier the name “trans fat”. however, 65% of wur and 80% of sggw students from other studies had heard this term (fig. 1). similar studies were conducted in canada, where since 2005 it has been obligatory to provide information on the content of tfas. on this basis, it was found that food selection based on tfas increased significantly (smed, 2012). a study conducted in 2007 among students of different fields of study (related to food and not related) in the u.s. (where since 2006 it has been obligatory to label food products containing tfas) showed that 92% of respondents were aware of the term “trans fat” table 1 nationality of wur students. country contribution (%) netherlands 38 spain 18 czech republic 10 finland 4 germany 4 hungary 4 usa 4 canada 4 china 4 france 2 greece 2 turkey 2 denmark 2 sweden 2 table 2 characteristics of the studied population. legends’:*fs – students from studies related to food science or human nutrition; **nfs – students from studies not related to food science. specification structure of population (%) wur sggw sex women 78 81 men 22 19 age avarage 24.6 24 sd 5.60 0.99 field of studies fs* 38 81 nfs** 62 19 degree of studies bechelor’s 16 8 master’s 84 92 4 ital. j. food sci., vol. 27 2015 fig. 1 students who had heard the term “trans fat”. sggw university of life sciences in warsaw; wur wageningen university; all – all of the respondents; fs – students from studies related to food science or human nutrition; nfs – students from studies not related to food science. fig. 2 sources of tfas indicated by students of (a) wur and (b) sggw. *all all of the respondents; * fs, nfs explanation under the table 2. (stampfer et al., 1991). based on this, it can be assumed that the subject of tfas is more widespread in the u.s. (also among students of subjects not related to food or nutrition). most of the respondents correctly indicate as the main source of tfas 3 groups of products: shortening, hard margarines and pastry products (fig. 2). the other correct answers related to natural sources of trans fatty acids (milk fat and dairy products) were selected much more often (30% more) by the students from wur (fig. 2a) than from sggw (fig. 2b). the aim of the following question was to verify whether respondents knew that food products might be a natural source of tfa developing naturally in bodies of ruminants. in research done by stampfer (1991) 59% of students considered confectionery products as a source of tfas in their diet. ital. j. food sci., vol. 27 2015 5 analysis of answers given by respondents concerning knowledge of the nutritional role of tfas and fat most tfas, mainly coming from industrial fats, have a negative influence on the human body. they contribute to increase of cardiovascular diseases. only in the case of conjugated linoleic acid (cla), which is present in the fat of ruminants, is it believed that the effect is positive (gebauer et al., 2007). therefore, the next question was to check the students’ knowledge about the impact of tfas on human health. at both universities more than two thirds of the students answered that tfas have an adverse effect on human health (fig. 3). similarly, in another survey (stampfer et al., 1991) 73% of american students considered tfas as negative food components for health. another question, testing knowledge in the field of fats with particular emphasis on tfas, considered nutritionally valuable elements which are supplied to the body from fat. as nutritionally beneficial elements, respondents chose the following answers: essential fatty acids (efas), vitamins and, unfortunately incorrectly, saturated fatty acids (sfas). all of the students who study food and nutrition and related fields at sggw selected efas and half of them vitamins (fig. 4a). among those students who study food science and nutrition at wur, 89% of them selected the answer for efas and 32% for vitamins. several people (12%) from the dutch university thought that tfas are nutritionally important (fig. 4b). fig 4 nutritionally valuable elements indicated by students of (a) sggw and (b) wur. explanation under the fig. 1. fig. 3 structure of responses to the question “are tfas good for one’s health?”. a no, b yes, c it depends on their origin, d i do not know; explanation under the fig.1. 6 ital. j. food sci., vol. 27 2015 results of the knowledge test from the test of knowledge in the field of fats, students from sggw in warsaw scored around 9 points and students from wur 8 points, from the 13 possible (fig. 5). polish students from studies related to food and nutrition sciences did not show any statistically significant differences (p < 0.05) in higher knowledge in the field of tfas and fats than the respondents from unrelated studies (fig. 5). however, differences were significant in the case of students from the dutch university. eating habits of respondents students of both universities were eating pastry and confectionary products at a similar frequency (table 3). it could be due to similar availability of these types of products in shops and vending machines at universities. women were consuming much more of these types of products than men. in the netherlands 80% and in poland 62% of female students declared that they consume sweet snacks at least several times a week. men had better eating habits, because two thirds of sggw male students and nearly all male students from wageningen university stated that they eat such products very rarely. men from the polish university were eating confectionary and pastry products more often than men studying in holland. researchers (urbańska and czarniecka-skubina, 2007) previously reported that 21% of high-school students were eating sweet snacks every day, while 31% consumed them several times a week. thus, it can be concluded that university students eat fewer sweets than high-school students. tfas develop during frying. the most sensitive to trans isomerization in heat treatment are oils rich in unsaturated fas. moreno et al. (1999) reported that the tfa content substantially increases at 150°c and at 200°c reaches 357% compared to the initial content of trans isomers, while at 300°c it reaches the level of 3026%. that is why, for frying, it is advisable to use fats with a high thermal resistance. almost all of the students declared that they use vegetable oil for this reason (94% of wur respondents and 90% sggw). flaczyk and korczak (2002) obtained a slightly different structure of answers of the question related to the fat used for frying. they found that, for frying meat, the most popular fat was lard (32% of answers) and vegetable oil (25%), for fish vegetable oil (91%), fig. 5 averages of achieved points from students’ test results. a, a – statistically not significant; a, a – statistically not significant; a, b statistically significant (p = 0,00291). *explanation under the fig. 1. table 3 frequency of pastry product consumption among sggw and wur students. frequency of consumption contribution of answers (%) wur sggw women men all women men all once a month / not at all 11 10 11 19 10 17 2-4 times a month 9 90 27 19 60 27 2-4 times a week 43 0 33 31 0 25 almost everyday 37 0 29 31 30 31 ital. j. food sci., vol. 27 2015 7 and for eggs table margarine (63%). to fry flourbased products 40% of the respondents favored the use of vegetable oils. students’ decisions related to purchase of pastry and confectionery were influenced by different factors (table 4). in the netherlands the most popular factors of purchase taken into consideration were price (29% of answers) and information on the package (24%). however, polish students indicated first of all the ingredients and the information on the packages (38%) and then the brands (27%). therefore the polish students appreciated above all quality of these products and were guided as well by the trust of previously eaten products of the brands. regardless of universities, one fifth of students claimed that they always chose the same products. in the following question the obtained answers among the students from both universities were different and statistically significant (p = 0.04654). in turn, the studies of krasnowska and salejda [2011] showed that 37% of respondents were influenced by the price of products and then by the expiration date and brand (each 26%). the results of this research are very different from the answers obtained by the students from the polish university. conclusions differences in the level of students’ knowledge, independently of university, were statistically not significant (p<0.05). taking into account the fact, the all of the examined population’s interest in nutrition aspects, it can be concluded that knowledge in the topic of trans fas was not satisfactory. most of the examined populations had heard the term ‘trans fats’ before and were aware of their negative influence on human health. some of the students, independently of the university, could not indicate all of the products constituting a potential source of tfas (around 30%). polish students were not aware of natural sources of tfas (less than 10%) compared to students from the dutch university (around 40%). polish students from studies not related to food and nutrition sciences had less knowledge in the topic of tfa than respondents in the usa and canada. based on this, it can be concluded that polish society should be further educated and food producers should be encouraged to label packages about nutritional characteristics of fat. eating habits related to consumption of pastry and confectionery products, which constitute a potential source of tfas, of both populations were similar. worrisome is the fact that women (independently of population) were consuming these kinds of products definitely too often. taking into account the level of knowledge of polish students (sggw) and from different retable 4 factors influencing students’ decisions of the purchase of confectionery and pastry products (p = 0.04654). product features contribution influencing the choice of answers (%) wur sggw ingredients and information on the package 24 38 price 29 10 brand 5 27 attractive package 20 4 always the same products 22 21 gions of the eu (studying in the netherlands – wur) finishing master’s degree studies (fs and nfs) it should be considered that there is still a need for education about the negative effect of tfas on health and to inform consumers which products can be a source of them in the diet. based on the obtained results it can be assumed that people coming from different eu regions who were not educated may have even less knowledge in this subject. moreover, eating habits of women should change as they consume sweet snacks far more often than men, which could be a potential source of undesired fatty acids in the diet. references bayir a. sirkecioğlu a.n. aksakal e. bayir m. haliloğlu h.i. güneş m. and aras n.m. 2011. changes in the fatty acids of neutral and polar lipids of silurus glanis and barbus capito during an annual cycle. ijfs. 2: 173. downes s.m. thow a.m. and leeder s.r. 2013. the effectiveness of policies for reducing dietary trans fat: a systematic review of the evidence. bull. world health organ.91: 262. efsa. 2004. opinion of the scientific panel on dietetic products, nutrition and allergies on a request from the commission related to the presence of trans fatty acids in foods and the effect on human health of the consumption of trans fatty acids (request n° efsa-q-2003-022). the efsa journal. 81: 1. flaczyk e. and korczak j. 2002. influence of selected factors on consumer behavior on edible fats market. acta sci. pol., technol. aliment. 1: 113. gebauer s.k. posta t.l. and kris-etherton p.m. 2007. the diversity of health effects of individual trans fatty acids isomers. lipids. 9: 787. goodman s. hammond d. pillo-blocka f. glanville t. and jenkins r. 2001. use of nutritional information in canada: national trends between 2004 and 2008. jneb. 43: 356. gurr m. 2000. the role of lipids in human nutrition. ch. 13. in „handbook of olive oil”. ed. j. harwood and r. aparicio. p. 521-563. publisher springer us. jasti s. and kovacs s. 2010. use of trans fat information of food labels and its determinants in a multiethnic college student population. jneb. 42: 307. krasnowska g. and salejda a. 2011. consumer knowledge about food product labeling. żywność. technologia. nauka. jakość. 74: 173. martin c. milinsk m. visentainer j. matsushita, m. and de-souza n. 2007. trans fatty acid-forming processes in foodsa review. anais da academia brasileira de ciências. 79: 343. 8 ital. j. food sci., vol. 27 2015 matthäus b. 2007. use of palm oil for frying in comparison with other high-stability oils. eur. j. lipid sci. technol. 109: 400. mcdonald r. and mossoba m. 1996. trans fatty acids: labelind, nutrition, and analysis, food lipids and health. chicago, min d. marcel dekker. moreno m. olivares d. lopez f. adelantado j. and reig f. 1999. determination of unsaturation grade and trans isomers generated during thermal oxidation of edible oils and fats by ftir. j. molec. struct. 482: 551. richter e.k. albash shawish k. sheeder m.r.l. and colombani p.c. 2009. trans fatty acids content of selected swiss foods: the trans swiss pilot study. j. food comp. anal. 22: 479. simopoulos a. 1996. trans fatty acids, handbook of lipids in human nutrition. ed. spiller g. borca raton, usa: crc press inc., 91. smed s.2012. financial penalties on foods: the fat tax in denmark. nutrition bulletin. 37: 142. stampfer m.l. sacks f.m. savini s. willett w.c. and hennekens c.h. 1991. a prospective study of cholesterol, apolipoproteins, and the risk of myocardial infarction. n. engl. j. med. 325: 373. urbańska i. and czarniecka-skubina e. 2007. food products consumption frequency of youth offered by school shops. żywność. technologia. nauka. jakość. 52: 193. żbikowska a. 2010. formation and properties of trans fatty acids – a review. pol. j. food nutr. sci .60: 107. paper received october 22, 2013 accepted april 13, 2014 ijfs#947_bozza ital. j. food sci., vol. 30, 2018 156 paper the effect of blanching pre-treatment on the drying kinetics, thermal degradation of phenolic compounds and hydroxymethyl furfural formation in pomegranate arils h. vardi̇n1 and f.m. yilmaz*2 1harran university, engineering faculty, food engineering dept., 63040, şanlıurfa, turkey 2adnan menderes university, engineering faculty, food engineering dept., 09010, aydın, turkey *corresponding author. tel.: +90 2562137503; fax +90 2562136686 e-mail address: fatih.yilmaz@adu.edu.tr abstract this study examined the effect of blanching pre-treatment on the drying kinetics, thermal degradation of phytonutrients and hydroxymethylfurfural (hmf) formation in pomegranate arils. pre-treated and untreated arils were dried in a cabinet dryer operated at temperatures of 55, 65 and 75°c. the efficiency of the drying process was assessed according to the effective moisture diffusivity and activation energy values. effective moisture diffusivities ranged from 0.59 × 10−9 to 5.62 × 10−9 m2 s−1 and activation energies for drying were 31.82 and 76.11 kj/mol for pre-treated and untreated arils, respectively. six thin-layer drying models were tested, and page and modified page models were found to be the most suitable. the final quality of pomegranate arils was evaluated according to their total phenolic and total anthocyanin contents, their antioxidant capacities and the rate of hmf formation. the blanching pre-treatment prior to drying produced higher retention of antioxidant compounds with less hmf content and superior sensory properties. sensory analysis results revealed that pre-treated arils were preferred to untreated arils. keywords: pomegranate, drying kinetics, antioxidants, hmf, thermal degradation, blanching ital. j. food sci., vol. 30, 2018 157 1. introduction the pomegranate is a native plant to an area covered by persia, anatolia, mesopotamia and india, and has been cultivated in the usa and mediterranean countries (mena et al., 2013). significant attention has been given to pomegranate fruits in recent years because of the desirable aroma, flavor and characteristic bright red color (shahbaz et al., 2014). pomegranate fruits are used for the manufacture of various food products and fruit juice concentrates. fresh pomegranates are harvested during late september, october and november. even though there is significant demand for fresh pomegranates during the whole year, geographical and seasonal restrictions limit the fresh pomegranate supply to the market. therefore, many food preservation techniques have been investigated to extend the shelf life of the pomegranate fruits such as canning, freezing, modified atmosphere packaging and controlled storage of the products in addition to drying (viuda-martos et al., 2012). traditionally dried pomegranate arils are called ‘anardana’, which is widely consumed in southern asia and persia. anardana is an alternative pomegranate product enabling people to serve dried arils the whole year round. anardana production keeps increasing together with increasing acres of pomegranate plantations in other parts of the world. drying is one of the oldest food preservation methods that have been used from ancient times up to the modern day. the drying process extends the shelf life of the products by lowering water activity, and therefore inhibiting microbial and biochemical decay of the food (kami̇loglu et al., 2014). however, the drying process may negatively affect food quality parameters such as the nutritional quality, the bioactive compound content, the color, and the texture (fazaeli̇ et al., 2013). therefore, it is necessary to investigate the optimum drying conditions for the production of high quality, desirable and shelf life –extended foods [akdaş and başlar, 2015; sturm et al., 2012). pre-treatment applications, and different drying methods and conditions, are currently being investigated for the production of high quality products (chaethong and pongsawatmani̇t, 2015; adedeji̇ et al., 2008]. the balance between low cost, fast production techniques and high quality of the final product should be established to ensure consumer acceptability. the consumer acceptability and the market value of the products are influenced by both qualitative and quantitative factors. in general, consumers tend to prefer foods at a reasonable price with a high functional compound content and improved color characteristics. especially in recent years, together with the improvements in welfare and education, most people from all over the world have become more and more demanding with regard to functional foods (siro et al., 2008). the major functional compounds in fruit and vegetable products are vitamins, pigments, flavonoids and phenolic acids (zulueta et al., 2007). phenolic compounds have been shown to be associated with inhibiting cardiovascular diseases, tumor formation and cancer development (acostaestrada et al., 2014; bondia-pons et al., 2009). however, food-processing steps can also exert negative effects on vulnerable food compounds such as vitamins, pigments and phenolic acids (chong et al., 2009; mousa et al., 2002). even though most of the drying process is completed in cabinet-type dryers in the food industry, there are few studies in published literature evaluating the bioactive-compound degradation kinetics and the formation of harmful compounds. thus information on the final quality parameters is essential for enabling better process control and the manufacture of dried goods with a higher antioxidant capacity. the objective of this study is to evaluate the drying kinetics of pomegranate arils and determine the proper drying conditions to produce dried arils with higher nutritional value. blanched and unblanched pomegranate arils were dried using cabinet dryers at different temperatures. ital. j. food sci., vol. 30, 2018 158 the association between the thermal-processing steps and the phytonutrient content of pomegranates were determined at different temperatures. the heat and mass transfer characteristics of the dried foods determine their drying mechanisms. hence, mathematical methods are useful in predicting the drying behavior of food commodities. this study also looked at mathematical models to determine which model best described the kinetics of the drying process in the context of degradation of phenolics and anthocyanins, as well as the formation of hmf during the process. 2. materials and methods 2.1. materials fresh pomegranates (punica granatum l., cv. hicaz) were purchased from a wholesale market hall in şanlıurfa, turkey, and immediately transported to the laboratory. fruits were kept at 4°c before commencement of the tests. folin-ciocalteu, gallic acid and dpph (1,1-diphenyl-2-picrylhydrazyl) were obtained from the merck co. (darmstadt, germany), and 5-(hydroxymethyl)furfural (hmf) and acetone from the sigma chemical co. (st. louis, mo, usa). all other chemicals used were of analytical grade. 2.2. preparation of pomegranate arils the pomegranates were peeled manually, and the arils were separated from the fruits. the arils were separated into two groups. half of the group was pre-treated by dipping into water with 0.1% citric acid at 80 ± 2°c for 2 minutes and afterwards immediately transferred to the cabinet dryer (i.e. pre-treated). the ratio of blanching water and pomegranate arils was 20 ml/g. after blanching, the arils were drained using soft filter papers. the other half of the arils was dried directly without any pre-treatment. 2.3. drying procedure the pre-treated and untreated arils were immediately transferred for cabinet drying. cabinet drying was carried out in the dryer (kendro laboratory products, germany) at three different temperatures (55, 65 and 75°c) at an air velocity of 1.2 m/s. during drying, moisture loss was recorded and almost 10 g of samples were removed from the dryer at thirty-minute intervals. the samples were stored at −20°c until the extraction procedure. 2.4. mathematical modeling page, modified page, henderson and pabis, wang and singh, two term and logarithmic models (table 1) were used for modeling the drying kinetics of the pomegranate arils. in these models, the moisture ratio (mr) was simplified to equation 1, below, instead of (m−me)/(m0−me), as the value of me is relatively small compared to m or m0. mr=m/m0 (1) where m is the moisture content at time t, m0 is the initial moisture content and me is the equilibrium moisture content. goodness of fits were determined according to the evaluation of r2 and root mean square error (rmse). higher values of r2 and smaller rmse values (equation 2) indicated a better fit of the experimental data to the model. the ital. j. food sci., vol. 30, 2018 159 correlation coefficient (r2) and root mean square error (rmse) were taken into consideration to select the best model. 𝑅𝑀𝑆𝐸 = ! ! (𝑀𝑅!"#.! − 𝑀𝑅!"#.!)! ! !!! !/! (2) table 1. models used for determining thin-layer drying curves. model equation reference page mr = exp(-ktn) (page, 1949) modified page mr = exp[-(kt)n] (overhults et al., 1973) henderson and pabis mr = a exp(-kt) (henderson and pabis, 1961) wang and singh mr = 1+at+bt28 (wang and singh, 1978) two term mr = a exp(k0t)+ b exp(k1t) (henderson, 1974) logarithmic mr = a exp(-kt) + c (yagci̇oglu et al., 1999) 2.5. computation of effective moisture diffusivity and activation energy fick’s second law was adapted to spherical shapes for unstable diffusion conditions as follows (crank, 1975): 𝑀𝑅 = ! !! ! !! ! !!! 𝑒𝑥𝑝 −𝑛!𝜋! !!""! !! ) (3) for extended drying periods (equation 4) can be further simplified as follows (doymaz, 2012): 𝑙𝑛(𝑀𝑅) = 𝑙𝑛( ! !! ) − ( !!!!""! !! ) (4) plotting ln(mr) versus time enables calculation of the effective moisture diffusivity and a straight line can be obtained with a slope of (k) as expressed in (equation 5) below: 𝐾 = ! !!!"" !! (5) the activation energy for diffusion was determined from the slope of the arrhenius-type equation (equation 6): 𝐷!"" = 𝐷!𝑒𝑥𝑝 − !! !" (6) 2.6. modeling the degradation of total phenolic and total anthocyanin contents thermal degradation of most bioactive compounds follows first order kinetics (equation 7): 𝐶! = 𝐶! 𝑥 𝑒𝑥𝑝 (±𝑘! 𝑥 𝑡) (7) ital. j. food sci., vol. 30, 2018 160 where ct and c0 are the total phenolic (tp) and total anthocyanin (ta) contents after heating time t and t=0 minutes, respectively, and k is the kinetic constant. the natural logarithms of the ratios of ct and c0 against time were plotted, and by using the slope of this graph, half-life (t1/2) was deduced according to equation 8: 𝑡!/! = !"! ! (8) 2.7. extraction of bioactive compounds the arils were firstly homogenised with 80% acetone (0.01% hcl) using a pestle and mortar. extraction was carried out in a shaking incubator (labline, usa) operated at 50°c and 180 rpm for 60 minutes. then, the slurry was centrifuged at 6000 rpm for 8 minutes (hitachi ct6e, taiwan), and the supernatant was collected in falcon tubes and stored at −40°c until analysis. 2.8. determination of total phenolic (tp) content the tp contents of the samples were determined by the folin-ciocalteu method (singleton and rossi, 1965) using the gallic acid standard curve. the absorbance of each sample was read at 750 nm in a spectrophotometer (libra, biochrom, uk) against the blank, and the results were expressed as mg of gallic acid equivalent per kilogram of pomegranate aril (mg gae kg−1). 2.9. determination of total anthocyanin (ta) content the ta contents of the samples were determined using a ph-differential method (giusti and wrolstad, 2001) by a uv-vis spectrophotometer (biochrom libra, uk). the pomegranate extracts were mixed with ph 1.0 (0.025 m potassium chloride) and ph 4.5 (0.4 m sodium acetate) buffers, and the absorbance values were recorded at 520 and 700 nm. the results were expressed as mg of cyanidin 3-glucoside per kilogram dry weight of the pomegranate aril. 2.10. determination of antioxidant capacity the antioxidant capacity of the samples was estimated by dpph (1,1-diphenyl-2-picrylhydrazyl) assay as described by karaaslan et al., 2004a. an 0.1 ml amount of various concentrations of the extracts diluted in ethanol was added to 2.9 ml of 0.1 mm of the dpph solution. the decrease in absorbance at 517 nm was measured after 30 minutes of incubation at room temperature. the inhibition concentration (ec50) – the amount of sample concentration (g/ml db) necessary to decrease the initial dpph concentration by 50% – was calculated after plotting the percentage inhibition versus the extract concentration curve. 2.11. determination of hydroxymethylfurfural (hmf) the hmf contents of the arils were determined using a spectrophotometric method (acar et al., 1999). briefly, 5 g of dried sample was crushed and diluted with distilled water up to 50 ml. then, 5 ml of this solution was mixed with p-toluidine solution (10 g/100 ml) and 1 ml of barbituric acid solution was added to the mixture after 2 minutes. ital. j. food sci., vol. 30, 2018 161 the absorbance of the mixture was noted on the spectrophotometer at 55 nm and calculations were done using the hmf standard calibration curve. 2.12. sensory analysis dried samples (pre-treated and untreated at three different temperatures for a total of 6 samples) were randomly coded and served to panelists. samples were analyzed organoleptically by 20 trained panelists in terms of color, shape, texture, flavor-aroma and overall acceptability. evaluation was scored on a ten-point scale (0–4, very bad – bad; 5–9, acceptable – excellent), according to gould (1977). 2.13. statistical analyses experimental data were subjected to analysis of variance (anova) using spss version 15.0 software (spss inc., chicago, il, usa) and p values less than 0.05 were taken into consideration. the duncan test was used as a post hoc test after applying the homogeneity test. the parameters of kinetic models were determined by using sigma plot software (sigma plot 10.0 windows version, spss inc.). all the experiments were repeated three times. 3. results and discussions 3.1. drying kinetics and modelling the initial moisture content of the fresh and pre-treated pomegranate arils were 79.82 ± 0.44% and 80.13 ± 0.67% (w.b.), respectively. the samples were dried until they had 16% (w.b.) moisture content by considering sensory properties and current literature data. a graphical representation of mr values versus time belonging to pre-treated samples is shown in fig. 1. figure 1. drying curve of pre-treated pomegranate arils (plotted by the page model) in cabinet dryer. (mr moisture ratio: the ratio of dry basis moisture content at any time to initial dry basis moisture content). ital. j. food sci., vol. 30, 2018 162 as expected, the drying temperature had a significant effect on the drying kinetics of the samples. the time required to dry pre-treated pomegranate arils in a cabinet dryer were 168, 204 and 237 minutes at 75, 65 and 55 °c, respectively, while for untreated samples the values were 244, 448 and 945 minutes at the same temperatures. it is clear that pretreatment application had a significant effect on the drying time of the arils. in a pomegranate drying study using an air circulating oven (v = 1.3 m/s), drying times were reported to be 330, 520 and 1020 minutes at 55, 65 and 75°c, respectively (başlar et al., 2014). six thin layer drying models were evaluated by fitting the experimental data and considering the highest r2 and lowest rmse values. the results showed that page and modified page models were the most relevant models in all drying conditions. model constants with statistical evaluations are demonstrated in table 2. mathematical models used in drying applications are useful for designing new or improved existing drying systems. such models are directly related to the temperature and velocity of the drying medium inside the mechanical dryer as well as the energy cost (babalis and belessiotis, 2004). 3.2. effective moisture diffusivity and activation energy of the drying process knowing the effective moisture diffusivity is necessary for designing and modeling mass transfer processes (sharma and prasad, 2004). the deff values of the pre-treated samples in the cabinet dryer ranged from 2.87 × 10−9 to 5.62 × 10−9 m2/s and the untreated samples from 0.59 × 10−9 to 2.92 × 10−9 m2/s (table 3). higher deff values were observed in pre-treated samples that are associated with faster removal of moisture and thus the faster drying of samples. similar effective moisture diffusivity results were demonstrated by studies investigating pomegranate aril drying (doymaz, 2012; başlar et al., 2014; karaaslan et al., 2014b; minaei et al., 2011;hii et al., 2009). the ln(deff) value was plotted against the reciprocal of absolute temperature to determine the activation energy and equation (6) was used to determine the activation energy. the activation energy may be defined as the energy barrier that must be overcome in order to activate moisture diffusion. therefore, determination and comparison of the ea values are important in drying applications (karaaslan et al., 2014b). the activation energy was 31.82 kj/mol for the pre-treated arils and 76.11 kj/mol for untreated arils. pre-treatment of the samples brought about a significant decrease in the activation energies of the samples. başlar et al.,(2014) reported the ea value as 44.798 kj/mol for cabinet drying of pomegranate arils. in a pomegranate aril drying study, pre-treated (dipping in alkali emulsion of ethyl oleate) samples had lower activation energy than the control group (doymaz, 2012). 3.3. the changes in total phenolic compounds and anthocyanin content anthocyanins are responsible for giving the characteristics red color to pomegranate arils, and the arils are rich in anthocyanins and other colorless phenolic compounds. numerous studies have showed the health benefits of phenolics present in pomegranate arils (karaaslan et al., 2014a). however, the stabilities of these compounds can easily be affected by temperature increases and the presence of oxygen (verbeyst et al., 2010). therefore, the determination of such substances is important for process optimization. the anthocyanin content of the samples declined from 824.65 ± 87.27 (mg kg−1, d.b.) to 813.83 ± 79.91 (mg kg−1, d.b.), and total phenolic compounds declined from 7433.04 ± 685.51 (mg kg−1, d.b.) to 6863.86 ± 630.27 (mg kg−1, d.b.) as pre-treatment was applied. ital. j. food sci., vol. 30, 2018 163 table 2. statistical data of the six thin-layer mathematical models applied to the drying data. as demonstrated in table 4, more anthocyanin and phenolic contents were preserved in the samples dried at 55°c compared to other samples, while the increase in temperature caused more phenolic and anthocyanin degradation. the ta concentration of the pretreated samples decreased to 19% in a cabinet dryer at 75°c, to 25% at 65°c, and to 29% at 55°c. the ta in untreated samples in a cabinet dryer declined to 13%, 19% and 22% under the same temperature regimes. a decrease of tp content of the arils was also observed. the tp content of the pre-treated samples in the cabinet dryer dropped to 36% in the cabinet dryer at 75°c, to 41% at 65°c and to 54% at 55°c. the tp content of the untreated condition t (°c) model r2 rmse k n a b k0 k1 c untreated 55 1 0.9914 0.0203 3.1471 1.9017 2 0.9914 0.0203 0.0010 1.8922 3 0.9124 0.0637 0.0014 1.0919 4 0.9897 0.0458 0.0004 5.3849 5 0.9114 0.0651 0.5394 0.5448 0.0009 0.0009 6 0.9108 0.0039 0.0029 6.2423 -5.348 65 1 0.9955 0.0309 4.6778 2.076 2 0.9955 0.0310 0.0080 2.076 3 0.9068 0.0779 0.0074 1.1079 4 0.9845 0.0614 0.0027 1.8017 5 0.9073 0.0774 0.5481 0.5561 0.0078 0.0078 6 0.8345 0.0547 0.0058 4.2418 -4.154 75 1 0.9873 0.0323 8.8847 1.8517 2 0.9873 0.0323 0.0058 1.8488 3 0.9271 0.0738 0.5724 1.1417 4 0.9577 0.0617 0.0029 2.1854 5 0.9303 0.0716 0.5673 0.5673 0.0060 0.0060 6 0.9668 0.0515 0.0073 5.2346 -4.132 pre-treated 55 1 0.9964 0.0183 6.0977 1.9132 2 0.9962 0.1830 0.0063 1.9132 3 0.9249 0.0770 0.0061 1.1105 4 0.9840 0.0369 0.0023 0.5421 5 0.9248 0.0762 0.5537 0.5487 0.0061 0.0061 6 0.8978 0.0184 0.6913 1.2710 -2.026 65 1 0.9960 0.0187 4.6787 2.0874 2 0.9960 0.0188 0.0081 2.0874 3 0.9528 0.0854 0.0799 1.1104 4 0.9852 0.0451 0.0034 1.8446 5 0.9061 0.0839 0.5572 0.5572 0.0071 0.0071 6 0.8632 0.1239 3.0045 -0.353 75 1 0.9816 0.0425 0.0014 1.4628 2 0.9816 0.0425 1.4631 1.4628 3 0.9611 0.0849 0.0110 1.0921 4 0.9633 0.0624 0.0082 1.4365 5 0.9616 0.0850 0.5473 0.5473 0.0103 0.0103 6 0.9684 0.0058 0.0068 1.3641 -0.285 ital. j. food sci., vol. 30, 2018 164 samples in the cabinet dryer declined to 22%, 28%, and 33% under the same conditions at 75, 65, 55°c, respectively (table 4). it is clear that both tp and ta are preserved better in pre-treated samples. başlar et al. (2014) reported increasing tp retention with increasing temperature. the tp retentions of pomegranate arils in their study were 78.61, 81.82 and 84.11% for 55, 65 and 75°c, respectively. bchir et al. (2012) reported that the phenolic content of the arils decreased to 40% (w.b.), and the anthocyanin content decreased to 25% (w.b.) for cabinet drying of pomegranates, which are much lower than our results. table 3. effective moisture diffusivities and activation energies of drying. experimental samples temp (°c) deff (m 2/s) ea (kj/mol) r2 untreated cabinet 55 0.59 ×10-9 76.11 0.9708 65 1.71 ×10-9 75 2.92×10-9 pre-treated cabinet 55 2.87 ×10-9 31.82 0.9844 65 3.77 ×10-9 75 5.62 ×10-9 table 4. effect of drying temperatures and pre-treatment application on half-life (t1/2), drying time and total phenolic (tp) – total anthocyanin (ta) contents of pomegranate arils dried in a cabinet dryer. pre-treated untreated temperature (°c) t1/2 (min) r 2 drying time (min) ct/c0 at drying time temperature (°c) t1/2 (min) r 2 drying time (min) ct/c0 at drying time kinetic results of tp degradation 55 211 0.982 237 0.54 55 629 0.958 945 0.33 65 173 0.964 204 0.41 65 190 0.984 448 0.28 75 140 0.938 168 0.36 75 147 0.965 244 0.22 kinetic results of ta degradation 55 124 0.974 237 0.29 55 793 0.988 945 0.22 65 75 0.988 204 0.25 65 154 0.970 448 0.19 75 53 0.990 168 0.19 75 83 0.975 244 0.13 3.4. thermal degradation kinetics of total phenolic compounds and anthocyanins the time-dependent tp and ta degradation data were used to develop an arrhenius model to predict the bioactive compounds’ degradation during the drying. table 5 shows that the model adequately fits the degradation kinetics (0.9131 < r2 < 0.9754). a higher ea value implies the increasing temperature dependence of tp–ta degradation. table 5 shows the ea, half-life (t1/2) and final tp–ta contents at the drying point. ta degradation was less sensitive to heat treatment under both these conditions compared to tp degradation. in addition, pre-treated samples have a higher degradation rate compared to untreated samples. however, the final tp and ta contents were higher in pre-treated samples despite the higher degradation rate they experienced (table 4). the final concentrations of tp and ta in untreated samples were lower than pre-treated samples, even if ea values were lower. so, it is important to evaluate drying kinetics and phytonutrient degradation kinetics in combination. also, the necessity to control the time ital. j. food sci., vol. 30, 2018 165 to terminate the drying at a desired point is crucial. başlar et al. (2014) reported higher retention of tp and ta with increasing temperature, while on the other hand they demonstrated higher degradation rates of tp and ta at higher temperatures. they also concluded that the combination of time and temperature in the drying process is important. table 5. activation energy (ea) values obtained from arrhenius model for total phenolic (tp) and total anthocyanin (ta) degradation. condition arrhenius model ea (kj mol-1) r2 tp degradation kinetic pre-treated 32.53 0.9428 untreated 72.65 0.9641 ta degradation kinetic pre-treated 41.60 0.9131 untreated 102.27 0.9754 3.5. antioxidant capacities of dried pomegranate arils the antioxidant capacities of the final products were significantly affected by pretreatment application. pre-treated arils had a higher antioxidant capacity than untreated arils (fig. 2). the resulting higher antioxidant capacity of the pre-treated arils is linked to the shorter drying operation, which may protect the bioactive compounds. the temperature also had a significant effect on the antioxidant capacities of dried arils (fig. 2). figure 2. antioxidant capacities of dried pomegranate arils depending on the drying conditions. a c b c, d c d 50 55 60 65 70 75 80 85 90 95 100 pretreated arils untreated arils ec 50 ( m g/ kg ) ital. j. food sci., vol. 30, 2018 166 the highest antioxidant capacity value belonged to the pre-treated arils dried at 55°c. the antioxidant capacities were decreased as temperature increases, and this may result from degradation of heat sensitive bioactive compounds. the correlation between the antioxidant capacity and antioxidant compounds of the dried pomegranates were evaluated individually. the correlations among the data were determined using pearson’s correlation coefficient. the correlations between the tpc and antioxidant capacity (r = 0.833) and ta and antioxidant capacity (r = 0.774) were found to be statistically significant (p < 0.01). 3.6. formation of hmf (hydroxymethylfurfural) several factors, such as dry matter content (or ˚bx), aw, processing or storage temperature may affect the hmf formation in the dried fruits and concentrated juices (lavelli and vantaggi, 2009). the content of hmf in the dried pomegranate arils is depicted in fig. 3. a higher hmf content was found in untreated samples in this study.. in addition, increasing temperature led to higher hmf content in the final product. hmf contents of the dried samples were between 5.22 and 16.34 mg/kg. the lowest hmf content was found in pre-treated samples dried at 55°c. the harsh effect of temperature increase on the hmf content of dried samples has been illustrated in other studies (zanoni̇ et al., 1999; pekke et al., 2013; wojdyło et al., 2014). figure 3. the effect of pre-treatment conditions and temperature on hmf formation. 3.7. sensory analysis the untreated and pre-treated samples had statistically significant differences in color, shape, texture and overall acceptability, while there were no statistically significant differences in flavor and aroma (p < 0.05). the pre-treated samples were more acceptable– excellent to the panelists across all sensory properties. apart from color, the temperature had no statistically significant effect on the sensory properties of the dried samples. the average score for the overall acceptability of pre-treated samples was 8.76, and 5.34 for untreated samples. pretreated arils untreated arils 55 °c 5,2 8,42 65 °c 5,88 11,48 75 °c 10,11 16,22 a b a c b, c d 0 5 10 15 20 h m f (m g/ kg ) ital. j. food sci., vol. 30, 2018 167 4. conclusions the use of controlled drying conditions and pre-treatment applications may assist the production of better quality dried arils. drying rates were considerably higher for pretreated arils under all drying conditions. the drying process was completed in a shorter time for pre-treated samples, and thus, pre-treated arils had a higher phenolic and anthocyanin content in comparison to untreated samples, even if the thermal degradation rate of the bioactive compounds were higher in the pre-treated samples. pre-treated samples had a higher antioxidant capacity and lower hmf content at all temperatures. this study is a good example that reflects the necessity of evaluating the kinetics of drying and the bioactive compounds’ degradation together. acknowledgements the authors wish to thank dr. m. karaaslan (harran university) for his valuable technical help and also would like to acknowledge all members of the food engineering dept. 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110:220. yagcioglu a., degirmencioglu a. and cagatay f. 1999. drying characteristics of laurel leaves under different drying conditions, in proceedings of the 7th international congress on agricultural mech. and energy, adana, turkey, 565. zanoni b., peri c., nani r. and lavelli v. 1999. oxidative heat damage of tomato halves as affected by drying. food. res. int. 31:395. zulueta a., esteve m.j., frasquet i. and frigola a. 2007. vitamin c, vitamin a, phenolic compounds and total antioxidant capacity of new fruit juice and skim milk mixture beverages marketed in spain. food chem. 103:1365. paper received june 24, 2017 accepted september 27, 2017 ijfs#975_bozza ital. j. food sci., vol. 30, 2018 128 paper phenolic compounds and antioxidant activity of wild grape (vitis tiliifolia) m. jiménez*a, n. juáreza, v.m. jiménez-fernándezb, j.l. monribot-villanuevac and j.a. guerrero-analcoc a instituto de ciencias básicas, universidad veracruzana, xalapa ver., méxico b facultad de instrumentación electrónica, universidad veracruzana, xalapa, ver., méxico c red de estudios moleculares avanzados, clúster biomimic®, instituto de ecología, a.c., xalapa, ver., méxico *corresponding author. tel.: +52 2288418900 e-mail address: maribjimenez@uv.mx abstract vitis tiliifolia is a tropical grape with a deep purple colour and a high content of pigments. total polyphenols content in the skin and pulp was 400.35 and 171.26 mg gae/g dry sample of vitis tiliifolia, respectively, which coincides with dpph radical scavenging for skin (91.39%) and in the pulp (19.57%). the predominant individual phenolic compounds found in the skin were quercetin-3-glucoside (39.86 µg/g), rutin (37.01 µg/g) and transresveratrol (32.88 µg/g). the dpph radical scavenging and reducing power revealed a high antioxidant activity. this study demonstrates that wild grape can thus be utilised as a novel functional resource. keywords: vitis, wild grapes, anthocyanins, food composition, polyphenols ital. j. food sci., vol. 30, 2018 129 1. introduction consumers have focused increased attention on functional foods, especially those containing antioxidants, which decrease reactive oxygen species (ros) (manach et al., 2005). it is reported that some fruits such as grapes can dramatically increase the balance between the production and manifestation of ros and a biological system’s ability to readily detoxify the reactive intermediates or to repair the resulting damage is interrupted (stagos et al., 2006). at the same time, grape extracts and wine have been recognised to contain polyphenol compounds that have beneficial effects on human health. it is known that grapes are anti-mutagenic, antineoplastic, reduce human low-density lipoprotein (ldl) oxidation and allergic inflammation, decrease cardiovascular diseases (lekakis et al., 2005), exhibit antimicrobial (jayaprakasha et al., 2001), antihypertensives (soares de moura et al., 2002), and antiulcer activities (cuevas et al., 2011). on the one hand, anthocyanins are a type of polyphenol, and it is reported that they present strong antioxidant activity, inhibit the growth of cancerous cells and inflammation, and act as vasoprotectors and anti-obesity agents, in addition to having effects on diabetes and cardiovascular disease prevention, as well as the improvement of visual and brain functions (tsuda, 2012). on the other hand, resveratrol (3,5,4-trihydroxy-trans-stilbene) is a natural polyphenolic that acts as a defense mechanism against deleterious microorganisms. these compounds are present in several fruits as grapes, and their manufactured products, especially red wine. anthocyanins are primarily located in the skin and have pharmacological benefits. it has been known to exert its protective effect against cardiovascular disease, ischemia-reperfusion injury and diabetes mellitus through the modulation of adipocyte/fibroblast biology, platelet activation, blood vessel function, oxidative stress, inflammation, serum glucose maintenance, cardiomyocyte biology, the maintenance of cell structure, and serum lipid activity, cause body fat loss, and confer protection against disease or injury (tsuda, 2012). there are many types of grapes that have been widely studied. however, vitis tiliifolia is a wild grape resource that has not yet been adequately recognised by researchers and winemakers. it is a small to very large climbing shrub with thick, woody stems that can be 10-35 m long and up to 20 cm in diameter, which commonly grows in wet to dry forest or thickets, often in pine-oak forest; it grows regularly around 1700 meters above sea level. vitis tiliifolia grows in the southern states of mexico and the antilles to colombia (fernandez, 2009). in mexico, it is located in the states of chiapas, colima, guerrero, hidalgo, nuevo leon, oaxaca, querétaro, san luis potosí, tabasco and veracruz, where it is known by different names such as: wild grape, gunhi, loobabi-chuli, uvilla, xocomecatl, tecamate and others, according to the region and growing area (arellano et al., 2003). flowers are seen from may to june and fruits are harvested from august to november (ibarra and sinaca, 1996). the fruits have been used as raw materials for juice and wine (arellano et al., 2003). fresh fruit is commonly used to make vinegar and soft drinks (fernandez, 2009), while the root and leaves are used empirically against haemorrhoids. therefore, these products may be useful as a source of potentially functional ingredients providing the opportunity to develop innovative added value products. however, the further application of this wild grape requires the evaluation of their composition and there is little information on the physicochemical and antioxidant properties of vitis tiliifolia. therefore, the aim of this work was to investigate the physicochemical properties and antioxidant activity in the pulp and skin of vitis tiliifolia fruit to provide sufficient experimental evidence for the antioxidant activity and potential for further development and utilisation of this species. ital. j. food sci., vol. 30, 2018 130 2. materials and methods 2.1. chemicals 2,2’-diphenyl-1-picrylhydrazyl (dpph), trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid), gallic acid, trans-resveratrol, folin-ciocalteu reagent and 2,4,6-tris(2pyridyl)-1,3,5-triazine (tptz) were purchased from sigma-aldrich (st. louis, mo, usa). 4-hydroxybenzoic acid, (+) catechin, vanillin acid, scopolin, chlorogenic acid, caffeic acid, (-) epicatechin, vanillin, 4-coumaric acid, quercetin 3-glucoside, ferulic acid and transcinnamic acid were purchased from extrasynthese (lyon, france). the rest of the standards were bought at sigma-aldrich (usa). the solvents used for the extraction was analytical grade and ms grade for the ultra-high performance liquid chromatography (uplc) procedures and other standards were also purchased from sigma-aldrich (usa). all the stock solutions, samples, solvents and reagents were filtered through 0.20 µm ptfe membrane filters (phenomenex, usa) before separation or injection in the instrument. 2.2. samples the samples of vitis tiliifolia were collected at "cafetal" ranch, located in the veracruz state, situated at 19° 37' 0.4 " north latitude and 96° 50' 2.7" west longitude, at an elevation of 734 meters above mean sea level. ten kg of grapes were harvested during the month of august of the years 2015 and 2016 with the optimum stage of maturity and with a concentration of soluble solids between 12 and 14 °brix. samples were washed, drained and subsequently, skins, seeds and pulp were directly obtained by manual separation. one part of the samples was frozen at -40°c for the analysis of the composition and physicochemical properties of the pulp and skin and the other part was subjected to lyophilization for the preparation of the extracts. 2.3. determination of some basic physicochemical parameters total nitrogen was determined by the micro-kjeldahl method and protein was calculated as nitrogen·6.25. oil was extracted for 24 h with diethyl ether in a soxhlet system. ash was determined by incineration in a furnace at 550°c and weight, moisture, titratable acids, reducing sugars and total dietary fibre and ph were determined following the aoac (2000) methods. the water activity was measured at 25ºc using aqualab 4 te (decagon 142 devices, pullman, wa, usa) and °brix were measured with a hand refractometer. the colour was measured with a colorimeter (colorflex v1-72 snhcx 1115 s/n: cx1115 hunter lab, usa) using parameters a0 (yellow-red), b0 (blue-green) and l0 (intensity and brilliance) on the scale of the system cie lab (international commission on illumination, vienna). browning index was determined according to the method reported by buera et al. (1986). equations 1 and 2 were used to calculate hue angle (h°) and chroma, respectively. 𝐻𝑢𝑒 𝑎𝑛𝑔𝑙𝑒 = tan!! 𝑏 𝑎 eq. (1) 𝐶ℎ𝑟𝑜𝑚𝑎 = 𝑎! + 𝑏! eq. (2) ital. j. food sci., vol. 30, 2018 131 2.4. chemical compounds and antioxidant analysis 2.4.1. extraction the dry sample pulp and skin (approximately 10 g each one) were mechanically homogenised with 10 ml of acidified methanol/0.1% hcl in a manual blender and sonicated in an ultrasonic bath (branson model 2510) for 30 min and agitated in a horizontal shaker at room temperature (24°c) for 1.5 h. then, the sample was centrifuged (hettich, mod. universal 32r) at 2200 g for 15 min. the supernatant was removed and the residue was re-extracted twice with 10 ml of a mixture of methanol: hcl 0.1 v/v according to chiou et al., (2014). the three supernatants were pooled and brought to a final volume of 100 ml with the same solvent used in the last two extractions. this concentration was considered by quantification of the components present in the sample. this extract was prepared in triplicate and used for the analysis of individual phenolic compounds, total phenolics, monomeric anthocyanins, and antioxidant activity. the identification and quantitation of individual phenolic compounds, it was established by ultra high performance liquid chromatography (agilent 1290 series) and dynamic multiple reaction monitoring (dmrm) following the protocol conditions of durandhulak et al. (2015). the chromatographic analysis were carried out on a zorbax sbc18 column (1.8 μm, 2.1 × 50 mm) (agilent technologies) with the column temperature at 40°c. the mobile phase consisted of (a) water containing 0.1% formic acid and (b) acetonitrile containing 0.1% formic acid. the gradient conditions of the mobile phase were: 0 min 1% b, 0.1-40 min linear gradient 1-40% b, 40.1-42 min linear gradient 40-90% b, 42.144 min isocratic 90% b isocratic, 44.1-46 min linear gradient 90-1 %b, 46.1-47 min 1% b isocratic (total run time 47 min). the flow rate was 0.1 ml/min, and 5 μl of sample injection volume. dmrm were obtained on an agilent 6460 triplequadropole (qqq) mass spectrometer. the esi source was operated in positive and negative ionization modes, desolvation temperature of 300°c, cone gas (n2) flow of 5 l/min, nebulizer 45 psi, sheath gas temperature 250°c, sheath gas flow of 11 l/min, capillary voltage (positive and negative) 3,500 v, nozzle voltage (positive and negative) 500 v. for quantitation of each phenolic compound a calibration curve in a concentration range of 0.3 to 30 µm was prepared (r2 values ≥ 0.97 were considered for the linearity range) and quantities were established by using masshunter workstation software version b.06.00 (agilent technologies) (table 1). the results were expressed as µg/g of sample (dry weight). 2.4.2. anthocyanins profile anthocyanins were identifying according to liang et al. (2008). twenty grams of dry methanol pulp and skin extracts were dissolved in 1 ml of meoh with 0.1% of formic acid (lcms grade, sigma). the samples were filtered in ptfe filters and 1 µl injected in a uplc-ms system (acquiti class-i coupled to mass spectrometer synapt g2 si, waters™) for high resolution mass analysis. the mobile phases were water (a) and acetonitrile (b), both with 0.1% of formic acid. the elution gradient was: at t= 0 minutes, 1% of b, then in 13 minutes changes from 1 to 80% of b. isocratic in 80% of b for 1 minute and finally change in 1 minute from 80 to 1% of b and remains for 5 minutes. the flow rate of the mobile phase was 0.3 ml/min and the column oven temperature was 40oc. the mass spectrometer was operated in positive mode, with capillary, sampling cone and source offset voltages of 3, 40 and 80 kv, respectively. the source and desolvation temperatures were 100 and 450oc, respectively. the gas flows of desolvation was 600 l/h and the nebulizer pressure was 6.5 bar. the data were analyzed with the waters masslynx ital. j. food sci., vol. 30, 2018 132 software v4.1 and the mass spectra compared with the public databases metlin and massbank and analyzed with the masslynx tool named massfragment (v4.1). table 1. protocol used in the analysis of the compounds was a dynamic mrm (multiple reaction monitoring). reference compounds precursor ion product ion retention time collision energy polarity r 2 linearity range (µm) gallic acid 168.9 125 1.5 10 negative 0.996 0.3 24 4-hydroxybenzoic acid 137.02 93.03 9.4 10 negative 0.997 0.3 24 (+)-catechin 291.1 139.03 11.3 10 positive 0.971 0.3 12 vanillic acid 169.04 151.04 12 10 positive 0.998 0.3 12 scopolin 355.1 193 12.2 20 positive 0.998 0.3 12 chlorogenic acid 353.08 191.05 12.3 10 negative 0.998 0.3 12 caffeic acid 179 135 12.5 10 negative 0.999 0.3 12 (-)-epicatechin 291.1 139.1 14.6 10 positive 0.998 0.3 12 vanillin 153 93 15.3 10 positive 0.998 0.3 12 4-coumaric acid 163.05 119 16.4 10 negative 0.996 0.3 12 quercetin 3,4-di-o-glucoside 627.15 303.04 17.7 10 positive 0.997 0.3 12 scopoletin 193.04 133.02 18.6 10 positive 0.995 0.3 12 ferulic acid 193.1 133.9 18.8 5 negative 0.998 0.3 12 rutin 611.16 465.1 20.4 10 positive 0.994 0.03 12 quercetin 3-d-galactoside 465.1 303.04 20.6 10 positive 0.999 0.3 12 quercetin 3-glucoside 465.2 303.04 20.9 10 positive 0.987 0.03 12 luteolin 7-o-glucoside 449.1 287.05 21.3 10 positive 0.993 0.3 12 kaemperol 3-o-glucoside 449.1 287.05 23.3 10 positive 0.986 0.03 12 2,4-dimethoxy-6methylbenzoic acid 197.08 79.05 23.4 10 positive 0.992 0.3 12 trans-resveratrol 229.08 135.04 25.9 10 positive 0.999 0.3 24 trans-cinnamic acid 147.01 103.05 28.7 10 negative 0.999 1.5 24 quercetin 303.05 153.1 29.4 35 positive 0.990 0.03 12 piperine 286.14 201.05 43.8 10 positive 0.981 0.03 12 the retention time variation allowed for the search of the compounds was 2 min in each case. the fragmentor voltage was 100 v and the cell accelerator voltage was 7 v for each compound. it was made a calibration curve for each compound in a concentration range of 0.03 to 30 µm. 2.4.3. total phenolic content total phenolic content was estimated using the folin-ciocalteu method (singleton and rossi, 1965). briefly, the grape extracts were mixed with folin-ciocalteu reagent, and sodium carbonate solution (10%) was added. the mixture was allowed to react at room temperature in the dark for 120 min, and then the absorbance was measured at 765 nm in a uv/vis spectrophotometer (jenway, model 6305, japan). the result was then referred to a calibration curve obtained with a similarly prepared set of different gallic acid concentrations, and was expressed as mg of gallic acid equivalent (gae) per g of dry sample (r2=0.980). ital. j. food sci., vol. 30, 2018 133 2.4.4. total flavonoid content each grape extract was analysed for total flavonoid content according to a previously reported colorimetric method with modifications (veluri et al., 2006). specifically, 10 mg of lyophilised grape extract or 1 ml of quercetin standard (sigma, st. louis, mo) was mixed with 0.3 ml of 0.7 mol/l sodium nitrite (nano2), 0.3 ml of 0.8 mol/l aluminium chloride (alcl3), and 2 ml of 1 mol/l sodium hydroxide (naoh). all samples were analysed in duplicate and compared against a blank at an absorbance of 510 nm in a uv/vis spectrophotometer (jenway, model 6305, japan). results were expressed as milligram quercetin equivalent per gram of dry sample (mg/g). 2.4.5. total monomeric anthocyanin content the total monomeric anthocyanin (tma) content was estimated using the ph differential method (wrolstad, 2001). here, 10 mg of grape extract was diluted with buffers at ph 1.0 and 4.5 to obtain the same dilution. absorbance was measured in a uv/vis spectrophotometer (jenway, model 6305, japan) at 510 and 700 nm in both ph 1.0 and 4.5 buffers. the tma content (expressed in terms of cyanidin-3-glucoside) was calculated using the following formula: 𝐴 = (𝐴!"# − 𝐴!"")!" !.! − (𝐴!"# − 𝐴!"")!! !.! (3) 𝑇𝑀𝐴 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 = (𝐴𝑥 𝑀𝑊𝑥 𝐷𝐹𝑥𝑉𝑥1000)/(𝜀 𝑥 1 𝑥 𝑀) (4) where mw is the molecular weight of cianindin-3-glucoside (449 g mol-1), df is the dilution factor, v is the extract volume, ε is the molar extinction coefficient of cyanindin-3glucoside (29,600), and m is the mass of vitis tiliifolia extracted. 2.4.6. condensed tannins determination the determination of condensed tannins was performed according to the method described by porter et al. (1986). the dry extract of the pulp or skin (200 mg) and ten ml of aqueous acetone (70%) were added and suspended in an ultrasonic water bath, then the content was centrifuged for 10 min at approximately 3000g at 4°c, then 0.50ml of the supernatant was diluted with 70% acetone, 3.0 ml of butanol-hcl reagent and 0.1 ml of the ferric reagent. this sample was boiled for 60 min and measure the absorbance at 550 nm was obtained in the cool sample. 2.4.7. determination of ascorbic acid content. ascorbic acid contents were carried out by using a colorimetric method. the extract (10 mg) was mixed with metaphosphoric acid (10 ml, 1.0%) for 45 min at r.t. and filtered through whatman no. 1 filter paper. the filtrate (1.0 ml) was mixed with 2,6dichlorophenolindophenol (9.0 ml) and the absorbance was measured within 30 min against a blank at 515 nm in a uv/vis spectrophotometer (jenway, model 6305, japan). ascorbic acid contents were calculated on the basis of the calibration curve of authentic ascorbic acid (r2= 0.927). the results were expressed as l g of ascorbic acid of extract. ital. j. food sci., vol. 30, 2018 134 2.4.8. dpph free radical scavenging capacity dpph free radical-scavenging capacity was estimated using the method of güder and korkmaz (2012). briefly, grape extracts and dpph methanol solution were mixed and kept in the dark for 30 min. the absorbance of the reaction mixture was measured at 517 nm in a uv/vis spectrophotometer (jenway, model 6305, japan). the calibration curve was made with standard solutions of gallic acid in the range 1-100 mg ml (r2=0.935). 2.4.9. reducing power the determination of the reducing power was made according to the method of jayaprakasha et al. (2001). 0.125 ml of the sample in methanol (1 mg/ml), 1.25 ml of phosphate buffer (200 mm, ph 6.6) and 1.25 ml of potassium ferricyanide (1%) were added. the mixture was incubated at 50°c for 20 minutes. then, 1.25 ml of 10% trichloracetic acid was added to the mixture, which was centrifuged at 650 g for 10 minutes. an aliquot of 2.5 ml was taken, 2.5 ml of distilled water and 0.5 ml of ferric chloride were added, and the absorbance measured at 700 nm in uv/vis spectrophotometer (jenway, model 6305, japan). solutions of trolox (6-hydroxy-2.5.7.8tetramethyl-chroman-2carboxylic acid) in a range of concentrations were used for calibration of the frap assay. the values were expressed as mg of trolox/l of seed extracts. all determinations were performed in triplicate. 2.5. statistical analysis data were subjected to anova and tukey tests (statistica 7.0 software) at a 0.05 level of significance. five samples (n=3) of pulp and skin were analysed. 3. results and discussions 3.1. general composition and physicochemical properties the grapes of vitis tiliifolia are round and dark violet; with an average weight around 0.15 g, their dimensions were: length 3.74 mm and width 3.14 mm. physicochemical parameters of the pulp and skin in vitis tiliifolia fruits are described in table 2. pulp and skin showed moisture content of 84.88 and 82.00%, respectively. reducing sugars, ash and total dietary fibre were the major components in the pulp and skin of the grape. proteins content varied from 0.45 to 0.95 % in the pulp and skin, respectively; both values were lower to those reported for red and white grape (bravo and saura-calixto, 1998). the grape had a weight, brix, titratable acidity percentage and dimensions lower than those reported for other species, but a similar ph to those reported by jiang-fei et al. (2012) for four varieties of grapes, three red varieties (junzi #1, junzi #2 and liantang) and one white variety (baiyu). these differences are due to these properties being influenced by cultivar, ripening stage and environmental factors (cordenunsi et al., 2002). fruit colour is a tool that is commonly used by winemakers as a selection parameter to define the optimal moment for harvesting during the wine production process. however, this parameter generally is estimated visually and there is not enough information about relations among fruit colour, different harvest dates and some chemical parameters of the vitis tiliifolia fruit (obreque-slier et al., 2012). in this work, colour parameters showed significant differences (p<0.05) for the pulp (l*=6.23, a*=17.13 and b*=2.28) and skin (l*=38.42, a*=10.28 and b*=4.74). hue value of the skin (h=24.71°) was located in the first ital. j. food sci., vol. 30, 2018 135 quadrant of the colour plane which corresponds to a red-violet colour. parameter a*and b* assumed a positive values, indicating a characteristic violet colour. these colour parameters has been associated with the colour of anthocyanins present in grapes. all colour parameters of this fruit exhibited significant differences with respect to the other grape varieties reported (pérez-magariño and gonzález-san josé, 2003), but similar values to those reported for fruits such as chagalapoli (joaquín-cruz et al., 2015). table 2. composition of pulp and skin of vitis tiliifolia fresh fruit. pulp skin moisture (%) 84.88±0.14ᵇ 82.00±0.96a brix (°) 12.7±0.17ᵇ 8.16±0.28a ph 3.20±0.10a 3.30±0.50a titratable acid (%) 3.00±0.20a 3.50±0.20b reducing sugars (%) 15.82±0.16ᵇ 4.41±0.31a protein (%) 0.45±0.10a 0.95 ±0.05b oil 0.37±0.10a 0.50 ±0.10b ash 0.28 ±0.01a 0.85± 0.07b total dietary fiber 0.73±0.25a 1.53±0.10b aw 0.98±0.06ᵇ 0.64±0.05 a color parameters l 6.23±1.69a 38.42±0.04b a 17.13±0. 57ᵇ 10.28±0.16a b 2.28±0.26a 4.74±0.09b hue angle (°) 82.37±1.12ᵇ 24.71± 0.74a chroma 17.28±0.52ᵇ 11.31±0.12a browning index 81.63±7.94b 26.3±1.91a results are expressed as the mean (n=3)±sd. note: diameter of single grape was calculated on the basis of the mean of random 100 grapes. values of other parameters are mean±sd values of three replicates. means followed by different letters in column are significantly different by tukey's test 5%. 3.2. chemical compounds and antioxidant properties the antioxidant and functional properties of the different types of grapes depend to a great extent on the bioactive compounds it possesses. so that, in order to determine the compounds that may be responsible for the high antioxidant activity, we investigated the chemical constituents in pulp and skin of the grape (table 3). twelve compounds were identified and quantified in the skin, and only two were found in the pulp (vanillin and quercetin-3-d-galactoside). the most abundant compounds identified in skin were: quercetin-3-glucoside (39.86 µg/g dry sample), rutin (37.01 µg/g dry sample) and transresveratrol (32.88 µg/g dry sample). the majority of these compounds contain double bonds in their aromatic ring structure, reported to be responsible for electron delocalisation, which is attributed to their radical scavenging activity (rice-evans et al., 1996). these compounds may contribute to the antioxidant activity of this grape. moreover, it has been reported that the bioactivity of the grape is strongly correlated with the composition and the presence of polyphenol compounds (burin et al., 2014), which form an important group of secondary metabolites that is abundant and play an important ital. j. food sci., vol. 30, 2018 136 role in the quality and nutritional value of grapes. some of these polyphenol are synthesised in the skin of the fruit (jeandet et al., 1991) and their concentration depend of several factors such as climate, geographical area of cultivation, growing conditions and storage conditions (gerogiannaki-christopoulou et al., 2006). table 3. phenolic compounds (µg/g dry sample) presents in pulp and skin from vitis tiliifolia grape. compounds pulp skin 4-hydroxybenzoic acid 0.16±0.06 (+)-catechin 8.29±0.35 vanillic acid 12.60±0.18 caffeic acid 3.68±0.15 (-)-epicatechin 5.17±0.06 vanillin 0.004±0.00 0.33±0.01 4-coumaric acid 3.37±0.21 rutin 37.01±0.13 quercetin-3-d-galactoside 1.86±0.82 13.91±0.29 quercetin-3-glucoside 39.86 ±1.36 trans-resveratrol 32.88±0.72 quercetin 22.08±0.67 data are expressed as means±sd (n=3). anthocyanins were tentatively identified based on their mass spectra fingerprint (exact mass values and fragmentation pattern) in high resolution compared with public metabolomics databases (table 4). overall, the v. tiliifolia skin presented higher level of anthocyanins compared to the pulp (fig. 1). in total, five and seven anthocyanins were identified in pulp and skin, respectively. the most abundant anthocyanin tentatively detected in the skin was malvidin 3-glucoside, while that in pulp was malvidin 3,5diglucoside. the result that malvidin derivatives were the major anthocyanins agreed with the data reported by liang et al. (2008). total polyphenols concentration found in the skin (400.35 mg gae/g dry sample) was higher than in the pulp (171.26 mg gae/g dry sample) (table 5). this difference in the total polyphenol concentration between pulp and skin might be attributed to the different inherent components present in each part of the grape. the total soluble polyphenolic content of our grape was higher than those of other fruits, such as apple, melon, peach, pear, prune and strawberry (ishiwata et al., 2004) and similar to those reported in previous works with other varieties of grapes grown in various parts of the world (burin et al., 2014), but lower than reported by the red grape variety (apostoulo et al., 2013). these differences probably depend on the variety of grape and are influenced by climatic and geographical factors, cultural practices, and the stage of ripeness (burin et al., 2014). by other hand, flavonoids are secondary metabolites presents in plants and fruit such as grapes, which possess biological activities and have an impact on human health. the flavonoid content of the pulp (17.22 mg qe/g dry sample) was lower than quantified in the skin (282.57 mg qe/g dry sample) of the grape. ital. j. food sci., vol. 30, 2018 137 table 4. anthocyanins profile by ultra high resolution liquid chromatography and high-resolution mass spectrometry (uplc-hrms-esi-qtof). rt (min) mass detected (m/z) formula fragments (m/z) tentative identification formula ion type mass calculated error (ppm) pulp 2.36 655.187 c29h35o17 493.1341, 331.0816, 287.0543 malvidin 3,5-diglucoside c29h35o17 [m] + 655.1874 -0.6 2.74 479.1181 c22h23o12 317.0662 petunidin-3-o-β-glucoside c22h23o12 [m] + 479.119 -1.9 3.09 493.1341 c23h25o12 331.0809, 287.0541 malvidin 3-o-glucoside c23h25o12 [m] + 493.1346 -1 3.77 757.1971 c36h37o18 449.1087, 287.0550 cyanidin 3-o-(6-o-p-coumaroyl)glucoside5-o-glucoside c36h37o18 [m+h] + 757.198 -1.2 4.02 801.2236 c38h41o19 639.1685, 493.1356, 331.0816, 287.0551 malvidin 3-o-(6-o-(4-o-caffeoyl-alpharhamnopyranosyl)-beta-glucopyranoside) c38h41o19 [m+h] + 801.2242 -0.7 skin 2.36 655.1872 c29h35o17 493.1346, 331.0818, 287.0546 malvidin 3,5-diglucoside c29h35o17 [m] + 655.1874 -0.3 2.69 479.1183 c22h23o12 317.0652 petunidin-3-o-glucoside c22h23o12 [m] + 479.119 -1.5 3.02 493.1347 c23h25o12 331.0818, 287.0551 malvidin 3-o-glucoside c23h25o12 [m] + 493.1346 0.2 3.73 757.1961 c36h37o18 287.0551 cyanidin 3-o-(6-o-p-coumaroyl)glucoside5-o-glucoside c36h37o18 [m+h] + 757.198 -2.5 4.01 801.2236 c38h41o19 639.1699, 493.1339, 331.0815, 287.0556 malvidin 3-o-(6-o-(4-o-caffeoyl-alpharhamnopyranosyl)-beta-glucopyranoside) c38h41o19 [m+h] + 801.2242 -0.5 4.18 463.1234 c22h23o11 301.0706 peonidin 3-o-glucoside c22h23o11 [m+h] + 463.124 -0.6 4.73 639.1705 c32h31o14 331.0813 malvidin 3-(6''-p-coumarylglucoside) c32h31o14 [m+h] + 639.1714 -1.4 ital. j. food sci., vol. 30, 2018 138 figure 1. chromatograms and structure of anthocyanins tentatively identified based on their mass spectra fingerprint (exact mass values and fragmentation pattern). these data were lower than those reported in other varieties of grapes (güder et al., 2014). similarly, the total monomeric anthocyanins content was significantly higher (p < 0.05) in skin (188.11 mg cy3/g dry sample) than in pulp (150.93 mg cy3/g dry sample), and these values were similar to the values of anthocyanins reported for other varieties of grapes (de pascual-teresa et al., 2010). vitis tiliifolia skin had a high ratio of anthocyanins/total polyphenols close at 0.5, whereas pulp had a ratio close 1.0 indicating than more than half of the polyphenols present in pulp and skin are anthocyanins. a high proportion of the total polyphenols content presents in the grape correspond to anthocyanins, which are considered important groups of plant pigments that contribute to the coloration and sensorial attributes and diverse biological properties; therefore, these are considered secondary metabolites with potential nutritional value, as chronic diseases can be reduced by the regular consumption of anthocyanins in the diet. anthocyanins are regarded as important nutraceuticals due to their antioxidant activity (kallithraka et al., 2005). at the same time, total tannins were analysed in the pulp and skin, with a higher concentration found in the skin (188.37 mg leucocyanidin/g dry sample) than in the pulp (60.26 mg leucocyanidin/g dry sample). the result in total tannins concentration was consistent with other research that has reported a higher concentration of tannins in the skin and seeds of grapes, playing a ital. j. food sci., vol. 30, 2018 139 relevant role to define the sensory characteristics of red wines, contributing to bitterness and astringency, in addition to providing antioxidant and antibacterial activity (figueroa-espinoza et al., 2015). by last, in the present work, the pulp and skin from vitis tiliifolia presented a content of ascorbic acid of 130.88 and 5.75 mg aa/g dry sample, respectively, which could contribute to the recommended dairy dietary intakes (0.04-0.09 g/day) suggested by the united kingdom food standards agency or the united states national academy of science (del bubba et al., 2009). ascorbic acid is a good reducing agent present in grape juices that is associate with the biosynthesis of tartaric acid (debolt et al., 2006). in plants, ascorbic acid as vitamin c provides protection against free radicals generated during photosynthesis and respiration processes, and is also involved in cell growth; in addition, it is a co-factor of several enzymes participating in the synthesis of anthocyanidins and several secondary metabolites (bravo and sauracalixto, 1998). table 5. antioxidant activity of dry pulp and skin of vitis tiliifolia. pulp skin total polyphenols (mg gae/g dry sample) 171.26±7.90a 400.35±5.90b total monomeric anthocyanins (mg cy3/g dry sample) 150.93±5.55a 188.11±3.15b total flavonoids (mg qe/g dry sample) 17.22±2.40a 282.57 ±2.20b condensed tannins (leucocyanidin/g dry sample) 60.26±0.34a 188.37±0.20b ascorbic acid (mg aa/g dry sample) 130.88±9.60ᵃ 5.75±1.20b dpph radical scavenging activity (%) 19.57±2.13ᵃ 91.39±3.04b frap (mg te/g dry sample) 40.67±1.17a 7.24±1.80b gae: gallic acid equivalents, cy3: cyanidin-3-glucoside, qe: quercetin equivalents, aa: ascorbic acid, te: trolox equivalents. results are expressed as the mean (n=3)±sd. means followed by different letters in column are significantly different by tukey's test 0.05. the presence of these compounds has been demonstrated confer antioxidant activity. one of the techniques used to evaluate this capacity is through the percentage inhibition of the dpph radical and reducing power. dpph is a stable free radical and the effect of antioxidants on dpph scavenging is thought to be due to their hydrogenor electrondonating abilities. in its radical form, dpph radical absorbs at 517 nm, but this absorbance value decreases in the presence of an antioxidant or a radical species due to the reaction between antioxidant molecules and the dpph radical (güder and korkmaz, 2012). the radical scavenging activity of pulp and skin showed values from 19.57% and 91.39%, respectively, at a concentration of 10 mg/ml, showing that the skin was highly antioxidant than pulp, which is consistent with the concentration of some polyphenols compounds, such as flavonoids, tannins and anthocyanins. therefore, the data obtained reveal that these compounds present in this grape act as free radical inhibitors that confer antioxidant activity. similarly, the reducing power measured by the frap value was 40.67 and 7.24 mg te/g of dry pulp and skin, respectively. the value of reducing power were lower than those reported by red globe grapes (tagliazucchi et al., 2010), but frap values were consistent with other antioxidant techniques evaluated. frap assay does not react fast with some antioxidants, such as glutathione, but some authors consider the frap assay to still be suitable for assessment of the antioxidant activity of fruit samples because only limited amounts of plant glutathione are absorbed by humans (schafer and buettner, 2001). on the other hand, it is reported that the antioxidant activity determined by this technique corresponds to approximately 55% of the bioavailability at ital. j. food sci., vol. 30, 2018 140 the end of digestion (tagliazucchi et al., 2010). therefore, these reports are based on an estimate that approximately 13% of the total antioxidant is used by the human body. the reducing properties are generally associated with the presence of reductones, which also react with certain precursors of peroxide, thus preventing peroxide formation; a higher absorbance of the reaction mixture indicates greater reducing power (pin-der, 1998). table 6 shows the correlation analysis in the pulp and skin of vitis tiliifolia grape. a linearly relation of dpph radical scavenging activity with anthocyanins (r2=0.728), polyphenols (r2=0.878) and condensed tannins (r2=0.680), suggesting a strong antioxidant effect of these mixtures of components from vitis tiliifolia pulp. instead, ascorbic acid had a positive correlation (r2=0.850) with reducing power, and revealed a moderately strong relationship between ferric ion reducing power and ascorbic acid content. it is reported that in several fruits including grapes, over 80% of the frap value was from vitamin c contribution (guo et al., 2003). the results on the antioxidant activity of vitis tiliifolia pulp seems to be due to the presence of polyphenols and anthocyanins which may act in a similar fashion as reductones by donating the electrons and reacting with free radicals to convert them to more stable products and terminate the free radical chain reaction (jayapraskasha et al., 2001), which may serve as significant evidence of their potential antioxidant activity. instead, in the skin, the antioxidant activity is mainly due to the presence of polyphenols, anthocyanins, resveratrol, tannins and ascorbic acid. table 6. correlation coefficient (r2) between antioxidant activity and chemical components presents in vitis tiliifolia pulp. dpph reducing power (frap) total polyphenols 0.878 0.650 total flavonoids 0.650 0.500 total monomeric anthocyanins 0.728 0.567 resveratrol 0.320 0.450 condensed tannins 0.680 0.720 ascorbic acid 0.576 0.850 correlation was statistically significant at p < 0.05. 4. conclusions the results of the present study showed that skin from this wild grape has a higher concentration of polyphenols than pulp. the more abundant individual polyphenols in skin were quercetin-3-glucoside, rutin and trans-resveratrol. instead, the pulp has a large amount of ascorbic acid. malvidin 3-glucoside and malvidin 3,5-diglucoside were the most abundant anthocyanins identify in skin and pulp, respectively. all this compounds confer a strong antioxidant activity comparable to other grape varieties, and may explain in part the benefits for human health. in addition, the skin has an intense violet blue color that could be exploited to obtain pigments that can be used as food colorant, as food additives or as food supplements. ital. j. food sci., vol. 30, 2018 141 acknowledgements the authors’ acknowledges the support to the project 124229 (l-idea), and the national council of science and technology (conacyt). references aoac, association of official analytical chemists. 2000. official methods of analysis. 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56:159-170. veluri r., singh r. p., liu z., thompson j. a., agarwal r. and agarwal c. 2006. fractionation of grape seed extract and identification of gallic acid as one of the major active constituents causing growth inhibition and apoptotic death of du145 human prostate carcinoma cells. carcinogenesis. 27(7):1445-1453. wrolstad r. 2001. characterization and measurement of anthocyanin pigments in plums. j. agric. food chem. 57:339403. paper received july 20, 2017 accepted september 20, 2017 ijfs#639_bozza ital. j. food sci., vol 29, 2017 424 paper effect of high frequency ultrasounds on lycopene and total phenolic concentration, antioxidant properties and α-glucosidase inhibitory activity of tomato juice f. bot*a§, m. anesea, g. hungerfordb and m.a. lemosc adipartimento di scienze agroalimentari, ambientali e animali, università di udine, via sondrio 2/a, 33100 udine, italy bhoriba ibh,133 finnieston street, glasgow g3 8hb, united kingdom cfood & drink division, school of science, engineering and technology, university of abertay dundee, bell street, dundee dd1 1hg, united kingdom *corresponding author. francesca.bot@uniud.it §current address: school of food and nutritional sciences, university college cork, cork, ireland, francesca.bot@ucc.ie abstract tomato juice was subjected to high frequency ultrasounds (378 and 583 khz) at increasing energy densities (up to 250 mj/m3). results relevant to the treatments at high frequency providing an energy density of 250 mj/m3 were compared with those obtained at 24 khz delivering the same energy density. lycopene and total phenolic concentration, as well as the α-glucosidase inhibitory activity of tomato juice, were not affected by ultrasound regardless the frequency and energy density. however, the antioxidant properties were negatively affected by high frequency ultrasounds. keywords: antioxidant properties, α-glucosidase inhibitory activity, high frequency ultrasounds, tomato juice, total phenolic concentration ital. j. food sci., vol 29, 2017 425 1. introduction the consumer’s interest towards functional food which claims to have health-promoting or disease-preventing properties is increasing. this has led to the study and development of processes able to preserve the food nutritional and sensory quality as well as to enhance the bioactivity of certain constituents (knorr et al., 2004). amongst these, ultrasound (us) processing, which is a non-thermal technology characterized by low energy consumption, has been found to be suitable for different applications in the food industry. ultrasound technology is widely used to emulsify, encapsulate ingredients, extract bioactive molecules and inactivate microorganisms (piyasena et al., 2003; canselier et al., 2002; soria and villamiel, 2010; kentish and ashokkumar, 2011; chemat et al., 2011; mane et al., 2015). ultrasounds can be considered as waves with a frequency range higher than 18 khz. during the wave propagation into a liquid medium, cavitation phenomenon occurs. in particular, cavitation involves the formation and violent collapse of small bubbles, generating shock waves associated to high local temperatures (1000-5000 k) and pressures (100-50000 bar) inside the collapsing bubbles (leighton, 1994). these extreme conditions lead to the occurrence of physical phenomena (microject, turbulence, shear forces) and formation of free radicals (gogate et al., 2003). high frequency (100 khz to 1000 khz) low power ultrasounds is used for food quality monitoring and diagnostic purposes. however, recent studies have found that free radicals generated by high frequency ultrasounds can react with bioactive compounds and enhance their functionality. in particular, hydroxyl radicals generated during ultrasonication can enhance the hydroxylation degree of food materials and consequently modify their antioxidant activity (ashokkumar et al., 2008). tomatoes, an important agricultural commodity worldwide, are well known for their nutritional properties. in fact, they are a good source of bioactive compounds such as carotenoids and phenolic compounds (giovanelli et al., 1999; lenucci et al., 2012; zanfini et al., 2017). lycopene is the most abundant carotenoid in tomatoes and its chemical structure confers important properties, such as oxygen-radical scavenging and quenching capacity (di mascio et al., 1989; shi and le maguer 2000; rao and rao, 2007). moreover, phenolic compounds, although present in lower amount, may also contribute to beneficial effects of tomato products, because of their high antioxidant activity and ability to inhibit carbohydrate hydrolysing enzymes, such as α-glucosidase (hanhineva et al., 2010; nair et al., 2013; tomas et al., 2017). the latter, located in the brush-border surface of intestinal cells, catalyses the hydrolysis of complex carbohydrates and disaccharides to absorbable monosaccharides (kim et al., 2010). as a consequence, the α-glucosidase inhibition can suppress the influx of glucose from the intestinal tract to blood vessels resulting in an important strategy in the management of postprandial hyperglycemia (kwon et al., 2008). nevertheless, the nutritional value due to carotenoids and phenolic concentration might be modified during processing. in fact, there are several studies dealing with the effect of thermal and non-thermal processing (high pressure homogenization, pulsed electric field and ultrasounds) on bioactive molecules naturally occurring in tomato derivatives in order to obtain fruit and vegetables derivatives able to accomplish desired nutritional functions (colle et al., 2010; odriozola-serrano et al., 2009; anese et al., 2015). however, the effect of high frequency ultrasound treatment on tomato properties has been less studied (golmohamadi et al., 2013). because of this, the aim of the present study was to evaluate whether the high frequency ultrasounds might modify nutritional functions of the main bioactive compounds in tomato extract. to achieve this aim, the objectives of this work were to investigate the effect of high frequency (378 and 584 khz) ital. j. food sci., vol 29, 2017 426 ultrasounds on bioactives present in tomato (lycopene and total phenolic compounds) and the effects of extracts on the antioxidant properties and α-glucosidase inhibitory activity. moreover, the treatment was performed for increasing length of time (up to 60 min), thus providing increasing energy densities (up to 250 mj/m3). the results obtained by providing the maximum energy density value were compared with those obtained for samples subjected to low frequency ultrasounds (24 khz). such investigation represents a preliminary study aimed to understand whether high frequency us could represent a suitable technological tool for steering tomato juice functionality. no industrial application was considered at this stage of the research. 2. materials and methods 2.1. sample preparation commercial pasteurized tomato juice (7.5 °brix) was sieved (20 mesh) to separate seeds and coarse particles, and submitted to ultrasound treatment. tomato juice not subjected to ultrasound treatment was used as a control. 2.2. sonication ultrasound treatments were conducted using two ultrasonic processors operating at either 378 khz or 583 khz, and 24 khz. 2.2.1. ultrasound treatment at 378 khz and 583 khz an ultrasonic processor (meinhardt ultraschlltechnik leipzig, germany) equipped with two transducers (operating at 378 khz and 584 khz) was used. aliquots of 250 ml of tomato juice were introduced into a glass reaction vessel (63 mm internal diameter) with a cooling jacket (wall thickness 5 mm) connected to a cryostatic bath (fisher scientific, isotemp thermostatic bath). samples were subjected to sonication for increasing length of time up to 60 min. during the ultrasound treatment, the temperature never exceeded 20 °c. 2.2.2. ultrasound treatment at 24 khz an ultrasonic processor (hieschler ultrasonics gmbh, mod. up400s, teltow, germany) with a titanium horn tip diameter of 22 mm operating at 24 khz was used. aliquots of 100 ml of tomato juice were introduced into 250 ml capacity (110 mm height, 60 mm internal diameter) glass vessels. the tip of the sonicator horn was placed in the centre of the tomato juice, with an immersion depth in the fluid of 50 mm. sample were subjected to sonication up to 3 min. during the ultrasound treatment the temperature never exceeded 40 °c. 2.3. determinations 2.3.1. power and energy density computation the power density (pv, w/m3) transferred from the ultrasounds probe to the sample was determined calorimetrically during preliminary trials by recording the temperature (t, k) increase during the treatment, following eq. (1) (raso et al., 1999). ital. j. food sci., vol 29, 2017 427 𝑃! 𝑇 = 𝑑𝑐! 𝜕𝑇/𝜕𝑡 (1) where d is the sample density (kg/m3), cp is the water heat capacity (4186 j/kg k). the energy density (mj/m3) was calculated by multiplying the power density value by the duration of the treatment (hulsmans et al., 2010). 2.3.2. lycopene concentration the lycopene extraction was performed following the procedure of sadler et al., (1990), with minor modification, under subdued light to prevent carotenoid degradation and isomerisation. aliquots of 25 ml of extraction solution (hexane:acetone:ethanol, 2:1:1 v/v/v) were added to 1 g of tomato juice. the mixture was stirred at room temperature for 20 min. reagent grade water (7.5 ml) was added and stirring was continued for 10 min. the hexane phase, containing lycopene, was separated from the polar phase using a separation funnel. immediately after extraction, the absorbance of lycopene was measured at 472 nm with a spectrophotometer (thermo fischer scientific, genesys 10s uv/vis spectrophotomer) using hexane as reference. the total lycopene concentration was calculated using the beer-lambert law, considering the extinction coefficient of lycopene in hexane equal to 1.8 x 105 l/mol cm. 2.3.3. total phenolic concentration the total quantity of phenolic components was measured by the folin-ciocalteau method (singleton and rossi, 1965). aliquots of 10 ml of methanol:water (1:1 v/v) were added to 1 g of tomato juice and the mixture was stirred for 5 min. the solution was filtered (ql10, size 150 mm) and 100 µl was added to 5 ml of a 1:10 dilution of folinciocalteau reagents and 0.9 ml of distilled water. after 5 min, aliquots of 3.5 ml of na2co3 (115 g/l) were added and the mixture left in the dark, at room temperature for 2 hours. the absorbance of the solution was measured at 765 nm. the optical density was compared to a standard curve prepared with 0 to 500 mg/l of gallic acid and the results were expressed as mg gae (gallic acid equivalents)/100 g. 2.3.4. optical microscopy tomato juice microstructure was analysed by using an optical microscope (leica dm 2000, leica microsystems, heerburg, switzerland). the pictures were taken by a digital camera (leica ec3, leica microsystems, heerburg, switzerland), using the leica suite las ez software (leica microsystems, heerburg, switzerland). 2.3.5. antioxidant activity the antioxidant activity was determined following the procedure of benzie and strain (1996) determined using ferric reducing antioxidant potential (frap). aliquots of 20 ml of acetone:water (80:20 v/v) mixture were added to 1 g of tomato juice and the mixture was stirred for 5 min. aliquots of 90 µl of this mixture were added to 3 ml of frap solution and incubated in a water bath at 37 °c for 4 min followed by measurement of absorbance at 593 nm against a blank. the optical density was compared to the standard curve for ferrous sulphate (feso4) solution, with concentrations between 0 and 1 mm. results were expressed as feii (mm) produced/100 g sample. ital. j. food sci., vol 29, 2017 428 2.3.6. α-glucosidase inhibitory activity the α-glucosidase inhibitory activity of tomato juice was determined spectrophotometrically (uv-2501pc, uv-vis recording spectrophotometer, shimadzu corporation, kyoto, japan) following the method of sing et al., (2014) with some modifications. the tomato juice was first centrifuged at 2200 g for 3 min at 20 °c. aliquots of 30 µl of 1 u/ml α-glucosidase in phosphate buffer (100 mm, ph 7.0) were introduced into 1 ml capacity cuvette in the presence of 100 µl of tomato extract and a volume of 100 mm phosphate buffer (ph 7.0), giving a final volume of 900 µl in the cuvette, and mixed thoroughly. after 10 min of incubation at 37 °c, the reaction was started by the addition of 100 µl of 5 mm 4-nitrophenylα-d-glucopyranoside (pngp) solution (sigma-aldrich, milano, italy) in 100 mm phosphate buffer (ph 7.0) as substrate. the release of pnitrophenol from pngp was monitored at 405 nm for 15 min at 37 °c. the changes in absorbance per min were calculated by linear regression, applying the pseudo zero order kinetic model. the eventual stationary phase was excluded from the regression of data. control samples were run in the absence of tomato extract. the inhibitory activity (ia%) of samples on α-glucosidase was calculated according to the following equation: ia% = 100 − !! !! ∙ 100 (2) where ks and kc are the constant rates (abs/min) of the enzymatic activity in the presence or in the absence of the inhibitor. 2.4. data analysis the results reported here are the averages of at least two measurements carried out on two replicated experiments (n ≥ 4). data are reported as mean value ± standard deviation. statistical analysis was performed using r v. 2.15.0 (the r foundation for statistical computing). bartlett’s test was used to check the homogeneity of variance, one way anova was carried out and tukey test was used to determine statistically significant differences among means (p < 0.05). 3. results and discussions 3.1. effect of ultrasounds on lycopene and total phenolic concentration of tomato juice table 1 shows lycopene and phenolic concentration of tomato juice subjected to high frequency ultrasounds (378 and 584 khz) with increasing energy density. no differences in lycopene concentration were found among the untreated and ultrasonically treated samples, regardless of the frequency and energy density. the total phenolic concentration was slightly affected by the application of high frequency ultrasounds. a significant increase of the phenolic concentration was observed for the tomato juice subjected to ultrasounds at 378 khz, providing an energy density of 250 mj/m3. however, from a practical point of view, this result does not appear to be relevant in terms of total phenolics recovery. to our knowledge, only golmohamadi et al. (2013) have investigated the effect of high frequency ultrasounds (490 khz) on the total phenolics in red raspberry puree. they found that ultrasonication did not affect the total phenolic concentration. however, results of golmohamadi et al. (2013) cannot be directly ital. j. food sci., vol 29, 2017 429 compared with those obtained in this study, due to discrepancies in the frequencies as well as the lack of information concerning the energy density applied. levels of lycopene and total phenolics presented in the tomato juice after treatment at 378 and 583 khz (250 mj/m3) were compared with those obtained at 24 khz with the same energy density (table 1). in fact, although the application of low frequency ultrasounds is commonly used in food processing, no data are present in the literature comparing the effect of high and low frequency ultrasounds at the same energy density. it can also be observed that the ultrasound treatment at 24 khz did not cause any significant change in lycopene concentration. this result is in agreement with previously published data (anese et al., 2013) relating to tomato juice ultrasonically treated at 24 khz with an energy density of 731 mj/m3. moreover, the low frequency ultrasound treatment did not significantly modify the total phenolic concentration of tomato juice. this result is, however, contrary to that of chemat et al. (2011) and the discrepancy may be attributed to differences in the process parameters. table 1. lycopene and total phenolic concentrations, antioxidant activity and α-glucosidase inhibitory activity of untreated and ultrasonically treated tomato juice. data are referred to increasing energy density provided at 24 khz, 378 khz and 583 khz. frequency (khz) time (min) energy density (mj/m3) lycopene (mg/g) total phenolic (gae mg/100 g) antioxidant activity (feii mm/100 g) α-glucosidase inhibitory activity (%) untreated 0 0 0.35±0.01a 192.1±4.0bc 25.0±2.7a 72±4a 24 3 250 0.36±0.01a 193.9±1.0bc n.d. 74±2a 378 15 15 0.35±0.01a 192.9±1.0bc 17.8±0.5b n.d. 60 59 0.35±0.02a 191.4±1.0bc 15.7±0.3b 78±2a 15 62 0.36±0.01a 205.0±2.0ab 19.2±0.5b n.d. 60 250 0.37±0.02a 212.2±6.0a 18.8±1.1b 78±2a 583 15 8 0.35±0.00a 197.1±3.0bc 18.7±0.6b n.d. 60 31 0.35±0.01a 195.0±2.0bc 17.8±2.2b 76±1a 15 62 0.33±0.00a 194.3±3.0bc 19.3±1.8b n.d. 60 250 0.35±0.01a 187.1±5.0c 18.7±0.3b 79±3a means with different letters within the same column are significantly different (p<0.05). n.d.: not determined. although the application of high and low frequency ultrasounds did not affect the levels of bioactive components, slight differences in the tomato microstructure were observed. fig. 1 shows the micrographs of tomato juice subjected to ultrasounds at 24 khz, 378 khz, 583 khz at an energy density of 250 mj/m3. when compared to the untreated tomato juice, the low frequency ultrasonically processed samples showed a partial disruption of cell membranes with carotenoids distributed into the matrix. however, no differences between the untreated sample and those processed at 387 and 583 khz were observed. the differences in the microstructure of samples treated at low and high frequency can be attributed to cavitation phenomena occurring during ultrasound treatment. in fact, during low frequency (24 khz) ultrasounds, transient cavitation phenomena are responsible for the rapid change in fluid pressure and temperature, which might cause cell wall disruption and carotenoids released. by contrast, microstreaming phenomena are ital. j. food sci., vol 29, 2017 430 generated by stable cavitation at high frequency (378 khz and 583 khz) ultrasounds; these are reported not to cause dramatic structure changes (mcclements, 1995). figure 1. images of tomato juice subjected to ultrasounds at 24 khz, 378 khz and 583 khz (at 250 mj/m3 corresponding to 3 min for ultrasonically treated sample at 24 khz and 60 min for 378 and 583 khz ultrasonically treated sample) along with an untreated sample. 3.2. effect of ultrasounds on antioxidant properties and α-glucosidase inhibitory activity of tomato juice table 1 shows the effect of high frequency ultrasounds at 378 khz and 583 khz on the antioxidant activity of tomato juice. as compared with the untreated sample, the application of the high frequency ultrasounds caused a significant reduction in the antioxidant activity. this seems to be in contradiction with the levels of lycopene and total phenolic compounds which were affected by ultrasounds. it should be kept in mind that these are not the only compounds with antioxidant properties present in tomato product. for instance, ascorbic acid is a well-known antioxidant and this vitamin has been destroyed by high frequency ultrasounds (golmohamadi et al., 2013; portenlänger and heunsiger, 1992). this result is in contrast with data reported ital. j. food sci., vol 29, 2017 431 by ashokkumar et al. (2008) for a phenol model aqueous solution subjected to 358 khz. the authors attributed the increase in antioxidant properties of the phenolic component in the model solution to the generation of oh. radicals due to the homolysis of water molecules and subsequent phenol hydroxylation. the decrease in antioxidant capacity observed under our experimental conditions may be attributed to the addition of the hydroxyl radicals in a non-preferred position of the aromatic ring. this has previously been observed for a cyanidin 3-glucoside model system (ashokkumar et al., 2008). these results suggest that controlled hydroxylation is needed to promote an increase in the antioxidant properties of phenolic compounds, which is indeed difficult to achieve. increasing attention has been recently paid towards the capability of phenolic compounds to control blood glucose levels related to type 2 diabetes (hanhineva et al., 2010; lordan et al., 2013). in particular, phenolics were found to inhibit digestive enzymes involved in starch breakdown, such as α-glucosidase. table 1 shows the α-glucosidase inhibitory activity of tomato juice subjected (or not) to ultrasounds at 24 khz, 378 khz and 583 khz at an energy density of 250 mj/m3. it can be observed that in our experimental conditions for the untreated sample α-glucosidase inhibition was about 72%. moreover, no significant changes in α-glucosidase inhibition were found when considering the ultrasound treated samples. 4. conclusions the results of this study showed that ultrasound treatments at 24, 378 and 583 khz had no effect on major bioactive compounds (i.e. lycopene and total phenolics) as well as on αglucosidase inhibitory activity of tomato juice regardless the frequency and the energy density. on the other hand, the antioxidant properties appear to be reduced by high frequency ultrasounds. thus, we conclude that high frequency ultrasound treatments do not seem to effectively alter the bioactive concentration or improve the functionality of tomato juice. acknowledgements this work was supported by a stsm grant from cost action fc1001 and the authors would like to acknowledge networking supported by the cost action fc1001. the authors would like to thank david bremner (university of abertay dundee) for providing the ultrasounds equipment. references anese m., mirolo g., beraldo p. and lippe g. 2013. effect of ultrasound treatments of tomato pulp on microstructure and lycopene in vitro bioaccessibility. food chem. 136:458. anese m., bot f., panozzo a., mirolo l. and lippe g. 2015. effect of ultrasound treatment, oil addition and storage time on lycopene stability and in vitro bioaccessibility of tomato pulp. food chem. 172:685. ashokkumar m., sunartio d., kentish s., mawson r., simons l., vilkhu k. and versteeg c. 2008. modification of food ingredients by ultrasound to improve functionality:a preliminary study on a model system. innov. food sci. emerg. technol. 9:155. benzie i.f.f. and strain j.j. 1996. 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j.a. 1965. colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. am. j. enol. vitic. 16:144. soria a.c. and villamiel m. 2010. effect of ultrasound on the technological properties and bioactivity of food. a review. trends food sci. tech. 21:323. tomas m., beekwilder j., hall r.d., sagdic o., boyacioglu d. and capanoglu e. 2017. industrial processing versus home processing of tomato sauce:effects on phenolics, flavonoids and in vitro bioaccessibility of antioxidants. food chem. 220:51. zanfini a., franchi g.g., massarelli p., corbini g. and dreassi e. 2017. phenolic compounds, carotenoids and antioxidant activity in five tomato (lycopersicon esculentum mill.) cultivars. ital. j. food sci. 29:90. paper received october 18, 2017 accepted march 15, 207 #588_o brien_bozza ital. j. food sci., vol 29, 2017 158 paper purification and identification of antioxidant peptides from gelatin hydrolysates of unicorn leatherjacket skin s. karnjanapratum1, y.c. o’callaghan2, s. benjakul1, m.b. o’keeffe3, r.j. fitzgerald3 and n.m. o’brien*2 1department of food technology, faculty of agro-industry, prince of songkla university. hat yai, songkhla 90112, thailand 2school of food and nutritional sciences, university college cork, cork, republic of ireland 3department of life sciences, university of limerick, limerick, republic of ireland *corresponding author. address: nob@ucc.ie abstract antioxidant peptides from a gelatin hydrolysate of unicorn leatherjacket skin prepared using a partially purified glycyl endopeptidase were purified using sephadex g-25 gel filtration, deae-cellulose anion-exchange and reverse phase high-performance liquid chromatography. the fractions with the highest abts radical scavenging activity were analyzed using uplc–esi-ms/ms to identify the peptide sequences therein. four of the identified peptides, glu-pro-gly-pro-val-gly (555.27 da), leu-pro-gly-pro-ala-gly (511.29 da), leu-asp-gly-pro-val-gly (557.30 da) and glu-gly-pro-leu-gly (472.24 da), were subsequently synthesized. glu-gly-pro-leu-gly exhibited the highest antioxidant activity (4.95 µmol te/g solid). therefore, peptides from unicorn leatherjacket skin gelatin hydrolysate could be further employed as functional food ingredient. keywords: gelatin hydrolysate, unicorn leatherjacket, antioxidant activity, identification, uplc, mass spectrometry ital. j. food sci., vol 29, 2017 159 1. introduction there is an increased interest in the isolation of natural antioxidants from different sources as the use of synthetic antioxidants is restricted due to the potential risks to human health (hraš et al., 2000). antioxidant peptides have been found in several foodstuffs such as milk (phelan et al., 2009), cereals (malaguti et al., 2014) and algae (cornish and garbary, 2010). fish protein hydrolysates, especially gelatin hydrolysates, have been shown to have the ability to scavenge free radicals (mendis et al., 2005), inhibit lecithin liposome peroxidation (karnjanapratum and benjakul, 2015a; 2015b) and reduce the oxidation of fe3+ to fe2+ (aleman et al., 2011). the antioxidant activity of protein hydrolysates is mainly governed by the parent protein sequence, the specificity of enzymes used and the conditions used in the hydrolysate preparation (peña-ramos and xiong, 2011). the amino acid sequences of several antioxidant peptides have been identified (suetsuna et al., 2000; saiga et al., 2003; fan et al., 2012), demonstrating an association between bioactivity and the amino acid sequence (shahidi and zhong, 2008). gelatin peptides have an abundance of glycine (gly), proline (pro) and hydroxyproline (hyp), which may contribute to their enhanced bioactivity in comparison with peptides isolated from other sources. pro residues have a scavenging effect on radicals and the percentage of hydroxylation has been correlated with antioxidant activity (aleman et al., 2011). antioxidant gelatin hydrolysates from unicorn leatherjacket skin have been produced using different methods. the autolysis-assisted process mediated by indigenous protease in combination with thermal or enzymatic hydrolysis was implemented to prepare antioxidant gelatin hydrolysates from unicorn leatherjacket skin (karnjanapratum et al., 2015b). glycyl endopeptidase (ge), isolated from papaya latex, was shown to yield gelatin hydrolysates from unicorn leatherjacket skin with higher antioxidant activity, compared to the crude extract from papaya latex (karnjanapratum et al., 2015a). gelatin hydrolysates from unicorn leatherjacket skin prepared using ge with autolysisassisted process also demonstrated antioxidant properties in in vitro cellular model systems (karnjanapratum et al., 2015). nevertheless, no information on the structure and sequence of potential antioxidant peptides from skin gelatin hydrolysates of unicorn leatherjacket exists. the aims of this study were to purify and identify the amino acid sequences of antioxidant peptides from gelatin hydrolysate of unicorn leatherjacket skin prepared using glycyl endopeptidase. 2. materials and methods 2.1. chemical 2,2′-azinobis (3-thylbenzothiazoline-6-sulfonic acid) (abts), 6-hydroxy-2,5,7,8tetramethyl-chroman-2-carboxylic acid (trolox) and other reagents were obtained from sigma chemical co. (dublin, ireland). 2,2'-azobis(2-amidinopropane) dihydrochloride (aaph) was purchased from fluka chemie (buchs, switzerland). all solvents were high performance liquid chromatography (hplc) grade and were procured from merck (darmstadt, germany). ital. j. food sci., vol 29, 2017 160 2.2. preparation of gelatin hydrolysate from unicorn leatherjacket skin 2.2.1 preparation of fish skins the skins of unicorn leatherjacket (aluterus monoceros) were obtained from a dock in songkhla, thailand. the skins were washed with iced tap water (0-2°c) and cut into small pieces (0.5×0.5 cm2). skins were subjected to alkaline pretreatment to remove noncollagenous proteins as per the method of kaewruang et al. (2013). the autolysis of pretreated skin was conducted following the method of karnjanapratum and benjakul (2015a). the resulting autolyzed skin was used as a substrate for preparation of the gelatin hydrolysate. 2.2.2 preparation of partially purified glycyl endopeptidase from papaya (carica papaya) latex the glycyl endopeptidase (ge) was fractionated from the latex using the method of karnjanapratum and benjakul (2014). an aqueous two phase system (atps) with 10% peg 6000 and 10% ammonium sulphate (nh4)2so4 was used for fractionation of ge. the obtained ge was stored at -40°c until use. 2.2.3 production of gelatin hydrolysates the antioxidant gelatin hydrolysate from skin of unicorn leatherjacket was prepared as described by karnjanapratum and benjakul (2015a). autolyzed skin solution (3%, w/v) was hydrolysed using ge (8%, w/w based on solid matter) at 40°c for 60 min. the resulting gelatin hydrolysate (gh) was lyophilized using a scanvac model coolsafe 55 freeze dryer (coolsafe, lynge, denmark) and was then stored at -20°c until use for analysis. 2.3. purification of antioxidant peptides from a previous study, it was found that gh demonstrated strong abts radical scavenging activity in comparison with ferrous chelating activity and ferric reducing antioxidant power (karnjanapratum et al., 2015a). abts radical scavenging activity was therefore selected to monitor antioxidative activity of purified peptide in each step. 2.3.1 size exclusion chromatography gh (2 ml, 80 mg/ml) was subjected to gel filtration (2.5 x 50 cm column) using sephadex g-25 (product number 17-0032-01, ge healthcare bio-science ab, uppsala, sweden) as described by karnjanapratum and benjakul (2015a). the fractions of 3 ml were collected. the absorbance of the eluent was recorded at 220 and 280 nm. all fractions were measured for abts radical scavenging activity. 2.3.2 anion-exchange chromatography the fractions exhibiting the highest antioxidant activity obtained from size exclusion chromatography were pooled, lyophilized and further subjected to anion-exchange chromatography (deae-cellulose, whatman, england) column (1.0×50 cm) coupled with a fraction collector (model 2128, bio-rad laboratories ltd.). the elution was carried out using a constant flow rate (0.5 ml/min) with a linear gradient of nacl (0-0.4 m). the ital. j. food sci., vol 29, 2017 161 absorbance at 220 and 280 nm was monitored and 3 ml fractions were collected. abts radical scavenging activity of all fractions was measured and the fractions exhibiting high antioxidant activity were pooled (peak b-1, b-2, b-3), desalted into di water using size exclusion chromatography (sephadex g-25, 2.5×50 cm column) and subsequently lyophilized. 2.3.3 high performance liquid chromatography (hplc) the fraction with the highest abts radical scavenging activity obtained from anionexchange chromatography was further separated using reversed phase(rp-hplc) on a 201tp c18 (4.6×250 mm) column (grace davision discovery science, epping, australia). the hplc system consisted of a spectra system p2000 pump (thermo electron corporation, wisconsin, united state), sample injector (spectra system as3000, thermo electron corporation) and a detector (spectra system uv6000lp, thermo electron corporation). the column was equilibrated with 0.1% (v/v) trifluoroacetic acid (tfa) in water (solvent a), and a linear gradient was developed using solvent a and solvent b (acetonitrile containing 0.1% (v/v) tfa). to separate the peptides, elution was performed with the following conditions: 0.0-10.0 min, 5% b; 10.0-60.0 min, 5.0-30.0% b; 60.0-70.0 min, 100% b; 70.0-80.0 min, 100% a, at a flow rate of 1.0 ml/min. the fractions were manually collected, based on the peaks of a220 and a280. each fraction was evaporated to dryness in a mivac centrifugal vacuum quattro concentrator (genevac ltd., ipswich, uk) and abts radical scavenging activity of each fraction was then determined. total activity/a220 of each fraction was then calculated. 2.4. abts radical scavenging activity the abts radical scavenging activity of gelatin hydrolysates was determined as described by binsan et al. (2008). the activity was expressed as μmol trolox equivalent (te)/g sample. 2.5. peptide identification using ultra performance liquid chromatography (uplc)-electrospray ionization (esi) mass spectrometry (ms) and tandem ms (ms/ms) samples were separated on an acquity uplc (waters, milford, ma, usa) and analyzed on an impact hd™ mass spectrometer (bruker daltonics, bremen, germany). mobile phase a was 0.1% formic acid (fa) in ms-grade h2o and mobile phase b was 0.1% fa in 80% ms-grade acetonitrile. peptides were separated on an acquity uplc beh 300 c18 rp column (1.7 µm×50 mm, waters). a flow rate of 0.25 ml/min with isocratic elution was used for 5 min at 100% mobile phase a, followed by gradient elution to 80% mobile phase b from 5 to 25 min. ms/ms analysis was performed using two different methods: (i) a broad range method targeting peptides having a wide range of molecular masses and (ii) a short peptide method specifically targeting peptides having low molecular masses, as developed by o’keeffe and fitzgerald (2015), was used. the impact hd™ (bruker daltonics) was calibrated using esi low molecular mass tune mix (agilent technologies, cork, ireland) for the broad range method while sodium formate (10 mm naoh, 0.2% formic acid in isopropanol) was used as calibrant for the short peptide method. mass spectra were acquired in positive ion mode and scans were performed for auto ms/ms between 100 and 2500 m/z for the broad range method and between 50 and 600 m/z for the short peptide method. ms/ms conditions were as follows: capillary voltage: 4500 v; collision gas: ital. j. food sci., vol 29, 2017 162 nitrogen; nebulizer pressure 1.8 bar; dry heater temperature: 220 ºc and dry gas flow: 8 l/min. specific broad range method conditions were: collision energy 7.0 ev; commission cell radio frequency (rf): 1500 vpp and transfer time: 100 µs. specific short peptide ms/ms conditions were: collision energy 5.0 ev; collision cell rf was stepped between 200 and 350 vpp (50% of the time each) and transfer time was stepped between 36.1 and 51.1 µs (50% of the time each). all ms/ms spectra were searched against the swissprot database, limited to phylum chordata using peaks studio 7.5 (bioinformatics solutions inc., waterloo, canada) and mascot (version 2.3, matrix science, london, uk). further peptide identification was carried out by de novo sequencing using peaks studio 7.5, data analysis (version 4.0, bruker daltonics) and biotools (version 3.2, bruker daltonics) software. 2.6. peptide synthesis the identified peptides with the typical collagen sequence (gly-pro-x motif) and with homology of peptide to collagen proteins ≥75% as well as ≥65% average local confidence (alc) were selected, in which the presence of gly at c-terminal, attributed to glycyl endopeptidase cleavage, would be the major criteria. the selected peptides were synthesized by dgpeptides co., ltd (hangzhou, zhejiang, china). the purity of the synthesized peptides was greater than 95% as determined by hplc. all peptides were assayed for abts radical scavenging activity as described previously and results were expressed as μmol trolox equivalent (te)/g peptide. 2.7. statistical analysis experiments were carried out in triplicate. the data were subjected to analysis of variance (anova). comparison of means was carried out by duncan’s multiple range test (steel and torrie, 1980). statistical analysis was performed using the statistical package for social science (spss 11.0 for windows, spss inc., chicago, il, usa). 3. results and discussion 3.1. purification of antioxidant peptides previous work (karnjanapratum and benjakul, 2015a), found that abts radical scavenging activity had the highest activity in comparison with other antioxidant assays used. additionally, this assay has been shown to measure the antioxidant activity of both hydrophilic and hydrophobic antioxidants (aghdam et al., 2011). therefore, the abts assay is suitable for measuring the antioxidant activity of gelatin peptides, which contain both hydrophobic and hydrophilic amino acids in the peptides sequence, as the hydrophilic–hydrophobic property of the peptide is a critical factor affecting its antioxidant activity (mendis et al., 2005). thus, the abts radical scavenging activity was carried out for screening and selecting the most antioxidant fraction from gelatin hydrolysate. initial isolation of the peptides with the highest antioxidant activity from gh was carried out using sephadex g-25 size exclusion chromatography. two fractions (a and b) were collected and abts radical scavenging activity of each was determined (fig. 1). fraction b, which contained smaller lower molecular mass peptides showed the higher antioxidant activity (82.46 µmol/g solid), compared to gh (65.49 µmol te/g solid) and fraction a ital. j. food sci., vol 29, 2017 163 (9.31 µmol te/g solid) (p<0.05). in general, lower molecular mass peptides possess higher antioxidant activities (sun et al., 2013; intarasirisawat et al., 2013; fan et al., 2012). figure 1. elution profile of gelatin hydrolysate (gh) from unicorn leatherjacket skin on a sephadex g-25 column (a) and abts radical scavenging activity of gh and its fractions (b). values represent mean ± sd. different letters on the bars indicate significant differences (p < 0.05) between values. fraction b obtained from sephadex g-25 size exclusion chromatography was further separated by deae-cellulose column. three fractions (b-1, b-2 and b-3) with abts radical scavenging activity were obtained from anion exchange chromatography (fig. 2). the results suggested that most of the peptides in fraction b were negatively charged and those with lower negative charge were dominant (fraction b-1). the peptides with higher negative charge (b-2 and b-3) were eluted with increasing concentrations of nacl. fraction b-2 showed the highest abts radical scavenging activity (p<0.05) (fig. 2b). a similar result was reported for antioxidant peptides from tilapia skin gelatin hydrolysates, in which highly charged peptides showed the highest antioxidant activity (zhange et al., 2012). peptide charge can vary depending on hydrolysis conditions such as the type of enzyme and substrate used. when peptides from a skipjack roe hydrolysate were separated by cation exchange, fractions with lower charges had stronger antioxidant ital. j. food sci., vol 29, 2017 164 properties (intarasirisawat et al., 2013). in order to remove salt from fraction b-2, a sephadex g-25 column was used. the desalted fraction was subsequently lyophilized. figure 2. elution profile of fraction b on a deae-cellulose column (a) and abts radical scavenging activity of fractions (b). values represent mean ± sd. different letters on the bars indicate significant differences (p < 0.05) between values. fraction b-2 was further fractionated by rp-hplc on a c-18 column using a linear gradient of acetonitrile containing 0.1% tfa. as shown in fig. 3, eighteen fractions were obtained from the rp-hplc column and their abts radical scavenging activities were evaluated. the highest antioxidant activity was observed in fraction b-2/4 (0.72 total activity/a220), followed by fraction b-2/8 (0.64 total activity/a220). the fractions with higher hydrophobicity were eluted with increasing acetonitrile concentration. it was reported that the more hydrophobic peptides fractionated from enzymatic hydrolysates of tilapia frame protein using semi-preparative c18 rp-hplc exhibited the highest antioxidant activity (fan et al., 2012). in contrast, the more hydrophilic peptides obtained from a gelatin hydrolysate of tilapia skin using rp-hplc column had the stronger antioxidant activity (zhang et al., 2012). in the present study, fraction b-2/4 and b-2/8, which demonstrated the highest antioxidant, activity were selected for peptide sequence identification using uplc-ms/ms. ital. j. food sci., vol 29, 2017 165 figure 3. elution profile of fraction b-2 on rp-hplc (a) and abts radical scavenging activity/a220 ratio of fractions (b). absorbance at 220 nm (-). absorbance at 280 nm (-). values represent mean ± sd. different letters on the bars indicate significant differences (p < 0.05) between values. 3.2 identification of antioxidant peptides the identification of peptide sequences within selected fractions obtained from rp-hplc (fraction b-2/4 and b-2/8) were determined by uplc-ms/ms. thirty peptides were identified with molecular weight range from 279.17 to 555.28 da in fraction b-2/4. in fraction b-2/8, twenty six antioxidant peptides observed had mw ranging from 366.20 to 897.37 da. two peptides were selected from each fraction followed the criteria as mentioned above (table 1). glu-pro-gly-pro-val-gly (555.27 da) and leu-pro-gly-proala-gly (511.29 da) were identified from fraction b-2/4 (fig. 4). another two peptides, leu-asp-gly-pro-val-gly (557.30 da) and glu-gly-pro-leu-gly (472.24 da), were ital. j. food sci., vol 29, 2017 166 identified from fraction b-2/8 (fig. 5). in this study, each identified peptide possessed a specifically arranged amino acid sequence typical of collagen and gelatin, where glycine strictly represents every third amino acid residue (li et al., 2007). moreover, the molecular weight of the peptides was similar to the antioxidant peptides isolated from tilapia gelatin hydrolysate (317.33-645.21 da) (zhang et al., 2012; sun et al., 2013). in addition, the selected peptides contained gly at the c-terminal, indicating that the glycyl endopeptidase activity used for preparing the gelatin hydrolysates specifically cleaves peptide bonds with gly at p1 position (karnjanapratum et al., 2014). table 1. abts radical scavenging activity of synthesized peptides peptide sequence molecular weight (da) abts radical scavenging activity (µmol te/g solid) glu-pro-gly-pro-val-gly 555.28 1.25±0.36b leu-pro-gly-pro-ala-gly 511.29 1.22±0.10b leu-asp-gly-pro-val-gly 557.30 1.36±0.12b glu-gly-pro-leu-gly 472.24 4.95±0.65a values represent the mean ± sd for three separated experiment. different letters in the same column indicate significant differences (p < 0.05) between values in the same column. 3.3. antioxidant activity of synthesized peptides selected peptides (glu-pro-gly-pro-val-gly, leu-pro-gly-pro-ala-gly, leu-asp-gly-proval-gly and glu-gly-pro-leu-gly) were synthesized for further study. all four synthetic peptides had antioxidant activity (table 1) with glu-gly-pro-leu-gly having the highest abts radical scavenging activity (p<0.05). the remaining three synthetic peptides all demonstrated similar antioxidant activity (p>0.05). therefore, the amino acid sequence played an important role in antioxidant activity. peptides rich in hydrophobic amino acids (gly, pro), which contribute substantially to free radical scavenging, were previously found in pacific cod skin gelatin hydrolysate (ngo et al., 2011). the side chain of gly consists of a single hydrogen atom and may confer high flexibility on the peptide bond. the pyrrolidine ring of pro tends to interrupt the secondary structure of the peptide, thus imposing conformational constraints (rajapakse et al., 2005; aleman et al., 2011). glu and leu acted as direct radical scavengers in a peptide purified from fish skin gelatin (zhang et al., 2009; mendis et al., 2005). it was noted that gh herein showed a higher abts radical scavenging activity (65.49 µmol te/g sample) (fig. 1b), compared with those of the synthesized peptides (1.22-4.95 µmol te/g sample). this suggests that several peptides and/or free amino acids identified within the antioxidant fractions may exert a synergistic effect on radical scavenging activity. ital. j. food sci., vol 29, 2017 167 figure 4. base peak chromatogram (bpc) of b-2/4 fraction separated by acquity uplc beh c18 column (a) and representative ms/ms spectra of peptides glu-pro-gly-pro-val-gly (b) and leu-pro-gly-pro-ala-gly (c). the x-axis shows the m/z of the precursor (pre) and fragment ions while the y-axis shows the relative intensity. the deduced sequence can be seen on the top left. ital. j. food sci., vol 29, 2017 168 figure 5. base peak chromatogram (bpc) of b-2/8 fraction separated by acquity uplc beh c18 column (a) and representative ms/ms spectra of peptides leu-asp-gly-pro-val-gly (b) and glu-gly-pro-leu-gly (c). the x-axis shows the m/z of the precursor (pre) and fragment ions while the y-axis shows the relative intensity. the deduced sequence can be seen on the top left. a similar result was reported for a patin (pangasius sutchi) protein hydrolysate prepared using alcalase and papain, in which the purified peptide obtained had a lower antioxidant activity, in comparison with a crude patin hydrolysate (najafian et al., 2013). therefore, the combination of peptides present in protein hydrolysates could produce higher bioactivity than the isolated peptides (aluko, 2015). ital. j. food sci., vol 29, 2017 169 4. conclusions gh from unicorn leatherjacket skin prepared using glycyl endopeptidase contained peptides possessing free radical scavenging activity. four antioxidant peptides were identified as glu-pro-gly-pro-val-gly (555.27 da), leu-pro-gly-pro-ala-gly (511.29 da), leu-asp-gly-pro-val-gly (557.30 da) and glu-gly-pro-leu-gly (472.24 da). gh and its peptides could be potential candidates for functional food ingredients or nutraceuticals which protect against free radical generation and related diseases. references aghdam m. n., dehghan g. and kafshboran h. r. 2011. comparative study of abts radical scavenging activity and flavonoid contents in several populations of teucrium polium. int. conf. life sci. technol. 3:55-58. aleman a., gimenez b., montero p. and gomez-guillen m. 2011. antioxidative activity of several marine skin gelatins. lwt food sci. technol. 44:407-413. aleman a., gimenez b., perez-santin e., gomez-guillen m.c. and montero p. 2011. contribution of leu and hyp residues to antioxidant and ace-inhibitory activities of 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x., wang x. and yao h. 2009. antioxidant activities of the rice endosperm protein hydrolysate: identification of the active peptide. eur. food res. technol. 229:709-719. paper received august 28, 2016 accepted november 5, 2016 ijfs#816_bozza ital. j. food sci., vol. 30, 2018 89 paper optimisation of the extraction of flavonoids from apples using response surface methodology m. liaudanskas*1,2, k. zymonė1, j. viškelis2 and v. janulis1 1department of pharmacognosy, faculty of pharmacy, lithuanian university of health sciences, lithuania 2institute of horticulture, lithuanian research centre for agriculture and forestry, lithuania *e-mail address: m.liaudanskas@yahoo.com abstract the ultrasound-assisted extraction of flavonoids from apple samples was modelled using response surface methodology. a three-level-three-factor central composite design using the response surface methodology (rsm) was employed to optimise three extraction variables, including temperature, extraction time and ultrasonic power, for the achievement of the highest extraction yield of the flavonoids from lyophilised apple samples. the optimised extraction conditions were 44.61ºc, an extraction time of 26.90 min, and ultrasonic power 480 w. the experimental yield of flavonoids was 6.58 mg g-1 expressed as rutin equivalent, which was close to the predicted yield (6.69 mg g-1). optimised extraction conditions were applied for the analysis of apple samples of six cultivars. keywords: apple, central composite design, flavonoids, hplc, response surface methodology, ultrasonic extraction ital. j. food sci., vol. 30, 2018 90 1. introduction apples play an important role in the human diet. they are one of the most consumed fruits in the whole world (wu et al., 2007, ceymann et al., 2012). based on data from the year 2014, approximately 84.63 million tonnes of apples are grown per annum. countries that grow the most apples are china (approximately 40.92 million tonnes per annum), the usa (approximately 5.19 million tonnes per annum) and poland (3.20 million tonnes per annum) (fao statistical database, 2017). apples are widely used in the food industry to produce various products and drinks (juice, wine, cider); they are also used unprocessed (marks et al., 2007, price et al., 1999). some of the most important biologically active substances in apples are phenolic compounds, which are attributed to natural antioxidants. oxidative stress causes changes in cell metabolism related to dna and protein damage as well as lipid peroxidation (cooke et al., 2003; pizzimenti et al., 2010). it can cause inflammatory processes, cardiac, vascular and other diseases (madamanchi et al., 2010). phenolic compounds neutralise reactive forms of oxygen and nitrogen (pandey and rizvi, 2009) and therefore are valuable for the treatment and prophylaxis of various diseases. qualitative and quantitative composition analyses of raw materials that accumulate phenolic compounds are important and relevant. the selection of extraction conditions is an important analytical step in developing the qualitative and quantitative analysis methodologies of phenolic compounds in multicomponent matrices. our developed and validated method of flavonoid and phenolic acid determination in apples is published in the paper by liaudanskas et al. (2014). in developing this method, the extraction parameter selection was empirical. it is relevant to compare flavonoid extraction yield when the samples are extracted using empirical extraction conditions and when conditions are selected based on statistical modelling. 2. materials and methods 2.1. plant material apple samples of the ligol cultivar were chosen for the extract optimisation analysis. the ligol cultivar (winter cultivar, bred in poland) is one of the main cultivars in commercial apple orchards in lithuania. optimised extraction conditions were applied for the analysis of the apple samples of different cultivars. the following apple cultivars were included in the comparable researches: aldas (early winter cultivar, bred in lithuania, recommended for ecological orchards), auksis (early winter cultivar, bred in lithuania,), connel red (winter cultivar, bred in usa), ligol, lodel (early winter cultivar, bred in lithuania) and rajka (early winter cultivar, bred in czech republic). the apple trees were grown in the experimental orchard (block 2, row 4, trees 21-40) of the institute of horticulture, lithuanian research centre for agriculture and forestry, babtai, lithuania (55°60′ n, 23°48′ e). the altitude of babtai town is 57 m above sea level. trees were trained as a slender spindle, and pest and disease management was carried out according to the rules of the integrated plant protection. the experimental orchard was not irrigated. tree fertilisation was performed according to soil and leaf analysis. in addition, nitrogen was applied before flowering at the rate of 80 kg ha-1, and potassium was applied after harvest at the rate of 90 kg ha-1. soil conditions of the experimental orchard were the following: clay loam, ph 7.3, humus 2.8%, p2o5 255 mg kg-1 and k2o 230 mg kg-1. ital. j. food sci., vol. 30, 2018 91 2.2. sample preparation apples were cut into slices of equal size (up to 1 cm in thickness), and the stalks and the seeds were removed. the apple slices were immediately frozen in a freezer (at -35°c) with air circulation. apple samples were lyophilised with a zirbus sublimator 3× 4×5/20 (zirbus technology, bad grund, germany) at a pressure of 0.01 mbar (condenser temperature, -85°c). the lyophilised apple slices were ground to a fine powder (about 100 µm) by using the knife mill grindomix gm 200 (retsch, haan, germany). loss on drying before analysis was determined by drying the apple lyophilisate in a laboratory drying oven to complete the evaporation of free water and volatile compounds (temperature 105°c) and by calculating the difference in raw material weight before and after drying (european pharmacopoeia, 2010). the data were recalculated for the absolute dry lyophilisate weight. 2.3. chemicals all solvents, reagents, and standards used were of analytical grade. acetonitrile, aluminium trichloride hexahydrate, hexamethylenetetramine and acetic acid were obtained from sigma-aldrich gmbh (buchs, switzerland), and ethanol from stumbras ab (kaunas, lithuania). hyperoside, rutin, quercitrin, phloridzin, procyanidin b1 and procyanidin b2, and chlorogenic acid standards were purchased from extrasynthese (genay, france); reynoutrin, (+)-catechin and (−)-epicatechin were purchased from sigmaaldrich gmbh (buchs, switzerland); and avicularin, procyanidin c1 and isoquercitrin were purchased from chromadex (santa ana, usa). in the study, we used deionised water that the crystal e hplc (adrona sia, riga, latvia) water purification system produced. 2.4. extraction a total of 2.5 g of lyophilised apple powder (exact weight) was weighed, added to 30 ml of ethanol (70%, v/v) and extracted in a sonorex digital 10 p ultrasonic bath (bandelin electronic gmbh & co. kg, berlin, germany). a total of 480 w is the maximum ultrasonic power that can be achieved by using the sonorex digital 10 p ultrasonic bath. the obtained extract was filtered through a paper filter, and the apple lyophilisate on the filter was washed twice with 10 ml of ethanol (70%, v/v) in a 50ml flask. then, the extract was filtered through a pvdf syringe filter with a pore size of 0.22 µm (carl roth gmbh, karlsruhe, germany). 2.5. determination of the total flavonoid content the total flavonoid content in the extracts of lyophilised apple samples was determined by applying the technique that urbonavičiūtė et al. (2006) described. it was calculated using the rutin calibration curve and was expressed as its equivalent (mg re/g) for absolute dry weight (dw). 2.6. high-performance liquid chromatography the qualitative and quantitative analyses of phenolic compounds were performed according to the previously validated and described high-performance liquid chromatography (hplc) method (liaudanskas et al., 2014). ital. j. food sci., vol. 30, 2018 92 2.7. experimental design and statistical analyses before the development of the study through the response surface methodology (rsm), the flavonoid yield from the apples of three extractants (ethanol, methanol, and acetone) of different concentrations were compared. it was determined that the highest yield of flavonoids after four hours of extraction was achieved by macerating apple samples with ethanol 70% (v/v); therefore, this extractant was chosen for further analyses. selection of extraction method: the efficacy levels of maceration and extraction in ultrasonic bath methods were compared, and it was determined that the yield was higher when sonification method was applied. the results for the extractant and extraction method selections are discussed more in depth in the paper by liaudanskas et al. (2014). in general, such multiple parameters as liquid/solid ratio, temperature, time, solvent polarity and ultrasonic power influence the efficiency of the extraction of a compound, and these are the primary extraction parameters that many other authors have referred to (tian et al., 2013; radojkovic et al., 2012; chen et al., 2012) in this study, three factors (or independent variables) were selected: temperature (20-60°c), extraction time (5-95 min) and ultrasonic power (48-480 w) (table 1). table 1. factors and levels for rsm, and central composite experimental design with the independent variables. run coded and non-coded variable levels total flavonoid content, mg re g-1 x1 temperature, °c x2 extraction time, min x3 ultrasonic power, w 1 0 40 0 50 0 264 6.889 2 0 40 0 50 -1 48 6.58 3 0 40 -1 5 0 264 4.199 4 0 40 1 95 0 264 6.312 5 -1 20 -1 5 -1 48 3.154 6 1 60 0 50 0 264 6.315 7 0 40 0 50 0 264 7.087 8 1 60 1 95 -1 48 5.618 9 1 60 1 95 1 480 5.963 10 -1 20 1 95 -1 48 4.682 11 1 60 -1 5 -1 48 4.102 12 -1 20 -1 5 1 480 3.555 13 0 40 0 50 0 264 6.794 14 0 40 0 50 0 264 7.175 15 0 40 0 50 0 264 6.821 16 -1 20 0 50 0 264 4.966 17 0 40 0 50 0 264 6.982 18 -1 20 1 95 1 480 5.406 19 1 60 -1 5 1 480 4.665 20 0 40 0 50 1 480 7.465 ital. j. food sci., vol. 30, 2018 93 a three-level-three-factor central composite design was employed to determine the optimal combination of flavonoid extraction variables from apple samples. table 1 represents the coded and non-coded values of the experimental variables and 20 experimental points. six replicates (1, 7, 13, 14, 15, 17) were used to evaluate the pure error. experimental data showed that response variables were fitted to a quadratic polynomial model. the general form of the quadratic polynomial model is presented in fig. 1, where y is the dependent variable; β0, βi, βii and βij are the regression coefficients for intercept, linearity, square and interaction respectively. xi and xj are the independent variables. figure 1. the general form of the quadratic polynomial model. design-expert® 6.0.8 software (stat-ease inc., minneapolis, minnesota, usa) was used to analyse the data, develop models and optimise the extraction conditions. the fitness of the quadratic polynomial model was inspected with the regression coefficient of r2. the pvalue was used to check the significance of the regression coefficient. all of the experiments (except extraction optimisation researches) were carried out in triplicate. means and standards errors were calculated with spss 20.0 software (chicago, usa). a single factor analysis of variance (anova) along with the post hoc tukey's hsd test was employed for statistical analysis. differences were considered to be significant at the p<0.05 level. 3. results and discussions 3.1. optimisation of extraction conditions of total flavonoids in apples the design matrix and the corresponding results of rsm experiments to determine the effects of the three independent variables, including temperature (x1), extraction time (x2) and ultrasonic power (x3), are shown in table 2. through multiple regression analysis of the experimental data, the model for predicted response y could be expressed with the following quadratic polynomial equation (in the form of coded values), presented in fig. 2. figure 2. the model for the predicted response y expressed by the quadratic polynomial equation (in the form of coded values). ital. j. food sci., vol. 30, 2018 94 statistical testing of the model was performed in the form of analysis of variance (anova). the anova for the fitted quadratic polynomial model of extraction of polysaccharides is shown in table 2. table 2. analysis of variance for fitted quadratic model of extraction of phenolic compounds. source sum of squares degree of freedom p-value model 32.32 9 <0.0001 significant residual 0.60 10 lack of fit 0.49 5 0.0698 not significant pure error 0.11 5 cor. total 32.92 19 r2 = 0.9818; 𝑅!"# ! = 0.9674; c.v. = 4.27%; adequate precision = 26.232. the results of the analysis of variance for the fitted quadratic model of extraction of phenolic compounds are presented in table 2. they indicated a high degree of correlation between the observed and predicted values. the lack of fit test determines whether a selected model is adequate for explaining the experimental data or whether another model should be reselected. the value of the lack of fit test indicated that the fitting model was adequate. an adequate precision is a measure of the signal-to-noise ratio, which when greater than 4 is considered to be adequate (canettieri et al., 2007). in addition, the value of adequate precision demonstrates an adequate signal. at the same time, a relatively low value of the coefficient of variation indicates a better precision and reliability of the experimental values. therefore, the model is adequate for prediction in the range of experimental variables. residual analysis of the response surface design was performed. a normal probability plot was applied to the residuals. the data points fell along a straight line, indicating that they were distributed normally. in addition, residual runs for analysis were used. it was determined that the order of observations did not influence the results. residuals versus predicted responses were plotted. the data points fell on both sides of the zero line, so no pattern could be concluded. the relationship between the actual and predicted values was evaluated. the data points fell along a straight line indicating close similarity between the two data points and the adequacy of the model. table 3. regression coefficients estimate and their significance test for quadratic model. source sum of squares degree of freedom p-value x1 2.40 1 <0.0001 x2 6.90 1 <0.0001 x3 0.85 1 0.0037 3.23 1 <0.0001 5.94 1 <0.0001 0.24 1 0.0717 x1x2 0.040 1 0.4337 x1x3 5.886×10 -3 1 0.7605 x2x3 1.378×10 -3 1 0.8825 ital. j. food sci., vol. 30, 2018 95 the significance of each coefficient measured using the p-value is listed in table 3. a smaller p-value means the corresponding variables are more significant. the p-value of the model is less than 0.0001, which indicates that the model is significant and can be used to optimise the extraction variables. the three independent variables (x1, x2, x3) and two quadratic terms (𝑋!! and 𝑋!!) significantly affect the extraction yield of flavonoids. the interaction among temperature (x1), extraction time (x2) and ultrasonic power (x3) did not affect the extraction yield of flavonoids significantly. the three-dimensional response surface is the graphical representation of the regression equation and is very useful for judging the relationship between independent and dependent variables. different shapes of the contour plots indicate whether the mutual interactions among the variables are significant or not. a circular contour plot means the interactions between the corresponding variables are negligible, whereas an elliptical contour suggests that the interactions between the corresponding variables are significant (muralidhar et al., 2001). the three-dimensional representation of the response surfaces generated via the model is shown in fig. 3–5. with these three variables, when two variables are depicted in three-dimensional surface plots, the third variable is fixed at the zero level. figure 3. response surface plot showing the effect of temperature (x1) and extraction time (x2). x3 (ultrasonic power) = 264 w. ital. j. food sci., vol. 30, 2018 96 figure 4. response surface plot showing the effect of temperature (x1) and ultrasonic power (x3). x2 (extraction time) = 50 min. figure 5. response surface plot showing the effect of extraction time (x1) and ultrasonic power (x3). x2 (temperature) = 40°c. it is found in figs. 3-5 that all of the three response surfaces are convex in shape, which indicates that the ranges of variables were chosen properly. as shown in fig. 3, the yield of extraction increases when extraction time and temperature are increased. based on the chosen model, the projected highest amount of phenolic compounds (6.69 mg g-1) is achieved when samples are extracted at 44.61ºc for 26.90 min. according to the applied model, a further increase of extraction time and temperature decreases the extraction yield. flavonoid extraction yield dependency on temperature and ultrasonic power is presented in fig. 4. the highest extraction yield is achieved when apple samples are extracted at highest ultrasound power (480 w), at 44.61ºc. extraction time and ultrasound power influence on flavonoid extraction yield is presented in fig. 5. the highest yield is achieved when the ultrasound power is maximal (480 w), and extraction time is 50 min. 3.2. optimization of extraction parameters and validation of the model through these three-dimensional plots, the suitability of the model equation for predicting the optimal response values was tested using the selected optimal conditions. the results (table 3) showed that the optimized conditions were ultrasonic temperature of 44.61ºc, extraction time of 26.90 min, and ultrasonic power 480 w. under these conditions, the predicted extraction yield of flavonoids was 6.69 mg g-1. however, considering the operability in actual production, the optimal conditions can be modified as follows: temperature of 45ºc, extraction time of 27 min, and ultrasonic power 480 w. under the modified conditions, the experimental yield of flavonoids was 6.58 mg re g-1 (n = 3), which was close to the predicted value. extraction yield was 4.1 mg re g-1 when extraction conditions were selected empirically (liaudanskas et al., 2014). when extraction conditions were optimized by statistical modelling method the yield was 37.69% higher than the yield when extraction conditions were selected empirically. ital. j. food sci., vol. 30, 2018 97 3.3. analysis of ethanol extracts of apple samples of different cultivars the chemical composition in fruits of different apple cultivars may vary significantly (ceymann et al., 2012; łata et al., 2005; wojdyło et al., 2008), therefore it is very important to determine the qualitative and quantitative composition of individual phenolic compounds in apples that are grown under lithuanian climatic conditions. optimized extraction conditions were applied for the hplc analysis of ethanol extracts of apple samples of six cultivars grown in lithuania: aldas, auksis, connel red, ligol, lodel and rajka using previously developed and validated hplc method (liaudanskas et al., 2014). these phenolic compounds of various groups were identified and quantified in analysed extracts: procyanidin b1, (+)-catechin, chlorogenic acid, procyanidin b2, (−)epicatechin, procyanidin c1, rutin, hyperoside, isoquercitrin, reynoutrin, avicularin, quercitrin and phloridzin (synonym phlorizin). apple sample chromatogram (cultivar lodel) is presented in fig. 6. total amount of phenolic compound in analysed apple sample extracts varied from 2.521 mg g-1 (cultivar connel red) to 6.430 mg g-1 (cultivar aldas). figure 6. chromatogram of the ethanol extract of apple sample (λ=280 nm, cultivar lodel). 1 procyanidin b1, 2 (+)-catechin, 3 chlorogenic acid, 4 procyanidin b2, 5 (−)-epicatechin, 6 procyanidin c1, 7 rutin, 8 hyperoside, 9 isoquercitrin, 10 reynoutrin, 11 avicularin, 12 quercitrin, 13 phloridzin. the highest total amount of quercetin glycoside 0.811 mg g-1 was determined in apple samples of cultivar aldas. it was 1.93 times higher than the lowest total amount of quercetin glycoside (0.420 mg g-1), determined in apple samples of cultivar auksis. quantitative composition of quercetin glycoside group compounds determined in apple samples is presented in table 4. hyperoside was a predominant quercetin glycoside group compound in apple samples of cultivars aldas, auksis, connel red, ligol and lodel. it amounted to 25.14-35.16% of the total amount of identified and quantified quercetin glycoside group compounds. van der sluis et al. indicated similar tendencies of a hyperoside quantitative composition variation. hyperoside amounted to 23-33% of the total identified quercetin glycosides in samples of cultivars that these authors analysed (van der sluis et al., 2001). the composition of apple samples of cultivar rajka stood out among the analysed cultivars, as the predominant compound in this cultivar was quercitrin. its amount was 1.66 times higher than the amount of hyperoside determined in the apple samples of this cultivar. quercetin glycoside rutin-hyperoside-isoquercitrin triplet (where the predominant compound is always hyperoside, and levels of rutin are the lowest) was characteristic in apple sample extracts of all analysed cultivars. this pattern was also established in studies of other scientists (marks et al., 2007; price et al., 1999; schieber et al., 2001). the ratio of rutin-hyperoside-isoquercitrin amount varies in the apple samples of different cultivars. it varied from 1:5.1:1.6 (cultivar connel red) to 1:11.9:1.2 (cultivar lodel). ital. j. food sci., vol. 30, 2018 98 monomeric ((+)-catechin and (−)-epicatechin) and oligomeric (procyanidin b1, procyanidin b2, and procyanidin c1) flavan-3-ols were determined in the apple samples. the highest total amount of identified and quantified flavan-3-ols (2.919 mg g-1) was determined in apple samples of the cultivar lodel. it was 2.55 times higher than the lowest amount (1.143 mg g-1), determined in the apple samples of cultivar connel red. in the scientific literature, it was noted that in apples, the amount of (−)-epicatechin is higher than (+)-catechin (wu et al., 2007; duda-chodak et al., 2010; kahle et al., 2005; panzella et al., 2013). the results of our analysis also confirm this. the ratio of (+)catechin and (−)-epicatechin in the samples of different cultivars varied from 1:4.3 (cultivar rajka) to 1:21.9 (cultivar lodel). in the paper by wojdyło et al., it is specified that in samples of apples grown in poland, the amount of (+)-catechin varies from 0.010 to 0.720 mg g-1, and (−)-epicatechin − from 0.066 to 2.760 mg g-1 (wojdyło et al., 2008) the amounts of procyanidin b2 and c1 were higher than the amount of procyanidin b1 was in the samples of all analysed cultivars. this predisposition of the quantitative composition variation of these compounds in apples was also presented by other authors (duda-chodak et al., 2011). the ratio of procyanidin b1, b2 and c1 amounts in samples of different cultivars varied from 1:5.4:3.4 (cultivar aldas) to 1:13.8:7.9 (cultivar auksis). the quantitative composition of flavan-3-ol group compounds identified in apple samples is presented in table 5. qualitative and quantitative analyses of dihydrochalcone group compounds are extremely important because the compounds of this group may be selected as chemotaxonomic indicators in apple cultivar taxonomy, for apple product identification and for the determination of apple juice and cider quality (schieber et al., 2001; alonso-salces et al., 2004; gosch et al., 2010). the amount of dihydrochalcone phloridzin in the apple samples of the analysed cultivars varied from 0.101 mg g-1 (cultivar rajka) to 0.268 mg g-1 (cultivar lodel) (table 5). similar results were presented by other authors as well-the amount of phloridzin in apple fruit comprises 2-6% of the total amount of quantified phenolic compounds (sanoner et al., 1999) the highest amount of chlorogenic acid (3.074 mg g-1) was determined in the apple samples of the cultivar aldas. it was 4.99 times higher than the lowest determined amount of this acid (0.616 mg g-1), determined in the apple samples of the cultivar rajka (table 6). wojdyło et al. analysed the apple samples of apples grown in poland and indicated similar amounts (0.015-2.960 mg g-1) of this acid (wojdyło et al., 2008). ital. j. food sci., vol. 30, 2018 99 table 4. quercetin glycoside quantitative variation in apple samples. substance quercetin glycoside amount (mg g-1, dry raw material) coefficient of variation, % aldas auksis connel red ligol lodel rajka hyperoside 0.274±0.005a 0.138±0.002c,d 0.152±0.003c 0.129±0.002d 0.154±0.003c 0.184±0.004b 31.10 isoquercitrin 0.053±0.002a 0.020±0.001c,d 0.047±0.002a,b 0.025±0.001c 0.016±0.001d 0.044±0.002b 45.92 rutin 0.036±0.001a 0.015±0.001b,c 0.030±0.002a 0.022±0.001b 0.013±0.001c 0.035±0.002a 39.69 avicularin 0.226±0.005a 0.120±0.002c 0.099±0.001d,e 0.087±0.001e 0.114±0.002c,d 0.139±0.003b 38.15 reynoutrin 0.080±0.003a 0.053±0.002b 0.039±0.001c 0.037±0.001c 0.043±0.001c 0.025±0.001d 40.92 quercitrin 0.142±0.003b 0.082±0.001e 0.103±0.002c,d 0.120±0.003c 0.098±0.001d,e 0.305±0.007a 58.30 different letters in the same row indicate statistically significant differences of individual substance amounts in apple samples of analysed cultivars (p<0.05). table 5. flavan-3-ols, chlorogenic acid and phloridzin quantitative variation in apple samples. substance flavan-3-ols, chlorogenic acid and phloridzin amount (mg g-1, dry raw material) coefficient of variation, % aldas auksis connel red ligol lodel rajka (+)-catechin 0.092±0.002b 0.077±0.002c 0.034±0.001d 0.033±0.001d 0.042±0.001d 0.121±0.003a 54.26 (–)-epicatechin 0.720±0.015b 0.448±0.009d 0.315±0.006e 0.311±0.004e 0.928±0.019a 0.525±0.010c 44.86 procyanidin b1 0.157±0.003a 0.170±0.004a 0.035±0.001d 0.064±0.001c 0.094±0.002b 0.097±0.002b 50.84 procyanidin b2 0.854±0.017c 0.990±0.021b 0.484±0.007e 0.676±0.014d 1.279±0.023a 0.834±0.015c 31.81 procyanidin c1 0.534±0.020a 0.366±0.012b 0.275±0.008c 0.258±0.011c 0.576±0.021a 0.249±0.007c 38.56 phloridzin 0.188±0.004b 0.124±0.002d 0.135±0.002d 0.157±0.003c 0.268±0.005a 0.101±0.002e 36.82 chlorogenic acid 3.074±0.068a 2.498±0.052b 0.773±0.013e 1.249±0.020d 1.629±0.035c 0.616±0.011e 59.41 different letters in the same row indicate statistically significant differences of individual substance amounts in apple samples of analysed cultivars (p<0.05). ital. j. food sci., vol. 30, 2018 100 the study results confirmed the hypothesis of ceymann et al., that apple cultivars can be classified based on what types of compounds phenolic acids or flavan-3-ols are predominant in the apple samples (ceymann et al., 2012). the predominant compound in the apple samples of the cultivars aldas and auksis was chlorogenic acid, and in the apple samples of other cultivars, it was flavan-3-ol group compounds. the coefficient of variation, which reflects the variation amplitude of every compound, was calculated to evaluate the variation of the quantitative composition of the phenolic compounds in the apple samples of different cultivars. it varied from 31.10% to 59.41% (tables 5 and 6). the highest calculated coefficient of variation was for chlorogenic acid, and the lowest was for hyperoside. 4. conclusions in this study, we applied rsm for the extraction of flavonoids from lyophilised apples. the results showed that the independent variables (temperature, extraction time and ultrasonic power), and the quadratic terms of temperature and extraction time had a statistically significant effect on the efficacy of apple flavonoid extraction. a second-order (quadratic) polynomial model was employed to optimize flavonoid extraction from lyophilised apple samples. the projected optimal extraction conditions following statistical modelling were as follows: temperature 44.61ºc, extraction time 26.90 min and ultrasonic power 480 w. the experimental yield of flavonoids was 6.58 mg re g-1, which was close to the predicted yield value of flavonoids 6.69 mg g-1. by applying rsm selected via statistical modelling, it was determined that the apple flavonoid extraction yield was 37.12% higher compared with sample extraction when extraction conditions were selected empirically. optimised extraction conditions were applied for the hplc analysis of the apple samples of six cultivars grown in lithuania. the highest total amount of identified phenolic compounds (6.430 mg g-1) was determined 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dekker m.a., jager a. and jongen w.m.f. 2001. activity and concentration of polyphenolic antioxidants in apple:effect of cultivar, harvest year, and storage conditions. j. agric. food chem. 48:3606-3613. wojdyło a., oszmiański j and laskowski p. 2008. polyphenolic compounds and antioxidant activity of new and old apple varieties. j. agric. food chem. 56:6520-6530. wu j., gao h., zhao l., liao x., chen f., wang z. and hu x. 2007. chemical compositional characterization of some apple cultivars. food chem. 103:88-93. paper received march 28, 2017 accepted september 8, 2017 ijfs#702 bozza ital. j. food sci., vol. 30, 2018 1 paper effect of spirulina (spirulina platensis) addition on textural and quality properties of cookies s. onacik-gür*, a. żbikowska and b. majewska division of fats & oils and food concentrates technology, department of food technology, faculty of food sciences, warsaw university of life sciences sggw *e-mail address: sylwia.onacik@gmail.com abstract nowadays scientists are looking for new food ingredients that are not seasonal and are rich in bioactive compounds, such as microalgae. the aim of this study was to enrich wholegrain cookies with microalgae (spirulina platensis) powder. 1%, 2% and 3% of spirulina was used to fortify the cookies. their physical, textural and sensory properties were analyzed. the addition of even small amounts of the microalgae (1%) changed color of the cookies significantly to intensive green. decrease of moisture content and hardness of the cookies was correlated with addition of microalgae powder. it was observed that hardness measured by sensory analysis increased with the spirulina content. microalgae had a negative impact on the overall sensory quality. keywords: microalgae, spirulina, biscuits, sensory analysis, texture analysis ital. j. food sci., vol. 30, 2018 2 1. introduction snacks such as cookies have low water content, which protects them from microbial spoilage and provides longer shelf life. cookies are very popular, easy-to-eat products consumed all over the world. as such, they could be great carriers of nutritionally valuable compounds. unfortunately, such products in the market very often contain fat rich in saturated fatty acids and trans fatty acids. however, products with wholegrain flour and dried fruits are becoming more popular due to the consumers’ increasing awareness of healthy life style. scientific papers report possibilities of using bioactive compounds such as: dietary fibers (onacik-gür et al., 2015; demirkesen, 2016; zbikowska et al., 2017), plant extracts (mildner-szkudlarz et al., 2009; kozłowska et al., 2014), fruit pomaces in bakery products (bajerska et al. 2016) and legume flours (cheng and bhat, 2016). addition of such ingredients may very often have adverse impact on textural properties and sensory characteristics. fibers and ingredients with high protein content may increase hardness of bakery products. fruit pomaces and plant extracts significantly change color of products. there are still a few papers presenting spirulina platensis as an ingredient increasing nutritional value of such products and its influence on textural properties and quality characteristics. microalgae such as chlorella spp., dunaliella spp., scenedesmus spp. and spirulina spp. are becoming more and more popular as new, highly nutritious food ingredient. they are rich in easily digestible protein, fat with a high content of unsaturated essential fatty acids, vitamins, minerals, carotenes and chlorophyll (pelizer et al., 2015; kay and barton, 1991). moreover, they contain fat rich in unsaturated fatty acids, half of which is γlinolenic acid (gla). gla is particularly important because it plays many important functions in the human body and it prevents and helps treatment of many diseases (białek and rutkowska, 2015). spirulina is also rich in a and b group vitamins (tang and suter, 2011). it has an especially high content of b12 vitamin, which is particularly important for vegans since it is one of very few sources of this vitamin for people who do not consume meat and dairy products. moreover, it may display anticancer activity because, according to an in-vitro assay, polysaccharides obtained from this microalga have strong scavenging effects in vitro on dpph and hydroxyl radicals (kurd and samavati, 2015). microalgae such as spirulina and chlorella are sold in europe as dietary supplements, without any kind of processing except drying. most of them are produced in asia. such supplements contain (per 100g): 55-70g of protein, 2-6g of fat, 0.6-1g of chlorophyll and 0.1-0.4g of carotenoids, minerals (calcium 0.5-1g, magnesium 0.2-0.6g, iron 30-100 mg, zinc 2-4 mg, selenium 10-30 μg) and vitamins (a 100-200 mg, 1.5-4 mg b1, 3-5 mg b2, 10-30 mg b3, 0.6-0.8 mg b6, 0.05-1.5 mg b12, 5-10 mg e) (liang et al., 2004) spirulina (cyanobacteria) occurs naturally in subtropical lakes. this microalga has a spiral shape and green-blue color. cyanobacteria were known and used by aztecs hundreds of years ago to produce cakes (habib et al., 2008). microalgae were used in other food systems such as: noodles (5g of chlorella and spirulina per 90g of wheat flour) (kumoro et al, 2016), gluten free bread (3 and 5 % of spirulina supplementation) (figueira et al., 2011), cookies (1 and 3% of isochrysis galbana) (gouveia et al., 2008). microalgae are used in food not only because of their bioactive compounds but also as salt and glutenreplacers. addition of even small amounts of spirulina and chlorella has a strong impact on sensory characteristics. it changes color, smell, taste and texture of a product (kumoro et al., 2016). that is why it can be assumed that addition of spirulina to cookie recipes may significantly influence both physical parameters and overall acceptability. the aim of this work was to analyse physical and sensory properties of cookies supplemented with different amounts of spirulina. ital. j. food sci., vol. 30, 2018 3 2. materials and methods 2.1. materials the research materials were cookies and cookie dough. the following ingredients were used in cookie production: flour mixture (1:1:1:1 of wheat ash 0.5%, wholegrain wheat ash 2%, wholegrain oat ash 2%, wholegrain barley ash 1.6%) 47.7% (młyny kruszwica, polska), palm fat 6.2% (bunge poland), high-oleic sunflower oil 24,9% (bunge poland), sugar 20.7%, baking powder 0.1%, rapeseed lecithin 1.8% (bunge poland), powder spirulina – spirulina platensis (myvita natural supplements), 5% water. the cookies were made in four variants: 0%, 1%, 2% and 3% addition of spirulina to the whole dough weight as a flour replacer. spirulina powder is rich in bioactive compounds and its daily intake should not exceed 2.5 g. palm fat, oil, lecithin and water were mixed for approximately 5 minutes with a kitchen processor braun multiquick (type 4644) until the emulsion was fully homogenized. sugar was then added and the whole dough was mixed for another 5 minutes. the remaining dry ingredients were mixed and kneaded until the consistency of dough became uniform. the dough was subsequently flattened with a rolling pin down to a 6 mm sheet and cut into 55mm diameter circular shapes. the cookies were baked in a convection oven (unox, italy) at 170°c for 10 minutes. the cookies were packed in polyethylene bags and stored for 9 weeks at room temperature without access to light. 2.2. density of dough density of the dough was measured by pressing the dough into a glass-weighting bottle with a capacity of 30 cm3. then the bottle with the dough was weighed and the density was calculated (onacik-gür et al., 2015). the above assay was repeated 3 times for each sample. 2.3. texture analysis of cookie dough the texture of cookie dough was calculated by penetration test using a texture analyzer ta.xt plus (stable microsystems, uk, 5 kg load cell). 110 g of the dough was formed into a ball and put on a metal dish from a dough preparation set a/dp. the firmness was measured with an edged cylinder with a diameter of 6 mm (p/6), which plunged the sample 20 mm deep with a test speed of 3mm/s. the firmness was defined as resistance to the penetration and measured by the maximum force (in newtons). the adhesive force was the maximum negative measured force needed to take the plunger out of the dough with a speed of 3 mm/s. the test was conducted in triplicate. 2.4. physical characteristics of cookies after baking and cooling down to ambient temperature, the thickness (t) and diameter (d) of cookies were measured. the spread ratio (d/t) was calculated by dividing the diameter by thickness (demirkesen, 2016). the density of cookies was calculated from the weight and volume of eight cookies. the volume was determined by rapeseed displacement method (rehmati and tehrabi, 2014). the above assay was performed 3 times for each sample. ital. j. food sci., vol. 30, 2018 4 2.4. cookie moisture 5 grams of crushed cookies were dried in a laboratory convection dryer (sup 100, poland) at 130 °c for 1 hour. samples were weighed before and after drying. the moisture of cookies was calculated from the difference and expressed in %. above assay was run in triplicate (onacik-gür et al., 2015). 2.6. texture of cookies texture analysis of cookies was conducted by a three-point bending test (hdp/3pb edge), carried out at ambient temperature with a ta.xt plus texture analyzer (stable microsystems, uk). the span length was 40 mm and the compression test speed was 3 mm/s. the end result was an average of 9 repetitions, hardness determined in n and fracturability in mm. 2.7. color the cookie and cookie dough color was measured with a chromameater konica minolta cr-200 in cie l*a*b* system. the color parameters were determined by: l* lightness (0black, 100-white), a* (-a* green, +a* red) and b* (-b* blue, +b* yellow). the cookies were scanned at three different points to determine the average as an end result. the following parameters were calculated based on the results (chroma – color saturation, bi – browning index, ∆e – total color differences) (bal et al., 2011): ∆𝐸 = (𝐿! − 𝐿!)! + (𝑎! − 𝑎!)! + (𝑏! − 𝑏!)! where: lc – parameter l* of control cookies, ls – parameter l* of analyzed cookies; ac – parameter a* of control cookies, as – parameter a* of analyzed cookies, bc – parameter b* of control cookies, bs – parameter b* of analyzed cookies. 𝐶ℎ𝑟𝑜𝑚𝑎 = 𝑎∗! + 𝑏∗! 𝐵𝐼 = 100 (𝑋 − 0.31) 0.17 where: 𝑋 = 𝑎∗ + 1.75 𝐿∗ 5.645𝐿∗ + 𝑎∗ − 3.01𝑏∗ 2.8. sensory analysis the profile method was used to determine sensory properties of the cookies. 20 individuals evaluated the cookies. all the panelists had completed sensory analysis classes and passed the sweet and salty threshold test and were trained for profiling method. the sensory analysis was conducted in laboratory conditions. each person was given one cookie of each variant and an evaluation card with instructions concerning the evaluation procedure. 10 cm unscaled line was used to rate each of the discriminants: typical odour ital. j. food sci., vol. 30, 2018 5 (typical for wholegrain cookies), algae odour, browning uniformity, dark color, hardness, crispiness, taste (typical for wholegrain cookies), algae taste and overall sensory quality (all sensory properties which influence acceptability and quality). intensity of the discriminants was increasing from left to right side of the line. samples were presented in the same containers in randomized order and labeled with three digit random numbers. first, the evaluators examined odour and appearance of the cookies. after the first bite the hardness of the product was evaluated. hardness is the first experienced sensation and then crispness, which is noticeable 10 seconds after the first bite. crispness is a sensation of brittleness in the mouth, when the teeth crack the product during mastication, with multiple fractures at low force loads. after chewing, soaking the bite in saliva and swallowing, when all the taste substances reached taste buds, the panelists evaluated the taste of cookies. in the end, the evaluators rated the overall quality of products, taking into account all the discriminants (laguna et al., 2013; onacik-gür et al., 2015). 2.9. statistical analysis statistical analysis was performed by means of a computer program statistica 12.0 (statsoft, usa). the data was subjected to anova including post hoc comparison tukey’s test, at the probability level α = 0.05 to determine significant differences. moreover, person’s correlation was carried out for the results, p-value ≤ 0.05. 3. results and discussions 3.1. physical properties and texture of cookie dough and cookies addition of spirulina did not have significant effect (table 1) on cookie dough and cookie density. no significant differences were observed in geometry of cookies, either. however, it was found that addition of microalgae powder decreased the spread ratio of cookies. other researchers found that addition of fiber may have such impact on spread ratio of cookies (gupta et al., 2011). table 1. physical properties of cookie dough and cookies. spirulina addition [%] cookie dough density [g/cm3] diameter [mm] thickness [mm] cookie density [g/cm3] spread ratio [d/t] water content [%] 0 1.08±0.04a 58.56±1.43a 8.02±0.28a 0.85±0.01a 7.31±0.12b 4.16±0.05 b 1 1.12±0.09a 58.86±1.06a 8.33±0.27a 0.91±0.01a 7.05±0.16a 4.05±0.05 b 2 1.11±0.09a 58.70±1.00a 8.31±0.21a 0.94±0.05a 7.06±0.15a 3.83±0.09 a 3 1.11±0.09a 58.64±1.18a 8.27±0.36a 0.93±0.11a 7.08±0.14a 3.79±0.06 a *a, b, c describes homogenous groups. p-value ≤ 0.05. however, the addition of microalgae decreased the moisture content of the baked product (table 1). decreasing water content in the product can be related to a partial reduction of flour, which was replaced by spirulina. wholegrain flours used to produce these cookies, since they were rich in fiber, which increased the water absorption (sobczyk, 2012). ital. j. food sci., vol. 30, 2018 6 addition of spirulina did not significantly affect the firmness and adhesive force of cookie dough. even when the enrichment level of microalgae powder increased, the dough had the same textural parameters (fig. 1). fig. 1. textural properties of cookie dough. * a, b describes homogenous groups, p-value ≤ 0.05. however, the addition of algae decreased the hardness of cookies. statistically significant differences were not observed among additions at the levels of 1, 2 and 3% of spirulina. however, a tendency was found showing that the hardness of cookies decreased in line with microalgae powder content (fig. 2). gouveia et al. (2007) arrived at opposite conclusions in their study, where the hardness of cookies was increasing with the microalgae level enrichment. in our study, microalgae powder was added to the recipe as a flour replacer, which is rich in fiber and absorbs high quantities of water, while spirulina is rich in protein (60%) (pelizer et al., 2015). a partial replacement of flour caused a decrease in fiber content, which significantly changed the water absorption. as a consequence, the structure was less compact and the hardness decreased (gouveia et al., 2008; sudha et al., 2007). shyu and sung (2010) observed a decrease in hardness with increasing addition of γ-polyglutamic acid obtained from bacillus spp. addition of spirulina did not change significantly the fracturability of cookies on the first day after baking. however, the difference was visible during storage. in the 6th and 9th weeks, it was found that the fracturability of cookies decreased with spirulina enrichment level (fig. 3). texture of cookies during the storage did not change significantly. products with 1% addition of spirulina had a slightly lower hardness in the 9th week. nonetheless, it was found that fracturability of cookies decreased at the time of storage. a a a a a ab a a -0,6 -0,4 -0,2 0 0,2 0,4 0,6 0,8 1 0% 1% 2% 3% spirulina content firmness [n] adhesive force [n] ital. j. food sci., vol. 30, 2018 7 fig. 2. hardness of cookies. * a, b describes homogenous groups, p-value ≤ 0.05. fig. 3. fracturability of cookies. * a, b, c, d, e describes homogenous groups, p-value ≤ 0.05. 3.2. color of cookies and cookie dough color parameters of the cookie dough and cookies changed statistically significantly depending on the amount of spirulina addition. parameters l* (white/black), a* (red/green) and b* (yellow/blue) were decreasing for cookie dough and cookies with increasing content of microalgae powder. it shows that baked products and dough were becoming more green-blue with increasing content of spirulina in the recipe. similar results were obtained by gouveia et al. (2008) who added microalgae isochrysis galbana to cookies. ∆e value increased in products before and after baking by adding higher amounts of spirulina. chroma indicates the saturation – intensity of color (bal et al., 2011) and the highest values were obtained for the control sample (without a spirulina addition) despite b ab a a ab ab a a b ab a a b a a a y = -0,622x + 7,235 r² = 0,86043 0 2 4 6 8 10 0% 1% 2% 3% h ar dn es s [n ] 1 day 3 weeks 6 weeks 9 weeks ab ab ab a cde bcd de de cd cde e de bcd de de de 50 51 52 53 54 55 56 57 58 59 60 0% 1% 2% 3% f ra ct ur ab ili ty [m m ] 1 day 3 weeks 6 weeks 8 weeks ital. j. food sci., vol. 30, 2018 8 a strong green color of samples with microalgae addition. the sample of cookie dough with the lowest addition of spirulina (1%) has the lowest color saturation value, which increased with larger quantities of this additive. color saturation results for the baked cookies were the reverse, as chroma decreased with increasing addition of spirulina. browning index (bi) indicates the purity of brown color, which is particularly important when products are dried (bal et al., 2011) or, in the case of cookies, baked. this index decreased with the addition of spirulina and rose after the same samples were baked (table 2). table 2. color parameters of cookie dough and cookies. spirulina addition color parameters δe chroma bi l* a* b* c oo ki e do ug h 0% 49.18±2.40 c 4.97±0.12 c 9.45±0.72 c 0 10.68 28.35 1% 39.80±2.20 b -0.39±0.66 b 0.48±0.28 a 14.04 0.62 0.48 2% 37.34±0.94 ab -0.96±0.49 a -1.70±0.49 b 17.31 1.95 -6.15 3% 36.58±0.73 a -1.84±0.26 a -2.51±0.64 b 18.66 3.11 -10.01 c oo ki e 0% 61.89±2.77 c 5.46±0.31 c 20.45±1.00d 0 21.17 45.83 1% 50.61±1.02 b 0.83±0.71 b 14.01±1.58 c 13.79 14.03 32.85 2% 45.89±1.68 a -0.35±0.44 a 9.62±1.07 b 20.18 9.63 22.37 3% 42.96±1.59 a -0.44±0.70 a 6.75±0.59 a 24.10 6.76 15.91 *a, b, c describes homogenous groups. p-value ≤ 0.05. 3.3. sensory analysis microalgae powder had a positive impact on uniformity of browning. color of the cookies became darker with the increasing addition of algae, which was confirmed in instrumental analysis. cookies with the highest 3% addition of spirulina were the hardest, while the control sample 0% (without addition) and the one with lowest addition of spirulina (1%) were the softest. the results were not confirmed with the instrumental texture analysis, where the hardness of cookies decreased with the content of microalgae powder. hardness in sensory analysis could be affected by the density and thickness of cookies. in the 3-point blend test, a cookie breaks when first touched by a probe, while biting teeth are going through the whole cookie. that is why in some studies it was found that hardness measured instrumentally is more correlated to crunchiness or crispiness than hardness of the first bite (kim et al., 2011). another explanation for the difference of sensory hardness perception in relation to instrumental hardness can be due to the fact that an instrumental test always measures the force in the middle of the cookie, while the first bite is usually taken from a side. in the case of crispness, no influence of spirulina addition was observed (table 3). fracturability, which is measured instrumentally, should by definition be comparable with crispness and no significant differences were found for both of them. the intensity of sensing the taste and odour of algae between cookies with 2% and 3% of spirulina was similar. ital. j. food sci., vol. 30, 2018 9 table 3. sensory analysis of cookies with spirulina. spirulina powder content typical odour algae odour browning uniformity dark color hardness crispness typical taste algae taste overall sensory quality 0% 7.08±2.05 b 0 a 6.38±2.82 a 3.46±0.82a 4.09±2.54a 7.35±1.57a 6.64±2.32a 0 a 7.24±1.66a 1% 5.62±2.2 ab 2.64±1.80 ab 6.22±2.60 a 4.79±1.08a 4.15±1.67a 6.73±1.90a 4.86±1.44a 4.11±1.66b 5.64±2.21ab 2% 3.88±1.95 a 4.55±1.14 b 7.25±1.68 a 7.10±1.68b 4.75±1.82a 7.72±1.15a 3.42±1.32ab 6.35±1.02b 5.48±2.38ab 3% 3.50±2.25 a 4.88±1.70 b 7.62±1.54 a 7.62±1.54c 5.8±2.40a 6.57±1.60a 3.09±1.97b 6.56±1.25b 5.10±1.99b *a, b, c describes homogenous groups, p-value ≤ 0.05. table 4. correlation of variables – properties of cookie dough and cookies, p-value ≤ 0.05. spirulina dd dc h d m fd fc δec chroma c bi c q hard dd 0,59 1,00 0,84 0,98 0,85 -0,56 0,73 -0,96 0,78 -0,73 -0,68 -0,89 0,31 dc 0,87 0,84 1,00 0,91 0,46 -0,90 0,90 -0,95 0,96 -0,95 -0,93 -0,96 0,63 h 0,66 0,98 0,91 1,00 0,79 -0,66 0,77 -0,99 0,84 -0,79 -0,75 -0,92 0,35 d 0,08 0,85 0,46 0,79 1,00 -0,05 0,26 -0,69 0,33 -0,25 -0,19 -0,51 -0,23 m -0,97 -0,56 -0,90 -0,66 -0,05 1,00 -0,93 0,76 -0,95 0,97 0,98 0,87 -0,87 fd 0,98 0,73 0,90 0,77 0,26 -0,93 1,00 -0,86 0,98 -0,99 -0,99 -0,96 0,87 fc -0,87 -0,96 -0,95 -0,99 -0,69 0,76 -0,86 1,00 -0,91 0,87 0,84 0,96 -0,49 δec 0,96 0,78 0,96 0,84 0,33 -0,95 0,98 -0,91 1,00 -0,99 -0,99 -0,98 0,79 chroma c -0,98 -0,73 -0,95 -0,79 -0,25 0,97 -0,99 0,87 -0,99 1,00 0,99 0,96 -0,84 bi c -0,99 -0,68 -0,93 -0,75 -0,19 0,98 -0,99 0,84 -0,99 0,99 1,00 0,94 -0,87 q -0,90 -0,89 -0,96 -0,92 -0,51 0,87 -0,96 0,96 -0,98 0,96 0,94 1,00 -0,70 hard 0,93 0,31 0,62 0,35 -0,22 -0,87 0,87 -0,74 0,79 -0,84 -0,87 -0,70 1,00 crisp -0,32 -0,40 -0,10 -0,26 -0,30 0,09 -0,42 0,29 -0,29 0,28 0,26 0,39 -0,39 legend: dd – density of cookie dough, dc – density of cookies, h – height of cookies, d – diameter of cookies, m – moisture content, fd – hardness of cookie dough, fr – fracturability of cookies, fc – hardness of cookies (measured instrumentally), ∆ec – total color difference of cookies, chroma c – color saturation of cookies, bi c – browning index of cookies, q – overall sensory quality, hard – hardness (sensory analysis), crisp – crispness. ital. j. food sci., vol. 30, 2018 10 spirulina addition has adversely influenced sensory quality of cookies. it caused a decrease in typical aroma, typical taste and overall sensory quality – acceptability. in the study by sharma and dunkwal (2012), the acceptability of biscuits with addition of 10% of spirulina powder did not result in low acceptability. scores obtained for the control biscuits and spirulina based biscuits were similar. in comparison, the microalgae content was much higher than in the study presented in this paper. high overall acceptability in the study conducted by sharma and dunkwal (2012) can be due to the fact that eating habits and flavor preferences are different in india and in poland. 3.4. correlation between qualitative parameters of cookie dough and cookies it was observed that addition of spirulina had an effect on the analyzed parameters of cookies and cookie dough. only crispness, cookie geometry and density of cookie dough were weakly correlated with the amount of microalgae powder addition. statistical analysis indicated a statistically significant correlation between hardness measured by texture analyzer, color parameters and overall sensory quality (p<0,05). color was strongly and significantly dependent on the spirulina content in the recipe. based on the statistical analysis, it can be assumed that crispness and fracturability of cookies with spirulina did not depend significantly on any of the analyzed variables (table 4). the variables describing properties of cookie dough had strongly correlated with those describing the baked product. this may indicate that by knowing properties of the cookie dough before baking, we can anticipate features of the final product. hardness of cookies measured with texture analyzer was correlated with spirulina content, which was also confirmed by linear regression (fig. 2). 4. conclusions spirulina has influence on most physical and sensory properties of cookies. its even minor addition had strong impact on change of color to green of the cookie dough and baked products. addition of microalgae caused a decrease in moisture content and hardness measured by texture analyzer. it was observed that the color and hardness of products had an impact on the overall sensory quality. more brown color and less green improved acceptance of a product. because of increasing consumers’ interest in novel food ingredients such as microalgae, production of cookies with spirulina addition may be a forward-looking development. due to high content of bioactive ingredients and low sensory acceptability 1% addition of spirulina powder seems to be optimum. unfortunately, larger quantities of spirulina caused significant changes in some parameters affecting quality of cookies. references bajerska j., mildner-szkuldarz s., górnaś p. and seglina d. 2016. the effects of muffins enriched with sour cherry pomace on acceptability, glycemic response, satiety and energy intake: a randomized crossover trial. j. sci. food agr. 96(7):2486-2493. bal l.m., kar a., satya s. and naik s.n. 2011. kinetics of colour change of bamboo shoot slices during microwave drying. int. j. food sci. tech. 46:827-833. białek m. and rutkowska j. 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(4):1365-1370. tang, g. and suter p.a. 2011. vitamin a, nutrition, and health value of algae: spirulina, chlorella, and dunaliella. j. pharm. nutr. sci. 1:111-118. paper received december 20, 2016 accepted march 20, 2017 ijfs#842_bozza ital. j. food sci., vol. 30, 2018 303 paper the impact of partial-fat substitutions with doum (hyphaenethebaica) dregs on the quality characteristics of beef patties a.m. el-anany*a and rehab f.m. alib,c adepartment of special food and nutrition researches, food tech. res. institute; agricultural research center, giza, egypt bbiochemistry department, faculty of agriculture, cairo university, giza, egypt cdepartment of food science and human nutrition, college of agriculture and veterinary medicine, alqassim university, qassim, kingdom of saudi arabia *e-mail address: malkelanany@gmail.com abstract the present investigation aimed to evaluate the impact of partial substitution of kidney fat (10, 20, 30, 40 and 50%) with equal amounts of doum dregs on the quality attributes of beef patties. doum dregs contain mainly crude fiber (59.6 g/100 g dm) followed by the total carbohydrate content (35.0 g/100 g dm). water and fat absorption capacities of doum dregs were 2.07 g and 2.51 ml/g of the sample, respectively. raw and cooked beef patties formulated with different levels of doum dregs had significantly (p ≤ 0.05) higher moisture, ash, fiber and carbohydrate contents as compared to control samples (without doum dregs incorporation). both cooked and uncooked beef patties manufactured with 10% doum dregs had the lowest content of fat 8.02 and 8.08%, respectively. the lowest energy contents were observed for both uncooked (150.60 kcal/100g) and cooked (179.0 kcal/100g) beef patties supplemented with 10% of doum dregs. reductions in cholesterol content in cooked burgers supplemented with various levels of doum dregs varied from 8.42 to 33.48%. significant(p ≤ 0.05) improvements in cooking properties were observed for beef patties incorporated with doum dregs. sensory evaluation results indicate that the highest overall acceptability scores were recorded for those samples formulated with 2, 4 and 6% of doum dregs. keywords: doum, fiber, burger, fat, dregs ital. j. food sci., vol. 30, 2018 304 1. introduction consumption of meat in developing countries has been constantly growing from a humble average annual per capita consumption of 10 kg in the 1960s to 26 kg in 2000 and it will reach about 37 kg in 2030, according to estimates of food and agriculture organization (fao, 2007). beef burgers are among the food that have attractiveness, ready-to-serve foods despite their failure to appear on everybody's plate due to their high fat, trans fatty acids, saturated fatty acids which can lead to obesity, type 2 diabetes and coronary disease (yilmaz and geçgel, 2007). studies demonstrated that these diets caused significant elevation in the levels of low-density lipoprotein cholesterol (ldl), which clogs the arteries (zoraida et al., 2011). nowadays there are increased demands for healthier meat products that hold high dietary fiber and low fats. epidemiologic studies have provided consistent evidence that dietary saturated fat intake has been associated with the risks of cardiovascular disease (modi et al., 2003). recent researches have been focused on the positive effects of using vegetarian sources to enhance nutritional and quality properties of meat products. (kumar and sharma, 2004). meat extenders are non-meat components, which are added into meat products for technical, nutritional and economic reasons (mansour, 2003). moringa seed flour in beef patties (al-juhaimi et al., 2016), wheat flour in buffalo meat burgers (modi et al., 2003), as well as potato flakes in beef patties (ali et al., 2011) have been applied as extenders. dietary fiber rich meat products are excellent meat substitutes due to their potential nutritional and functional impacts. dietary fiber intake through meat incorporated with fruits, vegetables and grains is linked with decreases in plasma and ldl cholesterol; decrease the hazard of major dietary problems such as obesity, diabetes, heart diseases and gastrointestinal disorders (schneeman, 1999). the doum palm (hyphaenethebaica) belongs to the family palmae and subfamily borassoideae. doum fruit, a desert palm native to egypt, subsaharan africa and west india; is commonly called “african doum palm” or ginger bread palm (dosumu et al., 2006). doum fruits contain high levels of protein and minerals. salih, 1991 reported that the fruit of doum contains 7.0% ash, 15.0% crude fiber, 0.5% fat, and 3.2% crude protein. minerals were found to be 0.13%, 0.18%, 0.09% and 3.02% for ca, mg, na and k respectively. doum powder was used for preparing cake, frozen yoghurt, fermented milk and ice-cream (seleem, 2015, abd el-rashid and hassan 2005). however, there are, to our knowledge, no sound published data about the use of doum fruit dregs in food products; therefore, the major objective of the current investigation was to evaluate the impact of partial substitution of kidney fat (10, 20, 30, 40 and 50%) with equal amounts of doum dregs on the quality characteristics of beef patties. 2. materials and methods doum palm (hyphaenethebaica) fruits were purchased from the local market in giza, egypt, washed with tap water several times, cut into small pieces, and dried in an electric air draught oven (isotemp oven, fisher scientific) at 50°c for 24 hours. fresh lean beef meat and kidney fat were obtained from metro market, el haram st., giza governorate, egypt. ital. j. food sci., vol. 30, 2018 305 2.1. methods 2.1.1 preparation of doum dregs dried and crushed pieces of doum fruits were ground in an electric grinder (braun model 1021), passed through a 150μm mesh sieve. doum powder was soaked in tap water (1: 20 w/v) and kept in a refrigerator at 4°c for 48 hours. the extract was passed through a single layer of muslin cloth to filter out the solid materials. the solid dregs were dried in an electric air draught oven (isotemp oven, fisher scientific) at 50°c for 24 hours. the dried dregs were packed in clean, dry glass containers and stored at 4°c for further use. five replicates of doum samples were subjected to chemical analysis 2.1.2 preparation of beef patties: the fresh lean beef meat and kidney fat portions were individually ground in meat grinder equipment (moulinex me605131). the ground lean beef (5% lipids), kidney fat (89% lipids), doum fiber and ice flakes have been used for the formulation of beef burgers (table 1). the control samples contain 65% of lean beef meat and 20% of beef fat. five levels of fat portions (10, 20, 30, 40 and 50%) were partially replaced by equal amounts of doum dregs. ground beef meat and the other ingredients were blended together by hand, then ground finally in meat grinder meat with 0.5 cm plate, and formed into beef burgers (100 g weight, 12 mm thickness and 100 mm diameter). formulated burgers were placed on plastic foam plats, wrapped with 10 microns polyethylene film and kept in freezer at 25°c until further analysis. five replicates of each beef burger formula were subjected to chemical analysis. 2.1.3 cooking procedure frozen burgers were cooked by using electric grill (kumtel, turkey) for 6 min on each side to ensure that the internal temperature of 70±5°c measured at the centre of beef patty using a digital thermometer, model 16454, pyrex-accessories robinson knife company, china. table 1. beef patties formulated with various levels of doum fibers fat replacement treatment* lean beef (g) kidney fat (g) doum dregs (g) ice flakes (g) control 65 20 0 10 10% 65 18 2 10 20% 65 16 4 10 30% 65 14 6 10 40% 65 12 8 10 50% 65 10 10 10 *all treatments were formulated with 2 g salt, 1.5 g spices mixture, 1 g sugar, 0.2 g tripolyphosphate, 0.3 g ascorbic acid. doum fiber was rehydrated with water, doum fiber /water (1:2, w/v). ital. j. food sci., vol. 30, 2018 306 2.2. analytical methods 2.2.1 proximate analysis of doum dregs the moisture, ash, protein, crude fibre, and fat contents of doum dregs were assessed using the official methods described by aoac (2005, methods 930.15, 923.03, 976.05, 962.09 and 920.85, respectively). total carbohydrate was calculated by difference. 2.2.2 functional properties of doum dregs the absorption capacities of water and oil were determined according to the procedures described by sosulski (1962) and sosulski et al. (1976), respectively, and the results were expressed as grams of water or milliliters of corn oil bound with one gram of doum dregs flour. emulsifying and foaming capacities were determined according to the methods of neto et al. (2001) and lawhon et al. (1972), respectively. 2.2.3 proximate composition and caloric values of beef patties moisture (oven drying method) (methods 930.15), protein (n × 6.25) (method 920.152), fat (ether extraction with soxhlet apparatus) (method 991.36.), ash content (method 923.03) and crude fibre contents (method 962.09) were determined using the official methods described by aoac (2005). carbohydrate contents were calculated by difference. total caloric (kcal/100g sample) were calculated according to mansour and khalil (1999), as follow, for fat (9 kcal g-1), protein (4.02 kcal g-1), and carbohydrates (3.87 kcal g-1). 2.2.4 cholesterol assay the content of cholesterol was determined according to the previous procedures described by turhan et al. (2007). petroleum ether was used to extract the fats from 5 gm of beef burger samples. the petroleum ether was removed by evaporation at 50°c. the extracted fat was weighed and subjected to saponification using aqueous ethanolic koh solution. the mixture solution was allowed to cool at ambient temperature, and 10 ml of petroleum ether was added. the mixture was shaken vigorously for 1 min. after the layers have separated, the ether layer was transferred into clean test tube, then the ether solvent was evaporated at 50°c. acetic acid saturated with ferrous sulfate and concentrated sulfuric acid were added to develop the chromophore for colorimetric analysis. the absorbance was then measured at 490 nm against the reagent blank. 2.3. determination of cooking properties 2.3.1 cooking yield cooking yield percentage was determined by calculating weight differences for samples before and after cooking according to the procedures described by khalil (2000). 2.3.2 fat retention the fat retention value represents the amount of fat retained in the product after cooking. fat retention was measured according to the procedures described by khalil (2000). ital. j. food sci., vol. 30, 2018 307 2.3.3 moisture retention the moisture retention was measured according to the equation described by khalil (2000). 2.4. sensory evaluation of cooked patties sensory evaluation of cooked beef burgers was conducted according to al-juhaimi et al. (2016). each sample of the six formulas was evaluated by ten of trained judges who are belonging to food technology research institute, agriculture research center, giza, egypt. all judges had previous experience in quality attributes. judges were both male and female in the age range of 25-40 years old. cooked patties were cut into 2 equal-sized parts and served randomly to each panelist at 50ºc. three cut of each formulated beef burger samples were served to the panelist. samples were evaluated in three sessions (each session with two formulas). the panelists were asked to evaluate the appearance, taste, juiciness, flavour and overall acceptability of beef patties using 10-point hedonic scale, where (10 = i like extremely, 1 = i dislike extremely). cups of drinking water were provided for judges to clean their mouth between samples. 2.5. statistical analysis results were expressed as the average of values±standard deviation (sd). results were analyzed of variance (anova) (p ≤ 0.05). results were analyzed by excel (microsoft office 2007) and spss software version 18.0 (spss inc., chicago, il, usa). 3. results and discussions 3.1. proximate composition of doum (hyphaenethebaica) dregs table 2 shows the proximate composition of doum (hyphaenethebaica) dregs. moisture content of dried doum dregs was 6.36%. moisture content and water activity are key factors affecting the storage, shelf life, and safety of foods (ahmed and ali, 2015). low levels of fat and protein (0.39 and 1.69%, respectively) were detected in doum dregs. doum dregs had adequate amounts of ash 3.32 g/ 100g dm. doum dregs contain mainly crude fiber (59.6 /100 g dm) followed by the total carbohydrate content (35.0/100 g dm). doum palm fruit is one of important sources, which supplies human with carbohydrates, fibers, and anti-hypertension compounds (dosumu et al., 2006). the high level of fiber in doum fruit make it as a potential ingredient for production of bakery products for improving their nutritive value, as well as their contributions to prevent the gastrointestinal problems beside its relevant role as natural anti-cancer agent (coimbra and jorge, 2011). 3.2. functional properties of doum (hyphaenethebaica) dregs the investigations of the functional characteristics of raw materials provide an advanced knowledge for their potential use in food products (ahmed and ali, 2015). table 2 shows water absorption, oil absorption, emulsifying and foaming capacities of doum (hyphaenethebaica) dregs. water absorption capacity (wac) of doum dregs was 2.07 g of h2o/g of doum dregs. the hydrophilic constituents such as carbohydrate and crude fiber in doum dregs may have contributed to the high water absorption capacity of the dregs. ital. j. food sci., vol. 30, 2018 308 this finding indicates that the doum dregs can be used as water binding agent in food processing industries. fat absorption capacity (fac) of doum dregs was 2.51 ml of oil/g of sample. this finding indicates that doum dregs could be useful in formulation of foods such as sausages and bakery products. emulsifying activity determines the capacity of substrate to form oil-in-water emulsion. the emulsifying capacity (ml /g) of dried doum dregs was 19.20. functional characteristics influenced by the ability of dietary fiber to bind with oil and water as well as gel-forming ability. borderias et al. (2005), reported that the addition of dietary fiber agents into fish-based products enhanced the functional properties of these products. doum dregs have a poor foaming capacity. the foaming capacity (%) of dried doum dregs was 9.10. the low levels of protein in doum dregs accounted for the low foaming capacity of doum dregs. the ability of the flours to form foam depends on the presence of the flexible molecules of protein that may reduce the surface tension of water (sathe et al., 1982). table 2. proximate composition and some functional properties of doum (hyphaenethebaica) dregs (n= 5)a. parameters mean±sd functional property mean±sd moisture (%) 6.36±0.95a water absorption capacity (g of h2o/g of sample) 2.07±0.11 fat (%) 0.39±0.02 fat absorption capacity (ml of oil/g of sample) 2.51±0.16 protein (%) 1.69±0.15 emulsifying capacity (ml /g) 19.20±0.96 ash (%) 3.32±0.31 foaming capacity (% vol. increase) 9.10±0.87 crude fiber (%) 59.6±3.08 carbohydratesb (%) 35.0±2.85 avalues are means±sd of three determinations bby difference. 3.3. effect of the addition of different levels of hydrated doum dregs on chemical composition of raw and cooked tested beef burgers the proximate composition of the uncooked and grilled patties as influenced by adding different levels of doum dregs as fat replacer is shown in table 3. the results indicate that beef patties incorporated with various levels of doum dregs were significantly higher (p≤ 0.05) in moisture content than those of control. moisture content of uncooked beef patties ranged from 60.55 to 67.09%. the lowest (p ≤ 0.05) content of moisture was recorded for control beef burgers (without doum dregs incorporation). moisture of uncooked beef patties was gradually and significantly increased with increasing the levels of doum dregs. beef patty formulated with 10% doum dregs significantly (p≤0.05) recorded the highest moisture content followed by those formulated with 8% doum dregs. the high content of fiber in doum dregs powder indicates the potentiality of use this powder as a good source of dietary fibre, which plays an important role in increasing water absorption capacity (wac). the high wac of fibers encourages their use as a functional ingredient in food formulations, in order to reduce syneresis and dehydration during the storage process, to modify texture and viscosity and to reduce energetic content of food products (besbes et al., 2008). similar trend was observed for cooked beef patties. in this regard, cooking process caused significant decreases in moisture content of beef patties. during cooking process, the moisture content of beef patties has been lost by evaporation (ali et al., 2011). moisture content was decreased by cooking process that in turn leads to increase the percentage of protein and dietary fiber in the cooked samples (turhan et al., 2007). the ital. j. food sci., vol. 30, 2018 309 highest decreases in fat and moisture content were observed for cooked control sample (without doum dregs incorporation), however the lowest reductions of moisture content were observed for those samples incorporated with 10 and 8% of doum dregs. these findings indicate that the incorporation of different levels of doum dregs into beef patties resulted in retention of more moisture during cooking due to a high waterbinding capacity of doum fibers. addition of doum fiber reduces drip and evaporation which resulting in significant increases in the moisture content of cooked patties, these increases in moisture content of beef patties increased significantly (p ≤ 0.05) as the level of doom dregs inclusion was increased. incorporation of dietary fiber into meat batters caused significant increases in the viscosity of these products, which was effective in retaining water in the meat products (yun-sang et al., 2015). fat content of uncooked beef patties ranged from 8.08 to 19.10%. the highest level of fat was recorded for uncooked control sample (without doum dregs incorporation). beef patties formulated with various levels of doum dregs had significantly (p≤0.05) the lower levels of fat than control samples (without doum dregs incorporation). fat content decreased proportionally with increasing the level of doum dregs used in raw beef patty formulation. beef burger samples incorporated with 10% and 8% of doum dregs had the lowest (p≤ 0.05) content of fat (8.08 and 9.33%, respectively). similar results were showed by al-juhaimi et al. (2016) for low-fat beef patties formulated with different levels of moringa seed flour, and by turhan et al. (2000) for beef patties incorporated with okara. cooking process caused marked decreases in fat content, while, the lowest decreases of fat content were observed for beef burgers incorporated with different amounts of doum dregs. these findings are in good agreement with those obtained by mansour and khalil (1999) who reported that low-fat patties retained more fat during cooking than higher fat patties. in this regard, kirchner et al. (2000) noted that more fat was lost at the 15% fat level than at the 5% fat level in beef burger samples. no significant differences (p≥0.05) were recorded for protein contents among raw beef burger samples. cooking process caused significant increases in protein contents of beef burger samples. loss of moisture during grilling process could leads to concomitant increase in the protein content (bassey et al, 2014). crude protein contents for cooked beef burger samples were significantly higher (p≤ 0.05) than raw samples (table 3). however, no statistically significant differences in protein content were observed among the cooked beef burger samples. significant increases in fiber and ash content in raw and uncooked beef patties formulated with hydrated doum dregs (table 3). fiber content of cooked beef burgers supplemented with 6, 8 and 10% of doum dregs were about 7.25, 8.47 and 8.55 times as high as that in control samples (without doum dregs incorporation). statistically the highest ash contents were recorded for cooked samples incorporated with 8 and 10% of doum dregs, however, the lowest value was recorded for control samples (without doum dregs incorporation). this finding could be attributed to the presence of high amounts of fiber and ash in doum dregs. doum palm fruit (hyphaenethebaica) powder packs a health punch of dietary fiber and minerals (abd el-rashid and hassan, 2005). low amounts of carbohydrates (0.22 -1.0%) were recorded for control beef burger samples and those samples incorporated with different levels of doum dregs (table 3). with increasing the consumer awareness on food safety systems, and health concerns, there is a rapidly increased demand for the reduction of saturated fats in meat products and its substitution by non-meat substrates, such as dietary fibers, polysaccharides and other non-carbohydrate components. (fuentes-zaragoza et al., 2010). dietary fibers poss more of advantages such as inhibiting hydrolysis, digestion and absorption in the human small intestine, improving fecal bulk, enhancing colonic fermentation, reducing postprandial blood glucose, and decreasing pre-prandial cholesterol levels (ktari et al., 2014). ital. j. food sci., vol. 30, 2018 310 table 3. chemical compositions of raw and cooked beef patties formulated with different levels of doum dregs (n= 5)a. parameter doum fiber level (%) 0 2 4 6 8 10 lsd at.05* raw beef patties moisture (%) 60.55±0.87d 62.09±0.95cd 63.48±1.51bcd 64.98±1.19abc 66.00±2.23ab 67.09±1.64a 2.62 fat (%) 19.10±0.66a 16.51±0.58b 14.10±1.08c 11.27±1.06d 9.33±0.63e 8.08±0.81e 1.47 protein (%) 18.01±0.65a 18.12±0.43a 18.15±0.86a 18.35±0.96a 18.61±1.12a 18.71±1.01a 1.54 ash (%) 1.86±0.02d 1.89±0.04d 2.09±0.08c 2.35±0.03b 2.69±0.04a 2.70±0.05a 0.08 crudefiber (%) 0.26±0.02e 1.06±0.08d 1.68±0.04c 2.46±0.10b 2.68±0.12a 2.71±0.09a 0.14 carbohydratesb (%) 0.22±0.01 d 0.33±0.02c 0.50±0.05b 0.59±0.08b 0.69±0.02a 0.71±0.08a 0.09 cooked beef patties moisture (%) 53.80±0.91c 54.10±1.58c 55.62±1.32bc 57.4±2.351abc 58.70±2.27ab 59.89±1.08a 2.98 fat (%) 17.94±0.91a 16.92±0.92a 14.23±0.63b 11.52±1.23c 9.34±0.84d 8.02±0.74d 1.59 protein (%) 25.21±0.45a 25.31±0.28a 25.37±0.38a 25.43±0.46a 25.54±0.49a 25.6±0.61a 0.81 ash (%) 2.21±0.02b 2.13±0.05ab 2.28±0.08ab 2.32±0.08ab 2.38±0.11a 2.40±0.09a 0.15 crude fiber (%) 0.36±0.07e 1.18±0.06d 1.83±0.09c 2.61±0.14b 3.05±0.12a 3.08±0.06a 0.17 carbohydratesb (%) 0.48±0.02 d 0.36±0.03c 0.67±0.05b 0.71±0.05b 0.99±0.08a 1.00±0.07a 0.09 avalues are means±sd of three determinations. means followed by the same letter are not significantly different (p≤0.05). bby difference. *least significant difference at p≤0.05 according to duncan’s multiple-range test. 3.4. energy content (kcal) of uncooked and cooked beef burgers incorporated with various levels of doum dregs table 4 illustrates the amount of energy of uncooked and cooked beef burgers incorporated with various levels of doum dregs. generally, energy content of cooked and uncooked beef patties decreased when fat content decreased or level of doum dregs increased (table 4). the highest energy content (264.65 and 245.15 kcal/100g, respectively) was recorded for control samples (full fat) in uncooked and cooked patties. lipids in diet are the source of energy, fat-soluble vitamins and essential fatty acids as well as improve the flavor and texture of food products. on the other hand, fat supplies the body with approximately more than double the calories of protein and carbohydrates (papadima and bloukas 1999). the lowest energy values were observed for both uncooked (150.60 kcal/100g) and cooked (179.0 kcal/100g) beef patties supplemented with 10% of doum dregs. these findings indicate that incorporation of doum dregs caused significant (p ≤0.05) reductions in energy values. reduction rates in energy content of uncooked beef burger supplemented with different levels of hydrated doum dregs ranged from 9.15 to 38.56%. while, it varied from 3.49 to 32.36% for cooked patties compared with their control samples. the reduction of caloric energy was associated with the reduction of fat content (mansour, 2003; ali et al., 2011). ital. j. food sci., vol. 30, 2018 311 table 4. energy content (kcal) and cholesterol content (mg/100 g, dry weight basis) of raw and cooked beef patties formulated with different levels of doum dregs (n= 5)a. trait doum dregs level (%) lsd at.05* 0 2 4 6 8 10 energy content (kcal) raw beef patties 245.15±046 a 222.70±0.36b 201.79±0.68c 177.47±0.81d 161.41±0.62d 150.60±0.6 f 1.34 cooked beef patties 264.65±0.49 a 255.41±0.43a 232.64±0.39b 208.64±0.59c 190.56±0.49d 179.00±0.46e 7.59 cholesterol content (mg/100 g, dry weight basis) raw beef patties 189.76±1.25 a 170.41±2.05b 154.12±1.89c 146.91±1.12d 145.39±0.93d 140.14±2.01e 2.85 cooked beef patties 219.50±3.18 a 201.00±2.11b 183.20±0.97c 168.15±0.88d 150.02±1.13e 146.13±1.07f 3.14 avalues are means±sd of three determinations. means in the same row with different letters are significantly different (p≤0.05). *least significant difference at p≤0.05 according to duncan’s multiple-range test. 3.5. cholesterol content (mg/100 g, dry weight basis) of raw and cooked beef patties formulated with different levels of doum dregs table 4 shows the cholesterol content of uncooked and cooked samples. control samples had significantly higher (p≤0.05) cholesterol content than beef patties formulated with different levels of doum dregs. cholesterol concentration of uncooked control sample (full fat) was about 1.11, 1.23, 1.29, 1.30 and 1.35 times as high as that in uncooked beef patties formulated with 2,4,6,8 and 10% of doum dregs, respectively. cholesterol concentrations of beef patties decreased (p ≤ 0.05) significantly with reducing fat levels or increasing the level of doum dregs (table 4). the lowest cholesterol content (140.14, mg/100 g, dry weight basis) was observed for uncooked beef patties formulated with 10% of doum dregs as fat replacer. mansour and khalil (1999) showed that the content of cholesterol of cooked and uncooked samples significantly decreased with addition of wheat fibers. candogan and kolsarici (2003) showed also that reducing fat content in frankfurters from 17.0% to 3.0% resulted in a significant reduction in cholesterol content varied from 50 to 56%. cooking process caused significant (p ≤ 0.05) increases in cholesterol content of beef patties. cooked control samples had significantly (p ≤ 0.05) the highest amount of cholesterol (219.50 mg/100 g, dry weight basis). the levels of cholesterol were markedly higher in cooked beef meat samples compared with the fresh beef samples, and these increases may be attributed to the loss of moisture content, which varies depending on the different cooking methods, leading to variation in cholesterol levels (badiani et al., 2002; turhan et al., 2007). in the respect, the transmission of cholesterol from the adipose tissue to muscle as a result of the cooking process represents a strong explanation for the higher cholesterol content in the cooked samples than fresh. particularly when the fat found in high amounts in the subcutaneous or intramuscular tissues (badiani et al., 2002). here again, cholesterol content of cooked patties decreased significantly with increasing the level of fat substitutes (doum dregs). mansour and khalil (2007) reported that the cholesterol content of cooked beef burger samples markedly decreased with addition of wheat fibers. the concentration of cholesterol of cooked samples which incorporated with various levels of doum dregs were gradually reduced from 201mg/100 g dw in beef burger samples blended with 2% to 146.13 mg/100 g dw in those samples blended with 10% of doum dregs, this means that the decreases in ital. j. food sci., vol. 30, 2018 312 cholesterol content in cooked beef patties formulated with various levels of doum dregs varied from 8.42 to 33.48%. dietary fibers act as potential therapeutic agents against cardiovascular diseases. they exert this action by acting as lowering agent of hyperlipidemia and hypocholesterolemia (bullock et al., 1995). 3.6. effect of replacing fat with different levels of doum dregs on some of cooking characteristics of cooked beef patties the cooking characteristics of beef patties formulated with different levels of doum dregs are shown in table 5. cooking yield of formulated beef patties ranged from 68.53 to 75.84%. beef patties formulated with different levels of doum dregs had significantly higher (p≤0.05) cooking yield than control samples. the lowest value (68.53%) of cooking yield was observed for control sample (without doum dregs incorporation). this loss in control beef patties might be attributed to the excessive fat separation and water release during cooking process (turhan et al., 2007). at the same time, the highest (75.12 and 75.84%) cooking yields were recorded for those samples incorporated with 8 and 10% of doum dregs, respectively. when fat substitution level increased, the yield of cooking increased (table 5). these increases in cooking yield may be attributed to the ability of doum fibers to hold moisture and fat during cooking process. table 5. effect of replacing fat with different levels of doum dregs on some of cooking characteristics of cooked beef patties (n= 5)a. doum dregs level % cooking yield moisture retention fat retention control (0) 68.53±1.33a 60.86±1.77a 64.36±1.68b 2 70.12±1.41ab 61.11±1.24a 71.86±1.20a 4 72.35±1.12bc 63.40±1.32b 73.01±0.63a 6 73.17±0.95c 64.66±0.93bc 74.79±1.15a 8 75.12±1.36d 66.80±0.28c 75.20±1.30a 10 75.84±0.86d 67.70±1.08c 75.27±1.66a lsd at.05* 2.11 2.12 2.34 aeach value in the table is the mean of three replicates and two determinations were conducted for each replicate. means in the same column with different letters are significantly different (p≤0.05). *least significant difference at p≤0.05 according to duncan’s multiple-range test. the results of moisture retention of beef patties formulated with doum dregs had the same trend of cooking yield. moisture retention of cooked beef patties varied from 60.86 to 67.70%. generally, beef patties with doum dregs had relatively higher values of moisture retention compared to control sample (without doum dregs incorporation). the lowest moisture retention (60.86%) was recorded for control sample. the highest moisture retention values (66.80 and 67.70%) were observed for beef patties incorporated with 8 and 10% doum dregs. these findings plainly indicate that the addition of doum dregs into beef burger formulas leads to increase the moisture retention during cooking process, this finding attributed to the capacity of doum dregs to bind more of water (table 2). similar findings were recorded by bullock et al., 1995 for low-fat beef patties formulated with modified food starch, by khalil, 2000 for beef burger incorporated with mixtures of ital. j. food sci., vol. 30, 2018 313 polydextrose, sugar beet, oat fiber, potato starch, and modified corn starch and by ali et al., 2011 for beef patties formulated with potato flaks. fat retention of formulated beef patties ranged from 64.36 to 75.27% (table 5). the lowest (64.36%) value of fat retention was recorded for control samples (without doum dregs incorporation). similar finding was observed by tornberg et al. (1989), who reported that fat was more easily separated from higher fat patties during cooking process. incorporation of various levels of doum dregs (as fat replacer) into beef patties caused significant (p ≤ 0.05) increases in fat retention values, these increases were gradually and significantly increased with increasing the level of doum dregs. the highest fat retention values (75.20 and 75.27%, respectively) were recorded for beef patties formulated with 8 and 10% of doum dregs. the addition of dietary fibers causes marked increase in fat retention because of their ability to bind more of moisture and fat in their matrix (wan et al., 2011). in this regard, tornberg et al. (1989) reported that the dense meat protein matrix of low-fat ground beef prevented fat migration. similar results were reported by mansour and khalil (1999); turhan et al. (2007), and wan rosli et al. (2011) who used high fibersubstrates such as wheat fibers; hazelnut pellicles and corn silk powder respectively to improve the quality attributes of beef patty formulations. 3.7. sensory evaluation sensory traits for cooked patties are shown in table 6. appearance of beef patties formulated with 2, 4 and 6% of doum dregs as fat replacer was substantially higher than those in other formulations and control sample (without doum dregs incorporation). beef patties with 8 and 10% had significantly (p < 0.05) the lowest appearance values (6.20 and 6.10, respectively). control sample and patties formulated with 2 and 4% doum dregs had significantly the highest value of taste. no significant differences were observed in taste values between control samples and those patties with 2 and 4% doum dregs. fat plays a good role in foods, it acts as a carrier of flavors and contributes to the strength of food, while the reduction of fat can significantly reduce the overall acceptability, aroma, juiciness and flavor intensity of meat products (kirchner et al., 2000). the taste values decreased gradually with increasing the inclusion percentages of doum dregs to 8 and 10% (p ≤ 0.05). these decreases may due to the presence of doum flavour. previous studies have shown significantly lower sensory scores for flavour in beef patties formulated with starch or gums sources (mansour 2003; ali et al., 2011; turhan et al., 2007). table 6. sensory characteristics of cooked beef patties formulated with different levels of doum dregs (n= 10)a. sensory trait doum dregs level (%) lsd at.05* 0 2 4 6 8 10 appearance 6.30c 6.45b 7.15a 7.20a 6.20d 6.10e 0.07 taste 7.55a 7.66a 7.60a 7.25b 6.50c 6.30d 0.08 flavour 6.85a 6.90b 6.82c 6.82c 6.80c 6.75d 0.02 juiciness 7.87ab 7.90b 7.93a 7.89ab 7.88ab 7.85ab 0.07 overall acceptability 7.14 d 7.22c 7.37a 7.29b 6.84e 6.75f 0.02 ameans in a line with different letters are significantly different (p≤0.05). *least significant difference at p≤0.05 according to duncan’s multiple-range test. ital. j. food sci., vol. 30, 2018 314 juiciness values were significantly the higher (p≤0.05) in beef patties formulated with different levels of doum dregs than control samples (without doum dregs incorporation). control samples (full fat) had significantly (p≤0.05) the lowest score (6.87) of juiciness, while, beef patties formulated with 4, 6, 8 and 10% doum dregs had significantly the highest (p≤0.05) scores were 7.93, 7.92, 7.94 and 7.91, respectively. this finding could be attributed to the ability of doum dregs to hold more of water (table 3). beef burgers formulated with white and red beeswings were significantly more tender and juicy than control sample (mansour and khalil, 1999). beef patties formulated with 2, 4 and 6% of doum dregs were rated higher (p<0.05) overall acceptability than control samples (without doum dregs incorporation), while, the lowest scores for acceptability were recorded for those samples formulated with 8 and 10% doum dregs. 4. conclusions the current study shows that addition of different levels of doum dregs (as fat replacer) into beef patties caused marked and significant (p≤0.05) increases in fiber, ash, cooking yield, moisture and fat retentions of formulated beef patties. the lowest energy contents were observed for both uncooked (150.60 kcal/100g) and cooked (179.0 kcal/100g) beef patties formulated with 10% of doum dregs. the decreases in cholesterol content in cooked beef patties formulated with various levels of doum dregs varied from 8.42 to 33.48%. the sensory evaluation results indicate that the highest scores of overall acceptability were recorded for beef burger samples formulated with 2, 4 and 6% of doum dregs. references abd el-rashid a. and hassan z.m.r. 2005. potential utilization and healthy effects of doum palm fruits in ice cream and sesame butter (tehena). alex j. food science and technology 2:29. ahmed f. and ali rehab f.m.a. 2015. effect of germination time on proximate 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gamma irradiation on trans fatty acid composition inground beef. food control, 18(6): 635. yun-sang c., hyun-wook k., ko-eun h., dong-heon s., tae-jeon j., young-boong k., ki-hong j. and cheon-jei k. 2015. effect of dietary fiber extracted from algelicakeiskeikoidz on the quality characteristics of chicken patties. korean j. food sci. an. 35(3):307. zoraida c.á., diego a., restrepo m. and cortés m.r. 2011. vegetable products as source of dietary fiber in the food industry: a review, revistafacultadnacional de agronomía,edellín. paper received april 19, 2017 accepted december 13, 2017 #507_lavelli_bozza ! ital. j. food sci., vol 28, 2016 542 review recovery of winemaking by-products for innovative food applications v. lavellia*, l. torrib, g. zeppac, l. fiorid and g. spignoe adefens, department of food, environmental and nutritional sciences, università degli studi di milano, via celoria 2, 20133 milano, italy buniversity of gastronomic sciences, piazza vittorio emanuele 9, 12042 bra, italy cdisafa, department of agricultural, forestry and food sciences, università degli studi di torino, l. go p. braccini 2, 10095 grugliasco, italy ddepartment of civil, environmental and mechanical engineering, università degli studi di trento, via mesiano 77, 38123 trento, italy einstitute of oenology and agro-food engineering, università cattolica del sacro cuore, via emilia parmense 84, 29122 piacenza, italy *corresponding author. tel.: +39 0250319172; fax: +39 0250316632 e-mail address: vera.lavelli@unimi.it abstract winemaking by-products are potential resources for second-generation biorefineries, i.e., biorefineries fed with biowaste to produce added-value products, particularly for the food sector. in fact, winemaking by-products are outstanding sources of oil, phenolic compounds and dietary fibre and possess numerous health benefits and multifunctional characteristics, such as antioxidant, colouring, antimicrobial and texturizing properties. the present review highlights promising developments for the conversion of winemaking by-products into novel food ingredients, as well as their use in innovative foods, focusing on the type of recovered ingredients, dosage, formulation and processing. in addition, the primary benefits of winemaking by-products to new foods are described. keywords: by-products, dietary fibre, grape phenolics, grape seed oil, winemaking ! ital. j. food sci., vol 28, 2016 543 1. introduction food supply chains have significant environmental impacts due to their use of resources and production of emissions, effluents and wastes. according to the european union (eu) commission council directive 2008/98/ec, “waste” is defined as “any substance or object, which the holder discards or intends or is required to discard”. the importance of food waste stretches from environmental pressures to economic and social impacts, including negative effects on food and nutrition security (otles et al., 2015). to meet the overall objective of increasing the sustainability of production chains, waste prevention/minimization is the main priority and best option, followed by reuse, recycling and energy recovery. alternatively, disposal (the use of landfills or incineration with low energy recovery) must be considered the worst environmental option. reuse and recycling strategies have drawn attention on the valorisation of by-products. a “byproduct” is defined as a product that must originate from a production process without being the main goal of production, be usable in the same production process or in a subsequent production or utilization process and be directly re-usable without further treatment outside normal industrial practices. moreover, a by-product must have a market value, and the final use should be integral without negatively impacting human health or the environment (galanakis, 2015). like all agro-food productions, the winemaking process generates a series of by-products that are important from both a quantitative and qualitative point of view and have been considered potential resources for second-generation biorefineries, i.e., biorefineries fed with biowaste to produce added-value products (scoma et al., 2016). in fact, grapes are one of the most cultivated fruits worldwide. according to the fao (www.faostat3.fao.org), 77 mt of grapes were produced throughout the world in 2013 (most of which were used in winemaking), along with 3.4 mt of grape pomace (otherwise referred to as grape marc). the latter represents, by weight, the primary winemaking solid by-product (60% on average), followed by lees (approximately 25%) and stalks (approximately 14%). minor solid by-products mainly include wine filtration residues. on average, depending on the grape variety and winemaking process, 100 kg of processed grapes generates 20-25 kg of pomace, a mixture of skins and seeds, 3-5 kg of stalks and 810 kg of lees (spigno, 2015). all of the aforementioned by-products pose serious environmental concerns because their production is typically concentrated in a limited time frame, and their high organic matter content prevents direct disposal into the soil, except for limited and regulated amounts. although these materials are re-used for other applications, being correctly considered as by-products, landfill additions and incineration are also conducted, depending on the country. conventional applications for winemaking by-products include: agronomic use, animal feed production and compost production for all residues; distillation for pomace and lees; tartaric acid manufacturing and the production of colouring additives and nutritional supplements from pomace, lees and filtration residues; oil recovery from seeds. agronomic use, animal feed production, composting and distillation, a relatively new approach, are not considered remunerative strategies (spigno, 2015). if properly recovered, winemaking by-products show a wide range of potential and remunerative applications in many industrial sectors, including cosmetics, pharmaceuticals, biomaterials and food (bordiga, 2015; yu and ahmedna, 2013). in fact, grape pomace is a rich source of both dietary fibre and various phenolic compounds (texeira et al., 2014). the amount of phenolic compounds that remain in the pomace depends on the initial, genetically dependent content of grapes, as well as the processing conditions and skin thickness, which is another genetically dependent parameter that is crucial for the maceration phase (battista et al., 2015). a study of various cultivars of ! ital. j. food sci., vol 28, 2016 544 vitis vinifera l. has revealed that the content of soluble proanthocyanidins in the skin ranges between 1.16 and 44.6 g/kg d. w., while the content of soluble proanthocyanidins in seeds ranges between 23.1 and 68.5g/kg d. w. (travaglia et al., 2011). the total anthocyanin content of red grape skins is in the range of 2.5-132 g/kg d. w. (kammerer et al., 2004; sri harsha et al., 2013). the presence of anthocyanins in the red grape seed fraction, due to mash constituents adhering to the seeds, is generally neglected or reported to be low (kammerer et al., 2004; lavelli et al., 2015a). however, a recent patent was focused on the extraction of anthocyanins from grape seeds, suggesting that their content deserves attention in a full recovery strategy (bi and rui, 2014). the total flavonol content of grape skins is in the range of 0.3-2.6 g/kg d. w. (sri harsha et al., 2013; sri harsha et al., 2014), whereas these compounds are generally less than 0.1 g/kg d. w. in grape seeds (maier et al., 2009b). compared to the above-mentioned phenolic compounds, phenolic acids and stilbenes are present in considerably lower amounts in winemaking by-products (kammerer et al., 2004). grape seeds contain oil with a high nutritional value. among various vegetable oils, grape seed oil shows the largest percentage of linoleic acid (c18:2 ≈70%). other major fatty acids present in grape seed oil include oleic acid (c18:1 ≈15%), palmitic acid (c16:0 ≈7%) and stearic acid (c18:0 ≈3%) (hanganu et al., 2012; fernandes et al., 2013; fiori et al., 2014). in addition to the interesting fatty acid profile, grape seed oil contains significant amounts of bioactive compounds such as tocopherols and tocotrienols, presenting a total tocol content up to 1208 mg/kg (beverige et al., 2005; crews et al., 2006; fiori et al., 2014). winemaking by-products are of particular interest for food uses when they are obtained via organic production because consumer preference is positively influenced by information on sustainable production practices (laureati et al., 2013). in particular, a great deal of interest in sustainability issues has been expressed for winemaking (laureati et al., 2014). the purpose of using winemaking by-products in foods may be fortification or enrichment. the distinction between these terms is not always recognized in scientific studies but has been clarified as follows: a fortified product is defined as a food containing additional nutrients, while an enriched product is defined as a food with additional novel nutrients or components not normally found in a particular food (siro’ et al., 2008). tartaric acid, enocyanine (e163) and grape seed oil are classical examples of successful commercial products obtained from winemaking by-products. additionally, in the last several years, grape seed and grape skin powders have been commercialized by different companies and promoted as highly nutritional ingredients to enrich conventional cereal flours and baked products with fibre, minerals, antioxidants, colour and aroma. the concept of antioxidant dietary fibre was first proposed by saura-calixto (1998), who set the criteria that 1 g of antioxidant dietary fibre should possess a free radical scavenging capacity equivalent to at least 50 mg of vitamin e and should contain more than 50% dry matter of dietary fibre from the natural constituents of the material. whole grape pomace, grape seeds and grape skin generally meet these criteria and are often referred to as antioxidant dietary fibre. enocyanine and grape antioxidant dietary fibre represent the two basic solutions for the reintroduction of grape pomace into the food chain, including indirect and partial use as concentrated extracts, or direct use as ground, dehydrated and micronized antioxidant dietary fibre. in both of the above-mentioned cases, a new production process must be implemented. fig. 1 shows a schematic depiction of the basic conventional process for antioxidant dietary fraction production. ! ital. j. food sci., vol 28, 2016 545 figure 1: scheme of the basic process for the production of grape pomace, skin or seed antioxidant dietary fibre. pomace collection should follow preliminary care selection to identify the best pomace for the production of food grade ingredients, based on the content of functional constituents (fibre, polyphenols and minerals), as well as possible contaminants (heavy metals, pesticides residuals and mycotoxins) (corrales et al., 2010; solfrizzo et al., 2012). washing and cleaning operations should be required, while the recovery of phenolic compounds would be reduced. if needed, skins and seeds can be separated by sieving, before or after drying. of course, drying is necessary to obtain a final powder but is also the most common stabilisation treatment. grape pomace has a high moisture content (greater than 60%) and undergoes rapid fermentation if not properly treated. low temperature preservation may precede the drying step for logistic and timing reasons. drying, which is an energy consuming process, should be reduced to a minimum because the thermal degradation of antioxidant compounds is detrimental to the nutritional profile. however, drying allows for the inhibition of enzymatic activity and can be considered a mild sanitization process. the operating temperature should not exceed 60°c to limit the degradation of phenolic compounds (amendola et al., 2010). in the production of antioxidant dietary fibre from skins and seeds, the seeds can be defatted in a previous step to recover the oil and produce a fibre-rich ingredient with limited rancidity issues. the final milling step must set the ideal particle size, depending on the expected application. if dried skins and seeds are destined for the production of an extract, a particle size range of 0.5 to 2 mm is acceptable. as outlined in the following paragraphs, the particle size should be less than 0.5 mm for use in bakery products or pasta. for applications into fruit-based and dairy products, an even lower particle size is required, which leads to additional energy consumption. fig. 2 shows the basic process for the production of grape pomace, skin or seed extracts (spigno, 2015). ! ital. j. food sci., vol 28, 2016 546 ! figure 2: scheme of the basic process for the production of extracts from grape pomace, skins or seeds. as indicated in the production of antioxidant dietary fibre, the operating temperature should be less than 60°c. different extraction techniques can be applied, such as conventional solvent extraction using food-grade solvents (amendola et al., 2010), or non-conventional solvents and systems for the development of sustainable and environmentally friendly processes. the ultrasound-assisted extraction has been successfully applied for the extraction of grape pomace phenolics, using water as a solvent and achieving high extraction yield with a short extraction time (marinelli et al., 2015). microwave-assisted solvent extraction using ethanol/water (pedroza et al., 2015) and high-pressure extraction using ethanol/water (paini et al., 2016), as well as the use of aqueous solutions of organic acids (tzima et al., 2015) have also been proposed. in general, all of these systems are characterized by low selectivity, and other compounds (such as sugars, minerals and organic acids) are co-extracted with the phenolic compounds, producing a crude extract. for food applications, the purification of extracts may be omitted without further increasing the production costs. the crude extract can then be simply concentrated to give a liquid extract or dried to give a powder extract. in this case, using a tailored approach (that takes into account the food category of the final target), the addition of suitable carrier materials (e.g., maltodextrins) can be exploited to increase and modify the stability and solubility of phenolic compounds (spigno et al., ! ital. j. food sci., vol 28, 2016 547 2013; lavelli et al., 2016b). to obtain an extract with a higher purity in total phenolic compounds or within a selected class of phenolic compounds, a purification step is required. adsorption resins (soto et al., 2011) and membranes (zagklis and paraskeva, 2015) are the most commonly investigated purification systems, along with other non-conventional approaches, such as the use of colloidal gas aphrons (spigno et al., 2015). in any case, microfiltration is also suggested as a non-thermal technology to produce crude extracts while promoting microbiological stability. independent of the production process used to obtain grape pomace antioxidant dietary fibre, phenolic extracts or seed oil, this review describes literature examples of innovative food applications into meat, fish, cereal, fruit-based and dairy products, with a focus on the type of recovered ingredient, dosage level and primary results achieved by the application. 2. phenolic extracts and antioxidant dietary fibre from grape skins and seeds as ingredients in innovative foods 2.1. functional effects new ingredients recovered from winemaking by-products have the potential to provide a wide range of food products with numerous health benefits (saura-calixto, 1998; texeira et al., 2014). moreover, as outlined in the following sections, these by-products possess multifunctional properties and could be used as natural antioxidants, colorants, antimicrobial agents and texturizers. 2.1.1 meat products due to the growing interest in convenience foods, ready-to-eat products such as dehydrated meat, frozen and precooked hamburgers, patties and meatballs have become a major category in the meat industry. the quality and shelf-life of these products is primarily dependent on the inhibition of lipid oxidation, which affects the colour, flavour, odour, texture and nutritional value of foods (fernandez et al., 1997). consequently, research efforts have been devoted to the application of winemaking by-products in various meat products to prevent lipid oxidation during precooking and storage under refrigerated or frozen conditions, representing natural alternatives to the use of synthetic antioxidants (table 1). in addition to auto-oxidation, microbial contamination is another serious factor that affects the quality and shelf-life of ready-to-eat meat products. however, only a few studies have investigated the antimicrobial properties of winemaking by-products in meat products (ahn et al., 2007). regarding chicken meat, when red grape skin extract powder was added to the dehydrated product at a level of 1 g/kgmeat, the content of hexanal and thiobarbituric acid reactive substances (tbars) formed during processing and storage at 22°c under air decreased. however, the efficacy was lower than those of rosemary extract and synthetic antioxidants, such as butylated hydroxyanisole and butylated hydroxytoluene (nissen et al., 2000). the antioxidant dietary fibre obtained from red grape pomace with particle sizes < 0.5 mm (total dietary fibre: 782 g/kg; soluble phenolics: 49.3 g/kg) has been applied to chicken hamburger, delivering fibre and imparting antioxidant effects during processing and refrigerated storage under air (sayago-ayerdi et al., 2009). alternatively, the extract obtained from the entire pomace (skins and seeds) has been proven to act as an antioxidant in uncooked and cooked chicken meat at a concentration corresponding to 60 mg phenolics/kgmeat during processing and frozen storage under vacuum (selani et al., 2011). ! ital. j. food sci., vol 28, 2016 548 table 1: applications of winemaking by-products as new food ingredients in meat products. food product recovered ingredient main results and references chicken meat (dehydrated) red grape skin extract powder (soluble tp: 1.60 mmol phenol eq./g) integration: 1 g/kgmeat decrease in hexanal and tbars content during processing and storage at 22°c in aluminized sachets sealed in air. lower efficacy than rosemary extract. nissen et al., 2000 chicken hamburger (uncooked and pre-cooked) red grape pomace antioxidant dietary fibre (particle size < 0.5 mm; tdf: 782 g/kg; soluble tp: 49.3 g gae/kg) integration: 5-20 g/kgmeat decrease in tbars content during processing and storage in polyvinyl chloride bags (otr: 13.500 cm3/m2d) at 4°c. high antioxidant activity and fibre content. sáyago-ayerdi et al., 2009 chicken meatballs (uncooked and pre-cooked) red and white grape pomace extract (soluble tp: 7.8-9.4 g gae/kg) integration: 60 mg tp/kgmeat decrease in tbars content during processing and storage at -18°c under vacuum. selani et al., 2011 pork patties (pre-cooked) red grape skin extract powder (soluble tp: 1.60 mmol phenol eq./g) integration: 0.2 g/kgmeat decrease in hexanal and tbars content during processing and storage at 4°c in polyethylene bags (otr > 2000 cm3/m2d). lower efficacy than rosemary extract. nissen et al., 2004 beef and pork patties (pre-cooked) grape seed extract powder (tp: 980 g/kg) integration: 0.1-0.2 g/kgmeat decrease in tbars content during processing and storage in polyvinyl chloride bags (otr: 880 cm3/m2d) at 4°c. rojas and brewer, 2007 pork patties (pre-cooked) grape seed extract powder (tp: 865 g/kg) integration: 0.05-1 g/kgmeat decrease in tbars content during processing and storage in barrier film packs (otr: 3 cm3/m2d) under 75% o2 and 25% co2, at 4°c. carpenter et al., 2007 beef ground meat (pre-cooked) grape seed extract powder (tp: not specified) integration: 10 g/kgmeat reduced numbers of escherichia coli o157:h7, salmonella typhimurium, listeria monocytogenes and aeromonas hydrophila during storage in bags (otr: not specified) at 4°c. ahn et al., 2007 beef sausage (pre-cooked) grape seed extract powder (tp: 800 990 g/kg) integration: 0.1-0.5 g/kgmeat decrease in tbars content during processing and storage in polyvinyl chloride bags (otr: 880 cm3/m2d) at -20°c. higher efficacy than ascorbate. kulkarni et al., 2011 otr: oxygen transmission rate; tbars: thiobarbituric acid reactive substances; tdf: total dietary fibre; tp: total phenolics (expressed as gae: gallic acid equivalents or phenol eq: phenol equivalents or pas: proanthocyanidins). considering pork meat, red grape skin extract powder added at a level of 0.2 g/kgmeat enhanced the oxidative stability of cooked patties during processing and refrigerated storage under air but showed lower efficacy than rosemary extract, as observed for chicken meat (nissen et al., 2004). grape seed extract appears to be more effective than grape skin extract. in fact, grape seed extract powder added at a level of 0.1-0.2 g/kgmeat had better antioxidant effects compared to rosemary oleoresin and oregano water extract during the processing and refrigerated storage under air of cooked beef and pork patties (rojas and brewer, 2007). grape seed extract powder is also an effective antioxidant in cooked pork patties during processing and frozen storage under a high-oxygen atmosphere, when added at a concentration as low as 0.05 g/kgmeat (carpenter et al., 2007). ! ital. j. food sci., vol 28, 2016 549 in ground beef, grape seed extract powder has been demonstrated to inhibit the growth of escherichia coli o157:h7, salmonella typhimurium, listeria monocytogenes and aeromonas hydrophila (ahn et al., 2007) at a level of 10 g/kgmeat during refrigerated storage. however, the required addition of grape seed extract is higher than the effective amount for the inhibition of meat oxidation. in fact, at an extract concentration of 0.5-1 g/kgmeat, grape seed extract powder prevented the oxidation of beef sausage during processing and frozen storage under air, more effectively than ascorbic acid (kulkarni et al., 2011). 2.1.2 fish products fish tissues have a high content of polyunsaturated fatty acids (pufa), which undergo degradation via auto-oxidation. the use of natural antioxidants has become as an effective strategy for controlling the stability of these products, either during the frozen storage of minced tissue or during the processing and refrigerated storage of pre-cooked fish-based products. for this purpose, winemaking by-products have also been considered (table 2). table 2: applications of winemaking by-products as new food ingredients in fish products. food product recovered ingredient main results and references atlantic mackerel minced muscle (uncooked) phenolic fractions of white grape pomace. integration: 0.1 g monomeric flavonoids or pas/kgfish longer induction period for the formation of peroxides and aldehydes during storage in erlenmeyer flasks under air at -10°c. maximum protection by pas with high degree of polymerization and percentage of galloylation. pazos et al., 2005 horse mackerel minced muscle (uncooked) white grape pomace antioxidant dietary fibre (particle size < 0.25 mm; tdf: 760 g/kg; soluble tp: 78 g gae/kg) integration: 20 40 g/kgfish inhibition of formation of conjugated dienes and trienes and tbars during storage in cryovac bb4l bags (otr: 30 cm3/m2d) at -20°c. significant antioxidant activity and high fibre content. sanchez-alonso et al., 2008 chub mackerel minced muscle (uncooked) red grape seed extract (soluble tp: 66 g gae/kg) integration: 20 g/kgfish inhibition of lipid hydroperoxides and tbars formation during storage in cartoon trays under air at -20°c. ozen et al., 2011 meagre sausage (pre-cooked) white grape skin antioxidant dietary fibre (particle size < 1 mm; tdf: 820 g/kg; soluble tp: 42 g gae/kg) integration: 30 g/kgfish inhibition of tbars formation and oxidation during storage in barrier bags (otr: < 2.1 cm3/m2d) at 2°c. significant antioxidant activity and high fibre content. antimicrobial effect on h2s producers and a reduction in total viable counts. ribeiro et al., 2013 otr: oxygen transmission rate; tbars: thiobarbituric acid reactive substances; tdf: total dietary fibre; tp: total phenolics (expressed as gae: gallic acid equivalents or sum of monomeric flavonoids or pas: proanthocyanidins). fractionated grape pomace phenolic compounds at a concentration of 0.1 g/kgfish have been proposed as inhibitors for fatty fish species, such as the muscle of atlantic mackerel (scomber scombrus) during frozen storage under air. the induction period for the formation ! ital. j. food sci., vol 28, 2016 550 of peroxides and aldehydes was significantly increased in samples treated with grape phenolic fractions, and the maximum protection was achieved using procyanidins with a high degree of polymerization and percentage of galloylation (pazos et al., 2005). nonfractionated grape seed phenolics also increased the oxidative stability of minced fish during frozen storage under air (ozen et al., 2011). the antioxidant dietary fibre obtained from white grape pomace with a particle size < 0.25 mm (total dietary fibre: 760 g/kg; soluble phenolics: 78 g/kg) at a concentration of 20-40 g/kgfish can also increase the oxidative stability of the minced muscle of horse mackerel (trachurus trachurus) during frozen storage under a low oxygen atmosphere (sanchezalonso et al., 2008). similarly, white grape skin antioxidant dietary fibre with a particle size < 1 mm (total dietary fibre: 820 g/kg; soluble phenolics: 42 g/kg), which was used at a concentration of 30 g/kgfish in precooked meagre (argyrosomus regius) sausage, showed antioxidant effects as well as antimicrobial effects on h2s producer counts and total viable counts, during refrigerated storage under a low oxygen atmosphere (ribeiro et al., 2013). 2.1.3 bakery products and pasta bread is a staple food, and fortification with polyphenols and dietary fibre derived from winemaking by-products has been investigated to improve the diet of consumers (table 3). grape seed extract powder added at a level of 0.6-2 g/kgbread greatly increased the antioxidant activity of the final product, despite the loss of phenolic compounds during processing due to either thermal treatment or interaction with the food matrix. interestingly, a decreased amount of n-(carboxymethyl) lysine, an advanced glycation end-product associated with health risks, was observed in bread containing grape seed extract. moreover, the addition of grape seed extract powder did not significantly affect the hardness of bread but did increase the darkness (peng et al., 2010). to obtain another vehicle for grape seed phenolics, the entire grape seed can be milled to fine particle sizes (< 0.150 mm). however, upon the addition of this ingredient (total dietary fibre and total phenolic contents not specified) to dough at a level of 25-100 g/kgflour, a decrease in loaf brightness and volume, along with an increase in hardness and porosity, was observed (hoye and ross, 2011). these effects were likely due to the inhibition of yeast activity, which reduced the gassing power. moreover, phenolics can inhibit the activity of endogenous amylases in dough, leading to inadequate maltose release for yeast activity during proofing (mildner-szkudlarz et al., 2011). because grape phenolics also inhibit mammalian α-glucosidase and α-amylase, white grape skin antioxidant dietary fibre with particle sizes < 0.250 mm (soluble phenolics: 20.0 g/kg) at a level of 100 g/kgflour has been used in functional flat bread for diabetic people (lavelli et al., 2016a). sourdough fermentation improves the textural properties of wheat and rye breads. hence, the fortification of mixed wheat-rye bread with red grape pomace dietary fibre (total dietary fibre: 593 g/kg; soluble phenolics: 58.9 g/kg) has been investigated (mildnerszkudlarz et al., 2011). an increase in hardness, gumminess and springiness was once again observed in wheat-rye mixed sourdough bread, but the cohesiveness and resilience did not change (mildner-szkudlarz et al., 2011). to increase the fibre and/or phenolic content, the fortification of brownies and biscuits with winemaking by-products has been achieved. in brownies, upon addition of 150-250 g/kgflour of red grape pomace antioxidant dietary fibre with particle size < 0.589 mm (total dietary fibre and total phenolic contents not specified), hardness and chewiness decreased, while springiness increased. hence, the fortification of brownies showed an opposite trend compared to bread fortification, likely due to the presence of fat (walker et al., 2014). ! ital. j. food sci., vol 28, 2016 551 table 3. application of winemaking by-products as new food ingredients in bakery products and pasta. food product recovered ingredient main results and references wheat bread grape seed extract powder tp: not specified integration: 0.6-2 g/kgbread decreased level of n-(carboxymethyl)lysine, an advanced glycated end-product related to health risk. no significant difference in hardness. increase in darkness. peng et al., 2010 wheat bread grape seed antioxidant dietary fibre (particle size < 0.150 mm; tdf and tp: not specified) integration: 25-100 g/kgflour decrease in loaf volume, and increase in hardness and porosity. hoye and ross, 2011 wheat bread white grape skin antioxidant dietary fibre (particle size < 0.25 mm; soluble tp: 1.5 g monomeric flavonoids/kg and 18.5 g pas/kg) integration: 100 g/kgflour inhibition of mammalian α-glucosidase and αamylase. lavelli et al., 2016a wheat-rye bread (sourdough) red grape pomace antioxidant dietary fibre (particle size not specified; tdf: 593 g/kg; soluble tp: 58.9 g gae/kg). integration: 40-100 /kgflour significant increase in tdf and tp. increase in hardness, gumminess and springiness. no change in cohesiveness and resilience. mildner-szkudlarz et al., 2011 brownies red grape pomace antioxidant dietary fibre (particle size < 0.589 mm; tdf and tp: not specified). integration: 150-250 g/kgflour decrease in firmness and chewiness and increase in springiness. walker et al., 2014 biscuits grape seed extract encapsulated in mesquite gum, zein and maltodextrin (soluble tp: 21 g gae/kg) integration: 0.6 g tp/kgdough increase in tp content and thermal stability partial masking of darkness davidov-pardo et al., 2012 biscuits white grape pomace antioxidant dietary fibre (particle size < 0.150 mm; tdf: 509 g/kg; soluble tp: 31 g gae/kg) integration: 100 300 g/kgflour significant increase in tdf and tp. decrease in hardness, brightness and yellowness mildner-szkudlarz et al., 2013 biscuits red grape marc extract (soluble tp: 2.1 g gae/l) integration: 450 ml/kgsemolina increase in tp and antioxidant activity increase in compounds derived from the maillard reaction, except pyrazines, and lipid oxidation pasqualone et al., 2014 pasta red grape pomace antioxidant dietary fibre (particle size < 0.811 mm; tdf: 689.5 g/kg; tp: not specified). integration: 25-75 g/kgflour increase in tp and antioxidant activity increase in cooking loss sant’anna et al., 2014 pasta red grape marc (soluble tp: 4.43 g gae/kg) integration: 300 g/kgsemolina increase in tp and antioxidant activity decrease in cooking loss marinelli et al., 2015 tdf: total dietary fibre content; tp: total phenolic content (expressed as gae: gallic acid equivalents or sum of monomeric flavonoids or pas: proanthocyanidins). regarding biscuits, the addition of white grape pomace antioxidant dietary fibre with particle size < 0.150 mm (total dietary fibre: 509 g/kg; soluble phenolics: 31 g/kg) up to 300 g/kgflour led to a decrease in hardness, brightness and yellowness (mildner! ital. j. food sci., vol 28, 2016 552 szkudlarz et al., 2013). in contrast, the incorporation of grape seed phenolics encapsulated with mesquite gum, zein and maltodextrin (soluble phenolics: 21 g/kg) partially masked the dark colour (davidov-pardo et al., 2012). alternatively, biscuits containing red grape extract (soluble phenolics: 2.1 g/l) at a level of 450 ml/kgsemolina displayed a particular red colour and aromatic profile and possessed greater contents of maillard-reaction compounds, except pyrazines, as well as higher amounts of compounds derived from lipid peroxidation than the control biscuits (pasqualone et al., 2014). the fortification of pasta with winemaking by-products is also promising. grape pomace antioxidant dietary fibre with particle size < 0.811 mm (total dietary fibre: 689.5 g/kg; total phenolic content not specified) formulated into pasta (25-75 g/kgflour) increased the total phenolic content and antioxidant activity but also caused a slight increase in the cooking loss at high levels of addition. this effect could be attributed to changes in the gluten protein network due to the interference of grape pomace fibre, which reduces the gluten strength and interrupts the overall structure of pasta (sant’anna et al., 2014). however, the incorporation of red grape marc extract (soluble phenolics: 4.43 g/kg) into pasta at a ratio of 1:10, w/v, led to an increase in the phenolic content and antioxidant activity, as well as improved cooking performance. in fact, the cooking loss decreased due to the presence of grape pomace phenolics, which were complexed with proteins around starch granules, encapsulating phenolic compounds during cooking and restricting excessive swelling and amylose diffusion. the fortified sample was also characterized by a low adhesiveness value due to the formation of a stronger gluten network in the presence of phenolics, which entrapped the starch granules, slowing down amylose release during cooking. however, the hardness of fortified pasta was similar to that of the control pasta (marinelli et al., 2015). 2.1.4 fruit-based products the replacement of synthetic additives with natural compounds such as winemaking byproducts and the development of functional foods are emerging trends in the fruit processing industry (table 4). when the phenolic extract of red grape pomace (soluble phenolics: 30 g/kg) was added to a model fruit gel at a concentration of 8.2 g/kgmixture (prior to concentration), a stable red colour and a marked increase in the antioxidant capacity were observed. these effects were maintained, even after storage for 24 weeks at room temperature, likely due to intermolecular associations between pectins and anthocyanins (maier et al., 2009a). the formulation of 0.01 g/kgjuice of white grape skin extract (phenolic content not specified) in model fruit juice containing lactobacillus rhamnosus, bifidobacterium lactis and lactobacillus paracasei improved the stability of probiotic bacteria during storage due to the presence of a more stable anaerobic environment (shah et al., 2010). red and white pomace extracts (soluble phenolics: 75-280 g/kg) were added to apple and orange juice to achieve a concentration of 20-100 g/kgjuice of phenolics, and antimicrobial effects toward microbial contaminants were observed, including zygosaccharomyces rouxii and z. bailii (sagdic et al., 2011). alternatively, the use of antioxidant dietary fibre with particle size in the range of 0.125-0.5 mm obtained from white grape pomace (total dietary fibre: 505 g/kg; soluble phenolics: 30 g/kg; insoluble phenolics: 139 g/kg) imparted multiple functional effects. when this antioxidantand fibre-rich ingredient was added to tomato puree at a level of 30 g/kgpuree, the reducing capacity and inhibitory effect of hyperglycaemia-induced damage increased due to the ability of grape phenolics to act as both oxygen-radical and carbonyl-radical scavengers (lavelli et al., 2014; torri et al., 2015). ! ital. j. food sci., vol 28, 2016 553 table 4: application of winemaking by-products as new food ingredients in fruit-based products. food product recovered ingredient main results and references model fruit gel red grape pomace extract soluble tp: 30 g gae/kg integration: 8.2 g /kgmixture (before concentration) brilliant red colour and strong antioxidant capacity during storage at room temperature. maier et al., 2009a model fruit juice containing probiotic bacteria white grape skin extract tp: not specified integration: 0.01 g/kgmixture improved stability of the probiotic bacteria lactobacillus rhamnosus, bifidobacterium lactis, and lactobacillus paracasei during storage. shah et al., 2010 orange and apple juices red and white pomace extracts (soluble tp: 75-280 g gae/kg) integration: 20-100 g/kgjuice antifungal activity towards zygosaccharomyces rouxii and z. bailii with variety-dependent efficacy. sagdic et al., 2011 tomato puree white grape skin antioxidant dietary fibre (particle size in the range of: 0.125-0.5 mm; tdf: 505 g/kg; soluble tp: 30 g flavonoids/kg; insoluble tp: 139 g pas/kg). integration: 30 g/kgpuree increase in reducing capacity and potential ability to inhibit hyperglycaemia-induced damage. lavelli et al.,2014; torri et al., 2015 tomato puree white grape skin antioxidant dietary fibre (particle size in the range of 0.125-0.5 mm; tdf: 505 g/kg; soluble tp: 22 g gae/kg). integration: 30 g/kgpuree increase in bostwick consistency, storage (g) and loss (g) moduli, and complex viscosity (η*). lavelli et al., 2015b apple-based fruit jelly red grape skin antioxidant dietary fibre (particle size in the range: 0.125 0.5 mm; tdf: 600 g/kg; soluble tp: 26 g flavonoids/kg). integration: 63 g/kgmixture (before concentration) increase in puncture energy, i.e., stronger texture. requires less dehydration. high antioxidant and fibre content. cappa et al., 2015 tdf: total dietary fibre content; tp: total phenolic content (expressed as gae: gallic acid equivalents or sum of flavonoids or pas: proanthocyanidins). in addition, the fibre content increased, which improved the bostwick consistency, storage and loss moduli and complex viscosity, providing a means to modulate the textural properties of puree (lavelli et al., 2015b). a similar antioxidantand fibre-rich ingredient obtained from red pomace with particle size in the range of 0.125-0.5 mm (total dietary fibre: 600 g/kg; soluble phenolics: 26 g/kg) added at a level of 63 g/kgmixture to applebased candy modified the textural properties, resulting in a stronger structure that required greater puncture and penetration energy. as a result, during candy processing, the dehydration step could be reduced (cappa et al., 2015). 2.1.5 dairy products the addition of fruit phenolics into food products has increased due to the emerging popularity of functional foods, including phenolic addition in the dairy sector (o’connell and fox, 2001). the fortification of phenolics into dairy products may increase the heat and foam stability of milk and enhance nutritional benefits (o’connell and fox, 2001). however, many phenolics are bitter and astringent (vidal et al., 2004), and humans instinctively reject bitter substances (drewnowski ! ital. j. food sci., vol 28, 2016 554 and gomez-carneros, 2000). although phenolic compounds interact with proteins during the cheesemaking process, these interactions are dependent on the ph and molar ratio and molecular properties of the polyphenols and involve both hydrophobic and hydrophilic bonds (felix da silva et al., 2015). the application of winemaking byproducts to the dairy sector is shown in table 5. table 5: application of winemaking by-products as new food ingredients in dairy products. food product recovered ingredient main results and references cheese single phenolic compounds and whole grape extract integration: 0.5 mg single phenolic or gae/mlmilk slightly reduced hydration capacity of cheese curd network. no variation in gel strength. less smooth and less dense internal structure of cheese curds. more granular outer surfaces than control cheese by sem microstructure analysis. han et al., 2011a cheese single phenolic compounds and whole grape extract integration: 0.5 mg single phenolic or gae/mlmilk high retention of grape phenolics in the curd. high radical scavenging activity. increase in gel-formation rate due to a slight decrease in ph. han et al., 2011b cheese red and white grape pomace antioxidant dietary fibre before and after distillation (particle size < 0.25 mm; tp: 3.64-16.0 g gae/kg). integration: 8 and 16 g/kgcurd highest radical scavenging activity and tp content, yielding 16 g/kg of grape powder after distillation. no effect on lactic bacteria or proteolysis. marchiani et al., 2015 yogurt and salad dressing red grape pomace antioxidant dietary fibre (particle size < 0.18 mm; tdf: 613.2 g/kg, tp: 67 g gae/kg). integration: 10-30 g/kgyogurt; 5-10 g/kgsalad dressing increase in tdf, tp and radical scavenging activity (but slight decrease in tp during storage at 4 °c). decrease in peroxide values for both yogurt and salad dressing. stable value for lactic acid percentage and syneresis during 3 weeks of storage at 4 °c for yogurt. tseng and zhao, 2013 yogurt grape seed extract tp: 76 150 g gae/kg integration: 50-100 mg tp/kgyogurt increase in tp (slight decrease in tp during storage at 4 °c). no change in ph or lactobacilli counts. chouchouli et al., 2013 yogurt red and white grape skin antioxidant dietary fibre (particle size < 0.25 mm; tdf: 345-481 g/kg). integration: 60 g/kgyogurt increase in acidity, total phenolic content and antioxidant activity with respect to control, but lower ph, syneresis and fat. lactic acid bacteria, phenolic content and antioxidant activity were stable during 3 weeks of storage. marchiani et al., 2016 milk grape seed extract powder tp: 842-927 g/kg integration: 2 g/lmilk phenolic content equal to that of one serving of fresh apple. increase in potential health benefit. axten et al., 2008 tdf: total dietary fibre content; tp: total phenolic content (expressed as gae: gallic acid equivalents). ! ital. j. food sci., vol 28, 2016 555 han et al. (2011a,b) studied the effect of single phenolic compounds (catechin, epigallocatechingallate, tannic acid, homovanillic acid, hesperetine and flavone) and natural extracts, such as whole grape extract, green tea extract and dehydrated cranberry powder as functional ingredients in cheese at a level of 0.5 mg of total phenolics/mlmilk. three gel-forming parameters were evaluated, including tlag (the lag-time before the beginning of milk coagulation), vmax (the maximum rate of gel formation) and tvmax (the time required to reach the maximum rate of gel formation). the results demonstrated that the addition of phenolic compounds to milk affected all of the gel-forming parameters. in fact, the change in ph due to the addition of phenolics resulted in faster gel formation in cheese samples fortified with single phenolic compounds than those fortified with natural extracts. in addition, several phenolic sources improved the antioxidant properties of cheese products. among the tested single phenolic compounds and natural extracts, the greatest radical scavenging activity was achieved using whole grape extracts, likely due to the improved retention of grape phenolics in the curd, representing a promising utilization of winemaking by-products. marchiani et al. (2015) used antioxidant dietary fibre obtained from three grape pomaces (barbera, chardonnay prior to distillation and chardonnay after distillation) with particle size < 0.25 mm (soluble phenolics: 3.64-16.0 g/kg) in semi-hard and hard cheeses (italian toma-like and cheddar) to increase the content of phenolic compounds. powders were added at two different concentrations (8 and 16 g/kgcheese), and the results showed that the amount and type of powder did not significantly affect the physicochemical parameters of cheese, except the ph. cheeses containing chardonnay powder after distillation showed the highest phenolic content and radical scavenging activity at the end of ripening. proteolysis and microbial counts did not show significant differences between fortified and control cheeses. tseng and zhao (2013) applied grape pomace antioxidant dietary fibre with particle size < 0.18 mm (total dietary fibre: 613.2 g/kg; soluble phenolics: 67 g/kg) at a level of 1030 g/kg to yogurt and 5-10 g/kg to salad dressing to enhance the nutritional value and improve the storability. the authors demonstrated that this ingredient can be used as an alternative source of antioxidants and dietary fibre to delay the oxidation of lipids during refrigerated storage. however, the total phenolic content and radical scavenging activity of fortified yogurts decreased slightly during storage, likely due to interactions between proteins and phenolic compounds. consequently, further research is necessary to determine the mechanism of the long-term retention of grape phenolics and radical scavenging activity of the aforementioned products. chouchouli et al. (2013) used grape seed extracts from two grape varieties (50-100 mg of phenolics/kgyogurt) to fortify full-fat and non-fat yogurt. the addition of the extract did not affect the ph or lactobacilli count, while fortified yogurts showed higher antioxidant and antiradical activity than control samples, even after 3-4 weeks of cold storage. however, the phenolic content and radical scavenging activity decreased during storage. the fortification of yogurt with up to 60 g/kgyogurt of red and white grape skin antioxidant dietary fibre with particle size < 0.25 mm (total dietary fibre: 345-481 g/kg) was performed by marchiani et al. (2016). in this case, a reduction in the total phenolic content did not occur during refrigerated storage, but the radical scavenging activity decreased. grape addition did not affect the growth of lactic acid bacteria. moreover, uht low fat milk was fortified with different grape seed extract powders to obtain a concentration of 2 g/lmilk of phenolics in the final product, representing the amount of phenolics ingested when consuming one serving of fresh apple (axten et al., 2008). ! ital. j. food sci., vol 28, 2016 556 2.2. sensory effects understanding the impact of new ingredients on consumers’ perception is considered a key step in new product development (verbeke, 2006; tuorila, 2007). therefore, including a sensoryand consumer-based approach in product innovation is strategic for determining the properties that have the greatest effect on consumer preference, as well as relevant attributes that drive product optimization (torri et al., 2016). 2.2.1 meat products the sensory effects of grape by-products on meat products have been thoroughly analysed in the literature. for chicken meat, grape skin extract added to dehydrated products (1 g/kgmeat) prevented changes in sensory attributes due to oxidation (hot wash, boiled chicken, subcutaneous fat and rancidity), with comparable efficacy to synthetic antioxidants and other natural antioxidants, such as rosemary, coffee and tea (nissen et al., 2000). in chicken hamburger, the use of grape antioxidant fibre (5-20 g/kgmeat) increased the lipid stability without affecting the desirability of the odour, flavour, and tenderness during 5 days of storage. only the colour was modified by the addition of grape skin extract (15-20 g/kgmeat), which did not affect the acceptability of samples. the sensory test comparing the effect of four different addition levels revealed that hamburgers preferred by consumers had the highest extract content (15-20 g/kgmeat) (sàyago-ayerdi et al., 2009). the incorporation of grape pomace extracts in raw and cooked chicken meatballs (60 mg of phenolics/kgmeat) also provided satisfactory results in terms of odour and flavour properties during frozen storage, which were not different from those observed using synthetic antioxidants (selani et al., 2011). regarding pork meat, the addition of grape skin extract (0.2 g/kgmeat) to cooked patties was not sufficient to reduce the intensity of rancid and linseed odours and flavours correlated to lipid oxidation indexes (tbars and hexanal content); thus, acceptable sensory properties for consumption could not be guaranteed (nissen et al., 2004). nevertheless, grape seed extract (0.1-0.2 g/kgmeat) has been proven to have a positive effect on both cooked pork and beef patties and was even more effective than rosemary oleoresin and oregano water-extracts. in fact, sensory evaluation performed over eight days of storage at 4°c demonstrated the efficiency of grape seed extract at controlling several negative sensory characteristics associated with a warmed-over flavour, such as rancidity, wet cardboard (for beef patties) and grassy (for beef and pork patties) odour descriptors (rojas and brewer, 2007). accordingly, the addition of grape seed extract (0.05-1 g/kgmeat) did not significantly affect the average scores obtained for cooked pork patties for any of the quality parameters evaluated (colour, flavour, texture, juiciness and off-flavour) over four days of storage at 4°c under a modified atmosphere (carpenter et al., 2007). in beef patties, grape seed extract (0.1-0.2 g/kgmeat) led to visual green discoloration (rojas and brewer, 2007). this effect was also observed in precooked beef sausage containing grape seed extract (0.3-0.5 g/kgmeat). moreover, grape seed extract improved the persistence of fresh cooked beef odour and flavour during the storage of fortified products with respect to the control group and prevented the formation of a rancid odour (kulkarni et al., 2011). 2.2.2 fish products very little information is available on the sensory effects of grape by-products in fortified fish products. in fact, only one paper out of those cited in the present review included a sensory evaluation of fortified fish sausage. the sensory description of this product ! ital. j. food sci., vol 28, 2016 557 showed that samples containing grape skin antioxidant dietary fibre (30 g/kgfish) were significantly darker, less elastic, cohesive, succulent and oily and possessed a more unpleasant texture and flavour than the control product. the unpleasant odour and flavour has been described as a sour note, but a rancid aroma or flavour did not develop after 98 days of storage at refrigerated temperatures (ribeiro et al., 2013). 2.2.3 bakery products and pasta among the food categories considered in this review, cereal products have been the most commonly investigated from a sensory perspective. by adding different amounts of grape seed extract to white bread (0.6-2 g/kgbread), significant alterations in quality attributes (sweetness, porosity, astringency and stickiness) were not detected (peng et al., 2010). the recommended replacement of hard red spring flour with grape seed antioxidant dietary fibre in bread was 50 g/kgflour. above this threshold, low sensory acceptance of astringency, sweetness, bitterness and overall liking was observed (hoye and ross, 2011). similarly, the overall acceptance for sourdough mixed rye bread decreased as the content of red grape pomace antioxidant dietary fibre increased from 40 to 100 g/kgflour, indicating that a maximum of 60 g/kgflour could be used to prepare acceptable products. higher levels were associated with a decrease in the volume, porosity and typical aroma of freshly baked breads and an increase in hardness, acidity and alcoholic, sharp and fruity notes (mildner-szkudlarz et al., 2011). regarding brownies, fortification with red and white grape pomace antioxidant dietary fibre at levels higher than those acceptable for bread, up to 150 g/kgflour, did not impact the sensory properties and acceptability of the products (walker et al., 2014). the acceptance of biscuits enriched with white grape pomace antioxidant dietary fibre was also dependent on the level of addition: 100 g/kgflour incorporation in wheat flour was adequate, while concentrations of 200 or 300 g/kgflour induced a fruity-acidic note and an intense brown colour, which was undesirable to consumers (mildner-szkudlarz et al., 2013). however, microencapsulation of grape seed extracts prevented a decrease in the likeability ratings by consumers (davidov-pardo et al., 2012). biscuits enriched with a red grape pomace extract (450 ml/kgsemolina) have been described by a trained panel of assessors as having a more intense colour, fruity odour and sour taste and lower friability than control samples. moreover, consumers were able to discriminate among biscuits samples based on both colour and taste. however, neither the modifications in sensory profiles nor the differences perceived during the affective test influenced the acceptability or willingness of the subjects to buy anthocyanin-enriched biscuits. regarding both the colour and taste, in a global evaluation, the number of consumers who preferred enriched biscuits was not significantly different than those who preferred control biscuits (pasqualone et al., 2013). in fettuccini pasta formulated with red grape pomace antioxidant dietary fibre (25-75 g/kgflour), the overall liking and acceptance of aroma, aftertaste, flavour and appearance decreased, regardless of the concentration of the ingredient (sant’anna et al., 2014). however, upon addition of red grape pomace extract (300 g/kgsemolina), the sensory properties of fortified, fresh-extruded, fresh-pasteurized and dry spaghetti pasta were as acceptable as control products (marinelli et al., 2015). 2.2.4 fruit-based products information regarding the effect of grape by-products on the sensory properties of fruitbased products is scarce. the incorporation of different granulometric fractions of white grape skins into either smooth or rough tomato puree (30 g/kgpuree) induced a clear increase ! ital. j. food sci., vol 28, 2016 558 in the textural attributes (crispiness and granularity), a decrease in the perceived homogeneity and a change in the vegetable odour notes (spicy hay). the intensity of these effects depended on the fraction particle size, which also influenced consumers’ preferences. a cluster of subjects was found to significantly prefer the smallest particle size fraction (< 0.125 mm), especially when combined with smooth tomato puree, while another group of consumers showed opposite preferences, preferring the largest particle size (0.250-0.500 mm) and rough tomato puree cells (lavelli et al., 2014; torri et al., 2015). 2.2.5 dairy products a limited number of applications of grape pomace have been investigated in dairy products, especially in terms of sensory effects; however, yogurt fortified with winemaking by-products has been investigated. fortification with grape seed extract amounts corresponding to 50-100 mg of total phenolics/kgyogurt did not result in any major defects in sensory properties (consistency, colour and flavour) compared to control samples (chouchouli et al., 2013). on the contrary, higher levels of addition strongly modified the sensory properties of yogurt, which was perceived as too sour by consumers, possessing an unpleasant flavour and grainy/sandy texture. however, significantly different hedonic scores were observed using by-products from different grape varieties, suggesting that chardonnay was more suitable than pinot noir and muscat (marchiani et al., 2016). the fortification of yogurt with red grape pomace in the range of 10 to 20 g/kgyogurt provided satisfying overall likeability values. nevertheless, lower likeability scores for flavour and texture were observed for the sample with the highest concentration of grape pomace (tseng and zhao, 2013). in salad dressing, the addition of red grape pomace antioxidant dietary fibre at a level of 10 g/kgmixture was best received by consumers (tseng and zhao, 2013). fortification with grape skin antioxidant dietary fibre clearly influenced the sensory properties of soft cow milk cheeses, especially the appearance and texture. in particular, the marbling aspect, granularity, sandiness, sourness and astringency (due to the presence of fibre and polyphenols from grape skin) negatively impacted the overall likeability of the cheese when the amount of barbera and chardonnay grape was greater than 8 and 16 g/kgcurd, respectively (torri et al., 2016). the addition of grape seed extracts to low fat uht milk had a significant effect on the product sensory attributes, tending to suppress sweetness and uht odour and flavour and to increase the perception of bitterness, sourness, astringency, odours and flavours (fresh raisin, honey, inka, ashy and tobacco), as well as chalkiness (axten et al., 2008). 3. grape seed oil extraction by green tecnologies grape seed oil extraction can be applied in parallel with the recovery of both antioxidant dietary fibre and phenolic extracts from grape skins and defatted grape seeds, thus making the overall recovery strategy more sustainable. with the recent technological advancements, “green technologies” have been proposed to replace the traditional oil recovery process by solvents. the recovery of grape seed oil requires the preliminary separation of seeds from other grape pomace constituents, including skins and stalks. separation occurs by mechanical devices, usually after grape pomace drying. the conventional “non-green” solvent extraction allows for nearly complete oil recovery. non-polar solvents are used for oil extraction, including n-hexane and petroleum ether, which are the most common extracting solvents and have the lowest cost. after extraction, ! ital. j. food sci., vol 28, 2016 559 the oil must be separated from the solvent in which it is dissolved, and the solvent may be recycled. separation is achieved by evaporating off the solvent. for n-hexane, evaporation occurs at 69 °c (the boiling point of n-hexane at ambient pressure). high quality oil is obtained by mechanical extraction performed at ambient temperature. unfortunately, mechanical extraction does not afford a high yield of oil, particularly for grape seeds, whose woody texture makes them mechanically resistant and, more importantly, whose oil content is reduced to the range of 4 to 17% (fernandes et al., 2013; fiori et al., 2014). to increase the oil recovery, mechanical extraction can be performed at higher temperatures: however, at increased extraction temperatures, some of the noble oil constituents, which are thermally unstable, tend to degrade. an emerging solvent for use in the food industry is high pressure co2, more precisely termed supercritical co2 (sc-co2) (duba and fiori, 2015a). a fluid is in the supercritical state when the actual temperature and pressure are higher, respectively, than the critical temperature (tc) and pressure (pc) of the fluid. for co2, tc and pc are 31°c and 73 bar, respectively. therefore, sc-co2 can be used while operating at temperatures only slightly higher than ambient temperature, making sc-co2 particularly interesting for thermally unstable compounds, which is often the case in the food sector. an important advantage in the use of supercritical fluids as solvents is the extreme ease in separating the solvent and solute after extraction: separation occurs by simple depressurization. when using sc-co2 for extracting grape seed oil, after flowing sc-co2 is contacted with a static bed of milled grape seeds, the mono-phase stream consisting of scco2 and extracted and dissolved grape seed oil is expanded through a back-pressure valve. after expansion, low pressure gaseous co2 separates from grape seed oil, which is recovered solvent-free. solvent-free defatted grape seeds can also be recovered and used for further food applications (lavelli et al., 2015a). the process and corresponding equipment for an industrial-scale plant for sc-co2 extraction of grape seed oil have been fully explained in the literature (fiori, 2010; freitas et al., 2013). the effect of process parameters on the extraction kinetics and yield of the sc-co2 extraction of grape seed oil has been recently outlined (duba and fiori, 2015b). other obvious advantages of using sc-co2 in the food sector are represented by the peculiarities of co2, which is non-toxic, non-flammable and inexpensive. unfortunately, the proposed process includes drawbacks of an economic nature. the solubility of grape seed oil in sc-co2 is lower than 10 goil/kgco2 for pressures lower than 350 bar (duba and fiori, 2016). to achieve relatively high solubility values for oil in sc-co2, the equipment must be operated at not less than 400 bar, preferably at 500-600 bar, which translates into high investment costs for the facility, where the extractors must be employed at high pressure (fiori, 2010). 4. conclusions the studies summarized in the present review demonstrate an increase in interest in potential food applications of winemaking by-products and provide a new production scenario for winemakers. winemaking by-products can be processed into various food ingredients, including antioxidant dietary fibres, crude phenolic extracts or encapsulated extracts, and applied to produce new foods. grape seed oil can be recovered using sc-co2 extraction. however, economic and regulatory factors prevent these applications from achieving large-scale application. first, the proposed applications imply new production cycles across winemaking and other food sectors. the establishment of these connections demand improved logistic organization, including appropriate technologies for the collection, storage, transportation and processing of grape pomace. investment costs for ! ital. j. food sci., vol 28, 2016 560 new processes are sometimes high. second, although food legislation strongly promotes the recycling of by-products, a recovery strategy based on value addition and by-product use in functional food production results in additional regulatory issues. actually, the application of by-products in foods generally leads to novel foods, which opens the debate regarding safety. fortification/enrichment with the appropriate amount of grape antioxidant dietary fibre only allows for the labelling of foods as fibre-rich, while other possible health claims must be substantiated by specific studies. thus, further scientific research is necessary to surpass economic and regulatory barriers and achieve significant advance towards the establishment of a biorefinery fed with winemaking by-products. acknowledgements this research was supported by ager (project number 2010-2222). references ahn j., grun i.u. and mustapha a. 2007. effects of plant extracts on microbial growth, color change, and lipid oxidation in cooked beef. food microbiol. 24: 7. amendola d., de faveri d.m. and spigno g. 2010. grape marc phenolics: extraction kinetics, quality and stability of extracts. j. food eng. 97:384. axten l.g., wohlers m.w. and wegrzyn t. 2008. using phytochemicals to enhance health benefits of milk: impact of polyphenols on flavor profile. j. food sci. 73: h122. battista f., tomasi d., porro d., caicci f., giacosa s. and rolle l. 2015. winegrape berry skin thickness determination: comparison between histological observation and texture analysis determination. ital. j. food sci. 27: 136. beverige t.h.j., girard b., kopp t. and drover j.c.g. 2005. yield and composition of grape seed oil extracted by supercritical carbon dioxide and petroleum ether: varietal effects. j. agric. food. chem. 53: 1799. bi n. and rui d. 2014. extracting anthocyanin from grape seed, comprises crushing the grape seed, adding modifying agent, soaking grape seed particle in an organic solvent, adding water to the degreased grape seed, and performing supercritical extraction. patent number: cn103351370-a. patent assignee: jurong chuncheng niansheng-grapery. bordiga m. 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fax: +39 0512096105 e-mail address: maurizio.canavari@unibo.it abstract in the last decade, the local food movement has achieved a growing popularity in the italian food system. nevertheless, the italian food market still lacks a shared definition and labels indicating the local origin of the food products. in this study, we explore the meaning of “local food” in the italian market using a qualitative approach. results from twenty-three individual semi-structured interviews show that the meaning of “local” should be explained more in terms of connection between a community traditions and a geographical area than in terms of food miles. keywords: food traditions, italian food market, local food, qualitative research ital. j. food sci., vol 29, 2017 506 1. introduction in the italian market, local food is defined with the expression "chilometro zero" (zero kilometers), since the first form of direct marketing was represented by the points of sales organized by producers within their farm, where the supply of food products to consumers occurred in the same location as the production (bugni, 2010). the popularity of local food products in italy has been considerably growing: 1141 farmers' markets (fms) organized by “campagna amica” (the most popular format of fms in italy) are recorded in 2016 (campagna amica, 2016). in addition, the presence of community supported agriculture (csa) and direct marketing outlets as open markets, solidarity purchasing groups, small shops and farm-shops has been significantly growing in the last few years (aldinucci, 2014; giuca, 2012; franco et al., 2015; pascucci et al., 2013; vasquez et al., 2017; wellner and theuvsen, 2015). with the so-called "de castro" decree1, currently in force since the 1st of january 2008, guidelines have been set for the realization of marketplaces exclusively dedicated to direct retailing by farmers. moreover, the veneto region, first in italy, on the 25th of july 2008 issued regional law number 7 aimed at promoting the consumption of regional products in public food services in order to support the local economy. in addition, other italian regions such as emilia-romagna and abruzzo are tending to follow the same approach (coldiretti, 2013). given the increased popularity of the local food movement, large retail chains started, as well, to highlight the origin of the products that have been locally produced. however, in the italian market, labels certifying the local origin of the products are not present yet and what is local or not is not yet regulated. admittedly, according to italian and international literature reviews, it is difficult to identify a shared definition of "local food" (bazzani and canavari, 2013:30). thus, the aim of the present study is to determine a definition of "local food" that can be shared throughout italy, where the variety of resources in different territories and an ancient culinary art tradition lead to a high diversification in food consumption. in particular, the main goal of the research is to establish whether “local” can be better interpreted in terms of physical distance (i.e., food miles) or in terms of belonging to local community and food traditions. we performed an explorative qualitative research, based on the use of semi-structured interviews. to the best of our knowledge, previous research related to the definition of local food was mostly based on an anthropological analysis of geographical and cultural conditions which lead to the starting up of local food networks (d’amico et al., 2013; cholette, 2011; giovannucci, et al., 2010; martinez et al., 2010; amilian et al., 2007, brunori, 2007; sonnino and marsden, 2006; dupuis and goodman, 2005; kirwan, 2004; hinrichs, 2003; barham, 2003; la trobe, 2001) or they were mainly focused on the description of consumers' perceptions towards local food (pencarelli et al., 2015; aprile et al., 2012; darby et al., 2008; zepeda and deal, 2009). therefore, this study represents one of the few attempts, the first one in italy, to explore the meaning of “local food” using a qualitative approach. we interviewed twenty-three participants purposely chosen among consumers, farmers and experts of the food system asking about their opinions on local food consumption. through the exploration of concepts such as food values, quality perception, attitudes towards origin certification, we were able to highlight the main issues related to the definition of "local food" and we attempted to draw a possible scenario of the development of "local food" labels in the italian market. in the following sections we describe the methodology, then we summarize and discuss the results, and finally we draw our conclusions. ital. j. food sci., vol 29, 2017 507 2. background on the concept of “local food” brunori (2007) suggested the distinction between "local food", "locality food" and "localist food". the term "local food" implies the instauration within a community of shortdistance relationships, based on food habits and food traditions. on the other hand, the definition "locality food" is mainly focused on the origin of a product from a particular place, giving less importance to the "community factor". finally, the concept of "localist food" implies consumers’ willingness to reconstruct local identities by the regular consumption of food products, although they do not belong to the rural traditions of that local area. hand and martinez (2010) stated that the re-valuation of local food was first supported by the slow food movement and defines whether a product is local or not on the basis of a maximum distance range of 100 km (approximately 60 miles) within which the consumption and production locations are situated (slow food, 2013). it is necessary to point out that the "slow food" association itself does not strictly respect this distance constraint. for example, at the earth market (the farmers' market organized by slow food) of bologna seafood products originate from the coastal area of the emiliaromagna region, which is more than 100 km away from the city of bologna (bazzani et al., 2016). moreover, the concept of "local" has been often associated with regional, national boundaries (costanigro et al., 2014; feagan, 2007; hu et al., 2012; lombardi et al., 2013; scarpa et al., 2005) or in terms of "traditional" food from a certain area (akaichi et al., 2012). some authors (amilien et al., 2007; aprile et al., 2012; barham, 2003; gracia, 2013) associate to "local food" the well-known french term "terroir". this term highlights the influence of social and cultural factors in determining consumers' food habits: "the territorial reputation of a product is more often derived from a mixture of messages rather than the actual geography" (amilien et al., 2007:55). this term also refers to the so-called post-modern consumer, who is interested in the symbolic or cultural value rather than in the functional and utility value of products and services (viganò et al., 2015). 3. materials and methods in order to account for the complexity and diversity of meanings embodied in the concept of "local food" we developed the study using an explorative qualitative analysis approach. this approach was chosen because it is more suitable for achieving a level of depth and understanding that is usually not easy to obtain with a quantitative survey based on statistical methods (molteni and troilo, 2012). interviews were chosen as the most appropriate tool for analysing the social, cultural contexts through which informants can build cultural meanings (denzin, 2001; moisander et al., 2009). we performed indepth interviews, supported by a semi-structured interview schedule, which served as a non-binding guideline for the interviewer. a convenience, non-probabilistic sample of twenty-three individuals was selected. three interviews were conducted by phone, the rest in person. the face-to-face interviews were performed in the cities of bologna and genoa. the selected sample consisted of six consumers, eight farmers and nine food market experts. we decided to interview different actors in the supply chain in order to have a broader interpretation of the issues related to the local food system. the consumers were recruited on the basis of their interest in the local food networks, indeed, four of them were regular farmers' markets shoppers and two of them were members of a csa initiative. all the interviewed farmers regularly participated in farmers' markets and the selected experts were mainly involved in direct marketing activities or certification bodies (table 1). ital. j. food sci., vol 29, 2017 508 table 1. description of survey participants. no. consumer/farmer/ expert location of the interview residence of the respondent activity 1 consumer bologna bologna regular farmers' markets shopper 2 farmer bologna siracusa farmer participating in direct marketing activities 3 farmer bologna borgo panigale (bo) farmer participating in direct marketing activities 4 farmer bologna crevalcore (bo) farmer participating in direct marketing activities 5 expert bologna bologna brand manager involved in the direct marketing of a wine company 6 consumer bologna treviso regular farmers' markets shopper 7 farmer bologna crespellano (bo) farmer participating in direct marketing activities 8 farmer bologna rocca di roffeno (bo) farmer participating in direct marketing activities 9 expert bologna imola (bo) member of coldiretti association 10 expert bologna bologna small retailer of local food products 11 farmer bologna casalfiumanese (bo) farmer participating in direct marketing activities 12 farmer bologna borgo panigale (bo) farmer participating in direct marketing activities 13 consumer bologna catania regular farmers' markets shopper 14 consumer bologna padua regular farmers' markets shopper 15 expert bologna bologna researcher at the university of bologna 16 expert bologna bologna farm assurance technical coordinator of a certification body 17 consumer bologna ravenna regular csa shopper 18 expert bologna bologna member of slow food association, bologna 19 expert telephone interview viterbo brand manager involved in the direct marketing of organic fresh fruit and vegetable company 20 expert telephone interview bologna general manager of italian vegetable seed company 21 expert genoa genoa general manager of a retail company 22 consumer genoa genoa regular csa shopper 23 farmer telephone interview lugo (ra) farmer participating in direct marketing activities source: data from the survey. the recruitment of consumers was the most demanding among the three categories of respondents, since most of the consumers contacted affirmed that they had insufficient knowledge of the topic and did not accept the invitation to participate in the survey. in contrast, most of the contacted farmers and experts agreed to take part in the research (table 2). the interviews were administered during summer 2013. once respondents were contacted, they were asked to take part in a research regarding the local food system. they were informed about the duration of the interview (30-45 minutes) and they were assured that their participation would be anonymous. finally, the interviews were scheduled according to respondents' availability. ital. j. food sci., vol 29, 2017 509 table 2. contacted and selected respondents. contacted accepted response rate (%) consumers 17 6 34 farmers 11 8 73 experts 12 9 75 total 40 23 57.5 source: data from the survey. as previously mentioned, the interviews were structured according to a semi-structured interview schedule that was not strictly followed in order to minimize researcher influence and other sources of bias (alvesson, 2003). therefore, general questions (open-ended questions) were posed to introduce the argument and, along the discussion, informants tended to be induced to raise issues that were considered important and relevant to the subject of interest (myers, 2009). all the interviews were recorded and transcribed verbatim. the transcribed interviews were analysed using, firstly, an open coding approach to examine the discrete parts. then, axial coding was applied for the re-assembly of the data in categories and subcategories, which were finally brought together using selective coding (strauss and corbin, 1998). 4. results the adoption of an explorative approach, based on the use of in-depth interviews, turned out to be appropriate for the aim of the research; we were able to collect a high variety of information that let us highlight the different aspects of the proposed topic. the semistructured interview guideline was also effective in helping the interviewee to initially face the problem using a wide-angle lens, then turning the discussion into more specific issues related to the local food system (fig. 1). the definition of food values and respondents' perception of quality was essential in introducing the concept of origin, since nearly the totality of the interviewees marked the important issues as environmental and biodiversity safeguards, suitability of land and local traditions (fig. 1). furthermore, the variety of issues mentioned in the interviews was also due to the choice of addressing different actors in the food supply chain. indeed, results show that, generally, consumers were more focused on aspects such as organoleptic features of the products and support to the local economy, while farmers were more focused on environmental safeguards and, finally, experts highlighted the hygienic-sanitary safety aspect and cultural factors related to food consumption. ital. j. food sci., vol 29, 2017 510 figure 1. issues extrapolated in the research. 4.1. food values respondents suggested different interpretations of the concept of food values: they referred to features such as organoleptic characteristics and nutritional value as well as to the environmental and ethical aspects related to the production and the supply of food products. taste was defined as the feature that mainly explained the value of a product. it is necessary to point out that, in the case of fresh food products, taste was mostly mentioned in combination with freshness and correct grade of ripeness; interviewees indicated “good products”, such as the ones that were harvested and sold within the day. seasonality, as well, was mentioned as an important value in the food system, since the consumption of seasonal products implies both a better organoleptic quality and the respect of natural cycles. furthermore, common opinion was that conventional agricultural techniques, early harvest and the post-harvest treatments, generally, were the main cause of quality loss, not just from the organoleptic, but also from the nutritional point of view. in fact, safety was pointed out as one of the primary factors in food consumption: a good food product is one that a “mom can give to his child without worrying whether it is healthy or not” (interviewed farmer) and that “does not contain poisons” (interviewed farmer). accordingly, several respondents stated that an important value was whether the product had been organically produced. one expert argued that the industrialized food system lead to the research of agricultural techniques aimed at the production of “attractive” foods on large scale and he highlighted the necessity to turn to the use of techniques that were focused on the protection of soil fertility and the "respect of ital. j. food sci., vol 29, 2017 511 nature". indeed, safeguarding biodiversity became a crucial aspect in the definition of the food values, in order to preserve the variety of the products, which are typical of the different italian regions. particularly, protecting the countryside was defined as a very important aspect, both from the environmental and the social-cultural point of view: "the respect of natural conditions of the countryside must be considered as an investment in improving our lifestyles, the economy of local farmers and the re-vitalization of rural areas" (interviewed expert). indeed, re-valorisation of the role of farmers and of rural culture has been defined as the crucial point in the italian food system, where the dominance of large retail chains tends more and more to large scale production and does not focus on peculiar characteristics (typicalities) of regional production, which represent the strength of products "made in italy". therefore, several interviewees argued that communication between farmers and consumers or information provided by labels and certifications are essential in a context where consumers are increasingly unaware of and less interested in food traditions. finally, price was mentioned as a value that had a relative importance, but did not outweigh the items previously mentioned; only one consumer suggested price as one of the main attributes in purchasing food. in most of the cases, interviewees agreed on the fact that price had to be consistent with organoleptic characteristics of the product and quality of production techniques used. therefore, an expert mentioned the slogan of slow food: "buono, pulito e giusto" (good, clean, and fair) in order to summarize the values that should be related to food consumption: food products must have a good taste, must comply with food safety regulations and environmental safeguard, and must be purchased at a price that is fair to consumers and profitable for farmers. 4.2. the definition of quality most of the interviewees mentioned the word “quality”, when they were asked to explain the values related to food products. the concept of quality was mainly interpreted in two different ways: some interviewees tended to be more focused on the definition of intrinsic characteristics such as taste, freshness and seasonality, while others referred mainly to cultural, geographical and environmental factors related to food consumption. some experts argued that quality is a subjective concept, it can be interpreted as the "satisfaction of the needs of those receiving the product" (interviewed expert): consumers, for example, tend to look for good taste, flavour, while large retail chains are more interested in characteristics such as colour, standard shape, and long shelf life. in this respect, quality is therefore interpreted as excellence or differentiation according to consumer preferences, but it can also be interpreted as standardization and compliance with customers’ contractual requirements. indeed, quality was also defined as the respect of standards, laws and regulations that control the food system, thus potentially encouraging producers to aim no higher, quality-wise, than the minimum compliance requirements. food safety was mentioned as a basic feature or a prerequisite that food products must achieve at every stage of the food supply chain, therefore it should not lead to any differentiation among food products available on the market. in some cases, however, safety has been associated with organic production that, on the other hand, has been identified as a feature strictly related to quality. first of all, it implies the absence of synthetic chemicals, which allegedly alter the taste, flavour and healthiness of products. secondly, but not less important, the continued use of artificial fertilizers (as it is linked to conventional agriculture) might encourage soil exploitation and damages to the food’s nutritional value. soil protection and use of sustainable agricultural techniques have been mentioned as crucial aspects in giving a definition of quality in the food system, but in this case interviewees, especially farmers and experts, highlighted the importance of the suitability of the land: "the land must do what it can do" (interviewed expert). fruits and ital. j. food sci., vol 29, 2017 512 vegetables should be grown in the most favourable soil and climate conditions, animals should be kept living in their natural habitat, their welfare should be respected, and food product processing (cheese and wine production, for example) should be applied where environmental conditions make a particular food product part of the community’s traditions. on the basis of these issues, quality can be interpreted as the respect of natural cycles and the safeguard of food typicality. regarding organoleptic characteristics, interviewees stated that taste was the main attribute in defining food quality: "it does not matter whether a product looks perfect, the important thing is that it tastes good!" (interviewed consumer). it is necessary to point out that respondents argued that a product is good and healthy when it is fresh, since avoidance of preservatives, and in the specific case of fresh fruits and vegetables, seasonality and sound harvesting time, allow products to develop their authentic aromas and flavours. 4.3. the importance of the origin of food products and attitudes towards geographical indications the origin of food products was one of the most recurrent factors in relation to the concept of quality. in this paragraph, we will describe motivations that lead interviewees to explain the importance of this issue and their opinion regarding geographical indications (gis). first of all, they reaffirm the importance of land suitability and potential: soil and environmental conditions of a certain area are crucial for producing particular kind of food products. respondents suggested the examples of pachino cherry tomatoes and of parma ham. a farmer argued that pachino cherry tomatoes are typical from an area of sicily where soils are characterized by a high salinity and a very dry climate, and it would be difficult to obtain their typical sweet flavour in different environmental conditions. one expert stated that parma ham would not achieve its distinctive taste if raw materials were not kept exposed to the right grade of humidity that prevails in the parma area. generally, interviewees pointed out the variety of climate conditions in italy, which determined the presence of different food traditions and their historical value in the different regions. some experts stated that the process of selection by the population of a certain area, generation by generation, resulted in the best food products that they could obtain. they had learned, over the years, how to grow them and process them. the introduction and development the of new varieties may cause confusion in local farmers and, therefore, result in lower quality products. for all these reasons, several informants agreed on the fact that, in a "world of growing indifference towards food traditions" (interviewed expert), it is necessary to educate consumers to "respect what the land can give" (interviewed expert) and to re-discover the value of agriculture’s role in the italian economy. accordingly, when interviewees were asked their opinion about gi certification, some of them affirmed that this kind of certification may be a starting point for re-building a connection between consumers and land and to the re-evaluation of rural areas. however they highlighted the need to give more information about their function and meaning. indeed, interviewed consumers affirmed that they could not give their opinion about gi certifications, since their knowledge of these certifications was not sufficient. on the other hand, several interviewees were sceptical regarding this kind of certification for different reasons: (1) they affirmed that it is not difficult to fake a food product, especially when they are unpackaged, (2) origin specification of a product does not provide crucial information such as agricultural techniques and treatments that have been used. one farmer suggested that collective self-certification within a community of farmers would be the appropriate tool to overcome these drawbacks. ital. j. food sci., vol 29, 2017 513 it is necessary to point out that in several cases, when interviewees were asked their opinion regarding the importance of food product origin, they referred to proximity. the argument concerning the advantages of shortening the distance where the food is produced and where it is consumed will be the subject of the next section. 4.4. local food and its role in the food market in italy, "local food" is widely defined as those products defined with the expression "chilometro zero" or "km0" (zero kilometers). general opinion was that this label may be misleading, since it is barely possible to purchase food products that were produced in a range lower than one kilometre (around half a mile). interviewees stated that "km0" may have been developed just to persuade consumers to buy these products. indeed, the interviewed consumers appreciated this expression, they affirmed that it explained clearly the concept of a food product sourced from a nearby location. most respondents suggested that the designation of a food product as local was closely related to the distance of the production area from the place of purchase, and that it should be defined in terms of miles; some of them suggested 50 km (30 miles) as a reasonable threshold. on the other hand, it is necessary to point out that, once interviewees had analysed the issue more deeply, they considered that food miles restrictions should depend on the kind of product. several of them suggested the example of oranges, which are mostly cultivated in the south of italy (in particular in sicily and calabria), but they are typically consumed all over the country, thus implying hundreds of miles of transportation. interviewees agreed on the fact that in this case italian oranges could be defined as a local product, whereas non-local products are those coming from other countries, such as spain or morocco. the same can be argued for olive oil: the interviewed farmers in bologna were aware of the fact that very few olive groves were present within a range of 50 km, since the city is located at the northern limit of the natural distribution area of the olive tree. however, acknowledging that extra-virgin olive oil is consumed in significant quantities in the area, they affirmed that, in this case, the original olive oil from emilia-romagna, or from neighbouring regions (e.g., tuscany), could be defined as "local". indeed, the term has been frequently combined with food traditions and land suitability: parmigiano-reggiano (parmesan) cheese, for example, is produced in an area that includes four different provinces of the region emilia-romagna, where similar environmental conditions and land configuration has induced the development of the same culinary traditions. hence, local food has been valued as a factor linking farmers and food products to a certain area (vandecandelaere et al., 2009) and, especially, bringing farmers closer to consumers. local food supply is generally limited to forms of short food supply chain such as farmers' markets, csa, or direct marketing, where consumers come into direct contact with producers. this aspect has been considered crucial in educating consumers to build a connection with their traditions and rural areas. thanks to the direct communication with farmers, consumers can obtain information about when and how to consume what they buy and especially about the agricultural techniques that have been used. they become an active participant of the local agriculture and are aware of helping the local economy to grow. indeed, the interviewed farmers stated that these forms of short supply chains are the only ways for small farmers to maintain their business in a food system that is dominated by large retail chains. local consumption has also been associated with environmental safeguards, because of the reduced need for transportation, and consequently of gas emissions along the supply chain, as well as favouring a reduced use of packaging. another common opinion was that local products were fresher compared to non-local ones and that the face-to-face ital. j. food sci., vol 29, 2017 514 relationships between farmers and consumers were an encouragement for producers to sell higher quality products. on the other hand, interviewees agreed on the fact that these forms of short food supply chains have some limitations: first of all, food products supplied directly from farmers may be subject to less stringent food safety controls in comparison to conventional food streams. in fact, several small producers who take part in farmers’ markets state that they cannot afford certifications. moreover, farmers’ markets and services organized by csa may sometimes take place in periods and locations that are not convenient to consumers. another inconvenience may be given by the difficulty in providing variety to consumers, supplying them all the kind of foods that a household may need. for these reasons, interviewees were asked about their opinion regarding the possibility, in large retail chains that some food products labelled as “locally produced” could be sold. in most cases, interviewees said they would appreciate this initiative, since it may also represent a way to teach less aware consumers on how to value-enhance their local, seasonal food products and to re-establish a connection with their food traditions. they suggested that a “local food label” should be mainly focused on farmers’ identification and it should tell their “story”: location and features of the farm where food has been produced, agricultural techniques used, how tradition suggests to consume the product, etc.; one expert suggested that the use of qr-codes may be appropriate to give this kind of information. on the other hand, the opinion of some interviewees was that information given to consumers in this way may not replace the information given directly by farmers. most of them were sceptical about integrity of food certifications, especially where labels would define the origin of a product. furthermore, interviewed farmers commented that large retail chains usually request amounts of products that small farmers are often unable to supply and that the reward that large retailers offer is not worth the higher cost of production. finally, one expert argued that large retail chains would be interested in promoting the consumption of local food products only within the framework of a marketing strategy in which these products were characterized by a local brand owned by the large retail chain. 5. conclusions the results show that the meaning of “local” must be explained more in terms of connection to a geographical area than in terms of food miles. some authors (amilien et al., 2007; barham, 2003; giovannucci et al., 2010) suggest that the meaning of local can be associated to geographical indications. our opinion is that the interpretation of "local" should be more related to the concept of belonging to a community within a certain area, where a culinary tradition has been preserved generation after generation. in accordance with brunori's classification regarding local food systems (brunori, 2007), we would rather associate the concept of geographical indications to the definition of "locality food" that is focused on the origin of a product from a particular place, while "local food" is more based on re-valuation of food traditions within a community. furthermore, according to our results, distance restrictions and tolerance in defining “local” strictly depend on the kind of product. therefore, the concept of local goes further than simply food miles, in cases when a food product is an expression of the identity of a region or of a country. indeed, in several cases, respondents associated consumption of local food to re-valorisation of italian food products and support to the national economy. accordingly, "local food" labels would differ from "food miles" labels, since the latter are mainly associated (perhaps naively, according to cholette, 2011) to environmental impacts due to food transportation. "local food" labels, instead, should highlight the ital. j. food sci., vol 29, 2017 515 connection between a community and the land it occupies, and provide information not just regarding environmental benefits related to local food consumption, but also regarding support to local economy, and safeguard/conservation of land biodiversity, food traditions and, especially, characteristics and activities of food-producing farms. the supply of local food products is mainly associated to forms of alternative food networks (kirwan, 2004; la trobe, 2001; martinez et. al., 2010; d’amico et al., 2013) and respondents agreed upon the fact that the introduction of labels which determine the local origin of the products in mainstream food outlets may educate to local consumption even the more "distracted" consumer. nevertheless, results show that different limitations would affect the supply of locally grown products at large retail chains’ outlets. in the first place, general opinion was that consumers do not usually have a good knowledge of the meaning of certifications and the addition of a label may mostly generate confusion between consumers. in the second place, small farmers, who are generally the main actors in supplying local food (goodman, 2004; renting et al., 2003) may not be able to satisfy the volume requirements of large retail chains and they may not have the economic advantages that they usually obtain through alternative food networks. finally, but not less important, quality and quantity of information given by a label could not replace information given by producers, and a lack of direct communication between farmers and consumers would imply a loss of the connection between urban and rural traditions that represents the main issue for local food networks. local food seems to command a strong experiential content, authenticity, and low standardization of products and services (pencarelli et al., 2015) therefore an innovative approach to marketing is required. in future studies, it would be interesting to propose the same research question in other countries, with different climate conditions, culture and food habits. our results suggest that, at least in italy, local is strictly related to food traditions, but diverging cultural environments may induce to the value-enhancement of different food values and, therefore, to a different interpretation of the meaning of local food. note 1decreto 20 novembre 2007, ministero delle politiche agricole alimentari e forestali, attuazione dell'articolo 1, comma 1065, della legge 27 dicembre 2006, n. 296, sui mercati riservati all'esercizio della vendita diretta da parte degli imprenditori agricoli. gazzetta ufficiale n. 301 del 29 dicembre 2007. references akaichi f., gil j.m. and nayga r.m. 2012. assessing the market potential for a local food product: evidence from a non-hypothetical economic experiment. british food journal 114(1):19-39. doi:10.1108/00070701211197347. aldinucci m. 2014. farmers' markets, 2013 di crescita. 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organic and local food consumer behaviour: alphabet theory. international journal of consumer studies 33(6):697-705. doi:10.1111/j.1470-6431.2009.00814.x. paper received january 2, 2017 accepted march 23, 2017 ijfs#955_bozza ital. j. food sci., vol. 30, 2018 641 paper effect of rosemary powder on the quality of dry ewe sausages i. essid*1, s. smeti2 and n. atti2 1department of animal resources, fisheries and food technologies, research unity ur-17agr01, national agronomic institute of tunisia, 43 avenue charles nicole, carthage university, tunisia 2animal and forage productions, national institute of agronomic research of tunisia, carthage university, tunisia *e-mail address: essidiness@gmail.com abstract the aim of this study was to evaluate the effect of rosemary powder on the quality of dry ewe sausages. color parameters (l, a* and b*), ph and microbiological profile were evaluated over the 6-day drying period. sausages with 4 % rosemary powder added showed the lowest values of total viable counts and total coliforms. ph values of dry ewe sausages with rosemary powder decreased significantly during drying. redness (a*) decreased significantly during the drying of control dry sausages against a stability in sausages added with rosemary powder. finally, incorporating rosemary significantly affected dry sausages acceptability. keywords: dry ewe sausage, microbiological quality, physicochemical quality, rosemary powder, sensory quality ital. j. food sci., vol. 30, 2018 642 1. introduction many countries have meat products whose tradition people wish to preserve as part of their history and culture. dry ewe sausages are one of the oldest known traditional meat products in tunisia. in the past, they were often homemade for religious celebrations. nowadays, most dry ewe sausages are produced all year round both in butcher’s shops and sausage manufacturing companies. the ewe meat and fat are finely ground and seasoned with salt and other spices. chemical additives are not used. the mixture is then stuffed into natural casings. sausages are dried naturally at ambient temperature until reaching the desired dehydration, and they are usually consumed without cooking. despite, their short shelf life (not exceeding 2 weeks at 4°c), traditional dry cured ewe sausages are considered safe due to several factors such as reduced water activity and the addition of salt and spices. sensorial attributes such as color and texture are linked to drying conditions (temperature and relative humidity). rosemary (rosmarinus officinalis l.) is one of the most well-known mediterranean lamiaceae. like other aromatic herbs and spices used in mediterranean cuisine, it is used not only to improve or modify the flavor of foods, but also for its antioxidant and antimicrobial effects (liu et al., 2009). the antioxidant activity of rosemary has been related with the presence of some phenolic diterpenes such as carnosic acid and carnosol (pizzocaro et al., 1994; georgantelis et al., 2007). in addition, several authors have reported that some compounds present in rosemary extract possess antimicrobial effects (del campo et al., 2000; mccarthy et al., 2001; balentine et al., 2006; lund et al., 2007). the widespread availability of the rosemary plant in tunisia makes it easy to use as a preservative in meat products. many studies have documented the effects of rosemary essential oils on the quality of meat products (stephanie et al., 2002; mielnik et al., 2003; ahn et al., 2007; garcía-diez et al., 2016); however, few have focused on the effects of rosemary powder. the aim of this work was to study the effect of rosemary powder on physicochemical, microbiological and sensorial characteristics of traditional dry ewe sausage. 2. materials and methods 2.1. plant material the leaves of rosmarinus officinalis l. were collected from ouesslatia, (semi-arid bioclimatic zone of tunisia) in june. specimens of the plant were submitted to the herbarium division of the national institute of research on rural engineering, water and forests where identification was confirmed in the laboratory of forest ecology. rosemary leaves were dried in an oven (ecocell drying oven, mmm med center, germany) at 60°c, ground with a grinder (moulinex, france) and then stored in a moisture-proof container until use. 2.2. meat sampling and sausage preparation the meat used was taken from adult fat-tail barbarine ewes. ewes were fed oat hay and concentrate until slaughter at 48 kg. the carcasses were cut into leg, lumbar region, flank, thoracic region, neck, and shoulder following the procedure of colomer-rocher et al. (1972). the shoulders were dissected into muscle, fat and bone; the muscles were conserved at -20°c until sausage preparation. ital. j. food sci., vol. 30, 2018 643 the sausage mixture was prepared according to the following formulation: 80 % muscle, 20 % tail fat, 3 % salt and 1 % paprika (kamy, tunisia). muscle and tail fat were minced and mixed in a rotating bowl meat cutter (rowenta, universo, germany). the sausage mixture was divided into 3 batches. to the first and the second batches, 2 and 4 % (levels are results of sensorial analysis) of rosemary powder were added, respectively, corresponding to the samples labeled rp2 and rp4. the third batch, without rosemary powder, acted as a control (c). natural salted casings were purchased from a local market in tunis and soaked in water prior to use. subsequently, the sausage mixture was manually stuffed into casings (20 cm of length and about 4 cm of diameter) at approximately 25 g each and then dried at ambient temperature for 6 days. relative humidity and temperature averaged 70 % and 18°c respectively. for sampling, five sausages were taken from each batch at days 0, 3 and 6 of drying for ph, color determination and microbiological analysis. chemical composition and sensorial analysis were done after 6 days of drying. 2.3. sausage color and ph analysis the ph was measured on sausages with a penetrating electrode connected to a portable ph-meter (hanna instruments, romania) after calibration with two buffers (7.00 and 4.00). a minolta cm-2006 d spectrophotometer (konica minolta holdings, inc, osaka, japan) was used to measure color directly on the surface. color coordinates were calculated in the cielab space (cie, 1986). the lightness (l*), redness (a*) and yellowness (b*) parameters were directly recorded (hunt et al., 1991). the hue angle (h*) and chroma (c*) indices were calculated as h* = tan−1 (b*/a*) x 57.29, expressed in degrees and c*= (a*2+ b*2)1/2. h* is the attribute of a color perception denoted by blue, green, yellow, red, purple, etc. c* is related to the quantity of pigments and high values represent a more vivid color and denote lack of greyness (miltenburg et al., 1992). 2.4. microbiological analysis for the microbiological analysis, 10 g of meat were collected aseptically from the center of each sausage, and diluted with 90 ml of sterile peptone water (accumix, belgium) using a stomacher 80 biomaster. serial 10-fold dilutions were prepared in sterile peptone water. appropriate dilution samples (1 or 0.1 ml) were poured or spread in duplicate on different growth media. total viable counts were determined on plat count agar (pca) (accumix, belgium) after 48 h of incubation at 30°c; yeasts and molds on sabouraud agar (accumix, belgium) after 5 days of incubation at 25°c and coliforms on desoxycholate agar (accumix, belgium) after 24 h at 37°c for total coliforms and 44 °c for fecal coliforms (guiraud, 1998). 2.5. chemical composition sausage samples were dried by lyophilization (christ, beta 1-8 ld plus); samples of dry matter were ground (1 mm screen) and used for subsequent analyses. total protein was determined using the kjeldahl method (id 942.01) and ash content was determined by ashing at 600°c for 8 h (id 942.05) according to aoac (1990). water activity (aw) of the sausage samples was determined at 25°c using a thermoconstanter aw sprint novasina th500 (switzerland). ital. j. food sci., vol. 30, 2018 644 2.6. sensory analysis after 6 days of drying, sausages were cooled at room temperature, then cut into 1x1 cm pieces. each piece was then coded and served in random order to a sensory panel, which consisted of 13 panelists with experience in sensorial evaluation. each parameter in the sensorial analysis was evaluated on a scale of 1 to 9 (1low intensity; 9high intensity). color, odor, flavor, tenderness and overall acceptance were scored; then, mean values were calculated for each parameter. 2.7. statistical analyses each parameter was measured three times and then averaged. all data were analyzed using a general linear model anova with treatment (rosemary powder incorporation) and time as factors. duncan’s multiple range test was used to determine any significant difference between mean values, and evaluations were based on a significance level of p < 0.05. all statistical analyses were done using sas (2002) version 8.2. 3. results and discussion 3.1. chemical composition moisture, protein and ash contents of the ewe sausages after drying are presented in table 1. results showed that incorporating rosemary powder significantly increased (p < 0.05) moisture in the traditional ewe sausages. our results are in agreement with of those of jung et al. (2015). indeed, rosemary powder appeared to improve water retention due to its antioxidant activity (estevez and cava, 2006). the antioxidant activity of rosemary powder for protein degradation moderated the loss of sulfhydryl groups and the generation of carbonyl compounds and then maintained water holding ability (jung et al., 2015). other studies have reported that moisture of dried sausages could be affected by both processing method and processing time (dalmis and soyer, 2008). the addition of rosemary powder did not affect protein and ash content. the protein content of dried ewe sausages was the same as that found by kovačević et al. (2010) who showed that protein content of traditional dry sausages ranged between 26 and 53%. water activity (table 1), an important factor for microorganism growth, was not affected by the addition of rosemary powder, which contrasted with the results found by jung et al. (2015). table 1. effect of rosemary powder incorporation on chemical composition of dry ewe sausages. chemical composition c rp2 rp4 s.e.m p-value moisture (%) 15.7 18.5 20.7 1.22 0.001 protein (% dm) 39.7 40.4 41.2 5.48 0.977 ash (% dm) aw 7.8 0.75 7.7 0.72 8.3 0.78 0.47 0.01 0.590 0.450 c: control sausage; rp2: sausage added with 2 % of rosemary powder; rp4: sausage added with 4 % of rosemary powder; s.e.m: standard error of means. ital. j. food sci., vol. 30, 2018 645 3.2. microbiological analysis and ph results of the microbiological analysis of the sausages during drying are presented in table 2. counts of total viable and coliform increased significantly during drying and were significantly affected (p <0.05) by the addition of rosemary powder. at the end of the drying period, sausages with 4 % rosemary powder added showed the lowest value of total viable counts (4.17 log cfu/g) and total coliforms (2.18 log cfu/g). since there is no significant difference in water activity values between samples of dry ewe sausages, this result confirmed the antimicrobial effect of rosemary, widely seen when added directly to meat and meat products (ahn et al., 2007; angioni et al., 2004; georgantelis et al., 2007; jung et al., 2015). the antimicrobial properties of rosemary, especially on gram positive bacteria are related to some non-polar components such as phenolic diterpenes. furthermore, phenolic diterpenes could inhibit gram negative bacteria when in combination with factors which can disturb cell membrane permeability and/or integrity such as ph values and nacl concentrations (liu et al., 2009). yeast, mold and fecal coliform counts were not affected either by the drying period or by the addition of rosemary powder. table 2. populations of microbial groups (cfu/g) during the drying of traditional ewe sausages. batch days of drying 0 3 6 total viable counts c 4.39±0.352ax 5.35±0.387ax 6.07±0.889bx rp2 3.98±0.18ay 4.78±0.94by 5.38±0.98cy rp4 3.77±0.27ay 3.74±0.47ay 4.17±0.95by molds & yeasts c 3.38±0.74ax 4.14±0.89ax 5.27±0.72bx rp2 3.58±0.83ax 5.37±0.74by 5.36±0.98bx rp4 3.26±0.46 a x 3.51±0.85ax 5.22±0.65bx total coliforms c 3.81±0.32ax 4.03±0.24abx 4.31±0.85bx rp2 4.29±0.46ay 3.23±0.63by 3.11±0.51by rp4 4.16±0.59ay 2.43±0.20az 2.18±0.76bz fecal coliforms c 2.61±0.68ax 2.37±0.99ax 2.44±0.51ax rp2 2.81±0.72ax 2.74±0.62ax 2.95±0.30ay rp4 2.38±0.23ax 2.12±0.37ax 2±0.56ax c: control sausage; rp2: sausage added with 2 % of rosemary powder; rp4: sausage added with 4 % of rosemary powder; values are the mean of three replicates; a, b et c: means in the same line for the same treatment with a different letter differ significantly (p<0.05); x, y et z: means in the same column for the same day with a different letter differ significantly (p<0.05). the potential of hydrogen (ph) is usually ranked among the technological characteristics because it greatly influences meat processing and conservation. table 3 shows that incorporating rosemary significantly affected ph values every day of drying except the first. for the control samples, the ph remained stable between day 0 and day 3 of drying, then significantly increased to a value of 5.83 on day 6 (p <0.05). this rise can be attributed to the increase of total viable counts and total coliforms, which cause protein and amino acid degradation resulting in ammonia formation and consequently an increase in ph (georgantelis et al., 2007). for rp2 and rp4 sausages, ph values significantly decreased during the drying period (p < 0.05). these lower values are probably due to ital. j. food sci., vol. 30, 2018 646 sausage acidification, although no sugar was added to the mixture. rosemary is an herb rich in carbohydrates (deef, 2007) and during the drying process, these sugars are used by bacteria to produce lactic acids responsible for the drop in ph. this was not the case in the control sausage because no rosemary powder was added. 3.3. sausage color table 3 shows that l* values, expressing brilliance in color, decreased significantly during the drying period (p<0.05). kovačević et al. (2010) reported that this loss of clarity could be attributed to drying time. this decrease in l* values was in fact due to water loss (sanabria et al., 2004). the results indicated that adding rosemary did not have a significant effect on the lightness of dry sausages. table 3. effect of rosemary powder incorporation on the ph and color parameters of dry ewe sausages. parameters batch days of drying 0 3 6 ph c 5.59±0.13ax 5.62±0.06abx 5.83±0.07bx rp2 5.58±0.01ax 5.47±0.12bxy 5.41±0.1aby rp4 5.55±0.06ax 5.48±0.16aby 5.44±0.07by l c 47.93±4.82ax 38.36±5.04bx 37.68±5.02bx rp2 46.29±3.65ax 38.37±5.6bx 38.11±4072bx rp4 43.51±3.66ax 36.36±3.83bx 36.12±4041bx a* c 17.98±3.22ax 15.21±2.84abx 12.08±1.94bx rp2 7.19±1.74ay 7.37±1.4ay 7.03±1.37ay rp4 5.83±1.49ay 3.51±0.22by 3.82±0.31bz b* c 27.66±3.53ax 25.76±2.71abx 21.15±2.78bx rp2 28.63±3.19ax 23.51±2.68bx 22.82±3.39bx rp4 23.43±3.95ay 22.11±3.18by 19.12±1.78ax c c 33.06±4.04ax 30.14±4.04abx 24.71±3.58bx rp2 29.59±2.63ax 24.71±3.06by 23.92±3.13bx rp4 24.27±2.6ay 19.46±3.49bz 22.44±1.75ax h c 56.96±4.15ay 60.21±4.5az 59.81±5.34az rp2 75.80±3.71ax 72.79±4.54ay 72.57±3.71 ay rp4 75.87±3.48ax 79.71±2.65ax 80.12±1.18 ax c: control sausage; rp2: sausage added with 2 % of rosemary powder ; rp4:sausage added with 4 % of rosemary powder; values are the mean of three replicates; a, b, c: means in the same line for the same treatment with a different letter differ significantly (p<0.05); x, y, z: means in the same column for the same day with a different letter differ significantly (p<0.05). redness (a*) decreased significantly during the drying of control sausages, probably due to the oxidation of myoglobin to metmyoglobin (balentine et al., 2006). sausages with 2 % rosemary powder (rp2) showed no change in redness over time (p > 0.05), versus a decrease in sausages rp4 between day 0 and day 3 followed by stability until day 6 of the drying process. this stability may be attributed to the phenolic components of rosemary, which exert an antioxidant effect inhibiting the oxidation of myoglobin and thus maintaining the red color of samples (balentine et al., 2006; smeti et al., 2013). ital. j. food sci., vol. 30, 2018 647 liu et al. (2009) found that adding rosemary powder to fresh chicken sausage increased a* and decreased l values. similarly, georgantelis et al. (2007) explained that many factors such as differences in the oxidation pattern of myoglobin under conditions of reduced enzymatic activity, storage temperatures, packaging methods, muscle type and light intensity and differences in the meat spices studied might contribute to the variations in rosemary color retention efficiency between different studies. results showed that values of b* decreased significantly for all sausage samples during drying (p <0.05), but values stayed relatively high (>19). kovačević et al. (2010) reported that the higher b* values in the sausages are probably related to the presence of yellow carotenoids (ß-carotene and cryptoxanthin) from paprika. with their combined polyene chain, which acts as a chromophore, carotenoids are often responsible for the bright colors of certain fruits and vegetables. this chain is also responsible for the instability of the carotenoids against oxidation, light and heat, which may in turn. this instability of the carotenoids may be the cause of a decrease yellowness index during drying (chanforan, 2010). on the other hand, rosemary powder did not affect the b* values of sausages. on day 0, not all types of sausages exhibited the same degree of saturation; the control samples had the highest chroma when compared to samples rp2 and rp4. thereafter, the control samples saturation level dropped throughout the drying period. this continuous decline was not observed in sausages with rosemary added, thus demonstrating the effectiveness of rosemary by-products against myoglobin oxidation (camo et al., 2008). 3.4. sensory analysis color is one of the most important points in sensory evaluation, helping the consumer accept or reject particular foods. figure 1 showed that rosemary powder significantly affected sausage color (p <0.001). sausages with rosemary powder were darker, which could increase consumer sensory preferences. our results differed from those of liu et al. (2009) who found that the addition of rosemary decreased color preferences of fresh chicken sausages during refrigerated storage. figure 1. sensory evaluation of dry ewe sausages. c: control sausage ; rp2: sausage added with 2 % of rosemary powder ; rp4: sausage added with 4 % of rosemary powder. 0 2 4 6 8 10 color odor flavor tenderness acceptabilit y c rp2 rp4 ital. j. food sci., vol. 30, 2018 648 figure 1 shows that incorporating rosemary significantly affected the odor of the sausages (p < 0.05). in fact, aromatic substances allowed tasters to distinguish between samples. similarly, it has been documented that certain compounds found in rosemary (verbenone, borneol, camphor) may give food a specific odor, even at low concentrations (carrillo et al., 2006). likewise, incorporating rosemary significantly affected sausage flavor (p < 0.05). however, sausage tenderness was similar between groups (p > 0.05). in addition, rosemary significantly affected the acceptability of dry sausages (p < 0.05) in favor to rp2 samples, which were the most appreciated by panelists. these results agreed with previous investigations showing a beneficial effect of rosemary by-products on the sensory quality of ewe meat products (djenane et al., 2002; djenane et al., 2003; balentine et al., 2006). 4. conclusions the effect of rosemary powder on the microbiological, chemical and sensory qualities of dry ewe sausages was investigated. the results obtained showed that rosemary powder added at 2 and 4 % improved microbiological quality by decreasing total viable and total coliform counts. similarly, moisture and ph were affected by adding rosemary powder. therefore, sausages added with 2 % of rosemary powder were the most appreciated by the panelists. this level of inclusion could be of interest for ewes’ sausage preparation. references ahn j., grun i., and mustapha a. 2007. effects of plant extracts on microbial growth, color change, and lipid oxidation in cooked beef. food microbiology 24:7-14. angioni a., barra a., cereti e., barile d., coïsson j. d., arlorio m., dessi s., coroneo v. and cabras p.c. 2004. chemical composition, plant genetic differences, antimicrobial and antifungal activity investigation of the essential oil of rosmarinus officinalis l. journal of agriculture and food chemistry 52:3530-3535. aoac. 1990. “association of official analytical chemists, official methods of analysis”, 15th ed. association of official analytical chemists, arlington, va, usa. balentine c.w., crandall p.g., o’bryan c.a., duong d.q. and pohlman f.w. 2006. the pre-and post-grinding application of rosemary and its effects on lipid oxidation and color during storage of ground beef. meat science 73:413421. camo j., beltrán j.a. and roncalés p. 2008. extension of the display life of lamb with an antioxidant active packaging. meat science 80:1086-1091. carrillo j.d. and tena m.t. 2006. determination of volatile compounds in antioxidant rosemary extracts by multiple headspace solid-phase microextraction and gas chromatography. flavour and fragrance journal 21:626-633. chanforan c. 2010. stabilité de microconstituants de la tomate (composés phénoliques, caroténoïdes, vitamines c et e) au cours des procédés de transformation: études en systèmes modèles, mise au point d'un modèle stoechio-cinétique et validation pour l’étape unitaire de préparation de sauce tomate. thèse de doctorat. université d’avignon et des pays de vaucluse, p. 399. colomer-rocher f., dumont b.l. and murillo n.l. 1972. descripción del despieceovino aragonés y definición de un despiece de referencia normalizado anales del instituto nacional de investigaciones agrarias, serie producción animal 3:79-108. dalmis u. and soyer a. 2008. effect of processing methods and starter culture (staphylococcus xylosus and pediococcus pentosaceus) on proteolytic changes in turkish sausages (sucuk) during ripening and storage. meat science 80:345-354. deef h.e. 2007. copper treatments and their effects on growth, carbohydrates, minerals and essential oils contents of rosmarinus officinalis l. world agriculture sciences 3:322-328. ital. j. food sci., vol. 30, 2018 649 del campo j., amiot m.j. and nguyen-the c. 2000. antimicrobial effect of rosemary extracts. journal of food protection 63:1359-1368. djenane d. sanchez-escalante a. beltrana j.a. and roncales p. 2003. extension of the shelf life of beef steaks packaged in a modified atmosphere by treatment with rosemary and displayed under uv-free lighting. meat science 64:417-426. djenane d., sanchez-escalanteb a., beltrana j.a., and roncales p. 2002. ability of a-tocopherol, taurine and rosemary, in combination with vitamin c, to increase the oxidative stability of beef steaks packaged in modified atmosphere. food chemistry 76:407-415. estevez m. and cava r. 2006. effectiveness of rosemary essential oil as an inhibitor of lipid and protein oxidation: contradictory effects in different types of frankfurters. meat sciences 72:348-355. garcía-díez j., alheiro j., pinto a.l., soares l., falco v., fraqueza m.j. and patarata l. 2016. behaviour of food-borne pathogens on dry cured sausage manufactured with herbs and spices essential oils and their sensorial acceptability. food control 59 262-270. georgantelis d., ambrosiadis i., katikou p., blekas g. and georgakis s.a. 2007. effect of rosemary extract, chitosan and α-tocopherol on microbiological parameters and lipid oxidation of fresh pork sausages stored at 4°c. meat science 76:172-181. guiraud j.p. 1998. microbiologie alimentaire, microbiologie des principaux produits laitiers. dunod-ed, paris, 65. hunt m.c., acton j.c., benedict r.c., calkins c.r., cornforth d.p., jeremiah l.e., olson d.g., salm c.p., savell j.w. and shivas, s. d. 1991. amsa guidelines for meat color evaluation. in: proceedings 44th annual reciprocal meat conference (3-17; 9-12 july, kansas state university, manhattan, ks). jung j.h., shim k.s. and shin d. 2015. effects of ripening duration and rosemary powder addition on salchichon modified sausage quality. asian-australasian journal of animal sciences 28:671-676. kovačević d., mastanjević k., šubarić, d., jerković, i. and marijanović z. 2010. physico-chemical, colour and textural properties of croatian traditional dry sausage (slavoniankulen) meso, rujan-listopad, 12. liu d.c., tsau r.t., lin y.c., jan s.s. and tan f.j. 2009. effect of various levels of rosemary or chinese mahogany on the quality of fresh chicken sausage during refrigerated storage. food chemistry 117:106-113. lund m.n., hviid, m.s. and skibsted l.h. 2007. the combined effect of antioxidants and modified atmosphere packaging on protein and lipid oxidation in beef patties during chill storage. meat science 76:226-233. mccarthy t.l., kerry j.p., kerry j.f., lynch p.b. and buckley d.j. 2001. evaluation of the antioxidant potential of natural food/plant extracts as compared with synthetic antioxidants and vitamin e in raw and cooked pork patties. meat science 58:45-52. mielnik m.b. aaby j. and skrede g. 2003. commercial antioxidants control lipid oxidation in mechanically deboned turkey meat. meat science 65:1147-1155. miltenburg g.a., wensing t., smulders f.j. and breukink h.j.1992. relationship between blood hemoglobin, plasma and tissue iron, muscle heme pigment, and carcass color of veal. journal of animal science 70:2766-2772. pizzocaro f., senesi e. and babbini g. 1994. effettoprotettivo di salvia e rosmarinofreschisu hamburger surgelati di carne bovina. industrie alimentari 33:289-294. sanabria c., martin-alvarez p.j. and carrascosa a.v. 2004. colour and moisture changes during the manufacture of iberian dry-cured ham caused by some biotic and abiotic factors. food science and technology international 10:269-275. sas. 2002. users guide, statistical analysis systems institute, version 8.2. cary, nc, usa: inc. smeti s., atti n., mahouachi m. and munoz f. 2013. use of dietary rosemary (rosmarinusofficinalis l.) essential oils to increase the shelf life of barbarine light lamb meat. small ruminant research 113:340-345. stephanie a.c., graham r.t., frank r.d. and nagendra p.s. 2002. antioxidant effects of rosemary extract and whey powder on the oxidative stability of wiener sausages during 10 months frozen storage. meat science 62:217-224. paper received july 9, 2017 accepted may 25, 2018 ijfs#960_bozza ital. j. food sci., vol. 30, 2018 317 paper sensory optimisation in new food product development: a case study of polish apple juice m. halagarda* and g. suwała department of food commodity science, cracow university of economics, 30-033 cracow, sienkiewicza 5, poland *e-mail address: michal.halagarda@uek.krakow.pl abstract a large variety of juices representing a wide range of sensory profiles can be found on the market. nevertheless, their manufacturers may not fully recognise the needs and demands of consumers. the aim of the study was to verify a new strategy for consumer product testing using apple juice as an example. the material comprised of 14 apple juices. the sensory analysis was carried out by a team of 130 assessors. the impact of individual physicochemical parameters on the results of the juice sensory assessment was tested with the use of generalised additive models (gam). the results of the research indicate that the most preferred apple juice is characterised by a balanced sweet and sour taste, low density as well as the colour described by a compromise between the high value of l* and low values of a* and b* parameters and a relatively low price. after the analysis of the map of preferences and its comparison with the results of the sensory assessment, it can be concluded that the products tested do not fully meet the consumer expectations. the research adds new insight into knowledge on new food product development. it shows that by examining the sensory desirability of products available on the market and by employing the gam analysis the characteristics of the most appreciated product can be determined. keywords: new food product development, apple juice, new product, consumer preference ital. j. food sci., vol. 30, 2018 318 1. introduction along with the technical and technological progress in the world, consumer needs and expectations are likewise gradually changing. for this reason, from the point of view of food industry companies, it is very important to continuously develop concepts for new products. in this way, they can quickly adapt to new market conditions and meet the needs of their customers. the consumer is more often seen not only as a user of products, but as a decision-maker on the market, and the traditional view of the consumer buying behaviour being associated with the assumptions of economic rationality has been replaced by behavioural theories of choice and preferences (halagarda, 2017; sagan 2011). rising competition and customer expectations have led juice producers to develop a range of technologies in order to improve production and make their goods more attractive (kudełka and głuszek, 2014). as a result, a large variety of juices can be found on the market: raw and not from concentrate, both cloudy and clear, as well as juices from concentrate, or traditional and organic products (kudełka and głuszek, 2014). in this respect, products that are commercially available represent a wide range of sensory profiles. nevertheless, their manufacturers may not fully recognize the needs and demands of consumers (moskowitz et al., 2006). according to fuller (2011), the results of the evaluations of the products available on the market should be used as an inspiration when generating ideas for new products that better correspond to the changing needs and expectations of consumers. cooper (2001) and lord (2008) state that competitive products are a valuable source of ideas for new products. based on competitive analysis, the data on the position of the product on the market and characteristics of products and the benefits they offer to customers as well as their weak and strong points can be determined (rudder, 2003). this in turn may allow to formulate the necessary criteria to be fulfilled by a newly developed product (cooper, 2001). it is, however, important to consider consumer needs at each stage of the product development or improvement process (van kleef et al., 2005; bogue and sorenson, 2008). nevertheless, consumer tests are paramount in their early stages and in the assessment of the product before its commercialisation. their execution involves significantly lower costs than those that may be incurred in connection with the failure of a new product (maltz and kohli, 1996). according to mattsson and helmersson (2007), the participation of consumers in the initial phase of the new food product development is one of the three factors determining market success. the unsatisfied needs of consumers create opportunities for the development of new products (lord, 2008). fulfilling expectations, especially sensory ones, leads to consumer satisfaction and increases the likelihood of product’s marketability (grunert, 2002). the obtained information may also reveal market niches. products developed to fill these niches have greater chances of success. moreover, if the consumer reports the demand for a particular food product, the risk of failure significantly decreases (andersen and munksgaard, 2009). the choice of a particular method or a research tool is not a simple task. despite the fact that the aim is to provide information that can be used to determine the parameters of the product and estimate the level of consumer satisfaction with the new product, the processes of data collection can differ and, accordingly, the respondents can articulate their needs and requirements in different ways. in addition, the purpose behind research is of high importance. the company's goals may involve, for example, an introduction of the product to existing markets or creation of new markets. more reliable research results are achieved when consumers can appeal to their experience with the products already ital. j. food sci., vol. 30, 2018 319 available on the market. the data achieved may provide information about the product benefits that are anticipated by consumers. however, if competitors fully recognize these anticipations, a reliance on such data may result in imitation products being developed (van kleef et al., 2005). physicochemical tests and/or descriptive analyses are used to characterise competitive products. consequently, data on sensory characteristics of the products are compared with their physicochemical parameters, and the degree of their consumer acceptance. (moskowitz et al., 2006; moskowitz et al., 2008). the aim of the study was to verify a new strategy for consumer product testing using apple juice as an example. to achieve this goal, the following hypothesis was set up: a comparison of the features desired by consumers with the results of physicochemical analyses constitutes valuable input data for the development process of a new product. 2. materials and methods 2.1. juice samples the research material comprised of 14 apple juices, selected from those available on the polish market, and divided into 3 groups. the first group was formed of five not form concentrate juices that were registered on the list of traditional products of the polish ministry of agriculture and rural development. the second group comprised of six conventional not from concentrate juices, of which: four were obtained in the pressing process, one via squeezing, whereas the sixth product had no declaration of the manufacturing technology. the last group consisted of three juices made from concentrate and manufactured by leading polish producers. for the purpose of sensory analysis and in accordance with the literature guidelines (baryłko-pikielna and matuszewska, 2009; iso 6658:2005), the juice samples were coded using 3 random digits. detailed data is shown in table 1. 2.2. sensory analysis the desirability analysis of the apple juices was carried out by a team of 130 assessors all regular consumers of the category. consumer tests were conducted in a sensory laboratory that was designed in accordance with the iso 8589:1988 standard. evaluations were performed under artificial daylight and at room temperature (controlled between 22 and 24°c) and with the use of recirculation air system. the consumers were given the 50 ml samples of the juices in glasses alongside plain water to prevent carryover of the taste of the former samples. a rest period of at least 30 seconds was scheduled between each sample test. data were collected through self-administered questionnaires previously explained to consumers. the desirability of colour, consistency, palatability, sweet palatability, sour palatability and the whole product were determined with the use of 9point hedonic scale and according to the iso 11136:2014 standard. in the first stage the consumers were given the samples for evaluation without knowing the price. additionally, both the internal (sensory attributes and physicochemical properties) and external (price) factors were taken into account in the assessment of the quality and desirability of the product (menichelli et al., 2012). therefore, in the second stage, the consumers were asked to verify their overall rating when given the price of the product they assessed. ital. j. food sci., vol. 30, 2018 320 table 1. the coding and prices of juice samples. no. juice price, [pln/l] code 1 nfcj – fresh, squeezed 10,00 251 2 fcj 4,24 423 3 fcj 3,79 513 4 nfcj – cold pressed 2,65 426 5 fcj 3,33 754 6 nfcj – pressed 4,27 896 7 rnfcj – cold pressed 7,40 712 8 rnfcj – cold pressed 5,33 381 9 rnfcj – pressed 6,30 219 10 nfcj – pressed 5,00 101 11 nfcj 3,50 411 12 rnfcj – cold pressed 6,00 129 13 nfcj – cold pressed 6,10 613 14 rnfcj – cold pressed 11,77 147 rnfcj not-from-concentrate juice, registered on the list of traditional products of the polish ministry of agriculture and rural development nfcj not-from-concentrate juice fcjjuice from concentrate 2.3. physicochemical analyses four juices from the same production batch that was later used in the consumer assessment were tested. all analyses were performed in triplicate for each juice sample. the analyses covered the following: 2.3.1 density the density expressed in grams per 100 ml and converted to kg/l was determined using the pycnometer method by measuring the mass of the determined sample volume at 20±0.5°c and equating it to the mass of the same volume of reference liquid (distilled water) 20±0.5°c (pn-en 1131:1999). 2.3.2 titratable acidity titratable acidity was determined using 25 ml of a sample. the solution was continuously stirred by a magnetic stirrer and titrated against standardized solution of 0.1 mol naoh to the end point (ph 8.1) using mettler toledo mp225 basic ph/mv/°c meter. the titratable acidity was expressed as mmol h+/l and calculated according to the following equation: 𝐶 = 1000×𝑉!×𝑐 𝑉! (where, v0: the volume of the sample in ml; v1: the volume of naoh used for sample titration in ml; c: the exact concentration of naoh in mol/l). the titratable acidity was also converted to amount of an anhydrous citric acid (gram per 1000 ml) of juice with the use of the following equation: ital. j. food sci., vol. 30, 2018 321 𝐶!" = 𝐶×0.064 (where, c: titratable acidity in mmol h+/l) (pn-en 12147:2000). 2.3.3 total sugars for the determination of total sugars, 25 ml of the test sample was transferred to a flask of 250 ml. 25 ml of distilled water was added and the sample was deproteinized with carrez i and ii mixture. then the sample was filtered through a paper filter. 25 ml of filtrate was taken into a 100 ml flask. subsequently, an inversion was performed with the use of 25 ml of distilled water, 5 ml of concentrated hcl and in the temperature of 6870°c. the sample was cooled, neutralized and the contents of the flask were filled up to 100 ml with distilled water. the resulting solution was used as a titrant against the mixture of fehling i and ii solutions (5 + 5 ml) with methylene blue as the end-of-reaction indicator. based on the amount of used solution, the total sugars content in the test sample was determined (pn-90/a-75101/07). 2.3.4 colour the colour was mapped by measuring l* a* b* parameters in the ciel*a*b* system (mclaren, 1976) with the use of minolta cm-3500d spectrophotometer. 2.3.5 sugars/acids ratio sugars/acids ratios were calculated according to the equation: 𝑆𝐴𝑅 = 𝑆 𝐴 (where, sar: sugar acids ratio; s: total sugars; a: titratable acidity expressed as an anhydrous citric acid). 2.4. statistical analysis the data obtained from the research were analysed with the use of r 3.2.2. statistical package supplied by r foundation for statistical computing (r core team, 2017). the impact of individual physicochemical parameters on the result of the juice sensory assessment was tested with the use of generalized additive models (gam) (wood, 2017). dimension reduction aimed at the implementation of the juices’ preference map was made using the first two principal components. juices were divided into groups using agglomerative hierarchical clustering (dendrogram) with a complete linkage and euclidean distance as a measure of the juices similarity. before the development of the dendrogram, the physicochemical parameters were standardised in order to have a mean of 0 and a standard deviation equal to 1. in this way each of the physicochemical parameters had the same contribution to the euclidean distance between the two juices. all of the tests were conducted at a significance level of α=0.05. in order to verify the degree of fulfilment of consumer expectations by traditional and conventional apple juices available on the polish market, a preference map was developed. each of the analysed juices was described by 8 parameters (colour parameters: l*, a*, b*, ital. j. food sci., vol. 30, 2018 322 density, sugar/acid ratio, content of sugars, titratable acidity and price). due to the difficulties in constructing an 8-dimensional map, it was decided that a two-dimensional one would be developed. 8 parameters were transformed into 2 that best described the tested juices. for this purpose, the principal component analysis (pca) was used. two components that explain most of the variance were selected. due to the different scales of selected variables, they were all standardised. 3. results and discussion 3.1. the results of sensory and physicochemical tests sensory evaluations are used when developing new products and improving the characteristics of the products available on the market. however, due to difficulties arising from the nature of the sensory evaluation they are unfortunately seldom used by small and medium sized food companies (carbonell-barrachina, 2007). instrumental methods certainly provide objective data, but because of sensory analyses more comprehensive knowledge about the characteristics of products perceived by the senses can be provided (murray et al., 2001; carbonell et al., 2008). the results of the sensory desirability assessment of apple juices are presented in table 2. they prove that the chosen research material showed variability in respect of consumer preferences of selected apple juice characteristics. the highest ratings for colour were achieved by two juices from concentrate (coded 513 and 754), whereas the lowest ratings by traditional not-from-concentrate juices (coded 381 and 219). consumers did not have any major objections to the consistency of the juice samples tested. they mostly appreciated the consistency of fresh juice (sample 251), traditional not-from-concentrate juices (sample 712) and juice from concentrate (sample 754). the apple juices samples received mostly high rates for palatability and sour palatability as well. consumers were highly satisfied with two juices: fresh (coded 251) and from concentrate (coded 423). the lowest rank was given to traditional not-from-concentrate juice (sample 129). similarly, sweet palatability was highly valued in most of the cases. again the exception was sample 129. the highest ranks were given to fresh juice (sample 251), juice from concentrate (sample 423) and traditional not-from-concentrate juices (sample 712). considering overall rating, three juices were most appreciated by consumers: fresh juice (coded 251) and two juices from concentrate (coded 423 and 513). the lowest rating was received by traditional not-from-concentrate juice (coded 129). considering the price of juice in overall rating two traditional not-from-concentrate juices were barely acceptable by consumers (samples coded 129 and 147). the best ratings were achieved by two juices: fresh (coded 251) and from concentrate (coded 423). the results of overall sensory acceptability ratings are in most of the cases similar to those presented in the literature. aguiar et al. (2012) obtained average ratings for consumer apple juices at the level of 5.5-6.0 points in a 9-point scale. włodarska et al. (2016) received overall acceptance ratings ranging from 4.1.to 6.3 also in a 9-point scale. lee et al. (2016) obtained significantly higher scores of the experienced quality of the coded samples of freshly squeezed apple juices compared to juices pasteurised with the use of different technological methods. similarly in this study the fresh juice (sample 251) received the highest overall rating, when omitting its price. therefore, the results of sensory analysis conducted in this study and similar tendencies presented in the literature prove proper selection of the research material. ital. j. food sci., vol. 30, 2018 323 table 2. the results of the consumer sensory desirability assessment of 14 apple juices selected for testing. juice colour consistency palatability sweet palatability sour palatability overall rating overall rating including the price χ(s.d) χ(s.d) χ(s.d) χ(s.d) χ(s.d) χ(s.d) χ(s.d) 251 5.54 (1.69) 6.46 (1.34) 6.92 (1.21) 6.92 (1.00) 6.23 (1.67) 6.77 (0.89) 5.23 (1.62) 423 5.38 (1.33) 5.62 (2.10) 5.92 (1.38) 5.92 (1.49) 6.46 (1.22) 6.08 (1.27) 6.92 (1.21) 513 7.38 (1.39) 5.92 (1.86) 5.54 (1.82) 5.92 (1.64) 5.85 (2.21) 6.00 (1.71) 6.38 (1.15) 426 5.31 (1.32) 5.46 (1.65) 4.85 (2.03) 5.08 (1.98) 4.77 (1.72) 5.15 (1.96) 5.85 (2.11) 754 7.31 (1.77) 6.23 (2.04) 4.46 (2.13) 4.77 (1.89) 5.31 (1.98) 5.31 (2.09) 5.54 (1.78) 896 6.23 (1.58) 5.38 (1.64) 5.38 (1.50) 5.54 (1.50) 5.77 (1.42) 5.85 (1.35) 5.31 (1.26) 712 6.08 (1.49) 6.46 (1.50) 5.23 (2.45) 6.23 (2.42) 5.54 (2.59) 5.77 (2.08) 4.85 (2.41) 381 3.15 (2.18) 5.38 (1.69) 5.38 (1.60) 4.31 (1.59) 4.85 (1.61) 4.69 (1.38) 3.62 (1.60) 219 3.69 (1.73) 5.46 (1.28) 4.00 (2.29) 4.92 (1.86) 4.62 (2.31) 4.08 (1.64) 4.00 (2.04) 101 5.00 (1.47) 6.15 (1.23) 4.77 (1.97) 5.08 (1.21) 4.46 (1.82) 5.15 (1.17) 4.69 (1.38) 411 5.08 (1.90) 6.46 (1.45) 4.00 (2.80) 5.38 (2.17) 4.54 (2.59) 4.62 (2.65) 5.46 (2.76) 129 5.85 (1.46) 4.62 (1.90) 2.31 (1.43) 2.85 (1.51) 3.00 (1.96) 2.62 (1.44) 2.77 (1.76) 613 5.00 (1.75) 5.38 (1.78) 4.15 (1.56) 5.31 (1.49) 4.54 (2.06) 4.54 (1.22) 4.15 (1.10) 147 4.62 (2.10) 5.00 (1.36) 3.92 (1.86) 4.62 (2.24) 4.08 (1.54) 4.08 (1.64) 2.85 (1.61) s.d. standard deviation. table 3 contains the results of the physicochemical examinations used as a reference for the results of sensory desirability evaluations. they prove the typicality of the products used for the research (aijn, 2007). so far, there have only been a handful of studies on the quality of apple juices. rødbotten et al. (2009) and jaros et al. (2009) showed that, as it is in the case of apples (daillant-spinnler et al., 1996; jaeger et al., 1998), for apple juices sweet and sour palatability are the most important parameters affecting consumer ratings. the sugar content in the product is a measure of the sweet palatability (magwaza and opara 2015; bett-garber et al., 2014). the strong correlation between sour palatability and titratable acidity was also proved by various researchers (corollaro et al., 2014; bonany et al., 2014; van der merwe et al., 2015). jaros et al. (2009), on the basis of principal component analysis for the instrumental data and procrustes analysis for sensory data, demonstrated that the key attributes determining the consumer ratings of juices are: sweetness and acidity of the juice, as well as sweet/sour relation, the cloudiness of the juice and its colour. in this study titratable acidity was chosen as a measure of sour palatability and the total sugar concentration as a measure of sweet palatability. three juices tested were characterised by the highest total sugars content: two traditional juices (coded 712 and 147) and a pressed not-from-concentrate juice (coded 101). the lowest concentrations of total sugars were detected in two regional not-from-concentrate juices (coded 129 and 219). two of the juices tested had the highest titratable acidity: juice from concentrate (coded 513) and not from concentrate juice (coded 411). the lowest acidity characterized two not-from-concentrate juices (coded 896 and 426). the extract to acidity ratio is often used as a measure of the maturity of raw materials and palatability of processed fruits (magwaza and opara, 2015). however, due to the importance of acidity and sweetness, in the case of apple juice, confirmed by rødbotten et al. (2009) and jaros et al. (2009), the total sugars to acids expressed as an ital. j. food sci., vol. 30, 2018 324 anhydrous citric acid ratio was chosen as a reference parameter for the overall palatability. the highest value of sugars/acids ratio characterised not-from-concentrate juice (coded 896), whereas the lowest regional not-from-concentrate juice (coded 219), juice from concentrate (coded 513) and not-from-concentrate juice (coded 411). table 3. the results of physicochemical examinations, chosen for comparison with the results of sensory analysis, of 14 apple juices selected for testing. juice colour consistency palatability sweet palatability sour palatability l* χ(s.d) a* χ(s.d) b* χ(s.d) density, [kg/ll] χ(s.d) sugars/acids ratio χ(s.d) total sugars, [g/ll] χ(s.d) titratable acidity, [mmol h+/lmval] χ(s.d) 251 69.49 (0.05) 1.91 (0.04) 26.46 (0.06) 1.043 (0.001) 25.01 (0.15) 95.22 (0.54) 59.5 (0.6) 423 93.98 (0.02) -1.03 (0.02) 24.64 (0.03) 1.046 (0.001) 23.99 (0.12) 101.31 (0.49) 66.0 (0.0) 513 88.09 (0.05) 2.56 (0.02) 40.84 (0.01) 1.046 (0.001) 18.81 (0.16) 100.495 (0.22) 83.5 (0.4) 426 41.68 (0.30) 12.71 (0.15) 49.37 (0.12) 1.049 (0.001) 42.81 (0.73) 109.596 (0.73) 40.0 (0.4) 754 93.56(0.03) -0.89 (0.01) 26.74 (0.05) 1.048 (0.001) 22.49 (0.18) 105.091 (0.83) 73.0 (0.6) 896 50.92 (0.22) 11.65 (0.23) 52.42 (0.15) 1.048 (0.001) 57.37 (0.72) 104.65 (0.65) 28.5 (0.8) 712 91.19 (0.13) -0.59 (0.05) 23.64 (0.09) 1.052 (0.000) 30.09 (0.58) 117.495 (0.50) 61.0 (0.4) 381 58.74 (0.04) 7.20 (0.01) 34.02 (0.04) 1.050 (0.001) 25.49 (0.14) 103.61 (0.24) 63.5 (0.4) 219 55.34 (0.10) 5.64 (0.10) 26.10 (0.38) 1.045 (0.001) 16.76 (0.03) 81.51 (0.15) 76.0 (0.4) 101 58.73 (0.13) 7.79 (0.03) 36.62 (0.06) 1.048 (0.001) 29.01 (0.39) 115.10 (0.33) 62.0 (0.0) 411 58.59 (0.04) 3.96 (0.02) 37.14 (0.01) 1.047 (0.000) 19.08 (0.01) 98.32 (0.57) 80.5 (0.6) 129 87.13 (0.17) -0.50 (0.05) 29.26 (0.16) 1.105 (0.001) 23.51 (0.27) 77.50 (0.23) 51.5 (0.8) 613 54.91 (0.11) 7.40 (0.10) 32.31 (0.33) 1.049 (0.001) 33.62 (0.08) 101.13 (0.23) 47.0 (0.4) 147 57.34 (0.18) 10.88 (0.05) 44.34 (0.03) 1.112 (0.001) 33.39 (0.21) 118.61 (0.56) 55.5 (0.4) s.d. standard deviation. the density was selected as a measure of juice consistency. the tested apple juice samples showed similar density. the exceptions were two regional not-from-concentrate juices (coded 147 and 129), which had the highest densities. the l* a* b* parameters mapped the colour of the apple juices tested. the highest value of l* parameter and therefore the highest lightness characterised two juices from concentrate (coded 423 and 754) and one traditional not-from-concentrate juice (coded 712). sample 426 (not-from-concentrate juice) had the lowest value of this parameter among all of the tested products. this sample on the other hand, had the highest values of the a* parameter (the highest share of green shade). the lowest values of a* parameter characterised juices from concentrate (coded 423 and 754). samples coded 896 and 426 (not-from-concentrate juices) had the highest values of b* parameter indicating yellow colour saturation. due to the fact that the composition of juice aroma is influenced by many chemical substances (mainly esters, c6 alcohols and aldehydes) (stangl and ziegler, 2014) research concerning this issue was omitted. nevertheless, an independent project on the subject is planned. ital. j. food sci., vol. 30, 2018 325 3.2. comparison of apple juices: similarities to check the similarity of the analysed samples of juices, a hierarchical cluster analysis was performed. the physicochemical parameters, which constituted a reference point for the results of the desirability evaluations, and the prices of the analysed juices were considered. the results were compared with the results of the sensory assessments (fig. 1). figure 1. clusters separated for the analysed samples of apple juice. on the basis of the dendrogram, five product clusters were distinguished. the juices of clusters: 1 and 3 were characterised by high consumer desirability. sample 129 shows a similarity to cluster 3 in terms of its physicochemical properties. despite the preferred characteristics of the product, it received a low score in consumer test. this was a result of the poor palatability. the consumers’ comments prove that the product was characterised by a peculiar insipid taste of negative associations, despite a rather good balance between the acid and sweet taste. however, volatile compounds were not analysed in the present study, and therefore, it was impossible to refer consumer opinions to specific physicochemical parameters. the last two clusters (4 and 5) comprised of products of medium to low consumer desirability. 3.3. an attempt to determine the parameters of the most desirable apple juice based on the parameters of juices available on the polish market the colour of the product significantly affects its perception. according to spence (2015), there is a strong relationship between colour and perception of the type and intensity of flavour. the chart (fig. 2) presenting the relationship between the value of the colour parameter l* and the consumer desirability is u-shaped. this means that the most preferred are the extreme values. the rating of the juices increases slightly faster on the right side of the graph. the ideal value of the l* parameter for apple juice determined by the gam method and based on the analysis of the research material was 93.89. therefore, it was found that consumers mostly prefer light coloured apple juices. however, the left part of the chart ital. j. food sci., vol. 30, 2018 326 indicates that there is a group of consumers who are in favour of a juice of lower lightness. this was also confirmed by the research of włodarska et al. (2016), in which a segment of consumers who prefer juices with low values o l* colour coordinate was identified. figure 2. the relationship between the colour parameter l* and consumer desirability of the apple juice colour determined with the use of gam model. the relationship between the colour parameter a* and the consumer desirability is linear (fig. 3). the lower the value of a* parameter the higher the desirability of the product. therefore it was found that consumers prefer apple juices of less perceptible greenish shade. the best value of the a* parameter for apple juice determined by the gam method and based on the analysis of the research material was -1.03. figure 3. the relationship between the colour parameter a* and consumer desirability of the apple juice colour determined with the use of gam model ital. j. food sci., vol. 30, 2018 327 the relationship between the value of the colour parameter b* and consumer desirability of the analysed apple juices is exponential (fig. 4). the higher the value, the greater the consumer acceptance of the juice. the respondents preferred a more saturated colour, with a more pronounced share of yellow tone. the application of the gam method made it possible to determine the most desirable value of the parameter b* within the range restricted by the parameters of the research material. it amounted to 52.42. the research results by czarnowska et al. (2014) also confirm that consumers prefer an intensive dark yellow colour of apple juice. figure 4. the relationship between the colour parameter b* and consumer desirability of the apple juice colour determined with the use of gam model according to the linear chart (fig. 5), showing the relationship between the density of apple juice and its consumer desirability, the lower the density of apple juice, the more the beverage is favoured by consumers. application of the gam method made it possible to determine the most appropriate density 1.043 kg/l. it was the lowest density among the identified values. therefore, it can be stated that consumers prefer clear juices. this conclusion is consistent with the data presented in the 2014 report of the european fruit juice association. according to it, 365 out of the 673 million litres of juices drunk by poles in 2013 were made on the basis of concentrate (anonymous, 2014). the not-fromconcentrate juices are not clarified by polish producers so as not to lower their nutritional value. the research by czarnowska et al. (2014) also confirms preferences for clear juices. on the other hand, włodarska et al. (2016) recognized consumer segment which showed preference for juices of higher density identified with cloudy juices which are believed to have greater health benefits. the relationship between the sugar/acid ratio and consumer desirability is linear (fig. 6). the higher the ratio, limited by the characteristics of the analysed samples, the better the rating the juice received. the most preferred value of sugar/acid ratio was 57.37. jaros et al. (2009) noticed that, when considering cloudy juices, the most preferred products had low sugar/acid ratio. they, however, indicated that there is an optimum of this ratio. therefore, in the range limited by samples analysed in this study the preferences may be rising with the increasing sugar/acid ratio. ital. j. food sci., vol. 30, 2018 328 figure 5. the relationship between density and consumer desirability of the apple juice consistency determined with the use of gam model figure 6. the relationship between sugars/acids ratio and consumer desirability of the apple juice palatability determined with the use of gam model according to research by jaros et al. (2009), the majority of the surveyed consumers preferred the juice, which was more acidic and less sweet. however, their research also demonstrated the existence of a large group of consumers who prefer less acidic, sweeter juices. similarly, rødbotten et al. (2009) showed variability of apple juice acidity preference between analysed consumer segments. this is in accordance with the results of this study, showing that the relationship (fig. 7) between the titratable acidity and the consumer desirability of apple juice is complex. the ideal acidity for apple juice, determined in this research with the use of the gam model based on the analysis of the research material, was 83.5 mmol h+/l. ital. j. food sci., vol. 30, 2018 329 figure 7. the relationship between titratable acidity and consumer desirability of sour palatability of apple juice determined with the use of gam model the chart (fig. 8) presenting the relationship between the sugar content and the consumer desirability is in the form of an inverted u. the respondents, to a certain degree, prefer the sweet taste of juice. in the present study the limit was the sugar content of 100.83 g/l of juice. this is in accordance with jaros et al. (2009) findings. in their research a correlation between consumer ratings and sweet palatability was found. they noticed that there is a sweetness intensity level limiting acceptance of an apple juice. similarly, rødbotten et al. (2009), showed consumer preference for apple juices of a high sugar content, noticing that there might be an upper limit of sweetness. figure 8. the relationship between sugar content and consumer desirability of sweet palatability of apple juice determined with the use of gam model. the relationship between the price of juice and its consumer desirability is non-linear (fig. 9). some respondents, most likely, identify the high quality of juice with its high price. however, at a given quality level, below which none of the tested products were found, ital. j. food sci., vol. 30, 2018 330 products with lower prices are generally preferred. these findings are in accordance with the results of shirai (2015) research. on the basis of the gam analysis, it was determined that most respondents prefer the lowest price of the presented alternatives 2.65 pln/l juice. figure 9. the relationship between the price and consumer desirability of apple juice determined with the use of gam model. based on the results of the gam analysis on 14 samples of apple juices available on the polish market it can be concluded that the products most preferred by the respondents should have the following colour parameters: l*=93.98, a*=-1.03, b*=52.42, density of 1.043 kg/l, sugar content equal to 100,827 g/l, titratable acidity of 83.5 mmol h+/l, sugar/acid ratio equal to 57.37 and the price of 2.65 pln/l or less. 3.4. the preference map of apple juices available on the polish market, with consideration of the ideal juice concept two principal components determined by pca explain 66.25% of the variance of the whole data set, thus the obtained 2-dimensional map uses 66.25% of the information contained in the 8 original parameters. the exact patterns of the components were as follows: first component: 0.4·l* 0.48·a* 0.44·b* 0.06·density 0.45·sugar/acid ratio 0.22· sugar content + 0.4·titratable acidity 0.04·price second component: 0.04·l* 0.01·a* 0.12·b* + 0.69·density 0.08·sugars/acids ratio 0.03·sugar content 0.1·titratable acidity + 0.1·price based on the determined pattern it can be stated that: the high values of the first component result mainly from the high titratable acidity and high values of the l* parameter and low of a* and b* parameters as well as low values of sugars/acids ratio; the high values of the second component are primarily related to high density and high price. ital. j. food sci., vol. 30, 2018 331 the values of the two components, calculated for each tested apple juice and the juice of the parameters most desired by the respondents are shown on the preference map (fig. 10). figure 10. a preference map for the analysed 14 apple juice samples, showing location of ideal juice concept among tested products. it was determined that the juice with the parameters most preferred by the consumers has: a first component being close to 0. the equation determining the first component shows a positive contribution of colour parameter l* and titratable acidity as well as negative contribution of a* and b* colour parameters and sugar/acid ratio. therefore, the optimal juice parameters are a compromise between parameters of positive and negative contribution. low second component, and thus low density and low price. the significance of the taste of the juice and its price is also confirmed by the results of the survey conducted by ucherek (2011). according kraus and popek (2013), the taste plays a pivotal role in the perception of food products by consumers. when comparing the preference map with the results of the consumer desirability evaluation it can be concluded that the most appreciated apple juices have a low value of the second main component. nevertheless, none of the analysed juices has a value of the first principal component at a level close to 0. this leads to the conclusion that the ital. j. food sci., vol. 30, 2018 332 examined products do not fully meet the expectations of consumers. however, the apple juices coded 411, 513 and 101 have the values of the parameters closest to the parameters of ideal juice concept among tested samples. juices 411 and 513 are characterized by low sugars/acids ratio, average total sugars content and high titratable acidity as well as moderate values of l* colour parameter. sample 101 has moderate sugars/acids ratio and titratable acidity as well as high total sugars content and high value of l* colour parameter. 4. conclusions the results of the research confirmed the diversity of sensory characteristics of apple juices available on the market in poland, as well as the typicality of physicochemical parameters of the analysed products. agglomerative hierarchical clustering allowed to distinguish five groups of products, of which two where highly preferred by consumers. thanks to gam analysis dependences between the analysed physicochemical parameters of juices and their consumer ratings were identified. it was shown that: the relationship between the value of the colour parameter l* and the consumer desirability is u-shaped, the relationships between the colour parameter a* and the consumer desirability as well as density and the consumer desirability are inversely proportional and linear, the relationship between the value of the colour parameter b* and consumer desirability is exponential, the relationship between the sugar/acid ratio and consumer desirability is proportional and linear, the relationships between the titratable acidity and the consumer desirability as well as between the price of juice and its consumer desirability are complex, whereas the relationship between the sugar content and the consumer desirability is in the form of an inverted u. on the basis of gam analysis it was also shown that in the range of variability conditioned by the physicochemical parameters of the research material the ideal juice concept should be characterized by the colour identified by the following coordinates: l*=93.98, a*=-1.03, b*=52.42, density of 1.043 kg/l, sugar content equal to 100,827 g/l, titratable acidity of 83.5 mmol h+/l, sugar/acid ratio equal to 57.37 and the price of 2.65 pln/l or less. the results of pca analysis allowed to indicate that the most preferred apple juice is characterized by a balanced sweet and sour taste, low density and a relatively low price as well as the colour described by the compromise between high value of the l* parameter and low values of the a* and b* parameters. after the analysis of the preference map and its comparison with the results of the sensory desirability assessment, it may be also concluded that the products tested do not fully meet consumer expectations. therefore, a newly developed product that would meet consumer requirements stands a good chance of being successful on the market. in this way it was shown that the comparison of the product features desired by consumers with its physicochemical parameters constitutes valuable input data that can be used during the product development 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2017. “generalized additive models: an introduction with r” 2nd ed. crc press, boca raton, florida. paper received july 4, 2017 accepted october 2, 2017 paper ital. j. food sci., vol. 27 2015 1 keywords: coating, edible protein films, permeability, smoked fish, texture the physicochemical properties of edible protein films s. oğur1*, n. erkan2 1 bitlis eren university, engineering-architecture faculty, department of food engineering, bes minare mah., ahmet eren bulvari, kampus yerleskesi, 13000 bitlis, turkey 2 istanbul university, faculty of fisheries, department of seafood processing and quality control, ordu cad., no: 200, 34470 laleli-istanbul, turkey *corresponding author: tel. (+90 0434) 2283377-241; fax (+90 0434) 2283378, email: sdogur@beu.edu.tr abstract in the present study, edible films from isolated or concentrated protein sources and from proteins of two different fish species were produced. the texture properties, light transmission (lt) and oxygen permeability (op) of producing films were determined. the cl film settled in the second range according to both tension test parameters, thus outclassing the other tested films. the wg film possessed the lowest lt, so making it more effective in protecting of food products from light than the other tested films. the spi film with the lowest op value can be used for the purpose of protecting of food products from harmful effects of oxidation. 2 ital. j. food sci., vol. 27 2015 1. introduction edible films and coatings are thin layers that can be eaten with food. they are derived from natural sources and are formed on the surface of a food for the purpose of protecting food and to prolong its shelf-life. for this agricultural function, protective coatings were improved as an alternative to commercial packaging materials such as glass, tin and polymer. edible films and coatings do not carry carcinogen risk and do not cause a waste problem, which are important problems with plastic-based food packaging. if edible films are prepared in appropriate conditions, they can perform all the functions of a useful package. edible films are prepared utilizing hydrocolloids (protein and polysaccharide), lipids and composites (hydrocolloid+lipid). edible protein films are separated, however, into two groups: plant origin proteins (corn zein, wheat gluten, soy protein, pea protein, sunflower protein, peanut protein and cotton protein, etc.) and animal origin proteins (keratin, collagen, gelatin, fish myofibrillar protein, egg white protein, casein and whey protein, etc.). edible protein films can be manufactured from isolates or concentrated protein products that are purified from various protein sources, and by the evaluation of processing waste products (rhim and ng, 2007). the use of edible films for food is very old. so far, the most important application of edible films and coatings is an emulsion, made from oil and waxes (in 1930) that protect the important features of fruits such as brightness and color, prevents complications such as softness, initialing paleness and for improving fungicides, to better control the maturation and to delay water loss (debeaufort et al., 1998; baldwin, 1999). the advantages of natural biopolymer films can be summarized as follows: they are edible and biodegradable; to supplement the nutritional value of foods; enhance organoleptic characteristics of food, such as appearance, odor, and flavor; reduce packaging volume, weight and waste; incorporate antimicrobial agents and antioxidants; extended shelf-life and improved quality of usually non-packaged items; control over inter-component migration of moisture, gases, lipids, and solutes; individual packaging of small particulate foods, such as nuts and raisins; function as carriers for antimicrobial and antioxidant agents; for microencapsulation and controlled release of active ingredients; and have a possible use in multilayer food packaging materials together with non-edible film. they are low-cost and abundant; annually renewable resources (krochta, 2002). despite the positive effects in the literature (gennadios et al., 1997), the industrial application of edible films is not very common yet. the theoretical, experimental studies related to different polymers will be used to compose edible films or coatings (using fresh, frozen, processed products and determining the permeability properties of films) are currently underway. in the present study were used soy protein isolates (spi), whey powder protein (wp), egg white powder protein (ewp), wheat gluten (wg), corn zein (z), cattle gelatin (g) and collagen (cl), as well as rainbow trout protein (rtp) and atlantic mackerel protein (mp) as the protein source. the texture properties, the light transmission and the oxygen permeability of edible films produced from these sources were established. 2. material and methods 2.1. edible protein film manufacturing the materials for the production of edible protein film were obtained from the smart chemicals company (izmir, turkey). the edible protein films were manufactured as explained below, by pouring a determined amount film of solution into a teflon pan (18 cm×18 cm), drying for a determined time and at a determined temperature. 2.1.1. soy protein isolates (spi) film 5 g spi (produced in germany and containing 90% protein), 100 ml distilled water and 2.5 ml glycerol (gly) was stirred for 15-20 minutes at 55-60ºc. this solution was then stirred for another 10-15 minutes at 75-80 ºc and filtered through a cloth, after the solution’s ph was adjusted to 10.5±0.1 with 2 m sodium hydroxide (naoh) solution (denavi et al., 2009). prepared film solution (70 ml) was poured into a teflon pan and dried for 24 hours at 35°c. 2.1.2. whey protein (wp) film 5 g wp (produced by lactoprot company in germany and containing 80% protein), 100 ml distilled water and 5 ml gly was stirred for 15-20 minutes at 55-60ºc. this solution then was stirred for another 15-20 minutes at 75-80 ºc and filtered through a cloth, after the solution’s ph was adjusted to 8.0±0.1 with 2 n naoh (sarikus, 2006). the wp film solution (70 ml) was poured into a teflon pan and dried for 48 hours at 35°c. 2.1.3. egg white powder protein (ewp) film 9 g ewp (produced in turkey and containing 80% protein), 100 ml distilled water, 4.5 ml gly was stirred for 5 minutes at room temperature. then the solution’s ph was adjusted to 11.25±0.1 with 1 n naoh. the solution was kept for 20 minutes in a water bath at 45 ºc and filtered through a cloth (gennadios et al., 1997). the prepared film solution (70 ml) was poured into a teflon pan and dried for 24 hours at 35°c. ital. j. food sci., vol. 27 2015 3 2.1.4. wheat protein (gluten-wg) film 7.5 g wg (produced in belgium and containing 75-82% protein), 55 ml 95% ethyl alcohol, 45 ml distilled water and 3.75 ml gly was stirred for 15 minutes at 55 ºc. the solution’s ph was adjusted to 4.0±0.1 with 50% acetic acid. the solution was filtered through a cloth, then stirred for another 15 minutes at 70 ºc (tanada-palmu, 2000). all the prepared film solution was poured into a teflon pan and dried for 48 hours at 45°c. 2.1.5. corn protein (zein-z) film the film solution was prepared from zein protein (produced by sigma aldrich in usa and containing 90% protein), by modifiying the method developed by baysal et al. (2009). 2.5 g z, 60 ml 95% ethyl alcohol, 1 ml gly was stirred for 30 minutes at 75-80 ºc (baysal et al., 2009). the ph of the prepared z film solution was measured as 5.8. the z film solution (40 ml) was poured into a teflon pan and dried for 24 hours at 45°c. 2.1.6. gelatin (g) film 2 g g (produced by rousselot company in argentina and containing 83% protein), 100 ml distilled water, 1.1 ml gly was stirred for 30 minutes at 55-60 ºc. the solution was then filtered through a cloth (thomazine et al., 2005). the ph of the prepared g film solution was determined as 5.1. the g film solution (130 ml) was poured into a teflon pan and dried for 48 hours at 45°c. 2.1.7. collagen (cl) film 3 g cl (produced in turkey and containing 90% protein), 200 ml 3% acetic acid, 1.5 ml gly was stirred for 30 minutes at 75-80 ºc (ho et al., 2001). when prepared according to this method, the ph of the cl film solution was 3.3. all the prepared film solution was cast into teflon pan and dried for 24 hours at 45°c. 2.1.8. rainbow trout protein (rtp) film rainbow trout (oncorhynchus mykiss) were obtained from the istanbul fish market. the fish were transported in ice to the laboratory. the gutted and beheaded fish were minced, after skinning. the fish mince was washed 2-3 times to remove the water-soluble proteins, blood and dirty components, by keeping it for 5-10 minutes in cold water (water:fish ratio of 4:1, w/w). following the washing process, fish mince was pressed through a cloth in order to remove water. prepared in this way, washed fish mince constituted the raw material for the edible fish film. previously produced edible film were tested for protein and moisture in this mince. the rtp ratio was found to be 18.48%, and the moisture ratio was found to be 73.35%. because the protein ratio of the fish mince should be 2%, it was adjusted by adding the necessary amount of distilled water and then gly at a ratio of 50% of the protein ratio was added. the mixture was blended and filtered. the solution’s ph was adjusted to 3 with 50% acetic acid, after the solution was stirred for 10-15 minutes at 75-80 ºc. the solution was stirred for another 10-15 minutes at 75-80 ºc (cuq et al., 1997). the prepared film solution (100 ml) was poured into a teflon pan and dried for 24 hours at 35°c. 2.1.9. atlantic mackerel protein (mp) film used in manufacturing of edible film, atlantic mackerel minces (scomber scombrus) were prepared by cleaning and washing, as in the case of rainbow trout flesh. the mp ratio was found to be 23.38%, and the moisture ratio was found to be 72.62%. the mp film manufacturing method was similar to the rtp film manufacturing method. the only difference was that less distilled water was added, as when adjusting the protein ratio was to 2%, because of the mp ratio was higher. the film solution’s ph was adjusted to 3 again (cuq et al., 1997). the mp film solution (100 ml) was poured into a teflon pan and dried for 24 hours at 35°c. 2.2. the texture profile of the films tension tests were made for the purpose of determining the resistance of the film against breakage and tensile forces. test measurement values were as follows: target type: distance, test type: tension, target value: 40-90 mm, trigger load: 0.04 n, test speed: 0.5 mm/second, return speed: 4.5 mm/second, probe type: ta3/100, fixture: ta-dga, load cell: 1500 g. the measured parameters were the tensile force (n) and the maximum elongation (mm). three measurements were taken from three protein films belong to each group. 2.3. the light transmission (lt) of the films the lt of edible protein films was measured according to the method in astm (2009) by using working visible position dual beam spectrophotometer at 560 nm, at 23±2 ºc. this test method covers the measurement of the transparency of plastic sheeting in terms of regular transmittance (t r ). although generally applicable to any translucent or transparent material, it is principally intended for use with nominally clear and colorless thin sheeting. three measurements were taken from three protein films belong to each group. the lt of the films was calculated with the formula below accord4 ital. j. food sci., vol. 27 2015 ing to light intensity value, measured by equipment (astm, 2009). t r = (i r /i 0 ) x 100 t r : light transmission, % i r : light intensity value of spectrophotometer bath with sample i 0 : light intensity value of spectrophotometer bath without sample 2.4. the oxygen permeability (op) of the films the op of the films was measured during 4 hours at 23±2 ºc by using systech mark gas permeability test equipment, at 170 bar o 2 of pressure (astm, 2010). three measurements were taken from three protein films belong to each group. 2.5. statistical analysis the resulting analysis data were evaluated by using an ibm spss statistics 20® program. the results were given as an average ± standard deviation. the one-way analysis of variance (one-way anova) was applied. in the data parametric assumptions for multiple comparisons occurred. the tukey test was used to locate the sources of the differences found within different groups in this test. p<0.05 variation was accepted as the significant discrepancy between the groups and the parameters. 3. results the texture parameters, the light transmission and the oxygen permeability values of edible protein films are presented in table 1. the lowest and the highest tensile force of edible protein films were 0.668±0.001 n (in the wp film), and 5.267±0.559 n (in the g film), respectively. the z film had the lowest maximum elongation (0.50±0.02 mm), and the ewp film had the highest maximum elongation (82.00±2.94 mm) (table 1). it was reported by sabato et al. (2007) that mechanical and barrier properties of proteinbased films were generally better than polysaccharide (pls)-based films. the properties of protein-based films were dependent on the various factors, such as the protein source, the ph of protein solution, the species and the amount of plasticizer, film thickness, the manufacturing conditions (temperature and relative humidity), and the structures, included film forming solution (enzymes, antimicrobials, etc.) (benjakul et al., 2008). temiz and yesilsu (2006) indicated that when made from wg strong films have mechanical properties like rubber. wg films, containing a low amount of gly, had a higher tensile resistance (tr) and lower elongation at break (eab) (tanada-palmu et al., 2000). when the wg film was prepared from three different flours (commercial bread flour, hard red winter flour and soft white flour), it had a smooth tissue and film resistance, measured as tr, and the ratio increased by 50% by adding cysteine (cross-linking agent) (rayas et al., 1997). when the mechanical and physical properties of the wg films, plasticized with gly, were examined at different temperature (20°c, 50°c and 80°c) and the relative humidity (35% and 70%), the tensile strength (ts) increased too, by increasing the drying temperature at 35% relative humidity. ts decreased, when the temperature increased, however, at 70% relative humidity. the film thickness decreased with increasing temperature (kayserilioglu et al., 2003). lieberman and gilbert (1973) reported that the mechanical properties of edible films were considerably affected by the ratio of the plasticizer, used. the composite films can be manufactured taking into account the different barriers and mechanical properties needed by different products and thus the effectiveness of the material was increased. the use of the plasticizers such as gly, polyethylene glycol (peg), sorbitol (sor), etc. in the film formulations or the composites is advantable 1 the texture parameters, the light transmission and the oxygen permeability values of edible protein films. edible protein tensile force maximum elongation light transmission oxygen permeability film (n) (mm) (%) (ml/mm/day) spi 2.883±0.155af 31.85±1.11ag 20.08±0.01a 17.10±0.01a wp 0.668±0.001b 5.69±1.03b 23.35±0.01b 322.00±0.01b ewp 1.972±0.117ac 82.00±2.94c 23.22±0.01c 45.40±0.01c wg 1.275±0.147bc 57.40±2.07d 13.29±0.01d 218.00±0.01d z 0.948±0.427bc 0.50±0.02b 19.92±0.01e 181.37±0.01e g 5.267±0.559d 35.12±1.88af 63.30±0.01f 45.50±0.01f cl 4.145±0.198de 68.03±1.57e 18.70±0.01g 105.10±0.01g rtp 3.559±0.720ef 39.94±0.03f 14.53±0.01h 97.10±0.01h mp 1.273±0.082bc 28.95±0.96g 39.35±0.01i 281.00±0.01i *different letters (a, b, c) in the same column indicate the significant difference (p<0.05). ital. j. food sci., vol. 27 2015 5 tageous to impart pliability and flexibility, which improves handling. utilization of the plasticizers reduces the brittleness of film by interfering with the hydrogen bonding between the lipid and hydrocolloid molecules (tharanathan, 2003). in the study of mariniello et al. (2003), whole soy flour and apple pectin were used as raw materials in order to obtain films, which due to their consistency could be perfectly handled. the films were also prepared in the presence of transglutaminase (t-gase). the latter films showed a smoother surface and higher homogeneity, as demonstrated by microstructural analyses, whereas studies on the mechanical properties indicated that t-gase increased their ts and reduced their flexibility. it was reasoned that by there was a possible use of the t-gase polymerized pectin-soy protein films as edible food or drug coatings. the proper ph value to prepare the spi film with good mechanical and barrier properties was 10. addition of peg as a plasticizer at 60% of spi weight gave better film properties comparing with other used plasticizers. cross-linking of the spi film by adding formaldehyde or glutaraldehyde at a different level into the film forming solution and the combination of spi with the starch, also caused a noticeable improvement in the mechanical and barrier properties of the films (soliman et al., 2007). according to denavi et al. (2009), the applied drying conditions when preparing the spi film affected the mechanical properties, with the optimal drying conditions of 70 ºc and 30% relative humidity and 60 ºc and 60% relative humidity. dried under these conditions, the spi films presented a higher ts, lower eab values. shih (1998) indicated that the ts and the eab belonging to spi was affected by ph. the mechanical properties of edible films improved with some of the applied methods. the mechanical properties of the cl film improved with glyceraldehyde and alcoholdiols; the ts of the cl film increased by exposing to uv beams; the ts of the z film improved with aldehydes; the ts of the wg film developed with keratin; the ts of the wp film improved by heat treatment; the ts of the spi film developed by heat treatment, uv and gamma radiation application, and by adding calcium chloride and calcium sulphate (mchugh and krochta, 1994). it was expressed by gennadios et al. (1997) that the ewp films are highly hydrophilic; they could be used for water-soluble packets (pouches) for ingredients in the food, chemical, and pharmaceutical industries similar to cellulose ether-based water soluble packets in commercial use. the mechanical and barrier properties of such films can be modified by varying the types and amounts of added plasticizers. when microbial t-gase was employed as the catalyst for preparing the chitosan (ch)-ovalbumin films, the mechanical resistance of the ch-ovalbumin films increased from 24 mpa to 35 mpa. the improved mechanical and solubility properties of these new materials confirmed that this enzymatic approach could be a useful tool for preparing edible films for food coating and pharmaceutical applications (di pierro et al., 2007). in the study conducted by kolodziejska and piotrowska (2007), the effect of gly on the mechanical and water barrier properties, as well as on the water solubility, of fish gelatinch films (4:1, w/w) cross-linked with t-gase or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (edc) was determined. the ts of the films decreased after modification of the components with t-gase or edc, by about 25% and 40% respectively. the elongations of the enzymatically modified films containing 20% of gly and of chemically modified films containing 15% of gly were, respectively, about 8 and 13 times higher than those of unplasticized films. however, the ts of plasticized films were, respectively, 2.5 and 5 times lower. these properties widen the practical applications of modified films as packaging material. however, the cross-linking of components with commercial preparations of t-gase, -different to modification with edcincreased the fragility of films. therefore, such films have should be plasticized. the increase in the proportion of gly caused a reduction of the puncture force, the ts, the modulus of elasticity, and an increase of the puncture deformation and the eab of the g film (thomazine et al., 2005). when the g film and the z film were prepared by incorporating nisin (ns) into the film-forming solutions, the z film with the ns of 12.000 iu/ml had an increase of 11.6 mpa in the ts compared with the control, but the g film had a slight increase resulting from the increase of the ns concentration. these results suggested that the incorporation of ns into the z film and the g film improved the physical properties of the films (ku and song, 2007). in the study of carvalho et al., (2008), the g films from the skins of nile perch, a warm-water fish species, were reported to exhibit stress and eab similar to that of bovine bone gelatin. the gelatin, extracted from halibut skins, showed a suitable filmogenic capacity, leading to transparent, weakly colored, water-soluble and highly extensible films. the intermediate evaporation step at 60 ºc in the industrial procedure for drying the g film induced thermal protein degradation, caused the resulting films to be significantly less resistant and more extensible. it was seen that the g film with predominance of lower-molecular-weight fractions was apparently more plasticized by sor molecules, favoring a higher extensibility and a lower resistance in the final film. the edible films were successfully prepared from fish skin gelatin of brownstripe red snapper (lutjanus vitta) and bigeye snapper (priacanthus macracanthus). the films with the greater protein content had a higher thickness and me6 ital. j. food sci., vol. 27 2015 chanical properties (ts and eab), but lower water vapor permeability than those with the lower protein content. the films without gly were mostly brittle, and became more flexible in the presence of gly. the ts generally decreased with increasing gly concentration from 25% to 75% (jongjareonrak et al., 2006). iwata et al. (2000) expressed that edible films prepared from watersoluble fish proteins, had better flexibility compared to most of the other protein films. the ph change, the various physical and chemical treatments and the properties of plasticizer affected the quality of fish protein film. lower mechanical properties were found in the films prepared from the lower quality washed fish mince (benjakul et al., 2008). the ts of the films was higher, when prepared at acidic (ph 2, 3) and alkaline (ph 11, 12) conditions. the film with the lowest ts was made at ph 7. the mechanical resistance of the myofibrillar protein (myfp) based films were substantially lower than the synthetic films -such as polypropylene (pp), polyethylene and polyvinyl dichloridebut was relatively close to low density polyethylene (ldpe) (shiku et al., 2003). gly and peg plasticizers gave flexible structure to the films prepared from water soluble fish protein. as the concentration of gly increased, ts decreased with the concomitant increase of eab. in contrast, peg showed more marked influence on ts than on eab. gly:peg ratio of 2:1 exhibited the maximum eab value. it was confirmed that myfp based films plasticized with the mixture of gly-peg showed lower ts (2.5-3.0 mpa) than with only gly (5.0 mpa) or with only peg plasticized films (7.0 mpa) (tanaka et al., 2001). parris and coffin (1997) observed that zbased films plasticized with a mixture of glypolypropylene glycol (ppg) showed lower ts and lower elastic modulus than with only gly plasticized films or ppg plasticized films. moreover, the films plasticized with the mixture of gly-peg caused higher eab (117.8%) than those with only gly (2.6%) or peg plasticized films (2.8%). the functional properties of the fish protein films produced by the thermo-melting techniques were similar to known protein films and the ts of the films were similar to ldpe (cuq et al., 1998). the films obtained from tilapia fish treated at 65°c/30 minutes were more resistant and more rigid than the films treated at 40°c/30 minutes (garcia and sobral, 2005). when the protein concentration was treated at 90°c/30 minutes, the filmogenic solution was 2 g of sarcoplasmic protein (srcp)-myfp/100 g filmogenic solution, the prepared films were more resistant than the respective films (sobral et al., 2005). cuq et al. (1995) expressed that myfp films produced from sardine had the higher tr than that obtained from other protein films, such as z, wg, spi and wp. the quality of alaska pollack surimi (srm) films was decreased in order to determine the influence of the srm quality on the ts, and the eab values. thawed alaska pollack srm was incubated at 30ºc for 20 minutes (slight protein denaturation) and at 30 ºc for 5 hours (complete protein denaturation) in order to decrease the srm quality. slight protein denaturation caused a decrease in the eab of the films and complete denaturation gave rise to a reduction of the ts, and the eab values (shiku et al., 2004). myfp-based films were developed from a film-forming solution based on fish mince and the influence of plasticizers on the protein film quality was investigated. when no plasticizer was introduced in formulation the films were relatively brittle and needed to be handled very carefully, and gly, sor and sucrose were added in various concentrations. plasticization of the myfp-based films induced a large decrease in film strength and elasticity and an increase in deformation properties (cuq et al., 1997). sor plasticized films were the most brittle, with the highest ts (3.14 mpa). in contrast, gly and peg plasticized films exhibited flexible structure, despite the low ts (2.13 mpa and 1.80 mpa, respectively). as plasticizer concentration increased, the ts decreased concomitant with an increase in eab (bourtoom et al., 2006). propylene glycol alginate (pgal) was incorporated into alaska pollack srm film in order to improve their mechanical properties. the ts of the pgal-srm films prepared at ph 11 were twice as high compared to the srm films prepared at ph 7. the eab was not affected by the incorporation of pgal. results revealed that the formation of cross-linkages between epsilon-amino groups of lysine residues in srm proteins and mannuronic acid esters of pgal above ph 10 was responsible for the increased ts of the films (weng et al., 2006). the effect on the ratios of myfp to srcp from round scad (decapterus maruadsi) muscle on the properties of the resulting films was investigated. the ts of the films decreased with an increased srcp content. the films prepared from myfp:srcp ratio of 10:0 (w/w) exhibited the highest ts. the eab of the films, prepared with srcp content greater than 30%, had the decreased eab (arthan et al., 2008). a comparison could not be made, due to no mention in the literature to studies involving similar textural values of edible protein films obtained from the texture analyzer equipment used in our study. moreover, when both tensile test parameters were evaluated, as seen clearly in fig. 1 and fig. 2, placing the cl film in the second range, this film group outclassed other film groups considerably. sarikus (2006) expressed that the cl film is used in sausage coating more than natural coatings. the most important advantages of these films are that they remain strong in producting process conditions, having a flexible structure with a high-tensility. the g protein film obtained the highest lt value (63.30%±0.01); the wg protein film obtained, however, the lowest lt value (13.29%±0.01) (table 1). ital. j. food sci., vol. 27 2015 7 because food products are exposed to light trigger oxidative reactions, the products were packed with non-light transmitting packaging material in order to protect them from the light. there are a limited number of studies mentioned in the literature about the lt value of edible films. shiku et al. (2003) expressed that myfp films, prepared from blue marlin flesh, have excellent barrier properties to uv light in the range of 200-280 nm regardless of ph. however, the uv barrier property of the films gradually became poor above the wavelength of 300 nm. in contrast, the myfp film prepared at ph 7 blocked the most light in the uv-visible range from 350 to 800 nm because of its semi-transparency. in our study, the lt of the wg film was measured as the lowest value (13.29%±0.01) at 560 nm/23±2 ºc. the rtp film possessed the second lowest lt value after the wg film (fig. 3). comparisons could not be made, as there are no simfig. 1 -the changes of the tensile force values of edible protein films. fig. 2 -the changes of the maximum elongation values of edible protein films. ilar studies in the research literature of lt measurements conducted at the same wavelength as in our study. however, it is possible to say that the wg film can be more effective protecting products from light than other protein films. the highest op value was observed at 322.00±0.01 ml/mm/day in the wp film, and the lowest op value was observed at 17.10±0.01 ml/mm/day in the spi film (fig. 4). the op value of the ewp film and the op value of the g film; the op value of the cl film and the op value of the rtp film was determined to be close to each other in. the diffusion of gas and water vapor into film is called transition. these gases and the water vapor were absorbed by a surface of the polymer and oscillated by the other surface of the polymer during transition. the films have different barrier properties according to component properties and manufacturing techniques. the polar polymers, such as protein and carbonhydrates, 8 ital. j. food sci., vol. 27 2015 show low gas permeability and high water vapor permeability. in contrast, whilst materials (containing apolar hydrocarbon such as lipid) constitute an excellent barrier against the water vapor transition, they are not an effective barrier to gas transition. the chemical structure and the shape of the transition or absorbed film matrix substance is effective at the speed of diffusion and transfer. for example, the small molecules diffuse faster than the big molecules or the polar molecules diffuse faster than the apolar molecules in the polar films (cagri-mehmetoglu, 2010). the rate of rancidity, causing lipid oxidation and brown coloration, as well as causing myoglobin oxidation in meats, could be reduced by using edible coatings with the low op (gennadios et al., 1997). it was implied by yilmaz et al. (2007) that wp-based edible film and coating slowed gas (o 2 , co 2 ) transfer. the op of the cl fig. 3 -the changes of the light transmission values of edible protein films. fig. 4 -the changes of the oxygen permeability values of edible protein films. film was <0.04 cm3.μm/m2.dk.pa; the op of the z film was 11.8 cm3.μm/m2.dk.pa; the op of the wg film was 3.9-6.1 cm3.μm/m2.dk.pa; the op of the spi film was 1.6-4.5 cm3.μm/m2.dk.pa; and the op of the wp film was 18.5-76.1 cm3. μm/m2.dk.pa (lieberman and gilbert, 1973). lin and zhao (2007) expressed that the o 2 permeability (7.84x10-19 m3.m/m2.s.pa) of the zbased coating was lower than the o 2 permeability of plastic films (such as ldpe, pp, polystyrene and polyvinylchloride) and of pls, pls/lipid composite coatings, but higher than the o 2 permeability (2.89x10-17 m3.m/m2.s.pa) of wg coatings. it was observed that the o 2 permeability of the wg coating was close to the o 2 permeability (2.25x10-17 m3.m/m2.s.pa) of ldpe. it was recognized in literature that the op of edible films was affected considerably by factors such as the preparation conditions (temperature and relative ital. j. food sci., vol. 27 2015 9 humidity); the type and the amount of plasticizers, the incorporation of various chemicals; heattreatments and ph levels. for example, mchugh and krochta (1994) specified that the gas permeability of the cl film decreased with formaldehyde and chrome tannic acid, whilst the op of the wp film and the spi film improved with heat treatment. the op (3.16 cm3.μm/m2.dk.paat 23 ºc and 50% relative humidity) of the wg film, made with 40% gly, was found to be similar to the op (3.82 cm3.μm/m2.dk.pa-at 23 ºc and %0 relative humidity) of the film obtained by gennadios et al. (2006) (mujica-paz and gontard, 1997). despite the fact that the op of all wg films was low, the op level could be raised by increasing the gly concentration. it was seen that temperature has less effect on op than the relative humidity. the low op of the wg films may be due to their polar nature and linear structure, leading to the high cohesive-energy density and the low free volume (tanada-palmu et al., 2000). shih (1998) expressed that the op of the wg film decreased by adding of mineral oil and dipping in ca+2. it was specified that the spi coatings were effective oxygen barriers (op; 3.14x10-19 m3.m/m2.s.pa) at low relative humidity conditions, especially as their high oxygen barrier properties made them suited to gave the facility their applications such as flavour and pharmaceutical microencapsulated agents or as coatings for fruit, vegetable and cheese. it was reported that the wp-based films also had excellent oxygen barrier properties (op; 1.13x10-18 m3.m/m2.s.pa) at low and medium relative humidity when they compared with synthetic polymers (lieberman and gilbert, 1973). the proper ph value for the preparation of spi film with good barrier properties was 10; the o 2 permeability of the spi film was 1.06 cm3.μm/m2.dk.pa at this ph value. the addition of peg as a plasticizer at 60% of spi weight made the film properties better, compared with other plasticizers, and the o 2 permeability of the spi film was 0.76 cm3.μm/m2.dk.pa. the cross-linking of the spi film by adding formaldehyde or glutaraldehyde at different levels into the film forming solution improved the barrier properties of the resulting films (soliman et al., 2007). denavi et al. (2009) found that the applied drying conditions used in spi film manufacturing affected the barrier properties of the film: the o 2 permeability of the film was 18.2 cm3.μm/m2.dk.pa dried at 70 ºc and 30% relative humidity and the o 2 permeability of the film was 47.6 cm3.μm/m2.dk.pa dried at 60 ºc and 60% relative humidity. in our study, the spi film had the lowest o 2 permeability (17.10±0.01 ml/mm/day) (table 1). the properties of spi film can be improved with application of additional methods in light of the literature (lieberman and gilbert, 1973; soliman et al., 2007; denavi et al., 2009) and it can be used for the purpose of protecting food products from the harmful effect of oxygen. 4. conclusions in our study of the mechanical properties of edible protein films, both tensile test parameters (tensile force and maximum elongation) were evaluated, placing of the cl film in the second range: this film group outclassed other film groups considerably. it was observed that the wg film possessed the lowest lt value. it is possible to say that the wg film is more effective in protecting products from light than other protein films. the lowest op in our study belonged to the spi film. the properties of the spi film -which can be improved with additional methods in light of the literaturecan be used for the purpose of protecting food products from the harmful effect of oxygen. abbreviations ch: chitosan pgal: propylene glycol alginate cl: collagen pls: polysaccharide eab: elongation at break pp: polypropylene edk: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimid ppg: polypropylene glycol ewp: egg white powder protein rtp: rainbow trout protein g: gelatin sor: sorbitol gly: glycerol spi: soy protein isolates ldpe: low density polyethylene srcp: sarcoplasmic protein lt: light transmission srm: surimi mp: mackerel protein t-gase: transglutaminase myfp: myofibrillar protein tr: tensile resistance naoh: sodium hydroxide ts: tensile strength ns: nisin wg: wheat gluten op: 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ijfs#778_bozza ital. j. food sci., vol. 30, 2018 75 paper influence of water activity on listeria monocytogenes growth in “salsiccia sarda” fermented sausage a. ruggeri1, v. carraro1, s. succa1, b. meloni1, a. sanna1, c. sanna1, a. espa2, a. pinna1, g. carrucciu1, m. grosso3 and v. coroneo*1 1department of medical sciences and public health, university of cagliari, via porcell 4, 09124 cagliari, italy 2service of food hygiene of animal origin, local health 8, via nebida 09100 cagliari, italy 3department of mechanical, chemical and materials engineering, university of cagliari, via marengo 2, 09123 cagliari, italy *corresponding author. tel.: +39 0706758379; fax +39 0706758373 *e-mail address: coroneo@unica.it abstract “salsiccia sarda” is catalogued as a ready-to-eat food (rte) for which actual european legislation proposes microbiological criteria for l. monocytogenes. this study evaluates the influence of water activity (aw) on l. monocytogenes growth in 180 "salsiccia sarda” samples. a challenge test was performed to determine the l. monocytogenes growth potential (δ). the highest values of δ were detected in samples with limited values of aw, showing that this product is frequently around the limits for the growth of this pathogen. our results provide some critical information about process parameter combinations that could lead to greater safety of this product and better l. monocytogenes control. keywords: challenge test, growth potential, legislation, listeria monocytogenes, ready to eat meat products, "salsiccia sarda", water activity ital. j. food sci., vol. 30, 2018 76 1. introduction the implementation of haccp (hazard analysis and critical control points) system is fundamental to minimize the percentage of microbiological risk associated with food consumption. listeria monocytogenes is a pathogen that may contaminate different foods and many areas of the food processing environment (barza, 1985; schlech, 2000; gaulin et al., 2003; macdonald et al., 2005; varma et al., 2007; jackson et al., 2011; aissani et al., 2012). this pathogen causes listeriosis in humans and animals; human listeriosis can cause serious illness in immunocompromised individuals, pregnant women, newborns and elderly people. there has been a gradual increase in cases of listeriosis over the past 5 years in european union countries (efsa, 2014). in italy, between 1993 and 2000, the number of cases of listeriosis increased (petruzzelli et al., 2010). l.monocitogenes was mostly found in ready-to-eat foods (rte) and was responsible for many outbreaks associated with the consumption of rte meat, poultry, dairy, fish and vegetable products (liu, 2006; lianou and sofos, 2007; chan and wiedmann, 2009; efsa, 2014; coroneo et al., 2016). the possible presence of pathogens is a critical issue when dealing with a wide variety of fermented, dried and semi-dried sausages. these products are classified as ready-to-eat foods (rte) in which the presence of l. monocytogenes can pose a health risk to consumers. with this regard, some recent listeriosis cases are linked to the consumption of rte meat products (phac, 2009). l. monocytogenes contamination tends to increase during the production process of meat products because their production requires different handling steps and exposure to contaminated surfaces in the processing environment. raw meat is an important contamination source and may be contaminated by l. monocytogenes from the slaughterhouse environment or during the meat processing. once the production plant has been contaminated, l. monocytogenes can survive on work surfaces and equipment and grow on the meat products due to its high ability to tolerate environmental stress factors (wide ranges of ph and aw, high salt concentration, presence of nitrite and nitrate, and refrigeration temperature) (petruzzelli et al., 2010; mureddu et al., 2014; meloni et al., 2014). sardinia has a long tradition of quality meat-products and "salsiccia sarda" is considered the sardinian salami par excellence. it is a fermented rte meat product included on the list of italian traditional food products. it is made from minced lean pork mixed with different ingredients (salt, pepper, fennel and herbs). the mixture is introduced into a natural pork casing and, subsequently, the products are first heated to 20-22°c for 4-6 hours and then dried for six days in a fermentation chamber. during the first day of drying, the products are stored at 20-22°c and 60% relative humidity. in the subsequent five days of drying, the temperature is gradually reduced to 15°c and the relative humidity is gradually increased to 70%. the product is then dried and finally subjected to ripening for a period ranging from 8 to 25 days. the "salsiccia sarda" marketed shows a ph value of 5.28 and aw ranging from 0.90 to 0.95 (greco et al., 2005). previous studies of traditional fermented meat products showed that the prevalence of l. monocytogenes was 10% in france (thévenot et al., 2005), 10.6% in chile (cordano and rocourt, 2001) and between 13% and 42% in italy (de cesare et al., 2007; meloni et al., 2009). petruzzelli et al. (2010) reported a l. monocytogenes prevalence of 45.7% in traditional salami samples from the marche region (central italy). “salsiccia sarda” is catalogued as a ready-to-eat food (rte) for which actual european legislation, regulation (ec) 2073/2005 as amended by regulation (ec) 1441/2007 (european commission, 2005; european commission, 2007), specifies microbiological criteria for l. monocytogenes. according to these regulations, the l. monocytogenes growth is not supported in rte products with ph <4.4 or aw ≤0.92 or with ph ≤5.5 and aw≤0.94. for rte products that meet these conditions, a criterion of risk acceptability was established ital. j. food sci., vol. 30, 2018 77 of 100 cfu (colony-forming unit)/g during the shelf-life. although several studies (meloni et al., 2009; meloni et al., 2012) have shown the presence of l. monocytogenes in 42% of fermented “salsiccia sarda”, the contamination levels are always lower than 100 cfu/g. many factors affect the growth capacity of l. monocytogenes in foods and the intrinsic and extrinsic properties (i.e. ph, nacl content, aw, food composition, competing microflora, antimicrobial constituents naturally present, growth temperature, atmospheric gases) are certainly the most important (beaufort et al., 2008). during the production process of fermented meat, l. monocytogenes can survive due to its ability to tolerate low ph conditions and high salt concentrations (farber and peterkin, 1991). its survival is also linked to the absence of specific procedures in the production process. one of the very important factors that influences the growth/survival of l. monocytogenes is the aw. its variation inhibits part of the aerobic flora and a selection of the lactic flora (lab) causing a decrease in ph during production. thus, careful monitoring of the aw parameter, together with the proper choice of the ripening time, are essential for the microbial safety and stability of fermented sausages which, if marketed with a low maturation level, may be microbiologically unsafe. the aim of the present study was to assess the presence of l. monocytogenes in 84 naturally contaminated samples of "salsiccia sarda" over a seven month period. moreover the growth of listeria monocytogenes in 180 experimentally spiked samples of "salsiccia sarda" was evaluated using a full factorial experimental design. this design evaluated the impact of storage times and temperatures, level of ripening, packaging conditions and type of sausage. 2. materials and methods during the period between december 2014 and june 2015, a total of 84 “salsiccia sarda” samples, collected from local manufacturing plants in sardinia, were examined. all of the samples were transported under a controlled temperature (4°c) and were subjected to analysis at the laboratory of food hygiene at the university of cagliari which operates in conformity with european standard uni cei en iso/iec 17025:2005. 2.1. microbiological analyses the presence/absence of l. monocytogenes was investigated using the international standard method uni en iso 11290-1:2005. twenty-five grams of "salsiccia sarda" samples were suspended in 225 ml of half fraser broth (microbiol diagnostici, uta, cagliari, italy) incubated at 30°c ± 1 °c for 24h ± 2 h (primary enrichment). afterwards, 0.1 ml of the primary enrichment was transferred into a 10 ml tube containing fraser broth (microbiol diagnostici, uta, cagliari, italy) and incubated at 37°c ± 1 °c for 48h ± 2 h (secondary enrichment). after incubation, primary and secondary enrichment broths were streaked onto agar listeria ottaviani agosti (aloa, microbiol diagnostici, cagliari, italy) and polymyxin acriflavin lithium chloride ceftazidime aesculin mannitol (palcam) agar (microbiol diagnostici, cagliari, italy) plates and incubated at 37°c, respectively. from the positive sample plates, up to 5 presumptive colonies were subcultured on tryptone soy yeast extract agar (tsyea, microbiol diagnostici, cagliari, italy) and incubated at 37°c for 24 h. species confirmation was obtained with the following tests: gram staining, catalase and oxydase test (microbiol diagnostici, cagliari, italy), haemolytic activity, camp tests on sheep blood agar (microbiol diagnostici, cagliari, italy) and the biochemical test api listeria® (biomérieux, marcy-l´etoile, france). in all biochemical reactions the reference strain l. monocytogenes atcc 35152 was used as positive. ital. j. food sci., vol. 30, 2018 78 2.2. molecular investigation detection of l. monocytogenes was performed according to the previously published pcr protocols described by coroneo et al. (2016). the dna extraction was performed using the dneasy merikon food kit (qiagen, hilden, germany). following the manual indications, twenty-five gram samples of "salsiccia sarda" were suspended in 225 ml of half fraser broth (microbiol diagnostici, cagliari, italy), and incubated at 30°c ±1°c for 24h ± 2 h. after the pre-enrichment step, 1ml of each sample was taken and centrifugated for three minutes at 11,000 x g. the l. monocytogenes dna was detected using the merikon l. monocytogenes kit (qiagen, hilden, germany). l. monocytogenes atcc 35152 was used as pcr-positive control in all amplifications and molecular grade water as negative control. the reaction was carried out with the stratagenetm mx3005pqpcr (stratagene, la jolla, ca, usa) as it follows: initial denaturation at 95°c for 5 min, followed by 40 cycles of denaturation at 95°c for 15 s, annealing at 60 °c for 23 s and extension at 72°c for 10 s. 2.3. challenge tests the present study used 180 "salsiccia sarda" samples which were produced in sardinia. the samples were characterized by two different ripening times (i.e., 12 days and 20 days) because these are the levels of ripening most commonly used to meet the market demands. the 180 samples consisted of 90 "salsiccia sarda" samples of pure pork (n.45 at 12 days and n.45 at 20 days of ripening) and 90 myrtle flavored "salsiccia sarda" samples (n.45 at 12 days and n.45 at 20 days of ripening). the ingredients of each product are shown in table 1. table 1. ingedients of “salsiccia sarda”samples. ingredients “salsiccia sarda” (%) “salsiccia sarda” myrtle flavored (%) meat and fat minced lean pork 87.0 87.0 pork back fat 8.0 8.0 additive salt 3 3 dextrose and sucrose 0.736 0.736 potassio nitrate (e252) 0.024 0.024 sodium ascorbate (e301) 0.040 0.040 garlic 0.15 0.15 ground pepper 0.25 0.25 other spice 0.8 0.8 myrtle flavor 1 starter p p p: presence. in this study the 45 samples tested at each ripening time belonged to three different batches (i.e., 15 samples/batch). "salsiccia sarda" samples were artificially contaminated with l. monocytogenes. the samples not inoculated were defined as blank samples (bs) and ital. j. food sci., vol. 30, 2018 79 used to evaluate the natural contamination of "salsiccia sarda" with l. monocytogenes. the testing points were: t0 which was the time of inoculation, and t1, t2, t3, t4, t5, which were respectively the examination points carried out every 45 days for a total of seven and a half months after inoculation. this storage time has been adopted in order to achieve an extreme condition for the purposes of the research. 2.4. inoculation of "salsiccia sarda", packaging and storage conditions the challenge test was carried out according to the technical guidance document prepared by eu community reference laboratory (crl) for l. monocytogenes (beaufort et al., 2008). three strains of l. monocytogenes were used in the study. the inoculum was composed by: l. monocytogenes reference strain atcc 35152 obtained from the american type culture collection and two were wild type strains (serovar 1/2a and 1/2c) previously recovered from the "salsiccia sarda" samples. the preparation of inoculum has been previously described (coroneo et al., 2016). the level of contamination was approximately 10-100 cfu/g, which was obtained contaminating 10g of salsiccia slices with 100 μl of inoculum at a concentration of 103 cfu/ml. colony counts were confirmed by plate count agar (pca, microbiol, ca, it). the inoculated "salsiccia sarda" samples (pure pork and myrtle flavored) were packaged under air (n=180) or modified atmosphere (map) (i.e., 30% co2 and 70% n2) (n=180) and then stored at three different temperatures, 4°c, 8°c and 25°c. the challenge tests were carried out in independent trials for each batch (a, b and c) performed one week apart. a full factorial design of the variables (i) temperature, (ii) time of analysis, (iii) ripening time, (iv) type of packaging and (v) type of salsiccia was accomplished. the related experimental design is reported in table 2, leading to 144 different combinations of the variables with three replicates. table 2. experimental design for challenge studies. variables temperature 4°c 8°c 25°c time of analysis t0 t1 t2 t3 t4 t5 level of ripening 12 d 20 d type of packaging normal modified atmosphere packaging (map) type of sausage normal myrtle flavored the detection and enumeration of l. monocytogenes was conducted according to international standard methods uni en iso 11290-1:2005 and uni en iso 11290-2:2005. the enumeration of l. monocytogenes was performed on an aliquot of the sample homogenized 1/10 with base fraser broth (microbiol diagnostici, cagliari, italy) and incubated at 20°c ± 2°c for 1 h ± 5 min. a volume of 1ml from each suspension was streaked onto three aloa plates and incubated at 37°c for 24 and 48 hours. presumptive colonies of l. monocytogenes were counted. the final results were expressed as log10cfu/g. 2.5. intrinsic properties for all the samples of "salsiccia sarda" intrinsic properties, ph and aw, were determined. the measurement of ph and aw was carried out using ph meter eutech instruments ph 510 (thermo fisher scientific, waltham, massachusetts, usa) and aqualab4te (decagon, ital. j. food sci., vol. 30, 2018 80 pullman, wa, usa), respectively. the aw measurement was performed at different points of the product according to the diagram in fig. 1 and to international standard methods uni 11302: 2009. figure 1. operation chart used for the aw measurement in salsiccia sarda samples. 2.6. growth potential the growth potential (δ) of l. monocytogenes was determined by the difference between the counts at the end (t5) (log10cfu/g) and at the beginning (time “0”) (log10cfu/g) of shelflife. the rte product was considered as supporting growth of the l. monocytogenes when δ was higher than 0.5 log10.the pathogen was considered not able to grow in a rte product when δ values were negative or lower than 0.5 log10 (beaufort et al., 2008). 2.7. statistical analysis all tests of the assessment of l. monocytogenes growth were run in triplicate and averaged. means( x ) and standard deviations (s) were computed for each experimental condition. the confidence interval is calculated as ( ) nstxxsetx nn 2 ,05.0,05.0 ±=±=µ where n= 3 is the number of replicates. analyses were performed using microsoft excel xp 2010 and matlab® 2015 equipped with the toolbox statistics. correlation amongst the variables has been estimated by resorting to spearman’s rank correlation coefficient (gibbons and wolfe, 2003). this statistic seems to be the most reasonable choice for our data since it is a non-parametric statistic that reveals to be more robust when dealing with non-linear relationships. a multiway analysis of variance (n-way anova) test is accomplished for testing the effects of the factors: i) storage temperature, which assumes three different levels; ii) measuring time, which is available at 5 different levels; iii) time of ripening (data available at two different levels); iv) absence/presence of myrtle (2 levels); v) type of packaging (2 levels); vi) measured ph and vii) measured aw. with regards to the latter two variables, the measured values of ph and aw are divided into classes of width 0.1 and 0.05 respectively. ital. j. food sci., vol. 30, 2018 81 this allows a finite number of levels of such factors (11 for the ph and 27 for the aw) to be considered for the statistical test. 3. results and discussions 3.1. conventional microbiological analysis in the present work, natural contamination of the salsiccia samples analyzed was never detected along the seven month observation period. our result is consistent with some literature in which several mediterranean-style dried fermented sausages could be included in the category of rte products that do not favor l. monocytogenes growth. there is, however, a great variability according to local traditions that influence fermentation and ripening (hospital et al., 2012; meloni, 2015). in fact, as reported by petruzzelli et al. (2010), a high frequency of isolation of l. monocytogenes was found in ciauscolo salami manufactured in the marche region. this type of salami is particularly exposed to the risk of contamination because of its peculiarities (short maturation period, high aw, rare use of additives and starter cultures). the microbiological results were confirmed by molecular analysis. 3.2. challenge test the challenge tests conducted in this study on the sausage samples subjected to different storage and packaging conditions show that l. monocytogenes was unable to survive and grow until the end of shelf life in both situations regarding packaging and refrigeration (with a significance level p<0.05). however, we also observed an increase of the pathogen concentration in a specific time of the shelf life in samples with a particular level of ripening (12 days), stored in certain packaging conditions (under air) and with aw values around 0.92. in fact, the l. monocytogenes concentration in “salsiccia sarda” samples at 12 days of ripening and stored at 4°c increased from 1.66 log10cfu/g at t0 to 3.9 log10cfu/g at t2 when aw values were equal to 0,922± 0.001 (table 3). table 3. results of spiked samples packaged under air and tested at 12 and 20 days of ripening. 12 d ay s storage temperature 4°c 8°c 25°c time l.m ph aw l.m ph aw l.m ph aw t0 1.66 5,2±0.22 0.924±0.007 1.6 5.4±0.40 0.924±0.017 1.4 5.3±0.12 0.921±0.005 t 1 1.48 5.3±0.12 0.931±0.005 1.6 5.7±0.50 0.931±0.000 2.17 5.7±0.07 0.925±0.015 t2 3.9 5,5±0.07 0,922±0.001 3.3 5.8±0.12 0.925±0.005 3.3 5.8±0.05 0.923±0.002 t3 3.07 5.7±0.42 0.930±0.001 1 5.8.0±0.25 0.909±0.000 <1 5.7±0.30 0.905±0.001 t4 <1 5.8±0.32 0.917±0.012 1 5.7±0.40 0.903±0.015 <1 5.9±0.27 0.890±0.015 t5 <1 5.8±0.27 0.912±0.010 <1 5.7±0.32 0.901±0.010 <1 5.8±0.25 0.881±0.004 δ -1.35 -1.6 -0.70 ital. j. food sci., vol. 30, 2018 82 20 d ay s t0 1.48 5.7±0.03 0.881±0.012 1.48 5.6±0.35 0.874±0.010 1.3 5.77±0.30 0.868±0.005 t 1 1.84 5.7±0.04 0.903±0.224 <1 5.8±0.01 0.872±0,248 <1 5.7±0.01 0.901±0.298 t2 <1 5.9±0.27 0.908±0.010 <1 5.9±0.02 0.891±0.298 <1 5.8±0.01 0.901±0.248 t3 <1 5.8±0,30 0.884±0.005 <1 5.7±0.27 0.881±0.010 <1 5.9±0.07 0.893±0.005 t4 <1 5.6±0.22 0.863±0.124 <1 5.8±0.22 0.865±0.020 <1 5.8±0.010 0.872±0.010 t5 <1 5.8±0.27 0.852±0.012 <1 5.8±0.02 0.862±0.007 <1 5.9±0.27 0.861±0.007 δ -1.17 -1.00 -0.99 l.m: l.monocytogenes concentration expressed as median log10cfu/g ; δ: growth potential calculated as the difference between the median l.m (log10cfu/g) at t5 and the median (log10cfu/g) at t0; t0: the time of inoculation; t1, t2, t3, t4, t5: the examination points carried out every 45 days for a total of seven and a half months after inoculation. data are shown as mean ( )xsetx 082.0)( ± of three different replications. the l. monocytogenes concentration decreased in later observations (t4 and t5) due to a decrease in aw and an increase in ph values (table 3). the same results were observed in samples at 12 days of ripening stored at 8°c and 25°c. in the salsiccia samples with 20 days of ripening, l. monocytogenes was able to survive only in samples stored at 4°c at time t1, whereas at higher temperatures (8°c and 25°c) it was not detected (table 3). in both map packed salsiccia samples, at 12 and 20 days of ripening, stored at 4°c, 8°c and 25°c, significant l. monocytogenes growth was not observed (tables 4). table 4. results of spiked samples map packaged and tested at 12 and 20 days of ripening. 12 d ay s storage temperature 4°c 8°c 25°c time l.m ph aw l.m ph aw l.m ph aw t0 1.7 5.3±0.45 0.926±0.022 1.00 5.0±0.42 0.914±0.005 1.4 5.2±0.30 0.912±0.002 t1 <1 5.24±0.02 0.924±0.005 <1 5.3±0,05 0.906±0.001 <1 5.0±0.32 0.907±0.002 t2 <1 5.53±0.12 0.916±0.010 <1 5.6±0.12 0.910±0.001 <1 5.4±0.27 0.901±0.001 t3 <1 5.5±0.15 0.910±0.002 <1 5.8±0.17 0.887±0.005 <1 5.5±0.05 0.893±0.050 t4 <1 5.6±0.22 0.909±0.124 <1 5.7±0.22 0.899±0.015 <1 5.6±0.30 0.882±0.012 t5 <1 5.4±0.15 0.892±0.007 <1 5.6±0.10 0.880±0.007 <1 5.5±0.27 0.871±0.007 δ -1.7 -0.7 -1.1 20 d ay s t0 1.48 5.6±0.32 0.854±0.010 1.48 5.6±0.22 0.882±0.020 1.48 5.7±0.22 0.891±0.020 t1 <1 5.7±0.30 0.852±0.010 1 5.7±0.30 0.853±0.010 1 5.7±0.30 0.870±0.010 t2 <1 5.6±0.25 0.863±0.348 <1 5.5±0.35 0.864±0.007 <1 5.5±0.32 0.885±0.010 t3 <1 5.7±0.22 0.872±0.010 <1 5.8±0.30 0.852±0.010 <1 5.8±0.27 0.874±0.007 t4 <1 5.8±0.2 0.864±0.007 <1 5.9±0.17 0.851±0.005 <1 5.7±0.22 0.882±0.010 t5 <1 5.8±0.00 0.853±0.149 <1 5.8±0.17 0.850±0.0149 <1 5.8±0.10 0.861±0.224 δ -1.25 -0.8 -1.13 l.m: l.monocytogenes concentration expressed as median log10cfu/g ; δ: growth potential calculated as the difference between the median l.m (log10cfu/g) at t5 and the median (log10cfu/g) at t0; t0: the time of inoculation; t1, t2, t3, t4, t5: the examination points carried out every 45 days for a total of seven and a half months after inoculation. data are shown as mean ( )xsetx 082.0)( ± of three different replications. ital. j. food sci., vol. 30, 2018 83 in myrtle flavored “salsiccia sarda” samples at 12 days of ripening and stored at 4°c, the l. monocytogenes concentration increased from 1.6 log10 cfu/g at t0 to 2.07 log10 cfu/g at t5. these results show that bacterial survival was greater as evidenced by δ = 0.47 log10 cfu/g (table 5). table 5. results of spiked samples myrtle flavored packaged under air and tested at 12 and 20 days of ripening. 12 d ay s storage temperature 4°c 8°c 25°c time l.m ph aw l.m ph aw l.m ph aw t0 1.6 5.4±0.12 0.923±0.005 1.6 5.5±0.45 0.927±0.017 1.74 5.4±0.27 0.916±0.007 t1 1.48 5.3±0.47 0.920±0.020 2.5 5.8±0.15 0.921±0.012 <1 5.6±0.50 0.908±0.124 t2 3.17 5.7±0.12 0.931±0.005 3.9 5.9±0.07 0.929±0.005 <1 5.6±0.05 0.907±0.050 t3 2.25 5.6±0.15 0.930±0.001 1 5.7±0.17 0.908±0.001 <1 5.8±0.17 0.896±0.007 t4 2.3 5.5± 0.30 0.922±0.015 1 5.8±0.15 0.905±0.017 <1 5.7± 0.15 0.891±0.020 t5 2.07 5.6±0.22 0.917±0.010 <1 5.7±0.02 0.891±0.010 <1 5.8±0.12 0.883±0.124 δ 0.47 -1.1 -1.26 20 d ay s t0 1.48 5.4±0.02 0.903±0.017 1.48 5.4±0.30 0.920±0.348 1.3 5.3±0.22 0.920±0.015 t1 <1 5.7±0.02 0.901±0.007 <1 5.4±0.22 0.901±0.273 <1 5.9±0.27 0.902±0.010 t2 <1 5.6±0.22 0.891±0.422 <1 5.2±0.25 0.885±0.248 <1 5.7±0.42 0.894±0.005 t3 <1 5.8±0.27 0.882±0.012 <1 5.4±0.22 0.863±0.075 <1 5.8±0.27 0.882±0.012 t4 <1 5.7±0.30 0.844±0.005 <1 5.5±0.30 0.857±0.012 <1 5.7±0.22 0.821±0.015 t5 <1 5.6±0.22 0.825±0.012 <1 5.7±0.20 0.842±0.007 <1 5.8±0.12 0.790±0.007 δ -1.00 -0.70 -1.05 l.m: l.monocytogenes concentration expressed as median log10cfu/g ; δ: growth potential calculated as the difference between the median l.m (log10cfu/g) at t5 and the median (log10cfu/g) at t0; t0: the time of inoculation; t1, t2, t3, t4, t5: the examination points carried out every 45 days for a total of seven and a half months after inoculation. data are shown as mean ( )xsetx 082.0)( ± of three different replications. in the spiked samples myrtle flavored map packaged and tested at 12 days of ripening, the l. monocytogenes concentration increased from 1.78 log10 cfu/g at t0 to 3.58 log10 cfu/g at t1, then decreased to 1.78 log10 cfu/g at t2. in all other types of samples l. monocytogenes survival and growth was not observed (tables 6). 3.3. statistical analysis the previous considerations can be ascertained in a more rigorous manner by resorting to a multiway anova test. this test has been carried out on the data in order to assess which factors significantly affect the l. monocytogenes growth. the results are reported in table 7 where the statistically significant factors are highlighted with an asterisk. it appears that the growth is strongly influenced by the factors (i) time of analysis (p-value = 1.8555e-7) and (ii) aw (p-value = 4.9e-3). it should be noted that the factor “level of ripening” shows a p-value = 0.0622. ital. j. food sci., vol. 30, 2018 84 table 6. results of spiked myrtle flavored samples map packaged and tested at 12 and 20 days of ripening. 12 d ay s storage temperature 4°c 8°c 25°c time l.m ph aw l.m ph aw l.m ph aw t0 1.78 5.4±0.35 0.927±0.012 1.4 5.2±0.07 0.916±0.002 1.84 5.4±0.01 0.918±0.004 t1 3.58 5.8±0.02 0.929±0.015 1 5.5±0.22 0.897±0.002 <1 5.8±0.17 0.909±0.006 t2 1.95 5.8±0.12 0.926±0.001 <1 5.6±0.01 0.889±0.001 <1 5.7±0.15 0.900±0.0002 t3 <1 5.7±0.17 0.914±0.001 <1 5.7±0.17 0.862±0.015 <1 5.8±0.5 0.770±0.05 t4 <1 5.8±0.15 0.906±0.020 <1 5.6±0.20 0.875±0.010 <1 5.6±0.25 0.821±0.006 t5 <1 5.7±0.10 0.895±0.005 <1 5.8±0.22 0.868±0.007 <1 5.7±0.22 0.810±0.03 δ -1.17 -1.30 -1.26 20 d ay s t0 1.3 5.2±0.35 0.921±0.002 1.48 5.3±0.22 0.916±0.012 1.3 5.3±0.22 0.918±0.005 t1 <1 5.3±0.27 0.901±0.010 <1 5.4±0.25 0.915±0.000 <1 5.8±0.32 0.902±0.012 t2 <1 5.8±0.25 0.896±0.015 <1 5.6±0.20 0.880±0.002 <1 5.7±0.27 0.894±0.012 t3 <1 5.7±0.35 0.886±0.017 <1 5.8±0.22 0.878±0.010 <1 5.6±0.32 0.883±0.015 t4 <1 5.9±0.30 0.884±0.012 <1 5.7±0.25 0.874±0.007 <1 5.7±0.27 0.876±0.012 t5 <1 5.7±0.25 0.867±0.012 <1 5.6±0.17 0.863±0.124 <1 5.8 ±0.01 0.862±0.075 δ -0.5 -1.30 -0.7 l.m: l.monocytogenes concentration expressed as median log10cfu/g ; δ: growth potential calculated as the difference between the median l.m (log10cfu/g) at t5 and the median (log10cfu/g) at t0; t0: the time of inoculation; t1, t2, t3, t4, t5: the examination points carried out every 45 days for a total of seven and a half months after inoculation. data are shown as mean ( )xsetx 082.0)( ± of three different replications table 7. effect of the variables on the l. monocytogenes concentration in “salsiccia sarda”. source sum of squares degree of freedom mean square f-test prob>fa temperature 1.2143e+06 2 6.0717e+05 0.7063 0.4943 time of analysis 3.6114e+07 5 7.2228e+06 8.4016 1.8555e-7* ph 6.9740e+06 10 6.9740e+05 0.8112 0.6180 aw 4.3245e+07 26 1.6633e+06 1.9347 0.0049* type of sausage 3.6356e+03 1 3.6356e+03 0.0042 0.9482 level of ripening 3.0113e+06 1 3.0113e+06 3.5026 0.0622 type of packaging 1.0974e+06 1 1.0974e+06 1.2765 0.2594 error 2.6049e+08 303 8.5969e+05 total 3.8097e+08 349 a to determine a significant influence of these variables on the l. monocytogenes growth we use n-way anova tests with a significance level p<0.05. thus, although it cannot be considered as relevant for a significance level of the test a=0.05, we cannot exclude, at least for the data here investigated that it might have some impact on the process. on the other hand, the other variables (i.e. packaging, ph, temperature and absence/presence of myrtle) do not seem to significantly affect the l. monocytogenes concentration. ital. j. food sci., vol. 30, 2018 85 fig. 2 reports the l. monocytogenes concentration with respect to the aw for the 5 different levels of time. it appears that the microbial concentration reveals a sudden increase for aw values close to 0.92 at each level of time. the region close to the critical value is zoomed in on in the insets. incidentally, it was found that the microbial growth may take place even at values slightly less than 0.92 (see insets on fig. 2c and 2e). figure 2. l. monocytogenes concentration with respect to the aw for the different levels of time. the spearman correlation coefficient between l. monocytogenes concentration and aw is computed at each different time and the corresponding point estimations are reported in table 8 together with their p-values. for comparison, the corresponding pearson correlation coefficients and the related p-values are reported in table 8. table 8. the spearman correlation coefficient values between l. monocytogenes concentration and aw at each different time of analysis. spearman coefficient pearson coefficient time r p-value r p-value 1 0.1683 0.1575 0.1844 0.1211 45 0.3716 0.0013 0.2322 0.0497 90 0.6977 9.7e-12 0.4559 5.71e-5 135 0.5919 5.43e-8 0.3434 0.0034 180 0.4272 1.82e-4 0.3447 0.0030 ital. j. food sci., vol. 30, 2018 86 calculation of the correlation coefficient at time t6 is meaningless since all the measurements of concentration are zero. it was found that the correlation coefficient is always significantly greater than zero except for the initial time. this further confirms that the aw is the main factor affecting the growth process. as a final remark, l. monocytogenes was never detected in blank samples (bs) during the challenge test. these results are consistent with previous studies that have shown a l. monocytogenes growth in samples of fermented sausages contaminated with about 5 log10cfu/g (sperandii et al., 2015). some manufacturers tend to reduce the ripening period to respond to market needs (hospital et al., 2012; meloni, 2015). these products with early ripening, as highlighted by our study, may have aw levels close to 0.92-0.94. this can increase risks associated with the l. monocytogenes growth during shelf-life, even in the presence of competitive microflora. our study showed that, in the presence of an improper ripening time, initial low levels of contamination in the product could however lead to a concentration that is potentially harmful to human health during the first 45 days of storage. 3.4. intrinsic properties among the intrinsic properties, our results showed that the evolution of aw values is the same of that of other typical italian fermented meat products (greco et al., 2005; petruzzelli et al. 2010; meloni et al., 2012; mataragas et al. 2015a, b). the aw decreased constantly for both situations regarding packaging and storage temperatures. its initial values at t0 were 0.924±0.003 for salsiccia samples at 12 days of ripening when packaged under air and stored at 4°c and at 8°c and 0.921±0.002 for those stored at 25°c. these values decrease to the time t5 with values equal to 0.912±0.010, 0.901±0.010, 0.881±0.004 for samples stored at 4°c, 8°c and 25°c respectively (table 3). in general, the sausages with 12 days of ripening showed the average aw levels typical of products able to support the l. monocytogenes growth in all storage conditions (at 4°c, 8°c and 25°c) (table 3,5) in salsiccia samples with 20 days of ripening the aw values are uniform in the products in both situations regarding packaging and storage temperatures. the initial values at t0 show a decrease of about 0.03-0.08 units at t5 time ((table 3, 4, 5, 6). in accordance with other authors (meloni et al., 2014) our results confirm, with reasonable certainty, the safety of products with a longer ripening time. as far as ph is concerned, our results have showed that the analyzed samples have similar values to those found in most mediterranean-style fermented sausages (meloni, 2015) and were close to 5.4-5.8 for both situations regarding packaging and storage temperatures. as reported in other studies (vermeulen et al., 2007; vermeulen et al., 2009; mataragas et al., 2015b;) the ph level showed a slight increase (0.3-0.5 units), during the experiment, in all sausages analyzed and for both situations regarding packaging and storage conditions. (tables 3, 4, 5, 6). in general, the aw and ph values were always within the limits of growth for l.monocytogenes. 4. conclusions previous studies have been carried out to evaluate the effect on l. monocytogenes growth and survival during the production of fermented sausages to evaluate the safety of the process (mataragas et al., 2015b). the realization, in this study, of an experimental protocol for a challenge test, specific for this traditional product, "salsiccia sarda", has ital. j. food sci., vol. 30, 2018 87 allowed us to obtain usable results for the definition of adequate product security during the shelf-life. in our study, by resorting to statistical tools, the aw value measured in these fermented sausages was demonstrated as a critical control point. through the challenge test, in conjunction with the aw measurements at each storage time, it has been possible to show that l. monocytogenes is able to replicate in a sausage with 12 days of ripening, only when the values of 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acetic acid. international journal of food microbiology 135:83-89. paper received february 21, 2017 accepted september 1, 2017 ijfs#996_bozza ital. j. food sci., vol. 30, 2018 170 paper an ancient crop revisited: chemical composition of mediterranean pine nuts grown in six countries a. zuleta1, a. weisstaub1, s. giacomino1, l. dyner1, v. loewe2, r. del río2 and m. lutz*3 1facultad de farmacia y bioquímica, universidad de buenos aires, argentina; 2instituto forestal, ministerio de agricultura, chile 3centro de investigación y desarrollo de alimentos funcionales, facultad de farmacia, universidad de valparaíso, gran bretaña 1093, valparaíso, chile *e-mail address: mariane.lutz@uv.cl abstract the aim of the study was to analyze the proximate composition of pine nuts harvested from 15 growing areas in chile, argentina, italy, spain, turkey, and israel. the main component was fat, followed by protein. pine nuts from chile and argentina were similar, zones with the highest thermal oscillation and rainfall. italian pine nuts had the highest fiber content, while spanish nuts had the highest fat content. israel presented the highest number of dry months, where pine nuts contained more protein and minerals, while nuts from turkey showed an intermediate position. minimum and maximum average temperatures, amount of dry months, and thermal oscillation affected the chemical composition. keywords: agro-climatic conditions, chemical composition, growing zone, pine nuts, pinus pinea ital. j. food sci., vol. 30, 2018 171 1. introduction pinus pinea l., also known as stone pine, is one of the nine most important tree nut species in the world. pine nut is the most expensive nut worldwide, while stone pine is one of the oldest fruit trees, as demonstrated by archaeological remains that evidence its cultivation in the pre-christian era (rottoli and castiglioni, 2011). a risk for depletion and degradation of european stone pine forests has appeared, in relation to emerging threats such as climate change (milano et al., 2015) and demographic dynamics (united nations, 2015). climate change has affected the production of pine nuts worldwide (mutke et al., 2005), and efforts are being made to improve the growth of valuable mediterranean forests in non-traditional areas. the species is endemic to the mediterranean basin, cultivated mainly in spain, portugal, italy, turkey, and tunisia. pine nuts are part of the traditional mediterranean diet, which is well recognized for reducing cardiovascular risk factors (rees et al., 2014; ros, 2015). as part of the mediterranean diet, pine nuts contribute to reducing risk factors of cardiovascular disease (cvd), type-2 diabetes, and some types of cancer (alasalvar and bolling, 2006; sabaté and ang, 2009; bao et al., 2013; estruch et al., 2013; sorlí et al., 2013). in general, nuts are energy-dense foods, since they contain 4.4 to 7.4 g kg-1 fat (ros and mataix, 2006; ryan et al., 2006; kornsteiner-krenn et al., 2013; usda, 2016). however, nut consumption improves blood lipid levels (sabaté et al., 2010) and reduces risk factors of cvd (kris-etherton et al., 2008). the cardioprotective constituents of pine nut oil include unsaturated fatty acids, phytosterols, tocopherols, and squalene, among other bioactives (wolff and bayard, 1995; maguire et al., 2004; alasalvar and bolling, 2006; bolling et al., 2011). moreover, although nuts are high fat and energy dense foods, their consumption has been associated with reduced body mass index (bmi) (bes-rastrollo et al., 2009; ibarrola-jurado et al., 2013; lutz and luna, 2016). pine nuts are not only a good source of fat, but they also contain high levels of proteins, they supply various vitamins (e, b6, niacin, folic acid), minerals (potassium, phosphorus, magnesium, zinc, iron, copper), and a variety of phytochemicals, including phenolic compounds (nergiz and dönmez, 2004; evaristo et al., 2010; bolling et al., 2011; lutz et al., 2016). evaristo et al. (2010) reported significant differences in the mineral profile and other chemical components of pine nuts grown in different regions, suggesting that environment and soil types have an important influence. vanhanen and savage (2013) also reported differences in minerals probably due to soil conditions, climate and growing practices. pine nuts supply has been extremely affected by leptoglossus occidentalis heideman, an insect spread in all the major producing countries, which represents an elevated economic impact in global markets (bloomberg business, 2013). on the other hand, given the high nutritional and outstanding organoleptic quality of pine nuts, its demand is increasing worldwide, reaching high prices (international nut and dried fruits, 2016), which represents an opportunity to produce the seeds in non-conventional growing areas, including the southern hemisphere. models at different scales have been developed to establish relationships between stone pine productivity and several variables, including climatic ones (calama et al., 2011), but no reference has been found on their impact on pine nut quality. the chemical characterization of pine nuts grown in different areas is important due to health, commercial, and genetic concerns. taking into account that most available composition data have been obtained from the main productive countries, the aim of the study was to assess the current pine nut proximate composition across six countries: three located in the traditional growing areas (italy, spain, and turkey), and three in areas where there is no current commercialization of pine nuts (israel, argentina, and chile), ital. j. food sci., vol. 30, 2018 172 accessible to authors for seed collection. the study focuses on the chemical composition, and the impact of climate variables on the composition of pine nuts harvested in these countries. 2. materials and methods 2.1. study area fifteen areas were selected for collecting pine nuts in italy, turkey, spain, israel, argentina, and chile. average climatic variables of locations from where pine nuts were grown are summarized in table 1, and their distribution is shown in fig. 1. figure 1. distribution of locations in six countries where pine nuts were collected. ital. j. food sci., vol. 30, 2018 173 table 1. location and mean climatic variables of harvest sites of pinus pinea l. nuts. location country annual rainfall (mm) annual average temperature (°c) dry months† (n°) maximum average temperature (°c) minimum average temperature (°c) thermal oscillation (°c) latitude longitude soil type miramar 660.7 15.1 5 22.1 8.0 14.1 38°16's 57°50'w dunes afforested with conifers, poor in organic matter. entisol (fredes et al., 2009) claromecó 758.4 14.5 4 21.2 7.9 13.3 38°51's 60°04'w clayey granular soils with 3-5% of organic matter, moderately acids. molisols. imperfect drainage (carbone and piccolo, 2002). mean argentina 709.6 14.8 5 21.7 8.0 13.7 north mz 383.7 14.1 9 21.9 7.5 14.3 33°26's 71°04'w alluvial origin (alfisols, mollisol, entisols) (albers, 2012). dry coast mz 648.3 13.6 8 21.0 7.0 14.0 35°14's 72°12'w granitic origin (alfisols, inceptisols (albers, 2012). south mz 1,047.0 13.2 7 19.8 7.5 12.3 37°54's 72°40'w volcanic origin (altisols, red clay) (albers, 2012). mean chile 693.0 13.6 8 20.9 7.3 13.5 ortal 830.0 14.7 6 20.6 8.9 11.7 33°05'n 35°50'e volcanic origin (orenstein et al., 2001). basaltic brown mediterranean soils and basaltic lithosols (dan et al., 1975). bar'ham 682.1 16.2 7 20.4 12.0 8.4 33°04'n 35°26'e volcanic origin (orenstein et al., 2001). pale rendzinas (dan et al., 1975), yatir 275.0 17.6 8 22.4 12.8 9.6 31°18'n 35°01'e aeolian origin loess with a clayloam texture, overlying chalk and limestone bedrock (naama et al., 2012). brown lithosols and loessial arid brown soil (dan et al., 1975) mean israel 595.7 16.2 7 21.1 11.2 9.9 ital. j. food sci., vol. 30, 2018 174 climatic data sources: argentine meteorological service (www.smn.gov.ar); chilean environmental information system (www.inia.cl); the israel meteorological service (www.ims.gov.il); italian army aeronautic meteorological service (www.meteoam.it); state meteorological agency (www.aemet.es); turkish meteorological service (www.mgm.gov.tr). †dry months were calculated as those with monthly rainfall/monthly potential evapotranspiration <0.5. table 1. continues. cecina (li) 833.6 14.5 4 19.8 9.1 10.7 43°19'n 10°30'e alluvial soils, sometimes with shallow water table (calcaric cambisols, fluvisols and gleysols), clay accumulation along the profile (costantini et al., 2004). feniglia (gr) 455.1 13.2 4 16.0 10.3 5.7 42°25'n 11°12'e sandy soils (piraino et al., 2012). calabria (rc) 546.8 18.3 6 22.5 14.1 8.3 39°19'n 16°21'e eroded soils (eutric and calcaric regosols) with accumulation of carbonates and soluble salts, rich in iron oxides and clay. volcanic soils (umbric andosols) (costantini et al., 2004). mean italy 611.8 15.3 5 19.4 11.2 8.2 cádiz spain 524.0 18.7 5 21.7 15.6 6.1 36°32'n 06°17'w litoral dunes (entisols). sandy, poor soils (muñoz and gracía, 2009). balikesir 576.8 14.6 6 20.5 8.9 11.5 39°39'n 27°53'e sandy-loamy soil texture, neutral ph, non-calcareous or slightly calcareous, salt-free and organic matter weak (yilmaz and satil, 2017). bagyuzu 743.2 13.1 5 18.9 8.8 10.1 39°18'n 26°58'e rough broken land (brown forest soil material) (oaeks and arikok, 1954). aydin 651.7 17.5 7 24.5 11.9 12.6 37°50'n 27°51'e alluvial and youthful soils (oaeks and arikok, 1954). mean turkey 657.2 15.1 6 21.3 9.9 11.4 ital. j. food sci., vol. 30, 2018 175 2.2. materials stone pine seeds were collected from planted trees (none corresponds to cultivars) in 2013/14 in turkey (n=3 zones), israel (n=3 zones), spain (n=1 zone), italy (n=3 zones), argentina (n=2 zones) and chile (n=3 zones) in 2013; a minimum of 500 pine nuts from 10 trees were harvested in each collection area. chilean stone pine seeds were collected from pinus pinea l. planted trees distributed in three macrozones located between 30.82°n and 38.99°s (loewe et al., 2015). 167 trees were sampled taking into account the macrozone size and variability of the most variable chemical contents. from each tree, 500 pine nuts were harvested. samples were harvested during winter since it corresponds to the maturation season according to abellanas and pardos (1989). once obtained, in-shell pine nuts were kept in plastic nets individually tagged at room temperature until manually shelled. for sample preparation, shelled nuts were dried at 40°c until moisture reached 4 g kg-1. all the seeds were ground with a kitchen processor (moulinex®) and sieved to 0.5 mm, and then frozen in sealed plastic bags at -20°c until analyses. 2.3. methods all reagents and solvents were analytical grade chemicals from merck (darmstadt, germany). proximate analyses were performed using aoac methodologies (aoac, 2012). protein content was determined by kjeldahl assay (aoac 920.54) using a nitrogen digestor dk6 (velp®) and a nitrogen distiller udk 129 (velp®), applying factor of 5.3 to convert nitrogen to proteins (greenfield and southgate, 1972). crude fat was assessed using the aoac method 920.39, moisture was determined using the aoac method 945.15, and ash was determined using the aoac method 942.05. total dietary fiber (tdf) was measured using the aoac enzymatic-gravimetric method 991.43 using the megazyme k-tdrf 05/12 kit supplied by megazyme®. 2.4. statistical analysis chemical analyses were done in triplicate; each replicate was quantified in duplicate, unless stated otherwise. all data given represent mean values + standard error (se). data were compared using heteroscedastic anova, and statistical significance was determined with an lsd test (p<0.05). the relative contribution of climatic variables to chemical components was estimated using cart (classification and regression trees) algorithms (breiman, 1999). as confirmatory analysis, the groups suggested by the identified climatic variables thresholds were also compared by anova. finally, a principal component analysis (pca) was applied, generating a biplot for chemical composition of pine nuts and climate variables of different countries. analyses were performed using the software infostat® and its interface with the software r® (di rienzo et al., 2014). 3. results and discussions in this comparative study, we took into consideration geographic zones and agro-climatic conditions that may affect the chemical composition of pine nut seeds, considering that loewe et al. (2016a) reported marked differences on stone pine cone productivity along the climatic gradient in chile, which could also be translated to chemical composition, as in fact has been determined by using a discriminant analysis by near infrared ital. j. food sci., vol. 30, 2018 176 spectroscopy (nirs) in stone pine nuts collected in different macrozones of chile (loewe et al., 2016b). the chemical composition of pine nuts collected in six countries is shown in table 2. table 2. chemical composition of pine nuts by location and country (g/100 g)1. location/country moisture protein lipids ashes total dietary fiber miramar 4.6 33.1 42.0 4.2 10.5 claromecó 3.0 31.2 41.6 4.6 9.1 argentina 3.8±0.8ab 32.1±0.9b 41.8±0.2a 4.4±0.2ab 9.8±0.7c north mz 4.1 34.9 42.3 4.7 11.6 dry coast mz 4.5 35.3 46.9 4.7 11.6 south mz 4.3 32.1 43.6 4.6 11.8 chile* 4.3±0.07a 34.1±0.5b 44.3±0.7a 4.7±0.03a 11.7±0.1b ortal 3.5 35.3 31.0 4.8 13.0 bar'ham 3.7 37.2 30.1 4.7 12.3 yatir 4.0 37.0 42.9 4.6 11.9 israel 3.7±0.12b 36.6±0.5a 34.7±4.1a 4.7±0.06a 12.4±0.3b cecina 5.2 33.2 37.0 4.2 14.7 feniglia 4.9 32.8 37.9 3.9 13.9 calabria 5.2 30.3 36.4 4.7 15.2 italy 5.1±1.0a 32.1±0.9b 37.1±0.4a 4.3±0.2ab 14.6±0.4a spain (cádiz) 4.8 33.8 45.3 4.1 12.4 balikesir 3.7 33.3 45.0 4.3 12.2 bgyuzu 4.0 34.0 43.5 4.0 12.6 aydin 4.0 37.0 40.1 4.1 14.4 turkey 3.9±0.1ab 34.8±1.1ab 42.9±1.4a 4.1±0.1b 13.1±0.7ab 1data are expressed as means±se (n=3). different letters in a column indicate statistically significant differences (p<0.05). *lutz et al. (2016). significant differences were found among countries for protein (p=0.0053), tdf (p=0.0003), ash (p=0.0008), and moisture (p=0.0001). the main chemical component in pine nut seeds is lipids. in the analyzed seeds, fats ranged from 34.7% (israel) to 45.3% (spain) (p<0.05). these values are in agreement with nergiz and dönmez (2004) and ryan et al. (2006), but lower than the fat content reported for this nut by kornsteinerkrenn et al. (2013) and esche et al. (2013), including the usda database (usda, 2016), which reports a mean value of 68.4%. according to the principal components analysis, the lipid content of the pine nuts was enhanced in chile by low minimum temperature, and across countries by high maximum temperatures. the lipid quality is relevant to the energetic and nutritive values of pine nuts, while the fatty acid profile, as well as phytosterols, phytostanols, tocopherols and other lipid bioactives contents play major roles in their healthy properties (kornsteiner et al., 2006; bolling et al., 2010; esche et al., 2013). ital. j. food sci., vol. 30, 2018 177 pine nuts are recognized as a good dietary source of proteins, and the average protein content ranges from 13% to 30% dry matter, depending on the pinus species (evaristo et al., 2010; usda, 2016). the protein content observed in the seeds collected from six countries ranged from 32.1% (italy and argentina) to 36.6% (israel), which are above the reported averages. in three chilean macrozones across 1,300 km with varying climatic conditions, proteins ranged from 32.1% to 35.3% (lutz et al., 2016). these results demonstrate that protein content can be significantly affected by the agro-climatic conditions in which the species grow. moisture was highest in italian pine nuts (5.1%), while it was lowest in the israeli samples (3.7%); ashes varied from 4.1% (spain and turkey) to 4.7% (israel and chile), and tdf varied between 9.8% (argentina) and 14.6% (italy). fig. 2 represents the biplot of the two principal components by country, which explained 66% of the variability. differences were observed between pine nuts grown in chile and argentina, and between turkey and israel, presenting a chemical composition that differs from italy and from spain. south american pine nuts, which grew in areas with the highest thermal oscillation and rainfall, showed similar chemical composition. italian pine nuts exhibited the highest tdf and moisture contents. spanish pine nuts showed a different composition, which would be related to the minimum average temperature and average temperature. israeli and turkish pine nuts showed a similar composition, exhibiting high protein content. mineral content related to the number of dry months, and lipids to the maximum average temperature. lipids content was superior when maximum average temperature was high. figure 2. biplot for chemical composition of pine nuts and climate variables according to country. maxt: maximum average temperature, at: average temperature, mint: minimum average temperature, dm#: dry month number, to: thermal oscillation, tdf: total dietary fiber. fig. 3 shows the biplot of the two principal components, explaining 55.6% of the variability. it also shows that the italian samples hold a high content of tdf and moisture, with calabria (it) and cádiz (sp) being characterized by a high minimum average temperature. south american pine nut samples grown in areas with high thermal oscillation –especially those from northern and central zones in chile – and rainfall – ital. j. food sci., vol. 30, 2018 178 especially those from the southern zone of chile showed a similar composition. turkish and israeli pine nuts showed some differences in the second component, being yatir (il) and aydin (tk) characterized by a high maximum average temperature and dry month number, and high protein content. bagyuzu (tk), an area with a high rainfall, holds a similar lipid content to the ones from southern chile. figure 3. biplot for chemical composition of pine nuts and climate variables by location. maxt: maximum average temperature, at: average temperature, mint: minimum average temperature, dm#: dry month number, to: thermal oscillation, tdf: total dietary fiber. in the cart analyses for each component (table 3), data were first split into two subsets based on the predictor variable (mint for tdf; to for moisture and dm# for ash) and its thresholds (8.4°c, 8.4°c, and 5.5 months, respectively). each subset, or node, for tdf was then analyzed independently using the same procedure (maxt, 22.4°c). top nodes are the most important to explain the chemical composition. interestingly, the influence of climate on some chemical components was observed. a significant negative influence of thermal oscillation on moisture was detected, as well as significant positive effects of dry months on minerals, and of minimum and maximum temperatures on tdf. in particular, the tdf content in the seeds of the six countries varied from 9.8% (argentina) to 14.6% (italy) (p<0.05). it was affected by climatic variables such as the minimum average temperature and maximum average temperature (p<0.05), increasing with minimal temperatures above 8.4°c and maximum temperatures above 22.4°c. a different situation was observed in the ash content, which increased in presence of longer dry periods (over 5.5 months) by 9.5%. fig. 4 depicts the effect of climatic variables on pine nut composition. across locations, tdf was significantly influenced by climatic variables, with an increase of 21.1% at a minimum average temperature above 8.4°c (13.2% vs 10.9%, p=0.0032), and an increase of 22.3% at a maximum average temperature above 22.4°c (14.8% vs 12.1%, p=0.0203). minerals were influenced by the number of dry months (p=0.0063), with an increase of -4,00 -2,00 0,00 2,00 4,00 cp 1 (30,2%) -4,00 -2,00 0,00 2,00 4,00 c p 2 (2 5, 4% ) aydin tk bagyuzu tk balikesir tk bar'ham il cádiz sp calabria it cecina it claromecó ar dry coast cl feniglia it miramar ar north cl ortal il south cl yatir il tdf lipids protein ash moisture rainf all at dm# maxt mint to aydin tk bagyuzu tk balikesir tk bar'ham il cádiz sp calabria it cecina it claromecó ar dry coast cl feniglia it miramar ar north cl ortal il south cl yatir il tdf lipids protein ash moisture rainf all at dm# maxt mint to ital. j. food sci., vol. 30, 2018 179 9.5% when dry months exceeded 5.5 (4.6% vs 4.2%). moisture was influenced by thermal oscillation, being 22.5% higher when thermal oscillation was below 8.4°c (4.9% vs 4.0%, p=0.0101). table 3. climatic variables that best explain total dietary fiber (tdf), moisture and ash determined by cart analyses. node predictor variable average content (g/100 g) n standard error total dietary fiber 1 mint≤8.4°c 10.9 5 1.33 2 mint>8.4°c 13.2 10 1.42 2.1 maxt≤22.4°c 12.1 8 0.90 2.2 maxt>22.4°c 14.8 2 0.33 moisture 1 to≤8.4°c 4.9 3 0.05 2 to>8.4°c 4.0 12 0.31 ash 1 dm#≤5.5 4.2 6 0.05 2 dm#>5.5 4.6 9 0.05 mint: annual average minimum temperature; maxt: annual average maximum temperature; to: thermal oscillation (annual average maximum absolute temperature minus annual average minimum absolute temperature); dm#: dry month number. ital. j. food sci., vol. 30, 2018 180 figure 4. climatic variables that influence chemical composition across fifteen localities distributed in six countries. each threshold was detected by cart analysis. different letters indicate statistically significant differences (p<0.05). thermal oscillation: annual average maximum absolute temperature minus annual average minimum absolute temperature. the soil nutrient content effect has been limitedly studied. in fact, in turkey, a positive correlation between nitrogen, phosphorus, calcium and manganese depletion was detected in needles and cone loss (kilci, 2013). in israel, malchi and shenker (2011) found that iron deficiency decreased root growth and induced a reduction in chlorophyll concentration on needles in soils with high concentration of calcium carbonate, being a high soil ph the cause for reduced iron absorption. however, no studies have been performed on the relationship among pine nut composition and soil contents. in our study, we observed a similar pine nut protein content when grown in sandy soils, around 33%. thus, the chemical composition of pine nuts grown in different regions could be explained, at least in part, by the environment and soil type variability between regions, which is in agreement with several authors (gómez-ariza et al., 2006; evaristo et al., 2013; lutz et al., 2016). future studies should also address the use of cultural practices such as fertilization on pine nut quality, considering that borrero (2004) reported an increase in pine nuts concentrations of fat, copper, magnesium and sodium in fertilized plots. 4. conclusions the study describes the proximate chemical composition of pine nuts harvested in six countries. the results obtained indicate that from a nutritional quality standpoint, all the analyzed seeds exhibited good nutritional properties, independently of the geographic zone where they were grown, justifying their inclusion in a healthy diet. the major components of pine nuts are lipids, protein and dietary fiber, while their carbohydrates ital. j. food sci., vol. 30, 2018 181 content is low, which make them a good choice in the prevention of diabetes, metabolic syndrome and other common non-transmissible diseases. the effect of the climatic conditions, soil quality and other environmental variables are usually not taken into consideration when average values are used in food composition databases. the results obtained in this study indicate significant differences among countries for protein, tdf, ash and moisture, variability probably related to climate and environmental conditions of the growing areas. relevant climatic variables were thermal oscillation for moisture, dry months for minerals, and minimum and maximum average temperature for tdf. the study reveals that pinus pinea l., a traditional ancient tree grown in the mediterranean basin, may also be successfully grown in south america, contributing to diversify agriculture, as pine nuts represent an opportunity for the global food industry as well. finally, the composition of the seeds collected from different countries, in various climatic conditions, constitutes relevant information that should be considered when food composition data are included in tables and reference data, shown as mean values. acknowledgements this work was supported by conicyt, project fondef d11i1134. authors acknowledge the support from universidad de buenos aires, argentina, and universidad de valparaiso, chile. we thank the assistance with seed collection to the jewish national fund (kkl) israel; bryfoods turkey; a. camporini and a.c. millanes, argentina; f. pelleri, italy, and d. ciavolino, spain. references abellanas b, and pardos j.a. 1989. seasonal development of female strobilus of stone pine (pinus pinea l.). ann. sci. for. 46 (suppl):s51-s53. alasalvar c. and bolling b.w. 2006. review of nut phytochemicals, fat-soluble bioactives, antioxidant components and health effects. brit. j. nutr. 113 (suppl s2):s68-s78. albers c. 2012. coberturas sig para la enseñanza de la geografía en chile. www.rulamahue.cl/mapoteca. universidad de la frontera. temuco aoac. 2012. official methods of analysis (19th ed). arlington, va, usa. 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received august 20, 2017 accepted october 10, 2017 ijfs#923_bozza ital. j. food sci., vol. 30, 2018 226 paper optimization of rice-field bean gluten-free pasta improved by the addition of hydrothermally treated rice flour a. diba, a. wójtowicz*b, l. benatallaha, m.n. zidounea, m. mitrusb and a. sujakc alaboratoire de nutrition et technologie alimentaire, institut de la nutrition, de l’alimentation et des technologies agro-alimentaires, université des frères mentouri, constantine, algeria bdepartment of thermal technology and food process engineering, university of life sciences, lublin, poland cdepartment of biophysics, university of life sciences, lublin, poland *corresponding author: tel./fax: +48 814610683 e-mail address: agnieszka.wojtowicz@up.lublin.pl abstract rice and field bean semolina was used to obtain proteinand fiber-enriched gluten-free pasta. the tests covered the effect of pre-gelatinized rice flour used as a gluten-free pasta improver. a central composite design was applied involving the water hydration level and pre-treated rice flour level. instrumental analyses of pasta (cooking loss, water absorption capacity, texture, hydration index, pasting and thermal properties, and microstructure) were carried out to assess the impact of experimental factors. the results showed that the application of hydrothermally treated rice flour improved the cooking and textural characteristics of pasta. the optimum recipe contained 5.845 g of pregelatinized rice flour and 59.266 ml of water, both selected based on the desirability function approach with the value of 0.775 corresponding to the optimum pasta properties. keywords: central composite design, gluten-free, pasta making, quality, starch, texture ital. j. food sci., vol. 30, 2018 227 1. introduction semolina from durum wheat is a preferred substrate for the manufacture of pasta products. the quality characteristics of pasta such as texture, cooking properties, color, and sensory properties are under the influence of several conditions (sissons, 2008; petitot et al., 2010). both raw material quality and technological process have a major impact on the final product quality (de noni and pagani, 2010). gluten protein plays an important role in determining the quality of pasta. wheat semolina pasta products are required to exhibit the best quality (marti and pagani, 2013). however, wheat pasta is not recommended for people with celiac disease because their immune system has a disorder in which the sentinel lesion is on enteropathy triggered by the ingestion of gluten proteins (leffler et al., 2017). it is known that the only possible therapy is a lifetime gluten-free diet, during which the damage to the intestinal mucosa regresses and the patient’s well-being improves considerably. to ensure the manufacture of proper quality gluten-free (gf) foodstuffs, the proposed formula must be of such quality characteristics that resemble conventional pasta. the degree of difficulty in producing gf products is closely associated with the technological role of gluten in the food-system (marti and pagani, 2013; larrosa et al., 2016). although the demand for better-tasting, better-textured, and healthier gf products offers great market opportunities for food manufacturers, the replacement of gluten functionality still presents a major technological challenge (chillo et al., 2007; sozer, 2009; loubes et al., 2016). having that in mind, pre-treated flour could be used as the improving agents and key components to offer the effect of gluten in gf pasta (marti and pagani, 2013). moreover, improvement of the functional properties of starches is an important step in the manufacturing of gf pasta. to achieve this objective, natural treatment of flour through the passage of heating and cooling cycles can be applied. this passage facilitates the forming of a rigid network based on retrograded starch, which gives the dough a suitable texture (chillo et al., 2009; marti et al., 2010; cabrera-chávez et al., 2012). the change in the organization of starch molecules by the application of physical and thermal treatment has been widely approved as a natural material with high food safety. the aim of this treatment is to promote the functional properties of starch without destroying its granular structure and, thus, lift any legislative limits as to its quantity in food (bemiller, 1997; zavareze and dias, 2011). the dominating properties of pregelatinized flour resulting from starch modifications under different treatments are associated with the re-organization of the macromolecular structure (lai and cheng, 2004). this re-organization of starch molecules in gf dough means better quality characteristics of the final product compared with those in durum semolina pasta. in addition, pre-gelatinized rice flour was used as a major component and bulking or thickening agent in several food preparations (moore et al., 1984; lai and cheng, 2004). besides, as demonstrated by yoenyongbuddhagal and noomhorn (2002), the thermal treatment of rice flour improves the quality characteristics of rice noodles. hormdok and noomhorm (2007) applied the hmt (heat-moisture treatment) and the ann (annealing) of rice starch for noodle preparation and obtained rice noodles with the desired features. in many countries, celiac patients suffer from the lack of gf products, which makes it difficult for them to follow the recommended diet. one of the methods to improve the situation of celiac patients is to design new gluten-free products (giuberti et al., 2016; bourekoua et al., 2016). also, as gf products are less available and less diverse, as well as being more expensive than gluten-rich food products, there is a strong urge to develop gf products that are technologically enhanced as well as economically sustainable. in ital. j. food sci., vol. 30, 2018 228 addition, there are fewer studies and information about the manufacture of traditional laminated pasta and its acceptability by consumers. moreover, the supplementation of rice-based laminated pasta with leguminous is envisaged in order to improve the nutritional value of the proposed formulation (giuberti et al., 2016) and enhance the machinability of dough as well as the final quality of pasta products. field bean is part of the leguminous group which is widely produced and consumed in the mediterranean area and has an excellent potential to be used in ever new recipes because of its high nutritional quality. this plant is a rich source of fiber and protein and supplements the protein intake from cereals and tubers for a more beneficial amino acid balance (micard et al., 2010; benatallah et al., 2012; caracciolo et al., 2016). also, proteins of field bean have such functional properties as solubility, water retention capacity, foaming capacity as well as producing an emulsifying effect that plays an important role in food formulation and processing (dakia et al., 2007; roy et al., 2010; boye et al., 2010). in addition, the need to achieve the appropriate quality and the possibility of production of more natural products for consumers with celiac disease are strong arguments behind the idea of a new formula of gf rice-field bean laminated pasta. for these reasons, the application of natural hydrothermal treatment of rice flour has been proposed. the main objective of this study was to investigate the effect of the addition of hydrothermally treated rice flour as an improver in the manufacture of gf laminated pasta from rice supplemented with field bean semolina (rfbs). the optimization of water and pre-gelatinized rice flour (pgrf) levels needed to ensure the improvement of selected properties of gf pasta products was done by means of the response surface methodology (rsm) and following the desirability function approach (dfa). 2. materials and methods 2.1. raw materials cereal and leguminous semolina used for making gf laminated pasta was of rice and field bean. rice (oryza sativa) semolina was provided by lubella sp. z o. o. s.k. (lublin, poland). field bean dehulled seeds (vicia faba minor) were applied as supplement for improvement of the protein content and amino acids balance in gf pasta. field bean seeds were purchased from alamir company (albehera, egypt) and ground. all semolina used in gf laminated pasta had particle sizes between 200 and 500 µm. rice flour (lubella sp. z o. o. s.k., lublin, poland) with particles smaller than 200 µm was used for pregelatinization. 2.1.1 chemical composition analysis the raw materials and pasta samples were analyzed for fat, ash, and protein content using the standard procedures of the aacc methods (1995). fat content was determined by soxhlet extraction with n-hexane (aacc 30-10), ash by incineration in a muffle furnace at 550°c (aacc 08-01), the kjeldahl procedure (aacc 46-10) was used for determining the protein content in three replications. fiber was determined with the scharrer-kürschner method using the procedure of aoac (2000). the method involved the acid digestion of the sample for the elimination of nutritive components; it was then flushed with ethyl alcohol and dried to dry matter. based on that, the amount of fiber was calculated (aoac 993.21). the measurements were performed in duplicate. ital. j. food sci., vol. 30, 2018 229 2.1.2 hydrothermal treatment of rice flour the heat treatment process of flour was performed according to the tangzhong method described by yvonne (2007) and bourekoua et al. (2016). pre-gelatinized rice flour (pgrf) was made by mixing water and flour (1 to 5) in an aluminum pan. the mixture was then heated on a heating plate for 8-9 min and continuously stirred with a spatula until the temperature reached 65°c. afterwards, the obtained paste was cooled down at ambient temperature for 1 h and stored in a fridge (4°c) for 24 h. some preliminary experiments were made in a laboratory and indicated that the cooling of the slurries for 24 h at 4°c produces better cooking and textural properties when added to the pasta formula than pgrf kept for 1 h or used immediately after heating. pre-gelatinized rice flour was added to pasta made with rice-field bean semolina by mixing with other ingredients of the formula after keeping at ambient temperature for 1 h. 2.2. experimental design a central composite design (ccd) was used, involving water hydration level (x1) and pregelatinized rice flour (stored in a fridge for 24 h) level (x2). the effects of two variables on the quality characteristics of laminated pasta were analyzed. the hydration range applied in the experimental design was determined by preliminary experiments using from 46.42 to 72.12 ml of water for 100 g of the recipe (46.42 ml/100 g is the minimum level of water necessary to make dough and 72.12 ml/100 g is the maximum level of water for obtaining sticky pasta). the level of pre-gelatinized rice flour was fixed from 0 to 11.70 g for 100 g of recipe based on the calculation of the water level added in each run. these values were incorporated into jmp software to determine the ccd matrix. the optimization of the studied formula was carried out using the rsm. the factorial section is a 22 test; the star section includes four tests. five replicates (runs 1, 4, 5, 10, 11, table 1) at the center of the design were used to estimate the pure error at the sum of square, for a total of 22+22+5=13 runs. table 1. factors, levels and code values used in the central composite design (ccd) for gluten-free ricefield bean semolina (rfbs) pasta. run hydration x1 (ml) pgrf x2 (g) code value code value 1 0 59.26 0 5.85 2 -1 50.18 1 9.98 3 0 59.26 1.414 11.7 4 0 59.26 0 5.85 5 0 59.26 0 5.85 6 -1 50.18 -1 1.71 7 1 68.35 1 9.98 8 0 59.26 -1.414 0 9 -1.414 46.42 0 5.85 10 0 59.26 0 5.85 11 0 59.26 0 5.85 12 1 68.35 -1 1.71 13 1.414 72.12 0 5.85 ital. j. food sci., vol. 30, 2018 230 a statistical analysis was performed for the experimental data for each response variable in order to select an optimized recipe using the desirability function approach (dfa). the dfa is a method of multi-criteria optimization showing some relationships between several responses. the desirability factor (d) varied from 0 to 1, where 1 meant a maximum satisfaction and 0 was complete refusal (larrosa et al., 2016; bourekoua et al., 2016). 2.3. pasta making according to the previous research and recommendations (fao, 1982; micard, 2010), the optimum combination of cereal and legume required to achieve an excellent nutritional balance is 65% of cereal and 35% of legume. the gf formulation (rice-field bean semolina: rfbs) studied in this work was based on a mixture in a ratio of 2:1 (w/w) of cereal and leguminous semolina in order to ensure a technological approach to processing and enable good machinability of laminated rice-based dough as well as providing a good amino acids balance (benatallah et al., 2012). the hydration range applied in the experimental design was determined by preliminary experiments (46.42 to 72.12 ml for 100 g of recipe). the level of pre-gelatinized rice flour was fixed up to 11.70 g for 100 g of the recipe. hydration and pgrf levels were expressed based on the rfbs blend. the basic recipe of rfbs consisted of 66.66 g of rice semolina, 33.33 g of field bean semolina, 2 g of salt and the amount of distilled water defined in the experimental design data. pasta produced without the addition of pgrf (run 8) was considered as control pasta (table 1). the optimum recipe was determined according to the desirability method performed by jmp software version 7 (sas, usa). the selected optimum pasta was also prepared according to the procedure adapted both for traditional and industrial production. in the first step, the ingredients were mixed for 15 min at 25°c using a kitchenaid mixer, model kpm5 (st. joseph, michigan, usa). the obtained dough was rounded, divided into balls of 50 g and covered with a sealed plastic wrap and then rested at 25°c for 1 h to let the starch hydrate. the dough was molded and passed through the reduction rolls of a pasta machine marcato ampia type 150 (campodarsego, italy) for four times from each pass and in all directions to produce a uniform dough sheet. the roller gap was maintained at 5 for the last sheeting. the final thickness of each dough sheet was 1.5 mm as determined with a caliper. finally, pasta samples were dried in an oven with air circulation at 40°c for 4 h until it reached the final moisture content below 12%. they were stored in sealed plastic bags at room temperature. 2.4. determination of pasta quality 2.4.1 cooking quality the cooking quality was determined according to the approved method 66-50 (aacc, 2000) with the modifications proposed by chillo et al., (2007). in brief, 10 g of dried pasta was boiled in 300 ml of distilled water for an optimal cooking time (oct), dripped for 3 min and weighed. the cooking water was evaporated by drying at 105°c overnight. the residue was weighed and the cooking loss (cl) was reported as a percentage of dry sample weight before cooking. the water absorption capacity (wac) was expressed as a percentage increase of pasta weight after cooking compared with the weight of uncooked pasta. the measurements were performed in triplicate. ital. j. food sci., vol. 30, 2018 231 2.4.2 hydration properties the hydration properties of the optimum pasta and control sample were determined at ambient temperature designating the water absorption index (wai), water solubility index (wsi), and swelling power (sp). ground pasta samples (0.7 g) were mixed in centrifugal tubes with 7 ml of distilled water. after 5 min of rest, the samples were hydrated for 10 min and mixed every minute for uniform reconstitution, followed by centrifugation (15000 rpm, 10 min, 21°c) in t24 centrifuge (veb mlw medizinetechnik, leipzig, germany). the supernatant was dried to constant weight at 105°c. the tests were carried out in four replications. the wai and wsi calculations were made according to the method described by wójtowicz and mościcki (2014). sp was calculated as proposed by lai and cheng (2004). 2.4.3 textural characteristics the texture characteristics of pasta were achieved using the universal testing machine zwick/roell bdo-fb0.5 th (zwick gmbh& co., ulm, germany). the cutting force (n) of single cooked strand of the optimum and control pasta was evaluated in five independent replications with a warner-bratzler blade (60 mm long, 3 mm thick, double face truncated at 45°), as described by wójtowicz and mościcki (2014). the measurements were carried out with a test speed of 8.33 mm/s using a 0.5 kn working head. otms ottawa cell was used for the evaluation of firmness, stickiness and cohesiveness of cooked pasta samples from each run of experimental design and for the optimum pasta properties (martinez et al., 2007). for firmness (f) (n), stickiness (s) (mj) and cohesiveness (mj) of cooked pasta products, a double-compression test was applied. 50 g of cooked and drained pasta was put in the otms chamber and compressed with a test speed of 3.3 mm/s. the testxpert® 10.11 software was used to record data in three independent replications. 2.4.4 pasting properties the pasting performance of hydrothermally treated slurries were determined after 1 h of cooling at room temperature and after 24 h storage at 4ºc. the properties were adjusted according to bouasla et al. (2017) using a brabender micro-visco amylograph (brabender ohg, duisburg, germany) under the constant measurement conditions: speed 250 rpm, sensitivity 235 cmg, and heating rate 7.5°c/min. the following characteristics were considered: pasting temperature (°c), temperature at the beginning of viscosity increase; initial viscosity (mpas), cold viscosity at 30°c; peak viscosity (pv) (mpas), the highest viscosity during the heating cycle; breakdown (bd) (mpas), corresponding to the difference between the pv and the viscosity at the end of the holding period at 93°c and an index of viscosity decrease during holding at 93°c; setback (mpas), corresponding to the final viscosity minus the viscosity at the end of the holding period at 93°c and an index of viscosity increase during the cooling cycle; final viscosity (mpas), i.e. viscosity reached at the end of the cooling period. 2.4.5 thermal characteristics for calorimetric measurements, the differential scanning calorimeter dsc star system (mettler toledo ag, greifensee, switzerland) was used. the calibration of the instrument was done by means of the indium standard before the evaluation of the sample. all the measurements were performed under nitrogen gas atmosphere. the instrument was ital. j. food sci., vol. 30, 2018 232 controlled with stare system. ground samples of dried optimum and control pasta (3-5 mg) with a granulation below 250 µm were precisely weighed in aluminum crucibles with a pin (volume 40 µl). the sample-encapsulating press was used for the sealing of powderfilled pans. the samples were thermally inserted into the instrument, equilibrated at 25°c for 10 min and then heated at the rate of 10°c/min up to 180°c. an empty aluminum pan was used as the reference in all recorded thermograms. during the measurement, the temperature was controlled with an accuracy of ±0.1ºc by means of the high precision thermoregulation system tc 100 mt (peter huber kältemaschinenbau ag, germany) (bourekoua et al., 2017). heat flow during the whole process of sample heating was recorded. the following features of thermogram were analyzed: onset temperature (t0), peak temperature corresponding to the maximum heat flow (tp) and endset temperature (tc). in order to compare the length of the transition, the difference between endset and onset temperatures (tr = tc-to) was calculated. the gelatinization enthalpy (δh) expressed per weight of dry sample (j/g) was assessed by integrating the area between the thermogram and the baseline under the peak. all thermal parameters were calculated using the evaluation mode of the stare system. thermal scans were performed in three independent replicates. 2.5. sensory evaluation the sensory assessment was carried out on the optimal selected pasta and control sample. pasta products were cooked in the optimum time and drained and placed in warm conditions until testing. the samples were served in a random order on a white ceramic plate to a panel of 55 untrained consumers (25-45 years old, 26 females and 29 males) who were the habitual consumers of pasta and were familiar with the definitions and references. they evaluated the products for appearance, taste, flavor, consistency, and stickiness on a 5-point scale (1 = poor, 5 = good) (wójtowicz and mościcki, 2014). for the preliminary tests of taste, five independent sessions were organized; each session involved a panel of 11 untrained consumers who were familiar with the terminology related to pasta (iso 11036). for the final analysis, a single session was performed with the same pasta cooking conditions involving the presentation of the sample and specific environmental conditions. the results and observations of final sensory analysis were taken into account. the overall acceptability was evaluated by the same panel, and each pasta sample was assessed using a verbal nine-point hedonic scale. the ratings were converted into numerical scores, where 1 was dislike extremely, 5 neither like, nor dislike, and 9 as like extremely. gf pasta was considered acceptable if the mean score for the overall quality was above 5 (wójtowicz and mościcki, 2014; bouasla et al., 2017). 2.6. pasta microstructure the pictures of dry optimum and control pasta were taken using a scanning electronic microscope (sem). a piece of dry pasta, attached to a carbon disc with a silver tape was sprayed with gold in the k-550x vacuum sublimator (emitech, ashford, england). the electron microscope vega lmu (tescan, warrendale, usa) was used to observe the surface and cross-section of the samples at different magnifications (×200, ×600 for surface, and ×200, ×600, ×1500 for cross-sections). the accelerating voltage of 30 kv was applied. ital. j. food sci., vol. 30, 2018 233 2.7. statistical analysis a second order complete polynomial equation was applied to fit the behavior of each measured variable as a function of dough composition (myers et al., 2009; larrosa et al., 2016) by using the jmp software version 7 (sas, usa). the models were used to determine response surfaces in statistica, version 10 (statsoft. inc., usa). a one-way anova analysis of variance was employed for the assessment of the effect of water (x1) and pre-gelatinized rice flour (x2) on dependent variables (y) with a 0.05 significance level. the model proposed for each response was: y = b0 + b1x1 + b2x2 + b11x1x1+ b22 x2x2+ b12x1x2 where: b0 is the value of the fitted response at the center point of design, that is (0,0); b1 and b2 are the linear regression terms; b11, b22 are the quadratic regression terms; and b12 is the cross-product regression term (interaction coefficients). optimization was performed with jmp, version 7 to find the best formula of gf rice-field bean pasta with the dfa method. to compare the measured parameters (cooking and textural parameters, hydration index, pasting and thermal properties) between the optimized recipe and the control pasta, the results were analyzed with the anova test at the level of significance of p≤0.05 followed by fisher’s lsd using statistica 10. 3. results and discussion 3.1. chemical composition table 2 shows the chemical composition of the raw materials and pasta samples. as expected, protein, fat, ash, and fiber increased significantly with the addition of field bean to rice-based pasta (p<0.05) because of the leguminous supplementation of rice flour (gimenez et al., 2012; giuberti et al., 2015; giuberti et al., 2016). table 2. proximate composition of raw materials and gluten-free pasta samples (g/100 g). protein fat ash fiber rice 6.72±0.02c 0.14±0.02c 0.42±0.026c 1.24±0.03c field bean 31.40±0.52a 1.38±0.07a 2.53±0.12a 10.88±0.18a rice pasta 6.90±0.02c 0.13±0.04c 0.37±0.05c 1.31±0.03c rice-field bean pasta 14.92±0.03b 0.61±0.03b 1.15±0.05b 4.31±0.10b a-cvalues followed by the same letter in the same column are not significantly different (p=0.05). the enrichment of our formula based on rice with field bean semolina causes an increase of the protein content by 116% and the fiber level in the recipe by 229%. celiac consumers have a low intake of dietary fiber (bouasla et al., 2017), so the proposed formula could be beneficial if having an increased fiber content. legumes supply an important dose of dietary fiber and protein with a good amino acids balance, and the supplementation of cereals with legumes could enhance the nutritional value of the final product. ital. j. food sci., vol. 30, 2018 234 3.1.1 pasting performance of pgrf the pasting properties of pgrf were investigated by the measurements through a viscoamylograph test. the viscograms of raw flour and pgrf were analyzed for comparison as shown in fig. 1. figure 1. pasting profile of native rice flour, and pgrf after 1h and 24 h of preparation and storage. legend: (______) temperature profile; (_______) rice flour; (………..) treated rice 24 h; (-) treated rice 1 h. the results showed that changes in viscosity depended on the treatment conditions. the brabender viscograms showed an increase in the iv of pre-gelatinized rice flour after 1 h and 24 h of preparation (54 mpas, 52 mpas, respectively) as a confirmation of partial gelatinization of starch under a hydrothermal treatment compared to untreated rice flour (17 mpas). the high iv for pgrf was attributed to a modification in the molecular organization of the starch during treatment, which resulted in the loss of granule integrity and breaking of the order of crystallinity of starch (lai and cheng, 2004; marti et al., 2013). the hydrothermal treatment of rice flour exhibited a significant lowering of pv for both treatments (370 mpas and 407 mpas, respectively, for treated rice for 1 h and 24 h of storage) compared to the raw material (606 mpas). the decrease in pv might be related to the limited swelling capacity and its effect was the increase in pt (78.2°c and 78.1°c, respectively, for treated rice flour for 1 h and 24 h) compared to untreated rice flour (70°c). this corresponds with the increased temperature needed to gelatinize undamaged starch granules, as found by hormdok and noomhorm (2007). moreover, the hydrothermal process induced the viscosity reduction during the cooling stage; the same behavior was observed in several studies (adebowale et al., 2005; hoover and vasanthan, 1994; jacobs et al., 1995; stute, 1992; bourekoua et al., 2016). after treatment, the progression and continuation of retrogradation of the starch molecules during storage will take place, which leads to a significant (p<0.05) increase in the viscosity of treated rice flour 24 h after the beginning of storage (827 mpas) compared to treated rice flour after 1 h (655 mpas). this phase is commonly described as the setback region and is related to the retrogradation and reordering of starch molecules. all these observations indicated that a ital. j. food sci., vol. 30, 2018 235 hydrothermal treatment may affect the granule rigidity and starch molecular reassociation. therefore, various treatment conditions applied would give the possibility to obtain a different form of pre-gelatinized flour with different pasting properties that should be used for various applications. 3.2. the effect of pre-gelatinized rice flour on the laminated gf pasta quality 3.2.1 verification of the fitted models for each model, linear, quadratic and interaction effects were calculated. regression coefficients are shown in table 3. adequacy of the model was verified by estimating the coefficient of determination (r2) and the lack-of-fit test. table 3. regression coefficients for the predictive models for cooking loss, water absorption capacity, firmness and stickiness. model term cl (%) wac (%) f (n) s (mj) x1 -0.3115 +0.0829 -0.0073* -0.0086* x2 -0.1631 -0.0755 -0.2998 -0.0028* x1x1 +0.4966 +0.4503 -0.0280* +0.1112 x2x2 +0.0056* -0.0323* -0.0192* +0.0009* x1x2 +0.6541 +0.7510 +0.6851 -0.0149* lack of fit ns ns ns ns f 3.87ns 3.39ns 6.05ns 14.96ns r2 0.73 0.71 0.81 0.91 cl: cooking loss, wac: water absorption capacity, f: firmness, s: stickiness, x1: water, x2: pgrf, x1x1, x2x2: quadratic coefficients, x1x2: interaction between coefficients, f: variance ficher-snedecor, r2: coefficient of determination, ns: not significant (p>0.05), * significant at p≤0.05. the statistical analysis proved that the fitting models were adequate because they yielded satisfactory values of r2, and the lack-of-fit test was not significant for all responses. if the values of a predicted model are not significantly different from real values, and the lackof-fit is not significant, the model is adequate (goupy, 2013). the results of the discussed experiment confirmed that the model was adequate to envisage the responses of cl, wac, f and s. 3.2.2 cooking quality the cl and wac represent the crucial parameters of the cooking quality of pasta. the effect of a range of water and pgrf on these responses was shown on fig. 2a and 2b, respectively. the cl of pasta samples ranged from 9.35 to 14.26%. the cl decreased with an increased addition of pgrf up to the incorporation level of 7 g/100 g, which raised the maximum incorporation as its quadratic effect was positive (p<0.05, table 3). moreover, the chart (fig. 2a) and the data in table 3 demonstrate the absence of the linear and quadratic effect of water on the value of cl. ital. j. food sci., vol. 30, 2018 236 as reported in a paper presented by bouasla et al. (2017), a cooking loss below 10% indicates good quality pasta. moreover, marti et al. (2013) reported a cooking loss for pasta ranging between 3.5 and 11.3%; cham and suwannaporn (2010) found the cl of rice noodles varying from 6.5 to 10.25%; and bouasla et al. (2017) presented the range of 3.5 5.93% of the cl for extruded gf pasta. according to these values, it could be concluded that pasta with an addition of pgrf has sufficient cooking quality as a gf product. figure 2. response surface of cl (a) and wac (b) of gluten-free rfbs pasta depend on hydration level and pgrf amount. as starch is the decisive component of gf pasta, its applicability can be noticeably enhanced by a convenient re-organization (bemiller, 1997). the cooking quality of pasta could be improved by the addition of treated flour containing an appropriate amount of modified starch (hormdok and noomhorm, 2007). the hydrothermal treatment of rice flour discussed in this study probably leads to the formation of a permanent and solid network of retrograded starch within gelatinized starch, which may produce the effect of lesser solubilization of amyloceous components and, therefore, results in a less cooking loss, as found also by resmini and pagani (1983). recently, marti and pagani (2013) announced that the incorporation of 50% of treated rice flour improved the characteristics of pasta in terms of cooking properties. as hypothesized by the above authors, pre-gelatinized flour could perform the role of a binding agent leading to the establishment of a new network around starch granules, thus increasing their resistance to cooking stress, as suggested by pagani (1986). the wac of rfbs laminated pasta ranged from 215.83 to 255.00% (fig. 2b). the wac grew with an increasing addition of pgrf up to the incorporation level of 6 g/100 g, which decreased with the maximum incorporation as its negative quadratic effect (p<0.05, table 3). in addition, the chart (fig. 2b) showed that the wac of the samples increased with an increasing amount of water. the wac is a primordial measured parameter, which depends on the fragments of damaged starch and the fragility of its granules (bouasla et al., 2017). the observed water uptake dynamics suggests that hydrothermal treatment conditions of rice flour in our study might possibly promote a less hydrophilic structure of starch resulting in low ital. j. food sci., vol. 30, 2018 237 water absorption, as suggested by marti et al. (2013). in addition, hormdok and noomhorm (2007) reported that rice starches thermally treated by both hmt and ann slightly reduced the wac of composite noodles. moreover, the low wac indicates a poor quality of cooked pasta due to chewy texture (wang et al., 2012). 3.2.3 textural characteristics the textural properties of pasta are related to the ability to maintain consistency after cooking (larrosa et al., 2016). the response surface obtained for the texture measurement showed that firmness ranged from 216.50 to 344.72 n (fig. 3a). the chart showed an increased firmness of samples along with the increasing amount of pgrf up to the incorporation level of 7 g/100 g, which decreased with the maximum incorporation as its negative quadratic effect (p<0.05, table 3). however, the chart (fig. 3a) showed a decreased firmness of samples with the increased amount of water as its negative linear and quadratic effect (p<0.05, table 3). as demonstrated by the results of bouasla et al. (2017), firmness of extruded gluten-free pasta supplemented with legumes ranged between 199.5 and 326.5 n. marti et al. (2013) reported that firmness of rice pasta ranged from 188 to 902 n., zhao et al. (2005) regarded firmness as work required to shear 4 cooked strands of spaghetti that ranged between 5.72 to 8.64 g/cm. the firmness characteristic of pasta can be evaluated with various methods, so the results are difficult to compare. figure 3. response surface of firmness (a) and stickiness (b) of gluten-free rfbs pasta depend on hydration level and pgrf amount. the response surface in fig. 3b for rfbs pasta showed the stickiness values varying from 9.10 to 25.65 mj. the chart demonstrated decreased stickiness of samples as the amount of pgrf and water increased. moreover, the addition of treated rice represents a negative linear effect, a positive quadratic effect and a positive interaction effect between the two variables (water and pgrf). also, water induced a negative linear effect on this parameter (p<0.05, table 3). heat treatment of rice flour seems to generate a higher regulated crystalline area in starch granules; this induced a lesser leaching of amylose fractions and, consequently, low ital. j. food sci., vol. 30, 2018 238 stickiness. moreover, the modification and re-organization of starch macromolecules as a result of the applied treatment gives rise to the formation of a rigid network. consequently, the strengthened starchy matrix could protect the amyloceous components from increased leaching and, thus, giving an improved texture as exhibited by the low cl values and high firmness. the hydrothermal treatment discussed above partially eliminates the swelling of granules by slowing down gelatinization and enhancing the stability of starch paste (hoover and vasanthan, 1994; hormdok and noomhorm, 2007) and, thus, enhancing the aptitude of cooking and consistency of rice pasta (yoenyongbuddhagal and noomhorn, 2002; marti et al., 2013). in addition, raina et al. (2005) showed that the application of pre-gelatinized rice flour activated the starch arrangements, which guaranteed a good textural quality of both uncooked and cooked rice pasta. fiorda et al. (2013) also reported a significant enhancement and elevated firmness of pasta after the incorporation of pre-gelatinized flour made from blends of cassava starch and bagasse, and the level of pre-gelatinized flour was the most significant component when it comes to pasta stickiness. 3.3. optimization and characteristics of optimum gf pasta 3.3.1. cooking and textural quality the optimum recipe was determined based the desirability method performed by jmp, version 7 (sas, usa). desirability is a useful approach for the optimization of multiple responses in order to select the best recipe. the main objective of optimization was to determine the levels of variables (x1, x2) that produce the best characteristics of gf pasta. good quality of pasta is a combination of high water absorption, low cooking loss, and good texture parameters (high firmness and low stickiness) (bruneel et al., 2010). the criteria used to obtain individual desirability functions, predicted responses and real results are presented in table 4. table 4. the criteria used to obtain desirability functions, predicted responses and individual desirability functions (di). parameter objective predicted response individual desirability values (di) experimental response cl (%) minimum 9.53 a 0.956 9.49 a wac (%) maximum 237.49 a 0.603 234.14 a f (n) maximum 338.09 a 0.891 339.85 a s (mj) minimum 9.14 a 1 9.05 a cl: cooking loss, wac: water absorption capacity, f: firmness, s: stickiness, aexperimental responses with the same superscript than predictive response indicate there are not significant differences (p>0.05). for optimization, the global desirability was selected, and the dfa method for selection of the optimum recipe involved the cl and s as minimized, the wac and f as maximized. based on such a desirability approach, the optimum amounts were found to be 5.845 g of pgrf and 59.266 ml of water for 100 g of rfbs with a value of 0.775. a hydration level is another key factor to be considered in order to obtain good quality pasta. this may be directly related to the fact that hydrating goes with formulation having an impact on the textural properties of pasta. moreover, in the opinion of larrosa et al. (2016), the amount of water used in pasta production should be optimized to achieve the acceptable ital. j. food sci., vol. 30, 2018 239 quality of pasta. it was demonstrated that the insufficient water content produces streaky and/or flaky dough sheet surfaces, resulting in softer texture of cooked pasta. using the above optimum levels of water in the formulation has also adverse quality effects, as dough will be stickier to handle and difficult to sheet tending to produce poor quality finished pasta. the cooking behavior, textural parameters (cutting force, firmness, stickiness and cohesiveness) of the optimized and control gf pasta are shown in table 5. table 5. characteristics of optimum and control gluten-free rfbs. parameters optimum pasta control pasta cooking quality cooking loss (%) 9.49±0.03a 14.26±0.01b water absorption capacity (%) 234.14±7.21a 217.11±0.10b textural parameters cutting force (n) 1.21±0.03a 0.62±0.03b firmness (n) 339.85±0.03a 295.50±1.12b stickiness (mj) 9.05±0.07a 25.00±0.5b cohesiveness (mj) 28.14±0.15a 14.12±0.15b pasting properties pasting temperature (°c) 77.50±0.04a 73.80±0.13b initial viscosity (mpa s) 18.00±0.10a 14.00±0.01b peak viscosity (mpa s) 195.00±0.02a 256.00±0.01b final viscosity (mpa s) 351.00±0.03a 456.00±0.26b setback (mpa s) 160.00±0.07a 205.00±0.02b breakdown (mpa s) 2.05±0.05a 5.00±0.07b hydration properties wai (g/g) 2.93±0.07a 1.15±0.21b wsi (%) 7.38±0.13a 10.85±0.13b sp (-) 3.22±0.95a 5.60±0.01b thermal characteristics to (°c) 50.80±0.04 a 46.93±0.11b tp (°c) 102.84±0.00 a 95.58±0.00b tc (°c) 171.57±0.02 a 169.76±0.24b tr (°c) 120.77±0.01 a 123.76±0.03b δh (j/g) 123.64±1.09a 180.70±1.23b a-bmeans with the same superscript within line are not significantly different (p > 0.05). as described by bruneel et al. (2010), good quality pasta should be characterized by the low cl and stickiness, the high wac and firmness. for commercial durum wheat spaghetti, they reported that the cooking loss ranged between 3.9 and 6.1%, stickiness between 0.2 and 119.9 mn/mm, water absorption capacity between 1.83 and 2.13 g/g dm, and firmness between 5.8 and 7.8 n/mm. the addition of hydrothermally treated rice flour reduced the cl significantly (9.49%) and improved the wac (234.14%) of optimum pasta, and these were found comparable or superior to the control pasta (14.26% of cl, 217.11% of wac, respectively) (p<0.05). for texture measurements, the optimized pasta exhibited higher and better values of cutting ital. j. food sci., vol. 30, 2018 240 force (1.21 n), f (339.85 n) and cohesiveness (28.14 mj) with lower s (9.05 mj) than the control pasta (0.62 n, 295.5 n, 14.12 mj, 25.00 mj, respectively). the presented results of gf pasta are difficult to compare with the durum pasta properties. a good and firm structure of pasta means less quantity of substances going into cooking water (padalino et al., 2013). a hydrothermal treatment of rice flour might stimulate the variation in the physical and chemical properties of starch. these modifications of starch organization are responsible for the new macromolecular structure, resulting in reduced stickiness and cooking loss and an improved texture. 3.3.2 pasting properties integrity of starch granules is commonly investigated by measurements of the pasting behavior of cereals before and after modification under specific treatment conditions. the results of pasting parameters presented in table 5 show some significant differences in the pasting characteristics of optimized formula (pt, iv, pv, fv, bd and set) in comparison with the control pasta without pgrf (p<0.05). higher pt, iv and lower pv, fv, bd and set were noted for the optimum pasta. the observed differences can be explained by the modification and alteration of rigidity of treated starch granules (marti et al., 2010). the reported increase in pasting temperature, a decrease in pv, fv and bd may be attributed to the alterations of the pasting properties in treated flour possibly due to the production of bonds between the chains in the amorphous regions of starch granules as well as modification of crystallinity during the hydrothermal treatment, as reported previously by other authors (adebowale and lawal, 2002; chung et al., 2009; hormdok and noomhorm, 2007; olayinka et al., 2008; zavareze and dias, 2011). additionally, the low pv of the optimum pasta may ensue from the limited starch swelling capacity (hagenimana et al., 2006). the strengthening of intra-granular bonds means that more heat is required for the structural disintegration of starch and formation of paste (olayinka et al., 2008; zavareze and dias, 2011). a reduction in bd after thermal treatment indicates a higher stability of starch exposed to continuous heating, as reported by adebowale et al. (2005). moreover, the lowest recorded value of bd may indicate that pgrf may have a good potential as a food improver for food exposed to heat and mechanical treatment. setback (set) indicating the retrogradation tendency was apparently lower in the optimized recipe than in the control (p<0.05). retrogradation is influenced by the amount of extracted amylose, the size of granules, and the presence of stiff, nondisintegrated swollen granules (lan et al., 2008). additional amylose–amylose and/or amylopectin–amylopectin chain interactions may occur upon the applied hydrothermal treatment, which can result in the cut-down of amylose extraction and decrease of retrogradation. a similar phenomenon of the modifications of pasting parameters of heat-moisture treated potato starch was reported by stute (1992). the reported alterations in pasting characteristics indicated some degree of re-association of starch molecules during the hydrothermal treatment. moreover, the high iv of sample incorporated with pgrf indicates a good wsi, consistent with the value of hydration parameters shown in table 5. this is combined with the effect of heat treatment on the properties of pre-gelatinized rice flour and dextrinization of starch molecules to a high extent, as reported by lai and cheng (2004). upon heating, the bonding force within starch granules can affect the swelling behavior, which explains the rapid increase in viscosity. moreover, the starch hydration properties were greatly affected by the thermal treatment as a consequence of macromolecular disorganization and degradation (nakorn et al., 2009; marti et al., 2013). ital. j. food sci., vol. 30, 2018 241 3.3.3 hydration properties starch solubility is the result of amylose leaching from starch during swelling as dissociating from and diffusing out of granules. this process represents a transition from the ordered to disordered structure of starch granules that occurs during heating in the presence of water (zavareze and dias, 2011). macromolecular disorganization and degradation is the result of alteration of starch hydration properties by the thermal treatment (nakorn et al., 2009). the hydration properties differed significantly between the optimized and control recipe (p<0.05) – the results are shown in table 5. a significant increase of the wai in the optimum sample as compared to the control results probably from a greater possibility of exposed hydrophilic groups to create bonds with water molecules during the formation of gel. in addition, this is the result of partial starch gelatinization occurring during the treatment, as suggested by lai and cheng (2004). moreover, the reduction in the sp and wsi were observed compared to the control sample without pgrf. waduge et al. (2006), jacobs et al. (1995) and hoover and vasanthan (1994) explained that a decrease in the sp may result from the increased crystallinity and interactions between amylose and amylopectin molecules. the authors also showed that this phenomenon could be attributed to enhanced intra-molecular bonds and modifications of the crystalline structure of starch. in addition, gomes et al. (2005) described the effect of increased molecular organization on the reduction of hydration properties of starch. also, the same group suggested that the reduction in the solubility of annealed starch was due to the enhancement of the formation of bonds between amylose and amylopectin or between amylopectin molecules, inhibiting the extraction of starch granules. the hydrothermal treatment applied in our work probably leads to the re-organization of the structure attributed to the changes in starch granule rigidity under treatment, which entails other specific interactions between starch molecules (hoover and manuel, 1996; lai, 2001) such as the formation of more ordered double helical amylopectin side-chain clusters and amylose–lipid complexes located within granules. a similar phenomenon of decreased swelling power and solubility was reported in other studies (hoover and manuel, 1996; olayinka et al., 2008). 3.3.4 thermal properties the differential scanning calorimetry (dsc) is useful in the characterization of starch gelatinization as it detects the temperature of the different stages of this process. the onset temperature (t0) indicates the beginning of the gelatinization process, peak temperature corresponds to the maximum heat flow (tp), thus indicating the temperature of main phase transition, and the endset temperature indicates the end of the process of gelatinization (tc). the difference between endset and onset temperatures is the information about the length of the gelatinization process. the thermal parameters characterizing the gelatinization process of the optimum and control pasta measured by the dsc are presented in table 5. the obtained parameters significantly varied between the control and optimized recipe (p<0.05). as shown in table 5, the optimum pasta was characterized by a higher onset, endset and peak temperatures (to=50.80, tc=171.57 and tp=102.84ºc) as compared to the control (46.93, 169.76 and 95.58ºc, respectively). this indicates that a higher temperature is needed to start the gelatinization process, and that the main transition occurs at a higher temperature. on the other hand, a significant decrease in the length of transition (gelatinization process) was observed – as indicated by the shortening of tr distance in the optimum sample. ital. j. food sci., vol. 30, 2018 242 crystalline and amorphous transitions influence the shape of experimental curves representing the gelatinization process (cham and suwannaporn, 2010). the analysis of thermal results showed some distinctive changes in the heat-treated internal granular structure of starch, which revealed a higher stability. similarly, the hydrothermal treatment of rice flour admixed to pasta produced a more homogenous structure of starch crystallite during melting, swelling and hydration and resulted in the formation of new kinds of starch crystallites displaying dissimilar heat stability, as indicated by hordmok and noomhorm (2007). besides, the neighboring amorphous region can govern the melting temperatures of starch crystallite indirectly. an addition of treated rice flour to pasta would increase the stability of amorphous regions during crystallite melting. as a consequence, a higher temperature is required to melt crystallites of pgrf. increased to, tp, and tc can be explained by the structural changes of starch granules and the associated interactions between amylose or amylose–lipid (hoover and vasanthan, 1994). adebowale et al. (2009) found that for starch granules, the hydration and swelling of the amorphous regions is driven by gelatinization, which involves the melting of crystalline regions and double helices. during the swelling of the amorphous regions, stress is induced on the crystalline regions as well as on the polymer chains and the former is removed from the starch crystallite surface. subsequently, treated starches require a higher temperature for the swelling and breaking of crystalline areas leading to the increased to, tp, and tc, which was also confirmed in our study. as an accompanying effect, a decrease in enthalpy of the temperature-induced gelatinization process was observed in the optimum sample (123.64 j/g) as compared to the control (180.70 j/g). it is obvious that the reduction in δh following the hydrothermal treatment indicated partial gelatinization of some fractions of molecules having smaller heat stability, as suggested by stute (1992) and hordmok and noomhorm (2007). also, the decrease in δh can be interpreted as a presence of disorganized double helices of starch granules revealing a crystalline and non-crystalline character under the treatment conditions. thus, some reductions in the fraction of unbend and melted double helices during gelatinization would be noticed after treatment. 3.4. sensory attributes the sensory characteristics of cooked gf pasta are reported in table 6. the sensory evaluation showed no significant difference (p>0.05) in taste, color and flavor between the gf control and the optimum pasta. the obtained results showed that the optimum recipe pasta was significantly better with the highest scores for appearance and stickiness. table 6. sensory evaluation and overall acceptability of gluten-free pasta. appearance1 color1 flavor1 taste1 stickiness1 overall acceptability2 control pasta 3.75±0.34 b 4.18±0.21a 3.45±0.34a 4.40±0.43a 3.17±0.40b 4.42±0.33b optimum pasta 4.45±0.46 a 4.22±0.31a 3.35±0.33a 4.42±0.42a 4.52 ±0.48a 6.42±0.24a 15-point scale, 29-point hedonic scale (n = 55). a-bmeans with the same superscript within a column are not significantly different (p > 0.05). ital. j. food sci., vol. 30, 2018 243 the best scores were recorded for appearance and stickiness of the optimum pasta, as confirmed by the texture measurements; it can be attributed to the enhancing effect of the hydrothermal treatment of rice flour added to the pasta recipe in our study. moreover, the selected optimum pasta gathered superior scores (values above 5) in the overall acceptability in comparison with the control sample without the addition of pgrf. 3.5. microstructure of dry pasta the microscopic pictures can be helpful in analyzing the appearance, texture or integrity of food products (gorinstein et al., 2004). the surface of traditional laminated gf pasta is presented in fig. 4. figure 4. sem surface of laminated pasta from control and optimized recipe at different magnifications; control pasta: a) x200, b) x600, optimum pasta: c) x200, d) x600. the analysis of the micrographs of pasta manufactured from the rfbs recipe without pgrf (fig. 4a) exhibits the presence of small cracks with a small amount of empty spaces, which suggests some lack of continuity due to the absence of gluten. also, the graphs showed the structure of poorly agglomerated starch granules and separate granules visible on the pasta surface (fig. 4b). the microstructure images, presented in fig. 4c, highlighted the differences in starch organization and collocation observed on the surface of traditional pasta laminated from the optimized recipe with the addition of pgrf. it was ital. j. food sci., vol. 30, 2018 244 characterized by a compact and homogeneous matrix. the agglomerates of starch and proteins are surrounded by pre-gelatinized starch, which forms a continuous phase responsible for the better results of the cl and a low quantity of starchy components passing into the cooking water. pre-gelatinized rice flour forms aggregates combine all pasta components, which is clearly visible at high magnification (fig. 4d). similar morphological features could be observed and confirmed by analyzing the cross-sectional microstructure of pasta (fig. 5). figure 5. cross-section of control rfbs pasta: a) x200, b) x600, c) x1500, and optimum pasta with the addition of pgrf: d) x200, e) x600, f) x1500 observed with sem. ital. j. food sci., vol. 30, 2018 245 fig. 5a and 5b show the irregular and discontinuous internal structure of rfbs pasta with a small amount of swollen starch granules and visible higher dimensions, and only few places are linked by a protein fibrous fraction that comes from field bean flour (fig. 5c). a more compact and uniform structure can be observed if pre-gelatinized rice flour was used as a pasta improver (fig. 5d). the incorporation of pgrf caused the ultra-structural reorganization of matrix and influenced the excellent microstructure. comparing the size and amount of visible starch granules, pasta with the addition of treated rice flour showed the lower amount of free starch and a more uniform distribution of components inside the tread (fig. 5e). starch granules are surrounded by a continuous phase of gelatinized starch responsible for the limited leaching of components during cooking and the enhanced consistency of pgrf-improved pasta (fig. 5f). 4. conclusions laminated pasta based on rice and enriched with field bean semolina provides an important quantity of dietary fiber and protein with a positive amino acids balance. the supplementation of cereals with legumes is likely enhance the nutritional value of glutenfree pasta products. optimization made by an experimental design showed that the amount of water and improver are the important factors having an influence on the quality of gluten-free pasta. the optimum formulation of rice and field bean containing 5.845 g of pre-gelatinized rice flour and 59.266 ml of water was selected based on the desirability function approach with the value of 0.775, which showed the optimum pasta properties. the hydrothermal treatment of rice flour resulted in the changes to the starch structure and the physical properties of pgrf pasta, such as the cooking and texture parameters, pasting and hydrations properties, thermal features, microstructure, and sensory profile. the obtained results also showed that the addition of pre-gelatinized flour induced significant differences (p<0.05) in all parameters in comparison with the control pasta. the pasta processed with the optimized formula was characterized by the improved cooking quality and texture. as regards the sensory evaluation, the optimum recipe showed acceptable scores for all the sensory attributes and the overall quality of glutenfree pasta. optimized gf rice and field bean pasta obtained using hydrothermally treated rice flour as the improver allow the manufacture of gluten-free laminated pasta with improved nutritional characteristics and appropriate quality 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a review. carbohydr. polym. 83:317-28. paper received june 10, 2017 accepted october 23, 2017 ijfs#980_bozza ital. j. food sci., vol. 30, 2018 37 paper homemade tomato sauce in the mediterranean diet: a rich source of antioxidants a. ricci, e. antonini and p. ninfali* department of biomolecular sciences, university of urbino “carlo bo”, via saffi 2, 61029 urbino, pu, italy *e-mail address: paolino.ninfali@uniurb.it abstract the basic ingredients used to make the italian soffritto were studied in order to define the polyphenol, antioxidant capacity and lycopene content of homemade or commercial tomato sauces, as well as their contribute in whole wheat or refined wheat pasta. the addition of aromatic herbs to sauces increased polyphenols and antioxidant capacity, with basil providing the biggest boost, whereas ready-made commercial tomato sauces showed the lowest antioxidant values. cooked whole wheat pasta with homemade tomato sauce offers an enormous amount of antioxidants, which could protect against oxidative stress. keywords: antioxidant activity, extra virgin olive oil, lycopene, polyphenols, tomato sauces, vegetables and aromatic herbs ital. j. food sci., vol. 30, 2018 38 1. introduction the mediterranean diet (med diet) is based on the daily consumption of fresh vegetables, aromatic herbs, whole grains and extra virgin olive oil (evoo), cooked quickly to conserve the molecular integrity of their nutrients (bertuccioli and ninfali, 2014). indeed, most of the beneficial effects of the med diet stem from the high level of vitamins, carotenoids and in particular polyphenols, found in homemade mediterranean cuisine (dragsted, 2003). soffritto (sauté) is the italian world that perfectly describes the process of gently cooking vegetables in oil to soften them and release their flavours. during the preparation of a sauté, onions, garlic, celery and carrots are chopped and gently sautéed in extra virgin olive oil (evoo) for some minutes. the addition of the tomato (solanum lycopersicum) sauce is another important step in the preparation. forty polyphenols and seven carotenoids have been identified in a typical med diet tomato sauce, whose composition has attracted the interest of food professionals for its nutritional and chemo preventive value (martí et al., 2016; shen et al., 2007; vallverdú-queralt et al., 2013). tomatoes contain several micronutrients, including vitamin c, vitamin e, folates, phenolic compounds and lycopene (vallverdú-queralt et al., 2012). beyond differences in their nutritional contents, due to the tomato cultivar and agronomic conditions, the antioxidant compounds and lycopene concentration found in processed tomato products is markedly affected by the average maturity stage of the bulk of processed tomatoes (cooperstone et al., 2015; ghasemzadeh et al., 2016; gómez et al., 2001; guine and goncalves, 2016; raffo et al., 2002; zanfini et al., 2016). in the med diet tomato sauce, the addition of aromatic herbs, mainly basil, oregano and marjoram, near the end of the cooking represents an important step able to enhance its taste and flavor (guine and goncalves, 2016). basil (ocimum basilicum) contains more than 200 bioactive compounds, including monoterpenes, phenolic acids, steroids, vitamins (a, c, e, k) and flavonoids (ghasemzadeh et al., 2016). oregano (origanum virens) mainly contains carvacrol, cinnamaldehyde and essential oils and marjoram (origanum marjorana) is rich in phenolic acids, flavonoids and essential oils (guine and goncalves, 2016). the med diet tomato sauce may play a key role in conferring higher antioxidant activity to the pasta dish. antioxidant activity is an important parameter in assessing the quality of products, as it measures the global antioxidant system of the product and appears to be closely related to the prevention of oxidative stress linked diseases (ghiselli et al., 2000). pasta is the popular worldwide product made with durum wheat semolina. remarkable total antioxidant capacity is attained when pasta is made with whole grain flour, due to the higher polyphenol content of whole grains compared to refined wheat (antonini et al., 2017; liu, 2007). the aim of this study was to evaluate the polyphenol, orac and lycopene values of four homemade tomato sauces containing evoo, fresh vegetables, tomato puree, and different types of aromatic herbs. these homemade sauces were compared with their commercially available industrially produced counterparts. whole wheat pasta was compared with refined wheat pasta, to rank their respective polyphenol and orac values. ital. j. food sci., vol. 30, 2018 39 2. materials and methods 2.1. chemicals folin-ciocalteu reagent, trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), aaph (2,20-azobis(2-methylpropionamidine) dihydrochloride), fluorescein (2-(3hydroxy-6-keto-xanthen-9-yl)benzoic acid) and caffeic acid were purchased from sigmaaldrich, inc. (st. louis, mo, usa). sodium carbonate (na2co3) was supplied by carlo erba reagents (milano, mi, italy). acetone, ethanol, ethylacetate, methanol, n-hexane were purchased from vwr international inc. (radnor, pa, usa). 2.2. samples fresh vegetables, garlic (cv. aglio bianco), celery (cv. plein blanc pascal), carrots (cv. flakkè 2), onions (dorata cv. dorata di parma; tropea cv. tropea rossa tonda record; suasa cv. cipolla di suasa), aromatic herbs (basil, oregano and marjoram), tomato puree (glass bottle, 750 g) and sunflower seed oil (plastic bottle, 1 l) were purchased from the local supermarket. artisanal evoo (cv. coratina) was supplied by a producer from apulia. 2.3. chemical quality parameters of evoo regarding the chemical quality parameters of evoo, free acidity (given as % of oleic acid) and peroxide values (meqo2/kg of oil) were evaluated according to the european economic community regulation no. 2568/91 and its later modifications (eec, 1991). 2.4. homemade tomato sauce preparation the sauces were prepared as follows (portions for 4 people). sauce 1. evoo (30 g), onion (dorata, 50 g), celery (25 g), carrots (25 g), garlic (2 g). the mixture was heated 10 min at 180°c, tomato puree (180 g) was then added and cooked for an additional 10 min. sauce 2. the same steps and ingredients as sauce 1 with the addition of basil (2 g), which was added at the same time as the tomato puree. sauce 3. the same steps and ingredients as sauce 1 with the addition of oregano (2 g), which was added at the same time as the tomato puree. sauce 4. the same steps and ingredients as sauce 1 with the addition of marjoram (2 g), which was added at the same time as the tomato puree. the sauces were cooked on electric or gas heating plates and the temperature was controlled by a thermometer. the evaporation of the water reduced the weigh from about 300 g to 200 g, thus allowing the dressing of 4 pasta dishes. eight samples of industrially produced sauces where purchased at a local supermarket. average composition was: tomato pulp (75%), tomato concentrate, onion, olive or seed oil, basil (2%), carrots, sugar, salt, celery, natural basil aromas. refined wheat pasta (rwp) was purchased from the local supermarket, whereas whole wheat pasta (wwp) was made by a local producer with whole grain (triticum durum, cv. odisseo), milled with a stone mill (sifting rate 15%), extruded using bronze dies and dried at 45°c. table 1 shows the nutritional data of rwp and wwp, as reported by the manufacture’s indications, as well as those of sauce 1 obtained with a nutritional software. ital. j. food sci., vol. 30, 2018 40 table 1. macronutrient composition and fiber in the typical med diet pasta dish. nutritional data per serving* sauce 1 (50 g) rwp (80 g) wwp (80 g) sauce 1 (50 g) + rwp (80 g) sauce 1 (50 g) + wwp (80 g) energy (kcal) 55 282 259 337 314 carbohydrates (g) 1.8 63.3 53.0 65.1 54.8 proteins (g) 0.6 8.7 10.7 9.3 11.3 lipids (g) 5.1 1.1 2.0 6.2 7.1 fiber (g) 0.8 2.2 5.1 3.0 5.9 *per serving: 50 g of sauce 1; 80 g of rwp or wwp. rwp, refined wheat pasta; wwp, whole wheat pasta. 2.5. lycopene extraction and assay the lycopene analysis was performed as previously described (davis et al., 2003; periago et al., 2004), with some modifications. briefly, 2 g of sauce was mixed with 4 ml of distilled water and homogenized for 5 min with a potter homogenizer. then 1 g of homogenate was extracted with 20 ml of hexane:acetone:ethanol (2:1:1) mixture. the solution was shaken for 10 min and then centrifuged at 1800 x g at 5°c for 10 min. the absorbance of the supernatant was measured at 472 nm. a calibration curve was prepared with lycopene pure powder (sigma-aldrich inc., st. louis, mo, usa). values were expressed as mg of lycopene per 100 g of sauce. 2.6. extraction of polyphenols from vegetables and sauces this analysis was performed following the procedure previously described (agbor et al., 2014), with some modifications. each sample (11 g) was homogenized with the potter homogenizer. then 0.5 g of homogenate was added to 8 ml of 50% methanol/water containing 1.2 m hcl. the sample was heated for 2h at 95°c and then centrifuged at 1000 x g at 5°c for 10 min. the supernatant (sn1) was brought to 10 ml with distilled water. 2m naoh (8 ml) was added to the pellet and the mixture was shaken for 2 h at room temperature and centrifuged at 1000 x g at 5°c for 10 min. the supernatant (sn2) was transferred into a flask and brought to 10 ml with distilled water. both supernatants were combined (sn1 + sn2), and this solution (20 ml) was used for the total polyphenol and antioxidant capacity determination. 2.7. extraction of polyphenols from oils this analysis was performed as previously reported (antonini et al., 2016a). briefly, the oil sample (3 g) was mixed with 5 ml of 80% methanol. the solution was vortexed for 2 min and then centrifuged at 1000 x g at 5°c for 10 min. the supernatant was transferred into a falcon tube at 4°c. the extraction was repeated twice, and the two supernatants were combined and preserved for the polyphenol and antioxidant capacity assay. 2.8. extraction of polyphenols from raw and cooked pasta whole wheat pasta (wwp) and refined wheat pasta (rwp) samples (80 g) were cooked for 10 min in 2 l of boiling water. raw and cooked pasta was freeze-dried and milled in a zm 200 ultracentrifugal mill with a 0.5 ring sieve (retsch, haan, germany). free and bound ital. j. food sci., vol. 30, 2018 41 polyphenols were extracted from the freeze dried material as previously reported (antonini et al., 2016b). the extracts were used for polyphenol and antioxidant capacity assays. 2.9. polyphenols assay the phenol compounds were assayed using the folin-ciocalteu method, as previously reported (singleton et al., 1999). the absorbance of the mixture was measured at 725 nm. a calibration curve was prepared with caffeic acid (sigma-aldrich inc., st. louis, mo, usa). for vegetables, polyphenol values were expressed as mg/100 g of fresh weight; for oils, as mg/kg; for sauce, as mg/100g of product. for raw and cooked pasta, total phenol values were obtained by the sum of free + bound phenols and expressed as mg/80 g dry weight (d.w.). moisture was determined in the raw and cooked pasta using a thermal balance (sartorius ma 40, gottingen, germany) after drying at 120°c to constant weight. 2.10. oxygen radical absorbance capacity (orac) assay the antioxidant capacity of phenols was determined by the orac method (ninfali et al., 2005; prior et al., 2003), using a fluostar optima plate reader fluorimeter (bmg labtech, offenburgh, germany) equipped with a temperature-controlled incubation chamber and an automatic injection pump. the incubator temperature was set at 37°c. the following mix was used for the hydrophilic orac (h-orac): 200 µl of 0.096 µm fluorescein sodium salt in 0.075 m na-phosphate buffer (ph 7.0), 20 µl of sample or trolox. the reaction was initiated with 40 µl of 0.33 m aaph. the blank was 0.075 m naphosphate buffer (ph 7.0). fluorescence was read at 485 nm ex. and 520 nm em. until complete extinction (ninfali et al., 2005). a calibration curve was made each time with the standard trolox in 0.075 m na-phosphate buffer (ph 7.0). the lipophilic orac (l-orac) for the antioxidant contribution of lycopene and liposoluble vitamins was measured as follows. the sauce was extracted with hexane (1:5 w/v) twice. after centrifugation at 1800 x g for 10 min, the supernatants were combined and dried under nitrogen flow, then re-suspended in 1 ml of 50% acetone, which was mixed with 7 ml of 7% hydroxypropyl β-cyclodextrin (kleptose hp oral grade, roquette, france) in 50% acetone (ou et al., 2013). after the incorporation of the lipophylic extract into the β-cyclodextrins by rotating overnight in the dark, the solution was centrifuged and read with the fluostar optima plate reader fluorimeter, with the same reaction mixture of the h-orac and the blank with 20 µl of 7% β-cyclodextrins in 50% acetone, diluted with phosphate buffer. a calibration curve was made each time with the standard trolox in β-cyclodextrins. for vegetables, orac values were expressed as µmol trolox equivalents (te)/100 g of fresh weight; for oils, as µmol te/kg; for sauce, as µmol te/100g of product. for raw and cooked pasta, total orac (free + bound phenols) values were expressed as µmol te/80 g d.w. 2.11. statistical analysis the chemical parameters of oils were detected in triplicate and values were expressed as the mean±sd. polyphenol and lycopene concentrations were measured in triplicate and results were the mean±sd. orac data were obtained by eight independent determinations for each sample and results were the mean±sd. statistical significance was ital. j. food sci., vol. 30, 2018 42 tested using student’s t test and one-way anova, with a p ≤ 0.05 indicating a significant difference between data sets (spss® 17.0 software, ibm, chicago, il, usa). 3. results and discussions 3.1. antioxidant content and activity of raw and sautéed vegetables we first investigated the total phenols and antioxidant capacity of the individual vegetables after they had been sautéed for 10 min in evoo to assess how each vegetable was stable during the sautéing process. results are reported in fig. 1. figure 1. polyphenols (a) and antioxidant capacity (b) in raw and evoo sautéed vegetables. the cooking time was 10 min at 180±5°c. (*) indicates statistically significant differences (p ≤ 0.05) between raw and sautéed vegetables (student’s t test). after the vegetables had been sautéed in evoo and placed on filter paper for few minutes to get rid of excess oil, their total phenols (fig. 1a), as well as their antioxidant capacity (fig. 1b), increased remarkably compared to their raw values. the increase was due to the penetration of the oil into the vegetable tissues and the evaporation of the water from ital. j. food sci., vol. 30, 2018 43 those tissues. moreover, the polyphenols bound to the fiber were freed by the thermal disruption of chemical bonds and new molecules, including maillard products, may have been formed (frati et al., 2016; nicoli et al., 1999; santos et al., 2013). in the three sautéed onion cultivars (dorata, tropea, suasa), there was an average increase of 70% in polyphenols and orac values compared to raw values, but no statistically significant difference between the three cultivars was observed (fig. 1). as onion is the major vegetable in the soffritto, possibly the study of other genetic varieties would be an important issue, for increasing the antioxidants in the sauté. 3.2. chemical and antioxidant parameters in evoo and seed oil after cooking in line with the best tradition of the med diet, we only used evoo for sautéing the vegetable mixture. however, as many people and many industrial companies use seed oil, we analyzed the chemical modifications of the evoo in comparison with sunflower seed oil after sautéing in the absence or in the presence of the vegetable mixture. table 2 shows the quality parameters of oils: acidity, peroxide number, polyphenols and orac. table 2. quality parameters of raw and sautéed evoo and sunflower seed oil in the presence or in the absence of the vegetable mixture. acidity (% oleic acid) peroxide number (meqo2/kg) polyphenols (mg/kg) orac (µmolte/kg) evoo raw 0.68±0.05 a 5.01±0.47 c 360±20 a 15,600±940 a sautéed alone† 0.66±0.07 a 22.17±1.02 a 200±11 c 8,580±500 c sautéed with vegetables‡ 0.60±0.05 a 10.02±0.98 b 250±15 b 9,900±600 b sunflower seed oil raw 0.11±0.02 b 1.02±0.14 c 20±0.3 a 678±40 a sautéed alone† 0.22±0.03 a 38.47±2.90 a 3.0±0.5 c 68±4 c sautéed with vegetables‡ 0.18±0.04 a 27.61±3.11 b 8.0±0.4 b 151±14 b a,cdifferent letters indicate, for each quality parameter, statistically significant differences among raw, sautéed alone or sautéed with vegetables evoo or sunflower seed oil (p ≤ 0.05; one-way anova). †evoo or sunflower seed oil were sautéed 15 min at 180±5°c in the absence of vegetables; ‡evoo or sunflower seed oil were sautéed 15 min at 180±5°c in the presence of the vegetable mixture (the same used to prepare the homemade tomato sauce: onion, celery, carrots and garlic). the acidity of raw sunflower seed oil was comparatively smaller than that of raw evoo, due to the neutralization step during refining (casal et al., 2010). nevertheless, in the sautéed evoo, the acidity remained unchanged, whereas it increased in the sautéed sunflower seed oil (table 2). concerning the peroxide number, table 2 shows that the thermal treatment led to a moderate increase in the evoo and a marked increase in the sunflower seed oil, due to the high polyunsaturated fatty acid concentration in the latter, highly susceptible to the oxidation (casal et al., 2010). moreover, the vegetables protected the oils from oxidation, limiting the increase of the peroxide number, with respect to the oils sautéed alone (table 2). ital. j. food sci., vol. 30, 2018 44 a similar trend was obtained for polyphenols and orac values, as the vegetables protected the oils from the decay of the two parameters during the thermal treatment (table 2). therefore, the evoo remains the preferred option for making sauces for three main reasons: 1) higher concentration of polyphenols (ninfali et al., 2002); 2) reduced phenol and orac losses in the presence of the vegetables; 3) higher stability of the peroxide index. 3.3. homemade and commercial tomato sauces and lycopene values after the addition of the tomato puree to the fried vegetables, we evaluated total orac, including both hydrophilic (h-orac) and lipophilic (l-orac) fraction, the polyphenol and lycopene values of the homemade (sauces 1-4) and industrially produced (commercial) sauces. results are reported in table 3. table 3. polyphenols, antioxidant capacity and lycopene in homemade and commercial sauces. polyphenols h-orac l-orac total orac lycopene (mg/100 g) (µmolte/100 g) (mg/100 g) sauce 1 207±9 c 10,184±509 c 886±44 c 11,070±303 c 18.72±1.12 a sauce 2 298±8 a 16,216±811 a 1,604±80 a 17,820±730 a 21.22±1.27 a sauce 3 250±12 b 13,800±690 b 1,200±110 b 15,000±403 b 17.92±1.43 a sauce 4 274±13 b 13,431±671 b 1,011±85 b 14,442±397 b 18.45±1.66 a commercial 134±18 d 6,595±330 d 573±29 d 7,168±353 d 13.70±1.20 b a,ddifferent letters indicate statistically significant differences among samples (p ≤ 0.05; one-way anova). commercial values are the average of 8 commercial ready-to-eat sauces; h-orac, hydrophilic orac; lorac, lipophilic orac; total orac, given by the sum of hand l-orac. the addition of tomato puree to the sautéed vegetables confers to the sauces many powerful antioxidants, including flavonoids present in the tomato puree (vallverdúqueralt et al., 2012), as well as lycopene, which largely contributes to the lipophilic antioxidant capacity (l-orac) (cano et al., 2003). the h-orac, which correlates to the ascorbic acid content (cano et al., 2003), contributed to the highest antioxidant capacity (table 3), representing more than 90% of total orac (wu et al., 2004). regarding the lycopene content, no statistically significant difference was observed among the homemade sauces (p > 0.05) (table 3). all of the aromatic herbs, namely basil, oregano, and marjoram, increased the polyphenol and antioxidant capacity values of the tomato sauce, with the sauce 2, with basil, showing the highest polyphenol and total orac values (table 3). basil leaves were found to yield the highest increase, possibly due to the herb’s high phenol and flavonoid contents (guine and goncalves, 2016; ninfali et al., 2005). the commercial sauces had significantly lower polyphenol, orac and lycopene values than do homemade sauces (table 3). although only eight commercial samples were assessed, our data show that homemade sauces have greater nutritional value than do their store-bought counterparts. the lower nutritional value of commercial sauces may be due to the use of seed oil instead of evoo, non homogeneously ripened fruits, concentrated ingredients stored for long periods, rough processing technologies in the sauce preparation (ninfali and bacchiocca, 2004). ital. j. food sci., vol. 30, 2018 45 hence, the ability to make homemade tomato sauces using fresh vegetables and aromatic herbs is part of the cultural background and has important health implications (hoffman and gerber, 2015; prior et al., 2007; valussi, 2012). the experiences linked to the food literacy project (https://dining.harvard.edu/foodliteracy-project), promoted by the harvard university and the lifelong learning by the european union commission, are focused on the increase of the consumers’ consciousness in the culinary practices and sustainable nutrition. in this light, table 4 summarizes the best ways to select, cook and store the vegetables to be used in the homemade sauce. 3.4. antioxidants in pasta with tomato sauce fig. 2 shows the concentration of free and bound polyphenols (fig. 2a) and orac (fig. 2 b) values in whole wheat pasta (wwp) and refined wheat pasta (rwp), under raw or cooked conditions. figure 2. free and bound polyphenols (a) and antioxidant capacity (b) in raw and cooked whole wheat pasta (wwp) and refined wheat pasta (rwp). polyphenol values are expressed as mg/80 g of raw and cooked pasta, on dry weight (d.w.). orac values are expressed as µmol of trolox equivalents (te)/80 g of raw and cooked pasta, on d.w. a,b different letters indicate statistically significant differences among raw and cooked rwp and wwp (p ≤ 0.05, student’s t test), for each component (free and bound). given that the average portion per meal, for a healthy adult, is 80 g of pasta (http://www.sinu.it/public/20141111_larn_porzioni.pdf), the values are reported per serving of raw and cooked pasta, on dry weight. concerning the raw pasta, wwp showed significantly higher total polyphenol (fig. 2a) and orac (fig. 2b) values than rwp. when the free and bound polyphenols were compared, the bound were predominant in wwp with respect to rwp, due to the loss of polyphenols in the refining process (antonini et al., 2017; panato et al., 2017). after cooking, total polyphenols and orac values remained almost unchanged in the wwp, whereas they were reduced by 50 and 70%, respectively, in the rwp. in the cooked rwp, the free polyphenols and orac were markedly reduced, as compared to the bound ones (fig. 2). this aspect is linked to the presence of the fiber which preserve the polyphenols during cooking (liu, 2007). ital. j. food sci., vol. 30, 2018 46 table 4. nutritional values of sauce ingredients with guidelines regarding their selection, storage and cooking. vegetable/ aromatic herb main antioxidants heat resistance freshness index seasonality storage references carrot carotenoids, hydroxycinnamic acids, anthocyanidins, vitamin c β-carotene is heat resistant and becomes more bioavailable through cooking in oil roots should be firm, smooth, bright in color all year up to two weeks if stored without leaves in the refrigerator (lemmens et al., 2011) (seljåsen et al., 2013) celery phenolic acids, flavonoids, stilbenoids, furanocoumarins, phytosterols, vitamin c, β-carotene heat for as little time as possible (5-15 min) crisp, tight and compact, stalks with pale bright green leaves all year fresh for 5-7 days in the refrigerator (yao et al., 2011) (ovodova et al., 2009) garlic allicin (thiosulfinate) heat for as little time as possible (5-15 min) plump with unbroken skin all year fresh for 1 month stored in a cool, dark place (martins et al., 2016) onion quercetin, vitamin c heat for as little time as possible (5-15 min) well shaped, no opening at the neck, with crisp, dry outer layer of skin all year fresh for four weeks in a dark ventilated space at room temperature (bystrická et al., 2013) tomato lycopene, β-carotene, hydroxycinnamic acids, flavonoids, vitamin c and e, folate lycopene is heat resistant and becomes more bioavailable through cooking in oil red, well shaped and smooth skinned from may to september up to two weeks at room temperature and out of direct exposure to sunlight, depending on ripeness (gómez et al., 2016) (vallverdúqueralt et al., 2012) basil phenolic acids, flavonoids, βcarotene, vitamins (a,c,e,k) essential oils add near the end of the cooking to retain its maximum essence and flavor the leaves should be deep green in color in the summer fresh, wrapped in a paper towel in the refrigerator; dried, in a sealed glass container in a cool, dark place (ghasemzadeh et al., 2016; lee et al., 2005) oregano carvacrol, cinnamaldehyde, essential oil add fresh near the end of the cooking; at the beginning if dried firm stems with fresh leaves, green in color all year fresh, wrapped in a paper towel in the refrigerator; dried, in a sealed glass container in a cool, dark place (teixeira et al., 2013) (guine and goncalves, 2016) marjoram see oregano ital. j. food sci., vol. 30, 2018 47 4. conclusions this study focuses on the contribution in terms of phenols, antioxidant capacity and lycopene of homemade tomato sauce, a staple of the med diet. for the sake of transparency, the food industry, which produces ready-made sauces, should rank the quality of the ingredients used in their sauces, as well as the impact of industrial processing technologies, used in their production by measuring polyphenols and antioxidant capacity before and after processing. the main 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g.r., holden j.m., haytowitz d.b., gebhardt s.e. and prior r.l. 2004. lipophilic and hydrophilic antioxidant capacities of common foods in the united states. j. agric. food chem. 52:4026. yao y. and ren g. 2011. effect of thermal treatment on phenolic composition and antioxidant activities of two celery cultivars. lwt food sci. technol. 44:181. zanfini a., franchi g.g., massarelli p., corbini g. and dreassi e. 2016. phenolic compounds, carotenoids and antioxidant activity in five tomato (lycopesicon esculentum mill.) cultivars. ital. j. food sci. 29:424. paper received july 24, 2017 accepted september 20, 2017 ijfs_2010.indd p u b l i c a t i o n s codon italian journal of food science, 2022; 34 (1): 1–12 issn 1120-1770 online, doi 10.15586/ijfs.v34i1.2010 1 effects of drying methods and acidic strength on physicochemical properties of potassium caseinate fatma maruddin1*, wahyu triputra hasim1, nursida1, ratmawati malaka1, hikmah muhammad ali1, muhammad taufik2, syahriana sabil3 1department of animal production, faculty of animal science, hasanuddin university, makassar, south sulawesi, indonesia; 2agricultural development polytechnic of gowa, south sulawesi, indonesia; 3animal science study program, faculty of agriculture, animal science and forestry, maros moslem university, maros, south sulawesi, indonesia *corresponding author: fatma maruddin, department of animal production, faculty of animal science, hasanuddin university perintis kemerdekaan street km 10, makassar, south sulawesi 90245, indonesia. email: fatma_maruddin@ yahoo.co.id/fatma_maruddin@unhas.ac.id received: 22 january 2021; accepted: 19 october 2021; published 1 january 2022 © 2022 codon publications open access paper abstract the aims of the present study were to characterize physicochemical characteristics and chemical structures by fourier transform infrared (ftir), and mark dissolved protein content, microstructure, and moisture content of potassium caseinate prepared by drying methods and acid strength. the experiment was arranged according to factorial complete randomized design with triplicates, while data from ftir and microstructure analysis was presented descriptively. the results demonstrated that acids and drying methods for preparing potassium caseinate could increase antioxidant activity, a* score (reddish) and b* score (yellowish). specifically, freeze-drying method coupled with acid treatments accounted for reducing moisture content but improved viscosity and microstructural properties. briefly, we could argue that drying techniques and acids established noticeable effects on the quality of potassium caseinate. keywords: acid; drying method; potassium caseinate; quality introduction potassium caseinate is derived from casein with aqueous potassium hydroxide (koh) used to dissolve it (de souza et al., 2010). casein itself is a milk protein and contains large amount of essential amino acids as commonly found in other types of casein products. the ph of potassium caseinate is close to neutral, which makes it favorable for food processing. casein is widely employed in various foods such as cheese, ice cream, edible film, and health supplements (badem and ucar, 2017; sarode et al., 2016). chemical composition and functional properties of milk protein and peptides (casein and whey) could be altered rermarkably by processing conditions (jimenez et al., 2012). quality of casein could be reduced by improper methods. a few changes in protein structure could be reponsible for release of other chemically bound molecules, but large changes may alter both quantity and quality of the protein. degradation of protein chains is able to deactivate particular protein that acts as antioxidant (pralea et al., 2011; winarno, 2008). in processing of casein, precipitation is considered important since it accounts for separation of casein from milk. the techniques used may vary from chemical to physical approaches, using chemical agents and control over processing conditions such as temperature, pressure, agitation, and holding time, which influence ph of the 2 italian journal of food science, 2022; 34 (1) maruddin f et al. materials and methods materials the materials used were fresh milk, ch3cooh pro-analysis grade, hcl pro-analysis grade, koh pro-analysis grade, alcohol, and distilled water. experimental procedures skimming was performed by separating cream from fresh milk using cream separator. then the skim was pasteurized at 85°c for 5 min. after being stored in refrigerator at 5°c for 24 h, the pasteurized skimmed milk was defatted under aseptic conditions. casein was precipitated from the skimmed milk using hcl 1n and ch3cooh 1n at 5% (w/v) and 10% (w/v) of the skimmed milk volume, respectively. under these conditions, ph of casein was in the range of 4.4–4.6. difference in concentration was due to difference in the acid strength of two acids. subsequently, filtration was performed to collect casein curd while whey was discarded. the obtained casein curd was then washed thrice using distilled water with the similar volume of discarded whey. at the last washing, the curd was weighed and added to distilled water in 1:1 (w/w) ratio. the ph of casein was also measured and adjusted to 6–7 using koh. finally, casein curd was filtered and dried. the oven-drying (ecocell sis-b2v/ ec111, d112457) was operated at 50°c for 48 h, while freeze-drying (christ freeze dryer, type alpha-2 ld) was performed at –40°c for 48 h, adopted from the methods described by badem and uçar (2017), kumaresan et  al. (2017), and sarode et al. (2016). parameters dpph (1,1-diphenyl-2-picrylhydrazyl) assay antioxidant activity was determined using dpph assay (pająk et al., 2014). dpph reagent (0.008 g) was mixed with methanol 50 ml. sample (1 g) was dissolved in 9-ml distilled water, and the sample solution (1 ml) was mixed with 0.8-ml methanol. this sample solution was prepared in duplicate. subsequently, 0.8 ml of each sample solution, 0.4-ml ethanol and 1.8-ml methanolic dpph solution, was mixed. after 60 min, absorbance was measured at 515 nm using ultraviolet–visible spectroscopy (uv−vis) spectrophotometer (shimadzu irp prestige-21). the antioxidant activity was calculated using eq. 1: dpph %( ) ( ) ,� � � a a a dpph sampel dpph 100 (1) solvent, protein conformation, and properties of the final product (jimenez et al., 2012). acid precipitation of casein could be a promising method, as commonly applied in food preparation by adding hydrochloric acid (hcl) and acetic acid (ch3cooh), often recognized as strong and weak acids, respectively. difference in acidity causes various effects to the extent that casein changes into a simple form of protein (triyono, 2010; vasbinder et al., 2004). further, incredible stage in casein processing is dehydration (djaeni et al., 2015; haque and roos, 2006), which is a final step for producing potassium caseinate. in this case, drying is applied to induce mass transfer process, including removal of water present in the product (sarode et al., 2016). in addition, drying in the processing of potassium caseinate is further addessed to extend storability of the product, resulting in dry matter, which easily modifies its chemical components. however, it must be considered that product should be dehydrated without reducing the quality of end product. the drying process is often carried out using oven and freeze dryer. even though oven-drying is less favorable for its undesired effects to color changes of the final product, it is affordable, easy to  operate, and available comfortably. oven-drying seems to be disadvantageous if applied to protein-rich foods, since it can incredibly reduce their quality (liu et al., 2008). as an alternative technique, freeze-drying is more favorable because it enables to minimize cellular damages because of heat. the main principle of freeze-drying is based on removal of ice from materials through the process of sublimation (ciurzyńska and lenart, 2011). the use of acids and drying methods in isolation of casein inevitably affects its physical and chemical properties. processing conditions allow to induce deformation of chemical bonds in functional groups of casein from other molecules. casein is also an important source of peptides that are active biologically. inactive peptides in protein chain can be turned into an active form and released through stimulation by using acid, enzyme, heating, mechanical treatments, and salts (felix da silva et al., 2018; winarno, 2008). however, the stimulating activities may cause some effects, such as changes in color and microscopic structure, shrinkage, porosity, and reduction of water-binding capacity as well as microscopic destruction. in addition, changes in chemical properties may also occur, such as antioxidative properties, protein content, and moisture content (raikos, 2010; witrowa-rajchert and lewicki, 2006). the present study aimed to uncover the effects of acids and drying techniques on the physiochemical properties of potassium caseinate regarding antioxidant activity, content of crude and dissolved proteins, moisture as well as color (l*, a*, and b*) and chemical properties determined by fourier transform infrared (ftir) analysis. italian journal of food science, 2022; 34 (1) 3 effect of drying methods and acids on the quality of potassium caseinate use, the color meter was callibrated to ensure its accuracy (maruddin et al., 2018). ftir analysis prior to ftir analysis, casein (0.2 g) was dissolved in distilled water. the solution was dropped in calcium fluoride (caf2) window, coupled with another caf2 window, ensuring that the solution was thoroughly spread onto the surface of window. the caf2 window was then set in a holder. analysis was performed using ftir (shimadzu irp prestige-21; sari, 2011). determination of dissolved protein content of dissolved protein was calculated using the lowry method (wikandari et al., 2011). casein (1.5  g) was added in 7.5-ml distilled water and centrifuged for 15 min to collect supernatant. the supernatant was boiled on hotplate, centrifuged for 15 min to collect the final supernatant. the final supernant (2 ml) was added to 1-ml trichloroacetic acid (tca) 10%, followed with centrifugation for 15 min. then the sample (0.1 ml) was mixed with distilled water (1.9 ml) and folin–ciocalteu reagent (fcr; 2.5 ml), and incubated for 10 min at room temperature. folin reagent (0.5 ml) was added again and incubated for 30 min till the color changes to blue. the absorbance was determined spectrophotometrically at 600 nm. bovine serum albumine (bsa) was used as a standard solution. the level of dissolved protein was calculated using eq. 4: dissolved protein %( ) ( ) � � � a a a sample control sample 100 (4) where asample is the absorbance of sampel solution and acontrol is the absorbance of bsa microstructure of casein microstructural feature of casein was observed using scanning electron microscope (sem; hitachi su 3500). sample was set with a double adhesive tape, then coated with gold using hitachi ion sputter mc 1000 in vacuum. microstructural scanning was carried out in sem at 20 kv. the image was recorded at different magnifications (20× to 2000×). determination of moisture content moisture content was determined using the method described by kumesan et al. (2017). porcelain was dried where adpph is the absorbance of dpph solution, and asample is the absorbance of sample solution. determination of crude protein crude protein was quantified by using the method described by aoac (2019). briefly, casein (1 g) was transferred into kjeldahl flask. subsequently, 1-g copper sulfate (cuso4) and 2.5-ml concentrated sulfuric acid (h2so4) were added to the flask, followed with thermal destruction at 100°c for 2 h. after destruction, the mixture was transferred into volumetric flask containing boiling chips. afterward, 50-ml demineralized water and 15-ml sodium hydroxide (naoh), 50% (w/v), were added prior to distillation. then distillate was collected in erlenmeyer containing 10-ml hcl, 0.02 n. four drops of each methyl red and methyl blue were added to reach a total volume of 40 ml, then titrated with naoh and standardized with oxalic acid (h2c2o4), 0.02 n. titration was stopped after purple color changed into green. the volume of naoh used was recorded. protein content was calculated using eq. 2: crude protein % n ( ) . , � �� �� � � � vs vb w 14 007 6 25 100 (2) where vs is the volume (ml) of the standardized acid used to titrate the sample, vb is the volume (ml) of the standardized acid used to titrate a reagent blank, and w is weight (g) of sample portion of standard viscosity viscosity was measured according to the method described by konstance and strange (1991). casein solution was prepared with 12% (w/v) casein powder blended with cold water using electric blender (miyako bl-152 pf_ap) for 10 min. the solution was heated at 95°c for 5 min and left to cool for analysis. the cooled solution was transferred into a glass beaker, and the viscosity was determined using viscometer (with spindle 3) at a speed of 50 rpm. viscosity was determined using eq. 3: viscosity value obtained in viscometer correction factor � � (3) color digital color meter test (t135) was used to detect color, expressed as l* (0–100, dark to white), a* (–60–+60, green to red), and b* (–60–+60, blue to yellow). prior to 4 italian journal of food science, 2022; 34 (1) maruddin f et al. hcl + oven-drying; and ch3cooh + freeze-drying and ch3cooh + oven. the highest antioxidant activity was attributed to ch3cooh + oven-drying, while the lowest one was found in ch3cooh + freeze-drying. the higher antioxidative activity represents stronger effect on free radical scavenging and vice versa. this suggests that sample contains a particular compound responsible for retardation of oxidation reaction. as explained by simanjuntak et al. (2004), free radicals require electrons from adjacent electron pairs, thus donation of electron is needed to stabilize the radicals. glab and boratynski (2017) discussed that free radicals could be in the form of atom, molecule, or compound containing one or more unpaired electrons, which make them highly reactive and unstable. the results demonstrated that average antioxidant activity of casein prepared from hcl was higher than ch3cooh. it is well known that hcl serves as a strong acid, capable of breaking down peptide bonds. as previously explained by felix da silva et al. (2018) and kusumaningtyas et al. (2015), peptides and amino acids resulted from protein degradation could exert antioxidative activities. interestingly, we also found that casein prepared from oven-drying showed a higher antioxidant activity in comparison to that prepared from freeze-drying. the in an oven for 15 min, desiccated and weighed (c). casein (5 g) was transferred into porcelain and weighed (b). the sample was then dried in an oven at 105°c for 2 h, desiccated and weighed (a). calculation of the moisture content was determined using eq. 5: moisture content %( ) ( ) ( ) � � � � b c b a 100 (5) results and discussion antioxidant properties of casein antioxidant is a compound capable of alleviating oxidation reaction through scavenging of radicals and active molecules (nahariah et al., 2014; pająk et al., 2014; winarsi, 2007). figure 1a shows the antioxidant activity of casein ranging from 55.74% to 73.12%. this finding is  in agreement with the previous result reported by pralea et al. (2011) that antioxidant activity of casein added with sodium ranged from 45% to 95%. analysis of variance demonstrated that antioxidant activity was affected by interaction of acids and drying methods (p < 0.01). additionally, statistical analysis demonstrated a significant difference between processes, that is, between: hcl + freeze-drying and figure 1. (a) antioxidant activity of casein treated with acids and drying methods. different superscripts above the bars depicted a significant difference (p < 0.01). (b) content of crude protein in casein treated with acids and drying methods. different superscripts above the bars depicted a significant difference (p < 0.01). (c) viscosity of casein treated with acids and drying methods. different superscripts above the bars showed a significant difference (p < 0.01). (a) (b) (c) italian journal of food science, 2022; 34 (1) 5 effect of drying methods and acids on the quality of potassium caseinate which is the major cause of protein destruction and lower crude protein content compared to freeze-drying. viscosity viscosity constitutes a key parameter related to the characteristic of a fluid. this is noteworthy that viscosity may be incredibly influenced by several factors such as protein conformation, hydration properties, group of hydrophobicity, and distribution of charges. as exhibited in figure 1c, the viscosity ranged from 8.33 centipoise [cp] to 58.33 cp. chairunnisa (1997) reported viscosity of other casein types, such as lactic casein (29.6 cp), co-precipitate casein (70.1 cp), and sodium caseinate (52.7 cp). the results suggest that several significant factors affect the viscosity of casein, such as interaction of acids and methods of drying (p < 001). statistical test revealed that types of acids used in freeze-drying showed a higher viscosity than those used in oven-drying. the viscosity tended to decrease due to change in temperature. however, the variability of viscosity in our experiment was considerably affected by acid types. high temperature (oven-drying) treatment can hydrolyze certain peptide bonds. the process changes the conformation of proteins by exposing hydrophobic proteins and resulting in decreased values of viscosity. broyard and gaucheron (2015) asserted that proteolysis resulted in low secondary peptides. the peptides are closely related to a noticeable reduction in viscosity. in addition, casein proteolysis because of the use of acid and temperature during processing causes an increase in exposed fat molecules. as stated by ting et al. (2016), viscosity was a result of internal friction between fat molecules present in a fluid. in general, the viscosity tends to decrease due to higher concentration of unsaturated fatty acids. conversely, viscosity rises when the solution is hydrogenated. raikos et al. (2009) investigated molecular weight of milk components with medium–high fat contents exposed to heat at 50, 95, and 125°c. heating at >95°c leads to unfolding of protein chains, which promotes denaturation. as a consequence, hydrophobic components (such as fat) were more exposed, then aggregating to form larger molecules. high temperature is responsible for alleviation of viscosity as it induces destruction of protein structure. we found that viscosity of casein dried using the freeze drying process was higher than that of oven-drying process. this is in line with the process reported by chairunnisa (1997) that heat treatment of milk protein isolates at 60°c reduces viscosity. such condition is oven-drying could have a high antioxidant ability because of exposure of sample to high temperature, which converts protein into antioxidative peptides. similarly, pralea et al. (2011) reported that heating method could alter antioxidant properties of food. exposure to heat could produce undesired effects on food quality, although some studies have revealed that antioxidant ability increases as more heat is provided. morales and babbel (2002) stated that correlation between antioxidant activities and heat level was due to formation of strong antioxidative components. such components are able to scavenge free radicals resulted from chemical reaction during heating. crude protein protein is one of the macronutrients found in foods. in this work, the content of crude protein in casein ranged from 48.96% to 53.54%, as depicted in figure 1b. this suggests that the range is much lower than standard set by codex alimentarius (2014), according to which the crude protein content must be at least 88.0%. statistical analysis established that interaction of acids and drying methods offered significant effects on the content of crude protein (p < 0.01). additionally, average content of crude protein in casein prepared by oven drying was higher than obtained by freeze-drying. this presumably relates to the ability of oven to remove water, which leads to increase in protein content of casein. kusnandar (2011) argued that level of protein was influenced by coagulating properties of casein, which losses its ability to bind water molecules. statistical data exhibit a more satisfied content of crude protein in casein treated with oven-drying in comparison to freeze-drying, while the use of ch3cooh + oven-drying is also more satisfied than ch3cooh + freeze-drying regarding content of crude protein. felix da silva et al. (2018) and sarode et al. (2016) reported that rate of denaturation and agglomeration of milk protein was noticeably controlled by heating and chemical conditions, that is, temperature and ph. these conditions must be emphasized in order to regulate rate of protein denaturation. the application of hcl produced a completely ionized casein, resulting in the enhancement of crude protein content. triyono (2010) found that hcl could ionize completely, thus able to degrade protein into smaller structures. as stated by malaka (2014), precipitation of milk protein could be induced by acids, reaching the isoelectric points. neutralization occurs if acid reacts with protein in milk, which in turn agglomerates to form a new complex. however, the use of hcl combined with high temperature (in oven-drying) promotes denaturation, 6 italian journal of food science, 2022; 34 (1) maruddin f et al. noticeably influenced by the chemical composition of products, using whey extract and casein isolate for fermented wine. the value of a* was –0.38–0.70 (greenish to reddish) in edible film made of dangke whey and carrageenan (maruddin et al., 2018), while casein-based edible film was reported to have an a* score of 4.59–5.20 (reddish; wahyuni, 2017). the results showed that the a* score of casein was affected by interaction between acids and drying methods. furthermore, the a* score of casein treated with hcl + freeze-drying tends to be greenish. meanwhile, the color of casein prepared with other processes was reddish. in addition, the use of hcl for casein preparation enables to reform the structure of proteins, which displayed greenish color with presence of riboflavin. as discussed by malaka (2014), one of pigments in milk is riboflavin, which is water-soluble and yellow-greenish in color. high temperature in oven-drying during preparation of casein turned a* value into reddish. this is caused by interaction between reducing sugars and amino acids, known as the maillard reaction, thus producing reddish color. kusnandar (2011) explained that the maillard reaction involved reaction of reducing sugars and amine groups that form simple proteins and release water molecules. presence of water activity in casein escalates rate of reaction. this is in line with the use of high temperature, which could alter color intensity of casein into brownish-reddish. regarding b* color, the score ranges from 12.83 to 20.32 (yellowish), as presented in table 1. the properties of b* are strongly linked to the characteristics of milk protein. in addition, the content of fat left from defatting process may also affect b* score. in general, the score of b* closely relates to carotenoid and riboflavin present in fat. associated with thermal aggregation within casein isolates as well as degradation of protein hydrophobicity. color the results revealed that the treatments (use of acids and drying techniques) significantly contributed to the color of casein, as presented in table 1. l* (dark to white) was found to differ from 81.52 to 91.57. in general, the color of casein tends to be white, linked to its basic color. regarding the base color of casein, cheng et al. (2019) and misawa et al. (2016) have argued that milk brightness is determined by the presence of nutritional components and the effect of processing. statistically, l* was significantly affected by interaction between acid types and drying methods (p < 0.01). further, we found that l* of the casein treated with ch3cooh + oven-drying was comparable with that treated with hcl + freeze-drying. processing treatments could alter the whiteness degree of casein. the use of hcl (strong acid) seems to more incredibly affect changes in protein structure than ch3cooh (weak acid). in terms of exposure to high temperature, oven-drying was able to reduce degree of whiteness, which could be linked to the maillard reaction. the reaction promotes interaction between reducing sugars and amino acids, leading to reduction of white intensity of casein. winarno (2008) explained that the maillard reaction represents a browning activity triggered by high temperature, promoting reaction between reducing sugars and primary amino groups. exposure to high temperature causes structural changes in protein, alleviating the intensity of white color. regarding a* color, the value was found in the range of –3.41–0.87, affected by the chemical composition of products and processing conditions. the range of a* was table 1. color intensity. color parameters drying methods averageacids freeze-drying oven-drying l* hydrochloric acid 90.92 ± 0.34b,c 81.52 ± 3.68a 86.22 ± 5.65a acetic acid 87.31 ± 0.52b 91.57 ± 1.86c 89.44 ± 2.63b average 89.12 ± 2.01 86.55 ± 6.09 a* hydrochloric acid –3.41 ± 0.42a 0.87 ± 0.42b –1.26 ± 2.37a acetic acid 0.54 ± 0.15b 0.60 ± 0.20b 0.57 ± 0.20b average –1.43 ± 2.18a 0.73 ± 0.35b b* hydrochloric acid 15.53 ± 0.42b 20.93 ± 0.32d 18.23 ± 2.97b acetic acid 12.82 ± 0.15a 16.94 ± 0.04c 14.88 ± 2.25a average 14.17 ± 1.50a 18.93 ± 2.19b note: different superscripts (a,b,c,d) following means in similar rows and columns showed significant difference (p < 0.05). different superscripts (a,b) following means in similar rows and columns showed significant difference (p < 0.05). italian journal of food science, 2022; 34 (1) 7 effect of drying methods and acids on the quality of potassium caseinate the destruction of bonds, more the expansion of molecules. this changes configuration of proteins, leading to an increase in yellow intensity of casein. the heat produced in oven-drying for casein preparation is able to unfold protein structures, thus uncovering fat molecules buried inside protein structures. this accounts for higher intensity of b* casein, displayed as bright yellow. broyard and gaucheron (2015) studied high temperature for hydrolyzing chemical chains of proteins, thereby inducing more intense hydrolytic activity of fats. ftir analysis of casein spectroscopic analysis using ftir is based on the characteristics of functional groups in proteins. the result is depicted in figure 2. the hcl + freeze-drying process casein is derived from skimmed milk. although fat content in casein is low, addition of acids as coagulant could remarkably alter the color of casein. skimmed milk originally appears bonewhite in color (umaroh, 2018), which is due to low concentration of fat in skim. meanwhile, presence of carotenoid and riboflavin in fat accounts for yellownish color of skim. chairunnisa (1997) found that the b* score of milk protein modified by lactic acid was 12.8−16.4, displayed as a bright yellowish color. statistical analysis showed that b* color was affected by interaction of acids and drying methods (p < 0.01). we found that hcl + oven-drying process resulted in bright yellow b* color of casein, while other processes produced casein with a dark yellow color. ch3cooh showed less destruction of protein than hcl. winarno (2008) demonstrated that application of strong acid could drastically alter polypeptide chains and protein molecules. more figure 2. ftir spectrum for casein prepared with: (a) hcl + freeze-drying; (b) hcl + oven-drying; (c) ch 3 cooh + freeze drying; (d) ch 3 cooh + oven-drying. (a) (b) (c) (d) 8 italian journal of food science, 2022; 34 (1) maruddin f et al. dissolved proteins dissolved proteins refer to a group of simple proteins resulting from degradation of casein after treatment with acids and freeze-drying methods. the content of dissolved proteins in casein was found in the range of 0.35– 0.89% (figure 3), being slightly lower than that reported by rahayu et al. (2013), reaching a range of 1.14–1.29% in casein treated with calcium chloride (cacl2). our experimental data statistically established that content of dissolved proteins was significantly affected by the given treatments (p < 0.01). duncan statistical test revealed that the highest content of crude protein was attributed to hcl + freeze-drying process and next to hcl + oven-drying process. a strong acid such as hcl can iozine completely; consequently, this promotes the conversion of uncharged protein molecules into positive charged molecules. thus, such condition increases the solubility of proteins. winarno (2008) reported that treatment with a strong acid enhanced protein solubility because of changes in charge of proteins. meanwhile, kusumaningtyas et al. (2015) reported that acid treatment was also effective in destroying complex of protein molecules, converting them into simple parts such as peptides and amino acids. furthermore, difference in the formation of dissolved proteins is also linked to the drying techniques. as mentioned previously, hcl coupled with freeze-dyring produced higher quantity of dissolved proteins than hcl coupled with oven-drying. previous studies (felix da silva et al., 2018; sarode et al., 2016; winarno, 2008) asserted that denaturation of proteins is achieved by using acid, alkali, heating, mechanical treatment, and salt. in this case, freeze-drying is considered as a mechanical treatment (low temperature) for casein, resulting in finer texture. for this reason, we could argue that each processing method caused different changes to protein features. denaturation of protein is highly essential to consider as it causes loss of protein conformation because of structural changes (deulgaonkar et al., 2012). moreover, our experimental data established a higher content of dissolved protein in casein prepared by oven-drying. this is in accordance with the results of felix da silva et al. (2018), which demonstrated that higher temperature would bring greater changes to protein destruction, until reaching a constant level. as mentioned by deulgaonkar et al. (2012), denaturation of protein could be induced by heat. in addition, ciurzyńska and lenart (2011) and raikos (2010) reported that denaturation of protein induced by heat caused drammatical changes in the structural properties of protein, from strong double-structure to weak opened-structure. (figure 2a) resulted in seven regions of absorbance, with two specific peaks of 2924.09 cm–1 and 2852.72 cm–1. the wave number of 3738.05 cm–1 refers to aromatic group (c-h). in addition, absorption peak of 2924.09 cm–1– 3475.73 cm–1 corresponds to methylene, while the peak range of 2538.32–2852.72 cm–1 refers to alkane (c-h bond). furthermore, hydroxyl groups (o-h) were found in the wave number range of 2331.94–2357.01 cm–1. as depicted in figure 2b, ftir spectrum was detected in six regions of absorbance, with two specific peaks at wave numbers of 2922.16 cm–1 and 2852.72 cm–1. the wave number of 3477.66 cm–1 corresponds to hydroxyl group (o-h), characterized by a wide region specific to the o-h group. absorption peak range of 2852.72–2922.16 cm–1 refers to methylene, which is a asymetric stretch vibration of c-h bonds. meanwhile, absorption peak range of 2368.59–2407.16 cm–1 is associated with hydroxyl group (o-h). figure 2c exhibits seven regions of absorbance with two sharp peaks, namely 2924.09 cm–1 and 2852.72 cm–1. the absorption peaks of 3741.70 cm–1, 3562.52 cm–1, and 3444.67 cm–1 correspond to hydroxyl group (o-h), while the wave number range of 2852.72–2924.09 cm–1 represents methylene, referring to asymetric stretch vibration of c-h bonds. in addition, the wave number range of of 2333.87–2363.87 cm–1 demonstrates hydroxyl group (o-h). figure 2d depicts nine regions of absorbance with two sharp peaks. the wave numbers of 3743.83 cm–1, 3564.45 cm–1, and 3444.87 cm–1 correspond to hydroxyl group (o-h). in addition, peak at the wave number range of 2852.72–2922.16 cm–1 indicates presence of methylene group with asymetric stretch vibration of c-h bonds, while several peaks detected at wave numbers of 2771.71 cm–1, 2573.04 cm–1 2358.94 cm–1, and 2330.91 cm–1 refer to hydroxyl group (o-h). figure 3. contents of dissolved proteins after treatment with acids and freeze-drying methods. different superscripts above the bars depicted a significant difference (p < 0.01). freeze-drying p ro te in t e rl a ru t (% ) oven-drying acetic acid 0.9 0.75 0.6 0.45 0.3 0.15 0 hydrochloric acid italian journal of food science, 2022; 34 (1) 9 effect of drying methods and acids on the quality of potassium caseinate microstructure microstructural analysis of casein by sem is depicted in figure 4. images in figure 4a seem to be solid, with absence of pores on their surfaces. meanwhile, figure 4c is found to have a more porous structure compared to figures 4b and 4d. this suggested that combination of ch3cooh and freeze-drying produced high porosity casein. the moisture content of casein in hcl + freeze-drying process was 5.94%, which was higher than other processes. dehydration process could be optimized through considering the factors retarding removal of water. these factors include amount and thickness of sample, drying temperature and time, and sample positioning in dryer. deulgaonkar et al. (2012) stated that changes in food matrix occurred during dehydration, including shrinkage, browning, and case hardening. figure 4. potassium caseinate produced by different processes: (a) hcl + freeze-drying; (b) hcl + oven-drying; (c) ch 3 cooh + freeze-drying; (d) hcl + oven-drying. (a) (b) (d)(c) moisture content moisture content represents the quantity of water in foods. kusnandar (2011) stated that chemical properties of water in food matrix differ remarkably, since water is bound in different manners. water in food is often trapped in cells or bound with other chemical components of foods. as depicted in figure 5, mositure content of casein was found in the range of 5.29–5.94%. the range must be according to the standards set by codex alimentarius (2014), that is, maximum mositure of 8.0%. statistical analysis revealed that acid types demonstrated a significant effect on moisture content (p < 0.01). the average moisture content in casein treated with hcl was higher than that treated with ch3cooh. in short, acid used in this experiment enables to hydrolyze macromolecules, 10 italian journal of food science, 2022; 34 (1) maruddin f et al. but improved viscosity and microstructural properties. combination of oven-drying and strong acid hcl was responsible for increase in l* (lightness) score and content of proteins and dissolved proteins as well as promoted changes in chemical features by ftir analysis. acknowledgements authors would like to thank ministry of research, technology, and higher education (kemenristekdikti), republic of indonesia, for funding this research under the scheme of stranas no. 123/ sp2h/ptnbh/dprm/2018. and scheme of terapan 7/e1/ kp/ptnbh/2021 we also express many thanks to lp2m, hasanuddin university for valuable support in research regulations (no. sp-dipa-042.06-1.401516/2018) and 752/un4.22./pt.02.00/2021. references aoac (association of official analytical chemists). 2019. dairy products. in: robert l. bradley, jr. 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plant tissues. int j food sci technol. 41(9): 1040–1046. https://doi.org/10.1111/j.1365-2621.2006.01164.x ijfs#968_bozza ital. j. food sci., vol. 30, 2018 102 paper effects of archaic olive and oil storage methods still used in southern tunisia on olive oil quality s. el-gharbi1,2, m. tekaya1, a. bendini3,4, e. valli*3,4, r. palagano3, t. gallina toschi3,4, m. hammami1 and b. mechri1 1laboratoire de biochimie, uscr spectrométrie de masse, lr-nafs /lr12es05, nutrition –aliments fonctionnels et santé vasculaire, faculté de médecine, université de monastir, 5019 monastir, tunisia 2faculty of science of gabes, university of gabes, 6072 gabes, tunisia 3department of agricultural and food sciences, alma mater studiorum, university of bologna, italy 4inter-departmental centre for agri-food industrial research, alma mater studiorum, university of bologna, italy *corresponding author. tel.: +39 0547 338121; fax +39 0547382348 *e-mail address: enrico.valli4@unibo.it abstract the present paper investigated how virgin olive oil quality is influenced by two different storage conditions that residents of gabes (southern tunisia) usually apply to fruits of the zarazi cultivar: long conservation as oil in glass bottles or traditional storage of olives as sun-dried fruits before processing for oil production. even if both storage conditions are associated with strong losses in the qualitative characteristics of olive oil, the changes observed were more accentuated for oil stored for two years after its production compared to the oil obtained from olives stored by traditional methods. keywords: fatty acid alkyl esters, olives, phenols, traditional storage, virgin olive oil ital. j. food sci., vol. 30, 2018 103 1. introduction long known to many generations in the mediterranean basin populations as essential to their diet, virgin olive oil is now widely appreciated around the world for its healthy and sensory properties. the nutritional benefits of virgin olive oil are firstly related to the fatty acid composition, mainly due to both the high content of oleic acid and the balanced ratio of saturated and polyunsaturated fatty acids (gargouri et al., 2015; ruiz-canela and martínez-gonzález, 2011); in addition, virgin olive oil presents considerable amounts of natural antioxidants. because of these natural characteristics, virgin olive oil is particularly resistant to storage and more suitable for cooking than other vegetable oils (pellegrini et al., 2001). it is also considered important in prevention of many diseases, such as cardiovascular disease, obesity, metabolic syndrome, type 2 diabetes and hypertension (nunez-cordoba et al., 2009). it is one of the few vegetable oils that can be consumed in the raw state and contains important nutritional elements (fatty acids, vitamins, sterols, etc.). extra virgin olive oil (evoo) is considered the highest quality virgin olive oil due to its organoleptic and chemical characteristics (jabeur et al., 2015). nevertheless, oxidation is the main cause of deterioration of evoos during the storage period. auto-oxidation follows a free radical mechanism, wherein a first result of the absorption of oxygen is the formation of hydroperoxydes (frankel, 2005). these labile compounds also decompose to produce a complex mixture of volatile molecules, such as aldehydes, ketones, hydrocarbons, alcohols and esters, among which some are directly responsible for perception of the rancid sensory defect (pristouri et al., 2010). the most important factors affecting the quality of olive oil during storage are environmental, especially temperature, light exposure and contact with oxygen (pristouri et al., 2010). much research has been carried out to study the effects of storage conditions and packaging materials on olive oil quality (dabbou et al., 2011; pristouri et al., 2010). moreover, one of the primary factors responsible for the low quality and stability of virgin olive oils is linked to the mishandling of the olives during the period between harvesting and processing. indeed, the storage of olives, which leads to degenerative processes in a short period of time, should be carried out by simple heaping in fruit piles, awaiting their transformation (rabiei et al., 2011). unfortunately, olive processing is often not well synchronized with crop harvests; in particular, for countries in which the production of olives is very high and concentrated in a short period, if the capacity of the local olive oil mills is not adequate to support such enormous amounts, olives are often piled into large heaps or in plastic sacks. moreover, olives can be stored in big cumuli outside the olive oil mill for periods that may range from weeks to months prior to oil extraction (kiritsakis et al., 1998). during this storage period, mechanical, physical, chemical and physiological alterations occur in the fruit, which can cause the rupture of cellular structures and subsequent negative chemical and sensory changes in the oils obtained (biasone et al., 2012; inarejos-garcía et al., 2010; vichi et al., 2009). the olive fruit deteriorates rapidly due to the combined action of pathogenic microorganisms and internal processes of senescence. both processes are accelerated by temperature increases due to fruit fermentation and mechanical damage due to compression. the degradation of fruit causes the loss of the texture of the flesh and tanning of the skin, and finally complete decomposition (garcía et al., 1996). virgin olive oils obtained from damaged olives usually present high acidity, low oxidation stability and high levels of oxidation, due to the increased peroxide value and specific extinction coefficients at 232 and 270 nm (garcía et al., 1996; inarejos-garcía et al., 2010). moreover, the content in fatty acid ital. j. food sci., vol. 30, 2018 104 ethyl esters also increases with storage time of olives due to fruit degradation (biedermann et al., 2008). in this context, herein we investigated the effects on olive oil quality of i) the olive oil immediately processed, and ii) of the olive oil and olive fruits stored for two-years following the traditional method of gabes. although some data have already been published on the effects of olive storage before oil extraction (biasone et al., 2012; inarejos-garcía et al., 2010; vichi et al., 2009), in the present investigation a particular interest was given to a traditional conservation procedure that the residents of gabes (southern tunisia) still apply to olive fruits of the zarazi cultivar. in fact, after harvesting, olive fruits are traditionally sun-dried, and when completely dried they are stored in plastic bags at room temperature and then processed to obtain olive oil. therefore, the objective of this investigation was to compare the deteriorative effects on the oil quality of two different conservation methods: conservation as oil or as intact fruits both carried out for a long period of time (two years). for this aim, different quality parameters were investigated, including composition in minor compounds, such as phenolics and fatty acid alkyl esters. 2. materials and methods 2.1. olive fruit samples the present study was carried out on olives picked up from o. europaea l. trees (cv. zarazi) grown in an orchard located in the region of gabes (southern tunisia: 33° 32’ n, 10° 06’ e). in particular, olive samples were harvested manually and were divided into two unequal lots: about 4 kg of olives were processed (see paragraph 2.3) directly after harvest (about 24 hours between harvest and start of processing) and the virgin olive oil obtained was divided into two samples: one used as a control sample (sample code: “tc”) and the other stored in dark glass bottles at room temperature for two years (sample code: “t1”). about 2 kg of olives were cleaned from leaves, washed and stored following a traditional method described later (see the section 2.2) for two years, oil (sample code: “t2”) was then extracted from dried fruit using the same lab-scale mill described in paragraph 2.3, in order to evaluate the effects of both conservation methods (as intact fruits or as oil) on virgin olive oil quality. 2.2. olive fruit conservation in this investigation, a traditional storage procedure that the residents of gabes still apply to olive fruits of zarazi cultivar was simulated. after harvesting, olive fruits were sundried (thrown singly upon a stone-floor, exposed to the sun in the open air for 10 to 15 days) and, once completely dried, they were stored in plastic bags at room temperature for two years, before processing to obtain the oil. 2.3. oil extraction olive oil was extracted under similar industrial extraction conditions using a bench hammer mill. a sample of 2 kg of olive fruits was firstly crushed with a small hammer crusher, and the paste was mixed at about 25°c for 30 min and centrifuged in a two-phase decanter (3500 rpm for 1 min). after extraction, oils were transferred into 250 ml amber glass bottles and stored in darkness at room temperature (25°c) for further analyses. ital. j. food sci., vol. 30, 2018 105 2.4. quality indices determination free acidity (fa, expressed as % oleic acid), peroxide value (pv, expressed as milliequivalents active oxygen meq o2 kg−1 oil) and extinction coefficients (k232 and k270) were determined by analytical methods described respectively in iso 660:2009, iso 3960: 2010 and iso 3656: 2011. 2.5. total phenol and o-diphenol content the total phenol and o-diphenol content of olive oil samples were quantified colorimetrically using an uv–vis 1800 spectrophotometer (shimadzu co., kyoto, japan), according to the procedures previously described (mateos et al., 2001; singleton et al., 1965). phenolic compounds were extracted in methanol/water (80:20, v/v), according to ioc/t.20/doc no 29. folin-ciocalteu reagent and sodium carbonate (15% p/v) were added to a suitable aliquot of the extract, and the absorption of the solution at 750 nm was measured to evaluate the total phenolic content, by a specific calibration curve built with different concentrations of gallic acid (sigma-aldrich, st. louise, mo, usa) (r2 =0.995). for o-diphenol content determination, 0.5 ml of the extract was dissolved in 5 ml of a methanol/water solution (50:50, v/v). four ml of the obtained solution were withdrawn, then 1 ml of sodium molybdate (5% in ethanol/water 50:50) was added. after vigorous mixing, they were centrifuged for 5 min at 3000 rpm. the concentration was determined colorimetrically at 370 nm. a specific calibration curve (r2 = 0.985) was built using gallic acid for the quantification. data were expressed as mg gallic acid per kg of oil for both total phenolic compounds and o-diphenols. 2.6. determination of phenolic compounds by hplc-dad-ms hplc analysis was performed using an agilent technologies 1100 series system equipped with an automatic injector, a diode array uv-vis detector (dad) and a mass spectrometer detector (msd) (lerma-garcia et al., 2009). a c18 column kinetex (100 cm x 3.00 mm x 2.6 µm; phenomenex, torrence ca, usa) was used, maintained at 40°c during the analyses, with an injection volume of 5 µl and a flow rate of 0.7 ml min-1. the wavelength was set at 280 nm for detection of phenolic acids, phenyl ethyl alcohols and secoiridoids. the mobile phase was a mixture of water/formic acid (99.5:0.5%, v/v) (solvent a) and acetonitrile (solvent b). a linear gradient was run from 95% (a) and 5% (b) to 80% (a) and 20% (b) during 3 min, it changed to 60% (a) and 40% (b) in 1 min, after 1 min it changed to 55% (a) and 45% (b), it changed to 40% (a) and 60% (b) after 4 min, and then in 1 min it becomes 100% (b), finally it changed to 95% (a) and 5% (b) in 3 min (13 min total time). phenolic compounds were identified by comparison of their relative retention times and maximum absorbance according to ioc/t.20/doc no 29, and by interpretation of their mass spectra (lerma-garcia et al., 2009). syringic acid (sigma-aldrich) was used as an internal standard for the quantification of identified phenols and results are expressed as mg of tyrosol per kg of oil (according to the ioc/t.20/doc no 29). 2.7. tocopherol determination oils were dissolved in a solution of isopropanol and the isomers of α-tocopherol, βtocopherol and γ-tocopherol were analysed as described (anwar et al., 2013). an agilent technologies 1100 series hplc apparatus (paolo alto, california, usa), comprising a hp pump series 1050 (darmstadt, germany) and a dad detector set at 292 nm was used. ital. j. food sci., vol. 30, 2018 106 the eluting solvents were methanol/water (90:10, v/v) acidified with 0.2% h3po4 (solvent a), and acetonitrile (solvent b). samples were eluted through a cosmosil column (π nap 4.6 mm × 150 mm x 5 μm; nacalai-tesque, kyoto, japan) according to the following gradient: 100% a maintained for 22 min; 100% b for 13 min; 100% a for 15 min (total run = 50 min). the injection volume was 20 μl. the flow rate was 1 ml min-1. the concentrations of different tocopherol isomers were expressed on a calibration curve constructed using solutions of α-tocopherol at different concentrations (r2 = 0.998). results are expressed in mg of α tocopherol per kg of oil. 2.8. oxidative stability measurements oxidative stability was measured by a rancimat 743 apparatus (metrohm ω, schweiz ag, zofingen, switzerland) following the method described (tura et al., 2007). air (20 l h-1) was passed through a sample (3 g) held at constant temperature (120°c). stability was expressed as the oxidation induction time (h). 2.9. determination of fatty acid alkyl esters (faae) by gas chromatographic analyses fatty acid alkyl esters were extracted following the methods described in ioc/t.20/ doc. no.28 (2009). subsequent separation of alkyl esters (methyl and ethyl esters) was performed on a gas chromatograph equipped with an injector port and a fid both set at 325°c. the capillary column was a zb–5ms (30 m length × 0.25 mm i.d. × 0.25-µm-film thickness; phenomenex) with a split ratio 1:10. helium was the carrier gas at a flow rate of 1 ml min-1. the oven temperature was programmed from 80°c (kept for 1 min) to 140°c at a rate of 15°c min-1, then raised to 325°c at a rate of 4.5°c min-1 and kept for 20 min. the amounts of alkyl esters were expressed as mg of methyl heptadecanoate (c17:0 me) per kg of oil. 2.10. statistical analysis the statistical package for social sciences (spss) program, release 16.0 for windows (spss, chicago, il, usa) was used for all statistical analyses. the results were expressed as mean±standard deviation (sd) of three measurements for each analytical determination. significant differences between the values of all parameters were considered at p< 0.05 according to the one-way anova post hoc comparisons (duncan’s test). 3. results and discussions 3.1. effect of storage on basic chemical quality parameters the basic chemical quality parameters of the olive oil samples were investigated and the results are shown in table 1. the values of fa, pv, k232 and k270 for the control sample (tc) were below the legal limits established by ioc for extra virgin olive oils (ioc/t.15/nc no. 3/rev. 10 november 2015). regarding the effects of storage, both methods of storage (t1 and t2) negatively affected oil quality (p <0.001) by increasing all four abovementioned parameters, which all exceeded the limits established by ioc for extra virgin olive oils. comparing the differences between the two storage methods, the increase of these parameters was more accentuated for the oil extracted from dried fruits (t2) compared to the oil stored for two years (t1). in both cases (t1 and t2), the adopted ital. j. food sci., vol. 30, 2018 107 storage methods led to the classification of the olive oil samples in the category of “lampante oils”. the results obtained for free acidity agree with a previous study (jabeur et al., 2015), which confirmed the increase of this parameter in olive oil over time. a similar result was reported by (méndez and falqué, 2007) after 3 and 6 months of storage. moreover, the acidity value was also related with the storage temperature and the percentage of damaged olives (nabil et al., 2012). the increase of oil acidity during storage of oils obtained from stored olives is likely the result of fungal lipase activity (kiritsakis et al., 1998). regarding the pv, in the present study the olive oil quality was affected by an increase of this parameter after both storage conditions. these results are in accordance with the study performed by méndez and falqué (2007) during 3 and 6 months that showed an increase of pv in olive oil during storage. as reported by servili and gianfrancesco (2002), the pv is basically affected by several factors that damage the fruits (e.g. olive fly attacks or improper systems of harvesting, transport and storage) or the oil (processing technology and oil storage conditions). finally, concerning the extinction coefficients, the k270 of olive oil increased more slowly than k232 over two years of storage. however, the highest increase of k232 and k270 took place in the oil obtained from stored olives (t2). these results are in accordance with those of (gutierrez et al., 1992; vichi et al., 2009) which suggested that k232 and k270 progressively increased during olive storage. the increase in the extinction coefficient values during storage could be due to the presence of conjugated dienes. as reported by frankel (1993), it is important to consider that the storage temperature can affect the formation of certain volatile compounds resulting in the formation and degradation of different hydroperoxydes formed by oxidation processes. in general, an increase in these parameters can affect the conservation of evoos, as well as their nutritive and organoleptic characteristics. table 1. quality indices (free acidity, peroxide value, k232 and k270) of zarazi olive oil not stored (tc), stored for two years (t1) and obtained from intact sun-dried olive fruits stored for two years before being processed (t2). quality indices tc t1 t2 fa (% oleic acid) 0.77±0.01c 1.68±0.04b 2.40±0.03a pv (meq o2 kg oil -1) 10.1±0.1c 21.4±0.5b 23.5±1.7a k232 1.85±0.34 c 2.89±0.48b 3.80±0.52a k270 0.05±0.01 c 0.2±0.01b 0.43±0.05a fa: free acidity; pv: peroxide value; k232 and k270: spectrophotometric indices. each value represents the mean of three determinations (n = 3) ± standard deviation. a-c different letters in the same row indicate significantly different values (p < 0.05) according to duncan test. 3.2. total phenols and o-diphenols as influenced by storage methods total phenols and o-diphenols were quantified in samples and the results are shown in fig. 1. statistical analysis showed a significant decrease in both total phenol and odiphenol contents compared to the control sample under the effect of t1 and t2 (p <0.001). concerning the o-diphenol concentration, it also dramatically decreased from 506.8 mg kg 1 (sample tc) to 82.0 mg kg-1 in t1 and to 80.1 mg kg-1 in t2 (fig.1 b). no significant differences were found between the two storage methods. ital. j. food sci., vol. 30, 2018 108 the decrease of total phenols with storage time is in accordance with other studies showing that, during storage, phenols undergo qualitative and quantitative variations due to decomposition and oxidation reactions (dabbou et al., 2011; gargouri et al., 2015). the decrease of the total phenol content in all samples during storage is due to hydrolysis and oxidation processes (sicari et al., 2010). the losses of total phenols could be also be the result of an increase in the oxidative state of fruit. in fact, the oxidation of polyphenols in olive fruits is the result of enhanced activity of oxidative enzymes such as polyphenoloxidase and peroxidase, which contribute to the impairment of health-related qualities and sensory characteristics of olive oil (clodoveo et al., 2007; servili et al., 2003). figure 1. total phenol (a) and o-diphenol (b) content in control olive oil (tc), in olive oil stored for two years (t1) and in olive oil obtained from intact sun-dried olive fruits stored for two years before being processed (t2). results are shown as mean ± sd (n = 3). a-b different letters indicate significantly differences at p < 0.05 according to duncan’s test. 0 100 200 300 400 500 600 700 800 tc t1 t2 to ta l p he no ls (m g kg -1 ) 0 100 200 300 400 500 600 700 tc t1 t2 odi ph en ol s (m g kg -1 ) ital. j. food sci., vol. 30, 2018 109 3.3. modification of phenolic profiles during different methods of storage the analysis of phenolic profiles allowed the separation and identification of several phenolic compounds (table 2). in all olive oil samples, secoiridoid derivatives were the most abundant. the major secoiridoids detected were oleuropein aglycone (3,4-dhpeaea) and its decarboxymethylated derivative (3,4-dhpea-eda). the results presented in table 2 showed significant differences in the concentration of a wide number of phenolic compounds among samples. in general, in both the methods tested, it is possible to observe a significant decrease of all phenolic compounds compared to control samples (tc). in fact, 3,4-dhpea-ea decreased significantly (p<0.001) by 69.87% in t1 and by 58.81% in t2, compared to tc. another compound (3,4-dhpea-eda) also decreased significantly (p<0.001) by 84.34% in t1 and 68.40% in t2 compared to tc. for the other phenolic compounds, t1 had the lowest content of tyrosol (p-hpea), decarboxymethyl ligstroside aglycon (p-hpea-eda) and luteolin, while t2 oil samples contained the lowest concentration of ligstroside aglycon (p-hpea-ea) and acetoxypinoresinol. on the other hand, hydroxytyrosol (3,4-dhpea) was not detected in t1 oil, while its concentration was 19.8 mg kg-1 and 0.9 mg kg-1, respectively in samples tc and t2. table 2. phenolic compounds (mg of tyrosol kg-1 oil) identified and quantified by hplc-dad-ms in the control olive oil (tc), stored for two years (t1) and obtained from intact sun-dried olive fruits stored for two years before being processed (t2). phenolic compounds tc t1 t2 3,4-dhpea 19.8±1.0a nd 0.9±1.5b p-hpea 14.9±0.7a 3.4±0.3c 7.8±0.3b p-hpea-ea 6.8±0.3ab 8.4±1.4a 5.6±0.7b 3,4-dhpea-ea 35.3±1.6a 10.6±1.5c 14.5±0.8b p-hpea-eda 15.9±0.9b 9.1±0.5c 19.5±0.4a 3,4-dhpea-eda 33.2±1.7a 5.2±1.1c 10.5±0.4b acetoxypinoresinol 26.5±1.7a 11.6±0.8b 10.0±0.2b luteolin 12.2±0.9 a 3.2±0.1c 5.3±0.5b 3.4-dhpea: hydroxytyrosol; p-hpea: tyrosol; p-hpea-ea: ligstroside aglycon; 3.4-dhpea-ea: oleuropein aglycon; p-hpea-eda: dialdehydic form of elenolic acid linked to tyrosol; 3.4-dhpea-eda: dialdehydic form of elenolic acid linked to hydroxytyrosol; nd: not detected. results as expressed as means ± sd (n = 3). a-c different letters in the same row show statistically significant differences (p < 0.05) according to duncan test. given the importance of the phenolic fraction, with regards to antioxidant activities (bendini et al., 2006), sensory properties and health benefits (bendini et al., 2007) of olive oil, the content of phenolic compounds could be an important quality control parameter of evoo. the trends of simple phenols, secoiridoids, lignans and flavones during storage are related to the stability and nature of their molecular structure. indeed, these compounds may undergo alterations due mainly to hydrolysis, oxidation and increase of decarboxymethylated derivatives during storage of evoo in the industry or after sales (lerma-garcia et al., 2009). the reduced contents of different phenolic compounds observed in this study could be related to hydrolytic processes that may occur in parallel with oxidation, which is in concordance with previous studies (pagliarini et al., 2000) that suggested aglycone esters undergo hydrolysis during long term storage. ital. j. food sci., vol. 30, 2018 110 several studies have focused on possible hydrolytic and oxidative degradation forms of phenolic compounds present in evoo during storage (lozano-sánchez et al., 2013). in addition, secoiridoids, 3,4-dhpea-eda and 3,4dhpea-ea, were the most affected phenolic compounds during the storage period. these results are in accordance with those of other authors (hachicha et al., 2015). the results of this study show that under oil storage conditions, almost all phenolic compounds (except p-hpea-ea and acetoxypinoresinol) were lost more in samples of stored oil (t1) than in those produced after fruit storage conditions (t2). it is not easy to provide an explanation for this phenomenon, but it is possible that when conserved as fruits, exposure of the oil to external environmental factors is avoided, which may cause alteration of phenolic profiles, such as availability of oxygen, presence of light and temperature (méndez and falqué, 2007; pristouri et al., 2010). 3.4. modification of the concentrations of tocopherol isomers as an effect of storage methods as reported in table 3, α-tocopherol was the most predominant isomer in olive oil samples and its content was 180.4 mg kg-1 in the control olive oil (tc), while the β and γ isomers were present at relatively minor concentrations (6.0 mg kg-1 and 17.7 mg kg-1, respectively). statistical analysis revealed a significant decrease of total tocopherol content as well as the individual concentrations of each isomer (p<0.001) as an effect of the two storage methods. comparing the latter two methods, we observed that t1 oil samples had the lowest content of total tocopherols compared to tc and t2, especially because of its significantly lower content of α-tocopherol. tocopherols play an important role as antioxidants in the oxidative stability of olive oil by helping to maintain its shelf-life and in preserving oils from rancidity by interrupting the chain reactions involved in the formation of hydroperoxydes (morelló et al., 2004). our study showed that the loss of α-tocopherol content was about 50% in t1, which confirms the data by krichene et al., (2010) who observed a strong reduction in α-tocopherol during storage. as described for most phenolic compounds in paragraph 4.3, α-tocopherol also decreased more in samples of stored oils (t1) than in those obtained from olives stored for two years (t2). however, for both storage methods, it appears that polyphenols and o-diphenols were much more sensitive to storage time than tocopherols. this is definitely attributable to the fact that polyphenols, particularly o-diphenols, are the preferred substrates for oxidation (blekas et al., 1995). table 3. tocopherols isomer contents (mg α-tocopherol kg-1) in the control olive oil (tc), in olive oil stored for two years (t1) and in olive oil obtained from intact sun-dried olive fruits stored for two years before being processed (t2). tocopherols (mg kg-1) α-tocopherol β-tocopherol γ-tocopherol total tocopherols tc 180.4±2.8a 6.0±0.4a 17.7±0.6a 204.2±3.0a t1 109.0±6.8c 5.3±0.6ab 13.1±0.9b 127.5±8.2c t2 140.4±2.4b 5.1±0.1b 13.3±0.2b 158.9±2.2b a-c different letters in the same column indicate significantly different values (p < 0.05) according to duncan test. results as expressed as means ± sd (n = 3). ital. j. food sci., vol. 30, 2018 111 3.5. effect of the two storage methods on oxidative stability oxidative stability was studied by the rancimat test (fig. 2). the stability (os) decreased significantly from 8.47 h (tc) to 1.50 h in t1 and to 2.77 h in t2, reaching a significant lower value in of the latter. as reported by some studies in the literature (garcía et al., 1996; gutierrez et al., 1992), oil stability is clearly affected by storage time and conditions. this is in accordance with our results: osi time decreased more in the stored oil (t1) than in the that obtained from olives stored for two years (t2). this is a quite interesting finding, since it confirms that the traditional storage method of the olives had a lesser negative effect on osi time than the storage of the oil. fig. 2 variation of oxidative stability of control olive oil (tc), olive oil stored for two years (t1) and olive oil obtained from intact sun-dried olive fruits stored for two years before being processed (t2). results are shown as mean ± sd (n = 3). a-c different letters indicate significantly differences at p < 0.05 according to duncan's test. 3.6. changes in fatty acid alkyl esters in olive oil samples following the two storage methods the experimental results of the amounts of faaes, fames, faees and faees/fames in oil extracted directly after harvesting (from fresh olives) and in oils stored after two years using the two different methods are summarized in table 4. for evoo, the concentration of the faees must be ≤35 mg kg -1 (ioc, 2015). the major fatty acid alkyl esters (faaes) present in olive oil are those corresponding to palmitic, oleic and linoleic acids. indeed, the amount of faees in sample tc did not exceed the quantity of 35 mg kg-1, thus indicating the classification of this olive oil as extra virgin. compared with the control oil, the content of faees showed significantly higher values in samples t1 and t2 (p <0.001) with 624.2 mg kg-1 and 62.6 mg kg-1, respectively. the two types of storage (t1 and t2) both negatively affected the quality of the oil. this leads to their classification as non-extra virgin olive oils. 0 1 2 3 4 5 6 7 8 9 10 tc t1 t2 o xi da tiv e st ab ili ty (h ) b c ital. j. food sci., vol. 30, 2018 112 fatty acid alkyl esters (faaes), mainly ethyl (faees) and methyl esters (fames), are formed by esterification of free fatty acids (ffas) with low molecular alcohols, such as methanol and ethanol. in good quality extra virgin olive oils, fames and faees are present in very small amounts (valli et al., 2013), while they are present in higher amounts in “lampante” olive oils, as confirmed in the current study. the high content of faaes in samples t1 and t2 can be due to the presence of specific components, such as free fatty acids (ffas) and low chain alcohols. these compounds are, respectively, originated through substantial liberation of ffas from triglycerides by lipolysis and by a parallel consistent formation of ethanol due to the aerobic metabolism of microorganisms and methanol due to the degradation of the pectins linked to the action of endogenous pectinmethyl-esterases (biedermann et al., 2008). jabeur et al., (2015) found that in lampante oils the amounts of ethyl esters were greater than those of methyl esters: in our study, this was only observed for sample t1. the results also highlighted that the change in the content of faaes, and particularly faee, that occurred in the stored oil (t1) was significantly higher than that in oils extracted from olives stored for two years (t2). in fact, (conte et al., 2014; gómez-coca et al., 2016) reported that the faee concentration is a quality parameter that reflects fruit quality at the moment of extraction, and the presence of ethyl esters are markers of fermentation (low quality of olive fruits and extracted oil). it could be assumed that sunlight drying of olives and subsequent long storage could lead to a higher liberation of methanol (from degradation of pectins) respect to a fermentative process, explaining the highest content of fames compared to faees for t2. on the other hand, the high content of faees in sample t1 could be explained by the low quality of olives before processing, as shown by the remarkable content of faaes as well as a free acidity value close to the legal limit in sample tc. table 4. fatty acid alkyl esters (mg i.s. kg-1) of olive oil not stored (tc), stored for two years (t1) and obtained from intact sun-dried olive fruits stored for two years before being processed (t2). tc t1 t2 σ fames 33.7±0.2c 185.2±9.8a 172.4±31.9b σ faees 23.6±3.6c 624.2±15.5a 62.6±8.7b faees/fames 0.7±0.1b 3.4±0.2 a 0.4±0.1c total faaes 53.5±8.1c 809.5±20.5 a 235.0±33.0b fames: fatty acid methyl esters; faees: fatty acid ethyl esters; faaes: fatty acid alkyl esters. each value represents the mean of the three determination (n = 3) ± standard deviation. a-c different letters show statistically significant differences (p < 0.05) according to duncan test. 4. conclusions overall, from this study it can be concluded that both the storage methods herein investigated had a strong negative influence on the quality of the olive oils. this is a crucial information to be communicated to the local olive oil producers residents in gabes (southern tunisia), in order to make them aware about the strong influence on the quality of the product. in particular, it should be underlined that the basic quality parameters of the stored oils exceeded the limits established by the ioc trade standard for virgin olive oils, thus declassifying the oil as “lampante”. a significant degradation (p<0.05) in phenolic compounds also occurred, being more accentuated in the oil conserved for two ital. j. food sci., vol. 30, 2018 113 years than in that obtained from stored and dried olive fruits. a similar trend was also seen in α-tocopherol content and in oxidative stability time (osi) of the samples. the high faaes level observed in both the two storage conditions suggested that the oils were obtained from olives of suboptimal quality, wherein fermentation and other types of degradations (oxidation) have occurred. however, the results of the current study highlight that some traditional methods, still applied in gabes for storage of olives and oil, exerted a notable negative effect on all parameters investigated. acknowledgements this study was supported by the ministry of higher education and scientific research in tunisia. we want to thank the members of lr-nafs /lr12es05 «nutrition-functional food and vascular health» and the laboratory staff of the department of agricultural and food sciences, alma mater studiorum – university of bologna (italy) for their contribution to this 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phenolic compounds of virgin olive oil during 12 months. j. am. oil chem. soc. 74:109. ital. j. food sci., vol. 30, 2018 115 servili m. and gian francesco m. 2002. contribution of phenolic compouns to virgin olive oil quality. eur. j. lipid sci. technol. 104:602-613. servili m., selvaggini r., taticchi a., esposto s. and montedoro g. 2003. volatile compounds and phenolic composition of virgin olive oil: optimization of temperature and time of exposure of olive pastes to air contact during the mechanical extraction process. j. agric. food chem. 51:7980-8. singleton v.l. and rossi j.a.j. 1965. colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. am. j. enol. vitic. 16:144-158. tura d., gigliotti c., pedò s., failla o., bassi d. and serraiocco a. 2007. influence of cultivar and site of cultivation on levels of lipophilic and hydrophilic antioxidants in virgin olive oils (olea europea l.) and correlations with oxidative stability. sci. hortic. (amsterdam) 112:108-119. valli e., bendini a., maggio rm., cerretani l., toschi t. g., casiraghi e. and lercker g. 2013. detection of low-quality extra virgin olive oils by fatty acid alkyl esters evaluation: a preliminary and fast mid-infrared spectroscopy discrimination by a chemometric approach. int. j. food sci. technol. 48:548-555. vichi s., romero a., gallardo-chacón j., tous j., lópez-tamames e. and buxaderas s. 2009. influence of olives’ storage conditions on the formation of volatile phenols and their role in off-odor formation in the oil. j. agric. food chem. 57:1449-1455. paper received july 21, 2017 accepted september 2, 2017 review ital. j. food sci., vol. 27 2015 1 keywords: biodegradable packaging, biopolymers, edible coating, fresh-cut fruits and vegetables, minimally processed foods, nanocomposites biodegradable packaging and edible coating for fresh-cut fruits and vegetables f. galgano1*, n. condelli1, f. favati2, v. di bianco1, g. perretti3 and m.c. caruso1 1 school of agricultural, forestry, food and environmental sciences, university of basilicata, viale dell’ateneo lucano 10, 85100 potenza, italy 2 department of biotechnology, university of verona, strada le grazie 15, 37134 verona, italy 3department of agriculture, food and environmental sciences, university of perugia, via s. costanzo n.c.n., 06126 perugia, italy *corresponding author: tel. +39 0971-205570, fax +39 0971-205503, email: fernanda.galgano@unibas.it abstract this work focuses on biodegradable packaging and edible coatings applied to fresh-cut fruits and vegetables and their effects on the product quality. practical applications are mainly limited to the use of biodegradable materials that, however, do not allow full control of the product moisture loss. better results can be achieved by the combined use of biodegradable packagings with edible coatings and recent research has shown that enrichment with silver-montmorillonite nanoparticles may be a promising technique. however, the actual utilization of these materials is still limited, due to the high costs of the raw materials and the limited production. 2 ital. j. food sci., vol. 27 2015 introduction in recent years, the establishment of new lifestyles, characterized by a lack of time in domestic preparations, has been accompanied by an evolution of the structure of food consumption and a destructuring of meals. therefore, the consumption of minimally processed fruits and vegetables has grown rapidly as a result of the consumer trend“rich in cash, poor in time” (martín-diana et al., 2007; conte et al., 2009). minimally processed fruits and vegetables were developed in the 1980s to respond to the emerging consumer demand for both convenience and high quality aspects (del nobile et al., 2008). these foodstuffs combine fresh-like and healthy characteristics with a minimal time of preparation before consumption (ragaert et al., 2004). in fact, their success is also due to the beneficial health effects for the presence of antioxidants that act as receptors of free radicals; in particular, ascorbic acid and β-carotene are the antioxidants present in the greatest quantities in fruits and vegetables (rico et al., 2007). the minimal processing operations (“mild technology”) necessary to produce fresh-cut foods, such as peeling, cutting, washing, treatments with sanitizing agents, drying, alter the physical integrity of these products, making them more perishable than the original raw materials (corbo et al., 2006). this is due to respiration, transpiration, enzymatic activity of the living tissue after harvest and processing and, at the same time, to proliferation of spoilage and pathogenic microorganisms (galgano et al., 2014; nguyen-the and carlin, 1994). the industrial process accelerates the degradation of the minimally processed foods and leads to biochemical changes such as: increasing of respiration that accelerates the oxidation processes; degradation of cell membranes and enzymatic browning; loss of tissue texture. the knowledge of factors influencing quality degradation after processing of fruits and vegetables is essential to develop technologies for shelflife extending and maintaining quality during processing and distribution (corbo et al., 2006). in order to reduce the microbiological, chemical and physical spoilage, it is possible to act on processing or, more usually, on packaging that represents a barrier to qualitative decay of the product (galgano et al., 2014). the packaging operation should establish inside the packaging an optimal atmosphere for the best preservation of the product. generally, low o 2 and elevated co 2 atmospheres, associated with low storage temperature, reduce product respiration rate, limiting, in this way, losses in fresh weight (watada et al., 1996). therefore, a proper combination of product characteristics and film permeability results in the evolution of an appropriate atmosphere within packages (smith et al., 1987). traditionally, food companies use polymeric films (polyethylene pe, polypropylene pp, polystyrene, ps) to package fresh fruits and vegetables because of their large availability at relatively low cost and their good mechanical performance, good barrier to oxygen, carbon dioxide (siracusa et al., 2008). nowadays, there is a growing trend in fresh fruits and vegetables packaging sector to replace the petrochemical based packaging films with more environmentally-friendly biodegradable materials (tharanathan, 2003). the extensive use of synthetic packaging films has led to serious ecological problems due to their total nonbiodegradability. therefore, biodegradability is not only a functional requirement but also an important environmental attribute. biologically-based packaging contains raw materials originated from agricultural sources, produced from renewable raw materials such as starch and bio-derived monomers (lucera et al., 2010). these materials represent a viable alternative because: they are obtained from renewable sources; they are recyclable and degradable; they are an opportunity to reduce costs. the applications range from the design of multilayer barrier coating consisting of biopolymers, to enrichment of the matrix of traditional plastics pp and pe with nanocomposite materials of natural origin. however, bio-packaging still represents a niche market because of the cost and poor overall performance of biodegradable films when compared to those of traditional plastic materials (del nobile et al., 2009 a). the present work focuses on the different packaging strategies for fresh-cut fruits and vegetables. in particular, the potential applications of biodegradable materials and edible coatings have been described, with their effects on the quality of these products. the application of nanotechnology, as a tool to improve the performance and thermal, barrier and mechanical properties of bio-polymers has also been assessed (azeredo, 2009). bio-based materials for food packaging the term “bio-based materials” (bbm) is assigned to packaging materials and to packaging produced from biological renewable raw materials (piergiovanni and mascheroni, 2007). polymers derived from renewable resources (biopolymers) are broadly classified according to method of production, as follows: 1. polymers directly extracted/removed from natural materials (mainly plants). examples are polysaccharides, such as starch and cellulose and proteins, like casein and wheat gluten, and lipids. ital. j. food sci., vol. 27 2015 3 2. polymers produced by “classical’’ chemical synthesis from renewable bio-derived monomers. a good example is polylactate, a biopolyester polymerised from lactic acid monomers. the monomer itself is produced via fermentation of carbohydrate feedstock. 3. polymers produced by microorganisms or genetically transformed bacteria. the best known bio-polymer types are the polyhydroxyalkanoates, mainly polyhydroxy butyrates and copolymers of hydroxy-butyrate (hb) and hydroxyvalerate (hv) (petersen et al., 1999). the compostability attribute is very important for biopolymer materials, because composting allows disposal of the packages in the soil, which is more energy efficient than recycling. during biological degradation water, carbon dioxide and inorganic compounds without toxic residues are produced. according to the european bioplastics, biopolymers made with renewable resources have to be biodegradable and especially compostable, so they can act as fertilizers and soil conditioners. whereas plastics based on renewable resources must not necessarily be biodegradable or compostable, the bioplastic materials do not necessarily have to be based on renewable materials, because the biodegradability is directly correlated to the chemical structure of the materials rather than the origin. in particular, the type of chemical bond defines whether and in what time the microbes can biodegrade the material. several synthetic polymers are biodegradable and compostable, such as starch, cellulose, which are naturally carbon-based polymers. viceversa, the bioplastics based on natural monomer, can lose their biodegradability through chemical modification like polymerization, such as for example nylon 9 type polymers obtained from polymerization of oleic acid monomer or polyamid 11 obtained from the polymerization of castor oil monomer (siracusa et al., 2008). the problems associated with renewable biopolymers are threefold: performance, processing and cost. although these factors are somewhat interrelated, problems due to “performance and processing’’ are more pronounced with polymers extracted directly from biomass (cellulose, starch, proteins). conversely, polymers belonging to categories 2 and 3 above, generally perform very well and are easily processed into films using standard plastic techniques, but tend to be expensive compared with synthetic analogues (petersen et al., 1999). polymers directly extracted/removed from natural materials polysaccharides the polysaccharides show effective gas barrier properties although they are highly hydrophilic and show high water vapor permeability in comparison with commercial plastic films. the main polysaccharides that can be included in edible coating formulations are starch and starch derivates, cellulose derivates, alginate, carrageenan, chitosan, pectin, and several gums. based on the molecular level, polysaccharides vary according to their molecular weight, degree of branching, conformation, electrical charge and hydrophobicity. variations in these molecular characteristics will lead to variations in the ability of different polysaccharides to form coatings, as well as to variations in the physicochemical properties and performance of the coatings formed. for example, the linear structure of some of these polysaccharides such as cellulose, amylose (a component of starch), and chitosan makes their films tough, flexible, transparent, and resistant to fats and oils (vargas et al., 2008). starch, a storage polysaccharide of cereals and legumes, is most commonly used in the formulation of edible coatings and films because it is inexpensive, abundant, biodegradable, and easy to use. films based on starch have moderate gas barrier properties. their mechanical properties are generally inferior to synthetic polymer films. when a plasticizer, such as water, is added starches exhibit thermoplastic behavior (krochta and mulder-johnston, 1997). starch-based thermoplastic materials have been commercialized over the last few years and today dominate the market of biobased, compostable materials (claus, 2000). the properties of the starch-based materials can be improved by destructuring the native conformation of the starch and adding synthetic substances. it is common practice to add copious amounts of synthetic polymer to the starch, such as polyvinyl alcohol (pva) or polycaprolactone. another important polysaccharide is chitosan, which is mainly obtained from crab and shrimp shells (hirano, 1999). films and coatings based on chitosan have selective permeability to gases (co 2 and o 2 ) and good mechanical properties. however, their uses are limited mainly because of their high water vapor permeability (butler et al., 1996; caner et al., 1998). moreover, chitosan shows antifungal and antibacterial properties, which are believed to have originated from its polycationic nature. an edible film with innovative characteristics has been obtained by combining the antimicrobial properties of chitosan with the property of self-sealing banana starch, variety kluai namwa. the presence of starch in the composite film makes water soluble and sealable bags or wraps possible, while the presence of chitosan gives them the antimicrobial property. the composite bags were found to protect asparagus, baby corn and chinese cabbage against staphylococcus aureus activity by serving as a good barrier and as an antimicrobial agent. the properties of composite 4 ital. j. food sci., vol. 27 2015 films were more similar to starch films than to films made solely from chitosan as the amount of banana flour was greater than chitosan in the films. these composite films were cheaper and less flexible than chitosan films. film extensibility was improved by the addition of banana flour and glycerol. banana flour addition enhanced wvp and solubility, although, the films were not completely smooth. the biodegradable and hot water soluble films can be used in food packaging and to reduce microbial counts thus extending shelf-life of fruit and vegetables in convenient bags (pitak and rakshit, 2011). cellulose is the most abundant natural polymer on earth and it is an essentially linear polymer of anhydroglucose. as a consequence of its chemical structure, it is highly crystalline, fibrous, and insoluble. many derivatives of cellulose have excellent properties of film-forming, but are simply too expensive for use on a large scale. finally, the similarity of cellulose and chitosan in primary structures has facilitated the formation of homogeneous composite films. möller et al. (2004) reported that 1% of chitosan in chitosan-hpmc (hydroxypropylmethylcellulose) composite films is effective against l. monocytogenes. alginates can also be used to prepare edible coatings and films. alginates are the salts of alginic acid, which is a linear copolymer of d-mannuronic and l-guluronic acid monomers. alginate coating formation is based on the ability of alginates to react with di-valent and tri-valent cations such as calcium, ferrum or magnesium, which are added as gelling agents (cha and chinnan, 2004). an original study (norajit et al., 2010) investigated the physical and antioxidant properties of alginate biodegradable film incorporating white, red and extruded white ginseng extracts. the major pharmacologically active constituents of ginseng are triterpene saponins called ginsenosides. the studies demonstrate that the ginseng extract can be successfully incorporated into biodegradable alginate films and retain excellent antioxidant activities. the incorporation of ginseng extract into the alginate films did not cause major changes in the moisture content values. on the other hand, significant reductions in tensile strength and elastic modulus values of ginsengalginate film have been found compared to those of the film without ginseng extract. galactomannans, natural polysaccharides commonly used in the food industry, mostly as a stabilizer, thickener and emulsifier, are one of the alternative materials that can be used for the production of edible films/coatings based on their edibility and biodegradability. these gums are mostly obtained from the endosperm of dicotyledonous seeds of numerous plants, particularly the leguminosae. the great advantage of galactomannans is their ability to form very viscous solutions at relatively low concentrations that are only slightly affected by ph, ionic strength and heat processing. the mechanical, barrier and rheological properties of galactomannan films/coatings may be used to improve the stability, safety, and quality of food products (cerqueira et al., 2011). lima et al. (2010) successfully blended collagen with two galactomannans from different species (a. pavonina and c. pulcherrima). the composition of films/coatings with different proportions of galactomannan, collagen and glycerol have been optimized based on the wettability of films, transport and mechanical properties; these films have been subsequently used to coat mangoes and apples. the results showed that the application of the coatings leads to a decrease of gas transfer rates of the fruits. for mangoes, a coating of a. pavonina galactomannan (0.5%), collagen (1.5%) and glycerol (1.5%) decreased oxygen consumption and carbon dioxide production in 28% and 11%, respectively. for apple, the oxygen consumption and carbon dioxide production decreased for both gases by approximately 50%, with the utilization of a coating of c. pulcherrima galactomannan (0.5%) and collagen (1.5%). proteins proteins that have received great attention for their capability of forming edible films and coatings include corn zein, wheat gluten (wg), soy protein, whey protein, casein, collagen/gelatin, pea protein, rice bran protein, cottonseed protein, peanut protein, and keratin (han and gennadios, 2005). casein based edible coatings are attractive for food applications due to their high nutritional quality, excellent sensory properties, and strong potential for providing food products with adequate protection against their surrounding environment. whey proteins have been subjected to intense investigation over the past decade or so. with the addition of plasticizer, heatdenatured whey proteins produce transparent and flexible water-based edible coatings with excellent oxygen, aroma, and oil barrier properties at low relative humidity. however, the hydrophilic nature of whey protein coatings causes them to be less effective as moisture barriers (vargas et al., 2008). the water vapor permeability limits their potential uses for food packaging and justifies the interest in natural hydrophobic substances, such as lipids. lipids edible lipids used to develop edible coatings are: beeswax, candelilla wax, carnauba wax, triglycerides, acetylated monoglycerides, fatty acids, fatty alcohols, and sucrose fatty acid esters. lipid coatings and films are mainly used for their hydrophobic properties, representing a good barrier to moisture loss. this factor is extremely important, as a great number of studies deal with the use of coatings on fresh fruits ital. j. food sci., vol. 27 2015 5 and vegetables to control their desiccation. in addition to preventing water loss, lipid-based coatings have been used to reduce respiration, thereby extending shelf life and to improve appearance by generating a shine on fruits and vegetables. in contrast, the hydrophobic characteristic of lipids forms thicker and more brittle films. consequently, they must be associated with film forming agents such as proteins or cellulose derivatives (debeaufort et al., 1993). polymers produced by “classical” chemical synthesis from renewable bio-derived monomers aliphatic polyesters belong to this category and are obtained from bio-derived monomers by means of classical polymerization procedures. one of the most promising biopolymer is the poly(lactic acid) (pla), which is derived from the controlled depolymerization of the lactic acid monomer, obtained from the fermentation of sugar feedstock, corn, etc., which are renewable resources readily biodegradable (cabedo et al., 2006). discovered in 1932 by carothers (jamshidian et al., 2010), it is a versatile polymer, recyclable and compostable, with high transparency, high molecular weight, good processability and water solubility resistance. in comparison to other biopolymers, the production of pla has numerous advantages including: a) production of the lactide monomer from lactic acid, which is produced by fermentation of a renewable agricultural source corn (usually based on the strain of lactobacillus); b) fixation of significant quantities of carbon dioxide via corn (maize) production by the corn plant; c) significant energy savings; d) the ability to recycle back to lactic acid by hydrolysis or alcoholysis; e) the ability to tailor physical properties through material modification (dorgan et al., 2000). pla is generally recognized as safe (gras) by the united state food and drug administration (fda). moreover, pla can be easily processed by conventional processing techniques used for thermoplastics like injection moulding, blow moulding, thermoforming and extrusion. pla can be used in a wide range of applications; the major pla application today regards packaging (nearly 70%). pla food packaging applications are ideal for fresh products, such as fresh meat (galgano et al., 2009) or fruits and vegetables. polymers produced by microorganisms: polyhydroxyalkanoates (pha) in recent years poly-β-hydroxyalkanoates (pha) have attracted a lot of attention as biocompatible and biodegradable thermoplastic materials with potential applications. poly(3-hydroxybutyrate) (phb) is one of the well-known biodegradable poly(hydroxy alkanoates) (pha). phb is a natural thermoplastic polyester and has many mechanical properties comparable to synthetically produced degradable polyesters such as the poly-l-lactides (freier et al., 2002). since 1925 phb has been produced by bacterial fermentation (rosa et al., 2004), which takes place in the presence of a wide variety of bacteria, as intracellular reserve material. at least 75 different genera of bacteria have been known to accumulate phb as intracellular granules. this polymer is synthesized under limited culture conditions and its production has most commonly been studied with microorganisms belonging to the genera alcaligenes, azobacter, bacillus and, pseudomonas (ugur et al., 2002). phb films are degraded by numerous microorganisms (bacteria, fungi and algae) in various ecosystems. when in contact with the polymer, the microorganisms secrete enzymes that break it into successively smaller segments, thereby reducing the average molecular weight (bucci et al., 2005). these polymers, alone or in combination with synthetic plastic or starch produce excellent packaging films. the polyalkanoates are more hydrophobic than the polysaccharide-based materials, resulting in a material with good moisture barrier properties, whereas the gas barriers are inferior (petersen et al., 1999). phb has been used in small disposable products and in packing materials. however, little is known about the application of phb to packaging for food products. this polymer is not used in cut-fruits and vegetables, so there is no sense to have it here, unless you write some possible uses in cut-fruits and vegetables. another biopolymer produced from microorganisms is the pullulan, extracted from cells of aureobasidium pullulans, tremella mesenterica and cyttaria harioti. it is characterized by high resistance to fats, good barrier to gases; moreover, it is completely biodegradable and can be processed with conventional techniques. chemically pullulan is a polymer of maltotriose used in combination with sorbitol and fatty acids esterified with sucrose to achieve edible coatings for fruits (piergiovanni and mascheroni, 2007). in a recent study the possibility of using new biodegradable material such as kefiran, a microbial polysaccharide obtained from the flora of kefir grains has been investigated (ghasemlou et al., 2011). it is a water-soluble polysaccharide containing approximately equal amounts of glucose and galactose (micheli et al., 1999), used in the food industry as a texturing agent and gelling agent. it could be a viable alternative to synthetic materials for food packaging, also because it has antibacterial and anticancer properties (maeda et al., 2004). the biopolymers fulfill environmental requirements, but show some limitations in terms of 6 ital. j. food sci., vol. 27 2015 performance, such as thermal resistance, mechanical properties and barrier properties, associated with high costs. so, more research needs to be carried out on the packaging material with a view to introducing, for instance, intelligent molecules or inorganic material, preferably in the form of nano-particles, in order to expand the range of properties of the materials. the protagonist of the twenty-first century is another scientific innovation that is affecting the packaging industry: nanotechnology. nanotechnology and nanocomposites the concept of nanotechnology was introduced by richard feyman in 1959 at a meeting of the american physical society (khademhosseini and langer, 2006). nanotechnology is defined as “the design, characterization, production and application of structures, devices and systems by controlling the shape and size at the nanometer scale” (royal society and royal academy of engineering, nanoscience and nanotechnologies, 2004). nanotechnology and its application in food science have recently been studied by several researchers. the use of nanoparticles, such as micelles, liposomes, nanoemulsions, biopolymeric nanoparticles aimed at ensuring food safety, are some novel nano-food applications. nanotechnology is also applicable in the context of food packaging: however, the use of edible and biodegradable polymers has been limited because of problems related to performance (such as brittleness, poor gas and moisture barrier), processing (such as low heat distortion temperature), and cost. the application of nanotechnology to these polymers may open new possibilities for improving not only the properties but also the cost-price-efficiency (sorrentino et al., 2007). the use of fillers with at least one nanoscale dimension (nanoparticles) produces nanocomposites. most reinforced materials present poor matrix–filler interactions, which tend to improve with decreasing filler dimensions. nanoparticles have proportionally a larger surface area than their microscale counterparts, which favors the filler–matrix interactions and the performance of the resulting material. polymer composites are mixtures of polymers with inorganic or organic fillers with certain geometries (fibers, flakes, spheres, particulates). a uniform dispersion of nanoparticles leads to a very large matrix/filler interfacial area, which changes the molecular mobility, the relaxation behavior and the consequent thermal and mechanical properties of the material. although several nanoparticles have been recognized as possible additives to enhance polymer performance, the packaging industry has focused its attention mainly on layered inorganic solid like clay and silicates, due to their availability, low cost and relative simple processability (azeredo, 2009). the most widely studied type of clay fillers is montmorillonite (mmt), a hydrated alumina-silicate layered clay consisting of an edgeshared octahedral sheet of aluminum hydroxide between two silica tetrahedral layers (weiss et al., 2006). mmt is an effective reinforcement filler, due to its high surface area and large aspect ratio (50–1000) (uyama et al., 2003). furthermore, the presence of this nanoclay in polymer matrix increases its degradation rate, due to hydroxyl groups of mmt (souza et al., 2013). mangiacapra et al. (2005) reported that it is possible to reduce the oxygen permeability by adding clay montmorillonite to pectins. also the combination of pla film with mmt-layered silicates may be useful to create a nanocomposite material with good barrier properties (souza et al., 2013). moreover, a starch/clay nanocomposite film have been obtained by dispersing mmt nanoparticles via polymer melt processing techniques (avella et al., 2005). the results related to mechanical characterization showed an increase of modulus and tensile strength. besides nano-reinforcements, nanoparticles can have other functions when added to a polymer, such as antimicrobial activity, enzyme immobilization, biosensing (azeredo, 2009). the incorporation of antimicrobial compounds into food packaging materials has received considerable attention. films with antimicrobial activity could help control the growth of pathogenic and spoilage microorganisms. an antimicrobial nanocomposite film is particularly desirable due to its acceptable structural integrity and barrier properties imparted by the nanocomposite matrix, and the antimicrobial properties contributed by the natural antimicrobial agents impregnated within (rhim and ng, 2007). the most common nanocomposites used as antimicrobial films for food packaging are based on silver, which is well known for its strong toxicity towards a wide range of microorganisms (liau et al., 1997), with high temperature stability and low volatility (kumar and münstedt, 2005). some mechanisms have been proposed for the antimicrobial property of silver nanoparticles (ag-nps): adhesion to the cell surface, degrading lipopolysaccharides and forming ‘‘pits’’ in the membranes, largely increasing permeability (sondi and salopek-sondi, 2004); penetration inside bacterial cell, damaging dna (li et al., 2008); and releasing antimicrobial ag+ ions by ag-nps dissolution (morones et al., 2005). moreover, ag-nps absorbs and decomposes ethylene (hu and fu, 2003), which may contribute to its effects on extending shelf life of fruits and vegetables. indeed, li et al. (2009) reported that a nanocomposite pe film with ag-nps can retard the senescence of jujube, a chinese fruit. an et al. (2008) reported that a coating containing ag-nps is efital. j. food sci., vol. 27 2015 7 fective in decreasing microbial growth and increasing shelf life of asparagus. the three new requirements of food packaging are the following: “bioactive, biodegradable and bionanocomposite”. however, there are many safety concerns about nanomaterials, as their size may allow them to penetrate into cells and eventually remain in the system. one can assume that the application of nanoparticles with an antimicrobial function (the nanoparticles of silver or copper) poses the risk the consumer’s direct exposure to metals. although the migration of metals from biodegradable nanocomposite film of starch / clay applied to vegetable products is considered to be minimal (avella et al., 2005), it is necessary to have more accurate information about the likely impact on human health. in fact, many studies and experiments relating to the use of nanoparticles are still in progress in order to avoid premature generalizations that could undermine the potential benefits obtainable from this technology. in the next section, the potential applications of this new category of materials (biodegradable, edible coating and nanocomposites) and their possible effects on the quality characteristics of fresh-cut fruit and vegetables is discussed. edible films and coatings for fresh-cut fruits and vegetables consumers usually judge the quality of freshcut fruit on the basis of appearance and freshness at the time of purchase (kader, 2002). however, minimal processing operations alter the integrity of fruits, with negative effects on product quality, such as browning, off-flavour development and texture breakdown. also, the presence of microorganisms on the fruit surface may compromise the safety of fresh-cut fruit. traditionally, edible coatings have been used in the fresh-cut industry as a strategy to reduce the deleterious effects that minimal processing imposes on intact vegetable tissues. an edible coating may be defined as a thin layer of material that covers the surface of the food and can be eaten as part of the whole product. therefore, the composition of edible coatings must be conform to the regulation that apply to the food product concerned (guilbert et al., 1995; vargas et al., 2008). edible coatings may contribute to extend the shelf-life of fresh-cut fruits by reducing moisture and solute migration, gas exchange, respiration and oxidative reaction rates, as well as by reducing or even suppressing physiological disorders (baldwin et al., 1996; park, 1999). edible coatings are capable of producing a modified atmosphere on coated fruits by isolating the coated product from the environment. coatings with selective permeability to gases are capable of decreasing the interchange of o 2 and co 2 between coated fruits and the environment (olivas and barbosa-cánovas, 2005). high co 2 concentration within fruit tissues also delays ripening by decreasing the synthesis of ethylene, a hormone essential for ripening (saltveit, 2003). lee et al. (2003) demonstrated that the respiration rate of apple slices decreases 20% when coated with a film based on whey protein. edible coating based on aloe vera reduced respiration rate and microbial spoilage in sliced kiwifruit (benítez et al., 2013). fresh-cut products are characterized by high water transpiration rates and the creation of a moisture and gas barrier may lead to weight loss and respiration rate reduction, with a consequent general delay of produce senescence (valencia-chamorro et al., 2011). coating apple slices with a carbohydrate/lipid bilayer film reduces water loss during storage between 12 to 14 times when compared to the water loss suffered by uncoated apple slices in similar storage conditions (wong et al., 1994). alginate coatings prevented water loss on fresh-cut apple (lee et al., 2003; fontes et al., 2007), papaya (tapia et al., 2007), pear (omsoliu et al., 2008a), and melon (oms-oliu et al., 2008b). chitosan coatings reduced water loss in sliced mango (chien et al., 2007) and carrageenan coatings prevented water loss on sliced banana (bico et al., 2009). the cassava starch edible coating with or without the calcium lactate was able to reduce weight loss in fresh-cut pineapple (bierhals et al., 2011), while the cassava starch edible coating with or without potassium sorbate decreased the respiration rate in freshcut strawberries (garcia et al., 2010). minimally processed pummelo (citrus maxima merr.), coated with starch-based coatings (derived from cassava and rice) had a lower weight loss of 4.8– 7.7% compared to the control (non-coated minimally processed pummelo) (kerdchoechuen et al., 2011). moreover, the combination of citric acid dipping and cassava starch or sodium alginate edible coatings was able to delay the quality deterioration of fresh-cut mangoes, decreasing the respiration rate of fruits with value up to 41% lower than the control fruit. although the citric acid increases the weight loss of the product, causing a partial dehydration of the vegetable tissue, the use of cassava starch and alginate coatings was able to hinder this undesirable effect of citric acid, presenting a lower weight loss than the coated samples (chiumarelli et al., 2011). in a previous work, chiummarelli et al. (2010) reported that this treatment provided a better maintenance of colour characteristics, due to the combined effect of citric acid dipping and cassava starch coating. citric acid delayed browning along the storage and the edible coatings acted as a gas barrier, decreasing the respiration rate of mango pieces and, consequently the formation of carotenoids. moreover, another important advantage of edible coating is the reduction of synthetic packag8 ital. j. food sci., vol. 27 2015 ing waste, because these coatings are composed of biodegradable raw material (dhall, 2013). the properties of edible coating depend primarily on molecular structure rather than molecular size and chemical constitution. specific requirements for edible films and coatings are as follows (arvanitoyannis and gorris, 1999): the coating should be water-resistant so that it remains intact and covers a product adequately, when applied; it should not deplete oxygen or build up excessive carbon dioxide. a minimum of 1–3% oxygen is required around a commodity to avoid a shift from aerobic to anaerobic respiration; it should reduce water vapor permeability; it should melt above 40°c without decomposition; it should be easily emulsifiable, non-sticky or should not be tacky, and have efficient drying performance; it should never interfere with the quality of fresh fruit or vegetable and not impart undesirable order; it should have low viscosity and be economical; it should be translucent to opaque but not like glass and capable of tolerating slight pressure. the ability of edible coatings to preserve the quality of fresh-cut products may vary from product to product, then it is necessary to consider the variety and maturity of the product, food surface coverage, storage conditions and composition and thickness of the coating (gonzález-aguilar et al., 2010). according to their components, edible films and coatings can be divided into three categories: hydrocolloids, lipids, and composites. hydrocolloids include proteins and polysaccharides, while lipids include waxes, acylglycerols, and fatty acids. composites contain both hydrocolloid components and lipids. their presence and abundance determine the barrier properties of material with regard to water vapor, oxygen, carbon dioxide, and lipid transfer in food systems. however, none of the three constituents can provide the needed protection by themselves and so are usually used in a combination for best results (mchugh et al.,1994; guilbert et al., 1996). the main objective of producing composite films is to improve the permeability or mechanical properties according to specific applications. these heterogeneous films are applied either in the form of an emulsion, suspension, or dispersion of the non miscible constituents, or in successive layers (multilayer coating or films), or in the form of a solution in a common solvent. several other compounds such as plasticizers and emulsifiers may be added to edible films and coatings to improve their mechanical properties and form stable emulsions when lipids and hydrocolloids are combined (valencia-chamorro et al., 2011). the application of edible coatings to fresh-cut fruits must address the problem regarding the difficult adhesion of materials to the hydrophilic surface of the sliced fruit. the layer-by-layer (lbl) electrodeposition can solve this problem (weiss et al., 2006). lbl assembly, which is performed by alternating the immersion of substrates in solutions of oppositely charged polyelectrolytes with rinsing steps, produces ultrathin polyelectrolyte multilayers on charged surfaces. a requirement for multilayer formation is that the addition of an opposite charged polyelectrolyte to a charged surface results in a charge reversal, which permits the successive deposition of oppositely charge polyelectrolytes. chitosan, poly-l-lysine, pectin, and alginate are the most common biopolymers that can be used in the formation of these multilayered structures (marudova et al., 2005; krzemiski et al., 2006; bernabé et al., 2005). in a study of mantilla et al. (2013), an alginatebased multilayered coating (developed using the layer-by-layer technique) enhanced the quality and shelf-life of fresh-cut pineapple extending its shelf-life to 15 days at 4°c. the main advantage of using edible films and coatings is that several active ingredients (such as antimicrobials, antibrownings, texture enhancer and nutraceuticals) can be incorporated into the polymer matrix and consumed with the food, thus enhancing safety or even nutritional and sensory attributes (rojas-graü et al., 2009). antimicrobial agents fresh-cut fruits are more perishable than their corresponding whole uncut commodities due to wounding during preparation (brecht, 1995). the physical and chemical barrier provided by the epidermis, which prevents the development of microbes on the fruit surface, is removed during processing. there are several categories of antimicrobials that can be potentially incorporated into edible films and coatings, including organic acids (acetic, benzoic, lactic, propionic, sorbic), fatty acid esters (glyceryl monolaurate), polypeptides (lysozyme, peroxidase, lactoferrin, nisin). eswaranandam et al. (2006) incorporated malic and lactic acid into soy protein coatings aiming to evaluate its effect on the sensory quality of fresh-cut cantaloupe melon, without studying the antimicrobial effect of the coating. in general, organic acids incorporated in films did not adversely affect the sensory properties of coated fruit. with reference to the use of natural antimicrobials, the development of coatings which use inherently antimicrobial polymers as a support matrix is very promising. for example, chitosan, produced from the deacetylation of crustacean chitin (poly-β-(1→4)-n-acetyl-d-glucosamine), is one of the most effective antimicrobial film ital. j. food sci., vol. 27 2015 9 forming biopolymers. chitosan is a cationic polysaccharide, which, among other antimicrobial mechanisms, promotes cell adhesion by the interaction of the positive-charged amines with the negative charges in the cell membranes, causing leakage of intracellular constituents (helander et al., 2001). chitosan based coatings were shown to protect highly perishable fruits like strawberries, raspberries, grapes and fresh-cutgreen pepper from fungal decay. (vargas et al., 2006; el gaouth et al., 1991; zhang and quantick, 1998; romanazzi et al., 2002; devlieghere et al., 2004; park et al., 2005, raymond et al., 2012). the application of chitosan edible coating on fresh-cut broccoli (moreira et al., 2011a) significantly reduced mesophilic and psychrotrophic counts and inhibited the growth of total coliform with respect to the control samples, throughout the whole storage period. similar results were reported by geraldine et al. (2008) working with minimally processed garlic. at the end of the storage, yeasts and molds were the most dominant flora and represented the largest part of the total aerobic count in broccoli. on the contrary, durango et al. (2006) founded an important fungicidal action of chitosan applied on minimally processed carrot. moreover, the application of mild heat shock enhanced the chitosan inhibition action (moreira et al., 2011b). from a sensory point of view, the chitosan inhibited the yellowing and opening florets of fresh-cut broccoli. moreover, chitosan-based edible coatings can also be used to carry other antimicrobial compounds such as organic acids (outtara et al., 2000), essential oils (zivanovic et al., 2005), spice extracts (pranoto et al., 2005), lysozyme (park et al., 2004) and nisin (pranoto et al., 2005; cha et al., 2003). essential oils (eos) (cinnamon, oregano, lemongrass) stand out as an alternative to chemical preservatives and their use in foods meets the demands of consumers for natural products. burt (2004) reported that hydrophobicity is an important characteristic of eos, which makes them able to pass through cell membranes and enter mitochondria, disturbing the internal structures and rendering the membranes more permeable. yet the application of eos in foods is limited due to their impact on organoleptic food properties, variability of their composition, and their variable activity in foods due to interactions with food components. nevertheless, the use of eos to control microbial growth in foods has been proposed for several products including freshcut fruits and vegetables. raybaudi-massilia et al. (2008b) studied the effect of malic acid and essential oils of cinnamon, palmarosa and lemongrass as natural antimicrobial substances incorporated into an alginate-based edible coating on the shelf-life of fresh-cut melon. the coating containing malic acid was effective in improving the shelf-life of fresh-cut melon from both the microbiological and physicochemical points of view in comparison with non-coated fresh-cut melon samples. the incorporation of the essential oils or their active compounds into the coating prolonged the microbiological shelf-life by more than 21 days in some cases, probably due to an enhanced antimicrobial effect of malic acid and the essential oils. however, some physicochemical characteristics, such as firmness and color, and also some sensory quality attributes were adversely affected, causing a significant reduction of fresh-cut melon shelf-life. in contrast, when malic or lactic organic acids incorporated in soy protein coatings were applied to fresh-cut cantaloupe, they did not adversely affect the sensory properties of the fruit after cold storage (eswaranandam et al., 2006). ayala-zavala et al. (2013), developed an edible pectin film enriched with the essential oil from cinnamon leaves and proved that this application can increase the antioxidant status and reduce bacterial growth of fresh-cut peach. chitosan coatings enriched with bioactive compounds (bee pollen, ethanolic extract of propolis, pomegranate dried extract, resveratrol)/essential oils (tea tree, rosemary, clove, oreganum, lemon aloe vera calendula) could be a good alternative for controlling not only the microorganisms present in broccoli, but also the survival of e. coli and l. monocytogenes inoculated in the product, without introducing deleterious effects on the sensory attributes of minimally processed broccoli (alvarez et al., 2013). rojas-grau et al. (2006, 2007a) have studied the effects of oregano, cinnamon, and lemongrass oils and their active compounds (carvacrol, cinnamaldehyde and citral) incorporated into apple puree and alginate apple puree edible films against escherichia coli o157:h7. in these works, oregano oil or its active compound, carvacrol, was most effective against e. coli o157:h7. in line with these preliminary studies, rojas-grau et al. (2007b) combined the efficacy of alginate and gellan edible coatings with the antimicrobial effect of eos (lemongrass, oregano oil and vanillin) to prolong the shelf-life of fresh-cut apples. a 4 log reduction of the inoculated population of listeria innocua in fresh-cut apple was observed when lemongrass or oregano oils were incorporated into an apple puree alginate edible coating. in addition, the coating reduced respiration rate and ethylene production of coated fresh-cut apples. in a later work (raybaudi-massilia et al., 2008a), the addition of cinnamon, clove and lemongrass essential oils or their active compounds, cinnamaldehyde, eugenol and citral into an alginate-based coating increased their antimicrobial effect, reduced the population of e. coli 0157:h7 by more than 4 log cfu/g and extended the microbiological shelflife of “fuji” apples for at least 30 days. according to these studies, azarakhsh et al. (2014), observed that the lemongrass incorporated into 10 ital. j. food sci., vol. 27 2015 an alginate-based coating for fresh-cut pineapple, reduces the microbial growth of the product during storage time. according to the institute of food science and technology (ifst), 106 cfu/g is considered the limit of acceptance for shelf-life of fruit-based products (bierhals et al., 2011). in fact, the coated samples with 0.3% and 0.5% lemongrass reached 106 cfu/g after 12 and 16 days, respectively. on the contrary, the application of tapioca starch/decolorized hsian-tsao leaf gum coatings on minimally processed carrots with antimicrobial agents (cinnamon oil and grape seed extract) had no beneficial effect on controlling the mesophile aerobics and psychrophiles. as a result, the respiration rate of the product increased (lai et al., 2013). instead, in the case of fresh-cut “fuji” apples, cinnamon oil incorporated in tapioca starch/decolorized hsian-tsao leaf gum (dhg) based edible coatings, significantly reduced the growth of microorganisms, respiration rate, co 2 and ethylene production (pan et al., 2013). mantilla et al. (2013) studied the effects of a multilayered edible coating with a microencapsulated antimicrobial complex (beta-cyclodextrin and transcinnamaldehyde) on the quality and shelf-life of fresh-cut pineapple. they reported that transcinnamaldehyde affects the pineapple flavour. however, the application of this antimicrobial coating extended the shelf-life of samples to 15 days at 4°c by inhibiting microbial growth. the same edible coating was applied on fresh-cut watermelon (sipahi et al., 2013) and similar results were obtained: this type of alginate coatings extended the shelf life of fresh-cut watermelon from 7 (control) to 12-15 days. a microencapsulated beta-cyclodextrin and trans-cinnamaldehyde complex was also incorporated into a multilayered edible coating made of chitosan and pectin. this coating extended the shelf life of fresh-cut papaya up to 15 days at 4°c while uncoated fruits did not last this long (< 7days). moreover, the coating reduced the losses of vitamin c and the total carotenoid content (brasil et al., 2012). antibrowning agents fresh-cut fruit processing operations can induce undesirable changes in colour and appearance of these products during storage and marketing. the phenomena is usually caused by the enzyme polyphenol oxidase (ppo), which in the presence of oxygen, converts phenolic compounds into dark colored pigments (zawistowski et al., 1991). application of antioxidant treatments such as dipping after peeling and/or cutting is the most common way to control browning of fresh-cut fruits. ascorbic acid is the most extensively used to avoid enzymatic browning of fruit due to the reduction of the o-quinones, generated by the action of the ppo enzymes, back to the phenolic substrates (mcevily et al., 1992). for example, the antioxidants citric and ascorbic acid were incorporated into methylcellulose-based edible coatings in order to control oxygen permeability and reduce vitamin c losses in apricots during storage (ayranci and tunc, 2004). the combination of chitosan coating and sodium chlorite dip treatment on pear slices adversely affected the quality of the fruit, accelerating the discoloration of cut surfaces and increasing the ppo activity. on the contrary, coating sodium chlorite-treated samples with carboxymethyl chitosan significantly prevented the browning reaction and inhibited ppo activity. (xiao et al., 2011). the effect of coatings in combination with anti-browning agents (1% chitosan; 2% ascorbic acid + 0.5% cacl 2 ) on minimally processed apple slices has been studied during storage by haiping et al. (2011). chitosan-coating treatments effectively retarded enzymatic browning on minimally processed apples during storage and effectively retarded tissue softening. as an alternative to ascorbic acid, several thiol-containing compounds, such as cysteine, nacetylcysteine, reduced glutathione and 4-hexylresorcinol have been investigated as inhibitors of enzymatic browning (gorny et al., 2002; son et al., 2001). these compounds react with quinones formed during the initial phase of enzymatic browning reactions to yield colorless additional products or to reduce o-quinones to odiphenols (richard et al., 1992). 4-hexylresorcinol, in combination with sodium erythorbate, was effective in maintaining the color of pear slices “anjou” (colelli and elia, 2009). furthermore, carboxylic acids (citric acid and oxalic acid) have also been suggested as effective antioxidant agents in fresh-cut fruits (jiang et al., 2004; pizzocaro et al., 1993; son et al., 2001). the incorporation of antibrowning agents into edible coatings applied on fresh-cut fruits has been studied by various authors. the application of alginate edible coating in conjunction with antibrowning agents (ascorbic and citric acid) to mango cubes increased vitamin c content compared to mango cubes treated only with alginate coating or control cubes, preserving the color of fresh-cut mangoes and increasing the antioxidant potential of cubes (robles-sánchez et al., 2013). moreover, perez-gago et al., (2006) reported a substantial reduction in browning of fresh-cut apples when using a whey protein concentrate-beeswax coating containing ascorbic acid, cysteine or 4-hexylresorcinol. they observed a significant improvement of the efficiency of antioxidant agents when incorporated into the coating formulation, being the most effective treatment with 0.5% cysteine. brancoli and barbosa-cánovas (2000) decreased surface discoloration of apple slices with maltodextrin and methylcellulose coatings including ascorbic acid. baldwin et al. (1996) found that a carboxymethyl ital. j. food sci., vol. 27 2015 11 cellulose-based coating with addition of several antioxidants, including ascorbic acid, reduced browning and retarded water loss of cut apple more effectively than an aqueous solution of antioxidants. furthermore, rojas-graü et al. (2008) observed that both alginate and gellan edible coatings containing n-acetylcysteine prevented apple wedges from browning during 21 days of storage. more recently, the application of konjac glucomann (polysaccharide derived from the tuber of konjac, amorphophallus konjac) with pineapple fruit extract represents an effective alternative to prevent browning of fresh-cut taaptimjaan rose apple fruit during storage. this coating enhanced total phenols and inhibited both ppo and peroxidase (pod) activities (supapvanich et al., 2012). or even, pullulan-based coating treatments in combination with antibrowning and antibacterial agents (1% pullulan; 0.8% glutathione + 1% chitooligosaccharides) effectively inhibited enzymatic browning, retarded tissue softening, inhibited microbial growth, decreased weight loss and respiration rate of the minimally processed apple slices (wu and chen, 2013). nutraceuticals nutraceuticals can also be incorporated into the formulation of edible coatings. despite the growing interest in incorporating nutraceutical compounds into food products, few studies have suggested their integration into edible films or coatings. in this sense, the concentration of nutrients added to the films/coatings must be carefully studied since it is important to know the effects on their basic functionality, namely on their barrier and mechanical properties. some studies have reported the effect of the addition of active compounds in the functionality of edible films. for instance, mei and zhao (2003) evaluated the feasibility of milk protein-based edible films to carry high concentrations of calcium (5 or 10% w/v) and vitamin e (0.1% or 0.2% w/v). they concluded that protein-based edible films can carry active compounds, although the film functionality can be compromised. in contrast, park and zhao (2004) reported that the water barrier property of the chitosan-based films was improved by increasing the concentration of mineral (5and 20% w/v zinc lactate) or vitamin e in the film matrix. nevertheless, the tensile strength of the films was affected by the incorporation of high concentrations of calcium or vitamin e, although other mechanical properties, such as film elongation, puncture strength, and puncture deformation, were not affected. several researchers have endeavoured to incorporate minerals, vitamins and fatty acids into edible film and coating formulations to enhance the nutritional value of some fruits and vegetables, where these micronutrients are present in low quantities. tapia et al. (2008) reported that the addition of ascorbic acid (1% w/v) to the alginate and gellan based edible coatings helped to preserve the natural ascorbic acid content in fresh-cut papaya, thus helping to maintain its nutritional quality throughout storage. in the last few years, attention has been paid to the addition of probiotics to obtain functional edible films and coatings. tapia et al. (2007) developed the first edible films for probiotic coatings on fresh-cut apple and papaya, observing that both fruits were successfully coated with alginate or gellan film-forming solutions containing viable bifidobacteria. in fact, values higher than 106 cfu/g bifidobacterium lactis bb-12 were maintained for 10 days during refrigerated storage of both papaya and apple pieces, demonstrating the feasibility of these polysaccharide coatings to carry and support viable probiotics on fresh-cut fruits. alginate offers the possibility of formulating a broad range of functional and innovative food products, increasing the nutritional properties of foods. recently, an edible alginate coating containing prebiotics such as oligofructose and inulin has been applied on fresh-cut apples wedges. fructan analysis showed that all prebiotics remained stable over the 14 day storage period and sensory and visual assessment indicated acceptable quality of apple wedges coated with prebiotics (rößle et al., 2011). the addition of prebiotics could be especially appealing to consumers as they are essential to human nutrition in the context of dietary guidelines. however, more studies are necessary to understand the interactions among active ingredients and coating materials when developing new edible film and coating, before they are applied to the surface of a real food system (rojas-graü et al., 2009). further progress comes from nanotechnology, a science that could provide new techniques for extending the shelf-life of foods. among the inorganic agents, silver nanoparticles have received great attention from the scientific world, due to the high biocidal effects towards many species of microorganisms (kim et al., 2007). in a study by costa et al. (2012), the effects of both active calcium-alginate coating loaded with silver-montmorillonite (ag-mmt) nanoparticles and film barrier properties on the shelf-life of freshcut carrots were assessed. the results of this study suggest that the active coating is the best treatment and could be used to control both dehydration and microbial spoilage of minimally processed carrots. moreover, the combination of coating with ag-mmt controlled the microbial growth better than the sole coating treatment. the combined use of proper packaging and active coating allowed the carrots to be kept in a good state of preservation thus prolonging the shelf-life to about 70 days, with respect to the uncoated samples (about 4 days). however, the 12 ital. j. food sci., vol. 27 2015 toxicological aspects of silver nanoparticles are well known (kiruba et al., 2010; lu et al., 2010); eu safety regulation limits the silver amount to 0.05 mg ag/kg of food (fernandez et al., 2009). the application of edible coating either on fresh-cut apples or on fruits and vegetables are respectively summarized in table 1 and in table 2 (a and b). table 1 application of edible coating on fresh-cut apple. food product coating composition main results references “gala” apple alginate alginate low weight loss; low enxymatic browning; greater firmness olivas et al. (2007) fresh-cut “fuji” apple cinnamon, clove, lemon grass essential oil or their their active compound cinnamaldehyde, eugenol and citral 4 log reduction of the e.coli o157:h7population; shelf-life extension at least 30 days raybaudi-massilia et al. (2008) fresh-cut apple wedges prebiotics, such as oligo fructose and inulin all prebiotics remained stable over the 14 days; good sensory characteristics rößle et al. (2011) fresh-cut “fuji” apple alginate and gellan essential oils: lemongrass, oregano oil and vanillin 4 log reduction of the inoculated population of listeria innocua; l owe r re s p i ra t i o n ra te a n d ethylene production rojas-graü et al. (2007b) fresh-cut apple alginate or gellan film-formi n g s o l u t i o n c o n t a i ning viable bifidobacteria higher values higher than 10^6 cfu/g of bifidobacterium lactis bb-12 were maintained for 10 days during refrigerate storage tapia et al. (2007) fresh-cut apple n-acetylcysteine enzymatic browning prevention during 21 days of storage rojas-graü et al. (2008) apple slices methylcellulose coatings maltodextrin and ascorbic acids. decreased surface discoloration of apples slices b r a n c o l i e t a l . (2000) fresh-cut apple whey protein concentrate beeswax ascorbic acid, cysteine or 4-hexyl-hexylresorcinol substantial reduction of browning perez-gago et al. (2006) apple “fuji” slices ascorbic acid and citric acid 20% decrease of the respiration rate; shelf-life extension to 2 weeks. lee et al. (2003) apple slices carbohydrate/lipid bilayer film carbohydrate/lipid bilayer film water loss reduction wong et al. (1994) fresh-cut “fuji” apple tapioca starch/decolorized hsian-tsao leaf gum cinnamon oil reduction of microbial growth, respiration rate, co 2 and ethylene production pan et al. (2013) fresh-cut taaptijaan rose apple konjac glucomannan pineapple fruit extract increase of total phenols; inhibi-tion of ppo and pod activities. supapvanich et al. (2012) fresh-cut apple slices pullulan pullulan; glutathione and chitooligosaccharides d e l ay o f t i s s u e s o fte n i n g, inhibition of microbial growth, decrease of weight loss and respiration rate. wu and chen (2013) fresh-cut apple slices chitosan ascorbic acid and cacl2 delay of enzymatic browning and tissue softening haiping et al. (2011) ital. j. food sci., vol. 27 2015 13 table 2a application of edible coating on fresh-cut fruits and vegetables. food product coating composition main results references sliced kiwifruit “haiward” aloe vera aloe vera reduced o 2 consumption and co 2 production; reduction of microbial spoilage benítez et al. (2013) sliced mango fresh-cut mango chitosan alginate and gellan chitosan ascorbic acid reduction of water loss increase of vitamin c bico et al. (2009) robles-sánchez et al. (2013) fresh-cut mango (pre-treated with citric acid dipping) sliced banana cassava starch-edible coating carrageenan cassava starch-edible coating carrageenan reduction of weight loss and respiration rate; better maintenance of colour characteristics reduction of water loss chiumarelli et al. (2010, 2011) bico et al. (2009) cantaloupe melon cube soy protein alginate soy protein, glycerol, malic acid, lactic acid malic acid and essential oils of cinnamon, palmarosa and lemongrass good sensory characteristics microbiological shelf-life extension. eswaranandam et al. (2006) raybaudi-massilia et al. (2008) fresh-cut papaya alginate and gellan chitosan and pectin ascorbic acid microencapsulated anti-microbial complex (beta-cyclodextrin and trans-cinnamaldehyde) preservation of the natural ascorbic acid content in papaya shelf-life extension to 15 days; reduction of vitamin c and total carotenoid content loss mantilla et al. (2013); sipahi et al. (2013) fresh-cut apple, papaya, pear, melon alginate alginate shelf-life extension to 15 days fontes et al. (2007); tapia et al. (2007); oms-oliu et al. (2008) fresh-cut pineapple, fresh-cut melon multilayered sodium alginate coating microencapsulated antimicrobial complex (beta-cyclodextrin and trans-cinnamaldehyde) prevention of water loss fontes et al. (2007); tapia et al. (2007); oms-oliu et al. (2008) fresh-cut pineapple; fresh-cut strawberries cassava starch-edible coating calcium lactate/ potassium sorbate reduction of weight loss reduction of respiration rate bierhals et al. (2011) garcia et al. (2010) biodegradable packaging and minimally processed fruits and vegetables fruits and vegetables are living organisms which continue to respire and transpire after harvesting, being characterized by a respiration rate; respiration and transpiration rates of fresh fruits and vegetables are often good indicators of their storage life; the higher the rate, the shorter the shelf-life (floros, 1993). moreover, the shelf-life of fresh-cut products is limited by microbiological deterioration and in order to reduce the effects of microbiological, chemical and physical events, it is possible to act on processing or, more usually, on packaging. the packaging operation should allow to establish inside the packaging an optimal atmosphere for the best preservation of the product. generally, low and elevated atmospheres, together with low storage temperature, reduce the product respiration rate limiting, in this way, losses in fresh weight (watada et al., 1996). the atmosphere that is established is the resultant of oxygen and carbon dioxide permeation through the walls of the packaging and of respiration 14 ital. j. food sci., vol. 27 2015 table 2b application of edible coating on fresh-cut fruits and vegetables. food product coating composition main results references minimally processed pummel cassava or rice starch cassava or rice starch lower weight loss of 4.8– 7.7% than the control kerdchoechuen et al. (2011) fresh-cut peach pectin essential oil from cinnamon leaves reduction of bacterial growth; better antioxidant status ayala-zavala et al. (2013) pear slices chitosan carboxymethyl chitosan sodium chlorite increase of ppo activity inhibition of ppo activity xiao et al. (2011) fresh-cut pineapple alginate lemongrass oil reduction of microbial growth azarakhsh et al. (2014) fresh-cut broccoli, garlic fresh-cut broccoli chitosan chitosan chitosan bioactive compounds (bee pollen, ethanoli c ex t ra c t o f p ro p o lis, pomegranatedried extract,resveratrol)/essential oils (tea tree, rosemary, clove, oreganum, lemon aloe vera calendula) reduction of mesophilic and psychrotrophic counts; inhibition of the yellowing and opening florets of fresh-cut broccoli better control of microorganisms (e. coli and l. monocytogenes); good sensory attributes moreira et al. (2011) geraldine et al. (2008) alvarez et al. (2013) fresh-cut carrots tapioca starch/decolorized hsian-tsao leaf gum active calcium-alginate coating loaded with sillver montmori-llonite (ag mmtt) nanoparticles cinnamon oil high biocidal effects no beneficial effect on controlling the mesophyles aerobics and psychrophiles good control of dehydration and microbial spoilage; of shelf-life extension to about 70 days, with respect to the uncoated samples lai et al. (2013) costa et al. (2012) by the cells, until reaching an equilibrium condition aimed to slow the senescence and avoid sensorial defects to the packaged product. either high co 2 or low o 2 concentrations could indirectly induce off-flavours by stimulating the growth of homoand hetero-fermentative bacteria and yeast, which produce organic acids, ethanol and volatile esters (carlin et al., 1989; kakiomenou et al., 1996). therefore, a proper combination of product characteristics and film permeability, which is a fundamental characteristic of the packaging materials, results in the evolution of an appropriate atmosphere within packages (smith et al., 1987). moreover, the packaged food actually interacts with the packaging material, changing the initial mechanical and barrier properties of the packaging materials (petersen et al., 1999). in alternative to the traditional polymeric materials, biodegradable films and coatings could be used. in fact, studies and experiments relating to possible applications of biodegradable materials, possibly combined with coating technique, to minimally processed fruits and vegetables are not lacking. results obtained by using biodegradable films for samples of iceberg lettuce are interesting: the film that is traditionally used to package fresh-cut salads is the bi-oriented polypropylene, which is associated with a level of senescence, linked to the metabolic activity of the vegetable, which is always higher than that achieved by salads packaged in biodegradable film. so, the potential application of biopolymers for the packaging of fresh-cut lettuce is justified (del nobile et al., 2008). also the rate of quality loss of minimally processed grapes in relationship with packaging film barrier properties has been assessed (del nobile et al., 2009a). in earlier work, del nobile et al. (2008b) investigated the influence of postharvest treatments (ethanol, chlorinated water and hot water) on the quality loss kinetics of freshly processed grapes packaged in biodegradable films. the results suggested that ethanol is the ital. j. food sci., vol. 27 2015 15 best solution to preserve the microbial stability of the fresh produce without affecting its respiration rate to any great extent. based on these results, clusters of table grapes (vitis vinifera cv. italia) were treated in ethanol solution prior to packaging in different bags made of two types of commercially available films (a multilayer film obtained by laminating nylon and a polyolefin layer (np); an oriented polypropylene film (opp)) and in three biodegradable polyesterbased films (nvt-100, nvt-50, nvt-35). results suggest that the respiratory activity of packed minimally processed produce is the main reason for its quality loss during storage. in fact, the best results in terms of grape quality were obtained using grapes with lower respiratory activity and high barrier films, such as np and nvt-100. from a sensory point of view, no undesirable after-taste developed within the fruit, when the grapes were packaged in the thicker film (np and nvt-100). also pla has been tested for fresh-cut fruits and vegetables packaging; the coating of pla silicon oxide (siox) improved the barrier oxygen and water vapor properties (peelman et al., 2013). in particular, the use of pla containers with lids and pouches composed of vc999 biopack pla films coated with a barrier of pure siox to preserve quality of fresh-cut pears after 9 days of storage in comparison with conventional polyethylene pe material, has been investigated. the results showed that color and firmness color of fresh-cut pears is better maintained with biodegradable packaging materials (krasnova et al., 2013) biodegradable materials do not always fulfill the physiological requirements of the specific fruit and/or vegetable packaged in them: in fact, poor moisture barrier properties, linked to the hydrophilic character that marks most of the bio-based-materials, is sometimes responsible for higher percentages of weight loss in products, such as zucchini (lucera et al., 2010), artichokes (del nobile et al., 2009b) lampascioni (muscari comosum) (conte et al., 2009c) and melon (conte et al., 2009b). often in several experiments, the technique of coating is used as a pretreatment to improve the performance of biodegradable materials and its effects on product quality. the coating treatment, containing sodium alginates, has been more effective than the dipping in delaying the metabolic activity associated to respiration rate in samples of lampascioni, subsequently packaged in ntv2, a film based biodegradable polyester (conte et al., 2009c). moreover, the browning process decreased, as well as, the microbial growth, since the availability of oxygen on the surface of the product was reduced. the same type of coating was also applied to fresh-cut artichokes (del nobile et al., 2009 b): also in this case, the coating together with packaging in ntv2 film seems to represent the better strategy to preserve the quality of artichokes, also from a microbiological point of view. interesting results also came from a study of fresh-cut cactus-pear: the shelf life of the minimally processed fruit, coated with an alginate and then packed with monolayer film (based on a blend of biodegradable polyesters), was prolonged to about 13 days, corresponding to an shelf-life increase of about 40%, compared to the control sample (untreated sample); on the contrary, produce immersion into either agar or fish protein strongly reduced the shelf life, most probably due to water migration from the surrounding hydro-gel to the fresh-cut produce (del nobile et al., 2009 c). also pla has been tested for fresh-cut fruits and vegetables packaging; the coating of pla silicon oxide (siox) improved the barrier oxygene and water vapor properties (peelman et al., 2013). in particular, the use of pla containers with lids and pouches composed of vc999 biopack pla films coated with a barrier of pure siox to preserve quality of fresh-cut pears after 9 days of storage in comparison with conventional polyethylene pe material, has been investigated. the results showed that color and firmness color of fresh-cut pears is better maintained with biodegradable packaging materials (krasnova et al., 2013) pla is also been used in combination with silver; silver ions have been incorporated into polylactic film, in order to evaluate the silver-infused pla films for inactivation of salmonella and feline calicivirus in vitro and on fresh-cut vegetables (martínez-abad et al., 2013). the results showed that in vitro the antibacterial and antiviral activity is more efficient than on food samples. anyway, in lettuce samples incubated a 4°c at 6 days of storage, 4 log cfu of salmonella was inactivated for film with 0.1% wt of silver ions and no viral infection has been found in the same conditions (martínez-abad et al., 2013). the results of the case studies on biodegradable packaging applied to fresh-cut fruits and vegetables and in some cases combined with edible coating are summarized in table 3. conclusions foodstuffs are dynamic systems with a limited shelf-life and specific requirements in terms of packaging. in order to ensure the highest standards of product quality, the right combination of product characteristics and packaging film is the basis for the development of appropriate storage conditions. this work focuses on the potential applications of biodegradable packaging and edible coating for minimally processed foods. the results highlight how the biodegradable films may have a more or less advantageous performance in relation to the physiological characteristics of 16 ital. j. food sci., vol. 27 2015 table 3 application and combination of biopolymer with coating for fresh-cut fruits and vegetables packaging. food product biopackaging property of materials main results reference iceberg lettuce polyester-based biodegradable film good gas barrier properties shelf-life extension del nobile et al. (2008) table grape (vitis vinifera) polyester-based biodegradable film /nylon and polyolefin layer high gas barrier good microbiological stability; good sensory characteristics del nobile et al. (2009) (ethanol pre-treatment before packaging) zucchini (cucurbita pepo) biodegradable co-extruded polyesters high permeability to water vapor high percentage of weight loss lucera et al. (2010) artichoke biodegradable monolayer film based on a blend of biodegradable polyesters permeability to water vapor high percentage of weight loss; good microbiological stability del nobile et al.( 2009) coating with sodium alginate (pre-treatment before packaging) barrier to water vapour shelf-life extension (200%) low water loss lampascioni (muscari comosum) polyester-based biodegradable film high permeability to water vapor high percentage of weight loss conte et al. (2009) coating with sodium alginate (pre-treatment before packaging) barrier to water vapour and oxygen lower water loss; low microbial growth. reduced browning process cactus pear fresh-cut pears (pyrus communis) monolayer film, based on a blend of biodegradable polyesters high permeability to water vapour high percentage of weight loss. del nobile et al. (2009) krasnova et al. (2013) coating with alginates (pre-treatment before packaging) pla films coated with a barrier of pure silicon oxide (siox) barrier to water vapour and other agents improved barrier properties shelf-life extension to about 13 days better maintenance of color an firmness with respect to conventional pe packaging melon blend of biodegradable polyester high permeability to water vapor high percentage of weight loss conte et al. (2009) complex matrices, such as fruit and vegetables, characterized by an active metabolism which significantly affects their conservation. certainly, biopolymers fulfill the environmental requirements but they have some limitations in terms of performance, like thermal resistance, barrier and mechanical properties. better results can be achieved by the combined use of biodegradable packagings with edible coatings. the use of edible films and coatings represents an environmentally-friendly technology that offers substantial advantages for shelf-life prolongation of many food products, including fruits and vegetables. the development of new natural edible films and coatings with either inherent microbicidal activity or the addition of antifungal ingredients (food preservatives, essential oils, antagonistic microorganisms, etc.) to improve the quality of fresh-cut fruits and vegetables is a technological challenge for the industry and a very active field of research worldwide. edible coatings can keep product characteristics, such as texture and hydration intact, and they can provide significant fruit senescence retardation. moreover, they serve as a carrier for a wide range of food additives, including anti-browning or antimicrobial agents. new challenges should focus on the development of tailor-made coatings, containing the most appropriate film forming constituents and active ingredients for fresh and minimally processed fruits and vegetables, according to specific industrial needs. new trends have shown that enrichment of biodegradable materials with silver-montmorillonite nanoparticles may be a promising technique. undoubtedly, biodegradation offers an attractive route to environmental waste management. however, the actual application of these packaging solutions to food is still limited, due to the high cost of raw materials and the smallscale production. in the future, when the quanital. j. food sci., vol. 27 2015 17 tities processed have increase to allow 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